Development of novel bisubstrate-type inhibitors of histone methyltransferase SET7/9

Article abstract of DOI:10.1016/j.bmc.2010.10.022

Histone modification, for example, by histone deacetylase (HDAC) and histone lysine methyltransferase (HMT), plays an important role in regulating gene expression. To obtain novel inhibitors as tools for investigating the physiological function of members of the HMT family, we designed and synthesized novel inhibitors, which are amine analogues of adenosylmethionine (AdoMet; the cofactor utilized in the methylation reaction) bearing various alkylamino groups coupled via an ethylene linker. The inhibitory activities of these compounds towards SET7/9, an HMT, were evaluated. It was found that introduction of an alkylamino group increased the inhibitory activity.

Full text of DOI:10.1016/j.bmc.2010.10.022

Bioorganic & Medicinal Chemistry 18 (2010) 8158–8166
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of novel bisubstrate-type inhibitors of histone methyltransferase
SET7/9
Shuichi Mori a, Kenta Iwase a, Naoko Iwanami b, Yujiro Tanaka a, Hiroyuki Kagechika a,b, Tomoya Hirano b,
⇑
a Graduate School of Biomedical Science, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
b Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Histone modification, for example, by histone deacetylase (HDAC) and histone lysine methyltransferase
(HMT), plays an important role in regulating gene expression. To obtain novel inhibitors as tools for
investigating the physiological function of members of the HMT family, we designed and synthesized
novel inhibitors, which are amine analogues of adenosylmethionine (AdoMet; the cofactor utilized in
the methylation reaction) bearing various alkylamino groups coupled via an ethylene linker. The inhib-
itory activities of these compounds towards SET7/9, an HMT, were evaluated. It was found that introduc-
tion of an alkylamino group increased the inhibitory activity.
Received 7 September 2010
Revised 6 October 2010
Accepted 7 October 2010
Available online 14 October 2010
Keywords:
Histone methyltransferase
Small molecule inhibitor
Bisubstrate inhibitor
Epigenetics
Ó 2010 Elsevier Ltd. All rights reserved.
SET7/9
1. Introduction
cofactors of the methylation reaction were also reported to be
inhibitors; for example, AzaAdoMet, an analogue of AdoMet in
Post-translational modification of histone protein plays an
important role in the regulation of gene expression, and can be
controlled by histone-modifying enzymes.1–3 Histone lysine meth-
ylation, which is regulated by histone lysine methyltransferase
(HMT) and histone demethylase, is one of those modifications,
and refers to covalent methylation of histone lysine tails to pro-
duce mono-, di- or trimethylated states.4,5 In the methylation reac-
tion, the methyl group of the cofactor adenosylmethionine
(AdoMet) is transferred to nitrogen of the substrate lysine residue,
yielding methylated lysine and adenosylhomocysteine (AdoHcy)
(Fig. 1). Recently, various reports have suggested that HMTs are in-
volved in the development of various diseases, including cancer.6,7
For example, E2H2, which targets Lys27 of histone 3 (H3K27), is
overexpressed in many different types of cancer.8 In addition,
substrates of some HMTs are not limited to histone.9 Tumor sup-
pressor protein p53 is methylated at K372 by SET7/9 or at K370
by Smyd2.10,11 Other substrates of SET7/9 include Tat protein of
which the bridging sulfur atom is replaced with nitrogen, and
Sinefungin inhibit AdoMet-dependent methyltransferases, such
as DNA methyltransferase.16,17 In this paper, we report the design
and synthesis of novel derivatives of these cofactors, together with
the results of assay of their inhibitory activities towards histone
methyltransferase SET7/9.
2. Results
2.1. Design of novel inhibitor candidates
The crystal structure of the ternary complex of human SET7/9
with histone substrate peptide and cofactor AdoHcy has been eluci-
dated.18 In this structure, the peptide substrate and cofactor bind on
opposite surfaces of the enzyme. The target lysine residue (Lys4) of
the substrate peptide is inserted into a narrow channel passing
through the enzyme, affording access to the cofactor ( Fig. 3a).
Cofactors or their analogues conjugated with a substructure of the
substrate could be utilized as bisubstrate inhibitors, which are able
to interact with both substrate-binding sites, in this case the cofac-
tor-binding site and the substrate peptide-binding site, to provide
high potency and selectivity.19 For example, ATP derivatives conju-
gated with substrate peptide have been reported as bisubstrate
kinase inhibitors,20 and a conjugate of substrate estradiol with
adenosine, which is a substructure of cofactor NADPH, has been
reported as an inhibitor of 17b-hydroxysteroid dehydrogenase.21,22
HIV-112 and estrogen receptor a 13
.
To elucidate the physiological function of HMTs, several HMT
inhibitors have been developed (Fig. 2). Chaetocin was identified
as a selective inhibitor of SUV39 from a library of natural
compounds.14 BIX-01294, a G9a inhibitor, was developed based on
screening of a large chemical library.15 In addition, derivatives of
⇑
Corresponding author. Tel.: +81 3 5280 8128; fax: +81 3 5280 8127.
E-mail address: hiraomc@tmd.ac.jp (T. Hirano).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.10.022

S. Mori et al. / Bioorg. Med. Chem. 18 (2010) 8158–8166
8159
Figure 1. Methylation of lysine residue by histone methyltransferase utilizing adenosylmethionine (AdoMet) as a cofactor.
adenosine. In a reported synthetic scheme of AzaAdoMet and its
derivatives, direct alkylation involving hydroxyl, halide, sulfonate
ester, amino and sulfonamide at the 5’ position has been uti-
lized.23,24 So, we first prepared the adenosine derivative with a me-
syl group (2b) from 2’,3’-isopropylidene adenosine (2a) using the
reported method.25 The derivative with an amino group (2c) at
the 5’ position was also prepared by means of a slight modification
of the reported method.26,27 For the synthesis of 1a, Boc-protected
N-methylaminoethylene with an amino (3a) or a nosylamido (3b)
group was prepared,28 for conjugation with compounds 2. How-
ever, the reaction of 3a or 3b with 2b under basic conditions did
not proceed, yielding various by-products. Mitsunobu reaction be-
tween the hydroxyl group of 2a and nosylamido compound 3b,
which has been applied for the synthesis of AzaAdoMet and other
5-aza derivatives of adenosine,29,30 also did not proceed. These re-
sults were thought to be due to the relatively low reactivity of the
5’ position of adenosine and our aminoethylene compounds 3.
