Development of a liver-targeted stearoyl-CoA desaturase (SCD) inhibitor (MK-8245) to establish a therapeutic window for the treatment of diabetes and dyslipidemia

Article abstract of DOI:10.1021/jm200319u

The potential use of SCD inhibitors for the chronic treatment of diabetes and dyslipidemia has been limited by preclinical adverse events associated with inhibition of SCD in skin and eye tissues. To establish a therapeutic window, we embarked on designing liver-targeted SCD inhibitors by utilizing molecular recognition by liver-specific organic anion transporting polypeptides (OATPs). In doing so, we set out to target the SCD inhibitor to the organ believed to be responsible for the therapeutic efficacy (liver) while minimizing its exposure in the tissues associated with mechanism-based SCD depletion of essential lubricating lipids (skin and eye). These efforts led to the discovery of MK-8245 (7), a potent, liver-targeted SCD inhibitor with preclinical antidiabetic and antidyslipidemic efficacy with a significantly improved therapeutic window.

Full text of DOI:10.1021/jm200319u

ARTICLE
pubs.acs.org/jmc
Development of a Liver-Targeted Stearoyl-CoA Desaturase (SCD)
Inhibitor (MK-8245) to Establish a Therapeutic Window for the
Treatment of Diabetes and Dyslipidemia
Renata M. Oballa,*,† Liette Belair,† W. Cameron Black,† Kelly Bleasby,‡ Chi Chung Chan,†
Carole Desroches,† Xiaobing Du,‡ Robert Gordon,† Jocelyne Guay,† Sebastien Guiral,† Michael J. Hafey,‡
Emelie Hamelin,† Zheng Huang,† Brian Kennedy,† Nicolas Lachance,† France Landry,† Chun Sing Li,†
†
Joseph Mancini,† Denis Normandin,† Alessandro Pocai,‡ David A. Powell,†Yeeman K. Ramtohul,
Kathryn Skorey,† Dan Sørensen,† Wayne Sturkenboom,† Angela Styhler,† Deena M. Waddleton,†
Hao Wang,† Simon Wong,† Lijing Xu,† and Lei Zhang†
†Merck Frosst Centre for Therapeutic Research, 16711 TransCanada Highway, Kirkland, Quꢀebec H9H 3L1, Canada
‡MerckResearch Laboratories, 126 East Lincoln Avenue, Rahway, New Jersey 07065-0900, United States
S Supporting Information
b
ABSTRACT: The potential use of SCD inhibitors for the
chronic treatment of diabetes and dyslipidemia has been limited
by preclinical adverse events associated with inhibition of SCD
in skin and eye tissues. To establish a therapeutic window, we
embarked on designing liver-targeted SCD inhibitors by utiliz-
ing molecular recognition by liver-specific organic anion trans-
porting polypeptides (OATPs). In doing so, we set out to target
the SCD inhibitor to the organ believed to be responsible for the
therapeutic efficacy (liver) while minimizing its exposure in the tissues associated with mechanism-based SCD depletion of essential
lubricating lipids (skin and eye). These efforts led to the discovery of MK-8245 (7), a potent, liver-targeted SCD inhibitor with
preclinical antidiabetic and antidyslipidemic efficacy with a significantly improved therapeutic window.
’ INTRODUCTION
there are reports of skin and eye abnormalities in the SCD1ꢀ/ꢀ
mice3,15 as well as in rodents treated with SCD1 inhibitors.8ꢀ10
These adverse events (AEs) consist of dry eye, squinting, and
alopecia and are believed to be due to mechanism-based deple-
tion of essential SCD-derived lubricating lipids. Thus, the main
challenge with developing a small molecule SCD inhibitor is to
achieve adequate therapeutic efficacy without affecting the hu-
man skin and eye functions. Given the nonlife threatening nature
of metabolic diseases, it is crucial to establish a sufficient
therapeutic window for SCD inhibitors for treating metabolic
syndromes before embarking on human clinical trials. One
potential way to achieve a therapeutic window for SCD inhibi-
tion is to target the SCD inhibitor to the organ believed to be
responsible for the therapeutic efficacy (liver) while minimizing
its exposure in the tissues associated with mechanism-based SCD
depletion of essential lubricating lipids (skin and eye).
One of the fundamental goals for pharmaceutical intervention
is to achieve the desired efficacy with as few side effects as
possible. This can be particularly challenging in cases where the
target enzyme or receptor is ubiquitously expressed and deemed
essential for certain physiological processes. One such target is
stearoyl-CoA desaturase-1 (SCD1). SCD1 is the key enzyme
involved in the synthesis of monounsaturated fatty acids and
catalyzes the installation of a cis-double bond at the Δ9 position
of long chain saturated fatty acyl-coenzyme A esters.1 The
monounsaturated lipid products (palmitoleoyl-CoA (C16:1)
and oleoyl-CoA (C18:1)) are key building blocks in the synthesis
of membrane phospholipids, cholesterol esters, and triglycerides.
It has been reported that rodents deficient in SCD1 either by
gene deletion,2ꢀ4 antisense oligonucleotide (ASO) treatment,5ꢀ7
or pharmacological inhibition8ꢀ12 are resistant to diet-induced
weight gain and have an improved insulin sensitivity, glucose
tolerance, and lipid profile. In humans, elevated SCD1 levels are
positively correlated with higher plasma triglycerides13 as well as
increased BMI and high insulin levels.14 On the basis of the
human and rodent findings, SCD1 represents an attractive novel
therapeutic target for the treatment of type II diabetes, dyslipi-
demia, obesity, and metabolic diseases. However, in addition to
the positive metabolic effects associated with SCD1 inhibition,
Two pivotal SCD1 knockdown experiments provided evi-
dence that it may be possible to develop a safe and effective SCD
inhibitor for therapeutic application. First, treatment of mice with
an SCD1 ASO resulted in ∼60ꢀ70% reduction of SCD1 mRNA
in liver and fat and prevented diet-induced obesity and improved
Received: March 19, 2011
Published: June 10, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of Tetrazole 4a
Scheme 2. Syntheses of Tetrazole 5 and Tetrazole Acetic
Acid 6a
a Reagents and conditions: (a) 8, K2CO3, DMF, 80 °C; (b) CuCN,
t-BuONO, CH3CN, 50ꢀ60 °C; (c) NaN3, NMP, 130 °C.
a Reagents and conditions: (a) 13, iPr2EtN, 1,4-dioxane; (b) NaN3,
NMP, 130 °C; (c) NaH, ethyl bromoacetate, DMF, ꢀ78 °C to rt;
(d) NaOH, THF/MeOH.
insulin sensitivity.5 Importantly, after 10 weeks of treatment with
the SCD1 ASO, the mice did not demonstrate the hair, skin, and
eye abnormalities observed either with SCD1 inhibitor-treat-
ment or in the global SCD1ꢀ/ꢀ mice. Second, Cre-lox technol-
ogy was used to generate mice with a liver-specific knockout of
SCD1 (LKO),4 and these mice were protected from high-
carbohydrate diet-induced adiposity and hepatic steatosis. Unlike
the global SCD1ꢀ/ꢀ mice showing alopecia and closed eyes, the
LKO mice were indistinguishable from both wild-type and Lox
mice (18 weeks).
In drug discovery, tissue-targeting refers to a process which
delivers a pharmaceutical agent (small molecule or biologic) to
the desired specific tissue. Targeting a pharmaceutical agent to
the liver (i.e., liver-targeting) can be highly effective to treat liver-
related diseases when it is essential to minimize exposure to other
tissues where adverse events can occur as a result of mechanism-
based or off target drug activity. To target a pharmaceutical agent
to the liver, the following approaches have been studied:
(i) nanoparticles to deliver incorporated therapeutic materials
such as small molecules, proteins, genes, and siRNAs,16
(ii) HepDirect cytochrome P450-activated prodrugs of certain
small molecules such as nucleotides,17 and (iii) utilization of
liver-specific transport proteins.18 The approach to liver-target-
ing outlined in this paper relies on the latter strategy. Described
herein will be the strategy used to design liver-targeting SCD
inhibitors and how this approach resulted in the discovery of the
lead compound MK-8245 (7), which maintains preclinical
pharmacological efficacy while significantly improving the ther-
apeutic window compared to systemically distributed SCD
inhibitors (i.e., 1).
hydride and ethyl bromoacetate generated two alkylated pro-
ducts, the major one bearing the ethylacetate at the N-2 position
of the tetrazole. Upon chromatographic separation of the two
alkylated products, the desired major product was subjected to
basic ester hydrolysis to generate the desired tetrazole acetic acid
6. The synthesis of the optimal liver-targeted tetrazole acetic acid
7 was prepared as outlined in Scheme 3. The key transformation
is an unprecedented three-component 1,3-dipolar cycloaddition
and nucleophilic substitution reaction between 13, 15, and 16 to
give the isoxazole intermediate 17 in ∼20% yield. The mechan-
ism of this transformation is still not clear. Because 3-bromoisox-
azole is quite resistant to nucleophilic substitution, it is
reasonable to hypothesize that the piperidine 13 and the dipole
precursor 16 may react prior to the cycloaddition reaction with
propiolate 15. The ester group in compound 1719 was converted
to the corresponding amide 18 by reaction with ammonium
hydroxide. The amide 18 was then dehydrated to the nitrile 19 by
reaction with trifluoroacetic anhydride. The nitrile 19 was
converted to the tetrazole 20 and finally to the desired tetrazole
acetic acid 7 in a similar manner as previously described for
compound 6.
’ RESULTS AND DISCUSSION
Strategy to Design a Liver-Targeted SCD Inhibitor. Our
goal was to increase drug exposure in liver (target organ) by
engaging active transport into heptocytes via the liver-specific
organic anion transporting polypeptides (OATPs). Our goal was
to incorporate key transporting elements into SCD inhibitors to
enable recognition by the OATP transport proteins while main-
taining SCD potency. Moreover, we also sought to decrease the
extent of unselective passive cell diffusion in order to minimize
exposures in off-target tissues and cells (skin and eye). Fortu-
nately, acidic moieties used to engage the active transporters
generally impart reduced passive cell penetration. Nonetheless,
we required a research operating procedure (ROP) that would
allow us to funnel potential compounds and help us identify
compounds which may exhibit a liver-targeting tissue distribu-
tion profile. The ROP to select for liver-targeted SCD inhibitors
is depicted in Figure 1 and can be summarized as follows:
’ CHEMISTRY
The synthesis of the thiadiazole tetrazole 4 was prepared as
described in Scheme 1. Starting with the bromo thiadiazole
amine 9, addition of the piperadine 8 under basic conditions
resulted in the formation of the amine 10. The amine 10 was then
converted to the nitrile 11 using standard conditions and, finally,
the tetrazole 4 was generated by reacting nitrile 11 with sodium
azide. The syntheses of tetrazole 5 and the tetrazole acetic acid 6
are described in Scheme 2. The bromo thiadiazole 12 was reacted
under basic conditions with the piperadine 13 to generate the
nitrile 14. Compound 5 was prepared by reacting nitrile 14 with
sodium azide. Subsequent reaction of the tetrazole 5 with sodium
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Scheme 3. Synthesis of MK-8245 (7)a
a Reagents and conditions: (a) KHCO3, EtOAc, H2O; (b) NH4OH, MeOH, THF; (c) TFAA, Et3N, CH2Cl2, 0 °C to rt; (d) NaN3, py HCl, NMP,
3
130 °C; (e) Et3N, ethyl bromoacetate, THF, reflux; (f) NaOH, EtOH, H2O; (g) HPLC separation.
on the right-hand side produced compounds completely devoid
of SCD inhibition (3, Figure 2). One of the first acidic moieties to
be appended was the tetrazole (4, Scheme 1). This resulted in a
significant loss of potency on the SCD1 rat enzyme (∼140-fold,
4 vs 1, Table 1) as well as being inactive in the HepG2 cell assay.
