Optimization of physicochemical properties for 4-anilinoquinazoline inhibitors of trypanosome proliferation

Article abstract of DOI:10.1016/j.ejmech.2017.10.007

Human African trypanosomiasis (HAT) is a deadly disease in need of new chemotherapeutics that can cross into the central nervous system. We previously reported the discovery of 2 (NEU-617), a small molecule with activity against T. brucei bloodstream prol

Full text of DOI:10.1016/j.ejmech.2017.10.007

Accepted Manuscript
Optimization of physicochemical properties for 4-anilinoquinazoline inhibitors of
trypanosome proliferation
Jennifer L. Woodring, Kelly A. Bachovchin, Kimberly G. Brady, Mitchell F. Gallerstein,
Jessey Erath, Scott Tanghe, Susan E. Leed, Ana Rodriguez, Kojo Mensa-Wilmot,
Richard J. Sciotti, Michael P. Pollastri
PII:
S0223-5234(17)30799-7
10.1016/j.ejmech.2017.10.007
EJMECH 9799
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 April 2017
Revised Date: 1 October 2017
Accepted Date: 3 October 2017
Please cite this article as: J.L. Woodring, K.A. Bachovchin, K.G. Brady, M.F. Gallerstein, J. Erath,
S. Tanghe, S.E. Leed, A. Rodriguez, K. Mensa-Wilmot, R.J. Sciotti, M.P. Pollastri, Optimization of
physicochemical properties for 4-anilinoquinazoline inhibitors of trypanosome proliferation, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.10.007.
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ACCEPTED MANUSCRIPT
TABLE OF CONTENTS GRAPHIC

ACCEPTED MANUSCRIPT
Optimization of Physicochemical Properties for 4-Anilinoquinazoline
Inhibitors of Trypanosome Proliferation
a
a
a
a
Jennifer L. Woodring, Kelly A. Bachovchin, Kimberly G. Brady, Mitchell F. Gallerstein,
b
b
c
b,e
d
Jessey Erath, Scott Tanghe, Susan E. Leed, Ana Rodriguez, Kojo Mensa-Wilmot, Richard
c
a
J. Sciotti, and Michael P. Pollastri.*
a
Northeastern University Department of Chemistry & Chemical Biology, 360 Huntington Avenue,
Boston, MA 02115 USA. Tel: 617-373-2703; E-mail: m.pollastri@neu.edu
b
New York University School of Medicine, Department of Microbiology, Division of Parasitology,
th
3
41 E. 25 St. New York, NY 10016 USA
c
Experimental Therapeutics, Walter Reed Army Institute for Research,2460 Linden Lane, Silver
Spring, MD, 20910
d
University of Georgia Department of Cellular Biology, Athens, GA USA
e
Anti-Infectives Screening Core, New York University School of Medicine, New York, NY 10010
Abstract
Human African trypanosomiasis (HAT) is a deadly disease in need of new
chemotherapeutics that can cross into the central nervous system. We previously reported the
discovery of 2 (NEU-617), a small molecule with activity against T. brucei bloodstream
proliferation. Further optimization of 2 to improve the physicochemical properties (LogP,
LLE,[1] and MPO score)[2] have led us to twelve sub-micromolar compounds, most importantly
1

ACCEPTED MANUSCRIPT
the headgroup variants 9i and 9j, and the linker variant 18. Although these 3 compounds had
reduced potency compared to 2, they all had improved LogP, LLE and MPO scores. Cross-
screening these analogs against other protozoan parasites uncovered 9o with potent activity
towards T. brucei, T. cruzi and L. major, while four others compounds (17, 18, 21, 26) showed
activity towards P. falciparum D6. This reinforces the effectiveness of lead repurposing for the
discovery of new protozoan disease therapeutics.
Keywords
Neglected tropical disease, human African trypanosomiasis, target class repurposing,
Trypanosoma brucei, Leishmania major, Trypanosoma cruzi, Plasmodium falciparum.
1
. Introduction
Human African trypanosomiasis (HAT) is a neglected tropical disease endemic in 36
countries of sub-Saharan Africa. The parasitic causative agent, Trypanosoma brucei, puts 65
million people at risk and is fatal if not treated. Current treatments for HAT are limited, requiring
administration by intramuscular or intravenous injection. Clinics needed to treat patients are not
easily accessible in the rural regions of Africa where HAT is endemic [3]. In addition, vaccines
are not available to prevent infection. As a result, new drugs are needed to control and eradicate
this disease.
Lead repurposing is the method of utilizing, as a starting point, well-established classes
of small molecules known for their efficacy against human targets with homologous function in
parasites, and re-optimizing the compounds for desired properties and anti-parasitic activity [4].
2

