CA1267500A - Compounds for detecting the presence of hydrolytic analytes in a test sample - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A novel compound useful in detecting leukocytes, esterase and protease in a test sample. The compound has the structure in which: A is an acid residue, R is lower alkyl, aryl, carboxyl, carboxyl ester, amido or cyano, R*
is H or lower alkyl, and X is O, S, or NR', in which R' is H, lower alkyl or aryl.

Description

~26.~0 NOVE~ COMPOUNDS FOR DETECTING THE
PRESENCE OF HYDROLYTIC ANALYTES
IN A TEST SAMPLE

CONTENTS

1. Introduction

2. Background of the Invention

3. Summary of the Invention

4. Definitions 4.1 Acid Residue 4.2 Aryl 4.3 Lcwer alkyl

5. Detailed Description of the Invention 5.1 The alcoholic moiety 5.2 The acyl moiety 5.3 Preparation of pyrroles

6. Experimental 6.1 General Information 6.2 Synthesis of the Compound 6.2.1 3-(N-tosyl-L-alaninyloxy)-5-phenyl-pyrrole 6.2.2 3-(N-tosyl-L-alaninyloxy)-5-phenyl-thiophene 6.Z.3 3-~N-tosyl-L-alaninyloxy)-l-met}lyl-5-phenylpyrrole 6.2.4 3-~N-tosyl -L- alaninyloxy)-5-~p-chlorophenyl)pyrrole f~ ~

~Z6, ~

6.3 Preparation and Use of Test Device Containing the Compound 6.3.1 3-~N-tosyl-L-alaninyloxy)-5-phenyl pyrrole 6.3.2 3-~N-tosyl-L-alaninyloxy)-5-phenyl thiophene 6.3.3 3-(N-tosyl-L-alaninyloxy)-l-methyl-5-phenylpyrrole 6.3.4 3-(N-tosyl-L-alaninyloxy)-5-~p-chlorophenyl)pyrrole 6.4 Evaluation of the Test Device . !

.~ ~L2~, r ~o 1. I~ITRODU~IO~

The present invention relates to novel compounds useful in assaying a test sample for the presence of certain analyte constitutents. Such analytes include leukocytes, esterase, and protease. The detection of leukocytes in urine is especially important in medical diagnostics.
The presence of an abnormal 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 2Q expenditure on the part of the clinician. Moreover, the traditional techniques suffer from the inadequacy that only intact cells are determined. Leukocytes occurring in the urinary system are subject to con-ditions which can ~avor 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 exami~a-tion techniques, erroneously low determinations and false ncgatives can result.
Of the two techniques of microscopic leuXocyte analysis - urine sediment and non-centrifuged, homo-genized urine - the former is clearly the most de-sirable. Although dependable results can inure to the ~z~

latter, urine sediment observation is used in an over-whelming majority of instances. It requires that the urine sample be centrifuged and the sediment isolated and subjected to microscopic inspection. The analyst then counts the number of leukocytes appearing in the viewing field. However, this task is further compli-cated by the presence of 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 of the sample and differing optical powers among microscope equipment, can lead to enormous errors in the ultimate determination.
It is thus apparent that a quick, facile 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 were intact or had been lysed, would indeed advance the state of the art by a quantum jump. The present invention provides such an advance. Moreover, it is based, not on the ability to see leukocytes, but on the enzymatic activity they exhibit, and therefore is substantially free of the inaccuracies described above.

2. BACKGROUND OP T71E I~YEl~TIO~

There exists in the prior art a body of ref-erences which disclose the us~ of certain esters which, when cleaved by enzymatic activity, result in 30- the formation of color or other detectable species.
Thus, British Patent No. 1,128,371 discloses the use `- MS-1335 .. : ., ' ~

6 ~ ~ 0 o~ indoxyl and thioindoxyl esters as useful chromogens in detecting hydrolytic enzymes in ~ody fluids. The enzymes cleave the ester to generate ree indoxyl, which subsequently oxidizes to form the dimeric pro-duct indigo, a readily observable blue dye. Suchactivity 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 reference to the enzyme to be detected. For example, it is stated that the acid radical can be acetate, or laurate or stearate for detection of esterase or lipase, respectively. For detecting enzymes such as phosphatase or sulfatase the acyl radical can be inorganic. Thus, the British Patent can be held to teach the use of chromogenic esters as substrates for determining esterolytic enzymes, such esters com-prising indoxyl or thioindoxyl as the alcoholic moiety of the ester, the acyl moiety being tailored to be conducive to the particular enzyme to be determined.
The effect of careful acyl radical selection is nowhere more clearly exemplified than 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. Soc. Exper. B~oZ. 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-Ilanine methyl ester.
Moreover, L-alanine-p-nitrophenol ester was similarly hydrolyzed to yield the yellow p-nitrophenol color-form.

lZ~ 0 Similarly, Sweetman et at., Jour. Hist. Soc., 22:327-339 teaches the use of l-naphthyl-N-acetyl-DL-alanine and l-naphthyl butyrate to demonstrate the presence of esterase, as well as l-naphthyl-N-acetyl-L-alanyl-L-alanyl.
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 tradi-tional chromogenic substrate for leukocytic esteraseactivity. Moreover the Boehringer patent teaches the equivalence of proteases and esterase in their estero-lytic penchants.

It is known that ester hydrolysis reactions can be activated by the presence of many nucleophilic agents, including many alcohols. Thus, the rate of hydrolysis of phenyl acetate and p-nitrophenyl acetate by esterase is increased 2.5 to 5.5 times upon ad-dition of methanol and butanol. Greenzaid and Jencks, Biochemistry, 10(7), 1210-1227 (1971). Moreover, the effect increases with the length of the n-alkyl group.
Wynne and Shalatin, Eur. J. Biochem., 31, 554-560 (1972).
In particular, this activation effect 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, Biochemistry, 12(11), 2025-2034 (1973). High molecular weight alcohols increase the 3n rate of esterase-induced hydrolysis of p-nitrophenyl t-BOC-L-tyrosinate. Ashe and Zimmer, Biochem. and Biophys. Res. Comm., 75(1), 194-199~1977). The dis-closure of U.S. Patent No. 4,299,917 describes other known ester hydrolysis activators such as certain metal complexes, pyramine derivatives and imidazoles.

