Discovery of 3-Ethyl-4-(3-isopropyl-4-(4-(1-methyl-1 H-pyrazol-4-yl)-1 H-imidazol-1-yl)-1 H-pyrazolo[3,4- b]pyridin-1-yl)benzamide (TAS-116) as a Potent, Selective, and Orally Available HSP90 Inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.8b01085

The molecular chaperone heat shock protein 90 (HSP90) is a promising target for cancer therapy, as it assists in the stabilization of cancer-related proteins, promoting cancer cell growth, and survival. A novel series of HSP90 inhibitors were discovered by structure-activity relationship (SAR)-based optimization of an initial hit compound 11a having a 4-(4-(quinolin-3-yl)-1H-indol-1-yl)benzamide structure. The pyrazolo[3,4-b]pyridine derivative, 16e (TAS-116), is a selective inhibitor of HSP90α and HSP90β among the HSP90 family proteins and exhibits oral availability in mice. The X-ray cocrystal structure of the 16e analogue 16d demonstrated a unique binding mode at the N-terminal ATP binding site. Oral administration of 16e demonstrated potent antitumor effects in an NCI-H1975 xenograft mouse model without significant body weight loss.

Full text of DOI:10.1021/acs.jmedchem.8b01085

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Article
Discovery of 3-Ethyl-4-(3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-
yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl)benzamide
(TAS-116) as a Potent, Selective, and Orally Available HSP90 Inhibitor
Takao Uno, Yuichi Kawai, Satoshi Yamashita, Hiromi Oshiumi, Chihoko Yoshimura, Takashi
Mizutani, Tatsuya Suzuki, Khoon Tee Chong, Kazuhiko Shigeno, Mitsuru Ohkubo, Yasuo Kodama,
Hiromi Muraoka, Kaoru Funabashi, Koichi Takahashi, Shuichi Ohkubo, and Makoto Kitade
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01085 • Publication Date (Web): 07 Dec 2018
Downloaded from http://pubs.acs.org on December 7, 2018
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Discovery of 3-Ethyl-4-(3-isopropyl-4-(4-(1-methyl-
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1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-
pyrazolo[3,4-b]pyridin-1-yl)benzamide (TAS-116)
as a Potent, Selective, and Orally Available HSP90
Inhibitor
1
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Takao Uno, * Yuichi Kawai, Satoshi Yamashita, Hiromi Oshiumi, Chihoko Yoshimura,
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Takashi Mizutani, Tatsuya Suzuki, Khoon Tee Chong, Kazuhiko Shigeno, Mitsuru Ohkubo,
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Yasuo Kodama, Hiromi Muraoka, Kaoru Funabashi, Koichi Takahashi, Shuichi Ohkubo,
and Makoto Kitade3
1Discovery and Preclinical Research Division, Taiho Pharmaceutical Co. Ltd., Tsukuba, Ibaraki
00-2611, Japan
3
2Formulation Research, CMC Division, Taiho Pharmaceutical Co. Ltd., Kawauchi-cho,
Tokushima, Tokushima 771-0194, Japan
3Chemical Technology Laboratory, CMC Division, Taiho Pharmaceutical Co. Ltd., Kamikawa-
machi, Kodama-gun, Saitama 367-0241, Japan
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Abstract
The molecular chaperone heat shock protein 90 (HSP90) is a promising target for cancer
therapy, as it assists in the stabilization of cancer-related proteins, promoting cancer cell growth
and survival. A novel series of HSP90 inhibitors were discovered by structure-activity
relationship (SAR)-based optimization of an initial hit compound 11a having a 4-(4-(quinolin-3-
yl)-1H-indol-1-yl)benzamide structure. The pyrazolo[3,4-b]pyridine derivative, 16e (TAS-116),
is a selective inhibitor of HSP90α and HSP90β among the HSP90 family proteins and exhibits
oral availability in mice. X-ray co-crystal structure of the 16e analog 16d demonstrated a unique
binding mode at the N-terminal ATP binding site. Oral administration of 16e demonstrated
potent antitumor effects in an NCI-H1975 xenograft mouse model without significant body
weight loss.
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Introduction
Heat shock protein 90 (HSP90) is an adenosine triphosphate (ATP)-dependent molecular
chaperone.1,2 ATP binds to the N-terminal of HSP90, and its hydrolysis facilitates folding of
substrate proteins, referred to as HSP90 clients. Inhibition of HSP90 leads to the destabilization
of HSP90 clients. HSP90 regulates more than two hundred HSP90 clients, including protein
kinases, steroid hormone receptors, and transcription factors.2-4 Many of the HSP90 clients are
oncoproteins that are involved in tumor development and survival. It is also known that HSP90 is
over-expressed in cancer cells and tumor tissues, where it exists in a highly activated state.5-8
Therefore, HSP90 is considered to play a crucial role in cancer cell growth and survival. Based
on these findings, ATP-competitive inhibitors acting at the N-terminal of HSP90 have been
developed as therapeutic agents for cancer. Although some HSP90 inhibitors have shown clinical
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efficacies, potential off-target and/or HSP90-related toxicities have proved problematic.3,4,9,10 For
1
1
12
13
instance, the first generation 1 (geldanamycin) analogs, such as 2 (17-AAG), 3 (17-DMAG),
and their derivative 414,15 (IPI-504), have shown excess hepatotoxicity probably because of their
quinone structure.11,16,17 Although, small second-generation analogs with different structures
have shown low hepatotoxicity, some analogs such as purine-based derivative 5 (BIIB021)18,19
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exhibited neurological toxicities in a phase I trial. In addition, visual disturbances have been
observed as an HSP90-related adverse event, although the frequency and degree varied among
2
1
22
the compounds. For instance, in a phase I trial of 7 (NVP-AUY922) derived from the natural
product 623 (radicicol), 43% of patients experienced visual disturbances.24 Further, clinical
2
5
development of 8 (PF-04929113) was discontinued because of ocular toxicity observed in a
26
phase I study. These results suggest that HSP90 inhibitors with different structures may exhibit
acceptable toxic profile with minimal off-target and HSP90-related toxicities. Alternatively, the
3
use of C- terminal inhibitor is another strategy to inhibit the HSP90 activity. The C-terminal
2
contains a nucleotide binding motif and mediates dimerization of HSP90. It has been reported
that N-terminal HSP90 inhibitors induce a heat shock response (HSR) that is potentially involved
in drug resistance upon HSP90 inhibition.27 During recent years, several C-terminal HSP90
inhibitors have been discovered. These inhibitors downregulate HSP90 clients and exhibit anti-
proliferative activity in several cancer cell lines without HSR.28,29 The clinical evaluation of
efficacy and toxicities of this class of compounds is the next challenge.
