Identification of a Novel Oxadiazole Inhibitor of Mammalian Target of Rapamycin

Article abstract of DOI:10.1002/bkcs.11965

We performed a biochemical screen against mTOR using in-house small molecule library. Two novel, structurally distinct hits were identified. Among them, a novel oxadiazole scaffold compound (2) suppressed the phosphorylation of both S6K1 and Akt1 in HeLa cells. Docking study suggested that 2 is ATP-competitive and shows a pi-pi interaction with Trp2239 and hydrogen bonds with Trp2239 and Thr2245. Through derivatization, a slightly more potent analogue (2a) was identified with IC₅₀ of 9.6 μM. Our study provides a starting point for discovery of novel potent mTOR inhibitors.

Full text of DOI:10.1002/bkcs.11965

Article
DOI: 10.1002/bkcs.11965
BULLETIN OF THE
S. Lim et al.
KOREAN CHEMICAL SOCIETY
Identification of a Novel Oxadiazole Inhibitor of Mammalian Target of
Rapamycin
Sunwoo Lim,†,‡,¶ Hyomin Lee,‡,§,¶ Euijung Kim,†,‡ and Wooyoung Hur‡,§,
*
†Department of Chemistry, Korea University, Seoul 02841, South Korea
‡Chemical Kinomics Research Center, Korea Institute of Science and Technology (KIST), 5 Hwarangro-
14-gil, Seoul 02792, South Korea
§University of Science and Technology (UST), Daejeon 34113, South Korea. *E-mail: whur@kist.re.kr
¶These authors contributed equally to this work.
Received November 6, 2019, Accepted December 19, 2019
We performed a biochemical screen against mTOR using in-house small molecule library. Two novel,
structurally distinct hits were identified. Among them, a novel oxadiazole scaffold compound (2)
suppressed the phosphorylation of both S6K1 and Akt1 in HeLa cells. Docking study suggested that 2 is
ATP-competitive and shows a pi-pi interaction with Trp2239 and hydrogen bonds with Trp2239 and
Thr2245. Through derivatization, a slightly more potent analogue (2a) was identified with IC50 of
9.6 μM. Our study provides a starting point for discovery of novel potent mTOR inhibitors.
Keywords: Mammalian target of rapamycin, mTOR, Inhibitor, Screening, 2,3-Dihydro-1,3,4-oxadiazole
Introduction
uncovered. mTORC2 is inhibited by a long-term treatment of
rapamycin, but the inhibition is cell-type dependent.10
Mammalian target of rapamycin (mTOR) is a serine/threo-
nine kinase that is convergence point for regulation of a wide
array of processes including energy homeostasis and cell
growth.1,2 mTOR exists in two distinct, but evolutionarily
conserved mTOR complexes: mTORC1 (mTOR Complex
1) and mTORC2 (mTOR Complex 2). Both complexes share
multiple components including mTOR, mLST8, Deptor,
Tti1, and Tel2.3–5 In addition, mTORC1 contains Raptor and
PRAS40, while mTORC2 has Rictor, Protor, and mSin1 as
specific constituents. Each complex is differentially regu-
lated, differentially sensitive to rapamycin, and has distinct
substrate specificity.
mTORC1 integrates signals from growth factors, cellular
stresses, and nutrients such as amino acids, and induces
protein translation through phosphorylation of S6K1
(Thr389) and 4EBP1, as well as the lipid synthesis by
upregulating SREBP1 and PPARγ.6 Also, mTORC1 sup-
presses autophagy through phosphorylation and inhibition
of the ULK1.7 The natural product mTORC1 inhibitor
Rapamycin forms a complex with FK506-binding protein
12 (FKBP12), which binds to the FRB domain of mTOR
and disrupts the mTORC1 complex formation.
mTOR is an important therapeutic target. Aberrantly
activated mTOR is implicated in various diseases includ-
ing cancers, diabetes, and neurodegenerative disorders.1,11,12
Activation of mTOR pathway is also a major compensatory
mechanism that causes resistance to targeted cancer therapy.13
Rapamycin analogues such as Temsirolimus and Everolimus
were FDA approved as anti-cancer drugs. However, their effi-
cacy is limited by the negative feedback mechanism induced
by the selective mTORC1 inhibition.14 Targeting the ATP
pocket of mTOR is a therapeutically more effective approach,
because it ablates the functions of the both mTOR complexes.
Numerous ATP-competitive mTOR inhibitors have been iden-
tified (Figure 1), many of which are in clinical evaluation for
cancers.15 Herein, we wish to report a novel scaffold mTOR
inhibitor identified from a small molecule screen.
