Synthesis and in vitro antiprotozoal evaluation of substituted phenalenone analogues

Article abstract of DOI:10.1016/j.bmc.2010.04.062

A set of derivatives encompassing structural modifications on the privileged phenalenone scaffold were assessed for their antiparasitic activities against the most clinically relevant forms of trypanosomiasis and leishmaniasis. Several compounds exhibited leishmanicidal effects at levels comparable or better than the reference drug pentamidine, while the parent phenalenone was shown to have a level of activity against Trypanosoma cruzi comparable to the marketed drug benznidazole.

Full text of DOI:10.1016/j.bmc.2010.04.062

Bioorganic & Medicinal Chemistry 18 (2010) 4530–4534
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro antiprotozoal evaluation of substituted
phenalenone analogues
Laura I. Rosquete a, M. Gabriela Cabrera-Serra b, José E. Piñero b, Patricia Martín-Rodríguez c,d
,
a,
Leandro Fernández-Pérez c,d, Javier G. Luis a, Grant McNaughton-Smith a,d, , Teresa Abad-Grillo
*
*
a Instituto Universitario de Bio-Orgánica ‘Antonio González’, Universidad de La Laguna, Avda. Fco., Sánchez 2, 38206 La Laguna, Tenerife, Canary Islands, Spain
b Instituto Universitario de Enfermedades Tropicales y Salud Pública de Las Islas Canarias, Laboratorio de Quimioterapias de Protozoos, Universidad de La Laguna,
Avda. Francisco Sánchez s/n, 38206 La Laguna, Tenerife, Canary Islands, Spain
c Department of Clinical Sciences, Molecular and Translational Endocrinology Group, University of Las Palmas de GC—Cancer Research Institute of the Canary Islands (ICIC), Spain
d CEAMED, SA, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
A set of derivatives encompassing structural modifications on the privileged phenalenone scaffold were
assessed for their antiparasitic activities against the most clinically relevant forms of trypanosomiasis
and leishmaniasis. Several compounds exhibited leishmanicidal effects at levels comparable or better
than the reference drug pentamidine, while the parent phenalenone was shown to have a level of activity
against Trypanosoma cruzi comparable to the marketed drug benznidazole.
Received 29 October 2009
Revised 15 April 2010
Accepted 21 April 2010
Available online 28 April 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Synthesis
Substituted phenalenones
Antiprotozoal activities
1. Introduction
keted drugs are effective. The emergence of drug-resistant para-
sites is also becoming an additional and major problem. Further
Trypanosomatids of the order Kinetoplastida are the causative
agents of several lethal parasitic diseases, such as Chagas’ disease
(Trypanosoma cruzi), and African sleeping sickness (Trypanosoma
brucei). The human protozoan parasite Leishmania is the causative
agent of leishmaniasis, a disease with a wide variety of clinical
manifestations, ranging from self-healing cutaneous lesions
(mostly from Leishmania tropica and Leishmania mexicana com-
plexes) to life-threatening visceral infections caused by different
species of the donovani complex (Leishmania donovani, Leishmania
infantum, and Leishmania chagasi). The reemergence of trypanoso-
miasis and leishmaniasis over the last two decades has become a
significant threat to human health and the economical develop-
ment of several developing nations. The drugs which have been
most frequently used to treat the leishmaniasis (the pentavalent
antimonials, Pentostam, and Glucantime1) are however, quite toxic
and in some areas resistance can be as high as 40%.2,3 Likewise
those used for trypanosomiasis, such as Nifurtimox (currently dis-
continued) and Benznidazole are still inadequate due to their
undesired side effects.4 Indeed, no vaccine or recommended drugs
are currently available to prevent these diseases. Moreover, once
the infection has progressed into its later stages none of the mar-
investigations are therefore urgently needed to discover new drugs
that are not only effective, but also affordable and readily available
for the treatment of these infectious diseases.
