Identification of a Hit Series of Antileishmanial Compounds through the Use of Mixture-Based Libraries

Article abstract of DOI:10.1021/acsmedchemlett.7b00045

From a screening campaign that included mixture-based libraries containing more than 6 million compounds, a lead series of bis-cyclic guanidines was identified as the most promising. Lead optimization resulted in the identification of potent (IC₅₀ < 500 nM) and selective compounds within this series as well as potent and selective monoguanidines.

Full text of DOI:10.1021/acsmedchemlett.7b00045

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Letter
The Identification of a Hit Series of Antileishmanial
Compounds Through the Use of Mixture-Based Libraries
Marc A. Giulianotti, Brian Andrew Vesely, Ala Azhari, Ashley Souza,
Travis LaVoi, Richard A. Houghten, Dennis E Kyle, and James W. Leahy
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00045 • Publication Date (Web): 10 Jul 2017
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The Identification of a Hit Series of Antileishmanial Com-
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Marc A. Giulianotti†,‡, Brian A. Vesely§, Ala Azhari§, Ashley Souza§, Travis LaVoi‡, Richard A.
Houghten‡, Dennis E. Kyle§* and James W. Leahy†,#,¶*
†
Department of Chemistry, University of South Florida, CHE 205, 4202 E. Fowler Avenue, Tampa, FL 33620, United
States.
‡
Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St. Lucie, Florida 34987, United States.
§ Department of Global Health, College of Public Health, University of South Florida, 3720 Spectrum Boulevard, Suite 304,
Tampa, Florida 33612, United States.
#
Florida Center of Excellence for Drug Discovery and Innovation, University of South Florida, 3720 Spectrum Boulevard,
Suite 303, Tampa, FL 33612, United States.
Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, MDC 7, 12901 Bruce B.
¶
Downs Boulevard, Tampa, FL 33612, United States.
KEYWORDS Leishmaniasis, Screening, Mixture-Based Libraries, Lead Optimization
ABSTRACT: From a screening campaign that included mixture-based libraries containing more than 6 million compounds, a lead
series of bis-cyclic guanidines was identified as the most promising. Lead optimization resulted in the identification of potent (IC50
00 nM) and selective compounds within this series as well as potent and selective monoguanidines.
<
5
Leishmaniasis is a disease caused by infection with Leish-
ranking sample Lib1 demonstrated potency in both of the afore-
mentioned leishmaniasis assays with no sign of cytotoxicity in
an uninfected macrophage counter screen. A positional scan-
ning library was then screened in order to develop an SAR pro-
file around the core bis-cyclic guanidine scaffold. From this
analysis, 27 individual compounds were selected as good can-
didates to validate the profile.
mania parasites, which are spread by the bite of phlebotomine
sand flies. The most severe form of the disease is visceral leish-
maniasis caused by the protozoan parasites Leishmania do-
1
,2
novani and Leishmania infantum. People suffering from vis-
ceral leishmaniasis experience cachexia, cytopenia, fever,
2
hepatosplenomegaly and death if not treated. It is estimated
that there are 200,000 to 400,000 new cases of visceral leish-
maniasis each year and >20,000 deaths a year due to the dis-
ease. Current therapeutics for patients with visceral leishmani-
Upon confirmation of the SAR trends with the individual
compounds, a lead optimization campaign was undertaken to
3
asis include liposomal amphotericin B (1), sodium stiboglucon-
tate (2), miltefosine (3), pentamidine (4) and paromomycin (5,
Figure 1). All of these therapeutics are less than ideal through
some combination of ease-of-use, efficacy, cost, safety, and
Figure 1. Currently Used Antileishmaniasis Agents
4
drug resistance. While much effort is underway on the devel-
opment of new therapeutics, there is a pressing need to continue
to identify novel compounds for the treatment of this disease.
