Exploration of novel 3-substituted azetidine derivatives as triple reuptake inhibitors

Article abstract of DOI:10.1021/jm3008294

Novel azetidines based on the 3-aryl-3-oxypropylamine scaffold were designed, synthesized, and evaluated as TRIs. Reduction of 1 followed by Swern oxidation and then Grignard reaction gave 3. The alkylation of 3 provided the corresponding azetidine derivatives 6, of which the two most promising, 6bd and 6be, were selected from 86 prepared analogues based on their biological profiles. Compound 6be showed activity in vivo in FST at 10 mg/kg IV or 20-40 mg/kg PO.

Full text of DOI:10.1021/jm3008294

Brief Article
pubs.acs.org/jmc
Exploration of Novel 3‑Substituted Azetidine Derivatives As Triple
Reuptake Inhibitors
Younghue Han,† Minsoo Han,†,‡ Dongyun Shin,†,§ Chiman Song,† and Hoh-Gyu Hahn*,†
†Organic Chemistry Laboratory, Korea Institute of Science and Technology, Seoul 136-791, Korea
‡Department of Chemistry, Korea University, Seoul 136-701, Korea
S
* Supporting Information
ABSTRACT: Novel azetidines based on the 3-aryl-3-oxypropylamine scaffold were designed, synthesized, and evaluated as
TRIs. Reduction of 1 followed by Swern oxidation and then Grignard reaction gave 3. The alkylation of 3 provided the
corresponding azetidine derivatives 6, of which the two most promising, 6bd and 6be, were selected from 86 prepared analogues
based on their biological profiles. Compound 6be showed activity in vivo in FST at 10 mg/kg IV or 20−40 mg/kg PO.
the design of novel compounds through the structure analysis
and molecular modification of the marketed reuptake trans-
porter-based antidepressants. We selected fluoxetine, atom-
oxetine, reboxetine, and duloxetine, which have the 3-aryl-3-
oxypropylamine scaffold in the structures. These compounds
significantly affect the central nervous system (CNS) by
monoamine reuptake inhibitory mechanism. For instance,
fluoxetine was the first selective serotonin uptake inhibitor to
reach the market with far fewer side effects than other
antidepressants.8 Atomoxetine and reboxetine are selective
noradrenaline reuptake inhibitors developed for the treatment
of either attention deficit and hyperactivity disorder or MDD.9
Furthermore, duloxetine has emerged as a dual acting SNRI
that offers improved efficacy and faster onset of action than
SSRIs.10 These compounds have the 3-aryl-3-oxypropylamine
scaffold and rotatable bonds, except reboxetine, in which the
corresponding bonds are locked into a cyclic ring (see Figure
1). On the basis of the characteristic common points of the
INTRODUCTION
■
Major depressive disorder (MDD) is a common and serious
illness with the potential to become the leading cause of
disability worldwide. Pathophysiologically, the cause of
depression is commonly associated with a deficiency of
monoamine neurotransmitters (serotonin (5-HT), norepi-
nephrine (NE), and dopamine (DA)) in the brain, and a
number of antidepressants aim to increase the levels of these
neurotransmitters in the synapses. Among various mono-
aminergic strategies for maintaining the concentration of
neurotransmitters, blocking the reuptake of neurotransmitters
by presynaptic 5-HT, NE and DA transporters (SERT, NET,
and DAT) has been an important strategy in modern
antidepressant therapy.1 Although many kinds of reuptake
inhibitor such as selective serotonin reuptake inhibitors (SSRIs)
and serotonin NE reuptake inhibitors (SNRIs) are commer-
cially available for the treatment of major depression, they may
take several weeks of treatment to affect any improvement in
symptoms, and some inhibitors present side effects such as
insomnia and sexual dysfunction.2 One strategy to improve the
efficacy and/or reduce the delay in the onset of their action is
the addition of a DA component to SSRIs or SNRIs. This is the
concept of triple reuptake inhibition, which blocks the synaptic
reuptake to all of 5-HT, NE, and DA. Recent study results
supported the effect of DA in depression.3 For example, D3-
preferring DA receptor agonists such as pramipexole showed
therapeutic efficacy in major depression.1a,4 DA reuptake
inhibitors such as bupropion enhanced the antidepressant
actions of SSRIs and SNRIs in humans.5 A few classes of triple
reuptake inhibitor (TRI), including DOV 216,3036 and GSK
372,475,7 showed positive phase II clinical effect. However, no
TRI is yet available in the market. Combination- and multiple-
drug therapy to inhibit the reuptake of 5-HT, NA, and DA may
raise some problems of pharmacokinetics (PK). Therefore,
TRIs as a single molecule are expected to become the next
generation of antidepressants and remain desirable.