Results and Discussion
A high-throughput screening against recombinant human
mTOR (amino acid 1362-end) was performed using ca. 2000
in-house compound library at a concentration of 30 μM.
Time-resolved FRET (fluorescence resonance energy transfer)
assay16 was employed for the enzyme-based screening, which
measured FRET signal between the terbium-labeled phospho-
specific 4EBP1 antibody and the GFP-labeled 4EBP1 that is
phosphorylated during mTOR kinase reaction. We excluded
known mTOR inhibitors and reactive compounds, then identi-
fied two hits (1, 2) (Figure 2). Compound 1 and 2 inhibited
human mTOR in vitro with IC50’s of 54 and 13 μM, respec-
tively (Tables 1 and 2).
Upon growth factor stimulation, mTORC2 is recruited to
PIP3 in the plasma membrane through the PH domain of
mSin1, and directly phosphorylates Ser473 of Akt1, promot-
ing cell proliferation and survival.2 The mTORC2-Akt path-
way is also responsible for the chaperone-mediated autophagy
in lysosome.8 Similarly, mTORC2 also phosphorylates the
hydrophobic motif (Ser422) of another SGC family kinase
SGK1,9 but its physiological functions are still being
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H
(a)
N
O
N
N
N
O
O
O
O
O
OH
HO
OH
1
2
Figure 2 Structures of two hit compounds identified from an
enzymatic mTOR inhibitor screen.
p-dihydroxy (1d, 1e) phenyl groups decreased the inhibi-
tory activity (Table 1), indicating that the predicted hydro-
gen bond between p-OH group and Cys2243 does not
exist, and instead the m-OH group may be involved in the
binding. Also, the phenoxy group of 1 was predicted to be
positioned as the aminopyridine ring of Torin2. Thus we
introduced p-NH2 group at the phenoxy group, expecting a
potential hydrogen bond with the side chain of Asp2195 as
shown in Torin2-mTOR interaction. However, this attempt
(1b, 1d–f) also failed to improve the inhibitory activity,
suggesting that our predicted binding model was incorrect.
To investigate the structure–activity relationship (SAR)
of 2, we first varied the p-tolyl moiety. We found that o-
tolyl analogue (2a) showed a slightly better inhibition
(IC50 = 9.6 μM), while m-chlorophenyl (2b) and
(b)
Table 1. in vitro mTOR inhibition by analogues of 1.
Compound
Ar
R
H
IC50 (μM)
1
54
Figure 1 Structures of representative mTOR inhibitors. Structures
of (a) rapalogues, (b) ATP-competitive inhibitors.
1a
1b
H
>100
>100
To evaluate the ability of 1 and 2 to inhibit mTOR in
cells, each compound was treated to HeLa cells at 10 and
100 μM for 1 h, and the phosphorylation levels of S6K1
(Thr389) and Akt1 (Ser473) were examined (Figure 3).
Compound 1 enhanced the phosphorylation of both S6K1
and Akt1 in cells. The unexpected upregulation of mTOR
downstream signaling may be due to its off-target effects. On
the other hand, compound 2 suppressed both p-S6K1 and p-
Akt1, indicating its inhibition of mTOR activity in cells.
We then prepared analogues of the two hit compounds
and measured their IC50’s. Analogues of 1 and 2 were syn-
thesized following Schemes 1,2 and 3, respectively.
NH2
1c
1d
1e
H
>100
>100
>100
NH2
NH2
We predicted that two hydrogen bonds between the
pyrazole moiety of 1 and G2238, V2240 at the hinge back-
bone and a hydrogen bond between p-OH group at the
trihydroxyphenyl moiety and Cys2243. Substitution of the
tri-hydroxyphenyl group in 1 with p-hydroxy (1a-c) or o,
1f
NH2
>100
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Table 2. in vitro mTOR inhibition by analogues of 2.
Compound
Ar1
Ar2
IC50 (μM)
2
13
Figure 3 Western blot analysis for phospho-levels of S6K1
(Thr389) and Akt1 (Ser473) in HeLa cells following treatment of
each compound at 10, 100 μM for 1 h.
2a
2b
2c
9.6
31
positioned for π–π interaction with the side chain of
Trp2239 within the hinge region, and an oxygen atom of
the benzodioxole forms a hydrogen bond with the amide
backbone NH of Val2240. The carbonyl oxygen of 2 is
located for a hydrogen bond with the side chain of
Thr2245.
The toluene group is positioned for hydrophobic interac-
tion with the side chain of Trp2239. This prediction model
was consistent with the SAR result described in Table 2.
