Optimisation of biphenyl acetic acid inhibitors of diacylglycerol acetyl transferase 1 - The discovery of AZD2353

Article abstract of DOI:10.1039/c2md20190a

Inhibition of diacylglycerol acetyl transferase 1 is of great interest in the treatment of diabetes, obesity and other diseases that constitute the metabolic syndrome. Small molecule inhibitors of the enzyme are often dogged with physicochemical property-

Full text of DOI:10.1039/c2md20190a

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Optimisation of biphenyl acetic acid inhibitors of
diacylglycerol acetyl transferase 1 – the discovery of
AZD2353†
Cite this: Med. Chem. Commun., 2013,
4, 159
*
Michael J. Waring, Alan M. Birch, Susan Birtles, Linda K. Buckett, Roger J. Butlin,
Leonie Campbell, Pablo Morentin Gutierrez, Paul D. Kemmitt, Andrew G. Leach,
Philip A. MacFaul, Charles O’Donnell and Andrew V. Turnbull
Inhibition of diacylglycerol acetyl transferase 1 is of great interest in the treatment of diabetes, obesity and
other diseases that constitute the metabolic syndrome. Small molecule inhibitors of the enzyme are often
dogged with physicochemical property-related problems such as poor solubility. Herein, the optimisation
of a series of biphenyl acetic acid inhibitors is reported. Focus on ligand efficiency and ligand lipophilicity
efficiency and a strategy based on conformational restriction led to compounds with excellent potency and
ADMET properties, culminating in the discovery of AZD2353.
Received 11th July 2012
Accepted 26th September 2012
DOI: 10.1039/c2md20190a
www.rsc.org/medchemcomm
inhibitor series previously disclosed.4 Herein, the programme of
work to improve potency to the level required for a candidate
drug, whilst maintaining these attractive features is described.
Introduction
Diacylglycerol acyl transferase-1 (DGAT1) is a membrane bound
enzyme found in the endoplasmic reticulum, notably in enter-
ocytes and adipocytes. It catalyses the acylation of diac-
ylglycerols with fatty acid-coenzyme A active esters to form
triglycerides. Clearly, this is of critical importance in lipid
biosynthesis and hence is a target of great interest in the search
for new treatments for diabetes, obesity and other diseases that
constitute the metabolic syndrome.1 Small molecule inhibitors
of DGAT1 have progressed into the clinic from Novartis (LCQ-
908, phase II), Pzer (PF-4620110, phase I – discontinued) and
AstraZeneca (AZD-7687, phase I – discontinued, structure
undisclosed) and preclinical compounds have been disclosed
by Roche (RO-6036), Abbott (A-922500) and AstraZeneca (AZD-
3988), amongst others (Fig. 1).
Results and discussion
The initial strategy to improve potency without signicant
increases in molecular size or lipophilicity was based on
conformational analysis. The three aryl rings of 1 were expected
to have little preference for any particular conformation and
constraining the unbound molecule in its, albeit unknown,
active conformation would likely improve potency by reducing
the entropic penalty of binding associated with loss of rota-
tional freedom. Accordingly, initial structural modications
As part of an ongoing drug discovery programme, the
biphenyl acetic acid 1 (Table 1) was identied as an inhibitor of
DGAT1, which was promising as an attractive start point due to
its low lipophilicity/high lipophilic ligand efficiency (LLE) and
small size/high ligand efficiency (LE).2 These features were of
critical importance for the optimisation of an acidic series in
particular due to the need to maintain low lipophilicity and
hence desirable physical properties and a small size to achieve
acceptable permeability for such polar molecules.3 A second
attractive feature was the very high solubility of the compounds
given the precedented solubility issues with a number of DGAT1
Cardiovascular and Gastrointestinal Innovative Medicines Unit, AstraZeneca R&D,
Mereside, Alderley Park, Maccleseld, Cheshire, SK10 4TG, UK. E-mail: mike.j.
waring@astrazeneca.com; Tel: +44 (0)1625 230942
† Electronic supplementary information (ESI) available: Experimental protocols
and references thereof are given in the ESI. See DOI: 10.1039/c2md20190a
Fig. 1 Known DGAT1 inhibitors.
