MXPA98004081A - 4,7-dialkoxy-n-acetylneuraminic acid derivatives and methods for detection of influenza type a and b viruses in clinical specimens - Google Patents

Abstract

Chromogenic and fluorogenic 4,7-dialkoxy-N-acetylneuraminic acid substrates of general formula (I) are provided wherein R1 and R2 are alkyl groups containing 1 to 4 carbon atoms and R3 is a chromogenic or fluorogenic group. These substrates can be used to detect influenza types A and B in clinical samples or specimens. More particularly, these 4,7-dialkoxy-N-acetylneuraminic acid substrates can be used to distinguish between various viruses having neuraminidase reactivity. Thus, influenza type A and B viruses can be distinguished from parainfluenza type 1, 2, 3 and 4, and mumps using the 4,7-dialkoxy-N-acetylneuraminic acid derivatives of this invention. Diagnostic methods employing these substrates are provided to identify influenza type A and B viruses in clinical specimens and to distinguish from other viruses having neuraminidase reactivity.

Description

DERIVATIVES OF ACID 4, 7-DIALCOXI-N-ACETILNEURAMIN CO AND METHODS FOR DETECTION OF INFLUENZA VIRUSES TYPES A AND B IN CLINICAL SAMPLES The present invention provides cholalogenic and fluorogenic 4,7-dialkoxy-N-acetylneuraminic acid substrates which can be used to detect influenza types A and B in clinical specimens or samples. More particularly, the present invention provides substrates of 4,7-dialkoxy-N-acetylneuraminic acid which can be used to distinguish between various viruses having neuraminidase activity. Thus, influenza viruses type A and B can be distinguished from para-influenza viruses type 1, 2, 3 and 4 and from mumps using the 4,7-dialkoxy-N-acetylneuraminic acid derivatives of this invention. Background of the Invention Infectious diseases are the most common reason for doctor's office visits. Viruses are responsible for more of these infections than all other groups of microorganisms combined. Of all the various infections caused by viruses, the respiratory viruses (influenza A and B, para-influenza 1, 2, 3 and 4, respiratory syncytial virus, and adenovirus) are the most prevalent as a group. The lethality of the influenza virus was discovered as early as 430 BC in the plague of Athens (Langmuir et al., New. Engl. J. Medicine 313 (1985) 1027). Influenza is the number one cause of acute respiratory diseases and a contributor to the sixth leading cause of mortality in the United States annually (Monthly Vital Statistics Report, vol.43, No. 6 (1995)). As a result, the development of diagnostic methods for viruses and viral infections has become increasingly important. The rapid diagnosis of viral infections has also become an integral part of good medical practice. Some viruses have definable antigens against which antibodies can be produced. Therefore, immunoassays have been widely used for the measurement of the presence of a virion. Where it is desirable to measure a larger group of virions, it may be possible to detect a particular component of the virus. For example, influenza viruses express surface glycoproteins having neuraminidase (sialidase) activity. The enzyme neuraminidase hydrolyzes substrates containing N-acetylneuraminic acid bound 2-ketosidically (Neu5Ac, also known as sialic acid). Neu5Ac consists of a backbone of nine carbon atoms, a carboxyl group and an N-acetyl group. The general structure, as well as the numbering system used to denote carbon atoms, are shown When a virion with neuraminidase activity is incubated with a chromogenic or fluorogenic glycoside of Neu5Ac, the enzyme will cut the chromogenic or fluorogenic aglycone from the substrate, and the reaction product will indicate the presence of a virion. Throughout this description, the N-acetyl group attached to the carbon at position 5 in the above formula will be denoted as AcH. A method for detecting the presence of a virus through the reaction of an enzyme with a chromogenic substrate for the enzyme is described in U.S. Patent No. 5,252,458, which is incorporated herein by reference. An assay for the direct measurement of influenza neuraminidase was developed by Yolken et al. (J. Infectious Diseases 142 ^ (1980) 516-523). Yolken et al. Used Neu5Ac 4-methylumbelliferyl-2-ketoside as a fluorescent substrate to measure neuraminidase activity in preparations containing small amounts of cultured virus as well as in some nasal lavage samples of human volunteers infected with the influenza virus. . Yolken and colleagues suggested that "the successful development of fluorometric enzyme assays for the detection of influenza neuraminidase could thus provide practical means of diagnosing influenza that are rapid enough to allow the institution of appropriate preventive and therapeutic interventions" . According to Yolken et al., The colorimetric assays were insufficiently insensitive for clinical applications. In contrast, Yolken et al. Noted that fluorometric assays could be suitable for detecting influenza neuraminidase in clinical samples. Pachucki et al. (J. Clinical Microbiolosy 26 (1988) 2664-2666) tested Neu5Ac 4-methylumbelliferyl-2-ketoside in clinical specimens collected from influenza patients. Due to its low sensitivity, the assay was not useful to detect neuraminidase directly and quickly in clinical specimens. However, the assay identified 91% of virus-positive isolates 25 hours after inoculation of tissue cultures. The use of modified Neu5Ac substrates can increase the specificity of the neuraminidase assay. In sialic acids, the carbon at position 4 (C-4) has been reported to play an important role in enzyme-substrate interactions. In addition, as it is known that salivary bacterial enzymes exhibit neuraminidase activity (Varki et al., J. Biol. Chem. 258 (1983) 12465-12471), it is essential to eliminate these undesirable interactions. It has already been shown that the 4-methoxy-Neu5Ac ketosides are resistant to certain bacterial sialidases, but are rapidly cleaved by certain viral sialidases (Beau et al., Eur. J. Biochem. 