For example, the bulky phenyl group attached to benzo[d]
imidazole moiety of 2e created a steric clash within the
hinge region. The oxygen in the coumarin group of 2j and
2k did not fit for the hydrogen bond with the hinge region.
Thus the benzodioxole moiety may not provide enough
rooms for derivatization, and instead the o-tolyl moiety of
2a is a better modification spot to enhance activity. The
ortho-methyl of the toluene moiety could be elaborated and
optimized for additional hydrophobic group that allows for
new interactions with the hydrophobic surface area com-
prised of F2182, V2183, F2184, and L2185.
>100
2d
2e
>100
>100
2f
>100
>100
>100
>100
2g
2h
2i
Conclusion
2j
>100
We identified two structurally distinct mTOR inhibitors
from in-house small molecule library screen. Among the
two hits, a novel oxadiazole scaffold compound 2 was able
to suppress the phosphorylations of mTORC1/2 substrates
(S6K1, Akt1) in cells. Docking study suggested that 2 binds
in the ATP-binding pocket of mTOR. Through derivatiza-
tion of 2, we identified more potent analogue (2a) that
inhibits mTOR with IC50 of 9.6 μM. Our results could be
elaborated to a discovery of new potent mTOR inhibitors.
2,5-dimethoxy phenyl derivatives showed
a
lower
(IC50 = 31 μM) and no inhibition, respectively (Table 2).
In addition, replacing the benzodioxole moiety with phe-
nyl group (2d) removed the inhibitory activity, suggesting
that the dioxolane ring in the benzodioxole moiety makes
an important contact with mTOR. We also introduced
para-chlorophenyl (2f-2h) or coumarin (2i, 2j) at Ar1 and
derivatized Ar2, but all of them showed IC50 > 100 μM.
We performed a docking study for 2 against the mTOR
active site based on the reported X-ray crystal structure
(Figure 4).17 The benzodioxole moiety is predicted to be
Experimental
General Information for Synthesis. Unless otherwise
described, all commercial reagents and solvents were pur-
chased from commercial suppliers and used without further
purification. All reactions were performed under N2 atmo-
sphere in flame-dried glassware. Structures of synthesized
1
compounds were confirmed by the H NMR spectra using
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Scheme 1 Synthesis of derivatives of compound 1.
a Bruker 400 MHz FT-NMR (Billerica, MA, USA). The
reaction progress was checked on precoated TLC Silica
gel 60 F254 glass plates from Merck (Darmstadt, Ger-
many) under UV light (254 nm). Silica gel (Kieselgel
60 Art. 9385, 230–400 mesh) was used for column
chromatography.
(32 mL). The mixture was stirred and refluxed for 20 h,
then cooled down to room temperature. The reaction solu-
tion was stirred at room temperature for 1 h until yellow
Representative Synthesis (Compound 1d) for Analogues
of 1
1-(2,4-Dimethoxyphenyl)-2-(4-nitrophenoxy)ethan-1-one
(3). 2-Bromo-1-(2,4-dimethoxyphenyl)ethan-1-one (5 g)
was added to
a mixture of 4-nitrophenol (3.34 g,
24.0 mmol) and K2CO3 (6.03 g, 43.7 mmol) dissolved in
DMF (44 mL), and the solution was stirred at room temper-
ature for 4 h. The mixture was transferred into a flask filled
with ice water (30 mL) and stirred at room temperature for
30 min. The solids were filtered to afford the corresponding
1
product (87–94% yield). H NMR (400 MHz, DMSO‑d6)
δ 8.22–8.11 (m, 2H), 7.79 (d, J = 8.8 Hz, 1H), 7.13–7.01
(m, 2H), 6.73 (d, J = 2.3 Hz, 1H), 6.69 (dd, J = 8.8,
2.3 Hz, 1H), 5.45 (s, 2H), 3.97 (s, 3H), 3.88 (s, 3H).
HRMS (ESI) m/z calculated for C16H16NO6 [M + H]+:
+
318.0978. Found: 318.0966.
1-(2,4-Dimethoxyphenyl)-3-(dimethylamino)-2-(4-
nitrophenoxy)prop-2-en-1-one
(4).
N,N-dimethylfor-
mamide dimethyl acetal (8.88 mL, 66.9 mmol) was added
to a solution of 3 (5.40 g, 22.3) dissolved in toluene
Scheme 2 Synthesis of derivatives of compound 2.
Scheme 3 Synthesis of 2e.
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atmosphere (1 atm, balloon) at room temperature for 4 h.
