WO2001012727A1

WO2001012727A1 - Detection of polyhydroxyl compounds

Info

Publication number: WO2001012727A1
Application number: PCT/GB2000/002685
Authority: WO
Inventors: Prakash Patel; Christopher James Ward; Tony David James
Original assignee: Avecia Limited
Priority date: 1999-08-13
Filing date: 2000-07-13
Publication date: 2001-02-22
Also published as: JP2003507390A; EP1210393A1; GB9919129D0; AU6168200A

Abstract

A compound of Formula (1): X-[-(CnH2n)n-NH-(CmH2m)-W-B(OH)2]p and salts thereof wherein: X is a chromophore or a fluorophore; W is an optionally substituted aromatic ring; n is 0, 1 or 2; m is 1 or 2; and p is 1 or 2. Also the use of compounds of Formula (1) in a method for detecting the presence and/or amount of polyhydroxyl compounds and a kit for detecting the presence and/or amount of polyhydroxyl compounds.

Description

DETECTION OF POLYHYDROXYL COMPOUNDS

The present invention relates to novel compounds, to a method for detecting the presence and/or amount of polyhydroxyl compounds and to a kit for detecting the presence and/or amount of polyhydroxyl compounds. .

Compounds containing multiple hydroxyl groups such as polyols, saccharides and sugars are widespread. They play important roles in living organisms in information transmission, energy metabolism and structure formation. For example, glucose is crucial as an energy source for a variety of cells. Glucose is stored in the liver as glycogen, which is released in body fluids as needed. The production and the consumption of glucose are finely balanced and controlled in normal and healthy body fluids and deviation from a known concentration range can be an indication of disease. Thus, the detection of glucose in the blood or the urine can provide valuable information for the diagnosis of such diseases as diabetes and adrenal insufficiency. Polyhydroxyl compound detection is also important in many other areas for example to monitor the status of fermentation processes. Glucose-sensors are commonly based on an enzyme such as glucose oxidase.

The glucose oxidase method is based on oxidizing glucose with oxygen and measuring the amount of resultant hydrogen peroxide produced by an appropriate means (such as by an electrode). These methods suffer since enzymes can irreversibly change in quality with time and cannot easily be recycled for repeated use. In addition the sample often cannot be provided for other measurement as the glucose or other substrate has been modified in some way during analysis.

Thus, other approaches for determining glucose have been developed such as linking a reporter chromophore to an aryl boronic acid which has a high affinity for vicinal hydroxyl groups. On reaction of aryl boronic acid with the hydroxyl groups the spectral characteristics of the reporter chromophore are altered. These changes in spectral characteristics are typically small bathochromic or hypsochromic shifts. Some larger changes in spectral characteristics have been seen with aryl boronic acid bound to a fluorescent reporter chromophore, reported in US 5,503,770, where it was shown that the presence of a tertiary amine in the vicinity of the boronic group led to a large increase in the flourescence of the compound on binding to a saccharide.

The present invention provides novel compounds in which a secondary amine is provided in the vicinity of the boronic acid group. These compounds display an unexpectedly large spectral shift on reaction with hydroxyl groups. This makes them particularly suitable for use in the detection of polyhydroxyl compounds. Thus, according to a first aspect of the present invention, there is provided a compound of Formula (1 ) and salts thereof: X-[-(C_nH_2n)-NH-(C_mH_2m)-W-B(OH)₂]_p

Formula (1 ) wherein:

X is a chromophore or a fluorophore;

W is an optionally substituted aromatic ring; n is 0, 1 or 2; m is 1 or 2; and p is 1 or 2.

Preferred chromophores X comprises an optionally substituted azo, disazo, anthraquinone, pyrroline, phthalocyanine, polymethine, aryl-carbonium, triphenodioxazine, diarylmethane, triarylmethane, anthraquinone, phthalocyanine, methine, polymethine, indoaniline, indophenol, stilbene, squarilium, aminoketone, xanthene, fluorone, acridene, quinolene, thiazole, azine, nigrosine, oxazine, thiazine, indigoid quinonioid, quinacridone, lactone, pyrroline, benzodifuranone, or indolene group or a combination of such groups.

