Discovery of an Orally Bioavailable Benzimidazole Diacylglycerol Acyltransferase 1 (DGAT1) Inhibitor That Suppresses Body Weight Gain in Diet-Induced Obese Dogs and Postprandial Triglycerides in Humans

Article abstract of DOI:10.1021/acs.jmedchem.7b00173

Modification of a gut restricted class of benzimidazole DGAT1 inhibitor 1 led to 9 with good oral bioavailability. The key structural changes to 1 include bioisosteric replacement of the amide with oxadiazole and α,α-dimethylation of the carboxylic acid, improving DGAT1 potency and gut permeability. Since DGAT1 is expressed in the small intestine, both 1 and 9 can suppress postprandial triglycerides during acute oral lipid challenges in rats and dogs. Interestingly, only 9 was found to be effective in suppressing body weight gain relative to control in a diet-induced obese dog model, suggesting the importance of systemic inhibition of DGAT1 for body weight control. 9 has advanced to clinical investigation and successfully suppressed postprandial triglycerides during an acute meal challenge in humans.

Full text of DOI:10.1021/acs.jmedchem.7b00173

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Article
Discovery of an orally bioavailable benzimidazole diacylglycerol
acyltransferase 1 (DGAT1) inhibitor that suppresses body weight gain
in diet induced obese dogs and postprandial triglycerides in humans
Katsumasa Nakajima, Ricardo Chatelain, Kevin B. Clairmont, Renee Commerford, Gary M. Coppola,
Thomas Daniels, Cornelia J. Forster, Thomas A. Gilmore, Yongjin Gong, Monish Jain, Aaron D Kanter,
Youngshin Kwak, Jingzhou Li, Charles Daniel Meyers, Alan D. Neubert, Paul Szklennik, Vivienne
Tedesco, James Thompson, David Truong, Qing Yang, Brian K. Hubbard, and Michael H Serrano-Wu
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 12 May 2017
Downloaded from http://pubs.acs.org on May 12, 2017
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Discovery of an orally bioavailable benzimidazole
diacylglycerol acyltransferase 1 (DGAT1) inhibitor
that suppresses body weight gain in dietꢀinduced
obese dogs and postprandial triglycerides in humans
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Katsumasa Nakajima,*, † Ricardo Chatelain,‡ Kevin B. Clairmont,‡ Renee Commerford,‡
Gary M. Coppola,† Thomas Daniels,‡ Cornelia J. Forster,† Thomas A. Gilmore,† Yongjin Gong,†
Monish Jain,ǁ Aaron Kanter,† Youngshin Kwak,† Jingzhou Li,† Charles D. Meyers,§
Alan D. Neubert,† Paul Szklennik,† Vivienne Tedesco,‡ James Thompson,‡ David Truong,‡
Qing Yang,‡ Brian K. Hubbard,‡ Michael H. SerranoꢀWu†
†Global Discovery Chemistry, ‡Cardiovascular and Metabolism, ǁPK Sciences and §Translational
Medicine, Novartis Institutes for Biomedical Research, 100 Technology Square, Cambridge, MA
02139
ABSTRACT
Modification of a gut restricted class of benzimidazole DGAT1 inhibitor 1 led to 9 with good
oral bioavailability. The key structural changes to 1 include bioisosteric replacement of the
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amide with oxadiazole and α,αꢀdimethylation of the carboxylic acid, improving DGAT1 potency
and gut permeability. Since DGAT1 is expressed in the small intestine, both 1 and 9 can suppress
postprandial triglycerides during acute oral lipid challenges in rats and dogs. Interestingly, only 9
was found to be effective in suppressing body weight gain relative to control in a dietꢀinduced
obese dog model, suggesting the importance of systemic inhibition of DGAT1 for body weight
control. 9 has advanced to clinical investigation and successfully suppressed postprandial
triglycerides during an acute meal challenge in humans.
