The evaluation of quinonoid compounds against Trypanosoma cruzi: Synthesis of imidazolic anthraquinones, nor-β-lapachone derivatives and β-lapachone-based 1,2,3-triazoles

Article abstract of DOI:10.1016/j.bmc.2010.03.029

In continuing our screening program of naphthoquinone activity against Trypanosoma cruzi, the aetiological agent of Chagas' disease, new β-lapachone-based 1,2,3-triazoles, 3-arylamino-nor-β-lapachones, 3-alkoxy-nor-β-lapachones and imidazole anthraquinones were synthesised and evaluated against bloodstream trypomastigote forms of the parasite. Compounds 2,2-dimethyl-3-(2,4-dibromophenylamino)-2,3-dihydro-naphtho[1,2-b]furan-4,5-dione, IC₅₀/24 h 24.9 ± 7.4 and 4-azido-3-bromo-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione with 23.4 ± 3.8 μM showed a trypanosomicidal activity higher than benznidazole. These results demonstrate the potential of naphthoquinone derivatives as novel structures for the development of alternative drugs for Chagas' disease.

Full text of DOI:10.1016/j.bmc.2010.03.029

Bioorganic & Medicinal Chemistry 18 (2010) 3224–3230
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The evaluation of quinonoid compounds against Trypanosoma cruzi: Synthesis
of imidazolic anthraquinones, nor-b-lapachone derivatives and
b-lapachone-based 1,2,3-triazoles
Eufrânio N. da Silva Júnior a,b, Tiago T. Guimarães a, Rubem F. S. Menna-Barreto c,
Maria do Carmo F. R. Pinto a, Carlos A. de Simone d,e, Claudia Pessoa g, Bruno C. Cavalcanti g, José R. Sabino f,
a
Carlos Kleber Z. Andrade b, Marilia O. F. Goulart d, Solange L. de Castro c, , Antônio V. Pinto
*
a Núcleo de Pesquisas de Produtos Naturais, UFRJ, 21941-971, Rio de Janeiro, RJ, Brazil
b Instituto de Química, Universidade de Brasília, 70910-970, Brasília, DF, Brazil
c Laboratório de Biologia Celular, Instituto Oswaldo Cruz, FIOCRUZ, 21045-900, Rio de Janeiro, RJ, Brazil
d Instituto de Química e Biotecnologia, UFAL, 57072-970, Maceió, Alagoas, Brazil
e Departamento de Física e Informática do Instituto de Física, USP, 13560-160, São Carlos, SP, Brazil
f Instituto de Física, UFG, 74001-770, Goiânia, GO, Brazil
g Departamento de Fisiologia e Farmacologia, UFC, 60430-270, Fortaleza, CE, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
In continuing our screening program of naphthoquinone activity against Trypanosoma cruzi, the aetiolog-
ical agent of Chagas’ disease, new b-lapachone-based 1,2,3-triazoles, 3-arylamino-nor-b-lapachones, 3-
alkoxy-nor-b-lapachones and imidazole anthraquinones were synthesised and evaluated against blood-
stream trypomastigote forms of the parasite. Compounds 2,2-dimethyl-3-(2,4-dibromophenylamino)-
2,3-dihydro-naphtho[1,2-b]furan-4,5-dione, IC50/24 h 24.9 7.4 and 4-azido-3-bromo-2,2-dimethyl-
Received 7 January 2010
Revised 11 March 2010
Accepted 12 March 2010
Available online 16 March 2010
3,4-dihydro-2H-benzo[h]chromene-5,6-dione with 23.4 3.8 lM showed a trypanosomicidal activity
Keywords:
higher than benznidazole. These results demonstrate the potential of naphthoquinone derivatives as
novel structures for the development of alternative drugs for Chagas’ disease.
Ó 2010 Elsevier Ltd. All rights reserved.
Naphthoquinones
Nor-b-lapachone
Chagas’ disease
Trypanosoma cruzi
Experimental chemotherapy
1. Introduction
ever, as also noted with other diseases, the development of new
drugs for Chagas’ disease is not of interest to pharmaceutical
Chagas’ disease, caused by the protozoan parasite Trypanosoma
cruzi, remains a major health problem in Latin America. The trans-
mission of this disease occurs mostly by vector, but it can also
be transmitted by blood transfusion and through congenital
pathways.1 Rarer instances of transmission occur by laboratory
accidents,2 organ transplantations3,4 and the ingestion of contam-
inated food with infected triatomines or their dejection.5,6
Recently, Chagas’ disease has also been recognised as an opportu-
nistic disease in HIV-infected individuals.7
Introduced in the 1960s and 1970s, nifurtimox and benznidaz-
ole are the nitroderivatives currently available for the treatment of
Chagas’ disease. While these drugs are effective for acute infec-
tions, the data regarding their use and efficacy during the chronic
phase are still controversial primarily due to the undesirable side
effects and poor indices of apparent cure from the disease.8 How-
industries in developed countries due to a lack of potential profits.9
In T. cruzi, the presence of a single mitochondrion and a specia-
lised region rich in DNA named the kinetoplast,10 make this orga-
nelle an extraordinary drug target.11 In addition, the T. cruzi
mitochondrion is known to be deficient in reactive oxygen, making
it especially sensitive to oxidative stress conditions.12 In this con-
text, since the microbicidal mechanism of quinones is mainly re-
lated to the generation of reactive oxygen species,13 these
molecules are interesting for medicinal chemistry studies.14
Among the bioactive quinones, lapachol (1) and b-lapachone (14)
originally isolated from the heartwood of trees of the Bignoniaceae
family (Tabebuia sp.)15 were selected as starting points for the
screening of trypanosomicidal drugs16 because they are easily
accessible in Brazilian flora and facile synthetic routes have been
previously developed when exploring the reactivity of 1,2-quinoi-
dal carbonyls.17
Taking in account that molecular hybridisation18 is a powerful
approach for the design of new compounds, quinone derivatives
* Corresponding author. Fax: +55 21 2260 44 34.
