Potent and selective 5-LO inhibitor bearing benzothiophene pharmacophore: Discovery of MK-5286

Article abstract of DOI:10.1016/j.bmcl.2010.10.024

The strategy and SAR studies that led to the discovery of a novel potent and orally available 5-lipoxygenase (5-LO) inhibitor 3-(4-fluorophenyl)-6-({4- [(1S)-1-hydroxy-1-(trifluoromethyl)propyl]-1H-1,2,3-triazol-1-yl}methyl) -1-benzothiophene-2-carboxamid

Full text of DOI:10.1016/j.bmcl.2010.10.024

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7440–7443
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent and selective 5-LO inhibitor bearing benzothiophene pharmacophore:
Discovery of MK-5286 q
Lianhai Li a, , Carl Berthelette a, Anne Chateauneuf b, Marc Ouellet b, Claudio F. Sturino a, Zhaoyin Wang a
⇑
a Department of Medicinal Chemistry, Merck Frosst Centre for Therapeutic Research, 16711 Trans Canada Hwy., Kirkland, Québec, Canada H9H 3L1
b Department of Biochemistry, Merck Frosst Centre for Therapeutic Research, 16711 Trans Canada Hwy., Kirkland, Québec, Canada H9H 3L1
a r t i c l e i n f o
a b s t r a c t
Article history:
The strategy and SAR studies that led to the discovery of a novel potent and orally available 5-lipoxyge-
nase (5-LO) inhibitor 3-(4-fluorophenyl)-6-({4-[(1S)-1-hydroxy-1-(trifluoromethyl)propyl]-1H-1,2,3-
triazol-1-yl}methyl)-1-benzothiophene-2-carboxamide ((S)-2l or MK-5286) were described.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 9 September 2010
Revised 1 October 2010
Accepted 5 October 2010
Available online 15 October 2010
Keywords:
5-Lipoxygenase
Inhibitor
Benzothiophene
MK-5286
Leukotrienes, a group of locally acting hormones produced in
living systems from arachidonic acid metabolism, are potent con-
tractile and inflammatory mediators in various diseases.1 The bio-
synthesis of these leukotrienes, including leukotriene B4 (LTB4),
LTC4, LTD4, and LTE4, begins with the epoxidation of arachidonic
acid by 5-lipoxygenase (5-LO) enzyme2 and 5-lipoxygenase-
activating protein (FLAP)3 to produce leukotriene A4 (LTA4). LTA4
is then converted to the other leukotrienes by subsequent
enzymatic steps. Indeed, inhibition of leukotriene biosynthesis by
a 5-LO inhibitor has been a validated strategy in searching for no-
vel therapeutic agents to treat various inflammatory conditions.4
Furthermore, it has been reported that 5-LO may be an important
contributor to the atherogenic process and cancer,5 and other oxi-
dative stress-linked pathological processes.6 Thus, a potent and
selective 5-LO inhibitor might also be useful for treatment of
diseases related to these pathogenic processes. Although there is
currently one marketed 5-LO inhibitor, Zileuton (ZYFLOÒ), for the
treatment of asthma,7 the search for 5-LO inhibitor that targets
better safety and efficacy profile never stopped. Herein we disclose
our work that led to the discovery of a potent and orally available
5-LO inhibitor bearing a benzothiophene scaffold.
we also observed that the 3-fluoro-5-(4-methoxytetrahydro-
2H-pyran-4-yl)phenol ether moiety in 1a had serious liabilities
related to oxidative metabolism resulting in high level of covalent
protein binding. We then found that this moiety could be replaced
by various groups that were more resistant to oxidative metabo-
lism and one such group was a triazole moiety as shown in 1b. Pre-
vious studies also showed that the 5-LO inhibitory activity
associated with 1b mainly resided on the enantiomer with an
(S)-configuration at the carbon center bearing a hydroxyl group.9
Thus, our further efforts were focused on identifying a suitable aryl
(Ar) group in 1b that enabled potent 5-LO inhibitory activity and
O
F
F
OH
N
O
Ar
N
N
O
F
Ar
F
1b
1a
As part of our previous efforts8 for the 5-LO inhibitor program,
we established that compounds with a common structure as repre-
sented by 1a exhibited 5-LO inhibitory activity (Fig. 1). In addition,
O
S
N
N
N
O
HO
F
F
q
F
In memory of legacy Merck Frosst Centre for Therapeutic Research.
