Synthesis, anticonvulsant activity and metabolism of 4-chlor-3-methylphenoxyethylamine derivatives of trans-2-aminocyclohexan-1-ol

Article abstract of DOI:10.1002/chir.22406

In this study, we report the synthesis, spectral characterization, antiepileptic activity and biotransformation of three new, chiral, N-aminoalkyl derivatives of trans - 2 aminocyclohexan-1-ol: 1 (R enantiomer), 2 (S enantiomer) and 3 (racemate). Antiepileptic activity of the titled compounds was studied using MES and scMet. Moreover, in this study, the biotransformation of 1, 2 and 3 in microbial model (Cunninghamella), liver microsomal assay as well as in silico studies (MetaSite) was evaluated. Studies have indicated that 1, 2 and 3 have good antiepileptic activity in vivo, comparable to valproate. Biotransformation assays showed that the most probable metabolite (indicated in every tested assays) was M1. The microbial model as well as in silico study showed no difference in biotransformation between tested enantiomers. However, in a rat liver microsomal study compound 1 and 2 (R and S enantiomer) had different main metabolite - M2 for 1 and M1 for 2. MS/MS fragmentation allowed us to predict the structures of obtained metabolites, which were in agreement with 1°alcohol (M1) and carboxylic acid (M2). Our research has shown that microbial model, microsomal assay, and computational methods can be included as useful and reliable tools in early ADME-Tox assays in the process of developing new drug candidates.

Full text of DOI:10.1002/chir.22406

CHIRALITY 27:163–169 (2015)
Synthesis, Anticonvulsant Activity and Metabolism of 4-chlor-3-
methylphenoxyethylamine Derivatives of Trans-2-aminocyclohexan-1-ol
1
PAULINA KUBOWICZ,1 HENRYK MARONA,2 AND ELŻBIETA PĘKALA
*
1Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, Jagiellonian University, Medical College, Cracow, Poland
2Department of Bioorganic Chemistry, Chair of Organic Chemistry, Faculty of Pharmacy, Jagiellonian University, Medical College, Cracow, Poland
ABSTRACT
In this study, we report the synthesis, spectral characterization, antiepileptic ac-
tivity and biotransformation of three new, chiral, N-aminoalkyl derivatives of trans – 2
aminocyclohexan-1-ol: 1 (R enantiomer), 2 (S enantiomer) and 3 (racemate). Antiepileptic activ-
ity of the titled compounds was studied using MES and scMet. Moreover, in this study, the bio-
transformation of 1, 2 and 3 in microbial model (Cunninghamella), liver microsomal assay as
well as in silico studies (MetaSite) was evaluated.
Studies have indicated that 1, 2 and 3 have good antiepileptic activity in vivo, comparable to
valproate. Biotransformation assays showed that the most probable metabolite (indicated in ev-
ery tested assays) was M1. The microbial model as well as in silico study showed no difference
in biotransformation between tested enantiomers. However, in a rat liver microsomal study com-
pound 1 and 2 (R and S enantiomer) had different main metabolite – M2 for 1 and M1 for 2.
MS/MS fragmentation allowed us to predict the structures of obtained metabolites, which were
in agreement with 1°alcohol (M1) and carboxylic acid (M2).
Our research has shown that microbial model, microsomal assay, and computational methods
can be included as useful and reliable tools in early ADME-Tox assays in the process of develop-
ing new drug candidates. Chirality 27:163–169, 2015. © 2014 Wiley Periodicals, Inc.
KEY WORDS: Cunninghamella; Biotransformation; Anticonvulsant activity; Liver microsomes;
Enantiomers
INTRODUCTION
AEDs.4 Therefore, there is an unmet need to develop AEDs
with lower toxicity and higher selectivity. In order to make
this development more rational, structural fragments that
may enhance anticonvulsant activity were identified.
The phenomenon of chirality plays a key role in the life of
every living organism. All proteins, amino acids, enzymes,
carbohydrates, and nucleosides are chiral compounds, which
means that they are not superimposable to their mirror im-
ages. They exist in nature under a single enantiomeric form.
