Semisynthesis, cytotoxicity, antimalarial evaluation and structure-activity relationship of two series of triterpene derivatives

Article abstract of DOI:10.1016/j.bmcl.2017.12.060

In this report, we describe the semisynthesis of two series of ursolic and betulinic acid derivatives through designed by modifications at the C-3 and C-28 positions and demonstrate their antimalarial activity against chloroquine-resistant P. falciparum (W2 strain). Structural modifications at C-3 were more advantageous to antimalarial activity than simultaneous modifications at C-3 and C-28 positions. The ester derivative, 3β-butanoyl betulinic acid (7b), was the most active compound (IC₅₀ = 3.4 μM) and it did not exhibit cytotoxicity against VERO nor HepG2 cells (CC₅₀ > 400 μM), showing selectivity towards parasites (selectivity index > 117.47). In combination with artemisinin, compound 7b showed an additive effect (CI = 1.14). While docking analysis showed a possible interaction of 7b with the Plasmodium protease PfSUB1, with an optimum binding affinity of ?7.02 kcal/mol, the rather low inhibition displayed on a Bacillus licheniformis subtilisin A protease activity assay (IC₅₀ = 93 μM) and the observed accumulation of ring forms together with a delay of appearance of trophozoites in vitro suggests that the main target of 3β-butanoyl betulinic acid on Plasmodium may be related to other molecules and processes pertaining to the ring stage. Therefore, compound 7b is the most promising compound for further studies on antimalarial chemotherapy. The results obtained in this study provide suitable information about scaffolds to develop novel antimalarials from natural sources.

Full text of DOI:10.1016/j.bmcl.2017.12.060

Accepted Manuscript
Semisynthesis, cytotoxicity, antimalarial evaluation and structure-activity rela-
tionship of two series of triterpene derivatives
Simone Tasca Cargnin, Andressa Finkler Staudt, Patrícia Medeiros, Daniel de
Medeiros Sol Sol, Ana Paula de Azevedo dos Santos, Fernando Berton Zanchi,
Grace Gosmann, Antonio Puyet, Carolina Bioni Garcia Teles, Simone Baggio
Gnoatto
PII:
S0960-894X(17)31234-9
https://doi.org/10.1016/j.bmcl.2017.12.060
BMCL 25516
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 October 2017
18 December 2017
25 December 2017
Please cite this article as: Cargnin, S.T., Staudt, A.F., Medeiros, P., de Medeiros Sol Sol, D., de Azevedo dos Santos,
A.P., Zanchi, F.B., Gosmann, G., Puyet, A., Garcia Teles, C.B., Gnoatto, S.B., Semisynthesis, cytotoxicity,
antimalarial evaluation and structure-activity relationship of two series of triterpene derivatives, Bioorganic &
Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.12.060
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Semisynthesis, cytotoxicity, antimalarial evaluation and structure-activity
relationship of two series of triterpene derivatives
Simone Tasca Cargnina, Andressa Finkler Staudta, Patrícia Medeirosb, Daniel
de Medeiros Sol Solb, Ana Paula de Azevedo dos Santosb, Fernando Berton
Zanchic, Grace Gosmanna, Antonio Puyetd, Carolina Bioni Garcia Telesb,
Simone Baggio Gnoattoa, *
Affiliations
aLaboratório de Fitoquímica e Síntese Orgânica, Faculdade de Fármacia,
UFRGS, Porto Alegre, RS, Brazil.
simonetc@gmail.com; dessastaudt@gmail.com; grace.gosmann@ufrgs.br;
simone.gnoatto@ufrgs.br.
bPlataforma de Bioensaios de Malária e Leishmaniose, FIOCRUZ, Porto Velho,
RO, Brazil.
carolinateles@fiocruz.br; patriciasmmedeiros@yahoo.com.br;
cLaboratório de Bioestatística e Bioinformática, FIOCRUZ, Porto Velho, RO,
Brazil.
fbzanchi@fiocruz.br
dDepartamento de Bioquímica
Complutense de Madrid, Spain.
apuyet@ucm.es
y
Biología Molecular IV, Universidad
* Corresponding author: Dr. Simone Baggio Gnoatto
Laboratório de Fitoquímica e Síntese Orgânica, Faculdade de Farmácia,
UFRGS, Porto Alegre, Brazil;
Tel.: +55 51 3308-5451; Fax: +55 51 3308-5313.
E-mail address: simone.gnoatto@ufrgs.br

