Identification, optimisation and in vivo evaluation of oxadiazole DGAT-1 inhibitors for the treatment of obesity and diabetes

Article abstract of DOI:10.1016/j.bmcl.2012.04.117

A novel series of DGAT-1 inhibitors was discovered from an oxadiazole amide high throughput screening (HTS) hit. Optimisation of potency and ligand lipophilicity efficiency (LLE) resulted in a carboxylic acid containing clinical candidate 53 (AZD3988), wh

Full text of DOI:10.1016/j.bmcl.2012.04.117

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3873–3878
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification, optimisation and in vivo evaluation of oxadiazole DGAT-1
inhibitors for the treatment of obesity and diabetes
⇑
William McCoull , Matthew S. Addie, Alan M. Birch, Susan Birtles, Linda K. Buckett, Roger J. Butlin,
Suzanne S. Bowker, Scott Boyd, Stephen Chapman, Robert D.M. Davies, Craig S. Donald, Clive P. Green,
Chloe Jenner, Paul D. Kemmitt, Andrew G. Leach, Graeme C. Moody, Pablo Morentin Gutierrez,
Nicholas J. Newcombe, Thorsten Nowak, Martin J. Packer, Alleyn T. Plowright, John Revill, Paul Schofield,
Chris Sheldon, Steve Stokes, Andrew V. Turnbull, Steven J.Y. Wang, David P. Whalley, J. Matthew Wood
Cardiovascular & Gastrointestinal Innovative Medicines Unit, AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of DGAT-1 inhibitors was discovered from an oxadiazole amide high throughput screening
(HTS) hit. Optimisation of potency and ligand lipophilicity efficiency (LLE) resulted in a carboxylic acid
containing clinical candidate 53 (AZD3988), which demonstrated excellent DGAT-1 potency (0.6 nM),
good pharmacokinetics and pre-clinical in vivo efficacy that could be rationalised through a PK/PD
relationship.
Received 3 April 2012
Revised 25 April 2012
Accepted 27 April 2012
Available online 2 May 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
DGAT
Oxadiazole
LLE
Obesity
PK/PD
N
N
Inhibition of diacylglycerol acyl transferase-1 (DGAT-1) is of
increasing interest as a mechanism for therapeutic treatment of dia-
betes, obesity and other diseases which together constitute meta-
bolic syndrome.1–3 DGAT-1 deficient (Dgatꢀ/ꢀ) mice are viable,
resistant to weight gain when fed a high-fat diet and show increased
insulin and leptin sensitivity.4 Ourselves,5 and others6–14 have re-
cently reported small molecule inhibitors of DGAT-1 from different
chemical series and the progress of some compounds into clinical
development supports the potential for therapeutic use.15–17 In this
Letter we report our discovery and optimisation of oxadiazole
amide inhibitors, leading to the clinical candidate 53 (AZD3988).
High throughput screening (HTS) of the AstraZeneca compound
collection was conducted and identified oxadiazole amide 1 (Fig. 1)
originating from a kinase directed screening library (although inac-
tive against a panel of kinases). This compound had moderate po-
O
O
N
H
N
N
F
O
H
1
Figure 1. HTS hit from high throughput screening.
N
N
N
O
N
N
Figure 2. Unsuccessful oxadiazole replacements.
N
N
O
tency as a DGAT-1 inhibitor (IC50 = 0.52
l
M)18 and was selective
M).19
O
R
N
N
O
H
against the related DGAT-2 enzyme (zero inhibition at 10
l
2 R = NH
The log D was measured20 at 3.0 resulting in an acceptable ligand
lipophilicity efficiency (LLE)21 start point of 3.2. Consequently,
improvement in potency and LLE were key aims throughout the
optimisation campaign.
3 R = NHC=O
Figure 3. Amido and anilino oxadiazole leads.
Initial chemistry efforts were focused on understanding the key
features necessary for potency within the core of the molecule. The
oxadiazole was systematically replaced by numerous heteroaryl
and phenyl ring systems (Fig. 2) but all resulted in significant
⇑
Corresponding author.
E-mail address: william.mccoull@astrazeneca.com (W. McCoull).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.117