In this milieu, we aimed to develop a novel N-terminal HSP90 inhibitor that is different from
the known N-terminal inhibitors, such as 1, 5, 6, and their derivatives, and with superior
druggability with respect to target selectivity and pharmacologic and pharmacokinetic profiles.
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O
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R
O
HCl
O
O
OH
H
N
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H
OH
OH
OH
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O
O
O
NH2
O
1
Geldanamycin
NH2
Hydroquinone hydrochloride form of 17-AAG
R=OMe
4
(
2 17-AAG (Tanespimycin)
Retaspimycin hydrochloride)/IPI-504
2
R=NHCH CH=CH2
3
17-DMAG (Alvespimycin)
R=NHCH CH NMe
2
2
2
N
Cl
N
OH
O
HN
O
OH
Cl
O
N
N
N
O
O
HO
H N
2
N
HO
H
H
O
N
OMe
O
5
BIIB021
6 Radicicol
7 Luminespib/NVP-AUY922
H
N
N
O
H N
2
H
N
N
Br
OR
HN
O
N
N
F3C
O
N
8
R=H
9
PF-04928473 (SNX-2112)
PF-04929113 (SNX-5422)
R=Glycine ester mesylate
10
Figure 1. Structure of selected small molecular HSP90 inhibitors 1−10.
Results and Discussion
Drug design strategies. Several X-ray co-crystal structures of small molecular HSP90
inhibitors in complex with N-terminal ATP binding site of human HSP90 have been
reported.30,31 In this study, we designed a novel scaffold as HSP90 inhibitor using these X-ray
co-crystal structures. Firstly, we investigated the X-ray co-crystal structure of the first and
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second generation HSP90 inhibitors to analyze the binding mode to the HSP90 N-terminal ATP
binding site.30,31 These binding modes have been commonly observed, forming a water-mediated
hydrogen bond network with Asp93 and Thr184. Furthermore, the second generation HSP90
inhibitor 8 analogs25,32,33 form hydrogen bonds with Tyr 139 and Lys 58 in addition to the above
two common hydrogen bonds. Secondly, we focused on the structures of 825,32 and 10, which
are different from natural products, such as 1 and 6 analogs, and purine analogs such as 5.
However, the X-ray co-crystal structures of 8 and 10, neither with each other nor with HSP90
individually, have been reported. Therefore, we speculated the binding mode of 8 and 10 based
on the previously reported X-ray co-crystal structure of 8 analogs (PDB entry: 3DOB,
3MNR)25,32,33 and made a superposition of 8 and 10 (Figure 2A). Figure 2A shows the simplified
scheme including four key hydrogen bonds of 8 and 10 with the HSP90 N-terminal ATP binding
site. The speculated binding mode common to 8 and 10 are as follows. (i) The carboxamide
group of 8 and pyrrolopyridine group of 10 act as adenine mimics with the amide group forming
hydrogen bonds with Asp93 and Thr184. (ii) The hydroxyl group of trans-4-
hydroxycyclohexylamino moiety forms hydrogen bonds with Lys58. (iii) The carbonyl group of
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8
and nitrogen atom of 3-quinoline group of 10 form a hydrogen bond with Tyr139. Through the
superposition of the speculated binding mode of compounds 8 and 10, we designed a hybrid
compound 11a by combining these key hydrogen bonds (Figure 2B). The hybrid compound 11a
exhibited moderate HSP90 binding inhibitory activity (IC = 9.5 M). The result encouraged us
50
to develop a novel HSP90 inhibitor. Subsequently, this compound was also reported by Mailliet
3
5
et al. Therefore, we devised two drug design strategies aiming to further enhance the HSP90
binding inhibitory activity, as shown in Figure 2C. Strategy 1 involved the introduction of a
substituent to fill the small hydrophobic pocket and the introduction of nitrogen atom into the
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indole core. This strategy was supported by a previous study by Huang et al., which reported
that the filling of small hydrophobic pocket contributed significantly to the HSP90 binding
inhibitory activity. Strategy 2 involved exploration of an alternative structure of 3-quinoline
group based on the SAR study.
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Figure 2. (A) Superposition of the speculated binding mode of 8 and 10, a hybridization strategy
used to design novel HSP90 inhibitors. (B) The structure and HSP90 binding inhibitory activity
of 4-(1H-indol-1-yl)benzamide hit 11a. (C) Drug design strategies to enhance the HSP90 binding
inhibitory activity.
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Strategy 1: Structure-activity relationship exploration of the indole core in the initial hit
1
1a. As mentioned above, the introduction of an alkyl chain at the C-3 position of the indole ring
in 11a with the intent to fill the small hydrophobic pocket formed by the residues Ala111,
Val138, and Tyr139 is likely to enhance the HSP90 binding inhibitory activity. To confirm this
hypothesis, we designed compound 11b, in which the C-3 position of the indole ring in 11a was
substituted with a methyl group. In addition, we also designed compound 12a, in which a
nitrogen atom was introduced into the indole ring of 11a. Table 1 shows the HSP90 inhibitory
activity of 11a and 12a, determined based on the binding of HSP90 by the alphascreen
competitive assay. The HSP90 binding inhibitory activity of 11b was significantly enhanced by
the introduction of a methyl group at the C-3 position of the indole ring. In addition, the indazole
derivative 12a, which is an N-inserted derivative of 11a, also exhibited good HSP90 binding
inhibitory activity. Based on these results, we aimed to further enhance the HSP90 binding
inhibitory activity and investigated the effect of introduction of various alkyl chains at the C-3
position in the indazole derivative 12a. Most alkyl-substituted indazole derivatives showed good
HSP90 binding inhibitory activity with IC values less than 100 nM. Among them, 12c with an
1
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3
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isopropyl group at the C-3 position in 12a presented the highest affinity toward HSP90. Based on
its potent HSP90 binding inhibitory activity, we selected the 3-isopropyl derivative 12c as the hit
compound for further optimization.
Table 1. Biological data of compounds with 3-alkyl and nitrogen substitution at the indole
core
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N
H
O
H N
2
HSP90
50
Compd
X
R1
a
IC (nM)
1
1a
1b
12a
2b
2c
CH
CH
N
H
Me
H
9524±4870
585±85
365±64
94±13
1
1
N
Me
i-Pr
1
N
67±16
aThe interaction of compounds with HSP90 was determined by the alphascreen competitive
assay. The IC values (nM) are shown as mean ± SE (n = 3).
