CN114057812A

CN114057812A - Neuraminidase substrate, kit containing substrate and application of kit

Info

Publication number: CN114057812A
Application number: CN202010937209.6A
Authority: CN
Inventors: 万士林; 何海音
Original assignee: Hefei Huajin Biotechnology Co ltd
Current assignee: Hefei Huajin Biotechnology Co ltd
Priority date: 2020-09-08
Filing date: 2020-09-08
Publication date: 2022-02-18

Abstract

The invention discloses a neuraminidase substrate, a kit containing the substrate and application thereof, wherein a compound of formula (II) or a salt thereof:

wherein R is¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹And R¹²Each independently is hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, hydroxy, or alkoxy; r⁹、R¹⁰、R¹¹And R¹²At least one of which is not a hydrogen radical. The compound of formula (II) and the compound of formula (III) of the invention are used as substrates of neuraminidase, and the compounds areThe sensitivity of detecting the neuraminidase is about 4 times that of IBX-4041, and the improvement of the sensitivity means that the using amount of a patient sample can be obviously reduced in the process of clinically detecting the neuraminidase, and the method is suitable for detecting the patient sample with low content of the neuraminidase. The compounds of formula (II), compounds of formula (III) or salts thereof according to the invention are more suitable as detection reagents for automated analyzers than IBX-4041.

Description

Neuraminidase substrate, kit containing substrate and application of kit

Technical Field

The invention relates to a reagent for in vitro detection, in particular to a neuraminidase substrate, a kit containing the neuraminidase substrate and application of the neuraminidase substrate in preparation of a reagent for detecting the activity of neuraminidase.

Background

Bacterial Vaginosis (BV) is one of the major causes of abnormal vaginal secretions in adult women. The vaginal flora is normally dominated by hydrogen peroxide-synthesizing lactobacilli, but in BV patients, overgrown gardnerella (also known as Haemophilus), Mycoplasma hominis, and anaerobic flora such as Bacteroides and Prevotella predominate, with the result that the normal metabolism of the body is disturbed (Hill, G.B. the microbiology of bacterial vaginosis. am. J.Obstet. Gynecol.1993,169: 450). BV can also cause endometritis, pelvic inflammation, venereal disease, HIV infection and other gynecological diseases if untreated in time (a. faro, S.; et al. variable flow and physiological inflammation disease. am. J. Obstet. Gynecol.1993,169: 47; b. taha, T.E. et al. HIV infection and disorders of variable flow reducing prognosis. J. acquired Imm. def. Syndr.1999,20: 52). However, if symptomatic antibiotics can be used in time, BV can be completely cured, so that the quick and accurate diagnosis of BV is the key to the treatment of BV and the prevention of other gynecological syndromes.

BV diagnosis has conventionally been carried out by the Amsel gold standard method and Nugent scoring method (Nugent, R.P.; et al.Reliablility of diagnosis of bacterial variation by a stabilized method of Gram stand interpretion.J.Clin.Microbiol.1991, 29: 297). The traditional methods have low sensitivity and long testing time, and take the Amsel gold standard method as an example, 3 of the following 4 indexes are considered to be clinically diagnosed as BV: (1) vaginal pH > 4.5; (2) vaginal secretion is increased, becomes thin like milk and has peculiar smell; (3) the amine test is positive, namely 10 percent KOH is added into vaginal secretion to generate fishy smell; (4) clue cells are visible in the vaginal smear. However, in practice, there are more problems, firstly, factors unrelated to infection, such as recent sexual intercourse, vaginal douche, menstruation or the presence of large amounts of cervical mucus and the like, can cause the vaginal pH to rise, or produce an unpleasant odor in the secretions; the identification of odor in the amine test varies from person to person; the identification of clue cells as the best marker for BV diagnosis is influenced by the quality of microscopic equipment, the collection of samples, the experience of operators and other factors, and has strong subjectivity, so that the judgment results are difficult to count and compare.

Many findings suggest that some bacterially produced enzymes, particularly neuraminidase, are important indicators of the onset of BV (Briseden, A.N.; et al., sialidases (neuroamididases) in bacterial vacuities and bacterial vacuities-associated microfluora.J.Clin. Microbiol.1992,30: 663). Sialidase cleaves alpha-ketose bonds to hydrolyze sialic acid residues from glycoproteins and glycolipids (Lee Y, Youn HS, Lee JG, An JY, Park KR, Kang JY, Ryu YB, Jin MS, Park KH, Eom SH. "Crystal Structure of the catalytic domain of Clostridium perfringens neuroamidinate in complex with a non-carbohydrate-based inhibitor,2- (cyclohexenylamino) ethanosulfonic acid", Biochem Biophys Commun.2017,486(2): 470-. Elevated levels of neuraminidase in vaginal secretions are closely associated with the pathogenesis of BV. Many research groups report that the diagnosis of BV based on neuraminidase activity has the advantages of high sensitivity, high specificity and high positive and negative correlation. It has been generally accepted by scientific research institutions and hospitals in the field to determine the activity of neuraminidase to achieve rapid and accurate diagnosis of BV.

For measuring the neuraminidase activity, a substrate for neuraminidase can be used. The enzyme substrate produces fluorescence or color after the enzymatic hydrolysis reaction, and therefore the activity of neuraminidase can be measured based on the fluorescence intensity, color intensity, and rate of fluorescence and color production after the enzymatic hydrolysis reaction (a. Fujii I, Iwabuchi Y, Teshima T, Shiba T, Kikuchi M. "X-Neu 5Ac: a novel substrate for chromogenic assay of neuronal activity in bacterial expression systems", Bioorganic & medicinal chemistry,1993,1(2): 147-9; b. Johnson SC, Saeed A, Luo M. 6512100B1).

