Discovery of diamide compounds as diacylglycerol acyltransferase 1 (DGAT1) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.01.025

Diamide compounds were identified as potent DGAT1 inhibitors in vitro, but their poor molecular properties resulted in low oral bioavailability, both systemically and to DGAT1 in the enterocytes of the small intestine, resulting in a lack of efficacy in vivo. Replacing an N-alkyl group on the diamide with an N-aryl group was found to be an effective strategy to confer oral bioavailability and oral efficacy in this lipophilic diamide class of inhibitors.

Full text of DOI:10.1016/j.bmcl.2016.01.025

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of diamide compounds as diacylglycerol acyltransferase 1
(DGAT1) inhibitors
a
a
b
a
Katsumasa Nakajima a, , Myriam April , Jason T. Brewer , Thomas Daniels , Cornelia J. Forster ,
Thomas A. Gilmore a, Monish Jain c, Aaron Kanter a, Youngshin Kwak a, Jingzhou Li a, Les McQuire a,
Michael H. Serrano-Wu a, Ryan Streeper b, Paul Szklennik a, James Thompson b, Bing Wang a
⇑
a Global Discovery Chemistry, Novartis Institutes for Biomedical Research, 100 Technology Square, Cambridge, MA 02139, USA
b Cardiovascular and Metabolism, Novartis Institutes for Biomedical Research, 100 Technology Square, Cambridge, MA 02139, USA
c Metabolism and Pharmacokinetics, Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Diamide compounds were identified as potent DGAT1 inhibitors in vitro, but their poor molecular prop-
erties resulted in low oral bioavailability, both systemically and to DGAT1 in the enterocytes of the small
intestine, resulting in a lack of efficacy in vivo. Replacing an N-alkyl group on the diamide with an N-aryl
group was found to be an effective strategy to confer oral bioavailability and oral efficacy in this lipophilic
diamide class of inhibitors.
Received 24 November 2015
Revised 8 January 2016
Accepted 9 January 2016
Available online xxxx
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
DGAT1 inhibitors
Diamide compounds
1,2,4-Triazole
Oral bioavailability
Triglyceride
While triglyceride is an important energy source, its excess
accumulation in the body contributes to the pathogenesis of car-
diovascular disease1 and pancreatitis.2 Inhibition of triglyceride
synthesis can offer a potential therapeutic opportunity towards
treatment of these diseases.
absorption of dietary triglyceride, DGAT1 inhibitors must be
absorbed into enterocytes, but systemic exposure is not required.10
Our high throughput screening using recombinant human
DGAT1 enzyme identified diamide compound 1 as a DGAT1 inhibi-
tor (Table 1). Despite excellent potency (DGAT1 IC50 = 0.007 l
M),11
Triglyceride synthesis involves two biochemical pathways: one
starts from monoacylglycerol, a metabolite of hydrolysis of dietary
triglyceride in the gut. The other starts from glycerol-3-phosphate.
Both routes ultimately provide a common intermediate diacylglyc-
erol, which is then coupled with fatty acyl-CoA to generate triglyc-
eride. This final step is catalyzed by two enzymes known as
diacylglycerol acyltransferases, DGAT13 and DGAT2.4 While DGAT2
knockout mice die shortly after birth,5 DGAT1 knockout mice are
healthy, resistant to diet-induced obesity and have improved insu-
lin sensitivity relative to wild-type.6 This phenotype of DGAT1
knockout mice has led a number of groups to develop DGAT1
inhibitors.7,8
1 was neither bioavailable nor efficacious when dosed orally to rats
at 10 mg/kg,12 suggesting that it does not reach the target in ente-
rocytes. Gut permeability and stability of 1 against CYP enzymes
while it resides in the enterocytes are unlikely the problem based
on Caco-2 and rat liver microsomal (RLM) clearance13 data, respec-
tively. Therefore poor stability and/or solubility of 1 in gastroin-
testinal fluid likely hindered 1 from reaching the target in the
enterocytes. A medicinal chemistry program to optimize 1 into
an orally efficacious DGAT1 inhibitor was then initiated. Although
systemic exposure for analogs of 1 is not required for efficacy, we
nevertheless measured it to confirm that molecules were being
absorbed into enterocytes.
DGAT1 is a membrane-bound protein localized to the endoplas-
mic reticulum. It is ubiquitously expressed, but is most abundant
in the enterocytes of the small intestine where it plays an impor-
tant role in dietary triglyceride uptake.3,9 In order to suppress
Our initial approach was to simply replace the ester groups of 1
with groups that were predicted to be more stable to hydrolytic
degradation. This provided potent DGAT1 inhibitors 2 and 3
(Table 1). In particular, 2 was stable in vitro in simulated gastric
fluid in the presence of pepsin (83 % remained after 1 h of
incubation). Nevertheless, after oral suspension administration
to rats, these analogs still showed poor systemic exposure
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2016.01.025
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Nakajima, K.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.01.025