Abstract
In this report, we describe the semisynthesis of two series of ursolic and
betulinic acid derivatives through designed by modifications at the C-3 and C-
28 positions and demonstrate their antimalarial activity against chloroquine-
resistant P. falciparum (W2 strain). Structural modifications at C-3 were more
advantageous to antimalarial activity than simultaneous modifications at C-3
and C-28 positions. The ester derivative, 3β-butanoyl betulinic acid (7b), was
the most active compound (IC50 = 3.4 µM) and it did not exhibit cytotoxicity
against VERO nor HepG2 cells (CC50 > 400 µM), showing selectivity towards
parasites (selectivity index > 117.47). In combination with artemisinin,
compound 7b showed an additive effect (CI = 1.14). While docking analysis
showed a possible interaction of 7b with the Plasmodium protease PfSUB1,
with an optimum binding affinity of -7.02 kcal/mol, the rather low inhibition
displayed on a Bacillus licheniformis subtilisin A protease activity assay (IC50 =
93 µM) and the observed accumulation of ring forms together with a delay of
appearance of trophozoites in vitro suggests that the main target of 3β-
butanoyl betulinic acid on Plasmodium may be related to other molecules and
processes pertaining to the ring stage. Therefore, compound 7b is the most
promising compound for further studies on antimalarial chemotherapy. The
results obtained in this study provide suitable information about scaffolds to
develop novel antimalarials from natural sources.
Keywords: Antimalarial; betulinic acid; Plasmodium falciparum; semisynthesis;
ursolic acid.

Malaria is a devastating disease that remains a significant public health
problem worldwide. There are more than 198 million cases of malaria and
584,000 deaths annually, mainly affecting children under 5 years of age.1
Among the five protozoan species of Plasmodium that cause human malaria,
Plasmodium falciparum is the most virulent, responsible, by far, for the greatest
morbidity and mortality, with several hundred million cases of clinical malaria
and deaths.1,2
The antimalarial drugs used for treatment depend on the species of malaria
parasite causing the infection, where the infection was acquired, pregnancy
status, and the severity of infection.1,2 Chloroquine (CQ) was the antimalarial of
choice for several decades due to its efficiency, safety, tolerance and low cost.
Nevertheless, because of its irrational use, CQ treatment has been a failure,
and the use of CQ to treat falciparum malaria is restricted to just a few
countries.1,3 Currently, the first-line treatment of uncomplicated malaria caused
by P. falciparum is artemisinin combination therapy (ACT).1 Combination
therapy has been adopted to prevent drug-resistant parasites; however, the
emergence and spread of drug-resistant malaria, mainly with artemisinin’s
efficacy declining, has been a major problem that hinders the control of
malaria.1,4,5
The high morbidity and mortality caused by malaria, the lack of licensed
vaccines, and the widespread resistance to artemisinin, together with the lack
of drugs safely administered to children and pregnant women are, in general,
the main issues that point to the real and critical requirement for discovering
new antimalarial medications.1,5,6 Despite the advances in malaria research
and the great efforts towards developing new effective and safe drugs, the
majority remain very limited in their mechanisms of action, and until now, no
innovative drug is commercially available, proving that more studies and novel
lead chemotypes are urgently needed.5,6
In the context of improving therapy against malaria infection, natural
products could be a potential source of new classes of drugs with high activity
and low toxicity, which can be further optimized by chemical modifications.7,8
Ursolic acid (UA) and betulinic acid (BA) (Fig. 1) are pentacyclic triterpenoids

extracted from several natural sources and have been reported to possess a
broad and promising spectrum of biological activities, including antimicrobial,
anticancer, and anti-inflammatory characteristics.9–11
Figure 1 - Chemical structures of ursolic acid (UA) and betulinic acid (BA).
(Source: ChemDraw Ultra® Software)
The antimalarial activity of these compounds and their derivatives was
previously assessed by our group, and they demonstrated to be promising lead
compounds. The UA and BA analogues bearing an acetyl group at C-3 and
piperazine moiety at C-28 were active on CQ-resistant (FcB1) and CQ-
sensitive (3D7) P. falciparum strains.12,13 Afterwards, two series of UA and BA
derivatives possessing several ester substituents at C-3 were evaluated
against CQ-sensitive (3D7) P. falciparum, and the derivatives with shorter side-
chains were more active.14 Two inactive BA-group compounds were selected
to verify the importance of C-3 groups on cytotoxicity when C-28 is linked to
piperazine. In both compounds, the piperazine moiety significantly increased
the antiplasmodial activity, showing satisfactory in vitro selectivity.15 However,
these compounds presented some in vivo toxicity attributed to the piperazine
moiety (unpublished data).
The triterpenes UA and BA were obtained by our group from natural
sources from southern Brazil, apple pomace (Malus domestica) and Platanus
acerifolia bark, with good yields (3.5% and 1.5%, respectively) using simple
and low-cost sources and methodologies.13,16 A large number of structural
modifications in the triterpenes are possible. Here, the semisynthesis

