NEW THIOUREA-BASED COMPOUNDS, PRODUCTION METHOD AND USE THEREOF TO DETECT GHB IN BEVERAGES

Abstract

The present invention relates to compound 1 or 2 or the composition comprising it. The present invention also relates to a process for obtaining compound 1 or 2, as well as to the use thereof for detecting GHB in beverages. An equipment or kit for use in detecting GHB in beverages is also included.

Claims

1. A compound of formula 1 or 2: ##STR00006##

2. A composition comprising the compound according to claim 1.

3. The composition according to claim 2, comprising the compound according to claim 1 and an organic solvent.

4. The composition according to claim 2, consisting of the compound according to claim 1 and an organic solvent.

5. The composition according to claim 3, wherein the solvent is dimethylsulfoxide (DMSO).

6. A process for obtaining the compound in claim 1 with a reaction scheme comprising: ##STR00007##

7. A process for detecting γ-hydroxybutyric acid (GHB) or a salt thereof in a beverage, comprising detecting the γ-hydroxybutyric acid (GHB) or a salt thereof using the compound according to claim 1.

8. The process according to claim 7, wherein the beverage is an alcoholic beverage with a mixture comprising a non-alcoholic beverage.

9. The process according to claim 7, wherein the beverage is a non-alcoholic beverage.

10. The process according to claim 8, wherein the alcoholic beverage is selected from gin, vodka, rum, whisky, white wine, vermouth and beer.

11. The process according to claim 8, wherein the non-alcoholic beverage is a soft drink or juice.

12. The process according to claim 11, wherein the soft drink is selected from tonic, orange soft drink, lemon soft drink, tea soft drink and a cola soft drink.

13. A process for detecting GHB or a salt thereof in a beverage using the compound according to claim 1, the process comprising: a) taking a drop of the beverage to be analyzed and dissolving the drop in an aqueous solution of weak base; b) adding an aliquot of the solution obtained in step a) to the compound 1 or 2, according to claim 1; c) observing a reaction produced in step b); and d) qualitatively determining the presence of GHB in the beverage to be analyzed according to the observation of step c).

14. A process for detecting GHB or a salt thereof in a beverage using the compound according to claim 1, the progress comprising: a) mixing an aqueous solution of a weak base with an organic solvent; b) adding the compound 1 or 2 according to claim 1; c) immediately after step b), adding a drop of the beverage to be analyzed; d) observing a reaction produced in step c); and e) qualitatively determining the presence of GHB in the beverage to be analyzed according to the observation of step d).

15. The process according to claim 14, wherein the organic solvent is dimethylsulfoxide.

16. The process according to claim 13, wherein the weak base is sodium, potassium, ammonium or calcium bicarbonate.

17. The process according to claim 13, wherein the weak base is at a concentration between 0.4 mM and 1 mM.

18. A process for detecting GHB or a salt thereof in a beverage using the compound according to claim 1, the process comprising the steps of: a) introducing a strip of non-woven fabric containing at least 50% polypropylene into a beverage sample; b) introducing the beverage-impregnated strip of step a) into a container containing the compound 1 or 2 according to claim 1; c) observing a reaction produced in the container; and d) qualitatively determining the presence of GHB in the beverage to be analyzed according to the observation of step c).

19. The process according to claim 18, wherein the compound 1 or 2 is at a concentration in a composition between 50 μM and 500 μM.

20. The process according to claim 18, wherein the beverage is an alcoholic beverage or a mixture comprising a non-alcoholic beverage.

21. The process according to claim 18, wherein the beverage is a non-alcoholic beverage.

22. The process according to claim 20, wherein the alcoholic beverage is selected from gin, vodka, rum, whisky, white wine, vermouth and beer.

23. The process according to claim 20, wherein the non-alcoholic beverage is a soft drink or juice.

24. The process according to claim 23, wherein the soft drink is selected from tonic, orange soft drink, lemon soft drink, tea soft drink and a cola soft drink.

