Potent, orally bioavailable, liver-selective stearoyl-CoA desaturase (SCD) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.06.017

Two structurally distinct series of SCD (Δ9 desaturase) inhibitors (1 and 2) have been previously reported by our group. In the present work, we merged the structural features of the two series. This led to the discovery of compound 5b (CVT-12,012) which

Full text of DOI:10.1016/j.bmcl.2009.06.017

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4070–4074
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent, orally bioavailable, liver-selective stearoyl-CoA desaturase
(SCD) inhibitors
b
c
b
a
Dmitry O. Koltun a, , Timur M. Zilbershtein , Vasily A. Migulin , Natalya I. Vasilevich , Eric Q. Parkhill ,
Andrei I. Glushkov b, Malcolm J. McGregor d, Sandra A. Brunn e, Nancy Chu f, Jia Hao f, Nevena Mollova f,
Kwan Leung f, Jeffrey W. Chisholm e, Jeff Zablocki a
*
a Department of Medicinal Chemistry, Gilead Sciences, Inc., 3172 Porter Dr., Palo Alto, CA 94304, USA
b ASINEX, NNT Ltd, 20 Geroev Panfilovtzev St. Bldg 1, Moscow 125480, Russia
c Zelinsky Institute of Organic Chemistry, Leninsky prospect 47, Moscow, 119991, Russia
d Accelrys, Inc., 10188 Telesis Court, Suite 100, San Diego, CA 92121, USA
e Department of Pharmacological Sciences, Gilead Sciences, Inc., 3172 Porter Dr., Palo Alto, CA 94304, USA
f Department of Pre-Clinical Development, Gilead Sciences, Inc., 3172 Porter Dr., Palo Alto, CA 94304, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Two structurally distinct series of SCD (D9 desaturase) inhibitors (1 and 2) have been previously reported
Received 18 April 2009
Revised 1 June 2009
Accepted 3 June 2009
Available online 13 June 2009
by our group. In the present work, we merged the structural features of the two series. This led to the
discovery of compound 5b (CVT-12,012) which is highly potent in a human cell-based (HEPG2) SCD assay
(IC50 = 6 nM). This compound has 78% oral bioavailability in rats and is preferentially distributed into
liver (76 times vs plasma) with relatively low brain penetration. In a five-day study (sucrose fed rats)
compound 5b significantly reduced SCD activity in a dose-dependent manner as determined by GC anal-
ysis of fatty acid composition in plasma and liver, and significantly reduced liver triglycerides versus the
control group (ꢀ50%).
Keywords:
Stearoyl-CoA
Desaturase
SCD
Ó 2009 Elsevier Ltd. All rights reserved.
Delta-9 desaturase
HEPG2
Microsomal assay
Hydroxyacetamide
Tissue partitioning
Tissue selectivity
Molecular overlay
Liver triglycerides
In the past decade, stearoyl-CoA desaturase (SCD,
ase) has emerged as a promising target for the potential treatment
of metabolic diseases.1–8 SCD catalyzes the
9-desaturation of
D
9 desatur-
ob/ob mice lacking SCD1 were lean and had increased energy
expenditure (Fig. 1).2
In humans, two isoforms of SCD, SCD1 and SCD5, have been
identified. SCD1 is primarily found in liver, adipose and skeletal
muscle9 while SCD5 is found primarily in the brain.10 The develop-
ment of isoform-selective compounds may be challenging due to
D
long-chain fatty acids, leading to the biosynthesis of monounsatu-
rated fatty acids: oleic (18:1n-9), palmitoleic (16:1n-7), and vaccenic
(18:1n-7, via the elongase). Monounsaturated fatty acids are major
components of triglycerides, cholesterol esters, and phospholipids.
