Discovery of 2-methyl-1-{1-[(5-methyl-1H-indol-2-yl)carbonyl]piperidin-4- yl}propan-2-ol: A novel, potent and selective type 5 17β-hydroxysteroid dehydrogenase inhibitor

Article abstract of DOI:10.1016/j.bmc.2013.06.025

Type 5 17β-hydroxysteroid dehydrogenase (17β-HSD5), also known as aldo-keto reductase 1C3 (AKR1C3), is a member of the aldo-keto reductase superfamily of enzymes and is expressed in the human prostate. One of the main functions of 17β-HSD5 is to catalyze the conversion of the weak androgen, androstenedione, to the potent androgen, testosterone. The concentration of intraprostatic 5α-dihydrotestosterone (DHT) in patients following chemical or surgical castration has been reported to remain as high as 39% of that of healthy men, with 17β-HSD5 shown to be involved in this androgen synthesis. Inhibition of 17β-HSD5 therefore represents a promising target for the treatment of castration-resistant prostate cancer (CRPC). To investigate this, we conducted high-throughput screening (HTS) and identified compound 2, which displayed a structure distinct from known 17β-HSD5 inhibitors. To optimize the inhibitory activity of compound 2, we first introduced a primary alcohol group. We then converted the primary alcohol group to a tertiary alcohol, which further enhanced the inhibitory activity, improved metabolic stability, and led to the identification of compound 17. Oral administration of compound 17 to castrated nude mice bearing the CWR22R xenograft resulted in the suppression of androstenedione (AD)-induced intratumoral testosterone production. Compound 17 also demonstrated good isoform selectivity, minimal inhibitory activity against either CYP or hERG, and enhanced pharmacokinetic and physicochemical properties.

Full text of DOI:10.1016/j.bmc.2013.06.025

Accepted Manuscript
Discovery of 2-methyl-1-{1-[(5-methyl-1H-indol-2-yl)carbonyl]piperidin-4-
yl}propan-2-ol: A novel, potent and selective type 5 17β-hydroxysteroid dehy‐
drogenase inhibitor
Kazushi Watanabe, Akio Kakefuda, Minoru Yasuda, Kentaro Enjo, Aya
Kikuchi, Takashi Furutani, Yoichi Naritomi, Yukio Otsuka, Minoru Okada,
Mitsuaki Ohta
PII:
S0968-0896(13)00556-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.06.025
BMC 10918
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
7 May 2013
11 June 2013
11 June 2013
Please cite this article as: Watanabe, K., Kakefuda, A., Yasuda, M., Enjo, K., Kikuchi, A., Furutani, T., Naritomi,
Y., Otsuka, Y., Okada, M., Ohta, M., Discovery of 2-methyl-1-{1-[(5-methyl-1H-indol-2-yl)carbonyl]piperidin-4-
yl}propan-2-ol: A novel, potent and selective type 5 17β-hydroxysteroid dehydrogenase inhibitor, Bioorganic &
Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.06.025
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Discovery of 2-methyl-1-{1-[(5-methyl-1H-indol-2-yl)carbonyl]piperidin-4-yl}propan-2-ol: A novel,
potent and selective type 5 17β-hydroxysteroid dehydrogenase inhibitor
Kazushi Watanabe a,*, Akio Kakefuda b, Minoru Yasuda a, Kentaro Enjo a, Aya Kikuchi a, Takashi Furutani
c, Yoichi Naritomi a, Yukio Otsuka a, Minoru Okada d, and Mitsuaki Ohtaa
a Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
b Licensing & Alliances, Astellas Pharma Inc., 2-5-1, Nihonbashi-honcho, Chuo-ku, Tokyo 103-8411,
Japan
c
Product and Portfolio Strategy, Astellas Pharma Inc., 2-5-1, Nihonbashi-honcho, Chuo-ku, Tokyo
103-8411, Japan
d Technology, Astellas Pharma Inc., 160-2 Akahama, Takahagi, Ibaraki 318-0001, Japan
*Address correspondence regarding this manuscript to:
Kazushi Watanabe
Drug Discovery Research
Astellas Pharma Inc.
21 Miyukigaoka, Tsukuba
Ibaraki 305-8585, Japan
Tel: +81-(0)29-863-7023
Fax: +81-(0)29-854-1519
E-mail: kazushi.watanabe@astellas.com

Abstract
Type 5 17β-hydroxysteroid dehydrogenase (17β-HSD5), also known as aldo-keto reductase 1C3
(AKR1C3), is a member of the aldo-keto reductase superfamily of enzymes and is expressed in the
human prostate. One of the main functions of 17β-HSD5 is to catalyze the conversion of the weak
androgen, androstenedione, to the potent androgen, testosterone. The concentration of intraprostatic
5α-dihydrotestosterone (DHT) in patients following chemical or surgical castration has been reported to
remain as high as 39% of that of healthy men, with 17β-HSD5 shown to be involved in this androgen
synthesis. Inhibition of 17β-HSD5 therefore represents a promising target for the treatment of
castration-resistant prostate cancer (CRPC). To investigate this, we conducted high-throughput screening
(HTS) and identified compound 2, which displayed a structure distinct from known 17β-HSD5 inhibitors.
To optimize the inhibitory activity of compound 2, we first introduced a primary alcohol group. We then
converted the primary alcohol group to a tertiary alcohol, which further enhanced the inhibitory activity,
improved metabolic stability, and led to the identification of compound 17. Oral administration of
compound 17 to castrated nude mice bearing the CWR22R xenograft resulted in the suppression of
androstenedione (AD)-induced intratumoral testosterone production. Compound 17 also demonstrated
good isoform selectivity, minimal inhibitory activity against either CYP or hERG, and enhanced
pharmacokinetic and physicochemical properties.

Key words
Type 5 17β-hydroxysteroid dehydrogenase (17β-HSD5); Aldo-keto reductase 1C3 (AKR1C3);
Castration-resistant prostate cancer (CRPC); Testosterone

1. Introduction
Prostate cancer (PC) was the most frequently diagnosed form of cancer and the second-most frequent
cause of mortality reported in male patients between 2005-2009.1 The advancement of prostate cancer is
dependent on androgen, testosterone (T) and 5α-dihydrotestosterone (DHT). Therefore, hormone therapy
such as surgical castration or administration of gonadotropin releasing hormone (GnRH) agonist is major
therapeutic method. However, prostate cancer can develop into castration-resistant prostate cancer
(CRPC), and the effect of hormone therapy declines progressively over time, positioning CRPC as the
most significant obstacle at present in the effective treatment of prostate cancer. In this regard, the
inhibition of the androgen biosynthesis route in the adrenal and prostate glands represents a potential
therapeutic target.
Abiraterone acetate,2 a 17α-hydroxylase/C17,20-lyase inhibitor, has been approved for the treatment of
CRPC in the US since April 2011. Abiraterone acetate works via the inhibition of dehydroepiandrosterone
(DHEA) production, a precursor of testosterone, in the adrenal gland.3 This approach has proven that
inhibition of the androgen biosynthesis route involved with the adrenal gland is effective against CRPC.
However, as abiraterone acetate is involved in biosynthesis of several steroids, such as
17OH-pregnenolone, and also acts as an inhibitor of upstream androgen biosynthesis,4 this treatment
could potentially cause side effects, such as increasing levels of mineralocorticoid, which requires
concomitant use of prednisone (Figure 1).