106 (1980) 531-540). Although modification of position 4 of N-acetylneuraminic acids provides specificity between certain viral and bacterial neuraminidase reactivity, it would still be desirable to obtain substrates that allow for greater specificity or differentiation between the various viral neuraminidase reactivities while maintaining the specificity between viral neuraminidase and viral reactivities. bacterial Such substrates, for example, would allow high specificity for particular types of viruses containing neuraminidase and would allow better and more targeted treatment regimens. Such substrates would also allow more acte surveillance of viral infections and more focused medical intervention, as appropriate. The substrates of N-acetylneuraminic acid 4, 7-modified chromogenic and fluorogenic of the present invention allow greater specificity or differentiation between the various viral neuraminidase reactivities while maintaining the specificity between viral and bacterial neuraminidase reactivities. SUMMARY OF THE INVENTION This invention relates to substrates of N-acetylneuraminic acid 4, 7-modified chromogenic and fluorogenic, which can be used for the detection and identification of influenza viruses in clinical specimens. More specifically, this invention relates to chromogenic and fluorogenic 4,7-dialkoxy-N-acetylneuraminic acid substrates which can be used for the detection and identification of influenza viruses in clinical specimens. These substrates of 4,7-modified N-acetylneuramminic acid can be used in diagnostic tests to distinguish between type A and B influenza viruses and other viruses possessing neuraminidase enzymes in clinical specimens. This invention also relates to diagnostic methods employing such substrates. As used herein, the terms "chromogenic and fluorogenic group" and "reporter or marker group" are intended to include, without limitation, molecules that exhibit absorbance or fluorescence. The term "color" similarly intended to include, without limitation, absorbance and fluorescence. It is an object of this invention to provide chromogenic and fluorogenic N-acetylneuraminic acid 4, 7-modified substrates useful in diagnostic methods for virus detection. Another objective of this invention is to provide a practical, convenient and cost-effective method for the detection of influenza viruses type A and B in clinical specimens. Another objective of this invention is to provide a practical, convenient and cost-effective diagnostic method that can distinguish between influenza viruses type A and B and other viruses having general neuraminidase reactivity in clinical specimens. Still another object of this invention is to provide a 4,7-dialkoxy-N-acetylneuraminic acid of the general formula:where Rx and R2 are alkyl groups containing 1 to 4 carbon atoms. Preferably, both Rx and R2 are methyl groups. Yet another objective of this invention is to provide a substrate of 4,7-dialkoxy-N-acetylneuraminic acid of the general formula: where Rx and R2 are alkyl groups containing 1 to 4 carbon atoms and R3 is a chromogenic or fluorogenic group. Preferably, both Rx and R2 are methyl groups and R3 is a chromogenic group. Yet another objective of this invention is to provide a method for detecting influenza viruses type A and B in a clinical sample of an individual suspected of having a viral respiratory infection, said method comprising: (1) incubating the clinical sample with a substrate of chromogenic or fluorogenic 4,7-dialkoxy-N-acetylneuraminic acid of the general formula: where R x and R 2 are alkyl radicals containing 1 to 4 carbon atoms and R 3 is a chromogenic or fluorogenic group that exhibits a distinctive and characteristic color when cut from the substrate or a salt of the substrate; (2) Observe the clinical sample incubated to determine if the distinctive and characteristic color is formed, where the distinctive and characteristic color formation indicates the presence of type A or B influenza viruses in the clinical sample. Yet another objective of this invention is to provide a method for detecting influenza viruses type A and B in a clinical sample of an individual suspected of having a viral respiratory infection, said method comprising: (1) dividing the clinical sample into a first portion and a second portion; (2) incubating the first portion with a first substrate of a 4,7-dialkoxy-N-acetylneuraminic acid of the general formula: where Rx and R2 are alkyl radicals containing 1 to 4 carbon atoms and R3 is a first chromogenic or fluorogenic group that exhibits a distinctive and characteristic first color when cut from the first substrate or a salt of the first substrate; (3) observe the first incubated portion to determine if the first distinctive and characteristic color is formed, where the formation of the first distinctive and characteristic color indicates the presence of type A or B influenza viruses in the clinical sample; (4) incubating the second portion with a second chromogenic or fluorogenic 4-alkoxy-N-acetylneuraminic acid substrate of the general formula: where R4 is an alkyl radical containing 1 to 4 carbon atoms and R5 is a second chromogenic or fluorogenic group that exhibits a second distinctive and characteristic color when cut from the second substrate or a salt of the second substrate; and (5) observing the second incubated portion to determine if the second distinctive and characteristic color is formed, where the formation of the second distinctive and characteristic color indicates the presence of reactive neuraminidase viruses in the clinical sample; where the presence of the first and second colors indicates the presence of type A or B influenza viruses alone or in combination with neuraminidase-reactive viruses other than type A and B influenza viruses in the clinical sample; where the presence of the second color and the absence of the first color indicate neuraminidase-reactive viruses other than type A and B influenza viruses in the clinical sample; and where the absence of the first and second colors indicates the absence of reactive neuraminidase viruses in the clinical sample. Other objects, advantages, aspects and characteristics of the present invention will be more evident when considering the following description and the appended claims. Detailed Description of the Invention The present invention relates to a 4,7-dialkoxy-N-acetylneuraminic acid of the general formula: where R? and R2 are alkyl groups containing 1 to 4 carbon atoms. R? and R2 may be the same or different alkyl groups. Higher alkyl groups (ie, when R contains 3 to 4 carbon atoms) can include linear and branched isomers. Preferably, Rx and R2 are alkyl groups containing 1 or 2 carbon atoms; more preferably, Rx and R2 are both methyl groups. The 4,7-dialkoxy-N-acetylneuraminic acid of the present invention can be prepared using