WO1999050438A2 - Chromogenic indole derivatives - Google Patents

Chromogenic indole derivatives Download PDF

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Publication number
WO1999050438A2
WO1999050438A2 PCT/EP1999/002174 EP9902174W WO9950438A2 WO 1999050438 A2 WO1999050438 A2 WO 1999050438A2 EP 9902174 W EP9902174 W EP 9902174W WO 9950438 A2 WO9950438 A2 WO 9950438A2
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Prior art keywords
cyclodextrin
chromogenic
product
compounds
group
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PCT/EP1999/002174
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French (fr)
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WO1999050438A3 (en
Inventor
Giorgio Zoppetti
Pasqua Oreste
Giovanni Cipolletti
Giancarlo Tamerlani
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Inalco S.P.A.
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Priority to AU36024/99A priority Critical patent/AU3602499A/en
Publication of WO1999050438A2 publication Critical patent/WO1999050438A2/en
Publication of WO1999050438A3 publication Critical patent/WO1999050438A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
    • C07D209/32—Oxygen atoms
    • C07D209/36—Oxygen atoms in position 3, e.g. adrenochrome
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
    • C07F9/572—Five-membered rings
    • C07F9/5728—Five-membered rings condensed with carbocyclic rings or carbocyclic ring systems

Definitions

  • Chromogenic compounds are substances capable to develop a colour in the visible spectrum due to a replacement reaction.
  • Chromogenic compounds are useful especially in the identification of biologic materials as cells and genetically manipulated micro-organisms. Their use in cloning is well known. Chromogenic compounds are also useful in the allocation of a specific DNA or RNA sequence. In this regard specific DNA or RNA sequences can be allocated on chromosomes or other genetic materials using nucleic acid probes. Currently, to identify and allocate nucleic acid probes on chromosomes or other genetic materials a technique preparing nucleic acid probes containing radioactive isotopes is used.
  • Chromogenic substrates are useful when used together with enzyme-antibody conjugates which are important to determine the enzyme tied by immunoabsorbance.
  • microbiology the presence of indicating micro-organisms is often used to determine the quality of various products. For example in water and food analysis the presence of coliforms or Escherichia coli is revealed using chromogenic substrates.
  • the systems that use the described chromogenic compounds are plates for bacteriology filled with aqueous culture media composed by classic ingredients like agar and various organic or salt nutrients suitable to the growth of the microorganism and added with one or more chromogenic compounds.
  • the poor solubility of chromogenic compounds in water is a limit for the preparation and efficiency of the system.
  • Chromogenic compounds in fact used in these systems have a solubility ranging from 0,0001 to 2%.
  • the use requires a concentration ranging from 0,004 to 0,04 for most of these products, but this is obtained operating often at high temperatures (with the risk of the degradation of the product) or with solvent mixtures starting from more concentrated solutions (0,1- 1 %).
  • R is selected from the group consisting of ⁇ -D-Galactose, ⁇ -D-Galactose, ⁇ -D-
  • Glucose ⁇ -D-Glucose, ⁇ -D-Galactosamine, ⁇ -D-Glucosamine, ⁇ -D-Glucuronic acid, ⁇ -D-Glucuronic acid sodium or cyclohexilammonium salt or a capryiate or sulphate or phosphate;
  • Chromogenic compounds according to the present invention can be used in biochemistry in general and particularly in biotechnology, diagnostic chemistry, microbiology and molecular biology.
  • the characteristics and advantages of the chromogenic compounds of the present invention will be further explained in the following detailed description.
  • the chromogenic compounds of the present invention are indole derivatives in form of complexes with cyclodextrins or with cyclodextrin derivatives and optionally in free form.
  • Said compounds are in general indole derivatives with a saccharidic or fatty acid or sulphate or phosphate group and with halogens and particularly said derivatives in their free form have the formula (I).
  • R 1 is selected from the group consisting of ⁇ -D-Galactose, ⁇ -D-Galactose, ⁇ -D- Glucose, ⁇ -D-Glucose, ⁇ -D-Galactosamine, ⁇ -D-Glucosamine, ⁇ -D-Glucuronic acid, ⁇ -D-Glucuronic acid sodium or cyclohexilammonium salt or a capryiate or sulphate or phosphate group;
  • the cyclodextrins used for the complexes are selected from the group comprising alpha, beta and gamma cyclodextrin, the O-dimethylated, O-trimethylated, O- hydroxymethylated and O-hydroxyethylated derivatives of cyclodextrin, the copolymer cyclodextrin-polivinylalcohol and the copolymer cyclodextrin-cellulose.
  • the preparation of the compounds in the complexed form is performed with the following method:
  • the fine powder of the chromogenic compound having a particle size less than 150 ⁇ m, is added with a cyclodextrin or its derivative in powder form or with an amount of water to obtain cyclodextrin concentration between 0.1 and 50%.
  • the chromogenic compound is added with an aqueous solution of clyclodextrin or its derivative having a concentration between 0.1 and 50%.
  • the weight ratio between the cyclodextrin or its derivative and the chromogenic compound is comprised between 2:1 and 500:1.
  • the obtained suspension is warmed up at a temperature comprised between 20 and 80 °C and kept under stirring from 10 minutes to 168 hours and then let cool at room temperature.
  • the solution is subsequently dried by evaporation under vacuum or preferably freeze dried.
  • the obtained complexes have a ratio (w/w) between the cyclodextrin or its derivative and the chromogenic compound comprised between 2:1 and 500:1.
  • the solubility in water of the chromogenic compound in the complexed form is from 2 to 50 times greater than to the solubility of the chromogenic compound itself.
  • complexes prepared in this way allow the use of the chromogenic compounds usually insoluble in water like the capryiate derivatives without the aid of solvents and a higher availability in the solution of the chromogenic compounds partially soluble in water like the derivatives with galactose, glucose, glucuronic acid, glucosamine or galactosamine with consequently better response on the system of the determination on plate of the presence of a certain micro organism.
  • Complexes and derivatives of the present invention can be used particularly as specific culture media additives for the diagnostics of micro-organisms as E. coli on plate, as described by Watkins et al. Appl. Environ. Microbiol. 54:1874-1875 (1988) and by Ley et al. Can J.
  • the present invention refers also to culture media containing such complexes and derivatives as additives.
  • EXAMPLE 1 Synthesis of the 6-Bromo-3-lndolyl- ⁇ -D-Galactopyranoside chromogenic compound.
  • the iron filings are filtered and the filtered solution is dry-concentrated.
  • the residue is taken back with water (20 ml) and methylene chloride (20 ml) and, under stirring, caustic soda is added to pH 12.
  • caustic soda is added to pH 12.
  • the iron hydroxide precipitates.
  • the dark suspension is filtered on a dicalite panel and washed with methylene chloride (50 ml).
  • the double phase is separated in a separatory funnel and the organic phase is anhydrified on sodium sulphate for 10 minutes. Subsequently the sodium sulphate is filtered, it is washed with 20 ml of methylene chloride and the organic solution is dry-concentrated. 3.7 g of dark fluid oil is obtained. Yield: 79.1 %.
  • the solution is left cooling spontaneously to room temperature.
  • the product crystallizes already at 55 °C. It is cooled to 5 °C and the suspension is left under stirring at this temperature for 15'. It is quickly filtered and washed with 5 ml of acetic acid cooled to 15 °C. A second washing is carried out with 10 ml of water.
  • the solid which weighs 2.5 g after drying in a vacuum stove for an hour at 50 °C, is discharged.
  • the solid is filtered and washed with 5 ml of water. After drying in a vacuum stove at 50 °C, the anhydrous product weighs 0.86 g.
  • the product in TLC is monospot.
  • the product has been characterized by 1H and 13C NMR.
  • the temperature, during the addition and for an hour after the end of this one, is maintained at 85-90 °C.
  • the suspension is left cooling to room temperature, it is filtered on dicalite and it is warmed with 20 ml of water. The filtered solution is concentrated under vacuum to a final volume equal to 50 ml.