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INTRODUCTION
Excess accumulation of triglycerides in the body may result in obesity or contribute to the
pathogenesis of cardiovascular disease1 and pancreatitis.2 Pharmacological inhibition of
triglyceride synthesis has great therapeutic potential towards treatment of these diseases. Of
several enzymes involved in triglyceride synthesis, two diacylglycerol acyltransferases, DGAT13
and DGAT2,4 catalyze the final committed step. While DGAT2 is essential for survival in mice,5
DGAT1 knockout mice have an attractive metabolic phenotype, being healthy, resistant to
dietꢀinduced obesity and with improved insulin sensitivity relative to wildꢀtype.6 DGAT1 has
emerged as an attractive drug discovery target for a number of research groups.7ꢀ9
While DGAT1 is expressed ubiquitously, it is most abundant in the enterocytes of the
small intestine where it is involved in resynthesizing triglycerides from fatty acids and
monoacylglycerol, the hydrolysis products of dietary triglycerides present in the gastrointestinal
tract.3,10 Consistent with this expression pattern, a recent genetic study11 in mice suggests that
loss of intestinal DGAT1 is responsible for the phenotype of DGAT1 wholeꢀbody knockout
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mice. Towards recapitulating this phenotype pharmacologically, we have reported benzimidazole
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inhibitor 1 that selectively targets intestinal DGAT1 with low oral bioavailability (Figure 1).12
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Figure 1. Benzimidazole DGAT1 inhibitor 1
Despite exposure of 1 being restricted to the small intestine after oral administration,
postprandial triglycerides were significantly suppressed compared to control during an oral lipid
challenge in rats. A robust suppression of postprandial triglycerides was similarly observed in
dogs with very low plasma exposure of 1 after oral administration at 1 mg/kg.12 1 was then dosed
orally to dietꢀinduced obese dogs at 5 mg/kg q.d. for 21 days. Unexpectedly, both treatment and
control groups (n = 6 per group) showed statistically similar body weight gains over baseline.
Systemic exposure of 1 on the last day of the study was measured to be low (AUC0ꢀ24h 70 nM*h),
suggesting that, as expected, DGAT1 inhibition by 1 was restricted to the intestine. In a separate
study using a mouse model of dietꢀinduced obesity, the group treated with 1 did show reduced
body weight gain compared to control (P ≤ 0.05) after oral administration at 30 mg/kg q.d. for
14 days. However, the higher dose led to significant plasma exposure of 1 (Cmax 350 nM, Cmin
71 nM, AUC0ꢀ24h 3081 nM*h), so we could not conclude that the body weight reduction was
based solely on inhibition of intestinal DGAT1. These observations raise the possibility that
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systemic exposure is required for a DGAT1 inhibitor to be efficacious against dietꢀinduced body
weight gain, suggesting a need for a potent DGAT1 inhibitor from the benzimidazole series
which has good oral bioavailability. Here we report details of the medicinal chemistry effort that
led to the discovery of such an inhibitor and its effectiveness in a chronic body weight study in
dogs as well as in an acute meal challenge study in humans.
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RESULTS AND DISCUSSION
Compound 1 may have a subꢀoptimal membrane permeability based on a 10ꢀfold
decrease in potency from DGAT1 measured biochemically (IC50 39 nM) to inhibition of
triglyceride synthesis in a C2C12 mouse myoblast cellular assay (IC50 390 nM) as well as a low
AꢀB value (0.7 x 10ꢀ6 cm/s) in the Cacoꢀ2 permeability assay. BꢀA/AꢀB ratio (12.9) in the Cacoꢀ2
assay also suggests 1 is an efflux substrate in the gut, resulting in low bioavailability after oral
administration. To improve membrane permeability and reduce efflux, our optimization strategy
focused on replacing the amide and the carboxylic acid. Oxazole and oxadiazole are widely
utilized bioisosteres of amides in drug design.13,14 In particular, they lack a NꢀH hydrogen, which
can lead to reduced solvation and improved membrane permeability.15 The effect of these
replacements on DGAT1 inhibition was first examined in benzimidazoles devoid of a propionic
acid (2ꢀ5)16,17 (Table 1), showing that both oxazole (3, IC50 132 nM) and oxadiazole (4, IC50
47 nM) were in fact more potent than the corresponding amide 2 (IC50 280 nM). When the
4ꢀsubstituent on the western phenyl ring was changed from a chloride to a methoxy group, a
slightly more potent analog 5 (IC50 23 nM) was obtained. The impact of oxadiazole on
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membrane permeability was investigated later when the propionic acid was attached on the
eastern phenyl ring.
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Table 1. DGAT1 IC50 data of compounds 2ꢀ5a
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Compounds
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5
DGAT1 IC50 (nM)
280 (1)
132 ± 2 (2) 47 ± 12 (4) 23 ± 5 (2)
aAverage values ± SD (The number of independent experiments. Each experiment was run in
duplicate)
In a related subꢀseries with a quinolineamide side chain, α,αꢀdimethylation of the
propionic acid was found to be well tolerated (DGAT1 IC50: 6, 14 nM; 7, 32 nM)16 (Table 2).
Both 6 and 7 showed similar IC50 values in C2C12 cells (6, 913 nM; 7, 954 nM), so
improvement of membrane permeability of 7 over 6 was not evident in C2C12 cells. However,
increase of AꢀB values from 6 (1.4 x 10ꢀ6 cm/s) to 7 (3.5 x 10ꢀ6 cm/s) as well as reduced efflux
ratio from 6 to 7 (BꢀA/AꢀB: 6, 10.1; 7, 3.5) in the Cacoꢀ2 assay suggested improved gut
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permeability of 7 over 6. These Cacoꢀ2 data nicely predicted increased oral bioavailability (6,
2%; 7, 23%) in a rat pharmacokinetic (PK) study (Table 3).
