A COMPOUND, A PROBE AND ASSOCIATED PROCESSES

Abstract

A COMPOUND, A PROBE, AND ASSOCIATED METHODS This disclosure proposes a compound of formula (I), in which A is an aryl nucleus comprising C10 to C20 atoms; R1 and R2 are independently selected from a C1-6 alkyl or a C6-12 aryl; or R1 and R2 combine to form a heterocyclyl nucleus having 3 to 8 atoms; m is in the range of 0 to 10; and n is in the range of 0 to 10. This disclosure further proposes a probe comprising the compound of formula (I) for the detection of a sensitizer. In addition, this disclosure proposes a method for detecting a sensitizer. Formula (I) Figure for abstract: none

Description

Title of the invention: A COMPOSITE, A PROBE AND ASSOCIATED METHODS FIELD OF INVENTION

[0001] This disclosure relates, in general, to the analysis and assessment of the safety of compounds or skin-reactive sensitizers. In particular, this disclosure relates to a compound, a probe, and a method for detecting skin sensitizers. CONTEXT OF THE INVENTION

[0002] Skin sensitization is an important evaluation criterion for assessing cosmetic risk, taking into account both the nature of the ingredients used and post-market surveillance. The regulatory measures necessary to prevent the induction and triggering of allergies are addressed in several regulatory requirements and in current market practices. The coexistence of regulatory needs and recent developments in skin sensitization testing protocols for chemicals, fragrances, and cosmetics allows for a correct estimation of risks and efficacy, as well as risk management without the use of in vivo procedures that present ethical issues.Due to the complexity of the biological mechanisms associated with skin sensitization, integrated approaches combining different chemical, biological and in silico processes are recommended to replace conventional animal testing.

[0003] Chemical methods are intended to characterize the potential of a sensitizer to induce early molecular initiation events. The presence of an electrophilic mechanistic domain is considered one of the essential chemical features for covalently binding to the biological target and inducing further haptenation processes. Current chemical assays rely on the quantification of model nucleophiles that have not reacted after incubation with the candidate sensitizer.

[0004] The compounds involved in allergic contact dermatitis (ACD) are generally electrophilic in nature. Existing techniques or assays employ the formation of covalent bonds between electrophiles and nucleophiles due to the interaction of skin sensitizers with proteins, peptides, and nucleophiles representing proteins or peptides. However, these techniques involve a longer incubation time and, moreover, do not identify pro-electrophiles.

[0005] Therefore, there is a need to develop a reactive compound to be used as a probe and a method to rapidly identify potential electrophilic skin sensitizers and to differentiate an electrophilic sensitizer from a pro-electrophilic sensitizer. Summary of the invention

[0006] In one aspect of this disclosure, a compound of formula (I), its solvates, stereoisomers, enantiomers, racemates or their salts are proposed

[0007] Formula (I)

[0008] in which A is an aryl nucleus comprising C10 to C20 atoms; • Ri and R2 are independently chosen from a C6 alkyl or a C6n aryl; or Ri and R2 combine together to form a heterocyclyl nucleus having 3 to 8 atoms; and • m is in a range from 0 to 10; and • n is in a range from 0 to 10.

[0009] In another aspect of this disclosure, a probe is proposed for detecting a sensitizer comprising the compound of formula (I) as disclosed herein.

[0010] In another aspect of this disclosure, a method for detecting a sensitizer is proposed, the method comprising: (a) mixing the compound of formula (I) or the probe, as disclosed herein, with the sensitizer in the presence of a base to obtain a first solution; (b) incubating the first solution for a period of 0.5 to 2 hours to obtain an incubated solution; (c) spectral analysis of the incubated solution to measure the compound of formula (I); and (d) identification of the sensitizer.

[0011] These features, aspects, and advantages, as well as others, of the present subject matter will be better understood with reference to the following description and the attached claims. The purpose of this summary is to present a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor to be used to limit the scope of the claimed subject matter. BRIEF DESCRIPTION OF THE FIGURES

[0012] The following drawings form part of this specification and are included to further illustrate aspects of this disclosure. The disclosure may be best understood by referring to the drawings in combination with the detailed description of the specific embodiments presented here.

[0013] Figure 1 schematically illustrates the chromatograms of incubated solutions of compound of formula (I) with (a) p-benzoquinone, (b) 2-methyl 4-isothiazolin 3-one, and (c) famesal, according to one embodiment of this disclosure.

[0014] Figure 2 schematically illustrates the chromatograms of solutions incubated with p-benzoquinone with (a) a compound of formula (I) (NNDNAC); and (b) N-(2-(l-naphthyl)acetyl)-L-cysteine ​​(NAC-ADRA), at a variable incubation time of 1 h and 24 h, in accordance with one embodiment of this disclosure. DESCRIPTION OF THE INVENTION

[0015] A person skilled in the art will be aware that this disclosure is subject to variations and modifications other than those specifically described. It should be understood that this disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions, and compounds designated or indicated in this patent memorandum, individually or collectively, and any combination of one or more of these steps or features.

