Discovery and structure activity relationship of the first potent cryptosporidium FIKK kinase inhibitor

Article abstract of DOI:10.1016/j.bmc.2017.01.036

FIKKs are parasite-specific protein kinases with distinctive sequence motifs and their biological roles have not been completely elucidated. Here, we report the first potent Cryptosporidium FIKK (CpFIKK) inhibitor. We identified 4b as a potent (IC50 = 0.2

Full text of DOI:10.1016/j.bmc.2017.01.036

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery and structure activity relationship of the first potent
cryptosporidium FIKK kinase inhibitor
Khan T. Osman a,b, Juntao Ye c, Zhihao Shi c,d, Christina Toker c, Diogo Lovato a, Rajiv S. Jumani e,
William Zuercher f, Christopher D. Huston e, Aled M. Edwards a, Mark Lautens c,
,
⇑
a,
Vijayaratnam Santhakumar a, , Raymond Hui
⇑
⇑
a Structural Genomics Consortium, Toronto M5G1L7, Canada
b Molecular Genetics, University of Toronto, Toronto M5S 1A8, Canada
c Department of Chemistry, University of Toronto, Toronto M5S 3H6, Canada
d China Pharmaceutical University, Jiangsu Sheng 210009, China
e Department of Medicine, Cellular, Molecular, and Biomedical Sciences Program, University of Vermont, Burlington 05405, USA
f UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 2759, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
FIKKs are parasite-specific protein kinases with distinctive sequence motifs and their biological roles
have not been completely elucidated. Here, we report the first potent Cryptosporidium FIKK (CpFIKK)
inhibitor. We identified 4b as a potent (IC50 = 0.2 nM) inhibitor of CpFIKK catalytic activity. In addition,
we identified both CpCDPK1 selective as well as dually acting CpFIKK-CDPK1 inhibitors from the same
structural class of compounds. We evaluated these CpFIKK inhibitors for inhibition of parasite growth
in vitro. The observed effects on parasite growth did not correlate with CpFIKK inhibition, suggesting that
CpFIKK may not be involved in parasite growth.
Received 16 December 2016
Revised 13 January 2017
Accepted 19 January 2017
Available online xxxx
Keywords:
Cryptosporidium
Kinase inhibitor
FIKK Kinase inhibitor
Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction
genetic manipulation and continuous culturing, are only now
becoming available for Cryptosporidium parasites.
Some of the most widespread infectious diseases are caused by
the protists in the phylum Apicomplexa - Plasmodium, Cryp-
tosporidium and Toxoplasma. In particular, Cryptosporidium para-
sites are responsible for cryptosporidiosis, an enteric disease
affecting millions of people worldwide but receiving much less
attention than malaria.1 Children under the age of five in under-
developed regions of the world are the most vulnerable population,
with hundreds of thousands dying every year and millions infected
and often re-infected.2–4 Developmental problems among this age
group of children are also associated with Cryptosporidium infec-
tion.5 There are also periodic outbreaks of cryptosporidiosis in
North America, Europe and across the world and it is a significant
veterinarian concern affecting ruminant and poultry operations.
Nitazoxanide is the only drug approved by the FDA to treat
cryptosporidiosis.3,5,6 Unfortunately, it has limited efficacy among
malnourished and immunocompromised children.4 Finding new
cryptosporidiosis drugs is challenging because existing animal
models are considered inadequate4, and techniques for identifying,
characterizing, testing and validating new drug targets, such as
We elected to explore parasite-specific protein kinases as
potential drug targets, because we could leverage the knowledge
and tools already available for the human kinome. Candidate par-
asite-specific kinases include calcium-dependent protein kinases
(CDPKs) – found only in specific protists and plants; FIKK kinases
(named FIKK after a conserved Phe-Ile-Lys-Lys motif in their amino
acid sequence), which are exclusively found in the Apicomplexa
phylum; and MAP kinases, three of which are present in
Cryptosporidium genomes despite the absence of a proper MAP
kinase signalling cascade.7–9 CpCDPK1 is the subject of an ongoing
drug discovery program, but chemical inhibition of CpCDPK1 does
not appear to correlate with parasite inhibition in vitro.10,11
To begin to explore the potential of FIKKs as drug discovery
targets, we recently characterized the enzymatic activity of
Plasmodium falciparum FIKK8 (PfFIKK8) and its C. parvum ortho-
logue (CpFIKK).9 Recombinant CpFIKK is a catalytically active
kinase and prefers serine in the substrate as phospho-acceptor
residue flanked by two arginines in the À3 and +3 positions. FIKKs
display quite divergent sequences from canonical kinases.12,13
Most CpFIKK and orthologues contain a small residue at the
gatekeeper position, which is a selectivity determinant of the inhi-
bitors in some protein kinases, including CpCDPK1.10,14,15
⇑
Corresponding authors.
E-mail address: santha.santhakumar@utoronto.ca (V. Santhakumar).
http://dx.doi.org/10.1016/j.bmc.2017.01.036
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Osman K.T., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.036

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K.T. Osman et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Most parasites, including Cryptosporidium parvum and Toxo-
against 300 human kinases tested, inhibiting only one kinase with
>25% inhibition at 100 nM (KDR, 33%) and two others with >50%
plasma gondii, have a single representative FIKK (CpFIKK and
TgFIKK, respectively) in their genomes.12,13,16 Most of the Plasmod-
ium genomes contain a single FIKK member as well, but the family
expanded to >20 members in three highly related species of Plas-
modium, Plasmodium falciparum, Plasmodium reichenowi and Plas-
modium gaboni . The reason for this expansion is not clear, but
multiple members from the Plasmodium FIKK family play roles in
erythrocyte infection by modifying erythrocyte membranes.17–19
Some of the non-orthologous FIKK members in Plasmodium falci-
parum are non-essential; a knock out attempt of the single FIKK
in Plasmodium bergheii (PbFIKK) was unsuccessful, suggesting that
it might be essential.20 Knock-out of TgFIKK in Toxoplasma gondii
did not affect the growth, but demonstrated almost complete elim-
ination of tissue-cyst formation,21 a process that occurs during
acute infection and is difficult to treat. Recombinant PfFIKK8 and
CpFIKK are catalytically active kinases and prefer similar substrate
sequences, but their biological roles remain to be determined.9
Potent, selective and cell permeable chemical probes would be
useful to understand the biological roles of FIKK kinases.
inhibition at 1 l
M (KDR, 67%; MAP4K4, 60%; P38A, 50%).23 Another
study reported that compound 4a inhibits of HsALK5 at 10 nM.24 In
order to obtain more potent and selective inhibitors, we systemat-
ically explored both the structure-activity-relationship (SAR) for
CpFIKK inhibition and the selectivity against CpCDPK1.
3. Chemistry
1,5-Naphthyridine aminothiazoles 4a–r were readily prepared
following a known procedure used for the preparation of com-
pound 4a as shown in Scheme 1.23 2-Methyl[1,5]naphthyridine
1a was reacted with the corresponding ester 2a or 2b in the pres-
ence of potassium bis(trimethylsilyl)amide to give the ketones 3a–
q, which were used in the subsequent step without purification.
Bromoketone intermediates, obtained by treating the ketones
3a–q with bromine, were immediately reacted with thiourea to
provide the aminothiazoles 4a–q. Following the similar procedure,
compound 4r was prepared starting from 1b (Scheme 1).
