Patent Application
20230286931
The present invention relates to new benzoxazole-based compounds, to a process for obtaining them and to their use in the detection of γ-hydroxybutyric acid (GHB) in beverages and urine.
γ-hydroxy butyric acid (GHB) is one of the drugs used in chemical submission. It is an odorless, colorless, slightly salty compound sometimes used for recreational purposes. However, this compound, when supplied without knowledge of the victim, has been used on numerous occasions in order to commit an offense. In many of them, it is used to void the will of the victims and carry out sexual assault. In general, the intake of the drug by the victim occurs through a beverage that has been contaminated with the product without noticing the product. Residence time in the body is 3-6 hours and its metabolites are excreted rapidly, so that it is very difficult to detect their presence in the body after the assault.
Four references addressing this subject have been found in the scientific literature (J. Forensic. Sci. 2004, 49, p. 379-387, Chem. Commun. 2013, 49, p. 6170-6172, Chem. Commun. 2014, 50, p. 2904-2906, J. Mat. Chem. B 2017, 8, p. 2736-2742).
The first case (J. Forensic. Sci. 2004, 49, p. 379-387) relates to an enzymatic reaction while the other three reviews are referring to chemical sensors.
Chem. Commun. 2014, 50, 2904-2906 describes the use of Compound A as chemosensor for detecting GHB. The interaction with the analyte was based on a hydrogen bonding interaction between the phenol group on the sensor and the carboxylate unit on GHB.
In addition, the same authors also described in Chem. Commun. 2013, 49, 6170-6172 the use of Compound B for detecting the corresponding lactone (GBL), but again, the interaction was related to the formation of a hydrogen bond. However, in any case, soft drinks alone or mixed with alcoholic beverages were not tested as interferents.
On the other hand, J. Mat. Chem. B 2017, 8, 2736-2742 describes the preparation of a complex of Ir (C) capable of detecting GHB. The complex showed luminescence quenching in the presence of analyte that was observed using UV light.
By following a different approach, Bravo D. T.; Harris, D. O. Parsons S. M. J. Forensic. Sci. 2004, 49, p. 379-387 described the use of γ-hydroxybutyrate dehydrogenase (GHB-DH) to detect GHB. Enzymatic oxidation of GHB by NAD+ is combined with reduction of a dye showing a colour change. Ethanol is an interferent that can be avoided by drying the sample to be studied.
The present inventors have been able to develop a reliable and easy-to-use procedure and device that enables visual determination of whether a beverage, whether alcoholic, non-alcoholic, or combined, has been contaminated with GHB prior to ingestion. Additionally, the ready-to-use device only needs a drop of the beverage under analysis so that in case the response is negative, it can be ingested substantially entirely without any problems. Likewise the present inventors have applied the same process for detecting GHB in urine.
In a first aspect, the present invention relates to a compound 1 or 2 according to claim 1.
In a second aspect, the present invention also relates to a composition comprising a compound 1 or 2 according to the first aspect.
In a third aspect, the present invention also relates to a process for obtaining a compound 1 or 2 according to the first aspect.
In a fourth aspect, the present invention relates to the use of a compound 1 or 2 according to the first aspect or a composition according to the second aspect for the detection of GHB in beverages or urine.
In a fifth aspect, the present invention relates to a procedure for detecting GHB in beverages.
In a sixth aspect, the present invention relates to a procedure for detecting GHB in urine.
In a seventh aspect, the present invention relates to an equipment or kit for use in detecting GHB in beverages and/or urine.
In a first aspect, the present invention relates to a compound of formula:
wherein R is
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 or consists of Compound 1 or 2, as defined above, and an organic solvent. DMF (dimethylformamide) and DMSO (dimethylsulfoxide) may be used as the organic solvent, although DMSO is preferably used.
In a third aspect, the present invention relates to a process for obtaining compound 1, wherein R is
comprising the step:
The reaction conditions are not particularly relevant as it can be carried out at room P and T, although an increase in temperature would likely increase the reaction rate.
The present invention also relates to a process for obtaining compound 2, wherein R is
comprising the step:
The reaction conditions are not particularly relevant as it can be carried out at room P and T.
In a fourth aspect, the present invention relates to the use of a composition, as defined above, or 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. When reference is made to alcoholic beverages, alcoholic distillates such as, for example, whiskey or gin, as well as alcoholic beverages that are not distilled, such as wine and beer, are included.
The present invention relates to the use of a composition, as defined above, or Compound 1 or 2, as defined above, in the detection of γ-hydroxybutyric acid (GHB) or a salt thereof in urine.
In a fifth aspect, the present invention relates to a process for detecting GHB or a salt thereof in beverages, comprising:
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).
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, most preferably 0.4 mM.
In another preferred embodiment, compound 1 or 2 is at a concentration from 50 μM to 500 μM, preferably from 50 μM to 100 μM.
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. If compound 1 reacts positively with GHB or a salt thereof, a color change (from yellow to red) will be observed, while if compound 2 reacts positively with GHB or a salt thereof, fluorescence will be observed (from dim green to intense yellow in distilled alcoholic beverages and from blue to green in combined beverages, soft drinks, or undistilled alcoholic beverages, such as beer, wine, or vermouth). Thus, one can conclude in step d) about the presence or not of GHB in the beverage being analyzed.