Figure 1. Design of 3-azetidine derivatives by rigidification of rotatable
bonds.
structures, we have designed novel 3-substituted azetidine
derivatives by modifying the 3-aryl-3-oxypropylamine scaffold.
As shown in Figure 1, rigidification of two bonds (α and β) of
the 3-aryl-3-oxypropanamine moiety via the formation of a new
cyclic scaffold that included these rotatable bonds afforded a
four-membered azetidine skeleton. Recently, azetidine sub-
stituted diphenyl ether derivatives with combined NE reuptake
inhibitor and 5-HT1A partial agonist pharmacology were
DESIGN
■
Our initial effort to explore target compounds for the
development of a novel TRI as an antidepressant focused on
Received: June 13, 2012
© XXXX American Chemical Society
A
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Journal of Medicinal Chemistry
Brief Article
reported.11 Additionally, the four characteristic structural
common points of five compounds6a,12−15 that show TRI
activity are: (a) they are basic due to the 2° or 3° amino moiety,
(b) the amino nitrogen atom presents in the cyclic ring, (c) one
or two aromatic groups are present, and (d) they have one or
more hydrogen bonding acceptors or donors.
Table 1. Percentage Inhibition of Activities of 3-Substituted
Azetidines 4 and 6 against HEK-hSERT, HEK-hNET, and
HEK-hDAT
% reuptake
inhibition, at 0.1 μM
entry
compd
R1
R2
SERT NET DAT
fluoxetine
nisoxetine
GBR12909
4a
44
9.9
1.9
1.7
2.0
3.3
4.0
3.9
4.6
16
4.7
78
5.8
9.8
29
SYNTHESES
■
The overall synthetic route of azetidine derivatives 6 is depicted
in Scheme 1. Aldehyde 2 was obtained in 80% yield from the
−5.9
0.2
3.5
1.5
4.5
7.7
10
1
2
3
4
5
6
7
C6H5
H
3.9
2.1
2.2
3.0
2.6
7.3
22
4b
C6H4(4-Cl)
C6H5
H
a
Scheme 1
6aa
CH3
6ab
C6H5
CH2CH3
6ac
C6H4(4-Cl) CH3
6ad
C6H4(4-Cl) CH2CH3
6ae
C6H3(3,4-di CH3
Cl)
47
8
6af
C6H3(3,4-di CH2CH3
Cl)
28
49
30
9
6ah
6ai
6aj
C6H5
C6H5
25
22
16
18
68
89
9.3
36
58
10
11
C6H4(4-Cl) C6H5
C6H3(3,4-di C6H5
Cl)
12
13
14
6ak
6al
C6H5
CH2C6H5
12
12
12
9.4
16
30
2.0
5.7
12
C6H4(4-Cl) CH2C6H5
6am
C6H3(3,4-di CH2C6H5
Cl)
a
Reagents and reaction conditions: (a) BH3·SMe, THF, 0°C; (b)
(COCl)2, DMSO, then TEA, CH2Cl2, −78 °C to 0 °C; (c) THF, 0°C;
(d) 1N HCl, MeOH, 60°C; (e) NaH, THF, reflux; (f) PPh3, DIAD,
THF, rt.
