Structure-activity relationship study of selective benzimidazole-based inhibitors of Cryptosporidium parvum IMPDH

Article abstract of DOI:10.1016/j.bmcl.2012.01.029

Cryptosporidium parasites are important waterborne pathogens of both humans and animals. The Cryptosporidium parvum and Cryptosporidium hominis genomes indicate that the only route to guanine nucleotides is via inosine 5′-monophosphate dehydrogenase (IMPDH). Thus the inhibition of the parasite IMPDH presents a potential strategy for treating Cryptosporidium infections. A selective benzimidazole-based inhibitor of C. parvum IMPDH (CpIMPDH) was previously identified in a high throughput screen. Here we report a structure-activity relationship study of benzimidazole-based compounds that resulted in potent and selective inhibitors of CpIMPDH. Several compounds display potent antiparasitic activity in vitro.

Full text of DOI:10.1016/j.bmcl.2012.01.029

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1985–1988
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship study of selective benzimidazole-based
inhibitors of Cryptosporidium parvum IMPDH
Sivapriya Kirubakaran a, , Suresh Kumar Gorla a, Lisa Sharling b, Minjia Zhang a, Xiaoping Liu b,
Soumya S. Ray d, Iain S. MacPherson a, Boris Striepen b,c, Lizbeth Hedstrom a,e,à, Gregory D. Cuny f,
⇑
a Department of Biology, Brandeis University, MS009, 415 South Street, Waltham, MA 02454, USA
b Center for Tropical and Emerging Global Diseases, University of Georgia, 500 D.W. Brooks Drive, Athens, GA 30602, USA
c Department of Cellular Biology, University of Georgia, 500 D.W. Brooks Drive, Athens, GA 30602, USA
d Center for Neurologic Diseases, Department of Neurology, Brigham Women’s Hospital and Harvard Medical School, 65 Landsdowne Street, Cambridge, MA 02139, USA
e Department of Chemistry, Brandeis University, MS009, 415 South Street, Waltham, MA 02454, USA
f Laboratory for Drug Discovery in Neurodegeneration, Brigham Women’s Hospital and Harvard Medical School, 65 Landsdowne Street, Cambridge, MA 02139, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Cryptosporidium parasites are important waterborne pathogens of both humans and animals. The Crypto-
sporidium parvum and Cryptosporidium hominis genomes indicate that the only route to guanine nucleo-
tides is via inosine 50-monophosphate dehydrogenase (IMPDH). Thus the inhibition of the parasite IMPDH
presents a potential strategy for treating Cryptosporidium infections. A selective benzimidazole-based
inhibitor of C. parvum IMPDH (CpIMPDH) was previously identified in a high throughput screen. Here
we report a structure–activity relationship study of benzimidazole-based compounds that resulted in
potent and selective inhibitors of CpIMPDH. Several compounds display potent antiparasitic activity
in vitro.
Received 12 December 2011
Revised 10 January 2012
Accepted 11 January 2012
Available online 24 January 2012
Keywords:
Cryptosporidium
Parasite
Protozoan
Inosine 50-monophosphate dehydrogenase
(IMPDH)
Ó 2012 Elsevier Ltd. All rights reserved.
Inhibitor
Benzimidazole
Structure–activity relationship
Cryptosporidiosis is a waterborne diarrheal disease caused by
protozoan parasites of the genus Cryptosporidium.1,2 While Crypto-
sporidium hominis is specific to humans, others such as Cryptospo-
ridium parvum infect humans and a wide range of animals and can
be transmitted zoonotically. Cryptosporidiosis is a major cause of
malnutrition in the developing world and can be life threatening
in immunocompromised patients. Cryptosporium oocysts are resis-
tant to commonly employed methods of water treatment, leading
to frequent outbreaks in the developed world. In addition, oocysts
are relatively easy to obtain, and therefore pose a credible biowar-
fare threat. No vaccines exist for Cryptosporidium infections and the
approved drugs are not particularly effective. Therefore, the tools
currently available to combat a massive outbreak are limited.
