Scaffold and Parasite Hopping: Discovery of New Protozoal Proliferation Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.9b00453

Utilizing a target repurposing and parasite-hopping approach, we tested a previously reported library of compounds that were active against Trypanosoma brucei, plus 31 new compounds, against a variety of protozoan parasites including Trypanosoma cruzi, Leishmania major, Leishmania donovani, and Plasmodium falciparum. This led to the discovery of several compounds with submicromolar activities and improved physicochemical properties that are early leads toward the development of chemotherapeutic agents against kinetoplastid diseases and malaria.

Full text of DOI:10.1021/acsmedchemlett.9b00453

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Letter
Scaffold and Parasite Hopping: Discovery
of New Protozoal Proliferation Inhibitors
Baljinder Singh, Jean A Bernatchez, Laura-Isobel McCall, Claudia M Calvet, Jasmin Ackermann, Julia M
Souza, Diane Thomas, Everton M Silva, Kelly A. Bachovchin, Dana M Klug, Hitesh B Jalani, SEEMA BAG,
Melissa J. Buskes, Susan E. Leed, Norma E Roncal, Erica C Penn, Jessey Erath, Ana Rodriguez, Richard
J Sciotti, Robert F Campbell, James McKerrow, Jair L Siqueira-Neto, Lori Ferrins, and Michael P. Pollastri
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00453 • Publication Date (Web): 10 Jan 2020
Downloaded from pubs.acs.org on January 13, 2020
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Chemical Biology
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Scaffold and Parasite Hopping: Discovery of New Protozoal Proliferation Inhibitors
Baljinder Singh,1 Jean A. Bernatchez,2 Laura-Isobel McCall,2 Claudia M. Calvet,2 Jasmin Ackermann,2
Julia M. Souza,2 Diane Thomas,2 Everton M. Silva,2 Kelly A. Bachovchin,1 Dana M. Klug,1 Hitesh B.
Jalani,1 Seema Bag,1 Melissa J. Buskes,1 Susan E. Leed,4 Norma E. Roncal,4 Erica C. Penn,4 Jessey Erath,3
Ana Rodriguez,3 Richard J. Sciotti,4 Robert F. Campbell,4 James McKerrow,2 Jair L. Siqueira-Neto,2 Lori
Ferrins,1* Michael P. Pollastri1
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1Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115
2Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla,
CA 92093
3Department of Microbiology, New York University School of Medicine, 430 E. 29th Street New York,
New York 10010
4Experimental Therapeutics, Walter Reed Army Institute of Research, 2460 Linden Lane, Silver Spring,
MD 20910
* Corresponding author
Abstract
Utilizing a target repurposing and parasite-hopping approach, we tested a previously-reported library of
compounds that were active against Trypanosoma brucei, plus 31 new compounds, against a variety of
protozoan parasites including Trypanosoma cruzi, Leishmania major, Leishmania donovani and
Plasmodium falciparum. This led to the discovery of several compounds with submicromolar activities and
improved physicochemical properties that are early leads towards the development of chemotherapeutic
agents against kinetoplastid diseases and malaria.
Keywords: Kinetoplastids, leishmaniasis, Chagas disease, malaria, protozoan parasite inhibitors, parasite-
hopping
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Neglected tropical diseases (NTDs) are a group of life-threatening diseases affecting a significant
population of the world. These diseases are especially devastating in developing and economically
disadvantaged countries. Most of the treatments available to treat NTDs have serious adverse effects.
Further, the issues of emerging resistance and a lack of affordable drugs is driving the search for new
treatments or vaccines; this search is a priority for the World Health Organization (WHO). Chagas disease
and leishmaniasis, caused by Trypanosoma cruzi and Leishmania spp., respectively, are among the 20
NTDs highlighted by the WHO as urgently needing new therapies.1-3
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Chagas disease, also known as American trypanosomiasis, is a life-threatening illness endemic
mostly in Latin American countries that is transmitted primarily by the kissing bug (Reduviidae family).
