New antimalarial and cytotoxic 4-nerolidylcatechol derivatives

Article abstract of DOI:10.1016/j.ejmech.2008.10.025

4-Nerolidylcatechol (1) was isolated from cultivated Pothomorphe peltata root on a multigram scale using straight-forward solvent extraction-column chromatography. New semi-synthetic derivatives of 1 were prepared and tested in vitro against multidrug-res

Full text of DOI:10.1016/j.ejmech.2008.10.025

European Journal of Medicinal Chemistry 44 (2009) 2731–2735
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
New antimalarial and cytotoxic 4-nerolidylcatechol derivatives
,
b
c
d
Ana Cristina da Silva Pinto a , Luis Francisco Rocha Silva , Bruno Coelho Cavalcanti , Marcia Rubia
´
´
b
e
d
d
´
´
Silva Melo , Francisco Celio Maia Chaves , Letıcia Vera Costa Lotufo , Manoel Odorico de Moraes ,
Valter Ferreira de Andrade-Neto f, Wanderli Pedro Tadei g, Claudia O. Pessoa d, Pedro Paulo
Ribeiro Vieira c, Adrian Martin Pohlit b
,
*
a
´
Universidade Federal do Amazonas, Campus Universitario, 69077-000 Manaus, AM, Brazil
b Laborato´rio de Princ´ıpios Ativos da Amazoˆnia, Coordenaç˜ao de Pesquisas em Produtos Naturais, Instituto Nacional de Pesquisas da Amazoˆnia, 69060-001 Manaus, AM, Brazil
c
´
˜
Laborato´rio da Gereˆncia de Malaria, Fundaçao de Medicina Tropical do Amazonas, 69040-000 Manaus, AM, Brazil
d Laboratorio de Oncologia Experimental, Universidade Federal do Ceara, 60431-970 Fortaleza, CE, Brazil
e Embrapa Amazonia Ocidental, 69010-970 Manaus, AM, Brazil
f Departamento de Microbiologia e Parasitologia, Universidade Federal do Rio Grande do Norte, 59072-970 Natal, RN, Brazil
g
´
´
˜
ˆ
´
ˆ
Laboratorio de Malaria e Dengue, Coordenaçao de Pesquisas em Ciencias da Saude, Instituto Nacional de Pesquisas da Amazonia, 69060-001 Manaus, AM, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
4-Nerolidylcatechol (1) was isolated from cultivated Pothomorphe peltata root on a multigram scale using
straight-forward solvent extraction-column chromatography. New semi-synthetic derivatives of 1 were
prepared and tested in vitro against multidrug-resistant Plasmodium falciparum K1 strain. Mono-O-methyl,
Received 14 June 2008
Received in revised form
16 October 2008
Accepted 20 October 2008
Available online 31 October 2008
mono-O-benzyl, O,O-dibenzyl and O,O-dibenzoyl derivatives 2–8 exhibited IC50 in the 0.67–22.52 mM
range. Mono-O-methyl ethers 6 and 7 inhibited the in vitro growth of human tumor cell lines HCT-8 (colon
carcinoma), SF-295 (central nervous system), LH-60 (human myeloblastic leukemia) and MDA/MB-435
(melanoma). In general, derivatives 2–8 are more stable to light, air and pH at ambient temperatures than
their labile, natural precursor 1. These derivatives provide leads for the development of a novel class of
antimalarial drugs with enhanced chemical and pharmacological properties.
Keywords:
Plasmodium falciparum
Human colon carcinoma
Human nervous system cancer
Melanoma
Ó 2008 Elsevier Masson SAS. All rights reserved.
Human myeloblastic leukemia
Semi-synthetic 4-nerolidylcatechol
derivatives
1. Introduction
vitro cytotoxic activity towards tumor cell lines as compared to 1 [7].
The only other O-substituted derivatives of 1 known in the literature
Pothomorphe spp. (caapeba, pariparoba) are traditionally used
plants for the treatment of malaria in the form of teas [1]. In vitro
and in vivo antimalarial activities of Pothomorphe spp. extracts
have been reported in Refs. [2–6] and we recently showed that
4-nerolidylcatechol (1), isolated from dried, ground Pothomorphe
peltata roots, presents significant in vitro cytotoxic [7] and anti-
plasmodial activities [8].
Compound 1 is labile under ordinary laboratory conditions or in
(alkaline) solution due a priori to the presence of highly reactive
catechol hydroxyls and the aromatic ring activating quaternary
carbon of the nerolidyl side chain. Early experience showed that
compound 1 underwent O,O-diacetylation in good yield. The
resulting diacetate exhibited greater stability and modulation of in
came as a result of an 8-step racemic synthesis of O,O-dimethyl-4-
nerolidylcatechol as part of studies on structural elucidation of 1 [9].
