Design and synthesis of prostate cancer antigen-1 (PCA-1/ALKBH3) inhibitors as anti-prostate cancer drugs

Article abstract of DOI:10.1016/j.bmcl.2014.01.008

A series of 1-aryl-3,4-substituted-1H-pyrazol-5-ol derivatives was synthesized and evaluated as prostate cancer antigen-1 (PCA-1/ALKBH3) inhibitors to obtain a novel anti-prostate cancer drug. After modifying 1-(1H-benzimidazol-2-yl)-3,4-dimethyl-1H-pyrazol-5-ol (1), a hit compound found during random screening using a recombinant PCA-1/ALKBH3, 1-(1H-5- methylbenzimidazol-2-yl)-4-benzyl-3-methyl-1H-pyrazol-5-ol (35, HUHS015), was obtained as a potent PCA-1/ALKBH3 inhibitor both in vitro and in vivo. The bioavailability (BA) of 35 was 7.2% in rats after oral administration. As expected, continuously administering 35 significantly suppressed the growth of DU145 cells, which are human hormone-independent prostate cancer cells, in a mouse xenograft model without untoward effects.

Full text of DOI:10.1016/j.bmcl.2014.01.008

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1071–1074
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of prostate cancer antigen-1 (PCA-1/ALKBH3)
inhibitors as anti-prostate cancer drugs
Syuhei Nakao a,b, Miyuki Mabuchi a, Tadashi Shimizu a,b, Yoshihiro Itoh a,b, Yuko Takeuchi a,b
,
Masahiro Ueda a,b, Hiroaki Mizuno a, Naoko Shigi c, Ikumi Ohshio c, Kentaro Jinguji c, Yuko Ueda c,
Masatatsu Yamamoto d, Tatsuhiko Furukawa d, Shunji Aoki b, Kazutake Tsujikawa c, , Akito Tanaka a,b, ,
⇑
a Advanced Medical Research Center, Hyogo University of Health Science, 1-3-6 Minatojima, Kobe 650-8530, Japan
b Department of Pharmacy, Hyogo University of Health Science, 1-3-6 Minatojima, Kobe 650-8530, Japan
c Laboratory of Molecular and Cellular Physiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Suita 565-0871, Japan
d Department of Molecular Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1-aryl-3,4-substituted-1H-pyrazol-5-ol derivatives was synthesized and evaluated as prostate
cancer antigen-1 (PCA-1/ALKBH3) inhibitors to obtain a novel anti-prostate cancer drug. After modifying
1-(1H-benzimidazol-2-yl)-3,4-dimethyl-1H-pyrazol-5-ol (1), a hit compound found during random
screening using a recombinant PCA-1/ALKBH3, 1-(1H-5-methylbenzimidazol-2-yl)-4-benzyl-3-methyl-
1H-pyrazol-5-ol (35, HUHS015), was obtained as a potent PCA-1/ALKBH3 inhibitor both in vitro and
in vivo. The bioavailability (BA) of 35 was 7.2% in rats after oral administration. As expected, continuously
administering 35 significantly suppressed the growth of DU145 cells, which are human hormone-inde-
pendent prostate cancer cells, in a mouse xenograft model without untoward effects.
Received 20 November 2013
Revised 2 January 2014
Accepted 6 January 2014
Available online 11 January 2014
Keywords:
PCA-1
ALKBH3
Inhibitor
Ó 2014 Elsevier Ltd. All rights reserved.
