Tri- and tetrasubstituted pyridinylimidazoles as covalent inhibitors of c-Jun N-terminal kinase 3

Article abstract of DOI:10.1021/acs.jmedchem.6b01180

The concept of covalent inhibition of c-Jun N-terminal kinase 3 (JNK3) was successfully transferred to our well validated pyridinylimidazole scaffold varying several structural features in order to deduce crucial structure-activity relationships. Joint ta

Full text of DOI:10.1021/acs.jmedchem.6b01180

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Article
Tri- and Tetra-substituted Pyridinylimidazoles as
Covalent Inhibitors of c-Jun N-Terminal Kinase 3
Felix Muth, Ahmed El-Gokha, Francesco Ansideri, Michael Eitel, Eva Doering, Adrian Sievers-
Engler, Andreas Lange, Frank M. Boeckler, Michael Lämmerhofer, Pierre Koch, and Stefan A. Laufer
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 15 Dec 2016
Downloaded from http://pubs.acs.org on December 16, 2016
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Tri- and Tetra-substituted Pyridinylimidazoles as Covalent Inhibitors of c-
Jun N-Terminal Kinase 3
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Felix Muth,a,‡ Ahmed ElꢀGokha,a,b,‡ Francesco Ansideri,a Michael Eitel,a Eva Döring,a Adrian Sieversꢀ
Engler,a Andreas Lange,a Frank M. Boeckler,a Michael Laemmerhofer,a Pierre Kocha,* and Stefan A.
Laufera,*
aDepartment of Pharmaceutical Chemistry, Institute of Pharmaceutical Sciences, Eberhard Karls Univerꢀ
sität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany.
bMenofia University, Chemistry Department, Faculty of Science, Menofia, Egypt.
Covalent kinase inhibitors, neurodegenerative disorders, cꢀJun Nꢀterminal kinase 3, JNK3, p38α mitoꢀ
genꢀactivated protein kinase, p38α MAP kinase
ABSTRACT: The concept of covalent inhibition of cꢀJun Nꢀterminal kinase 3 (JNK3) was successfully
transferred to our well validated pyridinylimidazole scaffolds varying several structural features in order
to deduce crucial structure–activity relationships. Joint targeting of the hydrophobic region I and methꢀ
ylation of imidazoleꢀN1 position increased the activity and reduced the number of offꢀtargets. The most
promising covalent inhibitor, the tetraꢀsubstituted imidazole 3ꢀacrylamidoꢀNꢀ(4ꢀ((4ꢀ(4ꢀ(4ꢀfluorophenyl)ꢀ
1ꢀmethylꢀ2ꢀ(methylthio)ꢀ1Hꢀimidazolꢀ5ꢀyl)pyridinꢀ2ꢀyl)amino)phenyl)benzamide (7), inhibits the JNK3
in the subnanomolar range (IC50 = 0.3 nM), shows high metabolic stability in human liver microsomes
and displays excellent selectivity in a screen against a panel of 410 kinases. Covalent bond formation to
Cysꢀ154 was confirmed by incubation of the inhibitors with wildꢀtype JNK3 and JNK3ꢀC154A mutant
followed by mass spectrometry.
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Introduction
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The cꢀJun Nꢀterminal kinase 3 (JNK3) belongs to the mitogenꢀactivated protein (MAP) kinase family,
which comprises ten members: p38 MAP kinases (p38α, β, γ, δ), JNKs (JNK1, 2, 3) and three extracelꢀ
lularꢀregulated kinases.1 The three genes jnk1, jnk2, jnk3 encode for a total of ten alternatively spliced
JNK isoforms with a molecular weight between 46 and 55 kDa.2, 3 Although structurally highly conꢀ
served, JNKs differ in tissue distribution fundamentally. JNK1 and JNK2 are ubiquitously expressed,
whereas JNK3 expression is primarily restricted to the heart, testis and brain.4
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Based on results from several knock out experiments5ꢀ7 and due to its specific tissue distribution, JNK3
has been identified as a promising target for potential treatment of neurodegenerative disorders such as
Huntington’s disease, Parkinson’s disease and Alzheimer’s disease.8
Within the last years, covalent inhibition of enzymes has experienced a resurgence.9 Carefully designed
irreversible inhibitors have been proven to be even more selective than their reversibly binding counterꢀ
parts.10, 11 With approval of the covalent inhibitor of the epidermal growth factor receptor afatinib in
2013,12 the design of irreversible inhibitors raised the attention of the protein kinase research communiꢀ
ty.
The recently highlighted high quality kinase probe JNKꢀINꢀ813 (1, Figure 1) was discovered by Zhang et
al.14 and provided the first highly selective covalent panꢀJNK inhibitor. The reported aminopyrimidineꢀ
based panꢀJNK inhibitors possess an electrophilic moiety and target a cysteine (Cysꢀ154, JNK3 numberꢀ
ing) conserved among all JNKs (in the hinge7 position) that is not present in other protein kinases.10
In the search for new covalent inhibitors, researchers mostly follow two different paths. Either, a nonꢀ
specific weak reversible binder is used as a lead structure or a noncovalent, relatively potent inhibitor
already optimized for the target kinase is chosen to be refined.14 In continuing efforts to enhance both
selectivity and activity of pyridinylimidazoleꢀbased JNK3 inhibitors, we applied the latter approach of
covalent targeting to our previously reported potent dual JNK3/p38α inhibitor 3 (Figure 1).15 This scafꢀ
fold was designed to align all moieties for optimal interaction with the active site of the enzymes. Comꢀ
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putational docking studies with inhibitor 3 as well as comparison of known crystal structures of similar
pyrimidinylimidazole inhibitors16 (PDBꢀcode: 1PMN and 4Z9L) bound to JNK3 suggest that the 4ꢀ
fluorophenyl ring of 3 is located in the hydrophobic region I of JNK3 and two hydrogen bond interacꢀ
tions are formed with Metꢀ149 positioned in the hinge region. Therefore, it provides a suitable starting
point to examine structure–activity relationships (SAR).
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Due to the fact that the hinge binding motifs present in 1 and in our 2ꢀaminopyridine scaffold are formꢀ
ing two hydrogen bonds to Metꢀ149 (Figures 1 and 2), derivatives of 3 bearing an appropriate side chain
at the pyridineꢀC2 position containing an electrophilic warhead should be able to target the unique cysꢀ
teine (Cysꢀ154).
Figure 1. PanꢀJNK inhibitors 1 and 2 and dual JNK3/p38α inhibitor 3; adata previously reported by Anꢀ
sideri and coworkers.15
This design hypothesis was supported by the comparing of the binding mode of JNKꢀINꢀ7 (2, Figure 1)
in the crystal structure with JNK3 (PDB code: 3V6S) and one of our designed inhibitors, compound 7,
computationally docked into the active site (Figure 2).
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Figure 2. Superimposition of 2 (grey) in crystal structure with JNK3 (PDB code: 3V6S, cyan) and 7
(salmon) covalently docked into JNK3 (PDB code: 1PMN, grey) using Schrödinger Glide.17 Polar conꢀ
tacts of 2 and JNK3 are depicted as orange dashes. The image was generated with PyMOL.18 Amino
acid sequence was partially hidden for clarity.
Herein, we report the synthesis of triꢀ and tetraꢀsubstituted pyridinylimidazoles (compounds 4ꢀ23) as
well as their evaluation as covalent JNK3 inhibitors (Figure 3). The effects of the 4ꢀfluorophenyl ring at
the imidazoleꢀC4 position as well as those of an imidazole Nꢀalkylation were evaluated. Moreover, the
influence of the substitution pattern of both phenyl rings A and B on the biological activity was investiꢀ
gated extensively. An acrylamideꢀtype electrophilic warhead was chosen for the design of the herein
presented compounds, since the α,βꢀunsaturated carbonyl group represents a soft electrophile, which
reacts preferentially with the soft thiol present in cysteine side chains.19 Furthermore, the presence of
this Michael acceptor in several launched covalent kinase inhibitors (afatinib, ibrutinib, osimertinib)20 as
well as in drug candidates (neratinib, dacomitinib)20, 21 allows the assessment of a general safety profile
for compounds bearing this moiety. However, in contrast to the N,Nꢀdimethylaminoꢀmethyl acrylamide
present in afatinib or neratinib as well as in compounds 1 and 2, the unsubstituted acrylamide was preꢀ
ferred as electrophilic warhead for our series of inhibitors. In fact, although the tertiary amine has provꢀ
en to improve properties as solubility and cellular halfꢀlife,22 it also significantly increases the reactivity
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of the acrylamide by catalyzing the Michael addition23 and therefore could be potentially responsible for
offꢀtarget side effects.
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Figure 3. Structural modifications of imidazole scaffold and depiction of the terminology used throughꢀ
out this article.
Results and Discussion
Chemistry. The synthesis of triꢀsubstituted imidazole derivatives 4 and 5 bearing a 4ꢀfluorophenyl moiꢀ
ety at the imidazoleꢀC4 position was accomplished in three steps starting from reported inhibitor 315
(Scheme 1). First, the primary amine was reacted with the respective nitrobenzoic acid using benzotriaꢀ
zolꢀ1ꢀylꢀoxytripyrrolidinophosphoniumꢀhexafluorophosphate (PyBOP) as a coupling reagent to yield
amides 24 and 25. Second, the nitro compounds 24 and 25 were reduced to the corresponding amines 26
and 27 using tin(II) chloride. Finally, the Michael acceptor system was introduced by reaction of the
anilines 26 and 27 with acryloyl chloride in the presence of DIPEA as a base. As a negative control for
evaluation of covalent binding to the enzyme, compound 6 (the saturated analog of 4) was synthesized
by reaction of 3 with 3ꢀpropionamidobenzoic acid and PyBOP as coupling reagent (Scheme S1, supꢀ
porting information).