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Table 2. In vitro data of compounds 6 and 7a
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Compounds
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7
DGAT1 IC50 (nM)
C2C12 cells IC50 (nM)
Cacoꢀ2: AꢀB (x 10ꢀ6 cm/s)
Cacoꢀ2: BꢀA (x 10ꢀ6 cm/s)
BꢀA/AꢀB ratio
14 ± 9 (752)
913 (1)
32 ± 4 (2)
954 (1)
3.5 (1)
12.4 (1)
3.5 (1)
1.4 ± 0.3 (4)
14.1 ± 2 (4)
10.0 ± 2 (4)
aAverage values ± SD (The number of independent experiments. Each experiment was run in
duplicate for DGAT1 IC50 and quadruplicate for C2C12 cells IC50)
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Table 3. Rat PK data of compounds 6 and 7a
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Compounds
iv dose (mg/kg)b
CL (mL/min/kg)
Vss (L/kg)
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6.9 ± 1
0.4 ± 0
1.7 ± 0.4
5232 ± 706
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5.5 ± 0.7
0.4 ± 0.07
0.8 ± 0.07
6111 ± 676
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T1/2 (h)
AUC0ꢀ8h (nM*h)
po dose (mg/kg)b
Cmax (nM)
333 ± 169
0.4 ± 0.1
405 ± 250
1717 ± 283
0.5 ± 0
4234 ± 1423
Tmax (h)
AUC0ꢀ8h (nM*h)
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F (%)
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23 ± 8
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aAverage values ± SD of 2 rats (iv dose) or 3 rats (po dose). bSolution administration, vehicle:
10% NMP, 30% PG, 6% ETPGS (for compound 6); 10% NMP, 40% PG, 5% ETPGS (for
compound 7).
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Our next molecular design combined the oxadiazole and α,αꢀdimethylated propionic acid
groups in one molecule (9) (Table 4). The corresponding αꢀunsubstituted propionic acid analog 8
was evaluated to confirm the impact of α,αꢀdimethylation of the carboxylic acid on oral
bioavailability in the oxadiazole series. Good DGAT1 potency was obtained with both of the
compounds (IC50: 8, 10 nM; 9, 17 nM). Potency for inhibition of triglyceride synthesis in C2C12
cells by 8 and 9 (IC50 181 and 151 nM, respectively) was also improved relative to 1 (IC50
390 nM). Potency shifts between the two assays for 8 (18ꢀfold) and 9 (9ꢀfold) were, however,
still comparably high to 1 (10ꢀfold), implying that neither oxadiazole ring (8, 9) nor
α,αꢀdimethylation of carboxylic acid (9) contributed to improved membrane permeability in
C2C12 cells. However, improvement of gut permeability from 8 to 9 was suggested based on
increase of AꢀB values from 8 (1.4 x 10ꢀ6 cm/s) to 9 (4.2 x 10ꢀ6 cm/s) as well as reduced efflux
ratio (BꢀA/AꢀB: 8, 6.6; 9, 2.4) in the Cacoꢀ2 assay. This improved in vitro data for 9 over 8 was
again reflected in a rat PK study, where 9 had improved oral bioavailability (90%) over 8 (2%)
(Table 5). The extent of the improvement was more dramatic than that of the carboxamide
analogs 6 and 7 (2% to 23%), suggesting that 9 might be more soluble in the intestinal
environment at the site of absorption than 7, though both compounds showed poor solubility in
vitro (<5 and 8 µM, respectively, in pH 6.8 buffer after 24 h incubation). Further characterization
for in vitro properties of 7 and 9 such as solubility and stability in simulated gastric or intestinal
fluids are necessary to better understand the outcome.
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Table 4. In vitro data of compounds 8 and 9a
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Compounds
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DGAT1 IC50 (nM)
C2C12 cells IC50 (nM)
Cacoꢀ2: AꢀB (x 10ꢀ6 cm/s)
Cacoꢀ2: BꢀA (x 10ꢀ6 cm/s)
BꢀA/AꢀB ratio
10 ± 4 (4)
181 (1)
1.4 (1)
9.2 (1)
6.6 (1)
17 ± 15 (42)
151 ± 31 (4)
4.2 (1)
10 (1)
2.4 (1)
aAverage values ± SD (The number of independent experiments. Each experiment was run in
duplicate for DGAT1 IC50 and quadruplicate for C2C12 cells IC50)
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Table 5. Rat PK data of compounds 8 and 9a
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Compounds
iv dose (mg/kg)b
CL (mL/min/kg)
Vss (L/kg)
8
9
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21 ± 3
0.7 ± 0.07
1.6 ± 0.3
1686 ± 206
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9.5 ± 2
0.6 ± 0
1.9 ± 0.4
3857 ± 626
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T1/2 (h)
AUC0ꢀ8h (nM*h)
po dose (mg/kg)b
Cmax (nM)
40 ± 9
0.5 ± 0
103 ± 25
3551 ± 548
1.2 ± 0.8
10376 ± 1235
Tmax (h)
AUC0ꢀ8h (nM*h)
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F (%)
2 ± 0.6
90 ± 10
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aAverage values ± SD of 2 rats (iv dose) or 3 rats (po dose). For compound 9, exposures at 6h
time point were below lower limit of quantification and measureable exposure was observed at
8h, and as a result, 6h data was not used for PK calculations. bSolution administration, vehicle:
10% NMP, 30% PG, 6% ETPGS (for compound 8); 10% NMP, 30% PEG400, 6% ETPGS (for
compound 9).
