Discovery of Tetralones as Potent and Selective Inhibitors of Acyl-CoA:Diacylglycerol Acyltransferase 1

Article abstract of DOI:10.1021/acsmedchemlett.7b00450

Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) plays an important role in triglyceride synthesis and is a target of interest for the treatment of metabolic disorders. Herein we describe the structure-activity relationship of a novel tetralone series of DGAT1 inhibitors and our strategies for overcoming genotoxic liability of the anilines embedded in the chemical structures, leading to the discovery of a candidate compound, (S)-2-(6-(5-(3-(3,4-difluorophenyl)ureido)pyrazin-2-yl)-1-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydronaphthalen-2-yl)acetic acid (GSK2973980A, 26d). Compound 26d is a potent and selective DGAT1 inhibitor with excellent DMPK profiles and in vivo efficacy in a postprandial lipid excursion model in mice. Based on the overall biological and developability profiles and acceptable safety profiles in the 7-day toxicity studies in rats and dogs, compound 26d was selected as a candidate compound for further development in the treatment of metabolic disorders.

Full text of DOI:10.1021/acsmedchemlett.7b00450

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Letter
Discovery of Tetralones as Potent and Selective
Inhibitors of Acyl-CoA: Diacylglycerol Acyltransferase 1
Mui Cheung, Raghuram Tangirala, Sridhar R Bethi, Hemant V
Joshi, Jennifer L Ariazi, Vijaya G Tirunagaru, and Sanjay Kumar
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00450 • Publication Date (Web): 16 Jan 2018
Downloaded from http://pubs.acs.org on January 17, 2018
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Discovery of Tetralones as Potent and Selective Inhibitors of Acylꢀ
CoA: Diacylglycerol Acyltransferase 1
Mui Cheung,*† Raghuram S. Tangirala,# Sridhar R. Bethi,# Hemant V. Joshi,# Jennifer L. Ariazi,† Vijaya
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G. Tirunagaru,# Sanjay Kumar†
†Virtual Proof of Concept Discovery Performance Unit, Alternative Discovery and Development, GlaxoSmithKline, 709
Swedeland Road, King of Prussia, PA 19406. #Collaborative Research, GVK Biosciences Private Limited, 28A, IDA, Nachaꢀ
ram, Hyderabad 500076, India.
KEYWORDS DGAT1 inhibitors, tetralones, GSK2973980A, acyltransferase, metabolic disorders, aniline, genotoxicity, mu-
tagenicity, Ames.
ABSTRACT: AcylꢀCoA:Diacylglycerol Acyltransferase 1 (DGAT1) plays an important role in triglyceride synthesis and is a tarꢀ
get of interest for the treatment of metabolic disorders. Herein we describe the structureꢀactivity relationship (SAR) of a novel teꢀ
tralone series of DGAT1 inhibitors and our strategies for overcoming genotoxic liability of the anilines embedded in the chemical
structures, leading to the discovery of a candidate compound, (S)ꢀ2ꢀ(6ꢀ(5ꢀ(3ꢀ(3,4ꢀdifluorophenyl)ureido)pyrazinꢀ2ꢀyl)ꢀ1ꢀoxoꢀ2ꢀ
(2,2,2ꢀtrifluoroethyl)ꢀ1,2,3,4ꢀtetrahydronaphthalenꢀ2ꢀyl)acetic acid (GSK2973980A, 26d). Compound 26d is a potent and selective
DGAT1 inhibitor with excellent DMPK profiles and in vivo efficacy in a postprandial lipid excursion model in mice. Based on the
overall biological and developability profiles and acceptable safety profiles in the 7ꢀday toxicity studies in rats and dogs, compound
26d was selected as a candidate compound for further development in the treatment of metabolic disorders.
Synthesis of triglycerides (TG) is a fundamental biochemical
pathway important for nutrient utilization and energy storage.
Excess TG has been linked to human diseases such as obesity,
insulin resistance, dyslipidemia, and nonalcoholic steatohepaꢀ
titis (NASH). AcylꢀCoA:Diacylglycerol acyltransferase 1
(DGAT1) catalyzes the final step in the TG biosynthetic pathꢀ
way. Mice lacking DGAT1 are viable and show reduced
plasma and tissue TG levels associated with increased sensiꢀ
tivity to insulin and leptin, and increased expression of leptinꢀ
regulated genes compared to wild type mice.1 DGAT1 defiꢀ
ciency protected against hepatic steatosis by reducing TG synꢀ
thesis and increasing fatty acid oxidation in adipose and skeleꢀ
tal muscle.2,3 Furthermore, DGAT1 deficient mice are reꢀ
sistant to dietꢀinduced obesity.4 Since DGAT1 is an attractive
therapeutic target for the treatment of metabolic disorders,
there has been intense interest over recent years in identifying
DGAT1 inhibitors.5ꢀ7
Our initial discovery effort was focused on tetrahydronaphthaꢀ
lene and tetralone series of DGAT1 inhibitors (Figure 2).13
Tetrahydronapthalene (Compound A) and tetralone (Comꢀ
pound B) emerged as interesting initial hits which showed
IC50 values of 5.1 and 2.8 ꢀM, respectively, in the human
DGAT1 (hDGAT1) fluorescenceꢀbased biochemical assay
(CPM assay).