However, compound 3 exhibited a modest SCD inhibition in the
rat hepatocyte assay which contains active OATPs (IC50 = 3467
nM). This demonstrated that appendage of an acidic moiety
could decrease passive cell penetration and engage in active
transport into a hepatocyte (assumption based on HepG2 vs
hepatocyte shift). To improve potency on the rat SCD enzyme
Figure 1. Strategy to identify liver-targeting SCD inhibitors. HepG2
cell SCD assay21 is a gauge of the extent of passive cell diffusion; rat
hepatocyte SCD assay identifies compounds which are actively trans-
ported into the liver; mouse tissue distribution is a measure of the extent
of liver-targeting.
assay, structureꢀactivity relationship (SAR) studies were per-
formed on the right-hand side phenoxy portion and, as has been
previously reported,22 2,5-dihalogen substitution resulted in a
significant 8-fold improvement in potency (5, rat enzyme IC50
=
34 nM, Scheme 2, Table 1). Compound 5 was significantly
shifted in the HepG2 assay (370-fold) and slightly more potent
in the hepatocyte assay (4-fold vs HepG2). A further increase in
potency could be obtained by appending an acetic acid side chain
onto the tetrazole heterocycle (6, rat enzyme IC50 = 7 nM,
Table 1). In this case, the acidic tetrazole moiety was effectively
replaced with a carboxylic acid. As with the tetrazoles 4 and 5,
acid 6 was >100-fold shifted in potency in the HepG2 cellu-
lar assay and more potent in the hepatocyte assay. Finally,
by replacing the middle thiadiazole ring with an isoxazole
(7, Scheme 3), the potency in both the rat enzyme and hepato-
cyte assays was further improved 2-fold (Table 1) while main-
taining a >300-fold shift in the HepG2 assay. This compound is
designated as 7 and was also determined to be a highly potent
inhibitor of the human SCD1 enzyme (IC50 = 1 nM).23 It should
be also noted that for all of the SCD inhibitors listed in this paper,
(i) determine if compounds are SCD inhibitors via an in vitro rat
microsomal enzyme assay,20 (ii) test active SCD inhibitors in two
cell assays, one devoid of active OATPs (HepG2 cell line21) and
one containing functional, active OATPs (rat hepatocyte), and
(iii) conduct mouse tissue distribution studies on those com-
pounds which are at least 4- to 5-fold more potent in the OATP
vs non-OATP cell assays.
The first task was to determine where an acidic moiety could
be appended onto existing SCD inhibitors to impart recognition
by OATPs, decrease passive cell diffusion while at the same time
maintaining SCD potency. Starting with our previously reported
SCD inhibitor MF-438 (1) (Figure 2),9 acidic moieties were
placed on either the right- or left-hand side of the molecule. It
became evident that acidic moieties were only tolerated on the
left-hand side of the molecule (2, Figure 2); appendage of acids
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Figure 2. Chemical structure of systemically distributed 1. Compounds of general structures 2 and 3 were assessed for potency vs the rat microsomal
SCD1 enzyme.20 Compounds of general structure 2 inhibited the rat SCD1 enzyme (IC50s < 1 μM), whereas compounds of general structure 3 were
inactive vs rat SCD1 (IC50s > 10 μM).
Table 1. In Vitro Potencies and Liver-Targeting Tissue Distribution Profiles of Key SCD Inhibitors
SCD inhibitor
assay
1
4
5
6
7
rat microsomal enzyme IC50 (nM)a,20
Hum HepG2 Cell IC50 (nM)a,21
rat hepatocyte cell IC50 (nM)a
HepG2/hepatocyte ratio
mouse [liver]/[Harderian gland] ratioa,b
a Data represent mean values of at least n = 3. b 6 h post 10 mg/kg PO dose.
2
276
34
7
3
21
>50000
3467
12588
3070
4:1
904
189
5:1
1066
68
157
0.14:1
1.5:1
>14:1
330:1
16:1
21:1
26:1
40:1
there were no significant differences in potencies between the rat,
mouse, and human SCD1 (i.e., for 7, the rat, mouse, and human
SCD1 enzyme IC50s were 3, 3, and 1 nM, respectively). More-
over, all of the SCD inhibitors listed in Table 1 were highly
selective over the Δ-5 and Δ-6 desaturases (i.e., >100000 μM vs
rat and human Δ5D and Δ6D as assessed in the HepG assay21).
Liver-Targeted Tissue Distribution Profile. To determine if
our strategy to select compounds based on their shift in the
HepG2 vs hepatocyte assays resulted in compounds which
exhibit a liver-targeted tissue distribution profile, the inhibitors
in Table 1 were dosed in mice (10 mg/kg, PO) and 6 h post dose
the animals were euthanized and the concentration of compound
in select tissues (plasma, liver, skin, and the eye-lubricating
Harderian glands) was measured. To simplify the discussion,
the liver-to-Harderian gland ratio was used as a measure of the
degree of liver-targeting. Starting with what we suspected to be a
systemically distributed compound (i.e., a compound that dis-
tributes readily to most tissues), 1, which is actually more potent
in the HepG2 assay vs hepatocyte assay, did show almost
equivalent levels of compound in the Harderian gland vs liver
(Table 1). We were gratified to observe that the tetrazole 3,
which is at least 14-fold more potent in the hepatocyte vs HepG2
assay, did demonstrate a liver-targeted tissue distribution profile.
As indicated in Table 1, the liver-to-Harderian gland ratio for 3
was >300:1, which is clearly superior to the 1.5:1 ratio obtained
with the nonacidic compound 1. Finally, the more potent acidic
SCD inhibitors (5, 6, and 7) also demonstrated a liver-targeted
tissue distribution profile as judged by their liver-to-Harderian
gland ratios.
targeting compound to be absorbed in the GI tract in order to
be delivered to the portal vein effectively. Thus, there still had to
be some degree of cell penetration (i.e., Papp > 1 ꢁ 10ꢀ6 cm/s
and <10 ꢁ 10ꢀ6 cm/s) and it was determined that compound
7 was moderately bioavailable in multiple species (F = 12%, 28%
and 40% in mice, rats and dogs, respectively). Moreover, it is
known that OATPs are expressed in the intestinal wall25,26 and
this may also be responsible for the absorption of compound 7.
To determine if mouse liver-targeting via OATPs would
translate to liver-targeting in other species,27 7 was dosed orally
to mice, rats, dogs, and rhesus monkeys and at 6 h post dose, the
animals were euthanized, and the concentration of compound in
select tissues (plasma, liver, skin, and Harderian glands) was
measured. Only rodents possess Harderian glands, and because
the corresponding eye-lubricating meibomian glands in dogs and
rhesus monkeys were too difficult to extract, skin was used as the
off-target tissue to measure liver-targeting in these higher species.
As depicted in Figure 3, in all species examined, 7 was distributed
mainly to the liver, with low exposure in tissues associated with
potential adverse events (i.e., skin and eye lubricating glands).
The liver-to-skin ratios were >30:1 in all four species.
OATP Transport Profile. To demonstrate that 7 is actively
transported via human OATPs, uptake studies were performed in
butyrate-treated stably transfected OATP1B1 and 1B3 MDCKII
cell lines. As shown in Figure 4, 7 was found to be a substrate of
both OATP1B1 and 1B3 and the uptake was inhibited by the
OATP inhibitor sulfobromophthalein28 (BSP). This data sug-
gests OATP1B1 and OATP1B3 likely contribute toward the liver
selectivity of 7 by mediating uptake into hepatocytes. The
potency of 7 was measured in both freshly isolated and cryopre-
served human hepatocytes and was found to be highly potent
(IC50 = 5 nM, Supporting Information Method 3) compared to
the non-OATP human HepG2 cell line (IC50 = 1066 nM),
further indicating active uptake transport of 7 by human OATPs.
Pharmacological Efficacy of the Liver-Targeted SCD
Inhibitor 7. To assess the diabetic efficacy, lipid efficacy, and
safety profile of a systemically distributed vs liver-targeted SCD
inhibitor, the following models were used: (i) acute oral glucose
tolerance in eDIO mice (oGTT) to assess glucose clearance and
(ii) chronic administration in eDIO mice to assess body weight
In addition to generating OATP substrate affinity, our liver-
targeting strategy required the minimization of passive cell
diffusion to prevent the penetration of any circulating SCD
inhibitor to off-target tissues. The most potent SCD inhibitor 7
was significantly shifted in the non-OATP HepG2 cellular assay
(∼350-fold), indicating that this compound should possess the
desired lack of cell penetration. To confirm this, the diffusion rate
of 7 was evaluated across monolayers of LLC-PK1 cells and the
P
app was determined to be 3.8 ꢁ 10ꢀ6 cm/s, which is indicative of
poor to moderate cell penetration24 (Supporting Information
Method 2). It should be noted that we required the liver-
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Figure 3. Plasma and tissue concentration ratios at 6 h post oral dosing of 7 in mouse, rat, dog, and rhesus monkey. (a) Concentration of 7 in tissues 6 h
post dose. Compound 7 was dosed orally (formulated in 0.5% methylcellulose) to male C57BL6 mice (n = 2, 10 mg/kg), male SpragueꢀDawley rats
(n = 3, 10 mg/kg), female rhesus monkeys (n = 2, 5 mg/kg), and male beagle dogs (n = 2, 5 mg/kg). (b) Ratio of tissue concentrations in liver vs tissues
associated with potential adverse events. ND = not determined.
Figure 4. Uptake of 7 into (a) OATP1B1 stably transfected MDCK11 cells and (b) OATP1B3 stably transfected MDCK11 cells. OATP1B1 and 1B3
were found to mediate the BSP sensitive uptake of 7, suggesting these transporters may play a role in the uptake of 7 into human hepatocytes. All data are
mean ( sem, n = 3.
effects and lipid profile as well as evaluating the adverse event
profiles in skin and eyes. Compounds 1 and 7 were given orally to
eDIO mice 1 h before administration of an oral glucose challenge,
and blood glucose levels were monitored for 2 h thereafter. As
shown in Figure 5, both the systemically distributed 1 and the
liver-targeted 7 improved glucose clearance dose-dependently to
a similar extent, with ED50 values of 3 and 7 mg/kg, respectively.
This was the first indication that selectively inhibiting liver SCD1
with a liver-targeted small molecule SCD inhibitor was effective
at lowering glucose levels.
