Sars for the antiparasitic plant metabolite pulchrol. 3. combinations of new substituents in a/b-rings and a/c-rings

Article abstract of DOI:10.3390/molecules26133944

The natural products pulchrol and pulchral, isolated from the roots of the Mexican plant Bourreria pulchra, have previously been shown to possess antiparasitic activity towards Try-panosoma cruzi, Leishmania braziliensis and L. amazonensis, which are protozoa responsible for Chagas disease and leishmaniasis. These infections have been classified as neglected diseases, and still require the development of safer and more efficient alternatives to their current treatments. Recent SARs studies, based on the pulchrol scaffold, showed which effects exchanges of its substituents have on the antileishmanial and antitrypanosomal activity. Many of the analogues prepared were shown to be more potent than pulchrol and the current drugs used to treat leishmaniasis and Chagas disease (miltefosine and benznidazole, respectively), in vitro. Moreover, indications of some of the possible interactions that may take place in the binding sites were also identified. In this study, 12 analogues with modifications at two or three different positions in two of the three rings were prepared by synthetic and semi-synthetic procedures. The molecules were assayed in vitro towards T. cruzi epimastigotes, L. braziliensis promastigotes, and L. amazonensis promastigotes. Some compounds had higher antiparasitic activity than the parental compound pulchrol, and in some cases even benznidazole and miltefosine. The best combinations in this subset are with carbonyl functionalities in the A-ring and isopropyl groups in the C-ring, as well as with alkyl substituents in both the A-and C-rings combined with a hydroxyl group in position 1 (C-ring). The latter corresponds to cannabinol, which indeed was shown to be potent towards all the parasites.

Full text of DOI:10.3390/molecules26133944

molecules
Article
SARs for the Antiparasitic Plant Metabolite Pulchrol.
3. Combinations of New Substituents in A/B-Rings
and A/C-Rings
Paola Terrazas 1,2 , Efrain Salamanca 3, Marcelo Dávila 2, Sophie Manner 1 , Alberto Gimenez 3 and
Olov Sterner 1,
*
1
2
3
Department of Chemistry, Centre for Analysis and Synthesis, Lund University, 22100 Lund, Sweden;
paola.terrazas_villarroel@chem.lu.se (P.T.); Sophie.Manner@chem.lu.se (S.M.)
Centre of Agroindustrial Technology, San Simón University, Cochabamba 3299, Bolivia;
marcelodavila@fcyt.umss.edu.bo
Institute for Pharmacological and Biochemical Sciences, San Andrés University, La Paz 3299, Bolivia;
efrain_salamanca@hotmail.com (E.S.); agimenez@megalink.com (A.G.)
Correspondence: Olov.Sterner@chem.lu.se; Tel.: +46-70-530-6649
*
Abstract: The natural products pulchrol and pulchral, isolated from the roots of the Mexican
plant Bourreria pulchra, have previously been shown to possess antiparasitic activity towards Try-
panosoma cruzi, Leishmania braziliensis and L. amazonensis, which are protozoa responsible for Chagas
disease and leishmaniasis. These infections have been classified as neglected diseases, and still
require the development of safer and more efficient alternatives to their current treatments. Recent
SARs studies, based on the pulchrol scaffold, showed which effects exchanges of its substituents have
on the antileishmanial and antitrypanosomal activity. Many of the analogues prepared were shown
to be more potent than pulchrol and the current drugs used to treat leishmaniasis and Chagas disease
(miltefosine and benznidazole, respectively), in vitro. Moreover, indications of some of the possible
interactions that may take place in the binding sites were also identified. In this study, 12 analogues
with modifications at two or three different positions in two of the three rings were prepared by
synthetic and semi-synthetic procedures. The molecules were assayed in vitro towards T. cruzi
epimastigotes, L. braziliensis promastigotes, and L. amazonensis promastigotes. Some compounds
had higher antiparasitic activity than the parental compound pulchrol, and in some cases even
benznidazole and miltefosine. The best combinations in this subset are with carbonyl functionalities
in the A-ring and isopropyl groups in the C-ring, as well as with alkyl substituents in both the A- and
C-rings combined with a hydroxyl group in position 1 (C-ring). The latter corresponds to cannabinol,
which indeed was shown to be potent towards all the parasites.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Terrazas, P.; Salamanca, E.;
Dávila, M.; Manner, S.; Gimenez, A.;
Sterner, O. SARs for the Antiparasitic
Plant Metabolite Pulchrol. 3.
Combinations of New Substituents in
A/B-Rings and A/C-Rings. Molecules
2021, 26, 3944. https://doi.org/
10.3390/molecules26133944
Academic Editors: Francesca
Mancianti and Valentina Virginia
Ebani
Received: 23 March 2021
Accepted: 21 June 2021
Published: 28 June 2021
Keywords: Trypanosoma cruzi; Leishmania braziliensis; Leishmania amazonensis; pulchrol; pulchral;
cannabinol; SARs
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Natural products have been one of the main sources for bioactive compounds used
to treat a wide range of diseases [1]. The chemical diversity, potential selectivity, and the
availability of traditional knowledge about the use of natural materials, played an im-
portant role in the development of modern drugs. Some examples are morphine isolated
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
from opium [
2]; taxol isolated from Taxus brevifolia [
3]; important antiparasitic drugs,
such as quinine, isolated from Cinchona officinalis [
Artemisia annua [5].
4]; and artemisinin extracted from
Natural products are often isolated in limited quantities and can be assayed in just a
few biological systems. In order to extend the bioactivity scope, synthetic routes can be
designed to obtain sufficient quantities of the product, and can also be used to prepare
Molecules 2021, 26, 3944. https://doi.org/10.3390/molecules26133944
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3944
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new derivatives and analogues, which may possess superior biological properties [
For diseases in which the mechanism of action and the drug target are not fully understood,
as is the case for leishmaniasis and Chagas disease [ ], derivatives can be prepared and
assayed towards cells or whole organisms to measure their activity [10 13], leading to the
1,6].
7–9
–
development of structure–activity relationships studies (SARs). These can be used to design
more potent and less toxic analogues, assuming that just one target is involved [11,14].
The vegetal specie, Bourreria pulchra, which is native in the Yucatan province in Mexico,
is traditionally used to treat cutaneous diseases, fevers, and infections [15]. The isolation of
the main chemical compounds from its roots yielded pulchrol (1a, see Figure 1), which was
shown to possess antiparasitic activity against Leishmania braziliensis, L. amazonensis and
L. mexicana promastigotes, as well as against Trypanosoma cruzi epimastigotes [16].
Figure 1. The 6H-benzo[c]chromene scaffold studied here (left), and structures of pulchrol (1a), cannabinol (5f) and
∆9-tetrahydrocannabinol (6).
Leishmania and Trypanosoma parasites are part of the family Trypanosomatidae, and are
responsible for leishmaniasis and Chagas disease, respectively. Both are considered ne-
glected diseases, but still affect millions of people in developing countries, mainly placed
in the tropical and subtropical regions of the world [7,17–21]. Leishmaniasis can appear as
cutaneous, mucocutaneous or visceral leishmaniasis, and around 700,000 to 1 million new
cases are diagnosed every year. The treatments for leishmaniasis (mainly amphotericin, mil-
tefosine and pentavalent antimonials) may give several toxic side effects and may require
hospitalization [22
tissue and eventually cause death. Twelve million people are affected by this disease and
the existing treatments are far from ideal (nifurtimox or benznidazole) [24 28]. Currently,
,23]. Likewise, Chagas disease is able to produce damage in the hearth
–
there are few validated drug targets for leishmaniasis and Chagas disease, and too little is
understood of the complex life cycle of these pathogens [9].
Pulchrol (1a) is one example of the many natural products that are based on a 6H-
benzo[c]chromene scaffold. This type of compound has been shown to possess different
kinds of biological activities [29–34]. The most studied benzo[c]chromenes are probably
the cannabinoids, isolated mainly from the plant Cannabis sativa, and known for their
affinity to the cannabinoid receptors CB1 and CB2. The natural product cannabinol (5f
see Figure 1), which possess the same skeleton as pulchrol, has been shown to be selective
for CB2, a receptor expressed on immune cells, macrophages, and in other peripheral
organs, while ∆9-tetrahydrocannabinol (THC, 6, see Figure 1), also isolated from C. sativa,
showed greater affinity for CB1, which is the receptor associated with the psychotropic
activity of C. sativa [35]. SARs including cannabinol (5f) have been studied previously,
and indicated that hydroxyl groups at positions 1 and 10, together with bulky alkyl sub-
stituents at position 3, are important for the affinity to CB1 [36]. In addition, shorter and
,
bulkier alkyl substituents at C-3 improved the affinity for the CB2 receptor [37,38]. Cannabi-
noids have also shown immunomodulatory properties in the treatment of psoriasis [39],
antinociceptive properties related to their capacity to induce vasorelaxation and release
neuropeptides [40,41], and antineoplastic activity on Lewis lung tumors [33].
The potential shown by pulchrol (1a) as an antiparasitic agent against T. cruzi and
the Leishmania species led to the development of a synthetic route [42,43] that yields