We have previously reported on the use of mixture-based
5-7
libraries to rapidly identify and prioritize active scaffolds. Us-
ing this approach, we evaluated a scaffold-ranking library con-
taining >6,000,000 compounds in a L. donovani axenic
amastigote assay as well as a high-content imaging screen uti-
lizing macrophages infected with L. donovani amastigotes. As
5
-7
previously described in detail, a scaffold-ranking library is a
collection of mixture samples where each mixture sample con-
tains only compounds with the same core scaffold, and there-
fore all of the compounds in a given mixture are close structural
analogs. Additionally, each compound in a specific mixture
sample is represented in approximately equal molar concentra-
tion. From this initial screen, the bis-cyclic guanidine scaffold-
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Table 1. Leishmaniasis Activity of R
1
Positional Scanning Study of Lib1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a.a. is the % inhibition at 5 M in the axenic amastigotes assay. i.m. is the IC50 in M in the infected macrophage assay. The structure
for this table is the same for each of the subsequent tables.
further explore the SAR profile around the scaffold. These ef-
forts resulted in the discovery of several new compounds pos-
sessing sub-micromolar IC50s in the L. donovani infected mac-
rophage assay. Among the hits is a monocyclic guanidine com-
pound, 64.
potential toxicity to the host mammalian cell. From these two
assays, a selectivity index (SI) was calculated by dividing the
IC50 obtained from the non-infected macrophage by the IC50
from the infected macrophage assay. From this infected macro-
phage screen, we found several scaffolds with attractive activity
(sub-micromolar IC50) and selectivity (> 50 SI) profiles, one of
We initiated our study by screening 28 of our scaffold-rank-
ing libraries at 4 doses (50, 25, 5, and 2.5 M) in an assay using
axenic L. donovani amastigote strains, CR6 and CS1 as de-
which was Lib1. We thus turned our attention to exploring a
positional scanning study on this scaffold.
8
Using our previously described synthetic methods,5-7 we
screened a 110 sample array around Lib1 (42 carboxylic acids
x 42 carboxylic acids x 26 amino acids). In this manner, we
were able to evaluate a total of 45,864 different bis-cyclic guan-
idine analogs, where each compound was replicated in 3 differ-
ent samples. The results of these screens are shown in Tables 1
scribed in Wang et al. Only scaffold-ranking sample Lib1
showed substantial activity at any of the doses tested, producing
9
>
90% inhibition at the 25 and 50 M doses.
While the axenic amastigote assay allows for a quick assess-
ment of a sample’s intrinsic anti-leishmanial activity, it does
have the weakness in that the effect of the host mammalian cell
cannot be taken into account. In order to assess the anti-leish-
manial efficacy of the scaffold-ranking samples against intra-
cellular parasites, a high content screening (HCS) assay with L.
donovani infected J774 macrophages was utilized. In addition,
a counter screen assay utilizing non-infected J774 macrophages
was performed on the scaffold-ranking samples to assess their
–
3. Although the libraries were evaluated at several concentra-
tions, the best range of results was apparent at 5 M, and the
percent inhibition at this concentration is shown. This data al-
11
1
0
lowed us to rank order the relative likelihood that active indi-
vidual compounds contain certain functionalities at a given po-
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Table 2. R
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Positional Scanning Study of Lib1
Table 4. Individually Synthesized Compound Activity
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3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a.a. is the % inhibition at 5 M in the axenic amastigotes assay.
i.m. is the IC50 in M in the infected macrophage assay.
sition and to begin to augur certain trends. For example, based
on the 5 M data, it is clear that the mixture defined with cyclo-
a.a. is the % inhibition in the axenic amastigotes assay. i.m. is
the infected macrophage assay. cytotox is the cytotoxicity in
J774 macrophage cells. SI is the selectivity index (cytotox/i.m.).
hexylbutyl at the R
more active (100%) than the one defined with isobutyl (0%) at
the same R position (Table 1 sample Lib1.022). This implies
that it is more likely to find active individual compounds with
cyclohexylbutyl defined at R than with isobutyl at R . Across
1
position (Table 1 sample Lib1.031), is
1
at the 5 M dose in the axenic amastigotes assay, demonstrating
these samples’ ability to inhibit the growth of the amastigotes
in both assay formats.