Recently, Sajiki’s group reported catalytic monoalkylation of
various primary amines using nitrile as an alkylating reagent.31
In their paper, Rh/C was utilized for monoalkylation of aliphatic
amines, and secondary aliphatic amines were mainly obtained by
using Pd/C. So, we initially utilized Rh/C catalyst for the monoalky-
lation of 2c with Boc-protected methylamino acetonitrile (5a).
However, this reaction did not proceed, whereas the reaction using
Pd/C afforded secondary amine 6a in moderate yield (Scheme 2).
These results were due to the low reactivity of our compounds,
as well as failure of the alkylating reaction shown in Scheme 1.
Compound 6a was further alkylated with iodinated oxazolidinone
(4),24 yielding tertiary amino compound 7a. Then, the oxazolidi-
none ring was opened under basic conditions to provide 8a, and
subsequent deprotection gave compound 1a. Compound 1e, which
does not have additional nitrogen, was similarly prepared starting
from compound 2c and pentylnitrile (5e). Reductive alkylation and
the introduction of oxazolidinone gave tertiary amino compound
7e. After ring opening and deprotection, compound 1e was
obtained.
Figure 2. Structures of HMT inhibitors.
So, based on the structure of the ternary complex, we designed no-
vel derivatives of AzaAdoMet, in which the nitrogen that replaces
the sulfur atom of AdoMet is coupled to various alkylamino groups
(R = methyl: 1a; n-propyl: 1b; n-hexyl: 1c; phenethyl: 1d) via an
ethylene linker as inhibitor candidates (Fig. 3b). The introduced
nitrogen is expected to form a hydrogen-bonding network similar
to that of the lysine residue of substrate peptide, involving
Tyr245 and a water molecule tightly bound to Tyr305. Further,
the alkyl group (R) is expected to interact with hydrophobic side
chains of amino acids that form the channel through the enzyme,
such as Leu267, Tyr305, Tyr335 and Tyr337. Namely, introducing
an alkylamino group is expected to result in enhanced interaction
with the substrate peptide-binding site. In addition, in order to
study the effect of the nitrogen atom, a derivative without such
nitrogen, 1e, was also designed (Fig. 3b).
Next, we tried to prepare compounds 1b, 1c and 1d in a similar
manner. However, in the case of 1b, reductive alkylation reaction
of 2c with the nitrile compound bearing the n-propyl group (7b)
did not proceed, and rearrangement of the Boc group occurred to
yield compound 10 (Scheme 3). Compound 5c with the n-pentyl
group behaved similarly to 5b. So we changed the reactive group
of this conjugation reaction from the nitrile group to diisobutylal-
uminium-imine complex with DIBAL-H, as reported by Van
2.2. Synthesis
The key step in the synthesis of compounds 1a–1e was thought
to be construction of tertiary amine structure at the 5’ position of

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S. Mori et al. / Bioorg. Med. Chem. 18 (2010) 8158–8166
Figure 3. Design of novel inhibitor candidates. (a) On the left, a schematic representation of the structure of SET7/9 ternary complex with histone peptide (shown in green)
and AdoMet is presented, and on the right, our strategy for the design of novel inhibitor candidates is shown. (b) Structure of novel SET7/9 inhibitor candidates 1a–1e.
compounds were further alkylated with oxazolidinone 4 to yield
compounds 7b–7d, then opening of the ozazolidinone ring gave
8b–8d and deprotection afforded 1b–1d.
2.3. Inhibitory activity towards SET7/9
The inhibitory activity of the synthesized compounds towards
histone methyltransferase SET7/9 was examined by utilizing
anti-monomethyl-histone H3 (Lys4) antibody, and the results are
shown in Figure 4. Compounds 1a–1d showed relatively strong
inhibitory activity compared with compound 1e, which lacks the
additional nitrogen. The inhibitory activities were little affected
by the nature of the alkyl group attached to the nitrogen, though
compound 1c with a n-hexyl group was slightly more potent than
the other compounds. Sinefungin, which is another derivative with
a branched nitrogen substituent at the 5’ position of adenosine,
also showed strong inhibitory activity, so a nitrogen atom around
this position seems to enhance inhibitory potency towards SET7/9.
3. Discussion
Compounds 1a–1d were designed as derivatives of AzaAdoMet
bearing an additional alkylamino group, which was expected to
interact with the enzyme in the same manner as the lysine side
chain of the substrate. Compared with 1e, which lacks the addi-
tional nitrogen atom, 1a–1d showed relatively potent inhibitory
activities towards SET7/9. The nature of the alkyl group attached
to this nitrogen had a small influence on the inhibitory activity.
From the reported crystal structure of the ternary complex of hu-
man SET7/9 with histone peptide and cofactor, the cofactor and
substrate peptide bind on opposite surfaces of the enzyme.18 The
side chain of the substrate lysine residue can access AdoMet
through a narrow channel connecting the two surfaces. In the case
of our compounds, it was expected that the alkylamino chain
would pass thorough this channel, and interact with amino acids
forming the channel. Compared with the methyl derivative (1a),
n-propyl derivative (1b) or phenethyl (1d)-conjugated compound,
the n-hexyl compound (1c) was slightly more potent. These results
presumably reflect the better hydrophobic interaction of the
Scheme 1. (a) Retrosynthetic analysis of compounds
construct the secondary amino structure.
1 and (b) attempts to
Wagene’s group.32 The conjugation of 2c with 5b– 5d via the reac-
tion with DIBAL-H proceeded in moderate yield, and secondary
amine compounds 6b–6d were obtained (Scheme 4). These

S. Mori et al. / Bioorg. Med. Chem. 18 (2010) 8158–8166
8161
Scheme 2. Synthesis of compounds 1a and 1e via reductive alkylation.
in order to obtain novel histone lysine methyltransferase inhibi-
tors. From our data in Figure 4, the position of nitrogen is an impor-
tant determinant of the inhibitory activity. Our synthetic scheme
via reductive amination should be useful to produce AzaAdoMet
derivatives substituted with various functional groups from corre-
sponding nitrile compounds. We are adopting this methodology in
further studies aimed at obtaining more potent and selective
inhibitors.
