Epidithiodiketopiperazine as a pharmacophore for protein lysine methyltransferase G9a inhibitors: Reducing cytotoxicity by structural simplification

Article abstract of DOI:10.1016/j.bmcl.2012.11.087

Chaetocin (1), a structurally complex epidithiodiketopiperazine (ETP) alkaloid produced by Chaetomium minutum, is a potent inhibitor of protein lysine methyltransferase G9a, which plays important roles in many biological processes. Here we present our syn

Full text of DOI:10.1016/j.bmcl.2012.11.087

Bioorganic & Medicinal Chemistry Letters 23 (2013) 733–736
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Epidithiodiketopiperazine as a pharmacophore for protein lysine
methyltransferase G9a inhibitors: Reducing cytotoxicity by structural
simplification
Shinya Fujishiro a, Kosuke Dodo a,b, Eriko Iwasa a,b, Yuou Teng a,b, , Yoshihiro Sohtome a,
Yoshitaka Hamashima a,c, Akihiro Ito a, Minoru Yoshida a, Mikiko Sodeoka a,b,
⇑
a RIKEN Advanced Science Institute, 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan
b Sodeoka Live Cell Chemistry Project, ERATO, JST, 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan
c School of Pharmaceutical Sciences, University of Shizuoka, 52-1, Yada, Suruga-ku, Shizuoka 422-8526, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Chaetocin (1), a structurally complex epidithiodiketopiperazine (ETP) alkaloid produced by Chaetomium
minutum, is a potent inhibitor of protein lysine methyltransferase G9a, which plays important roles in
many biological processes. Here we present our synthetic investigations to identify a simple prototype
G9a inhibitor structure based on structure–activity relationship (SAR) studies on chaetocin derivatives.
The simple derivative PS-ETP-1 (14) was found to be a potent G9a inhibitor with greatly reduced
cytotoxicity.
Received 23 October 2012
Revised 10 November 2012
Accepted 21 November 2012
Available online 5 December 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Methyltransferase inhibitor
Protein methylation
Epigenetics
Epidithiodiketopiperazines
Chaetocin
Protein lysine methyltransferases (PKMTs) dynamically regu-
late gene expression profiles through methylation of lysine resi-
dues of histone proteins.1 In contrast to invariant factors encoded
in the DNA sequence, epigenetic gene expression control based
on chromatin architecture is reversibly governed by acquired envi-
ronmental factors.2 It is becoming clear that PKMTs play critical
roles in a range of diseases, and so discovery of potent PKMT inhib-
itors is attracting increasing attention.3 G9a inhibitors are of par-
reported (Fig. 1).13,14 Because 1 exhibits various biological activi-
ties,15–18 structural development aimed at increasing its selectivity
for PKMTs is important. For example, Imhof and co-workers
reported that the cytotoxicity of 1 adversely effected their cellular
assays.13 However, little is known about the structure–activity
relationships (SAR) of 1 for PKMT-inhibitory activity,19 probably
because of the structural complexity of 1. The difficulties in syn-
thesizing 1 arise from (i) the high density of functionalities on
the ETP scaffold, and (ii) the dimeric structure connected with a
chiral quaternary carbon.20 In order to address these issues, we
recently established a strategy based on early oxidation of the
diketopiperazine (DKP) moiety to introduce the disulfide function-
alities under Lewis acidic conditions; this approach is critical to
avoid b-elimination of the hydroxymethyl group on ETPs. This
strategy was the basis of the first total synthesis of 1.21–23
ticular interest,4–7 because G9a is involved in
a variety of
biological processes, including tumor cell growth,8,9 gene silencing
in ES cells,10,11 and cocaine-induced neuronal responses.12 Here we
present our studies on the development of epidithiodiketopiper-
azine (ETP)-type G9a inhibitors as simplified prototype PKMT
inhibitors, based on structure–activity relationships (SAR) studies
of chaetocin (1) derivatives. The cytotoxicity and thioredoxin
reductase (TrxR)-inhibitory activity of the newly developed ETPs
are also discussed.
