Novel 1,2-dihydroquinazolin-2-ones: Design, synthesis, and biological evaluation against Trypanosoma brucei

Article abstract of DOI:10.1016/j.bmcl.2017.07.032

In 2014, a published report of the high-throughput screen of?>42,000 kinase inhibitors from GlaxoSmithKline against T. brucei identified 797 potent and selective hits. From this rich data set, we selected NEU-0001101 (1) for hit-to-lead optimization. Through our preliminary compound synthesis and SAR studies, we have confirmed the previously reported activity of 1 in a T. brucei cell proliferation assay and have identified alternative groups to replace the pyridyl ring in 1. Pyrazole 24 achieves improvements in both potency and lipophilicity relative to 1, while also showing good in vitro metabolic stability. The SAR developed on 24 provides new directions for further optimization of this novel scaffold for anti-trypanosomal drug discovery.

Full text of DOI:10.1016/j.bmcl.2017.07.032

Accepted Manuscript
Novel 1,2-dihydroquinazolin-2-ones: Design, synthesis, and biological evalua-
tion against Trypanosoma brucei
ThanhTruc Pham, Madeline Walden, Christopher Butler, Rosario Diaz-
Gonzalez, Guiomar Pérez-Moreno, Gloria Ceballos-Pérez, Veronica Gomez-
Pérez, Raquel García-Hernández, Henry Zecca, Emma Krakoff, Brian Kopec,
Ogar Ichire, Caden Mackenzie, Marika Pitot, Luis Miguel Ruiz, Francisco
Gamarro, Dolores González-Pacanowska, Miguel Navarro, Amy B. Dounay
PII:
S0960-894X(17)30728-X
http://dx.doi.org/10.1016/j.bmcl.2017.07.032
BMCL 25145
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Accepted Date:
8 June 2017
10 July 2017
Please cite this article as: Pham, T., Walden, M., Butler, C., Diaz-Gonzalez, R., Pérez-Moreno, G., Ceballos-Pérez,
G., Gomez-Pérez, V., García-Hernández, R., Zecca, H., Krakoff, E., Kopec, B., Ichire, O., Mackenzie, C., Pitot, M.,
Ruiz, L.M., Gamarro, F., González-Pacanowska, D., Navarro, M., Dounay, A.B., Novel 1,2-dihydroquinazolin-2-
ones: Design, synthesis, and biological evaluation against Trypanosoma brucei, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.07.032
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Novel 1,2-dihydroquinazolin-2-ones: Design, synthesis, and biological
evaluation against Trypanosoma brucei
ThanhTruc Pham,a Madeline Walden,a Christopher Butler,a Rosario Diaz-Gonzalez,b Guiomar
Pérez-Moreno, b Gloria Ceballos-Pérez,b Veronica Gomez-Pérez b, Raquel García-Hernández b,
Henry Zecca,a Emma Krakoff,a Brian Kopec,a Ogar Ichire,a Caden Mackenzie,a Marika Pitot,a
Luis Miguel Ruiz,b Francisco Gamarro,b Dolores González-Pacanowska,b Miguel Navarro,b Amy
B. Dounaya*
aColorado College, Department of Chemistry and Biochemistry, 14 E. Cache La Poudre,
Colorado Springs, CO 80903
bInstituto de Parasitología y Biomedicina "López-Neyra" Consejo Superior de
Investigaciones Científicas (CSIC), Granada 18100 Spain
Abstract
In 2014, a published report of the high-throughput screen of >42,000 kinase inhibitors from
GlaxoSmithKline against T. brucei identified 797 potent and selective hits. From this rich data
set, we selected NEU-0001101 (1) for hit-to-lead optimization. Through our preliminary
compound synthesis and SAR studies, we have confirmed the previously reported activity of 1
in a T. brucei cell proliferation assay and have identified alternative groups to replace the pyridyl
ring in 1. Pyrazole 24 achieves improvements in both potency and lipophilicity relative to 1,
while also showing good in vitro metabolic stability. The SAR developed on 24 provides new
directions for further optimization of this novel scaffold for anti-trypanosomal drug discovery.
African sleeping sickness, also known as human African trypanosomiasis (HAT),
threatens millions of people in sub-Saharan Africa.1 HAT is caused by the parasitic protozoa
Trypanosoma brucei, which are transmitted to humans by the bite of the tsetse fly. More than
95% of HAT cases occur in western and central Africa and are caused by T. b. gambiense; the
other HAT infections, in eastern and southern Africa, are caused by T. b. rhodesiense. The
disease occurs in two stages and is fatal if left untreated. In the first stage, parasites proliferate
in the hemolymphatic system, and symptoms are relatively non-specific, presenting a challenge
for early diagnosis and intervention. In the second stage, parasitic invasion of the central
nervous system (CNS) causes progressive neurological dysfunction, including convulsions,
serious sleep disturbance, and ultimately coma and death.
Current treatments for Stage II HAT are inadequate due to their poor safety profiles,
marginal efficacy, and complex dosing protocols.2,3 Additionally, drug-resistant infections have
emerged in recent years.4 The World Health Organization has identified the complexity of
current therapies as a major issue for sustaining ongoing disease control efforts.5 A safe, orally
1

