Structure-activity relationships of benzimidazole-based selective inhibitors of the mitogen activated kinase-5 signaling pathway

Article abstract of DOI:10.1016/j.bmc.2010.09.017

In a prior communication we identified a novel class of benzimidazole-based inhibitors of EGF-induced phosphorylation of ERK5. In this paper we examine the biological activity of several 1-isopropyl-4-amino-6-ether linked benzimidazole-based compounds for

Full text of DOI:10.1016/j.bmc.2010.09.017

Bioorganic & Medicinal Chemistry 18 (2010) 8054–8060
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationships of benzimidazole-based selective inhibitors
of the mitogen activated kinase-5 signaling pathway
Patrick T. Flaherty a, , Ishveen Chopra a, Prashi Jain a, Darlene Monlish b, Jane Cavanaugh b
⇑
a Division of Medicinal Chemistry, School of Pharmacy, Duquesne University, 600 Forbes Ave., Pittsburgh, PA 15282, United States
b Division of Pharmacology, School of Pharmacy, Duquesne University, 600 Forbes Ave., Pittsburgh, PA 15282, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
In a prior communication we identified a novel class of benzimidazole-based inhibitors of EGF-induced
phosphorylation of ERK5. In this paper we examine the biological activity of several 1-isopropyl-4-amino-
6-ether linked benzimidazole-based compounds for their ability to selectively inhibit EGF-mediated
ERK5 phosphorylation; potential utility of variation at the 6-position was indicated by the initial structural
feature survey. Modification of EGF-induced formation of pERK1/2 and pERK5 in HEK293 cells were ana-
lyzed by Western blot analysis. Subsequent analysis of selected compounds in a high-throughput multiple
kinase scan and the NCI 60-cell-line screen is presented.
Received 16 June 2010
Revised 1 September 2010
Accepted 7 September 2010
Available online 19 October 2010
Keywords:
MEK
MEK5
Ó 2010 Elsevier Ltd. All rights reserved.
ERK
ERK5
Benzimidazole
Hoechst 33258
1. Background
The complexity of the signaling pathway and the relevance of
the MAPK signaling pathway to human disease states including
The mitogen activated protein kinase (MAPK) signaling pathway
is a complex intersecting series of phosphorylation events that cou-
ple an extracellular signaling event such as growth factor binding,
chemical mitogen presence, or stress with an appropriate cellular
response such as MefC, Mef2A, or CREB upregulation.1–4 These cellu-
lar responses can be either cytosolic responses or nuclear responses.
The localization of the sub-cellular response is dependent on several
factors including the degree of phosphorylation of the final signaling
kinase known as extracellular signal-regulated kinase (ERK),
binding of signaling kinases and/or ERK to scaffold proteins, and
additional proteins associated with the phosphorylated ERK (pERK)
scaffold complex.5 Typically the sequence of signaling events pro-
ceeds as follows: (1) growth factor binding, (2) receptor dimeriza-
tion and autophosphorylation, (3) mitogen activated kinase kinase
kinase (MEKK) phosphorylation, (4) mitogen activated kinase
kinase (MEK) phosphorylation, and (5) resultant ERK phosphoryla-
tion. There are considerable cross-interactions between the
receptors, MEKK phosphorylation, MEK phosphorylation, and pERK
effects. The most specific interaction in these cascades is the MEK
mediated phosphorylation of ERK; for example, MEK5 is the only
kinase that can phosphorylate ERK5 and ERK5 is the only known
substrate for MEK5.6
cancers,5,7 specifically squamous cell carcinoma,8 prostate,9 and
breast cancer10—in the case of MEK5 mediated phosphorylation
of ERK5—suggest this would be a opportune area for the develop-
ment of new therapeutic agents. There have been two reports of
MEK5 inhibitors recently.11,12 One group11 presented a series of
compounds of ATP-competitive oxindole inhibitors, represented
by BIX02188 1, the second was our early communication12 where
compound 2 was identified as a novel inhibitor of EGF-mediated
ERK phosphorylation. This communication examined compounds
originally designed in the context benzimidazole-based ATP-site
competitive inhibitors for the enzyme cyclin-dependent kinase-5
(CDK5). The rationale for cross-assaying was that early MEK inhib-
itors were derived from existing CDK2 inhibitors and that both
MEK5 and CDK5 are ATP utilizing serine/threonine kinases. Analy-
sis of how structural variation over the entire benzimidazole core
inhibited the EGF-induced formation of pERK1/2 and pERK5, indi-
cated that a 1-isopropyl group was better than a larger substituent,
4-mono-benzyl amine compounds were better than 4-dibenzyl
amine compounds, and that a simple 6-methoxy compound was
more potent than more complicated alkoxy or benzyl derivatives;
see Figure 1. This last point stands in stark contrast to the emerging
SAR on 1-isopropyl-4-benzylamino-6-substitutedbenzimidazoles
at the enzyme CDK5.13–16 For this core to have activity at CDK5,
a hydrogen bond donor has been found essential for activity; this
same structural feature results in inactivity in EGF-mediated
⇑
Corresponding author. Tel.: +1 412 396 1847; fax: +1 412 396 4660.
