Synthesis, characterization and hplc analysis of the (1S,2S,5R)-diastereomer and the enantiomer of the clinical candidate ar-15512

Article abstract of DOI:10.3390/molecules26040906

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 AR-15512 has also been reported, revealing that epimerization processes at the C-1 can occur at specific stages of the synthesis. In order to confirm that the desired configuration of AR-15512 does not change throughout the process and to discard the presence of the enantiomer in the final product due to possible contamination of the initial starting material, both the enantiomer of AR-15512 and the diastereomer at the C-1 were synthesized and fully characterized. In addition, the absolute configuration of the (1S,2S,5R)-diastereomer was determined by X-ray crystallographic analysis, and new HPLC methods were designed and developed for the identification of the two stereoisomers and their comparison with the clinical candidate AR-15512.

Full text of DOI:10.3390/molecules26040906

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; rodriguez.arevalo@ub.edu (S.R.-A.); epujol@ub.edu (E.P.);
soniaabas88@gmail.com (S.A.)
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; cgaldeano@ub.edu
2
*
Correspondence: cescolano@ub.edu (C.E.); svazquez@ub.edu (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
AR-15512 has also been reported, revealing that epimerization processes at the C-1 can occur at
specific stages of the synthesis. In order to confirm that the desired configuration of AR-15512 does
not change throughout the process and to discard the presence of the enantiomer in the final product
due to possible contamination of the initial starting material, both the enantiomer of AR-15512 and
the diastereomer at the C-1 were synthesized and fully characterized. In addition, the absolute
configuration of the (1S,2S,5R)-diastereomer was determined by X-ray crystallographic analysis, and
new HPLC methods were designed and developed for the identification of the two stereoisomers
and their comparison with the clinical candidate AR-15512.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Rodríguez-Arévalo, S.;
Pujol, E.; Abás, S.; Galdeano, C.;
Escolano, C.; Vázquez, S. Synthesis,
Characterization and HPLC Analysis
of the (1S,2S,5R)-Diastereomer and
the Enantiomer of the Clinical
Candidate AR-15512. Molecules 2021,
26, 906. https://doi.org/10.3390/
molecules26040906
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
Published: 9 February 2021
1. Introduction
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
being evaluated in phase 2b clinical trials as an ophthalmic solution for the treatment of
dry eye, a chronic, highly prevalent, and age-related condition associated with pain and
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published maps and institutional affil-
iations.
limitations in performing daily activities [
sidered an unmet medical need, causing a significant socioeconomic burden in the general
population [ ]. AR-15512 acts as a potent and selective agonist of the TRPM8 (transient re-
1–4]. Indeed, dry eye disease is nowadays con-
5,6
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 [
ultimately lead to a restoration of tear film volume and a reduction of ocular discomfort
in patients suffering from dry eye syndrome [ ]. AR-15512, formerly known as AVX-012,
was developed by Avizorex Pharma, S. L. (Barcelona, Spain), a Spanish ophthalmic phar-
maceutical company working on new therapeutics for tackling dry eye syndrome [ ]. In
7,8]. Importantly, this stimulation can
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
4
9
November 2019, Avizorex and AR-15512 were acquired by Aerie Pharmaceuticals (Durham,
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, 906
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phase 2a studies performed by Avizorex to the aforementioned 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 menthol-derived cooling
agent under the name of WS-12 [12]. Since then, other patents and several published works
have followed, including studies confirming its exact stereochemistry and its binding to
the TRPM8 receptor [13].
AR-15512 is easily synthesized starting from (1R,2S,5R)-(
molecule terpenoid from peppermint oil, as previously reported (Scheme 1) [12
Hence, the first step is the reaction of the commercially available with zinc chloride in the
presence of hydrochloric acid to give (1S,2R,4R)-2-chloro-1-isopropyl-4-methylcyclohexane
). Then, treatment of this intermediate with magnesium in diethyl ether to prepare
the Grignard reagent followed by in situ reaction with CO2 provides the corresponding
carboxylic acid ( ). Finally, a reaction between and p-anisidine in the presence of the
−
)-menthol (
1
), a small
,
14,15].
