Design, synthesis, and testing of an 6-O-linked series of benzimidazole based inhibitors of CDK5/p25

Article abstract of DOI:10.1016/j.bmc.2010.11.022

Alzheimer's disease (AD) is a progressive neurodegenerative disease resulting in cognitive and behavioral impairment. The two classic pathological hallmarks of AD include extraneuronal deposition of amyloid β (Aβ) and intraneuronal formation of neurofibri

Full text of DOI:10.1016/j.bmc.2010.11.022

Bioorganic & Medicinal Chemistry 19 (2011) 359–373
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and testing of an 6-O-linked series of benzimidazole
based inhibitors of CDK5/p25
Prashi Jain a, Patrick T. Flaherty a, , Shuyan Yi a, Ishveen Chopra a, Gwenyth Bleasdell b, Josh Lipay b,
⇑
Yoan Ferandin c, Laurent Meijer c, Jeffry D. Madura b
a Department of Medicinal Chemistry, Mylan School of Pharmacy, Duquesne University, 600 Forbes Hall, Pittsburgh, PA 15282, USA
b Bayer School of Natural and Environmental Sciences, Department of Chemistry, Duquesne University, 600 Forbes Hall, Pittsburgh, PA 15282, USA
c CNRS, Station Biologique, 29680 Roscoff, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Alzheimer’s disease (AD) is a progressive neurodegenerative disease resulting in cognitive and behavioral
impairment. The two classic pathological hallmarks of AD include extraneuronal deposition of amyloid b
(Ab) and intraneuronal formation of neurofibrillary tangles (NFTs). NFTs contain hyperphosphorylated
tau. Tau is the major microtubule-associated protein in neurons and stabilizes microtubules (MTs). Cyclin
dependent kinase 5 (CDK5), when activated by the regulatory binding protein p25, phosphorylates tau at
a number of proline-directed serine/threonine residues, resulting in formation of phosphorylated tau as
paired helical filaments (PHFs) then in subsequent deposition of PHFs as NFTs. Beginning with the struc-
ture of Roscovitine, a moderately selective CDK5 inhibitor, we sought to conduct structural modifications
to increase inhibitory potency of CDK5 and increase selectivity over a similar enzyme, cyclin dependent
kinase 2 (CDK2). The design, synthesis, and testing of a series of 1-isopropyl-4-aminobenzyl-6-ether-
linked benzimidazoles is presented.
Received 14 September 2010
Revised 1 November 2010
Accepted 6 November 2010
Available online 6 December 2010
Keywords:
CDK5
Cyclin dependent kinase
Benzimidazole
1-Isopropyl benzimidazole
Ó 2010 Elsevier Ltd. All rights reserved.
1. Background
b-secretase (BACE)8,9,13–16 and -secretase14,15,17–22 represent major
c
current targets for potential therapeutic intervention. These hold
the hope of reducing the biosynthesis of Ab 42, preventing the
deposition of Ab plaques, and reducing associated toxicities. NFTs
are intraneuronal depositions of insoluble proteins. The major
component of these NFTs is the structural protein tau. Tau can be
phosphorylated at multiple residues, but phosphorylation of
Ser202 and/or Thr205 is the phospho-tau form that exists in NFT’s.
This hyperphosphorylated tau form is the major component of
neurofibrillary tangles and is present in all confirmed cases of
Alzheimer’s disease. The origin this hyperphosphorylated tau is
still a complex issue but a coherent theory is emerging.23–27
The current understanding of tau hyperphosphorylation is that
the following residues are phosphorylated in PHF tau: Thr181,
Ser199, Thr205, Thr121, Thr217, and Thr231. These phosphoryla-
tions can be accomplished by sequential action of PKA, CaMKII
and then GSK-3b, or by CDK5 and then GSK-3b, or by CDK5 alone.
Suggestions have been made to target multiple kinases, and several
groups are currently pursuing these strategies. Additionally, there
have been reports that initial priming of tau by CDK5 facilitates
additional phosphorylation by GSK-3b.28,29 CDK5 has also been
implicated in phosphorylation of p53, activation of the ERK signaling
pathway, MEF-2 activation, and peroxiredoxin30–33 up-regulation.
These later two results are particularly interesting as they could
provide a link to other neurodegenerative diseases with aberrant
tau phosphorylation. Additionally it appears that CDK5 mediated
Alzheimer’s disease is a progressive neurodegenerative disease
without a cure. It is estimated that 4.5 million Americans have
Alzheimer’s disease.1 This is twice as many as in 1980, but less than
the estimated 11 million persons whom are estimated to be af-
flicted by 2050. This increase in number represents a shift in age
demographics where increased life-expectancy increases the num-
ber of persons afflicted with neurodegenerative diseases. A parallel
increase in the cost of caring for persons with Alzheimer’s disease
is also anticipated. The average lifetime cost for an individual with
Alzheimer’s disease is currently $175,0002 and these costs will
only increase.
The pathological hallmarks for Alzheimer’s disease have been
known for some time.3–8 Post-mortem examination of persons
with Alzheimer’s disease exhibit two types of aberrant proteins:
amyloid b 42 (Ab 42 and neurofibrillary tangles (NFT). The extra
neuronal precipitation of Ab was initially recognized by the
birefringence induced by the amyloid-detecting histological stain
congo red. Currently inhibiting the generation and deposition of
Ab represents a very hopeful target for therapy of Alzheimer’s
disease.9–12 Specifically, inhibition of the processing enzymes
⇑
Corresponding author.
E-mail address: flahertyp@duq.edu (P.T. Flaherty).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.022

360
P. Jain et al. / Bioorg. Med. Chem. 19 (2011) 359–373
tau hyperphosphoryaltion may induce an increase of sAPP produc-
tion and perhaps Ab production.
the tissue distribution is mostly localized to the brain and the testis
(in rats).48–51 Second, CDK5 does not require phosphorylation of
the ‘T loop’ (specifically Ser159) to generate an active enzymatic
complex.48 In fact it has been shown that phosphorylation of this
residue prevents effector cyclins from binding. In the case of
CDK5, hydrophobic and ionic interactions assemble the cyclin/
CDK complex. CDK5 binds to and is activated by cyclin D, a mem-
brane bound protein p35, or by a 208 residue C-terminal proteo-
lytic product of p35:p25. The complex CDK5/p25 has consistently
been shown to hyperphosphorylate tau protein and produce cell
death.52–55 Administration of CIP, a 125-residue portion of p35,
was shown to selectively inhibit CDK5, reduce tau phosphoryla-
tion, and protect against Ab induced apoptosis. Tau replacement
strategies with the tubulin stabilizer paclitaxel also reduced Ab in-
duced apoptotic cell death mediated by tau hyperphosphoryla-
tion.7,56,57 This raises the possibility that CDK5 mediated tau
hyperphosphorylation causes both NFT formation and the loss of
cytoskeletal integrity that facilitates Fb mediated apoptotic cell
death. With the capability to clone and express CDK5 it was found
that CDK5/p25 but not the CDK5/p35 complex35,53–55,58–60 is capa-
ble of hyperphosphorylating tau, specifically at the Ser202 and
Thr205 residues.5
There are several X-ray crystal structures of CDK5 published
and deposited into the PDB. The first X-ray crystal structure was
obtained and deposited by Tarricone.48 This confirmed the phos-
phorylation state of Ser159 and established the nature of the
CDK/p25 interaction. The remaining structures by Mappelli47 are
highly similar with respect to the protein domains, but have differ-
ent inhibitors diffused into the ATP binding pocket. These com-
pounds, (R) roscovitine 1, aloisine A 2, and indirubin 30-oxime 3,
were obtained from other CDK SAR studies and inhibit other CDKs
and other kinases as well. R-roscovitine (Figs. 1 and 2) was
There are increasing indications that tau can be hyperphospho-
rylated by CDK5/p25 selectively5 in response to excitotoxic
events34 including Ab,35 and that increased soluble amyloid pre-
cursor protein (APP) synthesis (the peptide inappropriately direc-
ted toward Ab 42 in pathologic states) may be a response to
these events. Thus the development of selective CDK5/p25 inhibi-
tors would contribute significantly to an understanding of the role
of tau hyperphosphorylation in Alzheimer’s disease and could
provide early proof of concept for the therapeutic strategy of inhib-
iting CDK5/p25 to effect a disease-modifying treatment for neuro-
degenerative disease including Altzheimer’s disease.
Cyclin-dependent kinases (CDKs) have been known for some
time and are the focus of several drug-development programs.36–43
The most actively researched CDK is CDK2.40,44,45 CDK2 has been
examined in the context of anti-cancer agents. All CDK’s have several
layers of intrinsic inhibition and require multiple interactions for
activation. A PSTAIRE helix is found in the primary sequence of all
CDKs and is inserted between two beta-sheets domains of the
CDK. Displacement of this helix exposes the ATP binding site.46–48
Displacement of this helix is effected by the obligate cyclin; cyclin
binding repositions the PSTAIRE helix to an interface between the
cyclin and the CDK permitting both ATP binding and then substrate
binding. In many cases there may be several cyclins capable of
activating a given CDK. Different cyclins may confer different sub-
strate specificities upon the CDKs. This implies a different induced
active site unique to each dimeric complex. Additional phosphoryla-
tion of a Thr residue of an extended ‘T loop’ may also be required.
CDK5 has a PSSALRE sequence for the ‘PSTAIRE’ motif,48 this
provided the basis for its initial identification as a CDK. CDK5 is dis-
tinct in several ways from other members of the CDK family. First
ILE10, PHE82, ASP84
GLY85, ASP86, LYS 89
H2N
O
SH
CYS83
°
O
2.81 A
N
H
N
GLN130
H
H
N
O
°
3.25 A
6
N
N
H
O
N
N 2 N
H
°
9
2.83 A
VAL18, LYS33, PHE80,
VAL64, ALA143
Figure 1. Interactions between roscovitine 1 and CDK547 (from PDB: 1UNL).
NH
N
NH
O
HO
N
N
N
N
N
HO
NH
OH
N
H
NH
N
3
1
2
R (-) roscovitine
aloisine A
indirubin-3’-oxime
Figure 2. Known inhibitors that have been co-crystallized in CDK5/p25.47

P. Jain et al. / Bioorg. Med. Chem. 19 (2011) 359–373
361
originally developed by analog synthesis on the adenine core for
CDK2 inhibition39,47 and was found to bind deeply into the ATP
binding site of CDK5 and inhibited CDK5/p25 with an IC50 of
160 nM. Further kinetic experiments with several CDK5/p25 inhib-
itors identified different modes of enzyme inhibition46 for com-
pounds 2 and 3.
Several research groups are currently working on CDK5 inhibi-
tors.61,62 The sources for many of these compounds are prior pro-
jects exploring ATP-binding kinases. The following centroids have
been used for the development of CDK5 inhibitors: pyrrolopyrim-
idines, benzthiazoles, indazoles, aminoimidazoles, aminothiazoles,
and paullinones.42 Many of these compounds were prepared in the
context of CDK2 inhibitors and were likely identified via high-
throughput screens detecting CDK5 inhibition. We sought to sys-
tematically examine the benzimidazole scaffold, a known adenine
mimic, but one that is underutilized both in medicinal chemis-
try63–66 and in CDK inhibition67,68 specifically.
substitution has been extensively employed in the exploration of
receptor tyrosine kinases.69,70 The polar and non-polar ribose bind-
ing pockets can be further explored with various substitutions
from the C6 position of the benzimidazole core.
The molecular modeling was conducted in a manner parallel to
prior studies.71,72 The PDB file for 1UNL was uploaded, protons and
valencies were examined. The structure of roscovitine was both di-
rectly modified to the benzimidazole core or was computer docked
with the automated docking protocol MOE-dock.73 Amber 98 was
used as the forcefield for protein calculations, MMFF94 was used
as the forcefield for small molecule calculations. Complementary
results were obtained for both docking strategies. Two strategies
were utilized for analysis of the inhibitor–protein complex. Scoring
using a principle-components method,71,72 or using the standard
MOE evaluation that generated a calculated pKi, with the MMFF94
forcefield using triangulation for the final fit. Selected compounds
are presented in Table 1, and the values for this second scoring
function strategy are presented in Table 2.
The proposed compounds retained the isopropyl group found at
the 9-N atom of roscovitine but are designated 1-N on the benz-
imidazole core based CDK5 inhibitors. The ribose/triphosphate
binding pocket for CDK5 is the first area where we would like to
explore structural variations. As such, compounds 4–8 examined
point variations on the alkoxy chain resulting from movement of
the ethyl chain, deletion of the ethyl chain, or capping the hydroxyl
group with a methoxy group. Compounds 9–11 examine addition
of an ammonium group in the proximity of Asp86. This is prece-
dented by the work of Toogood.74–76 He was able to optimize
inhibitor activity at CDK4 by reaching across from the purine-binding
region to the magnesium ion binding site on the cusp of the
ribose binding pocket. Additionally this strategy anchored the
hydrogen-bonding motif that mimics the purine core with a bulky
aliphatic group (cyclopentyl); this is analogous to our incorpora-
tion of the 1-N isopropyl group. Compounds 12–15 explore the
possibility of including an alkyl or alkoxy side chain to access a
hydrophobic binding region proximal to the roscovitine binding
region and uniquely utilized by the butyl chain of aloisine A as
identified in its CDK5 cocrystal structure.47
2. Molecular modeling
(R)-Roscovitine is a moderately active inhibitor of CDK5 (IC50
160 nM). Although it was first designed and developed as a CDK2A
inhibitor (IC50 700 nM40), roscovitine has displays preferential
inhibition for CDK5/p25 over other CDKs.38,40 Additionally com-
pounds 2 and 3, although they have been co-crystalized with
CDK5,47 yet exhibit preferential inhibition of CDK2. Our goal was
to explore the utility of the benzimidazole core as a purine mimic
using molecular modeling and analysis of the published crystal
structure of (R)-roscovitine-CDK5/p25 (PDB ID: 1UNL) (Fig. 1)47
as a starting point. Based on 1UNL, we proposed that the most
significant bonding pattern between CDK5 and roscovitine is from
the purine N7 (H-bond acceptor) and the exocylic NH at C6 of ros-
covitine (H-bond donor) to the amide NH and the backbone amide
carbonyl of Cys83, respectively. Purine nitrogens N1, N3 and the
exocyclic NH at C2 are not within hydrogen bonding distance of
any polar functionality on CDK5. In addition, the floor of the
purine-binding region is derived from Leu133 and the top of the
purine-binding region is derived from Ile10. This suggests that
replacement of the purine of roscovitine with a benzimidazole
and the exocylic 2-NH at C2 on the purine with an ether linkage
(Fig. 3) would not only retain the key H-bonding patterns but also
add hydrophobic interaction with Ile10 and Leu133. This strategy
could increase selectivity as the exocylic NH at C2 of roscovitine
forms an important H-bond with Glu131 of CDK2. This sort of ether
3. Chemical discussion
To prepare the desired functionalization at the 1, 4, and 6 posi-
tions on the benzimidazole core (Table 1), synthesis began with the
previously described 1-isopropyl-4-nitro-6-methoxybenzimidazole
ILE10, PHE82, ASP84
GLY85, ASP86, LYS 89
H2N
O
SH
CYS83
°
3.16 A
O
N
O
H
N
GLN130
H
°
H
N
3.81 A
N
N
H
O
O
°
2.69 A
VAL18, LYS33, PHE80,
VAL64, ALA143
Figure 3. Interactions between benzimidazole analog 4 and CDK5 (derived from PDB: 1UNL47).

