Antimalarial t-butylperoxyamines

Article abstract of DOI:10.1016/S0960-894X(01)00396-1

Twelve t-butylperoxyamines (6-17) were synthesized as targeted antimalarials and evaluated for antimalarial activity in vivo against Plasmodium berghei in mice and in vitro against both chloroquine sensitive and chloroquine resistant strains of Plasmodium falciparum. Compound 8 was found to have highest potency with activity at 80 and 160 mg/kg dose in vivo and compound 11 exhibited highest efficacy in vitro.

Full text of DOI:10.1016/S0960-894X(01)00396-1

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2269–2272
Antimalarial t-Butylperoxyamines
N. Sundar,a V. T. Jacob,a Sujata V. Bhat,a,* Neena Valechab and Sukla Biswasb
aDepartment of Chemistry, Indian Institute of Technology, Powai, Bombay 400 076, India
bMalaria Research Centre, 22 Sham Nath Marg, Delhi 110 054, India
Received 13 November 2000; accepted 26 May 2001
Abstract—Twelve t-butylperoxyamines (6–17) were synthesized as targeted antimalarials and evaluated for antimalarial activity in
vivo against Plasmodium berghei in mice and in vitro against both chloroquine sensitive and chloroquine resistant strains of Plas-
modium falciparum. Compound 8 was found to have highest potency with activity at 80 and 160 mg/kg dose in vivo and compound
11 exhibited highest efficacy in vitro. # 2001 Elsevier Science Ltd. All rights reserved.
Naturally occurring peroxides have been found to exhibit
potent antimalarial activity; artemisinin1À5 (1), dihydro-
ascaridole (2) and an endoperoxide6 3 are a few
examples. However, simple peroxides such as hydrogen
peroxide7 (4) and t-butyl hydroperoxide8,9 (5) also show
activity, but are less potent than 1 and cause hemolysis
of uninfected erythrocytes at parasiticidal concentra-
tions. The efficacy of compounds 1–5 depends on the
observation that malaria infected red cells are selectively
damaged by oxidants.
The amines containing t-butoxy-peroxides are reported
to have antimalarial activity in vitro against Plasmo-
dium falciparum. Vennerstorm16 reported a series of
such peroxides with potential antimalarial activity
approximately one order of magnitude more potent
than t-butyl hydroperoxide (5) in vitro against the
chloroquine sensitive (D-6) and chloroquine resistant
(W-2) clones of P. falciparum. But all of these amine
peroxides were inactive in vivo against Plasmodium
berghei.
Recently, we have reported the antimalarial activity of
monoterpenic fragment analogues of aplasmomycin17,18
and of 3-hydroxy-3-aryl-2-methylene-propionic acid
derivatives.19 In continuation of our search for new
antimalarials, we report herein the synthesis of peroxy-
amines and their antimalarial activity in vitro against P.
falciparum and in vivo against P. berghei in mice.
It has been proved beyond doubt from structure–activity
studies that the endoperoxide moiety in artemisinin (1)
and its analogues is absolutely essential for antimalarial
activity,10 which is mediated by activated oxygen
(superoxide, H2O2 and/or hydroxy radicals) or carbon-
free radicals. It suggests an oxidative mode of
action.11À14 The increase in the potency of 1 with an
increase in oxygen tensions ranging from 3 to 30% and
decrease in the potency of 1 when co-administered with
reducing agents support this oxidative mode of action.15
Chemistry20
The compounds 6–13 were obtained from the corre-
sponding secondary amines by the treatment with
formaldehyde (33% aqueous) and t-butyl hydroper-
oxide (70% aqueous) in methanol at 4 ꢀC. Whereas the
compounds 14–17 were prepared by treatment of the
aldehydes, namely citronellal, furfural, thiophene-2-car-
boxaldehyde with primary amines, namely benzylamine
and ethanolamine to give corresponding imines. The
imines were then reduced using sodium borohydride to
give corresponding secondary amines. The secondary
amines thus obtained were then converted to amine per-
oxides by the above-mentioned procedure (Scheme 1). A
*Corresponding author. Tel.: +91-22-576-7154; fax: +91-22-576-
7152; e-mail: svbhat@chem.iitb.ernet.in
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00396-1

