Synthesis and biological evaluation of p38α kinase-targeting dialkynylimidazoles

Article abstract of DOI:10.1016/j.bmcl.2009.09.094

Based on the mild, thermal rearrangement of 1,2-dialkynylimidazoles to reactive carbene or diradical intermediates, a series of 1,2-dialkynylimidazoles were designed as potential irreversible p38 MAP kinase α-isoform (p38α) inhibitors. The synthesis of th

Full text of DOI:10.1016/j.bmcl.2009.09.094

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6293–6297
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of p38
dialkynylimidazoles
a
kinase-targeting
*
Jing Li, Tamer S. Kaoud, Christophe Laroche, Kevin N. Dalby, Sean M. Kerwin
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 July 2009
Revised 22 September 2009
Accepted 24 September 2009
Available online 27 September 2009
Based on the mild, thermal rearrangement of 1,2-dialkynylimidazoles to reactive carbene or diradical
intermediates, a series of 1,2-dialkynylimidazoles were designed as potential irreversible p38 MAP kinase
a
-isoform (p38
ity is reported. The 1-ethynyl-substituted dialkynylimidazole 14 is a potent (IC50 = 200 nM) and selective
inhibitor of p38 . Moreover, compound 14 covalently modifies p38 as determined by ESI-MS after 12 h
incubation at 37 °C. The unique kinase inhibition, covalent kinase adduct formation, and minimal CYP450
a) inhibitors. The synthesis of these dialkynylimidazoles and their kinase inhibition activ-
a
a
Keywords:
Diradical
Carbene
2D6 inhibition by compound 14 demonstrate that dialkynylimidazoles are a new, promising class of p38
a
inhibitors.
Ó 2009 Elsevier Ltd. All rights reserved.
MAPK
Inflammation
p38 MAP kinase (p38
kinases that serve as important mediators of inflammatory cyto-
a
) belongs to a family of serine/theronine
(ImPy) products, which may result from trapping of an initially-
formed diradical intermediate via aza-Bergman cyclization.13
Thermolysis under neutral conditions in non-halogenated sol-
vents affords products derived from trapping cyclopentapyrazine
(CyPP) carbene intermediates by H-atom abstraction, C–H bond
insertion, and alkene addition reactions.14–16 The CyPP carbene is
proposed to be derived from an intermediate cyclic cumulene that
results from collapse of the diradical.14 Non-covalent association
between DAIms and a kinase may facilitate the rate-determining
aza-Bergman cyclization.
kines including tumour necrosis factor alpha (TNFa) and interleu-
kin-1 beta (IL-1b).1,2 Elevated levels of the pro-inflammatory
cytokines are associated with a number of diseases, such as toxic
shock syndrome, rheumatoid arthritis, osteoarthritis, diabetes,
and inflammatory bowel disease.3 Therefore, inhibition of p38
a
is considered to be a potential therapeutic strategy.4 A number of
p38
inhibitors have been synthesized and characterized.5
Although these compounds show good inhibition of p38 , many
a
a
also inhibit other protein kinases with similar or greater potency.6
Therehasbeena growinginterestinirreversible inhibitorsof pro-
tein kinases,7 and a number of these drugs are in clinical trials.8
Advantages of irreversible kinase inhibition include increased selec-
tivity,9 duration,10 and therapeutic utility, especially against kinases
that are resistant to competitive, ATP-binding pocket-targeting
drugs.11 Additionally, irreversible inhibitors and related selective,
covalent kinase modifying small molecules are of interest as probes
for chemical genetics studies.12 While certain natural products and
ATP analogs irreversibly inhibit kinases,13 none are selective toward
The formation of reactive diradical and carbene intermediates
under mild conditions from DAIms has led us to propose that DAIms
can be designed to undergo kinase binding-induced cyclization and
covalent inactivation of specific kinase targets. Specifically, the
structural similarity between DAIms and the known p38
such as SB-20358017 and RWJ-6765718 (Fig. 1) has inspired the de-
sign and inhibition studies of p38 -targeting DAIms described here.
ainhibitors
a
An initial route to kinase-targeting dialkynylimidazoles is
shown in Scheme 2. The known 4(5)-(4-fluorophenyl)-5(4)-(4-pyr-
idyl)imidazole 119 was protected with trityl group. Interestingly,
this reaction only afforded one regioisomer, which was assigned
as the 5-(4-fluorophenyl)-4-(4-pyridyl)imidazole 2 based on COSY
and NOESY NMR. Compound 2 was deprotonated with n-BuLi at
0 °C, and quenched with I2 to give the 2-iodo-imidazole 3, which
was deprotected in aqueous TFA to afford 4. Coupling of the lith-
ium anion of imidazole 4, formed by deprotonation with LHMDS,
with phenyl(phenylethynyl)-iodonium tosylate20 afforded a 15%
yield of a 1:1 mixture of the regioisomeric N-alkynyl-2-iodoimi-
dazoles 5 and 6. The separated regioisomers were subjected to
p38
a. Thus, there is a need to develop selective and irreversible
inhibitors that target p38
a
. We have discovered a novel thermal
cyclization and rearrangement of 1,2-dialkynylimidazoles (DAIms)
(Scheme 1). Mild thermolysis of DAIms in the presence of chlori-
natedsolventsorHClleads totheisolationof imidazo[1,2-a]pyridine
* Corresponding author.
E-mail address: skerwin@mail.utexas.edu (S.M. Kerwin).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.094

