Discovery of novel serine palmitoyltransferase inhibitors as cancer therapeutic agents

Article abstract of DOI:10.1016/j.bmc.2018.04.008

We pursued serine palmitoyltransferase (SPT) inhibitors as novel cancer therapeutic agents based on a correlation between SPT inhibition and growth suppression of cancer cells. High-throughput screening and medicinal chemistry efforts led to the identification of structurally diverse SPT inhibitors 4 and 5. Both compounds potently inhibited SPT enzyme and decreased intracellular ceramide content. In addition, they suppressed cell growth of human lung adenocarcinoma HCC4006 and acute promyelocytic leukemia PL-21, and displayed good pharmacokinetic profiles. Reduction of 3-ketodihydrosphingosine, the direct downstream product of SPT, was confirmed under in vivo settings after oral administration of compounds 4 and 5. Their anti-tumor efficacy was observed in a PL-21 xenograft mouse model. These results suggested that SPT inhibitors might have potential to be effective cancer therapeutics.

Full text of DOI:10.1016/j.bmc.2018.04.008

Accepted Manuscript
Discovery of novel serine palmitoyltransferase inhibitors as cancer therapeutic
agents
Takuto Kojima, Yasutomi Asano, Osamu Kurasawa, Yasuhiro Hirata, Naoki
Iwamura, Tzu-Tshin Wong, Bunnai Saito, Yuta Tanaka, Ryosuke Arai, Kazuko
Yonemori, Yasufumi Miyamoto, Yoji Sagiya, Masahiro Yaguchi, Sachio
Shibata, Akio Mizutani, Osamu Sano, Ryutaro Adachi, Yoshinori Satomi,
Megumi Hirayama, Kazunobu Aoyama, Yuto Hiura, Atsushi Kiba, Shuji
Kitamura, Shinichi Imamura
PII:
S0968-0896(17)32376-3
https://doi.org/10.1016/j.bmc.2018.04.008
BMC 14291
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
26 December 2017
3 April 2018
3 April 2018
Please cite this article as: Kojima, T., Asano, Y., Kurasawa, O., Hirata, Y., Iwamura, N., Wong, T-T., Saito, B.,
Tanaka, Y., Arai, R., Yonemori, K., Miyamoto, Y., Sagiya, Y., Yaguchi, M., Shibata, S., Mizutani, A., Sano, O.,
Adachi, R., Satomi, Y., Hirayama, M., Aoyama, K., Hiura, Y., Kiba, A., Kitamura, S., Imamura, S., Discovery of
novel serine palmitoyltransferase inhibitors as cancer therapeutic agents, Bioorganic & Medicinal Chemistry (2018),
doi: https://doi.org/10.1016/j.bmc.2018.04.008
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Discovery of novel serine palmitoyltransferase inhibitors as cancer therapeutic agents
†
,
†a
†a
b
Takuto Kojima, * Yasutomi Asano, Osamu Kurasawa, Yasuhiro Hirata, Naoki Iwamura, Tzu-Tshin Wong, Bunnai
c
d
e
Saito, Yuta Tanaka, Ryosuke Arai, Kazuko Yonemori, Yasufumi Miyamoto, Yoji Sagiya, Masahiro Yaguchi, Sachio
a
f
g
h
a
a
Shibata, Akio Mizutani, Osamu Sano, Ryutaro Adachi, Yoshinori Satomi, Megumi Hirayama, Kazunobu Aoyama,
a
i
Yuto Hiura, Atsushi Kiba, Shuji Kitamura, and Shinichi Imamura*
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-Chome,
Fujisawa, Kanagawa 251-0012, Japan
†
Equally contributing authors
*
Corresponding authors
Tel: +81 466 32 1116; Fax: +81 466 29 4449; E-mail: takuto.kojima@takeda.com (T. Kojima)
E-mail: imamura@ncgg.go.jp (S. Imamura)
a
Present address: Axcelead Drug Discovery Partners Inc. 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa
2
51-0012, Japan
b
Present address: AGC Asahi Glass Research Center, 1150 Hazawa-cho, Kangawa-ku, Yokohama, Kanagawa 221-8755,
Japan
c
Present address: Senjyu Pharmaceutical Company Limited, 2-5-8 Hiranomachi, Chuo-ku, Osaka 541-0046, Japan
d
Present address: Cardurion Pharmaceuticals K.K., 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-0012,
Japan
e
Present address: Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi
4
66-8550, Japan
f
Present address: Chordia Therapeutics Inc, 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-0012, Japan
Present address: Shinsei Corporate Investment Limited, 2-4-3 Nihonbashi-muromachi, Chuo-ku, Tokyo 103-0022,
g
Japan
h
Present address: Shionogi & Co., Ltd, 3-1-8 Doshomachi, Chuo-ku, Osaka, 541-0045, Japan
i
Present address: National Center for Geriatrics and Gerontology, 7-430 Morioka-cho, Obu, Aichi 474-8511, Japan
Abstract
We pursued serine palmitoyltransferase (SPT) inhibitors as novel cancer therapeutic agents based on a correlation
between SPT inhibition and growth suppression of cancer cells. High-throughput screening and medicinal chemistry
efforts led to the identification of structurally diverse SPT inhibitors 4 and 5. Both compounds potently inhibited SPT
enzyme and decreased intracellular ceramide content. In addition, they suppressed cell growth of human lung
adenocarcinoma HCC4006 and acute promyelocytic leukemia PL-21, and displayed good pharmacokinetic profiles.
Reduction of 3-ketodihydrosphingosine, the direct downstream product of SPT, was confirmed under in vivo settings
after oral administration of compounds 4 and 5. Their anti-tumor efficacy was observed in a PL-21 xenograft mouse
model. These results suggested that SPT inhibitors might have potential to be effective cancer therapeutics.
Keywords: SPT; 3-KDS; antitumor efficacy

Introduction
Serine palmitoyltransferase (SPT) is a heterodimeric membrane protein localized in endoplasmic reticulum. It
catalyzes condensation of L-serine and palmitoyl-coenzyme A to generate 3-ketodihydrosphingosine (3-KDS) as shown
in Figure 1. SPT is an initial and rate-limiting enzyme in the de novo biosynthesis of sphingolipids such as ceramide
1
,2
and sphingomyelin. Sphingolipids are known to play important biological roles (i.e. cell membrane formation, signal
3
-8
transduction, plasma lipoprotein metabolism) and to be associated with various diseases including cancer. Intradermal
and intraperitoneal administrations of Myriocin (1, Fig. 2), an SPT inhibitorisolated from fungal extracts, suppressed
9
tumor formation in a mouse B16F10 melanoma xenograft model with decrease in sphingolipids. In addition, our
studies revealed an apparent correlation between SPT enzyme inhibition and growth suppression of a human lung
1
0
adenocarcinoma cell line (HCC4006) . Therefore, SPT could be an attractive target for cancer therapy.
11
Recently, SPT inhibitors 2 and 3 were reported as potential treatments for diabetes and dyslipidemia. In our
laboratories, to develop orally bioavailable SPT inhibitors, we conducted high-throughput screening (HTS) of in-house
compound libraries and subsequent optimization via medicinal chemistry. These efforts successfully identified
structurally diverse SPT inhibitors such as tetrahydropyrazolopyridine 4 and 3-phenylpiperidine 5 (Fig. 2), which
exhibited potent SPT inhibition with favorable pharmacokinetic profiles. In this report, we describe the design and
synthesis of these compounds as wells as their biological profiles as anti-cancer agents.
Figure 1. Biosynthesis pathway for sphingolipids and their chemical structures.

Figure 2. Reported SPT inhibitors 1-3 and our compounds 4 and 5.
Chemistry
1
2
The synthesis of 1,2,3,4-tetrahydroquinoline derivative 13 is shown in Scheme 1. Starting from compound 6 ,
protection of the amine 6 with a Boc group and following reduction of the ketone 7 gave alcohol 8. After conversion of
8
3
to azide 9 by reaction with DPPA, reduction of 9 with PPh and subsequent amidation yielded compound 11.
Deprotection of the Boc group and an acylation of the secondary amine 12 afforded target compound 13.
a
Scheme 1. Synthesis of 1,2,3,4-tetrahydroquinoline derivative 13
a
Reagents and conditions: (a) (Boc)
, THF, H
THF, rt, 94%; (f) 4 M HCl in EtOAc, rt, 91%; (g) 3,4-dimethoxybenzoyl chloride, Et
2
O, DMAP, THF, rt; (b) NaBH
O, 100 °C, then 4 M HCl in EtOAc, rt, 44%; (e) 2-chlorobenzoyl chloride, Et
N, THF, 50 °C, 83%.
4
, THF, MeOH, 0 °C to rt, 87% in 2 steps; (c) DPPA,
DBU, THF, rt, quant; (d) PPh
3
2
3
N,
3
The synthesis of 4,5,6,7-tetrahydro-1H-pyrazolo[4,3-b]pyridine derivative 4 started from commercially available
compound 14 (Scheme 2). Mitsunobu reaction of compound 14 proceeded in a regioselective manner to give
N1-isopropyl (15a) and N1-methyl (15b) derivatives, and hydrogenation of their nitro groups afforded amines 16a and
1
6b. Michael reaction of 16a and 16b with methyl acrylate gave compounds 17a and 17b, which were converted to
compounds 18a and 18b by benzylation. Bicyclic ring was constructed by intramolecular condensation of compounds

