Benzoylbenzimidazole-based selective inhibitors targeting Cryptosporidium parvum and Toxoplasma gondii calcium-dependent protein kinase-1

Article abstract of DOI:10.1016/j.bmcl.2012.06.050

Calcium-dependent protein kinase-1 (CDPK1) from Cryptosporidium parvum (CpCDPK1) and Toxoplasma gondii (TgCDPK1) have become attractive targets for discovering selective inhibitors to combat infections caused by these protozoa. We used structure-based design to improve a series of benzoylbenzimidazole-based compounds in terms of solubility, selectivity, and potency against CpCDPK1 and TgCDPK1. The best inhibitors show inhibitory potencies below 50 nM and selectivity well above 200-fold over two human kinases with small gatekeeper residues.

Full text of DOI:10.1016/j.bmcl.2012.06.050

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5264–5267
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Benzoylbenzimidazole-based selective inhibitors targeting Cryptosporidium
parvum and Toxoplasma gondii calcium-dependent protein kinase-1
Zhongsheng Zhang a, Kayode K. Ojo b, Steven M. Johnson c, Eric T. Larson a, Penqing He b,
Jennifer A. Geiger d, Alejandro Castellanos-Gonzalez e, A. Clinton White Jr. e, Marilyn Parsons d,
a,
Ethan A. Merritt a, Dustin J. Maly c, Christophe L. M. J. Verlinde a, Wesley C. Van Voorhis b, , Erkang Fan
⇑
⇑
a Department of Biochemistry, University of Washington, Seattle, WA 98195, USA
b Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, WA 98195, USA
c Department of Chemistry, University of Washington, Seattle, WA 98195, USA
d Seattle Biomedical Research Institute, Seattle, WA 98109, USA
e Infectious Disease Division, Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX 77555, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Calcium-dependent protein kinase-1 (CDPK1) from Cryptosporidium parvum (CpCDPK1) and Toxoplasma
gondii (TgCDPK1) have become attractive targets for discovering selective inhibitors to combat infections
caused by these protozoa. We used structure-based design to improve a series of benzoylbenzimidazole-
based compounds in terms of solubility, selectivity, and potency against CpCDPK1 and TgCDPK1. The best
inhibitors show inhibitory potencies below 50 nM and selectivity well above 200-fold over two human
kinases with small gatekeeper residues.
Received 20 March 2012
Accepted 15 June 2012
Available online 23 June 2012
Keywords:
Cryptosporidium parvum
Toxoplasma gondii
Calcium-dependent protein kinase-1
Enzyme inhibitor
Ó 2012 Elsevier Ltd. All rights reserved.
Selectivity
Cryptosporidium parvum and Toxoplasma gondii are apicom-
plexan protozoa that can infect humans and domestic animals.
Infection by C. parvum parasites is transmitted by water and causes
cryptosporidiosis.1 It results in debilitating diarrhea and may be
life-threatening for immune compromised people and malnour-
ished children. Nitazoxanide is the only approved drug available
to treat cryptosporidiosis, but it is too costly for routine use in
poorer countries.2 Toxoplasmosis caused by T. gondii is spread to
humans by ingestion of undercooked meat or oocysts from cat
feces in food or water. Human infection by this parasite is very
common and is lifelong.3 Infection by T. gondii can cause retinitis
in immunocompetent persons and serious fetal abnormalities dur-
ing pregnancy. Immunocompromised individuals may develop
fatal encephalitis. Most women of childbearing age in the United
States are susceptible to acute infection.4 Treatment options are
limited to a single first-line therapy (pyrimethamine-sulfadiazine),
and the need to be given lifelong in immunocompromised persons.
Both C. parvum and T. gondii are listed as biodefense agents due to
possible threats by food or water contamination. New therapies for
treating both parasite infections are needed.
Recently, the calcium-dependent protein kinase-1 (CDPK1)
found in both parasites was shown to be an attractive target for
drug discovery.5–7 That is because CpCDPK1 and TgCDPK1 are not
only associated with important calcium-regulated biological func-
tions such as secretion, invasion, and gliding motility,8–10 they also
offer unique opportunities for discovering selective inhibitors. First,
these two enzymes belong to a clade of CDPKs that are present in
plants but not in animals.11 Second, a unique sequence and struc-
tural feature of both enzymes is the small glycine residue at the
‘gatekeeper’ position adjacent to the ATP-binding cleft.5,12 This
structural feature allows for the design of selective inhibitors that
take advantage of the larger ATP-binding cavity near the gatekeeper
residue of the parasite enzymes as compared to human kinases, for
which a larger amino acid residue occupies the gatekeeper position
and leaves a smaller ATP-binding pocket.13,14 As a result, we were
able to show previously that pyrazolopyrimidine-based inhibitors
can be designed to optimally occupy the enlarged ATP-binding
pockets of these CDPK1s and selectively inhibit parasite enzymes
over human kinases.7,15 In this report, we describe structure-guided
optimization of compounds based on a different chemical scaffold
that can also selectively inhibit parasite CDPK1s.
Screening work by the Medical Structural Genomics of Patho-
genic Protozoa Consortium (http://www.msgpp.org) identified
methyl (5-benzoylbenzimidazol-2-yl)carbamate (1, mebendazole)
⇑
Corresponding authors.
E-mail addresses: wesley@u.washington.edu (W.C. Van Voorhis), erkang@u.wa-
shington.edu (E. Fan).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.050