2270
N. Sundar et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2269–2272
singlet at d 1.24–1.26 (9H) and a singlet at d 4.5–4.7
(2H) in the H NMR spectra confirmed the incorpora-
1
tion of the t-butyl and the methylene groups, respec-
tively, in the molecules.
Biological Evaluation21
Antimalarial activity
In vitro. The compounds 6–17 were evaluated against
both chloroquine sensitive (FJB D9) and chloroquine
resistant (FSH 14) strains of P. falciparum at different
doses starting from 25 mmol/well onwards with 2-fold
serial dilution. Doses were kept constant for all com-
pounds to have a comparative profile. Results obtained
from the in vitro schizont maturation inhibition and
parasite growth inhibition in the case of both chloro-
quine sensitive (FJB D9) and chloroquine resistant
(FSH 14) strains of P. falciparum are summarised in
Tables 1 and 2, respectively. Compound 11 was found
to exhibit maximum efficacy in vitro.
Scheme 1.
Table 1. In vitro antimalarial activity of compounds against chlor-
oquine sensitive P. falciparum strain FJB D9a
Compounds (mmol/well)
IC50
IC90
SMI
PGI
1.8
SMI
PGI
6
7
8
9
10
11
12
13
14
15
16
0.135
1.6
22
1.5
>25
0.058
>25
0.47
4.6
6.4
4.0
>25
25
1.45
>25
5.0
>25
0.15
>25
>25
21
>25
2.6
>25
22.5
>25
9.0
>25
3.0
>25
>25
>25
10.0
5.5
In vivo. The compounds 6–17 were evaluated for anti-
malarial activity in vivo against virulent strains of P.
berghei in mice using Rane’s schizontocidal method
described by Osdene et al.22 The dose range selected was
20, 40, 80, 160 mg/kg and a minimum of five mice per
dose were used. Artemisinin (20 and 40 mg/kg) was kept
as standard drug in trial for comparison. On compar-
ison of activities (Table 3) compound 8 was found to be
3.6
3.27.8
1.6
5.5
4.4
2.1
2.7
0.015
1.45
0.9
0.01
2.8
6.0
17
Artemisinin
4.8
0.03
0.028
aSMI, schizont maturation inhibition; PGI, total parasite growth
inhibition.

