Synthesis, antiprotozoal and cytotoxic activities of new N-(3,4-dimethyl-5-isoxazolyl)-1,2-naphthoquinone-4-amino derivatives

Article abstract of DOI:10.1016/j.farmac.2004.03.003

Three derivatives of N-(3,4-dimethyl-5-isoxazolyl)-1,2-naphthoquinone-4- amino (1), a compound which exhibits significant activity against Trypanosoma cruzi and Plasmodium falciparum but with cytotoxicity toward murine L-6 cells, were synthesized with the aim of ameliorating its cytotoxicity. The in vitro antiprotozoal and cytotoxic activities of the synthesized compounds were evaluated against T. cruzi, Trypanosoma brucei rhodesiense, P. falciparum and murine L-6 cells. The hydroxymethyl (2) and the oxime (3) derivatives were active against T. cruzi, with IC₅₀ values in a range comparable to those of 1 (IC₅₀: 0.65 μg/ml) and benznidazole (IC₅₀: 0.56 μg/ml) while the carboxymethyloxime (4) was inactive. Compounds 2 and 3 were cytotoxic toward L-6 cells, with IC₅₀ values identical to that of 1 (IC₅₀: 0.50 μg/ml). The results did not support the suggestion that 2 and 3 may be used as prodrugs of 1.

Full text of DOI:10.1016/j.farmac.2004.03.003

IL FARMACO 59 (2004) 431–435
www.elsevier.com/locate/farmac
Synthesis, antiprotozoal and cytotoxic activities of new
N-(3,4-dimethyl-5-isoxazolyl)-1,2-naphthoquinone-4-amino derivatives
N.R. Sperandeo a,*, M.C. Briñón a, R. Brun b
a Dpto. de Farmacia, Faculdad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba 5000, Argentina
b Swiss Tropical Institute, Antiparasite Chemotherapy, P.O. Box, 4002 Basel, Switzerland
Received 28 October 2003; accepted 6 March 2004
Available online 20 April 2004
Abstract
Three derivatives of N-(3,4-dimethyl-5-isoxazolyl)-1,2-naphthoquinone-4-amino (1), a compound which exhibits significant activity
against Trypanosoma cruzi and Plasmodium falciparum but with cytotoxicity toward murine L-6 cells, were synthesized with the aim of
ameliorating its cytotoxicity. The in vitro antiprotozoal and cytotoxic activities of the synthesized compounds were evaluated against T. cruzi,
Trypanosoma brucei rhodesiense, P. falciparum and murine L-6 cells. The hydroxymethyl (2) and the oxime (3) derivatives were active
against T. cruzi, with IC50 values in a range comparable to those of 1 (IC50: 0.65 µg/ml) and benznidazole (IC50: 0.56 µg/ml) while the
carboxymethyloxime (4) was inactive. Compounds 2 and 3 were cytotoxic toward L-6 cells, with IC50 values identical to that of 1 (IC50:
0.50 µg/ml). The results did not support the suggestion that 2 and 3 may be used as prodrugs of 1.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Antiprotozoal agents; Quinones; Trypanocidal activity; Cytotoxicity
1. Introduction
from this study may shed light on whether these principles
could be subsequently used in the design of compounds for in
vivo evaluation. Thus, in the present work, three new deriva-
tives of 1 (compounds 2–4, Scheme 1) were synthesized and
tested for antiprotozoal and cytotoxic activities against T.
cruzi, Trypanosoma brucei rhodesiense, P. falciparum and
murine L-6 cells, respectively.
The epidemiological importance of American trypanoso-
miasis (Chagas’ disease) in human pathology is widely
known. The prognosis of this disease is serious and the
choice of effective medicines is limited; consequently, more
research is absolutely necessary to discover and develop new
trypanocidal drugs.
Recent studies from our laboratory [1] have shown
that N-(3,4-dimethyl-5-isoxazolyl)-1,2-naphthoquinone-4-
amino (1) exhibited significant in vitro antiprotozoal activity
against Trypanosoma cruzi, the causative agent of Chagas’
disease; and Plasmodium falciparum, which is accompanied
by cytotoxicity toward murine L-6 cells.
