Thiadiazoles as new inhibitors of diacylglycerol acyltransferase type 1

Article abstract of DOI:10.1016/j.bmcl.2012.02.006

A novel class of DGAT1 inhibitors containing a thiadiazole core has been discovered. Chemical optimization lead to inhibitors of human DGAT1 with an appropriate ADME profile and that show in vivo activity in target tissues.

Full text of DOI:10.1016/j.bmcl.2012.02.006

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2497–2502
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Thiadiazoles as new inhibitors of diacylglycerol acyltransferase type 1
Patrick Mougenot a, , Claudie Namane a, Eykmar Fett a, Florence Camy a, Rommel Dadji-Faïhun a,
⇑
Gwladys Langot a, Catherine Monseau a, Bénédicte Onofri a, François Pacquet a, Cécile Pascal a,
Olivier Crespin a, Majdi Ben-Hassine a, Jean-Luc Ragot a, Thao Van-Pham a, Christophe Philippo a,
Florence Chatelain-Egger a, Philippe Péron a, Jean-Christophe Le Bail a, Etienne Guillot a,
Philippe Chamiot-Clerc a, Marie-Aude Chabanaud a, Marie-Pierre Pruniaux a,
a
Friedemann Schmidt b, Olivier Venier a, Eric Nicolaï a, , Fabrice Viviani
⇑
a Sanofi-aventis R&D, Early to Candidate Unit, 1 avenue Pierre Brossolette, 91385 Chilly-Mazarin, France
b Sanofi-aventis Deutschland GmbH, Lead Generation to Candidate Realization, Design & Informatics, Industriepark Hoechst, 65926 Frankfurt am Main, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of DGAT1 inhibitors containing a thiadiazole core has been discovered. Chemical optimiza-
tion lead to inhibitors of human DGAT1 with an appropriate ADME profile and that show in vivo activity
in target tissues.
Received 16 December 2011
Revised 31 January 2012
Accepted 2 February 2012
Available online 15 February 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Diacylglycerol acyltransferase type 1
DGAT-1 inhibitors
Triacylglycerides inhibition
Thiadiazole
Pharmacodynamic in vivo assay
Triacylglycerides (TG) are the principal form of energy storage
in eukaryotes. An imbalance in the metabolism of triacylglycerides
can participate in the pathogenesis of several metabolic disorders
such as obesity, insulin resistance and type 2 diabetes.1
DGAT-1, diacylglycerol acyltransferase type 1 catalyses the last
step of triacylglyceride biosynthesis, transforming diacylglycerol
and acyl-CoA into triacylglycerides. Two isoforms of diacylglycerol
acyltransferase have been described: DGAT-12 and DGAT-2.3
DGAT-1 and DGAT-2 show 12% homology in their amino acid se-
quences. In mammals, DGAT-1 is widely expressed, particularly
in the intestine, skeletal muscle, and testis while DGAT-2 is widely
expressed with high expression levels in the liver and adipose
tissues.
Potential beneficial therapeutic effects of DGAT-1 inhibition are
supported by DGAT-1 silencing studies. Indeed, DGAT-1 deficient
mice are resistant to diet-induced obesity, have lower plasma glu-
cose levels associated with an increase of insulin and leptin sensi-
tivity and are also protected against diet-induced hepatic
steatosis.4 In addition, DGAT-1 anti-sense oligonucleotide injection
reduces the fibrosis promoted by a methionine/choline deficient
diet.5 Moreover, small molecule DGAT-1 inhibitors improve phys-
iological parameters such as TG or glucose homeostasis in rodent
preclinical models of metabolic disorders.6
Bayer and Japan Tobacco first reported potent DGAT-1 inhibi-
tors in 2004.7 Despite a strong interest and the efforts of the phar-
maceutical industry, very few compounds have reached and are
currently undergoing clinical trials since that time, for example,
LCQ-908 (structure not disclosed) from Novartis in phase II, AZD-
7687 (structure not disclosed) from Astra-Zeneca and PF-
04620110 from Pfizer reported in phase I (Fig. 1).8
We aimed to identify new DGAT-1 inhibitors by combining li-
gand-based modeling from known DGAT-1 inhibitors,9 high
throughput parallel synthesis (HTPS) and rescaffolding chemistry
to generate leads amenable to optimization. Three lead series were
thus found with IC50s below 100 nM in a DGAT-1 enzymatic
assay.10
H2N
O
N
OH
O
N
N
O
⇑
Corresponding authors.