N-Acyl derivative 5a was prepared by treating 4a with acetyl
chloride in the presence of a base. 4a was treated with di-tertiary
butyl dicarbonate in the presence of a base and a catalytic amount
of DMAP to give the Boc intermediate 5b. Deprotonation of 5b fol-
lowed by exposure to methyl iodide and subsequent treatment of
the methylated intermediate with trifluoroacetic acid to give N-
methylderivative 6 (Scheme 2).
Here, we report a new series of small molecule, cell permeable
inhibitors targeting CpFIKK and describe their selectivity profiles.
This report is the first of any inhibitor of a FIKK kinase. Inhibition
of FIKK did not affect parasite growth, casting doubt on the suit-
ability of the FIKK kinases as anti-parasitic drug targets.
2. Hit identification
In order to identify inhibitors of CpFIKK, we screened a focused
library of ꢀ2500 known kinases inhibitors including PKIS from
4. Results and Discussion
GSK22 at 100
lM
concentration, using recombinant, purified
In order to understand the binding mode of 4a with CpFIKK and
improve CpFIKK activity of 4a, we tried to crystalize 4a with
CpFIKK but failed to obtain a co-crystal structure with good resolu-
tion. However, 4a is a known inhibitor of human HsALK5 kinase
(IC50 = 10 nM) and the crystal structure of a close analogue of 4a,
diarylpyrazole (compound 19 in the publication, Fig. 1), with the
HsALK5 (PDB ID 1VJY, Fig. 1) has been reported.24 4a and
diarylpyrazole share some common structural features; 4a and
diarylpyrazole has the same naphthyridine ring and 4a has
aminothiazole and phenyl ring in the place of pyrazole ring and
pyridyl rings respectively. In addition, similar compounds, the
CpFIKK kinase, in two orthogonal assays, differential static light
scattering (DSLS) and an enzyme assay. We identified compound
4a and 4j as potent inhibitors of CpFIKK kinase, with IC50 = 3 nM
and 220 nM respectively. Compound 4a was re-synthesized to con-
firm identity and tested for selectivity against 14 representative
members of parasite specific Cryptosporidium kinases (Supplemen-
tary Table 1) by DSLS. Compound 4a stabilized and inhibited only
CpCDPK1 significantly (IC50 = 84 nM) and all the other kinases
showed <2 °C shift and >10
lM IC50 in the biochemical assays.
Interestingly, compound 4a already showed good selectivity
R2
NH2
R3
R1
N
R1
CO2R5
R1
N
S
N
N
N
N
a
b, c
R4
+
O
N
R4
R2
R3
R2
3a-r
1a
R4
(R1=H)
1b (R1=Me)
2a
(R5=Me)
4a-r
R3
2b (R5=Et)
Scheme 1. Synthesis of 1,5-naphthyridine aminothiazoles. (a) KHMDS, THF, À78 °C to rt; (b) Br2, Dioxane, room temperature; (c) Thiourea, EtOH, reflux.
O
R6
NH
N
NH
N
S
NH2
N
S
N
S
a or b
c, d
N
N
N
N
Cl
N
Cl
Cl
5a
5b
(R6=Me)
(R6=O Bu)
6
4a
t
Scheme 2. Modifications on the primary amine functionality of 4a. (a) Boc2O, Et3N, DMAP (10 mol%), THF, rt; (b) Ac2O, Et3N, THF, 60 °C; (c) NaH, MeI, DMF, rt to 60 °C; (d)
TFA, dichloromethane, rt.
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K.T. Osman et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
Fig. 1. Binding mode of a close analogue of 4a. Diarylpyrazole (compound 19 in the publication) structure in HsALK5 (left). Selectivity pocket (right) in HsALK5 (PDB ID 1VJY)
diaryl substituted imidazoles, show similar binding modes with
MAP kinases MAPK1 and MAPK14 (P38a) (PDB ID 1bl7, 3erk,
1bmk, 1bl6). Therefore, we hypothesized the general binding mode
of 4a to CpFIKK would be similar to the binding mode of other imi-
dazole based kinase inhibitors and used the structure of HsALK5 to
design compounds to explore SAR. As the pairwise sequence iden-
tity between HsALK5 and CpFIKK is low (23%), we did not attempt
to build homology model. We used the overlay of the amino acid
sequence of CpFIKK, CpCDPK1 and HsALK5 to predict (Cluster
Omega for amino acid sequence alignment: http://www.ebi.ac.
uk/Tools/msa/clustalo and Prosite for kinase domain prediction:
http://prosite.expasy.org/) the corresponding amino acid residues
in CpFIKK and CpCDPK1 binding pockets (Fig. 2).
In this crystal structure of the corresponding diarylpyrazoles
with HsALK5 (Fig. 1), the N1 of the naphthyridine ring and the
backbone NH of His-283 makes a hydrogen bond in the hinge
region; this bond is critical for the activity of the majority of known
kinase inhibitors as well as for ATP binding. Accordingly, we did
not vary the naphthyridine ring extensively. In HsALK5 structure,
the pyridyl group of diarylpyrazole extends towards the selectivity
pocket and it is in a close vicinity of the so called ‘gatekeeper’ resi-
due S280. The residues predicted to be in the CpFIKK selectivity
pocket are significantly different compared to the ones in the
HsALK5 structure and the ones predicted to be in CpCDPK1. CpFIKK
predicted to have asparagine, alanine and arginine residues and
CpCDPK predicted to have leucine, methionine and leucine in place
of Y249, L260 and F262 in the HsALK5 structure respectively. In
addition, CpCDPK1 has smaller glycine as gate keeper compared
with serine in CpFIKK. Therefore, in order to identify a potent
and CpFIKK selective inhibitor, we focused our efforts on variations
of meta- and para- substitutions at the phenyl group, which can
extend towards these residues selectivity pocket as in the HsALK5
structure. All the compounds synthesized were tested for activity
against the CpFIKK kinase and for selectivity against CpCDPK1.
The results are summarized in Table 1.
At the meta position, only small hydrophobic groups are toler-
ated for CpFIKK activity indicating there is only a small hydropho-
bic pocket in CpFIKK occupied by the aryl group as observed in the
HsALK5 structure. Compared with the unsubstituted compound
(4c), the chloro group (4a) improved CpFIKK activity by about 15
fold and the methyl group (4c) significantly improved both FIKK
activity (IC50 = 0.2 nM) and selectivity (940 fold) against CpCDPK1
(Fig. 3). Larger alkyl groups, ethyl (4h) and isopropyl groups (4i)
resulted in complete loss of CpFIKK activity and interestingly, the
ethyl group substituent (4h) maintained CpCDPK1 activity indicat-
ing that the corresponding pocket in CpCDPK1 is slightly larger
than that of CpFIKK. Addition of fluoro (4d) and methoxy (4e)
groups decreased CpFIKK activity by 4–5 fold and cyano (4f) and
Fig. 2. Sequence alignment of HsALK5, CpCDPK1 and CpFIKK.
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K.T. Osman et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 1
Table 2
CpFIKK and CpCDPK1 activities of 4a–l.
CpFIKK and CpCDPK1 kinase activities of 4m–q.