Representative DGAT1 inhibitors (LCQ908,8,9 PFꢀ
04620110,10 and AZD768711,12) which have been evaluated in
clinical trials are shown in Figure 1. All three compounds posꢀ
sessed a phenyl cyclohexyl acetic acid functionality that is a
Figure 1. Structures of representative DGAT1 inhibitors.
privileged scaffold for DGAT1 inhibitors.
Pradigastat
(LCQ908) is the most advanced DGAT1 inhibitor and is curꢀ
rently in Phase III evaluation for the treatment of familial chyꢀ
lomicronemia syndrome.8,9 Herein, we report our efforts that
led to the discovery of a candidate compound, GSK2973980A
(26d), a novel, potent and highly selective DGAT1 inhibitor,
as a potential therapeutic agent for the treatment of metabolic
disorders.
Figure 2. Tetrahydronaphthalene (Compound A) and Tetralone
(Compound B) as DGAT1 hits.
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Limited SAR explorations suggested that meta substitution on
Page 2 of 7
stitution and lower molecular weight (MW) compared to triꢀ
fluoroethyl substitution. In addition, compounds 12o and 12p
have reasonable solubility and acceptable potency. Further
exploration of the D ring with these αꢀsubstituents is shown in
Table 2.
1
2
3
4
5
6
7
8
ring D improved potency in both Compounds A and B and
tetralones tend to have better potency than tetrahydronaphthaꢀ
lenes. Therefore, tetralone (Compound B) was selected for
lead optimization. There were two perceived challenges assoꢀ
ciated with the monoꢀsubstituted tetralone AB ring system: (1)
the presence of an enolizable ketone could result in racemizaꢀ
tion, and (2) the ketone functionality could be labile and subꢀ
jected to nucleophilic attack. We envisioned resolving these
issues by introducing a quaternary center at the αꢀposition of
ketone in the AB ring with the goal of improving stability of
the compound. In addition, we explored the SAR of the ring
D substitutions with the goal of improving potency and PK
profiles of DGAT1 inhibitors.
Table 1. SAR of the αꢀSubstituent of Tetralones 12oꢀs.
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Soluꢀ
hDGAT1
Compd
#
bility
(µg/m
L)b
clogPa
4.6
CPM IC50
Tetralone derivatives were synthesized using standard condiꢀ
R
H
MW
(nM)b
tions as described in Scheme 1.
Treatment of 6ꢀ
methoxytetralone 1 with glyoxalic acid in diglyme followed
by sulfuric acid under heating conditions provided olefin 2.
Reduction of the olefin 2 under Zn/AcOH conditions followed
by esterification resulted in ester 4. Alkylation of compound 4
followed by demethylation and subsequent ester hydrolysis
afforded acid 6. Reꢀesterification of acid 6 followed by conꢀ
version of the phenol to its corresponding triflate led to the
triflate 8. Suzuki coupling of triflate 8 with pꢀnitrophenyl boꢀ
ronic acid gave nitro compound 9 which was reduced to its
corresponding aniline 10. Aniline 10 was then converted to
urea 11 upon treatment with an appropriately substituted pheꢀ
nyl isocyanate. Finally, hydrolysis of esters of urea 11 with
LiOH provided the corresponding tetralone derivatives 12.
Compd
B
414.5
2820
58
12o
12p
12q
Me
Et
428.5
442.5
456.5
5.1
5.6
6.2
126
63
54
34
74
nꢀPr
16
84% @ 10
12r
F
432.5
4.8
31
ꢀM
12s
CF3CH2
496.5
5.5
3
30
aClogP is a calculated LogP value using Daylight model.
bValue is an average of at least n=2.
