Development of 5-Aminopyrazole-4-carboxamide-based Bumped-Kinase Inhibitors for Cryptosporidiosis Therapy

Article abstract of DOI:10.1021/acs.jmedchem.9b00069

Cryptosporidium is a leading cause of pediatric diarrhea worldwide. Currently, there is neither a vaccine nor a consistently effective drug available for this disease. Selective 5-aminopyrazole-4-carboxamide-based bumped-kinase inhibitors (BKIs) are effective in both in vitro and in vivo models of Cryptosporidium parvum. Potential cardiotoxicity in some BKIs led to the continued exploration of the 5-aminopyrazole-4-carboxamide scaffold to find safe and effective drug candidates for Cryptosporidium. A series of newly designed BKIs were tested for efficacy against C. parvum using in vitro and in vivo (mouse infection model) assays and safety issues. Compound 6 (BKI 1708) was found to be efficacious at 8 mg/kg dosed once daily (QD) for 5 days with no observable signs of toxicity up to 200 mg/kg dosed QD for 7 days. Compound 15 (BKI 1770) was found to be efficacious at 30 mg/kg dosed twice daily (BID) for 5 days with no observable signs of toxicity up to 300 mg/kg dosed QD for 7 days. Compounds 6 and 15 are promising preclinical leads for cryptosporidiosis therapy with acceptable safety parameters and efficacy in the mouse model of cryptosporidiosis.

Full text of DOI:10.1021/acs.jmedchem.9b00069

Subscriber access provided by Washington University | Libraries
Article
Development of 5-aminopyrazole-4-carboxamide-based
bumped-kinase inhibitors for Cryptosporidiosis therapy
Wenlin Huang, Matthew A. Hulverson, Ryan Choi, Samuel L.M. Arnold,
zhongsheng zhang, Molly McCloskey, Grant Whitman, Robert Hackman, Kasey
Rivas, Lynn K Barrett, Kayode Ojo, Wesley C. Van Voorhis, and Erkang Fan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00069 • Publication Date (Web): 04 Mar 2019
Downloaded from http://pubs.acs.org on March 5, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 40
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
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
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 40
1
2
3
4
5
6
7
8
9
Development of 5-aminopyrazole-4-carboxamide-based
bumped-kinase inhibitors for Cryptosporidiosis therapy
Wenlin Huanga, Matthew A. Hulversonb, Ryan Choib, Samuel L.M. Arnoldb, Zhongsheng
Zhanga, Molly C. McCloskeyb, Grant R. Whitmanb, Robert C. Hackmanc,d,e, Kasey L. Rivasb,
Lynn K. Barrettb, Kayode K. Ojob, Wesley C. Van Voorhisb*, Erkang Fana*
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
50
51
52
53
54
55
56
57
58
59
60
a Department of Biochemistry, University of Washington, Seattle, Washington 98195, United
States
b Department of Medicine, Division of Allergy & Infectious Disease, Center for Emerging & Re-
emerging Infectious Disease (CERID), University of Washington, Seattle, Washington 98109,
United States
c Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, United States
d Department of Pathology, University of Washington, Seattle, Washington 98195, United States
e Department of Laboratory Medicine, University of Washington, Seattle, Washington 98195,
United States
* Corresponding authors:
Wesley C. Van Voorhis; E-mail: wesley@uw.edu
Erkang Fan; E-mail: erkang@uw.edu
ABSTRACT
Cryptosporidium is a leading cause of pediatric diarrhea worldwide. Currently there is
neither a vaccine nor a consistently effective drug available for this disease. Selective 5-
aminopyrazole-4-carboxamide-based bumped-kinase inhibitors (BKIs) are effective in both in
vitro and in vivo models of Cryptosporidium parvum. Potential cardiotoxicity in some BKIs led to
the continued exploration of the 5-aminopyrazole-4-carboxamide scaffold to find safe and
effective drug candidates for Cryptosporidium. A series of newly designed BKIs were tested for
efficacy against C. parvum using in vitro and in vivo (mouse infection model) assays, and safety
issues. Compound 6 (BKI 1708) was found to be efficacious at 8 mg/kg dosed once daily (QD)
for 5 days with no observable signs of toxicity up to 200 mg/kg dosed QD for 7 days. Compound
15 (BKI 1770) was found to be efficacious at 30 mg/kg dosed twice daily (BID) for 5 days with
no observable signs of toxicity up to 300 mg/kg dosed QD for 7 days. Compounds 6 and 15 are
ACS Paragon Plus Environment

Page 3 of 40
Journal of Medicinal Chemistry
1
2
3
4
5
6
promising pre-clinical leads for cryptosporidiosis therapy with acceptable safety parameters and
efficacy in the mouse model of cryptosporidiosis.
7
8
9
INTRODUCTION
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
50
51
52
53
54
55
56
57
58
59
60
Cryptosporidiosis, caused by the apicomplexan parasite Cryptosporidium, is a major
contributor to global diarrheal morbidity and mortality, particularly in 6-24 month old
malnourished children, and is associated with growth stunting and developmental deficits in
children.1-3 In immunocompromised individuals, Cryptosporidium infection becomes persistent
and life threatening. Nitazoxanide, the only U.S. Food and Drug Administration-approved drug
for the treatment of cryptosporidiosis, has limited efficacy in immunocompromised patients and
malnourished children, and is only approved for use in children ≥1 year-old, leaving the most
vulnerable patient populations without effective treatment.4-6 Thus, new therapies with established
safety and efficacy in malnourished infants and immunocompromised individuals are urgently
needed for cryptosporidiosis.
Previous studies have shown that inhibition of apicomplexan calcium-dependent protein
kinases interfere with the parasites’ ability to invade or egress mammalian host cells.7-9 The
calcium-dependent protein kinase 1 of C. parvum (CpCDPK1) is a promising target for drug
development for cryptosporidiosis.10-12 A recent scientific report demonstrated that knocking down
CpCDPK1 with small interfering RNAs (siRNAs) significantly decreased parasite invasion and
growth.12 Moreover, there was an additive effect of CpCDPK1 knockdown in combination with
treatment of BKIs 1294 and 1517 in reducing the proliferation of C. parvum parasites.12 These
results suggest that CpCDPK1 is a target of BKIs.
BKIs have been developed to selectively target CpCDPK1, prevent the growth of C.
parvum in vitro and in vivo, and have proven anti-Cryptosporidium efficacy in the SCID/beige
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 40
1
2
3
4
5
6
7
8
9
immunocompromised mouse and the newborn calf C. parvum clinical models (Fig. 1).
Pyrazolopyrimidine (PP) based BKIs 1294 and 1369 exhibited anti-Cryptosporidium efficacy in
mouse and the newborn calf C. parvum clinical models.11,13,14 Pyrrolopyrimidine (PrP) based BKIs
are distinct therapeutic alternatives of PP inhibitors. PrP BKI 1649 potently inhibited CpCDPK1,
prevented the growth and proliferation of C. parvum in vitro, and reduced oocyst shedding in a
mouse model of cryptosporidiosis.15 5-aminopyrazole-4-carboxamide (AC) scaffold BKIs are also
potent inhibitors of C. parvum growth in vitro16, 17 and in the SCID/beige immunocompromised
mouse and the newborn calf C. parvum clinical models.12, 13
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
50
51
52
53
54
55
56
57
58
59
60
Despite their promising efficacies, some BKIs have shown toxicity issues that limit their
potential for human treatment. PP BKI 1294 has shown activity against the human ether-á-go-go
related gene (hERG) in vitro.18 While AC BKI 1517 did not have significant in vitro hERG
inhibition signal,19 it exhibits other indicator of potential cardiac toxicity, namely a significant
change in mean arterial pressure, when dosed in rats (data not shown). However, changes to the R
groups in the PP scaffold has been previously shown to decrease these cardiovascular issues when
compared to BKI 1294, leaving open the possibility of similarly eliminating such issues in the AC
scaffold. 20 The study presented here describes the iterative improvements for efficacy and safety
of the AC scaffold inhibitors which have resulted in preclinical leads, compounds 6 and 15 (BKIs
1708 and 1770, respectively) for cryptosporidiosis therapy. Compounds 6 and 15 potently inhibit
activity of CpCDPK1 and reduce oocyst shedding in C. parvum infected interferon-γ knock out
(IFN-γ KO) mice. In addition, both 6 and 15 are safe in that they lack mutagenic activity, are not
toxic to a variety of human cell lines, show substantial improvement over BKI 1294 in hERG
assays, and can be administered orally to mice up to 200 to 300 mg/kg of body weight for 7 days
with little to no signs of toxicity.
ACS Paragon Plus Environment

