Discovery of Novel and Potent Stearoyl Coenzyme A Desaturase 1 (SCD1) Inhibitors as Anticancer Agents

Article abstract of DOI:10.1016/j.bmc.2017.05.016

A lead compound A was identified previously as an stearoyl coenzyme A desaturase (SCD) inhibitor during research on potential treatments for obesity. This compound showed high SCD1 binding affinity, but a poor pharmacokinetic (PK) profile and limited chem

Full text of DOI:10.1016/j.bmc.2017.05.016

Accepted Manuscript
Discovery of Novel and Potent Stearoyl Coenzyme A Desaturase 1 (SCD1)
Inhibitors as Anticancer Agents
Keisuke Imamura, Naoki Tomita, Youichi Kawakita, Yoshiteru Ito, Kouji Ono,
Noriyuki Nii, Tohru Miyazaki, Kazuko Yonemori, Michiko Tawada, Hiroyuki
Sumi, Yoshihiko Satoh, Yukiko Yamamoto, Ikuo Miyahisa, Masako Sasaki,
Yoshinori Satomi, Megumi Hirayama, Ryuichi Nishigaki, Hironobu Maezaki
PII:
S0968-0896(17)30209-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.016
BMC 13734
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 February 2017
6 May 2017
8 May 2017
Please cite this article as: Imamura, K., Tomita, N., Kawakita, Y., Ito, Y., Ono, K., Nii, N., Miyazaki, T., Yonemori,
K., Tawada, M., Sumi, H., Satoh, Y., Yamamoto, Y., Miyahisa, I., Sasaki, M., Satomi, Y., Hirayama, M., Nishigaki,
R., Maezaki, H., Discovery of Novel and Potent Stearoyl Coenzyme A Desaturase 1 (SCD1) Inhibitors as Anticancer
Agents, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.05.016
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Title
Discovery of Novel and Potent Stearoyl Coenzyme A
Desaturase 1 (SCD1) Inhibitors as Anticancer Agents
Keisuke Imamuraa, Naoki Tomitab, Youichi Kawakita a, Yoshiteru Itoa, Kouji Onoa, Noriyuki Nii a, Tohru
Miyazaki a, Kazuko Yonemori a, Michiko Tawada a, Hiroyuki Sumi a, Yoshihiko Satoh a, Yukiko Yamamotoa,
Ikuo Miyahisa a, Masako Sasaki a, Yoshinori Satomi a, Megumi Hirayama a, Ryuichi Nishigaki a, Hironobu
Maezaki a
a
Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd., 26-1, Muraokahigashi 2-chome,
Fujisawa, Kanagawa 251-8555, Japan
b Corporate Finance Department, Takeda Pharmaceutical Company Ltd., 12-10, Nihonbashi 2-chome, Chuo-ku,
Tokyo 103-8668, Japan

Abstract
A lead compound A was identified previously as an stearoyl coenzyme A desaturase (SCD) inhibitor
during research on potential treatments for obesity. This compound showed high SCD1 binding affinity, but a
poor pharmacokinetic (PK) profile and limited chemical accessibility, making it suboptimal for use in
anticancer research. To identify potent SCD1 inhibitors with more promising PK profiles, we newly designed
a series of 'non-spiro' 4, 4-disubstituted piperidine derivatives based on molecular modeling studies. As a
result, we discovered compound 1a, which retained moderate SCD1 binding affinity. Optimization around 1a
was
accelerated
by
analyzing
Hansch–Fujita
and
Hammett
constants
to
obtain
4-phenyl-4-(trifluoromethyl)piperidine derivative 1n. Fine-tuning of the azole moiety of 1n led to compound
1o (T-3764518), which retained nanomolar affinity and exhibited an excellent PK profile. Reflecting the good
potency and PK profile, orally administrated compound 1o showed significant pharmacodynamic (PD) marker
reduction (at 0.3 mg/kg, bid) in HCT116 mouse xenograft model and tumor growth suppression (at 1 mg/kg,
bid) in 786-O mouse xenograft model.
In conclusion, we identified a new series of SCD1 inhibitors, represented by compound 1o, which
represents a promising new chemical tool suitable for the study of SCD1 biology as well as the potential
development of novel anticancer therapies.
Keywords
Stearoyl Coenzyme A desaturase 1, SCD1, 4, 4-disubstituted piperidine, cancer, antitumor efficacy,
T-3764518
Abbreviations
Ac: acetyl, ADME: absorption, distribution, metabolism, and excretion, aq.: aqueous, AUC: total area under
the blood concentration–time curve, bid: bis in die (twice per day), Boc: (tert-butoxy)carbonyl, Bu: butyl, Bz:
benzoyl, cLogP: calculate logarithm of the octanol-water partition coefficient, Cltotal: total body clearance,
CPME: cyclopentyl methyl ether, DI: desaturation index, DIPEA: N,N-diisopropylethylamine, DMA:
N,N-dimethylacetamide, DMF: N,N-dimethylformamide, DMSO: dimethyl sulfoxide, Et: ethyl, ER:
endoplasmic reticulum, Fu: unbound fraction, GI: growth inhibition, HBA: hydrogen bond acceptor, HBD:
hydrogen bond donor, IPA: 2-propanol, IPE: diisopropyl ether, LMS: liver microsomal stability, m: meta, Me:
methyl, MRT: mean residence time, MW: molecular weight, o: ortho, p: para, PC: phosphatidylcholine, PD:
pharmacodynamic, Pd/C: palladium on carbon, PK: pharmacokinetic, PMB: p-methoxybenzyl, Pr: propyl, po:
per os (oral administration), quant.: quantitative yield, rt: room temperature, SAR: structure-activity
relationship, sat.: saturated aqueous, SCD: stearoyl coenzyme A desaturase, TEA: triethylamine, tert: tertiary,
TFA: trifluoroacetic acid, THF: tetrahydrofuran, Ts: tosyl.

Corresponding Author
E-mail: keisuke.imamura@takeda.com
Introduction
Stearoyl coenzyme A desaturase 1 (SCD1) is an iron-containing endoplasmic reticulum (ER) enzyme,
which controls the rate-limiting step in the synthesis of unsaturated fatty acids from saturated fatty acids.1
Oleic and palmitoleic acids, which are the principal products of SCD1, are important components in lipid
synthesis such as in the production of phospholipids, triglycerides, cholesterol esters, wax esters, and
diacylglycerol.1 Hence, SCD1 is responsible for a wide range of biological effects related to energy storage
and signaling,2a as well as human physiology,2b and is regarded as a promising drug target for the treatment of
lifestyle-related diseases such as obesity and metabolic disease.3 Recently, it has been reported that SCD1
expression and activity are closely related to cancer pathogenesis and tumor malignancy,4 and furthermore, it
is believed that tumor cells obtain most of their required fatty acid from de novo synthesis.5 Thus, inhibition of
SCD1 function would be expected to induce an imbalance in fatty acid composition among the intracellular
lipids, leading to cell death via lipotoxicity and ER stress responses in certain types of cancer.6
Spirocycle-based SCD inhibitor A was originally identified by Takeda for the treatment of metabolic
disease.7 This compound shows potent binding affinity (IC50 = 6.5 nM) to SCD1 and has promising druglike
properties, such as low molecular weight (MW = 351.4), low lipophilicity (cLogP value = 0.38) and structural
rigidity (number of rotatable bond = 3). Based on a structural similarity of compound A with stearoyl-CoA,
the natural ligand of SCD1, and the reported binding mode of this ligand to SCD1,8 we show an overlay
model of their structures (Figure 1). According to this view, we hypothesized that these ligands could adopt a
similar binding mode to each other and occupy the catalytic center of the enzyme. This model suggests the
existence of an unoccupied space around the terminal fused phenyl ring of A and thus prompted new designs
for increasing potency by filling this space. Compound A exhibited moderate oral exposure (AUCpo = 299.1
ng/mL) and duration (MRTpo = 2.3 h) in mouse cassette dosing (1 mg/kg po). However, we still sought to
further increase exposure ad in vivo duration. In addition, the amenability of these compounds for chemical
modification was considered suboptimal. Therefore, we sought to develop a non-spirocyclic series for use as a
new lead series. We thus initiated synthetic studies aimed at the discovery of potent and orally available SCD1
inhibitors with long duration of PK, with the ultimate goal of using these compounds for anticancer therapy.
Figure 1. Structural similarity between stearoyl-CoA and compound A, and proposed key interactions

Chemistry
The compounds in this work were generally synthesized according to the route illustrated in Scheme 1. The
preparation of chloropyridazines 2 and piperidines 3 are described in Schemes 2–4.
Compounds 1 were synthesized by nucleophilic substitution of chloropyridazines 2 with piperidines 3 directly
or followed by deprotection of the O-Bz group by an alkaline hydrolysis.
Scheme 1. General method for compound synthesis in this reporta
aReagents and conditions: (a) 2a or 2c, 3, DIPEA, IPA or DMA; (b) aq. NaOH, THF, MeOH, rt, 58–95% (2
steps from 2a or 2c); (c) 2b or 2d, 3n, DIPEA, IPA, 150 °C, 1 h, microwave irradiation, 70–72%.
Chloropyridazine 2a was prepared by the reported procedure,7 and the synthesis of 1,3,4-oxadiazole
intermediate 2b is presented in Scheme 2-1. Condensation of pyridazine-4-one carboxylic acid 4 and O-
PMB-protected hydrazide 59 was performed via acyl chlorination of 4 to give diacyl hydrazide 6, which was
cyclized in the presence of p-TsCl and trimethylamine hydrochloride10 to afford O-PMB-1,3,4-oxadiazole 7.
The PMB group was removed by treatment with TFA to give the desired intermediate 2b. Synthesis of
1,2,4-thiadiazole intermediate 2c is illustrated in Scheme 2-2. O-Bz-hydroxyamidine 87 was predominantly
acylated to 9, which was reduced to amidine 10. Construction of the 1,2,4-thiadiazole ring was carried out by