E-mail address: solange@ioc.fiocruz.br (S.L. de Castro).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.029

E. N. da Silva Júnior et al. / Bioorg. Med. Chem. 18 (2010) 3224–3230
3225
were prepared by reaction of naphthoquinones with [1,2,3]-tria-
zoles or arylamines and evaluated as potential anti-T. cruzi com-
pounds. Recently, several triazole derivatives (nor-b-lapachone-
based 1,2,3-triazoles) were prepared from nor-lapachol (2), and
the most active was 2,2-dimethyl-3-(4-phenyl-1,2,3-triazol-1-yl)-
2,3-dihydro-naphtho[1,2-b]furan-4,5-dione,19 with a phenyl group
attached to the triazolic ring, which, due to its higher lipophylic
character, could allow better penetration through the parasite’s
plasma membrane as reported.19 The screening of a series of
ortho-naphthodihydrofuranquinones obtained from the reaction
of 3-bromo-nor-b-lapachone (3) with substituted arylamines re-
vealed two derivatives with a high activity on trypomastigote
forms better than that of benznidazole.20 Such hybrid molecules
obtained from quinones and triazoles with redox properties are
an interesting prospect for a medicinal chemistry program directed
toward the chemotherapy of Chagas’.
Continuing our studies on the chemical reactivity of quinones
from the Brazilian flora, we now focus on the search of new com-
pounds with trypanosomicidal activity. We synthesised and char-
acterised six novel derivatives (16–21) of lapachol (1) and two
imidazolic anthraquinones (25 and 27), that together with 13 other
compounds previously synthesised (4–13, 22, 24 and 26) were as-
sayed for their activity against infective bloodstream forms of T.
cruzi and cytotoxicity mammalian cells.
compounds 18–21 were obtained in racemic form, but the stereo-
chemistry trans were defined by X-ray crystallography.
Other types of naphthoquinones were included for trypanoso-
micidal activity screening. The first substance was derived from
the reaction of naphthoquinones with lead dioxide in acetic acid,
a type of reaction first described by Hooker in 1936.26 Using lapa-
chol (1), we obtained the corresponding alkoxyquinone 22
(Scheme 1), which was purified by recrystallisation in ethanol,
since the use of column chromatography on silica gel led to degra-
dation of this compound.27
The second series are the anthraquinone imidazoles 24–27
described here for the first time, except for compound 24 and
26.28 All are easily obtained from the reaction of 1,2-diamino-
anthraquinone (23) and substituted aldehydes (Scheme 2).
The structures of the compounds 16–21 and 25–27 were deter-
mined by 1H and 13C NMR and IR by comparing their spectroscopic
data with those of similar compounds already reported29 and to-
gether with high-resolution (electrospray ionisation) mass spectra.
For the novel compounds, b-lapachone-based 1,2,3-triazoles, the
chemical shift of the hydrogens of the pyranic ring appears in the
same region (d 5.00–6.00). In general, the signals of the hydrogens
of the triazole ring appear in the aromatic region (d 8.00) and the
others signals corresponding to the substitution pattern observed
for each substance.
The structures of compounds 16 and 17 were reconfirmed by X-
ray crystallography data studies (Fig. 1). The ring trans-substitution
is clearly observed in both cases.
2. Results and discussion
2.1. Chemistry
2.2. X-ray analysis
Lapachol (1) (2-hydroxy-3-(30-methyl-20-butenyl)-1,4-naphtho-
quinone) was extractedfrom theheartwood of Tabebuia sp. (Tecoma)
and purified by a series of recrystallisations. From this naphthoqui-
none, nor-lapachol (2) (2-hydroxy-3-(20-methyl-propenyl)-1,4-
naphthoquinone) was obtained by Hooker oxidation method,21 in
which the presence of molecular bromine undergoes cyclisation
and produces in situ 3-bromo-nor-b-lapachone (3). This bromo
derivative by reaction with arylamines led to the synthesis of aryla-
mino-nor-b-lapachones 4–10, as previously reported.22,23 When
compound 3 is submitted to nucleophilic substitution with alcohols,
alkoxy-nor-b-lapachones 11–13 were formed24 (Scheme 1). Only
three 3-alkoxy nor-b-lapachones were obtained due to difficulty in
removal of the solvent and reagents after the end of the reaction un-
der our laboratory conditions. Thereactions usingalcohols with high
boiling points failed due to the difficulty of product isolation.