⇑
Corresponding author at present address: 4201 Hugo, Pierrefonds, Québec,
Canada H9H2V6. Tel.: +1-514-4283837.
2a
E-mail addresses: lianhai_li@merck.com, lianhai.li@gmail.com (L. Li).
Figure 1. The lead generation.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.024

L. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7440–7443
7441
The 5-LO inhibitors described in this study were prepared as
shown in Schemes 1–4.10 The synthesis of alcohol 4 was achieved
by the addition of [(trimethylsilyl)ethynyl]lithium, which was
formed in situ by reaction of 3 with n-BuLi, to 1,1,1-trifluorobutan-
2-one (Scheme 1). The corresponding alcohol thus formed was
treated with TBAF to remove the TMS protective group to provide
the desired alcohol 4. Compound 5, the starting material used to
build the advanced intermediate 6, was prepared according to a lit-
erature procedure (Scheme 2).11 Compound 5 was esterified with
MeOH in the presence of DMAP to form the corresponding ester.
The ester thus formed was subjected to benzylic bromination reac-
tion with NBS, followed by a reaction with sodium azide to provide
an azide intermediate, which then underwent a click reaction with
compound 4 to give compound 6. The Suzuki coupling between 6
and phenylboronic acid catalyzed by a palladium catalyst formed
in situ from Pd(OAc)2 and DavePhos yielded the prototype com-
pound 2a. Compound 2a is a potent 5-LO inhibitor and could also
serve as a starting material for the preparation of other 5-LO inhib-
itors with a range of different functional groups substituted at the
2-position of the benzothiophene scaffold (Scheme 2). First, 2a
was reduced by DIBAL-H to form 2d. Oxidation of 2d with MnO2, fol-
lowed by a reaction with methyllithium provided compound 2f.
Compound2f was further transformedto compound 2g through oxi-
dation with MnO2. Carboxylic acid 2b was obtained by hydrolysis of
2a with aqueous NaOH solution. Tertiary alcohol 2e was formed by a
reaction of 2a with an excess amount of MeLi in THF at low temper-
ature. The 2-carboxamide derivatives, 2h, 2i, and 2j, were synthe-
sized by reacting 2a with an aluminum amide intermediate
formed in situ from Me3Al with the corresponding amine source
such as ammonium chloride, methylamine, or dimethylamine,
respectively (Scheme 3). Decarboxylation of 2b promoted by copper
powder in hot quinoline provided compound 2c. Alternatively,
intermediate 6 was transformed into nitrile 7 by a reaction sequence
including reduction with DIBAL-H, oxidation with MnO2, oxime for-
mation with hydroxylamine hydrogen chloride, and dehydration
with CDI (Scheme 4). The Suzuki coupling between compound 7
and a suitable arylboronic acid under the above-mentioned Suzuki
coupling conditions provided compound 8, which can be further
hydrolyzed to form amide 2k and 2l. When the racemic mixture of
2j, 2k, or 2l was submitted to chiral separation on a Chiralpak AD col-
umn, each of them was resolved to provide the desired enantiomer
(S)-2j, (S)-2k, or (S)-2l, respectively.