2
K. Nakajima et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 1
DGAT1 inhibitors 1–4 and their in vitro/rat PK data
Compounds
1
2
3
4
DGAT1 IC50
(
l
M)11
0.007
0.6
0.2
33
0.011
0.4
0.1
31
0.027
1.0
0.2
42
>10
1.1
0.2
53
Caco-2 A–B (Â10À6 cm/s)
Caco-2 B–A (Â10À6 cm/s)
RLM CLh (mL/min/kg)13
Equilibrium solubility in pH 6.8 buffer (
l
M)13
<5
<5
<5
<5
Rat PK
Dose (mg/kg)—suspensiona
10
0
—
—
10
1
5
10
5
—
—
5
536
—
Systemic exposure AUC0–24 h (nM * h/mg/kg)
Dose (mg/kg)—solutionb
Systemic exposure AUC0–8 h (nM * h/mg/kg)
4
—
a
n = 3, vehicle: 0.5% methylcellulose/0.1% Tween 80.
n = 3, vehicle: 10% ethanol, 10% Cremophore, 30% PEG400, 50% vitamin E TPGS (10%).
b
Table 2
Design of DGAT1 inhibitors 5–7 and their in vitro data
Compounds
5
6
7
DGAT1 IC50
Equilibrium solubility in pH 6.8 buffer (
(
l
M)11
>10
710
4.8
60
3.5
<5
l
M)13
(AUC0–24 h = <5 nM * h/mg/kg). This poor exposure could not be improved
by oral solution administration for 2 (AUC0–8 h = 4 nM * h/mg/kg).
As 2 and 3 are not efflux substrates (Caco-2) and moderately stable
in the gut (RLM CLh), their poor systemic exposure might be attrib-
Subsequent investigation of other triazole substituents on 6 led
to identification of more potent analogs 15–17, whose syntheses
are shown in Scheme 1. Methyl thiourea 8 was diacylated to give
9, which was condensed with alkyl hydrazine,15 providing amido-
triazole 10. Acid-catalyzed deacylation gave aminotriazole 11. This
was coupled with oxazolone 13, which was in turn prepared from
N-Cbz protected aminobutyric acid 12, furnishing 14. The N-Cbz
group of 14 was removed, and the resulting free amine was acy-
lated to give 15–17. Table 3 shows DGAT1 IC50 and solubility data.
Substitution of a methyl group of the t-butyl group (6) with a
4-fluorophenyl group (15) improved potency by 2–3 fold
uted to their continued poor solubility (1–3:<5 lM, in vitro
assay).13
Interestingly, however, without improvement of in vitro
solubility, 4 (DGAT1 IC50 = >10 M) was well absorbed after oral
suspension administration to rats (systemic exposure AUC0–24 h
l
=
536 nM * h/mg/kg) (Table 1). We hypothesized that facile
ionization of the more acidic NH proton of the N-aryl amide or
sulfonamide (4) compared to the N-alkyl amides (1–3) improved
the solubility in vivo and hence systemic exposure for 4. As the
two adjacent amides were an important pharmacophore for good
DGAT potency (1–3),14 a heteroaryl group such as 1,2,4-triazole
was then introduced into the Eastern amide (5–7) (Table 2). With
concomitant replacement of the cyclopentyl and the Western acyl
groups with less lipophilic groups, we were delighted to observe
(IC50 = 1.4
a 4-fluorophenyl group (16) was more effective, slightly improving
potency (IC50 = 0.76 M) with three less carbon atoms from 15.
Potency was further improved when a methyl group was added
to one of the two 4-fluorophenyl groups (17) (IC50 = 0.062
M).16
All of these analogs improved DGAT1 potency from 6 while they
maintained similar solubility (8–15 M) to 6 (60 M).
lM). Replacement of the whole t-butyl group (6) with
l
l
l
l
that 6 and 7 showed inhibition of DGAT1 (IC50 = 4.8 and 3.5
lM,
As 17 is not only a potent DGAT1 inhibitor but also has good
respectively) with an improvement of solubility for 6 (60 M).
l
gut permeability (Caco-2: A–B = 20 Â 10À6 cm/s; B–A = 20 Â 10À6 cm/s)
Please cite this article in press as: Nakajima, K.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.01.025

