Facile synthesis of tetracyclic azepine and oxazocine derivatives and their potential as MAPKAP-K2 (MK2) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.11.113

Facile synthesis of two new series of tetracyclic azepine and oxazocine analogs is described. These analogs were evaluated for their potential as MAPKAP-K2 (MK2) inhibitors and several were found to be potent at inhibiting MK2 with a non-ATP competitive binding mode.

Full text of DOI:10.1016/j.bmcl.2011.11.113

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1068–1072
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Facile synthesis of tetracyclic azepine and oxazocine derivatives
and their potential as MAPKAP-K2 (MK2) inhibitors
Ashwin U. Rao a, , Dong Xiao a, Xianhai Huang a, Wei Zhou a, James Fossetta b, Dan Lundell b, Fang Tian b,
⇑
Prashant Trivedi b, Robert Aslanian a, Anandan Palani a
a Department of Medicinal Chemistry, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
b Department of Immunology, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Facile synthesis of two new series of tetracyclic azepine and oxazocine analogs is described. These ana-
logs were evaluated for their potential as MAPKAP-K2 (MK2) inhibitors and several were found to be
potent at inhibiting MK2 with a non-ATP competitive binding mode.
Published by Elsevier Ltd.
Received 21 October 2011
Revised 26 November 2011
Accepted 28 November 2011
Available online 6 December 2011
Keywords:
Azepine
Oxazocine
Tetracyclic
MK2
Mitogen-activated protein kinases (MAPKs) belong to the Ser/Thr
kinase family, to control cytoskeletal remodeling and regulate the
cell cycle. Because of their biological roles, MAPK have been targeted
for various chronic inflammatory diseases such as rheumatoid
arthritis (RA), Alzheimer’s disease, atherosclerosis and cancer.1
MAPKAP-K2 (MK2), a direct downstream substrate of p38, plays a
crucial role in signaling and synthesis of proinflammatory cytokines,
based on empirical reports that rigidity in the molecule would ren-
der proper orientation in the binding pocket since the amide bear-
ing substituents are conformationally flexible and rotation about
the amide bond is extremely facile. With this effort we have iden-
tified structurally restricted compounds of type 2 and 3 with im-
proved activity (Fig 2).7 We hypothesized that further restricting
the conformation by combining the structural features of 2 and 3
resulting in tetracyclic derivatives may improve MK2 activity and
selectivity. We focused our attention on the development of a prac-
tical synthetic route to tetracyclic azepine derivatives 4 and oxazo-
cine derivatives 5.
The synthesis started with preparing the appropriate intermedi-
ate 8 as outlined in Scheme 1. Conversion of 5-(4-chlorophenyl)-
furan-2-carboxylic acid ethyl ester 9 to benzhydryl alcohol 12
was achieved by reacting 9 with Knochel base and trapping the
Mg intermediate with aldehyde 8 (Scheme 2).8 Reduction of the ni-
tro group was effected with Zn in acetic acid to afford 15 in 90%
yield. Triethylsilane reduction of 15 in TFA afforded the benzyl
derivative 18 in 59% yield. Upon treatment with LiHMDS, 18
smoothly underwent cyclization to give 21 in 78% yield. Likewise,
azepinones 22 and 23 were synthesized in a similar manner de-
scribed for 21 in 95% yield. The 4-fluorophenyl derivative 24 was
obtained from bromide 23 by Suzuki cross-coupling.9 Compound
23 provides an expedient way to incorporate other aryl groups
through changes on the left-hand side of the azepine core. Depro-
tection of the N-Cbz group with TMSI afforded the tricyclic analogs
25 and 26.
such as TNFa . MK2 knockout mice show a strong
, IL-6 and IFNc 2
reduction in disease incidence and disease severity in arthritis mod-
els.3 As MK2 is downstream of p38, its inhibition is expected to pro-
duce the same beneficial effect as p38 MAPK inhibition but with
reduced side effects. In spite of the strong rationale for MK2 inhibi-
tors for treating human diseases, direct proof of concept in clinical
settings has yet to be demonstrated. Several ATP-competitive inhib-
itors have been published recently, and efforts are continuing to
identify novel and more selective MK2 inhibitors.4 In contrast,
non-ATP competitive inhibitors may provide distinct advantages
with respect to these issues. In this study we wish to disclose our dis-
covery efforts in targeting this kinase with a non-ATP competitive
binding mode and our strategy towards further modification of
our lead 1 obtained from the high-throughput Automated Ligand
Identification System (ALIS)5 screening hit I leading to the tetracy-
clic series (Fig 1).
