2-Aryl benzimidazoles featuring alkyl-linked pendant alcohols and amines as inhibitors of checkpoint kinase Chk2

Article abstract of DOI:10.1016/j.bmcl.2007.09.098

A series of benzimidazole compounds containing pendant alcohol and amine moieties was found to be active against checkpoint kinase Chk2. These compounds were prepared to examine a potential hydrogen bond interaction with an active site residue and to inve

Full text of DOI:10.1016/j.bmcl.2007.09.098

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6467–6471
2-Aryl benzimidazoles featuring alkyl-linked pendant alcohols
and amines as inhibitors of checkpoint kinase Chk2
Danielle K. Neff,* Alice Lee-Dutra, Jonathan M. Blevitt, Frank U. Axe, Michael D. Hack,
Johnathan C. Buma, Raymond Rynberg, Anders Brunmark,
Lars Karlsson and J. Guy Breitenbucher
Johnson & Johnson Pharmaceutical Research & Development, L.L.C, 3210 Merryfield Row, San Diego, CA 92121, USA
Received 24 July 2007; revised 27 September 2007; accepted 28 September 2007
Available online 4 October 2007
Abstract—A series of benzimidazole compounds containing pendant alcohol and amine moieties was found to be active against
checkpoint kinase Chk2. These compounds were prepared to examine a potential hydrogen bond interaction with an active site res-
idue and to investigate replacement of a biaryl linker with an aliphatic system in an effort to improve solubility.
Ó 2007 Elsevier Ltd. All rights reserved.
The checkpoint kinase Chk2 is activated by ATM (atax-
ia-telangiectasia mutated) in response to DNA damage
and controls the downstream p53-dependent apoptosis
machinery.1 Deletion of Chk2 in mice blocks apoptosis
after lethal radiation in several cell types and increases
cell survival.2,3 Since tumor tissue is generally more
apoptosis resistant than normal cells, it is hypothesized
that inhibition of Chk2 would not protect these tissues.
Small molecule inhibitors of Chk2 may therefore have
utility in clinical situations, such as radio- and chemo-
therapy, where p53-dependent apoptosis triggered by
DNA insult leads to tissue toxicity.4–7
ther-linked biaryl moieties (Fig. 1).8,9 The role of the
distal aryl ring in binding was unknown in this system
and was thought to be a strong contributor to the poor
OH
Cl
O
O
N
N
H2N
H2N
S
O
N
H
N
H
1
2
IC50 = 2 nM
IC50 = 15 nM
Figure 1. Lead biaryl benzimidazole compounds.
Lead compounds from a series of benzimidazoles that
were identified as potent inhibitors of Chk2 are shown
in Figure 1.8,9 Molecular modeling suggested that the
high affinity of compound 1 is potentially due to a
hydrogen bonding interaction between an active site res-
idue (histamine 157) and the hydroxyl group of the ter-
minal phenol (Fig. 2). In an effort to exploit this possible
interaction toward increasing activity and to improve
overall solubility, analogs with pendant hydroxyl and
amine groups were desired.
Previous reports from this laboratory described the syn-
thesis of benzimidazole compounds with ether or thioe-
Keywords: Chk2; Cds1; Solubility; Aminoalcohol; Mitsunobu.
*
Figure 2. Compound 1 docked into an active site model of Chk2
showing hydrogen-bonding to Hist-157.
Corresponding author. Tel.: +1 858 320 3384; fax: +1 858 784
3116; e-mail: dneff1@prdus.jnj.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.098

