Discovery of piperazin-1-ylpyridazine-based potent and selective stearoyl-CoA Desaturase-1 inhibitors for the treatment of obesity and metabolic syndrome

Article abstract of DOI:10.1021/jm301661h

Stearoyl-CoA desaturase-1 (SCD1) catalyzes de novo synthesis of monounsaturated fatty acids from saturated fatty acids. Studies have demonstrated that rodents lacking a functional SCD1 gene have an improved metabolic profile, including reduced weight gain, lower triglycerides, and improved insulin response. In this study, we discovered a series of piperazinylpyridazine-based highly potent, selective, and orally bioavailable compounds. Particularly, compound 49 (XEN103) was highly active in vitro (mSCD1 IC₅₀ = 14 nM and HepG2 IC₅₀ = 12 nM) and efficacious in vivo (ED₅₀ = 0.8 mg/kg). It also demonstrated striking reduction of weight gain in a rodent model. Our findings with small-molecule SCD1 inhibitors confirm the importance of this target in metabolic regulation, describe novel models for assessing SCD1 inhibitors for efficacy and tolerability and demonstrate an opportunity to develop a novel therapy for metabolic disease.

Full text of DOI:10.1021/jm301661h

Article
pubs.acs.org/jmc
Discovery of Piperazin-1-ylpyridazine-Based Potent and Selective
Stearoyl-CoA Desaturase‑1 Inhibitors for the Treatment of Obesity
and Metabolic Syndrome
Zaihui Zhang,† Shaoyi Sun,† Vishnumurthy Kodumuru,† Duanjie Hou,† Shifeng Liu,† Nagasree Chakka,†
Serguei Sviridov,† Sultan Chowdhury,† David G. McLaren,† Leslie G. Ratkay,† Kuldip Khakh,†
Xing Cheng,† Heinz W. Gschwend,‡ Rajender Kamboj,† Jianmin Fu,† and Michael D. Winther*,†
†Xenon Pharmaceuticals, Inc., 3650 Gilmore Way, Burnaby, BC V5G 4W8 Canada
‡Consultant, 161 Meadowcroft Way, Santa Rosa, California 95403, United States
ABSTRACT: Stearoyl-CoA desaturase-1 (SCD1) catalyzes de
novo synthesis of monounsaturated fatty acids from saturated
fatty acids. Studies have demonstrated that rodents lacking a
functional SCD1 gene have an improved metabolic profile,
including reduced weight gain, lower triglycerides, and
improved insulin response. In this study, we discovered a
series of piperazinylpyridazine-based highly potent, selective,
and orally bioavailable compounds. Particularly, compound 49
(XEN103) was highly active in vitro (mSCD1 IC50 = 14 nM and HepG2 IC50 = 12 nM) and efficacious in vivo (ED50 = 0.8 mg/
kg). It also demonstrated striking reduction of weight gain in a rodent model. Our findings with small-molecule SCD1 inhibitors
confirm the importance of this target in metabolic regulation, describe novel models for assessing SCD1 inhibitors for efficacy
and tolerability and demonstrate an opportunity to develop a novel therapy for metabolic disease.
avoided in developing systemically administered compounds.
The beneficial phenotypes seen in these genetic models are also
observed in high fat diet-induced obese (DIO) mice treated
with antisense oligonucleotides (ASOs) directed toward
SCD1.8,9 In humans, a similar role for SCD1 can be postulated
because elevated SCD1 activity positively correlates with high
triglyceride levels in familial hypertriglyceridemia subjects,10
increased body mass index (BMI), and high plasma insulin
levels.11 Therefore, compounds that inhibit SCD1 may hold
significant therapeutic potential for the treatment of obesity,
type 2 diabetes, and other metabolic disorders.
No drug-like, selective SCD1 inhibitors were reported until
we published a series of patent applications in 2005.12−16 These
selective SCD inhibitors demonstrated that SCD1 activity could
be potently inhibited by small drug-like molecules. Since then, a
number of small-molecule SCD1 inhibitors have been
reported.17−31 Our program targeting the discovery of small-
molecule SCD1 inhibitors for the treatment of obesity and
metabolic syndrome started with a high throughput screening
campaign that identified a number of pyridin-2-ylpiperazine
compounds with micromolar activity against mouse SCD1.
Compound 1 (Figure 1) was the most potent identified from
the library screen, with mSCD IC50 of 5.1 μM. The initial hit-
to-lead effort was focused on developing the SAR of this
pyridin-2-ylpiperazine scaffold and a focused compound library
of 588 compounds (Figure 1) was initially synthesized.32,33
INTRODUCTION
■
Obesity and type 2 diabetes are emerging as two major global
health problems of the 21st century. Evidence published over
the past decade has shown that abnormal lipid metabolism is
closely related to occurrence of metabolic syndrome, obesity,
type 2 diabetes, and dyslipidemia.1 One potential target for
treatment of these diseases, which has recently received great
attention in the scientific community, is stearoyl-CoA
desaturase-1 (SCD1, commonly known as delta-9 desaturase,
D9D).2 SCD1 catalyzes de novo synthesis of monounsaturated
fatty acids (MUFA) from saturated fatty acids by introduction
of a cis double bond between carbons 9 and 10 positions of
stearic acid and palmitic acid. The products, mainly oleate and
palmitoleate, are key substrates for the synthesis of triglycerides,
wax esters, cholesterol esters, and phospholipids.3 SCD1 is the
major isoform present in lipogenic tissues (including liver and
adipose tissues) in rodents and humans and functions as a key
regulator of lipid and carbohydrate metabolism. SCD1-deficient
mice have been shown to be lean and hypermetabolic.4 In the
leptin-deficient model for obesity (ob/ob), mice with an
inactive SCD1 gene were significantly less obese than the ob/
ob controls and had markedly increased energy expenditure; in
addition, these mice had histologically normal livers with
significantly reduced triglyceride storage and very-low-density
lipoprotein (VLDL) production.4 The data support a model in
which SCD1 deficiency is associated with an activation of lipid
oxidation and reduction of triglyceride synthesis and storage.5−7
Genetic models also show significant skin and eye effects arising
from diminished SCD1 activity, an effect that needs to be
Received: November 9, 2012
Published: December 17, 2012
© 2012 American Chemical Society
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Figure 1. The structure of HTS hit (1) and the hit-to-lead process to identify compound 2 for lead optimization.
Several preferred amine (R1) and carboxylic acids (R2) were
identified, and compound 2 emerged as the most potent with
mSCD IC50 of 78 nM (Figure 1). Herein, we report on our lead
optimization efforts toward compound 2, which led to the
discovery of a novel, potent, selective, and orally bioavailable
piperazinylpyridazine inhibitor 49 (XEN103). The in vitro
potency and in vivo efficacy will also be reported herein.
(trifluoromethyl)benzoyl chloride, followed by the treatment
with trifluoroacetic acid to afford piperazin-1-yl(2-
(trifluoromethyl)phenyl)methanone 4a. Replacement of the
chloro group on methyl 5-chloropyrazine-2-carboxylate with
compound 4a via nucleophilic substitution generated the ester
intermediate which was subjected to base hydrolysis to afford
carboxylic acid 4b. The final compound 4 was obtained after
the amide formation between 4b and isoamylamine.
CHEMISTRY
The 5-(piperazin-1-yl)pyrimidine analogue was obtained
following Scheme 3. Starting from commercially available 5-
■
2-(Piperazin-1-yl)pyrimidine analogue 3 (Scheme 1) was
synthesized starting from ethyl 2-(methylsulfonyl)pyrimidine-
a
Scheme 3
a
Scheme 1
a
Reagents and conditions: (a) NaCN, 0.15 equiv DABCO, H2O/
DMSO (1/1), rt, 2 h; (b) 4a, Cs2CO3, NMP (0.25 M), 80 °C; (c) n-
hexylamine, RuCl2(PPh3)3, 1,2-dimethoxyethane, 160 °C.
a
Reagents and conditions: (a) tert-butyl piperazine-1-carboxylate,
DIPEA, 1,4-dioxane, 100 °C, 13 h; (b) TFA, DCM, rt, overnight; (c)
2-(trifluoromethyl)benzoyl chloride, DIPEA, DCM, 0 °C to rt, 6 h;
(d) LiOH, THF/MeOH, H2O, rt, 24 h; (e) isoamylamine, HOBt,
EDCI, DIPEA, DCM, rt, 15 h.
bromo-2-chloropyrimidine, replacement of the chloro group by
a nitrile group was achieved in the presence of catalytic
DABCO to generate compound 5a. Under nucleophilic
substitution reaction conditions, the bromo group of 5a was
replaced by compound 4a to afford nitrile compound 5b.
Treatment of nitrile compound 5b with n-hexylamine under
ruthenium catalyzed amidation conditions,34 afforded the
desired product 5.
For the synthesis of pyridine-3-ylpiperazine analogue 6
(Scheme 4), 5-hydroxypicolinic acid was treated with isoamyl
amine under the amide formation conditions, followed by the
treatment with triflate anhydride to afford compound 6a. Under
palladium catalyzed coupling reaction conditions, the triflate
group was replaced by tert-butyl piperazine-1-carboxylate and
subsequent removal of BOC group with TFA generated
5-carboxylate. Under nucleophilc substitution reaction con-
ditions, the methylsulfonyl group was replaced by tert-butyl
piperazine-1-carboxylate. The BOC protecting group was
removed with trifluoroacetic acid to afford the 2-(piperazin-1-
yl)pyrimidine core structure. Amide formation with 2-
(trifluoromethyl)benzoyl chloride yielded the ester intermedi-
ate 3a. Ester saponification and amide formation between the
carboxylic acid and isoamylamine yielded the final product 3.
For the synthesis of 5-(piperazin-1-yl)pyrazine analogue 4 as
outlined in Scheme 2, the sequence started from the amide
formation between tert-butyl piperazine-1-carboxylate and 2-
a
Scheme 2
a
Reagents and conditions: (a) tert-butyl piperazine-1-carboxylate, DIPEA, DCM; (b) 15% TFA, DCM, 1 h; (c) methyl 5-chloropyrazine-2-
carboxylate, DBU, Bu4NI, DMF, 80 °C, 70 h; (d) LiOH, MeOH/H2O, 19 h; (e) isoamylamine, EDCI, HOBt, DIPEA, DCM, 18 h.
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a
Scheme 4
a
Reagents and conditions: (a) isoamylamine, HOBt, EDCI, DIPEA, DCM, rt, 23 h; (b) Tf2O, pyridine, 0 °C to rt, 25 h; (c) tert-butyl piperazine-1-
carboxylate, Pd(OAc)2, BINAP, DIPEA, DCM, 98 °C, 23 h; (d) TFA, DCM, rt, 15 h; (e) 2-(trifluoromethyl)benzoyl chloride, DIPEA, DCM, 0 °C
to rt, 5 h.
compound 6b. The amide formation between 6b and 2-
(trifluoromethyl)benzoyl chloride afforded the desired product
6.
with 2-cyclopropylethylamine to generate compound 8a. Under
nucleophilic substitution reaction conditions, the chloro group
of 8a was replaced by piperazine to afford compound 8b.
Amide formation between 8b and appropriate acid chlorides or
appropriate carboxylic acids generated final compounds 8 and
36−57.
The piperazin-1-ylpyridazine analogues with different R1
groups were synthesized following Scheme 5. Starting from
Scheme 5. General Synthetic Route for Piperazin-1-
a
ylpyridazine Libraries with Different R1
RESULTS AND DISCUSSION
■
The potency of all compounds was assessed in a series of assays
beginning with a mouse liver microsomal assay as the primary
biochemical assay. Compounds showing significant inhibition
in this assay were then advanced for further characterization in
a human liver hepatocellular carcinoma (HepG2) cell-based
assay. In the mouse liver microsomal assay, the SCD1 activity
was determined by measuring decreased production of tritiated
water released from the (9,10-3H)-labeled stearoyl coenzyme A
substrate. In the human HepG2 cell-based assay, SCD1 activity
was assessed by determination of the amount of 14C-oleic acid
product formed from 14C stearate substrate. The activity against
desaturase-5 and desaturase-6 was also evaluated to determine
the desaturase selectivity. We initiated our structure−activity
relationship (SAR) exploration using compound 2 as the
starting point. Because of the novelty concerns associated with
the original pyridin-2-ylpiperazine core structure, our initial
analogue efforts were focused on the replacement of the
pyridin-2-yl moiety with alternative six-membered nitrogen-
containing heterocycles. The results summarized in Table 1
demonstrated that analogues with pyrimidine (compounds 3
and 5) and pyrazine (compound 4) replacements had
dramatically decreased the SCD1 activity. The pyridin-3-yl
replacement (compound 6) retained activity in mouse liver
microsomal assay, but it was more than 24-fold less potent in
human HepG2 assay. The best results were obtained with the
a
Reagents and conditions: (a) piperazin-1-yl(2-(trifluoromethyl)-
phenyl)methanone 4a, K2CO3, Bu4NI, 1,4-dioxane, reflux, 24 h; (b)
LiOH, THF/H2O, rt, 6 h; (c) thionyl chloride, DMF, reflux, 18 h;
R1NH2, Et3N, DCM, rt, 4 h.
methyl 6-chloropyridazine-3-carboxylate, replacement of the
chloro group with compound piperazin-1-yl(2-
(trifluoromethyl)phenyl)methanone 4a via nucleophilic sub-
stitution afforded the intermediate 7a, which was hydrolyzed
under basic conditions to generate the carboxylic acid 7b. The
amide formation between the acid chloride of 7b and
appropriate amines afforded the final compounds 7−35.