E-mail address: patrick.mougenot@sanofi-aventis.com (P. Mougenot).
Figure 1. PF-04620110 in phase I.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.006

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P. Mougenot et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2497–2502
N
N
S
O
O
N
H
OH
N
H
1
DGAT1 IC50=0.030µM
Figure 2. Lead compound 1 from aminothiadiazole series.
The most potent inhibitors belonged to a 2-aminothiadiazole
series represented by compound 1 which displayed an IC50 of
0.03 lM in the enzymatic assay (Fig. 2).
The synthesis of the aminothiadiazole 1 started from aniline 211
which reacted with thiocarbonyldiimazole and phenylacetic
hydrazide to afford intermediate 3. Cyclization of 3 produced the
aminothiadiazole 4. In the last step, ester hydrolysis gave the acid
compound 1 (Scheme 1).
Figure 3. Ligand-based alignment for compound 1 (shown in red) and compound
9a (shown in light blue), electrostatic negative isovalue maps9 (at level ꢁ0.9 for
each compound). Poor overlap of the thiadiazole scaffolds is observed. The
interatomic distance between the aniline hydrogen N(H) and the carboxylate
(C)OOH varies from 13.07 Å in compound 1 to 11.37 Å in compound 9a.
Compound 1 showed encouraging functional cellular activity,
with an IC50 of 0.30 lM in an assay measuring the ability of inhib-
itors to decrease TG synthesis in Chang liver cells12 but suffered
from a low value in the Caco-2 permeability model (0.4 ꢀ 10ꢁ7
-
cm sꢁ1). We suspected that the permeability issue was mainly
due to a too high polarity (TPSA = 132)13 of the molecule and in
particular the presence of the benzoylvaline moiety, but attempts
at its modulation failed to give better permeability properties (data
not shown). Since the presence of a carboxylic acid turned out to be
critical, we looked for potential benzoylvaline surrogates which
would retain good inhibitory activity and improve permeability,
including the grafting of the phenylcyclohexane acetic acid frag-
ment to the 2-aminothiadiazole scaffold, thus leading to com-
pound 9.
The synthesis of compound 9 started from the commercially
available intermediate 5. The phenol 5 gave the alkene 6 after a
Horner–Emmons reaction. The subsequent hydrogenation fol-
lowed by a triflation reaction led to the intermediate 8. Finally,
the Buchwald reaction afforded the expected compound 9 as a
mixture of cis/trans (35:65) stereoisomers (Scheme 2).
Figure 4. Ligand-based alignment for compound 1 (shown in red) and compound
10 (shown in black), electrostatic negative isovalue maps9 (at level ꢁ0.9 for each
compound). In contrast to compound 9a, the thiadiazole scaffolds of compound 1
and 10 overlap significantly, forming
a
a
shared negative patch alongside the
recognition feature. The interatomic
thiadiazole nitrogens which indicates
Surprisingly, compound 9 was completely inactive, with an IC50
distance between the aniline hydrogen N(H) and the carboxylate (C)OOH of
compound 10 is 11.38 Å.
above 10
lM in the DGAT-1 enzymatic assay.
O
O
O
O
a, b
H
N
O
O
H2N
H
N
N
H
N
H
2
3
4
HN
S
O
c
N
N
N
N
S
S
O
O
O
O
d
N
H
N
H
OH
O
N
H
N
H
1
Scheme 1. Synthesis of compound 1. Reagents and conditions: (a) 1,10-thiocarbonyldiimidazole, DMF, rt; (b) phenylacetic hydrazide, EtOH, reflux, overnight, 49%, two steps;
(c) H2SO4, 0 °C, 1 h; (d) LiOH, THF, MeOH, H2O, rt, 83% (two steps).
a
b
HO
HO
N
HO
O
O
O
7
5
6
9
O
O
c
N
S
d
TfO
N
H
O
OH
8
O
O
Scheme 2. Synthesis of compound 9. Reagents and conditions: (a) methyl diethylphosphonoacetate, NaH, THF, rt; (b) H2, Pd/C (10%), THF; (c) Tf2O, DIEA, CH2Cl2, 0 °C,
overnight, 49%, three steps; (d) 5-benzyl-1,3,4-thiadiazol-2-amine, Pd2dba3, S-Phos, NaOtBu, toluene, reflux, 7%.