362
P. Jain et al. / Bioorg. Med. Chem. 19 (2011) 359–373
Table 1
Proposed compounds
of acetate versus formate in the subsequent Philips cyclization
was examined in parallel experiments: (1) reductive alkylation
with sodium triformylborohydride then cyclization with a formic
acid HCl mixture, or (2) reductive alkylation with sodium triacet-
oxyborohydride then cyclization with an acetic acid and HCl
mixture, or finally (3) reductive alkylation with sodium triacetoxy-
borohydride then cyclization with a formic acid HCl mixture. From
these studies it was found that the relative rate of cyclization with
acetic acid was approximately 100 times slower versus formic acid.
No 2-methylbenzamidazole product was found in the collected
product when the sequence of sodium triacetoxyborohydride
mediated reductive alkylation followed by formic acid HCl Philips
NH
4
N
R
N
O
6
1
HO
HO
4
5
cyclization was employed.
A second improvement was the
utilization of BHT. Initial Philips cyclization on dimine 17 required
careful exclusion of oxygen to reduce oxidative polymerization,
but use of catalytic BHT dramatically increased yield, increased
purity, and stopped the presumably radical mediated oxidative
polymerization.
Hydrobromic acid catalyzed deprotection of the methyl phenyl
ether 18 afforded the corresponding phenol 19 in 93% yield under
microwave conditions. Other deprotective systems were examined
including conventional heating with HBr, BBr3, or BBr3 with ani-
sole. These conditions were all lower yielding.
6
7
HO
O
O
8
N
9
Alkylation of the 6-phenol of 19 was achieved via standard
Mitsunobu coupling conditions as shown in Schemes 2–5 or by
abstraction of the phenolic proton of 19 using sodium hydride in
DMF followed by addition of the required halide as shown in
Scheme 6. Selection of the alkylation method employed was deter-
mined by the commercial availability of the required electrophile.
Compound 18 was used directly to generate the methoxy com-
pound 15 as previously described.78 When Mitsunobu reaction
conditions were employed, it was found to be necessary to add
the DIAD in two portions to prolong the length of time that the
electrophile was available for reaction. In general the compounds
prepared via simple alkylation could be characterized as the 4-nitro
ether analogs, but the compounds prepared via the Mitsunobu
route typically co-eluted with the side product triphenylphosphine
oxide and it was found expedient to fully characterize the subse-
quent product after reduction. This conversion was efficiently
achieved with catalytic palladium on carbon under 50 psi of H2
with shaking to give amino derivatives 22a–k. Mono-alkylation
on the 4-amine was achieved using a single equivalent of benzal-
dehyde with sodium triacetoxyborohydride in dichloroethane.
Use of an acid catalyst or use of a more aggressive hydride source
resulted in dialkylation. This sequence of 6-O alkylation, 4-nitro
reduction, and finally 4-alkylation represents the optimal sequence
of reaction events. All possible variations were examined including
temporary protection of the 4-benzylamine. A requirement for
the 4-nitro group for a successful Mitsunbou alkylation on the
6-hydroxy of 19 can be explained as arising from the electron-
withdrawing nature of the nitro group on the normally electron
rich benzimidazole system and the consequent lowering of pKa of
the 6-hydroxyl group of 19. More acidic phenols are known to be
better substrates for the Mitsunobu reaction as this facilitates attack
on the transient electrophile. Although the TBS group was found
expedient to generate 4 as shown in Scheme 2, this strategy was
not successful in preparing compounds 5 and 6. Use of 20b to pre-
pare compound 5 was successful and permitted partial character-
ization of the intermediate ethers. Transient protection of the
more accessible 1-hydroxyl group of 1,2-butane diol, benzoylation
of its secondary 2-hydroxyl group, and finally deprotection
gave 20c79 and permitted synthesis of 6,79 a known compound.
Hydrolysis of the benzoate esters 23a and 23b under standard
conditions gave 5 and 6 as shown in Scheme 3. Scheme 4 presents
the synthesis of compounds 7–9. These compounds do not have
10
HN
N
11
12
13
14
15
Table 2
Scoring of proposed compounds
Compound
Calcd pKi Proposed side chain interactions
4
5
6
7
8
9
10
11
12
13
14
15
7.783
7.690
7.624
7.892
7.385
9.436
9.422
9.753
7.839
7.310
7.166
7.065
H-bonding with Gln130 and Asp144
Gln130
H-bonding with Asp144
Hydrophobic interactions with Gln130 backbone
Hydrophobic interactions with glycine-rich loop
Ionic interaction with Asp144
Ionic interaction with Asp144
Ionic interaction with Asp144
Hydrophobic interactions with glycine-rich loop
Hydrophobic interactions with glycine-rich loop
Hydrophobic interactions with Gln130 backbone
Hydrophobic interactions with Gln130 backbone
H-bonding with Gln130 amide
1 roscovitine 7.971
18.77 The key feature in the preparation of this compound was
regioselective alkylation on the 1-amine of 16 prior to cyclization.
This was affected by reductive alkylation with acetone on the more
nucleophilic amine which appears meta to the electron-withdrawing
nitro group. The initial synthesis77 utilized sodium triformyl-
borohydride generated in situ from sodium borohydride and
formic acid to effect the reductive alkylation with acetone then
crude 17 was cyclized to the corresponding benzimidazole 18. Sub-
sequently it was found that the more traditional reductive alkyl-
ation reagent sodium triacetoxyborohydride could be employed.
This improved procedure shown in Scheme 1 is presented in the
Section 6. The advantages of this second method are the commer-
cial availability and improved stability of sodium triacetoxyboro-
hydride. Sodium triformylborohydride was stable as
solution in THF at 0 °C, but not at 23 °C. The possible competition
a 1 M

P. Jain et al. / Bioorg. Med. Chem. 19 (2011) 359–373
363
NO2
NO2
NO2
NO2
NH2
ii
i
NH2
NH2
N
iii
N
N
O
NH
O
N
O
HO
16
17
18
19
Scheme 1. Synthesis of 1-isopropyl-4-nitro-6-hydroxybenzimidazole 19. Reagents and conditions: (i) Na(AcO)3BH, acetone, formic acid, THF, 23 °C, 12 h; (ii) formic acid,
concd HCl, BHT, 100 °C, 15 min; (iii) 48% HBr, 120 °C, 2.5 h, microwave.
O
TBS
OH
NH2
NO2
NO2
NH
20a
i
N
N
N
N
N
iv
ii, iii
N
N
HO
O
HO
N
O
HO
TBS
O
O
22a
21a
4
19
Scheme 2. Synthesis of 4 via Mitsunobu coupling with a TBS-protective group strategy. Reagents and conditions: (i) DIAD, PPh3, DMF, 23 °C, 12 h; (ii) Pd/C, 50 psi H2, EtOH,
23 °C, 5 h; (iii) HCl, EtOH; (iv) Na(AcO)3BH, DCE, benzaldehyde, 23 °C, 12 h.
O
OH
O
20b
O
OH
O
NH2
20c
NO2
NH
NH
NO2
N
N
iv
N
N
iii
N
N
N
N
N
N
i
ii
R
R
R
R
O
HO
O
O
O
19
22b R=
22c R=
O
O
23a
21b
21c
HO
R=
R=
R=
R=
O
O
O
O
O
O
5 R=
6 R=
O
O
O
O
O
23b
HO
O
O
O
O
O
O
O
Scheme 3. Synthesis of 6-ether derivatives via Mitsunobu coupling with benzoate-protective group strategy. Reagents and conditions: (i) DIAD, PPh3, DMF, 23 °C, 12 h; (ii)
Pd/C, 50 psi H2, EtOH, 23 °C, 5 h; (iii) Na(AcO)3BH, DCE, benzaldehyde, 23 °C, 12 h; (iv) 5% KOH/MeOH, 23 °C, 12 h.
exchangeable protons and were prepared without protective group
manipulation.
Isolation of the expressed protein utilized a glutathione-agarose
affinity column chromatography and kinase activity was assayed
in 25 mM Mops Buffer (pH 7.2) with 60 mM b-glycerophosphate,
5 mM EDTA, 15 mM MgCl2, and 1 mM DTT. The protein histone
Piperidine compounds 10 and 11 were prepared as shown in
Scheme 5. Compound 10 was prepared from BOC-protected
precursor 20g with acid-catalyzed deprotection in the final step.
Attempts to synthesize compound 11 via the intended Mitsunobu
coupling partner 20f were not synthetically tractable. However,
compound 21h could be prepared conveniently from the protected
precursor 21g by acid-catalyzed BOC deprotection followed by
Eschweiler–Clarke methylation. Reduction of 21h then reductive
alkylation gave 11.
H1 (Sigma; 1 mg/mL) was used as the substrate with 15
-33P] ATP (3000 Ci/mmol; 1 mCi/mL) to a final volume of
30 L. After a 10 min incubation period at 30 °C, 25 L aliquots of
lm
[c
l
l
l
supernatant were spotted onto 2.5 ꢀ 3 cm pieces of Whatman
P81 phosphocellulose paper. After 20 s the filter paper was washed
five times (for at least 5 min each time) with a solution of 10 mL
phosphoric acid/liter of H2O. The activity of the wet filter papers
was counted in the presence of 1 mL ACS (Amersham) scintillation
fluid to determine the amount of substrate phosphorylation. IC50
values were determined with probit analysis and experimentally
determined values which were inconsistent with symmetrical
binding curves were assigned semi-quantitative values. Of the
compounds assayed (Table 3), only one compound, 4, was deemed
4. Biological assay
The compounds were assayed using an established protocol39
with mammalian CDK5 and p25 cloned and stably co-expressed
and isolated as glutathione-S-transferase (GST) fusion proteins.

364
P. Jain et al. / Bioorg. Med. Chem. 19 (2011) 359–373
NH2
NO2
NO2
NH
N
N
N
N
iii
i
ii
N
N
R
R
N
N
HO
O
R
O
O
19
O
O
O
O
21d
R=
O
O
20d
R=
O
O
O
O
OH
OH
22d
R=
O
O
O
O
7
R=
21e
21f
R=
R=
20e
20f
R=
R=
22e
22f
R=
R=
8
9
R=
N
N
N
O
N
OH
R=
O
O
Scheme 4. Direct synthesis of 6-ether derivatives via Mitsunobu coupling. Reagents and conditions: (i) DIAD, PPh3, DMF, 23 °C, 12 h; (ii) Pd/C, 50 psi H2, EtOH, 23 °C, 5 h; (iii)
Na(AcO)3BH, DCE, benzaldehyde, 23 °C, 12 h.
OH
N
i
NH2
NH
BOC
NH
NO2
20g
N
N
N
N
N
N
N
N
iii
iv
H
ii
O
O
O
O
21g
N
22g
23c
N
10
N
N
BOC
BOC
BOC
NO2
N
N
iv, v
OH
HO
N
19
NH2
20h
NH
NO2
N
i
N
N
N
iii
ii
N
N
O
O
O
N
22h
11
N
N
21h
Scheme 5. Synthesis of 6-ether derivatives via Mitsunobu coupling requiring BOC-protective group strategy. Reagents and conditions: (i) DIAD, PPh3, DMF, 23 °C, 12 h; (ii)
Pd/C, 50 psi H2, EtOH, 23 °C, 5 h; (iii) Na(AcO)3BH, DCE, benzaldehyde, 23 °C, 12 h; (iv) 2 N HCl/EtOH, 23 °C, 2 h; (v) formic acid, formaldehyde, 120 °C, 6 min, microwave.
NO2
NO2
NH2
NH
N
N
N
N
i
N
N
ii
iii
N
N
RO
HO
RO
RO
19
22 i-k
21 i-k
12-14
R-I
24 a
24 b
I
I
I
24 c
Scheme 6. Synthesis of 6-ether derivatives via direct alkylation on the 6-pheonolic group. Reagents and conditions: (i) NaH, DMF, 20 °C, 2 h; (ii) Pd/C, 50 psi H2, EtOH, 23 °C,
5 h; (iii) Na(AcO)3BH, DCE, benzaldehyde, 23 °C, 12 h.

