The human Aurora kinase inhibitor danusertib is a lead compound for anti-trypanosomal drug discovery via target repurposing

Article abstract of DOI:10.1016/j.ejmech.2012.07.038

New drugs for neglected tropical diseases such as human African trypanosomiasis (HAT) are needed, yet drug discovery efforts are not often focused on this area due to cost. Target repurposing, achieved by the matching of essential parasite enzymes to those human enzymes that have been successfully inhibited by small molecule drugs, provides an attractive means by which new drug optimization programs can be pragmatically initiated. In this report we describe our results in repurposing an established class of human Aurora kinase inhibitors, typified by danusertib (1), which we have observed to be an inhibitor of trypanosomal Aurora kinase 1 (TbAUK1) and effective in parasite killing in vitro. Informed by homology modeling and docking, a series of analogs of 1 were prepared that explored the scope of the chemotype and provided a nearly 25-fold improvement in cellular selectivity for parasite cells over human cells.

Full text of DOI:10.1016/j.ejmech.2012.07.038

European Journal of Medicinal Chemistry 62 (2013) 777e784
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
The human Aurora kinase inhibitor danusertib is a lead compound
for anti-trypanosomal drug discovery via target repurposing
,
,
,
Stefan O. Ochiana a 1, Vidya Pandarinath b 1, Zhouxi Wang a 1, Rishika Kapoor b,
Mary Jo Ondrechen a, Larry Ruben b, Michael P. Pollastri a
,
*
a Northeastern University, Department of Chemistry and Chemical Biology, Hurtig 102, 360 Huntington Avenue, Boston, MA 02115, USA
b Southern Methodist University Center for Drug Discovery, Design and Delivery, Department of Biological Sciences, Dedman Life Science Building,
Room 113, 6501 Airline Drive, Dallas, TX 75275, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
New drugs for neglected tropical diseases such as human African trypanosomiasis (HAT) are needed, yet
drug discovery efforts are not often focused on this area due to cost. Target repurposing, achieved by the
matching of essential parasite enzymes to those human enzymes that have been successfully inhibited
by small molecule drugs, provides an attractive means by which new drug optimization programs can be
pragmatically initiated. In this report we describe our results in repurposing an established class of
human Aurora kinase inhibitors, typified by danusertib (1), which we have observed to be an inhibitor of
trypanosomal Aurora kinase 1 (TbAUK1) and effective in parasite killing in vitro. Informed by homology
modeling and docking, a series of analogs of 1 were prepared that explored the scope of the chemotype
and provided a nearly 25-fold improvement in cellular selectivity for parasite cells over human cells.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 2 April 2012
Received in revised form
19 July 2012
Accepted 20 July 2012
Available online 31 July 2012
Keywords:
Aurora kinase
Trypanosoma brucei
TbAUK1
Cell cycle
Neglected disease
Target repurposing
Sleeping sickness
1. Introduction
spread out over 14 days. Although a nifurtimoxeeflornithine
combination therapy decreases the dosing time and toxicity
The neglected tropical disease (NTD) human African trypano-
somiasis (HAT) is caused by the insect-borne protozoan parasite
Trypanosoma brucei, resulting in an estimated 50,000 new infec-
tions per year [1]. The disease progresses through two stages:
Stage I, the bloodstream form; and Stage II, where the parasites
have invaded the central nervous system. It is in this later stage
that patients begin to show the trademark symptoms of sleeping
sickness: lethargy, sleep disorder, and coma. HAT is invariably fatal
when not treated. Although there are drugs that can effectively
and safely treat HAT in the bloodstream infections, most patients
are diagnosed when symptoms appear in Stage II. The current
treatments for Stage II disease are melarsoprol, a toxic arsenical
agent that itself has a 5% mortality rate, and eflornithine, which
has toxic side effects and requires an extensive dosing regimen
[2,3], an acute need persists for safe, inexpensive and convenient
therapeutics.
HAT and other NTDs primarily affect the destitute in developing
nations. Consequently, the pharmaceutical industry has been slow
to invest in the highly resource-intensive drug discovery programs
necessary for the development of new therapies. As a result, the
identification of new drug candidates remains a bottleneck in the
process. The costly process of drug discovery can be accelerated,
and costs reduced, by “target repurposing”, where essential
enzyme targets in infectious agents are matched with human
homologs that have been pursued for other indications [4]. This can
accelerate drug discovery by informing new anti-infective
programs with the wealth of medicinal chemistry, structural
biology, and molecular and cellular biology knowledge that has
accumulated over the years in pursuit of inhibitors for the human
targets. In fact, eflornithine as a treatment for sleeping sickness was
uncovered using this approach [5e7], and recent repurposing
efforts have been reported for antimalarial [8] and anti-
trypanosomal [9] phosphodiesterase (PDE) and phosphoinositide-
3-kinase (PI3K) inhibitors [10].
Abbreviations: HAT, human African trypanosomiasis; BF, bloodstream form
T. brucei; cAMP, cyclic adenosine monophosphate.
* Corresponding author. Tel.: þ1 617 373 2703.
E-mail address: m.pollastri@neu.edu (M.P. Pollastri).
These authors contributed equally to this work.
