Discovery of Sustainable Drugs for Neglected Tropical Diseases: Cashew Nut Shell Liquid (CNSL)-Based Hybrids Target Mitochondrial Function and ATP Production in Trypanosoma brucei

Article abstract of DOI:10.1002/cmdc.201800790

In the search for effective and sustainable drugs for human African trypanosomiasis (HAT), we developed hybrid compounds by merging the structural features of quinone 4 (2-phenoxynaphthalene-1,4-dione) with those of phenolic constituents from cashew nut s

Full text of DOI:10.1002/cmdc.201800790

Accepted Article
Title: Discovery of sustainable drugs for neglected tropical
diseases: cashew nut shell liquid (CNSL)-based hybrids target
mitochondrial function and ATP production in Trypanosoma
brucei
Authors: Michela Cerone, Elisa Uliassi, Federica Prati, Godwin U.
Ebiloma, Leandro Lemgruber, Christian Bergamini, David G.
Watson, Thais de A. M. Ferreira, Gabriella Simões Heyn Roth
Cardoso, Luiz A. Soares Romeiro, Harry P. de Koning, and
Maria Laura Bolognesi
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ChemMedChem
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Discovery of sustainable drugs for neglected tropical diseases:
cashew nut shell liquid (CNSL)-based hybrids target
mitochondrial function and ATP production in Trypanosoma
brucei
Michela Cerone,†[a, b] Elisa Uliassi,[a] Federica Prati,[a] Godwin U. Ebiloma,[b, c] Leandro Lemgruber,[b, d]
Christian Bergamini,[a] David G. Watson,[e] Thais de A. M. Ferreira,[f] Gabriella Simões Heyn Roth
Cardoso,[f] Luiz A. Soares Romeiro,[f] Harry P. de Koning,[b] and Maria Laura Bolognesi[a]
[a]
[b]
M. Cerone, Dr. E. Uliassi, Dr. F. Prati, Prof. C. Bergamini, Prof. M. L. Bolognesi
Department of Pharmacy and Biotechnology
Alma Mater Studiorum - University of Bologna
Via Belmeloro 6, I-40126 Bologna, Italy
E-mail: marialaura.bolognesi@unibo.it
M. Cerone, Dr. G. U. Ebiloma, Dr. L. Lemgruber, Prof. H. P. de Koning
Institute of Infection, Immunity and Inflammation
University of Glasgow
GBRC, University Place, G128AT, Glasgow, UK
E-mail: Harry.De-Koning@glasgow.ac.uk
Dr. G. U. Ebiloma
Department of Biochemistry, Faculty of Natural Sciences
Kogi State University
[c]
[d]
P.M.B 1008, Anyigba, Kogi State, Nigria
Dr. L. Lemgruber
Wellcome Trust Centre for Molecular Parasitology
Institute of Infection, Immunity and Inflammation
University of Glasgow
GBRC, University Place, G128AT, Glasgow, UK
Dr. D. G. Watson
Strathclyde Institute of Pharmacy and Biomedical Sciences
University of Strathclyde
16, Richmond Street, G11XQ, Glasgow, UK
T. de A. M. Ferreira, G. S H. Roth Cardoso, Prof. L. A. Soares Romeiro
Department of Pharmacy, Health Sciences Faculty
University of Brasília
[e]
[f]
Campus Universitário Darcy Ribeiro, 70910-900, Brasília, DF, Brazil
Prof. H. P. de Koning ORCID https://orcid.org/0000-0002-9963-1827
Prof. M. L. Bolgnesi ORCID https://orcid.org/0000-0002-1289-5361
M. Cerone ORCID https://orcid.org/0000-0003-0535-3983
Dr. E. Uliassi ORCID https://orcid.org/0000-0002-0990-2532
Dr. F. Prati ORCID https://orcid.org/0000-0002-8483-7646
Prof. C. Bergamini ORCID https://orcid.org/0000-0003-4387-5117
Dr. G. U. Ebiloma ORCID https://orcid.org/0000-0003-4072-9800
Dr. L. Lamgruber ORCID https://orcid.org/0000-0003-2832-6806
Dr. D. G. Watson ORCID https://orcid.org/0000-0003-1094-7604
Dr. T. de A. M. Ferreira ORCID https://orcid.org/0000-0001-7552-1458
Dr. G. S H. Roth Cardoso ORCID https://orcid.org/0000-0001-7120-1086
Prof. L. A. Soares Romeiro ORCID https://orcid.org/0000-0001-5679-0820
Supporting information for this article is given via a link at the end of the document
Abstract: In a search for effective and sustainable drugs for human
African trypanosomiasis (HAT), we developed hybrid compounds by
merging structural features of quinone 4 with those of phenolic
constituents from cashew nut shell liquid (CNSL). CNSL is a waste
product from cashew nut processing factories, with great potential as
precursor of drugs. The synthesized compounds were tested
against Trypanosoma brucei brucei, including three multi-drug
resistant strains, T. congolense, and a human cell line. The most
potent activity was found against T. b. brucei, the HAT causative
agent. Shorter-chain derivatives 20 and 22 were more active than 4,
displaying rapid, micromolar trypanocidal activity, and no human
cytotoxicity. Preliminary studies probing their mode of action on
trypanosomes showed ATP depletion, followed by mitochondrial
membrane depolarization and mitochondrion ultrastructural damage.
This was accompanied by reactive oxygen species production. We
envisage that such compounds, obtained from renewable and
inexpensive material, might be promising, bio-based, sustainable hits
for anti-trypanosomatid drug discovery.
Introduction
Neglected tropical diseases (NTD) are a group of 17 highly
debilitating and potentially fatal poverty-related diseases. These
include protozoan, bacterial, and helminthic infections that prevail
in tropical and subtropical areas in 149 countries. Communities
living in poverty, lacking access to basic sanitation and in close
1
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contact with infectious disease vectors, domestic animals and
livestock, are those worst affected [1]. Notwithstanding the recent
re-emergence of interest in NTD, continuous research efforts are
needed to sustain any drug development pipeline in the medium
displayed low micromolar activity against T. b. brucei and
absence of toxicity on a human cell line. We therefore investigated
the mechanism by which this compound class exerts its
trypanocidal activity.
and long term [2]
.
Human African trypanosomiasis (HAT) is one of the most
neglected tropical diseases, endemic in sub-Saharan Africa.
Trypanosoma brucei rhodesiense (East and Southern Africa) and
T. b. gambiense (West and Central Africa) are the causative
protozoan parasites, which are transmitted to humans by tsetse
flies that are found only in Africa [3]. This disease, which is
disabling and fatal if left untreated, is a major cause of rural
underdevelopment and severely affects economies and
communities. The control and elimination of HAT, which are
declared goals of the WHO [1], would be a major step in the
reduction of the overall burden of tropical disease that continues
to limit development in sub-Saharan Africa [4]. Although HAT
transmission is limited to the tsetse belt, comprising much of sub-
Results and Discussion
Design rationale. CNSL mainly consists of phenolic lipids, i.e.
anacardic acids (1 in Fig. 1) (71.7%), cardanols (2) (4.7%), and
cardols (18.7%) (3) [12]. The pentadecyl alkyl side chain of 1-3 may
be saturated, mono-olefinic, di-olefinic or tri-olefinic with a high
percentage of the components having one or two double bonds
(Fig. 1), depending on the production method [12]. Although CNSL
components have been reported to possess a wide range of
biological activities, in many cases they are not potent enough to
be drug candidates [9]. To overcome this limitation, their use in
combination with standard drugs, and the design of new semi-
synthetic derivatives have been exploited [9]
.
Saharan Africa, the risk of HAT in travelers and migrants, albeit
[5]
low, cannot be overlooked
. Moreover, Animal African
Along these lines, we decided to develop a series of CNSL-based
hybrid compounds. In particular, building on the strategy that the
combinations of two different fragments into one covalently linked
trypanosomiasis (AAT) has an enormous impact on African
agriculture and food security. This condition is caused by related
trypanosome species including T. b. brucei, T. congolense, T.
evansi and T. vivax, and, not necessarily being dependent on
tsetse flies, has spread to much of South America, South Asia and
[13]
hybrid compound can convey synergy and increase potency
,
we combined the chemical features of CNSL derivatives with
those of a previously developed anti-trypanosomal hit compound
(4 in Fig. 2) [14]. Intriguingly, both 4 [14b] and a mixture of anacardic
acids [15], isolated from Brazilian CNSL, have been reported to
the Middle East [6]
.
HAT treatment today relies on five drugs (pentamidine (PMD),
suramin, melarsoprol, nifurtimox and eflornithine, see Fig. S1 for
chemical structures), which suffer from toxic side effects, lack of
efficacy, and development of resistance [1]. The management of
patients using these drugs is complex and risky, requiring the
support services of a well-trained staff [7]. Moreover, despite the
fact that almost all HAT control programs subsidize the cost of
drugs and hospitalization [8], the availability of quality medicinal
agents on a sustainable basis is an increasingly appreciated
public health care concept. Consequently, lowering the costs of
therapy by developing new drugs based on inexpensive
resources is a valuable approach to be pursued.
inhibit
dehydrogenase (GAPDH), an essential glycolytic enzyme and a
validated anti-trypanosomatid target [16]
trypanosomal
glyceraldehyde-3-phosphate
.
OH
OH
OH
O
OH
C15H31-2n
HO
C15H31-2n
3
C15H31-2n
1
2
Based on the above considerations, as well as on our continuous
interest in the NTD field, we explored the possibility of using
cashew nut shell liquid (CNSL) as a sustainable, low-cost starting
material for the development of new drugs against
trypanosomiases. CNSL, which is obtained as the by-product of
cashew nut processing, has proven to be one of the most versatile
food wastes for the production of functional materials and
chemicals [9]. However, its potential as a precursor of drugs has
been relatively under-explored. Being an inedible waste material,
it presents clear environmental, financial and ethical advantages
over synthetic drugs and even over natural products derived from
crops grown for that purpose [10]. In addition, the fact that East
Africa (Tanzania, Kenya and Mozambique) and West Africa
(Benin, Guinea Bissau and Ivory Coast, Nigeria) are among the
largest CNSL-producing countries, opens up the exciting
possibilities to engage endemic countries as crucial actors in NTD
drug discovery and development [11]. On this basis, we have
developed a new chemical library of CNSL-derived hybrids and
investigated their anti-trypanosomal potential. In particular, the
compounds were evaluated against wild-type (WT) and multi-drug
resistant African trypanosomes; T. b. brucei, is extremely closely
related to the human-infectious species, and T. congolense is the
principal agent causing AAT in Africa. Some of the compounds
n = 0
n = 1
n = 2
n = 3
Figure 1. Main components of CNSL.
Furthermore, thanks to the presence of a naphthoquinone moiety,
4 was shown to generate reactive oxygen species (ROS), a
mechanism that may further contribute to its multi-target
trypanocidal activity [14b]. In fact, 4 exhibited high potency against
T. b. rhodesiense (STIB 900 strain) (IC50 = 80 nM) and a promising
selectivity index (SI) of 74, with respect to L6 mammalian cells [14a]
In particular, we anticipated that overlapping 4 with CNSL
.
2
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Scheme 1. Synthesis of target compounds 8-22. Reagents and conditions: (i)
K2CO3, DMF, rt, 3 % - 65 %.
derivatives 5 and 6 (Fig. 2) through their common phenoxy moiety
could lead to hybrids with an improved anti-trypanosomal profile
and an improved sustainability. In addition, considering that the
presence of the long alkyl chain (C15) might limit drug-likeness due
to excessive lipophilicity (see predicted physico-chemical
properties in Table 1 and S1) and might give rise to surfactant
properties and non-specific activities, we also turned our attention
to the shorter-chain (C8) CNSL derivative 7. Following this design
strategy, the small combinatorial library of 8-22 was generated
(Fig. 2 and Scheme 1).