The piperazin-1-ylpyridazine analogues with different R2
groups were synthesized following Scheme 6. 6-Chloropyr-
idazine-3-carboxylic acid was converted to the acid chloride by
the treatment with thionyl chloride, followed by the reaction
a
Scheme 6. General Synthetic Route for Piperazin-1-ylpyridazine Libraries with Different R2 Groups
a
Reagents and conditions: (a) thionyl chloride, DMF (catalytic amount), chloroform, reflux, 1 h; (b) 2-cyclopropylethylamine, Et3N, 0 °C to rt, 30
min.; (c) piperazine, acetonitrile, reflux, 2 h; (d) R2COCl, Et3N, DCM, 0 °C to rt, 1 h; (e) R2COOH, HOBt, EDCI, DIPEA, DCM, rt, overnight.
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pyridazine replacement (compound 7), which improved the
potency by about 3-fold in both assays. Moreover, this
compound possessed reasonable drug-like properties such as
minimal or no detectable CYP inhibitory activity (<50%
inhibition at 10 μM for CYP3A4, 2C9, 2C19, and 2D6),
reasonable metabolic stability (57% remaining after 30 min
incubation in rat liver microsomes), good cell permeability (in
Caco-2 cells, Papp(a to b) 25 × 10−6 cm/s), and moderate
protein binding (78% bound). In addition, pyridazine
compound 7 demonstrated a robust in vivo pharmacodynamic
response (PD) inhibiting rat liver SCD1 activity by about 40%
at 4 h post final dose after two days of once daily dosing
intraperitoneally (ip) at 15 mg/kg.
Table 1. SAR of Analogues with the Replacement of
Pyridine-2-yl Moiety
Encouraged by the discovery of the pyridazine replacement,
our optimization efforts were then focused on the piper-
azinylpyridazine template to improve the SCD1 potency,
pharmaceutical properties, and more importantly, in vivo
activity. We first kept the right-hand side of the molecule
constant using the 2-trifluoromethylbenzoyl group and varied
the left-hand side amide with diverse amines. The results are
summarized in Table 2. It was clearly demonstrated that a
variety of structural modifications at R1 were well tolerated. For
cycloalkylalkyl groups, the length of the linker between the
a
b
IC50 data presented are means of n ≥ 2. ND = not determined.
Table 2. SAR and Properties of Piperazinylpyridazine Analogues with Different R1 Groups
a
a
b
c
compd
R1
mSCD1 IC50 (nM)
HepG2 IC50 (nM)
39
RLM stability
cell permeability
8
cycloproylethyl
25
8690
34
76
ND
63
24/22
ND
d
9
cyclopropylmethyl
3-cyclopropylpropyl
4-cyclopropylbutyl
3-hydroxy-3-methylbutyl
2-ethoxyethyl
ND
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
10
100
ND
ND
28
32/30
13/17
ND
10
42
10000
1250
25
ND
ND
49
ND
pentyl
22/22
20/18
ND
hexyl
21
24
14
heptyl
18
1100
3
ND
65
4-chlorophenyl
7
2/2
phenyl
83
125
41
9
13/10
34/34
16/15
ND
benzyl
6
97
phenethyl
18
18
56
3-phenylpropyl
69
125
660
15
ND
ND
92
2-phenylpropyl
150
19
ND
(R)-2-hydroxy-2-phenylethyl
(S)-2-hydroxy-2-phenylethyl
2-oxo-2-phenylethyl
pyridine-2-yl
22/28
23/29
22/23
ND
25
60
82
42
23
63
1405
352
11
ND
ND
70
ND
ND
92
pyridin-2-ylmethyl
1H-benzo[d]imidazol-2-yl
(5-methylpyrazin-2-yl)methyl
thiazol-2-ylmethyl
oxazol-4-ylmethyl
oxazol-2-ylmethyl
(5-methylfuran-2-yl)methyl
(5-(trifluoromethyl)furan-2-yl)methyl
(1H-pyrazol-3-yl)methyl
ND
25/23
ND
33
2249
325
ND
ND
68
ND
79
32
29/29
ND
272
500
15
ND
ND
37
ND
25/23
18/18
ND
31
124
ND
78
>1000
ND
a
b
IC50 data presented are means of n ≥ 2. Percentage of parent compound remaining analyzed by LC/MS/MS after a 30 min incubation period with
c
rat liver microsomes. Permeability was determined using Caco-2 cells. The data were expressed as Papp(a to b) (× 10−6 cm/s)/Papp(b to a) (×
d
10−6 cm/s). ND = not determined
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Table 3. SAR and Properties of Analogues of Piperazinylpyridazine with Different R2 Groups
a
b
IC50 data presented are means of n ≥ 2. Percentage of parent compound remaining analyzed by LC/MS/MS after a 30 min incubation period with
c
rat liver microsomes. Permeability was determined using Caco-2 cells. The data were expressed as Papp(a to b) (× 10−6 cm/s)/ Papp(b to a) (×
d
10−6 cm/s). ND = not determined.
terminal cyclopropyl group and the amide was important for
activity against SCD1. Cyclopropylmethyl compound 9 was
250−800-fold less potent than its counterparts, with a longer
linker (compounds 8, 10, and 11) in the mouse liver
microsomal assay. A similar trend was observed with alkyl
substituents (compounds 14, 15, and 16). Incorporation of a
hydroxyl group into the alkyl chain (compounds 12)
dramatically decreased the activity against SCD1 in mouse
liver microsomal assay (IC50 ∼ 10 μM). The compound
containing an oxygen heteroatom in the chain (compound 13)
was not well tolerated, and the activity was decreased by about
50-fold. Replacing the isoamyl group with a phenyl-containing
group afforded good potency against SCD1 in both assays.
However, the potency was dependent upon the chain length
between the phenyl group and the amide. Compounds 18−21
were all quite potent against SCD1 in both assays, but
compounds with a benzyl (19) and phenethyl (20) group were
more potent than those compounds with a phenyl (18) and 3-
phenylpropyl (21) group. The optimal chain length between
the phenyl group and the amide was found to be 2 or 3 carbon
atoms. The chlorophenyl compound (17) also displayed good
potency in both assays, however, its cell permeability was poor.
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Methyl substitution on the alkyl linker resulted in a decrease in
activity as demonstrated by compound 22. Incorporation of a
hydroxyl group (compound 23 (R enantiomer) and compound
24 (S enantiomer)) or a carbonyl group (compound 25) into
the linker was tolerated. Introduction of a pyridine-2-yl led to
decreased activity (compounds 26 and 27). Similar results were
obtained for pyridine-3-yl and pyridine-4-yl compounds (data
not shown). The 5-methylpyrazin-2-ylmethyl compound 29
retained activity in the mouse liver microsomal assay, however,
the cell-based HepG2 activity was decreased by more than 60-
fold. In contrast, the benzimidazol-2-yl compound 28 retained
good potency in both assays. In a series of five-membered
heterocycles represented by compounds 30−35, furan and
thiazole compounds retained good activity and oxazole
compounds had modest activity, while pyrazole compound
were much less active. Compounds that demonstrated potency
in cell-based assay were also evaluated for their selectivity
against delta-5 and delta-6 desaturases, and none of the
compounds tested in these assays demonstrated significant
inhibition of these two desaturases at 10 μM (data not shown).
To further investigate the SAR of the right-hand side of the
molecule (R2), the 2-cyclopropylethyl group was kept
unchanged and R2 was varied (Table 3). In contrast to the
left-hand side of the molecule, the tolerance for structural
modifications was much more stringent for R2. A dramatic
decrease in activity against SCD1 in the mouse liver
microsomal assay was observed with alkyl compounds 36−
37, cycloalkyl compounds 38−39, and heteroaryl compounds
40−41, however, the 5-methyl-2-(trifluoromethyl)furan-3-yl
analogue 42 retained good activity against SCD1 in both
assays. Because replacing the 2-trifluoromethylphenyl group
presented in compound 8 with the alkyl, cycloalkyl, and
heteroaryl groups did not provide any advantages, we therefore
returned to the original 2-trifluoromethylphenyl group to
explore the substituent effects. When the 2-trifluoromethyl
group was replaced by other electron-withdrawing groups, such
as −CN (43), −COOH (44), −F (45), or electron-donating
groups such as −N(CH3)2 (46), −OCH3 (50), and −CH3
(52), the activity against SCD1 was decreased by 28- to 400-
fold in the mouse liver microsomal assay. We explored the
effect of disubstitution with a series of compounds, 47−57. Our
findings, in general, support the notion that the presence of an
electron-withdrawing group in addition to the trifluoromethyl
group enhances the SCD1 inhibitory activity. For example,
compound 49, containing an F group para to the
trifluoromethyl group, demonstrated an enhanced activity by
∼2−3-fold in both assays. Incorporation of an electron-
donating group, such as the −N(CH3)2 group para to the 2-
Cl group (47), resulted in decreased activity. The correspond-
ing electron-withdrawing para-F compound (48) was 160-fold
more potent than 47. From this work, the 2-trifluoromethyl-
phenyl group was identified as the optimum group to achieve
good potency, while an extra electron-withdrawing group on
the phenyl ring further enhanced the activity. Compounds that
demonstrated potency in cell-based assay were also tested for
their selectivity against delta-5 and delta-6 desaturases, and the
inhibition of these two desaturases was not significant at 10 μM
(data not shown).
The SCD1 activities of the liver microsomes obtained from
these rats were determined, and the results are illustrated in
Figure 2. Many of our SCD1 inhibitors showed robust effects in
Figure 2. Effect of SCD1 inhibitors on rat liver SCD1 activity in rat
PD model. Lewis rats were fed with high carbohydrate diet for one
week before the study. The rats were fasted for 4 h prior to dosing.
The rats (n = 4) were dosed orally with compound at 1 mg/kg dose in
Tween 20 0.2%/CMC (1%) 99.8% formulation and sacrificed 4 h post
dosing. The livers were harvested and homogenized. The activities of
these liver microsomes were determined by the method as described in
the Experimental Section. Compound 7 was dosed ip at 15 mg/kg for
two days. Each bar represents the mean of at least four rats and the
standard deviation from the mean.
this acute PD model as early as 4 h postdose. These results
mark the first report on such an acute and robust effect of
SCD1 inhibitors in vivo without using a radioactive label and
thus demonstrate the utility of this rodent model for efficient in
vivo compound screening. In comparison to the early
compound 7, many compounds had dramatically improved
PD effects. Compounds 8, 10, 24, 25, 49, 51, and 57 all
decreased liver SCD1 activity by more than 60%. Although
most compounds showed in vivo PD effects, the in vitro to in
vivo activity correlation was poor. For example, compounds 28,
33, and 42 all had less than 100 nM potency in both in vitro
assays, but their in vivo activities were <30% inhibition of rat
liver SCD1 activity. Compound 17, which was the most potent
compounds in vitro (<10 nM), had only moderate effects in the
Lewis rat PD model (Figure 2). Possible explanations for the
activity disconnect include differences in metabolic stability,
permeability, PK profiles, and bioavailability.
To better understand what drives the in vivo PD response,
pharmacokinetic (PK) profiles were determined for several
potent SCD1 inhibitors including 8, 25, 28, 49, and 51 (Table
4). All compounds shown below had similar metabolic stability
in rat liver microsomes, however, their in vivo clearance rates
were quite different. Compound 25 had very low clearance, a
small volume of distribution, high plasma exposure, and good
bioavailability. It demonstrated a profound PD effect in the rat
PD model (Figure 2). Compound 28, on the other hand,
demonstrated high clearance, very low plasma exposure, and
reduced oral bioavailability, and a marginal PD effect was
observed in vivo (Figure 2). Compounds 8, 49, and 51, which
share structural similarity, possessed similar PK profiles and oral
bioavailability and they all demonstrated a similarly profound
PD effect in vivo (Figure 2). On the basis of the in vitro and in
vivo activity, as well as overall drug-like properties, compound
49 was chosen for further in vivo testing to evaluate its effects
on metabolic disease.
Compounds with IC50 <100 nM in cell-based assay were also
evaluated in an acute rat pharmacodynamic (PD) model to
determine the effect of these SCD1 inhibitors on the liver
SCD1 activity. In these studies, Lewis rats were dosed with 1
mg/kg compound orally and then sacrificed 4 h after dosing.
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To assess the impact of SCD1 inhibition by 49 on energy
balance and weight maintenance, an in vivo study utilizing
Zucker Fatty rats was conducted examining metabolic end
points. The Zucker Fatty rats are obese, hyperphagic,
hyperinsulinemia, and hypertriglyceridemia and show an age-
dependent decreased energy expenditure.35,36 In this study, 49
was administered orally to the rats twice daily for 15 weeks. The
effect of 49 on the following metabolic parameters was
evaluated at the end of the study: body weight (at the end of
12 weeks), body fat mass using dual-energy X-ray absorptiom-
etry (DEXA), metabolic rate, and resting oxygen consumption
by indirect calorimetry.
Oral administration twice daily with 1 mg/kg 49 to 20-week-
old Zucker Fatty rats resulted in a significant 36% reduction in
the total body weight gained over a 12-week period when
compared to the vehicle treated controls. The reduction in
body weight gain was observed within the first week of the
treatment and was maintained for the 12 weeks of measure-
ment (Figure 4).