Type 5 17β-hydroxysteroid dehydrogenase (17β-HSD5), also known as aldo-keto reductase 1C3
(AKR1C3), is one of the enzymes involved in androgen biosynthesis.5 17β-HSD5 is expressed in the
human prostate, with elevated levels of expression reported in prostate cancer.6 17β-HSD5 plays an
essential role in producing testosterone from DHEA secreted from the adrenal gland and in transforming
androstenedione (AD), a weak androgen, to testosterone, a potent androgen (Figure 1).5 As 17β-HSD5 is
involved in downstream androgen biosynthesis, compared to 17α-hydroxylase/C17,20-lyase, inhibition of
17β-HSD5 is expected to avoid putative side effects of abiraterone acetate, bypassing the need for
combination therapy with prednisone. From this point of view, the inhibition of 17β-HSD5 is an attractive
target for the treatment of CRPC, with 17β-HSD5 inhibitors having the potential to be effective and safe
therapeutics for the treatment of CRPC.
Surgical or chemical castration has been reported to reduce serum testosterone levels by 97%.7 In
contrast, the concentration of intraprostatic DHT is only reduced by 61%, compared to healthy men.
Since DHT is produced by 5α-reductase from testosterone in the prostate, testosterone should exist in the
prostate despite incorporation of testosterone from blood scarcely contributes to intraprostatic testosterone
concentration after castration. This is because testosterone can be synthesized through two steps from
DHEA which is secreted from the adrenal gland and incorporated into the prostate, and 17β-HSD5 is
involved in one of the two steps. These facts suggest that 17β-HSD5 would have about half of
contribution to intraprostatic testosterone concentration and therefore would have a large impact on
prostate cancer growth.

Two closely related isoforms of 17β-HSD5 have been identified that play a role in steroid
biosynthesis: AKR1C1 and AKR1C2.8 As both isoforms are involved in DHT metabolism, their inhibition
may lead to the accumulation of DHT in the prostate and thereby induce the development or progression
of prostate cancer.9,10 Therefore, 17β-HSD5 inhibitors with no inhibitory activity against both AKR1C1
and AKR1C2 are desirable for the treatment of prostate cancer. We therefore conducted extensive
research to identify novel, potent, isoform-selective, orally bioavailable, and nonsteroidal 17β-HSD5
inhibitors.
Previous reports have demonstrated that nonsteroidal anti-inflammatory drugs (NSAIDs), such as
fulfenamic acid (1), exert inhibitory activity against 17β-HSD5 (Figure 2).11 In addition, some NSAIDs
derivatives have also been reported to show selectivity for 17β-HSD5 versus COX-1,2, as well as AKR
isoforms in their inhibitory activity.12,13
Here, to identify a compound that inhibits 17β-HSD5, we conducted high-throughput screening
(HTS) using a 17β-HSD5 enzyme assay. We describe the discovery of novel structural class of 17β-HSD5
inhibitors.
2. Chemistry
Compounds 3-18a were synthesized, except where noted, by an amidation reaction between
indole-2-carboxylic acid 19 or 20 and the corresponding amine (Scheme 1). Compounds 8 and 10 were
synthesized by an amidation reaction between, indole-2-carboxylic acid 19 and the corresponding amine,

followed by reduction of the ester moiety with LiBH4 (Scheme 2). Compound 11, an azetidine derivative,
was synthesized by first reacting tert-butyl 3-(2-ethoxy-2-oxoethylidene)azetidine-1-carboxylate14 (11a)
under an acidic condition to remove the tert-butoxycarbonyl (Boc)-group and then conducting an
amidation reaction with indole-2-carboxylic acid 19 to generate ester 11b, which was reduced with LiBH4,
and hydrogenated (Scheme 3). Compound 15, a secondary alcohol derivative, was synthesized by first
reacting aldehyde 15a with methyl magnesium bromide to generate secondary alcohol 15b. The
Boc-group on secondary alcohol 15b was then deprotected and subjected to an amidation reaction with
indole-2-carboxylic acid 19 (Scheme 4). Compound 18, a carboxylic acid derivative, was synthesized by
hydrolyzing ester 18a with sodium hydroxide (Scheme 5). Amine 23 was synthesized by first reacting
benzyl 4-(2-ethoxy-2-oxoethyl)piperidine-1-carboxylate15 (21) with methyl magnesium bromide to
generate tertiary alcohol 22, followed by deprotecting the benzyloxycarbonyl group on tertiary alcohol 22
by hydrogenation (Scheme 6). Amine 26 was synthesized by first reacting ketone 24 with ethyl
(diethoxyphosphoryl) acetate and then hydrogenated to generate the ester 25, followed by deprotecting
the Boc-group on ester 25 by hydrochloric acid (Scheme 6).
3. Results and Discussion
For the evaluation of the synthesized compounds, we conducted a cell assay that detects testosterone
converted from androstenedione in 17β-HSD5 expressing HEK293 cells.16 Using HTS approach based on
a 17β-HSD5 enzyme assay for in-house chemical library, we identified compound 2 (Figure 3), which

exhibited both inhibitory activity against 17β-HSD5, and a unique structure from known 17β-HSD5
inhibitors. We evaluated the inhibitory activity of compound 2 against 17β-HSD5 in a HEK293 cell assay
and observed an IC50 value of 2800 nM. Based on structure-activity relationship (SAR) data obtained
from HTS results and subsequent our preliminary SAR study data, we introduced a primary alcohol group
to compound 2, which generated compound 3. We then evaluated the inhibitory activity of compound 3,
against 17β-HSD5, and noted an IC50 value of 37 nM. The potent inhibitory activity and the structural
novelty of compound 3 prompted us to carry out further structural modifications, in order to obtain
compounds with an enhanced pharmacological profile.
We first modified the carbon chain length of the 2-hydroxyethyl moiety attached to the piperidine
ring in compound 3 (Table 1), observing a significant reduction in inhibitory activity on either shortening
or lengthening the carbon chain of compound 3, which generated compounds 4 and 5, respectively. This
phenomenon suggests that carbon chain length may represent an important factor in exerting potent
inhibitory activity. Substitution of the piperidine ring of compound 3 with a piperazine ring, which
generated compound 6, also resulted in a considerable decrease in inhibitory activity (IC50 = 770 nM), as
did insertion of a basic nitrogen atom on the ring.
The removal of the methyl group on the indole ring of compound 3 yielded compound 7 and brought
about an 8-fold improvement of the inhibitory activity with an IC50 value of 4.5 nM. We therefore
conducted further modifications of the 4-(2-hydroxyethyl) piperidine moiety based on compound 7.
Compounds 8 and 9, with 2-hydroxyethyl groups on the 3- or 2-position of the piperidine, respectively,

