Method, composition and kit for psychoactive substances detection

Abstract

The present invention describes the development of a method for the detection of psychotropic substances having an isatin or indazole core. A further purpose of the invention is a kit for the determination of these psychotropic substances.

Description

[0001] METHOD, COMPOSITION AND KIT FOR PSYCHOACTIVE SUBSTANCES DETECTION

[0002] Technical field

[0003] The present invention describes a chemical-colorimetric method for the presumptive recognition of psychoactive or narcotic substances and their respective chemical classes.

[0004] Known art

[0005] The clandestine trade in narcotic drugs has always been a global plague that continues undaunted to take root from country to country, giving lifeblood to a market that acts in the shadow of legality.

[0006] Traffickers, smugglers, drug dealers are continuing over time to devise strategic alternatives to circumvent the control measures of the police, bypassing customs inspections to move between the borders of neighbouring countries, escaping raids, searches or, as in the case of the so-called "smart drugs", synthesizing new psychoactive compounds not yet identified and therefore not comprised in the tables of narcotic substances by the interested State.

[0007] It is clear that the placing on the market of new synthetic derivatives, often different from their predecessors due to slight changes in the chemical structure, grants drug traffickers the access to a profitable market that cannot be prosecuted by justice and therefore difficult to contain. Unfortunately, the continuous efforts to gradually identify the individual narcotic drugs illegally marketed are partially thwarted by the sudden introduction of new analogues, similar in activity but nevertheless not impeachable precisely because of the aforementioned variations in the structural skeleton.

[0008] It is precisely this situation, therefore, that triggers an incessant race against time between criminals who introduce new synthetic assets and the justice system that tries to recognize them and make them illegal.

[0009] In recent decades, more than 80% of the synthetic cannabinoids seized and identified by law enforcement have been attributed to three chemical classes to which they belong, different from each other due to the nature of the central aromatic ring constituting the molecules.

[0010] In particular, the three scaffolds used as building blocks on which to install the different functional groups are respectively: indole, indazole and isatin.

[0011] Indole is so far the most widely used building block and is therefore present in many synthetic derivatives, many of which have already been characterized and comprised in the list of psychoactive substances of the Italian Ministry of Health. The last two decades have instead seen the emergence of the indazole nucleus, a bio isostere of indazole, strategically chosen by clandestine producers as a synthetic alternative to remain temporarily within the boundaries of legality. In the last 2 / 3 years, however, the use of a new core that is little known and not very widespread even in nature for the synthesis of new narcotics has taken hold: isatin.

[0012] The lack of ubiquity is found mainly in medicine since Sunitinib, Ropinirole and Nintedanib are some of the few available examples of active ingredients existing to contain an isatin core. However, due to its attractive organic structure, isatin is arousing considerable interest for its versatility and is pushing a growing number of drug traffickers to implement it in the creation of new smart drugs.

[0013] However, it is necessary to keep in mind that the unequivocal and incontrovertible identification of a chemical substance, be it a flavoring, an herbicide or a narcotic, requires the use of particularly sophisticated over- the-counter equipment (e.g., UPLC-MS, IR, NMR) that produce responses in a rather long time and require the presence of highly qualified personnel.

[0014] It is evident that these devices are far from suitable for ascertaining, following inspections or roadside checks by police patrols, the hypothesis of a crime for possession or dealing in psychotropic substances, the times would be anything but rapid for the authorization of the seizure of these substances. Precisely for this reason, faster and easier tests have been developed to be performed in situ to assess whether the unknown substance subjected to the arrest of the police patrol is presumably narcotic and then proceed with the seizure (in case of positivity to the assay) for a definitive confirmation at appropriate chemical laboratories equipped with the accurate analytical instruments mentioned above.

[0015] For instance, the test developed by SIRCHIE (NARK II Synthetic Cannabinoid Reagent) and commonly used by law enforcement agencies to test suspect substances involves immersing a sample in a solution of sulfuric acid and potassium nitrite to induce a nitrosation reaction of electron-rich aromatic rings, preferably of an indole nature.

[0016] In the event of a reaction, the solution shows an evident change of color detectable to the naked eye which therefore confirms the hypothesis of crime and which, as previously mentioned, being a presumptive assay, will require confirmation from the chemical laboratory.

[0017] However, this reaction also involves positivity for substances with an indole core that are not narcotic drugs such as indomethacin or melatonin. This aspect is widely documented both in the international press (American press link: https: / / spectrumlocalnews.com / nys / central- ny / politics / 2023 / 11 / 30 / new-york-punished-2-000-prisoners-over-false- positive-druq-tests--report-finds; https: / / www.wbur.org / news / 2021 / 12 / 02 / department-of-correction-rnail- drug-testing-ruling) and in the scientific literature (Drug Test Anal. 2018; 10:95-108.)

[0018] Therefore, the need is felt to have a presumptive test available for the detection of psychotropic substances, a test that is easily performed without the need for specific technical preparation and without having to make use of sophisticated, expensive and / or bulky instruments that often require the presence of a qualified technician.

[0019] In the literature, some scientific papers have described the use of isatins, or their derivatives, as chemical sensors for the detection of fluorides in unknown samples.

[0020] In particular, paper

[0001] describes the use of isatin as a selective chemosensor for fluoride anions, while paper [2], for the same purpose, describes an isatin core structure (scheme A).

[0021] Scheme A

[0022] Again, paper [3] describes a further isatin derivative with a pentafluorinated aromatic pendant group that is used for the detection of acetate and fluoride anions, and whose structure is reported in scheme B.

[0023] Scheme B

[0024] In a further scientific paper [4], another derivative built on an isatin scaffold (Scheme C) to be used as a sensor for the detection of dihydrogen phosphate (H2PO4-), acetate, and fluoride anions is reported. Scheme C

[0025] In paper [5], an isatin scaffold (Scheme D) is presented which, in addition to responding to the presence of the fluoride anion, is also sensitive to temperature and irradiation variations.

[0026] Similar to what has just been described is the behaviour of the isatin-core compound (Scheme E) reported in paper [6],

[0027] In paper [7], it is demonstrated that the introduction of a carbonyl residue between the 2 nitrogen atoms on the hydrazonic system of the isatin derivative (Scheme F) does not change the tautomeric behaviour of these molecules with respect to fluoride and other anions.

[0028]

[0029] Scheme F

[0030] Finally, the structural analogue (scheme G) reported in paper [8] also has the same property with respect to the fluoride anion.

[0031] Scheme G

[0032] To the knowledge of the inventors, the compounds identified above have no psychotropic activity.

[0033] There is no indication in the cited known art to suggest that compounds with psychotropic activity can be detected by rapid tests without the aid of complex equipment.

[0034] Unless explicitly excluded in the description of this document, what is described in this paragraph is to be considered as part of the detailed description of the present invention.

[0035] Technical Problem

[0036] The presumptive tests currently made available by SIRCHIE or similar companies specialized in forensic chemistry are anything but selective and therefore subject to frequent false positives, especially considering that aromatic scaffolds (e.g. imidazoles, indoles, indazoles, pyrazoles) are widely present in organic compounds, natural and synthetic, for the most diverse functions and purposes.

[0037] Not only that, but even in the case of correct detection of a psychoactive substance, they do not provide any information about the class to which this compound belongs, thus making its recognition more complicated (in cases where the chemical structure is not yet known) in specialized laboratories and thus lengthening identification times.

[0038] Summary of the invention

[0039] It is an object of the present invention a method for the detection of psychotropic substances according to claim 1 .

[0040] Another object of the invention is a kit for the determ ination of psychotropic substances.

[0041] Another object of the invention is the use of a basic and / or fluorinated reagent for the determination of psychotropic substances in a sample.

[0042] These and other objects of the invention will be evident from the detailed description that follows, together with the attached figures.

[0043] Brief description of the Figures

[0044] The invention will be described below in at least one preferred embodiment for the sole purpose of explanation and not limiting the scope of the present invention with the help of the figures in the annex, wherein:

[0045] • Figure 1 : comparison between two vials related to example 1 wherein the bathochrome effect is evident;

[0046] • Figure 2: Comparison of two vials from example 2 showing the hyperchromic effect.

[0047] • Figure 3: Bathochromic effect on an isatin core compound.

[0048] • Figure 4: Hyperchromic effect on an indazole core compound.

