CA1238560A - Composition and test device for determining the presence of leukocytes, esterase and protease in a test sample - Google Patents

Abstract

ABSTRACT

A composition for determining the presence of leukocytes, esterase or protease in a test sample.
The composition comprises an ester having the struc-ture

Description

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COMPOSITION AND TEST DEVICE POR
DETE~MINING THE PRESENCE OF LEUKOCYTES, ESTERASE AND PROTEASE IN A TEST SAMPLE

CONTENTS

1. Introduction

2. Background of the Invention 2.1 Chromogenic Esters 2.2 Accelerators 2.3 Diazonium Salt Coupling Agents 2.4 Summary

3. Summary of the Invention

4. Definitions 4.1 N-blocked-amino acids and peptides 4.2 Aryl 4.3 Lower Alkyl 4.4 Suitable Bufer Substance 4.5 Accelerator 4.6 Fused Ring Systems

5. The Composition 5.1 The Ester 5,2 The Bu~fer 5.3 The Accelerator 5.4 The Diazonium Salt

6. The Test Device EHG/pt ~S-1332 rl ~ ~r ,.
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7. Experimental 7.1 General Information 7.2 Preparation of the Ester 7.2.1 Synthesis o~ 3-(N-tosyl-L-alaninyloxy)-5-phenylpyrrole 7.2.2 Synthesis of 3-(N-~osyl-L-alaninyloxy)-5-phenylthiophene 7.2.3 Synthesis of 3-(N-tosyl-L-alaninyloxy~-l-methyl-5-phenylpyrrole 7.2.4 Synthesis of 3-~N-tosyl-L-alaninyloxy)-5-(p-chlorophenyl)pyrrole 7.3 Preparation and Use of Various Test Devices 7.3.1 Test Device in which the ester is 3-(N-tosyl-L-alaninyloxy)-5-phenyl-pyrrole 7.3.2 Test device in which the ester is 3-CN-tosyl-L-alaninyloxy)-5-phen ~hiophene 7.3.3 Test device in which the ester is 3-(N-tosyl-L-alaninyloxy~-l-methyl -5-phenylpyrrole 7.3.4 Test Device in which the ester is 3-(N-tosyl-L-alaninyloxy~-5-(p-chlorophenyl)pyrrole 7.4 Evaluation o the Test Device , .
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.7. INTRODUCTIO~

The present invention relates to a no~el compo-sition and test device useful in assaying a tesk sample for the presence of analytes such as leukocyte cells, esterase and protease. The invention is par-ticularly useful in detecting leukocyte levels in body fluids such as urine, and reduces the laboratory pro-cedure for such assay from a cumbersome counting procedure requiring microscopic observationa to a rapid, facile dip-and-read operation.
The presence of an abnormally high level of leukocytes in a patient's urine is possibly indicative of such pathological conditions as kidney or urogenital tract infection or other dysfunction. Accordingly, accurate urinary leukocyte information can be an invaluable tool to the physician in diagnosis and treatment of such pathologies.
Traditionally, the medical profession has relied on visual determination techniques to count leukocyte population in urine sediment or uncentrifuged urine, a process requiring expensive equipment such as a cen-trifuge and microscope, as well as inordinate time expenditure on the part of the clinician. Moreover, the traditional techniques suffer from the disad-vantage that only intact cells are determined. Leuko-cytes occurring in the urinary system are subject to conditions which can favor extensive cell lysis. For example, it is known that in urines of abnormally high pH, leukocyte half life can be as low as 60 minutes.
Since lysed cells escape detection in visual examina-tion techniques, erroneo~sly low determina~ions and false negatives can result.

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'~238 Of the two techniques o~ microscopic leukocyte analysis - urine sediment and non-centriuged, homo-genized urine - ~he Eormer is clearly the most de-sirable. Although dependable results can inure to the latter, urine sediment observation is used in an overwhelming majority of instances. It requires that the urine sample be centrifuged and the sediment iso-lated and subjected to microscopîc inspection. The analyst then counts the number of leukocytes appearing in the viewing Eield. This task is further compli-cated by the presence oE other urinary components in the sediment such as epithelial cells and salt par-ticles. The varying content of sediment constituents, coupled with other complicating factors including non-homogeneity oE the sample and differing optical powersamong microscope equipment, can lead to enormous errors in the ultimate determination.
It is thus apparent that a quick, acile method . .
of leukocyte determination, one which would eliminate the need for time-consuming techniques, as well as cost-consuming equipment, and which would provide accurate responses to esterase, protease or leukocyte cells, whether the cells are intact or lysed, would indeed constitute a quantum advance in the state-of-the-art. The present invention provides such an advance. Moreover, it is based, not on the ability to see leukocytes, but on the enzymatic activity they sxhibit, and therefore is substantially ree o:E the inaccuracies described above.

2. ~AC}~GROU1~1D OP T~E INVE~ITION

Prior to the present invention, methods ~or determining hydrolytic analytes included chromogenic esters which, when hydrolyzed by esterase or protease, produced a colored alcoholic product, the intact ester being of a different color from the free alcohol. ~any o~ these systems included accelerator compounds and diazonium salt coupling agents.

2.1 Chromogenic Esters Thus, there exists in ~he prior art a body of references which disclose the use of certain esters which, when cleaved by enzymatic activity, result in the formation of color or other detectable species.
British Patent No. 1,128,371 discloses the use of indoxyl and thioindoxyl esters as useful chromogens in detecting hydrolytic enzymes in body fluids. The enzymes cleave the ester to generate free indoxyl, which subsequently oxidizes to form the dimeric pro-duct indigo, a readily observable blue dye. Such activity is said to be due to, among other enzymes, cholinesterase. This patent also teaches that, in addition to the indoxyl portion of the ester sub-strate, the acid radical is chosen with particular reerence to the enzyme to be detected. For example, it is stated that the acid radical can be acetate, laurate or stearate :Eo.r detect:ion o:E esterase or lipase, respectively. For detecting enzymes such as phosphatase or sulfatase the acyl radical can be inorganic. Thus, the British Patent teaches the use ~ of chromogenic esters as substrates ~or determining esterolytic enzymes, such esters comprising indoxyl or thioindoxyl as the alcoholic moiety o~ the ester, the acyl moiety being tailored to the particular enzyme to be determined.

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~3~5~3 The affect of careful acyl radical selection is nowhere more clearl~ exemplified ~han in two refer-ences which demonstrate esterase specificity for esters in which the acyl radical comprises an N-protected amino acid or peptide. Thus Janoff, et aZ.,Proc. ~oc. Exper. Bio~. Med. 136:1045-1049 ~1971) teaches that alanine esters are specific substrates for esterase obtained from human leukocytes. Speci-fically, this reference teaches that an extract of human leukocyte granules is capable of hydrolyzing n-acetyl-l-alanyl-L-alanyl-L-alanine methyl ester.
Moreover, L-alanine-p-nitrophenyl ester was similarly hydrolyzed to yield the yellow p-nitrophenol color-form.
Similarly, Sweetman et a~., Jour. Hist. Soc., 22:327-339 teaches the use of l-naphthyl N-acetyl-DL-alanine, 1-naphthyl N-acetyl-L-alanyl-L-alanyl-L-alanine and l-naphthyl butyrate to demonstrate the presence of esterase.
2Q United States Patent No. 4,278,763, assigned to Boehringer Mannheim GmbH, combines these teachings in arriving at the indoxyl or thioindoxyl esters of amino acids or peptides as still another example of a tra-ditional colorogenic substrate for leukocytic esterase activity. Like the Janoff and Sweetman references, the Boehringer patent teaches the equivalence of protease and esterase in their esterolytic penchants.

2 . 2 . A cae ~ era ~ors It is known that ester hydrolysis reactions can be actiYated by the presence of many nucleophilic agents, including a myriad of alcohols. Thus, the rate of hydrolysis of phenyl acetate and p-nitrophenyl acetate by esterase is increased 2.5 to 5.5 times upon addition of methanol or butanol. Greenzaid and Jencks, Bio~hemistry, 10(7), 1210-1222 ~1971). Moreover, the 31 ~3~5~t effect increases with the length of the n alkyl group.
Wynne and Shalatin, Eur. J. Biochem., 31:554-560 (1972).
In particular, this activation affect of alcohols has been observed with esters of amino acids. p-Nitrophenyl-N-acetyl L-alaninate hydrolysis is acti-vated (accelerated) by the presence of methanol.
Fastrez and Fersht, Bio~hemist~y, 12(11J~ 2025-2034 (1973). High molecular weight alcohols increase the rate of esterase-induced hydrolysis of p-nitrophenyl-t-BOC-L-tyrosinate. Ashe and Zimmer, Biochem. and Biophys. ~es. Comm., 75flJ, 194-199 (1977). The disclosure of U.S. Patent No. 4,299,917 describes other known ester hydrolysis activators such as cer-tain metal complexes, pyridine derivatives and imida-zoles.

2.3 Diazonium SaZt CoupZing Agents Also known is the use of certain diazonium salts to couple with phenols and pseudophenols to produce 20 azo dyes. Martinet and Dornier Compt. Rend., 170, 592 (1920~. Such a technique is used in an esterase analysis whereby indoxyl esters are hydrolyzed via esterase to produce indoxyl, which is in turn coupled with a diazonium salt to form the corresponding azo 25 dye. Holt and Hicks, J. CeZZ BioZ. 29, 261-366 (1966);
Gossrau, ~istoohemistry~ 57J 323-342 (1978); ~est German O~fenZegungschrift Nc. 30 17 721, filed May 9, 1980.

2.4 Summ~ry To summarize the background o~ technological developments leading up to the present invention, several methods are known for assaying hydrolytic enzymes and leukocyte cells in solution. ~or measur-ing leukocyte populatons, in urine, for example, microscopy has long been the preferred method. Thus, the technician was required to make a microscope slide ..