Previous work from our group had demonstrated that a set of
antifungal phytoalexins,5,6 based on a phenyl-phenalenone skele-
ton, possessed leishmanicidal activity.7 In order to further investi-
gate the effect of electronic and spatial changes on antiprotozoal
activity we have synthesized a series of core modified struc-
tures,8–11 as well as a set of new heteroaryl substituted phenale-
nones. Herein, we report their synthesis and their antiprotozoal
activity against two forms of Leishmania (Leishmania amazonensis,
L. donovani) and one form of Trypanosoma (T. cruzi).
2. Results and discussion
2.1. Synthesis
The heteroaryl-substituted phenalenones were prepared using
a general two step Michael addition-oxidation sequence. The re-
quired heteroaryl Grignards for the preparation of compounds 6,
7, and 10 were generated in situ via either direct lithiation and me-
tal exchange, or lithium–halogen exchange followed by metal ex-
change as shown in Table 1 (entries 1–3).
* Corresponding authors. Tel.: +34 922 318575; fax: +34 922 318571.
E-mail address: tereabad@ull.es (T. Abad-Grillo).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.062

L. I. Rosquete et al. / Bioorg. Med. Chem. 18 (2010) 4530–4534
4531
Table 1
Table 2
Het-MgBr preparation conditions
In vitro antiprotozoal activities of phenalenones (4–25) and cytotoxicity
MCF7a
Entry
Het-MgBr
Compound
IC50
(l
g/mL) S.D
%inhibition
(10 lM)
.
L.
L. donovani
T. cruzi
BuLi, MgBr Et O,
2
o
2
MgBr
O
1
1
2
3
amazonensis
O
S
THF, -25 C
4
5
6
7
8
27.30 1.31
—
8.39 1.03
14.50 2.5
—
53.17 1.86
3.73 0.04
—
0.83 0.14
42.83
—
—
—
65.07 1.19 2.62 0.86
—
—
—
—
—
1.02 0.48
—
—
0
—
—
—
—
—
—
—
—
nd
14
4
0
0
44
5
13
1
23
19
22
.
MgBr Et O,
2
2
MgBr
S
Li
I
o
THF, -25 C
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Control
.
BuLi, MgBr Et O,
2
2
N
N
MgBr
THF, RT
3
2
27.80 2.12 49.75 0.5
—
—
—
—
—
—
Michael-type addition of 2-furyl Grignard reagent 1 to phena-
len-1-one 4 (Scheme 1) furnished the phenolic compound 5 in
good yield. Treatment of this intermediate with DDQ gave the de-
sired phenalenone 6 in high yield. In the same manner, addition of
the 2-thienyl Grignard reagent 2 to 4 followed by oxidation with
DDQ afforded 7. Addition of the 2-pyridyl Grignard 3 gave the
hydroxyl intermediate 9 and the bis-pyridyl adduct 8. A solution
of 9 in chloroform converted cleanly to the oxidized phenalenone
system 10. Attempts to increase the poor yield of 10 by changing
reaction conditions or the method of Grignard generation12–16
were fruitless. The reaction of 4 with sodium cyanide in aqueous
DMF introduced a cyano group at the same position as the previous
aryls. In situ air oxidation afforded 11 directly.
9.04 1.06
—
—
—
29.90 0.96 48.35 2.1 14
—
—
—
—
—
—
—
—
—
—
15
17
nd
19
—
4.43 0.64
—
11.4 0.99 17
—
12
2
—
—
—
5.72b
5.72b
0.83c
90d
a
Cytotoxicity in MCF7 cells. Inhibition of cell growth. Data are expressed as %
inhibition = % activity in the presence of vehicle À % activity in the presence of
inhibitor.
b
Reference compound, pentamidine.