We also reported preliminary SAR studies on 1, focusing on
G9a-inhibitory activity,21,22 cell-death-inducing activity in human
leukemia HL-60 cells,24 and thioredoxin reductase (TrxR)-inhibi-
tory activity.25 We found that the disulfide structure in 1 plays
an important role in the G9a-inhibitory activity by comparison of
1 with S-deficient chaetocins 2, including both enantiomers.21,22
On the other hand, an enantiomer of chaetocin, ent-1, inhibited
G9a as potently as 1,21,22 while it displayed more potent apopto-
Chaetocin (1), a naturally occurring product isolated from fungi
of Chaetomium species, was the first inhibitor to have been
⇑
Corresponding author.
E-mail address: sodeoka@riken.jp (M. Sodeoka).
Present address: College of Biotechnology, Tianjin University of Science and
Technology, No. 29, 13th Street, Tianjin Economic-Technology Development Area,
Tianjin 300457, China.
sis-inducing activity than
1
in HL-60 cells.24 These results
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.087

734
S. Fujishiro et al. / Bioorg. Med. Chem. Lett. 23 (2013) 733–736
H
N
H
N
O
O
O
HO
Me
O
HO
Me
O
O
H
N
H
N
HO
Me
Me
OH
H
R
(b)
(b)
N
N
S2
O
O
O
S2
N
H
9
N
H
Boc
N
N
Me
OH
Me
OH
N
N
N
H
N
S2
O
OTBS
O
OTBS
(a)
N
H
N
H
N
H
N
H
7: R = Br
O
8: R = H
Chaetocin(1)
IC50 = 2.4 µM
S-deficient chaetocin (2)
IC50 > 50 µM
O
O
HO
Me
H
Me
OH
R
N
N
S2
H
H
O
HO
Me
O
HO
Me
N
H
N
H
Boc
H
N
N
H N
N
H
N
N
O
ent-9
OTBS
O
O
O
O
OTBS
(a)
S2
N
N
Me
OH
Me
OH
N
N
ent-7: R = Br
S2
O
O
ent-8: R = H
N
H
N
H
N
H
N
H
O
Scheme 2. Reagents and conditions: Synthesis of 9 and ent-9. (a) n-Bu3SnH, V-70,
benzene, rt (quant.); (b) H2S, BF3ꢀOEt2, CH2Cl2, ꢁ78 to ꢁ20 °C, I2, EtOAc (9: 27% in 2
steps, ent-9: 34% in 2 steps).
1
2
ent-chaetocin(ent- )
IC50 = 1.7 µM
ent-
IC50 > 50 µM
Figure 1. Structures and reported G9a-inhibitory activities22 of 1, ent-1, 2 and
ent-2.
oxidation. The bridged-monosulfide chaetocin 6 was synthesized
from chaetocin according to the reported protocol, using triphenyl-
phosphine as a reductant.26 These modified chaetocin derivatives
were tested for G9a-inhibitory activity by using a modified ELISA
assay (AlphaScreen™ system, PerkinElmer Inc.). The results are
summarized in Table 1. In this new assay system, the IC50 value
suggested that the cytotoxicity of 1 is not necessarily linked to
G9a-inhibitory activity.
Encouraged by these preliminary findings, we planned further
SAR studies on 1 in order to identify the essential core structure,
including functional groups, of chaetocin (1) for G9a inhibition.
We initially designed and synthesized TBS-protected chaetocin 4,
reduced chaetocin 5 with four thiol groups, and bridged-monosul-
fide 6. The designed compounds were synthesized using the previ-
of chaetocin was determined as 7.2
lM, which is slightly higher
than the value (IC50 = 2.4 M) previously obtained using the origi-
l
nal ELISA assay. A similar tendency was observed with the previous
SAR studies using 1, ent-1, 2 and ent-2.21 The TBS-protected
chaetocin 4 showed similar activity (IC50 = 7.2 lM) to that of chae-
tocin (1), implying that the hydroxyl groups in 1 are not critical for
G9a inhibition. On the other hand, 5 showed less potent inhibitory
activity than 1, and 6 was inactive. These results highlight the
importance of the disulfide functionalities in 1 for the G9a-
inhibitory activity.