administered drug that is effective against both stages and strains of HAT could pave the way
for elimination of the disease, a milestone that is within reach for the first time in history.6
To address the urgent need for new HAT medicines, a number of innovative new
academic-industrial partnerships have emerged. In one recent collaborative effort, a high-
throughput screen (HTS) of 42,444 kinase inhibitors from the GlaxoSmithKline (GSK) screening
collection against T. brucei was completed.7 This study identified 797 sub-micromolar T. brucei
growth inhibitors that were also >100-fold selective over HepG2 cells. The publication of this
rich HTS data set has provided researchers worldwide with an abundance of potential new
starting points for HAT drug discovery programs. We reviewed this HTS data set with a goal of
identifying a hit with attractive properties for development into an oral, brain-penetrant drug. To
ensure that the data from our follow-up to the GSK HTS are readily available to the broader
neglected disease research community, we report herein our progress on hit-to-lead
development of one new series.
From the hundreds of hits identified in the GSK screen, we selected NEU-0001101 (1,
Figure 1) for hit-to-lead optimization. One attractive feature of 1 is its low molecular weight (299
g/mol). Typically, during the optimization of a hit into a clinical candidate, the compound’s
molecular weight increases. Therefore, selecting a low MW hit allows for increasing the
molecular size without exceeding targeted CNS properties.8,9 Additionally, 1 has only one
hydrogen-bond donor (HBD). Minimizing the number of HBD groups in drug molecules also
correlates with improved brain-penetration, a requisite property for any Stage II therapeutic.7-10
The favorable MW and HBD, in addition to other calculated properties, contribute to the
excellent CNS multi-parameter optimization (MPO) score (5.6/6.0) for 1.7-8 Additionally, the
optimal ligand efficiency (LE)11 range for hit compounds is 0.25-0.35.12,13 With an LE = 0.37, 1
has excellent LE for a hit compound. Finally, synthetic tractability of a hit is a key consideration
for enabling rapid hit-to-lead follow-up studies. Based on our preliminary analysis of the
structure of 1, we anticipated that synthesis of this hit and additional analogs in this series would
be straightforward.
Figure 1. Structure and key data for HTS hit NEU-0001101 (1)
2

In order to initiate hit-to-lead medicinal chemistry studies, we devised a flexible, two-step
synthesis of 1 from commercially available 2-amino-5-bromobenzonitrile (2, Scheme 1).14 In
the first step, phenylmagnesium bromide (R1MgBr) addition to the nitrile followed by trapping of
the Grignard adduct with methyl chloroformate afforded 6-bromo-4-phenylquinazolin-2(1H)-one
(3). Whereas the previously reported protocol for this Grignard reaction used diethyl ether as
solvent,13 our use of THF improved both the yield and the safety profile for this step. Suzuki
coupling of 3 with a variety of boronic acids under microwave conditions allows for readily
diversifying this series via the R2 substituent.15 The simplicity and brevity of this modular
synthetic approach has enabled the rapid synthesis of analogs (4) in this series.
Scheme 1. General synthetic approach toward compounds 1 - 27. a) R1MgBr, THF; ClC(O)OMe
(12 – 91% yield) b) R2B(OH)2, Pd(PPh3)4, Na2CO3, DMF/H2O (10 – 89%).
Hit confirmation via resynthesis and retesting of the hit compound is a crucial first follow-up
step for any hit-to-lead medicinal chemistry effort.16-18 A new synthetic batch of 1 was prepared
and showed an approximately 2-fold reduction in potency (EC50 = 1.89 µM) in our T. brucei cell
proliferation assay compared to the initial GSK assay result (EC50 = 0.84 µM).6 This minor shift
may be attributed to a number of factors, including minor differences in the biological assay
protocols, as well as differences in sample purity between the new batch of 1 compared to the
original GSK sample collection batch. Despite the slight loss of potency in this retest, 1
remained an attractive compound for hit-to-lead optimization, due to its highly favorable drug-
like physicochemical properties and good LE.
Following our hit confirmation studies, we initiated structure-activity relationship (SAR)
exploration with a focus on modifying the pyridyl ring in 1. Unsubstituted 4-pyridyl groups
present a potential drug-drug interaction (DDI) risk.19 Thus, preliminary SAR studies explored
the effect of altering this pyridyl group, and all new analogs were evaluated in the T. brucei cell
proliferation assay (Table 1).
3