P. Mougenot et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2497–2502
2499
O
O
O
O
a
O
O
O
11
12
13
O
O
b
O
O
O
c
HO
O
O
14
O
O
d
O
O
e
N
S
N
S
N
N
N
H
N
H
OH
15
10
O
Scheme 3. Synthesis of compound 10. Reagents and conditions: (a) tert-butyl diethylphosphonoacetate, NaH, THF, RT, 18 h, 72%; (b) H2 (35 psi), Pd/C (10%), EtOH, 3 h, 86%;
(c) LiOH, THF, MeOH, H2O, rt, overnight, 50% after recrystallization from ethyl acetate; (d) 5-benzyl-1,3,4-thiadiazol-2-amine, EDC, HOBt, DIPEA, DMF, CH2Cl2, rt, 4 days, 68%;
(e) TFA, CH2Cl2, 92%.
O
N
O
N
S
N
S
N
N
OH
N
H
OH
21
10a
O
O
enz. DGAT1 IC50>10µM
cell. DGAT-1 ND
Figure 5. Compound 10a.
Figure 6. Compound 21.
To understand this loss of activity, we used a ligand-based 3D
alignment model9 to compare compounds 1 and 9a (trans isomer).
In Figure 3, the alignment of compounds 1 and 9a shows that,
although there is good shape similarity, the thiadiazole in 9a is
geometrically distorted compared to 1.
This observation prompted us to modify the linker between the
thiadiazole and the phenylcyclohexyl group. Of all the chemical
modifications proposed and filtered using ligand-based alignment,
we selected the amide group as the preferred linker. This choice
was supported by the ligand-based alignment for compounds 1
and 10 (with CO inserted) which showed that the amide linker re-
tains a high shape similarity and provides excellent matching of
the thiadiazoles (Fig. 4).
The synthesis of compound 10 is depicted in Scheme 3, starting
with the preparation of the ketone 11 as previously described.15
Subsequent Horner–Emmons reaction afforded the alkene 12.
Hydrogenation followed by hydrolysis and then recrystallization
gave intermediate 14 with the trans configuration. Finally, the cou-
pling reaction with commercially available 5-benzyl-1,3,4-thia-
diazol-2-amine followed by the cleavage of the tert-butyl ester
led to the expected carboxylic acid 10.
tive also displayed a similar level of activity in the cellular assay
(IC50 = 0.029 lM). There was also an improvement of Caco-2 per-
meability (112 ꢀ 10ꢁ7-cm sꢁ1). Compound 10 was also found sta-
ble in metabolic stability studies (< or equal to 5% transformation
in human or mouse hepatic microsomes).
Starting from compound 10, we first explored the effects of
modifying the acidic moiety. In the same manner as described in
Scheme 3, but using a cis/trans mixture of intermediate 14, and
HPLC separation of the final compound14, we obtained the cis iso-
mer of compound 10 (Fig. 5).
This cis cyclohexyl stereoisomer 10a was found to be 15 times
less potent (IC50 = 0.578
matic assay.
lM) than the trans isomer 10 in the enzy-
Replacing the cyclohexylacetic acid moiety by cyclohexylprop-
ionic acid gave rise to compound 10b and a 10-fold drop in activity
(IC50 = 0.35 lM).