SCD inhibition, based on studies using SCD knockdown with anti-
sense RNA and in mice lacking functional SCD, is expected to be
beneficial for the treatment of type II diabetes, obesity, and hyper-
triglyceridemia. In one such study, Ntambi and co-workers demon-
strated that SCD1ꢁ/ꢁ mice fed a high-fat diet had reduced body
weight and adiposity, increased oxygen consumption, energy
expenditure and improved insulin sensitivity.1 In another study,
OMe
N
O
H
R1
R2
N
R1
R2
N
H
N
O
HO
N
O
N
NHAc
2a R1 = CF3, R2 = Cl
2b
R
1 = CF3, R2 = Cl
1a
CVT-11,563, R1 = Cl, R2 = Cl
1b R1 = Cl, R2 = Cl
1c R1 = CF3, R2 = H
* Corresponding author. Tel.: +1 650 384 8581; fax: +1 650 384 7559.
E-mail address: dmitry.koltun@gilead.com (D.O. Koltun).
Figure 1. Structures of previously reported SCD inhibitors.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.017

D. O. Koltun et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4070–4074
4071
the homology between the two SCD isoforms. Furthermore,
mounting evidence suggests SCD1 inhibition in peripheral tissues
like skin, pancreas and macrophages may lead to side-effects.3,11,12
As a result our program focused on the discovery of tissue selective
non-brain penetrating compounds that could inhibit SCD primarily
in the liver and/or adipose tissue.
match for the 4-methoxyphenyl substituent. We experimented
by replacing it with a much smaller methyl group (Table 1).
We synthesized an initial set of compounds that shared features
of series 1 and 2 as shown in Schemes 1 and 2. The compound 5a
and all of the N-Boc protected intermediates (3) were prepared
from 4-bromo-2-fluoronitrobenzene following the previously re-
ported method.19 The Boc group in 3 was removed to yield the
amine 4 (Scheme 1). Reaction of 4 with the corresponding acid
chlorides and the subsequent hydrolysis of the esters produced
compounds 5b,22 5f, and 5h–j. Compounds 5–e were prepared by
the direct HATU coupling23 of the corresponding hydroxyacids.
Compound 5g was prepared by heating with phenyl carbamate
in the presence of N-methylmorpholine.24
The synthesis of series 9 compounds was carried out as shown
in Scheme 2 using 2-amino-4-nitrophenol as a starting material.
Reaction with chloroacetyl chloride, 2-bromopropionyl bromide,
or 2-bromo-2-methylpropionyl bromide followed by cyclization
yields the intermediates 7a–c.25 Intermediate 10 (NPhth = phthal-
imide) was prepared in one step from commercial 2-(2-bromoeth-
ylethyl)-isoindoline-1,3-dione via Finkelstein reaction.26 The
D
9 Desaturases belong to a class of enzymes that includes the
D
5 and 6 desaturases. All 3 fatty acid desaturases are localized
D
to the endoplasmic reticulum and are complexed with cytochrome
b5 and cytochrome b5 reductase.13 As a result, we needed to ensure
that SCD inhibitors acted specifically on the SCD enzyme and not
on cytochrome b5 or cytochrome b5 reductase. For this purpose
we counter-screened selected molecules for
inhibitory activity.
D5 and D6 desaturase
A number of small-molecule SCD inhibitors have been pub-
lished to date.14–18 In previous communications, we have disclosed
two chemically distinct classes of SCD inhibitors.19,20 In series 1,19
compound 1a has been shown to be extremely potent in the rat
microsomal SCD assay (IC50 0.6 nM) and in the human cell-based
SCD assay21 (HEPG2 IC50 0.05 nM). The difference in potency may
be explained by the structural difference between species-specific
isoforms, and/or potential differences resulting from cell-mem-
brane permeability. Representative compounds 1b and 1c also
demonstrated single-digit nanomolar potency in the HEPG2 SCD
assay, however that series exhibited poor oral bioavailability in ro-
dents. For example, compound 1b had no detectable plasma levels
following oral administration to rats.