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of a urine sample and coun~ the number of leukocyte cells in the ~ield o~ view of a microscope; a pro-cedure requiring an inordinate expenditure o~ time and expensive equipment such as a microscope and centri-fuge.
Chem.ical and biochemical techniques are rapidly challenging the microscope for assaying leukocytes in diagnostics and are time-honored tools in the research laboratory. Chromogenic esters which have ~ormed the keystone of chemical tests include the ~ollowing alcoholic and acyl moieties:

Alcoholic ~phenolic) Moieties Acyl Groups indoxyl acetate thioindoxyl butyrate p-nitrophenyl laurate a-naphthol stearate amino acid peptide Chemistries utilizing such esters have been abetted by various hydrolysis accelerators, as well as diazonium salt coupling agents. As accelerators, many alkanols have been employed, as have certain metal complexes, pyridine derivatives and imidazoles. Dia-zonium cations having appropriate anions ionically bound or associated therewith are well known as coupl-ing agents or such chemistries, whereby the alcohol.
(phenol~ ~ormed upon hydrolysis o~ the ester is coupled with the diazonium salt for ~orming an azo dye.

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3. SZ~MMAR~l OF THE I~VE~IO~

The present invention provides a new test com-position and device for determining the presence of leukocy~es, esterase or protease in a test sample. The composition comprises an ester having the structure R*~_____,O - A

R X

in which: A is an N-blocked-amino acid residue or an N-blocked-peptide acid residue; X is O, S, or NR'; R
is aryl, or lower alkyl; R* is H, or lower alkyl; and R' is H, lower alkyl or aryl. In addition, the com-position includes a suitable buffer substance. The composition can also include an accelerator (such as an alkanol having 1-15 carbon atoms), and/or a dia-zonium salt coupling agent.
The test device comprises a carrier matrix in-corporated with the ester and buffer, and can addi-tionally include the accelerator and/or coupling agent.
The method for using the composition o~ device comprises contacting a test sample suspected of con-taining leukocyte cells, esterase or protease with the composition or test device, and observing a ~etectable response.

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lZ3~S~) ~ . DEFI NI TI ONS

Certain terms used in the present discussion should at this point be mentioned to assure that the reader is a~are of their respective meanings. Thus, the following definitions are provided to clari~y the scope of the present invention, and to enable i~s formulation and use.

~ .1 The expressions "N-bloc~ed-amino acid re-sidue" and "N-blocked-peptide residue" require defi-nition on two counts. "N-blocked" refers to the chemistry of the amine group of an amino acid or peptide ~hereby a hydrogen bound to the nitrogen atom is replaced by a protective group such as acetyl, p-toluenesulfonyl (tosyl), tert-butyloxycarbonyl (t-BOC) and other N-protective groups known in the art.
By the terms "amino acid residue" and "peptide residue" is meant an amino acid or peptide molecule without the -OH o its carboxyl group.

4.2 By the expression "aryl" is meant any ring system containing aromaticity. Included by the term are such 5- and 6-membered rings as pyrrole, phenyl, and pyridyl, as well as used ring systems such as naphthyl. Thus, the aromatic ring system can be heter-ocyclic or homocyclic, and can be substitu~ed or unsubstituted, provided the substituent groups~s) not interfere with ability o the composition to hydrolyze in the presence o leukocy~e cells, esterase or pro-tease. Selection o such ~ubstituents is a routine laboratory determination, given the present disclosure.

4.3 The expression "lower alkyl", as used in the present disclosure, is an alkyl moiety containing about 1-6 carbon atomsO Included in the meaning of lower alkyl are methyl, ethyl, n-propyl, isopropyl, ~-butyl, sec-butyl, ~ert-butyl and all isomers of pentyl and hexyl. These can be unsubstituted, or they can be substituted provided they not interfere with the ability to the composition or test device to detect leukocyte cells, esterase or protease.

4.4 By "suitable buffer substance" is meant a buffer which, when contacted with an aqueous test sample, will provide a resultant pH of at least about 7. Preferably the buffer is one capable of producing a pH in the range o~ about 7-10 and, optimally, 8.5-9Ø Boric Acid-NaOH, Bicine ~N,N-bis[2-hydroxyethyl]-glycine, or CHES~2-~N-cyclohexylamino]ethanesulfonic acid) are exemplary of suitable buffer substances.

4.5 The expression "accelerator" relates to any compound which serves to increase the rate of hydro-lysis o the chromogenic esters described herein.Included are such chemically diverse substances as pyridine, imidazoie and their derivatives; metal complexes of the formula Dm ~B~CN)n(NO)p] in which D
is alkalî metal, B is a heavy metal ion, P is 0 or 1, 25 m is 2-5, n is 4-8, and m is the sum of n and the valence of B; and alcohols. Suitable alcohols have rom 1 to about 15 carbon atoms. Linear alcohols are preferred over branchea chain alcohols, although the latter are included within the scope of the invention.

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4.6 By "fused ring system" is meant two or more aromatic rings that share a pair of carbon atoms. For example, in the structure 1~.., G~ ~ ,B
I ~ D (II) G
both of G can together form the fused ring system ~III) so3-in which both of G together constitute ~CH34. Yet another example is ~0~`~ (IV) N
so3 in which both of G together constitute ~CH~CH-CH=N3.
Hence, the fused ring system is polynuclear, aromatic, and can be heterocyclic or homocyclic.

4.7 The expression "detectable response" is intended herein as meaning a change in or occurrence o~ a parameter in a test mean5 system which is capable of being perceived, either by direct observation or instrumentally; and which is a function of the pre-sence of a specific analyte in an aqueous test sample.
Some detectable responses are the change in or ap-pearance of color, fluorescence, reflectance, pH,chemiluminescence and infrared spectra.

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5. TH~ COMPOSI~ION

The composition of the present invention includes an ester and a buffer. While there is great latitude in choosing these ingredients, there are preferred embodiments of each which produce maximized results, i.e., a high degree of detectable response de~eloping in a short time. This optimization can be still further promoted by including an accelerator and/or a diazonium salt in the composition.

10 5.1 The Ester Both the composition and test device of the pre-sent in~ention include an ester of the formula I and a suitable buffer. The preferred ester is one in which X is NR' and the preferred R' substituent is H. An-other preferred embodiment is where the acyl moiety,A, is a N-blocked-amino acid residue in particular N-blocked-L-alaninate. It is especially preferred to use as the ester the compound ~ ~N ~ (V) where Ts is p-toluenesulEonyl, i.e., compound I in which A is N-Ts-L-alaninate, R* is H, R is phenyl, and X is NH.

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As is stated, supra, the buffer substance in-cludes a mul~itude of compounds. The preferred pH
range to be produced by the buffer is about 7-10, with

8.5-9 0 being the optimum pH range. To achieve this optimum pH range, it is presently preferred to use Boric acid-NaOH, Bicine ~N,N-bis~2-hydroxyethyl]glycine), or CHES~2-[N-cyclohexylamino]ethanesulfonic acid) as the buffer.

0 5. 3 The AcceZer~ator The composition of the present invention can in-clude, in addition to the ester and buffer, various accelerators such as are defined in paragraph 4.5.
Alcohols having been found to be especially useful in increasing esterase and protease catalyzed hydrolysis of the esters discussed herein. Alcohols having 8-15 carbon atoms are preferred for this purpose, while decanol, undecanol and dodecanol are preferred for use with the test device, primarily because of their low volatility as compared with alcohols of lower mole-cular weight.

5. ~ The Di,azoni,um Se?,t The composition can also include a diazonium salt as a coupling agent. Participation of the diazonium salt in the overall reaction scheme can be represented as follows:
R ___,O-A R ~ _ O
R ~ X ~ + ~IO-,~
R~r~ O R~ _____,OII
~ J + Ar-N -~ N ~ ~ (VI) R X R'A~ N N=N-Ar :

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in which ArN _N is the diazonium salt, and A, R, R*
and X are as defined su~ra. The reaction product VI
is an azo dye which can exhibit a deep, distinctive color.
The salt ArN --N can take on a plethora of mean-ings. Generically it is an aromatic diazonium salt, and has as its generic structure the following:
r R" +
R"~ ~ ~R"
R~ 1YO, R" D (VII) in which R", same or diff0rent, is H, lower alkyl or aryl, or in which two adjacent R" together form a fused ring system, with the proviso that one of R"
is -N _N, i.e., diazonium. Y is N or CR". D is an anion such as chloride, bromide, or other appropriate counterion to the diazonium moiety.
A diazonium salt which is self-contained, i.e., a zwitterion, serves as an excellent coupling agent.
Such compounds have the structure ~ D ~VIII) in which B is H, lower alkyl or OH; D is a covalen~ly bound anion; G, same or di-fferent, is H, lower alkyl or aryl, or in which ~oth of G toge~her form a fused ring system.

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Thus the zwitterion is a species of diazonium salt wherein the counterion to the diazonium group is covalently bound to the ring system. Examples of such anions include sulfonyl (SO3 ), carbonyl ~CO2 ), phosphonyl ~PO3 ), and others. It has been found most advantageous to use l-diazo-2-naphthol-4-sulfonate.

6 . THE TEST DEVI CE

The composition described above can be used by itself in determining leukocytes, esterase or pro-tease, or it can be incorporated with a carrier matrixto form a test device, thereby providing a tool for rapid, reliable estimation of the presence of the analyte. The carrier matrix is usually, but not neces-sarily, a porous substance such as filter paper.
Other art-recognized forms of carrier matrix materials are felt, porous ceramic strips, and woven or matted glass ~ibers (U.S. Pat. No. 3,8~6,247). Also sug-gested are the use of wood, cloth, sponge material and argillaceous substances ~U.S. Pat. No. 3,552,928).
Alternatively, the carrier matrix can be nonporous, such as various polymeric films, glass and the like.
.
All such carrier matrix ~naterials are feasible for use in the present invention, as are others. It has been ound that filter paper is especially suitable.
In a preferred method o~ preparing the device a piece of filter paper is wetted with an aqueous solu-tion of the buf~er. This first-dip solution can also contain various processing excipients such as a deter-gent, a sizing agent such as polyvinylpyrrolidone, and other inert ingredients.
The impregnated filter paper is then dried and wetted with a second-dip solution, in ac~tone or other .

