Design and synthesis of 2-amino-6-(1H,3H-benzo[de]isochromen-6-yl)-1,3,5- triazines as novel Hsp90 inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.11.036

A novel series of 2-amino-1,3,5-triazines bearing a tricyclic moiety as heat shock protein 90 (Hsp90) inhibitors is described. Molecular design was performed using X-ray cocrystal structures of the lead compound CH5015765 and natural Hsp90 inhibitor geldanamycin with Hsp90. We optimized affinity to Hsp90, in vitro cell growth inhibitory activity, water solubility, and liver microsomal stability of inhibitors and identified CH5138303. This compound showed high binding affinity for N-terminal Hsp90α (K_d = 0.52 nM) and strong in vitro cell growth inhibition against human cancer cell lines (HCT116 IC₅₀ = 0.098 μM, NCI-N87 IC₅₀ = 0.066 μM) and also displayed high oral bioavailability in mice (F = 44.0%) and potent antitumor efficacy in a human NCI-N87 gastric cancer xenograft model (tumor growth inhibition = 136%).

Full text of DOI:10.1016/j.bmc.2013.11.036

Bioorganic & Medicinal Chemistry 22 (2014) 892–905
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of 2-amino-6-(1H,3H-benzo[de]isochromen
-6-yl)-1,3,5-triazines as novel Hsp90 inhibitors q
a
a
a
b
Atsushi Suda a, , Ken-ichi Kawasaki , Susumu Komiyama , Yoshiaki Isshiki , Dong-Oh Yoon ,
Sung-Jin Kim b, Young-Jun Na b, Kiyoshi Hasegawa a, Takaaki A. Fukami a, Shigeo Sato a, Takaaki Miura a,
Naomi Ono a, Toshikazu Yamazaki a, Ryoichi Saitoh a, Nobuo Shimma a, Yasuhiko Shiratori a,
Takuo Tsukuda a
⇑
a Research Division, Chugai Pharmaceutical Co., Ltd, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan
b Discovery Research Center, C&C Research Laboratories, DRC Natural Sciences Campus, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon-si, Gyeonggi-do 440-746,
Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 2-amino-1,3,5-triazines bearing a tricyclic moiety as heat shock protein 90 (Hsp90)
inhibitors is described. Molecular design was performed using X-ray cocrystal structures of the lead com-
pound CH5015765 and natural Hsp90 inhibitor geldanamycin with Hsp90. We optimized affinity to
Hsp90, in vitro cell growth inhibitory activity, water solubility, and liver microsomal stability of inhibi-
Received 4 October 2013
Revised 15 November 2013
Accepted 16 November 2013
Available online 25 November 2013
tors and identified CH5138303. This compound showed high binding affinity for N-terminal Hsp90
(Kd = 0.52 nM) and strong in vitro cell growth inhibition against human cancer cell lines (HCT116
IC50 = 0.098 M, NCI-N87 IC50 = 0.066 M) and also displayed high oral bioavailability in mice
a
Keywords:
l
l
Structure-based drug design
Lead optimization
Antitumor agent
Hsp90 inhibitor
(F = 44.0%) and potent antitumor efficacy in a human NCI-N87 gastric cancer xenograft model (tumor
growth inhibition = 136%).
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
differences between these isoforms are poorly understood. Recent
studies showed that cancer cells need secretion of Hsp90a into the
Heat shock protein 90 (Hsp90) is an ATP-dependent molecular
chaperone that is abundantly expressed in eukaryotic cells and
essential for their survival. Hsp90 participates in the maturation
and stability of many regulatory and signaling proteins that control
cell proliferation and survival by facilitating normal protein folding
and guarding protein from misfolding and aggregation.1–3 In many
tumors Hsp90 is overexpressed, because cancer cells use Hsp90 to
maintain cellular homeostasis in a hostile tumor microenviron-
ment, such as hypoxia, low nutrition and acidosis.4 Because
Hsp90 becomes activated in cancer cells after forming a complex
with a series of co-chaperones,5 inhibitors that are selective to
activated Hsp90 could lead to an effective suppression of cancer
cell proliferation and survival, and Hsp90 is considered an
attractive molecular target for cancer therapies.4,6–9
extracellular matrix for invasion and metastasis.10 Hsp90 exists
predominantly as a homodimer in the cytoplasm and consists of
three main domains, namely, the N-terminal, middle, and C-termi-
nal domains. The N-terminal domain contains a binding site to
adenosine triphosphate (ATP), which needs to be hydrolyzed for
chaperone activity to occur. The middle domain, as well as having
a key role in binding many client proteins to Hsp90, also modulates
the ATP hydrolysis by interacting with the c-phosphate of the ATP
that is bound in the N-terminal pocket.11 The C-terminal domain,
which contains an additional ATP-binding site, is responsible for
the inherent dimerization of Hsp90.12
The N-terminal ATP-binding site is also the binding site of many
known Hsp90 inhibitors, such as the natural products geldanamy-
cin (GM, 1a)13 and radicicol (2),14 the semi-synthetic analog of GM
known as 17-allylamino-17-demethoxygeldanamycin (17-AAG,
1b),15 and many synthetic small molecules like those shown in
Figure 1 (3–6).16–19 These Hsp90 inhibitors interact with the N-ter-
minal ATP-binding site to prevent ATP binding, stop the chaperone
cycle, and finally lead to degradation of multiple oncogenic client
proteins involved in tumor progression. 17-AAG (1b) entered clin-
ical trial as an intravenous (iv) formulation in 1999 and is currently
in phase III trials. Several small molecule inhibitors are also
In humans, Hsp90 has two cytosolic isoforms, Hsp90
a (induc-
ible form) and Hsp90b (constitutive form), and the functional
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which per-
mits non-commercial use, distribution, and reproduction in any medium, provided
the original author and source are credited.
⇑
Corresponding author. Tel.: +81 550 87 8444.
E-mail address: sudaats@chugai-pharm.co.jp (A. Suda).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.036

A. Suda et al. / Bioorg. Med. Chem. 22 (2014) 892–905
893
O
O
O
N
R
O
O
O
Cl
O
HO
N
H
HO
H
O
N
O
OH
OH
O
O
OH
O
N
O
O
NH2
2, Radicicol
3, AUY-922
1a, Geldanamycin; R = OMe
1b, 17-AAG; R = NHCH2CH=CH2
N
N
N
Cl
N
N
HO
N
H2N
N
HO
N
N
O
N
OH
N
N
H
OH
O
O
5, STA-9090
4, AT-13387
6, CNF-2024
Figure 1. Known Hsp90 inhibitors.
undergoing clinical trials: iv formulations AUY-922 (3) and
AT-13387 (4) in phase II, and STA-9090 (5) in phase III, which have
the resorcinol component, and the oral (po) formulation CNF-2024
(6) in phase II.6
position forms a hydrogen bond (HB) with a water molecule that
is hydrogen-bound to the backbone of Phe138 and the side chain
of Asp51.20 Herein we report a transformation of the methyl group
on the sulfur atom at the triazine ring to other substituents to im-
prove affinity and physicochemical properties while retaining the
tricyclic moiety.
Although the majority of these small molecule inhibitors share
a similar core structure, using a different core structure may result
in a different profile with the potential for further improving effi-
cacy, pharmacokinetics and safety. We therefore focused our effort
on identifying novel orally bioavailable small molecules that would
expand the structural diversity of the small molecule inhibitors
presently available for this important pharmacological target.
We have already reported using a combination of fragment
screening, virtual screening, and structure-based drug design to
identify a new class of orally available Hsp90 inhibitor that binds
to the N-terminal ATP binding site, CH5015765 (7, Fig. 2).20 Com-
pound 7 showed a high binding affinity for N-terminal Hsp90
(Kd = 3.4 nM) and in vitro cell growth inhibition against human
2. Results and discussion
2.1. Chemistry
The preparation of compound 7 as the key intermediate is
shown in Scheme 1. Nitration of commercial 4-bromo-1,8-naph-
thalic anhydride (8) gave compound 9 as a 4:1 mixture of regioi-
somers (3-nitro/6-nitro derivatives). Since the undesired
regioisomer (6-nitro derivative) was more soluble than the desired
regioisomer (3-nitro derivative) in acetonitrile, most of the byprod-
uct was removed by filtering the product suspension in acetonitrile
(82%, 3-nitro/6-nitro derivative = 13:1). After reducing anhydride
with lithium aluminum hydride (LAH) (10, 99%), substitution of
bromo to cyano group gave compound 11 (85%). Reduction of nitro
group (12, 86%) followed by introduction of chloro group by
Sandmeyer reaction gave compound 13 (70%). Transformation of
cyano to amidino group (14, 95%) and the consequent 2-amino-
1,3,5-triazine ring formation gave compound 7 (88%).
The preparation of compounds 16a–l is shown in Scheme 2.
Sulfoxide 15 was prepared from compound 7 by m-chloroperben-
zoic acid (mCPBA) oxidation (78%). This sulfoxide 15 was coupled
with the corresponding alkylthiols and the following amidation
gave compounds 16a–l.
The preparation of compounds 18a–f is shown in Scheme 3.
Thiol 17 was prepared from compound 15 by treatment with
potassium thioacetate, followed by hydrolysis (92%). This thiol 17
was coupled with the corresponding alkyl halides and the
following amidation gave compounds 18a–f.
cancer cell lines (HCT116, IC50 = 0.46
M). However, its poor water solubility (solubility in fasted state
simulated intestinal fluid (FaSSIF) = 29 M) limited the oral bio-
lM; NCI-N87, IC50 = 0.57 -
l
l
availability (F = 4.5%) and produced only moderate tumor growth
inhibition (TGI = 54%) against the xenografted NCI-N87 mouse
model (po, 400 mg/kg, 11 qd), both of which aspects needed to
be improved.
As we already reported, a tricyclic (1H,3H-benzo[de]isochro-
men-6-yl) moiety makes hydrophobic interactions with the side
chains of Val150, Leu107, and Phe138, and an etheric oxygen at 3
a
NH2
N
Hsp90α Kd
:
3.4 nM
0.46 μM
0.57 μM
HCT116 IC50
NCI-N87 IC50
:
:
Cl
N
S
N
TGI (NCI-N87)b: 54% (po, 400 mg/kg, 11qd)
F (mouse)c:
Solubility in FaSSIF: 29μM
4.5%
O
Figure 2. Chemical structure of CH5015765 (7) and its biological, physicochemical,
2.2. Molecular design
and pharmacokinetic profiles. aValues were measured by surface plasmon reso-
nance (SPR) using human N-terminal Hsp90a.
bGrowth inhibitory effect of
Our strategy is depicted in Figure 3. Since GM (1a) forms a HB to
the side chain of Lys58 (PDB code: 1YET), we attempted to incorpo-
rate this interaction into our molecular design. We expected
compound 7 in a NCI-N87 tumor xenograft model. SCID mice bearing NCI-N87
cells were orally administered on a daily basis for 11 consecutive days (Days 11–
21). Mean tumor volume is shown. cOral bioavailability.