1
(
2
3
3
coupling reagent benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
(PyBOP), N,N-diisopropylethylamine (DIPEA) as a base and dimethylacetamide (DMA)
as a solvent furnishes AR-15512. Alternatively, amide formation can also be achieved via
the corresponding acid chloride of
3, readily prepared by reaction with SOCl2 and like
reagents, which reacts with the same p-anisidine 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 confirm that the synthetic
sequence indicated in Scheme 1 is stereochemically robust, the synthesis of the (1S,2S,5R)-
diastereoisomer of AR-15512 (neo-AR-15512, 4) was envisaged. Furthermore, in order to
discard the presence of the enantiomer of AR-15512 (ent-AR-15512,
uct coming from possible contamination of the initial (1R,2S,5R)-(
enantiomer (1S,2R,5S)-(+)menthol, a synthetic sequence was developed to access to the
5
−
) in the final prod-
)menthol with its
enantiomer of AR-15512 (Figure 1).
Figure 1. Structure and configuration of the target compounds in this work.
Here, we report for the first time the synthesis and full characterization of both
the (1S,2S,5R)-diastereoisomer, 4, and the enantiomer of AR-15512, 5. In addition, we
provide detailed information on the high-performance liquid chromatography (HPLC)
analytical methods that have been developed for the identification of both compounds and
comparison with the clinical candidate. Finally, X-ray crystallographic analysis has been
performed to confirm the absolute configuration of the (1S,2S,5R)-diastereoisomer 4.

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2. Results and Discussion
2.1. Synthesis of (1S,2S,5R)-diastereomer of AR-15512, neo-AR-15512, 4
As previously mentioned, the synthetic sequence depicted in Scheme 1 to approach
AR-15512 starts from the commercially available (1R,2S,5R)-( )-menthol ( ). In order to ac-
cess to (1S,2S,5R)-N-(4-methoxyphenyl)-5-methyl-2-(1-methylethyl)cyclohexanecarboxamide
(neo-AR-15512) , the diastereoisomer of AR-15512 at the C-1, an inversion in the configura-
tion of the stereocenter C-1 is required from the same starting material. For its obtention,
we took advantage of the fact that the synthesis of the carboxylic acid derivative ( ) had
−
1
4
9
already been published (Scheme 2) [17]. Worth to highlight, in this reference, the author
reported that the aldehyde (8) was prone to epimerization.
Scheme 2. Synthetic procedure reported for the carboxylic acid 9 [17].
Hence, starting from the known compound 9, the corresponding acid chloride (10
)
was prepared by treatment with oxalyl chloride in dry dichloromethane (DCM) and in the
presence of a catalytic amount of dimethylformamide (DMF) under argon. Intermediate 10
was used without further purification into the next synthetic step. Finally, the formation of
compound 4 was achieved in 80% overall yield by reaction of 10 with p-anisidine in dry
DCM as a solvent and in the presence of pyridine as a base (Scheme 3).
Scheme 3. Synthesis of neo-AR-15512, 4, from the known carboxylic acid 9.
2.1.1. Characterization and X-ray Structure Analysis of 4
The diastereoisomer of AR-15512 at the C-1, compound 4, was completely character-
ized by means of 1H, 13C, COSY and HSQC NMR experiments, infrared, elemental analysis,
specific optical rotation, and melting point (see Material and Methods section and Supple-
mentary Materials for further details). Moreover, the chiral identity of its three stereogenic
centers was unambiguously confirmed by X-ray crystallography of a monocrystal obtained
by recrystallization from ethyl acetate (Figure 2). Compound
4 was found to crystallize
in the orthorhombic system, space group P 21 21 21, with the following cell parameters:
a = 9.1371(2) Å, b = 10.3821(3) Å, c = 17.4893(7) Å, V = 1659.98(7) Å3 and Z = 4 (further
crystal data can be found in Supplementary Materials) [18].

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Figure 2. Crystal structure for (1S,2S,5R)-diastereomer of AR-15512, 4.
2.1.2. HPLC Method for the Analysis of AR-15512 and Its Diastereoisomer 4
(neo-AR-15512)
Importantly, once the diastereoisomer of AR-15512 at the C-1 was synthesized, an
HPLC method for its identification and differentiation from AR-15512 was designed and
developed. Thus, AR-15512 and
of both compounds separately and in a mixture by using an Ultrabase C-18 (Akady) 5
ODS column 250 mm 4.6 mm (particle size 5 M) as stationary phase and an acetonitrile,
4 were distinguished by comparison of the retention times
×
µ
water and trifluoroacetic acid (TFA) (0.1%) as a mobile phase, with a UV detector set at 254
nm (Table 1) (see Materials and Methods and Supplementary Materials for further details).
Table 1. Retention times of AR-15512 and its diastereomer 4.