ACCEPTED MANUSCRIPT
Our previous lead repurposing work has primarily focused on inhibitors of phosphodiesterases[5-
1
0] and protein kinases [11-16].
We previously reported that the human tyrosine kinase inhibitor lapatinib (1), shown in
Figure 1, prevents proliferation of bloodstream T. brucei with an EC50 of 1.54 µM [17, 18].
Further optimization studies, especially the “tail” region (in red), led to the discovery of 2 (NEU-
6
17), with a potency of 0.042 µM and improved parasite selectivity over 1 [12].
Figure 1 – Discovery of 2, and paths towards the optimization of physicochemical properties
.
Headgroup Region
O
O
F
Tail Region
N
F
MeO S
2
NH
HN
N
Cl
HN
N
Cl
O
N
N
O
Lapatinib (1)
NEU-617 (2)
T. brucei EC50: 1.54 µM
HepG2 TC50: 6.27 µM
T. brucei EC50: 0.042 µM
HepG2 TC50: >15 µM
Despite its excellent potency and selectivity, the physicochemical properties of 2 need
improvement to convert it into a lead drug (i.e., capable of reducing parasitemia 100-fold in a
mouse model of HAT) [18-20]. For example, 2 is highly lipophilic (LogP = 7.3), has high
molecular weight (541), and has low lipophilic ligand efficiency (LLE = 0.1) [1]. Entering the
central nervous system (CNS) is a requirement for treating stage 2 HAT, as trypanosomes have
crossed the blood-brain barrier. A multiple parameter optimization (MPO) score incorporates
several key physicochemical properties (molecular weight, LogP, hydrogen bond donors, polar
surface area, and pKa) in gauging the possibility of drugs crossing the blood-brain barrier. A
higher MPO score ranging from 4.0-6.0 suggests a compound is more likely to be brain penetrant
3

ACCEPTED MANUSCRIPT
[2]. With an MPO score of 2.8, compound 2 is predicted to be a poor CNS drug candidate, and
this was further confirmed by mouse pharmacokinetics experiments that were previously
reported [21]. To resolve the issues that we identified in 2, optimization efforts focused
specifically on the “headgroup” region (in blue in Figure 1) and the “tail” region (in red in
Figure 1).
2
. Results & Discussion
In the same report for the discovery of 2, we observed an enhanced potency by truncating
the headgroup of lapatinib-related analogs. For example, the truncated analog 4 (in blue in
Figure 2) had a 1.7-fold increase in potency and improved MPO and cLogP values over the
lapatinib-like headgroup in compound 3 [12]. Furthermore, the analysis of 5 (NEU-964) shows
the removal of the sulfonyl group, in line with the tail of 2 (in red in Figure 2), was more potent
than its predecessor 4, albeit not as potent as 2. This led to the hypothesis that a reduction in size
of the headgroup on 2, by replacing the aniline moiety with aminoheterocycles, could provide
potent analogs that inhibit trypanosome proliferation while potentially improving MPO scores
and LLE values.
Figure 2 – Progression of headgroup truncation as a means of improving potency and physicochemical properties
4

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To achieve our goals, we enumerated a virtual library (VL) in silico using 247
commercially available aromatic amines. We then prioritized the compounds by performing
shape and electrostatics similarity comparison to the headgroup of 2 using ROCS and EON
(
OpenEye Scientific Software) and Kpµµ predictive blood-brain barrier partitioning (predictions
provided by AstraZeneca) [22]. After removal of products with an ET_Combo score <1
meaning lack of shape/electrostatic similarity and undesirable Kpµµ), 121 remaining
(
compounds were filtered by limiting cLogP <3 and TPSA between 60-130 Å. This provided an
initial set of nine prioritized compounds for synthesis (see Table S.1 for library parameters). In
addition to the nine compounds, we also prepared analogs that walked the nitrogen atom(s)
around the heterocyclic ring to include all positional isomers of the pyridines, pyrimidines,
pyridazines, and pyrazines. We noted that the nine compounds from the original VL contained
only six-membered heteroaromatic head groups. Therefore, to round out the set, we added five-
membered heteroaromatic rings (pyrazoles, oxazoles, thiazoles, thiazines) and their
commercially available isomers. This established a complete library comprised of 26 analogs.
Synthesis of these new compounds proved surprisingly challenging when employing the
sequence we reported for preparation of 2 [12]. A new approach was therefore established as
shown in Scheme 1. Optimized Suzuki chemistry resulted in improved results with employment
5

ACCEPTED MANUSCRIPT
2 3 4
of Pd(dppf)Cl -DCM, rather than Pd(PPh ) to afford the desired intermediate 7 in moderate
yields. To prepare chloroquinazoline 8, we applied modified Appel conditions [23] which proved
more reliable in this case than thionyl chloride as previously reported. In lieu of standard SNAR
conditions (which showed irreproducible results) we utilized a modified Buchwald-Hartwig
coupling for aryl chlorides [24-26] for the installation of the headgroup to obtain compounds 9a-
x. Production of analogs utilizing nucleotide derivatives like cytosine required another set of
2 3
reaction conditions, utilizing K CO and acetone with microwave irradiation to produce 10a-b.
Scheme 1. Preparation of headgroup replacement analogs 9 and 10.
O
O
Cl
N
I
NH
(a)
(b)
N
NH
N
N
N
O
O
N
6
7
8
Ar
N
HN
N
N
(c)
O
Cl
9a-x
N
N
O
N
O
X
8
N
N
NH
(d)
HN
N
N
O
10a-b
X = H or F
Reagents and Conditions: (a) 4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)morpholine, 2M Na CO in
2
3
o
o
H O, Pd(dppf)Cl -DCM, DME/EtOH (3:2), 85 C, 6 h, 66%; (b) PPh , CCl , DCE, N , 70 C, 12 h, 97%; (c) Ar-NH ,
2
2
3
4
2
2
o
o
K CO , xantphos, Pd(OAc) , 1,4-dioxane, N , 105 C, 12 h, 5-78%; (d) Ar-NH , K CO , acetone, N , MW, 120 C, 1
2
3
2
2
2
2
3
2
h, 5-23%
6