1
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.038

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S.O. Ochiana et al. / European Journal of Medicinal Chemistry 62 (2013) 777e784
Drug repurposing programs can develop from target-based
screens, or phenotype driven screens. For example, while eflorni-
thine and PDE inhibitors have validated targets in parasites, the
PI3K inhibitors are potent antiparasitic compounds whose specific
mechanism of action is not yet known. In general, phenotype-
driven drug discovery programs have resulted in the majority of
new chemical entities introduced [11]. The observation is perhaps
surprising in this ongoing era of target-based drug discovery,
however it reflects the challenges and complexities of pivotal bio-
logical targets and pathways. Consequently, phenotype-based
optimization is frequently employed in human systems, even in
cases where much target/pathway knowledge exists. Thus, one
would expect that phenotype based optimization would be most
fruitful in studies of pathogens where their biological pathways are
not as well understood. This expectation has been borne out in the
fact that 70% of first-in-class anti-infective agents launched
between 1999 and 2008 were discovered via phenotype driven
approaches [11]! Further, these programs are enhanced when
starting from established chemical classes with prior knowledge
matching chemotype to cellular phenotype.
We have begun studying inhibitors of the trypanosomal cell
cycle, starting with established mammalian Aurora kinase inhibi-
tors. Aurora kinases are druggable targets that have been pursued
for the discovery of new antineoplastic agents [12]. As an integral
enzyme in cell division, these enzymes control events including
mitotic spindle assembly, chromosomal separation, and cytoki-
nesis. Since Aurora kinases are essential cell cycle mediating
enzymes and are implicated in malignancy [13], they have been
pursued as important targets in drug discovery, resulting in a wide
variety of inhibitors of Aurora that have successfully moved into
clinical trials [14]. An evaluation of the T. brucei kinome identified
three Aurora kinase paralogs [15]. RNAi revealed that TbAUK1, but
not TbAUK2 or TbAUK3, was required for mitotic progression [16].
Loss of TbAUK1 inhibits nuclear division, cytokinesis and growth in
cultured infectious bloodstream forms (BF) and insect stage pro-
cyclic forms [17]. Additionally, TbAUK1 is required for infection in
mice [18], and is susceptible to the Aurora kinase inhibitors hes-
peradin [18] and VX-680 [19]. Since hesperadin has not been
advanced to human clinical trials, and the further development of
VX-680 has been halted during Phase II clinical trials, we looked
toward other chemotypes that are currently still under clinical
development.
Fig. 1. Pyrazolopyrazole inhibitors of human Aurora kinases.
(Fig. 2). In this experiment, the AU1-tagged kinase was pulled down
with anti-AU1 Sepharose and used to phosphorylate myelin basic
protein (MBP). The upper panel of Fig. 2 is an autoradiogram and
the lower panel is a Coomassie stain of the same gel showing that
each lane had an equivalent amount of MBP loaded on the gel.
Hesperadin at 500 nM completely inhibited TbAUK1, and was used
here to show background kinase activity in the pull-down assay. At
500 nM, compounds 1 and 2 inhibited TbAUK1 to the same back-
ground level as hesperadin, while 5a did not.
We assessed growth inhibition of T. brucei brucei bloodstream
form (BF) trypanosomes (90-13 strain) with the Cell Titer BlueÒ end
point assay for the compounds shown in Table 1. Taken with the
TbAUK1 kinase data above, these results demonstrate that 1 and 2
can completely inhibit TbAUK1 activity at 500 nM and cell growth
with an effective concentration that inhibits cellular growth by 50%
(EC50) in a similar concentration range. Conversely, 5a neither
inhibits TbAUK1 activity nor has any significant effect on cell
growth. Compounds 5b and 5c show an activity that approximates
2.
For each of these compounds tested we measured inhibition of
the acute myelogenous leukemia cell line MOLT-4 [22]. This cell line
overexpresses Aurora kinases A and B when compared with unin-
duced peripheral blood mononuclear cells, and growth of this cell
line has been shown to be selectively blocked by Aurora kinase
inhibition [23]. MOLT-4 also shares with trypanosomes an ability to
grow in suspension culture and circulate through the blood and
lymph fluid.
Growth of MOLT-4 was indeed blocked by these inhibitors
(Table 1). However, we note a range of selectivity between T. b.
brucei and MOLT-4, providing confidence that the structural
features that give rise to human and trypanosomal activity are not
inextricably linked. This is an important finding, since the goal of
our Aurora inhibitor repurposing project is to modify existing
scaffolds to generate potent and selective inhibitors of trypano-
some growth.
2. Results & discussion
Inspired by the initial growth inhibition observed by hesperadin
and VX-680, we decided to assess the pyrrolopyrazole danusertib
(1, formerly PHA-739358) [20] and its predecessor analog PHA-
680632 (2, Fig. 1) [21]. This compound class is of particular
interest since 1 is well advanced into clinical trials, is parallel-
synthesis enabled, and its medicinal chemistry and structural
biology profiles are well established. Thus, we hypothesized that
this chemotype would afford an opportunity to explore rapidly the
structureeactivity relationships of the series.
We synthesized three additional analogs (Scheme 1, compounds
5aec) to compare simple replacements for the diethylphenyl urea
head group of 2. To verify that our lead compounds inhibit TbAUK1
activity, 1, 2 and 5a were each tested at 500 nM in an in vitro kinase
assay. Since efforts to produce catalytically active recombinant
TbAUK1 have proven fruitless, we resorted to the use of AU1-tagged
TbAUK1, immunoprecipitated from trypanosome homogenates.
Using this method, we have previously shown that hesperadin
inhibits TbAUK1 at 200 nM to the level of a background kinase [18].