According to the WHO, 8 million people worldwide are estimated to be infected by T. cruzi. It is responsible
for 41% of heart failure cases in Latin America4, 5 killing more than 10,000 people per year. More than 25
million people are at risk of acquiring Chagas disease.6 Leishmaniasis is a group of diseases caused by more
than 20 different species of the Leishmania parasite transmitted by the sandfly and affecting humans in 98
countries. Approximately 1.3 million new cases and 20,000 to 30,000 deaths occur annually due to
leishmaniases.7 Though not considered an NTD, malaria is a life-threatening tropical disease caused by five
species of protozoan Plasmodium parasites (mainly Plasmodium falciparum and P. vivax) that are
transmitted by the bite of infected female Anopheles mosquitoes. In 2016, an estimated 216 million cases
of malaria occurred worldwide leading to an approximately 445,000 malaria-related deaths worldwide.8
As part of our drug discovery program for tropical diseases, we have employed a target class
repurposing approach9 wherein we identified lapatinib as an antitrypanosomal agent.9 The synthetic re-
optimization of lapatinib and its related analogs for activity against T. brucei resulted in the discovery of
NEU-195310, 11 (1, Figure 1) which was further optimized to improve aqueous solubility as an anti-HAT
(human African trypanosomiasis) agent.12 Here we report the assessment of this family of compounds
against P. falciparum, Leishmania spp., and T. cruzi, along with some newly prepared analogs.
Head
N
Cl
N
HN
N
O
F
Ref 10, 11
Core
NH
Tail
N
O
N
NH
O
S
N
N
N
N
O
Lapatinib
NEU-1953 (1)
Aq. sol. = 44 M
Human PPB% = 87
T. brucei EC50 = 1.5 M
T. cruzi EC50 = 1.5 M
P. falciparum D6 EC50 = 0.53 M
L. major EC50 = >15 M
T. brucei EC50 = 0.43 M
T. cruzi EC50 = 6 M
P. falciparum D6 EC50 = 0.03 M
L. major EC50 = >15 M
Human liver microsome CLint = 179 L/min/mg protein
Rat hepatocytes CLint = 127 L/min/106 cells
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Figure 1. Optimization of lapatinib leading to the identification of NEU-1953 (1)10, 11 as a multi-parasite inhibitor
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8
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Synthesis. The compounds 17, 18, 22-27, 33-35, 39, 42-45, S21-S23, S25-S27, S31-S35 and S38-
S41 were synthesized using previously reported protocols10, 12, 13 with slight modifications as described in
the Supporting Information (Schemes S1-S7). All other compounds (1-16, 28-32, 36, 38, 40, 41, S1-S20,
S28-S30 and S36-S37) were procured from our internally-available T. brucei compound library.11, 12
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Trypanosoma cruzi. Screening the compounds against intracellular T. cruzi amastigotes revealed
12 compounds with submicromolar activity and an additional 25 compounds with low micromolar activity
(≤10 µM) (Tables 1-3 and Supporting Information Tables S1-S3). The hit selection criteria against T.
cruzi is EC50 <10 µM with a 10-fold selectivity over host cells.14 Considering 1 as a starting point (T. cruzi
EC50 = 6.0 µM),11 several modifications were made to understand the complete structure-activity
relationships (SAR) profile. Removal of the N-methyl from the piperazine (8, EC50 >50 µM) resulted in a
complete loss of activity; the N-methyl is slightly favored over N-ethyl (11, EC50= 7.7 µM) or N-propyl
(12, EC50= 7.6 µM). Further, 11 and 12 were found to be toxic against the host cell line (C2C12; Supporting
Information Table S7). The insertion of a bridging carbon in the piperazine ring (10) or increasing sp3
content by replacing the piperazine ring with the homopiperazine (9, EC50= 1.2 µM) and spirocyclic amines
(13, EC50= 0.52 µM and 14, EC50= 0.22 µM) led an increase in solubility, better ADME properties and
equipotent or improved potency against T. cruzi compared to 1. The N-atoms of the pyrimidine were not
necessary for potency (c.f. 2 and 18) but play an important role in the aqueous solubility (2, aq. sol.= 1.7
M and 18, aq. sol.= 4.1 M). The insertion of a hydrogen bond donor (-NH) in the tail region (6) abrogated
the potency. The potency and selectivity data of tail group modification and ortho-methyl substituted
compounds are listed in Table 1.