In contrast, in pharmaceutical industries almost 90% of chiral
drugs are marketed as racemates consisting of an equimolar
mixture of two enantiomers. This is due to the fact that
obtaining the enantiomers is much more difficult and more
expensive.
Enantiomers show completely identical physicochemical
properties, when they are in an achiral environment. But be-
cause the human body is a chiral environment, two different
enantiomers of one compound can display the pharmacoki-
netic processes – such as absorption, distribution, metabo-
While searching for new substances with anticonvulsant
activities, we directed our attention to derivatives of
aminocyclohexanol. Many pharmacological activities of ami-
nocyclohexanol have been described (such as secretolytic,5
analgetic,6 and antidepressant).7 Our former research proved
that aminoalkanol derivatives can also act as an anticonvulsant
- they provide protection against seizures, mainly MES-induced
seizures and at the same time exhibited low neurotoxicity.8–11
We have previously reported some derivatives of 6-methoxy-2-
methylxanthone with anticonvulsant activities: (R,S)- and (S)-
1-amino- propan-2-ol as well as (R,S)-1-aminobutan-2-ol which
showed values of ED50 in MES (mice, ip) of 37.4, 33.89, and
41.2mg/kg, respectively.11
Considering our former findings we decided to continue re-
search in the group of aminoalkanol derivatives. Herein we
report the synthesis, in vivo anticonvulsant activities and the
in vitro biotransformation of the trans-2-aminocyclohexan-1-
ol derivatives – compound 1, 2, and 3 (two enantiomers
and the racemate, respectively). Those compounds were
found to have anticonvulsant activity against a MES test in
lism, and excretion
– as well as pharmacology and
toxicology, in a stereoselective manner.1
It is known that metabolizing enzymes often display a pref-
erence for one enantiomer of a chiral drug over another,
resulting in enantioselectivity.2 Therefore, enantiomers in a
racemic drug should be treated as two different compounds,
and studies on every enantiomer – not only racemates – must
be carried out.3 Antiepileptic drugs are no exception.
Epilepsy is one of the most common chronic neurological
problems characterized by seizures. Antiepileptic drugs
(AEDs) exert their action by various mechanisms: they can
influence inhibitory or excitatory neurotransmitter systems
such as GABA or glutamic and aspartic acid, respectively, or
the ion transport across cell membranes. Although there are
a number of antiepileptic drugs, there is a significant group
of patients (up to 30%) who are resistant to the available
Contract grant sponsor: Jagiellonian University Medical College; Contract
grant number: Grant Nos. K/DSC/001415.
*Correspondence to: Elżbieta Pękala, Department of Pharmaceutical Bio-
chemistry, Faculty of Pharmacy, Jagiellonian University-Medical College, 9
Medyczna Street, 30-688 Krakow, Poland. E-mail: elzbieta.pekala@uj.edu.pl
Received for publication 12 August 2014; Accepted 15 October 2014
DOI: 10.1002/chir.22406
Published online 1 December 2014 in Wiley Online Library
(wileyonlinelibrary.com).
© 2014 Wiley Periodicals, Inc.

164
KUBOWICZ ET AL.
studies carried out at the National Institute of Health
(Rockville, USA). Rat hepatic microsomal fractions in the
presence of an NADPH-generating system were used to
evaluate the metabolism of titled compounds. The results
obtained from liver fraction applications were compared to
the results from the in silico study as well as to the microbio-
logical biotransformation assay in which the Cunninghamella
model was involved.
General Methods
Procedure for the synthesis of 2-(4-chlor-3-methylphenoxy)ethyl]
amino}cyclohexan-1-ol. In the aminolysis reaction, 2.5 g (0.01 mol)
of 1-bromo- 2-( 4’-chloro-3’-methylphenoxy)ethan, 1.2 g (0.01 mol) of
1RS,2RS-trans-2-aminocyclohexan-1-ol, 1.4 g (0.01 mol) of K2CO3, and
20 ml of DMF were used.
The mixture was heated for 5 h. The precipitated KBr was separated.