2-Br-1,4-naphthoquinone 23 was commercially available,
whereas methoxy- and hydroxy-naphthoquinones 24–27 were
prepared as previously reported [19]
.
On the other hand, the CNSL-based phenols 5-7 have been
synthesized starting from the cashew nut shells. Particularly, to
synthesize phenol 5, an extraction of 150 g of cashew nut shells
with ethanol yielded 60 g of natural CNSL (40 wt% based on the
nut shells). The acids were then separated from other phenolic
components by precipitation of their calcium salts using calcium
hydroxide followed by acidification, which led to 21 g (70 wt%
based on 30 g of the alcoholic extract) of the acid mixture 1. Next,
hydrogenation of 1 using palladium on carbon as catalyst allowed
affording 28 in 70% yield (Scheme 2).
OH
O
OH
O
OH O
(i)
(ii)
OH
OH
C15H31
OCH3
C15H31-2n
C15H31
1
28
5
Scheme 2. Synthesis of phenol 5. Reagents and conditions: (i) H2, Pd/C, EtOH,
rt, 70 %; (ii) H2SO4, CH3OH, reflux, 86 %.
OH
(i)
OH
C15H31
Figure 2. Design strategy to generate CNSL-based hybrids 8-22 (see Scheme
6
1 for structures).
OH
C15H31-2n
(ii)
2
Synthetic strategies for CNSL-based hybrids (8-22) and
CNSL-phenols (5-7). The synthesis of 8-22 was performed as
illustrated in Scheme 1. We exploited a previously developed one-
pot reaction to combine phenols 5-7 and 2-bromo-1,4-
naphtoquinones 23–27 [14a, 14c]. Phenols 5–7 were first treated with
K2CO3 at room temperature in DMF, and then the proper 2-bromo-
1,4-naphtoquinone (23–27) was added, to rapidly (2-3 h) afford
final compounds 8-22. Despite its utility in nucleophilic
substitution reaction, DMF is clearly not compatible with the
current drive towards more sustainable and environmental
friendly medicinal chemistry processes [17]. With this in mind, we
C8H16OH
7
Scheme 3. Synthesis of phenols 6-7. Reagents and conditions: (i) H2, Pd/C,
EtOH, rt, 90 %; (ii) (a) Ac2O, H3PO4, MW, 400 W, 73 %; (b) O3, (CH3)2CO,
−78 °C; (c) NaBH4, CH3OH, rt, 79 %.
Subsequently, 28 is converted to its corresponding methyl ester
5, by performing a classical esterification reaction (H2SO4 in
methanol). Towards the synthesis of CNSL-based phenols 6 and
7, our approach was based on distilled technical CNSL.
Commercially available technical CNSL is obtained by treating
hydrated shells at temperatures around 180-210oC, and contains
mainly the mixture of cardanols (2) – by decarboxylation of the
anacardic acids (1) – and cardols (3). Distillation of the technical
CNSL led to a heterogeneous mixture of cardanols 2, as the
primary component of CNSL. Hydrogenation of 2 using palladium
on carbon as catalyst led to the saturated cardanol 6 in 90% yield
(Scheme 3). Conversely, compound 7 has been synthesized
using a three-steps protocol: (a) protection of the phenol group by
acetylation of 2, (b) oxidative cleavage by ozonolysis, (c)
reduction of the resulting secondary ozonide to the corresponding
alcohol with sodium borohydride (Scheme 3).
substituted DMF, which has been categorized as a hazardous
solvent, with the safer DMSO.
were obtained (see Experimental Section).
[18]
Encouragingly, similar yield
R1
R1
R3
O
O
O
OH
R4
R3
R4
Br
O
(i)
+
R2
R2
O
5-7
23-27
8-22
5: R3=H; R4=C15H31
6: R3=CO2Me; R4=C15H31
7: R3=H; R4 =C8H16OH
23: R1=H; R2=H
8: R1=H; R2=H; R3=CO2Me; R4=C15H31
9: R1=OMe; R2=H; R3=CO2Me; R4=C15H31
10: R1=H; R2=OMe; R3=CO2Me; R4=C15H31
11: R1=OH; R2=H; R3=CO2Me; R4=C15H31
12: R1=H; R2=OH; R3=CO2Me; R4=C15H31
13: R1=H; R2=H; R3=H; R4=C15H31
24: R1=OMe; R2=H
25: R1=H; R2=OMe
26: R1=OH; R2=H
27: R1=H; R2=OH
14: R1=OMe; R2=H; R3=H; R4=C15H31
15: R1=H; R2=OMe; R3=H; R4=C15H31
16: R1=OH; R2=H; R3=H; R4=C15H31
17: R1=H; R2=OH; R3=H; R4=C15H31
18: R1=H; R2=H; R3=H; R4=C8H16OH
19: R1=OMe; R2=H; R3=H; R4=C8H16OH
20: R1=H; R2=OMe; R3=H; R4=C8H16OH
21: R1=OH; R2=H; R3=H; R4=C8H16OH
22: R1=H; R2=OH; R3=H; R4=C8H16OH
In vitro activity against T. b. brucei wild type and resistant
strains and T. congolense wild type strain. The parent
compound 4 and the CNSL-based hybrid derivatives 8-22 were
tested for effects on cell viability against bloodstream
trypomastigotes of the standard drug-sensitive T. b. brucei strain
427WT. The most active ones were further tested against the
3
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multi-drug resistant strain B48 and the drug-transporter deletion
mutant aqp2/aqp3-KO (Table 1). The current first-line drug PMD
was used as the reference compound. It should be noted that the
EC50 values here reported were produced using a different
species (T. b. brucei vs T. b. rhodesiense) and a different
the lipophilicity of the test compounds increasing cell viability. In
fact, significant differences can be appreciated among the longer-
and the shorter-chain subsets: the CNSL-based derivatives 8-12
and 13-17 showed no anti-trypanosomal activity against T. b.
brucei up to 200 µM.
resazurin-based protocol than that used in the previous report [14a]
,
in which the EC50 of 4 against T. b. rhodesiense was found to be
80 nM. The very robust protocol used here utilizes a much higher
cell density and consequently results in substantially higher EC50
values [20]. Using this protocol, only compounds 18-22, which
carry the shorter (C8) aliphatic chain, showed anti-trypanosomal
activity in the micromolar range (5.0 to 40.5 μM). Importantly,
under these conditions, 18-22 had higher activity than 4, which
displays an EC50 value of 48.7 µM. Crucially, compounds 18-22
showed no sign of human cytotoxicity: none of the compounds
displayed any effects on either the viability or growth of the human
foreskin fibroblast (HFF) cell line at the highest tested
concentration (200 µM). Notably, 18-22 displayed statistically
identical activity (P>0.05) against the aqp2/aqp3-KO cell line from
which the well-characterized drug transporter HAPT1/TbAQP2
has been deleted [21], resulting in a moderated level of PMD
resistance (Table 1). Indeed, there was no cross-resistance
detected even in the very highly multi-drug resistant cell line B48,
although it displayed 178-fold resistance to PMD in this series of
experiments (Table 1). Interestingly, compounds 18-22 were up
to 12-times more active against all strains than the starting
compound 4, suggesting an improvement of the activity in at least
some of the newly synthetized CNSL-based hybrids. This shows
that the shorter alkyl chain with a primary alcoholic end function,
together with the regioisomeric substitution on the
naphthoquinone moiety, enhances the anti-trypanosomal activity
of the test compounds (see below).
Anti-trypanosomal profile and time-to-kill determination. In
order to determine whether 18-22 inhibited growth or cell division
rather than causing cell death of T. b. brucei, cell growth curves
were performed by treating 427WT trypomastigotes with
concentrations corresponding to 0.5×, 1× and 2× the EC50 value,
using untreated cells as a control. Incubation with test compounds
at half the EC50 concentration (Fig. 3A) caused mostly some
delayed growth phenotype, with rates increasing after 10 or 20 h.
At 1× their respective EC50 concentration (Fig. 3B), the hybrid
compounds induced a consistent, rapid-onset reduction in cell
growth rate over 48 hours. Compounds 18 and 19 showed
trypanocidal activity at this concentration, sterilizing the culture in
2 and 8 h, respectively. Compound 22 appeared similarly to
rapidly reduce the cell density but, at this concentration, killed only
a proportion of the cell population before stabilizing; 20 and 21
reduced the growth rate substantially, with a nearly trypanostatic
effect over the first 12 h (Fig. 3B). At double the concentration (Fig.
3C), all the hybrids cleared the culture between the 4 h and 8 h
time points, with the cell populations rapidly declining between 2
and 4 h. The rapid time to kill displayed by 18-22 (and the lack of
cross-resistance with existing chemotherapy) is
a
clear
advantage towards any (pre)-clinical development of this scaffold,
and encourages further hit optimization efforts.
Activity of the compounds against T. brucei GAPDH. Based
on the reported activity of starting compound 4, which displays an
IC50 of 7.25 μM against GAPDH [14b], we first tested inhibition of
this enzyme by 8-22, at a fixed concentration of 10 μM, following
a previously reported protocol [14c]. However, none of the
compounds displayed significant inhibition of GAPDH activity
(<15% reduction observed, data not shown). One explanation for
this lack of inhibitory activity might be that the current series of
hybrids is partially or completely prevented from binding the active
site of the trypanosomal glycolytic enzyme because of steric
hindrance by the long alkyl chain. Limitations of solubility
prevented us from testing the series at higher concentrations.
However, it must be noted that we cannot exclude the possibility
The two most active compounds 20 and 22, and the parent
compound 4, were also tested against T. congolense IL3000 cell
line and compared with the standard drug against animal
trypanosomiasis, diminazene aceturate (DA in Fig. S1). All three
compounds were less active than DA, with EC50 values in the mid-
to-high micromolar range, as well as several-fold less active than
against T. brucei. Despite the small number of compounds tested,
the results with T. congolense suggest that this species may be
systematically less sensitive to this scaffold than T. brucei is.
Similarly, T. congolense is less sensitive than T. brucei to
suramin[6] and PMD[22], reflecting species differences in drug
target or accumulation.
of
trypanocidal effect, as compounds may accumulate to relatively
Structure-Activity Relationship (SAR) of trypanocidal activity. high concentrations within the parasite. Indeed, examples where
a low-affinity inhibition of GAPDH contributing to the
Our goal was to design and synthetize a small library of CNSL-
based hybrids, aiming for a synergistic inhibition of energy
metabolism in T. b. brucei, targeting mitochondrial functions and
GAPDH inhibition. Along the line of this rationale, we designed
the library of CNSL-based hybrids starting from quinone 4 and
longer (5 and 6) and shorter (7) CNSL derivatives. Intriguingly,
only C8-compounds 18-22 showed a promising anti-trypanosomal
a high level of accumulation include almost all the first-line
trypanocidal agents (Fig. S1), including DA[23] PMD[24]
melarsoprol[25], and suramin[26] and highly active experimental
,
,
therapies, including
a
recently described series of
bisphosphonium compounds [27] that strongly accumulate in the T.
b. brucei mitochondrion.
activity, whereas 8-17 (C15) were not effective up to
a
concentration of 200 µM. Notably, a significantly higher efficacy
was detected for 18-22 in comparison with 4, suggesting that, at
least for this subset, the proposed hybridization strategy was
successful. As expected by the predicted logP values (Table 1),
the nature of the chain dramatically affected activity, probably by
modulating cell partitioning. This might be due to the insertion of
the alkyl chain on the phenoxy ring that might positively modulate
4
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Table 1. EC50 (μM) against trypomastigotes of T. b. brucei (T. b. b.) and T. congolense (T. c.), human HFF cells, selectivity index and predicted logP values
for 8-22.