The b-lapachone-based 1,2,3-triazoles 18–21, described herein
for the first time, were obtained from b-lapachone (14), synthes-
ised by cyclisation of lapachol (1) in the presence of H2SO4. Sub-
stance 14 reacted with N-bromosuccinimide (NBS) in CCl4 with
benzoyl peroxide as initiator to produce 3,4-dibromo-b-lapachone
(15).24 Compound 15 was stirred overnight with sodium azide in
The molecular dimensions in each of the two structures are
essentially in agreement with expected values, in particular, those
found among the pyranic rings reported in the Cambridge Structural
Database,30 with exception of the compound 17 in which one of the
methyl groups is disordered over two sets of sites C13 and C130 with
refined occupancies of 0.843(2) and 0.157(3). However hydrogen
atoms of this methyl group do no participate in intermolecular
hydrogen bond in the packing. The pyranic ring in the structure of
16 adopts a twisted boat conformation. The structure of 17 also
adopts a half boat conformation, as assessed by the ring puckering
q2 = 0.769(1), q3 = ꢀ0.037(1), Q = 0.769(1) Å, h = 92.72(9), u =
209.76° for 16 and q2 = 1.10(3), q3 = ꢀ0.47(4), Q = 1.20(3) Å, h =
113.0(2), u = 0.0(2)° for 17.31 The molecular packing involves weak
intermolecular hydrogen contacts in both structures: Compound
16 presents one weak interaction between C6–H6ꢁ ꢁ ꢁO1i where C6–
H6 = 0.930 Å; H6ꢁ ꢁ ꢁO1i = 2.58(3) Å and C6–H6ꢁ ꢁ ꢁO1i = 139° [i =
ꢀx + 1, ꢀy, ꢀz]. Compound 17 presents two weak interaction be-
tween C2–H2ꢁ ꢁ ꢁO2i where C2–H2 = 0.980 Å; H2ꢁ ꢁ ꢁO2i = 2.42(1) Å;
C2–H2ꢁ ꢁ ꢁO2i = 143° [i = ꢀx ꢀ 1/2, ꢀy + 1/2, z + 1/2] and C2–
H2ꢁ ꢁ ꢁO3i where C2–H2 = 0.980 Å; H2ꢁ ꢁ ꢁO3i = 2.55(3) Å; C2–
H2ꢁ ꢁ ꢁO3i = 137° [i = ꢀx ꢀ 1/2, ꢀy + 1/2, z + 1/2].
dichloromethane and 16 (3-bromo-4-azide-a-lapachone) and 17
(3-bromo-4-azide-b-lapachone) were obtained. A hypothesis that
can be considered for the formation of the compounds 16 and 17
is that after the formation of the compound 15 (3,4-dibromo-b-
lapachone) may occur the exit of the bromine of the position 3,
by anchimeric assistance of the oxygen in position 1, resulting
in an unstable pyran based-oxygen carbenium ion that suffers
nucleophilic attack through its less hindered side and the stereo-
chemistry trans was observed for both compounds 16 and 17.
The formation of compound 16 occurs due to isomerisation of
3-bromo-4-azide-b-lapachone (17). After separating substances
16 and 17 by column chromatography, compound 17 was submit-
ted to azide-alkyne Huisgen’s cycloaddition, producing the new
1,2,3-triazolic compounds 18–21. This reaction catalysed by
Cu(I), which is known in the literature as ‘click chemistry’.25 The
2.3. Biological assays
We recently reported the synthesis of a series of 3-arylamino-
nor-b-lapachones substituted by arylamine moiety containing
electron withdrawing and electron donating groups20,22 and these
compounds were evaluated against T. cruzi trypomastigotes. Other
compounds we recently reported19 were the nor-b-lapachone-
based 1,2,3-triazoles with potent trypanosomicidal and antitumor
activities.
In this paper, the anti-T. cruzi activity of the b-lapachone-based
1,2,3-triazoles (18–21), 3-alkoxy-nor-b-lapachones (11–13) and 3-
arylamino-nor-b-lapachones (4–10) with other types of arylamino
rings, obtained from modification of the C ring (Scheme 3, Table 1),
are described.

3226
E. N. da Silva Júnior et al. / Bioorg. Med. Chem. 18 (2010) 3224–3230
4 - R = R = Br, R = R = H
1
3
2
4
O
5 - R = R = Cl, R = R = H
2
3
1
4
6 - R = NO , R = R = H, R = OMe
O
1
2
2
4
3
R1
R4
7 - R = Me, R = R = H, R = NO
1
2
3
4
2
H
8 - R = R = H, R = NO , R = F
N
R2
R3
1
4
2
2
3
9 - R = NO , R = R = R = H
1
2
2
3
4
O
10 - R = Me, R = R = H, R = NO
1
2
4
3
2
O
O
65-95%
O
O
NH
2
O
R
R
1
O
R
4
22, 90%
2
R
3
CH Cl
2
2
PbO
2
AcOH
O
O
O
O
O
O
OH
Br
OH
2
HO
R
Hooker
Oxidation
O
Br
CHCl
3
R
O
O
O
O
O
1
3
2
11 R = CH(CH )
12 R = CH CH(CH )
3 2
H SO
2
4
2
3 2
13 R =
O
O
O
O
NBS
CCl
Br
60-95%
4
O
Benzoyl Peroxide
Br
15
14
NaN
3
CH Cl
2
2
R
O
O
O
O
O
O
R
O
N
18 R = H
19 R = Br
20 R = F
O
CH Cl /H O 1:1
2
2
2
N
+
N3
Br
N
CuSO .5H O
Na-ascorbate
4
2
Br
21 R = OMe
Br
O
N3
16, 30%
17, 35%
40-65%
Scheme 1. Synthetic route for the preparation of 4–10, 11–13, 16–21 and 22.
R
R
3
R
2
CHO
CHO
R1
O
NH
O
O
HN
2
1
N
O
O
HN
NH
2
R2
N
R3
AcO Na+, AcOH
AcO-Na+, AcOH
Reflux
-
O
23
Reflux
24 - R1 = R2 = R3 = H
25 - R1 = R2 = H, R3 = Ph
27
26 - R1 = R2 = H, R3 = CH3
Scheme 2. Synthetic route for the preparation of the imidazolic anthraquinones 24–27.
The arylamino rings of 4–10 were substituted by electron with-
drawing (–Br, –Cl, –NO2 and –F) or electron donating groups
(–OMe, –Me), as well as the aromatic triazoles (–Br, –F and –OMe).
