New alkenyl derivative from Piper malacophyllum and analogues: Antiparasitic activity against Trypanosoma cruzi and Leishmania infantum

Article abstract of DOI:10.1111/cbdd.12986

Alkylphenols isolated from Piper malacophyllum (Piperaceae), gibbilimbols A and B, showed interesting activity against the parasites Trypanosoma cruzi and Leishmania infantum. In continuation to our previous work, a new natural product from the essential

Full text of DOI:10.1111/cbdd.12986

DR. JOÃO PAULO DOS-SANTOS FERNANDES (Orcid ID : 0000-0002-9089-273X)
Received Date : 03-Jan-2017
Revised Date : 17-Feb-2017
Accepted Date : 22-Mar-2017
Article type : Research Letter
New alkenyl derivative from Piper malacophyllum and analogues: Antiparasitic
activity against Trypanosoma cruzi and Leishmania infantum
Marina T. VARELAa, Marta L. LIMAb,c, Mariana K. GALUPPOb, Andre G.
TEMPONEb, Alberto de OLIVEIRAd, João Henrique G. LAGOe,* and João Paulo S.
FERNANDESa,
aDepartamento de Ciências Farmacêuticas, Universidade Federal de São Paulo, Rua São
Nicolau 210, Diadema-SP 09913-030, Brazil
bCentro de Parasitologia e Micologia, Instituto Adolfo Lutz, Av. Dr. Arnaldo 355, São Paulo-
SP 01246-902, Brazil
cInstituto de Medicina Tropical de São Paulo, Universidade de São Paulo, Av. Dr. Enéas
Carvalho de Aguiar 470, São Paulo-SP 05403-000, Brazil
dInstituto de Química, Universidade Federal de Uberlândia, Av. João Naves de Ávila, 2121,
Uberlândia-MG 38400-902, Brazil
eCentro de Ciências Naturais e Humanas, Universidade Federal do ABC, Av. do Estado,
5001, Santo André-SP 9210-580, Brazil
Corresponding authors. Tel.: +55-11-3385-4137; e-mails: joao.lago@ufabc.edu.br (J.H.G.L.) and
joao.fernandes@unifesp.br (J.P.S.F.)
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.12986
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Keywords: natural product derivatives, leishmanicidal, trypanocidal, gibbilimbol
analogues, SAR.
Abstract
Alkylphenols isolated from Piper malacophyllum (Piperaceae), gibbilimbols A and B,
showed interesting activity against the parasites Trypanosoma cruzi and Leishmania
infantum. In continuation to our previous work, a new natural product from the
essential oil of the leaves of P. malacophyllum was isolated, the 5-[(3E)-oct-3-en-1-
il]-1,3-benzodioxole, and also a new set of five compounds was prepared. The
antiparasitic activity of the natural product was evaluated in vitro against these
parasites, indicating potential against the promastigote/trypomastigote/amastigote
forms (IC50 32-83 µM) of the parasites and low toxicity (CC50 >200 µM) to
mammalian cells. The results obtained to the synthetic compounds indicated that the
new derivatives maintained the promising antiparasitic activity, but the cytotoxicity
was considerably lowered The amine derivative LINS03011 displayed the most
potent IC50 values (13.3 and 16.7 µM) against amastigotes of T. cruzi and L.
infantum, respectively, indicating comparable activity to the phenolic prototype
LINS03003, with 3-fold decreased (CC50 73.5 µM) cytotoxicity, leading the selectivity
index (SI) towards the parasites up to 24.5. In counterpart, LINS03011 has not
shown membrane disruptor activity in Sytox Green model. In summary, this new set
showed the hydroxyl is not essential for the antiparasitic activity, and its substitution
could decrease the toxicity to mammalian cells.
American trypanosomiasis (Chagas’ disease) and visceral leishmaniasis are among
the most important parasitic infections known as neglected tropical diseases.1 They
mainly affect developing and poor countries, especially in Latin America, Middle East
and Asia, and are caused by the protozoans of the genus Trypanosoma and
Leishmania, respectively.2-5 Although these diseases affect millions of people
worldwide,1 only few drugs are available for their treatment (amphotericin B,
benznidazole, miltefosine, nifurtimox, pentamidine and antimonials), which are poorly
effective and considerably toxic. Despite the urgent necessity for new drugs to treat
these conditions, low investments are directed to this aim, giving their “neglected”
status.6,7
Considering this background, the search for prototypes to drug discovery against
neglected diseases is an important research field, and natural products can be
considered good starting point. In this respect, two alkylphenol derivatives,
gibbilimbols A and B, isolated from the essential oil from the leaves of Piper
malacophyllum, showed promising activity against both of the parasites responsible
for Chagas’ disease and visceral leishmaniasis, Trypanosoma cruzi and Leishmania
(L.) infantum, respectively.8,9 This previous study8 revealed that gibbilimbol B was
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capable of affecting the permeability of the plasma membrane of L. (L.) infantum in a
similar way than amphotericin B, suggesting that this action could be result from the
similarity to the membrane phospholipids, being the hydroxyl group the “polar head”
and the alkyl chain the “lipophilic tail”. Moreover, gibbilimbol B is more potent than
the A isomer, also suggesting the functionalization closer to the aromatic ring would
improve the activity.