[0049] • Figure 5: Overlapping UV-Vis spectra of 4F-MDMB-BUTINACA without TBAF (grey line) and with TBAF (black line).

[0050] • Figure 6: Overlapping UV-Vis spectra of AB-CHMINACA without TBAF (grey line) and with TBAF (black line).

[0051] • Figure 7: UV-Vis spectra superimposed on 4F-MDMB-BICA on the addition of increasing amounts of TBAF showing no significant spectral changes.

[0052] • Figure 8: Comparative colorimetric tests to evaluate the efficacy of different acetonitrile bases in the presence of BZO- 4en-P0XIZID.

[0053] Definitions

[0054] • In this document, the terms "narcotic", "cannabinoid", "synthetic cannabinoid", "psychoactive substance", " drug", "psychoactive compound", "psychotropic substance", "psychoactive substance", are to be understood as synonyms of each other and will be used randomly without alluding to different semantic meanings or significances.

[0055] • In this document, the term "presumptive test" is to be understood as a non-conclusive test, i.e. the result of which may indicate that a sample does not contain the compound of interest (negative result) or that the compound may be present (positive result).

[0056] • In this document, the term "color variation" is to be understood as a change in the color of the solution wherein the test is performed, i.e. a change in the absorption wavelength, or as a change in the intensity of the color of the solution, i.e. a change in the concentration of the test substance, following a chemical reaction that has occurred between the presumed psychoactive substance and an appropriate reagent / reactant.

[0057] In this document, the terms "approximately" or "about" as used herein when referring to a measurable value such as a quantity, a time duration, and the like, are intended to encompass variations of ±20%, ±10%, ±5%, ±1 %, or ±0.1 % from the specified value, where such variations are appropriate to perform the methods described.

[0058] • In this document, the term 'C1-C20 alkyl' comprises linear, branched or cyclic alkyl groups having from 1 to 20 total carbon atoms. This definition also comprises cycloalkyls (e.g. but not limited to cyclohexyl, cyclopentyl) possibly substituted with linear or branched alkyl chains (e.g. but not limited to methyl cyclohexyl, propyl cyclopentyl), provided that the total number of carbon atoms does not exceed 20. The alkyl group can be mono- or poly-substituted and is able to bind, directly or through an alkyl chain, to other functional groups such as aromatic rings or nitrogen atoms.

[0059] • In this document, the terms 'alkenyl' and 'alkynyl' refer to unsaturated aliphatic groups containing one or more carboncarbon double bonds ('alkenyl') or one or more carbon-carbon triple bonds ('alkynyl'), respectively, having up to 20 carbon atoms in total. These groups can be linear, branched or cyclic, and comprise cyclic structures that may be substituted with linear or branched alkenyl or alkynyl chains. The groups can be monosubstituted or polysubstituted and can bind, directly or through aliphatic chains, to other functional groups such as aromatic rings, heteroaryls or nitrogen atoms.

[0060] Detailed Description

[0061] The present invention describes a method for the presumptive detection of narcotic drugs illegally and clandestinely produced, possessed, marketed or consumed.

[0062] The method according to the present invention allows the detection of the presumed psychoactive compound directly in loco, i.e. at the site of seizure, without having to resort to the use of sophisticated, expensive and / or bulky instruments that often require the presence of a qualified technician.

[0063] Given the simplicity of the method according to the present invention, law enforcement patrols can be easily trained to perform the presumptive test following inspections, searches, seizures and checks directly on the road.

[0064] The method according to the present invention comprises simple manual operations to be performed in appropriate sequence for the development of a chemical-colorimetric reaction between a sample containing the presumed narcotic substance and an appropriate reagent / reactant added.

[0065] The colorimetric test according to the method of the present invention allows to detect the positivity or negativity of the assay directly with the naked eye, i.e. visually without the use of instruments such as ultraviolet detectors.

[0066] The chromatic variation of the method according to the present invention is to be understood either as a real color variation of the solution wherein the test is performed, i.e. a variation in the absorption wavelength, or as a variation in the intensity of the color of the solution itself following a chemical reaction that occurred between the presumed psychoactive substance and a suitable reagent / reactant.

[0067] The method according to the invention can preferably be used as a support to the investigative activity for the detection of an alleged psychoactive substance.

[0068] Advantageously, the method according to the invention can be used for the discrimination of the class to which the alleged psychoactive substance belongs so as to drastically reduce the number of combinatorial possibilities for its recognition.

[0069] Furthermore, the method according to the present invention can be used in combination with the current and commonly used narco-tests (e.g., NARK II Synthetic Cannabinoid Reagent developed by SIRCHIE) in order to achieve further information on the class to which the alleged psychoactive substance belongs.

[0070] The method according to the present invention for the detection of presumed compounds with psychoactive activity having the general formula (I) comprises the steps of:

[0071] • Taking / preparing an aliquot of a suspected sample containing the presumed psychotropic substance, e.g. approx. 20-80 mg herbaceous (solid) material or approx. 50-500 pL liquid material;

[0072] • Preparing two containers (or vials) containing equal quantities of reagent / reactant as specified below;

[0073] • Dipping or adding this sample to be examined to one of the two containers containing the reagent / reactant (the other container will instead be used as a reference to make a color comparison);

[0074] • Waiting for said reagent to react with the alleged psychotropic substance;

[0075] • Visually observing (without the aid of optical instruments such as UV detectors) the occurrence of a chromatic variation of the solution with respect to the reference container, either as an increase in the intensity itself or as a variation in the perceived chromatic tone. If necessary, is possible to help oneself by using a chromatic scale or by placing the two containers side by side on a white background.

[0076] According to the method of the present invention, the drug test described is preferably to be used for presumptive analyses on samples suspected of containing unidentified substances and presumed narcotics.

[0077] These presumed narcotic substances are obtained, by way of example but not limited to, from liquid or solid samples, such as: solutions, capsules, tablets, pills, lozenges, pastilles, vials, ampoules, powders, dried plant material, chewable tablets, tabs or blocks; in any case, samples detained, sold, produced, consumed and marketed clandestinely.

[0078] Samples taken from widely consumed substances, such as, but not limited to, cut tobacco, powdered tobacco or sugar cubes, are also suitable for narcotic inspection according to the method of the present invention. In fact, to facilitate and naturalize consumption by reducing potential suspicions, psychoactive liquid formulations contained, for instance, in vials, ampoules, bottles or containers (often pocket-sized) are commonly dripped or sprayed on top of the aforementioned products so as to facilitate and mask the illicit trade. Sometimes these products are directly impregnated / sprinkled with said psychoactive liquid formulations or immersed in liquid solutions containing them.

[0079] According to the method of the present invention, the reactive solution comprises a reagent (or reactant) dissolved or dispersed within a suitable solvent to which said suspect sample must be added or immersed in order to induce a chemical reaction between said reagent and the alleged psychotropic substance (if any) contained in said sample.

[0080] This reactant is basic.

[0081] This basic reagent can be organic or inorganic in nature, preferably organic.

[0082] This reagent can be anionic or neutral, preferably anionic in the form of a salt.

[0083] This reagent may be chosen by way of example and not limited to from the group comprising any salt consisting of an organic cation and the fluoride anion or an organic cation and the hydroxide anion.

[0084] This basic reagent is preferably chosen among quaternary ammonium fluorides.

[0085] Preferably, quaternary ammonium fluoride is chosen among tetrabutylammonium fluoride (NBU4F or TBAF), tetrapropylammonium fluoride, tetraethylammonium fluoride, tetramethylammonium fluoride (TMAF), benzyltrimethylammonium fluoride (BTAF).

[0086] In a preferred embodiment, the basic reagent is tetrabutylammonium fluoride (TBAF).

[0087] This basic reagent can also be chosen among quaternary ammonium hydroxides.

[0088] Preferred, are tetramethylammonium hydroxide, tetraethylammonium hydroxide, tetrapropylammonium hydroxide, tetrabutylammonium hydroxide, tetrapentylammonium hydroxide, most preferred is tetrabutylammonium hydroxide.

[0089] According to the method of the present invention, the solvent constituting the reactive solution is capable of dissolving / solubilizing or dispersing said reactant.

[0090] This solvent can be aqueous or organic, preferably organic.

[0091] Said organic solvent can be polar or non-polar, preferably polar.

[0092] Said solvent, preferably organic and polar, can be protic or aprotic, more preferably aprotic.

