Phthalazinone inhibitors of inosine-5′-monophosphate dehydrogenase from Cryptosporidium parvum

Article abstract of DOI:10.1016/j.bmcl.2012.12.037

Cryptosporidium parvum (Cp) is a potential biowarfare agent and major cause of diarrhea and malnutrition. This protozoan parasite relies on inosine 5′-monophosphate dehydrogenase (IMPDH) for the production of guanine nucleotides. A CpIMPDH-selective N-aryl-3,4-dihydro-3-methyl-4-oxo-1- phthalazineacetamide inhibitor was previously identified in a high throughput screening campaign. Herein we report a structure-activity relationship study for the phthalazinone-based series that resulted in the discovery of benzofuranamide analogs that exhibit low nanomolar inhibition of CpIMPDH. In addition, the antiparasitic activity of select analogs in a Toxoplasma gondii model of C. parvum infection is also presented.

Full text of DOI:10.1016/j.bmcl.2012.12.037

Bioorganic & Medicinal Chemistry Letters xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Phthalazinone inhibitors of inosine-50-monophosphate dehydroge-
nase from Cryptosporidium parvum
Corey R. Johnson a, , Suresh Kumar Gorla b, Mandapati Kavitha b, Minjia Zhang b, Xiaoping Liu c,
Boris Striepen c,d, Jan R. Mead e, Gregory D. Cuny f,g, Lizbeth Hedstrom a,b,
⇑
a Department of Chemistry, Brandeis University, MS009, 415 South Street, Waltham, MA 02454, United States
b Department of Biology, Brandeis University, MS009, 415 South Street, Waltham, MA 02454, United States
c Center for Tropical and Emerging Global Diseases, University of Georgia, 500 D.W. Brooks Drive, Athens, GA 30602, United States
d Department of Cellular Biology, University of Georgia, 500 D.W. Brooks Drive, Athens, GA 30602, United States
e Atlanta VA Medical Center and Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, United States
f Laboratory for Drug Discovery in Neurodegeneration, Brigham & Women’s Hospital and Harvard Medical School, 65 Landsdowne Street, Cambridge, MA 02139, United States
g Department of Pharmacological and Pharmaceutical Sciences, University of Houston, College of Pharmacy, 549A Science and Research Bldg 2, Houston, TX 77204, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Cryptosporidium parvum (Cp) is a potential biowarfare agent and major cause of diarrhea and malnutri-
tion. This protozoan parasite relies on inosine 50-monophosphate dehydrogenase (IMPDH) for the pro-
Received 21 August 2012
Revised 10 December 2012
Accepted 13 December 2012
Available online xxxx
duction of guanine nucleotides.
A
CpIMPDH-selective N-aryl-3,4-dihydro-3-methyl-4-oxo-1-
phthalazineacetamide inhibitor was previously identified in a high throughput screening campaign.
Herein we report a structure–activity relationship study for the phthalazinone-based series that resulted
in the discovery of benzofuranamide analogs that exhibit low nanomolar inhibition of CpIMPDH. In addi-
tion, the antiparasitic activity of select analogs in a Toxoplasma gondii model of C. parvum infection is also
presented.
Keywords:
IMPDH
IMP dehydrogenase
Cryptosporidiosis
Antiparasitic
Ó 2012 Elsevier Ltd. All rights reserved.
Cryptosporidium species cause self-limiting gastrointestinal dis-
ease that can be chronic and fatal in immunosuppressed patients.1
Oocysts from these organisms are resistant to common methods of
water treatment, and therefore pose a potential biowarfare threat.
The drugs currently approved for the treatment of cryptosporidio-
sis are largely ineffective, elevating the need for new chemothera-
peutic agents to counter outbreaks. Genomic analysis of the most
common human pathogens, Cryptosporidium parvum and C. homi-
nis, revealed that these parasites have a streamlined purine salvage
pathway that relies on IMP dehydrogenase (IMPDH) for the pro-
duction of guanine nucleotides.2–4 This enzyme catalyzes the oxi-
dation of IMP to XMP with the production of NADH as outlined
in Scheme 1.5 Curiously, the Cryptosporidium IMPDH gene appears
A previously reported high throughput screening (HTS) cam-
paign identified several structurally distinct classes of selective
CpIMPDH inhibitors that bind to the NAD site.8,9 Structure–activity
relationship studies of triazole,10 benzimidazole9,11 and urea12
CpIMPDH inhibitors, exemplified by A110, C64, C97 and P96,
respectively, have also now been reported (Fig. 1). In addition, a
co-crystal structure of the benzimidazole inhibitor C64 with
CpIMPDH demonstrated a unique binding mode compared to
inhibitors bound to human IMPDH.9 The phthalazinone-based
inhibitor 1 was also identified by HTS, with moderate potency
(IC50 = 5.4
l
M) and good selectivity (IC50 >50
l
M vs both human
IMPDH1 and IMPDH2).8 Compound 1 (50
lM) did not display anti-
parasitic activity.8 Herein we report a structure–activity relation-
ship study of 1, and the antiparasitic activity of select analogs in
a Toxoplasma gondii model of C. parvum infection.