We wished to demonstrate that compounds 1 and 2 are able to
lower kinase activity to the same background level as hesperadin
We sought to gain a better understanding of the differences in
activity between these close-in danusertib analogs by development
of a homology model of TbAUK1 based on the human [21] and
mouse [24] Aurora A crystal structures (PDB ID 2BMC and 3D14,
respectively). Inhibitors were docked to TbAUK1, and to human
Aurora A in instances where there was no published ligand-enzyme
crystal structure. When comparing the TbAUK model (Fig. 3c) with
the published Aurora A/1 complex (Fig. 3a) [20] a similar ligand
pose is observed, and most of the key ligand-protein contacts are
retained. In both structures the pyrazinylphenyl tail is oriented
towards the solvent, and the key H-bonding interactions between

S.O. Ochiana et al. / European Journal of Medicinal Chemistry 62 (2013) 777e784
779
Scheme 1. Synthesis of analogs of 2. Reagents and conditions. a. benzoyl chloride, pyridine; b. phenylisocyanate, THF; c. p-toluenesulfonyl chloride, pyridine; d. 10% Et3N, methanol.
the pyrazolopyrrole core and the kinase hinge region are preserved.
However, the head group region of 1 assumes a flipped orientation
in the TbAUK1 binding site, placing the phenyl group into
a hydrophobic pocket towards the N-terminus lobe (Fig. 3c). This
flip is likely driven by Met113 in TbAUK1, which corresponds to
Thr217 in human Aurora A. The bulkier hydrophobic amino acid
methionine side chain forces the phenyl group of danusertib up
into the hydrophobic pocket. Notably, T217 mutation of human
Aurora A confers resistance to MLN8054 [25], so this amino acid
difference is likely to be an important consideration in the design of
selective TbAUK1 inhibitors.
Based on this analysis, we suspected that the planar urea moiety
of 2 is less able to accommodate the Thr-Met change in the protein,
leading to the decrease in potency against T. brucei cultures, and
a decrease in selectivity over MOLT-4 cells. This was supported by
docking experiments: The head group of 2 (selectivity ¼ 0.06) is
rigidly held against Met113, which is likely to lead to lower activity
against the parasite enzyme (compare Fig. 3b and c). Thus, noting
this geometric difference between 1 and 2, we hypothesized that
the tetrahedral geometry of the carbon atom adjacent to the
carbonyl group in the danusertib head group would enable the
head group flip of danusertib analogs, and that lipophilic side
chains at this position could also help drive potency and/or selec-
tivity by fitting into the adjacent lipophilic pocket. The reduced
potency of other planar substitutions (notably compounds 5aec)
supports this contention (Table 1).
clog P ꢀ 5.0 were retained, and a diverse subset of 50 analogs was
selected for docking into the TbAUK1 homology model. These
docking experiments provided a prioritization by which the top
twenty analogs would be synthesized first.
From the prioritized list, 19 compounds were successfully
synthesized and each was tested at 1 and 10
mM against two
different T. brucei BF cell lines (Lister 427 90-13 and AnTat1.1A).
Eleven compounds (8e18) showed greater than 60% inhibition at
1 mM and were selected for a full doseeresponse analysis against
the human infective T. b. rhodesiense, and MOLT-4 cells (Table 2 and
Fig. 4). The single concentration data for all 19 compounds and
doseeresponse data for 8e18 against T. b. brucei 90-13 is tabulated
in the Supporting information.
Of note from these screening results is that, while compounds of
improved potency over 1 have not yet been uncovered, an
improved selectivity ratio for killing T. b. rhodesiense over MOLT-4
cells has been attained. Indeed, 3-fold selectivity has been
observed in the case of compound 8; eight other analogs display
selectivity >1.0 over MOLT-4 cells.
The docked pose of 8, the most selective compound, is shown in
Fig. 3c. The head group of this compound attains a flipped confor-
mation similar to that observed with danusertib, but is extended
a little deeper into the lipophilic pocket. In this case, the naphthyl
head group provides lipophilic interactions with the top of the
binding pocket, where it also encounters Lys58 of TbAUK1 in
a geometry that could allow a favorable p-cation interaction [26]. In
To further test this hypothesis, we designed a library of dan-
usertib analogs that retained a tetrahedral geometry adjacent to the
carbonyl group. With an eye towards preparation of this library
using the chemistry shown in Scheme 2, a virtual library of analogs
was enumerated using 208 arylacetic acids (6) that were available
in pre-weighed quantities from a commercial vendor (ASDI, Inc).
Those product molecules with molecular weight <500 and
contrast, an adverse steric interaction of the naphthylene group of 8
is apparent upon docking to human Aurora A (Fig. 3b), a result that
is confirmed with a significantly reduced Glide docking score
(ꢁ6.469) compared to 1 (ꢁ10.29).
Besides providing a framework for explanation of the selectivity
observations of this compound series, the homology model has
proved to be useful for biasing compound library designs. The rank-
Table 1
Screening data summary of singleton analogs of 1 tested against T. b. brucei and
MOLT-4 cells.a
Compd T. b. bruceia EC50
(
m
M) MOLT-4b EC50
(
m
M) Selectivity MOLT-4/Tbb
c
1
2
5a
5b
5c
0.6
4.0
11.1
6.1
0.15
0.22
0.54
3.55
0.94
0.25
0.06
0.05
0.58
0.16
5.7
a
T.b.b. Lister 427 90-13.
MOLT-4 acute myelogenous leukemia cell line.
EC50 values were calculated from inhibition curves at a minimum of 8 different
b
c
Fig. 2. Inhibition of kinase activity by compounds in the inhibitor set.
drug concentrations tested in triplicate and using OriginPro 8.5 analysis software.