The filtrate was concentrated and 15 ml of 10%HCL was added. To sepa-
rate the free base –10%NaOH was added to the filtrated. The base was
crystallized from n-hexane (mp. 94-96 °C) with a 48% yield (3). To obtain
R and S enantiomers R or S trans-2-aminocyclohexan-1-ol were used re-
spectively. The S-2-(4-chlor-3-methylphenoxy)ethyl]amino}cyclohexan-1-
ol (compound 2) (mp. 72-74 °C) was obtained with a 42.5% yield and R-
2-(4-chlor-3-methylphenoxy)ethyl]amino}cyclohexan-1-ol (compound 1)
(mp. 72-74 °C) with a 52.5% yield. 1 and 2 were used as hydrochlorides
and their melting points were 163-165 °C and 161-163 °C respectively.
MATERIALS AND METHODS
Chemicals and Reagents
Chemical synthesis. Reagents: cyclohexene oxide, L-tartaric acid, 4-
chlor-3-methylphenol, K2CO3 were purchased from Sigma Aldrich. Sol-
vents were commercially available materials of reagent grade.
Melting points (mp) are uncorrected and were determined using a
Büchi SMP-20 apparatus (Büchi Labortechnik, Flawil, Switzerland).
Elemental analyses were performed on a Elementar Vario EL III
(Elementar Analysensysteme, Hanau, Germany).
1R,2R trans-{[2-(4-chlor-3-methylphenoxy)ethyl]amino}cyclohexan-
1-ol (compound 1). Anal. Calcd for C15H22ClNO2: C, 63,47; H, 7,83; N,
1
4,93. Found: C, 63,85; H, 7,83; N, 4,93. H NMR (300 MHz, chloroform -d)
The purity of the obtained compounds was confirmed by thin-layer
chromatography (TLC), carried out on precoated aluminum sheets (Silica
Gel, 60 F-254 Merck, Darmstadt, Germany) using the solvents indicated
below. Spots were visualized by UV light. Specific rotation was measured
on a Jasco P-2000 polarimeter (1% w/v solutions in chloroform, sodium
light 589 nm).
δ ppm 1.04 - 1.20 (m, 2 H) 1.20 - 1.34 (m, 1 H) 1.59 - 1.79 (m, 3 H) 1.91 (d,
J = 14.62 Hz, 2 H) 2.12 (d, J = 12.82 Hz, 1 H) 2.31 (s, 3 H) 2.87 - 2.97 (m, 1 H)
3.28 (br. s., 1 H) 3.46 (br. s., 1 H) 3.74 (tt, J = 10.13, 5.39 Hz, 1 H) 4.26 - 4.42
(m, 2 H) 5.00 (d, J = 6.41 Hz, 1 H) 6.74 (dd, J = 8.72, 3.08 Hz, 1 H) 6.85 (d,
J = 2.82 Hz, 1 H) 7.19 (d, J = 8.72 Hz, 1 H). Rf =0.8 (toluene/methanol 1:1).
[α]20546 = +5.2 [C 0.5; CHCl3]
1H NMR spectra were obtained in CDCl3 or DMSO-d6 with a Varian
Mercury-VX 300 NMR spectrometer (Varian Inc., Palo Alto, CA, USA),
a Bruker Avance II spectrometer (Brucker, Karlsruhe, Germany) with
500.13 MHz, or a Brucker Avance III spectrometer (Brucker, Karlsruhe,
Germany) with 600.2 MHz with TMS or DMSO as an internal standard,
respectively.