Chronic efficacy of the systemically distributed 1 and the liver-
targeting SCD inhibitor 7 was evaluated in a 4-week oral dosing
study in eDIO mice. Once-daily oral dosing was used for
compound 1 based on its 6.4 h plasma half-life in mouse,9
whereas bid dosing was required for 7 (mouse plasma half-life
= 0.7 h) to provide similar SCD inhibition over the course of the
experiment. Table 2 compares the mouse pharmacokinetics of
compounds 1 and 7 and also indicates that the terminal trough
liver levels of both compounds in the chronic eDIO mouse study
are similar and would provide near complete liver SCD inhibition
(see Supporting Information Results 2). As shown in Figure 6a,
compound 1 treatment resulted in a significant, dose-dependent
decrease in body weight (∼10% and 20% for the 1 and 5 mg/kg
treatment groups, respectively), comparable to the effect seen
with the CB1 inverse agonist AM251.29 On the other hand, the
liver-targeted inhibitor 7, dosed at 20 or 60 mg/kg bid, showed
only a modest prevention of body weight gain (∼5% BW gain
reduction, Figure 6b). The maximally efficacious dose of 1, based
on body weight effects, was 5 mg/kg, qd. For compound 7, the
maximally efficacious dose was 20 mg/kg bid based on body
weight and liver triglyceride reduction (60 mg/kg bid dose did
not provide improved efficacy results). Histological analysis
showed that hepatic steatosis was significantly reduced, which
was confirmed by a reduction of liver triglyceride levels (see
Supporting Information Results 3, Figure 3). In these eDIO
studies, there was no effect on food consumption with either
compound.
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Figure 5. Dose-dependent effect of 1 and 7 on oGTT in DIO mice. Mice that were fed a high fat diet (HFD) for at least 14 weeks and had an established
obesity state before the study were fasted for 16 h. Test compound was given 1 h before administration of oral glucose at 2 g/kg. (a) Compound 1 effect
on glucose AUC following doses of 3, 10, and 15 mg/kg and (b) compound 7 effect on glucose AUC following doses of 3, 10, and 30 mg/kg. HFD, high
fat diet; ND, normal diet; Veh, vehicle. n = 10ꢀ15 mice/group; mean ( sem.
inhibition in the off target tissues, the desaturation indices (DIs)
in the off-target tissues were compared to that obtained in the
liver. The DI is the ratio of SCD product-to-SCD substrate
([C18:1, oleic acid]/[C18:0, stearic acid]) and is a direct
measure of chronic SCD activity.13 A reduction in DI indicates
inhibition of the SCD enzyme and DI can be monitored in
plasma and tissues. For the systemically distributed 1 at a
maximally efficacious dose of 5 mg/kg qd, the liver SCD DI
was reduced by ∼56% (Figure 8a, not significant due to high
variation in vehicle-treated mice), while there was significant
reduction of SCD activity in both the Harderian gland (92% V DI,
Figure 8b) and skin (25% V DI, Figure 8c). These significant DI
reductions are consistent with the high exposure of 1 in all of
these tissues ([liver] = 10 μM, [Harderian gland] = 5 μM, [skin] =
4 μM).
In contrast, for the liver-targeted SCD inhibitor 7, while there
was very significant reduction in liver DI (63%) following 4
weeks of dosing 20 mg/kg bid in eDIO mice, there was no
significant reduction in Harderian gland DI (Figures 8d,e,
respectively; skin DI was not measured in the compound 7-
treated mice). The reduction of DI (liver) or lack thereof
(Harderian gland) was consistent with the exposure of 7 in these
tissues ([liver] = 5 μM, [Harderian gland] = 0.9 μM). It should
be noted that there was a small erosion of liver-targeting upon
chronic dosing of 7 as assessed by liver-to-Harderian gland ratio
(21:1 following single administration vs 5:1 after 4 weeks of
dosing). The liver-to-Harderian gland ratio for 7 reached steady
state (5:1) after ∼1 week of dosing, did not worsen upon chronic
dosing (4 weeks) and was sufficient in providing a therapeutic
window in the chronic eDIO mouse model (i.e., lack of harderian
gland SCD inhibition (Figure 8e) and no significant eye AEs
(Figure 7d)).
Table 2. Mouse Pharmacokinetics of Compounds 1 and 7
mouse
pharmacokineticsa
Cl
(mL/min/
kg)
plasma plasma trough liver
Cmax AUC levels from
Vd
t1/2
F
compd
(L/kg) (h) (%) (μM) (μM h) eDIO (μM)b
3
1c
7d
15
41
7
1
6.4 73
0.7 12
1.3
0.4
19
1
5
4
a Mouse PK parameters obtained following a 2 mg/kg IV and 10 mg/kg
PO dose of compounds 1 and 7 to fasted male C57BL6 mice.
Concentrations of compounds 1 and 7 were determined by LC-MS/
MS and values represent the mean of n = 3 animals. b Liver concentra-
tions were determined at trough at termination of the 4-week eDIO
mouse study at the 20 mg/kg bid dose of compound 7 and the 5 mg/kg
qd dose of compound 1. c Compound 1 was dosed IV as a solution in
60%PEG-200 (5 mL/kg) and PO as a suspension in 1% methocel
(10 mL/kg). d Sodium salt of compound 7 was dosed IV as a solution in
saline (1 mL/kg) and PO as a suspension in 0.5% methocel (10 mL/kg).
Evaluation of Skin and Eye Adverse Effects in Rodents
Treated with Systemic vs Liver-Targeted SCD Inhibitors. In
addition to evaluating efficacy end points in the chronic 4-week
eDIO mouse studies, comparison of the skin and eye adverse
effect profiles of a systemic vs liver-targeted SCD inhibitor was of
paramount importance. Notably, would a liver-targeted SCD
inhibitor impart a therapeutic window compared to a systemi-
cally distributed inhibitor? As previously reported for systemi-
cally distributed SCD inhibitors,8ꢀ10 both skin and eye
abnormalities were observed in compound 1-treated eDIO mice
(Figures 7a,b). In contrast to systemically distributed SCD
inhibitors, which induce significant skin and eye adverse effects
in the eDIO mouse within 5ꢀ10 days of treatment at therapeutic
doses, the liver-targeted 7 did not induce meaningful skin or eye
adverse events during the 4-week treatment period (Figures 7c,
d). To confirm that the improved adverse event profile observed
with 7 in the 4-week eDIO mouse study is due to lack of SCD
One of most well-documented classes of liver-targeting small-
molecule therapeutics are HMG-CoA reductase inhibitors
(statins). As with SCD inhibitors, distribution of statins to tissues
other than liver is linked to adverse events (myopathy in the case
of statins).30 Statins are liver-targeted by virtue of their affinity for the
liver-specific organic anion transporting polypeptides (OATPs),
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Figure 6. Effect on body weight following chronic dosing of (a) 1 vs (b) 7 in eDIO mice. eDIO mouse fed on HFD for at least 18 weeks prior to
initiation of study were dosed orally with vehicle, 1 (1 or 5 mg/kg, qd) or 7 (20 or 60 mg/kg, bid). Doses were selected to target sustained liver SCD
inhibition based on mouse liver PD results (Supporting Information Method 4). HFD, high fat diet; Veh, vehicle; n = 8ꢀ10 mice/group; mean ( sem.
Figure 7. Skin and eye adverse event profile of 1 (a and b) and 7 (c and d) in a 4-week eDIO mouse study. Skin scoring system: 0, normal; 1, small patch
(1ꢀ3 mm) of hair loss; 2, larger patch (3ꢀ 0 mm); 3, extensive patch with lesions. Eye scoring system: 0, normal; 1, squinting eye; 2, partial eye closure,
one eye; 3, both eyes closed. To lessen the severity and allow for completion of the study, eye lubrication (BNP: Neomycin and Polymyxin B Sulfates and
Bacitracin Ophthalmic Ointment, USP) was applied to the eyes of compound 1-treated mice starting on day 12. Veh = vehicle.
and it was strategized that targeting the same OATP transporters
would lead to the generation of a liver-targeted SCD inhibitor.
The fact that statins were substrates for the OATPs was not
known a priori and this liver-targeting feature was not knowingly
designed into the compounds to improve the therapeutic index.
In fact, realization that statins were OATP substrates occurred
after the first statin was already on the market. It was the goal of
our research efforts to implement an in vitro strategy to predict
the liver-targeting capabilities of a compound.
specific tissues. Two of the OATP transporters (1B1 and 1B3)
are specifically expressed in the liver, are localized to the
basolateral membrane of the hepatocytes, and facilitate the
uptake of drugs from the blood into hepatocytes.31 Drug sub-
strates of OATPs 1B1 and 1B3 include a large number of
structurally diverse compounds, such as statins (i.e., atorvastatin,
rosuvastatin, simvastatin acid, etc.), endothelin receptor antago-
nists (i.e., atrasentan), certain antibiotics (i.e., benzylpenicillin),
and angiotensin II receptor antagonists (i.e., olmesartan,
valsartan), just to name a few.31 One of the key structural features
that these drugs have in common is an acidic moiety, either a
Transporters can affect the tissue distribution of drugs and
thereby contribute to the selective distribution of drugs to
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Figure 8. Desaturation indices in tissues of eDIO mice treated with 1 or 7. eDIO mice fed on HFD for at least 18 weeks prior to initiation of study were
dosed orally with vehicle, 1 (5 mg/kg, qd, 3 days) or 7 (20 mg/kg, bid, 28 days). Following sacrifice, tissues were harvested and DI measurements were
obtained. (a and d) represent liver DI measurements in vehicle vs 1 or 7-treated mice, respectively; (b and e) represent Harderian gland (HG) DI
measurements in vehicle vs 1 or 7-treated mice, respectively; (c) represents skin DI measurements in vehicle vs 1-treated mice (skin DI for 7-treated
mice was not determined). n = 8ꢀ10 mice/group; mean ( sem.
carboxylic acid or tetrazole moiety. It was determined that adding
an acidic moiety to the left-hand side of systemically distributed
SCD inhibitors allowed for retention of SCD potency
(compounds 4, 5, 6, and 7, Table 1). However, simply adding
an acidic moiety on the left-hand side of the molecule did not
automatically impart recognition by the liver-specific OATPs,
as judged by the shift in potency in HepG2 vs hepatocyte assays
as well as in tissue distribution studies. Detailed SAR studies
regarding the correct positioning of the acidic moiety to max-
imize both SCD potency and liver-targeting will be the subject of
subsequent publications.
The optimization of liver-targeted SCD inhibitors depicted in
Table 1 involved the important use of the HepG2 and hepatocyte
SCD cellular assays. The principle reason for using cellular SCD
assays to gauge OATP substrate affinity is that they are very high
throughput. Assays to directly measure OATP substrate affinity
certainly exist (see Experimental Section) but are not sufficiently
high throughput to be able to test hundreds of compounds per
month. The goal of the HepG2 assay is to gauge the extent of
passive diffusion and compounds which are not potent in this
assay are desired to minimize diffusion and distribution to non-
liver tissues. Conversely, in the OATP hepatocyte cell assay,
compounds which are recognized by the OATPs will be actively
transported into the hepatocyte and should be more potent at
inhibiting intracellular SCD compared to the HepG2 assay. It
should be noted that there is only a loose correlation between
HepG2-to-hepatocyte potency shift and liver-targeting as mea-
sured by compound levels in mouse liver vs Harderian gland.
