Exploration of 3-aminoazetidines as triple reuptake inhibitors by bioisosteric modification of 3-α-Oxyazetidine

Article abstract of DOI:10.1021/ml500187a

For a development of broad spectrum antidepressant 3-aminoazetidine derivatives, two series of compounds were explored by bioisosteric modification of 3-α-oxyazetidine. We synthesized 166 novel 3-aminoazetidine derivatives in series A and B, starting from Boc-protected 3-azetidinone (3) and Boc-protected 3-azetidinal (9) respectively, through parallel syntheses. The inhibitory reuptake activities against serotonin (5-HT), norepinephrine (NE), and dopamine (DA) neurotransmitters were measured by the Neurotransmitter Transporter Uptake Assay Kit using the human embryonic kidney 293 (HEK293) cells stably transfected with the respective three kinds of human transporters (hSERT, hNET, and hDAT). Our study aimed to identify compounds having relative inhibitory activities against hSERT > hNET > hDAT. Lead optimization including microsomal stability, CYP, hERG assay, Ames test, BBB, and PK study resulted in the identification of compound 10dl as a candidate for further studies.

Full text of DOI:10.1021/ml500187a

Letter
pubs.acs.org/acsmedchemlett
Exploration of 3‑Aminoazetidines as Triple Reuptake Inhibitors by
Bioisosteric Modification of 3‑α-Oxyazetidine
Minsoo Han,†,‡ Chiman Song,† Nakcheol Jeong,‡ and Hoh-Gyu Hahn*,†
†Chemical Kinomics Research Center, Korea Institute of Science and Technology, Hwarangro 14-gil 5, Seoul 136-791, Korea
‡Department of Chemistry, Korea University, Anam-ro 145, Seoul 136-701, Korea
S
* Supporting Information
ABSTRACT: For a development of broad spectrum anti-
depressant 3-aminoazetidine derivatives, two series of
compounds were explored by bioisosteric modification of 3-
α-oxyazetidine. We synthesized 166 novel 3-aminoazetidine
derivatives in series A and B, starting from Boc-protected 3-
azetidinone (3) and Boc-protected 3-azetidinal (9) respec-
tively, through parallel syntheses. The inhibitory reuptake
activities against serotonin (5-HT), norepinephrine (NE), and
dopamine (DA) neurotransmitters were measured by the Neurotransmitter Transporter Uptake Assay Kit using the human
embryonic kidney 293 (HEK293) cells stably transfected with the respective three kinds of human transporters (hSERT, hNET,
and hDAT). Our study aimed to identify compounds having relative inhibitory activities against hSERT > hNET > hDAT. Lead
optimization including microsomal stability, CYP, hERG assay, Ames test, BBB, and PK study resulted in the identification of
compound 10dl as a candidate for further studies.
KEYWORDS: Depression, triple reuptake inhibitor, 3-aminoazetidines, bioisosterism
epression is one of the leading diseases worldwide.
improve cognitive performance.1,12 An important recent
DEpidemiological studies demonstrate that depressive development has been achieved with the discovery of triple
reuptake inhibitors (TRIs), broad spectrum antidepressants that
are capable of inhibiting the reuptake of 5-HT, NE, and DA by
one molecule.13,14 Therefore, TRIs working as a single molecule
are expected to become the next generation of antidepressants
and offer desirable therapeutic effects. Although many of the
discovered compounds such as TRI have balanced ratio of
potency for inhibition of reuptake of 5-HT, NE, and DA, recent
reports suggest that the potency for reuptake inhibition of 5-HT
and NE should be more important than those of DA for
antidepressant activity of TRI.15−17 Thus, our goal was to
identify the triple reuptake inhibitor having relative inhibitory
activities in the order 5-HT transporter (SERT) > NE
transporter (NET) > DA transporter (DAT).