BV blue (BVBlue) kit, a novel biochemical Diagnostic reagent, which was first developed and marketed according to the above-mentioned principles in 1999 by Griyphus Diagnostic, LLC. The Kit contains a substrate for neuraminidase, called IBX-4041(a. Osom BVBlue Kit instructions, Sekisui Diagnostics, LLC; B. Bradshaw C, Morton A, Garland S, Horvath L, Kuzevska I, Fairley C. "Evaluation Of a Point-Of-Care Test, BVBlue, and Clinical and Laboratory Criteria for Diagnosis Of Bacterial Vaginosis", J. Clinical Microbiology,2005,43(3):1304-8.), which produces blue under alkaline conditions after enzymatic hydrolysis, and the blue Kit made with this substrate is a reliable Clinical tool for the Diagnosis Of Bacterial Vaginosis and has therefore been widely used in various countries in recent years (Diakzil, Romanski B, Johnson S. 1925. BV. Microdiagnosis, R. Microb., Yorkii, R. Microdiagnosis, Inc. 3, Microdiagnosis Of bacteriological, Va. Microbiol. J. Microbiol. A. As shown in FIGS.

However, BV blue is a manually operated kit, which takes a long time to detect, uses a large amount of sample, and is cumbersome to operate. With the development of clinical diagnostic automation, there has been a trend toward implementing BV detection automation. The essence of automatic BV diagnosis is that an automatic analyzer is used for simultaneously measuring the indexes, namely, hydrogen peroxide, proline aminopeptidase, leukocyte esterase, beta-glucuronidase, pH value and the like while detecting the neuraminidase. Therefore, for the key index of neuraminidase, the automatic determination method not only requires the sensitivity to be obviously improved compared with the manual BV blue method, but also requires the good specificity to be maintained. To do this, the substrate used for the automated assay must be hydrolyzed by the neuraminidase enzyme to a specific color, e.g., blue or red, more rapidly than IBX-4041 in the BV blue kit, but not in the absence of the neuraminidase enzyme.

Disclosure of Invention

In order to solve the problems that the BV blue kit requires a large sample amount and is complicated to operate, the invention aims to provide a neuraminidase substrate, and the sensitivity of the substrate is improved when the substrate is used for detecting the activity of the neuraminidase and is 4 times or more than 4 times that of the BV blue kit; in addition, the present invention also provides a kit comprising the neuraminidase substrate; in addition, the invention also provides application of the preparation of the reagent for detecting the activity of the neuraminidase by adopting the neuraminidase substrate.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, there is provided a compound of formula (II):

wherein,

R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹and R¹²Each independently is hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, hydroxy, or alkoxy;

R⁹、R¹⁰、R¹¹and R¹²At least one of which is not a hydrogen radical.

Preferably, wherein R is¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹And R¹²Each independently selected from hydrogen, lower alkyl, hydroxy or lower alkoxy.

Preferably, wherein formula (II) is selected:

in a second aspect of the present invention, there is provided a use of the compound of formula (II) or a salt thereof as described above in the preparation of a kit for detecting neuraminidase.

In a third aspect of the present invention, there is provided a use of the compound of formula (II) or a salt thereof as described above in the preparation of a reagent for detecting bacterial vaginosis.

The fourth aspect of the invention provides a bacterial vaginosis determination kit, which comprises a dry chemical reaction device, a diluent and a color developing agent; the dry chemical reaction device is a reaction device containing a plurality of dry chemical reaction blind holes, wherein at least one blind hole is internally provided with a neuraminidase substrate reagent pad, and the neuraminidase substrate reagent pad is prepared by soaking the neuraminidase substrate reagent pad in a reagent containing the compound of the formula (II) or a salt thereof, and then drying the soaked substrate reagent pad under reduced pressure.

In a fifth aspect of the present invention, there is provided a bacterial vaginosis assay kit, comprising a dry chemical reaction device, a diluent and a color-developing agent, wherein the dry chemical reaction device is a reaction device comprising a plurality of dry chemical reaction blind holes, and a reagent comprising the compound of formula (II) or a salt thereof is placed in at least one of the blind holes, and is dried under reduced pressure to obtain the bacterial vaginosis assay kit.

In a sixth aspect of the present invention, there is provided a bacterial vaginosis assay kit, comprising a chemical reaction device, a diluent and a color-developing agent, wherein the chemical reaction device is a reaction device comprising a plurality of chemical reaction blind holes, and a reagent comprising the compound of formula (II) or a salt thereof is placed in at least one of the blind holes.

In a seventh aspect of the invention, there is provided a compound of formula (III):

wherein R is¹And R⁴Each independently is hydrogen, halo, methyl, ethyl, or n-propyl; r²、R³、R⁵、R⁶、R⁷And R⁸Each independently is hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, hydroxy, or alkoxy.

Preferably, the formula of formula (III) is:

in an eighth aspect of the present invention, there is provided a use of the compound of formula (III) or a salt thereof as described above in the preparation of a kit for detecting neuraminidase.

In a ninth aspect of the present invention, there is provided the use of a compound of formula (III) or a salt thereof as described above in the preparation of a reagent for detecting bacterial vaginosis.

The tenth aspect of the invention provides a bacterial vaginosis assay kit, which comprises a dry chemical reaction device, a diluent and a color developing agent; the dry chemical reaction device is a reaction device comprising a plurality of dry chemical reaction blind holes, wherein at least one blind hole is internally provided with a neuraminidase substrate reagent pad, and the neuraminidase substrate reagent pad is prepared by soaking the neuraminidase substrate reagent pad in a reagent containing the compound of the formula (III) or a salt thereof, and then drying the soaked substrate reagent pad under reduced pressure.

In an eleventh aspect of the present invention, there is provided a bacterial vaginosis assay kit, comprising a dry chemical reaction device, a diluent and a color-developing agent, wherein the dry chemical reaction device is a reaction device comprising a plurality of dry chemical reaction blind holes, and a reagent comprising the compound of formula (III) or a salt thereof is placed in at least one of the blind holes and dried under reduced pressure.

In a twelfth aspect of the present invention, there is provided a bacterial vaginosis assay kit, comprising a chemical reaction device, a diluent and a color-developing agent, wherein the chemical reaction device is a reaction device comprising a plurality of blind holes for chemical reaction, and a reagent comprising the compound of formula (III) or a salt thereof is placed in at least one of the blind holes.

In a thirteenth aspect of the invention, there is provided an intermediate compound of formula (I):

wherein R is¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹And R¹²Each independently is hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, hydroxy, or alkoxy.