a modified method of the reported procedure using thionyl chloride, 3-chloro-6-methylpyridazine and 10 to
give 2c.7 The synthesis of intermediate 2d is illustrated in Scheme 2-3. Commercially available methyl ester
11 was converted to hydrazide 12 in the presence of hydrazine hydrate, which was acylated using acyl
chloride to give diacyl hydrazide 13. Hydrazide 13 was cyclized with phosphorus pentasulfide, followed by
chlorination to give O-Ac-2-hydroxymethyl-1,3,4-thiadiazole 14. The acetyl group was removed by alkaline
hydrolysis to afford the desired intermediate 2d.
Scheme 2. Synthesis of key intermediate 2a
aReagents and conditions: (a) thionyl chloride, DMF, 80 °C, 1 h; (b) 5, pyridine, THF, 0 °C to rt, overnight,
48% (2 steps from 4); (c) TEA•HCl, TEA, p-TsCl, MeCN, 50 °C, 1 h, 90%; (d) TFA, anisole, rt, 1 h, 87%; (e)
acetic anhydride, acetic acid, 0 °C to rt, overnight, 90%; (f) Pd/C, H2 (balloon), EtOH, rt, overnight, 99%; (g)
thionyl chloride, 3-chloro-6-methylpyridazine, reflux, overnight; then 10, aq. NaOH, THF, −5 °C to 0 °C, 1 h,
71%; (h) hydrazine hydrate, MeOH, rt, overnight, 93%; (i) acetoxyacetyl chloride, dichloromethane, H2O,
0 °C to rt, 1.5 h, 78%; (j) phosphorus pentasulfide, THF, 50 °C, overnight, 34%; (k) phosphoryl chloride,
MeCN, 70 °C, 2 h, 34%; (l) aq. LiOH, THF, 0 °C to rt, 2 h, 85%.
Piperidines 3b, 3e, 3f, 3g, 3i and 3l were prepared by the reported procedures.11 Synthesis of
4-cyano-4-phenylpiperidine intermediates 3j, 3k and 3m are described in Scheme 3. The piperidine ring was
cyclized via nucleophilic substitution of commercially available benzyl cyanides 15 with
bis(2-chloroethyl)amine derivative 16 in the presence of sodium hydride. N-Boc deprotection under acidic
conditions gave the desired 3 in moderate to high yield.
Scheme 3. Synthesis of 4-cyano-4-phenylpiperidine intermediates 3j, 3k and 3ma

a
−40%
Reagents and conditions: (a) 16, NaH, DMF, 70 °C, overnight, 20
; (b) hydrogen chloride, CPME,
EtOAc, 50 °C, overnight, 86–94%.
4-Trifluoromethyl-4-phenylpiperidine intermediate 3n was synthesized in a manner similar to a reported
procedure, with modifications as shown in Scheme 4.11b Commercially available acetophenone 18n was
converted to enone 19n by Lewis acid-mediated Knoevenagel condensation. Further homologation of enone
19n was achieved by sequential Michael addition and nucleophilic substitution reaction using
2-cyanoacetamide to lead to 2,6-dioxo-4-piperidine-3,5-dicarbonitrile 20n. Removal of the cyano groups from
20n was conducted by acid and alkaline hydrolysis followed by decarboxylation to give dicarboxylic acid 22n.
Introduction of an amino moiety was achieved using urea to give cyclic imide 23n and it was converted to
N-Boc-4-trifluoromethyl-4-phenylpiperidine 24n via borane reduction followed by N-Boc protection. Finally,
4-trifluoromethyl-4-phenylpiperidine intermediate 3n was obtained by deprotection of 24n with acidic
conditions.
Scheme 4. Synthesis of 4-trifluoromethyl-4-phenylpiperidine intermediate 3na
aReagents and conditions: (a) ethyl cyanoacetate, TiCl4, pyridine, dichloromethane, 0 °C to rt, overnight, 91%;
(b) 2-cyanoacetamide, NaOAc, EtOH, rt, overnight; (c) H2SO4, H2O, acetic acid, 130 °C, overnight; (d) aq.
KOH, reflux, 4 h; (e) H2SO4, H2O, reflux, 3 h; (f) Ac2O, reflux, 3 h; (g) urea, 190 °C, 30 min, 41% (6 steps
from 19n); (h) BH3•SMe2, THF, 0 °C to reflux, overnight; (i) Boc2O, DIPEA, THF, rt, 2 h, 77% (2 steps from
23n); (j) aq. HCl, EtOAc, IPA, reflux, 5 h, 95%.
Result and discussion
In this work, newly synthesized compounds were evaluated for their binding affinity to SCD1 using
(5-(6-(1'H-spiro(1-benzofuran-3,3'-pyrrolidin)-1'-yl)pyridazin-3-yl)-1,2,4-oxadiazol-3-yl)[3H2]methanol

(3H-A) as a radiolabeled ligand.12 To measure the inhibitory activity in cells of our SCD1 inhibitors, we
established growth inhibitory assay in HCT116 cells.
With the aim of discovering a new scaffold alternative to the spiro series, we conducted a ligand-based
conformation study. As a result, we found 4-substituted 4-phenylpiperidine derivative 1a, a ring-opened
analogue of the spiro compound, which showed excellent overlay with compound A (Figure 2). As mentioned
above, the conformation study and overlay study of compound A with stearoyl-CoA also suggested the
existence of an unoccupied space around the 4-phenyl group that might accommodate an additional
substituent and offer a good opportunity to increase affinity as well as to adjust the physicochemical properties.
Therefore, we selected the corresponding 4-substituted 4-arylpiperidine as a candidate scaffold alternative to
the spiro series.
Figure 2. Overlay study of spiro compound A and designed compound 1a (compound A in cyan; designed
compound 1a in yellow).
Initially, we designed 4-cyano-4-phenylpiperidine derivative 1a in consideration of synthetic tractability
and evaluated compound potencies by SCD1 binding and cell growth assays. Thus, compound 1a was found
to exhibit moderate SCD1 binding affinity (IC50 = 190 nM) and GI activity (GI50 = 530 nM), indicating the
potential of this new design as a starting point; this level of binding affinity was over 40-fold weaker than for
compound A. Therefore, we initiated SAR studies around compound 1a as a new lead to identify more potent
inhibitors by utilizing the higher synthetic accessibility of this new series. First, SARs around the
4-substituted 4-aryl piperidine moiety were examined to identify the most suitable alternative scaffold to the
spiro series in terms of potency, as well as physical properties. As for the 4-position of the piperidine ring,
substituents with a variety of lipophilicities were introduced and the representative results are shown in Table
1. It was found that replacement of the cyano group with hydrophilic substituents such as amino 1b (IC50
=
220 nM), hydroxy 1c (IC50 = 130 nM), and methoxy 1d (IC50 = 110 nM) groups was tolerated in terms of
binding affinity. Remarkably, compound 1e with a lipophilic trifluoromethyl group exhibited more than 5-fold
enhancement in SCD1 binding affinity and 12-fold enhancement in growth inhibitory activity when compared
with 1a.
Table 1. SARs of 4-Substituted 4-Phenylpiperidines

a
b
ID
1a
1b
1c
1d
1e
R1
CN
IC50
GI50
190
220
130
110
38
530
530
NH2
OH
>1000
410
OMe
CF3
39
ahSCD1 Binding affinity (nM). bGI activity using HCT116 cells (nM).
Next, we examined substituent effects on the pendant phenyl group at the 4-position of the piperidine ring.
With the aim of not only enhancing binding affinity but also modulating the resulting ADME-tox profiles,
substituents with a wide range of size, lipophilicity, and electron effects were examined. As shown in Table 2,
mono chlorination at any position of the pendant phenyl group increased the binding affinity and GI activity
(1f–h). The potency increase was more than 4-fold larger for m-Cl 1g (IC50 = 15 nM) and p-Cl 1h (IC50 = 20
nM) than for o-Cl 1f (IC50 = 80 nM). In contrast, effects on cell GI were larger for p-Cl 1h (GI50 = 110 nM)
than for o-Cl 1f (GI50 = 240 nM) and m-Cl 1g (GI50 = 200 nM). Thus, the p-position was indicated as the most
favorable substitution position for obtaining both binding affinity and growth inhibitory activity. Next, in
order to elucidate substituent effects at the p-position of the terminal benzene ring, we focused on substituents
with a range of Hansch–Fujita π and Hammett σ values, which indicate the lipophilicity and electron-donating
or electron-withdrawing nature of the substituents, respectively.13 Replacement of p-Cl 1h (π = +0.70) with
less lipophilic groups such as p-H 1a (π = 0.00) and p-NO2 1i (π = +0.24) decreased SCD1 binding affinity
and GI activity (1h IC50 = 20 nM, GI50 = 110 nM; 1a IC50 = 190 nM, GI50 = 530 nM; 1i IC50 = 100 nM, GI50 =
170 nM), whereas replacement with more lipophilic groups such as p-tBu 1j (π = +1.68) and p-OCF3 1k (π =
+1.04) maintained or enhanced SCD1 binding affinity and GI activity (1j IC50 = 21 nM, GI50 = 37 nM; 1k IC50
= 11 nM, GI50 = 32 nM). These results support the hypothesis that the enzyme has space around the pendant
phenyl group and indicated the merit of occupying that space with lipophilic substituents at the p-position of
the phenyl group for enhancing potency. On the other hand, liver microsomal stability (LMS) of 1k with an
electron-withdrawing p-OCF3 group (σ = +0.35) increased more than 3-fold from that of 1j with an
electron-donating group p-tBu (σ = −0.20). These results suggest that substitution with electron-withdrawing
groups at the p-positon of the terminal benzene ring could improve the PK profile. Based on the results of
binding affinity and metabolic stability studies, our focus was shifted to the more lipophilic (π > 0) and
electron-withdrawing (σ > 0) region in the Craig plot,14 which was expected to lead to highly potent and orally
available SCD1 inhibitors with long PK duration. Indeed, derivatives with a p-CF3 moiety (1l π = +1.07) and
p-SF5 moiety (1m π = +1.23) exhibited higher SCD1 binding affinity and GI activity (1l IC50 = 27 nM, GI50 =
39 nM; 1m IC50 = 9.5 nM, GI50 = 8.4 nM) in proportion to their Hansch π values. Both compounds showed
excellent LMS, as expected. Taken together with the SAR data obtained at the 4-position of the piperidine
ring
and
the
p-position
of
the
terminal
benzene
ring,
we
identified