In our previous paper, we reported the 3-substituted azetidine
1 with similar relative inhibitory activities against 5-HT, NE, and
DA transporters (SERT ≅ NET ≅ DAT), which showed
antidepressant effect in the forced swimming test in mice at 10
mg/kg iv or 20−40 mg/kg po.18 On the negative side, compound
1 is a racemate, which required separation by either additional
chiral chromatography or by independent asymmetric synthesis.
In fact, we had been frustrated by the difficult chiral resolution
and the enantioselective synthesis. As a part of our continuing
efforts to advance these compounds, we focused on the
bioisosterism with removal of stereogenic center of 1.
disorders are highly prevalent; it is estimated that, in general,
the lifetime prevalence of major depression was 20% in women
and 10% in men. The incidence of depression has risen every year
since the early 20th century. There are probably many reasons for
this rise, though most studies point to significant socioeconomic
changes and complicated circumstances experienced by the
younger generation. Pathophysiologically, the cause of depres-
sion is commonly associated with a deficiency of monoamine
neurotransmitters (serotonin (5-HT), norepinephrine (NE),
and dopamine (DA)) in the brain, and a number of
antidepressants aim to increase the levels of these neuro-
transmitters in the synapses.1−4 Since the launch of the first
selective serotonin reuptake inhibitors (SSRIs) in the 1980s,
second generation antidepressants such as dual 5-HT and NE
reuptake inhibitors (SNRIs) or NE and DA reuptake inhibitors
(NDRIs) with enhanced properties have been substituted to
tricyclic derivatives or monoamine oxidase inhibitors.5,6
However, a very large percentage of patients treated with
SSRIs or SNRIs show partial responses and remission rate
around 30%.7 Additionally, the associated side effects such as
insomnia, sexual dysfunction, and elevated blood pressure
hamper their efficacy.8,9 In modern therapies for depression,
one important 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. Recent study results support the
effect of DA in depression.10 NDRI bupropion enhanced the
antidepressant actions of SSRIs and SNRIs in humans,5,11 and
suppression of DA reuptake enhances sexual function and may
Received: May 9, 2014
Accepted: July 10, 2014
Published: July 10, 2014
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Bioisosterism is an approach and strategy for the rational
modification of lead compounds into safer and more clinically
effective agents.19,20 The replacement carbon atom of template 1
with a nitrogen atom leads to 3-aminoazetidine 2 as shown in
Figure 1. Additionally, considering the characteristic structural
azetidine 7. Deprotection of the Boc group in the azetidine by the
treatment of trifluoroacetic acid (TFA) in methylene chloride at
room temperature or 1 N HCl in boiling methanol gave crude
product 8 as the corresponding TFA or HCl salt, respectively.
This was treated with 1 N aqueous sodium hydroxide followed by
purification via flash chromatography or crystallization to obtain
the compounds 8 in yields ranging from 26 to 96%.
The structures of prepared compounds were confirmed by 1H
and 13C nuclear magnetic resonance (NMR) spectroscopies and
high-resolution mass spectrometry (HRMS), and the purity was
determined by high-performance liquid chromatography
(HPLC). Seventy four analogues of the 3-substituted amino-
azetidine derivatives 8 (series A) were synthesized in this
manner. Further, 92 derivatives of 3-aminoazetidine 10 (series
B) were prepared from 3-azetidinal 9 by a similar synthetic
method.
The reuptake inhibitory activities against 5-HT, NE, and DA
neurotransmitter were measured by the Neurotransmitter
Transporter Uptake Assay Kit (Molecular Devices, Sunnyvale,
CA, USA) with the FDSS6000 96-well fluorescence plate reader,
a high throughput screening device (Hamamatsu Photonics,
Hamamatsu, Japan).22 In this study, the human embryonic
kidney 293 (HEK293) cells stably transfected with human
dopamine transporter (hDAT), human norepinephrine trans-
porter (hNET), or human serotonin transporter (hSERT) were
used for the assay. All the synthesized compounds were screened
at three concentrations (10, 1.0, and 0.1 μM), and the selected
compounds were further screened to obtain their IC50.