1S,2S trans-{[2-(4-chlor-3-methylphenoxy)ethyl]amino}cyclohexan-
1-ol (compound 2). Anal. Calcd for C15H22ClNO2: C, 63,47; H, 7,83; N,
1
4,93. Found: C, 63,42; H, 7,80; N, 4,97. H NMR (300 MHz, chloroform -d)
δ ppm 1.05 - 1.34 (m, 3 H) 1.60 - 1.81 (m, 3 H) 1.86 - 1.96 (m, 1 H) 2.12 (d,
J = 12.57 Hz, 1 H) 2.31 (s, 3 H) 2.86 - 2.98 (m, 1 H) 3.21 - 3.31 (m, 1 H) 3.49
(dt, J = 12.76, 5.03 Hz, 1 H) 3.74 (t, J = 10.13 Hz, 1 H) 4.25 - 4.42 (m, 2 H)
4.97 (br. s., 1 H) 6.74 (dd, J = 8.72, 3.08 Hz, 1 H) 6.85 (d, J = 2.82 Hz, 1 H)
7.19 (d, J = 8.72 Hz, 1 H). Rf =0.8 (toluene/methanol 1:1). [α]20546 = - 5.2 [C
0.5; CHCl3]
The UPLC-MS/MS system consisted of a Waters ACQUITY® UPLC®
(Waters Corporation, Milford, MA, USA) coupled to a Waters TQD mass
spectrometer (electrospray ionization mode ESI-tandem quadrupole).
Chromatographic separations were carried out using an Acquity UPLC
BEH (bridged ethyl hybrid) C18 column; 2.1 × 100 mm, and 1.7 μm parti-
cle size. The column was maintained at 40 °C, and eluted under gradient
conditions using from 95% to 0% of eluent A over 10 min, at a flow rate of
0.3 ml min-1. Eluent A: water/formic acid (0.1%, v/v); eluent B:
acetonitrile/formic acid (0.1%, v/v). 10 μL of each sample were injected.
Chromatograms were recorded using a Waters λ PDA detector. Spec-
tra were analyzed in 200-700 nm range with 1.2 nm resolution and a sam-
pling rate 20 points/s.
MS detection settings of the Waters TQD mass spectrometer were as
follows: source temperature 150 °C, desolvation temperature 350 °C,
desolvation gas flow rate 600 L h-1, cone gas flow 100 L h-1, capillary poten-
tial 3.00 kV, cone potential 20 V. Nitrogen was used for both nebulizing
and drying gas. The data were obtained in a scan mode ranging from
50 to 1000 m/z in 0.5 s time intervals; 8 scans were summed up to get
the final spectrum.
1RS,2RS trans-{[2-(4-chlor-3-methylphenoxy)ethyl]amino}cyclohexan-
1-ol (compound 3). Anal. Calcd for C15H22ClNO2: C, 63,47; H, 7,83; N,
4,93. Found: C, 63,38; H, 7,96; N, 4,93.1H NMR (300 MHz, chloroform -d) δ
ppm 0.92 - 1.08 (m, 1 H) 1.19 - 1.33 (m, 3 H) 1.68 - 1.78 (m, 2 H) 2.01 - 2.14
(m, 2 H) 2.20 - 2.30 (m, 1 H) 2.33 (s, 3 H) 2.83 (dt, J = 12.57, 4.87 Hz, 1 H)
3.19 (dt, J = 12.31, 5.90 Hz, 2 H) 4.01 (t, J = 5.26 Hz, 2 H) 6.67 (dd, J = 8.72,
2.82 Hz, 1 H) 6.78 (d, J = 2.82 Hz, 1 H) 7.21 (d, J = 8.72 Hz, 1 H). Rf =0.8 (tolu-
ene/methanol 1:1).
Pharmacology. Pharmacological tests were performed at the Epilepsy
Branch, National Institute of Neurological Disorders and Stroke, National
Institutes of Health (Rockville, USA).
All compounds were tested using MES, scMet and neurotoxicity as-
says, after intraperitoneal (ip) injection into male albino mice (Carworth
Farms No. 1) as suspensions in 0.5% methylcellulose at doses of 30,
100, and 300 mg/kg. Observations were carried out after 0.5 h and 4 h af-
ter compound’s administration.12–14
Collision activated dissociation (CAD) analyses were carried out with
energy of 20 eV, and all the fragmentations were observed in the source.
Consequently, the ion spectra were obtained by scanning from 50 to
500 m/z range. The data acquisition software was MassLynx V 4.1
(Waters).