The reaction mixture was poured through Celite cake filter,
washed with methanol, and the filtrate was concentrated in
vacuo. The mixture was then washed with CH2Cl2: MeOH
(9:1 v/v) to afford corresponding amino compound, 1d
1
(20–78% yield). H NMR (400 MHz, acetone‑d6) δ 12.09
(s, 1H), 11.01 (s, 1H), 8.39 (d, J = 2.2 Hz, 1H), 7.84 (d,
J = 8.5 Hz, 1H), 7.58 (s, 1H), 6.96–6.72 (m, 2H),
6.71–6.59 (m, 2H), 6.40 (d, J = 2.5 Hz, 1H), 6.33 (dd,
J = 8.6, 2.5 Hz, 1H), 4.44 (s, 2H). HRMS (ESI) m/z calcu-
Figure 4. Docking result for 2 (yellow) based on mTOR crystal
structure (PDB id: 4jsx). Three interacting residues in the ATP
binding pocket are labeled and two hydrogen bonds are noted in
yellow dotted lines.
lated for C15H14N3O3 [M + H]+: 284.1035. Found:
+
284.1026.
Representative Synthesis (Compound 2) for Analogues
of 2 (2, 2a–d, 2f–j)
Ethyl 2-(benzo[d][1,3]dioxol-5-yloxy)acetate (6). To a
solution of benzo[d][1,3]dioxol-5-ol (1.00 g, 7.2 mmol) in
DMF (14 mL) was added K2CO3 (2.00 g, 14.48 mmol),
and was stirred at room temperature for 10 min. Ethyl
chloroacetate (0.81 mL, 7.602 mmol) was added, and the
reaction mixture was stirred for 6 h at 60 ꢀC. The mixture
was cooled down to room temperature and quenched with
water, and the solution was extracted twice with CH2Cl2.
The organic layer was dried over anhydrous Na2SO4, fil-
tered, and concentrated in vacuo. The crude compound was
purified by flash chromatography (n-hexane:EtOAc 3:1 v/v)
to afford pale yellow solid (71–97% yield). 1H NMR
(400 MHz, chloroform‑d) δ 6.69 (d, J = 8.5 Hz, 1H), 6.53
(d, J = 2.6 Hz, 1H), 6.31 (dd, J = 8.5, 2.6 Hz, 1H), 5.92 (s,
2H), 4.54 (s, 2H), 4.26 (q, J = 7.2 Hz, 2H), 1.29 (t,
J = 7.1 Hz, 3H). HRMS (ESI) m/z calculated for
C11H12O5Na+ [M + Na]+:247.0582. Found: 247.0562.
solids were formed, and was filtered and washed with n-
hexane to afford the desired product as a yellow powder
1
(81–96% yield). H NMR (400 MHz, Choroform-d) δ 8.19
(d, J = 9.1 Hz, 2H), 7.19 (d, J = 8.1 Hz, 1H), 7.14 (d,
J = 8.9 Hz, 2H), 6.81 (s, 1H), 6.59–6.39 (m, 2H), 3.83 (s,
6H), 3.00 (s, 6H). HRMS (ESI) m/z calculated for
C19H21N2O6+ [M + H]+: 373.1400. Found: 373.1390.
3-(2,4-Dimethoxyphenyl)-4-(4-Nitrophenoxy)-1H-Pyrazole
(5). Hydrazine monohydrate (8.64 mL, 178.2 mmol) was
added to a stirring solution of 4 (6.10 g, 17.8 mmol) in eth-
anol (36 mL), and stirred at 65ꢀC for 4 h. The reaction mix-
ture was cooled down to room temperature, and extracted
with isopropanol: chloroform (1:4) and water. The organic
layer was washed with brine, dried over anhydrous MgSO4,
and concentrated under reduced pressure. The crude mate-
rial was then purified by flash chromatography (CH2Cl2:
MeOH 24:1 v/v) to afford the desired product (14–45%
yield). 1H NMR (400 MHz, chloroform‑d) δ 8.20 (d,
J = 8.8 Hz, 2H), 7.67 (d, J = 8.7 Hz, 1H), 7.60 (s, 1H),
7.10 (d, J = 8.9 Hz, 2H), 6.55 (s, 1H), 6.49 (d, J = 8.9 Hz,
1H), 3.96 (s, 3H), 3.81 (s, 3H). HRMS (ESI) m/z calculated
for C17H16N3O5+ [M + H]+: 342.1090. Found: 342.1079.
4-(4-(4-Nitrophenoxy)-1H-pyrazol-3-yl)benzene-1,3-diol.