the following general reaction scheme: The starting material 1 (Derivative 8, 9-O-isopropylidine-methyl ester-methyl ketoside of Neu5Ac) is prepared from Neu5Ac, as generally described in U.S. Patent No. 5, 556, .963, issued September 17, 1996 to the applicant (application Serial No. 08 / 286,573, filed Aug. 5 1994), which is incorporated herein by reference. Neu5Ac is commercially available (MediHerb Inc., 4540 S. Navajo # 1, Englewood, Colorado 80110, United States). It can also be synthesized enzymatically from N-acetyl-D-mannosamine and pyruvic acid using SCHEME 1 the procedure described by Kim et al., J. Am. Chem. Soc. 110 (1988) 6481, and illustrated by the following equation: The enzymatic reaction can be monitored by thin layer chromatography (TLC) and the product can be purified by ion exchange chromatography. Neu5Ac is first converted to an alkyl ester ketoside alkyl of the general formula: as described in U.S. Patent No. 5,556,963. The neighboring hydroxyl groups in C-8 and C-9 of this methyl ester ketoside methyl are protected by the formation of a ketal (1) by treatment with effective amounts of acetone and an acid catalyst to form the ketal. Suitable acidic catalysts include p-toluenesulfonic acid, salts of p-toluenesulfonic acid such as the pyridinium salt (PPTS) and other salts, ZnCl2, FeCl3, and the like. The preferred acid catalyst is the non-hygroscopic pyridinium salt of p-toluenesulfonic acid. The protected methyl ester cetoside methyl ester (1.) is then alkylated to form a mixture of compound 2. containing an alkoxy group in the 4-position and the compound 3. with alkoxy groups in both the 4-position and the 7-position. hydroxyl in C-4 and C-7 can be methylation, ethylation, propylation, or butylation, whereby the hydroxy group in C-4 in 2 is converted into a -OR group and the hydroxy groups in C-4 and C- 7 in 3. are converted into groups -OR where R is an alkyl radical containing 1 to 4 carbon atoms. Preferably, the alkylation at C-4 and / or C-7 is methylation or ethylation, whereby 4-methoxy or 4-ethoxy or 4,7-dimethoxy or 4,7-diethoxy derivatives are obtained. More preferably, alkylation at C-4 and / or C-7 is methylation, whereby 4-methoxy or 4,7-dimethoxy derivatives are obtained. The introduction of the higher alkyl groups at the C-4 and C-7 positions is generally slower than the methylation and the yields are somewhat lower. Further, chromogenic substrates with higher alkyl groups tend to be less susceptible to enzymatic cleavage than 4,7-dimethoxy-Neu5Ac, thereby resulting in less sensitive assays. However, for some assays and specific applications, such higher alkyl groups at C-4 and C-7 may be useful and even preferred. The alkylation in the free, more sterically hindered hydroxyl group in C-7 can be stimulated by controlling the reaction conditions, as immediately described hereinafter. According to the process, intermediate 1 is treated with an excess (generally greater than 1.5 molar equivalents) of an alkylating agent in a dispersion of about 80% sodium hydride. The alkylating agent is selected from the group consisting of dimethyl sulfate, diethyl sulfate, dipropyl sulfates, and dibutyl sulfates. The reaction is generally conducted at a temperature of from about 0 to about 30 ° C for about 10 minutes to about 48 hours. Preferably, the reaction temperature is in the range of about 0 to about 22 ° C. Longer reaction times are generally preferred when the higher alkoxy groups are formed at positions 4 and 7. In a preferred embodiment of the invention, a methylation reaction to form methoxy groups at C-4 and C-7 is conducted at a temperature of about 0 to about 22 ° C, for about 10 to about 30 minutes. In another preferred embodiment of the invention, an ethylation reaction to form ethoxy groups at C-4 and C-7 is conducted at a temperature of from about 0 to about 30 ° C, more preferably from about 0 to around 22 ° C, for about 1 to about 24 hours. The treatment of methyl ester cetoside protected ester 1 with excess alkylating agent (eg, dimethyl sulfate) produces a mixture of compound 2 containing an alkoxy group in the 4-position and compound 3_ with alkoxy groups in both positions 4 and 7. Usually, an excess of about 1.5 molar equivalents of alkylating agent is used. Preferably, about 1.5 to about 2.0 molar equivalents of the alkylating agent are used. Generally, the amount of the desired compound 3. is increased relative to compound 2 by increasing the amount of the alkylating agent. The reaction mixture resulting from such treatment generally contains the 4-alkoxy compound 2 as the major product and about 10 to 20% by weight of the 4,7-dialkoxy compound 3_. The partial separation of compounds 2 and 3 can be obtained, for example by column chromatography and subsequent crystallization from a mixture of acetone-hexane, which removes preferentially the 4-alkoxy compound 2. The resulting residue contains a mixture of the two compounds enriched with the 4,7-dialkoxy material; generally, the molar ratio of the two compounds is increased to at least about 1: 1. The removal of the ketal group of compound 3 is achieved by treatment with around 80% acetic acid. The hydrolysis of acetic acid can also result in partial acetylation at the C-9 hydroxyl group. Therefore, the product of the hydrolysis can be treated with sodium methoxide to remove any acetate groups in C-9. The final deprotection of 4. is carried out by alkaline treatment and subsequent acid hydrolysis to give the final 4,7-dialkoxy-Neu5Ac product (5_). Of course, the 4-alkoxy compound 2. present in the mixture will also be treated in a similar manner to result in the corresponding 4-alkoxy-Neu5Ac compound. The resulting 4,7-dialkoxy-Neu5Ac can be further used by coupling to any suitable reporter or marker group, including, for example, a chromogenic or fluorogenic labeling group. The preferred reporter or marker group is a chromogenic group, including, for example, 4-chloro-1-naphthol, 6-bromo-l-naphthol, and 5-bromo-4-chloro-indole. Modified 4,7-dialkoxy-Neu5Ac, chromogenic, can be incorporated into a neuraminidase assay useful for detecting influenza A and B viral neuraminidase activity in samples or clinical specimens. Methods for synthesizing and using such substrates of N-acetylneuraminic acid modified at positions 4,7, chromogenic, in viral assays, are similar to those described for the substrates modified at position 4, related, in PCT publication WO 