E-mail address: flahertyp@duq.edu (P.T. Flaherty).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.017

P. T. Flaherty et al. / Bioorg. Med. Chem. 18 (2010) 8054–8060
monophenyl is better than diphenyl
8055
N
NH
4
NH
O
N
N
H2N
N
O
6
Methoxy is better than
phenyl or 2-alkoxybutanol
1
H
O
isopropyl is better than cyclopentyl
1
2
Figure 1. Known inhibitors of EGF-induced ERK5 phosphorylation.
phosphorylation of ERK. In a parallel observation, 6-methoxy com-
pound 2 was inactive as a CDK5 inhibitor.
part of a structure-based-molecular modeling assisted program di-
rected at identifying new CDK5 inhibitors were counter-screed
against MEK5. As the SAR at MEK5 began to emerge, we recognized
that the alkoxy derivatives followed a pattern that had been ob-
served for known receptor tyrosine kinase (RTK) inhibitors such
as gefitinib and erlotonib.17–21 Examination of the side-chains pre-
viously utilized in RTK inhibitors suggested that compounds 7–10
should be prepared as these side-chains had displayed activity in
several RTK SAR studies.
The marked divergence of the SAR between CDK5 and
EGF-induced pERK5 formation derived from the presence of the
6-methoxy moiety of 2 encouraged further exploration of substitu-
tion at this position; see Figure 2. Our approach took a two-prong
strategy. First, selected 6-alkoxy compounds 3–6 prepared13 as a
Compounds 2–6 have been prepared and characterized previ-
ously.13,22 Synthesis for compounds 7–10 followed this sequence.
Selective reductive alkylation of the known precursor diamine, then
Philips cyclization in a mixed-acid system of formic acid and con-
centrated hydrochloric acid gave the previously described 13.22
See Scheme 1. 6-Methoxy deprotection proceeded in concentrated
HBr with microwave irradiation.22 Alkylation of the resultant phe-
nol 14 proceeded either via Mitsunobu alkylation utilizing the pre-
cursor alcohol 15b–d or by sodium phenolate attack on the alkyl
iodide 15a. The choice of method employed was determined by
commercial availability of the alkyl precursor. However the sodium
phenolate alkylation was far cleaner permitting direct isolation and
characterization of the ether product 16a. When the Mitsunobu cou-
pling protocol was employed, the resultant ethers 16b–d co-eluted
with attempted silica column chromatography with coupling side
products notably triphenylphosphine oxide. These compounds
were carried on as the crude as indicated in Section 3 and character-
ized via combustion analysis at subsequent synthetic steps.
Catalytic reduction of the 4-nitro group to the corresponding 4-ami-
no moiety was smoothly affected utilizing palladium on carbon as
the catalyst under 50 PSI in a shaker apparatus to give compounds
17a–d. Final reductive alkylation gave the 4-amine to the monoben-
zyl amines 7–10. All compounds were prepared as the 4-amino
mono benzyl derivatives based on prior observation12 of this feature
as a requirement for inhibition of EFG-mediated ERK5 phosphoryla-
tion. Mono alkylation was achieved routinely by the careful use a
single stoichiometric equivalent of benzaldehyde, the use of sodium
triacetoxborohydride as the hydride source, and the absence of an
acid catalyst normally employed to activate the intermediate imine
to the more reactive iminium species. For the new compounds
prepared, no additional protective group strategy was necessary.
Non-benzylated 4-amino derivatives were prepared during the
synthesis as intermediates, were tested, and are presented when
relevant. Compounds were prepared and tested as their free base
or as their hydrochloride salt on the basis of which form a generated
compound of purity adequate for combustion analysis.
NH
4
N
R
N
O
6
1
3-10
R
OH
7
8
3
4
5
O
O
O
O
O
O
9
O
O
O
10
O
N
6
O
O
NH2
NH2
N
N
N
N
O
O
O
11
17c
N
N
N
H
N
N
N
H
To determine the capacity for compounds 2–12 and 17c to inhi-
bit EGF-mediated ERK phosphorylation, HEK293 cells were treated
with 50 ng/mL of EGF to activate all MEK/ERK pathways. EGF was
added to begin the phosphorylation cascade. After 30 min an
12
OH
Figure 2. Compounds prepared and assayed for inhibition of EGF-induced ERK
phosphorylation.

8056
P. T. Flaherty et al. / Bioorg. Med. Chem. 18 (2010) 8054–8060
O2N
H2N
NH
O2N
O2N
e
d
a
N
N
N
N
N
N
N
N
b or c
N
N
R
R
R
O
O
O
HO
O
13
16 a-d
14
17 a-d
7-10
R
OH
15a
15b
15c
I
O
OH
O
N
OH
OH
15d
O
Scheme 1. Preparation of additional 6-alkoxy analogs of 1. Reagents and conditions: (a) 48% HBr, microwave irradiation, 120 °C, 2.5 h, 97%22; (b) NaH, DMF, 23 °C, 2 h, 53%;
(c) ROH (2.5 equiv), PPh3 (2.5 equiv), DIAD (2.5 equiv) 30–46%; (d) 50 psi H2, EtOH, 5% Pd/C, 23 °C, 12 h, 90–98%; (e) benzaldehyde (1.1 equiv), NaHB(OAc)3 (2.1 equiv), DCE,
23 °C, 63–65%.
aliquot of test compound, initially prepared as a 10 mmol solution
in DMSO, was added to generate a final concentration of 10 M in
high-throughput screen at Ambit biosciences23–25 (Kinomescreen).
These assays examine the capacity for test compounds to competi-
tively displace a solid-support ATP mimic from each specific
enzyme’s active site.24,25 Two specific branches of the kinase family
tree, or kinome, were selected for screening: RTKs and STEs. RTKs
were selected to survey possible interactions at the level of receptor
activation. STEs were selected as they represent the family of related
l
the cellular incubate. Phosphorylation of ERK1/2 by MEK1/2 and
phosphorylation of ERK5 by MEK5 were examined by Western blot
analysis for phosphorylated (i.e., activated) pERK. Differentiation
between pERK1/2 and pERK5 was cleanly provided by prior SDS–
PAGE separation on the basis of molecular weight; pERK1/2 is
approximately 44 kDa while pERK5 is much larger with a molecu-
lar weight of approximately 115 kDa. See Table 1.