T. b. b.
427-WT
T. b. b.
B48
T. b. b.
aqp2/aq3-KO
T. c. IL3000
Cmp
SIa
RFb
RFb
HFFc
logPd
WT
8
9
>200
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
10.88
>200
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nef
ne
ne
ne
ne
ndi
10.85
10.85
11.07
11.07
10.75
11.0
10
11
12
13
14
15
16
17
18
19
20
21
22
4
>200
nd
nd
>200
nd
nd
>200
nd
nd
>200
nd
nd
>200
nd
nd
>200
nd
nd
11.0
>200
nd
nd
11.22
11.22
5.64
>200
nd
nd
9.1 ± 1.4
15.0 ± 1.3
5.0 ± 0.3
40.5 ± 4.9
7.6 ± 0.7
48.7 ± 0.8
>21,98
>13.3
>40
8.6 ± 2.4
14.6 ± 1.7
5.3 ± 0.8
36.9 ±5.3
6.8 ± 0.1
63.0 ±0.4
0.95
10.1 ± 2.2
17.7 ± 1.5
6.2 ± 0.58
46.7 ± 7.7
7.3 ± 0.5
62.4 ± 0.8
1.1
nd
0.98
1.1
1.2
1.2
1.2
0.96
1.3
nd
5.62
18.8 ± 0.2
nd
5.62
>5
0.91
0.91
1.3
5.84
>26.3
nde
43.4 ± 0.2
108 ± 2
5.84
nd
0.0093
0.0001
±
PMDg
DAh
3.1 ± 0.7
nd
339
0.20 ± 0.01
nd
22.0
nd
nd
nd
nd
nd
0.17 ± 0.0003 nd
None of the EC50 values for the test compounds against the T. b. brucei (T. b. b.) 427-WT strain was significantly different in the B48 or aqp2/aqp3-KO strains.
In contrast, the EC50 for pentamidine was highly significantly different in the latter two strains compared with 427-WT (P<0.001; Student’s unpaired t-test; n=4
for all data points).
a Selectivity index (SI) = EC50(HFF)/EC50 (T. b. brucei WT)
b Resistance factor relative to WT
c Cytotoxic activity (EC50) on human foreskin fibroblast (HFF) cells; cytotoxic activity was observed up to 200 μM
d logP values were predicted with FAF-Drugs4 software (http://fafdrugs3.mti.univ-paris-diderot.fr)
e nd, not determined
f ne, no effect at 200 µM
g Pentamidine
h Diminazene aceturate
i The EC50 value of 4 is 9.84 ± 3.25 against human dermal fibroblasts (HDF) [14c]
.
5
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10.1002/cmdc.201800790
ChemMedChem
FULL PAPER
ATP and MMP determination as relevant evidence for a
mitochondrial mode of action. T. b. brucei 427WT BSF were
incubated with 20 and 22, at approximately 0.5×EC50
concentration, and the ATP content was determined at different
time points (Fig. 4). The FoF1-ATPase inhibitor oligomycin (see
Fig. S2 for the chemical structure) was used as positive control
and untreated cells as negative control. We found that both
compounds rapidly decrease the ATP content, with even 30 min
inducing a highly significant reduction in cellular ATP levels
(P<0.01), stabilizing at approximately 50% of untreated [ATP]
after 4-6 h (P<0.001). The response to 20 and 22 was similar not
just to each other, consistent with an identical mode-of-action, but
also highly similar to that of oligomycin, although the latter
depressed the ATP levels even further at the respective
concentrations used (Fig. 4). It should be noted that the observed
reduction in ATP does not correlate with cell death, which even at
2×EC50 does not occur in large number until after 4 h. The
observed reduction in ATP levels could be the result of
mitochondrial functions and therefore we next investigated the
timing of the same concentrations of 20 and 22 on the
mitochondrial membrane potential Ψm, using flow cytometry with
the fluorescent probe TMRE. Valinomycin (Fig. S2) was used as
the control for depolarization and troglitazone (Fig. S2), as the
1 0 0 0
1 0 0
1 0
A
B
C
1 8
1 9
2 0
2 1
2 2
U n tr e a te d
1
0
0
0
2
2
2
4
4
4
6
6
6
8
1 0 2 0 3 0 4 0 5 0
T im
e / h
1 0 0 0
1 0 0
1 0
1 8
1 9
2 0
2 1
1
2 2
U n tr e a te d
0 . 1
8
1 0 2 0 3 0 4 0 5 0
T im
e / h
control for hyperpolarization (Fig. 5) [31]
.
1 0 0 0
1 0 0
1 0
Both test compounds reduced Ψm slightly over the first hour
(P>0.05), an effect that was stronger at the 4 h (P<0.05 for 20)
and subsequent time points (P<0.01 for both compounds). The
Ψm is expressed as the percentage of cells displaying a
fluorescence of ≥500 arbitrary units (calibrated at 50% for
untreated cells at t=0), with a shift towards lower fluorescence
indicating a mitochondrial membrane depolarization (Fig. S3 in
the SI). It is clear from the time-dependency and magnitude of the
effects that cellular ATP depletion preceded the partial
depolarization of the mitochondrial inner membrane. We thus
conclude that the depolarization is likely to be the result of the
reduced ATP availability for the FoF1-ATPase, which maintains
the membrane potential of the mitochondrion of bloodstream T.
brucei, utilizing the ATP to pump protons out of the mitochondrial
matrix [32]. The primary cause of the ATP depletion may be the
inhibition of an essential mitochondrial function, and/or a step in
the glycolysis.
1 8
1 9
2 0
2 1
1
2 2
U n tr e a te d
0 . 1
8
1 0 2 0 3 0 4 0 5 0
T im
e / h
Figure 3. Cell counts of T. b. brucei cultures (bloodstream trypomastigotes),
seeded at 1×105 cells/ml, incubated in the presence of either 0.5× (Frame A) 1×
(Frame B) or 2× (Frame C) the EC50 concentration of the test compounds.
Where the lines stop, no live cells were detected at the next time point. Error
bars are SEM; where not shown fall inside the symbol.
Mode of action studies. Since the drug design of our series of
hybrids is based on the naphthoquinone framework that is
recognized as a privileged structure for the modulation of the
[28]
1 5
1 0
5
mitochondrial functions
and 4 itself acted at mitochondrial
targets through production of ROS [14b], we had reason to further
investigate their mode of action at the mitochondrial level in T. b.
brucei. Furthermore, the chemical structures of the newly CNSL-
based hybrids resemble that of ubiquinone (Fig. S2), which is an
essential carrier in the electron transport chain via a redox
reaction inner the mitochondrial membrane. Accordingly, we
initially aimed for a synergistic inhibition of the energy metabolism
for our hybrids, targeting the mitochondrial membrane enzymes
involved in electron transport (e.g. Trypanosome Alternative
Oxidase (TAO), FoF1-H+ ATPase), in addition to glycolysis
(GAPDH, located in the glycosome).[29] This because inhibition of
both of the essential arms of trypanosomal energy metabolism
would be expected to deliver synergistic effects. Despite the fact
that we were not able to demonstrate a GAPDH inhibitory activity
of our hybrids, we looked at the effects of the two most active
compounds 20 and 22 on ATP content and on the mitochondrial
membrane potential (MMP), as relevant parameters for an action
0
0
2
4
6
8
1 0
1 2
T im
e / h
Fig. 4. ATP content of bloodstream trypomastigotes of T. b. brucei 427-WT after
incubation with oligomycin (2.0 µg/ml; ■), 20 (2.2 µM; ▼), 22 (3.5 µM; ▲) or no
drug (control; ●). The data are average and SEM of three determinations.
on the trypanosomal energy metabolism [30]
.
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800790
ChemMedChem
FULL PAPER
The results show that under normal culture conditions
trypanosomes generate a steady amount of ROS, which is greatly
increased in the presence of H2O2. The level of ROS was dose-
dependently increased over an incubation period of up to 2 hours
with 20 and 22 (Fig. 6A, B, respectively). As suggested previously
for 4 [14b], 20 and 22 could be substrates of the electron transport
chain, leading to cycle of reduction by glycerol-3-phosphate
dehydrogenase (G3PD) followed by reaction with molecular
oxygen and the production of ROS. In normal condition G3PD
works like a shuttle between glycosomes and mitochondria,
keeping the redox balance and feeding the respiratory chain for
the ATP production [35], with TAO acting as electron acceptor,
oxidizing the ubiquinol pool formed in consequence of G3PD
activity [36]. Accordingly, the observed overproduction of ROS is
consistent with an ubiquinol-like reactivity for our hybrids [37]. This
would cause both molecular and structural damage to T. b. brucei
mitochondria (see section on Transmission Electron Microscopy,
1 0 0
8 0
6 0
4 0
2 0
0
below) [38]
.
0
2
4
6
8
1 0
1 2
8 0 0 0 0
6 0 0 0 0
4 0 0 0 0
2 0 0 0 0
0
T im e
/ h
A
a
b
Fig. 5. Mitochondrial membrane potential Ψm in bloodstream trypomastigotes
of T. b. brucei 427-WT was determined at various time points after incubation
with various test compounds: troglitazone (10 µM; ■), valinomycin (0.1 µM; ▲),
20 (2.4 µM; ▼), 22 (3.5 µM; □) or untreated (control; ●). Ψm was expressed as
the percentage of cells with 500 or more artificial units of fluorescence,
calibrated at 50% for the control group at time = 0 h. Data are the average and
SEM of three independent experiments. Valinomycin and troglitazone are used
as controls for depolarization and hyperpolarization, respectively.
c
d
e
f
0
3 0
6 0
T im e / m in
9 0
1 2 0
However, we re-tested compounds 18, 19, 20 and 22 against
427WT cells, in the presence and absence of 5 mM glycerol in the
medium, an addition that sensitizes the cells to TAO inhibitors
such as salicylhydroxamic acid (SHAM) and ascofuranone (Fig.
S2).[33] Indeed, we found that the cells were significantly
sensitized to SHAM in the presence of glycerol (P<0.001), but not
to the hybrid compounds (Table 2). Interestingly, compound 20
displayed significantly (P<0.001) less activity in the presence of
glycerol, whereas the EC50 values for the other test compounds
was unchanged. We conclude that 18, 19, 20 and 22 do not act
via direct inhibition of TAO.
8 0 0 0 0
6 0 0 0 0
4 0 0 0 0
2 0 0 0 0
0
B
a
b
c
d
e
f
Table 2. Investigation of the role of TAO in the trypanocidal activity of test
0
3 0
6 0
9 0
1 2 0
compoundsa.
T im e / m in
Control
control s427WT + 5 mM glycerol
s427WT
Fig. 6. Effect of 20 (A) and 22 (B) on ROS production in bloodstream forms of
T. b. brucei s427WT. Production of ROS was measured using the fluorescent
indicator dye DCFH-DH. (a) Cells incubated with 100 μM H2O2; CNSL-based
hybrids were at: 2×EC50 (b); 1.25×EC50 (c); 0.32×EC50 (d). Parasites incubated
without test compound (e); plain assay buffer without cells (f).