P. Jain et al. / Bioorg. Med. Chem. 19 (2011) 359–373
365
Table 3a
been overstated in their magnitude with this scoring system. The
parallel difficulties with the MOE scoring protocol suggests that
this system may have features not well accounted using this mod-
el. A desolvation parameter, though implicit in the scoring func-
tions, may need to be modified as these compounds were quite
hydrophobic. Finally, there may be the possibility of internal
hydrogen bonding from the N4 amine to the lone pair of the N3
nitrogen.
Biological data: (a) Inhibition of CDK5/p25
Compound
CDK5/p25 IC50 (lM)
4
5
6
7
8
9
10
11
12
13
14
15
13
>100
>100
>10
>100
>100
>100
>100
>100
>100
>100
>10
Of the compounds prepared only compound 4 had appreciable
affinity for CDK5. The activity of compound 4 is consistent with
our proposed binding orientation. The absolute requirement for
the terminal hydroxyl group is consistent with the prior SAR of
purine-based ATP-site inhibitors of CDK5. Capping the alcohol with
a methoxy group clearly removed any activity. Curiously removing
or moving the ethyl group also decreased activity. There are multi-
ple examples of X-ray crystal structures confirming the benzimid-
azole core acting as either a hydrogen bond donor or as a hydrogen
bond acceptor at the ATP binding site of a kinase.80–85 Additional
studies have examined the interaction of a benzimidazole core
with pendant substituents utilizing molecular modeling tech-
niques.86–89 In many of these cases an additional hydrogen-bond-
ing interaction from the benzimidazole was provided from the 5
or 2 position. Additional interactions were then subsequently ob-
tained by optimizing interactions from these positions. Possible
paths forward in the presented series include modification of the
4-benzyl group to optimize interactions with the enzyme surface,
modification on the N4 substituent to reduce possible internal
hydrogen bonding and increase the effectiveness of the H-bond
donor–acceptor pair, increasing the pKa of the 4-amino group, or
incorporation of new additional interactions with the enzyme sur-
face. Interestingly a series of benzimidazoles including compounds
4 and 15 were tested against another serine/threonine kinase,
MEK5.78 The early SAR from those studies or presented here sug-
gest a very divergent SAR for MEK5 versus CDK5: 4 is moderately
active against CDK5 but inactive against MEK5, 15 is inactive
against CDK5 but active against MEK5. Clearly additional work is
required. The observed activity is consistent with the proposed
binding orientation and the core postulate that a benzimidazole
core can mimic an adenine core.
1 (R) roscovitine
0.16
Compounds were first tested at 10 and 100
curves were run and IC50 values determined for those com-
pounds displaying more than 50% inhibition at 100 M.
lM. Dose–response
l
of adequate activity to carry on to analysis with other kinases.
Compound was assayed against CDK1/cyclin B, glycogen
synthase kinase 3 /b (GSK3), casein kinase 1 (CK1), DYRK1A, and
4
a
CDK2/cyclin A. Analysis of these data indicates an affinity for
DYRK1A and CDK2/cyclinA within an order of magnitude of the
affinity for CDK5.
5. Discussion
These compounds were significantly less active than predicted
with the Madura scoring protocol or the standard MOE evaluation.
The principal components protocol and derived from the interac-
tion of HIV protease inhibitors with their target aspartyl protease.
Additional contributions to binding affinity derived primarily from
hydrophobic interactions. This strategy of a retained interaction
and point variations had suggested favorable analogy for this pro-
ject. The most likely failing of this strategy were assumptions that
the hydrogen bond donor–acceptor pair represented by the 4 NH
proton and the 3-N lone pair on the benzimidazole core may have
Table 3b
Biological data: (b) Selectivity of inhibition for 4: dose–response curves run on selected kinases

366
P. Jain et al. / Bioorg. Med. Chem. 19 (2011) 359–373
6.3. 2-(4-Amino-1-isopropyl-1H-benzo[d]imidazol-6-yloxy)-
6. Experimental
butan-1-ol (22a)
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 reac-
tions were conducted in dry glassware and under an atmosphere
of argon unless otherwise noted. Microwave reactions were con-
ducted in a 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 uncorrected. All proton NMR spectra were obtained with
a 500 or 400 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 analyses were conducted by Atlantic
Microlabs and are 0.4 of theoretical. All HRMS mass spectral
analyses 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.
A dry one-necked flask was charged with 1-isopropyl-4-nitro-
1H-benzo[d]imidazol-6-ol 19 (334.0 mg, 1.5 mmol), 1-(tert-butyl-
dimethylsilyloxy)butan-2-ol 20a lit90 (817.5 mg, 4.0 mmol), Ph3P
(985.0 mg, 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
slow dropwise addition. The solution was stirred for 30 min at 0 °C
then 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 in one portion at 0 °C. The reaction was stirred for 30 min
at 0 °C, then at 23 °C for 12 h. The reaction mixture taken up into
10 mL of EA and 10 mL K2CO3 (aq, satd). The mixture was extracted
three times each with 10 mL portions of EA and the combined or-
ganic extracts were washed with NaCl (aq, satd) and dried over
Na2SO4. The extract was decanted and the solvent was removed
under reduced pressure to afford crude 21a. This crude material
was dissolved into 75 mL of EtOH and added to a Parr hydrogena-
tion vessel previously charged with 150.1 mg 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. The mixture was filtered
through a pad of Celite and then the pad was washed with an addi-
tional 25 mL of EtOH. A 10 mL portion of concd HCl was added to
the 100 mL of resultant filtrate at 0 °C and was stirred at 23 °C
for 12 h. The solvent was removed under reduced pressure and
the resultant material was taken up into 50 mL of 1 N HCl (aq)
(pH 1) and washed three times each with 10 mL portions of EA,
neutralized then adjusted with 6 N NaOH (aq) to a pH of 10. The
mixture was then extracted (3 ꢀ 15 mL) with Et2O, washed with
(2 ꢀ 5 mL) NaCl (aq, satd), and dried over Na2SO4. The organic
phase was decanted and the solvent was removed under reduced
pressure to give 264.6 mg (67%) of 22a as a pale brown, hygro-
scopic solid. Mp 35.5–36.9 °C. SiO2 TLC Rf 0.44 (CH2Cl2/5%
CH3OH/0.1% NH4OH). 1H NMR (CDCl3): d 7.75 (s, 1H), 6.36 (d,
J = 2.0 Hz, 1H), 6.23 (d, J = 2.0 Hz, 1H), 4.45 (m, 1H), 4.36 (s, 2H,
NH2), 4.26 (m, 2H), 3.47 (m, 1H), 2.08 (s, 1H, OH) 1.66 (m, 2H),
1.57 (d, J = 6.8 Hz, 6H), 0.98 (t, J = 7.6 Hz, 3H). Anal. Calcd for
6.1. 1-Isopropyl-6-methoxy-4-nitro-1H-benzo[d]imidazole (18)
A 500 mL one-neck flask was charged with 5-methoxy-3-nitro-
benzene-1,2-diamine 16 (5.0 g, 27.3 mmol), NaHB(OAc)3 (17.36 g,
81.9 mmol), THF (100 mL), acetone (7.93 g, 137 mmol), and formic
acid (3.77 g, 81.9 mmol). The mixture was stirred overnight. The
solvent was removed in vacuo, and the dark red residue was dis-
solved in formic acid (31 mL). BHT (20.0 mg, 0.10 mmol) was
added, and then the mixture was cooled to 0 °C. Concentrated
HCl (87 mL) was added, and the mixture was quickly heated to re-
flux with a heating mantle. After maintaining reflux temperature
for 15 min, the solvent was removed in vacuo at 80 °C. This
solution was cooled to ambient temperature, neutralized with
50% NaOH (aq) to pH 8 then extracted with EA (4 ꢀ 50 mL). The
combined extracts were washed with NaCl (aq, satd) (3 ꢀ 10 mL),
dried over Na2SO4, decanted, and then the solvent was removed
under reduced pressure to give a brown solid, which was subjected
to SiO2 column chromatography (Hex/EtOAc 1:1) to afford 4.50 g
(74%) of 18 as a yellow solid. Rf 0.32 (CH2Cl2/MeOH/NH4OH
100:10:0.1). Mp 127.2–128.1 °C. 1H NMR (400 MHz, CDCl3): d
8.12 (s, 1H), 7.21 (d, J = 2.3 Hz, 1H), 7.79 (d, J = 2.3 Hz, 1H),
4.58–4.65 (m, 1H), 3.95 (s, 3H), 1.65 (d, J = 6.8 Hz, 6H), consistent
with the literature.77
C
14H21N3O2ꢁ1H2O: C, 59.77; H, 8.24; N, 14.94. Found: C, 59.91; H,
7.93; N, 14.64.
6.4. 2-(4-(Benzylamino)-1-isopropyl-1H-benzo[d]imidazol-6-yl-
oxy)butan-1-ol hydrochloride (4)
Compound 22a (80.0 mg, 0.3 mmol) and NaBH(OAc)3
(650.0 mg, 3.0 mmol) were added to 2.5 mL of DCE. Benzaldehyde
(36 lL, 1.2 mmol) was added in one portion and the mixture was
6.2. 1-Isopropyl-4-nitro-1H-benzo[d]imidazol-6-ol (19)
stirred for 12 h at 23 °C. 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 treated with 5 mL 5% K2CO3
(aq) and extracted three times with 15 mL portions of Et2O. The
combined organic extracts were washed with 10 mL of NaCl (aq,
satd) and dried (Na2SO4). The extract was decanted and the solvent
was removed under reduced pressure. The crude material was
isolated on SiO2 and eluted with hexane/EA (1:4). The HCl salt
was prepared by the addition of 2 mL of ethereal HCl, then
recrystallized from EA to give 46.1 mg (40%) of a white solid.
Mp: 148.7–149 °C. SiO2 TLC Rf 0.5 (CH2Cl2/5% CH3OH/0.1% NH4OH).
1H NMR (CDCl3): d 8.79 (s, 1H), 7.69 (s, 1H, NH), 7.41 (d, J = 7.6 Hz,
2H), 7.31 (d, J = 7.2 Hz, 2H), 7.28 (m, 1H), 6.26 (d, J = 2.0 Hz, 1H),
6.07 (d, J = 2.0 Hz, 1H), 4.63 (m, 1H), 4.54 (s, 1H), 4.14 (m, 2H),
3.70 (m, 1H), 2.69 (s, 1H, NH) 1.66 (d, J = 6.8 Hz, 6H), 1.57
(m, 2H), 0.86 (t, J = 7.6 Hz, 3H). Anal. Calcd for C21H28ClN3-
O2ꢁ1/8H2O: C, 64.32; H, 7.26; N, 10.71. Found: C, 64.27; H, 7.30;
N, 10.61.
A 50 mL microwave reactor tube was charged with 1-isopropyl-
6-methoxy-4-nitro-1H-benzo[d]imidazole 18 (1.00 g, 4.25 mmol)
and 20 mL of 48% HBr sealed and then subjected to 250 W of
microwave irradiation for 2.5 h, maintaining an internal tempera-
ture of 120 °C. The solvent was removed under reduced pressure,
and the resulting yellow solid was dissolved in minimum amount
(2 mL) of H2O. Solid NaHCO3 was added in small portions until pH
6. The resultant yellow solid was collected on #1 Whatmann filter
paper, washed twice with 2.0 mL of ice-cold H2O, and then dried in
vacuo (150 torr, 12 h) to afford 0.92 g (97%) of 19 as a yellow
solid. Rf 0.36 (CH2Cl2/MeOH/NH4OH 100:10:0.1). Mp 206–207 °C.
1H NMR (400 MHz, DMSO-d6): d 10.16 (s, 1H), 8.45 (s, 1H), 7.51
(d, J = 2.0 Hz, 1H), 7.40 (d, J = 2.0 Hz, 1H), 4.68–4.75 (m, 1H), 1.50
(d, J = 6.8 Hz, 6H). Anal. Calcd for C10H11N3O3: C, 54.29; H, 5.01;
N, 19.00; O, 21.70. Found: C, 51.84; H, 4.73; N, 18.07 plus 0.131
HBr.