Molecules 2021, 26, 3944
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sufficient amounts of pulchrol to perform additional biological assays. This could also
be adapted for the preparation of a series of analogues with individual transformations
of the benzyl alcohol moiety in the A-ring [44], variations at positions 1, 2 and 3 in the
C-ring, and modifications at the only available position in the B-ring (C-6) [45]. In this
investigation, we prepared analogues with combinations of different functionalities in the
A-, B-, and C-rings (see Figure 2), inspired by some of the SARs observed in the previous
studies [44,45]. Cannabinol (5f) and its 3-methyl analogue 5e (see Figure 2) were also
prepared and assayed. Our main objective was to study the effect that several substituents
coexisting at different positions in the benzo[c]chromene skeleton may have in the activity
towards the parasites under study, and retrieve more information about the chemical
surroundings of the active site in which pulchrol and its analogues may be interacting.
0
Figure 2. Prepared derivatives. 1a R2 = OMe, R3 = H, R6 = Me, R6” = Me; 1b R2 = isopropyl, R3 = H,
0
0
0
R6 = Me, R6” = Me; 1c R2 = H, R3 = isopropyl, R6 = Me, R6” = Me; 1d R2 = OMe, R3 = H, R6 = Me,
0
0
R6” = H; 1e R2 = OMe, R3 = H, R6 = H, R6” = Me; 2a R2 = OMe, R3 = H, R6 = Me, R6” = Me;
2b R2 = isopropyl, R3 = H, R6 = Me, R6” = Me; 2c R2 = H, R3 = isopropyl, R6 = Me, R6” = Me;
2d R2 = OMe, R3 = H, R6 = Me, R6” = H; 2e R2 = OMe, R3 = H, R6 = H, R6” = Me; 3a R2 = OMe;
0
0
0
0
3b R2 = isopropyl; 4a R2 = OMe, R3 = H; 4b R2 = isopropyl, R3 = H; 4c R2 = H, R3 = isopropyl;
5a R1 = H, R2 = OMe, R3 = H; 5b R1 = H, R2 = isopropyl, R3 = H; 5c R1 = H, R2 = H, R3 = isopropyl;
5d R1 = H, R2 = OH, R3 = H; 5e R1 = OH, R2 = H, R3 = Me; 5f R1 = OH, R2 = H, R3 = n-pentyl.
See Experimental part for synthetic details.
2. Results
2.1. Preparation
In this study, analogues of pulchrol were prepared, containing combinations of two or
three modifications in the A-, B, and C-rings (see Figure 2). The synthetic routes used to
prepare the analogues were partially based on the already reported procedure that was
used to prepare pulchrol from biaryl intermediates [42
(see Figure 2) were previously reported [44 45], and were used as the starting material
to prepare the compounds from the -, -, - and
-series, containing a 10-aldehyde, 10-
,43]. The molecules in the 1-series
,
2
3
4
5
methyl ketone, 3-methylbutanoic acid ester, and 9-methyl functionalities, respectively (see
Figure 2). Compound 5f (cannabinol) and its analogue 5e were obtained through iodine-
mediated deconstructive annulation in a one-pot synthesis, using citral and resorcinol
analogues as the starting material [46]. In total, 12 molecules were prepared and assayed
in vitro against T. cruzi epimastigotes, as well as L. braziliensis and L. amazonensis pro-
mastigotes (see Table 1). Their antiparasitic activities were compared with those previously