Table 1. Effect of substituted pyridyl analogs on T. brucei growth inhibition and MRC5 cytotoxicity
Cmpd.
R
T. brucei
EC50 (µM)a
clogPb
T.
MRC5
(µM)
brucei
LLEc
2.38
1
5
1.89 ±0.09
5.32 ±0.8
3.35
3.48
>50
>50
1.80
6
7
8
5.35 ±0.5
5.29 ±0.66
5.07 ±0.05
3.35
3.48
3.86
1.93
1.80
1.44
>50
>50
>50
9
5.24 ±0.23
5.21 ±0.18
3.86
4.61
1.42
0.67
>50
>50
10
11
12
2.04 ±0.19
1.77 ±0.08
4.05
4.45
1.64
1.30
>50
>50
13
1.73 ±0.11
4.72
1.04
>50
a EC50 values are mean SD; n = 4 measurements; All SEM values are within 25%.
bCalculated partition coefficient;29 c(LLE = pEC50 – clogP)
4

These studies show that the addition of a methyl group (compound 5) or the shift of the
pyridyl nitrogen from the 4-pyridyl to 3-pyridyl (compounds 6 – 9) result in a <3-fold loss of
potency in the T. brucei assay. Furthermore, the addition of a flanking trifluoromethyl group
(compound 10 vs. 6) has no significant impact on potency. This suggests that a basic pyridyl
nitrogen is not essential to this series, or that the increased lipophilic interactions of the
trifluoromethyl group can offset the weakening of electrostatic interactions achieved through the
weakly basic pyridine. Larger substituents on the pyridyl ring are also tolerated, with the largest
groups (compounds 12 and 13) achieving comparable potency to the original hit compound.
However, it is important to note that the lipophilic ligand efficiency (LLE)20,21 in this series erodes
with the addition of these large, lipophilic substituents. Thus, from a standpoint of optimizing for
LLE, compound 1 remains the best in this preliminary evaluation of the pyridyl group.
Additionally, all new analogs in this series were tested in an MRC5 counterscreen to evaluate
cytotoxicity in a human cell line. These compounds show no activity in the MRC5 cell
proliferation assay, demonstrating that they are selective for T. brucei in their inhibition of
cellular proliferation.
To further explore the importance of the pyridyl group for activity in this series, we
synthesized a set of analogs in which the pyridyl ring was replaced by substituted phenyl groups
(Table 2). Direct replacement of the pyridyl group in 1 with a phenyl group (compound 14)
causes a ~2.5-fold loss in potency. This result further demonstrates that the pyridine nitrogen
may provide a modest potency boost, but it is not essential and can be replaced by other
aromatic groups to improve the drug safety profile of this chemical series. The mono-
fluorophenyl analogs (compounds 15 – 17) all showed comparable potency (EC50 = 3.71 – 4.29
µM). The addition of the 4-cyano substituent (compound 18) restored potency to match the
original pyridyl hit (1), perhaps due to a favorable hydrogen bonding interaction. The addition of
larger substituents on the phenyl group (compounds 19 – 21) did not provide any significant
changes in potency. In general, compounds 20 and 21, combined with the previous data on
compounds 11 and 12, demonstrate that large substituents at the para position on this aromatic
group are tolerated and potentially beneficial to potency in the T. brucei assay. Overall, the SAR
for substitution of this phenyl group is relatively flat (EC50 range: 1.71 – 5.7 µM), and no
dramatic potency improvements were achieved in this initial compound set. Evaluation of these
compounds in the MRC5 assay further demonstrates the selectivity across this series for T.
brucei antiproliferative activity over the human cell line.
5