P. Jain et al. / Bioorg. Med. Chem. 19 (2011) 359–373
367
6.5. 2-(4-(Benzylamino)-1-isopropyl-1H-benzo[d]imidazol-6-yl-
oxy)ethanol (21b)
1.07 (t, J = 7.39 Hz, 3H). HRMS (EI) calcd for C28H31N3O3: 457.2438
[M+H+], found 457.2438 [M+H+].
An oven dried one-neck flask was charged with 1-isopropyl-4-
nitro-1H-benzo[d]imidazol-6-ol 19 (166.0 mg, 0.75 mmol),
1-hydroxybutan-2-yl benzoate 20b (388 mg, 2.0 mmol), Ph3P
(493 mg, 1.9 mmol), and 4.0 mL of anhydrous DMF. The resulting
solution was cooled to 0 °C. Neat DIAD (0.2 mL, 1.4 mmol) was
added with dropwise at 0 °C. The solution was stirred for 30 min
at 0 °C for additional 2 h at rt. The reaction was then cooled to
0 °C and a second amount of DIAD (0.2 mL, 1.4 mmol) was added
at 0 °C. The reaction was stirred for 30 min at 0 °C then at rt for
12 h. After completion of reaction (TLC), 10 mL of EA and 10 mL
satd K2CO3 (aq) (pH >9) were added to the reaction mixture and
extracted (3 ꢀ 10 mL) with EA. The combined organic extracts
were washed with NaCl (aq, satd) (3 ꢀ 5 mL) and dried over
Na2SO4. The organic extract was decanted, and the solvent was re-
moved under reduced pressure to afford 3.30 g of 21b as an off-
white solid which was carried on directly to the next step. 1H
NMR (400 MHz, CDCl3): d 8.10 (s, 1H), 7.99–8.03 (m, 5H), 7.79 (d,
J = 2.27 Hz, H), 7.28 (d, J = 2.30 Hz, 1H), 5.38–5.44 (m, 1H), 4.55
(m, 1H), 4.20–4.40 (m, 2H), 1.71–1.79 (m, 2H), 1.58 (dd, J = 4.72,
6.73 Hz, 6H), 1.05 (t, J = 7.46 Hz, 3H).
6.8. 2-(4-(Benzylamino)-1-isopropyl-1H-benzo[d]imidazol-6-yl-
oxy)ethanol hydrochloride (5)
2-(4-(Benzylamino)-1-isopropyl-1H-benzo[d]imidazol-6-ylox-
y)ethyl benzoate 23a was dissolved into 2.0 mL of 5% KOH in
MeOH and stirred for 12 h. The reaction progress was assayed by
TLC. The reaction was quenched with 5 mL of NH4OH (satd, aq)
and extracted (3 ꢀ 10 mL) with EA. The combined EA extracts were
washed (1 ꢀ 5 mL) with NaCl (aq, satd) and dried over Na2SO4. Re-
moval of the solvent under reduced pressure gave 30 mg of 5 as an
off white solid, which was dissolved in 2.0 mL Et2O. A slight excess
of freshly prepared ethereal HCl was slowly added to form the cor-
responding hydrochloride salt. Recrystallization from EtOH gave
15 mg (65% yield) of 5 as a white solid. Mp 140–142 °C Rf 0.62
(CH2Cl2/MeOH/NH4OH 100:10:0.1). 1H NMR (MeOD) for
C21H27N3O2: d 9.23 (s, 1H), 7.42–7.46 (d, J = 7.24 Hz, 2H), 7.38–
7.35 (t, J = 7.4 Hz, 2H), 7.30 (t, J = 7.24 Hz, 1H), 6.67 (d,
J = 1.71 Hz, 1H), 6.37 (d, J = 1.85 Hz, 1H), 4.92 (m, 1H), 4.49 (s,
2H), 3.96–4.1 (ddd, J = 5.2, 9.6, 16.0 Hz, 2H), 3.80–3.86 (m, 1H),
1.68 (d, J = 6.70 Hz, 6H), 1.50–1.65 (m, 2H), 1.00–1.05 (t,
J = 7.44 Hz, 3H). Anal. Calcd for C21H28ClN3O2: C, 64.69; H, 7.24;
N, 10.78. Found: C, 60.60; H, 7.10; N, 9.90 plus 1.46% H2O.
6.6. 2-(4-(Benzylamino)-1-isopropyl-1H-benzo[d]imidazol-6-yl-
oxy)ethanol (22b)
6.9. 2-(1-Isopropyl-4-nitro-1H-benzo[d]imidazol-6-yloxy)ethyl
A Parr hydrogenation vessel was charged with 100 mg of Pd/C
(10% w/w) and then solution of crude 21g in 100 mL of EtOH was
added to the reaction vessel. After three vacuum/purge cycles with
H2, the reaction vessel was shaken at 50 psi on a Parr hydrogena-
tion apparatus for 12 h. The reaction mixture was filtered through
a pad of Celite and the pad was washed with an additional 10 mL of
EtOH. The solvent was removed under reduced pressure to afford a
resultant brown oil that was treated with 50 mL of 1 N HCl (aq) (pH
<1) and washed with EA (3 ꢀ 10 mL). The aqueous layer was then
basified (pH >9) with 5% K2CO3 (aq) and extracted (3 ꢀ 15 mL) with
EA. The combined EA layers were washed with NaCl (aq, satd)
(3 ꢀ 10 mL) and dried over Na2SO4. The solvent was removed un-
der reduced pressure to give 280 mg of 22b as a clear film which
was carried on to the next step directly. 1H NMR (400 MHz, CDCl3):
d 8.06 (dd, J = 1.31, 8.45 MHz, 2H), 7.74 (s, 1H), 7.55–7.57 (m, 1H),
7.48–7.51 (m, 2H), 6.58 (br s, 2H), 6.30 (d, J = 2.06 Hz, 1H), 6.21 (d,
J = 2.09 Hz, 1H), 5.34–5.41 (m, 1H), 4.43 (m, 1H), 4.14–4.23 (ddd,
J = 4.46 Hz, J = 10.26, 20.23 MHz, 2H), 1.89–1.95 (m, 2H), 1.54
(dd, J = 3.67, 6.74 Hz, 6H), 1.05 (t, J = 7.43 Hz, 3H).
benzoate (21c)
An oven dried one-neck flask was charged with 1-isopropyl-4-ni-
tro-1H-benzo[d]imidazol-6-ol 19 (334.0 mg, 1.5 mmol), 2-hydroxy-
ethyl benzoate 20c (665 mg, 4.0 mmol), Ph3P (985.0 mg, 3.8 mmol),
and 4.0 mL of anhydrous DMF. The resulting solution was cooled to
0 °C. Neat DIAD (0.4 mL, 2.8 mmol) was added with dropwise at 0 °C.
Then this solution was stirred for 30 min at 0 °C then for an addi-
tional 2 h at 23 °C. The reaction was cooled to 0 °C and a second
amount of DIAD (0.4 mL, 2.8 mmol) was added at 0 °C. The reaction
was stirred for 30 min at 0 °C then at rt for 12 h. After completion of
reaction (TLC), 10 mL of EA and 10 mL satd K2CO3 (aq) (pH >9) were
added to the reaction mixture and extracted with EA (3 ꢀ 10 mL).
The organic layers were combined and washed with NaCl (aq, satd)
(3 ꢀ 5 mL) then dried (Na2SO4). The organic layer was decanted and
the solvent was removed under reduced pressure to give 2.0 g of 21c
as a light brown oil which was carried on directly in the next step. 1H
NMR (400 MHz, CDCl3): d 8.13 (s, 1H), 8.01–8.02 (m, 5H), 7.85 (d,
J = 2.29 Hz, 1H), 7.29 (d, J = 2.29 Hz, 1H), 5.23 (m, 1H), 4.73–4.76
(dd, J = 4.13, 5.27 Hz, 2H), 3.81–3.84 (dd, J = 5.25, 6.19 Hz, 2H), 1.62
(d, J = 6.74 Hz, 6H).
6.7. 2-(4-(Benzylamino)-1-isopropyl-1H-benzo[d]imidazol-6-yl-
oxy)ethyl benzoate (23a)
6.10. 2-(4-Amino-1-isopropyl-1H-benzo[d]imidazol-6-yloxy)-
A dry round bottomflask was chargedwith 2-(4-amino-1-isopro-
pyl-1H-benzo[d]imidazol-6-yloxy)ethyl benzoate 22g (280 mg,
0.75 mmol), NaHB(OAc)3 (317.9 mg, 1.5 mmol), benzaldehyde
ethyl benzoate (22c)
A Parr hydrogenation vessel was charged with 115 mg of Pd/C
(10% w/w) then a solution of crude 21c (Th 1.5 mmol) in 75 mL
of EtOH. After three vacuum/purge cycles with H2, the reaction
vessel was subjected to hydrogenation at 50 psi for 12 h on a Parr
hydrogenation apparatus. The reaction mixture was filtered
through a pad of Celite, and then the pad was washed with an addi-
tional 10 mL of EtOH. The solvent was removed under reduced
pressure. The resultant brown oil was treated with 50 mL of 1 N
HCl (aq) (pH <1) and washed with EA (3 ꢀ 10 mL), this aqueous
layer was then basified with 5% K2CO3 (aq) (pH ꢂ9–10) and then
extracted with EA (3 ꢀ 15 mL). The combined EA layers were
washed with NaCl (aq, satd) (3 ꢀ 10 mL) and dried over Na2SO4.
The solvent was removed under reduced vacuum to give 0.110 g
of 22c as a colorless film which was carried on directly. Rf 0.45
(85 lL, 0.85 mmol, 1.15 equiv) and 5.0 mL of DCE. This mixture
was stirred at rt for 12 h, quenched with 10 mL NaHCO3 (satd, aq),
then extracted (3 ꢀ 10 mL) with EA. The combined organic extracts
were washed (2 ꢀ 5 mL) with NaCl (aq, satd) and dried over Na2SO4.
Evaporation of the solvent gave a brown solid, which was subjected
to SiO2 flash column chromatography (Hexanes/EtOAc 1:1) to afford
40 mg of 23a (11%) as a brown solid. Mp 116.1–116.9 °C Rf 0.71
(CH2Cl2/MeOH/NH4OH 100:10:0.1). 1H NMR (CDCl3) for C28H31
-
N3O3: d 8.07 (dd, J = 1.35, 8.39 Hz, 1H), d 7.71 (s, 1H), 7.54–7.59 (m,
1H), 7.39–7.46 (m, 4H), 7.29–7.35 (m, 2H), 7.26 (m, 1H), 6.26 (d,
J = 2.07 Hz, 1H), 6.08 (d, J = 2.05 Hz, 1H), 5.39 (m, 1H), 5.25 (t,
J = 5.51 Hz, 1H), 4.45–4.52 (m, 4H), 4.13–4.22 (ddd, 2H, J = 5.0,
10.3, 22.9 Hz, 2H), 1.89–1.94 (m, 2H), 1.59 (dd, J = 3.5, 6.8 Hz, 6H),

368
P. Jain et al. / Bioorg. Med. Chem. 19 (2011) 359–373
(CH2Cl2/MeOH/NH4OH 100:10:0.1). 1H NMR (CDCl3) for
19H21N3O3: d 8.075 (dd, J = 1.31, 8.37 Hz, 2H), d 7.76 (s, 1H),
7.65–7.71 (m, 2H), 7.54–7.59 (m, 2H), 7.42–7.51 (m, 4H), 6.33 (d,
J = 2.12 Hz, 1H), 6.25 (d, J = 2.14 Hz, 1H), 5.00 (td, J = 6.3, 12.6 Hz,
1H), 4.62–4.71 (dd, J = 4.3, 5.4 Hz, 2H), 4.37 (br peak, 2H) 4.33–
4.36 (dd, J = 4.3, 5.3 Hz, 2H), 1.58 (d, J = 6.76 Hz, 6H).
added at 0 °C. The reaction was stirred for 30 min at 0 °C then at
rt until the reaction was complete (TLC). The reaction mixture
was poured into 10 mL of EA and 10 mL satd K2CO3 (aq) (pH >9).
This was extracted with EA (3 ꢀ 10 mL) and the combined organic
extracts were washed with NaCl (aq, satd) (3 ꢀ 5 mL) and dried
(Na2SO4). The extract was decanted and the solvent was removed
under reduced pressure to give crude 21d which was carried on
directly. 1H NMR (400 MHz, CDCl3): d 8.10 (s, 1H), 7.83 (d,
J = 2.24 Hz, 1H), 7.36 (d, J = 2.23 Hz, 1H), 4.55–4.62 (sept,
J = 6.7 Hz, 1H), 4.37–4.43 (m, 1H), 3.42–3.49 (m, 2H), 3.37 (s, 3H),
1.72–1.78 (m, 2H), 1.61 (d, J = 6.78 Hz, 6H), 1.00 (t, J = 7.47 Hz, 3H).
C
6.11. 2-(4-(Benzylamino)-1-isopropyl-1H-benzo[d]imidazol-6-yl-
oxy)ethyl benzoate (23b)
A dry round bottom flask was charged with 2-(4-amino-1-iso-
propyl-1H-benzo[d]imidazol-6-yloxy)ethyl benzoate 22c (110 mg,
0.3 mmol), NaBH(OAc)3 (127.16 mg, 0.6 mmol), benzaldehyde
6.14. 1-Isopropyl-6-(1-methoxybutan-2-yloxy)-1H-benzo[d]-
imidazol-4-amine (22d)
(33
room temperature for 12 h. A second quantity of NaBH(OAc)3
(41.0 mg, 0.2 mmol, 0.66 equiv) and benzaldehyde (11 L,
lL, 0.33 mmol) and 5.0 mL of DCE. The mixture was stirred at
l
Crude 21d was dissolved into 25 mL of EtOH and added to a Parr
hydrogenation vessel previously charged with 90 mg of Pd/C (10%
w/w). After three vacuum/purge cycles with H2, the vessel was
charged to 50 psi with H2 and shaken for 12 h. The mixture was
then filtered through a pad of Celite, the pad was washed with
an additional 10 mL of EtOH, and then the solvent was removed
under reduced pressure. The resultant oil was treated with 50 mL
of 1 N HCl (aq) (pH <1) and then washed with EA (3 ꢀ 10 mL).
The aqueous phase was neutralized, made basic with 5% K2CO3
(aq) (pH ꢂ9–10), and then extracted with (3 ꢀ 15 mL) of EA. The
combined EA layers were washed with 1 N HCl (3 ꢀ 10 mL), basi-
fied with 50% NaOH (aq) (pH ꢂ12), and then extracted with Et2O
(3 ꢀ 15 mL). These combined Et2O layers were washed with NaCl
(aq, satd) (3 ꢀ 10 mL) and dried (Na2SO4). The organic phase was
decanted and the solvent was removed under reduced pressure
to afford 220.0 mg of crude 22d as a light brown film carried on di-
rectly. 1H NMR (400 MHz, CDCl3): d 7.75 (s, 1H), 6.38 (d, J = 2.07 Hz,
1H), 6.25 (d, J = 2.07 Hz, 1H), 4.42–4.52 (m, 1H), 4.33 (br s, 1H),
4.26–4.33 (td, J = 5.5, 10.9 Hz, 1H), 3.52–3.62 (ddd, J = 4.91, 10.17,
24.31 Hz, 2H), 3.41 (s, 3H), 1.73–1.82 (m, 2H), 1.58 (d, J = 6.76 Hz,
6H), 0.96–1.01 (t, J = 7.47 Hz, 3H).
0.11 mmol, 0.33 equiv) were added to the reaction mixture to drive
the reaction to completion over the next 3 h (TLC). The reaction
was quenched with 10 ml NaHCO3 (satd, aq) and the mixture
was extracted (3 ꢀ 15 mL) with EA. The combined organic extracts
were washed (2 ꢀ 5 mL) with NaCl (aq, satd) and dried (Na2SO4).
Evaporation of the solvent gave a brown solid, which was sub-
jected to SiO2 flash column chromatography (Hexanes/EtOAc 1:1)
to afford 40 mg of 23b (31%) as a white solid. Mp 141.5–142.8 °C.
Rf 0.7 (CH2Cl2/MeOH/NH4OH 100:10:0.1). 1H NMR (CDCl3) for
C26H27N3O3: d 8.07 (dd, J = 1.3, 8.4 Hz, 1H), d 7.73 (s, 1H), 7.55–
7.60 (m, 1H), 7.40–7.46 (m, 4H), 7.31–7.35 (m, 2H), 7.27 (m, 1H),
6.26 (d, J = 2.07 Hz, 1H), 6.10 (d, J = 2.04 Hz, 1H), 5.29 (t,
J = 5.76 Hz, 1H), 4.67 (dd, J = 4.33, 5.31 Hz, 2H) 4.45–4.52 (m, 3H),
4.23–4.28 (dd, J = 3.70, 5.95 Hz, 2H), 1.58 (d, J = 6.76 Hz, 6H). Anal.
Calcd for C26H27N3O3: C, 65.41; H, 7.49; N, 10.40. Found: C, 65.43;
H, 7.35; N, 10.33.
6.12. 2-(4-(Benzylamino)-1-isopropyl-1H-benzo[d]imidazol-6-yl-
oxy)ethanol (6)
2-(4-(Benzylamino)-1-isopropyl-1H-benzo[d]imidazol-6-yloxy)-
ethyl benzoate 23b was dissolved in 2.0 ml of 5% KOH in MeOH and
reaction mixture was stirred for 12 h. The reaction was quenched
with 5 mL of NH4Cl (satd, aq) and extracted (3 ꢀ 10 mL) with EA.
The combined EA extracts were washed with NaCl (aq, satd) and
dried (Na2SO4). Removal of the solvent under reduced pressure
afforded an off white solid that was subjected to SiO2 flash column
chromatography (Hexanes/EtOAc; 1:1) to afford 18 mg of 6 as a
white solid. Recrystallization from DCM/Hexanes provided 6 as
white needles in 56% yield. Mp 140–142 °C. Rf 0.65 (CH2Cl2/MeOH/
NH4OH 100:10:0.1). 1H NMR (CDCl3) for C19H23N3O2: d 7.74 (s,
1H), 7.41–7.43 (m, 2H), 7.31–7.36 (m, 2H), 7.25–7.29 (m, 1H), 6.23
(d, J = 2.11 Hz, 1H), 6.06 (d, J = 2.07 Hz, 1H), 5.33 (br peak, 1H),
4.45–4.55 (m, 3H), 4.08–4.14 (dd, 2H, J = 3.9, 5.2 Hz), 3.94–3.97
(dd, J = 3.4, 5.51 Hz, 2H), 1.59 (d, J = 6.75 Hz, 6H). Anal. Calcd for
6.15. N-Benzyl-1-isopropyl-6-(1-methoxybutan-2-yloxy)-1H-
benzo[d]imidazol-4-amine hydrochloride (7)
A dry round bottom flask was charged with 1-isopropyl-6-
(1-methoxybutan-2-yloxy)-1H-benzo[d]imidazol-4-amine
(200.0 mg, 0.72 mmol), NaHB(OAc)3 (307.3 mg, 1.45 mmol), benz-
aldehyde (80 L, 0.80 mmol) and 5.0 mL of DCE. The mixture was
stirred at room temperature for 24 h, then a second amount of
NaBH(OAc)3 (40.0 mg, 0.19 mmol) and benzaldehyde (25 L,
22d
l
l
0.25 mmol) were added to drive the reaction to completion over
the next 2 h. The reaction mixture was quenched with the addition
of 10 mL 5% K2CO3 (aq) (pH ꢂ9–10) and the mixture was extracted
with (3 ꢀ 10 mL) of EA. The combined extracts were washed with
5 mL of NaCl (aq, satd) and dried (Na2SO4). Removal of the solvent
under reduced pressure gave crude 7 as a brown solid, which was
subjected to SiO2 flash column chromatography (Hexanes/EtOAc/
Et3N 1:1:0.005) to afford 200 mg of 7 (76%) as a colorless oil. Con-
version of 7 to its corresponding hydrochloride salt was achieved
in 1.0 mL EA with gaseous HCl to give 220 mg of 7 as a white solid
mp 129–130 °C. Rf 0.7 (CH2Cl2/MeOH/NH4OH 100:10:0.1). 1H NMR
C19H23N3O2: C, 70.13; H, 7.12; N, 12.91. Found: C, 67.88; H, 6.93;
N, 12.34 plus 0.6% H2O.
6.13. 1-Isopropyl-6-(1-methoxybutan-2-yloxy)-4-nitro-1H-benzo-
[d]imidazole (21d)
An oven dried one-neck flask was charged with 1-isopropyl-4-
nitro-1H-benzo[d]imidazol-6-ol 19 (334.0 mg, 1.5 mmol), 1-meth-
oxybutan-2-ol 20d (0.5 ml, 4.0 mmol), Ph3P (985.0 mg, 3.8 mmol),
and 4.0 mL of anhydrous DMF. The solution was cooled to 0 °C.
Neat DIAD (0.4 mL, 2.8 mmol) was added with dropwise addition.
The solution was stirred for 30 min at 0 °C then the reaction was
stirred for an additional 2 h at rt. The reaction was then cooled
to 0 °C and a second quantity of DIAD (0.4 mL, 2.8 mmol) was
(CDCl3) for C22H29N3O2:
d 7.27 (s, 1H), 7.41–7.43 (m, 2H),
7.31–7.34 (m, 2H), 7.26 (m, 1H), 6.32 (d, J = 2.05 Hz, 1H), 6.10 (d,
J = 2.04 Hz, 1H), 5.30 (br peak, 1H), 4.45–4.52 (m, 3H), 4.23–4.28
(m, 1H), 3.49–3.59 (dd, J = 4.85, 10.16, 23.14 Hz, 2H), 3.37 (s, 3H),
1.70–1.74 (m, 2H), 1.59 (d, J = 6.76 Hz, 6H) 0.96–1.00 (t, J =
7.47 Hz, 3H). Anal. Calcd for C22H30ClN3O2: C, 65.41; H, 7.49; N,
10.40. Found: C, 65.43; H, 7.35; N, 10.33.

