Design, synthesis and anticonvulsant-analgesic activity of new N-[(phenoxy)alkyl]- and N-[(phenoxy)ethoxyethyl]aminoalkanols

Article abstract of DOI:10.1039/c6md00537c

New derivatives of N-[(phenoxy)alkyl]- and N-[(phenoxy)ethoxyethyl]aminoalkanols have been synthesized and evaluated for their anticonvulsant activity in maximal electroshock (MES), maximal electroshock seizure threshold (MEST), and pentylenetetrazol (PTZ) tests. Their neurotoxicity was evaluated via rotarod and chimney tests. The compounds exhibiting the most beneficial activity and protection indices were evaluated for analgesic activity using the formalin test for neurogenic pain. They were also evaluated for their influence on cytotoxic activity using in vitro cellular models (HepG2 and CRL-2534 cell lines). Experiments performed using MTT and neutral red cytotoxicity assays showed that all evaluated compounds were safe for normal, glial cells (astrocytes) and did not induce hepatotoxic effects. Based on the results from the in vitro studies, the safety of the evaluated compounds was inferred. The most promising compound in this research was 1-{2-[2-(2,3-dimethylphenoxy)ethoxy]ethyl}piperidin-3-ol hydrochloride. Additionally, in silico metabolism prediction for the compound has been performed.

Full text of DOI:10.1039/c6md00537c

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RESEARCH ARTICLE
Design, synthesis and anticonvulsant-analgesic
activity of new N-[(phenoxy)alkyl]- and
N-[(phenoxy)ethoxyethyl]aminoalkanols†‡
Cite this: DOI: 10.1039/c6md00537c
a
b
Anna Rapacz, Anna M. Waszkielewicz, Katarzyna Pańczyk,b Karolina Pytka,a
Paulina Koczurkiewicz,c Kamil Piska,c Elżbieta Pękala,c Bogusława Budziszewska,d
Beata Starek-Świechowiczd and Henryk Maronab
*
New derivatives of N-[(phenoxy)alkyl]- and N-[(phenoxy)ethoxyethyl]aminoalkanols have been synthesized
and evaluated for their anticonvulsant activity in maximal electroshock (MES), maximal electroshock seizure
threshold (MEST), and pentylenetetrazol (PTZ) tests. Their neurotoxicity was evaluated via rotarod and
chimney tests. The compounds exhibiting the most beneficial activity and protection indices were evalu-
ated for analgesic activity using the formalin test for neurogenic pain. They were also evaluated for their in-
fluence on cytotoxic activity using in vitro cellular models (HepG2 and CRL-2534 cell lines). Experiments
performed using MTT and neutral red cytotoxicity assays showed that all evaluated compounds were safe
for normal, glial cells (astrocytes) and did not induce hepatotoxic effects. Based on the results from the
in vitro studies, the safety of the evaluated compounds was inferred. The most promising compound in this
research was 1-{2-[2-(2,3-dimethylphenoxy)ethoxy]ethyl}piperidin-3-ol hydrochloride. Additionally, in silico
metabolism prediction for the compound has been performed.
Received 23rd September 2016,
Accepted 7th November 2016
DOI: 10.1039/c6md00537c
www.rsc.org/medchemcomm
mexiletine exhibits analgesic properties in neurogenic pain,
since it significantly and dose-dependently reduces the dura-
Introduction
Epilepsy is a set of neurological disorders characterized by
hypersynchronous discharges of neurons and expressing itself
as recurrent seizures. The disease affects approximately 1% of
the human world population, influencing physical, social,
and economic aspects of life. Approximately 35% of seizures
are considered pharmacoresistant, and this, when added to
the toxicity of antiepileptic drugs and their interactions, is a
great premise for the search of novel and safe therapies.1
Premises for the search of anticonvulsants among amino-
alkanols include the activity of known antiarrhythmic drugs,
such as propranolol and mexiletine, in the maximal electro-
shock (MES) test; propranolol is also useful in the treatment
of seizures in children.2–4 Moreover, it has been proven that
tions of the first and second phases of the formalin test.5
The mechanism of action in this case, although mexiletine is
a non-selective voltage-gated sodium channel blocker, may
possibly rely on interaction with δ1 opioid receptors.6
Aroxyalkyl and aroxyethoxyethyl derivatives of amino-
alkanols as potential anticonvulsant agents have been a sub-
ject of our interest for many years. Among these, we have syn-
thesized and evaluated appropriate derivatives of
4-methylphenol, 2,6-dimethylphenol,7–9 4-chloro-3-methyl-
phenol, and 2-chloro-5-methylphenol,10 as well as 4-chloro-2-
methylphenol.11 The most active derivatives in the MES test
include structures with substitution in the phenyl ring at
positions 2,6-(CH3)2 of the phenyl group, and aminoalkanols
in configuration 1,2 (derivatives of colamine), with the linker
being ethylene or ethoxyethylene. However, in the case of
hydroxyxanthone derivatives as analogs of the studied group
of compounds modified by the aroxyl moiety, the propylene
linker proved more beneficial.12
a Department of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University
Medical College, Medyczna 9 Str., 30-688 Krakow, Poland
b Department of Bioorganic Chemistry, Faculty of Pharmacy, Jagiellonian
University Medical College, Medyczna 9 Str., 30-688 Krakow, Poland.
E-mail: awaszkie@cm-uj.krakow.pl
c Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, Jagiellonian
University Medical College, Medyczna 9 Str., 30-688 Krakow, Poland
d Department of Biochemical Toxicology, Faculty of Pharmacy, Jagiellonian
University Medical College, Medyczna 9, 30-688 Krakow, Poland
† The authors declare no competing interests.
Fig. 1 presents structures, activity in MES (mice, ip), and
neurotoxicity of reference drugs (mexiletine, propranolol)
and
compounds:
(S)-(+)-2N-[(2,6-dimethylphenoxy)ethyl]-
aminobutan-1-ol hydrochloride (I),7 (D,L)-trans-2N-[(2,6-
dimethylphenoxy)ethyl]aminocyclohexan-1-ol (II),9 and (R)-2-
[(2,6-dimethylphenoxy)ethyl]aminopropan-1-ol (III). The last
one showed activity in MES, and also in audiogenic seizures
‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/
c6md00537c
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Fig. 1 Chemical structures and anticonvulsant activity of reference compounds.2–4,9,13,14
and in female mouse electroshock or 6 Hz seizures.11 The
ethoxyethyl analog of III—(R)-2-[(2,6-dimethylphenoxy)-
ethoxyethyl]aminopropan-1-ol—in the form of hydrochloride
(IV) also exerts activity in MES.12 The mechanism of action
of III and IV is probably binding to σ, 5-HT1A receptors, as
well as 5-HT transporters. Compound IV additionally binds to
5-HT2B receptors.13,14 It also exhibits analgesic properties in
the formalin test. Fig. 1 also presents compounds V–VII,13,14
since they were found to be active in the MES and/or 6 Hz
tests. These compounds differ structurally from the most ac-
tive ones, but they also possess beneficial properties and can
serve as additional references. Especially, VII contains
piperidin-4-ol, where nitrogen is contained in the heterocyclic
aliphatic ring instead of the secondary amine group, and such
a moiety has not been explored sufficiently.
The purpose of the present study is continuation of our
former research. We planned new structures in terms of vari-
ous substitutions in the phenyl ring (2,3-, 2,4-, 2,6-dimethyl,
2,4,6-trimethyl-, 2-chloro-5-methyl-, 2-chloro-6-methyl), and
aminoalkanols (so far beneficial: 2-aminopropan-1-ol,
1-aminopropan-2-ol, 2-aminobutan-1-ol, 1-aminobutan-2-ol, as
racemates and when appropriate – enantiomers, D,L-trans-2-
aminocyclohexan-1-ol, or 4-aminocyclohexan-1-ol, 2-amino1-
phenylethan-1-ol, as well as piperidin-3- or -4-ol), as well as
varying the length and type of the linker between the two
moieties (2–4 methylene units, possibly containing oxygen in
the middle and thus forming an ether moiety).
Results and discussions
Chemistry
The title compounds consist of a group of phenoxyethyl,
phenoxypropyl, phenoxybutyl, and (phenoxyethoxy)ethyl de-
rivatives of chiral or achiral aminoalkanols. Their chemical
structures are presented in Table 1. The structures of all com-
pounds
were
preliminarily
examined
using
the
Molinspiration online toolkit,15 in order to estimate their
physicochemical parameters, including octanol–water parti-
tion coefficient (milog P), molecular weight, and topological
polar surface area (TPSA) (Table 1). None of the estimated
values exhibited derogations of the rules for “drug-like”
compounds.16,17
All compounds were achieved by means of multistep
chemical synthesis (Scheme 1), according to formerly pub-
lished procedures.7,18,19 The first step constituted obtaining
substituted phenoxyalkyl halide (chloride or bromide) or
phenoxyethoxyethyl bromide, which were subsequently used
in the N-alkylation of the appropriate aminoalkanol for
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Table 1 Structures and physicochemical properties of compounds 1–28
Compd.
R
n
Z
Configuration
milog P
Molecular weight
263.38
TPSA (Å2)
41.49
1
2,4-(CH3)2
2
D,L-trans
3.36
2
3
2,6-(CH3)2
4
R,S
R,S
2.50
3.35
251.37
265.40
41.49
41.49
4
5
D,L-trans
3.88
3.41
291.44
291.44
41.49
41.49
trans
6
7
2,4,6-(CH3)3
2-Cl, 5-CH3
3
2
2.52
3.30
277.41
305.81
32.70
41.49
R,S
8
9
2.10
3.12
269.77
283.80
32.70
41.49
trans
10
11
12
4
R,S
3.07
3.07
3.61
271.79
271.79
285.81
41.49
41.49
41.49
R-(−)
R,S
13
R-(−)
3.61
285.81
41.49
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Table 1 (continued)
Compd.
R
n
Z
Configuration
milog P
Molecular weight
TPSA (Å2)
41.49
14
S-(+)
3.61
285.81
15
16
17
2-Cl, 6-CH3
R,S
3.05
3.05
4.11
271.79
271.79
311.85
41.49
41.49
41.49
S-(+)
D,L-trans
18
trans
3.64
311.85
41.49
Compd.
R
Z
Configuration
milog P
Molecular mass
293.41
TPSA (Å2)
41.93
19
2,3-(CH3)2
2.47
20
D,L-trans
3.23
307.43
50.72
21
22
2,4-(CH3)2
R,S
2.19
2.73
267.37
281.40
50.72
50.72
R-(−)
23
24
2,6-(CH3)2
R,S
R,S
2.93
2.61
329.44
301.81
50.72
50.72
2-Cl, 5-CH3
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Table 1 (continued)
Compd.
R
Z
Configuration
milog P
Molecular mass
301.81
TPSA (Å2)
50.72
25
R,S
2.96
26
27
2.00
3.19
313.82
349.86
41.93
50.72
2-Cl, 6-CH3
R,S
28
R-(−)
3.19
349.86
50.72
achievement of each title compound in the form of a base.
Compounds 4, 17, and 19 were achieved as salts by satura-
tion with gaseous HCl in acetone, while other compounds
were finally characterized as bases.
