New SIRT2 inhibitors: Histidine-based bleomycin spin-off

Article abstract of DOI:10.1016/j.bmc.2019.03.003

Bleomycin is considered to exert its antitumor activity via DNA cleavage mediated by activated oxygen generated from the iron complex in its chelator moiety. Spin-offs from this moiety, HPH-1Trt and HPH-2Trt, with anti-cancer activities were recently synt

Full text of DOI:10.1016/j.bmc.2019.03.003

Accepted Manuscript
New SIRT2 inhibitors: histidine-based bleomycin spin-off
Taha F.S. Ali, Halil I. Ciftci, Mohamed O. Radwan, Ryoko Koga, Takeo Ohsugi,
Yoshio Okiyama, Teruki Honma, Akiko Nakata, Akihiro Ito, Minoru Yoshida,
Mikako Fujita, Masami Otsuka
PII:
S0968-0896(19)30219-6
https://doi.org/10.1016/j.bmc.2019.03.003
BMC 14795
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
7 February 2019
28 February 2019
2 March 2019
Please cite this article as: Ali, T.F.S., Ciftci, H.I., Radwan, M.O., Koga, R., Ohsugi, T., Okiyama, Y., Honma, T.,
Nakata, A., Ito, A., Yoshida, M., Fujita, M., Otsuka, M., New SIRT2 inhibitors: histidine-based bleomycin spin-
off, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.03.003
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Bioorganic & Medicinal Chemistry
Research Article
New SIRT2 inhibitors: histidine-based bleomycin spin-off
Taha F. S. Ali , Halil I. Ciftci , Mohamed O. Radwana,c,d, Ryoko Kogaa,
a,b
a,c
e
f
f
g
h,i
Takeo Ohsugi , Yoshio Okiyama , Teruki Honma , Akiko Nakata , Akihiro Ito ,
Minoru Yoshidag,h,j, Mikako Fujita *, Masami Otsuka *
k,
a,
a
Department of Bioorganic Medicinal Chemistry, Faculty of Life Sciences, Kumamoto University,
5
-1 Oe-honmachi, Chuo-ku, Kumamoto, Kumamoto 862-0973, Japan
b
Medicinal Chemistry Department, Faculty of Pharmacy, Minia University, Minia 61519, Egypt
Department of Drug Discovery, Science Farm Ltd., 1-7-30 Kuhonji, Chuo-ku, Kumamoto,
c
Kumamoto 862-0976, Japan
d
Chemistry
of
Natural
Compounds
Department, Pharmaceutical and Drug
Industries Research Division, National Research Centre, Dokki 12622, Cairo, Egypt
e
Department of Laboratory Animal Science, School of Veterinary Medicine, Rakuno-Gakuen
University, 582 Bunkyodai-Midorimachi, Ebetsu, Hokkaido 069-8501, Japan
f
Center for Biosystems Dynamics Research, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama,
Kanagawa 230-0045, Japan
g
Seed Compounds Exploratory Unit for Drug Discovery Platform, RIKEN Center for Sustainable
Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
h
Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, 2-1
Hirosawa, Wako, Saitama 351-0198, Japan
i
School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi,
Hachioji, Tokyo 192-0392, Japan
j
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University
of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan
k
Research Institute for Drug Discovery, School of Pharmacy, Kumamoto University, 5-1 Oe-
honmachi, Chuo-ku, Kumamoto, Kumamoto 862-0973, Japan
*
Corresponding authors.
E-mail addresses: mfujita@kumamoto-u.ac.jp (M Fujita); motsuka@gpo.kumamoto-
u.ac.jp (M Otsuka).
1

ABSTRACT
Bleomycin is considered to exert its antitumor activity via DNA cleavage mediated by
activated oxygen generated from the iron complex in its chelator moiety. Spin-offs from
this moiety, HPH-1Trt and HPH-2Trt, with anti-cancer activities were recently
synthesized. In this paper, we developed inhibitors of nicotinamide adenine dinucleotide-
dependent deacetylase isoform 2 of Sirtuin protein (SIRT2), based on HPH-1Trt/HPH-
2
Trt, and aimed to generate new anti-cancer drugs. HPH-1Trt and HPH-2Trt had in vitro
anti-SIRT2 inhibitory activity with 50% inhibitory concentration (IC50) values of 5.5 and
.8 M, respectively. A structural portion of HPH-1Trt/HPH-2Trt, a tritylhistidine
8
derivative TH-1, had stronger activity (IC50 = 1.7 M), and thus, fourteen derivatives of
TH-1 were synthesized. Among them, TH-3 had the strongest activity (IC50 = 1.3 M).
Selective binding of TH-3 in the pocket of SIRT2 protein was confirmed with a molecular
docking study. Furthermore, TH-3 strongly lowered viability of the breast cancer cell line
MCF7 with an IC50 of 0.71 M. A structure-activity relationship study using cell lines
suggested that the mechanism of TH-3 to suppress MCF7 cells involves not only SIRT2
inhibition, but also another function. This compound may be a new candidate anti-cancer
drug.
2

1
. Introduction
Bleomycin is a clinically used antitumor antibiotic that exerts its effect via iron
binding, oxygen activation, and oxidative cleavage of genomic DNA.1-3 The pyrimidine-
histidine moiety is the key structural unit of iron binding and leads to oxygen activation.2
We previously attempted to simplify and symmetrize the bleomycin metal core to obtain
histidine-containing small molecules, i.e., PYML-6/8,4,5 HPH, and HGGH, (Figure 1)
which can activate oxygen as a metal complex. The HPH skeleton was further
transformed into zinc-binding SN-18-14 and anti-fibrotic HPH-15 , both of which contain
cysteamine.
6
7
15
Figure 1. Structures of bleomycin A2, PYML-6/8,4,5 HPH, and HGGH.7
6
3