Compound
Retention Time (min)
AR-15512
neo-AR-15512, 4
11.8
15.6
2.2. Synthesis of the Enantiomer of AR-15512, 5 (ent-AR-15512)
In pursuance of the enantiomer of AR-15512, (1S,2R,5S)-N-(4-methoxyphenyl)-5-
methyl-2-(1-methylethyl)cyclohexanecarboxamide 5, we first considered following the
synthetic route previously shown in Scheme 1, but starting from the corresponding
enantiomer, i.e., (1S,2R,5S)-(+)-menthol. However, an interesting alternative was found
based on the synthetic accessibility of the epimerization-prone aldehyde
8, which had
indeed been used for obtaining diastereomer
(see Scheme 2) [17].
4, according to the bibliographic reference
Therefore, considering the known lability of the hydrogen in the
hyde derivative we reasoned that following the same sequence depicted
in Scheme 2, but starting from the enantiomer of , (1S,2R,5S)-(+)-menthol (11), we may
gain access to the enantiomer of , the aldehyde (14). We hypothesized that epimerization
α-position of the alde-
8,
1
8
at C-1 of the intermediate aldehyde 14 should take place under acidic conditions as it
would produce the thermodynamically more stable diastereoisomer 15, in which all three
substituents (aldehyde, isopropyl and methyl) are in equatorial (Scheme 4). The latter
would then serve as an intermediate for the synthesis of the final compound 5.

Molecules 2021, 26, 906
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Scheme 4. Route of synthesis for 5, ent-AR-15512, and key epimerization of 14 to 15.
To our delight, the envisaged synthetic strategy turned out to be successful and
allowed us to synthesize . In this manner, the first synthetic step involved the treatment
5
of 11 with mesyl chloride in dry DCM and in the presence of triethylamine as a base
to yield the mesylate derivative (12) in 83% yield. In the next step, the inversion of the
configuration in the C-1 occurred by means of an SN2 reaction of the mesylate group by
cyanide. The reaction of mesylate 12 with potassium cyanide in dry acetonitrile and in
the presence of equimolecular quantities of 18-crown-6 furnished the nitrile derivative (13
)
as a unique product with an R configuration in the C-1 and in 42% yield. The following
step involved the reduction of the nitrile to the aldehyde 14 in nearly quantitative yield
with DIBAL-H in dry THF. As expected, the aldehyde 14 is susceptible to epimerization
by treatment with aqueous sulfuric acid furnishing in 87% yield its epimer 15 that has
the required stereochemistry in the three stereogenic centers to access the enantiomer
of AR-15512. Oxidation of 15 using the Jones reagent yielded carboxylic acid 16 in 62%
yield. Subsequently, treatment of 16 with oxalyl chloride in dry DCM and in the presence
of a catalytic amount of DMF provided acid chloride (17), which was then reacted with
p-anisidine in DCM under basic conditions to give the final compound, enantiomer 5, with
38% yield (Scheme 4).
2.2.1. Characterization of 5
The final compound
5
was fully characterized by 1H, 13C, COSY and HSQC NMR
experiments, infrared, elemental analysis, specific optical rotation, and melting point (see
Material and Methods section and Supplementary Materials for further details).
2.2.2. Chiral HPLC Method for the Analysis of AR-15512 and Its Enantiomer 5
(ent-AR-15512)
Just as we proceeded with the diastereomer
and developed to demonstrate that compound
candidate AR-15512 and to discern between them. In particular, the chiral HPLC method
was set up using a chiral column (Chiralpack IC 250 mm 4.6 mm (particle size 5 M))
4
, a specific HPLC method was designed
5
was indeed the enantiomer of the clinical
×
µ
as stationary phase and a mixture of hexane and 2-propanol as mobile phase, with a
UV detector set at 254 nm (for detailed information, see Materials and Methods and
Supplementary Materials). The retention times of the two compounds are shown below
(Table 2).

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Table 2. Retention times of AR-15512 and its enantiomer 5.
Compound
Retention Time (min)
AR-15512
ent-AR-15512, 5
12.0
10.0
3. Materials and Methods
3.1. Chemical Synthesis
3.1.1. General Methods
Commercially available reagents and solvents were used without further purification
unless stated otherwise. Column chromatography was performed on silica gel 60 (35–
70 µm) with the indicated solvent system. Thin-layer chromatography was performed with
aluminum-backed sheets with silica gel 60 F254 (Merck, ref 1.05554), and spots were visual-
ized with UV light and 1% aqueous solution of KMnO4. Melting points were determined
in open capillary tubes with an MFB 595010M Gallenkamp instrument. IR spectra were
performed in a Shimadzu IRAffinity-1S spectrophotometer, and only noteworthy IR absorp-
tions (cm−1) are listed. Optical rotations were measured on a PerkinElmer 341 polarimeter.