NH2
N
NH2
N
S
S
N
N
Ar
Ar
N
N
Cpds
Ar
CpFIKK
CpCDPK1
Cpds
Ar
CpFIKK
CpCDPK1
IC50 (nM)a
IC50 (nM)a
IC50 (nM)a
IC50 (nM)a
4a
4b
4c
4d
4e
4f
4g
4h
4i
m-ClPh
m-MePh
ph
3
0.3
84
190
7
9
4m
25
6
450 74
0.2 0.1
42 12
190 20
210 15
260 20
1500 230
350 70
74 15
Cl
m-FPh
220 40
180 20
4100 750
1200 620
>10,000
F
m-OMePh
m-CNPh
m-CF3Ph
m-EtPh
m-iPrPh
p-FPh
4n
140 50
73
19
9
2
>10,000
>10,000
4j
4k
4l
220 40
3260 820
>10,000
220 30
375 25
2600 370
F
p-ClPh
p-OMePh
4o
4p
2900 850
750 60
a
IC50 values were average of triplicate experiments.
>10,000
Cl
Cl
4q
>10,000
3500 340
Cl
Cl
a
IC50 values were average of triplicate experiments.
be at the open side of the pocket, was explored initially. However,
(4r) did not show any significant CpFIKK activity. Around the
aminothiazole region, pyrazole ring of the diarylpyrazole makes
hydrogen bond with D351 in the HsALK5 structure, and both
CpFIKK and CpCDPK1 predicted to have the same aspartic acid at
the corresponding position therefore; simple acetyl (5a) and
methyl (6) substitutions which can maintain hydrogen bonding
ability of the amino group were explored initially. Both modifica-
tions (5a and methyl 6) led to almost complete drop in CpFIKK
activity (Table 3). Potency drop of 4r, 5a and 6 may be due to
the steric effects of the substitutions. We did not explore further
SAR around the naphthyridine ring and around aminothiazole ring,
as we identified a potent and selective compound (4b) during our
initial SAR exploration.
Fig. 3. The enzymatic inhibition of CpFIKK and CpCDPK1 by 4b.
trifluoromethyl groups (4g) decreased CpFIKK activity
significantly.
At the para position, only the fluoro group was tolerated with a
moderate drop in CpFIKK activity (compare 4c and 4j) but this did
not improve selectivity against CpCDPK1. Addition of chloro (4k) or
methoxy (4l) groups led to > 100 fold drop (compare 4c and 4k, 4l)
in CpFIKK activity. The effect of di-substitution at the phenyl ring
was also explored (Table 2). As observed with 4j, introduction of
the para fluoro group (4m and 4n) to the most potent CpFIKK com-
pounds, 4a and 4b, lead to a moderate drop in CpFIKK activity and
no improvement in selectivity against CpCDPK1. Both dimethyl
(4o) and dichloro (4p and 4q) groups lead to significant drop in
FIKK activity, but dimethyl (4o) groups maintains potent CpCDPK1
activity. Observed SAR at the para position further supports the
hypothesis that there is only a small hydrophobic pocket occupied
by the aryl group in CpFIKK and the corresponding pocket CpCDPK1
is slightly larger than that of CpFIKK.
Table 3
CpFIKK and CpCDPK1 kinase activities of compounds 4r, 5a, and 6.
R
NH2
NH
S
S
N
N
N
N
N
N
Cl
Cl
4r
5a (R=COCH3)
6 (R=CH3)
F
As the two key residues around the hinge region, I211 and H283
are predicted to be identical in HsALK5 and CpFIKK and D281 of the
HsALK5 is replaced with a close analogue glutamic acid (Figs. 1 and
2) in CpFIKK, the binding pocket in this region are expected to be
similar. In the HsALK5 structure, the naphthyridine ring fits tightly
in the pocket in the hinge region and therefore only a small methyl
group at 8-position at the naphthiridine ring (4r), which is likely to
Compound
CpFIKK
CpCDPK1
IC50 (nM)a
IC50 (nM)a
4r
5a
6
>10,000
>10,000
3600 287
400 180
330 60
1510 195
a
IC50 values were average of triplicate experiments.
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K.T. Osman et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
4.1. Orthogonal confirmation of CpFIKK inhibitors
able.24 Therefore, the observed parasite growth inhibition neither
correlates with CpFIKK nor with CpCDPK1 inhibition in vitro.
CpFIKK selective inhibitors without significant CpCDPK1 activity
and selective inhibitors from different structural classes will be
needed to establish the involvement of CpFIKK in parasite growth.
In conclusion, our potent and CpFIKK inhibitor 4b, used at
appropriate concentrations, could serve as a useful in vitro tool
for understanding the biological role of FIKK in C. parvum. Even
though the CpFIKK inhibitors showed weak parasite growth inhibi-
tion, it is not likely that the inhibition is mediated by CpFIKK, as
parasite growth was unaffected by CpFIKK inhibitors at concentra-
tion predicted to inhibit activity significantly. As observed with the
TgFIKK knockout,21 the function of CpFIKK may not be growth, and
4b can be used as an in vitro tool to help determining the function
(s) of CpFIKK as assays that probe different aspects of the Cryp-
tosporidium life cycle become available. Selective inhibitors from
different structural classes would be needed to understand further
the involvement of CpFIKK inhibition in parasite growth.
DSLS was used to confirm the direct binding of representative
CpFIKK inhibitors identified by biochemical assay.26,27 All CpFIKK
inhibitors tested, including the most potent inhibitor 4b (Supple-
mentary Fig. 1), showed significant increase in thermal stabiliza-
tion and the inactive or weakly active compounds 4i, 4l and 4o
showed no significant thermal stabilization of the CpFIKK upon
binding (Supplementary Table 2).
4.2. Selectivity profiles of CpFIKK chemical probe (4b) against human
kinases
To understand the selectivity of the newly synthesized inhibi-
tors against the human kinome, we screened 4b against a panel
of 140 human kinases (ꢀ25% of the human kinome) at the Interna-
tional Centre for Kinase Profiling, MRC, UK.28 Only 6 protein
kinases showed >50% inhibition of activity when screened with
compounds at 500 nM (Supplementary Fig. 2). The complete list
of kinases and the percentage of inhibition are provided in the Sup-
plementary Table 3. Not surprisingly, since the parent compound
derived from a human kinome drug discovery program, ALK5
(TGFBR1), KDR (VEG-FR2) and P38A showed 86%, 59% and 91%
inhibition of activity, respectively, by 4b. These three kinases were
also inhibited by 4a in a previous study.23 The addition of 4b also
inhibited four other kinases, TAK1, RIPK2, PKD1 and Yes1, signifi-
cantly (60%, 70% and 91% and 47%, respectively at 500 nM).
5. Experimental section
5.1. Enzymatic inhibition assay
An NADH coupled LDH-PK ATPase assay was used to measure
the kinase activity according to previously described proce-
dures.9,25 The concentrations of proteins in the reaction were
1.2 nM (CpCDPK1) and 10 nM (CpFIKK). Inhibitor compounds were
solubilized at the stock concentration of 10 mM in 100% DMSO and
4.3. Evaluation of CpFIKK inhibitors in C. parvum growth inhibition
were applied in the reaction from 25 lM downward with 2-fold
dilutions. Syntide 2.0 (PLARTLSVAGLPGKK) for CpCDPK1 and P5
(RRRAPSFYRK) for CpFIKK were the substrates used as the phos-
phoacceptor peptides. The reaction solution contained 0.25 mM
of ATP and 0.1 mM of peptide substrates. The CpCDPK1 reaction
contained 2 mM CaCl2. Inhibition parameters were plotted and
calculated by using Sigmaplot.