Important trypanosomicidal compounds and structural features
were defined from present studies with comparisons of previously
reported data,19a,20 which include: (i) the difference of the activity
observed between lapachol (1) and nor-lapachol (2) is reproduced
in the case of b-lapachone (14) and nor-b-lapachone (28); and (ii)
the 3-substituted arylaminonaphthoquinones provided four deriv-
and nature of the substituents play important roles, particularly
for halogens in the arylamino ring. In the bromo derivatives [m-
Br (140.8 11.9
the second bromine atom led to increase of the biological activity
[2,4-Br, compound (4) (24.9 7.4 M)]; the same trend was
observed for the chloro derivatives [m-Cl (332.8 23.3 M); p-Cl
(384.4 52.5
M)]20 and 3,4-dichloro, compound (5) (43.8
4.2 M)]. The presence of the atom of fluor does not enhance the
trypanosomicidal activity as previously shown [m-F (>4000 M,
p-F (2517.9 169.8
M)].20 The substitution by nitro groups
enhances the anti-T. cruzi effect, except in p-substitution [o-NO2,
compound 9, (55.6 4.6 M); m-NO2 (86.3 4.6
M),20 p-NO2
(857.3 96.4
M)20]. As a second substituent, in the case of
lM); p-Br (952.5 71.1 l
M)],20 the presence of
l
l
l
l
l
atives in structural classes [4 (24.9 7.4
(59.6 13.2 M) and 9 (55.6 4.6 M)] with activity between
about 1.7 and 4.2 higher than that of the standard drug, benznidaz-
ole (103.6 0.6
M).19a In this class of compounds, the position
l
M), 5 (43.8 4.2
l
M), 6
l
l
l
l
l
l
l

E. N. da Silva Júnior et al. / Bioorg. Med. Chem. 18 (2010) 3224–3230
3227
Figure 1. ORTEP3 projection of the compounds (a): 16 and (b): 17, showing the atom numbering and 50% probability displacement ellipsoids.
O
O
O
O
Br
O
H
N
C
C ring modification
N
N
O
N
C
4
Br
O
O
O
Br
21
active
O
active
O
insertion of 1,2,3-triazole
group or azide group in
the C ring
24.9 µM*
Insertion of
groups
in the C ring
313.0 µM*
C
C
O
O
O
28
O
O
14
inactive
> 4.800 µM*
low activity
N N N
Br
O
391.5 µM*
C
C
O
O
17
12
active
23.4 µM*
active
47.2 µM*
Scheme 3. New compounds active against T. cruzi. *Mean values of IC50/24 h (see Table 1).
methoxy derivative [p-OMe (88.2 6.7
siderably increased [compound 6 (59.6 13.2
of a methyl group in position 2 of the arylamino ring stunts the
activity in compound 7 (284.3 60.9 M); however, this methyl
group is highly favourable in compound 10 (156.2 9.1 M). De-
l
M)],20 the activity is con-
M)]. The presence
The anthraquinone imidazoles 24–27 were not active against T.
cruzi trypomastigote forms. Previous studies have shown that p-
naphthoquinones are not active compounds against T. cruzi. Based
on previous results,17 we insert the imidazolic nucleus in attempt
to increase activity for p-naphthoquinones, but the obtained re-
sults were not promising.
l
l
l
spite the impossibility of rationalisation, it seems that the substitu-
tion in position 2 is favourable.
The cytotoxicity of the compounds against mammalian cells
(human peripheral blood mononuclear cells—PBMC) was also
investigated, and the IC50 values obtained for the majority of the
compounds were in the range of 2.1–10 lM, while for 7, 22, 24–
26 and benznidazole the values were higher than 12 lM.
Compound 12 (47.2 4.1 lM), from the 3-substituted alkoxy-
quinones, is two times more active than benznidazole,19a indicat-
ing that the insertion of alkoxy groups intensifies the activity of
nor-b-lapachone (28).
The key intermediate azide 17 used for the synthesis of b-
lapachone-based 1,2,3-triazoles 18–21 displays an important activ-
3. Conclusions
ity against the parasite, with an IC50/24 h of 23.4 3.8 lM, about
To obtain trypanosomicidal compounds, we synthesised and
evaluated the activity of nor-b-lapachones 3-arylamino and 3-alk-
oxy substituted, imidazolic anthraquinones and b-lapachone-
based 1,2,3-triazoles. In comparison with previous studies, we con-
cluded that in a nor-b-lapachones-3-arylamino-substituted com-
pound, the insertion of chlorine, bromine, nitro and methoxy
groups in the arylamino ring intensifies the activity, while fluorine
is harmful for trypanosomicidal activity. By the Alamar blue test, it
was observed that after 72 h of treatment, the IC50 values were
lower than those obtained for the trypanosomicidal activity, mon-
itored after 24 h. This result points to toxicity of the compounds for
four times more active than the standard drug and seventeen times
more active than its precursor, b-lapachone (14). Nor-b-lapachone
azide-substituted was recently reported19a to have important
activity with IC50/24 h of 50.2 3.8 lM, and this result highlights
the importance of the azide-substituted lapachones. In comparison
with b-lapachone (14), the substitution by a triazole ring appears to
be a promising approach. b-Lapachone-based 1,2,3-triazole 18 with
an IC50/24 h value of 313.0 26.4
compound 14. However, compound 21, with IC50/24 h of
106.1 19.0 M, presented a high lytic activity against T. cruzi that
was 3 times more responsive than b-lapachone (14).
lM is slightly more active than
l

3228
E. N. da Silva Júnior et al. / Bioorg. Med. Chem. 18 (2010) 3224–3230
Table 1
amounts of benzoyl peroxide. The reaction mixture was gently
heated to near vaporisation and the product formed, 15, was ob-
served by TLC. Compound 15 was treated with an excess of sodium
azide (2 mmol, 130 mg) at room temperature. The reaction system
was stirred for 24 h, filtered and the compounds 16 and 17 were
isolated by column chromatography over silica gel, using an eluent
as a gradient mixture of hexane/ethyl acetate with increasing
polarity. Compound 16 was isolated as an orange solid (108 mg,
0.3 mmol, 30% yield, mp 163–164).