[0093] Said solvent is preferably an organic, polar, and aprotic solvent and may be chosen by way of example but not limiting to the group comprising acetonitrile, dimethyl sulfoxide, dimethylacetamide (DMA), dimethylformamide (DMF), acetone, 1 ,4-dioxane, tetrahydrofuran (THF), hexamethylphosphoramide (HMPA), tetramethyl urea, ethyl acetate (EtOAc), butyl acetate, methyl isobutyl ketone, 1 ,3-dimethyl-3, 4,5,6- tetrahydro-2-pyrimidinone (DMPU), preferably acetonitrile.

[0094] In a preferred embodiment, the aprotic polar solvent is acetonitrile. In fact, the inventors observed that, compared to other polar aprotic solvents, this solvent is more able to enhance the chromatic variation, both in terms of intensity and in terms of shift of the absorption length.

[0095] According to the method of the present invention, this reactive solution for the detection of the presence of presumed psychotropic substances is contained within containers such as, but not limited to, glass vials, plastic tubes, disposable "Eppendorf" tubes or other suitable containers, preferably sealed and sealable

[0096] Preferably, the titre of the reactive solution, i.e. the concentration of the dissolved or dispersed reagent in the solvent, is known. Preferably, the titre of said reactive solution is between 0.1 and 10 mM.

[0097] Preferably, the molar ratio between the presumed substance of psychotropic activity having one of the general formulas (I), 1A and 1 B, and the base, preferably chosen between quaternary ammonium fluorides and quaternary ammonium hydroxides, is greater than 0.1 , preferably between 0.1 -20, more preferably between 0.1 -2.

[0098] It has been experimentally observed that, below the value of 0.1 , the chromatic variation is difficult to appreciate with the naked eye. It was also found that, beyond a molar ratio of 20, the effectiveness of the reaction reaches a plateau, without further appreciable improvements in the variation of the chromatic tone, understood both as an increase in the intensity of absorption and as a shift in the absorption wavelength.

[0099] According to a preferred embodiment, the reactive solution for the detection of the presence of presumed psychotropic substances is a solution of tetrabutylammonium fluoride (TBAF) dissolved in acetonitrile (ACN).

[0100] This reactive solution (ACN and TBAF) has a colour chosen between quaternary ammonium fluorides and natural pale yellow I straw yellow quaternary ammonium hydroxides.

[0101] This reactive solution is preferably dosed with a volume of about 200 pL.

[0102] The psychotropic substances that can be analyzed by the method of the present invention have the following general formula (I):

[0103] General formula (I) wherein indicates a single bond or a double bond, with the constraint that, when the C=Y bond is double, the C-X bond is single and vice versa, and the double bond can have E or Z configuration;

[0104] X, Y, independently of each other = N, C=O;

[0105] R1 =

[0106] • alkyl (linear or branched or cyclic) with length between Ci and C20 as methyl (Me-), ethyl (Et-), propyl (Pr-), isopropyl (iPr-), sec-butyl, 1 -pentyl, 2-pentyl, 1 -hexyl, 2-hexyl, 3-hexyl;

[0107] • alkenyl or alkynyl (linear or branched or cyclic) with length between Ci and C20, preferably C2-C20, and containing one or more unsaturation both terminal and internal to the chain such as 1 -pentenyl, 2-pentenyl, 1 -hexenil, 2-hexenyl, 2-pentynyl, 3- hexynyl;

[0108] • alkyl or alkenyl or alkynyl (linear or branched or cyclic) with length between Ci and C20, preferably C2-C20, and substituted with at least one halogen such as 5-Fluoropentyl, 4- Fluoropentyl, 4-Chlorobutyl;

[0109] • alkyl or alkenyl or alkynyl (linear or branched or cyclic) with a length between Ci and C20, preferably C2-C20, containing at least one functional group chosen among carbonyl, ester, amide, carboxylic acid, amine, ether, hydroxyl, halide and combinations thereof;

[0110] • cyclic alkyl with a ring size between C4 and Cs such as cyclopentyl, cyclohexyl, cycloheptyl;

[0111] • heterocyclic alkyl with a ring size between C4 and Cs, and containing heteroatoms such as N, S and 0, such as tetrahydrofuranyl, tetrahydropyranyl; phenyl (CeHs-) or aryl, possibly substituted with at least one group chosen by way of example but not limited to from the group comprising nitro group (-NO2), sulfonic group (-SO3H), phosphate group (-POs2-), alkyl group (linear or branched or cyclic) with a length between Ci and C20,

[0112] • acyl (linear or branched or cyclic) with length between Ci and C20, preferably C2-C20, such as acetyl (MeCO-), propanoyl (CH3CH2CO-), butanoyl (CH3CH2CH2CO-);

[0113] • acyl (RCO-) (linear or branched or cyclic) with a length between Ci and C20, preferably C2-C20, containing in the alkyl chain (R) at least one functional group chosen among carbonyl, ester, amide, carboxylic acid, amine, ether, hydroxyl, halide and combinations thereof;

[0114] • aroyl (ArCO-) wherein aryl (Ar) can be a pure phenyl, i.e. unsubstituted (CeHs-), or a substituted phenyl with one or more functional groups chosen as examples from fluorine (-F), chlorine (-CI), nitro group (-NO2), sulphonic group (-SO3H), alkoxy group such as methoxide (-OMe), ethoxide (-OEt), propoxide (-OPr), isopropoxide (-OiPr), butoxide (-OBut);

[0115] R2 =

[0116] • hydrogen (H)

[0117] • alkyl (linear or branched or cyclic) with length between Ci and C20, such as methyl (Me-), ethyl (Et-), propyl (Pr-), isopropyl (iPr-), sec-butyl, 1 -pentyl, 2-pentyl, 1 -hexyl, 2-hexyl, 3-hexyl;

[0118] • alkenyl or alkynyl (linear or branched or cyclic) with length between Ci and C20, preferably C2-C20, and containing one or more unsaturation both terminal and internal to the chain such as 1 -pentenyl, 2-pentenyl, 1 -hexenil, 2-hexenyl, 2-pentynyl, 3- hexynyl;

[0119] • alkyl or alkenyl or alkynyl (linear or branched or cyclic) with length between Ci and C20, preferably C2-C20, and substituted with at least one halogen such as 5-Fluoropentyl, 4- Fluoropentyl, 4-Chlorobutyl;

[0120] • alkyl or alkenyl or alkynyl (linear or branched or cyclic) with a length between Ci and C20, preferably C2-C20, containing at least one functional group chosen among carbonyl, ester, amide, carboxylic acid, amine, ether, hydroxyl, halide and combinations thereof;

[0121] • cyclic alkyl with a ring size between C4 and Cs such as cyclopentyl, cyclohexyl, cycloheptyl;

[0122] • heterocyclic alkyl with a ring size between C4 and Cs, and containing heteroatoms such as N, S and 0, such as tetrahydrofuranyl, tetrahydropyranyl;

[0123] • phenyl (CeHs-) or aryl, possibly substituted with at least one group chosen by way of example but not limited to from the group comprising nitro group (-NO2), sulfonic group (-SO3H), phosphate group (-PCs2-), alkyl group (linear or branched or cyclic) with a length between Ci and C20;

[0124] • acyl (linear or branched) with length between Ci and C20, preferably C2-C20, such as acetyl (MeCO-), propanoyl (CH3CH2CO-), butanoyl (CH3CH2CH2CO-);

[0125] R3 =

[0126] • hydrogen (H)

[0127] • alkyl (linear or branched or cyclic) with length between Ci and C20, such as methyl (Me-), ethyl (Et-), propyl (Pr-), isopropyl (iPr-), sec-butyl, 1 -pentyl, 2-pentyl, 1 -hexyl, 2-hexyl, 3-hexyl;

[0128] • alkenyl or alkynyl (linear or branched or cyclic) with length between Ci and C20, preferably C2-C20, and containing one or more unsaturation both terminal and internal to the chain such as 1 -pentenyl, 2-pentenyl, 1 -hexenil, 2-hexenyl, 2-pentynyl, 3- hexynyl; • alkyl or alkenyl or alkynyl (linear or branched or cyclic) with length between Ci and C20, preferably C2-C20, and substituted with at least one halogen such as 5-Fluoropentyl, 4- Fluoropentyl, 4-Chlorobutyl;