There is no information available on the biological activity of the
latter racemic mixture. On this basis and pursuing our interest in the
discovery of new anticancer and antimalarial agents, we have
recently focused our attention on semi-synthetic mono- and di-O-
benzyl, O-benzoyl and O-methyl 4-nerolidylcatechol derivatives,
prepared in simple one- or two-step procedures starting from 1, as
promising cytotoxic and antimalarial agents with greater inherent
stability than their natural precursor. In the present study, new
semi-synthetic derivatives 2–8 were synthesized and their in vitro
antimalarial activity towards the human malaria parasite Plasmo-
dium falciparum K1 strain, as well as in vitro cytotoxic activity
towards 4 human cell lines, were investigated. Also, structurally
analogous catechol (9) and nerolidol (10) were tested to provide
further insight into what may be important carbon skeleton/
connectivity features related to antimalarial and cytotoxic activities.
* Corresponding author. Tel.: þ55 92 3643 3177; fax: þ55 92 3643 3176.
E-mail address: ampohlit@inpa.gov.br (A.M. Pohlit).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.10.025

2732
A.C.S. Pinto et al. / European Journal of Medicinal Chemistry 44 (2009) 2731–2735
2. Chemistry
O
O
4-Nerolidylcatechol (1) was isolated on a multigram scale from
dried, ground roots of P. peltata (cultivated at Embrapa Amazonia
Ocidental in Manaus, Amazonas State, Brazil) using straight-
forward solvent extraction-column chromatography steps
which have been previously described [8]. In general, the 4-
nerolidylcatechol derivatives prepared (patent solicited 12/2007,
PCT/BR2007, 0003375) took advantage of the reactive catechol
hydroxyl groups present in 1 yielding mono- and di-O-derivatized
compounds depending upon the specific reactions employed. The
structures of derivatives were established based on spectroscopic
analyses. Structurally, these O-substituted derivatives offer
a simple range of steric, functional and electronic differences
around the aromatic nucleus which in comparison to 1, catechol
and nerolidol provide insight into qualitative structure–activity
requirements for both in vitro anti-plasmodial as well as in vitro
cytotoxic effects.
1 R1 = R2 = H
2 R1 = R2 = Bn
3 R1 = Bn; R2 = H
4 R1 = H; R2 = Bn
5 R1 = R2 = Bz
6 R1 = H; R2 = Me
7 R1 = Me; R2 = H
OR2
OAc
8
OR1
OAc
Fig. 1. Structures of 4-nerolidylcatechol (1), semi-synthetic derivatives 2–8 and
structural analogues, catechol (9) and nerolidol (10).
Four of the seven 4-nerolidylcatechol derivatives exhibited
significant in vitro anti-plasmodial activities (IC50 < 2 mg/mL).
Dibenzoyl derivative 5 was the most active antimalarial derivative
and presented an IC50 comparable to that of chloroquine and
natural precursor 1. Monobenzyl derivatives 3 and 4, and diepoxy
diacetyl mixture 8 exhibited important inhibition (IC50 ¼ 2.8–
3. Results and discussion
4.0 mM) while monomethyl derivative 7 was only partially active
and its structural isomer 6 was inactive.
In previous work, 4-nerolidylcatechol (1) was shown to exhibit
cytotoxic potential in five tumor cell lines with IC50 ranging from 6
Pure 4-nerolidylcatechol (1) is labile in ambient light, air, room
temperatures and in solution under mild alkaline conditions.
Within a few minutes to hours 1 in CD3OD/K2CO3 is converted to
a mixture of higher and lower polarity products having vinylic and
allylic hydrogens corresponding to internal olefin groups of the
terpenyl side chain, but no aromatic or terminal olefin hydrogens,
according to TLC and NMR analysis (A.M. Pohlit, A.C.S. Pinto,
unpublished results). This is evidence for the oxidation of the
catechol nucleus of 1 under alkaline conditions. There is also
evidence that oxidation of 1 may preferentially involve single
electron transfer and/or formation of radical intermediates. In this
vein, a quantitative study on the antiradical capacity of several
natural catechols and polyphenols revealed that 1 anomalously
underwent oxidation involving only one hydroxyl group (presum-
ably through loss of 1Hþ þ 1eꢀ per molecule of 1) whereas poly-
phenols in general were oxidized proportionally to the number (n)
of hydroxyl and alkyloxyl substituent groups (loss of nHþ þ neꢀ per
molecule) present on the aromatic nucleus [10]. This result is also
reproducible in other common systems used for quantitative
evaluation of antioxidant behavior, such as ABTSþ where rapid
oxidation kinetics and a TEAC of 0.50 are observed in aerobic and
anaerobic conditions, whereas other catechols exhibit the expected
TEAC value of 1 (D. Rettori, A.M. Pohlit, Pinto et al., unpublished
data). No conclusive mechanistic interpretation is available for
these results at the moment, but rapid formation of radical inter-
mediates might be a feasible explanation for these data and the
general lability of 1 under a number of different conditions.