Compound
EC50 (µM)
30.0 ± 0.08
16.8 ± 0.06
9.2 ± 0.04
25.4 ± 1.0
86.3 ± 0.4
EC50 (µM)
31.2 ± 0.6
16.3 ± 0.4
16.0 ± 0.2
28.2 ± 0.2
58.9 ± 0.6
R.F.
1.04
0.97
1.74
1.11
0.68
t-test
ns
18
19
ns
20
P<0.001
ns
22
SHAM
P<0.001
a All values are the average and SEM of three independent determinations; ns,
not significant; R.F., resistance factor; statistical significance was determined
using Student’s unpaired t-test.
CNSL-based hybrids cause damages of the mitochondria but
not the kinetoplast. Transmission electron microscopy (TEM)
was used to study the trypanosomes’ ultrastructure after
exposure to compound 22 at 1×EC50 (7.6 μM) in order to visualize
any damage to the mitochondrion. Based on the information
obtained from the ATP and Ψm determinations, TEM samples
were taken after 4 and 8 hours exposure to test compound, as
‘early’ and ‘late’ time points to define the effects on cellular
ultrastructure (Fig. 7). After 4 h of incubation with 20, some
mitochondria presented an irregular shape. Moreover,
membranous, electron-light structures had appeared in many
CNSL-based hybrids increase the production of reactive
oxygen species (ROS) in T. b. brucei. In order to test whether
our hybrids display a redox activity in T. b. brucei 427WT BSF
(similarly to what has been demonstrated for 4 [14b]), we assessed
the production of ROS in cells treated with 20 and 22 and in
control cells, using the ROS-sensitive fluorescent dye 2’,7’-
dichlorodihydrofluorescein diacetate (DCFH-DH) (Fig. 6) [34]
.
7
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10.1002/cmdc.201800790
ChemMedChem
FULL PAPER
mitochondria, resembling vacuoles (Fig. 7; 2nd row of images); no
other ultrastructural changes were evident, appearing to confirm
the mitochondrion as a main target for CNSL-based hybrids in
trypanosomes. Indeed, 20 caused further ultrastructure
abnormalities in the mitochondrial structure at 8 h, displaying
damage to the mitochondrial membrane, and the presence of
membranous and dense vesicles within the matrix (Fig. 7, third
row). However, the treated cells do not display any morphological
alteration to kinetoplasts, which completely maintain the original
disk-like structure [39]. Accordingly, this evidence stands for an
exclusive metabolic effect at mitochondrial level for the test
compounds, which do not interfere with both functioning and
replication of kinetoplasts.
kDNA as a potential target for our test hybrids, with the available
data all consistent with a mitochondrial target.
7 5
6 0
4 5
n s
2 2
2 0
4
I S M
n s
n s
1 0
8
6
* * *
4
2
0
1
1
1
1
T
S
T
S
T
S
T
A
R
R
R
R
W
W
W
W
M
M
M
M
7
7
7
7
2
2
2
2
S
I
I
I
I
4
s
4
4
4
s
s
s
Fig. 8. The bar chart compares the EC50 values for 20, 22, 4 and the control
drug isometamidium (ISM) obtained performing the in vitro activity against
bloodstream trypomastigotes of T. b. brucei s427WT and T. b. brucei ISMR1.
ns, not significant; ***, P<0.001 (Student t-test, unpaired).
Although we had observed no toxicity of these naphthoquinone
hybrids towards human cells, we next assessed whether they
might damage mitochondria in human cells. We therefore
determined the ATP content in treated and untreated HFF cell line
with a deliberately high concentration (200 μM) of 22. The
compound had only a very minor effect on cellular ATP levels,
even at this concentration, being approximately 26x trypanocidal
EC50, although the effect became significant at 8 h of incubation
(P<0.05). Oligomycin exhibited a strong effect even at 4 h
(P<0.0.1) (Fig. 9).
B
U n tr e a te d
1 0 0
C
2 2 ( 2 0 0  M )
*
O
lig o m y c in
* *
( 2  g / m L )
5 0
* *
0
0
4
8
0
4
8
0
4
8
T im
e / h
Fig. 7. Ultrastructure TEM images of untreated (A) and treated (B, 4 h; C, 8 h)
s427WT BSF with 22 at 1×EC50. m (mitochondrion); N, nucleus; F, flagellum; k,
kinetoplast; arrow, membranous structure within mitochondrion matrix. The bars
indicate 1 μm length.
Fig. 9. The bar chart depict the ATP content determination at different time point
(0, 4, 8, hours) in untreated and treated HFF cell line with 22 (200 µM). The
detection was carried out using the fire fly luciferase kit and measuring the level
of luminescence to calculate the concentration of ATP (nM). Oligomycin (2
µg/mL) is used as positive control for the ATP decreasing.
To confirm whether the CNSL-based hybrids exclusively target
the mitochondrial energetic pathway, rather than the
mitochondrial kDNA, we decided to test compounds 4, 20 and 22
against the isometamidium-adapted ISMR1 cell line of T. b. brucei
in parallel with the parental cell line 427WT. As ISMR1 is a
dyskinetoplastic cell line, and thus highly resistant to the drug
isometamidium (ISM, Fig. S2) and other kinetoplast-targeting
drugs [32], a primary effect on energy metabolism rather than
mitochondrial kDNA should not result in resistance to the test
compounds. Indeed, compounds 4, 20 and 22 did not show a
significant increase in the EC50 values, compared to the standard
WT strain 427, tested in parallel (Fig. 8). These data exclude
We also used TEM after exposure to the same concentration of
22 (200 μM) at two different time points (4 and 12 h) in order to
analyze whether ultrastructural changes similar to those in T.
brucei could be observed. The TEM images reveal that no
morphological damage occurs to mitochondria in human cells
after treatment with even high concentrations of 22, nor were any
other ultrastructural changes in the exposed HFF cells visible (Fig.
10). These results clearly show the absence of metabolic and
structural toxic effects at the human mitochondrial level, indicating
a highly species-specific mode of action for our CNSL-based
hybrids.
8
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10.1002/cmdc.201800790
ChemMedChem
FULL PAPER
anti-infective agents for use against NTD. The required chemistry
is simple, accessible and easily scaled up, making the entire
production process highly affordable and local. Taken together,
the collected data suggest that, in the case of compounds 20 and
22, this CNSL hybridization strategy led to an increased
trypanocidal activity up to 10-fold relative to the parent compound
4. This did not a priori signify that the new compounds necessarily
act similarly as 4, as the fusion with the CNSL-based fragments
affects both the pharmacokinetic and pharmacodynamic features
of the synthesized hybrids. In addition to the contribution in target
recognition, the long alkyl chain clearly affects the physico-
chemical properties, which are determinant for uptake by both
target and host cells, delivery to mitochondria, and interaction with
molecular targets.
Fig. 10. Transmission electron microscopy images of Human Foreskin
Fibroblast (HFF) cells cultured for 12 h in the absence (A) or presence of 200
µM of 20. ER, endoplasmic reticulum; N, nucleus; M, mitochondrion.
Metabolomics analysis. Metabolomics has successfully been
used to elucidate the mode of action of drugs against T. brucei [40]
.
We present clear evidence for mitochondrial targeting in
trypanosomes, especially the TEM pictures are convincing, with
mitochondrial damage but no other ultrastructural changes
observed after just 4 h incubation with 1×EC50 of 22. It is quite
possible that this localized damage is linked to the observed
production of ROS. However, ATP depletion precedes
mitochondrial abnormalities and the depolarization of Ψm can be
observed within 30 min, and we propose that the hybrids, as
intended, exert more than one trypanocidal effect, consistent with
the complex return of the metabolomics experiment, (although it
was not possible to derive any clear conclusion from the
metabolomics data, at this point). All this does not exclude a target
inside the mitochondrion, but it is not (principally) TAO or glycerol-
3-phosphate dehydrogenase (G3PDH), which together enable
the re-oxidation of glycolysis-produced NAD+ via the cyanide-
resistant, non-protonmotive oxidation of ubiquinol by TAO.[41] This
is because the activity of the hybrids was not enhanced in the
presence of glycerol. The target linked to rapid ATP depletion
could also be in glycosomes but we saw no evidence of
glycosomal damage in the TEM images, nor consistent inhibition
of GAPDH. The combination of cellular damage by ROS, while
depleting the ATP required for damage repair, is likely to
contribute to the rapid cell death observed at just 1-2× EC50.
It is probably because the kinetoplast is not involved in the
trypanocidal action of these hybrid compounds that there is no
cross-resistance with diamidines (PMD and DA) and
phenanthridine (isometamidium, ethidium) drugs. As these are
the mainstay of human (PMD) and especially veterinary
trypanosomiasis treatment, this is an important ‘plus’ for this
compound series. Even more important, the compounds
displayed excellent in vitro selectivity over human cells; in addition
to the lack of effect on growth rate of human fibroblasts, no effects
on mitochondrial ultrastructure or ATP content, even at very high
concentrations of compound were observed. This is especially
encouraging in light of the severe toxicity of current HAT
We therefore employed a metabolomics approach to further
investigate the mode of action of 22. T. b. brucei 427WT BSF
were incubated with and without 0.5×EC50 of 22 for 2 h in order to
identify specific metabolic changes caused by moderate exposure
to the compound (early effects). Samples were instantly cooled
down to block the intracellular metabolic activity, and processed
to extract the pool of intracellular metabolites. Analysis by LC-MS
showed a large number of metabolite differences between the
treated and non-treated cells, but without a clear pattern
designating a particular enzyme function or pathway, or indeed a
particular class of metabolites being affected apart from some
sphingolipid precursors which accumulated in the exposed cells
(Table 3), possibly indicating some direct or indirect interference
in this pathway. These results are consistent with a multi-target
mode of action (as reported for 4)[14b] or the result of general
damage such as caused by ROS, affecting many cellular
functions at once.
Table 3.Sphingolipid precursors elevated in the trypanosomes treated with 22
versus untreated controlsa.
M/Z
RT
Metabolite
P value
Treated/Control
302.3046
5.0
Sphinganine
0.034
1.654
[SP
(16:0)]
N-
N-
N-
N-
(hexadecanoyl)-
sphing-4-enine
538.5181
540.5348
566.5504
3.9
5.1
3.8
0.013
0.051
0.009
2.450
14.230
3.028
[SP
(16:0)]
(hexadecanoyl)-
sphinganine
[SP
(18:0)]
(octadecanoyl)-
sphing-4-enine
[SP
(18:0)]
(octadecanoyl)-
sphing-4-enine
sphing-8(Z)-enine-
19:0, ceramide
chemotherapy (Fig. S1) [42]
.
566.5493
578.5528
5.6
3.9
0.046
0.018
4.689
6.708
In conclusion, while further rounds of optimization are required
before these molecules can be turned into valuable leads for
trypanosomiasis, we have successfully demonstrated that CNSL
hybrids show promise as a template for the development of anti-
infective drugs on a sustainable basis. Importantly, the utilization
of CNSL and its components for combating trypanosomiasis
could be of high importance and economically feasible in
developing countries that cultivate and process cashew and, at
the same time, represent endemic areas. Further studies will
focus on extending SAR, optimizing for T. congolense, and testing
on veterinary trypanosome species that are more closely related
a
RT, retention time (min). Statistical significance was determined using
Student’s unpaired two-tailed t-test.