Anti-prostate cancer drug
Small compound
Bioavailability
Prostate canceris highlyserious when malignant, and developing
novel anti-prostate cancer drugs that are effective against both
androgen-dependent and independent types is now urgently
required.1 Tsujikawa et al. have reported a novel gene that encodes
a DNA and/or RNA-alkylating damage-repair enzyme called prostate
cancer antigen (PCA)-12 or AlkB homologue 3 (ALKBH3) that is
highly expressed in clinical prostate cancer cells; genetic inhibition
of the enzymatic activity by injecting siRNA effectively inhibited the
growth of androgen-independent prostate cancer cells, such as
DU145, that express high levels of PCA-1/ALKBH3.3,4 In addition, los-
ing PCA-1/ALKBH3 led to 3-methylcytosine accumulation and re-
duced cell proliferation in various cell lines.5 Therefore, a small,
orally available PCA-1/ALKBH3 inhibitor would be a novel and
clinically effective anti-prostate cancer drug, even for hormone-
independent varieties. Random screening using commercially avail-
able 17,000-compound libraries and recombinant PCA-1/ALKBH3
has been carried out to identify small PCA-1/ALKBH3 inhibitors; 1-
(1H-benzimidazol-2-yl)-3,4-dimethyl-1H-pyrazol-5-ol (1) is a hit
was confirmed with 76% inhibition at the same concentration using
re-synthesized 1 (Fig. 1). However, 1 demonstrated only a 0.8%
inhibitory effect against the proliferation of DU145 cells at 10 lM.
Accordingly, we focused our efforts to obtain a novel PCA-1/ALKBH3
inhibitor that is effective in cell assays and in vivo.
We report the design and synthesis of novel 1-ary-3,4-substi-
tuted-1H-pyrazol-5-ol derivatives and their PCA-1/ALKBH3
inhibitory activities and anti-cancer effects in vivo. Among them,
1-(5-methyl-1H-benzimidazol-2-yl)-4-benzyl-3-methyl-1H-pyr-
azol-5-ol (35, HUHS015) was a potent PCA-1/ALKBH3 inhibitor
that significantly suppressed the growth of DU145 cells in vitro
and in a mouse xenograft model. These small PCA-1/ALKBH3 inhib-
itors are highly important because PCA-1/ALKBH3 is associated
with numerous cancers, such as lung5,6 and pancreatic cancers.5,7
The substituted pyrazoles evaluated in this work were synthe-
sized by condensing N-substituted hydrazines (3) with ethyl aceto-
acetate derivatives (4)8 (Scheme 1). The assay methods for PCA-1
inhibitory activities and biological assays in vitro and in vivo of
these compounds were shown in Supplementary data.
compound with 61% inhibition at 10 lM, and its inhibitory activity
The deletion and replacement of the aromatic rings on the
benzimidazole portion of 1 generated only weak anti-PCA-1/
ALKBH3 activities, indicating the importance of the benzimidazole
(5–13, Table 1). Only compounds with a methyl group at the 5
⇑
Corresponding author. Tel.: +81 78 304 3067; fax: +81 78 304 2767.
E-mail address: tanaka-a@huhs.ac.jp (A. Tanaka).
These authors contributed equivalently for this work.
0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2014.01.008

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S. Nakao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1071–1074
Table 2
Me
Me
Me
N
N
N
N
PCA-1 inhibitory activities of 1-(substituted benzimidazol-2-yl)-5-hydroxy-3-
methyl-4-(subsituted benzyl) pyrazoles
N
N
H
N
N
H
R1
HO
HO
O
R2
Me
N
N
N
N
DMT1 blocker (2)
Hit compound (1)
H
HO
R2
Figure 1. Structure of PCA-1/ALKBH3 inhibitor obtained in our random screening
R1
PCA-1 inhibition IC50 (lM)
and previously known DMT1 blocker.
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
H
H
H
H
H
H
H
H
H
H
5-Me
5-Cl
2-Cl
3-Cl
4-Cl
2,4-Cl2
3,4-Cl2
4-CF3
4-F
4-MeO
4-Phenyl
4-tBu
H
H
H
H
H
H
H
>10 (À13%)
0.54
>10 (46%)
>10 (11%)
3.1
0.55
0.60
0.63
0.72
4.1
0.67
4.5
0.49
0.61
0.64
0.81
1.5
R3
O
O
N
R1
N
R1 NHNH2
R3
+
OEt
R2
R2
HO
3
4
5 - 52
Scheme 1.
