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

Article abstract of DOI:10.1016/j.bmcl.2009.04.004

We discovered a structurally novel SCD (Δ9 desaturase) inhibitor 4a (CVT-11,563) that has 119 nM potency in a human cell-based (HEPG2) SCD assay and selectivity against Δ5 and Δ6 desaturases. This compound has 90% oral bioavailability (rat) and excellent

Full text of DOI:10.1016/j.bmcl.2009.04.004

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3050–3053
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Orally bioavailable, liver-selective stearoyl-CoA desaturase (SCD) inhibitors
b
a
b
b
Dmitry O. Koltun a, , Natalya I. Vasilevich , Eric Q. Parkhill , Andrei I. Glushkov , Timur M. Zilbershtein ,
Elena I. Mayboroda b, Melanie A. Boze a, Andrew G. Cole c, Ian Henderson c, Nathan A. Zautke d, Sandra A.
Brunn d, Nancy Chu e, Jia Hao e, Nevena Mollova e, Kwan Leung e, Jeffrey W. Chisholm d, Jeff Zablocki a
*
a Department of Medicinal Chemistry, CV Therapeutics, Inc., 3172 Porter Dr., Palo Alto, CA 94304, USA
b ASINEX, NNT Ltd., 20 Geroev Panfilovtzev St. Bldg 1, Moscow 125480, Russia
c Department of Chemistry, Ligand Pharmaceuticals, Inc., Cranbury, NJ 08512, USA
d Department of Pharmacological Sciences, CV Therapeutics, Inc., 3172 Porter Dr., Palo Alto, CA 94304, USA
e Department of Pre-Clinical Development, CV Therapeutics, 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:
We discovered a structurally novel SCD (
potency in a human cell-based (HEPG2) SCD assay and selectivity against
D
9 desaturase) inhibitor 4a (CVT-11,563) that has 119 nM
5 and 6 desaturases. This
Received 18 March 2009
Revised 31 March 2009
Accepted 3 April 2009
Available online 8 April 2009
D
D
compound has 90% oral bioavailability (rat) and excellent plasma exposure (dAUC 935 ng h/mL). Addi-
tionally, 4a shows moderately selective liver distribution (three times vs plasma and adipose tissue)
and relatively low brain penetration. In a five-day study (high sucrose diet, rat) compound 4a signifi-
cantly reduced SCD activity as determined by GC analysis of fatty acid composition in plasma and liver.
We describe the discovery of 4a from HTS hit 1 followed by scaffold replacement and SAR studies focused
on DMPK properties.
Keywords:
Stearoyl CoA desaturase (SCD)
HEPG2 assay
Microsomal assay
Tissue partition
Ó 2009 Elsevier Ltd. All rights reserved.
Tissue distribution
Saturation index
Obesity and Type II Diabetes continue to expand at epidemic
rates and new medicines targeting novel mechanisms are urgently
signed not to be SCD1 isoform selective, but rather to be tested la-
ter for appropriate in vivo tissue distribution (including brain
penetration and pharmacodynamics).
needed. Stearoyl CoA desaturases (SCD’s), also known as
D9 fatty
acid desaturases are microsomal enzymes that convert coenzyme
A conjugates of saturated long-chain fatty acids into monounsatu-
rated fatty acids (primarily, stearic acid into oleic acid and palmitic
acid into palmitoleic acid). Deletion, mutation or inhibition of SCD
in mice and rats results in decreased hepatic triglyceride, resis-
tance to weight gain and improvements in insulin sensitivity and
glucose uptake.1–5 Thus, SCD inhibitors may present a therapy for
obesity, insulin resistance, and diabetes.6–8
D
9 Desaturases are localized to the endoplasmic reticulum and
complexed with cytochrome b5 and cytochrome b5 reductase. This
feature is shared with 5 and 6 desaturases. Therefore, it was
D
D
important to verify that our small-molecule inhibitors acted di-
rectly on SCD enzyme itself, and not on cytochrome b5 or cyto-
chrome b5 reductase. As
a
result, selected molecules were
screened against
D5 and D6 desaturases as a part of our discovery
paradigm.13
Two isoforms of SCD, SCD1 and SCD5 (also known as SCD2 in ro-
dents), have been identified in humans. SCD1 is primarily found in
liver, adipose and skeletal muscle and is well characterized9 com-
pared to SCD2/5 which is found primarily in the brain.10 The devel-
opment of isoform-selective compounds may be challenging due to
the high homology between SCD isoforms. In addition, SCD1 inhi-
bition in peripheral tissues like skin, pancreas and macrophages
may lead to side-effects.3,11,12 In light of these observations, the
development of tissue selective, non-brain penetrating compounds
may be desirable. The compounds described in this letter were de-
A large number of small-molecule SCD inhibitors have been
published to date by our group14 and others,15–19 therefore we pur-
sued an independent approach to discovering new structural ser-
ies. Using a rat microsomal
D9 desaturase assay derived from the
literature,20 we performed a screen of approximately 5.2 million
proprietary compounds. Initial hits were confirmed in a human
hepatocellular liver carcinoma cell (HEPG2)
D9 desaturase assay.
