EP1163360A2 - Zusammensetzung und gerät zur bestimmung von leukozyten in urin - Google Patents

Zusammensetzung und gerät zur bestimmung von leukozyten in urin

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Publication number
EP1163360A2
EP1163360A2 EP00904434A EP00904434A EP1163360A2 EP 1163360 A2 EP1163360 A2 EP 1163360A2 EP 00904434 A EP00904434 A EP 00904434A EP 00904434 A EP00904434 A EP 00904434A EP 1163360 A2 EP1163360 A2 EP 1163360A2
Authority
EP
European Patent Office
Prior art keywords
substituted
compound
salt
unsubstituted
alkenyl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00904434A
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English (en)
French (fr)
Inventor
Nam-Won Huh
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Medisys America Ltd
Original Assignee
Hypoguard America Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/365,322 external-priority patent/US6348324B1/en
Priority claimed from US09/365,592 external-priority patent/US6528652B1/en
Application filed by Hypoguard America Ltd filed Critical Hypoguard America Ltd
Publication of EP1163360A2 publication Critical patent/EP1163360A2/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/918—Carboxylic ester hydrolases (3.1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)

Definitions

  • This invention relates generally to compounds, compositions, devices, and methods useful for detecting the presence of leukocytes through the activity of leukocyte esterases and proteinases in urine.
  • a basic method to measure the number of leukocytes in urine by microscope has been widely available for some time.
  • the disadvantages of this method include the investment of time and money in obtaining and installing the appropriate instrumentation. Further, false negatives can be obtained when samples are allowed to sit too long before analysis.
  • indicator assays that are suitable for detecting leukocytes more easily, conveniently, and accurately.
  • Typical indicator assays use a specific chemical substance (e.g., a substrate) that is degraded by one or more enzymes present in leukocytes to create a product suitable for effectuating a visible color change.
  • leukocyte assay involves the use of peroxidase that is contained in a granular leukocyte.
  • This assay includes a filter paper stained with hydrogen peroxide and ⁇ -tolidine, which shows a colored oxidative product when contacted with leukocytes.
  • this assay is less than desirable because peroxidase can be dangerous and reductive materials in urine may make actual application impractical.
  • Other methods designed to confirm the presence of esterase and proteinase in leukocytes have also been developed. For example, one method uses a colorless or pale-colored ester compound as a substrate that is degraded by esterase into a colorless acid moiety and an alcoholic moiety.
  • This assay allows for the determination of leukocyte concentration by the naked eye. Yet the use of this assay is less than desirable because the diazonium salt may react with urobilinogen or bilirubin contained in urine. As a result, concentrations of leukocytes greater than 500 cells/ ⁇ l are often necessary to avoid a false-negative reading.
  • U.K. Patent No. 1,128,371 discloses other possible substrates, i.e., colorless indoxyl or thioindoxylesters. These substrates are degraded into indoxyl or thioindoxyl by esterase. Colored indigo or thioindigo may then be produced by reaction with oxygen in the air or by an oxidizer. Yet the use of an assay with these substrates is less than desirable because this is not sensitive and fails to detect leukocytes at a concentration less than 10,000 cells/ ⁇ l.
  • U.S. Patent No. 4,278,763 suggests indoxyl-type substrates by disclosing a method of using indoxyl or thioindoxylamino acid ester as a substrate.
  • U.S. Patent No. 4,704,460 discloses use of an amino acid ester of a pyrrole derivative as a substrate. When this derivative is degraded in the presence of diazonium salt, a change in sample color to deep violet results. Moreover, the addition of a nucleophilic alcohol, such as decanol, greatly facilitates reaction rate, and in turn, allows detection of leukocyte concentrations as low as 10 cells/ ⁇ l within 90 seconds. Yet this assay is less than desirable because the syntheses of these two substrates is very difficult.
  • the above-described assays are characterized by numerous disadvantages. Some disadvantages include the detection of false negatives, the interference of urobilinogen or bilirubin, the lack of sensitivity, and the requirement of less-than- desirable substrates. Thus, it would be desirable to identify new compounds and methods of using the compounds to detect the presence of leukocytes in urine.
  • the invention is directed to thiazole esters suitable for detecting leukocytes in urine, compositions containing thiazole esters, diagnostic devices suitable for detecting leukocytes in urine, and methods of using the thiazole esters or compositions thereof for detecting leukocytes in urine.