Also known is the use of certain diazonium salts to couple with phenols and pseudophenols to produce azo dyes. Martinet and Dornier Compt. Rend., 170, 592 (1920). Such a technique is used in an esterase analysis whereby indoxyl acetate is hydrolyzed via esterase to produce indoxyl, which is in turn coupled with a diazonium salt to form the corresponding azo dye. Holt and Hicks, J. Ce~ B~o~. 29, 361-366 (1966);
Gossrau, His~ochemistry, 57, 323-342 ~1978); West German OffenZegungschrift No. 31 17 7~1, filed May 9, 1980.
The dye industry, an art which is clearly not analogous to medical diagnostic assays, but which nevertheless has long been a reservoir of experimental organic chemistry procedures, provides some guidance as to certain amino acids and their cyclization reac-tions. Thus, it is known to form pyrroles through the addition of glycine to salts of ~-phenylglycidic acid, followed by treatment with hot acetic anhydride.
Madelung and Obermann, Ber./ 63/ 2870 (1930).

3. SUMMARY OF THE IDVENTION

The present invention provides a new compound which is useful in determining the presence of leuko-cytes, esterase and/or protease in a test sample. The inventio~ also relates to a method for preparing the compound. The compound is one having the structure R* O-A
~ (I) R X

in which:

C~O

A is an acid residue, R is lower alkyl, aryl, carboxyl, carboxyl ester, amido or cyano, R* is H or lower alkyl, and X is 0, S, or NR', in which R' is H, lower alkyl or aryl.
The presently claimed method relates to preparing the ester (I) wherein the structure is R* ,O-B
R~` N ~

in which B is an N-blocked amino acid residue or an N-blocked peptide residue, and wherein R is lower alkyl, aryl, carboxyl, carboxyl ester, amido or cyano and R*
is H or lower alkyl. The method comprises the se-quential steps of (a) forming a 3-hydroxypyrrole having the struc-ture R* OH
~ (III) R

R' (b) adding an acid halide of a N-blocked amino acid or a N-blocked peptide to the 3-hydroxypyrrole in the presence of an organic acid to form a product mix-ture, and (c) isolating the ester (II) from the product mixture.
The procedure of step (a) comprises the sequen-tial steps of: .
1. _ausing an aqueous mixture of a ketone, an alkali metal monopersulfate and a compound having the structure R~,COOH
R~ (IV) to react in the presence of a sufficient amount of alkali metal bicarbonate to main-tain the mixture at a pH of at least 7, thereby forming a first reaction mixture.
2. adding HOOC-CH2-NHR', where R' is as defined above, to the first mixture to form a second reaction mixture;
3. Removing water from the second mixture to form a substantially dried second mixture;
4. adding a carboxylic acid anhydride to the dried second mixture in the presence of an organic base to form a third reaction mix-ture;
5. sub~ecting the resultant mixture from step 4 to hydrolyzing conditions to produce a reac-tion mixture containing a 3-hydroxypyrrole;
and 6. isolating the 3-hydroxypyrrole;

4 . DE~I NI TI ONS

The following definitions are provided to clarify the scope of the present invention, and to enable its formulation and use.

lZ675C~

4.1 The e~pression "acid residue" includes derivative structures of ester-forming acids without their characteristic acidic -OH group. Thus, the term includes the acyl portion of the acids phos-phoric, sulfonic, carbonic, carboxylic, and otherester-forming -OH group-containing acids.
The terms "N-blocked-amino acid residue" and "N-blocked-peptide residue" require defini~ion on two counts. "N-blocked" reers to the chemistry of the amine group of an amino acid or peptide whereby a hydrogen bound to the nitrogen atom is replaced by a protective group such as acetyl, p-toluenesulfonyl (tosyl) and tert-butyloxycarbonyl ~t-BOC) and other N-protective groups known in the art.
By the expressions "amino acid residue" and "peptide residue" is meant an amino acid or peptide molecule without the -OH of 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 fused ring systems such as naphthyl. Thus, the aromatic ring system can be heterocyclic or homocyclic, and can be substituted or unsubstituted, provided the substituent group(s) not interfere with the operation or functioning of the claimed composition or test device in its capability of detecting leukocyte cells, esterase or protease.
Selection of such substituents is a routine laboratory determination, given the pre~ent disclosure.

3Q 4.3 The expression "lower alkyl", as used in the present disclosure, is an alkyl moiety containing about 1-6 carbon atoms. Included in the meaning of ' MS-1335 lower alkyl are methyl, ethyl, n-propyl, isopropyl, n-butyl. sec-butyl, te~t-butyl and all isomers of pentyl and hexyl. These can be unsubstituted, or they can be substituted provided that any such substituent group~s) not interfere with the operation or functioning of the composition or test device in its capability to detect leukocyte cells, esterase or protease. Selection of such substituents is a routine laboratory determina-tion, given the present disclosure.

5. DETAII,ED DESC~IPTION OF THE INVENTION

The compound claimed herein includes a broad range of esters, the alcoholic (phenolic) and acyl moieties of which can be chosen to suit particular needs. Thus, the alcoholic moiety can be tailored to provide a particular desired response, such as a particular color or light absorbance, and the acyl group can be selected in accordance with a particular analyte to be detected.

5.1 The AZcohoZic Moiety The alcoholic moiety of the ester is a pseudo-phenol in that it contains a heteroatom having a pair of electrons which can delocalize to produce aroma-ticity in concert with the double bonds in the ring.
Thus the heteroatom X can be oxygen, sulfur or nitro-gen. In the case where X is nitrogen, the atom can be unsubstituted i.e., NH, or it can be substituted witha lower alkyl oi aryl group. As a result of this aromatic character, the compound lends itself easily to coupling with a diazonium salt to form an azo dye, once the ester has become hydrolyzed.

