Orally active amino functionalized antimalarial 1,2,4-trioxanes

Article abstract of DOI:10.1016/j.bmcl.2003.10.051

Using readily available trioxanes 6a-b, a new series of amino functionalized 1,2,4-trioxanes 8a-e and 9a-e have been prepared and evaluated for antimalarial activity against multi-drug resistant Plasmodium yoelii in Swiss mice model. Several of these nove

Full text of DOI:10.1016/j.bmcl.2003.10.051

Bioorganic & Medicinal Chemistry Letters 14 (2004) 459–462
Orally active amino functionalized antimalarial 1,2,4-trioxanes§
Chandan Singh,a,* Heetika Malika and Sunil K. Purib
aDivision of Medicinal Chemistry, Central Drug Research Institute, Lucknow-226001, India
bDivision of Parasitology, Central Drug Research Institute, Lucknow-226001, India
Received 19 September 2003; accepted 23 October 2003
Abstract—Using readily available trioxanes 6a–b, a new series of amino functionalized 1,2,4-trioxanes 8a–e and 9a–e have been
prepared and evaluated for antimalarial activity against multi-drug resistant Plasmodium yoelii in Swiss mice model. Several of
these novel trioxanes are orally more active than the parent trioxanes 6a–b. Antimalarial activity of amino functionalized trioxane
9a, the most potent compound in the series, is very close to that of b-arteether.
# 2003Elsevier Ltd. All rights reserved.
1. Introduction
amino functionalized derivatives of artemisinin are
known,7 to the best of our knowledge, this is the first
report on amino functionalized synthetic 1,2,4-trioxanes.
Discovery of Artemisinin 1, as the active principle of the
Chinese traditional drug Artemisia annua, has opened
new possibilities in malaria chemotherapy.1 Artemisinin
and its more potent derivatives for example, artemether
2, arteether 3 and artesunic acid 4, are highly active
against both chloroquine-sensitive and resistant
malaria. These drugs are fast acting and are well suited
for the treatment of cerebral malaria caused by multi-
drug resistant Plasmodium falciparum.2 Peroxide group
present in the form of 1,2,4-trioxane, is essential for the
antimalarial activity of these drugs. Synthesis of a large
number of structurally simple trioxanes have been
reported, several of which have shown promising in vivo
antimalarial activity.3,4 Though chloroquine is known
to antagonise the antimalarial effects of artemisinin
derivatives,5 recently we and others have attempted to
improve the antimalarial activity of easily accessible
synthetic trioxanes by incorporating 4-aminoquinoline
moiety.6 These hybrid molecules ‘trioxaquines’ (proto-
type 5) prepared by reductive amination of trioxanes 6a–b
with 4-aminoquinolines 7, show only marginal improve-
ment in antimalarial activity over the parent trioxanes.
They also suffer from serious limitations such as poor
solubility both in water and oil.6 Herein we report the
preparation and antimalarial activity of 8a–e and 9a–e, a
new series of amino functionalized trioxanes which are
orally more active than the parent trioxanes 6a–b. While
2. Chemistry
b-Hydroxyhydroperoxides 11a–b prepared by photo-
oxygenation of allylic alcohols 10a–b were condensed
with 1,4-cyclohexanedione to furnish keto-trioxanes
6a–b in 42–51% yield.6 Reductive amination of 6a with
various amines in presence of NaBH(OAc)3 furnished
amino functionalized trioxanes 8a–e as inseparable
§CDRI Communication no.: 6461.
* Corresponding author. Tel.: +91-0522-2224273; fax: +91-0522-
2223405; e-mail: chandancdri@yahoo.com
0960-894X/$ - see front matter # 2003Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.10.051