780
S.O. Ochiana et al. / European Journal of Medicinal Chemistry 62 (2013) 777e784
Fig. 3. Comparison of (a) the human Aurora A/danusertib complex (PDB ID: 2J50, danusertib colored in green); (b) The predicted conformation of 2 (colored in purple), 8 (colored in
yellow), and danusertib (colored in green), docked into human Aurora A. and (c) in the TbAUK1 model. The colors in (c) are shown for side chain heteroatoms in Lys58 (blue) nd
Met113 (yellow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
ordering of compounds by the docking experiments was aligned
with the observed potency against trypanosome cells, and the
docking scores showed a good correlation (R2 ¼ 0.75 for T. brucei
rhodesiense and 0.72 for T. brucei brucei) with the cellular potency
values (Supporting information). This supports the hypothesis that
the cellular growth inhibition phenotype is, at least in part, medi-
ated by TbAUK1 inhibition.
inhibitors of parasite cell growth. While we acknowledge that we
have direct TbAUK1 inhibition data only for compounds 1, 2 and 5a,
this has provided a starting point to initiate inhibitor optimization
studies in the absence of conclusive trypanosome kinase inhibition
data. This is of great importance, given that the functional pathways
of the trypanosome kinome are only lightly understood at this
point in time. However, by starting the program with human
Aurora inhibitors and utilizing previous medicinal chemistry and
structural biology information, we have been able to drive cellular
selectivity away from a mammalian cell line, which is a key goal of
this project.
3. Conclusions
We report that danusertib (1) inhibits both TbAUK1 kinase
activity and growth of three different trypanosome strains,
including human infective T. b. rhodesiense. Notably, the EC50 value
we report for T. b. rhodesiense (150 nM) is consistent with the mean
EC50 averaged from 15 different cancer cell lines (123 nM) [27].
Because danusertib is an optimized human Aurora inhibitor, it is
not surprising that the structural changes we made decreased
potency against mammalian cells. However, while we also observe
loss of potency against trypanosomes for analogs of 1, the loss is not
as severe as that observed for mammalian cells. Consequently, we
observe an improved selectivity ratio ranging from 2.2 to 23.4-fold.
Furthermore, since the molecular model of TbAUK1 provides
a plausible structural basis for this improvement, we expect to be
able to develop compounds with further improvements in potency
and selectivity.
In summary, we have reported an initial structureeactivity
relationship study of growth inhibitors of T. brucei spp. that
mediate their effect, in whole or in part, by TbAUK1. Besides
a demonstration of the expedience provided by repurposing
historical medicinal chemistry and structural biology knowledge of
the homologous human enzymes, we have shown that compounds
with improved selectivity for parasite killing over host cells can be
designed based on the structure of the hypothesized enzyme
target. Further optimization of this compound chemotype for
potency against the parasites, continued selectivity over human
cells, and central nervous system exposure will be reported in due
course.
4. Materials and methods
A key aspect of the target repurposing approach for neglected
disease drug discovery is the opportunity to utilize the extensive
historic research performed in the process of developing a clinical
candidate. In this case, the chemical matter previously disclosed as
human Aurora inhibitors do indeed show great promise as
4.1. Chemistry
Unless otherwise noted, reagents were obtained from
SigmaeAldrich, Inc. (St. Louis, MO), and used as received. Reaction
Scheme 2. Synthesis of arylacetamide derivatives 8e18. Reagents and conditions: (a) oxalyl chloride, CH2Cl2, DMF. (b) 3, DMF, DIEA. (c)10% Et3N, MeOH.

S.O. Ochiana et al. / European Journal of Medicinal Chemistry 62 (2013) 777e784
781
Table 2
rotamers), 7.59e7.32 (m, 2H), 7.49e7.55 (m, 3H), 7.06 and 6.95 (2d,
J ¼ 8.79 Hz, 2H, rotamers), 4.95e4.82 (m, 4H), 4.50 (q, J ¼ 7.00 Hz,
2H), 3.34e3.41 (m, 4H), 2.57e2.64 (m, 4H), 2.32 and 2.38 (2s, 3H,
rotamers), 1.27 and 1.27 (2t, J ¼ 7.32, 9.28 Hz, 3H, rotamers). LCMS
found 503.01, [M þ H]þ.
Dose-response experiments on the parallel array of analogs of 1 tested against T. b.
rhodesiense and MOLT-4 cells.
Compd R1
Ar
T.b.r.b EC50 MOLT-4c
Selectivity
(
m
M)d
EC50
(
m
M)d MOLT/Tbr
1
8
9
OMe Phenyl
0.15
0.61
0.15
1.0
23.4
6.9
6.8
6.9
6.3
6.4
4.3
2.2
1.2
1.0
0.7
H
H
1-Naphthyl
2,3,6-Trifluorophenyl 0.32
OMe Phenyl
14.25
2.22
4.13
4.0
4.1.2. N-(5-Benzoyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-
4-(4-methylpiperazin-1-yl)benzamide (5a)
10a
11
12a
13
14
15
16
17
18
0.61
0.58
0.4
A solution of 4a (5.80 mg, 0.012 mmol) in MeOH (3 mL) and Et3N
(0.3 mL) was stirred at 30 ꢂC for 4 h. The reaction was concentrated.
The residue was dissolved twice in diethyl ether and concentrated
to produce a white solid, which was triturated with a mixture of
EtOAc (3 mL) and ether (0.3 mL) to give 5a (Yield: 99%). 1H NMR
H
3-ClePh
Phenyl
iPr
Me
H
H
H
2.5
4-Methylphenyl
3,5-Dimethylphenyl
2,5-Dimethylphenyl
2,3,5-Trifluorophenyl
3-(2-Methylindolyl)
3,5-Difluorophenyl
0.86
1.04
1.2
2
1.2
5.48
4.46
2.65
2.31
1.16
0.63
H
H
(500 MHz, d6-DMSO)
d
7.82 (7.91 minor) (d, J ¼ 8.5 Hz, 2H), 7.6e7.54
0.91
(m, 2H), 7.49e7.45 (m, 3H), 6.92 (6.98 minor) (d, J ¼ 9.0 Hz, 2H)
4.69e4.52 (m, 4H), 3.25e3.23 (3.29e3.27 minor) (m, 4H),
2.41e2.39 (2.44e2.43 minor) (m, 4H), 2.20 (2.21 minor) (s, 3H). The
sets of amide rotamer signals that were doubled at room temper-
ature and listed as minor were shown to coalesce at 140 ꢂC. LCMS
found 431.01, [M þ H]þ.
a
Indicates compounds tested as racemate.
