J. Yun et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3234–3237
3235
hDAT, entry 8 against hNET, entries 14–16 against three mono-
amine transporters). In contrast, the substitution of trifluorometh-
oxy group at R2 is a disadvantage to the inhibitory activities against
three monoamine transporters (entries, 9–11, 21, 22). An interest-
ing result was that some of the compounds with 2,4-difluoro-
phenyl moiety at R1 showed less inhibitory activities against
hNET in comparison with those against hSERT and hDAT (entries
23, 24, and see example 28, 30–35 in the Supplementary material).
Based on the initial results of reuptake inhibitory activities, 7 com-
pounds were selected to determine their IC50 values against three
monoamine reuptake transporters and for further testing of the
microsomal stability against human liver microsomes (percent
remaining after 0.5 h incubation using BD Gentest assay kit).
Table 2 lists the IC50 values and the microsomal stability of the
selected 7 compounds (detailed results were reported in Supple-
mentary data). Comparing the IC50 values of the selected 3-phene-
thylazetidines (entries 1–7) with six reference compounds for
reuptake assay, the selected compounds showed better reuptake
inhibitory potency than fluoxetine, GBR12909, DOV 216,303
against all the three transporters. Two compounds (entries 5 and
6) showed higher potency than nisoxetine against NE reuptake
transporter. Among the 7 compounds, none of them displayed bet-
ter activity than venlafaxine or duloxetine against serotonin reup-
take transporter but all of them showed more than 7 times better
activities than venlafaxine or duloxetine against NE and DA reup-
take transporters. Some compounds (entries 3–5, 7) showed rela-
tive potency among the three transporters in the order of
hNET J hSERT J hDAT, while the other compounds (entries 1,
2, 6) displayed relative potency among the three transporters in
the order of hNET J hDAT J hSERT. In general the 4-chloro-3-
fluorophenyl derivatives (entry 5 and 6) exhibited higher potency
against all three monoamine transporter (hSERT, hNET and hDAT)
than 3,4-dichlorophneyl derivatives. Microsomal stability data can
be beneficial in optimizing in vivo pharmacokinetic performance.
Most of the compounds were stable compared to mibefradil except
compound 2aq in the microsomal stability test.
R2
O
O
homologation
HN
R1
HN
3-substituted azetidine
2
1
3-phenethylazetidine
Figure 1. Design of 3-phenethylazetidines by homologation.
uct 2 as corresponding HCl salt. This was treated with 1 N aqueous
sodium hydroxide solution followed by purification through flash
chromatography to obtain the corresponding 3-phenethylazeti-
dines 2 in 23–65% isolated yield. The structures of prepared com-
pounds were confirmed by 1H NMR and 13C NMR spectroscopies.
A total of 49 analogues of 3-phenethylazetidines 2 were synthe-
sized in this manner.
The reuptake inhibitory activities against DA, NE, and 5-HT neu-
rotransmitter 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).13 In
this study, the human embryonic kidney 293 (HEK293) cells stably
transfected with human dopamine transporter (HEK-hDAT), human
norepinephrine transporter (HEK-hNET), or human serotonin trans-
porter (HEK-hSERT) were used for the assay. All the synthesized
compounds were assayed at three different concentrations
(10 lM, 1.0 lM and 0.1 lM). Some compounds were selected to
obtain their IC50 values based on the initial screening result. The
primary screening results of all the compounds are reported in Sup-
plementary data. We used fluoxetine (SSRI), nisoxetine (selective
NE reuptake inhibitor), GBR12909 (selective DA reuptake inhibitor),
venlafaxine (SNRI), duloxetine (SNRI) and DOV 216,303 (TRI) as ref-
erence compounds.
Based on reuptake inhibition assay and microsomal stability
test, four compounds (2aq, 2as, 2at, 2au) were selected for further
studies of CYP inhibition and hERG inhibition. CYP and hERG inhi-
bition tests play important role in the preclinical studies because
they can reduce attrition rate in later stage of drug development
process. Similar to microsomal test, CYP and hERG inhibition
assays can reduce dropout rate of drug candidate due to poor phar-
macokinetic and toxicity performance in clinical studies. The four
selected compounds were screened using five different isozyme
of CYP (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and
hERG channel. In particular, the data of 1A2, 2D6 and 3A4 can be
considered significant in this study because CYP2D6 and CYP3A4
are responsible for metabolism of a number of drugs (more than
The primary screening results of 26 selected compounds at a
concentration of 0.1 lM are summarized in Table 1. Some com-
pounds with 3,4-dichlorophenyl or 4-chloro-3-fluorophenyl moi-
ety at R1 showed good inhibitory activities against three
monoamine transporters (entries 1, 2, 8, 12–20) while a 2-naph-
thyl group at R1 showed low inhibitory activities (entries 25–26).
In general, the compounds with 4-chloro-3-fluorophenyl moiety
at R1 showed better inhibitory activities against three monoamine
transporters than those of the compounds with 3,4-dichlorophenyl
moiety at the same position (compare entries 1–11 with entries
12–21). In case of the compounds in which fluorine is substituted
in phenyl at R2 showed a tendency good activities (entry 5 against
HO
path B
1) (a)
Boc
R1 MgX
(c)
Boc
Boc
CHO
N
N
CO2CH3
N
R1
(e)
2) (b)
5
4
3
MsO
Boc
path A
N
(d)
R1
6
R2
R1
R2
O
O
Boc
HN
N
R1
(f)
(g)
2
7
Scheme 1. Reagents and reaction conditions: (a) LiAlH4, THF, À78 °C; (b) (COCl)2, DMSO, then TEA, CH2Cl2, À78 °C to 0 °C; (c) PhMgBr, THF, 0 °C; (d) substituted phenol, PPh3,
DIAD, THF, rt; (e) MsCl, TEA, CH2Cl2, 0 °C; (f) substituted phenol, NaH, THF, reflux; (g) 1 N HCl, MeOH, 60 °C and then aq 1 N NaOH.
Yun, Jun