Like other apicomplexan parasites, Cryptosporidium is unable to
synthesize purine nucleotides de novo. Instead, Cryptosporidium re-
lies on a highly streamlined purine salvage pathway.3–5 The parasite
obtains adenosine from the host, which is converted sequentially to
AMP and IMP. The enzyme inosine 50-monophosphate dehydroge-
nase (IMPDH) converts IMP to XMP (Scheme 1). XMP is subse-
quently converted to GMP. Cryptosporidium does not contain
guanine salvage enzymes, so this pathway appears to be the only
route to guanine nucleotides.
Interestingly, Cryptosporidium acquired its IMPDH gene by lat-
eral gene transfer from an e-proteobacterium and consequently
the enzyme is highly divergent from the host counterpart.6 Thus,
selective inhibition of Cryptosporidium IMPDH presents a potential
strategy for treating cryptosporidiosis with minimal effects on its
mammalian host.7–9 The benzimidazole analog C was identified in
a high throughput screen targeting the highly diverged NAD binding
site of C. parvum IMPDH (CpIMPDH; this protein is identical to C.
hominis IMPDH) (Fig. 1).7 Compound C is a moderately potent but
⇑
Corresponding author. Tel.: +1 617 768 8640; fax: +1 617 768 8606.
E-mail addresses: hedstrom@brandeis.edu (L. Hedstrom), gcuny@rics.bwh.
harvard.edu (G.D. Cuny).
Present address: Dana-Farber Cancer Institute, Department of Biological Chem-
istry and Molecular Pharmacology Harvard Medical School, Seeley G. Mudd building,
Room 628, 250 Longwood Ave., Boston, MA 02115, USA.
highly selective inhibitor for CpIMPDH (IC50 = 1.2
detectable activity against the human IMPDH1 and IMPDH2
(IC50 > 50 M).
lM) with no
à
l
Tel.: +1 781 736 2333; fax: +1 781 736 2349.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.029

1986
S. Kirubakaran et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1985–1988
Table 1
SAR of the aniline ring
O
O
NAD+
NADH
N
N
N
HN
N
N
N
HN
+ 2H+
+ H2O
N
R
S
O
N
H
N
-
R
Y
O
XMP
IMP
X
R
Scheme 1. The IMPDH reaction. R = ribose-50-phosphate.
Compound
X
Y
R
IC50 (nM)
IC50 (nM)
BSA
The structure of CpIMPDH in complex with IMP and the C deriv-
Ca
C39
C43
C9
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
4-OMePh
4-SMePh
4-i-PrPh
4-FPh
1200 200 n.d.c
ative C64 was recently solved.10 This structure revealed the pres-
ence of a cavity next to the aniline ring of C64, suggesting that
more potent inhibitors would be obtained if the 4-bromoaniline
group was replaced with bulkier groups. This information was used
to guide the design of C90 and C97.10 As expected, the substitution
of 2-naphthyl for the 4-bromoaniline increased potency, with C90
and C97 exhibiting values of IC50 of 7–8 nM (Table 1). Herein, we
120 40
ꢀ5000
69
n.d.
n.d.
200 20
330d
n.d.
n.d.
n.d.
n.d.
7
900 100
120 40
220 40
370 60
60 30
ꢀ5000
C10b
C11
C58
C14b
C40
C45
C20
C48
C86b
C93
C90b
C28
C79
C87
4-ClPh
4-CF3Ph
4-CNPh
4-BrPh
4-SO2MePh
4-OCF3Ph
2-ClPh
140 50
ꢀ5000
report
a more comprehensive structure–activity relationship
n.d.
(SAR) study for this class of inhibitors.
3-ClPh
490 40
30 10
30 10
1430d
The benzimidazole analogs were synthesized following the pro-
cedure outlined in Scheme 2. Various acetylamide derivatives 3
were prepared by treating substituted anilines 1 with bromo acetyl-
chlorides, 2, in dichloromethane (DCM) and in the presence of
catalytic amounts of 4-N,N-dimethylaminopyridine (DMAP).