T. brucei rhodesiense YTAT1.1 strain.
MOLT-4 acute myelogenous leukemia cell line.
b
c
d
EC50 values were calculated from inhibition curves at a minimum of 8 different
drug concentrations tested in triplicate and using OriginPro 8.5 analysis software.
4.1.3. Ethyl 3-(4-(4-methylpiperazin-1-yl)benzamido)-5-tosyl-5,6-
dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylate (4c)
solvents were purified by passage through alumina columns on
a purification system manufactured by Innovative Technology
(Newburyport, MA). NMR spectra were obtained on Varian NMR
systems, operating at 400 MHz or 500 MHz for 1H acquisitions.
LCMS analysis was performed using a Waters Alliance reverse-
phase HPLC, with single-wavelength UVevisible detector and LCT
Premier time-of-flight mass spectrometer (electrospray ionization).
All newly synthesized compounds were deemed >95% pure by
LCMS analysis prior to submission for biological testing. Published
methods were employed for preparation of danusertib (1) [20],
PHA-680632 (2) [21], 3 [21], and 5b [21].
To 0.013 g of 3 (0.029 mmol) was added pyridine (2 mL), and
then tosyl chloride (5.57 mg, 0.029 mmol). The reaction mixture
was stirred at rt for 16 h. The solvent was concentrated and the
crude product was purified via silica gel chromatography, eluting
with 0e10% MeOH in DCM to give 4c (Yield: 23%). 1H NMR
(500 MHz, d4-CD3OD)
d
7.83 (d, J ¼ 8.5 Hz, 2H), 7.80 (d, J ¼ 8.75 Hz,
2H), 7.42 (d, J ¼ 8.5 Hz, 2H), 7.00 (d, J ¼ 8.75 Hz, 2H), 4.65e4.63 (m,
4H), 4.44 (q, 2H), 3.39e3.37 (m, 4H), 2.62e2.60 (m, 4H), 2.41 (s, 3H),
2.36 (s, 3H), 1.41 (t, J ¼ 7.25 Hz, 3H). LCMS found 553.01, [M þ H]þ.
4.1.4. 4-(4-Methylpiperazin-1-yl)-N-(5-tosyl-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl)benzamide (5c) [28]
4.1.1. Ethyl 5-benzoyl-3-(4-(4-methylpiperazin-1-yl)benzamido)-
5,6-dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylate (4a)
To 0.015 g of 3 (0.034 mmol) was added pyridine (2.346 mL,
A solution of 4c (3.7 mg, 6.7 mmol) in MeOH (1 mL) and Et3N
(0.1 mL) was stirred at 30 ꢂC for 4 h. The solvent was concentrated.
The residue was dissolved twice in diethyl ether and concentrated
to produce a white solid, which was triturated with a mixture of
EtOAc (3 mL) and ether (0.3 mL) to give 5c (Yield: 93%). 1H NMR
29.1 mmol) and benzoyl chloride (7.95 mL, 0.069 mmol). The reac-
tion was stirred for 16 h. The reaction was concentrated and the
crude product was purified via silica gel chromatography, eluting
with 0e8% MeOH in DCM to give 4a (Yield: 21%). 1H NMR
(500 MHz, d4-CD3OD)
d
7.83 (d, J ¼ 9.0 Hz, 2H), 7.88 (d, J ¼ 8.5 Hz,
(500 MHz, methanol-d4)
d
7.89 and 7.78 (2d, J ¼ 8.79 Hz, 2H,
2H), 7.41 (d, J ¼ 8.5 Hz, 2H), 7.01 (d, J ¼ 9.0 Hz, 2H), 4.62e4.35 (m,
4H), 3.39e3.37 (m, 4H), 2.64e2.62 (m, 4H), 2.41 (s, 3H), 2.37 (s, 3H).
LCMS found 481.01, [M þ H]þ.
4.1.5. General procedure for library synthesis
To the various carboxylic acids (obtained from ASDI, Inc.,
0.076 mmol, 1 equiv.) was added dry DCM (0.5 mL), oxalyl chloride
(0.076 mmol,1 equiv.), and then 2 drops of DMF. The reactions were
placed in the shaker and reacted overnight at ambient temperature
for 24 h. A solution of the hydrochloride salt of 3 (0.048 mmol, 1
equiv.) in 1.5 mL of DMF was added, followed by DIEA (0.240 mmol,
5 equiv.). The vials were agitated in the shaker for 24 h at ambient
temperature. The solvent was removed in
a Genevac HT24
centrifugal evaporator, and the crude intermediates were carried
forward without purification. To each of the above crude products
was added 10% TEA in MeOH (2 mL). The reaction vials were then
placed in the shaker at 33 ꢂC overnight, and the solvent was
evaporated. All the crude products were purified via preparative
HPLC to >95% purity, and the desired products in the yields as
described below.
Fig. 4. Dose response of 1, 2, 12 and 18 against T. b rhodesiense cultures. YTat1.1 BF cells
were incubated for a 48 h period with varying drug concentrations. Cell viability was
monitored with the Cell Titer BlueÒ assay. Values plotted are the average ꢃ std dev of
2e3 independent experiments, each done in triplicate.