25. A kit or equipment comprising the compound according to claim 1 for detecting GHB or a salt thereof in a beverage, the kit or equipment comprising: a transparent container with an opening and a closure containing the compound 1 or 2 according to claim 1; a transparent container with an opening and a closure containing a weak base in the form of an aqueous solution; and a tool for collecting a sample of the beverage to be analyzed.

26. A kit or equipment comprising the compound according to claim 1 for detecting GHB or a salt thereof in a beverage, comprising a strip of non-woven fabric (NWF) with 50 to 100% polypropylene; a transparent container with an opening and a closure containing a composition comprising the compound 1 or 2 according to claim 1.

27. The kit or equipment according to claim 25 further comprising instructions for use.

28. The kit or equipment according to claim 25, wherein the weak base is sodium, potassium, ammonium or calcium bicarbonate.

29. The kit or equipment according to claim 25, wherein the weak base is at a concentration between 0.4 mM and 1 mM.

30. The kit or equipment according to claim 25, wherein the compound 1 or 2 is at a concentration in a composition between 50 μM and 100 μM.

31. The kit or equipment according to claim 25, wherein the beverage is an alcoholic beverage or a mixture comprising a non-alcoholic beverage.

32. The kit or equipment according to claim 25, wherein the beverage is a non-alcoholic beverage.

33. The kit or equipment according to claim 31, wherein the alcoholic beverage is selected from gin, vodka, rum, whisky, white wine, vermouth and beer.

34. The kit or equipment according to claim 31, wherein the non-alcoholic beverage is a soft drink or juice.

35. The kit or equipment according to claim 34, wherein the soft drink is selected from tonic, orange soft drink, lemon soft drink, tea soft drink and a cola soft drink.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0017] In a first aspect, the present invention relates to a compound of formula 1 or 2:

##STR00002##

[0018] In a second aspect, the present invention relates to a composition comprising Compound 1 or 2 as defined above. In a preferred embodiment, said composition comprises Compound 1 or 2 as defined above and an organic solvent. As the organic solvent, but not limited thereto, DMF (dimethylformamide) and DMSO (dimethylsulfoxide) may be used, although DMSO is preferably utilized.

[0019] In a third aspect, the present invention relates to a process for obtaining compound 1 or 2, comprising the following reaction scheme:

##STR00003##

[0020] The reaction conditions are not particularly relevant, as it may be carried out at room P and T.

[0021] In a fourth aspect, the present invention relates to the use of a composition, as defined above, or a compound 1 or 2, as defined above, in the detection of γ-hydroxybutyric acid (GHB) or a salt thereof in beverages. Such salts may include, but are not limited to, salts with alkali or alkaline earth metals, preferably sodium. In the present invention, when reference is made to beverages, as indicated below, it will be understood as alcoholic beverages, non-alcoholic beverages or mixtures thereof. Where reference is made to alcoholic beverages, alcoholic distillates such as, for example, whisky or gin, as well as alcoholic beverages that are not distilled, such as wine and beer, are included.

[0022] In a fifth aspect, the present invention relates to a process for detecting GHB or a salt thereof in beverages, comprising: [0023] a) taking a drop of the beverage to be analyzed and dissolving it in an aqueous solution of weak base; [0024] b) adding an aliquot of the solution obtained in step a) to the composition, as defined above, or alternatively to Compound 1 or 2, as defined above; [0025] c) observing the reaction produced; and [0026] d) qualitatively determining the presence of GHB in the beverage to be analyzed according to the observation of step c).

[0027] It should be noted that in step b) the composition of Compound 1 or 2 and the organic solvent may already be prepared or may be prepared at that point of time just before the addition of the aliquot of the solution obtained in step a).

[0028] In a preferred embodiment, said weak base in step a) is sodium bicarbonate, although any weak base such as, for example, potassium, ammonium, calcium bicarbonate could be used. In another preferred embodiment, the concentration of the aqueous solution of weak base is from 0.4 mM to 1 mM, even more preferably 0.4 mM.

[0029] In another preferred embodiment, Compound 1 or 2 is at a concentration between 50 μM and 100 μM, even more preferably 50 μM.

