Antileishmanial activity and cytotoxicity of ent-beyerene diterpenoids

Article abstract of DOI:10.1016/j.bmc.2018.11.030

We describe the in vitro activity of two natural isomeric ent-beyerene diterpenes, several derivatives and synthetic intermediates. Beyerenols 1 and 2 showed EC₅₀ of 4.6 ± 9.4 and 5.3 ± 9.4 μg/mL against amastigotes of L. (V) brazilensis, with

Full text of DOI:10.1016/j.bmc.2018.11.030

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Antileishmanial Activity and Cytotoxicity of ent-Beyerene Diterpenoids
Jilmar A. Murillo, Juan F. Gil, Yulieth A. Upegui, Adriana M. Restrepo, Sara
M. Robledo, Winston Quiñones, Fernando Echeverri, Aurelio San Martin,
Horacio F. Olivo, Gustavo Escobar
PII:
S0968-0896(18)31615-8
https://doi.org/10.1016/j.bmc.2018.11.030
BMC 14634
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 September 2018
8 November 2018
20 November 2018
Please cite this article as: Murillo, J.A., Gil, J.F., Upegui, Y.A., Restrepo, A.M., Robledo, S.M., Quiñones, W.,
Echeverri, F., San Martin, A., Olivo, H.F., Escobar, G., Antileishmanial Activity and Cytotoxicity of ent-Beyerene
Diterpenoids, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.11.030
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Antileishmanial activity and cytotoxicity of
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ent-beyerene diterpenoids
Murillo, J. A.; Gil, J. F.; Upegui, Y. A.; Restrepo, A. M.; Robledo, S. M.; Quiñones, W.; Echeverri, F.; San
Martin, A.; Olivo, H. F.; Escobar, G.
Universidad de Antioquia; Universidad de Antioquia; Universidad de Magallanes; The University of Iowa.

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Bioorganic & Medicinal Chemistry
Antileishmanial Activity and Cytotoxicity of ent-Beyerene Diterpenoids
Jilmar A. Murilloa, Juan F. Gila, Yulieth A. Upeguib, Adriana M. Restrepob, Sara M. Robledob, Winston
Quiñonesa, Fernando Echeverria, Aurelio San Martinc, Horacio F. Olivod, and Gustavo Escobara, 
aQOPN Grupo Química Orgánica de Productos Naturales, Instituto de Química, Universidad de Antioquia
bPECET, Instituto de Investigaciones Médicas, Facultad de Medicina, Universidad de Antioquia, Calle 70 No. 52-21, Postal Code 0500100, Medellín, Colombia
cFacultad de Ciencias, Universidad de Magallanes, Punta Arenas, Chile
dDivision of Medicinal and Natural Products Chemistry, The University of Iowa, Iowa City, IA 52242, USA
ARTICLE INFO
ABSTRACT
We describe the in vitro activity of two natural isomeric ent-beyerene diterpenes, several
derivatives and synthetic intermediates. Beyerenols 1 and 2 showed EC50 of 4.6±9.4 and 5.3±9.4
g/mL against amastigotes of L. (V) brazilensis, with SI of 5.1 and 7.7, respectively. Beyerenol
1 was synthesized from stevioside. In vivo experiments with bereyenols showed cure in 50% of
hamsters infected with L. (V) brazilensis topically applied as Cream I (beyerenol 1, 0.81%, w/w)
and Cream III (beyerenol 2, 1.96%, w/w). These results suggest that beyerenols are potential
candidates for cutaneous leishmaniasis chemotherapy by topical application. In vitro assays of
amastigotes of L. (V) brazilensis showed EC50 of 1.1±0.1 and 1.3±0.04 g/mL, with SI of 3.1
and 3.5 for hydrazone intermediates 10 and 11, respectively.
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
ent-beyerene diterpenoids
Leishmania brazilensis
antileishmanial
semisynthesis
2009 Elsevier Ltd. All rights reserved.
steviol
1. Introduction
treatment of wounds and inflammations and their extracts have
been studied as antibacterial and anti-inflammatory.12 Marginal
Leishmaniasis is an endemic parasitic disease, widely
distributed around the world and affecting a large economically
disadvantaged population that lives in rural areas of tropical and
subtropical countries.1,2 Available treatments include pentavalent
antimonials, miltefosine, pentamidine isothionate and
amphotericin B.3 The use of these products has various
difficulties such as the need for parenteral administration (except
for miltefosine that is administered orally), very long treatment
periods, high doses, serious side effects and high cost. The
landscape is complicated by the potential development of
resistance to some of these medicines.4,5 The pharmaceutical
companies are not interested to invest in developing new
chemical entities to control neither leishmaniasis nor other
parasite diseases and it is classified by the World Health
Organization as a neglected disease.6
activities have been reported against HIV-1, antimycobacterial
and antileukemic.8,9,13,14,15 There are some reports of active
diterpenes against leishmania, with structures as diverse as
clerodanes,16 labdanes,17 abietanes,18 and alkaloids,19 some of
them present interesting in vitro activities against different
species of leishmania, However, the leishmanicidal activity of
ent-beyerene diterpenoids has not been reported. In the present
work, the in vitro and in vivo leishmanicidal activities of two
isomeric compounds isolated from the Chilean plant Baccharis
tola (Figure 1) and some others obtained by semisynthesis were
evaluated. The therapeutic efficacy of natural compounds in
experimental cutaneous leishmaniasis (CL) model in hamsters is
also reported in the present work.
In the search for more and better drugs, natural products
represent a good alternative to discover new antileishmanial
agents.7 ent-Beyerene diterpenoids have been reported in species
of Asteraceae and Euphorbiaceae families, among others.8,9,10,11
In the first family, several species of Baccharis have been
reported as medicinal plants in Argentina, Bolivia, Chile and
Figure 1. ent-Beyer-15-en-18-ol (1) and ent-beyer-15-en-19-ol (2) from
P—er—u, —where they are used in traditional medicine for the
Baccharis tola.
 Corresponding author. Tel.: +0-000-000-0000; fax: +0-000-000-0000; e-mail: author@university.edu