Page 5 of 40
Journal of Medicinal Chemistry
1
2
3
4
5
6
RESULTS AND DISCUSSION
Molecular design and synthesis
7
8
9
We previously developed a number of highly promising AC scaffold BKIs that potently
block the enzymatic activity of Toxoplasma gondii calcium-dependent protein kinase 1
(TgCDPK1), thereby blocking the invasion of T. gondii into host cells.17,20 Many of these selective
inhibitors are also effective in both in vitro and in vivo models of cryptosporidiosis, especially the
previously reported BKI 1517.12,13,16 Unfortunately, BKI 1517 was found to have cardiotoxicity in
rats at doses very close to therapeutic doses for cryptosporidium efficacy (data not shown).
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
50
51
52
53
54
55
56
57
58
59
60
Recent studies on the PP BKIs suggest that higher fecal levels of this BKI series may be a
good proxy for elevated intraepithelial concentrations in the gastrointestinal (GI) tract and in vivo
efficacy.22 We also found that compound 7 (BKI 1608), bearing a hydroxyl aliphatic chain at the
N1 position, reduced systemic exposure, high fecal levels, and in vivo efficacy in neonatal mice.16
Thus, we investigated a series of AC BKIs with a hydroxyl aliphatic chain at the N1 position and
a fixed C3 position Ar group as ethoxy or cyclopropoxy substituted quinolone/naphthyl to identify
CpCDPK1 inhibitors that possess distinct physiochemical properties, favorable anti-parasitic
activity, and improved safety properties.
The synthesis of these AC BKIs followed previously reported procedures.16,17,20 As shown
in Scheme 1, a quinoline/naphthyl aldehyde with varied substitutions, was reacted with a hydrazine
in ethanol to give the hydrazone, which was then brominated by NBS. The intermediate was then
reacted with cyanoacetamide anion that was generated in situ to afford the final product. The
majority of the synthetic effort was spent on generating quinoline/naphthyl aldehydes and
hydrazines with desired substitutions. The synthesis of these key intermediates are reported in
supporting information.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 40
1
2
3
4
Scheme 1.
5
6
7
8
1 R
N N
2
H
N
O
H
H
NH
R N
2 HCl
N
b
N
R
c
5
3
N
R
H
Ar
NH2
Ar
a
4
Br
Ar
9
H
Ar
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
50
51
52
53
54
55
56
57
58
59
60
H2N
O
Reagents and conditions (a) DIPEA, EtOH, microwave, 70°C, 20 min; (b) NBS, DMF, 0°C, 20 min; (c)
cyanoacetamide, NaH, DMF, room temperature (r.t.), overnight.
In vitro compound screening
Multiple assays were used to determine the activity and selectivity of each compound, 1-
24, with various Ar and R group constituents and variations on the position of the nitrogen within
the quinoline group as compared to the naphthalene equivalents. Compounds were first tested for
activity against CpCDPK1 (Table 1). We were expecting all of the compounds to be potent
inhibitors of CpCDPK1 based on the SAR we found previously, as all the compounds bear an Ar
group as ethoxy or cyclopropoxy substituted quinolone/naphthyl which are optimal substitutions.
16, 17, 20
The various hydroxyl containing R groups were also expected to be well tolerated as this
functional group projects into the ribose binding pocket and the solvent.16 Indeed, all of the
compounds potently inhibit CpCDPK1 at < 0.01 μM. The result is consistent with the SAR we
found previously.16 BKIs that successfully blocked in vitro activity to CpCDPK1 were tested
against mammalian tyrosine kinase Src (c-Src), a representative mammalian kinase, to eliminate
compounds with potential off-target activities (Table 1). All compounds were then tested against
Nanoluciferase expressing (Nluc) C. parvum infected HCT-8 cells for in vitro cellular potency and
CRL-8155 and HepG2 mammalian cells for general off-target activity and toxicity (Table 1).
Cellular inhibition of C. parvum was consistently <1 μM for most compounds (22 out of the 27
total), regardless of R groups or the quinoline/naphthyl variation.
ACS Paragon Plus Environment

Page 7 of 40
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Due to the previous prevalence of human hERG activity and its associated cardiovascular
issues in BKIs of the PP scaffold,18 compounds that displayed sufficiently promising in vitro
potencies against CpCDPK1 and C. parvum cells and lacked off-target activities against c-Src and
mammalian cells were tested to determine their hERG IC50s in either the thallium-based or Qpatch
assays (Table 1). Additional in vitro screens were conducted to determine compound properties
that may best correlate to future in vivo efficacy. These included plasma protein binding in both
mouse and human plasma using dialysis membranes, and solubility at pH values present in the GI
tract (2.0 and 6.5) (Table 1).
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
50
51
52
53
54
55
56
57
58
59
60
Pharmacokinetic profiling in mice
A subset of compounds were tested to determine plasma exposure and fecal concentrations
after a single 10 or 25 mg/kg body weight oral dose (PO). Pharmacokinetic (PK) calculations were
performed to determine maximum concentration (Cmax), area under the curve (AUC), terminal
half-life (t½), and oral clearance (Table 2). Compound excreted in feces over a 24-hour period post
25 mg/kg PO dose were also measured (Table 2). None of the PK parameters correlated with
quinoline/naphthyl variants within the scaffold. There was no association between solubility and
the observed Cmax or AUC among the compounds, regardless of R groups or quinoline/naphthyl
variants. In addition, solubility did not correlate with fecal levels observed after oral dosing.
Efficacy in IFN-γ KO mouse model of C. parvum infection
A subset of 11 compounds, 3, 5, 6, 7, 9, 12, 15, 20, 21, 23, and 24, were selected for oral
dosing in IFN-γ KO mice infected with Nluc expressing C. parvum (Table 3). Since most
compounds were potent against both the CpCDPK1 enzyme and C. parvum in vitro, these
compounds were selected to represent a variety of properties, including varying solubility,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 40
1
2
3
4
5
6
7
8
9
systemic exposures, fecal exposures, and half-lives, across the different groups of
quinoline/naphthyl variants and the different R groups, in order to best determine which chemical
properties from Tables 1 or 2 would best predict efficacy within this mouse model for the AC
BKIs. Mice were dosed orally for 5 days with a dose based partially upon observed PK (Table 2).
Based on the observed PK of compound 15 and previous results with twice daily (BID) dosing of
AC compound 1517, the compound was dosed BID for the efficacy studies.12 All other compounds
were dosed once daily. Compounds 5, 6, 7, 9, 11, 15, 20, 23, and 24 all showed significant
reduction in oocyst shedding at various doses (Table 3). Compounds 3 and 21 failed to reduce
infection despite having a relatively high fecal concentration, suggesting a differential attribute
profile requirement for in vivo efficacy of the AC relative to the PP scaffold BKIs.20 It could also
mean that some AC compounds in feces may not be associated with solubilized compound in the
gastrointestinal tract.20, 22 The efficacious compounds showed wide variations in Cmax, AUC, t½,
and fecal concentration from their respective single dose oral mouse PKs (Table 2). There was no
observed correlation between efficacy and any of these PK parameters. While all compounds
efficacious in mice had solubilities >50 μM at both pH 2.0 and 6.5, compounds 3 and 21 had poor
solubility at least at one pH level and failed to be efficacious in the IFN-γ KO mouse model despite
reasonable plasma exposure for 21 (Tables 1 and 2). These results strongly suggest a certain
threshold of solubility may be required for in vivo efficacy for this class of compounds.
Additional compound evaluation
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
50
51
52
53
54
55
56
57
58
59
60
Based upon the above predicted importance of solubility in correlation to efficacy, three
additional AC compounds, 25, 26, and 27, were chosen for evaluation of in vitro and PK properties
(Tables 1 and 2) and efficacy to confirm correlation to solubility. Compounds 25, 26, and 27 have
unrelated R1 and R2 groups and all have the naphthyl variant in the central scaffold. All three had
ACS Paragon Plus Environment

Page 9 of 40
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
low oral clearance, relatively high t½, and good systemic exposure from oral PK (Table 2).
However, 25 had good solubility and low fecal exposure, while 26 and 27 showed poor solubility
and high fecal exposures (Tables 1 and 2). When tested in Nluc expressing C. parvum infected
IFN-γ KO mice, compound 25 was efficacious while 26 and 27 were not (Table 3). With the
inclusion of compounds 25, 26 and 27, a strong correlation between efficacy, as represented by
log reduction over untreated controls (Table 3), and solubility at pH 2.0 (r12=0.93, P<0.0001) and
pH 6.5 (r10=0.95, P<0.0001) can be observed. A pH of 2.0 is representative of the stomach, where
the compounds are first administered by oral gavage, while pH 6.5 is more representative of the
intestine. Compounds 3 and 27 have high solubility at pH 2.0, but low solubility at pH 6.5. They
should be highly solubilized in the stomach, but could be failing efficacy if they were partially
precipitating into the intestinal lumen with the decreased solubility at the more neutral intestinal
pH, thereby dropping the solubilized compound below concentrations that can enter infected
epithelial cells of the small or large intestine at sufficient levels to inhibit growth of the parasites.
Similarly, if compounds 21 and 26 were only solubilized at low levels in both the stomach and
intestine, they would not reach sufficient concentrations to enter infected epithelial cells and inhibit
parasite growth. However, efficient absorption of poorly soluble compounds may happen at sites
away from parasite infection in the GI track, leading to reasonable or high plasma exposures (such
as 21, 26, or 27). This could also explain how compounds 3, 21, 26, and 27 could reach high
concentrations in the feces or even plasma but not show efficacy, as the majority of the compound
would simply pass through the GI lumen in a precipitated state without ever reaching the site of
infection within the intestinal epithelial cells. This may be one possible explanation for the lack
of correlation (r10=-0.33, P>0.2) seen between fecal levels and efficacy, contrary to what was
previously observed with the PP scaffold of BKIs.20 Given the previously established necessity to
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
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 40
1
2
3
4
5
6
7
8
9
have BKI exposure throughout the entire GI tract in order to effectively treat cryptosporidiosis in
mice, it is clear that a threshold of solubility is required at all pHs present in the GI tract for a
compound to be efficacious.22 Future studies that further examine the relationship of AC
compound solubility and exposure to parasites and infection sites within the different sections of
the GI tract would certainly be warranted.
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
50
51
52
53
54
55
56
57
58
59
60
At this stage, many compounds were eliminated from further consideration for a variety of
individual shortcomings that might make them unsuitable as a clinical treatment candidate. Table
4 shows the reasoning as to why individual compounds were either removed from consideration
or moved to further testing.
Dose response and toxicity profiling
Based on their promising initial efficacy and in vitro testing results, compounds 6, 9, 15,
20, and 25 were tested using dose titrations in Nluc expressing C. parvum infected IFN-γ KO mice
to determine the lowest possible efficacious dose in mice, while lowering Cmax plasma levels to
assist in alleviating possible toxicity concerns. Compound 6 was initially efficacious at 60 mg/kg
dosed once daily (QD) for 5 days (Fig. 2A). Doses of 30, 15, and 8 mg/kg were also shown to be
efficacious when administered QD for 5 days, with 3 to 4-log reductions in infection for each.
Only the rates of decline varied among these doses, with the lowest doses taking slightly longer to
reach the lower limits of detection (LLOD) for this assay. Plasma levels of 6 at the lowest
efficacious dose of 8 mg/kg QD reached a Cmax of 2.6 ± 0.7 µM after the 4th dose. Compound 9
was initially efficacious at a dose of 60 mg/kg QD for 5 days (Fig. 2B). Although 9 showed an
improvement over untreated controls at 30 mg/kg BID, it unfortunately allowed infection levels in
mice to remain a full log above the LLOD (Fig. 2B). The high dose concentration of 9 could prove
to be a future liability as this compound contains a chiral center at the R group, which could drive
ACS Paragon Plus Environment