Scheme 3.
4. Conclusion
We have designed and synthesized histone lysine methytrans-
ferase (SET7/9) inhibitors, 1a–1d, consisting of AzaAdoMet substi-
tuted with various alkylamino groups. These compounds showed
relatively potent inhibitory activities, and are expected to be lead
compounds for development of more potent and selective
inhibitors.
n-hexyl group with side chains of amino acids forming the channel,
such as Leu267, Tyr305, Tyr335 and Tyr337. Sinefungin also
showed strong inhibitory activity, so in this case, the presence of
the nitrogen atom around this position of adenosine appeared to
enhance SET7/9 inhibition.
The in situ formation of bisubstrate inhibitors of protein
arginine methyltransferase (PRMT) from a AdoMet analogue and
peptide substrate has already been reported.33 Very recently,
Dowden’s group developed AzaAdoMet analogues with guanidine
functionality linked via various alkyl moieties as protein arginine
methytransferase inhibitors.34 Here, we have developed a versatile
synthetic approach to secondary amine-containing AzaAdoMet
analogues via reductive amination with various nitrile compounds
5. Experimental
5.1. General information
All reagents were purchased from Sigma–Aldrich Chemical Co.,
Tokyo Kasei Kogyo Co., Wako Pure Chemical Industries, and Kanto

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S. Mori et al. / Bioorg. Med. Chem. 18 (2010) 8158–8166
Scheme 4. Synthesis of compounds 1b–1d.
Figure 4. Inhibitory activities of compounds 1a–1e and sinefungin on SET7/9. Monomethylation of H3K4 by SET7/9 and AdoMet (2
1–100 M were detected with anti-monomethyl-histone H3 (Lys4), HRP-fused secondary antibody and substrate for HRP. The activity was quantified in terms of OD450,
shown as a percentage of the value with no inhibitor, taken as 100%. Values are the mean SD for separate experiments (n = 3).
lM), and its inhibition by each inhibitor at
l
Kagaku Co., Inc. Silica gel for column chromatography was pur-
chased from Kanto Kagaku Co., Inc. 1H and 13C NMR spectra were
recorded on Bruker Avance 500 instrument. The following abbrevi-
ations are used for spin multiplicity: s = singlet, d = doublet,
dd = doublet of doublet, t = triplet, q = quartet, m = multiplet,
br = broad, brs = broad singlet, br m = broad multiplet. Mass spec-
tral data was obtained on a Bruker Daltonics microTOF-2focus in
the positive and negative ion detection modes. Melting points,
determined on a Yanaco Micro Melting Point Apparatus, are uncor-
rected. Preparative reversed-phase HPLC was performed with a
5.2. Synthesis
5.2.1. Synthesis of compound 6a
Compound 2c (0.30 g, 0.98 mmol) was added to Pd/C (21 mg,
20 mol %) and 5a (0.83 g, 5.0 equiv) in methanol (5.0 ml). The flask
was purged with hydrogen. The mixture was stirred for 26 h at
room temperature, then filtered through a Celite bed and the fil-
trate was concentrated in vacuo. The residue was purified by flash
column chromatography (neat dichloromethane then 3% methanol
in dichloromethane) to afford 6a (0.37 g, 0.80 mmol, 82%) as a col-
orless foam.
reversed-phase column (Mightysil RP-18 GP 250–20 (5 lm)) on
JASCO UV-2070, PU-2080, PU-2080 and MX-2080-32 components.
Analytical thin layer chromatography (TLC) was performed on
Merck precoated analytical plates, 0.25 mm thick, Silica Gel 60
Compound 6a. 1H NMR (500 MHz, DMSO-d6) d 8.32 (s, 1H), 8.14
(s, 1H), 7.30 (s, 2H), 6.07 (d, J = 2.5 Hz, 1H), 5.44 (br, 1H), 4.94 (d,
J = 6.0, 3.0 Hz, 1H), 4.20–4.17 (m, 1H), 3.20 (br, 1H), 3.14–3.10
(m, 2H), 2.76–2.58 (m, 4H), 2.69 (s, 3H), 1.52 (s, 3H), 1.32 (s, 9H),
1.22 (s, 3H); 13C NMR (125 MHz, CD3OD) d 156.25, 156.01,
148.92, 140.54, 119.29, 114.20, 90.23, 85.29, 83.40, 82.47, 79.64,
50.74, 46.75, 33.47, 27.23, 26.09, 24.17; HRMS (ESI+) calcd for
F254. Preparative TLC separations were made on Merck precoated
analytical plates, 0.50 mm thick, Silica Gel 60 F254. All non-aqueous
reactions were carried out in oven-dried glass apparatus under a
slight positive pressure of argon. All solvents were used after hav-
ing been dried over molecular sieves 3A or 4A or commercially
available dehydrated solvents. For the assay of methyltransferase
activity, OD450 was recorded on a PerkinElmer 1420 multilabel
counter, ARVO MX. All other reagents were commercial products
and were used without further purification.
C
21H34N7O5 (M++H) 464.2616, found 464.2612.
5.2.2. Synthesis of compound 7a
N,N-Diisopropylethylamine (75.2 lmol, 2.0 equiv) was added to
a stirred solution of 6a (105 mg, 1.3 equiv) and 4 (100 mg,

S. Mori et al. / Bioorg. Med. Chem. 18 (2010) 8158–8166
8163
0.22 mmol) in acetonitrile (1.5 ml) at room temperature. The mix-
ture was stirred at 60 °C for two days, then the solvent was re-
moved in vacuo. The residue was purified by flash column
chromatography (3% methanol in dichloromethane) to afford 7a
(70.8 mg, 0.10 mmol, 46%) as a pale brown foam.
citric acid and extracted with ethyl acetate. The organic layer
was washed with brine, dried over magnesium sulfate and filtered.