Preferably, wherein formula (I) is selected:

compared with the prior art, the invention has the following beneficial effects: the compound of formula (II), the compound of formula (III) or the salt thereof is a substrate of neuraminidase, enzymatic hydrolysis reaction is carried out under the catalysis of the neuraminidase, and the hydrolysis product shows red, purple or blue; by reacting the compound of formula (II) and the compound of formula (III) with IBX-4041 of BV blue in the same amount, the compound of formula (II) and the compound of formula (III) are found to show weak positive when 2 muL of the neuraminidase working mother liquor is added, and show positive or strong positive when 4 muL or more of the neuraminidase working mother liquor is added; while IBX-4041 showed negative results when less than 8. mu.L of the neuraminidase working stock solution was added, weak positive results when 8. mu.L was added, and positive results when 16. mu.L and 32. mu.L were added, the compounds of formula (II) and (III) were used as substrates for neuraminidase, and the sensitivity for detecting neuraminidase was about 4 times that of IBX-4041. This improved sensitivity means that patient sample volumes can be significantly reduced in clinical testing of neuraminidase, and means that it is suitable for testing patient samples with low levels of neuraminidase. The compounds of formula (II), compounds of formula (III) or salts thereof according to the invention are more suitable as detection reagents for automated analyzers than IBX-4041.

Drawings

The invention is described in further detail below with reference to the following figures and detailed description:

FIG. 1 is a UV spectrum of Compound 1(1:1 acetonitrile/water);

FIG. 2 is a UV spectrum of Compound 1' (1:1 acetonitrile/water);

FIG. 3 is a UV spectrum of Compound 7 (1:1 acetonitrile/water);

FIG. 4 is a UV spectrum of Compound 8 (1:1 acetonitrile/water);

FIG. 5 is a UV spectrum of Compound 10 (1:1 acetonitrile/water);

FIG. 6 is a graph showing the color change after reaction of Compound 7 with different doses of neuraminidase;

FIG. 7 is a graph showing the color change after reaction of Compound 8 with different doses of neuraminidase;

FIG. 8 is a graph showing the color change following reaction of Compound 10 with different doses of neuraminidase;

FIG. 9 shows the color change after reaction of IBX-4041 with different doses of neuraminidase.

Detailed Description

Unless otherwise indicated, the following terms used in the specification and claims have the meanings discussed:

the term "substrate of an enzyme" or "substrate" as used herein is defined as a compound that participates in an enzyme-catalyzed reaction, by which the substrate may be converted into another compound or compounds.

"sialidase substrate" is defined as a compound that participates in a reaction catalyzed by a sialidase, (1) another compound or compounds can be produced by the reaction catalyzed by the sialidase; (2) the resulting compounds may exhibit color under certain conditions (e.g., basic conditions). Colors include, but are not limited to, red, purple, and blue.

The term "effective amount" is defined as an amount of an agent, combination of agents, substrate, or combination of substrates that produces the desired effect.

The term "clinical automated testing" or "automated testing" is defined as testing a patient sample using automated means, electronically or mechanically controlled or a combination of both, to obtain qualitative, semi-quantitative or quantitative analysis of the patient sample. The automated detection process may include all or part of the following exemplary procedures: (1) adding a reagent, (2) adding a patient sample, (3) uniformly mixing, (4) incubating, (5) adding a color-developing agent, (6) carrying out color comparison or development, (7) carrying out color processing, (8) carrying out data measurement, and (9) carrying out data processing.

"Hydrogen radical" is-H.

"halo" represents fluoro, chloro, bromo or iodo, preferably fluoro, chloro.

Unless otherwise specified, "alkyl" is defined as a straight or branched saturated carbon chain radical of one to about ten carbon atoms. Preferred alkyl groups are "lower alkyl" groups having from one to about five carbon atoms. Wherein one or more hydrogen atoms may also be substituted with a substituent selected from the group consisting of: acyl, amino, amido, acyloxy, alkoxycarbonyl, carboxy, carboxamido, cyano, halogen, hydroxy, nitro, mercapto, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl (including indolyl or substituted indolyl, etc.), alkoxy, aryloxy, sulfinyl, sulfonyl, oxo (oxo), guanidino, formyl, and amino acid side chains. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, t-butyl, isopropyl, and methoxymethyl.

"lower alkyl" is defined as a straight or branched saturated carbon chain radical of 1 to 4 carbon atoms, such as methyl, ethyl, propyl.

"n-propyl" is-CH₂CH₂CH₃。

"alkenyl" is defined as a straight or branched chain group having from 2 to about 10 carbon atoms, preferably from 3 to about 10 carbon atoms, and containing at least one carbon-carbon double bond. Wherein one or more hydrogen atoms may also be substituted with a substituent selected from the group consisting of: acyl, amino, amido, acyloxy, alkoxycarbonyl, carboxy, carboxamido, cyano, halogen, hydroxy, nitro, mercapto, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl (including indolyl or substituted indolyl, etc.), alkoxy, aryloxy, sulfinyl, sulfonyl, formyl, oxo, and guanidino. The double bond portion of the unsaturated hydrocarbon chain may be in either the cis or trans configuration. Examples of alkenyl groups include, but are not limited to, vinyl or phenylvinyl.

"cycloalkyl" or "cycloalkyl ring" is defined as a monocyclic, fused or polycyclic group containing a three to twelve membered saturated carbocyclic ring. Preferred cycloalkyl groups should contain a three to eight membered saturated carbocyclic ring system. Wherein one or more hydrogen atoms may also be substituted with a substituent selected from the group consisting of: acyl, amino, amido, acyloxy, alkoxycarbonyl, carboxy, carboxamido, cyano, halogen, hydroxy, nitro, mercapto, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl (including indolyl or substituted indolyl, etc.), alkoxy, aryloxy, sulfinyl, sulfonyl, and formyl. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclohexyl, and cycloheptyl.

"cycloalkenyl" refers to monocyclic, fused or polycyclic groups containing three to twelve membered unsaturated or partially unsaturated carbocyclic rings. Preferred cycloalkenyl groups should contain a three to eight membered unsaturated carbocyclic ring system. Wherein one or more hydrogen atoms may also be substituted with a substituent selected from the group consisting of: acyl, amino, amido, acyloxy, alkoxycarbonyl, carboxy, carboxamido, cyano, halogen, hydroxy, nitro, mercapto, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl (including indolyl or substituted indolyl, etc.), alkoxy, aryloxy, sulfinyl, sulfonyl, and formyl. Examples of cycloalkenyl groups include, but are not limited to, cyclopentenyl, cyclohexenyl, and cycloheptenyl.