1
1
8a and 18b in the presence of LiHMDS, and following decarboxylation under basic conditions afforded compounds
9a and 19b. Compounds 22a and 22b were obtained through oxime formation and its hydrogenation, followed by
protection with a Boc group. Deprotection of the benzyl group gave key intermediates 23a and 23b. After acylation of
the secondary amines, deprotection of the Boc group and amidation provided target compounds 26 and 27 as a
racemates (Scheme 3). Optical resolution of 26 was conducted by Chiralpak AD to provide 4 and 26R with ≥99.9%ee,
respectively. Absolute configuration of compound 4 was determined to be S by its X-ray crystallographic analysis.
a
Scheme 2. Synthesis of 4,5,6,7-tetrahydro-1H-pyrazolo[4,3-b]pyridine intermediates 23a and 23b
a
Reagents and conditions: (a) for 15a, DTBAD, PBu
1%; (b) Pd/C, H , MeOH, rt, 97-98%; (c) methyl acrylate, DMAP, DMF, 100 °C; (d) BnBr, K
in 2 steps; (e) NaHMDS, THF, 0 °C to rt, then NaOH aq., 70 °C, 91%-quant; (f) NH OH-HCl, pyridine, EtOH,
N, THF, rt; (i) Pd/C, H , AcOH, MeOH, rt,
3
, i-PrOH, THF, rt, 73%; for 15b, ADDP, PBu
3
, MeOH, toluene, rt,
6
2
2
CO
3
, DMF, rt, 68-76%
2
8
5
0-90 °C; (g) Raney Ni, H
9-93% in 2 steps.
2
, MeOH, rt, 58-71% in 2 steps; (h) Boc
2
O, Et
3
2
Scheme 3. Synthesis of 4,5,6,7-tetrahydro-1H-pyrazolo[4,3-b]pyridine derivatives 26 and 27, and chiral resolution of
a
racemic 26

a
Reagents and conditions: (a) 5,6-dimethoxynicotinic acid, HATU, Et
rt, 98%; (c) 2-(trifluoromethoxy)benzoic acid, HATU, Et N, DMF, rt, 70%; (d) 3,4-dimethoxybenzoyl chloride, pyridine,
rt, 98%; (e) 4 M HCl in CPME, MeOH, rt, 97%; (f) 2-chlorobenzoic acid, HATU, Et N, DMF, rt, 83%.
3
N, DMF, rt, 95%; (b) 4 M HCl in EtOAc, MeOH,
3
3
The synthesis of 3-phenylpiperidine derivative 31 is shown in Scheme 4. The amidation of 3-phenylpiperidine-4-one
1
3
hydrochloride 28 with 3,4-dimethoxybenzoyl chloride, subsequent condensation with hydroxylamine, and reduction
of the resulting oxime by Raney nickel gave cis/trans mixture of 4-amino-3-phenylpiperidine 30. Finally, amidation
with 2-chlorobenzoyl chloride afforded compound 31 (cis/trans = 10/3, racemate).
a
Scheme 4. Synthesis of 3-phenylpiperidine derivative 31
a
Reagents and conditions: (a) 3,4-(MeO)
2
C
6 3
H COCl, pyridine, 0 °C to rt, 71%; (b) NH
2 3
OH-HCl, Et N, EtOH, reflux,
9
9%; (c) Raney Ni, H , MeOH, rt, 89%; (d) 2-Cl-C
2
6
H
4
COCl, Et N, THF, 0 °C to rt, 83%.
3
Scheme 5 shows the synthesis of four stereoisomers of 31 (31a−d). Curtius rearrangement of commercially
available racemic trans carboxylic acid 32 followed by reaction with benzyl alcohol yielded carbamate 33, which was
converted to intermediate amine 34 by removal of the benzyloxycarbonyl group. The target compounds 31a−31d were
obtained through acylation of primary amine followed by deprotection of the Boc group and subsequent acylation of the
1
3
13
secondary amine. Cis isomers 31a and 31b were prepared from the corresponding chiral materials 34a and 34b ,
respectively, while trans isomers 31c and 31d were prepared from the racemic material 35 by chiral resolution at the
final step.

a
Scheme 5. Synthesis of 3-phenylpiperidine derivatives 31a−31d
a
Reagents and conditions: (a) DPPA, Et
3 2
N, toluene, then BnOH, reflux, quant; (b) Pd/C, H , MeOH, rt, 99%; (c)
2
-Cl-C COCl, Et N, THF, 0 °C to rt, 38-94%; (d) 4 M HCl in EtOAc, 0 °C to rt; (e) 3,4-dimethoxybenzoyl chloride,
6
H
4
3
pyridine, 0 °C to rt, 89-98% in 2 steps; (f) Chiral HPLC resolution, 67-70% in 3 steps.
The synthetic route to 3-phenylpiperidine derivative 5 is illustrated in Scheme 6. Condensation of diamine 36 with
glyoxal followed by hydrolysis of the ester group provided carboxylic acid 38. Amidation of the chiral
1
3
4
-aminopiperidine 34b afforded compound 39. Finally, target compound 5 was obtained through the removal of the
Boc group followed by acylation of the piperidine nitrogen. Absolute configuration of 5 was confirmed to be 3S,4R by
its X-ray crystallographic analysis.
a
Scheme 6. Synthesis of 3-phenylpiperidine derivative 5
a
Reagents and conditions: (a) glyoxal, MeOH, THF, rt, 98%; (b) 4 M NaOH aq., THF, MeOH, rt, 98%; (c)
3
1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxylic acid, WSC, HOBt, Et N, DMF, rt, 95%; (d) 4 M HCl in EtOAc,

3
rt., 98%; (e) 38, WSC, HOBt, Et N, DMF, rt, 59%.
Results and discussion
The synthesized compounds were evaluated for their SPT inhibitory activity (IC50) and growth inhibitory activity
against HCC4006 cells. The growth inhibition was assessed based on inflection point (IP), since the maximum
inhibition rate was around 50% in the HCC4006 growth assay.
Figure 3 highlights our medicinal chemistry approach to discover tetrahydropyrazolopyridine compound 4. Our
initial efforts for exploring SAR of HTS hit tetrahydroquinoline derivative 41 to avoid basic amine moiety led to the
identification of amide compound 13, which exhibited significant improvement in SPT inhibitory activity (IC50 = 650
nM vs 5.1 nM). Next, we focused on scaffold hopping of the central core ring system, in order to acquire drug-like
properties such as metabolic stability and oral bioavailability. In the course of screening of the core systems to reduce
lipophilicity, 4,5,6,7-tetrahydro-1H-pyrazolo[4,3-b]pyridine 27 was found to have favorable metabolic and
pharmacokinetic profiles (F = 28%) while keeping the activity (IC50 = 7.7 nM). It was selected as a lead compound for
the next optimization step. We optimized the lead 27 to improve inhibitory activity against HCC4006 cell growth based
on the SAR information - modification of both substituent on pyrazole ring and upper amide were effective to boost the
cellular activity. As a result of optimization, we identified compound 26 as the most promising compound, showing
enhanced inhibitory activity against SPT enzyme (IC50 = 1.2 nM) and HCC4006 cell growth (IP = 23 nM). Chiral
resolution of 26 afforded compound 4 as a eutomer and compound 26R as a distomer. The compound 4 potently
inhibited both SPT enzyme (IC50 = 1.2 nM) and HCC4006 cell growth (IP = 3.9 nM), and showed good
pharmacokinetic properties in mice (F = 53%).
Figure 3. Identification of compound 4 from HTS hit 41.
a
b
c
SPT inhibitory activity (n = 2). Growth inhibition of HCC4006 cells (n = 2). Metabolic clearance in [human/mouse]

d
e
hepatic microsomes (n = 2, μL/min/mg). Oral bioavailability in mice following cassette dosing (n = 3). NC: not
calculated because the measured plasma concentrations after oral administration were basically below the quantitation
limit and less than two available data points.
To expand the diversity of structure, we continued the generation of another chemotype from compound 13. SAR
study of the tetrahydropyrazolopyridine series revealed that introducing sterically bulky groups on the pyrazole nitrogen
was well tolerated and was rather preferred. This suggested that there would be a space to accommodate large
substituents. Therefore, we aimed to replace the tetrahydroquinoline (13) core by 3-phenlypiperidine (31) to occupy the
space. As expected, the designed compound 31 was found to show excellent inhibitory activity against SPT enzyme
(
IC50 = 1.2 nM) as well as HCC4006 cell growth (IP = 41 nM).
Figure 4. Design concept of 3-phenylpiperidine derivative 31.
a
b
SPT inhibitory activity (n = 2). Growth inhibition of HCC4006 cells (n = 2).
Since the prototype compound 31 was a mixture of four stereoisomers, preparation and evaluation of the individual
isomers were carried out. Consequently, compound 31b with 3S,4R absolute configuration was found as the most potent
isomer (Figure 5). Judging from the comparison of four isomers, stereo configuration of the 4-position (IC50 = 0.36 nM
vs 3000 nM) on the piperidine ring seemed to have a greater impact on potency than that of the 3-position (IC50 = 0.36
nM vs 5.0 nM). Our strategy for optimizing compound 31b to improve bioavailability was adjusting lipophilicity and
replacing dimethoxyphenyl ring of 31b with quinoxaline ring, which led to the identification of compound 5 as the
most promising compound among this series. It was surprising that the absolute configuration of the nitrogen
substituent of compound 4 and compound 5 are opposite, but superposition of both compounds is still inconclusive.

Figure 5. Identification of compound 5 from the prototype 31.
a
b
c
SPT inhibitory activity (n = 2). Growth inhibition of HCC4006 cells (n = 2). Metabolic clearance in [human/mouse]
d
e
hepatic microsomes (n = 2, μL/min/mg). Oral bioavailability in mice following cassette dosing (n = 3). NC: not
calculated because the measured plasma concentrations after oral administration were basically below the quantitation
limit and less than two available data points.
In vitro profiles of compounds 4 and 5 are summarized in Table 1. Both compounds 4 and 5 were as potent as
Myriocin in a human SPT enzyme inhibition assay. Compounds 4 and 5 were also tested in a ceramide content assay
1
4
using HCC4006 cells, measuring the ceramide content . In this assay, both compounds 4 and 5 exhibited inhibitory
activity almost equal to that of Myriocin, indicating that the compounds definitely suppressed de novo synthesis of
sphingolipids by SPT inhibition. Next, cancer cell growth assay was conducted. While we evaluated potency in growth
inhibition using HCC4006 cells during the compound optimization stage, our efforts to search for cell lines more
sensitive to SPT inhibitors led to the identification of PL-21 cells derived from an acute promyelocytic leukemia
(
maximum inhibition rate > 90%). Compounds 4 and 5 displayed potent growth inhibitory activity against both
HCC4006 and PL-21 cells on the same level with Myriocin. Once the potency of compounds 4 and 5 as SPT inhibitors
was validated in vitro, we then performed their in vivo evaluation.
a
Table 1. In vitro profiles of compounds 1, 4, and 5
Assays
1 (Myriocin)
4
5
SPT enzyme inhibition
0.13
0
.54 (0.44–0.66)
0.71 (0.53–0.95)
a,b,d
IC50 (nM)
(0.11–0.16)
Ceramide content, HCC4006
1
.5 (0.95–2.2)
2.4 (1.4–4.0)
3.9 (3.0–5.0)
2.3 (1.3–3.8)
11 (6.2–18)
a,b,e
cells IC50 (nM)
Growth inhibition, HCC4006
cells
2
.1 (1.0–4.5)