Table 2. Effect of substituted phenyl analogs on T. brucei growth inhibition and MRC5 cytotoxicity
Cmpd.
R
T. brucei EC50
(µM)a
4.88 ±0.58
3.71 ±0.55
clogPd
T. brucei
LLEe
MRC5
(µM)
>50
14
15
Ph
2-F-Ph
4.56
4.71
0.75
0.72
>50
16
17
18
3-F-Ph
4-F-Ph
4.29 ±0.29
3.88 ±0.25
1.71 ±0.17
4.71
4.71
4.42
0.66
0.71
1.35
>50
>50
>50
19
5.7 ±0.45b
3.64
1.61
>50
20
5.17 ±0.26c
0.83
>50
21
2.17 ±0.37
4.45
1.21
23.7
a EC50 values are mean SD; n = 4 measurements; All SEM values are within 25% unless otherwise noted.b SEM =
2.14;
c SEM = 1.84; dCalculated partition coefficient; 29 e (LLE = pEC50 – clogP)
Numerous analyses have demonstrated that highly lipophilic compounds experience a
significant rate of attrition due to ADME problems and toxicity.22-26 In an effort to reduce the
lipophilicity and improve the LLE of molecules in this series, the replacement of the pyridyl
group of 1 with alternative heteroaromatic groups was investigated (Table 3). Although
replacement of the pyridyl group with a more polar and less basic pyrimidine (compound 22)
resulted in a significant potency loss, isoxazole and pyrazole groups were advantageous
(compounds 23 – 27). In particular, pyrazoles 24 and 25 show improved LLE relative to our hit
compound 1 due to their improvements in both potency and cLogP. These improvements
suggest that the pyrazole achieves useful, polar drug-target interactions. Notably, the N-methyl
pyrazole 26 has 4- to 5-fold lower potency relative to pyrazoles 24 and 25, suggesting that their
6

potency is enhanced by their hydrogen bond donating capability. Likewise, the weaker potency
of N-methyl pyrazole 27 may be attributed to the absence of the hydrogen bond donor group or
the conformational change in the biaryl group induced by the N-methyl group. Among the
isomeric pyrazoles 24 and 25, we have chosen to focus on 24 for further optimization, due to its
higher selectivity for T. brucei over MRC5. A preliminary set of in vitro ADME assays confirms
that 24 achieves a desirable lipophilicity (LogD7.4 =2.2) and metabolic stability (HLM Clint = 19
µL/min/mg; Rat hepatocyte Clint<1 µL/min/106 cells). The modest aqueous solubility (8 µM) of
24 is one focus of ongoing efforts at further optimizing this series.
Table 3. Effect of pyrimidine, isoxazole, and pyrazole analogs on T. brucei growth inhibition and MRC5
cytotoxicity
Cmpd.
22
R
T. brucei EC50
(µM)a
clogPc
T. brucei
LLEd
MRC5
(µM)
10.04 ±2.95
2.13 ±0.63
1.04 ±0.06
2.64
3.26
3.25
2.36
2.41
>50
>50
>50
23
24
2.73
25
26
27
1.44 ±0.1
5.6 ±0.63b
2.87
2.99
2.97
2.26
22
>50
5.96 ±0.13
2.91
2.31
>50
a EC50 values are mean SD; n = 4 measurements; All SEM values are within 25% unless otherwise noted. b SEM =
1.72; c Calculated partition coefficient;29 d (LLE = pEC50 – clogP)
As another important step in our preliminary hit-to-lead optimization of 1, we have prepared
a number of analogs to identify the optimal growth vectors for improving upon the T. brucei
potency for this scaffold (Table 4). These studies have demonstrated that relocation of the aryl
substituent from the C6 position of the 1,2-dihydroquinazolin-2-one scaffold to the C5 or C7
7