  • this invention is directed to novel thiazole esters.
  • One thiazole ester according to this invention is of the formula:
  • A is an N-blocked amino acid residue or an N-blocked peptide chain, preferably N-blocked alanine or N-blocked polyalanine;
  • Ri is unsubstituted or substituted heteroaryl; alkenyl substituted with unsubstituted or substituted aryl; or alkenyl substituted with unsubstituted or substituted heteroaryl.
  • Ri may be thienyl, pyridyl, furyl, styryl, pyrrolyl, or indolyl.
  • Another thiazole ester according to this invention is of the formula:
  • A is an N-blocked amino acid residue or an N-blocked peptide chain, preferably N-blocked alanine or N-blocked polyalanine;
  • R 2 is unsubstituted or substituted fused hydrocarbyl rings in which at least one ring is aromatic.
  • R 2 may be naphthyl or anthryl.
  • Another thiazole ester according to this invention is of the formula:
  • A is an N-blocked amino acid residue or an N-blocked peptide chain, preferably N-blocked alanine or N-blocked polyalanine; and R 3 and Rt are each independently hydrogen, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted alkyl, unsubstituted or substituted alkenyl, unsubstituted or substituted alkoxy, amino, unsubstituted or substituted acyl, halo, nitro, cyano. -SO H, or hydroxy, with the proviso that R 3 and R 4 are not both hydrogen.
  • compositions that include a thiazole ester of formula I, II, or III.
  • Compositions of the invention may also include a diazonium salt such as 2-methoxy-4-morpholinobenzene diazonium chloride, zinc chloride double salt.
  • diagnostic devices that include a compound or composition of the invention. Such diagnostic devices include a substrate having a thiazole ester of formula I, II, or III deposited thereon.
  • the compounds, compositions, and diagnostic devices of the invention are also suitable for use in methods for detecting leukocytes in urine.
  • Methods for detecting leukocytes in urine include contacting a thiazole ester of the invention and a diazonium salt with a urine sample.
  • the compounds and compositions suitable for use in devices and methods of the invention are typically pharmaceutically acceptable.
  • compositions, diagnostic devices, and methods of the invention can be free of accelerating salt.
  • the invention is directed to detecting leukocytes in urine.
  • the presence of leukocytes is typically detected by detecting the presence of enzymes known in the art as leukocyte esterases and leukocyte proteinases that are present with leukocytes.
  • Compounds of the invention include thiazole esters that contain an ester functionality, which is hydrolyzed upon exposure to leukocytes in urine. The hydrolysis of the ester of a thiazole ester of the invention provides a useful diagnostic technique because thiazolyl can react with a diazonium salt to produce an azo dye.
  • the compounds according to this invention are thiazole esters that are suitable for use in compositions, diagnostic devices, and methods that can be used to detect leukocytes in urine.
  • One thiazole ester suitable for use in compositions and methods of the invention is of the formula: or a salt or solvated salt thereof, in which A is an N-blocked amino acid residue or an N-blocked peptide chain, preferably N-blocked alanine or N-blocked polyalanine (e.g., alanine-alanine, alanine-alanine-alanine, and the like), being blocked at the amino terminus by protecting groups known in the art, such as, for example, benzyloxycarbonyl, t-butoxycarbonyl, and -toluenesulfonyl; and Ri is unsubstituted or substituted heteroaryl; alkenyl substituted with unsubstituted or substituted aryl; or alkenyl substituted with unsubstituted or substituted heteroaryl.
  • A is an N-blocked amino acid residue or an N-blocked peptide chain, preferably N-blocked alanine or N
  • heteroaryl includes heterocyclic aromatic derivatives having at least one heteroatom, such as, for example, nitrogen, oxygen, phosphorus, or sulfur, and includes, for example, furyl, pyrrolyl, thienyl, oxazolyl, pyridyl, imidazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, and the like.
  • heteroaryl also includes fused rings in which at least one ring is aromatic, such as, for example, indolyl, purinyl, benzofuryl, benzothienyl, quinolyl, 4,5,6,7-tetrahydro-lH-indolyl, and the like.
  • fused rings that have a hydrocarbyl ring fused to a heterocyclic ring, such as, for example, benzothienyl and indolyl
  • the fused rings may be bonded to thiazolyl through either the hydrocarbyl ring or the heterocyclic ring.