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:
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:
Compound 1 or 2 is preferably at a concentration of 50 μM to 500 μM, preferably 50 μM to 100 μM.
The reaction is considered positive in GHB if in step c) a colorless to red color change is observed with compound 1 and an appearance of fluorescence is observed with compound 2.
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.
In a sixth aspect, the present invention relates to a process for detecting GHB or a salt thereof in urine, comprising:
To obtain positive results the urine sample should be analyzed between 0 and 3 hours after drinking the beverage.
In a preferred embodiment, said weak base in step b) 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 1.2 mM.
In another preferred embodiment, compound 1 or 2 is at a concentration of 0.5 mM to 1.5 mM, preferably 1 mM.
In step e) 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 urine sample to be analyzed. If compound 1 reacts positively with GHB or a salt thereof, a color change (from yellow to red) will be observed, while if compound 2 reacts positively with GHB or a salt thereof, fluorescence will be observed. Thus, one can conclude in step f) about the presence or not of GHB in the beverage being analyzed. If one wishes to quantitatively know the amount of GHB present in the sample, e.g., UV spectroscopy may be applied.
DMF (dimethylformamide) and DMSO (dimethylsulfoxide) may be used as, but not limited to, the organic solvent for step d), although DMSO is preferably used.
In a seventh aspect, the present invention relates to a kit or equipment for use in detecting GHB or a salt thereof in beverages and/or urine comprising:
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:
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.
As noted above herein, the weak base used for kit for beverages without strip detection and/or urine 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 when detecting beverages is 0.4-1 mM, even more preferably 0.4 mM. In the case of a urine sample, the concentration of the aqueous solution of weak base is preferably from 0.4 mM to 12 mM
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 from 50 μM to 500 μM, preferably from 50 μM to 150 μM, more preferably from 50 μM to 100 μM. In the case of a urine sample, compound 1 or 2 is preferably present at a concentration from 0.5 mM to 1.5 mM, preferably 1 mM.
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., Eppendorf™).
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.
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.
Preferably, said alcoholic beverage is selected from, by way of non-limiting example, gin, vodka, rum, whisky, white wine, vermouth, and beer.
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.
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.
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:
In a 50 L two-neck round-bottom flask, under inert atmosphere, 202 mg of 3-amino-2-naphthol (1.256 mmol) were dissolved along with 191 mg of 4-nitrophenyl isothiocyanate (1.047 mmol) in 13 mL of pyridine. Stirring was kept for 8 h at room temperature. The solvent was then evaporated, the solid dissolved in 35 mL of AcOEt and washed once with acidic water (about pH 5), once with NaHCO3 (sat) and once with NaCl (sat). The organic phase was dried over MgSO4, filtered and the solvent evaporated in vacuo. The crude was purified by silica-gel chromatography column using as eluent a 6:4 Hexane:AcOEt mixture. A slightly-orange yellow solid (Compound 1) was obtained in 35% yield.
In a 25 mL one-neck round-bottomed flask, 45 mg of 3-amino-2-naphthol (0.283 mmol) were dissolved along with 98 mg of fluorescein-5-thioisocyanate (0.283 mmol) in 3 mL of THF, 50 μL of Net3 were then added and allowed to stir at room temperature for 16 h. 34 μL of 30% H2O2 (0.565 mmol) and 1 mg of tetrabutylammonium iodide (0.003 mmol) were then added. An orange solid (Compound 2) was obtained in 79% yield.
Initially, beverages are contaminated with GHB at a concentration of 12 mM. 100 μL of beverage are then taken and mixed with 100 μL of NaHCO3 (1 mM or 0.4 mM). 30 μL of this solution were then mixed with 50 μL of sensor (from a 1 mM solution, in DMSO), previously dissolved in 920 μL of DMSO. The same process was followed for samples without GHB.
The results with Compound 1 and Compound 2 are seen in
The results of a new assay with compound 1 and 2 on Fanta® orange beverages and white wine are seen in
The assay was applied again with compound 1 and 2 but with the following beverages: rum, gin, vodka and whisky. The results of the color changes are seen in
Finally, the assay with compounds 1 and 2 was applied to the gin and tonic and whisky-cola beverage. The results are seen in
A C18 column (ExtraBond Cartridge C18 500 mg, 2 ml (Scharlau C18500-03 L)) was used through which 1 mL of methanol and 1 mL of water were initially passed and discarded. 5 mL of a urine sample from the subject was then passed and collected in a petri dish. The column was washed first with 3 mL of a 5% methanol solution in deionized water followed by 3 mL of methanol. Both wash liquors were collected in the same Petri dish where the urine sample had been collected.
The solution was lyophilized to dryness and re-dissolved in 1 mL of water.
10 μL of the prepared urine was mixed with 10 μL of a 1 mM NaHCO3 aqueous solution and 100 μL of a 1 mM sensor solution. The mixture was diluted to completion of 3 mL with DMSO. The color change was observable to the naked eye or more accurately by UV spectroscopy, which allowed to further quantify the amount of GHB present in the urine sample.
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, a color change into red occurred in the case of Compound 1 and strong fluorescence appeared in the case of Compound 2. The system detects all types of combinations provided that the concentration is above 35 μM.
| Number | Date | Country | Kind |
|---|---|---|---|
| P202030840 | Aug 2020 | ES | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/ES2021/070595 | 8/5/2021 | WO |