Fluoxetine (SSRI), nisoxetine (NRI), GBR12909 (DRI),
venlafaxine (SNRI), and duloxetine (SNRI) were selected as
references. Primary screening results of selected 3-amino-
azetidines series A and B at a concentration of 0.1 μM are
summarized in Tables 1 and 2, respectively (see the Supporting
Information for complete screening results).
Figure 1. 3-Aminoazetidines: series A and B designed by bioisosterism
of 3-α-oxyazetidine 1.
common points of “Rule of 7” for designing new antidepres-
sants,21 we have designed the novel 3-aminoazetidine series A
and B.
A synthetic route to 3-aminoazetidines, series A and B, is
summarized in Scheme 1. Intermediates 4 were prepared by
a
Scheme 1
At a glance, the biological activities data of the compound of
series A in Table 1 revealed that the reuptake inhibitory activities
against hSERT were higher than those against hNET and hDAT.
Comparing the reuptake inhibitory activities against hSERT
where n = 1, the presence of a naphthyl or a 3,4-dichlorophenyl
moiety in R2 increased the activities significantly, while it is a
disadvantage when these groups are present at R1 except
compound 8ax (entry 10) (from comparison of entries 1, 2, 14−
17 with entries 8, 9, and 11 in Table 1). In the case that these
groups are present at both R1 and R2 (8au and 8ax), decreased
activity was shown. In addition, no elevated inhibitory activities
were found when there is methylene spacer present between the
tertiary nitrogen and R2 group (n = 2 or 3) (compare entries 3
with 4, 5 and 9 with 6, 7 in Table 1). The screening results of 74
compounds in series A are reported in the Supporting
Information. In general, the compounds of series B shown in
Table 2 demonstrated relatively higher reuptake inhibitory
activity against the three monoamine transporters than those of
series A shown in Table 1. Most of the compounds of series B
showed high reuptake inhibitory activities against hSERT and
hNET and moderate activity against hDAT (Table 2).
Interestingly, the bulkiness of R1 seems to be a disadvantage
for reuptake inhibitory activities. Thus, the compounds of series
B when R1 is a bulky group such as 3,4-dichlorophenyl (entries
3−5 in Table 2), naphthyl (entries 10 and 11), or 4-
phenoxyphenyl (entries 12−14) showed less activity than
when R1 is a relatively small substituent such as cyclopropyl
(entry 9), cyclopentyl (entries 8 and 15), cyclohexyl (entries 6, 7
and 16), or phenyl (entries 1 and 2). The screening results of 92
a
Reagents and reaction conditions: (a) NaBH(OAc)3, AcOH, CH2Cl2,
r.t.; (b) toluene, 110°C, and then NaBH4, MeOH. r.t.; (c) K2CO3,
DMF, 150°C; (d) NaBH(OAc)3, AcOH, CH2Cl2, r.t.; (e) TFA,
CH2Cl2, r.t., and then aq. 1 N NaOH or 1 N HCl, MeOH, 60°C.
reductive amination of commercially available 3-azetidinone 3
and primary amine in the presence of sodium triacetoxyborohy-
dride (NaBH(OAc)3) in methylene chloride solution at room
temperature. The reaction proceeded smoothly where the R1 is
phenyl, substituted phenyl, or aryl group in moderate to high
yields (49−95%). In contrast, in the case that R1 is an alkyl,
cycloalkyl, or benzyl group, the reaction required drastic
conditions. Thus, heating 3 with alkyl, cycloalkyl, or benzylamine
in toluene at reflux under a Dean−Stark water trap, followed by
the treatment of sodium borohydride in methanol at room
temperature gave 4 in good yield (74−86%). N-Alkylation of 4
was accomplished by the reaction using the appropriate building
blocks 5 or 6. For instance, the reaction of 4 with benzyl halide 5
in the presence of potassium carbonate in boiling dimethylfor-
mamide (DMF) gave the corresponding benzyl azetidine 7.