showed considerably weaker inhibitory activities than compound 7 (respective IC50 = 2100 and 230 nM).
The pyrrolidine analogue 10 and azetidine analogue 11 also showed significant reductions in inhibitory
activity compared with compound 7 (respective IC50 = 1100 and 140 nM). Taken together, these results
indicate that modifying the position at which the 2-hydroxyethyl group is attached or reducing the ring
size of the piperidine moiety leads to a significant attenuation of inhibitory activity. Further, compound
1217, a linear alcohol derivative with the same number of carbon atoms between the amide moiety and the
hydroxyl group as compound 7, showed weaker inhibitory activity than compound 7 (IC50 = 1200 nM),
suggesting that a sterically-restricted structure, such as a piperidine ring, might be essential for the
exertion of potent inhibitory activity.
As
our
modification
of
the
4-(2-hydroxyethyl)piperidine
moiety
showed,
the
4-(2-hydroxyethyl)piperidine structure is optimal for exerting inhibitory activity. We therefore conducted
further modifications on the terminal hydroxyl moiety in an attempt to improve its metabolic stability, as
primary alcohols are known to be vulnerable to conjugative or oxidative metabolism (Table 2).
Compound 13, a methyl ether, showed relatively good inhibitory activity (IC50 = 33 nM), although the
potency was less than that of compound 7, possibly due to the proton of the hydroxyl moiety of 7 not
acting as a hydrogen bond donor but rather as an acceptor when binding to the enzyme. Compound 14, an
acetyl derivative, showed rather potent inhibitory activity (IC50 = 13 nM), which seems to support our
expectation that the existence of a hydrogen bond acceptor is preferable in attempting to achieve potent
inhibitory activity. While compound 15, a secondary alcohol derivative, displayed even more potent

inhibitory activity (IC50 = 12 nM), compound 16, a tertiary alcohol derivative, showed 2-fold more potent
inhibitory activity than compound 7 (IC50 = 2.1 nM). Similarly, compound 17, the 5-methyl indole
analogue of compound 16, exerted 13-fold more potent inhibitory activity than compound 7 (IC50 = 0.34
nM). In contrast, Compound 18, a carboxylic acid derivative, showed little inhibitory activity.
Both compounds 3 and 17 were tested for their inhibitory activity against testosterone conversion
from androstenedione using the human PC cell line CWR22R, which highly expresses 17β-HSD5 (Table
3). Compound 17 showed more potent inhibitory activity (IC50 = 1.9 nM) than compound 3 (IC50 = 58
nM), which is consistent with the results from the HEK293 cell assay.
Since compounds 3, 16, and 17 showed good membrane permeability and good solubility, the plasma
concentrations were evaluated after the oral administration of 30 mg/kg of each compound to mice (Table
3). Compound 3 was not detected in the plasma 1 h post-administration; however, the corresponding
carboxylic acid derivative 18 was detected. As we expected, compounds 16 and 17, which are both
tertiary alcohol derivatives, were detected in the plasma at 1 h post-administration. Further the plasma
concentration of compound 17 was 4-fold higher than that of compound 16 at 1 h post-administration and
was detected in the plasma as late as 4 h post-administration, indicating that metabolic stability of
compound 17 is improved by the presence of a methyl group at the 5-position of the indole ring.
Examination of the pharmacokinetic properties of compound 17 in rats showed the compound to
have good oral bioavailability (F = 65%) at a dose of 1 mg/kg in rats (Table 4). The half-life of compound
17 was 0.46 h after intravenous administration. The maximum drug concentration was observed 0.5 h

after oral administration, demonstrating that compound 17 was promptly absorbed from the intestine and
transported into the blood.
The inhibitory effect of compound 17 on AD-induced intratumoral testosterone production in prostate
cancer was evaluated in castrated nude mice bearing the CWR22R xenograft (Figure 4).18 Compound 17
orally administered at 1, 3, and 10 mg/kg inhibited testosterone production in a dose-dependent manner.
In addition, compound 17 exerted a complete suppression of testosterone production when orally
administered at 10 mg/kg.
Evaluation of the AKR1C2 inhibitory activity of compound 17 showed up to 800-fold selectivity
against AKR1C2 (Table 5). Since AKR1C1 and AKR1C2 differ by only a single amino acid at their
respective active sites, an inhibitor of AKR1C2 could potentially also act to inhibit AKR1C1.11,12
Compound 17 was also found to be clean with regard to various CYP enzyme inhibitory activities up to
25 μM and demonstrated minimal hERG inhibition up to 100 μM (Table 5).
4. Conclusion
In summary, we conducted SAR studies to identify novel, potent, selective, and orally bioavailable
17β-HSD5 inhibitors. We started our SAR studies with compound 2, an indole derivative discovered
using HTS, and successfully enhanced its inhibitory activity against 17β-HSD5 by introducing a hydroxyl
group. Further examination led to an improvement in both inhibitory activity and metabolic stability by
substitution with a tertiary alcohol, generating compound 17, which displayed potent inhibitory activity in

the PC cell line CWR22R and good isoform selectivity, and suppressed AD-induced intratumoral
testosterone production when orally administered to castrated nude mice bearing the CWR22R xenograft.
Further, compound 17 demonstrated minimal inhibitory activity against either CYP or hERG and
therefore is expected not to have a high risk of drug-drug interactions or unfavorable cardiovascular
effects. In addition, the pharmacokinetic properties and the physicochemical properties of compound 17
were preferable. The results of further studies on this compound will be reported in due course.