In order to understand the role of FIKK in C. parvum growth, we
evaluated the in vitro parasitic activity of 4 compounds (4a, 4b, 4d,
4o) having varying CpFIKK activity and selectivity against
CpCDPK1. 1-NA-PP1 which was reported to show parasite inhibi-
tion with EC50 = 2.56uM was used as a positive control and 4q
was used as a negative control.29 The compounds were added to
the host cell (HCT) prior to infection. All the CpFIKK and CpCDPK1
dual active compounds showed inhibition of parasite growth with
5.2. Cryptosporidium parvum growth assay
EC50 6 5.5 lM and the CpFIKK inactive compound 4q showed no
Compound inhibition of Cryptosporidium parvum growth was
tested using a previously developed microscopy-based assay.30
Human ileocecal adenocarcinoma (HCT-8) cells (ATCC) were cul-
tured in RPMI-1640 medium (Invitrogen) supplemented with
10% heat inactivated FBS (Sigma), 120 U/mL penicillin and
inhibition. If the mechanism of cell killing was mediated by CpFIKK,
we would expect the EC50 to correlate with the biochemical CpFIKK
IC50. However, we did not find this. For significant growth inhibi-
tion, the most CpFIKK active compounds 4a and 4b required con-
centrations that were 1840 and 12,800 fold higher than the
CpFIKK enzymatic inhibition IC50 respectively. Even though 4d
and 4o showed significant growth inhibition at concentrations that
are 6 and 1.3-fold higher than CpFIKK IC50 values, respectively, the
EC50 values of all the compounds tested were 6–204 fold of
CpCDPK1 inhibition IC50 (Table 4). Large drop off of the cell activity
is not likely due to the poor permeability or efflux. 4b and 4d
showed good permeability (Caco-2 permeability for 4b and 4d
are 11 and 16 10-6 cm/s respectively) and did not show efflux
(efflux ratio < 1 for both compounds). 4a is a known ALK5 inhibitor
and reported to show low nM activity in the ALK5 mediated effect
in the cellular assay and therefore 4a is also expected to perme-
120
l
g/mL streptomycin (ATCC). Three-fold increasing concentra-
M diluted in culture media
tions of compounds from 0.004 to 25
l
were added to 100% confluent HCT-8 cells in clear well, back-
walled 384-well plates (BD Falcon), followed by addition of
approximately 5500 excysted Cryptosporidium parvum Iowa strain
oocysts (Bunchgrass Farms, USA) per well. Oocysts were excysted
with 10 mM HCl for 10 min at 37° C, followed by 2 mM solution
of sodium taurocholate (Sigma) for 10 min at 16 °C and then
diluted in culture media and added to each well. After 48 h of
infection, the wells were washed three times with 111 mM
D-
galactose, fixed with 4% paraformaldehyde for 15 min at room
temperature, permeabilized with 0.25% Triton X100 in PBS for
10 min at 37 °C, washed three times with PBS containing 0.1%
Tween 20 and then blocked with 4% bovine serum albumin (BSA)
(Sigma) in PBS for 2 h at 37 °C. Parasitophorous vacuoles of Cryp-
Table 4
Parasite invasion inhibition profiles of CpFIKK inhibitors.
tosporidium parvum were stained with 1.33 lg/mL fluorescein
Cpds
EC50
(l
M)
Fold CpFIKK IC50
Fold CpCDPK1 IC50
labeled Vicia villosa lectin (Vector Laboratories) diluted in 1% BSA
in PBS with 0.1% Tween 20. After 1 h incubation at 37 °C, the
nuclear stain Hoechst 33258 (Anaspec) was added at 0.09 mM
diluted in water for another 15 min, followed by five washes with
PBS containing 0.1% Tween 20 before imaging. A Nikon Eclipse
Ti2000 epifluorescence microscope with an EXi blue fluorescent
4a
4b
4d
4o
4q
1-NA-PP1
5.5
2.6
1.3
3.9
1840
12,800
6
1.3
Not applicable
Not determined
66
14
6
204
Not applicable
1
No inhibition
4.1
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6
K.T. Osman et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
microscopy camera (QImaging, Canada) and NIS-Elements
Advanced Research software (Nikon, USA) was programmed to
focus on the center of each well and acquire a three-by-three com-
posite of 20X field images. Nuclei and parasite numbers were
quantified on exported .tif files using previously developed macros
and the batch function in ImageJ (National Institutes of Health).
dried over sodium sulfate, and evaporated. The crude product
was passed through a short silica gel column (eluent: hexane/ethyl
acetate = 2/1) to give a mixture of ketone and enol form of product
(0.3397 g), which was used in the next step directly.
To the flask containing the crude product were added anhy-
drous dioxane (12 mL) and bromine (90 lL, 1.64 mmol). The
resulting brown suspension was stirred at room temperature for
1 h and then evaporated to dryness. Thiourea (0.1174 g,
1.50 mmol) and ethanol (20 mL) were added, and the mixture
was heated at reflux overnight. The reaction mixture was allowed
to cool at room temperature and treated with aqueous ammonia
(0.88 M, 10 mL) with stirring. The mixture was evaporated, and
the residue was dissolved in CH2Cl2 (100 mL) and washed with
water (100 mL). The organic phase was extracted with 1 N
hydrochloric acid (2 Â 50 mL), the aqueous layers were combined,
basified with aqueous sodium hydroxide 35% (ca. 30 mL) until
pH > 10, and extracted with CH2Cl2. The organic phase was dried
over sodium sulfate and evaporated to give the crude product as
a brown powder. This powder was crystallized from CH2Cl2/ethyl
acetate to afford 4c (0.1856 g, 30% yield over 3 steps) as yellow
solid: mp 221–222 °C; 1H NMR (400 MHz, CDCl3) d = 8.85 (dd,
J1 = 4.2, J2 = 1.7 Hz, 1H, Ar-H), 8.30–8.27 (m, 1 H, Ar-H), 8.01 (dd,
J1 = 9.0, J2 = 0.9 Hz, 1 H, Ar-H), 7.60–7.56 (m, 3 H, Ar-H), 7.43–
7.39 (m, 4 H, Ar-H), 5.19 (s, 2 H, NH2); 13C NMR (100 MHz,
DMSO-d6) d = 168.9, 152.9, 151.3, 150.0, 143.1, 141.8, 136.1,
135.8, 135.6, 129.0, 128.7, 128.6, 125.0, 122.4, 120.9, 99.5; HRMS
5.3. General methods for chemical synthesis
Unless otherwise stated all reactions were set up under inert
atmosphere (argon) utilizing glassware that was flame-dried and
cooled under vacuum. Reactions were monitored using thin-layer
chromatography (TLC) on EMD Silica Gel 60 F254 plates. Visualiza-
tion of the developed plates was performed under UV light
(254 nm) or KMnO4 stain. Silica Gel Flash Column Chromatography
was performed on SILICYCLEÒ SILIAFLASHÒ P60, 40–63
lm silica
gel. Unless otherwise indicated, starting materials were obtained
from Sigma Aldrich, Strem, Alfa Aesar, or Combi-Blocks and used
without further purification. THF was freshly distilled over Na
under N2 prior to use. NMR characterization data were collected
at 300 K on a Bruker Avance III 400 or Varian Mercury 600 MHz
spectrometer (Funded by the Canadian Foundation for Innovation,
project number 19119, and the Ontario MRI). 1H NMR spectra were
internally referenced to TMS. 13C NMR spectra were internally ref-
erenced to the residual solvent signal. Data for 1H NMR are
reported as follows: chemical shift (d ppm), multiplicity (s = sin-
glet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad),
coupling constant (Hz), integration. Melting Point ranges were
determined on a FISHER-JOHNSÒ Melting Point Apparatus and
are reported uncorrected. High Resolution Mass Spectra (HRMS)
were obtained on a micromass 70S-250 spectrometer (EI) or an
AB SCIEX QSTARÒ Mass Spectrometer (ESI) or a JEOLÒ AccuTOF
medel JMS-T1000LC mass spectrometer equipped with an IONICSÒ
Direct Analysis in real Time (DART) ion source at ADVANCED
INSTRUMENTATION FOR MOLECULAR STRUCTURE (AIMS) in the
Department of Chemistry at the University of Toronto. All com-
pounds have a purity of greater than 95% by UV absorbance at
254 nm as determined by HPLC. Purity determination was con-
ducted by UV absorbance at 254 nm during tandem liquid chro-
matography/mass spectrometry (LCMS) using a Waters Acquity
separations module. Identity was determined via low resolution
mass spectrometry (LRMS) conducted in positive ion mode using
a Waters Acquity SQD mass spectrometer (electrospray ionization
source) fitted with a PDA detector. Mobile phase A consisted of
0.1% formic acid in water, while mobile phase B consisted of 0.1%
formic acid in acetonitrile. The gradient ran from 5% to 95% mobile
(DART) calculated for
305.08659.