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nonaqueous solvent, containing the ester and, if desired, the accelerator and/or diazonium salt. The twice-impregnated paper is then dried a second time, thus forming a test device sensitive to the presence of leukocytes or other analytes.
The dried, reagent-bearing carrier matrix can be mounted on a backing material if desired. In a preferred embodiment o~ the test device, a filter paper carrier matrix is incorporated with the com-position as described, supra, the matrix being affixedto one side to an elongated piece of transparent poly-styrene film. The matrix is secured to the film by any suitable means, such as double faced adhesive tape (Double Stick~ available from 3M Company), the other end of the polystyrene film serving as a handle. In use, such a device is held by the free end of the polystyrene film backing material and the matrix end is immersed into the test sample ~e.g., urine) and quickly removed. Any color formation or other detect-able response is observed after a predetermined timeand compared with a reference standard corresponding to responses to known concentrations of leukocytes or other analyte having esterase or protease activity. It has been ~ound that an incubation time of about 1-3 minutes is usually sufficient to enable color develop-ment to occur in the reagent-containing filter paper.

7 . EXPERI ME~TA L

The following examples are provided to further assist the reader in making and using the present in-vention. Thus, preferred embodiments are described inexperimental detail and analyzed as to the results~
The examples are meant to be illustrative only, and are in no way intended as limiting the scope of the invention described and claimed herein.

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7.1 Ge~eraZ Information In the ~ollowing experimental discussion abbre-viations are used as indicated:
g = gram kg = kilogram L = liter mL = milliliter M = molar mM = millimolar N = normal eq = equivalents mol = gram molecular formula (moles) mmol = gram molecular -formula x 10 3 (millimoles) aq = aqueous hr = hour TLC = thin layer chromatography .
In~rared (IR) spectra were obtained with a Perkin-Elmer Model 710B or 237 infrared spectrophotometer as solutions in CHC13 unless otherwise noted; the 1602 20 cm~l band of polystyrene ~ilm was used as an external calibration standard. Signals are reported as cm 1.
Proton magnetic resonance ( H NMR) spectra were obtained at 89.55 MHz using a JEOL FX-900 spectrometer or at 60 MHz using a Varian T-60 spectrometer; spectra 25 were obtained in CDC13 solution unless otherwise noted. Chemical shi~ts are reported in part per million down~ield ~rom the internal standard tetra--methylsilane.
Carbon-13 magnetic resonance (13C NMR) spectra 30 were obtained at 22.5 MHz using a JEOL FX9OQ spectro-meter with Fourier ~rans~orm and with ull proton broad-band noise decoupling; spectra were obtained in CDC13 solution unless otherwise noted. Carbon shi~ts 31~
are reported in parts per millon do~nield from the in~ernal stand.~rd tetr~ethylsilane~
Mass spectra ~IS) were o~tained on a Hewlett-Pac~ard $~85A spectrometer operating in ei~her an el~c~ron impact tEI~ or a ~a~t ~tom bombardment ~AB) ~ode. Hi~h-resolution mass spe~ra we~e ob~ained on an AEI MS-902 spect~omet~r.
Organic reagents ~ere obtained r~m Aldric~
Chemical Company and were used without purification, unless o~herwise noted. Inor~anic reagents were ACS
reagent grade from ~isher Scien~ific Company or o~}ler major vendor, Reaction sol~ents were ACS reagen~
grade; ~etrahydro~ran (~F) ~as HPLC grade fro~ J. T.
Baker Chemical Company. Brine reers to a satura~ed aqueous sodium ~hloride solutionL
Thin layer chromatography (TLC) ~as perormed using silica gel 60F-~S4 plates ~rom E. Merck, Column chromatography was perormed usin~ ~, J~erck Silica Gel 60 (70-230 mesh~. All mel~in~ points and boiling ~ points reported are u~eorre~ted, 7. 2 P~epar~tio~ of the E~er The following experiments were performed to il-l~strate the synthesis of th~ ester of ~he present in~ention, While these experiments relate to speciic starting ma~erials and end produc~s, it is ~elieved th~t the proced~res are applicable to a broad r~nge o~
species included within the ~eneric class of esters disclosed h~rein, 7, 2,1 ,~ynthesis of 3- (N-to~yt-L-ctIan*n~oso~) S-phenytp~r~oI~
The synthe~is of 3-~N-~osyl-L-alaninyloxy~-5-phenylpyrrole is illustrated in the following reaction sequen~e~

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COOH . ,COOI-I

Q' KHS05, 1120,~
acetone, NaH 3 ~

¦ KOI-I/EtOH

~COO ~( 2H20 ~10~ +Glycine ~ICH2COO K 100~ C

Pyridine 12~ ~
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0~1 _~0 - Ac (Z) ~ I O C ~ [~ t3 .

O-C-Cll-C~I I
~ rll~ _ Cl - c - Ic~l- C~13 p~ridine TFA ~,~ N~rs ';

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N-tosyl-L-alanine l~alanine (lOOg; 1.11 moles) was dissolved in 2.25 L of l N sodium hydroxide (aq), cooled to 5C and stirred while a solution of p-toluenesulfonyl chloride (218 g; l.ll moles) dissolved in 450 mL of toluene was added slowly. The mixture was stirred at ambient temperat~re for 20 hr. The layers were separated and the chilled aqueous layer acidified to pH l with con-centrated hydrochloric acid. The white solid title compound was collected by filtration, washed with water and dried. Yield ~78.5 g (66%) mp 134-5C. IR
(CHC13) cm 1 1726, 1340, 1165, 1095; lH NMR (DMSO-D6) 1.20 (d,J=7j3H), 2.40 (s,3H), 3.85 (p,J=8,lH), 6.4 (br s,lH)(C02H), 7 41 (d,¦JAB¦=8, 2H) and 7.75 (d,¦JAB¦=
8,2H) ~center of pattern: 7.58; aVAB=20.49Hz], 8.03 (br d,J=8,lH)(NH).

N-tosyl-L-alaninyl chloride Method A
..
A mixture of N-tosyl-L-alanine ~12.4 g; 0.05 mol) and thionyl chloride (25 mL) was heated for 90 minutes at 55C, and then concentrated on the rotary evapora-tor at 40C. The red solid residue was dissolved in 200 mL of boiling CC14, decolorized with 20 g of oven-dried Norit~ 211 ~American Norit Co., Inc.), fil~ered and chilled. The cream colored solid title product was collected by filtration, washed with hexane and dried. Yield 8 48 g (65%) with mp 101-101.5C.
IR ~CHC13~ cm 1 3360, 3260, 3025, 1775, 1605, 1350, 1170, 910; lH NMR (CDCl3) ~ 1.48 (d ~J=7,3H), 2.43 (s, 3H), 4.33 (p,J-8,1H), 5.~3 (br d,J=8,1H)(NH), 7.31 (d,¦JAB¦ 38, 2H~ and 7.76 (d,¦JAB¦=8,2H) ~center of pattern: 7.53; ~VAB=26.83Hz].
Anal. calcd. for CloH12ClN03S: C,45.89; H,4-62; N,5-35-Found: C,46.63; H,4.90; N,5.19.

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Method B
___ A mi~ture of N-tosyl-L-alanine ~3.1 g; 13 mmol) and thionyl chloride (6 mL) was heated for 90 min at 50C, then diluted wi~h 50 mL of dry hexane. The mix~ure ~as stirred rapidly, chilled and ~he solid product filtered. Yield 3.15 g (93%) mp 99-100C. The IR spectrum was identical to that o-f the recrystal-lized material prepared ~y Method A.

2-Hydroxy-3-~carbo2ymethyZaminol-hydrocinnamic ac~d Dipotassium saZt dihydrate (11 A stirred slurry of 1.0 kg of trans-cinnamic acid ~6.75 mol~ in 4.5 L acetone was treated first with NaHCO3 (2.47 kg; 29.4 mol; 4.36 eq) then carefully with water C4 5 L). The resulting thick mixture was lS treated dropwise, over 1.5-2.0 hr, with a solution of OXONE ~Dupont Co.) monosulfate compound (3.78 kg;
contains 1.825 eq of KHSO5) in 0.4 mM aqueous disodium ethylenediamine tetraacetic acid (EDTA) ~14.5 L;
prepared ~y dissolving 2.17 g disodium EDTA dihydrate in 14.5 L distilled water).lj2 During this addition the reaction temperature was maintained at 24-27C
using a water bath; the reaction pH was noted to be about 7.4. After the addition was completed the mixture was stirred an additional 0.5 hour then cooled to about 10C. The reaction was acidified with conc.
HCl ~~ 1.2 L) to pH ~ 2 while maintaining the tempera-ture at around 10C, and then treated with CH2C12 ~5.05 L) and stirred vigorously or 10 minutes.
Ater allowing the mixture to settle, the aqueous layer was decan~ed off and the organic layer, which 1. J,O. Edwards, et aZ, Photochem. Photoo~o?~ 30, 63 (1979)-2. R. Corci, et a~, J. Org. Chem. 45, 4758 ~1980).