4.1.5.1. 4-(4-Methylpiperazin-1-yl)-N-(5-(2-(naphthalen-1-yl)
acetyl)-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)benzamide
(8). (Yield: 30%) 1H NMR (500 MHz, d6-DMSO)
d 10.65 (s, 1H),

782
S.O. Ochiana et al. / European Journal of Medicinal Chemistry 62 (2013) 777e784
7.90e7.84 (m, 5H), 7.77 (d, J ¼ 8.5 Hz, 1H), 7.48e7.43 (m, 3H), 6.99
(d, J ¼ 9.5 Hz, 2H), 4.80e4.47 (m, 4H), 4.10e4.02 (m, 2H), 3.92 (d,
J ¼ 10.5 Hz, 2H), 3.15 (m, 2H), 2.56 (m, 4H), 2.31 (s, 3H). 13C NMR
(16). (Yield: 73%). 1H NMR (500 MHz, d4-methanol)
d 7.89e7.86 (m,
2H), 7.12e7.07 (m, 1H), 7.05e7.02 (m, 2H), 7.0e6.95 (m, 1H),
4.93e4.59 (m, 4H), 3.91 (d, J ¼ 5.5 Hz, 2H), 3.42e3.40 (m, 4H),
2.77e2.73 (m, 4H), 2.46 (s, 3H). LCMS found 499.01, [M þ H]þ.
(126 MHz, DMSO-d6) d 169.5, 169.5, 133.3, 133.0, 131.8, 129.3, 128.2,
127.7, 127.5, 127.4, 126.0, 125.5, 113.5, 53.9, 48.6, 46.3, 42.1, 40.1.
LCMS found 495.01, [M þ H]þ.
4.1.5.10. N-(5-(2-(2-methyl-1H-indol-3-yl)acetyl)-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-4-(4-methylpiperazin-1-yl)
4.1.5.2. 4-(4-Methylpiperazin-1-yl)-N-(5-(2-(2,3,6-trifluorophenyl)
benzamide (17). (Yield: 70%). 1H NMR (400 MHz, d6-DMSO)
d 10.84
acetyl)-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)benzamide
(d, J ¼ 8.0 Hz, 1H), 10.68 (d, J ¼ 6.4 Hz, 1H), 7.89 (m, 2H),
7.46 (d, J ¼ 8.0 Hz, 1H), 7.23e7.20 (m, 1H), 7.01e6.88 (m, 4H),
4.76e4.41 (m, 4H), 3.71 (s, 2H), 3.33e3.30 (m, 4H), 2.61e2.56 (m,
4H), 2.36 (d, J ¼ 16.0 Hz, 3H), 2.31 (s, 3H). LCMS found 498.01,
[M þ H]þ.
(9). (Yield: 62%). 1H NMR (400 MHz, d6-DMSO)
d 10.70 (s, 1H),
7.93e7.89 (m, 2H), 7.50e7.38 (m, 1H), 7.18e7.10 (m, 1H), 7.01 (d,
J ¼ 8.4 Hz, 2H), 6.56 (s, 1H), 4.86e4.45 (m, 4H), 3.87 (s, 3H),
3.40e3.30 (m, 4H), 2.70e2.63 (m, 4H), 2.38 (s, 3H). LCMS found
499.01, [M þ H]þ.
4.1.5.11. N-(5-(2-(3,5-Difluorophenyl)acetyl)-1,4,5,6-
4.1.5.3. N-(5-(2-methoxy-2-phenylacetyl)-1,4,5,6-tetrahydropyrrolo
tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-4-(4-methylpiperazin-1-yl)
[3,4-c]pyrazol-3-yl)-4-(4-methylpiperazin-1-yl)benzamide (10). (Yield:
benzamide (18). (Yield: 56%). 1H NMR (500 MHz, d4-methanol)
81%). 1H NMR (400 MHz, d6-DMSO)
d
10.67 (d, J ¼ 9.6 Hz, 1H), 7.89 (d,
d
7.89 (d, J ¼ 9.0 Hz, 2H), 7.07e7.05 (m, 2H), 6.96e6.93 (m, 2H),
J ¼ 12.0 Hz, 2H), 7.44e7.32 (m, 5H), 7.01 (d, J ¼ 12.0 Hz, 2H), 5.10 (d,
J¼ 12.0 Hz,1H), 4.82e4.39 (m, 4H), 3.40e3.35(m,4H),3.31(d,J¼ 3.2Hz,
3H), 2.80e2.65 (m, 4H), 2.48 (s, 3H). LCMS found 475.01, [M þ H]þ.
6.86e6.82 (m, 1H), 4.85e4.56 (m, 4H), 3.82 (s, 2H), 3.54e3.52
(m, 4H), 3.17e3.13 (m, 4H), 2.75 (s, 3H). LCMS found 481.01,
[M þ H]þ.
4.1.5.4. N-(5-(2-(3-Chlorophenyl)acetyl)-1,4,5,6-tetrahydropyrrolo
[3,4-c]pyrazol-3-yl)-4-(4-methylpiperazin-1-yl)benzamide
4.2. Computational chemistry
(11). (Yield: 27%). 1H NMR (500 MHz, d6-DMSO)
d
10.70 (s, 1H),
4.2.1. Virtual library design
7.92e7.89 (m, 2H), 7.35e7.21 (m, 4H), 7.01 (d, J ¼ 9.0 Hz, 2H),
4.76e4.44 (m, 4H), 3.76 (d, J ¼ 14.0 Hz, 2H), 3.37 (m, 4H), 2.72 (m,
4H), 2.42 (s, 3H). LCMS found 479.01, [M þ H]þ.
A virtual library of analogs was enumerated using Pipeline Pilot
based on 208 arylacetic acids that were available in pre-weighed
quantities from a commercial vendor (ASDI, Inc). Those product
molecules with molecular weight >500 and clog P ꢄ 5.0 were
removed, and SciTegic extended connectivity fingerprints (FCFP_6)
were calculated for the set of molecules. A subset of 50 maximally
diverse analogs was selected for docking in the TbAUK1 homology
model.