A mixture of 5 (770 mg, 2.3 mmol) and anhydrous CH2Cl2
was cooled down to −78 ꢀC, and 1.0 M boron tribromide
in CH2Cl2 (11.3 mL, 11.3 mmol) was added dropwise. The
mixture was stirred at room temperature for 3 h, then
quenched with ice-cold water and saturated aqueous
NaHCO3. The reaction mixture was extracted with iso-
propanol: chloroform (1:4), and the organic layer was
washed with brine, dried over anhydrous MgSO4, and was
purified by flash chromatography (CH2Cl2:THF 6:1 v/v) to
2-(Benzo[d][1,3]dioxol-5-yloxy)acetohydrazide (7). To a
solution of 6 (1.58 g, 7.0 mmol) in EtOH (18 mL) was
added hydrazine monohydrate (3.45 mL, 70.5 mmol). The
reaction mixture was stirred for 12 h at 60 ꢀC, cooled down
to room temperature, then EtOH was removed under
reduced pressure. The crude compound was washed with n-
hexane to obtain the desired product as white powder
(86–99% yield). 1H NMR (400 MHz, DMSO‑d6) δ 9.28 (s,
1H), 6.80 (d, J = 8.5 Hz, 1H), 6.66 (d, J = 2.5 Hz, 1H),
6.38 (dd, J = 8.5, 2.5 Hz, 1H), 5.96 (s, 2H), 4.39 (s, 2H),
4.31 (s, 2H). HRMS (ESI) m/z calculated for C9H10N2O4
Na + [M + Na]+: 233.0538. Found: 233.0517.
2-(Benzo[d][1,3]dioxol-5-yloxy)-N0-(4-methylbenzylidene)
acetohydrazide (8). To
0.71 mmol) in EtOH
a
mixture of
(4 mL)
7
was
(150 mg,
added
1
yield the corresponding product (11–56% yield). H NMR
4-methylbenzaldehyde (0.09 mL, 0.79 mmol) and few
drops of 37% aq. HCl solution. The reaction mixture was
stirred for 1 h at room temperature, then quenched with ice
water and saturated aqueous NaHCO3. The reaction mix-
ture was extracted with CH2Cl2, and the organic layer was
washed with brine, dried over anhydrous Na2SO4, and con-
centrated under reduced pressure. The resulting solid was
washed with Et2O and CH2Cl2 (9:1 v/v) to afford white
(400 MHz, DMSO‑d6) δ 7.73 (d, J = 8.7 Hz, 1H), 6.68 (d,
J = 2.3 Hz, 1H), 6.65 (dd, J = 8.7, 2.3 Hz, 1H), 4.71 (s,
2H), 3.92 (s, 3H), 3.86 (s, 3H). HRMS (ESI) m/z calculated
for C17H18N3O3+ [M + H]+: 312.1348. Found: 312.1348.
4-(4-(4-Aminophenoxy)-1H-pyrazol-3-yl)benzene-1,3-diol
(1d). To a suspension of Pd/C (10% wt, Pd in charcoal) in
MeOH (7 mL) was added the nitro compound (400 mg,
1.3 mmol). The mixture was hydrogenated under H2
1
solid (68–100% yield). H NMR (400 MHz, DMSO‑d6) δ
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11.47 (s, 1H), 8.13 (d, J = 135.4 Hz, 1H), 7.58 (d,
J = 7.9 Hz, 2H), 7.25 (t, J = 7.1 Hz, 2H), 6.81 (dd,
J = 14.3, 8.5 Hz, 1H), 6.67 (dd, J = 26.2, 2.6 Hz, 1H), 6.39
(ddd, J = 28.3, 8.5, 2.6 Hz, 1H), 5.96 (d, J = 6.0 Hz, 2H),
4.80 (d, J = 188.3 Hz, 2H), 2.33 (s, 3H). HRMS (ESI) m/z
J = 7.4, 1.3 Hz, 1H), 7.10 (dd, J = 9.3, 3.0 Hz, 1H), 3.86
+
(s, 3H). HRMS (ESI) m/z calculated for C16H16NO6 [M
+ H]+: 245.0926. Found: 245.0908.