On the other hand, oxygen-activating DNAcleavage and anti-cancer activities of HPH
were improved by introduction of one and two trityl group(s) into its imidazole rings,
resulting in HPH-1Trt and HPH-2Trt (Figure 2), respectively.16 DNA replication is
accelerated in cancer cells, and thus, DNA is always exposed, rendering cancer cells more
sensitive to drugs targeting DNA than normal cells. However, off-target effects of this
kind of drug are inevitable. Strategies to kill cancer cells by just damaging genomic DNA
using anti-cancer drugs are considered obsolete.
Recent strategies involve antitumor drugs that target specific proteins that are
important in cancer. For example, the antibody drug trastuzumab targets human epidermal
growth factor receptor 2,17 and the low molecular-weight drugs imatinib and afatinib
interact with the breakpoint cluster region in kinase-Abelson kinase (Bcr-Abl) and
epidermal growth factor receptor,18 respectively. These drugs are called molecular
targeted drugs, and are clinically used with fewer side effects than conventional
chemotherapy.19 One candidate for another new target is isoform 2 of Sirtuin protein
(SIRT2). Sirtuin proteins include seven isoforms in humans, and are nicotinamide adenine
+
dinucleotide (NAD )-dependent histone or non-histone deacetylases that control many
physiological functions.20 SIRT2 shuttles between the cytoplasm and nucleus, and
catalyzes deacetylation of various proteins, e.g., histone, tubulin, Forkhead box, and
2
1
p53. Some of these proteins are related to maintenance of cancer cells, and thus, SIRT2
is a target of cancer therapy.21,22 Various SIRT2 inhibitors have been reported,21-23
although none of them has entered the market or clinical trials.
We developed new SIRT2 inhibitors based on HPH-1Trt and HPH-2Trt. These
compounds contain dimethylaminopyridine, histidines, and trityl groups that could be
4

rearranged to fit various protein pockets including SIRT2. Here, we report synthesis and
activities of the compounds.
Figure 2. Structures of HPH-1Trt, HPH-2Trt, and trityl histidine derivatives synthesized
in this study.
2
. Results and discussion
5

1
6
In vitro SIRT2 inhibitory activities of already synthesized HPH-1Trt/HPH-2Trt were
examined with a fluorogenic assay. As shown in Table 1, these compounds showed single-
digit micromolar 50% inhibitory concentration (IC50) values: 5.5 and 8.8 M. Previous
X-ray crystal structural studies24-26 showed that the pocket in SIRT2 that accommodates
low molecular-weight inhibitors is long and narrow. Structures of known SIRT2 inhibitors
(
six are shown in Figure 3)27-32 are close to a linear shape. We then designed a new
compound, TH-1 (Figure 2), which is a structural portion of HPH-1Trt/HPH-2Trt that has
a more linear structure.
Table 1
In vitro inhibitory activity of trityl histidine derivatives against SIRT2.a
Compound
HPH-1Trt
HPH-2Trt
TH-1
IC50 against SIRT2 (M)
5.5 ± 0.5
8.8 ± 1.7
1.7 ± 0.0
TH-2
43.0 ± 0.8
1.3 ± 0.2
TH-3
TH-4
5.6 ± 0.1
TH-5
11.8 ± 0.3
16.4 ± 1.1
21.8 ± 1.0
10.2 ± 0.3
6.7 ± 1.5
TH-6
TH-7
TH-8
TH-9
TH-10
TH-11
TH-12
TH-13
TH-14
TH-15
5.9 ± 0.4
7.5 ± 1.6
2.5 ± 0.3
3.3 ± 0.8
20.4 ± 3.1
>100
6

SirReal2
0.21 ± 0.02
aFor HPH-1Trt, HPH-2Trt, and TH-1, the values were determined with the fluorogenic
assay. For the others and TH-1, the electrophoretic mobility shift assay was performed.
For both assays, TH-1 showed the same value.
27
28
Figure 3. Structures of previously reported SIRT2 inhibitors, AEM2, TM, Chroman-
2
9
30
31
32
4
-one analogue, SirReal2, RK-9123016 and NPD11033 .

TH-1 was synthesized by reductive amination of (S)-N -tritylhistidine methyl ester
(TH-2) and N,N-dimethyl-4-aminopyridin-2-carbaldehyde, both of which were
synthesized previously,16,33 in a 51% yield (Scheme 1). Then, in vitro SIRT2 inhibitory
activities of TH-1 and TH-2, which is the residual structure after deletion of TH-1 from
7

HPH-2Trt, were examined. The IC50 of TH-1 was 1.7 M, showing stronger activity than
that of the original compound HPH-1Trt/HPH-2Trt, although the activity of TH-2 was
weaker.
Scheme 1
To improve the activity of TH-1, we next synthesized 13 analogues with various aryl
groups instead of the pyridine moiety (TH-3-TH-13), a peptide instead of a secondary
amine (TH-14), and a bis-benzyl amine (TH-15) (Figure 2). TH-3-TH-13 were
synthesized with the same method as that for TH-1 using TH-2 and the corresponding
aldehyde (Scheme 1) in
a
31-75% yield. Among the aldehydes, 3-
previously,34
diethylamino)picolinaldehyde (2)35 was synthesized from the corresponding bromo-
(dimethylamino)benzaldehyde
was
synthesized
and
4-
(
3
6
derivative (1) (Scheme 2). The other aldehydes were commercially obtained. Except for
3
3
TH-6 , these derivatives are new compounds. Synthesis of TH-14 was performed by
reaction of TH-2 and picolinoyl chloride in a 24% yield (Scheme 1), and TH-15 was
synthesized using TH-2 and more than two equivalents of benzaldehyde in a 39% yield
(Scheme 1).
8

