Synthesis and Structure?Activity Relationships of Imidazopyridine/Pyrimidine- and Furopyridine-Based Anti-infective Agents against Trypanosomiases

Article abstract of DOI:10.1002/cmdc.202000616

Neglected tropical diseases remain among the most critical public health concerns in Africa and South America. The drug treatments for these diseases are limited, which invariably leads to fatal cases. Hence, there is an urgent need for new antitrypanosomal drugs. To address this issue, a large number of diverse heterocyclic compounds were prepared. Straightforward synthetic approaches tolerated pre-functionalized structures, giving rise to a structurally diverse set of analogs. We report on a set of 57 heterocyclic compounds with selective activity potential against kinetoplastid parasites. In general, 29 and 19 compounds of the total set could be defined as active against Trypanosoma cruzi and T. brucei brucei, respectively (antitrypanosomal activities <10 μM). The present work discusses the structure?activity relationships of new fused-ring scaffolds based on imidazopyridine/pyrimidine and furopyridine cores. This library of compounds shows significant potential for anti-trypanosomiases drug discovery.

Full text of DOI:10.1002/cmdc.202000616

Accepted Article
Title: Synthesis and structure-activity relationships of imidazopyridine/
pyrimidine- and furopyridine-based anti-infective agents against
trypanosomiases
Authors: Daniel Gedder Silva, Anna Junker, Shaiani M. G. de Melo,
Fernando Fumagalli, J. Robert Gillespie, Nora Molasky,
Frederick S. Buckner, An Matheeussen, Guy Caljon, Louis
Maes, and Flavio S. Emery
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000616
Link to VoR: https://doi.org/10.1002/cmdc.202000616
01/2020

10.1002/cmdc.202000616
ChemMedChem
FULL PAPER
Synthesis
and
structure-activity
relationships
of
imidazopyridine/pyrimidine- and furopyridine-based anti-infective
agents against trypanosomiases
[a]
[c]
Daniel G. Silva,*[a, b] Anna Junker, [b] Shaiani M. G. de Melo,
Fernando Fumagalli,
J. Robert
Gillespie, [d] Nora Molasky, [d] Frederick S. Buckner, [d] An Matheeussen, [e] Guy Caljon, [e] Louis Maes,[e]
and Flavio S. Emery* [a]
[a]
Dr. D. G. Silva, Ms. S. M. G. de Melo, Prof. F. S. Emery
QHeteM - Laboratório de Química Heterocíclica e Medicinal, School of Pharmaceutical Sciences of Ribeirão Preto - University of São Paulo, Ribeirão
Preto, São Paulo 14040-903, Brazil *corresponding authors: danielgedder@usp.br (D. G. Silva); flavioemery@usp.br (F. S. Emery)
[b]
Dr. A. Junker
European Institute for Molecular Imaging (EIMI), Westphalian Wilhelms-University Münster-D-48149, North Rhine-Westphalia, Germany
[c]
Prof. F. Fumagalli
Centro de Ciências da Saúde (CCS), Universidade Federal de Santa Maria (UFSM), Av. Roraima, 1000, Camobi, Santa Maria, Rio Grande do Sul,
97105-900, Brazil
[d]
J. R. Gillespie, N. Molasky, Prof. F. S. Buckner
Department of Medicine, University of Washington, Seattle, Washington 98195, United States
[e]
A. Matheeussen, Prof. G. Caljon, Prof. L. Maes
Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Universiteitsplein 1, Wilrijk B-2610, Belgium.
Supporting information for this article is given via a link at the end of the document.
Abstract: Neglected Tropical Diseases remain among the most
critical public health concerns in Africa and South America. The drug
treatments for these diseases are limited which leads to invariably
fatal cases. Hence, the need for new antitrypanosomal drugs
remains a current urgency. To address this issue, a large number
and diverse heterocyclic compounds were prepared. Straightforward
synthetic approaches tolerated pre-functionalized structures, giving
rise to a structurally diverse set of analogs. We report on a set of 57
heterocyclic compounds with selective activity potential against
kinetoplastid parasites. In general, 29 and 19 compounds of the total
set could be defined as active against Trypanosoma cruzi and
T. brucei brucei, respectively (antitrypanosomal activities < 10 μM).
The present work discusses the structure-activity relationships of
new fused ring scaffolds based on imidazopyridine/pyrimidine and
furopyridine cores. This library of compounds shows significant
potential for anti-trypanosomiases drug discovery.
A previous phenotypic high-throughput screening of a
700,000 compounds library performed by the Genomics Institute
of the Novartis Research Foundation (GNF) led to the
identification of 1035 compounds that inhibited in vitro growth of
T. brucei at concentrations below 3.6 µM and were non-toxic to
mammalian cells (Huh7). The lead compound A (thiazol-2-
ethylamine derivative) was one of the scaffolds selected for
optimization by researchers at the University of Washington and
[10]
North Carolina at Chapel Hill (UNC).
The most potent and
selective compound A against T. brucei showed poor metabolic
stability (Figure 1) as demonstrated by its short half-life in mice
[11]
and human liver microsomes.
To overcome the metabolic
[12]
stability issues, Patrick and coworkers
synthesized a new
series of compounds based on urea derivatives of 2-aryl-
benzothiazol-5-amines.
The most promising benzothiazole compound B was a
[12]
new lead against T. brucei (EC50 of 0.03 µM; Figure 1).
Guided by these previous studies Silva and coworkers [13] further
modified the benzothiazole core of B to the imidazopyrimidine
rings in compounds C and D. A basic nitrogen atom inserted at
the 6-position of the imidazopyrimidine core resulted in the most
significant increase in activity with a 1000-fold change in EC50
against T. brucei (C vs D). The main results of our previous
works [11-14] are summarized in Figure 1.
Based on promising in vitro activity, cytotoxicity, metabolic
stability, protein binding and pharmacokinetic (PK) properties,
the imidazo[1,2-c]pyrimidine derivative D was selected as a
candidate for in vivo follow-up studies in an acute mouse model
of T. cruzi infection (Tulahuen strain). After established infection,
Introduction
Trypanosomiases caused by the unicellular protozoan
parasites Trypanosoma brucei and T. cruzi are economically
significant obstacles to human welfare. Human African
Trypanosomiases (HAT) caused by T. brucei occurs in Sub-
Saharan Africa, while Chagas disease caused by T. cruzi is a
devastating disease in Latin America.
treatments available for these diseases are toxic, prone to
resistance, and poor/limited efficacy. [2-7] Developing a new drug
is a laborious challenge, and investments dedicated to the
identification of new chemical entities (NCEs) for the treatment
of infectious diseases are insufficient. [8, 9] Existing infrastructures
to prevent and combat Chagas disease and HAT are largely
inadequate and therefore, these have been classified as
Neglected Tropical Diseases (NTDs).
[1]
The current drug
mice were dosed twice daily for 5 days and monitored for 6
[13]
weeks using an in vivo imaging system (IVIS)
demonstrating
parasite inhibition comparable to benznidazole. Scaffold D had
alterations primarily at either of the two terminal rings, but the
1
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10.1002/cmdc.202000616
ChemMedChem
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[22]
SAR of the internal portion of the molecule remained largely
unexplored. [13]
resistant strains of mycobacteria for tuberculosis.
However,
this class of compound has not yet been employed for
antitrypanosomal agents.
To enlarge the chemical space of heterocycles as potential
anti-infective agents against trypanosomiases, we aimed to
explore, develop, and modify the central core of the hit
compound D (bottom of Figure 1) and perform SAR analysis
based on biological assays against T. cruzi, T. brucei, and
mammalian cells.
Results and Discussion
Designed library and synthesis of heterocycles
The biochemical targets or mechanisms of action of the hit
compound D are unknown. A new set of compounds was
designed by dividing the general scaffold (Figure 1) into regions
and specific insertion of modifications at the positions V-Z and in
regions R1-R3 on the central core of the fused ring. The
modifications to the general scaffold are shown in Schemes 1
and 2.
A relatively simple synthetic pathway (low cost, few steps
and good yields) was amenable to many functional groups,
giving rise to a structurally diverse set of analogs (Scheme 1).
Synthesis of the compounds 1-5, 7 and 16-19 started with a
condensation
reaction
of
the
appropriate
amino
pyrimidine/pyridine and bromoacetophenone, which resulted in
the intermediate endowed with the imidazopyrimidine or
imidazopyridine cores (I). Following the reaction with
triphosgene, a corresponding isocyanate was obtained, which
provided a way for the formation of urea derivatives D and 15 (II).
The reaction with the appropriate acyl chloride provided the
amide derivatives 8-14 (II). Upon Mannich reaction, we explored
aminoalkylations in region R2 of the imidazopyrimidine/pyridine
scaffold III (analogs 6, 20-40, 56 and 57).
Compounds 1-3 are imidazopyrimidine fragment-like
containing a free amine group (R1) and bearing different phenyl
groups at the 2-position of the imidazole portion of the backbone.
Compound 4 is an analog of compound 2 without the free amine
and the pyrimidine nitrogen at the position W. Compounds 5-7
are imidazopyridine fragment-like bearing 3,4-difluorophenyl (5
and 6) and phenyl groups (7) at the 2-position of the imidazole
region of the backbone (R3). In addition, compound 6 has a
(dimethylamine)methyl in region R2 and compound 7 has a
methyl group at the position R1 and X. Analogs 8-14 are
imidazopyrimidine amides retaining a phenyl substituent on the
same imidazole portion mentioned before and showing
modifications in region R1. Compound D and 15 have an
electron withdrawing substituents on the imidazopyrimidine
system (R3) with a 3-fluoropyrrolidinyl urea in region R1.
Figure 1. TOP: initial compound designed for the treatment of HAT (A).
BELOW: benzothiazole B and imidazopyrimidines C and D compounds.
Antitrypanosomal activities highlighted in green and orange. BOTTOM: set of
compounds planned, synthesized, and assayed against T. cruzi, T. brucei and
mammalian cells in the present work.
Drug discovery and development is a complex endeavor
since new candidates need to meet acceptable pharmacological
endpoints combined with favorable safety profiles. Moving from
target identification to lead generation is laborious, but our
compound D may represent a potential hit for trypanosomiases
[13] and was therefore studied further in the present work.
Compounds 16-19 have
a
6-methyl-2-phenylimidazo[1,2-
a]pyridine as the central core and diverse phenyl groups in
region R3. Additionally, a set of 20 alkylamine-type compounds
in the side chain of the imidazo[1,2-a]pyridine core ring was
synthesized. The synthetic route provided efficient ways to
introduce substituents via the Mannich reaction at region R2
(analogs 21-40). The hit compound D embedded with the 4-
fluorophenylpiperazinyl group resulted in compound 56.
Compounds 20 and 57 are side products of the Mannich
reaction, and we isolated and purified these compounds as well.
We took the opportunity to employ robust synthetic routes
Imidazopyridine/pyrimidine fused rings have been
employed in drugs such as antipsychotics, anxiolytics,
analgesics, and migraine therapeutics, demonstrating drug-like
[10, 15-17]
features associated with the core structures.
The
furo[2,3-b]pyridine has recently received extensive attention as a
[18-21]
useful pharmacophore in different therapeutic areas.
We
also identified promising selective bioactive compounds
containing furopyridine as a central core against different drug-
2
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Scheme 1. Synthetic routes and sets of modifications into the imidazopyridine/pyrimidine I (~21-92%), II (~54-86%) and Mannich-type scaffolds III (~38-97%).
Reagents and conditions: a) appropriate bromoacetophenone, NaHCO3, MeOH, reflux, 12 h; b) triphosgene, Et3N, CH2Cl2, 0°C and then appropriate 2° amine,
0°C to 25°C, 15 h or c) appropriate acyl chloride, Et3N, CH2Cl2, 0°C to 25°C, 16 h; d) appropriate amine, formaline, acetic acid, CH2Cl2, 18 h.
Scheme 2. Synthetic routes and sets of modifications into the furopyridine scaffold (V: ~14-41%; VI: 18-67%; VII: 87%; VIII: 44%). Reagents and conditions: e)
appropriated acyl chloride, DBU, DMAP, CH2Cl2, rt, overnight; f) mCPBA, CH2Cl2, rt, 48 h; g) appropriate 2° amine, PyBroP, DIPEA, MeCN, rt, 18 h; h) LiOH,
THF/EtOH/H2O, 55°C, 24 h; i) MeNH∙HCl, EDCI, HOBt, DMF, Et3N, 25°C, N2, 4 h.
to further alter the internal portion of the imidazopyridine core
and introduce different functional groups at different positions of
the general scaffold, which led to the fused ring furopyridine
derivatives 41-55 (Scheme 2). Recently our group reported a
concise strategy to synthesize and decorate this core through
addition, we functionalized this heteroaromatic core through
C−H amination (VI, compounds 45-53). Carboxylic acid
derivative 54 (VII) was obtained through ester hydrolysis. The
following coupling reaction afforded the amide derivative 55 (VII).
Analogs 41-45 are furopyridines bearing different phenyl groups
in region R3 of the backbone (2-position) and an ethyl ester
group in the side chain in region R2 of the furan moiety (3-
position). Compounds 46-55 have a methyl or isopropyl group in
C−H activation reaction. [23] The synthesis of furo[2,3-b]pyridines
[23]
started from pyridine-N-oxide derivative (IV).
Under mild,
metal-free conditions, we synthesized the furopyridines
derivatives 41-44 with appropriated acyl chlorides (V). In
3
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10.1002/cmdc.202000616
ChemMedChem
FULL PAPER
region R3 and N-cycloalkyl groups at different positions (R1, V or
X).
Fast analysis of the antitrypanosomal activities reveals that
14 compounds are totally inactive against both parasites (EC50-
values of T. cruzi and T. brucei > 20.0 µM). Additional 17
compounds are also inactive against T. brucei, but some of them
displayed high potencies against T. cruzi. Notably, this set of
compounds exhibited better T. cruzi than T. brucei activities.
Twenty-nine compounds showed anti-T. cruzi potency and 19
compounds showed anti-T. brucei potency lower than 10 µM. It
suggests that a more detailed analysis of the underlying
structure-activity relationships would be of interest.
Antitrypanosomal activities and cytotoxicity
[24]
Compounds 1-55 were tested in vitro against T. cruzi
and T. brucei using EC50 and EC90 protocols.
[25]
Assay results
are presented in Table 1. Additionally, we retested the hit
compound D and the new compounds 56 and 57 using IC50
protocols against T. cruzi and T. brucei (Table 1). Experimental
details are provided in Supporting Information and in the
literature. [24-26]
Table 1. Antitrypanosomal activities of heterocyclic compounds
T. cruzi
T. brucei
V, W, X = CH
R1
R2
R3
unless otherwise noted
EC50
EC90
(µM)*
EC50
EC90
(µM)*
> 20.0
> 20.0
(µM)**
> 20.0
> 20.0
(µM)**
1
2
NH2
NH2
H
H
phenyl
V = N
V = N
> 20.0
> 20.0
> 20.0
> 20.0
3,4-difluorophenyl
3-
3
NH2
H
V = N
> 20.0
> 20.0
> 20.0
> 20.0
trifluoromethylphenyl
4
5
H
H
H
3,4-difluorophenyl
3,4-difluorophenyl
3,4-difluorophenyl
phenyl
W = N
> 20.0
> 20.0
> 20.0
7.2
> 20.0
> 20.0
> 20.0
13.4
> 20.0
> 20.0
> 20.0
> 20.0
> 20.0
> 20.0
3.9
> 20.0
> 20.0
> 20.0
> 20.0
> 20.0
> 20.0
10.9
H
-
6
H
CH2N(CH3)2
-
7
CH3
H
H
H
H
H
H
H
H
X = CCH3
8
phenoxy
phenyl
-
-
-
-
-
-
-
> 20.0
> 20.0
9.8
> 20.0
> 20.0
> 20.0
> 20.0
7.3
9
4-nitrophenoxy
3-nitrophenoxy
benzyl
phenyl
10
11
12
13
14
phenyl
phenyl
14.2
> 20.0
12.3
> 20.0
> 20.0
> 20.0
> 20.0
cyclohexylmethyl
4-cyanophenyl
3,4-difluoro phenyl
phenyl
6.3
phenyl
10.3
> 20.0
> 20.0
4.4
phenyl
10.5
> 20.0
(3S)-3-
fluoropyrrolidin-1-yl
3-
15
H
-
0.08
0.09
0.03
0.05
trifluoromethylphenyl
16
17
18
19
20
21
22
23
-
-
-
-
-
-
-
-
H
phenyl
4-methylphenyl
4-chlorophenyl
3,4-difluorophenyl
3,4-difluorophenyl
phenyl
-
-
-
-
-
-
-
-
> 20.0
>5.0
> 20.0
> 5.0
> 20.0
5.9
> 20.0
> 20.0
> 20.0
> 20.0
> 20.0
> 20.0
> 20.0
> 20.0
H
H
>5.0
> 5.0
10.4
H
CH2OH
>5.0
> 5.0
12.3
>5.0
> 5.0
> 20.0
> 20.0
> 20.0
> 20.0
CH2N(CH3)2
> 20.0
> 20.0
> 20.0
> 20.0
> 20.0
> 20.0
3-(trifluoromethyl)phenylaminemethyl
pyrrolidin-1-ylmethyl
phenyl
phenyl
4
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FULL PAPER
T. cruzi
T. brucei
V, W, X = CH
R1
R2
R3
unless otherwise noted
EC50
(µM)*
EC90
(µM)*
EC50
EC90
(µM)**
> 20.0
> 20.0
> 20.0
9.9
(µM)**
24
25
26
27
28
-
-
-
-
piperidin-1-ylmethyl
morpholin-4-ylmethyl
phenyl
phenyl
phenyl
phenyl
-
-
-
-
15.0
13.4
8.6
> 20.0
> 20.0
> 20.0
9.7
> 20.0
> 20.0
> 20.0
10.1
(3S)-3-fluoropyrrolidin-1-ylmethyl
4-phenylpiperazin-1-ylmethyl
5.2
4-(2,3-dichlorophenyl)piperazin-1-
ylmethyl
-
phenyl
-
3.3
6.5
9.9
10.1
29
30
31
32
-
-
-
4-(4-chlorophenyl)piperazin-1-ylmethyl
4-phenylpiperazin-1-ylmethyl
phenyl
-
-
-
1.9
1.6
1.0
6.5
3.6
3.6
7.2
8.0
6.2
8.9
3,4-difluorophenyl
3,4-difluorophenyl
11.6
11.8
4-(4-fluorophenyl)piperazin-1-ylmethyl
4-[3-(trifluoromethyl)phenyl]piperazin-
1-ylmethyl)
-
-
-
-
-
-
-
3,4-difluorophenyl
4-chlorophenyl
-
-
-
-
-
-
-
1.6
1.1
1.7
1.0
1.5
2.6
1.8
4.1
3.3
3.3
5.9
4.6
12.8
33
34
4-(4-fluorophenyl)piperazin-1-ylmethyl
4-[3-(trifluoromethyl)phenyl]piperazin-
1-ylmethyl)
4-chlorophenyl
> 5.0
3.4
6.6
11.4
35
36
4-(4-fluorophenyl)piperazin-1-ylmethyl
4-methylphenyl
4-methylphenyl
3,4-difluorophenyl
3,4-difluorophenyl
6.9
7.8
4-[3-(trifluoromethyl)phenyl]piperazin-
1-ylmethyl)
> 5.0
> 5.0
> 5.0
5.2
> 20.0
> 20.0
> 20.0
37
38
4-cyclohexylpiperazin-1-ylmethyl
> 20.0
> 20.0
4-[2-(pyridin-3-yl)ethyl]piperazin-1-
ylmethyl
39
40
41
42
43
44
45
-
4-benzylpiperidin-1-ylmethyl
4-phenylpiperidin-1-ylmethyl
COOCH2CH3
3,4-difluorophenyl
3,4-difluorophenyl
phenyl
-
-
-
-
-
-
1.6
1.1
4.7
16.6
7.6
18.1
9.6
-
> 5.0
> 20.0
> 20.0
> 20.0
18.9
H
H
H
H
14.7
13.8
10.9
8.6
> 20.0
18.9
10.3
17.5
> 20.0
> 20.0
> 20.0
> 20.0
COOCH2CH3
4-fluorophenyl
3,4-difluorophenyl
4-methylphenyl
COOCH2CH3
COOCH2CH3
(3S)-3-
fluoropyrrolidin-1-yl
COOCH2CH3
COOCH2CH3
COOCH2CH3
4-methylphenyl
-
-
10.9
17.3
18.9
> 20.0
> 20.0
> 20.0
> 20.0
> 20.0
14.5
> 20.0
> 20.0
> 20.0
46
47
(3S)-3-
fluoropyrrolidin-1-yl
CH3
CH3
V = C-(3S)-3-
fluoropyrrolidin-1-yl
H
48
49
50
morpholin-4-yl
morpholin-4-yl
COOCH2CH3
COOCH2CH3
CH3
-
-
17.4
4.8
> 20.0
18.4
> 20.0
> 20.0
> 20.0
> 20.0
C(CH3)2
4-phenylpiperazin-
1-yl
COOCH2CH3
COOCH2CH3
COOCH2CH3
CH3
-
-
3.4
11.0
7.1
6.4
> 20.0
9.2
> 20.0
> 20.0
9.8
> 20.0
> 20.0
13.8
51
52
4-phenylpiperazin-
1-yl
C(CH3)2
C(CH3)2
X = C-4-
phenylpiperazin-1-yl
H
53
54
pyrrolidin-1-yl
pyrrolidin-1-yl
pyrrolidin-1-yl
COOCH2CH3
COOH
C(CH3)2
C(CH3)2
C(CH3)2
-
-
-
4.5
> 20.0
> 20.0
> 20.0
> 20.0
> 20.0
> 20.0
> 20.0
> 20.0
> 20.0
> 20.0
> 20.0
55
CONHCH3
***56
(3S)-3-
fluoropyrrolidin-1-yl
4-(4-fluorophenyl)piperazin-1-ylmethyl
3,4-difluorophenyl
3,4-difluorophenyl
3,4-difluorophenyl
-
-
-
10.42
18.97
0.73
-
-
-
0.95
1.21
0.21
-
-
-
***57
***D
(3S)-3-
fluoropyrrolidin-1-yl
CH2OH
H
(3S)-3-
fluoropyrrolidin-1-yl
Compounds 1-55: * the values are averages of triplicate data. Benznidazole was used as a control compound for the T. cruzi assay, with EC50 and EC90 values (average ± SEM) 0.65 ± 0.10
µM (n=5) and 1.64 ± 0.09 µM (n=5). ** Pentamidine was used as a control compound for the T. brucei assays, with EC50 and EC90 values (average ± SEM) of 1.24 ± 0.23 nM (n=5) and 6.20
± 2.12 nM (n=5). Compounds 56, 57 and D: ***activity is expressed as IC50-values in μM concentrations. The values of T. cruzi and T. brucei activities are averages of duplicate data
(average ± SD). Benznidazole (IC50 of 2.43 ± 0.45 µM) and Suramine (IC50 of 0.06 ± 0.01 µM) were used as reference drugs to measure T. cruzi and T. brucei activities, respectively.
Furthermore, cytotoxicity was evaluated in mammalian
cells (lymphocytic cells - CRL-8155 and hepatocellular cells -
HepG2) and selectivity index (SI) calculated for selected
compounds and compared to the published results for
compound D [13] (Table 2).
remaining compounds exhibited no considerable or detectable
toxicity to either cell line.
The selectivity ratio of compounds for T. cruzi and T.
brucei parasites over each of the two cell lines was calculated.
As an example, the most active compound 15 exhibited a
selectivity index (SI) >625 for T. cruzi and >1667 for T. brucei
against either cell line, comparable to the hit compound D.
Only compounds 28 and 52 exhibited low toxicity to the
CRL-8155 cell line, at 33.0 and 23.70 μM, respectively. The
5
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Table 2. Cytotoxicity and Selectivity index (SI) for Selected Compounds
T. cruzi
EC50 (µM)
0.09
0.08
>5.0
> 20.0
> 20.0
15.0
13.4
8.6
T. brucei
EC50 (µM)
0.02
Hep G2a,b
EC50 (µM)
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
-
SIe
CLR-8155c,d
EC50 (µM)
> 50.0
> 50.0
41.3
SIe
T. cruzi
556
625
10
3
T. brucei
T. cruzi
556
625
8
T. brucei
***D
15
17
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
38
39
40
44
45
49
50
52
53
2500
2500
1667
7
0.03
1667
8
5.9
> 20.0
> 20.0
> 20.0
> 20.0
> 20.0
9.9
3
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
33.0
3
3
3
3
3
3
3
3
3
3
4
3
4
3
6
3
6
3
5.2
10
15
26
31
49
30
43
29
-
5
10
10
26
31
42
30
43
29
50
32
28
30
45
6
5
3.3
9.9
5
3
1.9
7.2
7
49.0
7
1.6
8.0
6
> 50.0
42.9
6
1.0
6.2
8
7
1.6
3.3
15
8
> 50.0
> 50.0
> 50.0
> 50.0
47.6
15
8
1.1
5.9
1.7
6.6
8
8
1.0
6.9
-
7
1.50
1.8
5.2
-
-
-
9
> 20.0
16.6
40.6
23
30
45
6
2
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
23.7
3
1.6
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
> 50.0
3
3
1.1
7.6
7
7
8.6
17.5
3
3
10.9
4.8
> 20.0
> 20.0
> 20.0
9.8
5
3
5
3
10
15
7
3
10
15
3
3
3.4
3
3
7.1
5
2
4.5
> 20.0
11
3
> 50.0
11
3
a Human hepatocytes (HepG2). Compounds were tested in quadruplicate. b Quinacrine was included as a control compound, with EC50 and EC90 values (average ± SEM) of 14.34±4.51 μM
(n=3) and 18.74±7.05 μM (n=3). c Human lymphoblasts (CRL-8155). Compounds were tested in quadruplicate. d Quinacrine was included as
a control compound, with EC50 and
EC90 values (average ± SEM) of 5.47±1.97 μM (n=3) and 15.74±4.54 μM (n=3). e Selectivity index expressed as the ratio EC50 (cell line)/EC50 (T. cruzi or T. brucei), rounded to the nearest
integer. ***reference 13.
Compounds D, 56 and 57 were also screened against T. b.
rhodesiense, Leishmania infantum and cytotoxicity for MCR-5
were explored with 14 variants. At the left of Figure 2 the SAR
analysis for the region R1 is presented. At the bottom and top
right the SAR analysis for the region R2 and variants for the
region R3, respectively. Analogs 1-15 were assayed against T.
cruzi (highlighted in green), T. brucei (highlighted in orange) and
the results are expressed as EC50 (µM).
and PMM cells. Besides the already known biological activity of
[13]
the compound D against T. cruzi and T. brucei,
this
compound also exhibited high biological activity against T. b.
rhodesiense (IC50 of 0.11 μM). Compounds 56 and 57 showed
anti-T b. rhodesiense activities of 0.90 μM and 1.06 μM,
respectively. However, these three compounds were not potent
against L. infantum. Marginal cytotoxicity was observed only for
compound D against PMM cells (IC50 of 48.0 μM) and no
cytotoxicity against MRC-5 cell line.
Compounds 1 and 8-14 bear a phenyl group in region R3
and hydrogen atom in region R2. Thus, the evaluation of the
SAR was based on replacements in region R1. The addition of a
phenyl carbamate group (1 vs 8) or even the introduction of a
nitro group at para-position of the phenyl moiety (8 vs 9) had no
effect in the potency against both parasites. However, moving
the nitro group to meta-position enhanced the potency against T.
brucei by more than 5-fold (9 vs 10). Compound 10 showed an
EC50 value against T. brucei of 3.88 µM. Replacing the oxygen
atom in the phenyl carbamate group for a methylene linker (8 vs
11) resulted in low anti-T. cruzi activity (11, EC50 T. cruzi of
14.20 µM). It appears that the cyclic aliphatic amide is better
than an aromatic group in the region R1 for achieving potency
against T. cruzi (8 vs 12). Cyclohexylacetamide derivative 12
had an EC50 value against T. cruzi of 6.27 µM.
Structure-Activity Relationships (SAR)
We discussed the SAR of heterocyclic compounds based
on the wide range of biological activity obtained against T. cruzi
[24]
[25]
and T. brucei
for compounds D and 1-57. In order to
accomplish this in a feasible way, we divided and discussed the
substitutions systematically introduced into specific regions (R1-
R3) and positions (V-Z) of
3 distinct general scaffolds:
imidazopyrimidine (15 analogs), imidazopyridine (28 analogs)
and furopyridine (15 analogs). The following, we discuss the
most representatives SARs established in this work.
The attempts to optimize the imidazopyrimidine scaffold is
shown in Figure 2. The different regions of the molecule (R1-R3)
T. cruzi activity was not affected by the exploration of the
substitution pattern on the aromatic ring. Compounds 13 (R1 = 4-
cyanophenyl) and 14 (R1 = 3,4-difluoro phenyl) showed an EC50
value against T. cruzi around 10.0 µM, although the effect
6
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Figure 2. Modifications and EC50 µM of the imidazopyrimidine core ring. Compounds assayed against T. cruzi (highlighted in green)/T. brucei (highlighted in
orange). All the results are expressed as EC50 (µM); except for compounds D, 56 and 57 IC50 (µM). Green and orange arrows represent the improvement of the T.
cruzi and T. brucei activities, respectively. Green and orange dashed arrows represent the loss of the T. cruzi and T. brucei activities, respectively. Gray arrow
represents no improvement of the antitrypanosomal activities.
of the cyano group was more pronounced against T. brucei
(EC50 T. brucei of 4.45 µM) than against T. cruzi (EC50 T. cruzi of
10.33 µM).
lipophilicity (log-P), solubility and through hydrogen-bonding
[30]
interactions, the mode of binding of the pharmacophore.
In
these two compounds the combination of the best moiety
present in region R1 and R3 of analog D, plus the alteration of
the imidazo portion has not shown to be synergistic, i.e.
compounds 56 and 57 risen from this approach have shown
bioactivities lower than D. Aiming to explore and evaluate the
SAR for modifications only in region R2, a neutral scaffold was
chosen. That means an absence of substituents in region R1 and
an exchange of a basic nitrogen atom for a methyl group at the
6-position of the imidazopyrimidine core (Figure 2); which
Compounds
3
and 15 are analogs bearing 3-
trifluoromethylphenyl substituent in region R3 and hydrogen
atom in region R2. Substantial enhancement of activity was
achieved by the introduction of a 3-fluoropyrrolidinyl urea group
in region R1 of the imidazopyrimidine system. This substitution
enhanced the potency of compound 15 more than 250-fold
against T. cruzi and more than 660-fold against T. brucei, when
compared to compound 3 (R1 = NH2). Compound 15 exhibited
an EC50 value against T. cruzi of 0.08 µM and against T. brucei
of 0.03 µM (Figure 2).
turned into
a new scaffold 6-methylimidazo[1,2-a]pyridine
(Figure 3).
The same pattern can be observed for the amide
derivatives compared to the urea derivatives D and 15, the
presence of the urea group attached to the region R1 of the
fused ring system proved to be essential for bioactivity.
Compounds D and 15 are nanomolar inhibitors of T. cruzi and T.
brucei, while some of the amide derivatives showed moderate
activity only against one of these parasites.
The introduction of dimethylmethanamine (21), 3-
(trifluoromethyl)phenylaminemethyl (22) and pyrrolidinylmethyl
(23) as substituents in region R2, resulted in inactive compounds
(Figure 3). Replacements with piperidinylmethyl (24, EC50 of
15.04 µM), morpholinylmethyl (25, EC50 of 13.40 µM) and (3S)-3-
fluoropyrrolidinylmethyl groups (26, EC50 of 8.61 µM) showed
only low potency against T. cruzi (Figure 3, highlighted in green).
However, the insertion of a phenylpiperazinylmethyl moiety in
region R2 of the imidazole portion (Figure 3, bottom right)
showed to be promising to enhance antitrypanosomal activity
(16 vs 28 and 29). The introduction of chloride atoms at the
phenyl group attached at the piperazine at the 4- and 2,3-
According to the literature compounds bearing methyl-N,N-
substituted amines (phenylpiperazines) could be an attractive
group to increment potency for anti-infective agents against
[27-29]
trypanosomiases.
We then used the strategy of combining
chemical substructures found in the literature, with the highly
active compound D to derive new compounds, but keeping the
positions
enhanced
only
Anti-T.
cruzi
potency.
skeleton
unaltered.
Embedding
the
4-
Phenylpiperazines derivatives 28 and 29 exhibited EC50 values
of 3.31 and 1.90 µM, respectively. In fact, upon embedding
phenylpiperazinylmethyl in region R2 of the imidazole portion an
enhancement in potency for the T. brucei compared to T. cruzi
potencies was not observed. While some compounds showed
EC50 about 1.0 µM against T. cruzi, most of the compounds
showed EC50 above to 5.0 µM against T. brucei.
Bulky aminoalkyl groups in the side chain of
imidazopyridine derivatives are clearly required for better anti-T
cruzi activity. About 11 compounds bearing bulky aminoalkyl
groups achieved potency lower than 2 µM only against T. cruzi.
fluorophenylpiperazinylmethyl (R2) into the compound D with the
combination of 3-fluoropyrrolidinyl urea group in region R1 and
3,4-difluorophenyl group in region R3 did not improve the
antitrypanosomal activities (D vs 56). The activity of compound
56 decreased more than 4-fold against both parasites.
The incorporation of the hydroxymethyl functional group
(−CH2OH) in region R2 (57) resulted in similar antitrypanosomal
activities as compound 56. However, hydroxymethyl group can
be a versatile point of diversification to access other target
compounds; to alter physicochemical properties such as
7
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10.1002/cmdc.202000616
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Figure 3. Modifications of the 6-methyl-2-phenylimidazo[1,2-a]pyridine core ring. Compounds assayed against T. cruzi (highlighted in green)/T. brucei (highlighted
in orange). Results are expressed as EC50 (µM). Green arrows represent the improvement of the T. cruzi activity. Orange dashed arrows represent the loss of the
T. brucei activity. Gray arrows represent no improvement of the antitrypanosomal activities
Moreover, inserting electron-withdrawing groups (31 and 32) or
exchanging the aromatic portion of the piperazine (37-39) did
not modify the potency against T. cruzi (Figure 3, bottom left).
At the same time, the activities of compounds 37-39 reveal that
the presence of more flexible groups in region R2 did not
contribute to enhancing the potency against T. brucei. The
compounds display above 7 times better T. cruzi potency than
T. brucei potency.
Furthermore, the SAR were explored diversifying phenyl
groups in region R3 of the imidazole portion. The methylation
(17), chlorination (18) or difluorination (19) conferred a slight
improvement in potencies compared to 16 (Figure 3, top right).
A similar pattern was observed for the pairs of compounds
containing replacements of the aromatic ring attached in region
R3 (pairs: 31 vs 33; 32 vs 34; 31 vs 35; 32 vs 36, Table 1).
Subsequently, altering the core ring system from an
imidazopyridine/pyrimidine to a furo[2,3-b]pyridine allowed us to
expand the chemical space of new heterocyclic compounds as
potential antitrypanosomal agents. We selected substituents
from our previous SAR and we also introduced new
modifications into the core ring of furopyridines, such as aliphatic
groups in region R3 or functional groups in region R2. Figure 4
shown the selected SAR analysis for each region.
necessary for the activity of this class against T. cruzi (53 vs 54,
55). We further evaluated the amine fragments, such as
morpholine, phenylpiperazine and (3S)-3-fluoropyrrolidinyl, in
the activity of our furopyridine framework (46 and 48 vs 50).
Repeatedly in this SAR studies, the phenylpiperazine moiety has
appeared to be promising to achieve gains in potency against T.
cruzi (Figure 4, top left). Compound 50 (EC50 of 3.40 µM) is 5-
fold more potent than the derivatives containing (3S)-3-
fluoropyrrolidinyl (46, EC50 of 17.29 µM) and morpholinyl groups
(48, EC50 of 17.36 µM). The relocation of the (3S)-3-
fluoropyrrolidinyl or phenylpiperazine moiety did not result in any
benefits to antitrypanosomal activities (47 and 52). Meanwhile,
the phenylpiperazine attached at the X-position was the unique
replacement that resulted in T. brucei activity below 10 µM.
The fragment (3S)-3-fluoropyrrolidinyl which showed to be
an important feature in the imidazopyrimidine framework of this
series of compounds, did not display the same positive influence
in the activity of some furopyridines (45 and 47), no matter the
position placed. Probably, the absence of the functional urea
group in this furopyridine class of compounds could be an
explanation for no enhancement in the potency. In other words,
the imidazopyrimidine series of compounds required a functional
group containing a carbonyl group bonded to two nitrogen atoms.
For example, compounds D and 15 achieved nanomolar
potency against both parasites. In summary, our findings in the
SAR analysis can guide further studies with this class of
compounds.
Fluorination or methylation (41 vs 42-44) of the aromatic
ring attached to the 2-position of furopyridine (R3) had no impact
on the potency of this class of compounds. Despite that,
isopropyl and methyl groups appeared to be more promising
than aromatic groups in this region. Compound 49 and 50
exhibited EC50 values of 4.81 and 3.40, respectively (Figure 4,
top right). We also verified that the ester group in region R2 was
8
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FULL PAPER
Figure 4. Modifications of the furo[2,3-b]pyridine core ring. Compounds assayed against T. cruzi (highlighted in green)/ T. brucei (highlighted in orange). Results
are expressed as EC50 (µM). Green solid or dashed arrows represent the improvement or the loss of the T. cruzi activities, respectively. Gray arrow represents no
improvement of the antitrypanosomal activities.
Conclusion
Flavio da Silva Emery: 0000-0002-8652-7123
The imidazopyridines/pyrimidines and furopyridines
Author Contributions
planned and obtained herein allowed us to build a SAR study by
means of cell assays against T. cruzi, T. brucei, and mammalian
cells. By exploring the chemical diversity of three different
heteroaromatic cores and introducing various groups at eight
different positions of the general scaffold within this study, we
were able to enlarge the chemical space of heterocycles as
potential antitrypanosomal agents. The central core was
embedded with urea, amide, ester, and organic acid functions.
However, the presence of the urea group attached to the 7-
position (R1) of the fused ring system proved to be essential for
bioactivity (D and 15 are nanomolar inhibitors of T. cruzi and
T. brucei). We introduced the furopyridine derivatives as a new
core ring to be developed for antitrypanosomal agents. We
explored aromatic and aliphatic replacements and employed
different organic functions at the 3-position of the furan ring
portion, respectively. In addition, we embedded four different
groups at three positions of the pyridine portion. However, we
still need to combine promising substituents found through SAR
in order to improve the potency of furopyridines as
antitrypanosomal agents. The best replacements identified in
this SAR studies, will guide the design and selection of the novel
compounds that can be transitioned further into drug
development for these parasitic infections.
The manuscript was written through the contributions of all
authors. All authors have given approval to the final version of
the manuscript. DGS planned and synthesized 47 compounds.
SMGM and FF planned and synthesized 10 compounds.
Biological assays were performed by JRB, NM, and AM.
Acknowledgements
We are grateful to Fundacão de Amparo à Pesquisa do Estado
de São Paulo (FAPESP), Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001
and Conselho Nacional de Desenvolvimento Científico
e
Tecnológico
#88881.170193/2018-01
(CNPq)
for
(DGS),
fundings
[grant
numbers:
(DGS),
#2017/22001-0
#2017/21146-4 (FSE), #313834/2018-0 (FSE), #428527/2016-7
(FSE). We thank Professor Dr. Bernhard Wünsch for hosting Dr.
Silva in his Laboratory at the University of Münster (WWU) in
Germany and also for helpful discussions to develop this work.
Conflict of Interest
The authors declare no conflict of interest.
ORCID
Keywords: Anti-infectives • heterocyclic • T. brucei • T. cruzi • in
vitro
Daniel Gedder Silva: 0000-0001-8879-456X
Anna Junker: 0000-0001-5151-0930
Fernando Fumagalli: 0000-0002-0622-4481
J. Robert Gillespie: 0000-0002-5650-0333
Guy Caljon: 0000-0002-4870-3202
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Table of Contents
We have modified the core ring at eight different positions to obtain various new heterocyclic compounds as anti-infective agents.
The work explores the SAR against different Trypanosoma subtypes. The imidazopyridine/pyrimidine- and furopyridine-based
compounds demonstrate high antitrypanosomal activities on parasite cultures and show significant promise for trypanosomiases drug
discovery.
11
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