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56~

contained insoluble salts, was ~iltered through paper with suction. The ~iltered solids were washed with CH2Cl2 Cl.9 L~ and the aqueous layer extracted with this filtrate. T~e filtered solids were again washed with CH2Cl2 (3.15 L) and ~he aqueous layer e~tracted with this filtrate. The combined CH2C12 layers were extracted with a solution of KOH (593.3 g) in water (6.31 L) - gentle heating to about 40C is often re-quired to dissolve a solid which may separate during the base extraction. The CH2C12 layer was then ex-tracted with a solution of KOH (99 g) in water (1.5 L) and the combined base extracts treated with glycine (481.7 g; 6.416 mol; 0.95 eq); the organic layer was discarded.
The solution pH was adjusted to 11.5 with 25%
aqueous KOH then heated to boiling. Approximately 900 mL of low boiling liquid (acetone and water) was distilled off until the ~apor temperature reached 99C, following which, the mixture was refluxed for 2 hours. Ater cooling, the reaction mixture was ex-tracted with CH2C12 (3.15 L), the CH2C12 phase dis-carded and the aqueous phase evaporated to dryness under reduced pressure with a 70C ba~h. The residue was boiled in 95% EtOH (8.83 L) ~or 30 ~inutes, then allowed to cool slowly with stirring, whereupon the product separates as fine crystals. These were il-tered, washed with ~resh 95% EtOH (1.26 L) and dried in ~ 50-60C oven to give the title compound ~1.77 kg;
74.6%) as white crystals with mp - 120-2C (uncor- ~
rected).
IR (KBr) cm 1 3420 (br.~, 1590 (br.), 1~10, 1130, 710; iH NMR ~D2O-TSP) ~ 3.1 (s, 2H), 3.89 (d,¦JAB¦ =
4,lH) aild ~.52 (d,¦JAB¦=4,lH) (center of pattern:
4.21; ~VAB=18.83 Hz.), 4.68 (s, 6H, exchangable ~ ", ~l23~

protons~, 7.4 ~sl 5H); TLC Rf = 0.59 ~E~OH:lM tri-ethylammonium bicarbonate, 7:3) Anal. Calcd. for CllHl5NO7K2 C,37.59; H~4-30; N~3-99 Found: C,37.22; H,4.24; N,3.36 N-acetyZ-~-acetoxy-5-pheny~pyrro~e (2) A suspension of 2-hydroxy-3-~carboxymethylamino)-hydrocinnamic acid dipotassium salt dihydrate (1) (1.0 kg; 2.84 mol~ in pyridine ~3.0 L) was treated wi~h acetic anhydride (4.0 L) at ambient temperature under an inert gas atmosphere. A mild exothermic reaction ensued and the reaction temperature rose exponentially to 60-70C during a period of 1.5-2.5 hours. Once the reaction began to cool the mixture was heated to 120-123 C for 15 minutes, then allowed to cool to ambient temperature over 1 hour, during which time pyridinium acetate separated as crystals. The mixture was fil-tered through paper with suction and the salts washed with EtOAc until colorless; the filtrate was evaporated to dryness ~n vaauo.
The dark red residue was dissolved in EtOAc (3.0 L) washed three time with water Cl.o L) each), dried over MgSO4 and treated with Darco~-G60 (ICI Americas, Inc.~ (300 g). After stirring for 30 minutes the mixture was filtered through Celite~ (Johns-Manville) and evaporated to dryness ~n vaouo to give a reddish-orange oil. This oil was dissolved in warm 2-propa~ol ~1.2 L), then allowed to cool slowly to ambient temp-erature overnight, whereupon a solid separated. l`ho crystalline pr~duct was fil~ered, washed with 50%
aqueous 2-propanol and dried ~n vacuo to give the title compound ~417 g; 60%) with mp=58-60C ~uncor-rected). A portion was taken up in Et2Q, treated with ~ ~ .

'.

~.

Norit 211, ~iltered and concentrated under reduced pressure; on standing at 0C colorless tiny needles separated. These were filtered, washed with Et20/-Hexane (l:l) and ~acuum dr,ied to give the analytical sample with mp=60-62.5C (uncorrected).
IR ~CHCl3) cm 1 3020, 1760, 1730~ 1595, 1378, 1320, 1220 ~br.), 1030, 960, 903; H M~R ~CDC13) ~
2.23 ~s, 3H), 2.27 (s, 3H), 6.18 (d, J=2, lH), 7.35 (s, 5H), 7.42 (d, ~=2, lH); TLC R~= 0O56 (toluene:-dioxane, 4:1~.Anal- Calcd- for C14H13N3 C~ 69-12; H~5-38; N~5-76 Found: C,68.88; H,5.25; N,5.53 3-Hydroxy-S-p~enyZpyrroZe (3) A finely di~ided portion of N-acetyl-3-acetoxy-5-phenylpyrrole C2) (36.8 g; 0.15 mol) was ~reed ofoxygen by stirring in a flowing argon stream ~or 10 minutes, then suspended in deoxygenated MeOH (379 mL), cooled to -6 C cin a -15C methanol ~MeOH)/dry-ice bath) under an inert gas atmosphere and rapidly treated with an ice cold deoxygenated solution of 2N
NaOH ~300 mL). The reaction temperature rose imme-diately upon addition of base to 18 C, and after ~3 minutes the reaction mixture became Xomogeneous. As the reaction mixture cooled, compound ~3) separated as fine crystals. Ater 15 minutes a solution Gf cold deoxygenated 2M citric acid (150 mL) was added, the resulting mixture was stirred for 10 minutes, and then filtered. The solid was washed thoroughly with de-oxygenated water ~200 mJ~), taking care to minimize exposure of the product to air, then dried under va~u~m overnight to yield the title compound (22.3 g;
93.6~) as light pink tiny needles.
IR ~KBr) cm 1 3400, 3110, 2900, 1600, 1580, 1555, 1480, 126~, 1180, 742; 640; lH NMR (DMSO-D6) ~ 6.1 ~S-1332 ~ ` .

~23~15~3 ~m, lH), 6.3 (m~ lH), 7.0-7.7 (m, 5H), 8.0 ~s, lH), 10.4 ~br s~ lH); TLC R~ = 0.~0-0.2~ ~EtOH:CHC13, q ) .
Anal. Calcd. or CloH9NO: C,75.45; H,5.70; N,8.80 Found: C,75.30; H,5.69; N,8.67 3-(N-tosyZ-L-a~an?,nyZoxy)-S-phenyZpyr~oZe (4) A solution of anhydrous tetrahydrofuran ~THF, 450 mL), pyridîne ~43.8 mL; 0.542 mol; 1.2 eq) and tri-fluoroacetic acid ~85.0 mL; 1.10 mol; 2.4 eq), main-tained at 0C under an inert gas atmosphere, wastreated in one portion with 3-hydroxy-5-phenylpyrrole ~3) ~71.5 g; 0.45 mol; 1.0 eq) followed immediately by the dropwise addition, over 5-10 minutes of a solution of freshly prepared N-toysl-L-alaninyl chloride (141.0 g; 0.54 mol; 1.2 eq~ in anhydrous THF ~450 mL). The resulting mixture was stirred for 15 minutes at 0C.
The reaction was then quenched by addition of a solu-tion o 1.0 M aqueous citric acid ~315 mL) and EtOAc ~1.35 L). After brief mixing the phases were separated and the organic layer washed with a solution of aqueous NaCl C360 mL; 0.18 g NaCl per mL of water). The organic layer was next extracted twice with a solution of 5% aqueous NaHCO3 Cl.35 L each), and then washed with another portion o~ aqueous NaCl ~360 mL; 0.18 g z5 NaCl per mL o water~. The reddish brown organic layer was stirred at ambient temperature for 15 min-utes with MgSO4 ~101 g) and Darco-G60 ~143 g), then filtered through Celite and evaporated to dryness ~n vacuo from a 37C bath to give ~4) as a pinkish-white solid. The crude product was ground to a powder and dissolved in warm ~50C) THF ~250 mL), stirred vigor-ously and diluted with n-hexane C250 mL). The stir-ring was continued or 1 hour at ambient temperature ~ ...... ' .

;~ 7 ~2;~

as the l~roduct cr)~stalli;~ed. T]le solid ~ s filtercd, washed with toluene ~ca. 1. O L) un~ he ;~ rate ~ s colorless, thell drie~ ~n v~c~o o~erni~ht ~o ~ield the title compound (112 g; 65%) as a white powder ~ith mp = 154.5-155C)~
IR ~Cl~ cm 1 33S0, ~325, 1760, 1508, 132~, 1155, 770; 1H N~R (DMSO^d~) ~ 1.33 (d, J=7, ~H) ~ 2.36 (s, 3H~ 7 .13 (p, J-8, lH), 6.25 (m, 1l1), 6.73 (m, 1l1), 7~05-7.50 (m, 5~), 7.5-7.85 (~, 4~), 8.42 (d, J-8, 1~), 11-1~ (br s, lH); 13C NMR (D~5O-~6~ ppm 18.335, 21.001, Sl.370, 98.061, 108.336, 123.423, 126.~2~, 126.610, 128.560t lZ8.75~, 129.~01, 13~.3g7, 137.~00, 13g.380, 142.737, 169.919; ~u~D = -70 (c=l,ll, MeOI-I); TLC Rf ~
0.45 ~EtOAc~hexane, 1:1); TLC Rf = 0.40 (toluene:-diox~ne, 4:1).
Anal- Calcd- for C20H20~24S C,62.48; ~5-24; N77~9 Found: C,62.6~; H,5.27; N,7.30 7.2.2 Sy~the~ of 3~~ to~yta~an~ty~o~yJ-s pheny~l;hq,ophene (~J
A se~ies of e~peri~ents was conducted to prepare 3-hydroxy-5~phenylthiop]lene by ~inor modi~ications o the repo~ted literature procedu~es3'4 outlined on a foll~ing pa~e. The resultant ~droxy~hiophene was then acylated with N-tosyl-L-alaniny~ chloride to give the correspondin~ N-to5yl-~-alaninate ester in 46 yield (unoptimi2ed p~oced~re).

3. P. Friedl~nder an~ S, ~ielbasinski, Chem. ~r.
~5, 33~9 Cl~l2) 4. A~I~ Kosak, R.J.F. Pal~hak, W.A. Steele and C.M.
Selwitz, J. Amer. Chem. Soc. 7~, 4450 ~1954).
~S~1332 ~ .

.

