Discovery of (S)-4-isobutyloxazolidin-2-one as a novel leucyl-tRNA synthetase (LRS)-targeted mTORC1 inhibitor

Article abstract of DOI:10.1016/j.bmcl.2016.05.011

A series of leucinol analogs were investigated as leucyl-tRNA synthetase-targeted mTORC1 inhibitors. Among them, compound 5, (S)-4-isobutyloxazolidin-2-one, showed the most potent inhibition on the mTORC1 pathway in a concentration-dependent manner. Compound 5 inhibited downstream phosphorylation of mTORC1 by blocking leucine-sensing ability of LRS, without affecting the catalytic activity of LRS. In addition, compound 5 exhibited cytotoxicity against rapamycin-resistant colon cancer cells, suggesting that LRS has the potential to serve as a novel therapeutic target.

Full text of DOI:10.1016/j.bmcl.2016.05.011

Accepted Manuscript
Discovery of (S)-4-Isobutyloxazolidin-2-one as a Novel Leucyl-tRNA Synthe-
tase (LRS)-targeted mTORC1 Inhibitor
Suyoung Yoon, Jong Hyun Kim, Ina Yoon, Changhoon Kim, Sung-Eun Kim,
Yura Koh, Seung Jae Jeong, Jiyoun Lee, Sunghoon Kim, Jeewoo Lee
PII:
S0960-894X(16)30494-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.05.011
BMCL 23866
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 March 2016
28 April 2016
4 May 2016
Please cite this article as: Yoon, S., Kim, J.H., Yoon, I., Kim, C., Kim, S-E., Koh, Y., Jeong, S.J., Lee, J., Kim, S.,
Lee, J., Discovery of (S)-4-Isobutyloxazolidin-2-one as a Novel Leucyl-tRNA Synthetase (LRS)-targeted mTORC1
Inhibitor, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.05.011
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Discovery of (S)-4-Isobutyloxazolidin-2-one as a Novel
Leucyl-tRNA Synthetase (LRS)-targeted mTORC1 Inhibitor
Suyoung Yoon, Jong Hyun Kim, Ina Yoon, Changhoon Kim, Sung-Eun Kim, Yura Koh, Seung Jae Jeong,
Jiyoun Lee, Sunghoon Kim, Jeewoo Lee*

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Discovery of (S)-4-Isobutyloxazolidin-2-one as a Novel Leucyl-tRNA Synthetase
(LRS)-targeted mTORC1 Inhibitor
Suyoung Yoon a, Jong Hyun Kim b, Ina Yoon b, Changhoon Kim a, Sung-Eun Kim a, Yura Koh a, Seung Jae
Jeong b, Jiyoun Lee d, Sunghoon Kim b,c, Jeewoo Lee a,*
a Laboratory of Medicinal Chemistry, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea
bMedicinal Bioconvergence Research Center, College of Pharmacy, Seoul National University, Seoul 151-742, Korea
c Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University,
Seoul 151-742, Korea
d Department of Global Medical Science, Sungshin University, Seoul 142-732, Korea
ARTICLE INFO
ABSTRACT
Article history:
Received
A series of leucinol analogs were investigated as leucyl-tRNA synthetase-targeted
mTORC1 inhibitors. Among them, compound 5, (S)-4-isobutyloxazolidin-2-one,
showed the most potent inhibition on the mTORC1 pathway in a concentration-
dependent manner. Compound 5 inhibited downstream phosphorylation of mTORC1
by blocking leucine-sensing ability of LRS, without affecting the catalytic activity of
LRS. In addition, compound 5 exhibited cytotoxicity against rapamycin-resistant
colon cancer cells, suggesting that LRS has the potential to serve as a novel
therapeutic target.
Revised
Accepted
Available online
Keywords:
Leucyl-tRNA Synthetase
mTORC1 Inhibitor
Leucinol
2009 Elsevier Ltd. All rights reserved.
Mammalian target of rapamycin (mTOR) is
a
acid sufficiency.5,6 More specifically, it has been proposed that
leucyl-tRNA synthetase (LRS) activates the Rag GTPase in the
presence of leucine, and promotes the lysosomal translocation of
mTORC1 for activation.7,8 It has been also demonstrated that
certain leucine analogues, such as leucinol, inhibited leucine-
induced mTORC1 activation,9,10 probably by blocking leucine-
sensing ability of LRS,8 suggesting that LRS-targeted inhibitors
can suppress mTORC1 activity.
Overactive mTORC1 is associated with many pathological
conditions, including obesity, diabetes, neurodegenerative
diseases, and cancers.11-13 Rapamycin and its analogs, also called
rapalogs, have been widely used to study underlying
mechanisms of mTORC1 activation in the pathogenesis of these
diseases.14 While rapamycin is considered to be a highly
selective allosteric inhibitor of mTORC1, recent studies indicate
that it does not completely shut down all the effects of mTORC1,
implying the existence of ‘rapamycin-resistant’ or ‘rapamycin-
insensitive’ pathways.15 One possible explanation for this
phenomenon is that the kinase activity of mTORC1 changes
depending on the nutrient and growth factor levels, therefore
exhibiting differential sensitivity to rapamycin.16 mTORC1
serine/threonine protein kinase, regulating various signaling
processes that are crucial for cell survival, such as cell growth,
proliferation, metabolism and autophagy.1 mTOR forms two
structurally distinct protein complexes, mTOR complex 1
(mTORC1) and mTOR complex 2 (mTORC2); mTORC1
controls protein synthesis by sensing and integrating a wide
range of extracellular and intracellular signals such as growth
factors, nutrients, energy status, and various stressors, whereas
mTORC2 regulates the cytoskeleton and metabolism.2
Interestingly, amino acids, particularly leucine, regulate
mTORC1 activity via specific mechanisms independent of other
environmental signals.3,4 Although how amino acids control
mTORC1 activity has not been fully elucidated, the Rag family
of GTPases appears to regulate mTORC1 in response to amino
___________
* Corresponding author. Tel.: +82 2 880 7846; fax: +82 2 762 8322.
E-mail address: jeewoo@snu.ac.kr (J. Lee).
http://dx.doi.org/10.1016/j.bmcl.2015.04.024
0960-894X/ⓒ 2015 Elsevier Ltd. All rights reserved.