Preferred fluorophores represented by X are optionally substituted naphthyl, anthryl, pyrenyl, stilbene and phenanthryl or a combination of such groups. Preferably X is such that when the compound of Formula (1 ) is dissolved in a solvent the resultant solution is red, orange, yellow, green, blue, indigo or violet. It is preferred that the compound is of Formula (2) or a salt thereof:

A-N=N-D-(C_nH_2n)-NH-(C_mH_2m)-E-B(OH)₂

Formula (2) wherein:

A, D and E are each independently an optionally substituted aromatic heterocyclic or homocyclic group and m and n are as hereinbefore defined. In Formulae (1 ) and (2) it is preferred that n is 0 and m is 1

Preferred optionally substituted aromatic heterocyclic groups are diazine, thiazole, benzthiazole, benzdiazine, triazole, isoxazole, benzisoxazole, thiadiazole, oxadiazole, isothiazole, benzisothiazole, pyridiazine, triazine, oxazole, thiophene, benzoxazole, pyrimidine or pyridine. Preferred optionally substituted homocyclic groups are optionally substituted phenyl, naphthyl, pyrenyl, stilbenyl.

Preferably A is optionally substituted phenyl and D and E are each independently optionally substituted phenylene.

Preferably the optional substituents which may be present on X, A, D, E and W are each independently selected from alkyl (preferably C^-alkyl), alkoxy (preferably C,^- alkoxy), aryl (preferably phenyl), aryloxy (preferably phenoxy), polyalkylene oxide (preferably polyethylene oxide or polypropylene oxide), nitro, cyano, sulpho, halo, ureido, SO₂F, hydroxy, carboxy, ester; -NR¹R², -COR¹, -CONR-R², -NHCOR¹, phosphato, sulphato, carboxyester, sulphone, and -SO₂NR¹R² wherein R¹ and R² are each independently H or alkyl (especially C_1-4-alkyl). It is more preferred that the optional substituents on X, A, D and E are selected from methyl, ethyl, propyl, butyl, phenyl, methoxy, ethoxy, butoxy, nitro, phenoxy, carboxy, phosphato, sulpho, sulphato, cyano, carboxyester, sulphone, sulphonamide, ureido, SO₂ NR¹R² and NHCOR groups.

In view of the preferences expressed hereinbefore it is especially preferred that the compound is of Formula (3) or a salt thereof:

Formula (3) wherein: Z is NO₂, CO₂H, OCH₃ or SO₃H; and

R³ is H, C^-alkyl, OR⁴, NHCONH₂, NHCOR⁴, wherein R⁴ is H or alkyl. Preferably Z is para with respect to the azo (-N=N-) group. Preferred salts of the compounds described herein are alkali metal salts, especially lithium, sodium and potassium salts, ammonium and substituted ammonium salts and mixtures of the foregoing salts.

The compounds described herein may exist in tautomeric forms other than those shown in this specification. These tautomers are included within the scope of the present claims.

An advantage many of the compounds described in Formula (3) have over the tertiary amine compounds described in US 5, 503,770 is their ease of preparation. The compounds in Formula (3) may be prepared by a simple synthetic route in which an optionally substituted 2-formylarylboronic acid is reacted with an aniline (e.g. m-toluidine) to form the respective imine. The imine is reduced to the corresponding secondary amine using a sodium borohydride reduction and the resultant compound coupled with the diazonium salt of an optionally substituted aniline to give the desired compound of the invention.

According to a second aspect of the present invention there is provided a method for detecting the presence and/or amount of a polyhydroxyl compound in a test sample which comprises contacting the test sample with a compound according to the first aspect of the present invention and evaluating any change in the spectral characteristics of the compound. In the method of the present invention the change in the spectral characteristics of the compound is preferably evaluated visually or, more preferably, using a spectrophotometer, fluorimeter or similar device. On binding to a polyhydroxyl compound in the test sample the spectral properties of the compounds change, typically in terms of a change in fluorescent properties and/or a bathochromic or hypsochromic shift. There may also be an associated change in the intensity of light absorption.