[0030] In step c) it should be observed if a reaction occurs between compounds 1 or 2, alone or in the composition, with the possible GHB component or a salt thereof present in the beverage to be analyzed. Whether compound 1 and compound 2 react positively with GHB or a salt thereof, a color change (from pale yellow to orange) will be observed.

[0031] Thus, one can conclude in step d) about the presence or not of GHB in the beverage being analyzed.

[0032] In another alternative embodiment of the process, this may be slightly modified in the order of mixing, although the final result would be the same. In this regard, the alternative process comprises the steps of: [0033] a) mixing an aqueous solution of a weak base as defined above with the organic solvent as defined above; [0034] b) adding compound 1 or 2 as defined above; [0035] c) immediately after step b), adding a drop of the beverage to be analyzed; [0036] d) observing the reaction produced; and [0037] e) qualitatively determining the presence of GHB in the beverage to be analyzed according to the observation of step d).

[0038] In another alternative embodiment of the process, the visual detection is performed on a strip of a non-woven fabric (NWF) with 50 to 100% polypropylene, preferably with 100% polypropylene. In this case, the process for detecting GHB or a salt thereof in beverages comprises the steps of: [0039] a) introducing a strip of non-woven fabric containing at least 50% polypropylene into a beverage sample, typically between 3 and 5 seconds is enough; [0040] b) introducing the beverage-impregnated strip of step a) into a container containing the composition of the invention, as defined above, or alternatively Compound 1 or 2, as defined above, preferably the composition; [0041] c) observing the reaction produced in the container; and [0042] d) qualitatively determining the presence of GHB in the beverage to be analyzed according to the observation of step c).

[0043] Compound 1 or 2 is preferably at a concentration of 50 μM to 500 μM, preferably 50 μM to 100 μM.

[0044] The reaction is considered positive in GHB if in step c) a light yellow to intense orange change is observed with compounds 1 or 2.

[0045] This detection process with a fabric strip detects GHB in all types of pure beverages (alcoholic and non-alcoholic) and combined alcoholic and non-alcoholic beverages provided that it is at a concentration above 35 μM.

[0046] In a sixth aspect, the present invention relates to a kit or equipment for use in detecting GHB or a salt thereof in beverages comprising: [0047] a transparent container with opening and closure containing a composition as defined above, alternatively a Compound 1 or 2, as defined above, optionally wherein said container containing Compound 1 or 2 is accompanied by another container with the same characteristics but with organic solvent as defined above, [0048] a transparent container with opening and closure containing a weak base in the form of an aqueous solution: [0049] a tool for collecting a sample of the beverage to be analyzed.

[0050] In another embodiment, in the case of detecting GHB or a salt thereof in beverages by means of a strip of a non-woven fabric (NWF) with 50 to 100% polypropylene, the kit or equipment alternatively comprises: [0051] a strip of non-woven fabric (NWF) with 50 to 100% polypropylene; [0052] a transparent container with opening and closure containing a composition as defined above, alternatively a compound 1 or 2 as defined above, optionally wherein said container containing Compound 1 or 2 is accompanied by another container with the same characteristics but with organic solvent as defined above, i.e. DMF (dimethylformamide) or DMSO (dimethylsulfoxide) are preferably used, more preferably DMSO.

[0053] Optionally, such a kit or equipment according to any of the preceding embodiments (whether based on strips for detection or not) further comprises instructions for use.

[0054] As noted above herein, the weak base used for the beverage sample in the kit without strip detection is preferably sodium bicarbonate, although any weak base such as potassium bicarbonate, ammonium bicarbonate, calcium bicarbonate could be used. In another preferred embodiment, the concentration of the aqueous solution of weak base is 0.4-1 mM, even more preferably 0.4 mM.

[0055] Additionally, as noted herein above, in the case of beverage detection for any of the embodiments of the kit (whether based on strips for detection or not), Compound 1 or 2 is preferably present in the composition at a concentration between 50 μM and 500 μM, preferably from 50 μM to 150 μM, more preferably from 50 μM to 100 μM.