[0016] Definitions

[0017] For convenience, before describing this disclosure in more detail, certain terms used in the patent memorandum and examples are defined here. These definitions should be read in light of the rest of the disclosure and understood as by a person skilled in the art. The terms used here have meanings recognized and known to a person skilled in the art; however, for convenience and completeness, specific terms and their meanings are set out below.

[0018] The articles "un", "une", "la", "le" and "les" are used to designate one or more of one (i.e. at least one) grammatical object of the article.

[0019] The terms “include” and “comprising” are used in an inclusive and open sense, meaning that additional elements may be included. They are not intended to be interpreted as “consists solely of”.

[0020] The expression "at least one" is used to mean one or more and therefore includes individual components as well as mixtures / combinations.

[0021] Throughout this patent memorandum, unless the context otherwise requires, the term "include", and variations such as "includes" and "comprising", shall be understood to imply the inclusion of any element or step or group of elements or steps stated, but not the exclusion of any other element or step or group of elements or steps.

[0022] The term "including" is used to mean "including, but not limited to". "Including" and "including, but not limited to" are used interchangeably.

[0023] The meaning of the various terms used in the description will now be illustrated.

[0024] According to one embodiment of the present, the term "C,6 alkyl" refers to a straight-chain or branched C6 Ci alkyl which may optionally be substituted. Representative examples of alkyl groups include methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl and 1-ethyl-2-methylpropyl or the different isomers.

[0025] According to one embodiment hereof, the term "aryl" refers to groups of cyclic or polycyclic (spiro, fused, bridged, unfused) nuclei that satisfy Huckel's rule. Representative examples of aryl nuclei include benzene, naphthalene, chrysene, pyrene, anthracene, phenanthrene, or the like.

[0026] According to one embodiment of the present, the term "heterocycle" or "heterocyclic" or "heterocyclyl" includes "aromatic heterocycle", "non-aromatic heterocycle" or polycyclic or bicyclic (spiro, fused, bridged, unfused) nuclei in which the nucleus may be aromatic or non-aromatic, where the heterocyclic nucleus contains at least one heteroatom selected from nitrogen, oxygen and sulfur as well as one or more carbon atoms, with a total of three to eight atoms.

[0027] The term “solvates,” as used here, refers to a compound in which its crystal lattice contains one or more solvent molecules. Non-limiting examples of solvates include hydrates when the solvent is water, ammoniates when the solvent is ammonia, alkoxides when the solvent is an alcohol, and etherates when the solvent is an ether.

[0028] The term "salt," as used herein, refers to acid or base salts of compounds of formula (I). Non-limiting examples of acid salts include both inorganic acid salts, such as hydrochloric, sulfuric, phosphoric, diphosphoric, hydrobromic, hydroiodic, and nitric acids, and organic acid salts, such as citric, fumaric, maleic, malic, mandelic, ascorbic, oxalic, succinic, tartaric, benzoic, acetic, methanesulfonic, ethanesulfonic, benzenesulfonic, or p-toluenesulfonic acid. Non-limiting examples of basic salts include alkali metal salts of sodium hydroxides or of potassium and alkaline earth metal salts of calcium or magnesium hydroxides and organic bases, for example alkyl amines, arylalkyl amines and heterocyclic amines.

[0029] The compounds of formula (I) described herein may contain one or more chiral centers and / or double bonds and, consequently, may exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), regioisomers, enantiomers, or diastereomers. Accordingly, the chemical structures represented herein encompass all possible enantiomers and stereoisomers of the compounds illustrated or identified, including the stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures. The enantiomeric and stereoisomeric mixtures can be resolved into their enantiomeric or stereoisomeric components using separation techniques or chiral synthesis techniques well known to those skilled in the art.Compounds can also exist in several tautomeric forms, including the enol form, the keto form, and mixtures thereof. Consequently, the chemical structures schematically represented here encompass all possible tautomeric forms of the illustrated or identified compounds. It is also understood that certain isomeric forms, such as diastereomers, enantiomers, and geometric isomers, can be separated by physical and / or chemical processes by those skilled in the art. Furthermore, compounds can exist as mixtures, each having equal amounts of enantiomers to form racemates or a racemic mixture.

[0030] According to one embodiment of the present, the terms "sensitizer(s)" or "skin sensitizer(s)" or "skin sensitizing compound(s)" refer to substances that are reactive when applied to skin and can cause damage to the skin surface upon contact, due to the extent of their reactivity.

[0031] According to one embodiment hereof, the term “electrophile” refers to a compound that tends to attract or acquire electrons and is thus reactive in nature. For the purposes of this disclosure, the term “electrophile” refers to “haptens,” which are immunogenic compounds of an electrophilic nature. These haptens cause an allergic reaction upon contact with the skin. For the purposes of this disclosure, electrophiles are skin-sensitizing electrophilic compounds and are also referred to as haptens.

[0032] According to one embodiment hereof, the term "pro-electrophile" refers to a compound which, when subjected to certain external conditions, is converted into an electrophile. For the purposes of this disclosure, the term "pre-hapten" refers to Immunogenic compounds that are non-reactive in nature and are converted into haptens when subjected to certain external conditions. Pre-haptens, upon conversion to haptens, cause allergic skin reactions. For the purposes of this disclosure, pro-electrophiles are skin-sensitizing pro-electrophilic compounds and are also referred to as pre-haptens.