Table 4. Pharmacokinetic Parameters for SCD1 Inhibitors in
Lewis Rats (3 mg/kg IV and 10 mg/kg PO Doses Unless
Otherwise Stated)
a
a
IV
PO
b
c
d
e
f
g
h
i
compd t1/2
AUC
Vss
Cl
Cmax
Tmax
AUC
F
j
8
1.0
220
2.2
0.7
1.8
2.7
1.6
1.9
0.9
1.9
1.0
3.3
0.5
3.3
2.3
167
46
42
19
49
30
25
28
49
51
1.8
1.1
1.6
1.8
1238
0.28
1.3
1756
k
l
1.6
0.07
0.9
0.6
485
459
96.6
1.3
453
0.86
0.5
a
Average of at least two rats for IV and three rats for PO per
b
c
compound. Intravenous half-life (h). Area under the curve or total
drug exposure after intravenous dosing from t = 0 to t = ∞ (μM·h).
d
e
Intravenous volume of distribution at steady state (L/kg). Clearance
f
after intravenous dosing ((L/h)/kg). Maximum plasma concentration
after oral dosing (μM). Time from oral dosing to maximum plasma
concentration (h). Area under the curve or total drug exposure after
oral dosing from t = 0 to t = ∞ (μM·h). Oral bioavailability (%). PO
dose 5 mg/kg. Dose: IV 1 mg/kg and PO 2 mg/kg. IV dose 1 mg/
kg.
g
h
i
j
k
l
At the end of 15-week study, the rats were sacrificed and the
epididymal, retroperitoneal, and mesenteric fat pads were
collected and measured to provide the combined visceral fat
weights. The 49 treatment group demonstrated a 24%
reduction in combined visceral fat weight when compared to
the vehicle control group (Figure 5A; p < 0.001). All three
visceral fat pads measured were reduced in the 49 treatment
group. In addition, dual energy X-ray absorptiometry (DEXA)
measurements of bone mineral content (BMC), lean mass
(LM), and fat mass (FM) in the thoracovisceral area of the
carcasses were carried out. The 49 treatment group showed a
9% reduction in body fat percentage when compared to the
vehicle control group (Figure 5A; p = 0.002). The fat mass was
reduced by 20% in the 49 treatment group (Figure 5B; p <
0.0001). In contrast, lean mass and bone mineral content were
not reduced (Figure 5B).
Compound 49 was evaluated in the Lewis rat PD model in a
dose responsive manner at doses ranging from 0.1 to 1 mg/kg
(4 h post oral dosing sampling). The results illustrated in
Figure 3 indicate a clear dose related reduction of liver SCD1
activity with an ED50 estimated as 0.8 mg/kg.
To confirm that the effects observed with 49 were due to
SCD inhibition, plasma PD (as measured by desaturation
index) was evaluated. The 49 treatment group demonstrated a
significant reduction of C16 plasma DI by 73% and C18 plasma
DI by 61%, respectively, when compared to vehicle control
(Figure 6; p < 0.001).
Figure 3. Dose response of 49 on inhibition of liver SCD1 activity 4 h
after PO dosing in Lewis rats. Each bar represents the standard
deviation from the mean of at least four rats.
Figure 4. Total body weight change during 12 weeks of treatment with 49. Rats were weighed every day, and the change in weight from day zero was
calculated for each rat. Each bar represents the mean of 24 rats and the standard error from the mean. 49 was administered orally twice daily at a
dose of 1 mg/kg.
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Figure 5. Effect of 49 on combined visceral fat weight and body fat mass (A) and on fat mass and lean mass (B) after 15 weeks oral administration of
49 at 1 mg/kg twice daily. The percentage of fat mass over the sum of BMC, LM, and FM was calculated to provide the fat %. Each bar represents
the mean of 12 rats and the standard error from the mean.
Figure 6. Plasma DI changes after a 15-week study of Zucker Fatty rats with 49. Each bar represents the mean of 12 rats and the standard error from
the mean. 49 was administered twice daily at a dose of 1 mg/kg.
To evaluate the potential mechanism-based side effect of a
systemic SCD1 inhibitor, we also developed a clinical
observation model. In this model, rats were administered orally
once daily at a dose 20-fold of the efficacious dose determined
by the rat PD model for 11 days. Rats were examined daily for
general health and the specific observations on eyes and skin.
Compounds with high systemic exposure manifested mecha-
nism-based toxicity as early as day 3 of dosing as red eye and
dry skin symptoms. This model provided us an efficient means
to assess the mechanism-based toxicity of SCD1 inhibition.
The in vivo study with Zucker Fatty rats with orally
administration of 49 clearly demonstrated a significant
reduction of total body weight gain, with a concordant decrease
in visceral fat pad weight, without decreasing lean body mass. It
clearly demonstrate that small-molecule inhibitors with
favorable pharmacology are capable of recapitulating the
improved metabolic parameters observed in the genetically
SCD1 deficient animal. However, the systemic SCD1 inhibitors
can also recapitulate other features observed in the SCD1
knockout mouse such as dry skin and dry eye side effects.
Models were developed to assess the relative effects of SCD1
inhibitors on liver SCD1 (one of the sites of action associated
with beneficial metabolic changes) and the eye and skin
(adverse mechanism-based effects). The use of these models
allows effective screening of compounds that have maximum
effect in the liver yet do not appear to cause changes to eye and
skin. Examination of 49 by topical administration on sebaceous
glands as potential acne therapy will be reported in due course.
CONCLUSIONS
■
We identified a series of piperazinylpyridazine-based highly
potent, selective, and orally bioavailable SCD1 inhibitors. The
SAR analysis demonstrated that the 2-trifluoromethylphenyl
group was the optimal right-hand side group to achieve strong
inhibitory activity against SCD1 activities in both mouse liver
microsomal assay and cell-based HepG2 assay. In vivo studies
demonstrated that a diverse set of piperazinylpyridazine
compounds had an acute impact on the liver SCD1 activity
within 4 h after a single oral dose. This investigation led us to
identify a highly potent, selective, and efficacious SCD1
inhibitor: N-(2-Cyclopropylethyl)-6-(4-(5-fluoro-2-
(trifluoromethyl)benzoyl)piperazin-1-yl)-pyridazine-3-carboxa-
mide (49). Compound 49 demonstrated a very strong potency
in liver SCD1 inhibition (ED50 = 0.8 mg/kg). This highly
efficacious inhibition is likely due to a combination of strong
enzymatic inhibitory activity (IC50 = 14 nM) and good oral
bioavailability (F = 49%).
EXPERIMENTAL SECTION
■
General. All reagents were obtained from commercial sources and
were either used as supplied or purified using reported methods.
Melting points were determined on a Buchi hot-stage apparatus and
̈
1
are uncorrected. H NMR spectra were recorded on a Bruker Avance
300 spectrometer with chemical shifts (δ) reported in parts per million
1
(ppm) relative to the residual signal of the deuterated solvent. H
NMR data are tabulated in the following order: multiplicity (s, singlet;
d, doublet; t, triplet; m, multiplet; br, broad), coupling constants in
hertz, and number of protons. Mass spectra were obtained using a
Waters 2795/ZQ LC/MS system (Waters Corporation, Milford, MA).
HPLC analyses were performed at 25 °C on Agilent 1100 or 1200
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systems (Agilent Technologies, Santa Clara, CA) using an EMD
Chromolith SpeedROD RP-18e column (4.6 mm i.d. × 50 mm
length) (Merck KGaA, Darmstadt, Germany). The mobile phase
consisted of a gradient of component “A” (0.1% v/v aqueous
trifluoroacetic acid) and component “B” (acetonitrile) at a flow rate of
1 mL/min. The gradient program used was as follows: initial
conditions 5% B, hold at 5% B for 1 min, linear ramp from 5% to
95% B over 5 min, 100% B for 3 min, return to initial conditions for 1
min. Peaks were detected at a wavelength of 254 nm with an Agilent
photodiode array detector. All compounds reported herein exhibited
spectral data consistent with their proposed structures and had HPLC
purities in excess of 96% a/a. Chemical names were generated using
ChemBioDraw version 12 by CambridgeSoft.
Ethyl 2-(4-(2-(Trifluoromethyl)benzoyl)piperazin-1-yl)-
pyrimidine-5-carboxylate (3a). A mixture of ethyl 2-(methylsul-
fonyl)-pyrimidine-5-carboxylate (33 mg, 0.14 mmol), tert-butyl
piperazine-1-carboxylate (21 mg, 0.15 mmol), and DIPEA (26 μL,
0.15 mmol) in 1,4-dioxane 4 mL) was refluxed for 13 h and then
concentrated in vacuo. The residue was washed with water (3 × 10
mL) and dried in vacuo and dissolved in 15% TFA in DCM (2 mL).
The mixture was stirred at room temperature overnight. The volatiles
were removed in vacuo, and the residue was dissolved in DCM (4
mL), followed by the addition of DIPEA (122 μL, 0.7 mmol) and 2-
(trifluoromethyl)benzoyl chloride (22 μL, 0.15 mmol) in DCM (3
mL) at 0 °C. The resulting solution was stirred at room temperature
for 6 h, and EtOAc (40 mL) was added. The organic layer was
separated and washed with brine, dried over anhydrous Na2SO4, and
concentrated. The residue was purified by column chromatography to
afford 3a as a colorless gum (45 mg, 78% yield). MS (ES+) m/z 409.3
(M + H)+.
carboxylate (172 mg, 1.0 mmol), piperazine-1-yl-(2-trifluoromethyl-
phenyl)-methanone 4a (387 mg, 1.5 mmol), DBU (0.6 mL, 4.0
mmol), and Bu4NI (18.8 mg, 0.05 mmol) in DMF (3 mL) was heated
at 80 °C for 70 h and then diluted with EtOAc (100 mL). The
resulting mixture was washed with brine (4 × 100 mL), dried over
anhydrous MgSO4, and concentrated. The residue was dissolved in 4
mL of MeOH/water mixture (3:1), and LiOH (48 mg, 2.0 mmol) was
added. The reaction mixture was stirred at room temperature
overnight and then concentrated. The residue was diluted with
water (20 mL), and the pH of the resulting aqueous solution was
adjusted to ∼5 with 5% HCl solution. This solution was then extracted
with DCM (3 × 50 mL). The combined organic layer was washed with
brine, dried over anhydrous MgSO4, and concentrated. The residue
was purified by column chromatography to afford 4b as a white solid
(263 mg, 69% yield). MS (ES+) m/z 381.2 (M + H)+.
N-Isopentyl-5-(4-(2-(trifluoromethyl)benzoyl)piperazin-1-
yl)pyrazine-2-carboxamide (4). 5-(4-(2-(Trifluoromethyl)-
benzoyl)piperazin-1-yl)pyrazine-2-carboxylic acid 4b (38 mg, 0.10
mmol) was dissolved in DCM (3 mL), followed by the addition of
HOBt monohydrate (23 mg, 0.15 mmol), EDCI (28.8 mg, 0.19
mmol), DIPEA (0.5 mL, 2.9 mmol), and isoamylamine (17.5 μL, 0.15
mmol). The mixture was stirred at room temperature for 18 h and
diluted with EtOAc (20 mL). The organic layer was washed with 5%
HCl solution, brine, dried over anhydrous Na2SO4, and concentrated.
The residue was purified by preparative TLC to afford 4 as white solid
(3.5 mg, 8% yield). 1H NMR (300 MHz, CDCl3) δ 8.84 (s, 1H), 7.95
(s, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.63−7.55 (m, 2H), 7.46−7.42 (m,
1H), 7.35 (d, J = 7.2 Hz, 1H), 4.02−3.99 (m, 1H), 3.90−3.68 (m,
3H), 3.67−3.63 (m, 2H), 3.49−3.41 (m, 2H), 3.34−3.30 (m, 2H),
1.71−1.47 (m, 3H), 0.95 (d, J = 6.6 Hz, 6H). MS (ES+) m/z 450.1 (M
+ H)+.
5-Bromopyrimidine-2-carbonitrile (5a). To a vial containing
sodium cyanide (0.27 g, 5.43 mmol) and DABCO (0.087 g, 0.80
mmol) in 3 mL of water was added DMSO (3 mL) under nitrogen
atmosphere, followed by the addition of 5-bromo-2-chloropyrimidine
(1.00 g, 5.17 mmol) in DMSO (3 mL). The reaction mixture gradually
turned brown over 2 h. The reaction mixture was diluted with ethyl
acetate (75 mL), washed with water, 1 N HCl, saturated NaHCO3
solution, and brine, dried over MgSO4, filtered, and concentrated in
vacuo to yield the product as a yellow solid (0.86 g, 89% yield). MS
(ES+) m/z 183.9 (M + H)+.
5-(4-(2-(Trifluoromethyl)benzoyl)piperazin-1-yl)pyrimidine-
2-carbonitrile (5b). A mixture of 5-bromopyrimidine-2-carbonitrile
(0.86 g, 4.6 mmol) obtained above, CsCO3 (6.00 g, 18.0 mmol) and
piperazin-1-yl-(2-trifluoromethylphenyl)methanone 4a (1.35 g, 4.6
mmol) in 20 mL of anhydrous NMP was heated to 80 °C overnight.
After removal of the solvent, the residue was dissolved in ethyl acetate
(150 mL), and washed with 1 N HCl solution, water, saturated
NaHCO3 solution, and brine and then dried over anhydrous MgSO4.