Otherwise, the treatment of 4 with aldehyde 6 in the presence of
NaBH(OAc)3 in methylene chloride gave the corresponding
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ACS Medicinal Chemistry Letters
Letter
Table 1. Percentage Inhibition of Activities of Selected 3-Aminoazetidine Derivatives 8 (Series A) at HEK-hSERT, HEK-hNET,
and HEK-hDAT
a
% reuptake inhibition at 0.1 μM
entry
compd
R1
R2
n
hSERT
hNET
hDAT
Fluoxetine
Nisoxetine
GBR12909
venlafaxine
8ad
44
10
2
5
6
78
−6
9
10
29
6
56
95
99
19
11
53
29
30
13
59
11
11
100
86
100
100
100
40
1
cyclohexyl
C6H5
C10H7
C10H7
C6H5
C6H5
C6H5
C6H5
C6H5
C10H7
C6H5
C10H7
1
1
1
2
3
2
3
1
1
1
1
1
1
1
1
1
1
22
18
30
16
12
17
16
13
100
10
22
12
57
1
5
b
2
8af
5
3
8ak
8al
C6H5
10
8
4
C6H5
5
8am
8aq
8ar
8au
8aw
8ax
8ay
8ba
8bl
C6H5
8
6
C6H3(3,4-di Cl)
C6H3(3,4-di Cl)
C10H7
5
7
13
2
8
9
C6H3(3,4-di Cl)
C6H3(3,4-di Cl)
C6H3(3,4-di Cl)
C6H4(4-CH3)
CH2C6H5
9
10
11
12
13
14
15
16
17
4
C6H4(4-Cl)
C10H7
4
7
C10H7
13
14
21
22
12
8bv
8bz
8cf
cyclopropyl
cyclopentyl
C6H4(2-F)
cyclopropyl
C10H7
C10H7
21
47
9
C10H7
8cu
C6H3(3,4-di Cl)
a
b
Percent values are the means obtained at least three or four times. HCl salt.
Table 2. Percentage Inhibition of Activities of Selectied 3-Aminoazetidine Derivatives 10 (Series B) at HEK-hSERT, HEK-hNET,
and HEK-hDAT
a
% reuptake inhibition at 0.1 μM
entry
compd
R1
R2
n
hSERT
hNET
hDAT
Fluoxetine
Nisoxetine
GBR12909
venlafaxine
10ab
44
10
2
5
6
78
−6
9
10
29
6
56
95
90
82
44
43
100
63
100
100
16
29
9
1
C6H5
C6H5
C10H7
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
50
73
54
7
4
2
10ac
C6H4(4-Cl)
C6H5
10
18
5
3
10ai
C6H3(3,4-di Cl)
C6H3(3,4-di Cl)
C6H3(3,4-di Cl)
cyclohexyl
cyclohexyl
cyclopentyl
cyclopropyl
C10H7
4
10aj
C10H7
5
10ak
C6H4(4-Cl)
C10H7
15
49
85
28
57
40
55
27
7
12
7
6
10ar
7
10as
C6H4(4-Cl)
C10H7
1
8
10ax
5
9
10bd
C10H7
25
17
24
5
b
10
11
12
13
14
15
16
17
18
19
20
10bf
C6H4(4-Cl)
C6H4(3-CH3)
C10H7
b
10bg
C10H7
b
10bh
C6H4(4-OPh)
C6H4(4-OPh)
C6H4(4-OPh)
cyclopentyl
cyclohexyl
C6H5
b
10bi
C6H4(4-Cl)
C6H5
6
4
b
10bj
14
93
76
100
100
100
100
5
11
30
16
22
22
36
22
10 cd
10ce
C6H3(3,4-di Cl)
C6H3(3,4-di Cl)
C6H4(4-F)
C6H4(3-CH3)
C6H3(2,3-di Cl)
80
62
93
100
100
97
b
10cl
b
10cn
C6H5
b
10cq
C6H5
c
10dl
isopropyl
C6H3(3,4-di Cl)
a
b
c
Percent values are the means obtained at least three or four times. TFA salt. HCl salt.
compounds in series B are reported in the Supporting
Information.