460
C. Singh et al. / Bioorg. Med. Chem. Lett. 14 (2004) 459–462
mixture of diastereomers.8 A similar reductive amination
of 6b furnished amino functionalized trioxanes 9a–e
again as inseparable mixture of diastereomers (Scheme 1,
Table 1).9 Unlike trioxaquines 5, these amino functiona-
lized trioxanes are stable as free bases and are soluble in
groundnut oil, the most commonly used vehicle for anti-
malarial assessment of artemisinin analogues.
3. Antimalarial activity
Trioxanes 6a–b and amino functionalized trioxanes 8a–
e and 9a–e were tested against multi-drug resistant
Plasmodium yoelii in Swiss mice initially at 96 mg/kg
both by oral and intramuscular (im) routes.10 Com-
pound 9a which showed 100% protection at 96 mg/kg
by oral route was also tested at 48 mg/kg and 24 mg/kg.
b-Arteether 3, which provides 100% protection at 48
mg/kg by oral route, served as positive control. The
results are summarized in Table 2.
Scheme 1. Reaction conditions: (a) hn, O2, methylene blue, MeCN,
À10 to 0 ꢀC, 4 h. (b) 1,4-cyclohexanedione, concd HCl, 5 ꢀC, 18 h. (c)
RNH2, NaBH(OAc)3, CH2Cl2, rt, 4 h.
Table 1. Amino functionalized 1,2,4-trioxanes
Compd
Ar
R
Yield (%)
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
4-Biphenyl
4-Biphenyl
4-Biphenyl
4-Biphenyl
4-Biphenyl
Phenyl
81
61
62
88
78
84
91
44
61
41
4-Methoxyphenyl
3,5-Dichlorophenyl
4-Acetylaminophenyl
1-Naphthyl
4. Results and discussion
We have earlier tried to improve the antimalarial activ-
ity of trioxanes 6a–b by incorporating 4-aminoquinoline
moieties 7. But the resulting hybrid molecules ‘triox-
aquines’ (represented by prototype 5) showed only
marginal improvement in activity. These trioxaquines
were also unstable as free bases and had serious solubi-
lity problem. Amino functionalized trioxanes 8a–e and
Phenyl
4-Methoxyphenyl
3,5-Dichlorophenyl
4-Acetylaminophenyl
1-Naphthyl
Table 2. In vivo antimalarial activity against P. yoelii in Swiss mice
Compd
Dose (mg/kg/day)
Route
% Suppression on day 4a
Mice alive on day 28
Mean survival timeb (day)ÆSE
6a
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
48
24
96
96
96
96
96
96
96
96
96
48
24
48
24
—
oral
im
oral
im
oral
im
oral
im
oral
im
oral
im
oral
im
oral
oral
oral
im
oral
im
oral
im
oral
im
oral
im
oral
oral
oral
oral
—
7
99
92
100
95
60
930/5
630/5
100
62
77
98
100
44
100
100
98
72
94
57
65
46
49
90
0/5
0/5
0/5
1/5
0/5
0/5
12.4
10.8
2/5
0/5
0/5
0/5
0/5
0/5
5/5
3/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
5/5
1/5
2/5
0/5
0/15
7.2Æ0.38
11.6Æ1.08
10.2Æ0.58
17.7Æ1.97
12.2Æ0.73
13.4Æ1.50
Æ1.12
6b
8a
8b
8c
8d
8e
9a
Æ1.50
16.5Æ2.31
12.4Æ0.93
9.8Æ0.37
9.0Æ0.45
19.5Æ1.85
9.2Æ1.68
>28
14.5Æ2.5
13.4Æ1.14
10.7Æ1.25
11.1Æ1.06
8.4Æ2.4
9b
9c
10.2Æ2.03
7.0Æ0.55
8.4Æ0.75
11.6Æ1.69
14.8Æ1.06
6.6Æ0.24
>28
9d
9e
100
nil
b-Arteether
Chloroquine
Vehicle control
100
100
100
100
—
17.75Æ2.46
17.6Æ1.33
14.8Æ2.24
7.07Æ0.10
a Percent suppression=[(CÀT)/C]Â100; where C=parasitaemia in control group, and T=parasitaemia in treated group.
b MST calculated for the mice which died during 28-day observation period and the mice which survive beyond 28 days are excluded.