New mono- and di-O-substituted 4-nerolidylcatechol derivatives
2–8 exhibit greater stability in general than 4-nerolidylcatechol, as
would be expected, given the lower number (or absence) of phenol
groups in these derivatives. Despite the inert atmosphere used
during reactions, decomposition of 4-nerolidylcatechol (discussed
above) appears to be a factor under the mildly alkaline conditions
used in benzylation, benzoylation and methylation (with methyl
iodide), as well as methylation with diazomethane, leading to
relatively low yields.
to 13
m
g/mL [7]. Monomethyl derivative 6 presented good cytotoxic
g/mL) in human colon carcinoma,
potential (IC50, 14.4–20.7
m
central nervous system and melanoma tumor cell lines while its
structural isomer 7 inhibited human leukemic cells. Dibenzyl,
monobenzyl and dibenzoyl derivatives 2, 3 and 5, respectively,
were essentially inactive towards all tumor cell lines. Among
derivatives, cytotoxic potential was associated with small O-
substituent groups, such as CH3 and CH3CO. This is further
corroborated by the significant in vitro inhibition exhibited by
another derivative, O,O-diacetyl-4-nerolidylcatechol, to human
leukemia cell strains HL-60 and CEM [IC50, 6.17 (4.74–8.03) and 6.22
(4.83–8.02)
1.08–13.54
m
g/mL, respectively] [7]. The inherent cytotoxicity (IC50
,
m
g/mL) exhibited by catechol (9) in three tumor cell
lines is noteworthy as is the general absence of cytotoxicity
Table 1
In vitro IC50 values of 4-nerolidylcatechol (1), its semi-synthetic derivatives 2–8 and
compounds 9 and 10 to the K-1 strain of Plasmodium falciparum.
Compounds
P. falciparum
Mean IC50 values
mg/mL
mM
0.67a
(0.61–0.73)
22.52
(17.92–27.12)
3.86
(3.78–3.94)
2.84
(2.44–3.24)
0.67
(0.58–0.76)
–
–
1
2
3
4
5
0.21a
(0.19–0.4)
11.14
(8.85–13.43)
1.56
(1.48–1.64)
1.15
(0.95–1.35)
0.35
(0.3–0.4)
Ib
6
7
8
PAb
1.70
3.95
(3.63–4.27)
80.65
(1.56–1.84)
8.88
The antimalarial activity of semi-synthetic derivatives 2–8
(Fig. 1) was determined as the percentage reduction of parasite
growth versus untreated controls (Table 1). The concentration of
each derivative required for reduction of parasite growth by 50%
was expressed as median inhibition concentrations (IC50) after
probit analysis. The cytotoxic activity of each compound was
determined as the percentage inhibition of tumor cell growth
caused by each derivative relative to the inhibition presented by
doxorubicin-treated controls (Table 2).
9
(5.88–11.88)
PAb
(53.45–107.85)
–
10
Chloroquine diphosphate
0.46
0.89
(0.86–0.92)
0.012
(0.44–0.48)
0.004
Quinine salt
(0.002–0.006)
(0.006–0.018)
a
Ref. [8].
b
I – inactive (<50% inhibition) or PA – partially active (50–79% inhibition) at 50
g/mL.
and 2.5
m

A.C.S. Pinto et al. / European Journal of Medicinal Chemistry 44 (2009) 2731–2735
2733
Table 2
5. Experimental
In vitro IC50 values to the relative activities in 4 human tumor cell lines of 4-ner-
olidylcatechol (1) and semi-synthetic derivatives 2–8 and compounds 9 and 10.
5.1. Chemistry
Compounds
Inhibition of tumor cell lines (
mg/mL)
1H and 13C NMR spectra were recorded on a Varian (500 MHz)
spectrometer using CDCl3 as solvent. Accurate mass (þ)-ESI-tof-MS
spectra were recorded on a Bruker-Daltronics UltrOTof Mass
Spectrometer by direct infusion of sample dissolved in suitable
solvents into the ion source. Column chromatography was per-
formed on silica using Kiesegel 60 (230–400 mesh, Merck). All
reagents used in the present work were of analytical grade.