The synthesis of compound 10b is depicted in Scheme 4 starting
from the ketone 11. Subsequent reaction with trimethylsulfoxoni-
um iodide gave epoxide 16. Treatment with boron trifluoride
etherate afforded the aldehyde 17 which was then engaged in a
Horner–Emmons reaction to yield the trans alkene 18 after chro-
matography. Hydrogenation followed by a basic hydrolysis gave
The use of the amide linker restored DGAT-1 inhibition with
compound
10
displaying
good
enzymatic
activity
(IC50 = 0.036
lM). Moreover, contrary to compound 1, this deriva-
O
O
O
O
O
O
b
a
O
O
O
11
19
16
17
c
O
O
O
d
O
O
O
O
O
18
e
O
O
f, g
O
N
O
O
N
N
H
HO
S
10b
OH
20
Scheme 4. Synthesis of compound 10b. Reagents and conditions: (a) trimethylsulfoxonium iodide, NaH, THF, DMSO, 50 °C, 1 h, 46%; (b) BF3ꢂEt2O, CH2Cl2, rt, 3 h, 81%; (c) tert-
butyl diethylphosphonoacetate, NaH, THF, rt, 18 h; (d) H2, Pd/C (10%), EtOH, 3 h, 79%, two steps; (e) LiOH, THF, MeOH, H2O, rt, 4 h, 36%; (f) 5-benzyl-1,3,4-thiadiazol-2-amine,
PyBrop, DIEA, DMF, 2 day, 20%; (g) TFA, CH2Cl2, 3 h, 56%.

2500
P. Mougenot et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2497–2502
Table 1
intermediate 20. Finally, the coupling reaction followed by cleav-
Modulation of R moiety: compounds 10c–y
age of the tert-butyl group led to the carboxylic acid 10b.
Interestingly, with respect to modifying the amide linker, we
found that the N-methyl substituted compound 21 (Fig. 6) was
completely inactive.
To explore the effect of modifying the 5-substituent of the thia-
diazole ring, we prepared compounds 10c–y (Table 1). The syn-
thetic route to derivatives 10c–y was the same as for compound
10 (Scheme 3) using the appropriate 2-aminothiadiazole build-
ing-blocks.16 The synthesis of the aminothiadiazole intermediate
24 is described in Scheme 5.
N
H
N
N
S
R
O
OH
O
Enz. DGAT -1
Cell. DGAT -1
Compound
R
IC50 (µM)
IC50 (µM)
As shown in Table 1, while maintaining the trans-cyclohexyl-
acetic acid moiety, we assessed different modulations at the 5-po-
sition of the thiadiazole. Introduction of a substituent at the
benzylic position was tolerated (derivative 10c) or gave less active
compounds in the case of fluoro derivatives (10t and 10u). The
phenyl derivative 10d retained an enzymatic IC50 below 100 nM,
but cellular activity dropped to 588 nM. Replacing the benzyl
group by a large variety of substituents (10e–10j) failed to give
more active compounds. Finally, replacement of the phenyl moiety
of 10 (benzyl) or 10k (phenethyl) by a cyclopentyl moiety (10q and
10o, respectively) gave rise to more potent inhibitors in the enzy-
matic test and in the cellular assay.
10
0.036
0.162
0.029
ND
10c
0.085
4.77
0.97
1.19
0.588
ND
10d
10e
10f
10g
N
ND
ND
N
O
In order to guide further syntheses, 3D-QSAR studies were
undertaken using the CoMFA17 partial least squares (PLS) module
of SYBYL 8.1.18 Pipeline Pilot 7.519 was used to generate and regu-
larize 3D molecular structures, ligand-based molecular alignment
was performed9 and Gasteiger–Hückel charges were added in SYB-
YL. For CoMFA analyses, we used the built-in partial least squares
method in SYBYL 8.1 with pre-set parameters provided in the cor-
responding CoMFA module with a 2.0 Å grid spacing and a cut-off
at 30 kcal/mol. A consistent model with r2 = 0.91 using 3 main PLS
components (estimated variance as standard error of estimate,
SEE = 0.27) was derived on the representative dataset given in Ta-
ble 1. The PLS analysis was repeated 10 times with five randomly
selected cross-validation groups, resulting a slightly lower mean
O
>10µM
ND
ND
ND
S
10h
10i
10j
O
Br
2.84
1.19
0.070
0.218
0.59
ND
10k
10l
S
O
cross-validated r2c value of 0.73. A visual representation of the ste-
10m
10n
10o
10p
10q
10r
10s
0.047
0.040
0.019
0.065
0.024
0.426
0.498
0.107
ND
v
ric and electrostatic fields is presented in Figure 7a which shows
sterically favored (85%, green) and disfavored (15%, yellow), and
anion favored 15%, red) contours on compound 10o. Green colored
areas indicate substitution points where bulky groups tend to favor
protein-ligand interaction opposed to the yellow region, where ste-
ric interaction is disfavored. The red region indicates where nega-
tively charged groups are likely to be tolerated.