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D. K. Neff et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6467–6471
O
O
c
a or b
H
H
Y
OH
O
OH
Y = alkyl linker
O
O
Y OH
O
Y OH
O
N
N
HO
H2N
d
N
H
N
H
Scheme 1. Synthesis of alkyl-linked pendant alcohols. Reagents and conditions: (a) Bromo alkyl alcohol, K2CO3, DMF, rt; (b) Diol, DIAD, PPh3,
CH2Cl2, rt; (c) 3,4-Diaminobenzoic acid, Na2S2O5, DMF, 90 °C (d) i—HOBt, DICI, DMF; ii–Rink amide resin, CH2Cl2; iii—20% TFA/CH2Cl2.
solubility of these compounds. To exemplify this issue,
the solubility of 2 in simulated intestinal fluid (SIF,
pH 7) is 0.007 mM and in simulated gastric fluid
(SGF, pH 2) is 0.031 mM.10 It was hoped that incorpo-
ration of more flexible alkyl linkers might increase solu-
bility; such changes would also allow for the placement
of hydrogen bond donors and acceptors in different
areas of the active site. As a result, benzimidazoles con-
taining alkyl-linked alcohols, amines, and amides were
envisioned. The synthesis of such alkyl-linked pendant
alcohol analogs is shown in Scheme 1.11
tant for kinase inhibition. Reducing the molecular
weight of the compounds and incorporating a hydroxyl
group yielded increased solubility for compounds 3 and
5 at neutral pH and increased solubility for compounds
3–5 at low pH, when compared to compound 2.
The synthesis of alkyl-linked pendant amines is shown
in Scheme 2.
Aldehyde intermediates were accessed by combining 4-
hydroxybenzaldehyde and various aminoalcohols under
Mitsunobu conditions. These aldehydes were cyclized to
benzimidazoles as previously described. The resulting
carboxylic acid was readily converted to the primary
amide by reaction with carbonyldiimidazole in DMF
followed by addition of ammonium carbonate.
The necessary aldehyde precursors were obtained
through alkylation of 4-hydroxybenzaldehyde with de-
sired bromoalcohols or coupling of appropriate diols
under Mitsunobu conditions. Subsequent benzimidazole
formation was carried out using the resultant aldehydes
with 3,4-diaminobenzoic acid in the presence of sodium
metabisulfite. These intermediates were coupled onto
Rink amide resin, allowing impurities to be removed
by thorough rinsing before acidic cleavage from the re-
sin to provide the desired amides.
Though the resulting amine analogs (Table 2) lacked
notable SAR trends with regard to the pendant amine,
all compounds tested for solubility reached the maximum
measured level at both neutral and low pH levels. Second-
ary amines (entries 12, 15, 17, 18) were not significantly
more potent than tertiary amines (entries 9–11, 13, 14,
16). Additionally, changes in linker length between the
ether and amine, from two to four carbons, did not dras-
tically affect activity. Increasing the size and lipophilicity
of the molecule by substituting the amine moiety with
methyl and benzyl groups resulted in an increase in activ-
ity (entries 9 and 10). Changing substitution on the amine
from a dimethyl to a cyclohexyl group (entries 13 and 14
consecutively), which also increased the size of the mole-
cule, yielded no change in potency.
The resulting compounds (Table 1) retained reasonable
activity against Chk2.11 Though these compounds were
less potent than compound 1, the inhibition shown indi-
cated that a biaryl system was not necessary to retain
nanomolar activity against the target. Interestingly, the
100-fold difference in activity between the cis versus
trans configurations of the methyl cyclohexane analogs
(entries 7 and 8) implied that not only the presence
but also the position of the pendant alcohol was impor-
Table 1. Pendant alcohols
O
Y OH
O
N
H2N
N
H
Yb
SIFc (mM)
SGFc (mM)
a
Compound
Chk2 IC50
3
4
5
6
7
8
52
73
4
7
Ethane
1,3-Propyl
>0.400
0.007
0.139
ND
>0.400
0.185
>0.400
ND
72 23
1,3-Cyclopentane (cis/trans)
1,4-Cyclohexane (cis/trans)
1,4-Methyl-cyclohexane (cis)
1,4-Methyl-cyclohexane (trans)
80
35
8
2
ND
ND
ND
ND
400 20
a IC50 values expressed in nM SEM, all values are means of at least three replicated experiments.
b As described in Scheme 1.
c Compounds with solubility >0.4 mM are soluble above the upper limit of the assay.