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W. McCoull et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3873–3878
bility, typically less than 10
these analogues which were generally synthesized in a crystalline
form. Basic compound 15 did show higher solubility of 91 M but
high clearance in rat of greater than 100% liver blood flow pre-
cluded further optimisation of such compounds.
lM was a problematic feature for all
N
N
O
O
a, b
H
N
c
NH2
NH2
R2
R1
O
N
H
R1
N
H
N
R1
l
O
H
4
5
6
Increasing flexibility through introducing sp3 character was
used as a tactic to improve solubility and explore SAR (Table 2).
Unfortunately, solubility remained low although carbon, oxygen,
nitrogen or sulfur could be used as a linker with a suitably large
substituent to achieve good potency. Notably, increasingly lipo-
philic ethers such as compounds 20–22, gave increasing potency
suggesting a lipophilic binding interaction was responsible. Re-
duced potency of pyridine 23 is consistent with this hypothesis.
Lipophilicity was generally too high to be accurately measured,
only 20 and 23 returning log D values of 3.2 and 3.1 respectively,
thus further exploration to achieve our dual potency and LLE aims
was not justified
Scheme 1. General synthetic procedure for substituted oxadiazole amides.
Reagents: (a) MeOCOCOCl, pyridine, CH2Cl2; (b) NH2NH2, EtOH, reflux; (c) R2NCS,
PS-CDI.
potency loss (>10-fold). Similarly, methylation of either the amide
or aniline nitrogen resulted in loss of potency. The pharmacophore
was not tolerant to changes anywhere but the terminal fragments.
The only encouraging alteration to the amide-oxadiazole-ani-
2 (IC50 = 0.30 lM,
LLE = 3.4) to an amide 3 (IC50 = 5.2 M, LLE = 4.3) as shown in
Figure 3. Although potency was reduced, the bis-amide compound
3 showed improved LLE and both these sub-series were progressed
to lead expansion activities.
The compounds described in this Letter were prepared using
similar methods22,23 which allowed diversification of R1 and R2
(Scheme 1). Starting with anilines 4, reaction with methyl chloro-
oxoacetate followed by hydrazine afforded acyl-hydrazines 5
which were condensed with various isothiocyanates. Ring closure
to form oxadiazoles 6 was effected using polymer-supported car-
bodiimide (PS-CDI)
line core was conversion of the aniline
l
The aniline R2 group was extensively explored and a large vari-
ety of substitution was tolerated at several positions on the aryl
ring (Table 3). Notably, substitution at the 3-position often gave
Table 2
DGAT-1 potency for selected morpholine replacements
N
N
O
The amide-oxadiazole-aniline sub-series was initially explored
as a two-dimensional array, varying R1 and R2. Principal compo-
nent analysis24, using common descriptors such as clogP, polar
surface area (PSA) and hydrogen bond counts, was used to guide
design of a diverse set of compounds but all significant structural
deviations away from 1 resulted in a loss of potency (data not
shown). Consequently, more conservative SAR exploration was
conducted around the morpholino aryl side chain R1 (Table 1).
Addition of a single F (7) or Me (8) group on the arylamide-3-posi-
tion maintained potency but with lower LLE. However, at the aryla-
mide-2-position F (9) resulted in a loss of potency. Lowering
lipophilicity by conversion of the aryl group to a pyridine could
be tolerated with the 3-aza 10 improving potency whereas the 2-
aza compound 11 showed a significant decrease. Changing the
morpholine to more lipophilic groups, for example piperidine 12
or thiomorpholine 13, also improved potency. Although potent,
13 exhibited high in vitro clearance in human microsomes
O
N
H
N
R
F
H
X
a
Compd
X
R
h DGAT-1 IC50 (lM)
10
17
18
19
20
21
22
23
24
25
N
Morpholine
n-Bu
0.13
CH
CH
CH
N
N
N
N
N
CH
0.62b
0.31
c-Hex
Ph
MeO
n-BuO
PhO
0.12
8.0
0.060
0.0067
0.41
0.060
0.016
3-PyridylO
PhN
n-BuS
a
Mean values from P2 experiments unless otherwise stated.
n = 1.
b
(Clint = 258
(Cmax = 0.02
l
l
L/min/mg) and low oral exposure in rat
M from 2 mg/kg dose) thus was not progressed. Oxi-
Table 3
DGAT-1 potency, log D and LLE for selected R2 variations
dation of 13 to sulfoxide 14 reduced potency. Piperazines 15 and
16 were both tolerated and gave improved LLE. Low aqueous solu-
N
N
O
O
N
H
N
R
N
H
O
N
a
Compd
R
h DGAT-1 IC50
(lM)
log D
LLE
Table 1
DGAT-1 potency, log D and LLE for selected R1 variations
26
10
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
H
2-F
3-F
4-F
3,4-diF
2-Cl
3-Cl
0.62
0.13
0.15
0.84
2.9
2.7
3.2
3.0
2.9
2.9
>3.4
—
2.8
3.6
>4.2
>4.2
3.1
3.3
3.4
>4.0
—
3.3
4.2
3.6
3.1
3.6
3.0
—
N
N
O
Y
O
N
H
N
N
F
W
H
0.34
X
1.3b
a
Compd
X
Y
W
h DGAT-1 IC50
(l
M)
log D
LLE
0.092
0.19
4-Cl
—
1
7
8
9
10
11
12
13
14
15
16
CH
CF
CMe
H
CH
CH
CH
CF
CH
N
CH
CH
CH
CH
CH
O
O
O
O
O
O
CH2
S
S@O
NM e
NHAc
0.52
0.46
0.35
>30
0.13
11
0.19
0.023
3.7
0.46
0.54
3.0
3.6
4.0
3.4
2.7
2.4
>4.2
3.0
—
3.2
2.7
2.5
—
4.2
2.5
—
4.6
—
4.0
3.8
3-OMe
3-OEt
3-OPh
3-OCH2Ph
3-OCH2-2-pyridyl
3-OCH2-3-pyridyl
3-Me
0.73b
0.032b
0.0066b
0.013
0.54
3.3
3.9
—
—
N
3.2
2.4
3.4
—
CH
CH
N
N
N
2.1
0.17
3-Ph
3-CO2H
0.0057
>30b
—
2.4
2.5
CH
a
Mean values from P2 experiments unless otherwise stated.
n = 1.
b
a
Mean values from P2 experiments.