The following compounds 4b–4r were prepared according to
the above General Procedure.
C
17H12N4S [M+H+]: 305.08609, found:
5.4.2. 4-(3-Methylphenyl)-5-(1,5-naphthyridin-2-yl)-1,3-thiazol-2-
amine (4b)
2 mmol scale, 57% yield (3 steps), mp 205–207 °C. 1H NMR
(600 MHz, CDCl3) d = 8.85 (dd, J1 = 4.2, J2 = 1.6 Hz, 1 H, Ar-H),
8.29–8.27 (m, 1 H, Ar-H), 8.01 (dd, J1 = 9.0, J2 = 0.8 Hz, 1 H, Ar-H),
7.58 (dd, J1 = 8.5, J2 = 4.2 Hz, 1 H, Ar-H), 7.43–7.41 (m, 2 H, Ar-H),
7.34–7.33 (m, 1 H, Ar-H), 7.31–7.29 (m, 1 H, Ar-H), 7.26–7.23 (m,
1 H, Ar-H), 5.27 (s, 2 H, NH2), 2.38 (s, 3 H, CH3); 13C NMR
(100 MHz, DMSO-d6) d = 168.9, 153.0, 151.5, 150.0, 143.1, 141.8,
137.8, 136.0, 135.7, 135.6, 129.6, 129.4, 128.5, 126.1, 125.0,
122.4, 120.9, 21.0; HRMS (DART) calculated for C18H14N4S [M
+H+]: 319.10174, found: 319.10222.
5.4.3. 4-(3-Fluorophenyl)-5-(1,5-naphthyridin-2-yl)-1,3-thiazol-2-
amine (4d)
phase
(2.1 mm  50 mm, 130 Å, 1.7
was used with column temperature maintained at 25 °C. The sam-
ple solution injection volume was 2 L.
B
over 3 min at 0.5 mL/min. An Acquity CSH C18
2 mmol scale, 48% yield (3 steps), mp 205–206 °C; 1H NMR
(400 MHz, CDCl3) d = 8.87 (dd, J1 = 4.2, J2 = 1.7 Hz, 1H, Ar-H),
8.30–8.27 (m, 1H, Ar-H), 8.07 (dd, J1 = 8.9, J2 = 0.9 Hz, 1H, Ar-H),
7.59 (dd, J1 = 8.5, J2 = 4.2 Hz, 1H, Ar-H), 7.41 (d, J = 9.0, 1H, Ar-H),
7.38–7.31 (m, 3H, Ar-H), 7.15–7.10 (m, 1H, Ar-H), 5.25 (s, 2H,
lm) column (part no. 186005296)
l
Compound 1a, 4a, and 4j were prepared according to literature
procedures.1
NH2); 13C NMR (100 MHz, DMSO-d6) d = 169.0, 162.1 (d, JC-F
=
242.6 Hz), 152.6, 150.2, 149.4 (d,
JC-F = 2.3 Hz), 143.1, 141.8,
5.4. Experimental procedures
138.1 (d, JC-F = 8.0 Hz), 136.3, 135.7, 130.6 (d, JC-F = 8.3 Hz), 125.2
(d, JC-F = 2.7 Hz), 125.1, 122.5, 121.6, 115.7 (d, JC-F = 21.8 Hz),
5.4.1. General procedure: preparation of 4-phenyl-5-(1,5-
naphthyridin-2-yl)-1,3-thiazol-2-amine (4c)
115.5 (d,
J
C-F = 20.9 Hz); 19F NMR (377 MHz, DMSO-d6)
d = À112.2; HRMS (DART) calculated for
C
17H11FN4S [M+H+]:
To a flame-dried flask were added 1a (0.2893 g, 2 mmol), 2a
(0.25 mL, 2 mmol), and anhydrous THF (8 mL). Potassium bis
(trimethylsilyl)amide (1.0 M solution in THF, 4 mL, 4 mmol) was
added dropwise over 10 min under Ar atmosphere at À78 °C. This
mixture was stirred at À78 °C for 1 h and then at room tempera-
ture for 18 h. The reaction mixture was quenched by adding satu-
rated aqueous ammonium chloride and partitioned between ethyl
acetate and water. The aqueous phase was extracted with ethyl
acetate. The organic layers were combined, washed with water,
323.07667, found: 323.07620.
5.4.4. 4-(3-Methoxyphenyl)-5-(1,5-naphthyridin-2-yl)-1,3-thiazol-2-
amine (4e)
2 mmol scale, 16% yield (3 steps), mp 248–250 °C; 1H NMR
(400 MHz, CDCl3) d = 8.85 (dd, J1 = 4.2, J2 = 1.6 Hz, 1H, Ar-H),
8.29–8.27 (m, 1H, Ar-H), 8.03 (d, J = 9.0 Hz, 1H, Ar-H), 7.58 (dd,
J1 = 8.5, J2 = 4.2 Hz, 1H, Ar-H), 7.43 (d, J = 9.0 Hz, 1H, Ar-H), 7.32
(t, J = 7.8 Hz, 1H, Ar-H), 7.15–7.12 (m, 2H, Ar-H), 6.99–6.97 (m,
Please cite this article in press as: Osman K.T., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.036

K.T. Osman et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
1H, Ar-H), 5.22 (s, 2H, NH2); 3.79 (s, 3H, OCH3); 13C NMR (100 MHz,
DMSO-d6) d = 168.9, 159.3, 152.9, 151.0, 150.0, 143.0, 141.2, 137.1,
136.0, 135.6, 129.7, 125.0, 122.5, 121.3, 121.1, 114.7, 114.0, 55.1;
135.7, 134.6, 133.3, 130.9, 128.7, 125.1, 122.6, 121.3; HRMS (DART)
calculated for C17H11ClN4S [M+H+]: 339.04712, found: 339.04681.
HRMS (DART) calculated for
found: 335.09619.