  • the product results insoluble in chloroform.
  • the product has been characterized by 1H and 13C NMR (DMSO solvent).
  • the solution is acidified at pH 6 with concentrated hydrochloric acid.
  • the product which is filtered is crystallized, washed with 5 ml of water and dried in a vacuum stove at 50 °C.
  • the weight of the anhydrous product is 1.8 g.
  • the solution is added under stirring with 0.8 g (8.5 mmol) of chloroacetic acid and, immediately after, with a solution of 0.38 g (6.1 mmol) of potassium carbonate in deionized water (2 ml). After 5' 0.86 g (8.6 mmol) of potassium bicarbonate are added. It is warmed to 40 °C for 66 hours. At the end of the 66 hours the obtained suspension is cooled to 5 °C for 30'. The suspension is filtered and the product is washed with cold water. After drying in a vacuum stove at 50 °C the anhydrous product weighs 1.2 g.
  • bitterns are acidified to pH 6.7 with concentrated hydrochloric acid and cooled at 5 °C for an hour.
  • the precipitated solid is filtered and washed with 1 ml of water. After drying in a vacuum stove at 50 °C, the anhydrous product weighs
  • the product has been characterized by 1 H and 13C NMR spectra.
  • Step 1 Synthesis of 1-(N-Acetyl-6-Br-lndolylV2.3.4 Triacetyl- ⁇ -D-Glucuronic- Methyl-Ester Acid
  • Step 2 Synthesis of 6-Br-3-lndolyl- ⁇ -D-Glucuronide Cvclohexyl ammonium Salt 3 ml of anhydrous methanol, 0.47 g (0.82 mmol) of the product obtained in the step 1 ) are charged in a 25 ml little flask, equipped with a mechanical stirrer, thermometer and a capillary for nitrogen bubbling and nitrogen has been bubbled for 10'. The temperature is lowered to 10 °C and 0.03 ml (0.17 mmol) of a 30% solution of sodium methoxide in methanol are added to the white suspension. The suspension, from white, becomes gradually a yellow solution in an hour.
  • the product has been characterized by 1H and 13C NMR spectra.
  • Step 2) Synthesis of: 1-(5-Bromo-6-Chloro-1-H-lndol-3-vn-2.3.4.6-Tetra-Q-Acetyl- ⁇ -D-Galactopyranoside
  • the solution is filtered and concentrated to syrupy consistence, then 20 ml of ethanol are added and the product is crystallized at room temperature.
  • the suspension is cooled for 1 hour at 5 °C.
  • the product is filtered, washed and dried.
  • the product is characterized by Nuclear Magnetic Resonance to proton and Carbon 13.
  • Step 5 Preparation of 5,6-diBromo-3-lndolyl- ⁇ -D-Galactopyranoside 0.5 g of 5,6-diBromo-3-lndolyl- ⁇ -D-Galactopyranoside pentaacetate have been suspended in 5 ml of absolute ethanol and 0.05 ml of 40% benzyl trimethylammonium hydroxide solution in methanol have been then added.
  • EXAMPLE 9 Synthesis of the 6-Bromo-1-Methyl-lndol-3-yl- ⁇ -D- Galactopyranoside chromogenic compound.
  • EXAMPLE 10 Preparation of the complex with ⁇ -Cyclodextrin of the 6-Bromo-3- Indolyl- ⁇ -D-Galactopyranoside.
  • the obtained solution is then freeze-dried.
  • the obtained anhydrous complex is reconstituted with water in order to obtain the same initial concentration obtaining a clear solution.
  • This solution diluted 2000 times shows an absorbance at 229 nm equal to 1386 mAbs.
  • An enzymatic test with ⁇ -Galactoxidase has been carried out on the solution of the complex. 100 ⁇ l of a ⁇ -Galactoxidase (3000 LAU/ml Sigma) solution have been added to 10 ml of the solution observing a red colour development caused by the formation of the insoluble indigo blue.
  • EXAMPLE 11 Preparation of the complex with ⁇ -Cyclodextrin of the 6-Bromo-3- Indolyl- ⁇ -D-Glucuronide Cyclohexylammonium Salt. 10 mg of 6-Bromo-3-lndolyl- ⁇ -D-G!ucuronide Cyclohexylammonium salt prepared as described in the Example 2 are added with 885 mg of ⁇ -Cyclodextrin and 4 ml of water and treated as described in the Example 10. The obtained solution is freeze-dried. The obtained anhydrous complex is reconstituted with water at the same initial concentration obtaining a clear solution.
  • Galactoxidase has been carried out on the solution of the complex as described in the Example 10 producing red colour.
  • EXAMPLE 16 Preparation of the complex with ⁇ -Cyclodextrin of the 5-Bromo-4- Chloro-3-lndolyl-Glucoside.
  • EXAMPLE 20 Preparation of the complex with ⁇ -Cyclodextrin-O-dimethylated of the 6-Chloro-3-lndolyl-Caprylate. 10 mg of 6-Chloro-3-lndolyl-Caprylate are treated as in the Example 14. The obtained complex diluted 100 times shows an absorbance at 226 nm equal to 315 mAbs. The not complexed compound, treated in the same way but only with water, does not show an absorbance at 226 nm.

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Abstract

The present invention refers to indole derivatives suitable to be used as chromogenic compounds in free form or in complexed form with cyclodextrins or with cyclodextrin derivatives, usable in the diagnostic, microbiological, molecular-biological field and similar ones.

Description

INDOLE DERIVATIVES SUITABLE TO BE USED AS CHROMOGENIC COMPOUNDS STATE OF THE ART
Chromogenic compounds are substances capable to develop a colour in the visible spectrum due to a replacement reaction.
This characteristic makes them useful in the areas of biotechnology, diagnostic chemistry, microbiology, molecular biology and similar. Chromogenic compounds are useful especially in the identification of biologic materials as cells and genetically manipulated micro-organisms. Their use in cloning is well known. Chromogenic compounds are also useful in the allocation of a specific DNA or RNA sequence. In this regard specific DNA or RNA sequences can be allocated on chromosomes or other genetic materials using nucleic acid probes. Currently, to identify and allocate nucleic acid probes on chromosomes or other genetic materials a technique preparing nucleic acid probes containing radioactive isotopes is used.
It is therefore advisable due to environmental pollution reasons, to develop alternative non-isotopic techniques that can allocate and identify nucleic acid probes to eliminate the risk associated with radioactive materials. Chromogenic substrates are useful when used together with enzyme-antibody conjugates which are important to determine the enzyme tied by immunoabsorbance.
In microbiology, the presence of indicating micro-organisms is often used to determine the quality of various products. For example in water and food analysis the presence of coliforms or Escherichia coli is revealed using chromogenic substrates.
Currently various methods exist to determine, identify and count indicating microorganisms, with various degrees of accuracy and easiness. Some methods simply indicate the presence or absence of the micro-organism while others quantify it in the tested material. For instance the compound 5-Bromo-4-Chloro-3-lndolyl-β-D-Glucuronide (X-Gluc) is used to identify E. coli. When this substrate comes into contact with the enzyme β-Glucuronidase produced by E. Coli, X-Gluc forms an indigo precipitate that allows a quantitative determination of E. Coli. X-Gluc and its ability to identify E. Coli are described by Watkins, et al., Appl. Environ. Microbiol. 54: 1874-1875 (1988). A similar compound, Indoxyl-β-D-Glucuronide, that produces as well blue coloured E. Coli colonies, is described by Ley, et al., Can. J. Microbiol. 34: 690- 693 (1987).
