Novel 1-(2-aminopyrazin-3-yl)methyl-2-thioureas as potent inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2)

Article abstract of DOI:10.1016/j.bmcl.2009.04.088

Novel 1-(2-aminopyrazin-3-yl)methyl-2-thioureas are described as inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2). These compounds demonstrate potent in vitro activity against the enzyme with IC₅₀ values as low as 15 nM, and suppress expression of TNFα in THP-1 cells and in vivo in an acute inflammation model in mice. The synthesis, structure-activity relationship (SAR), and biological evaluation of these compounds are discussed.

Full text of DOI:10.1016/j.bmcl.2009.04.088

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3238–3242
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel 1-(2-aminopyrazin-3-yl)methyl-2-thioureas as potent inhibitors
of mitogen-activated protein kinase-activated protein kinase 2 (MK-2)
*
Songnian Lin , Matthew Lombardo, Sunita Malkani, Jeffrey J. Hale, Sander G. Mills, Kevin Chapman,
James E. Thompson, Wen Xiao Zhang, Ruixiu Wang, Rose M. Cubbon, Edward A. O’Neill, Silvi Luell,
Ester Carballo-Jane, Lihu Yang
Merck Research Laboratories, Rahway, NJ 07065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel 1-(2-aminopyrazin-3-yl)methyl-2-thioureas are described as inhibitors of mitogen-activated
protein kinase-activated protein kinase 2 (MK-2). These compounds demonstrate potent in vitro activity
against the enzyme with IC50 values as low as 15 nM, and suppress expression of TNFa in THP-1 cells and
in vivo in an acute inflammation model in mice. The synthesis, structure–activity relationship (SAR), and
biological evaluation of these compounds are discussed.
Received 25 March 2009
Revised 18 April 2009
Accepted 21 April 2009
Available online 24 April 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
MAPKAP kinase 2
MK-2
TNFa
Inflammation
Rheumatoid arthritis
Aminopyrazine
Thiourea
Tumor necrosis factor-
a
(TNF
a
) is a cytokine that is over-pro-
pyridones,8 tertrahydro-b-carbolines,9 tricyclic indole deriva-
tives,10 pyrrolo-pyrimidones,11 and benzothiophenes.12
duced in several inflammatory disease states such as rheumatoid
arthritis (RA).1 Anti-TNF
a
biologics have been very successful in
Our effort to develop small molecule MK-2 inhibitors began
with high throughput screening that identified several 1-(2-amino-
pyrazin-3-yl)methyl-2-thioureas as moderate MK-2 inhibitors as
represented by phenyl analog 1a (Fig. 1). Compound 1a exhibits
the treatment of several autoimmune diseases like RA, Crohn’s dis-
ease and psoriasis.2 Considerable effort has been devoted to the
search for biological targets which are amenable for modulation
with small molecules and a number of targets have been identified.
Among them, p38 mitogen-activated protein kinase (MAPK) is
most notable,3 with inhibitors of p38 MAPK demonstrating efficacy
in preclinical in vivo models as well as in RA patients in clinical
trials.4
a 2.0
7.9 M IC50 value in a cell-based assay that measures LPS-stimu-
lated TNF
production from THP-1 cells.14 This prompted us to
l
M IC50 value in an in vitro MK-2 enzyme assay13 and
l
a
start a chemistry program to further explore this class of com-
pounds. In this Letter, we would like to disclose a series of potent
MK-2 inhibitors discovered through this effort.
Compounds 1–12 in Figures 1 and 2 and Tables 1–7 were syn-
thesized according to Schemes 1 and 2. Substituted 2-aminopyra-
zin-3-carbamides 1315 were converted to corresponding cyanides
Mitogen-activated protein kinase-activated protein kinase 2
(MAPKAPK-2 or MK-2) is a direct substrate of p38 MAPK and plays
a critical role in the signal transduction pathway regulating the
production of TNFa 5
less TNF
.