NMR spectra were recorded in CDCl3 at 400 MHz (1H) and 100.6 MHz (13C). Chemical
shifts are reported in
δ values downfield from TMS or relative to residual chloroform
(7.26 ppm, 77.0 ppm) as an internal standard. Data are reported in the following manner:
chemical shift, multiplicity, coupling constants (J) in hertz (Hz), integrated intensity, and
assignment. Multiplicities are reported using the following abbreviations: s, singlet; dd,
doublet of doublets; ddd, doublet of a doublet of doublets; dddd, doublet of a doublet of a
doublet of doublets; m, multiplet; dm, double multiplet; br s, broad signal. Assignments
given for the NMR spectra of the compounds were carried out on the basis of COSY 1H/1H
(standard procedures) and COSY 1H/13C (gHSQC) experiments. The elemental analyses
were carried out in a Flash 1112 series ThermoFinnigan elemental microanalyzer (A5) to
determine C, H, and N. The two final compounds possessed purity
their elemental analyses.
≥
95%, as evidenced by
3.1.2. Synthesis of Intermediates 6–9
Compounds
dures [17].
6, 7, 8 and 9 were synthesized according to previously published proce-
3.1.3. Synthesis of (1S,2S,5R)-5-methyl-2-(1-methylethyl)cyclohexanecarbonyl Chloride, 10
The carboxylic acid (293 mg, 1.6 mmol) and a catalytic amount of dry DMF (6 L,
9
µ
0.08 mmol) were put in dry DCM (10 mL) under argon and cooled to 0 ◦C, followed by
the slow addition of oxalyl chloride (0.17 mL, 1.9 mmol) over a 10 min period. When the
reaction was completed (monitored by NMR), the solvent was removed in vacuo. A yellow
oil was obtained (323 mg, 100%) and was used in the next step without further purification.
Its 1H NMR spectrum was in agreement with the reported data [19].
3.1.4. Synthesis of neo-AR-15512, (1S,2S,5R)-N-(4-methoxyphenyl)-5-methyl-2-
(1-methylethyl)cyclohexanecarboxamide, 4
To a solution of p-anisidine (296 mg, 2.4 mmol) in dry DCM (8 mL) under argon,
pyridine (0.2 mL, 2.4 mmol) was added. The solution was cooled to 0 ◦C and treated with
the previously obtained compound 10 (323 mg, 1 mmol) dissolved in DCM (2 mL). The
reaction mixture was stirred at room temperature overnight. The mixture was filtered, and
the resulting filtrate was treated with 5 N aqueous NaOH until pH = 14 and subsequently
extracted with DCM (3
×
50 mL). The combined organic extracts were dried over anhydrous
Na2SO4, filtered and concentrated under reduced pressure to provide a yellow solid.
Purification by column chromatography on silica with 100% DCM as eluent gave pure
◦
◦
22
compound
4
as white crystals (360 mg, 78%), mp (ethyl acetate) 152 C (151 C [19]). [
α
]
D
22
= +17.6 (c 0.23, EtOH) ([
α
]
= +16.4 (c 1.0, EtOH) [19]). IR (KBr) ν: 527, 679, 822, 826,
D

Molecules 2021, 26, 906
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1
1036, 1165, 1246, 1510, 1520, 1643, 1651, 2920, 2938, 2949, 3289, 3412, 3480 cm−1. H NMR
(400 MHz, COSY, HETCOR, CDCl3), : 0.84 (d, J = 6.4 Hz, 3 H, 5C-CH3), 0.90 (m, 1 H,
4-Hax), 0.91 [d, J = 6.4 Hz, 3 H, CH(CH3)2], 0.94 [d, J = 6.8 Hz, 3 H, CH(CH3)2], 1.08 (ddd,
δ
0
00
J = 16.8 Hz, J = 8.0 Hz, J = 4.0 Hz, 1 H, 2-H), 1.25 (m, 1 H, 6-Hax), 1.67–1.73 (m, 2 H, 3-Ha,
5-H), 1.81 (dm, J = 12.8 Hz, 1 H, 4-Heq), 1.89–1.99 [m, 3 H, 3-Hb, 6-Heq, C (CH3)2], 2.67 (br
s, 1 H, 1-H), 3.78 (s, 3 H, OCH3), 6.84 [m, 2 H, 3(5)-ArH], 7.04 (br s, 1 H, NH), 7.39 [m, 2 H,
2(6)-ArH]. 13C NMR (100.6 MHz, CDCl3),
: 21.3 [CH3, CH( H3)2], 21.7 [CH3, CH( H3)2],
22.5 (CH3, 5C-CH3), 25.5 (CH2, 3-C), 27.1 [CH, H(CH3)2], 30.5 (CH, 5-C), 35.3 (CH2, 4-C),
H
δ
C
C
C
39.3 (CH2, 6-C), 44.3 (CH, 1-C), 46.8 (CH, 2-C), 55.3 (CH3, OCH3), 114.1 [CH, 3(5)-ArC],
121.7 [CH, 2(6)-ArC], 131.3 (C, 1-ArC), 156.2 (C, 4-ArC), 173.4 (C, CON). Anal. calcd for
C18H27NO2: C 74.70, H 9.40, N 4.84. Found: C 74.90, H 9.34, N 4.69.