As the compounds are ionisable their spectral properties can vary with the pH or their environment. Therefore the method is preferably performed at a constant pH preferably in the presence of a pH buffer. Suitable pH buffers are well known in the art, particularly including phosphate, pyrophosphate, acetate, carbonate and citrate and mixed buffers such as citrate/phosphate.

The polyhydroxyl compound which may be present in the test sample may be a single compound or a mixture of compounds. Preferred polyhydroxyl compounds contain at least 2 or more hydroxyl groups. Preferably at least two of the hydroxyl groups are vicinal hydroxyl groups. Examples of such polyhydroxyl compounds include saccharides, especially monosaccharides and disaccharides such as glucose, fructose, sucrose, mannose, arabinose, xylose, ribose, galactose, sorbitol.

The test sample preferably comprises or is derived from a human or animal body fluid, a plant extract or a microbial fermentation product, more preferably blood or urine. Usually such samples have been treated to remove coloured substances which could otherwise interfere with the method, for example blood may be centrifuged to remove red blood cells and urine may be decolorised with activated charcoal or some other substance which does not affect the quantity of polyhydroxyl compounds in the test sample.

The compound according to the invention used in the method is preferably in solution or attached to a water-insoluble carrier, preferably a cellulosic, glass or polymeric water-insoluble carrier. The carrier may be in any suitable form, for example as a flat support or beads.

The method of this second aspect of the invention preferably comprises the steps of: a) contacting a test sample with a compound according to the first aspect of the present invention, wherein the compound is in solution or attached to a water- insoluble carrier; b) evaluating the spectral characteristics of the compound when in contact with the test sample according to step a); c) evaluating the extent of any change in the spectral characteristics determined in step b) compared to the spectral characteristics of the compound when the compound is not in contact with the test sample.

The method may be performed using an automated polyhydroxyl compound detection system, for example where the compound and test sample are automatically introduced into a spectrophotometer or fluorimeter for evaluation of the spectral characteristics of the compound after and optionally before contact with the test sample. The amount of a polyhydroxyl compound may be measured by comparison with a series of predetermined standards or a calibration curve prepared by contacting the compound with a series of known concentrations of the polyhydroxyl compounds.

The spectral characteristics of a compound when the compound is not in contact may be evaluated as part of the method normally before step a) or the spectral characteristics may already be known to the user e.g. from a colour chart or the general literature or a manual resulting in no need for this initial spectral characteristic to be measured as part of the method.

An advantage of the method is that by using the compounds of the first aspect of the invention the bathochromic or hypsochromic shift observed on reaction with a polyhydroxyl compound is so large that the colour change may be clearly visible to the eye. Thus the reaction of these compounds provides the basis of a rapid colorimetric test for polyhydroxyl compounds by visual comparison with a colour chart, in a similar manner to how pH is determined using litmus paper by comparison with a colour chart. This can be a particular advantage in third world countries and in remote regions where there is no access to spectrometers or fiuorimeters. The visual evaluation is not as valuable or accurate as the use of electronic equipment, but it is better than no evaluation at all and may avoid considerable suffering of patients.

According to a fourth aspect of the invention there is provided a kit for the detecting the presence and/or amount of a polyhydroxyl compound which comprises: a) a compound according to the first aspect of the present invention, preferably in one or more pre-weighed portions or attached to a water-insoluble carrier; b) instructions for determining the presence and/or amount of a polyhydroxyl compound using the compound; and c) optionally a pH buffer.

Preferably the instructions for determining the presence and/or amount of a polyhydroxyl compound using component a) comprise the method according to the second aspect of the present invention. The invention is further illustrated by the following Examples.

Example 1

Preparation of the compound (1 )

(1 ) Step 1 - Preparation of compound (A)

(A) m-Toluidine (0.72 g, 6.67 mmol) and 2-formylbenzeneboronic acid (1.00 g, 6.67 mmol) were mixed in 50 ml of toluene. A Dean and Stark trap was fitted to permit the azeotropic removal of water, and the reaction mixture heated under reflux overnight (18 hours). After cooling, the solvent was removed in vacuo to give compound (A) (1.35 g, 92%) as a cream coloured solid: mpt 170-172°C.