c,d
IP (nM)
Growth inhibition, PL-21 cells
7.8 (6.3–9.3)
4.1 (3.4–4.7)
11 (9.8–13)
a,b,f
IC50 (nM)
Metabolic clearance, human
-
-
6
5
<1
60
g
(μL/min/mg)
Metabolic clearance, mouse
g
(μL/min/mg)
h
Solubility pH 6.8 (μg/mL)
PAMPA pH7.8 A to B (nm/sec)
-
-
24
8
167
203
a
b
Numbers in parentheses represent 95% confidence intervals. Concentration where dose-inhibition curve passes
c
d
e
f
g
through 50% inhibition. Concentration at the inflection point of dose-inhibition curve. n = 2. n = 4. n = 3.
h
Metabolic clearance was measured using liver microsomes and NADPH. Thermodynamic solubility in Japanese
Pharmacopoeia disintegration test solution 2 (pH 6.8).
Pharmacokinetic profiles of compounds 4 and 5 following cassette dosing to mice are summarized in Table 2. Both
compounds possessed appropriate volume of distribution and clearance rate. Moreover, compound 4 and 5 displayed
different pattern of the plasma concentration-time curve – compound 5 had 2-fold longer mean residence time and time
to peak concentration compared to compound 4. The oral bioavailability of compounds 4 and 5 were 53% and 56%,
respectively. The good pharmacokinetic properties in mice are presumably due to their appropriate drug-like properties,
including metabolic stability, aqueous solubility, and permeability as shown in Table 1.
a
Table 2. Pharmacokinetic parameters of compounds 4 and 5 in mice
Compd
iv (0.1 mg/kg)
po (1 mg/kg)
F (%)
Vdss
mL/kg)
CLtot
Cmax
Tmax
(h)
AUC
MRT
(h)
(
(mL/h/kg)
(ng/mL)
(ng·h/mL)

4
5
1128
750
954
507
309
282
0.5
1.2
572
1.4
2.7
53
56
1113
a
Pharmacokinetic parameters were obtained by cassette dosing. Data represent mean values (n = 3).
The in vivo PD profiles of compounds 4 and 5 are shown in Figure 6. Quantity of 3-KDS, the direct product of SPT,
in tumors was measured 8 h after oral administration of compounds 4 or 5 (10 mg/kg) in mouse HCC1806 (breast
cancer) xenograft model. As with HCC4006, HCC1806 was found to be a sensitive cell line to SPT inhibitor, and its
xenograft model was more easily prepared than that of HCC4006. As a result, both compounds 4 and 5 significantly
suppressed the level of 3-KDS in tumors 8 h after oral administration (about 40% suppression relative to vehicle).
Figure 6. 3-KDS levels in the tumors of HCC1806 xenografted mice were detected by LC/MS 8 h after oral
administration of compound 4 or 5 at 10 mg/kg. Data are expressed as mean ± SD (n = 5). ***p < 0.001 vs vehicle
(
Dunnett's test).
Antitumor efficacy study of compound 4 was carried out in a PL-21 xenograft mouse model. Oral administration of
compound 4 (10 and 30 mg/kg, Q2D, 2 weeks) exhibited antitumor efficacy with T/C values, which meaned the median
tumor weight in the treated group (T) divided by the median tumor weight in the control group (C), of 52% at 10 mg/kg
and 38% at 30 mg/kg without severe body weight loss (Figure 7). These data indicated that compound 4 is an orally
bioavailable SPT inhibitor with in vivo antitumor efficacy, which provides a useful tool for assessing the
pharmaceutical potential of SPT inhibitors especially against cancer. Significant antitumor effect of compound 5 was
15
also observed in the same in vivo model.

Figure 7. Mean tumor volume in PL-21 xenograft mice dosed with 4 or vehicle. Data are expressed as mean ± SD (n =
/group). Compound 4 was suspended in 0.5% aqueous methylcellulose solution and orally administered every other
6
day for two weeks. *** p < 0.001, **p < 0.01 vs vehicle (Dunnett's test). T/C values were as follows: 52% (10 mg/kg,
Q2D), 38% (30 mg/kg, Q2D).
3
. Conclusion
We established a good correlation between SPT inhibition and growth suppression of HCC4006 cell line and
discovered structurally diverse SPT inhibitors 4 and 5 by an HTS campaign and iterative compound design including
scaffold hopping. Both compounds 4 and 5 potently inhibited SPT enzyme activity and de novo synthesis of ceramide.
They suppressed growth of HCC4006 and PL-21 cancer cell lines in vitro at the same level as Myriocin. In addition,
compounds 4 and 5 displayed favorable pharmacokinetic profiles. Significant reduction of 3-KDS levels in HCC1806
xenograft mice was observed by oral administration of compounds 4 and 5 (10 mg/kg). Furthermore, antitumor efficacy
of an SPT inhibitor was demonstrated in a PL-21 xenograft mouse model. Oral administration of compound 4 (30
mg/kg, Q2D, 14 days) reduced tumor growth with a T/C value of 38%. These results along with recent reported efficacy
1
5
study of compound 5 show that SPT inhibitors might have the potential to be effective cancer therapeutics.
Acknowlwdgement
We thank Mitsuyoshi Nishitani for X-ray structure analysis.
Experimental Section
Chemistry
General Chemistry Information. All commercially available solvents and reagents for reactions were reagent-grade
and were used as purchased. Analytical thin layer chromatography (TLC) was performed on silica gel 60 F254 plates
(Merck) or NH TLC plates (Fuji Silysia Chemical Ltd.). Flash column chromatography separations were performed on
Purif-Pack (SI or NH, Fuji Silysia Chemical Ltd.). Melting points (mp.), determined on a SRS OptiMelt automated
melting point apparatus, are uncorrected. The proton nuclear magnetic resonance (1H NMR) spectra were recorded
using Bruker AVANCE II (300 MHz), Bruker AVANCE III (300 MHz, 400 MHz), or Varian mercury plus (300 MHz)
spectrometers with tetramethylsilane (TMS) as an internal standard. The NMR data are given as follows: chemical shift
(δ) in ppm, multiplicity (where applicable), coupling constants (J) in Hz (where applicable), and integration (where
applicable). Multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), dd (double doublets), dt,

(double triplet), dq (double quartet), br s (broad singlet), or m (multiplet). Mass spectra (MS) were acquired using an
Agilent LC/MS system (Agilent1200SL/Agilent6130MS), Shimadzu LC/MS system (LC-10ADvp high pressure
gradient system/LCMS-2010A) or Shimadzu UFLC/MS (Prominence UFLC high pressure gradient
system/LCMS-2020) operating in electron spray ionization mode (ESI+). The column used was an L-column 2 ODS
(3.0 x 50 mm I.D., 3 μm, CERI, Japan) with a temperature of 40 °C and a flow rate of 1.2 or 1.5 mL/min. Mobile phase
A was 0.05% TFA in ultrapure water. Mobile phase B was 0.05% TFA in acetonitrile which was increased linearly
from 5% to 90% over 2 minutes, 90% over the next 1.5 minutes, after which the column was equilibrated to 5% for 0.5
minutes. Elemental analyses were carried out by Takeda Pharmaceutical Company Ltd. Medicinal Chemistry Research
Laboratories. Each compound was confirmed to be 95% purity by either LC/MS or elemental analysis. Yields were
not optimized.
tert-Butyl 7-chloro-4-oxo-3,4-dihydroquinoline-1(2H)-carboxylate (7). Boc
2.1 g, 17.2 mmol) were added to a stirred solution of 6 (7.6 g, 41.8 mmol) in THF (100 mL) at room temperature, and
the mixture was stirred at room temperature for 14 h. The mixture was concentrated in vacuo, diluted with water, and
extracted with EtOAc. The organic layer was washed with water and brine, dried over MgSO , and filtered. The filtrate
2
O (10.1 mL, 44.1 mmol) and DMAP
(
4
was concentrated in vacuo, and the residue was purified by column chromatography (silica gel, eluted with 50% EtOAc
in hexane) to give 7 (13.1 g, quant) as a yellow oil. This product was used in the next step without further purification.
1
3
H NMR (300 MHz, CDCl ) δ 1.57 (9H, s), 2.70-2.81 (2H, m), 4.11-4.20 (2H, m), 7.12 (1H, dd, J = 8.5, 2.1 Hz), 7.87
(1H, d, J = 1.9 Hz), 7.93 (1H, d, J = 8.5 Hz).
tert-Butyl 7-chloro-4-hydroxy-3,4-dihydroquinoline-1(2H)-carboxylate (8). Sodium borohydride (1.6 g, 42.3 mmol)
was added to a solution of 7 (13.1 g) in THF (150 mL) and MeOH (75 mL) at 0 °C. The mixture was stirred at 0 °C for
1
h and at room temperature for 14 h. The mixture was quenched with water and concentrated in vacuo. The residue
was diluted with water and extracted with EtOAc. The organic layer was washed with water and brine, dried over
1
MgSO
4
, and filtered. The filtrate was concentrated in vacuo to give 8 (10.3 g, 87% yield in 2 steps) as a yellow jelly. H
3
NMR (300 MHz, CDCl ) δ 1.48-1.61 (9H, m), 1.71-1.85 (1H, m), 1.86-2.12 (2H, m), 3.47-3.75 (1H, m), 3.89-4.08 (1H,
m),4.65-4.78 (1H, m), 7.04 (1H, dd, J = 8.3, 2.1 Hz), 7.30 (1H, d, J = 8.3 Hz), 7.91 (1H, d, J = 1.9 Hz).
tert-Butyl 4-azido-7-chloro-3,4-dihydroquinoline-1(2H)-carboxylate (9). DPPA (4.5 mL, 21.1 mmol) was added to a
solution of 8 (5.0 g, 17.6 mmol) and DBU (3.1 mL, 21.1 mmol) in toluene (100 mL) at room temperature, and the
mixture was stirred at room temperature for 18 h. The mixture was quenched with water and extracted with EtOAc. The
4
organic layer was washed with water and brine, dried over MgSO , and filtered. The filtrate was concentrated in vacuo.
The residue was purified by column chromatography (silica gel, eluted with 50% EtOAc in hexane) to give 9 (6.37 g,
1
quant) as a yellow oil. H NMR (300 MHz, CDCl
3
) δ 1.55 (9H, s), 2.00-2.09 (2H, m), 3.49-3.65 (1H, m), 3.98-4.10 (1H,
m), 4.56 (1H, t, J = 4.5 Hz), 7.02-7.08 (1H, m), 7.16-7.23 (1H, m), 7.99 (1H, d, J = 2.1 Hz).
tert-Butyl 4-amino-7-chloro-3,4-dihydroquinoline-1(2H)-carboxylate hydrochloride (10). A mixture of 9 (5.4 g,
1
3
7.6 mmol) and PPh (8.3 g, 31.7 mmol) in water (40 mL) and THF (400 mL) was stirred at 100 °C for 2 h. The
mixture was concentrated in vacuo, diluted with water, and extracted with EtOAc. The organic layer was washed with
water and brine, dried over MgSO
4
, and filtered. To the filtrate was added 4 M HCl (EtOAc solution), and the resulting
1
precipitate was collected by filtration to give 10 (2.5 g, 44% yield) as a colorless powder. H NMR (300 MHz, CDCl
3
) δ
1
.49 (9H, s), 1.94-2.10 (1H, m), 2.10-2.24 (1H, m), 3.59-3.77 (1H, m), 3.79-3.92 (1H, m), 4.49 (1H, brs), 7.22 (1H, dd,
J = 8.3, 2.3 Hz), 7.55 (1H, d, J = 8.3 Hz), 7.88 (1H, d, J = 2.3 Hz), 8.60 (3H, brs).
tert-Butyl 7-chloro-4-((2-chlorobenzoyl)amino)-3,4-dihydroquinoline-1(2H)-carboxylate (11). To a mixture of 10
2.5 g, 7.83 mmol) and Et N (2.5 mL, 23.4 mmol) in THF (50 mL) was added 2-chlorobenzoyl chloride (1.1 mL, 8.67
mmol) at room temperature, and the mixture was stirred at room temperature for 2 h. The mixture was concentrated in
(
3