It has been found that various substituents at the 5-position of the ring provide enhanced color formation and storage stability. Thus, R can be lower alkyl, aryl, carboxyl, carboxyl ester, amide, cyano or other substituent, provided that any such substituent group not interfere with ~he operation or functioning of the CQmpOSitiOn or test device in its capability to detect leukocyte cells, esterase or protease. For the purpose of assaying for leukocytes, esterase or pro-tease, esters in which R is phenyl, or p-chlorophenyl are preferred.

5. 2 The AcyZ Moiety The acyl moiety A has equally broad scope, and is chosen with the particular purpose of the assay in lS mind. Where it is desired to assay a test sample for esterase or protease activity, such as measuring leukocytes in urine, A can be an amino acid or peptide residue. Preferred for such use is N-tosyl-L-alanine residue.
5. 3 PYeparation of the Pyrro1,es and Their Esters The invention also includes a novel method of synthesizing certain pyrrole esters of N-blocked amino acids and peptides (II). The first. step of the pro-cedure comprises the formation of a 3-hydroxypyrrole having the structure (III). This is accomplished by a multistep synthesis beginning with a compound of structure (IV) as the starting material. This com-pound is reacted in the presence of acetone or other ketone to form an epoxide compound of the structure ~LZ6~;JSCO

-R* COOH
0~ (V) R
The epoxidation is preferably achieved using an alkali metal monopersulfate such as KHSO5 which is available Commercially as O~one~ ~DuPont Company). It has been found that pH control in this stepl'2 is dramatically facilitated when the reaction is performed in the presence of excess alkali metal bicarbonate such as NaHC03, i.e., at a pH of at least 7. Preferably the reaction mixture is kept at a temperature in the range of about 20 to 30C, although this range is not lQ critical. Acidification of the resultant reaction mixture, followed by the addition of an organic sol-vent such as methylene chloride, serves to extract the epoxide into the organic phase.
Next the organic phase is washed with aqueous base. This step renders the epoxide water-soluble, thus transferring it to the aqueous phase. The aqueous phase is separated and a glycine added to produce a reaction mixture containing the double salt R*
H0-C C00 (VI
R-C-N - CH2-coo~
H R' 2a This step has been found to be best performed where the aqueous base has a pH range of about 10.5 to 12, with about 11.5 being the preferred pH. The tem-perature for this step is preferabl~ elevated to about l. J.O. Edwards, et aZ, Photochem. Photobio~. 30, 63 2~ (1979) 2. R. Curci, et a~, J. Org. Chem. ~5, 4758 ~1980) 1~ 0 100C, and a substantial amount of low boiling liquid removed (acetone and water). As the vapor temperature approaches 100C heating is maintained to continue reflux at or near 100C. Following cooling, washing with an organic solvent such as CH2C12 and evaporating to dryness (remova' of residual water), (VI) is iso-~ated from the residue by boiling in a solvent such as ethanol and allowing crystals to separate.
Next, compound (VI) is treated with a carboxylic acid anhydride to form R~ _ ~ O-Ac R*~____~ O-Ac R N ~ R N (VII) Ac R"
where R" is lower alkyl or aryl. Ac is acetate in the case where the anhydride is acetic anhydride. This step is conducted initially at ambient temperature and ultimately heated to about 90 to about 140C. A key facet of this step is the inclusion of an organic base such as pyridine. Preferably Compound ~VI) is sus-pended in the base, followed by the acid anhydride addition.
The thus-formed reaction mixture containing (VII) can then be evaporated to dryness to remove residual anhydride and solvent and the product (VII) recovered.
Treatment of (VII) under a hydrolyzing environ-ment leads to the formation of R* ~ (VIII) R N
R' Preferred as a hydrolyzing environment is a mixture of an alcohol such as methanol and aqueous base, such as 2N NaOH. It is preferred to mix (VII), the alcohol , MS-1335 :~2~ 0 and aqueous base at a low te~perature, s-lch as about -15C to about 10C. Compound (VIII) generally se-parates out of solution at this temperature and is isolated, such as by filtration, and purified.

5The second step in the procedure is the ester-ification of (VIII) to form the product R* O-B
~ (IX) R N
R' in which B is a N-blocked amino acid or peptide re-sidue. (VIII) is added to an anhydrous solvent, such as tetrahydrofuran (THF), diethyl ether and the like, containing an organic base such as pyridine and an organic acid such as trifluoroacetic, oxalic, citric, acetic or other carboxylic acid. Preferred for this step are pyridine and trifluoroacetic acid. Next an acyl halide of the desired blocked amino acid or peptide residue is added slowly. When the reaction is complete it is quenched with water, optionally con-taining a buffer such as citric acid and ethyl acetate, and the product (IX) recovered.

6. EXPERIMEP~TAL

The following examples are provided to further assist the reader in making and using the present invention. Thus, preferred embodiments are described in experimental uetail and analyzed as to their utility. The examples are illustrative only, and are in no way intended as limiting ihe scope of the in-vention described and claimed herein.

;~Z~.'5~0 6'.1 G~neraI Information In the following e~perimental discussion abbre-viations are used as indicated:

g = gram kg = kilogram L = liter mL = milliliter M = molar mM = millimolar N = normal eq = equi~alents mol = gram molecular formula ~moles) mmol = gram molecular formula x 10-3 ~millimoles) aq ~ aqueous hr = hour TLC ~ thin layer chromatography Infrared (IR) spectra were obtained with a Perkin-Elmer Model 710B or 237 infrared spectrophotometer as solutions in CHC13 unless otherwise noted; the 1602 2Q cm 1 band of polystyrene film was used as an external calibration standard. Signals are reported as cm 1, Proton magnetic resonance (lH 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 were obtained in CDC13 solution unless otherwise noted. Chemical shifts are reported in parts per mil-lion downfield from the internal standard tetramethyl-silane.
Carbon-13 magnetic ;esonance ~13C MMR) spectra were obtained at 22.5 MHz using a JEOL FX9OQ spectro-meter with Fourier transform and with full protonbroad-band noise decoupling; spectra were obtained in CDC13 solution unless otherwise noted. Carbon shifts are reported in parts per million downfield from the internal standard tetramethylsilane.