Step 2 - Preparation of Compound (B)

(B) Sodium borohydride (0.79 g, 20.9 mmol, 5 equivalent) was added slowly to the product of step 1 (1.00 g, 4.18 mmol) in dry methanol (50 ml). The mixture was left to stir at room temperature for 2 hours and then poured into ice-water (50 ml). Hydrochloric acid (1 M, 10 ml) was added slowly and the mixture left to stir for 30 minutes, whereupon the precipitate formed was collected by suction filtration to give compound (B) (0.82 g, 81%) as a white solid: mpt 72-73°C (decomposition).

Step 3 4-nitroaniline (0.10 g, 0.70 mmol) was mixed in water (10 ml), methanol (10 ml) and hydrochloric acid (10 ml, 5.0 M) and then cooled to 0-5°C in an ice-bath. A chilled solution of sodium nitrite (0.12 g, 1.74 mmol, 0.2 M) was added dropwise. Excess nitrite was destroyed by the addition of sulphamic acid.

The product of step 2 (ie compound (B)) (0.16 g, 0.66 mmol) was dissolved in methanol (10 ml) and dilute hydrochloric acid (1 ml, 1 M) then added dropwise to the reaction mixture, which quickly turned a red colour. Sodium acetate was added to raise the pH of the solution to 4 and this was then left to stir at 0-5°C for 3 hours. Sodium hydroxide (2 M) was slowly added to raise the pH to 7. The resultant precipitate was collected by suction filtration and dried in a dessicator overnight to give the title compound (1 ) (0.19 g, 74%) as a dark red solid; mp 120-122°C (decomposition); λ_max(0.05 M NaCI, 33% MeOH: 67% H₂O w/w)/nm 486 (ε/dm³ mol^" cm^"1 19000, pH 8.I55), 547 (21000, pH 11.51).

Example 2

Preparation of compound (2)

(2) The method of Example 1 was repeated except that in Step 3, 3-aminobenzoic acid (0.06 g, 0.41 mmol) was used in place of 4-nitroaniline (all other reagents were used in the same ratios), to give compound (2) (0.10 g, 61 %) as a red solid (Found: C, 64.9; H, 5.2;

N, 10.9. C₂₁H₂₀BN₃O₄ requires C, 64.8; H, 5.2; N 10.8%); mp 133-135°C (decomposition); λ_max(0.05 M NaCI, 33% MeOH: 67% H₂O w/w)/nm 406 (ε/dm³ mol^"1 cm^"1 19000, pH 8.48), 457 (18200, pH 11.50).

Example 3

Preparation of compound (3)

(3) 4-Aminobenzoic acid (0.11 g, 0.83 mmol) was mixed in water (10 ml) and hydrochloric acid (10 ml, 10 M) and then cooled to 0-5°C on an ice bath. A chilled solution of sodium nitrite (0.29 g, 4.15 mmol, 0.4 M) was added slowly and the excess nitrite was destroyed with sulphamic acid. The product of Example 1 , Step 2 (0.20 g, 0.83 mmol) was mixed in dilute hydrochloric acid (10 ml, 2 M) then added dropwise to the reaction mixture, which quickly turned a red colour. Sodium acetate was added to raise the pH of the solution to 4 and this was then left to stir at 0-5°C for 3 hours. Sodium hydroxide (2 M) was slowly added to raise the pH to 7. The resultant precipitate was collected by suction filtration, washed with water and air-dried. The solid was purified by dissolving it in methanol (20 ml) and filtering off the insolubles, before preciptiation using an excess of water. The precipitate was collected by suction filtration to give compound (3) (0.10 g, 31%) as an orangered solid (Found: C, 64.8; H, 5.0; N, 10.7. C₂₁H₂₀BN₃O₄ requires C, 64.8; H, 5.2; N 10.8%); mp 208-210°C (decomposition); λ_max(0.05 M NaCI, 33% MeOH: 67% H₂O w/w)/nm 418 (ε/dm³ mor¹ cm^'1 16000, pH 8.42), 469 (16100, pH 11.50).