[0033] All percentages, parts, and ratios are based on the total weight of the compositions in this disclosure, unless otherwise stated. Ratios, concentrations, quantities, and other numerical data may be presented herein in a range format. It should be understood that this range format is used solely for convenience and conciseness and should be interpreted flexibly as including not only the numerical values ​​explicitly cited as the range limits, but also as including all individual numerical values ​​or subranges encompassed within that range as if each numerical value and subrange were explicitly cited.For example, a duration range of approximately 0.5 to 2 hours should be interpreted as including not only the explicitly stated limits of approximately 0.5 to approximately 2 hours, but also sub-ranges, such as 0.5 to 0.75 hours, 1.25 to 1.75 hours and so on, as well as individual quantities, including fractional quantities, within the specified ranges, such as 0.5 h, 1.0 h, 1.25 h, and 1.75 h, for example.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as that commonly understood by a person skilled in the art to which this disclosure relates. Although all processes and materials similar or equivalent to those described herein may be used in the practice or testing of the disclosure, preferred processes and materials are now described.

[0035] This disclosure shall not be limited in scope by the specific embodiments described herein, which are intended for illustrative purposes only. Functionally equivalent products and processes are clearly within the scope of this disclosure, as described herein.

[0036] This disclosure proposes a compound of formula (I), a probe comprising compound of formula (I), and a method for detecting a sensitizer using compound of formula (I). This disclosure proposes an efficient chemical characterization tool for identifying potential skin sensitizers with rapid and high throughput. The probe and the method for detecting a sensitizer in this disclosure are suitable and provide low-cost approaches for the pre-screening of large chemical compounds. The method for detecting a sensitizer using the probe comprising compound of formula (I) is advantageous compared to time-consuming and costly in vitro and in vivo evaluations of skin sensitizing compounds. Furthermore, the method disclosed herein facilitates the distinction between pro-electrophilic sensitizers and electrophilic sensitizers which existing processes cannot achieve.

[0037] Embodiments of the present formula provide a compound of formula (I), its solvates, stereoisomers, enantiomers, racemates or their salts Formula (I)

[0038] in which A is an aryl nucleus comprising Cm to C2o atoms; • Ri and R2 are independently chosen from a C6 alkyl, or a C6 i2 aryl; or Ri and R2 combine together to form a heterocyclyl nucleus having 3 to 8 atoms; m is in a range of 0 to 10; and n is in a range of 0 to 10.

[0039] The compound of formula (I) of the present invention makes it possible, in particular, to detect a sensitizer with rapid yield and by an economical approach. The embodiments herein also provide a probe comprising a compound of formula (I).

[0040] The embodiments of the present also propose a method for detecting a sensitizer in order to categorize a pro-electrophile and an electrophile. Compound

[0041] The embodiments hereof propose a compound of formula (I), its solvates, stereoisomers, enantiomers, racemates or their salts, according to the present invention, Formula (I)

[0042] wherein A is an aryl nucleus comprising C10 to C20 atoms; • Ri and R2 are independently chosen from a C6 alkyl, or a C6 i2 aryl; or Ri and R2 combine together to form a heterocyclyl nucleus having 3 to 8 atoms; m is in a range of 0 to 10; and n is in a range of 0 to 10.

[0043] According to one embodiment of the present, it allows, in particular, the efficient detection of a sensitizer using the compound of formula (I).

[0044] According to a particular embodiment of the present, A is an aryl nucleus comprising C10 to C[8] atoms; Ri and R2 are independently chosen from a Ci_6 alkyl; m is in a range of 0 to 3; and n is in a range of 0 to 3.

[0045] According to a particular embodiment of the present, A is chosen from naphthalene, anthracene, chrysene, phenanthrene or pyrene; Ri and R2 are independently chosen from a Ci 6 alkyl; m is in a range of 0 to 3; and n is in a range of 0 to 3.

[0046] According to a more particular embodiment of the present, A is naphthalene, Ri and R2 are independently a Ci alkyl; m is 1; and n is 0.

[0047] According to one embodiment hereof, the compound of formula (I) acts as a nucleophile to react with reactive electrophilic or pro-electrophilic sensitizers and thereby detects reactive species. The compound allows for the specific detection of pro-electrophilic sensitizers in a short incubation time of 0.5 to 2 hours. Furthermore, the compound of formula (I), according to embodiments hereof, used in a reactivity probe, overcomes the drawbacks associated with time consumption, difficulties in the detection process, the cost of reagents, and high yield.

[0048] According to other embodiments hereof, the compound of formula (I), its solvates, stereoisomers, enantiomers, racemates, or their salts are proposed for the detection of skin-sensitizing compounds. The compound of formula (I), its solvates, stereoisomers, enantiomers, racemates, or their salts, according to other embodiments hereof, is used in a probe for the efficient, rapid, and convenient detection of sensitizers. Probe

[0049] The embodiments of the present provide a probe for detecting a sensitizer comprising the compound of Formula (I), according to the present invention.