Pharmacology
The title compounds have been subject to preliminary screening
of anticonvulsant activity via the maximal electroshock seizure
(MES) test. Among the derivatives of N-[(phenoxy)alkyl]amino-
alkanols 1–18, the most active compounds are 1: D,L-trans-2-[2-
(2,4-dimethylphenoxy)ethyl]aminocyclohexan-1-ol, which is a
2,4-dimethyl analog of reference compound II, 2: (R,S)-1-[4-
(2,6-dimethylphenoxy)butyl]aminopropan-2-ol, 6: 1-[3-(2,4,6-
trimethylphenoxy)propyl]piperidin-4-ol, 15: (R,S)-2-[4-(2-chloro-
6-methylphenoxy)butyl]aminopropan-1-ol, and 18: D,L-trans-4-
[4-(2-chloro-6-methylphenoxy)butyl]aminocyclohexan-1-ol.
Among these, the most favourable ED50 and protection indices
PI are observed for 6 and 15 (Table 2).
Among the N-[(phenoxyethoxy)ethyl]aminoalkanols, ED50 values
were derived for 19–21, 23, and 27: 1-{2-[2-(2,3-dimethylphenoxy)-
ethoxy]ethyl}piperidin-3-ol hydrochloride (19), D,L-trans-2-{2-[2-
(2,3-dimethylphenoxy)ethoxy]ethyl}aminocyclohexan-1-ol (20), and
(R,S)-2-{2-[2-(2,4-dimethylphenoxy)ethoxy]ethyl}aminopropan-1-
ol (21), as well as (R,S)-2-{2-[2-(2,6-dimethylphenoxy)ethoxy]-
ethyl}amino-1-phenylethan-1-ol (23), and (R,S)-2-{2-[2-(2-chloro-
6-methylphenoxy)ethoxy]ethyl}amino-1-phenylethan-1-ol (27).
ED50s of compounds have been presented with confidence
intervals of active doses. In the cases of 1–2, 6, 15, 20–21,
and 27, these CIs are largely covered, which suggests similar
pharmacological profiles. This is not surprising, given such
structural similarity, since we can observe that all these
Scheme
1 Synthesis of compounds 1–28 (Table 1). Reagents: (a)
Br–(CH2)n–Cl, K2CO3, TEBA, acetone; (b) Br–(CH2)n–Br, NaOH; (c)
Cl–(CH2)n–OH, K2CO3, acetone/EtOH; (d) Cl–CH2–CH2–O–CH2–CH2–
OH, K2CO3, acetone/EtOH; (e) PBr3; (f) Z (aminoalkanol) as in Table 1,
K2CO3, toluene or TEA, DMF; (g) HClgas, acetone.
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Table 2 Anticonvulsant properties (MES) and neurotoxicity (rotarod) of compounds 1–28 (mice, ip)
ED50 (confidence interval)
TD50 (confidence interval)
Dose
Lethality
in MES
Compd.
(mg kg−1
)
MESa
Rotarodb
(mg kg−1
)
PIc
Control
1
—
100
60
30
10
100
30
25
20
30
30
100
60
100
30
20
0/6
6/6
3/6
4/6
0/6
4/4
4/6
2/6
1/6
4/6
4/6
5/6
0/6
6/6
4/6
3/6
1/6
1/4
4/4
—
4/4
0/4
4/4
0/4
4/4
1/4
4/4
1/4
4/4
0/4
4/4
5/6
3/6
2/6
—
5/6
0/6
1/6
1/6
6/6
0/4
0/6
0/6
1/6
2/6
1/6
6/6
3/6
0/6
1/6
1/6
3/6
1/4
0/4
3/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
1/6
0/6
0/6
3/4
4/4
0/6
3/6
1/6
0/6
0/6
0/6
1/6
0/6
0/6
3/6
1/6
0/6
0/6
0/6
1/6
0/4
0/4
0/4
0/6
0/6
0/6
0/4
0/4
0/4
0/4
0/4
2/4
0/6
0/6
—
ED50 = 31.80 (24.12–41.93)
TD50 = 76.78 (63.19–93.29)
2.41
2
ED50 = 27.11 (22.38–32.83)
TD50 = 43.29 (34.19–54.81)
1.60
3.93
3
4
5
—
—
—
6
ED50 = 20.87 (12.56–34.69)
TD50 = 81.95 (70.45–95.32)
10
7
8
9
10
100
100
100
100
30
100
30
100
30
100
30
100
30
100
30
20
2/4
4/4
TOX
4/4
0/4
4/4
0/4
4/4
0/4
4/4
0/4
4/4
1/4
11
12
13
14
15
ED50 = 19.06 (13.84–26.25)
TD50 = 45.66 (35.84–58.17)
2.40
15
16
17
18
100
100
100
60
30
100
80
60
30
100
60
30
10
100
30
25
20
100
30
100
20
15
10
TOX
TOX
—
6/6
3/6
2/6
6/6
5/6
4/6
2/6
6/6
5/6
3/6
0/6
6/6
4/6
2/6
1/6
4/4
0/4
4/4
6/6
2/6
1/6
4/4
0/4
4/4
1/4
4/4
0/4
6/6
5/6
ED50 = 41.11 (32.54–51.94)
TD50 = 49.99 (44.75–55.84)
1.22
1.77
19
20
21
ED50 = 68.71 (63.95–73.82)
TD50 = 121.64 (116.53–126.99)
ED50 = 32.87 (26.21–41.21)
TD50 = 47.39 (38.69–58.04)
1.44
4.02
ED50 = 27.11 (22.38–32.83)
TD50 = 108.91 (101.81–116.50)
22
23
3/4
0/4
ED50 = 13.03 (11.58–14.65)
TD50 = 36.00 (31.43–41.22)
2.76
24
25
26
27
100
30
100
30
100
30
100
30
4/4
1/4
4/4
0/4
3/4
0/4
ED50 = 23.94 (21.29–26.92)
TD50 = 103.04 (67.57–157.14)
4.30
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Table 2 (continued)
ED50 (confidence interval)
TD50 (confidence interval)
Dose
Lethality
in MES
Compd.
(mg kg−1
)
MESa
Rotarodb
TOX
(mg kg−1
)
PIc
25
20
100
2/6
1/6
—
0/6
0/6
2/4
28
Phenytoin
ED50 = 6.65 (5.42–8.16)
10.34
4.09
4.44
TD50 = 68.73 (60.79–77.71)
ED50 = 10.40 (9.43–11.47)
TD50 = 42.52 (40.93–44.18)
15 min. after administration
TD50 = 46.20 (44.48–48.00)
30 min. after administration
Lacosamide
a
MES – maximal electroshock seizure test. No. of animals protected/no. of animals tested. b TD50 (mg kg−1) or number of mice in which motor
c
impairment was observed/number of mice tested (10 rpm). PI = TD50/ED50
.
compounds contain one methyl substituent in position 2 of
the phenyl ring and a second substituent – methyl in posi-
tions 4 or 6 or chlorine in position 6. An additional methyl
(in 2,4,6-trimethyl derivative 6) does not appear to influence
the activity. Within the aminoalkanol moieties, we can ob-
serve derivatives of colamine. In all cases, the linker used
represents all tested possibilities – ethylene, propylene, and
tetramethylene, as well as ethoxyethylene group.
It is worth commenting on the protection indices (PI) of
the active compounds, since it is not only their activity that
influences the behavior of the animals, but also the neurotox-
icity observed in the rotarod test and quantified by TD50
(Table 2). The most promising ratio between TD50 and ED50
was observed for compound 6, followed by 1, 15, 21, 23, and
finally 19; this ratio, although not the highest, is still compa-
rable to that of valproic acid (1.7).4
compound 27 given at a dose of 10 mg kg−1 increased it by
57% (p < 0.001). Compounds 1 and 6 only slightly elevated
the ECT (by 24 and 12%, respectively). Compounds 20 and 21
were not able to elevate the ECT at the tested doses. Refer-
ence drugs phenytoin (2.5 mg kg−1) and lacosamide (10 mg
kg−1) increased the ECT by 23% (p < 0.001) and above 25
mA, respectively (Table 3).
The scPTZ test employs chemically induced clonic seizures
and is related to human generalized absence seizures. The
most beneficial activity in the scPTZ seizures was observed in
the case of compound 21, which, at a dose of 100 mg kg−1
,
significantly prolonged the latency time to the first seizure
episode by 117% (p < 0.05). Moreover, compound 1 delayed
the onset of seizures by 58%, but this result was not statisti-
cally significant. Ethosuximide – the reference drug – delayed
the onset of seizures at a dose of 100 mg kg−1 by 140% (p <
0.001), and at a dose of 50 mg kg−1 by 124% (p < 0.01) (Table 4).
Antinociceptive activity was evaluated via the formalin
test, which is typically used as a tonic model of nociception.
For each selected compound, the respective ED50 value from
In the MEST test, two compounds, 19 and 27, caused a
significantly elevated electroconvulsive threshold (ECT) in
comparison to vehicle-treated mice. Compound 19 at a dose
of 30 mg kg−1 increased the ECT by 75% (p < 0.001), whereas
Table 3 Anticonvulsant properties of selected compounds in MEST test (mice, ip)
Compd.
Dose (mg kg−1
)
CS50 (confidence interval) (mA)a
Effect (%)
Control
—
—
—
1
20
9.33 (7.65–11.38)
23.90
Control: 7.53 (6.45–8.79)
9.15 (7.76–10.78)
Control: 8.13 (7.41–8.92)
13.18 (12.12–14.33)***
Control: 7.53 (6.45–8.79)
7.01 (6.26–16.72)
Control: 7.53 (6.45–8.79)
6.76 (3.77–12.11)
Control: 8.13 (7.41–8.92)
11.09 (6.14–19.20)***
Control: 7.08 (6.48–7.73)
7.46 (6.66–8.34)**
6
10
30
20
10
10
2.5
10
12.55
75.03
—
19
20
21
—
27
56.64
23.31
>413.22
Phenytoin
Lacosamide
Control: 6.05 (5.20–7.03)
>25****
Control: 6.05 (5.20–7.03)
a
Statistical analysis: unpaired t test **p < 0.01, ***p < 0.001, ****p < 0.0001; – lack of effect.
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Table 4 Anticonvulsant properties of selected compounds in PTZ (mice, ip)
Dose
Latency of tonic
Effect
(%)
Compound
(mg kg−1
)
seizures SEM (s)a
Control
—
—
—
1
100
921 361.9
57.71
Control: 584 145.8
576.4 73.69
Control: 562.8 83.17
275.4 45.14
Control: 584 145.8
564.8 197
Control: 584 145.8
596 224
Control: 584 145.8
1269 220.1*
Control: 584 145.8
575.8 66.22
Control: 562.8 83.17
219.6 12.77
30
2.42
—
6
100
100
80
19
20
21
—
2.05
117.23
2.31
—
100
30
27
100
100
50
Control: 584 145.8
1470 166.2***
Control: 614.9 61.92
1377 181.5**
Control: 614.9 61.92
Ethosuximide
139.06
123.94
a
Statistical analysis: univariate ANOVA and Dunnett's post hoc test; *p < 0.05, **p < 0.01, ***p < 0.001; − lack of effect.
Table 5 Analgesic activity of selected compounds in formalin test (mice, ip)
Compd.
Dose (mg kg−1
)
Time of paw licking SEM (s) (phase I)a
Time of paw licking SEM (s) (phase II)a
Control
1
6
19
20
21
27
—
32
21
69
33
27
24
43.89 6.64
79.89 11.45
35.75 5.86 (18.55% of effect)
32 9.32 (27.01% of effect)
37.86 7.08 (13.74% of effect)
32.71 8.32 (25.47% of effect)
67.25 13.04 (lack of effect)
40.13 4.29 (8.57% of effect)
43 10.99 (46.16% of effect)*
76 126.8 (4.87% of effect)
28.29 14.05 (64.59% of effect)*
23.86 8.33 (70.13% of effect)**
13.25 2.7 (83.41% of effect)***
72.63 15.17 (8.80% of effect)
a
Statistical analysis: unpaired t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
the MES test was used to establish its analgesic activity. In
the first (neurogenic) phase of the test, five compounds (1, 6,
19, 20, and 27) only slightly reduced the duration of the lick-
ing response by 9–27%. In the second (late) phase of the for-
malin test, four compounds (1, 19, 20, and 21) revealed a
prominent antinociceptive activity. They significantly reduced
the nocifensive response by 46–83%. The precise results are
summarized in Table 5.