In a previous report,9 we presented a set of eight gibbilimbol analogues to evaluate
the role of functionalization and molecular modifications in the “lipophilic tail”. This
set included substitution close to aromatic ring and lower homologues with six-atom
length instead of ten. These analogues were tested against trypomastigote and
amastigote forms of T. cruzi and promastigote and amastigote forms of L. (L.)
infantum, in addition to the evaluation of cytotoxicity to mammalian cells. The data
resulted in preliminary structure-activity relationship (SAR) information. LINS03003
(Figure 1) was the most promising molecule, exhibiting IC50 values from 28.6 to 1.8
μM against these parasites. However considerable cytotoxicity to mammalian cells
(CC50 23.5 μM) was verified. Other molecules of the series showed more potent IC50
values than the natural prototypes, but they showed higher cytotoxicity than both
gibbilimbols A and B indeed.
In continuation to our work exploiting the potential from natural and synthetic
compounds, the present work reports a new compound from the essential oil from
leaves of P. malacophyllum, 5-[(3E)-oct-3-en-1-il]-1,3-benzodioxole (1, Figure 1).
The structure of this compound was determined by detailed analysis of NMR and MS
spectral data (see Supplementary Information) and despite the novel proposed
structure, this compound are closely related to gibbilimbol B. Considering this
similarity, we decided to investigate the activity of this new natural product against T.
cruzi and L. (L.) infantum, as well as its cytotoxicity against NCTC mammalian cells.
The present report aims to deal with the cytotoxicity issue. All the compounds of the
previous set (and also gibbilimbols) present a phenol group, which is responsible for
toxic effects in several molecules,10-14 and sometimes can be classified as pan-assay
interference compounds (PAINS)15 due to its possible interference as redox cycler.
However, this phenolic hydroxyl would be part of the pharmacophore when
considering the membrane disruption hypothesis observed previously.8,9 Considering
this, and the results from compound 1, a new set of compounds (2-6, Figure 1) were
designed and synthesized with a new site of modifications, the 4-position substituent
on the aromatic ring, as an attempt to investigate the importance of the hydroxyl in
the biological activity. The proposed molecular modifications included the
methylation of the hydroxyl and its removal, represented by the methoxybenzene
and benzene groups, respectively.
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The compounds were synthesized in a similar fashion than the previous reported set
(Figures 2 and 3, see Supplementary Information).9 Compounds 2 and 3 were
prepared from the corresponding benzaldehydes and 1-octylamine by mixing them
together in equimolar amount under ultrasonic conditions. The reaction led to
moderate yields for both compounds. Compound 4 was then prepared using
compound 2 as starting material, which was converted to the corresponding amine
by reacting with NaBH4, with good yield.
The amide derivative 5 was synthesized from aminolysis of benzoyl chloride with 1-
octylamine, in presence of triethylamine (TEA). The product was then purified by
recrystalization in water:ethanol mixture, also with good yield. Finally, compound 6
was obtained through aldol condensation of anisaldehyde and 2-nonanone in basic
conditions, with adequate yields.
The antiprotozoal activity of the new set of analogues 1–6 was evaluated in vitro
against
trypomastigote/amastigote
forms
of
T.
cruzi
as
well
as
promastigote/amastigote forms of L. (L.) infantum using the method from Varela et
al.,9 except by use of bone marrow-derived mouse macrophages differentiated using
cytokine M-CFS obtained from L929 cell-conditioned medium. Benznidazole and
miltefosine were used as positive control against T. cruzi and L. (L.) infantum,
respectively. Cytotoxicity was determined in mammalian NCTC clone 929 cells as
previously described.9
The natural product 1 has shown interesting activity against all parasite forms, with
IC50 values ranging from 32 to 82 µM. It must be stressed this compound was more
active against the amastigote forms of the parasites, the most relevant form in the
diseases. This activity profile is very similar to the gibbilimbol B, however the
substitution of the phenol for the benzodioxole showed to improve the activity. In
addition, the cytotoxicity was quite low for this compound (>200 µM). Considering the
higher activity of the benzodioxole 1 in comparison to the phenolic gibbilimbols, it is
suggested the phenolic hydroxyl is not essential to the activity, and thus it is an
important site for structural exploitation to obtain analogues with improved activity as
well as reduced cytotoxicity.