N. Sundar et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2269–2272
2271
Table 2. In vitro antimalarial activity of compounds against chloroquine resistant P. falciparum strain FSH 14a
Compounds (mmol/well)
IC50
IC90
SMI
PGI
SMI
PGI
6
7
8
9
10
11
12
13
14
15
16
0.5
2.65
10.0
2.1
>25
0.064
>25
0.45
1.95
>25
5.0
>25
0.16
>25
0.925.0
5.6
>25
>25
>25
2.9
>25
24
>25
10
4.5
11.5
0.028
5.0
>25
>25
>25
2.15
>25
>25
>25
12
5.4
12.0
0.029
2.9
1.4
1.6
1.35
0.5
0.01
5.0
3.8
2.5
2.25
2.2
0.01
17
Artemisinin
aSMI, Schizont maturation inhibition; PGI, total parasite growth inhibition.
Table 3. In vivo antimalarial activity of compounds against P. berghei in mice
Compounds
Untreated control (days)
Mean survival time (days) Doses (mg/kg)
Remarks
20
40
80
160
6
7
8
9
10
11
12
13
14
15
16
6.16.25.2
6.27.0
.8161..2610.2
6.5
6.5
6.5
6.4
5.8
6.8
6.0
6.8
6.8
6.3
6.8
7I.n2active
.610.0
Active at 80 and 160 mg/kg
8.4
Acti1v2e at 160 mg/kg
14.102
7.6
8.6
9.80
Inac1ti2ve
8.80
9.80
Inactive
13.4
Active at 160 mg/kg
7.28.4
6.8
5.4
5.26.6
6.27.3
5.26.8
6.4
9.40 .2
8.210.2
11.215.0
10.8 .2Active at 11620 mg/kg
7.6
7.0
11.4
11.8
Inactive
Active at 160 mg/kg
0
Active at 160 mg/kg
7.210.8
6.6
12.8
.0
12
10.214.0
Inactive
Active at 160 mg/kg
Active from 40 mg/kg
17
Artemisinin
5.8
10.8
References and Notes
most potent with activity at 80 and 160 mg/kg dose. The
compounds 7, 9, 12, 14, 15 and 17 showed activity at
160 mg/kg dose level.
1. Haynes, R. K.; Vonwiller, S. C. Acc. Chem. Res. 1997, 30,
73.
2. Zhou, W. S.; Xu, X. X. Acc. Chem. Res. 1994, 27, 211.
3. Qinghaosu Antimalarial Coordinating Research Group
Chin. Med. J. 1979, 92, 811.
4. Klayman, D. L. Science (Washington, DC) 1985, 228, 1049.
5. Luo, X. D.; Shen, C. C. Med. Res. Rev. 1987, 7, 2 9.
6. Kamchonwongpaisan, S.; Nilanonta, C.; Tarnchompoo,
B.; Thebtaranonth, C.; Thebtaranonth, Y.; Yuthavong, Y.;
Kongsaeree, P.; Clardy, J. Tetrahedron Lett. 1995, 36, 1821.
7. Dockrell, H. M.; Playfair, J. H. J. Infect. Immunol. 1983,
39, 456.
In conclusion, t-butylperoxyamines were found to be
novel antimalarials among which the compound 8 has
the highest potency in vivo against P. berghei. Com-
pound 11 was found to exhibit maximum efficacy in
vitro against P. falciparum. The present study assumes
greater significance because of the facile synthesis of
these compounds and their antimalarial activity against
P. berghei in mice.
8. Clark, I. A.; Cowden, W. B.; Bucher, G. A. Lancet 1983, I,
234.
9. Clark, I. A.; Hunt, N. H.; Cowden, W. B.; Maxwell, L. E.;
Mackie, E. J. Clin. Exp. Immunol. 1984, 56, 524.
10. Gu, H. M.; Lu, B. F.; Qui, Z. X. Acta. Pharmacol. Sinica
1980, 1, 48.
11. Cumming, J. N.; Ploypradith, P.; Posner, G. H. Adv.
Pharmacol. 1997, 37, 253.
12. Trager, W.; Jenden, J. B. Science 1976, 193, 673.
13. Biswas, S.; Saxena, Q. B.; Roy, A.; Sharma, V. P. Ind. J.
Malariol. 1988, 25, 7.
Acknowledgements
The authors thank I.C.M.R., New Delhi and Lady Tata
Memorial Trust, Bombay for financial assistance;
RSIC, I.I.T., Bombay and T.I.F.R., Bombay for spec-
tral data; S. H. Kelkar Bros., Bombay for pro-
viding citronellal; and Dr. (Mrs.) V. R. Kamath of
Haffkine Institute, Bombay for in vivo antimalarial test
results.