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After observation of successful suppression of postprandial triglycerides in rats (data not
shown), 9 was tested acutely in dogs (Figure 2). Dogs were fasted for 20 h, then vehicle or 9
(1 mg/kg) was orally gavaged (n = 3 animals per group). After 30 min, a saline solution of heavy
cream, glucose and acetaminophen18 was orally gavaged. 9 suppressed postprandial triglycerides
robustly over the following 4 h, confirming successful inhibition of DGAT1 in vivo.
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Triangles indicate significant differences between vehicle and group of 9 (p ≤ 0.05).
Figure 2. Suppression of postprandial triglycerides by compound 9 in dogs (n = 3 per group)
In a subsequent chronic efficacy study, 9 was orally dosed to dogs at 5 mg/kg q.d. for
21 days (Figure 3). Upon high fat ad libitum feeding (free access to food and water), the group
treated with 9 (n = 6 animals) showed an 11% decrease in body weight gain compared to the
vehicle group (n = 6 animals) (P <0.005). Plasma levels of 9 were measured at 90 minutes after
the first and twentieth doses [738 ± 272 (n = 6) and 930 ± 38 nM (n = 3), respectively]. A full
24 h pharmacokinetic profile was obtained in a separate group of three dogs by the same dosing
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method, confirming high concentration of 9 in plasma (AUC0ꢀ24h 5270 ± 842 nM*h). Comparing
to the chronic study of 1 described earlier, it is reasonable to speculate that it is the significant
plasma exposure of 9 which contributed to decreased body weight gain in the dog model.
However, further investigation is required to understand relationship of in vitro potency to in
vivo efficacy since free concentration of 9 in plasma is low (plasma protein binding 99.1 ± 0.7%
in the dog) with unbound Cave of 9 as 2 nM, which is below in vitro potency value of 9 in
C2C12 cellular assay (IC50 151 nM). Gastrointestinal adverse events such as diarrhea and
steatorrhea were not observed in the treatment group with 9.
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Figure 3. Decreased body weight gain by compound 9 in dogs (n = 6 per group)
During selectivity and in vitro safety profiling, 9 was found to be a poor inhibitor of
DGAT2 in vitro (IC50 >10 ꢁM). It also showed a low risk of mutagenicity in the Ames assay19,20
as measured by increase of reverting mutations in histidineꢀdepleted Salmonella typhimurium
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strains (TA98 and TA100) in the presence and absence of S9 metabolic activation at
concentration of 9 up to 1000 ꢁg/well. This finding was particularly satisfying as we had
previously identified that a benzimidazole substituted with 2,6ꢀdimethylphenyl group at Cꢀ2, a
partial structure present in 6, can serve as a toxicophore for such a risk via generation of a
reactive metabolite of proposed structure 10 after metabolic oxidation (Scheme 1).21,22 Although
9 contains the same structural motif, observation of a metabolite 11 during biotransformation
studies on 923 implies a shift in metabolism from the benzimidazole moiety to the methoxy
group, avoiding the formation of 10.
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Scheme 1. Bioactivations of compounds 6 and 9
Encouraged by positive preclinical findings, 9 was advanced to clinical investigation.
Healthy human subjects first underwent a baseline (untreated) high fat meal test during which
plasma triglyceride levels were monitored for 9 h. On a separate day, the same subjects received
single oral doses of 9 (at 5, 15, 50, 150, 300 or 600 mg) (n = 8 per dose) or placebo (n = 12),
followed by an identical high fat test meal (Figure 4). When given with the high fat test meal, 9
suppressed postprandial plasma triglycerides in a dose dependent manner. At doses of 150 mg
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and above, 9 completely blunted the postprandial plasma triglyceride excursion. Gastrointestinal
adverse events such as diarrhea and nausea were also observed in the treatment group with 9.
The details of the adverse events with 9 will be described by a separate report which discusses
development of a preclinical model to predict clinical observations of gastrointestinal adverse
events with DGAT1 inhibitors.