Among the compounds active in MES, compounds 19–21
(ethoxyethyl derivatives) were the most active. All three are di-
methylphenol derivatives (2,3 in the cases of 19 and 20 and
2,4 in the case of 21). They are also derivatives of piperidin-3-
ol, D,L-trans-2-aminocyclohexan-1-ol, or R,S-2-aminopropan-1-
ol, respectively. Compound
1 is a derivative of 2,4-
dimethylphenol and D,L-trans-2-aminocyclohexan-1-ol with the
shortest linker tested – ethylene.
Table 6 The influence on spontaneous activity of selected compounds and neurotoxicity in the chimney test (mice, ip)
Spontaneous activity
Chimney test
Activity
SEMa
Effect
(%)
Time of escaping from
the chimney SEM (s)
Animals exhibiting loss
of coordination (%)
Compd.
Dose (mg kg−1
)
Control
1
6
19
20
21
27
—
32
21
69
33
27
24
1402 201.2
1287 295.5
1125 238.8
1192 213.0
1354 299.5
519.5 137.5**
779.7 175.4*
—
8.87 1.79
4.75 0.67
6.5 1.68
4.75 1.76
4.13 0.64
6.0 2.35
3.5 0.65
—
0
0
0
0
8.20
19.76
14.98
3.42
68.92
44.39
0
0
a
Statistical analysis: unpaired t test; *p < 0.05, **p < 0.01.
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Among the tested compounds, 21 and 27 at doses equal to
the respective ED50 values obtained in the MES test signifi-
cantly reduced the number of crossings registered with
photoresistor actometers by 69 (p < 0.01), and 44% (p <
0.05), respectively. The results indicated that these com-
pounds possess sedative properties. The other tested com-
pounds 1, 6, 19, and 20 did not significantly influence loco-
motor activity in mice. None of the tested compounds
affected motor coordination in the chimney test (Table 6).
Taking into consideration the structural aspects of the
studied compounds, substitution in the phenyl ring with
methyl moieties in positions 2,4, 2,6, and 2,4,6, as well as
2-chloro-6-methyl substitution, are confirmed to be beneficial
for anticonvulsant activity. Analgesic activity was the most
promising in the case of 2,3- or (2,4-dimethylphenoxy)-
ethoxyethyl derivatives (compounds 19–21), and this suggests
that the ethoxyethyl linker is beneficial with respect to the
anticonvulsant and analgesic activity. In case of 2,4-dimethyl
and 2-chloro-6-methyl derivatives, sedative properties have
been observed (i.e. (R,S)-2-{2-[2-(2,4-dimethylphenoxy)ethoxy]-
ethyl}aminopropan-1-ol (21) and (R,S)-2-{2-[2-(2-chloro-6-
Fig. 2 Viability of HepG2 cells incubated in the presence of selected
compounds in the concentration range 10–250 μM. The graph shows
results from MTT assay expressed as a percentage of the control
conditions SEM. Three independent experiments were performed.
rubicin was dosed at the same concentration as the evaluated
compounds (1, 6, 19, and 21) and exhibited a strong cytotoxic
effect on HepG2 cells.
The potential cytotoxic effect of compound 19 was exam-
ined by LDH release and MTT reduction assays. Exposure of
SH-SY5Y cells to compound 19 at concentrations from 10−8 to
10−4 M for 72 hours did not change the LDH-release (Fig. 6).
methylphenoxy)ethoxy]ethyl}amino-1-phenylethan-1-ol
respectively).
(27),
The test compound at concentrations from 10−8 to 10−4
M
The most beneficial in vivo pharmacological profile was
observed for four compounds (1, 6, 19, and 21). It is worth
mentioning
that
compound
1
is
a
D,L-trans-2-
aminocyclohexan-1-ol derivative similar to reference com-
pound II, which may contribute to the activity. Compound 21
is a derivative of R,S-2-aminopropan-1-ol, similar to III and IV,
which are R enantiomer derivatives. Compounds 6 and 19 are
derivatives of piperidin-3- or −4-ol, respectively. These amino-
alkanols were also present in the structures of other active
compounds discovered within our former research, including
reference compound VII.14
Analysis of cytotoxic activity of compounds 1, 6, 19, and 21
using in vitro cellular models
Fig. 3 Viability of HepG2 cells incubated in the presence of
doxorubicin (DOX) in the concentration range 10–200 μM. The graph
shows results from MTT assay expressed as a percentage of the
Data on the hepatotoxicity of some antiepileptic drugs (e.g.
valproic acid,20,21 pregabalin22,23) became the premise for the
determination of the potential hepatotoxicity of selected com-
pounds possessing anticonvulsant activity. Compounds 1, 6,
19, and 21 were evaluated for potential cytotoxicity against
human cancer cells (HepG2). Results obtained from two inde-
pendent viability tests investigating different mechanisms of
cell-mediated cytotoxicity clearly show that none of the ana-
lyzed compounds show a cytotoxic effect on hepatoma cells
(Fig. 2 and 4). Performed analyses proved that the tested
compounds do not affect cellular metabolism (MTT) and do
not have an influence on endosomal activity – an important
physiological process occurring in cells (neutral red uptake).
What is more, they are completely safe with respect to nor-
mal cells [astrocytes (glial cells)] derived from the nervous
system (Fig. 5). In addition, the cytotoxicity effect of a chemo-
therapeutic agent, doxorubicin (anthracycline antibiotic), on
HepG2 cells was evaluated as a positive control (Fig. 3). Doxo-
control conditions
performed.
SEM. Three independent experiments were
Fig. 4 Viability of HepG2 cells incubated in the presence of selected
compounds in the concentration range 10–250 μM. The graph shows
results from neutral red assay expressed as a percentage of the control
conditions SEM. Three independent experiments were performed.
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Fig. 5 Viability of CRL-2541 cells (astrocytes) incubated in the pres-
ence of selected compounds in the concentration range 10–250 μM.
The graph shows results from MTT assay expressed as a percentage of
Fig. 7 The effect of compound 19 on basal MTT reduction in SH-SY5Y
cells. Results are shown as a percentage of control cells incubated
with vehicle alone. The significance of differences between the means
was evaluated by Dunnett's test following a one-way analysis of vari-
ance; ***p < 0.001 vs. culture with vehicle. C-culture without vehicle,
V-culture with appropriate vehicle.
the control conditions
performed.
SEM. Three independent experiments were
also had no significant effect on MTT reduction, although an
increasing trend was observed (Fig. 7). Addition of hydrogen
peroxide alone significantly damaged cells by increasing
above twofold the release of LDH and attenuated MTT reduc-
tion by approx. 20% (Fig. 8 and 9). In the highest examined
concentration, 10−5 M, compound 19 statistically significantly
potentiated the damaging effects of hydrogen peroxide on
LDH release, while at lower concentrations it had no effect
on this parameter. At concentrations of 10−6 and 10−5 M, the
tested compound significantly potentiated the H2O2-induced
decrease in MTT reduction. Triton, used in all conducted as-
says as a positive control, strongly increased LDH release and
decreased MTT reduction.
The potential cytotoxic activity of compound 19 was also
tested in SH-SY5Y cell culture. This cell line is of human ori-
gin, possesses a dopaminergic phenotype, and is one of the
most broadly used models for screening of the putative neu-
rotoxic and neuroprotective activity of a wide range of
chemicals.24,25 Similarly, addition of hydrogen peroxide to
cell medium, as the source of detrimental reactive oxygen
species, is a well-validated and frequently used model for oxi-
dative stress. Moreover, both in the pathogenesis of neurode-
generative diseases and in the mechanism of action of most
neurotoxic substances, oxidative stress plays a major role.
The present study showed that the test compound at concen-
trations up to 10−4 M did not decrease the SH-SY5Y cell via-
bility, assessed by MTT reduction assay, and did not produce
a cytotoxic effect, defined by LDH release. Considering the
possible use of this compound in the future in clinics, its
lack of adverse action on the integrity of membranes and via-
bility of cells with neuronal phenotypes is important.
Neuroprotection is a favorable effect of antiepileptic drugs,
because seizures can lead to nerve cell damage. However,
compound 19, in the model used in the present study, did
not reduce hydrogen-peroxide-induced cell damage. This
Fig. 6 The effect of compound 19 on basal lactate dehydrogenase
(LDH) release from SH-SY5Y cells. Results are shown as a percentage
of control cells incubated with vehicle alone. The significance of differ-
ences between the means was evaluated by Dunnett's test following a
one-way analysis of variance; ***p < 0.001 vs. culture with vehicle.
C-culture without vehicle, V-culture with appropriate vehicle.
Fig. 8 The effect of compound 19 on H2O2-induced lactate
dehydrogenase (LDH) release from SH-SY5Y cells. The significance of
differences between the means was evaluated by Dunnett's test fol-
lowing a one-way analysis of variance; **p < 0.01; ***p < 0.001 vs.
culture with vehicle only; #p < 0.05; ###p < 0.001 vs. cells treated
with H2O2.
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drug evaluation process. By applying several methods, which
are based on different mechanisms of action, we can accu-
rately determine how the tested substance affects individual
cells and thus predict its potential side effects.
Analyses performed using two different cytotoxicity assays
show that the tested compounds (1, 6, 19, and 21) are safe.
They do not induce hepatotoxicity (as do many anti-epileptic
drugs) and are also safe for normal cells derived from the
nervous system (astrocytes). Compound 19 does not affect
the metabolic activity of cells, which was proven using MTT
assay (Fig. 7 and 9). In addition, neutral red assay showed
that the tested compound did not interfere with the endo-
somal activity of cells.
Fig. 9 The effect of compound 19 on H2O2-induced MTT reduction in
SH-SY5Y cells. The significance of differences between the means was
evaluated by Dunnett's test following a one-way analysis of variance;
*p < 0.05; **p < 0.01; p < 0.001 vs. culture with vehicle only; #p <
0.05; ###p < 0.001 vs. cells treated with H2O2.
In silico prediction of metabolism
Metabolism of compound 19 was predicted using the Meta-
Site program. The premise for the study is the possibility of
further research regarding these compounds, facilitated by in
silico methods. The MetaSite program was chosen for simula-
tion due to its high probability of finding correct metabolites
in further research: in about 85% of cases, the method pre-
dicts the correct site of metabolism within the first two
choices in the ranking list.26,27
should be noted. Nevertheless, the potential neurotoxic/
neuroprotective action of compound 19 was determined only
in one experimental in vitro model and reflected the short-
term (72 hour) exposure of SH-SY5Y cells to a test substance.
Therefore, further more detailed studies in that regard will
be useful.
The most probable results for compound 19 are presented
in Fig. 10, and they show that the two main sites of metabo-
lism are the methyl group in position 3 of the phenyl ring
In vitro cellular models are commonly used to determine
the cytotoxicity of tested compounds at an early stage of the
Fig. 10 The main results of in silico metabolism prediction for compound 19.