The solvent was removed in vacuo, and the residue was purified by
PTLC (9% methanol in dichloromethane) to afford the carboxylic
acid (10.7 mg, 16
lmol, 86%) as a white solid. A solution of the car-
Compound 7a: 1H NMR (500 MHz, CD3OD) d 8.23 (s, 2H), 7.36–
7.31 (m, 5H), 6.15 (d, J = 1.6 Hz, 1H), 5.48–5.44 (m, 2H), 5.25–5.11
(m, 3H), 4.98–4.96 (m, 1H), 4.53 (br, 1H), 4.34 (m, 2H), 3.26–3.16
(br, 2H), 2.74–2.53 (m, 6H), 2.70 (s, 3H), 2.03 (br, 2H), 1.58 (s,
3H), 1.41 (s, 9H), 1.37 (s, 3H); 13C NMR (125 MHz, CD3OD) d
172.49, 155.98, 155.87, 152.67, 148.80, 140.72, 136.51, 136.18,
128.06, 127.74, 127.36, 119.30, 114.00, 90.25, 85.42, 83.50, 83.29,
83.22, 79.56, 67.40, 67.12, 56.12, 51.48, 51.14, 34.04, 33.85,
27.37, 26.17, 24.31; HRMS (ESI+) calcd for C34H47N8O9 (M++H)
711.3461, found 711.3454.
boxylic acid (10.7 mg, 16
and trifluoroacetic acid (0.12 ml) was stirred for 1.5 h at 60 °C. The
reaction mixture was concentrated in vacuo. The residue was puri-
fied by HPLC to afford 2a (4.0 mg, 9.4
l
mol) in methanol (60 l), water (60 l)
l
l
lmol, 70%) as a white solid.
Mp 150 °C (acetonitrile–H2O, decomp.).
Compound 1a: 1H NMR (500 MHz, D2O) d 8.43 (s, 1H), 8.42 (s,
1H), 6.16 (d, J = 3.5 Hz, 1H), 4.79 (t, J = 4.0 Hz, 1H), 4.48–4.44 (m,
2H), 3.87 (dd, J = 9.5 3.5 Hz, 1H), 3.66–3.65 (m, 2H), 3.55–3.39
(m, 6H), 2.69 (s, 3H), 2.34–2.28 (m, 1H), 2.16–2.13 (m, 1H); 13C
NMR (125 MHz, D2O) d 171.50, 150.04, 148.11, 144.52, 143.45,
119.30, 90.19, 78.09, 73.05, 71.55, 55.86, 52.33, 51.49, 48.94,
5.2.3. Synthesis of compound 8a
48.74, 24.61; HRMS (ESI+) calcd for
425.2255, found 425.2255.
C
17H29N8O5 (M++H)
Sodium bicarbonate (17.0 mg, 1.2 equiv) was added to a stirred
solution of 7a (120 mg, 0.17 mmol) in methanol (1.0 ml) at room
temperature. The mixture was stirred for 7 h at 60 °C, then
quenched with saturated aqueous ammonium chloride and ex-
tracted with dichloromethane. The organic layer was dried over
magnesium sulfate and filtered. The solvent was removed in vacuo.
The residue was purified by flash column chromatography (2%
methanol in dichloromethane) to afford 8a (95.0 mg, 0.13 mmol,
79%) as a colorless foam.
5.2.6. Synthesis of compound 6b
Diisobutylaluminium hydride (1.0 M solution in toluene, 1.0 ml,
2.0 equiv) was added dropwise to a stirred solution of 5b (100 mg,
0.54 mmol) in diethyl ether (4.0 ml) over 5 min at À78 °C. The mix-
ture was stirred for 20 min at À78 °C, then methanol (0.5 ml) was
added and stirring was continued for 10 min at the same temper-
ature. Then, 2c (185 mg, 1.2 equiv) in methanol (3.0 ml) was added
dropwise to the reaction mixture over 5 min at À78 °C and stirring
was continued for 2 h at room temperature. Then, sodium borohy-
dride (38.2 mg, 2.0 equiv) was added and the reaction mixture was
stirred for 14 h at room temperature, then concentrated in vacuo.
The residue was partitioned between dichloromethane and satu-
rated aqueous sodium bicarbonate. The organic layer was dried
over magnesium sulfate and filtered. The solvent was removed in
vacuo, and the residue was purified by flash column chromatogra-
phy (2% methanol in dichloromethane then 5%) to afford 6b
Compound 8a: 1H NMR (500 MHz, MeOD) d 8.22 (s, 1H), 8.21
(s, 1H), 7.35–7.28 (m, 5H), 6.14 (d, J = 2.0 Hz, 1H), 5.49 (d,
J = 5.5 Hz, 1H), 5.09–5.05 (m, 2H), 5.04–5.03 (m, 1H), 4.31–4.29
(m, 2H), 3.69 (s, 3H), 3.17–3.15 (m, 2H), 2.83–2.51 (m, 9H) 1.97
(br, 1H), 1.65 (br, 1H), 1.58 (s, 3H), 1.41 (s, 9H), 1.37 (s, 3H); 13C
NMR (125 MHz, MeOD) d 173.27, 157.02, 156.00, 152.63, 152.62,
148.84, 140.64, 136.79, 128.06, 127.59, 127.38, 119.34, 114.12,
90.26, 85.50, 83.53, 83.17, 79.55, 66.21, 56.17, 52.30, 51.32,
48.08, 47.91, 33.88, 28.78, 27.42, 26.13, 24.26; HRMS (ESI+) calcd
for C34H49N8O9 (M++H) 713.3617, found 713.3634.
(53.0 mg, 0.11 lmol, 21%) as a colorless foam.