91/09972; Yolken et al., J. Infectious Diseases 142 (1980) 516-523; and Pachucki et al., J. Clinical Microbioloqy 26 (1988) 2664-2666, each incorporated herein by reference. Of course, the 7-dialkoxy-N-acetylneuraminic acids herein can be used to form other chromogenic and fluorogenic derivatives and can be used in other viral assays. Generally, the chromogenic or fluorogenic labeling group can be incorporated into 4,7-dialkoxy-Neu5Ac (5_) using the following reaction scheme (using 5-bromo-3-indolyl as an exemplary label group): SCHEME 2 Compound 5_ is first converted into the corresponding methyl ester derivative by treatment with concentrated trifluoroacetic acid and methanol, and the ester is then reacted with excess acetyl chloride to form the chloroacetate-methyl ester of 4,7-dialkoxycarbonyl ester. Neu5Ac (6.). Coupling 6. with the sodium salt of 5-bromo-3-indolyl gives the 5-bromo-indol-3-ol-4,7-dimethoxy-N-acetylneuraminic acid acetoxy methyl ester (7.). Deacetylation of 7. is effected by treatment with sodium methoxide in methanol to give compound 8. and subsequent treatment with sodium hydroxide gives the sodium salt of 5-bromo-3-indolyl-4,7-dimethoxy-N- acetylneuraminic (9.). Of course, the 4-alkoxy-Neu5Ac in the mixture undergoes similar coupling reactions to form the salt of 5-bromo-3-indolyl-4-methoxy-N-acetylneuraminic acid. The coupled 4,7-dialkoxy derivative 9. is then separated from the mixture of the two coupled compounds (ie, the 4-alkoxy and 4,7-dialkoxy derivatives) by high performance liquid chromatography (HPLC) using, by example, a C18 reverse plate silica column. The mixture of the coupled products can be dissolved in water and charged to the column. The products are separated by a gradient that is increased by methanol, with the appropriate fractions collected and formed in pond. The resulting purified 5-bromo-3-indolyl-4,7-dimethoxy-N-acetylneuraminic acid (9) can be dried and stored until used. Generally, the resulting product is essentially free of the 4-methoxy derivative. It is important that this mixture is essentially free of the 4-methoxy derivative as it is highly reactive with the mumps virus and other viruses containing neuraminidase. As noted above, the 4,7-dialkoxy chromogenic or fluorogenic derivatives of this invention are of the general formula: where Rx and R2 are alkyl groups containing 1 to 4 carbon atoms and R3 is a chromogenic or fluorogenic group. Rx and R2 may be the same or different alkyl groups. Preferably, both Rx and R2 are methyl groups and R3 is a chromogenic group. More preferably, R3 is 4-methylumbelliferyl, 3-cyanoumbeliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 5-bromo-3-indolyl, 3-indolyl, nitrophenylazophenyl, nitrophenylazoresorcinyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-1-naphthyl, or 6-bromo-2-naphthyl. Even more preferably, R3 is 4-methylumbelliferyl, 5-bromo-4-chloro-3-indolyl, or 5-bromo-3-indolyl. More preferred R3 is 5-bromo-3-indolyl. Simple salts of these substrates, such as the Na +, K + and NH4 + salts, can also be used.

Examples of chromogenic Neu5Ac 4,7-dialkoxy derivatives falling within the above formula include 4-methylumbelliferyl-4,7-dimethoxy-N-acetylneuraminic acid alpha-ketoside, 2-nitrophenyl-, 7-dimethoxy acid alpha-ketoside. N-acetylneuraminic acid 4-nitrophenyl-4,7-methoxy-N-acetylneuraminic acid alpha-ketoside, 3-cyanoumbeliferyl-4,7-dimethoxy-N-acetylneuraminic acid alpha-ketoside, alpha-3-acetic acid ketoside -resorufin-4, 7-dimethoxy-N-acetylneuraminic acid, 5-bromo-4-chloro-3-indolyl-4,7-dimethoxy-N-acetylneuraminic acid alpha-ketoside, 5-bromo-3-alpha-ketoside -indolyl-4,7-dimethoxy-N-acetylneuraminic acid, 3-indolyl-4,7-dimethoxy-N-acetylneuraminic acid alpha-ketoside, 2- [4- (4-nitrophenylazo) phenyl] -acetoside] - 4,7-dimethoxy-N-acetylneuraminic acid 2- [4- (4-nitrophenylazo) resor-cinyl] -4,7-dimethoxy-N-acetylneuraminic acid alpha-ketoside 3-methoxyphenyl-4 acid , 7-dimethoxy-N-acetylneuraminic or, 3-dimethylaminophenyl-4,7-dimethoxy-N-acetylneuraminic acid alpha-ketoside, 6-bromo-2-naphthyl-4,7-dimethoxy-N-acetylneuraminic acid alpha-ketoside, alpha-acetic acid ketoside -chloro-l-naphthyl-4,7-dimethoxy-N-acetylneuraminic, as well as the 4,7-diethoxy derivatives, 4,7-dipropyl and 4,7-dibutyl corresponding. Generally, 4,7-dimethoxy derivatives are preferred. If desired, "mixed" 4,7-dialkoxy derivatives (e.g., 4-methoxy-7-ethoxy) can be used. The 4,7-dialkoxy chromogenic or fluorogenic derivatives can be used in diagnostic tests of type A and B influenza viruses in clinical samples. Clinical samples that are tested in the invention will typically be pharyngeal, nasopharyngeal or respiratory secretions collected from patients suffering from or suspected of suffering from influenza, such as washing, swab or sputum specimens. The washing, expectoration or swab will preferably be combined with an aqueous buffer solution containing a stabilizer before mixing with the substrate. The buffer solution generally contains a buffer that maintains the pH at about 4 to 7, preferably 5.5 to 6.5, optionally about 0.1 to 10% by weight of nonionic detergent, a small amount (1-20 mM) of cation of alkaline earth metal (Ca, Mg, preferably Ca), and a sufficient amount of a stabilizer selected from the group consisting of alditols, monosaccharides and disaccharides to increase the thermal stability of the neuraminic acid in the sample. The volume of buffer solution combined with the sample will normally be 0.1 to 2 ml. The buffer can be organic or inorganic. Suitable shock absorbers include, for example, conventional organic acid buffers and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc. .), acetate buffers (eg, acetic acid-sodium acetate mixture), succinate buffers (eg, succinic succinate-monosodium mixture, succinic acid-sodium hydroxide mixture, succinic acid-succinate mixture disodium, etc.), tartrate buffers (for example, tartaric acid-tartrate mixture, mixture of tartaric acid-potassium tartrate, mixture of tartaric acid-sodium hydroxide, etc.), fumarate buffers (for example, mixture of fumaric acid-monosodium fumarate, mixture of fumaric acid-disodium fumarate, monosodium fumarate-fumarate disodium mixture), gluconate buffers (eg, gluconic acid-sodium gluconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-gluconate potassium mixture, etc. .), oxalate buffers (eg, oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g. Lactic acid-sodium