However, as depicted in Table 1, HepG2/hepatocyte ratios of
g4 do translate to significant and likely sufficient liver-targeting
as determined from the mouse tissue distribution analyses. In the
tissue distribution analysis, particular attention is focused on the
drug concentration in tissues where adverse events have been
observed in the SCD1ꢀ/ꢀ mice (the eye lubricating Harderian
gland and skin tissue). Focusing only on the plasma-to-liver ratio
can be misleading because many nontransported drugs reach
high concentrations in the liver due to rapid exchange of blood
constituents with the liver tissue. Drugs absorbed in the gastro-
intestinal tract are completely transported to the liver via the
portal vein, and after steady state has been reached, most
compounds do not demonstrate cell specificity (liver levels of
compound vs that in other tissues). For example, the liver-to-
plasma ratios for the systemically distributed 1 and the liver-
targeted compound 4 were very similar (14:1 for 1 vs 15:1 for 4)
and could not be used to discriminate these two compounds.
However, when comparing the liver-to-Harderian gland ratios,
there is clearly a difference (1.5:1 for 1 and >300:1 for 4), thereby
demonstrating the need to examine drug concentrations in the
off-target tissues to properly assess liver targeting. Our target
tissue distribution profile was determined to be a concentration
of drug in liver that fully inhibits SCD and that is at least 20-fold
higher than levels found in the off-target tissues (Harderian gland
and skin). The most potent liver-targeted SCD inhibitor 7 met
these criteria not only in mouse but also in rat, dog, and rhesus
monkey. Furthermore, compound 7 is likely to be liver-targeted
in humans due to the fact that it was found to be a substrate for
the human OATP1B1 and 1B3 (Figure 4) and was also very
potent in a human hepatocyte assay (IC50 = 5 nM). It has been
reported that there is a strong association between simvastatin-
induced myopathy and two tightly linked variants of the
OATP1B1 gene.32 This is likely due to impaired liver uptake of
simvastatin in those subjects with the OATP1B1 variants which,
in turn, leads to increased systemic exposure of the statin and
adverse events in the off target muscle tissue. The fact that 7 is
actively taken up in the liver by both OATP1B1 and OATP1B3
may mitigate the risk of skin and eye adverse events in patients
with the OATP1B1 variants. Nonetheless, the effect of
OATP1B1 polymorphisms and tolerability of compound 7 will
be followed up in the future with in vitro uptake tools and in
clinical studies.
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It was shown in Figure 5 that the liver-targeted inhibitor 7 was
as efficacious in the oGTT eDIO mouse model as the systemi-
cally distributed 1. In an attempt to understand the mechanism
by which hepatic SCD1 inhibition results in an improved oral
glucose tolerance in eDIO mice, a hyperinsulinemicꢀeuglycemic
glucose clamp experiment in overfed rats was carried out (see
Supporting Information Results 1). As shown in Supporting
Information Figure 1a, under hyperinsulinemicꢀeuglycemic
conditions, compound 7 significantly increased the glucose
infusion rate (GIR) required to maintain euglycemia, indicative
of improvement in whole body insulin sensitivity. This was
accompanied by a nonsignificant suppression in hepatic glucose
production (Supporting Information Figure 1b). These results
indicate that a liver-targeted small molecule SCD inhibitor (7)
improves whole body insulin sensitivity, consistent with the
literature report with a liver-targeted ASO against SCD1.6 In a
chronic eDIO mouse model, there were differences in efficacy
and tolerability noted between the systemically distributed SCD
inhibitor 1 and the liver-targeted 7. While efficacy on liver
triglyceride and liver steatosis was similar for 1 and 7, the effect
on body weight reduction was significantly different (20% vs 5%,
respectively, Figure 6). It is clear that the antiobesity effect with
liver-selective SCD inhibitors is very modest compared to that
obtained with systemically distributed inhibitors. In an attempt
to understand the obesity efficacy differences, the liver-targeted
SCD1 inhibitor 6 and the systemically distributed SCD1 inhi-
bitor 1 were compared head-to-head in the eDIO mouse model
and the following parameters were measured: (i) body weight
and (ii) inhibition of SCD activity (DI) in fat vs liver tissues. As
previously observed in Figure 6, the systemically distributed SCD
inhibitor 1 had a significant body weight loss (∼15%) vs vehicle-
treated mice whereas the liver-targeted SCD inhibitor 6 did not
promote significant body weight loss in eDIO mice (see Sup-
porting Information Results 4, Figure 4a). Both 1 and 6 had
similar inhibition of liver desaturation index (DI = [C18:1]/
[C18:0], direct measure of chronic SCD activity) at termination
of the 2-week study, indicating that similar levels of liver SCD
inhibition had been obtained (Supporting Information Figure
4b). Conversely, while there was significant inhibition of eWAT
DI for compound 1, there was no inhibition observed in eWAT
for the liver-targeted 6 (Supporting Information Figure 4c),
which confirms that liver-targeting prevents exposure of SCD
compounds to other tissues. This data suggests that robust
obesity efficacy likely requires inhibition of adipose SCD and is
in agreement with previously reported data.4
tissue as assessed by monitoring the DI in the Harderian gland
following chronic dosing in the eDIO mouse model (Figure 8e).
The level of DI reduction in the liver and Harderian gland in this
chronic study correlated with the high drug exposures of 7 in the
liver and the lower drug exposure in skin and eye. This indicated
that the liver-targeting SCD inhibitor 7 was successful at provid-
ing good SCD inhibition in liver while sparing inhibition in
tissues associated with adverse events.
’ CONCLUSION
In this report, a liver-targeting strategy was successfully
implemented which resulted in the discovery of 7, a potent,
liver-targeted SCD inhibitor with excellent in vitro and preclini-
cal in vivo efficacy. This approach utilized high throughput
cellular assays, which predicted the extent of liver-specific OATP
substrate affinity. Following confirmation of liver-targeting by
tissue distribution studies, the lower throughput OATP assays
could be used to confirm human OATP substrate affinity. While
liver-targeting OATP approaches have been used to improve the
therapeutic window of pharmaceutical agents such as statins, to
the best of our knowledge, this is the first report of how to
intentionally design a liver-targeting agent via OATP substrate
affinity. Liver-targeting allowed compound 7 to demonstrate
maximal liver SCD inhibition while sparing inhibition in the skin
and eye tissues associated with adverse events. This ultimately
resulted in an improved safety profile for the liver-targeted SCD
inhibitor 7 relative to the previously reported systemically
distributed inhibitor 1. Future studies involve the clinical evalua-
tion of 7 for the treatment of diabetes and dyslipidemia.
’ EXPERIMENTAL SECTION
General. All commercial chemicals were used without further
treatment. Anhydrous solvents were purchased from Sigma-Aldrich
(Milwaukee, WI), Acros Organics (Fisher Scientific, Nepeau, ON,
Canada), or American Chemicals Ltd. (Montreal, QC, Canada). TLC
analyses were performed on Merck 60 F254 silica gel glass-backed plates,
with spots detected visually under ultraviolet irradiation (254 nm).
Purification of the crude reaction mixtures was performed as indicated
using chromatography on silica gel using a CombiFlash apparatus
(Teledyne Isco, Lincoln, NE) equipped with columns of appropriate
size (Teledyne Isco, Lincoln, NE, or Silicycle, Quꢀebec City, QC,
Canada). Proton (1H NMR) magnetic resonance spectra were recorded
on a Bruker AM instrument operating at 400 or 500 MHz. All spectra
were recorded using residual solvent (CHCl3, acetone or DMSO) as
internal standard. Signal multiplicity was designated according to the
following abbreviations: s = singlet, d = doublet, dd = doublet of
doublets, t = triplet, m = multiplet, br s = broad singlet, br t = broad
triplet. Carbon (13C NMR) nuclear magnetic resonance spectra were
recorded on a Bruker AM instrument operating at 126 MHz. All spectra
were recorded using residual solvent (CHCl3 or DMSO) as internal
standard. High-resolution mass spectrometry (HRMS) was performed
in the ESI mode using an Agilent Technologies MSD TOF mass
spectrometer coupled to an Agilent Technologies 1200 series LC. All
melting points were determined on a Buchi 510 melting point apparatus
in open capillary tubes and are uncorrected. Chemical purity of
compounds was assessed by reversed-phase HPLC and all compounds
were determined to have g95% purity. Reversed-phase HPLC
purity determinations were performed at three different wavelengths
(λmax, 220 and 254 nm) on an Agilent Technologies 1100 series system
equipped with a Zorbax RxC18 column (150 mm ꢁ 4.6 mm, 5 μ) using
an acetonitrile/water gradient containing 0.1% formic acid. Reactions
With regards to tolerability and adverse event profile in the
chronic eDIO mouse studies, there were important differences
between the systemically distributed SCD inhibitor 1 and the
liver-targeted inhibitor 7. As reported in parts a and b of Figure 7,
compound 1 induced eye adverse events, followed a few days
later by skin adverse events. At the maximally efficacious dose of
7 (20 mg/kg bid), there was observed a liver-targeted tissue
distribution profile which correlated with liver TG efficacy
(Supporting Information Figure 3a), modest body weight loss
and no significant eye or skin adverse events during the course of
the 4-week study (Figures 7c,d). At a 3-fold higher dose of 7
(60 mg/kg bid), a clean adverse event profile was also observed,
which indicates that a therapeutic window could be obtained by
targeting the SCD inhibitor to the liver, consistent with reported
SCD1 gene intervention studies.4,5 The greatly improved AE
profile observed with the liver-targeted SCD inhibitor 7 was
consistent with no significant inhibition of SCD in the off-target
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were carried out with continuous stirring under a positive pressure of dry
nitrogen except where noted. The synthesis of 1 is described in
reference.9
suspension was cooled with an iceꢀwater bath, and the title compound
(4) was collected by filtration as a white solid (4.77 g, 85% yield). The
material was dried under high vacuum by heating at 50 °C for 1ꢀ2 h; mp
226ꢀ228 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.60ꢀ7.62 (m, 2H),
7.37 (d, 1H, J = 9.0 Hz), 7.08 (t, 1H, J = 7.5 Hz), 4.86ꢀ4.95 (m, 1H),
3.56ꢀ3.64 (m, 4H), 2.00ꢀ2.15 (m, 2H), 1.75ꢀ1.89 (m, 2H). 13C NMR
(126 MHz, DMSO-d6): δ 173.3, 155.1, 149.7 (broad peak), 143.3,
134.7, 127.5 (q, JCF = 5.1 Hz), 124.3 (q, JCF = 272 Hz), 120.9, 118.2 (q,
4-[2-(Trifluoromethyl)phenoxy]piperidine Hydrochloride (8). To a
solution of tert-butyl 4-hydroxypiperidine-1-carboxylate (25 g, 124
mmol), 2-hydroxy-benzotrifluoride (22 g, 136 mmol) and triphenylpho-
sphine (39 g, 149 mmol) in THF (250 mL) was added diethyl
azodicarboxylate (23.5 mL, 149 mmol) dropwise at 0 °C. The mixture
was then warmed to room temperature and stirred for 14 h. The mixture
was concentrated and diluted with Et2O, washed with 1 N NaOH and
water, and then dried over Na2SO4. The mixture was concentrated and
diluted with Et2O/hexanes (35:65). The solid was filtered, and the
filtrate was concentrated. The residue was purified by column chroma-
tography on silica gel (eluting with 35% Et2O/hexanes) to give tert-butyl
4-[2-(trifluoromethyl)phenoxy]-piperidine-1-carboxylate as a solid. Tri-
fluoroacetic acid (26.3 mL, 342 mmol) was added to a solution of tert-
butyl 4-[2-(trifluoromethyl)phenoxy]piperidine-1-carboxylate (29.5 g,
85 mmol) in CH2Cl2 (171 mL). The mixture was stirred at room
temperature for 16 h. The solvent was evaporated. The residue was
diluted with EtOAc (200 mL), washed with 2 N NaOH (3 ꢁ 100 mL),
brine, dried over Na2SO4, and evaporated to give 4-(2-trifluoromethyl)-
phenoxy)piperidine as an oil (21.3 g, 70% yield). The corresponding
hydrochloride (title compound) can be prepared by precipitation of an
ethereal solution (0.5ꢀ1M) of the free amine with 1 equiv of 4N HCl in
dioxane. 1H NMR (400 MHz, DMSO-d6) δ 9.20 (br, 2H), 7.58ꢀ7.61
(m, 2H), 7.32ꢀ7.34 (t, 1H), 7.06ꢀ7.08 (t, 1H), 4.87ꢀ4.90 (m, 1H),
3.06ꢀ3.08 (m, 4H), 1.92ꢀ2.14 (m, 2H), 1.81ꢀ1.91 (m, 2H); (m/z):
(M + H)+ 246.