15
6aq
C6H3(3,4-di CH2CH2CH3
Cl)
39
87
74
16
17
18
6bd
6be
6bs
C10H7
C10H7
C10H7
CH2CH2CH3
C6H5
77
64
28
87
90
79
48
64
68
C6H4(3,4-di
Cl)
reduction of commercially available 1 by the treatment of
borane−dimethyl sulfide complex in tetrahydrofuran (THF) at
0 °C, followed by Swern oxidation. Grignard reaction of 2 with
aryl magnesium halide in THF at 0 °C gave an intermediate,
secondary alcohol 3. Azetidinyl alkyl ether 5a (R2 = alkyl,
benzyl etc) was prepared by two synthetic methods. In the case
of R2 = alkyl, the secondary alcohol 2 was treated with either
alkyl halide or benzyl halide in the presence of sodium hydride
in THF at ambient temperature to give the corresponding alkyl
or benzyl ether 5a in high yields (48−87%). In the case of R2 =
phenyl or substituted phenyl, azetidinyl phenyl ether derivatives
5b were prepared successfully by Mitsunobu reaction. The
reaction of 3 with substituted phenol in the presence of
triphenylphosphine and diisopropyl azodicarboxylate in THF at
room temperature afforded the corresponding ether 5b in high
yields (75−91%). Deprotection of the Boc group in 5a or 5b by
the treatment of 1N HCl in boiling methanol gave the
corresponding 6. The products 6 could be simply obtained by
filtration from the reaction mixture with isolated yields ranging
19
20
6bu
6bv
C6H3(3-F,
4-Cl)
C6H5
49
41
76
81
30
29
C6H3(3-F,
4-Cl)
C6H4(2-F)
FDSS6000 96 well fluorescence plate reader, which is a high
throughput screening device (Hamamatsu Photonics, Hama-
matsu, Japan).16 Human embryonic kidney 293 (HEK293)
cells stably transfected with human DA transporter (HEK-
hDAT), human NE transporter (HEK-hNET), or human
serotonin transporter (HEK-hSERT) were used for the assay.
All the synthesized compounds were screened at three
concentrations (10, 1, and 0.1 μM), and the selected
compounds were further screened to obtain their IC50 values.
The primary screening results of all the compounds are
reported in SI. To support the following discussion on the
structure−activity relationship of the compounds, the primary
screening results of 20 selected compounds at a concentration
of 0.1 μM are summarized in Table 1.
We used fluoxetine, nisoxetine, and GBR12909 that are
selective SERT, NET, and DAT reuptake inhibitors,
respectively, as references. The biological activities data in
Table 1 revealed that the presence of an O-substituent such as
an alkyl, phenyl, or substituted phenyl group of the alcoholic
moiety increased the activities (from comparison of 4b with
6ac, 6ad, and 6ai). The azetidinyl ethers 6 showed significantly
elevated activities against three monoamines, where R1 is the
naphthyl moiety (6bd, 6be, 6bs). Another substituent effect on
the biological activities of the azetidinyl ethers 6 was the
bulkiness of R2. Thus, the azetidine derivative in which R2 is a
bulky group such as n-propyl or aryl showed higher activity
1
from 12% to 99%. The structures were confirmed by H and
13C NMR spectroscopy and HRMS. The obtained solids were
the corresponding hydrogen chloride salts of azetidine, which
could be used directly for TRI screening. We synthesized 86
analogues of 3-substituted azetidine derivatives in this manner.
The selected compounds are listed in Table 1, along with the
results of their biological activity tests (see Supporting
Information (SI) for all the prepared compounds).
BIOLOGICAL SCREENING
■
DA, NE, and serotonin neurotransmitter uptake activities were
measured using the Neurotransmitter Transporter Uptake
Assay Kit (Molecular Devices, Sunnyvale, CA, USA) with the
B
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Journal of Medicinal Chemistry
Brief Article
than that in which R2 is a small substituent such as methyl (6ae
(R2 = methyl) < 6af (R2 = ethyl) < 6aq (R2 = n-propyl)). In
contrast, the benzyl substituents in R2 decreased the activity
against the three transporters (compare 6ah with 6ak, 6ai with
6al, 6aj with 6am). On the basis of the initial results of reuptake
inhibition, 11 compounds were selected for further character-
ization of their IC50 against three monoamine reuptake and
human ether-a go-go-related gene (hERG) channel inhibitory
activities (IC50).17 Table 2 showed the data of five selected
compounds (detailed results are reported in SI).