processes were focused at the C-3 and C-28 positions, which are available
sites for feasible chemical modifications in these structures. In both triterpenes,
the hydroxyl group at the C-3 position was acylated using acetic (1a, 1b)13,
butyric (7a, 7b)14 and isobutyric anhydrides (10a, 10b)14. Two other derivatives
were obtained through the oxidation of this hydroxyl group (4a, 4b)17, totaling
eight derivatives with a substitution at the C-3 position alone. Diverse natural
species have antimalarial and concomitant anticancer activity. Knowing that
they are unlikely to be biologically closely related because malaria involves
protozoa parasite and cancer involves aberrant mammalian cells, it is equally
unlikely that they share the same disease target mechanisms.21 Due to the
cytotoxic profile of methyl and imidazole groups on different cancer cells
lines18–20, starting with C-3 modified triterpene derivatives (esters or ketone
groups) the C-28 carboxylic acid was replaced by methyl (2a, 2b; 5a, 5b; 8a,
8b; 11a,11b) and imidazole moieties (3a, 2b; 6a, 6b; 9a, 9b; 12a, 12b).
Therefore, twenty-four compounds in two groups of derivatives were
successfully prepared using triterpene skeletons (Scheme 1).
Scheme 1 - Synthesis of derivatives 1a - 12b. Reagents and conditions: (a)
dichloromethane, acetic anhydride, pyridine, rt., 24hs; (b) acetone, Jones
Reagent at 0 °C,
rt., 3hs; (c) dichloromethane, butyric anhydride, DMAP, rt.,
24hs; (d) dichloromethane, isobutyric anhydride, DMAP, rt., 24hs (e)

dichloromethane, K2CO3, iodomethane, N2 atmosphere, 55 °C, 48h; (f)
dichloromethane, oxalyl chloride, N2 atmosphere, 0 °C, 3h. - trimethylamine, 0
°C - imidazole, rt., 24hs. (Source: ChemDraw Ultra® Software)
P. falciparum CQ-resistant (W2 strain - IC50 CQ = 0.156 µM) was utilized in
the in vitro evaluation. The parasite culture was performed as previously
described by Trager and Jansen,22 and was maintained in fresh human
erythrocytes (O+ blood) suspended at a 2% hematocrit in complete medium at
37 °C. The use of human blood was approved by the UFRGS Research Ethics
Committee, under protocol number 1.242.369. An anti-P. falciparum assay was
performed,23 and the half-maximal drug inhibitory response (IC50) was
estimated by curve fivefold dilution, ranging from 100 – 3.12 µM, using
software from OriginLab Corporation®. The results were compared with drug-
free control wells (100% parasite viability), and artemisinin was tested in
parallel as an antimalarial control. In addition, the effects of 7b on the growth of
W2 P. falciparum were assessed in order to verify the plasmodial stages
affected by the compound. The test was performed with some modifications
that were described by Moneriz et al. (2011)24, using a parasitaemia of 2%. As
mentioned, the current treatment regimen for malaria relies on the concept of
artemisinin-based combination therapy (ACT). Therefore, given the consensus
that combination therapy is essential to the optimal control of malaria and the
attractiveness of further development of combinations,25 the in vitro antimalarial
efficacy of the combinations of artemisinin and triterpene compounds (7b and
7a) was assessed and the combination index (CI), which is the sum of the two
fractional inhibitory concentrations (FIC), was calculated.26 The in vitro
cytotoxicity was determined for 24, 48 and 72h, using VERO (African Green
Monkey Kidney, ATCC CCL-81) and HepG2 (Human Hepatocellular
Carcinoma, ATCC HB-8065) cells,27 and the concentration that inhibited cell
growth by 50% (CC50) was determined by curve eightfold dilution, ranging from
400 – 1.56 µM. The selectivity indexes (SI) were calculated in relation to
HepG2 cells (SI = CC50/IC50). The structure-activity relationship (SAR) study of
these two series of compounds was then evaluated. All the experiments were
performed in triplicate and with at least three independent cultures (n = 3).