4-Methoxy-N1-Phenylbenzene-1,2-Diamine (10). To
a
suspension of Pd/C (0.58 g, 10% wt, Pd in charcoal) in
MeOH (40 mL) was added 9 (5.76 g, 23.58 mmol). The
mixture was hydrogenated under H2 atmosphere (1 atm,
balloon) at room temperature for 5 h. The reaction mixture
was filtered through Celite and washed with methanol, and
the filtrate was concentrated in vacuo, washed with n-
hexane and CH2Cl2 (9:1 v/v) to afford gray solid as the
corresponding amino compound (97% yield). 1H NMR
(400 MHz, Chloroform‑d) δ 7.19 (t, J = 7.0 Hz, 2H), 7.02
(d, J = 8.5 Hz, 1H), 6.78 (td, J = 7.3, 1.5 Hz, 1H), 6.64
(dd, J = 7.5, 1.2 Hz, 2H), 6.38 (d, J = 2.7 Hz, 1H), 6.33
(ddd, J = 8.5, 2.8, 1.6 Hz, 1H), 5.03 (s, 1H), 3.88 (s, 2H),
3.79 (d, J = 1.2 Hz, 3H). HRMS (ESI) m/z calculated for
C16H16NO6+ [M + H]+: 215.1184. Found: 215.1183.
calculated for C17H17N2O4 [M + H]+: 313.1188. Found:
+
313.1176.
(5-((Benzo[d][1,3]dioxol-5-yloxy)methyl)-2-methyl-
1,3,4-oxadiazol-3(2H)-yl)(p-tolyl)methanone (2). To a
mixture of 8 (70 mg, 0.22 mmol) in acetic anhydride
(2 mL) was added triethylamine (0.09 mL, 0.67 mmol), and
was stirred for 2 h at 120 ꢀC. The reaction mixture was
cooled down to room temperature, and mixed with satu-
rated aqueous NaHCO3. The solution was extracted with
CH2Cl2, and the organic layer was washed with brine, dried
over anhydrous Na2SO4. The crude compound was purified
by flash chromatography (n-hexane:EtOAc = 6:1 v/v) to
afford desired product 2 (20–25% yield). 1H NMR
(400 MHz, chloroform‑d) δ 8.56 (s, 1H), 7.67 (d,
J = 8.2 Hz, 2H), 7.27 (d, J = 7.3 Hz, 2H), 6.70 (d,
J = 8.5 Hz, 1H), 6.57 (d, J = 2.5 Hz, 1H), 6.36 (dd,
J = 8.5, 2.6 Hz, 1H), 5.92 (s, 2H), 5.09 (s, 2H), 2.53 (s,
3H), 2.42 (s, 3H). 13C NMR (100 MHz, Chloroform‑d) δ
171.77, 169.49, 165.11, 153.53, 148.33, 142.98, 142.37,
130.10, 129.86, 129.72, 129.69, 128.52, 107.93, 106.25,
101.28, 98.66, 69.95, 26.13, 21.69. HRMS (ESI) m/z calcu-
5-Methoxy-1-Phenyl-1H-Benzo[d]Imidazole (11). To a
solution of 10 (4.00 g, 18.67 mmol) in 2-methoxyethanol
(47 mL) was added formamidine acetate (2.92 g,
28.00 mmol). The solution was stirred and refluxed for 8 h
at 80ꢀC, cooled down to room temperature, then water was
added, and the reaction mixture was extracted twice with
CH2Cl2. The organic layer was dried over anhydrous
Na2SO4, filtered, and concentrated in vacuo. The crude
compound was purified by flash chromatography (n-hex-
ane:THF = 4:1 v/v) to afford red to orange oil product
lated for C19H19N2O5 [M + H]+: 355.1294. Found:
+
355.1290.
1
(96% yield). H NMR (400 MHz, Chloroform‑d) δ 8.50 (s,
1-(5-((Benzo[d][1,3]dioxol-5-yloxy)methyl)-2-(o-tolyl)-
1,3,4-oxadiazol-3(2H)-Yl)ethan-1-one (2a). 1H NMR
(400 MHz, DMSO‑d6) δ 11.48 (s, 1H), 8.25 (s, 1H), 7.74
(d, J = 7.0 Hz, 1H), 7.30–7.20 (m, 3H), 6.79 (d, J = 8.5 Hz,
1H), 6.63 (d, J = 2.5 Hz, 1H), 6.35 (dd, J = 8.5, 2.6 Hz,
1H), 5.95 (s, 2H), 5.03 (s, 2H), 2.42 (s, 3H). 13C NMR
1H), 7.71–7.57 (m, 4H), 7.52 (d, J = 8.9 Hz, 1H),
7.52–7.44 (m, 1H), 7.31 (d, J = 2.4 Hz, 1H), 6.95 (dd,
J = 8.9, 2.5 Hz, 1H), 3.82 (s, 3H). HRMS (ESI) m/z calcu-
lated for C16H16NO6 [M + H]+: 225.1028. Found:
+
225.1039.
