molecules Article Synthesis, Characterization and HPLC Analysis of the (1S,2S,5R)-Diastereomer and the Enantiomer of the Clinical Candidate AR-15512 Sergio Rodríguez-Arévalo 1 , Eugènia Pujol 1 ,Sònia Abás 1 , Carles Galdeano 2 , Carmen Escolano 1,* and Santiago Vázquez 1,* 1 Laboratori de Química Farmacèutica (Unitat Associada al CSIC), Facultat de Farmàcia i Ciències de l’Alimentació, Institute of Biomedicine (IBUB), Universitat de Barcelona, Av. Joan XXIII, 27-31, 08028 Barcelona, Spain; [email protected] (S.R.-A.); [email protected] (E.P.); [email protected] (S.A.) 2 Department of Pharmacy and Pharmaceutical Technology and Physical Chemistry, Faculty of Pharmacy and Food Sciences, Institute of Biomedicine (IBUB), University of Barcelona, Av. Joan XXIII, 27-31, 08028 Barcelona, Spain; [email protected] * Correspondence: [email protected] (C.E.); [email protected] (S.V.); Tel.: +34-934-024-542 (C.E.); +34-934-024-533 (S.V.) Abstract: AR-15512 (formerly known as AVX-012 and WS-12) is a TRPM8 receptor agonist currently in phase 2b clinical trials for the treatment of dry eye. This bioactive compound with menthol-like cooling activity has three stereogenic centers, and its final structure and absolute configuration, (1R,2S,5R), have been previously solved by cryo-electron microscopy. The route of synthesis of Citation: Rodríguez-Arévalo, S.; AR-15512 has also been reported, revealing that epimerization processes at the C-1 can occur at Pujol, E.; Abás, S.; Galdeano, C.; specific stages of the synthesis. In order to confirm that the desired configuration of AR-15512 does Escolano, C.; Vázquez, S. Synthesis, not change throughout the process and to discard the presence of the enantiomer in the final product Characterization and HPLC Analysis due to possible contamination of the initial starting material, both the enantiomer of AR-15512 and of the (1S,2S,5R)-Diastereomer and the Enantiomer of the Clinical the diastereomer at the C-1 were synthesized and fully characterized. In addition, the absolute Candidate AR-15512. Molecules 2021, configuration of the (1S,2S,5R)-diastereomer was determined by X-ray crystallographic analysis, and 26, 906. https://doi.org/10.3390/ new HPLC methods were designed and developed for the identification of the two stereoisomers molecules26040906 and their comparison with the clinical candidate AR-15512. Academic Editors: Keywords: absolute configuration; AR-15512; diastereomer; enantiomer; HPLC analytical method Patrizia Ferraboschi and Silvia Schenone Received: 30 December 2020 Accepted: 5 February 2021 1. Introduction Published: 9 February 2021 AR-15512 ((1R,2S,5R)-N-(4-methoxyphenyl)-5-methyl-2-(1-methylethyl) cyclohexan ecarboxamide) is a bioactive compound with menthol-like cooling effects that is currently Publisher’s Note: MDPI stays neutral being evaluated in phase 2b clinical trials as an ophthalmic solution for the treatment of with regard to jurisdictional claims in dry eye, a chronic, highly prevalent, and age-related condition associated with pain and published maps and institutional affil- limitations in performing daily activities [1–4]. Indeed, dry eye disease is nowadays con- iations. sidered an unmet medical need, causing a significant socioeconomic burden in the general population [5,6]. AR-15512 acts as a potent and selective agonist of the TRPM8 (transient re- ceptor potential melastatin member 8) cold thermoreceptor. TRPM8 is a calcium-permeable ion channel that serves as the principal detector of cold in humans and is involved in Copyright: © 2021 by the authors. the regulation of tear production and blink rate [7,8]. Importantly, this stimulation can Licensee MDPI, Basel, Switzerland. ultimately lead to a restoration of tear film volume and a reduction of ocular discomfort This article is an open access article in patients suffering from dry eye syndrome [4]. AR-15512, formerly known as AVX-012, distributed under the terms and was developed by Avizorex Pharma, S. L. (Barcelona, Spain), a Spanish ophthalmic phar- conditions of the Creative Commons Attribution (CC BY) license (https:// maceutical company working on new therapeutics for tackling dry eye syndrome [9]. In creativecommons.org/licenses/by/ November 2019, Avizorex and AR-15512 were acquired by Aerie Pharmaceuticals (Durham, 4.0/). NC, USA), a company that has further progressed this clinical candidate from successful Molecules 2021, 26, 906. https://doi.org/10.3390/molecules26040906 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 11 drome [9]. In November 2019, Avizorex and AR-15512 were acquired by Aerie Pharma- ceuticals (Durham, NC, USA), a company that has further progressed this clinical candi- date from successful phase 2a studies performed by Avizorex to the aforementioned Molecules 