A medicinal chemistry effort was carried out to optimize this
encouraging hit into a lead by substitution of the secondary amide
in I with various alkyl and heteroaryl groups.6 We hypothesized
To construct the fourth ring in the molecule, we decided to acti-
vate the lactam with POCl3 to generate the reactive chloroiminium
⇑
Corresponding author.
E-mail address: ashwin.rao@merck.com (A.U. Rao).
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2011.11.113

A. U. Rao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1068–1072
1069
Cl
Cl
O
O
O
O
NH
NH
N
N
HN
N
1
I
N
MK2: IC50 = 5.5 µM
µM
MK2: IC50 = 0.11 µM
pHSP27: EC50 = 0.35 µM
pHSP27: EC50 = 2
Figure 1. ALIS hit-to-lead modification.
N O
N
Ar
O
O
O
O
N
N
Ar
N
O
O
O
O
3
2
4
5
Figure 2. Conformational restraint on hit-to-lead compound 1.
The synthesis of oxazocine derivative started with the conver-
sion of the commercially available 5-(4-chlorophenyl)-2-formylfu-
ran-3-carboxylic acid ethyl ester 35 to dihydroimidazole by
reacting with 1,2-ethylenediamine followed by NBS treatment
(Scheme 4).11 Oxidation with iodophenyl acetate12 afforded imid-
azole derivative 36, followed by the reduction of ester 36 using DI-
BAL-H to afford 37. N-Arylation of imidazole 37 with 3-fluoro-4-
iodonitro benzene in the presence of Cs2CO3 gave a 76% yield of
38 which upon treatment with t-BuOK gave a low yielding (25%)
nitro derivative 39. Reduction of nitro group using Zn in acetic acid
gave the aniline intermediate in moderate yields, which was sub-
sequently dialkylated with bis(2-bromoethyl)amine hydrogen bro-
mide and basic alumina under neat conditions to yield 40.
An alternate approach for the preparation of oxazocinone deriv-
atives using 35 required the preparation of 45 and this synthesis
was carried out with relative ease as shown in Scheme 5. Treat-
ment of 35 with TMPMgClÁLiCl and quenching with DMF provided
aldehyde 39. Reduction of aldehyde 41 to alcohol was achieved
using NaBH4 followed by the formation of the nitro derivative 43,
which was prepared using standard Mitsunobu conditions from
42, obtained by a similar route as in Scheme 1.13 Reduction of 43
to amine 44 followed by ring closure using LiHMDS generated
NO2
CHO
a
NO2
NCbz
N
HN
F
CHO
CbzN
6
8
7
Scheme 1. Synthetic route for the preparation of intermediate 8. Reagents and
conditions: (a) DIEA, DMF, 80 °C, 12 h, 76%.
ion intermediate 27 (Scheme 3). Thus POCl3 treatment of 21 gener-
ated the active intermediate 27 which upon reaction with amino-
acetaldehyde dimethyl acetal followed by a reflux under acidic
conditions conveniently led to the preparation of tetracyclic deriv-
ative 28. In a similar fashion, the chloroiminium ion intermediate
27 generated from 21, 22 and 24 were coupled with formic hydra-
zide to afford triazoloazepines (30–32). The analogous methyl
substituted imidazo- and triazolo-azepine (33 and 34) were ob-
tained by heating 27 with propargylamine and acetic hydrazide,
respectively.10 Considering the ring constraint with the tetracyclic
systems, these imidazole and triazole forming conditions are use-
ful techniques.