[0129] • alkyl or alkenyl or alkynyl (linear or branched or cyclic) with a length between Ci and C20, preferably C2-C20, containing at least one functional group chosen among carbonyl, ester, amide, carboxylic acid, amine, ether, hydroxyl, halide and combinations thereof;

[0130] • cyclic alkyl with a ring size between C4 and Cs such as cyclopentyl, cyclohexyl, cycloheptyl;

[0131] • heterocyclic alkyl with a ring size between C4 and Cs, and containing heteroatoms such as N, S and 0, such as tetrahydrofuranyl, tetrahydropyranyl;

[0132] • phenyl (CeHs-) or aryl, possibly substituted with at least one group chosen by way of example but not limited to from the group comprising nitro group (-NO2), sulfonic group (-SO3H), phosphate group (-POs2-), alkyl group (linear or branched or cyclic) with a length between Ci and C20;

[0133] • acyl (linear or branched or cyclic) with length between Ci and C20, preferably C2-C20, such as acetyl (MeCO-), propanoyl (CH3CH2CO-), butanoyl (CH3CH2CH2CO-);

[0134] • acyl (RC0-) (linear or branched or cyclic) with a length between Ci and C20, preferably C2-C20, containing in the alkyl chain (R) at least one functional group chosen among carbonyl, ester, amide, carboxylic acid, amine, ether, hydroxyl, halide and combinations thereof;

[0135] • aroyl (ArCO-) wherein aryl (Ar) can be a pure phenyl (CeHs-) or a substituted phenyl with one or more functional groups chosen by way of example from fluorine (-F), chlorine (-CI), nitro group (-NO2), sulphonic group (-SO3H), alkoxide group such as methoxide (-OMe), ethoxide (-OEt), propoxide (-OPr), isopropoxide (-OiPr), butoxide (-OBut).

[0136] According to a preferred embodiment, the method is conducted on the compounds of the general formula (I), except on the following compounds:

[0137] The method according to the present invention allows, through a colorimetric variation, to be understood either as a shift in the absorption wavelength or as an increase in the intensity of absorption, to identify the presence of presumed narcotic substances within suspect samples, preferably constituting the object of trafficking, sale, possession or illegal consumption, wherein these narcotic substances have a structural formula according to the general formula (I).

[0138] Preferably, the method of invention allows the identification of psychotropic substances having at least one aromatic core of an isatin nature (isatin core, 1 H-indole-2, 3-dione, scheme 1 , left) or of an indazole nature (indazole core, scheme 1 , right). isatin core indazole core

[0139] Scheme 1

[0140] Isatin class

[0141] The reactive solution according to the invention, in the presence of a presumed psychoactive substance having the general formula 1A indicated below and having a functionalized isatin core in position 3 with a hydrazine group bound to the residue of a benzoic acid, triggers a chemical reaction capable of generating an evident and easily identifiable chromatic variation of the solution. More specifically, the pale-yellow characteristic of the initial reactive solution evolves to a bright orange, visually perceptible and observable. The bathochrome effect (red-shift or increase in the A absorption wavelength) that occurs is peculiar and qualitatively indicative of the presence of the isatin aromatic nucleus. (Figure 1 ).

[0142] General Formula 1A

[0143] Wherein • hydrogen (-H)

[0144] • alkyl (linear or branched or cyclic) having a length between Ci and C10 as pentyl, 4-pentenyl, hexyl, butyl;

[0145] • alkenyl or alkynyl (linear or branched or cyclic) with length between Ci and C20, preferably C2-C20, and containing one or more unsaturation both terminal and internal to the chain such as 1 -pentenyl, 2-pentenyl, 1 -hexenil, 2-hexenyl, 2-pentynyl, 3- hexynyl;

[0146] • alkyl or alkenyl or alkynyl (linear or branched or cyclic) with length between Ci and C20, preferably C2-C20, and substituted with at least one halogen such as 5-Fluoropentyl, 4- Fluoropentyl, 4-Chlorobutyl;

[0147] • cycloalkyl having a length between C4 and C7 as cyclohexyl;

[0148] R5 =

[0149] • hydrogen (-H), fluorine (-F), chlorine (-CI), nitro group (-NO2), sulfonic group (-SO3H), alkoxide group as methoxide (-OMe), ethoxide (-OEt), propoxide (-OPr), isopropoxide (-OiPr), butoxide (-OBut);

[0150] R6 =

[0151] • hydrogen (-H), fluorine (-F), chlorine (-CI), nitro group (-NO2), sulfonic group (-SO3H), alkoxide group as methoxide (-OMe), ethoxide (-OEt), propoxide (-OPr), isopropoxide (-OiPr), butoxide (-OBut).

[0152] Synthetic narcotic drugs of isatin derivation commonly differ from each other for the structure of the organic chain linked to nitrogen at position 1 , i.e. for structural changes made by way of example and not limited to the length, branching, degree of unsaturation, the presence of heteroatoms of said chain or the presence of any stereogenic center or any substitutions on the aromatic ring.

[0153] Advantageously, these structural alterations of the chain are not such as to significantly influence the interaction of the molecule with visible radiation, thus standardizing the chromatic response of all potential narcotics belonging to the isatin class once exposed to the reactive solution.

[0154] In other words, regardless of the structural nature of the organic pendant group attached to the molecule on the nitrogen at position 1 , the definite bathochromic variation (AA) (which can range from pale yellow to bright orange) triggered by the acid-base reaction with the reactive solution will be common to all molecules belonging to the same isatin class, i.e. which can be described by the general formula A.

[0155] Consequently, in the event that the method according to the invention is applied for the examination of a substance suspected of containing psychoactive cannabinoid derivatives, a bathochromic shift will not only be an indication of the probable presence of such illegal synthetic derivatives but will be sufficient to deduce the belonging of the alleged narcotic to the class of isatins.

[0156] As will be expressed in more detail in the examples given in the Examples section, the inventors have developed some experimental tests for the validation of the invention method.

[0157] In particular, by way of example and representative of the isatin class, the compound with psychoactive activity BZ0-4en-P0XIZID was tested. Contact with the reactive solution (acetonitrile and TBAF) generated a clear change in the chromatic tone, which went from colourless to intense orange.

[0158] Indazole class

[0159] According to the invention, the reactive solution, in the presence of a presumed psychoactive substance of general formula 1 B indicated below, triggers a chemical reaction capable of generating, exclusively, an increase in the chromatic intensity of the solution.

[0160]

[0161] • alkyl (linear or branched or cyclic) with length between Ci and C20 as methyl (Me-), ethyl (Et-), propyl (Pr-), isopropyl (iPr-), sec-butyl, 1 -pentyl, 2-pentyl, 1 -hexyl, 2-hexyl, 3-hexyl;

[0162] • alkenyl or alkynyl (linear or branched or cyclic) with length between Ci and C20, preferably C2-C20, and containing one or more unsaturation both terminal and internal to the chain such as 1 -pentenyl, 2-pentenyl, 1 -hexenil, 2-hexenyl, 2-pentynyl, 3- hexynyl;

[0163] • alkyl or alkenyl or alkynyl (linear or branched or cyclic) with length between Ci and C20, preferably C2-C20, and substituted with at least one halogen such as 5-Fluoropentyl, 4- Fluoropentyl, 4-Chlorobutyl;

[0164] • alkyl or alkenyl or alkynyl (linear or branched or cyclic) with a length between Ci and C20, preferably C2-C20, containing at least one functional group chosen among carbonyl, ester, amide, carboxylic acid, amine, ether, hydroxyl, halide and combinations thereof;

[0165] • cyclic alkyl with a ring size between C4 and Cs such as cyclopentyl, cyclohexyl, cycloheptyl; • heterocyclic alkyl with a ring size between C4 and Cs, and containing heteroatoms such as N, S and 0, such as tetrahydrofuranyl, tetrahydropyranyl;

[0166] • phenyl (CeHs-) or aryl, possibly substituted with at least one group chosen by way of example but not limited to from the group comprising nitro group (-NO2), sulfonic group (-SO3H), phosphate group (-POs2-), alkyl group (linear or branched or cyclic) with a length between Ci and C20;

[0167] R2 =

[0168] • hydrogen (-H), alkyl (linear or branched or cyclic) with length between Ci and C20 as methyl (Me-), ethyl (Et-), propyl (Pr-), isopropyl (iPr-), sec-butyl, 1 -pentyl, 2-pentyl, 1 -hexyl, 2-hexyl, 3-hexyl;