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Scheme 1. Synthesis of tri-substituted pyridinylimidazoles 4 and 5 bearing a 4-fluorophenyl ring at
the imidazole-C4-position.a
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aReagents and conditions: (i) mꢀ or pꢀnitrobenzoic acid, PyBOP, DIPEA in dichloromethane, 5 h, rt;
(ii) SnCl2·2 H2O, EtOH, reflux, 6 h; (iii) acryloyl chloride, DIPEA, DMF, 0 °C to rt.
The tetraꢀsubstituted imidazoles 7-8 and 10-20 were prepared using a convergent synthetic strategy
starting from the corresponding nitrobenzoic acid and bromoaniline (Scheme 2). Compounds 28aꢀh
were synthesized in good yields by adding Oꢀ(benzotriazolꢀ1ꢀyl)ꢀN,N,N′,N′ꢀtetramethyluronium tetraꢀ
fluoroborate (TBTU) and the corresponding bromoaniline to a suspension of nitrobenzoic acid in diꢀ
chloromethane at mild conditions using N,Nꢀdiisopropylethylamine (DIPEA) as base. In order to reduce
the nitro group to a primary amine, compounds 28a-h were treated with tin(II) chloride in ethanol at
75 °C. The acrylamide warhead and its saturated counterpart were introduced using the corresponding
acid chlorides. Compounds 29a-h were suspended in dry 1,4ꢀdioxane and treated with the activated carꢀ
boxylic acid at mild conditions using DIPEA as base. In the last step of this synthetic route, a Buchꢀ
waldꢀHartwig cross coupling reaction was applied. Optimized conditions for the introduction of substiꢀ
tuted aryl moieties at the pyridineꢀC2 amino position have been previously described.24 The scaffold 4ꢀ
(4ꢀ(4ꢀfluorophenyl)ꢀ1ꢀmethylꢀ2ꢀ(methylthio)ꢀ1Hꢀimidazolꢀ5ꢀyl)pyridinꢀ2ꢀamine (31)25 and the side
chains 30a-m were coupled using BrettPhos Pd G1 precatalyst as the catalyst/ligand system and cesium
carbonate as base.19 Compound 9 was synthesized by performing the Buchwald−Hartwig aryl amination
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reaction with 28a and 31 followed by a reduction step (Scheme S2, supporting information).
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Scheme 2. Synthesis of final compounds 7-8 and 10-20 exemplified by synthesis of 7.a
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aReagents and conditions: (i) SnCl2 · 2 H2O, EtOH, 75 °C, 2.5 h; (ii) acryloyl chloride, DIPEA, 1,4ꢀ
dioxane, rt, 2 h; (iii) 31, Cs2CO3, BrettPhos precatalyst, 1,4ꢀdioxane(abs)/tertꢀbutanol 4:1, 125 °C, 5 h.
This synthetic strategy allows the introduction of the side chain using optimized BuchwaldꢀHartwig
cross coupling reaction conditions in the last step, thus providing a high degree of flexibility in terms of
varying the scaffold or the side chain.
As further modifications, compounds 21 and 22 bearing a small methyl moiety at the imidazoleꢀC4 poꢀ
sition were synthesized in three steps (Scheme 3) starting from N1ꢀ(4ꢀ(4ꢀmethylꢀ2ꢀ(methylthio)ꢀ1Hꢀ
imidazolꢀ5ꢀyl)pyridinꢀ2ꢀyl)benzeneꢀ1,4ꢀdiamine (32, for synthesis of imidazole 32, see Scheme S3,
supporting information). The buildup of the linker bearing the electrophilic warhead at the aniline funcꢀ
tion was conducted similarly to the strategy described for the triꢀsubstituted imidazoles bearing a 4ꢀ
fluorophenyl ring at the imidazole core. The conditions used to synthesize compound 23, the saturated
analog of 21, starting with precursor 32 are identical to those used for the synthesis of 6 (Scheme S4,
supporting information).
Scheme 3. Synthesis of tri-substituted pyridinylimidazoles 21 and 22 bearing a methyl moiety at
the imidazole-C4 position.a
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aReagents and conditions: (i) mꢀnitrobenzoic acid, SOCl2, pyridine, rt or pꢀnitrobenzoyl chloride, pyriꢀ
dine, rt; (ii) SnCl2 · 2 H2O, EtOH, reflux, 6 h; (iii) acryloyl chloride, DIPEA, DMF, ꢀ20 °C to rt, 3 h.
Biological evaluation. All synthesized inhibitors were evaluated in enzymeꢀlinked immunosorbent asꢀ
says (ELISA)26, 27 to determine the IC50ꢀvalues for JNK3 and p38α. Compounds 7 and 21 were examꢀ
ined for their metabolic stability and further screened in a panel against 410 kinases to determine their
selectivity within the kinome. Results are presented in Tables 1ꢀ4 as well as in Tables S1 and S4 (supꢀ
porting information).
Both examples of the triꢀsubstituted pyridinylimidazoles series bearing a 4ꢀfluorophenyl ring at the imꢀ
idazoleꢀC4 position (compounds 4 and 5) are very potent dual JNK3/p38α inhibitors displaying IC50ꢀ
values down to the picomolar range (Table 1). While no preference for either tested kinase was detectaꢀ
ble, compounds 4 and 5 deduced from compound 3 (Figure 1) revealed that the bulky side chain was not
only well tolerated by both enzymes, but also caused an increase in inhibitory activity that is distinct in
compound 4 having the Michael acceptor in metaꢀposition. However, within the series of 4ꢀ
fluorophenylꢀsubstituted imidazoles, the influence of the Michael acceptor system in terms of JNK3
inhibition is rather low, since the saturated analog of 4, compound 6, shows no significant decrease in
terms of JNK3 affinity.
Table 1. Biological activity of tri-substituted imidazoles 4-6.
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JNK3a
IC50 [nM]c
p38αb
Qd
Cmp
R
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SEM
0.04
IC50 [nM]c
0.8
SEM
0.11
meta
para
meta
0.6
3
~1
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6
0.07
0.07
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0.69
0.27
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aincubation time, 50 min; incubation time 60 min; IC50ꢀvalues are the mean of three experiments;
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dratio: IC50 (p38α)/IC50 (JNK3).
We could not observe any beneficial effect of introducing electrophilic warheads in terms of selectivity
regarding the triꢀsubstituted imidazoles bearing a 4ꢀfluorophenyl moiety. Therefore, as a parallel conꢀ
cept, we chose to alkylate the imidazoleꢀN1 position with the utmost simple substituent as prior investiꢀ
gations of our group correlate the Nꢀalkylation with a reduced p38α inhibition.28
It is crucial for the linker between the scaffold and the warhead to comprise the optimal length and angle
in order to orient the warhead ideally for the nucleophilic attack of the thiol to occur and simultaneously
retain the original binding mode of the scaffold. Therefore, we synthesized a broad variety of linkers,
altering the meta and para substitution pattern consequently. Furthermore, we introduced methyl groups
at different positions of the A and B ring to slightly increase the torsion angle.
In order to elucidate the contribution of each moiety on ring B of 7 and 8, we synthesized compound 9
(IC50: JNK3: 30 nM; p38α: 149 nM). The para/meta substitution pattern (whereupon para refers to ring
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A and meta to ring B) with the unsubstituted aniline, presenting a fivefold prevalence for JNK3 over
p38α, was rather encouraging since the covalent tag was yet to be introduced. The para/meta substituted
compound 7 (IC50: JNK3: 0.3 nM; p38α: 36 nM), bearing an acrylamide warhead, features a picomolar
inhibitory effect for JNK3 and 120ꢀfold preference compared to the IC50ꢀvalue of p38α, emerging as the
most potent inhibitor synthesized in this series. Surprisingly, we observed a lower IC50ꢀvalue for p38α
alongside the JNK3 inhibition compared to compound 9. The initial assumption, that the carbonyl oxyꢀ
gen of the acrylamide forms an additional hydrogen bond to p38α and thereby increases its inhibitory
effect was invalidated by compound 8. It shows a fourfold decreased inhibition for p38α compared to its
unsaturated counterpart, compound 7. More interestingly, JNK3 inhibition of compound 8 is significantꢀ
ly decreased compared to compound 7. This can be considered as a strong indication for the formation
of a covalent bond of 7 to the JNK3.