The systems that use the described chromogenic compounds are plates for bacteriology filled with aqueous culture media composed by classic ingredients like agar and various organic or salt nutrients suitable to the growth of the microorganism and added with one or more chromogenic compounds. The poor solubility of chromogenic compounds in water is a limit for the preparation and efficiency of the system. Chromogenic compounds in fact used in these systems have a solubility ranging from 0,0001 to 2%. The use requires a concentration ranging from 0,004 to 0,04 for most of these products, but this is obtained operating often at high temperatures (with the risk of the degradation of the product) or with solvent mixtures starting from more concentrated solutions (0,1- 1 %). Therefore only the compounds most soluble in water allow an easy preparation while the least soluble need complex procedures involving solvents. In any case the final result depends on the dissolved amount. Thus any attempt to increase the solubility of these chromogenic compounds leads to an easier use of the poorly soluble compounds with the elimination of solvents and preliminary warming, and to a higher concentration of the compound that causes the development of more intense colour and therefore the whole system becomes more efficient. SUMMARY We have found out that the problems of the state of the art are resolved using chromogenic compounds derived from indole in a form complexed with cyclodextrin or with cyclodextrin derivatives and optionally in their free form. Said compounds are generally indole derivatives with a saccharidic or fatty acid or sulphate or phosphate group and with halogens and particularly said derivatives in their free form have formula (I):
Figure imgf000005_0001
in which:
R, is selected from the group consisting of β-D-Galactose, α-D-Galactose, β-D-
Glucose, α-D-Glucose, β-D-Galactosamine, β-D-Glucosamine, β-D-Glucuronic acid, α-D-Glucuronic acid sodium or cyclohexilammonium salt or a capryiate or sulphate or phosphate;
R2 is selected form the group consisting of H, methyl, ethyl, benzyl, paranitrobenzyl; X = H, Br. CI; Y = H, Cl, Br; W = H, Br, Cl; provided that when W = Br, X is H or Br and Y is H.
Chromogenic compounds according to the present invention can be used in biochemistry in general and particularly in biotechnology, diagnostic chemistry, microbiology and molecular biology. DETAILED DESCRIPTION OF THE INVENTION
The characteristics and advantages of the chromogenic compounds of the present invention will be further explained in the following detailed description. The chromogenic compounds of the present invention are indole derivatives in form of complexes with cyclodextrins or with cyclodextrin derivatives and optionally in free form.
Said compounds are in general indole derivatives with a saccharidic or fatty acid or sulphate or phosphate group and with halogens and particularly said derivatives in their free form have the formula (I).
Figure imgf000005_0002
in which:
R1 is selected from the group consisting of β-D-Galactose, α-D-Galactose, β-D- Glucose, α-D-Glucose, β-D-Galactosamine, β-D-Glucosamine, β-D-Glucuronic acid, α-D-Glucuronic acid sodium or cyclohexilammonium salt or a capryiate or sulphate or phosphate group;
R2 is selected from the group consisting of H, methyl, ethyl, benzyl, paranitrobenzyl; X = H, Br, Cl; Y = H, Cl, Br; W = H, Br, Cl; provided that when W = Br, X is H or Br and Y is H.
The cyclodextrins used for the complexes are selected from the group comprising alpha, beta and gamma cyclodextrin, the O-dimethylated, O-trimethylated, O- hydroxymethylated and O-hydroxyethylated derivatives of cyclodextrin, the copolymer cyclodextrin-polivinylalcohol and the copolymer cyclodextrin-cellulose. The preparation of the compounds in the complexed form is performed with the following method:
The fine powder of the chromogenic compound, having a particle size less than 150 μm, is added with a cyclodextrin or its derivative in powder form or with an amount of water to obtain cyclodextrin concentration between 0.1 and 50%. Alternatively the chromogenic compound is added with an aqueous solution of clyclodextrin or its derivative having a concentration between 0.1 and 50%. The weight ratio between the cyclodextrin or its derivative and the chromogenic compound is comprised between 2:1 and 500:1. The obtained suspension is warmed up at a temperature comprised between 20 and 80 °C and kept under stirring from 10 minutes to 168 hours and then let cool at room temperature. The solution is subsequently dried by evaporation under vacuum or preferably freeze dried. If at the end of the treatment a suspension is obtained, a filtration is performed and the resulting solution is freeze dried or dried under vacuum. The obtained complexes have a ratio (w/w) between the cyclodextrin or its derivative and the chromogenic compound comprised between 2:1 and 500:1. The solubility in water of the chromogenic compound in the complexed form is from 2 to 50 times greater than to the solubility of the chromogenic compound itself.
The complexes prepared in this way allow the use of the chromogenic compounds usually insoluble in water like the capryiate derivatives without the aid of solvents and a higher availability in the solution of the chromogenic compounds partially soluble in water like the derivatives with galactose, glucose, glucuronic acid, glucosamine or galactosamine with consequently better response on the system of the determination on plate of the presence of a certain micro organism. Complexes and derivatives of the present invention can be used particularly as specific culture media additives for the diagnostics of micro-organisms as E. coli on plate, as described by Watkins et al. Appl. Environ. Microbiol. 54:1874-1875 (1988) and by Ley et al. Can J. Microbiol. 34:690-693 (1987), or for the determination of total coliforms, Salmonella spp, Salmonella typhi and paratyphi, Salmonella typhimurium, Enterobacter, Citrobacter, Candida albicans, Enterococci, Klebsiella pneumoniae and others.
Thus the present invention refers also to culture media containing such complexes and derivatives as additives.
Examples of chromogenic compounds useful as complexes in the present invention are shown here below in table 1 , in which the substituents R.,, R2, X, Y and W are referred to formula (I).
TABLE 1
Figure imgf000008_0001
TABLE 1 - continued
Figure imgf000009_0001
TABLE 1 - continued
Figure imgf000010_0001
The following Examples, related to the preparation of chromogenic compounds and complexes according to the present invention are reported for illustrative aim. EXAMPLE 1 : Synthesis of the 6-Bromo-3-lndolyl-β-D-Galactopyranoside chromogenic compound.
Step 1 ) Synthesis of 2-Amino-4-Bromotoluene
224 ml of water and 25 g (447 mmol) of iron filings are charged in a 0.5 I flask. The mixture is cooled to 5 °C, 41 ml of 98% sulphuric acid are added under stirring, it is warmed to 20 °C and 5.2 g (24.1 mmol) of 4-Bromo-2-Nitrotoluene and 78 ml of 96% ethanol are added. The temperature raises to 34 °C. The suspension is left for 16 hours under stirring. The starting product is not immediately solubilized but at the end of the 16 hours, in addition to the residual iron filings, a solution wherein the nitro-derivative completely reacted is obtained.
The iron filings are filtered and the filtered solution is dry-concentrated. The residue is taken back with water (20 ml) and methylene chloride (20 ml) and, under stirring, caustic soda is added to pH 12. During the alkalinization the iron hydroxide precipitates. The dark suspension is filtered on a dicalite panel and washed with methylene chloride (50 ml).
The double phase is separated in a separatory funnel and the organic phase is anhydrified on sodium sulphate for 10 minutes. Subsequently the sodium sulphate is filtered, it is washed with 20 ml of methylene chloride and the organic solution is dry-concentrated. 3.7 g of dark fluid oil is obtained. Yield: 79.1 %.
The product has been characterized by 1 H and 13C NMR spectra. 1H NMR (CD Cl3) δ: 6.93 - 6.79 (3,m), 3.64 (2,s), 2.11 (3,s). 13C NMR (CD CI3) δ:146.5, 132.2, 121.7, 121.5, 120.5, 117.9, 17.3. Step 2) Synthesis of 2-Acetamido-4-Bromotoluene
9.3 ml of glacial acetic acid and 3.7 g (19.9 mmol) of the product obtained in the step 1 ) are charged in a 25 ml flask, equipped with a thermometer, calcium chloride tube and mechanical stirrer. It is warmed to 37 °C and 2.6 ml (100%, 27.5 mmol, d: 1.082) of acetic anhydride are added in 10 seconds. An exothermia of 20 °C is detected. The temperature is maintained for 10' at 55 °C. The acetylate crystallizes at this temperature. Subsequently the temperature is taken to 60 °C and this temperature is maintained for 25' obtaining a solution. After 35' the reaction is complete. The solution is left cooling spontaneously to room temperature. The product crystallizes already at 55 °C. It is cooled to 5 °C and the suspension is left under stirring at this temperature for 15'. It is quickly filtered and washed with 5 ml of acetic acid cooled to 15 °C. A second washing is carried out with 10 ml of water. The solid which weighs 2.5 g after drying in a vacuum stove for an hour at 50 °C, is discharged.