Several compounds showed differential activity in the rat micro-
somal and human HEPG2 assays. This was not surprising given
the potential differences in binding between the different
species-specific isoforms, in addition to potential differences in
cell-membrane permeability. Consequently, we set out to find
compounds with high inhibitory potency in both assays.
* Corresponding author. Tel.: +1 650 384 8581; fax: +1 650 384 7559.
E-mail address: dmitry.koltun@cvt.com (D.O. Koltun).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.004

D. O. Koltun et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3050–3053
3051
Compound 1 (Fig. 1) was identified from the screen as a mod-
estly potent inhibitor of SCD (Table 1, IC50 3.2 M in rat micro-
somal/1.15 M in HEPG2 SCD assays) and selective against
and 6 desaturases (IC50’s > 30 M). Unfortunately, compound 1
had poor metabolic stability (Table 2 and 1.0% and 0.2% remaining
after a 30 min incubation with human and rat liver microsomes,
respectively). In an effort to improve the metabolic stability of
the hit, we replaced the central benzene core with a quinazolin-
4-one, a known ‘drug-like’ scaffold (Fig. 1).
Quinazolin-4-one analogs 2–5, 9, and 12–15, and related aza-
analogs 6 were synthesized as shown in Scheme 1. Commercial
carboxylic acids 7 were first coupled with the desired amine, cy-
clized to generate the quinazolin-4-one core,21 and reduced to af-
ford the corresponding anilines (8). Acylation of common
intermediate 8 (5-isomer, X = CH, R1 = 3,4-dichlorobenzyl) was
performed with the following reagents and resulted in com-
pounds: CbzCl (2a–e), phenoxyacetyl chloride (3a,b), acetyl chlo-
ride (9). Acylation of intermediate 8 (5-isomer, X = CH) bearing
appropriate R1 substituent with acetoxyacetyl chloride with subse-
quent hydrolysis of the ester provided compounds 4a–f. Similarly
compounds 5a–c were obtained from appropriately substituted
intermediates 8 (4-isomer, X = CH) and compounds 6a and b were
H
N
O
N
O
O
H
N
O
R1
O
Cl
Cl
l
N
N
H
O
l
D5
O
D
l
2
1
N
H
N
O
N
H
N
O
R1
R1
O
N
HO
N
O
O
N
3
4
O
H
N
O
N
R1
R1
O
N
HO
N
HO
O
N
H
N
N
6
5
Figure 1. Screening hit 1 and the follow-up series 2–6.
Table 1
Potency for SCD inhibition
R1
SCD IC50 (nM)
Rat
Microsomal
Human
HEPG2
O
O
N
O
1
3200
251
260
865
>30,000
250
1150
193
250
220
R1
R1
OH
NH2
N
H
N
N
5
5
5
4
2a
2b
2c
2d
2e
3,4-Dichlorobenzyl
4-Chlorobenzyl
3-Chloro-4-fluorobenzyl
2,4-Dichlorobenzyl
3,4-Dimethylbenzyl
a, b, c
R2
d
O2N
H2N
d
4X
4X
N
O
7
8
2
3
9
R2 = OBn
2 = CH2OPh
R2 = Me
X = CH, N
790
68
e
R
3a
3b
3,4-Dichlorobenzyl
4-Chlorobenzyl
268
7970
O
N
O
H
R1
R2
N
Cl
Cl
4a
4b
4c
4d
4e
4f
3,4-Dichlorobenzyl
4-Chlorobenzyl
4-Chloro-3-(trifluoromethyl)benzyl
3-(Trifluoromethyl)-benzyl
4-(Trifluoromethyl)-benzyl
3-Phenylbenzyl
261
1170
190
1360
3420
3850
119
110
H
N
N
5
R2
N
4X
N
O
O
10 R2 = CH2OAc; 11 R2 = CO2Et
4, 5, 6 R2 = CH2OH; 12 R2 = CO2H
13 R2 = (S)-CH(Me)OH
14 R2 = (R)-CH(Me)OH
15 R2 = CH2CH2OH
f
>1000
180
4
(
5
10 12 6
X = CH, X = N)
,
,
-
5a
5b
5c
3,4-Dichlorobenzyl
4-Chlorobenzyl
3-Chlorobenzyl
348
3120
2690
Scheme 1. Reagents and conditions: (a) amine, EDCꢁHCl, HOBt, (i-Pr)2NEt, CHCl3, rt,
24 h; (b) HC(OEt)3 reflux, 24 h; (c) NH2NH2ꢁH2O, Raney Ni, MeOH, 60 °C, 24 h; (d)
R2COCl, (i-Pr)2NEt, CH2Cl2, rt, 24 h; (e) carboxylic acid, HATU, (i-Pr)2NEt, CHCl3, rt;
(f) LiOHꢁH2O, H2O/THF/MeOH, rt, 24 h.