Steric bulk close to the thiadiazole scaffold is unfavorable (little
yellow patch, 10c, 10f, 10u). However, the red patch indicates that
polar linkers, particularly ethers and arylethers, are tolerated
(10m, 10y). Steric requirements distal to the thiadiazole scaffold
are beneficial (10i < 10s < 10p < 10q < 10o < 10n). The distal green
patch is addressed by alkyl–aryl derivatives of the scaffold (10,
10k, 10v, 10w), and particularly from benzyl derivatives. Although
amongst the most potent inhibitors, the cycloalkyl compounds
were discarded since some of them (10n and 10o) suffered from
high metabolic liability; they were therefore not considered for
optimization (data not shown).
0.012
0.091
0.033
ND
ND
10t
0.408
2.36
ND
ND
F
F
10u
Since the benzyl derivative 10 was the most potent inhibitor in
the enzymatic and cellular assays and displayed favorable meta-
bolic stability, we focused our chemical efforts on the synthesis
of substituted benzyl derivatives as illustrated below (Table 2).
The synthetic route for preparing derivatives 25–31 was the same
as for compound 10 (Scheme 3). A regression plot of the calculated
versus experimentally determined log(IC50) is given in Figure 7b.
For the external set of compounds 25–31, a predictive r2 of 0.70
was obtained.
10v
10w
10x
10y
0.066
0.050
0.838
0.044
0.173
0.181
ND
F
F
O
O
0.091
F
As shown in Table 2, introduction of a methoxy group in the
para position led to a 20-fold loss in activity against DGAT-1 in

P. Mougenot et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2497–2502
2501
O
N
N
O
a
b
H
N
NH2
NH2
OH
22
S
N
H
S
23
24
Scheme 5. Synthesis of compound 24. Reagents and conditions: (a) thiosemicarbazide, HOBt, EDC, CH2Cl2 1 h, (b) H2SO4, rt, 8%, two steps.
Table 3
Metabolic stability and intestinal permeability
Compound % Metabolic liability h/
m21
Caco-2 permeability
22
(10ꢁ7 cm sꢁ1
)
10
27
28
30
0/5
1/10
0/4
112
120
75
6/14
80
1.2
1.0
0.8
0.6
0.4
0.2
0.0
vehicle (water challenge)
vehicle (corn oil challenge)
10 (1 mg/kg)
Figure 7a. Coefficient map of the CoMFA steric and electrostatic fields mapped on
compound 10o.
27 (1 mg/kg)
28 (1 mg/kg)
30 (1 mg/kg)
Table 2
Variations of the benzylic derivatives: compounds 10, 25–31
*
*
*
**
N
O
**
H
N
N
OH
R
S
O
-107%
-120%
-101%
-95%
Compound
R
Enz. DGAT-1 IC50
(l
M)
Cell. DGAT-1 IC50 (lM)
10
25
26
27
28
29
30
31
H
0.036
0.728
0.600
0.030
0.045
5.28
0.029
0.142
0.131
0.046
0.035
ND
para-MeO
para-F
meta-F
ortho-F
para-Cl
meta-Cl
ortho-Cl
Figure 8. Lipid challenge assay.20
atom to the meta or ortho position (27 and 28) gave rise to com-
pounds with enzymatic and cellular activity similar to the unsub-
stituted compound 10. Like the corresponding methoxy and
fluorine derivatives, the p-chloro substitution resulted in a dra-
matic loss of activity. The best chloro derivative was the meta
substituted compound 30, then the ortho derivative, following
the same trend as the fluoro derivatives.
