Discovery of novel leucyladenylate sulfamate surrogates as leucyl-tRNA synthetase (LRS)-targeted mammalian target of rapamycin complex 1 (mTORC1) inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.06.034

According to recent studies, leucyl-tRNA synthetase (LRS) acts as a leucine sensor and modulates the activation of the mammalian target of rapamycin complex 1 (mTORC1) activation. Because overactive mTORC1 is associated with several diseases, including co

Full text of DOI:10.1016/j.bmc.2018.06.034

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel leucyladenylate sulfamate surrogates as leucyl-tRNA
synthetase (LRS)-targeted mammalian target of rapamycin complex 1
(
mTORC1) inhibitors
a,1
a,1
b
b
a
a
Suyoung Yoon , Dongxu Zuo , Jong Hyun Kim , Ina Yoon , Jihyae Ann , Sung-Eun Kim ,
a
a
a
d
b,c
a,⁎
Dasol Cho , Won Kyung Kim , Sangkook Lee , Jiyoun Lee , Sunghoon Kim , Jeewoo Lee
Laboratory of Medicinal Chemistry, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea
Medicinal Bioconvergence Research Center, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea
a
b
c
Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul 08826,
Republic of Korea
d
Department of Global Medical Science, Sungshin University, Seoul 01133, Republic of Korea
A B S T R A C T
According to recent studies, leucyl-tRNA synthetase (LRS) acts as a leucine sensor and modulates the activation
of the mammalian target of rapamycin complex 1 (mTORC1) activation. Because overactive mTORC1 is asso-
ciated with several diseases, including colon cancer, LRS-targeted mTORC1 inhibitors represent a potential
option for anti-cancer therapy. In this work, we developed a series of simplified leucyladenylate sulfamate
analogues that contain the N-(3-chloro-4-fluorophenyl)quinazolin-4-amine moiety to replace the adenine group.
We identified several compounds with comparable activity to previously reported inhibitors and exhibited se-
lective mTORC1 inhibition and anti-cancer activity. This study further supports the hypothesis that LRS is a
promising target to modulate the mTORC1 pathway.
1. Introduction
activity, and thus are not sufficient to suppress mTORC1 activity in
many pathological conditions. Moreover, partial suppression of
Mammalian target of rapamycin (mTOR) is a serine/threonine
protein kinase that plays a crucial role in cell metabolism, growth,
proliferation, and autophagy. mTOR exists as two structurally and
mTORC1 often leads to rapamycin-resistance in cancer cells; therefore,
small molecules targeting other possible regulators of the mTORC1
pathway represent an alternative strategy to overcome rapamycin re-
sistance.
functionally different multi-protein complexes, mTOR complex
mTORC1) and mTOR complex 2 (mTORC2). mTORC1 regulates pro-
tein synthesis by phosphorylating two major substrates, S6 kinase 1
S6K1) and the translational regulators eukaryotic translation initiation
1
(
Leucine plays a major role in controlling the amino acid-dependent
activation of the mTORC1 signaling pathway.8 Although the exact
mechanism by which leucine activates mTORC1 pathway has not been
completely elucidated, several recent studies revealed that leucyl-tRNA
synthetase (LRS) acts as an intracellular leucine sensor by directly
,9
(
factor 4E (elF4E)-binding protein 1 (4E-BP1), whereas mTORC2 reg-
ulates cell survival and metabolism.1 Notably, overactive mTORC1 has
recently been shown to be associated with various human diseases, such
binding to RagD GTPase, one of the key mediators of the amino acid-
2–4
10,11
as diabetes, neurodegenerative diseases, and cancers.
dependent mTORC1 signaling pathway.
LRS is a member of the
Rapamycin and its analogs are well known allosteric inhibitors of
mTORC1 and have been widely studied for use as anti-cancer agents.
Rapamycin and its derivatives, also called rapalogs, inhibit downstream
phosphorylation by binding to the FK506-binding protein 12 (FKBP12)
and interacting with the FKBP12-rapamycin binding (FRB) domain in
class I aminoacyl-tRNA synthetase (ARSs) family that catalyzes the
ATP-dependent ligation of leucine to the cognate tRNA during protein
biosynthesis. Notably, LRS acts as a GTPase-activating protein (GAP)
for Rag GTPase to activate mTORC1;10 hence, inhibitors of LRS sup-
press the hydrolysis of RagD-GTP to RagD-GDP, blocking mTORC1
activity. Moreover, one leucine analogue, leucinol, blocks the amino
acid-mediated activation of mTORC1 by inhibiting the leucine-sensing
5
mTORC1. Although many rapalogs have been developed for anti-
6
,7
cancer therapy, these agents appear to only partially inhibit mTORC1
⁎
Corresponding author.
E-mail address: jeewoo@snu.ac.kr (J. Lee).
These two authors contributed equally to this work.
1
https://doi.org/10.1016/j.bmc.2018.06.034
Received 18 April 2018; Received in revised form 25 June 2018; Accepted 25 June 2018
0968-0896/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Yoon, S., Bioorganic & Medicinal Chemistry (2018), https://doi.org/10.1016/j.bmc.2018.06.034

S. Yoon et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
maintain specific binding to LRS and exhibit improved lipophilicity and
better synthetic accessibility.15
In our continuing efforts to develop LRS-targeted mTORC1 in-
hibitors as potential anti-cancer agents, we aim to expand the library of
our simplified leucyladenylate analogues by exploring a hybrid scaffold
combining the structures of 1 and gefitinib (2, Iressa) (Fig. 1). Gefitinib
is a selective epidermal growth factor receptor (EGFR) tyrosine kinase
1
6
Fig. 1. Structures of (S)-2-hydroxy-4-methylpentanoyl adenylate sulfamate (1)
and gefitinib (2).
inhibitor that binds to the ATP-binding site of the kinase domain. This
new series of simplified leucyladenylate analogues contain the N-(3-
chloro-4-fluorophenyl)quinazolin-4-amine moiety of gefitinib, which
represents the adenine-mimicking group, combined with leucine sur-
rogates through various linkers, replacing the 5-O-sulfamoylribose
group (Fig. 2).