2272
N. Sundar et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2269–2272
14. Shalini, B.; Vishwakarma, R. A.; Jain, S. K. J. Chem.
Soc., Perkin Trans. 1 1998, 2163.
15. Krungkrai, S. R.; Yuthavong, Y. Trans. R. Soc. Trop.
Med. Hyg. 1987, 81, 710.
16. Vennerstorm, J. L. J. Med. Chem. 1989, 32, 64.
17. Balu, N.; Thomas, J. V.; Bhat, S. V. J. Med. Chem. 1991,
34, 2821.
18. Biswas, S.; Valecha, N.; Kundu, M. K.; Thomas, J. V.;
Balu, N.; Bhat, S. V. Ind. J. Exp. Biol. 1995, 33, 521.
19. Kundu, M. K.; Sundar, N.; Kumar, S. K.; Bhat, S. V.
Bioorg. Med. Chem. Lett. 1999, 9, 731.
1640 media enriched with 10% AB+ serum and supplemented
with 25 mM HEPES buffer and 25 mM NaHCO3. Parasite
culture was synchronised at ring forms using density gradient
method.13 Assay was done at 10% haematocrit containing
1% ring stage parasite in 96-well flat-bottom tissue culture
plate. Compounds were dosed in wells in duplicate at con-
centrations of 25, 10, 5, 2.5, 1, 0.5, 0.25, 0.1, 0.05, 0.025 and
0.01 mmol per well. Control culture was done in complete
media without any synthesised compounds. To determine the
activity of various compounds, assay was done in two sets.
The first set of bioassay was conducted to determine the effect
of schizont maturation after 24 h and the second set of assay
was conducted to determine the effect of total growth of the
parasites after 72h. The volume of culture media per well for
both sets of assay was 200 mL. Growth of the parasites from
duplicate wells of each concentration was monitored micro-
scopically in JSB strained14 smears by counting number of
schizonts per 200 asexual parasites and total number of para-
sites per 5000 RBCs. Percentage schizont maturation inhibi-
tion and total growth inhibition were calculated by the
formula; (1ÀNt/Nc)Â100 where Nt and Nc represent the
number of schizont in the test and control well, respectively.
Inhibitory concentrations at 50 and 90% were calculated.
Biological activity in vivo (Rane’s test). The compounds were
evaluated for their activity against virulent strains of P. ber-
ghei yoelli (NK 65) using Rane’s schizontocidal method
described by Osdane et al.22 Four-week-old mice weighing 20–
25 g each received intraperitoneal inoculum of 1Â10À6 para-
sitized P. berghei red cells. The test solutions of synthesised
compounds in distilled water were prepared by homogenisa-
tion with two drops of 1% Tween-80 and injected once sub-
cutaneously 72h post infection. A control group of infected
mice that was not administered any drug was kept as
untreated control. The dose range selected was 20, 40, 80 and
160 mg/kg and a minimum of five mice per dose were used.
Artemisinin (20 and 40 mg/kg), cycloguanil hydrochloride
(25 mg/kg) and DDS (20 mg/kg) were kept as standard drugs
in trial for comparison. Deaths occurring within 24 h of treat-
ment classified as death due to toxicity. All mice receiving
synthetic compounds showed survival time of 12–18 days.
Testing was evaluated by calculating mean survival time
(MST) of the treated and controlled group of mice.
20. Experimental: general procedures
Conversion of secondary amine to t-butylperoxyamines
To a solution of secondary amine (10 mmol) in methanol
(5 mL) at 0 ꢀC was added 70% aqueous t-butyl hydroperoxide
(1.08 g, 12mmol) followed by 37% aqueous formaldehyde
solution (0.36 g, 12mmol). The resulting mixture was stirred
for 4 h at 4 ꢀC, the mixture was extracted with solvent ether,
and the organic layer was dried using anhydrous K2CO3.
Removal of solvent in vacuo provided the peroxide as a clear
oil. The compound was further purified by column chromato-
graphy using neutral alumina with ethyl acetate and petroleum
ether as eluants.
Synthesis of imines. A mixture of amine (10 mmol) and alde-
hyde (10 mmol) in 20 mL of dry benzene was refluxed for 8 h
using Dean–Stark apparatus until all the water that had formed
was removed. Removal of solvent in vacuo gave the imine, which
was directly taken to the next step without purification.
Reduction of imine using NaBH4. To a solution of imine
(0.01 mol) in methanol, an alkaline solution of NaBH4 (2g) in
NaOH solution (2mL) and water (20 mL) was added dropwise
at such a rate that the temperature doesn’t rise above 50 ꢀC
and the mixture was stirred for 1 h. The solvent was then dis-
tilled off, diluted with water and the amine was extracted with
CHCl3. The organic layer was dried over anhydrous Na2SO4.
Removal of the solvent in vacuo and purification using alu-
mina gave the required amine.
21. Biological activity in vitro. Two P. falciparum strains were
obtained from well adapted in vitro culture lines. One chloro-
quine sensitive, FJB D9 (Jabalpur, Central India) and one
chloroquine resistant, FSH 14 (Shankagarh, North India) iso-
late collected from patients in 1990 and 1993, respectively,
were adapted and maintained in vitro by candle-jar techni-
que.12 Parasites were cultured in O+ erythrocyted in RPMI
22. Osdene, T. S.; Russel, P. B.; Rane, L. J. Med. Chem. 1967,
10, 431.