Compound 6b: 1H NMR (500 MHz, CD3OD) d 8.26 (s, 1H), 8.23
(s, 1H), 6.10 (d, J = 3.0 Hz, 1H), 5.37 (br, 1H), 4.98 (m, 1H), 4.33
(br, 1H), 3.45–3.34 (m, 4H), 3.18 (t, J = 7.5 Hz, 2H), 2.76 (t,
J = 7.0 Hz, 2H), 2.94–2.92 (m, 2H), 2.73–2.71 (m, 2H), 1.60 (s, 3H),
1.52–1.47 (m, 2H), 1.42 (s, 9H), 1.40 (s, 3H), 0.85 (t, J = 7.5 Hz,
5.2.4. Synthesis of compound 9a
Compound 8a (85.0 mg, 0.12 mmol) was added to methanol
(1.0 ml) and Pd/C (25.4 mg, 20 mol %) at 0 °C under an argon atmo-
sphere. The flask was purged with hydrogen. The mixture was stir-
red for one day at room temperature, then filtered through a Celite
bed and the filtrate was concentrated in vacuo. The residue was
purified by flash chromatography on a short column (7% methanol
in dichloromethane then 12%) to afford the amine (65.2 mg,
0.11 mmol, 95%) as colorless foam. To a stirred solution of the
amine (60.0 mg, 0.10 mmol) in dichloromethane was added di-t-
butyl bicarbonate (27.2 mg, 1.2 equiv) at room temperature. The
mixture was stirred for 1 h at room temperature, then concen-
trated in vacuo. The residue was purified by flash column chroma-
tography (2% methanol in dichloromethane) to afford 9a (48.0 mg,
3H); 13C NMR (125 MHz, CD3OD)
d 156.02, 152.58, 148.92,
140.56, 119.30, 114.28, 90.23, 85.42, 83.50, 82.48, 79.66, 50.82,
48.08, 47.06, 43.08, 27.23, 26.07, 24.15, 10.01; HRMS (ESI+) calcd
for C23H38N7O5 (M++H) 492.2929, found 492.2917.
5.2.7. Synthesis of compound 7b
N,N-Diisopropylethylamine (29.8
to a stirred solution of 6b (42.0 mg, 85
1.2 equiv) in acetonitrile (0.6 ml) at room temperature. The
mixture was stirred at 60 °C for two days, then the solvent
was removed in vacuo. The residue was purified by flash
column chromatography (50% ethyl acetate in n-hexane then
l
mol, 2.0 equiv) was added
mol) and 4 (38.5 mg,
l
70
l
mol, 68%) as a colorless foam.
Compound 9a: 1H NMR (500 MHz, CDCl3) d 8.26 (s, 1H), 8.23 (s,
1H), 6.16 (d, J = 2.5 Hz, 1H), 5.52 (d, J = 6.0 Hz, 1H), 5.05 (br, 1H),
4.32–4.29 (m, 1H), 4.22 (br, 1H), 3.69 (s, 3H), 3.18 (br, 2H), 2.84–
2.52 (m, 9H), 1.93 (br, 1H), 1.66 (br, 1H), 1.60 (s, 3H), 1.43 (s,
18H), 1.39 (s, 3H); 13C NMR (125 MHz, CD3OD) d 173.57, 156.57,
156.02, 152.60, 148.85, 140.67, 119.30, 114.07, 90.32, 85.52,
83.52, 83.24, 79.56, 79.14, 56.14, 52.07, 51.83, 51.18, 48.08,
33.87, 28.80, 27.36, 27.26, 26.08, 24.18; HRMS (ESI+) calcd for
neat ethyl acetate) to afford 7b (28.7 mg, 39 lmol, 46%) as a
colorless foam.
Compound 7b: 1H NMR (500 MHz, CD3OD) d 8.23 (s, 2H), 7.36–
7.31 (m, 5H), 6.15 (d, J = 2.5 Hz, 1H), 5.47 (br, 1H), 5.44 (d,
J = 4.0 Hz, 1H), 5.30 (br, 1H), 5.18–5.14 (br m, 2H), 4.96–4.95 (m,
1H), 4.33–4.27 (m, 2H), 3.12 (br, 2H), 3.02–2.97 (m, 2H), 2.76–
2.74 (m, 2H), 2.51 (br, 4H), 2.04 (br, 2H), 1.58 (s, 3H), 1.45–1.38
(m, 2H), 1.41 (s, 9H), 1.37 (s, 3H), 0.76 (t, J = 7.5 Hz, 3H); 13C
NMR (125 MHz, CD3OD) d 172.51, 167.86, 155.99, 155.80, 152.63,
148.82, 140.64, 130.91, 128.24, 128.11, 127.82, 119.27, 114.18,
90.14, 83.51, 83.49, 83.12, 79.50, 77.75, 67.37, 65.85, 56.24,
52.77, 21.29, 48.92, 27.37, 26.43, 24.20, 22.60, 10.04; HRMS
(ESI+) calcd for C36H51N8O9 (M++H) 739.3774, found 739.3796.
C
31H51N8O9 (M++H) 679.3774, found 679.3773.
5.2.5. Synthesis of compound 1a
A solution of 9a (12.7 mg, 19 mol) in 1,4-dioxane (0.15 ml) and
l
5 M aqueous sodium hydroxide (0.15 ml) was stirred for 4.5 h at
room temperature. The reaction mixture was quenched with 10%

8164
S. Mori et al. / Bioorg. Med. Chem. 18 (2010) 8158–8166
5.2.8. Synthesis of compound 8b
3H), 0.87 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz, CD3OD) d 172.89,
A
solution of 7b (27.5 mg, 37
l
mol) in tetrahydrofuran
155.98, 155.76, 152.67, 148.82, 140.62, 135.94, 128.25, 128.11,
128.01, 127.82, 119.28, 114.21, 90.12, 83.51, 83.46, 83.08, 77.76,
67.36, 56.26, 52.78, 52.00, 48.04, 47.87, 47.70, 31.26, 27.40,
27.34, 26.14, 26.08, 24.23,22.24, 12.95; HRMS (ESI+) calcd for
(0.15 ml) and 1 M aqueous sodium hydroxide (0.15 ml) was stirred
for 1.5 h at room temperature. The reaction mixture was quenched
with 10% aqueous citric acid and extracted with dichloromethane.
The organic layer was dried over magnesium sulfate and filtered.
The solvent was removed in vacuo, and the residue was purified
by PTLC (8% methanol in dichloromethane) to afford 8b (20.7 mg,
C
39H57N8O9 (M++H) 781.4243, found 781.4266.
5.2.12. Synthesis of compound 8c
28
l
mol, 77%) as a colorless foam.
Compound 8c was prepared from 7c (22.0 mg, 28 lmol) accord-
Compound 8b: 1H NMR (500 MHz, CD3OD) d 8.24 (s, 2H), 7.35–
ing to the procedure described for 8b in a yield of 51% (11.1 mg,
14 mol, colorless foam.).