Page 11 of 40
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
up manufacturing costs for a drug that is primarily aimed at resource-limited populations.
Compound 15 was originally tested at 60 mg/kg BID for 5 days and showed a 4-log reduction to
below the LLOD (Fig. 2C). Subsequent doses of 120 and 60 mg/kg QD, and 30, 15, and 5 mg/kg
BID were administered for 5 days (Fig. 2C). Dose concentrations of 120 mg/kg QD and 30 mg/kg
BID of 15 were also successful at significantly reducing oocyst shedding compared to untreated
controls, with reductions to below the LLOD. However, 60 mg/kg QD and 15 and 5 mg/kg BID
of 15 failed to provide an efficacious result (Fig. 2C). Plasma levels of 15 at the efficacious dose
of 30 mg/kg BID reached a Cmax of 0.51 ± 0.2 µM after the 7th dose. Compound 20 was efficacious
to the LLOD at 60 mg/kg QD for 5 days, but failed to provide a significant reduction from initial
infection levels at 10 or 20 mg/kg BID (Fig. 2D). The systemic exposure of 20 is relatively high
(Table 2), so a reduced dose would also be ideal here to mitigate possible toxicity concerns.
Compound 25 was efficacious at 60 mg/kg QD, and was further tested at 30 and 15 mg/kg QD for
5 days (Fig. 2E). The 30 mg/kg dose of 25 also reduced infection to below the LLOD, but the 15
mg/kg dose failed to reduce infections from their initial levels, despite showing an improvement
over the untreated controls (Fig. 2E). Plasma levels of 25 at the efficacious dose of 30 mg/kg QD
reached a Cmax of 28.8 ± 8.9 µM after the 4th dose.
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
50
51
52
53
54
55
56
57
58
59
60
Various signs of toxicity in in vivo models have been previously observed with oral dosing
of specific BKI analogs of the PP scaffold, including neurological,23 gastrointestinal, or other
general adverse reactions.14 Based on the findings of low efficacious dose concentrations,
compounds 6, 15, and 25 were moved to tolerability and toxicity testing in mice in order to
determine if these efficacious AC compounds would induce any similar signs of toxicity. No
adverse effects were seen during the highest concentration dosing in the mouse efficacies, so a
single oral dose was given to mice at escalating dose concentrations until signs of toxicity were
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 40
1
2
3
4
5
6
7
8
9
observed as described.23 Doses were started at 100 mg/kg for 6, 150 mg/kg for 15, and 100 mg/kg
for 25. This was increased to 200, 300, and 400 mg/kg for 6, 200, 300, 400, and 600 mg/kg for 15,
and 150, 200, and 250 mg/kg for 25. There were no observable signs of toxicity or distress from
any of these dose concentrations for 6, 15, or 25. However, escalation had to be stopped as all
three compounds reached their limit of solubility in the dosing solutions at these concentrations.
For compound 6, mice were dosed at 100 and 200 mg/kg QD for 7 days to determine
potential toxicity. Neither dose concentration showed any observable signs of toxicity during or
after dosing. Plasma concentrations sampled at pre-dose, 0.5 hour, and 1 hour post dose around
the 2nd and 4th doses were analyzed by LC-MS/MS (Table 5). Compound 15 was dosed at 150 and
300 mg/kg QD for 7 days to check for signs of toxicity. Neither dose concentration showed any
observable signs of toxicity after 7 days of dosing. Plasma concentrations sampled at pre-dose,
0.5 hour, and 1 hour post dose around the 2nd and 5th doses were analyzed by LC-MS/MS (Table
5). Compound 25 was dosed at 100 and 250 mg/kg QD for 7 days. No signs of toxicity were
observed at the 100 mg/kg dose after 7 days. Minor signs of toxicity were observed after the 7th
dose of 25 at 250 mg/kg, including slight lethargy and lack of grooming. Plasma levels were
collected at 2 hours after the 2nd dose, prior to the 3rd dose, 2 hours after the 5th dose and 24, 48
and 72 hours after the 7th dose and analyzed for compound concentration by LC-MS/MS (Table
5). Blood and organs were collected from all mice after the final dose. Histological analysis of all
major organs showed no abnormalities and CBC and serum chemistry results were all within the
normal range for mice for both 15 and 25 at both dose concentrations. For compound 6, CBC and
serum chemistry results showed a decrease in the white blood cell counts from 4.8 ± 1.2 K/µL in
the untreated controls to 1.5 ± 0.2 K/µL for the 100 mg/kg dosed mice and 1.5 ± 0.9 K/µL for the
200 mg/kg dosed mice. There was also a decrease in lymphocytes in the compound 6 treatments,
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
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 13 of 40
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
from 4,060 ± 1,266 ABS/µL for the untreated controls to 1,194 ± 71 ABS/µL for the 100 mg/kg
dosed mice and 1,301 ± 678 ABS/µL for the 200 mg/kg dosed mice. Though there were no visible
signs of abnormalities in any of the organs when compared to controls at either dose for compound
6, some amount of the compound and vehicle was observed to have precipitated and remained in
the stomach and small intestines for the 200 mg/kg dose, suggesting that solubility limits may be
insurmountable for this compound at higher dose concentrations. However, this is well above the
efficacious dose concentration of 8 mg/kg QD, allowing for a reasonably large therapeutic window
to continue this compound as a possible treatment candidate.
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
50
51
52
53
54
55
56
57
58
59
60
Compound 25 only left a small therapeutic window between maximum plasma
concentrations of 72.2 ± 5.2 µM in the toxicity study and 28.8 ± 8.9 µM at the efficacious 30
mg/kg QD dose. Since minor signs of toxicity were already appearing after 7 doses of 25, this high
systemic exposure would be a limiting factor for its development into a clinical drug for
cryptosporidiosis. However, compound 15 had a low maximum plasma concentration of 0.51 ±
0.2 µM at its efficacious dose of 30 mg/kg BID. This is over 30-fold lower than the maximum
plasma exposures of 30.5 ± 6.1 µM after the 2nd dose and 34.1 ± 8.6 µM after the 5th dose in the
multi-dose toxicity at 300 mg/kg QD. Since there were no signs of toxicity at this high dose of 15,
this compound remains a viable candidate with a wide therapeutic window that is worth further
exploration in large animal efficacies and other toxicity screens.
Conclusions
In the present study, we designed a series of AC-scaffold BKIs, which contain variable Ar
and R groups. Hydroxyl containing R groups were mainly investigated with the goal of finding
compounds that have distinct physiochemical properties compared with the non-hydroxyl
containing counterparts. The compounds were screened for potent inhibition of the CpCDPK1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 40
1
2
3
4
5
6
7
8
9
enzyme and in vitro cellular potency. Inhibitors were then screened for the lack of growth
inhibition of mammalian cell lines and minimal hERG activity. Inhibitors were further tested for
efficacy in a mouse model and compounds that showed no clinical signs of illness were moved to
a dose response study. A strong correlation between solubility and in vivo efficacy was found for
this class of compounds. Compounds 6 and 15 are both efficacious in the C. parvum infected IFN-γ
KO mouse model, are not toxic to a variety of human cell lines, are safely administered orally to
mice at up to 200 to 300 mg/kg of body weight for 7 days, and did not show cardiotoxicity issues
in the in vitro hERG assays. Compounds 6 and 15 are both emerging as potential pre-clinical leads
for cryptosporidiosis therapy.
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
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL METHODS
General Synthetic Methods. Unless otherwise stated, all chemicals were purchased from
commercial suppliers and used without further purification. Microwave irradiation was performed
on a CEM Discover System. Reaction progress was monitored by thin-layer chromatograph on
silica gel containing an inert binder and a fluorescent indicator (activated at 254 nm) coated
flexible sheet (J. T. Baker). Chromatography was performed using an automated flash
chromatography system, eluting on pre-packed silica gel columns with CH2Cl2/MeOH or
Hexane/Ethyl ester gradient solvent system. The purity of all final compounds was determined by
analytical LC-MS using an Onyx Monolithic C18 column (4.6 mm x 100 mm) (Phenomenex,
Torrance, CA) and eluting with CH3CN/H2O solvent system (+0.1% TFA). The products were
detected by UV at 220 nm. All compounds were determined to be >95% pure by this method. The
mass spectra were recorded with an Ion Trap Mass Spectrometer (Agilent, Santa Clara, CA). High
resolution mass spectrometry (HRMS) was performed on LTQ-Orbitrap (Thermo Fisher) using
electrospray source in positive ionization mode and Orbitrap resolution setting of 60,000.
ACS Paragon Plus Environment

