Discovery of a novel series of potent MK2 non-ATP competitive inhibitors using 1,2-substituted azoles as cis-amide isosteres

Article abstract of DOI:10.1016/j.bmcl.2014.05.024

A unified strategy was conceived and implemented to deliver conformationally constrained anilides based on their preferred cis-amide conformers. The imidazole/triazole mimicing amide bonds were designed, building upon an earlier discovery of a novel series of tricyclic lactams MK2 kinase inhibitors. This approach enabled rapid, modular synthesis of structurally novel analogs. The efficient SAR development led to the discovery of low molecular weight and potent MK2 non-ATP competitive inhibitors with good ligand efficiency, which led to improved permeability and oral exposure in rats.

Full text of DOI:10.1016/j.bmcl.2014.05.024

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a novel series of potent MK2 non-ATP competitive
inhibitors using 1,2-substituted azoles as cis-amide isosteres
a
a
a
a
a
Dong Xiao a, , Xiaohong Zhu , Michael Sofolarides , Sylvia Degrado , Ning Shao , Ashwin Rao ,
Xiao Chen a, Robert Aslanian a, James Fossetta b, Fang Tian b, Prashant Trivedi b, Daniel Lundell b,
Anandan Palani a
⇑
a Medicinal Chemistry, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
b In Vitro Pharmacology, 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:
A unified strategy was conceived and implemented to deliver conformationally constrained anilides
based on their preferred cis-amide conformers. The imidazole/triazole mimicing amide bonds were
designed, building upon an earlier discovery of a novel series of tricyclic lactams MK2 kinase inhibitors.
This approach enabled rapid, modular synthesis of structurally novel analogs. The efficient SAR develop-
ment led to the discovery of low molecular weight and potent MK2 non-ATP competitive inhibitors with
good ligand efficiency, which led to improved permeability and oral exposure in rats.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 25 February 2014
Revised 6 May 2014
Accepted 9 May 2014
Available online xxxx
Keywords:
cis-Amide bond
Amide bond isostere
MK2 inhibitors
Conformational constraint
Ligand efficiency
Tumor necrosis factor
a
(TNF
a
) antagonism using biologics has
the non-competitive mode of inhibition may have several advanta-
ges. Since 1a do not bind in the active site, they usually have
improved selectivity versus other kinases. Additionally, cellular
become a gold standard in the treatment of several autoimmune
diseases such as rheumatoid arthritis (RA), Crohn’s disease, and
psoriasis since the late 1990s.1 The huge commercial successes of
etanercept (EnbrelÒ), infliximab (RemicadeÒ), and adalimumab
(HumiraÒ) provided incentive for the search of new therapeutic
interventions using small molecules.2 In recent years, MAPK-
activated protein kinase 2 (MK2)3 has evolved to become one of
the most important therapeutic targets. The MK2 pathway has
ATP (2–5 mM) with high affinity for P38
a MAPK-activated MK2
(KM = 2
l
M)11 would not compete with these inhibitors, which
would potentially make it easier to achieve inhibitory efficacy.
Upon being identified as a hit, compound 1a had been subject to
preliminary SAR investigation. It was shown that the alkylated
analogs such as 1b and 1c exhibited significantly increased
potency (Fig. 1).9
As we reported earlier, we postulated that this dramatic
increase of potency of the N-alkyl analogs could be attributed to
the conformational change of the amide bond. It has been shown
that anilides in solution or solid states existed in cis-amides (E-
conformers),12 which were generally favored by 3 kcal/mol. Our
hypothesis was confirmed by the synthesis and evaluation of a
been implicated in the regulation of mRNA stability of TNF-a, along
with several other mediators with unknown immune responses.4 It
has been proposed that inhibition of the MK2 kinase would result
in the reduction of TNF-a
production.5 In animal models, a recent
report showed that MK2 knockout mice were viable and devoid
of rheumatoid arthritis.6 These findings prompted industry-wide
discovery research on MK2 inhibitors.7,8
We recently reported a lead generation strategy of MK2 inhibi-
tors9 by affinity selection-mass spectrometry-based screening.10
By using a MK2 construct in the presence of 100 mM ATP, a series
of non-ATP competitive inhibitors represented by 1a were identi-
fied. Compared with previously known ATP competitive inhibitors,
NH
N
O
1a (R = H): IC50 (MK2) 5.0-5.5 µM
O
Cl
N
R
1b (R = Me): IC50 (MK2) 0.6 µM
1c (R = E t): IC50 (MK2) 0.13
M
µ
1
⇑
Corresponding author. Tel.: +1 9087404484.