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(reactions 1–3), as well as the methylene in position 6 of
piperidinol (reactions 4–5). Reactions 1–3 on the methyl
group constitute aliphatic hydroxylation as well as carbonyla-
tion (oxidation), resulting in the formation of alcohol,
aldehyde, or carboxylic acid, respectively. Reactions 4 and 5
constitute N-dealkylation (oxidation), resulting in opening of
the piperidinol ring and formation of aldehyde or carboxylic
acid, respectively. All metabolites are less lipophilic (all exert
lower log P) than the parent compound. Such results were
expected, since they are typical for liver metabolism. The
obtained structures will be useful for future research regard-
ing 19 and the group of N-[(phenoxyethoxy)ethyl]aminoalkanol
derivatives.
achievement of 1–18) or phenoxyethoxyethyl halide (for
achievement of 19–28), which was synthesized by three
methods.
The first method resulted in obtaining phenoxyalkyl chlo-
rides by heating the appropriately substituted phenol with
1-bromo-3-chloropropane or 1-bromo-4-chlorobutane in ace-
tone in the presence of K2CO3 and TEBA.
Another method constituted achieving substituted
phenoxyalkyl and phenoxyethoxyethyl bromides in two steps.
At first, the appropriate phenol was heated with chloro-
alkanol (2-chloroethan-1-ol, 3-chloropropan-1-ol, 4-chloro-
butan-1-ol), or 2-(2-chloroethoxy)ethan-1-ol in a mixture of ace-
tone and ethanol in the presence of K2CO3. The crude
products were subsequently subject to bromination with the
use of PBr3.
Substituted phenoxybutyl bromides were alternatively
achieved by heating the appropriate phenol with 1,4-
dibromobutan19 in the presence of NaOH to limit the number
of synthesis steps. However, this method was the least
efficient.
In order to obtain the final products 1–28, aminolysis of
the achieved halides (0.03 mole) with the appropriate amino-
alkanols (0.025 mole) was performed. The reaction was car-
ried out in toluene, in the presence of K2CO3 as a proton ac-
ceptor or in DMF in the presence of TEA. Both methods
produce comparable yields. Compounds 4, 17, and 19 were
subsequently converted into hydrochlorides, by means of sat-
uration with gaseous HCl in acetone solution, and purified
by recrystallization from a mixture of acetone and MeOH (3 :
1, v/v). Other obtained amines were purified either by recrys-
tallization from n-hexane or by column chromatography
(compounds 20–28, silica gel, eluting with CH2Cl2/MeOH 70 :
30, v/v).
Conclusion
The presented research shows that there exist premises for
the search of an anticonvulsant as well as analgesic com-
pounds among the derivatives of N-[(phenoxy)alkyl]- or
N-[(phenoxy)ethoxyethyl]aminoalkanols. The results are con-
sistent with our former research, indicating the possibility of
finding active compounds among derivatives of 2,6-dimethyl,
and also 2,3-dimethyl and 2,4-dimethyl, as well as 2,4,6-
trimethylphenol, among aminoalkanols
– derivatives of
2-aminopropan-1-ol, and D,L-trans-2-aminocyclohexan-1-ol, as
well as piperidin-3- or -4-ol. Both moieties are linked in the
structures of active compounds by ethylene, propylene, tetra-
methylene, or ethoxyethyl. The linker varies among active
compounds, which suggests a premise for further investiga-
tion of its effect on the activity.
The most promising compound in this research was 1-{2-
[2-(2,3-dimethylphenoxy)ethoxy]ethyl}piperidin-3-ol hydrochlo-
ride (19), and this compound was subject to additional re-
search on astrocytes as well as the in silico prediction of me-
tabolism, which confirms that this compound may be of
interest in terms of further research. The presented results
are intended to facilitate further research regarding this com-
pound and the group of N-[(phenoxyethoxy)ethyl]aminoalkanol
derivatives.
Melting points (mp) were determined by means of a Büchi
SMP-20 apparatus (Büchi Labortechnik, Flawil, Switzerland)
and were uncorrected. Analytical TLC was carried out on
precoated plates (silica gel, 60F-254 Merck), and spots were
visualized with UV light.
The 1H and 13C NMR spectra for compounds 19 and 27
were obtained with a Bruker AVANCE III 600 spectrometer
(Bruker, Karlsruhe, Germany) (600.2 MHz for 1H and
150.94 MHz for 13C) at the Faculty of Chemistry,
Jagiellonian University in Krakow. The 1H NMR spectra for
other compounds and 13C NMR spectra for compounds 9,
11, 14, 18 were recorded at the Faculty of Pharmacy,
Jagiellonian University Medical College (Krakow, Poland)
with a Varian Mercury-VX 300 NMR spectrometer at 29 °C.
Chemical shifts were referenced against solvent lock signal.
Standard Varian pulse sequences were used for 2D experi-
ments. The 1H and 13C NMR spectra for compounds 4, 17,
and 19 were recorded in DMSO-d6 and for other com-
pounds in CDCl3. The results are presented in the follow-
ing format: chemical shift δ (ppm), multiplicity, number of
protons, J values in Hertz (Hz), protons' position. Multiplic-
ity abbreviations: s (singlet), br s (broad singlet), d (dou-
blet), dd (doublet of doublets), dt (doublet of triplets), dtd
Experimental section
Chemistry
Most of the reagents and solvents were commercially avail-
able materials of reagent grade and were purchased from Alfa
Aesar (purchased from Chemat, Gdansk, Poland), Sigma-Aldrich
(purchased from Sigma-Aldrich, Poznan, Poland) or Merck Sp.
z o.o. (Warsaw, Poland). D,L-trans-2-aminocyclohexan-1-ol (1, 4,
17, 20) was synthesized according to a previously published
procedure from cyclohexene oxide.28
Synthesis of the title compounds
The synthesis was performed according to previously pub-
lished procedures.7,18,19 The aim of the first step of the syn-
thesis was to obtain the appropriate phenoxyalkyl (for
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(doublet of triplets of doublets), t (triplet), tt (triplet of trip-
lets), m (multiplet).
The IR spectra of compounds 1, 6, and 21 were recorded
on a Jasco FT/IR 410 spectrometer (KBr pellets).
>CH–NH–), 2.20 (s, 6H, Ar–(CH3)2), 1.59–2.09 (m, 8H, pip),
1.19 (d, 4H, J = 8.2 Hz, –CH2–CH2–CH2–CH2–NH–); LCMS
[M + H]+ m/z calcd for C18H29O2N 292.22, found 292.33,
99.39%.
The purity of the obtained compounds was confirmed by
LCMS. The LCMS system consisted of a Waters Acquity
UPLC, coupled to a Waters TQD mass spectrometer (electro-
spray ionization mode ESI-tandem quadrupole). All the analy-
ses were carried out using an Acquity UPLC BEH C18, 1.7 lm,
2.1 × 100 mm column. A flow rate of 0.3 mL min−1 and a gra-
dient of 5–95% B over 10 min and then 100% B over 2 min
was used. Eluent A: water/0.1% HCO2H; eluent B: acetoni-
trile/0.1% HCO2H. LCMS data were obtained by scanning the
first quadrupole in 0.5 s in a mass range from 50 to 1000 Da;
eight scans were summed to produce the final spectrum.
Measurement of optical rotation ([α]25809.0) for compounds 11,
13, 14, 16, 22, and 28 was carried out using Jasco 2000.
D,L-trans-2-[2-(2,4-Dimethylphenoxy)ethyl]aminocyclohexan-
1-ol (1). Yield 60%; white solid; M. P. 106–108 °C; Rf = 0.64
D,L-trans-4-[4-(2,6-Dimethylphenoxy)butyl]aminocyclohexan-
1-ol (5). Yield 70%; white solid; M. P. 106–107 °C; Rf = 0.33
(MeOH/ethyl acetate 1 : 1); NMR H (300 MHz, CDCl3): δ 6.97–
7.02 (m, 2H, Ar-H3, Ar-H5), 6.87–6.93 (m, 1H, Ar-H4), 3.76 (t,
2H, J = 6.4 Hz, Ar–O–CH2–), 3.56–3.67 (m, 1H, >CH–OH),
2.66–2.74 (m, 2H, –CH2–NH–), 2.40–2.51 (m, 1H, >CH–NH–),
2.26 (s, 6H, Ar–CH3), 1.89–2.03 (m, 4H, –CH2–CH2–CH2–NH–),
1.77–1.89 (m, 2H, pip), 1.63–1.75 (m, 2H, pip), 1.08–1.38 (m,
4H, pip); LCMS [M + H]+ m/z calcd for C18H29O2N 292.22,
found 292.33, 98.88%.
1
1-[3-(2,4,6-Trimethylphenoxy)propyl]piperidin-4-ol
(6).
Yield 60%; white solid; M. P. 66–68 °C; Rf = 0.46 (MeOH/ethyl
1
acetate 1 : 1); NMR H (300 MHz, CDCl3): δ 6.81 (s, 2H, Ar–H),
3.77 (t, 2H, J = 6.2, Ar–O–CH2–), 3.62–3.74 (m, 1H, CH–OH),
2.76–2.88 (m, 2H, pip), 2.58 (t, 2H, J = 7.2, 2H, –CH2–N<),
2.23 (s, 9H, Ar–(CH3)3), 2.12–2.22 (m, 2H, pip), 1.87–2.00 (m,
4H, pip, –CH2–CH2–CH2–), 1.58–1.68 (m, 2H, pip); IR (KBr,
νmax cm−1): 3393, 3230, 2937, 2859, 2786, 2740, 2299, 1682,
1486, 1381, 1218, 1149, 1057, 979, 852, 784, 720, 648, 586,
431, 418; LCMS [M + H]+ m/z calcd for C17H27O2N 278.21,
found 278.24, 98.18%.
D,L-trans-2-[2-(2-Chloro-5-methylphenoxy)ethyl]amino-1-
phenylethan-1-ol (7). Yield 65%; white solid; M. P. 125–127
°C; Rf = 0.88 (MeOH/ethyl acetate 1 : 1); NMR 1H (300 MHz,
CDCl3): δ 7.27–7.42 (m, 5H, CH–Ar), 7.23 (d, 1H, J = 8.0 Hz,
H-4), 6.66–6.81 (m, 2H, H-3, H-6), 4.75 (dd, 1H, J = 9.0 Hz, J =
3.6 Hz, CH–Ar), 4.04–4.22 (m, 2H, –O–CH2–CH2), 3.08–3.13
(m, 2H, –O–CH2–CH2), 3.04 (dd, 1H, J = 12.3 Hz, J = 3.6 Hz,
–NH–CHH–CH–), 2.80 (dd, 1H, J = 12.1 Hz, J = 9.0 Hz, –NH–
CHH–CH–), 2.33 (s, 3H, Ar–CH3), 1.41–2.00 (m, 1H, NH);
LCMS [M + H]+ m/z calcd for C17H20O2NCl 306.12, found
306.10, 99.39%.
1-[2-(2-Chloro-5-methylphenoxy)ethyl]piperidin-4-ol (8). Yield
60%; white solid; M. P. 192–194 °C; Rf = 0.68 (MeOH/ethyl
acetate 1 : 1); NMR 1H (300 MHz, CDCl3): δ 7.20 (d, 1H, J =
7.95 Hz, J = 1.4 Hz, Ar-H3), 6.66–6.75 (m, 2H, Ar-H4, Ar-H6),
4.13 (t, 2H, J = 5.90 Hz, Ar–O–CH2), 3.70 (tt, 1H, J = 8.88 Hz, J
= 4.33 Hz, >CH–OH), 2.88–2.96 (m, 2H, pip-H2,6), 2.85 (t,
2H, J = 5.90 Hz, –CH2–N<), 2.32–2.39 (m, 2H, pip-H2,6), 2.30
(s, 3H, Ar–CH3), 1.86–1.97 (m, 2H, pip-H3,5), 1.53–1.67 (m,
2H, pip-H3,5); LCMS [M + H]+ m/z calcd for C14H20O2NCl
270.12, found 270.20, 98.89%.