"hydroxy" is-OH.

"alkoxy" is defined as an oxygen-containing group substituted with an alkyl, cycloalkyl, or heterocyclyl group. Examples include, but are not limited to, methoxy, t-butoxy, benzyloxy, and cyclohexyloxy.

"lower alkoxy" is defined as an oxygen-containing group substituted with lower alkyl and includes methoxy, ethoxy, propoxy.

"salts" refers to those salts that retain the biological potency and properties of the parent compound. These salts include, but are not limited to: a salt formed by replacing a proton of an acidic group (e.g., a carboxyl group, a phenolic hydroxyl group, a hydroxyl group, etc.) contained in the parent compound with a metal ion (e.g., an ion such as a sodium, potassium, lithium, etc., an ion such as a magnesium, calcium, etc., an alkaline earth metal, an ion such as an aluminum, iron, etc.) or a metal complex ion, or a salt or a complex compound formed with an organic base (e.g., ammonia, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, other alkylamines or cycloalkylamines, ethanolamine, diethanolamine, triethanolamine, trimethylolaminomethane, N-methylglucamine, etc.).

"color developer" is defined as an agent that develops color of a product after a biochemical reaction.

An "alkaline developer" is defined as an alkaline agent that develops the color of the product of a biochemical reaction.

"buffer" is defined as an agent that, when added to a solution in amounts of acid and base, retards the change in pH of the solution.

The present invention discloses compounds of formula (II) or salts thereof:

as a preferred embodiment, R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹And R¹²Each independently is hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, hydroxy, or alkoxy; r⁹、R¹⁰、R¹¹And R¹²At least one group is not a hydrogen group; preferably, R is¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹And R¹²Each independently selected from hydrogen, lower alkyl, hydroxy or lower alkoxy; more preferably, R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹And R¹²Each independently selected hydrogen radical or lower alkyl; more preferably, R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹And R¹²Each independently selected hydrogen, methyl, ethyl or propyl.

As another preferred embodiment, formula (II) is selected:

the present invention discloses compounds of formula (III) or salts thereof:

Preferably, R is¹And R⁴Each independently is hydrogen, lower alkyl; r²、R³、R⁵、R⁶、R⁷、R⁸Each independently is hydrogen, lower alkyl, hydroxy or lower alkoxy.

As another preferred embodiment, R⁴And R⁵Each independently is hydrogen, methyl, ethyl or n-propyl; r¹、R²、R³、R⁶、R⁷、R⁸Each independently is hydrogen, lower alkyl, hydroxy or lower alkoxy; preferably, R is¹、R²、R³、R⁶、R⁷、R⁸Each independently is hydrogen or lower alkyl; more preferably, R⁶、R⁷Each independently is lower alkyl; more preferably, R⁶、R⁷Each independently being methyl, ethyl or propyl.

As another preferred embodiment, formula (III) is selected

The compound of formula (II) or (III) or the salt thereof is a neuraminidase substrate, and the neuraminidase substrate and a color developing agent are matched for use, so that the existence and the activity of the neuraminidase in a system can be indicated.

The invention discloses a kit, which comprises a compound of formula (II) or a salt thereof (or a compound of formula (III) or a salt thereof) and a color developing agent, wherein the color developing agent is an alkaline color developing agent, and is preferably NaOH solution or KOH solution. The concentration of the color developer can be prepared according to actual conditions, such as the concentration of 1 mol/L.

The compound of formula (II) or formula (III) disclosed by the invention can be used for detecting the activity of neuraminidase, and further can be used for detecting bacterial vaginosis.

The level of neuraminidase in vaginal secretions is closely related to the onset of BV, and BV can be diagnosed quickly and accurately by determining the activity of the neuraminidase. In order to make BV diagnosis more accurate and reliable, hydrogen peroxide, proline aminopeptidase, leukocyte esterase, beta-glucuronidase, pH value and the like are measured while neuraminidase is detected.

The invention discloses a bacterial vaginosis assay kit which can be used for independently detecting the activity of neuraminidase and simultaneously detecting hydrogen peroxide, proline aminopeptidase, leukocyte esterase, beta-glucuronidase or pH value and the like. When the combined determination of hydrogen peroxide, proline aminopeptidase, leukocyte esterase, beta-glucuronidase and pH is desired, it can be carried out in the manner disclosed in the prior art.

For example, in the 'combined determination kit for bacterial vaginosis' disclosed in application No. 200610056827.x, a hydrogen peroxide concentration reagent pad, a leukocyte esterase concentration reagent pad and a neuraminidase activity reagent pad are respectively arranged in a dry chemical reaction device with blind holes, and when in use, a cotton swab is used for taking secretion from the fornix vaginae and then diluent is added for diluting the secretion; dripping a drop of diluted vaginal fluid into the hydrogen peroxide concentration reagent pad, the leucocyte esterase concentration reagent pad and the neuraminidase activity reagent pad respectively; adding 1 drop of color developing solution into the hole of the sialidase activity reagent pad; and finally standing at room temperature for 30min or standing in a water bath box at 37 ℃ for 15 min. Interpretation results: hydrogen peroxide index: normally red or purple, and abnormally blue; neuraminidase indices: the color is not developed normally, and the positive is the color development of red or purple; leukocyte esterase index: normally, no color development, and positive color development of blue or green. The kit can be used for simultaneously measuring the hydrogen peroxide concentration, the leukocyte esterase and the neuraminidase activity, and respectively reflects the genital tract micro-ecological environment, the secretion cleanliness and the pathogenic bacteria state of the bacterial vaginosis, so that the genital tract secretion detection is more comprehensive.