E-mail address: dong.xiao@merck.com (D. Xiao).
Figure 1. Linear MK2 non-ATP competitive inhibitors.
http://dx.doi.org/10.1016/j.bmcl.2014.05.024
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Xiao, D.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.05.024

2
D. Xiao et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
novel series of tricyclic lactams.8a The prototypical analog 2a
afforded similar MK2 inhibitory potency of 1c. Subsequent lead opti-
mization efforts led to the discovery of a potent 2b, which showed
The synthesis of the azole analogs were in general straight-
forward. The tetrazole and triazole analogs could be derived simply
for the linear amide analogs. For example, the tetrazole 5 can be
synthesized using standard PPh3/DEAD/TMSN3 procedure.14 Simi-
lar approaches could be used in the preparation of triazoles 6
and 7 (Scheme 1).15 For the imidazole series, we developed a differ-
ent synthesis, so the N-phenyl ring can be modified readily. In this
sequence, an imidazole ring 9 was constructed16,17 from the com-
mercial aldehyde 8. SNAr reactions with substituted nitrofluoro-
benzenes yielded intermediates 10. The nitro group was then
reduced to an amine 11, which was converted to piperazine using
solid Al2O3 promoted cyclization.18 The benzamidazole analog 13g
was synthesized in an identical manner (Scheme 2).
Based on earlier structural–activity relationship (SAR) in the
linear 1 and tricyclic lactam 2 series, the R1 groups were kept as
small electron-withdrawing groups such as chlorines and
nitriles.8a,9 Our first round of investigation was directed at elucida-
tion of the nature of azole cores. As shown in Table 1, the imidazole
analogs 13a showed threefold improved MK2 activity compared to
the lactam 2a. The 1,2,4-triazole 13c,d also were similarly potent.
good cell based antagonism in a LPS-induced TNF
a assay (Fig. 2).
Although 2b is a very potent analog, its ligand efficiency (LE)
was reduced to 0.27 due to the addition of a large pyrimidylben-
zene moiety. This low LE was a likely culprit for the low permeabil-
ity and rat oral exposure (see data in Table 5). The initial lactam
lead 2a actually had a better LE of 0.33. Therefore, the objective
of the next phase of optimization was to see if the biaryl moiety
could be deleted and the potency be regained elsewhere. Two of
the underexplored areas thus far were the piperazine and the aryl
group attached to it. However, the chemistry to modify these
groups in the tricyclic lactam series was difficult. It was envisioned
that the amide bond replacement using azoles13 might provide a
simplified series. The azole series (vide infra) would allow a rapid,
modular synthesis of analogs that target the structure–activity
relationship (SAR) of N-phenyl and piperazine fragments. Using
this approach, we hoped to improve potency without resorting to
the large biaryl sidechain (Fig. 3).
O
O
R
NH
Me
O
N
Cl
O
N
N
N
O
O
Cl
Cl
N
cis-amide
N
2a (R = H),
(E conformer)
favored by
MK2 IC50 = 79 nM; LE = 0.33
Z conformer
NH
ca. 3 kcal/mol
2b (R =
N
N
H
N
MK2 IC50 = 5.7 nM; LE = 0.27
Figure 2. Evolution of lead optimization strategy: tricyclic lactams constrained to cis-amide.
X
N
O
X
O
Me
R1
O
R1
N
N
N
SAR
SAR
1,2-azole
isosteres
E conformer
favored by
ca. 3 kcal/mol
N
Azoles
3
X = N, CH, etc
N
H
N
H
Figure 3. Evolution of lead optimization strategy: 1,2-azole as cis-amide isosteres.
N
O
N
N
N
NH
1) PPh3, DEAD, TMSN3
2) TFA
O
O
R1
N
R1
N
4
5
NBoc
NH
1) POCl3
1) NaH, O-(mesitylsulfonyl)hydroxylamine
2) NH2NHCHO
3) TFA
2) NH2CHO, ZnCl2, 160oC
N
N
N
N
N
N
O
O
R1
R1
N
N
6
7
NH
NH
Scheme 1. Synthesis of tetrazole and triazole analogs.