Scheme 2
Their in vitro SIRT2 inhibitory activities were examined using the electrophoretic
mobility shift assay (Table 1). TH-15 with two bulky aryl groups did not show any activity.
On the other hand, TH-6, TH-7, and TH-14 with unsubstituted phenyl or pyridinyl rings
showed weak activity (two-digit micromolar IC50 values). All compounds with IC50
values less than 10 M had a substituted phenyl or pyridine ring. The strongest inhibitor
was TH-3, with an IC50 of 1.3 M. This value is lower than that of TH-1, although it did
not reach the value of the control compound, SirReal2, which has a sub-micromolar value.
Furthermore, five compounds with different structures, TH-2, TH-5, TH-7, TH-10, and
TH-15, were chosen, and selectivity for inhibitory activities against SIRT1, 2, and 3 were
examined. Of the three SIRT isoforms, SIRT2 was suppressed the most, demonstrating
selectivity for SIRT2 (Table 2).
Table 2
In vitro inhibitory activity of trityl histidine derivatives against SIRT1, SIRT2, and SIRT3a
Relative enzyme activity (%)
Compound
SIRT1
95.9
SIRT2
36.1
38.7
5.1
SIRT3
76.2
TH-2
TH-5
TH-7
105.3
96.5
81.7
67.7
9

TH-10
TH-15
102.1
115.8
20.8
61.3
71.9
90.7
aFor SIRT1 and SIRT3, the fluorogenic assay was used, and the electrophoretic mobility
shift assay was used for SIRT2.
To gain a deeper insight into the structural mechanism of SIRT2 inhibition by this
class of compounds, in silico analysis of TH-3 within the SIRT2 active site was performed.
The top-scoring pose for TH-3 is shown in Figure 4. The diethyl amino group attached to
the pyridine ring of TH-3 partially occupies the selectivity pocket, which is comparable
to the 4,6-dimethylpyrimidine moiety of SirReal2 (Figure 4A). Detailed binding of TH-3
to the SIRT2 active site (Figure 4B,C) showed that the carbonyl group has two hydrogen
bonding interactions with the NH groups of Ile169 and Asp170, in a similar manner to
+
37
the nicotinamide part of NAD . Moreover, the pyridyl moiety interacts with the side
chain of Ile169 through a CH–π interaction. Furthermore, the trityl moiety of TH-3
protrudes from the hydrophobic cavity into the acetyl-lysine channel and blocks the
substrate binding site, similar to a SirReal2 derivative as described previously.38
Altogether, these hydrophobic and hydrogen bonding interactions suggest that TH-3 may
inhibit SIRT2 via a unique hybrid mechanism that includes a selectivity pocket, NAD+
competition, and/or acetyl substrate competition. These results confirm that TH-3 is a
promising SIRT2 inhibitor.
Figure 4. Binding of TH-3 to the SIRT2 active site (PDB code 4RMG) as predicted by
MOE. (A) Comparison of modeled binding of TH-3 (magenta sticks) and SirReal2 (green
sticks). (B) Detailed binding of TH-3 (magenta sticks) displaying hydrogen bonds (red
dashed line) and CH-π interactions (black dashed line) with the key amino acid residues
10

(green sticks). (C) 2D depiction of TH-3 binding interactions with the key amino acid
residues.
(A)
(B)
(C)
11

Finally, anti-cancer activities of HPH-1Trt/HPH-2Trt and selected derivatives (TH
compounds) were examined using several cell lines. The human breast cancer cell line
MCF7 is sensitive to SIRT2 inhibitors,28,31 and thus, the viability of MCF7 cells in the
presence of each compound was examined (Table 3). Among TH compounds, the
tendency for inhibition of in vitro SIRT2 activity (Table 1) and cell viability were similar:
TH-2 was weak, TH-5 and TH-10 were intermediate, and TH-1 and TH-3 were strong.
One exception is that the in vitro activity of TH-7 on SIRT2 was weaker than that of TH-
5
and TH-10, but the activity on cells was opposite. These results suggest that one
mechanism of TH compounds to suppress MCF7 viability is inhibition of SIRT2. Notably,
all TH compounds except TH-2 had stronger activity against MCF7 cells than the control
compound SirReal2. In particular, the IC50 values of TH-1 and TH-3 were sub-micromolar.
Furthermore, the effect of the compounds on the human T cell leukemia cell lines, K562
and MT-2, was examined. As shown in Table 3, all TH compounds have IC50 values of
1
0 M or less, and the tendency for inhibitory effects was inconsistent with that of in vitro
12

inhibition of SIRT2 activity. Furthermore, SirReal2 did not show cytotoxic activity. Thus,
the mechanism of TH compounds to inhibit the viability of K562 and MT-2 cells appears
independent of inhibition of SIRT2. One possible mechanism is DNA cleavage as shown
1
6
for HPH-1Trt/HPH-2Trt, which show inhibitory activity against K562 and MT-2 cells
that is similar to TH compounds (Table 3). TH compounds and HPH-1Trt/HPH-2Trt are
considered to have dual effects on MCF7 cells, including inhibition of SIRT2 and another
effect such as DNA cleavage, leading to sub-micromolar IC50 values for TH-1 and TH-3.
Table 3
Cytotoxic effects of trityl histidine derivatives on MCF7, K562, and MT-2 cell linesa
IC50 (M)
Compound
MCF7
K562
MT-2
HPH-1Trt
HPH-2Trt
TH-1
2.29 ± 0.43
1.72 ± 0.51
0.77 ± 0.08
49.1 ± 3.9
0.71 ± 0.06
11.9 ± 1.2
8.96 ± 1.03
9.87 ± 1.27
13.7 ± 2.2
6.51 ± 0.89
7.91 ± 1.12
7.53 ± 0.55
7.20 ± 0.81
6.27 ± 0.91
10.4 ± 1.1
6.36 ± 0.73
5.35 ± 0.59
>100
8.61 ± 1.05
8.86 ± 1.38
3.53 ± 0.44
3.13 ± 0.28
4.36 ± 0.62
6.81 ± 0.42
1.75 ± 0.11
1.37 ± 0.08
>100
TH-2
TH-3
TH-5
TH-7
TH-10
SirReal2
aCell viability was determined with the MTT assay.
3
. Conclusion
In this study, we developed new tritylhistidine derivatives as SIRT2 inhibitors based
on metal chelators from bleomycin. The bulky trityl group seems to hinder the methyl
ester from hydrolysis in a cell. Among the derivatives, TH-3 had the highest in vitro anti-
SIRT2 activity (IC50 = 1.3 M), although it did not reach that of an already known
13