lactate, mixture of lactic acid-sodium hydroxide, mixture of lactic acid-potassium lactate, etc.), acetate buffers (for example, mixture of acetic acid-sodium acetate, mixture of acetic acid-sodium hydroxide, etc.), malate buffers (eg, D, L-malic-malate disodium), phosphate buffers (eg, monosodium phosphate-sodium hydroxide mixture, trisodium phosphate-hydrochloric acid, etc.), (N-mo rfol ino) ethane sulphonic acid, [bis - (2-hydroxyethyl) imino] tris (hydroxymethyl) methane, N-2-acetamidoiminodiacetic acid, 1, 3- bis [tris (hydroxy-methyl) methylamino] propane, piperazin-N, N'-2-ethanesulfonic acid, N-2-acetamido-2-aminoethanesulfonic acid, 3- (N-morpholino) -2-hydroxypropanesulfonic acid, - (N-morpholino) propanesulfonic, 2- [tris (hydroxyethylpiperazine-N, -2-ethanesulfonic acid, 3- [tris- (hydroxymethyl) methylamino] -2-hydroxypropanesulfonic acid. Useful nonionic detergents in the buffer include, for example, Pluronics, such as polysorbate 20 or polysorbate 80, Triton X-100, NP-40 and alkyl glucosides such as C 8 and C 9 alkyl glucosides. The detergent is an optional component and facilitates the release of neuraminidase from the viral envelope. Stabilizers that are used in the buffer include, for example, trihydric or higher alditols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, mannitol, the hexose glucose and fructose and the disaccharide sucrose. These stabilizers can be used alone or in combination. In order to stabilize the activity of the neuraminidase-containing viruses, the stabilizers are added to the liquid formulation system / excipient in an amount of 0.2 to 2.1 M and, preferably, 0.6 to 2.0 M. Once mixed with the buffer solution, the sample can be stored for prolonged periods, preferably at 2 to 8 ° C, without significant loss of neuraminidase activity. The substrate will normally be added to the buffered, stabilized sample, in amounts ranging between 0.05 and 0.5 mM. The mixture is incubated at ambient to physiological temperature (i.e., around 18 to 40 ° C) for a sufficient time to allow any neuraminidase in the sample to react with the substrate. That time will normally be in the range of 1 minute to 4 hours, usually 5 to 120 minutes, and more usually 30 to 60 minutes. If there is neuraminidase activity in the sample, the marker or reporter group will be released from the substrate and the precipitate of the released marker or reporter will impart a characteristic color to the mixture. Especially for colorimetric derivatives, the resulting reaction mixture is preferably transferred to a collection device containing a porous membrane filter and an absorbent pad to be impregnated with solution. A similar collection device is shown in U.S. Patent Application Serial No. 08 / 479,789, pending (filed June 7, 1995), which is incorporated herein by reference. In the presence of neuraminidase influenza type A and B, the reporter group in Neu5Ac 4, 7-modified is released by the action of neuraminidase and the resulting reporter molecule is collected as a color precipitate on the porous filter membrane. The presence of such a colored product indicates a positive diagnosis of influenza types A and B; the absence of such a colored product indicates a negative diagnosis of influenza types A and B.

The concentration or collection of the colored precipitate increases the sensitivity of the diagnostic test. However, such collection devices are not necessary for the practice of the invention. The following table indicates the characteristic color generated when the neuraminidase reacts with various derivatives of Neu5Ac 4, 7-modified chromogenic or fluorogenic and releases the reporter molecule.

The present chollogenic and fluorogenic 4,7-dialkoxy Neu5Ac derivatives only exhibit the characteristic color in the presence of influenza viruses type A and B. They do not exhibit the characteristic color (ie, release of the reporter molecule does not occur) in the presence of para-influenza types 1, 2, 3 and 4, mumps, respiratory syncytial virus and / or adenovirus. Moreover, the chromogenic and fluorogenic 4,7-dialkoxy Neu5Ac derivatives do not exhibit reactivity to bacterial neuraminidase. To maintain the desired degree of selectivity, the 4,7-dialkoxy derivatives Neu5Ac must be essentially free of the corresponding 4-alkoxy Neu5Ac derivatives. The maximum levels of the 4-alkoxy Neu5Ac derivatives that allow acceptable selectivity can be determined experimentally using known strains of the various types of viruses. Generally, the levels of the 4-alkoxy derivatives Neu5Ac should be less than 5% by weight, and preferably less than about 0.5% by weight. A potentially more informative diagnostic test can be obtained by combining the reactivity to neuraminidase and the selectivity of the chromogenic and fluorogenic Neu5Ac 4,7-dialkoxy derivatives of the present and chromogenic and fluorogenic 4-alkoxy Neu5Ac derivatives in a single test. In such a system, the clinical sample is divided into two portions which are then incubated separately with the 4,7-dialkoxy and 4-alkoxy Neu5Ac derivatives, respectively. The presence and / or absence of the characteristic color in the two incubated samples can be used to more closely determine the type of virus, if any, present in the clinical sample. If only type A or B influenza viruses are present in the clinical sample, then both 4-alkoxy and 4,7-dialkoxy derivatives will exhibit the characteristic colors of their reporter molecules. If both type A or B influenza viruses and neuraminidase reactive viruses other than type A and B influenza viruses are present in the clinical sample, then each portion will exhibit the characteristic color of its reporter molecule. If only neuraminidase reactive viruses other than influenza viruses type A and B are present in the clinical sample, then only the 4-alkoxy derivatives will exhibit the characteristic color of their reporter molecule. If neuraminidase-reactive viruses are not present in the clinical sample, then no portion will exhibit the characteristic color of its reporter molecule. Accordingly, the present invention provides simple and rapid techniques for selectively diagnosing influenza, especially types A and B, which can be carried out in the clinic or doctor's office and allowing the physician to prescribe the appropriate therapy to treat the infection and / or the Prophylactic treatment appropriate for people in close contact with the infected patient. The following