The maximal electroshock test (MES). Seizures were elicited by
60 Hz alternating current at 50 mA (mice) or 150 mA (rats) delivered for
0.2 s via corneal electrodes. A drop of 0.9% NaCl solution was placed into
each eye prior to applying the electrodes. Protection in the MES test is
defined as the abolition of the hindlimb tonic extension component of
the seizure.12–14
Biological biotransformation. Cunninghamella blakesleeana DSM
1906 (which was a gift from A.J. Carnell, University of Liverpool, UK),
was propagated on a Potato-Dextrose-Agar (PDA) plate at 30 °C for
7 days. A fermentation basal medium (20 g D-glucose and 20 g corn step
liquor in 1000 ml of water pH 5.0) was seeded with a 200 μl spore suspen-
sion gained by wetting colonies on solid medium PDA with sterile water.
The media required for the growth of Cunnighamella strains were pur-
chased from BioShop (Canada) (Potato Dextrose Agar) and from
Sigma-Aldrich (St Louis, MO, USA) (CSL, Corn Steep Liquor).
The subcutaneous pentetrazole seizure test (scMet). The scMet
was conducted by subcutaneous administration of pentetrazole dissolved
in 0.9% NaCl solution at the dose of 85 mg/kg (mice) or 70 mg/kg (rats)
into animals. A minimal time of 30 min subsequent to sc administration of
pentetrazole was used for seizure detection. A failure to observe even a
threshold seizure (a single episode of clonic spasm of at least 5 s in dura-
tion) was regarded as protection.12–14
Rat liver microsomal fractions as well as components of NADPH
regenerating fraction (NADP+, glucose-6-phosphate, glucose-6-phosphate
dehydrogenase) were obtained from Sigma Aldrich.
Chirality DOI 10.1002/chir

N-AMINOALKYL DERIVATIVES OF AMINOCYCLOHEXANOL
165
Neurotoxicity (TOX). Neurological deficit was measured in mice by
the rotorod test. The mouse was placed on a 1 in. diameter knurled plastic
rod rotating at 6 rpm. Neurotoxicity was indicated by the inability of the
animal to maintain equilibrium on the rod for at least 1 min in each of
the three trials. In rats, the neurological deficit was indicated by ataxia
and loss of placing response and muscle tone.12–14
The biotransformation was carried out for 7 days and its progress was
followed using TLC and LC-MS/MS.
Rat liver microsomal transformation. Mixture reactions contained
substrate (20 μM 1, 2, or 3 in 100 mM potassium phosphate buffer,
pH 7.4), microsomes (0.5 mg/ml rat liver microsomes), NADPH-
regenerating system (NADP, glucose-6-phosphate, glucose-6-phosphate
dehydrogenase in 100 mM potassium phosphate buffer, pH 7.4), and po-
tassium phosphate buffer (100 mM, pH 7.4). Incubation mixtures contain-
ing microsomes, substrate, and buffer were pre-incubated at 37 °C for
15 min before the addition of the NADPH-regenerating system. The
resulting mixture was incubated for 60 min at 37 °C in a Thermoblock
(Eppendrof). The reaction was stopped by the addition of perchloric acid
(69–72%, by volume). Proteins were sedimented by centrifugation.15
After the biotransformation (either microbiological or using liver frac-
tions), samples were extracted with ethyl acetate. The water phase was
separated from the organic phase. In the following experiment, only the
organic phase was used, which was then dried over anhydrous sodium
sulfate and concentrated in vacuo.
For the identification of compounds 1, 2, and 3 and their possible bio-
transformation products TLC and UPLC-MS/MS were applied. TLC was
used only for microbiological assay and was performed using Merck alu-
minum plates coated with silica gel with a thickness of 0.2 mm. It was con-
cluded, as a result of many experimental studies, that the most preferred
developing system is a mixture of toluene and methanol (1:1). Observa-
tion of the chromatograms was carried out under UV light and after expo-
sure to iodine vapors.