inhibitor, SirReal2 (IC50 = 0.21 M). Interestingly, TH-3 had higher activity against the
breast cancer cell line, MCF7 (IC50 = 0.21 M), than SirReal2 (13.7 M). The structure-
activity relationship study suggests that the mechanism of TH-3 to suppress MCF7 cells
is not only SIRT2 inhibition, but also another function such as DNA cleavage.
Investigation of the details is in progress. This study provides clues for development of
new anti-cancer drugs.
4
. Experimental
4
4
.1. Synthesis
.1.1. General procedure pertaining to synthesis
Chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), Kanto
Chemical (Tokyo, Japan), Nacalai tesque (Kyoto Japan), Tokyo Chemical Industry
Tokyo, Japan) and Wako (Osaka, Japan). Thin layer chromatography (TLC) was
(
performed on precoated plates [Merck Millipore (Burlington, MA, USA) TLC sheets
silica 60 F254 and Fuji Silysia Chemical Ltd. (Kasugai, Japan) TLC sheets Chromatorex
NH silica]. Chromatography was carried out on Silica Gel 60 N (40–100 mesh) and NH
silica gel Chromatorex (NH, 100-200mesh) (Fuji Silysia Chemical Ltd.). NMR spectra
JEOLJNM-AL300MHz or BRUKER AVANCE 600MHz were referenced to TMS. Infra-
red spectra were recorded on a JASCO FT/IR-410 or JEOL JIR-6500W / PerkinElmer
Frontier. The samples were prepared as KBr discs or Film or using ATR method. Mass
spectra (EI) and high-resolution mass spectra (HRMS) were recorded by a JEOL
JMSDX303HF. HRMS were recorded by using positive fast atom bombardment (FAB)
with 3-nitrobenzyl alcohol as the matrix.
4
.1.2. General procedure for the synthesis of TH-1, and TH-3-TH15.

33
To a solution of (S)-N -tritylhistidine methyl ester (TH-2) (1.0 equivalent) in 20 ml
ClCH CH Cl was added the corresponding aryl aldehyde (1.0 equivalent) and
NaBH(OAc) (1.2 equivalent). The mixture was stirred at room temperature for 48 h
under argon atmosphere. Asolution of saturated aqueous NaHCO (15 ml) was added and
the organic layer was separated. The aqueous layer was extracted with CH Cl (20 ml x
). The combined organic layers were dried over Na SO and concentrated in vacuo. The
residue was purified on NH silica gel column (eluted with proper solvent system) to afford
2
2
3
3
2
2
3
2
4
14

the desired product.
α
τ
4
.1.3. (S)-N -((4-(dimethylamino)pyridin-2-yl)methyl)-N -tritylhistidine methyl
ester (TH-1)
As the aldehyde, N,N-dimethyl-4-aminopyridin-2-carbaldehyde16 was used. The
residue was purified on flash NH silica gel column (eluted with EtOAc) to afford TH-1
1
(
140 mg, 51.3%) as a colorless viscous oil. H NMR (300 MHz, CD
3
OD) δ 7.98 (d, J =
6
1
2
1
7
.1 Hz, 1H), 7.47 – 7.28 (m, 11H), 7.22 – 7.05 (m, 5H), 6.71 (s, 1H), 6.68 (d, J = 2.4 Hz,
H), 6.53 (dd, J = 6.1, 2.6 Hz, 1H), 3.78 (d, J = 14.1 Hz, 1H), 3.63 (m, 2H), 3.58 (s, 3H),
1
3
.99 (s, 6H), 2.91 (d, J = 6.8 Hz, 2H). C NMR (75 MHz, CD OD) δ 175.84, 159.76,
3
57.13, 149.26, 143.77, 139.59, 137.88, 131.00, 129.47, 129.38, 121.40, 106.56, 106.13,
6.87, 62.22, 53.91, 52.30, 39.26, 32.67. IR (ATR) 3314, 3058, 2924, 1732, 1599, 1444,
-1
+
1
158, 699 cm .MS (FAB) m/z 546.1 (M+H) ; HRMS (FAB). Calcd for C34
H
36
O
2
5
N :
5
46.2869. Found: 546.2874.
4
.1.4. 4-(diethylamino)picolinaldehyde (2)
To a solution of 2-bromo-4-(diethylamino)pyridine (1)36 (840 mg, 3.68 mmol) in dry
THF (60 mL) was added t-BuLi (5 mL of 1.6 M solution in pentane, 8 mmol) at -78 °C
over a period of 10 min. The solution was stirred for 30 minutes at -78 °C, and DMF (2
mL) was added slowly over 1 minute. The solution was stirred for further 2 hours at -
7
8 °C. The reaction mixture was warmed to room temperature and stirred at room
temperature overnight. A solution of H O-saturated aqueous NaHCO (1:1, 100ml) was
added and excess THF was evaporated under vacuum then extracted with CH Cl (50 ml
X 5) then brine (50 ml). The organic layer was dried over MgSO and concentrated in
vacuo. The residue was purified on silica gel column (eluted with n-Hexane : EtOAc =
2
3
2
2
4
1
:1) to afford 2 [29] (210 mg, 31.8%) as a yellow viscous oil.
α
τ
4
.1.5. (S)-N -((4-(diethylamino)pyridin-2-yl)methyl)-N -tritylhistidine methyl ester
(TH-3)
As the aldehyde, 4-(diethylamino)picolinaldehyde was used. The residue was
purified on NH silica gel column (eluted with EtOAc) to afford TH-3 (109 mg, 48.6%)
1
as a colorless viscous oil. H NMR (600 MHz, CDCl ) δ 8.08 (d, J = 6.0 Hz, 1H), 7.34
3
(d, J = 1.3 Hz, 1H), 7.32 – 7.28 (m, 9H), 7.11 (ddd, J = 5.1, 3.9, 2.0 Hz, 6H), 6.56 (d,
J=1.1Hz, 1H), 6.53 (d, J=2.6Hz, 1H), 6.32 (dd, J=6.0, 2.7Hz,1H), 3.83 (d, J=13.8Hz, 1H),
15