4-(trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidine (1n) as a new scaffold worthy of further study.
Table 2. SARs of 4-Substituted 4-Phenylpiperidines
a
b
ID
1a
1f
R1
R2
none
IC50
GI50
hLMSc
NSg
NSg
mLMSd
NSg
NSg
NSg
39
πe
σf
CN
CN
CN
CN
CN
CN
CN
CN
CN
CF3
190
80
530
240
200
110
170
37
±0.00
–
0.00
–
o-Cl
m-Cl
15
NSg
–
–
1g
1h
1i
p-Cl
20
−
14
6
+0.70
+0.24
+1.68
+1.04
+1.07
+1.23
+0.23
+0.78
−0.20
+0.35
+0.55
+0.68
–
p-NO2
p-tBu
p-OCF3
p-CF3
p-SF5
p-CF3
100
21
11
0
119
35
1j
11
32
−
12
2
1k
1l
27
39
33
9.5
8.4
1.3
7
24
1m
2.3
20
19
–
1n
b
c
d
ahSCD1 Binding affinity (nM). GI activity against HCT116 cells (nM). LMS in human (µL/min/mg). LMS
e
g
in mouse (µL/min/mg). Lipophilic parameter proposed by Hansch–Fujita. fHammett electronic constant. Not
shown.
As shown in Table 3, compound 1n showed good LMS (mLMS = 19 µL/min/mg; hLMS = 20 µL/min/mg)
in vitro. However, compared to compound A, the improvement of oral exposure (AUCpo = 432.2 ng/mL) and
plasma duration (MRTpo = 3.2 h) in a mouse cassette-dosing PK study (0.1 mg/kg iv; 1 mg/kg po) are limited.
Final optimization of the oxadiazole moiety was conducted to improve the PK profile. All of the designed
compounds 1o–q showed excellent binding affinity, GI activity, and LMS. Among the regioisomers of
oxadiazole and thiadiazole, 1,3,4-oxadiazole 1o and 1,2,4- thiadiazole 1p exhibited excellent PK profiles (1o
AUCpo = 3756.5 ng/mL, MRTpo = 4.2 h; 1p AUCpo = 1823.3 ng/mL, MRTpo = 4.2 h) represented by the
reduced Cltotal values. In addition, compound 1o demonstrated a higher fraction unbound in plasma (Fu =
0.07) than for compound 1p (Fu = 0.01). Therefore, compound 1o (T-3764518) was selected as a candidate
for further in vivo PD testing and efficacy studies.
Table 3. SARs for Compounds Varied at the 1,2,4-Oxadiazole Moiety
a
b
e
f
g
ID
R3
IC50
2.3
GI50
1.3
hLMSc
20
mLMSd Cltotal
19 926
AUCpo
432.2
MRTpo
3.2
Fuh
0.05
1n

4.7
1.2
2.5
2.7
1.3
1.1
14
10
33
−11
1
310
494
952
3756.5
1823.3
4.2
4.2
4.1
0.07
0.01
0.03
1o
1p
1q
−16
1191.1
b
c
d
ahSCD1 binding affinity (nM). GI activity against HCT116 cells (nM). LMS in human (µL/min/mg). LMS
e
in mouse (µL/min/mg). Cltotal (mL/h/kg) in a mouse cassette-dosing PK study (0.1 mg/kg iv; 1 mg/kg po).
fAUC (ng·h/mL) after oral administration in a mouse cassette-dosing PK study. gMRT (h) after oral
administration in mouse cassette-dosing PK study. hFu in plasma.
Compound 1o (T-3764518) was evaluated for in vivo effects in mice bearing HCT116 xenograft tumors.
The DI (desaturation index) of phosphatidylcholine (PC) was used as a PD marker for the evaluation of SCD1
inhibition in HCT116 xenograft tumors.15 As shown in Figure 3, administration of compound 1o decreased DI
(ratio of PC C36:3/C36:0) in tumor tissue at all the oral doses down to the lowest dose tested in this study (0.3
mg/kg, po, bid). Reflecting these significant in vivo PD changes, compound 1o also induced tumor growth
inhibition.6, 15
Compound 1o demonstrated tumor growth inhibition with a percent tumor growth inhibition (T/C) of
32.8% in a 786-O xenograft mouse model at an oral dose of 1 mg/kg, po, bid without severe toxicity (Figure
4). Overall, these data indicated 1o to be an orally bioavailable SCD1 inhibitor with in vivo antitumor efficacy,
demonstrating is to be a useful tool for assessing the pharmacology of SCD1 inhibition in medicine,
particularly in terms of developing new cancer therapies.
Figure 3. Results of in vivo PD test using HCT116 xenograft model mice. Dose level 0.3, 3, 30 mg/kg, po,
bid; ***P < 0.0005 by Williams’ test.
Figure 4. Result of in vivo efficacy test using 786-O xenograft model mice. Dose level 1 mg/kg, po, bid; *P <
0.005 by Student’s t-test.

Conclusion
In conclusion, with the aim of discovering highly potent and orally available SCD1 inhibitors for cancer
treatment, we designed novel 4-substituted-4-phenylpiperidine derivatives based on a ligand based overlay
model of stearoyl CoA with spirocycle-based SCD inhibitor A and 3D conformational studies. The compound
thus designed (1a) exhibited moderate binding affinity toward SCD1 as wells as moderate GI activity. Further
substituent modification guided by Hansch–Fujita constants (π) and Hammett (σ) constants resulted in the
identification of several inhibitors that showed high SCD1 binding affinity, potent GI activity in HCT116
cancer cell line, and an good PK profile. Notably, 1,3,4-oxadiazole analogue 1o (T-3764518) showed potent in
vivo PD response and tumor growth suppression without severe toxicity. These results indicate that our
4-substituted-4-phenylpiperidine-based SCD1 inhibitors, represented by compound 1o, have potential use not
only as chemical tools for basic pharmacology research into SCD1 function, but also as a new lead series of
clinical candidates for therapy in oncology.
Experimental section
General
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 60F254 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
1
automated melting point apparatus, are uncorrected. H NMR and 13C 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. Abbreviations are used as follows:
s = singlet, d = doublet, t= triplet, q = quartet, m = multiplet, br s = broad singlet. Coupling constants (J
values) are given in hertz (Hz). Mass spectra (MS) were acquired using an Agilent LC/MS system (Agilent
1200SL/Agilent 6130MS), Agilent LC/MS system (Agilent 1200SL/Agilent 6110MS), Agilent LC/MS

system (Agilent 1200SL/Agilent 6120MS), 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 mm × 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 MeCN,
which was increased linearly from 5% to 90% over 2 min, 90% over the next 1.5 min, after which the column
was equilibrated to 5% for 0.5 min. Elemental analyses were carried out by Sumika Chemical Analysis
Service, Ltd. Each compound was confirmed to be ≥95% purity by either LC/MS or elemental analysis.
Yields were not optimized.
6-Chloro-N'-(((4-methoxybenzyl)oxy)acetyl)pyridazine-3-carbohydrazide (6). To
a suspension of
6-oxo-1,6-dihydropyridazine-3-carboxylic acid 4 (57.7 g, 412 mmol) in thionyl chloride (580 mL) was added
DMF (5.32 mL) at room temperature. The mixture was stirred at 80 °C for 1 h. The mixture was concentrated
in vacuo and diluted with THF (650 mL). To the mixture was added pyridine (55 mL) and a solution of
2-((4-methoxybenzyl)oxy)acetohydrazide 5 (72.1 g, 343 mmol) in THF (650 mL) at 0 °C. The mixture was
stirred at room temperature under a dry atmosphere overnight. The mixture was poured into sat. NaHCO3 at
room temperature and extracted with THF–EtOAc. The organic layer was separated, washed with brine, dried
over Na2SO4, and concentrated in vacuo. The residue was triturated with EtOAc to give
6-chloro-N'-(((4-methoxybenzyl)oxy)acetyl)pyridazine-3-carbohydrazide 6 (57.0 g, 48%) as an off-white solid.
1
This product was subjected to the next reaction without further purification. H NMR (300 MHz, DMSO-d6)
δ: 3.76 (3H, s), 4.02 (2H, s), 4.53 (2H, s), 6.93 (2H, d, J = 8.5 Hz), 7.34 (2H, d, J = 8.5 Hz), 8.10–8.18 (1H,
m), 8.21–8.29 (1H, m), 10.12 (1H, d, J = 18.5 Hz), 11.01 (1H, br s).
3-Chloro-6-(5-(((4-methoxybenzyl)oxy)methyl)-1,3,4-oxadiazol-2-yl)pyridazine (7). To a suspension of
6-chloro-N'-(((4-methoxybenzyl)oxy)acetyl)pyridazine-3-carbohydrazide 6 (70.0 g, 0.199 mol) in MeCN (600
mL) was added trimethylamine hydrochloride salt (5.72 g, 59.8 mmol), Et3N (83.4 mL, 0.598 mol) and p-TsCl
(57.0 g, 0.299 mol) at room temperature. The mixture was stirred at 50 °C under N2 for 1 h. The mixture was
diluted with THF (300 mL), and the insoluble material was removed by filtration. The filtrate was poured into
water and extracted with EtOAc. The organic layer was washed with water and brine, dried over Na2SO4 and
concentrated in vacuo. The residue was purified by silica gel column chromatography to give
3-chloro-6-(5-(((4-methoxybenzyl)oxy)methyl)-1,3,4-oxadiazol-2-yl)pyridazine 7 (60.0 g, 90%) as a pale
yellow solid. This product was subjected to the next reaction without further purification. MS(ESI+) m/z:
333.0 (M+H)+. 1H NMR (300 MHz, DMSO-d6) δ: 3.74 (3H, s), 4.59 (2H, s), 4.87 (2H, s), 6.92 (2H, d, J = 8.7
Hz), 7.30 (2H, d, J = 8.5 Hz), 8.20 (1H, d, J = 9.1 Hz), 8.47 (1H, d, J = 9.1 Hz).
(5-(6-Chloropyridazin-3-yl)-1,3,4-oxadiazol-2-yl)methanol
(2b).
A
mixture
of
3-chloro-6-(5-(((4-methoxybenzyl)oxy)methyl)-1,3,4-oxadiazol-2-yl)pyridazine 7 (50.0 g, 0.150 mol), TFA
(150 mL) and anisole (50 mL) was stirred at room temperature for 1h. The reaction mixture was concentrated in