Molecules 2021, 26, 3944
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reported for the analogues in the
and 5a 44]. Figure 2 summarizes the structure types of the analogues prepared, while the
biological activities are given in Table 1.
1-series in addition to compounds 2a (pulchral), 3a, 4a,
[
Table 1. Antileishmanial and antitrypanosomal activity pulchrol derivatives, compared to the positive controls benznidazole
and miltefosine. See experimental for details about the assays.
L. amazonensis
T. cruzi
L. braziliensis
0
0
R6
R1
R2
R3
R6
Mol.
IC50 (µM)
IC50 (µM)
IC50 (µM)
1a
2b
2c
2d
2e
3b
4b
4c
5b
5c
5d
5e
5f
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OMe
i-Pr
H
OMe
OMe
i-Pr
i-Pr
H
i-Pr
H
OH
H
H
Me
H
i-Pr
H
H
H
H
i-Pr
H
i-Pr
H
Me
n-Pen
Me
Me
Me
Me
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
18.5 ± 9.6
10.7 ± 4.3
7.1 ± 1.4
59.2 ± 11.8
12.1 ± 4.6
17.8 ± 1.8
70.8 ± 19.7
118.0 ± 0.8
8.8 ± 1.0
77.7 ± 5.6
11.4 ± 3.6
17.8 ± 0.7
44.0 ± 1.6
80.6 ± 4.7
9.5 ± 4.1
125.8 ± 7.9
170.3 ± 7.9
3.4 ± 0.2
10.9 ± 3.8
13.6 ± 5.7
23.7 ± 8.6
50.3 ± 11.3
54.9 ± 0.2
5.9 ± 2.0
272.9 ± 0.00
63.3 ± 4.4
49.2 ± 15.0
311.6 ± 50.3
30.4 ± 2.9
15.7 ± 5.1
10.3 ± 0.6
25.9 ± 5.5
43.7 ± 8.2
49.6 ± 4.5
236.5 ± 48.8
33.3 ± 5.4
21.2 ± 2.4
14.2 ± 1.3
7.4 ± 0.6
Benznidazole
Miltefosine
19.2 ± 7.7
13.0 ± 1.2
10.8 ± 1.5
Tables 2 and 3 give the 1D 1H and 13C NMR shifts of the assayed compounds.
Table 2. Proton chemical shifts (in ppm) for the compounds prepared in this study, measured at 400 MHz. The assignments
were made with 2D NMR spectroscopy, COSY, HMQC and HMBC experiments.
0
Compd.
1-H
2-H
3-H
4-H
7-H
8-H
10-H
1 -H/H2/H3
2-OCH3
6-H/H2
6,6-CH3
1a
2b a
2c b
2d
7.26
7.65
7.73
7.31
7.32
7.65
7.57
7.64
7.59
7.63
7.20
-
-
-
6.81
7.15
-
6.87
6.87
7.13
7.11
-
7.10
-
6.70
-
-
6.89
6.90
6.85
6.94
6.94
6.89
6.88
6.82
6.89
6.82
6.83
6.43
6.43
7.23
7.41
7.40
7.34
7.34
7.32
7.23
7.22
7.15
7.12
7.12
7.15
7.14
7.30
7.79
7.77
7.81
7.81
7.85
7.28
7.24
7.12
7.08
7.12
7.07
7.07
7.68
8.24
8.19
8.17
8.17
8.32
7.71
7.66
7.58
7.51
7.45
8.17
8.16
4.74
10.07
10.05
10.07
10.07
-
5.17
5.14
2.43
2.39
2.39
2.39
2.38
3.85
-
-
3.87
3.87
-
-
-
-
-
-
-
-
-
-
-
1.61
1.65
1.66
1.63
1.63
1.64
1.62
1.63
1.63
1.62
1.60
1.60
1.59
6.94
-
-
-
-
6.90
-
6.88
-
6.28
6.29
5.25
5.27
2e
3b c
4b d
4c e
5b f
5c g
5d
-
-
-
-
-
-
-
-
5e h
5f i
a
-
b
c
Isopropyl signals at 2.95 and 1.30 ppm. Isopropyl signals at 2.90 and 1.27 ppm. Methyl signal at 2.66 ppm and isopropyl signals at 2.95
d
e
and 1.30 ppm. i-Butyl signals at 2.28, 2.16 and 0.99 ppm; and isopropyl signals at 2.94 and 1.30 ppm. i-Butyl signals at 2.26, 2.15 and
f
g
0.97 ppm; and isopropyl signals at 2.88 and 1.26 ppm. Isopropyl signals at 2.95 and 1.31 ppm. Isopropyl signals at 2.88 and 1.26 ppm.
h
i
Methyl signal at 2.26 ppm. n-Pentyl signals at 2.50, 1.61, 1.33, 1.31, 0.88 ppm.

Molecules 2021, 26, 3944
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Table 3. 13C NMR chemical shifts (in ppm) for compounds of series
were made with 2D NMR spectroscopy, COSY, HMQC and HMBC experiments.
1 to 5 determined at 100 MHz in CDCl3. The assignments
Compd.
C-1
C-2
C-3
C-4
C-4a
C-6
C-6a
C-7
C-8
C-9
C-10
C-10a
C-10b
C-10
2-OCH3
6,6-CH3/6-CH3
1a
2b a
2c b
2d
108.0 154.6
121.1 142.5
123.1 120.5
108.0 155.0
108.0 155.1
121.1 142.4
120.8 142.0
122.8 120.1
120.7 141.9
122.7 119.9
109.5 150.1
153.2 110.7
153.2 110.0
115.5
128.6
152.2
116.7
116.7
128.2
127.8
151.4
127.4
150.9
116.3
139.5
144.7
118.8
118.1
115.9
118.8
118.9
118.0
117.9
115.8
117.8
115.8
118.9
111.6
110.9
146.9
150.9
152.8
147.5
147.5
150.9
150.9
152.9
151.0
152.9
146.9
154.8
154.8
77.4
77.4
77.7
73.6
73.6
77.4
77.4
77.6
77.4
77.6
77.4
77.4
77.4
139.5
146.0
145.4
142.5
142.6
144.6
139.7
139.2
137.1
136.5
137.4
137.0
137.0
123.7
124.2
124.2
125.1
125.1
123.7
123.7
123.6
123.3
123.2
123.3
122.8
122.8
126.8
129.5
129.0
129.8
129.8
128.0
127.7
127.4
128.6
128.3
129.1
127.7
127.7
140.4
135.9
136.0
136.4
136.4
136.6
135.6
135.6
137.2
137.2
137.3
137.0
127.7
121.0
123.2
123.2
123.1
123.1
122.0
122.1
121.9
122.8
122.6
123.1
126.6
126.5
129.1
130.3
130.2
130.8
130.8
129.7
129.4
129.3
127.7
128.7
128.4
127.6
137.0
123.0
121.0
118.9
122.1
122.1
121.3
121.7
119.7
122.1
120.1
123.5
108.7
108.8
65.3
192.2
192.1
192.1
192.1
198.0
66.0
66.0
21.5
64.8
21.4
56.0
27.5
27.5
27.6
19.9
19.9
27.5
27.7
27.8
27.8
27.9
27.6
27.2
27.2
-
-
56.0
56.0
2e
3b c
4b d
4c e
5b f
5c g
5d
-
-
-
-
-
-
-
-
5e h
5f i
21.7
21.7
a
b
c
Isopropyl signals at 33.9 and 24.4 ppm. Isopropyl signals at 34.2 and 23.9 ppm. Methyl signal at 26.9 ppm and isopropyl signals at 33.9
and 24.4 ppm. d 3-Methylbutanoate signals at 173.1, 43.6, 25.9 and 22.6 ppm and isopropyl signals at 33.8 and 24.4 ppm. e 3-Methylbutanoate
f
g
signals at 173.1, 43.6, 25.9 and 22.6 ppm and isopropyl signals at 34.1 and 23.9 ppm. Isopropyl signals at 33.9 and 24.4 ppm. Isopropyl
signals at 34.1 and 24.0 ppm. Methyl signal at 21.4 ppm. n-Pentyl signals at 35.8, 31.6, 30.6, 22.7 and 14.2 ppm.
h
i
2.2. Selected Functionalities
The natural products pulchrol (1a) and pulchral (2a) have been studied in the past,
and both of them have been shown to be active against Trypanosoma and Leishmania par-
asites [44]. Pulchrol (1a) has been reported to be moderately active against L. braziliensis
and L. amazonensis promastigotes (IC50 59.2
has been shown to possess potent toxicity towards T. cruzi epimastigotes (IC50 18.5
comparable to that of the drug benznidazole (19.2 M) that currently is used as treatment
for Chagas disease. Meanwhile, pulchral (2a) has been shown to be active against all of
µM and IC50 77.7
µM, respectively), while it
µM),
µ
the three parasites (T. cruzi, IC50 24.2
29.8 µM) [44,45].
µM; L. braziliensis, IC50 24.2 µM; L. amazonensis, IC50
In previous investigations, we studied the effect that individual transformations in
the A-, B-, and C-rings have on the activity towards T. cruzi, L. braziliensis and L. amazo-
nensis [44,
45]. Initially, we reported the effect of the modifications at position 10 in the
A-ring, and concluded preliminarily that the benzyl alcohol functionality was important
for pulchrol’s activity towards all of the three parasites, possibly acting as a hydrogen bond
acceptor. It was also observed that the 10-carbonyl analogues were equipotent towards
T. cruzi compared to pulchrol, while they were more potent towards L. braziliensis and
L. amazonensis. On the other hand, the 9-methyl analogue was reported to possess con-
siderably less activity than pulchrol against all the parasites. Finally, the ester analogues
were shown to be, in general, more potent than pulchrol, with longer and branched alkyl
substituents showing considerable improvements [44]. The effects that modifications in the
B- and C-rings have on the antiparasitic activity were also investigated, and were mainly fo-
cused on the effects that variations in the lipophilicity may have in the antiparasitic activity.
The 6-monoalkyl (methyl and ethyl) derivatives, also as pure enantiomers, were less potent
towards the parasites compared to pulchrol, and a preference for 6,6-dialkyl analogues
was established. Alkyl substituents in the C-ring were shown to be beneficial for the
antiparasitic activity; most notably, longer and branched alkyl substituents at positions 2
and 3 increased the potency considerably [45].
3. Discussion
In this study, we analyse the effect that two or three transformations in the different
rings of the pulchrol scaffold may have on the antiparasitic activity. Most of the analogues
have transformations in the A- and C-rings, whereas just a pair have the A- and B-rings
modified. The 10-aldehyde, the 3-methyl butanoic acid ester, and the 9-methyl functionali-
ties in the A-ring were combined with isopropyl substituents at C-2 and C-3 in the C-ring
(
2b and 2c
;
4b and 4c; and 5b and 5c, respectively) to see how an increase in lipophilicity
0
would affect their antiparasitic activity. An analogue with the 1 -methyl ketone functional-