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Placebo (n = 12), each treatment group (n = 8)
Figure 4. Suppression of postprandial triglycerides by compound 9 in humans
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Chemistry
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9
Compounds 8 and 9 were synthesized as shown in Scheme 2. Acyl hydrazine 12 was coupled
with benzoic acid 13 to give diacyl hydrazine 14. Dehydration of 14 using Burgess reagent
provided oxadiazole 15. The nitro group was reduced by catalytic hydrogenation to give
dianiline compound 16. Condensation of 16 with benzaldehyde 1712 using potassium
peroxomonosulfate constructed benzimidazole 18. Reduction of the carbonꢀcarbon double bond
next to the ester group and subsequent hydrolysis of the ester furnished 8. To prepare
benzaldehyde 21, Negishi coupling of alkyl zinc iodide 2024 and aryl triflate 19 was found to be
efficient. 21 was then condensed with the dianiline 16, which was followed by hydrolysis of the
ester to give 9.
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Reagents and conditions: (a) HOBT, EDCI, Et3N, DMF, RT, 83 %; (b) Burgess reagent, THF,
microwave at 150 oC, 70 %; (c) H2 / PtO2, EtOHꢀEtOAc, RT, 1 atm, 67 %; (d) Potassium
peroxomonosulfate, DMFꢀwater, RT; (e) H2 / PdꢀC, EtOHꢀTHF; (f) 1N NaOH, MeOH, RT (41
% over 3 steps, dꢀf, for 8; 36 % over 2 steps for d and f for 9); (g) Pd(PPh3)4, tolueneꢀDMA, 50
oC, 99 %.
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Scheme 2. Syntheses of DGAT1 inhibitors 8 and 9
CONCLUSION
In summary, a potent DGAT1 inhibitor 9 with good oral bioavailability was identified by
optimization of gut restricted inhibitor 1. While both compounds robustly inhibit intestinal
DGAT1, chronic studies in dogs suggests that only 9, with good systemic exposure, is
efficacious against diet induced body weight gain in dogs. Expecting this outcome to translate
into humans, 9 was advanced to clinical investigation. A preliminary result from an acute meal
challenge was positive, demonstrating successful proof of concept for pharmacological inhibition
of DGAT1 by 9 in humans.
EXPERIMENTAL SECTION
Solvents and chemicals used were reagent grade. NMR spectra were recorded on a
Bruker Avance 400 MHz spectrometer. Chemical shifts (δ) are reported in parts per million
(ppm) relative to residual undeuterated solvent as internal reference: DMSOꢀd6 2.50 ppm, CDCl3
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7.26 ppm, CD3OD 3.31 ppm. Coupling constants (J) are reported in hertz (Hz). LCꢀMS spectra
were recorded using an Agilent 1100 series spectrometer. Column chromatography was
performed by an ISCO CombiFlash or a Biotage SP1 apparatus using disposable normal phase
silica gel columns. Microwave reactions were conducted using a Biotage initiator. The purity of
all target compounds was ≥95 % as determined by analytical HPLC (20 min), 1H NMR or
combustion analysis.
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3-(4-(6-(5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-yl)-1H-benzo[d]imidazol-2-yl)-3,5-
dimethylphenyl)propanoic acid (8). Potassium peroxomonosulfate (Oxone™) (824 mg, 1.34
mmol) was added to a solution of 16 (564 mg, 2.00 mmol) and 17 (436 mg, 2.00 mmol) in a
mixture of DMF (10 mL) and water (1 mL). The mixture was stirred at room temperature for 20
min and partitioned between ethyl acetate and water. The combined organic extract was dried
over MgSO4 and concentrated to give crude 18 (1.0 g). MS m/z 481 (M+1).
Palladium on carbon (5%, 300 mg) was added to a solution of crude 18 (1.0 g) in a mixture of
EtOH (20 mL) and THF (20 mL) and was stirred under hydrogen (1 atmosphere) at room
temperature for 3 h. The catalyst was removed by filtration through Celite. The filtrate was
concentrated and purified by column chromatography to give the hydrogenated product (600
mg). MS m/z 483 (M+1).
1N Aqueous NaOH (10 mL, 10 mmol) was added to a solution of the product above in MeOH
(10 mL). The mixture was stirred at room temperature for 2 h and acidified to pH 3~4 by
addition of 1N aqueous HCl. The precipitate formed was separated, washed with water, dried
over MgSO4 and evaporated under reduced pressure to give 8 as a white solid (380 mg, 41 %
over 3 steps). MS m/z 469 (M+1). 1H NMR (400 MHz, CD3OD) δ 8.40 (br s, 1 H), 8.15ꢀ8.08 (m,
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3 H), 7.81 (br s, 1 H), 7.16 (d, J = 9 Hz, 2 H), 7.10 (s, 2 H), 3.91 (s, 3 H), 2.93 (t, J = 8 Hz, 2 H),
2.55 (t, J = 8 Hz, 2 H), 2.15 (s, 6 H).