"a kit for the combined determination of aerobic and bacterial vaginosis" as disclosed in application No. 2015102549516, comprising a chromogenic agent and reaction means including a hydrogen peroxide reaction pad, a neuraminidase reaction pad, a leukocyte esterase reaction pad, a β -glucuronidase reaction pad and a coagulase reaction pad. When the kit is used, a straw is used for sucking sample liquid, 1 drop of sample liquid is respectively dropped on a hydrogen peroxide reaction pad, a neuraminidase reaction pad, a leukocyte esterase reaction pad, a beta-glucuronidase reaction pad and a coagulase reaction pad on a reaction device, the reaction device is placed in a 37 +/-1 ℃ water bath box or a 48 ℃ dry bath device for developing for 15 minutes, 1 drop of developing liquid A is added on the neuraminidase reaction pad, 1 drop of developing agent B is added on the coagulase reaction pad, and the result is judged and read after 30 seconds, so that the hydrogen peroxide, the neuraminidase, the leukocyte esterase, the beta-glucuronidase and the coagulase can be simultaneously detected to diagnose aerobic and bacterial vaginosis.

The invention discloses a bacterial vaginosis determination kit, which comprises a dry chemical reaction device, diluent and a color developing agent; the dry chemical reaction device is a reaction device comprising a plurality of dry chemical reaction blind holes, wherein at least one blind hole is internally provided with a neuraminidase substrate reagent pad, and the neuraminidase substrate reagent pad is prepared by soaking the neuraminidase substrate reagent pad in a reagent containing the compound of the formula (II) or a salt thereof (the compound of the formula (III) or a salt thereof) and drying the soaked reagent pad under reduced pressure.

The invention also discloses a bacterial vaginosis determination kit, which comprises a dry chemical reaction device, diluent and a color developing agent, wherein the dry chemical reaction device is a reaction device containing a plurality of dry chemical reaction blind holes, and a reagent containing the compound of the formula (II) or the salt thereof (the compound of the formula (III) or the salt thereof) is placed in at least one blind hole and is dried under reduced pressure to prepare the bacterial vaginosis determination kit.

The invention also discloses a bacterial vaginosis determination kit, which comprises a chemical reaction device, diluent and a color developing agent, wherein the chemical reaction device is a reaction device containing a plurality of chemical reaction blind holes, and a reagent containing the compound of the formula (II) or the salt thereof (the compound of the formula (III) or the salt thereof) is placed in at least one blind hole.

Wherein, the developer is alkaline developer, preferably NaOH solution or KOH solution.

As an embodiment, the kit further comprises a buffer solution, preferably a potassium acetate buffer solution.

The invention takes the formula (I) as an intermediate to prepare the compound shown in the formula (II) or the formula (III), and the compound shown in the formula (II) or the formula (III) is subjected to enzymatic hydrolysis reaction under the action of neuraminidase to generate the intermediate shown in the formula (I).

The invention discloses an intermediate compound shown as a formula (I) or a salt thereof:

as a preferred embodiment, R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹And R¹²Each independently is hydrogen, halo, alkyl, alkenyl, cycloalkyl, cycloalkenyl, hydroxy, or alkoxy; preferably, R is¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹And R¹²Each independently selected from hydrogen, lower alkyl, hydroxy or lower alkoxy; more preferably, R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹And R¹²Each independently selected hydrogen radical or lower alkyl; more preferably, R⁴、R⁵Each independentlySelected from hydrogen, methyl, ethyl or n-propyl.

As a preferred embodiment, R⁹、R¹⁰、R¹¹And R¹²At least one of which is not a hydrogen radical.

As another preferred embodiment, R⁹、R¹⁰、R¹¹And R¹²Each hydrogen radical is selected.

As another preferred embodiment, formula (I) is selected:

the compounds of the formula (I) are colorless in aqueous solutions at pH < 10.2 and develop a blue color in aqueous solutions at pH >12, pH10.2 to 12 being the transition range of the compounds of the formula (I) from colorless to blue.

Thus, the compounds of formula (I) of the present invention may be used as pH indicators. When used as a pH indicator, the intermediate compound of formula (I) or a salt thereof is usually diluted with water.

The preparation method of the intermediate compound shown as the formula (I) comprises the following steps:

reaction formula-1

The weight ratio of 2: 1 and 4-methylphthalic anhydride in the presence of sulfuric acid or a lewis acid such as tin tetrachloride (SnCl4) to produce a mixture of compounds 1 and 1'. The mixture was purified by chromatography to give pure 1 and 1' white powders.

Other compounds of formula (I) can be produced by heating to effect Friedel-Crafts condensation reaction by changing the substituent group of 2, 5-dimethylphenol and/or the substituent group of 4-methylphthalic anhydride.

The preparation of the compound of formula (II) is as follows:

reaction formula-2

N-acetyl-D-neuraminic acid methyl ester (3) with pure acetyl Chloride (CH)₃COCl) reaction to produce N-acetyl-4, 7, 8, 9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminic acid methyl ester (4). Compounds 4 and 1 are described in the reaction of compounds such as sodium methoxide (NaOCH)₃) The compound 5 can be coupled to generate 5 under the action of strong alkali, and the compound 5 can be converted into a final product 7 through alkaline hydrolysis.

Replacement of 1 in the above formula with 1' gives the final product 8, see equation-3.

Reaction formula-3

Other compounds in the formula (I) (wherein, R⁹、R¹⁰、R¹¹And R¹²Not simultaneously hydrogen) to couple with 4, and the product of this coupling reaction is hydrolyzed with base to yield the other compound of formula-II.

The preparation of the compound of formula (III) is as follows:

reaction formula-4

Other compounds in the formula (I) (wherein, R⁹、R¹⁰、R¹¹And R¹²And simultaneously hydrogen) to couple compound 1 with compound 4, and the product of this coupling reaction can be hydrolyzed with base to yield the other compound of formula-III.

The following preparation examples and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. They should not be considered as limiting the scope of the invention but merely as being illustrative and representative thereof.

EXAMPLE 1 Synthesis of Compounds 1 and 1

2, 5-dimethylphenol (1.2 g, 2.0 equivalents) and 4-methylphthalic anhydride (0.76 g, 1.1 equivalents) were placed in a 50 ml round bottom flask, mixed well, heated to 100 ℃ and gradually melted, and then anhydrous tin tetrachloride (0.6 g, 0.5 equivalents) was added in portions over 15 minutes with stirring. The mixture was stirred at 100 ℃ for 30 minutes to give a red oil.