HL-60
6.17a
(4.74–8.03)
I
HCT-8
12.17a
(8.38–17.67)
I
SF-295
MDA/MB-435
–
1
–
2
3
4
I
I
–
–
I
I
12.42
(10.41–15.62)
–
20.60
(17.70–23.99)
I
18.22
(16.64–19.95)
I
18.57
(15.92–21.67)
I
5
6
14.40
14.53
20.70
>25
(10.85–17.34)
18.84
(13.63–26.06)
17.41
(9.82–30.86)
1.08
(0.84–1.39)
–
(11.87–17.77)
>25
(15.57–27.51)
>25
5.1.1. Preparation of O,O-dibenzyl-4-nerolidylcatechol (2) and
mono-O-benzyl derivatives 3 and 4
7
8
9
>25
>25
>25
I
Semi-synthetic derivatives 2–4 (Fig. 1) were prepared from 1
(401.0 mg, 1.3 mmol) by treatment with C6H5CH2Br (432.0 mg,
2.5 mmol, 2 equiv), K2CO3 (705.4 mg, 5.1 mmol, 4 equiv), KI
(22.3 mg, 0.1 mmol), and DMF (2.4 mL) for 20–30 min under N2
(adapted from [17,18]). The reaction was stirred at room tempera-
ture for 18 h and then filtered. The filtrate was extracted with CHCl3
(3 ꢁ 5 mL). The combined extracts were washed with 1% NaOH
(5 mL) and water (5 mL), dried with MgSO4, filtered and concen-
trated using rotary evaporation. The product was purified by flash
column chromatography flash (using a 95:5 to 1:1 hexanes:AcOEt
gradient), yielding a mixture of benzylated derivatives 2 (slightly
viscous, yellowish-clear oil, 163.8 mg, 26.0%), 3 (viscous, clear oil,
46.1 mg, 7.3%) and 4 (viscous, clear oil, 40.6 mg, 6.4%) which were
separated by preparative TLC (hexanes:CHCl3, 1:4). 2: 1H NMR
17.47
(10.63–28.73)
13.54
(10.53–17.41)
I
0.04
22.85
(13.45–38.82)
12.92
(10.76–15.50)
10
I
Doxorubicin
0.02
(0.01–0.02)
0.25
(0.03–0.05)
I – inactive; – not tested.
a
Ref. [7].
observed for nerolidol (10). Based on these results, the catechol
moiety would appear to be relevant to the observed cytotoxicity in
4-nerolidylcatechol (1) and derivatives, while the nerolidyl side
chain would appear not to be a necessary structural element for the
observed cytotoxicity. In this light, it is interesting that synthetic 4-
salicylamidophenylalkyl catechols inhibit growth of murine colon
tumor cells in vitro, as well as 5-, 12- and 15-lipoxygenase in intact
cells and rabbit reticulocyte 15-lipoxygenase [11].
(500 MHz, CDCl3):
d 7.43–7.45 (m, 2H, OCH2Ph), 7.33–7.37 (m, 4H,
OCH2Ph), 7.27–7.31(m, 4H, OCH2Ph), 6.91 (m, 1H, Ar-H3), 6.32 (dd,
J ¼ 8.5, 2.0 Hz, 1H, Ar-H5) 6.87 (m, 1H, Ar-H6), 5.96 (dd, 1H, J ¼ 17.7,
10.5 Hz), 5.14 (s, 2H, OCH2Ph), 5.13 (s, 2H, OCH2Ph), 5.10 (m, 1H),
5.08 (m, 1H), 5.05 (dd, 1H, J ¼ 10.5, 1.5 Hz), 4.99 (dd, 1H, J ¼ 17.7,
1.5 Hz), 2.05 (m, 2H), 1.95 (m, 2H), 1.77 (m, 2H), 1.68 (s, 3H), 1.66 (m,
2H),1.60 (s, 3H),1.49 (s, 3H),1.30 (s, 3H). 13C NMR (125 MHz, CDCl3):
Nerolidol causes total inhibition of P. falciparum trophozoite
development at the schizont stage [12] and in vitro IC50 values in
the range 120–760 nM [13,14] have been reported for nerolidol
towards P. falciparum. Terpenoid compounds like nerolidol, farnesol
and linalol have been shown to strongly inhibit the biosynthesis of
both dolichol and the isoprenic side chain of ubiquinones, as well as
the isoprenylation of proteins in the intraerythrocytic stages of
P. falciparum in a specific manner, which does not affect overall
protein biosynthesis [14]. It is assumed that the observed inhibition
of P. falciparum by 4-nerolidylcatechol (1) and derivatives 2–8 is
related to a similar mechanism involving the nerolidyl side chain
eventhough in the present study, nerolidol (10) presented only
partial antimalarial activity (Table 1). Catechol (9) and nerolidol,
which exhibit carbon skeletal elements present in substances 1–8,
are, on a molar basis, much less active in vitro against P. falciparum
than 1, which leads to the supposition that the combined terpenyl
side chain and aromatic ring system of the nerolidylcatechyl
skeleton is important to the antimalarial activity of this class of
compound. In this vein, it is noteworthy that synthetic (mono- and
di-O-substituted) 4-allylcatechol derivatives (which share a 4-
(propen-3-yl)catechol subskeleton in common with compounds
1–8) have antimalarial properties [15]. Also, it is interesting that
catechol siderophores (ironchelators) such as dicatecholates with
long aliphatic chains (i.e. FR160) have similar in vitro IC50 values to
those observed for 4-nerolidylcatechol (1) and its derivatives 2–8 in
chloroquine resistant P. falciparum infected-erythrocytes [16].