MK-2 knockout mice produce significantly
a
when challenged with lipopolysaccharide (LPS),5a in
addition to being fertile, healthy, and resistant to developing dis-
eases in arthritis models.5,6 MK-2 inhibition therefore may provide
NH2
N
X
an effective treatment for TNFa-mediated diseases. Recently, sev-
N
N
eral structural classes of compounds have been reported to be
H
H
N
inhibitors of MK-2, among them aminocyanopyridines,7 pyrrolo-
Cl
1a: X =S
1b: X = O
* Corresponding author. Tel.: +1 732 594 0585; fax: +1 732 594 2840.
E-mail address: Songnian_lin@merck.com (S. Lin).
Figure 1. HTS hit 1a and its analog 1b.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.088

S. Lin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3238–3242
3239
Table 2
S
SAR on the aminopyrazine moiety
N
N
R
NH2
N
S
H
H
2a-l
N
N
H
H
6
N
R =
R2
5
NH2
R1
NH2
N
NH2
N
NH2
N
N
N
Compound
R1
R2
MK-2 IC50 (lM)
Ph
1a
4a
4b
4c
4d
4e
4f
4g
4h
4i
Cl
H
Me
Et
n-Pr
CF3
c-Pr
Ph
Cl
H
H
H
H
H
H
H
H
2.0
32
4.0
1.5
N
NH2
N
Me
Ph
Me
2d
2a
2b
2c
2.3
>50
0.47
ꢀ50
>50
>50
>50
NH2
N
NMe2
OEt
N
Cl
Cl
4j
Cl
SMe
2e
2i
2f
2g
2h
further explored the right-hand side aminopyrazine subunit. As
shown in Figure 2, aminopyrazine was replaced with aminopyrim-
idines (2a and 2b), aminopyridazine (2c), aminopyridine (2d), pyr-
idines (2e and 2f), quinolines (2j), isoquinolines (2i), naphthylenes
(2k), and benzenes (2g and 2h), and all these replacements
N
N
N
N
Me
2j
2k
2l
Figure 2. Preliminary SAR on aminopyrazine.
resulted in the loss of activity (IC50 > 40 lM). The deletion of the
2-amino group and the replacement of a 5-Cl with a 6-methyl
14 upon treatment with POCl3 in DMF followed by aqueous HCl.
Reduction of cyanides 14 with LiAlH4 afforded desired amines 15
in good yields. Treatment of amines 15 with various of amines in
the presence of 1,10-thiocarbonyldiimidazole (TCI), or directly with
isothiocyanides, provided corresponding 1-(2-aminopyrazin-3-yl)-
methyl-2-thioureas 1a and 3–12 in moderate to high yields
(Scheme 1). Urea 1b was prepared readily from amine 15a with
phenyl isocyanate, and compounds 2a–l were prepared straight-
forwardly from phenyl isothiocyanate 16 with corresponding
amines (Scheme 2).