P. Jain et al. / Bioorg. Med. Chem. 19 (2011) 359–373
369
6.16. 1-Isopropyl-6-(2-methoxyethoxy)-1H-benzo[d]imidazol-
4-amine (22e)
J = 2.0 Hz, 2H), 3.72 (t, J = 2.8 Hz, 2H), 3.40 (s, 3H), 1.64 (d,
J = 6.8 Hz, 6H). Anal. Calcd for C22H27N3O6ꢁ1/6H2O: C, 61.10; H,
6.37; N, 9.72. Found: C, 61.08; H, 6.31; N, 9.78.
A dry one-necked flask was charged with 1-isopropyl-4-nitro-
1H-benzo[d]imidazol-6-ol, 19 (334.0 mg, 1.5 mmol), 2-methoxy-
ethanol 20e (0.5 mL, 6.9 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 mixture 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 portion. The reaction was stirred for
30 min at 0 °C and 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 re-
moved under reduced pressure. Crude 21e was dissolved into
75 mL of EtOH and added to a Parr hydrogenation vessel previ-
ously charged with Pd/C (10% w/w, 119.8 mg). After three vac-
uum/purge cycles with H2, the vessel 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 to give a brown oil. This
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 neu-
tralized 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 extracts were washed with NaCl (aq, satd)
and dried over Na2SO4. The solvent was decanted and then re-
moved under reduced pressure to give 150.5 mg (40%) of 22e
as a pale brown, hygroscopic solid. Mp 32.9–34.1 °C. SiO2 TLC
Rf 0.31 (CH2Cl2/5% CH3OH/0.1% NH4OH). 1H NMR (CDCl3): d
7.75 (s, 1H), 6.31 (d, J = 2.0 Hz, 1H), 6.23 (d, J = 2.0 Hz, 1H),
4.48 (m, 1H), 4.14 (t, J = 6.4 Hz, 2H), 3.76 (t, J = 3.6 Hz, 2H),
3.46 (s, 3H), 1.57 (d, J = 6.8 Hz, 6H). Anal. Calcd for
6.18. 1-(Dimethylamino)butan-2-ol (20f)
An oven dried 250 mL round bottom flask was cooled under ar-
gon and charged with a 2 M solution of dimethylamine in THF
(30 mL, 60 mmol). The reaction mixture was cooled to ꢃ78 °C
and then 2 M n-BuLi in THF (25 mL, 50 mmol) was added slowly
over 15 min maintaining the internal temperature of the reaction
below ꢃ50 °C. The reaction was then permitted to warm to 23 °C
and then stirred at 23 °C for 1 h. The reaction mixture was cooled
to ꢃ78 °C and ( )2-ethyloxirane (4.0 mL, 46 mmol) was added over
5 min. Then the reaction mixture was permitted to warm to 23 °C
and then stirred for additional 12 h. A mixture of 3:1 NH4OH/NH4Cl
was added and the mixture was extracted (3 ꢀ 15 mL) with Et2O,
washed (3 ꢀ 10 mL) with NaCl (aq, satd), and then dried (Na2SO4).
The extract was decanted and then removed under reduced pres-
sure to give 1.23 g (88%) of 20f as a brownish-yellow oil. bp:
149.8–151.1 °C (lit91 152–153 °C). 1H NMR (CDCl3): d 3.46 (m,
1H), 2.51 (m, 2H), 2.31 (s, 6H), 1.51 (m, 2H), 0.95 (t, 3H).
6.19. N-Benzyl-6-(1-(dimethylamino)butan-2-yloxy)-1-iso-
propyl-1H-benzo[d]imidazol-4-amine hydrochloride (9)
A dry one-necked flask was charged with 1-isopropyl-4-ni-
tro1H-benzo[d]imidazol-6-ol 19 (334.0 mg, 1.5 mmol), 1-(dimeth-
ylamino)butan-2-ol 20f (468.6 mg, 4.0 mmol), Ph3P (985.0 mg,
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 slow drop-
wise addition. The solution was stirred for 30 min at 0 °C, the
ice-bath was removed, and 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 (0.4 mL, 2.8 mmol) was added in one
portion at 0 °C. The reaction was stirred for 30 min at 0 °C, then
at 23 °C for 12 h. The reaction mixture taken up into 10 mL of EA
and 10 mL of K2CO3 (aq, satd). This mixture was extracted three
times with 10 mL portions of EA and the combined organic extracts
were washed with 10 mL of NaCl (aq, satd) and then dried over
Na2SO4. The extract was decanted and the solvent was removed
under reduced pressure. The HCl salt was prepared with the addi-
tion of 2 mL of ethereal HCl. Crude 21f was dissolved into 75 mL of
EtOH and was added to a Parr hydrogenation vessel previously
charged with Pd/C (10% w/w, 91.3 mg). After three vacuum/purge
cycles with H2, the vessel was charged to 50 psi with H2 and sha-
ken for 5 h. The mixture was filtered through a pad of Celite and
then the pad was washed with an additional 25 mL of EtOH. A
10 mL portion of concd HCl was added to the filtrate at 0 °C. The
reaction was stirred at 23 °C for 12 h. The solvent was removed un-
der reduced pressure. The crude oil was taken up into 50 mL of 1 N
HCl (aq) (pH 1) and washed three times with 10 mL portions of EA,
neutralized and adjusted with 6 N NaOH (aq) to a pH of 10 and
then extracted three times with 15 mL portions of Et2O. The com-
bined Et2O layers were washed with NaCl (aq, satd) and dried
(Na2SO4). The organic phase was decanted and the solvent was re-
moved under reduced pressure to give crude 22f which was used
directly to the next step. 1H NMR (CDCl3): d 8.31 (s, 1H), 6.83 (d,
J = 2.0 Hz, 1H), 6.44 (d, J = 2.0 Hz, 1H), 6.42 (s, 1H, NH), 4.44 (m,
1H), 4.52 (s, 1H), 2.92 (s, 6H), 3.62 (m, 1H), 1.70 (m, 2H), 1.68 (d,
J = 6.8 Hz, 6H), 1.20 (m, 2H), 1.11 (t, J = 7.6 Hz, 1H).
C
13H19N3O2ꢁ1/5H2O: C, 61.74; H, 7.73; N, 16.61. Found: C,
61.99; H, 7.81; N, 16.26.
6.17. N-Benzyl-1-isopropyl-6-(2-methoxyethoxy)-1H-benzo[d]-
imidazol-4-amine oxalate (8)
1-Isopropyl-6-(2-methoxyethoxy)-1H-benzo[d]imidazol-4-
amine 22e (120.0 mg, 0.48 mmol) and solid NaBH(OAc)3
(279.7 mg, 1.4 mmol) were added to 2.5 mL of DCE. Benzalde-
hyde (53.6 lL, 0.5 mmol) was added in one portion and the mix-
ture 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% of 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 under reduced pres-
sure. The crude material was isolated on SiO2 and eluted with
hexanes/EA (1:3) to give 69.3 mg of a clear film. This was dis-
solved into 2.5 mL of EA, followed by addition of a solution of
anhydrous oxalic acid (20.1 mg, 0.22 mmol) in 2.5 mL of EA to
afford 82.6 mg (40%) of
8 as a white crystalline solid. Mp
164.6–165.1 °C. SiO2 TLC Rf 0.55 (CH2Cl2/5% CH3OH/0.1% NH4OH).
1H NMR (MeOD-d4): d 8.90 (s, 1H), 7.41 (d, J = 7.6 Hz, 2H), 7.33
(t, J = 7.2 Hz, 2H), 7.25 (t, J = 7.6 Hz, 1H), 6.56 (d, J = 2.0 Hz, 1H),
6.19 (d, J = 2.0 Hz, 1H), 4.88 (m, 1H), 4.47 (s, 2H), 4.11 (t,
6-(1-(Dimethylamino)butan-2-yloxy)-1-isopropyl-1H-benzo[d]
imidazol-4-amine, 22c, (84.0 mg, 0.39 mmol) and solid NaBH(OAc)3
(650.0 mg, 3.0 mmol) were added to 2.5 mL of DCE. Benzaldehyde