5. Experimental
Chemistry
1H NMR spectra were recorded on a JEOL JMN-EX-400, and the chemical shifts were expressed in
δ (ppm) values with trimethylsilane as an internal reference (s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, and br = broad peak). Mass spectra (MS) were recorded on a JEOL LX-200 or
Thermo Electron LQC Advantage or Thermo Electron TRACE DSQ spectrometer. Elemental analyses
were performed with Yanaco MT-6 (C, H, N) or Elementar Vario EL III (C, H, N) instruments, and results
were within 0.4% of theoretical values.
2-{1-[(5-Methyl-1H-indol-2-yl)carbonyl]piperidin-4-yl}ethanol (3)
To a solution of 5-methyl-1H-indole-2-carboxylic acid (20) (263 mg, 1.50 mmol) in THF (7 mL)
were added 2-(piperidin-4-yl)ethanol (213 mg, 1.65 mmol), 1-hydroxybenzotriazole (HOBt, 101 mg,
0.750 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) hydrochloride (316 mg, 1.65
mmol), followed by stirring at room temperature for 14 h. The reaction mixture was partitioned between
ethyl acetate and 0.5 M aqueous hydrochloric acid. The organic layer was washed with saturated aqueous
sodium hydrogen carbonate solution and saturated aqueous sodium chloride solution, dried over
anhydrous sodium sulfate, and then concentrated in vacuo. The residue was recrystallized from ethyl
acetate/acetonitrile (5 mL/2 mL) to give 3 (365 mg, 85.0%) as a beige powder. 1H NMR-DMSO-d6 (400
MHz) δ 1.11-1.19 (2H, m), 1.38-1.43 (2H, m), 1.73-1.76 (3H, m), 2.36 (3H, s), 2.98 (2H, s), 3.45-3.49
(2H, m), 4.37-4.44 (3H, m), 6.63 (1H, m), 7.00 (1H, m), 7.29 (1H, m), 7.36 (1H, s), 11.38 (1H, s). EI-MS:
m/z 286 [M]+. Anal. Calcd for C17H22N2O2: C, 71.30; H, 7.74; N, 9.78. Found: C, 71.10, H, 7.87; N, 9.67.

1-[(5-Methyl-1H-indol-2-yl)carbonyl]piperidin-4-ylmethanol (4)
Compound 4 was prepared in 73% yield from piperidin-4-ylmethanol using a similar procedure to
that of 3. 1H NMR-DMSO-d6 (400 MHz) δ 1.08-1.20 (2H, m), 1.65-1.78 (3H, m), 2.36 (3H, s), 2.99 (2H,
m), 3.29 (2H, m), 4.45 (2H, m), 4.52 (1H, m), 6.63 (1H, s), 7.00 (1H, d, J = 8.4 Hz), 7.29 (1H, d, J = 8.4
Hz), 7.36 (1H, s), 11.39 (1H, s). EI-MS: m/z 272 [M]+. Anal. Calcd for C16H20N2O2: C, 70.56; H, 7.40; N,
10.29. Found: C, 70.42; H, 7.41; N, 10.19.
3-{1-[(5-Methyl-1H-indol-2-yl)carbonyl]piperidin-4-yl}propanol (5)
Compound 5 was prepared in 73% yield from 3-(piperidin-4-yl)propan-1-ol using a similar procedure
to that of 3. 1H NMR-DMSO-d6 (400 MHz) δ 1.03-1.16 (2H, m), 1.22-1.29 (2H, m), 1.41-1.49 (2H, m),
1.54 (1H, m), 1.75 (2H, m), 2.36 (3H, s), 2.98 (2H, m), 3.39 (2H, m), 4.38 (1H, m), 4.43 (2H, m), 6.63
(1H, m), 7.00 (1H, m), 7.29 (1H, m), 7.36 (1H, s), 11.39 (1H, s). FAB-MS: m/z 301 [M+H]+. Anal. Calcd
for C18H24N2O2: C, 71.97; H, 8.05; N, 9.33. Found: C, 71.78; H, 8.08; N, 9.35.
2-{1-[(5-Methyl-1H-indol-2-yl)carbonyl]piperazin-4-yl}ethanol (6)
Compound 6 was prepared in 47% yield from 2-(piperazin-1-yl)ethanol using a similar procedure to
that of 3. 1H NMR-DMSO-d6 (400 MHz) δ 2.36 (3H, s), 2.43 (2H, m), 2.46-2.51 (4H, m), 3.53 (2H, m),
3.73 (4H, m), 4.45 (1H, m), 6.67 (1H, m), 7.01 (1H, m), 7.30 (1H, m), 7.36 (1H, s), 11.42 (1H, s).
FAB-MS: m/z 288 [M+H]+. Anal. Calcd for C16H21N3O2: C, 66.88; H, 7.37; N, 14.62. Found: C, 66.75;
7.47; N, 14.87.
2-[1-(1H-Indol-2-ylcarbonyl)piperidin-4-yl]ethanol (7)
Compound 7 was prepared in 85% yield from 1H-indole-2-carboxylic acid (19) using a similar
procedure to that of 3. 1H NMR-DMSO-d6 (400 MHz) δ 1.07-1.20 (2H, m), 1.41 (2H, m), 1.67-1.79 (3H,
m), 2.99 (2H, m), 3.47 (2H, m), 4.39 (1H, m), 4.43 (2H, m), 6.73 (1H, m), 7.03 (1H, m), 7.17 (1H, m),

7.41 (1H, m), 7.59 (1H, m), 11.52 (1H, s). EI-MS: m/z 272 [M]+. Anal. Calcd for C16H20N2O2: C, 70.56;
H, 7.40; N, 10.29. Found: C, 70.54; H, 7.48; H, 10.28.
2-[1-(1H-Indol-2-ylcarbonyl)piperidin-3-yl]ethanol (8)
To a solution of 1H-indole-2-carboxylic acid (19) (500 mg, 3.10 mmol) and ethyl
piperidin-3-ylacetate
(530
mg,
3.10
mmol)
in
DMF
(8
mL)
were
added
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) hydrochloride (610 mg, 3.18 mmol) and
1-hydroxybenzotriazole (HOBt, 430 mg, 3.18 mmol), followed by stirring at room temperature for 2 h.
0.5 M aqueous hydrochloric acid was added, followed by extraction with ethyl acetate. The organic layer
was washed with 0.5 M aqueous sodium hydroxide solution and saturated aqueous sodium chloride
solution, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to give ethyl
[1-(1H-indol-2-ylcarbonyl)piperidin-3-yl]acetate (519 mg, 53%). To a solution of the resulting ethyl
[1-(1H-indol-2-ylcarbonyl)piperidin-3-yl]acetate (392 mg, 1.25 mmol) in THF (7 mL) was added lithium
borohydride (30 mg, 1.38 mmol), followed by stirring at room temperature for 24 h. Water was added to
the reaction liquid, followed by extraction with ethyl acetate. The organic layer was washed with
saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and then concentrated
under reduced pressure. The resulting residue was purified by silica gel column chromatography
[hexane:ethyl acetate = 1:0-2:1], and then crystallized from diisopropyl ether to give 8 (92 mg, 27%) as a
1
white solid. H NMR-DMSO-d6 (400 MHz) δ 1.24 (1H, m), 1.32-1.52 (3H, m), 1.63-1.76 (2H, m), 1.87
(1H, m), 2.70-3.21 (2H, m), 3.46 (2H, m), 4.31 (2H, m), 4.43 (1H, m), 6.75 (1H, s), 7.03 (1H, dd, J = 7.6,