-~s~ ,, .',"
* Trade Mark 12~';tS~

Mass spectra (MS) were obtained on a ~ewlett-Packard 5935A spectrometer operating in either an electron impact (EI) or fast atom bombardment (FAB) mode. High-resolution mass spectra were ob~ained on an AEI ~IS-902 spectrometer.
Organic reagents were obtained from Aldrich Chemical Company and were used without purification, unless otherwise noted. Inorganic reagents were ACS
reagent grade from Fisher Scientific Company or other major vendor. Reac'ion solvents were ACS reagent grade; tetrahydrofuran (THF) was HPLC grade from J.T.
3aker Chemical Company. Brine refers to a saturated aqueous sodium chloride solution.
Thin layer chromatograph ~TLC) was performed using silica gel 60F-254 plates from E. Merck. Column chromatography was performed using E. Merck Silica Gel 60 (70-230 mesh). All melting points and boiling points reported are uncorrected.

6.2 Syn~hesis of the Compound 6.2.1 Synthesis of 3-~.V-tosyt-L-aZer.inyZo~yJ-5-phenyZpyl'rOIe (d) The synthesis of ~4) is illustrated in the fol-lowing reaction sequence:

, .
* Trade Mark ''1r;C~o - 1 g -~I~ COOII c~COOIl QJI L~L~S05, 1120~1Cl acetone, NaH 03 1(011 /EtOH

COO K
~10 ~CH-COO- K- KOH ~

\l~cH2coo K 100 C
Ac20 Pyridine 12~
\ ~ .

~O-A~ (3 O- C - CH- CH~ () Irjr N ' TIIL~ Cl-C-CIH-CH3 (~) ~ N ~ Il/ ~ts p~ridil~e ) IF~ N

~;!r N- tosy ~ - L-a Zc~nine L-alanine (lOOg; 1.11 moles) was dissolved in 2.25 L of 1 N sodium hydroxide (aq), cooled to 5C and stirred while a solution of p-toluenesulfonyl chloride (218 g; 1.11 moles) in 450 mL of toluene was added slowly. The mixture was stirred at ambient tempera-ture for 20 hr. The layers were separated and the chilled aqueous layer acidified to pH 1 with con-centrated hydrochloric acid. The white solid title compound was collected by fil~ration, washed with water and dried. Yield 178.5 g (66~) mp 134-5C. IR
(CHC13) cm 1 1726, 1340, 1165, 1095; lH NMR (DMSO-D6) 1.20 (d,J=7,3H), 2.40 (s,3H), 3.85 (p,J=8,1H), 6.4 (br s, lH)(C02H), 7.41 (d, JAB =8, 2H) and 7.75 (d, JAB =8,2H) [center of pattern: 7.58; ~VAB=20.49Hz], 8.03 (br d, J=8,1H)(NH).

N- tosy ?,-L-aZaniny ~ ch1,oride 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 tAmerican Norit Co., Inc.), filtered 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
(CHCl~) cm 1 3360, 3260, 3025, 1775, 1605, 1350, 1170, 910; H NMR (CDC13) ~ 1.48 ~d,J=7,3H), 2.43 (s, 3H), 4.33 (p,J=o,lH), 5.93 (br d,J=8,1H)(NH), 7.31 (d, JAB
=8~ 2H) and 7.76 (d, JAB =8,2H) [center of pattern:

7.53; QVAB=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.

12~ '`0 Method B
A mixture of N-tosyl-~-alanine C3.1 g; 13 mmol) and thionyl chloride (6 mL) was heated for 90 min at 50C, then diluted with 50 mL of dry hexane. The mixture was stirred rapidly, chilled and the solid product filtered. Yield 3.15 g (93%) mp 99-100C.
The IR spectrum was identical to that of the recrystal-lized material prepared by Method A.

2-Hydroxy-3(carbo~ymethyZamino)-hydrocinnam~c acid Dipotassium sa~t dihydrate fl) A stirred slurry of 1.0 kg of tra~s-cinnamic acid (6.75 mol) in 4.5 L acetone was treated first with NaHC03 (2.47 kg; 29.4 mol; 4.36 eq) then carefully with water (4.5 L). The resulting thick mixture was treated dropwise, over 1.5-2.0 hr, with a solution of OXONE monopersulfate compound (3.78 kg; contains 1.825 eq of KHSO5) in 0.4 mM aqueous disodium ethylene-diamine tetraacetic acid (EDTA) (14.5 L; prepared by dissolving 2.17 g disodium EDTA dihydrate in 14.5 L
distilled water). 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 hr then cooled to about 10C. The reaction was acidified with concentrated HCl (- 1.2 L) to pH = 2, while maintaining the tem-perature at around 10C, and then treated with CH2C12 (5.05 L) and stirred vigorously for 10 minutes.
After allowing 'he mixture to settle, the aqueous layer was decanted ~nd set aside and the organic layer, which contained insoluble salts, was filtered through paper with suction. The filtered solids were washed with CH2C12 (1.9 L) and the aqueous layer extracted with this filtrate. The filtered solids lZ~ 5C10 were again washed with CH2C12 (3.15 L) and the aqueous layer extracted 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 required to dissolve a solid which may separate during the base extraction. The CH2C12 layer was then extracted 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 or-ganic layer was discarded.
The aqeous solution pH was adjusted to 11.5 with25% aqueous KOH then heated to boiling. Approximately 900 mL of low boiling liquid (acetone and water) was distilled off until the vapor temperature reached 99C, following which, the mixture was refluxed for 2 hours. After 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 bath. The residue was boiled in 95% ethanol (EtOH) (8.83 L) for 30 minutes, then allowed to cool slowly wi~h stirring, whereupon the product separated as fine crystals.
These were filtered, washed with fresh 95% EtOH (1.26 L) and dried in a 50-60C oven to give the title compound (1.77 kg; 74.6%) as white crystals with mp =
120-2C (uncorrected).
IR (KBr) cm 1 3420 (br.), 1590 (br.), 1410, 1130, 710; lH NMR (D2O-TSP) ~ 3.1 (s, 2H), 3.89 (d, JAB
=4,1H) and 4.52 ~d, JAB =4,1H) (center of pattern:
4.21; ~VAB=18.83 Hz,), 4.68 (s, 6H, exchangable protons), 7.4 (s, SH); TLC Rf = 0.5S (EtOH:lM tri-ethylammonium bicarbonate, 7:3) A~al. Calcd. for CllH15NO7K2: C,37.59; H,4-30; N~3-99 Found: C,37.22; H,4.24; N,3.96 . . .
. ;. :.~. . . :
,~. . .
: ;',.'. ' ~' '' lZt~ O