ACCEPTED MANUSCRIPT
The biological data for the resulting headgroup replacement library is presented in Table
. Of the 26 compounds tested, nine had sub-micromolar potency against T. brucei. The most
1
potent were pyridine (9i, EC = 0.29 µM) and pyrimidine-substituted products (9j, EC = 0.32
50
50
µM). The isomers of the pyridines (9h, i, q) have activities within a ten-fold window ranging
from 0.29 µM to 2.1 µM. The pyrimidine isomers (9b, j, l) have a wider potency range from 0.32
µM to 7.0 µM. There seems to be no significant importance or pattern to the nitrogen atom
positioning, based on comparison of the pyridyl and pyrimidinyl data. Interestingly, methylation
of the 1,3-amino pyrimidine resulted in a three-fold loss of potency (9k, 1.0 µM) when compared
to 9j, and little difference was observed between the free amine 9t and the corresponding
acetamide 9r.
In addition, the thiazoles (9u, v, x), thiazine (9w), and oxazoles (9o-p) also showed
roughly equivalent potency with each other, within ~two-fold range. Overall, the triazines (9c,
d), pyrazole (9a), pyridazines (9e, f, m), pyrazines (9g, s), and cytosine derivatives (10a-b) were
not highly active against T. brucei. However, we note that the fluorinated cytosine (10b) showed
a significant (>50-fold) increase in potency compared to 10a.
Despite none of these compounds having improved potency over 2, and both pyridine 9i
and pyrimidine 9j were the only compounds with improved potency over the truncated 5 (NEU-
9
64), every compound had a significant improvement in lipophilicity (between 2-5 orders of
magnitude) and MPO score (between 1.5-3 orders of magnitude). Figure 3 clearly shows these
6 analogs (in red) had significantly improved physicochemical properties, with higher MPO
2
scores and lower LogP values, over the compounds that were simultaneously reported in the
SAR and discovery of 2 (orange) [12]. Accordingly, we observed an improvement in LLE from
7

ACCEPTED MANUSCRIPT
0
.1 (for 2) to as high as 3.2 (for 10b). In fact, the only compound to not have an improved LLE
from this library was compound 10a due to its inactivity in the assay.
Figure 3 – Representation of the improvements in physicochemical properties for the compounds in the headgroup
library (red) and linker/tail library (blue) over the compounds used in the SAR of 2 (orange)
8

ACCEPTED MANUSCRIPT
Table 1 – Biological screening data for the headgroup replacement library
T. brucei
T. brucei
Entry
R
LogP
MPO Score
a
EC (µM)
LLE
5
0
b
2
7.3
5.4
3.9
3.7
2.8
3.0
3.9
2.8
3.2
3.8
2.8
2.2
3.1
3.5
5.4
4.9
3.2
4.3
4.0
3.4
0.042
0.53
19.7
7.0
0.1
0.8
0.8
1.5
2.4
1.9
1.2
2.6
1.7
2.2
NEU-617
5
NEU-964
a
9
NEU-1939
b
9
NEU-1940
c
9
6.3
NEU-1942
d
9
12.6
7.1
NEU-1943
e
9
NEU-1944
f
9
3.8
NEU-1945
g
9
12.8
0.95
NEU-1946
h
9
NEU-1959
9

ACCEPTED MANUSCRIPT
T. brucei
T. brucei
Entry
R
LogP
MPO Score
a
EC50 (µM)
LLE
9
i
3.8
3.8
3.9
3.1
3.4
3.5
3.6
3.6
4.4
3.6
3.9
3.6
4.1
3.8
3.3
4.8
4.3
3.0
2.7
0.29
0.32
1.0
2.8
2.7
2.1
2.9
2.5
2.4
2.5
2.4
1.3
2.5
NEU-1960
9
j
NEU-1961
9
k
NEU-1963
9
l
0.97
1.4
NEU-1964
9
m
NEU-1965
9
n
1.2
NEU-1966
9
o
0.79
1.2
NEU-1967
9
p
NEU-1968
9
q
2.1
NEU-2071
3
.6
0.77
9
r
NEU-2072
9
s
3.6
3.5
3.3
3.2
1.7
1.2
2.2
2.4
NEU-2073
9
t
NEU-2075
1
0