vacuo, diluted with water, and extracted with EtOAc. The organic layer was washed with water and brine, dried over
MgSO
4
, and filtered. The filtrate was concentrated in vacuo, and the residue was purified by column chromatography
1
(
silica gel, eluted with 10-50% EtOAc in hexane) to give 11 (3.1 g, 94% yield) as a colorless solid. H NMR (300 MHz,
3
CDCl ) δ 1.55 (9H, s), 2.09-2.32 (2H, m), 3.65-3.81 (1H, m), 3.87-4.03 (1H, m), 5.29-5.39 (1H, m), 6.39 (1H, d, J = 7.5
Hz), 7.04 (1H, dd, J = 8.3, 2.3 Hz), 7.27-7.43 (4H, m), 7.67-7.75 (1H, m), 7.88 (1H, d, J = 2.1 Hz).
-Chloro-N-(7-chloro-1,2,3,4-tetrahydroquinolin-4-yl)benzamide hydrochloride (12). A mixture of 11 (3.1 g, 7.36
mmol) and 4 M HCl (EtOAc solution, 20 mL) was stirred at room temperature for 1 h. The mixture was treated with
2
Et
2
O and stirred at room temperature for 30 min. The precipitate was collected by filtration to give 12 (2.4 g, 91% yield)
1
as a colorless powder. H NMR (300 MHz, DMSO-d
6
) δ 1.87-1.98 (2H, m), 3.27 (2H, t, J = 5.7 Hz), 4.98-5.18 (1H, m),
6
.46 (2H, brs), 6.54-6.66 (2H, m), 7.13 (1H, d, J = 7.9 Hz), 7.32-7.53 (4H, m), 8.82 (1H, d, J = 8.3 Hz).
2-Chloro-N-(7-chloro-1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydroquinolin-4-yl)benzamide (13). Oxalyl chloride
(280 uL, 3.26 mmol) was added to a mixture of 3,4-dimethoxybenzoic acid (480 mg, 2.63 mmol) and DMF (3 drops) in
THF (15 mL) at room temperature, and the mixture was stirred at room temperature for 1 h. The mixture was
concentrated in vacuo, and the residue was suspended in THF (15 mL). To the suspension, 12 (790 mg, 2.21 mmol) and
Et
was concentrated in vacuo, diluted with water, and extracted with EtOAc. The organic layer was washed with aqueous 1
M HCl, aqueous saturated NaHCO , and brine, dried over MgSO , and filtered. The filtrate was concentrated in vacuo,
3
N (690 uL, 4.94 mmol) were added at room temperature, and the mixture was stirred at 50 °C for 3 h. The mixture
3
4
and the residue was purified by column chromatography (silica gel, eluted with 10-40% EtOAc in hexane) to give 13
1
(
891 mg, 83% yield) as a colorless solid. H NMR (300 MHz, CDCl
3
) δ 2.08-2.25 (1H, m), 2.28-2.49 (1H, m),
3
.76-3.89 (1H, m), 3.84 (3H, s), 3.91 (3H, s), 4.00-4.17 (1H, m), 5.33-5.49 (1H, m), 6.56 (1H, d, J = 8.0 Hz), 6.80 (1H,
+
d, J = 8.3 Hz), 6.98-7.15 (4H, m), 7.29-7.44 (4H, m), 7.68-7.78 (1H,m). LC-MS (ESI): m/z [M + H ] 484.9
Methyl 1-isopropyl-4-nitro-1H-pyrazole-5-carboxylate (15a). To a solution of 14 (10 g, 58.4 mmol), propan-2-ol
(22.0 mL, 292 mmol), and tributylphosphine (17.5 mL, 70.1 mmol) in dry THF (200 mL) was added dropwise a
solution of di-tert-butyl azodicarboxylate (17.5 g, 76.0 mmol) in dry THF (100 mL) at 20 °C. After being stirred at
room temperature overnight, the mixture was partitioned between EtOAc and water. The organic layer was separated,
2 4
washed with brine, dried over Na SO , filtered, and concentrated in vacuo. The residue was purified by column
chromatography (silica gel, eluted with 5-50% EtOAc in hexane) to give 15a (9.05 g, 73% yield) as a pale yellow oil.
1
3
H NMR (300 MHz, CDCl ) δ 1.53 (6H, d, J = 6.6 Hz), 4.03 (3H, s), 4.68 (1H, spt, J = 6.6 Hz), 8.05 (1H, s).
Methyl 1-methyl-4-nitro-1H-pyrazole-5-carboxylate (15b). To a solution of 14 (5 g, 29.2 mmol), methanol (5.93 ml,
1
1
46 mmol) and tributylphosphine (14.6 mL, 58.4 mmol) in toluene (500 mL) was added
,1'-(azodicarbonyl)dipiperidine (14.8 g, 58.4 mmol) at room temperature. The mixture was stirred at room temperature
overnight. The insoluble material was removed by filtration, and the filtrate was partitioned between EtOAc and water.
The organic layer was separated, washed with aqueous saturated NaHCO and brine, dried over Na SO , filtered, and
3
2
4
concentrated in vacuo. The residue was purified by column chromatography (silica gel, 30 g, eluted with 0-70% EtOAc
1
in hexane) to give 15b (3.28 g, 61% yield) as a pale yellow oil. H NMR (300 MHz, CDCl
3
) δ 4.03 (3H, s), 4.03 (3H, s),
8
.02 (1H, s).
Methyl 4-amino-1-isopropyl-1H-pyrazole-5-carboxylate (16a). To a solution of 15a (3.57 g, 16.8 mmol) in MeOH
60 mL) was added Pd/C (50% wet, 0.178 g), and the mixture was stirred at room temperature for 2 h under hydrogen
atmosphere. The insoluble materials were filtered off by using a pad of Celite, and the Celite was washed with MeOH.
(
4
The filtrate was concentrated in vacuo. The residue was diluted with EtOAc, washed with brine, dried over MgSO , and
1
filtered. The filtrate was concentrated in vacuo to afford 16a (3.02 g, 98% yield) as a brown oil. H NMR (300 MHz,
6
DMSO-d ) δ 1.30 (6H, d, J = 6.6 Hz), 3.79 (3H, s), 5.00 (2H, s), 5.21 (1H, dt, J = 13.1, 6.5 Hz), 7.05 (1H, s). LC-MS