370
P. Jain et al. / Bioorg. Med. Chem. 19 (2011) 359–373
(31
l
L, 1.2 mmol) was added in one portion and the mixture was
6.21. N-Benzyl-1-isopropyl-6-(piperidin-4-ylmethoxy)-1H-
benzo[d]imidazol-4-amine dihydrochloride (10)
stirred for 12 h. The reaction mixture was quenched with the
addition of 1 mL of NaHCO3 (aq, satd) and stirred for 10 min. The
mixture was taken up into 5% K2CO3 (aq) and then extracted
three times with 15 mL of Et2O. The combined organic extracts
were washed with NaCl (aq, satd) and then dried over Na2SO4.
The extract was decanted and the solvent was removed under re-
duced pressure. The crude material was isolated on SiO2 and eluted
with hexane/EA (1:4). The HCl salt of 9 was prepared with drop-
wise addition of 2 mL of ethereal HCl to give a white, hygroscopic
solid. Mp 88.2–89.5 °C. SiO2 TLC Rf 0.47 (CH2Cl2/5% CH3OH/0.1%
NH4OH). 1H NMR (MeOD-d4): d 9.31 (s, 1H), 7.42 (q, 2H), 7.35 (d,
J = 7.2 Hz, 2H), 7.29 (q, 1H), 6.83 (d, J = 2.0 Hz, 1H), 6.44 (d,
J = 2.0 Hz, 1H), 6.42 (s, 1H, NH), 4.44 (m, 1H), 4.52 (s, 1H), 2.92
(s, 6H), 3.62 (m, 1H), 1.70 (m, 2H), 1.68 (d, J = 6.8 Hz, 6H), 1.20
(m, 2H), 1.11 (t, J = 7.6 Hz, 1H). Anal. Calcd for
tert-Butyl 4-((4-(benzylamino)-1-isopropyl-1H-benzo[d]imida-
zol-6-yloxy)methyl)piperidine-1-carboxylate 23c (71.1 mg, 0.15
mmol) was dissolved into 50 mL EtOH and 5 mL of concd HCl at
0 °C. The reaction was stirred at 23 °C for 12 h. The solvent was
then removed under reduced pressure. Recrystallization from
MeOH afforded 60 mg (83%) of a white solid. Mp 218–220 °C. 1H
NMR (MeOD-d4): d 9.26 (s, 1H), 7.51 (s, 1H, NH) 7.42 (q, 2H),
7.35 (d, J = 7.2 Hz, 2H), 7.28 (q, 1H), 6.66 (d, J = 2.0 Hz, 1H), 6.42
(s, 1H, NH), 6.25 (d, J = 2.0 Hz, 1H), 4.48 (m, 1H), 3.95 (s, 1H),
3.43 (s, 2H), 3.30 (m, 1H), 3.01 (m, 2H), 2.14 (m, 2H), 2.13 (m,
2H), 1.58 (d, J = 6.8 Hz, 6H), 1.28 (m, 2H). Anal. Calcd for
C
23H32Cl2N4Oꢁ2.5H2O: C, 55.64; H, 7.51; N, 11.21. Found: C,
55.40; H, 7.38; N, 11.26.
C
23H34Cl2N4Oꢁ2.5H2O: C, 55.42; H, 7.89; N, 11.24. Found: C,
55.38; H, 7.62; N, 11.15.
6.22. 1-Isopropyl-6-((1-methylpiperidin-4-yl)methoxy)-4-nitro-
1H-benzo[d]imidazole (21h)
6.20. tert-Butyl-4-((4-(benzylamino)-1-isopropyl-1H-benzo[d]-
imidazol-6-yloxy)methyl)piperidine-1-carboxylate (21g)
Carbamate 21a (650.0 mg, 1.6 mmol) was dissolved into 30 mL
of EtOH and 3 mL of concd HCl was added dropwise at 0 °C. The
reaction was stirred at 23 °C until the reaction was complete as
determined by TLC. The solvent was removed under reduced pres-
sure and resulting solid was then dissolved into 50 mL of 1 N HCl
(aq) and washed three times with 10 mL portions of EA, neutral-
ized, then adjusted to a pH 10 with 6 N NaOH (aq). This mixture
was extracted three times with 15 mL portions of CHCl3. The com-
bined organic extracts were washed with a 5 mL aliquot of NaCl
(aq, satd) and then dried (Na2SO4). The organic phase was decanted
and the solvent was removed under reduced pressure. The crude
yellow solid (100 mg, 0.38 mmol) obtained was dissolved in
0.7 mL of formic acid, 1 mL of 38% aqueous formaldehyde, and then
subjected to 250 Watt microwave irradiation to a temperature of
120 °C for 5.5 min. After cooling the solvent was removed and
the resultant yellow oil was taken up into 10 mL K2CO3 (aq, satd)
and 10 mL EA. This was extracted three times with 10 mL portions
of EA, washed with 5 mL of NaCl (aq, satd), and then dried
(Na2SO4). The extract was decanted and the solvent was removed
under reduced pressure to give 150.2 mg (30%) of 21h as a brown
solid. Mp 205–211 °C. SiO2 TLC Rf 0.31 (CH2Cl2/5% CH3OH/0.1%
NH4OH). 1H NMR (CDCl3): d 8.42 (s, 1H), 7.77 (d, J = 2.0 Hz, 1H),
7.23 (d, J = 2.0 Hz, 1H), 4.64 (m, 1H), 3.98 (d, J = 5.6 Hz, 2H), 3.61
(d, J = 10.0 Hz, 1H), 2.74 (s, 3H), 2.68 (m, 2H), 2.06 (m, 2H), 1.95
(m, 2H), 1.65 (d, J = 6.8 Hz, 6H), 1.26 (m, 2H). Anal. Calcd for
A dry one-necked flask was charged with 1-isopropyl-4-nitro1H-
benzo[d]imidazol-6-ol 19 (334.0 mg, 1.5 mmol), tert-butyl 4-(hydro-
xymethyl)piperidine-1-carboxylate 20g (861.1 mg, 4.0 mmol), Ph3P
(985.0 mg, 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
slow dropwise addition. The solution was stirred for 30 min at 0 °C
then the reaction was stirred for an additional 2 h at 23 °C. The reac-
tion was cooled to 0 °C and an second quantity of DIAD (0.4 mL,
2.8 mmol) was added in one portion at 0 °C. The reaction was stirred
for 30 min at 0 °C, then at 23 °C for 12 h. The reaction mixture taken
up into a mixture of 10 mL of EA and 10 mL K2CO3 (aq, satd) and ex-
tracted (3 ꢀ 10 mL) with EA. The combined organic extracts were
washed with 10 mL of NaCl (aq, satd) and dried over Na2SO4. The ex-
tract was decanted and the solvent was removed under reduced
pressure. Crude 21a was dissolved into 75 mL of EtOH and added
to a Parr hydrogenation vessel previously charged with Pd/C (10%
w/w, 145.7 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 the pad was washed with
an additional 25 mL of EtOH. Crude 21g was carried directly to next
step. 1H NMR (CDCl3): d 7.61 (s, 1H), 6.18 (d, J = 2.0 Hz, 1H), 6.02 (d,
J = 2.0 Hz, 1H), 5.27 (s, 1H, NH), 4.47 (s, 1H), 4.14 (m, 1H), 3.78(d, 2H),
2.71 (m, 1H), 1.70 (m, 2H), 1.80 (m, 2H), 1.57 (d, J = 6.8 Hz, 6H), 1.46
(s, 9H), 1.30 (m, 2H), 1.25 (m, 2H).
C17H24N4O3: C, 61.43; H, 7.28; N, 16.86. Found: C, 61.45; H, 7.21;
N, 16.58.
Crude 21g (83.3 mg) and solid NaBH(OAc)3 (650.0 mg,
3.0 mmol) were added to 5 mL of DCE. Benzaldehyde (24 lL,
1.2 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) and extracted
three times with 15 mL portions of Et2O. The combined organic ex-
tracts were washed with NaCl (aq, satd) and then dried (Na2SO4).
The extract was decanted off and the solvent was removed under
reduced pressure. The crude material was isolated on SiO2 and
eluted with hexane/EA (1:4) to give 72.1 mg (10%) of 23c as a white
hygroscopic solid. Mp 81.1–81.5 °C. SiO2 TLC Rf 0.57 (CH2Cl2/5%
CH3OH/0.1% NH4OH). 1H NMR (CDCl3): d 7.61 (s, 1H), 7.41 (d,
J = 7.6 Hz, 2H), 7.31 (d, J = 7.2 Hz, 2H), 7.28 (q, 1H), 6.18 (d,
J = 2.0 Hz, 1H), 6.02 (d, J = 2.0 Hz, 1H), 5.27 (s, 1H, NH), 4.47 (s,
1H), 4.14 (m, 1H), 3.78 (d, 2H), 2.71 (m, 1H), 1.70 (m, 2H), 1.80
(m, 2H), 1.57 (d, J = 6.8 Hz, 6H), 1.46 (s, 9H), 1.30 (m, 2H), 1.25
(m, 2H). Anal. Calcd for C28H38Cl4N3Oꢁ1H2O: C, 67.71; H, 8.12; N,
11.28. Found: C, 68.09; H, 7.77; N, 11.26.
6.23. 1-Isopropyl-6-((1-methylpiperidin-4-yl)methoxy)-1H-
benzo[d]imidazol-4-amine (22h)
Compound 21h (148.3 mg, 0.4 mmol) was dissolved into 75 mL
of EtOH and added to a Parr hydrogenation vessel previously
charged with Pd/C (10% w/w, 8.1 mg). After three vacuum/purge
cycles with H2, the vessel was charged to 50 psi with H2 and sha-
ken for 5 h. The mixture was filtered through a pad of Celite and
then the pad was washed with an additional 25 mL of EtOH. A
10 mL portion of concd HCl was added to resultant filtrate at
0 °C. The solvent was removed under reduced pressure. The resul-
tant solid was dissolved into 10 mL K2CO3 (aq, satd) and extracted
three times with 10 mL portions of Et2O. The combined organic ex-
tracts were washed three times with NaCl (aq, satd) and then dried
over Na2SO4. The organic phase was decanted and the solvent was
removed under reduced pressure to give 76.7 mg (51.1%) of 22h as
a yellow, hygroscopic solid. Mp 37.0–38.5 °C. SiO2 TLC Rf 0.22