Taking into account that one way to ameliorate the cyto-
toxicity of a compound is to prepare prodrugs [2], it seemed
of interest to prepare some derivatives as candidate prodrugs
of 1 and to compare their biological and cytotoxic activities
with those of 1 and known standard compounds. The results
2. Experimental procedures
Melting points (m.p., uncorrected) were determined on a
Büchi 510.Yields of solids refer to crude products. IR (KBr),
mass (at 70 EV), and 1H NMR spectra (chemical shifts in d,
ppm downfield from TMS) were recorded on Nicolet 5-SXC
FTIR, Finningan 3300, and Bruker WP 80 SY spectrometers,
respectively. Microanalyses (C, H and N) were performed by
UMYMFOR, Buenos Aires, Argentina and the values
were 0.4 of theoretical ones. N-(3,4-dimethyl-5-isoxa-
zolyl)-4-amino-1,2-naphthoquinone (1) and 2-(3,4-dime-
thyl-5-isoxazolylamino)-N-(3,4-dimethyl-5-isoxazolyl)-1,4-
naphthoquinone-4-imine (5) were synthesized by
a
* Corresponding author.
E-mail address: nrscor@dqo.fcq.unc.edu.ar (N.R. Sperandeo).
previously reported procedure [3].
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.03.003

432
N.R. Sperandeo et al. / IL FARMACO 59 (2004) 431–435
O
O
CH2OH
CH2OH
CH O
2
N
Ar
O
N
O
2
O
N
O
NOH
H
Ar
NOCH2COOH
BrCH COOH/NaOH
2
H2NOH.HCl
1
N
H
Ar
H
Ar
3
4
Me
Me
O
NOH
NHAr
4' 3'
NHAr
Ar:
N
O
H2NOH.HCl
N
N
Ar
Ar
6
5
Scheme 1. Reagents and conditions for the synthesis of 2–4.
2.1. 3-Hydroxymethyl-N-hydroxymethyl-N-(3,4-dimethyl-5-
isoxazolyl)-4-amino-1,2-naphthoquinone (2)
2.2. N-(3,4-dimethyl-5-isoxazolyl)-4-amino-1,2-naphtho-
quinone-2-one oxime (3)
A suspension of 1 (0.299 g, 1.1 mmol), 20 ml of formalin
(40% formaldehyde in water), 0.015 g (0.11 mmol) of
K2CO3, 30 ml of ethanol, and 10 ml of water were stirred at
room temperature for 96 h. After that, the ethanol was re-
moved under reduced pressure and then 30 ml of water were
added. After standing at 5 °C overnight, the resulting precipi-
tate was collected by filtration, washed with water, and dried
to give 0.310 g of 2 (86% yield). The crude product was
recrystallized from ethanol. M.p.: 225–226 °C (dec.). dH
(DMSO-d6): 1.04 (3H, s, Me-4′); 1.89 (3H, s, Me-3′); 3.65
(2H, dd, N–CH2OH; dC: 62.84); 4.73 (2H, dd, 3-CH2OH; dC:
56.28); 6.04 (1H, t, OH); 7.70–8.10 (1H, m, OH); 7.79 (1H,
m, H-5); 7.88 (1H, m, H-6); 7.79 (1H, m, H-7); 7.97 (1H, m,
H-8) (J5,6: 7.45; J5,7: 2.64; J5,8: 0.54; J6,7: 8.42; J6,8: 3.24;
J7,8: 7.72; rms: 0.02). m/z (%): 328 (M+, 3); 102 (23); 68 (15);
42 (47); 31 (100). mmax/cm–1: 3329/3281 (OH); 1676,1628
(CO). Elemental analysis calc. (%) for C17H16N2O5: C,
62.19; H, 4.91; N, 8.53; found: C, 62.20; H, 5.10; N, 8.67.