c
a
b
O
O
O
X
X
X
O
O
O
OEt
OH
OEt
OEt
OH
H2N
O2N
9
X = 4-Cl-Ph
12
13
X = 4-Cl-Ph, 50%
X = 4-CN-Ph, 70%
15
X = 4-Cl-Ph, 90%
X = 4-CN-Ph, 95%
10
X = 4-CN-Ph
11 X = Br
16
14 X = Br, 73%
N
N
17 X = Br, 75%
NCbz
NCbz
O
O
O
X
O
O
O
NH
NH
d
f
X
X
OEt
H2N
21
22
N
X = 4-Cl-Ph, 78%
X = 4-CN-Ph, 95%
N
18
X = 4-Cl-Ph, 59%
X = 4-CN-Ph, 80%
25
26
X = 4-Cl-Ph, 61%
X = 4-CN-Ph, 53%
19
N
23 X = Br, 95%
NCbz
NH
20 X = Br, 52%
e
NCbz
24
X = 4-F-Ph, 89%
Scheme 2. Synthetic route for the preparation of azepinone derivatives 21–26. Reagents and conditions: (a) TMPMgClÁLiCl, 8, THF, À30 °C, 2 h; (b) Zn, AcOH, 1 h; (c) Et3SiH,
TFA, CH2Cl2, 1 h; (d) LiHMDS, THF, 0 °C, 3 h; (e) Pd(dppf)2Cl2ÁCH2Cl2, K3PO4, dioxane, 100 °C, 6 h, 80%; (f) TMSI, CH2Cl2, 1 h.

1070
A. U. Rao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1068–1072
O
Cl
NH+
O
O
NH
X
X
a
21
X = Cl
27
N
N
22 X = CN
24 X = F
NCbz
NCbz
d, c
e, c
f, c
b, c
N
N
N
N
N
N
N
N
N
Me
N
Me
N
O
O
O
N
O
X
X
X
X
N
N
NH
NH
NH
NH
34
X = Cl, 75%
30
33
X = Cl, 55%
X = Cl, 70%
28
X = Cl, 65%
31 X = CN, 17%
32 X = F, 67%
29 X = CN, 29%
Scheme 3. Synthetic route for the preparation of 27–34. Reagents and conditions: (a) POCl3, 100 °C, 2 h; (b) (i) aminoacetaldehyde dimethyl acetal, THF, reflux, 2 h; (ii) 4 N
HCl, reflux, 2 h; (c) TMSI, CH2Cl2, 1 h; (d) formic hydrazide, THF, reflux, 2 h; (e) (i) propargylamine, THF, reflux, 2 h; (ii) 4 N HCl, reflux, 2 h; (f) acetic hydrazide, THF, reflux, 2 h.
Cl
Cl
Cl
c
d
a, b
N
N
O
O
O
O
O
CHO
OEt
N
N
H
H
OEt
HO
35
36
37
Cl
Cl
Cl
N
N
O
e
f, g
N
N
O
O
N
N
F
HO
O
O
O2N
N
NO2
38
39
40
N
H
Scheme 4. Synthetic route for the preparation of 40. Reagents and conditions: (a) 1,2-ethylenediamine, NBS, CH2Cl2, 12 h, 55%; (b) PhI(OAc)2, K2CO3, DMSO, 12 h, 77%; (c)
DIBAL-H, THF, À78 °C, 84%; (d) 3-fluoro-4-iodonitro benzene, Cs2CO3, DMF, 65 °C, 12 h, 76%; (e) t-BuOK, THF, 6 h, 25%; (f) Zn, AcOH, 2 h, 54%; (g) bis(2-bromoethylamine)
hydrogen bromide, Al2O3, 120 °C, 12 h, 53%.
45, which could be a useful building block that can be functional-
ized to give desired tetracyclic products such as imidazo- and triaz-
olo-derivatives as discussed in Scheme 3. Removal of the Boc-
group with TFA afforded the tricyclic oxazocine derivative 46 in
affinity but with reduced cellular activity. On the other hand cyano
derivative 31 showed similar MK2 and cellular inhibition profiles
as 29. Compound 32 had no major effect on MK2 inhibition however
the fluoro substitution had a favorable effect on solubility. Interest-
ingly, the less soluble derivatives 29 and 31 showed improved cellu-
lar activity indicating that cellular potency depend upon various
additional parameters. The IC50 for MK2 inhibition for the oxazocine
derivative 40 was determined to be 18 nM with improved solubility
and was 6-fold more potent than the corresponding tricyclic analog
46. The mode of inhibition of MK2 by azepine 31 and oxazocine 40
with respect to peptide substrate, TAMRA peptide was determined
by analysis of MK2 activity in the presence of saturating ATP and
varying concentrations of peptide substrate and the inhibitor (Fig
3). The results identified a non-ATP competitive binding mode
throughout the series.16
75% yield.