examples are intended to illustrate the invention. Example 1. The synthesis of 4,7-dimethoxy-Neu5Ac was carried out using the reactions shown in Scheme 1. A cold solution (ice bath temperature) of the isopropylidene derivative 1 (1.2 g) in about 10 ml of dry acetonitrile was saturated with nitrogen. Sodium hydride (270 mg, 80% dispersion in oil) was added and the mixture was stirred for 20 minutes. Dimethyl sulfate (1 ml) was added and stirring continued for an additional 30 minutes using an ice bath to cool the mixture. The resulting mixture was filtered through Celite and the precipitate was washed with dry acetonitrile. The precipitate was evaporated and the residue was dried and extracted with acetone. After the filtration, the filtrate was evaporated again. The resulting residue was dried and subjected to chromatography on silica gel. Elution with methylene chloride / methanol (25: 1) removed various by-products. Continuous elution with the same solvent system provided a syrup containing 2 and 3., which was crystallized from acetone-hexane. 2 crystalline (177 mg) was collected. The filtrate contained a mixture of 2 and 3 (about 1: 1) but with a higher ratio of 3. than the original mixture. The filtrate containing 3. (1.22 g) was treated with 80% aqueous acetic acid (15 ml) at 85 ° C for one hour. The mixture was evaporated and co-evaporated with water. The residue was subjected to chromatography. Elution with methylene chloride / methanol (5: 1) removed a minor by-product. Continuous elution with the same solvent system gave the product 4. partially unprotected (0.87 g; 80% yield). The 4,7-dimethoxy-methyl ester ketoside (4_) (0.87 g) was treated with 1 M sodium hydroxide (3 ml) in methanol (5 ml) and water (5 ml) at room temperature for one hour . The mixture was neutralized with Dowex 50 (H +) resin; The resin was removed by filtration and washed with methanol. The combined filtrate was evaporated. The residue was treated with Dowex 50 (H +) resin (1.5 g) in 0.025 M hydrochloric acid at 100 ° C for two hours. The resin was removed by filtration and the filtrate collected and evaporated. The residue was dried under vacuum to give essentially 7,7-dimethoxy-Neu5Ac (5.) (0.71 g, 88% yield). Example 2. Synthesis of 5-bromo-3-indolyl-4,7-dimethoxy-Neu5Ac was carried out using the reactions shown in Scheme 2. Compound 5_ (1.0 g) was treated with trifluoroacetic acid (0.2 ml) in methanol (25 ml) overnight, at room temperature. The mixture was evaporated. The residue was dried and treated with acetyl chloride (5 ml) in methylene chloride (5 ml). The reaction mixture was stirred overnight at room temperature and then evaporated.

The residue was dried to provide the crude chloride (6.) (1.36 g). The crude product 6 was not further purified due to its instability. A suspension of the crude product 6. (0.7 g) and 1-acetyl-5-bromo-indole-3-ol (0.26 g) in acetone (5 ml) was saturated with nitrogen. A solution of 1 M sodium hydroxide (1 ml) was added. The reaction mixture was stirred under nitrogen for one hour. After evaporation, the residue was dried and subjected to chromatography on silica gel. Elution with methylene chloride / methanol (25: 1) removed the chromogen without reacting (0.128 g). Continuous elution with the same solvent system gave fractions containing mainly the coupled product 7_ (0.114 g). The coupled product 7. (0.114 g) was treated with 1 M sodium methoxide in methanol (0.1 ml) for 30 minutes at room temperature. After neutralization with Dowex 50 (H +) resin, the resin was removed and washed with methanol. The combined filtrate was evaporated. The residue was dried and subjected to chromatography on silica gel. Fractions containing mainly the coupled product 8. were stagnated and evaporated to give about 60 mg of 8. The coupled product 8. (60 mg) was treated with 1 M sodium hydroxide (0.5 ml) in 50% aqueous methanol ( 5 ml) for 30 minutes at room temperature. The mixture was neutralized with Dowex 50 (H +) resin. The resin was removed by filtration and washed with methanol. The combined filtrate was evaporated and the residue was purified by HPLC. The first fractions contained a non-coupled product. The coupled material appeared as a broad peak which consisted of both coupled 4-methoxy-Neu5Ac and coupled 4,7-dimethoxy-Neu5Ac (product 9.). As a minor amount of the coupled 4-methoxy derivative may render the diagnostic analysis nonspecific, only highly pure fractions containing 9 were stagnated. Upon evaporation, 9. highly purified (4 mg) was collected; the collected material contained less than about 5% by weight of the corresponding 4-alkoxy derivative. Example 3. This example demonstrates the specificity of 5-bromo-3-indolyl-4,7-dimethoxy-Neu5Ac (9.) of Example 2 in diagnostic tests of influenza A and B in cultured viruses. The viruses were cultured from fresh isolates of patients or cultures of frozen material using the appropriate cell line, medium and incubation conditions, suitable for the development and culture of the particular virus. The following cells were used: (1) RMK cells (Rhesus monkey kidney) for influenza and para-influenza viruses; (2) Hep-2 cells (human laryngeal carcinoma) for respiratory syncytial virus and adenovirus; and (3) VERO cells (African green monkey kidney) for mumps. All cells were cultured in almost confluent monolayers in tubes or flasks with minimal essential medium with 5-10% serum at 37 ° C. Infections with virus were carried out in the appropriate cell lines with minimal essential medium without serum at 37 ° C. Virus-infected cultures were monitored and confirmed by observing the appearance of characteristic cytopathic effects and by testing with immunofluorescence assays using monoclonal antibodies specific to the type of virus. The following characteristic cytopathic effects were observed for each of the viruses used, as follows: (1) Influenza A and B: areas of large cells, irregularly shaped, granular or vacuolated with progressive degeneration of the cell monolayer; (2) para-influenza 1: small cells rounded through the entire cell monolayer; (3) para-influenza 2: dark, granular and irregular syncytia that retract from the cell monolayer; (4) para-influenza 3: elongated cells, fusiform, which eventually retract and move away from the cell monolayer; (5) respiratory syncytial virus: large, irregularly shaped syncytia that appear as large, multi-nucleated cells with indistinct boundaries across the