The PK profiles of 6bd and 6be in rats were evaluated and
the PK parameters are shown in Table 3. Compounds 6bd and
6be exhibited a moderate bioavailability (F) of 5.7 and 5.8,
respectively.
Table 3. Pharmacokinetic (PK) Parameters of Compounds
6bd and 6be
6bd
6be
a
b
a
b
parameters
PO
IV
PO
IV
Cmax (ng/mL)
Tmax (h)
7.3
1.0
198.2
5.6
1.3
112.1
Table 2. IC50 Values of Monoamine Reuptake and hERG
Channel Inhibitory Activities of the Selected Azetidines
t1/2z (h)
4.8
0.7
210.7
214.6
4.7
3.3
1.4
153.3
159.7
6.4
AUCall (ngh/mL)
AUCinf (ngh/mL)
CLz/F (L/h/kg)
Vz/F (L/kg)
MRT (0 − t)
F (%)
23.9
49.4
46.5
248.9
2.2
17.9
41.7
48.7
241.0
2.0
reuptake assay (IC50, nM)
entry
compd
hSET
hNET
hDAT
hERG (IC50, μM)
4.8
13.1
fluoxetine
nisoxetine
GBR12909
6aq
150
700
3840
44
4410
18.0
1460
10
18400
1150
190
32
0.9
1.73
5.7
5.8
a
b
1
2
3
4
5
2.2
PO dose: 2 mg/kg. IV dose: 1 mg/kg.
6bd
6.8
12
72
0.98
0.83
0.18
1.56
6be
8.3
3.1
35
To verify the potential antidepressant effect in vivo of these
6bs
35
16
54
novel azetidine derivatives, we selected compound 6be for
profiling in the forced swimming test (FST) in mice, which is
sensitive to known antidepressant drugs. The FST was carried
out on mice according to the method of Porsolt.19 Compound
6be was administrated intravenously (IV) 0.5 h before the test
in mice at 2.5, 5.0, and 10.0 mg/kg. As clearly seen in the
Figure 2, 6be showed a dose-dependently reduced immobility
6bu
142
21
148
The IC50 values presented in Table 2 revealed the superiority
of the azetidine derivatives 6 as a superior novel scaffold for
application as three monoamine reuptake inhibitors in
comparison with that of three reference compounds (fluox-
etine, nisoxetine, and GRB12909). Comparing 6aq (entry 1)
with 6bd (entry 2), the naphthyl substituent increased the
hSET inhibitory activity approximately 6-fold. In general, the
naphthyl derivatives 6bd, 6be, and 6bs (entry 2, 3, and 4,
respectively) showed good potency against all three mono-
amines. The activity of 6bd (entry 2) decreased in the order
hSET > hNET > hDAT, while the others (entries 3, 4)
decreased in the order hNET > hSET ≈ hDAT. In addition,
compounds 6aq, 6bd, and 6be showed excellent biological
activities against three monoamine uptake inhibition as well as
hERG inhibition profiles. Having obtained the three mono-
amine uptake inhibitory profiles, we carried out human
cytochrome P450 (CYP) enzyme assay (percentage inhibition
at 10 μM for CYP1A2, CYP2D6, CYP2C9, and CYP3A4 using
VIVID CYP enzymes assay kit) and investigated the metabolic
stability at human liver microsomes (percentage remaining after
0.5 h using BD Gentest assay kit) of 37 compounds (detailed
results are reported in SI). Compounds 6be and 6bs in which
the naphthyl moiety is substituted in the azetidine scaffold
showed relatively low percentage inhibitory value for CYP2D6
and CYP3A4. Most of the compounds were stable against
human liver microsomes.