Page 15 of 40
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1H NMR spectra were recorded on Bruker 300 or 500 MHz spectrometers at ambient temperature.
13C NMR spectra were recorded on Bruker 126 MHz spectrometer at ambient temperature.
Chemical shifts are reported in parts per million (δ) referenced to the internal standards (1H signals:
13
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
50
51
52
53
54
55
56
57
58
59
60
7.26 ppm for CDCl3, 3.34 ppm for CD3OD and 2.50 ppm for (CD3)2SO; C signal: 39.51 for
(CD3)2SO) and coupling constants in Hz.
General Synthetic Procedure. Quinoline aldehyde (0.1 mmol, 1 eq), a corresponding hydrazine
hydrochloride (0.11 mmol, 1.1 eq) and DIPEA (0.11 mmol, 1.1 eq) were dissolved in EtOH (2
mL) in a microwave tube, purged with N2, heated to 70°C under microwave for 20 min. After
evaporation of the solvent, the product hydrozone was used for the next step directly.
Hydrazone obtained above (0.1 mmol, 1eq) was dissolved in 2 mL DMF and cooled to 0°C, then
NBS (0.11 mmol, 1.1 eq) dissolved in 1 mL DMF was added dropwise, the reaction mixture was
stirred at 0°C for 20 min.
In another flask, NaH (0.3 mmol, 3 eq) was added to a solution of 2-cyanoacetamide (0.2
mmol, 2 eq) in DMF. After stirring for 20 min, the solution was added to the above bromo-
hydrazone solution at 0°C, and the resulted reaction mixture was stirred at r.t. overnight. The
solvent was removed; the residue was extracted with CH2Cl2, dried over Na2SO4, filtered, and
concentrated in vacuo. The crude product was then purified via flash chromatography over silica,
eluting with a CH2Cl2/MeOH gradient. Further purification, if necessary, was performed via
preparatory RP-HPLC, eluting with H2O/CH3CN gradient (+0.1% TFA).
5-amino-3-(7-ethoxyquinolin-3-yl)-1-(3-hydroxy-2,2-dimethylpropyl)-1H-pyrazole-4-
carboxamide (1). Reported previously.21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 40
1
2
3
4
5-amino-3-(2-ethoxyquinolin-6-yl)-1-(3-hydroxy-2,2-dimethylpropyl)-1H-pyrazole-4-
5
6
carboxamide (2). Reported previously.21
7
8
9
5-amino-3-(6-ethoxynaphthalen-2-yl)-1-(3-hydroxy-2,2-dimethylpropyl)-1H-pyrazole-4-
carboxamide (3). 1H NMR (500 MHz, CDCl3) δ 7.94 (s, 1H), 7.81 – 7.75 (m, 2H), 7.58 (d, J =
8.6 Hz, 1H), 7.19 (d, J = 8.6 Hz, 1H), 7.15 (s, 1H), 4.17 (q, J = 6.6 Hz, 2H), 3.87 (s, 2H), 3.34 (s,
2H), 1.49 (t, J = 6.6 Hz, 3H), 1.05 (s, 6H). 13C NMR (126 MHz, DMSO) δ 166.2, 156.9, 151.6,
147.9, 134.0, 129.6, 128.9, 128.1, 127.5, 127.1, 126.9, 119.3, 106.5, 94.2, 67.4, 63.2, 52.6, 40.0,
37.7, 22.6, 14.6. MS (ESI) (M+H)+=383.6; HRMS (ESI) m/z: calculated for C21H26N4O3 [M +
H]+ 383.2078; observed 383.2083. HPLC analysis: 97.8% purity.
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
50
51
52
53
54
55
56
57
58
59
60
5-amino-3-(7-cyclopropoxyquinolin-3-yl)-1-(3-hydroxy-2,2-dimethylpropyl)-1H-pyrazole-4-
carboxamide (4). 1H NMR (500 MHz, MeOD) δ 9.28 (s, 1H), 9.22 (s, 1H), 8.28 (d, J = 9.2 Hz,
1H), 7.88 (s, 1H), 7.61 (dd, J = 9.2, 1.8 Hz, 1H), 4.20 – 4.13 (m, 1H), 3.95 (s, 1H), 3.32 (s, 1H),
1.06-1.00 (m, 8H), 0.94 – 0.88 (m, 2H). MS (ESI) (M+H)+= 396.5; HRMS (ESI) m/z: calculated
for C21H25N5O3 [M + H]+ 396.2030; observed 396.2026. HPLC analysis: 96.0% purity.
5-amino-3-(2-cyclopropoxyquinolin-6-yl)-1-(3-hydroxy-2,2-dimethylpropyl)-1H-pyrazole-4-
1
carboxamide (5). H NMR (500 MHz, CDCl3) δ 8.03 (d, J = 8.9 Hz, 1H), 7.99 (d, J = 8.5 Hz,
1H), 7.91 (s, 1H), 7.79 (d, J = 8.5 Hz, 1H), 6.94 (d, J = 8.9 Hz, 1H), 5.27-5.12 (m, 2H), 4.57-
4.48 (m, 2H), 3.86 (s, 2H), 3.34 (s, 2H), 1.04 (s, 6H), 0.90 – 0.80 (m, 4H). 13C NMR (126 MHz,
DMSO) δ 166.2, 162.6, 151.6, 147.4, 145.8, 139.6, 130.5, 129.7, 127.7, 127.0, 124.8, 113.1,
94.3, 67.4, 52.6, 50.0, 37.7, 22.6, 5.6. MS (ESI) (M+H)+= 396.6; HRMS (ESI) m/z: calculated
for C21H25N5O3 [M + H]+ 396.2030; observed 396.2027. HPLC analysis: 97.0% purity.
5-amino-3-(6-cyclopropoxynaphthalen-2-yl)-1-(3-hydroxy-2,2-dimethylpropyl)-1H-
pyrazole-4-carboxamide (6). 1H NMR (500 MHz, DMSO) δ 7.98 (s, 1H), 7.91 (d, J = 8.7 Hz,
ACS Paragon Plus Environment