The residue obtained after removal of the solvent was purified by
column chromatography. The product 5b was obtained as a white
solid (0.63 g, 38% yield). MS (ES+) m/z 362.2 (M + H)+.
N-Isopentyl-2-(4-(2-(trifluoromethyl)benzoyl)piperazin-1-
yl)pyrimidine-5-carboxamide (3). A solution of ethyl 2-(4-(2-
(trifluoromethyl)benzoyl)piperazin-1-yl)pyrimidine-5-carboxylate 3a
(45 mg, 0.11 mmol) in a mixture of THF/MeOH/water (3:1:1, 5
mL) was stirred at room temperature for 24 h and concentrated. The
residue was diluted with aqueous HCl solution and extracted with
DCM (5 × 20 mL). The combined organic layer was dried over
anhydrous Na2SO4 and concentrated. The residue was dissolved in
DCM (4 mL), followed by the addition of HOBt monohydrate (17
mg, 0.11 mmol), EDCI (21 mg, 0.11 mmol), DIPEA (38 μL, 0.22
mmol), and isoamylamine (13 μL, 0.11 mmol). The mixture was
stirred at room temperature for 15 h and diluted with DCM (50 mL).
The organic layer was washed with diluted HCl solution, brine, dried
over anhydrous Na2SO4, and concentrated. The residue was purified
by column chromatography to afford 3 as a colorless gum (46 mg, 94%
1
yield). H NMR (300 MHz, CDCl3) δ 8.68 (s, 2H), 7.74 (m, 1H),
7.71−7.54 (m, 2H), 7.35−7.32 (m, 1H), 6.13−6.10 (m, 1H), 3.97−
3.80 (m, 6H), 3.46−3.39 (m, 2H), 3.25−3.23 (m, 2H), 1.65−1.46 (m,
1H), 1.45−1.43 (m, 2H), 0.93 (d, J = 6.3 Hz, 6H). MS (ES+) m/z
450.0 (M + H)+.
Piperazine-1-yl-(2-trifluoromethylphenyl)methanone (4a).
To a stirred solution of tert-butyl piperazine-1-carboxylate (6.00 g,
32.2 mmol) and DIPEA (16.8 mL, 96.7 mmol) in DCM (350 mL) at 0
°C was added a solution of 2-trifluoromethylbenzoyl chloride (5.3 mL,
36.1 mmol). The resulting mixture was stirred at room temperature for
16 h, and water (80 mL) was added. The organic layer was washed
with water (2 × 150 mL), dried over Na2SO4, filtered, and
concentrated in vacuo to provide a yellow oil (11.50 g, 32.1 mmol).
This yellow oil was dissolved in 200 mL of 1:4 mixture of
trifluoroacetic acid and DCM, and the mixture was stirred at room
temperature for 4 h. After removal of the solvents, the residue was
dissolved in DCM (500 mL) and washed with 1 N NaOH (100 mL),
water (80 mL), and brine (80 mL). The organic layer was dried over
anhydrous Na2SO4, filtered, and concentrated to afford the product 4a
N-Hexyl-5-(4-(2-(trifluoromethyl)benzoyl)piperazin-1-yl)-
pyrimidine-2-carboxamide (5).
A mixture of 5-[4-(2-
trifluoromethylbenzoyl)piperazin-1-yl]-pyrimidine-2-carbonitrile 5b
(0.050 g, 0.14 mmol), hexylamine (0.015 mg, 0.15 mmol), water (5
μL, 0.28 mmol), and dichloro-tris(triphenylphosphine)ruthenium(II)
(4.0 mg, 0.004 mmol) in 1,2-dimethoxyethane (0.2 mL) in a sealed
tube was heated to 160 °C for three days. The reaction mixture was
diluted with dichloromethane and then loaded onto a preparative thin
layer chromatography plate developed using ethyl acetate/hexanes =
4:1 to afford the crude compound. The pure compound was obtained
as a white solid after recrystallization from isopropyl alcohol (8.9 mg,
1
14% yield). H NMR (300 MHz, CDCl3) δ 8.39 (s, 2H), 7.81−7.72
1
(m, 2H), 7.69−7.52 (m, 2H), 7.39−7.36 (m, 1H), 4.16−3.88 (m, 2H),
3.71−3.21 (m, 8H), 1.65−1.58 (m, 2H), 1.42−1.22 (m, 6H), 0.93 (t, J
= 6.9 Hz, 3H). MS (ES+) m/z 464 (M + H)+.
as an oil in 95% yield (7.93 g, 95% yield). H NMR (300 MHz,
CDCl3) δ 7.73−7.51 (m, 3H), 7.34−7.32 (m, 1H), 4.83 (br s, 1H),
4.15−4.07 (m, 1H), 3.91−3.78 (m, 1H), 3.48−2.91 (m, 6H). MS (ES
+) m/z 259.1 (M + H)+.
5-(4-(2-(Trifluoromethyl)benzoyl)piperazin-1-yl)pyrazine-2-
carboxylic Acid (4b). A mixture of methyl 5-chloropyrazine-2-
5-Hydroxy-N-isopentylpicolinamide (6a). To a solution of 5-
hydroxypicolinic acid (103 mg, 0.74 mmol) in DCM (6 mL) was
added DIPEA (0.13 mL, 1.48 mmol), HOBt monohydrate (100 mg,
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0.74 mmol), and EDCI (142 mg, 0.74 mmol). The resulting mixture
was stirred at room temperature for 15 min, followed by the addition
of isoamylamine (68 μL, 0.74 mmol). The reaction mixture was stirred
at room temperature for 23 h, and DCM (50 mL) was added. The
organic layer was washed with diluted HCl solution, brine, dried over
anhydrous Na2SO4, and concentrated. The residue was purified by
column chromatography to afford 6a as a colorless gum (0.86 g, 56%
yield). MS (ES+) m/z 209.1 (M + H)+.
6-(Isopentylcarbamoyl)pyridin-3-yl trifluoromethanesulfo-
nate (6b). To a solution of 5-hydroxy-N-isopentylpicolinamide 6a
(82 mg, 0.39 mmol) in pyridine (2 mL) was added Tf2O (74 μL, 0.4
mmol) at 0 °C. The resulting mixture was stirred at 0 °C for 10 min
and then at room temperature for 25 h, follwed by the addition of
water (50 mL). The mixture was extracted with ether (3 × 40 mL).
The combined organic layer was washed with water, brine, dried over
anhydrous MgSO4, and concentrated. The residue was purified by
column chromatography to afford 6b as colorless oil (50 mg, 38%
yield). MS (ES+) m/z 341.2 (M + H)+.
tert-Butyl 4-(6-(Isopentylcarbamoyl)pyridin-3-yl)piperazine-
1-carboxylate (6c). A 50 mL flask was charged with Cs2CO3 (67 mg,
0.21 mmol), Pd(OAc)2 (1.7 mg, 0.0074 mmol), and BINAP (6.9 mg,
0.011 mmol) and flashed with argon for 30 min. A solution of 6-
(isopentylcarbamoyl)pyridin-3-yl trifluoromethanesulfonate 6b (50
mg, 0.15 mmol) and tert-butyl piperazine-1-carboxylate (33 mg, 0.18
mmol) in toluene (2 mL) was added via a syringe. The reaction
mixture was heated at 98 °C for 19 h and then cooled to room
temperature. Toluene (20 mL) was added, and the mixture was
filtered. The filtrate was concentrated in vacuo, and the residue was
purified by column chromatography to provide 6c as a white solid (9
mg, 14% yield).
was dissolved in dichloromethane (2 mL). This solution was added
dropwise at room temperature to a solution of 2-aminopyridine (38
mg, 0.40 mmol) and triethylamine (0.1 mL) in dichloromethane (2
mL). The reaction mixture was stirred at room temperature for 4 h
and diluted with EtOAc (100 mL) and washed with water (3 × 10
mL) and brine (2 × 10 mL). The organic layer was dried over Na2SO4
and evaporated to provide a crude product, which was purified by
column chromatography (hexane/EtOAc = 1:1 to pure EtOAc) to
1
afford 26 as a white solid (44 mg, 37% yield). H NMR (300 MHz,
CDCl3) δ 10.30 (s, 1H), 8.35 (m, 2H), 8.09 (d, J = 9.3 Hz, 1H), 7.75−
6.69 (m, 2H), 7.65−7.52 (m, 2H), 7.35 (d, J = 7.2 Hz, 1H), 7.09−6.96
(m, 2H), 4.10−4.02 (m, 1H), 3.92−3.71 (m, 5H), 3.37−3.34 (m, 2H).
MS (ES+) m/z 457.3 (M + H)+.
The general procedure described for 26 was followed for the
preparation of compounds 7 and 9−35, using the appropriate amines
for amide formation.
N-Isopentyl-6-(4-(2-(trifluoromethyl)benzoyl)piperazin-1-
yl)pyridazine-3-carboxamide (7). White solid (73% yield). 1H
NMR (500 MHz, CDCl3) δ 8.05 (d, J = 9.6 Hz, 1H), 7.98 (t, J = 6.4
Hz, 1H), 7.77−7.73 (m, 1H), 7.66−7.62 (m, 1H), 7.59−7.55 (m, 1H),
7.39−7.35 (m, 1H), 7.00 (d, J = 9.6 Hz, 1H), 4.08−4.04 (m, 1H),
3.90−3.87 (m, 2H), 3.82−3.64 (m, 3H), 3.51−3.47 (m, 2H), 3.38−
3.34 (m, 2H), 1.72−1.68 (m, 1H), 1.52−1.49 (m, 2H), 0.95 (d, J = 7.1
Hz, 6H). MS (ES+) m/z 450.3 (M + H)+.
N-(Cyclopropylmethyl)-6-(4-(2-(trifluoromethyl)benzoyl)-
piperazin-1-yl)pyridazine-3-carboxamide (9). White solid (31%
yield). 1H NMR (500 MHz, CDCl3) δ 8.16−7.88 (m, 2H), 7.75 (d, J =
7.8 Hz, 1H), 7.68−7.46 (m, 2H), 7.18 (d, J = 7.8 Hz, 1H), 7.00 (d, J =
9.3 Hz, 1H), 4.17−3.64 (m, 6H), 3.21−3.12 (m, 4H), 1.07−1.00 (m,
1H), 0.61−0.44 (m, 2H), 0.26−0.20 (m, 2H). MS (ES+) m/z 434.4
(M + H)+.
N-Isopentyl-5-(4-(2-(trifluoromethyl)benzoyl)piperazin-1-
yl)picolinamide (6). This compound was synthesized analogously to
compound 3 and was isolated as a white solid (10.4 mg, 97% yield).
1H NMR (300 MHz, CDCl3) δ 8.15−8.14 (m, 1H), 8.06 (s, 1H),
N-(3-Cyclopropylpropyl)-6-(4-(2-(trifluoromethyl)benzoyl)-
piperazin-1-yl)pyridazine-3-carboxamide (10). White solid (28%
1
yield). H NMR (400 MHz, CDCl3) δ 8.04 (d, J = 9.3 Hz, 1H), 7.89
7.76−7.72 (m, 1H), 7.63−7.58 (m, 2H), 7.37−7.34 (m, 1H), 7.24−
7.19 (m, 2H), 4.10−3.95 (m, 4H), 3.49−3.36 (m, 4H), 3.23−3.19 (m,
2H), 1.73−1.66 (m, 2H), 1.55−1.53 (m, 1H), 0.96 (d, J = 6.5 Hz,
6H). MS (ES+) m/z 449.0 (M + H)+.
(t, J = 5.8 Hz, 1H), 7.73 (t, J = 7.4 Hz, 1H), 7.65 (t, J = 7.3 Hz, 1H),
7.58 (t, J = 7.6 Hz, 1H), 7.38 (d, J = 7.2 Hz, 1H), 6.99 (d, J = 9.3 Hz,
1H), 4.08−3.67 (m, 6H), 3.54−3.46 (m, 2H), 3.39−3.31 (m, 2H),
1.77−1.66 (m, 2H), 1.34−1.23 (m, 2H), 0.72−0.62 (m, 1H), 0.45−
0.36 (m, 2H), 0.06−0.04 (m, 2H). MS (ES+) m/z 462.2 (M + H)+.
N-(4-Cyclopropylbutyl)-6-(4-(2-(trifluoromethyl)benzoyl)-
piperazin-1-yl)pyridazine-3-carboxamide (11). White solid (21%
yield). 1H NMR (300 MHz, CDCl3) δ 8.03 (d, J = 9.4 Hz, 1H), 7.88−
7.84 (m, 1H), 7.72 (d, J = 7.5 Hz, 1H), 7.60−7.56 (m, 2H), 7.34 (d, J
= 7.5 Hz, 1H), 6.98 (d, J = 9.4 Hz, 1H), 4.05−4.02 (m, 1H), 3.89−
3.62 (m, 5H), 3.48−3.31 (m, 4H), 1.64−1.41 (m, 4H), 1.21−1.19 (m,
2H), 0.61−0.59 (m, 1H), 0.39−0.30 (m, 2H), −0.06−0.02 (m, 2H).
MS (ES+) m/z 476.1 (M + H)+.