ACCEPTED MANUSCRIPT
T. brucei
T. brucei
Entry
R
LogP
MPO Score
a
EC50 (µM)
LLE
9
u
5.0
4.3
3.5
3.8
2.5
2.7
3.4
5.3
3.3
3.9
1.1
1.0
1.5
2.6
2.4
NEU-2079
9
v
1.4
NEU-2080
9
w
0.94
0.64
>50
NEU-2081
9
x
NEU-2082
0a
c
1
nd
NEU-1941
0b
NEU-2074
2
.7
4.5
1.2
3.2
1
a
Compounds showing >75% growth inhibition at 5 µM for T.b. brucei were tested for EC50 values. T.b. brucei EC50
b
c
values are the result of duplicate experiments, within ± 24%. Previously reported data.[12] nd=not calculated due
to inactivity towards T.b. brucei
We also conducted an exploration on the tail region of 2 (in red, Figure 1). Since
previous work detailed by Patel et al. focused primarily on potency and the SAR of the
morpholine ring [12], we focused on exchanging the phenyl ring for bioisosteres to lower the
LogP. Specifically, the phenyl linker in 2 was replaced with pyrimidines, triazoles, and an
acyclic amide linker. We avoided the common thiophene bioisostere due to a lack of
improvement in LogP when compared to 2.
1
1

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Synthesis for these new compounds expands on the previously synthesized intermediate
1
1 [12], shown in Scheme 2. The boronic ester 12 was synthesized from a Miyaura borylation
reaction with the aryl iodide, followed by a Chan-Lam coupling [27, 28] to obtain the azide 13.
Both reactions proceeded in good yields. These three intermediates (11, 12, 13) were used to
synthesize the 5 analogs in the phenyl replacement library. Scheme 3 illustrates the synthetic
route to the pyrimidine-linked analogs 17 and 18, which were synthesized starting with
commercially available 2,4-dichloropyrimidine 14. Chloride displacement with morpholine
afforded the two isomers 15 and 16 in a 1 to 13 ratio, which were readily separated by column
chromatography. Suzuki coupling of the boronic ester 12 provided the two final isomers 17 and
1
8.
The amide-linked compound 21 was synthesized from commercially available 2-chloro
acetamide 19, shown in Scheme 4. As previously reported [29], the acetamide underwent in situ
halogen exchange and subsequent displacement with morpholine to afford intermediate 20. This
intermediate reacted with the aryl iodide 11 in a copper-catalyzed Buchwald amidation reaction
to produce the final compound 21.
Lastly, the triazole linkers were synthesized through methods shown in Schemes 5 and 6.
In Scheme 5, the 1,2,3-triazole 23 was synthesized from a Huisgen cyclization reaction with
intermediate 13 and the respective alkyne 22 adapted from Amegadzie et al. [30]. Use of the
ruthenium catalyst selectively promotes formation of the 1,5 product whereas a copper catalyst
promotes 1,4 products or a mixture of the two isomers [31]. Finally, in Scheme 6 the
commercially available dibromo-1,2,4-triazole 24 is mono-substituted with morpholine to
synthesize intermediate 25, which then undergoes a Suzuki coupling with the aryl boronic ester
1
2 to afford the final product 26.
1
2

ACCEPTED MANUSCRIPT
Scheme 2. Synthesis of azide intermediate 13.
O
O
O
F
F
F
HN
Cl
HN
N
Cl
HN
N
Cl
I
(a)
Bpin
(b)
N
3
N
N
N
N
11
12
13
o
Reagents and conditions: (a) B
2
pin
2
, KOAc, Pd(dppf)Cl
2
-DCM, DMF, 85 C, 12 h, 82%; (b) NaN
3
2
, Cu(OAc) ,
o
MeOH, 55 C, 12 h, 88%.
Scheme 3. Synthesis of analogs 17 and 18.
O
F
Cl
N
Cl
Cl
(b)
X
Y
HN
N
Cl
(a)
N
N
N
N
N
N
N
N
N
O
N
Cl
O
14
O
1
1
7: X=C, Y=N, 9%
8: X=N, Y=C, 28%
1
5
16
7%
92%
o
Reagents and conditions: (a) morpholine, DIPEA, iPrOH, 0 C to RT, 12 h; (b) 12, 2M Na CO in H O, Pd(PPh ) ,
2
3
2
3 4
o
DME/EtOH (3:2), 85 C, 12 h.
Scheme 4. Synthesis of 21.
O
F
HN
N
Cl
O
H
(a)
O
O
(b)
N
Cl
NH2
N
N
N
NH2
O
O
1
9
2
0
21
o
Reagents and conditions: (a) morpholine, K
, RT, 18 h, 37%.
2
CO
3
, NaI, EtOH, 85 C, 6 h, 91%; (b) 11, Cs
2
3
CO , DMEDA, CuI, THF,
N
2
1
3

ACCEPTED MANUSCRIPT
Scheme 5. Preparation of analog 23.
O
Cl
F
N
N
N
HN
N
N
O
(a)
N
N
O
23
2
2
o
Reagents and conditions: (a) 13, Cp*RuCl (PPh ) , 1,4-dioxane, N , MW, 150 C, 30 min, 13%.
2
3 2
2
Scheme 6. Preparation of compound 23.
o
2 3 2
Reagents and conditions: (a) morpholine, pyridine, MW, 210 C, 10 min, 52%; (b) 12, 2M Na CO in H O,
o
Pd(PPh
3
)
4
, DME/EtOH (3:2), 85 C, 12 h, 5%.
Biological screening data for the five phenyl replacement analogs are shown in Table 2.
The most potent analog of the series is the pyrimidine compound 18 with an EC of 0.41 µM, a
5
0
1
0-fold drop in potency compared to 2. Interestingly, the 2-morpholine substituted isomer was
active while the 4-morpholine (17) was less-so (~3.5-fold). The amide linked analog (21) was 8-
fold less active than 18 possibly due to lack of structural rigidity provided by the aromatic
linkers. Both the 1,2,3-triazole 23 and the 1,2,4-triazole 26 had sub-micromolar potencies with
EC50 values of 0.61 and 0.78 µM, respectively. As shown in Figure 3, these linker replacement
1
4