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Compound 10 (IC50: JNK3: 2 nM; p38α: 2 nM), which features the para/meta substitution pattern and a
methyl group in ortho position at the B ring, inhibits both kinases in the low single digit nanomolar
range. The methyl group at that position seems to increase the potency on p38α drastically. The saturatꢀ
ed analog 11 (IC50: JNK3: 17 nM; p38α: 38 nM) lacking the electrophilic warhead supports this asꢀ
sumption since JNK3 and p38α inhibition are alike. The inversion of the para/meta substitution pattern
led to compound 18 (IC50: JNK3: 4 nM; p38α: 26 nM) exhibiting a low preference for JNK3. The introꢀ
duction of an ortho methyl group at the A ring, which was described by Zhang et al.14 to be the key deꢀ
terminant for selectivity, bred compound 20 (IC50: JNK3: 2 nM; p38α: 3 nM). Since we could not obꢀ
serve a selectivity increase with respect to JNK3 and p38α inhibition, we introduced the ortho methyl
group to the para/para substituted compound 16 (IC50: JNK3: 8 nM; p38α: 35 nM) and its propioꢀ
namide analog 17. However, this did not yield the desired effect.
Table 2. Biological Activities of tetra-substituted imidazoles 7-20.
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JNK3a
p38αb
IC50 [nM]c
Qd
120
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10
Cmp
R
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IC50 [nM]c
SEM
0.03
SEM
4.38
0.3
36
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21.18
2.71
0.08
3.25
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6.28
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
25
4
3
0.24
0.07
26
1.43
8.60
7
18
19
100
33
2
0.65
3
0.34
1
20
aincubation time, 50 min; incubation time 60 min; IC50ꢀvalues are the mean of three experiments;
b
c
dratio: IC50 (p38α)/IC50 (JNK3).
Judged by the small difference in inhibition between compounds 14 (IC50: JNK3: 10 nM; p38α: 47 nM)
and 15 (IC50: JNK3: 27 nM; p38α: 17 nM) it appears that compound 14 does not establish a covalent
bond with JNK3. Remarkably, compound 15 bearing the propionamide moiety is better tolerated by
p38α compared to 14, which stands in contrast to the prior observation. The meta/meta substituted 12
(IC50: JNK3: 3 nM; p38α: 217 nM) features a 72ꢀfold preference for JNK3 compared to the IC50ꢀvalue
of p38α. Additionally, the almost 70ꢀtimes lower JNK3 inhibition of 13 (IC50: JNK3: 175 nM; p38α:
241 nM) and the almost constant p38α inhibition suggest the successful formation of a covalent bond
between compound 12 and Cysꢀ154. The unfavorable ortho methylation at the B ring seen with comꢀ
pounds 19 and 20 (increasing p38α inhibition) is in agreement with a similar observation in compounds
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Journal of Medicinal Chemistry
16 and 17.
1
2
3
4
5
6
7
8
Since our efforts of optimizing the linker did not result in a JNK3 inhibitor exceeding 120ꢀfold preferꢀ
ence over p38α, we chose to replace the 4ꢀfluorophenyl ring of compounds 4-6 by a small methyl group
leading to compounds 21-23.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. Biological activity of tri-substituted imidazoles 21-23.
JNK3a
IC50 [nM]c
p38αb
IC50 [nM]c
Qd
Cmp
R
SEM
0.02
SEM
62
meta
para
meta
2
1,952
5,920
976
30
21
22
23
200
253
12
107
ꢀꢀꢀ
8.7
>10,000
>39
b
c
aincubation time, 50 min; incubation time 60 min; IC50ꢀvalues are the mean of three experiments;
dratio: IC50 (p38α)/IC50 (JNK3).
The rationale behind this modification is the fact that numerous examples of known JNK selective inꢀ
hibitors are not targeting the hydrophobic region I (for recent reviews on JNK inhibitors, see Koch et
al.29 and Gehringer et al.30). The most selective JNK3 inhibitor in this series (Table 3), compound 21,
shows an IC50ꢀvalue in the low nanomolar range and is 976ꢀfold selective against p38α (IC50: JNK3: 2
nM; p38α: 1,952 nM). Comparison of 21 and 22 demonstrates that the position of the electrophilic warꢀ
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head on ring B is crucial for improving both, JNK3 activity and selectivity. Compound 22 (IC50: JNK3:
200 nM), having the acrylamide in paraꢀposition is 100ꢀfold less active than its isomer 21, which has
the same substituent in metaꢀposition. Compound 23 lacks the Michael acceptor system and shows a
tremendous drop (>125ꢀfold) in JNK3 inhibition compared to 21. The structure activity relationships
(SAR) of the triꢀsubstituted pyridinylimidazoles series, which carry a small methyl moiety at the imidꢀ
azoleꢀC4 position, indicate the formation of covalent bond between JNK3 and the inhibitor 21. Comꢀ
pound 22, presenting a para/para substitution pattern, apparently does not establish a covalent bond to
the cysteine in JNK3, since its inhibitory effect was similar to the saturated compound 23, unable to
form a covalent bond.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
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Inhibitors 7 and 21, displaying the best inhibition profile within the series, were further investigated. In
order to confirm the formation of a covalent bond to Cysꢀ154, the enzymeꢀinhibitor complex was anaꢀ
lyzed by mass experiments. Both compounds were incubated with JNK3 and subsequently the unbound
and reversibly bound ligand were removed both by chromatography and by application of a high declusꢀ
tering potential prior to ionization (for further details, see supporting information). These two passages
are aimed at disrupting reversible electrostatic interactions, allowing only the detection of covalently
bound ligand. The expected mass shift of ~ 578 Da and ~ 484 Da in case of incubation with inhibitor 7
and 21, respectively, could be measured on the intact protein level using LCꢀµESIꢀQTOF mass specꢀ
trometry (for further details, see: Table S2 and Figures S1 – S3a,b,d, supporting information). Moreoꢀ
ver, performing the same experiment with compounds 8 and 23, the saturated analogs of 7 and 21, no
mass shift could be observed, since the noncovalent inhibitors do not sustain the binding to JNK3 during
the liquid chromatographyꢀmass spectrometry (LCꢀMS) experiment (Figures S3c and S3e, supporting
information). Interestingly, MS analysis carried out on compound 14 (Figure S4a, supporting inforꢀ
mation), which bears the electrophilic warhead in paraꢀposition and had been assumed not capable of
binding covalently, displayed the mass shift of ~578 Da relative to the irreversible bond with the target.
However, the shifted peak had a significantly decreased intensity compared to the mass shift of the
aforementioned compounds 7 and 21, revealing that the para/para substitution pattern of the linker can
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Journal of Medicinal Chemistry
still permit the formation of the covalent bond, although with a nonꢀfavorable efficiency. As expected,
incubation of compound 15, the noncovalent counterpart of 14, did not give rise to any enzyme mass
increase (Figure S4b, supporting information). Finally, the analogous MS experiment was performed by
incubating compounds 7, 14 and 21 with a JNK3 mutant, where the reactive Cysꢀ154 was replaced by
alanine (Figure S5, supporting information). Results for the three compounds tested showed absence of
mass shift, proving our hypothesis both on the formation of the covalent bond and on the specific amino
acid (Cysꢀ154) targeted.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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26
27
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30
31
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Table 4. Off-target activity of compounds 21 and 7 in a panel of 410 kinases.
21
7
at 1 µM
at 0.5 µM
CKꢀ1ꢀδ
CKꢀ1ꢀγ2
CKꢀ1ꢀγ3
CLK4
MAP3K10
MAP3K11
MKK7
PKCꢀnu
SGK1
TAOK2
TSF1
ZAK
TIE2ꢀwt
MAPKAP2
CKꢀ1ꢀδ
10 – 29 %a
30 – 50 %a
aResidual activity.
Covalent inhibitors 7 and 21 were further tested in a panel of 410 kinases, including 70 diseaseꢀrelevant
protein kinase mutants and 13 lipid kinases (Table S1, supporting information) to evaluate their selectivꢀ
ity within the kinome. At a testing concentration of 1 µM, which represents 500ꢀtimes its IC50ꢀvalue,
imidazole 21 inhibited only 15 kinases (including all JNK isoforms) out of the tested kinase panel, reꢀ
sulting in a low S (50) score of 0.037. This parameter is defined as the portion of kinases, which are
inhibited to more than 50 %, in relation to all tested kinases.31 Notably, the tetraꢀsubstituted imidazole 7
reveals a remarkable selectivity profile. Besides the JNKs, 7 inhibits only three other protein kinases
(Table 4) at a screening concentration of 0.5 µM (representing more than 1,500ꢀfold its IC50ꢀvalue) and
possesses an excellent S (50) score of 0.015. The slightly changed selectivity is mainly attributed to the
methylation of the imidazoleꢀN1 position and specific interactions within the hydrophobic region I, the
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soꢀcalled selectivity pocket, which is not targeted by compound 21. As expected, no selectivity was seen
1
2
3
within the JNKꢀfamily since the targeted cysteine is conserved among all isoforms.
4
5
6
7
8
All final compounds were checked for being potential panꢀassay interference compounds (PAINS) using
the ZINC Patterns tool32 which led to no hits. Since acrylamides might interfere with intracellular proꢀ
teins in a nonꢀdiscriminatory manner,33 most promising covalent inhibitor 7 (10 µM) was incubated with
the nonspecific thiol glutathione (present in almost all cells, 5 mM) under similar conditions used in the
kinase activity assay (max. concentration 10 µM, 54 min, 37°C). Subsequent high performance liquid
chromatography (HPLC) analysis revealed only low depletion of 7 (Table S3; supporting information).