3.1.5. Synthesis of (1S,2R,5S)-(+)-menthol Mesylate, 12
A solution of (1S,2R,5S)-(+)-menthol (11) (15 g, 96 mmol) in dry DCM (188 mL) under
◦
argon was cooled to 0 C, and to that methane sulfonyl chloride (9.3 mL, 120 mmol)
was added in one portion. The mixture was stirred for 20 min, followed by a dropwise
addition of triethylamine (17.5 mL, 124.8 mmol) to the cold solution. The mixture was
◦
stirred for 24 h at 20 C, whereupon complete consumption of starting material was shown
by TLC analysis on SiO2 using 15% ethyl acetate in hexane as eluent. The mixture was
diluted with DCM (200 mL) and washed with water (2
×
100 mL), saturated aqueous
NaHCO3 (100 mL) and saturated aqueous NaCl (50 mL). The organic solution was dried
over anhydrous Na2SO4, filtered, and the solvent removed in vacuo. The pale yellow oil
was chromatographed on silica using 10% ethyl acetate in hexane as eluent to give 12 as
a colorless oil (18.6 g, 83%). Its 1H NMR spectrum was in agreement with the reported
1
data for ent-12
[
17]. H (400 MHz, CDCl3),
δ
: 0.83 (d, J = 6.8 Hz, 3 H, 5C-C
H
3), 0.87 (m, 1
H, 4-Hax), 0.92 [d, J = 6.8 Hz, 3 H, CH(CH3)2], 0.93 [d, J = 6.8 Hz, 3 H, CH(CH3)2], 1.04 (m,
1 H), 1.20 (m, 1 H), 1.30–1.49 (complex signal, 2 H), 1.57–1.69 (complex signal, 2 H), 2.00 (m,
1 H), 2.19 (dm, J = 12.0 Hz, 1 H), 3.00 (s, 3 H, SO2C
H
3), 4.54 (ddd, J = 10.8 Hz, J0 = 10.8 Hz,
J00 = 4.4 Hz, 1 H, 1-H).
3.1.6. Synthesis of (1R,2R,5S)-2-isopropyl-5-methylcyclohexane-1-carbonitrile, 13
To a solution of the mesylate 12 (5.76 g, 24.6 mmol) in dry acetonitrile (70 mL) under
argon was added potassium cyanide (8 g, 123 mmol) and 18-crown-6 (6.5 g, 24.6 mmol)
and the resulting mixture was heated to reflux for 48 h. The mixture was poured into
DCM (100 mL). The yellow solution was washed with water (5
×
30 mL) to remove excess
cyanide and 18-crown-6. The organic phase was washed with saturated aqueous NaCl
(50 mL) and dried over anhydrous Na2SO4. The solution was filtered, and the solvents
were removed in vacuo. The resulting orange oil was chromatographed on silica with
5% ethyl acetate in hexane as eluent, affording compound 13 as a pale yellow oil (1.68 g,
1
42%). Its 1H NMR spectrum was in agreement with the reported data for ent-13
[
20]. H
(400 MHz, CDCl3), δ: 0.85 (m, 1 H), 0.92 (d, J = 6.8 Hz, 3 H, 5C-CH3), 0.96 [d, J = 6.4 Hz,
6 H, CH(CH3)2], 1.00 (m, 1 H), 1.15 (ddd, J = 13.2 Hz, J0 = 12.0 Hz, J00 = 4.0 Hz, 1 H), 1.28
(m, 1 H), 1.59 (m, 1 H), 1.68–1.82 (complex signal, 2 H), 1.89 (m, 1 H), 1.99 (dq, J = 13.2 Hz,
J0 = 2.8 Hz, 1 H), 3.06 (m, 1 H, 1-H).