C
18H14N4OS [M+H+]:335.09666,
5.4.10. 4-(4-Methoxyphenyl)-5-(1,5-naphthyridin-2-yl)-1,3-thiazol-
2-amine (4l)
2 mmol scale, 22% yield (3 steps), mp 220–222 °C; 1H NMR
(400 MHz, DMSO-d6) d = 8.83 (dd, J1 = 4.2, J2 = 1.6 Hz, 1H, Ar-H),
8.24–8.22 (m, 1H, Ar-H), 8.05 (dd, J1 = 9.0, J2 = 0.8 Hz, 1H, Ar-H),
7.69 (dd, J1 = 8.5, J2 = 4.2 Hz, 1H, Ar-H), 7.51 (s, 2H, NH2), 7.48–
7.44 (m, 2H, Ar-H), 7.35 (d, J = 9.0 Hz, 1H, Ar-H), 7.01–6.97 (m,
2H, Ar-H), 3.81 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6)
d = 168.8, 159.5, 153.2, 151.2, 149.9, 143.1, 141.8, 136.0, 135.5,
130.4, 128.0, 125.0, 122.4, 120.1, 114.0, 55.2; HRMS (DART) calcu-
lated for C18H14N4OS [M+H+]: 335.09666, found: 335.09727.
5.4.5. 4-(3-Cyanophenyl)-5-(1,5-naphthyridin-2-yl)-1,3-thiazol-2-
amine (4f)
2 mmol scale, 21% yield (3 steps), mp 237–239 °C; 1H NMR
(400 MHz, DMSO-d6) d = 8.90 (dd, J1 = 4.2, J2 = 1.7 Hz, 1H, Ar-H),
8.30–8.27 (m, 1H, Ar-H), 8.11 (dd, J1 = = 9.0, J2 = 0.9 Hz, 1H, Ar-
H), 7.96–7.95 (m, 1H, Ar-H), 7.81–7.79 (m, 1H, Ar-H), 7.71–7.69
(m, 1H, Ar-H), 7.62 (dd, J1 = 8.6, J2 = 4.2 Hz, 1H, Ar-H), 7.52–7.48
(m, 1H, Ar-H), 7.35 (d, J = 8.9 Hz, 1H, Ar-H), 5.25 (s, 2H, NH2); 13C
NMR (100 MHz, DMSO-d6) d = 169.0, 152.3, 150.3, 148.4, 143.1,
141.9, 136.9, 136.6, 135.7, 133.9, 132.5, 132.3, 129.9, 125.2,
122.6, 121.9, 118.5, 111.7; HRMS (DART) calculated for
5.4.11. 4-(3-Chloro-4-fluorophenyl)-5-(1,5-naphthyridin-2-yl)-1,3-
thiazol-2-amine (4m)
C
18H11N5S [M+H+]:330.08134, found: 330.08137.
2 mmol scale, 34% yield (3 steps), mp 335–337 °C; 1H NMR
(400 MHz, CDCl3) d = 8.89 (dd, J1 = 4.2, J2 = 1.7 Hz, 1H, Ar-H), 8.30–
8.27 (m, 1H, Ar-H), 8.10 (dd, J1 = 9.0, J2 = 0.9 Hz, 1H, Ar-H), 7.70
(dd, J1 = 7.1, J2 = 2.2 Hz, 1H, Ar-H), 7.60 (dd, J1 = 8.5, J2 = 4.2 Hz, 1H,
Ar-H), 7.45–7.40 (m, 2H, Ar-H), 7.16 (t, J = 8.7 Hz, 1H, Ar-H), 5.16
(s, 2H, NH2); 13C NMR (100 MHz, DMSO-d6) d = 169.0, 157.4
(d, JC-F = 247.1 Hz), 152.5, 150.4, 148.3, 143.2, 141.9, 136.6, 135.9,
133.5 (d, JC-F = 4.0 Hz), 131.2, 130.0 (d, JC-F = 7.3 Hz), 125.3, 122.7,
121.6, 119.8 (d, JC-F = 17.8 Hz), 117.2 (d, JC-F = 21.1 Hz); 19F NMR
(377 MHz, DMSO-d6) d = À116.0; HRMS (DART) calculated for
5.4.6. 5-(1,5-Naphthyridin-2-yl)-4-(3-(trifluoromethyl)phenyl)-1,3-
thiazol-2-amine (4g)
2 mmol scale, 40% yield (3 steps), mp 183–185 °C; 1H NMR
(400 MHz, DMSO-d6) d = 8.87 (dd, J1 = 4.2, J2 = 1.6 Hz, 1H, Ar-H),
8.26–8.24 (m, 1H, Ar-H), 8.11 (dd, J1 = 9.0, J2 = 0.9 Hz, 1H, Ar-H),
7.89 (s, 1H, Ar-H), 7.84–7.78 (m, 2H, Ar-H), 7.72 (dd, J1 = 8.5,
J2 = 4.2 Hz, 1H, Ar-H), 7.66–7.63 (m, 3H, two from NH2, one from
Ar-H), 7.34 (d, J = 9.0 Hz, 1H, Ar-H); 13C NMR (100 MHz, DMSO-
d6) d = 169.0, 152.4, 150.3, 148.8, 143.1, 141.9, 136.53, 136.50,
C
17H1035ClFN4S [M+H+]: 357.03770, found: 357.03856.
135.8, 133.0, 129.6, 129.3 (q, JC-F = 31.5 Hz), 125.6 (q, JC-F
=
3.9 Hz), 125.4, 125.19, 125.16, 122.7, 121.7; 19F NMR (377 MHz,
DMSO-d6) d = À62.7; HRMS (DART) calculated for C18H11F3N4S
[M+H+]: 373.07348, found: 373.07321.
5.4.12. 4-(4-Fluoro-3-methylphenyl)-5-(1,5-naphthyridin-2-yl)-1,3-
thiazol-2-amine (4n)
2 mmol scale, 64% yield (3 steps), mp 203–204 °C; 1H NMR
(400 MHz, DMSO-d6) d = 8.84 (dd, J1 = 4.2, J2 = 1.6 Hz, 1H, Ar-H),
8.25–8.22 (m, 1H, Ar-H), 8.07 (dd, J1 = 9.0, J2 = 0.8 Hz, 1H, Ar-H),
7.70 (dd, J1 = 8.5, J2 = 4.2 Hz, 1H, Ar-H), 7.54 (s, 2H, NH2), 7.49–
7.46 (m, 1H, Ar-H), 7.37–7.30 (m, 2H, Ar-H), 7.19 (dd, J1 = 9.8,
J2 = 8.4 Hz, 1H, Ar-H), 2.25 (s, 3H, CH3); 13C NMR (100 MHz,
DMSO-d6) d = 168.8, 160.7 (d, JC-F = 243.4 Hz), 152.9, 150.3, 150.0,
143.1, 141.8, 136.2, 135.6, 132.3 (d, JC-F = 5.3 Hz), 131.9 (d,
JC-F = 3.6 Hz), 128.4 (d, JC-F = 8.2 Hz), 125.0, 124.6 (d, JC-F = 17.3 Hz),
122.4, 120.8, 115.1 (d, JC-F = 22.4 Hz), 14.1 (d, JC-F = 3.2 Hz); 19F
NMR (377 MHz, DMSO-d6) d = À117.2; HRMS (DART) calculated
for C18H13FN4S [M+H+]: 337.09232, found: 337.09270.