N-(3-Hydroxy-3-methylbutyl)-6-(4-(2-(trifluoromethyl)-
benzoyl)piperazin-1-yl)pyridazine-3-carboxamide (12). White
solid (46% yield). 1H NMR (500 MHz, CDCl3) δ 8.30 (br s, 1H), 8.05
(d, J = 9.8 Hz, 1H), 7.78−7.74 (m, 1H), 7.68−7.62 (m, 1H), 7.60−
7.55 (m, 1H), 7.40−7.35 (m, 1H), 6.98 (d, J = 9.8 Hz, 1H), 4.07−4.04
(m, 1H), 3.91−3.85 (m, 2H), 3.81−3.69 (m, 3H), 3.67−3.60 (m, 2H),
3.40−3.32 (m, 3H), 1.80 (t, J = 7.5 Hz, 2H), 1.30 (s, 6H). MS (ES+)
m/z 466.0 (M + H)+.
Methyl 6-(4-(2-(Trifluoromethyl)benzoyl)piperazin-1-yl)-
pyridazine-3-carboxylate (7a). To a solution of 6-chloro-
pyridazine-3-carboxylic acid methyl ester (4.36 g, 25.3 mmol) in 1,4-
dioxane (100 mL) was added 1-(2-trifluoromethylbenzoyl)piperazine
hydrochloride (7.80 g, 26.5 mmol), K2CO3 (10.14 g, 73.4 mmol), and
Bu4NI (0.071 g, 0.19 mmol). The reaction mixture was heated to
reflux for 24 h and then concentrated. The residue was washed with
1
water and ether to afford 7a as a white solid (8.67 g, 87% yield). H
NMR (300 MHz, CDCl3) δ 7.92 (d, J = 9.3 Hz, 1H), 7.77 (d, J = 7.5
Hz, 1H), 7.64−7.51 (m, 2H), 7.34 (d, J = 7.5 Hz, 1H), 6.89 (d, J = 9.3
Hz, 1H), 4.11−3.35 (m, 6H), 3.33 (t, J = 5.1 Hz, 2H). MS (ES+) m/z
395.1 (M + H)+.
6-(4-(2-(Trifluoromethyl)benzoyl)piperazin-1-yl)pyridazine-
3-carboxylic Acid (7b). To
a solution of 6-[4-(2-
trifluoromethylbenzoyl)piperazin-1-yl]pyridazine-3-carboxylic acid
methyl ester (8.38 g, 21.2 mmol) in THF (100 mL) and water (50
mL) was added lithium hydroxide monohydrate (1.80 g, 42.9 mmol).
The reaction mixture was stirred at room temperature for 6 h, and the
pH of the mixture was adjusted to ∼5 with 5% hydrochloric acid at 0
°C. THF was removed in vacuo. Ethyl acetate (100 mL) was added to
the residue and a white solid precipitated. The solid was isolated by
filtration and washed with ether and dried to afford 7b as a white solid
N-(2-Ethoxyethyl)-6-(4-(2-(trifluoromethyl)benzoyl)-
piperazin-1-yl)pyridazine-3-carboxamide (13). White solid (25%
1
yield). H NMR (500 MHz, CDCl3) δ 8.18 (t, J = 5.6 Hz, 1H), 8.07
(d, J = 9.6 Hz, 1H), 7.78−7.74 (m, 1H), 7.67−7.63 (m, 1H), 7.61−
7.57 (m. 1H), 7.40−7.36 (m, 1H), 7.00 (d, J = 9.6 Hz, 1H), 4.09−4.03
(m, 1H), 3.93−3.89 (m, 2H), 3.83−3.65 (m, 5H), 3.62−3.58 (m, 2H),
3.54−3.50 (q, 2H), 3.38−3.33 (m, 2H), 1.20 (t, J = 8.1 Hz, 3H). MS
(ES+) m/z 452.0 (M + H)+.
N-Pentyl-6-(4-(2-(trifluoromethyl)benzoyl)piperazin-1-yl)-
pyridazine-3-carboxamide (14). White solid (94% yield); mp
123−125 °C. 1H NMR (300 MHz, CDCl3) δ 8.04 (d, J = 9.6 Hz, 1H),
7.86−7.82 (m, 1H), 7.80−7.70 (m, 1H), 7.68−7.52 (m, 2H), 7.40−
7.32 (m, 1H), 6.99 (d, J = 9.6 Hz, 1H), 4.06−4.00 (m, 1H), 3.91−3.69
(m, 5H), 3.50−3.30 (m, 4H), 1.62−1.55 (m, 2H), 1.37−1.31 (m, 4H),
0.84 (t, J = 6.9 Hz, 3H). MS (ES+) m/z 450.2 (M + H)+.
1
(15.4 g, 98% yield). H NMR (300 MHz, CDCl3) δ 8.02 (d, J = 9.3
Hz, 1H), 7.75−7.72 (m, 1H), 7.66−7.5 (m, 2H), 7.37−7.35 (m, 1H),
7.01 (d, J = 9.3 Hz, 1H), 4.13−4.03 (m, 1H), 3.95−3.64 (m, 5H),
3.38−3.34 (m, 2H). MS (ES+) m/z 381.1 (M + H)+.
N-(Pyridin-2-yl)-6-(4-(2-(trifluoromethyl)benzoyl)piperazin-
1-yl)pyridazine-3-carboxamide (26). To a solution of 6-[4-(2-
trifluoromethylbenzoyl)piperazin-1-yl]pyridazine-3-carboxylic acid
(0.10 g, 0.26 mmol) in chloroform (10 mL) was added thionyl
chloride (0.5 mL) and a drop of DMF. The reaction mixture was
refluxed for 18 h, and the volatiles were removed in vacuo. The residue
577
dx.doi.org/10.1021/jm301661h | J. Med. Chem. 2013, 56, 568−583

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Article
N-Hexyl-6-(4-(2-(trifluoromethyl)benzoyl)piperazin-1-yl)-
pyridazine-3-carboxamide (15). White solid (35% yield). 1H NMR
(300 MHz, CDCl3) δ 8.02 (d, J = 9.6 Hz, 1H), 7.85 (t, J = 5.7 Hz,
1H), 7.74−7.70 (m, 1H), 7.57−7.54 (m, 2H), 7.36−7.32 (m, 1H),
6.97 (d, J = 9.6 Hz, 1H), 4.02−3.96 (m, 1H), 3.90−3.64 (m, 5H),
3.48−3.28 (m, 4H), 1.61−1.56 (m, 2H), 1.31−1.27 (m, 6H), 0.85 (t, J
= 6.9 Hz, 3H). MS (ES+) m/z 464.0 (M + H)+.
N-Heptyl-6-(4-(2-(trifluoromethyl)benzoyl)piperazin-1-yl)-
pyridazine-3-carboxamide (16). White solid (41% yield). 1H NMR
(300 MHz, CDCl3) δ 8.05 (d, J = 9.9 Hz, 1H), 7.85 (t, J = 5.7 Hz,
1H), 7.74−7.70 (m, 1H), 7.60−7.57 (m, 2H), 7.36−7.33 (m, 1H),
7.00−6.96 (m, 1H), 4.05−4.00 (m, 1H), 3.94−3.64 (m, 5H), 3.47−
3.28 (m, 4H), 1.61−1.57 (m, 2H), 1.32−1.25 (m, 8H), 0.84 (t, J = 6.3
Hz, 3H). MS (ES+) m/z 478.2 (M + H)+.
7.2 Hz, 1H), 4.83 (d, J = 5.4 Hz, 2H), 4.17−3.66 (m, 6H), 3.37 (t, J =
5.1 Hz, 2H). MS (ES+) m/z 471.0 (M + H)+.
N-(1H-Benzo[d]imidazol-2-yl)-6-(4-(2-(trifluoromethyl)-
benzoyl)piperazin-1-yl)pyridazine-3-carboxamide (28). White
1
solid (31% yield); mp 231−232 °C. H NMR (300 MHz, CDCl3) δ
8.07 (d, J = 9.6 Hz, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.66−7.49 (m, 4H),
7.35 (d, J = 7.5 Hz, 1H), 7.24−7.19 (m, 2H), 6.97 (d, J = 9.6 Hz, 1H),
4.15−4.03 (m, 1H), 3.91−3.71 (m, 5H), 3.34 (t, J = 4.8 Hz, 2H). MS
(ES+) m/z 496.5 (M + H)+.
N-((5-Methylpyrazin-2-yl) methyl)-6-(4-(2-(trifluoromethyl)
benzoyl) piperazin-1-yl) pyridazine-3-carboxamide (29).
White solid (71% yield); mp 149−151 °C (ethyl acetate/hexanes).
1H NMR (300 MHz, CDCl3) δ 8.67−8.62 (m, 1H), 8.46 (s, 1H), 8.34
(s, 1H), 8.00 (d, J = 9.3 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.61−7.49
(m, 2H), 7.31 (d, J = 7.8 Hz, 1H), 6.95 (d, J = 9.3 Hz, 1H), 4.74 (d, J
= 5.7 Hz, 2H), 4.07−3.62 (m, 6H), 3.30 (t, J = 5.1 Hz, 2H), 2.49 (s,
3H). MS (ES+) m/z 486.4 (M + H)+.
N-(4-Chlorophenyl)-6-(4-(2-(trifluoromethyl)benzoyl)-
piperazin-1-yl)pyridazine-3-carboxamide (17). White solid (67%
1
yield). H NMR (300 MHz, CDCl3) δ 9.79 (s, 1H), 8.08 (d, J = 9.6
N-(Thiazol-2-ylmethyl)-6-(4-(2-(trifluoromethyl)benzoyl)-
piperazin-1-yl)pyridazine-3-carboxamide (30). White solid (62%
yield); mp 153−154 °C (ethyl acetate/hexanes). 1H NMR (300 MHz,
CDCl3) δ 8.55 (t, J = 6.0 Hz, 1H), 8.02 (d, J = 9.3 Hz, 1H), 7.75−7.50
(m, 4H), 7.25−7.23 (m, 1H), 6.94 (d, J = 9.3 Hz, 1H), 4.96 (d, J = 6.0
Hz, 2H), 4.11−3.69 (m, 6H), 3.32 (t, J = 5.1 Hz, 2H). MS (ES+) m/z
477.4 (M + H)+.
N-(Oxazol-4-ylmethyl)-6-(4-(2-(trifluoromethyl)benzoyl)-
piperazin-1-yl)pyridazine-3-carboxamide (31). White solid (20%
yield); mp 76−78 °C (EtOAc/hexanes). 1H NMR (300 MHz, CDCl3)
δ 8.25 (t, J = 5.7 Hz, 1H), 8.01 (d, J = 9.3 Hz, 1H), 7.82 (s, 1H), 7.72
(d, J = 7.8 Hz, 1H), 7.64−7.51 (m, 3H), 7.34 (d, J = 7.8 Hz, 1H), 6.97
(d, J = 9.3 Hz, 1H), 4.58 (d, J = 5.7 Hz, 2H), 4.12−3.64 (m, 6H),
3.34−3.30 (m, 2H). MS (ES+) m/z 461.2 (M + H)+.
N-(Oxazol-2-ylmethyl)-6-(4-(2-(trifluoromethyl)benzoyl)-
piperazin-1-yl)pyridazine-3-carboxamide (32). White solid (22%
yield); mp 94−97 °C. 1H NMR (300 MHz, CDCl3) δ 8.40 (d, J = 5.7
Hz, 1H), 8.01 (d, J = 9.3 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.62−7.44
(m, 3H), 7.33 (d, J = 7.8 Hz, 1H), 7.03−6.89 (m, 2H), 4.77 (d, J = 5.7
Hz, 2H), 4.11−3.66 (m, 6H), 3.37−3.30 (m, 2H). MS (ES+) m/z
461.2 (M + H)+.
N-((5-Methylfuran-2-yl)methyl)-6-(4-(2-(trifluoromethyl)-
benzoyl)piperazin-1-yl)pyridazine-3-carboxamide (33). White
solid (87% yield). 1H NMR (300 MHz, CDCl3) δ 8.02 (t, J = 5.7 Hz,
1H), 7.98 (d, J = 9.6 Hz, 1H), 7.71−7.49 (m, 3H), 7.32 (d, J = 7.2 Hz,
1H), 6.96 (d, J = 9.6 Hz, 1H), 6.16 (d, J = 2.7 Hz, 1H), 5.83 (d, J = 2.7
Hz, 1H), 4.54 (d, J = 2.7 Hz, 1H), 4.03−3.62 (m, 6H), 3.32−3.26 (m,
2H), 2.20 (s, 3H). MS (ES+) m/z 474.4 (M + H)+.
Hz, 1H), 7.82−7.55 (m, 5H), 7.36−7.28 (m, 3H), 7.02 (d, J = 9.6 Hz,
1H), 4.10−4.00 (m, 1H), 3.93−3.68 (m, 5H), 3.35 (t, J = 5.1 Hz, 2H).
MS (ES+) m/z 490.1 (M + H)+.
N-Phenyl-6-(4-(2-(trifluoromethyl)benzoyl)piperazin-1-yl)-
pyridazine-3-carboxamide (18). White solid (99% yield). 1H NMR
(300 MHz, CDCl3) δ 8.18 (t, J = 5.4 Hz, 1H), 8.07 (dd, J = 9.6, 0.9
Hz, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.65−7.50 (m, 2H), 7.37−7.25 (m,
6H), 6.99 (d, J = 9.6 Hz, 1H), 4.08−4.00 (m, 1H), 3.90−3.64 (m,
5H), 3.33 (t, J = 5.4 Hz, 2H). MS (ES+) m/z 456.3 (M + H)+.