Here, we describe the synthesis of newly designed compounds and
their inhibitory effects on the mTORC1 signaling pathway. In addition,
we evaluated leucylation activity of selected mTORC1 inhibitors to
investigate their mechanism of action and their cytotoxicities in various
types of cancer cells to assess their anti-cancer activity.
2. Results and discussion
2.1. Chemistry
The syntheses of the N-(3-chloro-4-fluorophenyl)quinazolin-4-
amine containing fragments (4, 5, 6 and 8) began with compound 3,
1
7
which was prepared using previously reported procedures (Scheme
). Compound 3 was reacted with sulfamoyl chloride prepared in a
quantitative yield from chlorosulfonyl isocyanate according to a pre-
1
1
8
viously reported procedure to synthesize intermediate 4. A hydro-
xyethyl linker was introduced under basic conditions to yield inter-
mediate 5, which was then subsequently sulfamoylated to generate
intermediate 6. O-alkylation of compound 3 with 1,2-dibromoethane
produced compound 7, which was then reacted with an aqueous am-
monia solution to produce a primary amino group.
Fig. 2. Newly designed simplified leucyladenylate analogues.
ability of LRS without affecting its catalytic function.12,13
As shown in our recent study, (S)-2-hydroxy-4-methylpentanoyl
adenylate sulfamate (1) is a leucyladenylate sulfamate surrogate that
selectively inhibits LRS-mediated mTORC1 activation without affecting
the catalytic activity of LRS. Compound 1 also exhibits specific anti-
tumor activity against colon cancer cells expressing hyperactive
mTORC1, suggesting that LRS-targeted mTORC1 inhibitors may re-
present a novel treatment option for human colorectal cancer.14 In
addition, simplified analogues of the leucyladenylate sulfamates
The compounds lacking the ethyl linker (11, 12, 15 and 16) were
synthesized using the method illustrated in Scheme 2. Compound 3 was
coupled with N-Cbz leucine and O-benzyl-protected (2S)-hydro-
xyisocaproic acid (HICA, l-leucic acid) to afford compounds 9 and 10,
respectively. Compound 4 was conjugated with N-Cbz leucine or O-
acetyl-protected HICA to produce compounds 13 and 14, respectively.
Scheme 1. Synthesis of the N-(3-chloro-4-fluorophenyl)quinazolin-4-amine-containing intermediates. Reagents & conditions: (a) sulfamoyl chloride, DMA, 0 °C to r.t.,
overnight; (b) 2-bromoethanol, K CO , DMF, r.t., 12 h; (c) 1,2-dibromoethane, K CO , DMF, r.t., 12 h; (d) NH OH, DMF, 50 °C, 24 h.
2
3
2
3
4
2

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Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Scheme 2. Synthesis of the hybrid analogues 11, 12, 15, and 16. Reagents & conditions: (a) cyanuric chloride, TEA, acetone, N-Cbz leucine for 9, O-benzyl-protected L-
leucic acid for 10, 0 °C to r.t., 2 h; (b) Pd/C, H , 2 M NH in MeOH, r.t., overnight for 11, BBr , CH Cl , -78 °C to r.t., 12 h for 12; (c) DCC, DMAP, CH Cl , N-Cbz
leucine for 13, O-acetyl-protected L-leucic acid for 14, r.t., 2 h; (d) Pd/C, H , 2 M NH in MeOH, r.t., overnight for 15, 0.02 M NaOMe, 0 °C to r.t., 2 h for 16.
2
3
3
2
2
2
2
2
3
Scheme 3. Synthesis of the hybrid analogues with an ethyl linker, 21–24, 27, and 28. Reagents & conditions: (a) cyanuric chloride, TEA, acetone, N-Cbz leucine for 17
and 19, O-acetyl-protected L-leucic acid for 18 and 20, 0 °C to r.t., 2 h; (b) Pd/C, H , 2 M NH in MeOH, r.t., overnight for 21 and 23, 0.02 M NaOMe, 0 °C to r.t., 2 h
for 22 and 24; (c) DCC, DMAP, CH Cl , N-Cbz leucine for 25, O-acetyl-protected L-leucic acid for 26, r.t., 2 h; (d) Pd/C, H , 2 M NH in MeOH, r.t., overnight for 27,
.02 M NaOMe, 0 °C to r.t., 2 h for 28.
2
3
2
2
2
3
0
The O-benzyl- or O-acetyl-protected HICA were prepared by benzyla-
tion or acetylation of commercially available l-leucic acid, respectively.
Deprotection of the α-amino or α-hydroxyl group of compounds 9, 10,
screened our compounds by employing the cell-based kinase assay used
in our previous studies.1
4,15,19
We pretreated HEK293 cells with each
final compound at one fixed concentration (200 μM) together with ra-
pamycin (100 nM) and leucinol (800 μM) for comparison, and then
activated mTORC1 by treating the cells with leucine for 10 min. As
shown in Fig. 3, the rapamycin and leucinol pretreatment blocked
leucine-induced S6K phosphorylation, as the phosphorylated S6K band
(pS6K) displayed a weak intensity. Among the hybrid analogues
without an ethyl spacer, compounds with a sulfonamide linker (15 and
1
3 and 14 yielded the final compounds 11, 12, 15 and 16, respectively.
Compounds with an ethyl linker (21–24, 27 and 28) were synthe-
sized from the N-(3-chloro-4-fluorophenyl)quinazolin-4-amine-con-
taining fragments using the methods shown in Scheme 3. Compounds
17–28 were synthesized using the same procedures described in
Scheme 2.