Z. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5264–5267
5265
Table 1
O
OH
H
N
H
N
Enzyme inhibition data for compounds 4–6
a
NH
O
NH
O
ID R1 in Scheme 2
TgCDPK1
(IC50 M)
CpCDPK1
(IC50 M)
Human
kinase (IC50,
N
O
N
O
,
l
,
l
2
1
lM)
1
3
0.67
0.23
0.92
0.60
5.0 (Abl)
>10 (Src)
ꢀ10 (Abl)
>10 (Src)
O
O
H
N
NH2
b
NH2
3
N
Reaction conditionNs:H(2a) NaBH4, THF, rt.; (b) BrCN, H2O.
OH
4a
>3
>3
H
N
4b
>3
>3
Scheme 1. Synthesis of 2 and 3 for initial SAR studies.
NH
2
5a
0.14
0.15
OH
5b
0.38
0.13
0.59
0.18
OH
5c
0.094 (Abl)
0.10 (Abl)
O
O
O
NH
H
N
5d
0.13
0.18
NH
NH
2
b
a
S
H
N
H
N
2
5e
0.12
0.18
0.16
0.13
0.010 (Abl)
O
H
N
H
N
4a-b
c
NH2
0.55 (Abl)
>10 (Src)
Br
NH
6a
N
N
R
1
6b
6c
0.069
0.022
0.091
0.072
NH2
O
O
NH
2
H
N
N
5a-e
1
d
e
NH
O
1
3
6d
6e
6f
0.060
0.041
0.018
0.16
0.12
0.11
0.082
0.13
NH
NH
R
NH
H
N
NH
O
6a-i
N
R
1
NH
OH
HN
Reaction conditions: (a) potassium ethyl xanthate, H2O/EtOH, refluxing;
(b) HBr/Br2, HOAc; (c) R1-NH2, pyridine, microwave irradiation, 120 °C, 40
min, Boc is removed by TFA/DCM if necessary; d) R1-NH2, acetonitrile,
microwave irradiation, 100 °C, 2 h. Boc is removed by TFA/DCM if
necessary; e) R1-COOH, HATU DIPEA, microwave irradiation, 70 °C, 20
min, THF/DCM. Boc is removed by TFA/DCM if necessary.
6g
NH2
6h
6i
0.52
0.14
1.41
0.32
H
N
NH2
N
Scheme 2. Synthesis of alkyl, acyl, and aminoacylated derivatives of compound 3.
R
1
O
O
O
NH
F
F
c, d
HO
a, b
NH
2
NO
2
NO
2
R
O
1
R
O
1
N
N
N
N
f, g
NH
O
e
NH
2
NH
2
8
7a-r
Reaction conditions: (a) thionyl chloride, microwave, 85 °C, 30 min; (b)
benzene, AlCl3, microwave, 65 °C, 10 min; (c) R1-NH2, K2CO3, acetonitrile,
microwave irradiation, 75 °C, 30 min; (d) Zn powder, ammonium formate,
MeOH, 5 min, rt; (e) BrCN, K2CO3, acetonitrile, microwave irradiation, 70
°C, 20 min; (f) Boc-β-analine, HATU, DIPEA, microwave irradiation, 70°C,
20 min, THF/DCM; (g) TFA/DCM, 20 min, rt.
Scheme 3. Synthesis of N1-substituted compounds 7 and 8.