To a solution of 1 (0.362 g, 1 mmol) in ethanol (80 ml),
1.39 g (20 mmol) of HCl.NH2OH in water (10 ml) was
added, and the mixture was heated under reflux for 4 h. After
that, the ethanol was removed under reduced pressure and
50 ml of cold water were added. The resulting precipitate was
filtered off, washed with water, and dried to give 0.263 g of 3
(93% yield). The crude product was recrystallized from ben-
zene. M.p.: 211–212 °C (dec.). dH (DMSO-d6): 1.84 (3H, s,
Me-3′); 2.16 (3H, s, Me-4′); 7.16 (1H, s, H-3); 7.61 (1H, m,
H-7); 7.72 (1H, m, H-6); 8.12 (1H, m, H-8); 8.20 (1H, m,
H-5) (J5,6: 7.54; J5,7: 1.38; J5,8: 0.84; J6,7: 7.12; J6,8: 1.67;
J7,8: 7.88; rms: 0.12); 8.93 (1H, br s, NH); 12.65 (1H, br s,
NOH). m/z (%): 283 (M+, 48); 266 (M-OH, 3); 96 (52);
68 (100). mmax/cm–1: 3325 (NH); 3187 (N–OH); 1644 (CO);
971 (=N–OH). Elemental analysis calc. (%) for
C15H13N3O3: C, 63.60; H, 4.63; N, 14.83; found: C, 63.83:
H, 4.85; N, 14.93.

N.R. Sperandeo et al. / IL FARMACO 59 (2004) 431–435
433
2.3. N-(3,4-dimethyl-5-isoxazolyl)-4-amino-1,2-naphto-
quinone-2-[O (carboxymethyl)]-oxime (4)
hypoxanthine incorporation assay [6] was used. Briefly, in-
fected human red blood cells were exposed to serial drug
dilutions in microtiter plates for 72 h. Viability was assessed
by measuring the incorporation of [3H]-hypoxanthine during
the final 24 h of incubation by liquid scintillation counting.
Counts were expressed as percentage of the control and
presented as sigmoidal inhibition curves. IC50 values were
calculated by linear interpolation selecting values above and
below the 50% mark according to Hills et al. [7].
A mixture of 0.278 g (2 mmol) of bromoacetic acid and
1 g of crushed ice was alkalinized with aqueous NaOH (40%)
and added to a solution of 3 (0.283 g, 1 mmol) in ethanol
(50 ml). The mixture was stirred at room temperature for 4 d,
then was cooled and acidified with aqueous HCl (37%). The
resulting precipitate was filtered off, washed with water, and
dried to give 0.259 g of 4 (76% yield), which was purified by
recrystallization from methanol–water. M.p.: 197–198 °C
(dec.). dH (DMSO-d6): 1.85 (3H, s, Me-3′); 2.17 (3H, s,
Me-4′); 4.88 (2H, s, CH2); 7.03 (1H, s, H-3); 7.66 (1H, m,
H-7); 7.74 (1H, m, H-6); 8.12 (1H, m, H-8); 8.15 (1H, m,
H-5) (J5,6: 7.17; J5,7: 1.35; J5,8: 0.17; J6,7: 7.61; J6,8: 1.84;
J7,8: 7.27; rms: 0.07); 9.20 (s, 1H, NH). m/z (%): 341 (M+, 9);
267 (8); 225 (100); 102 (32); 96 (34); 68 (66); 42 (35).
mmax/cm–1: 3324 (NH); 2915–2500 (COOH); 1727,1656
(CO). Elemental analysis calc. (%) for C17H15N3O5: C,
59.82; found: C, 59.96 and HRMS m/z calc. for C17H15N3O5:
341.1012 [M]+; found: 341.0994.
3.4. Cytotoxicity toward L-6 cells
The determination of the cytotoxicity was performed with
L-6 rat skeletal myoblast cells according to a previously
reported procedure [1]. Briefly, L-6 cells were seeded in
96-well microtiter plates at a density of 105/ml in MEM
supplemented with 10% heat-inactivated fetal bovine serum.
A threefold serial dilution ranging from 90 to 0.123 µg/ml in
test medium was added. The plates were incubated as de-
scribed for the antitrypanosomal assay. After 70 h, Alamar
Blue (10 µl) was added to each well and incubation continued
for a further 2–4 h. The plate was then processed in the same
way as described for T. b. rhodesiense.
3. Biological screenings
3.1. T. cruzi
Rat skeletal myoblasts (L-6 cells) were seeded in 96-well
microtiter plates at 2000 cells/well/100 µl in RPMI 1640 me-
dium with 10% fetal bovine serum and L-glutamine (2 mM).