894
A. Suda et al. / Bioorg. Med. Chem. 22 (2014) 892–905
NO2
Br
NO2
NO2
Br
N
Br
O
a
b
c
d
O
O
O
O
O
O
O
9
10
11
8
NH2
N
NH2
Cl NH
NH2
Cl
N
N
Cl
N
e
f
g
N
S
O
O
O
O
12
13
14
7 (CH5015765)
Scheme 1. Synthesis of compound 7. Reagents and conditions: (a) HNO3, H2SO4, 4 °C, 2.5 h, 82%; (b) LiBH4, BF3ÁOEt2, THF, 40 to 50 °C, 2 h, 99%; (c) CuCN, DMF, 120 °C, 1 h,
85%; (d) Na2S2O4, THF-water, reflux, 30 min, then HCl aq. (5 M), rt, 1 h, 86%; (e); CuCl2, t-BuONO, CH3CN, rt, 1 h, 70%; (f) NH4Cl, Al(CH3)3, toluene, reflux, 21 h, 95%; (g)
dimethyl cyanodithioiminocarbonate, DIPEA, THF, reflux, 3 h, 88%. Abbreviations: aq, aqueous solution; DIPEA, N,N-diisopropylehylamine; DMF, N,N-dimethylformamide; rt,
room temperature; THF, tetrahydrofuran.
NH2
N
NH2
N
NH2
N
Cl
N
Cl
N
Cl
N
a
b or c (for 16l),
d (for 16d-l)
R
N
S
N
S
O
N
S
O
O
O
7 (CH5015765)
15
16
Compd
R
Compd
R
Compd
R
O
O
H
N
16e
16a
16b
NH2
16i
*
*
N
*
O
O
NH
O
O
OH
16f
16j
N
NH2
NH2
N
H
*
*
*
*
*
O
O
NH2
OH
O
16c
16d
16g
16h
16k
16l
*
*
O
NH2
*
N
H
NH2
*
O
Scheme 2. Synthesis of compound 16a–l. Reagents and conditions: (a) mCPBA, CH2Cl2, rt, 3 h, 78%; (b) RSH, base (TEA, DIPEA or K2CO3), solvent (THF, THF-DMF, or DMF), rt;
(c) 4,4-dithiobutyric acid, tributylphosphine, DIPEA, DMF-water, rt; (d) amine (NH4Cl, CH3NH2ÁHCl, (CH3)2NHÁHCl, or 2-aminoethan-1-ol), HOBT, EDCÁHCl, DIPEA, DMF, rt.
Abbreviations: mCPBA, m-chloroperbenzoic acid; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HOBT, 1-hydroxybenzotriazole; TEA, triethylamine.
affinity and physicochemical properties to be improved by intro-
ducing a HB acceptor via methylene carbons on the sulfur atom
at the triazine ring. As an option to improve the physicochemical
properties, introducing a solubilizing group on a HB acceptor was
also attempted.
We analyzed the superimposed structures of 1a and compound
7 in Hsp90a (Fig. 4) and found that two to three methylene carbons
could be acceptable because the side chain of Lys58, located near
the solvent-exposed region, is flexible. First we synthesized deriv-
atives having various HB acceptors with two methylene carbons.
ties, and the in vitro metabolic stability. As described in an intro-
duction, Hsp90 is important for invasion and metastasis of
cancer cells. We thought that the utilization of Hsp90 as an in-
dex for lead optimization was important for identification of a po-
tent antitumor agent. We first examined acetylamino (16a) and
mesylamino (18a) as HB acceptors aiming to Lys58. As expected,
introducing these HB acceptors increased affinity to Hsp90
(Kd = 0.82 and 0.43 nM, respectively) by 4- to 8-fold in compari-
son with that of compound 7. With regards to water solubility,
a
a
16a increased it by 30-fold (856
lM) but 18a did not show any
improvement (<31 M). Water solubility can be described by
l
2.3. Structure–activity relationships
some physicochemical properties such as pKa, polar surface area
(PSA), and the general solubility equation using LogP (the molar
octanol–water partition coefficient).21 In this case, we expected
the weak acidic property of the mesylamino group (calculated
pKa = 9.08) to affect water solubility negatively in FaSSIF at pH
6.5. In order to improve water solubility with sub-nanomolar
Tables 1–3 summarize the affinity to Hsp90a, the in vitro cell
growth inhibitory activity against an HCT116 colorectal cancer
cell line (a KRAS mutant cell line) and an NCI-N87 gastric cancer
cell line (a HER2-positive cell line), the physicochemical proper-

A. Suda et al. / Bioorg. Med. Chem. 22 (2014) 892–905
895
NH2
N
NH2
NH2
N
Cl
N
Cl
N
N
Cl
N
a
b, c (for 18f)
R
N
S
O
N
SH
N
S
O
O
O
15
17
18
Compd
R
Compd
R
O
H
*
*
N
O
O
18d
18e
18f
18a
18b
S
*
N
H
O O
H
O
N
O
NH2
*
*
N
H
O
O
O
NH2
18c
NH2
*
Scheme 3. Synthesis of compound 18a–f. Reagents and conditions: (a) potassium thioacetate, DMF, rt, 19 h, and then NaOH aq (2 M), rt, 1.5 h, 92%; (b) RBr/Cl, DIPEA, DMF,
70–80 °C, (c) NH4Cl, HOBT, EDCÁHCl, DIPEA, DMF, rt.
*
Lys58
Val150
Ile96
*
*
H
H
N H
H
N
Solvent-exposed region
Cl N 2
N
Leu107
*
7
HB
acceptor
Methylene
carbons
Solubilizing
group
4
6
N
S
8
O
5
13
Phe138
( Option )
3 O
1
*
N
H
O
H
H
*
H
H
O
Hydrophobic interaction(s)
Hydrogen bond
N
Asn51
*
Figure 3. Design of derivatives having an aliphatic side chain on the S atom.
(773
(Kd = 1.4 and 1.5 nM, respectively), 16b had significantly im-
proved water solubility (3320 M). This basic functional group
is assumed to be ionized in FaSSIF. Human liver microsomal clear-
lM) to 16a. Even though its Kd values were not improved
Thr184
Leu48
Asp93
l
ance (LM CL) of 16b (30
of acetylamino 16a (19
l
L/min/mg protein) was higher than that
Val150
Phe138
Gly97
l
L/min/mg protein). Although sub-nano-
Ile96
molar level affinity was retained (Kd = 0.54 nM), the hydroxyl 16c
showed poor water solubility (<32 M). Carbamoyloxy (18c) and
carboxamide (16d) showed acceptable water solubility (362 and
113 M, respectively) with high binding affinity (Kd = 0.46 and
0.66 nM, respectively). 18c and 16d also showed potent in vitro
cell growth inhibition (NCI-N87 IC50 = 0.036 and 0.056 M,
respectively) and sufficient stability (human LM CL = 20 and 7.0
L/min/mg protein). After judging all the differences, we used
Asn51
l
Gly137
Tyr139
Met98
l
Lys58
l
Leu107
l
these two acceptors for further modification.
Introduction of the acetylamino group (16e) to compound 16a
decreased affinity (Kd = 4.4 nM) and cellular IC50 (NCI-N87
Lys112
IC50 = 1.0
slightly improved affinity (Kd = 0.60 nM) and cellular IC50 (NCI-
N87 IC50 = 0.032 M), but water solubility decreased (53 M).
The compound with a chiral methyl group (16g) showed almost
the same activity as the racemate (16f). These data suggested
hydrophobic subsitituents are favorable on a methylene carbon,
but introducing the geminal dimethyl group (18f) as a hydrophobic
substituent caused a significant decrease in affinity (Kd = 41 nM)
lM) by 10-fold. Introduction of the methyl group (16f)
Figure 4. The superimposed structures of GM (1a) and CH5015765 (7) in Hsp90
a
.
,
Atom types were distinguished as separate colors: gray for CHsp90 , pink for C1a
l
l
a
green for C7, red for O, blue for N, yellow for S, and purple for Cl.
level affinity, ureido (18b), dimethylamino (16b), and hydroxyl
(16c) were prepared. Although the calculated LogP (cLogP) of
18b is lower than that of 16a, 18b showed similar solubility

896
A. Suda et al. / Bioorg. Med. Chem. 22 (2014) 892–905
Table 1
Binding affinity, in vitro cell growth inhibitory activity, water solubility and in vivo antitumor activity of derivatives having various HB acceptors attached on an aliphatic side
chain
NH2
Cl
N
N
S
N
(CH2)n
R
O
Solubilityb
(
lM)
cLogPc
PSAd (Å2)
LM CL (
l
L/min/mg protein)
Mouse
a
Compd
n
R
Kd (nM)
IC50
HCT116
(
l
M)
NCI-N87
Human
16a
18a
18b
16b
16c
18c
16d
2
2
2
2
2
2
2
NHAc
0.82
0.43
1.4
0.13
0.34
0.23
0.30
0.14
0.076
0.12
0.13
0.90
0.23
0.29
0.094
0.036
0.056
856
<31
773
3320
<32
2.25
2.34
1.85
2.98
2.31
2.43
1.78
85.1
101.3
107.5
64.1
75.8
104.4
95.7
19
26
13
30
22
24
13
32
44
8.8
82
18
20
7.0
NHSO2Me
NHCONH2
NMe2
OH
OCONH2
CONH2
1.5
0.54
0.46
0.66
362
113
a
b
c
Values were measured by SPR using human N-terminal Hsp90a.
Solubility in FaSSIF. (The use of FaSSIF may over-predict the solubility in physiological in vivo conditions).
cLogP was calculated by the PCModels software.
Molecular PSA was calculated by the TPSA software.
d
Table 2
Binding affinity, in vitro cell growth inhibitory activity, and water solubility of carboxamide derivatives and carbamoyloxy derivatives
NH2
Cl
N
N
S
R
N
O
Solubilityb
(lM)
cLogPc
PSAd (Å2)
LM CL (
l
L/min/mg protein)
a
Compd
R
Kd (nM)
Cell IC50
HCT116
(
l
M)
NCI-N87
Human
Mouse
O
16e
16f
4.4
1.2
1.0
ND
53
0.88
2.20
119.0
95.3
ND
11
ND
15
*
*
NH2
NHAc
O
0.60
0.048
0.032
NH2
[RAC]
O
O
O
16g
18f
0.33
41
0.034
6.4
0.026
7.3
34
2.20
2.65
95.4
95.0
53
49
*
NH2
ND
ND
ND
*
*
NH2
O
18d
18e
1.8
1.6
0.15
0.15
0.14
0.12
249
ND
2.71
2.71
96.1
95.8
40
37
N
H
O
*
O
ND
ND
N
H
a
b
c
Values were measured by SPR using human N-terminal Hsp90
Solubility in FaSSIF.
cLogP was calculated by the PCModels software.
Molecular PSA was calculated by the TPSA software.
a.
d
and cellular IC50 (NCI-N87 IC50 = 7.3
l
M). The geminal dimethyl
In the case of cyclic carbamoyl derivatives (18d and e), affinity
(Kd = 1.8 and 1.6 nM, respectively) and cellular IC50 (NCI-N87
group was considered to cause a conformation that is unfavorable
for binding to the protein and to expose one of the methyl groups
to the solvent-exposed region.
IC50 = 0.14 and 0.12
lM, respectively) were decreased in compari-
son to those of compound 18c. These results suggested that there is