6a
6b
3,4-Dichlorobenzyl
4-Chlorobenzyl
208
2580
49
Table 2
Calculated, in vitro, and in vivo DMPK parameters for the selected compounds
O
O
N
O
H
N
H
BnO
R1
R2
N
R1
R2
R1
R2
H
N
N
PhO
N
N
5
4X
N
O
O
HO
N
O
2
3
4
5
6
5-isomer, X = CH
4-isomer, X = CH
5-isomer, X = N
R1, R2
MLogPc
Caco-2 Papp
Met. stability
% after 30 min
Pharmacokineticsb
a
ꢀ106 cm/s
dAUC (PO)
(ng h/mL)
Cmax (PO)
%F
CLp (IV)
t1/2 (IV)
A?B
B?A
Human
Rat
(ng/mL)
(mL/min/kg)
(h)
1
3.18
4.09
3.88
3.69
2.60
2.94
2.60
2.42
n.d.
1.7
0.8
n.d.
0.7
0.3
1.0
94
56
61
85
78
82
100
0.2
72
56
46
75
88
42
88
n.d.
1.2 1.2
0
0.21 0.02
935 90
451 123
195 68
415 54
n.d.
1.05 0.15
0
1.29 0.27
360 111
439 118
53 20
53.5 18
n.d.
n.d.
n.d.
n.d.
90
29
23
56
n.d.
n.d.
n.d.
n.d.
16.6 3.8
12.9 2.3
29.0 1.2
22.7 3.1
n.d.
n.d.
n.d.
n.d.
1.46 0.16
1.38 0.16
2.20 0.21
1.44 0.05
2a
2c
3a
4a
4c
5a
6a
3,4-Cl2
3-Cl-4-F
3,4-Cl2
3,4-Cl2
3-CF3-4-Cl
3,4-Cl2
1.5
0.7
39.7
42.4
33.3
9.3
47.3
39.6
40.8
47.1
3,4-Cl2
a
Caco-2 permeability data was obtained by Absorption Systems, Inc., Exton, PA, USA.
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
b
clearance; F—oral bioavailability, t1/2—elimination half-life (0–1); n.d.—not determined.
TM
c
Calculated by Accord software, Accelrys, Inc., San Diego, CA, USA.

3052
D. O. Koltun et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3050–3053
obtained from appropriately substituted intermediates 8 (5-iso-
mer, X = N). Reaction of 8 (5-isomer, X = CH, R1 = 3,4-dichloroben-
zyl) with oxalic acid monoethyl ester chloride and subsequent
hydrolysis provided analog 12. Direct HATU coupling22 of 8 (5-iso-
mer, X = CH, R1 = 3,4-dichlorobenzyl) with 1(S)-, 1(R)-, and 2-
hydroxypropionic acids provided analogs 13-15.
In a newly-designed scaffold 2 the Cbz-group on the western
portion and the substituted benzyl group on the eastern portion
were retained, and the oxy-pyridyl group on the southern end
was left out. The resulting compound 2a (IC50 251 nM in the rat
microsomal/193 nM in the HEPG2 SCD assays) not only exceeded
the potency of the initial hit, but also had very good metabolic sta-
bility (94% and 72% remaining after 30 min incubation with human
and rat liver microsomes, respectively, Table 2).
chloro substituent in 4a (dose-adjusted AUC = 451 vs 935 ng h/
mL).
As a part of our SAR effort we tested isomeric series 5 and aza-
series 6. In each case 3,4-dichlorobenzyl analogs (5a and 6a) were
similar to 4a in terms of SCD activity and microsomal stability (Ta-
ble 1). At the same time, both 5a and 6a showed higher IV clear-
ance (29.0 and 22.7 mL/min/kg, respectively) and lower oral
bioavailability (23% and 56%, respectively) than 4a (Table 2).