5
0
At this point of the study, the 3-isopropyl derivative 12c represented the most advanced analog
and demonstrated potent HSP90 binding inhibitory and tumor cell growth inhibitory activities in
SK-BR-3 cells (HER2-overexpressing human breast cancer cell line); however, it evidently
exhibited hERG inhibitory activity (Table 2). Therefore, with the aim to lower the hERG
inhibitory activity of 12c, we examined the reduction in lipophilicity by introducing a nitrogen
atom into the indazole ring. Table 2 shows the HSP90 binding inhibitory and tumor cell growth
inhibitory activities of 12c in SK-BR-3 cells, as well as its in vitro hERG inhibitory activity.
Compound 13a, in which a nitrogen atom was inserted at the X position of indazole in 12c,
showed highly attenuated HSP90 binding and cellular activities in SK-BR-3 cells compared with
those of 12c. In addition, compound 13b, in which a nitrogen atom was inserted at the Y
position, did not exhibit lowered lipophilicity and hERG inhibition. Furthermore, 13b also
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exhibited significantly attenuated cellular activity in SK-BR-3 cells. On the contrary, compound
1
3c, in which a nitrogen atom was inserted at the Z position, showed equally potent HSP90
binding inhibitory and cellular activities in SK-BR-3 cells when compared with those of 12c. In
addition, the pyrazolo[3,4-b]pyridine derivative 13c showed lower hERG inhibitory activity than
that of indazole 12c. Further, nitrogen substitution at the Z position lowered the LogD value of
the resulting pyrazolo[3,4-b]pyridine analog. Based on these results, we decided to perform SAR
study of the pyrazolo[3,4-b]pyridine 13c.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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6
7
8
9
0
Table 2. Biological data of compounds with nitrogen substitution at the indazole core
N
X
N
OH
Y
N
Z
N
H
O
H N
2
hERG
%Inhibition (at
HSP90
SK-BR-3
IC50 (nM)
Compd
X
Y
Z
LogDc
a
b
IC (nM)
5
0
d
1
0 M)
60±3
35±2
57±4
37±3
1
2c
13a
3b
3c
CH
N
CH
CH
N
CH
CH
CH
N
67±16
470±55
72±10
58±3
39±10
1721±351
473±430
54±16
3.9±0.3
3.3±0.1
4.0±0.2
3.4±0.1
1
CH
CH
1
CH
aThe interaction of compounds with HSP90 was determined by the alphascreen competitive
assay. The IC values (nM) are shown as mean ± SE (n = 3).
5
0
bSK-BR-3 cells were cultured with the compounds for 72 h. Cell survival was then measured and
the IC value was calculated. The IC values (nM) are shown as mean ± SE (n = 3).
5
0
50
c
36
LogD value at pH 7.4. The LogD values (nM) are represented as mean ± SD (n = 3).
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dThe hERG inhibitory activity of compounds at a concentration of 10 M. The values of hERG
percent inhibition are shown as mean ± SE (n = 3).
Strategy 2: Structure-activity relationship exploration of an alternative structure of the 3-
quinoline group. To further enhance the HSP90 binding and cellular activities, we examined the
effect of introduction of other C-4 substituents in place of 3-quinoline group. Table 3 shows the
HSP90 binding inhibitory and growth inhibitory activities in SK-BR-3 cells. Naphthalene 14a
and phenyl 14b, in which the nitrogen atom at the N-3 position of the 3-quniline group was
removed, significantly decreased the HSP90 binding and cellular activities. The results suggested
that the nitrogen atom in the 3-quinoline group is essential for the formation of hydrogen bond
with Tyr139, similar to that of our speculated binding mode (Figure 2A). Therefore, we
examined the effect of introduction of various substituted pyridine groups. 3-Pyridine 14c and its
0
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9
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9
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5
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9
0
6
- and 5-methoxy substituted derivatives 14d and 14e, and other bicyclic 3-pyridine derivatives
4f and 14g, other than the 3-quinoline group, tended to exhibit reduced potencies against
1
HSP90 binding and tumor cell growth. On the contrary, thiophene 14h and furan 14i with sulfur
and oxygen atoms at the C-4 position, respectively, did not exhibit measurable HSP90 binding
and cellular activities. In contrast, imidazole 15a showed marginally higher inhibitory activity
against HSP90 binding than that by 14h and 14i. In addition, compound 15b with a phenyl group
at the C-4 position of imidazole showed significantly enhanced inhibitory activity against HSP90
binding and tumor cell growth. Based on these findings, the 4-phenylimidazole derivative 15b
was selected for further optimization as a candidate with an alternative structure of the 3-
quinoline group.
Table 3. Preliminary structure and biological data of 3-quinoline derivatives
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R1
N
OH
N
N
N
H
O
H N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
HSP90
SK-BR-3
Compd
HSP90
50
SK-BR-3
IC50 (nM)
Compd
R1
R1
a
b
a
b
IC (nM)
IC50 (nM)
IC (nM)
5
0
N
N
N
1
3c
58±3
>10000
>10000
166±6
54±16
14f
14g
14h
14i
789±132
750±123
>10000
>10000
1132±272
N
14a
>10000
>10000
464±69
423±105
>10000
S
1
4b
N
O
1
4c
>10000
OMe
N
N
1
4d
1168±87
1358±228
15a
1620±386
7300±981
N
MeO
N
1
4e
183±29
451±110
15b
N
132±24
276±81
N
aThe interaction of compounds with HSP90 was determined by the alphascreen competitive
assay. The IC values (nM) are shown as mean ± SE (n = 3).
5
0
bSK-BR-3 cells were cultured with the compounds for 72 h. Cell survival was then measured and
the IC value was calculated. The IC values (nM) are shown as mean ± SE (n = 3).
5
0
50
Exploration of an alternative structure of the 3-quinoline group. To further enhance the
HSP90 binding inhibitory and growth inhibitory activities in SK-BR-3 cells, we examined the
effect of introduction of other C-4 substituents in place of the phenyl group in 15b. Table 4
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
shows HSP90 binding inhibitory and growth inhibitory activities in SK-BR-3 cells. The 4-
methoxy phenyl derivative 15c retained the HSP90 binding and cellular activities, but the 4-
phenoxyphenyl derivative 15d showed reduced potencies against HSP90 binding and tumor cell
growth. Various heterocyclic derivatives 15e-k have a tendency to increase the inhibitory
activity against HSP90 binding and tumor cell growth. Among them, compound 15k with a
methyl pyrazole group showed the most potent growth inhibitory activity in SK-BR-3 cells.