4.1.5.5. N-(5-(3-Methyl-2-phenylbutanoyl)-1,4,5,6-tetrahydropyrrolo
[3,4-c]pyrazol-3-yl)-4-(4-methylpiperazin-1-yl)benzamide (12). (Yield:
67%). 1H NMR (500 MHz, d4-methanol)
d 7.90e7.86 (m, 2H), 7.46e7.40
(m, 2H), 7.35e7.30 (m, 2H), 7.28e7.22 (m, 1H), 7.04 (t, J ¼ 8.5 Hz, 2H),
5.03 (d, J ¼ 12.0 Hz, 1H), 4.95e4.44 (m, 4H), 3.50e3.40 (m, 4H),
2.86e2.81 (m, 4H), 2.53 (s, 3H), 2.46e2.40 (m, 1H), 1.08 (d, J ¼ 7.0 Hz,
4.2.2. Homology modeling
3H), 0.71 (t, J¼ 4.5 Hz, 3H).13C NMR (126 MHz, d6-DMSO)
d
172.4,172.4,
The protein sequence of TbAUK1 (Tb11.01.0330) was searched
against the PDB (http://www.rcsb.org/) using PSI-BLAST [29].
Models were built for a portion of the catalytic domain (residues
28e219). TbAUK1 shares 43% sequence identity with human Aurora
A and 41% sequence identity with human Aurora B, with 88%
sequence coverage by these crystal structures. Homology modeling
of the kinase catalytic domain was performed with the YASARA
suite of programs [30,31]. The final obtained model was a hybrid
based on human Aurora A crystal structure (PDB ID 2bmc) [21] and
a mouse Aurora A X-ray crystal structure (PDB ID 3d14) [24] as
templates. The resulting hybrid model was refined by energy
minimization for 500 ps using an explicit solvent molecular
dynamics simulation with a YAMBER3 force field in YASARA [32].
The refinement consists of a short steepest descent minimization to
remove the largest intermolecular and intramolecular clashes, then
a second steepest descent minimization with all potential energy
terms, followed by a simulated annealing procedure. The quality of
the model was examined using PROCHECK [33] and MolProbity
[34] and was found to be of sufficiently good quality (see
Supplementary information).
153.6,139.6,139.6,130.0,129.9,129.2,129.1,129.1,127.5,114.2, 57.4, 57.1,
54.6, 46.9, 45.6, 32.6, 32.4, 22.2, 22.1. LCMS found 487.01, [M þ H]þ.
4.1.5.6. 4-(4-Methylpiperazin-1-yl)-N-(5-(2-(p-tolyl)propanoyl)-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)benzamide (13). (Yield:
68%). 1H NMR (400 MHz, d6-DMSO)
d
10.62 (d, J ¼ 9.6 Hz, 1H),
7.89e7.85 (m, 2H), 7.21e7.17 (m, 2H), 7.13e7.10 (m, 2H), 7.70e6.97
(m, 2H), 4.82e4.77 (m, 1H), 4.48e3.88 (m, 4H), 3.40e3.30 (m,
4H), 2.61e2.55 (m, 4H), 2.32 (s, 3H), 2.23 (s, 3H), 1.30 (d, J ¼ 6.8 Hz,
3H). LCMS found 473.01, [M þ H]þ.
4.1.5.7. N-(5-(2-(3,5-Dimethylphenyl)acetyl)-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-4-(4-methylpiperazin-1-yl)
benzamide (14). (Yield: 75%). 1H NMR (400 MHz, d6-DMSO)
d 10.69
(s,1H), 7.88 (d, J ¼ 8.4 Hz, 2H), 6.98 (d, J ¼ 8.4 Hz, 2H), 6.87e6.84 (m,
3H), 4.72e4.43 (m, 4H), 3.62 (d, J ¼ 8.00 Hz, 2H), 3.32e3.29 (m, 4H),
2.53e2.48 (m, 4H), 2.27 (s, 3H), 2.23 (s, 6H). LCMS found 473.01,
[M þ H]þ.
4.1.5.8. N-(5-(2-(2,5-Dimethylphenyl)acetyl)-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-4-(4-methylpiperazin-1-yl)
4.2.3. Compound docking
benzamide (15). (Yield: 74%). 1H NMR (400 MHz, d6-DMSO)
d
10.70
The model TbAUK1 structures were prepared using the Maestro
9.1 protein preparation wizard (Schrodinger, LLC, 2010, New York,
NY) before docking, bond orders were assigned and the orientation
of hydroxyl groups, amide groups of the side chains of Asn and Gln,
and the charge state of histidine residues were optimized. A
restrained minimization of the protein structure was performed
using the default constraint of 0.3 Å RMSD and the OPLS 2001 force
field [35].
(s, 1H), 7.90 (d, J ¼ 8.8 Hz, 2H), 7.04e6.99 (m, 3H), 6.94e6.92 (m,
2H), 6.56 (s, 1H), 4.76e4.46 (m, 4H), 3.66 (d, J ¼ 4.0 Hz, 2H),
3.40e3.30 (m, 4H), 2.70e2.60 (m, 4H), 2.38 (s, 3H), 2.22 (s, 3H), 2.16
(s, 3H). LCMS found 473.01, [M þ H]þ.