Anticonvulsant quantification. Anticonvulsant quantification, that is,
the dose of drug required to produce the biological responses in 50% of
animals (ED50), and the respective 95% confidence intervals, were deter-
mined for selected compounds displaying sufficient antiepileptic activity
and low neurotoxicity in the primary evaluations. The details of these pro-
cedures have been published elsewhere.12–14
Microbiological transformation. Biotransformation was carried out in
a 50 ml fermentation medium, which was inoculated with a 200 μl suspen-
sion of corresponding Cunninghamella species. Microorganisms were
cultured in 30 °C for 48 h. Flasks were shaken for the whole time. After
2 days 250 μl of the stock solution, which was prepared by dissolving
100 mg of 1, 2, or 3 in 1 ml H2O, was added to the fermentation medium.
In silico studies. Computational simulation of metabolism of the title
compound was carried out by using MetaSite software, demo version 2.1.0.
(2005), Molecular Discovery Ltd. MetaSite is a computational procedure to
predict metabolism issues related to cytochrome-mediated reactions in phase
I metabolism. The methodology uses 3-D maps of interaction energies
between the protein, chemical probes, and the 3-D structure of compounds
to be analyzed. The MetaSite procedure is completely automated and does
not require any user assistance. All the work can be handled and submitted
in a batch queue. The basic concept of MetaSite is to compare the interaction
patterns inside the protein with the 3-D structure of the ligand. The informa-
tion obtained from a friendly user interface can be easily translated into deci-
sions in the drug discovery process.16
RESULTS AND DISCUSSION
Chemistry. Title compounds 1, 2, and 3 were obtained by
N-alkylation of trans-2-amino-1-cyclohexanol with 3-methyl-4-
chlorphenoxyethyl bromide in the presence of DMF and
K2CO3.
Scheme 1. The synthesis of compounds 1, 2 and 3.
TABLE 1. The results of anticonvulsant activity evaluation in mice (ip)
MESa scMetb
TOX
Compd.no.
Dose mg/kg
0.5 h
4 h
0.5 h
4 h
0.5 h
4 h
*ASP class
1
1
30
100
300
30
100
300
30
1/1
3/3
–
0/1
0/3
–
0/1
0/1
–
0/1
0/1
–
0/4
1/8
4/4
0/4
3/8
4/4
0/4
2/8
4/4
0/2
0/4
–
2
3
0/1
3/3
–
0/1
1/3
–
0/1
0/1
–
0/1
0/1
–
0/2
0/4
–
1
1
1/1
3/3
–
0/1
1/3
–
0/1
0/1
–
0/1
0/1
–
0/2
0/4
–
100
300
MES, maximal electroshock seizure.
aNumber of animals protected/number of animals tested in the MES; ScMet, subcutaneous pentylenetetrazole seizure;
bNumber of animals protected/number of animals tested in the scMet tests; TOX toxicity screen: the minimum dose of compound whereby toxicity was exhibited.
*The ASP classification (Anticonvulsant Screening Project) is as follows: 1, anticonvulsant activity at 100 mg/kg or less; 2,– anticonvulsant activity at doses grater
than 100 mg/kg; 3, compound inactive at 300 mg/kg; 4, toxicity at doses 30 mg/kg.
The compound was not tested.
Chirality DOI 10.1002/chir

166
KUBOWICZ ET AL.
TABLE 2. The results of anticonvulsant quantification in in vivo study
1
2
Compd. no.
Dose mg/kg
TD50
ED50(MES)
38.34a
PI(MES)
1
30
100
30
100
300
66.03a
66.03ascMet
71.04a
71.04ascMet
>250b
34.45a
>500b
47.8a
1.722a
<1.722ascMet
2.839a
>90.00ascMet
25.05a
>150ascMet
99.25b
6.48a
3
<0.474ascMet
2.519b
PHT
CBZ
VPA
6.60a
32.2b
>22b
9.85a
4.90a
361b
3.57b
101b
483a
287a
1.70a
859b
395b
2.2b
1Dose of the compound which produces toxicity in 50% of tested animals; 2Dose of the compound which gives protection against seizures toxicity
in 50% of tested animals.
aValues for mice after i.p. administration.
bValues for rats after p.o. administration.
not determined,
PHT: phenytoin, BZ: carbamazpine, VPA: valproate.