Table 1. Inhibition profile of quinolines 1-18 with modified tail regions against T. cruzi, L. major and P. falciparum D6
N
N
HN
R2
R1
N
R3
1-18
T. cruzi
L. majorc
P. falciparum D6
Entry
1
R1
R2
R3
EC50 (µM)a / SIb
EC50 (µM) / SId
EC50 (µM)e / SIf
N
-H
-H
6.0 / 5.7
>15
0.026 / >1461
N
N
N
N
N
2
3
-H
-H
-H
-H
6.1 / 6.7
>20
>23
0.074 / >513
0.054 /402
N
H
N
N
N
5.3 / 4.2
N
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Table 1. Inhibition profile of quinolines 1-18 with modified tail regions against T. cruzi, L. major and P. falciparum D6
4
5
6
N
N
HN
R2
7
8
9
R1
N
R3
1-18
10
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T. cruzi
L. majorc
P. falciparum D6
Entry
4
R1
R2
R3
EC50 (µM)a / SIb
EC50 (µM) / SId
EC50 (µM)e / SIf
N
N
N
N
N
H
-H
-H
>50
17
3.3 / 6.1
7.9 / 2.2
0.18 / 111
0.13 / 131
N
N
H
N
5
6
-H
-H
-H
-H
H
N
N
47
1.7 / 14
0.077 / 312
0.15 / >207
N
N
N
N
N
N
7
O
N
-H
-H
>50
0.35 / >89
O
N
8
9
-H
-H
-H
-H
-H
-H
>50
>5.2
0.13 / >300
0.024 / 1375
0.052 / >712
N
N
HN
N
1.2 / 1.6
6.1 / >1.6
24 / 1.4
2.6 / >14
N
N
N
N
(R)
10
N
N
N
(R)
N
N
N
N
11
12
-H
-H
-H
-H
7.7 / 0.05
24 / 1.0
0.029 / 862
N
N
N
7.6 / 0.0075
4.3 / >8.4
0.023 / >1565
N
N
13
14
-H
-H
-H
-H
0.52 / 19
0.22 / 1.9
>23
>24
0.030 / 533
N
N
N
N
N
0.058 / >414
N
N
N
15
16
17
-CH3
-H
-H
22
25
45
1.8 / 3.3
>24
0.18 / 33
0.069 / 139
>4.3
N
N
N
N
-CH3
-H
N
N
N
N
N
N
-H
0.73 / >45
O
S
O
N
N
18
-H
-H
0.37 / 1.0
5.5 / 1.7
0.010 / 960
N
nd = not determined; All SEM values within 25%; SI=C2C12 or NIH3T3 TC50/T. cruzi EC50; All r2 values are >0.8 unless noted otherwise;
a
b
c
dSI=HepG2 TC50/L. major EC50; eAll r2 values are >0.9 unless noted otherwise; fSI= HepG2 TC50/P. falciparum D6 EC50
Further modifications were performed on the head group, and some cross-over compounds (19-32 and
S24-S30) with different head and tail combinations were prepared (Table 2 Supporting Information
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Table S2). Replacement of the pyrazine with saturated rings led to the identification of 28 and 29 with EC50
values of 2.1 and 1.2 µM, respectively, against T. cruzi. In general, substituted anilines such as 22 (EC50=
0.060 µM) and 23 (EC50= 0.080 µM) were preferred over heterocyclic head groups but a decrease in
aqueous solubility was observed (Supporting Information, Table S8).
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Table 2. Inhibition profile of quinolines 19-32 with cross-over head and tail combinations against T. cruzi, L. major, L. donovani
and P. falciparum D6
R2
HN
R1
N
19-32
T. cruzi
L. majorc
L. donovani
P. falciparum D6
Entry
19
R1
R2
EC50 (µM)a / SIb
EC50 (µM) / SId
EC50 (µM)e / SIf
EC50 (µM)g / SIh
F
N
Cl
0.030 / 290
>1.5
0.023 / 1739
0.018 / 167
N
N
O
N
N
N
20
21
nd
nd
>23
nd
nd
0.024 / >1500
0.019 / 947
N
N
N
N
N
N
N
9.2 / 2.0
N
N
N
N
OCF3
22
23
0.060 / 38
0.080 / 12
8.2 / 0.8
1.6 / 4.3
0.085 / 7.8
>10
0.0060 / 233
0.0060 / 1133
N
N
N
N
O
N
N
Cl
N
N
N
OCF3
O
S
24
25
0.13 / 40
0.16 / 21
0.22 / 7.3
1.3 / 1.4
>10
>10
0.14 / 11
N
O
Cl
N
O
S
0.051 / 35
N
O
N
N
N
26
27
>10
>22
>24
>10
>10
0.025 / >1320
0.039 / 487
N
N
N
N
1.3 / 7.5
N
N
N
N
N
28
29
2.1 / 1.9
>10
>24
>25
>10
4.0
0.024 / 125
0.025 / 600
N
N
N
N
N
N
O
N
N
N
O
30
31
1.2 / 3.2
> 10
>24
8.7
0.026 / 615
0.053 / 47
N
N
N
N
N
>2.2
>10
N
N
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Table 2. Inhibition profile of quinolines 19-32 with cross-over head and tail combinations against T. cruzi, L. major, L. donovani
and P. falciparum D6
R2
HN
7
8
9
R1
N
19-32
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
T. cruzi
L. majorc
L. donovani
P. falciparum D6
Entry
32
R1
R2
EC50 (µM)a / SIb
EC50 (µM) / SId
EC50 (µM)e / SIf
EC50 (µM)g / SIh
N
(R)
> 10
>2.4
>10
0.037 / >97
N
N
(R)
N
OH
nd = not determined; All SEM values within 25%; SI=C2C12 TC50/T. cruzi EC50; All r2 values are >0.8 unless noted otherwise; SI=HepG2
TC50/L. major EC50; eAll SEM values within 25%; fSI=B10R TC50/L. donovani EC50; gAll r2 values are >0.9 unless noted otherwise; hSI= HepG2
TC50/P. falciparum D6 EC50
a
b
c
d
In an attempt to explore the SAR around the scaffold, a range of quinolinimines, open chain
compounds, isocryptolepines and C-6 substituted quinolines were synthesized and the potency and
selectivity data are listed in Table 3 and Supporting Information Table S3. Compound 33 (EC50 = 0.33
µM) was the most potent compound with improved and acceptable physicochemical and ADME properties.