This observation together with the rather clean profile of acrylamide compounds 7 and 21 in the selecꢀ
tivity screening show no evidence of unpredictable interactions with other cysteine side chains.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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33
34
35
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Inhibitors 7 and 21 were further tested for their metabolic stability in male human liver microsomes
(HLM). Both covalent inhibitors undergo a partial biotransformation. Nevertheless, both tested comꢀ
pounds are exhibiting 13 % and 31 % degradation over a time span of 180 min and 190 min, respectiveꢀ
ly, and can therefore be considered to be metabolically stable (Table S4 and Figures S6 and S7, supportꢀ
ing information). In agreement with prior findings,24 the metabolic degradation leads most likely to the
oxidation of the imidazoleꢀC2 sulfur.
Conclusion.
We successfully transferred the concept of covalently inhibiting the JNKs to our pyridinylimidazole
scaffold, which was elaborated for JNK interaction. Compounds 7 and 21 with their para/meta substituꢀ
tion pattern comprise the optimal linker length and angle for the electrophilic warhead to form a covaꢀ
lent bond to the Cysꢀ154. Incubation of JNK3 with compounds 7 and 21 followed by mass experiments
substantiated the assumption of a covalent bond formation. Both covalent inhibitors are metabolically
relatively stable when exposed to HML. Targeting the hydrophobic region I with a 4ꢀfluorphenyl ring
combined with a methylation of the imidazoleꢀN1 position reduced the number of off targets, resulting
in compound 7 with a remarkable selectivity profile.
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Journal of Medicinal Chemistry
Experimental Section.
Chemistry
1
2
3
4
5
6
General. The purity of all tested compounds is ≥ꢁ95ꢁ% and was determined via reverse phase HPLC.
7
8
9
General procedure for the reduction of nitro group of compounds 24, 25, 33 and 34 using tin(II)
chloride (General Procedure A)
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12
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20
21
22
23
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In 100 mL roundꢀbottom flask, the nitro compound was dissolved in ethanol. Tin(II) chloride was added
and the reaction mixture was refluxed for 6 h. After cooling to rt, a saturated solution of sodium bicarꢀ
bonate (20 mL) was added. The aqueous layer was extracted with ethyl acetate (3 x 10 mL) and the
combined organic layer was dried over anhydrous Na2SO4. The solvent was evaporated under reduced
pressure and the crude product was purified by flash chromatography. (SiO2, dichloromethane/ethanol,
95:05 to 90:10).
General Procedure for the preparation of final compounds 4, 5, 21 and 22 (General Procedure B).
The respective primary amine 26, 27, 35 or 36 was dissolved in DMF (2 mL) and placed in a roundꢀ
bottom flask under argon atmosphere. DIPEA was added in one portion followed by dropwise addition
of acryloyl chloride diluted in dry dichloromethane (1 mL) at 0 °C (in case of preparation of 4 and 5) or
in dry DMF (0.2 mL) at ꢀ20 °C (in case of preparation of 21 and 22). The reaction mixture was further
stirred for 2 h at rt, then poured over ice and stirred for 30 min. If the crude product precipitated, the
precipitate was filtered off and purified by column chromatography. In case of preparation of 21, the
crude product did not precipitate. Therefore, the aqueous phase was extracted with ethyl acetate (3 x 10
mL). The combined organic layers were dried over Na2SO4, the solvent was evaporated under reduced
pressure and the residue was purified with flash chromatography.
General procedure for synthesis of amides 28a-h (General Procedure C)
To a stirred suspension of corresponding nitrobenzoic acid (1.1 eq) in dichloromethane (9 mL / mmol
aniline), TBTU (1.1 eq), the corresponding bromoaniline (1 eq) and DIPEA (2 eq) were added at 0 °C.
The yellowish suspension cleared up as it was allowed to warm to room temperature and was subseꢀ
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quently stirred for 3 h until complete consumption of the respective bromoaniline was detected by TLC.
The reaction mixture was poured on a pad of silica and eluted with a mixture of ethyl acetate and petrol
ether 50/50.
1
2
3
4
5
6
7
8
9
General procedure for synthesis of 29a-h by reduction of nitrobenzene analogs 28a-h (General
Procedure D)
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12
13
14
15
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20
21
22
23
24
25
26
27
28
29
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60
To a suspension of tin(II) chloride (5 eq) in ethanol (100 mL / mmol nitrobenzene) was added the correꢀ
sponding nitrobenzene (1eq) and the reaction mixture was heated to 75 °C for 2.5 h until complete conꢀ
version was detected by TLC. The solvent was evaporated under reduced pressure. The white residue
was treated with saturated NaHCO3 solution and brine. The aqueous layer was extracted with ethyl aceꢀ
tate (3x 60 mL). The combined organic phases were dried over Na2SO4 und evaporated under reduced
pressure to yield the desired title compound as a white solid, which was used without further purificaꢀ
tion.
General procedure for synthesis of compounds 30a-m via amide coupling (General Procedure E)
To a suspension of corresponding aniline 29 (1 eq) in 1,4ꢀdioxane (12 mL / mmol aniline), DIPEA (2.5
eq) was added followed by the corresponding acid chloride (1.1 eq) at 0 °C. The mixture was allowed to
warm up to room temperature and was stirred for 2 h until complete conversion was detected by TLC.
After removing all volatiles under reduced pressure, methanol was added and the mixture was evapoꢀ
rated again. This process was repeated for three times and the solid obtained was used without further
purification.
General procedure for synthesis of final compounds 7,8,10-20 via Buchwald-Hartwig cross cou-
pling reaction (General Procedure F)
BrettPhos precatalyst (2.5ꢁmol %) was added to a dry mixture of tertꢀbutanol and 1,4ꢀdioxane (1:4;
54ꢁmL / mmol 31) in an argon flushed glass tube. Compound 31 (1 eq) was added to the reaction mixꢀ
ture followed by cesium carbonate (2.5 eq) and the respective aryl bromide 30 (1.1 eq). The reaction
mixture was heated to 125 °C and stirred for 2 h after sealing the glass tube tightly. Another 2.5 mol %
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Journal of Medicinal Chemistry
BrettPhos precatalyst was added and the reaction mixture was heated to 125 °C and stirred for additionꢀ
al 3 h until HPLC confirmed complete conversion. The reaction mixture was allowed to cool to room
temperature, filtered and washed with ethyl acetate (20 mL). All volatiles were removed under reduced
pressure and the residue was purified by silica flashꢀchromatography.
1
2
3
4
5
6
7
8
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3-Acrylamido-N-(4-((4-(4-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-
yl)amino)phenyl)benzamide (4)
Compound 4 was synthesized according to general procedure B, using compound 26 (80 mg, 0.16
mmol), DIPEA (30 µL, 0.17 mmol) and acryloyl chloride (16 µL, 0.19 mmol). Purification by flash
chromatography (SiO2, dichloromethane/ethanol, 92:08) afforded 35 mg (40 %) of a light yellow solid
product. 1H NMR (250 MHz, DMSOꢀd6) δ 12.67 (br. s, 1H), 10.35 (s, 1H), 10.14 (s, 1H), 8.97 (br. s,
1H), 8.21 ꢀ 7.96 (m, 2H), 7.96 ꢀ 7.87 (m, 1H), 7.73 ꢀ 7.40 (m, 8H), 7.38 ꢀ 7.13 (m, 2H), 7.12 ꢀ 6.77 (m,
1H), 6.68 (d, J = 4.6 Hz, 1H), 6.48 (dd, J = 9.9, 17.0 Hz, 1H), 6.30 (dd, J = 2.3, 17.0 Hz, 1H), 5.88 ꢀ
13
5.72 (m, 1H), 2.62 (s, 3H). C NMR (101 MHz, DMSOꢀd6) δ 164.9, 163.3, 161.9 (d, J = 247.0 Hz),
156.3, 147.2, 142.8, 141.9, 139.1, 137.9, 135.9, 134.7, 132.0, 131.7, 130.7 (d, J = 8.9 Hz), 129.5 (d, J =
3.5 Hz), 128.7, 127.1, 122.3, 122.0, 121.0, 118.8, 118.2, 115.7 (d, J = 23.0 Hz), 112.4, 111.8, 107.0,
15.0; HPLC tR = 6.25 min. ESIꢀHRMS: calculated: 565.1817 [M+H]+, found: 565.1812.
4-Acrylamido-N-(4-((4-(4-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-
yl)amino)phenyl)benzamide (5)
Compound 5 was synthesized according to general procedure B, using compound 27 (78 mg, 0.15
mmol), DIPEA (30 µL, 0.17 mmol) and acryloyl chloride (15 µL, 0.18 mmol). Purification by flash
chromatography (SiO2, dichloromethane/ethanol, 92:08) afforded 22 mg (27 %) of a light yellow solid
product. 1H NMR (400 MHz, DMSOꢀd6) δ 12.68 (br. s, 1H), 10.44 (s, 1H), 10.03 (s, 1H), 8.99 (br. s,
1H), 8.12 (br. s, 1H), 8.02 ꢀ 7.89 (m, 2H), 7.85 ꢀ 7.74 (m, 2H), 7.62 (d, J = 8.3 Hz, 2H), 7.57 (d, J = 8.6
Hz, 2H), 7.51 (br. s, 2H), 7.31 (br. s, 1H), 7.23 (br. s, 1H), 7.12 ꢀ 6.74 (m, 1H), 6.74 ꢀ 6.60 (m, 1H), 6.49
(dd, J = 10.1, 17.0 Hz, 1H), 6.31 (d, J = 17.2 Hz, 1H), 5.81 (d, J = 10.1 Hz, 1H), 2.63 (s, 3H). 13C NMR
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(101 MHz, DMSOꢀd6) δ 164.3, 163.4, 161.3 (d, J = 246.2 Hz), 156.3, 147.7, 147.1, 142.9, 142.7, 141.7,
137.6, 131.6, 130.6 (d, J = 5.5 Hz), 129.7, 128.4, 127.4, 126.8, 121.0, 118.5, 118.4, 118.3, 115.7 (d, J =
26.7 Hz), 111.8, 107.1, 99.9, 15.0. HPLC tR = 6.11 min. ESIꢀHRMS: calculated: 565.1817 [M+H]+,
found: 565.1804.