d
148.3, 147.2, 147.0, 140.9, 137.5, 134.8, 131.3, 128.4, 128.3, 127.7,
127.6, 127.5, 127.3, 124.5, 124.3, 119.7, 115.1, 114.5, 111.5, 71.7
(OCH2Ph), 71.4 (OCH2Ph), 43.9, 41.1, 39.7, 26.7, 25.7, 24.9, 23.1, 17.7,
15.9. m/z 517.2958 [M þ Na]þ (C35H42NaO2þ, mcalc ¼ 517.3077). Rf
0.36 (hexanes:Et2O, 95:5). 3: 1H NMR (500 MHz, CDCl3):
d 7.38–
7.40 (m, 2H, OCH2Ph), 7.41–7.42 (m, 1H, OCH2Ph), 7.34–7.37 (m, 2H,
OCH2Ph), 6.94 (d, 1H, J ¼ 3.0 Hz, Ar-H3), 6.85 (d, 1H, J ¼ 8.5 Hz, Ar-
H6), 6.77 (dd, J ¼ 8.5, 2.5 Hz, 1H, Ar-H5), 6.00 (dd, 1H, J ¼ 10.7,
17.4 Hz), 5.58 (s,1H, OH), 5.12 (m,1H), 5.08 (s, 2H, OCH2Ph), 5.07 (m,
1H), 5.05 (dd, 1H, J ¼ 6.0, 1.2 Hz), 5.03 (dd, 1H, J ¼ 17.4, 1.2 Hz), 2.04
(m, 2H), 1.95 (m, 2H), 1.84 (m, 2H), 1.76 (m, 2H), 1.68 (s, 3H), 1.60 (s,
3H), 1.52 (s, 3H), 1.35 (s, 3H). 13C NMR (125 MHz, CDCl3):
d 147.0,
145.3, 143.8, 141.4, 136.5, 134.9, 131.3, 127.8, 128.7, 128.3, 124.5,
124.4, 113.5, 118.0, 111.6, 111.4, 71.2 (OCH2Ph), 43.8, 41.2, 39.7, 26.7,
25.7, 24.9, 23.2, 17.7, 15.9. m/z 405.2789 [M þ H]þ (C28H37O2þ,
mcalc ¼ 405.2788), Rf 0.36 (hexanes:Et2O, 9:1) 4: 1H NMR (500 MHz,
CDCl3):
d 7.41–7.42 (m, 1H, OCH2Ph), 7.38–7.40 (m, 2H, OCH2Ph),
7.34–7.36 (m, 2H, OCH2Ph), 6.89 (d, 1H, J ¼ 2.2 Hz, Ar-H3), 6.87 (d,
1H, J ¼ 8.2 Hz, Ar-H6), 6.83 (dd, J ¼ 8.2, 2.2 Hz, 1H, Ar-H5), 5.99 (dd,
1H, J ¼ 17.6, 11.7 Hz), 5.51 (s, 1H, OH), 5.13 (m, 1H), 5.08 (s, 2H,
OCH2Ph), 5.07 (m, 1H), 5.05 (dd, 1H, J ¼ 11.7, 1.3 Hz), 5.02 (dd, 1H,
J ¼ 17.6,1.3 Hz), 2.05 (m, 2H),1.96 (m, 2H),1.83 (m, 2H),1.70 (m, 2H),
1.68 (s, 3H), 1.60 (s, 3H), 1.52 (s, 3H), 1.34 (s, 3H). m/z 405.2787
[M þ H]þ (C28H37Oþ2 , mcalc ¼ 405.2788), Rf 0.30 (hexanes:Et2O, 9:1).
4. Conclusion
5.1.2. Preparation of O,O-dibenzoyl-4-nerolidylcatechol (5)
Compound 1 (300.1 mg, 1 mmol) was dissolved in dry C5H5N
(2.0 mL) or Et3N under N2 atmosphere and stirring, followed by
dropwise addition of C6H5COCl (337.5 mL, 2.9 mmol, 3 equiv). The
solution was maintained at 80 ꢂC with stirring for 1 h. Then the
reaction was stirred and allowed to come to room temperature over
Several semi-synthetic 4-nerolidylcatechol derivatives were
identified herein with significant antimalarial and cytotoxic
potential. Syntheses of new derivatives are underway in light of the
qualitative structure–activity relationships suggested herein.