3.1.7. Synthesis of (1R,2R,5S)-2-isopropyl-5-methylcyclohexane-1-carboxaldehyde, 14
A solution of nitrile 13 (1.41 g, 8.5 mmol) in dry THF (10 mL) under argon was cooled
to 0 ◦C. Diisobutylaluminum hydride (17.5 mL,◦1.2 M in toluene, 21.3 mmol) was added
◦
dropwise. The resulting solution was stirred at 0 C for 4 h, then allowed to warm to 25 C,
and stirred for 18 additional hours. To quench, 200 mL of diethyl ether and 100 mL of 2%
aqueous H2SO4 were cooled to 0 ◦C. The reaction mixture was added in 5 mL aliquots
to the two-phase mixture with stirring between additions. After complete addition, the
two-phase mixture was further acidified with 4 mL of 2% aqueous H2SO4. The organic
phase was separated, and the aqueous phase was extracted with diethyl ether (3
×
150 mL).

Molecules 2021, 26, 906
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The organic phases were combined and washed with saturated aqueous NaCl (100 mL),
then dried over anhydrous Na2SO4, filtered, and the solvents removed in vacuo. 14 was
isolated as a pale yellow oil (1.41 g, 99%). Due to its instability upon chromatography, this
compound was used without further purification. Its 1H NMR was in agreement with the
reported data for ent-14 [17].
3.1.8. Synthesis of (1S,2R,5S)-2-isopropyl-5-methylcyclohexane-1-carboxaldehyde, 15
A solution of aldehyde 14 (0.84 g, 5 mmol) in diethyl ether (50 mL) was cooled to
◦
0 C, followed by a dropwise addition of 25% aqueous H2SO4 (18 mL) to the cold solution.
The resulting solution was stirred at 0 ◦C for 4 h and at room temperature overnight. The
organic phase was separated, and the aqueous phase was extracted with diethyl ether
(3
×
100 mL). The organic phases were combined and washed with saturated aqueous
NaCl (100 mL), then dried over anhydrous Na2SO4, filtered, and the solvents removed in
vacuo. Aldehyde 15 was isolated as a pale orange oil (732 mg, 87%). Due to its instability
upon chromatography, 15 was used in the next step without further purification. Its 1H
NMR was in agreement with previously reported data [21].
3.1.9. Synthesis of (1S,2R,5S)-2-isopropyl-5-methylcyclohexane-1-carboxylic Acid, 16
To a solution of the aldehyde 15 (732 mg, 4.35 mmol) in 30 mL of diethyl ether and
◦
cooled to 0 C, whereupon an excess of Jones reagent (8 mL) was transferred dropwise via
pipet until the reaction solution remained orange colored. This mixture was vigorously
stirred at room temperature for 60 min. A few drops of 2-propanol were then added until
the mixture remained dark green colored and the solvents were removed under reduced
pressure. The green solid residue was dissolved in water (50 mL), and this mixture was
extracted with diethyl ether (4
under reduced pressure until about 50 mL of the solvent remained. Then, it was washed
with 2 N aqueous NaOH (3
50 mL), the combined basic layers acidified at 0 ◦C with
conc. HCl until the pH was adjusted to about 2. The white precipitate thus formed was
extracted with diethyl ether (3 40 mL); the combined ethereal layers were successively
×
50 mL). The combined organic layers were concentrated
×
×
washed with one portion of water and saturated aqueous NaCl and dried over anhydrous
Na2SO4. The dried ethereal solution was filtered through Celite® to remove any remaining
chromium impurities, and the solvent was then removed in vacuo to give yellow crystals
of acid 16 (500 mg, 62%). Its 1H NMR was in agreement with previously reported data [22].
3.1.10. Synthesis of (1S,2R,5S)-2-isopropyl-5-methylcyclohexane-1-carbonyl Chloride, 17
The carboxylic acid 16 (0.45 g, 2.44 mmol) and a catalytic amount of dry DMF (9 µL,
0.12 mmol) were put in dry DCM (15 mL) under argon and cooled to 0 ◦C, followed by
the slow addition of oxalyl chloride (0.25 mL, 2.93 mmol) over a 10 min period. When
the reaction was completed (monitored by NMR), the solvent was removed in vacuo.