Under stirring, slowly and at 5 °C, the washing liquids are pooled in acetic acid and in water. The acetylate which is maintained 1 hour under stirring precipitates.
At the end of this period of time the solid is filtered and washed with 5 ml of water. After drying in a vacuum stove at 50 °C, the anhydrous product weighs 0.86 g.
Total yield: 79%.
The product in TLC is monospot.
The product has been characterized by 1H and 13C NMR.
1H NMR (CD CL δ: 8.03 (l,s), 7.2-6.9 (3,m), 2.2 (6,s). 13C NMR (CD Cl3) δ: 132.1 , 128.5, 128.4, 126.3, 126.1 , 120.3, 24.8, 17.8
Step 3) Synthesis of 2-Acetamido-4-Bromobenzoic Acid
63 ml of a 0.25 M aqueous solution of eptahydrated magnesium sulphate and
3.56 g (15.6 mmol) of the product obtained in the step 2) are charged in a 100 ml four-neck flask, equipped with a reflux refrigerator, thermometer and mechanical stirrer. The suspension is warmed to 85 °C and 7.5 g (47.4 mmol) of potassium permanganate are added at portions in 2.5 hours.
The temperature, during the addition and for an hour after the end of this one, is maintained at 85-90 °C.
The suspension is left cooling to room temperature, it is filtered on dicalite and it is warmed with 20 ml of water. The filtered solution is concentrated under vacuum to a final volume equal to 50 ml.
Some drops of sulphuric acid are added to the precipitation of the product. The mixture is maintained under stirring for 10', then it is filtered and the product is washed with water. At the end the product which weighs 2.65 g after drying in a stove at 45 °C, is discharged.
Yield: 66%.
The product results insoluble in chloroform. The product has been characterized by 1H and 13C NMR (DMSO solvent).
Η NMR (DMSO) δ: 11.1 (l,s), 8.74 (l,s), 7.9 (l,dd,8.6Hz/1.1 Hz), 7.35 (l,d,8.6Hz),
2.16 (3,s). 3C NMR (DMSO) δ: 169, 142, 133.1 , 127.8, 125.7, 122.4, 115.5. Step 4) Synthesis of 2-Amino-4-Bromobenzoic Acid
5.4 ml of deionized water, 1.3 g (90.5%, 21 mmol) of potassium carbonate and
2.65 g of the product obtained in the step 3) are charged in a 20 ml little flask, equipped with reflux refrigerator, thermometer and mechanical stirrer.
The solution is reflux warmed for 6 hours. At the end of the 6 hours the reaction in ethyl acetate/acetic acid-100/6.5 is controlled. The reaction is complete.
The solution is acidified at pH 6 with concentrated hydrochloric acid.
The product which is filtered is crystallized, washed with 5 ml of water and dried in a vacuum stove at 50 °C. The weight of the anhydrous product is 1.8 g.
Yield: 81 %. The product has been characterized by 1H and 13C NMR.
1H NMR (DMSO) δ: 7.6 (l,d,8.5Hz), 6.98 (l,d,1.5Hz), 6.65 (l,d,8.5Hz), 3.35.
13C NMR (DMSO) δ: 169.2, 152.7, 133.3, 127.6, 118.4, 117.6, 109.2.
Step 5) Synthesis of N-(5-Bromo-2-Carbonylphenvπ-Glvcine
4 ml of deionized water, 0.5 g (90.5%, 8.3 mmol) of potassium carbonate and 1.8 g (8.3 mmol) of the product obtained in the step 4) are charged in a 20 ml little flask, equipped with reflux refrigerator, mechanical stirrer and thermometer.
The solution is added under stirring with 0.8 g (8.5 mmol) of chloroacetic acid and, immediately after, with a solution of 0.38 g (6.1 mmol) of potassium carbonate in deionized water (2 ml). After 5' 0.86 g (8.6 mmol) of potassium bicarbonate are added. It is warmed to 40 °C for 66 hours. At the end of the 66 hours the obtained suspension is cooled to 5 °C for 30'. The suspension is filtered and the product is washed with cold water. After drying in a vacuum stove at 50 °C the anhydrous product weighs 1.2 g.
The bitterns are acidified to pH 6.7 with concentrated hydrochloric acid and cooled at 5 °C for an hour. The precipitated solid is filtered and washed with 1 ml of water. After drying in a vacuum stove at 50 °C, the anhydrous product weighs
0.3 g. A new TLC control is carried out on this product resulting as the former product.
Total yield: 65%.
The product has been characterized by 1 H and 13C NMR spectra.
1H NMR (D20+NaOD) δ: 7.54 (l,d), 6.72 (1 ,dd), 6.66 (l,s), 3.68(2,s). 13C NMR (D20+NaOD) δ: 181.6, 176.2, 153, 136, 129.1 , 121.2, 121 , 116.9, 50. Step 6) Synthesis of 6-Bromo-3-lndolyl-1.3-Diacetate
14 ml (148.4 mmol) of acetic anhydride, 1.5 g (5.5 mmol) of the product obtained in the step 5) and 0.5 g (6.1 mmol) of sodium acetate are charged in a 20 ml little flask, equipped with thermometer, reflux refrigerator, mechanical stirrer and calcium chloride tube. It is warmed to 135 °C for 20'. The reaction is complete. The mixture is cooled to 15 °C and kept under stirring at 15 °C for an hour. The solid is filtered and washed with 3 ml of acetic acid saturated with sodium acetate and previously cooled to 15 °C. Then a second washing is carried out with 10 ml of cold water. The sodium acetate dissolves. The solid is dried in a vacuum stove at 50 °C. The weight of the anhydrous product is 1 g. Partial yield: 62 %.
The bitterns and the washing liquids in acetic acid are slowly added, under stirring, to the aqueous washing liquids at 5 °C. The mixture is concentrated at small volume, added with 4 ml of water and cooled for an hour at 5 °C. The solid which weighs 0.4 g after the washing with water and drying in a vacuum stove, is filtered. Total yield: 86 %.
The product has been characterized by 1H and 13C NMR spectra. 1H NMR (CD Cl3) δ: 8.7 (l,s), 7.7 (l,s) 7.5-7.3 (2,s), 2.6 (3,s), 2,4 (3,s). 13C NMR (CD Cl3) δ: 169, 168.1 , 134.8, 133.8, 127.6, 122.9, 120.5, 120.3, 119.1 , 1 14.1 , 24.2, 21.4.
Step 7^ Synthesis of N-Acetyl-6-Bromo-lndolyl-3-OI
6 ml of 75% sulphuric acid are charged in a 10 ml flask and cooled to 15 °C. 1.4 g (4.7 mmol) of the product obtained in the step 6) are added in portions and, under stirring, it is left reacting for an hour. After an hour 5 ml of water are dripped, in 20', with subsequent precipitation of the product which is filtered, washed with 5 ml of water, with 2% sodium acetate to neutrality and again with water in an equal volume with respect to the sodium acetate solution one. It is dried in a vacuum stove to 50 °C to constant weight.
1.18 g of monoacetylated product are obtained.
Yield: 98%.
The solid has been characterized by 1H and 13C NMR spectra. 1H NMR (CD Cl3) δ: 8.8 (l,s), 7.6 (l,d,8.1 Hz), 7.36 (l.dd), 4.3 (2,s), 2.33 (3,s).
13C NMR (CD Cl3) δ: 133.2, 128.3, 125, 122.3, 56.7, 24.7.
Step 8) Synthesis of 1-(N-Acetyl-6-Bromo-lndolvn 2.3.4.6-Tetraacetyl-β-D-
Galactopyranoside
For the synthesis of this product we refer to the article of the Journal of Medical Chemistry (7-574, 1964). The N-6-Br-lndol-3-OI (1.18 g, 4.6 mmol) has been reacted first with tetraacetyl-β-D-galactopyranosyl bromide and then with soda 1 N.