The derivatives were evaluated against two cells lines: mammalian cells
(VERO) and hepatocellular cancer cells (HepG2). HepG2 cells show similarity
to human liver cells, and can be useful as an alternative model in the screening
for cytochrome P450 (CYP450) enzyme inducers early in the drug discovery
process.28 For this reason, it is very important to assess a drug’s cytotoxicity
not just in mammalian, but also in HepG2 cells. The CC50 concentration of the
compounds, excluding UA and 4b, was > 400 µM for both cells lines,
presenting, therefore, low toxicity in the tested conditions. UA presented
inexpressive antimalarial activity (IC50 = 50.64 µM) and cytotoxicity against
VERO (CC50 24h = 98.08 µM; CC50 48h = 101.64; CC50 72h = 99.91), and
HepG2 cell lines (CC50 24h = 101.32 µM; CC50 48h = 100.44 µM; CC50 72h =
113.62 µM), thus being nonselective (SI = 2.24). The oxidized derivative, 4b,
presented cytotoxicity on the HepG2 cell line alone after 72h (CC50 72h =
150.50 µM). Therefore, considering its antimalarial activity, it was selective
towards the parasites (SI = 19.12) (Table 1).
The IC50 values of the derivatives against P. falciparum (W2) were in the
range of 3.4 and > 100 µM (Table 1). The compound modified at the C-3
position alone, 3β-butanoyl betulinic acid (7b), was the most active compound
(IC50 = 3.4 µM), corroborating with that previously reported on a CQ-sensitive
strain (IC50 = 5.0 µM).14 Compound 7b was the most active against CQ-
resistant P. falciparum (W2), and was also the most active against the sensitive
strain (3D7), which elucidates its effectiveness as an antimalarial agent and
suggests low levels of cross-resistance to CQ. Taking into account the
resistance index, calculated from the ratio of the IC50 value of the sensitive and
resistant strains of P. falciparum (W2/3D7), the ratio found for compound 7b
was 0.68, lower than that observed for CQ (4.01) (data not shown).
The butyric and isobutyric ester derivatives (7a, 7b; 10a, 10b) possess the
same number of carbons in the side-chain at the C-3 position (four-carbons);
nevertheless their spatial conformations are completely different, reflecting in
different fits for the molecular targets, and consequently, different profiles of
antimalarial activity (Table1). This fact, however, did not affect their cytotoxicity
towards VERO and HepG2 cells.

A ketone moiety at the C-3 position was also active, especially oxidized BA
(4b) (IC50 = 7.87 µM). Interestingly, the oxidation, which exchanges a donor
grouping for a hydrogen bond acceptor, did not demonstrate a strong
difference in antimalarial activity, but after 72 h, showed some cytotoxic activity
against HepG2 cells. On the other hand, a functional group at C-28 that is
capable of acting as a hydrogen bond donor (COOH) was most important for
the antimalarial activity, while the presence of a group that cannot donate a
hydrogen bond (methyl group - COOCH3) was not favorable at this position,
annulling the activity (IC50 > 100 µM). For these compounds, the cytotoxic
potential was not affected. Although the imidazole ring did not affect the
cytotoxicity, it showed interesting behavior in its antiplasmodial activity,
sometimes increasing or decreasing the IC50 value of the initial scaffold (Table
1). The methyl group is acyclic, while imidazole is an aromatic heterocycle. The
spatial structures of the compounds are, therefore, different, and could
influence their biological activity.
It is generally accepted that biological efficacy is not due to in vitro
cytotoxicity when SI ≥ 10.29 Twelve compounds showed selectivity indexes ≥
10 (Table 1), which indicated high selectivity indexes for these compounds
towards parasites. It is worth mentioning that compound 7b presents the best
selectivity index (> 117.47). Combination interactions of triterpene derivatives
(7a, 7b) and artemisinin were done using the “1 point” Chou – Talalay
approach, which is used to investigate the possible synergy at a minimum of
one combination ratio.26 A combination index (CI) ≤ 1 was considered to
indicate synergy, 1 < CI ≤ 2 additivity and CI > 2 antagonism.26,30 The effect of
7a and 7b with artemisinin was additive, with CI = 1.05 and CI = 1.34,
respectively (Fig. 2). Moreover, the dose reduction indexes (DRI) were
favorable,31 almost 7.0 and 4.0 for 7a and 7b, respectively, and approximately
1.0 for artemisinin. Artemisinin’s DRI indicates that 7a and 7b did not affect its
antimalarial activity, but artemisinin decreased the dose of 7b by almost 7 and
4-fold in order to achieve the same therapeutic effect.