to have been obtained from an e-proteobacterium via horizontal
gene transfer,6 and is structurally distinct from the host ortholog
(the C. parvum and C. hominis enzymes are identical and will be de-
noted as CpIMPDH). Consequently, CpIMPDH displays unique enzy-
matic properties compared with the host counterparts.7
Compound 1 and its analogs were prepared following a four-
step sequence as depicted in Scheme 2 beginning with the Wittig
olefination of phthalic anhydride, 2, in the presence of carbeth-
oxymethylidenetriphenylphosphorane in chloroform to produce
(E)-ethyl-2-(3-oxoisobenzofuran-1(3H)-ylidene)acetate, 3. Addi-
tion of hydrazines in refluxing ethanol generated the phthalazi-
none acetoesters 4. Hydrolysis of the esters produced
phthalazinone acetic acids 5.13,14 Finally, coupling of the acetic acid
moiety with various aromatic amines in DMSO afforded 6.
⇑
Corresponding author. Tel.: +1 781 736 2333; fax: +1 781 736 2349.
E-mail address: hedstrom@brandeis.edu (L. Hedstrom).
Present address: Department of Chemistry and Biochemistry, University of the
Sciences, 600 South 43rd Street, Philadelphia, PA 19104, United States.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.037
Please cite this article in press as: Johnson, C. R.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2012.12.037

2
C. R. Johnson et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
N
O
N
N
N
O
2H+
O
NAD+ NADH
E = IMPDH
O
H2O
--O3PO
N
O
N
O
--O3PO
--O3PO
N
E
NH
HO
OH
N
NH
N
NH
HO
OH
HO
OH
S
E-SH
O-
IMP
E-XMP*
XMP
Scheme 1. IMPDH catalyzed conversion of IMP to XMP via intermediate E-XMP⁄.
Cl
Cl
O2N
OMe
N
N
O
N
N
N
S
O
N
O
N
H
NH
N
N
NH
N
O
O
NH
N
HO
R
O
A110
C64 R = 4-Br-Ph
C97 R = 2-naphthyl
1
P96
Figure 1. Optimized CpIMPDH inhibitors A110, C64, C97, P96 and HTS identified CpIMPDH inhibitor 1.
The synthesized derivatives were evaluated for CpIMPDH inhi-
(15) was not tolerated. Replacement of the electron donating group
at the 4-position with electron withdrawing groups improved po-
tency. For example, the 4-chloro and 4-bromo analogs (16 and 17)
demonstrated IC50 values of 230 and 130 nM, respectively. How-
ever, other electron withdrawing groups (18–21) generally were
not as well tolerated. Also, transposition of the chlorine from the
4-position to the 3-position (23) resulted in loss of activity. Di-sub-
stitution at the 3,4-positions with chlorine and bromine atoms (24
and 25) resulted in increased potency. Furthermore, other 3,4-di-
substituted analogs with either an electron-donating or an elec-
tron-withdrawing group at the 3-position were also active. Inter-
estingly, in a number of cases substituents at the 3-position that
alone were not tolerated when combined with electron withdraw-
ing groups (especially Cl or Br) at the 4-position resulted in potent
CpIMPDH inhibitors (e.g., 26, 27 and 37). However, transposition of
the substituents from the 3-position to the 2-position (39–42) re-
sulted in poor inhibitors. In addition, several other di- and tri-sub-
stitutions (43–46) were also detrimental. Encouraged by the
results of 3,4-disubstitution, the 2-naphthyl analog 47 was pre-
pared and demonstrated an IC50 of 63 nM. Similar increases in
CpIMPDH activity by introduction of 3,4-disubstitution or incorpo-
ration of 2-naphthylene had also been observed in the structure–
activity relationship studies resulting in A110 and C97.10,11 Finally,
analogs that lack methylation of the phthalazinone nitrogen atom
(48 and 49) were found to be active indicating that this substituent
is not necessary for CpIMPDH inhibitory activity.
bition using an assay that monitored the production of NADH in
the presence of varying inhibitor concentrations.10 In order to eval-
uate the effects of non-specific binding, inhibition was also deter-
mined in the presence of 0.05% fatty acid free bovine serum
albumin (BSA). None of the compounds inhibited human IMPDH2
(IC50 >5
lM).