After 24 h, 5000 trypomastigotes of T. cruzi (Tulahuen strain
C2C4 containing the b-galactosidase (Lac Z gene)) were
added in 100 µl per well with serial drug dilutions. The plates
were incubated at 37 °C in 5% CO2 for 4 d. After 96 h, the
substrate (CPRG/Nonidet) was added to the wells. The color
reaction that developed during the following 2–4 h was read
photometrically at 540 nm. Optical density values were ex-
pressed as percentage of the control, and IC50 values were
calculated from the sigmoidal inhibition curve.
4. Results
4.1. Chemistry
Reaction of 1 with formaldehyde in basic medium yielded
only the bis-hydroxymethyl derivative 2 (Scheme 1). Treat-
ment of 1 with hydroxylamine hydrochloride at reflux
yielded the mono-oxime 3 as the only product. Subsequent
reaction of 3 with bromoacetic acid in basic medium yielded
4 (Scheme 1). Structures 2–4 were verified by analytical and
spectroscopic data obtained by 1H NMR (including the
checking of the assignments by spectral simulation with the
LAOCOON PC program [8]), IR and MS. In addition, and in
order to assign unequivocally the two CH2OH groups of 2,
3.2. T. b. rhodesiense
Minimum essential medium (MEM, 50 µl) supplemented
according to Baltz et al. [4] with 2-mercaptoethanol and 15%
heat-inactivated horse serum was added to each well of a
96-well microtiter plate. Serial drug dilutions were added to
the wells followed by a trypanosome suspension (T. b. rhod-
esiense STIB 900, 50 µl). After incubation for 72 h at 37 °C
under a 5% CO2 atmosphere, Alamar Blue (10 µl) was added
to each well and incubation continued for a further 2–4 h. The
plate was then read with a Millipore Cytofluor 2300 using an
excitation wavelength of 530 nm and an emission wave-
length of 590 nm [5]. Fluorescence development was ex-
pressed as percentage of the control, and the IC50 values
determined.
1
the H NMR analysis was assisted by means of spin–spin
decoupling experiments. Thus, the non-irradiated spectrum
showed two double doublets centered at dH 3.65 (N–CH2OH)
and dH 4.73 (3-CH2OH) which were assigned according to
Shoolery’s effective shielding constants [9]. Irradiation of
the NCH2 protons led to the collapse of the OH triplet at dH
6.04 into an enhanced singlet. Similarly, irradiation of the dH
6.04 signal caused enhancement and simplification of the
double doublet at dH 3.65, showing that the OH at dH 6.04 is
attached to the N–CH2 group. Then, the other OH proton that
falls within the aromatic signals is bonded to the 3-CH2
moiety.
It is remarkable that the 1H NMR spectrum of 3 exhibited
only one =N–OH proton (at dH 12.65, the typical range of
oximinoketones) [10] pointing out that 3 was configuration-
ally pure as also indicated by TLC results. In addition, the IR
spectrum of 3 showed only one C=O absorption at 1644 cm–1
3.3. P. falciparum
Antiplasmodial activity was determined using the P. fal-
ciparum strains K1 and NF54. A modification of the [3H]-

434
N.R. Sperandeo et al. / IL FARMACO 59 (2004) 431–435
Table 1
In vitro antiprotozoal and cytotoxic activities of 2–4 against T. cruzi, T. b. rhodesiense, P. falciparum and murine L-6 cells a
Compound
T. cruzi
T. b. rhodesiense
P. falciparum
(K1)
0.067d
0.0015e
0.11
Cytotoxicity
L-6 cells
(NF54)
0.003d
0.0028e
0.16
Standard
0.56 b
–
0.0061 c
–
–
Standard
–
1
2
3
4
5
0.65
0.83
0.68
11.89
12.85
0.47
1.14
1.24
32.16
0.92
0.5
0.5
0.5
17
51
0.64
0.8
2.49
2.46
3.41
5.41
0.12
0.19
Standards:
a The figures reported are IC50 values in µg/ml. IC50: the concentration required to give 50% inhibition.
b Benznidazole.
c Melarsoprol.
d Chloroquine.
e Artemisinin.
which was attributed to a 1-CO group since in isoxazolyl-
naphthoquinones [11,12] the 1-CO was found at ca.