2-(4-chlor-3-methylphenoxy)ethanol was synthesized from
most interesting anticonvulsant results were observed for 1
and 3. For these compounds advanced quantitative tests
(ED50 and TD50) were performed. Compound 1 displayed
anti-MES activity with a protective index (TD50/ED50) of 1.72
(mice, i.p.), and compound 3 – 2.83 (mice, i.p.) corresponding
with PI for valpronate of 1.7 (mice, i.p.) (Table 2).18
4-chlor-3-methylphenol by O-alkylation with 2-bromoethanol
in the presence of methanolate sodium. 3-methyl-4-
chlorophenoxyethyl bromide was synthesized by bromination
of 2-(4-chlor-3-methylphenoxy)ethanol by tribromo phosphine
(Scheme 1).
Racemic trans-2-amino-1-cyclohexanol was obtained from
cyclohexene oxide in reaction with 25% ammonia. The
racemic trans-2-amino-1-cyclohexanol was separated into its
optical active S-(+) and R-(-) enantiomers, using diastereoiso-
meric pairs of salts (hydrogen (+)-tartrates). The process was
possible due to the difference in solubility of the both isomers
in ethanol and methanol.17
Since compounds 1 and 2 are enantiomers of 3, it could be
expected that one of the enantiomers should be chosen for
further development. The screening results in MES reveal
that enantiomer 1 (1R,2R) is more active (in the dose of
30 mg/kg) and less neurotoxic (in the dose of 100 mg/kg)
0.5 h after administration, giving promises for the choice of
1 for development. However, further quantitative analysis of
TD50, ED50 and PI showed that 3 (racemate) has the most fa-
vorable ED50 and PI (Table 2). Moreover, the costs of provid-
ing the racemate is much lower than the costs of synthesizing
the enantiomer.
Anticonvulsant screening. The new compounds (1, 2, 3)
were tested in vivo by using three screens: MES, ScMet
(anticonvulsant test), and TOX (neurotoxicity) (Table 1). The
Fig. 1. A: The LC-MS/MS spectrum of 3. MS spectra from 3 (m/z 284) eluted at 4.38 minutes. B: The LC-MS/MS spectrumof mtabolite of 1, 2 and 3 (and its
proposed structure) obtained after biotransformation carried out by Cunninghamella blakesleeana DSM 1906. MS spectra from metabolite (m/z 300) eluted at
3.17 minutes.
Chirality DOI 10.1002/chir

N-AMINOALKYL DERIVATIVES OF AMINOCYCLOHEXANOL
167
which corresponds to the molecular weight (283) of the par-
ent molecule. In addition, in every analyzed spectra of the bio-
transformation products the protonated ion [M + H]+ at m/z
300 (M1) was noticed (Figure 1 B), which is in agreement
with the molecular formula C15H22ClNO3. MS/MS spectrum
and fragmentation analysis for metabolite M1 is shown in
Figure 2. The proposed hydroxylation at the methylphenyl
moiety is in agreement with the main metabolite of mexiletine
– hydroxymethylmexiletine.19 Comparison with mexiletine is
due to the structural similarity of this drug and the tested
compounds.
M1 was absent in the spectra from the samples obtained in
“the zero day” of biotransformation. The ion at m/z 300 ap-
peared predominantly in the spectra obtained after biotrans-
formation of every tested compound (Figure 3).
Liver microsomes biotransformation study. The biotransforma-
tion of 1, 2, and 3 by rat liver microsomes was investigated.
The presence of possible metabolites was investigated in all
incubations using UPLC-MS/MS. The protonated ion [M
+ H]+ at m/z 300 (M1) was present in every mass spectra of
analyzed samples. However, incubations of two different en-
antiomers with the same concentrations of rat liver micro-
somes exhibited a distinct pattern of metabolism.
The presented studies indicate that the most of the tested
compound disappeared from the reaction mixture during
the biotransformation assay. When compound 1 was used (R
enantiomer) the protonated ion [M+H]+ at m/z 314 (retention
time 2.8min) appeared predominantly (M2). Ion m/z 314 is in
agreement with the molecular formula C15H20ClNO4 and struc-
ture of carboxylic acid. MS/MS spectrum and fragmentation
analysis for metabolite M2 is shown in Figure 4. The proton-
ated ion [M+H]+ at m/z 342 (M4) was also present.