D,L-trans-4-[2-(2-Chloro-5-methylphenoxy)ethyl]aminocyclohexan-
1-ol (9). Yield 70%; white solid; M. P. 114–116 °C; Rf = 0.57
(CH2Cl2/MeOH 7:3); NMR 1H (300 MHz, CDCl3): δ 7.21 (d,
1H, J = 8.0 Hz, Ar–H), 6.68–6.75 (m, 2H, Ar–H), 4.11 (t, 2H,
J = 5.3 Hz, 2H, –O–CH2–CH2–), 3.54–3.70 (m, 1H, CH–OH),
3.04 (t, 2H, J = 5.3 Hz, –CH2–NH–), 2.53 (tt, 1H, J = 10.5 Hz, J =
3.5 Hz, –NH–CH), 2.31 (s, 3H, Ar–CH3), 1.99 (dd, 4H, J =
8.7 Hz, J = 2.1 Hz, (CH2)2 cyclohex), 1.59 (br s, 1H, NH),
1.11–1.40 (m, 4H, (CH2)2 cyclohex); 13C (75 MHz, CDCl3): 153.91
(Ar-C1), 137.90 (Ar-C5), 129.72 (Ar-C3), 122.31 (Ar-C4), 119.93
1
(MeOH/ethyl acetate 1 : 1); NMR H (300 MHz, CDCl3): δ 6.91–
6.96 (m, 2H, Ar-H3, Ar-H5), 6.72 (d, 1H, J = 7.9 Hz, Ar-H6),
4.03 (t, 2H, Ar–O–CH2–), 3.14–3.23 (m, 2H, CHH–NH–, OH),
2.83–2.89 (m, 1H, –CHH–NH–), 2.24–2.26 (m, 1H, >CH–NH–),
2.25 (s, 3H, Ar–CH3), 2.19 (s, 3H, Ar–CH3), 0.97–2.15 (m, 8H,
cyclohex); IR (KBr, νmax cm−1): 3318, 3270, 3064, 2928, 2856,
1629, 1451, 1351, 1110, 1068, 967, 894, 884, 857, 661; LCMS
[M + H]+ m/z calcd for C16H25O2N 264.19, found 264.28,
99.27%.
(R,S)-1-[4-(2,6-Dimethylphenoxy)butyl]aminopropan-2-ol
(2). Yield 62%; white solid; M. P. 70–71 °C; Rf = 0.47 (MeOH/
1
ethyl acetate 1 : 1); NMR H (300 MHz, CDCl3): δ 6.99 (d, 1H, J
= 7.2 Hz, Ar-H4), 6.58–6.70 (m, 2H, Ar-H3, Ar-H5), 4.12 (dt,
1H, J = 6.3 Hz, J = 3.3 Hz, –CH–CH3), 3.96 (t, 2H, J = 5.8 Hz,
Ar–O–CH2–), 2.86–3.00 (m, 2H, –CH2–CH2–NH–), 2.68 (dd,
2H, J = 12.1 Hz, J = 9.9 Hz, –CH2–CH–), 2.30 (s, 6H, Ar–CH3),
1.79–2.00 (m, 4H, –CH2–CH2–CH2–NH–), 1.42 (d, 1H, J = 6.3
Hz, –OH), 1.16–1.28 (m, 3H, –CH–CH3); LCMS [M + H]+ m/z
calcd for C15H25O2N 252.19, found 252.21, 100.00%.
(R,S)-2-[4-(2,6-Dimethylphenoxy)butyl]aminobutan-1-ol (3).
Yield 70%; white solid; M. P. 49–51 °C; Rf = 0.42 (MeOH/ethyl
1
acetate 1 : 1); NMR H (300 MHz, CDCl3): δ 6.98–7.02 (m, 2H,
Ar-H3, Ar-H5), 6.87–6.94 (m, 1H, Ar-H4), 3.77 (t, 2H, J = 6.4
Hz, Ar–O–CH2–), 3.62 (dd, 1H, J = 10.5 Hz, J = 4.1 Hz, –CHH–
OH), 3.28 (dd, 1H, J = 10.5 Hz, J = 6.4 Hz, –CHH–OH), 2.73–
2.83 (m, 1H, –NH–CH<), 2.51–2.69 (m, 2H, –CH2–NH–), 2.27
(s, 6H, Ar–(CH3)2), 1.81–1.92 (m, 2H, Ar–O–CH2–CH2–), 1.66–
1.77 (m, 2H, –CH2–CH2–NH–), 1.35–1.57 (m, 2H, –CH2–CH3),
0.92 (t, 3H, J = 7.3 Hz, 3H, –CH2–CH3); LCMS [M + H]+ m/z
calcd for C16H27O2N 266.21, found 266.28, 100.00%.
D,L-trans-2-[4-(2,6-Dimethylphenoxy)butyl]aminocyclohexan-
1-ol hydrochloride (4). Yield 60%; white solid; M. P. 181–183
°C; Rf = 0.32 (MeOH/ethyl acetate 1 : 1); NMR 1H (300 MHz,
+
DMSO-d6): δ 8.39–8.81 (m, 2H, NH2 ), 6.98–7.02 (m, 2H, Ar-
H3, Ar-H5), 6.85–6.92 (m, 1H, Ar-H4), 5.50 (d, 1H, J = 5.3 Hz,
OH), 3.72 (t, 2H, J = 5.9 Hz, Ar–O–CH2–), 3.48 (br s, 1H,
>CH–OH), 3.01 (br s, 2H, –CH2–NH–), 2.70–2.82 (m, 1H,
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(Ar-C2), 114.76 (Ar-C6), 70.52 (>CH–OH), 68.90 (Ar–O–CH2–),
55.62 (–NH–CH<), 46.08 (–CH2–NH–), 33.96 ((CH2)2–OH),
31.22 ((CH2)2–NH), 21.33 (Ar–CH3); LCMS [M + H]+ m/z
calcd for C15H22O2NCl 284.14, found 284.17, 98.22%.
(S)-2-[4-(2-Chloro-5-methylphenoxy)butyl]aminobutan-1-ol
(14). Yield 60%; white solid; M. P. 57–58 °C; Rf = 0.48
(MeOH/ethyl acetate 1 : 1); NMR H (300 MHz, CDCl3): δ 7.21
1
(d, 1H, J = 8.0 Hz, Ar-3H), 6.72 (s, 1H, Ar-6H), 6.67–6.71 (m,
1H, Ar-4H), 4.02 (t, 2H, J = 6.2 Hz, –O–CH2–), 3.62 (dd, 1H, J
= 10.5 Hz, J = 4.1 Hz, CH–CHH–OH), 3.27 (dd, 1H, J = 10.5
Hz, J = 6.4 Hz, CH–CHH–OH), 2.72–2.83 (m, 1H, –NH–CH),
2.50–2.68 (m, 2H, –CH2–NH), 2.31 (s, 3H, Ar–CH3), 1.84–1.94
(m, 2H, –O–CH2–CH2–), 1.69 (dt, 2H, J = 14.9 Hz, J = 7.2 Hz,
–CH2–CH2–NH), 1.34–1.57 (m, 2H, –CH2–CH3), 0.91 (t, 3H, J =
7.4 Hz, –CH2–CH3); 13C (75 MHz, CDCl3): δ 156.17 (Ar-C1),
137.79 (Ar–C5), 129.74 (Ar-C3), 121.90 (Ar-C4), 119.78 (Ar-C2),
114.34 (Ar-C6), 68.81 (Ar–O–CH2–), 62.54 (–NH–CH<), 60.19
(>CH–CH2–), 46.40 (–CH2–NH–), 27.23 (–CH2–CH2–NH–),
26.95 (Ar–O–CH2–CH2–), 24.47 (–CH2–CH3), 21.32 (Ar–CH3),
(R,S)-2-[4-(2-Chloro-5-methylphenoxy)butyl]aminopropan-
1-ol (10). Yield 65%; white solid; M. P. 52–54 °C; Rf = 0.47
1
(MeOH/ethyl acetate 1 : 1); NMR H (300 MHz, CDCl3): δ 7.22
(d, 1H, J = 8.0 Hz, Ar-H4), 6.59–6.80 (m, 2H, Ar-H3, Ar-H6),
4.04 (t, 2H, J = 6.3 Hz, –O–CH2–), 3.59 (dd, 1H, J = 10.5 Hz,
J = 4.1 Hz, –CH2–OH), 3.24 (dd, 1H, J = 10.5 Hz, J = 6.9 Hz,
–CH2–OH), 2.73–2.93 (m, 2H, –CH2–NH–CH–), 2.62 (dt, 1H,
J = 11.3 Hz, J = 7.1 Hz, –CH2–NH–CH–), 2.32 (s, 3H, Ar–CH3),
1.82–2.02 (m, 2H, –O–CH2–CH2–), 1.70 (quin, 2H, J = 7.4 Hz,
–CH2–CH2–NH–), 1.58 (br s, 1H, NH), 1.07 (d, 3H, J = 6.4 Hz,
–CH–CH3); LCMS [M + H]+ m/z calcd for C14H22O2N 272.14,
found 272.26, 99.60%.
10.44 (–CH2–CH3); LCMS [M + H]+ m/z calcd for C15H24O2NCl
20.0
589
(R)-2-[4-(2-Chloro-5-methylphenoxy)butyl]aminopropan-1-
ol (11). Yield 55%; white solid; M. P. 56–57 °C; Rf = 0.44
286.15, found 286.10, 99.63%; [α]
= +12.49°.
(R,S)-2-[4-(2-Chloro-6-methylphenoxy)butyl]aminopropan-
1-ol (15). Yield 70%; white solid; M. P. 67–69 °C; Rf = 0.47
(MeOH/ethyl acetate 1 : 1); NMR 1H: δ 7.20 (dd, 1H, J = 7.9
Hz, J = 1.5 Hz, Ar-H3), 7.04–7.09 (m, 1H, Ar-H5), 6.89–6.96
(m, 1H, Ar-H4), 3.92 (t, 2H, J = 6.4 Hz, Ar–O–CH2–), 3.58 (dd,
1H, J = 10.5 Hz, J = 4.1 Hz, –CHH–OH), 3.23 (dd, 1H, J = 10.5
Hz, J = 7.0 Hz, –CHH–OH), 2.74–2.87 (m, 2H, –CH2–NH–),
2.62 (dt, 1H, J = 11.3 Hz, J = 7.3 Hz, –NH–CH<), 2.30 (s, 3H,
Ar–CH3), 1.83–1.94 (m, 2H, Ar–O–CH2–CH2–), 1.67–1.78 (m,
2H, –CH2–CH2–NH–), 1.06 (d, 3H, J = 6.4 Hz, >CH–CH3);
LCMS [M + H]+ m/z calcd for C14H22O2NCl 272.14, found
272.21, 100.00%.