Page 17 of 40
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2H), 7.58 (d, J = 11.6 Hz, 2H), 7.22 (dd, J = 8.9, 2.0 Hz, 1H), 6.35 (s, 2H), 5.09 (t, J = 5.0 Hz,
1H), 4.03 – 3.94 (m, 1H), 3.80 (s, 2H), 3.21 (d, J = 4.9 Hz, 2H), 0.93 (s, 6H), 0.88 (q, J = 6.1 Hz,
2H), 0.74 (s, 2H). 13C NMR (126 MHz, DMSO) δ 166.7, 157.0, 151.5, 147.8, 133.8, 129.6,
129.0, 128.3, 127.5, 127.1, 126.9, 118.8, 107.9, 94.2, 67.4, 52.6, 50.9, 37.6, 22.5, 5.9. MS (ESI)
(M+H)+= 395.6; HRMS (ESI) m/z: calculated for C22H26N4O3 [M + H]+ 395.2078; observed
395.2074. HPLC analysis: >99.0% purity.
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
50
51
52
53
54
55
56
57
58
59
60
5-amino-3-(7-ethoxyquinolin-3-yl)-1-(4-hydroxybutan-2-yl)-1H-pyrazole-4-carboxamide
(7). Reported previously.21
5-amino-3-(2-ethoxyquinolin-6-yl)-1-(4-hydroxybutan-2-yl)-1H-pyrazole-4-carboxamide
(8). 1H NMR (500 MHz, MeOD) δ 8.18 (d, J = 8.8 Hz, 1H), 7.98 (d, J = 1.8 Hz, 1H), 7.91 (d, J =
8.6 Hz, 1H), 7.78 (dd, J = 8.6, 1.9 Hz, 1H), 6.99 (d, J = 8.8 Hz, 1H), 4.59 – 4.47 (m, 3H), 3.60
(ddd, J = 10.8, 5.9, 4.9 Hz, 1H), 3.42 (ddd, J = 10.8, 8.7, 5.2 Hz, 1H), 2.18 (ddt, J = 14.4, 9.8, 5.0
Hz, 1H), 2.04 – 1.92 (m, 1H), 1.50 (d, J = 6.7 Hz, 3H), 1.46 (t, J = 7.1 Hz, 3H). MS (ESI)
(M+H)+= 370.8; HRMS (ESI) m/z: calculated for C19H23N5O3 [M + H]+ 370.1874; observed
370.1871. HPLC analysis: >99.0% purity.
5-amino-3-(6-ethoxynaphthalen-2-yl)-1-(4-hydroxybutan-2-yl)-1H-pyrazole-4-carboxamide
(9). 1H NMR (500 MHz, MeOD) δ 7.96 (s, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 8.9 Hz,
1H), 7.57 (dd, J = 8.4, 1.5 Hz, 1H), 7.30 (d, J = 2.3 Hz, 1H), 7.21 (dd, J = 8.9, 2.3 Hz, 1H), 4.58
– 4.47 (m, 1H), 4.20 (q, J = 7.0 Hz, 2H), 3.60 (dt, J = 10.8, 5.4 Hz, 1H), 3.42 (ddd, J = 10.8, 8.8,
5.2 Hz, 1H), 2.18 (ddt, J = 14.4, 9.8, 5.0 Hz, 1H), 2.04 – 1.91 (m, 1H), 1.54 – 1.43 (m, 6H). 13C
NMR (126 MHz, DMSO) δ 166.40, 156.9, 150.3, 148.1, 134.0, 129.6, 129.2, 128.1, 127.5,
127.3, 126.8, 119.3, 106.5, 94.5, 63.2, 57.6, 48.2, 38.3, 20.1, 14.6. MS (ESI) (M+H)+= 369.5;
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 40
1
2
3
4
5
6
HRMS (ESI) m/z: calculated for C20H24N4O3 [M + H]+ 369.1921; observed 369.1918. HPLC
analysis: >99.0% purity.
7
8
9
5-amino-3-(2-cyclopropoxyquinolin-6-yl)-1-(4-hydroxybutan-2-yl)-1H-pyrazole-4-
carboxamide (10). 1H NMR (500 MHz, MeOD) δ 8.24 (d, J = 8.9 Hz, 1H), 8.02 (s, 1H), 7.97 (d,
J = 8.6 Hz, 1H), 7.83 (dd, J = 8.6, 1.7 Hz, 1H), 7.06 (d, J = 8.9 Hz, 1H), 4.55 (dt, J = 9.7, 6.6 Hz,
1H), 4.49 (ddd, J = 9.0, 6.2, 3.0 Hz, 1H), 3.61 (dt, J = 10.7, 5.4 Hz, 1H), 3.43 (ddd, J = 10.8, 8.9,
5.3 Hz, 1H), 2.19 (ddt, J = 14.3, 9.7, 4.9 Hz, 1H), 1.99 (tt, J = 8.8, 5.7 Hz, 1H), 1.51 (d, J = 6.7
Hz, 4H), 0.93 – 0.87 (m, 2H), 0.85-0.79 (m, 2H). MS (ESI) (M+H)+= 382.8; HRMS (ESI) m/z:
calculated for C20H23N5O3 [M + H]+ 382.1874; observed 382.1871. HPLC analysis: 95.0%
purity.
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
50
51
52
53
54
55
56
57
58
59
60
5-amino-3-(7-cyclopropoxyquinolin-3-yl)-1-(4-hydroxybutan-2-yl)-1H-pyrazole-4-
carboxamide (11). 1H NMR (500 MHz, MeOD) δ 8.97 (d, J = 2.1 Hz, 1H), 8.49 (d, J = 1.9 Hz,
1H), 7.94 (d, J = 9.0 Hz, 2H), 7.76 (d, J = 2.3 Hz, 1H), 7.33 (dd, J = 9.0, 2.4 Hz, 1H), 4.61 – 4.52
(m, 1H), 4.02 (dq, J = 9.0, 2.9 Hz, 1H), 3.65 – 3.58 (m, 1H), 3.43 (ddd, J = 10.9, 8.7, 5.2 Hz,
1H), 2.21 (qd, J = 9.8, 5.0 Hz, 1H), 2.06 – 1.95 (m, 1H), 1.53 (d, J = 6.7 Hz, 3H), 0.98 – 0.92 (m,
2H), 0.86 – 0.79 (m, 2H). MS (ESI) (M+H)+= 382.5; HRMS (ESI) m/z: calculated for
C20H23N5O3 [M + H]+ 382.1874; observed 382.1872. HPLC analysis: 97.0% purity.
5-amino-3-(6-cyclopropoxynaphthalen-2-yl)-1-(4-hydroxybutan-2-yl)-1H-pyrazole-4-
carboxamide (12). 1H NMR (500 MHz, MeOD) δ 7.98 (s, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.86 (d,
J = 8.9 Hz, 1H), 7.60 (d, J = 2.1 Hz, 1H), 4.54 (ddd, J = 9.6, 6.7, 4.9 Hz, 1H), 3.96 (dq, J = 9.0,
2.9 Hz, 1H), 3.61 (dt, J = 10.8, 5.4 Hz, 1H), 3.43 (ddd, J = 10.9, 8.7, 5.2 Hz, 1H), 2.19 (ddt, J =
14.4, 9.8, 5.0 Hz, 1H), 2.07 – 1.94 (m, 1H), 1.12 – 0.50 (m, 2H). 13C NMR (126 MHz, DMSO) δ
166.0, 157.0, 149.9, 147.7, 133.6, 129.8, 129.7, 128.9, 127.5, 127.3, 126.8, 118.9, 107.9, 94.5,
ACS Paragon Plus Environment

Page 19 of 40
Journal of Medicinal Chemistry
1
2
3
4
5
6
57.40, 50.87, 48.00, 38.20, 20.14, 5.98. MS (ESI) (M+H)+= 381.5; HRMS (ESI) m/z: calculated
for C21H24N4O3 [M + H]+ 381.1921; observed 381.1923. HPLC analysis: 98.0% purity.
7
8
9
5-amino-3-(7-ethoxyquinolin-3-yl)-1-(4-hydroxy-2-methylbutan-2-yl)-1H-pyrazole-4-
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
50
51
52
53
54
55
56
57
58
59
60
1
carboxamide (13). H NMR (500 MHz, MeOD) δ 9.25 (d, J = 1.5 Hz, 1H), 9.21 (s, 1H), 8.27
(d, J = 9.2 Hz, 1H), 7.59 (dd, J = 9.2, 2.0 Hz, 1H), 7.49 (d, J = 1.5 Hz, 1H), 4.39 – 4.32 (m, 2H),
3.56 (t, J = 7.1 Hz, 2H), 2.27 (t, J = 7.1 Hz, 2H), 1.72 (s, 6H), 1.54 (t, J = 7.0 Hz, 3H). MS (ESI)
(M+H)+= 384.8; HRMS (ESI) m/z: calculated for C20H25N5O3 [M + H]+ 384.2030; observed
384.2026. HPLC analysis: 96.2 % purity.
5-amino-3-(2-ethoxyquinolin-6-yl)-1-(4-hydroxy-2-methylbutan-2-yl)-1H-pyrazole-4-
carboxamide (14). 1H NMR (500 MHz, MeOD) δ 8.19 (d, J = 8.7 Hz, 1H), 7.98 (s, 1H), 7.90 (d,
J = 8.5 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.00 (d, J = 8.8 Hz, 1H), 4.55 (q, J = 6.8 Hz, 2H), 3.60
(t, J = 6.8 Hz, 2H), 2.28 (t, J = 6.8 Hz, 2H), 1.73 (s, 6H), 1.47 (t, J = 6.8 Hz, 3H). MS (ESI)
(M+H)+= 384.6; HRMS (ESI) m/z: calculated for C20H25N5O3 [M + H]+ 384.2030; observed
384.2031. HPLC analysis: 96.5% purity.
5-amino-3-(6-ethoxynaphthalen-2-yl)-1-(4-hydroxy-2-methylbutan-2-yl)-1H-pyrazole-4-
carboxamide (15). 1H NMR (500 MHz, MeOD) δ 7.95 (s, 1H), 7.88 (d, J = 8.3 Hz, 1H), 7.84 (d,
J = 8.9 Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.30 (s, 1H), 7.20 (d, J = 8.9 Hz, 1H), 4.20 (q, J = 6.7
Hz, 2H), 3.60 (t, J = 6.9 Hz, 2H), 2.27 (t, J = 6.9 Hz, 2H), 1.72 (s, 6H), 1.49 (t, J = 6.7 Hz, 3H).
13C NMR (126 MHz, DMSO) δ 166.7, 156.8, 150.4, 145.8, 133.9, 129.5, 129.1, 128.0, 127.5,
127.2, 126.8, 119.2, 106.5, 95.7, 63.1, 60.1, 57.0, 42.5, 27.2, 14.5. MS (ESI) (M+H)+= 383.5;
HRMS (ESI) m/z: calculated for C21H26N4O3 [M + H]+ 383.2078; observed 383.2075. HPLC
analysis: 98.9% purity.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 40
1
2
3
4
5
6
7
8
9
5-amino-3-(7-cyclopropoxyquinolin-3-yl)-1-(4-hydroxy-2-methylbutan-2-yl)-1H-pyrazole-4-
carboxamide (16). 1H NMR (500 MHz, CDCl3) δ 9.03 (s, 1H), 8.29 (s, 1H), 7.82 (s, 1H), 7.77
(d, J = 8.9 Hz, 1H), 7.26 (d, J = 8.9 Hz, 1H), 5.85 (s, 2H), 5.24 (s, 2H), 3.97-3.92 (m, 1H), 3.77
(t, J = 6.0 Hz, 2H), 2.29 (t, J = 6.0 Hz, 2H), 1.76 (s, 6H), 0.96-0.91 (m, 2H), 0.91-0.87 (m, 2H).
MS (ESI) (M+H)+= 396.5; HRMS (ESI) m/z: calculated for C21H25N5O3 [M + H]+ 396.2030;
observed 396.2030. HPLC analysis: 96.9% purity.
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
50
51
52
53
54
55
56
57
58
59
60
5-amino-3-(2-cyclopropoxyquinolin-6-yl)-1-(4-hydroxy-2-methylbutan-2-yl)-1H-pyrazole-4-
carboxamide (17). 1H NMR (500 MHz, MeOD) δ 8.22 (d, J = 8.9 Hz, 1H), 8.00 (d, J = 1.7 Hz,
1H), 7.95 (d, J = 8.6 Hz, 1H), 7.82 (dd, J = 8.6, 1.7 Hz, 1H), 7.04 (d, J = 8.9 Hz, 1H), 4.48 (tt, J
= 6.3, 3.0 Hz, 1H), 3.60 (t, J = 7.2 Hz, 2H), 2.28 (t, J = 7.2 Hz, 2H), 1.73 (s, 6H), 0.93 – 0.86 (m,
2H), 0.84 – 0.76 (m, 2H). MS (ESI) (M+H)+= 396.7; HRMS (ESI) m/z: calculated for
C21H25N5O3 [M + H]+ 396.2030; observed 396.2027. HPLC analysis: 97.5% purity.
5-amino-3-(6-cyclopropoxynaphthalen-2-yl)-1-(4-hydroxy-2-methylbutan-2-yl)-1H-
pyrazole-4-carboxamide (18). 1H NMR (500 MHz, MeOD) δ 7.97 (s, 1H), 7.92 (d, J = 8.4 Hz,
1H), 7.85 (d, J = 8.9 Hz, 1H), 7.61 – 7.55 (m, 2H), 7.22 (dd, J = 8.9, 2.2 Hz, 1H), 4.00 – 3.91 (m,
1H), 3.61 (t, J = 7.2 Hz, 2H), 2.28 (t, J = 7.2 Hz, 2H), 1.73 (s, 6H), 0.93-0.87 (M, 2H), 0.82 –
0.75 (m, 2H). MS (ESI) (M+H)+= 395.8; HRMS (ESI) m/z: calculated for C22H26N4O3 [M + H]+
395.2078; observed 395.2075. HPLC analysis: 96.0% purity.
5-amino-3-(7-ethoxyquinolin-3-yl)-1-(1-hydroxy-2-methylpropan-2-yl)-1H-pyrazole-4-
1
carboxamide (19). H NMR (500 MHz, MeOD) δ 9.28 (d, J = 1.3 Hz, 1H), 9.23 (s, 1H), 8.28
(d, J = 9.2 Hz, 1H), 7.61 (dd, J = 9.2, 2.2 Hz, 1H), 7.50 (s, 1H), 4.37 (q, J = 7.0 Hz, 3H), 3.89 (s,
2H), 1.66 (s, 6H), 1.56 (t, J = 7.0 Hz, 4H). MS (ESI) (M+H)+= 370.5; HRMS (ESI) m/z:
ACS Paragon Plus Environment