Reference compound, benznidazole.
c
d
Reference compound, adriamycin; — signifies an IC50 >100
not determined.
lg/mL; nd signifies
2.2. Antiprotozoal activity
A total of 21 phenalenone-based compounds were investigated
for their activities against the clinically relevant forms of trypano-
somiasis and leishmaniasis (namely T. cruzi, L. amazonesis, and
L. donovani), the results of which are illustrated in Table 2.
While only the parent phenalenone-1-one 4 exhibited signifi-
tive than the parent compound 4 and fivefold more active than
the positive control, pentamidine. Interestingly, compound 23,
which possesses the 4-methoxyphenyl group on carbon 4, also
exhibited activity that was comparable to the positive control,
indicating that this change in the relative positions between the
carbonyl and the substituted phenyl moiety was tolerated. The
addition of the 4-methoxyphenyl group to the b-carbon of the
enone system (compound 25) however, rendered a compound void
of activity against any of the parasitic types. Addition of either, a
hydroxyl (compound 16) or methoxy group (compound 19), next
to the carbonyl of 13 resulted in abolishment of all antiparasitic
activity, suggesting that hydrophilic groups were not tolerated in
this region of the molecule. Similarly the diol 21 was also found
to be void of activity.
cant potency against T. cruzi (IC50 = 2.6 lg/mL), several of the
substituted compounds possessed enhanced activities in a selec-
tive manner against L. amazonensis. While the addition of either a
bulky 2,5-dimethylphenyl (compound 14), a 2-pyridyl (compound
10) or an unsubstituted phenyl group (compound 12) at position 9
proved to be detrimental to activity against L. amazonensis, the
addition of a 2-furyl (compound 6) or a 2-thienyl group (compound
7) afforded compounds with modest IC50’s. The corresponding
4-methoxyphenyl, compound 13, however, was 30-fold more ac-
O
HO
DDQ, CH Cl
2
2
O
O
2-Furyl-MgBr (1),
THF
5 (73%)
6 (88%)
O
1). 2-Thienyl-MgBr (2),
THF
S
2
1
O
3
2). DDQ, CH Cl
7 (90%)
2
2
4
9
N
4
O
HO
HO
2-Pyridyl-MgBr (3),
N
N
N
+
THF
10 (18%)
8 (3%)
9
O
NC
NaCN, NH Cl
4
DMF: H O
2
11( 19%)
Scheme 1.

4532
L. I. Rosquete et al. / Bioorg. Med. Chem. 18 (2010) 4530–4534
R1
CH3
O
R1
4. Experimental
O
R2
O
R2
R3
R3
4.1. Chemistry
CH3
15 R1=OH, R2=H, R3=OH
16 R1=OH, R2=H, R3=OCH3
17 R1=OH, R2=R3=H
14
12 R1=R2=R3=H
4.1.1. General
13 R1=R2=H, R3=OCH3
1H and 13C NMR spectra were recorded on Bruker AMX400,
AMX300 and WP200SY. IR spectra were taken on a Perkin–Elmer
1600 (FTIR) spectrophotometer. High resolution mass spectra were
run on a VG-Micromass ZAB-2F at 70 eV. Melting points were re-
corded in a BÜCHI B-540. Optical rotations were recorded in a
polarimeter Perkin–Elmer 343. Organic solvents used were dried
by standard methods. All reactions were performed under an
atmosphere of argon unless otherwise specified. Commercially ob-
tained reagents were used without further purification. All reac-
tions were monitored by TLC with Merck 60 F254 Silica Gel
coated plates. Flash column chromatography was carried out using
230–240 mesh silica gel at increased pressure.
18 R1=OCH3, R2=R3=H
19 R1=OCH3, R2=H, R3=OCH3
OCH3
R1
R1
O
R2
O
R2
O
R3
25
22 R1=OH, R2=H
23 R1=H, R2=OCH3
24 R1=R2=OH
20 R1=OH, R2=OCH3, R3= H
21 R1=R2=OH, R3=OCH3
Figure 1. Compounds included in in vitro antiprotozoal study.
4.1.2. Synthetic procedures
Of significant interest was the level of activity displayed by the
cyano compound 11 against both forms of Leishmania. It possessed
activity equal to that of pentamidine against L. amazonensis while
being fivefold more active than the control against L. donovani, pos-
4.1.2.1. 9-Furan-2-yl-9H-phenalen-1-ol (5). n-BuLi (0.33 mL,
0.825 mmol, 2.5 M) in THF was added dropwise to a solution of fur-
an (0.06 mL, 0.825 mmol, 1 M) in THF at À25 °C. After 0.5 h this
solution was added to
a
mixture of MgBr2ÁOEt2 (156 mg,
sessing an IC50 of 1 lg/mL.