Conclusions
In this report, we explore the utilization of a waste product of food
production, the CNSL, as a cheap and abundant source of new
9
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10.1002/cmdc.201800790
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FULL PAPER
2-Hydroxy-6-pentadecylbenzoate (29) To a solution of heterogeneous
mixture of anacardic acids 1 (5 g, 14.35 mmol for average molecular wt
344) in ethanol (50 mL), was added with 10% palladium on carbon (0.20
g, 2 mol %) and shaken in a Parr apparatus, under hydrogen atmosphere
(60 psi) during 6 h. After, the reaction mixture was filtered in a sintered
funnel and the filtrate was concentrated under reduced pressure. The
residue was purified by chromatography with silica gel (hexane and then
hexane: ethyl acetate 20%) as eluent, to afford the title compound 6 as a
white solid. Yield: 70%. mp 81-83 oC. IR (KBr) máx cm-1: 3326 (OH); 2954
(asCH3); 2920 (asCH2); 2850 (sCH2); 1610 (C=O); 1560, 1542, 1498 and
1466 (C=C); 1287(asC-O); 1086 (sC-O). 1H-NMR (300 MHz, CDCl3): δ 0.89
(t, J = 6.5 Hz, 3H); 1.26 (m, 24H); 1.57-1.62 (m, 2H); 3.00 (t, J = 7.7 Hz,
2H); 6.79 (d, J = 7.4 Hz, 1H); 6.89 (d, J = 8.01 Hz, 1H); 7.37 (t, J = 7.9 Hz,
1H). 13C-NMR (75 MHz, CDCl3): δ 14.3, 22.9, 29.6-30.0, 32.1, 32.2, 36.7,
110.6, 116.1, 123.0, 135.6, 148.1, 163.8, 176.5.
to T. brucei: T. evansi and T. equiperdum, which, being
dyskinetoplastic, are not treatable with DA or isometamidium, and
not geographically restricted to the tsetse belt of Africa [6]
.
Experimental Section
General chemical methods. All of the commercial available reagents and
solvents were used as purchased from Sigma-Aldrich and Tedia without
further purification. The cardanol mixture (2) was purchased from Resibras
(Fortaleza, Brazil). Reactions were followed by analytical thin layer
chromatography (TLC), performed on precoated TLC plates (layer 0.20
mm silica gel 60 with a fluorescent indicator UV254, from Merck and F254
Silicycle plates). Developed plates were air-dried and analyzed under a
UV lamp (UV 254/365 nm) or visualized by exposure to iodine stain. The
oxidative cleavages were performed using a Brasil Ozônio BrO3 ozonizer,
whereas the catalytic hydrogenations were performed using a Parr
Shaker Hydrogenation Apparatus 3916. Column chromatography
purifications were performed using Sigma-Aldrich silica gel grade 9385, 60
Å, 230-400 mesh and Silicycle silica gel type G60, 60 Å, 70-230 mesh.
Melting points were determined on a Quimis MQAPF 302 apparatus and
are uncorrected. Infrared (IR) spectra were obtained on a Perkin Elmer –
Spectrum BX infrared spectrophotometer. Nuclear magnetic resonance
(NMR) experiments were run on Varian VXR 400 (400 MHz for 1H; 100
MHz for 13C) and on Bruker Avance DRX500 and DRX300 (500 and 300
MHz for 1H; 125 and 75 MHz for 13C, respectively) instruments. 1H- and
13C-NMR spectra were acquired at 300 K using deuterated chloroform
(CDCl3) as solvent. Chemical shifts (δ) are reported in parts per million
(ppm) relative to the residual solvent peak as internal reference, and
coupling constants (J) are reported in hertz (Hz). The spin multiplicities are
reported as s = singlet, br s = broad singlet, d = doublet, dd = doublet of
doublet t = triplet, q = quartet, and m = multiplet. Mass spectra were
recorded on a Waters ZQ 4000 apparatus, and electrospray ionization
(ESI) in positive mode was applied. Compounds were named following
IUPAC rules as applied by ChemDraw Professional (version 15.0). All
tested compounds possess a purity of at least 95% as assessed by HPLC.
HPLC parameters were the following: MeOH (eluent A) and H2O with
0.05% trifluoroacetic acid (eluent B); flow rate 1.0 mL/min; elution type
isocratic with 50% of eluent A and 50% of eluent B; detection UV-Vis Abs
at 254 nm; Kinetex® 5μm EVO C18 100 Å, LC Column 150 x 4.6 mm (see
section Compound Purity and Table S2 in SI for details).
Methyl 2-hydroxy-6-pentadecylbenzoate (5) A mixture of 0.2 g of 28
(0.574 mmol) and 1.5 mL of H2SO4 in methanol was stirred and refluxed
for 18 hours. After, the reaction was washed with brine (10 mL), extracted
with dichloromethane (3 x 10 mL) and dried with anhydrous sodium sulfate.
The solvent was evaporated under reduced pressure and the product was
purified by chromatography on silica gel (hexane and then hexane: ethyl
acetate 20%) to afford the title compound 5 as a white solid. Yield: 86%.
IR (KBr) max cm-1: 3433 (OH), 2917 (as CH2), 2851 (S CH2), 1663 (C=O),
1451 (C=C), 1250, 1203 (as C-O). 1H-NMR (500 MHz, CDCl3): δ 0.89 (t, J
= 6.8 Hz, 3H), 1.27-1.32 (m, 29H), 1.51-1.55 (m, 2H), 2.89 (t, J = 7.8 Hz,
2H), 3.96 (s, 1H), 6.73 (d, J = 7.3 Hz, 1H), 6.84 (d, J = 8.1 Hz, 1H), 7.27-
7.31 (m, 1H), 11.06 (s, 1H). 13C-NMR (125 MHz, CDCl3): δ 14.2, 22.9,
29.5-30.1, 32.1, 32.3, 36.8, 52.3, 112.0, 115.8, 122.6, 134.3, 146.4, 162.8,
172.1.
3-Pentadecylphenol (6). To a solution of heterogeneous mixture of
cardanols 2 (monoene, diene, and triene (10 g, 33.05 mmol for average
molecular wt 300) in ethanol (40 mL), was added with 10% palladium on
carbon (0.25 g, 2 mol %) and shaken in a Parr apparatus, under hydrogen
atmosphere (60 psi) during 4 h. After, the reaction mixture was filtered in
a sintered funnel and the filtrate was concentrated under reduced pressure.
The residue was purified by chromatography with silica gel using a mixture
of hexane: dichloromethane (1:1) as eluent, to afford the title compound 6
as a white solid. Yield: 90%. mp 44-45 oC. 1H-NMR (500 MHz, CDCl3): 
0.92 (t, J = 6.8 Hz, 3H), 1.29-1.33 (m, 24H), 1.60-1.61 (m, 2H), 2.58 (t, J =
7.7 Hz, 2H), 6.67-6.69 (m, 2H), 6.79 (d, J = 7.4 Hz, 1H), 7.16 (t, J = 7.7 Hz,
1H). 13C-NMR (125 MHz, CDCl3):  14.3, 22.9, 29.6-29.9, 31.5, 32.2, 36.0,
112.8, 115.6, 121.2, 129.5, 145.2, 155.6.
Chemistry
Synthesis of phenols 5-7
3-(8-Hydroxyoctyl)phenol (7) An Erlenmeyer flask containing a solution
of 12 g of mixture of cardanols 2 (monoene, diene and triene) (39.4 mmol)
distilled acetic anhydride (12 mL) and phosphoric acid (12 drops) was
placed inside an unmodified household microwave oven and irradiated for
3 minutes (3 x 1') at a power of 400 W. After, the residue was extracted
with ethyl acetate (3 x 15 mL) and the combined organic fractions washed
with solution of 5% sodium bicarbonate (20 mL), 10% hydrochloric acid
solution (20.0 mL), brine (20 mL), and dried over anhydrous sodium sulfate.
After evaporation of the solvent at reduced pressure, the reaction mixture
was purified by chromatography on a silica gel (dichloromethane) affording
the desired compound in 73% yield. Then, 10.00 g of the mixture of
acetylated cardanols was diluted with dichloromethane (20 mL) and
methanol (20 mL) in a ozonolysis flask of 250 mL. The flask was adapted
to the ozonator with a stream of ozone for one 1.5 h, in bath of dry
ice/acetone. Next, the secondary ozonide was reduced with 5.9 g of
sodium borohydride (158.7 mmol) in 60 mL of methanol. At the end of
addition of sodium borohydride, the reaction remained for six hours under
stirring. Then, the mixture was acidified with concentrated hydrochloric
acid to pH = 3, and it was extracted with ethyl acetate (3 x 30 mL). The
combined organic fractions were washed with brine (30 mL) and dried over
sodium sulfate. After evaporation of the solvent, the product was purified
by chromatography on silica gel (dichloromethane/chloroform, 5: 5 and
then chloroform/ethanol, 9: 1) leading to the title compound 7 as colorless
oil. Yield: 79%. IR (KBr) max cm-1: 3351, 2929, 2855, 1589, 1456. IV (KBr)
Natural CNSL extraction. In a Soxhlet extraction system were added 150
g of cashew nutshell and absolute ethanol (400 mL). The system was
maintained under reflux for 4 hours. Then, the collecting flask was changed
and a new extraction was carried out under the same conditions. In both
cases, the solvent was evaporated under reduced pressure, providing 60
g of natural CNSL as a brown liquid in 40% yield relative to the nutshell
mass.
Mixture of anacardic acids (1) from natural CNSL In a flask were added
30 g of natural CNSL (~86.08 mmol), 15 g Ca(OH)2 (202.44 mmol) and
methanol (180 mL) and water (30 mL). The reaction system remained in
reflux and agitation for 3 hours. Then, the mixture was cooled to room
temperature and filtered. The solid obtained was washed with ethyl acetate
to remove the other components of the CNSL. The calcium salts formed
were treated with 50% hydrochloric acid solution to pH 1.0 to liberate the
mixture of anacardic acids, which were extracted with ethyl acetate (3 x 50
mL). The combined organic phases were washed with brine and dried over
anhydrous sodium sulfate. After removal of the solvent under reduced
pressure, the mixture was purified on a silica gel chromatography column,
eluted in a mixture of hexane and 20% ethyl acetate, affording the mixture
of anacardic acids (1) as a brown oil in 70% yield.
10
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800790
ChemMedChem
FULL PAPER
silica gel with a mixture of petroleum ether/ethyl acetate/toluene (7: 2: 1),
as eluent. Compound 11 was obtained as an orange-yellow waxy solid.
Yield: 65%. 1H-NMR (400 MHz; CDCl3): δ 0.84-0.87 (m, 2H), 1.23-1.28 (m,
27H), 2.66 (t, J = 8 Hz, 2H), 3.79 (s, 3H), 5.95 (s, 1H), 6.95 (d, J = 8 Hz,
1H), δ 7.19 (d, J = 8 Hz,1H), 7.24-7.27 (m, 1H), 7.40 (t, J = 8 Hz, 1H), 7.58-
7.65 (m, 2H), 11.76 (s, 1H). 13C-NMR (100 MHz; CDCl3): δ 14.09, 22.66,
29.33, 29.45, 29.50, 29.63, 29.64, 29.66,31.24, 31.90, 33.65, 52.35,
114.26, 114.78, 118.96, 124.05, 126.60, 127.90, 131.17, 131.98, 137.14,
144.00, 149.83, 159.80, 161.93, 166.42, 183.97, 184.29. MS (ESI+) m/z
C33H42O6: 557 [M+ + Na+], 573 [M+ + K+].

máx cm-1: 3351 ( OH); 2929 (as CH2); 2855 (s CH2); 1589, 1456 (C=C). 1H-
NMR (300 MHz, CDCl3):  1.30 (s, 8H), 1.53-1.59 (m, 4H), 2.54 (t, J = 7.6
Hz, 2H), 3.66 (t, J = 6.6 Hz, 2H), 6.65 (dd, J = 8.1 Hz, J = 2.5 Hz, 1H), 6.67
(s, 1H), 6,71 (d, J = 7.6 Hz, 1H), 7.19 (d, J = 7.8 Hz, 1H). 13C-NMR (75
MHz, CDCl3):  25.8, 29.2, 29.4, 29.5, 31.3, 32.7, 35.9, 63.2, 112.8, 115.6,
120.8, 129.5, 144.9, 156.0.