P. Jain et al. / Bioorg. Med. Chem. 19 (2011) 359–373
371
(CH2Cl2/5% CH3OH/0.1% NH4OH). 1H NMR (CDCl3): d 7.73 (s, 1H),
6.23 (d, J = 2.0 Hz, 1H), 6.17 (d, J = 2.0 Hz, 1H), 4.64 (m, 1H), 3.98
(d, J = 5.6 Hz, 2H), 3.61 (d, J = 10.0 Hz, 1H), 2.74 (s, 3H), 2.68 (m,
2H), 2.06 (m, 2H), 1.95 (m, 2H), 1.65 (d, J = 6.8 Hz, 6H), 1.26 (m,
2H). Anal. Calcd for C17H26N4O: C, 67.52; H, 8.67; N, 18.53. Found:
C, 67.45; H, 8.41; N, 18.58.
2H), 0.97 (t, J = 7.2 Hz, 3H). Anal. Calcd for C14H21N3O: C, 67.98;
H, 8.56; N, 16.99. Found: C, 68.23; H, 8.54; N, 16.82.
6.27. N-Benzyl-6-butoxy-1-isopropyl-1H-benzo[d]imidazol-4-
amine (12)
Compound 22i (110.0 mg, 0.44 mmol) and solid NaBH(OAc)3
(282.8 mg, 1.3 mmol) were suspended in 2.5 mL of DCE. Benzalde-
hyde (54.0 lL, 0.5 mmol) was added in one portion and the mix-
6.24. N-Benzyl-1-isopropyl-6-((1-methylpiperidin-4-yl)meth-
oxy)-1H-benzo[d]imidazol-4-amine hydrochloride (11)
ture 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 combined extracts were washed with three
5 mL portions of NaCl (aq, satd) and then dried (Na2SO4). The
extract was decanted and the solvent was removed under reduced
pressure. The crude material was isolated on SiO2 with hexanes/EA
(1:3). This material was recrystallized from EA and hexanes to give
75.0 mg (50%) of 12 as a white crystalline solid. Mp 64.5–65.9 °C.
SiO2 TLC Rf 0.61 (CH2Cl2/5% CH3OH/0.1% NH4OH). 1H NMR (CDCl3):
d 7.70 (s, 1H), 7.41 (d, J = 7.6 Hz, 2H), 7.31 (t, J = 7.2 Hz, 2H), 6.20 (d,
J = 2.0 Hz, 1H), 6.05 (d, J = 2.0 Hz, 1H), 5.23 (m, 1H), 4.48 (d,
J = 6.8 Hz, 2H), 3.95 (t, J = 6.8 Hz, 2H), 1.77 (m, 2H), 1.58 (d,
J = 6.8 Hz, 6H), 1.51 (m, 2H), 0.97 (t, J = 7.6 Hz, 3H). Anal. Calcd
for C21H27N3Oꢁ1/7H2O: C, 74.18; H, 8.09; N, 12.36. Found: C,
74.17; H, 7.98; N, 12.37.
Compound 22h (63.7 mg, 0.16 mmol) and solid NaBH(OAc)3
(650.0 mg, 3.0 mmol) were added to 5 mL of DCE. Benzaldehyde
(18 lL, 1.2 mmol) was added in one portion and the mixture was
stirred for 12 h. The reaction mixture was quenched with the addi-
tion of 1 mL of NaHCO3 (aq, satd) and stirred for 10 min. The mix-
ture was taken up into 5% K2CO3 (aq) and then extracted three
times with 15 mL portions of Et2O. The combined organic extracts
were washed with 10 mL 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 EA. The HCl salt was prepared from 2 mL of ethe-
real HCl to give 38.1 mg (46%) of 9 as a white hygroscopic solid. Mp
92.1–92.7 °C. SiO2 TLC Rf 0.39 (CH2Cl2/5% CH3OH/0.1% NH4OH). 1
H
NMR (MeOD-d4): d 9.26 (s, 1H), 7.51 (s, 1H, NH) 7.42 (q, 2H), 7.35
(d, J = 7.2 Hz, 2H), 7.28 (q, 1H), 6.66 (d, J = 2.0 Hz, 1H), 6.25 (d,
J = 2.0 Hz, 1H), 4.48 (m, 1H), 3.95 (s, 1H), 3.43 (d, 2H), 3.30 (m,
1H), 3.01 (m, 2H), 2.88 (s, 3H), 2.14 (m, 2H), 2.13 (m, 2H), 1.58
(d, 6H), 1.28 (m, 2H). Anal. Calcd for C24H34Cl2N4Oꢁ4H2O: C,
53.63; H, 7.88; N, 10.42. Found: C, 53.31; H, 7.59; N, 10.31.
6.28. 1-Isopropyl-4-nitro-6-propoxy-1H-benzo[d]imidazole (21j)
A dry 100 mL one-necked flask was charged with 1-isopropyl-4-
nitro-1H-benzo[d]imidazol-6-ol, 19, (250.0 mg, 1.1 mmol), 1-iodo-
propane 23j (0.22 mL, 2.30 mmol), and 2 mL of DMF. NaH (81.4 mg,
3.4 mmol) was added at 0 °C in two portions. This solution was
stirred at 0 °C for 30 min and at 23 °C for an additional 2 h. The
reaction mixture was poured into a mixture of 10 mL EA and
10 mL of K2CO3 (aq, satd). The mixture was extracted three times
with 10 mL portions of EA and the combined extracts were washed
with 10 mL of NaCl (aq, satd) and dried (Na2SO4). The extract was
decanted and the solvent was removed under reduced pressure.
The crude material (180.9 mg) was isolated on SiO2 and eluted
with hexanes/EA (1:3). Recrystallization from EA gave 163.6 mg
(55%) of 23j as a yellow solid. Mp 121.1–121.9 °C. SiO2 TLC Rf
0.51 (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),
4.05 (q, J = 6.8 Hz, 2H), 1.89 (m, 2H), 1.64 (d, J = 6.8 Hz, 6H), 1.09
(t, J = 7.2 Hz, 3H). Anal. Calcd for C13H17N3O3: C, 59.30; H, 6.51;
N, 15.96. Found: C, 59.21; H, 6.54; N, 15.58.
6.25. 6-Butoxy-1-isopropyl-4-nitro-1H-benzo[d]imidazole (21i)
A dry 100 mL one-necked flask was charged with 1-isopropyl-4-
nitro-1H-benzo[d]imidazol-6-ol, 19 (400.0 mg, 1.8 mmol), 1-iodo-
butane, 23i, (0.3 mL, 2.72 mmol), and 2.5 mL of DMF. NaH
(130.2 mg, 5.4 mmol) was added at 0 °C in two portions. This solu-
tion was stirred at 0 °C for 30 min and then at 23 °C for an addi-
tional 2 h. The reaction mixture was poured into a mixture of
10 mL EA and 10 mL K2CO3 (aq, satd). The mixture was extracted
(3 ꢀ 10 mL) with EA and the combined extracts were washed with
(2 ꢀ 5 mL) of NaCl (aq, satd) then dried (Na2SO4). The organic
phase was decanted and the solvent was removed under reduced
pressure. The residue was isolated on a SiO2 column with hex-
anes/EA (1:2). Recrystallization from EA and hexane gave
200.6 mg (40%) of 21i as a yellow solid. Mp 73.9–74.2 °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), 4.08 (t, J = 6.4 Hz, 2H), 1.84 (m, 2H), 1.64 (d, J = 6.8 Hz,
2H), 1.53 (m, 6H), 1.01 (t, J = 7.2 Hz, 3H). Anal. Calcd for
C14H19N3O3: C, 60.63; H, 6.91; N, 15.15. Found: C, 60.67; H, 6.92;
6.29. 1-Isopropyl-6-propoxy-1H-benzo[d]imidazol-4-amine (22j)
N, 15.14.
Compound 21j (150.4 mg, 0.57 mmol) was dissolved into
75 mL of EtOH and added to a Parr hydrogenation vessel previ-
ously charged with Pd/C (10% w/w, 7.7 mg). After three vac-
uum/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 the pad was washed with an additional
25 mL of EtOH. The solvent was removed under reduced pressure
to give 119.6 mg (90%) of 22j as a pale brown, hygroscopic solid.
Mp 30.2–30.9 °C. SiO2 TLC Rf 0.32 (CH2Cl2/5% CH3OH/0.1%
NH4OH). 1H NMR (CDCl3): d 7.74 (s, 1H), 6.27 (d, J = 2.0 Hz, 1H),
6.20 (d, J = 2.0 Hz, 1H), 4.47 (m, 1H), 3.93 (t, J = 6.4 Hz, 2H), 1.83
(m, 2H), 1.59 (d, J = 6.8 Hz, 6H), 1.05 (t, J = 7.2 Hz, 3H). Anal. Calcd
for C13H19N3Oꢁ1/5H2O: C, 65.91; H, 8.25; N, 17.74. Found: C,
66.11; H, 8.21; N, 17.39.
6.26. 6-Butoxy-1-isopropyl-1H-benzo[d]imidazol-4-amine (22i)
Compound 21i (170.3 mg, 0.61 mmol) was dissolved into 75 mL
of EtOH and added to a Parr hydrogenation vessel previously
charged with Pd/C (10% w/w, 8.5 mg). After three vacuum/purge
cycles with H2, the vessel was charged to 50 psi with H2 and sha-
ken 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 re-
moved under reduced pressure to give 133.4 mg (88%) of 22i as a
pale brown, hygroscopic solid. Mp 33.9–35.1 °C. SiO2 TLC Rf 0.34
(CH2Cl2/5% CH3OH/0.1% NH4OH). 1H NMR (CDCl3): d 7.74 (s, 1H),
6.25 (d, J = 2.0 Hz, 1H), 6.19 (d, J = 2.0 Hz, 1H), 4.47 (m, 1H), 3.96
(t, J = 6.4 Hz, 2H), 1.78 (m, 2H), 1.57 (d, J = 6.8 Hz, 6H), 1.56 (m,

372
P. Jain et al. / Bioorg. Med. Chem. 19 (2011) 359–373
6.30. N-Benzyl-1-isopropyl-6-propoxy-1H-benzo[d]imidazol-4-
amine (13)
ture was stirred for 12 h. The reaction mixture was quenched
with the addition of 1 mL of NaHCO3 (aq, satd) then stirred for
an additional 10 min. The mixture was taken up into 25 mL of 5%
K2CO3 (aq, satd) and extracted three times with 15 mL portions
of Et2O. The combined extracts were washed (3 ꢀ 5 mL) with NaCl
(aq, satd) and then dried (Na2SO4). The extract was decanted and
the solvent was removed under reduced pressure. The crude mate-
rial was isolated on SiO2 eluted with hexane/EA (1:3). This material
was recrystallized from EA and hexanes to give 77.6 mg (50%) of 14
as a white crystalline solid. Mp: 124.1–124.7 °C. SiO2 TLC Rf 0.57
(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), 6.19 (d, J = 2.0 Hz,
1H), 6.03 (d, J = 2.0 Hz, 1H), 5.25 (m, 1H), 4.46 (m, 2H), 4.02 (q,
J = 6.8 Hz, 2H), 1.57 (d, J = 6.8 Hz, 1H), 1.39 (t, J = 7.2 Hz, 3H). Anal.
Calcd for C19H23N3O: C, 73.76; H, 7.49; N, 13.58. Found: C, 73.49; H,
7.12; N, 13.64.
Compound 22j (100.0 mg, 0.43 mmol) and solid NaBH(OAc)3
(272.6 mg, 1.3 mmol) were suspended in 2.5 mL of DCE. Benzalde-
hyde (53.0 lL, 0.5 mmol) was added in one portion and the mix-
ture 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 por-
tions of Et2O. The combined extracts were washed three times with
5 mL portions of NaCl (aq, satd) and then dried (Na2SO4). The
extract was decanted and the solvent was removed under reduced
pressure. Crude 13 was isolated on SiO2 and eluted with hexanes/
EA. This material was recrystallized from EA and hexanes gave
77.0 mg (55%) of 13 as
a
white crystalline solid. Mp
102.4–104 °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), 7.31 (t,
J = 7.2 Hz, 2H), 6.19 (d, J = 2.0 Hz, 1H), 6.05 (d, J = 2.0 Hz, 1H),
5.23 (m, 1H), 4.46 (d, J = 6.8 Hz, 2H), 3.90 (t, J = 6.4 Hz, 2H), 1.80
(m, 2H), 1.57 (d, J = 6.8 Hz, 6H), 1.02 (t, J = 7.6 Hz, 3H). Anal. Calcd
for C20H25N3O: C, 74.35; H, 7.90; N, 12.97. Found: C, 74.27; H, 7.79;
N, 12.99.
Acknowledgments
The vectors for CDK5 and p25 were developed by Dr. L. H. Tsai
(Harvard Medical School, Department of Pathology) and were used
with his permission. We would like to thank Mr. Tim Holtzer for
acquisition and analysis of High Resolution Mass Spectra, and
NIH (NINDS): 1R15NS057772, NSF Research Experiences for
Undergraduates (REU): CHE-0354052, and an NSF equipment grant
for the NMR (NMR: CHE 0614785). This research was also supported
by grants from the ‘Association France-Alzheimer Finistère’ (L.M.),
the ‘Association pour la Recherche sur le Cancer’ (ARC-1092)
(L.M.), the ‘Ligue Nationale contre le Cancer’ (Comité Grand-Ouest)
(L.M.).
6.31. 6-Ethoxy-1-isopropyl-4-nitro-1H-benzo[d]imidazole (21k)
A dry 100 mL one-necked flask was compound 19 (250.0 mg,
1.1 mmol), ethyl iodide, 23k, (0.2 mL, 2.05 mmol) and 2 mL of
DMF. NaH (81.4 mg, 3.4 mmol) was added at 0 °C in two portions.
This suspension 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 5 mL of NaCl (aq, satd) and then dried (Na2SO4). The extract
was decanted and the solvent was removed under reduced pressure.
The residue was loaded onto a SiO2 column and eluted with hexanes/
EA (1:3). Recrystallization from EA and hexane gave 169.0 mg
(60%) of 21k as a yellow solid. Mp 82.7–82.9 °C. SiO2 TLC Rf 0.52
(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), 4.16 (q,
J = 6.8 Hz, 2H), 1.64 (d, J = 6.8 Hz, 6H), 1.48 (t, J = 7.2 Hz, 3H). Anal.
Calcd for C12H15N3O3ꢁ1/5H2O: C, 57.00; H, 6.14; N, 16.62. Found: C,
56.93; H, 6.00; N, 16.49.
References and notes
1. Hebert, L. E.; Scherr, P. A.; Bienias, J. L.; Bennett, D. A. Arch. Neurol. 2003, 60,
1119.
2. Ernst, R. L.; Hay, J. W. Am. J. Public Health 1994, 84, 1261.
3. Dimakopoulos, A. C. Curr. Alzheimer Res. 2005, 2, 19.
4. Ferrer, I.; Gomez-Isla, T.; Puig, B.; Freixes, M.; Ribe, E.; Dalfo, E.; Avila, J. Curr.
Alzheimer Res. 2005, 2, 3.
5. Higuchi, M.; Iwata, N.; Saido, T. C. Biochim. Biophys. Acta 2005, 1751, 60.
6. Iwata, N.; Higuchi, M.; Saido, T. C. Pharmacol. Ther. 2005, 108, 129.
7. Michaelis, M. L.; Seyb, K. I.; Ansar, S. Curr. Alzheimer Res. 2005, 2, 227.
8. Thompson, L. A.; Bronson, J. J.; Zusi, F. C. Curr. Pharm. Des. 2005, 11, 3383.
9. De Strooper, B.; Vassar, R.; Golde, T. Nat. Rev. Neurol. 2010, 6, 99.
10. Citron, M. Nat. Rev. Drug Disc. 2010, 9, 387.
11. Panza, F.; Solfrizzi, V.; Frisardi, V.; Capurso, C.; D’Introno, A.; Colacicco, A. M.;
Vendemiale, G.; Capurso, A.; Imbimbo, B. P. Drugs Aging 2009, 26, 537.
12. Burns, A. Lancet Neurol. 2009, 8, 4.
6.32. 6-Ethoxy-1-isopropyl-1H-benzo[d]imidazol-4-amine (22k)
13. Tang, X.; Wang, X.; Gong, X.; Tong, M.; Park, D.; Xia, Z.; Mao, Z. J. Neurosci. 2005,
25, 4823.
Compound 21k (150.0 mg, 0.60 mmol) was dissolved into
75 mL of EtOH and added to a Parr hydrogenation vessel previously
charged with Pd/C (10% w/w, 8.1 mg). After three vacuum/purge
cycles with H2, the vessel was charged to 50 psi with H2 and sha-
ken for 5 h. The mixture was filtered through a pad of Celite and
the pad was washed with an additional 25 mL of EtOH. The solvent
was removed under reduced pressure to give 128.0 mg (97%) of
22k as a pale brown, hygroscopic solid. Mp 37.2–38.0 °C. SiO2
TLC Rf 0.30 (CH2Cl2/5% CH3OH/0.1% NH4OH). 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 (q, J = 6.8 Hz, 2H), 1.57 (d, J = 6.8 Hz, 1H), 1.42 (t,
J = 7.2 Hz, 3H). Anal. Calcd for C12H17N3Oꢁ1/3H2O: C, 63.98; H,
7.90; N, 18.05. Found: C, 63.83; H, 7.77; N, 17.78.
14. Taru, H.; Yoshikawa, K.; Suzuki, T. FEBS Lett. 2004, 567, 248.
15. Marks, N.; Berg, M. J. Neurochem. Res. 2010, 35, 181.
16. De Kimpe, L.; Scheper, W. Curr. Med. Chem. 2010, 17, 198.
17. Campbell, W. A.; Wolfe, M. S.; Xia, W. Amyloid Precursor Protein 2005, 51.
18. Churcher, I.; Beher, D. Curr. Pharm. Des. 2005, 11, 3363.
19. Bergmans Bruno, A.; De Strooper, B. Lancet Neurol. 2010, 9, 215.
20. Wu, W.-L.; Zhang, L. Drug Dev. Res. 2009, 70, 94.
21. Wolfe Michael, S. Semin. Cell Dev. Biol. 2009, 20, 219.
22. Cole, S. L.; Vassar, R. J. Biol. Chem. 2008, 283, 29621.
23. Meraz-Rios, M. A.; Lira-De Leon, K. I.; Campos-Pena, V.; De Anda-Hernandez, M.
A.; Mena-Lopez, R. J. Neurochem. 2010, 112, 1353.
24. Wolfe, M. S. J. Biol. Chem. 2009, 284, 6021.
25. Spires-Jones, T. L.; Stoothoff, W. H.; de Calignon, A.; Jones, P. B.; Hyman, B. T.
Trends Neurosci. 2009, 32, 150.
26. Rissman, R. A. J. Alzheimer Dis. 2009, 18, 453.
27. Hanger, D. P.; Anderton, B. H.; Noble, W. Trends Mol. Med. 2009, 15, 112.
28. Li, B. S.; Ma, W.; Jaffe, H.; Zheng, Y.; Takahashi, S.; Zhang, L.; Kulkarni, A. B.;
Pant, H. C. J. Biol. Chem. 2003, 278, 35702.
29. Hooper, C.; Killick, R.; Lovestone, S. J. Neurochem. 2008, 104, 1433.
30. Gillardon, F.; Steinlein, P.; Burger, E.; Hildebrandt, T.; Gerner, C. Proteomics
2005, 5, 1299.
6.33. N-Benzyl-6-ethoxy-1-isopropyl-1H-benzo[d]imidazol-4-
amine (14)
31. Gong, X.; Tang, X.; Wiedmann, M.; Wang, X.; Peng, J.; Zheng, D.; Blair, L. A.;
Marshall, J.; Mao, Z. Neuron 2003, 38, 33.
32. Smith, P. D.; Mount, M. P.; Shree, R.; Callaghan, S.; Slack, R. S.; Anisman, H.;
Vincent, I.; Wang, X.; Mao, Z.; Park, D. S. J. Neurosci. 2006, 26, 440.
Compound 22k (110.0 mg, 0.50 mmol) and NaBH(OAc)3
(318.9 mg, 1.5 mmol) were suspended in 2.5 mL of DCE. Benzalde-
hyde (61.0 lL, 0.6 mmol) was added in one portion and the mix-