16) exhibited comparable activity to leucinol, whereas compounds with
an ester linker (11 and 12) showed no activity. In contrast, the hybrid
analogs with an ethyl spacer displayed an opposite trend, in which the
analogs with an ester or an amide linker (21–24) generally showed
more potent activity, whereas compounds with a sulfonamide linker
2
.2. Biological activity
We examined the effects of the synthesized compounds on the
mTORC1 pathway by determining the leucine-induced phosphorylation
of S6K, a major mTORC1 substrate, using Western blotting. We
(
27 and 28) had completely lost their activities, suggesting that the
3

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Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Fig. 5. Inhibition of catalytic leucylation by compounds 16, 21, 22 and 23.
phosphorylation in
a dose-dependent manner to similar extents,
whereas compound 23 only inhibited the phosphorylation when ap-
plied at 200 μM. Compounds 16, 21 and 22 strongly inhibited S6K
phosphorylation at 100 μM, showing a significant reduction in pS6K
band intensities, but compound 23 did not inhibit S6K phosphorylation
at the same concentration. Because compounds 16 and 22 appeared to
be the most potent compounds based on the band intensities, the hy-
droxyl group in the leucyl side chain may be crucial for activity. On the
other hand, the cellular expression levels of S6K, LRS, mTOR, and
pmTOR were not significantly altered by these compounds at any
concentrations.
Fig. 3. Inhibition of leucine-induced mTORC1 activation in HEK293 cells
treated with fixed concentrations of each compound.
distance between the leucyl side chain and the adenine binding site is
critical for mTORC1 inhibition. Additionally, for compounds with a
sulfonamide linker, compound 16, which has α-hydroxyl group,
showed more potent inhibition than its α-amino group-containing
counterpart (15); however, for compounds with an ethyl amino linker,
the compound with an α-amino group (23) showed more potent in-
hibition than the compound with an α-hydroxyl group (24), suggesting
that the binding interactions may be altered by the linker structures,
likely involving hydrogen-bond formation. When we examined the
expression levels of other related proteins, such as S6K, LRS, mTOR,
and pmTOR, their levels were consistent in cells treated with each of
the compounds, supporting our hypothesis that these compounds sup-
pressed the mTOR pathway by selectively interacting with LRS.
Next, we selected compounds 16, 21, 22, and 23, which appeared
to be more potent than leucinol, and determined the dose-dependent
inhibition of mTORC1 (Fig. 4). Compounds 16 and 22 inhibited S6K
We performed aminoleucylation assays with compounds 16, 21, 22
and 23 to further investigate the mechanism by which these hybrid
analogs inhibit the catalytic activity of LRS. As shown in Fig. 5, none of
the tested compounds exerted a significant effect on leucylation activity
when applied in the low micromolar range. In this case, > 90% of the
enzyme activity remained, except for the reaction with compound 21.
Based on this result, these compounds selectively acted on the LRS-
associated mTORC1 pathway in the same manner as leucine and leu-
cinol without significantly affecting the catalytic activity of LRS, sup-
porting our hypothesis that these simplified adenylate analogues se-
lectively bind to LRS and suppress LRS-mediated mTORC1 activation.
We next performed the sulforhodamine B (SRB) colorimetric assays
20
to assess the anticancer activity of compounds 16, 21, 22 and 23.
Compounds 16, 21, 22 and 23 were administered to six different types
Fig. 4. Dose-dependent inhibition of leucine-induced mTORC1 activation in HEK293 cells by compounds 16, 21, 22 and 23.
4

S. Yoon et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
of cancer cell lines in parallel with etoposide as a positive control. As
shown in Table 1, all compounds showed moderate cytotoxicity, dis-
playing IC50 values in the low micromolar range against various cancer
cell lines. Interestingly, when tested against the normal cell line, MRC5,
compounds 16 and 23 showed little or no cytotoxicity, whereas com-
pounds 21 and 22 exhibited a similar level of cytotoxicity as in cancer
cells. We postulate that the cytotoxicity of compounds 21 and 22 is
probably due to the inhibition of the catalytic activity of LRS. Overall,
these observations supported the hypothesis that the specific inhibition
of LRS-mediated activation of the mTOR pathway resulted in selective
cytotoxicity towards cancer cells.
4.2. Procedure
4.2.1. 4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl
sulfamate (4)
A solution of compound 3 (200 mg, 0.625 mmol) in dimethylace-
tamide (4 mL) was treated with freshly prepared sulfamoyl chloride
(108 mg, 0.938 mmol) at 0 °C and gradually warmed to room tem-
perature. After stirring for 2 h, the reaction mixture was diluted with
H
2
O and extracted with EtOAc several times. The combined organic
layers was washed with H O, dried over MgSO and concentrated in
vacuo. The residue was purified by silica gel column chromatography to
2
4
1
obtain compound 4 (187 mg) in 75% yield as a pale yellow solid.;
H
NMR (300 MHz, DMSO) δ 8.88 (s, 1H), 8.56 (s, 1H), 7.95 (dd, 1H,
3
. Conclusions
J = 6.78, 2.22 Hz), 7.63 (m, 1H), 7.54 (t, 1H, J = 8.97 Hz), 7.25 (s,
1
H), 3.99 (s, 3H); MS (ESI) m/z 399 [M + H]+
.
In summary, we developed a new series of simplified leucyladeny-
late sulfamate analogues that inhibited LRS-mediated mTORC1 acti-
vation. In this new series, we introduced the N-(3-chloro-4-fluor-
ophenyl)quinazolin-4-amine group to replace the adenine moiety and
incorporated various linker structures to study SARs. Compound 16
contains a leucylquinazoline sulfamate and showed a comparable in-
hibitory effect to leucinol, whereas compounds 21–23, which contain
an ethyl spacer with an ester or an amide linkage between the leucyl
side chain and the quinazoline group, also exerted potent inhibitory
effects. These compounds did not affect the levels of other related
proteins, such as LRS and mTOR, nor did they affect the catalytic ac-
tivity of LRS. Furthermore, these compounds showed general cyto-
toxicity towards various cancer cell lines, suggesting that these agents
may serve as a potential therapeutic agents for the treatment of cancer.