In order to examine the effect of the dimeric structure of chae-
tocin (1), we next synthesized monomeric ETP derivatives. As
shown in Scheme 2, both enantiomers of monomeric derivatives
of 1 were synthesized from diketopiperazine 7 and ent-7, the
dimerization precursors for 1 and ent-1.21,22 After reduction of
the Br group in 7 and ent-7 using n-Bu3SnH and V-70, the resulting
8 and ent-8 were treated with H2S in the presence of BF3ꢀOEt2 to
afford the corresponding 9 and ent-9. As shown in Table 1, mono-
ously reported synthetic intermediate
3 (Scheme 1). In the
synthesis of 4 using the procedure with H2S in the presence of
BF3ꢀOEt2, we found that controlling the reaction temperature
(ꢁ78 to ꢁ20 °C) is important to avoid deprotection of the TBS
groups on 3. Subsequent iodine oxidation of the crude mixture pro-
moted S–S formation to give the TBS-protected chaetocin 4, albeit
with low yield. Reduced chaetocin 5 was isolated using a proce-
dure similar to the original one, but without the iodine-mediated
H
N
TBSO
Me
O
H
N
O
S2
N
Me
meric ETP 9 (IC50 = 3.3 lM) showed slightly stronger G9a-inhibi-
Boc
N
TBSO
O
O
S2
(a)
(b)
O
H
N
tory activity than the parent chaetocin (1), indicating that the
dimeric ETP structure of 1 is not necessary for G9a inhibition. It
is also noteworthy that ent-9 showed weaker activity
(IC50 = 23.5 lM). These results indicate that the chirality of the
ETP framework affects the G9a-inhibitory activity.
N
H
N
N
H
O
O
HO
Me
O
OTBS
N
HO
Me
OH
OH
N
4
N
H
Boc
H
N
HO
O
O
N
H
N
OTBS
Finally, simple ETP derivatives derived from proline and serine,
13, 14 (PS-ETP-1) and 15, were synthesized (Scheme 3) and evalu-
O
O
HS
Me
3
N
N
HS
Me
SH
ated. First, the DKP 10 was prepared from N-Cbz-L-proline and
SH
N
N
N
H
L-serine-methyl ester in 5 steps. Then, to increase the oxidation state
(c)
N
H
of the DKP, 10 was subjected to radical bromination followed by
treatment with phosphate buffer. Tribromination and subsequent
hydrolysis proceeded to give the racemic diol 11.27 After removal
of the undesired bromo group in 11 by means of radical reduction,
the hydroxyl groups were replaced with thiols to give the corre-
sponding di-thiol 13 as a single diastereomer. Finally, 13 was sub-
jected to I2-mediated oxidation, and careful separation afforded
PS-ETP-1 (14) and its trisulfide derivative 15. As shown in Table
1, the sulfur-bridged derivatives PS-ETP-1 (14) and 15 showed sim-
OH
5
6
H
N
H
N
HO
Me
O
HO
Me
O
S
H
N
H
N
O
O
S
S2
(d)
N
Me
OH
Me
OH
N
S2
O
O
N
H
N
H
N
N
H
O
O
H
1
Chaetocin( )
ilar G9a-inhibitory activity [14: IC50 = 5.2
lM, 15: IC50 = 5.3
lM].
Dithiol derivative 13 was less potent (IC50 = 13.1
l
M). These SAR
Scheme 1. Reagents and conditions: Synthesis of chaetocin derivatives 4, 5 and 6.