,. : , ~ .
,:

~2385~;~

~COOC211 ~ ~0 a. Na2S
1~ C112C(~()II
pll=8 . 7 , 2 ~ S ~ COOII

NaOH
~120 MeOH
Cl - c- f~l - c~13 OH ~ O-C-C~I-C113 (8) C~12C12 pyridine (~) -;

~23~5~

3-Pher~yZ-1, 2-dithia-3-cycZoper~ten-5-one f S) A suspension of 10 g of ethyl cinnamate ~56 82 mmol) and 10 g of sulfur was heated at 250G for four hours in a 50 mL flask equipped with a distillation head and receiver to remove ethanol produced during the reaction. The reaction mixture was then allowed to cool to 100C and added to 500 mL of refluxing ethanol.
The resulting precipitate was filtered and succes-sively triturated with 500 mL of boiling acetone and twice with 500 mL por~ions of ethanol. The combined supernatants were concentrated to a black solid, which was crystallized from methanol to give dark brown needles. A second recrystallization from methanol using Norit and filtration through Celite gave 2.023 g of light yellow needles mp 113-115C.
IR (KBr) cm 1 1650, 1550, 1390, 1350, 1130, 770;
lH NMR ~60m Hz, CDC13) ~ 6.92 ~s, lH), 7.58 ~m,5H);
TLC Rf = 0.5 (dichloromethane).
Anal. Calcd. for CgH6O2S C,55.64; H,3.11 Found: C,55.53; H,3.47 o~s-4-Ke~o-6-phenyZ-3,7-d~th3a-S-nonenedioia acid ~6) A molten solution of 35.48 g of sodium sulfide nonahydrate ~148 mmol) at 94C was treated w~th 6.65 g of 3-phenyl-1,2-dithia-3-cyclopenten-3-one (5) t34.23 mmol) added portionwise over five minutes. After fifteen minutes, the mixture was added to an ice-cold solution containing 43.6 g of bromoacetic acid l314 mmol) in 60 mL of H20 adjusted to pH ~.7 with sodium carbonate. The resulting solution was maintained at 0C, pH 8.7 for 45 minutes, and was then filtered.
The supernatent was maintained at 0C and acidified to pH 3.7 with a 5N HCl solution. The resulting mixture was stirred overnight at 5C. The supernatant was then decanted, and the resulting oil triturated with 6C~ `

ether. The oil was evaporated with ~oluene until 6.98 g of a colorless foam was obtained (65%)~ This material was used without urther purification.
An analytical sample was obtained from the ether supernatant, which upon concentration, successive evaporation with acetic acid and toluene, and tri-turation with ether, gave tan crystals. mp = 142.5-150C.
IR~KBr) cm 1 1705, 1655; lH NMR (60 MHz, DMSO-D6) ~ 2 06 (s, CH3CO2H impurity) 3.30 (s, 2H), 3.77 (s, 2H), 5.67 (m, 2H)(OH), 6.37 ~s, lH), 7.43 ~m, 5H); TLC
Rf = 0 85 (chloroform:methanol:acetic acid, 5:5:1).
Anal. Calcd. for C13H12S2O5: C, 50-00; H~ 3-88 Found: C, 50.26; H, 3.98 3-HydYoxy-S-phenyZthiophene Aaetate t7J
A vigorously stirred suspension of 3.40 g of crude eis - 4-keto-6-phenyl-3,7-dithia-5-nonenedioic acid ~6) (10.9 mmol), 3.40 g of sodium acetate ~41.5 mmol), and 30 mL of acetic anhydride was heated to reflux for one hour. The mixture was allowed to cool and was then filtered and evaporated to give a black oil. This residue was dissolved in 75 mL of ethyl acetate and extracted three times with 50 mL portions o ice-cold saturated sodium bicarbonate solution, The organic layer was then washed with brine, dried over sodium sulfate, filtered, and evaporated to give 2.826 g of a black solid. The crude product was purified by evaporative distillation at 120-140C and 0Ol mm to give 1.235 g of a light orange oil which solidi~ied upon standing ~52~).
IR cm 1 1700, 1745; lH NMR ~60 MHz, CDC13) ~
2.23 (S, 3H), 7.03 ~d,J~2Hz, lH), 7.13 (d,J = 2Hz, lH); 7.23-7.73 (m, SH); MS (EI, DIP) m/e 218 (M , 12.6~); TLC Rf = 0.48 (hexane:ethyl acetate, 5:1).

~23~

Anal. Calc. for C12Hloso2 l/2 H2O C~63 41; H~4 88 Found: C,63.78; H,4.86 3-Hydro~y-5-phenyZthiophene ~8) A mixture of 2.126 g of 3-hydroxy-5-phenylthio-phene acetate ~7~ ~9.74 mmol) and 80 mL of methanol under an argon atmosphere was treated with 11 mL of lN
NaOH. After 20 minutes, the reaction was quenched by the addition of 11 mL of lN HCL, e~aporated at 25C, 12 mm, to approximately ~0 mL ~olume, and treated with 100 mL of ethyl acetate. The organic layer was separated, washed with brine, dried over sodium sul-fate, filtered, and evaporated to give a black solid.
This residue was dissolved in 75 mL of ethyl acetate and dried over MgSO4. Filtration and evaporation gave a black solid which was triturated four times with hot hexane to give upon cooling a total o 837 mg of a yellow solid, mp 74-75C (49%). The combined mother liquors were concentrated to give 0.87 g of a solid which was chromatographed over 100 g of SiO2 eluted with a hexane:ethyl acetate ~7:1) solvent mixture.
Obtained ater recrystallization was an additional 380 mg o product. mp 73.5-74C. The combined yield was thus 1.217 g ~71%). mp 74.5-75C (Lit3'4 75C, 78C).
IR cm 1 3380-, 1635; lH NMR (90 MHz, CDC13) ~
3.81 ~s, 2H), 6.57 (s, lH), 7.2-7.7 ~m, 5H); MS (EI) m/e 176.0 (70.7%); TLC Rf = 0.23 (hexane:ethyl acetate, 1:5).
Anal. Calcd. for CloH8OS: C, 68.15; H,4.57 Found: C,68.05; H,4.70 3-(N-tosyl-L-aZan~nyZo~y)-5-phænyZth~ophene (9) A solution containing 440 mg of 3-hydroxy-5-phenylthiophene ~8~ (2.5 mmol~ in 20 mL o dichloro-methane and 0.61 mL o pyridine (7.5 mmol) at 0C

. . .

under an argon atmosphere was treated with a solution containing 1.314 g of N-tosyl-~-alaninyl chloride ~5 mmol) in 10 mL o~ dichloromethane added dropwise o~er a period of five minutes. The reaction was allowed to stir ~or 0.5 hour at 0C, and was then poured into 100 mL of chloroform. The mixture was then successively extracted with 50 mL portions of lN citric acid, water, ice-cold sodium bicarbonate solution, water, and brine. The mixture was then dried over sodium sulfate, filtered, and evaporated to give 1.78 g of a brown oil. Attempted crystallization from toluene after treatment with 1.78 g of Norit was unsuccessful.
The residue was then chromatographed on a 200 g column of SiO2 eluted with dichloromethane at a flow rate of 10 mL/minute. Fractions containing the product were pooled and concentrated to give 951 mg of a reddish oil. The product was crystallized from toluene.
Successive recrystallizations from toluene ga~e a total of 463 mg of product as light yellow sol.id, (46%). mp 85-87C.
IR ~KCl) cm 1 1735, 1330, 1150; lH NMR ~90 MHZ, CDCl3) ~ 1.53 (d, J = 7 Hz, 3H), 1.62 ~s, 3H), 4.23 (m, lH), 5.32 ~d, Ja9Hz, lH), 6.84 ~d, J = 1.4 Hz, lH), 6.88 ~d, J = 1.4Hz, lH), 7.23-7.83 ~m, 9H); MS
~FAB) m/e 402 tM ~ 1, 15%); TLC Rf ~ 0.20 ~hexane-ethyl acetate, 4:1).
Anal. Calcd. for C20HlgNO~S C,59.83; H,4.77; N,3.59 Found: C,59,60; H,4.77; N,3.43 .. ..

.

~2~

7.2.3 Synthesis o~ 3-(~7-tosyZ-~-alaninyZoxy)-l-methyZ-5-phenyZpyrroZe ~13) A series of experiments was conducted to prepare the captioned ester corresponding to compound (I) in which A is N-tosyl-L-alaninyl, R is phenyl, R* is H, X
is NR' and R' is CH3. The reaction sequence is as follows:

COO K
O ~ HO y COO K

NH(CH_)CH2COOH ~ N'-~COO K

l ~c2o ~ Et3N

OH

(IZ~ ~O ,}__ ~ ~ )H3 1l'" ~
CF3COOH Cl-C-ICH-CH3 pyridine ~ N~
THF }1 Ts O
0- C - C~l- Cl~l I ~ 1 3 H Ts ~3~S~

2-Hydroxy-3-fN-methyZca~bo~ymethy~am~noJ-hydroc~nnamic ~cid dipotassium saZt (10) A mixture of ~-phenylglycidic acid potassium salt (30 g; 0.15 mole), N-methylglycine (13.2 g; 0.15 mole), distilled water ~119 ml) and KOH solution (9N;
22.3 ml) was heated to reflux for 3 hours to give a light yellow solution. The reaction mixture was evaporated to dryness under reduced pressure at 70C.
The residue was then crystallized from 95% EtOH (100 ml) to give a white solid which, after drying over-night under reduced pressure at 110C, yielded 30.8 g of white solid (10) (yield 63%).
IR (KCl~ cm 1 3360 (br), 1580, 1405, 705; lH NMR
(CD30D) ~ 2.30 (s, 3H), 2.98 (s, 2H), 3.70 (d, J=3 Hz, lH), 4.48 (d, J=3 Hz, lH), 4.92 (s, lH), 7.40 (s, 5H);
TLC Rf = 0.51 (EtOH:lM triethylammonium bicarbonate, 7:3). (Product had no melting point less than 270C) 3 Acetoxy-l-me~hyZ-S-phenyZpy~roZe (11) A mixture of-2-hydroxy-3-(N-methylcarboxymethyl-amino)-hydrocinnamic acid dipotassium salt (10) (15.2 g, 46 mmole), acetic anhydride (173 ml) and triethyl-amine (308 ml) was heated at 90C for 19 hrs. The reaction mixture 7 which became deep brown in color, was filtered and the solid washed with ether. The filtrate was evaporated under reduced pressure to give a deep brown residue, which was taken up in ether (300 ml) and water i~00 ml). The layers were separated-and the ether layer washed with another portion of water (200 ml). The ether solution was then dried over MgSO4, iltered and concentrated under reduced pres-sure to give 10.7 g of brown residue which was puri-fied by evaporative distillation (120-135C; 0.03 tor-) and crystallization rom ether to yield 3.0 g of white crystals ~11) (yield 30%~ mp = 64-65C.