The quinolinimine analog of 1 (39) possessed better aqueous solubility and in vitro ADME properties
but led to a complete loss in potency. These quinolinimine modifications led to the identification of two
low micromolar compounds (37, EC50= 3.0 µM and 38, EC50= 2.1 µM).
In an attempt to increase the conformational flexibility of the quinoline series, we synthesized a series
of open chain analogs (75-83) by breaking one of the central core rings. Only two compounds (42 and 43)
from the open chain series were found to have micromolar activity against T. cruzi. Alternatively,
isocryptolepine analogs (84-86) were designed and synthesized to lock the free rotation of the head group.
This strategy identified compounds 44 (EC50= 0.24 µM) and 45 (EC50= 0.21 µM) as potent T. cruzi
inhibitors but the aqueous solubility was decreased.
The general SAR trends observed for this series against T. cruzi and a representative best compound
with favorable ADME properties are summarized in Figure 2. The selectivity index (SI) of all compounds
tested against T. cruzi was determined with reference to their toxicity against host cells (C2C12 or NIH3T3).
Whilst there was no particular trend observed for toxicity against host cells several active compounds (13,
19, 22, 23, 24, 25 and 33) have SI values above our targeted threshold (SI > 10).
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Table 3. Inhibition profile of C-6 substituted quinolines (33-35), quinolinimines (36-39), pseudoring/open ring analogs (40-43)
and isocryptolepines (44-45) against T. cruzi, L. major, L. donovani and P. falciparum D6
5
6
7
8
R2
R2
R2
O
HN
N
HN
R2
HN
R1
R4
R1
N
33-35
R2
R1
N
9
R3
40-43
R1
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
R3
44-45
L. majorc
36-39
T. cruzi
L. donovani
P. falciparum
D6
Entry
33
R1
R3
R4
EC50
EC50 (µM) EC50 (µM)e /
(µM)a / SIb
/ SId
SIf
EC50 (µM)g / SIh
(R)
O
--
--
0.33 / >30
>22
nd
0.40 / >80
S
(R)
N
O
O
OH
O
O
O
34
35
--
--
--
--
1.9 / 0.89
4.7 / 1.3
10 / 0.81
>21
>21
nd
>10
0.030 / 307
0.075 / >280
0.12 / 300
N
S
N
O
O
S
N
O
O
N
N
N
N
7136
-CH3
--
2.3 / 16
0.020 / 265
N
N
N
N
N
N
N
N
N
37
38
39
40
41
-CH2CH3
-CH2CH2CH3
-CH3
--
--
3.0 / 1.0
2.1 / 1.4
>10
23 / >1.5
1.5 / >22
2.3 / >10
0.36 / >97
>4.3
>10
>10
>10
>10
>10
0.17 / >200
0.087 / >379
0.18 / 133
>23
N
N
N
N
N
N
N
N
--
N
N
N
O
O
N
-OH
-H
-H
>10
Cl
N
O
-OH
>10
0.088 / >74
N
N
Cl
O
N
N
N
42
43
-F
-F
5.5 / 1.6
3.9 / 1.6
>24
nd
nd
4.6 / >5.2
N
Cl
N
-OH
-H
>2.2
0.071 / >310
N
Cl
N
F
N
44
-H
--
--
0.24 / 6.7
>5.0
nd
0.046 / >161
N
N
N
N
45
-CH3
--
--
0.21 / 2.4
0.44 / 10
0.64 / 3.6
0.037 / 127
N
N
N
nd = not determined; aAll SEM values within 25%; bSI=C2C12 TC50/T. cruzi EC50; cAll r2 values are >0.8 unless noted otherwise; dSI=HepG2
TC50/L. major EC50; eAll SEM values within 25%; fSI=B10R TC50/L. donovani EC50; gAll r2 values are >0.9 unless noted otherwise; hSI= HepG2
TC50/P. falciparum D6 EC50
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Substituted phenyl groups
favored over heterocycles
N-methylation eliminates potency
Representative compound
N
N-atoms not necessary for activity
but removal kills solubility
O
T. cruzi EC50 = 0.33 M
Replacement with aliphatic
amines tolerated
N
O
S
HN
N
N
C2C12 TC50 = >10 M
Selectivity index = >30
Aq. sol. = 137 M
HN
N
Increasing sp3 content
tolerated
O
OH
Ring opening reduce activity
N
Human PPB% = 72
Human liver microsome CLint = 43 L/min/mg protein
Rat hepatocytes CLint = 30 L/min/106 cells
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N
N
NEU-5535 33
(
)
o-methylation not tolerated
N
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N-methyl favored over
other substitutions
-NH insertion unfavorable
Figure 2. SAR trends for T. cruzi summarized around 1 and representative best compound from the series (33).