ACCEPTED MANUSCRIPT
compounds (in blue) had LogP values about 1-3 units lower and relatively unchanged MPO
scores when compared to 2 and its related SAR analogs (in orange). This translated to only a
modest improvement in LLE (values of 0.8 and 1.1 for the amide/1,2,4-triazole and 1,2,3-
triazole, respectively, compared to 0.1 for compound 2).
1
5

ACCEPTED MANUSCRIPT
Table 2 – Biological assay results of the linker replacement library.
T. brucei
T. brucei
Entry
R
LogP
MPO Score
a
EC (µM)
LLE
5
0
7
6
6
.3
.5
.5
2.8
0.042
1.4
0.1
b
2
1.8
1.8
-0.6
-0.1
1
7
NEU-1008
0.41
1
8
NEU-1011
2
1
4.7
1.7
1.8
3.1
0.8
1.1
NEU-1009
5
5
.1
.3
0.61
2
3
NEU-2083
1.1
0.78
0.8
2
6
NEU-2078
a
Compounds showing >75% growth inhibition at 5 µM for T.b. brucei were tested for EC50 values. T.b. brucei EC50
b
values are the result of duplicate experiments, within ± 24%. Previously reported data.[12]
1
6

ACCEPTED MANUSCRIPT
With promising head and tail replacements in hand, we prepared the cross-over analogs
3
3a-d, which consisted of the most potent pyrimidine linker/tail replacement of pyrimidine 18
and the 2-aminopyrimidine (9j) or 4-aminopyridine (9i) from the improved headgroup
replacement library. A redesigned synthetic route was utilized (Scheme 7), as the sequence
described for previous analogs proved to be low-yielding in these cases. Mono-bromide
displacement on 2,4-dibromopyrimidine with morpholine provided isomers 28a-b that were
chromatographically separable. Preparation of the boronate ester 30 was accomplished using
Miyuara borylation conditions, followed by a Suzuki coupling with the pyrimidine regioisomers
2
8a and 28b to provide intermediates 31a-b. Condensation with formamide afforded the
aminoquinazoline core (32a-b), which was arylated under Buchwald-Hartwig conditions to
obtain the desired compounds 33a-d [32, 33].
Scheme 7. Synthesis of headgroup/linker crossover analogs 33.
1
7

ACCEPTED MANUSCRIPT
Reagents and conditions: (a) morpholine, K CO , THF, RT, 24 h; (b) B pin , KOAc, Pd(dppf)Cl2-DCM, 1,4-
2
3
2
2
dioxane, MW, 130°C, 1 h, 88%; (c) 31a or 31b, Cs CO , Pd(PPh ) , 1,4-dioxane/H O (3:1), MW, 130°C, 10 min;
2
3
3 4
2
(
d) formamide, MW, 200°C, 1 h; (e) 2-chloropyrimidine or 4-chloropyridine, Pd
70°C, 1-2 h, 30-67%.
2
3
(dba) , DPPF, 1,4-dioxane, MW,
1
Screening results for the cross-over compounds 33a-d are shown in Table 3. While all
compounds showed at least 2-fold improved computed properties (lower LogP and higher MPO
scores) over 2, only 33d showed activity against T. brucei cultures at 5.7 µM (LLE=2.2). This
suggests that while we were able to observe compounds with active tail replacements
(maintaining the large headgroup of 2) and active headgroup replacements (retaining the original
tail of 2), these active compounds could potentially be mediating their effects via different
targets, such that combining these structural changes abrogates some activity. Target validation
efforts are underway to further explain this result.
1
8

ACCEPTED MANUSCRIPT
Table 3 – Biological assay data for crossover compounds 33.
R
N
X
Y
HN
N
N
Z
O
MPO
Score
1.8
T.brucei
T. brucei
Entry
X
Y
Z
R
LogP
EC (µM) LLE
5
0
C
C
C
7.3
0.042
0.1
O
2
F
Cl
c
3
3a
NEU-4436
3b
NEU-4444
3c
NEU-4446
3d
N
N
C
C
C
C
N
N
N
N
N
N
2.9
2.9
2.9
3.0
4.3
4.4
4.3
4.4
>10.0
>10.0
>10.0
5.70
nd
c
3
nd
c
3
nd
3
2.2
NEU-4445
a
Compounds showing >75% growth inhibition at 5 µM for T.b. brucei were tested for EC50 values. T.b. brucei EC50
b
c
values are the result of duplicate experiments, within ± 25%. Previously reported data.[12] nd=not calculated due
to inactivity towards T.b. brucei
1
9