  • heteroaryl includes a 5-membered ring.
  • heteroaryl includes a 6-membered ring.
  • Such heteroaryl groups may be unsubstituted or substituted on the ring by, for example, aryl, heteroaryl, alkyl, alkenyl, alkoxy, amino, acyl, halo, nitro, cyano, -SO 3 ⁇ , or hydroxy, in which such substituents may further be substituted by aryl, heteroaryl, alkyl, alkenyl, alkoxy, amino, acyl, halo, nitro, cyano, -SO 3 H, or hydroxy.
  • heteroaryl is substituted with alkyl, such as methyl, ethyl, propyl, or butyl.
  • heteroaryl is substituted with alkoxy, such as methoxy, ethoxy, propoxy, or butoxy.
  • heteroaryl is substituted with aryl or heteroaryl.
  • aryl includes aromatic hydrocarbyl, such as, for example, phenyl, including fused aromatic rings, such as, for example, naphthyl, anthryl, and the like.
  • alkyl includes a straight or branched saturated aliphatic hydrocarbon chain having from 1 to 4 carbon atoms, such as, for example, methyl, ethyl, propyl, isopropyl (1-methylethyl), butyl, t-butyl (1,1-dimethylethyl), and the like.
  • alkenyl includes an unsaturated aliphatic hydrocarbon chain having from 2 to 4 carbon atoms, such as, for example, ethenyl, 1-propenyl, 2- propenyl, 1-butenyl, 2-methyl- 1-propenyl, and the like.
  • the above alkyl or alkenyl groups may optionally be interrupted in the chain by a heteroatom, such as, for example, a nitrogen or oxygen atom, forming an alkylaminoalkyl or alkoxyalkyl group, for example, methylaminoethyl or methoxymethyl, and the like.
  • a heteroatom such as, for example, a nitrogen or oxygen atom, forming an alkylaminoalkyl or alkoxyalkyl group, for example, methylaminoethyl or methoxymethyl, and the like.
  • alkoxy includes alkyl as defined above joined to an oxygen atom having from 1 to 4 carbon atoms in a straight or branched chain, such as, for example, methoxy, ethoxy, propoxy, isopropoxy (1-methylethoxy), butoxy, t- butoxy (1,1-dimethylethoxy), and the like.
  • amino includes a substituent of the formula -N(R 5 ) 2 in which each R 5 is independently hydrogen or alkyl.
  • alkyl is as defined above.
  • alkenyl substituted with unsubstituted or unsubstituted aryl or “alkenyl substituted with unsubstituted or substituted heteroaryl” includes alkenyl as defined above, and such groups are substituted with unsubstituted or substituted aryl or unsubstituted or substituted heteroaryl, respectively.
  • aryl and heteroaryl are as defined above. Examples of suitable substituted alkenyls include styryl, cinnamyl, furylethenyl, pyridylethenyl, andthienylpropenyl.
  • alkenyl substituted with unsubstituted or substituted aryl is alkenyl substituted with unsubstituted or substituted phenyl.
  • This phenyl in some embodiments, is substituted with alkoxy, such as methoxy, ethoxy, propoxy, or butoxy.
  • alkenyl substituted with unsubstituted or substituted heteroaryl is alkenyl substituted with a nitrogen-containing ring, oxygen-containing ring, or sulfur-containing ring.
  • Ri groups for thiazole esters suitable for use in compositions and methods of the invention include 2-thienyl; 4-pyridyl; 2-furyl; ⁇ -styryl; 1,2- dimethyl-4-pyrrolyl; and 3-indolyl. It should be appreciated that one skilled in the art, having read this specification, would understand that a heteroatom may be at any one of several positions in a ring.
  • Another thiazole ester suitable for use in compositions and methods of the invention is of the formula: or a salt or solvated salt thereof, in which A is an N-blocked amino acid residue or an N-blocked peptide chain, preferably N-blocked alanine or N-blocked polyalanine (e.g., alanine-alanine, alanine-alanine-alanine, and the like), being blocked at the amino terminus by protecting groups known in the art, such as, for example, benzyloxycarbonyl, t-butoxycarbonyl, andjo-toluenesulfonyl; and R 2 is unsubstituted or substituted fused hydrocarbyl rings in which at least one ring is aromatic.