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3-(4-(6-(5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-yl)-1H-benzo[d]imidazol-2-yl)-3,5-
dimethylphenyl)-2,2-dimethylpropanoic acid (9). This compound was prepared from 16 and
21 using the methods described for the synthesis of 18 and the final step of the synthesis of 8.
MS m/z 497 (M+1). 1H NMR (400 MHz, CD3OD) δ 8.55ꢀ8.45 (m, 1H), 8.25ꢀ8.18 (m, 3 H), 7.95ꢀ
7.85 (m, 1 H), 7.26 (d, J = 8 Hz, 2 H), 7.15 (s, 2 H), 4.02 (s, 3 H), 2.99 (s, 2 H), 2.26 (s, 6 H),
1.30 (s, 6 H). Anal. Calcd for C29H28N4O4: C, 70.14 %; H, 5.68 %; N, 11.28 %. Found: C, 69.86
%; H, 5.62 %; N, 11.14 %.
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4-Amino-N'-(4-methoxybenzoyl)-3-nitrobenzohydrazide (14). HOBT (608 mg, 4.50 mmol)
and EDCI (920 mg, 4.80 mmol) were added to a solution of 13 (728 mg, 4.00 mmol) in DMF (30
mL). The mixture was stirred at room temperature for 10 min. Triethylamine (1.7 mL, 12 mmol)
and 12 (664 mg, 4.00 mmol) were added. The mixture was stirred at room temperature for 3 h.
Water was added, and the resulting suspension was filtered. The yellow solid was separated,
washed with water and dried under vacuum to give 14 (1.1 g, 83 %). MS m/z 331 (M+1). 1H
NMR (400 MHz, DMSOꢀd6) δ 10.46 (s, 1 H), 10.36 (s, 1 H), 8.70 (d, J = 2 Hz, 1 H), 7.97ꢀ7.87
(m, 5H), 7.12 (d, J = 8 Hz, 1 H), 7.09 (d, J = 8 Hz, 2 H), 3.87 (s, 3 H).
4-(5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-yl)-2-nitroaniline (15). A solution of 14 (750 mg,
2.27 mmol) and Burgess reagent (1.62 g, 6.81 mmol) in THF (15 mL) was heated at 150 oC in a
microwave reactor for 20 min. The mixture was concentrated and purified by column
chromatography to give 15 as a yellow solid (500 mg, 70 %). MS m/z 313 (M+1). 1H NMR (400
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MHz, DMSOꢀd6) δ 8.65 (d, J = 2 Hz, 1 H), 8.10ꢀ8.00 (m , 5H), 7.23 (d, J = 8 Hz, 1 H), 7.19 (d, J
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6
= 8 Hz, 2 H), 3.90 (s, 3 H).
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4-(5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-yl)benzene-1,2-diamine (16). A slurry of PtO2 (817
mg, 3.60 mmol) in EtOAc (15 mL) was added to a suspension of 15 (5.6 g, 18 mmol) in EtOH
(150 mL). The mixture was stirred under hydrogen (1 atmosphere) at room temperature for 3
days. The catalyst was removed by filtration through Celite. The filtrate was concentrated to give
16 as a grey solid (4.9 g, 97 %). MS m/z 283 (M+1). 1H NMR (400 MHz, DMSOꢀd6) δ 8.00ꢀ7.95
(m, 2 H), 7.25 (d, J = 2 Hz, 1 H), 7.19ꢀ7.13 (m, 3 H), 6.62 (d, J = 8 Hz, 1 H), 5.27 (br s, 2 H),
4.84 (br s, 2 H), 3.86 (s, 3 H).
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Methyl 3-(4-formyl-3,5-dimethylphenyl)-2,2-dimethylpropanoate (21). ZnꢀCu couple (1.3 g)
was stirred in toluene (16 mL) and dimethylacetamide (2 mL) at room temperature under
nitrogen for 15 min. 3ꢀIodoꢀ2,2ꢀdimethyl propionate methyl ester (800 mg, 3.31 mmol) was
added, and the reaction mixture was heated at 80 °C for 1.5 h under nitrogen. It was cooled to 50
°C and 19 (800 mg, 2.82 mmol) and a solution of Pd(PPh3)4 (240 mg, 0.208 mmol) in toluene (4
mL) were added. The mixture was heated at 50 oC for 18 h, cooled to room temperature, diluted
with EtOAc and quenched with triethylamine (0.8 mL). Insoluble materials were removed by
filtration, and the filtrate was concentrated. The residue was partitioned between EtOAc and 1N
aqueous HCl. The combined organic extract was dried over MgSO4, concentrated and purified
by column chromatography to give 21 (690 mg, 99 % based on 19). MS m/z 249 (M+1). 1H
NMR (400 MHz, CDCl3) δ 10.57 (s, 1 H), 6.82 (s, 2 H), 3.67 (s, 3 H), 2.81 (s, 2 H), 2.57 (s, 6
H), 1.19 (s, 6 H).