We were also interested in further exploring the SAR around the
hydroxyacetamide substituent. While keeping the 3,4-dichlorob-
enzyl substituent constant, we replaced the hydroxyacetyl group
in the western portion with unsubstituted acetyl (10, R2 = methyl,
Table 3), oxalyl (12), 1(S)-hydroxypropionyl (13), 1(R)-hydroxypro-
pionyl (14), and 2-hydroxypropionyl (15) derivatives. None of the
above mentioned compounds presented any advantage over the
parent analog 4a in terms of potency of SCD inhibition.
Next, we undertook an analoging effort around the R1 substitu-
ent in scaffold 2 and looked at a number of substituted benzyl
groups resembling the 3,4-dichlorobenzyl group, namely, 4-chlo-
robenzyl (2b), 3-chloro-4-fluorobenzyl (2c), 2,4-dichlorobenzyl
(2d), and 3,4-dimethylbenzyl (2e) groups. Compound 2c had po-
tency for SCD inhibition somewhat comparable to 2a (IC50
865 nM in rat microsomal/220 nM in HEPG2 SCD assays) and
was also moderately stable in liver microsomes (56% in each hu-
man and rat). Unfortunately both 2a and 2c showed low or no
measurable plasma levels after oral dosing in rats (Table 2).
While the 3,4-dichlorobenzyl substituent on the eastern side
appeared near-optimal, we turned our attention to the western
side and attempted to modify the Cbz substituent. We postulated
that Cbz group could be a site of metabolic cleavage and that by
replacing it with a bioisostere we might be able to improve the
pharmacokinetic properties. For that purpose, we switched the
western portion from benzyloxycarbonyl in 2 to phenoxyacetyl
in 3. In 3a, we were able to maintain potency (IC50 268 nM in the
rat microsomal/68 nM in the HEPG2 SCD assays) and microsomal
stability (61% and 46% with human and rat liver microsomes,
respectively) but failed to improve the oral exposure.
Table 3
Elaboration of hydroxyacetyl motif in compound 4a
H
N
O
R
Cl
Cl
N
O
N
R
SCD IC50 (nM)
Rat
Microsomal
Human
HEPG2
4a
10
12
13
14
15
Hydroxymethyl
Methyl
Carboxylic acid
1(S)-Hydroxyethyl
1(R)-Hydroxyethyl
2-Hydroxyethyl
267
3860
890
>30,000
>30,000
11,600
79
>1000
Since compounds of series 2 and 3 are highly lipophilic
(M logP > 3.5) and were found to have low Caco-2 permeability
(Table 2), we assumed that poor absorption rather than metabo-
lism may be responsible for low oral exposure. In subsequent de-
sign, we proceeded to reduce the molecular weight and
introduced a hydrophilic group. The phenyl group was removed
from the phenoxyacetyl western portion of series 3 and replaced
with a free hydroxyl resulting in series 4 (Fig. 1). The compound
4a23 retained good SCD inhibitory potency (IC50 261 nM in rat
microsomal/119 nM in HEPG2 SCD assays), good microsomal sta-
bility (85% and 75% with human and rat liver microsomes, respec-
8000
6000
4000
2000
0
tively), and selectivity against
D
5
and
D
6
desaturases
(IC50’s > 30 M). Caco-2 permeability of 4a was dramatically im-
l
proved compared to 2a, 2c, and 3a (Table 2). The pharmacokinetic
data of compound 4a demonstrated good oral plasma exposure as
expressed by the area under curve (dose-adjusted AUC = 935 ng h/
mL) and Cmax (360 ng/mL), and high oral bioavailability (90%). This
suggests that GI absorbtion may have been indeed the limiting fac-
tor since Caco-2 screen was predictive of oral PK properties. The
moderate intravenous plasma clearance (16.6 mL/min/kg) and ter-
minal half-life of about 1.5 h were expected to be sufficient to en-
able a twice-a-day dosing regimen in a potential proof of concept
study.
Within series 4, we tested some additional benzyl substituents
on the eastern portion, and found 4-chloro-3-(trifluoro-
methyl)benzyl analog (4c) to have SCD inhibitory activity (IC50
190 nM in the rat microsomal/110 nM in the HEPG2 SCD assays)
and good microsomal stability (85% and 75% with human and rat
liver microsomes, respectively) similar to 4a. However, the intro-
duction of the bulkier, more lipophilic trifluoromethyl group (4c)
reduced the oral exposure approximately 2-fold compared to the
Figure 2. Tissue partitioning of compound 4a (PO, rat, 10 mg/kg, 2 h post dose,
mean SEM).
