CA1055954A - Halogenated sulphophthaleins - Google Patents

Abstract

ABSTRACT

Octahalosulphophthaleins are provided of formula -

Description

The present invention is concerned with novel indicators for u~e in diagnoqtic agent~, for the detection of protein in body fluids.

This application is a division of Canadian Patent Application S.N. 248,333 filed March 12, 1976.

The detection of protein in body fluids, especially in urine, i8 of outstanding importance for the diagnosis of kidney diseases, and rapid diagnostic agents for the detection and determination of protein in urine have been developed.
These diagnostic agents are usually test papers which have been impregnated with a buffer substance and with a so-called protein error indicator. Protein error indicatorq are pH indicators, the pK value of which i9 displaced in the presence of protein. Depending upon the direction in which the pK value is displaced by protein, the buffer present mu~t give a pH value which lies above or below the pK value and preferably just outside the colour change region of the indicator.
Those indicator~ are preferred which, upon dipping into a protein-free urine, are present in a lightly coloured form 90 that the presence of protein leads to a more or less complete change-over of the indicator into the more strongly coloured form and thus gives a sensitive colour change.
The best known of these protein error indicators are tetrabro phenol-phthalein ethyl ester and tetrabromo-phenol blue (octabromophenolsulphophthalein). A whole series of such protein test papers are described in the literature, most of which only differ in the use of additional materials, such as anionic wetting agents, coloured materials, inorganic sulphates and the like and which, in general, permit a sensitive protein detection.

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` lOSS9S4 However, all the known test papers suffer from the serious disadvantage that they react with the metabolites of pharmaceuticalq which frequently occur in the urine, in the same way as with protein quch metabolites, for example, include quinine, quinidine, chloroquine and other nitrogen-containing compounds.
The present invention provides novel octahalosulpho-phthaleins useful as indicatorq in diagnostic agents for detect-ing the presence of proteins in body fluids.
According to the.invention there is provided octahalo-sulphophthaleinq of formula -x2 X3 xl~cJ~x4 X
X

and the enol tautomer thereof wherein X is bromine or chlorine and each of Xl, X2, X3 and X4 is a halogen atom selected from the group consisting of b~omine and chlorine, provided at least one of Xl, X2, X3 and X4 is different from X and provided that when X is chlorine at least one of Xl, X2, X3 and X is chlorine.
According to a still further aspect.of the invention there is provided a process for the prep æ ation of a novel octahalosulphophthalein, as defined above, wherein an appropriate tetrahalobenzene-sulphocarboxylic anhydride is reacted with phenol or an appropriate 2-halophenol and the resultant phenolsulphophthalein subsequently chlorinated or brominated.