5266
Z. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5264–5267
Table 2
inhibitory potency (IC50
: 0.23 lM against TgCDPK1, 0.60 lM
Enzyme inhibition data for compounds 7 and 8
against CpCDPK1), indicating that the carbonyl function of the car-
bamate is not critical for inhibition. In addition, a crystal structure
of 1 bound to TgCDPK1 revealed that the binding pocket of CDPK1
has more room to accommodate hydrophilic substitutions around
the 2-aminobenzimidazole function of 3, as this group is pointing
towards open solvent. Therefore, a variety of alkyl, acyl, and ami-
noacyl substituted variants of 3 were prepared.
The synthesis of derivatives of 3 is shown in Scheme 2. Reaction
of 2-bromobenzimidazole with substituted amines gave alkylated
2-amino derivatives 4. Reaction of 1 with an amine produced urea
derivatives 5, and direct acylation of 3 generated amide analogs 6.
Enzyme inhibition data for compounds 4–6 is summarized in
Table 1. In general, addition of a variety of hydrophilic groups
through urea (5) or amide (6) linkages resulted in improvements
to the inhibition of parasite CDPK1s, with several analogs having
IC50 of less than 100 nM (see data for 6b–6f). However, selectivity
over human kinases with smaller gatekeeper residues, such as Abl
and Src (both contain threonine gatekeeper) was minimal, and
occasionally reversed undesirably (5e). Therefore, extension from
the 2-amino group of 3 did not yield significant improvements in
terms of selectivity.
In order to improve selectivity, a design approach validated in
the pyrazolopyrimidine class of inhibitors was taken into consider-
ation for the optimization of the benzoylbenzimidazoles. For the
pyrazolopyrimidine class of compounds, we found that selective
inhibition of parasite CDPK1 can be significantly enhanced by
simultaneously occupying the binding pockets close to the gate-
keeper residues, and a pocket in the enzyme that recognizes the ri-
bose moiety of ATP.16 For this reason, we designed compounds 7
(synthesized according to Scheme 3) with a variety of substituents
to fill the ribose binding pocket. In addition, one example of com-
pound 8, which combines features of 7 and 6 was also synthesized
for comparison.
ID
R1 in Scheme 3
TgCDPK1
(IC50 M)
CpCDPK1
(IC50 M)
Human kinase
(IC50 M)
,
l
,
l
, l
>10 (Src)
>10 (Abl)
7a
7b
7c
7d
0.015
0.15
0.035
0.26
NH
N
>10 (Src)
>10 (Abl)
NH
0.020
0.030
0.017
0.026
>10 (Src)
>10 (Abl)
N
H
N
7e
7f
1.00
0.41
0.66
0.029
0.87
0.26
0.79
0.030
H
N
N
7g
7h
>10 (Src)
>10 (Abl)
N
H
7i
0.068
0.071
N
7j
0.065
0.12
0.095
0.12
N
H
7k
NH
7l
0.067
0.063
0.23
0.13
7m
7n
7o
7p
7q
0.16
0.22
0.043
0.18
0.30
0.39
0.036
0.17
As shown in Table 2, this design approach of simultaneously
occupying the two pockets described above generally improved
both efficacy and selectivity of the inhibitors compared to the start-
ing compound 1 or 3. The best inhibitors contain a piperidine or a
piperizine ring bridged by 1 or 2 carbons from the benzimidazole
N1 position, such as 7a, 7c, or 7p. These inhibitors all have IC50 below
50 nM against both parasite CDPK1s, and are >200-fold selective
over human Src and Abl. The substitution on N1 of 7 is not compat-
ible to acylation on the 2-amino group as compound 8 lost inhibitory
potency when compared to 7a. In addition, the addition of hydro-
philic groups into 7 in general improved the solubility of this class
N
O
N
>10 (Src)
>10 (Abl)
N
N
N
N
F
7r
0.19
0.95
N
of inhibitors to >50
lM compared to ꢀ4
lM of the starting hit 1.
7s
0.26
0.57
0.83
0.86
The best inhibitors were also tested in cell-based assays for
antiparasitic activity and host cell toxicity using described proce-
dures.5,15,16 We found low toxicities against mammalian cells
8
>10 (Src)
NH
(EC50 generally >30
lM). Unfortunately, none of the compounds
showed activity below 1
lM against either C. parvum or T. gondii.
from a commercial library as a binder to TgCDPK1. Mebendazole 1
is an approved drug with broad spectrum anthelmintic activities.
Enzyme inhibition assay confirmed that 1 was inhibitory to both
The exact causes for the lack of cellular activity are still under
investigation but may arise from poor cell permeability, selective
export by molecular pumps, or intracellular inactivation.
In summary, using structure-based design, we synthesized a
series of benzoylbenzimidazole based inhibitors of CpCDPK1 and
TgCDPK1 that have low nM potency and good selectivity against
human kinases that have small gatekeeper residues. This offers a
new chemical scaffold upon which anti-cryptosporidiosis and
anti-toxoplasmosis drugs may be discovered.
parasite CDPK1 enzymes (IC50
: 0.67 lM against TgCDPK1,
0.92 M against CpCDPK1). A crystal structure of TgCDPK1 bound
l
to 1 reveals that compound 1 binds at the same site as the pyraz-
olopyrimidine class of inhibitors to TgCDPK1,16 which serves as the
starting point of our structure-based optimization of benzoylbenz-
imidazole-based compounds.
An initial structure activity relationship (SAR) study was per-
formed to probe the importance of functional groups in 1 for inhi-
bition of CDPK1 activity. As shown in Scheme 1, reduction converts
the ketone group in 1 to hydroxyl group in 2, but leads to loss of
Acknowledgments
This work is supported by the National Institutes of Health
grants R01AI089441 (E.A.M. and W.C.V.V.) and R01GM086858
(D.J.M.). J.A.G. was supported by a training grant from the National
inhibition towards both parasite CDPK1 enzymes (IC50 >3
lM).
Compound 3, without the carbamate moiety in 1, retains the

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We thank Dr. Suzanne Scheele for technical assistance.
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