At the end of the process 1.3 g (2.23 mmol) corresponding to a yield equal to 49% are obtained.
The product has been characterized by 1 H and 13C NMR spectra. 1H NMR (CD Cl3) δ: 8.6 (l,s), 7.43-7.32 (2,m) 7.11 (l,s), 5.6-5.48 (2,m), 5.13
(1 ,dd,10.5Hz/3.3Hz), 4.98 (l,d,8Hz), 4.25-4.05 (3,m), 2.20 (3,s), 2.11 (3,s), 2.08
(3,s), 2.03 (3,s). 3C NMR (CD Cl3) δ: 170.7, 170.6, 170.5, 169.8, 168.4, 141.8, 134.5, 127.5, 123.3,
120.7, 120.1 , 119.3, 109.9, 101.9, 72.1 , 71 , 69, 67.4, 62.2, 24.3, 21.2, 21.1 , 21 Step 9) Synthesis of 6-Br-3-lndolyl-β-D-Galactopyranoside
13 ml of absolute ethanol and 1.3 g (2.23 mmol) of the product obtained in the step 8) are charged in a 20 ml flask. Nitrogen has been bubbled for 10' and 0.07 ml (0.7 mmol) of 10 M soda are added. The obtained solution is left under stirring for an hour. After an hour the reaction is complete. The solution is neutralized with 0.04 ml of glacial acetic acid and maintained for 10' under stirring. 10 ml of water are added, the product crystallizes and it is filtered and washed with 1 ml of water.
After drying in a vacuum stove at 45 °C 0.52 g of product are obtained.
Yield: 61.3 %.
The product has been characterized by 1H and 13C NMR spectra. H NMR (DMSO) δ: 10.71 (l,s), 7.6-7.49 (2,m), 7.11 (2,m) 5.24 (1 ,d,5Hz), 4.83
(l,d), 4.66 (l,m), 4.56-4.49(2,m). 3.8-3.3 (6,m).
13C NMR (DMSO) δ: 137.6, 134.3, 121.2, 119.7, 119.1 , 114.5, 114.3, 112.4, 105.1 , 75.9, 73.6, 70.8, 68.5, 60.8.
EXAMPLE 2: Synthesis of the 6-Bromo-3-lndolyl-β-D-Glucuronide
Cyclohexylammonium Salt chromogenic compound.
The N-Acetyl-6-Bromo-lndol-3-OI obtained as described in the Example 1 (step 1 - 7) is treated as follows:
Step 1 ) Synthesis of 1-(N-Acetyl-6-Br-lndolylV2.3.4 Triacetyl-β-D-Glucuronic- Methyl-Ester Acid
As reported in the article of the Chemical Pharmaceutical Bulletin (32-8- 1759/1763, year 1975), for the synthesis of the methyl ester, the N-Acetyl-6-Br- lndol-3-OI (1.18 g, 4.6 mmol) has been reacted first with sodium methoxide and then with 1-Br-1-Deoxy-2,3,4-Tri-0-Acetyl-α-D-Glucopyranuronate. At the end of the process 0.47 g of the product are obtained, with a yield equal to 20%, which is characterized by 1 H and 13C NMR spectra. H NMR (CD Cl3) δ: 8.63 (l,s), 7.43-7.32 (2,m), 7.16 (l,s), 5.36 (3,m), 5.11-5.08 (1 ,m), 4.23-4.19 (l,m) 3.76 (3,s), 2.58 (3,s), 2.11 (3,s), 2.09 (3,s), 2.06 (3,s).
13C NMR (CDCI3) δ: 170.5, 169.7, 169.6, 168.6, 167.2, 141.3, 134.5, 127.5, 123.2, 120.7, 120.3, 119.3, 110.7, 101.2, 73.2, 72.2, 71.4, 69.4, 53.5, 24.3, 21.1 , 21. Step 2) Synthesis of 6-Br-3-lndolyl-β-D-Glucuronide Cvclohexyl ammonium Salt 3 ml of anhydrous methanol, 0.47 g (0.82 mmol) of the product obtained in the step 1 ) are charged in a 25 ml little flask, equipped with a mechanical stirrer, thermometer and a capillary for nitrogen bubbling and nitrogen has been bubbled for 10'. The temperature is lowered to 10 °C and 0.03 ml (0.17 mmol) of a 30% solution of sodium methoxide in methanol are added to the white suspension. The suspension, from white, becomes gradually a yellow solution in an hour. The temperature is taken to 10 °C and 0.08 ml (0.84 mmol) of 10 M soda are added and then the temperature is left to return to 20 °C. After 2 hours the suspension is neutralized with IR 120 anionic resin (4.7 theoretical equivalents). After 10' the suspension is filtered, the resin is washed with methanol and the filtrate is dry concentrated. A dirty white product is obtained which is taken back with 3 ml of acetone.
A solution of cyclohexylamine (0.1 ml, 0.89 mmol) in acetone (5 ml) is separately prepared and therein nitrogen has been bubbled. The acetonic solution of the product is added in 10' to this solution of cyclohexylamine always under nitrogen starting from a temperature equal to 10 °C. A white product crystallizes which after
10' from the addition has been filtered. The solid is washed with 1 ml of acetone and it is put to dry into a vacuum stove at 55 °C. 0.37 g of product are obtained. Yield: 91 %.
The product has been characterized by 1H and 13C NMR spectra.
1H NMR (DMSO) δ: 10.85 (l,s), 7.62 (1 ,d,8.4Hz), 7.5 (l,d,1.5Hz), 7.11-7.05 (2,m),
4.6 (l,d), 3.45-3.15 (5,m), 2.9 (1.m), 2-1 (11 ,m).
13C NMR (DMSO) δ: 172.8, 137.5, 134.3, 121.2, 119.8, 119, 114.4, 114.3, 112.4, 104.2, 76.9, 74.2, 73.6, 72.5, 49.4. 30.9, 24.9, 24.1.
EXAMPLE 3: Synthesis of the 5-Bromo-6-Chloro-1 -Methyl 3-lndolyl-3-yl-β-D-
Galactopyranoside chromogenic compound.
Step 1 ) Synthesis of 1-(1-Acetyl-5-Bromo-6-Chloro-lndol-3-yl-2.3.4.6-Tetra-Q-
Acetyl-β-D-Galactopyranoside For the synthesis of this product we refer to the article of the Journal of Medical
Chemistry (7-574, 1964).
2.3 g (7.9 mmol) of 1-Acetyl-5-Bromo-6-Chloro-lndol-3-OI are dissolved in acetone, cooled to 0 °C and they have been reacted under stirring for 16 hours with 4.3 g (10.3 mmol) of 1-Bromo-2,3,4,6-Tetra-0-Acetyl-α-D-Galactopyranoside in presence of 1 N NaOH. The product is recovered completely evaporating the acetone and washing the residual solid with water and subsequently with ethanol.
2.4 g (3.8 mmol) of product are obtained. Yield: 48%.
The product has been characterized by 1H and 13C NMR spectra. Step 2) Synthesis of: 1-(5-Bromo-6-Chloro-1-H-lndol-3-vn-2.3.4.6-Tetra-Q-Acetyl- β-D-Galactopyranoside
2.4 g (3.8 mmol) of the product obtained in the step 1 ) are suspended in 60 ml of methanol, then they are added to 0.37 g (3.8 mmol) of sodium acetate and they are maintained at a temperature equal to 45 °C for 20 minutes. At the end of the reaction 35 ml of H2θ are added under stirring. The suspension is maintained for 2 hours at room temperature and then the solid is filtered, washed with demineralized water and dried. 1.7 g (2.95 mmol) of product are obtained. Yield: 77.5%.
The product is characterized by Nuclear Magnetic Resonance to proton and
Carbon 13.