position significantly reduces T. brucei activity in this series. Although large, lipophilic
substituents at the C7 (R1) position can partially restore activity (e.g., compounds 31 and 32),
these compounds have low LLE values. Thus, our efforts to optimize this series have focused
on using the C6 growth vector for optimizing the left-hand side of the scaffold.
Table 4. Effect of C5 (R2) and C7 (R1) substituents on 1,2-Dihydroquinazolin-2-one scaffold on T. brucei
growth inhibition and MRC5 cytotoxicity
Cmpd.
28
R1
R2
T. brucei EC50
(µM)a
cLogPb
T. brucei
LLEc
MRC5
(µM)
H
14.18 ±0.67
>40
3.35
3.35
2.64
1.5
--
>50
>50
>50
29
30
31
H
H
H
>40
--
4.87 ±0.815
4.45
0.86
>50
32
33
H
4.65 ±0.88
>40
4.71
2.91
0.63
--
>50
>50
H
H
34
>40
4.42
--
>50
a EC50 values are mean SD; n = 4 measurements; All SEM values are within 25%.
b Calculated partition coefficient;29 c(LLE = pEC50 – clogP)
In preliminary efforts to improve aqueous solubility in this series, we have briefly explored
strategies for increasing the overall fraction of sp3 character (Fsp3) in our molecules (Table 5),
which has been shown to correlate with improved physical properties and clinical development
potential.27 Replacement of the C6 phenyl group with a methyl group (compound 35) improves
aqueous solubility, but with complete loss of T. brucei activity. Likewise, insertion of saturated
heterocyclic groups at the C6 position on the 1,2-dihydroquinazolin-2-ones scaffold (compounds
8

36 - 38) significantly improves aqueous solubility but also eliminates or reduces compound
potency in our T. brucei assays.
Table 5. Effect of C4 and C6 alkyl, heteroalkyl, and substituted aromatic analogs on T. brucei growth
inhibition, kinetic aqueous solubility, and MRC5 cytotoxicity
Cmpd.
R1
R2
T. brucei EC50
(µM)a
>40
clogPb
3.43
T. brucei
LLEc
Aq. Sol.
(µM)d
80
MRC5
(µM)
>50
35
36
Ph
Ph
CH3
--
>40
2.87
--
>1000
>50
37
38
Ph
Ph
>20
2.38
2.81
--
>1000
324
>25
>50
14.1 ±0.37
2.05
39
40
3.14 ±0.28
28.99 ±6.82
5.76 ±0.71
5.95 ±1.45
3.77
3.10
3.47
2.58
1.74
1.44
1.77
2.65
13
N.D.
5
>50
>50
>50
>50
41
42
74
a EC50 values are mean SD; n = 4 measurements; All SEM values are within 25%.
b Calculated partition coefficient;29 c(LLE = pEC50 – clogP); dKinetic aqueous solubility measured using
chemiluminescent nitrogen detection (CLND) method.
Alternate C4 groups have also been explored with the goal of reducing the overall planarity of
the system using either ortho-substituted phenyl or cycloalkyl groups (compounds 39 – 42).
The C4 cyclobutyl analog 42 produced the best solubility improvement in this series (aqueous
solubility = 74 µM), albeit with a nearly 6-fold loss of potency in the T. brucei assay relative to
the C4-phenyl analog 24. Notably, due to the improved lipophilicity of 42 (cLogP = 2.58; LogD7.4
=2.3), its efficiency (LLE = 2.65) approaches that of pyrazole 24 (LLE = 2.73). Taken together,
9

these studies suggest that maintaining an aromatic heterocycle such as the pyrazole at the C6
position may be crucial for achieving potent activity in the T. brucei assay for this series.
However, insertion of small cycloalkyl groups such as cyclobutyl at the C4 position allows for
improving aqueous solubility without significant loss of T. brucei activity. Based on these
outcomes, future efforts will focus on appending additional polar or ionizable groups to the
scaffold of 24 or 42 to further improve potency and solubility while maximizing LLE.
In an effort to identify additional applications for our novel 1,2-dihydroquinazolin-2-ones in
the treatment of other neglected tropical diseases, we have also screened all new compounds
in this series against T. cruzi and L. donovani (Table 6). Many proteins are highly conserved
among T. brucei, T. cruzi, and Leishmania sp.,28 so effective inhibitors against one
trypanosomatid species could potentially aid in identifying new drug leads against other species.
Several compounds in the series (compound 8, 9, 12, 13, 15, 16, 17, 21, 25, 39) yield T. cruzi
EC50 values below 10 µM, with all of these compounds demonstrating modest selectivity for T.
brucei over T. cruzi. To date, pyrazole 39 is the most potent T. cruzi cell proliferation inhibitor
(EC50 = 5.28 µM) in this series. No compounds in this 1,2-dihydroquinazolin-2-one series have
shown significant activity in the L. donovani intracellular proliferation assay. As part of our
ongoing effort to optimize new lead compounds in this series against T. brucei, we will continue
to screen against T. cruzi and L. donovani, as well as relevant human cell lines to assess
selectivity.
10