0.030
0.082
0.057
ND
9
8
7
6
5
4
3
In order to assess the potential of this series in a proof-of-con-
cept (POC) study in a pharmacological animal model, we selected
the most potent benzyl derivatives (10, 27, 28, 30) which all dis-
played very good metabolic stability and high intestinal permeabil-
ity values (Table 3), and therefore were amenable to in vivo testing.
The pharmacodynamic effect following DGAT-1 inhibition was
evaluated using an acute lipid challenge test where plasma triglyc-
erides (TG) levels were measured after administration of an oral
bolus of corn oil to fasted male C57Bl/6 J mice.20 The administra-
tion of corn oil induced a significant rise in plasma TG levels 1 hour
after the lipid bolus compared to mice treated with water (+60%; p
<0.05). Tested at 1 mg/kg po 1 h before lipid challenge, compounds
10, 27, 28 and 30 inhibited by 95% (p <0.05), 107% (p <0.01), 120%
(p <0.01) and 101% (p <0.05), respectively, the increase in plasma
TG (Fig. 8). Compounds concentrations in plasma were found to
be in agreement with their respective IC50 values, as observed for
compound 10 that reached concentrations in plasma of
79.1 14.8 ng/ml (181 34 nM) at 1 h post-administration.
In conclusion, the combination of in silico analysis, virtual
screening, parallel synthesis and ligand alignment methods has
been successful in the discovery of new very potent heterocyclic
DGAT-1 inhibitors. Lead optimization produced several potent
inhibitors with favorable in vitro ADME properties. Advanced lead
Training
External
3
4
5
6
7
8
9
)
experimental log(IC50
Figure 7b. Regression plot of the CoMFA predictions for the set of training
compounds (Table 1) and the external prediction set (Table 2).
the enzymatic assay. The replacement of the p-methoxy moiety by
a p-fluorine atom led to a slight improvement. Moving the fluorine

2502
P. Mougenot et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2497–2502
in
a superposition with one conformer per compound. The best ranked
compounds have been evaluated in a POC study in mice and shown
to completely block TG production at 1 mg/kg after a lipid chal-
lenge. Further optimisation has been carried out based on these
first results and will be disclosed in due course.
alignment—as determined by the maximum sum of Scores—is being reported.
Electrostatic maps were generated with VIDA 2.0 (a product of OpenEye
Scientific Software Inc., Santa Fe, New Mexico).
10. Compound activity (0.000038 to 10 lM, final concentration) was measured in a
phase-partition based assay using a recombinant human DGAT-1 enzyme
(Kristie, Analytical Biochemistry, 2006, 358). Briefly, the assay was performed
in 96-well Isoplate, in a 50
sucrose, 150 M 1,2-di-(cis-9-octadecenoyl)-sn-glycerol, 40
CoA (1 Ci/ml), 10 mM MgCl2 and 0.25 g of microsomal proteins from Sf9
insect cells overexpressing human DGAT1. The reaction was initiated by the
addition of DGAT1 microsomes and carried out for 60 min at 25 °C, then
Acknowledgments
l
l final volume, with 100 mM Hepes, 250 mM
l
lM 3H-octanoyl-
l
l
The authors would like to thank Jochen Görlitzer, Hans Matter,
Kurt Ritter and Hans-Ludwig Schäfer for supporting the program.
stopped with 10
ll of acetic acid (3.3% final concentration). After 45 min of
further incubation at room temperature, 60
l
l
of BetaPlate Scint cocktail
References and notes
(Perkin–Elmer) were added in order to partition lipids from the aqueous phase.