P. Jain et al. / Bioorg. Med. Chem. 19 (2011) 359–373
373
33. Verdaguer, E.; Alvira, D.; Jimenez, A.; Rimbau, V.; Camins, A.; Pallas, M. Br. J.
Pharmacol. 2005, 145, 1103.
67. Arienti, K. L.; Brunmark, A.; Axe, F. U.; McClure, K.; Lee, A.; Blevitt, J.; Neff, D. K.;
Huang, L.; Crawford, S.; Pandit, C. R.; Karlsson, L.; Breitenbucher, J. G. J. Med.
Chem. 2005, 48, 1873.
68. Sondhi Sham, M.; Singh, N.; Kumar, A.; Lozach, O.; Meijer, L. Bioorg. Med. Chem.
2006, 14, 3758.
69. Dussault, I.; Bellon, S. F. Anti-Cancer Agents Med. Chem. 2009, 9, 221.
70. Petrelli, A.; Giordano, S. Curr. Med. Chem. 2008, 15, 422.
71. Zhou, Z.; Madura, J. D. Proteins: Structure, Function, Bioinform. 2004, 57, 493.
72. Zhou, Z.; Madura, J. D. J. Chem. Info. Comput. Sci. 2004, 44, 2167.
73. MOE: Molecular Operating Environment (MOE), version 2008.10; Chemical
Computing Group, Inc.: Montreal, 2008.
74. Booth, R. J.; Dobrusin, E. M.; Toogood, P. L.; Vanderwel, S. N. Application: WO
Patent 2001-US2657, 2001070741; 2001, p 119.
75. Toogood, P. L.; Harvey, P. J.; Repine, J. T.; Sheehan, D. J.; VanderWel, S. N.; Zhou,
H.; Keller, P. R.; McNamara, D. J.; Sherry, D.; Zhu, T.; Brodfuehrer, J.; Choi, C.;
Barvian, M. R.; Fry, D. W. J. Med. Chem. 2005, 48, 2388.
34. O’Hare, M. J.; Kushwaha, N.; Zhang, Y.; Aleyasin, H.; Callaghan, S. M.; Slack, R.
S.; Albert, P. R.; Vincent, I.; Park, D. S. J. Neurosci. 2005, 25, 8954.
35. Zheng, Y. L.; Kesavapany, S.; Gravell, M.; Hamilton, R. S.; Schubert, M.; Amin,
N.; Albers, W.; Grant, P.; Pant, H. C. EMBO J. 2005, 24, 209.
36. Knockaert, M.; Blondel, M.; Bach, S.; Leost, M.; Elbi, C.; Hager, G. L.; Nagy, S. R.;
Han, D.; Denison, M.; Ffrench, M.; Ryan, X. P.; Magiatis, P.; Polychronopoulos,
P.; Greengard, P.; Skaltsounis, L.; Meijer, L. Oncogene 2004, 23, 4400.
37. Knockaert, M.; Greengard, P.; Meijer, L. Trends Pharmacol. Sci. 2002, 23, 417.
38. Meijer, L.; Bisagni, E.; Legraverend, M. WO19961129. Patent 96-FR1905,
9720842; 1997, 52.
39. Meijer, L.; Borgne, A.; Mulner, O.; Chong, J. P. J.; Blow, J. J.; Inagaki, N.; Inagaki,
M.; Delcros, J. G.; Moulinoux, J. P. Eur. J. Biochem. 1997, 243, 527.
40. Meijer, L.; Raymond, E. Acc. Chem. Res. 2003, 36, 417.
41. Senderowicz, A. M. Oncogene 2003, 22, 6609.
42. Sielecki, T. M.; Boylan, J. F.; Benfield, P. A.; Trainor, G. L. J. Med. Chem. 2000, 43,
1.
43. Songyang, Z.; Lu, K. P.; Kwon, Y. T.; Tsai, L. H.; Filhol, O.; Cochet, C.; Brickey, D.
A.; Soderling, T. R.; Bartleson, C.; Graves, D. J.; DeMaggio, A. J.; Hoekstra, M. F.;
Blenis, J.; Hunter, T.; Cantley, L. C. Mol. Cell. Biol. 1996, 16, 6486.
44. Senderowicz, A. M. Cancer Biol. Ther. 2003, 2, S84.
45. Stein, P. A.; Toret, C. P.; Salic, A. N.; Rolls, M. M.; Rapoport, T. A. J. Cell Sci. 2002,
115, 3389.
46. Ahn, J. S.; Radhakrishnan, M. L.; Mapelli, M.; Choi, S.; Tidor, B.; Cuny, G. D.;
Musacchio, A.; Yeh, L. A.; Kosik, K. S. Chem. Biol. 2005, 12, 811.
47. Mapelli, M.; Massimiliano, L.; Crovace, C.; Seeliger, M. A.; Tsai, L. H.; Meijer, L.;
Musacchio, A. J. Med. Chem. 2005, 48, 671.
76. VanderWel, S. N.; Harvey, P. J.; McNamara, D. J.; Repine, J. T.; Keller, P. R.; Quin,
J., III; Booth, R. J.; Elliott, W. L.; Dobrusin, E. M.; Fry, D. W.; Toogood, P. L. J. Med.
Chem. 2005, 48, 2371.
77. Moore, M. D.; Jain, P.; Flaherty, P. T.; Wildfong, P. L. D. Acta Crystallogr., Sect. E:
Struct. Rep. Online 2008, E64, o1336.
78. Flaherty, P. T.; Chopra, I.; Jain, P.; Yi, S.; Allen, E.; Cavanaugh, J. Bioorg. Med.
Chem. Lett. 2010, 20, 2892.
79. Chen, Y.; Wang, P. G. Tetrahedron Lett. 2001, 42, 4955.
80. Sabat, M.; VanRens, J. C.; Laufersweiler, M. J.; Brugel, T. A.; Maier, J.;
Golebiowski, A.; De, B.; Easwaran, V.; Hsieh, L. C.; Walter, R. L.; Mekel, M. J.;
Evdokimov, A.; Janusz, M. J. Bioorg. Med. Chem. Lett. 2006, 16, 5973.
81. Ni, Z.-J.; Barsanti, P.; Brammeier, N.; Diebes, A.; Poon, D. J.; Ng, S.; Pecchi, S.;
Pfister, K.; Renhowe, P. A.; Ramurthy, S.; Wagman, A. S.; Bussiere, D. E.; Le, V.;
Zhou, Y.; Jansen, J. M.; Ma, S.; Gesner, T. G. Bioorg. Med. Chem. Lett. 2006, 16,
3121.
48. Tarricone, C.; Dhavan, R.; Peng, J.; Areces, L. B.; Tsai, L. H.; Musacchio, A. Mol.
Cell. 2001, 8, 657.
49. Monaco, E. A., 3rd Curr. Alzheimer Res. 2004, 1, 33.
50. Monaco, E. A., 3rd; Vallano, M. L. Curr. Med. Chem. 2003, 10, 367.
51. Monaco, E. A., 3rd; Vallano, M. L. Front Biosci. 2005, 10, 143.
52. Chae, T.; Kwon, Y. T.; Bronson, R.; Dikkes, P.; Li, E.; Tsai, L. H. Neuron 1997, 18,
29.
53. Patrick, G. N.; Zukerberg, L.; Nikolic, M.; de la Monte, S.; Dikkes, P.; Tsai, L. H.
Nature 1999, 402, 615.
82. McBride, C. M.; Renhowe, P. A.; Gesner, T. G.; Jansen, J. M.; Lin, J.; Ma, S.; Zhou,
Y.; Shafer, C. M. Bioorg. Med. Chem. Lett. 2006, 16, 3789.
83. Moriarty, K. J.; Winters, M.; Qiao, L.; Ryan, D.; DesJarlais, R.; Robinson, D.; Cook,
B. N.; Kashem, M. A.; Kaplita, P. V.; Liu, L. H.; Farrell, T. M.; Khine, H. H.; King, J.;
Pullen, S. S.; Roth, G. P.; Magolda, R.; Takahashi, H. Bioorg. Med. Chem. Lett.
2008, 18, 5537.
54. Tsai, L. H.; Delalle, I.; Caviness, V. S., Jr.; Chae, T.; Harlow, E. Nature 1994, 371,
419.
55. Zheng, Y. L.; Li, B. S.; Amin, N. D.; Albers, W.; Pant, H. C. Eur. J. Biochem. 2002,
269, 4427.
84. Trujillo, J. I.; Kiefer, J. R.; Huang, W.; Thorarensen, A.; Xing, L.; Caspers, N. L.;
Day, J. E.; Mathis, K. J.; Kretzmer, K. K.; Reitz, B. A.; Weinberg, R. A.; Stegeman,
R. A.; Wrightstone, A.; Christine, L.; Compton, R.; Li, X. Bioorg. Med. Chem. Lett.
2009, 19, 908.
56. Michaelis, M. L.; Dobrowsky, R. T.; Li, G. J. Mol. Neurosci. 2002, 19, 289.
57. Zhang, M.; Li, J.; Chakrabarty, P.; Bu, B.; Vincent, I. Am. J. Pathol. 2004, 165, 843.
58. Mermillod, P.; Tomanek, M.; Marchal, R.; Meijer, L. Mol. Reprod. Dev. 2000, 55,
89.
85. Ramurthy, S.; Subramanian, S.; Aikawa, M.; Amiri, P.; Costales, A.; Dove, J.;
Fong, S.; Jansen, J. M.; Levine, B.; Ma, S.; McBride, C. M.; Michaelian, J.; Pick, T.;
Poon, D. J.; Girish, S.; Shafer, C. M.; Stuart, D.; Sung, L.; Renhowe, P. A. J. Med.
Chem. 2008, 51, 7049.
59. Patrick, G. N.; Zhou, P.; Kwon, Y. T.; Howley, P. M.; Tsai, L. H. J. Biol. Chem. 1998,
273, 24057.
60. Tsai, L. H.; Lee, M. S.; Cruz, J. Biochim. Biophys. Acta 2004, 1697, 137.
61. Helal, C. J.; Kang, Z.; Lucas, J. C.; Gant, T.; Ahlijanian, M. K.; Schachter, J. B.;
Richter, K. E. G.; Cook, J. M.; Menniti, F. S.; Kelly, K.; Mente, S.; Pandit, J.; Hosea,
N. Bioorg. Med. Chem. Lett. 2009, 19, 5703.
86. McBride, C. M.; Renhowe, P. A.; Heise, C.; Jansen, J. M.; Lapointe, G.; Ma, S.;
Pineda, R.; Vora, J.; Wiesmann, M.; Shafer, C. M. Bioorg. Med. Chem. Lett. 2006,
16, 3595.
87. Neff, D. K.; Lee-Dutra, A.; Blevitt, J. M.; Axe, F. U.; Hack, M. D.; Buma, J. C.;
Rynberg, R.; Brunmark, A.; Karlsson, L.; Breitenbucher, J. G. Bioorg. Med. Chem.
Lett. 2007, 17, 6467.
62. Strobel, H.; Nemecek, C.; Lesuisse, D.; Ruf, S.; El-Ahmad, Y.; Guessregen, S.;
Lebrun, A.; Ritter, K.; Benard, D.; Hittinger, A.; Bouchard, H. Application: EP
Patent 2004-2919041621536; 2006, p 82.
63. Hwang, J.-S.; Kregler, O.; Schilf, R.; Bannert, N.; Drach, J. C.; Townsend, L. B.;
Bogner, E. J. Virol. 2007, 81, 11604.
64. Hwang, J.-S.; Schilf, R.; Drach, J. C.; Townsend, L. B.; Bogner, E. Antimicrob.
Agents Chemother. 2009, 53, 5095.
65. Townsend, L. B.; Revankar, G. R. Chem. Rev. 1970, 70, 389.
66. Townsend, L. B.; Wise, D. S. Parasitol. Today 1990, 6, 107.
88. Sessions, E. H.; Smolinski, M.; Wang, B.; Frackowiak, B.; Chowdhury, S.; Yin, Y.;
Chen, Y. T.; Ruiz, C.; Lin, L.; Pocas, J.; Schroeter, T.; Cameron, M. D.; Lo Grasso,
P.; Feng, Y.; Bannister, T. D. Bioorg. Med. Chem. Lett. 2010, 20, 1939.
89. Peddibhotla, S.; Shi, R.; Khan, P.; Smith, L. H.; Mangravita-Novo, A.; Vicchiarelli,
M.; Su, Y.; Okolotowicz, K. J.; Cashman, J. R.; Reed, J. C.; Roth, G. P. J. Med. Chem.
2010, 53, 4793.
90. Irlapati, N. R.; Adlington, R. M.; Conte, A.; Pritchard, G. J.; Marquez, R.; Baldwin,
J. E. Tetrahedron 2004, 60, 9307.
91. Sard, H.; Duffley, R. P.; Razdan, R. K. Synth. Commun. 1983, 13, 813.