Inhibitor 1 exhibited an IC50 of 1.0
lM upon resynthesis. Grati-
fyingly, the activity was retained in the presence of 0.05% BSA
(IC50 = 970 nM) as shown in Table 1. However, moving the electron
donating group to the 2- or 3-position resulted in loss of activity (7
and 10). Replacing the 4-OMe with a variety of other electron
donating substituents (11–14) was also detrimental. Likewise,
replacing the phenyl with an amine (8) or alkyl group (9) or insert-
ing a methylene between the benzene and amide nitrogen atom
O
CO2Et
b
OEt
R
O
a
N
N
O
O
O
O
O
2
3
4
Inspired by the results with 47, a variety of fused heterocycles
were explored as replacements of the 2-naphthylene (Table 2).
Although the 5-benzofuranyl analog 50 had reduced activity and
the 5-benzoxazolyl analog 51 was inactive, the 2-methyl-5-ben-
zofuranyl derivative 52 retained activity with an IC50 of 64 nM.
Furthermore, incorporation of an additional ring (53) resulted in
enhanced potency. Unsaturation of the ring further increased
activity with 54 demonstrating an IC50 of 4 nM. However, the reg-
ioisomer 55 and two carbazoles 56 and 57 were not active.
Compounds with IC50 values less than 30 nM were candidates
for evaluation of antiparasitic activity in a Toxoplasma gondii model
of C. parvum infection.15 In this model, the endogenous T. gondii
IMPDH and hypoxanthine-guanine-xanthine phosphoribosyltrans-
O
O
R1
OH
d
c
N
H
N
N
N
N
R
R
O
O
6
5
Scheme 2. General procedure for the synthesis of phthalazinone derivatives of 1.
Reagents and conditions: (a) Ph3PCHCO2Et, CHCl3, reflux, 16 h; (b) NH2NH2 or
NH2NHMe, EtOH, reflux, 3 h; (c) 3 M NaOH/THF, reflux, 2 h followed by acidificat-
ion; (d) R1-NH2, EDC, HOBt, DIPEA, DMSO, 3–5 h.
Please cite this article in press as: Johnson, C. R.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2012.12.037

C. R. Johnson et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
3
Table 1
Table 2
SAR of anilide and nathylide phthalazinone inhibitors
SAR of heterotricyclic anilide phthalazinone inhibitors
O
O
R1
N
R
N
H
H
N
N
N
N
R
Me
O
O
Compound
R
R1
(ꢀ)-BSA (nM)
1000 200
>5000
>5000
>5000
>5000
>5000
>5000
>5000
(+)-BSA (nM)
Compound
R
(ꢀ)-BSA (nM)
(+)-BSA (nM)
85 12
1
7
8
9
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
4-OMePh
2-OMePh
NH2
t-Bu
3-OMePh
4-OEtPh
4-OCF3Ph
4-MePh
4-i-PrPh
CH2(4-OMePh)
4-ClPh
4-BrPh
4-FPh
4-CNPh
4-CF3Ph
4-SO2MePh
3-CF3Ph
3-ClPh
3,4-di-ClPh
3-Cl-4-BrPh
3-CF3-4-BrPh
3-CF3-4-ClPh
3-CF3-4-CNPh
3-Cl-4-CNPh
3-CF3-4-FPh
3-CF3-4-OMePh
3-CF3-4-NH2Ph
3-F-4-ClPh
3-Me-4-ClPh
3-Me-4-BrPh
3-Me-4-CNPh
3-OMe-4-ClPh
3,4-di-CNPh
2-CF3-4-ClPh
2-CF3-4-BrPh
2-F-4-BrPh
2,4-BrPh
930 180
ND
ND
ND
ND
ND
ND
ND
ND
50
93 15
>5000
O
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
51
52
ND
O
O
>5000
>5000
Me
64 11
72 15
ND
230 50
130 30
>5000
950 90
>5000
>5000
>5000
>5000
280 50
130 20
ND
940 90
ND
ND
ND
ND
53
54
55
20
4
5
150 20
30 10
ND
O
1
55
30
24
13
54
6
1
2
1
5
95
75
48
25
52
2
2
4
5
6
O
O
>5000
>5000
290 40
150 20
>5000
>5000
200 30
470 120
150 30
ND
56
57
ND
ND
ND
N
H
240 30
96 19
140 10
460 90
79
9
70 10
400 100
33
1
40
7
>5000
>2500
>5000
>5000
1100 200
>5000
>5000
>5000
>5000
>5000
>2500
ND
ND
1700 100
ND
ND
ND
ND
ND
140 20
N
Et
Assays as described Ref. 10 All values are average of three independent determi-
nations unless otherwise stated. ND, not determined.