4
.2.2. 2-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)
oxy)ethan-1-ol (5)
A solution of compound 3 (200 mg, 0.625 mmol) in DMF (5 mL) was
treated with K
.88 mmol) and then stirred at room temperature for 12 h. The reaction
mixture was diluted with H O and extracted with EtOAc several times.
The combined organic layers was washed with H O, dried over MgSO
2 3
CO (260 mg, 1.88 mmol) and 2-bromoethanol (234 mg,
1
2
2
4
and concentrated in vacuo. The residue was purified by silica gel column
chromatography to obtain compound 5 (68 mg) in 30% yield as a white
solid.; 1H NMR (300 MHz, CD
OD) δ 8.42 (s, 1H), 8.04 (dd, 1H,
3
J = 6.60, 2.58 Hz), 7.74 (s, 1H), 7.69–7.63 (m, 1H), 7.25 (t, 1H,
J = 8.97 Hz), 7.18 (s, 1H), 4.48 (t, 1H, J = 6.06 Hz), 3.99 (s, 3H), 3.71
(
t, 1H, J = 6.03 Hz); MS (ESI) m/z 364 [M + H]+
.
4. Experimental section
4
.2.3. 2-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)
oxy)ethyl sulfamate (6)
By following the procedure for the synthesis of 4, compound 6 was
prepared in 75% yield as a white solid.; 1H NMR (300 MHz, CD
OD) δ
.36 (s, 1H), 7.91 (dd, 1H, J = 6.57, 2.55 Hz), 7.65 (s, 1H), 7.60–7.56
m, 1H), 7.16 (t, 1H, J = 8.97 Hz), 7.09 (s, 1H), 4.49–4.46 (m, 2H),
4
.1. General
All chemical reagents were commercially available. Melting points
3
were determined on a melting point Buchi B-540 apparatus and are
uncorrected. Silica gel column chromatography was performed on silica
gel 60, 230–400 mesh, Merck. H and C NMR spectra were recorded
on a JEOL JNM-LA 300 at 300 MHz, Bruker Analytik, DE/AVANCE
Digital 400 at 400 MHz and 100 MHz, Bruker Analytik, DE/AVANCE
Digital 500 at 500 MHz, JEOL JNM-ECA-700 at 175 MHz, respectively.
8
(
4
1
13
+
.38–4.35 (m, 2H), 3.91 (s, 3H); MS (ESI) m/z 443 [M + H]
.
4.2.4. 6-(2-Bromoethoxy)-N-(3-chloro-4-fluorophenyl)-7-
4
Chemical shifts are reported in ppm units with Me Si as a reference
methoxyquinazolin-4-amine (7)
A solution of compound 3 (200 mg, 0.625 mmol) in DMF (5 mL) was
treated with CO (260 mg, 1.88 mmol) and 1,2-dibromoethane
352 mg, 0.936 mmol) and then stirred at room temperature for 8 h.
The reaction mixture was diluted with H O and extracted with EtOAc
several times. The combined organic layers was washed with H O,
dried over MgSO and concentrated in vacuo. The residue was purified
by silica gel column chromatography to obtain compound 5 (102 mg) in
standard. Mass spectra were recorded on a VG Trio-2 GC–MS instru-
ment and a 6460 Triple Quad LC–MS instrument. All final compounds
were purified to > 95% purity, as determined by high-performance li-
quid chromatography (HPLC). HPLC was performed on an Agilent 1120
Compact LC (G4288A) instrument using an Agilent TC-C18 column
K
2
3
(
2
2
(
4.6 mm × 250 mm, 5 μm), λ = 250 nm, flow rate 1.0 mL/min, Mobile
4
phase (60:40) buffer/acetonitrile (buffer: 5 mol% ammoinium acetate
aqueous solution. According to the HPLC analyses, final compounds 16,
1
4
8
5% yield as a white solid.; H NMR (300 MHz, CD
3
OD) δ 8.44 (s, 1H),
2
1–23 showed a purity of ≥95%.
.00 (dd, 1H, J = 6.60, 2.58 Hz), 7.74 (s, 1H), 7.69–7.63 (m, 1H), 7.25
(
3
t, 1H, J = 8.97 Hz), 7.18 (s, 1H), 4.50 (t, 1H, J = 6.06 Hz), 4.00 (s,
Table 1
+
H), 3.81 (t, 1H, J = 6.03 Hz); MS (FAB) m/z 427 [M + H]
.
Relative inhibitory effects of compounds 16, 21, 22 and 23 on the growth of
various cancer cell lines.a
4
.2.5. 6-(2-Aminoethoxy)-N-(3-chloro-4-fluorophenyl)-7-
IC50 (μM) A549 HCT116 K562 MDA-MB-
31
SK-HEP-1 SNU638 MRC5
2
methoxyquinazolin-4-amine (8)
A solution of compound 7 (100 mg, 0.234 mmol) in DMF (3 mL) was
treated with an excess amount of NH OH at room temperature and
gradually heated to 50 °C. After stirring for 24 h, the reaction mixture
was cooled and concentrated under reduced pressure to afford com-
1
2
2
2
6
1
2
3
4.38
3.59
3.16
2.79
4.04
3.97
3.12
4.24
1.06
4.10
4.01
3.89
3.83
0.76
6.23
3.65
2.05
4.59
1.53
3.30
1.94
1.39
2.21
0.63
4.94
3.86
3.41
2.97
1.05
18.78
1.87
1.12
> 20
11.73
4
Etoposide 0.30
pound 8 (80 mg) in 94% yield as a yellow oil, which was directly used
for the next reaction.; H NMR (300 MHz, CD
1
3
OD) δ 8.41 (s, 1H), 8.14
a
A549, lung cancer cells; HCT116, colon cancer cells; K562, leukemia cells;
MDA-MB-231, breast cancer cells; SK-Hep-1, liver cancer cells; SNU638, sto-
mach cancer cells; MRC5, normal lung epithelial cells.
(
s, 1H), 8.01–7.99 (m, 1H), 7.71–7.65 (m, 1H), 7.24 (t, 1H,
J = 8.97 Hz), 7.16 (s, 1H), 4.35–4.24 (m, 4H), 3.99 (s, 3H). MS (ESI) m/
z 363 [M + H]+
.