V-acetyZ-3-ace~o~cy-5-phenyZpyrroZe (2J
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 with 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-la 123 C for 15 minutes, then allowed to cool to ambienttemperature 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 ethyl acetate (EtOAc) until colorless; the fil-trate was evaporated to dryness under vacuum.
The dark red residue was dissolved in EtOAc (3.0L) washed three time with water (1.0 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-Mannville) and evaporated to dryness under vacuum to give a reddish-orange oil. This oil was dissolved in warm 2-propanol (1.2 L), then allowed to cool slowly to ambient temperature overnight, whereupon a solid separates. The crystalline product was filtered, washed with 50% aqueous 2-propanol and dried to give the title compound (417 g; 60%) with mp=58-60C
(uncorrected). A portion was taken up in diethyl ether (Et20), treated with Norit 211, filtered and con-centrated under reduced pressure; on standing at 0C
colGrless tiny needles separated. These were fil-tered, washed with Et20:Hexane ~1:1) and vacuum dried to give the analytical sample with mp=60-62.5C
(uncorrected).

12t~ 0 IR (CHC13) cm 1 3020, 1760, 1730, 1595, 1378, 1320, 1220 (br.~, 1030, ~60, 903; lH NMR ~CDC13) ~
2.23 ~s, 3H), 2.27 ~s, 3H), 6.18 ~d, J=2, lH), 7.35 ts, 5H), 7.42 td, J=2, lH); TLC Rf = 0.56 ~toluene:
dioxane, 4:1).
Anal. Calcd. for C14H13NO3: C,69.12; H,5-38; N,5-76 Found: C,68.88; H,5.25; N,5.53 3-Hydroxy-S-pheny~pyrro~e (3) A finely divided portion of N-acetyl-3-acetoxy-5-phenylpyrrole ~2) (36.8 g; 0.15 mol) was freed of oxygen by stirring in a flowing argon stream for 10 minutes, then suspended in deoxygenated methanol ~MeOH) ~379 mL), cooled to -6 C (in 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 immediately upon addition of base to 18 C, and after ~3 minutes the reaction mixture became homogeneous.
As the reaction mixture cooled, compound 3 separated as fine crystals. After 15 minutes a solution of 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 mL), taking care to minimize exposure of the product to air, then dried under vacuum overnight to yield the title compound ~22.3 g;
93.6%) as light p-;lk tiny needles.
IR ~KBr) cm 1 3400, 3110, 2900, 1600, 1580, 1555, 1480, 1268, 1180, 742, 640; lH NMR ~DMSo-D5) ~ 6.1 (m, lH), 6.3 ~m, lH), 7.0-7.7 (m, 5H), 8.0 (s, lH), 10.4 (br s, lH); TLC Rf = 0.20-0.28 tEtOH:CHC13, 1:9) -Anal. Calcd. for CloHgNO: C,75.45; H,5.70; N,8.80 Found: C,75.30; H,5.69; N,8.67 ~2~ C~O

3-(~v-~osyz-L-aza7~yzoxy~-5-phenyzpyrroze (~1 A solution of anhydrous tetrahydrofuran ~THF, 450 mL), pyridine ~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-lQ minutes of a soluti~n 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 of 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 ~360 mL; 0.18 g NaCl per mL of water). The organic layer was next extracted twice with a solution of 5~ aqueous NaHCO3 ~1.35 L each), and then washed with another portion of aqueous NaCl ~360 mL; 0.18 g 2Q NaCl per mL of water). The reddish brown organic layer was stirred at ambient temperature for 15 min-utes with MgS04 ~101 g) and Darco-G60 ~143 g), then filtered through Celite and evaporated to dryness under vacuum from a 37C bath to give ~4) as a pinkish-white solid. The crude product was ground to a powderand dissolved in warm ~50C) THF (250 mL), stirred vigorously and diluted with n-hexane t250 mL). The stirring was continued for 1 hour at ambient tem- -perature as the product crystallized. The solid was filtered, washed with t~l:ene ~about 1 L) until the filtrate was colorless, then dried overnight to yield the title compound ~112 g; 65~) as a white powder with mp - 154.5-155C.

~M~ * Trade Mark :, .

~a2~ 0 IR (KCl) cm 1 3350, 3325, 1760, 1508, 1320, 1155, 770; 1H NMR (DMSO-d6) ~ 1.33 (d, J=7, 3H), 2.36 (s, 3H), 4.13 (p, J=~, lH), 6.25 (m, lH), 6.73 (m, lH), 7.05-7.50 (m, 5H), 7.5-7.85 (m, 4H), 8.42 (d, J=8, lH), 11.18 (br s, lH); 13C NMR (DMSO-d6) ~ 18.335, 21.001, 51.370, 98.061, 108.336, 123.423, 126.024, 126.610, 128.560, 128.756, 129.601, 13~.397, 137.600, 138.380, 142.737, 169.919; ~]D = ~70 (c=l.ll, MeOH);
TLC Rf = 0.45 (EtOAc:hexane, 1:1); TLC Rf = 0.40 (toluene:dioxane, 4:1).
Anal- Calcd- for C20H20N24S C,62.48; H~5-24; N~7-29 Found: C,62.62; H,5.27; N,7.30 6.2.2 Synthesis of 3-(~-tosy~-L-a~aninyIoxyJ-5-pheny~thiophene (9) A series of experiments was conducted to prepare 3-hydroxy-5-phenylthiophene by minor modifications of the reported literature procedures3'4 outlined on the following page. The resultant hydroxythiophene was then acylated with N-tosyl-L-alaninyl chloride to give the corresponding N-tosyl-L-alaninate ester in 46%
yield (unoptimized procedure).