Given that these compounds utilized a structural feature derived
from the SAR of RTKs17–19,21 a concern was that these compounds
could be exerting their effects via inhibition of these kinases. The
capacity for compound 9 to inhibit the EGF-mediated phosphoryla-
tion of MEK5 in an isoform selective manner and the ability of the
different compounds to differentially affect phosphorylation of dif-
ferent ERK isoforms is inconsistent with this postulate. However, to
further examine this possibility, compound 9 was submitted to a
kinases that include the various MEK isoforms.24 At a 10
lm concen-
tration no reasonable affinity was identified for the RTKs examined.
See Supplementary data. Curiously the capacity for compound 9 to
compete for the MEK5 ATP binding site was not seen. This raises
questions regarding the specific site(s) of activity for compounds
2, 9, and 12 either on MEK5 or perhaps on another biological target
mediating the EGF-induced phosphorylation of MEK5. One possibil-
ity is that these compounds could be allosteric inhibitors of MEK5.
Other possibilities include the possibility of interaction with other
yet unidentified biological targets.
Compounds 2 and 12 have previously12 been submitted to the
NCI’s 60-cell-line screen26 (single dose inhibition study). Compare
profile analysis identified compound 12 as being the most similar
compound in the NCI’s open access small molecule database to
compound 2. Interestingly both compounds contain a benzimid-
azole core. Compound 12 is the known DNA minor groove binding
dye Hoechst 33258.27 This commercially available compound was
examined in the assay for EGF-mediated ERK phosphorylation and
was shown to inhibit both pERK1/2 and pERK5 formation by about
30%. Curiously when compound 9, a selective inhibitor of pERK5
formation, was tested in the NCI 60-cell-line screen and analyzed
using the compare profile, it exhibited a much lower similarity to
either the methoxy compound 2 or Hoechst 33258 than they
displayed amongst themselves; see Supplementary data.
Table 1
Cellular assay of inhibition of EGF-mediated formation of pERK isoforms
Compounds
pERK1/2 (%)
Inhibition (%)
pERK5 (%)
Inhibition (%)
2
3
4
5
6
7
8
9
46
246
313
153
170
100
0
487
9
82
0
78
18
0
0
100
54
15
37
76
95
115
78
25
50
100
58
32
70
78
73
0
85
63
24
5
Activation
Activation
Activation
Activation
0
100
Activation
92
18
100
22
Activation
22
75
51
0
42
68
30
22
27
100
0
10
11
17c
12
Staurosporine
U0126
NT
82
2. Discussion
100
100
0
+
100
Modification of the 6-alkoxy substituent on the 1-isopropyl-4-
benzylaminobenzimidazole core did modify the observed
inhibition of EGF-mediated phosphorylation of ERK. This SAR is
divergent from that observed for CDK5. Of the compounds exam-
Average of three experiments, error 5%.
NT = no treatment with EGF.
+ = treatment with EGF.

P. T. Flaherty et al. / Bioorg. Med. Chem. 18 (2010) 8054–8060
8057
ined compounds 3, 4, 7, and 9 displayed the most selectivity for
inhibition of EGF-induced ERK5 phosphorylation versus inhibiting
pERK1/2 formation. Significantly these compounds did not inhibit
MEK1/2 as did the previously identified compound 2. The morpho-
lino-derivative, 10, was noticeably inactive consistent with a cat-
ionic group ablating modification of pERK levels. It is unclear if
this is a direct effect or an artifact of cellular transport. Of these
compounds 3 and 9 were equipotent within the limitations of
the assay (63% and 50% inhibition of EGF-induced formation of
pERK5, respectively). Given that the 4-amino debenzylated com-
pounds 11 and 17c also displayed activity it was a concern if this
could be a complicating factor as these compounds were examined
in further studies with in vivo systems. Compound 11, the 4-amio-
debenzylated derivative of 2, displays inhibition of both pERK1/2
and pERK5 as did its 4-aminobenzyl congener 2. Compound 17 is
devoid of the ability to block EGF induction of pERK1/2 and is
the 4-amino debenzylation derivative of compound 9. Of note is
that a considerable number of compounds could actually increase
pERK1/2 levels. It is know that inhibition of MEK1/2 can result in
increased MEK5 levels, but modification of MEK1/2 levels by inhib-
iting MEK5 levels has not been shown. This upregulation of pERK1/
2 appears to be independent of the decrease in pERK5 levels and
suggests a secondary interaction for these compounds mediating
this effect rather than a direct compensatory mechanism. Addi-
tional work is required to better understand this upregulation of
pERK1/2 by these compounds. In an attempt to better understand
the role of selective inhibition of MEK5 versus MEK1/2, compound
9 was selected for additional testing; this compound would have
the greatest decrease in MEK5 relative to MEK1/2. The determina-
tion of inhibition of pERK1/2 and pERK5 utilized is a cellular assay
so it is likely that the differential inhibition of pERK isoforms
represents a direct effect on the biological target rather than an
artifact arising from differential membrane permeability or biolog-
ical partitioning.
to more closely examine this series of compounds in isolated en-
zymes including MEK5 assays which will permit a kinetic analysis
and a better determination of the nature of these compounds’ ob-
served in vivo inhibition of EGF-mediated phosphorylation of
MEK5. A pull-down analysis of the potential biological site(s) of
interaction should assist in determining the basis for attenuation
of EGF-mediated pERK5 formation and is planned. Additional anal-
ysis of this series using in vitro models more representative of na-
tive human tumors will also be examined.