. .

~l238~

IR (CHCL3) cm l 2990, 17507 1570, 1518, 14~2, 1375, 1230 (br), 1024, 910, 700; lH NMR (CDCl3) ~
2.20 (s, 3H), 3.5$ (s, 3H), 6.10 (d, J=2 Hz, lH)7 6.75 (d, J=2 Hz, lH), 7.35 (s, SH); TLC R~ = 0 58 (hexane:
EtO~c 7:3) Anal. Calcd. for C13H13NO2: C,72.54; H,6-10; N,6-44;
Found: C,72.57; H,6.09; N,6.51 3-(N-TosyZ-~-a~aninyZo~y)-l-methyZ-5-phenyZpyrroZe (13) To a mixture of deoxygenated methanol (15.5 ml) and 3-acetoxy-1-methyl-S-phenylpyrrole (11) ~1.3 g, 6 2 mmole), under argon, was added deoxygenated NaOH
~2N, 12.5 ml). The reaction mixture was stirred in an ice-bath for 15 minutes. Then deoxygenated citric acid (2M, 7 ml) was added and the resulting mixture was stirred in an ice bath ~or 8 minutes. The reac-tion mixture was concentrated under reduced pressure, then 20 ml of water was added and was extracted twice with ethylacetate (EtOAc) ~50 ml). The EtOAc layers were combined, dried over MgSO4, filtered and con-centrated under reduced pressure to give 3-hydroxy-1-methyl-5-phenylpyrrole (12) as an orange residue.
Under argon, a cold solution o-f anhydrous THF ~12.4 ml~, pyridine (0.6 ml, 7.4 mmole, 1.2 eq) and tri-fluoroacetic acid (1.2 ml, 15 mmole, 2.4 eq) was added to the orange residue, followed immediately by the addition of a solution nf freshly prepared N-tosyl-L-alaninyl chloride (1.2 g, 7.4 mmoie, 1.2 eq) in an--hydrous THF (12.4 ml). The resulting mixture was stirred for one hr at 0C. Then the reaction was quenched by the addition of aqueous citric acid (lM, 5ml) and ~tOAc (30 ml). A~ter a brief mixing, the layers were separated and the organic layer was successively washed with saturated NaCl solution, ...

~22~15~j~
twice with 5% NaHCO3 solution and again with saturated NaCl solution. The EtOAc e~tract was then dried over MgSO4, treated with Norit 211, ~iltered and concen-trated under reduced pressure to give the crude pro-duct ~13) as an orange residue. This was dissolved inhexane:EtOAc ~ 5 ml) and chromatographed on a column ~Si02, 100 g) by elution with hexane:EtOAc ~7:3) to give 1 g of (13) as a thick light orange oil.
A portion o-f this crude product was further purified by semi-preparative HPLC (column, IBM silica, 1 cm x 25 cm; mobile phase, hexane:EtOAc 8:2; flow rate, 4.0 ml/min; pressure, 0.2 psi) to yield a honey color thick oil ~13).
IR (film) cm 1 3260, 2950, 1760, 1520, 1350, 1170, 770; lH NMR (DMSO-d6) ~ 1.28 (d, J=7 Hz, 3H), 2.36 (s, 3H), 3.58 (s, 3H), 5.85 (d, J=2 Hzg lH), 6.15 (m, lH), 6.74 (d, J=2 Hz, lH), 7.30-7.80 (m, 9H), 8.37 (d, J=8 Hz, lH); 13C NMR (DMSO-d6) ~ 18.205, 20.936, 34.917, 51.240, 100.598, 11~.148, 126.544, 127.000, 128.105, 128.560, 129.601, 130.901, 132.202, 135.714, 138.315, 142.672, 169.724; TLC Rf= 0.52 (toluene:dioxane 4:1); High-resolution mass spectrum~ C21H22N2O4S
requires m/e 398.1300, found m/e 398.1297.

7.2.4 Synthesis o~ 3-(N-tosyZ-L-aZaninyZo~y)-5- (p-chZorophenyZ)pyrroZe (18) A series of experiments was conducted to prepare the captioned ester compound corresponding to compound (I) in which A is N-tosyl-L-alaninyl, R is p-chloro-phenyl, R* is ~l, X is NR' and R' is H. The reaction sequence is as follows:

~l23~

C0011 ~C0011 ~D ~IS05, 112(' ~IC~ ,[~ (1~) Cl ~ ~ - > ~ Cl Glycine pH = 11.5 \ / '.

OAc HO ~ COO K
G~ ~ AC2 ' ~ N '~ COO K 2H20 l~ ) I pyridine ~ l~JJ ¦
Cl~ A~ 121-2 Cl `~ H
~16) . ~15) NaOH

H20 . . .

01'1 1l O - C - C~l - Cl I
l~f Cl - C- C~l - C~l 1~ 1 2 3 Cl~ ~ 3 J~ N~ ~ ~T
Cl~ ~ COOI I
( 1 7 ) ~7 ( l v T~l~' ' ~ .

.,. :s ~2~ 0 trans-~-(p-ChZorophenyZ)gZyc~d~c acid (1~) To a stirred slurry of p-chlorocinnamic acid ~68 5 g; 0.375 mol) in 260 mL of acetone was added NaHCO3 (137 g; 1.63 mol~, followed by slow addition of 260 mL of water. To this mixture was added, over 2.5 hours at 22-27C, a mixture of OXONE (211 g; 0.343 mol), 120 mg of disodium EDTA and 805 mL of water.
After ive hours the mixture was acidified with 70 mL
of cold 12 N HCL, to bring the pH down to about 2.5, and it then was extracted with 700 mL of ethyl acetate.
The extract was washed with brine, dried with MgSO4, filtered, and the filtrate was evaporated to dryness under vacuum. The white solids were crystallized from ethyl acetate: mp 121-5C. (72.2 g; 97% yield). lH
NMR ~CDC13/DMSO-D6) ~ 7.3 ~m, 4H), 4.05 (d, J=2, lH), and 3.4 ~d, J=2, lH).
Anal. Calcd. for CgH7C103 C,54.43; H,3.55; C1,17.85 Found: C,54.53; H,3.80; Cl,17.91 2-~ydroxy-3-(ca~boxymethyZaminoJ-p-~hZorohydrocinnamic acia' d~potassium sa~t dihydrate (15) To a solution of KOH (85%) (46.7 g; 0.709 mol) and 400 mL of water was added glycine (25.9 g; 0.345 mol) followed by trans-~-p-chlorophenylglycidic acid (14) (72.2 g; 0.3635 mol). This mixture was heated at 100C for two hours, cooled to room temperature and sufficient KOH added to raise the pH to 12. The turbid solution was extracted three times with ethyl acetate, which extract was then discarded; the clear aqueous solution ~about 500 mL) was evaporated under vacuum to dryness using a 70 water bath. The soli.ds were than dissolved in about 350 mL of hot ethanol, filtered, and the filtrate chilled in an ice bath for several hours. The crystallized solids were collected by filtration and washed with some cold ethanol: mp 93-5C with decarboxylation at 185C (57.2 g; 41%).

. .

~3~

lH NMR ~D20-TSP) ~ 7.4 (s, 4H), 4.7 (s, 6H, ex-changable protons), 4.4 ~d, J=4, lH), 4.05 (d, J=4, lH), and 3.1 (s, 2H).
Anal. Calcd. for CllHloclNo5K2-2H2o: C,34-24; H,3-66;
N, 3.63 Found: C,34.40; H,4.03;N,3.~2 N-~cety1~-3-acetoxy-5-~p-chZo~opheny~JpyrroZe (16) To the 2-hydroxy-3-~carboxymethylamino)-p-chlorohydrocinnamic acid dipotassium salt dihydrate lQ (15) ~10 g; 0.02591 mol) was added acetic anhydride ~40 mL) and pyridine ~30 mL). This mixture was gently heated to 35C at which point the solution exothermed to 67 then began to fall, whereupon heating was again resumed. The mixture was heated at 121-2 ~internal temperature) for one hour then cooled. To the reac-tion mixture was added about 30 mL of ethyl acetate which precipitated most the pyridinium acetate salt;
this salt was collected by ~iltration and washed with a small amount of ethyl acetate. The filtrate was then evaporated under vacuum to an oil and ice water added. The product was extracted with ether and the ether extracts were successively washed twice with cold dilute aq. citric acid, cold water, three times with cold dilute aq. NaHCO3, cold water and brine, followed by drying over MgSO4 and filtering. The fil-trate was treated with 10 g of Darco, stirred for 20 minutes and then filtered. The iltrate was evaporated under vacuum to an oil. To the oil was added 25 mL-of 2-propanol. The resultant solution yielded, with chilling anu rcratching, pale yellow crystals: mp 69-71~C ~3.4 g; ~7%); TLC Rf=0.61 (toluene:dioxane, 95:5). An analytical sample was recrystallized from 2-propanol, but no change in mp was observed.