Compound 8c: 1H NMR (500 MHz, CD3OD) d 8.24 (s, 2H), 7.35–
7.26 (m, 5H), 6.21 (br, 1H), 5.45 (d, J = 5.5 Hz, 1H), 5.10–5.07 (m,
3H), 4.50–4.40 (br m, 1H), 4.21–4.14 (br m, 1H), 3.27–2.81 (br m,
10 H), 2.06 (br, 1H), 1.80 (br, 1H), 1.59 (s, 3H), 1.48–1.32 (m, 2H),
1.40 (s, 9H), 1.37 (s, 3H), 0.78 (t, J = 7.5 Hz, 3H); 13C NMR
(125 MHz, CD3OD) d 175.14, 156.95, 156.24, 156.02, 152.66,
148.71, 140.74, 136.81, 128.05, 127.55, 127.36, 119.37, 114.39,
90.44, 83.75, 83.58, 83.02, 79.85, 66.17, 55.65, 53.29, 52.31,
52.04, 43.37, 27.31, 26.05, 24.14, 21.00, 10.00; HRMS (ESI+) calcd
for C35H51N8O9 (M++H) 727.3774, found 727.3779.
l
7.27 (m, 5H), 6.20 (br, 1H), 5.44 (d, J = 5.0 Hz, 1H), 5.07 (br, 3H),
4.48–4.39 (br m, 1H), 4.21–4.14 (br m, 1H), 3.20–2.81 (br m,
10H), 2.05 (br, 1H), 1.80 (br, 1H), 1.59 (s, 3H), 1.41 (s, 9H), 1.37
(s, 3H), 1.28–1.16 (br m, 8H), 0.88 (t, J = 6.5 Hz, 3H); 13C NMR
(125 MHz, CD3OD)
d 175.09, 156.90, 156.01,152.66, 148.72,
140.68, 136.82, 128.04, 127.54, 127.33, 119.35, 114.39, 90.40,
83.57, 82.82, 79.80, 66.14, 55.87, 55.63, 52.07, 48.03, 43.27,
31.24, 28.34, 27.96, 27.32, 26.06, 26.00, 24.15, 22.23, 12.93; HRMS
(ESI+) calcd for C38H57N8O9 (M++H) 769.4243, found 769.4256.
5.2.9. Synthesis of compound 1b
Compound 8b (18.0 mg, 25 lmol) was added to methanol
(0.3 ml) and Pd/C (2.6 mg, 10 mol %) at 0 °C under an argon atmo-
sphere. The flask was purged with hydrogen, and stirred for 20 h at
room temperature. The reaction mixture was filtered through a
Celite bed and concentrated in vacuo. The residue was purified
by PTLC (12% methanol in dichloromethane) to afford the amine
5.2.13. Synthesis of compound 1c
Compound 1c was prepared from 8c (11.0 mg, 14
ing to the procedure described for 1b in a yield of 56% (two steps,
lmol) accord-
4.0 mg, 8.2
decomp.).
lmol, white solid. Mp 195 °C (acetonitrile–H2O,
(6.2 mg, 11
(6.2 mg, 11
l
l
mol, 42%) as a colorless foam. A solution of the amine
mol) in methanol (80 l), water (80 l) and trifluoro-
Compound 1c 1H NMR (500 MHz, D2O) d 8.39 (s, 1H), 8.38 (s,
1H), 6.11 (d, J = 4.0 Hz, 1H), 4.73 (t, J = 5.0 Hz, 1H), 4.42–4.37 (m,
2H), 3.83–3.81 (m, 1H), 3.54 (br, 2H), 3.40–3.29 (m, 6H), 2.86–
2.82 (m, 2H), 2.26–2.23 (m, 1H), 2.11–2.07 (m, 1H), 1.43–1.39
(m, 2H), 1.18–1.10 (m, 6H), 0.76 (t, J = 7.0 Hz, 3H); 13C NMR
(125 MHz, D2O) d 172.98, 150.62, 148.20, 145.82, 142.99, 119.16,
89.72, 79.19, 72.96, 71,47, 55.51, 53.00, 52.87, 48.59, 47.91,
41.83, 30.29, 25.30, 25.15, 25.14, 21.64, 13.13; HRMS (ESI+) calcd
for C22H39N8O5 (M++H) 495.3038, found 495.3056.
l
l
acetic acid (0.16 ml) was stirred for 2 h at 60 °C. The reaction mix-
ture was concentrated in vacuo. The residue was purified by HPLC
to afford 2d (2.8 mg, 6.2
lmol, 59%) as a white solid. Mp 168 °C
(acetonitrile–H2O, decomp.).
Compound 1b: 1H NMR (500 MHz, D2O) d 8.40 (s, 1H), 8.39 (s,
1H), 6.12 (d, J = 3.5 Hz, 1H), 4.77 (t, J = 4.5 Hz, 1H), 4.45–4.42 (m,
2H), 3.83–3.82 (m, 1H), 3.62 (br, 2H), 3.46–3.29 (m, 6H), 2.88 (t,
J = 7.5 Hz, 2H), 2.27–2.26 (m, 1H), 2.12–2.10 (m, 1H), 1.52 (m,
2H), 0.82 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz, D2O) d 173.08,
151.13, 148.26, 146.20, 142.95, 119.21, 89.65, 79.32, 72.87, 71.57,
55.61, 52.91, 52.71, 49.48, 48.68, 42.02, 25.39, 18.91, 9.95; HRMS
(ESI+) calcd for C19H33N8O5 (M++H) 453.2568, found 453.2580.
5.2.14. Synthesis of compound 6d
Compound 6d was prepared from 5d (50 mg, 0.19 mmol) and 2c
(118 mg, 2.0 equiv) according to the procedure described for 6b in
a yield of 22% (23.0 mg, 42 lmol, colorless foam).