5.4.7. 4-(3-ethylphenyl)-5-(1,5-naphthyridin-2-yl)-1,3-thiazol-2-
amine (4h)
2 mmol scale, 51% yield (3 steps), mp 183–184 °C; 1H NMR
(400 MHz, DMSO-d6) d = 8.82 (dd, J1 = 4.2, J2 = 1.6 Hz, 1H, Ar-H),
8.25–8.22 (m, 1H, Ar-H), 8.02 (d, J = 8.9 Hz, 1H, Ar-H), 7.68 (dd,
J1 = 8.5, J2 = 4.2 Hz, 1H, Ar-H), 7.56 (s, 2H, NH2), 7.37–7.25 (m, 5H,
Ar-H), 2.59 (q, J = 7.6 Hz, 2H, CH2), 1.14 (t, J = 7.5 Hz, 3H, CH3);
13C NMR (100 MHz, DMSO-d6) d = 169.0, 153.1, 151.5, 150.0,
144.1, 143.1, 141.8, 135.9, 135.7, 135.6, 128.6, 128.5, 128.3,
126.3, 125.0, 122.5, 120.9, 28.1, 15.6; HRMS (DART) calculated
for C19H16N4S [M+H+]: 333.11739, found: 333.11770.
5.4.13. 4-(3,4-Dimethylphenyl)-5-(1,5-naphthyridin-2-yl)-1,3-
thiazol-2-amine (4o)
5.4.8. 4-(3-Isopropylphenyl)-5-(1,5-naphthyridin-2-yl)-1,3-thiazol-2-
amine (4i)
2 mmol scale, 32% yield (3 steps), mp 209–210 °C; 1H NMR
(400 MHz, DMSO-d6) d = 8.82 (dd, J1 = 4.2, J2 = 1.6 Hz, 1H, Ar-H),
8.25–8.22 (m, 1H, Ar-H), 8.03 (dd, J1 = 9.0, J2 = 0.9 Hz, 1H, Ar-H),
7.69 (dd, J1 = 8.5, J2 = 4.2 Hz, 1H, Ar-H), 7.51 (s, 2H, NH2), 7.36–
7.33 (m, 2H, Ar-H), 7.23–7.17 (m, 2H, Ar-H), 2.28 (s, 3H, CH3),
2.23 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) d 168.8, 153.2,
151.6, 149.9, 143.1, 141.8, 136.9, 136.5, 135.9, 135.6, 133.3,
130.0, 129.6, 126.4, 125.0, 122.4, 120.5, 19.31, 19.29; HRMS (DART)
calculated for C19H16N4S [M+H+]: 333.11739, found: 333.11726.
2 mmol scale, 38% yield (3 steps), mp 225–226 °C; 1H NMR
(400 MHz, DMSO-d6) d = 8.83 (dd, J1 = 4.2, J2 = 1.6 Hz, 1 H, Ar-H),
8.25–8.22 (m, 1H, Ar-H), 8.03 (dd, J1 = 9.1, J2 = 0.9 Hz, 1H, Ar-H),
7.70 (dd, J1 = 8.5, J2 = 4.2 Hz, 1H, Ar-H), 7.55 (s, 2H, NH2), 7.39–
7.38 (m, 1H, Ar-H), 7.35–7.29 (m, 4H, Ar-H), 2.89 (m, 1H, CH),
1.17 (d, J = 6.9 Hz, 6H, two CH3); 13C NMR (100 MHz, DMSO-d6)
d = 168.9, 153.0, 151.6, 150.0, 148.5, 143.1, 141.8, 135.9, 135.6,
135.6, 128.6, 127.0, 126.9, 126.5, 125.0, 122.5, 120.8, 33.3, 23.7;
HRMS (DART) calculated for C20H18N4S [M+H+]: 347.13304, found:
347.13446.
5.4.14. 4-(3,4-Dichlorophenyl)-5-(1,5-naphthyridin-2-yl)-1,3-thiazol-
2-amine (4p)
5.4.9. 4-(4-Chlorophenyl)-5-(1,5-naphthyridin-2-yl)-1,3-thiazol-2-
amine (4k)
2 mmol scale, 28% yield (3 steps), mp 262–264 °C; 1H NMR
(400 MHz, DMSO-d6) d = 8.87 (dd, J1 = 4.2, J2 = 1.6 Hz, 1H, Ar-H),
8.26 (dt, J1 = 8.4, J2 = 1.2 Hz, 1H, Ar-H), 8.16 (dd, J1 = 8.8,
J2 = 0.9 Hz, 1H, Ar-H), 7.80 (d, J = 2.0 Hz, 1H, Ar-H), 7.73 (dd,
J1 = 8.5, J2 = 4.2 Hz, 1H, Ar-H), 7.67 (d, J = 8.3 Hz, 1H, Ar-H), 7.62
(s, 2H, NH2), 7.50 (dd, = 8.3, J2 = 2.1 Hz, 1H, Ar-H), 7.41 (d,
J = 9.0 Hz, 1H, Ar-H); 13C NMR (100 MHz, DMSO-d6) d = 169.0,
152.4, 150.4, 147.8, 143.1, 141.9, 136.7, 136.2, 135.8, 131.3,
2 mmol scale, 44% yield (3 steps), mp 225–226 °C; 1H NMR
(400 MHz, CDCl3) d = 8.87 (dd, J1 = 4.3, J2 = 1.7 Hz, 1H, Ar-H), 8.29
(dd, J1 = 8.6, J2 = 1.5 Hz, 1H, Ar-H), 8.08–8.06 (m, 1H, Ar-H), 7.59
(dd, J1 = 8.5, J2 = 4.2 Hz, 1H, Ar- H), 7.54–7.52 (m, 2H, Ar-H),
7.43–7.38 (m, 3H, Ar-H), 5.16 (s, 2H, NH2); 13C NMR (100 MHz,
DMSO-d6) d = 169.0, 152.7, 150.2, 149.6, 143.1, 141.9, 136.4,
Please cite this article in press as: Osman K.T., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.036

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K.T. Osman et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
131.2, 130.9, 130.8, 129.3, 125.2, 122.9, 121.9; HRMS (DART) calcu-
lated for C17H1035Cl2N4S [M+H+]: 373.00815, found: 373.00781.
5.5.2. 4-(3-chlorophenyl)-N-methyl-5-(1,5-naphthyridin-2-yl)-1,3-
thiazol-2-amine (6)
To a solution of 5b (0.1560 g, 0.35 mmol) in anhydrous DMF
(5 mL) was added NaH (0.0213 g, 0.53 mmol, 60%) at 0 °C. The
reaction mixture was stirred at 0 °C for 0.5 h before MeI
(0.1513 g, 1.05 mmol) was added. The mixture was then heated
to 60 °C and stirred overnight. After the reaction mixture was
cooled to room temperature, saturated aqueous ammonium chlo-
ride was added to quench the reaction. The mixture was parti-
tioned between CH2Cl2 and water. The aqueous phase was
extracted with CH2Cl2. The organic layers were combined, washed
with brine, dried over sodium sulfate, and evaporated. The crude
product was passed through a short silica gel column (eluent: hex-
ane/ethyl acetate = 1/2) to give light yellow product (0.0450 g).