Table 6. Compared activities of generated analogs on kinetoplastid parasites growth inhibition and
mammalian cell lines.
MRC5
EC50
(µM)
L.
donovani
THP-1
EC50
T.
brucei
LLE
T.
cruzi
LLE
T. brucei
EC50 (µM)a
T. cruzi EC50 L6 EC50
(µM)a
L.d.
LLE
Cmpd.
(µM)a
EC50 (µM)a (µM)a
1
1.89 ±0.09
5.32 ±0.8
>50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
23.76
> 50
10.94 ±2.65
>50
>5
2.38
1.8
1.62
1.3
1.96
1.82
1.96
1.82
1.44
1.44
0.69
1.25
0.85
0.58
0.74
0.59
0.59
0.59
0.88
1.66
0.85
0.85
2.66
5
16.84 ±2.26
15.84 ±0.54
14.85 ±1.45
8.94 ±1.91
9.56 ±1.41
18.42 ±1.52
13.97 ±0.73
6.29 ±0.62
7.19 ±0.38
> 20
>50
>50
>50
>50
>50
> 50
50
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
50
6
5.35 ±0.5
1.93
1.8
1.45
1.35
1.19
1.16
0.13
0.81
0.75
0.42
0.14
0.36
0.32
0.35
0.34
0.66
0.37
0.63
2.06
7
5.29 ±0.66
5.07 ±0.05
5.24 ±0.23
5.21 ±0.18
2.04 ±0.19
1.77 ±0.08
1.73 ±0.11
4.88 ±0.58
3.71 ±0.55
4.29 ±0.29
3.88 ±0.25
1.71 ±0.17
5.7 ±0.45
8
1.44
1.42
0.67
1.64
1.3
9
10
11
12
13
14
15
16
17
18
19
20
21
22
> 50
> 16
> 50
1.04
0.75
0.72
0.66
0.71
1.35
1.61
0.83
1.21
2.36
55.82
±5.22
8.63 ±0.63
9.48 ±1.59
8.73 ±0.64
17.41 ±1.71
> 20
23.03
50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
5.17 ±0.26
2.17 ±0.37
10.04 ±2.95
15.06 ±1.01
8.3 ±0.33
> 20
22.73
±4.08
> 50
11

23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
2.13 ±0.63
1.04 ±0.06
1.44 ±0.1
5.6 ±0.63
5.96 ±0.13
14.18 ±0.67
> 40
> 50
> 50
22
11.91 ±0.84
13.33 ±0.82
8.33 ±1.67
> 20
> 50
>50
> 50
>50
> 50
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
> 2.5
>5
>5
>5
>5
>5
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 25
> 50
> 50
> 50
> 50
> 50
2.41
2.73
2.97
2.26
2.31
1.5
1.67
1.62
2.21
1.71
1.85
1.35
1.35
2.06
0.25
-0.01
1.79
0.28
1.27
1.83
2.31
1.89
1.51
1.6
2.04
2.05
2.43
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 25
> 50
> 50
> 50
> 50
> 50
17.42 ±0.72
> 20
2.39
1.96
1.96
2.66
0.85
0.59
2.39
0.88
1.87
2.43
2.92
2.5
7.73
±0.72
> 20
1.05
1.76
0.86
0.63
1.49
-0.02
0.97
1.53
2.01
2.05
1.74
1.44
1.77
2.65
> 40
> 20
4.53
±0.2
4.87 ±0.815
4.65 ±0.88
> 40
> 20
> 20
> 20
> 50
> 50
> 50
> 50
> 25
> 50
> 50
> 50
> 50
> 50
> 40
> 20
> 40
> 20
> 40
> 20
> 20
> 10
14.1 ±0.37
3.14 ±0.28
28.99 ±6.82
5.76 ±0.71
5.95 ±1.45
> 20
5.28 ±0.23
20
1.53
2.21
1.84
2.72
20
1.23
2.2
16.89 ±3.27
a EC50 values are mean SD; n = 4 measurements; All SEM values are within 25%.
12