3. P. Friedlander and S. Kielbasinski, Chem. Ber.
~5, 3389 (1912).
4. A.I. Kosak, r.J.F. Palchak, W.A. Steele, and C.M. Selwitz, J. Amer. Chem. Soc. 76, 4450 (1954) , ~

r ~C~

COOC2 5 . ~ O
S ~s,S (5) a. Na2S
b. BrCH2COOil q . ` p~-l=8.7 ;C-CM3 ~5 COOH

NaOH

MeOH
1l .Cl-C-CIH-CH3 , /~
N O-C-CH-CH
~;~(8) C:! Cl ~J H Ts pyr id ine ~ g lZ~.~C`O

3-PhenyZ-1,2-dit~i~-3-cyclopenten-5-one ~5J
A suspension of 10 g of ethyl cinnamate ~56.8Z
mmol) and 10 g of sulfur was heated at 250C for four hours in a S0 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 S00 mL of boiling acetone and twice with S00 mL portions of ethanol. The combined supernatants were concentrated to a black solid, whichwas crystallized from methanol to give dark brown needles ~5). A second recrystallization from methanol using Norit and filtration through Celite gave 2.023 g lS of light yellow needles mp 113-115C.
IR (KBr) cm 1 1650, 1550, 13~0, 1350, 1130, 770;
lH NMR (60m Hz, CDC13) ~ 6.92 ~s, lH), 7.58 (m,SH);
TLC Rf = 0.5 (dichloromethane).
Anal. Calcd. for CgH6O2S C,55.64; H,3.11 Found: C,55.53; H,3.47 c~s-4-Keto-6-pheny~-3~7-dithia-5-nonenedioic acid ~6) A molten solution of 35.48 g of sodium sulfide nonahydrate (148-mmol) at 94C was treated with 6.65 g of 3-phenyl-1,2-dithia-3-cyclopenten-5-one ts) (34.23 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 (314 mmol) in 60 mL of H20 adjusted to pH 8.7 with sodium carbonate. The resulting solution was maintained at 3Q 0C, pH 8.7 for 45 minutes, and was then filtered.
The filtrate 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 ~2~;~;JS~iO

ether. The oil l~as evaporated with toluene until 6.98 g of a colorless foam ~as obtained (65%). This material was used without further 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 ~rystals. 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, 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-Hydroxy-S-phenyZthiophene Acetate (7) A vigorously stirred suspension of 3.40 g of crude cis-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 a 50 mL portion of 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 (7) was purified by evaporative distillation at 120-140~C-and 0.1 mm to give 1.235 g of a light orange oil which solidified upon stand ng (52%).
IR cm 1 1700, 17~5; 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, 5H); MS (EI, DIP) m/e 218 (M~, 12.6%);
TLC Rf = 0.48 (hexane:ethyl acetate, 5:1).

, ~

12ti, 5~!o Anal. Calc for C12Hloso2- 1/2 H2O C~63-41; H~4-88 Found: C,63.78; H,4.86 3-Hydro~y-5-phenyIthiophene (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, evaporated at 25C, lZ mm Hg, to approximately 50 mL volume, 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 of 837 mg of a yellow solid ~8), 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 after recrystallization was an additional 380 mg of 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 Fc md: C,68.05; H,4.70 3-(~-tosyZ-L-aZaninyZoxy)-5-phenyZthiophene ~9) A solution containing 440 mg of 3-hydroxy-5-phenylthiophene (~) (2.5 mmol) in 20 mL of dichloro-methane and 0.61 mL of pyridine (7.5 mmol) at 0C
under an argon atmosphere was treated with a solution ; containing 1.314 g of N-tosylalaninyl chloride (5 . .
, ~2~S~

mmol) in 10 mL of dichloromethane added dropwise over a period of five minutes. The reaction was allowed to stir for 0.5 hour at 0C, and was then poured into lO0 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 Z00 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 gave a total of 463 mg of product (9) as light yellow solid, (46%). mp 85-87C.
IR (KCl) cm 1 1735, 1330, 11~50; lH NMR (90 MHZ, CDC13) ~ 1.53 (d, J=7 Hz, 3H), 1.62 (s, 3H), 4.23 (m, lH), 5.32 (d, J=9Hz, 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 (M + 1, 15%); TLC Rf = 0.20 (hexane:ethyl acetate, 4:1).
Anal. Calcd. for C20HlgNO4S C,59.83; H,4-77; N~3-59 - Found: C,59.60; H,4.77; N,3.43 ~ MS-1335 Jtj(~O

6.2.3 Synthesis of 3-~-tosyZ-L-a~aniny~oly)-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:

COOK
O ~ HO ~ COOK

NH~CH3)CHzCOOH ~ N'^~COOK
KOH/~120 . IW l I AC2 ' Et3N
,.
~"~,OH ~O ~CR3 O
CF3COOH Cl-C-CH-CH3 pyridine ~ N~
THF H Ts \ / 1l _____,O-C-CH-CH
N~ (13) Cll3 . .
.~.~ ,, .

3~26 ~S~) 2-Hydro~y-3-tN-~n2t~y~arbo2~methy~amino)-~yd~oei~namic ac~d dipotassium sa~ ~10~
A mixture of ~-phenylglycidic acid potassium salt (30 g; 0.15 mole), N-methylglycine (13.2 g; 0.15 mole), ~istilled 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 (S7 5H);
TLC Rf = 0.51 (EtOH:lM triethylammonium bicarbonate, 7:3). (Product had no melting point less than 270C).

3~Acetoxy-l-methyZ-S-pheny~pyrro~e (11) A mixture of 2-hydroxy-3-(N-methylcarboxymethyl-Z 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, 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 (200 ml). The layers were separated-and the ether layer washed with another portion of water (200 ml). The ether solution was then dried over 3Q MgSO4, filtered and concentrated under reduced pres-sure to give 10.7 g of brown residue which after Kugelrohr distillation and cryslallization from ether yielded 3.0 g of white crystals (11) (yield 30%) mp =
64-65C.