7.6 Hz), 7.17 (1H, dd, J = 7.6, 7.6 Hz), 7.41 (1H, d, J = 7.6 Hz), 7.60 (1H, d, J = 7.6 Hz), 11.52 (1H, s).
FAB-MS: m/z 273 [M+H]+. Anal. Calcd for C16H20N2O2: C, 70.56; H, 7.40; N, 10.29. Found: C, 70.54; H,
7.33; N, 10.14.
2-[1-(1H-Indol-2-ylcarbonyl)piperidin-2-yl]ethanol (9)
Compound 9 was prepared in 19% yield from 1H-indole-2-carboxylic acid (19) and
2-(piperidin-2-yl)ethanol using a similar procedure to that of 3. 1H NMR-DMSO-d6 (400 MHz) δ 1.44
(1H, m), 1.55-1.84 (6H, m), 1.96 (1H, m), 3.07 (1H, m), 3.43 (2H, m), 4.30 (1H, m), 4.51 (1H, m), 4.78
(1H, m), 6.79 (1H, s), 7.03 (1H, dd, J = 7.6, 7.6 Hz), 7.16 (1H, dd, J = 7.6, 7.6 Hz), 7.40 (1H, d, J = 7.6
Hz), 7.58 (1H, d, J = 7.6 Hz), 11.52 (1H, s). FAB-MS: m/z 273 [M+H]+. Anal. Calcd for C16H20N2O2: C,
70.56; H, 7.40; N, 10.29. Found: C, 70.59; H, 7.37 ; N, 10.14.
2-[1-(1H-Indol-2-ylcarbonyl)pyrrolidin-3-yl]ethanol (10)
Compound 10 was prepared in 43% yield from ethyl pyrrolidin-3-ylacetate (26) using a similar
procedure to that of 8. 1H NMR-DMSO-d6 (400 MHz) δ 1.47-1.71 (3H, m), 1.99-2.39 (2H, m), 3.08-4.07
(6H, m), 4.50 (1H, s), 6.95 (1H, s), 7.03 (1H, m), 7.18 (1H, m), 7.44 (1H, m), 7,63 (1H, m), 11.51 (1H, s).
FAB-MS: m/z 259 [M+H]+. Anal. Calcd for C15H18N2O2.0.1H2O: C, 69.26; H, 7.05; N, 10.77. Found: C,
69.13; 6.92; N, 10.67.
Ethyl [1-(1H-indol-2-ylcarbonyl)azetidin-3-ylidene]acetate (11b)
To a solution of tert-butyl 3-(2-ethoxy-2-oxoethylidene)azetidine-1-carboxylate14 (11a, 237 mg, 0.98
mmol) in CHCl3 (4 mL) was added trifluoroacetic acid (0.1 mL, 1.30 mmol), followed by stirring at room
temperature for 1 h. 4 M hydrochloric acid/ethyl acetate (2 mL, 8.00 mmol) was added to the mixture,
and the mixture was stirred at room temperature for 2 h. The mixture was concentrated under reduced

pressure to give the residue. To a solution of the resulting residue in DMF (5 mL) was added
1H-indole-2-carboxylic acid (19, 180 mg, 1.12 mmol), 1-hydroxybenzotriazole (HOBt, 160 mg, 1.18
mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) hydrochloride (230 mg, 1.20 mmol),
and triethylamine (410 uL, 2.94 mmol) at 0 °C, followed by stirring at room temperature for 2 h. The
reaction mixture was partitioned between ethyl acetate and 0.5 M aqueous hydrochloric acid. The organic
layer was washed with 1 M aqueous sodium hydroxide solution and saturated aqueous sodium chloride
solution, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to give 11b
(96 mg, 34%) as a colorless oil. 1H NMR-DMSO-d6 (400 MHz) δ 1.24 (3H, m), 4.15 (2H, m), 4.89 (2H,
m), 5.33 (2H, m), 5.98 (1H, s), 6.89 (1H, m), 7.06 (1H, m), 7.21 (1H, m), 7.45 (1H, m), 7.64 (1H, m),
11.67 (1H, s). ESI-MS: m/z 285 [M+H]+.
2-[1-(1H-Indol-2-ylcarbonyl)azetidin-3-yl]ethanol (11)
To a solution of ethyl [1-(1H-indol-2-ylcarbonyl)azetidin-3-ylidene]acetate (11b, 94 mg, 0.33 mmol)
in THF (2 mL) was added lithium borohydride (20 mg, 0.92 mmol), followed by stirring at room
temperature for 24 h. Water was added to the reaction liquid, followed by extraction with ethyl acetate.
The organic layer was washed with saturated aqueous sodium chloride solution, dried over anhydrous
sodium sulfate, and then concentrated under reduced pressure to give the residue. To a solution of the
residue in methanol (2 mL) was added 10% palladium on carbon (wet, contains 50% water, 10 mg),
followed by stirring under a hydrogen atmosphere at room temperature for 24 h. Insoluble material was
removed by filtration through Celite, and the filtrate was concentrated under reduced pressure. The
residue was purified by silica gel column chromatography [chloroform:methanol = 1:0-10:1], and then
solidified from diisopropyl ether to give 11 (25 mg, 31%) as a white powder. 1H NMR-DMSO-d6 (400
MHz) δ 1.78 (2H, m), 2.80 (1H, m), 3.45 (2H, m), 3.74 (1H, m), 4.17 (2H, m), 4.48 (1H, m), 4.60 (1H,
m), 6.79 (1H, m), 7.04 (1H, m), 7.19 (1H, m), 7.43 (1H, m), 7.61 (1H, m), 11.55 (1H, s). ESI-MS: m/z