Table 1 – Antiplasmodial and cytotoxic activity of compounds UA, 1a-12a;
BA, 1b-12b. The results represent three independent experiments (n=3).

Figure 2 - Normalized isobologram of triterpene derivatives. (A)
Isobologram of 7a; (B) Isobologram of 7b. (Source: Rstudio® Program)
The incubation of synchronized cultures of P. falciparum (W2) with 7b at
100 µM prevents an increase in parasitaemia, leading to parasite cell death if
the compound is present for up to 72 hours (Fig. 3). The addition of 7b at ring-
stage results in the accumulation of ring forms and the delayed appearance of
trophozoites, suggesting that its inhibition occurs as early as the ring stage.
However, the compound, as well as other compounds of the terpene class,
may display multiple-target activity, affecting sequential processes occurring at
ring-trophozoite and schizont stages.32

Figure 3 - A. Total parasitaemia reduction after 24, 36, 48 and 72 hours of
treatment with compound 7b; B. Effect of 7b treatment on different P.
falciparum erythrocytic stages: (A) Control; (B) Artemisinin; (C) Compound 7b
(100 µM). Bars show the percentage of rings (grey); trophozoites (black) and
schizonts (hatched) forms observed after 24, 36, 48 and 72 hours of treatment
with compound 7b. Data collected by microscopic inspection of smears
accounts for a total of 1,000 erythrocytes.

In order to identify another possible parasite target of the derivatives, in
silico docking analysis of two selected compounds (7b and 9b) in Plasmodium
falciparum subtilisin-like serine protease-1 (PfSUB1) were performed using
AutoDock 4.2.33 This enzyme has emerged as a promising antimalarial drug
target, since it is a key enzyme in both erythrocytes egress and invasion
processes15 and it has been reported as a target for other pentacyclic
triterpenoids.32 7b showed optimum binding affinity with a molecular target,
with a binding energy of −7.02 kcal/mol as compared to MRT12113 (-6.32
kcal/mol), a highly selective inhibitor of PfSUB134 and the carboxylic acid and
ester group of seems to be important for the activity in this target. Since the in
vitro antimalarial activity of 7b was seventeen-fold higher than the activity of
the compound with an imidazole ring (9b), we expected and found a worse
binding energy (-5.87 kcal/mol). Therefore, 7b has more affinity for PfSUB1
than 9b (Fig. 4). To analyze the possible inhibition of 7b on PfSUB1 activity, an
in vitro assay was performed using the closely related Bacillus licheniformis
subtilisin A. The inhibition kinetics yielded a rather high IC50 = 93 µM (Fig. 5)
suggesting that either 7b targets other proteins and processes of the
intraerithrocytic cycle of Plasmodium, or the conditions in vivo differ from the
assay with a higher binding affinity. Docking studies simulate the binding of a
flexible ligand to a rigid biological receptor and not foresee all other factors that
are involved.35 It may also not be discarded that 7b may show higher specificity
for PfSUB1 than for subtilisin A.

Figure 4 - Result of docking between PfSUB1 from P. falciparum (PDB code
4LVN) (orange-red ribbons) and compound 7b (predominantly light blue and
stick representation). The closest amino acids were highlighted. The dashed
green lines are hydrogen bonds between the target and 7b. (Source: UCSF
Chimera®)
Figure 5 - Dose-response curve of compound 7b on B. licheniformis subtilisin A
(Sigma-Aldrich). The protease inhibition assay was performed by using
EnzCheck Peptidase/protease assay kit form Molecular Probes as indicated by
manufacturer, with concentration of 7b ranging from 5 to 0.01 mM. The resulting
data was plotted as fluorescence vs. log 7b concentration. (Source: GraphPad®
software)