Furthermore, 15k showed more potent inhibitory activity against HSP90 binding and tumor cell
growth than those of the 3-quinoline derivative 13c. With Strategies 1 and 2, we succeeded in
identifying the most potent methyl pyrazole derivative 15k.
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Biological data of compounds with 4-substitution at the imidazole ring
R1
N
N
N
OH
N
N
N
H
O
H N
2
HSP90
50
SK-BR-3
IC50 (nM)
HSP90
50
SK-BR-3
IC50 (nM)
Compd
R1
Compd
15g
R1
a
b
a
b
IC (nM)
IC (nM)
N
N
15b
132±24
96±15
276±81
91±14
73±8
387±19
MeO
PhO
O
S
1
5c
256±8
15h
146±7
1
5d
643±119
503±80
15i
87±15
145±23
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N
N
1
5e
48±8
55±7
41±20
15j
52±9
103±16
36±6
N
N
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
15f
165±26
15k
54±10
aThe interaction of compounds with HSP90 was determined by the alphascreen competitive
assay. The IC values (nM) are shown as mean ± SE (n = 3).
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bSK-BR-3 cells were cultured with the compounds for 72 h. Cell survival was then measured and
the IC value was calculated. The IC values (nM) are shown as mean ± SE (n = 3).
5
0
50
Optimization of the benzamide group. At this point of the study, the methyl pyrazole
derivative 15k represented the most advanced analog and demonstrated potent inhibitory activity
against HSP90 binding and tumor cell growth. However, compound 15k did not exhibit
measurable oral exposure in mice (Table 5), although it had good metabolic stability (data not
shown). This suggested that the low oral exposure in mice might be attributed to the large
molecular weight (MW) and topological polar surface area (TPSA), high LogD value, and low
solubility at neutral pH (pH 7.4). Therefore, to improve its oral exposure in mice, we examined
the optimization of benzamide substituents in order to reduce MW, TPSA, and LogD value, and
to improve solubility at pH 7.4. Table 5 shows the HSP90 binding inhibitory and growth
inhibitory activities in SK-BR-3 cells, and also their MW, TPSA, LogD, solubility at pH 7.4, and
oral exposure in mice (AUC0-6h (M*h)). Although the cellular activity of compound 16a,
without the 4-aminocyclohexanol group, was highly attenuated, it showed a certain level of oral
exposure in mice. The result suggested that the reduction in MW, TPSA, and LogD value of 16a
might be important for improving oral exposure. Compound 16b, having a 4-aminomethyl group
with minimized 4-aminocyclohexanol structure, showed attenuated cellular activity when
compared with that of 15k. On the contrary, 16c having a 3-aminomethyl group at R position
3
showed HSP90 binding inhibitory activity similar to that of 15k, and the cellular activity also
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increased when compared with that of 16a and 16b. Furthermore, by shifting the substitution
position from C-4 to C-3, the LogD value of 16c decreased significantly. In addition, the oral
exposure of 16c in mice was similar to that of compound 16a. Based these results, we
successfully acquired a favorable derivative with oral exposure in mice by minimizing the 4-
aminocyclohexanol group, thus reducing the MW, TPSA, and LogD value. In addition, these
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results suggested that the position of substitution is better in R than in R . To further improve
3
4
their oral exposure in mice, we examined the introduction of other R substituents in place of 3-
3
aminomethyl group. Compound 16d, in which the aminomethyl group was converted to methyl
group as the smallest alkyl group, with reduced TPSA and increased LogD value, showed
attenuated inhibitory activity against HSP90 binding and tumor cell growth when compared with
those of 16c. In contrast, the solubility of 16d at pH 7.4 was highly improved and its oral
exposure in mice was also highly improved when compared with those of 16c. Compound 16e
with ethyl group showed increased inhibitory activity against HSP90 binding and tumor cell
growth. In particular, the oral exposure of 16e in mice was further improved when compared
with that of 16d. Based on these results, we succeeded in obtaining 16e with favorable oral
exposure in mice by optimizing the balance of MW, TPSA, LogD value of 15k, and improving
the solubility of 15k at pH 7.4. These results suggest that the 4-aminocyclohexanol group in the
methylpyrazole derivative 15k has negligible effect against the HSP90 binding inhibitory
activity when compared with that of 16e. That is, it suggests that the contribution of interactions,
which might be formed by the 4-(1H-imidazol-4-yl)-1-methyl-1H-pyrazole and 3-
ethylbenzamide groups of 16e, is more important for the HSP90 binding inhibitory activity. In
addition, these results also suggest that the solubility at pH 7.4 greatly affects oral exposure in
mice.
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Table 5. Biological data of compounds with substitution at the benzamide ring
N
N
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
N
R3
R4
O
H N
2
Solubility
HSP90
50
SK-BR-3 AUC0-6h
IC (nM) IC50 (nM) (M*h)
MW TPSA LogDa pH 7.4
Compd R3
R4
c
d
e
b
(M)
OH
1
5k
-H
-H
-H
539.64 134.51 3.6±0.2 3±1
426.48 102.25 3.0±0.3 4±1
54±10
204±14
131±37
58±6
36±6
N.D.f
N
H
1
6a
-H
1636±323
773±129
419±105
1137±336
330±119
5.00
N.T.g
1
6b
-NHMe 455.53 114.28 3.0±0.3
<2
16c -NHMe
-H
-H
-H
455.53 114.28 1.9±0.2 15±2
2.69
37.69
90.1
1
6d -Me
440.51 102.25 2.1±0.2 >170
98±10
1
6e
-Et
454.54 102.25 2.4±0.2 >170
69±11
a
b
36
LogD value at pH 7.4. The LogD values (nM) are shown as mean ± SD (n = 3).
37
Solubility at pH 7.4. Solubility (M) is shown as mean ± SD (n = 3).
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cThe interaction of compounds with HSP90 was determined by the alphascreen competitive
assay. The IC values (nM) are shown as mean ± SE (n = 3).
5
0
dSK-BR-3 cells were cultured with the compounds for 72 h. Cell survival was then measured and
the IC value was calculated. The IC values (nM) are shown as mean ± SE (n = 3).