Molecules 2021, 26, 3944
6 of 14
ity in the A-ring and an isopropyl substituent at position 2 was also prepared (3b) to see if
any differences would arise with respect to the corresponding 10-aldehyde analogue (2b).
The 9-methyl functionality was also combined with a hydroxyl substituent at C-2 in the
C-ring (5d), and the natural compound cannabinol (5f) and its analogue 5e were prepared,
both of them containing a 9-methyl substituent in the A-ring, a hydroxyl group at C-1,
and an alkyl group at C-3 in the C-ring; cannabinol (5f) with an n-pentyl substituent and
5e with a methyl substituent at C-3. Finally, the 10-aldehyde functionality was combined
with the 6-monomethyl substituent in the B-ring, as a pair of enantiomers (2d and 2e),
to evaluate whether the carbonyl group helps to improve their activity or hamper it in
comparison with the already reported benzyl alcohol enantiomers 1d and 1e [45].
3.1. Antiparasitic Activities towards Trypanosoma Cruzi Epimastigotes
It has previously been reported that the transformation of the benzyl alcohol func-
tionality in pulchrol (1a, IC50 = 18.5
3a, IC50 = 21.3 M) was not beneficial for the antiparasitic activity towards T. cruzi [44],
while the replacement of the methoxy functionality (in 1a) for an isopropyl group in po-
sitions 2 and 3 in the C-ring was shown to improve the IC50 values for this parasite (1b
IC50 = 12.4 M and 1c, IC50 = 14.2 M, respectively) [45]. In order to evaluate the effect
µ µM and
M) to a 10-carbonyl group (2a, IC50 = 24.2
µ
,
µ
µ
that combinations involving the functionalities mentioned before may have in the antit-
rypanosomal activity, analogues with an aldehyde functionality in the A-ring and with
an isopropyl substituent at positions 2 or 3 in the C-ring were prepared and found to be
considerably more active (2b, IC50 = 10.7
previously reported corresponding benzyl alcohols 1b and 1c
by a change in orientation in a hypothetical binding site, by the combined functionalities,
possibly enhancing the hydrophobic interactions of the isopropyl substituents in 2b and 2c
The methyl ketone 3b, substituted with an isopropyl group at C-2, was actually the most po-
tent compound towards T. cruzi (IC50 = 3.4 M) of all the analogues prepared in this study,
showing six-fold higher activity than the positive control benznidazole (IC50 = 19.2 M),
µM; and 2c, IC50 = 7.1
µM) compared to the
[45]. This could be caused
.
µ
µ
indicating possible interactions between the methyl substituent in the ketone and the
active site.
Contrary to the improvements shown by the analogues substituted with an isopropyl
group in the
a 3-methylbutanoic acid ester) were less potent towards T. cruzi (4b, IC50 = 10.9
4c, IC50 = 13.6 M) compared to the corresponding 2-methoxy ester 4a (IC50 = 4.2
2
- and
3
-series, 2- and 3-isopropyl analogues of the
4
-series (combined with
M;
M),
µ
µ
µ
which has previously been reported to possess better activity than the positive control
benznidazole [44].
Among the analogues of the
A-ring, analogue 5a (with a methoxy group at C-2, IC50 = 51.1
shown to possess less potency than pulchrol (1a) [44]. Similarly, in this study analogue 5d,
with a hydroxyl functionality at C-2, showed comparable activity (IC50 = 54.8 M) to 5a
5-series, which contain a 9-methyl substituent in the
µM) has previously been
µ
towards T. cruzi. It is possible that both analogues, 5a and 5d, interact with the binding
site in a rotated position, in which the methoxy group in 5a and the hydroxy group in 5d
act as hydrogen bond acceptors where pulchrol’s benzyl alcohol usually interacts. On the
other hand, more lipophilic analogues, 5b and 5c, with isopropyl substituents at positions 2
and 3 (IC50 = 23.7
pulchrol (1a, IC50 = 18.5
an alkyl substituent at C-3 and, in addition, a hydroxyl functionality at C-1, were shown
to be the most potent (5f, IC50 = 7.4 M; 5e, IC50 = 5.9 M) compounds from the -series
µM and IC50 = 50.3
µM, respectively) were found to be less toxic than
µ
M) However
.
, cannabinol (5f) and its analogue 5e, which also has
µ
µ
5
towards T. cruzi. Analogue 5e, with a methyl substituent at C-3 instead of the n-pentyl
group in cannabinol (5f), is slightly more active than 5f and possesses one of the highest
activities towards T. cruzi among all the derivatives prepared in this project, possibly due
to interactions between the hydroxyl group at C-1 and the target protein.
Finally, the aldehyde enantiomers 2d and 2e, substituted with a single methyl group
at position 6 in the B-ring, possess considerably lower antiparasitic activity compared to