N-Benzyl-6-(4-(2-(trifluoromethyl)benzoyl)piperazin-1-yl)-
pyridazine-3-carboxamide (19). White solid (98% yield). MS (ES
+) m/z 470.6 (M + H)+.
N-Phenethyl-6-(4-(2-(trifluoromethyl)benzoyl)piperazin-1-
yl)pyridazine-3-carboxamide (20). White solid (82% yield). 1H
NMR (300 MHz, CDCl3) δ 8.03 (d, J = 9.6 Hz, 1H), 7.94−7.90 (m,
1H), 7.75−7.72 (m, 1H), 7.65−7.57 (m, 2H), 7.36−7.09 (m, 6H),
6.97 (d, J = 9.6 Hz, 1H), 4.11−4.00 (m, 1H), 3.90−3.65 (m, 7H),
3.36−3.30 (m, 2H), 2.91 (t, J = 7.2 Hz, 2H). MS (ES+) m/z 484 (M +
H)+.
N-(3-Phenylpropyl)-6-(4-(2-(trifluoromethyl)benzoyl)-
piperazin-1-yl)pyridazine-3-carboxamide (21). White solid (15%
yield). 1H NMR (500 MHz, CDCl3) δ 8.05 (d, J = 9.3 Hz, 1H,), 7.93
(t, J = 6.3 Hz, 1H), 7.74 (d, J = 7.6 Hz, 1H), 7.63 (t, J = 7.4 Hz, 1H),
7.56 (t, J = 7.4 Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H), 7.29−7.13 (m, 5H),
6.92 (d, J = 9.3 Hz, 1H), 4.12−3.29 (m, 10H), 2.68 (t, J = 5.7 Hz,
2H,), 2.02−1.83 (m, 2H).
N-(2-Phenylpropyl)-6-(4-(2-(trifluoromethyl)benzoyl)-
piperazin-1-yl)pyridazine-3-carboxamide (22). White solid (63%
yield). 1H NMR (500 MHz, CDCl3) δ 7.97 (d, J = 9.5 Hz, 1H), 7.82−
7.78 (m, 1H), 7.70−7.67 (m, 1H), 7.59−7.55 (m, 1H), 7.52−7.49 (m.
1H), 7.32−7.28 (m, 1H), 7.25−7.22 (m, 2H), 7.20−7.12 (m, 3H),
6.92 (d, J = 9.5 Hz, 1H), 3.98 (m, 1H), 3.80 (m, 2H), 3.74−3.60 (m,
4H), 3.53 (m, 1H), 3.28 (br s, 2H), 3.02−2.98 (m, 1H), 1.28 (d, J =
6.7 Hz, 3H). MS (ES+) m/z 474.4 (M + H)+.
(R)-N-(2-Hydroxy-2-phenylethyl)-6-(4-(2-(trifluoromethyl)-
benzoyl)piperazin-1-yl)pyridazine-3-carboxamide (23). White
solid (65% yield). 1H NMR (500 MHz, CDCl3) δ 8.26 (t, J = 9.3 Hz,
1H), 8.01 (d, J = 9.6 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.65−7.53 (m,
2H), 7.46−7.21 (m, 6H), 6.96 (d, J = 9.6 Hz, 1H), 4.94−4.90 (m.
1H), 4.05−3.98 (m, 1H), 3.90−3.52 (m, 7H), 3.30 (t, J = 5.1 Hz, 2H).
MS (ES+) m/z 500.0 (M + H)+.
(S)-N-(2-Hydroxy-2-phenylethyl)-6-(4-(2-(trifluoromethyl)-
benzoyl)piperazin-1-yl)pyridazine-3-carboxamide (24). White
solid (64% yield). 1H NMR (500 MHz, CDCl3) δ 8.27 (t, J = 9.3 Hz,
1H), 8.01 (d, J = 9.6 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.65−7.53 (m,
2H), 7.41−7.23 (m, 6H), 6.98 (d, J = 9.3 Hz, 1H), 4.95−4.91 (m,
1H), 4.13−4.00 (m, 1H), 3.90−3.53 (m, 7H), 3.32 (t, J = 5.1 Hz, 2H).
MS (ES+) m/z 522.0 (M + Na)+.
6-(4-(2-(Trifluoromethyl)benzoyl)piperazin-1-yl)-N-((5-
(trifluoromethyl)furan-2-yl)methyl)-pyridazine-3-carboxamide
(34). White solid (77% yield); mp 151−152 °C (EtOAc/hexanes). 1H
NMR (300 MHz, CDCl3) δ 8.22 (t, J = 6.0 Hz, 1H), 8.01 (d, J = 9.3
Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.64−7.51 (m, 2H), 7.34 (d, J = 7.8
Hz, 1H), 6.98 (d, J = 9.3 Hz, 1H), 6.69−6.68 (m, 1H), 6.34−6.33 (m,
1H), 4.66 (d, J = 6.0 Hz, 2H), 4.14−3.66 (m, 6H), 3.33 (t, J = 5.4 Hz,
2H). MS (ES+) m/z 528.2 (M + H)+.
N-((1H-Pyrazol-3-yl)methyl)-6-(4-(2-(trifluoromethyl)-
benzoyl)piperazin-1-yl)pyridazine-3-carboxamide (35). White
1
solid (37% yield); mp 102−103 °C (EtOAc/hexanes). H NMR (300
MHz, CDCl3) δ 8.43 (t, J = 6.0 Hz, 1H), 8.03 (d, J = 9.6 Hz, 1H), 7.72
(d, J = 7.8 Hz, 1H), 7.61−7.49 (m, 3H), 7.34 (d, J = 7.8 Hz, 1H), 6.97
(d, J = 9.6 Hz, 1H), 6.25 (d, J = 1.8 Hz, 1H), 5.39 (br s, 1H), 4.68 (d, J
= 6.0 Hz, 2H), 4.06−3.65 (m, 6H), 3.39−3.33 (m, 2H). MS (ES+) m/
z 460.2 (M + 1)+.
N-(2-Oxo-2-phenylethyl)-6-(4-(2-(trifluoromethyl)benzoyl)-
piperazin-1-yl)pyridazine-3-carboxamide (25). To a solution of
(R)-N-(2-hydroxy-2-phenylethyl)-6-(4-(2-(trifluoromethyl)-benzoyl)-
piperazin-1-yl)pyridazine-3-carboxamide 23 (0.52 g, 1.03 mmol) in
DCM (10 mL) was added Dess−Martin periodinate (0.53 g) in one
portion under stirring in a cold water bath. After stirring in a cold
water bath for 15 min, the reaction mixture was warmed to room
temperature and stirred for 2 h and then was diluted with diethyl ether
(20 mL). The mixture was poured into a solution of sodium
thiosulfate (1.18 g, 7.44 mmol) in saturated aqueous sodium
N-(Pyridin-2-ylmethyl)-6-(4-(2-(trifluoromethyl)benzoyl)-
piperazin-1-yl)pyridazine-3-carboxamide (27). White solid (48%
1
yield); mp 179−181 °C. H NMR (300 MHz, CDCl3) δ 8.81 (t, J =
5.1 Hz, 1H), 8.59 (d, J = 4.8 Hz, 1H), 8.12 (d, J = 7.2 Hz, 1H), 7.78−
7.50 (m, 4H), 7.37 (t, J = 8.4 Hz, 1H), 7.21−7.13 (m, 1H), 6.93 (d, J =
578
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1
biocarbonate (29 mL). The mixture was extracted with EtOAc (100
mL). The organic layer was washed with saturated aqueous NaHCO3
(2 × 15 mL) and water (2 × 15 mL). The combined aqueous washes
were then extracted with EtOAc (2 × 80 mL). The combined organic
phases were dried over anhydrous Na2SO4 and filtered. The filtrate
was concentrated in vacuo. The residue was purified by column
chromatography eluted with hexane/EtOAc (1:1), hexane/EtOAc
(1:2), and EtOAc (100%). The product was obtained as a white
mide (41). White solid (35% yield). H NMR (300 MHz, CDCl3) δ
8.77 (s, 1H), 8.08 (d, J = 9.6 Hz, 1H), 7.97 (t, J = 6.3 Hz, 1H), 7.01 (d,
J = 9.6 Hz, 1H), 4.06−3.68 (m, 6H), 3.55 (q, J = 6.9 Hz, 2H), 3.39 (t,
J = 5.4 Hz, 2H), 1.50 (q, J = 6.9 Hz, 2H), 0.76−0.71 (m, 1H), 0.48−
0.42 (m, 2H), 0.1−0.05 (m, 2H).
6 - ( 4 - ( 2 - C y a n o b e n z o y l ) p i p e r a z i n - 1 - y l ) - N - ( 2 -
cyclopropylethyl)pyridazine-3-carboxamide (43). White solid
(26% yield). 1H NMR (300 MHz, CDCl3) δ 8.05 (d, J = 9.6 Hz, 1H),
7.97 (t, J = 5.7 Hz, 1H), 7.76−7.72 (m, 1H), 7.69−7.66 (m, 1H),
7.58−7.55 (m, 1H), 7.53−7.43 (m, 1H), 6.99 (d, J = 9.6 Hz, 1H),
4.03−3.94 (m, 2H), 3.88−3.85 (m, 4H), 3.58−3.51 (m, 4H), 1.52−
1.47 (m, 2H), 0.78−0.65 (m, 1H), 0.48−0.37 (m, 2H), 0.16−0.02 (m,
2H). MS (ES+) m/z 405.2 (M + H)+.
1
powder (0.26 g, 51% yield); mp 196−198 °C. H NMR (300 MHz,
CDCl3) δ 8.75−8.70 (m, 1H), 8.06−7.97 (m, 3H), 7.74 (d, J = 7.5 Hz,
1H), 7.66−7.47 (m, 5H), 7.36 (d, J = 7.5 Hz, 1H), 6.99 (d, J = 9.6 Hz,
1H), 4.96 (d, J = 4.8 Hz, 2H), 4.11−4.02 (m, 1H), 3.92−3.70 (m,
5H), 3.42−3.27 (m, 2H). MS (ES+) m/z 498.3 (M + H)+.
N-(2-Cyclopropylethyl)-6-(4-(2-fluorobenzoyl)piperazin-1-
yl)pyridazine-3-carboxamide (45). White solid (20% yield). 1H
NMR (300 MHz, CDCl3) δ 8.04 (d, J = 9.6 Hz, 1H), 7.98 (t, J = 5.7
Hz, 1H), 7.47−7.4 (m, 2H), 7.26−7.21 (m, 1H), 7.15−7.09 (m, 1H),
6.99 (d, J = 9.6 Hz, 1H), 3.95−3.78 (m, 6H), 3.58−3.50 (m, 4H),
1.54−1.47 (m, 2H), 0.78−0.69 (m, 1H), 0.48−0.42 (m, 2H), 0.11−
0.05 (m, 2H).
6-Chloro-N-(2-cyclopropylethyl)pyridazine-3-carboxamide
(8a). To a suspension of 6-chloropyridazine-3-carboxylic acid (3.70 g,
23.0 mmol) in chloroform (30 mL) was added thionyl chloride (5
mL), followed by the addition of a drop of DMF. The resulting
reaction mixture was heated to reflux for 11 h, and the volatiles were
removed in vacuo. The residue was dissolved in dichloromethane (100
mL), and the resulting solution was cooled to 0 °C, followed by the
addition of Et3N (5.5 mL, 39.0 mmol) and 2-cyclopropylethylamine37
(2.50 g, 29.3 mmol). The reaction mixture was stirred at room
temperature for 30 min, diluted with ethyl acetate, washed with water,
saturated NaHCO3, and brine, dried over anhydrous Na2SO4, and
filtered. The filtrate was concentrated, and the residue was
recrystallized from ethyl acetate and hexane to afford 8a as a white
solid in 84% yield (4.40 g). 1H NMR (300 MHz, CDCl3) δ 8.26 (d, J
= 9.0 Hz, 1H), 8.14 (br s, 1H), 3.64−3.53 (m, 2H), 1.62−1.50 (m,
2H), 0.80−0.67 (m, 1H), 0.56−0.51 (m, 2H), 0.18−0.02 (m, 2H).
N-(2-cyclopropylethyl)-6-(piperazin-1-yl)pyridazine-3-car-
boxamide (8b). A solution of 6-chloro-N-(2-cyclopropylethyl)-
pyridazine-3-carboxamide (7.80 g, 34.0 mmol) and piperazine (8.93
g, 103 mmol) in acetonitrile (100 mL) was heated to refluxed for 2 h.
The solvent was removed in vacuo, and the residue was dissolved in
water (100 mL) and then extracted with dichloromethane (5 × 100
mL). The combined extracts were dried over anhydrous Na2SO4 and
filtered. The filtrate was concentrated to provide 8b as a white solid
N-(2-Cyclopropylethyl)-6-(4-(2-(dimethylamino)benzoyl)-
piperazin-1-yl)pyridazine-3-carboxamide (46). White solid (61%
1
yield). H NMR (300 MHz, CDCl3) δ 8.04 (d, J = 9.5 Hz, 1H), 7.96
(t, J = 5.9 Hz, 1H), 7.36−7.25 (m, 2H), 7.05−6.94 (m, 3H), 4.17−
4.05 (m, 1H), 3.98−3.72 (m, 5H), 3.60−3.43 (m, 3H), 3.37−3.24 (m,
1H), 2.80 (s, 6H), 1.53−1.49 (m, 2H), 0.80−0.73 (m, 1H), 0.47−0.42
(m, 2H), 0.12−0.07 (m, 2H).