Step 3) Synthesis of: 1-(5-Bromo-6-Chloro-1-Methyl-lndol-3-vn-2.3.4.6-Tetra-Q- Acetyl-β-D-Galactopyranoside
1.7 g (2.95 mmol) of the product obtained in the step 2) are dissolved in 20 ml of
DMF at room temperature and they are added with 1 g (7.2 mmol) of potassium carbonate, 0.35 ml (5.6 mmol) of methyl iodide and 0.05 g (0.15 mmol) of tetrabutylammonium bromide. The temperature is taken to 40 °C and maintained for 15 hours.
15 ml of methylene chloride and then 15 ml of demineralized water are added to the suspension. The suspension is maintained under stirring for 2 hours and then the phases are separated. The organic phase is anhydrified with sodium sulphate.
The solution is filtered and concentrated to syrupy consistence, then 20 ml of ethanol are added and the product is crystallized at room temperature. The suspension is cooled for 1 hour at 5 °C. The product is filtered, washed and dried.
1 g (1.7 mmol) of product is obtained. Yield: 57.4%.
The product is characterized by Nuclear Magnetic Resonance to proton and Carbon 13.
1H NMR (CD Cl3) δ:7.75 (l,s), 7.35 (l,s), 6.83 (l,s), 5.6-5.4 (2,m), 5.2-5 (l,m), 4.79 (l,d,8Hz), 4.3-4.1 (2,m), 4.1-3.9 (l,m), 3.66 (3,s) , 2.18 (3,s), 2.17 (3,s), 2.04 (3,s),
2 (3,s).
13C NMR (CDCI3) δ:170.7, 170.6, 170.5, 169.8, 135.8, 133.9, 128.5, 122.7, 121.1 , 118.6, 113, 111.3, 103.3, 71.7, 71.3, 69.3, 67.5, 61.9, 33.4, 21.3, 21.1 , 21.
Step 4) Synthesis of: 5-Bromo-6-Chloro-1-Methyl-lndol-3-yl-β-D-
Galactopyranoside
1 g (1.7 mmol) of the product obtained in the step 3) is suspended in 10 ml of absolute ethanol and 0.1 ml of a solution of benzyl trimethyl ammonium hydroxide at 40% in methanol are added to the suspension at room temperature. The suspension is left under stirring at room temperature for 30 minutes and then the solid is filtered, washed and dried. 0.65 g (1.59 mmol) of product are obtained. Yield: 93.5%.
The product is characterized by Nuclear Magnetic Resonance to proton and
Carbon 13.
1H NMR (DMSO) δ: 8 (l,s), 7.78 (l,s), 7.21 (l,s), 5.31 (l,d,0.6Hz), 4.86 (l,d,3.3Hz), 4.66 (l,m), 4.5 (2,m), 3.8-3.3, (9,m).
13C NMR (DMSO) δ: 135.8, 133.2, 125.9, 122.5, 120.7, 118.9, 111.9, 110.8, 105.5,
75.9, 73.4, 70.8, 68.4, 60.7, 33.
EXAMPLE 4: Synthesis of the 5-Bromo-4-Chloro-1-Methyl-lndol-3-yl-β-D-
Galactopyranoside chromogenic compound. This product is obtained as described in the Example 3 starting from 1-Acetyl-5-
Bromo-4-Chloro-lndol-3-OI with a comparable final yield.
1H NMR (DMSO) δ:7.5-7.3 (2,m),7.22 (l,s), 4.7-4.3 (5,m), 3.8.3.3 (9,m).
13C NMR (DMSO) δ: 136.1 , 133.7, 125.7, 123.5, 118, 117.5, 112, 110.7, 104.5,
75.9, 73.7, 70.8, 68.4. 60.7, 33. EXAMPLE 5: Synthesis of the 5,6-diBromo-3-lndolyl-β-D-Galactopyranoside chromogenic compound.
Step 1 ) Preparation of N-(2-Carboxy-4.5-Dibromophenviy-Glvcine
2.74 g (0.01 mol) of N-(5-Bromo-2-Carboxyphenyl)-Glycine have been suspended in 20 ml of glacial acetic acid and 1.5 g of Br diluted in 10 ml of glacial acetic acid have been dripped for an hour under good stirring at a temperature equal to 25-30
°C. After a overnight stirring at room temperature 0.82 g of sodium acetate have been added and the mixture has been maintained under stirring for 1 additional hour and then it has been filtered. The solid product has been washed with 10 ml of acetic acid and then with water and finally it has been vacuum dried. 2.5 g of N-(2-Carboxy-4,5-diBromophenyl)-Glycine have been obtained.
Step 2) Preparation of 1-Acetyl-3-Acetoxy-5.6-diBromoindole
2.5 g of the product obtained in the step 1 ) have been suspended in 25 ml of acetic anhydride, 0.6 g of anhydrous sodium acetate have been added and the mixture has been reflux warmed for 1 hour. Then the mixture has been cooled to 0 °C and filtered. The solid product has been washed with acetic acid and with water and vacuum dried. 1.75 g of product have been obtained.
Step 3) Preparation of Acetyl-5.6-diBromo-3-Hydroxyindole
1.75 g of the product obtained in the step 2) have been added in little portions under stirring to 5 ml of 90% sulphuric acid. After 1 hour of stirring at room temperature the reaction mixture has been poured in a mixture of 15 ml of water and 15 g of ice. After 30' of stirring the mixture has been filtered and the solid product has been washed with water and then with 8% sodium bicarbonate solution and finally again with water. The product has been vacuum dried.
1.5 g of product have been obtained. Step 4) Preparation of 5.6-diBromo-3-lndolyl-β-D-Galactopyranoside-Pentaacetate
A suspension of 1.5 g of the product obtained in the step 3) in 75 ml of acetone and 2.3 g of tetraacetyl-β-galactopyranosyl bromide, has been cooled to 0 °C and degassed with nitrogen for 30'. Then 5.33 ml of 1 N sodium hydroxide have been dripped and the mixture has been maintained under stirring at 0 °C for 16 hours. Then the mixture has been vacuum concentrated to a complete removal of the solvent. The residual has been suspended in H2θ, filtered and washed repeatedly with water and finally with ethanol. The product has been vacuum dried.
1.5 g of product have been obtained.
Step 5) Preparation of 5,6-diBromo-3-lndolyl-β-D-Galactopyranoside 0.5 g of 5,6-diBromo-3-lndolyl-β-D-Galactopyranoside pentaacetate have been suspended in 5 ml of absolute ethanol and 0.05 ml of 40% benzyl trimethylammonium hydroxide solution in methanol have been then added.
After 16 hours of stirring at 30 °C the mixture has been cooled and filtered. The solid product has been washed with ethanol and dried. 0.3 g of product have been obtained.
1H NMR (DMSO)δ : 10.9(l,d), 8 (l,s), 7.7 (l,s), 7.19 (l,d,2Hz), 5.3 (l,d,4.6Hz), 4.85
(l,d), 4,69 (l,m), 4.6-4.4 (2,m), 3.8-3.3 (6,m). 3CNMR (DMSO) δ: 136.6, 133, 122.2, 121 , 116.6, 115.7, 114.5, 112.6, 105.4,
75.9, 73.4, 70.7, 68.4, 60.7. EXAMPLE 6: Synthesis of the 6-Chloro-1-Methyl-lndol-3-yl-β-D-
Galactopyranoside chromogenic compound. This product is obtained as described in the Example 3 starting from 1-Acetyl-6-
Chloro-lndol-3-OI with a comparable final yield.
1H NMR (DMSO) δ: 7.62 (1 ,d,4.2Hz), 7.52 (1 ,s), 7.12 (1 ,s), 7.01
(1 ,dd,4.2Hz 0.9Hz), 5.28 (1 ,d,3.4Hz), 4.87 (1 ,d,5Hz),4.67 (l,m), 4.6-4.4 (2,m), 3.8- 3.3 (9,m).
13C NMR (DMSO) δ: 136.8, 134.3, 126.8, 119.5, 119.1 , 118.7, 117, 109.7, 105.2,
75.9, 73.6, 70.8, 68.5,60.7, 32.8.