[0050] According to one embodiment hereof, the term "probe" refers to a system for analyzing the properties of a target by reversible binding to the target and modifying its function. According to another embodiment hereof, the probe is a molecular probe used to detect sensitizers by reacting with them. The sensitizers, according to embodiments hereof, are an electrophile or a pro-electrophile.

[0051] According to other embodiments of the present, the sensitizer is an electrophilic or pro-electrophilic skin sensitizing compound.

[0052] According to other embodiments of the present, the probe comprises a compound of formula (I), its solvates, stereoisomers, enantiomers, racemates or their salts, according to the present invention, Formula (I)

[0053] • in which A is an aryl nucleus comprising C10 to C20 atoms; • Ri and R2 are independently chosen from a Ci_6 alkyl, or a C6 i2 aryl; or Ri and R2 combine together to form a heterocyclyl nucleus having 3 to 8 atoms; m is in a range of 0 to 10; and n is in a range of 0 to 10.

[0054] According to a particular embodiment of the present, the probe comprises a compound of formula (I), in which A is an aryl nucleus comprising C10 to C10 atoms; Ri and R2 are independently selected from a C10 alkyl; m is in a range of 0 to 3; and n is in a range of 0 to 3.

[0055] According to a particular embodiment of the present, the probe comprises a compound of formula (I), in which A is selected from naphthalene, anthracene, chrysene, phenanthrene or pyrene; Ri and R2 are independently selected from a Ci_6 alkyl; m is in a range of 0 to 3; and n is in a range of 0 to 3.

[0056] According to other embodiments of the present, the probe comprises a compound of formula (I), in which A is naphthalene, Ri and R2 are independently a Ci alkyl; m is 1; and n is 0.

[0057] According to other embodiments of the present, the probe comprises a compound of formula (I) with other non-reactive components that facilitate the detection of skin-sensitizing compounds. The non-reactive components are generally inert components that can be used during the formulation of the probe.

[0058] According to other embodiments hereof, a use of the probe comprising the compound of formula (I) for the detection of a sensitizer is disclosed. The probe exhibits rapid and efficient detection of electrophilic and pro-electrophilic sensitizers. The probe, according to embodiments hereof, detects a sensitizer by a method as disclosed herein. Method

[0059] Embodiments of the present include a method for detecting sensitizers. The method includes the detection of sensitizers, in particular electrophilic and pro-electrophilic sensitizers.

[0060] The embodiments herein propose a method for detecting a sensitizer using the compound of formula (I) as disclosed herein. According to one embodiment herein, the method for detecting a sensitizer comprises: (a) mixing the compound of formula (I) with the sensitizer in the presence of a base to obtain a first solution; (b) incubating the first solution for a period of 0.5 to 2 hours to obtain an incubated solution; (c) spectral analysis of the incubated solution to measure the compound of formula (I); and (d) identifying the sensitizer.

[0061] The embodiments herein propose a method for detecting a sensitizer using the compound of formula (I) as disclosed herein. According to one embodiment herein, the method for detecting a sensitizer comprises: (a) mixing the compound of formula (I), in which A is naphthalene, Ri and R2 are independently a Ci alkyl group; m is 1; and n is 0, with the sensitizer in the presence of a base to obtain a first solution; (b) incubating the first solution for a period of 0.5 to 2 hours to obtain an incubated solution; (c) spectral analysis of the incubated solution to measure the compound of formula (I); and (d) identifying the sensitizer.

[0062] According to one embodiment of the present, the base is selected from 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,4-diazabicyclo[2.2.2]octane (TED), 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD), 4-dimethylaminopyridine, pyridine, piperazine, or combinations thereof. According to a particular embodiment of the present, the base is 1,8-diazabicyclo[5.4.0]undec-7-ene. According to another particular embodiment of the present, the base is the 1,5-diazabicyclo[4.3.0]non-5-ene.

[0063] According to another embodiment of this article, the incubation of the first solution is carried out for a period of 0.5 to 2 hours. According to a particular embodiment of this article, the incubation of the first solution is carried out for a period of 0.75 to 1.5 hours. According to a more particular embodiment of this article, the incubation of the first solution is carried out for a period of 1 hour.

[0064] According to other embodiments of this product, the sensitizer is identified by measuring the depletion of the compound of formula (I). According to other embodiments of this product, the compound of formula (I), being a nucleophile, reacts with an electrophilic sensitizer, leading to the depletion of the compound of formula (I).

[0065] According to another embodiment of the present, the spectral analysis of the incubated solution is carried out by chromatographic detection and by fluorescence. According to a In a particular embodiment of this text, spectral analysis is performed by liquid chromatography followed by fluorescence detection. In a more specific embodiment of this text, spectral analysis is performed by liquid chromatography followed by fluorescence detection to measure the presence of compound Formula (I) in the incubated solution and correlate it with the depletion of compound Formula (I). The depletion of compound Formula (I) categorizes the sensitizer as electrophilic or pro-electrophile.