Compound 6d 1H NMR (500 MHz, CD3OD) d 8.29 (s, 1H), 8.24 (s,
1H), 7.28–7.23 (m, 2H), 7.17–7.11 (m, 3H), 6.17 (d, J = 3.0 Hz, 1H),
5.48 (br, 1H), 5.01 (m, 1H), 4.35–4.32 (m, 1H), 3.39–3.36 (m, 2H),
3.25–3.16 (br m, 2H), 2.91 (br, 2H), 2.77 (br, 2H), 2.68 (br, 2H),
1.61 (s, 3H), 1.39 (s, 3H), 1.37 (s, 9H); 13C NMR (125 MHz, CD3OD)
d 156.01, 152.59, 148.91, 140.53, 139.07, 128.54, 128.06, 125.88,
119.30, 114.24, 90.22, 85.23, 83.43, 82.42, 79.63, 53.36, 50.73,
49.24, 47.86, 27.19, 26.07, 24.16; HRMS (ESI+) calcd for
5.2.10. Synthesis of compound 6c
Compound 6c was prepared from 5c (150 mg, 0.62 mmol) and
2c (382 mg, 2.0 equiv) according to the procedure described for
6b in a yield of 14% (45.5 mg, 85 lmol, colorless foam).
Compound 6c: 1H NMR (500 MHz, CD3OD) d 8.27 (s, 1H), 8.22 (s,
1H), 6.15 (d, J = 2.5 Hz, 1H), 5.47 (br, 1H), 5.00 (dd, J = 6.5, 3.5 Hz,
1H), 4.34–4.31 (m, 1H), 3.26–3.18 (m, 2H), 3.12–3.08 (m, 2H),
2.90 (d, J = 6.0 Hz, 2H), 2.71–2.67 (m, 2H), 1.59 (s, 3H), 1.48–1.44
(m, 4H), 1.37 (s, 3H), 1.31 (s, 9H), 1.30–1.20 (m, 4H), 0.88 (t,
J = 6.5 Hz, 3H); 13C NMR (125 MHz, CD3OD) d 156.02, 152.59,
148.92, 140.53, 119.31, 114.23, 90.21, 85.31, 83.45, 82.45, 79.49,
50.80, 48.11, 47.94, 47.77, 31.24, 27.29, 26.06, 24.21, 22.22,
12.93; HRMS (ESI+) calcd for C26H44N7O5 (M++H) 534.3398, found
534.3385.
C
28H40N7O5 (M++H) 554.3085, found 554.3076.
5.2.15. Synthesis of compound 7d
Compound 7d was prepared from 6d (37.5 mg, 68 lmol) and 4
(30.5 mg, 1.2 eq) according to the procedure described for 7b in a
yield of 53% (28.6 mg, 36 mol, colorless foam).
l
Compound 7d 1H NMR (500 MHz, CD3OD) d 8.23 (s, 1H), 8.22 (s,
1H), 7.34–7.07 (m, 10H), 6.14 (d, J = 2.5 Hz, 1H), 5.46 (br, 1H), 5.43
(d, J = 4.0 Hz, 1H), 5.20 (br, 1H), 5.18–5.14 (br m, 2H), 4.95–4.93 (m,
1H), 4.32–4.29 (m, 2H), 3.18–2.48 (m, 12H), 2.00 (br, 2H), 1.57 (s,
3H), 1.40 (s, 9H), 1.35 (s, 3H); 13C NMR (125 MHz, CD3OD) d
172.90, 171.55, 155.97, 155.62, 152.68, 148.82, 140.55, 139.08,
135.92, 128.59, 128.25, 128.02, 127.83, 127.61, 125.80, 119.28,
114.16, 90.16, 83.51, 83.12, 82.81, 79.33, 77.76, 67.38, 66.31,
60.10, 56.30, 53.38, 52.81, 34.51, 34.00, 27.27, 24.21; HRMS
(ESI+) calcd for C41H53N8O9 (M++H) 801.3930, found 801.3954.
5.2.11. Synthesis of compound 7c
Compound 7c was prepared from 6c (25.2 mg, 47
(35.4 mg, 2.0 equiv) according to the procedure described for 7b
lmol) and 4
in a yield of 58% (21.5 mg, 28 lmol, colorless foam).
Compound 7c: 1H NMR (500 MHz, CD3OD) d 8.23 (s, 2H), 7.37–
7.32 (m, 5H), 6.15 (d, J = 2.5 Hz, 1H), 5.45–5.44 (br, 2H), 5.26 (br,
1H), 5.22–5.19 (br m, 2H), 4.96–4.95 (m, 1H), 4.34–4.30 (m, 2H),
3.15–3.00 (br m, 4H), 2.76 (br, 2H), 2.58–2.53 (br m, 4H), 2.04
(br, 2H), 1.58 (s, 3H3H), 1.45–1.38 (m, 8H), 1.41 (s, 9H), 1.38 (s,

S. Mori et al. / Bioorg. Med. Chem. 18 (2010) 8158–8166
8165
5.2.16. Synthesis of compound 8d
Compound 8d was prepared from 7d (27.0 mg, 34
according to the procedure described for 8b in a yield of 61%
(16.2 mg, 21 mol, colorless foam).
5.2.21. Synthesis of compound 1e
Compound 1e was prepared from 8e (9.0 mg, 14
ing to the procedure described for 1b in a yield of 35% (2 steps,
l
mol)
lmol) accord-
l
2.1 mg, 4.6 mol, white solid).
l
Compound 8d: 1H NMR (500 MHz, CD3OD) d 8.24 (s, 1H), 8.21
(s, 1H), 7.34–7.26 (m, 5 H), 7.25–7.22 (m, 2H), 7.16–7.11 (m, 3H),
6.20–6.17 (br, 1H), 5.43 (d, J = 6.0 Hz, 1H), 5.06 (br, 3H), 4.47–
4.37 (br m, 1H), 4.18–4.12 (br m, 1H), 3.27–3.15 (br m, 4H),
2.93–2.68 (br m, 8H), 2.02 (br, 1H), 1.77 (br, 1H), 1.58 (s, 3H),
1.40–1.35 (m, 9H), 1.34 (s, 3H); 13C NMR (125 MHz, CD3OD) d
176.26, 156.91, 155.99, 152.66, 148.69, 140.70, 139.03, 136.79,
128.61, 128.12, 128.05, 127.57, 127.39, 125.93, 125.85, 119.35,
114.38, 90.48, 84.67, 83.59, 83.01, 79.97, 66.18, 64.86, 56.58,
55.63, 52.19, 52.01, 49.02, 34.35, 27.18, 26.06, 24.14; HRMS
(ESI+) calcd for C40H53N8O9 (M++H) 789.3930, found 789.3943.