Scheme 1. General Synthesis of Tetralone Derivatives.a
We first explored electronic effect of small substituents at the
R5 meta position of the D ring, based on our prior knowledge,
for both αꢀmethyl and αꢀethyl tetralones. αꢀEthyl analogues
(12aꢀe) were consistently more potent against hDGAT1 than
the corresponding αꢀmethyl analogues (12jꢀn) regardless of
the electronic effect of the substituents. In addition, analogues
with electronꢀwithdrawing substituents (CF3 and Cl) showed
significant improvement in potency over the electronꢀdonating
substituents (Me and –OMe) for the corresponding αꢀmethyl
analogues (12j and 12l vs 12k and 12m) and αꢀethyl anaꢀ
logues (12a and 12c vs 12b and 12d). We also noted that para
substitution (12fꢀg) maintained similar hDGAT1 potency to
the corresponding meta substitution (12aꢀc); however, the
orthoꢀsubstituted CF3 analog (12i) showed significant reducꢀ
tion in hDGAT1 potency when compared to the corresponding
paraꢀ (12f) and meta-substituted analog (12a).
aReagents and conditions: (a) Glyoxalic acid, H2SO4, diglyme;
(b) Zn, AcOH; (c) MeSO3H, EtOH; (d) Alkyl halide, NaH, DMF;
(e) Aq. HBr; (f) CH3SO3H, EtOH; (g) Tf2O, CH2Cl2, Et3N; (h) 4ꢀ
Nitrophenyl boronic acid, Pd(PPh3)4, Cs2CO3, DioxaneꢀH2O; (i)
FeꢀNH4Cl, EtOHꢀH2O; (j) R1PhNCO, Et3N, THF; (k) LiOH,
THFꢀH2O.
All the tetralone derivatives tested thus far in the biological
assays were racemates. Therefore, in order to determine if
there was any stereo preference, the most potent racemic anaꢀ
logues (12a and 12g) were chosen for separation. The R and S
enantiomers of 12a and 12g were obtained by separation of the
corresponding ethyl ester precursors 11a and 11g by chiral
semiꢀprep HPLC followed by ester hydrolysis of esters (Rꢀ
11a, Sꢀ11a, and Rꢀ11g, Sꢀ11g) to acids (Rꢀ12a, Sꢀ12a, and Rꢀ
12g, Sꢀ12g). The absolute stereochemistry was determined
and assigned by the ab initio Vibrational Circular Dichroism
(VCD) analysis of the corresponding ethyl ester precursors (Rꢀ
11a, Sꢀ11a, and Rꢀ11g, Sꢀ11g).14 Biological activities of the
racemates (12a and 12g) and their corresponding enantiomers
in S and R configurations are shown in Table 3.
As shown in Table 1, we evaluated both the steric and elecꢀ
tronic effect at the αꢀposition of the tetralones as racemic mixꢀ
tures. Interestingly, electron donating groups such as methyl
(12o), ethyl (12p) and nꢀpropyl (12q) substitutions showed
significant improvement in enzyme potency while electron
withdrawing fluoro group (12r) was less active. Interestingly
trifluoroethyl substitution (12s) resulted in a very potent comꢀ
pound. We selected methyl and ethyl substitutions at the αꢀ
position as preferred substituents for further optimization beꢀ
cause of lower lipophilicity (clogP) compared to nꢀpropyl subꢀ
Table 2. SAR of the D Ring Substituent of the Tetralones
12aꢀn.
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ACS Medicinal Chemistry Letters
potent than Sꢀ12g. In addition compounds were more potent
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4
5
6
7
8
against the human DGAT1 compared with the rat DGAT1
enzyme in the LE assays. Both S enantiomers showed similar
inhibition in a cell based assay using C2C12 mouse myoblast
cells, and the data was consistent with the potency observed in
the rat DGAT1 LE assay. Compound Sꢀ12a has better solubilꢀ
ity compared to Sꢀ12g; therefore, Sꢀ12a was selected for furꢀ
ther profiling. Compound Sꢀ12a showed >1000ꢀfold selectiviꢀ
ty over human DGAT2, ACAT1 (SOAT1) and ACAT2
(SOAT2) in the LE assay. Based on the overall potency, seꢀ
lectivity and developability profiles, compound Sꢀ12a was
selected for further profiling for DMPK properties and in vivo
pharmacodynamic models. Table 4 summarized the PK propꢀ
erties of Sꢀ12a in mouse, rat and dog. Compound Sꢀ12a was a
low to moderate clearance compound with excellent oral bioaꢀ
vailability across species.
hDGAT1
CPM IC50
(nM)a
Solubility
(µg/mL)a
Compd#
R
R4
R5
R6
9
12a
12b
12c
12d
12e
12f
12g
12h
12i
Et
Et
H
H
CF3
Me
Cl
H
H
3
16
6
39
6
10
11
12
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59
60
Et
H
H
101
6
Et
H
OMe
F
H
50
32
20
2
Table 4. Pharmacokinetics Properties of Compound Sꢀ12a
in male mouse, rat and dog (n=3).