5-{4-[2-(Trifluoromethyl)phenoxylpiperidin-l-y1}-1,3,4-thiadiazol-
2-amine (10). To a solution of 4-[2-(trifluoromethyl)phenoxylpiperidine
hydrochloride (8) (5.5 g, 19.5 mmol) in DMF (50 mL) was added
5-bromo-1,3,4-thiadiazol-2-amine(9) (3.3 g, 18.3mmol) and K2CO3 (9.1
g, 65.8 mmol). The reaction was heated at 80 °C with stirring overnight.
After cooling, the salt was removed by filtration and the filtrate was
evaporated in vacuo. The residue was triturated with EtOAc to afford the
title compound (4.4 g, 70% yield). 1H NMR (400 MHz, DMSO-d6): δ
7.57ꢀ7.60 (m, 2H), 7.29ꢀ7.35 (m, 1H), 7.03ꢀ7.05 (m, 1H), 6.46 (s,
2H), 4.84 (br s, 1H), 3.22ꢀ3.30 (m, 4H), 1.91ꢀ2.01 (m, 2H), 1.68ꢀ1.78
(m, 2H); (m/z): (M + H)+ 345.
5-{4-[2-(Trifluoromethyl)phenoxy]piperidin-1-yl}-1,3,4-thiadiazole-2-
carbonitrile (11). To a suspension of 5-{4-[2-(trifluoromethyl)phenoxy]-
piperidin-1-yl}-1,3,4-thiadiazol-2-amine (10) (10.0 g, 29.0 mmol) in
CH3CN (150 mL) was added CuCN (5.3 g, 59.2 mmol) and t-BuONO
(90%) (8 mL, 60.0 mmol) at room temperature. The reaction mixture was
heated at 50ꢀ60 °C for 2 h until TLC indicated disappearance of the
starting material. The reaction mixture was diluted with EtOAc and mixed
with silica gel. The resulting suspension was filtered through a pad of silica
gel by eluting with 100% EtOAc. The filtrate was concentrated in vacuo to
afford the crude product, which was purified by column chromatography on
silica gel (eluting with 2:1 hexanes/EtOAc) to afford the title compound
(4.2 g, 41% yield) as a yellow oil. 1H NMR (400 MHz, CDC13): δ 7.61 (d,
1H), 7.50 (t, 1H), 6.93ꢀ7.06 (m, 2H), 4.86 (br s, 1H), 3.77ꢀ3.83 (m, 4H),
2.01ꢀ2.20 (m, 4H); (m/z): (M + H)+ 355.
J
CF = 29.4 Hz), 115.1, 71.6, 47.1 (2C), 29.4 (2C). HRMS (ESI) (m/z):
(M + H)+ calcd for C15H15F3N7OS, 398.1005; found, 398.0997.
5-Bromo-1,3,4-thiadiazole-2-carbonitrile (12). To a suspension of
ethyl 5-amino-1,3,4-thiadiazole-2-carboxylate (10 g, 58 mmol) in
CH3CN (180 mL) was added CuBr2 (25.7 g, 115 mmol). The mixture
turned dark green and was further stirred for 15 min at room tempera-
ture. t-BuONO, 90% (13.8 mL, 115 mmol) was added dropwise over
15ꢀ20 min. The mixture became slightly warm, and gas was evolved
after 5 min and then throughout the addition. After completion of the
addition and gas evolution subsided, the mixture was heated at 60 °C for
30 min. Solvent was then evaporated in vacuo. Water and EtOAc were
added, and the mixture was agitated in the flask until the dark-green
color disappeared. The organic phase became light brown, and the
aqueous was green with insoluble material. The whole mixture was
filtered through Celite and washed with EtOAc. The EtOAc layer was
separated, washed with dilute brine solution, dried (Na2SO4), and
concentrated to give ethyl 5-bromo-1,3,4-thiadiazole-2-carboxylate
(13.5 g, 98% yield). 1H NMR (400 MHz, acetone-d6): δ 4.52
(q, 2H), 1.43 (t, 3H). To a solution of ethyl 5-bromo-1,3,4-thiadia-
zole-2-carboxylate (13.5 g, 56.9 mmol) in THF (50 mL) at room
temperature was added NH4OH (28 wt %, 39.6 mL, 164 mmol). The
mixture was stirred at room temperature overnight, and a precipitate
appeared in the aqueous layer. Volatile materials were removed in vacuo.
The mixture was diluted with water and the precipitate was collected,
washed with water, and dried under vacuum to afford 5-bromo-1,3,4-
thiadiazole-2-carboxamide (9.3 g, 79% yield). 1H NMR (400 MHz,
acetone-d6): δ 7.99 (s, 1H), 7.55 (s, 1H). To a solution of 5-bromo-
1,3,4-thiadiazole-2-carboxamide (11 g, 53 mmol) and Et3N (17.1 mL,
122 mmol) in THF (106 mL) at 0 °C was added TFAA (17 mL,
58 mmol). The mixture was then warmed to room temperature and
stirred for 30 min. Solvent was evaporated in vacuo. The residue was
diluted with water. The precipitate was collected, washed with water, and
dried to give the title compound (7 g, 70% yield). 13C NMR (75 MHz,
CDC13): δ 77.3, 109.0, 141.7.
4-(2-Bromo-5-fluorophenoxy)piperidine (13). To a solution of tert-
butyl 4-hydroxypiperidine-l-carboxylate (50.6 g, 251 mmol) and di-tert-
butyl azodicarboxylate (71.0 g, 308 mmol) in THF (350 mL) was added
2-bromo-5-fluorophenol (36 mL, 324 mmol). The mixture was cooled
to ꢀ78 °C, and a solution of triphenylphosphine (81.5 g, 311 mmol) in
CH2C12 (130 mL) was added via a cannula. The reaction was then
warmed to room temperature and stirred overnight. The solvents were
removed under vacuum, and the crude oil was dissolved in EtOH
(200 mL). The solution was cooled to ꢀ78 °C and treated with 4 M
HC1 in 1,4-dioxane (450 mL). The reaction was warmed to room
temperature and stirred 24 h. After this time, the solvents were removed
under vacuum. The salts were neutralized with 1 N NaOH (750 mL) and
extracted with a mixture of Et2O:hexanes (1:1) several times. The
organic layers were combined and concentrated to dryness. The crude
material was treated with heptane (1 L), and a white precipitate was
filtered and discarded. The heptane layer was diluted with Et2O and
treated with 4 M HC1 in 1,4-dioxane (100 mL). The resulting
precipitate was collected by filtration and washed three times with
Et2O:hexanes (1:1). The salts were again neutralized with 1 N NaOH
(500 mL) and extracted with a mixture of Et2O:hexanes (1:1) several
times. The organic layers were combined, washed with brine, dried
(MgSO4), filtered, and concentrated. The crude material was dissolved
1-[5-(2H-Tetrazol-5-yl)-1,3,4-thiadiazol-2-yl]-4-[2-(trifluoromethyl)-
phenoxy]-piperidine (4). A suspension of 5-{4-[2-(trifluoromethyl)-
phenoxy]piperidin-1-yl}-1,3,4-thiadiazole-2-carbonitrile (11) (4.99 g,
14.1 mmol), NaN3 (4.65 g, 71.5 mmol), and pyridinium hydrochloride
(3.43 g, 29.7 mmol) in NMP (50 mL) was heated at 130 °C for 18 h. The
reaction mixture was cooled to room temperature and poured into
aqueous 0.5 N HC1, extracted with EtOAc, and washed three times with
aqueous 0.5 N HC1 and with aqueous brine solution. The organic layer
was dried (Na2SO4) and filtered. Evaporation of the solvent was
followed by trituration in a mixture of MeOH/Et2O/heptane (1:1:6)
(v/v) with stirring for 4 h at room temperature. After this time, the
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in heptane (2 L), washed four times with 1 N NaOH (250 mL) and
brine, and dried (MgSO4). The organic layer was filtered and concen-
trated to dryness to afford the title product as a colorless oil (54 g, 78%
fluorophenoxy)piperidin-1-yl]-1,3,4-thiadiazol-2-yl}-1H-tetrazol-1-yl)-
acetate. H NMR (500 MHz, DMSO-d6): δ 7.64 (dd, 1H), 7.29 (dd,
1H), 6.82 (td, 1H), 5.79 (s, 2H), 4.94ꢀ4.89 (m, 1H), 4.19 (q, 2H),
3.86ꢀ3.80 (m, 2H), 3.75ꢀ3.69 (m, 2H), 2.12ꢀ2.06 (m, 2H), 1.901.84
(m, 2H), 1.20 (t, 3H).
1
1
yield). H NMR (500 MHz, acetone-d6): δ 7.58 (dd, 1H), 7.00 (dd,
1H), 6.70 (td, 1H), 4.64ꢀ4.58 (m, 1H), 3.12ꢀ3.06 (m, 2H), 2.73ꢀ2.66
To
a solution of ethyl (5-{5-[4-(2-bromo-5-fluorophenoxy)-
(m, 2 H), 2.02ꢀ1.94 (m, 2H), 1.69ꢀ1.60 (m, 2H).
piperidin-1-yl]-1,3,4-thiadiazol-2-yl}-2H-tetrazol-2-yl)acetate (2.57 g,
5.02 mmol) in a mixture of THF/MeOH (30 mL) (2:1) (v/v) was
added 1 N NaOH (10 mL) at room temperature. After 5 min, the
reaction mixture was poured into 1 N HCl, extracted with EtOAc,
washed with brine, dried (Na2SO4), filtered, and concentrated. The
residue was triturated with a mixture of Et2O/hexanes to give the desired
material (6) as a white solid (2.23 g, 92% yield); mp: 243ꢀ245 °C
(decomp). 1H NMR (400 MHz, DMSO-d6): δ 13.89 (br s, 1H), 7.65
(dd, 1H, J = 8.8, 6.3 Hz), 7.30 (dd, 1H, J = 9.0, 3.0 Hz), 6.83 (td, 1H, J =
9.0, 3.0 Hz), 5.84 (s, 2H), 4.95ꢀ4.88 (m, 1H), 3.86ꢀ3.78 (m, 2H),
3.74ꢀ3.66 (m, 2H), 2.15ꢀ2.07 (m, 2H), 1.92ꢀ1.84 (m, 2H). 13C NMR
(126 MHz, DMSO-d6): δ 172.8, 167.6, 162.8 (d, JCF = 244 Hz), 158.3,
5-[4-(2-Bromo-5-fluorophenoxy)piperidin-1-yl]-1,3,4-thiadiazole-2-
carbonitrile (14). To a solution of 4-(2-bromo-5-fluorophenoxy)-
piperidine (13) (14.4 g, 52.7 mmol) in 1,4-dioxane (80 mL) was added
N,N-diisopropylethylamine (20 mL, 115 mmol), followed by 5-bromo-
1,3,4-thiadiazole-2-carbonitrile (12) (10.0 g, 52.7 mmol). The mixture
was stirred 1 h at room temperature. The reaction mixture was then
poured into saturated aqueous NH4C1, extracted with EtOAc, washed
with brine, dried (Na2SO4), filtered, and concentrated under reduced
pressure. The crude material was purified by column chromatography on
silica gel (eluting with a gradient from 10 to 40% EtOAc/hexanes) to
afford the desired product as a colorless oil (15.6 g, 77% yield). 1H NMR
(500 MHz, acetone-d6): δ 7.63 (dd, 1H), 7.14 (dd, 1H), 6.78 (td, 1H),
5.04ꢀ4.99 (m, 1H), 4.00ꢀ3.95 (m, 2H), 3.89ꢀ3.84 (m, 2H), 2.27ꢀ
2.21 (m, 2H), 2.11ꢀ2.05 (m, 2H).