The 5-LO inhibitors reported herein were evaluated for their
potency to inhibit the oxidation of arachidonic acid by recombi-
nant human 5-LO (H5-LO),8 and the production of LTB4 in calcium
ionophore-stimulated HWB.12 To address the hERG binding issue
observed in other related series of 5-LO inhibitors, all compounds
were also evaluated for its potential to block the voltage-gated
potassium channel encoded by the human ether-a-go-go gene
(hERG) as measured in vitro by using a MK-499 displacement bind-
ing assay. As shown in Table 1, compound 2a exhibits good and
moderate activities (entry 1) in human 5-LO enzyme assay and
LTB4 whole blood assay, respectively. However, it also shows mod-
erate activity in the hERG binding assay. Our goal of SAR study was
to find a compound that was potent in both human 5-LO enzyme
assay and LTB4 whole blood assay but with diminished hERG
potassium channel binding activity. Numerous compounds were
prepared in the series and data in Table 1 only summarizes the re-
sults of representative compounds that illustrated the SAR trends
and key compounds that were selected for further evaluation.
Among all of the substituents made to the benzothiophene phar-
macophore, the most impact on structure–activity relationship
(SAR) came from substituents at the 2-position.
a, b, c
TMS
HO
CF3
3
4
Scheme 1. Reagents and conditions: (a) n-BuLi, THF, À78 °C; (b) EtCOCF3, THF,
À78 °C to rt; (c) TBAF, THF, rt.
Cl
O
S
N
O
O
S
N
N
a-d
e
Cl
Cl
CF3
5
6
HO
N
O
O
S
N
N
N
S
g
N
N
CF3
OH
2a
HO
CF3
HO
2e
f
i
N
OH
S
N
N
O
S
N
N
N
OH
CF3
CF3
2d
HO
2b
HO
h, g
N
S
N
N
N
S
h
N
N
OH
O
CF3
CF3
HO
HO
2f
2g
Scheme 2. Reagents and conditions: (a) MeOH, DMAP, DCM; (b) NBS, BzOOBz, CCl4,
80 °C; (c) NaN3, DMF, rt; (d) 4, CuI, DIPEA, THF, rt; (e) PhB(OH)2, Pd(OAc)2,
DavePhos, dioxane/H2O, rt; (f) DIBAL, THF, À78 °C; (g) MeLi, THF, À78 °C; (h) MnO2,
DCM; (i) NaOH, THF/MeOH/H2O, reflux.
N
O
S
N
N
a
NR1R2
2a
CF3
HO
2h: R1, R2 = Me, Me
2i: R1, R2 = Me, H
2j: R1, R2 = H, H
N
N
S
N
b
2b
CF3
HO
2c
Scheme 3. Reagents and conditions: (a) Me3Al, NH4Cl (MeNH2 or Me2NH), C6H6,
80 °C; (b) Cu (powder), quinoline, 140 °C.
had other desirable properties such as high bioavailability and
good metabolic stability. After a quick screen of several heteroaryl
groups, we identified a benzothiophene scaffold as represented in
compound 2a as the best Ar group in 1b. Thus compound 2a served
as a prototype for our subsequent SAR studies which were focused
on the substituents of the benzothiophene scaffold.
The apparent liability of compound 2a is its instability towards
hydrolysis, either chemically or enzymatically. The corresponding
carboxylic acid 2d, the more stable hydrolysis product, was found

7442
L. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7440–7443
N
S
N
N
a-d
CN
e
6
Cl
CF3
CN
7
HO
S
N
N
N
NH2
O
S
N
N
f
N
Ar
CF3
Ar
CF3
HO
8
HO
2k (Ar = 3-FC6H4)
2l (Ar = 4-FC6H4)
N
NH2
N
N
S
NH2
O
S
N
g
N
N
O
Ar
Ar
CF3
CF3
HO
HO
(S)-2k (Ar = 3-FC6H4)
(S)-2l (Ar = 4-FC6H4)
(R)-2k (Ar = 3-FC6H4)
(R)-2l (Ar = 4-FC6H4)
Scheme 4. Reagents and conditions: (a) DIBAL, THF, À78 °C; (b) MnO2, DCM; (c) NH2OHÁHCl, NaOAc, EtOH, rt; (d) DCI, DCM, rt; (e) ArB(OH)2, Pd(OAc)2, DavePhos,
dioxane/H2O, rt; (f) Na2CO3Á1.5H2O2, acetone, reflux; (g) Chiralpak AD.