D. K. Neff et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6467–6471
6469
O
O
H
R1
O Z N
R2
a
b
H
OH
O
O
R1
O Z N
R2
R1
O Z N
R2
N
N
HO
c
H2N
N
H
N
H
Scheme 2. Synthesis of alkyl-linked pendant amines. Reagents and conditions: (a) Aminoalcohol, DIAD, PPh3, CH2Cl2, rt; (b) 3,4-Diaminobenzoic
acid, Na2S2O5, DMF, 90 °C; (c) i—CDI, DMF, rt; ii—(NH4)2CO3.
Table 2. Pendant amines
O
R1
R2
N
N
H
H2N
O Z N
Compound
Chk2 IC50
710 120
Linker lengthb
2
SIFc (mM)
ND
SGFc (mM)
ND
a
-O-Z-NR1R2
O
N
9
N
O
10
11
12
13
14
15
16
17
18
109
174
2
7
2
2
2
3
3
3
3
3
4
ND
ND
O
N
>0.400
>0.400
>0.400
>0.400
ND
>0.400
>0.400
>0.400
>0.400
ND
O
180 15
176 16
NH
O
N
110
3
O
O
N
710 340
233 50
158 11
231 + 15
NH
O
O
>0.400
ND
>0.400
ND
N
NH
O
>0.400
>0.400
NH
a IC50 values expressed in nM SEM, all values are means of at least three replicated experiments.
b Number of carbons present between ether linkage and basic amine.
c Compounds with solubility >0.4 mM are soluble above the upper limit of the assay.
The promising activity of the benzyl substituted tertiary
amine (entry 10), along with the encouraging solubility
data for related compounds, led to the exploration of
fluoroacetic acid in methylene chloride, after which they
were submitted to reductive amination conditions to
yield the desired tertiary amine products.
a set of benzyl-substituted piperidine compounds
synthesized by the route shown in Scheme 3. N-Boc-
aminoalcohol intermediates were deprotected with tri-
Varying linker length and moving the nitrogen around the
piperidine ring did not reveal a preferred location for the
O
O
N
N
H2N
H2N
a
O
n
O
n
N
H
N
H
N
N
Boc
R3
˚
Scheme 3. Synthesis of benzylated piperidine analogs. Reagents and conditions: (a) i—20% TFA, CH2Cl2; ii—NaBH(OAc)3, aryl aldehyde, 4 A MS,
DMF.

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D. K. Neff et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6467–6471
Table 3. Substituted piperidine analogs
O
2
N
1
H2N
3
O
n
6
N
H
N
4
5
R3
a
Compound
Chk2 IC50
37
57 10
R3b
H
nb
N locationb
SIF (mM)
SGF (mM)
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
2
2
2
2
2
4
4
4
4
4
0.267
ND
ND
>0.400
ND
ND
4-CH3
4-OMe
4-Cl
55
14
2
8
ND
ND
ND
ND
180 150
70
3,4-di-Cl
H
3
>0.400
ND
ND
>0.400
ND
ND
70 16
67 20
4-CH3
4-OMe
4-Cl
60
176 148
45
8
ND
ND
ND
ND
3,4-di-Cl
H
5
0.155
0.097
0.084
0.115
0.229
0.005
0.003
<0.001
0.002
0.015
>0.400
>0.400
>0.400
>0.400
0.039
0.110
0.084
>0.400
0.179
0.013
61 25
74 47
53 42
114 50
90 40
4-CH3
4-OMe
4-Cl
3,4-di-Cl
H
90
5
4-CH3
4-OMe
4-Cl
95 14
82 22
157 33
3,4-di-Cl
a IC50 values expressed in nM SEM, all values are means of at least three replicated experiments.
b As described in Scheme 3.
nitrogen. The results shown in Table 3 demonstrate that
substitution on the benzyl group was well tolerated: 4-
chloro substitution was generally best, while 3,4-dichloro
substitution yielded the lowest activity. Though almost all
of the compounds tested for solubility in Table 3 exceeded
the solubility of the biaryl series (represented by com-
pound 2 [SIF 0.007 mM, SGF 0.031 mM]), these com-
pounds did not retain the solubility of the smaller, more
simple pendant amines shown in Table 2.
pendant alcohol compounds shown in Table 1. Notably,
the benzamide analogs were more active than their acet-
ylated counterparts. Unfortunately the removal of the
basic amine resulted in reduced solubility, especially evi-
dent in compounds 40 and 41, which have no basic
amine and an additional aryl group.
In summary, efforts to achieve greater solubility for
benzimidazole Chk2 inhibitors were successful. The
addition of pendant alcohols and amines as well as
reduction in compound size seem to play a role in
increasing the overall solubility of these molecules.
Exploration of a potentially beneficial hydrogen bond
interaction through the display of these pendant alco-
hols and amines resulted in relatively flat SAR with
respect to the Chk2 kinase; moreover, complete removal
of hydrogen bonding capability was not detrimental to
activity, as evidenced through the preparation of a series
of amide analogs. Furthermore, compounds with ali-
phatic linkers revealed that the biaryl system in previ-
ously described Chk2 inhibitors is not required to
achieve enzyme inhibition below 100 nM.
In order to gain further knowledge about the ability of
these benzimidazole analogs to participate in hydrogen
bonding, acetylated and benzoylated analogs were syn-
thesized by the route shown in Scheme 4. Again, N-
Boc-aminoalcohol intermediates were deprotected under
acidic conditions. The resulting secondary amines were
coupled with various anhydrides using DMAP in
DMF to form the desired amide products.
The results shown in Table 4 illustrate that hydrogen
bond donating ability was not necessary to retain activ-
ity, as the benzamide analogs were equipotent to the
O
O
N
N
H2N
H2N
a
O
n
O
n
N
H
N
H
N
N
O
R4
Boc
Scheme 4. Synthesis of benzoylated piperidine analogs. Reagents and conditions: (a) i—20% TFA, CH2Cl2; ii—(R4CO)2O, DMAP, DMF.