Among the five new analogues synthetized, only the compound 4 showed significant
activity against the trypomastigote form of T. cruzi, with IC50 value of 21 µM. The
remaining compounds showed poor activity against this form of the parasite (>100
µM). However, three compounds showed considerable activity against the
amastigote form of T. cruzi, being the compound 4 the most active (IC50 13 µM). As
observed in the previous results from our group,9 the amine group in the “tail” moiety
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leads to promising activity, especially against the amastigote form. Molecules 2 and
6 also showed interesting activity, but were less active against the trypomastigotes
than compound 4. According to Drugs for Neglected Disease initiative (DNDi) the
intracellular amastigotes are the most important target to drug discovery studies, but
candidates that eliminate the trypomastigotes are of great interest, due to
recrudescence of the parasite.16 In our study, compound 4 fulfilled these
requirements, also showing a selectivity towards T. cruzi with a SI value of 5.5
(considering the intracellular amastigotes).
Similar activity profile was observed when the compounds were tested against L.
infantum parasite. The amine compound 4 was again the most active against both
forms of the parasite, promastigotes and amastigotes, being more active against the
promastigotes. On the other hand, the prototype LINS03003 was more active against
amastigote form of L. infantum than against the promastigotes. Compounds 2 and 3
were also active against both forms of L. infantum, showing IC50 values in the range
of 28-40 µM. The amide derivative 5 lacks considerable activity against both
parasites, as previously observed to the phenolic analogues, although it showed no
considerable cytotoxicity indeed.
Maybe the most important finding in this work is the low cytotoxicity of this new set of
gibbilimbol analogues. Practically all the compounds showed decreased toxicity to
mammalian cells than the previous set from our group.9 With exception of compound
4, which exhibited a CC50 value of 74 µM, the compounds showed CC50 >200 µM.
Although compound 4 was considerably cytotoxic, the selectivity indexes towards the
parasites were over than 3.6, and up to 25 considering the L. infantum
promastigotes. Perhaps this low cytotoxicity can be attributed to the absence of
phenolic hydroxyl in this new set, corroborating with the hypothesis of the toxicity of
this group. Moreover, the compounds suggest the hydroxyl is not essential to the
biological activity albeit it increases the activity, especially against T. cruzi
trypomastigotes. As example, when comparing the methoxyl compound 6 to its
hydroxyl analogue LINS03001,9 the activity against T. cruzi amastigote is almost the
same, but the cytotoxicity is quite low when the methoxy group is present. The same
behavior can be observed for compound 4, which exhibited CC50 3-fold increased
than the hydroxyl molecule LINS03003. On the other hand, the benzodioxole 1
suggests that additional substitution in the 3-position of the aromatic ring may
improve the activity keeping the cytotoxicity low, and in future works we should
prepare synthetic 3-substituted derivatives.
Considering the gibbilimbols could act through membrane disruption,8 the compound
4 was studied using the fluorescent probe Sytox Green in late growth-phase (non-
stationary) of promastigotes of L. infantum8,17,18 (Figure 4). Sytox Green (Molecular
Probes) is a high-affinity nucleic acid stain that penetrates cells with compromised
plasma membranes and enhances its fluorescence by more than 500-fold upon
nucleic acid binding. The non-ionic surfactant Triton X-100 (Tx100) was used as the
positive control, resulting in the maximal intensity of fluorescence. Fluorescence was
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measured in a microplate reader with excitation and emission wavelengths of 485
and 520 nm, respectively. Gibbilimbol B showed to promote the penetration of the
dye, suggesting the disruption of the plasma membrane permeability of Leishmania.8
However, compound 4 showed no increased fluorescence inside the cells at the
EC50 value, suggesting other mechanism than gibbilimbol against Leishmania, which
caused disruption of the plasma membrane. At elevated concentration (EC90)
compound 4 slightly increased the dye fluorescence; possibly the hydroxyl of
gibbilimbol B plays an important role in this action, acting as the “polar head” to
guide its membrane insertion. Since compound 4 do not present the hydroxyl group,
this action in the membrane was reduced. Further studies must be done to confirm
this statement.
Natural products are always reported as inspiring molecules to achieve novel
antiparasitic derivatives. Several works are reported reporting semi-synthetic
molecules to evaluate the role of substituents in the activity. Morais et al. reported
the promising activity of natural and semi-synthetic masticadienoic acid derivatives
against T. cruzi and L. infantum, achieving good IC50 values for both parasites.19
Aphidicolin was also used to prepare some semi-synthetic derivatives to assess the
antileishmania activity in several species.20 However, natural products are frequently
very complex structures which difficult the synthesis of cheap bioactive compounds,
an important requirement for future drugs to neglected diseases. The compounds
presented herein are much simpler structures in comparison to these cited
examples, allowing the easy design of several synthetic analogues to improve the
activity profile.