245 [M+H]+. Anal. Calcd for C14H16N2O2.0.1H2O: C, 68.33; H, 6.64; N, 11.38. Found: C, 68.28; H, 6.60 ;
N, 11.27.
2-[1-(1H-Indol-2-ylcarbonyl)piperidin-4-yl]-1-methoxyethane (13)
Compound 13 was prepared in 5% yield from 1H-indole-2-carboxylic acid (19) and
4-(2-methoxyethyl)piperidine using a similar procedure to that of 3. 1H NMR-DMSO-d6 (400 MHz) δ
1.15 (2H, m), 1.48 (2H, m), 1.64-1.80 (3H, m), 2.99 (2H, m), 3.23 (3H, s), 3.38 (2H, m), 4.43 (2H, m),
6.73 (1H, m), 7.03 (1H, m), 7.17 (1H, m), 7.40 (1H, m), 7.60 (1H, m), 11.52 (1H, s). FAB-MS: m/z 287
[M+H]+. Anal. Calcd for C17H22N2O2.0.1H2O: C, 70.85; H, 7.76; N, 9.72. Found: C, 70.77; H, 7.65; N,
9.63.
1-[1-(1H-Indol-2-ylcarbonyl)piperidin-4-yl]acetone (14)
Compound 14 was prepared in 47% yield from 1H-indole-2-carboxylic acid (19) and
1-(piperidin-4-yl)acetone19 using a similar procedure to that of 3. 1H NMR-DMSO-d6 (400 MHz) δ 1.15
(2H, m), 1.71 (2H, m), 2.01-2.12 (4H, m), 2.43 (2H, m), 3.03 (2H, m), 4.40 (2H, m), 6.73 (1H, m), 7.03
(1H, m), 7.17 (1H, m), 7.41 (1H, m), 7.59 (1H, m), 11.52 (1H, s). FAB-MS: m/z 285 [M+H]+. Anal.
Calcd for C17H20N2O2.0.1H2O: C, 71.35; H, 7.12; N, 9.79. Found: C, 71.20; H, 7.10; N, 9.80.
tert-Butyl 4-(2-hydroxypropyl)piperidine-1-carboxylate (15b)
To a solution of tert-butyl 4-(2-oxoethyl)piperidine-1-carboxylate (15a, 250 mg, 1.10 mmol) in THF
(5 mL) was added 1.4 M methyl magnesium bromide in toluene-THF (1.2 mL, 1.68 mmol), followed by
stirring at room temperature for 2 h. Water was added to the reaction liquid, followed by extraction with
ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride solution, dried over
anhydrous sodium sulfate, and then concentrated under reduced pressure. The residue was purified by
silica gel column chromatography [chloroform:methanol = 1:0-10:1] to give 15b (236 mg, 88%) as a

colorless oily substance. 1H NMR-CDCl3 (400 MHz) δ 0.99-1.78 (20H, m), 2.70 (2H, m), 3.93 (1H, m),
4.08 (2H, m). FAB-MS: m/z 244 [M+H]+.
1-[1-(1H-Indol-2-ylcarbonyl)piperidin-4-yl]propan-2-ol (15)
To a solution of 15b (233 mg, 0.96 mmol) in ethyl acetate (4 mL) was added 4 M hydrochloric
acid/ethyl acetate (3 mL, 12.0 mmol), followed by stirring at room temperature for 2 h. The reaction
liquid was concentrated under reduced pressure to give 4-(2-hydroxypropyl)piperidine hydrochloride. To
a solution of the 4-(2-hydroxypropyl)piperidine hydrochloride and 1H-indole-2-carboxylic acid (19, 155
mg, 0.96 mmol) in DMF (5 mL) were added triethylamine (0.15 ml, 1.08 mmol),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) hydrochloride (190 mg, 0.99 mmol) and
1-hydroxybenzotriazole (HOBt, 130 mg, 0.96 mmol), followed by stirring at room temperature for 24 h.
0.5 M aqueous hydrochloric acid was added to the reaction liquid, followed by extraction with ethyl
acetate. The organic layer was washed with 0.5 M aqueous sodium hydroxide solution and saturated
aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and then concentrated under
reduced pressure. The residue was purified by silica gel column chromatography [chloroform:methanol =
1
1:0-10:1], and then solidified from diisopropyl ether to give 15 (49 mg, 18%) as a white solid. H
NMR-DMSO-d6 (400 MHz) δ 1.02-1.24 (6H, m), 1.34 (1H, m), 1.69-1.83 (3H, m), 2.81-3.18 (2H, m),
3.72 (1H, m), 4.36 (1H, d, J = 4.8 Hz), 4.42 (2H, m), 6.73 (1H, m), 7.03 (1H, m), 7.17 (1H, m), 7.40 (1H,
m), 7.59 (1H, m), 11.52 (1H, s). FAB-MS; m/z 287 [M+H]+. Anal. Calcd for C17H22N2O2: C, 71.30; H,
7.74; N, 9.78. Found: C, 71.10; H, 7.76; N, 9.71.

1-[1-(1H-Indol-2-ylcarbonyl)piperidin-4-yl]-2-methyl-propan-2-ol (16)
Compound 16 was prepared in 3% yield from 1H-indole-2-carboxylic acid (19) and
2-methyl-1-(piperidin-4-yl)-2-propanol (23) using a similar procedure to that of 3. 1H NMR-DMSO-d6
(400 MHz) δ 1.11-1.21 (8H, m), 1.34 (2H, d, J = 5.6 Hz), 1.76-1.88 (3H, m), 3.03 (2H, m), 4.11 (1H, s),
4.37 (2H, m), 6.72 (1H, m), 7.03 (1H, m), 7.17 (1H, m), 7.40 (1H, m), 7.59 (1H, m), 11.51 (1H, s).
FAB-MS: m/z 301 [M+H]+. Anal. Calcd for C18H24N2O2: C, 71.97; H, 8.05; N, 9.33. Found: C, 71.83; H,
8.08; N, 9.23.
2-Methyl-1-{1-[(5-methyl-1H-indol-2-yl)carbonyl]piperidin-4-yl}propan-2-ol (17)
Compound 17 was prepared in 71% yield from 2-methyl-1-(piperidin-4-yl)-2-propanol (23) using a
similar procedure to that of 3. 1H NMR-DMSO-d6 (400 MHz) δ 1.08-1.21 (8H, m), 1.33 (2H, d, J = 5.6
Hz), 1.75-1.88 (3H, m), 2.36 (3H, s), 3.01 (2H, m), 4.11 (1H, s), 4.37 (2H, m), 6.62 (1H, m), 6.99 (1H, m),
7.29 (1H, m), 7.36 (1H, m), 11.37 (1H, s). FAB-MS: m/z 315 [M+H]+. Anal. Calcd for C19H26N2O2: C,
72.58; H, 8.33; N, 8.91. Found: C, 72.46; H, 8.23; N, 8.81.
Ethyl {1-[(5-methyl-1H-indol-2-yl)carbonyl]piperidin-4-yl}acetate (18a)
Compound 18a was prepared in 94% yield from ethyl piperidin-4-ylacetate using a similar procedure
to that of 3. 1H NMR-DMSO-d6 (400 MHz) δ 1.12-1.28 (5H, m), 1.75 (2H, m), 2.02 (1H, m), 2.29 (2H,
m), 2.36 (3H, s), 3.02 (2H, m), 4.07 (2H, m), 4.42 (2H, m), 6.64 (1H, m), 7.00 (1H, m), 7.29 (1H, m),
7.36 (1H, m), 11.40 (1H, s). FAB-MS: m/z 329 [M+H]+.
{1-[(5-Methyl-1H-indol-2-yl)carbonyl]piperidin-4-yl}acetic acid (18)
To a solution of 18a (600 mg, 1.83 mmol) in ethanol/THF (6 mL/3 mL) was added 1 M aqueous