Table 1
Potency for SCD inhibition in 5a–j
N
N
R3
O
F3C
N
H
In the series 2,20 compounds with good oral absorption were
identified by lowering the molecular weight and lipophilicity of
the initial leads, in addition to incorporating a key hydroxyaceta-
mide group. In rats, representative compounds 2a and 2b (CVT-
11,563) were shown to have 29% and 90% oral bioavailability,
respectively. Compound 2b has also been found to be distributed
into the liver with moderate selectivity (threefold versus both plas-
ma and adipose tissue) and showed in vivo inhibition of SCD activ-
ity in a five-day study using rats fed a high sucrose diet.
In this work, we set out to modify the highly potent series 1 and
exploit the features from the more bioavailable series 2. A superpo-
sition of molecules 1b and 2b (Fig. 2) revealed a striking overlap
between the bulky, lipophilic substituted benzyl groups containing
Cl and/or CF3 substituents. Another ovelapping feature was the
presence of the NHAc group in series 1 that we modified to incor-
porate the OH group from CVT-11,563 (2b). We did not find a
5a-j
HN
O
R1
R1
R3
SCD IC50 (nM)
Rat
Microsomal
Human
HEPG2
1a
1b
1c
2a
2b
0.6
110
93
190
261
0.05
8.6
33
110
119
5a
5b
5c
5d
5e
5f
Me
CH2OH
Me
Me
Me
Me
Me
Me
Me
162
38
6.1
1(S)-Hydroxyethyl
1(R)-Hydroxyethyl
2-Hydroxyethyl
( )-CH(OH)CH2OH
NH2
8000
3900
1100
268
15
68
>1000
5g
5h
5i
5j
CH2OH
CH2OH
CH2OH
CF3
i-Pr
t-Bu
1.9
2800
>3000
2.7
NO2
N
R3
a
ref. 19
Br
F
R2
N
H
N
O
3
NHBoc
N
R3
N
N
R3
O
b, c
or
d or e
R2
N
H
N
O
R2
N
H
4
5b-j
NH2
HN
O
OAc
AcO
Cl
R1
Figure 2. Three dimensional superimposition of 1b (green) and 2b (CVT-11, 563,
white). This picture was produced using the ‘Molecular Overlay’ feature of Accelrys
Discovery Studio v2.0, which gives an automated optimum overlay of a number of
small molecules using equally weighted steric and electrostatic fields, and flexible
rotatable bonds. Pharmacophore features were added manually to show possible
common hydrogen bond acceptor atoms (arrows) and hydrophobic/aromatic
regions (centroid).
O
(
)-6
Scheme 1. Reagents and conditions: (a) TFA, CH2Cl2, rt, 24 h; (b) AcOCH2COCl or
( )-6, (i-Pr)2NEt, CH2Cl2, rt, 24 h; (c) LiOHꢂH2O, H2O/THF/MeOH, r.t., 24 h; (d)
R1CO2H, HATU, (i-Pr)2NEt, CHCl3, rt, 24 h; (e) PhOCONH2, N-methylmorpholine,
THF, reflux, 2 h.

4072
D. O. Koltun et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4070–4074
R4
was carried out in two steps (one pot). The aniline was heated with
OH
O
R5
a
b
3,4-dichlorobenzaldehyde in ethanol in the presence of silicon tet-
raethoxide, and then sodium borohydride was added. This was fol-
lowed by the ester hydrolysis to yield compounds 9a–c.
Compound 5b displayed the highest potency in both the
microsomal and the HEPG2 SCD assays (IC50 38 nM and 6.1 nM,
respectively) compared to the other methyl-substituted com-
pounds (5a–g, Table 1). This suggests that, similarly to CVT-
11,563 (2b)20 the hydroxyacetamide functional group appears to
be optimal for SCD inhibitory potency.
Having found that the methyl group is an acceptable substitu-
ent at the R3 position, we proceeded to study the influence of the
other R3 substituents (5h–j, Table 1). This SAR study revealed that
the trifluoromethyl group was preferred as compound 5h showed
superior potency in both assays (HEPG2 SCD IC50 1.9 nM, miscoso-
mal SCD IC50 2.7 nM) while methyl (5b), isopropyl (5i), and tert-bu-
tyl (5j) analogs were significantly less potent.