1
1
all three of these positions, oxygenated substituents dramati-
cally reduced activity at the 5 M dose. Also, there appears to
2
be little preference for stereochemical specificity at the R po-
In order to confirm some of the SAR trends that were ob-
served in the positional scanning library data as well as to iden-
tify individual hit compounds with antileishmanial activity, a
set of previously synthesized bis-cyclic guanidines were se-
sition (Table 2), where diastereomeric mixtures (e.g.
Lib1.046/Lib1.054 and Lib1.049/Lib1.057) demonstrated
nearly identical activities. In addition to the axenic amastigote
data in Tables 1 to 3, the IC50 data from the HCS infected mac-
rophage assay is also provided. The infected macrophage data
does not correlate perfectly with the axenic amastigote data,
which is consistent with what we observed in our initial scaf-
fold-ranking studies. While both assays measure the ability of a
sample to inhibit the growth of the amastigotes at a given dose,
the infected macrophage assay requires the samples to penetrate
the macrophage first in order to get to the amastigote. Regard-
less, a number of the samples that produced sub 2.5 M IC50 in
the infected macrophage assay also produced a >80% response
1
2
lected for testing. The compounds were synthesized as previ-
ously described incorporating 3 different functionalities in each
1 2 3
of the R , R and R positions resulting in 27 structural analogs
(Table 4). These different functionalities, when incorporated
into the positional scanning library, demonstrated a range of ac-
tivities in the anti-Leishmania proliferation assays (Tables 1 to
3) therefore making them a good set to test the SAR, which is
shown in Table 4. In general, when cyclohexylbutyl was fixed
in the R
1
position (Compounds 15 to 23), the compounds
showed an increased response in the axenic amastigote assay (6
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Table 3. R
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a.a. is the % inhibition at 5 M in the axenic amastigotes assay. i.m. is the IC50 in M in the infected macrophage assay.
out of 9 showed > 65% inhibition at 2.5 M) relative to the other
compounds (2 out of 18). These results are consistent with the
data generated from the positional scanning library data (Table
We also pursued additional studies around compounds 6 and
8, which differed only about the R position, but showed con-
3
siderable activity in the infected macrophage assay (Table 6).
While we again observed some compounds with acceptable ac-
tivity and SI profiles, we were pleased to note that monosubsti-
tuted analog 50 was quite potent. We therefore synthesized and
analyzed some analogs around this compound. Desfluoro ana-
log 55 gave an excellent profile and indicated a preference for
meta-substitution (see 54, 56 and 57). Saturation of the ring re-
vealed that 59 was another analog with an excellent profile.
1
) where Lib1.031 showed greater activity than Lib1.041 or
Lib1.007. Of those 9 individual compounds (15-23), the com-
pounds where R was R-2-naphthylmethyl were the inactive an-
2
alogs (18-20). Again, this is consistent with the positional scan-
ning data (Table 2) showing that Lib1.066 was much less active
than Lib1.063 or Lib1.067. Using these trends as well as the
range of activity in the infected macrophage and cytotoxicity
13
assays, we initiated lead optimization activities.
Encouraged by the identification of multiple monosubsti-
tuted analogs with submicromolar activity and low cytotoxicity,
we wanted to determine the necessity of having two guanidine
units. We were further struck by the prospect that the butyl sub-
The first two candidates we selected for further exploration
were 21 and 26, both of which showed submicromolar activity
in the infected macrophage assay with dramatically different ac-
tivities in the axenic amastigote assay. In both cases, we pre-
pared a series of deletion analogs (in hopes of decreasing their
lipophilicity) as well as inverting the stereocenter built into the
initial scaffold (33 and 40, Table 5). In both cases, this scaffold
change resulted in a significant decrease in the activity. For the
series around 21, most of the deletion compounds resulted in a
significant deleterious effect on the infected macrophage activ
ity, with the sole exception being 34. However, the good activ-
ity and SI profiles for 42 and 43 suggested that we might be able
to focus our attention on monosubstituted analogs. Toward this
end, while biphenyl compound 46 was not particularly active,
the truncated phenethyl compound 47 showed excellent activity
and selectivity.
stitution at R
(Figure 2). In this regard, we could consider the R
2
could serve as a surrogate for the scaffold linker
and R sub-
1
3
stituents together. Toward this end, we synthesized a series of
monoguanidine analogs with and without the butyl substituent
(Table 7), which clearly showed that the butyl group improved
1 3
Figure 2. Similarity of R and R
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Table 5. Analogs of 21 and 26
ACS Medicinal Chemistry Letters
Table 6. Analogs of 6 - 8
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
i.m.
cytotox
i.m.
cytotox
ID
*
R
1
R
2
R
3
SI
ID
*
R
1
R
2
R
3
SI
IC50 (M) IC50 (M)
IC50 (M) IC50 (M)
6
S
0.6
5.0
0.3
0.6
2.5
0.9
3.5
48
50
28
50
9
81
21 S
0.7
8.9
25
50
36
7
8
S
S
10
88
79
4
33 R
6
34 S
H
1.8
3.5
2.5
5.2
27
6
16
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
48 R
35 S
H
H
49 S
H
H
36 S
H
50
24
20
5
50 S
H
H
7
9
37 S
H
H
38 S
39 S
H
H
H
H
10
13
50
1
51 S
11
3
4.8
10
52 S
H
H
1.3
5.0
22
50
17
10
26 S
0.9
3.8
4.2
0.7
1.2
22
21
13
23
50
25
6
53 S
H
H
H
40 R
54 S
H
H
3.5
0.9
50
50
14
56
41 S
H
3
55 S
42 S
H
H
33
43
56 S
H
H
H
H
H
H
H
H
3.9
1.2
1.8
0.6
22
16
19
17
6
43 S
H
57 S
58 S
13
10
28
44 S
H
H
3.5
10
50
50
50
20
14
5
45 S
H
H
H
59 S
46 S
47 S
H
H
4.9
0.6
10
36
60 S
H
H
H
H
5.4
4.4
14
6
3
1
i.m. is the infected macrophage assay. cytotox is the cytotoxi-
city in J774 macrophage cells. SI is the selectivity index (cyto-
tox/i.m.).
61 S
i.m. is the infected macrophage assay. cytotox is the cytotoxi-
city in J774 macrophage cells. SI is the selectivity index (cyto-
tox/i.m.).
the activity. Furthermore, the optimal activity of 47 was closely
reflected in analog 64, which showed an excellent SI and was
more potent in our infected macrophage assay as miltefosine
3), a clinically-used antileishmanial agent that is used as our
standard.
In summary, using our scaffold ranking and positional scan-
ning libraries, we were able to identify a series of guanidine-
based compounds that possessed potent antileishmanial activity
in multiple assays as well as selectivity over human macro-
phages, demonstrating that activity is unlikely to arise through
simple promiscuous interactions. These compounds are struc-
turally distinct from any of the currently used antileishmaniasis
agents as shown in Figure 1, and thus represent a novel oppor-
tunity for lead optimization. We were subsequently able to use
these libraries to focus in on compounds such as 64 that retain
not only excellent activity (an IC50 of 1.13 M in the infected
macrophage assay when run in quadruplicate with a standard
deviation of 0.81 M) and selectivity (an SI of 23 over J774
macrophages) but a reasonable molecular weight (245 g/mol)
and physicochemical properties (e.g. cLogP = 3.26, tPSA = 39).
Work toward the further optimization of these compounds with
optimal pharmacokinetic and pharmacodynamics properties as
well as attempts to ascertain their target or mechanism of action
is underway and will be reported in due course.
(
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Table 7. Monoguanidines
the Discovery of nAChR-Selective Compounds Suitable for Optimiza-
tion Studies. J. Med. Chem. 2013, 56, 10103-10117.
Houghten, R. A.; Pinilla, C.; Giulianotti, M. A.; Appel, J. R.;
Dooley, C. T.; Nefzi, A.; Ostresh, J. M.; Yu, Y.; Maggiora, G. M.; Me-
dina-Franco, J. L.; Brunner, D.; Schneider, J. Strategies for the use of
mixture-based synthetic combinatorial libraries: scaffold ranking, di-
rect testing in vivo, and enhanced deconvolution by computational
methods. J. Comb. Chem. 2008, 10, 3−19.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
.
7
.
Rideout, M. C.; Boldt, J. L.; Vahi-Ferguson, G.; Salamon,
P.; Nefzi, A.; Ostresh, J. M.; Giulianotti, M.; Pinilla, C.; Segall, A. M.
Potent antimicrobial small molecules screened as inhibitors of tyrosine
recombinases and Holliday junction-resolving enzymes. Mol. Diversity
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
011, 15, 989−1005.
8. Wang, M. Z.; Zhu, X.; Srivastava, A.; Liu, Q.; Sweat, J. M.;
Pandharkar, T.; Stephens, C. E.; Riccio, E.; Parman, T.; Munde, M.;
Mandal, S.; Madhubala, R.; Tidwell, R. R.; Wilson, W. D.; Boykin, D.
W.; Hall, J. E.; Kyle, D. E.; Werbovetz, K. A., Novel arylimidamides
for treatment of visceral leishmaniasis. Antimicrob. Agents Chemother.
2
010, 54, 2507-16.
9. Screening was conducted on 28 different scaffold libraries
that contained > 6 million compounds. These data are provided in the
Supplementary Information. In order to show concentrations of mix-
tures in terms of molarity, we used an average molecular weight of 500
g/mol for each compound in the mixture. For example, we considered
a 5 g/mL to equate to a 10 M solution. Actual concentrations were
used for the evaluation of single compounds.
i.m. is the infected macrophage assay. cytotox is the cytotoxi-
city in J774 macrophage cells. SI is the selectivity index (cyto-
tox/i.m.).
10.
Siqueira-Neto, J. L.; Moon, S.; Jang, J.; Yang, G.; Lee, C.;
Moon, H. K.; Chatelain, E.; Genovesio, A.; Cechetto, J.; Freitas-Jr, L.
H., An image-based high-content screening assay for compounds tar-
geting intracellular Leishmania donovani amastigotes in human mac-
rophages. PLoS Neglected Trop. Dis. 2012, 6, e1671.
ASSOCIATED CONTENT
Supporting Information
11.
Santos, R. G.; Appel, J. R.; Giulianotti, M. A.; Edwards, B.
The Supporting Information is available free of charge on the ACS
Publications website.
S.; Sklar, L. A.; Houghten, R. A.; Pinilla, C., The mathematics of a
successful deconvolution: a quantitative assessment of mixture-based
combinatorial libraries screened against two formylpeptide receptors.
Molecules 2013, 18, 6408-24.
Experimental details for the preparation and characterization of all
compounds and how the assays were performed (PDF)
12.
Fleeman, R.; LaVoi, T. M.; Santos, R. G.; Morales, A.;
Nefzi, A.; Welmaker, G. S.; Medina-Franco, J. L.; Giulianotti, M. A.;
Houghten, R. A.; Shaw, L. N., Combinatorial Libraries As a Tool for
the Discovery of Novel, Broad-Spectrum Antibacterial Agents Target-
ing the ESKAPE Pathogens. J. Med. Chem. 2015, 58, 3340-3355.
AUTHOR INFORMATION
Corresponding Author
1
3.
To facilitate screening, each sample was run as single data
Corresponding Author
points, with miltefosine (3) as a control on each plate. The IC50 for this
standard is 1.43 ± 0.20 M, and no plates contained significant outliers
for the control.
*Phone: (813) 974-4642. Email jwleahy@usf.edu.
Funding Sources
This work was supported in part by the Florida Drug Discovery
Acceleration Program, State of Florida Department of Department
of Health. We also gratefully acknowledge support for this re-
search from the University of South Florida Office of Research
and Innovation and the Florida Center of Excellence for Drug
Discovery and Innovation.
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ACS Paragon Plus Environment

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