Table 3
SAR on the phenyl ring
NH2
N
S
R
N
N
H
H
N
Cl
We began the SAR study on compound 1a with a preliminary
exploration of the minimal structural features that are required
for MK-2 activity. A simple replacement of the central thiourea lin-
ker with an urea linker resulted in compound 1b (Fig. 1), which is
Compound
R
MK-2 IC50 (lM)
5a
5b
5c
5d
5e
5f
4-Me
4-Cl
4-MeO
4-i-Pr
4-t-Bu
4-Br
2.7
3.7
1.9
2.6
13.7
3.1
inactive at 40 lM concentration in the MK-2 enzyme assay. We
5g
5h
5i
5j
5k
5l
4-NO2
4-CN
4-Tetrazole
4-BnO
4-Ac
4-EtOC(@O)–
4-NMe2
4-(Morphorin-1-yl)
4-AcNH–
4-NH2
4-BnOC(@O)NH–
2-Me
2-Cl
2-MeO
2-F
2-MeS
2-Ph
3-Me
3-Cl
3-MeO
9.9
6.9
>40
2.7
6.9
10.7
7.0
17.3
5.2
2.0
0.46
3.6
1.1
2.6
2.0
2.5
>20
9.3
11.3
14.5
2.5
Table 1
Preliminary SAR on phenyl moiety
S
NH2
N
R
N
N
5m
5n
5o
5p
5q
6a
6b
6c
6d
6e
6f
7a
7b
7c
8a
8b
8c
8d
8e
H
H
N
Cl
Compound
R
MK-2 IC50 (lM)
1a
3a
3b
3c
3d
3e
3f
3g
3h
3i
Ph
Et
i-Pr
t-Bu
c-Pr
2.0
20
13
2.5
12
53
35
36
>50
22
>50
91
c-Pentyl
c-Heptanyl
Bn
PhCH2CH2
MeOCH2CH2
Et2NCH2CH2CH2
Benzoyl
1-Naphthyl
2,4-Di-MeO
2,4-Di-Cl
3,4-Di-MeO
3,4-Di-Cl
3,5-Di-Cl
4.3
ꢀ40
ꢀ40
27
3j
3k
3l
3.2

3240
S. Lin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3238–3242
Table 4
Table 6
SAR on C4 position of phenyl ring
Selected SAR on both phenyl group and aminopyrazine
4
3
S
NH2
N
R1
R1
S
NH2
N
N
H
N
H
N
N
N
H
H
N
R2
R2
Compound
R1
R2
Et
MK-2 IC50
(l
M)
THP-1 IC50
4.0
(lM)
Compound
R1
R2
MK-2 IC50 (lM)
O
O
O
5q
9a
9b
9c
9d
9e
9f
9g
BnOC(@O)NH–
MeOC(@O)NH–
EtOC(@O)NH–
n-BuOC(@O)NH–
i-BuOC(@O)NH–
t-BuOC(@O)NH–
i-BuOC(@O)N(Ph)–
MeNHC(@O)NH–
t-BuNHC(@O)NH–
t-BuOC(@O)NH–
PhOC(@O)NH–
EtOC(@O)N(Me)–
t-BuOC(@O)O–
EtC(@O)NH–
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
0.46
0.94
0.31
0.23
0.15
0.19
1.5
C4
C4
C4
12a
0.14
O
O
O
N
H
12b
12c
n-Pr
c-Pr
0.045
0.103
3.0
3.2
N
H
1.7
9h
Cl
0.70
0.21
0.73
10.0
2.9
13.3
2.3
1.5
N
H
10a
10b
10c
10d
10e
10f
10g
10h
10i
Me
Me
Me
Me
Me
Me
Me
Me
Me
O
O
12d
12e
12f
12g
C3
C3
C3
n-Pr
n-Bu
c-Pr
c-Pr
0.020
0.030
0.015
0.083
0.75
3.8
HN
n-PrC(@O)NH–
n-BuC(@O)NH–
BnC(@O)NH–
C4
O
O
3.8
0.58
PhC(@O)NH–
HN
C4
O
O
group on aminopyrazine (2l) led to inactive compounds as well
(IC50 > 40 M). In contrast, the left-hand phenyl group is more
l
1.7
HN
amenable to the modifications as can be seen in Table 1. Replace-
ments with sizable alkyl groups (3a–c), cycloalkyl groups (3d–f),
methoxyethanyl group (3i), benzyl group (3g), or a naphthyl group
(3l) retain a certain level of activity, albeit most of them are weaker
than that of the original phenyl group. The key structural features
of 1a, that is, the aminopyrazine subunit, the phenyl group, and
the thiourea linker, were therefore kept in our further SAR
explorations.