~ I - 2 _ In another a_pect of the invention there is provided in a method for detecting protein in body fluids wherein a Aample of the fluid is contacted with a diagnostic agent con-taining an indicator effective to show a perceptible colour change in the pre~ence of protein, the improvement wherein the indicator is a novel octahalosulphophthalein as defined above.
The novel indicators may be employed in diagnoqtic agents, for example, test papers, in which disturbance due to nitrogen-containing compounds does not occur or is small enough to be neglected, without the detection sensitivity for protein being reduced in comparison with the known test papers.
Such diagno_tic agents for the detection of protein in body fluids may suitably compri_e an ab~orbent carrier impregnated with the novel pH indicator with protein error, a buffer for the indicator and at least one linear or branched chain polypropylene glycol which i9 immiscible with water and which optionally contains lower oxyalkylene groups other than oxypropylene.
It will be understood that each component of the diagnostic agent iq employed in an effective amount having regard to its function in the diagnostic agent. For example, it will be apparent that the indicator should be present-in an amount effective to show a perceptible colour change to the naked eye and that,the buffer should be present in an amount to maintain the pH value constant. Suitable amounts of each component can be readily determined and are illustrated in the specification below, The diagnostic agents may be prepared in a proce-~s com-pri~ing impregnating an absorbent carrier with the indicator, buffer and polypropylene glycol.
The diagno~tic agents may be employed in a method for detecting the presence of a protein, for example, albumin, ~ _ 3 -in body fluids, for example, urine, which comprises contacting a sample of the fluid with the diagnostic agent and evaluating any colour change in the diagnostic agent, as an indication of the absence or presence of protein in the sample.
The absorbent carrier is preferably filter paper but other carriers, for example, fibre fleece, asbestos or the like, can alqo be uqed.
The polypropylene glycols are, in particular, the linear polypropylene glycols, as well as block polymers of propylene oxide and ethylene oxide and also branched chain compound~ in which propylene oxide i9 polymerised on to poly-hydroxy alcohols, for example, trimethylolpropane, glycerol and pentaerythritol, and theqe can be further modified with ethylene oxide. These polypropylene glycols suitably have a molecular weight of from about 500 to about 10,000.
Polypropylene glycols of this type are known and are used technically for a large variety of purposes, for example, .. ..
as lubricants, hydraulic fluids, solvents, raw materials for the production of polyurethanes, wetting agents and the like.
The effect exerted by these polyprow lene glycols in the diagnostic agent was not to have been foreseen and is also most surprising because the water-soluble representatives of this group of compounds, for example, polypropylene glycol with a molecular weight of about 400 or pure polyethylene gly-cols do not act in the desired manner.
It i~ noteworthy that test papers with the desired properties can be produced with the polypropylene glycols only with protein error-indicators of the octahalosulphophthalein group, In the case of other protein error indicators, for example, tetrabromophenolphthalein ethyl ester, test papers are obtained which admittedly do not react with nitrogen bases but in which the reaction with protein is also very considerably weakened.
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lOS5954 U_eful indicators in the diagnostic agents include, for example, octabromophenol-sulpho-phthalein (tetrabromophenol blue), octachlorophenol-sulphophthalein (tetrachlorophenol blue), as well as the mixed halogenated analogues, for example, 3',3"-5',5"-tetrabromophenol-3,4,5,6-tetrachlorosulphophthalein, 3',3",5',5"-tetrachlorophenol-3,4,5,6-tetrabro sulphophthalein and 3',3"-dichloro-5',5"-dibromophenol-3,4,5,6-tetrachloro-_ulphophthalein.
Whereas the first three compounds are known from the literature, the other indicator_ are new and are the subject of thi_ invention. The indicators can be prepared according to - known methodq, for example, by the reaction of the known tetra-halobenzene-sulpho-carboxylic anhydrides with phenol or 2-halo-phenols in the presence of Lewis acids, for example, tin tetrachloride, and chlorination or bromination of the resultant phenol-sulphophthaleins in inert solvents, for example, with chlorine or bromine in glacial acetic acid.
Those indicators are especially preferred which have four chlorine atoms in the 3',3",5',5"-poQition because they are even less disturbed by nitrogen ba-qes than the corresponding -bromo compounds.
Protein test papers need a strong buffer which keeps the pH value constant even when the test papers are dipped into body fluids which possibly have a different pH value so that a change of the indicator clearly depends upon a displace-; ment of the pK value due to protein and not upon a change of the pH value. Generally speaking, in the case of sulpho-phthalein indicators, the buffer is adjusted to a pH value which lies somewhat below the pH change region of the indicator in order that the indicator iQ present completely in the less coloured acidic form. A better sensitivity towards very small concentrations of protein is obtained when the pH value of the ~ _ 5 _ lOS5954 buffer lies in the change region of the indicator. However, the result of that is that, after dipping into urine, a part of the indicator changes and the negative coloration is more difficult to differentiate from a slight protein coloration.
A further unexpected property of the polypropylene glycols when used i~ that they suppress this commencing indicator change without substantially influencing the sensitivity tow æ ds protein.
By the "change region" of the indicator, there is to be understood, in general, the pH region of, in each case, one unit above and below the pK value in pure water. For the protein test papers according to the present invention, pH
values are preferably selected which lie about 1.0 units below up to about 0.5 units above the pK value of the indicators employed. Since these lie in the region of 3.5 to 4.0, the usable pH range extends from about pH 2.5 to about pH 4.5. In the case of lower values, a weakening of the protein reaction generally occurs and in the case of higher values a strengthen-ing of the reaction with the nitrogen bases and with normal urine. The preferred pH value, which depends not only upon the indicators used but also upon the nature of the polypropylene glycol used in the diagnostic agent and upon the other reagents present, is easily determined by simple serial experiments in which the pH value and the amount of the buffer is so varied that the indicator, upon dipping into protein-free urine, still just shows a pure "acid" colour.