Activity of napththoquinones and precursors on trypomastigote forms of T. cruzi
Compounds
IC50/24 ha
(lM)
4
5
6
7
8
9
10
11
12
13
16
17
18
19
20
21
24.9 7.4
43.8 4.2
59.6 13.2
284.3 60.9
526.2 80.5
55.6 4.6
156.2 9.1
268.3 29.2
47.2 4.1
558.5 77.1
248.3 29.1
23.4 3.8
313.0 26.4
439.6 31.6
219.8 27.2
106.1 19.0
233.3 29.2
>2000
>4000
>4000
>4000
410.8 53.5
1280.6 167.2
391.5 16.5
>4800
IR mmax (cmꢀ1, KBr): 1682 (C@O), 1652 (C@O), 2105 (N3); 1H
NMR (300 MHz, CDCl3) d: 8.18–8.11 (2H, m), 7.82–7.70 (2H, m),
5.02 (1H, d, J = 5.1 Hz), 4.15 (1H, d, J = 5.1 Hz), 1.65 (3H, s), 1.64
(3H, s); 13C NMR (75 MHz, CDCl3) d: 183.3 (C@O), 178.9 (C@O),
153.4 (C0), 134.4 (CH), 133.5 (CH), 131.6 (C0), 130.7 (C0), 126.5
(CH), 126.3 (CH), 116.1 (C0), 80.7 (C0), 58.8 (CH), 54.0 (CH), 25.4
(CH3), 25.0 (CH3). EI-HRMS (m/z) [M+Na]+ 383.9953. Calcd for
[C15H12BrN3O3Na]+: 383.9959.
22
24
25
26
Compound 17 was isolated as an orange solid (126 mg,
0.3 mmol, 35% yield, mp 186–187).
IR mmax (cmꢀ1, KBr): 1654 (C@O), 1702 (C@O), 2107 (N3); 1H
NMR (300 MHz, CDCl3) d: 8.14 (1H, dd, J = 7.5; 1.2 Hz), 7.88 (1H,
dd, J = 7.8; 1.2 Hz), 7.72 (1H, td, J = 7.5; 1.4 Hz), 7.26 (1H, td,
J = 7.5; 1.2 Hz), 4.96 (1H, d, J = 5.1 Hz), 4.17 (1H, d, J = 5.1 Hz),
1.71 (3H, s), 1.67 (3H, s); 13C NMR (75 MHz, CDCl3) d: 178.6
(C@O), 178.3 (C@O), 162.2, 135.4, 132.3, 131.0, 130.6, 129.3,
125.4, 110.3, 81.9, 59.2, 54.3. EI-HRMS (m/z) [M+Na]+ 383.9946.
Calcd for [C15H12BrN3O3Na]+: 383.9959.
27
Lapachol (1)b
Nor-lapachol (2)b
b-Lapachone (14)b
Nor-b-lapachone (28)b
Benznidazolec
103.6 0.6
a
b
c
Mean SD of at least three independent experiments.
Ref. 16a.
Ref. 19a.
4.3. General procedure for the synthesis of the b-lapachone-
based 1,2,3-triazoles 18–21
PBMC, however since compounds 4 and 17 were about 4 times
more active than benznidazole, they will be further investigated
in relation to the in vivo toxicity monitoring hepatic, renal and car-
diac muscle marker. The compounds, 3-bromo-4-azide-b-lapach-
one (17) and b-lapachone-based 1,2,3-triazoles were recognised
as important prototypes and can aid in the development of new
anti-T. cruzi agents.
CuSO4ꢁ5H2O (9.3 mg, 0.04 mmol), sodium ascorbate (22 mg,
0.11 mmol) and the desired substituted phenylacetylene (see be-
low) were added to a solution of 17 (299 mg, 0.83 mmol) in
12 mL CH2Cl2/H2O (1:1). The mixture was stirred at room temper-
ature for 12 h and was monitored by TLC. The organic phase was
extracted with CH2Cl2, dried over Na2SO4 and concentrated under
reduced pressure. The obtained residue was purified by column
chromatography on silica gel, using an eluent as a gradient mixture
of hexane/ethyl acetate with increasing polarity. The alkynes used
were the following: to synthesise 18, phenylacetylene; for 19, 1-
bromo-4-ethynylbenzene; for 20, 1-ethynyl-4-fluorobenzene and
for 21, 1-ethynyl-4-methoxybenzene.
4. Experimental protocols
4.1. General procedures
Melting points were determined in a capillary Thomas Hoover
apparatus (Thomas Co., Philadelphia, PA, USA) and are uncorrected.
Analytical grade solvents were used. Column chromatography was
performed on silica gel (Acros Organics 0.035–0.070 mm, pore
diameter ca. 6 nm). Infrared spectra were recorded on a Perkin–El-
mer FT-IR Spectrometer (Perkin–Elmer Inc., Wellesley, MA, USA).
1H and 13C NMR were recorded at room temperature using a Varian
Mercury Plus 300 instrument (Varian, Palo Alto, CA, USA) in the
solvents indicated with TMS as an internal standard. Chemical
shifts (d) are given in ppm and coupling constants (J) are reported
in Hertz. High-resolution mass spectra (electrospray ionisation)
were obtained using a MicroTOF Ic—Bruker Daltonics. (Bruker–
Franzen Analytic, Bremen, Germany). Elemental analysis was per-
formed in a Perkin–Elmer CHN 2400 analyser (Perkin–Elmer Inc.,
Wellesley, MA, USA).
4.3.1. 3-Bromo-2,2-dimethyl-4-(4-phenyl-1,2,3-triazol-1-yl)-
3,4-dihydro-2H-benzo[h] chromene-5,6-dione (18)
Using phenylacetylene (84 mg, 0.83 mmol), 18 was obtained as
an orange solid (300 mg, 0.6 mmol, 65% yield, mp 184–185 °C).