(a) H2S, BF3ꢀOEt2, CH2Cl2, ꢁ78 to ꢁ20 °C, I2, EtOAc (6% in 2 steps); (b) H2S, BF3ꢀOEt2,
CH2Cl2, ꢁ78 °C to rt (27%); (c) H2S, BF3 ꢀOEt2, CH2Cl2, ꢁ78 °C to rt, I2, EtOAc (44% in 2
steps); (d) PPh3, CH2Cl2 (40%).
studies are consistent with the results obtained for chaetocin (1)
and tetra-thiols 5. Thus, the simple ETP structure appears to be
available as a pharmacophore for G9a inhibitors.

S. Fujishiro et al. / Bioorg. Med. Chem. Lett. 23 (2013) 733–736
735
Table 1
G9a-inhibitory activity of chaetocin derivatives
Compound
IC50
7.2
>100
6.4
(
lM)
Compound
IC50
3.3
(lM)
O
Chaetocin (1)
S-Deficient Chaetocin (2)
ent-Chaetocin (ent-1)
ent-2
Me
H
N
N
S2
N
H
>100
N
H
O
O
OTBS
Me
9
H
N
TBSO
Me
O
H
N
H
S2
O
S2
N
H
N
Me
N
7.2
N
H
23.5
S2
O
9
OTBS
O
N
H
ent-
N
H
O
OTBS
4
H
O
HO
O
O
HS
H
N
N
Me
SH
N
N
O
O
HS
N
N
HS
Me
SH
Me
SH
N
27.1
13.1
O
13
OH
N
H
N
OH
H
5
6
H
N
O
HO
O
S
H
N
Me
OH
S2
O
S
N
N
N
Me
OH
Me
N
>100
5.2
5.3
O
14
O
N
H
N
H
O
O
Me
OH
N
S3
O
15
O
H
N
HCl
O
100
80
60
40
20
0
H
Me
(a)–(e)
H2N
H
CO2Me
OH
N
H
OH
N
Cbz
O
10
OTBS
3
O
O
O
Br
HO
N
HO
N
Me
OH
10
30
Me
OH
(f)
(g)
N
N
OTBS
O
( )-11
OTBS
( )-12
O
O
O
HS
Me
OH
Me
Me
SH
(i)
N
N
(h)
N
Figure 2. Cytotoxicity of chaetocin derivatives in HL-60 cells. HL-60 cells were
treated with chaetocin derivatives for 4 h, and cell viability was determined by
alamarBlue assay.25
S3
S2
N
N
N
O
O
OH
O
OH
( )-13
( )-14
PS-ETP-1
( )-15
the newly developed simple derivatives 13–15 was significantly
reduced, compared with that of the parent chaetocin (1)/ent-1 or
monomeric ETPs 9/ent-9 at the concentrations we tested.
Thioredoxin reductase (TrxR) was also reported to be a target of
chaetocin (1), and TrxR-inhibitory activity was suggested to be
associated with the cytotoxic effects of 1.28 To examine the selec-
tivity between G9a and TrxR, we finally examined the TrxR-inhib-
itory activity of PS-ETP-1 (14) and 15. Enzymatic reaction of TrxR
Scheme 3. Reagents and conditions: Synthesis of 13, 14 and 15. (a) EDCꢀHCl, HOBt,
Et3N, CH2Cl2; (b) TBSCl, imidazole, DMF (94% in 2 steps); (c) H2, 10% Pd/C, EtOH; (d)
NH4OH aq, MeOH (99%, in 2 steps); (e) MeI, NaH, DMF (91%); (f) NBS, AIBN, CCl4,
reflux, phosphate buffer (pH 7), MeCN (64% in 2 steps); (g) n-Bu3SnH, AIBN,
benzene, reflux (83%); (h) H2S, BF3ꢀOEt2, CH2Cl2, ꢁ78 °C to rt (13: 87%); (i) I2, EtOAc
(14: 65% in 2 steps, 15: 5% in 2 steps).