6294
J. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6293–6297
H
H
N
N
R2
H-atom donor
R1
R
H
N
N
N
N
N
N
N
N
R2
N
N
R2
R2
RH
R
R2
R2
R1
R
R1
R1
R1
R
R
R1
DAIm
CyPP carbene
N
HCl
R2
N
N
R1
H
N
R2
R1
ImPy
Cl
Scheme 1. Thermal cyclization and rearrangement of 1,2-dialkynylimidazoles.
ers 8a–c, in contrast to reported 1-substituted pyridylimidazole
p38
inhibitors.28 Interestingly, the 1-ethynyl-substituted analog
14 is a potent inhibitor of p38
F
F
a
N
N
O
N
a
. Compound 14 completely inhibits
OH
N
H
N
N
N
N
N
H
N
Ph
RWJ-67657
Figure 1. Examples of 4,5-diarylimidazole p38 inhibitors.
N
N
b
a
I
N
SB-203580
N
N
CPh3
CPh3
a
F
F
F
2
1
3
I
N
N
N
Sonogashra coupling with various terminal acetylene partners to
provide the regioisomeric dialkynylimidazoles 7 and 8. The regio-
chemical assignments within this series were made based on the
X-ray crystal structure of 7b shown in Figure 2.21
F
N
5
Although providing access to select kinase-targeting dialkyny-
limidazoles, the synthetic route shown in Scheme 2 suffers from a
number of limitations associated with the alkynyliodonium cou-
pling reaction. Only the phenylethynyl and TMS-ethynyl iodonium
reagents could be employed in this coupling,22 and even in these
cases, theyields are poor and mixturesof regioisomers are produced.
An improved synthetic route to these dialkynylimidazoles
employing the recently reported copper-catalyzed N-alkynylation
of imidazoles with bromoalkynes was devised (Scheme 3). Treating
4-fluorophenylimidazole 923 with TIPS-protected bromo-acetylene
in the presence of catalytic CuI and 2-acetyl-cyclohexanone as li-
gand affords a 9:1 mixture of regioisomeric alkynylimidazoles
10b and 10a, respectively, in 79% yield.22 Iodination of the 2-posi-
tion of 10b affords the 2-iodoimidazole 11, which undergoes Sono-
gashira coupling with O-TIPS-protected homo-propargyl alcohol to
give the dialkynylimidazole 12 in 73% yield.24 Deprotonation of 12
with n-BuLi followed by iodine quench affords the 5-iodoimidazole
13 in 74% yield. A final Suzuki–Miyaura coupling of the 5-iodo
imidazole 13 with pyridine-4-boronic acid followed by TBAF
deprotection gives the dialkynylimidazole 14.25 Mild thermolysis
of 14 at 80 °C under acidic conditions in the presence of chloride
afforded 15, the product of HCl addition to the diradical, in 50%
yield.
Ph
N
c
d
+
I
N
H
Ph
N
F
4
N
N
I
F
6
N
N
e
7a: R = H (9 %)
7b: R = CH2OH (77 %)
7c: R = CH2CH2OH (40 %)
R
5
N
F
Ph
Ph
N
8a: R = H (16 %)
8b: R = CH2OH (48 %)
8c: R = CH2CH2OH (62 %)
N
N
e
6
R
All of these 1,2-dialkynylimidazoles were assayed against p38
MAPK at a fixed time-point of 60 min (Table 1).26 Compounds 7a–c
and 8a–c display modest inhibition at 10 M concentration. In this
series there is little difference in activity between the 1-alkynyl-5-
fluorophenyl regioisomers 7a–c and the 1-alkynyl-5-pyridylisom-
a
F
6b
l
Scheme 2. Reagents and conditions: (a) Et3N, Ph3CCl, CH2Cl2 (58%); (b) (i) n-BuLi;
(ii) I2, THF, 0 °C (60%); (c) TFA, H2O (83%); (d) LHMDS, PhI+CCPhTsOꢀ; (e) RCCH,
Pd(PPh3)4, CuI, Et3N.

J. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6293–6297
6295
Table 1
In vitro activity of 1,2-dialkynylimidazoles against p38
a
Compound
p38a % inhibition (@ 10 l
M)a
7a
8a
7b
8b
7c
8c
14
19
28
63
83
53
75
100
a
Tests were carried out in duplicate.
Dialkynylimidazole 14 (100
lM) was incubated with non-phos-
phoryated p38 (5 M) at 37 °C in 50 mM HEPES, 10 mM MgCl2,
a
l
2 mM DTT, 1 mM EGTA, pH 7.5 for 12 h, followed by extensive dial-
ysis, and the sample was analyzed by ESI-MS. A new peak in the
mass spectrum at m/z = 41,896, which corresponds to addition of
a single molecule of 14 (MW = 331) to p38a, was observed
Figure 2. X-ray crystal structure of dialkynylimidazole 7b.
(ꢁ25% adduct) (Fig. 3). Under identical conditions but with 1 mM
DTT present, the adduct was the predominant species observed
(Supplementary data).
A common concern for pyridinylimidazole MAPK inhibitors
such as RWJ 67657 and SB-203580 is their inhibition of cyto-
chrome P450 (CYP450) enzymes, which may be linked to hepato-
toxicity.29 Interestingly, the dialkynylimidazole 14 displays a
much lower level of inhibition of CYP450 2D6 (4% inhibition at
p38
comparison, the IC50 of 14 against p38b (5.4
Dialkynylimidazole 14 was also assayed at concentration of 20
against a panel of 53 additional human kinases. Only one kinase,
(MAPK4/HGK) was strongly inhibited (>90% inhibition at 20 M,
IC50 = 4.2 M), while six additional kinases were moderately inhib-
ited (between 50% and 90% inhibition, see Supplementary data).
The cyclized 15 also inhibited p38 (IC50 = 370 nM).
a
at 10
lM (Table 1), and has an IC50 for p38
a
of 200 nM.27 In
lM) is >25-fold higher.
l
M
l
10
l
M) compared to SB-203580 (78% inhibition at 10
lM).
l
In summary, novel p38 -targeting dialkynylimidazoles were de-
a
signed, synthesized and evaluated. Although 1-phenethynyl-substi-
a
N
H
N
F
F
F
TIPS
N
a
10a (minor)
+
N
H
F
9
N
N
N
N
b
I
H
TIPS
TIPS
10b (major)
11
F
F
N
N
OTIPS
d
N
N
OTIPS
c
I
TIPS
TIPS
12
13
F
F
N
N
OH
e,f
N
N
g
OH
N
N
Cl
H
14
15
Scheme 3. Reagents and conditions: (a) BrCCTIPS, CuI, AcC, Cs2CO3, dioxane, 50 °C overnight followed by reflux for 4 h (79%, 1:9 10a/10b); (b) (i) n-BuLi; (ii) I2, THF, ꢀ78 °C
(91%); (c) TIPSOCH2CH2CCH, Pd(PPh3)4, CuI, Et3N (73%); (d) (i) n-BuLi; (ii) I2, THF, ꢀ78 °C (74%); (e) pyridine 4-boronic acid, Pd(PPh3)4, K2CO3 (41%); (f) TBAF, THF, ꢀ78 °C
(89%); (g) Me4NCl, TfOH, DMF, 80 °C, 5 days (50%).

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J. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6293–6297
Figure 3. (a) ESI-MS spectrum of unphosphorylated p38
for 12 h at 37 °C, followed by extensive dialysis.
a
incubated for 12 h at 37 °C; (b) ESI-MS spectrum of unphosphorylated p38a incubated with dialkynylimidazole 14
tuted dialkynylimidazoles 7a–c and 8a–c are only modest inhibitors
of p38 , the 1-ethynyl-substituted dialkynylimidazole 14 is a potent
zation/trapping of 14 to afford 15 (5 days at 80 °C), indicating that
the kinase may facilitate the cyclization of 14. Further studies on
a
and selective inhibitor. Commensurate with the increased facility of
rearrangementof 1-ethynyl-substituteddialkynylimidazols relative
to 1-phenethynyl analogues,16 compound 14 forms a covalent
adduct with p38
mation (12 h at 37 °C) are much milder than those required for cycli-
the site and mechanism of this covalent modification of p38a by 1-
ethynyl-substituted dialkynylimidazoles are on-going. The unique
kinase inhibition, covalent kinase adduct formation, and minimal
CYP450 2D6 inhibition by compound 14 demonstrate that dialkyny-
a. a adduct for-
30 However, the conditions for p38
limidazoles are a new, promising class of p38a inhibitors.

J. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6293–6297
6297
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