3
.71–3.66 (m, 2H), 3.57 (s, 3H), 3.33 (q, J = 7.1 Hz, 4H), 3.00 – 2.92 (m, 2H), 1.14 (t, J
1
3
=
7.1 Hz, 6H). C NMR (151 MHz, CDCl ) δ 174.91, 159.52, 152.85, 149.49, 142.62,
3
1
5
5
38.66, 137.31, 129.91, 128.11, 128.07, 119.51, 104.89, 104.48, 75.26, 61.45, 53.94,
-1
1.65, 43.89, 32.48, 12.51. IR (ATR) 3317, 1732, 1597, 1444, 700 cm . MS (FAB) m/z
+
74.4 (M+H) ; HRMS (FAB). Calcd for C36
H
40
O
2
N : 574.3182. Found: 574.3201.
5
α
τ
4
.1.6. (S)-N -(3-(dimethylamino)benzyl)-N -tritylhistidine methyl ester (TH-4)
As the aldehyde, 3-(dimethylamino)benzaldehyde34 was used. The residue was
purified on NH silica gel column (eluted with n-Hexane : EtOAc = 2:1) to afford TH-4
1
(
163 mg, 59%) as a colorless viscous oil. H NMR (600 MHz, CDCl ) δ 7.35 (d, J = 1.4
3
Hz, 1H), 7.33 – 7.28 (m, 9H), 7.12 (m, 7H), 6.68 (d, J = 1.6 Hz, 1H), 6.65 – 6.59 (m, 2H),
.57 (d, J = 1.3 Hz, 1H), 3.80 (d, J = 12.9 Hz, 1H), 3.67 – 3.62 (m, 2H), 3.61 (s, 3H), 2.93
6
1
3
(dd, J = 6.5, 1.7 Hz, 2H), 2.91 (s, 6H). C NMR (151 MHz, CDCl ) δ 175.19, 150.94,
3
1
42.65, 140.86, 138.64, 137.45, 129.92, 129.13, 128.14, 128.11, 119.52, 116.80, 112.74,
1
6
11.52, 75.27, 61.07, 52.65, 51.67, 40.81, 32.46. IR (ATR) 2946, 1730, 1602, 1443, 746,
-1
+
99 cm . MS (FAB) m/z 545.3 (M+H) ; HRMS (FAB). Calcd for C35
H
37
O
2
N : 545.2917.
4
Found: 545.2881.
α
τ
4
.1.7. (S)-N -(4-(dimethylamino)benzyl)-N -tritylhistidine methyl ester (TH-5)
As the aldehyde, 4-(dimethylamino)benzaldehyde was used. The residue was
purified on NH silica gel column (eluted with n-Hexane : EtOAc = 2:1) to afford TF-5
1
(
156 mg, 57.3%) as a yellow viscous oil. H NMR (600 MHz, MeOD) δ 7.37 – 7.33 (m,
1
3
2
1
3
0H), 7.14 – 7.09 (m, 6H), 7.08 (d, J = 8.4 Hz, 2H), 6.68 (d, J = 8.4 Hz, 2H), 6.66 (s, 1H),
.64 (d, J = 12.5 Hz, 1H), 3.61 (s, 3H), 3.57 (t, J = 6.5 Hz, 1H), 3.50 (d, J = 12.5 Hz, 1H),
1
3
.89 – 2.86 (m, 7H). C NMR (151 MHz, MeOD) δ 175.67, 151.65, 143.63, 139.50,
37.66, 130.85, 130.45, 129.33, 129.26, 121.17, 114.18, 76.83, 61.49, 52.32, 52.29, 41.14,
-1
2.44. IR (ATR) 3318, 1732, 1614, 1521, 1444, 746, 699 cm . MS (FAB) m/z 545.3
+
(
M+H) ; HRMS (FAB). Calcd for C35
H
37
O
2
4
N : 545.2917. Found: 545.2928.
α
τ
4
.1.8. (S)-N -(pyridin-2-ylmethyl)-N -tritylhistidine methyl ester (TH-7)
As the aldehyde, picolinaldehyde was used. The residue was purified on NH silica
gel column (eluted with CH
2
Cl
2
: MeOH = 20:1) to afford TH-7 (350 mg, 70%) as a pale
) δ 8.49 (dd, J = 4.8, 0.7 Hz, 1H), 7.58 (td,
1
yellow viscous oil. H NMR (600 MHz, CDCl
3
J = 7.7, 1.8 Hz, 1H), 7.36 (d, J = 1.3 Hz, 1H), 7.32 – 7.29 (m, 10H), 7.14 – 7.09 (m, 7H),
16