0.605 mmol) in THF (5.5 mL) at À25 °C. The reaction mixture was
allowed to warm to room temperature and stirring was continued
for a further 1 h. A solution of 4 (100 mg, 0.55 mmol) in THF
(5.5 mL) was then added and the mixture was heated at reflux for
3 h. The reaction was quenched with saturated aqueous NH4Cl
(10 mL) and extracted with Et2O (3 Â 10 mL). The combined organic
layers were washed with water (2 Â 10 mL), brine (10 mL) and then
dried (Na2SO4). Filtration and evaporation of the solvent afforded
the crude product. Column chromatography (hexane/EtOAc, 9:1)
gave 5 (99.3 mg, 73%) as a yellowish oil. IR (CHCl3) mmax 2348,
2.3. Cellular cytotoxicity
The ability of these phenalenone-based compounds to inhibit
cell growth in a human breast adenocarcinoma cell line (MCF7)
was used as a measure of cytotoxicity. In general the compounds
possessed only low levels of inhibition (<25% inhibition at
10
concentration (0% at 1.8
Compound 23 was also non-cytotoxic at a concentration (17% at
2.9 g/mL) similar to its IC50 against L. amazonensis, while the more
lM). The parent phenalenone 4 showed no cytotoxicity at a
l
g/mL) similar to its IC50 against T. cruzi.
1637, 1239, 841 cmÀ1 1H NMR (300 MHz, CDCl3) d 7.72–7.68 (m,
.
2H), 7.41 (br s, 1H), 7.37–7.32 (m, 1H), 7.24–7.20 (m, 2H), 6.83 (d,
J = 9.6 Hz, 1H), 6.37 (m, 2H), 6.22 (m, 1H), 6.15 (m, 1H), 5.53 (br s,
1H). 13C NMR (75 MHz, CDCl3) d 156.1, 152.2, 141.8, 129.5, 129.1,
128.7, 128.3, 127.9, 127.7, 127.4, 124.2, 123.9, 118.8, 116.2, 110.9,
106.3, 36.9. EIMS: m/z 246 (100.0), 248 [M]+ (37.7). HRMS: calcd
for C17H12O2 248.0837, found 248.0774.
l
potent and selective antiprotozoal, compound 13, did not show
signs of inhibiting cell growth at a concentration three fold greater
(13% at 2.9 lg/mL) than its IC50 against L. amazonensis. The only
compound in this series to show a modest inhibition of cell growth
was compound 11. At a concentration twofold higher than its IC50
against L. donovani (10 lM or 2.1 lg/mL), it inhibited cell growth
by 44% (Fig. 1).
4.1.2.2.
9-(2-Furyl)-1H-phenalen-1-one
(6). DDQ
(41 mg,
0.18 mmol) was added to a solution of 5 (44.1 mg, 0.18 mmol) in
CH2Cl2 (1.8 mL) and the reaction mixture was heated at reflux for
3 h. The reaction was quenched with saturated aqueous NH4Cl
(3 mL), and extracted with Et2O (3 Â 3 mL). The combined organic
layers were washed with water (2 Â 10 mL), brine (10 mL) and
then dried (Na2SO4). Filtration and evaporation of the solvent affor-
ded the crude product. Column chromatography (hexane/EtOAc,
8:2) gave 6 (39 mg, 88%) as a yellowish oil. IR (CHCl3) mmax 1726,
1237, 843 cmÀ1. UV–Vis (EtOH) kmax 255, 278, 291, 381 nm. 1H
3. Conclusions
Screening of a collection of modified phenalen-1-one based
structures has lead to the identification of several new and potent
antiprotozoal agents. Interestingly, the majority of the active com-
pounds were more active against L. amazonensis than the L. dono-
vani strain. Of the active compounds, 23 possessed a level of
activity comparable to the positive control pentamidine, while
compound 13 was significantly more potent than the control, pos-
NMR (300 MHz, CDCl3)
d 8.16 (d, J = 8.5 Hz, 1H), 7.98 (d,
J = 8.0 Hz, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.75 (d, J = 7.0 Hz, 1H),
7.68 (d, J = 9.7 Hz, 1H), 7.62–7.56 (m, 2H), 7.18 (d, J = 3.3 Hz, 1H),
6.71 (d, J = 9.7 Hz, 1H), 6.60 (dd, J = 3.3, 1.5 Hz, 1H). 13C NMR
(75 MHz, CDCl3) d 185.4, 153.1, 143.4, 140.0, 134.5, 134.0, 131.7,
131.4 (Â2), 130.2, 129.7, 128.5, 128.4, 126.5, 125.7, 111.9, 111.7.