Radioactivity of the upper organic phase was determined using a MicroBeta
counter (Perkin–Elmer). As confirmation of the screening, IC50s were assessed
through dose–response in monoplicate. Each run was validated with
reference compound JT-553 described in the
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37 °C, 5% CO2. Then, the substrate (Glycerol [14C] 0.4
lCi/ml final) was added
into each wells and the plates were incubated 6 h at 37 °C, 5% CO2. Incubation
medium was removed and the cells were washed twice with PBS. After
trypsination, cells were centrifuged for 5 minutes at 1300g and the
supernatant was discarded. Lipids were extracted from cell pellets by adding
400
were sonicated and filtered (0.45
(Waters 2695 with a SunFireÒ column C18 3
l
l of a methanol/dichloromethane/TFA (50:50:0.1%) solution. The samples
l
m). The samples were analysed by HPLC
l
m 4.6 ꢀ 75 mm), with a mobile
phase 5% (H2O + 0,1%TFA), 70% methanol, 25% dichloromethane with a flow of
1,5 ml/min, coupled to a flow scintillation analyzer (Perkin–Elmer radiomatic
625T) to separate and quantify the relative amount of all 14C-acylglycerides
formed.
13. Program ACD9 was used (a product of ACD/Labs).
14. Separation on Chiralcel OJ-H 250⁄21 mm (5
lm) with a mobile phase CO2
(MeOH + 0.5% d’isopropylamine).
15. Bovy, P. R.; Cecchi, R.; Croci, T.; Venier, O. WO2003/099772.
16. (a) Aminothiadiazole used in the synthesis are commercially available.; (b)
Fett, E.; Mougenot, P.; Namane, C.; Nicolaï E. and Philippo, C. WO2010/086551.
17. (a) Cramer, R. D.; Patterson, D. E.; Bunce, J. E. J. Am. Chem. Soc. 1988, 110, 5959;
(b) Clark, M.; Cramer, R. D.; Jones, D. M.; Patterson, D. E.; Simeroth, P. E.
Tetrahedron Comp. Meth. 1990, 3, 47.
18. SYBYL, Tripos International, 1699 South Hanley Rd., St. Louis, Missouri, 63144,
USA.
19. Pipeline Pilot 7.5, Accelrys, 10188 Telesis Court, San Diego CA 92121.
20. Lipid challenge assay: On the day of the experiment, mice were orally treated
(10 mL/kg) with vehicle (0.5% methylcellulose/DMSO 1%), compounds 10, 27,
28 and 30 at 1 mg/kg (n = 10 mice per group) 1 h prior to the lipid challenge. At
T0, mice were treated with 100 lL of corn oil or water. One hour after the lipid
challenge (or 2 h after the treatment), blood collection was performed
(intracardiac puncture) under isoflurane anesthesia. Measurement of plasma
triglycerides was performed using the triglyceride determination kit from
Sigma–Aldrich One way ANOVA followed by a Dunnett test was used to
establish statistical significance between vehicle and treated mice. A p value
below 0.05 was considered as significant (⁄p <0.05; ⁄⁄p <0.01 versus water-
treated vehicle group). All the protocols used in this study were validated by
the local SANOFI ethical committee.
9. For generating
a
high-quality 3D alignment we employed our internal
combinatorial analysis of ROCS 3D
application MARS, which performs
a
shape-based alignments. The program ROCS (3.1.1. (a product of OpenEye
Scientific Software Inc., Santa Fe, New Mexico)) was used, which performs
shape-based overlays of conformers for target molecules to the query molecule
in one or multiple conformations. The ROCS algorithm maximizes the rigid
overlay of atom-centered Gaussian functions and thereby maximizes the
overlap between a query molecule and a single conformation of a target
21. Compound is incubated 20 min with human or mouse hepatic microsomal
fraction. Liability % result corresponds to disappearance of tested compound at
the end of the experiment.
22. (a) Grès, M.-C.; Julian, B.; Bourrié, M.; Meunier, V.; Roques, C.; Berger, M.;
Boulenc, X.; Berger, Y.; Fabre, G. Pharm. Res. 1998, 15, 726; (b) Grandi, D. D.;
Press, B. Curr. Drug Metab. 2008, 9, 893.
molecule. The MARS procedure starts from
a given conformer and
systematically scores each conformer of new compounds by aligning them to
the optimally matched conformer. The quality of the alignment is measured on
the basis of ROCS ComboScores, taking both shape and atomic features into
account. This results into a matrix of similarity scores between each conformer
to the others. The scores matrix is analysed to optimize alignment sets to result