The reaction product was cooled to room temperature and dissolved in methanol (5.0 ml), and this solution was stirred with 1.0M sodium hydroxide (20 ml) to give a blue solution, and after filtration, the filtrate was neutralized to pH 7.0 with 3.6% hydrochloric acid, and then a precipitate was obtained by centrifugation (4000 rpm). The precipitate was washed twice with water and dried in vacuo to give a pale yellow solid (1.95 g) which was a mixture of 1 and 1' as indicated by HPLC analysis (ca. 2: 3).

The above mixture (520 mg) was dissolved in a small amount of methanol, followed by reverse phase high pressure liquid phase separation to obtain compound 1(210.2 mg) and 1' (285.6 mg), both amorphous white powders. A chromatographic column: phenomenex carbon-18 chromatography column (10um,250x 30mm, 120A); solvent system linear gradient (40% to 100% acetonitrile/water) + 0.01% trifluoroacetic acid at a flow rate of 20 ml/min for 25 min.

Physicochemical properties of compound 1:

molecular weight: 388; mass Spectrometry (ESI, positive polarity) m/z 389.2 (MH)⁺)；

The ultraviolet spectrogram is shown in figure 1(1:1 acetonitrile + water);

nmr hydrogen spectrum data (chemical shift, 400 mhz, 1:1 deuterated methanol/deuterated dimethyl sulfoxide):

δ_H 7.78(1H,br d,8.0Hz),7.74(1H,br d,8.0Hz),7.25(1H,br s),6.70(2H,s),6.62(2H,s),2.49(3H,s),2.02(6H,s),1.95(6H,s)。

physicochemical properties of compound 1':

The ultraviolet spectrum is shown in FIG. 2(1:1 acetonitrile + water);

nmr hydrogen spectrum data (chemical shift, 400 mhz, deuterated methanol):

δ_H 7.69(1H,br s),7.58(1H,br d,8.0Hz),7.28(1H,br d,8.0Hz),6.68(2H,s),6.57(2H,s),2.48(3H,s),2.02(6H,s),1.96(6H,s)。

EXAMPLE 2 color change of Compounds 1 and 1' with different pH values

Hydrochloric acid (pH <7.0) or sodium hydroxide (pH >7.0) is used in combination with distilled water to make an aqueous solution having a pH of 1.0 to 14.0. During the formulation, a Corning 430pH meter was used to measure pH.

Compounds 1 and 1 'were dissolved in each of the above solutions (concentration of compounds 1 and 1' was 50 μ g/ml), respectively, and then color was observed and the results were recorded in table 1 below.

TABLE 1

From table 1, it is understood that compounds 1 and 1' are colorless in an aqueous solution having a pH of 10.0 or less, deep blue in an aqueous solution having a pH of 12.0 or more, and transition from light blue to deep blue in an aqueous solution having a pH of 10.2 to 12.0.

EXAMPLE 3 Synthesis of Compound 7

The synthetic route is shown in a reaction formula-2.

N-acetyl-D-neuraminic acid methyl ester white powder (3, 1.2 g) was suspended in 10.0 ml (1:20) acetic acid (CH)₃COOH)/acetyl Chloride (CH)₃COCl), the suspension was stirred at room temperature for 16 hours and the reaction was complete. The resulting colorless solution was evaporated at room temperature under reduced pressure to give methyl N-acetyl-4, 7, 8, 9-tetra-O-acetyl-2-chloro-2-deoxy-D-neuraminic acid as a white solid (4, 1.65 g).

Compound 1(205.0 mg, 1.0 eq) and compound 4(125.0 mg, 0.4 eq) were dissolved in anhydrous tetrahydrofuran (THF, 4.0 ml) to make solution A, and sodium methoxide powder (NaOCH) was added₃82 mg, 2.5 eq) was dissolved in anhydrous tetrahydrofuran (2 ml) to make solution B. Solution B was slowly added to solution A at low temperature with stirring (. about.10 ℃ C., 10 minutes) and the combined solution (dark blue) continued atStirring at room temperature for 30 minutes gave 5. The reaction product was neutralized with acetic acid (orange yellow) and evaporated to dryness at 40 ℃ under reduced pressure, and then dissolved in methanol (2.0 ml). The methanol solution was mixed with 0.3M aqueous LiOH (3.0 ml), followed by stirring at room temperature for 20 hours to hydrolyze to give final product 7.

The alkaline hydrolysate is neutralized with acetic acid, evaporated to-3.0 ml at-40 deg.c under reduced pressure and centrifuged to remove white flocculent precipitate (unreacted 1). The supernatant was extracted three times with ethyl acetate (2 ml) to wash the residual 1, and then the product 7 was extracted with n-butanol (2 ml). The n-butanol solution was extracted with water (0.5 ml) to remove salts, evaporated to dryness at 40 ℃ under reduced pressure and finally dried in vacuo to give 7 as a white powder (23.6 mg).

Physicochemical properties of compound 7:

molecular weight: 679; mass spectrum (ESI, positive polarity) m/z 680.2 (MH)⁺)；

The ultraviolet spectrum is shown in FIG. 3(1:1 acetonitrile + water);

nmr hydrogen spectrum data (chemical shift, 400 mhz, deuterated methanol):

δ_H 7.77(1H,br d,8.0Hz),7.74(1H,d,8.0Hz),7.24(0.5H,s),^a1 7.20(0.5H,s),^a27.22(1H,br s),6.80(1H,s),6.65(0.5H,s),^b1 6.64(0.5H,s),^b2 6.57(1H,s),3.86(1H,m),3.83(1H,m),3.81(1H,m),3.75(1H,m),3.74(1H,m),3.65(0.5H,dd,11.3,5.5Hz),^c13.64(0.5H,dd,11.3,5.5Hz),^c23.56(1H,dd,8.8,1.5Hz),2.85(0.5H,dd,12.5,3.9Hz),^d12.84(0.5H,dd,12.5,3.9Hz),^d2 2.47(3H,s),2.09(3H,s),2.01(6H,s),2.00(3H,s),1.98(1H,m),1.92(3H,s)。

in addition: due to the presence of two asymmetric rotamers in compound 7, there are 4 hydrogen spectral signals (a, b, c, d) that split into two sets of signals of intensity (1: 1), denoted a1 and a2, b1 and b2, c1 and c2, and d1 and d2, respectively.