General procedure for the synthesis of CNSL-based hybrids (8-22).
To a solution of the proper phenol derivative (5-7) (0.1 mmol and 0.3 mmol
for 7) in dry DMF (0.04 M), K2CO3 (0.1 mmol and 0.3 mmol for 7) was
added. The resulting mixture was stirred for half an hour at room
temperature. For phenol 7, the reaction was conducted at 0 °C to avoid the
formation of side products. Subsequently, the suitable 2-bromo-1,4-
naphtoquinone (23-27) was added (0.1-0.3 mmol) to the reaction mixtures,
which were stirred at room temperature for further 2-3 hours. Then, LiCl
5% solution was added (10 mL), whereas the reaction mixtures involving
26 and 27, were previously acidified with HCl 2N solution (pH ≅ 5), until
the chemical toning changes from blue-green to orange. Next, the obtained
mixtures were extracted with EtOAc (10 mL x 3). The organic extracts were
collected, dried over Na2SO4 and the solvent was evaporated under
vacuum. The crude residue was purified by chromatography on silica gel
or/and purified by crystallization, when required.
Methyl 2-((5-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)oxy)-6-
pentadecylbenzoate (12). The title compound was obtained according to
the general procedure using 5 and 27, and purified by chromatography on
silica gel with a mixture of petroleum ether/ethyl acetate (8.5: 1.5) as
eluent. Compound 12 was obtained as an orange-yellow solid. Yield: 30%.
1H-NMR (400 MHz; CDCl3): δ 0.84-0.88 (m, 2H), 1.24-1.28 (m, 28H), 2.67
(t, J = 8 Hz, 2H), 3.78 (s, 3H), 5.89 (s, 1H), 6.95 (d, J = 8 Hz, 1H), 7.18-
7.28 (m complex, 2H), 7.41 (t, J = 8 Hz, 1H), 7.59 (t, J = 8 Hz, 1H), 7.70
(d, J = 8 Hz, 1H) 12.08 (s, 1H). 13C-NMR (100 MHz; CDCl3): δ 14.08,
22.66, 29.34, 29.45, 29.50, 29.63, 29.66, 31.24, 31.89, 33.64, 52.36,
113.77, 114.22, 118.96, 119.59, 125.24, 126.59, 127.96, 131.01, 131.178,
135.64, 144.04, 149.84, 160.75, 161.10, 166.37, 167.537,178.70,190.80.
MS (ESI+) m/z C33H42O6: 557 [M+ + Na+], 573 [M+ + K+].
Methyl
2-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)oxy)-6-
pentadecylbenzoate (8) The title compound was obtained according to
the general procedure using 5 and 23, and purified by chromatography on
silica gel with a mixture of petroleum ether/ethyl acetate (9:1), as eluent.
Compound 8 was obtained as a yellow waxy solid. Yield: 61%. 1H-NMR
(400 MHz; CDCl3): δ 0.85 (t, J = 6.8 Hz, 2H), 1.23-1.28 (m, 27H), 2.66 (t,
J = 8.4 Hz, 2H), 3.77 (s, 3H), 5.96 (s, 1H), 6.95 (d, J = 8.4 Hz, 1H), 7.18
(d, J =7.6 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.72-7.75 (m, 2H), 8.04-8.06
(m, 1H), 8.15-8.16 (m, 1H). 13C-NMR (100 MHz; CDCl3): δ 14.08, 22.65,
29.32, 29.34, 29.45, 29.50, 29.62, 29.66, 31.24, 31.89, 33.62, 52.30,
114.18, 119.04, 126.20, 126.67, 126.71, 127.76, 131.03, 131.10, 131.96,
133.48, 134.34, 143.91, 149.96, 160.16, 166.47, 179.40, 184.88. MS
(ESI+) m/z C33H42O5: 541 [M+ +Na+]; 557 [M+ +K+].
2-(3-Pentadecylphenoxy)naphthalene-1,4-dione (13). The title
compound was obtained according to the general procedure using 6 and
23, and purified by crystallization from EtOH/H2O. Compound 13 was
obtained as a yellow waxy solid. Yield: 21%. 1H-NMR (400 MHz; CDCl3):
δ 0.89 (t, J = 8 Hz, 2H), 1.27-1.34 (m, 27H), 2.65 (t, J = 4 Hz, 2H), 5.98 (s,
1H), 6.95-6.97 (m, 2H), 7.14 (d, J = 8 Hz, 1H), 7.36 (t, J =4 Hz, 1H), 7.76-
7.80 (m complex, 2H), 8.07-8.09 (m, 1H), 8.22-8.24 (m, 1H). 13C-NMR
(100 MHz; CDCl3): δ 14.15, 14.16, 22.27, 29.24, 29.39, 29.47, 29.58,
29.68, 29.71, 29.72, 31.19, 31.95, 35.72, 113.27, 118.13, 120.88, 126.23,
126.75, 130.05, 131.11, 131.99, 133.52, 134.45, 145.96, 152.60, 160.64,
180.07, 185.11. MS (ESI+) m/z C31H40O3: 483 [M+ + Na+].
Methyl
2-((8-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)oxy)-6
8-Methoxy-2-(3-pentadecylphenoxy)naphthalene-1,4-dione (14). The
title compound was obtained according to the general procedure using 6
and 24, and purified by chromatography on silica gel with a mixture of
petroleum ether/ethyl acetate (6: 4), as eluent. Compound 14 was
obtained as a yellow waxy solid. Yield: 65%. 1H-NMR (400 MHz; CDCl3):
δ 0.84-0.87 (t, J = 8 Hz, 2H), δ 1.23-1.27 (m, 27H), δ 2.60 (t, J = 7.6 Hz,
2H), δ 4.02 (s, 3H), δ 5.86 (s, 1H), δ 6.88-6.90 (m, 2H), δ 7.08 (d, J = 8
Hz, 1H), δ 7.26-7.32 (m, 2H), δ 7.64-7.71 (m, 2H). 13C-NMR (100 MHz;
CDCl3): δ 14.08, 22.65, 29.19, 29.32, 29.41, 29.53, 29.63, 29.66, 31.13,
31.89, 35.68, 56.51, 111.38, 117.51, 118.13, 118.84, 118.96, 120.89,
126.48, 129.89, 134.31, 34.43, 145.77, 152.89, 160.21, 161.22, 178.33,
184.98. MS (ESI+) m/z C32H42O4: 513 [M+ +Na+], 529 [M+ +K+].
pentadecylbenzoate (9) The title compound was obtained according to
the general procedure using 5 and 24, and purified by chromatography on
silica gel with a mixture of ethyl acetate/petroleum ether/toluene (6: 3: 1),
as eluent. Compound 9 was obtained as a yellow waxy solid. Yield: 60%.
1H-NMR (400 MHz; CDCl3): 0.85 (t, J = 7.2 Hz, 2H), 1.23-1.27 (m, 27H),
2.64 (t, J = 8 Hz, 2H), δ 3.76 (s, 3H), 4.00 (s, 3H), 5.87 (s, 1H), 6.92 (d, J
= 8 Hz, 1H), 7.15 (d, J = 8 Hz, 1H), 7.24-7.28 (m, 1H), 7.37 (t, J = 8.4 Hz,
1H) 7.61-7.70 (m, 2H). 13C-NMR (100 MHz; CDCl3): δ 14.08, 22.65, 29.32,
29.35, 29.44, 29.50, 29.63, 29.66, 31.24, 31.89, 33.57, 52.32, 54.50,
112.19, 117.51, 118.83, 118.87, 119.06, 126.76,127.54, 130.97, 134.28,
135.43, 143.71, 150.13, 160.18, 160.69, 166.52, 177.77, 184.85. MS
(ESI+) m/z C34H44O6: 571 [M+ + Na+], 587 [M+ + K+].
5-Methoxy-2-(3-pentadecylphenoxy)naphthalene-1,4-dione (15). The
title compound was obtained according to the general procedure using 6
and 25, and purified by chromatography on silica gel with a mixture of
petroleum ether/ethyl acetate (6: 4), as eluent. Compound 15 was obtained
as a yellow waxy solid. Yield: 30%. 1H-NMR (400 MHz; CDCl3): δ 0.84-
0.87 (m, 2H), δ 1.23-1.27 (m, 27 H), δ 2.60 (t, J =8 Hz, 2H), δ 3.97 (s, 3H),
δ 5.87 (s, 1H), δ 6.90-6.92 (m, 2H), δ 7.08 (d, J = 8 Hz, 1H), δ 7.29-7.31
(m, 2H), δ 7.66 (t, J = 8 Hz, 1H), δ 7.85 (d, J = 8 Hz, 1H).
13C-NMR (100 MHz; CDCl3): δ 14.09, 22.66, 29.19, 29.32, 29.41, 29.52,
29.63, 29.66, 31.10, 31.89, 35.68, 56.54, 115.74, 118.09, 118.70, 119.38,
119.64, 120.84, 126.47, 129.92, 133.40, 134.39, 145.80, 152.70, 158.57,
159.40, 180.27, 180.51. MS (ESI+) m/z C32H42O4: 513 [M+ +Na+], 529 [M+
+K+].
Methyl 2-((5-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)oxy)-6-
pentadecylbenzoate (10). The title compound was obtained according to
the general procedure using 5 and 25, and purified by chromatography on
silica gel with a mixture of ethyl acetate/petroleum ether/toluene (5: 4: 1),
as eluent. Compound 10 was obtained as a yellow waxy solid. Yield: 53%.
1H-NMR (400 MHz; CDCl3): δ 0.84-0.87 (m, 2H), 1.23-1.28 (m, 27H), 2.65
(t, J = 8 Hz, 2H), 3.75 (s, 3H), 3.98 (s, 3H), 5.89 (s, 1H), 6.94 (d, J = 8.5
Hz, 1H), 7.16 (d, J = 7.6 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.38 (t, J = 8
Hz, 1H), 7.66 (t, J = 8 Hz, 1H), 7.83 (d, J = 7.2 Hz, 1H). 13C-NMR (100
MHz; CDCl3): δ 14.10, 22.67, 29.33, 29.35, 29.47, 29.51, 29.63, 29.65,
29.67, 31.24, 31.90, 33.61, 52.26, 56.56, 116.37, 118.68, 119.06,
119.37,119.61, 126.69, 127.55, 131.06, 133.32, 134.44, 143.79, 150.05,
158.16, 159.44, 166.55, 179.69, 184.47. MS (ESI+) m/z C34H44O6: 571
[M+ + Na+], 587 [M+ + K+].
8-Hydroxy-2-(3-pentadecylphenoxy)naphthalene-1,4-dione (16). The
title compound was obtained according to the general procedure using 6
and 26, and purified by crystallization from EtOH/H2O. Compound 16 was
obtained as an orange waxy solid. Yield: 45%. 1H-NMR (400 MHz; CDCl3):
δ 0.86-0.89 (m, 2H), 1.25-1.31 (m, 27H), 2.63 (t, J = 7.6 Hz, 2H), 5.95 (s,
Methyl
2-((8-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)oxy)-6-
pentadecylbenzoate (11) The title compound was obtained according to
the general procedure using 5 and 26, and purified by chromatography on
11
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10.1002/cmdc.201800790
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FULL PAPER
1H), 6.92-6.94 (m, 2H), 7.12 (d, J = 7.6 Hz, 1H), 7.28 (m, H), 7.35 (t, J =
8 Hz, 1H), 7.58-7.66 (m, 2H), 11.79 (s, 1H). 13C-NMR (100 MHz; CDCl3):
δ 14.04, 22.63, 29.16, 29.30, 29.38, 29.50, 29.60, 29.64, 31.07, 31.87,
35.66, 113.86, 114.32, 117.94, 118.90, 120.69, 123.98, 126.79,
130.06,132.01, 137.15, 146.02, 152.51, 160.23, 161.97,184.06, 184.78.