2021, 26, x FOR PEER REVIEW 2 of 11 phase 2b, with a topline readout expected in the third quarter of 2021 [10,11]. Curiously, this molecule was first described in the literature in 1971, when it was patented as a men- Molecules 2021, 26, 906 thol-derived cooling agent under the name of WS-12 [12]. Since then, other patents2 ofand 11 dromeseveral [9]. published In November works 2019, have Avizorex followed, and including AR-15512 studies were acquiredconfirming by itsAerie exact Pharma- stereo- ceuticachemistryls (Durham, and its binding NC, USA) to ,the a company TRPM8 receptor that has [13]. further progressed this clinical candi- datephase fromAR 2a- studies15512 successful is performed easily phase synthesized by2a Avizorexstudies starting performed to the from aforementioned (1 byR,2 AvizorexS,5R)-(−)- phasementholto the 2b, aforementioned (1 with), a small a topline mol- phasereadoutecule 2b,terpenoid expected with a fromtopline in the peppermint readout third quarter expected oil, as of previously 2021 in the [10 third,11 reported]. quarter Curiously, (Scheme of 2021 this 1) molecule[10,11]. [12,14,15] Curiously, was. Hence, first thisdescribedthe moleculefirst step in thewasis the literature first react describedion in of 1971, the in commercially the when literature it was availablein patented 1971, when 1 as with a it menthol-derived zincwas chloridepatented in as the coolinga men- pres- tholagentence-derived underof hydrochloric thecooling name agent ofacid WS-12 underto give [12 the]. Since(1 nameS,2R then,,4 ofR) -WS other2-chloro-12 patents [12].-1-isopropyl Since and severalthen,-4- methylcyclohexaneother published patents works and severalhave(2). Then, followed, published treatment including works of this have studies intermediate followed, confirming withincluding its magnesium exact studies stereochemistry in confirming diethyl ether and its itstoexact prepare binding stere theo- to chemistrytheGrignard TRPM8 andreagent receptor its binding followed [13]. to bythe inTRPM8 situ reaction receptor with [13]. CO 2 provides the corresponding car- boxylicARAR-15512- 15512acid ( 3is). is easilyFinally, easily synthesized synthesizeda reaction startingbetween starting from 3 fromand (1 Rp (1-,2anisidineRS,5,2RS,5)-(R−)-() -inmenthol− the)-menthol presence (1), a ( 1small ),of athe small mol- cou- eculemoleculepling terpenoid reagent terpenoid frombenzotriazol frompeppermint peppermint-1-yl oil-oxytripyrrolidinophosphonium, as oil,previously as previously reported reported (Scheme (Scheme hexafluorophosphate1) [12,14,15]1)[ 12. Hence,,14,15]. theHence,(PyBOP first thestep), N first ,isN -the stepdiisopropylethylamine react is theion reaction of the commercially of the (DIPEA) commercially available as a base available 1 and with dimethylacetamide1 zincwith chloride zinc chloride in the (DMA) inpres- the encepresenceas a ofsolvent hydrochloric of hydrochloric furnishes acid AR acid -15512.to togive give Alternatively, (1 (1S,2S,2RR,4,4RR)-)-2-chloro-1-isopropyl-4-methylcyclohexane2- chloroamide- formation1-isopropyl can-4- methylcyclohexanealso be achieved via ((2the).). Then, Then,corresponding treatment treatment acidof of this thischloride intermediate intermediate of 3, readily with with magnesium prepared magnesium by in reactiondiethyl in diethyl etherwith ether toSOCl prepare to2 prepareand thelike Grignardthereagents, Grignard reagent which reagent reacts followed followed with by the in by same situ in situ reactionp-anisidine reaction with in with CO appropriate2 COprovides2 provides base the correspondingand the correspondingsolvent. car- boxyliccarboxylic acid acid (3). (Finally,3). Finally, a reaction a reaction between between 3 and3 andp-anisidinep-anisidine in the in presence the presence of the of cou- the plingcoupling reagent reagent benzotriazol benzotriazol-1-yl-oxytripyrrolidinophosphonium-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate hexafluorophosphate (PyBOP(PyBOP),), N,,NN--diisopropylethylaminediisopropylethylamine (DIPEA) (DIPEA) as as a a base base and dimethylacetamide (DMA) (DMA) asas a a solvent solvent furnishes furnishes AR AR-15512.-15512. Alternatively, Alternatively, amide formation can also be achieved via thethe corresponding corresponding acid acid chloride chloride of of 3,, readily prepared by reaction with SOClSOCl2 and like reagents,reagents, which reacts reacts with the same p--anisidineanisidine in appropriate base and solvent. Scheme 1. Structure of the product AR-15512 and its reported synthetic procedure. Bearing in mind both the chemical structure of AR-15512 and its synthesis, it has not been fully discarded whether epimerization processes at the C-1 can take place during its obtention [16]. Therefore, in order to rule this out and to