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Table 1 Chloro-substitution of R1 to R4
DGAT1 ACAT1
Sola/
Log D mM
R1 R2 R3 R4 pIC50
pIC50
LLE LE
Fig. 2 Optimal torsional angles for 2 about the critical rotatable bonds.
1
2
3
4
5
H
Cl
H
H
H
H
H
Cl
H
H
H
H
H
Cl
H
H
H
H
H
6.2
5.2
7.0
8.2
<4.5
<4.5
5.0
5.9
<4.5
0.3
0.6
0.6
0.5
0.3
1100 5.9
0.32
0.25
0.34
0.40
0.29
520
330
510
—
4.6
6.4
7.7
5.6
Cl 5.9
a
Crystalline solubility.
were concerned with the introduction of a chloro-substituent in
the R1-4 positions (Table 1) to perturb the torsional energy
prole of the molecule.
Since it had been observed previously within the project that
DGAT1 inhibitors oen carried ACAT1 activity,5 despite no
ACAT1 activity being observed for 1, all compounds were
assayed for both activities simultaneously.
The R1 and R4 chloro derivatives 2 and 5 lost DGAT1 potency
relative to 1. The R2 chloro 3 gained almost an order of
magnitude but the most marked effect was observed with R3
chloro 4, which exhibited a two log unit increase in potency
relative to 1, achieving the level of activity required for a drug
candidate. It is notable within this set that changing only the
position of chloro substitution can alter the observed activity by
three orders of magnitude overall with little change in log D and
constant molecular weight (LLE and LE ranges of 2.4 and 0.15
units respectively).
The conformational proles computed with B3LYP/6-31G*
for rotation about
a phenyl-pyrazine bond (with fully
substituted pyrazine) for model compounds with and without
chlorine to mimic 2 were computed (Fig. 2 and 3). These show
that if the change in LLE between 1 and 2 is due to a confor-
mational effect then the bioactive conformation is likely to
Fig. 3 Torsional profiles computed with B3LYP/6-31G* for rotation of the indi-
cated dihedral.
involve a twist about this bond of no more than ꢀ30ꢁ. In this severe enough to require a signicant margin between the two
range of dihedral angles, the model for 1 has a low energy while activities for a compound to be safe enough to progress.6 This
that of the model for 2 is signicantly higher. The effect of margin was set minimally at 100-fold, with an ideal compound
either R2 or R3 being Cl on the torsion linking the two phenyl having a margin signicantly in excess of this gure.
rings is likely to be very similar; both favour a twist of about
To follow up these encouraging potency improvements, the
ꢀ60ꢁ and at conformations (twist less than about 45ꢁ) are effect of varying the R2 and R3 substituents was investigated. In
excluded. That 4 has a higher LLE than 3 suggests that the Cl in the R2 position (Table 2), the methyl derivative 6 was broadly
the R3 position is able to form a constructive interaction with equivalent to 3 and the uoro derivative 7 showed equipotency
the DGAT enzyme. The drop in LLE caused by adding a Cl to R4 with a slightly improved ratio to ACAT1 and increased LLE.
is consistent with a bioactive conformation that has the More polar substituents methoxy-(8) and cyano-(9) led to
C–CO2H bond at an angle of ꢀ60ꢁ twist, the region in which the reduced potency but the magnitude of decrease was not
R4 ¼ H has an energy that is substantially lower than that of consistent with the drop in log D. The cyano derivative actually
R4 ¼ Cl.
showed increased LLE and a loss of measurable ACAT1 activity.