MS (ESI+) m/z C31H40O4: 499 [M+ +Na+], 515 [M+ +K+].
1.26-1.32 (m, 11H), 1.54-1.64 (m complex, 8H), 2.64 (t, J = 7.6 Hz, 2H),
3.63 (t, J = 6.6 Hz, 2H), 5.95 (s, 1H), 6.93-6.95 (m, 2H), 7.13 (d, J = 7.6
Hz, 1H), 7.2-7.29 (m, 1H), 7.36 (t, J = 7.6 Hz, 1H), δ 7.59-7.67 (m, 2H),
11.80 (s, 1H). 13C-NMR (100 MHz; CDCl3): δ 25.64, 28.98, 29.24, 29.32,
31.01, 32.74, 35.62, 63.00, 113.82, 114.32, 118.02, 118.94, 120.72,
124.02, 126.83, 130.11, 131.99, 137.21, 145.91, 152.49, 160.27, 161.98,
184.12, 184.79. MS (ESI+) m/z C24H26O5: 417 [M+ +Na+], 433 [M+ +K+].
5-Hydroxy-2-(3-pentadecylphenoxy)naphthalene-1,4-dione (17). The
title compound was obtained according to the general procedure using 6
and 27, and purified by chromatography on silica gel with a mixture of
petroleum ether/ toluene/ ethyl acetate (8.5: 1: 0.5), as eluent. Compound
17 was obtained as an orange waxy solid. Yield: 3%. 1H-NMR (400 MHz;
CDCl3): δ 0.85 (t, J = 7.2 Hz, 2H), 1.23-128 (m, 27H), 2.64 (t, J = 7.6 Hz,
2H), 5.88 (s, 1H), 6.93-6.95 (m, 2H), 7.14 (d, J = 8 Hz, 1H), 7.30 (d, J =
8.4 Hz, 1H), 7.36 (t, J = 8 Hz, 1H), 7.62 (t, J = 8 Hz, 1H), 7.75 (d, J = 8 Hz,
1H), 12.13 (s, 1H). 13C-NMR (100 MHz; CDCl3): δ 14.05, 22.64, 29.16,
29.30, 29.39, 29.50, 29.61, 29.64, 31.07, 31.87, 35.65, 112.85, 114.19,
117.95, 119.59, 120.71, 125.24, 126.83, 130.05, 131.10, 135.56, 146.01,
152.53, 161.09, 161.19, 179.23, 190.90. MS (ESI+) m/z C31H40O4: 499 [M+
+Na+].
5-Hydroxy-2-(3-(8-hydroxyoctyl)phenoxy)naphthalene-1,4-dione (22).
The title compound was obtained according to the general procedure using
7 and 27, and purified by chromatography on silica gel with a mixture of
ethyl acetate/n-hexane/toluene (5: 4: 1). Compound 22 was obtained as
an orange-yellow waxy solid. Yield: 28%. 1H-NMR (400 MHz; CDCl3): δ
1.30-1.40 (m, 12H), 1.52-1.59 (m complex, 5H), 2.61 (t, J = 7.6 Hz, 2H),
3.61 (t, J = 6.6 Hz, 2H), 5.87 (s, 1H), 6.91-6.92 (m, 2H), 7.11 (d, J = 7.6
Hz, 1H), 7.24-7.33 (m, 2H), 7.58 (t, J = 8 Hz, 1H), 7.71 (d, J =7.2 Hz, 1H),
12.09 (s, 1H). 13C-NMR (100 MHz; CDCl3): δ 25.65, 29.00, 29.25, 31.02,
32.73, 35.62, 63.01, 112.82, 114.17, 118.01, 119.65, 120.72, 125.30,
126.87, 130.10, 131.07, 135.62, 145.91, 152.49, 161.07, 161.19, 179.26,
190.53. MS (ESI+) m/z C24H26O5: 417 [M+ +Na+], 433 [M+ +K+].
2-(3-(8-Hydroxyoctyl)phenoxy)naphthalene-1,4-dione (18). The title
compound was obtained according to the general procedure using 7 and
23, and purified by chromatography on silica gel with a mixture of n-
hexane/ethyl acetate (5:5), and then by crystallization from n-
hexane/EtOH. Compound 18 was obtained as an orange-yellow waxy
solid. Yield: 25%. 1H-NMR (400 MHz; CDCl3): δ 1.32-1.52 (m, 10H), 1.53-
1.61 (m, 6H), 2.63 (t, J = 7.6 Hz, 2H), 3.63 (t, J = 6.4, 2H), 5.96 (s, 1H),
6.93-6.95 (m, 2H), 7.11 (d, J = 7.6 Hz, 1H), δ7.35 (t, J = 8 Hz, 1H), 7.74-
7.78 (m, 2H), 8.05-8.07 (m, 1H), 8.19-8.21 (m, 1H). 13C-NMR (100 MHz;
CDCl3): δ 14.09, 22.61, 25.64, 28.97, 29.24, 29.32, 31.01, 31.55, 32.72,
35.62, 62.96, 113.21, 118.10, 120.81, 126.18, 126.70, 126.75,
130.04,131.07,131.94, 133.48, 134.39, 145.78, 152.59, 160.60, 179.16,
185.07. MS (ESI+) m/z C24H26O4: 401 [M+ +Na+].
Prediction of physicochemical properties and Pan Assay
Interference Compounds (PAINS) analysis
The online server FAF-Drugs3[43] (http://fafdrugs3.mti.univ-paris-diderot.fr)
was used to predict physicochemical properties of 8-22 (Table S1). These
include: number of rotatable bonds, hydrogen-bond acceptors (HBAs),
hydrogen-bond donors (HBDs), MW, log P, log D (at pH 7), total polar
surface area (tPSA), flexibility, and aqueous solubility (log Sw).
FAF-Drugs3[43] was also used to screen 8-22 for known classes of pan
assay interference compounds (PAINS). As expected, 8-22 were flagged
as potential PAINS, due to the presence of the quinone sub-structure and
the long alkyl chain. However, in spite the potential for these compounds
to interfere with non-cellular assays,[44] we found no activity against
isolated TbGAPDH protein. Furthermore, the fact that the trypanocidal
activity is limited to quinones with C8 alkyl chain (18-22) points to a high
degree of specificity and interaction with well-defined target(s) rather than
a nonspecific interaction. In addition, 20 and 22 showed several-fold lower
trypanocidal activity when evaluated against T. congolense (IL3000 WT).
These observations led us to believe that the current subset does not
behave as PAINS.
2-(3-(8-Hydroxyoctyl)phenoxy)-8-methoxynaphthalene-1,4-dione
(19). The title compound was obtained according to the general procedure
using 7 and 24, and purified by chromatography on silica gel with a mixture
of ethyl acetate/petroleum ether/toluene/EtOH (5.5: 3: 1: 0.5). Compound
19 was obtained as an orange-yellow waxy solid. Yield: 20%. 1H-NMR
(400 MHz; CDCl3): δ 1.32 (s, 10H), 1.54-1.62 (m complex, 6H), 2.62 (t, J
= 7.6 Hz, 2H), 3.63 (t, J = 6.4 Hz, 2H), 4.04 (s, 3H), 5.87 (s, 1H), 6.91-6.92
(m, 2H), 7.19 (d, J = 7.6 Hz, 1H), 7.29-7.33 (m, 2H), 7.68-7.71 (m, 2H).
13C-NMR (100 MHz; CDCl3): δ 25.61, 28.94, 29.20, 29.27, 30.94, 32.74,
35.59, 56.50,62.96, 111.43, 117.59, 118.12, 118.85, 120.84, 126.44,
129.89, 134.36, 135.35, 145.64,152.99, 160.25, 161.22, 178,23, 184.94.
MS (ESI+) m/z C25H28O5: 431 [M+ +Na+], 447 [M+ +K+].
Biological evaluation
Organisms and culture media. Only bloodstream trypomastigotes of T.
b. brucei were used throughout this study. The drug-sensitive wild type
strain Trypanosoma brucei brucei Lister 427 (427-WT)[25a] was used
alongside three multi-drug resistant strains: B48, ISMR1, and aqp2/aqp3-
KO. B48 was created from 427-WT after deletion of the TbAT1 gene,
encoding for the P2 drug transporter,[25b] and adaptation to increasing
concentrations of pentamidine.[45] The aqp2/aqp3-KO strain was
generated from wild type T. b. brucei 2T1 cells after knockout of the locus
2-(3-(8-Hydroxyoctyl)phenoxy)-5-methoxynaphthalene-1,4-dione
(20). The title compound was obtained according to the general procedure
using 7 and 25, and purified by chromatography on silica gel with a mixture
of ethyl acetate/n-hexane/toluene (7: 2: 1). Compound 20 was obtained as
an orange-yellow waxy solid. Yield: 34%. Compound 20 was also
synthesized from 7 (0.3 mmol), 25 (0.3 mmol), and K2CO3 (0.3 mmol) in
dry DMSO (0.04 M), and purified according to the above-reported
procedure. Yield: 31%. 1H-NMR (400 MHz; CDCl3): δ 1.23-1.29 (m, 8H),
1.51-1.63 (m, 6H), 2.60 (t, J =7.6, 2H), 3.61 (t, J = 6.8 Hz, 2H), 3.97 (s,
3H), 5.86 (s, 1H), δ 6.90-6.91 (m, 2H), 7.07 (d, J = 7.6 Hz, 1H), 7.29-7.33
(m, 2H), 7.66 (t, J = 8 Hz, 1H), 7.84-7.86 (m, 1H). 13C-NMR (100 MHz;
CDCl3): δ 25.62, 28.94, 29.24, 29.32, 30.97, 32.75, 35.59, 56.53,62.96,
115.44, 118.14, 118.72, 119.33, 119.65, 120.85, 126.52, 129.97, 133.38,
134.43, 145.66, 152.69, 158.61, 159.41, 180.27, 184.67. MS (ESI+) m/z
C25H28O5: 431 [M+ +Na+], 447 [M+ +K+].
encoding for the aquaglyceroporin
2 and 3 channels, resulting in
melarsoprol-pentamidine cross resistance.[46] ISMR1 is an isometamidium
resistant clone obtained from T. b. brucei 427WT that lost their kinetoplast
DNA and express an FoF1-ATP synthase mutation.[32] All the T. b. brucei
strains were cultured as described[47] in standard HMI-9 medium,
supplemented by 10% of heat inactivated Fetal Bovine Serum (FBS), 14
μL/L of β-mercaptoethanol, and 3.0 g/L of sodium hydrogen carbonate
(pH=7.4). Parasites were cultured in vented flasks at 37 °C and 5% CO2
atmosphere and they were passaged every 3 days. Bloodstream forms of
T. congolense savannah-type strain IL3000 were cultured in basal MEM
medium, supplemented by 10% fresh goat serum, 14 μL of β-
mercaptoethanol, 800 μL of 200 mM glutamine solution, and 10 mL of
penicillin/streptomycin solution per liter of medium (pH = 7.3).[48] T.
congolense were cultured in 6-well plates at 34 °C and 5% CO2.