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D. Xiao et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
R
N
1) NBS
H2N
N
F
NO2
O
N
CHO
R1
O
O
N
H
NH2
R
R1
R1
DIEA, 80 o
C
2) PhI(OAc)2
10
9
8
NO2
Cl
N
N
O
N
NH-HCl
R1
O
N
R
Cl
R1
Zn, HOAc
basic Al2O3
120 o
R
N
C
12
11
NH2
N
H
Scheme 2. Synthesis of imidazole analogs.
Table 1
Table 2
MK2 assay19 data of azole analogs
MK2 assay data of N-phenyl analogs
X
N
Y
N
R2
N
O
N
O
R3
N
NC
R1
N
14
13
NH
NH
Entry
R2
R3
IC50 (MK2) (nM)
LE
Entry
R1
X
Y
IC50 (MK2) (nM)
LE
14a
14b
14c
14d
14e
F
Cl
CH3
Br
CF3
H
H
H
H
H
16.2
13.0
17.4
22.3
31.3
0.34
0.35
0.34
0.34
0.30
13a
13b
13c
13d
13e
13f
Cl
CH
CH
N
CH
CH
CH
CH
CH
N
25.5
84.7
0.36
0.32
0.37
0.35
0.32
0.29
CN
Cl
14.3
CN
Cl
N
23.3
N
N
14f
H
43.5
0.29
CH
N
157.0
450.1
CN
14g
14h
H
H
Cl
Br
7.7
35.7
0.36
0.33
Cl
13g
95.4
0.29
MK2 inhibition data represented the average values of duplicates or triplicates.
MK2 inhibition data represented the average values of duplicates or triplicates.
aromatic ones, such as pyridines. The substituted piperidines 15
were synthesized by the Cu catalyzed C–N coupling reactions. Anal-
ogous reactions were used to synthesize the morpholines (18k and
18l in Table 3). The pyridine analogs 17 were assembled from Suzuki
coupling with intermediate arylbromides 16, which in turn were
synthesized from the corresponding anilines 11 (Scheme 3).
The 3- and 4-piperidine analogs 18a and 18b showed similar
activity to the piperazine analog 13a while transposing the nitro-
gen to 2-position caused loss of MK2 potency. Given the much
lower basicity of the 3-, and 4-pyridine analogs 18d and 18e, it
was not surprising to observe 100 fold less potency. Data of 18f–
18i were quite intriguing. Being one order lower in basicity to that
of analogs 18b, they showed a small but consistent trend toward
lower potency in the chlorophenyl series. Both enantiomers of
morpholines 18g and 18h exhibited comparably potency. This data
indicated the lack of opportunity for further potency gain using
chirally differentiated amines. Substitutions at the benzylic posi-
tion in 18i to 18l were generally tolerated, which provided addi-
tional handle to influence basicity and other properties of the
analogs.21 Taken together, the SAR confirmed the need of a basic
amine for MK2 potency and carbon linkage to the phenyl ring were
acceptable (Table 3).
However, the 1,2,4-triazole 13e, the tetrazole 13f and the benzami-
dazole analog 13g all showed reduced potency. Taken together,
this SAR trend may indicate some subtle preference at the Y posi-
tion for the enzyme interactions. Nevertheless, this SAR confirmed
the utility of 1,2-positioned imidazole and 1,2,4-triazole as
cis-amide isosteres with good LE, which paved way for further opti-
mizations (Table 1).
Our next objective was to investigate the SAR of the N-phenyl
ring. As shown in Table 2, analogs 14a–c with small R2 substituents
ortho to the imidazole provided consistent improvement of
potency. As the groups increased in size, in 14d–14f, the potency
started to decline. For the R3 position meta to the imidazole, a
chloro-substitution in 14g led to most improved potency com-
pared with 13b while the bromo analog 14h was four time less
potent. Now we achieved single digit potency with good ligand
efficient of 0.36 without resorting to the large biaryl sidechain pre-
sented in the lactam 2b (Table 2).