5

S. Yoon et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
4.2.6. 4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl
(ESI) m/z 512 [M + H]+
.
((benzyloxy)carbonyl)-l-leucinate (9)
Compound 9 was prepared by following the previous procedure
4.2.13. 4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl
(S)-(2-hydroxy-4-methylpentanoyl)sulfamate (16)
reported in reference 15 in 32% yield as a white solid.; 1H NMR
(
(
1
300 MHz, CD
3
OD) δ 8.51 (s, 1H), 8.03 (s, 1H), 8.01 (m, 1H), 7.66–7.63
Compound 16 was prepared by following the previous procedure
m, 1H), 7.37–7.20 (m, 7H), 5.14 (s, 2H), 4.55 (m, 1H), 3.92 (s, 3H),
reported in reference 15 in 46% yield as
a
white solid,
mp = 215–217 °C; H NMR (400 MHz, DMSO) δ 9.60 (s, 1H), 9.46 (s,
H), 8.46 (s, 1H), 8.20 (dd, 1H, J = 6.78, 2.58 Hz), 7.85–7.79 (m, 1H),
.85 (m, 2H), 1.03 (t, 1H, J = 6.24 Hz); MS (ESI) m/z 567 [M + H]+
.
1
2
4
2
.2.7. 4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl (S)-
-(benzyloxy)-4-methylpentanoate (10)
Compound 10 was prepared by following the previous procedure
7.76 (s, 1H), 7.40 (t, 1H, J = 9.18 Hz), 7.20 (s, 1H), 3.96 (s, 3H), 3.15
(d, 1H, J = 4.38 Hz), 1.98 (m, 1H), 1.75–1.55 (m, 2H), 0.93 (m, 6H);
13
C NMR (100 MHz, DMSO) δ 169.75, 167.9, 157.3, 154.4, 148.8,
reported in reference 15 in 58% yield as a white solid.; 1H NMR
146.8, 146.4, 138.2, 121.5, 118.6, 118.3, 114.2, 112.7, 108.5, 103.0,
+
(
(
4
300 MHz, CD
m, 1H), 7.41–7.22 (m, 7H), 4.55 (d, 1H, J = 11.70 Hz), 4.33 (m, 1H),
.01 (s, 3H), 1.88 (m, 1H), 1.23 (t, 1H, J = 7.14 Hz), 0.99 (d, 3H,
3
OD) δ 8.54 (s, 1H), 8.17 (s, 1H), 8.02 (m, 1H), 7.70–7.65
68.2, 56.1, 43.4, 25.28, 24.41, 22.50; MS (FAB) m/z 513 [M + H]
HRMS (FAB) m/z calcd for C21 22ClFN
S [M + H]+ 513.0933,
found: 513.1021. Anal.; HPLC 97%.
;
H
4 6
O
+
J = 6.42 Hz), 0.91 (d, 3H, J = 6.39 Hz); MS (ESI) m/z 524 [M + H]
.
4.2.14. 2-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
4.2.8. 4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl
l-
yl)oxy)ethyl ((benzyloxy)carbonyl)-l-leucinate (17)
leucinate (11)
Compound 17 was prepared by following the previous procedure
reported in reference 15 in 37% yield as a white solid.; H NMR
1
Compound 11 was prepared by following the previous procedure
reported in reference 15 in 58% yield as a white solid.; 1H NMR
3
(300 MHz, CD OD) δ 8.45 (s, 1H), 8.01 (dd, 1H, J = 6.75, 2.73 Hz),
(
300 MHz, DMSO) δ 9.68 (s, 2H), 9.47 (s, 1H), 8.46 (s, 1H), 8.20 (dd,
H, J = 6.78, 2.58 Hz), 7.83 (m, 1H), 7.77 (s, 1H), 7.40 (t, 1H,
J = 8.97 Hz), 7.20 (s, 1H), 3.96 (s, 3H), 3.60 (m, 1H), 1.44 (m, 1H),
7.75 (s, 1H), 7.68 (m, 1H), 7.27–7.16 (m, 7H), 5.03 (s, 2H), 4.60–4.40
(m, 4H), 4.23 (t, 1H, J = 6.96 Hz), 3.97 (s, 3H), 1.68 (m, 1H),
1.59–1.54 (m, 2H), 0.88 (d, 3H, J = 6.39 Hz), 0.85 (d, 3H,
1
1
.22 (s, 2H), 0.89–0.82 (m, 6H); MS (ESI) m/z 433 [M + H]+
.
J = 6.39 Hz); MS (ESI) m/z 611 [M + H]+
.
4
2
.2.9. 4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl (S)-
-hydroxy-4-methylpentanoate (12)
4.2.15. 2-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)oxy)ethyl (S)-2-acetoxy-4-methylpentanoate (18)
A cooled solution of compound 10 (52.4 mg, 0.1 mmol) in CH
2 mL) at -78 °C was slowly treated with BBr (1.0 M, 0.15 mL,
.15 mmol) and gradually warmed to room temperature. After stirring
2
Cl
2
Compound 18 was prepared by following the previous procedure
reported in reference 15 in 43% yield as a white solid.; H NMR
1
(
0
3
3
(300 MHz, CD OD) δ 8.45 (s, 1H), 8.00 (dd, 1H, J = 6.78, 2.58 Hz),
for 30 min, the reaction mixture was cooled in ice-bath and then
quenched with MeOH. The reaction mixture was concentrated in vacuo
and the residue was purified by silica gel flash column chromatography
7.76 (s, 1H), 7.69–7.64 (m, 1H), 7.25 (t, 1H, J = 8.97 Hz), 7.18 (s, 1H),
5.02–4.95 (m, 2H), 4.64–4.50 (m, 2H), 4.42 (m, 1H), 4.00 (s, 3H), 2.06
(s, 3H), 1.79–1.57 (m, 3H), 0.96–0.86 (m, 6H); MS (ESI) m/z 535
1
[M + H]+
to afford compound 12 (26 mg) in 60% yield as a white solid.; H NMR
.