ACCEPTED MANUSCRIPT
We have previously reported the value of screening our libraries against multiple
kinetoplastid and protozoan parasites [14, 34]. Most importantly, our cross-screening campaign
of lapatinib-derived analogs has produced multiple active compounds against one or more
parasites in a single library [15]. We therefore tested our new analogs against the causative
agents for Chagas disease (Trypanosoma cruzi), leishmaniasis (Leishmania major), and malaria
(Plasmodium falciparum). The results are displayed in Table 4.
Of the 35 new compounds, the oxazole analog 9o was the only analog found to be active
against T. cruzi and L. major amastigotes with EC50 values of 0.060, and 0.67 µM, respectively;
further studies of this compound against these pathogens are underway and will be reported in
due course. Interestingly, other researchers have also denoted the ability of oxazole compounds
to show activity against related kinetoplastids T. brucei, T. cruzi, and leishmaniasis [35]. Nearly
all of the linker replacements showed activity towards P. falciparum D6 with the pyrimidines 17
and 18 (EC50 = 0.93 and 0.069 µM, respectively), amide 21 (0.53 µM), and 1,2,4-triazole 26
(0.21 µM) being the most potent analogs from all the compounds screened against P. falciparum
D6. As seen with T. brucei, the 2-morpholine substituted pyrimidine 18 was 13.5-fold more
active than its isomer 17 against P. falciparum. The cross-over analog 33b with pyrimidines in
both the linker (same stereochemistry as 18) and headgroup replacements had an EC of 0.31
50
µM against P. falciparum D6. Only 9c showed meaningful toxicity to the HepG2 cell line with a
TC50 of 4 µM. Additional screening data, such as that for L. major promastigotes and P.
falciparum strains W2 and C235, can be found in the Supporting Information (Tables S.2 and
S.3)
2
0

ACCEPTED MANUSCRIPT
Table 4 – Cross-screening data for all compounds.
L. major
P. falciparum
T.b. brucei T. cruzi
Amastigotes D6
NIH 3T3 HepG2
a
a
b
c
Entry EC50 (µM) EC50 (µM) EC50 (µM)
EC50 (µM)
0.23
1.87
2.87
3.21
>15
TC50 (µM)
>50
TC50 (µM)
d
2
5
9
9
9
9
9
9
9
9
9
9
9
9
0.042
0.53
19.7
7.0
1.8
7.98
>15
>15
>15
>15
>15
>15
8.78
>15
>15
>15
>15
>15
>15
>30
>30
>30
>30
4.0
>50
>50
10.6
8.9
>100
>100
>50
a
b
c
d
e
f
6.3
39
12.6
7.1
27.2
13.9
>50
>50
31.5
3.4
2.60
5.59
1.01
6.62
>15
>50
>20
>30
>30
>30
>30
>30
>30
>30
>30
>50
3.8
>100
>100
38
g
h
i
12.8
0.95
0.29
0.32
1.0
>15
>100
>100
>100
22
j
32.2
23.9
22.5
>15
k
l
>15
0.97
14.17
2
1

ACCEPTED MANUSCRIPT
L. major
P. falciparum
T.b. brucei T. cruzi
Amastigotes D6
NIH 3T3 HepG2
a
a
b
c
Entry EC50 (µM) EC50 (µM) EC50 (µM)
EC50 (µM)
10.56
5.71
TC50 (µM)
>50
TC50 (µM)
9
9
9
9
9
9
9
9
9
9
9
9
1
1
1
1
m
n
o
1.4
27.0
6.5
>15
>30
>30
22
1.2
>15
>50
0.79
1.2
0.06
2.7
0.67
12.50
5.22
22.70
24.13
25.09
4.96
6.60
25.61
25.68
>15
5.16
>100
12
p
q
r
13.45
5.22
>30
>30
>30
>20
>30
13.4
14.6
>30
>30
>30
>30
>20
>20
d
2.1
>20
>50
11.0
13.9
5.1
nd
d
0.77
1.7
22.70
>15
nd
d
s
nd
d
t
1.2
8.16
nd
u
v
1.1
4.17
13
1.4
2.2
>15
8.1
d
w
x
0.94
0.64
>50
1.2
14.7
>50
>50
>20
4.8
>15
nd
d
7.56
nd
0a
0b
7
>15
>100
d
23.90
>3
>20
nd
1.4
0.93
36
47
8
0.41
24.4
>15
0.069
2
2

ACCEPTED MANUSCRIPT
L. major
P. falciparum
T.b. brucei T. cruzi
Amastigotes D6
NIH 3T3 HepG2
a
a
b
c
Entry EC50 (µM) EC50 (µM) EC50 (µM)
EC50 (µM)
0.53
TC50 (µM)
9.1
TC50 (µM)
2
2
2
3
3
3
3
1
3.1
6.9
8.11
>20
>20
>20
>30
>30
14.8
>30
3
0.61
0.78
>10.0
>10.0
>10.0
5.7
>15
7.6
6.34
6.05
>50
6
1.88
0.21
>50
3a
3b
3c
3d
15
25.88
25.95
25.88
25.95
25.88
0.31
>50
>50
>50
0.37
>50
11.12
1.04
>50
>50
a
Compounds showing >75% growth inhibition at 5 or 10 µM for T.b. brucei or T. cruzi respectively, were tested for EC50 values. T.b. brucei EC50 values are the
result of duplicate experiments, within ± 24%. T. cruzi EC50 values are the result of duplicate experiments, within ± 24%, with the exception of 2 (± 50%) and 6p
b
2
c
2
(
± 100%). Compounds screened against L. major amastigotes had r values >0.75. Compounds tested in duplicate against P. falciparum (D6 strain) had r
d
values >0.80. Previously reported data.[12, 15]
2
3