EIMS: m/z 246 [M]+ (100.0), 205 (82.6), 189 (91.7). HRMS: calcd
for C17H10O2 246.0688, found 246.0699.
sessing an IC50 <1 lg/mL. Neither compound showed signs of cyto-
toxicity at these concentrations. Compound 11 was the only
compound from this collection that significantly inhibited both
forms of Leishmania. It was shown to be more active than the
positive control in both strains. Unlike the other compounds in
the series, 11 inhibited cell growth in human MCF7 cells at concen-
trations in the same range as its antiprotozoal activity. Compounds
13 and 23 represent interesting candidates for further develop-
ment, while further SAR explorations around 11 may lead to
compounds with a higher therapeutic window. Given the issues
associated with the therapeutic agents currently used for the
treatment of T. cruzi infections, further development of the parent
compound phenalenone-1-one (4) is also warranted.
4.1.2.3. 9-(2-Thienyl)-1H-phenalen-1-one (7). 2-Thienyllithium
(0.825 mL, 0.825 mmol) was added dropwise to a stirring solution
of MgBr2ÁEt2O (156.2 mg, 0.605 mmol) in THF (1.21 mL) at À25 °C.
The reaction mixture was allowed to warm to room temperature.
After 1 h a solution of 4 (100 mg, 0.55 mmol) in THF (5.5 mL)

L. I. Rosquete et al. / Bioorg. Med. Chem. 18 (2010) 4530–4534
4533
was added and the reaction was heated at reflux for 3 h. The reac-
tion was quenched with saturated aqueous NH4Cl (10 mL) and ex-
tracted with Et2O (3 Â 10 mL). The combined organic layers were
washed with water (2 Â 10 mL), brine (10 mL) and then dried
(Na2SO4). Filtration and evaporation of the solvent afforded the
intermediate alcohol that was used without further purification.
It was dissolved in CH2Cl2 (6.18 mL) and DDQ (140.7 mg,
0.618 mmol) was added. The reaction mixture was heated at reflux
for 3 h and then quenched with saturated aqueous NH4Cl (10 mL),
and extracted with Et2O (3 Â 10 mL). The combined organic layers
were washed water (2 Â 10 mL), brine (10 mL) and then dried
(Na2SO4). Filtration and evaporation of the solvent afforded the
crude product. Column chromatography (hexane/EtOAc, 7.5:2.5)
gave 7 (130 mg, 90%) as a yellowish oil. IR (CHCl3) mmax 1637,
1238, 835 cmÀ1. UV–Vis (EtOH) kmax 208, 255, 371 nm. 1H NMR
(300 MHz, CDCl3) d 8.13 (d, J = 8.3 Hz, 1H), 8.00 (d, J = 8.2 Hz, 1H),
7.77–7.54 (m, 3H), 7.45 (dd, J = 5.0, 1.2 Hz, 1H), 7.20 (dd, J = 3.5,
1.2 Hz, 1H), 7.13 (dd, J = 5.0, 3.5 Hz, 1H), 6.63 (d, J = 9.7 Hz, 1H).
13C NMR (75 MHz, CDCl3) d 185.7, 143.4, 140.5, 139.9, 133.7,
132.4, 132.0, 131.8 (Â2), 130.2, 128.3, 127.1, 126.8, 126.7, 126.6,
126.1 (Â2). EIMS: m/z 261 [MÀ1]+ (100.0), 262 (45.70). HRMS:
calcd for C17H10OS 262.0452, found 262.0418.