5
0
50
eBalb/cA male mice (n = 2) were orally administered at a dose of 50 mg/kg.
fNot detected at all time points (lower limit of quantification = 0.03 M).
gNot tested.
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0
Structural analysis and discussion of compound 16d. To rationalize the structural basis for the
potent HSP90 binding affinity of 3-ethyl-4-(3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-
imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl)benzamide 16e,38 we tried to acquire its X-ray
co-crystal structure in complex with N-terminal ATP binding site of human HSP90. However,
we were unable to obtain it. Therefore, we determined the X-ray co-crystal structure of 16d as
1
6e (TAS-116) analog in complex with N-terminal ATP binding site of human HSP90 (Figure
3). As expected, the carboxamide group behaved as an adenine mimic and formed a water-
mediated hydrogen bond network with Asp93 and Thr184. The phenyl group is sandwiched
between Met98 and Asn51. This suggests that the alkyl group in the benzamide ring at C-3
position might lead to appropriate twist conformation against pyrazolo[3,4-b]pyridine ring. The
pyrazolo[3,4-b]pyridine core occupies a hydrophobic region formed by Leu107, Phe138, and
Trp162. In addition, the isopropyl group at the C-3 position in the pyrazolo[3,4-b]pyridine core
fills the small hydrophobic pocket formed by Ala111, Tyr139, and Val136. This helical specific
pocket formed by the rearrangement of Ile110-Gly114 residues has also been previously
observed in other HSP90 inhibitors.30,39 Furthermore, the nitrogen atom at the N-2 position of
pyrazolo[3,4-b]pyridine forms a water-mediated hydrogen bond network with Asn51. As
expected, the nitrogen atom at the N-3 position of the imidazole group at the C-4 position of
pyrazolo[3,4-b]pyridine forms a hydrogen bond with Tyr139. Moreover, the methylpyrazole
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group introduced at the imidazole C-4 position occupies a hydrophobic subpocket with Phe22,
Ile26, Ile104, Gly108, and Phe170 (Figure 3B). These results suggest that the enhancement of
HSP90 binding inhibitory activity and the binding mode of 16d observed by X-ray co-crystal
structure analysis are correlated. Based on these results, the 16e (TAS-116) analog 16d was
considered to have a unique binding mode that has not been observed in the typical first and
second generation HSP90 inhibitors.30 The hydrogen bond with Tyr139 and water-mediated
hydrogen bond network with Gln23 were observed in other HSP90 inhibitors by Vallée et al.40
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Figure 3. (A) Co-crystal structure of compound 16d bound to the N-terminal ATP binding site
of human HSP90. (B) The molecular surface representation of the co-crystal structure of
compound 16d in hydrophobic (red) to non-hydrophobic (white) gradient (PDB:5ZR3).
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Based on its promising HSP90 inhibitory activity and oral availability profile, the in vitro
4
1
profile and pharmacokinetic (PK) properties of compound 16e was characterized. Compound
16e selectively binds to HSP90 than to the highly homologous HSP90 family proteins
GRP94 and TRAP1, as determined by the fluorescence polarization competitive binding assay
with geldanamycin-FITC.42,43 In addition, 16e did not inhibit other ATPases, such as HSP70 and
HSP48, among different protein kinases tested. Regarding the toxicity of 16e toward non-cancer
44
cell lines, the IC value of normal human stromal cells was more than 5 M. Furthermore, 16e
5
0
demonstrated good metabolic stability in isolated hepatocytes of rodent and non-rodent species
(data not shown). Moreover, 16e did not inhibit the major human cytochrome P450 enzyme in
vitro at in vivo effective concentrations of 16e. It also showed good bioavailability in rodent and
non-rodent species. Further, 16e was distributed less in the eye tissues than in the plasma of rats;
consequently, 16e did not cause any detectable ocular toxicity, while 7 induced photoreceptor
41
cell death in rats. These results suggest that the novel orally available HSP90 selective
inhibitor 16e has a favorable profile as a potential clinical candidate.
In vivo pharmacology of 16e. Based on the favorable in vitro profiles and PK properties,
compound 16e was further evaluated in a human tumor xenograft model. Shown in Figure 4A is
4
5
the in vivo antitumor activity of 16e in an NCI-H1975 (EGFR L858R/T790M) xenograft model
of mice, and the mean body weight change (BWC) (%) is shown in Figure 4B. Reflecting its
potent HSP90 inhibitory activity, compound 16e exhibited potent efficacy (tumor/control ratio
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[
T/C]): 27%) after the daily oral administration of 16e for 14 days at a dose of 10 mg/kg without
significant body weight loss in mice. The level of tumor proteins at 12 h after the last
administration of 16e for 3 days at a dose of 10.0 mg/kg, determined by western blot analysis, is
shown in Figure 4C. Compound 16e induced a marked decrease in the level of EGFR, thus,
inhibiting downstream signaling, such as phospho-AKT, phospho-RPS6, and phospho-
MAPK1/3. These results suggest that 16e has antitumor effects in an EGFR-driven tumor model.
In addition, these results suggested that 16e demonstrates its antitumor effects by decreasing the
level of mutant EGFR, thereby, inhibiting the mutant EGFR-activated AKT and MAPK 1/3
signaling pathways, which are involved in the survival and proliferation of tumors. Therefore,
16e might be used to develop an effective chemotherapy for HSP90 client-driven cancer such as
lung cancer-expressing mutant EGFR.
1
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5
5
5
5
5
5
6
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1
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
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8
9
0
1
2
3
4
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6
7
8
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0
1
2
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4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. (A) Antitumor efficacy of 16e (TAS-116) in athymic nude mice bearing NCI-H1975
human lung cancer xenografts. (B) Body weight change of the tumor-bearing mice administered
1
6e (TAS-116). (C) Pharmacodynamic activity of 16e (TAS-116) in the NCI-H1975 tumors.