entire cell monolayer; (6) adenovirus: large, rounded, picnotic cells, which are eventually added to rounded cell sheets and detached from the culture vessel; and (7) mumps: syncytia with vacuolation and cellular degeneration throughout the cell monolayer. Commercial-type monoclonal antibodies specific to the virus type were used in fluorescence assays to confirm the culture of each virus in the inoculated cultures. The cells of the infected cultures were fixed with methanol in glass slide holders and then incubated at 37 ° C with monoclonal antibodies marketed with fluorescein. Direct and indirect fluorescence assays were used. For the direct assays, the fixed cells, infected with the virus, were incubated for 30 minutes with monoclonal antibodies marinated with fluorescein isothiocyanate (FITC), specific to the type of virus. For indirect assays, the fixed cells, infected with the virus, were incubated for 30 minutes with unlabelled monoclonal antibodies, specific to the type of virus, followed by incubation for an additional 30 minutes with monoclonal antibodies marinated with secondary FITC. The lens holders were covered with mounting medium and examined using a fluorescent microscope. A positive virus result was verified by the presence of a characteristic apple green stain of the virus. The presence of progressive and characteristic cytopathic effects (together with confirmation of specific accompanying virus with fluorescence assay) was an indication that the particular inoculated virus had proliferated maximally. After reaching maximum growth of the virus and confirmation of the presence of the appropriate virus, the culture fluids were harvested for evaluation with the chromogenic substrates of 4-methoxy-N-acetylneuraminic acid and 4,7-dimethoxy-N-acetylneuraminic acid. The virus-containing culture fluids (0.1 ml) were mixed with the chromogenic substrates in 1 or 2 ml of a solution containing excipients and buffer designed to achieve optimal pH and optimal neuraminidase activity. The buffer / excipient solution contained 35 mM malic / malate buffer with 10 mM calcium chloride, 0.85% sodium chloride, 0.1% mannitol, 0.5% methanol, and 0.1% methyl paraben. The combination of substrate and buffer at a pH of about 5.4 was determined to provide specific detection of neuraminidase producing viruses with clearly negative results for viruses that do not produce neuraminidase and negative cultures on viruses. The test solutions were incubated at 37 ° C for about one hour, after which the reaction was stopped by the addition of 0.2 ml of a buffer concentrate that changed the pH to around 9 and helped increase the chromogen precipitation released. The entire reaction mixture was then transferred to a collection device containing a selectively porous membrane filter and an absorbent pad to be impregnated with the solution, whereby the precipitate is concentrated. The collection device is described in U.S. Patent Application Serial No. 08 / 479,789, pending (filed June 7, 1995). The appearance of a precipitate of appropriate color indicated a positive reaction, whereas the absence of such an appropriate color precipitate indicated a negative reaction. Using the 5-bromo-3-indolyl chromogen, the characteristic color for a positive test was blue. The following viruses were used to test the substrate of 5-bromo-3-indolyl-4-methoxy-N-acetylneuraminic acid (BI-4-MeONeu5Ac) and the substrate of 5-bromo-3-indolyl-4, 7- acid. dimethoxy-N-acetylneuraminic acid (BI-4, 7- (MeO) 2Neu5Ac).

The following results were obtained using these various virus cultures. The virus cultures were developed at their highest titers (determined by the observance of characteristic cytopathic effects of extensive viruses -about 90 to 100% - in the cellular lamina, and confirmation with fluorescence assays of bright green apple spotting, specific to virus, in most cells) to ensure that any reaction differences are not due to non-detectable viral concentrations.

A rating scale of 1+ to 3+ was used, based on the intensity of the reaction color, to indicate relative levels of detectable neuraminidase activity. The classification colors were matched with color samples from a standard reference source (The Pantone Color Specific Book 747XR). A rating of 1+ (light color) indicates a low positive, 2+ (moderate color) indicates a moderate positive, and 3+ (deep color) indicates a high positive. A negative result is indicated by the absence of the characteristic color. An indeterminate result is indicated if a very tenuous trace or suggestion of the characteristic color can be present by visual observation. As can be seen from these results, influenza A neuraminidase type A and B viruses were the only organisms tested that gave a positive reaction with substrates of both 4-methoxy-Neu5Ac and 4,7-dimethoxy-Neu5Ac, undiluted all dilutions tested. For para-influenza types 1, 2 and 3 and the mumps virus, the 4-methoxy-Neu5Ac substrate gave both negative and positive reactions, depending on the dilution. The substrate of 4,7-dimethoxy-Neu5Ac gave a negative reaction with para-influenza types 1, 2 and 3 and the mumps virus undiluted and in all dilutions. Viruses that do not contain neuraminidase (ie, respiratory syncytial virus and adenovirus) produced negative results with both substrates. Likewise, negative controls on viruses without viruses were appropriately negative. These results clearly demonstrate the specificity of chromogenic substrates of 4,7-dialkoxy-N-acetylneuraminic acid for influenza viruses type A and B. Example 4. Results similar to those reported in Example 3 were obtained using 5-bromo-4 -chloro-indole-3-ol and 4-methylumbelliferone as reporter molecules in 4-methoxy-Neu5Ac and 4,7-dimethoxy-Neu5Ac. It is expected that numerous modifications and variations in the practice of the invention will occur to those skilled in the art upon consideration of the detailed description of the preceding invention. Accordingly, such modifications and variations are intended to be included within the scope of the following claims.

Claims (21)

1. CLAIMS 1. A 4,7-dialkoxy-N-acetylneuraminic acid of the general formula: where R1 and R2 are alkyl groups containing 1 to 4 carbon atoms.
2. 2. A 4,7-dialkoxy-N-acetylneuraminic acid, as defined in claim 1, wherein both Rx and R2 are methyl groups.
3. 3. A 4,7-dialkoxy-N-acetylneuraminic acid, as defined in claim 1, wherein both Rx and R2 are ethyl groups.