1
(MeOH/ethyl acetate 1 : 1); NMR H (300 MHz, CDCl3): δ 7.21
(d, 1H, J = 8.0 Hz, Ar-4H), 6.72 (s, 1H, Ar-6H), 6.66–6.71 (m,
1H, Ar-3H), 4.02 (t, 2H, J = 6.3 Hz, –O–CH2–), 3.58 (dd, 1H,
J = 10.4 Hz, J = 4.2 Hz, –CH–CHH–OH), 3.23 (dd, 1H, J = 10.4
Hz, J = 7.1 Hz, –CH–CHH–OH), 2.74–2.86 (m, 2H, –CH2–
NH–), 2.61 (dt, 1H, J = 11.5, J = 7.2, –CH<), 2.31 (s, 3H,
Ar–CH3), 1.84–1.94 (m, 2H, –O–CH2–CH2–), 1.64–1.75 (m, 2H,
–CH2–CH2–NH–), 1.06 (d, 3H, J = 6.4 Hz, –CH–CH3); 13C
(75 MHz, CDCl3): δ 154.10 (Ar-C1), 137.79 (Ar-C5), 129.73
(Ar-C3), 121.87 (Ar-C4), 119.75 (Ar-C2), 114.30 (Ar-C6), 68.79
(>CH–), 65.47 (Ar–O–CH2–), 54.26 (>CH–CH2–OH), 46.56
(–CH2–NH–), 27.16 (–CH2–CH2–NH–), 26.96 (Ar–O–CH2–
(S)-2-[4-(2-Chloro-6-methylphenoxy)butyl]aminopropan-1-ol
(16). Yield 70%; white solid; M. P. 71–73 °C; Rf = 0.55
CH2–), 21.35 (Ar–CH3), 17.38 (>CH–CH3); LCMS [M + H]+ m/z
20.0
1
calcd for C14H22O2NCl 272.14, found 272.08, 99.04%; [α]
−16.73°.
=
(MeOH/ethyl acetate 1 : 1); NMR H (300 MHz, CDCl3): δ 7.19
589
(dd, 1H, J = 8.1 Hz, J = 1.4 Hz, Ar-H3), 7.06 (dd, 1H, J = 7.6
Hz, J = 0.6 Hz, Ar-H5), 6.93 (t, 1H, J = 7.7 Hz, Ar-H4), 3.92 (t,
2H, J = 6.2 Hz, Ar–O–CH2–), 3.61 (dd, 1H, J = 10.6 Hz, J = 4.0
Hz, –CHH–OH), 3.29 (dd, 1H, J = 10.6 Hz, J = 7.1 Hz, –CHH–
OH), 2.77–2.92 (m, 2H, –CH2–NH–), 2.66 (dt, 1H, J = 11.3 Hz,
J = 7.1 Hz, –NH–CH<), 2.30 (s, 3H, Ar–CH3), 2.03 (br s, 2H,
NH, OH), 1.84–1.95 (m, 2H, –O–CH2–CH2–), 1.72–1.83 (m, 2H,
–CH2–CH2–NH–), 1.09 (d, 3H, J = 6.4 Hz, >CH–CH3); LCMS
(R,S)-2-[4-(2-Chloro-5-methylphenoxy)butyl]aminobutan-1-
ol (12). Yield 60%; white solid; M. P. 60–62 °C, Rf = 0.53
(MeOH/ethyl acetate 1 : 1); NMR H (300 MHz, CDCl3): δ 7.21
1
(d, 1H, J = 8.2 Hz, Ar-H3), 6.66–6.74 (m, 2H, Ar-H4, Ar-H6),
4.02 (t, 2H, J = 6.2 Hz, Ar–O–CH2–), 3.62 (dd, 1H, J = 10.5 Hz,
J = 4.1 Hz, –CHH–OH), 3.27 (dd, 1H, J = 10.5 Hz, J = 6.4 Hz,
–CHH–OH), 2.60–2.83 (m, 2H, –CH2–NH–), 2.50–2.58 (m, 1H,
–NH–CH<), 2.32 (s, 3H, Ar–CH3), 1.83–1.95 (m, 2H, Ar–O–
CH2–CH2–), 1.63–1.75 (m, 2H, –CH2–CH2–NH–), 1.34–1.57 (m,
2H, –CH2–CH3), 0.88–0.96 (m, 3H, –CH2–CH3); LCMS [M + H]+
m/z calcd for C15H24O2NCl 286.15, found 286.17, 100.00%.
(R)-2-[4-(2-Chloro-5-methylphenoxy)butyl]aminobutan-1-ol
(13). Yield 60%; white solid; M. P. 58–60 °C, Rf = 0.53
[M + H]+ m/z calcd for C14H22O2NCl 272.14, found 272.32,
20.0
589
98.32%; [α]
= +15.61°.
D,L-trans-2-[4-(2-Chloro-6-methylphenoxy)butyl]aminocyclohexan-
1-ol hydrochloride (17). Yield 60%; white solid; M. P. 175–177
1
+
°C; NMR H (300 MHz, DMSO-d6): δ 8.45–8.93 (m, 2H, NH2 ),
7.25–7.29 (m, 1H, Ar-H3), 7.15–7.20 (m, 1H, Ar-H5), 6.99–7.05
(m, 1H, Ar-H4), 5.52 (d, 1H, J = 5.1 Hz, OH), 3.85 (t, 2H, J =
5.9 Hz, Ar–O–CH2–), 3.50 (dt, 1H, J = 9.7 Hz, J = 4.9 Hz, >CH–
OH), 3.01 (br s, 2H, –CH2–NH–), 2.68–2.83 (m, 1H, –NH–
CH<), 2.26 (s, 3H, Ar–CH3), 2.01–2.09 (m, 1H, cyclohex),
1.76–1.93 (m, 4H, cyclohex), 1.65 (d, 2H, J = 13.3 Hz, Ar–O–
CH2–CH2–), 1.24–1.46 (m, 2H, Ar–O–CH2–CH2–CH2–), 1.12–
1.23 (m, 3H, cyclohex); LCMS [M + H]+ m/z calcd for
C17H26O2NCl 312.17, found 312.28, 98.95%.
1
(MeOH/ethyl acetate 1 : 1); NMR H (300 MHz, CDCl3): δ 7.21
(d, 1H, J = 8.0, Ar-H3), 6.66–6.74 (m, 2H, Ar-H4, Ar-H6), 4.02
(t, 2H, J = 6.2 Hz, Ar–O–CH2–), 3.64 (dd, 1H, J = 10.6 Hz, J =
4.0 Hz, –CHH–OH), 3.30 (dd, 1H, J = 10.6 Hz, J = 6.5 Hz, –CH
H–OH), 2.63–2.87 (m, 2H, –CH2–NH–), 2.52–2.63 (m, 1H, –CH
<), 2.31 (s, 3H, Ar–CH3), 2.09 (br s, 2H, NH, OH), 1.84–1.96
(m, 2H, –O–CH2–CH2–), 1.66–1.77 (m, 2H, –CH2–CH2–NH–),
1.36–1.60 (m, 2H, –CH2–CH3), 0.92 (t, 3H, J = 7.4 Hz, –CH2–
CH3); LCMS [M + H]+ m/z calcd for C15H24O2NCl 286.15,
D,L-trans-4-[4-(2-Chloro-6-methylphenoxy)butyl]aminocyclohexan-
1-ol (18). Yield 70%; white solid; M. P. 95–97 °C; Rf = 0.41
20.0
589
found 286.35, 100.00%; [α]
= −11.98°.
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1
(MeOH/ethyl acetate 1 : 1); NMR H (300 MHz, CDCl3): δ 7.19
(dd, 1H, J = 8.0 Hz, J = 1.3 Hz, Ar-H3), 7.06 (dd, 1H, J = 7.6
Hz, J = 0.9 Hz, Ar-H5), 6.92 (t, 1H, J = 7.7 Hz, Ar-H4), 3.91 (t,
2H, J = 6.4 Hz, Ar–O–CH2), 3.62 (tt, 1H, J = 10.5 Hz, J = 4.0
Hz, >CH–OH), 2.73 (t, 2H, J = 1.0 Hz, –CH2–NH–), 2.45 (tt,
1H, J = 10.7 Hz, J = 3.7 Hz, –NH–CH<), 2.29 (s, 3H, Ar–CH3),
1.90–2.01 (m, 4H, cyclohex), 1.81–1.89 (m, 2H, Ar–O–CH2–
CH2–), 1.65–1.77 (m, 2H, cyclohex), 1.23–1.39 (m, 3H, –CH2–
CH2–NH–), 1.07–1.21 (m, 2H, cyclohex); 13C (75 MHz, CDCl3):
δ 153.43 (Ar–C1), 133.29 (Ar-C6), 129.50 (Ar-C5), 127.94 (Ar-
C3), 124.41 (Ar-C4), 72.57 (>CH–OH), 70.60 (Ar–O–CH2–),
56.05 (–NH–CH<), 47.22 (–CH2–NH–), 34.07 ((CH2)2CH–OH),
31.36 ((CH2)2CH–NH–), 28.12 (Ar–O–CH2–CH2–), 27.13
(–CH2–CH2–NH–), 16.47 (Ar–CH3); LCMS [M + H]+ m/z calcd
for C17H26O2NCl 312.17, found 312.09, 100.00%.
OH), 2.88–2.97 (m, 1H, >CH–NH–), 2.67–2.81 (m, 2H, –CH2–
NH–), 2.25 (s, 3H, Ar–CH3(4)), 2.20 (s, 3H, Ar–CH3(2)), 1.05 (d,
3H, J = 6.4 Hz, >CH–CH3); IR (KBr, νmax cm−1): 3434, 3300,
3126, 2961, 2924, 2879, 1612, 1506, 1459, 1379, 1256, 1226,
1132, 1066, 1028, 952, 801, 556, 538; LCMS [M + H]+ m/z calcd
for C15H25O3N 268.19, found 268.40, 100.00%.
(R)-2-{2-[2-(2,4-Dimethylphenoxy)ethoxy]ethyl}aminobutan-
1-ol (22). Yield 55%; white solid; M. P. 46–48 °C; Rf = 0.75
1
(MeOH/ethyl acetate 1 : 1); NMR H (300 MHz, CDCl3): δ 6.88–
6.98 (m, 2H, Ar-H3, Ar-H5), 6.72 (d, 1H, J = 8.0 Hz, Ar-H6),
4.06–4.12 (m, 2H, Ar–O–CH2–), 3.80–3.85 (m, 2H, Ar–O–CH2–
CH2–), 3.65–3.70 (m, 2H, –CH2–O–CH2–), 3.58–3.64 (m, 1H,
>CH–CHH–OH), 3.31 (dd, 1H, J = 10.8 Hz, J = 6.4 Hz, >CH–
CHH–OH), 2.85–2.98 (m, 1H, –CHH–NH–), 2.70–2.80 (m, 1H,
–CHH–NH–), 2.52–2.62 (m, 1H, –CH<), 2.25 (s, 3H, Ar–CH3
(para)), 2.20 (s, 3H, Ar–CH3 (ortho)), 2.09–2.17 (m, 1H, NH),
1.39–1.53 (m, 2H, –CH2–CH3), 0.91 (t, 3H, J = 7.4 Hz, –CH2–
CH3); LCMS [M + H]+ m/z calcd for C16H27O3N 282.20, found
282.43, 100.00%; [α]25809.0= −9.64°.