8-Hydroxy-2-(3-(8-hydroxyoctyl)phenoxy)naphthalene-1,4-dione (21).
The title compound was obtained according to the general procedure using
7 and 26, and purified by chromatography on silica gel with a mixture of
ethyl acetate/n-hexane/toluene (5: 4: 1). Compound 21 was obtained as
an orange-yellow waxy solid. Yield: 13%. 1H-NMR (400 MHz; CDCl3): δ
In Vitro drug susceptibility assay. The drug susceptibilities of
bloodstream form trypanosomes of T. b. brucei 427WT, B48, aqp2/aqp3-
KO and ISMR1 strains were determined by using the resazurin (Alamar
blue) viability indicator dye following the previously described protocol,[49]
slightly adapted for T. congolense. In brief, the assays were performed in
12
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10.1002/cmdc.201800790
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96-well plates with 2×105 cell/well for T. b. brucei or 5×105 cell/well for T.
congolense, in their respective culture media. First, 200 μL of test
compounds’ solutions (400 μM, in T. brucei or T. congolense medium as
appropriate) was added to the first well of each 12-well row, from which
doubling dilutions were conducted over one row per test compound.
Trypanosomes (100 μL in each well) were added and the plates were
incubated for 48 h at 37 °C and 5% CO2, followed by addition of 20 μL
Alamar blue solution (125 mg/mL of resazurin sodium salt (Sigma-Aldrich)
in phosphate-buffered saline (PBS)) followed by 24 h of incubation at 37 °C
and 5% CO2. Pentamidine (Sigma-Aldrich) (for 427WT, B48, aqp2/aqp3-
KO), diminazene aceturate (Sigma-Aldrich; for T. congolense) and
isometamidium (gift from Merial France) (for ISMR1) were used as
ATP concentrations in each sample. The ATP content was measured at 0,
0.5, 1, 2, 4, 8 and 12 h of incubation with test sample.
The same procedure was used to determine the ATP content of HFF cells,
using 200 μM of the test compounds for incubation times of 0, 4 and 8 h,
1×106 HFF cells per sample and a centrifugation speed for pelleting the
cells of 800×g for 5 min. Oligomycin (2 µg/ml) was used as a positive
control as described.[50]
Mitochondrial Membrane Potential Assay on T. b. brucei. Changes in
mitochondrial membrane potential (Ψm) after incubation of trypanosomes
with the test compounds were determined using Fluorescence Activated
Cell Sorting (FACS) with the indicator dye tetramethylrhodamine ethyl
ester (TMRE), as described[34] with minor modifications. Cell suspensions
of T. b. brucei 427WT trypomastigotes were incubated at 0.5×EC50
concentration with test compound and 1×107 cells were transferred at each
time point (0, 0.5, 1, 2, 4, 8 and 12 h) into a microfuge tube and centrifuged
at 2000×g for 10 min at room temperature. The pellet was washed once in
1 mL of PBS (pH 7.4) and resuspended in 1mL of PBS containing 200 nM
TMRE; the cells then were incubated at 37 °C for 30 min and subsequently
placed on ice for another 30 min before analysis on a Becton Dickinson
FACSCalibur using a FL2-height detector, and CellQuest and FlowJo
software. Valinomycin (Sigma-Aldrich; 100 nM) and troglitazone (Sigma-
Aldrich; 10 μM) were used as controls for mitochondrial membrane
depolarization and positive hyperpolarization, respectively.[51]
trypanocidal positive controls. Fluorescence was detected using
a
FLUOstar Optima (BMG Labtech, Durham, NC, USA) at wavelength of 540
nm (excitation), 590 nm (emission). EC50 values were calculated by non-
linear regression using an equation for a sigmoidal dose-response curve
with variable slope using Prism 5.0 (GraphPad Software Inc., San Diego,
CA, USA).
Growth Curve. T. b. brucei 427WT cells were grown to mid-log-phase in
standard HMI-9/FBS medium distributed in in 6-well plates at 1×105
cell/mL and incubated with two different concentrations of test compounds
(EC50, 2×EC50) for 48h at 37 °C and 5% CO2. Untreated parasites, used
as control, were grown in parallel. The cells were counted by using
hemocytometer cell counter (cell counting/mL×104) at 0, 2, 4, 8, 12, 24, 28,
32, 36, 48 h of incubation. Each experiment was performed as two
independent replicates, and each sample was counted at least twice.
Transmission Electron Microscopy Assay on T. b. brucei and HFF
cells. TEM of bloodstream trypomastigotes (427WT) and of HFF was
performed essentially as previously described.[52] Briefly, cell cultures were
adjusted to 2.5×106 cell/mL and incubated in the presence or absence of
test compounds at EC50 concentration. Cells were fixed overnight at 4°C
in 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate
buffer (pH 7.4). Samples were washed with 0.1 M phosphate buffer (pH
7.4), post-fixed in 1% osmium tetroxide for 1 h on ice, and washed with the
phosphate buffer. Cells were next incubated in 0.5% uranyl acetate
solution for 30 min, washed with distilled water and dehydrated in
increasing concentrations of acetone (30, 50, 70, 90 and 100%). Cells
were embedded in epoxy resin; thin sections of 50-60 nm were observed
in a Tecnai T20 (FEI) at 200 kV.
Cytotoxicity assay on Human Foreskin Fibroblast (HFF). Toxicity of
test compounds to mammalian cells was carried out using the resazurin
assay previously described[31] with slight modifications as follows. HFF
human cells were grown in a culture medium containing 500 mL of
Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma), 50 mL New-Born
Calf Serum (NBCS) (Gibco), 5 mL Penicillin/Streptomycin (Gibco), and 5
mL of L-Glutamax (200 nM, Gibco), at 37 °C and 5% CO2 in vented flasks
and passaged at 80-85% of confluence. For the cytotoxicity assay, cells
were suspended at 3×105 cell/mL and 100 μL aliquots were added to each
well of a 96-well plate. The plate was incubated at 37 °C and 5% CO2 for
24 hours to allow cells adhesion. Serial test compounds dilution was
performed in a different 96-well plate and 100 μL of each dilution was
transferred to each well containing cells, resulting in exposure of cells to
200 - 0.2 µM of test compound. Phenylarsine oxide (Sigma-Aldrich) was
used as positive control and drug-free incubation as negative control. The
plates were incubated at 37 °C and 5% CO2 for an additional 30 h, at which
point 10 μL of resazurin solution (125 mg/mL in PBS) was added, followed
by a final incubation for 24 h. The plates were read and the data analyzed
as described above for the trypanosome assay.
Radical Oxygen Species (ROS) Assay on T. b. brucei. The production
of ROS when T. b. brucei 427WT trypomastigotes incubated with different
concentration (2.5×EC50; 1.25×EC50; 0.3×EC50) of test compound was
assessed using the indicator dye 2,7-dichlorodihydrofluorescein diacetate
(DCFH-DH; Sigma-Aldrich). This assay was performed in a 96-well black-
bottomed well plate; 200 μL of each test compound solution, at 5×EC50
concentration in assay buffer pH 7.3, was added to the well in the first
column of the plate and doubling dilutions were carried out across the row,
after which 3×106 cell in 100 μL of assay buffer were added to each well,
immediately followed by 2 μL of 1 mM DCFH-DH, under minimal light
conditions. The plates were incubated in a FLUOstar OPTIMA fluorimeter
at 37 °C 5% CO2 and the fluorescence was monitored at 485 nm for the
excitation, 520 nm for the emission for 3 h, taking readings of each well
every 2 min. Three wells were included in the plate as controls: (i) 3×106
cell/well in assay buffer without test compound; (ii) 3×106 cell/well in assay
buffer with 2 μL of 10 mM H2O2; (iii) assay buffer without cells and test
compound.
ATP assay on T. b. brucei and HFF cells. Changes in cellular ATP levels
due to the exposure of trypanosomes to the test compounds (0.5×EC50
concentration) were monitored using the Molecular Probes ATP
Determination Kit (A22066, Invitrogen Detection Technologies), based on
the luciferin-luciferase bioluminescent enzymatic reaction. Bloodstream
trypomastigote cultures of T. brucei s427WT were incubated with and
without test compound, and, at each predetermined incubation time, 107
cells of each sample were transferred into a microfuge tube and
centrifuged at 2000×g for 10 min at 4 °C. The pellet was washed twice with
1 mL of 50 mM Tris-HCl (pH 7.4) containing 0.1 mM DTT, and then re-
suspended in 200 μL of the same buffer. The cells were lysed by sonication
on ice (twice for 10 s separated by 30 s), using a Soniprep 150 (MSE) at
8 μm amplitude. The samples were centrifuged at 10,000×g for 10 min at
4 °C and the supernatant was collected, instantly frozen in liquid nitrogen,
and stored at -80 °C. Oligomycin (2.0 μg/mL) was used as positive control.
ATP levels were quantified using the contents of the kit following the
manufacturer’s instructions. 90 μL of standard reaction solution was added
to each well of a 96-well plate and the background luminescence was
recorded in a FLUOstar OPTIMA fluorimeter; 10 μL of each sample was
then added to each well. The plate was incubated at 28 °C for 15 min and
the luminescence was measured, including a standard curve with an ATP
concentration ranging from 1 nM to 1 μM to allow the calculation of the
Metabolomics Assay in T. b. brucei. The experiment was performed to
detect changes in the intracellular metabolic pathway in T. b. brucei
427WT BSF after 2 hours incubation with the most active test compound,
at a concentration of 0.5×EC50, and compared with a DMSO drug free
control. Parasites were grown to 1.5×106 cell/mL in order to have a mid-
log phase culture at the end of the incubation time. At the end of the
incubation, the cell density was adjusted and 1×108 cells were transferred
to a 50 mL centrifuge tube. The samples were quenched by rapidly cells
cooling to 4 °C in a dry ice/ethanol bath and centrifuged at 1250×g for 10
min at the same temperature. 10 μL of supernatant (spent medium) was
collected, 200 μL of CMW (Chloroform/Methanol/Water 1:4:1) was added
and the mixture stored at -80 °C until LC-MS analysis. The rest of the
supernatant was discarded; the cell pellet was transferred to a microfuge
13
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10.1002/cmdc.201800790
ChemMedChem
FULL PAPER
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Acknowledgements
This work was supported by the University of Bologna (Grant RFO
2016). LASR was supported by the University of Brasilia (CNPq
#401864/2013-8). GUE was supported by a personal studentship
from the Tertiary Education Trust Fund of Nigeria (TETFUND-
2011/2012). The Wellcome Trust Centre for Molecular
Parasitology is supported by core funding from the Wellcome
Trust (grant number 085349). MLB has been supported by a grant
of Visiting Professor (PVE–CNPq #401864/2013-8) from the
programme “Ciência sem Fronteiras”, Brazil. LASR is a senior
fellow of the National Council of Research (CNPq, DT2
#310385/2014-8, Brazil). MC thanks the University of Bologna for
a post-lauream fellowship that enabled her to work in the
laboratory of HPdK.
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All authors declare no conflict of interest.
Keywords: antiprotozoal agents • cashew nut shell liquid •
hybrid drugs • natural products • trypanosomiasis
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10.1002/cmdc.201800790
ChemMedChem
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We synthesized a novel and sustainable series of anti-trypanosomal hybrids, derived from cashew nut shell liquid (CNSL), which target
the energy metabolism in T. brucei, altering the ATP level, mitochondrial membrane potential and radical oxygen species production.
Compound 20 showed low µM EC50, no drug-resistance and a good selectivity index.
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