Compound 1e: 1H NMR (500 MHz, D2O) d 8.33 (s, 2H), 6.07 (d,
J = 4.0 Hz, 1H), 4.80 (t, J = 4.5 Hz, 1H), 4.43–4.39 (m, 2H), 3.72–
3.71 (m, 1H), 3.66–3.61 (m, 1H), 3.57–3.55 (m, 1H), 3.42–3.37
(m, 2H), 3.15–3.11 (m, 2H), 2.25–2.20 (m, 1H), 2.09 (br, 1H), 1.50
(br, 2H), 1.04 (br, 6H), 0.66 (br, 3H); 13C NMR (125 MHz, D2O) d
171.76, 150.04, 148.08, 144.52, 143.58, 119.26, 90.08, 72.95,
72.89, 71.75, 54.72, 53.92, 51.62, 50.77, 30.29, 25.11, 24.62,
22.59, 21.54, 13.07; HRMS (ESI+) calcd for C20H34N7O5 (M+H+)
452.2616. found 452.2628.
5.3. Assay for inhibitory activity towards SET7/9
A mixture of biotin-conjugated histone H3 peptide (residue
1–21) (Upstate), adenosylmethionine (AdoMet), human recombi-
nant SET7/9 (Funakoshi) and a test compound in reaction buffer
(50 mM Tris, 100 mM NaCl, 1 mM EDTA, 4 mM DTT, pH 8.8) was
incubated in a plastic tube for 1 h. The solution was added to a
streptavidin-coated microplate (BD Science), and incubated for
45 min. Each well was washed with PBST, blocked by 3% skim milk
in PBST, and anti-monomethyl-histone H3 (Lys4) antibody (Up-
state) was added. The plate was left for 1 h. The wells were washed
again with PBST, then anti-rabbit IgG horseradish peroxidase-
linked antibody (Abcam) was added and the plate was left for
30 min. The wells were washed with PBST, substrate for HRP
(TMB) (BioRad) was added to each well and the plate was left for
20 min. After addition of diluted sulfuric acid to stop the reaction,
the microplate was scanned to determine OD450.
5.2.17. Synthesis of compound 1d
Compound 1d was prepared from 8d (15.2 mg, 19
according to the procedure described for 1b in a yield of 42%
lmol)
(2 steps, 4.1 mg, 8.0
H2O, decomp.).
lmol, white solid). Mp 186 °C (acetonitrile–
Compound 1d: 1H NMR (500 MHz, D2O) d 8.38 (s, 1H), 8.31 (s,
1H), 7.30–7.23 (m, 3H), 7.07 (d, J = 7.5 Hz, 2H), 6.08 (d, J = 4.0 Hz,
1H), 4.65 (m, 1H), 4.41–4.35 (m, 2H), 3.85–3.83 (m, 1H), 3.58–
3.34 (m, 8H), 3.11 (t, J = 7.5 Hz, 2H), 2.67 (t, J = 7.5 Hz, 2H), 2.28–
2.25 (m, 1H), 2.11–2.09 (m, 1H); 13C NMR (125 MHz, D2O)
d 172.77, 149.36, 147.84, 144.97, 135.74, 129.03, 128.49, 127.46,
118.97, 89.69, 78.92, 73.01, 71.36, 55.38, 53.21, 52.67, 48.51,
48.80, 41.43, 31.10, 25.23; HRMS (ESI+) calcd for C24H35N8O5
(M++H) 515.2725, found 515.2716.
5.2.18. Synthesis of compound 6e
Acknowledgements
Compound 6e was prepared from 5e (0.19 ml, 5.0 equiv) and 2c
(100 mg, 0.33 mmol) according to the procedure described for 6a
in a yield of 73% (93 mg, 0.24 mmol, colorless oil).
The work described in this paper was partially supported by
Grants-in-Aid for Scientific Research from The Ministry of Educa-
tion, Science, Sports and Culture, Japan (Grant Nos. 19201044 to
H.K., 19790090 and 22790106 to T.H.). T.H. also thanks the Moch-
ida Memorial Foundation and Astellas Foundation for financial
support. We also thank Dr. Takeshi Umehara, RIKEN Systems and
Structural Biology Center, for valuable discussions on biological
assays.
Compound 6e: 1H NMR (500 MHz, MeOD) d 8.28 (s, 1H), 8.21 (s,
1H), 6.16 (d, J = 2.5 Hz, 1H), 5.52 (m, 1H), 5.01 (m, 1H), 4.36–4.33
(m, 1H), 2.87–2.83 (m, 2H), 2.53–2.46 (m, 2H), 1.59 (s, 3H), 1.38
(s, 3H), 1.34–1.20 (m, 8H), 0.87 (t, J = 6.0 Hz, 3H); HRMS (ESI+)
calcd for C19H31N6O3 (M++H) 391.2452, found 391.2458.
5.2.19. Synthesis of compound 7e
Compound 7e was prepared from 6e (90 mg, 0.23 mmol) and 4
(103 mg, 1.2 equiv) according to the procedure described for 7b in
References and notes
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Compound 7e: 1H NMR (500 MHz, MeOD) d 8.24 (s, 1H), 8.22 (s,
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5.2.20. Synthesis of compound 8e
Compound 8e was prepared from 7e (27.0 mg, 34 lmol) accord-
ing to the procedure described for 8b in a yield of 96% (52 mg,
83 mol, colorless foam).
Compound 8e: 1H NMR (500 MHz, CD3OD) d 8.26 (s, 1H), 8.25
l
(s, 1H), 7.36–7.27 (m, 5H), 6.30 (s, 1H), 5.46 (d, J = 6.0 Hz, 1H),
5.17 (br, 1H), 5.07 (s, 2H), 4.64–4.62 (br m, 1H), 3.97 (dd, J = 7.0,
4.5 Hz, 1H), 3.69–3.63 (br m, 1H), 3.48–3.45 (br m, 1H), 3.18–
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1.89–1.87 (br m, 1H), 1.60 (s, 3H), 1.38 (s, 3H), 1.29 (br, 2H),
1.21–1.07 (br m, 6H), 0.84 (t, J = 7.0 Hz, 3H); HRMS (ESI+) calcd
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