4.1.5.9. 4-(4-Methylpiperazin-1-yl)-N-(5-(2-(2,3,5-trifluorophenyl)
acetyl)-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)benzamide

S.O. Ochiana et al. / European Journal of Medicinal Chemistry 62 (2013) 777e784
783
4.3. Biology
expression vector pHD496 containing an NH3-AU1 epitope tagged
TbAUK1. Logarithmically growing cultures (1 ꢅ108 cells total) were
4.3.1. Cell cultures [36]
treated with the proteasome inhibitor MG132 (16 mM) for 3 h to
The bloodstream form (BF) trypanosomes used in this study
include: T. b. rhodesiense YTat1.1, a variant derived from UGANDA/
60/TREU 164 (ETat 3) and kindly provided by C. Patton; T. b. brucei
AnTat1.1A, kindly provided by E. Pays; and T. b. brucei Lister 427
strain 90-13 from G. A. M. Cross. The YTat1.1 and AnTat1.1A BF cells
were culture adapted from rat blood in HMI-9 medium [36] with
10% FBS (Atlanta Biologicals) and 10% Serum PlusÔ (SAFC Biosci-
ences) at 37 ꢂC and 5% CO2. The 90-13 cells were grown in HMI-9
increase the cellular content of TbAUK1. The remaining steps were
all performed at 4 ꢂC. The cells were washed twice in PBS with
Dulbecco’s salts (Gibco), and then incubated for 15 min in 400 mL of
lysis buffer (50 mM Hepes, pH 7.4, 100 mM KCl, 25 mM NaF, 0.5%
NP-40, 1 mM Na3VO4, 1 mM DTT and 100 nM okadaic acid) con-
taining protease inhibitor cocktail (Sigma, P-8340). The lysate was
centrifuged at 10,000ꢅ g for 15 min. A 50% slurry of Sephadex G-25
beads was prepared in lysis buffer and 80
mL was added to the
medium supplemented with G418 (2.5
m
g mLꢁ1) and hygromycin
supernatant. After 2 h, the pre-cleared supernatant was collected
and combined with 50 L of a 50% slurry of AU1-conjugated beads.
B (5
m
g mLꢁ1). The human T lymphocytic cell line CRL-1582Ô
m
(MOLT-4, obtained from ATCC) was grown in RPMI 1640 medium
with 20% FBS, penicillin (100 units mLꢁ1) and streptomycin
(0.1 mg mLꢁ1) at 37 ꢂC and 5% CO2. Cell densities for trypanosomes
and mammalian cells were maintained in the range of
The beads had previously been washed once in 1ꢅ kinase buffer
(20 mM Hepes, 150 mM KCl, 5 mM NaF, 5 mM MgCl2, and 1 mM
DTT, pH 7.4). The beads were incubated overnight at 4 ꢂC, washed,
collected by centrifugation and suspended in 50
The beads were the source of kinase for the kinase reactions. Kinase
reactions were in 50 L volumes containing 10 L of beads, 25
L 2ꢅ
kinase buffer and 160 g of myelin basic protein (Sigma). Drugs
were added in 2 L DMSO and incubated for 5 min before the
reaction was initiated with 8 M ATP (4 Ci of [
32P]ATP). After
a 30 min incubation at 30 ꢂC, the reaction was stopped with the
addition of 50
mL of kinase buffer.
5 ꢅ 104e1 ꢅ 106 cells mLꢁ1
.
m
m
m
4.3.2. Cell viability screen
m
Inhibitor sets were dissolved in DMSO and stored at ꢁ80 ꢂC as
m
a 500x dilution series. The final concentrations ranged from 50 nM
m
m
g-
to 8 mM, and DMSO was constant at 0.2%. Each drug was tested at
a minimum of 8 concentrations in triplicate. Log phase BF
trypanosomes were counted with a hemocytometer and seeded at
an initial density of 1 ꢅ105 cells mLꢁ1 in a volume of 1.5 mL in a 24
well plate format. The cultures were incubated with the inhibitor
sets for 4-doubling times (48 h). At the end of this time, trypano-
m
L 2ꢅ Laemmli buffer and boiled. The products were
separated by SDS-PAGE and gels were exposed to X-ray film for
24 h.
Acknowledgments
somes were concentrated by centrifugation, suspended in 100 mL of
medium and transferred to 96-well flat bottom microtiter plates
(Costar UV transparent plates). The end-point Cell Titer BlueÒ (CTB)
assay was used to test for cell viability (Promega). CTB reagent
Funding from the National Institutes of Health (R01AI082577,
R15AI082515), the National Science Foundation (MCB-0843603),
the Gerald J. Ford Research Fellowship, and Northeastern University
is gratefully acknowledged. The funders played no role in the
research study design. We appreciate free academic licenses to the
OpenEye suite of software and to Pipeline Pilot (Scitegic).
(20
mL) was added to each well and the plates were incubated at
37 ꢂC and 5% CO2 for 3 h. Fluorescence was measured at 560Ex
/
590Em using the SPECTRAmax GEMINI XPS Microplate Spectroflu-
orometer (Molecular Devices). Background was calculated from
wells containing CTB and media without cells, while 100% growth
was calculated from cells with DMSO and without drugs. The
background fluorescence was subtracted from all the data points.
The EC50 values were computed from the inhibition curves with
OriginPro 8.5. The CTB end-point assay measures non-selective
dehydrogenases, where the total activity is proportional to cell
number. Because Aurora kinase inhibitors tend to be trypanostatic
rather than trypanolytic [18], and because the non-dividing
cells continue to grow in size and cell activity, the assay tends
to overestimate values for EC50 (i.e. it underestimates the potency
of the drugs). The CTB end-point assay was linear out to 180 min
and at trypanosome cell densities in the range of
Appendix A. Supplementary information
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2012.07.
038. These data include MOL files and InChiKeys of the most
important compounds described in this article. Biological activity
and structural data is also provided as a shared public data set at
www.collaborativedrug.com.
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