[0169] • alkenyl or alkynyl (linear or branched or cyclic) with length between Ci and C20, preferably C2-C20, and containing one or more unsaturation both terminal and internal to the chain such as 1 -pentenyl, 2-pentenyl, 1 -hexenil, 2-hexenyl, 2-pentynyl, 3- hexynyl;

[0170] • alkyl or alkenyl or alkynyl (linear or branched or cyclic) with length between Ci and C20, preferably C2-C20, and substituted with at least one halogen such as 5-Fluoropentyl, 4- Fluoropentyl, 4-Chlorobutyl;

[0171] • alkyl or alkenyl or alkynyl (linear or branched or cyclic) with a length between Ci and C20, preferably C2-C20, containing at least one functional group chosen among carbonyl, ester, amide, carboxylic acid, amine, ether, hydroxyl, halide and combinations thereof;

[0172] • cyclic alkyl with a ring size between C4 and Cs such as cyclopentyl, cyclohexyl, cycloheptyl;

[0173] • heterocyclic alkyl with a ring size between C4 and Cs, and containing heteroatoms such as N, S and 0, such as tetrahydrofuranyl, tetrahydropyranyl;

[0174] • phenyl (CeHs-) or aryl, possibly substituted with at least one group chosen by way of example but not limited to from the group comprising nitro group (-NO2), sulfonic group (-SO3H), phosphate group (-POs2-), alkyl group (linear or branched or cyclic) with a length between Ci and C20;

[0175] • acyl (linear or branched or cyclic) with length between Ci and C20, preferably C2-C20, such as acetyl (MeCO-), propanoyl (CH3CH2CO-), butanoyl (CH3CH2CH2CO-);

[0176] R3 =

[0177] • hydrogen (H)

[0178] • alkyl (linear or branched or cyclic) with length between Ci and C20 as methyl (Me-), ethyl (Et-), propyl (Pr-), isopropyl (iPr-), sec-butyl, 1 -pentyl, 2-pentyl, 1 -hexyl, 2-hexyl, 3-hexyl;

[0179] • alkenyl or alkynyl (linear or branched or cyclic) with length between Ci and C20, preferably C2-C20, and containing one or more unsaturation both terminal and internal to the chain such as 1 -pentenyl, 2-pentenyl, 1 -hexenil, 2-hexenyl, 2-pentynyl, 3- hexynyl;

[0180] • alkyl or alkenyl or alkynyl (linear or branched or cyclic) with length between Ci and C20, preferably C2-C20, and substituted with at least one halogen such as 5-Fluoropentyl, 4- Fluoropentyl, 4-Chlorobutyl;

[0181] • alkyl or alkenyl or alkynyl (linear or branched or cyclic) with a length between Ci and C20, preferably C2-C20, containing at least one functional group chosen among carbonyl, ester, amide, carboxylic acid, amine, ether, hydroxyl, halide and combinations thereof;

[0182] • cyclic alkyl with a ring size between C4 and Cs such as cyclopentyl, cyclohexyl, cycloheptyl;

[0183] • heterocyclic alkyl with a ring size between C4 and Cs, and containing heteroatoms such as N, S and 0, such as tetrahydrofuranyl, tetrahydropyranyl;

[0184] • phenyl (CeHs-) or aryl, possibly substituted with at least one group chosen by way of example but not limited to from the group comprising nitro group (-NO2), sulfonic group (-SO3H), phosphate group (-POs2-), alkyl group (linear or branched or cyclic) with a length between Ci and C20;

[0185] • acyl (linear or branched or cyclic) with length between Ci and C20, preferably C2-C20, such as acetyl (MeCO-), propanoyl (CH3CH2CO-), butanoyl (CH3CH2CH2CO-);

[0186] • acyl (RC0-) (linear or branched or cyclic) with a length between Ci and C20, preferably C2-C20, containing in the alkyl chain (R) at least one functional group chosen among carbonyl, ester, amide, carboxylic acid, amine, ether, hydroxyl, halide and combinations thereof;

[0187] • aroyl (ArCO-) wherein aryl (Ar) can be a pure phenyl (CeHs-) or a substituted phenyl with one or more functional groups chosen by way of example from fluorine (-F), chlorine (-CI), nitro group (-NO2), sulphonic group (-SO3H), alkoxide group such as methoxide (-OMe), ethoxide (-OEt), propoxide (-OPr), isopropoxide (-OiPr), butoxide (-OBut).

[0188] More specifically, there is no bathochromic (red shift) or hypochromic (blue-shift) effect in the solution, the variation in wavelength (AA), if present, is imperceptible to the naked eye, thus making the color of the solution (understood as the absorption wavelength) unaltered. What happens instead is an immediate increase in the chromatic intensity of the solution (hyperchromic effect, figure 2), which will go from a straw yellow (pale, faint) to a canary yellow (bright, intense).

[0189] Most of the molecules belonging to the class of indazoles differ in the nature of the organic pendant group anchored to nitrogen in position 1 , i.e. in the changes that are made by way of example and not limiting in terms of length, branching, presence of heteroatoms, unsaturations, (hetero)aromatic groups as well as stereogenic centres on the chain in position 1 but also on any substitutions on the aromatic ring.

[0190] As in the case of the class of isatins, these changes are not such as to significantly affect the interaction of the molecular chromophore with the incoming light radiation. Also in this case, once the acid-base reaction between the reactive solution and the presumed indazole-based psychotic has taken place, the colorimetric response will be identical for all molecules belonging to the class of indazoles and will consist of a visually perceptible increase in colour intensity.

[0191] Consequently, in the event that the method according to the invention is applied for the examination of a sample or a substance suspected of containing psychoactive cannabinoid derivatives, a variation in color intensity from pale yellow to bright yellow will not only be an indication of the probable presence of illegal synthetic derivatives but will be sufficient to deduce that the alleged narcotic belongs to the class of indazoles.

[0192] As will be expressed in more detail in the example forms reported in the Examples Section, the inventors performed some experimental tests for the validation of the invention method on the class of indazole compounds.

[0193] In particular, by way of example and representative of the indazole class, the following compounds with psychoactive activity have been tested;

[0194] • ADB-BUTINACA;

[0195] • 4F-MDMB-BUTINACA;

[0196] • AB-CHMINACA.

[0197] The interaction of each of the three compounds with the acetonitrile and TBAF reactive solution resulted in a marked increase in color intensity (hyperchromic effect). The effect has been scientifically documented with the aid of spectrophotometric analysis. Indole class

[0198] The method according to the present invention has not been found suitable for the recognition of presumed synthetic psychoactive derivatives belonging to the class of indoles. By subjecting any indolebased narcotic to the steps provided for by the method according to the invention, no chromatic variation is in fact perceived, neither in terms of intensity nor in terms of wavelength.

[0199] By way of example, an experimental test conducted in the presence of a substance with psychoactive activity of an indole nature, i.e. 4F-MDMB- BICA, has been reported in the Examples Section. The absence of the moieties characteristic of the indazole and isatin rings has in fact kept the coloration of the solution unchanged.

[0200] Advantageously, however, the use of the method according to the present invention in combination with the presumptive assays commonly used by law enforcement agencies can be extremely useful in identifying the class to which the alleged psychoactive compound belongs. In fact, the majority of drug tests in the hands of law enforcement are absolutely not selective and do not provide any indication of the structural nature of the psychoactive compound under examination. Therefore, since the three most widely used classes of synthetic derivatives are the isatinic, indazole and indole ones, the positivity to the common and non-selective narco-test supplied to the road patrols combined with the negativity to the test according to the method of the present invention is already indicative of the class to which the alleged drug in question belongs. It is precisely the negativity to the assay according to the method of the present invention that excludes the belonging of the presumed psychotic molecule to the isatin and indazole classes, drastically increasing the probability that it belongs to the indole class.