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2. Isolation, synthesis of beyerenol and analogs
this synthetic route and the use of low cost reagents, it was
possible to obtain several grams of the compound of interest.
Natural products have been a rich source of bioactive
molecules for the development of new drugs; however, they have
several drawbacks, among them the low concentrations present in
the plant and the extensive purification processes. For these
reasons, many investigations do not advance until stages that
allow to define their potential as a new medicine and thus be able
to establish QSAR relationships to optimize their effects. On the
other hand, more and better drugs are needed to control neglected
parasitic diseases because those that are currently available have
many limitations. In this work we analyze the leishmanicidal
activity of several natural diterpene molecules, as well as some
structural analogues.
Diterpenes are a large group of natural molecules with
pharmacological activities and very diverse chemical structures.
In this group are the ent-beyerenes, compounds which have not
been reported to possess antiparasitic activity. ent-Beyer-15-en-
18-ol (1) and ent-beyer-15-en-19-ol (2) were isolated from the B.
tola plant as previously reported.20 Four derivatives were
prepared from the major diastereomer, beyerenol 1, Figure 2.
Beyerenol 1 was hydrogenated in the presence of Pd/C in ethyl
acetate to furnish beyerol 3. Beyerenol 1 was oxidized with Jones
reagent to give beyerenoic acid 4, and this acid was oxidized with
m-CPBA to obtain the oxirane beyeroic acid 5 as a single isomer.
Finally, beyerenol
1
was oxidized with m-CPBA in
Figure 3. Synthesis of beyerenol 1 from stevioside.
dichloromethane to obtain oxirane 6. These simple derivatives (3
– 6) obtained from the natural product were valuable to obtain
initial relationships between structure and antiparasitic activity.
Some simple modifications were made to the cyclopentanone
ring of isosteviol ethyl ester 9 following literature procedures,
Figure 4. The ketone function of isosteviol 9 was converted to the
corresponding oxime 13 on treatment with hydroxylamine and
potassium acetate in 96% yield.25 Oxime 13 underwent a
Beckman fragmentation reaction when heated with tosyl chloride
in DMF providing nitrile 14 in 87% yield.26 Isosteviol ethyl ester
9 was reduced with sodium borohydride to give alcohol 15 as a
single isomer in 96% yield.25 The synthesis of isosteviol 8 and its
ethyl ester 9 were prepared according to the method reported in
the literature as well as for compounds 13, 14 and 15.
Figure 2. Some derivatives from ent-beyer-15-en-18-ol (1).
The promising leishmanicidal activity of beyerenol 1, its low
concentration in the Chilean plant B. tola16 and its extremely
difficult isolation, forced the search for a source that would allow
us to generate more material. Stevioside, a natural sweetener
widely used in the food industry emerged as an ideal candidate,
given its accessibility, low cost and structural similarity to the
molecule of interest. 21,22 To our knowledge, this is the first report
of a synthesis of beyerenol 1.
Isosteviol (8), generated by acid hydrolysis of stevioside (7),23
was initially esterified to ethyl ester 9 and then transformed into
the corresponding tosylhydrazone 11, which underwent a
Bamford-Stevens reaction employing basic conditions and
microwave irradiation to ethyl beyerenoate 12, Figure 3.24 This
ethyl beyerenoate 12 was reduced with LiAlH4 to furnish the
desired beyerenol 1 in 52% overall yield. The same synthetic
sequence was carried out with isosteviol, without the need to
prepare the ethyl ester, and the overall yield was 38%. Through
Figure 4. Synthesis of isosteviol ester derivatives 13 – 15.
3. Materials and methods
3.1 In vitro cytotoxicity in human U937 macrophages
Human promonocytic cells U937 (ATCC CRL-1593.2
previously cultured in RPMI-1640 (Invitrogen) enriched with