~2~i ~ 5~

IR (CHCL3) cm 1 2990, 1750, 1570, 1518, 1482, 1375, 1230 ~br.~, 1024, ~10, 700; 1H NMR tCDC13) ~
2.20 C5, 3H), 3.58 Cs, 3H), 6.10 (d, J=2 Hz, lH), 6.75 (d, J=2 Hz, lH), 7.35 (s, SH); TLC Rf = 0.58 ~hexane:
EtOAc 7:3) Anal. Calcd. for C13H13NO2: C,72.54; H,6.10; N,6-Found: C,7~.57; H,6.09; N,6.51 3 - (~1- tosy ?,-L-a Zani~y Zo:~:y ~ -1 -me thy Z - 5 - pheny Z pyrro Ze (13J
lQ To a ~ixture of deoxygenated methanol (15. 5 ml) and 3-acetoxy-1-methyl-5-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 for 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 MgS04, 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 of 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 of freshly prepared N-tosyl-L-alaninyl chloride (1.2 g, 7.4 mmole, 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 aqeuous citric acid (lM,5ml) and EtOAc (30 ml). After a brief mixing, the layers were separated and the organic layer was suc-cessively washed with saturated NaCl solution, twice ~Ztj7S~) with 5% NaHCO3 solution and again with saturated NaCl solution. The EtOAc extract was then dried o~er MgS04, treated with Norit 211, filtered and concen-trated under reduced pressure to give the crude pro-duct (13) as an orange residue. This was dissolved inhexane:EtOAc (1:1) (5 ml) and chromatographed on a column ~SiO2, 100 g) by elution with hexane:EtOAc (7:3) to give 1 g of (13) as a thick light orange oil.
A portion of this crude product was further purified by semi-preparati~e HPLC ~column~ IB~ 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 Hz, lH), 6.15 (m, lH), 6.74 (d, J=2 Hz, lH), 7.30-780 (m, 9H), 8.37 (d, J=8 Hz, lH); 13C NMR.(DMSO-d6) ppm 18.205, 20.936, 34.917, 51.240, 100.598, 113.148, 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.

~.2.4 Synthe~is of 3-fN-tosyZ-L-aZaninyZoxy)-5-(p-chZorophenyZ)pyrroZe ~18J
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 H, X is NR' and R' is H. The reaction sequence is as follows:

:lZf~ îÇ5~v I~ coo~ ~COO~I

Cl ~D ~ ~ HCl ~ (14) ~OH, H2O
glyc ine ~ pH=ll . 5-12 . 0 OAc HO ~ COO K
~3' Ac2O~ N ~ COO K 2H2O
~ J I pyridine l~ J
C 1 ~ Ac 1 21 - 2 C l ~~ H
~1 6) ~1 5) NaOH

\ / ~ O

Cl~ ~ ~JG H Ts ~17)pyridine ~18) THF

~Z6 J 5C~O

tra~s~ -ChZorophen~JgZycidic acid (14) 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.
~fter five 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-Hydroxy-3-(carboxymethy~amino)-p-chZorohydrocin~amic acid dipotassium saZt dihydrate fl5) 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 (1~) (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 ex~racted 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 solids 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%).

12~ 5(~0 1H ~IR (D20-TSP) ~ 7.4 (5, 4H), 4.4 (d, J-~, 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,3.42 N-Ace~yZ-3-ace~oxy-5-(p-chZoropheny~)pyrroZe fl6) To the 2-hydroxy-3-(carboxymethylamino)-p-chlorohydrocinnamic acid dipotassium salt dihydrate ~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 121-2C (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 filtration 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 aqueous citric acid, cold water, three times with cold dilute aq. NaHCO3, cold water and brine, followed by drying over MgSO4 and filtering.
The filtrate was treated with 10 g of Darco, stirred for 20 minutes and then filtered. The filtrate was evaporated under vacuum to an oil. To the oil was added 25 mL of 2-propanol. The resultant solution yielded, with chilling and scratching, pale yellow 3Q crystals: mp 69-71C (3.4 g; 47%); TLC ~f = 0.61 (toluene:dioxane, 95:5). An analytical sample was recrystallized from 2-propanol but no cllange in mp was observed.

:~2~ 5~0 - 3~ -IR (KCl) cm 1 1755 (C=0, ester) and 1730 (C=0, amide); lH NMR (CDC13) ~ 7.4 (m, 5H), 6.2 (d, J=2, lH), 2.4 (s, 3H) and 2.3 (s, 3H).
Anal. Calcd. for C14H12ClNO3: C,60.55; H,4-36; N~5-04 Found: C,60.65; H,4.55; N,5.07 ~-Hydroxy-5-fp-chIorophenyZ)pyrroZe ~17) A sample of N-acetyl-3-acetoxyl-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. A~ 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 deoxy-genated solution of citric acid (4.2 g; 0.02 mol) in 15 mL H20. The temperature rose briefly to 5C. After 0.5 hr. stirring at -5C, the solids were collected by filtration and washed with cold deoxygena~ed H2O.
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-tN-tosyZ-L-aZan~nyZoxy)-5-~p-chZorophenyZ)pyrroZe ~18) To N2 deoxygenated THF tl5 mL) was added pyridine (0.65 mL; 0.008 mol), trifluroracetic acid (1.27 mL;
0.0164 mol), and 3-hydro,;y-5-(p-chlorophenyl)pyrrole (17) (1. 3 g; 0.0065 mol). The solution was chilled to 0C to -4C and a N2 deoxygenated chilled (0 to -4C) 126~;J5~0 solution of N-tosyl-L-alaninyl chloride (2.1 g; 0.008 mol) in 15 mL of THF was added over lO 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 ~nd filtered. The filtrate was treated with 2 g of Darco and stirred for ten minutes, filtered and the filtrate concentrated 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 113-5Cs TLC Rf=0.47 (Et2O); IR (KCl) cm 1 1740 (C=O, 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 (d, 3H); MS (EI, DIP) m/e 418 2Q (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 6.3 Preparation and Use of Test Devices Containing the Compound A series of experiments was conducted to prepare test devices containing the present invention in which the ester substrates of paragraph 7.1, supra, were tested for responsiveness to leukocytes in urine. The devices comprised a small square of filter paper containing the assay reagents, the paper mounted at one end of a polystyrene film strip. The filter paper was impregnated with buffer, the ester an accelerator and a diazonium salt coupling agent. Each of the devices tested was found to exhibit a positive test for leukocytes in urine.