154.6 (d, JCF = 10.7 Hz), 144.8, 134.3 (d, JCF = 9.9 Hz), 109.6 (d, JCF
=
22.6 Hz), 107.5 (d, JCF = 4.3 Hz), 104.2 (d, JCF = 26.8 Hz), 72.8, 54.5,
47.2 (2C), 29.4 (2C); the regioconfiguration of the acetic acid moiety
was assigned by a 15N gHMBC experiment (JHN = 5 Hz) on the sodium
salt of 5 with observation of three long-range couplings between the
methylene protons at δ 5.00 ppm and tetrazole nitrogens at δ 393.8,
314.9, and 303.0 ppm (15N-benzamide external ref δ 110 ppm) and was
confirmed by X-ray crystallography. HRMS (ESI) (m/z): (M + H)+
calcd for C16H16BrFN7O3S, 486.0178; found, 486.0182.
Ethyl 3-[4-(2-bromo-5-fluorophenoxy)piperidin-1-yl]isoxazole-5-
carboxylate (17). To a suspension of 4-(2-bromo-5-fluorophenoxy)-
piperidine (13) (20 g, 73 mmol), hydroxycarbonimidic dibromide (16)
(13 g, 64 mmol), and ethyl propiolate (15) (38 g, 422 mmol) in EtOAc/
H2O (390 mL/65 mL) was added KHCO3 (19.4 g, 194 mmol). The
mixture was stirred for 12 h at room temperature. The organic layer was
then separated, dried over anhydrous Na2SO4, and concentrated. Chro-
matography over silica gel and elution with hexanes/EtOAc (1:1) and
swishing in hexanes/Et2O (3:2) to give the title compound as a white
powder (5.0 g, 19% yield). 1H NMR (400 MHz, acetone-d6): δ 7.62 (dd,
1H), 7.11 (dd, 1H), 6.97 (s, 1H), 6.76 (td, 1H), 4.91ꢀ4.86 (m, 1H),
4.39 (q, 2H), 3.71ꢀ3.63 (m, 2H), 3.49ꢀ3.41 (m, 2H), 2.17ꢀ2.09
(m, 2H), 1.99ꢀ1.88 (m, 2H), 1.38 (t, 3H).
4-(2-Bromo-5-fluorophenoxy)-1-[5-(2>H-tetrazol-5-yl)-1,3,4-thia-
diazol-2-yl]piperidine (5). A suspension of 5-[4-(2-bromo-5-fluorophe-
noxy)piperidin-l-y1]-1,3,4-thiadiazole-2-carbonitrile (14) (15.6 g, 40.7
mmol), NaN3 (13.2 g, 204 mmol), and pyridinium hydrochloride
(9.47 g, 82.0 mmol) in NMP (70 mL) was heated at 130 °C for 4 h.
The reaction mixture was cooled to room temperature and poured into
aqueous 0.5 N HC1, extracted with EtOAc, and washed three times with
aqueous 0.5 N HC1 and with aqueous brine solution. The organic layer
was dried (Na2SO4) and filtered. Evaporation of the solvent was
followed by trituration in a mixture of MeOH/Et2O/heptane (1:1:6)
(v/v). After filtration of the solid, the material was triturated again in this
solvent system to finally afford the desired product (5) as a white solid
(15.3 g, 88% yield); mp: 213-215 °C. 1H NMR (400 MHz, acetone-d6):
δ 7.64 (dd, 1H, J = 9.0, 6.5 Hz), 7.16 (dd, 1H, J = 9.0, 3.0 Hz), 6.79 (td,
1H, J = 9.0, 3.0 Hz), 5.04ꢀ5.00 (m, 1H), 4.02ꢀ3.94 (m, 2H), 3.89ꢀ3.81
(m, 2H), 2.29ꢀ2.20 (m, 2H), 2.12ꢀ2.03 (m, 2H). 13C NMR (126
MHz, DMSO-d6): δ 173.3, 162.8 (d, JCF = 243 Hz), 154.6 (d, JCF = 10.9
Hz), 149.7 (broad peak), 143.3, 134.2 (d, JCF = 10.0 Hz), 109.6 (d, JCF
=
22.7 Hz), 107.5 (d, JCF = 3.0 Hz), 104.2 (d, JCF = 26.4 Hz), 72.8, 47.2
(2C), 29.4 (2C). HRMS (ESI) (m/z): (M + H)+ calcd for
C14H14BrFN7OS, 428.0122; found, 428.0131.
3-[4-(2-Bromo-5-fluorophenoxy)piperidin-1-yl]isoxazole-5-carbox-
amide (18). To a suspension of ethyl 3-[4-(2-bromo-5-fluorophenoxy)-
piperidin-1-yl]isoxazole-5-carboxylate (17) (2.0 g, 4.84 mmol) in
MeOH (10 mL) and THF (10 mL) was added ammonium hydroxide
(28 wt %, 20 mL, 144 mmol) at room temperature. The mixture was
stirred at room temperature overnight stirring. A precipitate appeared
after about 15 min and became more abundant after overnight. Volatile
materials were then removed in vacuo. The residue was diluted with
water and extracted with EtOAc. The EtOAc extract was washed with
diluted brine, dried (Na2SO4), and concentrated. The residue was
triturated in Et2O/hexanes (1:1) to give the title compound as a white
powder (1.7 g, 91% yield). 1H NMR (400 MHz, acetone-d6): δ 7.62 (dd,
1H), 7.52 (s, 1H), 7.11 (m, 2H), 6.80ꢀ6.71 (m, 2H), 4.91ꢀ4.85 (m,
1H), 3.69ꢀ3.61 (m, 2H), 3.46ꢀ3.38 (m, 2H), 2.17ꢀ2.09 (m, 2H),
1.99ꢀ1.88 (m, 2H).
(5-{5-[4-(2-Bromo-5-fluorophenoxy)piperidin-1-yl]-1,3,4-thiadia-
zol-2-yl}-2H-tetrazol-2-yl)acetic Acid (6). A solution of 4-(2-bromo-5-
fluorophenoxy)-1-[5-(2H-tetrazol-5-yl)-1,3,4-thiadiazol-2-yl]piperidine
(5) (4.54 g, 10.66 mmol) in DMF (20 mL) was treated with NaH (60%
in oil) (512 mg, 12.80 mmol) at ꢀ78 °C. The mixture was warmed to
0 °C for 10ꢀ15 min, cooled again to ꢀ78 °C, and ethyl bromoacetate
(1.6 mL, 14.37 mmol) was added dropwise. The final reaction mixture
was warmed and stirred at room temperature overnight. The reaction
mixture was poured into aqueous 1 N HCl and extracted with EtOAc.
The organic layers was washed with 1 N HCl and brine, dried (Na2SO4),
and filtered. Solvents were removed in vacuo to afford the crude product,
which was purified by column chromatography on silica gel (eluting with
a gradient from 10 to 50% EtOAc/hexanes). The two isomers were
triturated with a mixture of Et2O/heptane. The more polar isomer
(Rf = 0.3 (50% EtOAc/hexanes)), obtained as an off-white solid (2.60 g,
48% yield), was the desired isomer ethyl (5-{5-[4-(2-bromo-5-fluor-
ophenoxy)piperidin-1-yl]-1,3,4-thiadiazol-2-yl}-2H-tetrazol-2-yl)acetate.
1H NMR (500 MHz, DMSO-d6): δ 7.64 (dd, 1H), 7.29 (dd, 1H),
6.82 (td, 1H), 5.98 (s, 2H), 4.93ꢀ4.89 (m, 1H), 4.24 (q, 2H), 3.84ꢀ3.77
(m, 2H), 3.72ꢀ3.66 (m, 2H), 2.13ꢀ2.06 (m, 2H), 1.901.83 (m, 2H),
1.24 (t, 3H).
3-[4-(2-Bromo-5-fluorophenoxy)piperidin-1-yl]isoxazole-5-carbo-
nitrile (19). Trifluoroacetic anhydride (0.8 mL, 5.7 mmol) was added to
a suspension of 3-[4-(2-bromo-5-fluorophenoxy)piperidin-1-yl]isoxazole-
5-carboxamide (18) (1.6 g, 4.2 mmol) and Et3N (1.5 mL, 10.8 mmol) in
CH2Cl2 (20 mL) at iceꢀwater bath temperature. After addition was
completed, the cooling bath was removed and the mixture was stirred
at room temperature for 2 h. The mixture was quenched with water
(10ꢀ15 mL), followed by saturated NaHCO3 (15ꢀ20 mL), and
extracted with CH2Cl2 (2 ꢁ 30 mL). The combined CH2Cl2 extracts
The less polar isomer (Rf = 0.5 (50% EtOAc/hexanes)), obtained as a
white solid (2.55 g, 47% yield), was ethyl (5-{5-[4-(2-bromo-5-
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were washed with diluted brine (40 mL), dried (Na2SO4), and con-
centrated. Chromatography over silica gel and elution with hexanes/
EtOAc (4:1) gave the title compound as a colorless gum (1.4 g, 92%
yield). 1H NMR (400 MHz, acetone-d6): δ 7.62 (dd, 1H), 7.33 (s, 1H),
7.10 (dd, 1H), 6.76 (td, 1H), 4.93ꢀ4.86 (m, 1H), 3.71ꢀ3.63 (m, 2H),
3.52ꢀ3.44 (m, 2H), 2.18ꢀ2.09 (m, 2H), 1.99ꢀ1.90 (m, 2H).
regioconfiguration of the acetic acid moiety of the nondesired isomer of
7 was assigned by a similar 15N gHMBC experiment with observa-
tion of two long-range couplings between methylene protons at δ
5.66 ppm and tetrazole nitrogens at δ 374.8 and 228.1 ppm and was
further corroborated by observation of strong NOE between the
methylene protons and isoxazole proton in a 1D ROESY experiment
(mix = 500 ms). HRMS (ESI) (m/z): (M + H)+ calcd for C17H17BrFN6O4,
469.0455; found, 469.0455.