vacuo. The residue was poured into sat. NaHCO3 at room temperature and extracted with EtOAc. The organic
layer was separated, washed with water and brine, dried over MgSO4 and concentrated in vacuo. The residue
was triturated with IPE to give (5-(6-chloropyridazin-3-yl)-1,3,4-oxadiazol-2-yl)methanol 2b (27.6 g, 87%) as
1
a light brown solid; MS(ESI+) m/z: 212.9 (M+H)+. H NMR (300 MHz, DMSO-d6) δ 4.83 (2H, s), 6.10 (1H,
br s), 8.14–8.27 (1H, m), 8.43–8.53 (1H, m). 13C NMR (76 MHz, DMSO-d6) δ 53.7, 129.1, 130.1, 146.6,
157.7, 161.6, 167.7.
2-(Acetoxyamino)-2-iminoethyl benzoate (9). To a solution of 2-amino-2-(hydroxyimino)ethyl benzoate 8
(68.0 g, 0.350 mol) in acetic acid (200 mL) was slowly added acetic anhydride (53.6 g, 0.525 mol) at 0 °C.
The mixture was stirred at room temperature overnight. The mixture was concentrated to give
2-(acetoxyamino)-2-iminoethyl benzoate 9 (74.0 g, 0.313 mol, 90%) as colorless oil. This product was
subjected to the next reaction without further purification. MS(ESI+) m/z: 237.7 (M+H)+.
2-Amino-2-iminoethyl benzoate acetic acid salt (10). A mixture of 2-(acetoxyamino)-2-iminoethyl benzoate
9 (30.0 g, 0.127 mol) and 10% Pd/C (3.0 g) in EtOH (200 mL) was hydrogenated under balloon pressure at
room temperature overnight. The catalyst was removed by filtration. The filtrate was concentrated in vacuo to
give 2-amino-2-iminoethyl benzoate acetic acid salt 10 (30.0 g, 99%) as white solid. This product was
subjected to the next reaction without further purification. MS(ESI+) m/z: 179.7 (M+H)+.
(5-(6-Chloropyridazin-3-yl)-1,2,4-thiadiazol-3-yl)methyl
benzoate
(2c).
A
mixture
of
3-chloro-6-methylpyridazine (20.0 g, 0.156 mol) and thionyl chloride (114 mL) was refluxed overnight. The
mixture was cooled to room temperature, concentrated in vacuo and evaporated twice with toluene
azeoptropically. The residue was suspended in THF (300 mL). To the suspension was added
2-amino-2-iminoethyl benzoate acetic acid salt 10 (18.6 g, 78.1 mmol) and stirred at −5 °C. Then sodium
hydroxide (50% in water, 42 mL) was added drop wise to the mixture at −5 °C. After being stirred at 0 °C for
1 h, the reaction mixture was extracted with EtOAc and concentrated in vacuo. The residue was purified by
column chromatography to give (5-(6-chloropyridazin-3-yl)-1,2,4-thiadiazol-3-yl)methyl benzoate 2c (18.4 g,
1
71%) as a white solid; MS(ESI+) m/z: 333.0 (M+H)+. H NMR (300 MHz, DMSO-d6) δ 5.74 (2H, s),
7.51–7.64 (2H, m), 7.66–7.78 (1H, m), 8.00–8.12 (2H, m), 8.15–8.25 (1H, m), 8.35–8.46 (1H, m). 13C NMR
(76 MHz, DMSO-d6) δ 62.2, 127.1, 128.8, 129.0, 129.4, 130.7, 133.7, 151.7, 158.1, 165.2, 172.0, 185.2. Anal.
Calcd for C14H9ClN4O2S·0.1H2O: C, 50.26; H, 2.77; N, 16.75. Found: C, 50.43; H, 2.97; N, 16.74.
6-Methoxypyridazine-3-carbohydrazide (12). A solution of methyl 6-methoxypyridazine-3-carboxylate 11
(4.50 g, 26.8 mmol) and hydrazine hydrate (80% in water, 13.4 g, 214 mmol) in MeOH (50 mL) was stirred at
room temperature for overnight. The mixture was added water (10 mL) and concentrated in vacuo to remove
MeOH. The residue was freeze-dried and washed with petroleum ether to give
6-methoxypyridazine-3-carbohydrazide 12 (4.20 g, 93%) as a yellow solid. This product was subjected to the
next reaction without further purification. MS(ESI+) m/z: 169.0 (M+H)+.

2-(2-((6-Methoxypyridazin-3-yl)carbonyl)hydrazino)-2-oxoethyl acetate (13). Acetoxyacetyl chloride
(16.3 g, 119 mmol) was added to a solution of 6-methoxypyridazine-3-carbohydrazide 12 (20.0 g, 119 mmol)
in dichloromethane (250 mL)–water (10 mL) at 0 °C. The mixture was allowed to warm to room temperature
for 1.5 h. The mixture was concentrated in vacuo at room temperature. The residue was added water (20 mL)
and
freeze-dried.
The
solid
was
washed
with
petroleum
ether
to
give
2-(2-((6-methoxypyridazin-3-yl)carbonyl)hydrazino)-2-oxoethyl acetate 13 (25.0 g, 78%) as a light yellow
solid. This product was subjected to the next reaction without further purification. MS(ESI+) m/z: 269.0
(M+H)+.
(5-(6-Chloropyridazin-3-yl)-1,3,4-thiadiazol-2-yl)methyl
acetate
(14).
A
solution
of
2-(2-((6-methoxypyridazin-3-yl)carbonyl)hydrazino)-2-oxoethyl acetate 13 (25.0 g, 93.3 mmol) and
phosphorus pentasulfide (21.4 g, 112 mmol) in THF (600 mL) was stirred at 50°C for overnight. The mixture
was cooled to room temperature, quenched with sat. NaHCO3 and extracted with EtOAc. The organic layer
was washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by silica gel
column chromatography to give (5-(6-hydroxypyridazin-3-yl)-1,3,4-thiadiazol-2-yl)methyl acetate (7.90 g,
34%) as a yellow solid. A solution of (5-(6-hydroxypyridazin-3-yl)-1,3,4-thiadiazol-2-yl)methyl acetate (7.90
g, 31.3 mmol) and phosphoryl chloride (9.60 g, 62.7 mmol) in MeCN (200 mL) was heated to 70 °C for 2 h.
The mixture was cooled to room temperature, quenched with water at 0 °C and extracted with EtOAc. The
organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified
by silica gel column chromatography to give (5-(6-chloropyridazin-3-yl)-1,3,4-thiadiazol-2-yl)methyl acetate
14 (2.90 g, 34%) as a yellow solid. This product was subjected to the next reaction without further purification.
MS(ESI+) m/z: 271.0 (M+H)+.
(5-(6-Chloropyridazin-3-yl)-1,3,4-thiadiazol-2-yl)methanol
(2d).
To
a
solution
of
(5-(6-chloropyridazin-3-yl)-1,3,4-thiadiazol-2-yl)methyl acetate 14 (1.60 g, 5.93 mmol) in THF (50 mL) was
added lithium hydroxide (1 M in water, 14.8 mL) at 0°C. The mixture was stirred at 0°C for 1 h and the
mixture was allowed to warm to room temperature for 1 h. After removing the solvent, the mixture was
extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo.
The
residue
was
crystallized
from
MeOH–dichloromethane
to
give
(5-(6-chloropyridazin-3-yl)-1,3,4-thiadiazol-2-yl)methanol 2d (1.16 g, 85%) as a yellow solid; MS(ESI+) m/z:
1
229.2 (M+H)+. H NMR (300 MHz, DMSO-d6) δ 4.88–5.05 (2H, m), 6.34–6.53 (1H, m), 8.11–8.21 (1H, m),
8.48–8.58 (1H, m). 13C NMR (76 MHz, DMSO-d6) δ 58.4, 127.0, 130.2, 152.1, 157.3, 165.3, 176.6.
tert-Butyl 4-(4-(tert-butyl)phenyl)-4-cyanopiperidine-1-carboxylate (17j). Sodium hydride (60% in oil,
3.46 g, 86.6 mmol) was added to a solution of tert-butyl bis(2-chloroethyl)carbamate 16 (10.0 g, 41.3 mmol)
and 2-(4-(tert-butyl)phenyl)acetonitrile 15j (5.00 g, 28.9 mmol) in DMF (300 mL) at 70 °C. The mixture was
stirred at 70 °C under N2 overnight. The mixture was poured into sat. NH4Cl at room temperature and

extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over MgSO4 and
concentrated in vacuo. The residue was purified by column chromatography to give tert-butyl
1
4-(4-(tert-butyl)phenyl)-4-cyanopiperidine-1-carboxylate 17j (3.50 g, 35 %) as a pale yellow oil; H NMR
(300 MHz, CDCl3) δ 1.32 (9H, s), 1.43–1.52 (9H, m), 1.85–2.19 (4H, m), 3.12–3.35 (2H, m), 4.17–4.41 (2H,
m), 7.35–7.46 (4H, m).
4-(4-(tert-Butyl)phenyl)piperidine-4-carbonitrile hydrochloride salt (3j). A mixture of hydrogen chloride
(4 M in CPME, 2.55 mL) and tert-butyl 4-(4-(tert-butyl)phenyl)-4-cyanopiperidine-1-carboxylate 16j (3.50 g,
10.2 mmol) in EtOAc (10 mL) was stirred at 50 °C under N2 overnight. The precipitate was collected by
filtration to give 4-(4-(tert-butyl)phenyl)piperidine-4-carbonitrile hydrochloride salt 3j (2.68 g, 9.61 mmol,
94 %) as a white solid; 1H NMR (300 MHz, DMSO-d6) δ 1.29 (9H, s), 2.29–2.46 (4H, m), 3.00–3.18 (2H, m),
3.42–3.55 (2H, m), 7.38–7.56 (4H, m), 9.31 (2H, br s).
tert-Butyl
4-cyano-4-(4-(trifluoromethoxy)phenyl)piperidine-1-carboxylate
(17k).
tert-Butyl
4-cyano-4-(4-(trifluoromethoxy)phenyl)piperidine-1-carboxylate 17k was prepared by a method similar to
that described for tert-butyl 4-(4-(tert-butyl)phenyl)-4-cyanopiperidine-1-carboxylate 17j using
1
(4-(trifluoromethoxy)phenyl)acetonitrile 15k. Yield: 40% as a white solid; H NMR (300 MHz, DMSO-d6)
δ 2.32–2.47 (4H, m), 2.99–3.19 (2H, m), 3.42–3.59 (2H, m), 7.46–7.58 (2H, m), 7.63–7.76 (2H, m), 9.28 (2H,
br s).
4-(4-Trifluoromethoxyphenyl)piperidine-4-carbonitrile
hydrochloride
salt
(3k).
4-(4-Trifluoromethoxyphenyl)piperidine-4-carbonitrile hydrochloride salt 3k was prepared by a method
similar to that described for 4-(4-(tert-butyl)phenyl)piperidine-4-carbonitrile hydrochloride salt 3j using
tert-butyl 4-cyano-4-(4-(trifluoromethoxy)phenyl)piperidine-1-carboxylate 17k. Yield: 86% as a white solid;
1H NMR (300 MHz, DMSO-d6) δ 2.32–2.47 (4H, m), 2.99–3.19 (2H, m), 3.42–3.59 (2H, m), 7.46–7.58 (2H,
m), 7.63–7.76 (2H, m), 9.28 (2H, br s).
tert-Butyl
4-cyano-4-(4-(pentafluorosulfanyl)phenyl)piperidine-1-carboxylate
(17m).
tert-Butyl
4-cyano-4-(4-(pentafluorosulfanyl)phenyl)piperidine-1-carboxylate 17m was prepared by a method similar to
that described for tert-butyl 4-(4-(tert-butyl)phenyl)-4-cyanopiperidine-1-carboxylate 17j using
1
(4-(pentafluorosulfanyl)phenyl)acetonitrile 15m. Yield: 20% as white solid; H NMR (300 MHz, CDCl3) δ
1.49 (9H, s), 1.84–2.18 (4H, m), 3.01–3.40 (2H, m), 4.11–4.57 (2H, m), 7.53–7.64 (2H, m), 7.78–7.87 (2H,
m).
4-(4-(Pentafluorosulfanyl)phenyl)piperidine-4-carbonitrile
hydrochloride
salt
(3m).
4-(4-(Pentafluorosulfanyl)phenyl)piperidine-4-carbonitrile hydrochloride salt 3m was prepared by a method
similar to that described for 4-(4-(tert-butyl)phenyl)piperidine-4-carbonitrile hydrochloride salt 3j using
tert-butyl 4-cyano-4-(4-(pentafluorosulfanyl)phenyl)piperidine-1-carboxylate 17m. Yield: 89% as a white

solid; 1H NMR (300 MHz, DMSO-d6) δ 2.26–2.47 (4H, m), 3.01–3.21 (2H, m), 3.45–3.62 (2H, m), 7.72–7.82
(2H, m), 8.03–8.13 (2H, m), 8.87–9.16 (2H, m).
Ethyl 2-cyano-4,4,4-trifluoro-3-(4-(trifluoromethyl)phenyl)but-2-enoate (19n). TiCl4 (32.9 g, 174 mmol)
was added drop wise to a solution of 2,2,2-trifluoro-1-(4-(trifluoromethyl)phenyl)ethanone 18n (20 g, 82.6
mmol) and ethyl cyanoacetate (10.3 g, 90.9 mmol) in dichloromethane (400 mL) at 0 °C. The mixture was
stirred for 20 min at 0 °C and then pyridine (6 mL) was added drop wise slowly, keeping the internal
temperature below 5 °C. After the addition, the ice bath was removed. Then reaction was stirred for 30 min at
room temperature. Then an additional pyridine (20 mL) was added and the mixture was stirred at room
temperature overnight. The mixture was diluted with dichloromethane and washed with hydrogen chloride
(2M in water), water and brine. The organic layer was dried over MgSO4 and concentrated in vacuo. The
residue
was
purified
by
silica
gel
column
chromatography
to
give
ethyl
2-cyano-4,4,4-trifluoro-3-(4-(trifluoromethyl)phenyl)but-2-enoate 19n (25.2 g, 91%) as light yellow oil. This
product was subjected to the next reaction without further purification as a mixture of cis and trans isomers.
Major isomer: 1H NMR (300 MHz, DMSO-d6) δ 1.09 (3H, t, J = 7.2 Hz), 4.13 (2H, q, J = 7.2 Hz), 7.40 (2H, d,
J = 8.0 Hz), 7.73 (2H, d, J = 8.0 Hz). Minor isomer: δ 1.42 (3H, t, J = 7.2 Hz), 4.44 (2H, q, J = 7.2 Hz), 7.60
(2H, d, J = 8.0 Hz), 7.80 (2H, d, J = 8.0 Hz).
2,6-Dioxo-4-(trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidine-3,5-dicarbonitrile (20n). To a
solution of ethyl 2-cyano-4,4,4-trifluoro-3-(4-(trifluoromethyl)phenyl)but-2-enoate 19n (116 g, 0.344 mol) in
EtOH (1000 mL) was added 2-cyanoacetamide (50.7 g, 0.603 mol) and NaOAc (98.9 g, 1.21 mol) at room
temperature. The mixture was stirred at room temperature overnight. The solvent was removed under reduced
pressure and the residue was treated with water. The aqueous layer was added hydrogen chloride (12M in
water) to adjust the pH rose to 3, and extracted with EtOAc. The organic layer was dried over Na2SO4 and
concentrated
in
vacuo
to
give
2,6-dioxo-4-(trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidine-3,5-dicarbonitrile 20n (135 g, crude) as
a yellow oil. This product was used for the next step without further purification. MS(ESI−) m/z: 374.0
(M−H)−.
9-(Trifluoromethyl)-9-(4-(trifluoromethyl)phenyl)-3,7-diazabicyclo[3.3.1]nonane-2,4,6,8-tetraon (21n). A
mixture of 2,6-dioxo-4-(trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidine-3,5-dicarbonitrile 20n (135
g, crude) in H2SO4 (529.6 g, 5.40 mol)–water (400 mL)–acetic acid (400 mL) was stirred at 130 °C overnight.
After cooling, the reaction mixture was diluted with water and extracted with EtOAc. The organic layer was
washed
with
brine,
dried
over
MgSO4
and
concentrated
in
vacuo
to
give
9-(trifluoromethyl)-9-(4-(trifluoromethyl)phenyl)-3,7-diazabicyclo[3.3.1]nonane-2,4,6,8-tetraon 21n (99.4 g,
crude) as a grey solid. This product was used for the next step without further purification. MS(ESI−) m/z:
393.2 (M−H)−.

3-(Trifluoromethyl)-3-(4-(trifluoromethyl)phenyl)pentanedioic
acid
(22n).
A
solution
of
9-(trifluoromethyl)-9-(4-(trifluoromethyl)phenyl)-3,7-diazabicyclo[3.3.1]nonane-2,4,6,8-tetraon 21n (99.4 g,
crude) in potassium hydroxide (20% in water, 549 g) was refluxed for 4 h. After cooling to 0 °C, H2SO4 (220
g, 2.34 mol)–water (500 mL) was added drop wise to the reaction mixture. Then the resulting mixture was
refluxed for additional 3 h. After cooling, the mixture was extracted with EtOAc. The organic layer was dried
and concentrated in vacuo to give 3-(trifluoromethyl)-3-(4-(trifluoromethyl)phenyl)pentanedioic acid 22n
(79.6 g, crude) as a grey solid. This product was used for the next step without further purification. MS(ESI−)
m/z: 343.2 (M−H)−.
2,6-Dioxo-4-(trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidine
(23n).
A
solution
of
3-(trifluoromethyl)-3-(4-(trifluoromethyl)phenyl)pentanedioic acid 22n (79.6 g, crude) in acetic anhydride
(507.9 g, 4.98 mol) was refluxed 3 h. The reaction mixture was concentrated in vacuo to give desired
intermediate. The mixture of intermediate and urea (14.6 g, 243 mmol) was stirred at 190 °C under N2 for 30
min. After cooling to room temperature, the reaction mixture was dissolved in dichloromethane and filtered
off the resulting precipitate. The filtrate was concentrated in vacuo. The residue was purified by silica gel
column
chromatography
followed
by
recrystallization
to
give
2,6-dioxo-4-(trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidine 23n (45.5 g, 41% in 6 steps from 19n).
1
This product was used for the next step without further purification. MS(ESI−) m/z: 342.2 (M−H)−. H NMR
(400 MHz, DMSO-d6) δ 3.38 (2H, d, J = 16.4 Hz), 3.51 (2H, d, J = 16.4 Hz), 7.83–7.89 (4H, m), 11.03 (1H,
s).
tert-Butyl 4-(trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidine-1-carboxylate (24n). The mixture
of borane dimethyl sulfide complex (2M in THF, 360 mL, 720 mmol) and
2,6-dioxo-4-(trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidine 23n (39.0 g, 120 mmol) was refluxed
overnight. After cooling to 0 °C, hydrogen chloride (6M in water, 210 mL) was added drop wise to the
mixture. This mixture was refluxed for 1 h, then cooled with ice bath and added sodium hydroxide (8 M in
water, 190 mL). Then this mixture was extracted with EtOAc. The organic layer was dried and concentrated in
vacuo to give desired intermediate. Boc2O (26.7 g, 122 mmol) was added to a solution of above mentioned
intermediate and DIPEA (18.6 g, 144 mmol) in THF (350 mL) at room temperature.The mixture was stirred at
room temperature for 2 h and then poured in to sat. NaHCO3. The mixture was extracted with EtOAc. The
organic layer was dried and concentrated in vacuo. The residue was purified by silica gel column
chromatography to give tert-butyl 4-(trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidine-1-carboxylate
24n (36.5 g, 77% in 2 steps from 23n) as a white solid. This product was used for the next step without further
purification. 1H NMR (400 MHz, CDCl3) δ 1.44 (9H, s), 2.06–2.14 (2H, m), 2.45–2.49 (2H, m),
2.61–2.78(2H, m), 3.94–4.15 (2H, m), 7.58 (2H, d, J = 8.0 Hz), 7.69 (2H, d, J = 8.8 Hz).
4-(Trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidine hydrochloride salt (3n). To a solution of
tert-butyl 4-(trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidine-1-carboxylate 24n (6.40 g, 16.1 mmol)