As buffers, there can be used all those which, in the specified range, possess a good buffering capacity, for example, mixtures of citric acid, malic acid, tartaric acid and the like with their alkali metal or ammonium salts.
Although some of the polypropylene glycols used in the diagnostic agents possess surface-active properties, it can, ~ _ 6 -nevertheles~, be desirable to add conventional ten3ides for the purpose of better distribution. For this purpose, it is particularly preferred to use wetting agents, especially ethoxylated fatty alcohols and phenols containing 1 to 4 oxyethylene groups. Anionic wetting agents strengthen the reaction with the nitrogen bases, whereas cationic tenside~
bring about a strong falsely positive indicator reaction if they are not used in conjunction with very acidic buffers which inhibit the protein reaction. Therefore, these two cla~se~ of tensides are not suitable.
Swelling materials and thickening agents can also be present, which retard the bleeding out of the reagents from the wettened test paper. However, it might be necessary to test whether these are compatible with the buffer substances used. Thu~, for example, hydroxyethyl- and hydroxy-propyl-cellulose have proved to be useful.
Furthermore, complex forming agents, especially magne~ium sulphate, can be added to the reagents.
The polypropylene glycols as well as the other com-ponents, are conveniently employed in the following amounts,referred to 100 ml. of impregnation solution:
polypropylene glycol: 0.5 to 5 g., preferably 1 to 2 g., buffer 10 to 30 g., prefera~ly 15 to 20 g., indicator 0.02 to 0.2 g., preferably 0.05 to 0.1 g.:
surface-active adjuvant 0.0 to 1.0 g., preferably 0.2 to 0.5 g.
A~ solvents for the components, there can be used mixture~ of water and lower alcoholq in which all the components are coluble. However, the absorbent carrier can also be first impregnated with an aqueous buffer solution and thereafter with a solution of the other components in an organic solvent.

~ - 7 -. .._ The teQt papers obtained can be u~ed as such or can be mounted in known manner on to handle~ or, preferably, can be sealed between synthetic resin films and fine-mesh materials.
The following Examples are given for the purpose of illustrating diagnoQtic agents, the effectiveness with regard to the influence of nitrogen bases being illustrated in that the amount of quinine is given, the coloration of which simulates a content of 5 mg.% albumin (upper limit of normal excretion).
Thus, the greater is this amount, then the less is the test disturbed by the presence of the quinine. The disturbance due to o'ther nitrogen bases, for example, quinidine, chloroquine benzydamine and the like, is of the same order of magnitude as that of quinine.
Exam~le 1:
Filter paper (SchIeicher & Sch~ll 2316)* is success-ively impregnated with the following two solutions and, after each impregnation, dried at 60C.:
Solution 1:
Citric acid monohydrate 20 g.
Ammonia, 25% aqueous solutionabout 10 ml.
Distilled water ad 100 ml.
The solution is adju~ted to a pH value of 4.1.
Solution 2: -3',3",5',5"-tetrachlorophenol-3,4,5,6-tetrabromosulphophthalein (pK = 3.9) 50 mg.