W. McCoull et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3873–3878
3875
increased potency. Increasing lipophilic bulk at this position gave
increasing potency within an ether series (compounds 33–36)
and again attempts to introduce polarity generally resulted in a
loss of potency, for example compounds 37 and 38. Introduction
of a carboxylic acid was not well tolerated in compound 41.
The bis-amide sub-series afforded the opportunity to achieve
higher solubility by means of an ionisable centre. The pKa of such
compounds was typically in the range 5–7, depending on other
substituents and resulted in solubilities generally greater than
10 lM. Modest DGAT -1 potency improvements could be made
but were generally accompanied with increased lipophilicity
and decreased LLE (Table 4). High metabolic clearance, particu-
larly in rat (e.g., 43 showed 62% liver blood flow and 3% bioavail-
ability), led us to discontinue work in this series.
Introduction of an ionisable centre, particularly a carboxylic
acid remained a key goal to obtain acceptable solubility and high
LLE. Disclosure of a potent acidic series in the patent literature25
encouraged us to continue to explore the incorporation of an acid
group into the R1 side chain (Table 5). Modestly potent butanoate
46 indicated that this would be possible and biphenyl containing
acids 47–49 maintained potency relative to the biphenyl non-acid
Table 4
DGAT-1 potency, log D and LLE for selected bis-amides
N
N
O
O
O
N
H
R
N
N
H
O
a
Compd
R
h DGAT-1 IC50
0.37
(l
M)
log D
LLE
4.5
Cl
42
1.9
Cl
43
44
0.52
0.32
3.0
3.2
3.3
3.3
Cl
Cl
45
0.18
3.7
3.0
a
Mean values from P2 experiments.
Table 5
DGAT-1 potency, log D and LLE for selected acids
N
N
O
O
N
H
N
R
F
H
a
Compd
R
h DGAT-1 IC50
(lM)
log D
LLE
46
47
HO2C
3.3b
—
—
—
—
HO2C
HO2C
0.21b
48
0.07
—
—
HO2C
HO2C
HO2C
49
50
0.08
1.3
1.5
—
5.6
—
51
52
0.0044
0.035
2.4
2.1
5.9
5.4
HO2C
a
Mean values from P2 experiments unless otherwise stated.
n = 1.
b