Leishmania major. Screening against L. major intracellular amastigotes resulted in several compounds
with potency ≤10 µM, the potency and selectivity data of all compounds tested are listed in Tables 1-3 and
Supporting Information Tables S1-S3. The SAR trends observed for L. major were different from that of
T. cruzi. Starting with modifications around 1 (L. major EC50 >15 µM), the insertion of a hydrogen bond
donor (-NH) in the tail region (3, 4 and 6) was favored and resulted in improved potency against L. major
with 6 showing improved aqueous solubility (129 µM) as well as other in vitro ADME properties.11 The
removal of nitrogen atoms from the pyrimidine of 6 (EC50= 1.7 µM) resulted in a reduction in potency (5,
EC50= 7.9 µM).11 Ortho-methylation of the aromatic core resulted in entirely different responses,
methylation at C-6 resulted in improved potency (15, EC50= 1.8 µM), whereas methylation at C-8 resulted
in a loss of potency (16, EC50 >24 µM). The replacement of N-methyl in the tail region with the carbamate
(7, EC50= 0.35 µM), N-propyl (12, EC50= 4.3 µM) and N-methylsulfonyl (17, EC50= 0.73 µM) resulted in
an enhancement of potency against L. major.
Three quinolinimines were active in the micromolar range; 36 (EC50= 2.3 µM), 38 (EC50= 1.5 µM)
and 39 (EC50= 2.3 µM) were most promising. Analogs bearing substituted anilines (23, EC50= 1.6 µM and
24, EC50= 0.22 µM ) resulted in improved potency but at the cost of significantly reduced solubility.
Replacement of the pyrazine with saturated rings and the variation of 6-substituted quinolines did not result
in any active compounds. One compound from the isocryptolepines (45, EC50= 0.44 µM) and open chain
analogs (40, EC50= 0.36 µM) were found to be sub-micromolar inhibitors of L. major. The general SAR
trends observed against L. major and representative best compound with improved ADME properties are
summarized in Figure 3. The SI of all compounds tested against L. major was determined with reference
to their toxicity against HepG2 cells.
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Representative compound
Substituted phenyl groups
favored over heterocycles
-NH insertion favorable
Removal of N-atoms
N
N
Replacement with aliphatic
amines not favored
HN
N
reduce potency
N
HN
N
Increasing sp3 content
increased potency
N
N
Ring opening and
isocrypolepines are tolerated
NEU-2213 17
(
)
N
N
N
O
N
L. major EC50 = 0.73 M
HepG2 TC50 = >33 M
Selectivity index = >45
Aq. sol. = 0.06 M
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S
N
N
Core modification to
quinolinimines are tolerated
O
N
Human PPB% = 95
C-6 methylation favored
over C-8 methylation
Replacement of N-methyl with
carbamate, sulfonyl, N-propyl
improved potency
Human liver microsome CLint = 44 L/min/mg protein
Rat hepatocytes CLint = 28 L/min/106 cells
Figure 3. SAR trends for L. major around 1 and representative best compound from the series (17).