~i,, , 2 ~

IR ~KCl) cm l 1755 ~C=0, ester) and ~730 ~C=0, amide); lH NMR (CDCl3) ~ 7.4 ~Tn~ SH), 6.2 ~d, J-2, lH), 2.4 ~s, 3H) and 2.3 ~s, 3H).
Anal. Calcd. for Cl4Hl2ClNO3: C,60.55; H,4.36; H,5-04 Found: C,60.65; H,4.55; N,5.07 3-hydro~y-5-~p-chZorophenyZ)pyrroZe (l~J
A sample of N-acetyl-3-acetoxy-5-p-chlorophenyl-pyrrole ~16) ~2.8 g; 0.01 mol) was deoxygenated for ten minutes with a stream of N2. The solids were then dissolved in deoxygenated methanol ~30 mL) which was then chilled to -8C. At once was added a cold de-oxygenated solution of NaOH ~1.6 g; 0.04 mol) in 20 mL
H20, which solution was then heated briefly to 15C
and then immediately cooled to -5C; after 25 minutes the clear solution was treated with a cold deoxygenated solution o~ citric acid (4.2 g, 0.02 mol) in 15 mL H20 and the temperature rose briefly to 5C. After 0.5 hour of stirring at -5C the solids were collected by filtration and washed with cold deoxygenated H20.
The pale green product was dried under vacuum at room temperature over P2O5 for several days ~1.3 g; 68%);
TLC Rf=0.19 (CHC13:EtOH, 9:1); IR (KCl) showed no evidence for C~O absorption.
Anal. Calcd. for CloH8ClNO 1/6H20: C,61.08; H,4.27;
N,7.12 Found: C,61.36; H,4.44;
N,6.85 3-fN-bo~yZ-L-aZaninyZo~y)-5-tp-ahZorophenyZ)pyrroZe ~l~J
To N2 deoxygenated THF (15 mL) was added pyridine (0.65 mL; 0.008 mol), tri~luroracetic acid ~1.27 mL;
0,0164 mol), and 3-hydroxy-5-p-chlorophenylpyrrole ~17) ~1.3 g; 0.0065 mol). The solution was chilled to 0C to -4C and a N2 deoxygenated, chilled ~0C to -4C) ~ - . .

~238~

solution of N-tosyl-L-alaninyl chloride (2.1 g; 0.008 mol) in 15 mL of THF was added over 10 minutes. After maintaining the mixture at 0C :for one hour, a mixture of ice and 100 mL of 1 N citric acid was added. This mixture was extracted with ethyl acetate and the extract washed once with cold brine, twice with cold dilute NaHCO3, and once with cold brine, following which, it was dried over MgSO4 and filtered. The filtrate was treated with 2 g of Darco and stirred for ten minutes, filtered and the filtrate concentra~ed under vacuum to a reddish-brown oil. A second treat-ment with 1.3 g Darco afforded a light reddish oil.
The oil was dissolved in toluene:cyclohexane (4:1) and placed in the refrigerator overnight. Light salmon crystals were obtained. (1.45 g; 53%); mp 113-5~C;
TLC Rf=0.47 (Et20); IR (KCl) cm 1 1740 (C=0, ester);
H NMR (CDC13) ~ 8.4 (br s, lH), 7.8-7.2 (m, 8H), 6.7 ~m, lH), 6.2 (m, lH), 5.5 ~d, J=9, lH), 4.2 (p, J=8, lH), 2.4 (s, 3H), 1.4 (dj 3H~; MS (EI, DIP) m/e 418 (M~, 2.3%) and 420 (M , 0.8%).
Anal. Calcd. for C20HlgClN2O4S: C,57.34; H,4.57;
N,6.69 Found: C,57.53; H,4.58; N,6.67 7.3 Preparation and Vse of Various Test Devices A series of Experiments was conducted to prepare test devices of the present invention in which the ester substrates o paragraph 7.1, su~ra, were tested for responsiveness to leukocytes in urine. The de-vices comprised a small square of filter paper con-taining the assay reagents, the paper mounted at one end of a polystyrene film strip. The filter paper was impregnated with buffer, the ester an ac~elerator and a diazonium salt coupling agent. Each of the devices tested was found to exhibit a positive test for leuko-cytes in urine.

7.3.1 Test device in which the ester is 3-(~~tosyZ~ ZaninyZoxyJ-5-p~enyZpyrroZe f~l A test device, sensi~ive to the presence of leukocytes in urine, was prepared. The device com-prised a small square of filter paper mounted at one end of an oblong strip of polystyrene film. The paper was impregnated with various ingredients including a chromogenic ester, an accelerator and a diazonium salt. A 2 inch wide strip o~ ~aton and Dickman ~205 filter paper was immersed in an aqueous solution containing the following:
0.4 M borate-NaOH buffer pH=8.6 2.0~ (w/v) polyvinylpyrrolidone K-30 0.2% (w/v) Bioterge AS-40 0.25 M NaCl The paper was then dried for 7 minutes in an Overly Air Foil paper dryer,at 175-200F at an airflow pres-sure of 1 inch of H20. Next, the dried paper was immersed in an acetone solution containing 2.0~ (v/v) n-decanol 0.75 mM 2-methoxy-4-morpholinobenzene diazonium chloride O.S mM 3-(N-tosyl-L-alaninyloxy~-S-phenylpyrrole Following this impregnation the paper was dried ~or 10 minutes in a ventilated Hotpack~ oven at 130F. An o~f-white test paper was obtained.
A piece of the dried, .impregnated paper was c~t to a square measuring 0.2 inches on a side and mounted at one end of an axially oriented polystyrene strip measuring 4 inches by 0 2 inches. Mounting the paper to the strip was achieved using Double Stick double faced adhesive (3M Company).

, .

.
.
:

i66~

7.3.2 Test device in ~hieh the ester is 3-~N-tosyz-L-azaninyzoxy~-s-pheny~thiophene ~9~
A test device sensitive to the presence of leuko-cytes in urine was prepared, wherein 3-~N-tosyl-L-alaninyloxy~-5-phenylthiophene was used as the in-dicator. A piece of filter paper ~Eaton ~ Dikeman #205) was immersed in an aqueous first solution containing the following:
0.4 M borate-NaOH buffer (pH = 8 5) 0.4 M NaCl 1.5~ ~w/v) polyvinylpyrrolidone ~K-30) The impregnated paper was dried in a forced air oven for 30 minutes at 70C, whereupon it was permitted to cool to room temperature and impregnated with a second dip solution comprising an acetone solution containing:
0.75 mM 3(-N-tosyl-L-alaninyloxy)-5-phenylthio-phene 0.75 mM 2-methoxy-4-morpholinobenzene diazonium chloride, zinc chloride double salt 0.5% ~v/v) n-decanol The doubly impregnated pap0r was then dried in a forced air oven for 5 minutes at 50C.
The dried paper was cut into squares measuring 0.2 inches on a side and mounted at the end of a polystyrene film measuring 0.2 by 3.25 inches. Mount-ing was accomplished using Double Stick, a double faced adhesive from 3M Company The test device was stored in bottles of 100 each, together with silic-a gel and molecular sieves to provide dessication.

:
, ~

1~385~
- '~4 -7. 3. 3 Test device in which the ester is 3-(IV-tosyZ-L-aZaninyZo~y)-l -methyZ-s-pheny Zpyrro Ze tl 3~
Test devices were prepared following the proce-dure in experiment 7.3.2 in which the ester indicatorwas 3-(N-tosyl-L-alaninyloxy)-l-methyl-5-phenylpyrrole and the coupling agent was l-diazonaphthalene-4-sulfonate. The aqueous first dip solution contained:
0.4 M boric acid 2.0% ~w/v) polyvinylpyrrolidone (K-30) O.Z% ~v/v) Bioterge AS-40 0,25 M NaCl Prior to impregnation of the filter paper, the solu-tion was titrated with NaOH to a pH of 9Ø
The second dip solution in acetone contained:
0.75 mM l-diazonaphthalene-2-sulfonate 1.3 mM l-methyl-3-~N-tosyl-L-alaninyloxy)-5-phenylpyrrole 1.5~ ~v/v) dodecanol Pollowing impregnation in the aqueous first dip, thepaper was dried for about 5 minutes at about 80C, and for about 5 minutes at 70C following impregnation in the acetonic second dip.
The dried paper was mounted as in experiment 7.2.2.

7.3.~ Test devioe in whiah the ester i8 3-tN-tosyZ-L-aZaninyZo~y)-5- (p-ahZoYophenyZ) pyrroZe (18) TesL devices were prepared as in Experiment 7.3.3 except that the acetone solution contained, in place of the phenylpyrrole, 1.3 mM 3-CN-tosyl-L-alaninyl-oxy)-5-(p chlorophenyl)pyrrole.

`':.. , ;

. : :

. ~

~23135~

7. ~ E~aZuation of tt~e Test ~evice The tes~ devices prepared in the experiments of paragraph 7.3 were subjected to evaluation of their ability to detect leukocytes present in urine.
Test samples were prepared from a normal human ~rine pool. One sample served as a blank and leuko-cytes isolated from freshly drawn blood were added to two additional urine samples to yield concentrations of 0, 10 and 75 leukocytes/~L, respectively.
Test devices were quickly immersed in and removed from a test sample. Two minutes later the devices were observed using a spectrophotometer to measure ~reflectance at different wavelengths from 400-700 nm (nanometers).
The data show that all of the test devices de-monstrated clearly discernable differences in light reflectance corresponding to different leukoctye levels in the test samples. The data are presented in the following table:

~3S~

Leukocyte % Refleckance Experimen~ No. Concentration at 555 nm ~cells/~L) __ 7.3.1* 0 7,3.2 ~ 65 . . . ~
7.3.3 0 67 7.3.4 0 61 5 *Visual observation: purple color formed at 10-12 cells/~L; blank gave no color change , , .
` ' ' ~ ~ ;; ' ' ' ' ' .

Claims (94)

The embodiments of the invention in which an ex-clusive property or privilege is claimed are defined as follows:

1. A composition for determining the presence of leukocytes, esterase or protease in a test sample, the composition comprising an ester having the structure in which:
A is an N-blocked-.alpha.-L-amino acid residue or an N-blocked-.alpha.-L-peptide acid residue, X is O, S or NR', R is aryl or lower alkyl group having 1 to 6 carbon atoms, R* is H or lower alkyl group having 1 to 6 carbon at oms, and R' is H, lower alkyl group having 1 to 6 carbon atoms or aryl; and a suitable buffer substance which when in contact with an aqueous test sample will provide a resultant pH of at least about 7.