Various 2-substituted benzimidazoles 6 were prepared by condens-
ing o-phenylene diamine 4 with aromatic aldehydes followed by
oxidation in the presence of sodium metabisulfite using a slight
modification of published procedures.11 Finally, 2-substituted benz-
imidazoles were coupled with the acetylamides 3 in the presence of
potassium carbonate to yield benzimidazoles 7. CpIMPDH inhibiton
was measured by monitoring the production of NADH in the pres-
ence of varying inhibitor concentrations.9 Inhibition was also deter-
mined in the presence of 0.05% fatty acid free bovine serum albumin
(BSA) in order to evaluate the effects of non-specific binding. Grati-
fyingly, none of the CpIMPDH inhibitors displayed activity against
3,4-DiClPh
3-CN, 4-ClPh
2-Naph
90
2
60 20
20 10
n.d.
80 20
300 70
7
4
1-(4-Cl)Naph
ꢀ5000
CH(CH3) 4-ClPh
CH(CH3) 3,4-
60 10
240 40
(OCH2CH2)Ph
4-ClPh
4-ClPh
C24
C18
NH
CH(iPr)
ꢀ5000
ꢀ5000
n.d.
n.d.
CH2NH CH2
All values are average of three independent determinations unless otherwise stated.
Data from.7
a
Data from.10
b
c
n.d. = not determined.
n = 1.
d
human IMPDH2 (IC50 > 5 lM). Selected compounds were also
O
evaluated for antiparasitic activity.12
NH2
Br
The first region of the molecule examined was the anilide sub-
stituent. Replacing the 4-methoxy of C with a thiomethyl (C39)
resulted in a 10-fold increase in activity (Table 1). However, a
branched aliphatic group (C43) at the same position resulted in de-
creased activity. Interestingly, replacing the 4-methoxy with elec-
tron withdrawing groups (C9–C11, C58, C14, C45) resulted in
compounds with increased activity, except for sulfone C40. Larger
more hydrophobic groups such as chlorine (C10) and bromine
(C14) were best. Translocation of the chlorine from the 4-position
to either the 3- or 2-positions (C20, C48) was detrimental. Several
compounds containing electron withdrawing groups in the 3- and
4-positions (C86 and C93) also demonstrated potent inhibitory
activity. Surprisingly, addition of a chlorine to the 2-naphthyl
(C28) was not tolerated. Introduction of a methyl onto the methy-
lene between the amide carbonyl and the imidazole resulted in in-
HN
O
DMAP, CH2Cl2
R2
Br
+
Cl
0 °C - rt, 3 h, 90%
R1
R2
R1
2
1
3
NH2
NH2
N
Na2S2O5
R3
R3
CHO
+
N
H
DMF, rt, 8 h, 70%
6
5
4
N
R3
N
K2CO3
DMF, rt, 8 h
R2
3 + 6
O
N
N
N
NH
R1
S
7
O
NH
Scheme 2. General procedure for synthesizing analogs of C.
C
MeO
creased potency (C79 vs C10). Increasing the steric bulk of the
methyl group to i-Pr (C24) was detrimental. Finally, inserting a
Figure 1. CpIMPDH selective inhibitor C identified by HTS.

S. Kirubakaran et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1985–1988
1987
Table 3
methylene between the amide NH and the phenyl ring (C18) was
Relative affinity of CpIMPDH inhibitors based on docking
not tolerated.
experiments13
Subsequently, the SAR of the 4-thiazolyl ring was examined (Ta-
ble 2). As reported previously, changing the connectivity to a 2-
thiazolyl increased activity for several analogs (C61 vs C10, C64
vs C14, C74 vs C79) and retained potent activity for another analog
(C97 vs C90).10 The 5-thiazolyl was also comparatively active (C67
vs C61). In addition, several other heterocycles (C62, C100, C16)
also retained potent activity. However, the 2-pyrrolyl (C65) and
2-oxazolyl (C69) derivatives demonstrated reduced potency. Like-
wise, replacing the thiazole ring by various phenyls (C17, C31, C59)
or a methyl (C38) resulted in significant losses in activity.