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W. McCoull et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3873–3878
Table 7
Pharmacokinetic parameters for 53a
Species
Clp (mL/min/kg)
Vdss (L/kg)
IV half-life (h)
po half-life (h)
po Cmax
(l
M)
Bioavailability (%)
Mouse
Rat
Dog
4.6
1.1
2.5
0.99
0.35
0.36
4.9
3.4
1.8
4.7
5.8
5.7
1.9
20
1.5
>100
>100
32
a
Compound was dosed IV at 0.5 mg/kg (mouse), 2 mg/kg (rat) and 1 mg/kg (dog) and po at 1 mg/kg (mouse) in 5% DMSO: 95% hydroxypropyl b cyclodextrin; po at 5 mg/kg
(rat) and 1 mg/kg (dog) in hydroxypropyl methylcellulose (HPMC) with 0.1% polysorbate.
19. Saturation of either ring resulted in decreased potency in 50 but
significantly increased potency in 51 relative to the non-acid 18.
Subsequent to this work, the acid fragment R in 51 has been re-
ported as a privileged structure contained within several DGAT-1
inhibitors.5,12,13,26 Des-homologue 52 was also potent but the phe-
Aqueous solubility of crystalline 53 is lower than ideal but is coun-
terbalanced by good cellular permeability, resulting in good phar-
macokinetic profiles determined in vivo in three pre-clinical
species: mouse, rat and dog (Table 7). Oral exposure was substan-
tial in all species. The rat and mouse pharmacokinetic profiles were
characterised by high oral bioavailability and low clearance while
more moderate bioavailability was observed in dog.
nyl-c-hexyl-ethanoate group gave
a clear potency and LLE
advantage.
Our previous R2 aniline SAR could be mapped onto a new acid-
containing sub-series and 53 (AZD3988) was quickly identified as
having a good overall balance of properties, most notably achieving
our key aims of high potency and LLE (Table 6). Excellent potency
in inhibiting triacylglycerol (TAG) synthesis in human HuTu 80
cells, relevant to the gut as a target organ, was determined. 53
showed no significant activity against DGAT-2, the hERG encoded
In an oral lipid tolerance test (OLTT), fasted rats were adminis-
tered compound 53 at a range of doses 2 h prior to a bolus dose of
corn oil and plasma TAG levels were measured 1.5 h later. Figure 4a
shows that 53 was highly efficacious in reducing plasma TAG
potassium channel (>30
CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (all >30
l
M), and cytochrome P450 enzymes
M).
l
Figure 5a. Pharmacodynamic effects of 53. Dose related reduction in adipose
triacylglycerol synthesis as determined by changes in the ratio of radiolabel
incorporation into TAG and DAG. Mean values SEM (⁄p <0.05, ⁄⁄⁄p <0.001).
Figure 4a. Pharmacodynamic effects of 53. Dose related reduction in plasma TAG
excursion at 1.5 h post bolus corn oil administration. Mean values SEM (⁄⁄p <0.01,
⁄⁄⁄p <0.001).
Figure 4b. PK/PD analysis of rat OLTT (plasma TAG vs free compound concentra-
tions in plasma) for 53. A direct response (Emax sigmoidal) model was used to fit the
PK/PD data.
Figure 5b. PK/PD analysis of rat adipose TAG synthesis assay (adipose tissue
TAG:DAG ratio vs free compound concentrations in plasma) for 53.
response (Emax sigmoidal) model was used to fit the PK/PD data.
A direct