3. Experimental
All solvents and reagents were used as received unless noted
otherwise. Tetrahydrofuran (THF) was distilled from Na–benzophe-
none ketyl radical under a blanket of argon prior to use. All reactions
were conducted in dry glassware and under an atmosphere of argon
unless otherwise noted. Microwave reactions were conducted in
sealed tube and utilized a multimode Milestone Start apparatus
for irradiation with power and control parameters as noted. Melting
points were determined on a MelTemp apparatus and are uncor-
rected. All proton NMR spectra were obtained with an 500 MHz
Oxford spectrospin cryostat, controlled by a Bruker Avance system,
and were acquired using Bruker Topsin 2.0 acquisition software.
Acquired FIDs were analyzed using MestReC 3.2. Elemental analyzes
were conducted by Atlantic Microlabs and are 0.4 of theoretical. All
HRMS mass spectral analyzes were conducted at Duquesne
University with a nano ESI chip cube TOF HRMS and are 0.004 of
theoretical. All 1H NMR spectra were taken CDCl3 unless otherwise
noted and are reported as ppm relative to TMS as an internal
standard. Coupling values are reported in Hertz.
3.1. 6-Isopropoxy-1-isopropyl-4-nitro-1H-benzo[d]imidazole
(16d)
The relatively low ATP-site affinity of these compounds in the
high-throughput ATP-site assay raises some interesting possibili-
ties. The previously reported series of oxindole compounds,11 of
which 1 is an example, did display an ATP-competitive nature in
a similar high-throughput assay and did attenuate pERK5 in vivo.
It is possible that the benzimidazole-derived compounds pre-
sented may be exerting their attenuation of EGF-mediated pERK5
formation by binding to a site other than the ATP site on MEK5.
This possibility would be consistent with the inability of the iso-
lated enzyme and an ATP-competitive assay to detect a previously
indentified in vivo inhibitor of EGF-mediated pERK5. Recently a
kinetic analysis of MEK1/2 has been published,28 and it has long
been known that allosteric inhibitors of MEK1/2 exist.1,3,4,7,29 An
X-ray crystal structure of such a complex has been deposited in
the PDB: 1S9J. This confirms the necessity to conduct both isolated
enzyme and in vivo kinase assays to adequately characterize the
origin of EGF-mediated MEK5 inhibition.
The differential activity in the NCI’s 60-cell screen and the activ-
ity of Hoescht 33258 in attenuating the EGF-mediated formation of
pERK5 again suggests some interesting possibilities. Hoescht
33258 is a known DNA minor groove binder and finds routine util-
ity as an assay dye for DNA. The marked difference of compound 9
from compound 1 in the 60-cell-line screen could arise from either
the selective inhibition of MEK5 or perhaps from the observed
upregulation of pERK1/2. Regardless the conversion of the 6-alkoxy
substituent does modify the selectivity for the inhibition of pERK5
formation relative to pERK1/2 formation and may underlie the
observed difference in these cancer cell types.
A dry one-necked flask was charged with 1-isopropyl-4-nitro-
1H-benzo[d]imidazol-6-ol 14 (300.0 mg, 1.36 mmol), 2-iodopro-
pane 15a (0.27 mL, 2.72 mmol), and 2.5 mL of DMF. NaH
(163.2 mg, 6.8 mmol) was added at 0 °C in two portions. This solu-
tion was stirred at 0 °C for 30 min and at 23 °C for an additional 2 h.
The reaction mixture was poured into 10 mL EA and 10 mL K2CO3
(aq, satd). The mixture was extracted three times each with 10 mL
portions of EA and the combined extracts were washed with NaCl
(aq, satd) and dried over Na2SO4. The extract was decanted and the
solvent was removed under reduced pressure. The crude material
(177.1 mg) was isolated on SiO2 and eluted with hexanes/EA.
Recrystallization from EA and hexane gave 154.0 g (51.3%) of 16a
as a yellow solid. Mp: 78.9–80.1 °C. SiO2 TLC Rf 0.55 (CH2Cl2/5%
CH3OH/0.1% NH4OH). 1H NMR (CDCl3): d 8.11 (s, 1H), 7.79 (d,
J = 2.0 Hz, 1H), 7.21 (d, J = 2.0 Hz, 1H), 4.61 (m, 1H), 4.57 (m, 2H),
1.64 (d, J = 6.8 Hz, 6H), 1.40 (d, J = 6.8 Hz, 6H). Anal. Calcd for
C
13H17N3O3Á1/6 H2O: C, 58.63; H, 6.56; N, 15.78. Found: C, 58.85;
H, 6.40; N, 15.94.
3.2. 6-Isopropoxy-1-isopropyl-1H-benzo[d]imidazol-4-amine
(17a)
6-Isopropoxy-1-isopropyl-4-nitro-1H-benzo[d]imidazole 16a
(110 mg, 0.42 mmol) was dissolved into 75 mL EtOH and added
to a Parr hydrogenation vessel previously charged with Pd/C (10%
w/w, 6.2 mg). After three vacuum/purge cycles with H2, the vessel
was charged to 50 psi with H2 and shaken for 5 h. The mixture was
filtered through a pad of Celite and then washed with an additional
25 mL of EtOH. The solvent was removed under reduced pressure.
95.2 mg (97.7%) of 17a as a pale brown, hygroscopic solid. Mp:
34.2–35.0 °C. SiO2 TLC Rf 0.33 (CH2Cl2/5% CH3OH/0.1% NH4OH).