Page 21 of 40
Journal of Medicinal Chemistry
1
2
3
4
5
6
calculated for C19H23N5O3 [M + H]+ 370.1874; observed 370.1870. HPLC analysis: 95.0%
purity.
7
8
9
5-amino-3-(2-ethoxyquinolin-6-yl)-1-(1-hydroxy-2-methylpropan-2-yl)-1H-pyrazole-4-
carboxamide (20). 1H NMR (500 MHz, MeOD) δ 8.19 (d, J = 8.8 Hz, 1H), 7.99 (s, 1H), 7.91 (d,
J = 8.6 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.00 (d, J = 8.8 Hz, 1H), 4.55 (q, J = 7.0 Hz, 2H), 3.89
(s, 2H), 1.65 (s, 6H), 1.47 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO) δ 166.5, 161.8,
151.0, 145.7, 139.3, 130.4, 129.6, 127.6, 126.7, 124.5, 113.4, 95.8, 68.4, 62.4, 61.2, 23.5, 14.3.
MS (ESI) (M+H)+= 370.8; HRMS (ESI) m/z: calculated for C19H23N5O3 [M + H]+ 370.1874;
observed 370.1871. HPLC analysis: 98.3% purity.
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
50
51
52
53
54
55
56
57
58
59
60
5-amino-3-(6-ethoxynaphthalen-2-yl)-1-(1-hydroxy-2-methylpropan-2-yl)-1H-pyrazole-4-
carboxamide (21). 1H NMR (500 MHz, MeOD) δ 8.12 (s, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.91 (d,
J = 8.9 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.35 (s, 1H), 7.26 (dd, J = 8.9, 1.4 Hz, 1H), 4.21 (q, J =
7.0 Hz, 2H), 3.93 (d, J = 1.1 Hz, 2H), 1.71 (d, J = 1.8 Hz, 6H), 1.48 (t, J = 7.0 Hz, 3H). 13C
NMR (126 MHz, DMSO) δ 166.6, 156.9, 151.1, 146.6, 146.2, 134.0, 129.6, 129.1, 128.1, 127.6,
127.3, 126.8, 119.3, 106.5, 95.7, 68.4, 63.2, 62.4, 23.6, 14.6. MS (ESI) (M+H)+= 369.7; HRMS
(ESI) m/z: calculated for C20H24N4O3 [M + H]+ 369.1921; observed 369.1919. HPLC
analysis: >99.0% purity.
5-amino-3-(7-cyclopropoxyquinolin-3-yl)-1-(1-hydroxy-2-methylpropan-2-yl)-1H-pyrazole-
4-carboxamide (22). 1H NMR (500 MHz, MeOD) δ 9.31 (s, 1H), 9.23 (s, 1H), 8.32 – 8.25 (m,
1H), 7.86 (d, J = 13.1 Hz, 1H), 7.62 (d, J = 9.0 Hz, 1H), 4.20-4.13 (m, 1H), 3.90 (s, 2H), 1.67 (s,
6H), 1.06-1.01 (m, 2H), 0.95-0.89 (m, 2H). MS (ESI) (M+H)+= 382.5; HRMS (ESI) m/z:
calculated for C20H23N5O3 [M + H]+ 382.1874; observed 382.1871. HPLC analysis: 98.4%
purity.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 40
1
2
3
4
5
6
7
8
9
5-amino-3-(2-cyclopropoxyquinolin-6-yl)-1-(1-hydroxy-2-methylpropan-2-yl)-1H-pyrazole-
4-carboxamide (23). 1H NMR (500 MHz, MeOD) δ 8.92 (d, J = 9.2 Hz, 1H), 8.35 (d, J = 1.6
Hz, 1H), 8.18 (dd, J = 8.7, 1.8 Hz, 1H), 8.00 (d, J = 8.7 Hz, 1H), 7.82 (d, J = 9.2 Hz, 1H), 4.61
(dq, J = 8.9, 3.1 Hz, 1H), 3.89 (s, 2H), 1.65 (s, 6H), 1.12 – 1.05 (m, 4H). 13C NMR (126 MHz,
DMSO) δ 166.6, 162.6, 151.1, 145.9, 145.8, 139.6, 130.6, 129.9, 127.7, 126.9, 124.8, 113.1,
95.8, 68.4, 62.5, 50.0, 23.6, 5.6. MS (ESI) (M+H)+= 382.7; HRMS (ESI) m/z: calculated for
C20H23N5O3 [M + H]+ 382.1874; observed 382.1870. HPLC analysis: >99.0% purity.
5-amino-3-(6-cyclopropoxynaphthalen-2-yl)-1-(1-hydroxy-2-methylpropan-2-yl)-1H-
pyrazole-4-carboxamide (24). 1H NMR (500 MHz, MeOD) δ 8.12 (s, 1H), 8.01 (d, J = 8.5 Hz,
1H), 7.92 (d, J = 9.0 Hz, 1H), 7.65 – 7.60 (m, 2H), 7.27 (dd, J = 8.9, 2.3 Hz, 1H), 3.97 (ddd, J =
9.0, 5.9, 2.8 Hz, 1H), 3.93 (s, 2H), 1.71 (s, 6H), 0.91 (dt, J = 11.7, 5.9 Hz, 2H), 0.80 – 0.76 (m,
2H). 13C NMR (126 MHz, DMSO) δ 166.6, 157.0, 151.1, 146.5, 133.8, 129.7, 129.3, 128.4,
127.7, 127.3, 126.9, 118.9, 107.9, 95.7, 68.4, 62.5, 51.0, 23.6, 6.0. MS (ESI) (M+H)+= 381.7;
HRMS (ESI) m/z: calculated for C21H24N4O3 [M + H]+ 381.1921; observed 381.1918. HPLC
analysis: 98.0% purity.
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
50
51
52
53
54
55
56
57
58
59
60
5-amino-1-(tert-butyl)-3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrazole-4-carboxamide
(25). Reported previously.21
5-amino-1-(tert-butyl)-3-(6-(prop-2-yn-1-yloxy)naphthalen-2-yl)-1H-pyrazole-4-
carboxamide (26). 1H NMR (500 MHz, CDCl3) δ 8.00 (s, 1H), 7.84 (dd, J = 13.9, 8.7 Hz, 2H),
7.65 (dd, J = 8.4, 1.1 Hz, 1H), 7.29 (s, 1H), 7.25 (dd, J = 8.9, 2.4 Hz, 1H), 5.72 (s, 2H), 5.32 (s,
2H), 4.85 (d, J = 2.3 Hz, 2H), 2.60 (t, J = 2.3 Hz, 1H), 1.72 (s, 9H). MS (ESI) (M+H)+= 363.8;
HRMS (ESI) m/z: calculated for C21H22N4O2 [M + H]+ 363.1816; observed 363.1813. HPLC
analysis: 97.6% purity.
ACS Paragon Plus Environment