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Chemistry
The initial hit compound 11a and 3-substitued indole derivative 11b were synthesized by the
method shown in Scheme 1. Commercially available 4-bromoindole 17a-b were reacted with 3-
quinolineboronic acid under Suzuki–Miyaura cross-coupling condition46 involving catalytic
tetrakis(triphenylphosphine)palladium (0) (Pd(PPh ) ) and sodium carbonate (Na CO ) in 1,2-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
4
2
3
dimethoxyethane (DME) and H O at 100 °C for 14 h to obtain substituted indoles 18a-b, with a
2
yield of 50% and 87%, respectively. Ullmann type coupling reaction of intermediates 18a-b with
2
-amino substituted benzonitrile (19) was carried out in the presence of catalytic copper (I)
iodide (CuI), N,N′-dimethylethylene diamine (DMEDA), and cesium carbonate (Cs CO ) in 1,4-
2
3
dioxane at 110 °C for 14 h47,48 to obtain the corresponding benzonitriles 20a-b, with a good yield
(95% and 96%). The hydrolysis of the obtained benzonitriles 20a-b with 4 M sodium hydroxide
4
9–51
(NaOH) and 30% hydrogen peroxide (H O ) in a mixture of dimethyl sulfoxide (DMSO)
and
2
2
ethanol (EtOH) resulted in the desired benzamide compounds 11a-b, with a yield of 65% and
7%, respectively.
9
Scheme 1. Synthesis of indole derivatives 11a-ba
I
OH
N
H
CN
N
1
9
Br
N
R1
R1
(a)
(b)
R1
OH
N
N
H
N
1
7a: R =H
1
N
H
1
7b: R =Me
H
1
1
8a-b
R4
2
1
0a-b: R = CN
1a-b: R = CONH
4
(c)
4
2
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Reagents and conditions: (a) 3-quinolineboronic acid, Pd(PPh ) , Na CO , DME, H O, 100 °C,
3
4
2
3
2
1
4 h, 18a: 50%, 18b: 87%; (b) 19, CuI, DMEDA, Cs CO , 1,4-dioxane, 110 °C, 14 h, 20a: 95%,
2 3
2
0b: 96%; (c) 4 M NaOH aq., 30% H O aq., DMSO, EtOH, 0 °C-rt, 2.5 h, 11a: 65%, 11b: 97%.
2 2
To examine the substitution effects of the nitrogen atom at the C-6 position (12a) in 11a and the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
introduction of methyl (12b), and isopropyl (12c) moieties as representative alkyl substituents at
the C-3 position, the compounds were synthesized as shown in Scheme 2. The synthesis of
derivatives 12a-c commenced with 3-quinolineboronic acid under Suzuki–Miyaura cross-
coupling
condition46
involving
catalytic
[1,1′-
bis(diphenylphosphino)ferrocene]dichloropalladium (II) (Pd(dppf)Cl .CH Cl ), and Na CO in
2
2
2
2
3
1
,4-dioxane and H O at 100 °C for 3 h to obtain 3-quinoline-substituted indazoles 22a-c (56%–
2
94%). Ullmann-type coupling reaction of the intermediates 22a-c with 2-amino substituted
benzonitrile (19) was carried out in the presence of catalytic CuI, DMEDA, and tripotassium
4
8
phosphate (K PO ) as a base in 1,4-dioxane at 110 °C for 24 h to obtain the corresponding
3
4
benzonitriles 23a-c (64%–98%). The hydrolysis of the obtained benzonitriles 23a-c with 4 M
4
9–51
NaOH and 30% H O in a mixture of DMSO
and EtOH resulted in the desired benzamide
2
2
compounds 12a-c (66%–84%).
Scheme 2. Synthesis of indazole derivatives 12a-ca
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9
I
OH
N
H
CN
N
1
9
Br
N
R1
N
R1
N
(a)
(b)
R1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OH
N
N
H
N
21a-c
N
H
N
H
2
2a-c
R2
2
1
3a-c: R = CN
2a-c: R = CONH
2
(
c)
2
2
Series of a to c in compounds mean: a: R =H, b:R =Me, c:R =i-Pr
1
1
1
a
Reagents and conditions: (a) 3-quinolineboronic acid, Pd(dppf)Cl .CH Cl , Na CO , 1,4-
dioxane, H O, 100 °C, 3 h, 56%–94%.; (b) 19, CuI, DMEDA, K PO , 1,4-dioxane, 110 °C, 24 h,
64%–98%; (c) 4 M NaOH aq., 30% H O aq., DMSO, EtOH, 0 °C-rt, 2.5 h, 66%–84%.
2
2
2
2
3
2
3
4
2
2
The 1H-pyrazolopyridine derivatives 13a-c with substitution of the nitrogen atom in 12c were
synthesized as shown in Scheme 3. Lithiation of commercially available 2,4-dichloropyridine
(
24a), 3,5-dibromopyridine (24b), and 2-fluoro-3-iodopyridine (24c) was carried out in the
presence of lithium diisopropylamide (LDA) in tetrahydrofuran (THF) at -78 °C, followed by
alkylation with isobutyl anhydride to obtain 1-(2,4-dihalopyridin-3-yl)-2-methylpropan-1-one
intermediates. Finally, intramolecular cyclization with hydrazine monohydrate was carried out to
obtain C3-isopropyl substituted 1H-pyrazolopyridines (25a-c) (17%–42%). Compound 25c was
obtained by the halogen dance reaction52 of 24c. The obtained 4-halo-3-isopropyl-1H-
pyrazolopyridines (25a-c) were reacted with 3-quinolineboronic acid under Suzuki–Miyaura
4
6
cross-coupling condition involving catalytic Pd(dppf)Cl .CH Cl , and Na CO in 1,4-dioxane
2
2
2
2
3
and H O at 100 °C to obtain 3-quinoline substituted pyrazolopyridines (26a-c) (76%–92%). In
2
the next step, Ullmann type coupling reaction of 3-quinoline substituted pyrazolopyridines 26a-c
with 2-amino substituted benzonitrile (19) was carried out in the presence of catalytic CuI,
4
8
DMEDA, and K PO in 1,4-dioxane at 110 °C for 24 h to obtain the corresponding
3
4
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
benzonitriles 27a-c (81%–95%). The hydrolysis of the obtained benzonitriles 27a-c with 4 M
4
9–51
NaOH and 30% H O in a mixture of DMSO
and EtOH resulted in the desired benzamide
2
2
compounds 13a-c (28%–91%).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Synthesis of pyrazolopyridine derivatives 13a-ca
I
OH
N
N
H
CN
1
(d)
9
R1
Z
R1
Z
N
(a)
(c)
X
Y
X
Y
X
Y
N
N
OH
N
N
Z
R1
X
Y
H
N
N
24a-b
25a-c
Z
H
N
H
2
4a: X=N, Y=CH, Z=CH 25a: X=N, Y=CH, Z=CH, R =Cl
1
26a-c
R4
R =Cl
25b: X=CH, Y=N, Z=CH, R =Br
1
1
2
4b: X=CH, Y=N, Z=CH 25c: X=CH, Y=CH, Z=N, R =I
1
27a-c: R = CN
4
(e)
R =Br
1
13a-c: R = CONH
4
2
1
1
3a: X=N, Y=CH, Z=CH
3b: X=CH, Y=N, Z=CH
I
(a)
13c: X=CH, Y=CH, Z=N
N
F
24c
a
Reagents and conditions: (a) 1) 24a-c, DIPA, n-BuLi, THF, -78 °C, 15 min; 2)
o
isobutylanhydride, -78 °C, 1 h; 3) N H aq., 60–110 C, 1–24 h, 17%–42%; (d) 3-
2
4
quinolineboronic acid, Pd(dppf)Cl .CH Cl , Na CO , 1,4-dioxane, H O, 100 °C, 3 h, 76%–97%;
2
2
2
2
3
2
(
d) 19, CuI, DMEDA, K PO , 1,4-dioxane, 110 °C, 24 h, 81%–95%; (e) 4 M NaOH aq., 30%
3 4
H O aq., DMSO, EtOH, 0 °C-rt, 2.5 h, 28%–91%.