Et
H
H
38
46
100
36
26
72
103
75
40
Et
H
H
CF3
Cl
Me
H
Species (Strain)
Et
H
H
Mouse
Rat
Parameter
Dog
Et
H
H
10
126
32
63
16
200
(C57B (Spragueꢀ
(Beagle)
L/6J)
2.2/2.7
42.3
2.4
Dawley)
1/2
Et
CF3
H
H
Dose IV/PO (mg/kg)
CLp (mL/min/kg)
1.8/5.3
4.6
12j
Me
Me
Me
Me
CF3
Me
Cl
H
6.3
12k
12l
H
H
V
SS (L/kg)
1.0
0.6
IV T1/2 (h)
2.6
3.9
4.6
H
H
Oral Bioavailability F (%)
54
87
149
12m
H
OMe
H
Pvalue <0.001
12n
Me
H
F
H
63
13
Mean Baseline TG (0 h)
Vehicle Control (2.5 h)
PF-01620110 - 0.3 mg/kg
S-12a - 0.03 mg/kg
S-12a - 0.1 mg/kg
#
500
aValue is an average of at least n=2.
S-12a - 0.3 mg/kg
S-12a - 1 mg/kg
S-12a - 3 mg/kg
*
400
19%
Table 3. DGAT1 activities and solubility of racemates 12a
and 12g and their corresponding enantiomers (Sꢀ12a, Rꢀ12a,
and Sꢀ12g, Rꢀ12g).
*
33%
S-12a - 10 mg/kg
300
200
100
0
*
60%
*
71%
hDGAT1
CPM
rDGAT
1 LE
C2C12
Inh. at
Soluꢀ
bility
Comꢀ
pound
#
hDGAT1
LE IC50
(nM)a
*
91%
*
94%
*
99%
IC50
IC50
300 nM (µg/m
(nM)a
(nM)a
(%)a
62
L)a
39
Groups
12a
(Rac)
3
8
158
# - Compared with Mean Baseline control (0 h)
* - Compared with Vehicle control (2.5 h)
Values in bar are percent inhibition
Sꢀ12a
Rꢀ12a
2.5
1
126
631
75
27
78
88
Figure 3. Dose response of compound Sꢀ12a and PFꢀ01620110 in
126
251
reducing TG following lipid challenge in mice.15
12g
(Rac)
2
NDb
NDb
72
100
To investigate the in vivo effects of DGAT1 inhibition, comꢀ
pound Sꢀ12a was evaluated in an acute postprandial lipid exꢀ
cursion model which measured plasma triglycerides (TG) folꢀ
lowing a corn oil bolus challenge in male Swiss Albino mice
(Figure 3).15 Overnight fasted animals were orally adminisꢀ
tered with various doses (0.03, 0.1, 0.3, 1, 3 and 10 mg/kg) of
compound Sꢀ12a. Thirty minutes later, mice were challenged
with oral bolus of corn oil (5 mL/kg), and the plasma TG was
measured after 2.5 hours following the corn oil challenge. As
shown in Figure 3, compound Sꢀ12a was highly efficacious
resulting in statistically significant reduction in plasma TG
levels in a doseꢀdependent manner with an estimated ED50 of
0.23 mg/kg. This inhibition occurred at low plasma concentraꢀ
tion of compound Sꢀ12a (7.89 ng/mL at 3 hour post dose with
Sꢀ12g
Rꢀ12g
2
8
158
83
5
158
NDb
1260
NDb
0.4
a Value is an average of at least n=2. bND = not determined.
Interestingly, the S enantiomers were approximately 50ꢀ to 80ꢀ
fold more potent than their corresponding R enantiomers (Taꢀ
ble 3). Since hDGAT1 potency measured by fluorescenceꢀ
based CPM assay was similar for analogues Sꢀ12a and Sꢀ12g,
we evaluated these compounds in a more sensitive radiometric
LE assay in order to further discriminate between these two
compounds. In hDGAT1 LE assay, Sꢀ12a was ~8ꢀfold more
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0.3 mg/kg, a dose closest to the estimated ED50 dose). This is
likely due to relatively higher local unbound concentration in
the gut compared to the plasma concentration, resulting in
greater DGAT1 inhibition in enterocytes than what would be
anticipated from the plasma concentration. PFꢀ04620110 was
used as a reference and showed 94% inhibition of TG levels at
0.3 mg/kg.