In summary, through our compound synthesis and SAR studies, we have confirmed the
previously reported activity of 1 (NEU-0001101) in a T. brucei cell proliferation assay and have
demonstrated that this hit can be modified to replace the pyridine ring, a potential drug safety
liability. Through the synthesis of a number of C5-, C6-, and C7-substituted analogs, we have
also shown that the C6 position presents the best growth vector for further elaboration of this
scaffold. Furthermore, we have shown that replacement of the pyridine with a pyrazole ring
(Compound 24) improves potency, lipophilicity, and LLE relative to the original hit, while
maintaining good in vitro metabolic stability. Additionally, placement of the cyclobutyl at the C4
position (Compound 42) has improved aqueous solubility without dramatic loss of T. brucei
activity. Based on these outcomes, our ongoing hit-to-lead optimization studies are exploring
additional modifications of the C6 pyrazole and C4 cyclobutyl groups to further improve potency
and solubility while maximizing LLE.
Acknowledgements
We thank Michael P. Pollastri and Lori Ferrins at Northeastern University for compound logistics
and data management support and manuscript editing suggestions; AstraZeneca for performing
in vitro ADME assays; Tanya Cervantes for LC-MS and NMR technical support. Funding was
provided by the Boettcher Foundation’s Webb-Waring Biomedical Research Program, Boettcher
Collaboration Grant, Research Corporation Cottrell College Science Award, and NSF-MRI CHE-
1429567. Biological assays were funded by the National Institute of Allergy and Infectious
Diseases (MPP and MN, R01AI114685), Spanish Ministerio de Economía, Industria y
Competitividad, (SAF2015-71444-P for MN, SAF2015-68042-R for FG and SAF2016-79957-R
for DG-P), Junta de Andalucía (FG, CTS-7282) and Subdirección General de Redes y Centros
de Investigación Cooperativa (RICET) (MN RD16/0027/0019, DG-P RD16/0027/0014).
References and Notes
1. World Health Organization. Trypanosomiasis, Human African (Sleeping Sickness);
http://www.who.int/mediacentre/factsheets/fs259/en/; Accessed 28.04.17.
2. Kuepfer I, Schmid C, Allan M, Edielu A, Haary EP, Kakembo A, Kibona S, Blum J, Burri C.
Safety and Efficacy of the 10-Day Melarsoprol Schedule for the Treatment of Second Stage
Rhodesiense Sleeping Sickness. PLOS Neglected Trop Dis. 2012;6:e1695.
3. Schmid C, Richer M, Bilenge CMM, Josenando T, Chappuis F, Manthelot CR, Nangouma A,
Doua F, Asumu PN, Simarro PP, Burri C. Effectiveness of a 10-Day Melarsoprol Schedule for
13

the Treatment of Late-Stage Human African Trypanosomiasis: Confirmation from a Multinational
Study (Impamel II). J Infect Dis. 2005;191:1922–1931.
4. Baker N, de Koning HP, Mäser P, Horn D, Drug Resistance in African Trypanosomiasis: The
Melarsoprol and Pentamidine Story. Trends in Parasitology. 2013;29:110–118.
5. World Health Organization. Human African Trypanosomiasis: Research
http://www.who.int/trypanosomiasis_african/research/en/; Accessed 28.04.17.
6. Gjini E. Human African Trypanosomiasis: What Are the Prospects for Control? Human
Parasitic Diseases. 2017;9:1–10.
7. Diaz R, Luengo-Arratta SA, Seixas JD, Amata E, Devine W, Cordon-Obras C, Rojas-Barros
DI, Jimenez E, Ortega F, Crouch S, Colmenarejo G, Fiandor JM, Martin JJ, Berlanga M,
Gonzalez S, Manzano P, Navarro M, Pollastri MP. Identification and Characterization of
Hundreds of Potent and Selective Inhibitors of Trypanosoma Brucei Growth from a Kinase-
Targeted Library Screening Campaign. PLoS Neglected Trop Dis. 2014;8:e3253.
8. Wager TT, Chandrasekaran RY, Hou X, Troutman MD, Verhoest PR, Villalobos A, Will Y.
Defining Desirable Central Nervous System Drug Space through the Alignment of Molecular
Properties, in Vitro ADME, and Safety Attributes. ACS Chem Neurosci. 2010;1: 420-434.
9. Wager TT, Hou X, Verhoest PR, Villalobos A. Moving beyond Rules: The Development of a
Central Nervous System Multiparameter Optimization (CNS MPO) Approach to Enable
Alignment of Druglike Properties. ACS Chem Neurosci. 2010;1:435-449.
10. Hitchcock SA, Pennington LD. Structure−Brain Exposure Relationships. J Med Chem. 2006;
49:7559–7583.
11. LE = 1.4(-logEC50)/#Heavy atoms)
12. Hopkins AL, Groom CR, Alex A. Ligand Efficiency: A Useful Metric for Lead Selection. Drug
Discovery Today. 2004;9:430–431.
13. Hopkins AL, Keserü GM, Leeson PD, Rees DC, Reynolds CH. The Role of Ligand Efficiency
Metrics in Drug Discovery. Nat Rev Drug Discov. 2014; 13:105–121.
14. Bergman J, Brynwolf A, Elman B, Vuorinen E. Synthesis of Quinazolines. Tetrahedron.
1986; 42:3697-3706.
15. Suzuki coupling protocol was adapted from Adams ND, Darcy MG, Dhanak D, Duffy KJ,
Fitch DM, Knight SD, Newlander KA, Shaw AN. PCT Int. Appl. 2006; WO 2006113432.
16. Rankovic Z, Jamieson C, Morphy R. High Throughput Screening Approach to Lead
Discovery. In: Rankovic Z, Morphy R., eds. Lead Generation Approaches in Drug Discovery.
Hoboken, NJ: John Wiley & Sons Inc:, 2010:21–71.
17. Frearson JA, Collie IT. HTS and Hit Finding in Academia – from Chemical Genomics to
Drug Discovery. Drug Discovery Today. 2009;14:1150–1158.
14