ACCEPTED MANUSCRIPT
Lastly, most compounds were also screened for their ADME properties (Supporting
Information Table S.4). A few representative compounds are shown below in Table 5. While
majority of the compounds that were screened had human plasma protein binding of 90-99%,
only 33a and 33c had values below 90%. All but 6 of the screened compounds had desired
human microsome clearance of <90 µL/min/mg, whereas only 10 out of the 27 compounds had
6
the desired rat hepatocytes clearance of <55 µL/min/10 cells. Despite almost all analogs having
a slightly improved aqueous solubility over 2, only 9a and 33c were significantly improved and
over the targeted value of 100 µM (288 and >1000 µM respectively). Even though all
compounds had reduced lipophilicity (LogP) values by design, this did not translate into
increased aqueous solubility across all of the analogs. Therefore, it can be concluded that
aqueous solubility is not dependent on LogP and these two factors do not correlate in this
chemical series.
Table 5 – ADME properties for selective compounds.
Entry
LogP
Aqueous
Solubility
Human
Plasma
Protein
Human
Microsome
Clint
Rat Hepatocytes
Clint 6
(µM)
(µL/min/10 cells)
Binding %
(µL/min/mg)
2
7.31
<1
99
63.0
44.5
99.5
47.2
25.4
NEU-617
9
a
3.87
3.78
3.75
288.0
2.9
93.9
98.8
93.8
60.9
80.2
33.3
NEU-1939
9i
NEU-1960
9
j
16.7
NEU-1961
2
4

ACCEPTED MANUSCRIPT
Entry
LogP
Aqueous
Solubility
Human
Plasma
Protein
Human
Microsome
Clint
Rat Hepatocytes
Clint 6
(µM)
(µL/min/10 cells)
Binding %
(µL/min/mg)
9
o
3.62
2.3
<1
99.0
50.9
46.9
NEU-1967
a
a
1
8
6.45
2.89
2.89
99.8
88
nd
nd
NEU-1011
33a
29.0
71.3
10.1
20.3
26.5
NEU-4436
33c
>1000
86
NEU-4446
a
Not determined (nd)
3
. Conclusions
We initially set out to improve the physicochemical properties of 2 by designing
compounds with improved LogP and MPO scores in order to achieve a better drug candidate
with higher LLE values. We specifically targeted the headgroup region of the molecule by
exploring truncated or small aminoheterocycles generated by in silico design, as well as
replacement of the tail region with other polar moieties. From these libraries, we discovered 12
compounds with sub-micromolar EC50 values against T. brucei, all of which had significantly
improved LogP and MPO scores over 2, and almost all had improved LLE values. Of those
compounds, the pyridine 9i (EC = 0.29 µM) and pyrimidines 9j (0.32 µM) and 18 (0.41 µM)
5
0
were utilized in the design of cross-over compounds, matching the best and most potent
components of each library, though despite their improved LogP and MPO scores, there was a
2
5

ACCEPTED MANUSCRIPT
significant reduction in potency. Throughout this series, aqueous solubility remains a significant
challenge to be surmounted, noting that the poor solubility does not correlate with LogP.
In addition to screening against T. brucei, all compounds were screened against related
protozoan parasites T. cruzi, L. major, and P. falciparum. Interestingly, analog 9o not only
showed sub-micromolar potency towards T. brucei (EC = 0.79 µM), but it was potent against
5
0
both T. cruzi and L. major amastigotes (EC50 = 0.06 and 0.67 µM, respectively). Plus 4 out 5
compounds (17, 18, 21, 26) in the tail optimization library and cross-over compound 33b
displayed sub-micromolar activity towards P. falciparum D6. We continue optimization efforts
in this chemical series against these four protozoan pathogens, with an eye towards improved
pharmacokinetics and in vivo efficacy.
4
. Materials and Methods
4
.1 Chemical synthesis.
Unless otherwise noted, reagents were obtained from Sigma-Aldrich, Inc. (St. Louis, MO) or
Fisher Scientific, and used as received. NMR spectra were obtained on a Varian NMR system,
operating at 400 MHz and 500 MHz for 1H acquisitions. Spectrum were analyzed using
ACD/Labs NMR processing software. GCMS analysis was performed using an Agilent HP 6890
gas chromatograph with an Agilent HP5973 transmission quadrupole mass spectrometer
(electron impact). LCMS analysis was performed using a Waters e2795 Alliance reverse-phase
HPLC-MS and 3.5 µm Waters SunFire C18 4.6x50mm or Waters SunFire 3.5 µm C8 4.6x50mm
columns, with single-wavelength Waters 2489 UV-visible detector (λ = 254 nm) or
multiwavelength Waters 2996 photodiode array detector (λ = 200-600 nm) and Waters
LCTPremier time-of-flight mass spectrometer (electrospray ionization) or MicroMass ZQ single
2
6