Some ACAT1 activity was observed with the more potent These LLE changes are possibly suggestive of specic interac-
DGAT1 inhibitors 3 and 4. Whilst this activity was only weak, the tions being made by these substituents with the protein in
potential consequences of ACAT1 inhibition were believed to be addition to their effects on lipophilicity and conformation.
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Table 2 Varying substitution in the R2 position
Table 4 a-Methyl substitution
ACAT1/
DGAT1 ACAT1 DGAT1
Sola/
Log D mM
ACAT1/
DGAT1 ACAT1 DGAT1
Sola/
R2
pIC50
pIC50
ratio
LLE LE
R5 R6 pIC50
pIC50
ratio
Log D mM
LLE LE
3
6
7
8
9
Cl
Me
F
OMe 5.2
CN 6.5
7.0
6.9
6.9
5.0
5.1
4.7
4.5
<4.5
96
55
180
4.5
>92
0.6
0.5
0.3
0.0
ꢂ0.4
330 6.4
0.34
0.33
0.34
0.25
0.30
>1200 6.4
930 6.6
410 5.2
560 6.9
1
H
H
H
6.2
6.7
<4.5
<4.5
4.8
>52
>150
19
0.3
0.7
0.7
1100
>1300 6.0 0.33
>1300 5.4 0.30
5.9 0.32
12 Me
13
H
Me 6.1
a
Crystalline solubility.
a
Crystalline solubility.
Accordingly, the effect of methyl substitution in this position
was investigated (Table 4). In this case, gratifyingly, the DGAT1
and ACAT1 potencies do indeed show orthogonal SAR with the
S-enantiomer 12 showing increased DGAT1 potency (0.5 log
increase, constant LLE) relative to 1, and the R-enantiomer 13
showing similar DGAT1 potency to 1 and introducing measur-
able ACAT1 activity.
However, both of these more polar substituents decreased the
overall log D of the molecule to such an extent that compro-
mised permeability was observed (for example, 8 and 9 had A to
B Papp values in MDCK cells of 59 and 12 nm sꢂ1 respectively)
and hence were not followed up.
With this information in hand, modications of the R3
position were selected so as not to lower the log D signicantly
(Table 3). In this case, chloro substitution proved to be optimal
with methyl (10) and uoro (11) both showing a loss of potency,
LLE and ACAT1 selectivity.
A further position of substitution likely to have an effect on
conformation and hence potency was the methylene substit-
uent a-to the carboxyl group. Substitution in this position would
also obviously introduce chirality to the molecule and possibly
affect the ACAT1 selectivity. In addition, phenylacetic acids are
known to sometimes have a propensity to form reactive acyl
glucuronide metabolites7 and it was postulated that a-substi-
tution would decrease the risk associated with this potential
issue.
The addition of this methyl group shis the conformational
minimum into one of two regions that were hypothesised to be
the bioactive conformation based on the effect of adding Cl at
R4. By desymmetrising the rotational prole, the methyl group
favours two of the four potential positions of the acid group and
so provides an entropic advantage for the correct enantiomer
and an enthalpic disadvantage for the other enantiomer.
These modications gave condence that a candidate suit-
able for development could be found within this series and that
a combination of the exemplied substituents was likely to
deliver the required prole. However, key considerations for
selection of a development candidate were ACAT1 selectivity,
predicted dose to man and acyl glucuronide risk. All of these
factors were impossible to predict from existing SAR and hence
an empirical approach to nding the best compound was
adopted. The entire matrix of suitable R2, R3 and R5 substitu-
ents was enumerated computationally and predictive potency
and log D data generated using Free–Wilson substructure based
models (see ESI†).8 Candidates of appropriate predicted potency
and log D were selected for synthesis and proled using in vitro
potency and ADMET, in vivo pharmacokinetic and in vitro
metabolite identication studies.