ded the crude product. Column chromatography (hexane/EtOAc,
9:1) gave 11 (22 mg, 19%) as an orange amorphous solid. IR (CHCl3)
m
max 2221, 1639, 843 cmÀ1. UV–Vis (EtOH) kmax 259, 308, 318, 372,
401 nm. 1H NMR (300 MHz, CDCl3) d 8.3 (d, J = 8.3 Hz, 1H), 8.10 (d,
J = 8.2 Hz, 1H), 8.00 (d, J = 8.3 Hz, 1H), 7.80 (d, J = 6.9, 1H), 7.70 (d,
J = 9.8 Hz, 1H), 7.70 (dd, J = 8.2, 7.0 Hz, 1H), 6.80 (d, J = 9.8 Hz, 1H).
13C NMR (75 MHz, CDCl3) d 182.6, 141.2, 134.1, 133.7, 132.5, 131.4,
131.3, 129.0, 128.9 (Â2), 128.1, 127.1, 118.4, 112.2. EIMS: m/z 150
(20.2), 177 (100.0), 205 [M]+ (62.6). HRMS: calcd for C14H7NO
205.0527, found 205.0500.
4.1.2.6. 9-(2, 6-Dimethyl-phenyl)-1H-phenalen-1-one (14). To a
stirred solution of 4 (100 mg, 0.55 mmol) in THF (5.55 mL) was
added 0.7 mL of 2,6-dimethylphenyl-magnesium bromide
(0.71 mmol, 1.0 M solution in THF). The reaction mixture was stir-
red and heated at reflux for 3 h. The reaction mixture was cooled to
room temperature, and quenched with an aqueous saturated solu-
tion of NH4Cl (2 mL) and extracted with EtOAc (3 Â 5 mL). The
combined organic extracts were washed with water (3 Â 5 mL),
brine (5 mL) and dried (Na2SO4). Removal of solvent under vacuo
followed by silica gel column chromatography (EtOAc/hexanes,
1:9) afforded compound 14 (100 mg, 63%) as a yellow solid; mp
154.6–155.1 °C; IR (thin film, NaCl) mmax 1621, 1553, 1351, 1238,
1178, 841, 692, 654, 601 cmÀ1; UV–Vis (EtOH) kmax 216, 254,
311, 346, 360, 391 nm. 1H NMR (400 MHz, CDCl3) d 8.32 (d,
J = 8.3 Hz, 1 H), 8.11 (dd, J = 8.2, 1.0 Hz, 1 H), 7.83 (dd, J = 7.0,
1.0 Hz, 1 H), 7.75 (d, J = 9.7 Hz, 1 H), 7.67 (dd, J = 8.2, 7.0 Hz, 1 H),
7.49 (d, J = 8.3 Hz, 1 H), 7.28–7.18 (m, 3 H), 6.60 (d, J = 9.7 Hz, 1
H), 1.93 (s, 6H); 13C NMR (100 MHz, CDCl3) d 185.5, 146.6, 142.3,
140.5, 134.6, 133.5 (Â2), 131.7, 131.6, 131.1, 130.6, 130.0, 128.5,
128.4, 127.4 (Â2), 126.7, 126.3 (Â2), 20.5 (Â2); EIMS: m/z 134.20
(17.36), 239.08 (16.62), 252.08 (26.57), 267.09 (39.77), 269.08
(100.00), 270.10 (39.43), 284.12 [M]+ (14.32); HRMS: calcd for
C21H16O 284.1201, found 284.1202.