A. Suda et al. / Bioorg. Med. Chem. 22 (2014) 892–905
897
Table 3
Binding affinity, in vitro cell growth inhibitory activity, and water solubility of carboxamide derivatives
NH2
Cl
N
N
S
N
(CH2)n
R
O
Solubilityb
(l
M)
cLogPc
PSAd (Å2)
LM CL (
Human
l
L/min/mg protein)
a
Compd
n
R
Kd (nM)
Cell IC50
HCT116
(
l
M)
NCI-N87
Mouse
16h
16i
16j
16k
16l
2
2
2
1
3
CONHMe
CONMe2
CONH(CH2)2OH
CONH2
0.66
2.5
1.3
1.3
0.48
0.086
0.31
0.29
0.27
0.098
0.069
0.31
0.16
0.16
0.066
1370
35
2310
86
2.25
2.46
1.27
1.29
2.27
84.9
76.3
101.0
95.5
11
30
8.7
11
14
24
18
10
7.6
14
CONH2
646
95.8
a
b
c
Values were measured by SPR using human N-terminal Hsp90
Solubility in FaSSIF.
cLogP was calculated by the PCModels software.
Molecular PSA was calculated by the TPSA software.
a.
d
no advantage to introducing substituents to a methylene carbon or
to forming cyclic carbamoyl groups.
(Kd = 1.3 nM), and compound 16l with three methylene carbons,
showed improved water solubility (646 M) and retained affinity
l
The effect that different substituents had on the amide nitrogen
of compound 16d was examined. Methylamide (16h) significantly
improved water solubility by 12-fold (1370 lM) in comparison to
compound 16d while retaining affinity (Kd = 0.66 nM). However,
dimethylamide (16i) decreased affinity (Kd = 2.5 nM) and water
(Kd = 0.48 nM). In these three compounds (16d, 16k, and 16l), the
numbers of rotatable bonds correlate to water solubility.22 In con-
clusion, compound 16l excels in the series of compounds by its
in vitro efficacy and physicochemical properties. In addition, com-
pound 16l reduced the phosphorylation and protein level of multi-
ple Hsp90 client proteins9 such as EGFR, HER2, Raf1, and AKT
(Fig. 5). This result indicates that our compound inhibits prolifera-
tion of cancer cells by inducing degradation of multiple Hsp90 cli-
ent proteins.
solubility (35
(Kd = 1.3 nM), although water solubility was improved
(2310 M). From the above examples, various HB acceptors are
lM). 2-Hydroxyethylamide (16j) decreased affinity
l
acceptable from the viewpoint of efficacy, and unsubstituted and
N-methyl-substituted carboxamide groups have an advantage in
terms of water solubility and liver microsomal stability. Finally,
the number of methylene carbons was optimized. As expected
from the analysis of the X-ray cocrystal structure, compound 16k
with one methylene carbon showed a slight decrease in affinity
2.4. X-ray cocrystal structure
The X-ray cocrystal structure of compound 16l and Hsp90
(PDB: 3WHA) reveals that the butyramide side chain contributes
to the improved affinity to Hsp90. The methylene groups at 4-
and 2-position of the butyramide side chain (C-4 and C-2, respec-
tively) is located at a distance (d) of lipophilic interactions with
16l
the methylene group at
c-position (C-c) in the side chain of
Met98 (d(C-4ÁÁÁC-
c
Met98) = 3.5 Å) and with the methyl group on
0
0.04 0.2
1
5 μM
b-carbon (C-bCH3) in the side chain of Ile96 (d(C-2ÁÁÁC-bCH3
EGFR
HER2
Raf1
Ile96) = 3.5 Å), respectively. The amide group of the butyramide
Leu48
Thr184
Asp93
pAKT
AKT
Asp54
Asn51
Phe138
2.8
Val150
2.8
2.6
Gly97
2.8
pERK
ERK
Ile96
3.5
2.9
3.5
PARP
Hsp70
GAPDH
Met98
Tyr139
2.9
Leu107
Lys58
Figure 6. An X-ray structure of N-terminal Hsp90
a in complex with 16l. The
numbering of the residues corresponds to those in the X-ray cocrystal structure
Figure 5. Degradation of multiple Hsp90 client proteins induced by compound 16l
in NCI-N87 cells. NCI-N87 cells were treated with the indicated concentration of
compound 16l for 24 h before lysing and analysis by Western blotting.
(gray CHsp90 , green Cligand, red O, blue N, yellow S, Purple Cl). Hydrogen bonds are
indicated by black dashes and lipophilic interactions are indicated by purple dashes.
Detailed binding mode of compound 16l (PDB code: 3WHA).
a

898
A. Suda et al. / Bioorg. Med. Chem. 22 (2014) 892–905
Table 4
Pharmacokinetic parameters of 7 and 16l in nude mice
Administration
Dose (mg/kg)
t1/2 (h)
tmax (h)
Cmax
(lg/mL)
AUCinf
(
lgÁh/mL)
CL or CL/F (mL/h/kg)
Fa (%)
7
iv
po
iv
10
200
10
0.250
2.62
0.963
0.944
—
0.5
—
—
3.92
—
36.7
8.55
7.65
24.9
54.8
1170
26100
403
—
4.5
—
(CH5015765)
16l
(CH5138303)
po
50
0.75
938
44.0
a
Oral bioavailability.
in mice demonstrated that compound 16l had improved PK pro-
files of total systemic exposure (AUC), plasma clearance (CL), and
oral bioavailability (F) in comparison to those of compound 7
(Table 4).
2.6. Efficacy in tumor xenograft studies
Compound 16l was further evaluated in a human NCI-N87 gas-
tric cancer xenograft mouse model (Fig. 7). Mice bearing NCI-N87
were orally treated with compound 16l once daily for 11 days at
0.62, 1.85, 5.56, 16.7, and 50 mg/kg, and their body weight and
tumor volume were measured twice a week. Compound 16l
showed potent antitumor efficacy with TGI of 136% and a median
effective dose (ED50) of 3.9 mg/kg without significant loss of body
weight.
3. Conclusion
In summary, we identified novel 2-amino-6-(1H,3H-
benzo[de]isochromen-6-yl)-1,3,5-triazines as Hsp90 inhibitors by
modifying the lead compound 7 (CH5015765). We improved the
binding affinity together with the physicochemical properties of
water solubility, liver microsomal stability and in vitro cell growth
inhibition. Of the resulting compounds, compound 16l (CH5
138303) demonstrated high oral bioavailability in mice and potent
antitumor efficacy in a human NCI-N87 gastric cancer xenograft
mouse model.
Figure 7. Growth inhibitory effect of compound 16l on NCI-N87 tumor xenograft
model. SCID mice bearing NCI-N87 cells were treated orally with the indicated
doses of compound 16l on a daily basis for 11 consecutive days (days 11–21) at
0.62 mg/kg (TGI = 27%), 1.85 mg/kg (TGI = 34%), 5.56 mg/kg (TGI = 53%), 16.7 mg/kg
(TGI = 90%), and 50 mg/kg (TGI = 136%). Mean tumor volume and mean body weight
change of mice are shown.
4. Experiments
4.1. Materials and measurements
Organic synthesis reactions were conducted using commer-
cially available reagents without further purification. Room tem-
perature refers to the range of 20–25 °C. All moisture-sensitive
reactions were performed under a nitrogen or argon atmosphere.
Concentration or solvent removal under reduced pressure was car-
ried out using a rotary evaporator, unless otherwise stated. Thin
layer chromatography (TLC) analytical separations were conducted
with silica gel 60 F254 precoated TLC plates or NH TLC plates, each
manufactured by E. Merck. Standard silica gel column chromatog-
raphy was employed as a method of purification using the indi-
cated solvent mixture.
NMR analyses were carried out using a JNM-EX270 (270 MHz),
a JNM-GSX400 (400 MHz), a JNM-A500 (500 MHz) (all manufac-
tured by JEOL), or an ARX-300 (300 MHz) manufactured by Bruker.
Chemical shifts are reported in parts per million (d). The deuterium
lock signal of the sample solvent was used as a reference, and cou-
pling constants (J) are given in hertz (Hz). The splitting pattern
abbreviations are as follows: s, singlet; d, doublet; t, triplet; q,
quartet; br, broadened; and m, unresolved multiplet due to the
field strength of the instrument. High performance liquid chroma-
tography-mass spectrometric data were obtained using a Micro-
side chain does not interact with the amino group of Lys58
strongly. As seen in the cocrystal structure, there are two alterna-
tive conformations of Lys58, depending on whether the amino
group at
e
-position (N-
e
) in the side chain of Lys58 is located at
Lys58ÁÁÁO) =
a distance of one HB from the amide oxygen (d(N-
e
2.9 Å) or not. This means the HB between Lys58 and the amide
is partial. On the other hand, the amide group forms HBs to the
protein via water molecules. Namely, the amide nitrogen forms
a HB to the carbonyl oxygen in the main chain of Asn51 via a
water molecule and the amide oxygen forms HBs to the carbonyl
oxygen in the main chain of Asp54 via two water molecules. In
the X-ray cocrystal structure of GM and Hsp90, the water mole-
cules are excluded by GM (PDB code: 1YET). Hence, introducing
the polar substituents improves the affinity by forming a HB net-
work of water molecules (Fig. 6).
2.5. Pharmacokinetics
Since compound 16l showed high binding affinity, strong
in vitro cell growth inhibition, and sufficient in vitro metabolic sta-
bility, we conducted in vivo profiling. The pharmacokinetic study
mass ZMD coupled with
performance liquid chromatography system or a Micromass ZQ
a Waters 996–600E gradient high