(
2
1
300 MHz, CD
3
OD) δ 8.44 (s, 1H), 8.02 (s, 1H), 7.92 (dd, 1H, J = 6.60,
.58 Hz), 7.57 (m, 1H), 7.20 (s, 1H), 7.16 (t, 1H, J = 8.97 Hz), 4.41 (m,
H), 3.88 (s, 3H), 1.91 (m, 1H), 1.73–1.68 (m, 2H), 0.94 (dd, 6H,
4.2.16. Benzyl
(S)-(1-((2-((4-((3-chloro-4-fluorophenyl)amino)-7-
methoxyquinazolin-6-yl)oxy)ethyl)amino)-4-methyl-1-oxopentan-2-yl)
carbamate (19)
+
J = 6.75, 2.73 Hz); MS (ESI) m/z 434 [M + H]
.
Compound 19 was prepared by following the previous procedure
1
4.2.10. 4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl
reported in reference 15 in 33% yield as a white solid.; H NMR
(
((benzyloxy)carbonyl)-l-leucyl)sulfamate (13)
Compound 13 was prepared by following the previous procedure
3
(300 MHz, CD OD) δ 8.46 (s, 1H), 8.05 (dd, 1H, J = 6.78, 2.58 Hz),
7.79 (s, 1H), 7.68 (m, 1H), 7.28–7.16 (m, 7H), 5.02 (s, 2H), 4.24 (m,
2H), 4.13 (m, 1H), 3.98 (s, 3H), 3.69 (m, 2H), 1.79–1.57 (m, 3H), 0.91
reported in reference 14 in 45% yield as a white solid.; 1H NMR
(
300 MHz, CD
3
OD) δ 8.45 (s, 1H), 8.19 (s, 1H), 7.95 (dd, 1H, J = 6.78,
.55 Hz), 7.20 (s, 1H), 7.17–7.13 (m, 6H), 4.90 (d, 2H, J = 4.02 Hz),
.15–4.08 (m, 1H), 3.98 (s, 3H), 1.72 (m, 1H), 1.56–1.49 (m, 2H), 0.99
(t, 6H, J = 6.39 Hz); MS (ESI) m/z 611 [M + H]+
.
2
4
(
4.2.17. (S)-1-((2-((4-((3-Chloro-4-fluorophenyl)amino)-7-
methoxyquinazolin-6-yl)oxy)ethyl)amino)-4-methyl-1-oxopentan-2-yl
acetate (20)
d, 6H, J = 6.57 Hz); MS (ESI) m/z 646 [M + H]+
.
4
.2.11. (S)-1-((((4-((3-Chloro-4-fluorophenyl)amino)-7-
Compound 20 was prepared by following the previous procedure
reported in reference 15 in 28% yield as a white solid.; H NMR
1
methoxyquinazolin-6-yl)oxy)sulfonyl)amino)-4-methyl-1-oxopentan-2-yl
acetate (14)
(300 MHz, CD OD) δ 8.36 (s, 1H), 7.96 (dd, 1H, J = 6.78, 2.58 Hz),
3
Compound 14 was prepared by following the previous procedure
7.71 (s, 1H), 7.61 (m, 1H), 7.16 (t, 1H, J = 8.97 Hz), 7.10 (s, 1H), 4.92
(m, 1H), 4.17 (m, 2H), 3.92 (s, 3H), 3.59 (m, 2H), 2.00 (s, 3H), 1.64 (m,
1H), 1.48 (m, 2H), 0.82 (t, 6H, J = 6.42 Hz); MS (ESI) m/z 519
reported in reference 14 in 49% yield as a white solid.; 1H NMR
(
300 MHz, CD
3
OD) δ 8.50 (s, 1H), 8.14 (s, 1H), 8.00 (dd, 1H, J = 6.78,
.76 Hz), 7.71–7.67 (m, 1H), 7.25 (t, 1H, J = 8.97 Hz), 7.24 (s, 1H),
.00 (s, 3H), 2.09 (s, 3H), 1.70–1.58 (m, 3H), 0.90 (dd, 6H, J = 6.60,
2
4
4
[M + H]+
.
+
.95 Hz); MS (ESI) m/z 555 [M + H]
.
4.2.18. 2-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)oxy)ethyl l-leucinate (21)
4.2.12. 4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl (l-
Compound 21 was prepared by following the previous procedure
leucyl)sulfamate (15)
reported in reference 15 in 54% yield as
a
white solid,
1
Compound 15 was prepared by following the previous procedure
mp = 199–202 °C; H NMR (400 MHz, CD OD) δ 8.44 (s, 1H), 8.00 (dd,
3
reported in reference 15 in 52% yield as a white solid.; 1H NMR
1H, J = 6.78, 2.58 Hz), 7.75 (s, 1H), 7.69–7.66 (m, 1H), 7.26 (t, 1H,
J = 6.42 Hz), 7.19 (s, 1H), 4.26 (m, 2H), 4.00 (s, 3H), 3.99 (m, 2H),
(
(
1
300 MHz, CD
t, 1H, J = 8.61 Hz), 4.04 (s, 3H), 3.98 (dd, 1H, J = 10.44, 3.12 Hz),
.85–1.75 (m, 1H), 1.70–1.46 (m, 2H), 0.93 (t, 6H, J = 6.57 Hz); MS
3
OD) δ 8.34 (s, 1H), 7.69–7.64 (m, 3H), 7.16 (s, 1H), 7.12
1
3
3.69 (m, 1H), 1.75–1.50 (m, 3H), 0.92 (t, 6H, J = 6.21 Hz);); C NMR
(100 MHz, DMSO) δ 169.75, 156.57, 156.00, 154.32, 153.91, 152.68,
6

S. Yoon et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
1
1
48.05, 147.01, 123.37, 122.23, 118.80, 116.56, 108.68, 107.37,
4.2.24. 2-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)oxy)ethyl (l-leucyl)sulfamate (27)
02.55, 66.70, 62.30, 53.81, 43.40, 38.54, 38.16, 25.28, 24.41, 22.50;
+
MS (FAB) m/z 477 [M + H]
26ClFN
5%.