6
6
1
.59 (d, J = 1.2 Hz, 1H), 3.97 (d, J = 14.3 Hz, 1H), 3.82 (d, J = 14.3 Hz, 1H), 3.68 (t, J =
1
3
.5 Hz, 1H), 3.60 (s, 3H), 3.01 – 2.92 (m, 2H). C NMR (151 MHz, CDCl
59.76, 149.25, 142.62, 138.72, 137.24, 136.51, 129.91, 128.14, 128.11, 122.21, 121.96,
19.55, 75.29, 61.38, 53.50, 51.76, 32.42. IR (ATR) 3320, 3058, 2923, 1733, 1591, 1444,
3
) δ 174.84,
1
-1
+
1
157, 700 cm .MS (FAB) m/z 503.4 (M+H) ; HRMS (FAB). Calcd for C32
H
31
O
2
4
N :
5
03.2447. Found: 503.2441.
α
τ
4
.1.9. (S)-N -((5-bromopyridin-2-yl)methyl)-N -tritylhistidine methyl ester (TH-8)
As the aldehyde, 5-bromopicolinaldehyde was used. The residue was purified on NH
silica gel column (eluted with n-Hexane : EtOAc = 1:2) to afford TH-8 (204 mg, 70.6%)
1
as a colorless viscous oil. H NMR(600MHz, MeOD)δ 8.48(d, J=1.9Hz, 1H), 7.86(dd,
J=8.3, 2.4Hz, 1H), 7.38–7.34(m,10H), 7.32(d, J=8.3Hz, 1H), 7.16–7.10(m, 6H), 6.72(d,
J=1.2Hz, 1H), 3.87(d, J=14.7Hz, 1H), 3.73(d, J=14.7Hz, 1H), 3.62–3.58(m, 4H), 2.93
13
(
dd, J = 14.4, 6.5 Hz, 1H), 2.89 (dd, J = 14.4, 6.9 Hz, 1H). C NMR (151 MHz, MeOD)
δ 175.53, 159.38, 150.81, 143.64, 140.83, 139.50, 137.68, 130.87, 129.35, 129.25, 125.26,
21.27, 120.00, 76.84, 61.98, 53.01, 52.34, 32.58. IR (ATR) 3313, 1732, 1465, 1444, 699
1
-1
+
cm . MS (FAB) m/z 581.5 (M+H) ; HRMS (FAB) Calcd for C32
H
30
O
2
4
N Br: 581.1552.
Found: 581.1537.
α
τ
4
.1.10. (S)-N -((3-bromopyridin-4-yl)methyl)-N -tritylhistidine methyl ester (TH-9)
As the aldehyde, 3-bromoisonicotinaldehyde was used. The residue was purified on
NH silica gel column (eluted with n-Hexane : EtOAc = 1:2) to afford TH-9 (105 mg,
1
3
6.2%) as a colorless viscous oil. H NMR (600 MHz, MeOD) δ 8.58 (s, 1H), 8.36 (d, J
=
5.0 Hz, 1H), 7.48 (dd, J = 5.0, 0.5 Hz, 1H), 7.39 (d, J = 1.4 Hz, 1H), 7.38 –7.34(m,
9
1
H),7.17-7.10(m, 6H),6.74(d, J=1.3Hz, 1H),3.92(d, J=16.0Hz, 1H),3.74(d, J=16.0Hz,
H),3.63(s, 3H), 3.60 (dd, J = 7.1, 6.3 Hz, 1H), 2.95 (dd, J = 14.5, 6.1 Hz, 1H), 2.90 (dd,
1
3
J = 14.4, 7.2 Hz, 1H). C NMR (151 MHz, MeOD) δ 175.64, 151.90, 150.62, 149.02,
1
5
43.57, 139.42, 137.88, 130.81, 129.30, 129.22, 125.66, 123.10, 121.19, 76.76, 62.20,
-1
2.35, 51.37, 32.76. IR (ATR) 3056, 1732, 1583, 1443, 698 cm . MS (FAB) m/z 581.7
+
(
M+H) ; HRMS (FAB) Calcd for C32
H
30
O
2
N
4
Br: 581.1552. Found: 581.1516.
α
τ
4
.1.11. (S)-N -((6-bromopyridin-3-yl)methyl)-N -tritylhistidine methyl ester (TH-10)
As the aldehyde, 6-bromonicotinaldehyde was used. The residue was purified on NH
silica gel column (eluted with n-Hexane : EtOAc = 1:2) to afford TH-10 (170 mg, 59.1%)
17

1
as a colorless viscous oil. H NMR (600MHz, MeOD)δ 8.23 (d, J=2.3Hz, 1H),7.61(dd,
J=8.2, 2.3Hz, 1H), 7.48 (d, J=8.2Hz, 1H), 7.37 (s, 1H), 7.37– 7.33 (m, 9H), 7.15 – 7.08
(
m, 6H), 6.68 (s, 1H), 3.78 (d, J = 13.8 Hz, 1H), 3.63 (d, J = 13.9 Hz, 1H), 3.60 (s, 3H),
.54 (t, J = 6.7 Hz, 1H), 2.89 (dd, J = 14.5, 6.8 Hz, 1H), 2.86 (dd, J = 14.5, 6.9 Hz, 1H).
13C NMR (151 MHz, MeOD) δ 175.74, 151.03, 143.62, 141.18, 140.70, 139.42, 137.84,
3
1
3
36.73, 130.84, 129.34, 129.25, 129.15, 121.16, 76.81, 62.04, 52.31, 32.59. IR (ATR)
-1
+
312, 1731, 1562, 1445, 699 cm . MS (FAB) m/z 581.3 (M+H) ; HRMS (FAB). Calcd
for C32
H
30
O
2
N
4
Br: 581.1552. Found: 581.1539.
α
τ
4
.1.12. (S)-N -((6-bromopyridin-2-yl)methyl)-N -tritylhistidine methyl ester (TH-11)
As the aldehyde, 6-bromopicolinaldehyde was used. The residue was purified on NH
silica gel column (eluted with n-Hexane : EtOAc = 2:1) to afford TH-11 (250 mg, 53.8%)
1
as a colorless viscous oil. H NMR (600 MHz, MeOD) δ 7.59 (t, J = 7.7 Hz, 1H), 7.43 (d,
J = 7.8 Hz, 1H), 7.39 (d, J = 1.4 Hz, 1H), 7.37 – 7.33 (m, 10H), 7.16 – 7.13 (m, 6H), 6.72
(d, J = 1.3 Hz, 1H), 3.86 (d, J = 14.8 Hz, 1H), 3.73 (d, J = 14.8 Hz, 1H), 3.62 – 3.59 (m,
13
4
H), 2.94 (dd, J = 14.4, 6.5 Hz, 1H), 2.89 (dd, J = 14.4, 7.0 Hz, 1H). C NMR (151 MHz,
MeOD) δ 175.50, 162.28, 143.64, 142.37, 140.70, 139.58, 137.65, 130.89, 129.34, 129.24,
1
1
5
27.71, 122.53, 121.32, 76.86, 61.96, 53.12, 52.36, 32.61. IR (ATR) 3316, 1732, 1581,
-1
+
554, 699 cm . MS (FAB) m/z 581.3 (M+H) ; HRMS (FAB). Calcd for C32
H
30
O
2
4
N Br:
81.1552. Found: 581.1530.
α
τ
4
.1.13. (S)-N -(quinolin-2-ylmethyl)-N -tritylhistidine methyl ester (TH-12)
As the aldehyde, quinoline-2-carbaldehyde was used. The residue was purified on
NH silica gel column (eluted with n-Hexane : EtOAc = 2:1) to afford TH-12 (86 mg,
1
3
1.1%) as a low melting orange powder. H NMR (600 MHz, MeOD) δ 8.19 (d, J = 8.5
Hz, 1H), 7.92 – 7.89 (m, 1H), 7.88 – 7.85 (m, 1H), 7.68 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H),
7
7
1
6
1
1
1
.56–7.51 (m, 1H), 7.50 (d, J=8.5Hz, 1H), 7.38 (d, J=1.4Hz, 1H), 7.32–7.28 (m, 9H),
.13–7.09 (m, 6H), 6.74 (d, J= 1.4Hz, 1H), 4.11(d, J=14.7Hz, 1H), 3.97(d, J=14.7Hz,
H), 3.69 (t, J=6.7Hz,1H), 3.60 (s, 3H), 2.98 (dd, J=14.5, 6.2Hz, 1H), 2.93 (dd, J = 14.6,
1
3
.9 Hz, 1H). C NMR (151 MHz, MeOD) δ 175.49, 161.03, 148.39, 143.60, 139.48,
38.33, 137.69, 130.89, 130.84, 130.67, 129.31, 129.22, 129.11, 129.06, 128.93, 128.85,
27.54, 121.77, 121.27, 76.84, 62.17, 54.09, 52.36, 32.56. IR (ATR) 3146, 1732, 1598,
-1
+
444, 746, 699 cm . MS (FAB) m/z 553.3 (M+H) ; HRMS (FAB). Calcd for
: 553.2604. Found: 553.2651.
C
36
H
33
O
2
N
4
18