Table 3 Varying substitution in the R3 position
Some of the best compounds emerging from this exercise
illustrate the effects of combining the benecial R2, R3 and R5
substituents (Table 5). The R2, R3 dichloro derivative 14 shows
similar potency to 4, illustrating that the potency benets of the
individual chloro substituents do not necessarily add up. This
could be a conformational effect; one chloro substituent adja-
cent to the biphenyl bond induces a certain amount of twist,
having two chloros causes the twist to be enhanced, presumably
beyond that which is optimal.
ACAT1/
DGAT1 ACAT1 DGAT1
Sola/
Log D mM
R3
Cl
pIC50
pIC50
ratio
LLE LE
4
8.2
5.9
5.7
5.4
190
100
150
0.5
0.4
0.1
510
>440 7.3
7.5
7.7
0.4
10 Me 7.7
0.37
0.37
11
F
7.6
—
a
Crystalline solubility.
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Table 5 Best compounds resulting from substituent combinations
4
14
15
16/AZD2353
DGAT1 pIC50
ACAT pIC50
8.2
5.9
8.1
6.0
7.9
5.2
8.1
5.3
ACAT1/DGAT1 ratio
Log D7.4
190
0.5
110
1.0
550
1.1
580
1.0
LLE
7.7
7.1
6.8
7.1
LE
0.4
510
89
0.38
520
170
0.2, 0.9, 0.3
1.2 (560)
0.5
100
9.4 (1000)
0.4
0.37
570
190
0.6, 1.0, 0.8
1.8 (300)
0.6
98
3.0 (290)
1.0
0.37
270
140
0.3, 1.4, 0.7
2.4 (700)
1.3
100
1.9 (140)
0.4
Solubility/mM
MDCK Papp/nm sꢂ1
%Free (rat, dog, human)
0.5, 2.2, 1.5
Rat Cl (Clu)/mL minꢂ1 kgꢂ1 1.0 (210)
Rat Vss/L kgꢂ1
Rat F%
0.6
88
Dog Cl (Clu)/mL minꢂ1 kgꢂ1 5.2 (240)
Dog Vss/L kgꢂ1
Dog F%
0.5
14
24
73
37
Fig. 4 Dose response of 16 for inhibition of triglyceride excursion in a rat oral
lipid tolerance test. Data show concentration of plasma triglycerides (TAG). Labels
show the % reduction from vehicle dosed animals.
Fig. 5 Dose response of 16 for inhibition of triglyceride synthesis. Data show the
ratio of plasma triglycerides (TAG) to diglycerides (DAG). Labels show the %
reduction from vehicle dosed animals.
Combining R3 chloro with R5 methyl (15) retains potency
with reduced ACAT1 activity relative to 4. This change, whilst acceptable level (0.4 mg kgꢂ1, twice daily) and was selected for
clearly benecial overall, is not consistent with that observed development, being given the identier AZD2353.
with the pair 1 and 12 previously where an increase in DGAT1
The acyl glucuronide formed from 16 was shown to be more
potency was observed with the R5 methyl substituent. A stable than those derived from ibuprofen or diclofenac.9
combination of all three substituents (16, R2 ¼ uoro, R3 ¼ Coupled with the low dose, this minimised the risk of this
chloro, R5 ¼ methyl) also showed retained DGAT1 potency and generating reactive metabolite issues.
reduced ACAT1 activity.