+
(
ESI): m/z [M + H ] 183.9
Methyl 4-amino-1-methyl-1H-pyrazole-5-carboxylate (16b). The compound 16b was prepared from 15b by the same
+
method as that described for 16a. Colorless amorphous powder (97% yield). LC-MS (ESI): m/z [M + H ] 156.2
Methyl 4-(benzyl(3-methoxy-3-oxopropyl)amino)-1-isopropyl-1H-pyrazole-5-carboxylate (18a). A mixture of 16a
(2.92 g, 15.9 mmol), methyl acrylate (13.7 g, 159 mmol), and DMAP (0.389 g, 3.19 mmol) in DMF (20 mL) was stirred
o
at 100 C for 3 days. The resulting mixture was cooled to room temperature, and the excess methyl acrylate was
removed in vacuo. To the residue were added benzyl bromide (2.84 mL, 23.9 mmol) and K
The mixture was stirred at room temperature for 8 h. The mixture was diluted with water (200 mL) and extracted with
EtOAc (200 mL). The organic layer was washed with brine, dried over MgSO , and filtered. The filtrate was
2 3
CO (3.30 g, 23.9 mmol).
4
concentrated in vacuo, and the residue was purified by column chromatography (silica gel, eluted with 0-20% EtOAc in
1
hexane) to give 18a as a pale yellow oil (4.34 g, 76% yield in 2 steps). H NMR (300 MHz, DMSO-d
6
) δ 1.34 (6H, d, J
=
6.6 Hz), 2.40-2.47 (2H, m), 3.22 (2H, t, J = 7.3 Hz), 3.50 (3H, s), 3.80 (3H, s), 4.14 (2H, s), 5.12-5.25 (1H, m),
+
7
.17-7.26 (1H, m), 7.27-7.31 (4H, m), 7.41 (1H, s). LC-MS (ESI): m/z [M + H ] 360.0
Methyl 4-(benzyl(3-methoxy-3-oxopropyl)amino)-1-methyl-1H-pyrazole-5-carboxylate (18b). The compound 18b
1
was prepared from 16b by the same method as that described for 18a. Yellow oil (68% yield in 2 steps). H NMR (500
MHz, CDCl
3
): δ 2.51 (2H, t, J = 7.5 Hz), 3.34 (2H, t, J = 7.5 Hz), 3.62 (3H, s), 3.89 (3H, s), 4.10 (3H, s), 4.21 (2H, s),
+
7
.25-7.33 (6H, m). LC-MS (ESI): m/z [M + H ] 332.0
4-Benzyl-1-isopropyl-5,6-dihydro-1H-pyrazolo[4,3-b]pyridin-7(4H)-one (19a). To a solution of 18a (4.24 g, 11.8
mmol) in dry THF (80 mL) was added dropwise NaHMDS in THF (6.83 mL, 13.0 mmol) at room temperature. After 1
h, NaHMDS in THF (3.10 mL, 5.90 mmol) was added dropwise. After 30 min, additional NaHMDS in THF (1.86 mL,
3
7
.54 mmol) was added dropwise. After 1 h, 2 M NaOH (59.0 mL, 118 mmol) was added, and the mixture was stirred at
0 °C. After 3 h, the mixture was poured into water (150 mL), and extracted with EtOAc (150 mL + 50 mL). The
4
organic layer was washed with brine, dried over MgSO , and filtered. The filtrate was concentrated in vacuo to afford
1
1
9a as a pale yellow oil (2.89 g, 91% yield). H NMR (300 MHz, DMSO-d
6
) δ 1.33 (6H, d, J = 6.6 Hz), 2.45-2.53 (2H,
+
m), 3.12-3.19 (2H, m), 4.30 (2H, s), 5.16 (1H, quin, J = 6.6 Hz), 7.25-7.42 (6H, m). LC-MS (ESI): m/z [M + H ] 270.3
4-Benzyl-1-methyl-5,6-dihydro-1H-pyrazolo[4,3-b]pyridin-7(4H)-one (19b). The compound 19b was prepared from
1
1
8b by the same method as that described for 19a. Yellow solid (quant in 2 steps). H NMR (500MHz, CDCl
3
): δ 2.51
(2H, t, J = 7.0 Hz), 3.15 (2H, t, J = 7.0 Hz), 4.01 (3H, s), 4.18 (2H, s), 6.95 (1H, s), 7.20-7.31 (5H, m). LC-MS (ESI):
+
m/z [M + H ] 242.2
4-Benzyl-1-isopropyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-b]pyridin-7-amine (21a). A mixture of 19a (900 mg, 3.34
mmol), pyridine (1.32 g, 16.7 mmol), and hydroxylamine hydrochloride (1.16 g, 16.7 mmol) in EtOH (20 mL) was
stirred at 80-90 °C for 6 h. The mixture was concentrated in vacuo. The residue was diluted with aqueous saturated
NaHCO
filtrate was concentrated in vacuo to afford 20a as a pale yellow solid. A mixture of 20a and Raney nickel (slurry in water,
g) in MeOH (20 mL) was stirred under hydrogen atmosphere at room temperature for 2 h. The insoluble materials were
filtered off and washed with MeOH-water (3:1). The filtrate was concentrated in vacuo. The residue was diluted with
aqueous saturated NaHCO , and extracted with EtOAc, washed with brine, dried over MgSO , and filtered. The filtrate
3 4
, and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO , and filtered. The
8
3
4
was concentrated in vacuo, and the residue was purified by column chromatography (silica gel, eluted with 0%- 100%
+
EtOAc in hexane) to give 21a as colorless oil (643 mg, 71% yield in 2 steps). LC-MS (ESI): m/z [M + H ] 271.0
4-Benzyl-1-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-b]pyridin-7-amine (21b). The compound 21b was prepared
1
from 19b by the same method as that described for 21a. Colorless solid (58% yield in 2 steps). H NMR (500MHz,
6
DMSO-d ): δ 1.65-1.69 (1H, m), 1.74 (2H, d, J = 6.5 Hz), 1.82-1.85 (1H, m), 2.68-2.80 (2H, m), 3.72 (3H, s), 3.89-3.91

(1H, m), 3.94 (1H, d, J = 14.0 Hz), 4.14 (1H, d, J = 14.0 Hz), 6.87 (1H, s), 7.23-7.26 (1H, m), 7.31-7.34 (4H, m).
+
LC-MS (ESI): m/z [M + H ] 243.0
tert-Butyl (1-isopropyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-b]pyridin-7-yl)carbamate (23a). To a solution of 21a
(
811 mg, 3 mmol) and Et
mmol) at room temperature, and the mixture was stirred at room temperature for 1 h. The mixture was diluted with water
at room temperature and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO , filtered, and
3 2
N (0.836 mL, 6.00 mmol) in dry THF (10 mL) was added dropwise Boc O (0.766 mL, 3.30
4
concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluted with 20-50% EtOAc in
hexane) to give 22a as a beige solid. A mixture of 22a (1.06 g, 2.86 mmol) and 10% Pd/C (319 mg) in MeOH (20 mL) and
AcOH (2 mL) was stirred under hydrogen atmosphere at room temperature overnight. The catalyst was removed by
filtration, and the filtrate was concentrated in vacuo. The residue was diluted with aqueous saturated NaHCO
3
and
, filtered, and concentrated
) δ 1.24-1.45 (17H,
extracted with EtOAc-THF (3/1). The organic layer was washed with brine, dried over MgSO
4
1
in vacuo to afford 23a as a white solid (780 mg, 93% yield in 2 steps). H NMR (300 MHz, DMSO-d
6
m), 1.63-1.81 (2H, m), 2.89-3.03 (2H, m), 4.23-4.43 (2H, m), 4.72-4.83 (1H, m), 6.87 (1H, s), 7.39 (1H, d, J = 9.2 Hz).
+
LC-MS (ESI): m/z [M + H ] 281.0
tert-Butyl (1-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-b]pyridin-7-yl)carbamate (23b). The compound 23b was
1
prepared from 21b by the same method as that described for 23a. Colorless solid (59% yield in 2 steps). H NMR (300
MHz, CDCl
3
) δ 1.47 (9H, s), 1.86-2.09 (2H, m), 2.89-3.05 (1H, m), 3.11 (1H, brs), 3.24 (1H, dt, J = 12.2, 3.7 Hz),
+
4
.71-5.06 (2H, m), 7.03 (1H, s). LC-MS (ESI): m/z [M + H ] 253.0
tert-Butyl
(4-(5,6-dimethoxynicotinoyl)-1-isopropyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-b]pyridin-7
-
yl)carbamate (24a). A mixture of 23a (1.40 g, 4.99 mmol), 5,6-dimethoxynicotinic acid (1.19 g, 6.49 mmol), HATU
(
2.47 g, 6.49 mmol), and Et N (2.09 mL, 15.0 mmol) in dry DMF (15 mL) was stirred at room temperature overnight.
3
The mixture was diluted with water at room temperature and extracted with EtOAc. The organic layer was separated,
washed with brine, dried over MgSO , filtered, and concentrated in vacuo. The residue was purified by column
4
chromatography (NH silica gel, eluted with 10-50% EtOAc in hexane) to give 24a (2.11 g, 95% yield) as a colorless
+
solid. LC-MS (ESI): m/z [M + H ] 446.1
N-(4-(5,6-Dimethoxynicotinoyl)-1-isopropyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-b]pyridin-7-yl)-2-(trifluorometho
xy)benzamide (26). To a solution of 24a (2.10 g, 4.71 mmol) in MeOH (20 mL) was added 4 M HCl (EtOAc solution,
1
1.8 mL, 47.1 mmol) at room temperature, and the mixture was stirred at room temperature overnight. The mixture was
concentrated in vacuo, and the residue was washed with EtOAc-IPE to give
7-amino-1-isopropyl-6,7-dihydro-1H-pyrazolo[4,3-b]pyridin-4(5H)-yl)(5,6-dimethoxypyridin-3-yl)methanone
tri-hydrochloride 25a (2.11 g, 98% yield) as a colorless powder. A mixture of 25a (2.11 g, 4.64 mmol),
-(trifluoromethoxy)benzoic acid (1.24 g, 6.03 mmol), HATU (2.29 g, 6.03 mmol), and Et N (2.59 mL, 18.6 mmol) in
dry DMF (15 mL) was stirred at room temperature under N for 3 h. The mixture was diluted with aqueous saturated
NaHCO at room temperature and extracted with EtOAc. The organic layer was separated, washed with brine, dried
over MgSO , filtered, and concentrated in vacuo. The residue was purified by column chromatography (NH silica gel,
(
2
3
2
3
4
eluted with 50-100% EtOAc in hexane) and triturated with EtOAc-IPE to give 26 (1.74 g, 70% yield) as a colorless
1
powder. H NMR (300 MHz, CDCl
3
) δ 1.42 (3H, d, J = 6.6 Hz), 1.48 (3H, d, J = 6.6 Hz), 2.25 (2H, d, J = 3.0 Hz), 3.56
(1H, brs), 3.91 (3H, s), 4.07 (3H, s), 4.22 (1H, d, J = 10.4 Hz), 4.47 (1H, dt, J = 13.0, 6.6 Hz), 5.53-5.68 (1H, m), 6.79
(1H, d, J = 8.5 Hz), 7.25-7.27 (1H, m), 7.28-7.36 (1H, m), 7.39-7.50 (1H, m), 7.51-7.62 (1H, m), 7.89 (1H, d, J = 1.9
+
Hz), 7.99 (1H, dd, J = 7.7, 1.9 Hz), 8.22 (1H, brs). LC-MS (ESI): m/z [M + H ] 534.2, Anal. Calcd for C25
H
26
F
N
3 5
O
5
: C,
5
6.28; H, 4.91; N, 13.13. Found: C, 56.30; H, 4.99; N, 13.00.
tert-Butyl (4-(3,4-dimethoxybenzoyl)-1-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-b]pyridin-7-yl) -carbamate

