A COMPOUND, A PROBE AND RELATED METHODS

Abstract

A COMPOUND, A PROBE AND RELATED METHODS The present disclosure provides a compound of formula (I), wherein A is an aryl ring comprising C10 to C20 atoms; R1 and R2 are independently selected from C1-6 alkyl, or C6-12 aryl; or R1 and R2 combine together to form a heterocyclyl ring having 3 to 8 atoms; m is in a range of 0 to 10; and n is in a range of 0 to 10. The present disclosure further provides a probe comprising the compound of formula (I) for the detection of a sensitizer. Furthermore, the present disclosure provides a method for detecting a sensitizer. Formula (I) Figure for abstract: none

Description

Title of the invention: A COMPOUND, A PROBE AND RELATED METHODS FIELD OF THE INVENTION

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

[0002] Skin sensitization is an important endpoint for cosmetic risk assessment, taking into account both the nature of the ingredients used and post-marketing 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, fragrance materials and cosmetics allows for proper risk and efficacy estimation, as well as risk management without the use of ethically problematic in vivo methods.Due to the complexity of biological mechanisms associated with skin sensitization, integrated approaches combining different chemical, biological and in silico methods are recommended to replace classical 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 to covalently bind to the biological target and induce further haptenation processes. Current chemical assays rely on the quantification of model nucleophiles that have not reacted after incubation with the candidate sensitizer.

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

[0005] Therefore, there is a need to develop a reactive compound for use as a probe and a method for rapidly identifying potential electrophilic skin sensitizers and for differentiating an electrophilic sensitizer from a pro-electrophile sensitizer. Summary of the invention

[0006] In one aspect of the present disclosure, there is provided a compound of formula (I), its solvates, stereoisomers, enantiomers, racemates or salts thereof

[0007] Formula (I)

[0008] in which A is an aryl ring comprising Cio to C2o atoms; • R1 and R2 are independently selected from C1-6 alkyl or C6-n aryl; or R1 and R2 combine together to form a heterocyclyl ring 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 the present disclosure, there is provided a probe for detecting a sensitizer comprising the compound of formula (I) as disclosed herein.

[0010] In another aspect of the present disclosure, there is provided a method of detecting a sensitizer, 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 time of 0.5 to 2 hours to obtain an incubated solution; (c) spectrally analyzing the incubated solution to measure the compound of formula (I); and (d) identifying the sensitizer.

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

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

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

[0014] Figure 2 schematizes the chromatograms of the incubated solutions of 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 an embodiment of the present disclosure. DESCRIPTION OF THE INVENTION

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

[0016] Definitions

[0017] For convenience, before describing the present disclosure in more detail, certain terms used in the patent specification and examples are defined herein. These definitions should be read in light of the remainder of the disclosure and understood as by a person skilled in the art. The terms used herein have the meanings recognized and known to those skilled in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

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

[0019] The terms “include” and “comprising” are used in the 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 specification, unless the context otherwise requires, the term "comprise", and variations such as "comprises" and "comprising", shall be understood to imply the inclusion of a recited element or step or group of elements or steps, 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 herein, the term "C,6 alkyl" refers to straight or branched chain C1-C6 alkyl which may be optionally substituted. Representative examples of alkyl 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 herein, the term "aryl" refers to cyclic or polycyclic ring groups (spiro, fused, bridged, unfused) that satisfy Huckel's rule. Representative examples of aryl rings include benzene, naphthalene, chrysene, pyrene, anthracene, phenanthrene, or the like.

[0026] According to one embodiment herein, the term "heterocycle" or "heterocyclic" or "heterocyclyl" includes "aromatic heterocycle", "non-aromatic heterocycle" or polycyclic or bicyclic ring compounds (spiro, fused, bridged, non-fused) in which the ring may be aromatic or non-aromatic, wherein the heterocyclic ring 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 herein, 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 acid, 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 base salts include alkali metal salts of sodium or potassium and alkaline earth metal salts of calcium or magnesium hydroxides and organic bases, e.g., 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, therefore, may exist as stereoisomers, such as double bond isomers (i.e., geometric isomers), regioisomers, enantiomers, or diastereomers. Accordingly, the chemical structures depicted 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 may be resolved into their enantiomeric or stereoisomeric components using separation techniques or chiral synthesis techniques well known to those skilled in the art.The compounds may also exist in several tautomeric forms including the enol form, the keto form, and mixtures thereof. Accordingly, the chemical structures diagrammed herein encompass all possible tautomeric forms of the compounds illustrated or identified. It is also understood that certain isomeric forms such as diastereomers, enantiomers, and geometric isomers may be separated by physical and / or chemical methods by those skilled in the art. In addition, the compounds may exist as a mixture having each of the enantiomers in equal amounts to form racemates or a racemic mixture.