A. Suda et al. / Bioorg. Med. Chem. 22 (2014) 892–905
899
coupled with a Waters 2525 high performance liquid chromatogra-
4.2.3. 7-Nitro-3-oxatricyclo[7.3.1.05,13]trideca-1(13),5,7,9,11-
pentaene-8-carbonitrile (11)
phy system using the positive electrospray ionization technique
(ESI+), both using a mobile phase of CH3CN/water with 0.05%
TFA. The purity of all tested compounds is P95% unless explicitly
stated otherwise. High resolution mass spectra (HRMS) of ESI+
were obtained using an LTQ Orbitrap XL (Thermofisher scientific)
with an ACQUITY ultra performance liquid chromatography sys-
tem (Waters), or a QSTAR XL (AB Sciex) with Agilent 1100 (Agi-
lent). Fast atom bombardment (FAB) and electron impact (EI)
mass spectra were obtained using a JMC-GCmate II (JEOL). The
abbreviations of mass spectrometry are as follows: m/z is the
mass-to-charge ratio; M is the molecular weight of the molecule
itself; and M+ is the molecular ion.
8-Bromo-7-nitro-3-oxatricyclo[7.3.1.05,13]trideca-
1(13),5,7,9,11-pentaene (10, 50.0 g, 170 mmol) and copper(I) cya-
nide (18.3 g, 204 mmol) were dissolved in anhydrous N,N-dimeth-
ylformamide (500 mL). Then, the flask was purged with nitrogen to
replace air. The mixture was stirred at 120 °C for 1 h, and then
cooled to room temperature. The resulting material was added to
10% aqueous ammonia (1.4 L) and stirred for 30 min. The resulting
mixture was diluted with water (1.2 L) and the precipitates were
collected by filtration. The precipitates were washed with 5% aque-
ous ammonia (1.0 L) and water successively. The precipitates were
suspended in acetonitrile (200 mL) with stirring at room tempera-
ture overnight, collected by filtration, washed with acetonitrile
(20 mL, twice) to yield the title compound as a yellow solid
(34.7 g, 85%). 1H NMR (300 MHz, CDCl3) d: 5.13 (2H, s), 5.16 (2H,
s), 7.53 (1H, d, J = 7.4 Hz), 7.84 (1H, dd, J = 8.3, 7.4 Hz), 8.15 (1H,
s), 8.42 (1H, d, J = 8.3 Hz). HRMS (ESIÀ) m/z: [MÀH]À calcd for
C13H7N2O3, 239.04622; found, 239.04526.
4.2. Synthetic procedures
4.2.1. 8-Bromo-7-nitro-3-oxatricyclo[7.3.1.05,13]trideca-1(13),5,7,
9,11-pentaene-2,4-dione (9)
8-Bromo-3-oxatricyclo[7.3.1.05,13]trideca-1(13),5,7,9,11-pent-
aene-2,4-dione (8, 126.2 g, 456 mmol) was dissolved in sulfuric
acid (620 mL). The reaction vessel was cooled in an ice bath, and
a mixed solution of fuming nitric acid (65.5 mL) and sulfuric acid
(84.8 mL) was added dropwise while keeping the internal temper-
ature below 4 °C. Then, the reaction mixture was stirred for 2.5 h
while keeping the internal temperature below 4 °C. Ice water
(1.5 L) was slowly added to the reaction mixture, and stirred at
room temperature for 30 min. Water (1.5 L) was added to the
aqueous mixture, and the resulting precipitates were filtered. The
precipitates were suspended in acetonitrile (1.0 L). The suspension
was stirred at room temperature overnight and filtered (3-nitro/6-
nitro derivative = 6:1 from 1H NMR analysis). After washing with
acetonitrile, the resulting precipitates were again suspended in
acetonitrile (1.0 L). The same treatment was repeated, and the pre-
cipitates were dried under reduced pressure to yield the title com-
4.2.4. 7-Amino-3-oxatricyclo[7.3.1.05,13]trideca-1(13),5,7,9,11-
pentaene-8-carbonitrile (12)
7-Nitro-3-oxatricyclo[7.3.1.05,13]trideca-1(13),5,7,9,11-pentae-
ne-8-carbonitrile (11, 50.0 g, 208 mmol) was suspended in tetrahy-
drofuran (680 mL). The suspension was heated to reflux and
sodium dithionite (109 g, 624 mmol) in water (500 mL) was added
to the solution under refluxing for over 1 h. After addition, the mix-
ture was stirred under refluxing for 30 min. Insoluble materials
were removed by filtration when the resulting mixture was cooled
to around 60 °C. 5 N hydrochloric acid (510 mL) was added to the
filtrate and the resulting mixture was stirred at room temperature
for 1 h. The pH was adjusted to approximately 10 by dropwise
addition of 5 N aqueous sodium hydroxide solution (650 mL) over
1.5 h in a water bath. The mixture was diluted with water (800 mL)
and stirred for 30 min. The precipitates were collected by filtration,
washed with water until washings became neutral, and dried un-
der reduced pressure. The title compound was obtained as a pale
yellow solid (37.5 g, 86%). 1H NMR (300 MHz, CDCl3) d: 4.75 (2H,
br s), 4.93 (2H, s), 4.99 (2H, s), 6.60 (1H, s), 7.02 (1H, d,
J = 7.2 Hz), 7.50 (1H, dd, J = 8.4, 7.2 Hz), 7.79 (1H, d, J = 8.4 Hz).
HRMS (ESI+) m/z: [M+H]+ calcd for C13H11N2O, 211.0865; found,
211.0871.
pound as
a pale yellow solid (120 g, 82%, 3-nitro/6-nitro
derivative = 13:1 from 1H NMR analysis). 1H NMR (300 MHz,
DMSO-d6) d: 8.19 (1H, dd, J = 7.2, 8.8 Hz), 8.74 (1H, d, J = 8.8 Hz),
8.83 (1H, d, J = 7.2 Hz), 8.92 (1H, s). HRMS (EI+) m/z: [M]+ calcd
for C12H479BrNO5, 320.9273; found, 320.9260, [M]+ calcd for
C
12H481BrNO5, 322.9252; found, 322.9250.
4.2.2. 8-Bromo-7-nitro-3-oxatricyclo[7.3.1.05,13]trideca-1(13),5,
7,9,11-pentaene (10)
4.2.5. 7-Chloro-3-oxatricyclo[7.3.1.05,13]trideca-1(13),5,7,9,11-
pentaene-8-carbonitrile (13)
Tetrahydrofuran (2.3 L) was added to 8-bromo-7-nitro-3-
oxatricyclo[7.3.1.05,13]trideca-1(13),5,7,9,11-pentaene-2,4-dione
(9, 120 g, 372.6 mmol), and stirred thoroughly to prepare a suspen-
sion. The suspension was cooled in an ice-water bath. Lithium
borohydride (16.3 g, 745.2 mmol) and trifluoroborane ether com-
plex (141.6 mL, 1118 mmol) were added to the suspension succes-
sively while keeping the internal temperature below 4 °C. The
resulting reaction mixture was stirred at 40–50 °C for 2 h. The mix-
ture was cooled to room temperature, and then water (500 mL)
was added and extracted twice with ethyl acetate (1.5 L). The or-
ganic layer was washed with brine (500 mL), dried over anhydrous
magnesium sulfate, and then concentrated under reduced pres-
sure. The crude product was suspended in a mixed solvent of n-
hexane and ethyl acetate (5:1, 1.2 L). The mixture was stirred at
room temperature for 2 h, and filtered. After washing with n-hex-
ane–ethyl acetate (5:1), the mixture was dried under reduced pres-
sure to yield the title compound as a pale yellow solid (108 g, 99%).
1H NMR (300 MHz, DMSO-d6) d: 5.05 (2H, s), 5.08 (2H, s), 7.59 (1H,
d, J = 7.0 Hz), 7.81–7.86 (2H, m), 8.24 (1H, d, J = 8.3 Hz). HRMS
(ESIÀ) m/z: [MÀH]À calcd for C12H779BrNO3, 291.96148; found,
291.96067, [MÀH]À calcd for C12H781BrNO3, 293.94058; found,
293.93925.
To a suspension of copper(II) chloride (21.5 g, 160 mmol) in
anhydrous acetonitrile (1.1 mL) was added tert-butyl nitrite
(24 mL, 200 mmol) at room temperature under nitrogen atmo-
sphere. 7-Amino-3-oxatricyclo[7.3.1.05,13]trideca-1(13),5,7,9,11-
pentaene-8-carbonitrile (12, 28.0 g, 133 mmol) was added portion-
wise thereto over 30 min. The resulting mixture was stirred at
room temperature for 1 h. 1 N hydrochloric acid (1.1 L) and water
(1.1 L) were added slowly to the mixture successively, and the
resulting mixture was stirred for 20 min. The precipitates were col-
lected by filtration, washed with water, and dried under reduced
pressure. The resulting residue was roughly purified by passing
through silica gel (100 g) using dichloromethane (1.5 L). The eluent
containing the desired product was concentrated to give the crude
product (26.4 g). The crude product was purified by crystallization
from acetonitrile (1.0 L). The crystals were collected by filtration,
washed with cold acetonitrile (50 mL, twice), and dried under re-
duced pressure. The title compound was obtained as a yellow solid
(21.3 g, 70%). 1H NMR (300 MHz, CDCl3) d: 5.05 (2H, s), 5.07 (2H, s),
7.27 (1H, s), 7.32 (1H, d, J = 7.2 Hz), 7.68 (1H, dd, J = 8.7, 7.2 Hz),
8.07 (1H, d, J = 8.7 Hz). HRMS (ESI+) m/z: [M+H]+ calcd for C13H9
35-
ClNO, 230.0367; found, 230.0362, [M+H]+ calcd for C13H937ClNO,