6-(4-(2-Chloro-5-(dimethylamino)benzoyl)piperazin-1-yl)-N-
(2-cyclopropylethyl)pyridazine-3-carboxamide (47). White solid
(53% yield). 1H NMR (300 MHz, CDCl3) δ 8.04 (d, J = 9.6 Hz, 1H),
7.96 (t, J = 5.8 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 6.94 (d, J = 9.6 Hz,
1H), 6.66 (dd, J = 9.0, 3.0 Hz, 1H), 6.55 (d, J = 3.0 Hz, 1H), 4.14−
4.00 (m, 1H), 3.92−3.71 (m, 5H), 3.58−3.32 (m, 4H), 2.93 (s, 6H),
1.52 (q, J = 6.9 Hz, 2H), 0.75−0.69 (m, 1H), 0.48−0.42 (m, 2H),
0.11−0.05 (m, 2H). MS (ES+) m/z 457.4 (M + H)+.
6-(4-(2-Chloro-5-fluorobenzoyl)piperazin-1-yl)-N-(2-
cyclopropylethyl)pyridazine-3-carboxamide (48). White solid
(94% yield); mp 194−196 °C. 1H NMR (300 MHz, CDCl3) δ 8.05 (d,
J = 9.4 Hz, 1H), 7.96 (t, J = 6.0 Hz, 1H), 7.41−7.37 (m, 1H), 7.11−
7.03 (m, 2H), 7.00 (d, J = 9.4 Hz, 1H), 4.07−3.70 (m, 6H), 3.53−3.34
(m, 4H), 1.55−1.46 (m, 2H), 0.79−0.68 (m, 1H), 0.48−0.42 (m, 2H),
0.11−0.06 (m, 2H). MS (ES+) m/z 432.2 (M + H)+.
1
(8.2 g, 88% yield). H NMR (300 MHz, CDCl3) δ 7.90−7.87 (m,
2H), 6.89 (d, J = 9.2 Hz, 1H), 3.78−3.50 (m, 6H), 3.12−2.90 (m,
4H), 1.77−1.49 (m, 2H), 0.83−0.60 (m, 1H), 0.51−0.36 (m, 2H),
0.15−0.01 (m, 2H).
N-(2-Cyclopropylethyl)-6-(4-(2-(trifluoromethyl)benzoyl)-
piperazin-1-yl)pyridazine-3-carboxamide (8). 2-Trifluoromethyl-
benzoic chloride (0.33 mL, 2.23 mmol) was added to a solution of N-
(2-cyclopropylethyl)-6-(piperazin-1-yl)pyridazine-3-carboxamide (0.55
g, 2.00 mmol) and Et3N (0.4 mL, 2.87 mmol) in dichloromethane (10
mL) at 0 °C. The resulting mixture was stirred for 5 min, and the
cooling bath was removed. The stirring was continued for 1 h at room
temperature. The reaction mixture was diluted with ethyl acetate (100
mL), washed with water, saturated NaHCO3, and brine, dried over
anhydrous Na2SO4, and evaporated. The residue was purified by flash
chromatography eluted with ethyl acetate to afford 8 as a white solid
(0.83 mg, 92% yield); mp 125−127 °C. 1H NMR (300 MHz, CDCl3)
δ 8.05 (d, J = 9.3 Hz, 1H), 7.96 (t, J = 5.7 Hz, 1H), 7.74 (d, J = 7.8 Hz,
1H), 7.65−7.52 (m, 2H), 7.35 (d, J = 7.8 Hz, 1H), 6.99 (d, J = 9.3 Hz,
1H), 4.08−4.00 (m, 1H), 3.97−3.65 (m, 5H), 3.62−3.57 (m, 2H),
3.38−3.22 (m, 2H), 1.55−1.46 (m, 2H), 0.80−0.67 (m, 1H), 0.48−
0.42 (m, 2H), 0.10−0.07 (m, 2H). MS (ES+) m/z 448.2 (M + H)+.
The general procedure described for 8 was followed for the
preparation of the following compounds, using the appropriate acid
chlorides or acid bromide for the coupling.
N-(2-Cyclopropylethyl)-6-(4-(5-fluoro-2-(trifluoromethyl)-
benzoyl)piperazin-1-yl)pyridazine-3-carboxamide (49). White
1
solid (65% yield); mp 177−179 °C. H NMR (300 MHz, CDCl3) δ
8.05 (d, J = 9.6 Hz, 1H), 7.97 (t, J = 5.7 Hz, 1H), 7.77−7.72 (m, 1H),
7.26−7.2 (m, 1H), 7.08−7.05 (m, 1H), 6.99 (d, J = 9.3 Hz, 1H),
4.05−3.99 (m, 1H), 3.89−3.69 (m, 5H), 3.58−3.51 (m, 2H), 3.35 (t, J
= 5.1 Hz, 2H), 1.53−1.46 (m, 2H), 0.76−0.69 (m, 1H), 0.48−0.42 (m,
2H), 0.10−0.05 (m, 2H). MS (ES+) m/z 466.5 (M + H)+.
N-(2-Cyclopropylethyl)-6-(4-(5-fluoro-2-methoxybenzoyl)-
piperazin-1-yl)pyridazine-3-carboxamide (50). White solid (61%
1
yield). H NMR (300 MHz, CDCl3) δ 8.03 (d, J = 9.5 Hz, 1H), 7.96
(t, J = 5.9 Hz, 1H), 7.10−6.98 (m, 3H), 6.86−6.84 (m, 1H), 4.03−
3.98 (m, 1H), 3.92−3.75 (m, 10H), 3.53−3.37 (m, 2H), 1.53−1.49
(m, 2H), 0.80−0.72 (m, 1H), 0.49−0.44 (m, 2H), 0.15−0.10 (m, 2H).
MS (ES+) m/z 428.1 (M + H)+.
6-(4-(5-Chloro-2-(trifluoromethyl)benzoyl)piperazin-1-yl)-N-
(2-cyclopropylethyl)pyridazine-3-carboxamide (51). White solid
(58% yield). mp 164−166 °C. 1H NMR (300 MHz, CDCl3) δ 8.07 (d,
J = 9.3 Hz, 1H), 7.96 (t, J = 5.8 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.54
(d, J = 7.6 Hz, 1H), 7.02 (d, J = 9.3 Hz, 1H), 4.07−4.00 (m, 1H),
3.87−3.72 (m, 5H), 3.66−3.60 (m, 2H), 3.42−3.35 (m, 2H), 1.54−
1.50 (m, 2H), 0.79−0.68 (m, 1H), 0.48−0.43 (m, 2H), 0.14−0.08 (m,
2H). MS (ES+) m/z 482.1 (M + H)+.
N-(2-Cyclopropylethyl)-6-(4-(3,3,3-trifluoro-2-methyl-2-
(trifluoromethyl)propanoyl)piperazin-1-yl)pyridazine-3-car-
boxamide (36). White solid (35% yield). 1H NMR (300 MHz,
CDCl3) δ 8.02 (d, J = 9.5 Hz, 1H), 7.96 (br s, 1H), 6.80 (d, J = 9.5 Hz,
1H) 3.91−3.75 (m, 8H), 3.60−3.57 (m, 2H), 1.58−1.48 (m, 5H),
0.78−0.63 (m, 1H), 0.48−0.43 (m, 2H), 0.11−0.04 (m, 2H). MS (ES
+) m/z 468.2 (M + H)+.
N-(2-Cyclopropylethyl)-6-(4-(2,5-dimethylbenzoyl)-
piperazin-1-yl)pyridazine-3-carboxamide (52). White solid (56%
1
yield). H NMR (300 MHz, CDCl3) δ 8.05 (d, J = 9.6 Hz, 1H), 7.96
(t, J = 5.8 Hz, 1H), 7.16−7.11 (m, 2H), 7.03−6.97 (m, 2H), 4.12−
3.65 (m, 6H), 3.60−3.54 (m, 2H), 3.43−3.38 (m, 2H), 2.23 (s, 3H),
6-(4-(2-Chloro-4-(trifluoromethyl)pyrimidine-5-carbonyl)-
piperazin-1-yl)-N-(2-cyclopropylethyl)pyridazine-3-carboxa-
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Journal of Medicinal Chemistry
Article
2.22 (s, 3H), 1.54−1.50 (m, 2H), 0.82−0.69 (m, 1H), 0.48−0.42 (m,
2H), 0.11−0.05 (m, 2H). MS (ES+) m/z 408.3 (M + H)+.
N-(2-Cyclopropylethyl)-6-(4-(2,5-difluorobenzoyl)piperazin-
0.50−0.45 (m, 2H), 0.15−0.05 (m, 2H). MS (ES+) m/z 400.0 (M +
H)+.
N-(2-Cyclopropylethyl)-6-(4-(2-(trifluoromethyl)furan-3-
carbonyl)piperazin-1-yl)pyridazine-3-carboxamide (40). White
solid (71% yield). 1H NMR (300 MHz, CDCl3) δ 8.04 (d, J = 10.6 Hz,
1H), 7.96 (t, J = 6.0 Hz, 1H), 7.56 (d, J = 6.0 Hz, 1H), 7.00 (d, J =
10.6 Hz, 1H), 6.54 (s, 1H), 3.90−3.70 (m, 6H), 3.60−3.50 (m, 4H),
1.49 (q, J = 7.0 Hz, 2H), 0.79−0.66 (m, 1H), 0.50−0.47−0.41 (m,
2H), 0.09−0.04 (m, 2H). MS (ES+) m/z 438.0 (M + H)+.
N-(2-Cyclopropylethyl)-6-(4-(5-methyl-2-(trifluoromethyl)-
furan-3-carbonyl)piperazin-1-yl)pyridazine-3-carboxamide
(42). White solid (53% yield); mp 128−130 °C. 1H NMR (500 MHz,
CDCl3) δ 8.08 (d, J = 9.5 Hz, 1H), 7.99 (t, J = 5.0 Hz, 1H), 7.01 (d, J
= 9.5 Hz, 1H), 6.15 (s, 1H), 3.94 −3.89 (m, 2H), 3.82−3.77 (m, 4H),
3.60−3.52 (m, 4H), 2.39 (s, 3H), 1.54−1.50 (m, 2H), 0.80−0.71 (m,
1H), 0.49−0.45 (m, 2H), 0.13−0.08 (m, 2H). MS (ES+) m/z 452.0
(M + H)+.
2-(4-(6-((2-Cyclopropylethyl)carbamoyl)pyridazin-3-yl)-
piperazine-1-carbonyl)benzoic Acid (44). Step 1: To a solution of
monomethyl phthalate (142 mg, 0.78 mmol) in DCM (10 mL) was
added DIPEA (0.2 mL, 1.15 mmol), HOBt monohydrate (116 mg,
0.86 mmol), and EDCI (0.16 mmL, 1.38 mmol). The resulting
mixture was stirred for 15 min, followed by the addition of N-(2-
cyclopropylethyl)-6-(piperazin-1-yl)pyridazine-3-carboxamide (184
mg, 0.66 mmol). The reaction mixture was stirred for 18 h and
diluted with ethyl acetate (100 mL), washed with water and brine,
dried over anhydrous Na2SO4, and concentrated. Purification by flash
chromatography (ethyl acetate) and recrystallization from ethyl acetate
and hexanes afforded methyl 2-(4-(6-((2-cyclopropylethyl)-
carbamoyl)pyridazin-3-yl)piperazine-1-carbonyl)benzoate as a white
solid (241 mg, 83% yield). MS (ES+) m/z 438.1 (M + H)+.
Step 2: LiOH.H2O (66 mg, 1.57 mmol) was added to a solution of
methyl 2-(4-(6-((2-cyclopropyl-ethyl)carbamoyl)pyridazin-3-yl)-
piperazine-1-carbonyl)benzoate (230 mg, 0.52 mmol) in THF (10
mL) and water (5 mL). The reaction mixture was stirred at room
temperature overnight. THF was removed in vacuo. The residue was
dissolved in ethyl acetate (100 mL) and neutralized by the addition of
5% HCl solution, washed with brine, dried over anhydrous Na2SO4,
and concentrated. The residue was recrystallized from dichloro-
methane and hexanes to afford 44 as a white solid (107 mg, 42%
yield). 1H NMR (300 MHz, CDCl3) δ 8.67 (br s, 1H), 9.07−7.87 (m,
3H), 7.54 (t, J = 7.5 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 7.26−7.24 (m,
1H), 6.95 (d, J = 9.6 Hz, 1H), 4.12−3.67 (m, 6H), 3.56−3.42 (m,
2H), 3.38−3.27 (m, 2H), 1.55−1.40 (m, 2H), 0.77−0.64 (m, 1H),
0.50−0.34 (m, 2H), 0.13−0.01 (m, 2H). MS (ES+) m/z 424.2 (M +
H)+.
1
1-yl)pyridazine-3-carboxamide (53). White solid (53% yield). H
NMR (400 MHz, CDCl3) δ 8.08 (d, J = 7.6 Hz, 1H), 8.00 (t, J = 5.7
Hz, 1H), 7.17−7.11 (m, 3H), 7.03 (d, J = 7.6 Hz, 1H), 4.02−3.92 (m,
2H), 3.85−3.83 (m, 4H), 3.59−3.5 (m, 4H), 1.54−1.50 (m, 2H),
0.74−0.69 (m, 1H), 0.46−0.40 (m, 2H), 0.09−0.04 (m, 2H).