The capacity of these compounds to uniquely raise or lower the
level of ERK5 phosphorylation might not be mediated directly at
the level of MEK5 phosphorylation of ERK5, but may arise from a
yet unidentified biological target. Current studies are underway

8058
P. T. Flaherty et al. / Bioorg. Med. Chem. 18 (2010) 8054–8060
1H NMR (CDCl3): d 7.75 (s, 1H), 6.27 (d, J = 2.0 Hz, 1H), 6.20 (d, J =
2.0 Hz, 1H), 4.47 (m, 1H), 4.05 (s, 2H, NH2, 2H), 1.57 (d, J = 6.8 Hz,
6H), 1.35 (d, J = 6.8 Hz, 6H). Anal. Calcd for C13H19N3OÁ1/4H2O: C,
65.66; H, 8.29; N, 17.67. Found: C, 65.73; H, 7.90; N, 17.62.
C14H21N3O2: C, 62.29; H, 6.59; N, 9.47. Found: C, 62.13; H, 6.57;
N, 9.36.
3.5. N-Benzyl-6-(2-ethoxyethoxy)-1-isopropyl-1H-benzo[d]imi-
dazol-4-amine oxalate (8)
3.3. N-Benzyl-6-isopropoxy-1-isopropyl-1H-benzo[d]imidazol-
4-amine (7)
6-(2-Ethoxyethoxy)-1-isopropyl-1H-benzo[d]imidazol-4-amine
17b (90.0 mg, 0.34 mmol) and solid NaBH(OAc)3 (217.3 mg,
6-Isopropoxy-1-isopropyl-1H-benzo[d]imidazol-4-amine 17a
1.0 mmol) were added to 2.5 mL of DCE. Benzaldehyde (53.6 lL,
(83.4 mg, 0.36 mmol) and solid NaBH(OAc)3 (227.38 mg, 1.07
mmol) were added to 2.5 mL of DCE. Benzaldehyde (43.8 lL,
0.5 mmol) was added in one portion and the mixture was stirred
for 12 h. The reaction mixture was quenched with the addition of
1 mL of NaHCO3 (aq, satd) and then stirred for an additional
10 min. The mixture was taken up into 50 mL of 5% K2CO3 (aq,
satd) and then extracted three times with 15 mL portions of
Et2O. The combined extracts were washed with NaCl (aq, satd)
and then dried over Na2SO4. The extract was decanted and the sol-
vent was removed under reduced pressure. The crude material was
isolated on SiO2 and eluted with hexanes/EA. The isolated product
(50.2 mg, 0.14 mmol) was dissolved in 2.5 mL of EA, followed by
addition of the solution of anhydrous oxalic acid (15.5 mg,
0.16 mmol) into 2.5 mL of EA. The solvent was removed under re-
duced pressure to afford 60.6 mg (40%) of 8 as a white crystalline
solid. Mp: 158.7–158.8 °C. SiO2 TLC Rf 0.57 (CH2Cl2/5% CH3OH/
0.43 mmol) was added in one portion and the mixture was stirred
for 12 h. The reaction mixture was quenched with the addition of
1 mL of NaHCO3 (aq, satd) and stirred for an additional 10 min.
The mixture was taken up into 25 mL of 5% K2CO3 (aq, satd) and
then extracted three times with 15 mL portions of Et2O. The com-
bined extracts were washed three times with 5 mL portions of NaCl
(aq, satd) and then dried over Na2SO4. The extract was decanted
and the solvent was removed under reduced pressure. The crude
material was isolated on SiO2 and eluted with hexanes/EA. This
material was recrystallized from EA and hexanes gave 75.7 mg
(65.5%) of 7 as a white crystalline solid. Mp: 101.6–101.9 °C. SiO2
TLC Rf 0.53 (CH2Cl2/5% CH3OH/0.1% NH4OH). 1H NMR (CDCl3): d
7.70 (s, 1H), 7.41 (d, J = 2.0 Hz, 2H), 7.31 (t, J = 2.0 Hz, 2H), 6.20
(d, J = 2.0 Hz, 1H), 6.05 (d, J = 2.0 Hz, 1H), 5.23 (m, 1H), 4.48 (s,
2H), 3.95 (m, 1H), 1.58 (d, J = 6.8 Hz, 6H), 1.31 (d, J = 6.0 Hz, 6H).
Anal. Calcd for C20H25N3O: C, 74.27; H, 7.79; N, 12.99. Found: C,
74.13; H, 7.71; N, 12.99.
0.1% NH4OH). 1H NMR (MeOD-d4):
d 8.87 (s, 1H), 7.41 (d,
J = 7.6 Hz, 2H), 7.33 (t, J = 7.2 Hz, 2H), 7.26 (t, J = 7.2 Hz, 1H), 6.56
(d, J = 2.0 Hz, 1H), 6.24 (d, J = 2.0 Hz, 1H), 4.80 (m, 1H), 4.46 (s,
2H), 4.11 (t, J = 4.4 Hz, 2H), 3.76 (t, J = 1.6 Hz, 2H), 3.56 (q, 2H),
1.63 (d, J = 6.8 Hz, 6H), 1.19 (t, J = 7.2 Hz, 3H). Anal. Calcd for
C29H23N3O6: C, 62.29; H, 6.59; N, 9.47. Found: C, 62.13; H, 6.57;
3.4. 6-(2-Ethoxyethoxy)-1-isopropyl-1H-benzo[d]imidazol-4-
N, 9.36.