Scheme 1. Synthesis of leucinol analogs
Reagents & conditions: (a) cat. conc. H2SO4, MeOH, reflux, 12 h, 99% for N-Cbz, 45% for N-Boc; (b) NaBH4, EtOH, 0 oC→r.t, 56% for N-Cbz, 55% for N-Boc;
(c) Pd/C, H2, MeOH, r.t, overnight, 89%; (d) 1, 1’-carbonyldiimidazole, DMF, r.t, 12 h, 39%; (e) MsCl, TEA, MC, 0 oC→r.t, 2 h, 90%; (f) 10% Pd/C, H2,
MeOH, r.t, 12 h, 89%; (g) NaN3, DMF, 80 oC, 4 h, 78%; (h) 10% Pd/C, H2, MeOH, r.t, 12 h, 33%; (i) 1,1’-carbonyldiimidazole, MC, r.t, 12 h, 54%; (j) NaH,
MeI, DMF, 0 oC→r.t, 12 h; (k) benzyl chloroformate, K2CO3, acetonitrile, 0 oC→r. t, 5 h, 65%; (l) 10% Pd/C, H2, 2M NH3 in MeOH, r.t, 12 h, 99%; (m) PPh3,
cat. H2O, THF, r.t, 12 h, 83%; (n) MsCl, TEA, MC, r.t, 1 h, 89%; (o) 10% Pd/C, H2, 2M NH3 in MeOH, r.t, 12 h, 79%; (p) acetic anhydride, 80 oC, 12 h, 99%;
(q) 10% Pd/C, H2, 2M NH3 in MeOH, r.t, 12 h, 98%; (r) NaCN, 15-crown-5, DMF, 60 oC, 48 h, 73%; (s) 10% Pd/C, H2, MeOH, r.t, 12 h, 70%; (t) 6N HCl,
reflux, 12 h, 99%; (u) BH3·THF, THF, reflux, overnight, 15%; (v) Dess-Martin periodinane, MC, r.t, 2 h, 61% for N-Cbz, 80% for N-Boc; (w) hydroxylamine
hydrochloride, Na2CO3, MeOH, r.t, 12 h, 66% for N-Cbz, 67% for N-Boc; (x) 10% Pd/C, H2, MeOH, r.t, 12 h, 86%.
inhibitors that do not bind mTOR, but block other regulators
such as LRS, would provide a powerful tool to study mTORC1
specific pathways and related disorders. In addition, since
rapamycin resistance is regarded as one of the contributing
factors to poor efficacy of rapalogs in cancer therapy,17,18 these
inhibitors have the potential to offer a new therapeutic option.
To develop LRS-targeted mTORC1 inhibitors, we first
designed and synthesized a series of leucinol analogs. Leucinol 1
and compounds 2-12 were prepared by following the pathway
described in Scheme 1. Compounds 2 and 6 were prepared by
carbonylation or methylation from leucinol respectively, which
was prepared from commercially available N-Cbz-leucine by 3
steps. Compounds 3, 8, and 10 were synthesized from the
mesylated product of N-Cbz-leucinol; compound 3 was obtained
via deprotection of the N-Cbz group, and compounds 8 and 10
were prepared by displacing the mesylate with a nitrile group
followed by reduction or reductive hydrolysis respectively.
Displacement of the mesylate with sodium azide yielded an
azido intermediate, which was then used to prepare compounds
4, 5, 7, and 9. Hydrogenation in the presence of 10% palladium
afforded diamine compound 4, and carbonylation of compound 4
yielded a cyclic analog, compound 5; selective reduction of the
azide produced the N-Cbz protected monoamine intermediate,
and subsequent methanesulfonylation or acylation, followed by
deprotection produced compound 7 and 9 respectively. To
prepare compound 11, N-Cbz protected leucinol was oxidized to
aldehyde by Dess-Martin periodinane, and subsequent
condensation with hydroxylamine, followed by deprotection
yielded compound 11. Compound 12 was prepared from N-Boc
protected leucine by applying the same procedure carried out for
compound 11.
Compound 13 was synthesized from commercially available
imidazole-2-carboxaldehyde (Scheme 2); the aldehyde group
was converted to a nitrile group which was then reacted with
isopropyl magnesium chloride to produce an imidazole
intermediate containing an isobutyl ketone group. The ketone
group was completely reduced by using Ni-Al catalyst to
generate compound 13.
Scheme 2. Synthesis of imidazole analog of leucinol
Reagents & conditions: (a) i) hydroxylamine hydrochloride, pyridine, r.t, 2 h,
ⅱ) acetic anhydride, 80 oC→r.t, 5 h, 80%; (b) isopropylmagnesium chloride,
o
THF, 0 C
→r.t, 3 h, 77%; (c) Ni
-Al alloy, H2O, reflux, 48 h, 50%.
Leucinol analogs with a chiral α-methyl group were
synthesized by following the pathway described in Scheme 3. N-
Dibenzyl protected leucinol was oxidized to aldehyde for
subsequent Grignard reaction, providing a racemic alcohol
mixture of R- and S- isomers with a ratio of 9:1. Final
debenzylation of purified enantiomers yielded compounds 14 (R-
isomer) and 15 (S-isomer). The stereochemistry of each
compound was assigned based on the previous report.19