In order to further analyze the SAR results, select molecules
were docked using GLIDE (Schrödinger Inc.) into a CpIMPDH model
based on the previously determined co-crystal structure. Free en-
ergy perturbation (FEP) calculations were then determined (calcu-
lated as DDG relative to inhibitor C) and the results were highly
correlated (r2 = 0.93) to the observed IC50 determinations (Table
3, Fig. 2).13 Inhibitor potency appears to be driven largely by two
major contributions: (1) a hydrogen bond between E329 of
CpIMPDH and the amide NH of the inhibitors; (2) an entropic effect
of displacing water molecules from the binding cavity by large
Compound
DDG relative to C (kcal/mol)
C
0
C39
C43
C10
C48
C20
C86
C90
C40
C11
C28
ꢁ2.49
4.8
ꢁ2.34
ꢁ1.39
3.33
ꢁ4.12
ꢁ5.87
3.99
ꢁ1.51
5.77
Table 2
SAR of thiazole ring
N
R1
R3
N
O
NH
R2
Compound R1
R2
R3
H
IC50 (nM) IC50 (nM) BSA
Figure 2. Correlation of calculated relative affinity with experimental values.
N
Ca
4-OMePh
1200
n.d.c
S
N
C61b
C64b
4-ClPh
4-BrPh
4-ClPh
3,4-DiClPh
2-Naph
H
H
30 10
50 10
27d
hydrophobic substituents. The presence of strong electron with-
drawing groups on the arylamide increases potency by increasing
the strength of the E329-NH H-bond provided no steric clashes
are present. Thus the balance between conformational state and
electron withdrawing ability appears critical for determining the
final potency of the inhibitors. For example, 3,4-dichloro substi-
tuted analog C86 is predicted to be more potent than the 3-chloro
analog C48. For the 2-chloro analog C20 a steric clash is predicted
to change the orientation of the phenyl ring lowering the inductive
effect of the chlorine substituent weakening the E329-NH bond.
For the 2-naphthyl analog C90, displacement of ordered water
molecules from the active site of the protein is entropically favored
resulting in DDG relative to C of ꢁ5.87 kcal/mol and an IC50 value
of 7 nM.
Compounds with a value of IC50 less than 30 nM were candi-
dates for testing in a Toxoplasma gondii model of C. parvum infec-
tion.12 Preference was given to compounds that displayed little
non-specific binding as judged by changes in the value of IC50 in
the presence of BSA. Wild type T. gondii expresses a eukaryotic
IMPDH that is resistant to the CpIMPDH inhibitors. In contrast, in
the T. gondii/CpIMPDH model parasite, the endogenous IMPDH
gene has been replaced with CpIMPDH. In addition, the gene
encoding hypoxanthine-guanine-xanthine phosphoribosyltrans-
ferase was knocked out, so this strain relies on the activity of
CpIMPDH to obtain guanine nucleotides. Both T. gondii strains ex-
press yellow fluorescent protein enabling easy monitoring of para-
site proliferation. T. gondii strains were cultured in human foreskin
fibroblasts immortalized with hTERT, so this assay also reports on
host cell toxicity. Compounds C64, C84, C90, C91 and C97 all
28
9
4
5
C74
Me 23
H
H
30
6
S
C84b
C97b
18
8
50 20
20 20
3
N
N
C67
C62
4-ClPh
4-ClPh
H
H
35
9
60d
S
S
20 20
60d
C100
4-ClPh
H
35
8
42d
N
Me
C16
C85
C91
C92
4-ClPh
3,4-DiClPh
2-Naph
3-CN, 4-ClPh
H
H
H
H
43
22
9
5
90 30
N
40
7
8
3
14
5
22 10
40 10
120d
C65
C69
4-ClPh
4-ClPh
H
H
80 10
HN
O
N
170 10
230 10
C17
C31
C59
C38
Ph
4-ClPh
4-ClPh
4-ClPh
4-ClPh
H
H
H
H
210 30
450 20
870 20
ꢀ5000
280 30
n.d.