1
2
3
4
5
6
7
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9
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N-(4-((4-(4-(4-Fluorophenyl)-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-yl)amino)phenyl)-3-
propionamidobenzamide (6)
Under argon atmosphere, compound 3 (100 mg, 0.23 mmol) was suspended in dry dichloromethane (10
mL) and DIPEA (54 µL, 0.31 mmol) was added. Finally, a suspension of 3ꢀpropionamidobenzoic acid
(54 mg, 0.28 mmol) and PyBOP (161 mg, 0.31 mmol) in dry dichloromethane (10 mL) was added and
the reaction mixture was stirred at rt for 18 h. The reaction mixture was concentrated at reduced presꢀ
sure and the crude was purified twice by flash column chromatography (SiO2, dichloromethane/ethanol,
97:03 to 80:20) yielding product 6 (30 mg, 21 %). 1H NMR (300 MHz, DMSOꢀd6) δ 12.67 (br. s, 1H),
10.11 (br. s, 1H), 10.06 (s, 1H), 9.08 ꢀ 8.87 (m, 1H), 8.17 ꢀ 7.94 (m, 2H), 7.84 (d, J = 8.0 Hz, 1H), 7.67 ꢀ
7.15 (m, 10H), 7.10 ꢀ 6.76 (m, 1H), 6.73 ꢀ 6.64 (m, 1H), 2.63 (s, 3H), 2.36 (q, J = 7.5 Hz, 2H), 1.11 (t, J
13
= 7.5 Hz, 3H); CꢀNMR (101 MHz, DMSOꢀd6) δ 172.2, 165.1, 163.1, 161.4 (d, J = 244.9 Hz), 156.3,
148.0, 147.2, 142.9, 142.0, 139.4, 138.9, 137.9, 137.4, 135.9, 134.7, 133.8, 132.5, 132.1, 130.7 (d, J =
8.0 Hz), 129.5 (d, J = 6.0 Hz), 128.6, 126.8, 126.2, 121.7, 121.0, 118.6, 118.5, 118.2, 115.8 (d, J = 22.0
Hz), 115.3 (d, J = 22.0 Hz), 112.4, 111.8, 111.4, 107.3, 107.0, 29.5, 15.1, 9.6. The peaks of the carbon
are double due to the amide tautomerism. HPLC tR = 5.47 min. ESIꢀHRMS: calculated: 567.1973
[M+H]+, found: 567.1957.
3-Acrylamido-N-(4-((4-(4-(4-fluorophenyl)-1-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-
yl)amino)phenyl)benzamide (7)
According to general procedure F, 7 was synthesized from 31 and 30a. Eluent: ethyl acetate/nꢀhexane
45:55 to 75:25. Another flashꢀchromatography was performed to purify the product; eluent: dichloroꢀ
1
methane/methanol 100:0 to 92:8 afforded 31ꢁ% yield. H NMR (200 MHz, DMSOꢀd6) δ 10.34 (s, 1H),
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Journal of Medicinal Chemistry
10.15 (s, 1H), 9.12 (s, 1H), 8.26 (d, J = 5.4 Hz, 1H), 8.15 (s, 1H), 7.92 (d, J = 7.8 Hz, 1H), 7.70 – 7.59
(m, 5H), 7.53 – 7.40 (m, 3H), 7.14 (t, J = 8.9 Hz, 2H), 6.78 – 6.69 (m, 2H), 6.55 – 6.17 (m, 2H), 5.84 –
1
2
3
4
5
6
13
5.72 (m, 1H), 3.43 (s, 3H), 2.65 (s, 3H). CꢁNMR (101 MHz, DMSOꢀd6) δ 165, 163.3, 161.0 (d, J =
7
8
9
243.7 Hz), 156.5, 148.3, 143.5, 139.1, 138.9, 137.3, 136.8, 135.9, 132.5, 131.7, 130.6 (d, J = 2.9 Hz),
128.7, 128.3 (d, J = 8.0 Hz), 128.3, 127.1, 122.3, 122.0, 121.0, 118.8, 118.4, 115.1 (d, J = 21.5 Hz),
114.8, 111.1, 31.5, 15.3. ESIꢀHRMS: calculated: 578.1900 [M+H]+, found: 578.1876. HPLC tR = 5.87
min.
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N-(4-((4-(4-(4-Fluorophenyl)-1-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-
yl)amino)phenyl)-3-propionamidobenzamide (8)
According to general procedure F, 8 was synthesized from 31 and 30j. Eluent: ethyl acetate/nꢀhexane
45:55 to 75:25. Another flashꢀchromatography was performed to purify the product; eluent: dichloroꢀ
1
methane/methanol 100:0 to 92:8 afforded 62ꢁ% yield. HꢁNMR (200 MHz, CDCl3) δ 8.33 – 8.15 (m,
2H), 7.98 (d, J = 10.6 Hz, 2H), 7.72 (d, J = 8.6 Hz, 1H), 7.55 – 7.28 (m, 6H), 7.23 – 7.12 (m, 2H), 7.04
(s, 1H), 6.93 (t, J = 8.6 Hz, 2H), 6.67 – 6.58 (m, 2H), 3.44 (s, 3H), 2.65 (s, 3H), 2.33 (q, J = 7.7 Hz,
2H), 1.17 (t, J = 7.5 Hz, 3H). 13CꢁNMR (101 MHz, DMSOꢀd6) δ 172.2, 165.1, 161.0 (d, J = 243.4 Hz),
156.4, 148.2, 143.6, 139.5, 138.9, 137.2, 136.8, 135.9, 132.6, 130.5 (d, J = 2.9 Hz), 128.6, 128.3 (d, J =
7.9 Hz), 128.3, 121.8, 121.7, 121.0, 118.5, 115.2 (d, J = 21.4 Hz), 114.8, 111.1, 31.6, 29.5, 15.3, 9.6.
ESIꢀHRMS: calculated: 580.2057 [M+H]+, found: 580.2054. HPLC tR = 7.55 min.
3-Amino-N-(4-((4-(4-(4-fluorophenyl)-1-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-
yl)amino)phenyl)benzamide (9)
Compound 9 was synthesized from S1, zinc dust (6 eq) and ammonium formate (6 eq) in ethanol (62
mL / mmol S1). The mixture was heated under reflux for 4 h until complete reduction of the nitro group
was detected by TLC. The mixture was filtered through a pad of celite, which was washed with ethanol.
All volatiles were removed under reduced pressure and the residue was purified using flash chromatogꢀ
raphy. Eluent: ethyl acetate/nꢀhexane 45:55 to 75:25 afforded 75ꢁ% yield. 1H NMR (200 MHz, CDCl3) δ
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8.24 (d, J = 5.7 Hz, 1H), 7.83 (s, 1H), 7.57 – 7.37 (m, 5H), 7.24 – 7.10 (m, 6H), 7.05 – 6.74 (m, 4H),
6.69 – 6.59 (m, 2H), 3.46 (s, 3H), 2.68 (s, 4H). 13CꢁNMR (50 MHz, CDCl3) δ 165.8, 161.8 (d, J = 245.9
Hz), 156.7, 149.1, 146.9, 144.5, 140.1, 138.6, 136.2, 136, 133.4, 130.1 (d, J = 3.2 Hz), 129.5, 128.9 (d, J
= 7.9 Hz), 128.1, 121.4, 121.3, 118.1, 116.2, 115.6, 115.1 (d, J = 21.4 Hz), 113.7, 109, 31.8, 16. ESIꢀ
HRMS: calculated: 524.1795 [M+H]+, found: 524.1761. HPLC tR = 4.13 min.