IR mmax (cmꢀ1 KBr): 1700 (C@O), 1652 (C@O); 1H NMR
,
(300 MHz, CDCl3) d: 8.13–8.08 (2H, m), 8.09 (1H, s), 7.95–7.92
(1H, m), 7.87–7.82 (1H, m), 7.79–7.71 (1H, m), 7.68–7.59 (1H,
m), 7.45–7.39 (2H, m), 7.35–7.29 (1H, m), 5.72 (1H, d, J = 8.8 Hz),
5.07 (1H, d, J = 8.8 Hz), 1.78 (3H, s), 1.69 (3H, s); 13C NMR
(75 MHz, CDCl3) d: 177.8 (C@O), 176.5 (C@O), 162.7 (C0), 146.7
(C0), 135.1 (CH), 132.2 (CH), 130.6 (C0), 130.4 (C0), 129.1 (CH),
128.6 (CH), 128.0 (CH), 125.7 (CH), 125.2 (CH), 122.7 (CH), 110.3
(C0), 83.4 (C0), 58.9 (CH), 54.3 (CH), 24.4 (CH3), 20.7 (CH3). EI-HRMS
(m/z) [M+Na]+ 486.0404. Calcd for [C23H18BrN3O3Na]+: 486.0429.
The compounds 4–10, 11–13, 22, 24 and 26 were synthesised as
previously described.22,27,28b
4.3.2. 3-Bromo-4-[4-(4-bromo-phenyl)-1,2,3-triazol-1-yl]-2,2-
dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (19)
Using 1-bromo-4-ethynylbenzene (148 mg, 0.83 mmol), 19 was
obtained as an orange solid (280 mg, 0.5 mmol, 52% yield, mp 185–
186 °C).
4.2. Synthesis of 4-azido-3-bromo-2,2-dimethyl-3,4-dihydro-
2H-benzo[g]chromene-5,10-dione (16) and 4-azido-3-bromo-
2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (17)
IR mmax (cmꢀ1 KBr): 1701 (C@O), 1647 (C@O); 1H NMR
,
b-Lapachone (14) (242 mg, 1 mmol) in carbon tetrachloride
(80 mL) was reacted with NBS (352 mg, 2 mmol) and catalytic
(300 MHz, CDCl3) d: 8.13–8.09 (1H, m), 8.09 (1H, s), 7.95–7.88

E. N. da Silva Júnior et al. / Bioorg. Med. Chem. 18 (2010) 3224–3230
3229
(1H, m), 7.78–7.76 (3H, m), 7.66–7.59 (1H, m), 7.56–7.50 (2H, m),
5.70 (1H, d, J = 9.0 Hz), 5.04 (1H, d, J = 9.0 Hz), 1.79 (3H, s), 1.68
(3H, s); 13C NMR (75 MHz, CDCl3) d: 177.8 (C@O), 176.5 (C@O),
162.7 (C0), 145.7 (C0), 135.2 (CH), 132.3 (CH), 131.8 (C0), 130.6
(C0), 130.4 (C0), 129.3 (C0), 129.2 (CH), 127.3 (CH), 125.3 (CH),
122.9 (CH), 121.9 (C0), 110.2 (C0), 83.5 (C0), 59.1 (CH), 54.2 (CH),
27.4 (CH3), 20.6 (CH3). EI-HRMS (m/z) [M+Na]+ 563.9538. Calcd
for [C23H17Br2N3O3Na]+: 563.9534.
127.5, 126.9, 125.6, 121.7, 119.4. Anal. Calcd for C27H16N2O2: C.
80,99; H. 4,03; N. 7,00. Found: C, 81.05; H, 4.09; N, 7.08.
4.4.2. 2-Naphthalen-1-yl-1H-anthra[1,2-d]imidazole-6,11-dione
(27)
Using 1-naphthaldehyde (172 mg, 1,1 mmol), 27 was obtained
as a yellow solid (190 mg, 0.51 mmol, 51% yield, mp 263–267 °C).
IR mmax (cmꢀ1, KBr): 3600, 3487, 3443, 3420, 3060, 3010, 2923,
2853, 1670, 1606, 1583, 1519, 1504, 1488, 1475, 1438, 1329, 1294,
1214, 1182, 1154, 1063, 1003, 855, 804, 772, 715, 563, 488. 1H
NMR (300 MHz, CDCl3) d: 8.86 (1H, d, J = 8.23 Hz), 8.37 (1H, dd,
J = 7.17, 1.49 Hz), 8.30 (1H, d, J = 8.38 Hz), 8.29–8.22 (2H, m)
8.08–7.93 (3H, m), 7.87–7.75 (2H, m), 7.69–7.55 (3H, m). 13C
NMR (75 MHz, DMSO-d6) d: 182.9, 182.5, 157.7, 134.3, 134.1,
133.2, 133.0, 130.7, 129.6, 128.3, 128.1, 127.1, 126.6, 126.1,
125.7, 124.9. Anal. Calcd for C25H14N2O2: C, 80.20; H, 3.77; N,
7.48. Found: C, 80.25; H, 3.81; N, 7.52.
4.3.3. 3-Bromo-4-[4-(4-fluoro-phenyl)-1,2,3-triazol-1-yl]-2,2-
dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (20)
Using 1-ethynyl-4-fluorobenzene (99 mg, 0.83 mmol), 20 was
obtained as an orange solid (192 mg, 0.4 mmol, 40% yield, mp
182–183 °C).