This crude product was dissolved in CH2Cl2 (2 mL) in a flask, TFA
(0.5 mL) was added, then the mixture was stirred at room temper-
ature for 3 h and monitored by TLC. Aqueous sodium carbonate
was added, and the solution was extracted with ethyl acetate,
the organic layers were combined, washed with brine, dried over
sodium sulfate, and evaporated. The crude product was purified
by silica gel chromatograph (eluent: hexane/ethyl acetate = 2/1)
to give a light yellow product (0.0184 g, 15% yield over 3 steps):
mp 254–256 °C; 1H NMR (400 MHz, CDCl3) d = 8.85 (dd, J1 = 4.2,
J2 = 1.6 Hz, 1H, Ar-H), 8.29–8.27 (m, 1H, Ar-H), 8.04 (dd, J1 = 9.1,
J2 = 0.9 Hz, 1H, Ar-H), 7.63 (t, J = 1.9 Hz, 1H, Ar-H), 7.58 (dd,
J1 = 8.5, J2 = 4.2 Hz, 1H, Ar-H), 7.46–7.40 (m, 2H, Ar-H), 7.37–7.33
(m, 2H, Ar-H), 5.92 (d, J = 5.3 Hz, 1H, NH), 3.02 (s, 3H, -CH3); 13C
NMR (100 MHz, DMSO-d6) d = 169.5, 152.5, 150.2, 149.6, 143.1,
141.9, 137.9, 136.4, 135.7, 133.3, 130.6, 128.8, 128.7, 127.8,
5.4.15. 4-(3,5-dichlorophenyl)-5-(1,5-naphthyridin-2-yl)-1,3-thiazol-
2-amine (4q)
2 mmol scale, 19% yield (3 steps), mp 253–255 °C; 1H NMR
(400 MHz, DMSO-d6) d = 8.88 (dd, J1 = 4.2, J2 = 1.6 Hz, 1H, Ar-H),
8.26–8.24 (m, 1H, Ar-H), 8.17 (dd, J1 = 9.0, J2 = 0.9 Hz, 1H, Ar-H),
7.73 (dd, J1 = 8.5, J2 = 4.2 Hz, 1H, Ar-H), 7.67 (t, J = 2.0 Hz, 1H, Ar-
H), 7.63 (s, 2H, NH2), 7.56 (d, J = 1.9 Hz, 2H,, Ar-H), 7.40 (d,
J = 9.0 Hz, 1H,, Ar-H). 13C NMR (100 MHz, DMSO-d6) d = 169.0,
152.2, 150.5, 147.4, 143.2, 141.9, 139.0, 136.7, 135.9, 134.2,
128.1, 127.7, 125.3, 122.9, 122.3; HRMS (DART) calculated for C17
-
H1°Cl2N4S [M+H+]: 373.00815, found: 373.00818.
5.4.16. 4-(3-Chloro-4-fluorophenyl)-5-(8-methyl-1,5-naphthyridin-2-
yl)-1,3-thiazol-2-amine (4r)
2 mmol scale, 11% yield (3 steps), mp 242–244 °C; 1H NMR
(400 MHz, CDCl3) d = 8.72 (d, J = 4.4 Hz, 1H, Ar-H), 8.07 (d,
J = 8.9 Hz, 1H, Ar-H), 7.70 (dd, J1 = 7.1, J2 = 2.2 Hz, 1H, Ar-H),
7.47–7.39 (m, 3H, Ar-H), 7.17 (t, J = 8.6 Hz, 1H, Ar-H), 5.19 (s, 2H,
NH2), 2.76 (s, 3H, CH3); 13C NMR (150 MHz, DMSO-d6) d = 168.9,
157.2 (d, JC-F = 246.8 Hz), 151.1, 149.8, 147.9, 145.0, 142.7, 141.2,
136.9, 133.6 (d, JC-F = 3.9 Hz), 131.1, 129.8 (d, JC-F = 7.4 Hz), 125.1,
122.2, 122.0, 119.6 (d, JC-F = 17.6 Hz), 117.1 (d, JC-F = 21.0 Hz),
16.3; 19F NMR (377 MHz, DMSO-d6) d = À114.6; HRMS (DART) cal-
culated for C18H12ClFN4S [M+H+]: 371.05335, found: 371.05372.
5.4.17. Preparation of N-Acetyl-4-(3-chlorolphenyl)-5-(1,5-
naphthyridin-2-yl)-1,3- thiazol-2-amine (5a)
125.2, 122.3, 121.4, 30.70; HRMS (DART) calculated for C18H13
-
To a flame-dried flask were added 4a (0.2068 g, 0.6 mmol),
acetic anhydride (0.1225 g, 1.2 mmol), triethylamine (0.1214 g,
1.2 mmol) and anhydrous THF (10 mL). The mixture was stirred
at 60 °C overnight. The solvent was evaporated and the crude pro-
duct was purified by silica gel chromatography (eluent: hexane/
ethyl acetate = 3/1) to give 5a (0.1484 g, 65%) as a light yellow
solid: mp 261–262 °C; 1H NMR (400 MHz, DMSO-d6) d = 12.44
(bs, 1H, NH), 8.93 (dd, J1 = 4.2, J2 = 1.6 Hz, 1H, Ar-H), 8.39–8.37
(m, 1H, Ar-H), 8.23 (dd, J1 = 9.0, J2 = 0.9 Hz, 1H, Ar-H), 7.77 (dd,
J1 = 8.5, J2 = 4.2 Hz, 1H, Ar-H), 7.63 (t, J = 1.7 Hz, 1H, Ar-H), 7.54–
7.44 (m, 4H, Ar-H), 2.21 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-
d6) d = 169.2, 158.7, 152.2, 151.2, 146.5, 143.2, 142.1, 137.1,
137.0, 136.3, 133.4, 130.6, 128.79, 128.77, 127.8, 127.1, 125.4,
123.3, 22.6; HRMS (DART) calculated for C19H1335ClN4OS [M+H+]:
381.05768, found: 381.05823.
ClN4S [M+H+]: 353.06277, found: 353.06289.
Acknowledgments
JY was supported by Collaborative Research and Training Expe-
rience grant from the Natural Sciences and Engineering Research
Council of Canada. The SGC is a registered charity (number
1097737) that receives funds from AbbVie, Bayer Pharma AG,
Boehringer Ingelheim, Canada Foundation for Innovation, Eshel-
man Institute for Innovation, Genome Canada, Innovative Medici-
nes Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen,
Merck & Co., Novartis Pharma AG, Ontario Ministry of Economic
Development and Innovation, Pfizer, SaoPaulo Research Founda-
tion-FAPESP, Takeda, and the Wellcome Trust.
A. Supplementary material
5.5. Preparation of 4-(3-chlorolphenyl)-N-methyl-5-(1,5-
naphthyridin-2-yl)-1,3- thiazol-2-amine (6)
Additional figures illustrating inhibition of host invasion,
Kinome selectivity profiles of 4b, and 1H, 13C, and 19F NMR spectra
of new compounds. Supplementary data associated with this arti-
cle can be found, in the online version, at http://dx.doi.org/10.
1016/j.bmc.2017.01.036.
5.5.1. tert-Butyl-4-(3-chlorophenyl)-5-(1,5-naphthyridin-2-yl)-1,3-
thiazol-2-ylcarbamate (5b)
To a flame-dried flask were added 4a (0.2393 g, 0.7 mmol),
ditertbutyl dicarbonate (0.1850 g, 0.84 mmol), triethylamine
(0.0857 g, 0.84 mmol), 4-dimethyl aminopyridine (0.0086 g,
0.07 mmol) and anhydrous THF (6 mL). This mixture was stirred
at room temperature for 20 h. Then the solvent was evaporated
and the crude product was passed through a short silica gel column
(eluent: hexane/ethyl acetate = 3/1) to give the product (0.1763 g,
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