α
τ
4
.1.14. (S)-N -(quinolin-3-ylmethyl)-N -tritylhistidine methyl ester (TH-13)
As the aldehyde, quinoline-3-carbaldehyde was used. The residue was purified on
NH silica gel column (eluted with n-Hexane : EtOAc = 2:1) to afford TH-13 (125 mg,
4
8
7
5.2%) as a low melting white powder. 1H NMR (600MHz, MeOD) δ
.78(d,J=2.1Hz,1H),8.17 (d, J=1.2Hz, 1H), 8.00 (d, J=8.3Hz, 1H), 7.83–7.79 (m, 1H),
.70 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.55 (ddd, J = 8.1, 6.9, 1.1 Hz, 1H), 7.37 (d, J = 1.4
Hz, 1H), 7.30 – 7.26 (m, 9H), 7.11 – 7.06 (m, 6H), 6.68–6.65 (m, 1H), 3.98
(d,J=13.7Hz,1H),3.84(d, J=13.7Hz, 1H), 3.63 (t, J=6.8Hz, 1H), 3.57(s, 3H), 2.95–2.91
1
3
(
m, 1H), 2.89 (dd, J = 14.1, 6.5 Hz, 1H). C NMR (151 MHz, MeOD) δ 175.77, 152.41,
1
1
1
47.88, 143.55, 139.39, 137.84, 136.88, 134.27, 130.79, 130.78, 130.70, 129.45, 129.28,
29.21, 129.07, 128.90, 128.14, 121.22, 76.76, 62.11, 52.31, 50.11, 32.64. IR (ATR) 3312,
-1
+
731, 1493, 1444, 699 cm . MS (FAB) m/z 553.3 (M+H) ; HRMS (FAB). Calcd for
Na: 575.2423. Found: 575.2437.
C
36
H
32
O
2
N
4
α
τ
4
.1.15. (S)-N -picolinoyl-N -tritylhistidine methyl ester (TH-14)
3
3
A mixture of TH-2 (412 mg, 1 mmol) and Triethyl amine (0.28 mL, 2 mmol) in 50
ml CH Cl was cooled to 0°C. A solid picolinoyl chloride hydrochloride (178 mg, 1.0
mmol) was added slowly and the reaction mixture was stirred at 0 °C for 2 h then at room
temperature overnight. The solvent was washed with saturated NaHCO solution (2 x 20
mL) and the aqueous layer was extracted with CH Cl (2 × 30 mL). The combined organic
extracts were dried over Na SO and concentrated under vacuo, and the residue was
2
2
3
2
2
2
4
purified on a flash silica gel column (eluted with EtOAc) to afford TH-14 (124 mg, 24%)
1
as a colorless viscous oil. H NMR (600 MHz, MeOD) δ 8.57 (ddd, J = 4.7, 1.5, 0.9 Hz,
1
1
1
H), 8.07 – 8.03 (m, 1H), 7.96 (td, J = 7.7, 1.7 Hz, 1H), 7.57 (ddd, J = 7.6, 4.8, 1.2 Hz,
H), 7.37 (d, J = 1.3 Hz, 1H), 7.34 – 7.24 (m, 9H), 7.06 (dd, J = 8.4, 1.1 Hz, 6H), 6.72 (s,
H), 4.93 (dd, J = 7.9, 4.8 Hz, 1H), 3.71 (s, 3H), 3.21 (dd, J = 14.7, 4.8 Hz, 1H), 3.13 (dd,
1
3
J = 14.7, 8.0 Hz, 1H). C NMR (151 MHz, MeOD) δ 173.00, 166.18, 150.47, 149.90,
1
5
43.58, 139.72, 138.81, 137.06, 130.82, 129.28, 129.18, 127.98, 123.26, 121.43, 76.82,
-1
3.81, 52.96, 31.15. IR (ATR) 3058, 1742, 1672, 747, 699 cm . MS (FAB) m/z 517.5
+
(
M+H) ; HRMS (FAB). Calcd for C32
H
29
O
3
4
N : 517.2240. Found: 517.2250.
α
α
τ
4
.1.16. (S)-N ,N -dibenzyl-N -tritylhistidine methyl ester (TH-15)
To a solution of TH-233 (147 mg, 0.36 mmol) in 20 ml ClCH
CH Cl was added
2
2
19