Page 4 of 7
of RHS anilines in Ames assay.18 Since screening anilines in
all five bacterial strains in Ames assay are time and resource
demanding, we initiated screening of anilines in a single bacteꢀ
rial strain (TA98), which is known to be sensitive to aromatic
amines, to get a quick go/no go decision. This approach alꢀ
lowed us to increase throughput of the assay, to reduce comꢀ
pound amount needed for testing, and to speed up turnaround
time for generating the data. If aniline was tested negative in
TA98, it would then be evaluated in the full Ames panel to
confirm its nonꢀmutagenic profile. Since the RHS aniline
(21g) was positive in Ames with and without S9, it suggested
that the mutagenicity of the aniline may not be related to
eHOMO measurement as eHOMO required oxidation.
1
2
3
4
5
6
7
8
Since compound Sꢀ12a showed excellent selectivity, stability
and in vivo efficacy, additional selectivity profiling of comꢀ
pound Sꢀ12a were initiated. These included cardiac ion chanꢀ
nels (hERG, Nav1.5 and Cav1.2) and a panel of 43 targeted
pharmacologic receptors, ion channels and transporter assays.
Compound Sꢀ12a showed overall good selectivity profile
(>500ꢀfold over hDGAT1 CPM IC50) across panels of assays
evaluated.
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11
12
13
14
15
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21
22
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24
25
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59
60
Table 5. In silico DEREK and eHOMO prediction and
TA98 Ames results of RHS anilines (21aꢀg, 24aꢀd).
One concern about the scaffold is the potential hydrolysis or
degradation of the urea functional group to the corresponding
anilines which could be mutagenic (Figure 4).16
Comꢀ
pound
#
DEREK/eHOMO/
R
X
Y
Z
TA98
21g
CH3
CF3
CH
CH
CH
+/+/+
(Rac)
21f
CH
CꢀF
CꢀF
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
ꢀ/+/+
+/+/+
ꢀ/+/+
+/ꢀ/ꢀ
21a CH3
21b CF3
Figure 4. Hydrolysis of urea Sꢀ12a and the corresponding
anilines.
CH
21c
H
CꢀCl
CꢀCl
CꢀCl
N
Compound Sꢀ12a or the leftꢀhand side (LHS) mꢀ
trifluoromethyl aniline were not mutagenic when tested in the
bacterial mutation screening assay (Ames test) with Salmonel-
la typhimurium TA1535, TA1537, TA98, TA100 and Esche-
richia coli WP2uvrA (pKM101) in the presence and absence
of the metabolic activating S9ꢀmix.17 However, the rightꢀhand
side (RHS) aniline 21g (racemic, potential degradant of raceꢀ
mic 12a) was mutagenic with and without S9 in the Ames test.
Sꢀenantiomer of αꢀethyl aniline 21g was not soluble enough to
be tested in the Ames test. However, 21g was predicted to be
positive in the in silico DEREK screen. In addition, Sꢀ
enantiomer of similar aniline (the αꢀmethyl analog) was posiꢀ
tive in both Ames test and DEREK screen. While we did not
observe aniline 21g in vivo, we could not rule out with certainꢀ
ty that metabolismꢀdriven hydrolysis would not occur in huꢀ
man. Therefore, we opted to address potential genotoxicity
issue of the anilines by identifying Ames negative aniline
fragments.
21d CH3
+/ꢀ/ꢀ
21e
24a
24b
CF3
CH3
CF3
ꢀ/ꢀ/+
+/ꢀ/+
CH
CH
CH
N
N
N
CH
N
ꢀ/ꢀ/+
ꢀ/ꢀ/+
ꢀ/ꢀ/ꢀ
24c CH3
24d CF3
N
As shown in Table 5, there was poor correlation between in
silico prediction and the TA98 Ames result. In general, αꢀ
trifluoroethyl analogues (21b, 21e, 21f, 24b, 24d) were preꢀ
dicted to be negative in DEREK. Chloroꢀcontaining anilines
(21cꢀe), pyridinyl anilines (24aꢀb) and pyridazinyl anilines
(24cꢀd) were predicted to be negative based on eHOMO
measurement. Pyridazinyl anilines (24cꢀd) were consistent in
both DEREK and eHOMO prediction and were predicted to be
nonꢀmutagenic. Unfortunately, most analogues tested were
positive in the TA98 Ames test regardless of the results from
the in silico prediction with the exception of two chloroꢀ
containing anilines (21c, 21d) and one pyridazinyl aniline
(24d). TA98 negative anilines (21c, 21d, and 24d) were furꢀ
It is known that electron deficient anilines reduce the risk of