ACCEPTED MANUSCRIPT
quadropole mass spectrometer (electrospray ionization). Preparative HPLC was performed using
a Waters FractionLynx and 5 µm Waters Symmetry C8 30x50mm or 5 µm Waters Xbridge OBD
RP18 30x50mm columns, with single-wavelength Waters 2489 UV-visible detector (λ = 254
nm) and MicroMass ZQ mass spectrometer (electrospray ionization). Gradients for the LCMS
analysis and preparative HPLC were water or acetonitrile, both with 0.1% v/v formic acid. Purest
fractions from the preparative HPLC and Biotage Isolera One reverse phase chromatography
with Biotage SNAP C18 columns provided the percent yields reported and any impure fractions
were discarded. All newly synthesized compounds were deemed >95% pure by LCMS and NMR
analysis.
6
-(3-morpholinophenyl)quinazolin-4(3H)-one (7). 6-iodoquinazolin-4(3H)-one (0.672 g, 2.469
mmol), 4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)morpholine (1.785 g, 6.17
mmol), a 2 M aqueous solution of sodium carbonate (7.41 mL, 14.81 mmol), [1,1’-
bis(diphenylphoshpino)ferrocene] dichloropalladium(II) dichloromethane adduct (0.141 g, 0.173
o
mmol), dimethoxyethane (5 mL), and ethanol (3.3 mL) were added together and heated at 85 C
for 6 h. The reaction mixture was concentrated under reduced pressure, filtered through celite,
and the product was collected by silica column chromatography (dichloromethane/methanol).
The collected fractions were concentrated under reduced pressure. A mixture of 70%
hexanes/30% toluene was added and the precipitated product was collected by vacuum filtration
1
to obtain a 66% yield as a tan solid. H NMR (500 MHz, DMSO-d ) δ ppm 3.21 (m, 4 H), 3.76
6
(m, 4 H), 7.00 (dd, J = 8.3, 2.4 Hz, 1 H), 7.17 (d, J = 7.8 Hz, 1 H), 7.25 (m, 1 H), 7.36 (t, J = 7.8
Hz, 1 H), 7.74 (d, J = 8.3 Hz, 1 H), 8.12 (m, 2 H), 8.31 (d, J = 2.0 Hz, 1 H), 12.31 (br. s, 1 H).
+
LCMS found 308.1, [M+H] .
2
7

ACCEPTED MANUSCRIPT
4
-(3-(4-chloroquinazolin-6-yl)phenyl)morpholine (8). A flask with septum containing both 7
(0.350 g, 1.137 mmol) and triphenylphosphine (0.597 g, 2.274 mmol) was purged with nitrogen
three times. Anhydrous 1,2-dichloroethane (12 mL) was added and allowed to stir for 5 mins,
followed by the addition of carbon tetrachloride dropwise (0.329 mL, 3.410 mmol). The reaction
o
was stirred at 70 C for 12 h. The reaction mixture was concentrated under reduced pressure and
purified by silica column chromatography (hexanes/ethyl acetate) to obtain the product in 97%
1
yield as a yellow solid. H NMR (500 MHz, DMSO-d
6
) δ ppm 3.25 (m, 4 H), 3.79 (m, 4 H), 7.09
(d, J = 7.3 Hz, 1 H), 7.24 (d, J = 7.3 Hz, 1 H), 7.34 (s, 1 H), 7.39 (t, J = 7.8 Hz, 1 H), 7.79 (d, J =
8
.3 Hz, 1 H), 8.18 (dd, J = 8.5, 2.2 Hz, 1 H), 8.34 (d, J = 2.0 Hz, 1 H), 8.39 (s, 1 H). LCMS
+
found 326.1, [M+H] .
N-(2-aminopyridin-3-yl)acetamide [36]. To a solution of pyridine-2,3-diamine (0.207 g, 1.896
o
mmol) and pyridine (0.307 mL, 3.79 mmol) in anhydrous THF (7 mL) cooled at 0 C was added
o
acetic anhydride (0.197 mL, 2.086 mmol) dropwise. The reaction mixture stirred at 0 C for 30
mins and then at room temperature for 12 h. The product was purified by silica column
chromatography (ethyl acetate/methanol) to obtain the product in 61% yield as a light brown
1
solid. H NMR (500 MHz, DMSO-d
6
) δ ppm 2.05 (s, 3 H), 5.75 (s, 2 H), 6.54 (dd, J = 7.8, 4.9
Hz, 1 H), 7.59 (dd, J = 7.3, 1.5 Hz, 1 H), 7.76 (dd, J = 4.6, 1.7 Hz, 1 H), 9.11 (s, 1 H). LCMS
+
found 151.7, [M+H] .
Reaction Conditions for Buchwald Library. To a flame-dried flask was added 8 (0.046 g,
0
.142 mmol), xantphos (0.016 g, 0.028 mmol), potassium carbonate (0.392 g, 2.83 mmol),
palladium(II) acetate (0.003 g, 0.014 mmol), and the respective amine (0.170 mmol). The flask
was purged with nitrogen three times and then anhydrous dioxane (1.5 mL) was added under
o
nitrogen. The reaction was refluxed at 105 C for 12 h while under nitrogen, maintaining a
2
8