IR mmax (cmꢀ1 KBr): 1701 (C@O), 1652 (C@O); 1H NMR
,
(300 MHz, CDCl3) d: 8.14–8.09 (1H, m), 8.09 (1H, s), 7.95–7.92
(1H, m), 7.84–7.71 (3H, m), 7.68–7.60 (1H, m), 7.15–7.06 (2H,
m), 5.70 (1H, d, J = 9.0 Hz), 5.05 (1H, d, J = 9.0 Hz), 1.79 (3H, s),
1.69 (3H, s); 13C NMR (75 MHz, CDCl3) d: 177.8 (C@O), 176.5
(C@O), 162.8 (C0), 145.9 (C0), 133.2 (CH), 132.3 (CH), 130.6 (C0),
130.4 (C0), 129.1 (CH), 127.6 (CH), 127.5 (CH), 126.7 (C0,
J = 3.3 Hz), 125.3 (CH), 122.6 (CH), 115.8 (CH), 115.5 (CH), 110.3
(C0), 83.5 (C0), 59.0 (CH), 54.3 (CH), 27.4 (CH3), 20.7 (CH3). EI-HRMS
(m/z) [M+Na]+ 504.0324. Calcd for [C23H17BrFN3O3Na]+: 504.0335.
4.5. Trypanosomicidal activity
Stock solutions of the compounds were prepared in dimethyl-
sulfoxide (DMSO) with the final concentrations in the experiments
never exceeding 0.1%. Preliminary experiments showed that at
concentrations up to 0.5% DMSO had no deleterious effect on the
parasites. Bloodstream trypomastigotes from the Y strain32 were
obtained at the peak of parasitemia from infected albino mice, iso-
lated by differential centrifugation and resuspended in Dulbecco’s
modified Eagle’s medium (DMEM) to a parasite concentration of
107 cells mLꢀ1 in the presence of 10% of mouse blood. This suspen-
4.3.4. 3-Bromo-4-[4-(4-methoxy-phenyl)-1,2,3-triazol-1-yl]-2,2-
dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (21)
Using 1-ethynyl-4-methoxybenzene (109 mg, 0.83 mmol), 21
was obtained as an orange solid (271 mg, 0.5 mmol, 55% yield,
mp 187–188 °C).
sion (100 lL) was added in the same volume to each compound
previously prepared at twice the desired final concentrations. Cell
counts were performed in Neubauer chamber, and the trypanoso-
micidal activity was expressed as IC50, corresponding to the con-
centration that leads to lysis of 50% of the parasites.
IR mmax (cmꢀ1 KBr): 1707 (C@O), 1654 (C@O); 1H NMR
,
(300 MHz, CDCl3) d: 8.10–8.06 (1H, m), 8.01 (1H, s), 7.94–7.88
(1H, m), 7.79–7.69 (3H, m), 7.65–7.58 (1H, m), 6.97–6.90 (2H,
m), 5.71 (1H, d, J = 8.8 Hz), 5.04 (1H, d, J = 8.8 Hz), 3.83, (3H, s),
1.76 (3H, s), 1.68 (3H, s); 13C NMR (75 MHz, CDCl3) d: 177.9
(C@O), 176.5 (C@O), 162.7 (C0), 159.5 (C0), 146.6 (C0), 135.2 (CH),
132.3 (CH), 130.6 (C0), 130.5 (C0), 129.1 (CH), 127.1 (CH), 125.2
(CH), 125.1 (CH), 123.1 (C0), 122.0 (CH), 114.1 (CH), 110.4 (C0),
83.4 (C0), 58.9 (CH), 55.2 (CH3), 54.3 (CH), 27.4 (CH3), 20.8 (CH3).
EI-HRMS (m/z) [M+Na]+ 516.0553. Calcd for [C24H20BrN3O4Na]+:
516.0535.
4.6. Inhibition of PBMC proliferation
To investigate the selectivity of the compounds toward a nor-
mal proliferating cell, the Alamar blue assay was performed using
peripheral blood mononuclear cells (PBMC).22 After 24 h of plating,
the compounds were added to each well and incubated for 72 h.
Doxorubicin was used as positive control. Twenty-four hours be-
fore the end of the incubation, 10 lL Alamar Blue (Resazurin, Sig-
4.4. General procedure for the synthesis of the antraquinone
imidazoles 25 and 27
ma–Aldrich Co) (0.312 mg/mL) was added to each well. The
absorbance was measured using a multiplate reader (DTX 880
Multimode Detector, Beckman CoulterÒ) and the drug effect was
quantified as the percentage of control absorbance at 570 nm
and 595 nm. The percent of reduced Alamar Blue is expressed as
ALW—(AHW ꢂ R0) ꢂ 100. R0 is the correction factor obtained by
the ratio AOLW/AOHW, corresponding AOLW to the difference be-
tween the absorbance of Alamar Blue in culture medium and that
of the medium alone at the lower wavelength and AOHW to the va-
lue obtained at the higher wavelength.
A
solution of 1,2-diamino-anthraquinone (23) (238 mg,
1 mmol) in 5 mL acetic acid, sodium acetate (106 mg, 1,3 mmol)
and the desired aldehyde (see below) were stirred in reflux. TLC
was used to monitor the end of the reaction. After the addition of
water, the formed precipitate was filtered, and the product was
purified by column chromatography using an eluent as a mixture
of dichloromethane/ethyl acetate with the ratio 5:1.
4.4.1. 2-Biphenyl-4-yl-1H-anthra[1,2-d]imidazole-6,11-dione
(25)
4.7. X-ray crystallographic analysis
Using 4-phenylbenzaldehyde (200 mg, 1.1 mmol), 25 was ob-
tained as a yellow solid (196 mg, 0.49 mmol, 49% yield, mp 269–
270 °C).