(
24b). To a solution of 23b (800 mg, 3.17 mmol) in pyridine (10 mL) was added 3,4-dimethoxybenzoyl chloride (700
mg, 3.49 mmol) at room temperature, and the mixture was stirred at room temperature overnight. The mixture was
diluted with water at room temperature and concentrated in vacuo. The residue was diluted with water at room
4
temperature and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO , filtered, and
concentrated in vacuo. The residue was purified by column chromatography (NH silica gel, eluted with 50-100%
1
EtOAc in hexane) to give 24b (1.29 g, 98% yield) as a colorless solid. H NMR (300 MHz, CDCl
3
) δ 1.47 (9H, s), 2.09
(
(
(
2H, d, J = 3.6 Hz), 3.36-3.56 (1H, m), 3.81 (3H, s), 3.90 (3H, s), 3.92 (3H, s), 4.11 (1H, brs), 4.92 (1H, brs), 4.96-5.09
+
1H, m), 6.87 (1H, d, J = 8.1 Hz), 7.00-7.11 (2H, m). LC-MS (ESI): m/z [M + H ] 417.0
7-Amino-1-methyl-6,7-dihydro-1H-pyrazolo[4,3-b]pyridin-4(5H)-yl)(3,4-dimethoxyphenyl)methanone
di-hydrochloride (25b). To a solution of 24b (1.29 g, 3.10 mmol) in MeOH (10 mL) was added 4 M HCl (CPME
solution, 7.74 mL, 31.0 mmol) at room temperature, and the mixture was stirred at room temperature overnight. The
mixture was concentrated in vacuo, and the residue was collected by filtration, and triturated with EtOAc to give 25b
+
(
1.17 g, 97% yield) as a colorless powder. LC-MS (ESI): m/z [M + H ] 317.0.
2
-Chloro-N-(4-(3,4-dimethoxybenzoyl)-1-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-b]pyridin-7-yl)benzamide
27). A mixture of 25b (645 mg, 1.66 mmol), 2-chlorobenzoic acid (285 mg, 1.82 mmol), HATU (819 mg, 2.15 mmol),
and Et N (0.69 mL, 4.97 mmol) in DMF (5.0 mL) was stirred at room temperature for 5h. The mixture was diluted with
water at room temperature and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO
(
3
4
,
filtered, and concentrated in vacuo. The residue was purified by column chromatography (NH silica gel, eluted with
1
3
0-100% EtOAc in hexane) and triturated with IPE to give 27 (623 mg, 83% yield) as a colorless powder. H NMR
3
(300 MHz, CDCl ) δ 2.13-2.40 (2H, m), 3.50 (1H, t, J = 11.0 Hz), 3.85 (3H, s), 3.90 (3H, s), 3.92 (3H, s), 4.03-4.30 (1H,
m), 5.54 (1H, dt, J = 7.9, 4.0 Hz), 6.61 (1H, brs), 6.88 (1H, d, J = 8.1 Hz), 6.99-7.12 (2H, m), 7.31-7.49 (3H, m),
+
7
.62-7.74 (1H, m). LC-MS (ESI): m/z [M + H ] 455.0
(
S)-N-(4-(5,6-Dimethoxynicotinoyl)-1-isopropyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-b]pyridin-7-yl)-2-(trifluorom
ethoxy)benzamide (4). Optical resolution of 26 (300 mg, 0.56 mmol) was conducted by SFC (CHIRALPAK AD, 50 mm
ID × 500 mm L, CO
2
/MeOH = 700/300). The obtained solid (tR2) was recrystallized from EtOAc-IPE to give 4 (123
1
mg) as colorless crystals. 99.9%ee. H NMR (300 MHz, CDCl
3
) δ 1.42 (3H, d, J = 6.4 Hz), 1.48 (3H, d, J = 6.6 Hz),
2
5
.16-2.30 (2H, m), 3.45-3.66 (1H, m), 3.91 (3H, s), 4.07 (3H, s), 4.12-4.35 (1H, m), 4.47 (1H, quin, J = 6.6 Hz),
.56-5.67 (1H, m), 6.78 (1H, d, J = 8.5 Hz), 7.24-7.28 (1H, m), 7.32 (1H, d, J = 8.3 Hz), 7.41-7.50 (1H, m), 7.52-7.61
+
(
1H, m), 7.89 (1H, d, J = 1.7 Hz), 8.00 (1H, dd, J = 7.7, 1.9 Hz), 8.23 (1H, brs). LC-MS (ESI): m/z [M + H ] 534.3,
26 3 5 5
Anal. Calcd for C25H F N O : C, 56.28; H, 4.91; N, 13.13. Found: C, 56.15; H, 4.97; N, 13.08. mp: 211-212 °C.
1-(3,4-Dimethoxybenzoyl)-3-phenylpiperidin-4-one (29). To a solution of 28 (1.0 g, 5.71 mmol) in pyridine (10 mL)
was added 3,4-dimethoxybenzoyl chloride (1.37 g, 6.85 mmol) at 0 °C, and the mixture was stirred at room temperature
overnight. The volatile components were evaporated in vacuo, and the residue was partitioned between EtOAc and
aqueous 1 M HCl. The organic layer was separated, washed with aqueous saturated NaHCO
Na SO , filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluted
with 30-100% EtOAc in hexane) to give 29 (1.38 g, 71% yield) as a pale yellow solid. H NMR (CDCl
3
and brine, dried over
2
4
1
3
) : 2.65 (2H,
brs), 3.55-3.83 (3H, m), 3.90 (3H, s), 3.91 (3H, s), 4.26-4.65 (2H, m), 6.87 (1H, d, J = 8.7 Hz), 7.01-7.10 (2H, m), 7.15
+
(
2H, d, J = 6.2 Hz), 7.28-7.41 (3H, m). LC-MS (ESI): m/z [M + H ] 340.3
(
4-Amino-3-phenylpiperidin-1-yl)(3,4-dimethoxyphenyl)methanone (30). To a solution of 29 (1.35 g, 3.98 mmol)
and Et
room temperature. After being refluxed for 2 h, the mixture was concentrated in vacuo. The residue was partitioned
between EtOAc and water, and the organic layer was separated, washed with brine, dried over Na SO , filtered, and
3
N (0.83 mL, 5.97 mmol) in EtOH (50 mL) was added hydroxylamine hydrochloride (415 mg, 5.97 mmol) at
2
4

concentrated in vacuo to give oxime derivative (1.40 g, 3.95 mmol, 99%) as a colorless solid. A mixture of the oxime
1.35 g, 3.81 mmol) and Raney Ni (slurry in water, 10 g, Kawaken NDHT-90) in MeOH (40 mL) was stirred under
(
hydrogen atmosphere at room temperature overnight. The catalyst was removed by filtration, and the filtrate was
concentrated in vacuo. The residue was purified by column chromatography (NH silica gel, eluted with 50-100%
+
EtOAc in hexane) to give 30 (1.15 g, 89% yield) as a colorless amorphous solid. LC-MS (ESI): m/z [M + H ] 341.2
2
-Chloro-N-(1-(3,4-dimethoxybenzoyl)-3-phenylpiperidin-4-yl)benzamide (31). To a solution of 30 (500 mg, 1.47
mmol) and Et N (0.307 mL, 2.20 mmol) in THF (10 mL) was added 2-chlorobenzoyl chloride (0.223 mL, 1.76 mmol)
at 0 °C. After being stirred at room temperature overnight, the mixture was partitioned between EtOAc and water. The
organic layer was separated, washed with aqueous 1 M HCl, aqueous saturated NaHCO , and brine, dried over Na SO
3
3
2
4
,
filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluted with 0-100%
+
EtOAc in hexane) to give 31 (582 mg, 83% yield) as a colorless amorphous solid. LC-MS (ESI): m/z [M + H ] 479.0,
4
81.1
tert-Butyl trans-4-(((benzyloxy)carbonyl)amino)-3-phenylpiperidine-1-carboxylate (33). To a solution of 32 (1.0 g,
.27 mmol) in toluene (25 mL) was added DPPA (0.774 mL, 3.60 mmol) followed by Et N (0.50 mL, 3.60 mmol) at
3
3
room temperature, and the mixture was stirred at reflux for 1 h. Benzyl alcohol (0.409 mL, 3.93 mmol) was added to the
mixture at room temperature, and the mixture was stirred at reflux for 5 h. The mixture was quenched with water at
2 4
room temperature and extracted with EtOAc. The organic layer was washed with water and brine, dried over Na SO ,
filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluted with
1
1
0-100% EtOAc in hexane) to give 33 (1.35 g, quant) as a colorless oil. H NMR (DMSO-d
6
) : 1.46 (9H, s), 2.10-2.27
(
1H, m), 2.46-2.64 (1H, m), 2.67-2.98 (2H, m), 3.83-4.03 (1H, m), 4.11-4.30 (2H, m), 4.47 (1H, brs), 4.70 (1H, d, J =
.8 Hz), 4.95 (2H, s), 7.09-7.49 (10H, m).
tert-Butyl trans-4-amino-3-phenylpiperidine-1-carboxylate (34). A mixture of 33 (1.3 g, 3.17 mmol) and 10% Pd-C
5
(200 mg) in MeOH (30 mL) was stirred under hydrogen atmosphere at room temperature overnight. The catalyst was
1
removed by filtration, and the filtrate was concentrated in vacuo to give 34 (864 mg, 99% yield) as a colorless oil. H
NMR (CDCl
.01 (1H, td, J = 10.7, 3.9 Hz), 3.99-4.35 (2H, m), 7.03-7.42 (5H, m).
tert-Butyl (3R,4S)-4-(2-chlorobenzamido)-3-phenylpiperidine-1-carboxylate (35a). To a solution of 34a (200 mg,
.72 mmol) and Et N (0.151 mL, 1.09 mmol) in THF (10 mL) was added 2-chlorobenzoyl chloride (0.110 mL, 0.87
mmol) at 0 °C. After being stirred at room temperature overnight, the mixture was partitioned between EtOAc and water.
The organic layer was separated, washed with aqueous 1 M HCl, aqueous saturated NaHCO , and brine, dried over
Na SO , filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluted with
-50% EtOAc in hexane) to give 35a (282 mg, 94% yield) as a colorless amorphous solid. H NMR (CDCl
brs), 1.80-2.04 (2H, m), 3.24 (1H, q, J = 4.8 Hz), 3.37-4.07 (4H, m), 4.63 (1H, tt, J = 8.2, 4.2 Hz), 5.99 (1H, brs),
3
) : 1.46 (9H, s), 1.74 (3H, brs), 1.83-2.04 (1H, m), 2.39 (1H, td, J = 11.0, 4.1 Hz), 2.66-2.93 (2H, m),
3
0
3
3
2
4
1
0
3
) : 1.45 (9H,
7
2
0
.18-7.37 (8H, m), 7.46-7.53 (1H, m).
-Chloro-N-((3R,4S)-1-(3,4-dimethoxybenzoyl)-3-phenylpiperidin-4-yl)benzamide (31a). A mixture of 35a (279 mg,
.67 mmol) and 4 M HCl (EtOAc solution, 10 mL) was stirred at room temperature for 2 h. The volatile components
were evaporated in vacuo to give amine derivative as a colorless solid. To a solution of the amine derivative (230 mg)
in pyridine (10mL) was added 3,4-dimethoxybenzoyl chloride (158 mg, 0.79 mmol) at 0 °C, and the mixture was stirred
at room temperature overnight. The volatile components were evaporated in vacuo, and the residue was partitioned
between EtOAc and aqueous 1 M HCl. The organic layer was separated, washed with aqueous saturated NaHCO
brine, dried over Na SO , filtered, and concentrated in vacuo. The residue was purified by column chromatography
silica gel, eluted with 30-100% EtOAc in hexane) to give 31a (279 mg, 89% yield in 2 steps) as pale yellow crystals.
3
and
2
4
(