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Enzymatic assay for the inhibition of human DGAT1 activity. The assay was performed in
384ꢀwell format, using membrane preparations from Sf9 insect cells expressing recombinant
human (His)6DGAT1 (1.25 µg / 50 µl reaction), diolein (50 µM) and oleoylꢀCoA (50 µM) in
50 mM HEPES, 5 mM MgCl2, 1 mM sodium acetate, 10% ethanol, pH 7.5. After a 30 min
incubation at room temperature, the reaction was extracted with 50 µl butanol and the triolein
ammonium ion adduct was measured by LC/MS/MS using a Micromass Quattro Micro API
mass spectrometer. Test compounds were dissolved in the appropriate volume of DMSO to a
final concentration of 10 mM. A 10ꢀpoint, 3ꢀfold dose response was used to evaluate compound
potency.
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Cellular assay for inhibition of intracellular triglyceride synthesis. C2C12 cells were seeded
18 hours prior to assay in 96ꢀwell plates in Dulbecco’s Modified Eagle Medium (DMEM)
containing 5 mM glucose and 10 % fetal bovine serum. Following 18 hours incubation at 37 °C,
medium was replaced with DMEM containing 5 mM glucose, 250 ꢁM oleate complexed to BSA,
and DGAT1 inhibitors at the specified concentrations in dimethyl sulfoxide to a final
concentration of 0.5 % for 2 hours at 37 °C. The medium was removed at the end of the
incubation and 150 ꢁl/well of 1ꢀbutanol containing the internal standard, tripalmitolein at 0.2
ꢁM, was added. The plates were sealed and left at room temperature for at least 10 min.
Butanolic extracts (85 ꢁl/well) were transferred to 384ꢀwell LCꢀMS plates and centrifuged at 209
x g for 5 min prior to loading on the LCꢀMS. Samples were analyzed by LCꢀMS using an Agilent
1100 Series LC and Micromass Quattro Micro API mass spectrophotometer with an ESI+
source. For doseꢀresponse determinations, the LC/MS/MS was run in the MUX mode, using a
Gilson QuadꢀZ 215 autosampler. Compound potency was determined by an 11ꢀpoint, 3ꢀfold dose
response.
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Rat Pharmacokinetic studies. The studies used male Sprague−Dawley rats weighing 200ꢀ300 g
obtained from Harlan Laboratories (Indianapolis, IN) and were approved by Institutional Animal
Care and Use Committee of Novartis. Animals were administered intravenously (n = 2) or orally
(n = 3) with a compound as described in Tables 3 and 5. Blood samples were obtained from the
jugular vein catheter of each animal at 0.083 (iv dose only) and 0.25, 0.5, 1, 2, 4, 6 and 8 h after
dosing, transferred to an EDTA coated tube and centrifuged to yield plasma samples for analysis
by LC−MS/MS.
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Acute meal challenge in dogs for 9. Male Beagle dogs (Marshall Farms, North Rose, NY)
weighing 7ꢀ12 kg were fed a standard chow diet (Harlan 8653 diet [fat: 9.95 grams %; 19% kcal
from fat; 4.02 kcal/gram]) and water was provided ad libitum. Dogs were maintained in an
environment with temperature and humidity appropriate for the species and kept on a 12ꢀhour
light/dark cycle. All procedures were performed in accordance with the standards of the US
Department of Health and Human Services and were approved by the Novartis Animal Care and
Use Committee (Protocol DB 9018). Animal husbandry and maintenance were in accordance
with the National Institute of Health Guide for the Care and Use of Laboratory Animals.
Dogs were studied on two separate days, using a paired study design. For both study days, dogs
were fasted 20 hours prior to study initiation. Vehicle or 9 (1 mg/kg suspended in 0.5 %
carboxymethyl cellulose) was orally gavaged at 2.5 mL/kg (n = 3 animals per group). After a
period of 30 minutes (t = 0 hr), a solution of heavy cream (8 ml/kg, 2.6 g/kg), glucose (1 g/kg)
and acetaminophen (20 mg/kg) in saline (2 ml/kg) was orally gavaged. Subsequent blood
samples were obtained after ingestion of the meal. Food was not provided during 6 hours after
the meal. The incremental area under the curve (AUC0ꢀ4h) of the plasma triglyceride response
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during the vehicle phase of the study averaged 35400 ± 8863 mg/dL·min, whereas it was reduced
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to 1,846 ± 754 mg/dL·min after administration of 9 (p ≤ 0.05, paired tꢀtest, oneꢀtailed).