EXAMPLE 4 Synthesis of Compound 8

The synthetic route is shown in a reaction formula-3.

By a similar preparation method to that of example 3, compound 1' (202.0 mg) was substituted for compound 1 to give 8 as a white powder (29.5 mg).

Physicochemical properties of compound 8:

The ultraviolet spectrum is shown in FIG. 4(1:1 acetonitrile + water);

nmr hydrogen spectrum data (chemical shift, 400 mhz, deuterated methanol):

δ_H 7.70(1H,br s),7.59(1H,d,8.0Hz),7.31(0.5H,br d,8.0Hz),^a1 7.29(0.5H,br d,8.0Hz),^a2 7.23(0.5H,s),^b1 7.21(0.5H,s),^b2 6.80(1H,s),6.65(0.5H,s),^c1 6.64(0.5H,s),^c2 6.56(1H,s),3.85(1H,m),3.83(1H,m),3.81(1H,m),3.76(1H,m),3.73(1H,m),3.65(0.5H,dd,11.3,5.5Hz),^d1 3.64(0.5H,dd,11.3,5.5Hz),^d2 3.555(0.5H,8.8,1.7Hz),3.552(0.5H,8.8,1.7Hz),2.85(0.5H,dd,12.8,3.5Hz),^e1 2.84(0.5H,dd,12.8,3.5Hz),^e22.48(3H,s),2.08(3H,s),2.005(6H,s),1.99(3H,s),1.98(1H,m),1.92(3H,s)。

in addition: due to the presence of two asymmetric rotamers in compound 8, there are 5 hydrogen spectral signals (a, b, c, d, e) that split into two sets of signals of intensity (1: 1), denoted a1 and a2, b1 and b2, c1 and c2, d1 and d2, and e1 and e2, respectively.

EXAMPLE 5 Synthesis of Compound 10

The synthetic route is shown in a reaction formula-4.

By a similar preparation method to that of example 3, compound 2(205 mg) was substituted for compound 1 to give 10 of a white powder (33.2 mg).

Physicochemical properties of compound 10:

molecular weight: 609; mass Spectrometry (ESI, positive polarity): m/z 610.2 (MH)⁺)；

The ultraviolet spectrum is shown in FIG. 5(1:1 acetonitrile + water);

nmr hydrogen spectrum data (chemical shift, 500 mhz, deuterated methanol):

δ_H 7.90(1H,d,7.5Hz),7.78(1H,br dd,7.5,7.5Hz),7.64(0.5H,br d,7.5Hz),^a17.63(0.5H,br d,7.5Hz),^a2 7.62(1H,br dd,7.5,7.5Hz),7.25(2H,br d,7.5Hz),7.19(2H,br d,7.5Hz),7.06(2H,br d,7.5Hz),6.75(2H,br d,7.5Hz),3.83-3.79(4H,m),3.73(1H,m),3.62(1H,m),3.53(1H,br d,7.5Hz),2.82(1H,br dd,12.5,4.0Hz),2.00(3H,s),1.92(1H,dd,12.5,12.5Hz)。

in addition: due to the presence of two asymmetric rotamers in compound 10, the hydrogen spectrum signal (a) splits into two sets of signals with intensities (1: 1), denoted as a1 and a 2.

Example 6 enzymatic hydrolysis and color development reaction of neuraminidase 1, and enzymatic hydrolysis and color development reaction of Compounds 7, 8 and 10 are shown in reaction formula-5

Reaction formula-5

Neuraminidase catalyzes the hydrolysis of substrates 7, 8, 10 to yield hydrolysates 1, 1' and 2, respectively. These hydrolysates show red, purple or blue colour in aqueous alkaline solutions at pH > 12.0. Whereas 7, 8, 10, which is not hydrolyzed by neuraminidase, shows a pale yellow color in the same aqueous alkaline solution. Therefore, these compounds are incubated with a patient sample in a buffer of pH 5-7 for a fixed period of time (typically about 30-36 ℃ C., 1-15 minutes) and then a basic solution (e.g., 1.0M KOH) is added, and the color change is used to determine the amount of neuraminidase contained in the sample. For 7 and 8, the blue color after reaction indicates that the sample has high enzyme content and fast catalytic hydrolysis reaction; light yellow indicates very low enzyme content without significant enzymatic hydrolysis; while green (mixed color of blue and yellow) indicates that enzymatic hydrolysis is occurring, but the enzyme content is low and the enzymatic hydrolysis reaction is slow. For 10, the purple-red color after the reaction indicates that the content of the neuraminidase in the sample is high; light yellow indicates very low enzyme content without significant enzymatic hydrolysis; a light purple red color indicates that enzymatic hydrolysis has occurred, but the enzyme content is lower and the enzymatic hydrolysis reaction is slower.

2. Process for enzymatic hydrolysis and color reaction of neuraminidase

(1) Materials:

solution A-Compound 7(10.0 mg) was dissolved in 0.5M potassium acetate buffer (pH 6.0,20 ml).

Solution Ether-Compound 8(10.0 mg) was dissolved in 0.5M potassium acetate buffer (pH 6.0,20 mL).

Solution C-Compound 10(10.0 mg) was dissolved in 0.5M potassium acetate buffer (pH 6.0,20 mL).

BV blue reagent-available from Sekisui Diagnostics, LLC, USA, product number 87905QW, containing substrate IBX-4041(0.5 mg/ml), 0.5M potassium acetate buffer, pH 6.0.

The structural formula of IBX-4041 is as follows:

neuraminidase solution (. alpha.2-3, 6,8 neuroamididase, # P0720s,50000 units/ml) -available from New England Biolab Inc., N.Y..

Neuraminidase working stock solution (250 units/ml) -prepared by diluting the above neuraminidase solution 200-fold with distilled water. Alkali solution-1.0M aqueous sodium hydroxide solution.

(2) The experimental steps are as follows:

(1) adding 0,2, 4, 8, 16 and 32 microliters (uL) of neuraminidase working mother liquor into the solution A (0.5 milliliter multiplied by 6 tubes), vibrating uniformly, keeping the temperature of each reaction tube at 30 ℃ for 12 minutes, then adding 50 microliters of alkali solution, shaking uniformly and generating color. The volume and final color of the neuraminidase working mother liquor for each reaction tube are shown in FIG. 6.