Two series of UA and BA derivatives were successfully prepared with the
purpose of investigating the structure-activity relationship of the compounds. It
is possible to conclude that compounds that were acylated and oxidized at C-3
presented relevant activity, and the insertion of an imidazole ring at C-28
seems to be not favorable to activity, increasing or not increasing the IC50
value. In contrast, the insertion of a methylester group at C-28 abolished the
antimalarial activity. None of the compounds presented significant cytotoxic
activity against mammalian and hepatic cells. The BA derivative modified at the
C-3 position alone, 3β-butanoyl betulinic acid (7b), presented the best in vitro
antimalarial activity against CQ-resistant P. falciparum (W2), being 3.76-fold
more effective than its natural precursor. Similarly, this potent activity was
previously demonstrated effective against a strain of CQ-sensitive P.
falciparum (3D7), suggesting that the action is not affected by the CQ
resistance mechanism. Moreover, 7b was featured in the in vitro activity,
showing high selectivity towards parasites (SI > 117.47), and presenting an
additive effect when associated with artemisinin. Compound 7b probably has
multiple-target activity, leading to the accumulation of ring forms and the
delayed appearance of trophozoites in vitro, and in silico, suggesting
interacting with PfSUB1; however, it was not able to inhibit subtilisin’s activity in
vitro. Therefore, the results obtained in this study could provide suitable
information for the development of new antimalarial compounds derived from
natural sources.
Acknowledgements
S.T.C. and A.F.S. performed the reactions and analysis of the compounds;
S.T.C, D.M.S., A.P.A.S., C.B.G.T. and A.P. carried out the biological assays;
F.B.Z performed the docking analysis; P.S.M., G.G. and S.B.G. coordinated the
study and provide financial support; and S.T.C. and S.B.G. drafted and edited
the manuscript.

Funding
The authors are grateful to the Brazilian agencies CAPES and CNPq for the
financial support and student's fellowships, and LRNANO (UFRGS) for spectra
data. The authors are also grateful to the Graduate Program in Pharmaceutical
Sciences – UFRGS and Malaria and Leishmaniasis Bioassays Platform –
Fiocruz (Rondônia).
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Figure Captions
Figure 1 - Chemical structures of ursolic acid (UA) and betulinic acid (BA).
(Source: ChemDraw Ultra® Software)
Figure 2 - Normalized isobologram of triterpene derivatives. (A) Isobologram of
7a; (B) Isobologram of 7b. (Source: Rstudio® Program)
Figure 3 - A. Total parasitaemia reduction after 24, 36, 48 and 72 hours of
treatment with compound 7b; B. Effect of 7b treatment on different P.
falciparum erythrocytic stages: (A) Control; (B) Artemisinin; (C) Compound 7b
(100 µM). Bars show the percentage of rings (grey); trophozoites (black) and
schizonts (hatched) forms observed after 24, 36, 48 and 72 hours of treatment
with compound 7b. Data collected by microscopic inspection of smears
accounts for a total of 1,000 erythrocytes.
Figure 4 - Result of docking between PfSUB1 from P. falciparum (PDB code
4LVN) (orange-red ribbons) and compound 7b (predominantly light blue and
stick representation). The closest amino acids were highlighted. The dashed
green lines are hydrogen bonds between the target and 7b. (Source: UCSF
Chimera®)
Figure 5 - Dose-response curve of compound 7b on B. licheniformis subtilisin A
(Sigma-Aldrich). The protease inhibition assay was performed by using
EnzCheck Peptidase/protease assay kit form Molecular Probes as indicated by
manufacturer, with concentration of 7b ranging from 5 to 0.01 mM. The resulting
data was plotted as fluorescence vs. log 7b concentration. (Source: GraphPad®
software)
Scheme 1 - Synthesis of derivatives 1a - 12b. Reagents and conditions: (a)
dichloromethane, acetic anhydride, pyridine, rt., 24hs; (b) acetone, Jones
Reagent at 0 °C,
rt., 3hs; (c) dichloromethane, butyric anhydride, DMAP, rt.,
24hs; (d) dichloromethane, isobutyric anhydride, DMAP, rt., 24hs (e)
dichloromethane, K2CO3, iodomethane, N2 atmosphere, 55 °C, 48h; (f)
dichloromethane, oxalyl chloride, N2 atmosphere, 0 °C, 3h. - trimethylamine, 0
°C - imidazole, rt., 24hs. (Source: ChemDraw Ultra® Software)

Table Captions
Table 1 – Antiplasmodial and cytotoxic activity of compounds UA, 1a-12a; BA,
1b-12b. The results represent three independent experiments (n=3).

Graphical abstract