W. McCoull et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3873–3878
3877
Table 8
PK/PD parameters for model fit for 53 in the rat OLTT
Parameter
Estimate
Units
Stderr%
IC50
Gamma
E0
0.00005
2.4
3.2
l
—
mM
mM
M
17
30
6.8
11
Imax
2.3
excursion in this model, exhibiting 40%, 66% and 73% inhibition at
doses of 0.03, 0.1 and 0.2 mg/kg respectively. Fig. 4b shows the
relationship between plasma TAG and free compound levels in
plasma for 53 in the rat OLTT assay. A direct response PK/PD model
(Emax sigmoidal model) was used to successfully fit the PK/PD data
showing a clear correlation between compound levels in plasma
and an effect in the OLTT. PK/PD parameters for the model fit are
Figure 6. Percentage reduction in body weight compared to start weight in DIO
mice receiving high energy cafeteria diet. Day 0 is first treatment day; mice
weighed and dosed at 6:00 and 16:00 each day. In mice receiving 53, statistical
differences from DIO controls were seen at all time points (⁄⁄⁄p <0.001).
shown in Table 8. The fact that the in vivo IC50 (0.00005
lM) is
10-fold lower than the in vitro IC50 (0.0005 M) is compatible with
l
the mouse DIO study for 53, free compound concentrations in plas-
ma were maintained >20-fold above the in vitro IC50 (0.0011 lM)
the idea that local inhibition of DGAT-1 in the gut (and not the sys-
temic exposure) could be the main driver for efficacy in the OLTT
assay.
for the whole dosing interval. At these compound concentrations,
based on the rat PK/PD knowledge, it is expected that compound
53 has fully inhibited DGAT-1 both in the gut and in the adipose
tissue. These effects on body weight appear specific and consistent
with the mechanism of action. Accordingly 53 had no effects on
body weight at similar or even higher exposures in lean animals,
and effects on food intake were only observed in animals fed a high
fat diet.
In summary, the novel oxadiazole DGAT-1 inhibitor 1 was iden-
tified and SAR exploitation enabled a design strategy to improve
both potency and LLE leading to the clinical candidate 53
(AZD3988), which displayed good pharmacokinetics and demon-
strated in vivo efficacy in obesity related models. Results from
the further development of 53 (AZD3988) as a drug to treat meta-
bolic disease will be reported in due course.
Imax ꢁ Cc
E ¼ E0 ꢀ
ICc50 þ Cc
Compound 53 also showed good effects on adipose TAG synthe-
sis (expressed as TAG:DAG ratio in adipose tissue) giving 33%, 65%,
78% and 78% decrease in the TAG:DAG ratio in adipose tissue at 0.1,
0.3, 1 and 3 mg/kg, respectively (Fig. 5a). Fig. 5b shows the rela-
tionship between adipose TAG synthesis and free compound levels
in plasma for 53 in the rat. A direct response PK/PD model (Emax
sigmoidal model) was used to succesfully fit the PK/PD data show-
ing a clear correlation between free compound levels in plasma
and an effect in the adipose TAG:DAG ratio. PK/PD parameters
for the model fit are shown on Table 9.
The in vivo IC50 (0.0003
very good agreement with the in vitro IC50 (0.0005
larger than the one measured in the OLTT assay (0.00005
l
M) in the adipose tissue assay is in
M), and it is
M) for
Acknowledgments
l
l
Teresa Collins and Mark Denn are acknowledged for expert
technical assistance in generating DMPK data. Usha Chauhan’s ex-
pert technical assistance in generating enzyme inhibition data is
also acknowledged.
this compound. These differences in in vivo IC50 between OLTT
and TAG synthesis are expected as we used free compound concen-
tration in plasma as a surrogate for compound concentration at the
target. In the OLTT assay we believe that local compound concen-
trations in the gut are driving effects and therefore using systemic
exposure as a surrogate will underestimate the compound concen-
trations at the target leading to a smaller IC50. For the adipose TAG
synthesis assay that does not seem to be the case and the observed
in vivo IC50 using free compound concentration in plasma is in very
good agreement with the in vitro one.
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E ¼ E0 ꢀ
ICc50 þ Cc
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Table 9
PK/PD parameters for model fit for 53 in the rat adipose TAG synthesis assay
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Estimate
Units
Stderr%
IC50
Gamma
E0
0.0003
4.2
5.2
lM
7.8
42
4.9
7.1
—
—
—
Imax
4.1

3878
W. McCoull et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3873–3878
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18. Assay protocols can be found in Ref. 5