[0066] According to other embodiments of the present, the sensitizer detection method disclosed herein is proposed for the pre-screening of a cosmetic composition. The method disclosed herein is used as an analytical assessment technique for a composition to understand the reactivity of the constituent compounds that lead to skin sensitization.

[0067] Although the subject has been described in great detail with reference to certain examples and implementations thereof, it is understood that other implementations are possible and included within the scope of the present invention. EXAMPLES

[0068] The disclosure will now be illustrated by the following examples, which are intended to illustrate the operation of the disclosure and are not intended to impose any restrictive limitations on the scope of this disclosure. Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as that commonly understood by a person skilled in the art of this disclosure. Although processes and materials similar or equivalent to those described herein may be used in the practice of the disclosed processes and compositions, examples of processes, devices, and materials are described herein. It should be understood that this disclosure is not limited to the particular processes and experimental conditions described, as these processes and conditions may be applicable. Example 1

[0069] Compound of formula (I)

[0070] The compound of formula (I) of this disclosure, NN dimethyl N-(2-(l-naphthyl)acetyl)-L-cysteine ​​(NNDNAC) having naphthalene as A, Ri and R2 being independently a Ci alkyl; m being 1; and n being 0, was prepared by the process described herein. Formula (I)

[0071] Preparation of the compound of Formula (I)

[0072] The compound of formula (I) was prepared by the process illustrated in diagram 1. Sr NHiCi MeOH;H2O 75°C. 2 h Step 1 MeWaH THF 6G ' C, '■ B h Step 2 oo Piî{aliyi jC!2. Xamphes DMaH; Xvtèrsë 140°C, 6 p.m. Step 3 LiOH aq; THFt. 4 p.m. -J.,,-..,™ ; ho. TFA.Ipr,Si o 'V Tit UOH-HjO ■ 71 9: Trt DCM. your, I8h 7-------- --------------L -.,- -------------H J HATU, D1PEA " VY « 8t»pe « 5 H Step 7 '[f DMF. ta, 16 h 8- 0 8 Step 5 WaPhAC (2 g)

[0073] Diagram 1

[0074] Step 1: 5-bromonaphthalen-l-amine (2):

[0075] To a stirred solution of 1-bromo-5-nitronaphthalene (1), (20.0 g, 79.36 mmol, 1.0 eq.) in aqueous methanol (200 mL), Fe (22.16 g, 396.82 mmol, 5.0 eq.) followed by ammonium chloride (42.44 g, 793.6 mmol, 10 eq.) were added at room temperature to obtain a reaction mixture. The reaction mixture was stirred at 75 °C for 1 h to obtain a reaction mass. The progress of the reaction was monitored by thin-layer chromatography (TLC). After completion of the reaction, the reaction mass was cooled to room temperature, filtered through Celite, and the filtrate was evaporated under reduced pressure. The residue was soaked with ice water (50 mL) and extracted with ethyl acetate (EtOAc). The organic phase was dried over sodium sulfate (Na2SO4) and evaporated under reduced pressure to yield 5-bromonaphthalen-l-amine (2) (15 g, 85%) as an off-white solid. Br MeOH / HO 75°C, 2 h Step 1 Br

[0076] Step 2: 5-bromo-N,N-dimethylnaphthalen-l-amine (3):

[0077] To a stirred solution of 5-bromonaphthalen-l-amine (2) (15.0 g, 67.56 mmol, 1.0 eq.) in dimethylformamide (DMF) (75 mL), NaH (sodium hydride, 8.1 g, 222.7 mmol, 3.0 eq.) was added and stirred at 0 °C. After 30 minutes, methyl iodide (12.58 mL, 222.70 mmol, 3.0 eq.) was added, and the reaction mixture was stirred at room temperature for 16 h to obtain a reaction mass. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mass was quenched with ice-cold water (50 mL) and extracted with EtOAc. The organic layer was dried over Na₂SO₄ and evaporated under reduced pressure to obtain crude material. This crude material was purified by Combi Flash on a 40 g column using 1.5% EtOAc in hexane to obtain 5-bromo-N,N-dimethylnaphthalen-1-amine (3) (12.5 g, 74%) as a white solid. LC-MS: (M+l)=252.1 L Br Mel / NaH THF 60 °C, 4 PM Step 2 Br

[0078] Step 3: 2-(5-(dimethylamino)naphthalen-l-yl)methyl acetate (5):

[0079] To a stirred solution of 5-bromo-N,N-dimethylnaphthalen-l-amine (3) (12.0 g, 48.0 mmol, 1.0 eq.) in xylene (100.0 mL), potassium 3-methoxy-3-oxopropanoate (4) (11.23 g, 72.0 mmol, 1.5 eq.), and DMAP (4-dimethylaminopyridine, 0.585 g, 4.8 mmol, 0.1 eq.) were added. The solution was degassed with nitrogen gas at room temperature for 10 minutes before and after the addition of Pd(Allyl)2Cl2(allylpalladium chloride, 0.35 g, 0.96 mmol, 0.02 eq.), and Xanthphos (4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene, 1.66 g, 2.88 mmol, 0.06 eq.). The reaction mixture was then stirred at 140 °C for 16 h. The reaction progress was monitored by TLC. After completion of the reaction, the reaction mass was filtered through a Celite bed. The filtrate was diluted with EtOAc and washed with water. The organic layer was washed with a saturated sodium bicarbonate solution, brine, dried over sodium sulfate, and concentrated under reduced pressure. The crude product was purified by Combi Flash using 20% ​​EtOAc in hexane to obtain 2-(5-(dimethylamino)naphthalen-l-yl)methyl(5) acetate (6.5 g, 55%) as a yellow liquid. LC-MS: (M+l)=244.10 Step 3