1
H NMR (DMSO-d
6
) : 1.51-1.90 (1H, m), 1.90-2.13 (1H, m), 3.16-3.30 (1H, m), 3.35-3.50 (1H, m), 3.76 (7H, brs),
4
.12 (2H, brs), 4.47-4.76 (1H, m), 6.81-7.12 (4H, m), 7.12-7.51 (8H, m), 8.57 (1H, d, J = 8.9 Hz). LC-MS (ESI): m/z
+
[
M + H ] 479.2, 481.1
tert-Butyl (3S,4R)-4-(2-chlorobenzamido)-3-phenylpiperidine-1-carboxylate (35b). The compound 35b was
1
prepared from 34b by the same method as that described for 35a. A colorless amorphous powder (38% yield): H NMR
(
CDCl
3
) : same as 35a. LC-MS (ESI) same as 35a.
2-Chloro-N-((3S,4R)-1-(3,4-dimethoxybenzoyl)-3-phenylpiperidin-4-yl)benzamide (31b). The compound 31b was
1
prepared from 35b by the same method as that described for 31a. A colorless amorphous powder (98% in 2 steps): H
NMR (CDCl ) : same as 31a. LC-MS (ESI) same as 31a.
3
tert-Butyl trans-4-(2-chlorobenzamido)-3-phenylpiperidine-1-carboxylate (35). The compound 35 was prepared
1
from 34 by the same method as that described for 35a. A colorless amorphous powder (82% yield): H NMR (CDCl
3
) :
1
.47 (9H, s), 2.26-2.45 (1H, m), 2.61-2.76 (1H, m), 2.76-2.90 (1H, m), 2.90-3.06 (1H, m), 4.17-4.38 (2H, m), 4.38-4.59
(1H, m), 5.84 (1H, d, J = 8.3 Hz), 7.11-7.40 (10H, m).
2-Chloro-N-((3R,4R)-1-(3,4-dimethoxybenzoyl)-3-phenylpiperidin-4-yl)benzamide (31c).
Trans racemate,
2-chloro-N-(trans-1-(3,4-dimethoxybenzoyl)-3-phenylpiperidin-4-yl)benzamide, was prepared from 35 by the same
method as that described for 31a. Chiral resolution of the trans racemate was conducted by chiral preparative HPLC
1
(
CHIRALPAK AS-H, 20 x 250 mm, CO
DMSO-d
2
(770) / MeOH (230), tR1). A colorless crystals (70% yield): H NMR
(
6
)  1.37-1.68 (1H, m), 1.82-2.17 (1H, m), 2.70-2.90 (1H, m), 2.90-3.30 (2H, m), 3.78 (6H, s), 3.61-4.06 (1H,
m), 4.42 (2H, d, J = 10.9 Hz), 6.88 (1H, d, J = 6.8 Hz), 6.92-7.05 (3H, m), 7.10-7.49 (8H, m), 8.27 (1H, d, J = 8.9 Hz).
+
LC-MS (ESI): m/z [M + H ] 479.2, 481.1.
2-Chloro-N-((3S,4S)-1-(3,4-dimethoxybenzoyl)-3-phenylpiperidin-4-yl)benzamide
(31d).
Trans
racemate,
2-chloro-N-(trans-1-(3,4-dimethoxybenzoyl)-3-phenylpiperidin-4-yl)benzamide, was prepared from 35 by the same
method as that described for 31a. Chiral resolution of the trans racemate was conducted by chiral preparative HPLC
1
(CHIRALPAK AS-H, 20 x 250 mm, CO
2
(770) / MeOH (230), tR2). A colorless crystals (67% yield): H NMR
(
DMSO-d ) : same as 31c, LC-MS (ESI) same as 31c.
6
Methyl 8-chloroquinoxaline-6-carboxylate (37). Glyoxal (40% aqueous solution, 20.0 mL, 175 mmol) was added to a
solution of 36 (23.4 g, 117 mmol) in MeOH (160 mL) and THF (80 mL) at room temperature. The mixture was stirred
at room temperature overnight. The organic solvent was evaporated in vacuo, and the resulting solid was collected,
1
washed with water, and dried in vacuo to give 37 (25.5 g, 98% yield) as a light brown powder. H NMR (300 MHz,
DMSO-d
-Chloroquinoxaline-6-carboxylic acid (38). To a solution of 37 (25.5 g, 115 mmol) in THF (200 mL) and MeOH
200 mL) was added aqueous 4 M NaOH (100 mL, 400 mmol) at room temperature. After being stirred at room
temperature overnight, the mixture was concentrated in vacuo. The residue was dissolved in water and neutralized with
6
) δ 3.97 (3H, s), 8.39 (1H, d, J = 1.7 Hz), 8.59 (1H, d, J = 1.7 Hz), 8.94-9.48 (2H, m).
8
(
6
9
M HCl (pH = 5). The precipitate was collected by filtration, washed with water and dried in vacuo to give 38 (23.3 g,
1
8% yield) as light brown powder. H NMR (300 MHz, DMSO-d
6
) δ 3.75 (1H, brs), 8.42 (1H, d, J = 1.7 Hz), 8.50 (1H,
d, J = 1.5 Hz), 9.09 (2H, s).
tert-Butyl (3S,4R)-4-(1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamido)-3-phenylpiperidine-1
carboxylate (39). To a solution of 34b (5 g, 18.1 mmol), 1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxylic acid
4.21 g, 21.7 mmol), and HOBt (3.67 g, 27.1 mmol) in DMF (60 mL) was added EDC (4.76 mL, 27.1 mmol) at 0 °C.
-
(
After being stirred at room temperature overnight, the mixture was concentrated in vacuo. The residue was partitioned
between EtOAc and water, and the organic layer was separated, washed with brine, dried over Na SO , filtered, and
concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluted with 10-100% EtOAc in
2
4

1
hexane) to give 39 (7.76 g, 95% yield) as a colorless amorphous solid. H NMR (300 MHz, DMSO-d
6
) δ 1.41 (9H, brs),
1
6
1
.60-1.91 (2H, m), 3.14 (1H, d, J = 3.6 Hz), 3.37-3.51 (1H, m), 3.58-3.87 (3H, m), 3.90 (3H, s), 4.49 (1H, brs),
.80-7.47 (6H, m), 8.31 (1H, d, J = 8.9 Hz).
-Methyl-N-((3S,4R)-3-phenylpiperidin-4-yl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide hydrochloride (40).
A mixture of 39 (7.75 g, 17.1 mmol) and 4 M HCl (EtOAc solution, 50 mL, 200 mmol) was stirred at room temperature
1
for 3 h, and the mixture was concentrated in vacuo to give 40 (6.50 g, 98% yield) as a colorless solid. H NMR (300
6
MHz, DMSO-d ) δ 1.87 (1H, d, J = 12.6 Hz), 2.23 (1H, ddd, J = 14.1, 10.1, 4.1 Hz), 3.13-3.34 (3H, m), 3.45 (1H, dt, J
=
13.4, 3.9 Hz), 3.83 (3H, s), 4.05 (1H, t, J = 13.0 Hz), 4.66 (1H, dd, J = 9.7, 3.1 Hz), 7.08-7.34 (5H, m), 7.40 (1H, s),
+
8
.75 (1H, d, J = 9.8 Hz), 9.08 (1H, brs), 9.65 (1H, brs). LC-MS (ESI): m/z [M + H ] 353.2
N-((3S,4R)-1-(8-Chloroquinoxaline-6-carbonyl)-3-phenylpiperidin-4-yl)-1-methyl-3-(trifluoromethyl)-1H-pyrazol
e-5-carboxamide (5). To a solution of 40 (6.50 g, 16.72 mmol), 38 (3.49 g, 16.7 mmol), and HOBt (3.39 g, 25.1 mmol)
in DMF (150 mL) was added EDC (4.40 mL, 25.1 mmol) at 0 °C. After being stirred at room temperature for 3 h, the
mixture was concentrated in vacuo. The residue was partitioned between EtOAc and water, and the organic layer was
2 4
separated, washed with brine, dried over Na SO , filtered, and concentrated in vacuo. The residue was purified by
column chromatography (silica gel, eluted with 30-100% EtOAc in hexane) to give 5 (5.39 g, 59% yield) as a colorless
amorphous solid. The obtained amorphous (1.50 g) was recrystallized from EtOAc-heptane to give 5 (1.18 g) as
1
colorless crystals. H NMR (300 MHz, DMSO-d
6
) δ 1.10-1.40 (2H, m), 1.51-2.19 (2H, m), 3.42 (2H, brs), 3.62 (2H, d,
J = 13.6 Hz), 3.89-4.07 (1H, m), 4.09-4.44 (1H, m), 4.62 (1H, brs), 6.84-7.57 (6H, m), 7.87-8.23 (2H, m), 8.46 (1H, d, J
+
=
9.0 Hz), 8.87-9.26 (2H, m). LC-MS (ESI): m/z [M + H ] 543.1. Anal. Calcd for C26
H
22ClF
3 6
N O
2
: C,57.52; H,4.08;
N,15.48. Found: C,57.39; H,3.98; N,15.37. mp: 129-131 °C.
X-ray structure analysis
26 3 5 5
Crystal data for compound 4: C25H F N O , MW = 533.51; crystal size, 0.12 x 0.10 x 0.02 mm; colourless, plate;
triclinic, space group P1, a = 5.00246(8) Å, b = 22.5164(3) Å, c = 24.0321(5) Å, α = 109.6190(15)°, β = 92.2852(15)°,
3
3
-1
γ = 90.7764(12)°, V = 2546.69(8) Å , Z = 4, Dx = 1.391 g/cm , T = 100 K, μ = 0.967 mm , λ = 1.54187 Å, R
wR = 0.178, Flack ParameterDD = -0.08(10). All measurements were made on a Rigaku XtaLAB P200 diffractometer
using graphite monochromated Cu-Kαradiation. The structure was solved by direct methods with SIR2008 and was
1
= 0.064,
2
1
6
2
17
refined using full-matrix least-squares on F with SHELXL-2014/7. All non-H atoms were refined with anisotropic
displacement parameters. CCDC 1511207 for compound 4 contains the supplementary crystallographic data for this
paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via
http://www.ccdc.cam.ac.uk/Community/Requestastructure/Pages/DataRequest.aspx?.
3 6 2
Crystal data for compound 5: C26H22ClF N O , MW = 542.95; crystal size, 0.06 x 0.05 x 0.04 mm; colourless, block;
triclinic, space group P1, a = 10.2798(3) Å, b = 10.6917(3) Å, c = 11.8027(3) Å, α = 103.716(2)°, β = 91.308(2)°, γ =
3
3
-1
9
0
6.677(2)°, V = 1249.97(6) Å , Z = 2, Dx = 1.442 g/cm , T = 100 K, μ = 1.878 mm , λ = 1.54187 Å, R
1
= 0.088, wR
2
=
1
8
.233, Flack Parameter = 0.043(13). All measurements were made on a Rigaku XtaLAB P200 diffractometer using
16
graphite monochromated Cu-Kαradiation. The structure was solved by direct methods with SIR2008 and was refined
2
17
using full-matrix least-squares on F with SHELXL-2014/7. All non-H atoms were refined with anisotropic
displacement parameters. CCDC 1511402 for compound 5 contains the supplementary crystallographic data for this
paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via
http://www.ccdc.cam.ac.uk/Community/Requestastructure/Pages/DataRequest.aspx?.