Molecules 2021, 26, 3944
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pulchrol (1a, IC50 = 18.5
µM). They are also less potent compared to their synthetic precur-
sors, the benzyl alcohols 1d and 1e [45], indicating that a possible change in orientation
may disrupt hydrophobic interactions around position 6 in the B-ring.
3.2. Antiparasitic Activities towards Leishmania Braziliensis Promastigotes
The opposite to what has been reported for T. cruzi, the transformation of pulchrol’s
benzyl alcohol to a 10-carbonyl functionality (2a and 3a) has been shown to consider-
ably increase the antiparasitic activity towards L. braziliensis (2a, IC50 = 24.2
IC50 = 28.3 M) [44]. Likewise, isopropyl substituents in the C-ring (1b and 1c) also in-
crease the potency (1b, IC50 = 18.1 M; and 3a, IC50 = 19.1 M) compared to pulchrol (1a
IC50 = 59.2 M) [45]. In this study, the aldehydes 2b and 2c (with isopropyl substituents at
C-2 and C-3, respectively were prepared and found to be more toxic towards L. braziliensis
2b, IC50 = 12.1 M; and 2c, IC50 = 17.8 M) than their benzyl alcohol precursors (1b and 1c).
As for T. cruzi, the 2-isopropyl ketone 3b was the most potent compound (IC50 = 8.8 M) in
this investigation. Furthermore, the 10-carbonyl analogues 2b and 3b are also more potent
than the positive control miltefosine (IC50 = 13.0 M). Possibly, the increase in potency
µM; and 3a,
µ
µ
µ
,
µ
)
(
µ
µ
µ
µ
shown by the carbonyl analogues combined with alkyl substituents in the C-ring, may be
due to better hydrophobic interactions with a target protein, produced by a change in the
orientation of the alkyl groups.
Similar results to those obtained for T. cruzi were observed for the analogues from
the 4-series; the 2- and 3-isopropyl esters 4b and 4c were less active towards L. braziliensis
(
4b, IC50 = 272.9
µM; and 4c, IC50 = 63.3
µM) than the previously reported ester 4a (with a
methoxy substituent at C-2, IC50 = 13.1
µM) [44]. The opposite to T. cruzi, analogue 4b was
inactive, indicating a possible limitation in the volume of the lipophilic pocket where the
3-alkyl substituents interact.
The transformation of the benzyl alcohol moiety in pulchrol (1a) to a 9-methyl sub-
stituent, as in 5a, has previously been determined to be unfavourable for the activity (1a
IC50 = 59.2 M; and 5a, IC50 = 69.6 M) [44]. In this investigation, the 9-methyl/2-isopropyl
analogue 5b and the 9-methyl/2-hydroxy analogue 5d are more potent towards L. brazilien-
sis (5b, IC50 = 49.2 M; and 5d, IC50 = 30.4 M), opposite to T. cruzi. On the other hand,
the 9-methyl/3-isopropyl analogue 5c was shown to be essentially inactive (IC50 = 312 M),
,
µ
µ
µ
µ
µ
suggesting again that there is a limit in the volume around positions 2 and 3. Similar to the
results obtained for T. cruzi, 5e and cannabinol (5f) were found to possess higher activity
(IC50 = 15.7 µM and 10.3 µM, respectively) than 5a and pulchrol (1a). However, the longer
chain at C-3 in cannabinol (5f) seems to be better for the activity against L. braziliensis
compared to 5e, with a methyl group at C-3, while the opposite is true for T. cruzi.
As with T. cruzi, the aldehyde enantiomers 2d and 2e were found to be less potent
(IC50 = 70.8 µM and 118 µM, respectively) compared to pulchrol (1a), although they are
more potent than their corresponding benzyl alcohol enantiomers 1e and 1f (IC50 = 156
and 129 µM, respectively) [45].
µM
3.3. Antiparasitic Activities towards Leishmania Amazonensis Promastigotes
For L. amazonensis, as with L. braziliensis, pulchral (2a), and the analogues 1b and 1c
(substituted with an isopropyl group at C-2 and C-3 in the C-ring, respectively) have been
reported to be more potent than pulchrol [44,45]. Similar to T. cruzi and L. braziliensis,
the combination of the 9-aldehyde functionality with isopropyl substituents at positions 2
and 3 (2b and 2c, respectively) is beneficial for the antiparasitic activity, and the highest
activities were shown by the aldehyde 2b (IC50 = 11.4
µM), and the methyl ketone 3b
(IC50 = 9.5 M), which are equipotent with the positive control miltefosine (IC50 = 10.8
µ
µM).
All the ester analogues (4a to 4b) were more toxic than pulchrol towards L. amazonensis,
and as with the other parasites, the previously reported 4a (with a methoxy substituent on
C-2) was still the most potent (IC50 = 14.5 µM) among the esters.
The analogues possessing a 9-methyl substituent were similarly as potent towards L. ama-
zonensis as towards L. braziliensis. Most of the 9-methyl analogues (5b, 5d, 5e and 5f) are more

Molecules 2021, 26, 3944
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potent than pulchrol (1a, IC50 = 77.7
less active (IC50 = 236.5
are cannabinol (5f, IC50 = 14.2
tiomers 2d (IC50 = 44.0 M) and 2e (IC50 = 80.6
6,6-dimethyl aldehyde 2a (pulchral, IC50 = 29.8
L. braziliensis and T. cruzi.
µ
M), while the 9-methyl/3-isopropyl analogue 5c is much
µ
M) towards L. braziliensis. The most potent compounds in the
M) and its analogue 5e (IC50 = 21.2 M). Finally, the enan-
M) were still less potent than the corresponding
M) [44], similar to what was observed for
5-series
µ
µ
µ
µ
µ
4. Materials and Methods
4.1. General
1H NMR spectra (400 MHz) and 13C NMR spectra (100 MHz) (See Supplementary
Materials) were recorded with a Bruker Avance II (Bruker Biospin AG, Industriestrasse 26,
8117 Fällanden, Switzerland) in CDCl3. The individual 1D signals were assigned using
2D NMR experiments (COSY, HSQC, HMBC). The chemical shifts are given in ppm with
the solvent signal as reference (7.27 ppm for 1H and 77.0 for 13C). Infrared spectra were
recorded with a Bruker Alpha-P FT/IR instrument (Bruker Biospin AG, Industriestrasse
26, 8117 Fällanden, Switzerland) with a Diamond ATR sensor as films, and the intensities
are given as vw (very weak), w (weak), m (medium), s (strong) and vs (very strong).
High-resolution mass spectra (HRMS) were recorded with Waters XEVO-G2 QTOF equip-
ment (Waters Corp, Milford, Worcester County, MA, USA), with electrospray ionization
(ESI). Synthetic reactions were monitored by TLC using alumina plates coated with silica
gel and visualized using either UV light and/or spraying/heating with vanillin/H2SO4.
Flash chromatography was performed with silica gel (35–70 µm, 60 Å). THF was distilled
from sodium, acetonitrile was distilled from CaH2 and other reaction solvents were dried
with Al2O3. Commercially available compounds were obtained from Aldrich.
4.2. Synthetic Procedures
Methyl 4-(hydroxymethyl)-2-iodobenzoate (intermediate in the synthesis of 1a–1e): BH3-
THF (1 M, 47.1 mL, 47.1 mmol) was slowly added to a stirred solution of 1-methyl-2-
iodoterephthalate (4.8 g, 15.7 mmol) in dry THF (250 mL) at 0 ◦C. After 30 h, saturated
aqueous NaHCO3/H2O was added, and the aqueous phase was extracted with ethyl
acetate (3
×
250 mL) before drying (Na2SO4) and removal of solvent under reduced
pressure. Purification by column chromatography (SiO2, 4:6 heptane/ethyl acetate) gave
4.06 g (89%) of the pure product as yellow crystals, identical to that previously reported [42].
Methyl 4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-iodobenzoate (intermediate in the syn-
thesis of 1a–1e): TBDPSCl (4.3 mL, 16.7 mmol) was added to a stirred solution of methyl
4-(hydroxymethyl)-2-iodobenzoate (4.06 g, 13.9 mmol) in pyridine (80 mL) at rt. After 24 h,
saturated aqueous NH4Cl/H2O was added and the aqueous phase was extracted with
diethyl ether (3
×
200 mL), then the organic phase was washed with brine (2
×
500 mL)
before drying (Na2SO4) and removal of solvent under reduced pressure. Purification by
column chromatography (SiO2, 20:2 heptane/ethyl acetate) gave 4.2 g (57%) of the pure
product as white crystals, identical to that previously reported [42].
General procedure for Suzuki coupling (intermediates in the synthesis of 1a–1e): corre-
sponding boronic acid (1.5 equiv), K2CO3 (5 equiv) and Tetrakis(triphenylphosphine)pallad-
ium(0) (0.17 equiv) were added to a stirred solution of methyl 4-(((tert-butyldiphenylsilyl)o-
xy)methyl)-2-iodobenzoate (1 equiv) dissolved in 4:1 DME/water (15 mL), the mixture
◦
(contained in a microtube) was degasified under vaccuum/N2 at
−
78 C five times. The mi-
crowave reaction conditions were 100 ◦C, high pressure, and 10 s of pre-stirring. After 30
to 60 min in the microwave reactor, the mixture was filtered through a plug of celite and
washed with ethyl acetate (250 mL) before drying (Na2SO4) and removal of solvent un-
der reduced pressure. Purification by column chromatography (SiO2, 20:3 heptane/ethyl
acetate) gave the pure products.
0
0
0
Methyl 5-(((tert-butyldiphenylsilyl)oxy)methyl)-2 ,5 -dimethoxy-[1,1 -biphenyl]-2-carboxylate
(intermediate in the synthesis of 1a): the pure product was obtained as an orange wax
(yield 94%) identical to that previously reported [42].