[0080] Step 4: 2-(5-(dimethylamino)naphthalen-l-yl)acetic acid. Lithium salt (6):

[0081] To a stirred solution of 2-(5-(dimethylamino)naphthalen-l-yl)methyl acetate (5) (6.00 g, 24.69 mmol, 1.0 eq.) in a tetrahydrofuran-methanol-water mixture (THF-MeOH-H2O in a 3:1:1 ratio, 50.0 mL), aqueous lithium hydroxide (LiOH·H2O, 5.18 g, 123.45 mmol, 5.0 eq.) was added. The total reaction mixture was then stirred at room temperature for 16 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mass was concentrated under reduced pressure. The residue was diluted with water and washed with EtOAc. The aqueous layer was acidified with hydrochloric acid (HCl)IN until the pH reached 8, then extracted with EtOAc. The resulting organic layer was washed with brine, dried over sodium sulfate, and concentrated under reduced pressure to obtain a lithium salt of 2-(5-(dimethylamino)naphthalen-l-yl)acetic acid, (6) (6.3 g, crude) in the form of a yellow liquid.LC-MS :(M+l)=230,l .

[0082] Step 5: N-(2-(5-(dimethylamino)naphthalen-l-yl)acetyl)-S-trityl- Methyl L-cysteinate (8):

[0083] To a stirred solution of 2-(5-(dimethylamino)naphthalen-l-yl)acetic acid, lithium salt (6) (2.50 g, 10.91 mmol, 1.0 eq.) in DMF (20.0 mL) and HATU (6.23 g, 16.36 mmol, 1.5 eq.) were added and stirred at room temperature. After 10 min, methyl S-trityl-L-cysteinate (7) (3.96 g, 10.91 mmol, 1.0 eq.) followed by DIPEA (5.71 mL, 32.77 mmol, 3.0 eq.) were added and the reaction mixture was stirred at room temperature for 16 h. The progress of the reaction was monitored by TLC. After the reaction was complete, the reaction mass was concentrated under reduced pressure, washed with n-pentane, and concentrated again under reduced pressure. The crude oil has was purified by combi flash using 30-35% EtOAc in hexane to obtain methyl N-(2-(5-(dimethylamino)naphthalen-l-yl)acetyl)-S-trityl-L-cysteinate (8) (5.19 g, 80%) as a yellow solid. LC-MS: (M+1) = 589.3. HO J Xi O 6 O '' .-K ..î OS X 7 NHj < f5”' > O ; , ..............................* AXA A. ■ As HATU.DIPEA ■" YN' DMF.ta.15h 8 H Step 5

[0084] Step 6: N-(2-(5-(dimethylamino)naphthalen-l-yl)acetyl)-S-trityl-L-cysteine ​​(9):

[0085] To a stirred solution of methyl N-(2-(5-(dimethylamino)naphthalen-l-yl)acetyl)-S-trityl-L-cysteinate (8) (1.0 g, 1.70 mmol, 1.0 eq.) in THF-MeOH-H₂O (in a 3:1:1 ratio, 30.0 mL), LiOH·H₂O (0.21 g, 5.1 mmol, 3.0 eq.) was added. The entire reaction mixture was then stirred at room temperature for 3 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mass was concentrated under reduced pressure. The resulting residue was diluted with water and washed with EtOAc. The aqueous layer was then acidified with HCl at pH 8 and extracted with EtOAc. The organic layer was washed with brine, dried on sodium sulfate, filtered and concentrated under reduced pressure.The crude product was purified by Combi Flash using 45-50% EtOAc in hexane to obtain N-(2-(5-(dimethylamino)naphthalen-l-yl)acetyl)-S-trityl-L-cysteine ​​(9) (700 mg, 72%) as an off-white solid. LC-MS: (M + 1) = 575.2. LiOH.HjO Step 6

[0086] Step 7: (2-(5-(dimethylamino)naphthalen-l-yl)acetyl)-L-cysteine ​​(Dimethylamino NaPhAc):

[0087] To a stirred solution of N-(2-(5-(dimethylamino)naphthalen-l-yl)acetyl)-S-trityl-L-cysteine ​​(9) (1.0 g, 1.74 mmol, 1.0 eq.) in DCM (dichloromethane, 15.0 mL), triisopropyl silane (1.79 mL, 8.7 mmol, 5.0 eq) was added, followed by the addition of TFA (trifluoroacetic acid, 5.32 mL, 8.70 mmol, 40 eq) at 0 °C and stirring at room temperature for 30 min. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mass was concentrated under reduced pressure and washed with concentrated n-Pantane under reduced pressure. crude compound was purified by titration with ethyl acetate and diethyl ether to obtain (2-(5-(dimethylamino)naphthalen-l-yl)acetyl)-L-cysteine ​​(Formula (I) Dimethylamino NaPhAc, NNDNAC) (1.9 g, 82%) as a white solid.