Compound 17 was obtained as a yellow oil (493 mg, 100%). Its 1H NMR was in agreement
with the reported data for ent-17 [19].
3.1.11. Synthesis of ent-AR-15512, (1S,2R,5S)-N-(4-methoxyphenyl)-5-methyl-2-
(1-methylethyl)cyclohexanecarboxamide, 5
To a solution of the p-anisidine (451 mg, 3.66 mmol) in dry DCM (16 mL) under
argon, pyridine (0.3 mL, 3.66 mmol) was added. The solution was then cooled to 0 ◦C and
treated with the corresponding acid chloride 17 (493 mg, 2.44 mmol) dissolved in DCM
(4 mL). The reaction mixture was stirred at room temperature overnight. The mixture
was filtered, and the resulting filtrate treated with 5 N aqueous NaOH until pH = 14 and
subsequently extracted with DCM (3
over anhydrous Na2SO4, filtered and concentrated under reduced pressure to provide a
yellow solid. The resulting solid was chromatographed on silica with 100% DCM as eluent.
×
50 mL). The combined organic extracts were dried
The final compound
5 was obtained as white crystals (245 mg, 38%), mp (ethyl acetate)
178.6 ◦C. [α]22D = +56.0 (c 0.25, EtOH).

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22
The specific optical rotation of AR-15512 was also measured [
α
]
=
−
57.6 (c 0.25,
D
22
EtOH) ([
α
]
=
−
58.6 (c 1.0, EtOH) [19]). IR (KBr)
ν
: 725, 833, 1032, 1165, 1236, 1302, 1510,
D
1539, 1645, 1651, 2909, 2967, 3044, 3231 cm−1. H (400 MHz, COSY, HETCOR, CDCl3),
δ:
1
0.83 (d, J = 6.8 Hz, 3 H, 5C-CH3), 0.91 [d, J = 6.0 Hz, 3 H, CH(CH3)2], 0.92 [d, J = 6.8 Hz,
3 H, CH(CH3)2], 0.99 (m, 1 H, 4-Hax), 1.07 (m, 1 H, 3-Hb), 1.29 (m, 1 H, 6-Hax), 1.30 (m, 1 H,
5-H), 1.61 (dddd, J = 11.2 Hz, J0 = 11.2 Hz, J00= 2.8 Hz, J000 = 2.8 Hz, 1 H, 2-H), 1.70 (m, 1 H,
3-H), 1.75 (m, 1 H, 4-Heq), 1.82 [m, 1 H, C
(ddd, J = 11.2 Hz, J0 = 11.2 Hz, J00 = 3.2 Hz, 1 H, 1-H), 3.78 (s, 3 H, OCH3), 6.84 [m, 2 H,
3(5)-ArH], 7.12 (br s, 1 H, NH), 7.42 [m, 2 H, 2(6)-ArH]. 13C NMR (100.6 MHz, CDCl3),
16.3 (CH3, 5C-CH3), 21.4 [CH3, CH( H3)2], 22.3 [CH3, CH( H3)2], 24.0 (CH2, 3-C), 28.8
[CH, H(CH3)2], 32.3 (CH, 5-C), 34.5 (CH2, 4-C), 39.4 (CH2, 6-C), 44.6 (CH, 2-C), 50.6 (CH,
H(CH3)2], 1.87 (dm, J = 11.6 Hz, 1 H, 6-Heq), 2.12
δ:
C
C
C
1-C), 55.5 (CH3, OCH3), 114.1 [CH, 3(5)-ArC], 121.7 [CH, 2(6)-ArC], 131.1 (C, 1-ArC), 156.3
(C, 4-ArC), 174.0 (C, CON). Anal. calcd for C18H27NO2: C 74.70, H 9.40, N 4.84. Found: C
74.62, H 9.26, N 4.64.