Based on the primary screening results, we decided to focus
on compounds 2, 5, 11, and 13, and further determined the
inhibitory effects on leucine-induced mTORC1 activation at the
various concentrations by immunoblotting. As shown in Figure
2, all four compounds inhibited S6K phosphorylation in a dose-
dependent manner while their apparent potency decreases in the
order of 5 > 13 > 11 > 2. The cyclic analogs, compounds 5 and
13 appear to be more potent than leucinol, however, another
cyclic derivative compound 2 only differs from compound 5 by
one atom, and yet exhibits significantly lower activity.
Additionally, we examined the effect of each compound on
mTORC2 activation by using Akt as the marker.1 All tested
compounds including rapamycin and leucinol do not affect the
cellular levels of Akt or phosphorylated Akt (pAkt) indicating
that biological effects of these compounds are indeed mTORC1
specific.
Scheme 3. Synthesis of α-methyl analogs of leucinol
Reagents & conditions: (a) benzyl bromide, K2CO3, aq. MeOH, 65 oC, 2 h,
41%; (b) oxalyl chloride, DMSO, TEA, MC, -78 oC, 2 h; (c) CH3MgI, ether,
0 oC→r.t, 3 h, 60% (R:S=9:1); (d) 10% Pd/C, H2, MeOH. r.t, overnight, 62%
for 14, 47% for 15.
As a primary screening, we first assessed the effect of each
compound on leucine-induced phosphorylation of S6 Kinase
(S6K) by immunoblotting. S6K is one of the mTORC1
substrates, and pretreatment with leucinol appears to block
phosphorylation of S6K by inhibiting LRS.8 We treated HEK293
cells with each compound at 1 mM concentration, along with
rapamycin as a control at 100 nM; and then activated mTORC1
by treating cells with leucine for 10 min. As demonstrated in
Figure 1, leucine treatment induces phosphorylation of S6K,
whereas pretreatment with rapamycin or deprivation of leucine
blocked the phosphorylation. Pretreatment of leucinol also
inhibited the phosphorylation to some extent, while compounds,
2, 5, 11, and 13 appeared to inhibit the phosphorylation more
effectively than leucinol at the same concentrations. It is
interesting to note that all four compounds have multiple
heteroatoms that can participate in additional hydrogen bond
interactions compared to leucinol. It is also noticeable that
compounds 2, 5, and 13 are all cyclic analogs. Introduction of an
additional methylene group or chiral α-methyl group did not
generate any desirable activity. Addition of a relatively bulky
functional group such as methanesulfonyl, mesyl, and acetyl
group at the alcohol position also do not produce inhibitory
effects, suggesting that leucine binding site is highly specific and
only afford a small sized residue.
Figure 2. Dose-dependent inhibition of LRS-mediated mTORC1 activation
of selected leucinol derivatives on HEK293 cells. Rapamycin was treated at
100 nM, leucinol was at 800 µM, and compounds 2, 5, 11, and 13 were
treated at 100, 200, 400, and 800 µM.
To investigate the mechanisms of leucine analogs in LRS-
mediated mTORC1 activation, we examined the effects of
compounds 5 and 13 on the catalytic activity of LRS. More
specifically, since LRS catalyzes leucylation in the presence of
tRNA, we carried out leucylation assays on these compounds. As
shown in Figure 3, both compounds 5 and 13 are poor inhibitors
of aminoleucylation, having IC50 values in the high millimolar
range. Given that both compounds inhibited phosphorylation of
S6K at micromolar concentrations, our result indicates that these
leucine analogs do not affect catalytic activity of LRS, which is
in agreement with the previously reported observation that LRS
activates mTORC1 signaling via leucine recognition without
involving tRNA charging.8
Figure 1. Inhibition of LRS-mediated mTORC1 activation of synthesized
leucinol analogues on HEK293 cells
Figure 3. Aminoacylation activity of compound 5 and 13