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A. Suda et al. / Bioorg. Med. Chem. 22 (2014) 892–905
232.0337; found, 232.0342, [M+NH4]+ calcd for C13H1235ClN2O,
247.0632; found, 247.0640.
4.2.9. N-{2-[(4-Amino-6-{7-chloro-3-oxatricyclo[7.3.1.05,13]tri
deca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazin-2-yl)sulfanyl]
ethyl}acetamide (16a)
4.2.6. 7-chloro-3-oxatricyclo[7.3.1.05,13]trideca-1(13),5,7,9,11-
pentaene-8-carboximidamide (14)
4-{7-Chloro-3-oxatricyclo[7.3.1.05,13]trideca-1(13),5,7,9,11-
pentaen-8-yl}-6-methanesulfinyl-1,3,5-triazin-2-amine (15, 5.8 g,
To a suspension of ammonium chloride (31.4 g, 699 mmol)
in toluene (70 mL) was added dropwise a solution of 2 N trim-
ethylaluminum in toluene (350 mL, 699 mmol) over 1 h at room
temperature. The resulting solution was stirred for 30 min and
7-chloro-3-oxatricyclo[7.3.1.05,13]trideca-1(13),5,7,9,11-pentae-
ne-8-carbonitrile (13, 8.03 g, 35.0 mmol) was added thereto. The
mixture was stirred while heating under reflux for 21 h, and
then cooled to room temperature. A saturated aqueous potas-
sium sodium tartrate solution (1 L) and 3 N aqueous sodium
hydroxide solution (500 mL) were added to the reaction mixture
successively, and then extracted with ethyl acetate (2.0 L, twice).
The organic layers were combined, washed with water (500 mL,
twice) and brine (500 mL) successively, and dried over anhy-
drous magnesium sulfate. The inorganic salts were removed by
filtration, and the filtrate was concentrated under reduced
pressure. The title compound was obtained as a yellow solid
(8.15 g, 95%). 1H NMR (300 MHz, DMSO-d6) d: 5.01 (4H, s),
6.65–6.90 (3H, br s), 7.32 (1H, d, J = 7.4 Hz), 7.36 (1H, s), 7.57
(1H, dd, J = 7.4, 8.3 Hz), 7.79 (1H, d, J = 8.3 Hz). HRMS (ESI+)
m/z: [M+H]+ calcd for C13H1235ClN2O, 247.06327; found,
247.06268, [M+H]+ calcd for C13H1237ClN2O, 249.06032; found,
249.05964.
16.1 mmol) was dissolved in a mixture of tetrahydrofuran
(400 mL) and N,N-dimethylformamide (30 mL). N-(2-Sulfanyleth-
yl)acetamide (3.4 mL, 32.1 mmol) and triethylamine (6.7 mL,
48.2 mmol) were added to the solution at room temperature. The
resulting mixture was stirred for 15 h at room temperature and
then the solvent was removed by evaporation. The resulting resi-
due was dissolved in ethyl acetate (2.0 L), washed with water
(1.0 L, twice) and brine (1.0 L) successively, dried over anhydrous
magnesium sulfate, and filtered. The filtrate was concentrated by
evaporation and the resulting residue was purified by recrystalliza-
tion from ethanol (80 mL). The solids were dissolved with a mix-
ture of ethyl acetate (2.0 L) and methanol (100 mL) washed with
water (1.0 L, 3 times). The organic layer was diluted with ethanol
(1.0 L) and concentrated by evaporation. The resulting solids were
triturated in ethanol (50 mL), collected by filtration, and dried un-
der reduced pressure. The title compound was obtained as a white
solid (4.8 g, 72%). 1H NMR (300 MHz, DMSO-d6) d: 1.77 (3H, s), 3.10
(2H, t, J = 6.6 Hz), 3.31 (2H, m), 5.04 (2H, s), 5.05 (2H, s), 7.34 (1H, d,
J = 6.5 Hz), 7.41 (1H, d, J = 8.4 Hz), 7.45 (1H, s), 7.52 (1H, dd, J = 8.4,
6.5 Hz), 7.81 (1H, br s), 7.83 (1H, br s), 8.07 (1H, t, J = 6.2 Hz). MS
(ESI+) m/z: 416, 418 [M+H]+. HRMS (ESI+) m/z: [M+H]+ calcd for
C
C
19H1935ClN5O2S, 416.09425; found, 416.09423, [M+H]+ calcd for
19H1937ClN5O2S, 418.09130; found, 418.09126.
4.2.7. 4-{7-Chloro-3-oxatricyclo[7.3.1.05,13]trideca-1(13),5,7,9,11
-pentaen-8-yl}-6-(methylsulfanyl)-1,3,5-triazin-2-amine (7)
4.2.10. 4-{7-Chloro-3-oxatricyclo[7.3.1.05,13]trideca-1(13),5,7,9,
11-pentaen-8-yl}-6-{[2-(dimethylamino)ethyl]sulfanyl}-1,3,5-
triazin-2-amine (16b)
To
a
solution of 7-chloro-3-oxatricyclo[7.3.1.05,13]trideca-
1(13),5,7,9,11-pentaene-8-carboximidamide (14, 5.8 g, 23.9 mmol)
in ethanol (60 mL) were added dimethyl cyanodithioiminocarbon-
ate (3.8 g, 26.3 mmol) and N-ethyldiisopropylamine (8.3 mL,
47.8 mmol) at room temperature. The resulting mixture was stir-
red under refluxing ethanol for 3 h and then cooled in an ice-water
bath. The precipitates were collected by filtration, washed with
cold ethanol (100 mL), and dried under reduced pressure. The title
compound was obtained as a yellow solid (7.1 g, 88%). 1H NMR
(300 MHz, DMSO-d6) d: 2.45 (3H, s), 5.04 (2H, s), 5.05 (2H, s),
7.34 (1H, d, J = 6.9 Hz), 7.40 (1H, d, J = 8.4 Hz), 7.44 (1H, s), 7.52
(1H, dd, J = 6.9, 8.4 Hz), 7.79 (2H, br s). MS (ESI+) m/z: 345, 347
[M+H]+. HRMS (ESI+) m/z: [M+H]+ calcd for C16H14ON435ClS,
345.0571; found, 345.0573, [M+H]+ calcd for C16H14ON437ClS,
347.0542; found, 347.0542.
4-{7-Chloro-3-oxatricyclo[7.3.1.05,13]trideca-1(13),5,7,9,11-pen-
taen-8-yl}-6-methanesulfinyl-1,3,5-triazin-2-amine (15, 50.0 mg,
0.139 mmol) and 2-(dimethylamino)ethanethiol hydrochloride
(39.3 mg, 0.277 mmol) was dissolved in N,N-dimethylformamide
(3.5 mL). Potassium carbonate (57.5 mg, 0.416 mmol) was added
to the solution and the resulting mixture was stirred for 1 h at
room temperature. The reaction mixture was diluted with ethyl-
acetate and the solution was washed with water and brine succes-
sively. The organic layer was dried over anhydrous magnesium
sulfate. The inorganic salts were removed by filtration and the fil-
trate was concentrated under reduced pressure. The crude product
was purified by silica gel column chromatography (dichlorometh-
ane/methanol = 10:1 to 4:1). The obtained product was triturated
in a mixture of dichloromethane and n-hexane (1:2, 10 mL),
washed with n-hexane, and dried in a vacuum. The title compound
was obtained as a light brown solid (29.2 mg, 52%). 1H NMR
(300 MHz, DMSO-d6) d: 2.12 (6H, s), 2.50 (2H, t, J = 7.2 Hz), 3.15
(2H, t, J = 7.2 Hz), 5.05 (4H, s), 7.34 (1H, d, J = 6.9 Hz), 7.40 (1H, d,
J = 8.4 Hz), 7.45 (1H, s), 7.52 (1H, dd, J = 8.4, 6.9 Hz), 7.79 (2H, br
s). MS (ESI+) m/z: 402, 404 [M+H]+. HRMS (ESI+) m/z: [M+H]+ calcd
for C19H2135ClN5OS, 402.11499; found, 402.11427, [M+H]+ calcd for
4.2.8. 4-{7-Chloro-3-oxatricyclo[7.3.1.05,13]trideca-1(13),5,7,9,11
-pentaen-8-yl}-6-methanesulfinyl-1,3,5-triazin-2-amine (15)
To a solution of 4-{7-chloro-3-oxatricyclo[7.3.1.05,13]trideca-
1(13),5,7,9,11-pentaen-8-yl}-6-(methylsulfanyl)-1,3,5-triazin-2-
amine (7, 7.1 g, 20.6 mmol) in dichloromethane (70 mL) was
added 3-chloroperbenzoic acid (5.7 g, 32.9 mmol) at room tem-
perature. After the reaction mixture was stirred at room temper-
ature for 3 h, the precipitate was collected by filtration and
washed with dichloromethane (100 mL). The resulting precipitate
was suspended in dichloromethane (1.5 L), and stirred at room
temperature for 1 h. The precipitates were collected by filtration,
washed with dichloromethane, and dried under reduced pres-
sure. The title compound was obtained as a light brown solid
(5.8 g, 78%). 1H NMR (300 MHz, DMSO-d6) d: 2.88 (3H, s), 5.05
(2H, s), 5.06 (2H, s), 7.32–7.40 (1H, m), 7.49–7.58 (3H, m), 8.38
(1H, br s), 8.53 (1H, br s). MS (ESI+) m/z: 361, 363 [M+H]+. HRMS
(ESI+) m/z: [M+H]+ calcd for C16H1435ClN4O2S, 361.05205; found,
361.05145, [M+H]+ calcd for C16H1437ClN4O2S, 363.04910; found,
363.04838.
C
19H2137ClN5OS, 404.11204; found, 404.11119.
4.2.11. 2-[(4-Amino-6-{7-chloro-3-
oxatricyclo[7.3.1.05,13]trideca-1(13),5,7,9,11-pentaen-8-yl}-
1,3,5-triazin-2-yl)sulfanyl]ethan-1-ol (16c)
4-{7-Chloro-3-oxatricyclo[7.3.1.05,13]trideca-1(13),5,7,9,11-pen-
taen-8-yl}-6-methanesulfinyl-1,3,5-triazin-2-amine (15, 50.0 mg,
0.139 mmol) was dissolved in tetrahydrofuran (5.0 mL).
2-Hydroxyethanethiol (19.5
lL, 0.278 mmol) and triethylamine
(97.0 L, 0.700 mmol) was added successively to the solution at
l
0 °C and the resulting mixture was stirred for 16 h at 15 °C. The sol-
vent was removed by evaporation and the resulting residue was
purified by silica gel column chromatography (dichloromethane/

A. Suda et al. / Bioorg. Med. Chem. 22 (2014) 892–905
901
ethylacetate = 10:1 to 1:1). The title compound was obtained as a
light brown solid (31.0 mg, 59%). 1H NMR (300 MHz, DMSO-d6) d:
3.14 (2H, d, J = 6.5 Hz), 3.62 (2H, dt, J = 5.5, 6.5 Hz), 4.91 (1H, t,
J = 5.5 Hz), 5.04 (2H, s), 5.05 (2H, s), 7.34 (1H, d, J = 7.8 Hz), 7.40
(1H, d, J = 8.0 Hz), 7.44 (1H, s), 7.52 (1H, dd, J = 8.0, 7.8 Hz), 7.78
(2H, br s). MS (ESI+) m/z: 375, 377 [M+H]+. HRMS (ESI+) m/z:
[M+H]+ calcd for C17H1635ClN4O2S, 375.06770; found, 375.06695,
[M+H]+ calcd for C17H1637ClN4O2S, 377.06475; found, 377.06383.
4.2.14. 3-[(4-Amino-6-{7-chloro-3-oxatricyclo[7.3.1.05,13
]
trideca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazin-2-yl)sulfanyl]
-2-methylpropanamide (16f)
Compound 16f was prepared from compound 15 and 2-methyl-
3-sulfanylpropanoic acid instead of 3-sulfanylpropanoic acid in the
same manner as that described for the preparation of compound
16d (94%, a white solid).
1H NMR (270 MHz, DMSO-d6) d: 1.09 (3H, d, J = 6.5 Hz), 2.51–
2.67 (1H, m), 3.10–3.19 (2H, m), 5.05 (4H, s), 6.89 (1H, br s), 7.34
(1H, d, J = 7.3 Hz), 7.36 (1H, s), 7.42 (1H, d, J = 8.9 Hz), 7.45 (1H,
s), 7.51 (1H, dd, J = 8.9, 7.3 Hz), 7.82 (2H, br s). MS (ESI+) m/z:
416, 418 [M+H]+. HRMS (ESI+) m/z: [M+H]+ calcd for C19H1935ClN5-
O2S, 416.09425; found, 416.09409, [M+H]+ calcd for C19H1937ClN5-
O2S, 418.09130; found, 418.09110.
4.2.12. 3-[(4-Amino-6-{7-chloro-3-oxatricyclo[7.3.1.05,13]tride
ca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazin-2-yl)sulfanyl]
propanamide (16d)
To the solution of 4-{7-chloro-3-oxatricyclo[7.3.1.05,13]trideca-
1(13),5,7,9,11-pentaen-8-yl}-6-methanesulfinyl-1,3,5-triazin-2-
amine (15, 8.07 g, 22.4 mmol) in N,N-dimethylformamide
(81.0 mL) was added 3-sulfanylpropanoic acid (4.9 mL, 55.9 mmol)
and N-ethyldiisopropylamine (11.7 mL, 67.1 mmol) at room tem-
perature successively. The resulting mixture was stirred at room
temperature for 1 h. The reaction mixture was poured into a mix-
ture of 0.5 N aqueous potassium hydrogen sulfate solution
(800 mL) and water (400 mL) and the resulting aqueous solution
was stirred at room temperature for 0.5 h. The precipitates were
collected by filtration, washed with water (400 mL), dried under
reduced pressure. 3-[(4-amino-6-{7-chloro-3-oxatricyclo[7.3.1
.05,13]trideca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazin-2-yl)sulfa-
nyl]propanoic acid was obtained as a white solid (8.86 g, 98%). 1H
NMR (300 MHz, DMSO-d6) d: 2.68 (2H, t, J = 6.9 Hz), 3.18 (2H, t,
J = 6.9 Hz), 5.04 (2H, s), 5.05 (2H, s), 7.34 (1H, d, J = 6.9 Hz), 7.42
(1H, d, J = 8.0 Hz), 7.45 (1H, s), 7.52 (1H, dd, J = 8.0, 6.9 Hz), 7.87
(2H, br s), 12.40 (1H, br s). MS (ESI+) m/z: 403, 405 [M+H]+.
4.2.15. (2S)-3-[(4-Amino-6-{7-chloro-3-oxatricyclo[7.3.1.05,13
trideca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazin-2-yl)sulfanyl]
-2-methylpropanamide (16g)
]
Compound 16g was prepared from compound 15 and (2S)-2-
methyl-3-sulfanylpropanoic acid instead of 3-sulfanylpropanoic
acid in the same manner as that described for the preparation of
compound 16d (78%, a white solid). 1H NMR (400 MHz, DMSO-
d6) d: 1.10 (3H, d, J = 7.1 Hz), 2.54–2.67 (1H, m), 3.10–3.19 (2H,
m), 5.05 (2H, s), 5.05 (2H, s), 6.88 (1H, br s), 7.34 (1H, d,
J = 7.1 Hz), 7.36 (1H, br s), 7.42 (1H, d, J = 8.2 Hz), 7.45 (1H, s),
7.53 (1H, dd, J = 8.2, 7.1 Hz), 7.81 (2H, br s). MS (ESI+) m/z: 416,
418 [M+H]+.
4.2.16. 3-[(4-Amino-6-{7-chloro-3-oxatricyclo[7.3.1.05,13
]
trideca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazin-2-yl)sulfanyl]
-N-methylpropanamide (16h)
To
a
solution
of
3-[(4-amino-6-{7-chloro-3-oxatricy-
clo[7.3.1.05,13]trideca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazin-2-
yl)sulfanyl]propanoic acid (6.85 g, 17.0 mmol), ammonium chlo-
ride (2.73 g, 51.0 mmol), 1-hydroxybenzotirazole (6.89 g,
51.0 mmol), and 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide
hydrochloride (9.78 g, 51.0 mmol) in N,N-dimethylformamide
(68.5 mL) was added N-ethyldiisopropylamine (11.9 mL,
68.0 mmol) at room temperature. The resulting mixture was stir-
red at room temperature for 20 h. The reaction mixture was diluted
with ethyl acetate (1.2 L), washed with a saturated sodium hydro-
gen carbonate aqueous solution (200 mL, twice), water (200 mL,
twice) and brine (200 mL) successively, dried over anhydrous mag-
nesium sulfate, and filtered. The filtrate was concentrated and the
resulting residue was purified by silica gel column chromatogra-
phy (dichromethane/methanol = 20:1) to yield the title compound
as a white solid (5.63 g, 82%). 1H NMR (300 MHz, DMSO-d6) d: 2.49
(2H, t, J = 6.9 Hz), 3.21 (2H, t, J = 6.9 Hz), 5.05 (4H, s), 6.96 (1H, br s),
7.35 (1H, d, J = 6.9 Hz), 7.38 (1H, br s), 7.42 (1H, d, J = 7.7 Hz), 7.46
(1H, s), 7.52 (1H, dd, J = 7.7, 6.9 Hz), 7.86 (2H, br s). MS (ESI+) m/z:
402, 404 [M+H]+. HRMS (ESI+) m/z: [M+H]+ calcd for C18H1735ClN5-
O2S, 402.07860; found, 402.07776, [M+H]+ calcd for C18H1737ClN5-
O2S, 404.07565; found, 404.07475.
Compound 16h was prepared from compound 15 and methyl-
amine hydrochloride instead of ammonium chloride in the same
manner as that described for the preparation of compound 16d
(75%, a white solid). 1H NMR (270 MHz, CD3OD) d: 2.67 (2H, t,
J = 7.1 Hz), 2.71 (3H, s), 3.39 (2H, t, J = 7.1 Hz), 5.06 (4H, s), 7.28
(1H, d, J = 6.8 Hz), 7.35 (1H, s), 7.41 (1H, d, J = 8.6 Hz), 7.49 (1H,
dd, J = 8.6, 6.8 Hz). MS (ESI+) m/z: 416, 418 [M+H]+. HRMS (ESI+)
m/z: [M+H]+ calcd for C19H1935ClN5O2S, 416.09425; found,
416.09359, [M+H]+ calcd for C19H1937ClN5O2S, 418.09130; found,
418.09054.
4.2.17. 3-[(4-Amino-6-{7-chloro-3-oxatricyclo[7.3.1.05,13
]
trideca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazin-2-yl)sulfanyl]
-N,N-dimethylpropanamide (16i)
Compound 16i was prepared from compound 15 and dimethyl-
amine hydrochloride instead of ammonium chloride in the same
manner as that described for the preparation of compound 16d
(33%, a white solid). 1H NMR (270 MHz, CDCl3) d: 2.80 (2H, t,
J = 7.3 Hz), 2.90 (3H, s), 2.92 (3H, s), 3.37 (2H, t, J = 7.3 Hz), 5.06
(2H, s), 5.07 (2H, s), 5.60 (2H, br s), 7.20 (1H, dd, J = 4.9, 3.1 Hz),
7.24 (1H, s), 7.45 (1H, d, J = 4.9 Hz), 7.45 (1H, d, J = 3.1 Hz). MS
(ESI+) m/z: 430, 432 [M+H]+. HRMS (ESI+) m/z: [M+H]+ calcd for C20-
H2135ClN5O2S, 430.10990; found, 430.10919, [M+H]+ calcd for C20-
H2137ClN5O2S, 432.10695; found, 432.10626.
4.2.13. (2R)-3-[(4-Amino-6-{7-chloro-3-oxatricyclo[7.3.1.05,13
]
trideca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazin-2-yl)sulfanyl]
-2-acetamidopropanamide (16e)
Compound 16e was prepared from compound 15 and (2R)-2-
acetamido-3-sulfanylpropanoic acid instead of 3-sulfanylpropa-
noic acid in the same manner as that described for the preparation
of compound 16d (31%, a white solid). 1H NMR (270 MHz, CD3OD)
d: 1.97 (3H, s), 3.27 (1H, dd, J = 14.0, 9.2 Hz), 3.79 (1H, dd, J = 14.0,
4.6 Hz), 4.78 (1H, dd, J = 9.2, 4.6 Hz), 5.06 (4H, s), 7.28 (1H, d,
J = 6.4 Hz), 7.35 (1H, s), 7.42–7.53 (2H, m). MS (ESI+) m/z: 459,
461 [M+H]+. HRMS (ESI+) m/z: [M+H]+ calcd for C20H2035ClN6O3S,
459.10006; found, 459.09941, [M+H]+ calcd for C20H2037ClN6O3S,
461.09711; found, 461.09648.
4.2.18. 3-[(4-Amino-6-{7-chloro-3-oxatricyclo[7.3.1.05,13
]
trideca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazin-2-yl)sulfanyl]
-N-(2-hydroxyethyl)propanamide (16j)
Compound 16j was prepared from compound 15 and 2-amino-
ethanol instead of ammonium chloride in the same manner as that
described for the preparation of compound 16d (90%, a white
solid).
1H NMR (270 MHz, CDCl3) d: 2.66 (2H, t, J = 7.2 Hz), 3.26–3.32
(2H, m), 3.35 (2H, t, J = 7.2 Hz), 3.61 (2H, t, J = 4.6 Hz), 5.06 (2H, s),
5.07 (2H, s), 5.82 (1H, br s), 6.03 (1H, br s), 6.31 (1H, br t), 7.21 (1H,