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3-Acrylamido-N-(4-((4-(4-(4-fluorophenyl)-1-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-
yl)amino)phenyl)-2-methylbenzamide (10)
According to general procedure F, 10 was synthesized from 31 and 30l. Eluent: ethyl acetate/nꢀhexane
45:55 to 75:25. Another flashꢀchromatography was performed to purify the product; eluent: dichloroꢀ
1
methane/methanol 100:0 to 92:8 afforded 12ꢁ% yield. HꢁNMR (400 MHz, DMSOꢀd6) δ 10.25 (s, 1H),
9.65 (s, 1H), 9.13 (s, 1H), 8.25 (d, J = 5.1 Hz, 1H), 7.67 – 7.52 (m, 5H), 7.51 – 7.41 (m, 2H), 7.33 –
7.24 (m, 2H), 7.15 (t, J = 8.9 Hz, 2H), 6.77 – 6.71 (m, 2H), 6.61 – 6.51 (m, 1H), 6.31 – 6.22 (m, 1H),
13
5.81 – 5.74 (m, 1H), 3.43 (s, 3H), 2.65 (s, 3H), 2.24 (s, 3H). CꢁNMR (101 MHz, DMSOꢀd6) δ 167.2,
163.4, 161.0 (d, J = 243.3 Hz), 156.5, 148.3, 143.5, 138.9, 138.9, 137.2, 136.8, 136.6, 132.7, 131.6,
130.6 (d, J = 3.1 Hz), 129.4, 128.26 (d, J = 8.0 Hz), 128.3, 126.7, 126.4, 125.5, 124.1, 120.2, 118.5,
115.2 (d, J = 21.5 Hz), 114.8, 111.1, 31.5, 15.3, 14.6. ESIꢀHRMS: calculated: 592.2057 [M+H]+, found:
592.2049. HPLC tR = 6.60 min.
N-(4-((4-(4-(4-Fluorophenyl)-1-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-
yl)amino)phenyl)-2-methyl-3-propionamidobenzamide (11)
According to general procedure F, 11 was synthesized from 31 and 30b. Eluent: ethyl acetate/nꢀhexane
1
45:55 to 75:25 afforded 63ꢁ% yield. HꢁNMR (200 MHz, DMSOꢀd6) δ 10.23 (s, 1H), 9.36 (s, 1H), 9.12
(s, 1H), 8.25 (d, J = 5.5 Hz, 1H), 7.71 – 7.52 (m, 4H), 7.52 – 7.39 (m, 3H), 7.30 – 7.06 (m, 4H), 6.80 –
6.66 (m, 2H), 3.43 (s, 3H), 2.65 (s, 3H), 2.37 (q, J = 7.3 Hz, 2H), 2.22 (s, 3H), 1.11 (t, J = 7.5 Hz, 3H).
13CꢁNMR (50 MHz, DMSOꢀd6) δ 172.1, 167.3, 161.0 (d, J = 243.7 Hz), 156.5, 148.3, 143.5, 138.9 (d, J
= 2.9 Hz), 137.1, 137.0, 136.76, 132.8, 130.6, 130.5, 129.5, 128.3, 128.2 (d, J = 8.0 Hz), 126.6, 125.4,
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123.8, 120.2, 118.5, 115.1 (d, J = 21.5 Hz), 114.8, 111.0, 31.5, 28.8, 15.3, 14.5, 9.8. ESIꢀHRMS: calcuꢀ
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lated: 594.2213 [M+H]+, found: 594.2231. HPLC tR = 7.27 min.
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3-Acrylamido-N-(3-((4-(4-(4-fluorophenyl)-1-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-
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yl)amino)phenyl)benzamide (12)
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According to general procedure F, 12 was synthesized from 31 and 30c. Eluent: ethyl acetate/nꢀhexane
45:55 to 75:25. Another flashꢀchromatography was performed to purify the product; eluent: dichloroꢀ
1
methane/methanol 100:0 to 92/8 afforded 30ꢁ% yield. H NMRꢁ(400 MHz, DMSOꢀd6) δ 10.37 (s, 1H),
10.24 (s, 1H), 9.26 (s, 1H), 8.26 (d, J = 5.2 Hz, 1H), 8.15 (s, 1H), 8.10 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H),
7.65 (d, J = 7.4 Hz, 1H), 7.54 – 7.41 (m, 4H), 7.29 – 7.10 (m, 4H), 6.81 (s, 1H), 6.77 (d, J = 5.2 Hz,
1H), 6.52 – 6.41 (m, 1H), 6.30 (d, J = 16.9 Hz, 1H), 5.79 (d, J = 10.6 Hz, 1H), 3.44 (s, 3H), 2.65 (s,
3H). 13CꢁNMR (101 MHz, DMSOꢀd6) δ 165.5, 163.3, 161.1 (d, J = 243.6 Hz), 156.3, 148, 143.7, 141.4,
139.4, 139.1, 136.8, 135.9, 131.7, 130.4 (d, J = 2.6 Hz), 128.75, 128.62, 128.33 (d, J = 8.0 Hz), 128.2,
127.2, 122.5, 122.2, 118.9, 115.2 (d, J = 21.5 Hz), 115.2, 114.0, 113.5, 111.6, 110.8, 31.6, 15.3. ESIꢀ
HRMS: calculated: 578.1900 [M+H]+, found: 578.1885. HPLC tR = 6.38 min.
N-(3-((4-(4-(4-Fluorophenyl)-1-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-
yl)amino)phenyl)-3-propionamidobenzamide (13)
According to general procedure F, 13 was synthesized from 31 and 30h. Eluent: ethyl acetate/nꢀhexane
45:55 to 75:25, afforded 62ꢁ% yield.1HꢁNMR (400 MHz, DMSOꢀd6) δ 10.21 (s, 1H), 10.07 (s, 1H), 9.21
(s, 1H), 8.27 (d, J = 5.2 Hz, 1H), 8.09 (d, J = 9.2 Hz, 2H), 7.85 – 7.80 (m, 1H), 7.63 – 7.58 (m, 1H),
7.56 – 7.50 (m, 1H), 7.49 – 7.40 (m, 3H), 7.27 – 7.18 (m, 2H), 7.14 (t, J = 8.9 Hz, 2H), 6.81 (s, 1H),
6.78 – 6.73 (m, 1H), 3.44 (s, 3H), 2.65 (s, 3H), 2.35 (q, J = 7.5 Hz, 2H), 1.10 (t, J = 7.5 Hz, 3H).
13CꢁNMR (101 MHz, DMSOꢀd6) δ 172.1, 165.5, 161 (d, J = 243.5 Hz), 156.4, 148.2, 143.5, 141.5,
139.4, 139.3, 138.9, 136.8, 135.8, 130.5 (d, J = 3.0 Hz), 128.5, 128.2 (d, J = 7.8 Hz), 121.8, 118.6,
115.1, 115.1 (d, J = 21.4 Hz), 113.9, 113.4, 111.5, 110.6, 31.5, 29.5, 15.3, 9.5. ESIꢀHRMS: calculated:
580.2057 [M+H]+, found: 580.2056. HPLC tR = 7.19 min.
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4-Acrylamido-N-(4-((4-(4-(4-fluorophenyl)-1-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-
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3
yl)amino)phenyl)benzamide (14)
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According to general procedure F, 14 was synthesized from 31 and 30e. Eluent: ethyl acetate/nꢀhexane
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45:55 to 75:25. Another flashꢀchromatography was performed to purify the product; eluent: dichloroꢀ
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methane/methanol 100:0 to 92:8 afforded 32ꢁ% yield. HꢁNMR (400 MHz, CDCl3/methanolꢀd4) δ 8.13
(d, J = 5.2 Hz, 1H), 7.86 (d, J = 8.6 Hz, 2H), 7.72 (d, J = 8.6 Hz, 2H), 7.54 (d, J = 8.6 Hz, 2H), 7.40 –
7.34 (m, 2H), 7.20 (d, J = 8.7 Hz, 2H), 6.97 (t, J = 8.7 Hz, 2H), 6.66 (s, 1H), 6.60 (d, J = 5.3 Hz, 1H),
6.43 – 6.36 (m, 2H), 5.75 (dd, J = 7.5, 4.2 Hz, 1H), 3.53 (s, 3H), 2.60 (s, 3H). 13CꢁNMR (101 MHz,
CDCl3/methanolꢀd4) δ 166.9, 165.4, 162.6 (d, J = 246.4 Hz), 157.4, 148.9, 145.1, 142.0, 140.3, 139.2,
136.9, 134.1, 131.3, 130.6, 130.1 (d, J = 3.2 Hz), 129.8 (d, J = 8.0 Hz), 129, 128.7, 128.1, 122.4, 121.5,
120, 115.6, 115.6 (d, J = 21.6 Hz), 110.3, 32.4, 16.7. ESIꢀHRMS: calculated: 578.1900 [M+H]+, found:
578.1894. HPLC tR = 5.70 min.
N-(4-((4-(4-(4-Fluorophenyl)-1-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-
yl)amino)phenyl)-4-propionamidobenzamide (15)
According to general procedure F, 15 was synthesized from 31 and 30k. Eluent: ethyl acetate/nꢀhexane
45:55 to 75:25. Another flashꢀchromatography was performed to purify the product; eluent: dichloroꢀ
1
methane/methanol 100:0 to 92:8 afforded 48ꢁ% yield. HꢁNMR (400 MHz, DMSOꢀd6) δ 10.15 (s, 1H),
10.01 (s, 1H), 9.13 (s, 1H), 8.26 (d, J = 5.2 Hz, 1H), 7.92 (d, J = 8.7 Hz, 2H), 7.73 (d, J = 8.7 Hz, 2H),
7.67 – 7.57 (m, 4H), 7.51 – 7.43 (m, 2H), 7.15 (t, J = 8.9 Hz, 2H), 6.77 – 6.71 (m, 2H), 3.43 (s, 3H),
2.65 (s, 3H), 2.37 (q, J = 7.5 Hz, 2H), 1.10 (t, J = 7.5 Hz, 3H). 13CꢁNMR (101 MHz, DMSOꢀd6) δ 172.3,
164.4, 161.0 (d, J = 243.5 Hz), 156.5, 148.3, 143.5, 142.1, 138.9, 137.1, 136.8, 132.7, 130.6 (d, J = 3.1
Hz), 129.1, 128.4, 128.3, 128.2 (d, J = 7.9 Hz), 121.0, 118.4, 118.1, 115.1 (d, J = 21.5 Hz), 114.8,
111.1, 31.5, 29.5, 15.2, 9.4. ESIꢀHRMS: calculated: 580.2057 [M+H]+, found: 580.2054. HPLC tR =
7.04 min.