Table 1
In vitro activity and hERG binding affinity of selected analogs of 5-LO inhibitors 2
N
S
N
N
R
Ar
CF3
HO
a
Entry
Compd
Ar
R
IC50 (nM)
hERG Kia
(lM)
Human 5-LO
HWB
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
2a
2b
2c
2d
2e
2f
2g
2h
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
–COOMe
–COOH
–H
7.9
>10,000
40
19
198
88
260
>20,000
598
64
392
161
84
1840
263
70
32
31
21
154
>20,000
6.3
>20
n/a
13
2.6
3.8
4.4
>20
>20
17
–CH2OH
–C(OH)Me2
–CH(OH)Me
–COMe
–CONMe2
–CONHMe
–CONH2
–CONH2
–CONH2
–CONH2
–CONH2
–COOH
11
1890
223
55
28
39
55
1160
>10,000
2i
2j
Ph
Ph
Ph
(S)-2j
(S)-2k
(S)-2lb
(R)-2l
(S)-2m
16
14
13
11
3-F-phenyl
4-F-phenyl
4-F-phenyl
4-F-phenyl
>20
a
IC50 and Ki values represent an average of at least three independent titrations.
The compound was assigned as MK-5286.
b
to be completely inactive. The complete removal of substituent at
the 2-position led to compound 2c with more than two-fold loss
of 5-LO activity when compared to 2a, indicating that a substituent
at the 2-position is helpful in improving 5-LO inhibiting activity.
Therefore, we prepared compounds 2d, 2e, 2f, and 2g in hope to
identify the intrinsic SAR trend associated with the series. The anal-
ysis of the SAR trend of these compounds clearly indicated that a
small substituent bearing polar group, as shown by 2d, was both
beneficial to the improved 5-LO inhibiting activity and diminished
hERG potassium channel binding activity. The good 5-LO inhibiting
activity by 2g suggests that a planar carbonyl group also helps.
These observations prompted us to prepare three amide bearing
compounds 2h, 2i, and 2j. This led to the identification of primary
amide group as a superior substituent for both high 5-LO inhibiting
activity and low hERG potassium channel binding affinity. As we
expected, enantiomer (S)-2j possesses most of 5-LO inhibitory
activity as compared to the racemate 2j. To follow up on this, we
carried out further SAR study by introducing substituent on the
phenyl group substituted at the 3-position of the benzothiophene
scaffold. The purpose of this practice is two-fold: (1) to further im-
prove the 5-LO inhibition potency and (2) to minimize the potential
oxidative metabolism at this part of the molecules. As shown in
Table 1 (entries 12 and 13), introduction of a fluorine at the 3- or
4-position of the phenyl moiety slightly improved the 5-LO inhibit-
ing activity but showed little impact on hERG potassium channel
binding affinity. Based on the HWB activity, (S)-2l was selected
for further evaluation. (S)-2l shows no activity against 12-LO,
15-LO, and FLAP (IC50 >20 lM), and retain low affinity to hERG
potassium channel. These properties of (S)-2l prompted us to per-
form intensive PK studies on it. We found that (S)-2l exhibits a good
pharmacokinetic profile in pre-clinical species such as rat (5 mg/kg
IV, 20 mg/kg PO, F = 114% and t1/2 = 7.9 h, Cmax = 19.4 lM), dog

L. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7440–7443
7443
(5 mg/kg IV, 4 mg/kg PO, F = 120% and t1/2 = 29 h, Cmax = 6.7
and rhesus monkey (5 mg/kg IV, 4 mg/kg PO, F = 66% and t1/
2 = 22 h, Cmax = 1.4 M), respectively. It is worth mentioning that
lM),
References and notes
1. (a)For a review and references therein, see: Leukotrienes and Lipoxygenases;
Rokach, J., Ed.; Elsevier: Amsterdam, 1989; (b) Peters-Golden, M.; Henderson,
W. R., Jr. N. Engl. J. Med. 2007, 357, 1841.
2. Rådmark, O.; Werz, O.; Steinhilber, D.; Samuelsson, B. Trends Biochem. Sci. 2007,
32, 332.