sodium hydroxide solution (2.74 mL, 2.74 mmol), followed by stirring at room teperature for 14 h. The
reaction mixture was concentrated in vacuo. To the residue was added 1 M aqueous hydrochloric acid
(2.74 mL) and water, followed by stirring at 0 °C for 10 min. The resulting precipitate was filtered and
washed with water to give 18 as a beige powder. The suspension of 18 (251 mg, 0.836 mmol) in
acetonitrile /water/ethanol (1:1:1, 12 mL) was stirred under reflux for 10 min, then at room temperature
for 1 h. The precipitate was filtered and washed with EtOH to give 18 (173 mg, 69%) as a beige powder.
1H NMR-DMSO-d6 (400 MHz) δ 1.19 (2H, m), 1.77 (2H, m), 1.99 (1H, m), 2.21 (2H, m), 2.36 (3H, s),
3.02 (2H, m), 4.42 (2H, m), 6.64 (1H, m), 7.00 (1H, m), 7.29 (1H, m), 7.36 (1H, m), 11.39 (1H, s), 12.13
(1H, s). ESI-MS: m/z 301 [M+H]+. Anal. Calcd for C17H20N2O3: C, 67.98; H, 6.71; N,9.33. Found: C,
67.83; H, 6.83; N, 9.31.
Benzyl 4-(2-hydroxy-2-methylpropyl)piperidine-1-carboxylate (22)
To a solution of benzyl 4-(2-ethoxy-2-oxoethyl)piperidine-1-carboxylate15 (21, 8.9 g, 29.145 mmol)
in THF (100 mL) was added 1.4 M methyl magnesium bromide in toluene-THF (46 mL, 64.4 mmol) at
0 °C, followed by stirring at room temperature for 3 h. 1 M aqueous ammonium chloride solution was
added to the reaction liquid, followed by extraction with ethyl acetate. The aqueous layer was extracted
with ethyl acetate. The combined organic layers were washed with saturated aqueous sodium chloride
solution, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to give 22
(8.5 g, 100%) as a colorless oily substance. 1H NMR-CDCl3 (400 MHz) δ 1.11-1.31 (7H, m), 1.35-1.58
(4H, m), 1.66 (1H, m), 1.78 (2H, m), 2.81 (2H, m), 4.13 (2H, m), 5.12 (2H, s), 7.27-7.43 (5H, m). CI-MS:
m/z 292 [M+H]+.

2-Methyl-1-(piperidin-4-yl)-propan-2-ol (23)
To a solution of 22 (8.5 g 29.171 mmol) in methanol (120 mL) was added 10% palladium on carbon
(wet, contains 50% water, 500 mg), followed by stirring under a hydrogen atmosphere at room
temperature for 24 h. Insoluble material was removed by filtration through Celite, and the filtrate was
1
concentrated under reduced pressure to give 23 (5.6 g, quantitative) as a white solid. H NMR-CDCl3
(400 MHz) δ 1.15-1.31 (8H, m), 1.42 (2H, m), 1.61 (1H, m), 1.78 (2H, m), 2.02 (1H, m), 2.64 (2H, m),
3.06 (2H, m), 3.48 (1H, s). EI-MS: m/z 157 [M]+.
tert-Butyl 3-(2-ethoxy-2-oxoethyl)pyrrolidine-1-carboxylate (25)
To a suspension of sodium hydride (60%, 420 mg, 10.5 mmol) in DMF (15 mL) was added dropwise
a solution of ethyl (diethoxyphosphoryl)acetate (2470 mg, 11.0 mmol) in DMF (5 mL) at 0 °C, and the
mixture was stirred at room temperature for 0.5 h. To the reaction mixture was added dropwise a solution
of tert-butyl 3-oxopyrrolidine-1-carboxylate (24, 1850 mg, 10.0 mmol) in DMF (5 mL) at 0 °C, and the
mixture was stirred at room temperature for 1 h. To the reaction mixture was added 1 M aqueous
ammonium chloride solution at 0 °C, and the mixture was partitioned between ethyl acetate and water.
The organic layer was washed with saturated aqueous sodium chloride solution, dried over anhydrous
sodium sulfate, and then concentrated under reduced pressure to give an orange oil. To a solution of the
oil obtained above in ethanol (30 mL) was added 10% palladium on carbon (wet, contains 50% water,
255 mg), followed by stirring under a hydrogen atmosphere at room temperature for 2 h. Insoluble

material was removed by filtration through Celite, and the filtrate was concentrated under reduced
pressure. The residue was purified by silica gel column chromatography [n-hexane:ethyl acetate = 2:1] to
1
give 25 (2210 mg, 86%) as a colorless oil. H NMR-CDCl3 (400 MHz) δ 1.26 (3H, t, J = 9.6 Hz), 1.46
(9H, s), 1.54 (1H, m), 2.06 (1H, m), 2.38 (2H, m), 2.56 (1H, m), 2.94 (1H, m), 3.29 (1H, m), 3.45 (1H, m),
3.60 (1H, m), 4.14 (2H, q, J = 9.6 Hz). FAB-MS: m/z 258 [M+H]+.
Ethyl pyrrolidin-3-ylacetate (26)
To a solution of 25 (2200 mg, 8.55 mmol) in ethyl acetate (10 mL) was added 4 M hydrochloric
acid/ethyl acetate (10.7 mL, 42.8 mmol), followed by stirring at room temperature for 24 h. The reaction
mixture was concentrated in vacuo and the residue was made alkaline with 1 M NaOH at 0 °C (pH 10).
The mixture was partitioned between CHCl3 and saturated aqueous sodium chloride solution, dried over
anhydrous sodium sulfate, and then concentrated under reduced pressure to give 26 (1170 mg, 87%) as a
1
slightly yellow oil. H NMR-CDCl3 (400 MHz) δ 1.26 (3H, t, J = 9.6 Hz), 1.38 (1H, m), 1.99 (1H, m),
2.34-2.55 (4H, m), 2.89-2.99 (2H, m), 3.15 (1H, m), 4.14 (2H, q, J = 9.6 Hz). EI-MS: m/z 157 [M]+.
AKR1C enzyme assay by monitoring NADPH oxidation
All assays were performed on SAFIRE spectrophotometer by monitoring the change in absorbance of
NADPH at 340 nm. The potency of the compounds was determined by measuring their ability to inhibit
NADPH-dependent reduction of 9,10-phenanthrenequinone (9,10-PQ) catalyzed by human AKR1C as