2734
A.C.S. Pinto et al. / European Journal of Medicinal Chemistry 44 (2009) 2731–2735
48 h. Cold H2O (3 mL) was added to the reaction mixture which was
then extracted with CHCl3 (3 ꢁ 5 mL) and water (2 ꢁ 5 mL), in an
alternate fashion. The CHCl3 phase was washed with HCl 0.1 N,
diluted NaHCO3, water (5 mL) and saturated NaCl and then dried
with anhydrous Na2SO4. The product was purified by column
chromatography flash (hexanes:AcOEt, 97:3), yielding dibenzoy-
lated derivative 5 (viscous, yellowish-clear oil, 73.6 mg, 14.7%), Rf
room temperature with stirring for 11 days. After this period, the
reaction mixture was transferred to a separation funnel and
washed with aqueous Na2S2O3, followed by aqueous NaHCO3 and
water. The organic phases were dried with anhydrous Na2SO4
(method adapted from Ref. [20]). The crude product was purified by
chromatography (hexanes:AcOEt, 3:2 to 1:1) yielding a diastereo-
meric mixture of diepoxy derivatives 8 (viscous, yellowish-clear oil,
77.2 mg, 56.3%), Rf 0.36 (hexanes:AcOEt, 1:1). 1H NMR (400 MHz,
0.33 (hexanes:acetone, 9:1) 1H NMR (500 MHz, CDCl3):
d 8.04–8.09
(m, 4H, COOPh), 7.52–7.56 (m, 4H, COOPh), 7.36–7.40 (m, 2H,
COOPh), 7.32–7.35 (m, 2H, Ar-H3, Ar-H6), 7.31 (dd, J ¼ 8.5, 2.0 Hz,1H,
Ar-H5), 6.06 (m, 1H, J ¼ 17.2, 10.2 Hz), 5.17 (m, 1H), 5.14 (dd, 1H,
J ¼ 10.2, 1.0 Hz), 5.11 (dd, 1H, J ¼ 17.2, 1.0 Hz), 5.08 (m, 1H), 2.06 (m,
2H), 1.96 (m, 2H), 1.86 (m, 2H), 1.84 (m, 2H), 1.68 (s, 3H), 1.60 (s, 3H),
CDCl3): d 7.79 (m, 1H, Ar-H3), 7.44 (m, 1H, Ar-H6), 7.10 (m, 1H, Ar-
H5), 5.97 (m, 1H), 5.10 (m, 2H), 3.31 (m, 2H), 2.27 (2, 3H, COOMe),
2.26 (s, 3H, COOMe), 2.05 (m, 2H), 1.91 (m, 2H), 1.81 (m, 2H), 1.75
(m, 2H), 1.57 (s, 3H), 1.51 (s, 3H), 1.35 (s, 3H), 1.23 (s, 3H). m/z
431.2437 [M þ H]þ (C25H35O6þ, mcalc ¼ 431.2428).
1.55 (s, 3H), 1.43 (s, 3H). 13C NMR (125 MHz, CDCl3):
d 164.3 (C]O),
164.2 (C]O), 146.7, 146.1, 142.0, 140.3, 135.2, 133.5, 131.3, 130.1,
129.0, 128.9, 124.8, 128.4, 124.4, 124.2, 122.8, 121.9, 112.5, 44.3, 41.1,
39.7, 26.7, 25.7, 25.0, 23.2, 17.7, 15.9. m/z 545.2632 [M þ Na]þ
(C35H38NaOþ4 , mcalc ¼ 545.2662).
5.2. In vitro anti-plasmodial activity
P. falciparum strain K1 (MRA-159, ATCC) was used in our study
and was acquired from MR4 (Malaria Research and Reference
Reagent Resource Center, Manassas, Virginia, US). P. falciparum was
cultivated by modification of the Trager and Jensen method [21].
The procedures used for assaying antimalarial activity are
described in Ref. [8]. Briefly, in a preliminary screen, active
compounds inhibited the growth of parasites by 80–100%, partially
active (PA) compounds by 50–79% and inactive (I) compounds by
5.1.3. Preparation of 1 and 2-O-methyl-4-nerolidylcatechols
(6 and 7)
Semi-synthetic derivatives 6 and 7 (Fig. 1) were prepared from 1
by two methods, the first by treatment of 1 (410.0 mg, 1.3 mmol)
with CH2N2 (10 mL) of an Et2O solution prepared by decomposi-
tion/distillation of Diazald (5.5 g) in Et2O (30 mL) at room
temperature for 30 min. The solvent was removed by rotary evap-
oration and products were purified by column chromatography
(gradient of hexanes:acetone (9:1 to 1:1)) to yield monomethyl
derivatives 6 (viscous, transparent oil, 33.0 mg, 7.7%) and 7 (viscous,
transparent oil, 36.0 mg, 8.4%) separated by preparative TLC (hex-
anes:acetone, 9:1). The second method was adapted from Ref. [19]:
to a solution of 1 (50 mg, 0.2 mmol) in (CH3)2CO (5 mL) was added
< 50% at concentrations of 50 and 2.5 mg/mL. Active compounds
were further evaluated at different dilutions for probit analysis.
5.3. In vitro antitumour activity
The human tumor cell lines used were HCT-8 (human colon
carcinoma), SF-295 (human nervous system), MDA/MB-435
(melanoma) and HL-60 (human myeloblastic leukemia) which
were donated by the Mercy Children’s Hospital (United States).