O2N
NH2
O2N
N
H
7a c
-
O
R4
R4
O
N
O
N
R5
R5
c, d, e, f, g
Cl
Cl
O2N
O
N
O
H
8a c
-
9a c
-
NPhth
NPhth
HN
O
Br
I
h
OH
NPhth
10
Scheme 2. Reagents and conditions: (a) ClCH2COCl, or BrCH(Me)COBr, or
BrCMe2COBr, acetone, 0 °C to rt, followed by Et3N, H2O, reflux, 3 h; (b) 10, NaH,
DMF, 0 °C to rt, 24 h; (c) NH2NH2ꢂH2O, EtOH, 60 °C, 5 h; (d) AcOCH2CO2H, TBTU, (i-
Pr)2NEt, THF, rt; (e) Zn, AcOH, 55 °C, 4 h; (f) 3,4-dichlorobenzaldehyde, Si(OEt)4,
EtOH, reflux, 3 h, followed by NaBH4, EtOH, 0 °C to r.t., 3 h; (g) K2CO3, H2O/MeOH;
(h) 4 equiv NaI, acetone, reflux, 5 h.
The oxygen-containing compound 9a (Table 2) led to an
improvement in potency in both the microsomal and the HEPG2
SCD assays (IC50 1.4 nM and 0.5 nM, respectively) when compared
to series 5. Introduction of the methyl substituents (9b and c)
slightly decreased potency for SCD inhibition in the rat microsomal
assay (IC50 80 nM and 12 nM, respectively).
Compounds 5b, 5h, and 9a–c were selected for the follow-up in
the microsomal stability assays (Table 3), were only 5b exhibited
>50% stability after the 30 min incubation in both human and rat
liver microsomal assay. Surprisingly, 9a was the more stable com-
pound among 9a–c, since the introduction of the methyl groups
next to the ether moiety was intended to improve metabolic stabil-
ity. In a rat PK study, 5b and 9a both demonstrated good oral bio-
availability (78% and 39%, respectively). It appears that the oral
absorption of 5b was not affected by a significant Pgp efflux, which
was expected based on Caco-2 assay results (Papp A?B
6.8 ꢃ 106 cm/s, B?A 48.2 ꢃ 106 cm/s, efflux ratio of 7). The plasma
clearance of both 5b and 9a was high (88 and 56 mL/min/kg,
respectively) with elimination half-life of approximately 1 h for
each compound.
Table 2
Potency for SCD inhibition in 9a–c
R4
O
R5
Cl
Cl
N
H
N
O
HN
O
9a-c
OH
R4, R5
SCD IC50, nM
Rat microsomal
38
Human HEPG2
5b
6.1
0.5
9a
9b
9c
H, H
1.4
80
12
( )-H, Me
Me, Me
coupling of intermediates 7a–c and 10 provided compounds 8a–c
and was followed by treatment with hydrazine to remove the
phthalimide protecting group, TBTU-assisted amide formation,27
and nitro group reduction. The subsequent reductive amination
We confirmed that 5b was selective for
D9 desaturase (D5
desaturase and
D6 desaturase IC50 >30 lM in the rat microsomal
assay).13 Next, we investigated the tissue partitioning of compound
5b (Fig. 3). Acute single dose PK experiments in rat (5 mg/kg) dem-
Table 3
In vitro, and in vivo DMPK parameters for the selected compounds
R4
O
N
N
N
Me
O
N
N
CF3
O
R5
Cl
Cl
F3C
F3C
N
H
O
N
H
N
H
9a-c
5b
5h
HN
O
HN
O
HN
O
OH
OH
OH
R4, R5
Met. stability
Pharmacokineticsa
% After 30 min
dAUC (PO) (ng h/mL)
Cmax (PO) (ng/mL)
%F
CLp (IV) (mL/min/kg)
Vdb (L/kg)
t1/2 (IV) (h)
Human
Rat
1b
2a
2b
5b
5h
9a
9b
9c
83
78
85
56
19
32
54
88
75
54
77
14
0
0
n.d.
29
90
78
n.d.
39
n.d.
n.d.
n.d.