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Methyl 5-(((tert-butyldiphenylsilyl)oxy)methyl)-50-isopropyl-20-methoxy-[1,10-biphenyl]-2-
carboxylate (intermediate in the synthesis of 1b): the pure product was obtained as colorless
wax (yield 97%) identical to that previously reported [45].
Methyl 5-(((tert-butyldiphenylsilyl)oxy)methyl)-20-methoxy-40-isopropyl-[1,10-biphenyl]-2-
carboxylate (intermediate in the synthesis of 1c): the pure product was obtained as a
yellowish wax (yield 38%) identical to that previously reported [45].
General procedure for organo-lithic addition (intermediate in the synthesis of 1a
Corresponding organo-lithic reagent (4 equiv) was added to a stirred solution of the
Suzuki coupling product (1 equiv) in dry THF (70 mL), at 0 or
78 ◦C, depending on the
organo-lithic reagent. After 12 h, saturated aqueous NH4Cl/H2O was added, and the
aqueous phase was extracted with ethyl acetate (3 100 mL) before drying (Na2SO4) and
–1e):
−
×
removal of solvent under reduced pressure. Purification by column chromatography (SiO2,
20:4 heptane/ethyl acetate) gave the pure product.
2-(5-(((tert-butyldiphenylsilyl)oxy)methyl)-20,50-dimethoxy-[1,10-biphenyl]-2-yl)propan-2-ol
(intermediate in the synthesis of 1a): the pure product was obtained as a yellowish wax
(yield 65.2%) identical to that previously reported [42].
2-(5-(((tert-butyldiphenylsilyl)oxy)methyl)-50-isopropyl-20-methoxy-[1,10-biphenyl]-2-yl)pro-
pan-2-ol (intermediate in the synthesis of 1b): the pure product was obtained as a transpar-
ent wax (yield 78.5%) identical to that previously reported [45].
2-(5-(((tert-butyldiphenylsilyl)oxy)methyl)-20-methoxy-40-isopropyl-[1,10-biphenyl]-2-yl)pro-
pan-2-ol (intermediate in the synthesis of 1c): the pure product was obtained as a transparent
wax (yield 64%) identical to that previously reported [45].
General procedure to prepare compounds 1a–1c: HI (55%, 10 equiv) was added to a stirred
solution of the corresponding starting material in acetonitrile (25 mL), at rt. After 30 min,
saturated aqueous Na2S2O3 (25 mL) was added, and the aqueous layer was extracted with
ethyl acetate (3
×
50 mL), before drying (Na2SO4) and removal of solvent under reduced
pressure. TBAF (1 M, 1.1 equiv) was added to the crude product in THF (150 mL). After 3 h,
aqueous saturated NaHCO3 (50 mL) was added, and the aqueous layer was extracted with
ethyl acetate (3
×
50 mL), before drying (Na2SO4) and removal of solvent under reduced
pressure. Purification by column chromatography (SiO2, 1:1 heptane/ethyl acetate) gave
the pure product.
(2-methoxy-6,6-dimethyl-6H-benzo[c]chromen-9-yl)methanol (1a): the pure product was
obtained as a yellowish wax (yield 77%) identical to that previously reported [42].
(2-isopropyl-6,6-dimethyl-6H-benzo[c]chromen-9-yl)methanol (1b): the pure product was
obtained as a transparent wax (yield 88%) identical to that previously reported [45].
(3-isopropyl-6,6-dimethyl-6H-benzo[c]chromen-9-yl)methanol (1c): the pure product was
obtained as a transparent wax (yield 85%) identical to that previously reported [45].
5-(((tert-butyldiphenylsilyl)oxy)methyl)-20,50-dimethoxy-[1,10-biphenyl]-2-carbaldehyde (in-
termediate in the synthesis of 1d and 1e): Morpholine (0.2 mL, 2.2 mmol) was◦added
to a solution of DIBALH (1 M, 1.1 mL, 1.1 mmol) in dry THF (30 mL) at 0 C. Af-
ter 3 h, methyl 5-(((tert-butyldiphenylsilyl)oxy)methyl)-20,50-dimethoxy-[1,10-biphenyl]-
2-carboxylate (600 mg, 1.1 mmol) in dry THF (20 mL) was added, 10 min later, DIBALH
(1 M, 1.1 mL, 1.1 mmol) was added again at 0 ◦C. After 4 h, aqueous HCL (1 N, 20 mL)
was added, and the aqueous phase was extracted with diethyl ether (3
×
50 mL) before
drying (Na2SO4) and removal of solvent under reduced pressure. Purification by column
chromatography (SiO2, 20:4 heptane/ethyl acetate) gave the pure product as a yellowish
wax (89.1 mg, 16%) identical to that previously reported [45].
General procedure to prepare compounds01d and 1e: MeLi (3 M, 2 equiv) was added
0
0
to 5-(((tert-butyldiphenylsilyl)oxy)methyl)-2 ,5 -dimethoxy-[1,1 -biphenyl]-2-carbaldehyde
(1 equiv) in dry THF (5 mL) at 0 ◦C. After 6 h, saturated aqueous NH4Cl/H2O was added,
and the aqueous phase was extracted with ethyl acetate (3
×
20 mL) before drying (Na2SO4)
and removal of solvent under reduced pressure. PBr3 (0.34 equiv) was added to the crude
product (1 equiv) in dichloromethane (10 mL) at rt. After 2 h, LiI (3 equiv) was added at
rt. After 12 h, saturated aqueous Na2S2O3/H2O was added, and the aqueous phase was