;
HRMS (FAB) m/z calcd for
Compound 27 was prepared by following the previous procedure
[M + H]+ 477.1627, found: 477.1716. Anal.; HPLC
reported in reference 15 in 65% yield as a white solid.; H NMR
1
C
9
23
H
4
O
4
(300 MHz, CD
.89 (s, 1H), 7.73 (m, 1H), 7.22 (t, 1H J = 8.97 Hz), 7.14 (s, 1H), 4.48
m, 4H), 3.98 (s, 3H), 3.60 (m, 1H), 1.56–1.75 (m, 3H), 0.91 (m, 6H);
3
OD) δ 8.44 (s, 1H), 8.06 (dd, 1H, J = 6.77, 2.76 Hz),
7
(
4.2.19. 2-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
+
MS (ESI) m/z 556 [M + H]
.
yl)oxy)ethyl (S)-2-hydroxy-4-methylpentanoate (22)
Compound 22 was prepared by following the previous procedure
4
.2.25. 2-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
reported in reference 15 in 54% yield as
mp = 196–203 °C; H NMR (400 MHz, DMSO) δ 11.05 (s, 1H), 8.81 (s,
H), 8.20 (s, 1H), 8.03 (m, 1H), 7.73 (m, 1H), 7.65 (t, 1H, J = 8.79 Hz),
a
white solid,
1
yl)oxy)ethyl (S)-(2-hydroxy-4-methylpentanoyl)sulfamate (28)
Compound 28 was prepared by following the previous procedure
1
7
1
1
1
3
1
reported in reference 15 in 77% yield as a white solid.; H NMR
.31 (s, 1H), 4.24 (m, 2H), 4.00 (s, 3H), 3.84 (m, 2H), 2.18 (m, 1H),
1
3
(300 MHz, CD
.90 (s, 1H), 7.70 (m, 1H), 7.20 (t, 1H, J = 9.15 Hz), 7.12 (s, 1H), 4.80
m, 1H), 4.47 (m, 4H), 3.98 (s, 3H), 1.80 (m, 1H), 1.55–1.42 (m, 2H),
3
OD) δ 8.43 (s, 1H), 8.06 (dd, 1H, J = 6.78, 2.76 Hz),
.76 (m, 2H), 0.87 (m, 6H); C NMR (100 MHz, DMSO) δ 169.75,
56.57, 156.00, 154.32, 153.91, 152.68, 148.05, 147.01, 123.37,
22.23, 118.80, 116.56, 108.68, 107.37, 102.55, 67.90, 55.81, 43.40,
7
(
+
+
0.86 (dd, 6H, J = 6.60, 1.65 Hz); MS (ESI) m/z 557 [M + H] .
8.54, 38.16, 25.28, 24.41, 22.50; MS (FAB) m/z 478 [M + H] ; HRMS
+
(
4
FAB) m/z calcd for C23
H
25ClFN
3
O
5
[M + H] 478.1467, found:
Acknowledgments
78.1556. Anal.; HPLC 96%.
This research was supported by the Global Frontier Project grant
NRF-2012M3A6A4054928) of National Research Foundation funded
by the Ministry of Education, Science and Technology of South Korea.
4
.2.20. (S)-2-Amino-N-(2-((4-((3-chloro-4-fluorophenyl)amino)-7-
(
methoxyquinazolin-6-yl)oxy)ethyl)-4-methylpentanamide (23)
Compound 23 was prepared by following the previous procedure
reported in reference 15 in 55% yield as
a
white solid,
References
1
mp = 198–202 °C; H NMR (400 MHz, DMSO) δ 8.49 (s, 1H), 8.12 (s,
1
7
H), 7.99 (dd, 1H, J = 6.78, 2.58 Hz), 7.75 (s, 1H), 7.71–7.66 (m, 1H),
.25 (t, 1H, J = 8.97 Hz), 7.16 (s, 1H), 4.25 (m, 2H), 3.98 (s, 3H), 3.80
1
2
3
4
5
6
7
. Laplante M, Sabatini DM. MTOR signaling in growth control and disease. Cell.
2012;149:274–293.
. Zoncu R, Efeyan A, Sabatini DM. MTOR: from growth signal integration to cancer,
diabetes and ageing. Nat Rev Mol Cell Biol. 2011;12:21–35.
. Bove J, Martinez-Vicente M, Vila M. Fighting neurodegeneration with rapamycin:
mechanistic insights. Nat Rev Neurosci. 2011;12:437–452.
. Chiang GG, Abraham RT. Targeting the mTOR signaling network in cancer. Trends
Mol Med. 2007;13:433–442.
. Benjamin D, Colombi M, Moroni C, Hall MN. Rapamycin passes the torch: a new
generation of mTOR inhibitors. Nat Rev Drug Discovery. 2011;10:868–880.
. Hudes G, Carducci M, Tomczak P, et al. Temsirolimus, interferon alfa, or both for
advanced renal-cell carcinoma. N Engl J Med. 2007;356:2271–2281.
. Motzer RJ, Escudier B, Oudard S, et al. Efficacy of everolimus in advanced renal cell
carcinoma: a double-blind, randomised, placebo-controlled phase III trial. Lancet.
(
m, 1H), 3.73 (m, 2H), 1.75–1.58 (m, 3H), 0.90 (dd, 6H, J = 6.21,
1
3
5
1
1
2
.31 Hz);
C NMR (175 MHz, DMSO) δ 169.75, 156.57, 156.00,
54.32, 153.91, 152.68, 148.05, 147.01, 123.37, 122.23, 118.80,
16.56, 108.68, 107.37, 102.55, 67.66, 55.81, 54.86, 38.54, 38.16,
5.28, 24.41, 22.50; MS (FAB) m/z 476 [M + H] ; HRMS (FAB) m/z
+
+
calcd for C23
H
5
27ClFN O
3
[M + H] 476.1786, found: 476.1876. Anal.;
HPLC 95%.