4.1.2.4. 1, 9-Di-pyridin-2-yl-1H-phenalen-1-ol (8) and 9-(2-
pyridyl)-1H-phenalen-1-one (10). To a solution of EtMgBr (3 mL,
3.00 mmol, 1 M) at room temperature was added 2-iodopyridine
(0.23 mL, 2.13 mmol). After 45 min a solution of 4 (500 mg,
2.77 mmol) in THF (27.7 mL) was added and the reaction was
heated at reflux for 3 h. The reaction was quenched with saturated
aqueous NH4Cl (5 mL) and extracted with Et2O (3 Â 5 mL). The
combined organic layers were washed with water (2 Â 10 mL),
brine (10 mL) and then dried (Na2SO4). Filtration and evaporation
of the solvent afforded the crude products. Column chromatogra-
phy (hexane/EtOAc, 9:1) gave 8 (26.8 mg, 3%) and 9 as a yellowish
amorphous solids. A solution of 9 in chloroform in air converted
cleanly to 10 (102.3 mg, 18%) as a yellowish amorphous solid.
Compound (8): IR (CHCl3) mmax 2923, 1585, 1432, 1257,
4.2. Biology/material and methods
670 cmÀ1
.
1H NMR (300 MHz, CDCl3) d 8.49 (br d, J = 4.4 Hz, 2H),
4.2.1. Antiparasitic activities. General
7.73–7.67 (m, 3H), 7.61 (dd, J = 8.0, 1.6 Hz, 1H), 7.54 (d, J = 8.2 Hz,
2H), 7.23–7.14 (m, 5H), 6.83 (d, J = 9.7 Hz, 1H), 6.15 (d, J = 9.7 Hz,
1H). 13C NMR (75 MHz, CDCl3) d 164.2, 156.0, 147.6, 137.3, 131.4,
129.3, 128.6, 128.3, 128.1, 128.0, 126.1, 124.5, 123.8, 122.7,
122.1, 121.3, 117.4, 58.7, 31.7, 29.4, 29.1, 22.5, 13.9. EIMS: m/z
228 (33.4), 256 (39.7), 257 (46.4), 258 (100.0), 336 [M]+ (15.6).
HRMS: calcd for C23H16N2O 336.1263, found 336.1247.
For the in vitro studies, samples were dissolved in dimethyl
sulphoxide (DMSO; Sigma) to prepare a working solution of
10 mg/mL. Further dilutions were made with RPMI 1640 medium
to the final highest concentration of DMSO on 1.5%, which was
not toxic to the parasites.
4.2.2. Leishmanicidal activity
Compound (9): 1H NMR (300 MHz, CDCl3)
d
8.73 (br d,
L. donovani (MHOM/IN/90/GE1F8R) and L. amazonensis (MHOM/
BR/77/LTB0016) strains were used in this study. Cultures were
handled as previously described.17 L. donovani promastigotes were
adapted for culture in RPMI 1640 liquid medium (Gibco-BRL) sup-
plemented with 20% heat inactivated fetal bovine serum, vitamins
and amino acids, at 26 °C. Logarithm phase cultures of promastig-
otes were used for experimental purposes. L. amazonensis strain
promastigotes, obtained from amastigotes forms isolated from
mouse lesions, were maintained at 26 °C in Schneider’s medium
(Schneider’s Insect Medium; Sigma Cell Culture, St. Louis, MO,
USA) containing a 10% heat inactivated fetal calf serum, 100 U of
J = 4.5 Hz, 1H), 7.73–7.67 (m, 1H), 7.58–7.53 (m, 2H), 7.43 (d,
J = 7.9 Hz, 1H), 7.20–7.13 (m, 4H), 7.00 (d, J = 9.7 Hz, 1H), 6.14
(dd, J = 9.7, 4.7 Hz, 1H), 5.67 (br d, J = 4.7 Hz, 1H).
Compound (10): IR (CHCl3) mmax 1638, 1239, 848, 670 cmÀ1
.