2,4-Di-ClPh
3,5-Di-ClPh
3,4,5-Tri-ClPh
3,4,5-Tri-FPh
2-Naphthyl
3-CF3-4-ClPh
3-CF3-4-BrPh
Table 3
Antiparasitic activity
63
40
32
3
1
6
61
1
Selectivityb
a
Compound
EC50
(
l
M)
H
130
8
Toxo/WT
Toxo/CpIMPDH
Assays as described Ref. 10 All values are average of three independent determi-
nations unless otherwise noted (⁄two determinations). ND, not determined.
26
27
53
54
4
23
1
3⁄
4
0.3 0.2
0.4 0.3
0.5 0.2
0.4 0.1
14
60
2
1⁄
1
3
7
Assays as described Ref. 15 Values are the average and standard deviations of three
independent determinations unless otherwise stated (⁄denotes average and range
of two determinations).
ferase genes have been knocked out and the CpIMPDH gene in-
serted to create T. gondii/CpIMPDH, a model parasite that relies
on CpIMPDH for the production of guanine nucleotides. Both
wild-type and T. gondii/CpIMPDH were cultured in human foreskin
fibroblasts immortalized with hTERT, so this assay also reports on
host cell toxicity. Compounds 26, 27, 53 and 54 all displayed sub-
micromolar activity against T. gondii/CpIMPDH (Table 3). However,
only 27 displayed selectivity P30 versus the wild-type strain,
strongly indicating that antiparasitic activity results from the inhi-
bition of CpIMPDH.
a
T. gondii RH (Toxo/WT) should be resistant to CpIMPDH inhibitors while T.
gondii/CpIMPDH (Toxo/CpIMPDH) should be sensitive.
b
Selectivity = EC50(Toxo/WT)/EC50(Toxo/CpIMPDH).
Compound 27 displayed good stability in mouse liver micro-
somes (T1/2 = 79 min). This compound was advanced into the IL12
knockout mouse model of C. parvum infection.16–19 Disappointingly,
no antiparasitic activity was observed with once per day oral dosing
of 27 (250 mg/kg) for 7 days. It may be that alternative dosing re-
gimes or modifications in formulations could improve efficacy
in vivo. Additional optimization of pharmacokinetic properties
The inhibition of CpIMPDH by 27 was characterized further.
Whereas 1 is a mixed inhibitor of CpIMPDH with respect to NAD+
(Kis = 1.8
(Kis = Kii = 3.4 0.2 nM; Fig. 2).
lM, Kii = 7 l
M),8 27 is a pure noncompetitive inhibitor
Please cite this article in press as: Johnson, C. R.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2012.12.037

4
C. R. Johnson et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
References and notes
1. Huang, D. B.; White, A. C. Gastroenterol. Clin. North Am. 2006, 35, 291.
2. 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.
3. Abrahamsen, M. S.; Templeton, T. J.; Enomoto, S.; Abrahante, J. E.; Zhu, G.;
Lancto, C. A.; Deng, M.; Liu, C.; Widmer, G.; Tzipori, S.; Buck, G. A.; Xu, P.;
Bankier, A. T.; Dear, P. H.; Konfortov, B. A.; Spriggs, H. F.; Iyer, L.;
Anantharaman, V.; Aravind, L.; Kapur, V. Science 2004, 304, 441.
4. Xu, P.; Widmer, G.; Wang, Y.; Ozaki, L. S.; Alves, J. M.; Serrano, M. G.; Puiu, D.;
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. Hedstrom, L. Chem. Rev. 2009, 109, 2903.
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.; Li, C.; Riera, T.; Hedstrom, L.; Striepen, B. J. Biol. Chem. 2004,
279, 40320.
8. 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.
9. 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.
10. 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.