compounds being mutagenic.16 We decided to explore substiꢀ
tution of the Cꢀring with electronꢀwithdrawing groups and
replacement of Cꢀring with heteroaryls. In addition, we decidꢀ
ed to reꢀevaluate αꢀtrifluoroethyl substitution for mutagenicity
potential as compound 12s was a potent DGAT1 inhibitor
(Table1). In order to expedite discovery of nonꢀmutagenic
anilines, we utilized in silico mutagenicity prediction models
(DEREK and eHOMO) to prioritize synthesis and evaluation
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ACS Medicinal Chemistry Letters
ther evaluated in the full panel of Ames assay and were conꢀ
IC50 values of >10 ꢀM across CYP1A2 (phenacetin), CYP2C9
(diclofenac), CYP2C19 (Sꢀmephenytoin), CYP2D6 (DEX)
and CYP3A4 (midazolam), and demonstrated no timeꢀ
dependent CYP inhibition at up to 50 ꢀM of 26d and no PXR
activation with EC50 >50 ꢀM. 26d did not form glutathione
conjugates in the presence of 5 mM GSH containing NADPH
in human liver microsomes at up to 10 ꢀM of 26d. Moreover,
26d had high protein binding across species with plasma proꢀ
tein binding of 99.98%, 99.9%, 99.95% in human, mouse and
rat, respectively. Compound 26d exhibited good PK profiles in
rat (CL = 3.3 mL/min/kg, Vdss = 0.42 L/kg, iv T1/2 = 2.6 h, %F
= 48). In addition, it was a low clearance compound with
plasma clearances of 13.3 and 5.1 mL/min/kg in mouse and
dog, respectively, and had good oral bioavailability of 41 and
68%F in mouse and dog, respectively. Based on the overall
developability profiles, compound 26d (GSK2973980A) was
nominated as a development candidate for the treatment of
obesity. Full in vitro and in vivo biological profiles of comꢀ
pound 26d have been reported recently.19
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firmed to be nonꢀmutagenic. Although in silico mutagenicity
predictions were not correlative to the Ames result, our apꢀ
proaches including using TA98 as a triage for Ames testing
did result in the discovery of nonꢀmutagenic anilines in a timeꢀ
ly manner.
Armed with the nonꢀmutagenic RHS anilines in hand, we then
performed a mixꢀandꢀmatch effort with the Ames negative
LHS anilines to identify potential preꢀcandidates without genꢀ
otoxic liabilities. αꢀEthyl (21d) and αꢀtrifluoroethyl RHS
anilines (24d) were selected for mixꢀandꢀmatch because of the
potency improvement over the αꢀmethyl RHS aniline (21c).
The most potent compounds with nonꢀmutagenic anilines are
shown in Table 6.
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26
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CONCLUSION
Table 6. DGAT1 activities of Ames negative aniline containꢀ
ing tetralones 26aꢀd.
Starting with tetralone Compound B with low ꢀM human
DGAT1 potency, we successfully identified a promising lead
Sꢀ12a with potency improvement over 1000ꢀfold, along with
excellent selectivity, pharmacokinetic profiles and in vivo effiꢀ
cacy. Unfortunately, compound Sꢀ12a contained RHS mutaꢀ
genic, Ames positive aniline which was a synthetic intermediꢀ
ate and a potential degradant. To expedite discovery of nonꢀ
mutagenic anilines, we utilized in silico DEREK and eHOMO
prediction to help prioritize anilines for synthesis and testing
in Ames. We then employed a miniꢀscreen of a single bacteriꢀ
al strain (TA98), which is known to be sensitive to aromatic
amines, as a first pass to improve turnaround time for mutaꢀ
genicity evaluation. TA98 negative anilines were then asꢀ
sessed in full Ames panel for nonꢀmutagenic potential. Altꢀ
hough in silico mutagenicity predictions were not predictive of
the Ames outcome, we were able to quickly identify nonꢀ
mutagenic anilines using this approach. This led us to the
identification of (S)ꢀ2ꢀ(6ꢀ(5ꢀ(3ꢀ(3,4ꢀdifluorophenyl)ureido)ꢀ
pyrazinꢀ2ꢀyl)ꢀ1ꢀoxoꢀ2ꢀ(2,2,2ꢀtrifluoroethyl)ꢀ1,2,3,4ꢀ
Co
mpo
und
#
hDGA
T1 LE
IC50
Soluꢀ
bility
(µg/m
L)a
R
Y
Z
R5
R6
(nM)a
26a
26b
26c
26d
CH3
CH3
CF3
CF3
CꢀCl
CꢀCl
N
CH
CH
N
CF3
F
H
F
8
20
3
151
125
56
CF3
F
H
F
N
N
3
134
aValue is an average of at least n=2.