1-Phenyl-1H-Benzo[d]Imidazol-5-Ol (12). A mixture of
11 (3.80 g, 2.3 mmol) and anhydrous CH2Cl2 (34 mL) was
cooled down to −78 ꢀC, and 1.0 M boron tribromide in
CH2Cl2 (33.89 mL, 33.89 mmol) was slowly added. The
mixture was stirred at room temperature for 4 h, then
quenched with ice water and saturated aqueous NaHCO3 at
(100 MHz, chloroform‑d)
δ 171.80, 169.45, 164.30,
153.51, 148.34, 142.40, 139.00, 131.88, 131.24, 127.60,
126.40, 107.93, 106.30, 101.30, 98.69, 70.10, 30.33, 26.18,
+
19.73. HRMS (ESI) m/z calculated for C19H19N2O5 [M
+ H]+: 355.1294. Found: 355.1293.
ꢀ
Synthesis of Compound 2e
0 C. The reaction mixture was extracted with CH2Cl2, and
4-Methoxy-2-nitro-N-phenylaniline (9). To a solution of
4-methoxy-2-nitroaniline (5.00 g, 29.74 mmol) in DMF
(59 mL) was added copper iodide (0.56 g, 2.97 mmol),
K2CO3 (12.33 g, 89.21 mmol), and iodobenzene (8.09 mL,
59.47 mmol). The reaction mixture was stirred for 24 h at
125 ꢀC, cooled down to room temperature, then copper was
removed by passing the reaction mixture through a pad of
Celite. The filtrate was extracted twice with CH2Cl2 and
water. The organic layer was dried over anhydrous Na2SO4,
filtered, and concentrated under reduced pressure. The
crude compound was purified by flash chromatography (n-
hexane:EtOAc = 93:7 v/v) to afford the desired product in
a form of red oil (81% yield). 1H NMR (400 MHz,
Chloroform‑d) δ 9.37 (s, 1H), 7.67 (d, J = 3.0 Hz, 1H),
7.42 (dd, J = 8.4, 7.3 Hz, 2H), 7.31–7.25 (m, 3H), 7.22 (tt,
the organic layer was washed with brine, dried over anhy-
drous MgSO4, and was purified by flash chromatography
(CH2Cl2:MeOH = 20:1 v/v), and the resulting solid was
washed once more with CH2Cl2 to yield the corresponding
product (67% yield). 1H NMR (400 MHz, DMSO‑d6) δ
10.21 (s, 1H), 9.67 (s, 1H), 7.85–7.75 (m, 2H), 7.76–7.66
(m, 2H), 7.65 (dd, J = 7.3, 1.5 Hz, 1H), 7.59 (d,
J = 9.0 Hz, 1H), 7.22 (d, J = 2.2 Hz, 1H), 7.08 (dd,
J = 9.0, 2.3 Hz, 1H). HRMS (ESI) m/z calculated for
C16H16NO6+ [M + H]+: 211.0871. Found: 211.0988.
Ethyl 2-((1-Phenyl-1H-Benzo[d]Imidazol-5-Yl)Oxy) Ace-
tate (13). To a solution of 12 (1.90 g, 9.04 mmol) in DMF
(23 mL) was added K2CO3 (2.50 g, 18.08 mmol), and was
stirred at room temperature for 10 min. Ethyl chloroacetate
(1.16 mL, 9.49 mmol) was added, and the reaction mixture
Bull. Korean Chem. Soc. 2020
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BULLETIN OF THE
Article
Novel mTOR Inhibitor
KOREAN CHEMICAL SOCIETY
was stirred for 6 h at 60 ꢀC. The mixture was cooled down
to room temperature and quenched with water, and the solu-
tion was extracted twice with CH2Cl2. The organic layer was
dried over anhydrous Na2SO4, filtered, and concentrated in
vacuo. The crude compound was purified by flash chroma-
tography (n-hexane:THF = 2:1 v/v) and light beige solid was
afforded as product (71% yield). 1H NMR (400 MHz,
Chloroform‑d) δ 8.07 (d, J = 2.7 Hz, 1H), 7.59–7.53 (m,
2H), 7.49 (ddd, J = 8.5, 3.7, 1.6 Hz, 2H), 7.44 (dt, J = 8.8,
2.5 Hz, 2H), 7.29 (d, J = 2.4 Hz, 1H), 7.07 (dq, J = 9.1,
2.3 Hz, 1H), 4.70 (s, 2H), 4.29 (qt, J = 7.2, 2.4 Hz, 2H),
1.31 (td, J = 7.2, 4.0 Hz, 3H). HRMS (ESI) m/z calculated
for C16H16NO6+ [M + H]+: 297.1239. Found: 297.1250.