[0056] In a preferred embodiment, such transparent containers with opening and closure, according to any of the embodiments of the kit (whether based on strips for detection or not), are microtubes, graduated or not graduated, of polypropylene (e.g., Eppendorfrm).

[0057] In another preferred embodiment of the non-strip based kit, said tool for collecting the sample can be a dropper or a small spoon. In the case of the strip-based kit, said tool is not necessary.

[0058] In a more preferred embodiment, in the use, process, or kit or equipment, as defined in the foregoing aspects, the beverage is an alcoholic beverage. In another more preferred embodiment, said alcoholic beverage is admixed with a non-alcoholic beverage, preferably a soft drink or juice. In another more preferred embodiment, said beverage is only a non-alcoholic beverage, preferably a soft drink or a juice.

[0059] Preferably, said alcoholic beverage is selected from, byway of non-limiting example, gin, vodka, rum, whisky, white wine, vermouth, and beer.

[0060] Preferably, said soft drink or juice is selected from, by way of non-limiting example, tonic, orange soft drink, lemon soft drink, cola drink, tea drink and any fruit juice.

[0061] It should be noted that any combination of previous embodiments or instances or examples encompassed by the present invention is understood by one skilled in the art as being feasible in the context of the present invention.

[0062] The various particular instances or examples recited above, as well as the following examples, are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

EXAMPLES

Example 1. Synthesis of Compound 1

[0063] ##STR00004##

Synthesis of Compound 4

[0064] In 50 mL of MeOH, 500 mg of 3 (3.30 mmol) and 842 mg of Na.sub.2CO.sub.3 (7.91 mmol) were stirred for 30 min and then 910 μL of Boc.sub.2O (3.96 mmol) was added dropwise. Stirring was continued at room temperature until the next day. The MeOH was then removed under vacuum and about 30 mL of deionized water was added. It was extracted with AcOEt (3×20 mL), the organic layer was dried over anhydrous MgSO.sub.4, filtered and the solvent removed under vacuum. 635 mg of compound 4 was obtained in 89% yield.

Synthesis of Compound 5

[0065] 365 mg of 4-(trifluoroacetyl)benzoic acid (1.67 mmol), 202 mg (1.67 mmol) of DMAP and 300 μL (1.67 mmol) of EDC were mixed in 10 mL of dry THF under inert atmosphere for 30 min. 300 mg of 4 (1.39 mmol, 0.139 M) was then dissolved in 10 mL of dry THF and added dropwise. The reaction was kept for 2 days at room temperature and under argon atmosphere. The solvent was then removed under vacuum, the resulting yellow oil was dissolved in 20 mL of AcOEt and the organic layer was washed with 1% HCl (2×10 mL), NaHCO.sub.3 (sat) (2×10 mL) and NaCl (sat) (10 mL). The organic layer was dried with anhydrous MgSO.sub.4, filtered, and the solvent removed under vacuum. 524 mg of compound 5 (91% yield) was isolated.

Synthesis of Compound 6

[0066] In 5 mL of dry DCM, 201 mg of 5 (0.48 mmol) was dissolved with 1 mL of TFA (13.06 mmol) under inert atmosphere. The mixture was stirred for 15 min and the solvent removed under vacuum. The colorless oil was dissolved in 15 mL of AcOEt and the organic layer was washed with Na.sub.2CO.sub.3 (sat) (2×10 mL) and NaCl (sat) (10 mL), dried over anhydrous MgSO.sub.4 and filtered. After evaporating the solvent, the compound 6 as a white powder (121 mg, 80% yield) was obtained.

Synthesis of Compound 1

[0067] 52 mg of 4-nitrophenyl isothiocyanate (0.29 mmol) was mixed with 110 mg of 6 (0.35 mmol) in 5 mL of pyridine under inert atmosphere for 2 days. The solvent was then removed and the resulting yellow oil was dissolved in 15 mL of AcOEt. The organic layer was washed with 1 M HCl (10 mL), NaHCO.sub.3 (sat) (10 mL) and NaCl (sat) (10 mL), dried over anhydrous MgSO.sub.4 and filtered. The solvent was removed under vacuum and the reaction crude was purified by chromatographic column using Hexane:AcOEt. AcOEt as eluent (from 7:3 to 6:4). 68 mg of compound 1 was obtained (47% yield).