451 123
935 90
148 8.6
n.d.
122 85
n.d.
439 118
360 111
374 147
n.d.
427 88
n.d.
12.9 2.3
16.6 3.8
88.1 7.5
n.d.
53.0 6.4
n.d.
1.46 0.13
2.06 0.24
7.22 0.96
n.d.
4.89 0.47
n.d.
1.38 0.16
1.46 0.16
0.95 0.07
n.d.
1.07 0.13
n.d.
H, H
( )-H, Me
Me, Me
2.3
3.1
1.5
9.6
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
a
Mean and standard deviation, based on n = 3 (male Sprague–Dawley rats, 1.5 mg/kg PO dose, 1 mg/kg IV dose). dAUC—AUC (0–1) adjusted to 1 mg/kg dose; CLp—plasma
clearance (0–1); Vdb—terminal phase volume of distribution; F—oral bioavailability, t1/2—elimination half-life (0–1); n.d.—not determined.

D. O. Koltun et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4070–4074
4073
40
Plasma
Liver
Adipose
100
10
30
Brain
Muscle
1
*
20
0.1
0.01
IC100
10
IC50
0
0.001
Vehicle
5b
0
6
12
18
24
(h)
Figure 5. Effect of SCD inhibitor 5b on liver triglycerides after 5 days of BID oral
treatment. High-carbohydrate fed male Sprague–Dawley rats, vehicle (n = 5)
compound 5b (20 mg/kg, n = 6). Samples were collected 4 h after last dose.
Mean SEM, *p <0.05 by unpaired t-test (actual p = 0.012).
Figure 3. Time course for compound 5b tissue partitioning (PO, rat, 5 mg/kg, n = 3,
mean values, SEM omitted).
onstrated selective partitioning into the liver. At 2 h post oral dose
the 5b levels measured in liver were 76, 75, 549 and 973-fold high-
er than those in plasma, muscle, adipose and brain, respectively.
The concentration of 5b remained above the IC100 (100% inhibition
level) in the liver and above the IC50 level in plasma, muscle, and
adipose tissue beyond 12 h (the IC100 and IC50 values are based
on the HEPG2 data).
duced the investigational compound 5b, a potent SCD inhibitor in
the microsomal assay (IC50 = 38 nM) and the human cell-based as-
say (HEPG2 IC50 = 6.1 nM) that is orally bioavailable (78%) and
strongly partitions to the liver. In 5 day in vivo efficacy studies,
compound 5b significantly reduced SCD products in the plasma
and the liver as well as reducing liver TG’s by ꢀ50%. Long-term ani-
mal studies are under way to further investigate the effect of com-
pound 5b (CVT-12,012) in models of obesity, fatty liver diseases
and diabetes.
These tissue and plasma levels were deemed sufficient to en-
able further in vivo efficacy studies.
Sprague–Dawley rats were kept on a high sucrose diet for
4 weeks (Fig. 4) prior to being dosed orally (BID) with compound
Acknowledgments
5b for 5 days. The
D9 desaturation products [palmitoleic acid
(16:1n-7) and vaccenic acid (18:1n-7)] were measured 4 h after
the final dose following extraction, transmethylation, and GC anal-
ysis of the fatty acid methyl esters.28,29
We would like to thank Dr. Brent Blackburn of Gilead Sciences
(Palo Alto, CA) for valuable discussions and Dr. Andrew Cole of Li-
gand Pharmaceuticals (Cranbury, NJ) for reviewing this manu-
script. We also would like to thank Dr. Dmitry Genis, Ms. Olga
Krasavina, Ms. Irina Khrustaleva, Dr. Alexey Streletskiy, Dr. Alexey
Stepanov and Dr. Roman Kombarov of ASINEX for their support of
the project.
Palmitoleic and vaccenic acids combined represent a better
marker of SCD activity than oleic acid (18:1n-9); as they are virtu-
ally absent from non- animal fat diets and originate from de novo
liogenesis and SCD mediated desaturation. In this five-day study,
we found a dose-dependent reduction in SCD product fatty acids
in both plasma and liver at all three dose levels (5, 10, and
20 mg/kg) thus demonstrating that compound 5b has in vivo SCD
inhibitory properties.