1-{2-[2-(2,3-Dimethylphenoxy)ethoxy]ethyl}piperidin-3-ol
hydrochloride (19). Yield 65%; white solid; M. P. 138–140 °C;
Rf = 0.29 (MeOH/ethyl acetate 1 : 1); NMR 1H (600.2 MHz,
DMSO-d6): δ 10.67 (br s, 1H, NH+), 7.00 (dd, 1H, J = 8.2 Hz, J
= 7.4 Hz, Ar-H5), 6.78 (d, 1H, J = 8.2 Hz, Ar-H6), 6.75 (d, 1H, J
= 7.4 Hz, Ar-H4), 4.79 (br s, 1H, OH), 4.10 (ddd, 2H, J = 6.1
Hz, J = 4.7 Hz, J = 3.3 Hz, Ar–O–CH2–), 3.91 (t, 2H, J = 5.2 Hz,
–O–CH2–CH2–NH–), 3.88 (br s, 1H, >CH–OH), 3.80 (ddd, 2H,
J = 6.1 Hz, J = 4.7 Hz, J = 3.3 Hz, –O–CH2–CH2–O–), 3.65 (br s,
2H, pip), 3.41 (br s, 2H, pip), 3.22 (t, 2H, J = 5.2 Hz, –O–CH2–
CH2–NH–), 2.21 (s, 3H, Ar–CH3 (3)), 2.09 (s, 3H, Ar–CH3 (2)),
1.98 (br s, 2H, pip), 1.74 (br s, 2H, pip); 13C (150.94 MHz,
DMSO-d6): δ 157.01 (Ar-C1), 137.82 (Ar-C3), 126.33 (Ar-C5),
125.14 (Ar-C2), 122.88 (Ar-C4), 110.52 (Ar-C6), 69.80 (Ar–O–
CH2–CH2–O–), 68.22 (Ar–O–CH2–CH2–O–), 65.55 (–O–CH2–
CH2–NH+–), 64.67 (CH–OH), 55.50 (–O–CH2–CH2–NH+–),
51.32 (piperidine-C2), 49.26 (piperidine-C6), 31.78 (piperi-
dine-C4), 29,98 (piperidine-C5), 20.00 (Ar–CH3 (3)), 11.86 (Ar–
CH3 (2)); LCMS [M + H]+ m/z calcd for C17H27O3N 294.20,
found 294.21, 100.00%.
D,L-trans-2-{2-[2-(2,3-Dimethylphenoxy)ethoxy]ethyl}-
aminocyclohexan-1-ol (20). Yield 60%; white solid; M. P. 84–
86 °C, Rf = 0.46 (MeOH/ethyl acetate 1 : 1); NMR 1H (300
MHz, CDCl3): δ 7.00–7.06 (m, 1H, Ar-H3), 6.78 (d, 1H, J = 7.6
Hz, Ar-H4), 6.71 (d, 1H, J = 8.2 Hz, Ar-H2), 4.07–4.14 (m, 2H,
Ar–O–CH2–), 3.80–3.86 (m, 2H, Ar–O–CH2–CH2–), 3.67 (dd,
2H, J = 5.9 Hz, J = 4.7 Hz, –CH2–O–CH2–), 3.08–3.20 (m, 1H,
>CH–OH), 3.03 (dt, 1H, J = 12.2 Hz, J = 5.9 Hz, –CHH–NH–),
2.65 (dt, 1H, J = 12.3 Hz, J = 4.7 Hz, –CHH–NH–), 2.27 (s, 3H,
Ar–CH3 (3)), 2.18–2.25 (m, 1H, >CH–NH–), 2.16 (s, 3H, Ar-
CH3 (2)), 1.97–2.11 (m, 2H, pip), 1.66–1.77 (m, 2H, pip), 1.32–
1.48 (m, 1H, NH), 1.18–1.31 (m, 3H, pip), 0.86–1.04 (m, 1H,
pip); LCMS [M + H]+ m/z calcd for C18H28O3N 308.22, found
308.35, 98.56%.
(R,S)-2-{2-[2-(2,6-Dimethylphenoxy)ethoxy]ethyl}amino-1-
phenylethan-1-ol (23). Yield 60%; white solid; M. P. 108–110
1
°C; Rf = 0.86 (CH2Cl2/MeOH 7 : 3); NMR H (300 MHz, CDCl3):
δ 7.27–7.38 (m, 5H, >CH–Ar), 6.97–7.03 (m, 2H, Ar-H3, Ar-
H5), 6.88–6.95 (m, 1H, Ar-H4), 4.81 (dd, 1H, J = 9.2 Hz, J = 3.3
Hz, >CH–Ar), 3.91–3.96 (m, 2H, Ar–O–CH2–), 3.78–3.84 (m,
2H, Ar–O–CH2–CH2–O–), 3.74 (t, 2H, J = 5.1 Hz, –CH2–CH2–
NH–), 2.93–3.05 (m, 3H, –CH2–CH2–NH–), 2.79 (dd, 2H, J =
12.1 Hz, J = 9.5 Hz, –NH–CH2–CH<), 2.27 (s, 6H, Ar–(CH3)2);
LCMS [M + H]+ m/z calcd for C20H27O3N 330.20, found
330.42, 97.22%.
(R,S)-1-{2-[2-(5-Chloro-2-methylphenoxy)ethoxy]ethyl}-
aminobutan-2-ol (24). Yield 70%; white solid; M. P. 67–68 °C;
Rf = 0.57 (MeOH/ethyl acetate 1 : 1); NMR 1H (300 MHz,
CDCl3): δ 7.23 (d, 1H, J = 8.0 Hz, Ar-H4), 6.76 (s, 1H, Ar-H6),
6.72 (dd, 1H, J = 8.1 Hz, J = 1.2 Hz, Ar-H3), 4.18 (t, 2H, J = 1.0
Hz, Ar–O–CH2–), 3.82–3.95 (m, 2H, Ar–O–CH2–CH2–), 3.70 (t,
2H, J = 5.1 Hz, –CH2–O–CH2–), 3.51 (dtd, 1H, J = 9.3 Hz, J =
6.3 Hz, J = 3.1 Hz, –CH2–CH–OH), 2.79–2.90 (m, 2H, –CH2–
CH2–NH–), 2.72–2.79 (m, 1H, –NH–CHH–CH–), 2.43 (dd, 1H, J
= 12.1 Hz, J = 9.5 Hz, –NH–CHH–CH–), 2.32 (s, 3H, Ar–CH3),
1.64 (br s, 2H, NH, OH), 1.35–1.53 (m, 2H, –CH–CH2–CH3),
0.96 (t, 3H, J = 7.6 Hz, –CH–CH2–CH3); LCMS [M + H]+ m/z
calcd for C15H24O3NCl 302.15, found 302.12, 99.51%.
(R,S)-2-{2-[2-(2-Chloro-5-methylphenoxy)ethoxy]ethyl}-
aminobutan-1-ol (25). Yield 65%; white solid; M. P. 57–59 °C;
Rf = 0.67 (MeOH/ethyl acetate 1 : 1); NMR 1H (300 MHz,
CDCl3): δ 7.21 (d, 1H, J = 8.0 Hz, Ar-H3), 6.69–6.78 (m, 2H,
Ar-H4, Ar-H6), 4.15–4.20 (m, 2H, Ar–O–CH2–), 3.90 (dd, 2H,
J = 5.6 Hz, J = 4.1 Hz, Ar–O–CH2–CH2–), 3.82 (t, 2H, J = 5.1
Hz, –CH2–CH2–NH–), 3.72 (dd, 1H, J = 11.5 Hz, J = 3.6 Hz,
–CHH–OH), 3.47 (dd, 1H, J = 11.5 Hz, J = 6.7 Hz, –CHH–
OH), 3.00–3.11 (m, 1H, –NH–CH<), 2.86–2.97 (m, 2H, –CH2–
NH–), 2.70–2.78 (m, 1H, NH), 2.31 (s, 3H, Ar–CH3), 1.50–1.63
(m, 2H, –CH2–CH3), 0.88–0.97 (m, 3H, –CH2–CH3); LCMS
[M + H]+ m/z calcd for C15H24O3NCl 302.15, found 302.18,
98.85%.
(R,S)-2-{2-[2-(2,4-Dimethylphenoxy)ethoxy]ethyl}aminopropan-
1-ol (21). Yield 60%; white solid; M. P. 52–54 °C; Rf = 0.36
1
(MeOH/ethyl acetate 1 : 1); NMR H (300 MHz, CDCl3): δ 6.90–
6.96 (m, 2H, Ar-H3, Ar-H5), 6.72 (d, 1H, J = 7.6 Hz, Ar-H2),
4.07–4.12 (m, 2H, Ar–O–CH2–), 3.80–3.84 (m, 2H, Ar–O–CH2–
CH2–), 3.64–3.69 (m, 2H, –CH2–O–CH2–), 3.56 (dd, 1H, J =
10.5 Hz, J = 4.1 Hz, –CHH–OH), 3.21–3.28 (m, 1H, –CHH–
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1-{2-[2-(2-Chloro-5-methylphenoxy)ethoxy]ethyl}piperidin-
seizures in the MES test. At least three groups of animals
were injected with various doses of tested compounds and
ED50 values were calculated with 95% confidence intervals by
probit analysis.30
4-ol (26). Yield 60%; white solid; M. P. 70–72 °C; Rf = 0.53
1
(MeOH/ethyl acetate 1 : 1); NMR H (300 MHz, CDCl3): δ 7.21
(d, 1H, J = 8.0 Hz, Ar-3H), 6.68–6.76 (m, 2H, Ar-H4, Ar-H6),
4.16 (t, 2H, J = 4.4 Hz, –O–CH2–), 3.84–3.90 (m, 3H, –CH2–O–,
–CH–OH), 3.01 (br s, 2H, –CH2–CH2–N–), 2.83 (br s, 2H, –CH2
–N–), 2.31 (s, 3H, Ar–CH3), 2.11 (br s, 2H, –N–CH2–), 1.72 (br
s, 4H, –N–CH2–, –CH2–CH–OH), 1.47 (br s, 2H, –CH2–CH–
OH); LCMS [M + H]+ m/z calcd for C16H24O3NCl 314.15, found
314.15, 99.05%.
Minimal motor impairment was established in mice by a
standard rotarod procedure.31 Mice were trained to balance
on an accelerating rotarod (Rotarod apparatus Panlab/Har-
vard Apparatus, LE 8200, Spain). Proper experimentation was
conducted at least 24 h after the training trial. On the test
day, mice were intraperitoneally (ip) pretreated with the test
compound and tested on a rotarod revolving at 10 rotations
per minute. Neurotoxicity was indicated by the inability of
the animal to maintain equilibrium on the rod for one min-
ute. For the selected, most potent compounds, the neurotoxic
effect was expressed as a TD50 value, representing the doses
at which the compound resulted in minimal motor impair-
ment in 50% of the animals in the rotarod test. At least three
groups of animals were injected with various doses of tested
compounds and TD50 values were calculated with 95% confi-
dence intervals by probit analysis.30
In the maximal electroshock seizure threshold test (MEST
test), electroconvulsions were produced by alternating current
(duration of the stimulus: 0.2 s; 50 Hz) delivered via auricular
electrodes by an electroshock generator (Rodent Shocker,
Type 221, Hugo Sachs, Germany), according to the procedure
previously described.32 Full tonic extension of both hind
limbs was taken as the endpoint. Mice not displaying hind-
limb tonic extension were considered protected from seizure.
The current intensity was established according to an ‘up-
and-down’ method previously described by Socała et al.33 The
current intensity was lowered or raised by 0.06-log intervals
depending on whether the previously stimulated animal did
or did not exert tonic hind-limb extension, respectively. The
convulsive threshold was evaluated as CS50, defined as cur-
rent strength (in mA) required to induce tonic hind-limb ex-
tension in 50% of the animals tested, using the log-probit
method.30
ScPTZ-induced seizures were performed by subcutaneous
injection of pentylenetetrazole (PTZ, 85 mg kg−1). This pro-
duced clonic convulsions lasting for at least 5 s, with accom-
panying loss of the righting reflex. PTZ was administered 0.5 h
after injections of tested compounds and observations were
carried out for 30 min. In the control groups, the first epi-
sodes of clonic convulsions were recorded between 4–16 min
in the observation period. The absence of clonic convulsions
within the observed time period was interpreted as the com-
pound's ability to protect against PTZ-induced seizures.34
Antinociceptive activity in the formalin hind paw test was
examined as previously described.35,36 Twenty microliters of a
5% formalin solution were injected intraplantarly (ipl) into
the right hind paw of the mouse. Immediately after formalin
injection, the animals were placed individually into glass bea-
kers and were observed for the next 30 min. Time (in s) spent
on licking or biting the injected hind paw in selected inter-
vals, 0–5 and 15–30 min after injection was measured in each
experimental group and was an indicator of nociceptive
(R,S)-2-{2-[2-(2-Chloro-6-methylphenoxy)ethoxy]ethyl}-
amino-1-phenylethan-1-ol (27). Yield 70%; white solid; M. P.