Page 23 of 40
Journal of Medicinal Chemistry
1
2
3
4
5-amino-1-(cyclopropylmethyl)-3-(6-ethoxynaphthalen-2-yl)-1H-pyrazole-4-carboxamide
5
6
(27). Reported previously.16
7
8
9
Pharmacokinetic analysis in adult mice. Mouse oral PK studies were performed as previously
described using three female BALB/c mice (10-12 weeks old) in each group.24 Each group
received an individual BKI at a dose of 10 or 25 mg/kg of body weight dissolved in 3% ethanol/7%
Tween 80/90% saline by oral gavage. Blood samples were taken by tail bleeding into heparinized
tubes at designated time points and centrifuged to obtain plasma. The samples were frozen at -
20°C. The test compounds were extracted from the plasma samples using acetonitrile/0.1% formic
acid with an internal standard. A standard mix of all test compounds was prepared for comparison
and quantification. The compounds were quantified by LC-MS/MS analysis. PK calculations of
Cmax, time at which Cmax is observed (Tmax), AUC, oral clearance, and half-life were performed
using Pharsight Phoenix WinNonlin software (Certara, St. Louis, MO, USA).
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
50
51
52
53
54
55
56
57
58
59
60
Fecal concentration analysis in adult mice. Fecal concentrations were determined as previously
described.20 Three female BALB/c mice (10-12 weeks old) were used in each group. Each group
received an individual BKI at a dose of 25 mg/kg of body weight dissolved in 3% ethanol/7%
Tween 80/90% saline by oral gavage. Feces were collected for 24 h, weighed and soaked overnight
in 3 mL of DPBS per gram of feces. Feces were homogenized and an internal standard added. The
test compounds and internal standard were extracted from the fecal samples using acetonitrile. The
samples were centrifuged for 30 min at 2000 g. Supernatant was removed and concentrated by
repeated evaporation in a Savant SPD121P SpeedVac Concentrator (Thermo Scientific, Waltham,
MA, USA) and reconstitution in acetonitrile. A standard mix of all test compounds was prepared
for comparison and quantification. The compounds were quantified by LC-MS/MS analysis and
the fecal concentration was calculated.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 40
1
2
3
4
5
6
7
8
9
Plasma protein binding. Plasma protein binding was determined as previously described.24
Dialysis membrane sheets (MW cutoff 3.5 kDa) (HTDialysis, LLC, Gales Ferry, CT, USA) were
soaked for 1 h in DPBS and then in 20% ethanol for 30 min. The membrane was clamped between
two Teflon plates containing a row of opposing wells. Test compound in DMSO was added to 0.12
mL of serum to a concentration of 5 μM; a small aliquot was taken as a 100% recovery standard
and the solution was placed on one side of the membrane. The well on the other side of the
membrane was charged with an equal volume of DPBS, and the block was placed on an orbital
shaker for 18 h at 37°C. An aliquot was taken from each side of the membrane. All aliquots were
matrix adjusted, internal standard in acetonitrile was added to each aliquot, and the samples were
centrifuged to precipitate protein. Test compound in the supernatants was quantified by LC-
MS/MS analysis to determine the concentration on each side of the membrane and the total
recovery of test compound from the device. A control dialysis was carried out with dialysis buffer
on both sides of the membrane and test compound on one side to ensure that equilibration across
the membrane was achieved. The fraction of compound bound to protein was calculated as
bound/(unbound + bound).
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
50
51
52
53
54
55
56
57
58
59
60
CpCDPK1 enzyme inhibition of BKIs. C.p CDPK1 enzyme inhibition and IC50 determination of
BKIs was determined by measuring ATP consumption in a coupled reaction for phosphate group
incorporation to biotinylated Syntide-2 (BioSyntide-2) peptide substrate (Biotin-C6-
PLARTLSVAGLPGKK) (American Peptide Company, Inc. Sunnyvale, CA) in the presence or
absence of inhibitors as previously described.10 All assays were performed in a buffered solution
containing 1 mM EGTA (pH 7.2), 10 mM MgCl2, 20 mM HEPES pH 7.5 (KOH), 0.1% BSA and
enzyme activation reagent containing 2 mM CaCl2. The final reaction volume of 25 µL contained
20 µM BioSyntide-2, 2.1 µM CpCDPK1, with or without serial dilutions of inhibitors. The
ACS Paragon Plus Environment

Page 25 of 40
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
reaction was initiated with the addition of ATP at 10 µM final concentration. Internal positive and
negative controls were included in each assay plate. Reaction mixture was incubated at 30°C and
90 rpm for 90 min. Subsequent changes to initial ATP concentration were measured by a non-
radioactive Kinaseglo® luciferase reagent (Promega, Madison, WI) as a luminescence readout
using an EnVision Multilabel Plate Reader (Perkin Elmer, Waltham, MA).
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
50
51
52
53
54
55
56
57
58
59
60
BKI inhibition of Nanoluciferase-expressing C. parvum growth. Inhibition of C. parvum
growth and EC50 determinations were performed with a Nluc-expressing C. parvum parasite in
HCT-8 cells as previously described.20, 25 Briefly, oocysts were propagated in IFN-γ KO mice and
isolated from stools by sucrose suspension and cesium chloride gradient. HCT-8 cells were seeded
to a 96 or 384-well plate and allowed to grow for 72 h to reach 90-100% confluence. Oocysts were
activated by 10-15 min incubation in 10% bleach (0.6% sodium hypochlorite) at room temperature
and then washed with DPBS. 500-2000 oocysts per well, depending on the plate type and volume
per well used, were applied to plates with RPMI-1640 medium supplemented with 10% heat
inactivated horse serum and 1% penicillin/streptomycin at the same time as compound addition.
The compound and the oocysts were left in place for the full 72 h growth time. The amount of
luciferase-induced light emission was determined by lysing the cell monolayer, adding Nano-
Glo® luciferase reagent (Promega, Madison, WI, USA), and reading on an EnVision Multilabel
Plate Reader (Perkin Elmer, Waltham, MA, USA). Controls included infected wells with no
addition of BKI or any other inhibitor and the background was taken as wells where no C. parvum
was added. The percentage of growth versus infected cells with no inhibitor was calculated for
each concentration of BKI and the half maximal effective concentration (EC50) values were
determined by a sigmoidal dose response with variable slope using GraphPad Prism version 6.07
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 40
1
2
3
4
5
6
(GraphPad Software, La Jolla, California, USA). Assays were performed 2-3 times, and were
expressed as the mean of the values determined.
7
8
9
Efficacy in adult interferon-γ knock-out mice. IFN-γ KO mice (B6.129S7-Ifngtm1Ts/J, Jackson
Laboratories), aged 8-10 weeks, were infected by oral gavage (PO) with 1,000 to 10,000 UGA1
Nluc-expressing C. parvum oocysts in 0.1 mL DPBS as previously described.20 Beginning on day
6 post infection (p.i.), mice were dosed PO with BKI suspended in 0.2 mL oral vehicle (3%
ethanol/7% Tween 80/90% saline) or vehicle only once daily for 5 days. Mice were moved to clean
cages after each dose and fecal collection. Feces were collected daily and weighed from each group
during dosing and twice weekly out to 20 to 21 days p.i. Each fecal sample was checked for
luminescence on day of collection and RLU readings were normalized to fecal sample weights.
Controls groups were dosed with vehicle only and 60 or 100 mg/kg of BKI 1369 to establish an
appropriate dynamic range between untreated and successfully treated infections for different
experiments.14 Mice were euthanized if they showed weight loss of ≥20% or other prominent signs
of toxicity, strong discomfort, or distress according to the University of Washington’s Institutional
Animal Care and Use Committee guidelines.
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
50
51
52
53
54
55
56
57
58
59
60
Animal ethics statement. All animal experiments conducted at the University of Washington,
USA were approved by the Institutional Animal Care and Use Committee. All animals used in
these studies were handled in strict accordance with practices made to minimize suffering.
ASSOCIATED CONTENT
Supporting Information Available: This material is available free of charge via the internet at
http://pubs.acs.org.
Synthesis and characterization data for all intermediate compounds;
ACS Paragon Plus Environment