:~2~

6. 31 Tes~ device in whict~ the ester is 3~
tosy~-L-aZaniny~oxy)-5-p~eny~pyrro~e (4) A test device, sensitive to ~he presence of leukocytes in urine, was prepared. The device com-prised a small square of filter paper mounted at oneend 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 of Eaton and Dickman #205 lQ 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 water. 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 0.5 mM 3-(N-tosyl-L-alaninyloxy)-5-phenylpyrrole Following this impregnation the paper was dried for 10 minutes in a ventilated Hotpack~ oven at 130F. An off-white test paper was obtained.
A piece of the dried, impregnated paper was cut 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~.

~;~6 ~t~

6.3.2 Test device ~ which the este~ is ~
tosyZ-L-~ZaninyZo~)-5-phenyZthiophene f9) 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-NaO~ buffer (pH = 8.5) 0.4 M NaCl lo 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-lL-alaninyloxy)-5-phenylthiophene 0.75 mM 2-methoxy-4-morpholinobenzene diazonium chloride, zinc chloride double salt 2Q 0.5% (v/v) n-decanol The doubly impregnated paper 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 poly-styrene film strips measuring 0.2 by 3.25 inches.
Mounting was accomplished using Double Stick, a doubie faced adhesive from 3M Company. The test devices were stored in bottles of lO0 each, together with silica gel and molecular sieves to provide des-3Q sication.

lZ6~ 0 6.3.3 Test device in which the ester is 3-(N-tosy~-L-a~aninyZo~yJ-1-methyZ-5-phenyZ
pyrroZe (13J
Test devices were prepared following the proce-dure in experiment 6.3.1 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) 0.2~ (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:
O.75 mM l-diazo-2-naphthol-4-sulfonate 1.3 mM 3-(N-tosyl-L-alaninyloxy)-l-methyl-5-phenylpyrrole 1.5% (v/v) dodecanol Following impregnation in the aqueous first dip, the paper 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 6.3.1.

6.3.4 Test device in which the ester is 3-(N-tosyZ-L-aZaninyZo~yJ-5-(p-chZorophenyZJ
pyrroZe fl8) Test devices were prepared as in Experiment 6.3.1 3Q except that the acetone solution contained, in place of the phenylpyrrole, 1.3 mM 3- (N-tosyl-L-alaninyloxy)-5-~p-chlorophenyl)pyrrole.

.

::~2ti~;~XC~O

6. 4 Eval,uo~tion of the Test ~evice The test devices prepared in the abo~e experi-ments were subjected to evaluation of their ability to detect leukocytes present in urine.
Test samples were prepared from a normal human urine 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 leukocyte levels in the test samples. The data are presented in the following table.

Leukocyte% Reflectance Conc.entrationat 555 nm Experiment No. ~cells/~L) 6.3.1*

6.3.2 0 65 6.3.3 0 67 6.3.4 0 61 _ *Visual observation: purple color formed at 10-12 cells/~L; blank gave no color change . .,

Claims (15)

The embodiments of the invention in which an exclusive property or privilege is claimed are de-fined as follows:

1. A compound having the structure in which:
A is -COCH3 or ,where Ts is tosyl;
R is a lower alkyl group having 1 to 6 car-bon atoms, phenyl or chlorophenyl;
R* is H or a lower alkyl group having 1 to 6 carbon atoms; and X is NR', in which R' is H or a lower alkyl group having 1 to 6 carbon atoms.

2. The compound 3-(N-tosyl-L-alaninyloxy)-5-phenyl pyrrole.

3. The compound 3-(N-tosyl-L-alaninyloxy)-1-methyl-5-phenyl pyrrole.

4. The compound 3-acetoxy-1-methyl-5-phenyl pyrrole.

5. The compound 3-(N-tosyl-L-alaninyloxy)-5-(p-chlorophenyl)pyrrole.

6. A method for preparing an ester having the structure in which:
B is -COCH3 or , where Ts is tosyl;
R is a lower alkyl group having 1 to 6 car-bon atoms, phenyl or chlorophenyl;
R* is H or a lower alkyl group having 1 to 6 carbon atoms; and R' is H or a lower alkyl group having 1 to 6 carbon atoms;
the method comprising the sequential steps of:
a) forming a 3-hydroxypyrrole having the structure wherein R, R* and R' are as defined above, by the sequential steps of:
1) reacting an aqueous mixture of a ketone, an alkali metal monoper-sulfate and a compound having the structure wherein R and R* are as defined above, in the presence of a suf-fieient amount of alkali metal bi-carbonate to maintain the mixture at a pH of at least 7 to form a first reaction mixture, 2) adding HOOC-CH2-NHR' to the first reaction mixture to form a second reaction mixture, R' being as de-fined above, 3) drying the second reaction mixture, 4) adding a symmetrical, lower alkyl carboxylic acid anhydride to the dried second reaction mixture in the presence of organic base to form a third reaction mixture, 5) hydrolyzing the resultant third re-action mixture to produce a reaction mixture containing a 3-hydroxypyr-role, and 6) isolating the 3-hydroxypyrrole;
b) adding an acid halide having the acyl group -COCH3 or -COCH-CH3, where Ts is tosyl, to the 3-hydroxypyrrole in the presenee of a earboxylie acid; and e) isolating the resulting ester.

7. The method of claim 6 in which R is phenyl.

8. The method of claim 6 in which R is phenyl and R* is H.

9. The method of claim 6 in which the pH of the first reaction mixture is raised to a pH in the range of 9.6 to 12.

10. The method of claim 6 in which methanol, water and alkali metal hydroxide are employed to hydrolyze the third reaction mixture.

11. The method of claim 10 which comprises about 0.1 to about 4N alkali. metal hydroxide, water and methanol,.

12. The method of claim 6 in which the acid halide is acid chloride.

13. The method of claim 6 in which the car-boxylic acid is trifluoroacetic acid.

14. The compound 3-(N-tosyl-L-alaninyloxy)-5-phenyl thiophene.

15. The compound 3-hydroxy-5-phenylthiophene acetate.