in EtOAc (20 mL) was added hydrogen chloride (12.1M in water, 4.0 mL, 48.4 mmol) and IPA (8.0 mL).
Then this mixture was refluxed for 5 h. The reaction mixture was concentrated in vacuo. The residue was
crystallized from diethyl ether to give 4-(trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidine
1
hydrochloride salt 3o (5.10 g, 95%) as a white solid; H NMR (400 MHz, DMSO-d6) δ 2.32–2.38 (2H, m),
2.54–2.58 (2H, m), 2.78–2.82 (2H, m), 3.29–3.43 (2H, m), 7.86 (4H, d, J = 8.8 Hz), 9.09–9.31 (2H, m).
1-(6-(3-(Hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-phenylpiperidine-4-carbonitrile
(1a).
The mixture of 4-phenylpiperidine-4-carbonitrile hydrochloride salt 3a (200 mg, 0.900 mmol),
(5-(6-chloropyridazin-3-yl)-1,2,4-oxadiazol-3-yl)methyl benzoate 2a (200 mg, 0.630 mmol), DIPEA (0.650
mL, 3.73 mmol) and IPA (4.0 mL) was heated at 150°C for 1 h under microwave irradiation. After cooling to
room temperature, the mixture was stirred at room temperature for 1h. The precipitate was collected and
washed with IPA to give desired intermediate. Sodium hydroxide (2 M in water, 1.0 mL) was added to a
solution of the above mentioned intermediate in THF (20 mL)–MeOH (10 mL) at room temperature. The
mixture was stirred at room temperature under N2 for 10 min. The mixture was poured into water at room
temperature and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried
over MgSO4 and concentrated in vacuo. The residue was crystallized from EtOAc–hexane to give
1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-phenylpiperidine-4-carbonitrile 1a (200 mg,
0.552 mmol, 87 % in 2 steps from 2a) as a colorless crystals; mp 195–199 °C. MS(ESI+) m/z: 363.2 (M+H)+.
1H NMR (300 MHz, DMSO-d6) δ 2.07–2.22 (2H, m), 2.26–2.40 (2H, m), 3.28–3.45 (2H, m), 4.60–4.70 (2H,
m), 4.74–4.90 (2H, m), 5.75–5.83 (1H, m), 7.34–7.65 (6H, m), 8.01–8.12 (1H, m). 13C NMR (76 MHz,
DMSO-d6) δ 34.7, 42.0, 42.3, 54.7, 112.3, 121.7, 125.6, 127.7, 128.2, 129.0, 138.5, 139.5, 159.2, 170.5, 172.9.
Anal. Calcd for C19H18N6O2·0.1H2O: C, 62.66; H, 5.04; N, 23.08. Found: C, 62.60; H, 5.27; N, 23.05.
(5-(6-(4-Amino-4-phenylpiperidin-1-yl)pyridazin-3-yl)-1,2,4-oxadiazol-3-yl)methanol
(5-(6-(4-Amino-4-phenylpiperidin-1-yl)pyridazin-3-yl)-1,2,4-oxadiazol-3-yl)methanol 1b was prepared by a
method similar to that described for
(1b).
1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-phenylpiperidine-4-carbonitrile 1a using 3b.
1
Yield: 94% (in 2 steps from 2a) as a colorless crystals; mp 194–198 °C. MS(ESI+) m/z: 353.1 (M+H)+. H
NMR (300 MHz, DMSO-d6) δ 1.64–1.81 (2H, m), 1.90–2.10 (4H, m), 3.55–3.76 (2H, m), 4.27–4.48 (2H, m),
4.55–4.75 (2H, m), 5.70–5.89 (1H, m), 7.16–7.24 (1H, m), 7.28–7.37 (2H, m), 7.41–7.47 (1H, m), 7.51–7.59
(2H, m), 7.93–8.05 (1H, m). 13C NMR (76 MHz, DMSO-d6) δ 37.4, 40.7, 52.3, 54.7, 111.6, 125.0, 126.0,
127.5, 127.9, 137.6, 150.6, 159.2, 170.4, 173.1. Anal. Calcd for C18H20N6O2·0.1H2O: C, 61.04; H, 5.75; N,
23.73. Found: C, 61.09; H, 5.98; N, 23.80.
1-(6-(3-(Hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-phenylpiperidin-4-ol
1-(6-(3-(Hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-phenylpiperidin-4-ol 1c was prepared by a
method similar to that described for
1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-phenylpiperidine-4-carbonitrile 1a using 3c.
(1c).

1
Yield: 68% (in 2 steps from 2a) as a colorless crystals; mp 181–185 °C. MS(ESI+) m/z: 354.1 (M+H)+. H
NMR (300 MHz, DMSO-d6) δ 1.70–1.84 (2H, m), 1.92–2.09 (2H, m), 3.41–3.58 (2H, m), 4.46–4.57 (2H, m),
4.62–4.69 (2H, m), 5.27 (1H, s), 5.73–5.83 (1H, m), 7.18–7.27 (1H, m), 7.29–7.38 (2H, m), 7.42–7.56 (3H,
m), 7.95–8.04 (1H, m). 13C NMR (76 MHz, DMSO-d6) δ 37.3, 40.7, 54.8, 70.0, 111.7, 124.7, 126.4, 127.5,
127.9, 137.8, 149.2, 159.3, 170.5, 173.1. Anal. Calcd for C18H19N5O3·0.1H2O: C, 60.87; H, 5.45; N, 19.72.
Found: C, 60.95; H, 5.72; N, 19.66.
(5-(6-(4-Methoxy-4-phenylpiperidin-1-yl)pyridazin-3-yl)-1,2,4-oxadiazol-3-yl)methanol
(5-(6-(4-Methoxy-4-phenylpiperidin-1-yl)pyridazin-3-yl)-1,2,4-oxadiazol-3-yl)methanol 1d was prepared by a
method similar to that described for
(1d).
1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-phenylpiperidine-4-carbonitrile 1a using 3d.
1
Yield: 95% (in 2 steps from 2a) as a colorless crystals; mp 187–190 °C. MS(ESI+) m/z: 368.1 (M+H)+. H
NMR (300 MHz, DMSO-d6) δ 1.87–2.03 (2H, m), 2.08–2.21 (2H, m), 2.97 (3H, s), 3.32−3.47 (2H, m),
4.39–4.56 (2H, m), 4.60–4.69 (2H, m), 5.72–5.84 (1H, m), 7.24–7.51 (6H, m), 7.96–8.05 (1H, m). 13C NMR
(76 MHz, DMSO-d6) δ 33.7, 40.4, 49.5, 54.8, 75.2, 111.8, 125.8, 127.3, 127.6, 128.4, 138.0, 143.7, 159.4,
170.5, 173.1. Anal. Calcd for C19H21N5O3: C, 62.11; H, 5.76; N, 19.06. Found: C, 61.90; H, 5.90; N, 19.21.
(5-(6-(4-Phenyl-4-(trifluoromethyl)piperidin-1-yl)pyridazin-3-yl)-1,2,4-oxadiazol-3-yl)methanol
(5-(6-(4-Phenyl-4-(trifluoromethyl)piperidin-1-yl)pyridazin-3-yl)-1,2,4-oxadiazol-3-yl)methanol 1e
prepared by method similar to that described
(1e).
was
for
a
1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-phenylpiperidine-4-carbonitrile 1a using 3e.
1
Yield: 79% (in 2 steps from 2a) as a colorless crystals; mp 221–225 °C. MS(ESI+) m/z: 406.1 (M+H)+. H
NMR (300 MHz, DMSO-d6) δ 2.00–2.19 (2H, m), 2.68–2.81 (2H, m), 2.84–3.01 (2H, m), 4.46–4.60 (2H, m),
4.61–4.69 (2H, m), 5.73–5.82 (1H, m), 7.36–7.57 (4H, m), 7.64–7.74 (2H, m), 7.96–8.07 (1H, m). 13C NMR
(76 MHz, DMSO-d6) δ 34.7, 42.0, 42.3, 54.7, 112.3, 121.7, 125.6, 127.7, 128.2, 129.0, 138.5, 139.5, 159.2,
170.5, 172.9. Anal. Calcd for C19H18F3N5O2·0.1H2O: C, 56.05; H, 4.51; N, 17.20. Found: C, 56.22; H, 4.81; N,
17.16.
4-(2-Chlorophenyl)-1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)piperidine-4-carbonitri
le
4-(2-chlorophenyl)-1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)piperidine-4-carbonitrile 1f
was prepared by method similar to that described for
(1f).
a
1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-phenylpiperidine-4-carbonitrile 1a using 3f.
1
Yield: 63% (in 2 steps from 2a) as a colorless crystals; mp 187–190 °C. MS(ESI+) m/z: 397.1 (M+H)+. H
NMR (300 MHz, DMSO-d6) δ 2.05–2.25 (2H, m), 2.59–2.75 (2H, m), 3.34–3.48 (2H, m), 4.60–4.72 (2H, m),
4.75–4.93 (2H, m), 5.69–5.88 (1H, m), 7.39–7.66 (5H, m), 8.03–8.13 (1H, m). 13C NMR (76 MHz,
DMSO-d6) δ 32.8, 40.4, 41.6, 54.8, 112.3, 119.9, 127.8, 127.9, 128.1, 130.2, 131.6, 132.5, 135.1, 138.7, 159.3,
170.6, 172.9. Anal. Calcd for C19H17ClN6O2·0.1H2O: C, 57.25; H, 4.35; N, 21.08. Found: C, 57.30; H, 4.57; N,