The final set of SAR efforts were focused on the piperazine moi-
ety. This investigation had three purposes. The first was to explore if
the potency can be further improved. The second was to reduce the
electron density on the piperazine linked phenyl ring, by using a car-
bon linkage, therefore mitigating potential oxidative metabolism of
the phenyl ring.20 The third was to define the effect of basicity of the
distal nitrogen on MK2 inhibition. The analogs planned included
saturated heterocycles such as piperidines and morpholines and
In summary, our initial vision of using azole replacement of
cis-amide bond was successful. The imidazole and 1,2,4-triazole
isosteres proved to be useful surrogates for the original amide
bond. Subsequent systematic investigations of the SAR of the sub-
stitutions on the N-phenyl ring and the piperazine moiety were
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4
D. Xiao et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 3
120
MK2 assay data of carbon linked analogs
% Inhibition @
N
100
80
60
40
20
0
N
100uM ATP
1uM ATP
O
R2
R1
R2
18
Entry
R1
pKa (calcd)
IC50 (MK2) (nM)
36.7
Cl
Cl
NH
NH
18a
10.1
18b
9.9
26.0
HN
Cl
Cl
Cl
18c
18d
18e
9.4
5.2
4.6
160.0
3730
3390
N
10 -13 10 -12 10 -11 10 -10 10 -9
10 -8
10 -7
10 -6 10-5
10-4
10-3
Mole
N
Figure 4. Inhibition data of compound 13a in different ATP concentrations.
NH
with the imidazole/triazole series retaining the non-ATP competi-
tive characteristics of the amide/lactam series.
Cl
Cl
18f
8.8
8.4
65.0
55.3
O
O
Given the non-ATP competitive nature of the 1,2-imidazole/
triazole series, it was not surprising to find the high kinase selectivity
as existed in the progenitor linear series. For example, the test
screen of 13a on an in-house kinase selectivity panel shows the
excellent selectivity retained against 24 sampled kinases (Table 4).
The compound 13a was further profiled in several key assays.
The data were listed alongside of that of 2b (Table 5). To make a
comparison, 13a was four fold less active in in vitro assay. In the
cell-based biochemical assay, using a MK2 downstream target
HSP2722 in THP-1 cells, 13a showed EC50 of 2139 nM. It was also
active in inhibiting LPS-stimulated hTNFa23 with EC50 of 800 nM.
Although with similar PPB, 13a showed much improved perme-
ability and rat oral exposure24 over 2b. These improvements were
likely due to a favorable LE of 13a.
18g
NH
NH
Cl
18h
8.4
74.9
F
Cl
18i
18j
18k
18l
9.0
9.6
9.0
9.6
66.5
25.8
24.9
26.0
NH
HO
F
Cl
NH
NH
NH
CN
CN
HO
MK2 inhibition data represented the average values of duplicates or triplicates.
In conclusion, a unified strategy was conceived and imple-
mented to deliver conformationally constrained anilides based on
their preferred cis-conformers. Our earlier discovery of a novel ser-
ies of tricyclic lactams proved the constrained cis-conformers were
indeed potent MK2 kinase inhibitors. Built upon this finding, the
imidazole/triazole mimics of the anilide bonds were designed
and synthesized. This approach enabled rapid, modular synthesis
of structurally novel analogs. The efficient SAR development led
to the discovery of low molecular weight, potent MK2 non-ATP
competitive inhibitors with good ligand efficiency. The key com-
pound 13a showed improvements of permeability and in vivo
exposure in rat PK studies.
also concluded. It appeared that the changes in the phenyl ring
provided the most gain in potency. In the end, a series of novel,
potent MK2 kinase inhibitors with good ligand efficiency was
discovered.
Mindful of the substantial structural changes from linear
amides and lactams to the azole analogs, we would like to assess
the mode of inhibition and the related key question of kinase
selectivity of this series. Compound 13a was tested in the MK2
enzyme assay in the presence of 1
lM and 100 lM ATP. As shown
in Figure 4, no shift in potency was observed. This was consistent
N
R
N
O
N
1)
I
NBoc
R1
O
N
H
R1
CuCl, 1,10-phenothroine
2) TFA
R
9
15
1) DIEA
N
H
F
NO2
2) HOAC, Zn
3) iBuNO2, CuBr
N
O
N
N
N
R1
O
R1
Suzuki coupling
N
16
17
Br
Scheme 3. Synthesis of carbon linked analogs.