In a separate five-day study (Fig. 5), compound 5b (20 mg/kg
PO, BID) was administered to rats that were kept on a high sucrose
diet for 4 weeks and liver triglycerides were measured. Adminis-
tration of 5b conferred a 50% decrease in liver triglycerides com-
pared to vehicle.
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6
***
***
***
***
***
***
0
5b (mg/kg BID)
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Figure 4. Effect of SCD inhibitor 5b on plasma and liver desaturation index (5 day,
PO BID, mean SEM). High-carbohydrate fed male Sprague–Dawley rats, vehicle (V,
n = 6), compound 5b, 5 mg/kg (n = 6), 10 mg/kg, (n = 5), 20 mg/kg (n = 6), Samples
***
p <0.001 by unpaired t-test.
were collected 4 h after last dose.

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25. Synthesis of 6-Nitro-4H-benzo[1,4]oxazin-3-one (7a). To
a solution of 2-
amino-4-nitrophenol (4 g, 26 mmol) in acetone (50 mL) at 0 °C chloroacetyl
chloride (3.3 g, 29 mmol) was added dropwise and the reaction mixture was
allowed to warm up to room temperature. At that time a precipitate was
observed and Et3N (5.8 g, 57 mmol) was added slowly. The reaction mixture
was stirred for additional 30 min, diluted with water (50 mL), and heated to
reflux for 3 h, then kept overnight at room temperature. The precipitate was
filtered, and washed consequetively with water and methanol. The product 6-
Nitro-4H-benzo[1,4]oxazin-3-one (7a) was obtained after drying as a grey
solid (4.3 g, 85 %). 1H NMR (300 MHz, DMSO-d6): d 11.07 (br s, 1H); 7.83 dd,
J = 2.2, 8.8 Yz, 1H), 7.72 d, J = 2.2 Yz, 1H), 7.14 d, J = 8.8 Yz, 1H), 4.77 (s, 2H).
26. Maloney, D. J.; Hecht, S. M. Org. Lett. 2005, 7, 4297.
21. Talamo, B. R.; Bloch, K. Anal. Biochem. 1969, 29, 300.
22. Compound 5b: 1H NMR (400 MHz, DMSO-d6): d 8.21 (t, J = 5.9 Hz, 1H), 7.87 (s,
1H), 7.76 (d, J = 7.4 Hz, 1H), 7.49–7.68 (m, 2H), 7.42 (d, J = 9.0 Hz, 1H), 7.15 (t,
J = 6.1 Hz, 1H), 6.75 (br s, 1H), 6.71 (br d, J = 9.0 Hz, 1H), 5.54 (t, J = 5.7 Hz, 1H),
4.57 (d, J = 6.3 Hz, 2H), 4.14 (t, J = 7.0 Hz, 2H), 3.84 (d, J = 5.5 Hz, 2H), 3.28 - 3.37
(m, 2H), 2.32 (s, 3H). 13C NMR (100 MHz, DMSO-d6): d 173.44, 155.34, 150.23,
150.02, 141.72, 134.94, 132.19, 130.19, 129.81, 129.47 (q, J = 31.1 Hz), 125.00,
124.84 (q, J = 3.8 Hz), 124.79 (q, J = 272.3 Hz), 124.08 (q, J = 3.6 Hz), 111.70,
93.85, 61.91, 45.86, 40.98, 35.80, 21.01. Anal. C, H, N.
27. Kim, S. J.; Kim, B. H. Nucleic Acids Res. 2003, 31, 2725.
28. Lepage, G.; Roy, C. C. J. Lipid Res. 1984, 25, 1391.
29. Lepage, G.; Roy, C. C. J. Lipid Res. 1986, 27, 114.
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Chan, C.; Convery, M. A.; Crowe, M. C.; Dayal, S.; Diallo, H.; Kelly, H. A.; King, N.