benzaldehyde (110 μl, 1.08 mmol) and NaBH(OAc)
was stirred at room temperature for 48 h under argon atmosphere. A solution of saturated
aqueous NaHCO (15 ml) was added and the organic layer was separated. The aqueous
layer was extracted with CH Cl (20 ml X 3). The combined organic layers were dried
3
(230 mg, 1.08 mmol). The mixture
3
2
2
over Na
2
SO
4
and concentrated in vacuo. The residue was purified on NH silica gel column
1
(
eluted with EtOAc) to afford TH-15 (160 mg, 75.1%) as a low melting white solid. H
NMR (600 MHz, MeOD) δ 7.41 – 7.34 (m, 9H), 7.30 (s, 1H), 7.22 – 7.11 (m, 16H), 6.60
s, 1H), 3.88 (d, J = 13.8 Hz, 2H), 3.73 (t, J = 7.6 Hz, 1H), 3.70 (s, 3H), 3.49 (d, J = 13.8
(
1
3
Hz, 2H), 3.00 – 2.89 (m, 2H). C NMR (151 MHz, MeOD) δ 174.04, 143.74, 140.70,
1
5
39.17, 138.57, 130.92, 129.94, 129.36, 129.25, 129.21, 128.02, 120.74, 76.82, 62.66,
-1
5.74, 51.63, 29.48. IR (ATR) 3059, 2947, 1727, 1492, 1444, 1156, 744, 696 cm .MS
+
(
FAB) m/z 592.4 (M+H) ; HRMS (FAB). Calcd for C40
H
38
O
2
3
N : 592.2964. Found:
5
92.2965.
4
.2. Molecular docking study
2
4
The Sirt2 X-ray structure Sirt2-SirReal2-NAD (PDB code 4RMG) was used in the
present study. The protein structure was prepared by using the Structure Preparation
module in MOE 2014.09 (Molecular Operating Environment, Version 2014.09, Chemical
Computing Group Inc., Montreal, Canada). Water molecules and ligand atoms except the
zinc ion and the conserved water molecule bridging the interaction between Pro94 and
the carbonyl group of SirReal2 were removed from the structure. Docking studies were
3
2
performed using the rigid-receptor method . The co-crystallized ligand (SirReal2) was
defined as the center of the binding site. Thirty docking poses were calculated for each
3
9
ligand. All other options were left at their default values. The generated docking poses
were visualized using MOE 2014.09.
4
4
.3. Biological Assays
.3.1. In vitro inhibitory activities against SIRT1, 2, and 3
Deacetylase activities of sirtuins including SIRT1, 2, and 3 were measured with an
electrophoretic mobility shift assay or a fluorogenic assay as described previously.31,32
For the electrophoretic mobility shift assay to measure SIRT2 activity, recombinant
SIRT2 proteins were incubated with a carboxyfluorescein (FAM)-labeled fluorescent
peptide (FAM-RHKK(Ac)LM) and 1 mM NAD in 50 μL assay buffer (25 mM Tris-HCl
[
pH 9.0], 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl , 0.1 mg/mL BSA) in 384-well plates.
2
After 60 min at 37 °C, the reaction was stopped by adding nicotinamide (final
concentration 10 mM) in 50 μL stop buffer (100 mM HEPES [pH 7.5], 10 mM EDTA,
20

0
.25% CR-3). The samples were analyzed using a LabChip EZ Reader II (PerkinElmer,
Waltham, MA, USA). Percent conversion is defined as 100 × P/(P+S), where P and S are
peak heights of product and peptide substrate, respectively. For fluorogenic assays to
measure SIRT1, 2, and 3 activities, recombinant sirtuin proteins were incubated with
aminomethylcoumarin (AMC)-conjugated acetylated lysine peptides (RHKK(Ac)-MCA
for SIRT1 and SIRT2; VSTPVK(Ac)-MCA for SIRT3) and 1 mM NAD in 20 μL assay
buffer (50 mM Tris-HCl [pH 9.0], 4 mM MgCl , 0.2 mM DTT) at 37 °C for 60 min. The
2
reaction was stopped by the addition of 20 μL trypsin (20 mg/mL) at 37 °C for 15 min.
The released AMC was measured using a fluorescence plate reader. The IC50 values were
determined as the means with standard deviation calculated from at least three
independent dose–response curves using Origin software (OriginLab, Northampton, MA,
USA).
4
.3.2. Cell culture
The human breast cancer cell line MCF7 (provided by the RIKEN BRC through the
National Bio-Resource Project of the MEXT/AMED, Japan),40 human chronic
41
42
myelogenous leukemia cell line K562, and human T cell leukemia cell line MT-2 were
maintained in RPMI1640 medium (Wako) supplemented with 10% heat-inactivated fetal
bovine serum.
4
.3.3. Cytotoxic activities
3
MCF7 cells (1 × 10 cells) were seeded in wells of a 24-well plate, and then
compounds were added. The MTT assay43 (MTT was purchased from Dojindo,
Kumamoto, Japan) was performed 4 days later. For experiments with K562 and MT-2
5
cells, nearly the same conditions were used, except that 1 × 10 cells/well were plated,
and the incubation time was 1 day.
Acknowledgements
We are grateful to the Screening Committee of Anticancer Drugs supported by Grant-in-
Aid for Scientific Research on Innovative Areas, Scientific Support Programs for Cancer
Research (No. 221S0001), from The Ministry of Education, Culture, Sports, Science and
Technology, Japan (MEXT), for assistance with the evaluation of compounds. This work
was supported by a Grant for Joint Research Project with Science Farm Ltd. The study
was also supported in part by a Grant-in-Aid for Challenging Exploratory Research to
M.O. (24659048). MCF7 cells were provided by the RIKEN BRC through the National
21

Bio-Resource Project of the MEXT/AMED, Japan.
Declarations of interest
The authors declare no conflict of interest.
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25

Graphical Abstract
HPH-2Trt
TH-2
TH-1
TH-3
SIRT2 IC50 = 8.8mM
MCF-7 IC50 = 1.72mM
SIRT2 IC50 = 43.0mM SIRT2 IC50 = 1.7mM
MCF-7 IC50 = 49.1mM MCF-7 IC50 = 0.77mM
SIRT2 IC50 = 1.3mM
MCF-7 IC50 = 0.71mM