l
the vehicle used for PO administration of (S)-2l was 50% PEG 400
at 4 mL/kg/day. With this formulation, inhibitor (S)-2l was dosed
to studied animals as a clear solution. Attempts to administer the
inhibitor as a fine suspension in 0.5% methocel and other vehicles
resulted in <10% bioavailability across all studied species. In the
PK studies, we also observed the formation of a circulating metab-
olite (S)-2m in all studied species. Thus (S)-2m was prepared13 and
evaluated. As presented in Table 1, (S)-2m shows no measurable
activities to 5-LO and hERG. Both inhibitor (S)-2l and metabolite
(S)-2m were also evaluated in the MDS Pharma PanLab Screen,
in vitro and in vivo covalent protein binding assay, ancillary phar-
macology study (CV dog at PO 3 and 10 mg/kg dose; mouse CNS
at PO 100 mg/kg dose). No signal that might suggest potential lia-
bility was identified. Based on all these findings inhibitor (S)-2l
was selected to advance into pre-clinical development.
3. Evans, J. F.; Ferguson, A. D.; Mosley, R. T.; Hutchinson, J. H. Trends Pharmacol.
Sci. 2008, 29, 72.
4. Pergola, C.; Werz, O. Expert Opin. Ther. Patents 2010, 20, 355.
5. See (a) Mehrabian, M.; Allayee, H.; Wong, J.; Shih, W.; Wang, X.-P.; Shaposhnik,
Z.; Funk, C. D.; Lusis, A. J. Circ. Res. 2002, 91, 120; (b) Avis, I.; Hong, S. H.;
Martinez, A.; Moody, T.; Choi, Y. H.; Trepel, J.; Das, R.; Jett, M.; Mulshine, J. L.
FASEB J. 2001, 15, 2007.
6. Yun, M. R.; Park, H. M.; Seo, K. W.; Lee, S. J.; Im, D. S.; Kim, C. D. Free Radic. Res.
2010, 44, 742.
7. Berger, W.; De Chandt, M. T.; Cairns, C. B. Int. J. Clin. Pract. 2007, 61,
663.
8. Ducharme, Y.; Blouin, M.; Brideau, C.; Chateauneuf, A.; Gareau, Y.; Grimm, E. L.;
Juteau, H.; Laliberte, S.; MacKay, B.; Masse, F.; Ouellet, M.; Salem, M.; Styhler,
A.; Friesen, R. W. ACS Med. Chem. Lett. 2010, 1, 170. and references therein.
9. Unpublished results.
10. Berthelette, C.; Dufresne, C.; Li, L.; Wang, Z. WO2007016784, 2007; 73pp.
11. Higa, T.; Krubsack, A. J. J. Org. Chem. 1975, 40, 3037.
12. Brideau, C.; Chan, C.; Charlson, S.; Denis, D.; Evans, J. F.; Ford-Hutchinson, A.
W.; Fortin, R.; Gillard, J. W.; Guay, J.; Guevremont, D.; Hutchinson, J. H.; Jones,
T. R.; Leger, S.; Mancini, J. A.; McFarlane, C. S.; Pickett, C.; Piechuta, H.; Prasit, P.;
Riendeau, D.; Rouzer, C. A.; Tagari, P.; Vickers, P. J.; Young, R. N.; Abraham, W.
M. Can. J. Physiol. Pharmacol. 1992, 70, 799.
To conclude, we discovered that substituted benzothiophene
was able to serve as a novel scaffold of 5-LO inhibitors. Through
extensive SAR studies on the series, a potent and selective
5-LO inhibitor 3-(4-fluorophenyl)-6-({4-[(1S)-1-hydroxy-1-(tri-
fluoromethyl)propyl]-1H-1,2,3-triazol-1-yl}methyl)-1-benzothio-
phene-2-carboxamide ((S)-2l or MK-5286) was identified.
13. By hydrolysis of (S)-2l with NaOH.