Compound 16 showed dose proportional effects on triglyc-
All four of these compounds showed excellent ADME proles eride excursion in rat oral lipid tolerance (OLT) tests (Fig. 4)10,11
with very high solubility and permeability characteristics. This and on triglyceride synthesis (Fig. 5),10,12 achieving maximal
translated into desirable pharmacokinetic proles in both the efficacy at 1–3 mg kgꢂ1 in both cases. PK-PD analysis indicates
rat and the dog. However, for acidic series such as these, human that the IC50 values generated for OLT (Fig. 6) and triglyceride
dose predictions are very sensitive to small changes in phar- synthesis (Fig. 7) are in very good agreement with the level of
macokinetic parameters (and associated errors) and hence not inhibition of recombinant human DGAT1, inhibition in rat liver
all apparently good proles translate into desirable predictions. microsomes and with a functional cell assay measuring
Aer rigorous scaling using a number of methods, 16 was triglyceride synthesis in the HuTu80 human gastrointestinal
deemed the compound most suitable for human dosing at an cell line (Table 6). This shows that 50% inhibition of DGAT1 in
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Scheme 1 Synthesis of 16. Reagents and conditions: (a) K3PO4, DME/EtOH/H2O,
80 ꢁC; 91%; (b) PhN(CF3SO2)2, K2CO3, THF, 120 ꢁC; 75%; (c) Pd(dppf)Cl2$DCM,
LiCl, K3PO4, DME/MeOH/H2O, 65 ꢁC; 55%; (d) (CH2O)n, K2CO3, DMF, 0 ꢁC to rt;
100%; (e) CH3SO2Cl, Et3N, THF, rt; 55%; (f) KOH, t-BuOH, rt; 67%; (g) (S)-
Ru(OAc)2(BINAP), H2, MeOH, 5 bar, 35 ꢁC; 35%.
Fig. 6 PK/PD showing the relationship between free plasma concentration and
plasma TAG concentration for 16 in the OLT assay.
Synthesis
The synthesis of 16 commenced with sequential palladium
mediated couplings of 17, rstly to install the pyrazine moiety
and then, aer conversion of the phenol to the triate, the phenyl
acetic acid to afford 18 (Scheme 1). The a-methyl group was
installed in an enantioselective manner by interception of the
enolate formed from 18 with formaldehyde to afford the alcohol
19. The hydroxyl group of 19 was then eliminated to form the
acrylate 20 which was saponied to acid 21. Finally, 21 was
reduced by stereoselective hydrogenation in the presence of (S)-
Ru(OAc)2(BINAP) as catalyst to afford homochiral 16. The abso-
lute conguration of 16 was established by X-ray crystallography.
Conclusions
In summary, it has been shown that substituents which provide
conformational restriction provided a highly effective means of
optimising this series avoiding ination of molecular size or
lipophilicity. Signicant improvements in potency and selec-
tivity were achievable with this strategy resulting in dramatic
increases in LE and LLE. Finally, Free–Wilson analysis provided
an effective means of predicting the best compounds within the
series resulting in the identication of 16 as a highly potent,
selective inhibitor of DGAT1 with promising in vivo activity and
a candidate for progression to the clinic.
Fig. 7 PK/PD showing the relationship between free plasma concentration and
plasma TAG to DAG ratio for 16 in the TAG synthesis assay.
Table 6 Comparison of in vivo derived pIC50 values for 16 with those from
isolated enzyme, microsomal and whole cell assays
In vivo OLT pIC50
In vivo TAG synthesis pIC50
DGAT1 pIC50
Rat microsomal pIC50
HuTu80 TAG synthesis pIC50
9.3
8.7
8.1
8.4
8.4
Acknowledgements
The research was sponsored by AstraZeneca. All authors were
employees of AstraZeneca at the time of the conduct of the
adipose tissue or in the GI tract can be achieved if the free research. The authors thank Craig Donald, Neal Fazakerley,
plasma concentration of 16 is maintained at the in vitro IC50 Mark D. Fenwick, Adele Lloyd, Adrian Pickup, Wendy Snelson
value.
and Rolf P. Walker for the synthesis and purication of test
Compound 16 showed a benign safety prole with no compounds, Usha Chauhan for the testing of compounds in
signicant activity in a panel of more than 100 in vitro secondary biological assays and colleagues in physical chemistry, DMPK
pharmacology assays and no signicant histopathological and safety assessment for the provision of secondary ADMET
ndings in in vivo safety studies.
data.
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and M. J. Waring, Med. Chem. Commun., DOI: 10.1039/
c2md20187a.
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