K. Nakajima et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
O
a
R2
R2
R1
O
R2 NHNH2
b
O
S
O
S
Cl
N N
N
N N
N
R1
R1
N
NH
R1
H2N
R1
HN NH2
R1
N
c
H
8
9
10
11
R2
BnO
HN
O
BnO
O
O
N
11
O
O
N
O
R1
BnO
15-17
HN
OH
N
N
N
d
e
f
H
14
13
12
F
F
F
O
O
O
N
N
N
N
CF3
O
N
CF3
O
N
CF3
O
F
HN
HN
HN
F
F
N
N
N
N
N
H
N
H
H
15
16
17
Scheme 1. Synthesis of DGAT1 inhibitors 15–17. Reagents and conditions: (a) NaHCO3, CH2Cl2, H2O, rt, 80–96%; (b) Et3N or i-Pr2EtN, EtOH, 60–70 °C; (c) 6 N aqueous HCl,
EtOH, 60–70 °C, 83–95% over 2 steps; (d) EDC, CH2Cl2, rt, 41%; (e) Toluene, 60–80 °C, 68–77%; (f) (i) 33% HBr in acetic acid, rt or Et3SiH, PdCl2, i-Pr2EtN, CH2Cl2, 35 °C; (ii) 1-
(trifluoromethyl)cyclopropane carboxylic acid, HATU, i-Pr2EtN, DMF or CH2Cl2, rt, 31–90%.
and metabolic stability (RLM CLh = 13 mL/min/kg),13 its pharma-
Table 3
DGAT1 inhibitors 15–17 and their in vitro data
cokinetic properties were investigated in rats (Table 4). Despite
only moderate improvement of solubility (15 M) from 1-3
(<5 M), 17 was highly orally bioavailable (76%), consistent with
our hypothesis described above for improved systemic exposure
of 4. 17 was also found to be orally efficacious in a rat meal
challenge model,17 significantly suppressing absorption of orally
administered triglyceride into plasma (Fig. 1).
l
Compounds
15
16
17
l
DGAT1 IC50
Equilibrium solubility in pH 6.8 buffer (
(
l
M)11
1.4
12
0.76
8
0.062
15
l
M)13
In summary we have identified an effective strategy to optimize
lipophilic diamide compounds with poor molecular properties into
an orally bioavailable DGAT1 inhibitor by replacing an N-alkyl
group on the diamide with an N-heteroaryl group. Thus, a novel
amidotriazole compound (17) was identified as a potent inhibitor
of DGAT1 with in vivo efficacy.
Table 4
Sprague Dawley rat pharmacokinetic data of 17
Intravenous administration (0.5 mg/kg, n = 2)a
Plasma clearance (mL/min/kg)
Volume of distribution (L/kg)
Half-life (h)
7
0.9
1.8
2308
AUC0–7 h (nM * h)
Oral administration (1.5 mg/kg, n = 3)a
Cmax (nM)
Tmax (h)
AUC0–7 h (nM * h)
F (%)
Acknowledgements
1638
0.5
5014
76
The authors would like to thank Drs. Greg Bebernitz and Jason
Elliott for their helpful suggestions to this manuscript.
a
Vehicle: 5% 0.1 N HCl, 20% PG, 50(20%) Cremophore EL, 100 mM pH 5 citrate
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area above baseline.
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K. Nakajima et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
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might be a bioactive conformation of this scaffold, thus requiring two adjacent
amides for potent DGAT1 activity. Upon titration with increased amount of
acetone, the free NH proton of the Western amide was shifted downfield, but
chemical shift of NH proton of the Eastern amide was not affected. Based on
detailed NMR studies of a series of related compounds, we confirmed the
formation of an intramolecular hydrogen bond between the NH proton of the
Eastern amide and carbonyl oxygen of the Western amide.
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11. DGAT1 assay was performed in 384-well format, using membrane
preparations from Sf9 insect cells expressing recombinant human (His)
6DGAT1 (1.25
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
butanol and the triolein ammonium ion adduct measured by LC/MS/MS using a
Micromass Quattro Micro API mass spectrometer. IC50 values are the average of
at least duplicate experiments.
l
g/50
l
l reaction), diolein (50
l
M) and oleoyl-CoA (50
l
M) in
15. Yu, Y.; Ostresh, J. M.; Houghten, R. A. Tetrahedron Lett. 2003, 44, 7841.
16. Although 17 has not been tested in DGAT2 assay in vitro,
amidotriazole analogs (DGAT1 IC50 = <0.3 M) structurally related to 17 were
found to be poor inhibitors of DGAT2 (IC50 = >10 M).
a series of
ll
l
l
17. Compound 17 was suspended in 0.5 % carboxymethylcellulose and dosed
orally to rats at 10 mg/kg. At four hours post-dose of 17, an intralipid–glucose
meal was given to rats by oral gavage, and plasma triglyceride levels were
monitored during the following 2 h.
12. Plasma triglyceride levels were measured at 2, 4 and 6 h after oral gavage of 1
in corn oil in SD rats. Absorption of triglyceride into plasma was not
suppressed by 1 compared to the control group (corn oil).
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