Molecules 2021, 26, 3944
10 of 14
extracted with ethyl acetate (3
under reduced pressure. TBAF (2 equiv) was added to the crude product in THF (25 mL)
at rt. After 5 h, saturated aqueous NaHCO3/H2O was added, and the aqueous phase was
×
20 mL) before drying (Na2SO4) and removal of solvent
extracted with ethyl acetate (3
×
25 mL) before drying (Na2SO4) and removal of solvent
under reduced pressure. Purification by column chromatography (SiO2, 1:1 heptane/ethyl
acetate) and the enantiomers were separated using a semipreparative HPLC (Chiralpack B
column, 96:4 hexane/isopropanol).
(2-methoxy-6-methyl-6H-benzo[c]chromen-9-yl)methanol (1d): the pure product was ob-
tained as a yellowish wax (yield 3%), αD20-17.8 identical to that previously reported [45].
(2-methoxy-6-methyl-6H-benzo[c]chromen-9-yl)methanol (1e): the pure product was ob-
◦
tained as a yellowish wax (yield 3%),
α
D20 +23.8 identical to that previously reported [45].
General procedure to obtain compounds 2b
–2e: Dess–Martin periodinane 15% (2 equiv)
was added to a stirred solution of corresponding starting material (1 equiv) in CH2Cl2 at rt.
After five hours, saturated aqueous Na2S2O3/H2O was added and the aqueous phase was
extracted three times with CH2Cl2 before drying (Na2SO4) and removal of solvent under
reduced pressure. Purification by column chromatography (SiO2, 20:1 CH2Cl2/methanol).
2-isopropyl-6,6-dimethyl-6H-benzo[c]chromene-9-carbaldehyde (2b): The pure product was
obtained as a transparent wax (85.7 mg, yield 88%). 1H NMR and 13C NMR data in
Tables 2 and 3, respectively. HRMS-ESI+ (m/z): [M + Na]+ calcd for C19H20O2, 303.1361;
found 303.1355.
3-isopropyl-6,6-dimethyl-6H-benzo[c]chromene-9-carbaldehyde (2c): the pure product was
obtained as a transparent wax (15.3 mg, yield 77%). 1H NMR and 13C NMR data in
Tables 2 and 3, respectively. HRMS-ESI+ (m/z): [M + Na]+ calcd for C19H20O2, 303.1361;
found 303.1357.
2-methoxy-6-methyl-6H-benzo[c]chromene-9-carbaldehyde (2d): the pure product was ob-
tained as a yellowish wax (5.4 mg, yield 96%). 1H NMR and 13C NMR data in Tables 2 and 3,
respectively. HRMS-ESI+ (m/z): [M + Na]+ calcd for C16H14O5, 309.0738; found 309.0739
(+2O due to oxidation).
2-methoxy-6-methyl-6H-benzo[c]chromene-9-carbaldehyde (2e): the pure product was ob-
tained as a yellowish wax (6.7 mg, yield 95%). 1H NMR and 13C NMR data in Tables 2 and 3,
respectively. HRMS-ESI+ (m/z): [M + Na]+ calcd for C16H14O5, 309.0739; found 309.0739
(+2O due to oxidation).
1-(2-isopropyl-6,6-dimethyl-6H-benzo[c]chromen-9-yl)ethan-1-one (3b): MeMgI (3 M, 0.24 mL,
0.7 mmol) was added to 2b (65 mg, 0.23 mol) in dry ethyl ether (5 mL), at 0 ◦C. After 20 h,
saturated aqueous NH4Cl/H2O was added, and the aqueous phase was extracted with
diethyl ether (3
×
5 mL). The organic product was washed with brine once before drying
(Na2SO4) and removal of solvent under reduced pressure. Celite (250 mg) and PCC (63 mg,
0.25 mmol) were added to the crude product in dry CH2Cl2 (5 mL) under nitrogen, at rt.
After 12 h diethyl ether (10 mL) was added and the mixture was filtered over a plug of
silica gel and washed with ethyl acetate (50 mL) before removal of solvent under reduce
pressure. Purification by column chromatography (SiO2, 20:4 hept/ethyl acetate) gave
30.0 mg, 44% as a transparent wax.%). 1H NMR and 13C NMR data in Tables 2 and 3,
respectively. HRMS-ESI+ (m/z): [M + Na]+ calcd for C20H22O2, 317.1514; found 317.1517.
General procedure to obtain compounds 4b–4c: Isovaleric anhydride (1.5 equiv), DMAP
(1.2 equiv), and Et3N (1.5 equiv) were added to a stirred solution of appropriate starting ma-
terial (1 equiv) in CH2Cl2 (25 mL) at rt. After three hours, saturated aqueous NH4Cl/H2O
was added and the aqueous phase was extracted with CH2Cl2 (3
×
25 mL) before dry-
ing (Na2SO4) and removal of solvent under reduced pressure. Purification by column
chromatography (SiO2, 20:4 heptane/ethyl acetate) gave the desired products.
(2-isopropyl-6,6-dimethyl-6H-benzo[c]chromen-9-yl)methyl 3-methylbutanoate (4b): The
1
pure product was obtained as a transparent wax (64.9, yield 53%). H NMR and 13C NMR
data in Tables 2 and 3, respectively. HRMS-ESI+ (m/z): [M + Na]+ calcd for C24H30O3,
389.2093; found 389.2099.

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(3-isopropyl-6,6-dimethyl-6H-benzo[c]chromen-9-yl)methyl 3-methylbutanoate (4c): Thep-
1
ure product was obtained as a transparent wax (15.2, yield 59%). H NMR and 13C NMR
data in Tables 2 and 3, respectively. HRMS-ESI+ (m/z): [M + Na]+ calcd for C24H30O3,
389.2093; found 389.2091.
General procedure to obtain compounds 5a–5c: Et3SiH (5 equiv) and PdCl2 (2 equiv) were
added to a stirred solution of corresponding starting material (1 equiv) in EtOH. After 3h,
ethyl acetate was added and the mixture was filtered through a plug of celite and washed
with ethyl acetate before drying (Na2SO4) and removal of solvent under reduced pressure.
Purification by column chromatography (SiO2, 20:1 heptane/ethyl acetate).
2-Methoxy-6,6,9-trimethyl-6H-benzo[c]chromene (5a): the pure product was obtained as
a yellowish wax (yield 92%) identical to that previously reported [44].
2-isopropyl-6,6,9-trimethyl-6H-benzo[c]chromene (5b): the pure product was obtained
1
as a transparent wax (105 mg, yield 89%). H NMR and 13C NMR data in Tables 2 and 3,
respectively. HRMS-ESI+ (m/z): [M + Na]+ calcd for C19H22O, 289.1568; found 289.1563.
3-isopropyl-6,6,9-trimethyl-6H-benzo[c]chromene (5c): the pure product was obtained
1
as a transparent wax (16 mg, yield 85%). H NMR and 13C NMR data in Tables 2 and 3,
respectively. HRMS-ESI+ (m/z): [M + Na]+ calcd for C19H22O, 289.1568; found 289.1565.
6,6,9-trimethyl-6H-benzo[c]chromen-2-ol (5d): Sodium ethanethiolate (132 mg, 1.57 mmol2)
was added to a stirred solution of 5a (0.157 mmol) in DMF (4 mL), and reacted in a mi-
◦
crowave reactor at 160 C. After one hour, saturated aqueous NH4Cl/H2O was added and
the aqueous phase was extracted with ethyl acetate (3
×
100 mL) before drying (Na2SO4)
and removal of solvent under reduced pressure. Purification by column chromatography
(SiO2, 20:4 heptane/ethyl acetate) gave 27 mg (72%) of the pure product as an orange
1
wax.%). H NMR and 13C NMR data in Tables 2 and 3, respectively. HRMS-ESI+ (m/z):
[M + H]+ calcd for C16H17O2, 241.1229; found 241.1231.
General procedure to obtain compounds 5e and 5f: Citral (1 equiv) and n-butylamine
(1 equiv) were added to the corresponding resorcinol analogue (1 equiv) in toluene at
110 ◦C. After 12 h, DOWEX 50WX8 (200 mg) was added to the stirred mixture at rt. After
10 min the mixture was filtered over a plug of celite. Iodine (2 equiv) was added to the
filtrate at 110 ◦C. After 3 h, saturated aqueous Na2S2O3/H2O was added and the aqueous
phase was extracted three times with EtOAc before drying (Na2SO4) and removal of solvent
under reduced pressure. Purification by column chromatography (SiO2, 100:5 heptane/
ethyl acetate).
3,6,6,9-tetramethyl-6H-benzo[c]chromen-1-ol (5e): the pure product was obtained as an
1
orange wax (26 mg, yield 18%). H NMR and 13C NMR data in Tables 2 and 3, respectively.
HRMS-ESI+ (m/z): [M + Na]+ calcd for C17H18O2, 277.1204; found 277.1201.
6,6,9-trimethyl-3-pentyl-6H-benzo[c]chromen-1-ol (5f): the pure product was obtained
as a brownish wax (6.7 mg, yield 4%). 1H NMR and 13C NMR data in Tables 2 and 3,
respectively. HRMS-ESI+ (m/z): [M + Na]+ calcd for C21H26O2, 333.1830; found 333.1825.
4.3. Biological Assays
4.3.1. Evaluations against Leishmania Parasites
Promastigotes of Leishmania (Leishmania): L. amazonensis, clone 1, NHOM-BR-76-
LTB-012 (Lma, donated by the Paul Sabatier Université, France) and Leishmania (Viannia):
L. braziliensis M2904 C192 RJA (M2904, donated by Dr. Jorge Arévalo from Universidad
Peruana Cayetano Heredia, Peru) [47]. All strains were cultured in Schneider’s insect
medium (pH 6.2), supplemented with 10% FBS and incubated at 26 ◦C. Medium changes
were made every 72 h to maintain a viable parasitic population. Leishmanicidal activ-
ity was determined according to Williams with some modifications [48]. Samples were
dissolved in DMSO (maximum final concentration 1%) at 10 mg/mL. Promastigotes in
logarithmic phase of growth, at the concentration 3
(100 L/well) in 96-well flat bottom microtiter plates. Samples with different concen-
trations (3.1–100 g/mL) were added (100 L). Miltefosine (3.1–100 g/mL) was used
as control drug [49]. Assays were performed in triplicates. The microwell plates were
×
106 parasites/mL, were distributed
µ
µ
µ
µ