  • A is an N-blocked amino acid residue or an N-blocked peptide chain, preferably N-blocked alanine or N-blocked polyalanine (e.g., alanine-alanine, alanine-
  • Fused hydrocarbyl rings include at least two hydrocarbyl rings that maintain at least one bond in common between the rings, for example, naphthyl, anthryl, 5,6,7,8-tetrahydronaphthyl, phenanthrenyl, triphenylenyl, lH-fluorenyl, and the like.
  • Fused hydrocarbyl rings may be substituted by, for example, aryl, heteroaryl, alkyl, alkenyl, alkoxy, amino, acyl, halo, nitro, cyano, -SO ⁇ , or hydroxy, in which such substituents may further be substituted by aryl, heteroaryl, alkyl, alkenyl, alkoxy, amino, acyl, halo, nitro, cyano, -SO H, or hydroxy. These substituents are as defined above.
  • fused hydrocarbyl rings contain at least one 5- membered ring. In other embodiments, fused hydrocarbyl rings contain at least one 6-membered ring. In still some embodiments, fused hydrocarbyl rings are substituted on at least one ring with alkyl, such as methyl, ethyl, propyl, or butyl. In other embodiments, fused hydrocarbyl rings are substituted on at least one ring with alkoxy, such as methoxy, ethoxy, propoxy, or butoxy. In one embodiment, R 2 is 1 -naphthyl.
  • Another thiazole ester suitable for use in compositions and methods of the invention is of the formula:
  • A is an N-blocked amino acid residue or an N-blocked peptide chain, preferably N-blocked alanine or N-blocked polyalanine (e.g., alanine-alanine, alanine-alanine-alanine, and the like) being blocked at the amino terminus by protecting groups known in the art, such as, for example, benzyloxycarbonyl, t-butoxycarbonyl, and ?-toluene sulfonyl; and R 3 and R are each independently hydrogen, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted alkyl, unsubstituted or substituted alkenyl, unsubstituted or substituted alkoxy, amino, unsubstituted or substituted acyl, halo, nitro, cyano, -SO 3 H, or hydroxy, with the
  • R 3 and Ri groups may be further substituted by aryl, heteroaryl, alkyl, alkenyl, alkoxy, amino, acyl, halo, nitro, cyano, -SO 3 H, or hydroxy.
  • aryl aryl
  • alkyl alkenyl
  • alkoxy alkyl
  • amino amino
  • R 3 and t is methoxy, ethoxy, propoxy, or butoxy
  • A is an N-blocked alanine residue.
  • R 3 is hydrogen and R t is methoxy, ethoxy, propoxy, or butoxy.
  • methoxy may be substituted at the ortho position and the compound of formula III may be:
  • methoxy may be substituted at, for example, the para or the meta position and the compound of formula III may be:
  • a composition of the invention includes a thiazole ester of formula I, II, or III.
  • Such a composition may also include a diazonium salt such as 2-methoxy-4- morpholinobenzene diazonium chloride, zinc chloride double salt.
  • compositions of the invention may be free of salts known in the art to have an accelerating action.
  • salts that give an accelerating action include salts of monovalent and divalent cations of the alkali metals and alkaline earth metals, such as, for example, Li + , Na + , K + , and Mg "1-1" as well as their typical anions.
  • a diagnostic device suitable for detecting leukocytes in urine includes a substrate suitable for supporting a thiazole ester of the invention, such as, for example, filter paper, filtration membrane, and other inert carriers. These substrates are known in the art.
  • the diagnostic device also includes one or more thiazole esters of formula I, II, or III. This compound is included with the substrate by, for example, sedimenting the derivative onto the substrate.
  • a novel diagnostic device may be prepared by combining the thiazole ester, an accelerator, and a diazonium salt.
  • Accelerators suitable for use in compositions and methods of the invention are known in the art and include, for example, octanediol and decanol.
  • Diazonium salts suitable for use in compositions and methods of the invention are known in the art and include compounds that may be used for color- developing technology, such as, for example, l-diazo-8-naphtol-3,6-disulfonic acid, chloride, zinc chloride double salt; 6-diazo-l-naphtol-3-sulfonic acid, chloride double salt; and 2-methoxy-4-morpholinobenzene diazonium chloride, zinc chloride double salt.
  • the diazonium salt used in accordance with the invention has no interaction with urobilinogen and/or bilirubin.