4. 4. A substrate of 4,7-dialkoxy-N-acetylneuraminic acid of the general formula: where Rx and R2 are alkyl groups containing 1 to 4 carbon atoms and R3 is a chromogenic or fluorogenic group.
5. 5. A substrate of 4,7-dialkoxy-N-acetylneuraminic acid, as defined in claim 4, wherein both R? as R2 they are methyl groups and R3 is a chromogenic group.
6. 6. A substrate of 4,7-dialkoxy-N-acetylneuraminic acid, as defined in claim 5, wherein R3 is selected from the group consisting of 4-methylumbelliferyl, 3-cyanoumbeliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 5-bromo-3-indolyl, 3- indolyl, nitrophenylazophenyl, nitrophenylazoresorcinyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-1-naphthyl, and 6-bromo-2-naphthyl.
7. 7. A substrate of 4,7-dialkoxy-N-acetylneuraminic acid, as defined in claim 6, wherein R3 is 5-bromo-3-indolyl.
8. 8. A substrate of 4,7-dialkoxy-N-acetylneuraminic acid, as defined in claim 6, wherein R3 is 5-bromo-3-indolyl.
9. 9. A substrate of 4,7-dialkoxy-N-acetylneuraminic acid, as defined in claim 8, wherein R3 is selected from the group consisting of 4-methylumbelliferyl, 3-cyanoumbeliferyl, 2-nitrophenyl, 4-nitrophenyl, -resorufine, 5-bromo-4-chloro-3-indolyl, 5-bromo-3-indolyl, 3-indolyl, nitrophenylazophenyl, nitrophenylazoresorcinyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-1-naphthyl, and bromo-2-naphthyl.
10. 10. A method for detecting influenza viruses type A and B in a clinical sample of an individual suspected of having a viral respiratory infection, said method comprising: (1) incubating the clinical sample as an acid substrate 4, 7- chromogenic or fluorogenic dialkoxy-N-acetylneuraminic acid of the general formula: where R x and R 2 are alkyl radicals containing 1 to 4 carbon atoms and R 3 is a chromogenic or fluorogenic group that exhibits a distinctive and characteristic color when cut from the substrate or a salt of the substrate; (2) observe the incubated clinical sample to determine if the distinctive and characteristic color is formed, where the distinctive and characteristic color formation indicates the presence of type A or B influenza viruses in the clinical sample.
11. 11. A method as defined in claim 10, wherein both Rx and R2 are methyl groups and R3 is a chromogenic group.
12. 12. A method as defined in claim 11, wherein R3 is selected from the group consisting of 4-methylumbeli-feryl, 3-cyanoumbeliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro -3-indolyl, 5-bromo-3-indolyl, 3-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 5-bromo-3-indolyl, 3-indolyl, nitrophenylazophenyl, nitrophenylazoresorcinyl, -methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-1-naphthyl, and 6-bromo-2-naphthyl.
13. 13. A method as defined in claim 12, wherein R3 is 5-bromo-3-indolyl.
14. 14. A method as defined in claim 10, wherein both Rt and R2 are ethyl groups and R3 is a chromogenic group.
15. 15. A method as defined in claim 14, wherein R3 is selected from the group consisting of 4-methylumbelliferyl, 3-cyanoumbeliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3. -indolyl, 5-bromo-3-indolyl, 3-indolyl, nitrophenylazophenyl, nitrophenylazoresorcinyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-1-naphthyl, and 6-bromo-2-naphthyl.
16. 16. A method for detecting influenza viruses type A and B in a clinical sample of an individual suspected of having a viral respiratory infection, said method comprising: (1) dividing the clinical sample into a first portion and a second portion; (2) incubating the first portion as a first substrate of chromogenic or fluorogenic 4,7-dialkoxy-N-acetylneuraminic acid of the general formula: where R? and R 2 are alkyl radicals containing 1 to 4 carbon atoms and R 3 is a first chromogenic or fluorogenic group that exhibits a distinctive and characteristic first color when cut from the first substrate or a salt of the first substrate; (3) observe the first incubated portion to determine if the first distinctive and characteristic color is formed, where the formation of the first distinctive and characteristic color indicates the presence of type A or B influenza viruses in the clinical sample; (4) incubating the second portion with a second chromogenic or fluorogenic 4-alkoxy-N-acetylneuraminic acid substrate of the general formula: where R4 is an alkyl radical containing 1 to 4 carbon atoms and R5 is a second chromogenic or fluorogenic group which exhibits a second distinctive and characteristic color when a second distinctive and characteristic color is exhibited when it is cut from the second substrate or a second salt substratum; and (5) observing the second incubated portion to determine if the second distinctive and characteristic color is formed, where the formation of the second distinctive and characteristic color indicates the presence of reactive neuraminidase viruses in the clinical sample; where the presence of the first and second colors indicates the presence of type A or B influenza viruses alone or in combination with neuraminidase-reactive viruses other than type A and B influenza viruses in the clinical sample; where the presence of the second color and the absence of the first color indicates neuraminidase-reactive viruses other than influenza viruses type A and B in the clinical sample; and where the absence of the first and second colors indicates the absence of reactive neuraminidase viruses in the clinical sample.
17. 17. A method as defined in claim 16, wherein both R1 and R2 are methyl groups and R3 is a first chromogenic group.
18. 18. A method as defined in claim 17, wherein R4 is a methyl group and R5 is a second chromogenic group.
19. 19. A method as defined in claim 17, wherein R3 and R4 are independently selected from the group consisting of 4-methylumbelliferyl, 3-cyanoumbeliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4- chloro-3-indolyl, 5-bromo-3-indolyl, 3-indolyl, nitrophenylazophenyl, nitrophenylazoresorcinyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-1-naphthyl, and β-bromo-2-naphthyl.
20. 20. A method as defined in claim 19, wherein R3 is 5-bromo-3-indolyl.
21. 21. A method as defined in claim 8, wherein R3 and R4 are independently selected from the group consisting of 4-methylumbelliferyl, 3-cyanoumbeliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4- chloro-3-indolyl, 5-bromo-3-indolyl, 3-indolyl, nitrophenylazophenyl, nitrophenylazoresorcinyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-1-naphthyl, and 6-bromo-2-naphthyl.