Rat SCD Hepatocyte Assay. Male SpragueꢀDawley rats were fed
a high carbohydrate diet (Ren’s Feed & Supplies Ltd. no. 7576) for 2
days to obtain SCD1-induced liver (175ꢀ200 g). Rat hepatocytes were
isolated by collagenase perfusion. Cells were plated, 100 μL of 4 ꢁ 106
cells/mL cells per well on a 96-well plate (NUNC). Then 1.2 μL of
compound was added to each well and incubated for 15 min at 37 °C/5%
CO2. Then 20 μL of 14C-stearic acid (final concentration 0.5 μCi/ml)
was added and incubated for 1 h, shaking in an Eppendorf shaker set at
400 rpm with 37 °C/5% CO2. Hepatocytes were spun down and washed
three times with PBS buffer to remove the tracer in medium. Complete
hydrolysis was done by incubation at 75 °C for 1 h with 2N NaOH. Cell
lysate was acidified with phosphoric acid and then the lipids were
extracted with acetonitrile. The quantification of the resulting radiola-
beled oleic acid and stearic acid in the final organic phase was done
by HPLC, using a C18-reverse phase column (Zorbax extended C18
(4.6 mm ꢁ 75 mm), eluting with a 3% water (0.1% formic acid)/
acetonitrile to 100%. Acetonitrile gradient (0.1% formic acid) at a flow
rate of 2 mL/min in 4 min, detection via a Packard Flow scintillation
analyzer.
Tissue Distribution Analysis Method. Compound 7 was
administered orally to fed C57BL6 mice (n = 2), male SpragueꢀDawley
rats (n = 3), female rhesus monkeys (n = 2), or fasted male beagle dogs
(n = 2) using 0.5% methylcellulose as vehicle and 6 h post dose, the
animals were sacrificed, tissues were harvested, and the concentration of
7 in tissues was analyzed following the procedure outlined below.
For Mice and Rats. Requested tissues (less than 0.2 g) were weighed
and put in a blue 96-well plate for tissues (strips of 12, attached, 1.1 mL
microtubes in microracks from National Scientific Supply Co.) and
frozen at ꢀ78 °C. Tissues were thawed and diluted with 3.5 volumes of a
2.5:1 MeCN:H2O mixture containing internal standard. To this mixture
was added 125ꢀ150 mg of silicon carbide chips and a small (4 mm)
stainless steel bead (bead must be added to the 96-well plate prior to the
addition of the tissue). The 96-well plate was capped (Marsh bio plug
caps, polyethylene for microtubes, strips of 12) and shaken at
1500ꢀ1700 strokes per min using a GenoGrinder for 20 min. The plate
was then centrifuged for 15 min and the supernatant was transferred to a
mass spec vial and the concentration of 7 was determined by LC-MS.
Quantification involved the preparation of a standard curve using blank
plasma instead of tissue (i.e., 10 μL of blank plasma +10 μL of MeCN
(with internal standard) containing different concentrations of 7 +
10 μL of water + 15 μL of MeCN (with internal standard). Because of
the small size of mouse Harderian glands (i.e., ∼10 mg/gland), a similar
procedure was used, except 2 volumes of water and 4 volumes of
acetonitrile were added to the well containing the Harderian gland (final
concentration was adjusted based on increased initial dilution). For skin,
tissue homogenization was not performed with the GenoGrinder.
Instead, to an 8 mL vial containing a weighed piece of shaved skin was
added 3.5 volumes of a 2.5:1 MeCN:H2O mixture containing internal
standard. The skin was then homogenized using a tissue tearor from
Biospec Products Inc., followed by centrifugation and analysis as above.
For Dogs and Rhesus Monkeys. Liver and skeletal muscle samples
(approx 1 g) were processed by hand using a tissue tearor from Biospec
Products Inc. Four spatially separated pieces of liver lobe (1 g) were
obtained during necropsy and processed separately. The results repre-
sent the average of the four samples, and all four samples were found to be
within (2-fold of the average. All skin and eyelid samples (approx 1 g)
4-(2-Bromo-5-fluorophenoxy)-1-[5-(1H-tetrazol-5-yl)isoxazol-3-yl]-
piperidine (20). A mixture of 3-[4-(2-bromo-5-fluorophenoxy)piperidin-1-
yl]isoxazole-5-carbonitrile (19) (1.4 g, 3.82 mmol), pyridine hydrochloride
(0.9 g, 7.79 mmol), and sodium azide (1.3 g, 20.00 mmol) in NMP (12 mL)
was stirred and heated at 130 °C bath for 2 h. After cooling, the mixture was
diluted with water and acidified with 1 N HCl to precipitate the tetrazole.
The whole mixture was then extracted with EtOAc. The EtOAc extract was
washed three times with water, dried (Na2SO4), and concentrated. The
residue was triturated with Et2O to give the title compound as a light-brown
powder (1.4 g, 89% yield). 1H NMR (400 MHz, acetone-d6): δ 7.63 (dd,
1H), 7.12 (m, 2H), 6.76 (td, 1H), 4.93ꢀ4.88 (m, 1H), 3.77ꢀ3.69(m, 2H),
3.55ꢀ3.47 (m, 2H), 2.21ꢀ2.13 (m, 2H), 2.02ꢀ1.92 (m, 2H).
Ethyl (5-{3-[4-(2-bromo-5-fluorophenoxy)piperidin-1-yl]isoxazol-
5-yl}-2H-tetrazol-2-yl)acetate (21). A mixture of 4-(2-bromo-5-
fluorophenoxy)-1-[5-(1H-tetrazol-5-yl)isoxazol-3-yl]piperidine (20)
(1.2 g, 2.93 mmol), ethyl bromoacetate (0.45 mL, 4.04 mmol), and
Et3N (0.75 mL, 5.38 mmol) in THF (12 mL) was refluxed for 2 h. After
cooling, the mixture was diluted with water and extracted with EtOAc.
The EtOAc extract was washed with water, dried (Na2SO4), and
concentrated. Thecrudematerialwaspurifiedbycolumnchromatography
on silica gel (Combi-Flash apparatus, 40 g SiO2 column, eluting with
25ꢀ40% EtOAc in hexanes for 20 min, 35 mL/min, 18 mL/fraction) to
give the title compound as a colorless gum (1.1 g, 76% yield), also
containing about 20%ofthepositionalisomer ethyl(5-{3-[4-(2-bromo-5-
1
fluorophenoxy)piperidin-1-yl]isoxazol-5-yl}-1H-tetrazol-1-yl)acetate. H
NMR (400 MHz, acetone-d6): δ 7.63 (dd, 1H), 7.14ꢀ7.09 (m, 1H), 7.03
(s, 1H), 6.76 (td, 1H), 5.84 (s, 2H), 4.93ꢀ4.87 (m, 1H), 4.34ꢀ4.26 (m,
2H), 3.77ꢀ3.68 (m, 2H), 3.55ꢀ3.46 (m, 2H), 2.21ꢀ2.12 (m, 2H),
2.03ꢀ1.91 (m, 2H), 1.30 (t, 3H).
(5-{3-[4-(2-Bromo-5-fluorophenoxy)piperidin-1-yl]isoxazol-5-yl}-
2H-tetrazol-2-yl)acetic acid (7). A mixture of ethyl (5-{3-[4-(2-bromo-
5-fluorophenoxy)piperidin-1-yl]isoxazol-5-yl}-2H-tetrazol-2-yl)acetate
(21), containing about 20% of the positional isomer ethyl (5-{3-[4-(2-
bromo-5-fluorophenoxy)piperidin-1-yl]isoxazol-5-yl}-1H-tetrazol-1-yl)-
acetate (0.175 g, 0.354 mmol) in EtOH (2 mL) and 1 M NaOH solution,
(1.77 mL, 1.77 mmol) was stirred at room temperature for 3 h. Volatile
solvents were removed in vacuo. The residue was diluted with water,
acidified with 1 M HCl, and extracted with dichloromethane. The
combined organic layers were dried over anhydrous Na2SO4, filtered,
and concentrated to give the crude product, which was purified by
preparative HPLC. HPLC conditions were: (a) column: YMC-Pack
ODS-AQ 250 mm ꢁ 20 mm; (b) eluant: solvent A, acetonitrile with
0.075% TFA, and solvent B, water with 0.075% TFA; gradient: 0ꢀ
15 min, 44% A in B to 74% A in B, then 15ꢀ18 min; 74% A in B to 100%
A. The title compound (7) was obtained from the fraction eluting at
about 15.5 min (0.083 g, 50% yield); mp: 204.1 ( 0.9 °C. 1H NMR (500
MHz, DMSO-d6): δ 13.90 (s, 1H), 7.60 (dd, J = 8.8, 6.3 Hz, 1 H),
7.25ꢀ7.19 (m, 2 H), 6.77 (td, J = 8.5, 2.7 Hz, 1 H), 5.83 (s, 2 H),
4.82ꢀ4.78 (m, 1 H), 3.59ꢀ3.53 (m, 2 H), 3.43ꢀ3.33 (m, 2 H),
2.03ꢀ1.97 (m, 2 H), 1.78ꢀ1.71 (m, 2 H). 13C NMR (126 MHz,
DMSO-d6): δ 167.6, 167.2, 162.8 (d, JCF = 244 Hz), 157.8, 155.8, 154.7
(d, JCF = 10.8 Hz), 134.2, (d, JCF = 10.0 Hz), 109.5 (d, JCF = 22.7 Hz),
107.5 (d, JCF = 3.7 Hz), 104.1 (d, JCF = 26.6 Hz), 97.4, 73.4, 54.5, 44.2
(2C), 29.3 (2C); the regioconfiguration of the acetic acid moiety
was assigned by a 15N gHMBC experiment (JHN = 5 Hz) with
observation of three long-range couplings between the methylene
protons at δ 5.83 ppm and tetrazole nitrogens at δ 386.2, 305.5, and
282.3 ppm (15N-benzamide external ref δ 110 ppm). In comparison, the
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Journal of Medicinal Chemistry
ARTICLE
were first frozen under liquid nitrogen and pulverized by hand with a
hammer to a fine powder. The pieces were transferred and accurately
weighted into a vial and diluted with 3.5 volumes of a 2.5:1 MeCN:H2O
mixture containing internal standard and further processed using the
tissue tearor. The suspensions were then centrifuged, and the concen-
tration of 7 in the supernatant was quantified using LC-MS.
time-dependent (data not shown) and significantly higher than in the
control MDCKII cells (Figure 4a,b). OATP1B1 and OATP1B3
mediated uptake of [14C]-7 (10 μM) was significantly inhibited by
bromosulphothalein (BSP), a known inhibitor of OATP transporters.
Uptake of 1 μM [3H]E217βG (OATP1B1 studies) or 10 nM
[3H]CCK8 (OATP1B3 studies) confirmed the functionality of these
assays.
OATP1B1 and OATP1B3 Recombinant Cell Line Uptake
Assays. MDCKII cells were provided by Dr. A. H. Schinkel (The
Netherlands Cancer Institute) and were used under license agreement.
OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3) cDNAs were
amplified from human liver cDNA libraries (A. Demartis, IRBM, and
R. Stocco, Merck Frosst, Montreal). OATP1B1 was cloned into the
expression vector pcDNA3.1-hygro (Invitrogen, CA) and stably trans-
fected into MDCKII cells (originally provided by Dr. P. Borst, The
Netherlands Cancer Institute). OATP1B3 was cloned into the
pcDNA5/FRT vector (Invitrogen) and stably transfected into Flp-In
MDCKII cells (MDCKII cells stably transfected with Flp Recombinase
Target site). MDCKII cell line was cultured in 96-well transwell culture
plates (Millipore, MA). Five μM [14C]-7 (see Synthesis in Supporting
Information Method 1) was prepared in HBSS with 10 mM HEPES.
Substrate solution (150 μL) was added to either the apical (A) or the
basolateral (B) compartment of the culture plate, and buffer (150 μL)
was added to the compartment opposite to that containing the
compound. At t = 3 h, 100 μL of sample was taken out from both sides
and scintillant (5 mL, ReadySafe, Beckman Coulter, CA) was added.
Radioactivity was determined by liquid scintillation counting in a LS6500
multipurpose liquid scintillation counter (Beckman Coulter, CA).
[3H]Prazosin (5 μM) was used as the positive control. The experiment
was performed in triplicate.
oGTT Study in eDIO Mice with 1 and 7. Male C57/Bl6 mice
were fed a high fat diet (Bio-Serv F3283) for at least 14 weeks starting at
6 weeks of age. The obese mice were fasted for 16 h, and the SCD
inhibitors 1 and 7 were given orally to eDIO mice 1 h before
administration of oral glucose (2 g/kg). Blood glucose levels were
monitored at t = ꢀ60 min, 0 min, 20 min, 40 min, 60 and 120 min post
glucose challenge.
Chronic eDIO Mouse Studies with 1, 7, and 6. Male C57/Bl6
mice were fed a high fat diet (Bio-Serv F3283) for at least 18 weeks
starting at 6 weeks of age. One week prior to dosing of compound, the
eDIO mice were dosed with vehicle (1% methocel, bid) for 7 days. Prior
to initiation of the study, the mice were randomized according to body
weight, and on day 1, the mice were dosed either with vehicle, SCD
inhibitor, or positive control (AM25129) (n = 8ꢀ10/group). Dosing
continued for either 28 days (1 and 7, parts a and b of Figure 6) or
14 days (1 and 6, Figure 4 in Supporting Information Results 4). Body
weight, food consumption, and skin and eye tolerability were monitored
twice per week. Becaue of dry eyes, mice treated with compound 1 were
administered eye lubrication (Neomycin and Polymyxin B Sulfates and
Bacitracin Ophthalmic Ointment, USP, sterile: active ingredients per g
Bacitracin Zinc 400 IU, Neomycin Sulfate (equivalent to 3.5 mg of
neomycin base) 5 mg and Polymyxin B Sulfate 5000 IU) daily from day
12 onward to allow for completion of study (hence why the severity of
reported eye abnormalities in Figure 7b diminished after day 12). At
termination of the study, animals were euthanized and plasma and
tissues (liver, skin, eWAT, Harderian glands) were collected for desa-
turation index measurements, determination of SCD inhibitor concen-
tration, and triglyceride/steatosis measurements (liver only).
The BꢀA/AꢀB ratio was calculated by dividing the Papp from B to A
by the Papp from A to B at t = 3 h:
PappðB f AÞ
B ꢀ A=A ꢀ B ratio ¼
PappðA f BÞ
OATP1B1 and OATP1B3 mediated uptake was determined in MDCKII
cells stably transfected with OATP1B1 or OATP1B3 cDNAs. Twenty-
four hours prior to the experiment, cells were treated with 10 mM
sodium butyrate (Sigma-Aldrich, St. Louis, MO) to increase OATP
expression. Cells were dislodged with trypsin EDTA (Invitrogen) and
resuspended in HBSS with 10 mM Hepes, pH 7.4. Cells were then
suspended in 96 deep well plates at a density of 0.2ꢀ0.6 ꢁ 106 cells/well.
Uptake was initiated by the addition of the indicated concentrations of
[14C]-7 or the positive control substrates [3H]E217βG (for OATP1B1
studies) or [3H]CCK8 (for OATP1B3 studies) in HBSS. Cells were
incubated for the indicated time at 37 °C, then uptake was stopped,
followed by immediate centrifugation at 1800g at 4 °C (Eppendorf,
model 5180R; Hamburg, Germany) and washing of the cell pellets with
phosphate buffered saline (PBS, Invitrogen), three times. Cell pellets
were resuspended in 50% acetonitrile (Sigma-Aldrich), scintillation fluid
was added (Scintisafe Econo 2; Fisher Chemicals, NJ) and radioactivity
was determined by liquid scintillation counting in a LS6500 multi-
purpose scintillation counter (Beckman Coulter, CA). The experiment
was performed in triplicate.
Method for Tissue Desaturation Index Determination.
Liver, fat, Harderian gland, eWAT, and skin were collected for desatura-
tion index determination. Liver, fat, eWAT, and Harderian glands were
weighed and collected in strips of 12 attached microtubes in microracks
(National Scientific, Claremont, California). Enough cold Dulbecco’s
Phosphate Buffer Saline (DPBS; Cellgro, Manassas, Virginia) was added
to liver and fat to obtain a final concentration of 25 mg/100 μL. A fixed
amount of 100 μL of cold DPBS was added to the Harderian glands.
Three medium size (5/3200) stainless steel beads (MetalFini, St-Laurent,
Quꢀebec) were added for homogenization. All tissues were mechanically
milled using the GenoGrinder 2000 (ATS Scientific, Burlington,
Ontario). Tough tissues like skin could not be properly homogenized
using a GenoGrinder. Pieces of weighed shaved skin were collected in
microcentrifuge tubes. The fresh or cryopreserved skin tissues were then
transferred to 8 mL glass tubes. Enough 10N NaOH (J.T. Baker,
Phillipsburg, New Jersey) was added to obtain a final concentration of
25 mg/500 μL. The skin samples were then hydrolyzed overnight at
100 °C. Upon cooling, 500 μL of hydrolyzed skin were transferred to
new 8 mL glass tubes. Aliquots (100 μL) of fresh or cryopreserved liver,
fat, eWAT, and Harderian gland tissue homogenates were also hydro-
lyzed by transferring them to 8 mL glass tubes (VWR), adding 500 μL of
10N NaOH, and submitting them to 100 °C for 1 h. Once cooled to
room temperature, 1 mL of isooctane (Sigma-Aldrich, Milwaukee,
Wisconsin) was added to all samples to conduct the first extraction.
Samples were mixed thoroughly and centrifuged to separate the
isooctane layer from the aqueous lower phase. The top layers of
isooctane containing the undesirable lipids were removed and discarded.
The surface of the aqueous phase was washed by gently adding 500 μL of
isooctane on the side of the tube. Again, this isooctane was removed and
For uptake studies stably transfected MDCKII cells, data were
expressed in nmoles ꢁ 10ꢀ1/106 cells and pmol/106 cells, respectively.
For uptake studies into membrane vesicles, data were expressed in
pmol/mg protein. For substrate kinetic determinations, the transporter
mediated component was calculated by subtracting uptake into parental
cells from transporter expressing cells. This transporter mediated
component was analyzed by nonlinear regression analysis in order to
determine Km data, using Prism 4.0 (Graphpad, San Diego, CA). Data in
Figure 4 are mean ( SE.
The uptake of [14C]-7 (10 μM) into MDCKII cells stably transfected
with the human uptake transporters OATP1B1 or OATP1B3 was
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discarded. Samples were acidified to pH 2.0 by adding 700 μL of formic
acid (Acros Organics, Geel, Belgium) and mixed. The extraction of the
fatty acids was carried on by adding 500 μL of isooctane and thoroughly
mixing. The solvent layers of isooctane containing the fatty acids were
collected after centrifugation and transferred to new 8 mL glass tubes
containing 1 mL of saturated NaCl solution with 1% formic acid. The
purpose of this step is to remove all residual aqueous solution from the
isooctane before gas chromatography (GC) analysis. Once mixed and
centrifuged, an aliquot of 100 μL was transferred to GC vials
(Chromatographic Specialties, Brockville, Ontario), containing 300 μL
conical inserts with springs (Fisher Scientific, Ottawa, Ontario). The
fatty acids were derivatized to their corresponding methyl esters with
20 μL of diazomethane solution prepared in-house and analyzed on an
Agilent Technologies (Mississauga, Ontario) 6890N GC system
equipped with a flame ionization detector (FID) using a HP-88 column
(100 m length, 0.25 mm ID, 0.2 μM film), also from Agilent Technol-
ogies. Samples (2 μL) were injected in split mode using ratio up to 25:1.
Helium was used as carrier gas at a flow rate of 2 mL/min. Hydrogen,
provided by a Parker hydrogen generator (Cleveland, Ohio), was used as
auxiliary gas for the flame ionization detector. The injector and detector
temperatures were set at 250 and 280 °C, respectively. The initial oven
temperature was set at 120 °C, increased to 165 °C at a rate of 10 °C/min,
increased to 210 °C at a rate of 5 °C/min, and increased to 230 °C at a
rate of 5 °C/min.
interfering ribonucleic acid; ROP, research operating procedure;
HepG2, human hepatocellular carcinoma cell line; SAR, structure
ꢀactivity relationship; LLC-PK1, renal epithelial cell line originally
derived from porcine kidneys; MDCKII, MadinꢀDarby canine
epithelium kidney cell line; BSP, sulfobromophthalein; eDIO, estab-
lished diet-induced obesity; oGTT, oral glucose tolerance test; CB1,
cannabinoid receptor 1; DI, desaturation index; HMG-CoA reduc-
tase, 3-hydroxy-3-methyl-glutaryl-CoA reductase; eWAT, epididy-
mal white adipose tissue; TG, triglyceride; AUC, area under
the curve; HFD, high fat diet; ND, normal diet; veh, vehicle;
1H NMR, proton nuclear magnetic resonance; 13C NMR, carbon
nuclear magnetic resonance; PBS, phosphate buffer solution; ND,
not determined
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’ ASSOCIATED CONTENT
Supporting Information. The synthesis of [14C]-radio-
S
b
labeled 7; the method for Papp determination; the human SCD
hepatocyte assay method; the mouse liver SCD pharmaco-
dynamic (PD) assay method; mouse liver triglyceride and
steatosis analysis methods; the rat hyperinsulenemic-euglycemic
glucose clamp method and results for 7; mouse liver PD results
for 1 and 7; mouse liver triglyceride and steatosis results for 7;
chronic eDIO mouse study with 1 and 6. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (604) 484-3336. Fax: (604) 484-3459. E-mail: renataoballa@
dhanni.com.
’ ACKNOWLEDGMENT
We thank Jingchao Dong, Dejun Liu, and Gaoyun Xie at WuXi
AppTec for preliminary work on the synthesis of 7.
’ ABBREVIATIONS USED
SCD1, stearoyl-CoA desaturase-1; OATPs, organic anion trans-
porting polypeptides; C16:1, palmitoleic acid; C16:0, palmitic
acid; C18:1, oleic acid; C18:0, stearic acid; ASO, antisense oligonu-
cleotide; BMI, body mass index; AE, adverse event; mRNA, messen-
ger ribonucleic acid; LKO, liver-specific knockout; siRNA, small
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