When compound 2 (S enantiomer) was analyzed in rat liver
microsomes model, two metabolites were detected. First,
with the protonated ion [M + H]+ at m/z 300 (M1, retention
time 3.16 min), second - M3 - with the protonated ion [M
+ H]+ at m/z 320 (retention time 2.5 min). M3 was produced
in majority. There were no M2 and M4 metabolites.
Fig. 2. MS/MS spectrum and fragmentation analysis for metabolite M1.
Microbiological transformation study. In our biotransformation
study the filamentous fungus Cunninghamella blakesleeana
DSM 1906 was used. During the study, evidence that the
strain is able to carry out the biotransformation of all the
tested compounds, was obtained. The samples from all cul-
tures were taken in subsequent days of biotransformation
and TLC was performed.
It was noticed that on the seventh day of biotransformation
no new derivatives appeared. After completion of the reaction
samples were taken to investigate the structure of the ob-
tained derivatives of 1, 2, and 3. UPLC-MS/MS spectra were
carried out. In every mass spectrum of 1, 2. and 3 the proton-
ated molecule [M + H]+ at m/z 284 (Figure 1 A) was detected,
In the case of the biotransformation of compound 3 (race-
mate), metabolites M1-M4 were present. M3 was the pre-
dominant metabolite. The parent molecule was also present.
Fig. 3. Chromatograms obtained in the microbiological model. Analysis of Cunninghamella biotransformation samples incubated with 3 at time A) 0 and B) after
7 days of biotransformation, using a LC-MS/MS operating in scan mode. For 1 and 2 chromatogram profiles are similar.
Chirality DOI 10.1002/chir

168
KUBOWICZ ET AL.
Fig. 4. MS/MS spectrum and fragmentation analysis for metabolite M2.
In the rat microsomes model, the obtained metabolites
were different depending on the used enantiomer (Figure 5).
This difference was not noticeable when the Cunninghamella
model was used.
In silico study. In our in silico study, MetaSite software was
used to predict 1, 2, and 3 metabolic transformation and in
particular to help in the identification of the metabolite struc-
ture. MetaSite is able to predict human regioselective metab-
olism using only the 3-D structure of the given compound.
Fig. 5. Chromatograms obtained in the microsomal biotransformation
model. Analysis of rat liver microsomes biotransformation samples incubated
with A: 1, B: 2 and C: 3 after 60 min of biotransformation, using a LC-MS/MS
operating in scan mode. Obtained metabolities (M1-M4) as well as parent
mplecules (1, 2 and 3) are indicated.
Fig. 6. Metabolic simulation of tested compounds performed by MetaSite.
Chirality DOI 10.1002/chir

N-AMINOALKYL DERIVATIVES OF AMINOCYCLOHEXANOL
169
The recognition of the site of metabolism could be a signifi-
cant step in designing new compounds with a better pharma-
cokinetic profile. Labile compounds can be stabilized when
the site of metabolism is known by adding stable groups at
metabolically susceptible positions.
The most probable pathways of the CYP2D6 metabolism
predicted by the MetaSite program are shown in the Figure 6.
CYP2D6 was chosen because mexiletine, the structure of
which is similar to the structure of tested compounds, is
mainly metabolized by CYP2D6.20
Katarzyna Kieć-Kononowicz for coordination of the coopera-
tion between the National Institute of Neurological Disorders
and Stroke and the Faculty of Pharmacy Jagiellonian Univer-
sity Medical College.
The publication is supported by Jagiellonian University
Medical College (Grant Nos. K/DSC/001415).
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ACKNOWLEDGMENTS
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The authors are grateful to J. Stables Ph.D for providing the
pharmacological data through the Antiepileptic Drug Devel-
opment program at the National Institutes of Neurological
Disorders and Stroke (Rockville, USA) and Professor
Chirality DOI 10.1002/chir