Leishmania donovani. Based on the positive results against L. major, we tested selected analogs
against L. donovani, one of the species that causes visceral leishmaniasis (Tables 2-3 and Supporting
Information, Tables S2, S3 andS7). The observed activity trends were not similar across the two species
(albeit we note that the host cell lines utilized were different for each species). Screening against L.
donovani resulted in three compounds with low micromolar inhibition (EC50 ≤10 µM) and four compounds
with sub-micromolar potency (EC50 <1 µM). Replacing the pyrazine of 1 with tetrahydropyran resulted in
an active compound against L. donovani (29, EC50= 4.0 µM). Replacing the piperazine with the
homopiperazine and the pyrazine with 4-(trifluoromethoxy)aniline resulted in 22 with improved potency
(EC50= 0.085 µM) though this was associated with toxicity against host cells (TC50= 0.66 µM; SI=8).
Further, modifying the core to the quinolinimine led to 36 (EC50= 0.02 µM); and different head and tail
combinations identified 19 (EC50= 0.023 µM) as the most potent compounds among all those tested. The
general SAR trends observed for this series against L. donovani and the representative best compound with
favorable ADME properties are summarized in Figure 4. The SI of all compounds tested against L.
donovani was determined with reference to their toxicity against host cells (B10R).
Representative compound
Diverse SAR trends with N-heterocyles,
phenyl groups and aliphatic amines
N
N
N
N
N-atoms required for
activity
N
N
NEU-4438 36
(
)
HN
Ring opening not tolerated
L. donovani EC50 = 0.020 M
B10R TC50 = 5.3 M
Selectivity index = 265
N
N-methyl homopiperazine
favored over N-methyl piperazine
N
N
N
N
Aq. sol. = 882 M
N
Quinolinimines generally favored
over quinolines
N
N
Human PPB% = 15
N
Human liver microsome CLint = 22 L/min/mg protein
Rat hepatocytes CLint = 13 L/min/106 cells
No effect of carbon length (-NCH3)
Figure 4. SAR trends for L. donovani around 1 and representative best compound from the series (36).
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Based upon the high potency against L. donovani, two compounds (19 and 36) were selected for in
vivo efficacy studies in an acute infection model in both female and male mice at 50 mg/kg b.i.d for 14
days. The results are summarized in Supporting Information, Figure S1 and Table S4. Compound 36 has
a better ADME and pharmacokinetic profile12 over 19 but neither compound showed any significant effect
on reducing the parasitemia in mice.
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Plasmodium falciparum. We have also recently reported the medicinal chemistry optimization of
lapatinib where 1 was found to be a potent inhibitor of P. falciparum.10 Continuing our program on
antimalarial drug discovery and based upon our previous observations that 4-anilinoquinolines have activity
against P. falciparum,10, 15 this library of compounds was also tested against three different strains of P.
falciparum D6 (chloroquine sensitive, mefloquine resistant), W2 (chloroquine resistant, mefloquine
sensitive), and C235 (chloroquine, mefloquine, and pyrimethamine resistant). The screening against P.
falciparum resulted in 42 compounds with low nanomolar activity (<0.1 µM), 28 compounds with sub-
micromolar activity (0.1-1 µM) and eight compounds with low micromolar activity (1-10 µM). The
inhibition profile of all tested compounds against P. falciparum D6 and the associated SI values is presented
in Tables 1-3 and Supporting Information Tables S1-S3; and the tabulated values against W2 and C235
are provided in the Supporting Information, Table S5. As previously reported, 110 was active across all
three strains of P. falciparum (D6 EC50= 0.026 µM, W2 EC50= 0.062 µM, and C235 EC50= 0.037 µM). The
pyrimidine nitrogen atoms were not necessary for potency (2, D6 EC50= 0.074 µM). Variations to the N-
alkyl on the piperazine (N-ethyl, 11, D6 EC50= 0.029 µM; N-propyl, 12, D6 EC50= 0.023 µM) was favored
over the free -NH (8, D6 EC50= 0.13 µM) in terms of potency with little or no effect on the solubility.
Increasing the sp3 content resulted in a significant increase in solubility and maintained the potency (9, D6
EC50= 0.03 µM). Further, the replacement of the pyrazine with other amines such as methylated pyrazines
(20 and 21), pyridines (26 and 27), and substituted anilines (22 and 23) were tolerated and led to the best
compound in the series i.e. 21 with excellent potency (D6 EC50= 0.02 µM) and favorable ADME properties.
Ortho-methylation of the aromatic core at C-8 (16) was favored over the C-6 position (15). Replacing the
pyrazine head group with saturated rings (28, 29, 30, 31 and 32 ), C-6 substituted quinolines (34 and 35),
scaffold modification with quinolinimine (38), isocryptolepines (44 and 45) and open chain analogs (41
and 43) were also successful and resulted in several compounds with low nanomolar to sub-micromolar
potency across all three strains of P. falciparum. The general SAR trends observed for this series against
P. falciparum D6 and a representative best compound with favorable ADME properties are summarized in
Figure 5.