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D. Xiao et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
5
Table 4
In-house kinase screening data of 13a
Kinase
IC50
Kinase
IC50
FLT3
PLK3
CSNK1D
NEK2
PKCA
IRAK4
AKT1
IKKB
CDK2
RSK2
ROCK2
JAK2
(
l
M)
>30
KDR
>30
>30
MET
>30
>30
TSSK2
>30
>30
MST2
>30
>30
LCK
>30
>30
IGF1R
>30
>15
ERK2
>30
>30
CHK1
>30
>30
EGFR
>30
>30
EPHB4
>30
>20
GSK3B
>30
>30
CAMK4
>30
(l
M)
Table 5
Comparison of key data of 2b and 13a
MW
CLogP
IC50 (MK2) (nM)
LE
TNF
a
(IC50) (nM)
HSP27 (IC50) (nM)
PPB (hu) (%)
Caco (nm/s)
Rat AUC (10 mpk, po) (lM h)
2b
13a
562
404
5.9
6.5
5.7
25.5
0.27
0.34
1493
800
ND
2139
98.4
97.2
<1
50
0
13.3
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Acknowledgment
The authors thank Drs. Malcolm MacCoss and William Greenlee
for stimulating discussion and their support of this research
program.
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Falorni, M.; Giacomelli, G.; Porcheddu, A. Tetrahedron Lett. 1999, 40, 8701; (j)
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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: 1Â inhibitor, 1% DMSO, 100
l
M ATP, 50 pM MK2 and
L of 1:400 diluted
100 nM substrate. Reaction was stopped by adding 60
l
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).
20. Metabolism simulation of compound 13a indicated that the electron-rich
phenyl ring attached to piperazine could be a major metabolic site. Simulation
software: MetaSite 4.0.2 (http://www.moldiscovery.com/index.php, Molecular
Discovery Ltd). Also see: Cruciani, G.; Carosati, E.; De Boeck, B.; Ethirajulu, K.;
Mackie, C.; Howe, T.; Vianello, R. J. Med. Chem. 2005, 48, 6970.
21. Morgenthaler, M.; Schweizer, E.; Hoffmann-Roder, A.; Benini, F.; Martin, R. E.;
Jaeschke, G.; Wagner, B.; Fischer, H.; Bendels, S.; Zimmerli, D.; Schneider, J.;
Diederich, F.; Kansy, M.; Muller, K. ChemMedChem 2007, 2, 1100.
22. LPS Induced Phospho-HSP27 Serine 78 assay: Bring the THP-1 cells to Log-phase
by passing the cells at proper density a day previous to the assay (1 Â 105 cells/
mL). Spin to collect cells and suspend in fresh complete-RPMI-1640 medium at
cell density of 2.5 Â 106 cells/mL. Add equal volume of RPMI-1640 without FBS
containing 40 nM okadaic acid (final: 100,000 cells/80 lL/well). Plate 100,000
cells/80
Add 10
incubate at 37 °C for 60 min. Add 10
and incubate at 37 °C for 60 min. Add 25
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 4 °C. MesoScale pHSP27
l
l
L into flat-bottom cell culture plate and incubate at 37 °C for 60 min.
L of 10Â diluted-compound in 1% DMSO in 5% FBS-RPMI medium and
L of 10Â LPS in 5% FBS-RPMI medium
l
l
L 5Â cell lysis buffer containing 5Â
S78 Assay 10–20
23. Assay of TNF secretion from THP1 cells: THP1 cells were obtained from ATCC
(Cat. No. TIB-202) and cultured in medium consisting of RPMI1640
supplemented with 2 mM -glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L
glucose, 10% fetal bovine serum, 10 mM HEPES, 1 mM sodium pyruvate and
lL cell lysate was used in this assay.
a
L
50 lM 2-mercapatoethanol. Cells were plated into the wells of 96-well culture
plates at 0.5 Â 105 cells/well for TNF
a, then cultured for 60 min to allow pre-
equilibration, and the treated with varying concentrations of compound 2s for
30 min prior to addition of LPS (Sigma–Aldrich, Cat. No. L2654) at 1
After 3 h of culture for TNF assays, supernatants were removed and secreted
cytokines were measured using commercially available kits and
manufacturer’s protocols. Secreted TNF was specifically measured by an
lg/mL.
a
a
ELISA method (R&D Systems; Cat. No. DTA00C).
24. The rat (Sprague–Dawley) PK assay was run at 10 mpk using oral dosing with
20% HPBCD as vehicle. AUC was determined from 0 to 6 h. For Ref.: Cox, K. A.;
Dunn-Meynell, K.; Korfmacher, W. A.; Broske, L.; Nomeir, A. A.; Lin, C. C.;
Cayen, M. N.; Barr, W. H. Drug Discovery Today 1999, 4, 232.
Please cite this article in press as: Xiao, D.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.05.024