  • Methods for manufacturing a device according to this invention are known in the art.
  • a first solution containing boric acid and polyvinylpyrrolidone is deposited onto a substrate, such as a filter paper.
  • a second solution containing a thiazole ester of the invention and a diazonium salt, such as 2-methoxy-4- morpholinobenzene diazonium chloride, zinc chloride double salt is deposited onto the substrate.
  • Both solutions deposited onto the substrate may be free of a salt known in the art to have an accelerating action as described above.
  • a thiazolone derivative and an alanine or polyalanine derivative may be used as starting materials.
  • Thiazolones may be synthesized by reacting carboxymethyl thiobenzimidate hydrobromide and pyridine. As illustrated in the following scheme, the synthesis of a thiazole ester of the invention includes the reaction of a thiazolone derivative of the general formula (2), and an amino acid chloride of the general formula (3).
  • Methods of the invention include detecting the presence of leukocytes in urine.
  • a urine sample is contacted with a thiazole ester of the invention and a diazonium salt in the presence of an accelerator. If leukocytes are present, then a reaction between thiazolyl and the salt produces an azo dye having a violet color.
  • a method is directed to contacting a urine sample with a substrate, for example, filter paper, that has a first solution of boric acid and poly vinyl pyrrolidone deposited thereon and then a second solution of a thiazole ester of formula I, II, or III; a diazonium salt; and an accelerator deposited thereon. This method may be carried out free of an accelerating salt. When a urine sample containing leukocytes contacts such a substrate, then a violet color, which appears on the substrate, is observed.
  • N-(p- toluenesulfonyl)-alanine is first synthesized. 50.0 g ofp-toluenesulfonyl chloride was added to 100 ml of 90 °C toluene with stirring. The j-toluenesulfonyl chloride solution in toluene was slowly added to 25.0 g of L-alanine dissolved in 500 ml of IN NaOH cooled to 5 °C and stirred for 24 hours. The resulting aqueous layer is separated and cooled to below 5 °C and with the addition of concentrated hydrochloric acid, pH was adjusted to 1.5. After standing in the refrigerator for 3 to 4 hours, the white solid crystals, so formed, were filtered, twice washed with water, and dried.
  • the blocked starting material is next produced by reacting N-(p- toluenesulfonyl)-alanine and oxalyl chloride.
  • HC1 gas is bubbled through a solution of 10 g of 4-cyanopyridine and 50 g of mercaptoacetic acid in approximately 20 ml of ether and 30 ml of chloroform for 24 hours. During the reaction 30 ml of ether and 50 ml of chloroform are added. The crystallized product, so formed, is filtered, washed with a solvent system of 1 : 1 ether and chloroform, and dried.
  • HC1 gas was bubbled through a solution of 25 g of 1-naphthonitrile and 25 g of mercaptoacetic acid in approximately 150 ml chloroform for 24 hours.
  • the crystallized product, so formed, was filtered, washed with 200 ml chloroform, and dried.
  • HC1 gas was bubbled through a solution of 5 g of l,5-dimethyl-2- pyrrolecarbonitrile and 5 g of mercaptoacetic acid in approximately 50 ml of ether/chloroform (1 : 1) for 24 hours.
  • the crystallized product, so formed, was filtered, washed with 100 ml ether/chloroform (1:1), and dried.
  • HC1 gas is bubbled through a solution of 5 g of 3-cyanoindole and 5 g of mercaptoacetic acid in approximately 50 ml of ether/chloroform (1 :1) for 24 hours.
  • the crystallized product, so formed, is filtered, washed with 100 ml ether/chloroform (1 :1), and dried.
  • HCl gas was bubbled through a solution of 5 g of 2-furonitrile and 5 g of mercaptoacetic acid in approximately 50 ml of ether/chloroform (1 : 1) for 24 hours.
  • the crystallized product, so formed, was filtered, washed with 100 ml ether/chloroform (1:1), and dried.
  • HCl gas was bubbled through a solution of 25 g of cinnamonitrile and 25 g of mercaptoacetic acid in approximately 150 ml of ether/chloroform (1 :1) for 24 hours.
  • the crystallized product, so formed, was filtered, washed with 200 ml ether/chloroform (1:1), and dried.