3.2. X-ray Crystallographic Analysis of neo-AR-15512, 4
A colorless needle-like specimen of C18H27NO2 (M = 289.42 g/mol), compound
approximate dimensions 0.046 mm 0.101 mm 0.576 mm, was used for the X-ray
crystallographic analysis. The X-ray intensity data were measured with a D8 Venture
system equipped with a multilayer monochromator and a Cu microfocus ( = 1.54178 Å)
4,
×
×
λ
at 100.2 K. The frames were integrated with the Bruker SAINT software package using a
narrow-frame algorithm. The structure was solved and refined using the Bruker SHELXTL
Software Package, using the space group P 21 21 21, with Z = 4 for the formula unit
C18H27NO2 (orthorhombic system). The final cell constants of a = 9.1371(2) Å, b = 10.3821(3)
Å, c = 17.4893(7) Å, V = 1659.98(7) Å3, are based upon the refinement of the XYZ-centroids
of reflections above 20 σ(I). Data were corrected for absorption effects using the multiscan
method (SADABS). For further information, refer to Supplementary Materials [18].
3.3. HPLC Equipment and Methods
3.3.1. HPLC Equipment
All analyses were carried out with a Waters HPLC system with a 1525 binary pump
coupled to a UV detector Waters 2489 set at 254 nm and using Waters Breeze software.
3.3.2. HPLC Method for Identification of AR-15512 and neo-AR-15512, 4
Sample concentration. Solid sample 1 mg AR-15512 in 1 mL acetonitrile; solid sample
1 mg compound
4 in 1 mL acetonitrile; and solid sample mixture 1 mg AR-15512/1 mg
compound 4 in 2 mL acetonitrile.
Chromatographic conditions. The employed column was a Ultrabase C-18 (Akady) 5
ODS 4.6 mm
temperature of 25 ◦C. The injection volume was 10
set at = 254 nm. The mobile phase consisted of acetonitrile + 0.1% TFA and water + 0.1%
×
250 mm (5 µ
m), with a constant flow of 1.0 mL min−1 and a controlled
µL, and the detection wavelength was
λ
TFA. A gradient method was developed and is detailed in Table 3.
Table 3. Detailed gradient for the developed method.
Time (min)
% Acetonitrile + 0.1% TFA
% Water + 0.1% TFA
0
0.5
3
15
20
22
25
5
5
65
65
95
95
5
95
95
35
35
5
5
95

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3.3.3. Chiral HPLC Method for Identification of AR-15512 and ent-AR15512, 5
Sample concentration. Solid sample 1 mg AR-15512 in 1 mL 2-propanol; solid sample
1 mg compound in 1 mL 2-propanol; and solid sample mixture 1 mg AR-15512/1 mg
compound in 2 mL 2-propanol; then, from the first two solutions, a new solution was
5
5
prepared with 0.75 mL of AR-15512 solution and 0.25 mL of compound 5 solution.
Chromatographic conditions. The employed column was a Chiralpack IC (Daicel Chem-
ical Ind., Ltd., Tokyo, Japan) 5 ODS 4.6 mm
×
250 mm (5
µ
m), with a constant flow of
L, and
λ = 254 nm. The mobile phase consisted of isocratic 90%
1.0 mL min−1 and a controlled temperature of 25 ◦C. The injection volume was 10
µ
the detection wavelength was
hexane and 10% 2-propanol.
4. Conclusions
In this work, two stereoisomers of AR-15512, a TRPM8 agonist currently in clinical
trials for the treatment of the very common ophthalmological disorder dry eye, were
synthesized and fully characterized. On one hand, its diastereomer at the C-1 was obtained,
and its absolute configuration was confirmed by X-ray crystallographic analysis. On the
other hand, and to our knowledge, we report for the first time a synthetic sequence to
access the enantiomer of AR-15512. In addition, we provide two different HPLC methods
designed and developed to identify both stereoisomers and to differentiate them from the
clinical candidate AR-15512.
1
Supplementary Materials: The following are available online, Figure S1. H, 13C, HSQC and COSY
NMR and IR spectra of compound
of compound , Tables S1–S5. X-ray crystallographic data for compound
4
, Figure S2. 1H, 13C, HSQC and COSY NMR and IR spectra
5
4, Figure S3. HPLC
chromatograms of compounds AR-15512 and
4, Figure S4. HPLC chromatograms of compounds
AR-15512 and 5.
Author Contributions: C.E. and S.V. designed and supervised the work; S.R.-A. performed the
chemical synthesis and full characterization; S.A. and C.G. performed HPLC chromatography and
analyzed the analytical data; E.P. wrote and edited the manuscript. All authors have read and agreed
to the published version of the manuscript.
Funding: This research was funded by AVIZOREX PHARMA, S. L.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest. The funder had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
Sample Availability: Samples of the compounds are available from the authors.
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