D. K. Neff et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6467–6471
6471
Table 4. Amide analogs
O
2
N
1
H2N
3
O
n
6
N
H
N
O
4
R4
5
R4b
nb
N locationb
SIF (mM)
SGF (mM)
a
Compound
Chk2 IC50
39
40
41
42
43
44
45
46
39 18
52 51
Ph
Ph
1
2
2
0
1
1
2
2
4
2
4
3
3
4
2
4
ND
ND
0.018
0.003
ND
0.028
0.003
ND
23
3
Ph
100 20
140 31
78 16
292 40
86 10
CH3
CH3
CH3
CH3
CH3
ND
ND
ND
ND
0.193
0.002
>0.400
0.087
a IC50 values expressed in nM SEM, all values are means of at least three replicated experiments.
b As described in Scheme 4.
9. McClure, K. J.; Huang, L.; Arienti, K. L.; Axe, F. U.;
Brunmark, A.; Blevitt, J.; Breitenbucher, G. J. Bioorg.
Med. Chem. Lett. 2006, 16, 1924.
References and notes
˚
1. Falck, J.; Mailand, N.; Syljuasen, R. G.; Bartek, J.; Lukas,
J. Nature 2001, 410, 842.
10. Equilibrium solubility: Solubility samples were prepared
by mixing 400 lmol of compounds (solids dissolved in
DMSO) with either 1 mL of SGF (0.1N HCl, 0.2% NaCl,
pH 1.2) or FasSIF (Galia et al. Pharm. Res. 15(5), 698–
705, 1998) for at least 3 days at room temperature. The
resulting mixtures were then filtered and the supernatants
were analyzed by UV-HPLC against external standards.
11. Activity of inhibitors was determined by incubating
inhibitory compounds with recombinant full-length
Chk2: 5 nM recombinant human Chk2, 50 mM Hepes
(pH 7.4), 100 mM NaCl, 10 mM MgCl2, 25 lM synthetic
peptide substrate (Biotin-SGLYRSPSMPENLNRPR,
1 lM ATP, 50 lCi/mL [g-33P]) ATP, and a protease
inhibitor mixture. The reaction mixtures were incubated at
37 °C for 3 h, and the peptide substrate was captured on
streptavidin conjugated to agarose beads. The agarose
beads were washed repeatedly with a 0.1% solution of
Tween-20 in phosphate-buffered saline, pH 7.4. Enzyme
activity at different Chk2 inhibitory compound concen-
trations was determined by measuring the amount of
radioactive phosphate bound to the substrate peptide by
scintillation counting.
2. Takai, H.; Naka, K.; Okada, Y.; Watanabe, M.; Harada,
N.; Saito, S.; Anderson, C. W.; Appella, E.; Nakanishi,
M.; Suzuki, H.; Nagashima, K.; Sawa, H.; Ikeda, K.;
Motoyama, N. EMBO J. 2002, 21, 5195.
3. Hirao, A.; Cheung, A.; Duncan, G.; Girard, P.; Elia, A. J.;
Wakeham, A.; Okada, H.; Sarkissian, T.; Wong, J. A.;
Sakai, T.; de Stanchina, E.; Bristow, R. G.; Suda, T.;
Lowe, S. W.; Jeggo, P. A.; Elledge, S. J.; Mak, T. W. W.
Mol. Cell Biochem. 2002, 22, 6521.
4. Ahn, J.; Urist, M.; Prives, C. DNA Cell Biol. 2004, 3,
1039.
5. Sharma, V.; Hupp, C. D.; Tepe, J. J. Curr. Med. Chem.
2007, 14, 1061.
6. Pommier, Y.; Sordet, O.; Rao, V. A.; Zhang, H.; Kohn,
K. W. Curr. Pharm. Des. 2005, 11, 2855.
7. Larson, G.; Yan, S.; Chen, H.; Rong, F.; Hong, Z.; Wu, J.
Z. Bioorg. Med. Chem. Lett. 2007, 17, 172.
8. Arienti, K. L.; Brunmark, A.; Axe, F. U.; McClure, K.;
Lee, A.; Blevitt, J.; Neff, D.; Huang, L.; Crawford, S.;
Pandit, C. R.; Karlsson, L.; Breitenbucher, J. G. J. Med.
Chem. 2005, 48, 1873.