4-ClPh
4-FPh
Me
1300d
n.d.
All values are average of three independent determinations unless otherwise stated.
Data from.7
a
Data from.10
b
c
n.d. = not determined.
n = 1.
d

1988
S. Kirubakaran et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1985–1988
Supplementary data
Table 4
Antiparasitic activity of selected compounds
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.01.029.
a
Compound
T. gondii model
EC50 M)
C. parvum modelb
(l
Selectivity
Toxo/WT
>23
Toxo/CpIMPDH
EC50 (lM)
References and notes
C64
C84
C90
C91
C97
0.3 0.1
0.7 0.3
0.6 0.1
0.3 0.2
0.5 0.4
>73
5
9
9
30
0.7 0.2c
1.7 0.8c
0.9 0.5
n.d.
1. Fayer, R. Vet. Parasitol. 2004, 126, 37.
3
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2. Huang, D. B.; White, A. C. Gastroenterol. Clin. North Am. 2006, 35, 291.
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Lancto, C. A.; Deng, M.; Liu, C.; Widmer, G.; Tzipori, S.; Buck, G. A.; Xu, P.;
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Anantharaman, V.; Aravind, L.; Kapur, V. Science 2004, 304, 441.
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Manque, P.; Akiyoshi, D.; Mackey, A. J.; Pearson, W. R.; Dear, P. H.; Bankier, A.
T.; Peterson, D. L.; Abrahamsen, M. S.; Kapur, V.; Tzipori, S.; Buck, G. A. Nature
2004, 431, 1107.
5. Striepen, B.; Pruijssers, A. J.; Huang, J.; Li, C.; Gubbels, M. J.; Umejiego, N. N.;
Hedstrom, L.; Kissinger, J. C. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 3154.
6. Striepen, B.; White, M. W.; Li, C.; Guerini, M. N.; Malik, S. B.; Logsdon, J. M., Jr.;
Liu, C.; Abrahamsen, M. S. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6304.
7. Umejiego, N. N.; Gollapalli, D.; Sharling, L.; Volftsun, A.; Lu, J.; Benjamin, N. N.;
Stroupe, A. H.; Riera, T. V.; Striepen, B.; Hedstrom, L. Chem. Biol. 2008, 15, 70.
8. Umejiego, N. N.; Li, C.; Riera, T.; Hedstrom, L.; Striepen, B. J. Biol. Chem. 2004,
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9. Maurya, S. K.; Gollapalli, D. R.; Kirubakaran, S.; Zhang, M.; Johnson, C. R.;
Benjamin, N. N.; Hedstrom, L.; Cuny, G. D. J. Med. Chem, 2009, 52, 4623.
10. MacPherson, I. S.; Kirubakaran, S.; Gorla, S. K.; Riera, T. V.; D’Aquino, J. A.;
Zhang, M.; Cuny, G. D.; Hedstrom, L. J. Am. Chem. Soc. 2010, 132, 1230.
11. Yang, D.; Fokas, D.; Li, J.; Yu, L.; Baldino, C. M. Synthesis 2005, 47.
12. Sharling, L.; Liu, X.; Gollapalli, D. R.; Maurya, S. K.; Hedstrom, L.; Striepen, B.
PLoS Negl. Trop. Dis. 2010, 4, e794.
2.7 0.9c
17
9
<0.8d
All values are the average of three independent trials unless otherwise stated.
T. gondii model.12 Toxo/WT, strain with endogenous IMPDH; Toxo/CpIMPDH,
a
strain that depends on CpIMPDH. Selectivity = EC50(Toxo/WT)/EC50(Toxo/
CpIMPDH).
b
C. parvum in vitro infection model.
Two determinations.