Figure 2. Effect of compound 6be on immobility in the forced
swimming test (FST) model in ICR mice (pretreatment time = 30
min). (a) IV dose: 2.5, 5.0, and 10.0 mg/kg. (b) PO dose: 10, 20, and
40 mg/kg.
time at 5.0 and 10.0 mg/kg, which was statistically significant
compared to the vehicle. (Figure 2a). The positive control,
venlafaxine, showed a similar reduction in immobility with that
of 6be at 10 mg/kg dosing. Oral administration (PO) of 6be
also showed dose-dependent reductions in immobility at 20
and 40 mg/kg (Figure 2b) (see SI for the detailed results).
In the next step of the characterization, the drug develop-
ment of a novel scaffold was related to the in vivo behavior
activity of the compounds using an animal model. On the basis
of their overall balanced profiles in terms of IC50 values against
three monoamine uptake inhibitory activity, CYP assay data,
metabolic stability, and hERG inhibition data, we chose 6bd
and 6be for further brain−blood barrier (BBB) study and
pharmacokinetic (PK) investigation. Because BBB permeabil-
ity18 is one of the factors relevant to the success of CNS-
targeted drugs, brain penetration was also evaluated. Com-
pounds 6bd and 6be showed adequate brain to plasma ratio of
0.86 and 2.70, respectively (see SI for detailed results).
SUMMARY
■
We designed a novel class of 3-substituted azetidine derivatives
6 and thereby achieved the study goal of creating a single
molecular entity with triple activities as 5-HT, NE, and DA
reuptake transporters. The bulkiness of the substituent at
oxygen (R2) and the presence of the naphthyl moiety at R1
were clearly important for these activities. To verify the
potential antidepressant effect in vivo of these novel azetidine
derivatives, we selected compound 6be for FST profiling in
mice. This compound showed a dose dependent reduction of
immobility at 5 and 10 mg/kg IV. A similar result was obtained
C
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Journal of Medicinal Chemistry
Brief Article
by oral administration of 6be. These 3-substituted azetidine
derivatives therefore may offer an important new scaffold to act
as single-molecule TRIs for application as the next generation
of antidepressants.
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional experimental procedures and biological screening
method, the yields, melting point, purity, H and 13C NMR
1
data for all the compounds, HRMS data for the representative
compounds, and the detailed results of biological assay. This
material is available free of charge via the Internet at http://
pubs.acs.org.
EXPERIMENTAL SECTION
■
Preparation of the 3-Substituted Azetidines 6. Synthesis of
tert-Butyl 3-Formylazetidine-1-carboxylate (2). To a solution of tert-
butyl-3-(hydroxymethyl)azetidine-1-carboxylic acid (1) (1.0 g, 5.0
mmol) in THF (20 mL) at 0 °C was added borane−dimethyl sulfide
complex (10 M solution in THF, 1.5 mL, 15 mmol) under N2
atmosphere. The reaction mixture was stirred at the same temperature
for 4 h. 1N HCl (20 mL) was added to the mixture while stirring, and
then the reaction mixture was extracted with methylene chloride. The
organic extract was washed with brine and dried over MgSO4. The
solvent was removed by evaporation, and the residue was used
immediately in the next step. To a solution of dimethyl sulfoxide (1.1
mL, 15 mmol) dissolved in methylene chloride (20 mL) at −78 °C
was added oxalyl chloride (1.3 mL, 15 mmol) under N2 atmosphere.
The reaction mixture was stirred for 10 min, and then a solution of the
prepared primary alcohol above, dissolved in methylene chloride (2.0
mL), was added. After the resulting mixture was stirred for an
additional 30 min at the same temperature, triethylamine (6.3 mL, 45
mmol) was added. The reaction mixture was stirred for 1 h and
allowed to warm to reach room temperature. The resulting reaction
mixture was washed with brine and then dried over MgSO4. The
solvent was removed, and the crude product was purified by flash
chromatography on silica gel (methylene chloride:methyl alcohol =
9:1) to obtain 2 (0.74 g). Yield 80%; yellow oil. 1H NMR (300 MHz,
CDCl3) δ 1.44 (s, 9H, CO2C(CH3)3), 3.39−3.30 (m, 1H, CH-
(CH2)2), 4.11−4.05 (m, 4H, CH(CH2)2), 9.85 (s, 1H, CHO).