amine (17b)
3.6. 1-Isopropyl-6-(3-methoxypropoxy)-1H-benzo[d]imidazol-
A dry one-necked flask was charged with 1-isopropyl-4-nitro-
1H-benzo[d]imidazol-6-ol 14 (334.0 mg, 1.5 mmol), 2-ethoxyetha-
nol 15b (0.52 mL, 5.4 mmol), Ph3P (985.0 g, 3.8 mmol), and 4 mL of
anhydrous DMF. This solution was cooled to 0 °C. Neat DIAD
(0.4 mL, 2.8 mmol) was added with drop-wise addition over
15 min. The solution was stirred for 30 min at 0 °C and then the
ice-bath was removed. The reaction was stirred for an additional
2 h at 23 °C. The reaction was then cooled to 0 °C and an additional
quantity of DIAD (0.4 mL, 2.8 mmol) was added at 0 °C in one por-
tion. The reaction was stirred for 30 min at 0 °C, then at 23 °C for
12 h. The reaction mixture was poured into 10 mL EA and 10 mL
K2CO3 (aq, satd), extracted three times with 10 mL portions of EA
and then the combined organic extracts were washed with NaCl
(aq, satd) and dried over Na2SO4. The extract was decanted and
the solvent was removed under reduced pressure to give 4.9 g of
crude 16b. Crude 16b was dissolved into 75 mL of EtOH and added
to a Parr hydrogenation vessel previously charged with Pd/C (10%
w/w, 120.2 mg). After three vacuum/purge cycles with H2, the ves-
sel was charged to 50 psi with H2 and shaken for 5 h on a Parr
hydrogenation apparatus. The mixture was filtered through a pad
of Celite and then washed with an additional 25 mL of EtOH. The
solvent was removed under reduced pressure. The crude oil was
poured into 50 mL of 1 N HCl (aq) (pH 1) and washed three times
with 10 mL portions of EA. The aqueous phase was neutralized and
then basified with 6 N NaOH (aq) to a pH of 10 and then extracted
three times with 15 mL portions of Et2O. The combined Et2O ex-
tracts were washed with NaCl (aq, satd) and dried over Na2SO4.
The solvent was decanted and then removed under reduced pres-
sure to give 119.0 mg (30%) of 17b as a pale brown, hygroscopic so-
lid was obtained. Mp: 37.4–38.0 °C. SiO2 TLC Rf 0.31 (CH2Cl2/5%
CH3OH/0.1% NH4OH). 1H NMR (CDCl3): d 7.74 (s, 1H), 6.31 (d,
J = 2.0 Hz, 1H), 6.23 (d, J = 2.0 Hz, 1H), 4.47 (m, 1H), 4.14 (t,
J = 5.2 Hz, 2H), 3.80 (t, J = 5.2 Hz, 2H), 3.62 (q, J = 6.8 Hz, 2H), 1.57
(d, J = 6.8 Hz, 6H), 1.25 (t, J = 7.2 Hz, 3H). Anal. Calcd for
4-amine (17c)
A dry one-necked flask was charged with 1-isopropyl-4-nitro-
1H-benzo[d]imidazol-6-ol 14 (334.0 mg, 1.5 mmol), 3-methoxy-
propan-1-ol 15c (0.52 mL, 5.4 mmol), Ph3P (985.0 g, 3.8 mmol),
and 4 mL of anhydrous DMF. This solution was cooled to 0 °C. Neat
DIAD (0.4 mL, 2.8 mmol) was added with drop-wise addition over
15 min. The solution was stirred for 30 min at 0 °C and then the
ice-bath was removed. The reaction was stirred for an additional
2 h at 23 °C. The reaction was then cooled to 0 °C and an additional
quantity of DIAD (0.4 mL, 2.8 mmol) was added at 0 °C in one por-
tion. The reaction was stirred for 30 min at 0 °C, then at 23 °C for
12 h. The reaction mixture was poured into 10 mL EA and 10 mL
K2CO3 (aq, satd), extracted three times with 10 mL portions of EA
and then the combined organic extracts were washed with NaCl
(aq, satd) and dried over Na2SO4. The extract was decanted and
the solvent was removed under reduced pressure to give 4.5 g of
crude 16c. Crude 16c was dissolved into 75 mL of EtOH and added
to a Parr hydrogenation vessel previously charged with Pd/C (10%
w/w, 120.1 mg). After three vacuum/purge cycles with H2, the ves-
sel was charged to 50 psi with H2 and shaken for 5 h on a Parr
hydrogenation apparatus. The mixture was filtered through a pad
of Celite and then the pad was washed with an additional 25 mL
of EtOH. The solvent was removed under reduced pressure. The
crude oil was poured into 50 mL of 1 N HCl (aq) (pH 1) and washed
(3 Â 10 mL) with EA. This was neutralized then basified with 6 N
NaOH (aq) to a pH 10 and then extracted (3 Â 15 mL) with Et2O.