EXAMPLE 7: Synthesis of the 4-Chloro-1-Methyl-lndol-3-yl-β-D-
Galactopyranoside chromogenic compound. This product is obtained as described in the Example 3 starting from 1-Acetyl-4-
Chloro-lndol-3-OI with a comparable final yield.
1H NMR (DMSO) δ: 7.4-6.9(4,m), 5-4.5 (5,m), 3.8-3.3 (9,m).
13C NMR (DMSO) δ: 136.3, 135.2, 124.1 , 122.3, 119.2, 116.2, 108.9, 104.4, 75.9,
73.9, 70.9, 68.5, 60.7, 32.9. EXAMPLE 8: Synthesis of the 5,6-diBromo-1-Methyl-lndol-3-yl-β-D-
Galactopyranoside chromogenic compound.
This product is obtained as described in the Example 3 starting from 1-Acetyl-5,6- diBromo-lndol-3-OI already described in the Example 4 step 3) with a comparable final yield. 1H NMR (DMSO) δ: 8.02 (l,s), 7.92 (l,s), 7.21 (l,s), 5.35 (l,d), 4.9 (l,d), 4.7 (l,m),
4.6-4.4 (2,m), 3.8-3.3 (9,m).
13C NMR (DMSO) δ: 135.7, 133.5, 122.3,121.1 , 118.9, 116.1 , 115.1 , 112.8, 105.5,
75.9, 73.4, 70.7, 68.3, 60.7, 33.
EXAMPLE 9: Synthesis of the 6-Bromo-1-Methyl-lndol-3-yl-β-D- Galactopyranoside chromogenic compound.
This product is obtained as described in the Example 3 starting from 1-Acetyl-6-
Bromo-lndol-3-OI with a comparable final yield.
1H NMR (DMSO) δ: 7.66-7.56 (2,m), 7.15-7.12 (2,m), 5.4-4.4 (5,m), 3.8-3.3 (9,m). 3C NMR (DMSO) δ: 136.8, 134.7, 121.3, 119.9, 119.3, 117, 114.9, 112.7, 105.2, 75.9, 73.6, 70.8, 68.4,60.7. 32.8.
EXAMPLE 10: Preparation of the complex with α-Cyclodextrin of the 6-Bromo-3- Indolyl-β-D-Galactopyranoside.
200 mg of 6-Bromo-3-lndolyl-β-D-Galactopyranoside prepared according to the Example 1 are added with 1.77 g of α-Cyclodextrin and 5 ml of water. The suspension is taken to 60 °C and maintained for 10 minutes and then it is cooled to room temperature.
The obtained solution is then freeze-dried. The obtained anhydrous complex is reconstituted with water in order to obtain the same initial concentration obtaining a clear solution. This solution diluted 2000 times shows an absorbance at 229 nm equal to 1386 mAbs. The not complexed 6-Bromo-3-lndolyl-β-D-Galactopyranoside, treated in the same way but only with water, produces a suspension which is filtered and the clear suspension which is obtained shows an absorbance at 229 nm equal to 286 mAbs. An enzymatic test with β-Galactoxidase has been carried out on the solution of the complex. 100 μl of a β-Galactoxidase (3000 LAU/ml Sigma) solution have been added to 10 ml of the solution observing a red colour development caused by the formation of the insoluble indigo blue.
EXAMPLE 11 : Preparation of the complex with α-Cyclodextrin of the 6-Bromo-3- Indolyl-β-D-Glucuronide Cyclohexylammonium Salt. 10 mg of 6-Bromo-3-lndolyl-β-D-G!ucuronide Cyclohexylammonium salt prepared as described in the Example 2 are added with 885 mg of α-Cyclodextrin and 4 ml of water and treated as described in the Example 10. The obtained solution is freeze-dried. The obtained anhydrous complex is reconstituted with water at the same initial concentration obtaining a clear solution. This solution, 1000 times diluted with H2θ, shows an absorbance at 228 nm equal to 2220 mAbs. The not complexed 6-Bromo-3-lndolyl-β-D-Glucuronide Cyclohexylammonium salt, treated in the same way but with water only, produces a suspension which is filtered and the clear solution shows an absorbance at 228 nm equal to 1742 mAbs. An enzymatic test with β-Glucuronidase has been carried out on the solution of the complex. 100 μl of a β-Glucuronidase (100 LAU/ml Sigma) solution have been added to 10 ml of the solution observing a red colour development caused by the formation of the insoluble indigo blue.
EXAMPLE 12: Preparation of the complex with β-Cyclodextrin of the 5-Bromo-4-
Chloro-N-Methyl-3-lndolyl-β-D-Galactoside.
5 mg of 5-Bromo-4-Chloro-N-Methyl-3-lndolyl-β-D-Galactoside prepared as described in the Example 3 are treated as described in the Example 10 but with 50 mg of β-Cyclodextrin dissolved in 5 ml of H2θ. The obtained solution, diluted 25 times with water, shows an absorbance at 232 nm equal to 2843 mAbs. The not complexed 5-Bromo-4-Chloro-N-Methyl-3-lndolyl-β-D-Galactoside, treated in the same way but only with water, produces a suspension which is filtered and the clear solution shows an absorbance at 232 nm equal to 933 mAbs. An enzymatic test with β-Galactoxidase has been carried out on the solution of the complex as described in the Example 10 observing a emerald green colour development caused by the formation of the insoluble indigo blue. EXAMPLE 13: Preparation of the complex with β-Cyclodextrin of the 5-Bromo-4- Chloro-3-lndolyl-Gal.
5 mg of 5-Bromo-4-Chloro-3-l ndolyl-Gal are treated as described in the Example 10 but with 50 mg of β-Cyclodextrin dissolved in 5 ml of water. The obtained solution, diluted 25 times with water shows an absorbance at 229 nm equal to 2874 mAbs. The not complexed 5-Bromo-4-Chloro-3-lndolyl-Gal, treated in the same way but only with water, produces a suspension which is filtered and the clear solution shows an absorbance at 229 nm equal to 1029 mAbs. An enzymatic test with β-Galactoxidase has been carried out on the solution of the complex as described in the Example 10 observing a blue colour development caused by the formation of the insoluble indigo blue. EXAMPLE 14: Preparation of the complex with β-Cyclodextrin-O-dimethylated of the 5-Bromo-4-Chloro-3-lndolyl Capryiate.
10 mg of 5-Bromo-4-Chloro-3-lndolyl Capryiate are weighed and added to 10 ml of an aqueous solution containing 330 mg of β-Cyclodextrin-O-dimethylated. The suspension is warmed to 50 °C and maintained for 3 hours and then cooled to room temperature. Then the solution is filtered and freeze-dried. The obtained anhydrous complex is reconstituted with water in order to obtain the same initial concentration obtaining a clear solution.
This clear solution diluted 1 :100 shows an absorbance at 232 nm equal to 148 mAbs.
The not complexed 5-Bromo-4-Chloro-3-lndolyl Capryiate, treated in the same way but only with water, produces a suspension which is filtered and the clear solution does not show absorbance at 232 nm.
EXAMPLE 15: Preparation of the complex with α-Cyclodextrin of the 6-Chloro-3-
Indolyl-Gal.
100 mg of 6-Chloro-3-lndolyl-Gal are added to 5 ml of an aqueous solution containing 885 mg of α-Cyclodextrin. The suspension is warmed to 60 °C and left under stirring for 60 minutes. The obtained clear solution is cooled to room temperature and then it is diluted 1000 times for the UV measure. The obtained complex shows an absorbance at 227 nm equal to 1836 mAbs. The not complexed compound, treated in the same way but only with water, produces a suspension which is filtered and the clear solution diluted 1000 times shows an absorbance at 227 nm equal to 864 mAbs. An enzymatic test with β-
Galactoxidase has been carried out on the solution of the complex as described in the Example 10 producing red colour.
EXAMPLE 16: Preparation of the complex with β-Cyclodextrin of the 5-Bromo-4- Chloro-3-lndolyl-Glucoside.