J = 2.5 Hz, 1H), 7.05 (dd, J = 8.9, 2.5 Hz, 1H), 5.29 (s, 2H),
2.78 (s, 1H), 2.47 (s, 3H), 2.41 (s, 3H). HRMS (ESI) m/z cal-
culated for C16H16NO6 [M + H]+: 427.1770. Found:
+
427.1785.
Biological Materials. HeLa cells were maintained in
DMEM, 10% FBS (Sigma Aldrich, St.Louis, MO, USA)
supplemented with penicillin/streptomycin. Antibodies
against phospho-Akt1 (Ser473), Akt1, phospho-S6K1
(Thr389), and S6K1 were from Cell Signaling Technol-
ogy (Danvers, MA, USA). The recombinant human
mTOR (1362-end) protein was from Sigma Aldrich.
in vitro mTOR Kinase Assay. in vitro mTOR activity was
assayed using the Lanthascreen™ time-resolved FRET assay
(Thermo Fisher Scientific, Waltham, MA, USA). Recombinant
mTOR (3 nM) was incubated with serially diluted compounds
(100 nL) for 30 min in 5 μL of kinase buffer (25 mM HEPES,
pH 7.4, 8 mM MgCl2, 6 mM MnCl2, 4 mM DTT) in a
384-well low-volume white plate (Corning). Then an equal
volume of the kinase buffer was added containing 0.6 μM
GFP-4EBP1 and 20 μM ATP. After incubation at rt. for
90 min, 5 μL of stop solution containing 45 mM EDTA and
4.5 nM Tb-labeled phospho-4EBP1 (Thr46) antibody. After
30 min, the FRET signal (FL520/FL495 ratio) between Tb and
GFP was measured using Envision plate reader (PerkinElmer,
Waltham, MA, USA). Each assay was duplicated and IC50
values were calculated using Prism7 software (GraphPad).
Docking Study. Crystal structure of mTOR (PDB: 4JSX)
was retrieved from the Protein Data Bank. Molecular dock-
ing studies and molecular dynamics simulation studies of
the mTOR complex with compound 2 were performed
using Maestro and Pymol software.
2-((1-Phenyl-1H-benzo[d]imidazol-5-Yl)oxy)
acet-
ohydrazide (14). To a solution of 13 (1.35 g, 4.56 mmol)
in EtOH (13 mL) was added hydrazine monohydrate
(2.21 mL, 45.56 mmol). The reaction mixture was stirred
for 12 h at 60 ꢀC, cooled down to room temperature, then
EtOH was removed under reduced pressure. The crude
compound was washed with n-hexane to obtain the desired
1
product as white powder (86% yield). H NMR (400 MHz,
DMSO‑d6) δ 9.39 (s, 1H), 8.51 (s, 1H), 7.70–7.57 (m,
4H), 7.53 (d, J = 8.9 Hz, 1H), 7.49 (tt, J = 7.2, 1.5 Hz,
1H), 7.31 (d, J = 2.4 Hz, 1H), 7.04 (dd, J = 8.9, 2.4 Hz,
1H), 4.55 (s, 2H), 4.34 (s, 2H). HRMS (ESI) m/z calculated
for C16H16NO6+ [M + H]+: 283.1195. Found: 283.1206.
N0-(4-Methylbenzylidene)-2-((1-phenyl-1H-benzo[d]
imidazol-5-Yl)oxy)acetohydrazide (15). To a mixture of
14 (1.00 g, 4.76 mmol) in EtOH (16 mL) was added
4-tolualdehyde (0.62 mL, 5.23 mmol) and few drops of acetic
acid. The reaction mixture was stirred for 1 h at room temper-
ature, then quenched with ice water and saturated aqueous
NaHCO3. The reaction mixture was extracted with CH2Cl2,
and the organic layer was washed with brine, dried over anhy-
drous Na2SO4, and concentrated under reduced pressure. The
resulting solid was washed with Et2O and CH2Cl2 (5:1 v/v) to
afford the corresponding product as a white solid (68% yield).
1H NMR (400 MHz, DMSO‑d6) δ 11.55 (s, 1H), 8.51
(d, J = 8.1 Hz, 1H), 8.16 (d, J = 130.6 Hz, 1H), 7.75–7.51
(m, 7H), 7.51–7.46 (m, 1H), 7.39–7.20 (m, 3H), 7.05
(ddd, J = 30.3, 8.9, 2.5 Hz, 1H), 4.96 (d, J = 188.4 Hz, 2H),
Acknowledgments. This work was supported by Korea
Institute of Science and Technology (2E30240).
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© 2020 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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BULLETIN OF THE
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Novel mTOR Inhibitor
KOREAN CHEMICAL SOCIETY
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