[0088] The compound obtained of Formula (I) was characterized by NMR and mass spectrum.

[0089] 1H NMR (400 MHz, Methanol-d4) α 8.18 (d, j=8, 1H), 8.03 (d, j=8 Hz, 1H), 7.60 at 7.76 (m, 4H), 4.61 (dd, J=4.4 Hz, 6.8 Hz, 1H), 4.17 (s, 2H), 3.30 (s, 6H), 2.93 (ddd, 4.4 Hz, 17.2 Hz, 34 Hz, 2H).

[0090] 13 C NMR (100 MHz, methanol-d4) ô 173.0, 172.4, 143.2, 134.4, 134.1, 129.9, 127.7, 127.1, 126.5, 125.4, 121.0, 117.1, 55.5, 46.5, 40.7, 26.2

[0091] Mass: 332 (M+) 333 (M+l); 331 (MH). HG, G n Tft TFA, lpr3Si if*'- O ï I r he i to DCM ta. 4 p.m. XKA U 1 \ Step? H 4S Dimethylamino NaPhAc {2 g) Example 2 Sensitizer detection method

[0092] The compound of formula (I) (NNDNAC) was used as a probe to detect skin sensitizing compounds.

[0093] The method for detecting a sensitizer was carried out by mixing the compound of formula (I) (NNDNAC) with a test compound (p-benzoquinone, 2-methyl-4-isothiazolin 3-one, farnesal, cinnamyl alcohol, lactic acid, p-phenylenediamine, 4-aminophenol) in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (base) to obtain a first solution. The first solution was then incubated for approximately 1 hour to obtain an incubated solution. The incubated solution was subjected to spectral analysis by liquid chromatography followed by fluorescence measurement using DAD / FLD detectors (diode array detector / fluorescence detector) to measure the depletion of the compound of formula (I). The peak intensity of compound Formula (I) was correlated with the % depletion of compound Formula (I) (probe compound, NNDNAC). Based on the % depletion of compound Formula (I), the test compounds were identified as sensitizing.A 100% depletion of the Formula (I) compound (absence of an NNDNAC peak in the chromatogram) showed that the test compound was a highly reactive sensitizer. Figure 1 (a, b, and c) schematically represents the chromatograms of incubated solutions comprising the test compounds p-benzoquinone, 2-methyl-4-isothiazolin 3-one, and farnesal, respectively, showing a depletion of 100%, 98%, and 3% of the probe compound (NNDNAC), respectively.

[0094] The test compounds were also subjected to existing assays such as the direct cysteine-peptide reactivity assay (Cys-DPRA) and the NAC-amino acid derivative reactivity assay (NAC-ADRA) in accordance with OECD 442C guidelines, and the % depletion of the corresponding probe compounds (cysteine ​​derivative (peptide) and N-acetylcysteine) was measured. Other techniques such as the Keratinosens and local lymph node (LLNA) assays were also performed on the test compounds, and the results are summarized in Table 1 below.

[0095] [Tables 1] Test compound % Depletion of compound / probe pep tide Keratinosens EC3 (mM) LLNA EC3 (mM) NNDNAC Cys-DPR A NAC-A DRA p-benzoquinone 100 100 100 32.77 0.01 2-methyl-4 isothiazo lin 3-one 98 100 100 29.56 1.9 Farnesal 3 15-55 20-40 >2000 12 Cinnamyl alcohol 1 0 0 >2000 21 Lactic acid 0 0 0 >2000 NC p-phenylenediamine 0 98.6 100 ND 0.16 4-Aminophenol 0 100 100 ND ND

[0096] ND-Not Determined

[0097] According to Table 1, p-benzoquinone and 2-methyl 4-isothiazolin-3-one were observed to be potent sensitizers with existing methods. Detection and identification of the test compounds using NNDNAC (Formula (I)) as the probe compound of the present invention also showed complete depletion of the probe compound (NNDNAC) after 1 h of incubation, which was consistent with existing methods. Therefore, it should be understood that the method of the present invention is equivalent to conventionally employed techniques, but with a much shorter incubation time. Furthermore, Farnesal was observed to be a moderate sensitizer with Cys-DPRA and NAC-ADRA, while the method of the present invention showed minimal depletion of the probe compound (NNDNAC) after 1 h of incubation, suggesting that Farnesal was a weak sensitizer. This conclusion was well supported by KeratinoSens data and LLNA. Cinnamyl alcohol and lactic acid were found to be the least reactive or non-reactive in the present process and were consistent with existing processes.