5 mg of 5-Bromo-4-Chloro-3-lndolyl-Glucoside are added with 5 ml of an aqueous solution containing 75 mg of β-Cyclodextrin and treated as in the Example 10.
This complex diluted 100 times shows an absorbance at 229 nm equal to 521 mAbs. The not complexed compound, treated in the same way but only with water, shows an absorbance at 229 nm equal to 57 mAbs.
EXAMPLE 17: Preparation of the complex with α-Cyclodextrin of the 5-Bromo-4-
Chloro-3-lndolyl-Glucuronide sodium salt.
200 mg of 5-Bromo-4-Chloro-3-lndolyl-Glucuronide sodium salt are added with
250 mg of α-Cyclodextrin and 1 ml of water and treated as in the Example 10. The obtained complex diluted 20,000 times shows an absorbance at 229 nm equal to
535 mAbs. The not complexed product, treated in the same way but only with water, shows an absorbance at 229 nm equal to 219 mAbs. An enzymatic test with β-Glucuronidase has been carried out on the solution of the complex as described in the Example 11 , obtaining a precipitate of blue colour.
EXAMPLE 18: Preparation of the complex with α-Cyclodextrin of the 5-Bromo-4-
Chloro-3-lndolyl-Glucuronide Cyclohexylammonium Salt . 300 mg of 5-Bromo-4-Chloro-3-lndolyl-Glucuronide Cyclohexylammonium salt are added with 10 ml of an aqueous solution containing 1000 mg of α-Cyclodextrin and treated as in the Example 10. The obtained complex diluted 2,000 times shows an absorbance at 229 nm equal to 800 mAbs. The not complexed compound, treated in the same way but only with water, shows an absorbance at 229 nm equal to 105 mAbs. An enzymatic test with β-Glucuronidase has been carried out on the solution of the complex as described in the Example 11.
EXAMPLE 19: Preparation of the complex with β-Cyclodextrin of the 5-Bromo-4-
Chloro-3-lndolyl-Glucosaminide.
5 mg of 5-Bromo-4-Chloro-3-lndolyl-Glucosaminide are treated as in the Example 16. The obtained complex diluted 100 times shows an absorbance at 229 nm equal to 660 mAbs. The not complexed product, treated in the same way but only with water, shows an absorbance at 229 nm equal to 90 mAbs.
EXAMPLE 20: Preparation of the complex with β-Cyclodextrin-O-dimethylated of the 6-Chloro-3-lndolyl-Caprylate. 10 mg of 6-Chloro-3-lndolyl-Caprylate are treated as in the Example 14. The obtained complex diluted 100 times shows an absorbance at 226 nm equal to 315 mAbs. The not complexed compound, treated in the same way but only with water, does not show an absorbance at 226 nm.

Claims

1. Chromogenic compounds derived from indole with a saccharidic or fatty acid or sulphate or phosphate group and with alogens, having formula (I):
Figure imgf000026_0001
(I) wherein:
R╬╣ is selected from the group consisting of ╬▓-D-Galactose, ╬▒-D-Galactose, ╬▓-D-
Glucose, ╬▒-D-Glucose, ╬▓-D-Galactosamine, ╬▓-D-Glucosamine, ╬▓-D-Glucuronic or ╬▒-D-Glucuronic acid sodium or cyclohexylamine salt, Capryiate, Sulphate or
Phosphate; R2 is selected from the group consisting of H, methyl, ethyl, benzyl and paranitrobenzyl;
X is selected from the group consisting of H, Br and Cl;
Y is selected from the group consisting of H, Cl and Br;
W is selected from the group consisting of H, Br and Cl; with the condition that when W = Br the meaning of X is H or Br and Y is H.
2. Compounds as claimed in claim 1 , characterized in that the compounds themselves are in a form complexed with cyclodextrins or cyclodextrin derivatives.
3. Compounds as claimed in claim 2, characterized in that said cyclodextrins and said cyclodextrin derivatives are selected from the group consisting of alpha, beta and gamma cyclodextrin, O-dimethylated, O-trimethylated, O-hydroxymethylated and O-hydroxyethylated cyclodextrin derivatives, the cyclodextrin-polyvinyl alcohol copolymer and the cyclodextrin-cellulose copolymer.
4. Compounds as claimed in claim 2, characterized in that in the complexed form the ratio by weight between said cyclodextrin or its derivative and chromogenic compound ranges from 2:1 to 500:1.
5. Compounds as claimed in claim 2, characterized in that in said complexed form the solubility in water of the chromogenic compound is from 2 to 50 times greater than the solubility of the chromogenic compound alone.
6. Use of the chromogenic compounds as defined in any claim from 1 to 5, as additives for culture media suitable to the determination of micro-organisms selected from the group comprising Escherichia Coli, total coliforms, Salmonella spp, Salmonella typhi and paratyphi, Salmonella typhimurium, Enterobacter, Citrobacter, Candida albicans, Enterococci and Klebsiella pneumoniae.
7. Culture media for the determination of micro-organisms, containing a chromogenic compound as defined in the claims from 1 to 6.
8. Process for the preparation of chromogenic compounds in form complexed with cyclodextrin or with cyclodextrin derivatives as defined in the claims from 2 to 5, characterized in that: a) the chromogenic compound in powder form is added with a cyclodextrin or one of its derivatives in powder form and with an amount of water obtaining a suspension; b) the suspension obtained in the step a) is warmed to a temperature ranging from
20 to 80 ┬░C under stirring for a time ranging from 10 minutes to 168 hours obtaining a solution, after filtration if necessary; c) the solution obtained in the step b) is cooled to room temperature and vacuum evaporated or freeze-dried.
9. Process as claimed in claim 8, characterized in that said chromogenic compound used in the step a) has a granulometry lower than 150 micrometers.
10. Process as claimed in claim 8, characterized in that the aqueous suspension obtained in the step a) has a concentration of cyclodextrin or of cyclodextrin derivative ranging from 0.1 to 50%.
1 1. Process as claimed in claim 8, characterized in that the ratio by weight between cyclodextrin or cyclodextrin derivative and the chromogenic compound in the step a) ranges from 2:1 to 500:1.
PCT/EP1999/002174 1998-03-30 1999-03-30 Chromogenic indole derivatives WO1999050438A2 (en)

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WO2006089889A1 (en) * 2005-02-22 2006-08-31 Alain Rambach Detecting a microorganism strain in a liquid sample
WO2007023185A1 (en) * 2005-08-26 2007-03-01 Alain Rambach Detection of salmonella lactose+
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JP4794627B2 (en) * 2005-08-26 2011-10-19 アラン ランバック Detection of Salmonella lactose +
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US8268580B2 (en) 2008-01-24 2012-09-18 Pilots Point Llc Medium for detecting the presence or absence of pathogenic staphylococci
US8420347B2 (en) 2008-01-24 2013-04-16 Pilots Point Llc Test mixture for detecting the presence or absence of methicillin resistant Staphylococcus aureus directly in a first generation test sample
US8765395B2 (en) 2008-01-24 2014-07-01 Pilots Point Llc Method and medium for detecting the presence or absence of methicillin resistant Staphylococcus aureus in a first generation biological test sample
US8268579B2 (en) 2008-01-24 2012-09-18 Pilots Point Llc Method and medium for detecting the presence or absence of pathogenic staphylococci in a first generation test sample
US8546103B2 (en) 2009-06-27 2013-10-01 Pilots Point, LLC Method for detecting the presence or absence of pathogenic Staphylococci in a test sample, including test mixture with micro particles
US8846336B2 (en) 2009-06-27 2014-09-30 Pilots Point Llc Test mixtures for detecting the presence or absence of target microbes
US8524468B2 (en) 2009-12-22 2013-09-03 Pilots Point Llc Method for detecting the presence or absence of methicillin resistant staphylococcus aureus (MRSA) in a test sample
CN108409815A (en) * 2018-03-26 2018-08-17 郑州安图生物工程股份有限公司 A kind of indoles glycoside substrate and preparation method and the application in the detection of aerobic population vaginitis
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