Molecules 2021, 26, 3944
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◦
incubated for 72 h at 26 C. After incubation, a solution of XTT (1 mg/mL, [2,3-bis[2-
methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide]) in PBS (pH 7.0 at 37 ◦C)
with PMS (0.06 mg/mL phenazine methosulfate) was added (50 µL/well), and incubated
for 3 h at 26 ◦C. All assays were carried out as triplicates. The optical density of each well
was measured with a Synergy HT microplate reader at 200–450 nm. The IC50 values were
calculated using the Gen5 program (BioTek).
4.3.2. Evaluations against Trypanosoma cruzi
Cultures of Trypanosoma cruzi (epimastigotes, donated by the Parasitology Department
of INLASA, Tc-INLASA) were maintained in medium LIT (liver infusion tryptose) (pH 7.2),
◦
supplemented with 10% FBS and incubated at 26 C. The LIT medium was prepared
with NaCl, 40 g/L; KCl, 4 g/L; Na2HPO4, 80 g/L; tryptose, 50 g/L; 10% liver infusion
broth, 50 mL/L; 40% glucose solution, 10 mL/L; penicillin, 200 units/mL; streptomycin,
200 units/mL; and neomycin, 200 units/mL. Medium changes were made every 72 h to
maintain a viable parasitic population. Trypanocidal activity was determined according to
Muelas-Serrano with some modifications [50]. Samples were dissolved in DMSO (maxi-
mum final concentration 1%) at 10 mg/mL. Epimastigotes in logarithmic phase of growth,
at a concentration of 3
flat bottom microtiter plates. Samples at different concentrations (3.1–100
×
106 parasites/mL, were distributed (100
µ
L/well) in 96-well
µg/mL) were
added (100 µL). Benznidazol (3.1–100 µg/mL) was used as the control drug. Assays were
performed in triplicates. The microwell plates were incubated for 72 h at 26 ◦C. After in-
cubation, a solution of XTT (1 mg/mL) in PBS (pH 7.0 at 37 ◦C) with PMS (0.06 mg/mL)
◦
was added (50
µ
L/well) and incubated for 4 h at 26 C. All assays were carried out as
triplicates. The optical density of each well was measured with a Synergy HT microplate
reader at 200–450 nm. The IC50 values were calculated using the Gen5 program (BioTek).
5. Conclusions
Twelve compounds with chemical transformations of the benzyl alcohol functionality
in the A-ring, and with modifications either in the C-ring or the B-ring, were prepared
and assayed towards T. cruzi, L. braziliensis and L. amazonensis. Although Trypanosoma and
Leishmania species belong to the same order (Trypanosomatida), they apparently possess
different molecular targets in their different life stages, and the ways they infect humans
are not fully understood. Moreover, the active site or active sites on which pulchrol
acts on each of the parasites are unknown; therefore, the bioactivity results reported in
this work just provide some suggestions of SARs and the surrounding near a possible
binding site. The effects of the antiparasitic activity that lipophilic groups in the C-ring
may have was studied by testing analogues substituted with isopropyl groups at position 2
and 3, combined with different transformations of pulchrol’s benzyl alcohol functionality.
The combination of a 10-carbonyl group in the A-ring was found to be beneficial for the
activity towards all of the three parasites; however, the 3-isopropyl analogues showed
slightly better leishmanicidal activity than the 2-isopropyl analogues. The latter are more
potent towards T. cruzi, suggesting that differences in the binding sites of the protein
targets may exist. The most interesting antiparasitic activity was showed by the analogue
containing a 10-methyl ketone functionality in the A-ring and the isopropyl group at
position 2. Moreover, all the carbonyl/isopropyl analogues were more potent than the
analogues containing just the carbonyl functionality in the A-ring or just the isopropyl
group in the C-ring, indicating that the combination may improve the orientation in the
binding site.
Supplementary Materials: The following are available online: 1D and 2D NMR spectra of all
isolated compounds.
Author Contributions: P.T is a PhD student and has planned and carried out the synthesis of
the reported compounds, and written the manuscript; E.S. has carried out the biological assays,
supervised by A.G.; M.D. has supervised the work of P.T. at UMSS; S.M. has supervised the work

Molecules 2021, 26, 3944
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of P.T. in Lund; while O.S. has supervised the work of P.T. in Lund and compiled the manuscript.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Swedish International Development Agency (SIDA,
grant No 2017-258), and a Ph.D. scholarships for P.T. from SIDA is gratefully acknowledge.
Institutional Review Board Statement: Not applicable, as the study did not involve humans or animals.
Informed Consent Statement: Not applicable, as the study did not involve humans or animals.
Data Availability Statement: All data are available in this publication and in the Supplementary Materials.
Conflicts of Interest: The authors declare no conflict of interest. The sponsors had no role in the
design, execution, interpretation, or writing of the study.
Sample Availability: Samples of the compounds are not available from the authors, but all informa-
tion about how to prepare them are given in the paper.
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