Polypropylene glycol, average molecular weight 1200(Polyglykol P1200) 2 g.
Methanol ad 100 ml.
The test papers give a yellow reaction with normal urine and with albumin-containing urines give green to blue-green coloration of increasing intensity.

Urines with a quinine content of about 100 mg.% give the same green coloration as urines with 5 mg.% aIbumin.
*trademark ~ ~, A test paper with the same composition but without the polypropylene glycol gives a green reaction with normal urine.
The green coloration of 5 mg.% albumin cannot be differentiated with certainty from this negative coloration. Therefore, com-parison is carried out with the reaction of 25 mg.%albumin:
even about 25 mg.% quinine simulates this amount of protein.
In the case of commercially available rapid tests, even 2 - 5 mg.% quinine ~imulates the presence of 5 mg.%
albumin.
Exam~le 2:
Filter paper (Schleicher & Sch~ll 2316)* is success-ively impregnated with the following two solutions and dried at 60C.:
Solution 1:
Citric acid monohydrate 20 g.
Ammonia, 25% aqueous solutionabout 6 ml.
Distilled water ad 100 ml.
The solution is adjusted to a pH value of 3.1.
Solution 2:

3',3",5',5",3,4,5,6-octabromophenol-sulphophthalein (tetrabromophenol blue) 50 mg.
(pK = 3.6) Polypropylene glycoll average molecular weight 2000 (Polyglyk~l P 2000)1 g.

Nonyl-phenol, etherified with 1 - 2 oxyethylene radicals (Antarox C0 210)* 0.4 g.
Methanol ad 100 ml.
These two solutions can also be made up with half the amounts of solvent and combined before impregnation.
The te~t paper gives a yellow reaction with normal urine and with albumin-containing urines gives green colorations -of increasing intensity. Urines with a quinine content of about 50 mg.% give the same greenish colorations as urines with 5 mg.% albumin.

*trademark - 9 -lOS5954 A teqt paper with the same composition but without the polypropylene glycol gives a pale greenish reaction with normal urine.
About 10 mg.% quinine simulate, in the case of this test paper, 5 mg.% albumin.
If, instead of the nonyl phenol etherified with 1 - 2 oxyethylene radicals (Antarox C0 210)*, there is used 0.4 g. coconut alcohol etherified with 2 oxyethylene radicals (Genapol C 020)* or 0.2 g. tributyl phenol etherified with 104 oxyethylene radicals (Sapogenate T 040)*, then practically identical test papers are obtained.
Examl~le_3:
Filter paper (Schleicher & Sch~ll 2316)* is first impregnated with a 15% aqueous solution of sodium dihydrogen citrate (pH 3.5) and dried at 60C. It is then impregnated with one of the following solutions and dried at 60C.:

a) 3',3",5',5",3,4,5,6-octachlorophenol-sulphophthalein 50 n~.
Propylene glycol (~ee following Table)1 g.
20Methanol ad 100 ml.
The propertie~3 of these test papers correspond sub-stantially to those of Example 1.

b) 3',3"-dibromo 5',5"-dichlorophenol-3,4,5,6-tetrachlorosulphophthalein50 mg.
Desphen 7200* 1 g.
Methanol ad 100 ml.
The properties of these test papers correspond sub-stantially to those of Example 2.

c) 3',3",5',5"-tetrabromophenol-3,4,5,6-tetrachiorosulphophthalein 50 mg.

Desmophen 7200* 1 g.
Methanol ad 100 ml.
The properties of these test papers correspond sub-stantially to those of Example 2.
*trademark '~,, - 10 _ ~,.