N-(4-((4-(4-(4-Fluorophenyl)-1-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-
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yl)amino)phenyl)-3-propionamidobenzamide (16)
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According to general procedure F, 16 was synthesized from 31 and 30f. Eluent: ethyl acetate/nꢀhexane
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45:55 to 75:25. Another flashꢀchromatography was performed to purify the product; eluent: dichloroꢀ
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methane/methanol 100:0 to 92:8 afforded 37ꢁ% yield. HꢁNMR (200 MHz, CDCl3/methanolꢀd4) δ 8.46
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(s, 1H), 8.30 – 8.17 (m, 2H), 7.83 – 7.69 (m, 2H), 7.66 – 7.49 (m, 3H), 7.45 – 7.27 (m, 3H), 7.14 – 7.06
(m, 1H), 6.92 (t, J = 8.8 Hz, 2H), 6.62 (d, J = 5.1 Hz, 1H), 6.51 – 6.19 (m, 4H), 5.79 – 5.67 (m, 1H),
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3.45 (s, 3H), 2.63 (s, 3H), 2.16 (s, 3H). CꢁNMR (50 MHz, CDCl3/methanolꢀd4) δ 166.4, 164.8, 162(d,
J = 246.3 Hz), 157.9, 148.4, 144.5, 141.5, 140.1, 138.7, 135.8, 133.8, 133.5, 130.8, 130, 129.5 (d, J =
3.2 Hz), 129.2 (d, J = 8.1 Hz), 128.4, 128.2, 127.8, 125.0, 123.2, 119.4, 119.2, 115.0 (d, J = 21.5 Hz),
114.7, 108.4, 32, 17.7, 16.2. ESIꢀHRMS: calculated: 592.2057 [M+H]+, found: 592.2068 . HPLC tR =
6.90 min.
N-(4-((4-(4-(4-Fluorophenyl)-1-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-yl)amino)-3-
methylphenyl)-4-propionamidobenzamide (17)
According to general procedure F, 17 was synthesized from 31 and 30i. Eluent: ethyl acetate/nꢀhexane
45:55 to 75:25; afforded 72ꢁ% yield. 1HꢁNMR (200 MHz, DMSOꢀd6) δ 10.23 (s, 1H), 9.36 (s, 1H), 9.11
(s, 1H), 8.25 (d, J = 5.2 Hz, 1H), 7.68 – 7.53 (m, 4H), 7.52 – 7.39 (m, 3H), 7.33 – 7.03 (m, 4H), 6.79 –
6.65 (m, 2H), 3.43 (s, 3H), 2.65 (s, 3H), 2.37 (q, J = 7.5 Hz, 2H), 2.21 (s, 3H), 1.11 (t, J = 7.5 Hz, 3H).
13CꢁNMR (50 MHz, CDCl3/methanolꢀd4) δ 174.5, 169, 162.1 (d, J = 246.4 Hz), 156.9, 148.6, 144.7,
140, 138.8, 138.6, 136.2, 135.9, 133.9, 130.9, 129.7 (d, J = 3.2 Hz), 129.3 (d, J = 8.0 Hz), 128.4 (d, J =
0.7 Hz), 127.6, 126.2, 125.1, 121.4, 121.0, 115.2, 115.2 (d, J = 21.5 Hz), 109.4, 32.1, 29.6, 16.3, 14.6,
9.8. ESIꢀHRMS: calculated: 594.2213 [M+H]+, found: 594.2213. HPLC tR = 6.68 min.
4-Acrylamido-N-(3-((4-(4-(4-fluorophenyl)-1-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-
yl)amino)phenyl)benzamide (18)
According to general procedure F, 18 was synthesized from 31 and 30g. Eluent: ethyl acetate/nꢀhexane
45:55 to 75:25. Another flashꢀchromatography was performed to purify the product; eluent: dichloroꢀ
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1
methane/methanol 100:0 to 92:8 afforded 26ꢁ% yield. HꢁNMR (200 MHz, CDCl3/methanolꢀd4) δ 7.93
(d, J = 5.3 Hz, 1H), 7.70 – 7.43 (m, 5H), 7.21 – 7.10 (m, 2H), 7.07 – 6.89 (m, 2H), 6.89 – 6.79 (m, 1H),
6.72 (t, J = 8.8 Hz, 2H), 6.54 (s, 1H), 6.39 (d, J = 5.2 Hz, 1H), 6.17 (d, J = 5.6 Hz, 2H), 5.58 – 5.47 (m,
1H), 3.33 (s, 3H), 2.38 (s, 3H). 13CꢁNMR (50 MHz, CDCl3/methanolꢀd4) 166.4, 164.8, 161.9 (d, J =
246.3 Hz), 156.5, 148.1, 144.4, 141.5, 140.5, 139.6, 138.9, 138.5, 130.7, 129.8, 129.4 (d, J = 3.2 Hz),
129.1 (d, J = 8.0 Hz), 129.0, 128.3, 128.1, 127.5, 119.2, 115.7, 115.3, 115, 114.9 (d, J = 21.6 Hz),
112.5, 110.4, 31.8, 16.1. ESIꢀHRMS: calculated: 578.1900 [M+H]+, found: 578.1900. HPLC tR = 5.95
min.
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3-Acrylamido-N-(3-((4-(4-(4-fluorophenyl)-1-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-
yl)amino)phenyl)-2-methylbenzamide (19)
According to general procedure F, 19 was synthesized from 31 and 30d. Eluent: ethyl acetate/nꢀhexane
45:55 to 75:25. Another flashꢀchromatography was performed to purify the product; eluent: dichloroꢀ
1
methane/methanol 100:0 to 92:8 afforded 30ꢁ% yield. HꢁNMR (400 MHz, DMSOꢀd6) δ 10.35 (s, 1H),
9.65 (s, 1H), 9.21 (s, 1H), 8.25 (d, J = 5.2 Hz, 1H), 8.07 (s, 1H), 7.62 – 7.50 (m, 2H), 7.50 – 7.42 (m,
2H), 7.33 – 7.25 (m, 2H), 7.24 – 7.18 (m, 2H), 7.18 – 7.09 (m, 2H), 6.80 (s, 1H), 6.75 (d, J = 5.2 Hz,
1H), 6.62 – 6.50 (m, 1H), 6.26 (d, J = 17.1 Hz, 1H), 5.77 (d, J = 10.3 Hz, 1H), 3.43 (s, 3H), 2.69 – 2.60
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(m, 3H), 2.23 (s, 3H). CꢁNMR (101 MHz, DMSOꢀd6) δ 167.7, 163.5, 161.0 (d, J = 243.6 Hz), 156.5,
148.2, 143.6, 141.6, 139.4, 138.9, 136.8, 136.6, 130.6 (d, J = 3.0 Hz), 130.5, 129.4, 128.7, 128.3 (d, J =
7.8 Hz), 128.2, 126.7, 126.5, 125.6, 124.1, 115.2, 115.1 (d, J = 21.4 Hz), 113.7, 112.6, 111.5, 109.8,
31.6, 15.3, 14.6. The missing signal is most probably overlaid by another signal. ESIꢀHRMS: calculatꢀ
ed: 592.2057 [M+H]+, found: 592.2045. HPLC tR = 6.32 min.
3-Acrylamido-N-(4-((4-(4-(4-fluorophenyl)-1-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-
yl)amino)-3-methylphenyl)benzamide (20)
According to general procedure F, 20 was synthesized from 31 and 30m. Eluent: acetate/nꢀhexane 45:55
to 75:25. Another flashꢀchromatography was performed to purify the product; eluent: dichloroꢀ
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1
methane/methanol 100:0 to 92:8 afforded 43ꢁ% yield. HꢁNMR (400 MHz, DMSOꢀd6) δ 10.36 (s, 1H),
10.16 (s, 1H), 8.31 (s, 1H), 8.18 – 8.12 (m, 2H), 7.92 (d, J = 8.0 Hz, 1H), 7.67 – 7.58 (m, 2H), 7.52 –
7.37 (m, 5H), 7.18 – 7.10 (m, 2H), 6.69 (dd, J = 5.2, 1.2 Hz, 1H), 6.55 – 6.42 (m, 2H), 6.30 (dd, J =
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17.0, 1.9 Hz, 1H), 5.82 – 5.74 (m, 1H), 3.42 (s, 3H), 2.63 (s, 3H), 2.17 (s, 3H). CꢁNMR (101 MHz,
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DMSOꢀd6) δ 165.1, 163.3, 161.0 (d, J = 243.6 Hz), 157.8, 148.5, 143.4, 139.1, 139.1, 136.8, 135.9,
135.1, 134.3, 131.9, 131.7, 130.6 (d, J = 3.0 Hz), 128.7, 128.4, 128.3 (d, J = 8.0 Hz), 127.1, 124.2,
122.5, 122.3, 122.1, 118.8, 118.4, 115.1 (d, J = 21.4 Hz), 114.3, 109.4, 31.5, 18.2, 15.4, 15.3. ESIꢀ
HRMS: calculated: 592.2057 [M+H]+, found: 592.2064. HPLC tR = 6.60 min.