All the compounds showed good potency against hDGAT1
enzyme. Compounds 26a and 26c exhibited poor oral bioaꢀ
vailability (23 and 25%F, respectively) while compounds 26b
and 26d demonstrated modest to good oral bioavailability (51
and 48%F, respectively) in SpragueꢀDawley male rats. Comꢀ
pounds 26b and 26d were evaluated for inhibition of TG in a
postprandial lipid excursion model in Swiss Albino mice.15
Compound 26d showed excellent inhibition of TG in the modꢀ
el with near complete inhibition at 1 mg/kg when dosed oralꢀ
ly.19 Specifically, as shown in Table 1S in the supporting inꢀ
formation, 26d showed 76% TG inhibition at 0.3 mg/kg with
terminal plasma drug level of 36 ng/mL, and 97.6% TG inhibiꢀ
tion at 1 mg/kg with terminal plasma drug level of 106 ng/mL.
tetrahydronaphthalenꢀ2ꢀyl)acetic acid (GSK2973980A, 26d)
as a development candidate compound with suitable biological
and developability profile for further progression. Full biologꢀ
ical profiles of 26d have been reported recently.19
ASSOCIATED CONTENT
Supporting Information
Experimental section on assays and methods, and procedures for
the synthesis of all compounds described herein. PK (%F in rats)
and in vivo efficacy (% inhibition of TG and terminal plasma level
in mice) of tetralones 26aꢀd is in Table 1S. The Supporting Inꢀ
formation is available free of charge on the ACS Publications
website.
Compound 26d was confirmed to be nonꢀmutagenic in the
Ames test. Since 26d was a potent human DGAT1 inhibitor
with good solubility, pharmacokinetic properties and in vivo
efficacy, additional selectivity and developability data were
generated to evaluate its suitability as a preꢀcandidate comꢀ
pound. Compound 26d exhibited >2900ꢀfold selectivity over
human DGAT2, ACAT1 (SOAT1) and ACAT2 (SOAT2)
enzymes (IC50 values of >10 ꢀM) in radiometric assays, and
an IC50 value of 77 nM in the cellꢀbased C2C12 assay. In
addition, 26d had an excellent cardiac ion channel profile with
IC50 values of >10 ꢀM in the hERG, Nav1.5 and Cav1.2
QPatch assays, and exhibited good selectivity profile (>700ꢀ
fold over hDGAT1 LE IC50) across a panel of selectivity asꢀ
says. Compound 26d showed minimal CYP inhibition with
AUTHOR INFORMATION
Corresponding Author
* mui.h.cheung@gsk.com
Present Addresses
N/A
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SmallꢀMolecule Diacylglycerol Acyltransferase (DGAT) Inꢀ
Author Contributions
hibitors. ChemMedChem. 2014, 9, 2410ꢀ2424.
(7) Ohshiro, T.; Tomoda, H. Acyltransferase Inhibitors: A Patent
Review (2010ꢀPresent). Expert Opin. Ther. Pat. 2015, 25,
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(8) Meyers, C.; Gaudet, D.; Tremblay, K.; Amer, A.; Chen, J.;
Aimin, F. The DGAT1 Inhibitor LCQ908 Decreases Triglycꢀ
eride Levels in Patients with the Familial Chylomicronemia
Syndrome. J. Clin. Lipidol. 2012, 6. 266ꢀ267.
(9) Meyers, C.D.; SerranoꢀWu, M.; Amer, A.; Chen, J.; Erik, R.;
Commerford, R.; Hubbard, B.; Brousseau, M.; Li, L.; Meihui,
P.; Chatelain, R.; Dardik, B. The DGAT1 Inhibitor Pradigasꢀ
tat Decreases Chylomicron Secretion and Prevents Postpranꢀ
dial Triglyceride Elevation in Humans. J. Clin. Lipidol. 2013,
7, 285.
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L. Litchfield, J.; Piotrowski, D. W.; Munchhof, M. J.; Manꢀ
ion, T. B.; Zavadoski, W. J.; Walker, G. S.; McPherson, R.
K.; Tapley, S.; Sugarman, E.; GuzmanꢀPerez, A.; DaSilvaꢀ
Jardine, P. Discovery of PFꢀ04620110, a Potent, Selective
and Orally Bioavailable Inhibitor of DGATꢀ1. ACS Med.
Chem. Lett. 2011, 2, 407ꢀ412.
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C.; Knutsson, M.; Eriksson, J. W. Proof of Mechanism for
the DGAT1 Inhibitor AZD7687: Results from a Firstꢀtimeꢀinꢀ
human Singleꢀdose Study. Diabetes Obes. Metab. 2013, 15,
136ꢀ143.
(12) Denison, H.; Nilsson, C.; Lofgren, L.; Himmelmann, A.; Marꢀ
tensson, G.; Knutsson, M.; AlꢀShurbaji, A.; Tornqvist, H.;
Eriksson, J. W. Diacylglycerol Acyltransferase 1 Inhibition
with AZD7687 Alters Lipid Handling and Hormone Secretion
in the Gut with Intolerable Side Effects: A Randomized Cliniꢀ
cal Trial. Diabetes Obes. Metab. 2014, 16, 334ꢀ343.