5-Br
position of benzimidazole (12) demonstrated slightly more potent
anti-PCA-1/ALKBH3 activity. Structurally similar 1-aryl-3,4-substi-
tuted-pyrazol-5-ol compounds, such as the 1-(2-pyridyl)-pyrazol-
5-ol derivative (2), were recently reported to block divalent metal
transporter 1 (DMT1, Fig. 1).9 In these DMT1 blockers, the neigh-
boring basic nitrogen in the aryl ring was vital for metal chelation
and DMT1 inhibitory activities. However, a similar 2-pyridyl deriv-
ative (11) examined in this study revealed only weak PCA-1/
ALKBH3 inhibition and attenuated inhibitory activities against
DU145 cell proliferation (data not shown). The basicity of the N
atom on the benzimidazole studied in this work is weak, and its
metal chelating ability should be poor. Therefore, we proposed that
metal chelation contributed little to the PCA-1/ALKBH3 inhibitory
activities. This result was favorable for developing a clinically use-
ful anti-cancer drug because metal chelators often cause adverse
effects or toxicity in vivo.
5-CF3
5-tBu
5-Phenyl
5-(4-Cl-phenyl)
5-(4-Me-phenyl)
4-Me
4,5-Me2
5-Me
5-Me
5-Me
5-Me
5-Me
5-Me
H
H
H
2.1
5.9
>10 (À47%)
4-CF3
4-MeO
3,4-Cl2
2-Cl
4-Cl
4-F
0.57
0.68
0.72
0.91
0.75
0.78
3.7
5.3
5-Me
5-Me
4-tBu
4-Phenyl
(14–24, Table 1). Introducing a phenyl (15) or naphthyl (16) group
on the methyl group at the 4 position of the pyrazole generated
potent PCA-1/ALKBH3 inhibitory effects, while the compounds
Based on the above results, we fixed 1H-benzimidazol-2-yl at
the 1 position of the pyrazole ring and evaluated 1-(substituted-
1H-benzimidazole-2-yl)-3,4-substituted-1H-pyrazol-5-ol derivatives
Table 1
PCA-1 inhibitory activities of 1,3,4-substituted-5-hydroxy-pyrazoles
R3
R2
N
R1
N
HO
R1
R2
R3
PCA-1 inhibition IC50 (lM)
5
6
7
8
Benzimidazol-2-yl
Phenyl
Benzthiazol-2-yl
Benzoxazol-2-yl
1-Me-benzimidazol-2-yl
2-Pyrimidyl
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
4.0
>10 (20%)*
>10 (À3%)
>10 (0%)
>10 (21%)
>10 (À4%)
>10 (À4%)
2.6
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
2-Pyridyl
5-Me-benzimidazol-2-yl
4-Me-benzimidazol-2-yl
Benzimidazol-2-yl
Benzimidazol-2-yl
Benzimidazol-2-yl
Benzimidazol-2-yl
Benzimidazol-2-yl
Benzimidazol-2-yl
Benzimidazol-2-yl
Benzimidazol-2-yl
5-Me-benzimidazol-2-yl
5-Cl-benzimidazol-2-yl
5-Me-benzimidazol-2-yl
>10 (À28%)
3.6
Benzyl
0.48
0.75
4.3
15.0
2-Naphtylmethyl
CH2COOMe
CH2COOH
H
Me
Benzyl
Me
Phenyl
Phenyl
7.2
>10 (À11%)
>10 (2%)
3.3
2.0
5.8
*
PCA-1/ALKB3 inhibition (%) at 10 mM are shown in parenthesis.

S. Nakao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1071–1074
1073
Table 3
Inhibitory activities of DU145 cells proliferation on dish (2-D assay) and in agar (3-D assay), and serum concentration at 1 h after oral administration (32 mg/kg)
Inhibition (%) on DU145 proliferation
Serum concentration at 1 h (lg/mL)
2D assay
3D assay
10
l
M (%)
1
l
M (%)
10
lM (%)
1
lM (%)
15
12
35
36
38
41
49
47
19
54
NT
84
63
46
42
À11
35
86
78
70
À1
90
69
81
87
92
42
3.8
34
84
90
91
2.8
NTa
1080
81
154
20
100
90
7
72
a
NT: not tested.
without the methyl (14) group or with a carboxyl moiety (17 and 18)
demonstrated similar activities. Derivatives with a phenyl ring at
the 3 position (19–24) exhibited weaker activities. Accordingly, we
fixed the methyl and benzyl groups at the 3 and 4 positions, respec-
tively, for further studies.