[0201] The method according to the invention described in this document is to be understood as a test for psychotropic substances that is particularly quick and simple to perform, indicative for the recognition of the class to which the presumed doping substance belongs and therefore useful in an initial phase of screening or broad-spectrum surveillance of the clandestine trafficking routes. However, like the other presumptive assays, it requires definitive confirmation at specialized analytical laboratories equipped with high-performance instrumentation in the recognition of unknown analytes (e.g., NMR, IR, UPLC-MS). The common practice for the recognition of a psychoactive substance is to compare the spectrometric and spectrophotometric data acquired by the alleged substance with large databases in order to ascertain a possible positive match with an already known psychoactive substance. In case of negative finding, the method according to the present invention offers the possibility of simplifying the process of exact identification of the molecule under examination as, by indicating the respective chemical class, the number of combinatorial possibilities is drastically reduced. In fact, the definition of the class to which the psychotropic substance belongs makes it possible to significantly streamline the work of structural investigation of the unknown molecule, thus speeding up the timing of incrimination of those guilty of use, production, possession or distribution of the substance and making the process of updating the list of psychotropic substances banned by the Ministry of Health faster.

[0202] Another advantage offered by the method according to the present invention is the high selectivity. The chromatic variation (to be understood either as a bathochrome effect or as a hyperchromic effect) that follows the chemical reaction between the presumed narcotic and the reagent used is highly specific and although it is linked to a visual perception, it is difficult to emulate by potential interferers contained in the sample.

[0203] An additional object of the present invention is to describe a composition suitable for the presumptive detection of the psychoactive agent in a suspect sample and for the determination (direct or indirect) of the chemical class to which it belongs.

[0204] The composition according to the present invention comprising, in one embodiment, a reagent / reactant preferably inorganic, basic and soluble in an organic solvent, even more preferably tetrabutylammonium fluoride (TBAF) dissolved / dispersed in an organic solvent, preferably non-polar aprotic, even more preferably acetonitrile, wherein the solution of said reagent / reactant in said solvent is of known titre, and a suspect sample immersed or solubilized in it.

[0205] In a preferred embodiment, the composition according to the invention is a solution of about 200 pM TBAF in acetonitrile in contact with 20-80 mg (or approx. 50-500 pL) of suspect specimen.

[0206] According to a preferred embodiment, the psychotropic substance is added to the reactive solution in such a way that the molar ratio between said psychotropic substance and the base is between 1 :10 and 1 :200.

[0207] The invention can be made in the form of a ready-to-use kit comprising:

[0208] • Two containers, preferably glass but anyway transparent, of any shape (e.g., cylindrical / spherical / rectangular)

[0209] • Reagents: e.g. approx. 200 pM solution of TBAF in acetonitrile to fill both containers (reference container and test container)

[0210] • Legend with a Color Scale to evaluate the test result

[0211] • Other possible components to better read the test, such as a white screen;

[0212] • Instructions for use.

[0213] EXAMPLES

[0214] The following examples are to be understood as embodiments that are purely illustrative and not limiting the scope of the present invention.

[0215] Materials and methods

[0216] Acetonitrile (CAS 75-05-8) and tetrabutylammonium fluoride (TBAF) (CAS 429-41 -4) were purchased from Sigma Aldrich. N-[(1 S)-1 - (aminocarbonyl)-2,2-dimethylpropyl]-1 -butyl-1 H-indazole-3-carboxamide (CAS 2682867-55-4) and (Z)-N'-(2-oxo-1 -(pent-4-en-1 -yl) indolin-3- ylidene) benzohydrazide (CAS 1048973-64-3) were purchased from Cayman Chemical. All UV-Vis spectra were acquired with a spectrophotometer from Jasco mod. V-670. Samples of narcotic substances are real samples obtained from seizures by the judicial authorities.

[0217] Example 1

[0218] Two vials containing 500 pL of a 200 pM solution of TBAF in Acetonitrile are prepared for visual comparison. Vial number 1 is not opened as it is the reference for comparison. To vial number 2 is added the sample consisting of 20 mg containing a psychoactive substance of a isatin nature (BZO-4en-POXIZID, IIIPAC nomenclature: (Z)-N'-(2-oxo-1 -(pent-4-en-1 - yl) indolin-3-ylidene)benzohydrazide)), with formula E1.

[0219] Formula E1

[0220] Immediately after the addition of the sample to be analyzed, a clear color variation (from colourless to intense orange) is noticed, indicating the positivity of the sample to psychoactive substances of isatin nature. This variation in the absorption wavelength of the compound BZO-4en- POXIZID, as far as perceptible to the naked eye, was additionally instrumentally measured (UV-Vis spectrophotometer Jasco mod. V-670) recording an absorption spectrum of the compound before and after the addition of TBAF (figure 3).

[0221] Example 2

[0222] Similarly to example 1 , two vials each containing 500 pL of a 200 pM solution of TBAF in Acetonitrile are visually compared. Vial number 1 is not opened as it is the reference for comparison. To vial number 2 is added the sample consisting of 20 mg containing a psychoactive substance of indazole nature (ADB-BUTINACA, IIIPAC: N-[(1 S)-1-(aminocarbonyl)- 2,2-dimethylpropyl]-1-butyl-1 H-indazole-3-carboxamide) with the formula E2.

[0223] Formula E2

[0224] Immediately after the addition of the sample to be analyzed, a clear intensification of the color (from colourless to intense yellow) is noticed, indicating the positivity of the sample to psychoactive substances of an indazole nature. The variation in the absorption intensity of the compound ADB-BUTINACA, as far as perceptible to the naked eye, was also instrumentally measured (UV-Vis spectrophotometer Jasco mod. V-670) recording an absorption spectrum of the compound before and after the addition of TBAF (figure 4).

[0225] Example 3

[0226] Two vials are prepared, each containing 500 pL of a 200 pM solution of TBAF in acetonitrile (ACN), following the procedure in Example 1. The reference vial remains sealed. In the second vial, 25 mg of sample containing 4F-MDMB-BUTINAC (IUPAC nomenclature: Methyl (S)-2-(1- (4-fluorobutyl)-1 H-indazole-3-carboxamide)-3,3-dimethylbutanoate), a psychoactive substance with formula E3 belonging to the class of indazoles, are added.

[0227]

[0228] Formula E3

[0229] Immediately after the addition of the sample, there is an increase in color intensity, with a transition from the colourless solution to canary yellow, easily detectable with the naked eye, confirming the positivity for psychoactive substances of indazole nature. The characteristic hyperchromic effect has been documented by UV-Vis spectrophotometric analysis (Jasco mod. V-670), recording the absorption spectra before and after the addition of the TBAF reagent (Figure 5).

[0230] Example 4

[0231] Similar to the previous examples, two identical vials containing 500 pL of TBAF 200 pM acetonitrile solution are prepared. In the test vial, 25 mg sample containing AB -CH MIN AC A (IIIPAC nomenclature: N-[(2S)-1 - Amino-3-methyl-1 -oxobutan-2-yl]-1 -(cyclohexylmethyl) indazole-3- carboxamide), a psychoactive compound (formula E4) of the indazole class.

[0232]

[0233] Formula E4

[0234] An increase in color intensity occurs immediately, with a change from colourless solution to yellow, indicative of the presence of indazole psychoactive substances. The hyperchromic variation was quantified by UV-Vis spectrophotometry, confirming the characteristic increase in absorbance without a significant shift in the maximum wavelength (Figure 6).

[0235] Example 5

[0236] Following the same procedure as in the previous examples, two comparison vials containing 500 pL of reactive solution (200 pM TBAF in acetonitrile) are prepared. A 25 mg sample containing 4F-MDMB-BICA (IIIPAC nomenclature: methyl 2-[[1 -(4-fluorobutyl) indole-3-carbonyl] amino]-3,3-dimethylbutanoate), a synthetic cannabinoid (with formula E5) of the indole family, is added to the test container.

[0237] Formula E5

[0238] No color variation is observed in the solution, which remains unchanged in comparison with the reference vial. There is neither a bathochromic effect (wavelength shift) nor a hyperchromic effect (increase in intensity). UV-Vis spectrophotometric analysis (Jasco mod. V-670) confirms the absence of significant changes in the absorption spectrum before and after the addition of the TBAF reagent, demonstrating the negative response characteristic of indole-based psychoactive compounds (Figure 7).

[0239] Example 6

[0240] In the present embodiment, the inventors performed in parallel a series of colorimetric tests to identify which was the most effective basic anion in interacting with a molecule with psychoactive activity having an isatin nucleus, i.e. the BZO-4en-POXIZID. (or simply poxizid).