Example 2. Synthesis of Compound 2

[0068] ##STR00005##

Synthesis of Compound 8

[0069] In 10 mL of MeOH, 500 μL of 7 (8.31 mmol) and 1400 μL of Net.sub.3 (9.97 mmol) were mixed and then 2300 μL of Boc.sub.2O (9.97 mmol) was added dropwise. Stirring was kept at room temperature for 16 h. The MeOH was then removed under vacuum and the colorless oil dissolved in 30 mL of AcOEt. The organic layer was washed with deionized water (2×15 mL) and NaCl (sat) (15 mL), dried over anhydrous MgSO.sub.4, filtered, and the solvent was removed under vacuum. 676 mg of compound 8 (50% yield) was obtained.

Synthesis of Compound 9

[0070] 807 mg of 4-(trifluoroacetyl)benzoic acid (3.70 mmol), 452 mg of DMAP (3.70 mmol) and 660 μL of EDC (3.70 mmol) were dissolved in 10 mL of dry THF under inert atmosphere. After 45 min stirring, 500 mg of 8 was dissolved in 10 mL of dry THF and added thereto. Stirring was kept at room temperature for 2 days. The solvent was then removed under vacuum and the resulting yellow oil was dissolved in 30 mL AcOEt. The organic layer was washed with 1% HCl (15 mL), NaHCO.sub.3 (sat) (2×15 mL) and NaCl (sat) (15 mL), dried over anhydrous MgSO.sub.4 and filtered. 1 g of compound 9 was obtained in 90% yield.

Synthesis of Compound 10

[0071] In 4.5 mL of dry DCM, 1 g of 9 (2.77 mmol) and 1 mL of TFA (13.06 mmol) were mixed for 15 min under inert atmosphere. The solvent was then removed and compound 10 was isolated (616 mg, 60% yield).

Synthesis of Compound 2

[0072] 566 mg of 10 (2.17 mmol, 0.109 M) and 323 mg of 4-nitrophenyl isothiocyanate (1.81 mmol) were dissolved in 20 mL of pyridine under inert atmosphere. Stirring was kept at room temperature for 2 days. The solvent was then removed under vacuum and the resulting yellow oil dissolved in 30 mL of AcOEt. The organic layer was washed with 1 M HCl (15 mL), NaHCO.sub.3 (sat) (2×15 mL) and NaCl (sat) (15 mL), dried over anhydrous MgSO.sub.4, filtered and the solvent removed under vacuum. The crude reaction was purified by chromatography column using Hexane:AcOEt (from 7:3 to 6:4) as eluent. 400 g of compound 2 was isolated in 50% yield.

Example 3. Assays on Real Samples

[0073] Firstly, the beverages were contaminated with GHB at a concentration of 24 mM. An aliquot of 100 μL was then taken and mixed with 100 μL of NaHCO.sub.3 (1 mM or 0.4 mM). 30 μL of this solution were then taken and mixed with 50 μL of a solution of Compound 1 or 2 (1 mM in DMSO) and 920 μL of DMSO. The same process was followed for samples without GHB.

[0074] Results are seen in FIGS. 1-3 for different beverages (FIG. 1 for Coca-Cola®, Beer, Fanta Orange, White Wine; FIG. 2 for Rum, Vodka, Gin, Whisky; FIG. 3 for gin and tonic, and tonic) for compound 2. Although not seen in the figures, the same results were obtained with compound 1.

Example 4. GHB Detection in Beverages Through a Polypropylene Strip

[0075] A strip of a 100% polypropylene non-woven fabric (NWF) of 0.5×3.0 cm was introduced into the beverage for three seconds and then brought to a vial containing a 125 μM concentration sensor solution in DMSO. Upon contact of the strip with the sensor, the color change from light yellow to intense orange occurred for both compounds 1 and 2 The system detects all types of combinations provided that the concentration is above 35 μM.