2
2
We then focused on the 3-quinoline group of compound 13c at the C-4 position. We selected a
3-isopropyl-1H-pyrazolo[3,4-b]pyridine in the terminal core structure, and carried out further
optimization of the 3-quinoline group at the C-4 position.
The synthesis of the C-4 aryl derivatives 14a-i is shown in scheme 4. The obtained intermediate
25c was treated with sodium hydride (NaH) in N,N-dimethylformamide (DMF), and then
coupled with 2,4-difluorobenzonitrile at 60 °C to obtain 2-fluorobenzonitrile 28 as a mixture of
the desired product and C-2 substituted regioisomer 28a. The obtained mixture of 28 and 28a
was reacted with trans-4-aminocyclohexanol in the presence of N,N-diisopropylethylamine
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(
DIPEA) in DMSO to obtain the desired intermediate 29 by column chromatography on silica
4
6
gel with 15% yield in two steps. In the next step, Suzuki–Miyaura cross-coupling reaction of
29 with various boronic acids or boronic acid pinacol esters mediated by catalytic Pd(PPh ) and
3
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Na CO in DME and H O was carried out at 100 °C for 4 h to obtain 4-substituted benzonitriles
2
3
2
3
0a-i (82%-quant.). Finally, the hydrolysis of the obtained benzonitriles 30a-i with 4 M NaOH
4
9–51
and 30% H O in a mixture of DMSO
and EtOH resulted in the desired benzamide
2
2
compounds 14a-i (75%–97%).
Scheme 4. Synthesis of pyrazolo[3,4-b]pyridine derivatives 14a-ia
I
I
I
I
N
N
OH
(
a)
N
(b)
N
N
N
N
N
N
N
CN
N
N
H
N
F
H
2
5c
CN
OH
F
CN
2
8
28a
29
R1
N
OMe
N
MeO
14e:
N
N
R =14a:
14b:
14c:
14d
:
N
1
(
c)
N
N
H
N
S
O
R2
N
N
14f:
14g:
14h:
14i:
3
0a-i: R = CN
2
(d)
14a-i: R = CONH
2
2
aReagents and conditions: (a) 2,4-difluorobenzonitrile, NaH, DMF, 60 °C, 1 h; (b) trans-4-
aminocyclohexanol, DIPEA, DMSO, 125–150 °C, 8 h, 15%, 2 steps; (c) Boronic acids or
boronic acid pinacol esters, Pd(PPh ) , Na CO , DME, H O, 100 °C, 4 h, 82%-quant; (d) 4 M
3
4
2
3
2
NaOH aq., 30% H O aq., DMSO, EtOH, rt, 30 min, 75%–97%;
2
2
The synthesis of the 4-aryl-1H-imidazole derivatives 15a-k is shown Scheme 5. Ullmann type
coupling reaction of the obtained intermediate 29 with various 4-aryl-1H-imidazoles mediated by
copper (II) oxide (CuO) nanopowder under basic conditions using potassium carbonate (K CO )
2
3
5
3
in DMF was carried out at 120 °C for 20 h to obtain 4-substituted benzonitriles 31a-k (45%–
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
9
0%). Finally, the hydrolysis of the obtained benzonitriles 31a-k with 4 M NaOH and 30% H O
2
2
in a mixture of DMSO49–51 and EtOH resulted in the desired benzamide compounds 15a-k (50%-
quant.).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 5. Synthesis of imidazole derivatives 15a-ka
R1
N
I
N
N
(a)
OH
N
N
N
OH
N
N
N
H
N
CN
H
R2
29
31a-k: R = CN
2
(b)
1
5a-k: R = CONH
2
2
MeO
PhO
N
N
N
H
R = 15a:
15b:
15c:
15d:
15j:
15e:
15k:
15f:
1
N
O
S
N
N
N
15g:
15h:
15i:
N
a
Reagents and conditions: (a) imidazoles, CuO nanopowder, K CO , DMF, 120 °C, 20 h, 45%–
2
3
9
0%; (b) 4 M NaOH aq., 30% H O aq., DMSO, EtOH, 0 °C-rt, 2.5 h, 50%-quant.
2 2
To examine the substituent effects on the benzamide group, 2- or 3-substituted benzamide
derivatives (16a-e), other than the 4-aminocyclohexanol group, were synthesized by the method
shown in Scheme 6. N-Protection of the obtained intermediate 25c with p-
methoxybenzylchloride (PMBCl) in the presence of NaH in DMF at room temperature for 1 h
yielded PMB protected pyrazolopyridine 32 with 91% yield. Ullmann type coupling reaction of
the obtained compound 32 and 4-(1H-imidazol-4-yl)-1-methyl-1H-pyrazole dihydrochloride (33)
was carried out in the presence of catalytic copper (I) oxide (Cu O), 4,7-dimethoxy-1,10-
2
5
4
phenanthroline, polyethylene glycol (PEG), and Cs CO in DMSO at 110 °C for 24 h to obtain
2
3
the desired 4-(1H-imidazol-4-yl)-1-methyl-1H-pyrazole intermediate (34) , with a yield of 62%.