21.15.
4-(3-Chlorophenyl)-1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)piperidine-4-carbonitri
le
4-(3-Chlorophenyl)-1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)piperidine-4-carbonitrile 1g
was prepared by method similar to that described for
(1g).
a
1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-phenylpiperidine-4-carbonitrile 1a using 3g.
1
Yield: 74% (in 2 steps from 2a) as a colorless crystals; mp 194–197 °C. MS(ESI+) m/z: 397.1 (M+H)+. H
NMR (300 MHz, DMSO-d6) δ 2.10–2.24 (2H, m), 2.28–2.40 (2H, m), 3.26–3.43 (2H, m), 4.59–4.70 (2H, m),
4.74–4.90 (2H, m), 5.72–5.83 (1H, m), 7.41–7.62 (4H, m), 7.64–7.68 (1H, m), 8.02–8.11 (1H, m). 13C NMR
(76 MHz, DMSO-d6) δ 34.5, 42.0, 42.4, 54.8, 112.3, 121.3, 124.7, 125.8, 127.7, 128.3, 130.9, 133.8, 138.6,
141.9, 159.2, 170.6, 173.0. Anal. Calcd for C19H17ClN6O2: C, 57.51; H, 4.32; N, 21.18. Found: C, 57.50; H,
4.62; N, 21.33.
4-(4-Chlorophenyl)-1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)piperidine-4-carbonitri
le
4-(4-chlorophenyl)-1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)piperidine-4-carbonitrile 1h
was prepared by method similar to that described for
(1h).
a
1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-phenylpiperidine-4-carbonitrile 1a using 3h.
1
Yield: 76% (in 2 steps from 2a) as a colorless crystals; mp 194–195 °C. MS(ESI+) m/z: 397.1 (M+H)+. H
NMR (300 MHz, DMSO-d6) δ 2.07–2.22 (2H, m), 2.26–2.37 (2H, m), 3.27–3.42 (2H, m), 4.60–4.70 (2H, m),
4.74–4.88 (2H, m), 5.73–5.83 (1H, m), 7.49–7.57 (3H, m), 7.59–7.67 (2H, m), 8.01–8.12 (1H, m). 13C NMR
(76 MHz, DMSO-d6) δ 34.6, 42.0, 42.1, 54.8, 112.3, 121.4, 127.7, 127.8, 129.0, 132.9, 138.6, 138.6, 159.2,
170.6, 173.0. Anal. Calcd for C19H17ClN6O2: C, 57.51; H, 4.32; N, 21.18. Found: C, 57.49; H, 4.60; N, 21.35.
1-(6-(3-(Hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-(4-nitrophenyl)piperidine-4-carbonitrile
(1i). 1-(6-(3-(Hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-(4-nitrophenyl)piperidine-4-carbonitrile
1i
was
prepared
by
a
method
similar
to
that
described
for
1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-phenylpiperidine-4-carbonitrile 1a using 3i.
1
Yield: 89% (in 2 steps from 2a) as a colorless crystals; mp 211–214 °C. MS(ESI+) m/z: 408.1 (M+H)+. H
NMR (400 MHz, DMSO-d6) δ 2.17–2.29 (2H, m), 2.31–2.41 (2H, m), 3.31–3.45 (2H, m), 4.62–4.69 (2H, m),
4.80–4.91 (2H, m), 5.75–5.82 (1H, m), 7.51–7.60 (1H, m), 7.87–7.96 (2H, m), 8.04–8.11 (1H, m), 8.27–8.34
(2H, m). 13C NMR (101 MHz, DMSO-d6) δ 34.3, 41.9, 42.8, 54.7, 112.3, 120.9, 124.0, 127.5, 127.7, 138.6,
146.4, 147.2, 159.2, 170.5, 172.9. Anal. Calcd for C19H17N7O4·0.8H2O: C, 54.10; H, 4.44; N, 23.25. Found: C,
53.97; H, 4.35; N, 23.19.
4-(4-(tert-Butyl)phenyl)-1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)piperidine-4-carbo
nitrile
(1j).

4-(4-(tert-Butyl)phenyl)-1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)piperidine-4-carbonitril
1j was prepared by method similar to that described for
e
a
1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-phenylpiperidine-4-carbonitrile 1a using 3j.
1
Yield: 83% (in 2 steps from 2a) as a colorless crystals; mp 227–231 °C. MS(ESI+) m/z: 419.2 (M+H)+. H
NMR (400 MHz, DMSO-d6) δ 1.28 (9H, s), 2.05–2.21 (2H, m), 2.26–2.37 (2H, m), 3.29–3.41 (2H, m),
4.60–4.70 (2H, m), 4.75–4.88 (2H, m), 5.74–5.84 (1H, m), 7.42–7.59 (5H, m), 8.01–8.12 (1H, m). 13C NMR
(101 MHz, DMSO-d6) δ 30.9, 34.2, 34.7, 41.8, 42.0, 54.7, 112.3, 121.8, 125.3, 125.7, 127.7, 136.6, 138.5,
150.6, 159.2, 170.5, 172.9. Anal. Calcd for C23H26N6O2: C, 66.01; H, 6.26; N, 20.08. Found: C, 65.86; H,
6.45; N, 20.29.
1-(6-(3-(Hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-(4-(trifluoromethoxy)phenyl)piperidine-
4-carbonitrile
1-(6-(3-(Hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-(4-(trifluoromethoxy)phenyl)piperidine-4-ca
rbonitrile 1k was prepared by method similar to that described for
(1k).
a
1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-phenylpiperidine-4-carbonitrile 1a using 3k.
1
Yield: 78% (in 2 steps from 2a) as a colorless crystals; mp 190–193 °C. MS(ESI+) m/z: 447.0 (M+H)+. H
NMR (400 MHz, DMSO-d6) δ 2.10–2.25 (2H, m), 2.29–2.40 (2H, m), 3.27–3.42 (2H, m), 4.61–4.70 (2H, m),
4.77–4.92 (2H, m), 5.75–5.83 (1H, m), 7.43–7.50 (2H, m), 7.52–7.59 (1H, m), 7.70–7.79 (2H, m), 8.02–8.12
(1H, m). 13C NMR (101 MHz, DMSO-d6) δ 34.6, 42.0, 42.1, 54.8, 112.3, 119.9, 121.4, 121.5, 127.7, 128.0,
138.6, 138.9, 147.9, 159.2, 170.5, 172.9. Anal. Calcd for C20H17F3N6O3: C, 53.81; H, 3.84; N, 18.83. Found: C,
53.91; H, 4.14; N, 18.96.
1-(6-(3-(Hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-(4-(trifluoromethyl)phenyl)piperidine-4-
carbonitrile
1-(6-(3-(Hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-(4-(trifluoromethyl)phenyl)piperidine-4-carb
onitrile 1l was prepared by method similar to that described for
(1l).
a
1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-phenylpiperidine-4-carbonitrile 1a using 3l.
1
Yield: 79% (in 2 steps from 2a) as a colorless crystals; mp 186–189 °C. MS(ESI+) m/z: 431.0 (M+H)+. H
NMR (300 MHz, DMSO-d6) δ 2.13–2.29 (2H, m), 2.29–2.43 (2H, m), 3.29–3.44 (2H, m), 4.62–4.70 (2H, m),
4.79–4.91 (2H, m), 5.73–5.84 (1H, m), 7.50–7.59 (1H, m), 7.78–7.91 (4H, m), 8.03–8.12 (1H, m). 13C NMR
(76 MHz, DMSO-d6) δ 35.0, 42.5, 43.2, 55.3, 112.9, 121.7, 124.5, 126.5, 127.4, 128.3, 129.4, 139.1, 144.6,
159.8, 171.1, 173.5. Anal. Calcd for C20H17F3N6O2·0.1H2O: C, 55.58; H, 4.01; N, 19.45. Found: C, 55.69; H,
4.16; N, 19.45.
1-(6-(3-(Hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-(4-(pentafluorosulfanyl)phenyl)piperidi
ne-4-carbonitrile
1-(6-(3-(Hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-(4-(pentafluorosulfanyl)phenyl)piperidine-4-
carbonitrile 1m was prepared by method similar to that described for
(1m).
a

1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-phenylpiperidine-4-carbonitrile 1a using 3m.
1
Yield: 58% (in 2 steps from 2a) as a colorless crystals; mp 228–232 °C. MS(ESI+) m/z: 489.1 (M+H)+. H
NMR (300 MHz, DMSO-d6) δ 2.12–2.27 (2H, m), 2.29–2.40 (2H, m), 3.28–3.44 (2H, m), 4.61–4.68 (2H, m),
4.78–4.91 (2H, m), 5.73–5.81 (1H, m), 7.49–7.60 (1H, m), 7.79–7.91 (2H, m), 7.96–8.12 (3H, m). 13C NMR
(76 MHz, DMSO-d6) δ 34.9, 42.5, 43.1, 55.3, 112.9, 121.5, 127.0, 127.1, 127.8, 128.3, 139.1, 144.4, 159.7,
171.1, 173.5. Anal. Calcd for C19H17F5N6O2S·0.2H2O: C, 46.38; H, 3.56; N, 17.08. Found: C, 46.31; H, 3.85;
N, 16.90.
(5-(6-(4-(Trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidin-1-yl)pyridazin-3-yl)-1,2,4-oxadiazol-3
-yl)methanol
(5-(6-(4-(Trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidin-1-yl)pyridazin-3-yl)-1,2,4-oxadiazol-3-yl)m
ethanol 1n was prepared by method similar to that described for
(1n).
a
1-(6-(3-(hydroxymethyl)-1,2,4-oxadiazol-5-yl)pyridazin-3-yl)-4-phenylpiperidine-4-carbonitrile 1a using 3n.
1
Yield: 80% (in 2 steps from 2a) as a colorless crystals; mp 189–192 °C. MS(ESI+) m/z: 474.1 (M+H)+. H
NMR (300 MHz, DMSO-d6) δ 2.03–2.28 (2H, m), 2.73–3.00 (4H, m), 4.48–4.60 (2H, m), 4.62–4.69 (2H, m),
5.71–5.83 (1H, m), 7.36–7.49 (1H, m), 7.82–7.91 (2H, m), 7.92–7.99 (2H, m), 8.00–8.08 (1H, m). 13C NMR
(75 MHz, DMSO-d6) δ 27.2, 39.9, 46.7, 54.8, 112.2, 122.2, 125.6, 129.1, 127.6, 128.9, 130.3, 138.2, 138.5,
159.4, 170.5, 172.9. Anal. Calcd for C20H17F6N5O2: C, 50.75; H, 3.62; N, 14.79. Found: C, 50.82; H, 3.91; N,
14.88.
(5-(6-(4-(Trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidin-1-yl)pyridazin-3-yl)-1,3,4-oxadiazol-2
-yl)methanol (1o). The mixture of 4-(trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidine hydrochloride
salt 3n (12.00 g, 36.0 mmol), (5-(6-chloropyridazin-3-yl)-1,3,4-oxadiazol-2-yl)methanol 2b (7.50 g, 35.3
mmol), DIPEA(12 mL, 68.9 mmol) and IPA(110 mL) was heated at 150°C for 1 h under microwave irradiation.
After cooling to room temperature, the mixture was stirred at room temperature overnight. The precipitate was
crystallized
from
EtOH
(300
mL)–water
(500
mL)
to
give
(5-(6-(4-(trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidin-1-yl)pyridazin-3-yl)-1,3,4-oxadiazol-2-yl)m
ethanol 1o (12.00 g, 72%) as colorless crystals; mp 208–211 °C. MS(ESI+) m/z: 474.1 (M+H)+. 1H NMR (300
MHz, DMSO-d6) δ 2.06–2.28 (2H, m), 2.70–3.04 (4H, m), 4.43–4.59 (2H, m), 4.70–4.81 (2H, m), 5.88–6.10
(1H, m), 7.35–7.52 (1H, m), 7.78–8.10 (5H, m). 13C NMR (75 MHz, DMSO-d6) δ 26.6, 39.4, 46.1, 53.1,
112.3, 121.7, 125.1, 125.3, 126.2, 128.3, 129.8, 137.6, 137.8, 158.9, 162.1, 165.9. Anal. Calcd for
C20H17F6N5O2: C, 50.75; H, 3.62; N, 14.79. Found: C, 50.70; H, 3.91; N, 14.93.
(5-(6-(4-(Trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidin-1-yl)pyridazin-3-yl)-1,2,4-thiadiazol-3
-yl)methanol
(1p).
The
mixture
of
DIPEA
(0.472
mL,
2.70
0.90
mmol),
mmol),
(5-(6-chloropyridazin-3-yl)-1,2,4-thiadiazol-3-yl)methyl benzoate 2c
(300 mg,
4-(trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidine hydrochloride salt 3n (331 mg, 0.99 mmol) in
DMA (5 mL) was stirred at 80 °C for 16 h. The mixture was poured into hydrogen chloride (0.1M in water) and

extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4 and
concentrated in vacuo to give desired intermediate. Sodium hydroxide (2M in water, 1 mL, 2.0 mmol) was
added to a solution of the above mentioned intermediate in THF (20 mL)–MeOH (20 mL) at room temperature.
The mixture was stirred at room temperature under N2 for 30 min. The mixture was poured into hydrogen
chloride (0.1M in water) at room temperature and extracted with EtOAc. The organic layer was separated,
washed with water and brine, dried over MgSO4 and concentrated in vacuo. The residue was crystallized from
EtOAc-hexane
to
give
(5-(6-(4-(trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidin-1-yl)pyridazin-3-yl)-1,2,4-thiadiazol-3-yl)m
1
ethanol 1p (335 mg, 76%) as a colorless crystals; mp 199–201 °C. MS(ESI+) m/z: 490.1 (M+H)+. H NMR
(300 MHz, DMSO-d6) δ 2.05–2.24 (2H, m), 2.73–2.99 (4H, m), 4.44–4.61 (2H, m), 4.67–4.79 (2H, m),
5.58–5.73 (1H, m), 7.41–7.51 (1H, m), 7.80–8.05 (5H, m). 13C NMR (75 MHz, DMSO-d6) δ 27.7, 40.5, 47.2,
60.8, 113.5, 122.7, 125.3, 126.1, 126.3, 129.4, 130.9, 138.7, 144.7, 160.4, 177.5, 186.7. Anal. Calcd for
C20H17F6N5OS: C, 49.08; H, 3.50; N, 14.31. Found: C, 49.04; H, 3.76; N, 14.39.
(5-(6-(4-(Trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidin-1-yl)pyridazin-3-yl)-1,3,4-thiadiazol-2
-yl)methanol (1q). The mixture of 4-(trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidine hydrochloride
salt 3n (50 mg, 0.15 mmol), (5-(6-chloropyridazin-3-yl)-1,3,4-thiadiazol-2-yl)methanol 2d (30 mg, 0.13
mmol), DIPEA (0.100 mL, 0.57 mmol) and IPA (4 mL) was heated at 150 °C for 1 h under microwave
irradiation. The reaction mixture was concentrated in vacuo. The residue was purified by column
chromatography
to
give
(5-(6-(4-(trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)piperidin-1-yl)pyridazin-3-yl)-1,3,4-thiadiazol-2-yl)m
ethanol 1q (45 mg, 0.092 mmol, 70%) as a colorless crystals; mp 199–202 °C. MS(ESI+) m/z: 490.1 (M+H)+.
1H NMR (300 MHz, DMSO-d6) δ 2.02–2.26 (2H, m), 2.73–2.97 (4H, m), 4.43–4.57 (2H, m), 4.83–4.95 (2H,
m), 6.21–6.33 (1H, m), 7.41–7.50 (1H, m), 7.81–7.91 (2H, m), 7.93–8.01 (2H, m), 8.06–8.16 (1H, m). 13C
NMR (75 MHz, DMSO-d6) δ 27.6, 40.5, 47.2, 58.9, 113.8, 122.7, 125.3, 126.1, 126.3, 129.4, 130.9, 138.7,
144.7, 160.1, 167.7, 174.3. Anal. Calcd for C20H17F6N5OS: C, 49.08; H, 3.50; N, 14.31. Found: C, 49.08; H,
3.75; N, 14.51.
Human SCD1 binding assay using radiolabeled ligand
Radioligand binding assays were performed in a total volume of 200 µl in 96-well plates in a buffer
containing 10 mM Tris-HCl (pH 7.5), 100 mM KCl, 3 mM MgCl2, 10 mM NaF, 0.005% Tween-20 and 1 mM
glutathione. The liver microsomes and various concentrations of test compounds were incubated with 3.0 nM
(5-(6-(1'H-spiro(1-benzofuran-3,3'-pyrrolidin)-1'-yl)pyridazin-3-yl)-1,2,4-oxadiazol-3-yl)[3H2]methanol
(3H-A) for 120 min. Bound ligands on microsomes were captured on GF/B filter plates (PerkinElmer)
pretreated with 0.3% polyethyleneimine and were separated from free ligands using rapid filtration with a
Filtermate Harvester (PerkinElmer) followed by five washes in 300 µL of 10 mM Tris–HCl buffer (pH 7.5).
Filter plates were then dried and radioactivity was measured after adding 25 µL of Microscint-O
(PerkinElmer) using a TopCount liquid scintillation counter (PerkinElmer). DMSO and 10 µM nonlabeled

compound A were used as 100% and 0% controls, respectively. IC50 values of test compounds were
determined by fitting the following three-parameter logistic equation to the data; % control = bottom + (top −
]−log IC50
bottom)/(1 + 10(log[
)).
??
Cell viability (GI) assay
HTC116 cells were seeded at 9 × 102 cells/well in 384-well cell culture plates (Corning, Corning, NY) and
cultured 24 h. Then, test compounds were diluted in growth medium to the desired final concentration and
added to the cells. After 72 h of incubation, cell proliferation was measured with a CellTiter-Glo®
Luminescent Cell Viability Assay (Promega Corporation, Madison, WI). GI50 values of test compounds were
determined by fitting the Hill equation to the data, where bottom was fixed to 0, by using GraphPad Prism
software (GraphPad, San Diego, CA, USA).
In vivo pharmacodynamics (PD) testing and lipidomics
Mice were housed and maintained within the facility at Takeda Pharmaceutical Company Shonan Research
Center in accordance with the Takeda Experimental Animal Care and Use Committee approved protocol.
Athymic nude mice were injected with 5 × 106 HCT116 cells. When the tumor volumes reached
approximately 200 mm3, mice were randomly assigned to control or treatment groups. The compounds were
orally administered to the mice in 0.5% methylcellulose (Shin-Etsu Chemical Co., Ltd., Tokyo, Japan).
Tumors were collected 16 hours after twice administration of compound 1o. Tumors were homogenized in
isopropanol (100 mg/mL) for lipidomics, using TissueLyzerII (Qiagen), and centrifuged at 21500 g for 5 min.
The supernatants were applied to lipidomics analysis.
Each 5 µL sample was injected onto a XBridge BEH C18 column (2.1 ×30 mm, 2.5 µm, Shiseido, Kyoto,
Japan) maintained at 60°C, and the metabolites were separated by gradient elution of mobile phase A, 0.01%
acetic acid, 1 mM NH3 and 10 µM EDTA-2Na in MilliQ water, and of mobile phase B, 0.001% acetic acid
and 0.2 mM NH3 in ethanol/isopropanol (1:1), with the flow rate set to 0.5 mL/min. The eluate was introduced
to an Orbitrap XL Mass Spectrometer (Thermo Fisher Scientific inc., San Jose, CA, USA), and the raw
LC/MS data were processed by Expressionist Refiner MS software (ver.8.2, Genedata AG, Basel,
Switzerland). Desaturation Index (DI) was calculated as the ratio of the unsaturated lipid molecule (PC C36:3)
to the saturated lipid molecule (PC C36:0).
In vivo efficacy testing and statistical analysis
All experimental procedures were approved by Shanghai Medicilon Inc., which was fully accredited by
Association for Assessment and Accreditation of Laboratory Animal Care International. Athymic nude mice
(BALB/cA Jcl-nu/nu) and 786-O Human renal cell adenocarcinoma cell line were purchased from Shanghai
SINO-British SIPPR/BK Lab Animal Ltd (Shanghai, China) and ATCC (American Type Culture Collection),
respectively. Seven weeks old female balb/c nu/nu mice were inoculated subcutaneously in the flank (cell
suspension, 1:1 with matrigel) with 3.0 × 106 786-O cells. Tumor growth was monitored with vernier calipers.
The mean tumor volume was calculated using the formula Volume = Width2 × Length/2. When the mean

tumor volume reached approximately 130 mm3, the animals were randomized into 8 treatment groups
(n=8/group). Mice were then dosed with 0.5% methylcellulose or compound 1o at 1 mg/kg over a 28 day
period. The dosing volume for each mouse was 10 mL/kg. Tumor growth was measured twice per week.
Tumor growth inhibition was calculated on Day 28 of treatment.
All statistical analyses were carried out using SAS System, Release 9.3 (SAS Institute Inc
[Cary, NC, USA]).
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Acknowledgements
We sincerely appreciate Drs. Hiroshi Miyake, Takahito Hara, Tomoyasu Ishikawa, Yuichi Hikichi, for
intellectual support during this work. We also appreciate Drs. Yasuhiro Imaeda, Douglas R. Cary and Satoru
Nishizawa for helpful suggestions during the preparation of this manuscript. We would like to express our
appreciation to Kazunobu Aoyama for supporting in PK studies; Takeshi Watanabe for helping in drug safety.
Graphical Abstract