18. Dahlin JL, Nissink JWM, Strasser JM, Francis S, Higgins L, Zhou H, Zhang Z, Walters MA.
PAINS in the Assay: Chemical Mechanisms of Assay Interference and Promiscuous Enzymatic
Inhibition Observed during a Sulfhydryl-Scavenging HTS. J Med Chem. 2015; 58:2091–2113.
19. Kaul KL, Novak RF. Inhibition and Induction of Rabbit Liver Microsomal Cytochrome P-450
by Pyridine. J Pharmacol Exp Ther. 1987; 243:384–390.
20. Edwards MP, Price DA. Role of Physicochemical Properties and Ligand Lipophilicity
Efficiency in Addressing Drug Safety Risks. In: Macor JE. ed. Annual Reports in Medicinal
Chemistry, Vol. 45. New York, NY: Academic Press; 2010:380–391.
21. Perola E. An Analysis of the Binding Efficiencies of Drugs and Their Leads in Successful
Drug Discovery Programs. J Med Chem. 2010;53:2986–2997.
22. Gleeson MP, Hersey A, Montanari D, Overington J. Probing the Links between in Vitro
Potency, ADMET and Physicochemical Parameters. Nat Rev Drug Discov. 2011;10:197–208.
23. Hughes JD, Blagg J, Price DA, Bailey S, DeCrescenzo GA, Devraj RV, Ellsworth E, Fobian
YM, Gibbs ME, Gilles RW, Greene N, Huang E, Krieger-Burke T, Loesel J, Wager T, Whiteley
L, Zhang Y. Physiochemical Drug Properties Associated with in Vivo Toxicological Outcomes.
Bioorg Med Chem Lett 2008;18:4872–4875.
24. Johnson TW, Dress KR, Edwards M. Using the Golden Triangle to Optimize Clearance and
Oral Absorption. Bioorg Med Chem Lett. 2009;19:5560–5564.
25. Waring MJ. Defining Optimum Lipophilicity and Molecular Weight Ranges for Drug
candidates—Molecular Weight Dependent Lower Log D Limits Based on Permeability. Bioorg
Med Chem Lett 2009; 19:2844–2851.
26. Waring MJ, Johnstone CA. Quantitative Assessment of hERG Liability as a Function of
Lipophilicity. Bioorg Med Chem Lett. 2007; 17:1759–1764.
27. Lovering F, Bikker J, Humblet C. Escape from Flatland: Increasing Saturation as an
Approach to Improving Clinical Success. J Med Chem. 2009;52:6752–6756.
28. Merritt C, Silva LE, Tanner AL, Stuart K, Pollastri MP. Kinases as Druggable Targets in
Trypanosomatid Protozoan Parasites. Chem Rev. 2014; 114:11280–11304.
29. Calculator Plugins were used for cLogP calculation, ChemAxon
(http://www.chemaxon.com).
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GRAPHICAL ABSTRACT
16