99–100 °C; Rf = 0.68 (CH2Cl2/MeOH 1 : 1); NMR 1H (600.2
MHz, CDCl3): δ 7.32–7.35 (m, 2H, Ar-H2′, Ar-H6′), 7.29–7.31
(m, 2H, Ar-H3′, Ar-H5′) 7.26–7.28 (m, 1H, Ar-H3), 7.19–7.23
(m, 1H, Ar-H4′), 7.16–7.19 (m, 1H, Ar-H5), 7.03 (dd, 1H, J =
7.8 Hz, J = 7.6 Hz, Ar-H4), 5.23 (d, 1H, J = 4.4 Hz, OH), 4.58–
4.62 (m, 1H, >CH–OH), 4.00 (t, 2H, J = 4.8 Hz, Ar–O–CH2–),
3.70 (t, 2H, J = 4.8 Hz, Ar–O–CH2–CH2–), 3.53 (t, 2H, J = 5.5
Hz, –O–CH2–CH2–NH–), 2.71 (t, 2H, J = 5.5 Hz, –O–CH2–CH2–
NH–), 2.61–2.68 (m, 1H, –NH–CH2–CH<), 2.26 (s, 3H, Ar–CH3
(6)), 1.78 (br s, 1H, NH); 13C (150.94 MHz, CDCl3): δ 152.79
(Ar-C1), 144.55 (Ar-C1′), 133.30 (Ar-C6), 129.84 (Ar-C5), 127.81
(Ar-C3′, Ar-C5′), 127.67 (Ar-C3), 126.65 (Ar-C4′), 126.63 (Ar-
C2), 125.82 (Ar-C6′, Ar-C2′), 124.79 (Ar-C4), 71.84 (Ar–O–
CH2–), 71.55 (>CH–OH), 70.29 (–O–CH2–CH2–NH–), 69.36
(Ar–O–CH2–CH2–), 57.59 (–NH–CH2–CH<), 48.56 (–O–CH2–
CH2–NH–), 15.93 (Ar–CH3); LCMS [M + H]+ m/z calcd for
C19H24O3NCl 350.15, found 350.17, 100.00%.
(R)-2-{2-[2-(2-Chloro-6-methylphenoxy)ethoxy]ethyl}amino-
1-phenylethan-1-ol (28). Yield 60%; white solid; M. P. 56–58
°C; Rf = 0.76 (MeOH/ethyl acetate 1 : 1); NMR 1H (300 MHz,
CDCl3): δ 7.27–7.40 (m, 5H, >CH–Ar), 7.19 (d, 1H, J = 7.7 Hz,
Ar-H3), 7.03–7.08 (m, 1H, Ar-H5), 6.90–6.97 (m, 1H, Ar-H4),
4.81 (dd, 1H, J = 9.5 Hz, J = 3.6 Hz, >CH-), 4.06–4.11 (m, 2H,
Ar–O–CH2–), 3.81–3.86 (m, 2H, Ar–O–CH2–CH2–), 3.74 (t, 2H,
J = 5.0 Hz, –O–CH2–CH2–NH–), 2.97–3.05 (m, 2H, –O–CH2–
CH2–NH–), 2.90–2.96 (m, 1H, NH), 2.79 (dd, 2H, J = 12.2 Hz, J
= 9.1 Hz, –NH–CH2–CH<), 2.30 (s, 3H, Ar–CH3); LCMS [M +
H]+ m/z calcd for C19H24O3NCl 350.15, found 350.37, 97.94%;
20.0
589
[α]
= −22.29°.
Pharmacology
The MES test was performed according to a procedure de-
scribed by Löscher et al.29 Briefly, the mice received an elec-
trical stimulus of sufficient intensity (25 mA, 500 V, 50 Hz,
0.2 s) delivered via auricular electrodes by an electroshock
generator (Rodent Shocker, Type 221, Hugo Sachs, Germany)
to induce maximal seizures. The endpoint was the tonic ex-
tension of the hind limbs. In vehicle-treated mice, the proce-
dure caused immediate hind-limb tonic extension. Mice not
displaying hind-limb tonic extension were considered to be
protected from seizures. For selected compounds exhibiting
potent anticonvulsant activity, the ED50 was evaluated, de-
fined as the dose of a drug protecting 50% of animals against
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behavior. In mice, an intraplantar injection of diluted forma-
lin produces a biphasic nocifensive behavioral response (i.e.,
licking or biting the injected hind paw). The acute nocicep-
tive phase lasts for the first 5 min, whereas the second in-
flammatory phase occurs between 15 and 30 min after forma-
lin injection.
Locomotor activity was measured with photoresistor
actometers (Ugo Basile, Italy) connected to a counter for the
recording of light-beam interruptions. Mice were individually
placed in plastic cages for a 30 min habituation period, and
then the number of light-beam crossings was counted for a
30 min session. The cages were cleaned with 70% ethanol af-
ter each mouse.
In case of the neutral red assay, HepG2 cells were seeded
in 96-well plates (Corning) at a density of 5 × 104 cells per
well. After 24 h, the culture medium was replaced with the
same medium (control) or medium containing tested com-
pounds at concentrations from 10 to 250 μM. The cells were
incubated in the presence of compounds for 24 h, and then
cell viability was determined by neutral red staining (Sigma
Aldrich) according to a procedure described in the litera-
ture.38 The optical density (OD) of each well was determined
at 570 nm on a plate reader (BIOTEK). The number of living
cells was directly proportional to the absorbance of the
samples.
One day before the experiment, the cells were seeded at
15000 cells per well in 96-well plates in the medium
containing a reduced amount of serum (1% FBS). The SH-
SY5Y cells were treated with compound 19 (dissolved in
ddH2O) at concentrations of 10−8, 10−7, 10−6, 10−5, 5 × 10−5,
and 10−4 M for 72 hours. In order to assess the effect of the
test compound on oxidative stress-induced cell damage, com-
pound 19 was added at concentrations of 10−8, 10−7, 10−6,
and 10−5 M for 72 hours alone or with hydrogen peroxide (0.5
mM). H2O2 was added for the last 24 hours of culture, i.e. 48
hours after the addition of compound 19. The control cul-
tures were supplemented with the same amount of an appro-
priate vehicle. For LDH release and MTT reduction assays as
a positive control, triton was used.
The chimney test was performed as previously described.37
Previously trained and selected animals were placed in a 25
cm long and 2.5 cm in diameter, horizontally located tube,
which was reversed in such a way that the mice were able to
leave it only by climbing up backward until they reached the
other end. The inability of mice to perform the test within 60
seconds indicated motor impairment.
The experimental protocol was approved by the First Local
Ethics Committee on Animal Testing at the Jagiellonian Uni-
versity in Krakow (No 15/2014, 73/2014 and 108/2014) and
was in accordance with the European Communities Council
Directive of 24 November 1986 (86/609/EEC).
Cytotoxicity analysis
Cytotoxicity was quantified by measuring the efflux of lac-
tate dehydrogenase (LDH) into the culture media 72 h after
treatment with compound 19 (alone or with hydrogen perox-
ide). LDH activity was determined in medium using a cytotox-
icity detection kit (Roche Diagnostic GmbH). The amount of
colored hydrazone, formed in the reaction of pyruvic acid
with 2,4-dinitrophenylhydrazine, was inversely proportional to
the LDH activity in the sample and could be quantified by
measuring the absorbance at 490 nm. Data were normalized
as a percent of the LDH released by vehicle-treated cells
(100%), i.e. cells cultured in the absence of compound 19
and without H2O2.
The viability of SH-SY5Y cells was measured, as described
previously,39 by determining the cellular reducing capacity,
estimated through the extent of MTT reduction to the insolu-
ble intracellular formazan, which depends on the activity of
intracellular dehydrogenases and is independent of changes
in the integrity of the plasma membrane. After 72 h culture
with the test compound (alone or with hydrogen peroxide)
the medium was removed and SH-SY5Y cells were incubated
with MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium
bromide; 0.15 mg mL−1 in PBS) for 1 h at 37 °C. After incu-
bation, when purple colored precipitates were visible under
the microscope, the supernatants were aspirated, and the
formazan precipitates were solubilized by the addition of
100 μL of dimethylsulfoxide (DMSO) per well. Absorbance
was measured at 570 nm in a plate-reader (Multiscan,
Labsystem). The results were expressed as a percentage of
control cells incubated in the absence of compound 19 and
H2O2.
The SH-SY5Y neuroblastoma cell line was obtained from the
American Type Culture Corporation (ATCC). Cells were cul-
tured in Dulbecco's modified Eagle's medium (DMEM,
Gibco-BRL) supplemented with 10% fetal bovine serum (FBS,
Gibco-BRL), 100 units per mL of penicillin, and 100 μg mL−1
of streptomycin (Sigma Co.), and kept in a humidified atmo-
sphere of 5% CO2/95% O2 at 37 °C.
An astrocyte cell line was obtained from the American
Type Culture Corporation (ATCC, CRL-2541), and human
hepatoma HepG2 (Sigma Aldrich, 85011430) were used in the
study. The cells were cultured under standard conditions (37
°C, 5% CO2), in DMEM low glucose medium (astrocytes) and
MEM medium (hepatoma), all supplemented with 10% FBS
and antibiotics.
The cytotoxic effect was determined with the use of both
MTT (Cayman) and neutral red assay. Briefly, in order to per-
form MTT assay, the cells were seeded at a density of 1 × 104
in 96-well plates. Following overnight culture, the cells were
then treated with increasing doses of compounds (1, 6, 19,
21) and incubated for 24 h. Following cell exposure to each
drug for 24 h in 96-well plates, 10 μL MTT reagent was added
to each well, and after 3 hours of incubation (37 °C, 5%
CO2), the medium was aspirated and the formazan produced
in the cells appeared as dark crystals in the bottom of the
wells. Next, the crystal-dissolving solution (Cayman) was
added to each well. Then, the optical density (OD) of each
well was determined at 570 nm on a plate reader (BIOTEK).
The number of metabolically active cells is directly propor-
tional to the absorbance of the samples.
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Statistical analysis
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The data were obtained in three independent experiments,
each performed in 3–4 wells and were presented as a percent-
age of the control SEM. The significance of differences be-
tween the means was evaluated by Dunnett's test, following a
one-way analysis of variance (ANOVA).
Metabolism prediction
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The MetaSite 5.1.1 Mass 3.2.1 computer program (Molecular
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the tools option of the program, a site of metabolism was
chosen, then liver metabolism. In the case of a metabolite
identification tool, the minimum mass was limited by 50 g
mol−1 and redundant metabolites (below 30%) were ignored.
All common CYP reactions were taken into consideration.
20 M. M. Schmid, R. W. Freudenmann, F. Keller, B. J.
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Acknowledgements
The work was financed by the National Science Centre, Po-
land, decision on grant no DEC-2013/11/B/NZ7/04834 and
DEC-2015/17/N/NZ7/00966.
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