[0241] Figure 8 shows the results of six vials containing 1 mM of BZO-4en- POXIZID in acetonitrile together with several basic anions (having tetrabutylammonium as a counterion). In particular, from left to right:

[0242] Vial 1 (reference solution with 1 mM poxizid): remains colourless

[0243] Vial 2 (1 mM poxizid + TBAF): Produces intense yellow coloration

[0244] Vial 3 (1 mM poxizid + TBA Acetate): No color variations

[0245] Vial 4 (1 mM poxizid + TBACI): No color variations

[0246] Vial 5 (1 mM poxizid + TBABr): Shows no color change

[0247] Vial 6 (1 mM poxizid + TBAOH): Shows a weaker but detectable yellow response

[0248] This demonstrates selectivity for basic anions, with fluoride showing the strongest response and hydroxide showing a weaker but still detectable response.

[0249] References

[0250] [1] Haider, Azeem, Ahmed, Mukhtiar, Faisal, Muhammad and Naseer, Muhammad Moazzam. "Isatin as a simple, highly selective and sensitive colorimetric sensor for fluoride anion" Heterocyclic Communications, vol. 26, no. 1 , 2020, pp. 14-19. https: / / doi.Org / 10.1515 / hc-2020-0003 [2] A. Haider et al. I Measurement 154 (2020) 107457

[0251] [3] RSC Adv., 2016,6, 109742-109750, https: / / doi.Org / 10.1039 / C6RA22396F

[0252] [4] K. Liu et al. I Sensors and Actuators B 226 (2016) 465-470, https: / / doi.Org / 10.1016 / j.snb.2O15.12.019

[0253] [5] isovsky, P.; Csicsai, K.; Donovalova, J.; Sandrik, R.; Sokolik, R.; Gaplovsky, A. Effect of a =X-NH-Fragment, (X = C, N), on Z / E Isomerization and ON / OFF Functionality of Isatin Arylhydrazones, ((Arylamino)Methylene)lndolin-2-Ones and Their Anions. Molecules 2020, 25, 3082. https: / / doi.Org / 10.3390 / molecules25133082

[0254] [6] J. Filo, et al., Tautomeric photoswitches: anion-assisted azo / azine-to- hydrazone photochromism, RSC Adv., 2019,

[0255] [7] Jakusova K, Donovalova J, Cigan M, Gaplovsky M, Garaj V, Gaplovsky A. Isatinphenylsemicarbazones as efficient colorimetric sensors for fluoride and acetate anions - anions induce tautomerism. Spectrochim Acta A Mol Biomol Spectrosc. 2014 Apr 5; 123:421 -9.

[0256] [8] M. Cigan, et al., Isatin phenylhydrazones: anion enhanced photochromic behaviour, f Photochem. Photobiol. Sci.,2015.

Claims

CLAIMS1. A method for the detection of compounds with psychoactive activity having the general formula (I):General formula (I) wherein: indicates a single bond or double bond, with the constraint that, when the C=Y bond is double, the C-X bond is single and vice versa, and the double bond can have E or Z configuration;X, Y, independently of each other = N, C=O;R1 =• linear, branched or cyclic alkyl, with a length between Ci and C20;• alkenyl or alkynyl, linear, branched or cyclic, with a length between Ci and C20 and containing one or more unsaturation both terminal and internal to the chain;• alkyl or alkenyl or alkynyl, linear, branched or cyclic, with a length between Ci and C20 and substituted with at least one halogen;• alkyl or alkenyl or alkynyl, linear, branched or cyclic, with alength between Ci and C20, and containing at least one functional group chosen among carbonyl, ester, amide, carboxylic acid, amine, ether, hydroxyl, halide and combinations thereof;• cyclic alkyl with a ring size between C4 and Cs;• heterocyclic alkyl with a ring size between C4 and Cs;• phenyl (CeHs-) or aryl, not substituted or substituted with at least one group chosen among the nitro group (-NO2), sulfonic group (-SO3H), phosphate group (-POs2-), linear, branched or cyclic alkyl group, with a length between Ci and C20,• acyl (RCO) linear, branched or cyclic, with a length between Ci and C20;• linear, branched or cyclic acyl, with a length between Ci and C20 containing at least one functional group chosen among carbonyl, ester, amide, carboxylic acid, amine, ether, hydroxyl, halide and combinations thereof;• aroyl (ArCO-), wherein aryl (Ar) can be a pure phenyl (CeHs-) or a substituted phenyl with one or more functional groups chosen among fluorine (-F), chlorine (-CI), nitro group (-NO2), sulphonic group (-SO3H), alkoxide group such as methoxide (-OMe), ethoxide (-OEt), propoxide (-OPr), isopropoxide (-OiPr), butoxide (-OBut);R2 =• hydrogen (-H);• linear, branched or cyclic alkyl, with a length between Ci and C20;• alkenyl or alkynyl, linear, branched or cyclic, with a length between Ci and C20 and containing one or more unsaturation both terminal and internal to the chain;• alkyl or alkenyl or alkynyl, linear, branched or cyclic, with a length between Ci and C20 and substituted with at least one halogen;• alkyl or alkenyl or alkynyl, linear, branched or cyclic, with a length between Ci and C20 and containing at least one functional group chosen among carbonyl, ester, amide, carboxylic acid, amine, ether, hydroxyl, halide and combinations thereof;• cyclic alkyl with a ring size between C4 and Cs;• heterocyclic alkyl with a ring size between C4 and Cs;• phenyl (CeHs-) or aryl, not substituted or substituted with at least one group chosen among the nitro group (-NO2), sulfonic group (-SO3H), phosphate group (-POs2-), linear, branched or cyclic alkyl group, with a length between Ci and C20;• linear acyl, branched or cyclic, with a length between Ci and C20;R3 =• hydrogen (H)• linear, branched or cyclic alkyl, with a length between Ci and C20;• alkenyl or alkynyl, linear, branched or cyclic, with a length between Ci and C20 and containing one or more unsaturation both terminal and internal to the chain;• alkyl or alkenyl or alkynyl, linear, branched or cyclic, with a length between Ci and C20 and substituted with at least one halogen;• alkyl or alkenyl or alkynyl, linear, branched or cyclic, with a length between Ci and C20 containing at least one functional group chosen among carbonyl, ester, amide, carboxylic acid,amine, ether, hydroxyl, halide and combinations thereof;• cyclic alkyl with a ring size between C4 and Cs such as cyclopentyl, cyclohexyl, cycloheptyl;• heterocyclic alkyl with a ring size between C4 and Cs, and containing heteroatoms such as N, S and 0, such as tetrahydrofuranyl, tetrahydropyranyl;• phenyl (CeHs-) or aryl, possibly substituted with at least one group chosen by way of example but not limited to from the group comprising nitro group (-NO2), sulfonic group (-SO3H), phosphate group (-PCs2-), alkyl group, linear, branched or cyclic, with a length between Ci and C20;• acyl linear, branched or cyclic, with a length between Ci and C20;• linear, branched or cyclic acyl (RC0-), with a length between Ci and C20 containing at least one functional group chosen among carbonyl, ester, amide, carboxylic acid, amine, ether, hydroxyl, halide and combinations thereof;• aroyl (ArCO-) wherein aryl (Ar) can be a pure phenyl (CeHs-) or a substituted phenyl with one or more functional groups chosen among fluorine (-F), chlorine (-CI), nitro group (-NO2), sulphonic group (-SO3H), alkoxide group such as methoxide (-OMe), ethoxide (-OEt), propoxide (-OPr), isopropoxide (-OiPr), butoxide (-OBut); said method comprising the steps of:• taking a portion of a sample suspected of containing the presumed psychoactive compound of formula (I);• preparing two containers with equal quantities of a reactive solution comprising a base chosen among organic cation fluorides and organic cation hydroxides, and a solvent chosen among aprotic polar solvents;• dipping or adding this sample aliquot to one of the two said containers;• waiting for the reactive solution to react with the presumed compound with psychoactive activity;• visually observing the occurrence of a chromatic variation of the solution to be understood either as a shift in the absorption wavelength or as an increase in the intensity of absorption.

2. The method according to the previous claim wherein the compound with psychoactive activity has the general formula 1A• hydrogen (H)• linear, branched or cyclic alkyl, having a length between Ci and C10;• linear, branched or cyclic alkenyl or alkynyl, with a length between Ci and C20 and containing one or more unsaturation both terminal and internal to the chain;• alkyl or alkenyl or alkynyl, linear, branched or cyclic, with a length between Ci and C20 and substituted with at least onehalogen, preferably fluorine;• cycloalkyl with a length between C4 and C7;R5 =• hydrogen (-H), fluorine (-F), chlorine (-CI), nitro group (-NO2), sulfonic group (-SO3H), alkoxide group as methoxide (-OMe), ethoxide (-OEt), propoxide (-OPr), isopropoxide (-OiPr), butoxide (-OBut);R6 =• hydrogen (-H), fluorine (-F), chlorine (-CI), nitro group (-NO2), sulfonic group (-SO3H), alkoxide group as methoxide (-OMe), ethoxide (-OEt), propoxide (-OPr), isopropoxide (-OiPr), butoxide (-OBut).