902
A. Suda et al. / Bioorg. Med. Chem. 22 (2014) 892–905
dd, J = 4.6, 3.6 Hz), 7.25 (1H, s), 7.46 (1H, d, J = 4.6 Hz), 7.46 (1H, d,
J = 3.6 Hz). MS (ESI+) m/z: 446, 448 [M+H]+. HRMS (ESI+) m/z:
[M+H]+ calcd for C20H2135ClN5O3S; 446.10481; found, 446.10466,
[M+H]+ calcd for C20H2137ClN5O3S; 448.10186; found, 448.10172.
zin-2-amine (15, 4.55 g, 11.1 mmol) in N,N-dimethylformamide
(100 mL) was added potassium thioacetate (2.54 g, 22.3 mmol) at
room temperature. The resulting mixture was stirred at room tem-
perature for 19 h and then the reaction mixture was cooled in an
ice-water bath. To the cold solution was added 2 N aqueous so-
dium hydroxide solution (100 mL) and the resulting mixture was
stirred at room temperature for 1.5 h. The reaction mixture was di-
luted with water (100 mL) and the pH of the solution was adjusted
to approximately 5 to 6 with 1 N aqueous hydrochloric acid
(185 mL). The aqueous mixture was diluted with water (400 mL)
and the resulting mixture was stirred at room temperature for
1 h. The precipitates were collected by filtration, washed with
water (50 mL, three times), and dried under reduced pressure.
The title compound was obtained as a brown solid (3.75 g, 92%).
1H NMR (300 MHz, DMSO-d6) d: 5.03 (2H, s), 5.06 (2H, s), 7.39
(1H, d, J = 5.8 Hz), 7.50 (1H, s), 7.55–7.64 (2H, m), 8.11 (1H, br s),
8.23 (1H, br s), 13.43 (1H, br s). MS (ESI+) m/z: 331, 333 [M+H]+.
HRMS (ESI+) m/z: [M+H]+ calcd for C15H1235ClN4OS, 331.04149;
found, 331.04138, [M+H]+ calcd for C15H1237ClN4OS, 333.03854;
found, 333.03854.
4.2.19. 2-[(4-Amino-6-{7-chloro-3-oxatricyclo[7.3.1.05,13
]
trideca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazin-2-yl)sulfanyl]
acetamide (16k)
Compound 16k was prepared from compound 15 and 2-sulfa-
nylacetic acid instead of 3-sulfanylpropanoic acid in the same
manner as that described for the preparation of compound 16d
(87%, a white solid). 1H NMR (300 MHz, DMSO-d6) d: 3.78 (2H, s),
5.04 (2H, s), 5.05 (2H, s), 7.12 (1H, br s), 7.34 (1H, d, J = 7.7 Hz),
7.41 (1H, d, J = 8.5 Hz), 7.44 (1H, s), 7.44 (1H, br s), 7.52 (1H, dd,
J = 8.5, 7.7 Hz), 7.81 (1H, br s), 7.83 (1H, br s). MS (ESI+) m/z: 388,
390 [M+H]+. HRMS (ESI+) m/z: [M+H]+ calcd for C17H1535ClN5O2S,
388.06295; found, 388.06216, [M+H]+. HRMS (ESI+) m/z: [M+H]+
calcd for C17H1537ClN5O2S, 390.06000; found, 390.05913.
4.2.20. 4-[(4-Amino-6-{7-chloro-3-oxatricyclo[7.3.1.05,13
]
trideca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazin-2-yl)sulfanyl]
butanamide (16l)
4.2.22. N-{2-[(4-Amino-6-{7-chloro-3-oxatricyclo[7.3.1.05,13]tri
deca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazin-2-yl)sulfanyl]
ethyl}methanesulfonamide (18a)
4, 4-dithiobutyric acid (2.75 g, 7.62 mmol) was dissolved in N,N-
dimethylformamide (25 mL) and water (2.7 mL) under nitrogen
atmosphere. Tri-n-butylphosphine (2.7 mL, 10.8 mmol) was added
to the solution and the resulting mixture was stirred for 1.5 h at
room temperature. To the obtained mixture were added N-ethyldi-
isopropylamine (4.0 mL, 23.2 mmol) and 4-(5-chloro-1H,
3H-benzo[de]isochromen-6-yl)-6-methanesulfinyl-1,3,5-triazin-2-
ylamine (2.77 g, 10.6 mmol) in N,N-dimethylformamide (14 mL)
successively, and stirring was continued for 2 h. The mixture was di-
luted with water (100 mL) and 1 N HCl (20 mL), and extracted with
ethyl acetate (100 mL, three times). The 0rganic layers were com-
bined and washed with water (100 mL) and brine (100 mL) succes-
sively, and then dried over anhydrous sodium sulfate. After
removal of inorganic salts by filtration, the filtrate was concentrated
in vacuo giving crude 4-[4-amino-6-(5-chloro-1H,3H-benzo[de]iso-
chromen-6-yl)-1,3,5-triazin-2-ylsulfanyl]-butyric acid as a crude
yellow oil (9.77 g). MS (ESI+) m/z: 417, 419[M+H]+.
To
the
solution
of
4-amino-6-{7-chloro-3-oxatricy-
clo[7.3.1.05,13]trideca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazine-
2-thiol (17, 399 mg, 1.21 mmol) and N-(2-chloroethyl)methanesul-
fonamide (381 mg, 2.41 mmol) in N,N-dimethylformamide (20 mL)
was added N-ethyldiisopropylamine (631 lL, 3.14 mmol) at room
temperature. The resulting mixture was stirred for 2.5 h at 70 °C.
After cooling to room temperature, the mixture was extracted be-
tween ethylacetate and water. The organic layer was washed with
water and brine successively, dried over anhydrous magnesium
sulfate, and filtered. The filtrate was concentrated by evaporation
and the resulting residue was purified by preparative HPLC to give
the title compound (383 mg, 86%) as a white solid. 1H NMR
(270 MHz, DMSO-d6) d: 2.84 (3H, s), 3.16 (2H, t, J = 6.6 Hz), 3.24–
3.28 (2H, m), 5.04 (2H, br s), 5.05 (2H, br s), 7.21 (1H, br s), 7.34
(1H, d, J = 6.8 Hz), 7.42 (1H, d, J = 8.3 Hz), 7.45 (1H, s), 7.52 (1H,
dd, J = 8.3, 6.8 Hz), 7.78 (1H, br s), 7.83 (1H, br s). MS (ESI+) m/z:
452, 454 [M+H]+. HRMS (ESI+) m/z: [M+H]+ calcd for C18H1935ClN5-
O3S2, 452.06124; found, 452.06032, [M+H]+ calcd for C18H1937ClN5-
O3S2, 454.05828; found, 454.05725.
The obtained crude product was dissolved in N,N-dimethyl-
formamide (50 mL). To the solution were added ammonium
chloride
(1.24 g,
23.1 mmol),
1-ethyl-3-(3-dimethylamino
propyl)carbodiimide (4.43 g, 23.1 mmol), 1-hydroxybenzotriazole
monohydrate (3.54 g, 23.1 mmol), and N-ethyldiisopropylamine
(6.6 mL, 38.5 mmol) successively at room temperature. The mix-
ture was stirred overnight at the same temperature. The reaction
mixture was poured into water (450 mL) and extracted with ethyl
acetate (300 mL, three times). The organic layers were combined
and washed with water (100 mL) and brine (100 mL) successively,
and then dried over anhydrous sodium sulfate. After removal of
inorganic salts by filtration, the filtrate was concentrated in vacuo
and the resulting residue was purified by silica gel column chroma-
tography (dichromethane/methanol = 30:1 to 15:1) to yield the
title compound as a white solid (1.52 g, 81% in 2 steps). 1H NMR
(400 MHz, DMSO-d6) d: 1.81–1.89 (2H, m), 2.16 (2H, t, J = 7.6 Hz),
3.05 (2H, t, J = 7.0 Hz), 5.05 (2H, s), 5.06 (2H, s), 6.75 (1H, br s),
7.28 (1H, br s), 7.34 (1H, d, J = 6.7 Hz), 7.41 (1H, d, J = 8.5 Hz),
7.45 (1H, s), 7.52 (1H, dd, J = 8.5, 6.7 Hz), 7.79 (1H, s), 7.80 (1H,
s). MS (ESI+) m/z: 416, 418 [M+H]+. HRMS (ESI+) m/z: [M+H]+ calcd
for C19H19O2N535ClS, 416.0942; found, 416.0942, [M+H]+ calcd for
4.2.23. {2-[(4-Amino-6-{7-chloro-3-oxatricyclo[7.3.1.05,13]tri
deca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazin-2-yl)sulfanyl]
ethyl}urea (18b)
Compound 18b was prepared from compound 17 and 2-chloro-
ethylurea instead of N-(2-chloroethyl)methanesulfonamide in the
same manner as that described for the preparation of compound
18a (55%, a white solid). 1H NMR (400 MHz, DMSO-d6) d: 3.10
(2H, t, J = 6.4 Hz), 3.25 (2H, m), 5.04 (2H, br s), 5.05 (2H, br s),
5.48 (2H, br s), 6.18 (1H, t, J = 5.5 Hz), 7.34 (1H, d, J = 6.9 Hz),
7.42 (1H, d, J = 8.2 Hz), 7.45 (1H, s), 7.52 (1H, dd, J = 8.2, 6.9 Hz),
7.82 (2H, br s). MS (ESI+) m/z: 417, 419 [M+H]+. HRMS (ESI+) m/z:
[M+H]+ calcd for C18H1835ClN6O2S, 417.08950; found, 417.08949,
[M+H]+ calcd for C18H1837ClN6O2S, 419.08655; found, 419.08637.
4.2.24. 2-[(4-Amino-6-{7-chloro-3-
oxatricyclo[7.3.1.05,13]trideca-1(13),5,7,9,11-pentaen-8-yl}-
1,3,5-triazin-2-yl)sulfanyl]ethyl carbamate (18c)
C
19H19O2N537ClS, 418.0913; found, 418.0913.
To a solution of 2-bromoethan-1-ol (10.0 mL, 141.1 mmol) in
N,N-dimethylformamide (140 mL) was added 4-nitrophenyl chloro-
formate (32.0 g, 158.8 mmol) and N-ethyldiisopropylamine
(34.4 mL, 197.5 mmol) at 0 °C successively. The resulting mixture
was stirred at 0 °C for 1.5 h. To the mixture was added 28%
4.2.21. 4-Amino-6-{7-chloro-3-oxatricyclo[7.3.1.05,13]trideca-
1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazine-2-thiol (17)
To the suspension of 4-{7-chloro-3-oxatricyclo[7.3.1.05,13
]
trideca-1(13),5,7,9,11-pentaen-8-yl}-6-methanesulfinyl-1,3,5-tria-