N-(2-Cyclopropylethyl)-6-(4-(2,5-dichlorobenzoyl)piperazin-
1
1-yl)pyridazine-3-carboxamide (54). White solid (54% yield). H
NMR (300 MHz, CDCl3) δ 8.05 (d, J = 9.6 Hz, 1H), 7.96 (t, J = 5.8
Hz, 1H), 7.38−7.30 (m, 3H), 6.97 (d, J = 9.6 Hz, 1H), 4.12−3.70 (m,
6H), 3.60--3.23 (m, 4H), 1.54−1.50 (m, 2H), 0.80−0.67 (m, 1H),
0.51−0.38 (m, 2H), 0.16−0.06 (m, 2H). MS (ES+) m/z 448.2 (M +
H)+.
6-(4-(2,5-Bis(trifluoromethyl)benzoyl)piperazin-1-yl)-N-(2-
cyclopropylethyl)pyridazine-3-carboxamide (55). White solid
(50% yield). 1H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 7.6 Hz, 1H),
7.99 (t, J = 5.7 Hz, 1H), 7.92−7.90 (m, 1H), 7.85−7.84 (m, 1H), 7.65
(s, 1H), 7.02 (d, J = 7.6 Hz, 1H), 4.13−4.08 (m, 1H), 3.95−3.71 (m,
5H), 3.57−3.55 (m, 2H), 3.38−3.36 (m, 2H), 1.57−1.44 (m, 2H),
0.8−0.7 (m, 1H), 0.48−0.46 (m, 2H), 0.16−0.08 (m, 2H).
N-(2-Cyclopropylethyl)-6-(4-(3-fluoro-2-(trifluoromethyl)-
benzoyl)piperazin-1-yl)pyridazine-3-carboxamide (56). White
solid (31% yield). 1H NMR (300 MHz, CDCl3) δ 8.05 (d, J = 9.6 Hz,
1H), 7.97 (t, J = 5.7 Hz, 1H), 7.65−7.59 (m, 1H), 7.29 (d, J = 9.6 Hz,
1H), 7.12 (d, J = 7.8 Hz, 1H), 6.99 (d, J = 9.6 Hz, 1H), 4.05−3.99 (m,
1H), 3.89−3.72 (m, 5H), 3.54 (q, J = 6.6 Hz, 2H), 3.35 (t, J = 5.1 Hz,
2H), 1.53−1.50 (m, 2H), 0.76−0.71 (m, 1H), 0.48−0.42 (m, 2H),
0.10−0.05 (m, 2H).
N-(2-Cyclopropylethyl)-6-(4-(4-fluoro-2-(trifluoromethyl)-
benzoyl)piperazin-1-yl)pyridazine-3-carboxamide (57). White
solid (83% yield). 1H NMR (300 MHz, CDCl3) δ 8.05 (d, J = 9.6 Hz,
1H), 7.97 (t, J = 5.7 Hz, 1H), 7.46−7.42 (m, 1H), 7.39−7.29 (m, 2H),
6.97 (d, J = 9.6 Hz, 1H), 4.07−4.01 (m, 1H), 3.89−3.67 (m, 5H),
3.58−3.51 (m, 2H), 3.36−3.32 (m, 2H), 1.53−147 (m, 2H), 0.76−
0.69 (m, 1H), 0.48−0.42 (m, 2H), 0.10−0.06 (m, 2H). MS (ES+) m/z
466.1 (M + H)+.
N-(2-Cyclopropylethyl)-6-(4-(4,4,4-trifluorobut-2-enoyl)-
piperazin-1-yl)pyridazine-3-carboxamide (37). To a solution of
4,4,4-trifluorobut-2-enoic acid (62 mg, 0.44 mmol) in DCM (6 mL)
was added DIPEA (0.18 mL, 1.03 mmol), followed by the addition of
HOBt (68.7 mg, 0.51 mmol) and EDCI (0.10 mL, 0.57 mmol). The
resulting mixture was stirred for 15 min, and N-(2-cyclopropylethyl)-6-
(piperazin-1-yl)pyridazine-3-carboxamide (0.10 g, 0.36 mmol) was
added. After stirring overnight at room temperature, the reaction
mixture was diluted with EtOAc (120 mL), washed with water (4 × 12
mL) and brine (2 × 15 mL), dried over anhydrous Na2SO4, and
concentrated in vacuo. Purification by column chromatography
afforded the product as a white solid (28.5 mg, 20% yield). 1H
NMR (500 MHz, CDCl3) δ 8.09 (d, J = 9.8 Hz, 1H), 8.00 (br s, 1H),
7.02−6.98 (m, 2H), 6.83−6.79 (m, 1H), 3.96−3.88 (m, 4H), 3.80−
3.76 (m, 4H), 3.61−3.55 (m, 2H), 1.54−1.51 (m, 2H), 0.78−0.63 (m,
1H), 0.52−0.34 (m, 2H), 0.12−0.02 (m, 2H). MS (ES+) m/z 398.0
(M + H)+.
Preparation of Mouse Liver Microsomes. Male ICR mice, on a
high-carbohydrate, low-fat diet, under light halothane (15% in mineral
oil) anesthesia were sacrificed by exsanguination during periods of
high enzyme activity. Livers were immediately rinsed with cold 0.9%
NaCl solution, weighed, and minced with scissors. All procedures were
performed at 4 °C unless specified otherwise. Livers were
homogenized in a solution (1:3 w/v) containing 0.25 M sucrose, 62
mM potassium phosphate buffer (pH 7.0), 0.15 M KCl, 1.5 mM N-
acetyleysteine, 5 mM MgCl2, and 0.1 mM EDTA using four strokes of
a Potter−Elvehjem tissue homogenizer. The homogenate was
centrifuged at 10400g for 20 min to eliminate mitochondria and
cellular debris. The supernatant was filtered through a three-layer
cheesecloth and centrifuged at 105000g for 60 min. The microsomal
pellet was gently resuspended in the same homogenization solution,
and the protein concentration was measured using bovine serum
albumin as the standard.
The general procedure described for 37 was followed for the
preparation of the following compounds, using the appropriate
carboxylic acid for amide formation.
N-(2-Cyclopropylethyl)-6-(4-(2-(trifluoromethyl)-
cyclopropanecarbonyl)piperazin-1-yl)pyridazine-3-carboxa-
1
mide (38). White solid (31% yield). H NMR (300 MHz, CDCl3) δ
8.03 (d, J = 9.6 Hz, 1H), 7.98 (br s, 1H), 7.00 (d, J = 9.6 Hz, 1H),
3.97−3.57 (m, 10H), 2.22−1.98 (m, 1H), 1.67−1.63 (m, 1H), 1.52−
1.49 (m, 2H), 1.28−1.24 (m, 2H), 0.77−0.73 (m, 1H), 0.48−0.44 (m,
2H), 0.011−0.08 (m, 2H). MS (ES+) m/z 412.0 (M + H)+.
N-(2-Cyclopropylethyl)-6-(4-(2-methylcyclohexane-
carbonyl)piperazin-1-yl)pyridazine-3-carboxamide (39). White
solid (60% yield). 1H NMR (300 MHz, CDCl3) δ 8.08 (d, J = 9.6 Hz,
1H), 8.00 (t, J = 5.7 Hz, 1H), 7.00 (d, J = 9.3 Hz, 1H), 3.98−3.60 (m,
10H), 2.75−2.68 (m, 1H), 2.05−1.98 (m, 1H), 1.90−1.42 (m, 9H),
1.26−1.24 (m, 1H), 0.95 (d, J = 6.6 Hz, 3H), 0.80−0.72 (m, 1H),
SCD1 Mouse Liver Microsomal Activity Assay. Assay method-
ology is based on the method first described by Talamo and Bloch.38
3
SCD1 activity was measured using radiolabeled substrate, [9,10 H]-
stearoyl-Coenzyme A, for conversion into oleoyl-Coenzyme A and
release of tritiated water. Reactions were initiated by the addition of 60
μg of microsomal protein to assay plates containing 0.20 μCi of the
substrate at a final concentration of 33 μM with 2.0 mM NADH in a
33 mM Na-phosphate buffer (pH 7.4). Plates were incubated for 20
580
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Journal of Medicinal Chemistry
Article
min at 25 °C, and the reactions were stopped using 10 μL of 60%
perchloric acid. Reactions were transferred to multiscreen filter plates
coated wih 0.5% charcoal to bind the unreacted labeled substrate, and
the released tritiated water was collected and counted in a Microscint
40 scintillation counter.
carcasses was carried out with a Norland pDEXA machine equipped
with Sabre small animal analysis software (Orthometrics).
AUTHOR INFORMATION
■
Corresponding Author
Cell-Based SCD1 Activity Assay. The method was based on the
published procedure by Obukowicz.39 Human liver hepatocellular
carcinoma cells (HepG2) were grown to 50−60% confluency and
washed with phosphate-buffered saline prior to incubation with test
compounds and radiolabeled stearic acid (15 μCi) for 24 h at 37 °C.
The cells were treated with 5N NaOH to saponify the substrate and
product fatty acids. Fatty acids were extracted with hexane and
separated by thin layer chromatography. Plates were read on an instant
imager to detect the substrate and product, and the percent conversion
was determined. The effect of the test compounds on SCD1 activity,
as assessed by the conversion of radiolabeled stearic acid (18:0) into
oleic acid (cis-9-octadecenoic acid 18:1-n9), was evaluated against a
control to determine the percent of residual activity.
Plasma Desaturation Index (DI) Determination. Plasma lipids
were extracted following a modified Folch procedure, and the different
lipid classes contained in the extract were separated by thin layer
chromatography. The desired lipid classes were hydrolyzed to form
free fatty acids, which were subsequently derivatized for analysis by gas
chromatography. GC analysis was performed to provide a profile of
the amounts of the individual fatty acids contained in the lipid fraction.
The quantitative amount of each individual fatty acid was determined
by comparison to a known amount of internal standard added at the
extraction stage. The plasma DI was calculated based on the ratio of
substrate and product as described.9
Delta-5 and delta-6 Desaturase Activity. This method was
based on the procedure reported by Obukowicz.39 Microsomes for the
measurement of delta-5 and delta-6 desaturase activity were prepared
as described above. Delta-6 desaturase activity was measured using
radiolabeled linoleic acid (cis,cis-9,12-octadecadienoic acid, 18:2-n6) as
a substrate for conversion into γ-linolenic acid (all cis-6,9,12-
octadecatrienoic acid, 18:3-n6). Thin layer chromatography was then
used to separate the substrate and product following saponifaction and
extraction of fatty acids as described above. The effects of the test
compounds on delta-5 and delta-6 desaturase activities were evaluated
against a solvent control to determine the percent of residual activity.
Efficacy Study in Zucker Fatty Rats. Twenty-week-old male
Zucker obese fa/fa rats purchased from Harlan Sprague−Dawley Inc.
(Indianapolis, Indiana) were quarantined for approximately one week.
Two rats were housed per cage in a separate, controlled room.
Temperature was between 18 and 25 °C and relative humidity
between 45 and 65%. There was a 12 h light/dark cycle and rats had
access to filtered tap water and rodent Teklad 2018 diet was fed ad
libitum throughout the study unless specified. After quarantine period,
rats were randomly divided into two groups of 24 rats each with
equivalent average total body weights. These two groups were
randomly assigned to 49 treatment group (1 mg/kg) or vehicle control
group. 49 was suspended in a mixture of 1% CMC (low viscosity),
0.1% Tween 20, 10% propylene glycol, and 88.9% water. Rats were
administered orally twice a day at 10 a.m. and 4 p.m., respectively, for
15 weeks. The body weight was measured each day for each rat. At the
end of the 15 weeks of treatment, the rats were fasted for 4 h and
received the final dose of 49 or vehicle 2 h prior to being euthanized
by exsanguination. Following termination, dual energy X-ray
absorptiometry (DEXA) measurement of bone mineral content
(BMC), lean mass, and fat mass in the thoracovisceral area of the
carcasses was measured.
*Phone: 604-484-3361. E-mail: mwinther@xenon-pharma.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank our colleagues at Xenon Pharmaceuticals, Inc., who
assisted in the technical conduct of the research, including:
Catherine Zaborowska, Mary Huber, Maryann Mattice, Chris
Radomski, Joseph Sanghara, Pritpaul Samra, Rainbow Kwan,
Ivana Rajlic, Darren Daley, Caroline Hall, Audrey Wang,
Annick Legendre, Robert Fraser, and Monica Mork. We also
thank our colleagues at the former Discovery Partners
International, ChemRx Division, Wenbao Li, Mikhail Kon-
dratenko, Chi Tu, Melwyn Abreo, and Mark W. Holladay, and
at Novartis AG, Natalie Dales, Bryan Burkey, Andreas Billich,
and Brian Hubbard.
ABBREVIATIONS USED
■
SCD1, stearoyl coenzyme A desaturase 1; C16:1, palmitoleic
acid; C16:0, palmitic acid; C18:1, oleic acid; C18:0, stearic acid;
HepG2, human hepatocellular carcinoma cell line; ip, intra-
peritoneal; SAR, structure−activity relationship; DI, desatura-
1
tion index; AUC, area under the curve; H NMR, proton
nuclear magnetic resonance; PBS, phosphate buffer solution;
ND, not determined; DEXA, dual energy X-ray absorptiometry
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