[0030] According to one embodiment herein, 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 its reactivity.

[0031] According to one embodiment herein, the term "electrophile" refers to a compound that has a tendency to attract or acquire electrons and is thus reactive in nature. For the purposes of the present 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 the disclosure, electrophiles are skin-sensitizing electrophilic compounds and are also referred to as haptens.

[0032] According to one embodiment herein, 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 into haptens cause allergic reactions on the skin. For the purposes of this disclosure, pro-electrophiles are skin-sensitizing pro-electrophile compounds and are also referred to as pre-haptens.

[0033] All percentages, parts, and ratios are based on the total weight of the compositions of the present disclosure unless otherwise indicated. Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that this range format is used solely for convenience and brevity and is to be flexibly interpreted as including not only the numerical values ​​explicitly cited as the limits of the range, 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 about 0.5 to 2 hours should be interpreted to include not only the explicitly stated limits of about 0.5 to about 2 hours, but also subranges, 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 commonly understood by a person skilled in the art to which this disclosure pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the disclosure, the preferred methods and materials are now described.

[0035] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for exemplary purposes only. Functionally equivalent products and methods are clearly within the scope of the disclosure as described herein.

[0036] The present disclosure provides a compound of formula (I), a probe comprising the compound of Formula (I), and a method for detecting a sensitizer using the compound of Formula (I). The present disclosure provides an efficient chemical characterization tool for identifying potential skin sensitizers with rapid and high throughput. The probe and method for detecting a sensitizer of the present disclosure are suitable and provide inexpensive approaches for pre-screening large chemical compounds. The method for detecting a sensitizer using the probe comprising the compound of Formula (I) is advantageous over time-consuming and expensive in vitro and in vivo evaluations of skin sensitizing compounds. Furthermore, the method disclosed herein facilitates the distinction between pro-electrophile sensitizers and electrophilic sensitizers which existing methods fail to achieve.

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

[0038] in which A is an aryl ring comprising atoms Cm to C2o; • R1 and R2 are independently selected from C1-6 alkyl, or C6-12 aryl; or R1 and R2 combine together to form a heterocyclyl ring 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 in particular allows for the detection of a sensitizer with rapid yield and by an economical approach. Embodiments herein also provide a probe comprising a compound of formula (I).

[0040] Embodiments herein also provide a method of detecting a sensitizer to categorize a pro-electrophile and an electrophile. Compound

[0041] Embodiments herein provide a compound of formula (I), its solvates, stereoisomers, enantiomers, racemates or their salts, according to the present invention, Formula (I)

[0042] in which A is an aryl ring comprising Cio to C20 atoms; • R1 and R2 are independently selected from C1-6 alkyl, or C6-12 aryl; or R1 and R2 combine together to form a heterocyclyl ring 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 invention, it makes it possible, in particular, to effectively detect a sensitizer using the compound of formula (I).

[0044] According to a particular embodiment herein, A is an aryl ring comprising C10 to C[8] atoms; R1 and R2 are independently selected from C1_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 herein, A is selected from naphthalene, anthracene, chrysene, phenanthrene or pyrene; R 1 and R 2 are independently selected from C 1 -C 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 hereof, A is naphthalene, R 1 and R 2 are independently C 1 alkyl; m is 1; and n is 0.

[0047] According to one embodiment herein, the compound of formula (I) acts as a nucleophile to react with reactive electrophilic or pro-electrophilic sensitizers and then detects reactive species. The compound enables 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 herein, employed in a reactivity probe overcomes the disadvantages associated with time consumption, difficulties in the detection method, cost of reagents and high yield.

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

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

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

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

[0052] According to other embodiments herein, 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 ring comprising Cio to C2o atoms; • R1 and R2 are independently selected from C1-6 alkyl, or C6-12 aryl; or R1 and R2 combine together to form a heterocyclyl ring 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 herein, the probe comprises a compound of formula (I), wherein A is an aryl ring comprising C10 to C18 atoms; R1 and R2 are independently selected from C1-6 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 herein, the probe comprises a compound of formula (I), wherein A is selected from naphthalene, anthracene, chrysene, phenanthrene or pyrene; R 1 and R 2 are independently selected from C 1-6 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 herein, the probe comprises a compound of formula (I), wherein A is naphthalene, R 1 and R 2 are independently C 1 alkyl; m is 1; and n is 0.

[0057] According to other embodiments herein, 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 inert components, in general, that may be employed during the formulation of the probe.

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

[0059] Embodiments herein include a method for detecting sensitizers. The method includes detecting sensitizers, particularly electrophilic and pro-electrophile sensitizers.

[0060] Embodiments herein provide 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) spectrally analyzing the incubated solution to measure the compound of Formula (I); and (d) identifying the sensitizer.