C4
O
C4
1.4
N
N
H
H
We then focused the SAR effort on the right hand side of com-
pound 1a, with emphasis on the C5 and C6 positions of the amino-
pyrazine subunit. The results are summarized in Table 2. C6
position was found not tolerant to bulky groups, as substitutions
with alkoxyl, amino, or mercaptan groups all led to inactive com-
pounds (4h–j). Deletion of chlorine substitution at C5 position
(4a), or replacing it with a phenyl group (4g), resulted in ca. 15–
25-fold weaker activity. While replacement of the C5 chlorine with
a small alkyl group such as a methyl, an ethyl or a n-propyl group
retains a similar level of potency (4b–d), replacement with a tri-
fluoromethyl group completely loses the activity (4e). Introduction
of a cyclopropyl group at C5 position, however, significantly im-
proves the potency, which led to the identification of the first com-
pound in this series that possesses sub-micromolar activity (4f,
IC50 = 0.46
lM).
The SAR exploration on the left side phenyl subunit of compound
1a utilizing the initial 5-Cl right-hand side was carried out simulta-
neously. A number of compounds with mono- or di-substitution on
the phenyl ring were synthesized, and wide-ranged substituents
such as halides, alkyl, aryl, alkoxy, amino, cyano, nitro, acyl, carbox-
ylic, tetrazole, and amido groups were investigated. As shown in Ta-
ble 3, mono-substitution on the 4-position (5a, 5b, and 5c, 4-Me, Cl,
and MeO, respectively) or the 2-position (6a, 6b, and 6c, 2-Me, Cl,
and MeO, respectively) of the phenyl group is clearly preferred over
3-position (7a, 7b, and 7c, 3-Me, Cl, and MeO, respectively), with 3–
10-fold differences in potency. Similarly, 2,4-disubstitution (8a and
8b, 2,4-di-MeO and Cl, respectively) exhibits 6–15-fold better activ-
ity compared with 3,4-disubstitution (8c and 8d, 3,4-di-MeO and
Cl, respectively) or 3,5-disubstitution (8e, 3,5-di-Cl). Overall, how-
ever, most of these modifications resulted in similar or weaker
activities compared with that of 1a, which has an unsubstituted
phenyl group. The highlight of this SAR study, however, was discov-
ered when a benzyl carbamate group was incorporated at the C4
position, which resulted in over 4-fold improvement in potency
Table 5
SAR on the bi-cyclic left-hand moiety
R1
R2
S
NH2
N
N
N
H
H
N
Me
Compound
R1–R2
MK-2 IC50 (lM)
11a
11b
11c
11d
11e
11f
3-OC(@O)NH-4
3-NHC(@O)NH-4
3-CH2C(@O)NH-4
3-SC(@O)NH-4
3-NHC(@O)O-4
3-NHC(@O)N(Me)-4
0.21
15
27
15
14
>20
(5q, IC50 = 0.47 lM).

S. Lin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3238–3242
3241
synthesized as showed in Table 6. As expected, many of these com-
pounds show further improved activity, with value of IC50 in the
MK-2 enzyme assay as low as 15 nM (12f). In the THP-1 cellular as-
say, these compounds were all active and exhibited up to 10-fold
NH2
N
O
NH2
N
1) POCl3/DMF
85 oC
NC
LiAlH4
H2N
N
R2
N
2) HCl (2N), reflux
34-52%
THF, rt
44-77%
R2
improved potency (12d, IC50 = 0.78
lM) compared to compound
R1
R1
1a (IC50 = 7.9
l
M) in reducing LPS-stimulated TNF production
a
13
14
from THP-1 cells. As is clearly evident from literature,7–12 there
are few known potent MK-2 inhibitors shown to possess submi-
cromolar cellular potency. These thioureas, therefore, represent a
novel class of highly potent MK-2 inhibitors.
NH2
S
NH2
N
R3-NCS, or
R3-NH2, TCI
R3
H2N
N
N
H
N
H
Compounds 12b, 12d, and 12f were selected for further study in
vivo. In an acute mouse inflammation model (mouse LPS model),16
all three compounds were found to be highly active (10 mg/kg, iv),
N
N
R2
R2
DIEA, CH2Cl2, rt
15-99%
R1
R1
with 62%, 70%, and 75% inhibition of TNFa production, respectively
15
for 12b, 12d, and 12f, one and half hours after LPS challenge.17
In summary, we have discovered a novel series of MK-2 inhibi-
tors based on 1-(2-aminopyrazin-3-yl)methyl-2-thiourea. Exten-
sive SAR studies were carried out on both the left-hand phenyl
moiety and the right hand aminopyrazine moiety of initial HTS
lead compound 1a, which resulted in the identification of potent
inhibitors with IC50 values as low as 15 nM. These compounds sup-
1a, 3 - 12
Scheme 1. Synthesis of compounds 1a, 3–12. See Table 2 for R1 and R2.