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Representative compound
Subsititution on pyrazine ring and
replacement with anilines tolerated
N-methylation kills potency
N
N
N
N-atoms not necessary for activity
but removal kills solubility
Replacement with aliphatic amines
tolerated and enhanced solubility
HN
N
N
HN
N
NEU-4439 21
(
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Increasing sp3 content tolerated
and it improved solubility
P. falciparum D6 EC50 = 0.019 M
HepG2 TC50 = 18 M
Ring opening and quinolinimine tolerated
but overall quinoline core favored
N
N
Selectivity index = 947
N
N
N
Aq. sol. = >1000 M
N
N
C-8 methylation favored
over C-6 methylation
Human PPB% = 77
N
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Human liver microsome CLint = 72 L/min/mg
Rat hepatocytes CLint = 31 L/min/106 cells
N-alkyl essential for activity; N-CH3,
N-C2H5 and N-C3H7 are euipotent
-NH insertion unfavorable
Figure 5. SAR trends for P. falciparum D6 around 1 and representative best compound from the series (21).
A comparison of activity between P. falciparum D6, W2 and C235 (Figure 6) revealed most
compounds tested had activities within 3-fold of the D6 potency value. Of those tested, 20% of the
quinolines (14 of 69 compounds) were less potent against the W2 strain than D6, as was one of the
quinolinimines (representing 25% of those tested) though this represents a small sample size and more data
is required to understand the trend for this scaffold. Further, 4% of the quinolines (3 of 69 compounds)
were less potent against the C235 strain than D6. This suggests a lack of sensitivity to chloroquine resistance
for the quinolines presented herein. The open chain and isocryptolepine scaffolds all had potency values
within 3-fold against each strain tested.
Figure 6. Comparison of potency (compounds with EC50 ≤0.5 µM shown) between: (A) P. falciparum W2 vs D6, and (B) P.
falciparum C235 vs D6. The blue line signifies a 3-fold change in potency between P. falciparum D6 and the strain in question,
the black line signifies compounds that are equipotent with P. falciparum D6 and the strain in question.
Utilizing a parasite-hopping approach, we have screened a previously reported library of T. brucei
inhibitors along with newly synthesized compounds against T. cruzi, L. major, L. donovani and three strains
of P. falciparum. Assessment of these compounds produced 13 submicromolar active compounds against
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T. cruzi (EC50 <1µM), five submicromolar active compounds against L. major (EC50 <1µM), four
submicromolar active compounds against L. donovani (EC50 <1µM) and 42 low nanomolar compounds
against P. falciparum D6 (EC50 <0.1µM). Further, the cross-screening resulted in identification of several
potent compounds with good selectivity index and improved ADME profile. Compounds 19 and 36 were
tested in an efficacy model of L. donovani infection; both compounds failed to translate in vitro potency to
in vivo activity. Work is ongoing to improve compound properties toward discovery of antiparasitic agents
that are effective in animal efficacy studies.
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Supporting Information
Details of synthetic chemistry, biological assay protocols, biological data of all tested compounds, in vitro
cell toxicity data, in vitro ADME properties and other biological data (annotated with NEU registry
numbers) are contained in the Supporting Information.
Author Information
Corresponding Author:
*E-mail: l.ferrins@northeastern.edu
Tel: 617-373-3715
Funding Sources
Funding from the National Institutes of Health (R01AI082577, R56AI099476, R01AI124046,
R21AI127594, R01AI126311 to M.P.P) is gratefully acknowledged.
Notes
The authors declare no competing financial interest. Material has been reviewed by the Walter Reed Army
Institute of Research. There is no objection to its presentation and/or publication. The opinions or
assertions contained herein are the private views of the author, and are not to be construed as official, or as
reflecting true views of the Department of the Army or the Department of Defense.
ACKNOWLEDGMENT
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We are grateful to AstraZeneca for performing the in vitro ADME experiments tabulated in Table S5 in
the Supporting Information. A free academic license to OpenEye Scientific Software and ChemAxon for
their suites of programs is gratefully acknowledged.
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References
1.
Gyapong, J. O. An Overview of Neglected Tropical Diseases in Sub-Saharan Africa. In Neglected
Tropical Diseases - Sub-Saharan Africa; J. Gyapong; B. Boatin Eds.; Springer International Publishing:
Cham, 2016; pp. 1-14.
2.
Mackey, T. K.; Liang, B. A.; Cuomo, R.; Hafen, R.; Brouwer, K. C.; Lee, D. E. Emerging and
reemerging neglected tropical diseases: a review of key characteristics, risk factors, and the policy and
innovation environment. Clin. Microbiol. Rev. 2014, 27, 949-979.
3.
Field, M. C.; Horn, D.; Fairlamb, A. H.; Ferguson, M. A. J.; Gray, D. W.; Read, K. D.; De Rycker,
M.; Torrie, L. S.; Wyatt, P. G.; Wyllie, S.; Gilbert, I. H. Anti-trypanosomatid drug discovery: an ongoing
challenge and a continuing need. Nat. Rev. Microbiol. 2017, 15, 217.
4.
5.
Bocchi, E. A. Heart failure in South America. Curr. Cardiol. Rev. 2013, 9, 147-156.
Vieira, J. L.; Tavora, F. R. F.; Sobral, M. G. V.; Vasconcelos, G. G.; Almeida, G. P. L.; Fernandes,
J. R.; da Escossia Marinho, L. L.; de Mendonca Trompieri, D. F.; De Souza Neto, J. D.; Mejia, J. A. C.
Chagas Cardiomyopathy in Latin America Review. Curr. Cardiol. Rep. 2019, 21, 8.
6.
2017. http://www.who.int/chagas/en/
7. Zulfiqar, B.; Shelper, T. B.; Avery, V. M. Leishmaniasis drug discovery: recent progress and
challenges in assay development. Drug Discov. Today 2017, 22, 1516-1531.
8. WHO World Malaria Report 2017. Access date
http://www.who.int/malaria/publications/world-malaria-report-2017/en/
9. Klug, D. M.; Gelb, M. H.; Pollastri, M. P. Repurposing strategies for tropical disease drug
discovery. Bioorg. Med. Chem. Lett. 2016, 26, 2569-2576.
10. Mehta, N.; Ferrins, L.; Leed, S. E.; Sciotti, R. J.; Pollastri, M. P. Optimization of Physicochemical
World Health Organization: Chagas disease (American trypanosomiasis). Access date 20 January
20
December
2017.
Properties for 4-Anilinoquinoline Inhibitors of Plasmodium falciparum Proliferation. ACS Infect. Dis. 2018,
4, 577-591.
11.
Ferrins, L.; Sharma, A.; Thomas, S. M.; Mehta, N.; Erath, J.; Tanghe, S.; Leed, S. E.; Rodriguez,
A.; Mensa-Wilmot, K.; Sciotti, R. J.; Gillingwater, K.; Pollastri, M. P. Anilinoquinoline based inhibitors of
trypanosomatid proliferation. PLoS Negl. Trop. Dis. 2018, 12, e0006834.
13
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2
3
4
12.
Bachovchin, K. A.; Sharma, A.; Bag, S.; Klug, D. M.; Schneider, K. M.; Singh, B.; Jalani, H. B.;
Buskes, M.; Mehta, N.; Tanghe, S.; Momper, J. D.; Sciotti, R. J.; Rodriguez, A.; Mensa-Wilmot, K.;
Pollastri, M. P.; Ferrins, L. Improvement of Aqueous Solubility of Lapatinib-derived Analogs:
Identification of a Quinolinimine as a Lead for Human African Trypanosomiasis Drug Development. J.
Med. Chem. 2019, 62, 665-687.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13.
Devine, W.; Thomas, S. M.; Erath, J.; Bachovchin, K. A.; Lee, P. J.; Leed, S. E.; Rodriguez, A.;
Sciotti, R. J.; Mensa-Wilmot, K.; Pollastri, M. P. Antiparasitic Lead Discovery: Toward Optimization of a
Chemotype with Activity Against Multiple Protozoan Parasites. ACS Med. Chem. Lett. 2017, 8, 350-354.
14.
Katsuno, K.; Burrows, J. N.; Duncan, K.; Hooft van Huijsduijnen, R.; Kaneko, T.; Kita, K.;
Mowbray, C. E.; Schmatz, D.; Warner, P.; Slingsby, B. T. Hit and lead criteria in drug discovery for
infectious diseases of the developing world. Nat. Rev. Drug Discov. 2015, 14, 751-758.
15.
Devine, W.; Woodring, J. L.; Swaminathan, U.; Amata, E.; Patel, G.; Erath, J.; Roncal, N. E.; Lee,
P. J.; Leed, S. E.; Rodriguez, A.; Mensa-Wilmot, K.; Sciotti, R. J.; Pollastri, M. P. Protozoan Parasite
Growth Inhibitors Discovered by Cross-Screening Yield Potent Scaffolds for Lead Discovery. J. Med.
Chem. 2015, 58, 5522-5537.
Table of contents graphic:
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