(a)
300
200
100
0
*** p<0.005
by t-test
Control
The substituent effects on the phenyl group in 15 were evalu-
ated (25–34, Table 2). However, no enhancement of anti-PCA-1/
ALKBH3 activities was observed. In particular, the derivative with
an ortho-chloro substituent (25 and 28) greatly decreased the
inhibitory activity. Therefore, a benzyl group was used at the 4 po-
sition, and the derivatives bearing benzimidazole ring substituents
were evaluated (35–44, Table 2). Some derivatives with substitu-
tion at the 5 position, such as 35 and 37–40, exhibited potent
PCA-1/ALKBH3 inhibitory activity, while compounds with substit-
uents at the 4 position, such as 43 and 44, displayed reduced activ-
ity. Of these compounds, 35 (HUHS015) was selected for further
study because it potently inhibited PCA-1/ALKBH3 and had good
physical properties. Finally, the substituents on the phenyl ring
of 35 were varied (45–52, Table 2). The inhibitory activities of
those derivatives were similar to that of 35 and therefore were
not pursued.
***
***
***
HUHS015(35)
0
2
4
6
8
10
Days
(b)
HUHS015(35)
110
100
90
Control
Next, we selected several compounds and tested the prolifera-
tion inhibition against DU145 cells and the oral availability in rats
(Table 3). The proliferation inhibition of DU145 was examined in
both an ordinary anchorage-dependent assay on dishes and an
anchorage-independent assay using soft-agar because the inhibi-
tory activity against the latter cells might be crucial to clinical effi-
cacy.10 Compounds with weak PCA-1/ALKBH3 inhibitory activity,
such as 12, demonstrated weak proliferation inhibition against
DU145, while those with sub-micromolar IC50 values, such as 15,
35, 38, and 49, exhibited more potent effects. We are uncertain
about the potency of 36 in the DU145 assays because its IC50 value
0
2
4
6
8
10
Days
Figure 2. (a) Inhibitory effect of HUHS015 (35, 32 mg/kg/day, open circle) on the
growth of subcutaneously implanted DU145 (nude mice) was examined (n = 6).
Compound 35 or saline was administered for 6 days (arrows), and the tumor
volume was measured for 10 days. Black circles demarcate the control data (0.5%
methylcellulose). (b) The mice administered with 35 (white circle) gained more
body weight than the control mice (black circle). No apparent toxicity or side effects
were found in the mice during the experiment. The values represent means SE
(n = 6).
during the PCA-1/ALKBH3 enzyme assay was 4.5 lM or weaker.
without limiting weight gains, even after a 6-day continuous
administration.11 Therefore, the synthesizing small molecule
inhibitor of PCA-1/ALKBH3, a clinically identified novel target,
demonstrated promising results for developing an anti-prostate
cancer drug without mechanism-oriented side effects. Now further
modifications of 35 to obtain more potent inhibitors and combina-
tion studies with 35 and clinically used drug such as docetaxel are
in progress, and will be reported in the near future.
In conclusion, we synthesized 1,3,4-substituted-1H-pyrazol-5-
ol derivatives to identify orally active PCA-1/ALKBH3 inhibitors
and selected 1-(5-methyl-1H-benzimidazol-2-yl)-4-benzyl-3-
methylpyrazol-5-ol (35, HUHS015) for further analysis. Compound
35 exhibited potent inhibition against DU145 proliferation without
observable side effects after subcutaneous administration in a
xenografted mouse model. The results for 35 (HUHS015) in the
xenograft mouse model, when combined with previously reported
PCA-1/ALKBH3 knock-out mice study,12 in which mice lacking
function of ALKBH3 gene are viable without overt phenotypes
Consequently, 36 was eliminated from further studies because
these inconsistent effects might cause unexpected side effects in
future studies. To select a compound for additional in vivo studies,
we also measured the blood concentration 1 h after each com-
pound was orally administered (32 mg/kg). Compound 12 exhib-
ited high serum concentrations relative to the other derivatives.