was quantified in the presence of these compounds (10 lM) based
Reducing the cytotoxicity of ETPs is also an important consider-
ation for the development of selective PKMTs inhibitors. Thus, we
next examined the cytotoxicity of these compounds using human
leukemia HL-60 cells (Fig. 2).26 We found that the cytotoxicity of
on the reduction of 5,50-dithiobis(2-nitrobenzoic) acid (DTNB)
using a Thioredoxin Reductase Assay Kit (Cayman). As shown in
Figure 3, PS-ETP-1 (14) and 15 showed almost no inhibition of

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S. Fujishiro et al. / Bioorg. Med. Chem. Lett. 23 (2013) 733–736
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Figure 3. Thioredoxin reductase (TrxR)-inhibitory activity of chaetocin derivatives.
TrxR reaction was quantified in the presence of chaetocin derivatives (10
lM) based
on the change in absorbance (405 nm).25
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TrxR, whereas chaetocin (1) was inhibitory. Because 1 was re-
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redoxin (Trx),28 which has both disulfide bridges and indole
structure in the TrxR-binding site,29 we speculated that elimina-
tion of the indole structure from 1 might have contributed to
reducing its TrxR-inhibitory activity, perhaps accounting for the
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In conclusion, we have identified the simple ETP derivative PS-
ETP-1 (14)30 as a novel prototype for G9a inhibitors. This finding
may also be helpful in the design of other PKMT inhibitors, which
are complementary to peptide substrate–competitive inhibitors.5,6
In addition, the cytotoxicity and TrxR-inhibitory activity of 14 were
significantly reduced, compared with those of chaetocin (1). Fur-
ther structural developments of ETPs aimed at constructing enan-
tiomerically distinct environments with improved selectivity, as
well as application to cellular assays, are ongoing in our research
group.
Acknowledgments
25. Sodeoka, M.; Dodo, K.; Teng, Y.; Iuchi, K.; Hamashima, Y.; Iwasa, E.; Fujishiro, S.
Pure Appl. Chem. 2012, 84, 1369.
26. Cherblanc, F.; Lo, Y.-P.; De Gussem, E.; Alcazar-Fuoli, L.; Bignell, E.; He, Y.;
Chapman-Rothe, N.; Bultinck, P.; Herrebout, W. A.; Brown, R.; Rzepa, H. S.;
Fuchter, M. J. Chem. Eur. J. 2011, 17, 11868.
27. Similar overbromination of DKP under radical conditions was reported. See,
Caballero, E.; Avendaño, C.; Menéndez, J. C. Tetrahedron 1999, 55, 14185.
28. Tibodeau, J. D.; Benson, L. M.; Isham, C. R.; Owen, W. G.; Bible, K. C. Antioxid.
Redox Signal. 2009, 11, 1097.
We thank Ms. Akiko Nakata for G9a inhibition assay. This work
was supported in part by Scientific Research (C) from the Ministry
of Education, Culture, Sports, Science and Technology, and Project
Funding from RIKEN.
References and notes
29. Fritz-Wolf, K.; Kehr, S.; Stumpf, M.; Rahlfs, S.; Becker, K. Nat. Commun. 2011, 2,
383. http://dx.doi.org/10.1038/ncomms1382.
30. PS-ETP-1 (14): 1H NMR (500 MHz, CDCl3) d 2.21–2.41 (m, 1H), 3.04 (ddd,
J = 8.1, 10.4, 14.4 Hz, 1H), 3.16 (s, 3H), 3.55 (dd, J = 5.7, 9.5 Hz, 1H), 3.62 (m,
1H), 3.91 (ddd, J = 3.4, 8.1, 11.5 Hz, 1H), 4.25 (dd, J = 9.5, 12.6 Hz, 1H), 4.36 (dd,
J = 5.7, 12.6 Hz, 1H); 13C NMR (125 MHz, CDCl3) d 23.1, 27.2, 32.8, 46.2, 60.9,
74.7, 76.3, 163.3, 166.6; HRMS (ESI) (m/z) calcd. for C9H12N2O3S2Na [M+Na]+:
283.0187, found: 283.0182.
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