NH2
N
O
NH2
N
Ph-NCO
press the expression of TNFa in THP-1 cells and in vivo in an acute
H2N
N
N
inflammation model in mice.18
H
H
N
DIEA, CH2Cl2, rt
98%
N
Cl
Cl
References and notes
15a
1b
1. Camussi, G.; Lupia, E. Drugs 1998, 55, 613.
2. (a) Richard-Miceli, C.; Dougados, M. Bio. Drugs 2001, 15, 251; (b) Braun, J.;
Sieper, J. Bio. Drugs 2003, 17, 187; (c) Olson, N. J.; Stein, C. M. N. Eng. J. Med.
2004, 350, 2167; (d) Mousa, S. A.; Goncharuk, O.; Miller, D. Exp. Opin. Biol. Ther.
2007, 7, 617.
3. (a) Chen, Z.; Gibson, T. B.; Robinson, F.; Slicvestro, L.; Pearson, G.; Xu, B.;
Wright, A.; Vanderbilt, C.; Cobb, M. Chem. Rev. 2001, 101, 2449; (b) Pargellis, C.;
Regan, J. Curr. Opin. Invest. Drugs 2003, 4, 566.
S
NCS
H2N
R
N
N
R
DIEA, CH2Cl2, rt
4-96%
H
H
16
2a-l
4. (a) Haddad, J. Curr. Opin. Invest. Drugs 2001, 2, 1070; (b) Lee, M. R.; Dominguez,
C. Curr. Med. Chem. 2005, 12, 2979; (c) Ding, C. Curr. Opin. Invest. Drugs 2006, 7,
1020. and references cited therein.
Scheme 2. Synthesis of compound 1b and 2. See Figure 2 for R.
5. (a) Kotlyarov, A.; Neininger, A.; Schubert, C.; Eckert, R.; Birchmeier, C.; Volk, H.
Nat. Cell Biol. 1999, 1, 94; (b) Neininger, A.; Kontoyiannis, D.; Kotlyarov, A.;
Winzen, R.; Eckert, R.; Volk, H. J. Biol. Chem. 2002, 277, 3065.
6. Hegen, M.; Gaestel, M.; Nickerson-Nutter, C. L.; Lin, L.-L.; Telliez, J.-B. J.
Immunol. 2006, 177, 1913.
7. Anderson, D. R.; Hegde, S.; Reinhard, E.; Gomez, L.; Vernier, W. F.; Lee, L.; Liu, S.;
Sambandam, A.; Snider, P. A.; Masih, L. Bioorg. Med. Chem. Lett. 2005, 15, 1587.
8. Anderson, D. R.; Meyers, M. J.; Vernier, W. F.; Mahoney, M. W.; Kurumbail, R.
G.; Caspers, N.; Poda, G. I.; Schindler, J. F.; Reitz, D. B.; Mourey, R. J. J. Med. Chem.
2007, 50, 2647.
9. (a) Trujillo, J. I.; Meyers, M. J.; Anderson, D. R.; Hegde, S.; Mahoney, M. W.;
Vernier, W. F.; Buchler, I. P.; Wu, K. K.; Yang, S.; Hartmann, S. J.; Reitz, D. B.