They were cultivated in RPMI 1640 medium which was supple-
mented with 10% bovine fetal serum and 1% antibiotics and
maintained in an incubator at 37 ꢂC and an atmosphere containing
5% CO2. Samples were diluted in DMSO at a stock concentration of
5 mg/mL. The cytotoxicity of the samples was evaluated using the
MTT method [22]. The cells were plated in 96-well test plates in the
following densities: 0.7 ꢁ 105 (HCT-8), 0.6 ꢁ 105 (SF-295) and
0.3 ꢁ 106 (HL-60). The samples were incubated for 72 h at a single
K2CO3 (76.9 mg, 0.5 mmol, 3.5 equiv) and CH3I (25 mL, 8.1 mmol,
2.5 equiv). The reaction mixture was heated under reflux for 2 h.
The mixture was concentrated and extracted three times with
equal volumes of CHCl3. After concentration, the combined extracts
were purified by flash column chromatography (gradient of hex-
anes:acetone, 9:1 to 1:1) to yield monomethyl derivative mixture
(5.3 mg, 9.7%). 6, Rf 0.39 (hexanes:CHCl3, 2:3): 1H NMR (500 MHz,
CDCl3):
d
6.85 (d, 1H, J ¼ 1.5 Hz, Ar-H3), 6.83 (d, 1H, J ¼ 7.2 Hz, Ar-
H6), 6.81 (dd, J ¼ 7.2,1.5 Hz,1H, Ar-H5), 6.01 (dd,1H, J ¼ 17.7, 9.5 Hz),
5.46 (s, OH), 5.11 (m, 1H), 5.09 (m, 1H), 5.06 (dd, 1H, J ¼ 9.5, 1.0 Hz),
5.04 (dd, 1H, J ¼ 17.7, 1.0 Hz), 3.87 (s, 3H, OMe), 2.04 (m, 2H), 1.95
(m, 2H), 1.86 (m, 2H), 1.76 (m, 2H), 1.67 (s, 3H), 1.59 (s, 3H), 1.52 (s,
concentration (100 mg/mL). Absorbance was measured with the aid
of a plate spectrophotometer operating at 550 nm.
The experiments were analyzed using averages and the corre-
sponding confidence intervals based on the non-linear regression
generated using GraphPad Prism. Each sample was tested in tripli-
cate in two independent experiments. An intensity scale was used
to evaluate the cytotoxic potential of the tested samples.
3H), 1.36 (s, 3H). 13C NMR (125 MHz, CDCl3):
d 147.4, 146.3, 143.8,
139.8, 135.2, 131.5, 124.8, 124.6, 119.6, 114.0, 111.7, 109.8, 56.1 (OMe),
44.3, 41.4, 39.9, 26.9, 25.9, 25.3, 23.5, 17.9, 16.1. m/z 329.2476
[M þ H]þ (C22H33O2þ, mcalc ¼ 329.2475). 7, Rf 0.45 (hexanes:CHCl3,
2:3): 1H NMR (500 MHz, CDCl3):
d
6.92 (d, 1H, J ¼ 1.7 Hz, Ar-H3),
6.78 (d, J ¼ 1.7 Hz, 2H, Ar-H5, Ar-H6), 6.00 (dd, 1H, J ¼ 17.1, 11.0 Hz),
5.51 (s, OH), 5.09 (m, 1H), 5.07 (dd, 1H, J ¼ 11.0, 1.0 Hz), 5.07 (m, 1H),
5.03 (dd,1H, J ¼ 17.1,1.0 Hz), 3.87 (s, 3H, OMe), 2.04 (m, 2H),1.95 (m,
2H), 1.86 (m, 2H), 1.77 (m, 2H), 1.68 (s, 3H), 1.59 (s, 3H), 1.52 (s, 3H),
Acknowledgements
This work was supported by FAPEAM: (PIPT 006/2003, CBA-
UFAM 1577/2005), and scholarships for study POSGRAD and PIBIC/
INPA. PPG-7/CNPq (no. 557106/06), MCT/CNPq/PPBio (no. 48.0002/
04-5). The authors thank Dr. Massayoshi Yoshida (CBA) for NMR
spectra, J.C. Tomaz and Prof. Norberto Lopes (USP) for ESI-HRMS
and Prof. Daniel Rettori (UNIBAN) for helpful comments on this
manuscript.
1.34 (s, 3H). 13C NMR (125 MHz, CDCl3):
d 147.3, 145.3, 144.7, 141.3,
131.5, 135.1, 124.8, 124.6, 118.2, 113.5, 111.7, 110.4, 56.2 (OMe), 44.0,
41.4, 39.9, 26.9, 25.9, 25.2, 23.4, 17.9, 16.1. m/z 329.2498 [M þ H]þ
(C22H33Oþ2 , mcalc ¼ 329.2475).