14
Regarding MK2 inhibition (IC50
)
and/or cellular activity
(EC50),15 the conformationally restricted tetracycles proved to be
clearly superior to the non-cyclized compound 1 and tricyclic ana-
logs 25, 26 and 46 (Table 1). Thus, a comparison of 1 and azepine
28 revealed that the former was more potent in cell-based activity,
while the cyclized analog 28 was 16-fold more potent against
MK2. We attributed the lack of cellular activity due to their differ-
ences in solubility (20 lM vs >200 lM). A similar level of potency
improvement was observed while comparing 28 with the tricyclic
analogs 25 and 26. The corresponding cyano derivative 29 restored
cellular activity at high nanomolar concentration (EC50 = 0.26
Compounds 30 and 33 with chloro substitution showed high MK2
l
M).
In conclusion, we have demonstrated practical synthetic routes
for generating tetracyclic azepine and oxazocine pharmacophores

A. U. Rao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1068–1072
Cl
1071
Cl
Cl
a
O
b, c
d
OEt
O
O
O
O
O
OEt
OEt
O
CHO
NO2
35
41
43
N
BocN
Cl
O
O
O
OH
NH
e
O
NO2
Cl
OEt
N
O
O
NH2
N
NR
44
42
45
46
R = Boc
BocN
f
N
R = H
BocN
Scheme 5. Synthetic route for the preparation of oxazocinone core 45. Reagents and conditions: (a) TMPMgClÁLiCl, DMF, THF, 2 h, 15%; (b) NaBH4, MeOH, 2 h, 70%; (c) DIAD,
PPh3, 42, THF, 12 h, 65%; (d) Zn, AcOH, 2 h, 95%; (e) LiHMDS, THF, 0 °C, 3 h, 65%; (f) TFA, CH2Cl2, 75%.
Table 1
Acknowledgments
Conformationally restricted MK2 inhibitors
We thank Dr. Chad Bennett for comments on the preparation of
the manuscript and Drs. Malcolm MacCoss and Ismail Kola for their
support and encouragement.
Compd
MK2a IC50 (nM)
p-HSP27b EC50
(
l
M)
Solubilityd
(lM)
1
25
26
28
29
30
31
32
33
34
40
46
110
96 20
0.35
>200
NDc
NDc
20
<16
10
16
33
20
10
NDc
110 10.0
7.0 0.23
2.9 0.31
9.3 0.92
3.8 0.55
15 0.57
9.2 0.95
880 22
18 3.2
110 11
NDc
1.5 0.14
0.26 0.04
2.1 0.38
0.26 0.06
NDc
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was carried in 384 well black reaction plate at room temperature in dark. Mix
5
lL of 4Â inhibitor in 4% DMSO, 5 lL of 400 lM ATP and 5 lL of 200 pM MK2
kinase and incubate for 30 min. Reaction started by adding 5 mL of 400 nM
TAMRA labeled peptide and incubating 30 min in dark. The final
concentrations are: 1x inhibitor, 1% DMSO, 100
lM ATP, 50 pM MK2 and
100 nM substrate. Reaction was stopped by adding 60
lL of 1:400 diluted
Progressive Binding Reagent in 1Â Progressive Binding Buffer A and incubating
30 min in dark. Read plate at Analyst HT 96-384 Plate Reader (LJL BioSystem)
equipped with Fluorescence Polarization module (Excitation wavelength
530 nm and Emission wavelength 580 nm).
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a day previous to the assay
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80
incubate at 37 °C for 60 min. Add 10
in 5% FBS-RPMI medium and incubate at 37 °C for 60 min. Add 10
LPS in 5% FBS-RPMI medium and incubate at 37 °C for 60 min. Add 25
Cell Lysis Buffer containing 5Â Halt Inhibitors and incubate on ice for 30 min.
Transfer cell lysate to glass fiber filter plate and stack the filter plate on top of a
96-well storage plate. Spin stacked filter-storage plate at 3500 rpm for 5 min at
l
L/well). Plate 100,000 cells/80
l
l
L into flat-bottom cell culture plate and
L of 10Â diluted-compound in 1% DMSO
L of 10Â
L 5Â
l
l
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4 °C. MesoScale pHSP27 S78 Assay 10–20
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lL cell lysate was used in this assay.