Page 27 of 40
Journal of Medicinal Chemistry
1
2
3
4
NMR spectra for final compounds;
5
6
Molecular formula strings of studied compounds including assay data.
7
8
9
Corresponding authors:
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
50
51
52
53
54
55
56
57
58
59
60
For Wesley C. Van Voorhis: E-mail: wesley@uw.edu; For Erkang Fan: E-mail: erkang@uw.edu
ACKNOWLEDGEMENTS
The authors thank Geno De Hostos and Robert Choy from PATH Drug Solutions (San Francisco,
CA, USA) for their consultation in the drug development pathway and James J. Lynch, Dale J.
Kempf, and Gail M. Freiberg (AbbVie) for establishing hERG inhibition of the BKIs. Research
studies reported in this publication were supported by National Institute of Allergy and Infectious
Diseases (NIAID) of the National Institutes of Health (NIH), USA, under award numbers
R01AI089441, R01AI111341 and R01HD080670. The work was also supported by award number
2014-06183 from the United States Department of Agriculture National Institute of Food and
Agriculture.
CONFLICT OF INTEREST
Dr. Wesley Van Voorhis is an officer and owns stock in ParaTheraTech Inc., a company that is
trying to bring BKIs to the animal health market. He helped design the experiments and edited the
paper, but did not have a role in performing or interpreting the results.
Abbreviations List: AUC, area under plasma concentration time curve; ATP, adenosine
triphosphate; CDPK1, calcium-dependent protein kinase 1; Cmax, maximum plasma concentration;
CpCDPK1, Cryptosporidium parvum CDPK1; DMF, dimethylformamide; EC50, half maximal
effective concentration; hERG, human Ether-à-go-go-Related Gene; IC50, half maximal inhibitory
concentration; PK, pharmacokinetic; Tmax, time at which maximum concentration is reached.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 40
1
2
3
4
5
REFERENCES
6
7
8
9
1. Checkley, W.; White, A. C.; Jaganath, D.; Arrowood, M. J.; Chalmers, R. M.; Chen, X.-
M.; Fayer, R.; Griffiths, J. K.; Guerrant, R. L.; Hedstrom, L.; Huston, C. D.; Kotloff, K.
L.; Kang, G.; Mead, J. R.; Miller, M.; Petri, W. A.; Priest, J. W.; Roos, D. S.; Striepen,
B.; Thompson, R. C. A.; Ward, H. D.; Van Voorhis, W. A.; Xiao, L.; Zhu, G.; Houpt, E.
R., A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for
cryptosporidium. The Lancet Infectious Diseases 2015, 15 (1), 85-94.
2. Kotloff, K. L.; Nataro, J. P.; Blackwelder, W. C.; Nasrin, D.; Farag, T. H.; Panchalingam,
S.; Wu, Y.; Sow, S. O.; Sur, D.; Breiman, R. F.; Faruque, A. S. G.; Zaidi, A. K. M.; Saha,
D.; Alonso, P. L.; Tamboura, B.; Sanogo, D.; Onwuchekwa, U.; Manna, B.;
Ramamurthy, T.; Kanungo, S.; Ochieng, J. B.; Omore, R.; Oundo, J. O.; Hossain, A.;
Das, S. K.; Ahmed, S.; Qureshi, S.; Quadri, F.; Adegbola, R. A.; Antonio, M.; Hossain,
M. J.; Akinsola, A.; Mandomando, I.; Nhampossa, T.; Acácio, S.; Biswas, K.; O'Reilly,
C. E.; Mintz, E. D.; Berkeley, L. Y.; Muhsen, K.; Sommerfelt, H.; Robins-Browne, R.
M.; Levine, M. M., Burden and aetiology of diarrhoeal disease in infants and young
children in developing countries (the Global Enteric Multicenter Study, GEMS): a
prospective, case-control study. The Lancet 2013, 382 (9888), 209-222.
3. Platts-Mills, J. A.; Babji, S.; Bodhidatta, L.; Gratz, J.; Haque, R.; Havt, A.; McCormick,
B. J. J.; McGrath, M.; Olortegui, M. P.; Samie, A.; Shakoor, S.; Mondal, D.; Lima, I. F.
N.; Hariraju, D.; Rayamajhi, B. B.; Qureshi, S.; Kabir, F.; Yori, P. P.; Mufamadi, B.;
Amour, C.; Carreon, J. D.; Richard, S. A.; Lang, D.; Bessong, P.; Mduma, E.; Ahmed, T.;
Lima, A. A. A. M.; Mason, C. J.; Zaidi, A. K. M.; Bhutta, Z. A.; Kosek, M.; Guerrant, R.
L.; Gottlieb, M.; Miller, M.; Kang, G.; Houpt, E. R., Pathogen-specific burdens of
community diarrhoea in developing countries: a multisite birth cohort study (MAL-ED).
The Lancet Global Health 2015, 3 (9), e564-e575.
4. Rossignol, J.-F. A.; Ayoub, A.; Ayers, M. S., Treatment of diarrhea caused by
Cryptosporidium parvum: A prospective randomized, double-blind, placebo-controlled
study of Nitazoxanide. The Journal of Infectious Diseases 2001, 184 (1), 103-106.
5. Amadi, B.; Mwiya, M.; Musuku, J.; Watuka, A.; Sianongo, S.; Ayoub, A.; Kelly, P.,
Effect of nitazoxanide on morbidity and mortality in Zambian children with
cryptosporidiosis: a randomised controlled trial. The Lancet 2002, 360 (9343), 1375-
1380.
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
50
51
52
53
54
55
56
57
58
59
60
6. Sparks, H.; Nair, G.; Castellanos-Gonzalez, A.; White, A. C., Treatment of
Cryptosporidium: What we know, gaps, and the way forward. Current Tropical Medicine
Reports 2015, 2 (3), 181-187.
7. Lourido, S.; Tang, K.; Sibley, L. D., Distinct signalling pathways control Toxoplasma
egress and host‐cell invasion. The EMBO Journal 2012, 31 (24), 4524-4534.
8. Ojo, K. K.; Larson, E. T.; Keyloun, K. R.; Castaneda, L. J.; DeRocher, A. E.; Inampudi,
K. K.; Kim, J. E.; Arakaki, T. L.; Murphy, R. C.; Zhang, L.; Napuli, A. J.; Maly, D. J.;
Verlinde, C. L. M. J.; Buckner, F. S.; Parsons, M.; Hol, W. G. J.; Merritt, E. A.; Van
Voorhis, W. C., Toxoplasma gondii calcium-dependent protein kinase 1 is a target for
selective kinase inhibitors. Nature Structural &Amp; Molecular Biology 2010, 17, 602.
ACS Paragon Plus Environment

Page 29 of 40
Journal of Medicinal Chemistry
1
2
3
4
5
6
9. Kieschnick, H.; Wakefield, T.; Narducci, C. A.; Beckers, C., Toxoplasma gondii
attachment to host cells Is regulated by a calmodulin-like domain protein kinase. Journal
of Biological Chemistry 2001, 276 (15), 12369-12377.
7
8
9
10. Murphy, R. C.; Ojo, K. K.; Larson, E. T.; Castellanos-Gonzalez, A.; Perera, B. G. K.;
Keyloun, K. R.; Kim, J. E.; Bhandari, J. G.; Muller, N. R.; Verlinde, C. L. M. J.; White,
A. C.; Merritt, E. A.; Van Voorhis, W. C.; Maly, D. J., Discovery of potent and selective
inhibitors of CDPK1 from C. parvum and T. gondii. ACS Medicinal Chemistry Letters
2010, 1 (7), 331-335.
11. Castellanos-Gonzalez, A.; White, J. A. C.; Ojo, K. K.; Vidadala, R. S. R.; Zhang, Z.;
Reid, M. C.; Fox, A. M. W.; Keyloun, K. R.; Rivas, K.; Irani, A.; Dann, S. M.; Fan, E.;
Maly, D. J.; Van Voorhis, W. C., A novel calcium-dependent protein kinase inhibitor as a
lead compound for treating Cryptosporidiosis. The Journal of Infectious Diseases 2013,
208 (8), 1342-1348.
12. Castellanos-Gonzalez, A.; Sparks, H.; Nava, S.; Huang, W.; Zhang, Z.; Rivas, K.;
Hulverson, M. A.; Barrett, L. K.; Ojo, K. K.; Fan, E.; Van Voorhis, W. C.; White, J. A.
C., A novel calcium-dependent kinase inhibitor, bumped kinase inhibitor 1517, cures
Cryptosporidiosis in immunosuppressed mice. The Journal of Infectious Diseases 2016,
214 (12), 1850-1855.
13. Schaefer, D. A.; Betzer, D. P.; Smith, K. D.; Millman, Z. G.; Michalski, H. C.;
Menchaca, S. E.; Zambriski, J. A.; Ojo, K. K.; Hulverson, M. A.; Arnold, S. L. M.;
Rivas, K. L.; Vidadala, R. S. R.; Huang, W.; Barrett, L. K.; Maly, D. J.; Fan, E.; Van
Voorhis, W. C.; Riggs, M. W., Novel bumped kinase inhibitors are safe and effective
therapeutics in the calf clinical model for Cryptosporidiosis. The Journal of Infectious
Diseases 2016, 214 (12), 1856-1864.
14. Hulverson, M. A.; Choi, R.; Arnold, S. L. M.; Schaefer, D. A.; Hemphill, A.; McCloskey,
M. C.; Betzer, D. P.; Müller, J.; Vidadala, R. S. R.; Whitman, G. R.; Rivas, K. L.; Barrett,
L. K.; Hackman, R. C.; Love, M. S.; McNamara, C. W.; Shaughnessy, T. K.; Kondratiuk,
A.; Kurnick, M.; Banfor, P. N.; Lynch, J. J.; Freiberg, G. M.; Kempf, D. J.; Maly, D. J.;
Riggs, M. W.; Ojo, K. K.; Van Voorhis, W. C., Advances in bumped kinase inhibitors for
human and animal therapy for cryptosporidiosis. International Journal for Parasitology
2017, 47 (12), 753-763.
15. Vidadala, R. S. R.; Golkowski, M.; Hulverson, M. A.; Choi, R.; McCloskey, M. C.;
Whitman, G. R.; Huang, W.; Arnold, S. L. M.; Barrett, L. K.; Fan, E.; Merritt, E. A.; Van
Voorhis, W. C.; Ojo, K. K.; Maly, D. J., 7H-pyrrolo[2,3-d]pyrimidin-4-amine-based
inhibitors of calcium-dependent protein kinase 1 have distinct inhibitory and oral
pharmacokinetic characteristics compared with 1H-pyrazolo[3,4-d]pyrimidin-4-amine-
based inhibitors. ACS Infectious Diseases 2018, 4 (4), 516-522.
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
50
51
52
53
54
55
56
57
58
59
60
16. Huang, W.; Choi, R.; Hulverson, M. A.; Zhang, Z.; McCloskey, M. C.; Schaefer, D. A.;
Whitman, G. R.; Barrett, L. K.; Vidadala, R. S. R.; Riggs, M. W.; Maly, D. J.; Van
Voorhis, W. C.; Ojo, K. K.; Fan, E., 5-Aminopyrazole-4-carboxamide-based compounds
prevent the growth of Cryptosporidium parvum. Antimicrobial Agents and Chemotherapy
2017, 61 (8), e00020-17.
ACS Paragon Plus Environment