  • HCl gas was bubbled through a solution of 11 g of 3 -methoxy benzonitrile and 10 g of mercaptoacetic acid in approximately 75 ml of ether for 24 hours.
  • the crystallized product, so formed, was filtered, washed with 200 ml ether, and dried.
  • the residue, so formed, is dissolved in 100 ml of acteone and 100 ml of hexane is added. After standing in the refrigerator for 1 hour, dark semisolid impurities are decanted off and the remaining solvent evaporated. The amorphous solid, so formed, is dissolved in a minimum of ethylacetate, the resulting solution is then applied to a silica column and eluted with a 2: 1 ethylacetate/hexane solvent system. Fractions containing the desired product are collected, the solvent is evaporated, and the resulting residue is dissolved in 20 ml of acetone followed by the addition of 50 ml hexane. After standing in the refrigerator the product, so formed, is filtered and dried.
  • the product was identified by a proton NMR analysis. A sample of the product was dissolved in acetone- ⁇ (19mg/0.7 ml) and a proton spectrum was collected following AMRI SOP INS-041 using a Bruker® 300 MHz NMR Spectrometer (Bruker-Physik AG, Germany). The identity of the product was further confirmed by infrared spectral analysis by first mixing 263 mg of potassium bromide with 1.7 mg of the product and then grinding them in a Wig L Bug® (Crescent Dental Manufacturing Co., Illinois). The ground mixture was pressed into a disk and an infrared spectrum was collected by a Spectrum 1000 IR Spectrometer following AMRI SOP INS-026. The sample was scanned between 4000 and 400 cm -1 .
  • the residue, so formed, is dissolved in 100 ml of acteone and 100 ml of hexane is added. After standing in the refrigerator for 1 hour, dark semisolid impurities are decanted off and the remaining solvent evaporated. The amorphous solid, so formed, is dissolved in a minimum of ethylacetate, the resulting solution is then applied to a silica column and eluted with a 2: 1 ethylacetate/hexane solvent system. Fractions containing the desired product are collected, the solvent is evaporated, and the resulting residue is dissolved in 20 ml of acetone followed by the addition of 50 ml hexane. After standing in the refrigerator the product, so formed, is filtered and dried.
  • the identity of the product was further confirmed by infrared spectral analysis by first mixing 204 mg of potassium bromide with 1.3 mg of the product and then grinding them in a Wig L Bug® (Crescent Dental Manufacturing Co., Illinois). The ground mixture was pressed into a disk and an infrared spectrum was collected by a Spectrum 1000 IR Spectrometer following AMRI SOP INS-026. The sample was scanned between 4000 and 400 cm -1
  • the product was identified by two proton NMR analyses. For the first analysis, a sample of the product was dissolved in acetone-c? 6 (1 lmg/0.7 ml) and a proton spectrum was collected following AMRI SOP INS-041 using a Bruker® 300 MHz NMR Spectrometer (Bruker-Physik AG, Germany). For the second analysis, a proton spectrum was collected using a Bruker® 500 MHz NMR Spectrometer. The identity of the product was further confirmed by infrared spectral analysis by first mixing 235 mg of potassium bromide with 2.4 mg of the product and then grinding them in a Wig L Bug® (Crescent Dental Manufacturing Co., Illinois). The ground mixture was pressed into a disk and an infrared spectrum was collected by a Spectrum 1000 IR Spectrometer following AMRI SOP INS-026. The sample was scanned between 4000 and 400 cm -1 .
  • EXAMPLE 21 Preparation of a Leukocyte Diagnostic Device
  • a test strip containing a compound of the invention was prepared.
  • a small type of testing paper in a regular square was attached to the end of a polystyrene strip, sedimented, and dried with the following two mixing solutions successively.
  • the first solution 100 ml of aqueous solution
  • K-10 polyvinyl pyrrolidone
  • a second solution (100 ml of acetone) contained 0.06% (w/v) of 2— (2- thienyl)-4-(N-tosyl-L-alanyloxy) thiazol or 2-( ⁇ -styryl)-4-(N-tosyl-L- alanyloxy) thiazol; 0.05% (w/v) 2-methoxy-4-morpholinobenzene diazonium chloride zinc chloride disalts and 1.0% (w/v) n-decanol.
  • the sedimented paper was dried by heating at 50° C for 5 minutes.
  • Each assay was conducted by contacting a test strip (manufactured as described above) with a urine sample.