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3-Acrylamido-N-(4-((4-(4-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-
yl)amino)phenyl)benzamide (21)
Compound 21 was prepared according to general procedure B starting from 35 (87.5 mg, 0.20 mmol),
DIPEA (46 µL, 0.26 mmol) and acryloyl chloride (22 µL, 0.24 mmol). Purification: flash column chroꢀ
matography (SiO2, dichloromethane/ethanol, 92:08). Yield: 40 mg (41 %) as a light yellow solid prodꢀ
uct. 1H NMR (250 MHz, DMSOꢀd6) δ 12.28 (s, 1H), 10.35 (s, 1H), 10.13 (s, 1H), 8.99 (s, 1H), 8.14 (s,
1H), 8.08 (d, J = 5.4 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.71 ꢀ 7.59 (m, 5H), 7.52 ꢀ 7.38 (m, 1H), 7.15 (s,
1H), 6.98 (d, J = 5.6 Hz, 1H), 6.56 ꢀ 6.38 (m, 1H), 6.36 ꢀ 6.20 (m, 1H), 5.86 ꢀ 5.70 (m, 1H), 2.55 (s,
3H), 2.42 (s, 3H); 13C NMR (101 MHz, DMSOꢀd6) δ 164.9, 163.3, 156.3, 147.2, 143.1, 139.1, 138.2,
137.0, 135.9, 134.0, 131.8, 131.7, 128.7, 127.1, 122.2, 122.0, 121.1, 118.8, 118.3, 117.9, 111.0, 105.9,
15.5, 11.4. HPLC tR = 5.29 min. ESIꢀHRMS: calculated: 485.1754 [M+H]+, found: 485.1738.
4-Acrylamido-N-(4-((4-(4-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-
yl)amino)phenyl)benzamide (22)
Compound 22 was synthesized according to general procedure B, using compound 36 (100 mg, 0.23
mmol), DIPEA (53 µL, 0.3 mmol) and acryloyl chloride (23 µL, 0.28 mmol). Purification by flash
chromatography (SiO2, dichloromethane/ethanol, 98:08) to afford 20 mg (22 %) of a light yellow solid
product. 1H NMR (250 MHz, DMSOꢀd6) δ 12.29 (br. s, 1H), 10.42 (s, 1H), 10.01 (s, 1H), 8.98 (s, 1H),
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8.09 (d, J = 5.4 Hz, 1H), 7.99 ꢀ 7.92 (m, J = 8.8 Hz, 2H), 7.84 ꢀ 7.76 (m, J = 9.0 Hz, 2H), 7.70 ꢀ 7.59 (m,
4H), 7.15 (s, 1H), 6.98 (d, J = 5.1 Hz, 1H), 6.48 (dd, J = 9.9, 17.0 Hz, 1H), 6.30 (dd, J = 2.2, 17.1 Hz,
1H), 5.76 ꢀ 5.85 (m, 1H), 2.55 (s, 3H), 2.42 (s, 3H). HPLC tR = 2.66 min. ESIꢀHRMS: calculated:
485.1754 [M+H]+, found: 485.1752.
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N-(4-((4-(4-Methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-yl)amino)phenyl)-3-
propionamidobenzamide (23)
Under argon atmosphere, compound 32 (80 mg, 0.26 mmol) was suspended in a mixture of dry diꢀ
chloromethane (10 mL) and DIPEA (0.054 mL, 0.31 mmol) was added. Finally, a solution of 3ꢀ
propionamidobenzoic acid (54 mg, 0.28 mmol) and PyBOP (162 mg, 0.31 mmol) in (5:1) mixture of
dichloromethane/DMF (6 mL) was added. The reaction was stirred at rt for 6 h. The reaction mixture
was poured in water and the aqueous layer was extracted 3 times with ethyl acetate. The organic layers
were washed with saturated NaCl solution, dried over anhydrous Na2SO4 and concentrated at reduced
pressure. The residue was finally purified twice by flash column chromatography (SiO2, dichloroꢀ
1
methane/ethanol, 92:08 to 85:15) yielding 27 mg of product (21%). H NMR (300 MHz, DMSOꢀd6) δ
10.15 (s, 1H), 10.06 (s, 1H), 9.17 (br. s, 1H), 8.10 (br. s, 1H), 8.07 (d, J = 5.6 Hz, 1H), 7.83 (d, J = 7.9
Hz, 1H), 7.73 ꢀ 7.58 (m, 5H), 7.49 ꢀ 7.40 (m, 1H), 7.15 (s, 1H), 7.01 (d, J = 5.4 Hz, 1H), 2.57 (s, 3H),
2.43 (s, 3H), 2.36 (q, J = 7.5 Hz, 2H), 1.11 (t, J = 7.4 Hz, 3H); 13C NMR (101 MHz, DMSOꢀd6) δ 172.2,
165.1, 155.8, 146.0, 143.2, 142.9, 139.8, 139.4, 137.3, 135.8, 132.6, 128.6, 121.7, 121.1, 118.8, 118.5,
113.8, 113.4, 111.0, 105.9, 29.5, 15.4, 9.6, 8.6. HPLC tR = 4.12 min. ESIꢀHRMS: calculated: 487.1911
[M+H]+, found: 487.1896.
N-(4-((4-(4-(4-Fluorophenyl)-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-yl)amino)phenyl)-3-
nitrobenzamide (24)
In 100 mL roundꢀbottom flask, mꢀnitrobenzoic acid (341.5 mg, 2.00 mmol), PyBOP (1.28 g, 2.40
mmol) and DIPEA (463 µL, 2.60 mmol) were mixed together in 30 mL dichloromethane. The mixture
was stirred for 1 h at rt. Then, amine 3 (800 mg, 2.00 mmol) was added to the reaction mixture in one
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Journal of Medicinal Chemistry
portion and the reaction stirred for 5 h at rt. The solvent was evaporated under vacuum, then ethyl aceꢀ
tate (50 mL) was added and washed with water (3 x 15 mL). The combined water layers were extracted
with ethyl acetate one time. The combined organic layers were dried over anhydrous Na2SO4. The solꢀ
vent was evaporated and the crude product was purified by flash chromatography (SiO2, dichloroꢀ
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methane/ethanol, 95:05 to 90:10) to afford 462 mg (42%) of an orange solid product. H NMR (400
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MHz, DMSOꢀd6) δ 12.69 (br. s, 1H), 10.48 (br. s, 1H), 9.05 (br. s, 1H), 8.80 (br. s, 1H), 8.50 ꢀ 8.30 (m,
2H), 8.01 (br. s, 1H), 7.89 – 7.75 (m, 1H), 7.64 (br. s, 4H), 7.52 (br. s, 2H), 7.30 (br. s, 2H), 7.13 ꢀ 6.75
(m, 1H), 6.70 (br. s, 1H), 2.63 (s, 3H). 13C NMR (101 MHz, DMSOꢀd6) δ 162.7, 162.2 (d, J = 249.5
Hz), 156.2, 147.7, 147.1, 142.9, 142.1, 139.6, 138.2, 136.5, 134.0, 131.6 (d, J = 4.6 Hz), 130.6 (d, J =
7.3 Hz), 130.1, 129.6, 128.2, 125.9, 122.3, 121.3, 118.3, 115.8 (d, J = 24.8 Hz), 112.0, 107.3, 15.0.
HPLC tR = 6.87 min. FABꢀMS: (m/z) calculated: 541.15 [M+H]+; found: 541.2.
N-(4-((4-(4-(4-Fluorophenyl)-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-yl)amino)phenyl)-4-
nitrobenzamide (25)
Compound 25 was synthesized similarly to 24 using pꢀnitrobenzoic acid (310 mg, 1.85 mmol), PyBOP
(1.1 g, 2.02 mmol) and DIPEA (353 µL, 2.02 mmol) and amine 3 (660 mg, 1.85 mmol) affording 637
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mg (70%) of an orange solid product. H NMR (400 MHz, DMSOꢀd6) δ 12.69 (br. s, 1H), 10.46 (br. s,
1H), 9.06 (br. s, 1H), 8.46 ꢀ 8.29 (m, 2H), 8.26 ꢀ 8.13 (m, 2H), 8.03 (br. s, 1H), 7.75 ꢀ 7.57 (m, 4H), 7.52
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(br. s, 2H), 7.28 (br. s, 2H), 7.07 (br. s, 1H), 6.76 ꢀ 6.62 (m, 1H), 2.63 (d, J = 2.3 Hz, 3H). C NMR
(101 MHz, DMSOꢀd6) δ 163.2, 161.7 (d, J = 248.9 Hz), 156.2, 149.0, 142.2, 140.8, 138.2, 131.7 (d, J =
1.8 Hz), 129.1 (d, J = 2.4 Hz), 129.0, 123.6, 123.4, 122.5, 121.4, 121.2, 120.8, 119.0, 118.3, 115.7 (d, J
= 22.2 Hz), 112.1, 107.3, 15.0. HPLC tR = 6.80 min. FABꢀMS: (m/z) calculated: 541.15 [M + H]+;
found: 541.2.
3-Amino-N-(4-((4-(4-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-
yl)amino)phenyl)benzamide (26)
Compound 26 was synthesized according to general procedure A, using compound 24 (160 mg, 0.30
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