4.2.21. (S)-N-(2-((4-((3-Chloro-4-fluorophenyl)amino)-7-
2
008;372:449–456.
methoxyquinazolin-6-yl)oxy)ethyl)-2-hydroxy-4-methylpentanamide (24)
8
. Stipanuk MH. Leucine and protein synthesis: mTOR and beyond. Nutr Rev.
2007;65:122–129.
Compound 24 was prepared by following the previous procedure
reported in reference 15 in 57% yield as a white solid.; 1H NMR
9. Dodd KM, Tee AR. Leucine and mTORC1: a complex relationship. Am J Physiol
Endocrinol Metab. 2012;302:E1329–1342.
(
7
300 MHz, CD
.60 (m, 1H), 7.15 (t, 1H, J = 8.97 Hz), 7.09 (s, 1H), 4.18 (t, 2H,
J = 5.49 Hz), 3.97 (m, 1H), 3.92 (s, 3H), 3.62 (m, 2H), 1.75 (m, 1H),
.37–1.45 (m, 2H), 0.82 (dd, 6H, J = 6.68, 4.05 Hz); MS (ESI) m/z 477
3
OD) δ 8.36 (s, 1H), 7.94 (d, J = 6.96 Hz), 7.73 (s, 1H),
1
0. Han JM, Jeong SJ, Park MC, et al. Leucyl-tRNA synthetase is an intracellular leucine
sensor for the mTORC1-signaling pathway. Cell. 2012;149:410–424.
1
1. Bonfils G, Jaquenoud M, Bontron S, Ostrowicz C, Ungermann C, De Virgilio C.
Leucyl-tRNA synthetase controls TORC1 via the EGO complex. Mol Cell.
1
[
2
012;46:105–110.
+
M + H]
.
12. Lynch CJ, Fox HL, Vary TC, Jefferson LS, Kimball SR. Regulation of amino acid-
sensitive TOR signaling by leucine analogues in adipocytes. J Cell Biochem.
2
000;77:234–251.
4
.2.22. 2-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
13. Wang X, Fonseca BD, Tang H, et al. Re-evaluating the roles of proposed modulators
of mammalian target of rapamycin complex 1 (mTORC1) signaling. J Biol Chem.
yl)oxy)ethyl (((benzyloxy)carbonyl)-l-leucyl)sulfamate (25)
2
008;283:30,482–30,492.
Compound 25 was prepared by following the previous procedure
1
4. Yoon S, Kim JH, Kim S-E, et al. Discovery of Leucyladenylate Sulfamates as Novel
reported in reference 14 in 46% yield as a white solid.; 1H NMR
Leucyl-tRNA Synthetase (LRS)-targeted Mammalian Target of Rapamycin Complex 1
(mTORC1) Inhibitors. J Med Chem. 2016;59:10,322–10,328.
(
7
(
300 MHz, CD
.91 (s, 1H), 7.67 (m, 1H), 7.20–7.11 (m, 7H), 4.94 (s, 2H), 4.41–4.37
m, 4H), 4.08 (m, 1H), 3.96 (s, 3H), 1.75–1.45 (m, 3H), 0.86 (m, 6H);
3
OD) δ 8.43 (s, 1H), 8.01 (dd, 1H, J = 6.78, 2.58 Hz),
1
5. Yoon S, Kim JH, Koh Y, et al. Discovery of simplified leucyladenylate sulfamates as
novel leucyl-tRNA synthetase (LRS)-targeted mammalian target of rapamycin com-
plex 1 (mTORC1) inhibitors. Bioog Med Chem. 2017;25:4145–4152.
+
16. Barker AJ, Gibson KH, Grundy W, et al. Studies leading to the identification of
ZD1839 (IRESSA): an orally active, selective epidermal growth factor receptor tyr-
osine kinase inhibitor targeted to the treatment of cancer. Bioorg Med Chem Lett.
MS (ESI) m/z 690 [M + H]
.
2
001;14:1911–1914.
4
.2.23. (S)-1-(((2-((4-((3-Chloro-4-fluorophenyl)amino)-7-
1
7. Kumar N, Chowdhary A, Gudaparthi O, Patel NG, Soni SK, Sharma R. A simple and
highly efficient process for synthesis of Gefitinib and its intermediate. Indian J. Chem.
2014;53B:1269–1274.
8. Lukkarila JL, da Silva SR, Ali M, et al. Identification of NAE inhibitors exhibiting
potent activity in leukemia cells: exploring the structural determinants of NAE spe-
cificity. ACS Med Chem Lett. 2011;2:577–582.
methoxyquinazolin-6-yl)oxy)ethoxy)sulfonyl)amino)-4-methyl-1-
oxopentan-2-yl acetate (26)
1
Compound 26 was prepared by following the previous procedure
reported in reference 14 in 58% yield as a white solid.; 1H NMR
(
300 MHz, CD
3
OD) δ 8.41 (s, 1H), 8.04 (dd, 1H, J = 6.78, 2.76 Hz),
.92 (s, 1H), 7.76–7.71 (m, 1H), 7.17 (t, 1H, J = 8.97 Hz), 7.10 (s, 1H),
.83–4.79 (m, 1H), 4.44 (m, 4H), 3.98 (s, 3H), 1.74–1.55 (m, 3H), 0.87
19. Yoon S, Kim C, Kim S-E, et al. Discovery of (S)-4-isobutyloxazolidine-2-one as a Novel
Leucyl-tRNA Synthetase (LRS)-targeted mTORC1 Inhibitor. Bioorg Med Chem Lett.
7
4
2
016;26:3038–3041.
2
0. Vichai V, Kirtikara K. Sulforhodamine B colorimetric assay for cytotoxicity screening.
(
dd, 6H, J = 9.54, 6.60 Hz); MS (ESI) m/z 599 [M + H]+
.
Nat Protoc. 2006;1:1112–1116.
7