1647 cm–1, while the 2-CO was situated at ca. 1680 cm–1.
This assignment was also supported by mechanistic consid-
erations as the bis-isoxazolyl analog of 1 (compound 5,
Scheme 1) under the same reaction conditions, did not yield
the oxime 6, but 3 was obtained as a pure isomer (TLC and
1H NMR evidence). The formation of 3 from 5 can be
rationalized on the basis that 5 in acidic and neutral media
undergo irreversible hydrolysis to 1 following the typical
mechanism for imines [3].
5. Discussion
The validity of the suggestion that 2–4 may be prodrugs of
1 to ameliorate its cytotoxicity was considered by evaluating
the data of Table 1.A comparison of the bioactivities of 1 and
2 indicates that the two additional CH2OH groups, despite
their steric hindrance, cause little change in the anti-T. cruzi
and cytotoxic activities. On the other hand, if 3 is a prodrug of
1, then the rate of release of the 2-CO group may be reflected
by different cytotoxicities as observed for other ketones [2].
Examination of the data (Table 1) reveals identical cytotox-
icity for 1 and 3, suggesting that 3 is active per se; hence, it
could not be a prodrug of 1. Compound 4, which is not
cytotoxic, may be a prodrug inactive in vitro but becoming
active after in vivo administration. However, to be useful,
the =NOCH2CO2H group must enable selective in vivo re-
lease of 1 (or 3) in trypanosomes [13,14].
The chemical modifications studied here indicated that the
main structural requirement for the anti-T. cruzi and cyto-
toxic activities of 1 is the presence of the 2-keto moiety or a
group sterically or electronically similar (=NOH). The deac-
tivation of the 1,2-naphthoquinone ring through the substitu-
tion of the 2-CO group by a carboxymethyloxime (4) or an
aminoisoxazolyl (5) moiety reduces or abolishes both the
anti-T. cruzi and cytotoxic activity, probably since they
modified the redox properties of the naphthoquinone ring or
they interfere to some extent with the naphthoquinone ability
to bind to DNA [14,15].
4.2. Biological and cytotoxic activities
The results of the biological evaluation of 2–4 against T.
cruzi, T. b. rhodesiense, P. falciparum and murine L-6 cells
are presented in Table 1, which also contains data for the
compounds 1 and 5 for comparison.
Compounds 2 and 3 exhibited good inhibitory activity
against T. cruzi, with IC50 values in a range comparable to
those of 1 (IC50: 0.65 µg/ml) and benznidazole (0.56 µg/ml).
In contrast, 3 was inactive with an IC50 value similar to that
of 5.
Activity of 2 and 3 against T. b. rhodesiense was less
pronounced than 1, while 4 was found to be inactive. These
results provide additional evidence that this family of com-
pounds is not active against this parasite [1] as none of the
tested compounds appeared more effective than the standard
drug melarsoprol (IC50: 0.0061 µg/ml).
In terms of their antimalarial activity, 2–4 were found to
be inactive against P. falciparum strains K1 (resistant to
chloroquine and pyrimethamine) and NF54 (sensitive to all
known drugs). Their IC50 values were in a range (0.80–
5.4 µg/ml) outside the threshold set for activity (0.5 µg/ml).
Compounds 2 and 3 displayed identical cytoxicity to that
of 1 (IC50: 0.50 µg/ml), indicating that their good antipara-
sitic activities could also lack specificity. In contrast, 4 was
less cytotoxic than 1, with an IC50 value ca. three times lower
than that of 5 (IC50: 51 µg/ml) which showed no cytotoxicity
for the L-6 cells [1].
In summary, the results reveal good inhibitory activity in
vitro of 2 and 3 against T. cruzi, which encourages further
investigations in order to circumvent the cytotoxic properties
of these potential trypanocidal agents.
Acknowledgements
The authors thank SECyT-UNC and the UNDP/World
Bank/WHO Special Program for Research and Training in
Tropical Diseases (TDR) for financial support. The technical
assistance of Elke Gobright and Christian Scheurer is highly
acknowledged.