  • the test strip showed the appearance of a violet color.
  • the initial tint of violet color appeared within about 10 seconds and gradually darkened until the reaction was complete, which was about 1 minute.
  • the completed reaction showed a degree of color change that was suitable not only for identifying whether leukocytes were present in the urine sample but also for semiquantitatively determining the concentration of the leukocytes present.
  • EXAMPLE 22 Preparation of a Leukocyte Diagnostic Device
  • a test strip containing a composition of the invention is prepared.
  • a small type of testing paper in a regular square is attached to the end of a polystyrene strip, sedimented, and dried with the following two mixing solutions successively.
  • the first solution 100 ml of aqueous solution
  • K-10 polyvinyl pyrrolidone
  • a second solution (100 ml of acetone) contains 0.06% (w/v) of 2-(4-pyridyl)-4-(N- tosyl-L-alanyloxy) thiazol; 2-(l-naphthyl)-4-(N-tosyl-L-alanyloxy) thiazole; 2- (1 ,2-dimethyl-4-pyrrolyl)-4-(N-tosyl-L-alanyloxy) thiazol; 2-(2-furyl) -4-(N- tosyl-L-alanyloxy) thiazol; or 2-(3-indolyl)-4-(N-tosyl-L-alanyloxy) thiazol; 0.05% (w/v) 2-methoxy-4-morpholinobenzene diazonium chloride zinc chloride disalts and 1.0% (w/v) n-decanol.
  • the sedimented paper is dried by heating at 50° C for 5
  • Each assay is conducted by contacting a test strip (manufactured as described above) with a urine sample.
  • the test strip shows the appearance of a violet color.
  • the initial tint of violet color appears within about 10 seconds and gradually darkens until the reaction is complete, which is about 1 minute.
  • the completed reaction shows a degree of color change that is suitable not only for identifying whether leukocytes are present in the urine sample but also for semiquantitatively determining the concentration of the leukocytes.
  • a test strip containing a compound of the invention was prepared.
  • a small type of testing paper in a regular square was attached to the end of a polystyrene strip, sedimented, and dried with the following two mixing solutions successively.
  • the first solution 100 ml of aqueous solution
  • K-10 polyvinyl pyrrolidone
  • a second solution (100 ml of acetone) contained 0.06% (w/v) of 2-(3- methoxyphenyl)-4-(N-tosyl-L-alanyloxy) thiazole or 2-(2-methoxyphenyl)-4- (N-tosyl-L-alanyloxy) thiazole; 0.05% (w/v) 2-methoxy-4-morpholinobenzene diazonium chloride zinc chloride disalts and 1.0% (w/v) n-decanol.
  • the sedimented paper was dried by heating at 50° C for 5 minutes.

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  • Plural Heterocyclic Compounds (AREA)
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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
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EP00904434A 1999-01-21 2000-01-20 Zusammensetzung und gerät zur bestimmung von leukozyten in urin Withdrawn EP1163360A2 (de)

Applications Claiming Priority (9)

Application Number Priority Date Filing Date Title
US365592 1989-06-13
US11661399P 1999-01-21 1999-01-21
US116613P 1999-01-21
US14338399P 1999-07-12 1999-07-12
US143383P 1999-07-12
US09/365,322 US6348324B1 (en) 1999-01-21 1999-07-30 Composition and device for detecting leukocytes in urine
US365322 1999-07-30
US09/365,592 US6528652B1 (en) 1999-01-21 1999-07-30 Composition and device for detecting leukocytes in urine
PCT/US2000/001333 WO2000043535A2 (en) 1999-01-21 2000-01-20 Composition and device for detecting leukocytes in urine

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EP1163360A2 true EP1163360A2 (de) 2001-12-19

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JP (1) JP2002535342A (de)
AU (1) AU2619500A (de)
CA (1) CA2359689A1 (de)
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WO (1) WO2000043535A2 (de)

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KR980010425A (ko) * 1996-07-30 1998-04-30 안홍원 뇨중 백혈구 및 단백질 분해 효소 검출을 위한 조성물 및 측정기구

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AU2619500A (en) 2000-08-07
JP2002535342A (ja) 2002-10-22
WO2000043535A2 (en) 2000-07-27
CA2359689A1 (en) 2000-07-27
WO2000043535A3 (en) 2000-11-30

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