Average growth inhibition 80 10% at 0.8 lM.
c
d
displayed sub-micromolar activity against T. gondii/CpIMPDH
(Table 4). C64 and C97 displayed selectivity P30 versus the
wild-type strain, strongly indicating that antiparasitic activity re-
sults from the inhibition of CpIMPDH.
Compounds C64, C84, C90 and C97 were also tested in an in vitro
model of C. parvum infection.12 Importantly, all four compounds are
approximately two orders of magnitude more potent than paromo-
mycin, the standard control for anticryptosporidial activity (litera-
13. Protein data base (PDB) file 3KHJ was used to generate a model for the protein
bearing the missing side chains, loops and hydrogen atoms. The loop positions
were refined using PRIME, IMP and C64 were modeled and the overall
structure was minimized. GLIDE (Schrödinger Inc.) was used to calculate a grid
around C64 with a single positional constraint on the carbon attached to the
benzimidazole nitrogen. The grid was restricted to a size similar to C64. The
inhibitor structures were converted to 3-D coordinates, pre-processed using
ligprep to generate tautomers and ionization states at pH 2.0–7.0 and stored as
sd files. GLIDE calculations were carried out in standard precision mode with
ture paromomycin EC50 values are 65–130 l
M7,12,14–16). The
potencies of C64, C84, C90 and C97 were similar to that observed
in the T. gondii model (Table 4).
In conclusion, a SAR study of benzimidazole-based CpIMPDH
inhibitors revealed that variations to the aniline and to the hetero-
cycle attached to the 2-position of the benzimidazole could be al-
tered in order to increase inhibitory activity, while retaining
excellent selectivity over human IMPDH2. The benzimidazole-
based CpIMPDH inhibitors described herein could serve as useful
molecular probes for studying C. parvum and other related organ-
isms in addition to providing lead compounds for the development
of effective treatments of cryptosporidiosis.
the same single constraint. GLIDE automatically generated
a pool of
conformation for each inhibitor (ꢀ5000 each) and the best pose was chosen
based on total number of productive interactions (both Van der Walls and
electrostatic) with the protein. No water molecules were included in this
calculation. The structural overlay of the docked inhibitors showed < 1.5 Å
deviation from C64 indicating that GLIDE was able to reproduce the
conformation of the inhibitors in the active site of CpIMPDH. For FEP
calculations, DESMOND molecular dynamics engine (D.E Shaw Research and
Schrödinger Inc.) with the OPLS2005 force field was used. In the current setup,
a 22-window scheme was adopted for both A (WT) and B (B) states to achieve
reasonably high accuracy. A simulation production time of 44 ns (2 ns ꢂ 22
windows) was used for all the calculations and the jobs were run on an in-
house 24 node 3.4 GHz Beowulf cluster. Inhibitor C (IC50 = 1200 nM) was used
as the initial structure (k = 0) to generate other structure in the series using
alchemical FEP mutations.
Acknowledgments
This work was supported by funding from the National Institute
of Allergy and Infectious Diseases (U01AI075466) to L.H. G.D.C.
thanks the New England Regional Center of Excellence for Biode-
fense and Emerging Infectious Diseases (NERCE/BEID), and the
Harvard NeuroDiscovery Center for financial support. B.S. is a
Georgia Research Alliance Distinguished Investigator. IC50 data
for these compounds are maintained using ChemAxon, http://
www.chemaxon.com/.
14. Perkins, M. E.; Wu, T. W.; Le Blancq, S. M. Antimicrob. Agents Chemother. 1998,
42, 843.
15. Woods, K. M.; Nesterenko, M. V.; Upton, S. J. FEMS Microbiol. Lett. 1995, 128, 89.
16. You, X.; Arrowood, M. J.; Lejkowski, M.; Xie, L.; Schinazi, R. F.; Mead, J. R. FEMS
Microbiol. Lett. 1996, 136, 251.