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Compounds as Diacylglycerol Acyltransferase Inhibitors.
PCT. Int. Appl. (2012) WO2012162127.
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The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manuꢀ
script.
Funding Sources
This research was supported by GlaxoSmithKline Pharmaceutical
Company. The authors declare the following competing financial
interests: MC, JLA and SK are currently employed by Glaxoꢀ
SmithKline and are stockholders of GlaxoSmithKline.
9
ACKNOWLEDGMENT
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We thank Collaborative Research at the GVK Biosciences for the
syntheses and profiling of compounds in biological and DMPK
assays. We thank Amanda Giddings, Angela White, Jordi
MunozꢀMuriedas, Bibi Satter, Paul Hastwell and Sharon Robinꢀ
son for supporting in silico and in vitro genotoxicity evaluation of
compounds. We thank Brian Donovan for providing the cardiac
ion channel selectivity data and Dan Hassler for providing
DGAT2, ACAT1 and ACAT2 selectivity data. We thank Douglas
Minick for the VCD stereochemistry determination of the comꢀ
pounds.
ABBREVIATIONS
ACAT, acylꢀCoenzyme A cholesterol acyltransferase; CL, clearꢀ
ance;
CPM,
7ꢀdiethylaminoꢀ3ꢀ(4ꢀmaleimidophenyl)ꢀ4ꢀ
methylcoumarin; CYP, cytochrome P450; DAG, diacylglycerol;
DGAT, acylꢀCoA:diacylglycerol acyltransferase; DEREK, deꢀ
ductive estimation of risk from existing knowledge; DMPK, disꢀ
tribution, metabolism and pharmacokinetics; eHOMO, energy of
the highest occupied molecular orbital; GSH, glutathione; hERG,
human Ether-à-go-goꢀRelated Gene; iv, intravenous; LE, lipid
extraction; MC, methylcellulose; NADPH, nicotinamide adenine
dinucleotide phosphate; PK, pharmacokinetics; SOAT, sterol Oꢀ
acyltransferase; T1/2, half live; TG, triglyceride or triglycerol or
triacylglycerol; VCD, vibrational circular dichroism; Vdss, volꢀ
ume of distribution at steady state.
(14) Vogt, F. G.; Spoors, G. P.; Su, Q.; Andemichael, Y. W.;
Wang, H.; Potter, T. C.; Minick, D. J. A Spectroscopic and
Computational
Study
of
Stereochemistry
in
2ꢀ
Hydroxymutilin. J. Mol. Struct. 2006, 797, 5ꢀ24.
(15) Zhang, X. D.; Yan, J. W.; Yan, G. R.; Sun, X. Y.; Ji, J.; Li, Y.
M.; Hu, Y. H.; Wang H. Y. Pharmacological inhibition of
diacylglycerol acyltransferase 1 reduces body weight gain,
hyperlipidemia, and hepatic steatosis in db/db mice. Acta
Pharmacol. Sin. 2010, 31, 1470ꢀ1477.
(16) Birch, A. M.; Groombridge, S.; Law, R.; Leach A. G.; Mee,
C. D.; Schramm, C. Rationally Designing Safer Anilines: The
Challenging Case of 4ꢀAminobiphenyls. J. Med. Chem. 2012,
55, 3923ꢀ3933.
(17) Maron, D. M.; Ames, B. N. Revised Methods for the Salmoꢀ
nella Mutagenicity Test. Mutat. Res. 1983, 113, 173ꢀ215.
(18) Fioravanzo, E.; Bassan, A.; Pavan, M.; MostragꢀSzlichtyng,
A.; Worth, A. P. Role of In Silico Genotoxicity Tools in the
Regulatory Assessment of Pharmaceutical Impurities. SAR
QSAR Environ. Res. 2011, 23, 257ꢀ277.
(19) Kumar, S.; Tirunagaru, V. G.; Ariazi, J.; Awasthi, A.; Jayaꢀ
raman, V. B.; Arumugam, P.; Yanamandra, M.; Mitra, S.;
Tiwari, S.; Tangirala, R. S.; Werner, T.; Thomson, D.; Berꢀ
gamini, G.; Cheung, M. A Novel AcylꢀCoA: Diacylglycerol
Acyltransferase 1 (DGAT1) inhibitor, GSK2973980A, Inhibꢀ
its Postprandial Triglycerides and Reduces Body Weight in a
Rodent DietꢀInduced Obesity Model. J. Pharm. Res. Int.
2017. DOI: 10.9734/JPRI/2017/36835.
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Table of Contents
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