[0098] Furthermore, the results of the existing Cys-DPRA and NAC-ADRA processes, in which the incubation time was approximately 24 hours, led to the conclusion that test compounds such as p-phenylenediamine and 4-aminophenol were strong electrophilic sensitizers. However, the results obtained from the process of the present invention (NNDNAC as the probe compound) indicated that these compounds were pro-electrophiles and only caused strong peptide depletion when the molecules underwent auto-oxidation during the prolonged incubation time in the existing processes (NAC-ADRA and Cys-DPRA). Thus, the present process was able to identify pro-electrophiles from electrophiles.

[0099] Table 2 shows the test results for the detection of skin sensitizing compounds using the method of the present invention compared to the existing NAC-ADRA assay, at different incubation times of 1 hour and 24 hours.

[0100] [Tables2] Test compound % Depletion of probe compound / peptide NNDNAC NAC-ADRA Incubation time 1h 24 h 1h 24 h p-benzoquinone 100 100 4 100 2-methyl-4-isothiazolin-3-one 98 100 4.8 100 Cinnamyl alcohol 1 2 0 0 Lactic acid 0 0 0 0 p-phenylenediamine 0 100 0 100 4-aminophenol 0 100 0 100

[0101] According to Table 2, it can be observed that the existing assay (NAC-ADRA) showed that not all test compounds depleted the peptide after 1 hour of incubation, but after 24 hours of incubation, the test compounds did deplete the peptide. Thus, the existing assay was only effective with a 24-hour incubation time. However, the method of the present invention showed a variable percentage depletion of the probe compound depending on the incubation time for the different test compounds, which facilitated categorization into electrophilic and pro-electrophile.

[0102] Figure 2 (a and b) schematically illustrates chromatograms of incubated p-benzoquinone solutions under varying incubation times using the process of the present invention and NAC-ADRA respectively.

[0103] The percentage of depletion of the probe compound (NNDNAC) was maximal at both 1 h and 24 h incubation times for p-benzoquinone and 2-methyl 4-isothiazolin 3-one, confirming that these compounds are electrophiles. Thus, the present method using the probe compound (NNDNAC) detected electrophilic skin sensitizers within a 1-hour incubation time and confirmed that the present method is time-efficient compared to existing assays that required a 24-hour incubation time.

[0104] Furthermore, the p-phenylenediamine and 4-aminophenol compounds showed no depletion of the probe compound (NNDNAC) at 1 h of incubation, whereas said compounds depleted the probe compound at 24 h of incubation time, which confirmed that these compounds are pro-electrophiles due to their auto-oxidation.

[0105] Thus, the present method is advantageous because it requires minimal incubation time and is effective in distinguishing pro-electrophiles from electrophiles. ADVANTAGES OF THIS DISCLOSURE

[0106] The present invention provides a compound of formula (I) and a probe comprising the compound of formula (I) for rapid analysis of sensitizers requiring a shorter incubation time. The present invention also provides a method for preparing the compound of formula (I).

[0107] The present invention provides a practical, economical, and time-efficient method for detecting skin sensitizing compounds. The probe of the present method is capable of detecting and identifying pro-electrophilic and electrophilic sensitizers.

Claims

1. Demands Compound of formula (I), its solvates, stereoisomers, enantiomers, racemates or their salts G

2.

3.

4.

5.

6. Formula (I) in which A is an aryl nucleus comprising C10 to C20 atoms; - Ri and R2 are independently chosen from a Ci6 alkyl, or a C612 aryl; or Ri and R2 combine together to form a heterocyclyl nucleus having 3 to 8 atoms; - m is in a range from 0 to 10; and - n is in a range from 0 to 10. Compound according to claim 1, wherein A is selected from naphthalene, anthrene, chrysene, phenanthrene or pyrene; R1 and R2 are independently a C,6 alkyl; m is in a range of 0 to 3; and n is in a range of 0 to 3. Compound according to any one of claims 1 and 2, wherein A is naphthalene, Ri and R2 are independently a Ci alkyl; m is 1; and n is 0. Probe for detecting a sensitizer comprising the compound of Formula (I) according to any one of claims 1 to 3. Probe according to claim 4, wherein the sensitizer is an electrophilic or pro-electrophilic skin sensitizing compound. Method for detecting a sensitizer, the method comprising: a. mixing the compound of formula (I) according to any one of claims 1 to 3 or the probe according to claim 4, with the sensitizer in the presence of a base to obtain a first solution; b. Incubating the first solution for a period of 0.5 to 2 hours to obtain an incubated solution; c. spectral analysis of the incubated solution to measure the compound of Formula (I); and d. identification of the sensitizer.

7. A method according to claim 6, wherein the spectral analysis is carried out by chromatographic detection and by fluorescence, and / or wherein the identification of the sensitizer is done by measuring the depletion of the compound of formula (I).

8. A method according to claim 7, wherein the exhaustion of the compound of formula (I) categorizes the sensitizer.

9. A method according to any one of claims 6 to 8, wherein the base is selected from 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo(4.3.0)non-5-ene (DBN), 1,4-diazabicyclo[2.2.2]octane (TED), 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD), 4-dimethylaminopyridine, pyridine, piperazine, or combinations thereof.

10. A method according to any one of claims 6 to 9, wherein the sensitizer is an electrophile or a pro-electrophile.