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Chronic dog body weight study for 9. Dogs were offered a high fat diet (fat: 24 grams %; 45%
kcal from fat; 4.8 kcal/gram) at the onset of daily oral dosing with either vehicle (gelatin capsule
containing 0.5% carboxymethyl cellulose) or 9 (sodium salt) (5 mg/kg q.d.; gelatin capsule with
compound suspended in water) (n = 6 animals per group). Throughout the study, dogs were
allowed ad libitum access to the high fat diet. Body weight was measured every three to five
days. At the beginning of the study and prior to the initiation of the high fat diet feeding period,
both groups of dogs had similar body weights (vehicle group: 9.50 ± 0.59 kg vs. 9 group: 9.43 ±
0.28 kg). Body weight in the vehicle group by day 21 was 11.87 ± 0.66 kg (25% increase). The
9ꢀtreated group achieved a final body weight of only 10.53 ± 0.54 kg (p ≤ 0.01; 11.7% increase
over 21 days). The rate of highꢀfat dietꢀinduced weight gain between the two groups also differed
significantly (p ≤ 0.005, twoꢀway ANOVA). Thus, the body weight increase of 2.37 ± 0.21 kg
from baseline after 21 days in the vehicleꢀtreated group was significantly greater than that
observed in the 9ꢀtreated group (1.1 ± 0.43 kg; p ≤ 0.0001) with the difference between groups
reaching significance by day 13 and remaining so thereafter (Bonferroni’s postꢀtest; p ≤ 0.05).
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Acute meal challenge in humans for 9. The study protocol and all amendments were reviewed
by an accredited Institutional Review Board. The study was conducted according to the ethical
principles of the Declaration of Helsinki. The single ascending dose study was randomized,
doubleꢀblind, and placeboꢀcontrolled which assessed 6 single oral doses of 9 (n = 8 subjects per
dose) or matching placebo (n = 12 subjects) under fasting and fat tolerance test (FTT) conditions.
Subjects were overweight or obese, but otherwise healthy males or females of nonꢀchildbearing
potential, aged 18 to 55 years old. Sequential cohorts of 10 subjects each were enrolled and
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randomized in a 4:1 ratio of 9 to placebo. Each subject participated in two treatment periods. All
eligible subjects were admitted to the study center for treatment Period 1 (P1) on Day ꢀ2 for
baseline assessments, where they remained domiciled until discharge on Day 4. On the morning
of baseline FTT day (Day ꢀ1), subjects were served a high fat (~1000 kcal meal, 55% fat, ~30%
carbohydrate & ~15% protein) test meal of fried eggs in butter, buttered toast, bacon and hash
browns (Meal 1) to profile baseline postprandial triglycerides response. After an overnight fast,
on the morning of Day 1, subjects received a single oral dose of 9 or matching placebo and
remained fasting for 4 hours for pharmacokinetic and safety assessments of 9. Subjects were
discharged on Day 4. Following a ≥7 day washout period, subjects returned to the study center
for treatment Period 2 on Day ꢀ1 for baseline safety assessments. The following morning (Day
1), after an overnight fast, subjects were administered the same oral single dose of 9 or matching
placebo they received on P1, Day 1. Approximately one hour after drug administration subjects
underwent a second FTT (~1000 kcal meal, 55% fat) meal (Meal 2), identical to Meal 1.
Subjects remained domiciled for 72 hours following dose administration for serial PK and PD
blood collections and safety assessments and were discharged after completion of all assessments
on Day 4.
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ASSOCIATED CONTENT
Supporting Information
Dog and mouse chronic efficacy data (body weight) of 1 and pharmacokinetic data of 9 in
humans. Molecular Formula Strings.
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AUTHOR INFORMATION
Corresponding Author
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*Phone: 617ꢀ871ꢀ7459. Eꢀmail: katsumasa.nakajima@novartis.com.
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Dr. Jason Elliott for his helpful suggestions for this manuscript, Drs. Susanne
Glowienke and James Mangold for providing Ames and biotransformation data of 9,
respectively, and members of MAPꢀADME group for profiling support. We thank Erik
Rocheford, Tara Mullarkey and Lisha Li for generating body weight data for 1 in a dietꢀinduced
obese mouse model.
ABBREVIATIONS
AUC, area under the curve; Cave, average concentration; DGAT, diacylglycerol acyltransferase;
EDCI, 1ꢀEthylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide; HOBT, Hydroxybenzotriazole; PG,
propylene glycol; PK, pharmacokinetics; PTGS, Tocopherylpolyethylene glycol 1000 succinate.
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REFERENCES
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Cases, S.; Smith, S.J.; Zheng, Y.ꢀW.; Myers, H.M.; Lear, S.R.; Sande, E.; Novak, S.;
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encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis.
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Cases, S.; Stone, S.J.; Zhou, P.; Yen, E.; Tow, B.; Lardizabal, K.D.; Voelker, T.; Farese,
R.V., Jr. Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related
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