11. Kirubakaran, S.; Gorla, S. K.; Sharling, L.; Zhang, M.; Liu, X.; Ray, S. S.;
Macpherson, I. S.; Striepen, B.; Hedstrom, L.; Cuny, G. D. Bioorg. Med. Chem. Lett.
2012, 22, 1985.
12. Gorla, S. K.; Kavitha, M.; Zhang, M.; Liu, X.; Sharling, L.; Gollapalli, D. R.;
Striepen, B.; Hedstrom, L.; Cuny, G. D. J. Med. Chem. 2012, 55, 7759.
13. Mylari, B. L.; Larson, E. R.; Beyer, T. A.; Zembrowski, W. J.; Aldinger, C. E.; Dee,
M. F.; Siegel, T. W.; Singleton, D. H. J. Med. Chem. 1991, 34, 108.
14. Niebel, C.; Lokshin, V.; Sigalov, M.; Krief, P.; Khodorkovsky, V. Eur. J. Org. Chem.
2008, 3689.
15. Sharling, L.; Liu, X.; Gollapalli, D. R.; Maurya, S. K.; Hedstrom, L.; Striepen, B.
PLoS Negl. Trop. Dis. 2010, 4, e794.
16. Campbell, L. D.; Stewart, J. N.; Mead, J. R. J. Parasitol. 2002, 88, 1014.
17. Ehigiator, H. N.; Romagnoli, P.; Borgelt, K.; Fernandez, M.; McNair, N.; Secor, W.
E.; Mead, J. R. Parasite Immunol. 2005, 27, 17.
18. Sun, X. E.; Sharling, L.; Muthalagi, M.; Mudeppa, D. G.; Pankiewicz, K. W.;
Felczak, K.; Rathod, P. K.; Mead, J.; Striepen, B.; Hedstrom, L. J. Biol. Chem. 2010,
285, 15916.
Figure 2. Inhibition of CpIMPDH by 27.
may also be necessary for this compound series in order to achieve
in vivo efficacy.
In conclusion, a SAR study of phthalazinone-based CpIMPDH
inhibitors revealed that expansion of the aniline ring could in-
crease inhibitory activity, while maintaining selectivity relative
to the human orthologs. The phthalazinone-based CpIMPDH inhib-
itors described herein could serve as lead compounds for the devel-
opment of effective treatments of cryptosporidiosis as well as
useful molecular probes for studying Cryptosporidium parasites.
Acknowledgments
This work was supported by funding from the National Institute
of Allergy and Infectious Diseases (U01 AI075466 and U01
AI075466S1) to L.H. G.D.C. thanks the New England Regional Cen-
ter of Excellence for Biodefense and Emerging Infectious Diseases
(NERCE/BEID) and Harvard NeuroDiscovery Center for financial
support. IC50 data for these entire compounds were maintained
using ChemAxon, http://www.chemaxon.com/. J.R.M. would like
to thank Ms. Nina McNair for technical support. C.R.J. would like
to thank Drs. Sivapriya Kirubakaran and Jihan Khan for insightful
and helpful suggestions.
19. The anticryptosporidial activity of 27 was assessed in the IL-12 knockout
mouse model, which resembles the acute human disease. Mice were
inoculated with 1000 oocysts and treated by gavage with vehicle (10%DMSO/
corn oil), vehicle plus 250 mg/kg 27, or 2000 mg/kg paromomycin starting 4 h
post infection. Treatment was given once daily for 7 days and mice sacrificed
on day 8 (peak infection). Parasite load was quantified by FACS assays by the
presence of the oocysts in the feces at days 0, 4 and 7. Fecal pellets from the
mice were collected and homogenized in adjusted volumes of 2.5% potassium
dichromate. Aliquots (200 ll) of vortexed samples were processed over micro-
scale sucrose gradients. The oocyst-containing fraction was collected and
washed. Purified oocysts were incubated with a fluorescein-labeled oocyst-
specific monoclonal antibody (OW5O-FITC) for 20 min. Samples were adjusted
to 600 ll and assayed assayed with a 102-s sampling interval (100 ll) using
logical gating of forward/side scatter and OW5O-FITC fluorescence signal on a
Becton Dickinson FACScan flow cytometer. Flow cytometry data were
evaluated by analysis of variance (Microsoft Excel; Microsoft Corporation,
Redmond, WA).
Supplementary data
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/j.bmcl.
2012.12.037.
Please cite this article in press as: Johnson, C. R.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2012.12.037