[0061] Embodiments herein provide 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), wherein A is naphthalene, R 1 and R 2 are independently C 1 alkyl; 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) spectrally analyzing the incubated solution to measure the compound of Formula (I); and (d) identifying the sensitizer.

[0062] According to one embodiment herein, 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-dimethylamino pyridine, pyridine, piperazine, or combinations thereof. According to a particular embodiment herein, the base is 1,8-diazabicyclo[5.4.0]undec-7-ene. According to another particular embodiment herein, the base is 1,5-diazabicyclo[4.3.0]non-5-ene.

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

[0064] According to other embodiments herein, the identification of the sensitizer is done by measuring the depletion of the compound of formula (I). According to other embodiments herein, the compound of formula (I), being a nucleophile, reacts with an electrophilic sensitizer, and leads to the depletion of the compound of Formula (I).

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

[0066] According to still other embodiments herein, there is provided a use of the method for detecting a sensitizer as disclosed herein for pre-screening a cosmetic composition. The method as disclosed herein is used as a technique for analytically assessing a composition to understand the reactivity of the constituent compounds that lead to skin sensitization.

[0067] Although the subject matter has been described in considerable 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 restrictively impose any limitations on the scope of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure pertains. Although methods and materials similar or equivalent to those described herein may be used in practicing the disclosed methods and compositions, the exemplary methods, devices, and materials are described herein. It is to be understood that this disclosure is not limited to the particular methods and experimental conditions described, as such methods and conditions may apply. Example 1

[0069] Compound of formula (I)

[0070] The compound of formula (I) of the present disclosure, NN dimethyl N-(2-(l-naphthyl)acetyl)-L-cysteine ​​(NNDNAC) having A as naphthalene, R 1 and R 2 independently being C 1 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 Scheme 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, 4:08 p.m.- 0 8 Step 5 WaPhAC (2 g)

[0073] Diagram 1

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

[0075] To a stirred solution of l-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 quenched with ice-cold water (50 mL) and extracted with ethyl acetate (EtOAc). The organic phase was dried over sodium sulfate (Na2SO4) and evaporated under reduced pressure to afford 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 min, 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 was extracted with EtOAc. The organic layer was dried over Na2SO4 and evaporated under reduced pressure to obtain crude material. The 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.l Br Mel / NaH THF 60°C, 16 h Step 2 Br

[0078] Step 3: 2-(5-(dimethylamino)naphthalen-1-yl)methyl acetate (5):

[0079] To a stirred solution of 5-bromo-N,N-dimethylnaphthalen-1-amine (3) (12.0 g, 48.0 mmol, 1.0 eq.) in xylene (100.0 mL) was added 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.). The solution was degassed with nitrogen gas at room temperature for 10 min 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 bed of Celite. The filtrate was diluted with EtOAc and washed with water. The organic layer was washed with saturated sodium bicarbonate solution, brine, dried over sodium sulfate, and concentrated under reduced pressure. The resulting crude was purified by Combi Flash using 20% ​​EtOAc in hexane to obtain methyl 2-(5-(dimethylamino)naphthalen-1-yl)acetate (5) (6.5 g, 55%) as a yellow liquid. LC-MS: (M+1)=244.10 Step 3

[0080] Step 4: 2-(5-(dimethylamino)naphthalen-1-yl)acetic acid. Lithium salt (6):

[0081] To a stirred solution of methyl 2-(5-(dimethylamino)naphthalen-1-yl)acetate (5) (6.00 g, 24.69 mmol, 1.0 eq.) in tetrahydrofuran-methanol-water (THF-MeOH-H2O in a ratio of 3:1:1, 50.0 mL), aqueous lithium hydroxide (LiOH.H2O, 5.18 g, 123.45 mmol, 5.0 eq.) was added. Then, the total reaction mixture was stirred at room temperature for 16 h. The reaction progress 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 1N hydrochloric acid (HCl) until the pH reached 8 and then extracted with EtOAc. The resulting organic layer was washed with brine, dried over sodium sulfate, and concentrated under reduced pressure to obtain 2-(5-(dimethylamino)naphthalen-1-yl)acetic acid lithium salt, (6) (6.3 g, crude) as 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-1-yl)acetic acid, lithium salt (6) (2.50 g, 10.91 mmol, 1.0 eq.) in DMF (20.0 mL), HATU (6.23 g, 16.36 mmol, 1.5 eq.) was 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 completion of the reaction, the reaction mass was concentrated under reduced pressure, washed with n-pentane and concentrated under reduced pressure. The crude was 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 À. ■ 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-1-yl)acetyl)-S-trityl-L-cysteinate (8) (1.0 g, 1.70 mmol, 1.0 eq.) in THF-MeOH-H 2 O (in a ratio of 3:1:1, 30.0 mL), LiOH.H2O (0.21 g, 5.1 mmol, 3.0 eq.) was added. Then, the whole reaction mixture was stirred at room temperature for 3 h. The reaction progress was monitored by TLC. After completion of the reaction, the reaction mass was concentrated under reduced pressure. The obtained residue was diluted with water and washed with EtOAc. Then, the aqueous layer was acidified with 1N HCl pH~8 and was extracted with EtOAc. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure.The crude 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 reaction progress was monitored by TLC. After completion of the reaction, the reaction mass was concentrated under reduced pressure, washed with concentrated n-Pantane under reduced pressure. The The 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 obtained compound of Formula (I) was characterized by NMR and mass spectrum.