N.R. Sperandeo et al. / IL FARMACO 59 (2004) 431–435
435
[8] A.A. Bothner-by, S. Castellano, in: D.F. De Tar (Ed.), Computer
Program for Chemists, 1, W.A. Benjamin Inc., New York, 1968
Library of Congress, Catalog number 68. 19706.
[9] B.P. Dayley, J.N. Shoolery, The electron withdrawal power of sub-
stituent groups, J. Am. Chem. Soc. 77 (1955) 3977–3981.
[10] G.G. Kleinspehn, J.A. Jung, S.A. Studniarz, The chemical shift of the
hydroxyl proton of oximes in dimethyl sulfoxide, J. Org. Chem. 32
(1967) 460–463.
[11] A.E. Fernández, M.M. de Bertorello, R.H. Manzo, Synthesis and
spectroscopic properties of 1,2-naphthoquinone-4-aminoisoxazoles,
Anales Asoc. Quím. Argent. 70 (1982) 49–60 and references therein.
[12] N.R. Sperandeo, M.M. de Bertorello, M.C. Briñón, Synthesis and
some physicochemical properties of 2-hydroxy-N-(3,4-dimethyl-5-
isoxazolyl)-1,4-naphthoquinone-4-imine derivatives, J. Pharm. Sci.
83 (1994) 332–334.
[13] R.W. Grady, S.H. Bobstein, S.R. Meshnick, P.C. Ulrich, A. Cerami,
J. Amirmoazzami, et al., The in vitro trypanocidal activity of
N-substituted p-benzoquinone imines: assessment of biochemical
structure–activity relationships using the Hansch approach, J. Cell.
Biochem. 25 (1984) 15–29.
References
[1] N.R. Sperandeo, R. Brun, Synthesis and biological evaluation of
pyrazolylnaphthoquinones as potential antiprotozoal and cytotoxic
agents, Chem. Biochem. 4 (2003) 69–73.
[2] J.R. Dimmock, K.M. Advikolanu, H.E. Scott, M.J. Duffy, R.S. Reid,
J.W. Quail, et al., Evaluation of cytoxicity of some Mannich bases of
various aryl and arylidene ketones and their corresponding arylhydra-
zones, J. Pharm. Sci. 81 (1992) 1147–1153.
[3] M.R. Longhi, M.M. de Bertorello, M.C. Briñón, Isoxazoles V: chemi-
cal stability of diisoxazolylnaphthoquinone in aqueous solution, J.
Pharm. Sci. 78 (1990) 408–412.
[4] T. Baltz, D. Baltz, C. Giroud, J. Crockett, Cultivate in a semi-defined
medium of animal infective forms of T. brucei, T. equiperdum, T.
evansi, T. rhodesiende and T. gambiense, EMBO J. 4 (1985) 1273–
1277.
[5] B. Raez, M. Iten,Y. Grether, R. Kaminsky, R. Brun, The Alamar Blue
assay to determine drug sensitivity of African trypanosoma (T. b.
rhodesiense) in vitro, Acta Trop. 68 (1997) 139–147.
[6] H. Matile, J.R.L. Pink, Plasmodium falciparum malaria parasite cul-
tures and their use in immunology, in: I. Lefkovits, B. Pernis (Eds.),
Immunological Methods, Academic Press, San Diego, 1990, pp. 221–
234.
[14] M.N.S. de Tarlovsky, S.G. Goijman, M.P. Molina Portela,
A.M.O. Stoppani, Effects of isoxazolylnaphthoquinone-imines on
growth and oxygen radical production in Trypanosoma cruzi and
Crithidia fasciculata, Experientia 46 (1990) 502–505.
[7] M. Hills, C. Hudson, P.G. Smith, Working paper No. 2.8.5 for the
informal consultation on epidemiology of drug resistance of malaria
parasites, World Health Organization, Geneva, 1986.
[15] F. Leteurtre, G. Kohlhagen, D.D. Paull,Y. Pommier, Topoisomerase II
inhibition and cytotoxicity of the anthrapyrazoles Dup937 and
DuP941, J. Natl. Cancer Inst. 86 (1994) 1239–1244.