Since mTORC1 is found to be overactive in many cancers,
we evaluated cytotoxicity of compound 5 in cancer cells. In
particular, mTORC1 is reported to be hyperactive in human
colorectal cancer,20 however, rapamycin appears to be mostly
ineffective.21 We selected a human colon cancer cell line
(SW620) which is known to be rapamycin resistant,22 to see
whether blocking LRS can solve the resistance issue. After
treating cells with each compound, we performed cytotoxicity
assays by using CellTox Green fluorescent dye which stains the
DNA from dead cells (Figure 4). As we expected, rapamycin did
not exhibit significant cytotoxicity while leucinol seemed to be
marginally cytotoxic even at the high concentrations; however,
leucine-sensing ability of LRS in the mTORC1 pathway has the
potential to serve as a novel therapeutic target.
Acknowledgments
This work was supported by the Global Frontier Project grant
(NRF-2012M3A6A4054928) of National Research Foundation
funded by the Ministry of Education, Science and Technology of
Korea
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leucine-sensing ability of LRS. These analogs act as
a
competitive inhibitor of leucine in LRS-mediated activation of
mTORC1, thereby inhibiting downstream phosphorylation of
S6K. In vitro mTORC1 inhibition study showed that compound
5, which contains an imidazolidin-2-one side chain, most
effectively inhibited leucine-mediated mTORC1 activation
among them; however, compound 5 did not suppress mTORC2
activation, nor did it affect catalytic activity of LRS.
Furthermore, compound 5 exhibited much higher cytotoxicity
than leucinol against rapamycin-resistant colon cancer cells,
implying that effective inhibition of leucine-sensing in the
mTORC1 pathway may play an important role in conferring
cytotoxicity. Taken together, we believe that the blockade of