Plasma
Liver
Adipose
2500
2000
1500
1000
500
0
5
10
15
20
Time after dose (h)
Figure 3. The time course of tissue partitioning for 4a (PO, rat, 5 mg/kg, mean
SEM).

D. O. Koltun et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3050–3053
3053
tigate the effect of compound 4a (CVT-11,563) in models of obesity
and diabetes.
0.25
0.20
0.15
0.10
0.05
0.00
Liver
Plasma
Acknowledgement
p<0.001
We would like to thank Dr. Brent Blackburn of CV Therapeutics
for valuable discussions. We would like to thank Dr. Dmitry Genis,
Ms. Olga Krasavina, Ms. Irina Khrustaleva and Dr. Roman Komba-
rov of ASINEX for their support in various aspects of the project.
p=0.007
***
**
Vehicle
4a
Vehicle
4a
References and notes
Figure 4. Effect of SCD inhibitor 4a on plasma and liver desaturation index (5 day,
PO BID, 10 mg/kg, mean SEM) (male Sprague–Dawley rats, high-carbohydrate diet,
n = 10 (control) and n = 8 (treatment), tested for significance in unpaired t-test, data
collected 4 h after last dose).
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Next we investigated the tissue partitioning of compound 4a.
We found 4a to selectively partition into the liver. At 2 h post oral
dose (10 mg/kg) it had three times higher levels in liver than plas-
ma and adipose tissue (Fig. 2). After oral dosing at 5 mg/kg (Fig. 3)
we found that preferential hepatic partitioning of the compound
was maintained to at least 24 h and the compound was cleared
similarly from plasma (t1/2 ꢂ 5 h), liver, (t1/2 ꢂ 5.6 h) and adipose
(t1/2 ꢂ 6.4 h). Based on the oral half-life and significant drug levels
(plasma112 ng/mL and liver 753 ng/g) at 12 h post dose the com-
pound appeared suitable for BID dosing. In brain, significantly low-
er levels of 4a were detected the (283 ng/g, 2 h post 10 mg/kg oral
dose) (Fig. 2). This finding at least partially alleviates the concern
for potential inhibition of SCD2/5 in the brain.
We carried out an in vivo efficacy study in Sprague–Dawley rats
that were kept on a high carbohydrate diet for 4 weeks (Fig. 4).
Compound 4a was dosed orally (BID) for 5 days and the desatura-
tion index [SCD product/substrate, palmitoleic acid (16:1nꢃ7)/pal-
mitic acid (16:0)] measured 4 h after the final dose by extraction,
esterification, and GC analysis of methyl esters.24,25 A similar trend
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was observed for other indexes of
D9 desaturation, however our
experience has been that the C16 ratio is less influenced by dietary
and stored oleic acid (18:1nꢃ9), since 16:1nꢃ7 is a low abundant
fatty acid and absent from non-animal fat diets. In this 5-day study
we found a highly significant reduction in SCD product fatty acids
in both plasma and liver, thus demonstrating the in vivo SCD inhib-
itory properties of compound 4a.
In conclusion, by changing the benzene ring core in hit 1 with
quinazolin-4-one (2), we achieved good metabolic stability and
improved SCD inhibitory properties. By reducing the molecular
weight and introducing the hydroxyacetamide group, we created
compounds with high oral bioavailability (90% in case of 4a).
Caco-2 has proven useful as a screening tool, since in series 2–4,
cell permeability was found to be predictive of oral exposure. In
an in vivo efficacy study compound 4a demonstrated the highly
significant reduction in the amount of SCD product in plasma
and liver. Long-term animal studies are under way to further inves-
17. Zhao, H.; Serby, M. D.; Smith, H. T.; Cao, N.; Suhar, T. S.; Surowy, T. K.; Camp, H.
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23. Compound 4a 1H NMR (400 MHz, DMSO-d6): d 8.6 4 d, J = 2.4 Yz, 1H), 8.52 (s,
1H), 8.10 (dd, J = 8.8, 2.4 Hz, 1H), 7.71 (d, J = 1.6 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H),
7.62 (d, J = 8.8 Hz, 1H), 7.37 (dd, J = 8.0,1.6 Hz, 1H), 5.19 (s, 2H), 4.06 (s, 2H). 13C
NMR (100 MHz, DMSO-d6): d 171.35, 160.02, 146.46, 143.89, 137.89, 137.51,
131.10, 130.79, 130.34, 129.97, 128.15, 127.79, 126.69, 121.82, 114.99, 79.15,
61.91. Anal. C, H, N.
24. Lepage, G.; Roy, C. C. J. Lipid Res. 1984, 25, 1391.
25. Lepage, G.; Roy, C. C. J. Lipid Res. 1986, 27, 114.