Example 4

Preparation of compound (4)

(4) The method of Example 3 was repeated except that p-anisidine (0.61 g, 4.98 mmol, 6 equivalents) was used in place of 4-aminobenzoic acid. Purification was performed by washing the product (in dichloromethane) with 10% sodium hydrogen carbonate solution (w/w) to remove residual acetic acid, to give compound (4) (0.11 g, 35%) as an orange- brown solid: mp 102°C (decomposition); λ_max(0.05 M NaCI, 33% MeOH: 67% H₂O w/w)/nm 400 (ε/dm³ mol^"1 cm^'1 14600, pH 8.42), 453 (13900, pH 11.47).

Example 5

Preparation of compound (5)

(5)

The method of Example 1 was repeated with sulphanilic acid (0.08 g, 0.44 mmol) in place of 4-nitroaniline (all other reagents were used in the same ratios) to give compound (5) (0.05 g, 27%) as a dark red solid: mp 219°C (decomposition); λ_max(0.05 M NaCI, 33% MeOH: 67% H₂O w/w)/nm 421 (ε/dm³ mol^'1 cm^"1 19300, pH 8.18), 476 (19800, pH 11.52). Example 6

Preparation of the compound (6)

(6)

Step 1 - Preparation of compound (A) 0

(A)

Aniline (0.15 g, 1.63 mmol) and 2-formylbenzeneboronic acid (0.24 g, 1.63 mmol) were 5 mixed in absolute ethanol (40 ml) and anhydrous benzene (4 ml). A Dean-Stark trap was fitted to permit the azeotropic removal of water and the reaction mixture heated under reflux for 7 hours. After cooling, the solvent was removed in vacuo to yield a cream coloured solid. The product (A) was used directly in the next step without further purification or analysis. 0

Step 2 - Preparation of Compound (B)

5 (B)

Product (A) from step 1 was mixed in methanol (45 ml) and sodium borohydride (0.12 g, 3.26 mmol, 2 equivalents.) was added slowly to the solution. The mixture was stirred at room temperature for 2 hours and then poured on to concentrated hydrochloric acid (5 ml) on ice. The solvent was removed and the solid product was washed with chloroform and o then water to yield an off-white solid, (B) (0.33 g, 84 %).

Step 3 Compound (B) from step 2 (0.33 g, 1.2 mmol) was mixed in N,N- dimethylformamide (5 ml) and tetracyanoethylene (0.16 g, 1.2 mmol) was added slowly. The reaction mixture was heated to 55 °C for 1 hour. After cooling, the mixture was poured on to ice-water (10 ml) and the precipitate collected by filtration. The precipitate was recrystallised from acetic acid to yield Compound 6 as a red solid (0.25 g, 0.76 mmol, 63 %), mpt 127-129°C (decomposition); λ_max 520 nm (pH 7.8 0.0026 M phosphate buffer in 33% MeOH: 67% H₂O w/w) ε/dm³ mol^"1 cm^"1, 31296 (19800, pH 11.52).

Absorption titration of compound (1) with D-Glucose at pH 11.3

The UV-visible absorption spectra of compound (1 ) (5.66 x 10^"5 mol dm^"3) in a pH 11.3 buffer (0.01000 mol dm^"3 DKI, 0.002771 mol dm^"3 NaHCO₃, 0.002771 mol dm^"3 Na₂CO₃ in 52.1 % methanol: 47.9% water) was recorded using a spectrophotometer. D- glucose was added in increments of 1.35, 2.41 , 2.93, 3.56, 4.45, 5.74, 7.69, 14.7, 25.6, 90.2 x 10^"3 mol dm^"3 with the spectra being rerecorded after each addition. As the titration proceeded the lambda max shifted from 509nm for compound (1 ) alone to 564 nm for compound (1) saturated with D-glucose. This represents a spectral shift of 55 nm which corresponds to a clear visible colour change from an indigo shade to orange.