Bioorg. Med. Chem. Lett. 2007, 17, 4657; (b) Wu, J.-P.; Wang, J.; Abeywardane,
A.; Andersen, D.; Emmanuel, M.; Gautschi, E.; Goldberg, D. R.; Kashem, M. A.;
Lukas, S.; Mao, W.; Martin, L.; Morwick, T.; Moss, N.; Pargellis, C.; Patel, U. R.;
Patnaude, L.; Peet, G. W.; Skow, D.; Snow, R. J.; Ward, Y.; Werneburg, B.; White,
A. Bioorg. Med. Chem. Lett. 2007, 17, 4664.
10. (a) Goldberg, D. R.; Choi, Y.; Cogan, D.; Corson, M.; DeLeon, R.; Gao, A.;
Gruenbaum, L.; Hao, M. H.; Joseph, D.; Kashem, M. A.; Miller, C.; Moss, N.;
Netherton, M. R.; Pargellis, C. P.; Pelletier, J.; Sellati, R.; Skow, D.; Torcellini, C.;
Tseng, Y.-C.; Wang, J.; Wasti, R.; Werneburg, B.; Wu, J. P.; Xiong, Z. Bioorg. Med.
Chem. Lett. 2008, 18, 938; (b) Xiong, Z.; Gao, D. A.; Cogan, D. A.; Goldberg, D. R.;
Hao, M.-H.; Moss, N.; Pack, E.; Pargellis, C.; Skow, D.; Trieselmann, T.;
Werneburg, B.; White, A. Bioorg. Med. Chem. Lett. 2008, 18, 1994.
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Scheufler, C.; Velcicky, J.; Waelchli, R.; Huppertz, C. Bioorg. Med. Chem. Lett.
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To follow up with the 4-carbamate SAR of compound 5q, a series
of 4-amino derivatives was synthesized and evaluated for their
inhibitory activity against MK-2, utilizing both 5-Cl and 5-Me
right-hand side (Table 4). Alkyl and aryl carbamates were first ex-
plored, and all compounds prepared in this series exhibit improved
potency over compound 1a (9a–e and 10a–b, IC50 = 0.15–0.94
tert-Butyl (9e and 10a, IC50 = 0.19 and 0.21 M, respectively) and
iso-butyl carbamates (9d, IC50 = 0.15 M) were identified to be
lM).
l
l
the most potent moieties. Replacement of the carbamate moiety
with a urea moiety was found well-tolerated with only slight com-
promise in activity (9g and 9h, IC50 = 1.7 and 0.70 lM, respectively).
However, the free NH is found to be necessary to keep the high
potency for the carbamate series, as substituting it with a phenyl
group (9f) or a methyl group (10c), or replacing it with an oxygen
(10d) resulted in significant loss of potency. Replacing the carba-
mate with an amide moiety was also investigated. As observed ear-
lier with compound 5o (Table 3), the aliphatic amides do not have
much influence on activity (10e–h, Table 5). Aromatic amides, that
is, benzamide (10i, IC50 = 0.58
lM) and 2-furanyl amide (10j,
IC50 = 0.61 M), however, do improve the activity markedly com-
l
12. a Anderson, D. R.; Meyers, M. J.; Kurumbail, R. G.; Caspers, N.; Poda, G. I.; Long,
S. A.; Pierce, B. S.; Mahoney, M. W.; Mourey, R. J. Bioorg. Med. Chem. Lett. 2009,
doi: 10.1016/j.bmcl.2009.02.15.; b Anderson, D. R.; Meyers, M. J.; Kurumbail, R.
G.; Caspers, N.; Poda, G. I.; Long, S. A.; Pierce, B. S.; Mahoney, M. W.; Mourey, R.
J.; Parikh, M. D. Bioorg. Med. Chem. Lett. 2009, doi: 10.1016/j.bmcl.2009.02.17.
pared to compounds 4b and 1a.