On the basis of the initial results of reuptake inhibitory
activities, 45 compounds were selected for further character-
ization of their IC50 against the three monoamine transporters
and the microsomal stability against human liver microsomes
(percentage remaining after 0.5 h using BD Gentest assay kit).
Table 3 represents the data of the 16 selected compounds, which
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ACS Medicinal Chemistry Letters
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Table 3. IC50 Values of Monoamine Reuptake Inhibitory Activities and Percentage Remaining of Microsomal Stability (M.S.) for
the Selected Compounds, Series A and B
a
reuptake assay (IC50, nM)
relative ratio of IC50
entry
compd
hSERT
hNET
hDAT
hNET/hSERT
hDAT/hSERT
M.S (% remaining) after 30 min
Fluoxetine
Nisoxetine
GBR12909
venlafaxine
duloxetine
mibefradil
8ab
150
4410
20.0
1460
5720
515
18400
1150
190
29.4
0.0
122
1.6
700
3840
36.1
10.4
0.4
0.0
15700
977
158
49.5
436
93.9
64
99
26
73
49
3
1
42.2
6.2
518
269
333
597
130
179
531
479
87.9
29.1
44.8
1.6
533
498
678
696
406
415
394
265
684
540
150
441
187
327
118
330
12.3
43.4
28.0
26.0
6.4
12.6
80.4
57.0
30.3
20.1
6.2
2
8ad
3
8af
11.9
23.0
20.2
67.4
99.9
114
8.4
4
8ba
5
8bk
6
8cg
2.7
66
64
100
23
9
7
8ch
5.3
3.9
8
8cu
4.2
2.3
9
10ab
10.5
2.0
81.4
37.0
5.2
10
11
12
13
14
15
16
10ac
14.6
28.8
5.2
10 cd
10ck
1.6
3
0.3
84.8
10.4
28.8
10.6
43.8
55
27
18
61
74
10cl
17.9
11.4
11.1
7.6
34.0
24.2
17.6
45.2
1.9
10cn
2.1
10cq
1.6
10dl
6.0
a
The values are the means obtained at least three or four times.
Table 4. IC50 Values of Human CYP Enzyme Activities and hERG Channel Inhibitory Activities for the Selected Compounds
CYP450 (IC50, μM)
entry
compd
1A2
2D6
2C9
3A4
2C19
hERG (IC50, μM)
hERG IC50 /hSERT IC50
a
positive control
duloxetine
8ab
32.6
5.3
25.1
1.58
0.13
0.01
0.02
13.7
2.1
4.2
2.5
17.9
4.1
29.1
6.89
3.13
0.46
3.34
12.9
0.36
0.44
32.7
0.44
0.81
1.33
3.64
0.5
1
2
3
4
5
6
7
8
205
12.7
9.39
13.9
19.3
52.7
0.57
10.6
1.75
0.27
0.07
7.06
8.08
5.4
8af
8cg
8ch
8cu
0.5
4.71
41
10ck
1.88
0.62
2.34
0.12
32.7
1.10
10cq
0.8
1.88
5.5
169
733
10dl
1.02
a
Positive control: α-naphthoflavone for 1A2, sulfaphenazole for 2C9, quinidine for 2D6, ketoconazole for 3A4, and miconazole for 2C19.
show good reuptake inhibitory potency against the three
monoamine transporters in comparison with the reference
standards (the data for the 45 compounds are reported in the
Supporting Information). From series A, on the basis of the data
of IC50 value, the relative ratio of IC50 (hNET/hSERT and
hDAT/hSERT), and the microsomal stability, five compounds
(8ab, 8af, 8cg, 8ch, and 8cu) were selected as candidates for the
next experiments. Additionally, the compounds of series B (entry
9−16 in Table 3) exhibited high potency against hSERT and
hNET compared with that against hDAT. Among the
compounds of series B, 10ck, 10cq, and 10dl showed most
potent activity against hSERT and hNET as well as good
microsomal stability.