However, compounds with relatively high molecular weights, such
as 41, exhibited poor oral absorbability. Therefore, smaller com-
pounds were more bioavailable. Consequently, we selected 35
(HUHS015) for further studies because it exhibited an adequate le-
vel of serum concentration after oral administration and potent
inhibition during both the enzymatic and cell assays. We measured
the preliminary bioavailability (BA) value for 35 after oral admin-
istration to rats, revealing a 7.2% BA value (Figs. S1 and S2). This
BA value was sufficient for continuing the in vivo studies.
The growth inhibition demonstrated by 35 (32 mg/kg) was
examined in a mouse xenograft model bearing DU145 after subcu-
taneous injection (Fig. 2); potent growth inhibition was observed

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S. Nakao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1071–1074
5. Dango, S.; Mosammaparast, N.; Sowa, M. E.; Xiong, L. J.; Wu, F.; Park, K.; Rubin,
M.; Gygi, S.; Harper, J. W.; Shi, Y. Mol. Cell 2011, 44, 373.
6. Tasaki, M.; Shimada, K.; Kimura, H.; Tsujikawa, K.; Konishi, N. Br. J. Cancer 2011,
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and histological changes, demonstrated that small PCA-1/ALKBH3
inhibitors would be effective drugs against prostate cancer without
mechanism-based side effects or toxicity.
7. Yamato, I.; Sho, M.; Shimada, K.; Hotta, K.; Ueda, Y.; Yasuda, S.; Shigi, N.;
Konishi, N.; Tsujikawa, K.; Nakajima, Y. Cancer Res. 2012, 72, 4829.
8. The representative procedure for synthesizing the compounds studied in this work
Acknowledgments
is as follows:
A mixture of 2-hydrazino-5-methyl-benzimidazole (3, 1.0 g,
6.17 mmol) and ethyl 2-acetyl-3-phenylpropanoate (4, 1.4 mL, 6.59 mmol) in
acetic acid (20 mL) was stirred for 2 h at ambient temperatures. To a mixture of
acetonitrile (100 mL) and water (100 mL) was added the reaction mixture.
After stirring at ambient temperatures, the resulting precipitates were
collected by filtration and washed with acetonitrile in water (1:1). The
precipitates was purified by recrystallization from ethanol (95 mL) to
generate 3-methyl-1-(5-methyl-1H-benzimidazol-2-yl)-4-benzyl-1H-pyrazol-
5-ol (35, 0.64 g, 32.6%). The other compounds studied in this work (5–52) were
This research was supported in part by a Grant-in-Aid for Scien-
tific Research (C) provided by the Program for Promotion of Funda-
mental Sciences in Health Sciences of the National Institute of
Biomedical Innovation (NIBIO) and by a Grant-in-aid from the
Knowledge Cluster Initiative (Second Stage) of the Ministry of Edu-
cation, Culture, Sports, Science, and Technology of Japan.
prepared in
a similar manner. The typical experimental procedure for
synthesizing and evaluating the compounds used in this study, in addition to
the analytical data and the estimated purity (HPLC) of the biologically relevant
compounds, are provided in Supplemental data and Table S1, respectively.
9. Cadieux, J. A.; Zhang, Z.; Mattice, M.; Brownlie-Cutts, A.; Fu, J.; Ratkay, L. G.;
Kwan, R.; Thompson, J.; Sanghara, J.; Zhong, J.; Goldberg, Y. P. Bioorg. Med.
Chem. Lett. 2012, 22, 90. 5-hydroxy-3-methyl-1-pyridin-2-yl-1H-pyrazole-4-
carboxylic acid phenylamide, a representative compound in this work showed
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.01.008.
References and notes
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