The combined Et2O extracts were washed with 5 mL of NaCl (aq,
satd) and dried (Na2SO4). The solvent was decanted and then re-
moved under reduced pressure to give 159.0 mg (40%) of 17c as
a pale brown, hygroscopic solid. Mp: 43.1–44.7 °C. SiO2 TLC Rf
0.32 (CH2Cl2/5% CH3OH/0.1% NH4OH). 1H NMR (CDCl3): d 7.74 (s,
1H), 6.28 (d, J = 2.0 Hz, 1H), 6.19 (d, J = 2.0 Hz, 1H), 4.47 (m, 1H),
4.06 (t, J = 6.4 Hz, 2H), 3.57 (t, J = 6.4 Hz, 2H), 3.36 (s, 3H), 2.06

P. T. Flaherty et al. / Bioorg. Med. Chem. 18 (2010) 8054–8060
8059
(m, 2H), 1.57 (d, J = 6.8 Hz, 6H). Anal. Calcd for C14H21N3O2Á1/3H2O:
the addition of 1 mL of NaHCO3 (aq, satd) and then stirred for an
additional 10 min. The mixture was taken up into 50 mL of 5%
K2CO3 (aq, satd) and then extracted three times with 15 mL por-
tions of Et2O. The combined extracts were washed with NaCl (aq,
satd) and then dried over Na2SO4. The extract was decanted and
the solvent was removed under reduced pressure. The crude mate-
rial was isolated on SiO2 and eluted with hexanes/EA. Recrystalli-
zation from EA and hexane gave 60.0 mg (46.8%) of 16 as a white
solid. Mp: 186.0–186.7 °C. SiO2 TLC Rf 0.41 (CH2Cl2/5% CH3OH/
0.1% NH4OH). 1H NMR (CDCl3): d 7.70 (s, 1H), 7.40 (d, J = 7.6 Hz,
2H), 7.32 (t, J = 7.2 Hz, 2H), 7.29 (d, J = 7.2 Hz, 1H), 6.20 (d,
J = 2.0 Hz, 1H), 6.04 (d, J = 2.0 Hz, 1H), 5.23 (m, 1H), 4.06 (m, 2H),
4.04 (t, J = 6.4 Hz, 2H), 3.67 (m, 4H), 3.34 (m, 2H), 2.46 (m, 2H),
2.03 (m, 4), 1.57 (d, J = 6.8 Hz, 6H). Anal. Calcd for C14H21N3O2Á
2H2O: C, 55.40; H, 7.40; N, 10.83. Found: C, 55.10; H, 7.13; N, 11.20.
C, 62.43; H, 8.11; N, 15.60. Found: C, 62.64; H, 8.15; N, 15.38.
3.7. N-Benzyl-1-isopropyl-6-(3-methoxypropoxy)-1H-
benzo[d]imidazol-4-amine (9)
1-Isopropyl-6-(3-methoxypropoxy)-1H-benzo[d]imidazol-4-
amine 17c (120.0 mg, 0.46 mmol) and solid NaBH(OAc)3 (289.8 mg,
1.4 mmol) were added to 2.5 mL of DCE. Benzaldehyde (53.6 lL,
0.5 mmol) was added in one portion and the mixture was stirred
for 12 h. The reaction mixture was quenched with the addition of
1 mL of NaHCO3 (aq, satd) and then stirred for an additional
10 min. The mixture was taken up into 5% K2CO3 (aq, satd) and then
extracted three times with 15 mL portions of Et2O. The combined
extracts were washed with NaCl (aq, satd) and then dried over
Na2SO4. The extract was decanted and the solvent was removed un-
der reduced pressure. The crude material was isolated on SiO2 and
eluted with hexanes/EA. Recrystallization from EA and hexane gave
101.4 mg (63%) of 9 as a white crystalline solid. Mp: 75.7–75.9 °C.
SiO2 TLC Rf 0.58 (CH2Cl2/5% CH3OH/0.1% NH4OH). 1H NMR (CDCl3):
d 7.70 (s, 1H), 7.40 (d, J = 7.6 Hz, 2H), 7.32 (t, J = 7.2 Hz, 2H), 7.29
(d, J = 7.2 Hz, 1H), 6.20 (d, J = 2.0 Hz, 1H), 6.04 (d, J = 2.0 Hz, 1H),
5.23 (m, 1H), 4.46 (m, 2H), 4.04 (t, J = 6.4 Hz, 2H), 3.55 (t,
J = 6.0 Hz, 2H), 3.34 (s, 3H), 2.03 (m, 2H), 1.57 (d, J = 6.8 Hz, 6H). Anal.
Calcd for C21H27N3O2: C, 71.36; H, 7.70; N, 11.89. Found: C, 71.22; H,
7.71; N, 11.89.
3.9. EGF-induced phosphorylation of ERK1/2 and ERK5
Western blot analysis. Equal amounts of protein (60 lg) from
each treatment were separated on 8% SDS gels and transferred to
PVDF membrane [Millipore, Danvers, MA] for Western blot analy-
sis. Blots were blocked in 5% non-fat milk in 1ÂTBS/0.1%Tween/
0.02% NaN3 for 1 h and then incubated overnight at 4 °C in primary
antibody (1:1000 pERK1/2, 1:500 pERK5, Cell Signaling). Blots
were washed with 1ÂTBS/0.1%Tween for 30 min and then incu-
bated with secondary antibody (1:1000, horseradish peroxidase-
conjugated goat anti-rabbit, Upstate). Proteins were visualized
with enhanced chemiluminescence (Upstate). Films were scanned
and quantified using MATLAB, v.7.1 (Mathworks, Natick, MA).
3.8. N-Benzyl-1-isopropyl-6-(3-morpholinopropoxy)-1H-
benzo[d]imidazol-4-amine hydrochloride (16)
A dry one-necked flask was charged with 1-isopropyl-4-nitro-
1H-benzo[d]imidazol-6-ol 14 (249.8 mg, 1.1 mmol), 3-morpholi-
nopropan-1-ol 15d (0.45 mL, 3.3 mmol), Ph3P (737.2 g, 2.75 mmol),
and 2 mL of anhydrous DMF. This solution was cooled to 0 °C. Neat
DIAD (0.32 mL, 1.65 mmol) was added with drop-wise addition
over 15 min. The solution was stirred for 30 min at 0 °C and then
the ice-bath was removed. The reaction was stirred for an addi-
tional 2 h at 23 °C. The reaction was then cooled to 0 °C and an
additional quantity of DIAD (0.22 mL, 1.1 mmol) was added at
0 °C in one portion. The reaction was stirred for 30 min at 0 °C, then
at 23 °C for 12 h. The reaction mixture was poured into 10 mL EA
and 10 mL K2CO3 (aq, satd), extracted three times with 10 mL por-
tions of EA and the combined organic extracts were washed with
NaCl (aq, satd) and dried over Na2SO4. The extract was decanted
and the solvent was removed under reduced pressure to give crude
16d which was dissolved into 75 mL of EtOH and added to a Parr
hydrogenation vessel previously charged with Pd/C (10% w/w).