isozymes of CYP and hERG channel inhibition, and the data are
summarized in Table 4. Compounds 8ab, 8af and 8cg are at a
disadvantage for use of CYP2D6, and compound 10ck showed
low IC50 for CYP3A4 to rule out further study. Then, three
compounds (8cu, 10cq, and 10dl) were screened against hERG
channel assay. As a result, we selected compound 10dl for
blood−brain barrier (BBB) and pharmacokinetics (PK) studies
for the following two reasons. First, the IC50 values of compound
10dl against CYP1A2, CYP2D6, and CYP3A4 were higher than
those of duloxetine.23 Second, compound 10dl showed the
highest safety margin (733-fold) of hERG IC50 to target IC50
(hERG IC50/hSERT IC50).24−26 Additionally, compound 10dl
did not have promutagenic or mutagenic effects on Salmonella
typhimurium strains TA98 and TA100 in the bacterial reverse
mutation assay (see Supporting Information).
The next step was to examine the selected compounds against
cytochrome P450 (CYP) and human ether-a go-go-related gene
(hERG) potassium channel inhibition. Having obtained the
reuptake inhibitory activities (IC50) and the microsomal stability
profile, eight compounds were selected and screened against five
Finally, the selected compound 10dl showed adequate brain-
to-plasma ratio (B/P = 2.09 at 3 h) in the BBB study and good
PK profile (Table 5).
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ACS Medicinal Chemistry Letters
Letter
Table 5. Pharmacokinetic Parameters of Compound 10dl
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compd 10dl
parameters
po (10 mg/kg, n = 5) iv (5.0 mg/kg, n = 5)
AUC0−∞ (μg·min/mL)
AUClast (μg·min/mL)
17.0
16.3
100.2
0.11
48
60.0
55.8
136.1
T
C
T
1/2 (min)
max (μg/mL)
max (min)
CL (mL/min/kg)
MRT (min)
168.0
123.7
27611
0.03
V
ss (mL/kg)
Ae (%)
0.02
28.4
F (%)
In summary, we have explored novel 3-aminoazetidine
derivatives series A and B by bioisosteric modification of 3-α-
oxyazetidine as a triple reuptake inhibitor for development of an
antidepressant. A focused library of 3-aminoazetidines composed
of 166 compounds was constructed through parallel syntheses.
The synthesized compounds were screened against three kinds
of human transporters (hSERT, hNET, and hDAT). Compound
10dl was selected, having relative inhibitory activities sequen-
tially against hSERT > hNET > hDAT through cell-based in vitro
assay, microsomal stability, CYP, hERG assay, Ames test, BBB,
and PK studies. Further studies are in progress and will be
reported soon.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures and biological screening method,
yields, melting points, 1H and 13C NMR data for all the
compounds, purity and HRMS data for the representative
compounds, and detailed results of biological assay. This material
is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(H.-G.H.) Tel: +82-2-958-5139. Fax: +82-2-958-5189. E-mail:
hghahn@kist.re.kr.
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Funding
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generation antidepressants. Prim. Psychiatry 2008, 15, 50−56.
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This work was supported by Korea Drug Development Fund and
Korea Institute of Science and Technology.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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We thank J. Sung for in vitro assay; and Dr. E. Lim, M. K. Ko, and
H. S. Jang for microsomal stability test, CYP assay, and PK study.
We also thank Prof. S. H. Cheon for helpful suggestions.
ABBREVIATIONS
■
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;
NDRI, norepinephrine dopamine reuptake inhibitor; TRI, triple
reuptake inhibitor; HEK, human embryonic kidney; hSERT,
human serotonin transporter; hNET, human norepinephrine
transporter; hDAT, human dopamine transporter; hERG,
human ether-a go-go-related gene; CYP, cytochrome P450;
BBB, blood−brain barrier; PK, pharmacokinetics
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