2. The composition of claim 1 in which X is NR'.

3. The composition of claim 1 in which A is an N-blocked amino acid.

4. The composition of claim 1 in which A is N-blocked-L-alaninate.

5. The composition of claim 1 in which A is N-blocked-L-alaninate, R is phenyl, X is NR' and both R' and R? are H.

6. The composition of claim 1, 2 or 3 in which the composition further comprises an accelerator.

7. The composition of claim 4 or 5 in which the composition further comprises an accelerator.

8. The composition of claim 1, 2 or 3 in which the composition further comprises an accelerator which is an alcohol having 1 to 15 carbon atoms.

9. The composition of claim 4 or 5 in which the composition further comprises an accelerator which is an alcohol having 1 to 15 carbon atoms.

10. The composition of claim 1 in which the com-position further comprises a diazonium salt coupling agent.

11. The composition of claim 2 in which the com-position further comprises a diazonium salt coupling agent.

12. The composition of claim 3 in which the com position further comprises a diazonium salt coupling agent.

13. The composition of claim 4 in which the com-position further comprise,s a diazonium salt coupling agent.

14. The composition of claim 5 in which the com-position further comprises a diazonium salt coupling agent.

15. The composition of claim 10, 11 or 12 in which said diazonium salt is a zwitterion having the struc-ture in which:
D- is an anion, G, same or different, is H, lower alkyl group having 1 to 6 carbon atoms or aryl, or in which both G together form a fused ring system, and in which B is H or OH.

16. The composition of claim 13 or 14 in which said diazonium salt is a zwitterion having the struc-ture in which:
D- is an anion, G, same or different, is H, lower alkyl group having 1 to 6 carbon atoms or aryl, or in which both G together form a fused ring system, and in which B is H or OH.

17. The composition of claim 10, 11 or 12 in which said salt is l-diazo-2-naphthol-4-sulfonate.

18. The composition of claim 13 or 14 in which said salt is l-diazo-2-naphthol-4-sulfonate.

19. The composition of claim 1 in which the com-position further comprises an accelerator and a dia-zonium salt coupling agent.

20. The composition of claim 2 in which the com-position further comprises an accelerator and a dia-zonium salt coupling agent.

21. The composition of claim 3 in which the com-position further comprises an accelerator and a dia-zonium salt coupling agent.

22. The composition of claim 4 in which the com-position further comprises an accelerator and a dia-zonium salt coupling agent.

23. The composition of claim 5 in which the com-position further comprises an accelerator and a dia-zonium salt coupling agent.

24. The composition of claim 19, 20 or 21 in which the accelerator is an alcohol having 1 to 15 carbon atoms.

25. The composition of claim 22 or 23 in which the accelerator is an alcohol having 1 to 15 carbon atoms.

26. The composition of claim 19, 20 or 21 in which the accelerator is an alcohol having from about 8 to 15 carbon atoms and the diazonium salt is 1-diazo-2-naphthol-4-sulfonate.

27. The composition of claim 22 or 23 in which the accelerator is an alcohol having from about 8 to 15 carbon atoms and the diazonium salt is 1-diazo-2-naphthol-4-sulfonate.

28. A test device for determining the presence of leukocytes, esterase or protease in a test sample, the device comprising a carrier matrix incorporated with an ester having the structure in which:
A is N-blocked-.alpha.-L-amino acid residue or N-blocked-a-L-peptide residue, R is aryl or lower alkyl group having 1 to 6 carbon atoms, X is O, S, or NR', R' is H, lower alkyl group having 1 to 6 carbon atoms or aryl, and R* is H or lower alkyl yroup having 1 to 6 carbon at-oms;
and a suitable buffer substance which when in contact with an aqueous test sample will provide a resultant pH of at least about 7.

29. The test device of claim 28 in which X is NR'.

30. The test device of claim 28 in which A is N-blocked amino acid residue.

31. The test device of claim 28 in which A is N-blocked-L-alaninate.

32. The test device of claim 28 in which A is N-blocked-L-alaninate, R is phenyl, X is NR' and both R*
and R' are H.

33. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 28, 29 or 30 and observing a detect-able response.

34. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 31 or 32 and observing a detectable response.

35. The test device of claim 28 wherein the car-rier matrix is additionally incorporated with an ac-celerator.

36. The test device of claim 29 wherein the car-rier matrix is additionally incorporated with an ac-celerator.

37. The test device of claim 30 wherein the car-rier matrix is additionally incorporated with an ac-celerator.

38. The test device of claim 31 wherein the car-rier matrix is additionally incorporated with an ac-celerator.

39. The test device of claim 32 wherein the car-rier matrix is additionally incorporated with an ac-celerator.

40. The test device of claim 35, 36 or 37 in which the accelerator is an alcohol having 8 to 15 carbon atoms.

41. The test device of claim 38 or 39 in which the accelerator is an alcohol having 8 to 15 carbon atoms.

42. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 35 and observing a detectable response.

43. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 36 and observing a detectable response.

44. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 37 and observing a detectable response.

45. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 38 and observing a detectable response.

46. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 39 and observing a detectable response.

47. The test device of claim 35 in which the ac-celerator is an alcohol having 1 to 15 carbon atoms.

48. The test device of claim 36 in which the ac-celerator is an alcohol having 1 to 15 carbon atoms.

49. The test device of claim 37 in which the ac-celerator is an alcohol having 1 to 15 carbon atoms.

50. The test device of claim 38 in which the ac-celerator is an alcohol having 1 to 15 carbon atoms.

51. The test device of claim 39 in which the ac-celerator is an alcohol having 1 to 15 carbon atoms.

52. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 47 and observing a detectable response.

53. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 48 and observing a detectable response.

54. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 49 and observing a detectable response.

55. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 50 and observing a detectable response.

56. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 51 and observing a detectable response.

57. The test device of claim 28 wherein the car-rier matrix is additionally incorporated with a dia-zonium salt coupling agent.

58. The test device of claim 29 wherein the car-rier matrix is additionally incorporated with a dia-zonium salt coupling agent.

59. The test device of claim 30 wherein the car-rier matrix is additionally incorporated with a dia-zonium salt coupling agent.

60. The test device of claim 31 wherein the car-rier matrix is additionally incorporated with a dia-zonium salt coupling agent.

61. The test device of claim 32 wherein the car-rier matrix is additionally incorporated with a dia-zonium salt coupling agent.

62. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 57, 58 or 59 and observing a detecable response.

63. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 60 or 61 and observing a detectable response.

64. The test device of claim 57 in which the salt is 1-diazo-2-naphthol-4-sulfonate.

65. The test device of claim 58 in which the salt is 1-diazo-2-naphthol-4-sulfonate.

66. The test device of claim 59 in which the salt is 1-diazo-2-naphthol-4-sulfonate.

67. The test device of claim 60 in which the salt is 1-diazo-2-naphthol-4-sulfonate.

68. The test device of claim 61 in which the salt is 1-diazo-2-naphthol-4-sulfonate.

69. The test device of claim 57 in which the dia-zonium salt is a zwitterion having the structure in which:
B is H or OH, D- is an anion, G, same or different, is H, lower alkyl group having 1 to 6 carbon atoms or aryl, or in which both G together form a fused ring structure.

70. The test device of claim 58 in which the dia-zonium salt is a zwitterion having the structure in which:

B is H or OH, D- is an anion, G, same or different, is H, lower alkyl group having 1 to 6 carbon atoms or aryl, or in which both G together form a fused ring structure.

71. The test device of claim 59 in which the dia-zonium salt is a zwitterion having the structure in which:
B is H or OH, D- is an anion, G, same or different, is H, lower alkyl group having 1 to 6 carbon atoms or aryl, or in which both G together form a fused ring structure.

72. The test device of claim 60 in which the dia-zonium salt is a zwitterion having the structure in which:
B is H or OH, D- is an anion, G, same or different is H, lower alkyl group having 1 to 6 carbon atoms or aryl, or in which both G together form a fused ring structure.

73. The test device of claim 61 in which the dia-zonium salt is a zwitterion having the structure in which:
B is H or OH, D- is an anion, G, same or different, is H, lower alkyl group having 1 to 6 carbon atoms or aryl, or in which both G together form a fused ring structure.

74. The test device of claim 69 in which the car-rier matrix is additionally incorporated with an ac-celerator.

75. The test device of claim 70 in which the car-rier matrix is additionally incorporated with an ac-celerator.

76. The test device of claim 71 in which the car-rier matrix is additionally incorporated with an ac-celerator.

77. The test device of claim 72 in which the car-rier matrix is additionally incorporated with an ac-celerator.

78. The test device of claim 73 in which the car-rier matrix is additionally incorporated with an ac-celerator.

79. The test device of claim 74 in which the ac-celerator is an alcohol having 1 to 15 carbon atoms.

80. The test device of claim 75 in which the ac-celerator is an alcohol having 1 to 15 carbon atoms.

81. The test device of claim 76 in which the ac-celerator is an alcohol having 1 to 15 carbon atoms.

82. The test device of claim 77 in which the ac-celerator is an alcohol having 1 to 15 carbon atoms.

83. The test device of claim 78 in which the ac-celerator is an alcohol having 1 to 15 carbon atoms.

84. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 64, 65 or 66 and observing a detect-able response.

85. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 67 or 68 and observing a detectable response.

86. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 79, 80 or 81 and observing a detect-able response.

87. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 82 or 83 and observing a detectable response.

88. The test device of claim 79 in which the ac-celerator is an alcohol having 8 to 15 carbon atoms.

89. The test device of claim 80 in which the ac-celerator is an alcohol having 8 to 15 carbon atoms.

90. The test device of claim 81 in which the ac-celerator is an alcohol having 8 to 15 carbon atoms.

91. The test device of claim 82 in which the ac-celerator is an alcohol having 8 to 15 carbon atoms.

92. The test device of claim 83 in which the ac-celerator is an alcohol having 8 to 15 carbon atoms.

93. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 88, 89 or 90 and observing a detect-able response.

94. A method for determining the presence of leu-kocytes, esterase or protease in a test sample, the method comprising contacting the sample with the test device of claim 91 or 92 and observing a detectable response.