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The PMB group of 34 was treated with TFA and anisole55 to obtain pyrazolopyridine
intermediate 35, with a yield of 88%. The condensation of intermediate 37 with commercially
available 4-fluorobenzonitriles was carried out in the presence of Cs CO in DMF at 50–120 °C
2
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
for 0.5–14 h to obtain 3-substituted benzonitriles 36a, 36d, and 36e (81%–91%). Ullmann-type
coupling reaction of the intermediate 37 with 4-iodo-2-(methylamino)benzonitrile was carried
out in the presence of catalytic CuI, DMEDA, and K PO as a base in 1,4-dioxane at 110 °C for
3
4
4
8
2
4 h to obtain the corresponding benzonitrile 36b (36%). Finally, the hydrolysis of the obtained
4
9–51
benzonitriles 36a, 36b, 36d, and 36e with 4 M NaOH and 30% H O in a mixture of DMSO
2
2
and EtOH resulted in the desired benzamide compounds 16a, 16b, 16d, and 16e (45%–95%),
respectively.
Scheme 6. Introduction of various benzamide groups to the 1H-imidazole-1-yl-3-isopropyl-
a
1
H-pyrazolo[3,4-b]pyridine scaffold 16a, 16b, 16d, 16e
N
N
2HCl
N
N
N
N
N
N
N
H
33
16a: R =H, R =H
1 2
16b: R1=H, R2=NHMe
I
N
N
(b)
(d) or (e)
1
6d: R =Me, R =H
1 2
N
N
N
16e: R =Et, R =H
N
1
2
N
N
R
N
R1
R2
5
N
2
3
5c: R = H
N
5
N
(a)
R
5
2: R = PMB
5
3
4: R = PMB
5
(c)
R4
35: R = H
5
36a,b,d,e: R = CN
4
(f)
1
6a,b,d,e: R = CONH
4 2
a
Reagents and conditions: (a) PMBCl, NaH, DMF, 0 °C-rt, 2 h, 91%; (b) 33, Cu O, 4,7-
2
dimethoxy-1,10-phenanthroline, Cs CO , PEG, DMSO, 110 °C, 24 h, 62%; (c) TFA, anisole,
2
3
reflux, 5 h, 88%; (d) benzonitriles, Cs CO , DMF, 50–120 °C, 0.5–14 h, 81%–91%; (e) 4-iodo-
2
3
2
-(methylamino)benzonitrile, CuI, DMEDA, K PO , 1,4-dioxane, 110 °C, 24 h, 36%; (f) 4 M
3 4
NaOH aq., 30% H O aq., DMSO, EtOH, 0 °C-rt, 2.5 h, 45%–95%.
2
2
The preparation of 3-amino substituted derivative 16c has been summarized in Scheme 7. The
condensation of intermediate 35 with commercially available 4-chrolo-3-nitrobenzonitrile was
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carried out in the presence of Cs CO in DMF at 80°C for 14 h to obtain 3-nitrobenznitrile 37
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(
70%). The reduction of the nitro group with iron powder and ammonium chloride (NH Cl) in
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THF, EtOH, and H O at 80 °C for 4 h, yielded 39% of 3-amino benzonitrile 38. The
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monomethylation of 3-amino benzonitrile 38 was carried out in the presence of copper (II)
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acetate (Cu(OAc) ), methylboronic acid, and pyridine in 1,4-dioxane at 120 °C for 2 h to obtain
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-methylamino benzonitrile 39 (38%). Finally, the hydrolysis of the obtained benzonitrile 39
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9–51
with 4 M NaOH and 30% H O in a mixture of DMSO
and EtOH resulted in the desired
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benzamide compound 16c with 83% yield.
Scheme 7. Introduction of 3-(methylamino)benzamide group to the 1H-imidazole-1-yl-3-
a
isopropyl-1H-pyrazolo[3,4-b]pyridine scaffold 16c
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
(a)
(b)
(c)
N
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N
N
N
N
N
N
N
N
NO2
NH2
NHMe
N
N
N
H
CN
CN
R4
9: R = CN
35
37
38
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(d)
6c: R = CONH
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a
Reagents and conditions: (a) 4-chloro-3-nitrobenzonitrile, Cs CO , DMF, 80 °C, 14 h, 70%; (b)
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Fe, NH Cl, THF, EtOH, H O, 80 °C, 4 h, 39%; (c) methylboronic acid, Cu(OAc) (II), pyridine,
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,4-dioxane, 120 °C, 2 h, 38%; (d) 4 M NaOH aq., 30% H O aq., DMSO, EtOH, 0 °C, 2 h,
3%.
2 2
Conclusions
Compound 16e (TAS-116), a potent and highly selective HSP90  inhibitor with a unique
binding mode was discovered by SAR-based optimization of an initial hit compound 11a. The
main findings of this study are as follows. (i) The introduction of an isopropyl group at the C-3
position of the initial hit 11a significantly increased the HSP90 binding inhibitory activity. (ii)
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The introduction of nitrogen atom at the C-2 position of the initial hit 11a also significantly
increased the HSP90 binding inhibitory activity. (iii) The introduction of nitrogen atom into the
indazole ring of 12c resulted in lower lipophilicity and hERG inhibitory activity than those by
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2c. (iv) The discovery of 4-(1H-imidazol-4-yl)-1-methyl-1H-pyrazole group, as an alternative
structure of 3-quinoline group, maximized the HSP90 inhibitory activity and tumor cell growth
inhibitory activity in SK-BR-3 cells. (v) Compound 16e with favorable oral exposure in mice
was discovered by optimizing the balance among the MW, TPSA, and LogD value and by
improving the solubility of 15k at pH 7.4. (vi) The X-ray co-crystal structure analysis of TAS-
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16 analog 16d demonstrated a unique binding mode.
Compound 16e also exhibited good oral absorption in mice. The oral administration of 16e on a
daily basis was well tolerated, resulting in potent antitumor effects by decreasing the amount of
mutant EGFR in the NCI-H1975 (human lung cancer) xenograft model. Phase II clinical trial to
evaluate the safety and efficacy of 16e (TAS-116) is in progress.58
Experimental Section
Measurement of HSP90-binding activity. The HSP90-binding activity was measured by the
AlphaScreen competitive assay. The His-tagged HSP90α NTD was added to a 384-well plate
containing the test compounds. After reaction at room temperature for 2 h, biotin-labeled
geldanamycin was added, and the mixture was incubated for a further 1 h. The bead mixture
containing both nickel-chelated acceptor beads and streptavidin-coated donor beads was added.
After incubation for 1 h in dark, the assay plate was measured in a PerkinElmer Envision plate
reader (PerkinElmer Corporation, Waltham, MA, USA). The IC value (nM), the concentration
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