Synthesis of the Azetidine Derivatives 3 (General Procedure). To
a solution of phenylmagnesium bromide (0.5 M solution in THF, 42
mL, 21 mmol) in THF (22 mL) at 0 °C was added a solution of the
aldehyde 8 (1.3 g, 7.0 mmol) dissolved in freshly distilled THF (15
mL) under N2 atmosphere. The reaction mixture was sirred for 3 h,
poured onto aqueous NH4Cl solution (15 mL), and stirred at room
temperature for 0.5 h and then extracted with methylene chloride. The
organic extracts were washed with brine and then dried over MgSO4.
The solvent was removed, and the crude product was purified by flash
chromatography on silica gel (n-hexane:ethyl acetate = 2:1) to obtain
3 (57−86% yields).
AUTHOR INFORMATION
Corresponding Author
■
*Phone: +82-2-958-5139. Fax: +82-2-958-5189. E-mail:
hghahn@kist.re.kr.
Present Address
§College of Pharmacy, Gachon University of Medicine and
Science, Yeonsu-gu, Incheon 406−799, Korea
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. S. H. Ahn in the Korea Research Institute of
Chemical Technology for BBB experiments and Dr. E. Lim for
CYP assay. We also thank Prof. C.-G. Jang in Sungkyunkwan
University for FST test. This work was financially supported by
the Korea Institute of Science and Technology.
ABBREVIATIONS USED
■
MDD, major depressive disorder; 5-HT, serotonin; NE,
norepinephrine; DA, dopamine; SERT, serotonin transporter;
NET, norepinephrine transporter; DAT, dopamine transporter;
SSRI, selective serotonin reuptake inhibitor; SNRI, serotonin
norepinephrine reuptake inhibitor; TRI, triple reuptake
inhibitor; CNS, central nervous system; HEK, human
embryonic kidney; hERG, human ether-a go-go-related gene;
CYP, cytochrome P450; BBB, brain−blood barrier; PK,
pharmcokinetics; FST, forced swimming test; IV, intravenous
injection; PO, per oral
Synthesis of the Azetidine Derivatives 4a and 4b (General
Procedure). Synthesis of 5a (by the Reaction of 3 with Alkyl or
Benzyl Halide). To a suspension of sodium hydride (1.2 mmol) in
THF (15 mL) at 0 °C was added 10 (0.60 mmol) dissolved in THF
(5.0 mL). The alkyl halide or benzyl bromide (1.20 mmol) was added
to the reaction mixture and then heated to reflux for 18 h. The
reaction mixture was cooled, poured onto water (5.0 mL), and then
extracted with methylene chloride. The organic extract was washed
with brine and dried over MgSO4. The solvent was removed, and the
residue was purified by flash chromatography on silica gel (n-
hexane:ethyl acetate = 1:1) to obtain the corresponding alkyl or benzyl
ether 5a (48−87% yields).
Synthesis of 5b (by the Reaction of 3 with Phenol Derivatives).
To a solution of 3 (0.45 mmol) in THF (15 mL) at 0 °C was added
sequentially triphenylphosphine (0.90 mmol), phenol (0.90 mmol),
and diisopropyl azodicarboxylate (0.90 mmol). The cooling bath was
removed, and the reaction mixture was stirred at room temperature for
2 h. The solvent was removed by evaporation, and the residue was
purified by flash chromatography on silica gel (n-hexane:ethyl acetate
= 4:1) to obtain the corresponding phenyl ether 5b (75−91% yields).
Synthesis of the Azetidine Derivatives 6(or 4) (General
Procedure). To a solution of 5 (or 3) (0.35 mmol) in methanol (15
mL) was added aqueous 1N HCl (5 mL). The reaction mixture was
stirred at 60 °C for 12 h. The solvent was removed, and the resulting
solid was washed with methylene chloride and ethyl acetate and then
dried in air to afford the corresponding 6 (or 4) (12−99% isolated
yields).
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