previously described.20 Purified enzyme (a final concentration of 10 μg/mL), 9,10-PQ (4 μmol/L),
NADPH (200 μmol/L), and a test compound were mixed in 100 mM potassium phosphate buffer (pH 6.0)
to give a total volume of 80 μL, and then react at room temperature for 10 to 20 min. Subsequently, the
change in absorbance of NADPH at 340 nm was measured. The amount of oxidation of NADPH in the
presence of the compound was obtained as a relative value with respect to the amount of NADPH in the
absence of the enzyme set at 0% and the amount of NADPH in the absence of the compound set at 100%.
The IC50 values were calculated using Sigmoid-Emax model non-linear regression.
Cell-based 17β-HSD5 enzyme assay
CWR22R cells and HEK293 cells stably expressing human 17β-HSD5 were seeded in 96-well plates
at 2 ×104 cells/100 μL/well in RPMI-1640 medium supplemented with 10% FBS and in D-MEM medium
supplemented with 10% FBS, respectively. After overnight incubation at 37°C in 5% CO2,
androstenedione (a final concentration of 300 nmol/mL) and a test compound were added to each well.
Four hours after incubation, the cell supernatants were removed to measure testosterone concentration
using DELFIA Testosterone reagents® (PerkinElmer Life Sciences Inc. MA, USA) according to
manufacturer’s instructions. The inhibitory effect was expressed as the concentration inducing 50%
inhibition (IC50) of conversion from AD to testosterone. The IC50 values were calculated using
Sigmoid-Emax model non-linear regression.

In vivo model.
Male BALB/c athymic nude mice (Charles River Japan, Inc) aged 4-6 weeks old were used for the
CWR22R xenograft model and were castrated before tumor implantation. An established tumor from a
host mouse was minced into small fragments, and digested with protease (0.1%). The digested cells were
dispersed in RPMI 1640 medium with 20% FBS and then mixed with Matrigel® (1:1 v/v). About 100 μL
of the mixture was subcutaneously injected into each flank of castrated mice. About 3 weeks after
transplantation, mice carrying CWR22R tumors with similar sizes were selected and randomly divided
into five groups (n = 4 for each group). In this study, compound 17 was administered orally.
Androstenedione was injected into xenografts (1 ng/100 mm3 tumor) 1 h after administration of
compound 17. The tumor tissues were removed and weighed for measurement of intratumoral
testosterone 2 h after administration of compound 17.
Detection of intratumoral testosterone concentration in CWR22R xenograft
The removed tumor tissues were homogenated with 200mmol/L phosphate buffer (pH 7.4), and
testosterone were extracted by tert-Butyl Methyl Ether. Then, testosterone concentrations of the
reconstituted extracts were determined using DELFIA Testosterone reagents® (PerkinElmer Life Sciences
Inc. MA, USA) according to manufacturer’s instructions.
Concentration of test compounds in mice plasma

Test compounds were administered in ICR mice by oral administration. Blood samples were
collected 1 or 4 h after administration, and then the test compounds were extracted from mice plasma by
acetonitrile and measured by HPLC.
Rat pharmacokinetic study
The pharmacokinetic properties of compound 17 were evaluated in male Sprague-Dawley rats (aged
8 weeks). Compound 17 was administered at 1 mg/kg intravenously in a solution of saline containing
15% DMF and 20% polyethylene glycol 400 and orally in a solution of water containing 10% DMF and
10% propylene glycol. Blood samples were taken at multiple points up to 24 h after a single
administration of compound 17 and centrifuged at 16,000 g for 2 min at 4 °C to obtain plasma. The
concentrations of compound 17 in rat plasma were determined using high-performance liquid
chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) after sample preparation.
PAMPA
The PAMPA Evolution instrument from pION Inc. was used in this study. In PAMPA, a “sandwich”
is formed from a 96-well microtiter plate (pION Inc., PN 110163) and a 96-well filter plate (Millipore,
IPVH), such that each composite well is divided into two chambers: donor at the bottom and acceptor at
the top, separated by a 125 μm-thick microfilter disc (0.45 μm pores) and coated with a 20%(w/v)
dodecane solution of a lecithin mixture (pION Inc., PN 110669). Drug samples were introduced as 10mM
DMSO stock solutions in a 96-well polypropylene microtiter plate. The robotic liquid handling system
draws a 5 μL aliquot of the DMSO stock solution and mixes it into an aqueous buffer solution including
10%(v/v) of DMSO so that the final typical sample concentration is 50 μM. The drug solutions were

filtered using a 96-well filter plate (Corning, PVDF), and added to the donor compartments. The donor
solutions were adjusted in pH 6.5 (NaOH-treated universal buffer, pION Inc., PN 110151), while the
acceptor solution had the same pH 7.4 (pION Inc., PN 110139). The plate sandwich was formed and
allowed to incubate at 25 °C for 2 h in a humidity-saturated atmosphere. On completion of the prescribed
incubation time, the sandwich plates were separated and both the donor and acceptor compartments were
assayed for the amount of material present by comparison with the UV spectrum (270–400 nm) obtained
from reference standards. Mass balance was used to determine the amount of material remaining in the
membrane barrier and permeability (Pe) was calculated using PAMPA Evolution software (pION Inc.).
Solubility test in pH 6.8 buffer solution with the precipitation method
To 13 μL of a 10 mM DMSO solution of a test compound that had been prepared in advance was
added exactly 1 mL of a second liquid (pH 6.8) for a disintegration test of Japanese Pharmacopoeia,
followed by shaking at 25 °C for 20 h, thereby giving a sample stock solution. Next, using a filter
impregnated with 200 μL of the sample stock solution, 200 μL of a fresh sample stock solution was added
for filtration to obtain a liquid, which was taken as a sample solution. Separately to this, to 10 μL of the
10 mM DMSO solution of the test compound was added accurately 1 mL of methanol, followed by
stirring, thereby giving a standard solution. 10 μL portions each of the sample solution and standard
solution were tested by liquid chromatography, and the ratio of the peak area of the sample solution to the
peak area of the standard solution was determined, thereby calculating the solubility.
Acknowledgments
We thank Dr. Hidenori Azami and Dr. Hiroyuki Koshio for their helpful support in preparing this
manuscript, and to the members of the Division of Analytical Science Laboratories for elemental analysis
and spectral measurements.

References and notes
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