3. The method according to claim 1 wherein the compound with psychoactive activity has the general formula 1 BGeneral formula 1 B wherein,R7 =• linear, branched or cyclic alkyl, with a length between Ci and C20;• linear, branched or cyclic alkenyl or alkynyl, with a length between Ci and C20 and containing one or more unsaturationboth terminal and internal to the chain;• alkyl or alkenyl or alkynyl, linear, branched or cyclic, with a length between Ci and C20 and substituted with at least one halogen;• alkyl or alkenyl or alkynyl, linear, branched or cyclic, with a length between Ci and C20 and containing at least one functional group chosen among carbonyl, ester, amide, carboxylic acid, amine, ether, hydroxyl, halide and combinations thereof;• cyclic alkyl with a ring size between C4 and Cs;• heterocyclic alkyl with a ring size between C4 and Cs;• phenyl (CeHs-) or aryl, not substituted or substituted with at least one group chosen among the nitro group (-NO2), sulfonic group (-SO3H), phosphate group (-POs2-), linear, branched or cyclic alkyl group, with a length between Ci and C20;R2 =• hydrogen (-H);• linear, branched or cyclic alkyl, with a length between Ci and C20;• alkenyl or alkynyl, linear, branched or cyclic, with a length between Ci and C20 and containing one or more unsaturation both terminal and internal to the chain;• alkyl or alkenyl or alkynyl, linear, branched or cyclic, with a length between Ci and C20 and substituted with at least one halogen;• alkyl or alkenyl or alkynyl, linear, branched or cyclic, with a length between Ci and C20 containing at least one functional group chosen among carbonyl, ester, amide, carboxylic acid, amine, ether, hydroxyl, halide and combinations thereof;• cyclic alkyl with a ring size between C4 and Cs;• heterocyclic alkyl with a ring size between C4 and Cs;• phenyl (CeHs-) or aryl, not substituted or substituted with at least one group chosen among the nitro group (-NO2), sulfonic group (-SO3H), phosphate group (-POs2-), alkyl group, linear, branched or cyclic, with a length between Ci and C20;• acyl linear, branched or cyclic, with a length between Ci and C20;R3 =• hydrogen (H)• linear, branched or cyclic alkyl, with a length between Ci and C20;• alkenyl or alkynyl, linear, branched or cyclic, with a length between Ci and C20 and containing one or more unsaturation both terminal and internal to the chain;• alkyl or alkenyl or alkynyl, linear, branched or cyclic, with a length between Ci and C20 and substituted with at least one halogen;• alkyl or alkenyl or alkynyl, linear, branched or cyclic, with a length between Ci and C20 and containing at least one functional group chosen among carbonyl, ester, amide, carboxylic acid, amine, ether, hydroxyl, halide and combinations thereof;• cyclic alkyl with a ring size between C4 and Cs;• heterocyclic alkyl with a ring size between C4 and Cs;• phenyl (CeHs-) or aryl, not substituted or substituted with at least one group chosen among the nitro group (-NO2), sulfonic group (-SO3H), phosphate group (-POs2-), linear, branched or cyclic alkyl group, with a length between Ci and C20;• acyl linear, branched or cyclic, with a length between Ci and C20;• acyl (RCO-), linear, branched or cyclic, with a length between Ci and C20 and containing in the alkyl chain (R) at least one functional group chosen among carbonyl, ester, amide, carboxylic acid, amine, ether, hydroxyl, halide and combinations thereof;• aroyl (ArCO-) wherein aryl (Ar) can be a pure phenyl (CeHs-) or a substituted phenyl with one or more functional groups chosen among fluorine (-F), chlorine (-CI), nitro group (-NO2), sulfonic group (-SO3H), alkoxide group such as methoxide (-OMe), ethoxide (-OEt), propoxide (-OPr), isopropoxide (-OiPr), butoxide (-OBut).

4. The method according to anyone of claims 1 -3 wherein the sample suspected of containing the compound with psychoactive activity is in an amount between approximately 20-80 mg of solid material or between approximately 50-500 pL of liquid material.

5. The method according to anyone of claims 1 -4 wherein the base is chosen between quaternary ammonium fluorides or quaternary ammonium hydroxides.

6. The method according to anyone of claims 1 -5 wherein the base is tetrabutylammonium fluoride (TBAF).

7. The method according to anyone of claims 1 -6 wherein the solvent of the reactive solution is chosen among: acetonitrile, dimethyl sulfoxide, dimethylacetamide (DMA), dimethylformamide (DMF), acetone, 1 ,4- dioxane, tetrahydrofuran (THF), hexamethylphosphoramide (HMPA), tetramethylurea, ethyl acetate (EtOAc), butyl acetate, methyl isobutyl ketone, 1 ,3-dimethyl-3,4,5,6-tetrahydro-2-pyrimidinone (DMPII) and mixtures thereof.

8. The method according to anyone of claims 1 -7 wherein the solvent of the reactive solution is acetonitrile.

9. The method according to anyone of claims 1 -8 wherein the concentration of the base in the reactive solution is in the range 0.1 -10 mM.

10. The method according to anyone of the previous claims wherein the molar ratio of said compound to psychoactive activity and said base is greater than 0.1 , preferably between 0.1 and 20.

11. A reagent mixture for the colorimetric determination of psychotropic substances according to the method of anyone of claims 1 -9, comprising a base chosen among quaternary ammonium fluorides and quaternary ammonium hydroxides, preferably tetrabutylammonium fluoride; a solvent chosen among aprotic polar solvents, preferably chosen among dimethyl sulfoxide, dimethylacetamide (DMA), dimethylformamide (DMF), acetone, 1 ,4-dioxane, tetrahydrofuran (THF), hexamethylphosphoramide (HMPA), tetramethylurea, ethyl acetate (EtOAc), butyl acetate, methyl isobutyl ketone, 1 ,3-dimethyl- 3,4,5,6-tetrahydro-2-pyrimidinone (DMPU) and mixtures thereof; and a compound with psychoactive activity having at least one isatin or indazole nucleus of general formula (I), 1 A or 1 B according to anyone of claims 1 -3 such that the molar ratio of said compound with psychoactive activity to the base is greater than 0.1 , preferably between 0.1 -20.

12. Use of a reactive solution comprising a base chosen among quaternary ammonium fluorides and quaternary ammonium hydroxides, preferably tetrabutylammonium fluoride, a solvent chosen among aprotic polar solvents, preferably chosen among dimethyl sulfoxide, dimethylacetamide (DMA), dimethylformamide (DMF), acetone, 1 ,4- dioxane, tetrahydrofuran (THF), hexamethylphosphoramide (HMPA), tetramethylurea, ethyl acetate (EtOAc), butyl acetate, methyl isobutyl ketone, 1 ,3-dimethyl-3,4,5,6-tetrahydro-2-pyrimidinonone (DMPU) and mixtures thereof for the detection of compounds with psychoactive activity having an isatin or indazole nucleus of general formula (I), 1A or 1 B according to anyone of claims 1 -3.

3. A kit for executing the method according to anyone of claims 1 -10, said kit comprising:• Two transparent, reusable or disposable containers containing effective doses in equal quantities of a reactive solution comprising a base chosen among quaternary ammonium fluorides and quaternary ammonium hydroxides, preferably tetrabutylammonium fluoride, a solvent chosen among aprotic polar solvents, preferably chosen among dimethyl sulfoxide, dimethylacetamide (DMA), dimethylformamide (DMF), acetone, 1 ,4-dioxane, tetrahydrofuran (THF), hexamethylphosphoramide (HMPA), tetramethyl urea, ethyl acetate (EtOAc), butyl acetate, methyl isobutyl ketone, 1 ,3-dimethyl-3,4,5,6-tetrahydro-2- pyrimidinone (DMPU) and mixtures thereof;• A legend with a color scale to evaluate the outcome of the test;• Instructions for use.

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