As a further extension of the SAR on the 4-amino series, we also
synthesized several compounds where 3- and 4-substituents are
joined to form a ring (Table 5). Very intriguingly, none of these
compounds show good potency against MK-2 in the in vitro en-
zyme assay, except cyclic carbamate compound 11a. Compound
13. Protocol of the MK-2 enzyme assay: The assay is started by mixing 12.5
0.5 nM MK-2 with 12.5 of biotinylated HSP27 72-90 (biotin-LC
AYSRALSRQLSSGVSEIRH) and 1.0 M ATP/30
Ci/mL 33P-ATP with or without
L of compound titration in kinase assay buffer (KB), which includes 50 mM
lL of
lL
4
l
lM
-
l
11a exhibits an IC50 of 0.21
lM, which is over 70-fold better than
1 l
Hepes (pH 7.5), 10 mM MgCl, 1 mM DTT, and 1 mg/mL BSA. The reaction was
incubated at room temperature for 30 min. The reaction was quenched by
addition of 125 lL of quench solution (PBS, pH 7.5, 50 mM EDTA, and 0.1%
Triton-100) containing 800 pmol/mL SPA bead to the above reaction mixture.
The plate was spun at 2000 rpm for 5 min, and counted at top counter (Packard).
that of other closely related compounds.
Having identified potent moieties on both the left-hand side
and the right-hand side of compound 1a, we then incorporated
these moieties onto the same molecules. Selected compounds were

3242
S. Lin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3238–3242
14. For protocol of the THP-1 assay, see: Natarajan, R. S.; Wisnoski, D. D.; Singh, S.
B.; Stelmach, J. E.; O’Neill, E. A.; Schwartz, C. D.; Thompson, C. M.; Fitzgerald, C.
E.; O’Keefe, S. J.; Kumar, S.; Hop, C. E. C. A.; Zaller, D. M.; Schmatz, D. M.;
Doherty, J. B. Bioorg. Med. Chem. Lett. 2003, 13, 273.
LCK). The pharmacokinetic properties of these two compounds were evaluated
in rat and the results are shown below in Table 7.
15. Shepard, K. L.; Mason, J. W.; Woltersdorf, O. W.; Jones, J. H.; Cragoe, E. J. J. Med.
Chem. 1969, 12, 280. and references cited therein.
16. Mouse LPS challenge assay: 12-week-old female Balb/c mice were dosed with a
MK-2 inhibitor (iv, 10 mg/kg in DMSO/Cremophor/saline) or vehicle, just prior
Table 7
Pharmacokinetic properties of 12b and 12d in rat
Compound
Cl (mL/min/kg)
T1/2 (h)
AUCN (
lM h kg/mg)
F (%)
to injection of 10
800 mg/kg -galactosamine (sigma) in saline. Animals were euthanized 90 min
later, and plasma TNF was measured by ELISA. See also: O’Keefe, S. J.;
lg/mouse LPS (Escherichia coli Sero-type 0111:b4, sigma) and
D
12b
12d
2.5
12.9
0.8
0.3
3.3
0.3
19
8
a
Mudgett, J. S.; Cupo, S.; Parsons, J. N.; Chartrain, N. A.; Fitzgerald, C.; Chen, S.-L.;
Lowitz, K.; Rasa, C.; Visco, D.; Luell, S.; Carballo-Jane, E.; Owens, K.; Zaller, D. M.
J. Biol. Chem. 2007, 282, 34663.
17. To further support that the activity of these compounds was primarily due to
MK-2 inhibition, the kinase selectivity of compounds 12b and 12d was
investigated by screening against a panel of 33 kinases, including related
MAPKAP kinases, p38 MAP kinases, CDK2, and other kinases associated with
18. Modeling effort is being undertaken to further understand how these novel
inhibitors interact with MK-2 in light of the recently available information on
the binding site of MK-2 (see Refs. 7, 9a, 10a, and 12). The results of this study,
together with the results of our continuous SAR effort to develop novel
aminopyrazine-based non-thiourea MK-2 inhibitors, will be communicated
separately in the near future.
TNF
inhibition at 1
>70% inhibition at 10
a
production. Of these kinases targeted for screening, only two had >70%
M for compound 12d (MK-5 and AMPK) and only four had
M for compound 12b (RPS6KA1, RPS6KA5, AMPK, and
l
l