~055954 TABLE

Commercial I Chemical composition according average hydroxy~
name to the manufacturer mol.wt. number (Trademark) . . _ Polyglykol linear polypropylene glycol4000 Desmophen branched polypropylene glycol3800 about 4 7200 modified with ethylene oxide Desmophen - branched polypropylene glycol 3100 about 4 7100 modified with ethylene oxide Des phen partially branched polypro- 3500 about 4 3800 pylene glycol Des phen - branched polypropylene glycol 3000 about 56 3400 modified with ethylene oxide Pluracol branched polypropylene glycol 6300 about 27 TPE 6542 based on trimethylolpropane modified with ethylene oxide Pluracol branched polypropylene glycol 2600 about 6 TP 2540 based on trimethylolpropane Pluracol branched polypropylene glycol 3800 about 29 MK 73 based on glycerol Pluracol branched polypropylene glycol 4500 about 3 MK 92 based on trimethylolpropane Pluronic linear polypropylene glycol 3800 L 101 modified with ethylene oxide up to an amount of 10% I , ExamDle 4:
Filter paper (~chleicher & Sch~ll 2316)* is success-ively impregnated with the following two solutions and dried at 60C.:
Solution 1:
Malic acid 15 g.
6N aqueous sodium hydroxide solution about 16 ml.
Hydroxyethylcellulose (Natrosol250 G)* 2 g.
Distilled water ad 100 ml.
The solution is adjusted to a pH value of 3.5.

*trademark . . "., Solution 2:

Tetrabromophenol blue 0.6 g.
Polyglycol P 1200* 3 g.
Chloroform ad 100 ml.
The properties of this test paper correspond sub-stantially to those of Example 2.
Example 5:
3',3"-Dichlorophenol-3,4,5,6-tetrachlorosulPhophthalein.
25.7 g. (0.2 mol) o-chlorophenol are mixed with 45 g.
(0.14 mol) tetrachloro-o-sulphobenzoic anhydride, 9 ml. (20.4 g.) tin tetrachloride are added thereto and the reaction mixture is heated for 12 hours, while stirring, on an oil bath at 120 - 130C. Thereafter, excess chlorophenol is removed with steam and the residue is purified by repeatedly dissolving in 4N aqueous sodium carbonate solution and precipitating with hydrochloric acid and finally recrystallized from glacial acetic acid. There are obtained 5.3 g. (47YO of theory) pink coloured 3',3"-dichlorophenol-3,4,5,6-tetrachlorosulphophthalein which contains 1 mol acetic acid of crystallisation; m.p. 244 -245C. (molecular weight: ClgH8C1605S.C2H402 = 621.13).
In the same manner but with use of o-bromophenol instead of o-chlorophenol, there is obtained 3',3"-dibromo-phenol-3,4,5,6-tetrachlorosulphophthalein which, after recrystallisation from glacial acetic acid, also contains 1 mol acetic acid of crystallisation, m.p. 172 - 173C.
Example 6:
3',3"-Dibromophenol-3,4,5,6-tetrachlorosulphophthalein.
4.9 g. (0.01 mol) phenol-3,4,5,6-tetrachlorosulpho-phthalein are dissolved in 50 ml. glacial acetic acid and a solution of 1.1 ml. (3.37 g.) bromine (O.04 g. atom) in 50 ml. glacial acetic acid added thereto dropwise at 20C., *trademark while stirring. Stirring i~ thereafter continued for 3 hours.
The crystals formed are filtered off with suction and recrystallised from glacial acetic acid. There are obtained 3.9 g. (55% of theory) 3',3"-dibro phenol-3,4,5,6-tetra-chlorosulphophthalein; m.p. 173 - 174C. The compound contains 1 mol acetic acid of crystallisation (molecular weight:

ClgH8Br2cl40ss ~C2H402 Example 7:

3',3"-Dibromo-5',5"-dichlorophenol-3,4,5,6-tetrachloro-sulphoPhthalein.
3.55 g. (0.005 1) 3',3"-dibromophenol-3,4,5,6-tetrachlorosulphophthalein are suspended in 50 ml. glacial acetic acid. To this suspension is slowly added, while stirring, a solution of 0.94 g. (0.025 g. atom) chlorine in 50 ml. glacial acetic acid. After stirring for several hours, there are obtained 3.8 g. (90.5% of theory) of colour-less crystals of 3',3"-dibromo-5',5"-dichlorophenol-3,4,5,6-tetrachlorosulphophthalein; m.p. 265 - 268C. The compound crystallises with 2 mol acetic acid of crystallisation (molecular weight: ClgH6Br2C1605S.2 C2H402 = 839.01).
The same compound can also be prepared by the bromination of 3',3"-dichlorophenol-3,4,5,6-tetrachloro-sulphophthalein (obtainable by the chlorination of phenol-3,4,5,6-tetrachlorosu-lphophthalein). The yield is 6~ of theory.
Example 8:
~ 3',3",5',5"-Tetrachlorophenol-3,4,5,6-tetrabromosulPho-_ . .
phthalein.
13.8 g. (0.02 mol) phenol-3,4,5,6-tetrabro sulpho-phthalein are suspended in 100 ml. glacial acetic acid and, while stirring, a solution of 3.6 g. chlorine (about 0.1 g.

atom) in 30 ml. glacial acetic acid is added thereto dropwise ~ - 13 _ at anibient temperature. The reaction mixture is thereafter stirred for several hours and the beige-coloured crystals formed are filtered off with suction. After crystallisation thereof from glacial acetic acid/water (9:1), there are obtained 11 g. (58.3% of theory) 3',3",5',5"-tetrachlorophenol-3,4,5,6-tetrabromosulphophthalein in the form of colourless crystals, m.p. 203 - 204C. (decomp.). The compound crystallises with 2 mole acetic acid of crystallisation and 1 mole water of crystallisation (molecular weight:
ClgH6Br4C1405S . 2 CH3COOH . H2O = 945.9).
In an analogous manner, from phenol-3,4,5,6-tetra-chlorosulphophthalein there is obtained, by chlorination in glacial acetic acid, 3',3",5',5"-tetrachlorophenol-3,4,5,6-tetrachlorosulphophthalein; m.p. 277 - 278C. The compound crystallises with 1 mole acetic acid of crystallisation (molecular weight: ClgH6C1805S.C2H4O2 = 690).

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Claims (7)

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

1. Octahalosulphophthaleins of formula- and the enol tautomer thereof wherein X is bromine or chlorine and each of X1, X2, X3 and X4 is a halogen atom selected from the group consisting of bromine and chlorine. provided at least one of X , X , X3 and X is different from X and provided that when X is chlorine at least one of X1, X2, X3 and X4 is chlorine.

2. 3',3",5',5"-Tetrachlorophenol-3,4,5,6-tetrabromo-sulphophthalein according to claim 1.

3. 3',3"-Dibromo-5',5"-dichlorophenol-3,4,5,6-tetra chlorosulphophthalein according to claim 1.

4. A process for the preparation of a octahalosulpho-phthalein as defined in claim 1 wherein an appropriate tetra-halobenzenesulphocarboxylic anhydride is reacted with phenol or an appropriate 2-halophenol and the resultant phenolsulpho-phthalein subsequently chlorinated or brominated.

5. In a method for detecting the presence of protein in body fluids wherein a sample of the fluid is contacted with a diagnostic agent containing an indicator effective to show a perceptible colour change in the presence of protein, the improvement wherein said indicator is an octahalosulpho-phthalein of formula- and the enol tautomer thereof wherein X is bromine or chlorine and each of X1, X2, X3 and X4 is a halogen atom selected from the group consisting of bromine and chlorine, provided at least one of X1, X2, X3 and X4 is different from X and provided that when X is chlorine at least one of X1, X2, X3 and X4 is chlorine.

6. The improvement of claim 5 wherein said indicator is 3', 3", 5', 5"-tetrachlorophenol-3,4,5,6-tetrabromosulpho-phthalein.

7. The improvement of claim 5 wherein said indicator is 3', 3"-dibromo-5',5"-dichlorophenol-3,4,5,6-tetrachloro-sulphophthalein.