A. Suda et al. / Bioorg. Med. Chem. 22 (2014) 892–905
903
ammonia solution in water (17.5 mL) at 0 °C and the resulting mix-
ture was stirred for 20 min. Water (300 mL) was added to the reac-
tion mixture and the pH was adjusted to approximately 4 by
addition of 1 N hydrochloric acid. The aqueous solution was ex-
tracted with ethyl acetate (200 mL, three times). The organic layers
were combined, washed with 0.1 N hydrochloric acid (100 mL),
water (100 mL), 0.5 N aqueous sodium hydroxide solution
(100 mL, four times), water (100 mL, three times), and brine
(100 mL) successively, dried over anhydrous sodium sulfate, and fil-
tered. The filtrate was concentrated by evaporation and dried under
reduced pressure to yield 2-bromoethyl carbamate along with
4-nitrophenol (5.42 g). The crude product was dissolved in N,N-
dimethylformamide (130 mL), and to the solution was added 4-ami-
no-6-{7-chloro-3-oxatricyclo[7.3.1.05,13]trideca-1(13),5,7,9,11-
pentaen-8-yl}-1,3,5-triazine-2-thiol (17, 2.90 g, 8.77 mmol) and N-
ethyldiisopropylamine (14 mL, 80.4 mmol) at room temperature.
The resulting mixture was stirred at 70 °C for 2 h. Water (300 mL)
was added to the reaction mixture and the pH was adjusted to
approximately 4 by addition of 1 N hydrochloric acid. The aqueous
solution was extracted with ethyl acetate (200 mL, three times).
The organic layers were combined, washed with 0.2 N hydrochloric
acid (100 mL), water (100 mL, twice), and brine (100 mL) succes-
sively, dried over anhydrous sodium sulfate, and filtered. The filtrate
was concentrated by evaporationand theresultingresidue waspuri-
fied by preparative HPLC (0.05% trifluoroacetic acid in acetonitrile/
0.05% trifluoroacetic acid in water = 35:65) to yield the title com-
pound as a white solid (1.33 g, 36%). 1H NMR (270 MHz, DMSO-d6)
d: 3.27 (2H, t, J = 6.4 Hz), 4.13 (2H, t, J = 6.4 Hz), 5.05 (4H, br s), 6.55
(2H, br s), 7.33–7.55 (4H, m), 7.86 (2H, br s). MS (ESI+) m/z: 418,
420 [M+H]+. HRMS (ESI+) m/z: [M+H]+ calcd for C18H1735ClN5O3S,
418.07351; found, 418.07325, [M+H]+ calcd for C18H1737ClN5O3S,
420.07056; found, 420.07023.
methyl)oxirane in the same manner as that described for the prep-
aration of compound 18d (79% in ethyl acetate, 15%, colorless oil).
1H NMR (270 MHz, CDCl3) d: 3.52–3.58 (1H, m), 3.63–3.80 (3H, m),
4.81–4.91 (1H, m), 5.56 (1H, br s). MS (ESI+) m/z: 136, 138 [M+H]+.
Compound 18e was prepared from compound 17 and (5R)-5-
(chloromethyl)-1,3-oxazolidin-2-one instead of N-(2-chloro-
ethyl)methanesulfonamide in the same manner as that described
for the preparation of compound 18a (60%, a light brown solid).
1H NMR (400 MHz, DMSO-d6) d: 3.26 (1H, dd, J = 8.8, 6.6 Hz),
3.38 (1H, dd, J = 14.0, 6.0 Hz), 3.47 (1H, dd, J = 14.0, 6.0 Hz), 3.57
(1H, dd, J = 8.8, 8.8 Hz), 4.79–4. 87 (1H, m), 5.05 (2H, s), 5.06 (2H,
s), 7.35 (1H, d, J = 7.1 Hz), 7.43 (1H, d, J = 8.8 Hz), 7.45 (1H, s),
7.53 (1H, dd, J = 8.8, 7.1 Hz), 7.57 (1H, s), 7.91 (2H, s). MS (ESI+)
m/z: 430, 432 [M+H]+. HRMS (ESI+) m/z: [M+H]+ calcd for C19H17
35
ClN5O3S, 430.07351; found, 430.07340, [M+H]+ calcd for C19H17
37
ClN5O3S, 432.07056; found, 432.07043.
4.2.27. 3-[(4-Amino-6-{7-chloro-3-oxatricyclo[7.3.1.05,13]tri
deca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazin-2-yl)sulfanyl]-
2,2-dimethylpropanamide (18f)
To
the
solution
of
4-amino-6-{7-chloro-3-oxatricy-
clo[7.3.1.05,13]trideca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazine-
2-thiol (17, 50 mg, 0.151 mmol) and 3-bromo-2,2-dim-
ethylpropioic acid (55 mg, 0.302 mmol) in N,N-dimethylformam-
ide (1.0 mL) was added N-ethyldiisopropylamine (79 lL,
0.453 mmol) at room temperature. The resulting mixture was stir-
red at 80 °C for 1 h. To the reaction mixture were added 0.5 N
aqueous potassium hydrogen sulfate solution (2.5 mL) and water
(3.0 mL) successively and the resulting aqueous solution was stir-
red at room temperature for 1 h. The precipitates were collected by
filtration, washed with water (3.0 mL, three times), and dried
under reduced pressure. 3-[(4-Amino-6-{7-chloro-3-oxatricy-
clo[7.3.1.05,13]trideca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazin-2-
yl)sulfanyl]-2,2-dimethylpropanoic acid was obtained along with
2,2-dimethyl-3-sulfanylpropanoic acid as a brown solid (54 mg).
1H NMR (400 MHz, DMSO-d6) d: 1.18 (6H, s), 3.36 (2H, s), 5.05
(2H, s), 5.06 (2H, s), 7.32 (1H, d, J = 7.1 Hz), 7.37 (1H, d,
J = 8.8 Hz), 7.45 (1H, s), 7.52 (1H, dd, J = 8.8, 7.1 Hz), 7.83 (1H, br
s), 7.85 (1H, br s), 12.50 (1H, br s). MS (ESI+) m/z: 431, 433 [M+H]+.
4.2.25. (5S)-5-{[(4-Amino-6-{7-chloro-3-
oxatricyclo[7.3.1.05,13]tri deca-1(13),5,7,9,11-pentaen-8-yl-
1,3,5-triazin-2-yl)sulfanyl]methyl}-1,3-oxazolidin-2-one (18d)
To
a
solution of (2S)-2-(chloromethyl)oxirane (0.813 g,
8.79 mmol) in water (70 mL) was added potassium cyanate
(1.43 g, 17.63 mmol) at room temperature and the resulting mix-
ture was refluxed with stirring for 19 h. The reaction mixture
was allowed to cool to room temperature and extracted with ethyl
acetate (50 mL, three times). The organic layers were combined,
dried over anhydrous sodium sulfate, and filtered. The filtrate
was concentrated by evaporation to yield (5S)-5-(chloromethyl)-
1,3-oxazolidin-2-one along with ethyl acetate as colorless oil
(87% in ethyl acetate, 0.242 g, 18%,). 1H NMR (270 MHz, CDCl3) d:
3.52–3.58 (1H, m), 3.63–3.80 (3H, m), 4.81–4.91 (1H, m), 5.56
(1H, br s). MS (ESI+) m/z: 136, 138 [M+H]+.
Compound 18d was prepared from compound 17 and (5S)-5-
(chloromethyl)-1,3-oxazolidin-2-one instead of N-(2-chloro-
ethyl)methanesulfonamide in the same manner as that described
for the preparation of compound 18a (62%, a light brown solid).1H
NMR (400 MHz, DMSO-d6) d: 3.26 (1H, dd, J = 6.6, 8.8 Hz), 3.38 (1H,
dd, J = 14.0, 6.0 Hz), 3.47 (1H, dd, J = 14.0, 6.0 Hz), 3.57 (1H, dd,
J = 8.8, 8.8 Hz), 4.79–4. 87 (1H, m), 5.05 (2H, s), 5.06 (2H, s), 7.35
(1H, d, J = 7.1 Hz), 7.43 (1H, d, J = 8.8 Hz), 7.45 (1H, s), 7.53 (1H,
dd, J = 8.8, 7.1 Hz), 7.57 (1H, s), 7.91 (2H, s). MS (ESI+) m/z: 430,
432 [M+H]+. HRMS (ESI+) m/z: [M+H]+ calcd for C19H1735ClN5O3S,
430.07351; found, 430.07277, [M+H]+ calcd for C19H1737ClN5O3S,
432.07056; found, 432.06954.
To
a
solution
of
3-[(4-amino-6-{7-chloro-3-oxatricy-
clo[7.3.1.05,13]trideca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazin-2-
yl)sulfanyl]-2,2-dimethylpropanoic acid (25 mg, 0.0581 mmol),
ammonium chloride (16 mg, 0.290 mmol), 1-hydroxybenzotirazole
(49 mg, 0.290 mmol), and 3-(3-dimethylaminopropyl)-1-ethylcar-
bodiimide hydrochloride (56 mg, 0.292 mmol) in N,N-dimethyl-
formamide (1.0 mL) was added N-ethyldiisopropylamine (60 lL,
0.348 mmol) at room temperature. The resulting mixture was stir-
red at room temperature for 1 h. The reaction mixture was diluted
with ethyl acetate (10 mL), washed with water (5.0 mL, twice), sat-
urated sodium hydrogen carbonate aqueous (5 mL, twice), and
brine (5 mL) successively, dried over anhydrous magnesium sul-
fate, and filtered. The filtrate was concentrated and the resulting
residue was purified by silica gel column chromatography (dichlo-
romethane/methanol = 99:1 to 9:1) to yield the title compound as
a white solid (15 mg, 60%). 1H NMR (400 MHz, DMSO-d6) d: 1.16
(6H, s), 3.31 (2H, s), 5.05 (2H, s), 5.06 (2H, s), 6.95 (1H, s), 7.19
(1H, s), 7.35 (1H, d, J = 7.1 Hz), 7.36 (1H, d, J = 8.2 Hz), 7.45 (1H,
s), 7.52 (1H, dd, J = 8.2, 7.1 Hz), 7.79 (1H, s), 7.81 (1H, s). MS
(ESI+) m/z: 430, 432 [M+H]+. HRMS (ESI+) m/z: [M+H]+ calcd for
C20H2135ClN5O2S, 430.10990; found, 430.10928, [M+H]+ calcd for
C
20H2137ClN5O2S, 432.10695; found, 432.10637.
4.2.26. (5R)-5-{[(4-Amino-6-{7-chloro-3-oxatricyclo[7.3.1.05,13
]
trideca-1(13),5,7,9,11-pentaen-8-yl}-1,3,5-triazin-2-yl)sulfanyl]
methyl}-1,3-oxazolidin-2-one (18e)
4.3. Surface plasmon resonance direct binding assay for Hsp90
a
(5R)-5-(Chloromethyl)-1,3-oxazolidin-2-one was prepared
from (2R)-2-(chloromethyl)oxirane instead of (2S)-2-(chloro-
Measurements were performed on a Biacore2000 at a flow rate
of 30 L/min, 25 °C in 50 mM Tris-based saline, pH 7.6, 0.005%
l