(2) Adding a certain volume of neuraminidase working mother liquor into the solution B (0.5 ml × 6 tubes), shaking uniformly, keeping the temperature of each reaction tube at 30 ℃ for 12 minutes, adding an alkali solution (50 microliters), and shaking uniformly to generate color. The volume and final color of the neuraminidase working stock solution for each reaction tube are shown in FIG. 7.

(3) Adding a certain volume of neuraminidase working mother liquor into the solution C (0.5 ml × 6 tubes), shaking uniformly, keeping the temperature of each reaction tube at 30 ℃ for 12 minutes, adding an alkali solution (50 microliters), and shaking uniformly to generate color. The volume and final color of the neuraminidase working stock solution for each reaction tube are shown in FIG. 8.

(4) Adding a certain volume of neuraminidase working mother liquor into a BV blue reagent (BVblue,0.5 ml. times.6 tube), shaking uniformly, keeping the temperature of each reaction tube at 30 ℃ for 12 minutes, adding an alkali solution (50 microliters), and shaking uniformly to obtain a color. The volume and final color of the neuraminidase working mother liquor for each reaction tube are shown in FIG. 9.

(3) Results of the experiment

The results shown in FIGS. 6-9 can be summarized in Table 2.

TABLE 2

Note that the colors in the table above are based on visual observations of the physical objects.

As can be seen from FIGS. 6-9 and Table 2, the BV blue group was negative when less than 8. mu.l of the neuraminidase working stock solution was added, weakly positive when 8. mu.l was added, and positive when 16 and 32. mu.l were added; the color reactions of both compounds 7 and 8 were very similar, with 2. mu.l of the neuraminidase working stock solution added showing weak positivity, and 4. mu.l or more of the neuraminidase added showing positive or strong positivity; compound 10 showed weak to positive on addition of 2 microliters of the neuraminidase working stock solution, while showed positive or strong positive on addition of 4 microliters or more of the neuraminidase working stock solution.

Thus, the sensitivity of compounds 7 and 8 is about 4 times that of the BV blue active ingredient IBX-4041, while the sensitivity of compound 10 is more than 4 times that of IBX-4041.

The foregoing detailed description is given by way of example only, to better enable one of ordinary skill in the art to understand the patent, and is not to be construed as limiting the scope of what is encompassed by the patent; any equivalent alterations or modifications made according to the spirit of the disclosure of this patent are intended to be included in the scope of this patent.

Claims

1. A compound of formula (II):

wherein,

R⁹、R¹⁰、R¹¹and R¹²At least one of which is not a hydrogen radical.

2. A compound of formula (II) or a salt thereof as claimed in claim 1 wherein R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹And R¹²Each independently selected from hydrogen, lower alkyl, hydroxy or lower alkoxy.

3. A compound of formula (II) or a salt thereof as claimed in claim 1 wherein formula (II) is selected from:

4. use of a compound of formula (II) or a salt thereof as claimed in any one of claims 1 to 3 in the manufacture of a kit for the detection of neuraminidase.

5. Use of a compound of formula (II) or a salt thereof as claimed in any one of claims 1 to 3 in the manufacture of a reagent for the detection of bacterial vaginosis.

6. A bacterial vaginosis determination kit is characterized by comprising a dry chemical reaction device, diluent and a color developing agent; the dry chemical reaction device is a reaction device comprising a plurality of dry chemical reaction blind holes, wherein at least one blind hole is provided with a neuraminidase substrate reagent pad, and the neuraminidase substrate reagent pad is prepared by drying under reduced pressure after being soaked with a reagent containing a compound of formula (II) or a salt thereof according to any one of claims 1 to 3.

7. A bacterial vaginosis assay kit, which is characterized by comprising a dry chemical reaction device, a diluent and a color-developing agent, wherein the dry chemical reaction device is a reaction device comprising a plurality of dry chemical reaction blind holes, a reagent containing the compound of the formula (II) or the salt thereof according to any one of claims 1 to 3 is placed in at least one of the blind holes, and the reagent is dried under reduced pressure to prepare the reagent kit.

8. A bacterial vaginosis assay kit, which comprises a chemical reaction device, a diluent and a chromogenic agent, wherein the chemical reaction device is a reaction device comprising a plurality of chemical reaction blind holes, and a reagent comprising a compound of formula (II) or a salt thereof according to any one of claims 1 to 3 is placed in at least one of the blind holes.

9. A compound of formula (III):

10. A compound of formula (III) according to claim 9, or a salt thereof, formula (III) being:

11. use of a compound of formula (III) or a salt thereof as claimed in any one of claims 9 to 10 in the manufacture of a kit for the detection of neuraminidase.

12. Use of a compound of formula (III) or a salt thereof as claimed in any one of claims 9 to 10 in the manufacture of a reagent for the detection of bacterial vaginosis.

13. A bacterial vaginosis determination kit is characterized by comprising a dry chemical reaction device, diluent and a color developing agent; the dry chemical reaction device is a reaction device comprising a plurality of dry chemical reaction blind holes, wherein at least one blind hole is provided with a neuraminidase substrate reagent pad, and the neuraminidase substrate reagent pad is prepared by soaking the dry chemical reaction blind hole in a reagent containing the compound of the formula (III) or a salt thereof according to any one of claims 9 to 10, and drying the reagent pad under reduced pressure.

14. A bacterial vaginosis assay kit, which comprises a dry chemical reaction device, a diluent and a color-developing agent, wherein the dry chemical reaction device is a reaction device comprising a plurality of dry chemical reaction blind holes, a reagent containing the compound of formula (III) or a salt thereof according to any one of claims 9 to 10 is placed in at least one of the blind holes, and the reagent is dried under reduced pressure to obtain the bacterial vaginosis assay kit.

15. A bacterial vaginosis assay kit, which comprises a chemical reaction device, a diluent and a chromogenic agent, wherein the chemical reaction device is a reaction device comprising a plurality of chemical reaction blind holes, and a reagent comprising a compound of formula (III) or a salt thereof according to any one of claims 9 to 10 is placed in at least one of the blind holes.

16. An intermediate compound of formula (I):

17. An intermediate compound of formula (I) or a salt thereof as claimed in claim 16 wherein formula (I) is selected from: