Design, synthesis, molecular modelling, and in vitro evaluation of tricyclic coumarins against Trypanosoma cruzi

Article abstract of DOI:10.1111/cbdd.13420

Chagas disease is caused by infection with the parasite protozoan Trypanosoma cruzi and affects about 8 million people in 21 countries in Latin America. The main form of treatment of this disease is still based on the use of two drugs, benznidazole and nifurtimox, which both present low cure rates in the chronic phase and often have serious side-effects. Herein, we describe the synthesis of tricyclic coumarins that were obtained via NHC organocatalysis and evaluation of their trypanocidal activity. Molecular docking studies against trypanosomal enzyme triosephosphate isomerase (TIM) were carried out, as well as a theoretical study of the physicochemical parameters. The tricyclic coumarins were tested in vitro against the intracellular forms of Trypanosoma cruzi. Among the 18 compounds tested, 10 were more active than the reference drug benznidazole. The trypanocidal activity of the lead compound was rationalized by molecular docking study which suggested the strong interaction with the enzyme TIM by T.?cruzi and therefore indicating a possible mode of action. Furthermore, the selectivity index of eight tricyclic coumarins with high anti-T.?cruzi activity was above 50 and thus showing that these lead compounds are viable candidates for further in vivo assays.

Full text of DOI:10.1111/cbdd.13420

DR JASON GUY TAYLOR (Orcid ID : 0000-0001-6844-6736)
Article type : Research Article
Design, Synthesis, Molecular Modelling and In Vitro Evaluation of Tricyclic
Coumarins Against Trypanosoma Cruzi
Gleicekelly Silva Coelhoa, Josimara Souza Andradea, Viviane Flores Xavierc, Policarpo
Ademar Sales Juniorb, Barbara Caroline Rodrigues de Araujoa, Kátia da Silva Fonsecac,
Melissa Soares Caetanoa, Silvane Maria Fonseca Murtab, Paula Melo de Abreu Vieirac,
Claudia Martins Carneiroc and Jason Guy Taylora*
a
Chemistry Department, ICEB, Federal University of Ouro Preto, Campus Universitário Morro do
Cruzeiro, 35400-000, Ouro Preto-MG, Brazil.
b René Rachou Institute, FIOCRUZ,30190-002, Belo Horizonte-MG, Brazil.
c
Immunopathology Laboratory, NUPEB, Federal University of Ouro Preto, Campus Universitário
Morro do Cruzeiro, 35400-000, Ouro Preto-MG, Brazil.
* e-mail address of the corresponding author: jason@iceb.ufop.br
Abstract
Chagas disease is caused by infection with the parasite protozoan Trypanosoma cruzi and affects
about 8 million people in 21 countries in Latin America. The main form of treatment of this disease
are still based on the use of two drugs, benznidazole and nifurtimox, which both present low cure
rates in the chronic phase and often have serious side effects. Herein, we describe the synthesis of
tricyclic coumarins that were obtained via NHC organocatalysis and evaluation of their trypanocidal
activity. Molecular docking studies against trypanosomal enzyme triosephosphate isomerase (TIM)
were carried out, as well as a theoretical study of the physicochemical parameters. The tricyclic
coumarins were tested to in vitro against the intracellular forms of Trypanosoma cruzi. Amongst the
18 compounds tested, 10 were more active than the reference drug benznidazole. The trypanocidal
activity of the lead compound was rationalized by molecular docking study which the suggested
strong interaction with the enzyme TIM by T. cruzi and therefore indicating a possible mode of
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doi: 10.1111/cbdd.13420
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action. Furthermore, the selectivity index of 8 tricyclic coumarins with high anti T.cruzi activity was
above 50 and thus showing that these lead compounds are viable candidates for further in vivo assays.
Keywords: Chagas disease; coumarins; Trypanosoma cruzi; triosephosphate isomerase;
isocoumarins; organocatalysis; tricyclic, in silico; in vitro.
1 Introduction
Neglected Tropical Diseases comprise a group of 17 different types of infections
that affect people living on low incomes in mainly developing countries, causing both
economic and health problems in patients and their communities. Chagas disease, discovered
in 1909 by the Brazilian sanitary doctor Carlos Ribeiro Justiniano Chagas, also called
American trypanossomiasis, is an infectious disease caused by the protozoan parasite
Trypanossoma cruzi (T. cruzi) (family Trypanosomatidae, order Kinetoplastida), through
direct contact with contaminated feces and/or urine of insects known as barbers (family
Reduviidae, subfamily Triatominae).[1,2] Recently, it was estimated that 6 to 7 million people
are infected with Chagas disease worldwide, mostly in Latin America, where it is
endemic.[3,4] Human infection also occurs in nonendemic areas because of growing
international immigration and nonvectorial transmission routes such as blood transfusion,
organ transplantation, and congenital infection.[5] T. cruzi has four developmental stages: the
replicative epimastigote and amastigote stages and the infective non-replicative metacyclic
and bloodstream trypomastigote stages.[1] Chagas disease is characterized by for two phases:
acute and chronic. The infection can lead to death due to cardiac arrhythmias or progressive
heart failure caused by the destruction of the heart muscle and nervous system.[1,2] Currently,
only two medicines are approved for the treatment of Chagas disease: nifurtimox (NFX) or
benznidazole (BZ), both of which were released for sale in the early 1970s. [6] Both NFX and
BZ cause undesirable side effects and present low cure rates mainly in the chronic phase of
disease.[7] Although BZ and NFX drugs can reach a 76% cure rate in acute cases of Chagas
disease, their efficiency is dramatic lower in chronic infections where the percentage of cure
is around 8%.[6,7]
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Given the negative economic and social impact caused by Chagas disease, lack of
vaccines, the limitations of benznidazoles efficacy in the chronic phase and the existence of
naturally resistant T. cruzi strains, the search for new drugs has become increasingly
necessary to treat this disease. Coumarins (1,2-benzopirone or benzopiran-2-one), are
heterocyclic compounds presents in many plants (Angiospermas, Apiaceae, Rutaceae,
Asteraceae),[8] fungi (Agaricus sp, Armillariella tabescens, Penicillium)[9] and bacteria
(Streptomyces spheroides, Streptomyces niveus).[10] The isocoumarins are structurally similar
to coumarins, differing only by the orientation of the lactone. Reyes-Chilpa et al. isolated
trypanocidal coumarins from the leaves and bark of Calophyllum brasiliense and Mammea
americana (Clusiaceae), both found in America (Figure 1). In 2013, Lago et al. isolated and
elucidated the antichagasic coumarin soulamarin (Figure 1) from the bark and stem of
Calophyllum brasiliense (Clusiaceae).[11]
Peacock et al. isolated the coumarin chalepine (Figure 2) from Pilocarpus
spicatus and elucidated the mode of binding of the molecule with the T. cruzi enzyme
GAPDH which is important for disrupting the glycolytic pathway.[12] In the search of
molecules that can serve as leads in the design of a new drug for the treatment of Chagas’
disease, our attention was drawn to brevifolin carboxylate derivatives (Figure 1) isolated
from Geranium bellum Rose. These compounds were able to inactivate triosephosphate
isomerase from T. cruzi (TcTIM) in a species-specific manner.[13] In view of the spectrum of
biological activity of naturally occurring coumarins (isolated from plants, fungi and animals),
several research groups have searched for new semi-synthetic or synthetic coumarins that
contain a spectrum of biological activity better than the natural analogue (Figure 2).
For example, Munoz et al., described the syntheses of 3-carboxyamidocoumarins
and evaluation in vitro of antitrypanossomal activity these compounds against epimastigotes
and trypomastigotes of T. cruzi. [14] In the same year, Vazquez-Rodriguez et al. described the
synthesis of new coumarin-chalcone hybrids and the evaluated their antitrypanossomal
activity against epimastigotes, trypomastigotes and amastigotes of T. cruzi.[15] Ryan et al.
recently described the synthesis of hydroxyl substituted 3-arylcoumarins and evaluated the
antichagasic profile of these compounds against the epimastigote form.[16] In order to meet
this deficit in new drug candidates for Chagas disease, we have prepared and tested 18
tricyclic coumarins against T. cruzi, evaluated their cytotoxic effect against L929 cells and
determined the selectivity index.
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2 Results and Discussion
2.1 Synthesis of tricyclic coumarins
Brevifolin carboxylate derivatives are substituted [6,6,5] tricyclic compounds
consisting of an isocourmarin fused to a pentanone ring. Although these compounds were not
evaluated for their trypanocidal activity, they were shown to be highly selective inhibitors of
triosephosphate isomerase from T. cruzi (TcTIM). These preliminary results inspired us to
study structurally related compounds in order to uncover more potent molecules against
T.cruzi. To this end, we synthesised [6,6,5] tricyclic coumarins in order to explore structure
activity relationships with TcTIM and determine if inactivation of this enzyme indeed
translates into anti T.cruzi activity. The synthetic design considered substitution at the phenyl
rings or the introduction of heteroaromatic and organometallic moietys to examine their role
and trends in terms of electronic and steric contributions. Synthesis of the target compounds 3
(Scheme 1) began with preparation of α,β-unsaturated aldehydes 1 by the Wittig reaction,
which afforded substituted cinnamaldehydes in good yield (37-80%).
The requisite
chalcones 2 were obtained (44-86%) by cross-aldol condensation between substituted 2-
hydroxyacetophenones and aromatic aldehydes. All of the intermediates prepared are known
compounds and were confirmed by comparison of their physical and spectroscopic data with
those reported in the literature.
Next, cinnamaldehydes were reacted with chlacones by a NHC-organocatalyzed
homoenolate annulation reaction to furnish tricyclic coumarins 3a-r in good to excellent
yields as shown in scheme 1. Using X-ray diffraction techniques, the relative stereochemistry
of the products formed by this reaction had been shown by Biju et al. to be predominantly
trans (trans diastereoisomer). In total, 18 coumarins were obtained of which, 15 are novel
compounds that were not previously described in the literature. In the case of coumarin 3r, a
hydrogenolysis of 3q was carried out in the presence of ammonium formate and palladium
(Pd/C 5%) to cleave the benzyl group and liberate the free hydroxyl. Compounds 3a-r were
characterized by FT-IR, HRMS and 1H and 13C nuclear magnetic resonance spectroscopy. In
the infrared spectrum, the carbonyl group displays a distinctive band at approximately 1716
1
cm-1 corresponding to the lactone carbonyl group. In the H NMR spectrum, all tricyclic
coumarins exhibited a characteristic double doublet resonance corresponding to a methylene
hydrogen (δ = 3.32 ppm) and a doublet resonance corresponding to a methine hydrogen (δ =
13
4.41 ppm) located at the same position as the R2 substituent. The key C NMR signals
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include the carbonyl carbon (δ = 158.6 ppm) and 3 other signals corresponding to sp3 carbons
that form part of the cyclopentene moeity (δ = 53.4, 59.1 and 39.5 ppm). The molecular
formula assignment was further supported by HRMS data and GC-MS was also carried out to
evaluate product purity. It has been well documented that for pharmacological active racemic
mixtures, one enantiomer is mainly responsible for the desired biological action (eutomer)
and the other inactive (distomer). Although the enantioselective version of the NHC-
catalyzed reaction between enals and hydroxy chalcones had not yet been reported, an
attempt at the asymmetric reaction was carried out in the hope of obtaining an
enantiomerically pure sample for biological testing. The quiral triazol 4 has successfully been
applied to the asymmetric synthesis of cyclopentanones[17,18] and the asymmetric
intramolecular Stetter reaction.[19,20] Compound 4 (Figure 3) was therefore employed as
catalyst (10 mol%) for the homoenolate annulation reaction but unfortunately, no reaction
occurred and only the starting materials were recovered.
Nevertheless, with the racemic compounds in hand, in vitro bioassays using
trypomastigote and amastigote forms of Y-strain T. cruzi were carried out.
2.2 Molecular docking in Human Triosephosphate isomerase (4POC) enzyme and T.
cruzi Triosephosphate isomerase enzyme (1TOC).
Computational simulation and molecular docking studies are an important research
technique for evaluating molecular interactions between the protein and the ligand, since the
3D structure of the protein has been elucidated. Through molecular docking it is possible to
predict the most stable structure of the protein-ligand complex, and also to calculate the
[21]
energy of interaction
based on preliminary study of Gayosso-De-Lucio et. al.,[13] that
described the inactivation of the T. cruzi triosephosphate isomerase (TIM) enzyme by
isocoumarins isolated from hellum rose geranium, this study was performed with molecular
docking in silico of new coumarin compounds. The objective was to investigate the enzyme-
compound interaction and thus predict which groups are responsible for the interaction with
the enzyme and determine the key amino acid residues participate in this interaction.
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Three-dimensional structures of human and tripanossomal triosephosphate
isomerase (TIM) enzymes were obtained from "Protein Data Bank" (PDB) with the 4POC
and 1TCD codes, respectively. With the Molegro Virtual Docker software (MVD), molecular
docking calculation of the compounds were carried out in both TIM enzymes in order to
identify the mode of the ligand interactions by estimating the number of solutions and the
power associated with its interactions with the protein. The binding sites between the
coumarins and TIM enzymes were determined by a prediction algorithm based on a 3D box,
through the MVD software. The volume of the cavity is 35.33 Å3 (TIM human) and 37,38 A3
(TIM T. cruzi). The interaction energy values and hydrogen bonds were calculated for the
enzyme-compound complex, and verified which groups are responsible for activity. Table 1
contains the amino acids that participate in the enzyme - compound interaction and the
energy interaction values.
Notably, compound 3p containing a styryl substituent interacted more strongly (-
143.12 kcal mol-1) with T. cruzi TIM enzyme than any other tricyclic coumarin as indicated
by having the lowest energy value of compounds 3a-r. Although the compound in question
does not perform hydrogen bonding interactions with the amino acid residues of the active
site of the enzyme, it stabilizes, as well as other compounds, performing π stacking
interactions with Tyr103 residue that appears to be of great importance for a good ligand-
enzyme interaction (Figure 4).
The second best result was observed for compound 3e containing a p-
methoxyphenyl group. The interaction energy was calculated to be -139.35 kcal mol-1 and the
existence of a hydrogen bond between the methoxy group and the amino acid residue Arg99
was observed (Figure 5).
Compound 3e in spite of having hydrogen interactions behaved similarly to 3p in
that they both share a common π stacking type interaction with amino acid residue Tyr103.
These two compounds are best stabilized in the active site of T. cruzi TIM enzyme adopted
very similar conformations, which can be seen in the following overlapping figure (Figure 6).
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Interestingly, the compounds 3j, 3k and 3m containing heterocyclic groups did not
show good interactions with the human TIM enzyme which indicated potentially low toxicity
for these coumarins. It is further noted that the compounds 3j and 3n, containing either a
furan or pyridine moiety presented good interaction with the T. cruzi TIM enzyme which
suggests these coumarins could be more selective. Observing the behaviour of the coumarins
in the active site of T. cruzi TIM, it was possible to observe that the compounds 3m and 3k
do not perform intermolecular π stacking interactions with amino acid residues Tyr 103 but
perform intramolecular π stacking interaction that do not favour its stabilization in the
enzyme (Figure 7).
Interestingly, compound 3n containing two pyridine rings showed lowest stability in
the active site of human TIM enzyme (-30.75 kcal mol-1) amongst the evaluated compounds.
Given this large difference in interaction energy presented by compound 3n in relation to
human and tripanossomal enzymes, this compound stands out as a potential ligand since it is
expected to be highly selective. Coumarin 3r interacts with T. cruzi TIM enzyme by
hydrogen bonding between the free hydroxyl group and amino acids residues Arg99 and
Glu78, indicating that the presence of hydroxyl help stabilize the compound in the active site
of the enzyme (Figure 8). Moreover, 3r also showed good interaction with the human TIM
enzyme, a difference of 3.44 kcal mol-1. This ligand adopts at the binding site a conformation
in which its non-fused aromatic rings are on opposite sides not performing intramolecular π
stacking interaction but intermolecular π stacking interaction once again with the amino acid
residue Tyr103.
2.3 Evaluation of in vitro anti T. cruzi activity
Once the final products were purified and fully characterized we carried out in vitro bioassays
against trypomastigote and intracellular amastigote of β-galactosidase transfected Tulahuen
strain of T. cruzi. There have been different approaches taken towards the in vitro evaluation
of anti T. cruzi activity. The use of the epimastigote form present in the midgut vector can be
employed for an in vitro screening method,[21,22] but we have opted for a methodology that
simultaneously evaluates trypomastigote forms that are initially present in the blood after
entering through the bite wound and intracellular amastigotes forms present in the vertebrate
host during the acute and chronic phases of the disease.[23,24] This approach is in accordance
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with the guidelines proposed by the Fiocruz Program for Research and Technological
Development on Chagas Disease and the Drugs for Neglected Diseases Initiative
(DNDi).[23,24] Furthermore, the whole cell-based screening methodology is also attractive
given that bioactive compounds can be tested for anti T. cruzi activity in infected cells whilst
at the same time monitoring their effects on both amastigotes and trypomastigotes in the same
system. Benznidazole was used as positive control against T. cruzi and cytotoxicity was
determined in mammalian L929 cells (Table 2). Amongst the 18 compounds tested, ten of
these were more active than reference drug benznidazole and eight had selectivity indexes
above 50. In all cases, the tested compounds were more toxic and less selective than
reference compound benznidazole. Nevertheless, following the guidelines suggested by
Fiocruz and DNDi,[25] compounds with a selectivity index equal to or greater than 50 are
highly selective and are recommended for in vivo testing. Therefore, 8 of the 18 tricyclic
coumarins tested would be suitable lead compounds for in vivo testing. Initially, compound
3a was evaluated for trypanocidal activity and this result used for comparison in order to
assess structure activity relationships. The IC50 for 3a (2.5 µM) was slightly better than the
reference compound and this initial preliminary result encouraged us to investigate other
analogues bearing substituents capable of forming stronger intermolecular interactions with
the TcTim which is our intended target for bioactivity. The most selective compound was
coumarin 3e (SI = 262) which differs from 3a (SI = 128) by containing an anisole substituent
instead of a benzene at the R1 position of the cyclopentene portion of the molecule.
Similarly, compound 3l, which also bears an anisole substituent on the cyclopentene ring but
in this case at the R2 position, demonstrated almost identical anti T. cruzi activity to 3e but
with significantly greater cytotoxicity and ultimately lower selectivity. The improvement in
T.cruzi activity observed for coumarins bearing a metoxy group suggests some important
hydrogen bonding interaction with TcTIM. The importance of methoxy substituents was also
noted for the trypanocidal activity of coumarin-chalcone hybrid molecules.[13] The
chlorobenzene group present in 3g resulted in compound that was equipotent to benznidazole
but four times more toxic than 3a thus making the presence of a chloro atom undesirable.
Compound 3p which contains a styryl group at the R1 position, was the most potent
coumarin tested against T. cruzi, with IC50 value of 0.46 ± 0.12 μM and selectivity index SI =
83.7. This tryponacidal activity represents an increase in potency of 6 fold in comparison
with 3a and 3p is also approximately 10 times more potent than benznidazole. According to
the molecular docking study, this compound presented one of the best molecular interaction
energies with TcTim (-143.12 kcal mol-1) and therefore may explain the trend in structure
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activity relationships that occurred during in vitro testing. Some promising biological
[26,27]
activities of ferrocenes against T.cruzi have been reported in the literature
and this
motivated us to synthesis and test an analogous compound 3o in this study. The presence of a
ferrocene did not significantly improve either trypanocidal activity or selectivity when
compared to 3a but the in vitro results for this compound were still promising and it should
be noted that cytotoxicity was reasonably low in vitro. Our lead compounds 3e and 3p were
subjected to in vitro bioassays using the epimastigote form present in the midgut vector and
presented IC50 values of 103µM and 109 µM respectively. Surprisingly, the impressive
typanocidal activities observed against amastigotes and trypomastigotes were not replicated
against epimastigotes. Several studies have demonstrated a higher resistance of the T. cruzi
[28,29]
epimastigote form to drugs, when compared to amastigote and trypomastigote forms
and it should be emphasized that amastigotes and trypomastigotes are the relevant forms to
human infection. The potential of compounds 3a-r as candidates for new drugs was also
evaluated by using Linpinski parameters, which considers the physicochemical drug
descriptors of the molecular properties for the synthesized compounds as calculated by
Molinspiration software. The partition coefficient (LogP: octanol/water partition coefficient)
describes the equilibrium distribution between two liquid phases such as octanol and water
and the total polar surface area (TPSA) is a measure of the extent of the molecules exposed
polar area. The risk of toxicity related to mutagenic and tumorigenic effects were determined
by utilizing "OSIRIS property explorer". The results show that compounds 3j-k and 3m-o
satisfy Linpinski's rule of five with no violations.[30] The other compounds violate the rule by
presenting lipophilicity (Log P) greater than 5.0, which could cause problems with oral
bioavailability. All compounds showed TPSA values lower than 90 Å2, values obtained range
from 30-60 Å2, indicating that these compounds would have a good permeability in the
plasma cell membrane and across the blood brain barrier. The risk of theoretical toxicity was
estimated by the OSIRIS program and this program classifies the risks of toxicity as either
high risk (HR), medium risk (MR), low risk (LR) or no risk (NR). Assessment of the toxicity
revealed that none of the compounds presented potential irritant effects. Only compound 3o
containing the ferrocene group presented high risk for mutagenic effect and low risk for
tumorigenic effect and all other coumarins did not present any of these risks.
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3 Conclusion
The NHC organocatalysis strategy was applied to the synthesis of 18 tricyclic
coumarins, of which 15 are novel compounds. All compounds satisfy the Lipinski rule
parameters, and theoretically, all have adequate oral availability. Molecular modeling studies
showed that the compounds are stabilized at the active site of the TcTIM T. cruzi enzyme by
intermolecular interactions and in particular, the presence of a styryl substituent showed
strongest interaction with TcTIM. The anti T. cruzi acitivity of 18 tricyclic coumarins were
evaluated in vitro against amastigote and tripomastigote forms of parasite. Most of the
evaluated coumarins presented promising activity against the intracellular forms of T. cruzi,
with ten compounds presenting IC50 values lower than the benznidazole. Regarding the
selectivity index, a criterion analyzed for the screening of possible drugs for subsequent in
vivo tests, eight compounds are suitable for in vivo assays. Further studies to determine
experimentally the mechanism of action of the tricyclic coumarins and in vivo evaluation of
our lead compounds are ongoing.
4 Experimental Section
All commercial reagents were used as received. Anhydrous solvents were purchased
from Sigma Aldrich. Flash column chromatography was performed using silica gel 200-400
Mesh. TLC analyses were performed using silica gel plates, using ultraviolet light (254 nm),
phosphomolybdic acid or vanillin solution for visualization. Melting points are uncorrected
and were recorded on a Buchi B-540 apparatus. For NMR data, the chemical shifts are
reported in δ (ppm) referenced to residual solvent protons and 13C signals in deuterated
chloroform. Coupling constants (J) are expressed in Hertz (Hz). Infrared spectra were
obtained on a Thermo Scientific Nicolet 380 FT-IR apparatus (600–4000 cm−1, Nicolet
Instrument Corp., Madison, WI, USA) using attenuated total reflection (ATR). Mass spectra
were obtained by GC-MS, Shimadzu QP-2010 Plus model (Shimadzu, Kyoto, Japan) and
High Resolution Mass Spectra were obtained on a Shimadzu HPLC-ESI-IT-TOF. SMILES
notations of the tricyclic coumarins derivatives were inputted into an online software and
subjected to molecular properties prediction by Molinspiration software (software version
v2015.01).
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4.1 Typical procedure for synthesis of tricyclic coumarins 3a-r: Chalcone (1 mmol),
cinamaldehyde (1 mmol), 1,3-Bis(2,4,6-trimethylphenyl)imidazolium chloride (10 mol%),
were added to the Schlenk tube and the atmosphere was purged with nitrogen. Thereafter,
1,8-diazabicyclo[5.4.0]undec-7-ene (20 mol%) and anhydrous tetrahydrofuran (4 mL) were
added with the aid of a syringe and the reaction mixture being kept under stirring for 12 hours
at room temperature. The solvent was evaporated with the aid of rotary evaporator and the
crude mixture was purified by flash column chromatography on silica gel, eluting with ethyl
acetate and hexane.
trans-2,3-diphenyl-2,3-dihydrocyclopenta[c]chromen-4(1H)-one (3a): Product obtained as a
white solid in 49%, mp. 124-126ºC; Rf: 0.2 (ethyl acetate/hexane 2:8); IR (cm-1): 3026, 2941,
1718, 1618, 1452, 1274, 1043, 761, 700; 1H-NMR (400 MHz, CDCl3): δ 3.33 (dd, J 8.0 Hz, J
13
20.0 Hz, 1H), 3.82 (m, 2H), 4.52 (d, J 4 Hz, 1H), 7.61 (m, 14H); C-NMR (75 MHz,
CDCl3): δ 39.5, 53.4, 59.1, 116.9, 118.3, 124.2, 125.0, 126.8, 126.9, 127.0, 128.6, 128.7
128.8, 131.4, 142.3, 144.5, 154.7, 155.5, 158.8. EI m/z: 260 (100%), 247 (50%), 203 (70%),
189 (30%), 130 (20%), 91 (40%); HRMS (ESI-TOF) m/z [M + Na] + Calcd for C24H18NaO2:
361.1199; Found: 361.1186.
trans-3-(2-chlorophenyl)-2-phenyl-2,3-dihydrocyclopenta[c]chromen-4(1H)-one
(3b):
Product obtained as a pale yellow oil in 74%; Rf: 0.58 (ethyl acetate/hexane 2:8); IR (cm-1):
1
3024, 2921, 1720, 1606, 1452, 1216, 1039, 746, 696; H-NMR (300 MHz, CDCl3): δ 3.30
(dd, J 9.0 Hz, J 21.0 Hz, 1H), 3.74 (m, 1H), 5.00 (d, J 3,0 Hz, 1H), 7,61 (m, 14H); 13C-NMR
(75 MHz, CDCl3): δ 39.3, 52,2, 55.4, 117.0, 118.2, 124.3, 125.0, 126.8, 127.0, 127.9, 128.1,
128.7, 130.1, 131.6, 133.9, 139.3, 144.4, 154.8, 156.5, 158.6; EI m/z: 372 (5%), 337 (100%),
+
259 (10%), 202 (10%), 144 (10%), 91 (20%); HRMS (ESI-TOF) m/z [M + H] calcd for
C24H18ClO2: 373.0990; Found: 373.0962.
trans-3-(3-methoxyphenyl)-2-phenyl-2,3-dihydrocyclopenta[c]chromen-4(1H)-one
(3c):
Product obtained as a pale yellow solid in 76%, mp. 146-148ºC; Rf: 0.2 (ethyl acetate/hexane
1
1:9); IR (cm-1): 2943, 1730, 1608, 1452, 1384, 1272, 1043, 756, 698; H-NMR (300 MHz,
CDCl3): δ 3.29 (dd, J 6 Hz, J 18 Hz, 1H), 3.79 (m, 5H), 4.47 (d, J 6 Hz, 1H), 7.60 (m, 13H);
13C-NMR (75 MHz, CDCl3): δ 39.4, 53.2, 55.1, 59.1, 115.1, 113.1, 116.9, 118.3, 119.4,
124.2, 125.0, 126.7, 126.9, 128.5, 128.8, 129.7, 131.5, 144.0, 144.6, 154.7, 155.6, 158.8,
159.7; EM m/z: 368 (100%), 353 (10%), 337 (5%), 260 (80%), 218 (30%), 145 (20%), 91
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(40%); HRMS (ESI-TOF) m/z [M+Na] + calcd for C25H20NaO3: 391.1305; Found: 391.1274.
trans-3-(3-bromophenyl)-2-phenyl-2,3-dihydrocyclopenta[c]chromen-4(1H)-one
(3d):
Product obtained as a pale yellow oil in 80%; Rf: 0.3 (ethyl acetate/hexane 1:9); IR (cm-1):
1
2914, 1716, 1608, 1454, 1390, 1068, 1216, 1043, 756, 698, 684; H-NMR (300 MHz,
CDCl3): δ 3.32 (dd, J 6 Hz, J 18 Hz, 1H), 3.58 (m, 2H), 4.45 (d, J 6 Hz, 1H), 7.60 (m, 13H);
13C-NMR (75 MHz, CDCl3): δ 39.5, 53.4, 58.8, 117.0, 118.1, 122.8, 124.3, 125.1, 126.0,
126.8, 127.8, 128.9, 130.1, 130.2, 131.7, 143.9, 144.7, 154.8, 155.9, 158.6; EM m/z: 417
(60%), 337 (80%), 260 (40%), 246 (50%), 202 (50%), 189 (20%), 91 (100%); HRMS (ESI-
TOF) m/z [M+H] + calcd for C24H18BrO2: 417.0485; Found: 417.0468.
trans-2-(4-methoxyphenyl)-3-phenyl-2,3-dihydrocyclo-penta[c]chromen-4(1H)-one
(3e):
Product obtained as a white solid in 65%; Rf: 0.5 (ethyl acetate/hexane 2:8); IR (cm-1): 3024,
1
1722, 1606, 1452, 1384, 1178, 1249, 1033, 750, 680; H-NMR (300 MHz, CDCl3): δ 3.27
(dd, J 6 Hz, J 18 Hz, 1H), 3.77 (m, 2H), 3.80 (s, 3H), 4.46 (d, J 6 Hz, 1H), 7.59 (m, 13H);
13C-NMR (75 MHz, CDCl3): δ 39.5, 52.8, 55.2, 59.3, 114.2, 116.9, 118.4, 124.2, 125.0,
126.8, 127.0, 127.8, 128.6, 131.4, 136.5, 142.4, 154.8, 155.5, 158.5, 158.6; EM m/z: 368
(90%), 353 (5%), 337 (5%), 260 (100%), 207 (40%), 121 (40%), 91 (20%); HRMS (ESI-
TOF) m/z [M+H]+ calcd for C25H21O3: 369.1485; Found: 369.1478.
trans-2-(4-fluorophenyl)-3-phenyl-2,3-dihydrocyclopenta[c]chromen-4-(1H)-one
(3f):
Product obtained as a white solid in 43%, mp: 1135-137ºC; Rf: 0.5 (ethyl acetate/hexane 1:9);
IR (cm-1): 3064, 1724, 1604, 1452, 1390, 1226, 1037, 754, 700; 1H-NMR (300 MHz, CDCl3):
13
δ 3.27 (dd, J 6 Hz, J 18 Hz, 1H), 3.80 (m, 2H), 4.44 (d, J 3 Hz, 1H), 7.60 (m, 13H); C-
NMR (75 MHz, CDCl3): δ 39.5, 52.8, 115.8-115.3 (d, JC-F 21.0 Hz), 117.0, 118.2, 124.3,
125.0, 127.0, 128.4 – 128.3 (d, JC-F 7,5 Hz), 128.4, 128.7, 140.1 – 140.0 (d, JC-F 3 Hz); 142.1,
154.7, 155.3, 158.7, 163.3 – 160.1 (d, JC-F 243,7 Hz); EM m/z: 356 (100%), 278 (30%), 260
+
(70%), 220 (10%), 203 (40%), 91 (20%). HRMS (ESI-TOF) m/z [M+H] calcd for
C24H18FO2: 357.1285; Found: 357.1258.
trans-8-chloro-2,3-diphenyl-2,3-dihydrocyclopenta[c]chromen-4-(1H)-one (3g): Product
obtained as a white solid in 49%, mp: 183-185ºC; Rf: 0.6 (ethyl acetate/hexane 2:8); IR (cm-
1
1): 3029, 1720, 1631, 1492, 1263, 1031, 730, 696, 669; H-NMR (300 MHz, CDCl3): δ 3.30
(dd, J 9 Hz, J 24 Hz, 1H), 3.77 (m, 2H), 4.50 (d, J 6 Hz, 1H), 7.52 (m, 13H); 13C-NMR (75
MHz, CDCl3): δ 39.3, 53.2, 59.2, 118.3, 119.4, 124.5, 126.7, 127.0, 128.7, 128.9, 129.5,
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129.8, 131.3, 142.0, 144.2, 153.1, 154.2, 158.1; EM m/z: 372 (100%), 337 (10%), 294 (80%),
+
218 (40%), 202 (90%), 91 (40%). HRMS (ESI-TOF) m/z [M+H] calcd for C24H18ClO2:
373.0990; Found: 373.0993.
trans-8-chloro-3-(4-clorophenyl)-2-phenyl-2,3-dihydrocyclopenta[c]chromen-4-(1H)-one
(3h): Product obtained as a beige solid in 68%, mp: 218-220ºC; Rf: 0.7 (ethyl acetate/hexane
1
2:8); IR (cm-1): 3066, 1728, 1604, 1488, 1257, 1022, 765, 727, 696; H-NMR (300 MHz,
CDCl3): δ 3.28 (dd, J 3 Hz, J 15 Hz, 1H), 3.74 (m, 2H), 4.45 (d, J 3 Hz, 1H), 7.53 (m, 13H);
13C-NMR (75 MHz, CDCl3): δ 39.3, 53.5, 59.6, 118.3, 119.3, 124.5, 126.8, 127.2, 128.4,
128.9, 128.9, 129.0, 129.2, 129.6, 131.5, 140.3, 143.4, 153.1, 154.5, 158.0; EM m/z: 406
(20%), 371 (20%), 294 (10%), 281 (20%), 207 (100%), 91 (15%). HRMS (ESI-TOF) m/z
[M+Na] + calcd for C24H16Cl2NaO2: 429.0424; Found: 429.0425.
trans-8-chloro-3-(4-methoxyphenyl)-2-phenyl-2,3-dihydrocyclopenta[c]chromen-4-(1H)-one
(3i) Product obtained as a white solid in 85%, mp: 103-105ºC; Rf: 0.6 (ethyl acetate/hexane
1
2:8); IR (cm-1): 2925, 1724, 1612, 1510, 1377, 1174, 1245, 1027, 821, 765, 702; H-NMR
(300 MHz, CDCl3): δ 3.25 (dd, J 3 Hz, J 15 Hz, 1H), 3.74 (m, 2H), 3.78 (s, 3H), 4.44 (d, J 3
13
Hz, 1H), 7.51 (m, 12H); C-NMR (75 MHz, CDCl3): δ 39.3, 53.4, 55.2, 58.4, 114.1, 118.3,
119.5, 124.5, 126.7, 126.9, 128.0, 128.8, 129.5, 130.0, 131.2, 134.1, 144.1, 153.1, 153.9,
158.1, 158.6; EM m/z: 402 (100%), 387 (5%), 371 (5%), 324 (40%), 294 (80), 266 (30%),
+
232 (20%), 207 (15%), 162 (20%), 91 (20%). HRMS (ESI-TOF) m/z [M+H] calcd for
C25H20ClO3: 403.1101; Found: 403.1109.
trans-2-(furan-2-yl)-8-methoxy-3-phenyl-2,3-dihydrociyclopenta[c]chromen-4(1H)-one (3j):
Product obtained as a yellow oil in 67%; Rf: 0.6 (ethyl acetate/hexane 2:8); IR (cm-1): 2923,
1
1720, 1573, 1494, 1272, 1074, 1199, 1012, 738, 702; H-NMR (300 MHz, CDCl3): δ 3.36
(dd, J 3 Hz, J 15 Hz, 1H), 3.77 (m, 2H), 3.88 (s, 3H), 4.61 (d, J 6 Hz, 1H), 6.10 (d, J 3 Hz,
1H), 6.31 (d, J 3 Hz, 1H), 6,96 (d, J 3 Hz, 1H), 7.37 (m, 8H); 13C-NMR (75 MHz, CDCl3): δ
29.6, 36.5, 46.5, 55.8, 105.1, 107.51, 110.2, 117.9, 118.7, 118.7, 127.0, 127.1, 128.7, 141.8,
149.1, 154.6, 156.0, 156.1, 158.8; EM m/z: 358 (100%), 329 (50%), 290 (30%), 262 (20%),
+
67 (5%), 55 (15%). HRMS (ESI-TOF) m/z [M+Na] calcd for C23H18NaO4: 381.1097;
Found: 381.1062.
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trans-2-(1-methyl-1H-pyrrol-2-yl)-3-phenyl-2,3-dihydrocyclo-penta[c]chromen-4(1H)-one
(3k): Product obtained as a yellow oil in 36%; Rf: 0.7 (ethyl acetate/hexane 3:7); IR (cm-1):
1
3425, 2916, 1722, 1631, 1454, 1386, 1043, 775, 694; H-NMR (300 MHz, CDCl3): δ 3.35
(m, 4H), 3.75 (m, 2H), 4.47 (d, J 6 Hz, 1H), 6.57 (m, 3H), 7.57 (m, 9H); 13C-NMR (75 MHz,
CDCl3): δ 33.9, 38.1, 44.8, 58.0, 104.6, 106.9, 116.9, 118.3, 122.3, 124.2, 124.9, 127.2,
128.4, 128.8, 131.4, 135.2, 142.0, 154.7, 154.9, 158.8; EM m/z: 341 (80%), 264 (5%), 260
(80%), 165 (50%), 94 (40%), 81 (100%), 77 (20%); HRMS (ESI-TOF) m/z [M+H] + calcd for
C23H20NO2: 342.489; Found: 342.1469.
trans-2-(2-chlorophenyl)-3-(4-methoxyphenyl)-2,3-dihydrocyclo-penta[c]chromen-4-(1H)-
one (3l): Product obtained as a yellow oil in 89%; Rf: 0.3 (ethyl acetate/hexane 1:9); IR (cm-
1
1): 2918, 1722, 1608, 1450, 1245, 1035, 1176, 1107, 831, 750, 678; H-NMR (300 MHz,
CDCl3): δ 3.25 (dd, J 6 Hz, J 21 Hz, 1H), 3.83 (m, 4H), 4,15 (m, 3H), 4,57 (d, J 6 Hz, 1H),
13
7.58 (m, 12H); C-NMR (75 MHz, CDCl3): δ 38.2, 49.4, 55.0, 56.4, 114.1, 116.9, 118.4,
124.5, 125.0, 127.3, 127.6, 128.0, 128.1, 128.2, 129.0, 129.9, 131.4, 133.6, 134.1, 141.7,
154.7, 154.9, 158.8; EM m/z: 402 (100%), 387 (5%), 367 (30%), 335 (5%), 290 (40%), 259
+
(60%), 232 (20%), 145 (40%), 77 (25%); HRMS (ESI-TOF) m/z [M+H] calcd for
C25H20ClO3: 403.1101; Found: 403.1091.
trans-2-(furan-2-yl)-3-phenyl-2,3-dihydrocyclopenta[c]chromen-4(1H)-one (3m): Product
obtained as a white solid in 59%, mp: 130-131ºC; Rf: 0.46 (ethyl acetate/hexane 3:7); IR (cm-
1
1): 2925, 1720, 1608, 1454, 1390, 1037, 1216, 1012, 750; H-NMR (300 MHz, CDCl3): δ
3.39 (d, J 3 Hz, J 18 Hz, 1H), 3.70 (m, 2H), 4.61 (d, J 3 Hz, 1H), 6.32 (m, 2H), 7.58 (m,
13
10H); C-NMR (75 MHz, CDCl3): δ 36.4, 46.6, 55.8, 105.2, 110.2, 116.9, 118.3, 124.2,
124.9, 127.0, 127.1, 128.2, 128.7, 131.4, 141.9, 154.7, 155.1, 156.0, 158.8; EM m/z: 328
(100%), 260 (60%), 126 (10%), 91 (40%); HRMS (ESI-TOF) m/z [M+H] + cald for C22H17O3:
329.1172; Found: 329.1175.
trans-2,3-di(2-pyridinyl)-2,3-dihydrocyclopenta[c]chromen-4(1H)-one
(3n):
Product
obtained as a white solid in 91%, mp: 135-136ºC; Rf: 0.26 (ethyl acetate/hexane 2:8); IR (cm-
1
1): 3008, 1722, 1591, 1434, 1218, 1390, 1037, 752; H-NMR (300 MHz, CDCl3): δ 3.78 (m,
2H), 4.27 (dd, J 3 Hz, J 6 Hz, 1H), 4.85 (d, J 3 Hz, 1H), 7.08 (d, 1H, J 6 Hz), 7.66 (m, 9H),
13
8.6 0 (d, J 3 Hz, 1H), 8.64 (d, J 3 Hz, 1H); C-NMR (75 MHz, CDCl3): δ 37.9, 52.9, 59.3,
116.8, 118.7, 122.0, 122.0, 122.7, 123.8, 124.1, 125.1, 128.0, 131.2, 136.5, 136.6, 149.8,
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149.9, 154.7, 155.9, 159.0, 161.0, 162.2; EM m/z: 340 (80%), 262 (100%), 1455 (5%), 78
(70%); HRMS (ESI-TOF) m/z [M+Na] + cald for C22H16N2NaO3: 363.1104; Found: 363.1108.
trans-2-(ferrocenyl)-3-phenyl-2,3-dihydrocyclopenta[c]chromen-4(1H)-one (3o): Product
obtained as a yellow solid in 48%, mp: 184-185ºC; Rf: 0.32 (ethyl acetate/hexane 1:9); IR
1
(cm-1): 3080, 1724, 1629, 1490, 1388, 1035, 765; H-NMR (300 MHz, CDCl3): δ 3.29 (dd, J
13
3 Hz, J 18 Hz, 1H), 3.74 (m, 2H), 4.14 (m, 9H), 4.40 (d, J 3 Hz, 1H), 7.61 (m, 10H); C-
NMR (75 MHz, CDCl3): δ 36.4, 46.6, 55.8, 65.8, 67.1, 67.7, 67.8, 68.4, 92.2, 116.9, 118.4,
124.1, 124.9, 126.9, 127.3, 128.7, 129.0, 131.3, 142.6, 154.6, 154.9, 158.8; HRMS (ESI-
TOF) m/z [M+H] + cald for C28H23FeO2: 447.1042; Found: 447.1041.
trans-3-phenyl-2-(E)-styryl-2,3-dihydrocyclopenta[c]chromen-4(1H)-one
(3p):
Product
obtained as a yellow oil in 59%; Rf: 0.44 (ethyl acetate/hexane 2:8); IR (cm-1): 2916; 1718,
1608, 1452, 1384, 1037, 1242, 1029, 742; 1H-NMR (300 MHz, CDCl3): δ 3.10 (dd, J 6 Hz, J
18Hz, 1H), 3.62 (m, 2H), 4.31 (d, J 3 Hz, 1H), 6.39 (d, J 6 Hz, 1H), 7.57 (m, 15H); 13C-NMR
(75 MHz, CDCl3): δ 37.4, 51.7, 56.9, 116.9, 118.4, 124.2, 125.0, 125.5, 126.2, 126.9, 127.2,
128.5, 128.6, 129.8, 130.5, 131.4, 132.4, 136.8, 141.8, 154.6, 155.5, 158.9; EM (m/z): 364
(60%), 273 (40%), 260 (100%), 202 (10%), 180 (40%), 91 (60%); HRMS (ESI-TOF) m/z
[M+Na] + cald for C26H20NaO2: 387.1356; Found: 387.1360.
trans-2-(4-benzyloxy)phenyl-3-phenyl-2,3-dihydrocyclopenta[c]chromen-4-(1H)-one (3q):
Product obtained as a yellow oil in 49%; Rf: 0.32 (ethyl acetate/hexane 2:8); IR (cm-1): 2920,
1724, 1608, 1452, 1386, 1178, 1243, 1026, 748; 1H-NMR (300 MHz, CDCl3): δ 3.28 (dd, J 6
Hz, J 18 Hz, 1H), 3.79 (m, 2H), 4.47 (d, J 6 Hz, 1H), 5.07 (s, 2H), 7.58 (m, 18H); 13C-NMR
(75 MHz, CDCl3): δ 41.0, 52.8, 59.3, 70.0, 115.1, 116.9, 118.3, 124.2, 125.0, 127.0, 127.5,
127.9, 128.0, 128.6, 128.7, 131.4, 136.8, 136.9, 142.2, 154.7, 155.7, 157.7, 158.8; HRMS
(ESI-TOF) m/z [M+H] + cald for C31H25O3: 445.1803; Found: 444.1814.
4.2 Typical procedure for synthesis of tricyclic coumarin 3r: To a Schlenk Tube was
added compound 3q (1 mmol), palladium acetate (5 mol%) and Pd/C 5% (1 equiv.). Next, the
atmosphere was purged with N2. Ammonium formate (15 mmol.) dissolved is methanol was
added by syringe and the temperature was raised 60ºC and maintained for 2-3 hours. The
mixture was cooled to room temperature, concentrated and the crude mixture purified by
flash column chromatography eluting with ethyl acetate and hexane.
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trans-2-(4-hydroxyphenyl)-3-phenyl-2,3-dihydrocyclopenta[c]chromen-4-(1H)-one
(3r):
Product obtained as a white solid in 26%, mp: 196-197; Rf: 0.5 (ethyl acetate/hexane 1:9); IR
(cm-1): 3282, 2925, 1693, 1606, 1454, 1388, 1056, 1224, 763; 1H-NMR (300 MHz, CDCl3): δ
3.25 (dd, J 6 Hz, J 18 Hz, 1H), 3.76 (m, 2H), 4.43 (d, J 6 Hz, 1H), 7.56 (m, 13H); 13C-NMR
(75 MHz, CDCl3): 39.6, 52.8, 59.3, 115.6, 117.0, 118.3, 124.3, 125.0, 126.9, 127.0, 128.0,
128.5, 128.7, 131.4, 136.5, 142.3, 154.5, 154.7, 155.7, 159.0; HRMS (ESI-TOF) m/z [M+H] +
cald for C24H19O3: 355.1329, Found: 355. 1328.
4.3 Anti-Trypanosoma cruzi activity assay (Amastigotes and Trypomastigotes)
The in vitro anti-T. cruzi activity was evaluated on L929 cells (mouse fibroblasts)
infected with Tulahuen strain of the parasite expressing the Escherichia coli β-galactosidase
as reporter gene according to the method described previously [25]. Briefly, for the bioassay,
4,000 L929 cells were added to each well of a 96-well microtiter plate. After an overnight
incubation, 40,000 trypomastigotes were added to the cells and incubated for 2 h. Then the
medium containing extracelullar parasites was replaced with 200 μl of fresh medium and the
plate was incubated for an additional 48 h to establish the infection. For IC50 determination,
the cells were exposed to each synthesized compound at serial decreasing dilutions and the
plate was incubated for 96 h. After this period, 50 μl of 500 μM chlorophenol red beta-D-
galactopyranoside (CPRG) in 0.5% Nonidet P40 was added to each well, and the plate was
incubated for 16 to 20 h, after which the absorbance at 570 nm was measured. Controls with
uninfected cells, untreated infected cells, infected cells treated with benznidazole at 3.8 μM
(positive control) or DMSO 1% were used. The results were expressed as the percentage of T.
cruzi growth inhibition in compound-tested cells as compared to the infected cells and
untreated cells. The IC50 values were calculated by linear interpolation. Quadruplicates were
run in the same plate, and the experiments were repeated at least once.
4.4 Anti-Trypanosoma cruzi activity assay (Epimastigotes)
In this study, epimastigotes of the Y strain from T. cruzi were used. Epimastigotes
obtained in exponential growth phase were washed with LIT medium 2 times in sterile PBS
at pH 7.2 (1500 g) for 10 min at 4 °C. The number of parasites was determined in a Neubauer
chamber. Next, the parasites were suspended in LIT medium, which was further
supplemented with 10% FBS (Fetal Bovine Serum inactivated at 56 °C), and the
concentration of epimastigotes adjusted to 5 × 106 epimastigotes/mL. With the goal of
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screening samples with possible biological activity against T. cruzi, compounds were
incubated in 48 well Nunc plates containing 800 μL of the suspension of parasites and 200
μL of the test compounds at different concentrations (40, 20, 10 and 1.6 μg/mL) diluted in
sterile DMSO for 72 h in duplicate. As a negative control, the parasites were incubated in the
absence of test compound and in the presence of 1% DMSO. Benznidazole (40, 20, 10 and
1.6 μg/mL) was used as positive control in tests against T. cruzi. The activity was determined
by counting in a Neubauer chamber and subsequent statistical evaluation. The results are
expressed as IC50. The tests were repeated 2 times with the objective of evaluating the
maintenance of the activity of the compounds and reproducibility of results.
4.5 In vitro cytotoxic test of trypanocidal compounds
The active compounds were tested in vitro for determination of cellular toxicity against
uninfected L-929 cells using the alamarBlue® dye. The cells were exposed to compounds at
increasing concentrations starting at IC50 value for T. cruzi. After 96 h of incubation with the
tested compounds, the alamarBlue® was added and the absorbance at 570 and 600 nm
measured after 4-6 h. The cell viability was expressed as the percentage of difference in the
reduction between treated and untreated cells. IC50 values were calculated by linear
interpolation and the selectivity index (SI) was determined based on the ratio of the IC50
value in the host cell divided by the IC50 value of the parasite. Quadruplicates were run in the
same plate, and the experiments were repeated at least once.
4.6 Molecular Docking Studies
The enzyme from both organisms, T. cruzi and Human, including water of crystallization
and ligands, was obtained from the ‘‘Protein Data Bank’’ (PDB). The codes are, respectively
1TCD and 4POC. Three-dimensional structures of the coumarins were obtained from Spartan
Pro. The compounds were docked in the active sites using Molegro Virtual Docker 2006
(Thomsen and Christensen, 2006), which predicts the most likely conformation that a ligand
will adopt for binding to a macromolecule.
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Acknowledgements
This work was supported by the Brazilian funding agency Fundação de Amparo à Pesquisa
do Estado de Minas Gerais (FAPEMIG) under research grant project code APQ-01629-16.
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior - Brasil (CAPES) - Finance Code 001. Authors gratefully acknowledge the generous
financial support from the Universidade Federal de Ouro Preto (UFOP), FAPEMIG and the
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors thank
the Program for Technological Development of Tools for Health-PDTIS-FIOCRUZ for use
of its facilities. The authors would also like to thank Prof. Dr. Robson Jose de Cassia Afonso
(UFOP) and Ananda Lima Sanson (UFOP) for the excellent mass spectroemtry service.
Policarpo Ademar Sales Junior is research fellow supported by Programa de Pós-graduação
em Ciências da Saúde, Fiocruz Minas (CAPES/PNPD).
List of the figure legends
Figure 1: Coumarin and isocoumarin natural products with in vitro activity against T. cruzi.
Figure 2: Synthetic coumarin compounds with in vitro activity against T. cruzi.
Scheme 1: Synthesis of tricyclic coumarins 3a-r.
Figure 3: Chiral catalyst 4.
Figure 4: - π stacking interaction between the compound (3p) and amino acid residues
Tyr103.
Figure 5: Interactions between the compound (3e) (red) and TIM T. cruzi enzyme.
Figure 6: Overlap of the conformations adopted by the compounds 3e and 3p in the active
site of T. cruzi TIM enzyme.
Figure 7: View of intramolecular π stacking interactions for compounds 3m (pink) and 3k
(blue)
Figure 8: (a) Hydrogen and π stacking interactions of the compound 3r; (b) more stable
conformation adopted by compound 3r in T. cruzi TIM enzyme.
Table 1 - Interaction energy values for enzyme-ligand complex (kcal mol-1).
Table 2 - In vitro trypanocidal activity, cytotoxicity, selectivity index and physicochemical
properties of bioactive tricyclic coumarins.
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Conflicts of interest: There are no conflicts of interest to declare.
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Table 1 - Interaction energy values for enzyme-ligand complex (kcal mol-1).
Human TIM (Enzyme 4POC) T. cruzi TIM (Enzyme 1TCD)
Amino
acid
residues
Compound
Interaction energy
(kcal mol-1)
Amino acid
residues
Interaction energy
(kcal mol-1)
Asn65
Arg98
Arg98
Arg98
Asn65
Asn65
Arg98
Arg98
Asn65
Arg98
Asn65
Arg98
Asn65
Arg98
Ile78
Asn65
Arg98
Asn65
Gly76
Gly112
Asn65
Thr45
Asn65
Asn65
Arg98
Asn65
Arg98
Thr45
Asn65
Arg98
Ile78
-141.09
-133.62
-
(3a)
-56.11
-45.53
-109.44
-113.15
-126.41
-135.63
-
-
-
(3b)
(3c)
(3d)
-109.80
-86.92
-139.35
-116.38
-118.70
Arg99
(3e)
(3f)
(3g)
-
-
-144.60
-150.79
-121.06
-74.83
-74.83
-129.77
-129.77
-132.24
-76.47
-
(3h)
(3i)
-
Arg99
Arg99
(3j)
(3k)
-78.72
-80.33
-30.75
-110.14
-47.49
-63.65
-83.68
-121.22
-143.12
-92.47
-
(3l)
Lys113
(3m)
(3n)
(3p)
(3q)
-
-
-
Glu78
Arg99
-134.31
-137.75
(3r)
Tyr67
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Table 2 - In vitro trypanocidal activity, cytotoxicity, selectivity index and physicochemical properties of
bioactive tricyclic coumarins.
In vitro Activity
Lipinski's rule of five
M
Risk Toxicity
Cytotox
icity
CC50(μ
M) ±
2.0
Violat
lo ions
M
T
I
R
E
Trypan
ocide
TP
SA
(A2
)
Volu
me
Compo
und
H
H
W
N
RB
SI
g
IC50(μ
M) ± 0.2
BA BD (g.m
P
A3
ol-1)
1
2
8
6
6
2
6
2
0
338. 5.
13 54
1
30.
21
311.
02
2
N
R
N
R
N
R
L
R
2.5
320
3a
2
3
2
3
0
0
0
0
372. 6.
09 17
368. 5.
14 58
416. 6.
04 33
368. 5.
14 60
1
1
1
1
30.
21
39.
45
30.
21
39.
45
324.
56
336.
56
328.
90
336.
56
2
3
2
3
N
R
N
R
N
R
N
R
N
R
N
R
N
R
N
R
N
R
N
R
N
R
N
R
L
R
L
R
L
R
L
R
2.4
6.1
160
160
3b
3c
3d
Inactive 320
-
2
6
2
2
8
2
3
0.6
160
3e
2
2
2
3
4
0
0
0
0
0
356. 5.
12 71
372. 6.
09 20
406. 6.
05 87
402. 6.
10 25
358. 4.
12 36
1
1
1
1
0
30.
21
30.
21
30.
21
39.
45
315.
95
324.
56
338.
09
350.
10
2
2
2
3
3
N
R
N
R
N
R
N
R
N
R
N
R
N
R
N
R
N
R
N
R
N
R
N
R
N
R
N
R
N
R
L
R
L
R
L
R
L
R
N
R
1.4
3.4
40
80
3f
3g
3h
3i
Inactive 250
-
5
1
1
1
3
3.1
2.6
160
294
52.
59
318.
13
3j
3
3
3
4
2
0
0
0
0
0
341. 4.
14 29
402. 5.
10 76
328. 4.
11 33
340. 2.
12 73
446. 4.
09 02
0
1
0
0
0
35.
15
39.
45
43.
35
55.
99
312.
95
350.
10
292.
59
302.
71
2
3
2
2
4
N
R
N
R
N
R
N
R
H
R
N
R
N
R
N
R
N
R
L
N
R
N
R
N
R
N
R
N
R
N
R
L
R
N
R
N
R
L
9.3
80
9
3k
3l
6
4
0.6
40
80.0
67.0
320
80
4
1
3m
3n
1
3
3
8
3
30.
21
394.
80
2.4
320
R
R
3o
2
3
3
-
0
0
1
-
364. 6.
14 20
444. 7.
17 19
354. 5.
1
1
1
-
30.
21
39.
45
50.
44
-
338.
44
408.
21
319.
04
-
3
5
2
-
N
R
N
R
N
R
-
N
R
N
R
N
R
-
N
R
N
R
N
R
-
N
R
L
R
L
R
-
0.4
39
3p
3q
3r
81.0
4.2
350
20
4
5
12
-
06
-
6
2
5
3.8
2381
Bz
IC50: 50% inhibitory concentration. CC50: 50% cytotoxic concentration determined using mammalian L929
cells.
SI: selectivity index calculated from CC50/IC50. LogP: octanol/water partition coefficient, TPSA: total polar
surface area, HBA = hydrogen bond acceptors, HBD = hydrogen bond donors, NRB = number of rotatable
bonds, M = mutagenicity, T = tumorogenicity, I = skin irritation, RE = reproductive effect, NR = no risk, LR =
low risk, MR = medium risk, HR = high risk
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Figure 1: Coumarin and isocoumarin natural products with in vitro activity against T. cruzi.
Figure 2: Synthetic coumarin compounds with in vitro activity against T. cruzi.
.
Figure 3: Chiral catalyst 4.
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Figure 4: - π stacking interaction between the compound (3p) and amino acid residues
Tyr103.
Figure 5: Interactions between the compound (3e) (red) and TIM T. cruzi enzyme.
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Figure 6: Overlap of the conformations adopted by the compounds 3e and 3p in the active
site of T. cruzi TIM enzyme.
Figure 7: View of intramolecular π stacking interactions for compounds 3m (pink) and 3k
(blue)
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Figure 8: (a) Hydrogen and π stacking interactions of the compound 3r; (b) more stable
conformation adopted by compound 3r in T. cruzi TIM enzyme.
Scheme 1: Synthesis of tricyclic coumarins 3a-r
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