Claims

1. A compound of Formula (1 ) and salts thereof:

X-[-(C_nH_2n)_n-NH-(C_mH_2m) -W-B(OH)J_p

Formula (1 )

wherein: X is a chromophore or a fluorophore;

2. A compound according to claim 1 wherein the chromophore represented by X is an optionally substituted azo, disazo, anthraquinone, pyrroline, phthalocyanine, polymethine, aryl-carbonium, triphenodioxazine, diarylmethane, triarylmethane, anthraquinone, phthalocyanine, methine, polymethine, indoaniline, indophenol, stilbene, squarilium, aminoketone, xanthene, fluorone, acridene, quinolene, thiazole, azine, nigrosine, oxazine, thiazine, indigoid quinonioid, quinacridone, lactone, pyrroline, benzodifuranone, or indolene group or a combination of such groups.

3. A compound according to claim 1 wherein the fluorophore represented by X is an optionally substituted naphthyl, anthryl, pyrenyl, stilbene and phenanthryl or a combination of such groups.

4. A compound as claimed in any one of claims 1 to 3 of Formula (2) or a salt thereof: A-N=N-D-(C_nH_2n)-NH-(C_mH_2m)-E-B(OH)₂

Formula (2) wherein:

A, D and E are each independently optionally substituted aromatic heterocyclic or homocyclic groups and m and n are as hereinbefore defined.

5. A compound according to claim 4 wherein A, D and E are each independently optionally substituted phenylene groups.

6. A compound according to claim 4 and claim 5 wherein the substituents on A, D and E are each independently selected from H, alkyl, alkoxy, aryl, aryloxy, heteroaryl, polyalkylene oxide, nitro, cyano, sulpho, halo, ureido, SO₂F, hydroxy, carboxy, ester; -NR R², -COR¹, -CONR¹R², -NHCOR¹, phosphate, sulphate, carboxyester, sulphone, sulphonamide, SO₂ NR¹R², and -SO₂NR¹R² wherein R¹ and R² are each independently H or alkyl.

7. A compound according to any one of the preceding claims of Formula (3) or a salt thereof:

Formula (3)

wherein: Z is NO₂, CO₂H, OCH₃ or SO₃H; and

R³ is H or C^-alkyl, OR⁴, NHCONH₂, NHCOR⁴, wherein R⁴ is H or alkyl.

8. A method for detecting the presence and/or amount of a polyhydroxyl compound in a test sample which comprises contacting the test sample with a compound according to any one of claims 1 to 7 and evaluating any change in the spectral characteristics of the compound.

9. A method according to claim 8 which is performed at a constant pH in the presence of a pH buffer.

10. A method according to claim 8 or 9 wherein the change in spectral characteristics is evaluated visually or by using a spectrophotometer or fluorimeter.

11. A method according to any one of claims in claim 8 to 10 wherein the test sample comprises or is derived from a human or animal body fluid, a plant extract or microbial fermentation product.

12. A method according to any one of claims 8 to 11 wherein the compound is bound to a water-insoluble carrier.

13. A method according to any one of claims 8 to 12 wherein the polyhydroxyl compound is a monosacharride or disaccharide.

14. A method according to any one of claims 8 to 13 which comprises the steps of : a) contacting a test sample with a compound according to any one of claims 1 to 7, wherein the compound is in solution or attached to a water-insoluble carrier; b) evaluating the spectral characteristics of the compound when in contact with the test sample according to step a); c) evaluating the extent of any change in the spectral characteristics determined in step b) compared to the spectral characteristics of the compound when the compound is not in contact with the test sample.

15. A kit for the detecting the presence and/or amount of a polyhydroxyl compound which comprises: a) a compound according to any one of claims 1 to 7; b) instructions for determining the presence and/or amount of a polyhydroxyl compound using the compound; and c) optionally a pH buffer.

16. A kit according to claim 15 wherein the instructions for determining the presence and/or amount of a polyhydroxyl compound using component a) comprise the method according to any one of claims 8 to 14.