X-ray data collection for compounds 16 and 17 was accom-
plished on a Smart APEXII diffractometer and Enraf–Nonius Kap-
paCCD area-detector diffractometer, respectively. Data collection
strategy and frames integration were conducted using SAINT PLUS
and COLLECT program for the crystallographic study.33 Integration
and scaling of the reflections were performed with the HKL Den-
zo-Scalepack system of programs.34 The structures of 16 and 17
were resolved by direct methods with SHELXS-97.35 The models were
refined by full-matrix least squares on F2 with SHELXL-97.35 The
programs SHELXL-97 and ORTEP-336 were used within WinGX37 to
IR mmax (cmꢀ1, KBr): 3334, 1664, 1645, 1609, 1562, 1540, 1476,
1437, 1420, 1327, 1290, 1214, 1199, 1153, 1060, 1008, 843, 772,
738, 717, 702, 667, 651, 596, 509. 1H NMR (300 MHz, DMSO-d6)
d: 13.10 (1H, s), 8.50 (2H, d), 8.16–8.22 (2H, m), 8.03–8.10 (2H,
q), 7.75–7.92 (6H, m), 7.38–7.52 (3H, m). 13C NMR (75 MHz,
DMSO-d6) d: 183.9, 183.0, 158.1, 150.1, 143.1, 139.9, 135.1,
135.0, 133.9, 133.7, 133.7, 129.8, 129.5, 128.8, 128.6, 127.6,

3230
E. N. da Silva Júnior et al. / Bioorg. Med. Chem. 18 (2010) 3224–3230
Pinto, M. C. F. R.; Santos, R. H.; de Castro, S. L.; Pinto, A. V. Journal. Med. Chem.
2002, 45, 2112–2115; (d) Moura, K. C. G.; Emery, F. S.; Neves-Pinto, C.; Pinto, M.
C. F. R.; Dantas, A. P.; Salomão, K.; de Castro, S. L.; Pinto, A. V. Journal. Braz.
Chem. Soc. 2001, 12, 325; (e) Moura, K. C. G.; Salomão, K.; Menna-Barreto, R. F.
S.; Emery, F. S.; Pinto, M. C. F. R.; Pinto, A. V.; de Castro, S. L. Eur. Journal. Med.
Chem. 2004, 39, 639; (f) Pinto, A. V.; Menna-Barreto, R. F. S.; de Castro, S. L. In
Recent Progress in Medicinal Plants; Govil, J. N., Ed.; Phytomedicines; Studium
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prepare materials for publication. Crystallographic data for com-
pound 16 and 17 have been deposited with the Cambridge Crystal-
lographic Data Centre as Supplementary Publication No. CCDC
754510 and 754041, respectively. Copies of the data can be ob-
tained free of charge by application to CCDC, 12 Union Road, Cam-
bridge CH21EZ, UK (fax: +44 1223 336 033 or e-mail:
deposit@ccdc.cam.ac.uk).
17. (a) Pinto, A. V.; Pinto, M. C. F. R.; de Oliveira, C. G. T. An. Acad. Bras. Cien. 1982,
54, 107; (b) Pinto, A. V.; Pinto, M. C. F. R.; Aguiar, M. A.; Capella, R. S. An. Acad.
Bras. Cien. 1982, 54, 115; (c) Pinto, A. V.; Ferreira, V. F.; Pinto, M. C. F. R.; Mayer,
L. U. Synth. Commun. 1985, 15, 1181; (d) Lopes, C. C.; Lopes, R. S. C.; Pinto, A. V.;
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Acknowledgments
This research was supported by grants from the Conselho Nac-
ional de Desenvolvimento Científico e Tecnológico (CNPq), FIO-
CRUZ, Fundação Carlos Chagas Filho de Amparo à Pesquisa do Rio
de Janeiro (FAPERJ), CAPES, UFRJ, UFAL, USP, UFC and UnB. The
authors are also indebted to the PRONEX-FAPERJ and to PRONEX-
CNPq-FAPEAL. Dr. Eufrânio N. da Silva Júnior thanks the CNPq for
post doctoral grant (No. 151558/2009-4).
18. Viegas, C., Jr; Danuello, A. C.; Bolzani, V. S.; Barreiro, E. J.; Fraga, C. A. M. Curr.
Med. Chem. 2007, 14, 1829.
19. (a) da Silva Júnior, E. N.; Souza, M. C. B. V.; Pinto, A. V.; Pinto, M. C. F. R.;
Ferreira, V. F.; Menna-Barreto, R. F. S.; Silva, R. S. F.; Teixeira, D. V.; de Simone,
C. A.; de Castro, S. L. Eur. Journal. Med. Chem. 2008, 43, 1774; (b) da Silva Júnior,
E. N.; de Moura, M. A. B. F.; Pinto, A. V.; Pinto, M. C. F. R.; de Souza, M. C. B. V.;
Araújo, A. J.; Pessoa, C.; Costa-Lotufo, L. V.; Montenegro, R. C.; de Moraes, M. O.;
Ferreira, V. F.; Goulart, M. O. F. Journal. Braz. Chem. Soc. 2009, 20, 635.
20. da Silva Júnior, E. N.; de Souza, M. C. B. V.; Fernandes, M. C.; Menna-Barreto, R.
F. S.; Pinto, M. C. F. R.; Lopes, F. A.; de Simone, C. A.; Andrade, C. K. Z.; Pinto, A.
V.; Ferreira, V. F.; de Castro, S. L. Bioorg. Med. Chem. 2008, 16, 5030.
21. Fieser, L. F.; Fieser, M. Journal. Am. Chem. Soc. 1948, 70, 3215.
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V.; Montenegro, R. C.; de Moraes, M. O.; Pinto, M. C. F. R.; de Simone, C. A.;
Ferreira, V. F.; Goulart, M. O. F.; Andrade, C. K. Z.; Pinto, A. V. Journal. Med. Chem.
2010, 53, 504.
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M. O. F.; Pessoa, C.; Costa-Lotufo, L.; Montenegro, R. C.; Moraes, M. O.; Ferreira,
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