[0089] 1 H NMR (400 MHz, Methanol-d4) ô 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 Û 1 \ Step? H 4 S 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 about 1 hour to obtain an incubated solution. The incubated solution was subjected to spectral analysis by liquid chromatographic analysis followed by fluorescence measurement by DAD / FLD detectors (diode array detector / fluorescence detector) to measure the depletion of the compound of Formula (I). The peak intensity of the compound of Formula (I) was correlated with the % depletion of the compound of Formula (I) (probe compound, NNDNAC). Based on the % depletion of the compound of Formula (I), the test compounds were identified as sensitizers.A 100% depletion of the compound of Formula (I) (absence of NNDNAC peak in the chromatogram) showed that the test compound was a highly reactive sensitizer. Figure 1 (a, b and c) schematically shows the chromatograms of the incubated solutions comprising the test compounds p-benzoquinone, 2-methyl-4-isothiazolin 3-one and farnesal, respectively, showing 100%, 98% and 3% depletion 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), the NAC-amino acid derivatives reactivity assay (NAC-ADRA) according to OECD 442C guidelines, and the % depletion of the corresponding probe compounds (cysteine ​​derivative (peptide) and N-acetyl cysteine) was measured. Other techniques such as the Keratinosens and local lymph node assay (LLNA) 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] From Table 1, p-benzoquinone and 2-methyl 4-isothiazolin-3-one were observed to be strong sensitizers with existing methods. Detection and identification of 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 deduction 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 method and were consistent with existing methods.

[0098] Furthermore, the results of the existing Cys-DPRA and NAC-ADRA methods in which the incubation time was about 24 hours, led to the conclusion that the test compounds such as p-phenylenediamine and 4-aminophenol were strong electrophilic sensitizers. However, the results obtained from the method of the present invention (NNDNAC as a probe compound) indicated that these compounds were pro-electrophiles and caused strong peptide depletion only when the molecules underwent autoxidation during the prolonged incubation time in the existing methods (NAC-ADRA and Cys-DPRA). Thus, the present method 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 24h 1h 24h 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] From Table 2, it can be observed that the existing assay (NAC-ADRA) showed that not all test compounds depleted the peptide at 1 h of incubation, but at 24 hours of incubation time, the test compounds depleted the peptide. Thus, the existing assay was only effective with an incubation time of 24 hours. However, the method of the present invention showed a variable % depletion of the probe compound depending on the incubation time for the different test compounds, which facilitated the categorization into electrophile and pro-electrophile.

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

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

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

[0105] Thus, the present method is advantageous because it required minimal incubation time and is effective in distinguishing pro-electrophiles from electrophiles. ADVANTAGES OF THE PRESENT 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 incurring a shorter incubation time. The present invention also provides a process 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-electrophile sensitizers and electrophile sensitizers.

Claims

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

2.

3.

4.

5.

6. Formula (I) wherein A is an aryl ring comprising C10 to C20 atoms; - R1 and R2 are independently selected from C1-6 alkyl, or C6-12 aryl; or R1 and R2 combine together to form a heterocyclyl ring having 3 to 8 atoms; - m is in a range from 0 to 10; and - n is in a range from 0 to 10. A compound according to claim 1, wherein A is selected from naphthalene, anthrene, chrysene, phenanthrene or pyrene; R i and R2 are independently C, 6 alkyl; m is in a range of 0 to 3; and n is in a range of 0 to 3. A compound according to any one of claims 1 and 2, wherein A is naphthalene, R1 and R2 are independently C1-alkyl; m is 1; and n is 0. A sensitizer detection probe comprising the compound of Formula (I) according to any one of claims 1 to 3. A probe according to claim 4, wherein the sensitizer is an electrophilic or pro-electrophile skin sensitizing compound. A method of 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. incubation of 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 and fluorescence detection, 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 depletion 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), l,5-diazabicyclo(4.3.0)non-5-ene (DBN), l,4-diazabicyclo[2.2.2]oct ane (TED), 7-methyl-l,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), l,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD), 4-dimethylamino pyridine, 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.