In summary, the results presented herein demonstrated that this new set of
gibbilimbol analogues also showed considerable activity but with improved selectivity
towards the parasites, possibly due to the absence of the toxicophoric phenolic
group. These molecules present important information to guide the design of
molecules with improved activity, and in fact a new set of derivatives based on the
amine derivatives 4 and LINS03003, in addition to the characteristics of compound 1
are in progress by our group to investigate the role of other substituents in these
molecules.
Acknowledgements
The authors would like to thank CNPq (grant no. 455411/2014-0) and FAPESP
(2015/11936-2, 2013/50228-8, 2012/18756-1) for providing financial support and
fellowship to M.T.V. (2016/00195-4). A.G.T. and J.H.G.L. thanks to CNPq for the
scientific research award.
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References and notes
[1] World Health Organization (WHO). Working to overcome the global impact of
neglected tropical diseases: first WHO report on neglected tropical diseases.
Geneva: WHO, 2010.
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J. Kato, Exp. Parasiltol. 2012, 132, 383.
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Kita, C. E. Mowbray, D. Schmatz, P. Warner, B. T. Slingsby, Nat. Rev. Drug
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Scotti, R. M. F. Sousa, A. C. C. Araujo, A. Oliveira, S. A. L. Morais, P. Sartorelli,
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Figures captions
gibbilimbol A
gibbilimbol B
NATURAL
PRODUCTS
5-[(3E)-oct-3-en-1-il]-1,3-benzodioxole (1)
SYNTHETIC ANALOGUE - LINS03003
T. cruzi trypomastigotes IC50 17 μM - amastigotes IC50 5.5 μM
L. infantum promastigotes IC50 28 μM - amastigotes IC50 1.8 μM
NCTC cells CC50 23 μM
LINS03009 (2)
LINS03010 (3)
LINS03011 (4)
LINS03012 (5)
LINS03013 (6)
Figure 1. Natural products, lead compounds and the new set of LINS03 molecules.
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(c)
6
(a)
(b)
4
2: R = H
3: R = OMe
Figure 2. Synthesis of LINS03 compounds 2-4 and 6. Reagents and conditions: (a)
octylamine, THF, 1 h, ))); (b) NaBH4, MeOH, 4 h; (c) 2-nonanone, NaOH 40%, EtOH,
12 h.
5
Figure 3. Synthesis of compound 5. Reagents and conditions: octylamine, TEA,
CH2Cl2, 60 °C, 6 h.
Figure 4. Effect of compound 4 on the membrane of Leishmania promastigotes
using the fluorescent probe Sytox Green. Promastigotes of the control group were
treated with 0.5% MeOH.
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Tables
Table 1. Antiparasitic activities of the natural product 1, and of the synthetic derivatives 2–6 against trypomastigote/amastigote
forms of T. cruzi and promastigote/amastigote forms of L. (L.) infantum.
IC50 µM (±SEM)
T. cruzi
amastigotes
32.6±1.5
50.0±18.3
>100
CC50 µM (±SEM)
NCTC cells
SIa
Compounds
T. cruzi
trypomastigotes
67.3±2.6
>100
L. (L.) infantum
promastigotes
82.5±5.2
28.3±1.8
27.8±1.3
3.0±0.06
>100
L. (L.)infantum
amastigotes
51.6±4.6
40.4±15.8
34.9±2.8
16.7±3.5
>100
TCT
TCA
LIP
LIA
1
2
3
4
>200
>200
>200
73.5±1.9
>200
>2.9
-
-
3.6
-
>6.1
>4.0
-
5.5
-
>2.4
>7.0
>7.2
24.5
-
>3.9
>4.9
>5.7
4.4
-
>100
20.7±0.9
>100
13.3±1.4
>100
5
6
>100
102.5
75.3
ND
26.2±6.1
NA
NA
ND
5.2±0.3
>100
NA
>100
135.7
94.9
7.4±0.9
ND
>200
-
>7.6
-
-
-
-
-
-
gibbilimbol Ab
gibbilimbol Bb
miltefosine
benznidazole
224.6
254.1
241.4b
>200
2.2
3.4
-
1.7
2.7
32.6
-
100.4
2.5
14.5
-
16.7b
16.4±2.1
ND
16.0
51.9
IC50: 50% inhibitory concentration determined after 48 h and 72 h treatment for extracellular and intracellular forms, respectively
NA: not active
ND: not determined
CC50: 50% cytotoxic concentration determined after 48 h treatment
aSI: selectivity index calculated from CC50/IC50 (TCT: T. cruzi trypomastigotes, TCA: T. cruzi amastigotes, LIP: L. (L.) infantum promastigotes, LIA: L. (L.)
infantum amastigotes)
bAvailable from Varela et al.9
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