(A)
(
B)
Figur
e
8.
(A)
ORTE
P
of
compo
und 4,
thermal ellipsoids are drawn at 30%
probability. (B) ORTEP of compound 5, thermal ellipsoids are drawn at 20% probability.
Biology
Preparation of human SPT2 enzyme
cDNA encoding human SPT1, human SPT2, and human ssSPTa were isolated by polymerase chain reaction (PCR) with
primers and subcloned to make expression vectors. For preparation of SPT2 enzyme, FreeStyle293 cells (Life
Technologies, Carlsbad, CA) were transfected with expression plasmids for human SPT1, human SPT2, and human
ssSPTa and cultured for 3 days. Cells were homogenized in 50 mM Hepes buffer (pH 7.5) containing 250 mM sucrose,
5
mM EDTA, 5 mM DTT, and Complete, EDTA-free (Roche Applied Science, Penzberg, Upper Bavaria, Germany).
Cell homogenates were centrifuged and the supernatant was recovered. Total membrane fractions were isolated by
ultracentrifugation. Pellets were re-suspended in 50 mM Hepes buffer (pH 7.5) containing 5 mM EDTA, 5 mM DTT,
and Complete, EDTA-free and stored at -80°C. The protein concentration was determined with the CBB Protein Assay.
Enzyme assay
The enzyme reaction was run in assay buffer, which consisted of 100 mM Hepes (pH 8.0), 2.5 mM EDTA, 5 mM DTT,
and 0.01% bovine serum albumin (fatty acid free) in a 384-well assay plate at a volume of 20 μL format. Briefly, 5 μL
of a tested compound and 10 μL of 100 μg/mL SPT2-expressed-membrane dissolved in the assay buffer, were mixed
and incubated for 60 minutes. Then, the enzyme reaction was started with the addition of 5 μL of substrate solution
containing 2 mM L-serine and 20 μM palmitoyl-CoA in the assay buffer. After 15-minute incubation at room
temperature, the reaction was terminated by adding 20 μL of 2% formic acid. Then, the reaction was added with 40 μL
of acetonitrile containing 600 nM C17-sphinganine as internal standard. High-throughput online solid phase extraction
was performed using a RapidFire® 300 (Agilent Technologies, Santa Clara, CA). Mass spectrometric analysis was
TM
performed using an API-4000 triple quadrupole mass spectrometer (AB SCIEX, Framingham, MA) in positive SRM
mode. The SRM transitions for 3-ketodihydrosphingosine as a reaction product and C17-sphinganine were set as
3
00.5/270.3 and 288.4/60.2, respectively. Analytical data was acquired using Analyst software version 1.5.0 (AB
SCIEX, Framingham, MA) and 300.5/270.3 was divided by 288.4/60.2 for calibration. IC50 values for the test

compounds were calculated using XLfit software (IDBS, London, UK).
Growth inhibition assay
HCC4006 cells were cultured in RPMI medium containing 10% fetal bovine serum and penicillin/streptomycin. Cells
were dispensed into a 384 well culture plate at a density of 250 cells/well in 40 μL of the culture medium and cultured
overnight. The cells were treated with 10 μL of test compounds and cultured for 5 days. The medium was removed and
replaced with 30 μL of CellTiter-Glo Luminescent Cell Viability Assay reagent (Promega, Fitchburg, WI).
Luminescence was measured with EnVision (PerkinElmer, Waltham, MA). IP values (concentration at the inflection
point of dose-response curve) for the test compounds were calculated using XLfit software.
Cassette dosing test to mice
Test compounds were administered intravenously (0.1 mg/kg, 10 compounds in one) or orally (1 mg/kg, 5
compounds in one) by cassette dosing to non-fasted mice. After administration, blood samples were collected and
centrifuged to obtain the plasma fraction. The plasma samples were deproteinized by mixing with acetonitrile
followed by centrifugation. The compound concentrations in the supernatant were measured by LC/MS/MS.
References
(
(
(
1) Merrill, A. H.; Jones, D. D. An update of the enzymology and regulation of sphingomyelin metabolism. Biochim.
Biophys. Acta.1990, 1044, 1–12.
2) Hanada, K. Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism. Biochim. Biophys. Acta. 2003,
1
632, 16–30.
3) Schissel, S. L.; Schuchman, E. H.; Williams, K. J,; Tabas, I. Zn2+-stimulated sphingomyelinase is secreted by
many cell types and is a product of the acid sphingomyelinase gene. J. Biol. Chem. 1996, 271, 18431–18436.
4) Futerman, A. H.; Hannun, Y. A.; The complex life of simple sphingolipids. EMBO Rep., 2004, 5, 777–782.
5) Pettus, B. J.; Bielawska, A.; Subramanian, P.; Wijesinghe, D. S.; Maceyka, M.; Leslie, C. C.; John H. E.; Jessica
F.; Patrick R.; Yusuf, A. H.; Charles, E. C. Ceramide 1-phosphate is a direct activator of cytosolic phospholipase
A2. J. Biol. Chem., 2004, 279, 11320–11326.
(
(
(
(
(
(
6) Modrak, D. E.; Gold, D. V.; Goldenberg, D. M.; Sphingolipid targets in cancer therapy. Mol. Cancer Ther., 2006, 5,
2
00–208.
7) Ogretmen, B.; Hannun, Y. A. Biologically active sphingolipids in cancer pathogenesis and treatment. Nat. Rev.
Cancer, 2004, 4, 604–616.
8) Kok, J. W.; Sietsma, H. Sphingolipid metabolism enzymes as targets for anticancer therapy. Curr. Drug Targets,
2
004, 5, 375–382.
9) Youn-Sun, L.; Kyeong-Mi, C.; Seunghyun, L.; Dong-Mi, S.; Kwang-Sik, Y.; Yong, L.; Yong-Moon, L.; Jin-Tae,
H.; Yeo-Pyo, Y.; Hwan-Soo, Y. Myriocin, a serine palmitoyltransferase inhibitor, suppresses tumor growth in a
murine melanoma model by inhibiting de novo sphingolipid synthesis. Cancer Biology & Therapy, 2012, 132,
9
2–100.
(
(
10) Sano, O; Kazetani, K; Adachi, R; Kurasawa, O; Kawamoto, T; Iwata, H. Using a biologically annotated library to
analyze the anticancer mechanism of serine palmitoyl transferase (SPT) inhibitors. FEBS open bio., 2017, 7,
4
95–503.
11) Genin, M. J.; Valcarcel, I. G.; Holloway, W. G.; Lamar, J.; Mosior, M.; Hawkins, E.; Estridge, T.; Weidner, J.;
Seng, T.; Yuek, D.; Adams, L. A.; Weller, J.; Reynolds, V. L.; Brozinick, J. T. Imidazopyridine and
Pyrazolopiperidine Derivatives as Novel Inhibitors of Serine Palmitoyl Transferase. J. Med. Chem. 2016, 59,
5
904−5910.

(
(
12) Werbel, L, M; Kesten, S, J; Turner, W, R. Structure–activity relationships of antimalarial indolo[3,2-c]quinolines
1,2]. Eur. J. Med. Chem. 1993, 28, 837–852.
13) Shirai, J; Yoshikawa, T; Yamashita, M; Yamamoto, Y; Kawamoto, M; Tarui, N; Kamo, I; Hashimoto, T; Ikeura,
Y. Design, structure–activity relationship, and highly efficient asymmetric synthesis of
[
3
-phenyl-4-benzylaminopiperidine derivatives as novel neurokinin-1 receptor antagonists. Bioorg. Med. Chem.
011, 19, 6430–6446.
2
(
(
(
14) Adachi, R; Asano, Y; Ogawa, K; Oonishi, M; Tanaka, Y; Kawamoto, T. Pharmacological characterization of
synthetic serine palmitoyltransferase inhibitors by biochemical and cellular analyses. Biochem. Biophys. Res.
Commum. in press.
15) Yaguchi, M; Shibata, S; Satomi, Y; Hirayama, M; Adachi, R; Asano, Y; Kojima, T; Hirata, Y; Mizutani, A; Kiba,
A; Sagiya, Y. Antitumor activity of a novel and orally available inhibitor of serine palmitoyltransferase. Biochem.
Biophys. Res. Commum. 2017, 484, 493–500.
16) Burla, M. C.; Caliandro, R., Camalli, M.; Carrozzini, B.; Cascarano, G. L.; De Caro, L., Giacovazzo, C.; Polidori,
G. Siliqi, D.; Spagna R. IL MILIONE: a suite of computer programs for crystal structure solution of proteins. J.
Appl. Cryst 2007, 40, 609.
(
(
17) Sheldrick, G.M. A short history of SHELX. Acta Cryst. A 2008, 64, 112.
18) Flack, H. D. On enantiomorph-polarity estimation. Acta Cryst. A 1983, 39, 876.