904
A. Suda et al. / Bioorg. Med. Chem. 22 (2014) 892–905
Tween20 and 1% DMSO. Biotinylated N-terminal Hsp90
was coupled on a streptavidin-coated surface of a sensorchip (Type
SA, GE healthcare) to a density of ca. 2000 RU.
a
(9–236)
HER2, Raf1 and GAPDH [Santa Cruz Biotechnology], and Hsp70
[Stressgen]) at 4 °C, and then washed with 0.1% Tween20 in TBS
buffer (TBS-T buffer; Nacalai Tesque). The membranes were incu-
bated for 1 h at room temperature with secondary antibodies (Cell
Signaling Technology or Invitrogen), washed with TBS-T buffer,
and then the bands were detected with ECL Plus (GE Healthcare)
followed by LAS-4000 (Fujifilm).
4.4. In vitro cell growth assay
HCT116 cell line (CCL-247, a colorectal cancer cell line) and NCI-
N87 cell line (CRL-5822, a gastric cancer cell line) were purchased
from ATCC. All cell lines were cultured according to the supplier’s
instructions. Cells suspended in a medium were added to solutions
containing various concentrations of the test substance, and the
cells were cultured at 37 °C in 5% CO2. Four days later, Cell Count-
ing Kit-8 solution (Dojindo Laboratories) was added and absor-
bance at 450 nm was measured with Microplate-Reader iMark
(Bio-Rad Laboratories). Antiproliferative activity was calculated
by the formula (1ÀT/C) Â 100 (%), where T represents the absor-
bance of drug-treated cells and C that of untreated control cells
at 450 nm. The 50% inhibition concentration (IC50) values were cal-
culated using Microsoft Excel 2007.
4.8. Xenograft model and in vivo antitumor study
All animal studies were approved by the Chugai Institutional
Animal Care and Use Committee. Cancer cells (0.5–1 Â 107) were
implanted subcutaneously into the right flank of athymic nude
(BALB/c nu/nu) mice (CAnN.Cg-Foxn1<nu>/CrlCrlj nu/nu, Charles
River Laboratories). Tumor volume was calculated using the for-
mula TV = ab2/2, where a and b represent tumor length and width,
respectively. Once the TV had reached approximately 200–
300 mm3, animals were randomized into each group (n = 4 or 5),
and treatment was initiated. CH5164840 (16) was dissolved in a
vehicle of 10% DMSO/10% Cremophor EL/0.02 N HCl in water and
then orally administered to mice. TGI was calculated using the for-
mula TGI = [1À(TtÀT0)/(CtÀC0)] Â 100 (%), where T (tumor volume
of treated group, T0 on day 0 or Tt on day t) and C (tumor volume
of control group, C0 on day 0 or Ct on day t) represent mean tumor
volume.
4.5. Lyophilized solubility assay
Samples were prepared in triplicate from 10 mM DMSO stock
solutions. After evaporation (1 h) of DMSO with a centrifugal vac-
uum evaporator, the compounds were dissolved in FaSSIF, and sha-
ken for 2 h. The solutions were filtered using a microtiter filter
plate (Whatman UNIFILTER) and the filtrate was analyzed by UV
measurement or HPLC-UV. In addition, a four point calibration
curve was prepared from the 10 mM stock solutions and used to
determine the solubility of the compounds. The results are ex-
4.9. Pharmacokinetics
Pharmacokinetic studies of test compounds were performed in
female athymic nu/nu mice following an oral (po) or intravenous
(iv) route. The test compounds were dissolved in vehicles
(CH5015765 (7): 10% DMSO/10% Cremophor EL/4% glucose in
water, CH5164840 (16): 10% DMSO/10% Cremophor EL/0.025 N
HCl in water). Blood samples were collected from the orbital cavity
using a heparinized capillary or by heart puncture using a dispos-
able syringe with a 25G needle under isoflurane anesthesia at the
scheduled time points of 2, 15 min, 1, 2, 4, 7 and 24 h following
iv dosing and at those of 30 min, 1, 2, 4, 7 and 24 h following po
dosing. Blood samples were immediately centrifuged by a hemat-
ocrit centrifuge (Model 3220, Kubota) at 12,000 rpm for 3 min or
by a centrifuge at 3000 rpm for 10 min at 4 °C (LX-130, Tomy Sei-
ko) to separate the plasma. Plasma samples were immediately fro-
zen on dry ice and stored at À80 °C until analysis. The plasma
pressed in lM.
4.6. Liver microsomal stability assay
Mouse or human microsome incubations were conducted by an
automated procedure implemented on a Biomek FX (Beckman
Coulter). Compounds (5 lM) were incubated in liver microsomes
(1.0 mg protein/mL) in a 50 mM potassium phosphate buffer (pH
7.4) at 37 °C. NADPH as a cofactor was produced by a generating
system (glucose 6-phosphate, 3.2 mM; b-NADP, 2.6 mM; MgCl2,
6.5 mM). NADPH from the generating system was added to the
pre-warmed liver microsomes containing the test compound. Ali-
quots (50
60 min and transferred into 100
l
L) were taken at four defined time points within
L of methanol containing an
l
sample (5 lL) was added with 35 lL of CH3CN/MeOH/water
internal standard. Concentration of each compound was analyzed
by liquid chromatography-tandem mass spectrometry (LC-MS/
MS) using an ODS-3 RP 18 column (GL Sciences). Quantitative
detection was achieved on an API 365 (AB SCIEX) using ESI. Con-
centrations were determined by ratio of test compound and inter-
nal standard peaks and given as a percentage of the concentration
measured at the first time point (substrate depletion). Intrinsic
(15:15:5 v/v) including an internal standard, and then thoroughly
mixed to precipitate plasma protein. The suspensions were trans-
ferred to 96-well MultiScreen filtration plates (0.45 lm) and fil-
tered by centrifugation at 1000g for 5 min at 4 °C (LX-130, Tomy
Seiko). The filtrate was applied to an LC-MS/MS system (API3000,
AB SCIEX) to measure concentrations of the compounds. The lower
limit of quantification (LLOQ) was 1 ng/mL. The pharmacokinetic
parameters were calculated by non-compartmental analysis using
Watson ver. 6.3 (Thermo Fisher Scientific).
clearance (CL, in
lL/min/mg microsomal protein) is the rate con-
stant of the first-order decay of the test compound, normalized
for the protein concentration in the incubation.
Acknowledgement
4.7. Western blotting
We thank Osamu Kondo and Toshiyuki Mio for their helpful dis-
cussions; Kiyoaki Sakata, Toshihiko Fujii, Fumie Sawamura, and
Masami Hasegawa for biological assays; Masami Kohchi, Jun-ichi
Shiina, Young-Jin Kwon, Toshihiko Yokoyama, Kihito Hada, and
Satoshi Niizuma for chemical synthesis; Miho Nagayasu for PK
analysis, Keiji Nii and Kiyoshi Yoshinari for physicochemical
assays; and Hitomi Suda and Yuichiro Ishiguro for mass spectrom-
etry measurements. We also thank Hiroshi Koyano and Editing Ser-
vices (Chugai Pharmaceutical, Co., Ltd) for their help with
preparation of the manuscript.
NCI-N87 cells were treated with various concentrations (0, 0.04,
0.2, 1, 5 lM) of the test substance for 24 h and lysed with cell lysis
buffer (Cell Signaling Technology) supplemented with protease
inhibitor cocktail (Roche Diagnostics) and phosphatase inhibitors
(Sigma–Aldrich). Equivalent amounts of cell lysate were resolved
by SDS–PAGE and transferred to PVDF membranes (Millipore).
Membranes were blocked with Blocking One (Nacalai Tesque)
incubated overnight with primary antibodies (pS473-Akt, Akt,
pT202/Y204-ERK, ERK, and Cleaved-PARP [Cell Signaling], EGFR,

A. Suda et al. / Bioorg. Med. Chem. 22 (2014) 892–905
15. Schulte, T. W.; Neckers, L. M. Cancer Chemother. Pharmacol. 1998, 42, 273.
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