UV–Vis (EtOH) kmax 212, 251, 315, 361, 394 nm. 1H NMR
(300 MHz, CDCl3) d 8.72 (br d, J = 4.6 Hz, 1H), 8.23 (d, J = 8.3 Hz,
1H), 8.06 (d, J = 7.6 Hz, 1H), 7.80–7.63 (m, 5H), 7.43 (d, J = 7.9 Hz,
1H), 7.35–7.31 (m, 1H), 6.59 (d, J = 9.7 Hz, 1H). 13C NMR
(75 MHz, CDCl3) d 185.4, 160.5, 149.2, 145.1, 140.6, 135.7, 133.9,
131.9, 131.6, 131.5, 130.0, 129.8, 128.3, 127.8, 126.5, 126.2,
123.1, 121.8. EIMS: m/z 256 (100.00), 257 [M]+ (58.09). HRMS:
calcd for C18H11NO 257.0841, found 257.0840.
penicillin/mL and 100 lg of streptomycin/mL. Subcultures were
made in the late-log phase of growth and parasites were used no
later than the fifth passage.
4.1.2.5. 9-Oxo-9H-phenalene-1-carbonitrile (11). NaCN (108 mg,
2.20 mmol) was added to a solution of 4 (100 mg, 0.55 mmol)
and NH4Cl (88 mg, 1.65 mmol) in DMF/H2O (1:1, 5.5 mL) at room
temperature. The reaction was heated at reflux for 5 h. The reaction
was then extracted with CH2Cl2 (3 Â 15 mL). The combined organic
layers were washed with water (2 Â 10 mL), brine (10 mL) and
then dried (Na2SO4). Filtration and evaporation of the solvent affor-
The inhibition of promastigotes growth in vitro was assessed
using a quantitative colorimetric assay with the oxidation–reduc-
tion indicator Alamar BlueÒ Assay.18 Briefly, promastigotes were
serially diluted in 200 ll RPMI 1640 medium without phenol red
and supplemented with 20% (L. donovani) or 10% (L. amazonensis)
heat-inactivated fetal bovine serum in 96-well plates. To these
wells were added parasites (106/well), and the drug concentration

4534
L. I. Rosquete et al. / Bioorg. Med. Chem. 18 (2010) 4530–4534
to be tested. After addition of 10% of Alamar BlueÒ, the plates were
incubated at 26 °C. After 72 h, the plates were analyzed on a Micro-
plate Reader Model 680 (Biorad, Hercules, CA) using a test wave-
Acknowledgments
This work has been subsidized by the Ministerio de Ciencia y
Tecnología (BQU2001-3721). We thank the M.C.T. for fellowship
support to L.I.R.
length of 570 nm and
a reference wavelength of 630 nm.
Percentage of inhibition and 50% inhibitory concentrations (IC50
)
were calculated by linear regression analysis with 95% confidence
limits. All experiments were performed three times each in dupli-
cate, and the mean values were also calculated. A paired two-tailed
t-test was used for analysis of the data. Values of P <0.05 were con-
sidered significant.
References and notes
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4.2.3. Antitrypanosomal activity
Y strain T. cruzi epimastigotes were grown at 28 °C in liver infu-
sion tryptone (LIT) medium supplemented with 10% (v/v) heat-
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4.2.4. Cytotoxicity activity
MTT proliferation assay: MCF-7 cells were purchased from ATCC
and cultured in DMEN containing 10% FBS. For 3-(4,5 dimethylthi-
azol-2-yl)-2,5-diphenyltetrazolium (MTT) (Sigma–Aldrich, St.
Louis, MO) analysis, cells were plated in 96-well plates at 10,000
cells/well. Twenty-four hours after plating, vehicle (0.1% DMSO,
final concentration) or compound was added to cells at indicated
doses. Seventy-two hours following compound addition, MTT
was added to each well (0.5 mg/mL, final concentration) and plates
were incubated for an additional 3 h at 37 °C. Medium was then
aspirated and the formazan product was solubilized in SDS–HCl
(20% SDS; HCl 0.02 M). The absorbance of each well was measured
at 570 nm using a microplate reader.