After three vacuum/purge cycles with H2, the vessel was charged
to 50 psi with H2 and shaken for 5 h on a Parr hydrogenation appa-
ratus. The mixture was filtered through a pad of Celite and washed
with an additional 25 mL of EtOH. The solvent was removed under
reduced pressure. The crude oil was poured into 50 mL of 1 N HCl
(aq) (pH 1) and washed three times with 10 mL portions of EA. The
aqueous phase was neutralized and then basified with 6 N NaOH
(aq) to a pH of 10 and then extracted three times with 15 mL por-
tions of Et2O. The combined Et2O extracts were washed with NaCl
(aq, satd) and dried over Na2SO4. The solvent was decanted and
then removed under reduced pressure to give 100 mg of 17d as a
crude product. 1H NMR (CDCl3): d 7.29 (d, J = 7.2 Hz, 1H), 6.20 (d,
J = 2.0 Hz, 1H), 6.04 (d, J = 2.0 Hz, 1H), 5.23 (m, 1H), 4.06 (m, 2H),
4.04 (t, J = 6.4 Hz, 2H), 3.67 (m, 4H), 3.34 (m, 2H), 2.46 (m, 2H),
2.03 (m, 4H), 1.57 (d, J = 6.8 Hz, 6H).
3.10. High-throughput assay of compound 9
This compound was submitted for analysis for solid-support
bound ATP analog displacement according to previously described
protocols. Full results are disclosed in Supplementary data.
NCI 60-cell-line screens were conducted according to their rou-
tine procedure. Results of single dose treatment are presented in
Supplementary data.
Acknowledgment
Tim Holtzer for acquisition and analysis of High Resolution
Mass Spectral analysis, and NIH (NINDS): 1R15NS057772, NSF
Research Experiences for Undergraduates (REU): CHE-0354052,
Duquesne University for start-up funds, and an NSF equipment
grant for the NMR (NMR: CHE 0614785).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.09.017.
References and notes
1. Bogoyevitch, M. A.; Fairlie, D. P. Drug Discov. Today 2007, 12, 622.
2. Chen, Z. et al Chem. Rev. 2001, 101, 2449.
3. Sebolt-Leo pold, J. S.; Herrera, R. Nat. Rev. Cancer 2004, 4, 937.
4. Sebolt-Leopold, J. S.; Herrera, R.; Ohren, J. F. Recent Results Cancer Res. 2007, 172,
155.
5. Nishimoto, S.; Nishida, E. EMBO Rep. 2006, 7, 782.
6. Kamakura, S.; Moriguchi, T.; Nishida, E. J. Biol. Chem. 1999, 274, 26563.
7. English, J. M.; Cobb, M. H. Trends Pharmacol. Sci. 2002, 23, 40.
8. Sticht, C. et al Neoplasia (Ann Arbor, MI, USA) 2008, 10, 462.
9. Dudderidge, T. J. et al Br. J. Cancer 2007, 96, 1384.
10. Montero, J. C. et al PLoS One 2009, 4.
11. Tatake, R. J. et al Biochem. Biophys. Res. Commun. 2008, 377, 120.
12. Flaherty, P. T. et al Bioorg. Med. Chem. Lett. 2010, 20, 2892.
13. Flaherty, P. T., et al., Bioorg. Med. Chem., submitted for publication.
14. Flaherty, P. T., et al., Abstracts of Papers, 236th ACS National Meeting,
Philadelphia, PA, United States, Aug 17–21, 2008, p COMP-378.
Crude 17d (100.0 mg, 0.31 mmol) was dissolved into 2.5 mL of
DCE. NaBH(OAc)3 (199.5 mg, 0.93 mmol) and benzaldehyde
(31.0
lL, 0.31 mmol) were added in one portion and the mixture
was stirred for 12 h. The reaction mixture was quenched with

8060
P. T. Flaherty et al. / Bioorg. Med. Chem. 18 (2010) 8054–8060
15. Jain, P., et al., Abstract of Papers, 238th ACS National Meeting, Washington, DC,
United States, Aug 16–20, 2009, p MEDI-365.
16. Jain, P., et al., Abstracts of Papers, 236th ACS National Meeting, Philadelphia, PA,
United States, Aug 17–21, 2008, p MEDI-061.
23. Fabian, M. A. et al Nat. Biotechnol. 2005, 23, 329.
24. Karaman, M. W. et al Nat. Biotechnol. 2008, 26, 127.
25. Lockhart, D. J.; Zarrinkar, P. P.; Treiver, D. K. U.S. Patent US19943 2004113556,
2004.
17. Albert, D. H. et al Mol. Cancer Ther. 2006, 5, 995.
18. Chu, X.-J. et al J. Med. Chem. 2006, 49, 6549.
19. Dai, Y. et al J. Med. Chem. 2007, 50, 1584.
26. Shoemaker, R. H. Nat. Rev. Cancer 2006, 6, 813.
27. Bazhulina, N. P. et al J. Biomol. Struct. Dyn. 2009, 26, 701.
28. VanScyoc, W. S. et al Biochemistry 2008, 47, 5017.
29. Simard, J. R. et al J. Am. Chem. Soc. 2009, 131, 13286.
20. Jasinghe, V. J. et al J. Hepatol. 2008, 49, 985.
21. Sun, L. et al J. Med. Chem. 1999, 42, 5120.
22. Moore, M. D., et al., Acta Crystallogr., Sect. E: Struct. Rep. Online 2008, E64: pp
o1336.