5.1.4. Preparation of O,O-diacetyl 6,10-diepoxy derivatives 8
Compound 1 (150 mg, 0.5 mmol) was treated with Ac2O (1 mL)
and C5H5N (1 mL) under N2 and magnetic stirring at room
temperature for 24 h. The O,O-diacetyl derivative (171.4 mg; 90.2%)
obtained was dissolved in CH2Cl2 and the reaction flask was placed
in a NaCl–H2O(s) bath (ꢀ5 ꢂC) and then treated with m-CPBA
(220.5 mg, 1.3 mmol, 1.2 equiv) in CH2Cl2 (10 mL) with stirring
under N2. After 2 h, the reaction mixture was allowed to warm to
References
[1] W. Milliken, Plants for Malaria; Plants for Fever, Kew, Royal Botanic Gardens,
United Kingdom, 1997, 83–86.
[2] C.Z. Amorim, B.E. Gomes, C.A. Flores, R.S.B. Cordeiro, Braz. J. Med. Biol. Res. 19
(1986) 569A.
[3] C.Z. Amorim, C.A. Flores, B.E. Gomes, A.D. Marques, R.S.B. Cordeiro, J. Ethno-
pharmacol. 24 (1988) 101–106.

A.C.S. Pinto et al. / European Journal of Medicinal Chemistry 44 (2009) 2731–2735
2735
[4] Y.L. Adami, Estudo in vivo e in vitro da potencial atividade antimala´rica de
Pothomorphe peltata e Pothomorphe umbellata (L.) Miq., Fundaça˜o Oswaldo
Cruz, Rio de Janeiro, Brazil, 1995, 102 pp.
[5] M.F. Ferreira-da-cruz, Y.L. Adami, E.C. Espinola-Mendes, Exp. Parasitol. 94
(2000) 243–247.
[12] N.P. Lopes, M.J. Kato, E.H.A. Andrade, J.G.S. Maia, M. Yoshida, A. Planchart,
A.M. Katzin, J. Ethnopharmacol. 67 (1999) 313–319.
[13] C.S. Macedo, M.L. Uhrig, E.A. Kimura, A.M. Katzin, FEMS Microbiol. Lett. 207
(2002) 13–20.
[14] H.R. Goulart, E.A. Kimura, V.J. Peres, A.S. Couto, F.A.A. Duarte, A.M. Katzin,
Antimicrob. Agents Chemother. 48 (2004) 2502–2509.
[15] H. Murakami, S. Tamura, T. Horii, Saneigen F.F.I. Inc., Jpn. Kokai Tokkyo Koho,
Patent No. JP 2005200332, Japan, July 28, 2005, 20 pp.
[16] B. Pradines, J.M. Rolain, F. Ramiandrasoa, T. Fusai, J. Mosnier, C. Rogier,
W. Daries, E. Baret, G. Kunesch, J. Le Bras, D. Parzy, J. Antimicrob. Chemother.
50 (2002) 177–187.
[17] S.K. Sharma, M.J. Miller, S.M. Payne, J. Med. Chem. 32 (1989) 357–367.
[18] M. Bouktaib, S. Lebrun, A. Atmani, C. Rolando, Tetrahedron 58 (2002)
10001–10009.
[19] H. Yoo, J.Y. Lee, J.H. Park, B.Y. Chung, Y.S. Lee, Il Farmaco 58 (2003)
1243–1250.
[20] P. Tassignon, P. Waard, D. Wit, D. Buyck, Ind. Crops Prod. 3 (1995) 273–280.
[21] W. Trager, J.B. Jensen, Science 193 (1976) 673–675.
[22] T. Mosmann, J. Immunol. Methods 65 (1983) 55–63.
[6] F. Sala-neto, J.S. Da Silva, R.O. Pires, N.P. Nascimento, C. Brenner, J.P. Boubli,
C.E. Tosta, Anais do 2o Simposio de Malaria, Rev. Soc. Bras. Med. Trop. 25 (1992)
92.
´
´
[7] A.C.S. Pinto, C. Pessoa, L.V.C. Lotufo, M.O.M. Moraes, M.E. Moraes, B.C. Cavalcanti,
S.M. Nunomura, A.M. Pohlit, Rev. Bras. Pl. Med. 8 (2006) 205–211.
[8] V.F. Andrade-Neto, A.M. Pohlit, A.C.S. Pinto, E.C.C. Silva, K.L. Nogueira,
M.R.S. Melo, M.C. Henrique, R.C.N. Amorim, L.F.R. Silva, M.R.F. Costa,
R.C.S. Nunomura, S.M. Nunomura, W.D. Alecrim, M.G.C. Alecrim, F.C.M. Chaves,
P.P.R. Vieira, Mem. Inst. Oswaldo Cruz 102 (2007) 359–365.
[9] R.C. Anand, H. Ranjan, Bull. Chem. Soc. Jpn. 58 (1985) 791–792.
[10] E.L. Bastos, C.R. Eckert, P. Romoff, W.J. Baader, J. Agric. Food Chem. 51 (2003)
7481–7488.
[11] J. Simpson, R. Forrester, M.J. Tisdale, D.C. Billingtony, D.L. Rathbone, Bioorg.
Med. Chem. Lett. 13 (2003) 2435–2439.