C. Singh et al. / Bioorg. Med. Chem. Lett. 14 (2004) 459–462
461
9a–e, on the other hand, are stable as free bases and are
freely soluble in groundnut oil, the most commonly
used vehicle for antimalarial testing of artemisinin deri-
vatives. As can be seen from Table 2, several of these
novel compounds are orally more active than the parent
trioxanes. Thus, while trioxane 6a is almost completely
inactive at 96 mg/kg by oral route, all the amino func-
tionalized trioxanes derived from it except 8d show 93–
100% clearance of parasitaemia on day 4 by oral route.
Amino functionalized trioxane 8c, which shows 100%
inhibition of parasitaemia on day 4, also provides 40%
protection in 28-day survival assay. Keto-trioxane 6b
shows 92% inhibition of parasitaemia on day 4 when
given orally at 96 mg/kg. Its derivative 9a, shows 100%
clearance of parasitaemia at 96 mg/kg by oral route and
all the treated mice survived beyond day 28. Even at 48
mg/kg by oral route 9a shows 100% clearance of para-
sitaemia on day 4 and 60% of treated mice survived
beyond day 28. Thus activity profile of 9a by oral route
is very close to that of b-arteether. Rests of the com-
pounds derived from 6b have either comparable activity
(9b and 9e) or are less active than 6b. However none of
the amino functionalized trioxanes is more active than
the parent trioxanes 6a–b by im route. Thus introduc-
tion of the amino moiety improves absorption by oral
route only.
Chem. Lett. 2002, 12, 1913. (d) Singh, C.; Gupta, N.; Puri,
S. K. Bioorg. Med. Chem. Lett. 2003, 13, 3447.
4. For other in vivo active trioxanes: (a) Peters, W.; Robin-
son, B. L.; Rossier, J. C.; Jefford, C. W. Ann. Trop. Med.
Parasitol. 1993, 87, 1. (b) Peters, W.; Robinson, B. L.;
Rossier, J. C.; Misra, D.; Jefford, C. W. Ann. Trop. Med.
Parasitol. 1993, 87, 9. (c) Posner, G. H.; Jeon, H. B.;
Parker, M. H.; Krasavin, M.; Paik, I.-H.; Shapiro, T. A.
J. Med. Chem. 2001, 44, 3054. (d) Posner, G. H.; Jeon,
H. B.; Ploypradith, P.; Paik, I.-H.; Borstnik, K.; Xie, S.;
Shapiro, T. A. J. Med. Chem. 2002, 45, 3824.
5. Fivelman, Q. L.; Walden, J. C.; Smith, P. J.; Folb, P. I.;
Barnes, K. I. Trans. R. Soc. Trop. Med. Hyg. 1999, 93,
429.
6. (a) Singh, C.; Malik, H.; Puri, S. K. Bioorg. Med. Chem.
2003, communicated. (b) Dechy-Cabaret, O.; Benoit-
Vical, F.; Robert, A.; Magnaval, J.-F.; Seguela, J.-P.;
Meunier, B. C. R. Chimie. 2003, 6, 153.
7. (a) Hindley, S.; Ward, S. A.; Storr, R. C.; Searle, N. L.;
Bray, P. G.; Park, B. K.; Davies, J.; O’Neill, P. M. J.
Med. Chem. 2002, 45, 1052. (b) Eckstein-Ludwig, U.;
Webb, R. J.; Van Goethem, I. D. A.; East, J. M.; Lee,
A. G.; Kimura, M.; O’Neill, P. M.; Bray, P. G.; Ward,
S. A.; Krishna, S. Nature 2003, 424, 957.
8. In all cases the ratio of the two isomers as measured by
HPLC (Shimadzu-CLC Reversed phase C18 column;
MeOH:H2O 80:20, Flow rate 0.8 mL/min) is around 60:40.
9. Selected spectral data: Compound 6a: FT-IR (KBr, cmÀ1
)
1717.4; 1H NMR (200 MHz, CDCl3) d 2.05 (t, 2H,
J=7.06 Hz), 2.32–2.67 (m, 6H), 3.85 (dd, 1H, J=11.87,
2.91 Hz), 3.96 (dd, 1H, J=11.87, 10.16 Hz), 5.31 (dd, 1H,
J=10.16, 2.91 Hz), 5.36 and 5.53 (2Âs, 2Â1H), 7.32–7.40
(m, 5H); 13C NMR (50 MHz, CDCl3) d 27.79 (t), 33.57 (t),
36.79 (t), 36.95 (t), 63.72 (t), 80.83 (d), 101.50 (s), 117.10
(t), 126.81 (2Âd), 128.71 (d), 129.04 (2Âd), 138.84 (s),
143.59 (s), 210.00 (s); FABMS (m/z) 275 (M++1). Anal.
calculated: C 70.05%, H 6.61%; found C 69.77%, H
5. Conclusion
Using easily available trioxanes 6a–b, for the first time
we have prepared a series of amino functionalized
trioxanes 8a–e and 9a–e in good yield. Several of these
trioxanes show better activity by oral route than the
parent trioxanes. The activity profile of 9a, the most
potent compound of the series, is very close to that of b-
arteether, one of the clinically useful antimalarial drugs
belonging to artemisinin class.
6.86%. Compound 8c: FT-IR (KBr, cmÀ1
) 1590.7,
3418.0; 1H NMR (200 MHz, CDCl3) d 1.47–2.01 (m, 7H),
2.58–2.86 (bm, 1H), 3.34 (bs, 1H), 3.70 (bm, 1H, NH),
3.78 (dd, 1H, J=11.88, 3.78 Hz), 3.85–4.04 (m, 1H), 5.26
(dd, 1H, J=10.70, 3.78 Hz), 5.32 and 5.51 (2Âs, 2Â1H),
6.42 and 6.43(2 Âs, 1H), 6.63(s, 1H), 7.25–7.39 (m, 6H);
FABMS (m/z) 420, 422 and 424 (M++1). Anal. calcu-
lated: C 62.86%, H 5.51%, N 3.33%; found C 62.59%, H
Acknowledgements
5.46%, N 2.89%. Compound 8e: FT-IR (Neat, cmÀ1
)
1
1581.8, 3423.9; H NMR (200 MHz, CDCl3) d 1.43–2.17
(m, 7H), 2.60–2.90 (bm, 1H), 3.60–3.65 (bm, 1H), 3.80
(dd, 1H, J=11.90, 2.77 Hz), 3.89–4.06 (m, 1H), 4.24 (bs,
1H, NH), 5.28 (dd, 1H, J=9.82, 2.77 Hz), 5.33 and 5.51
(2Âs, 2Â1H), 6.63(d, 1H, J=7.28 Hz), 7.19–7.47 (m, 9H),
7.75–7.79 (m, 2H); FABMS (m/z) 402 (M++1). Anal.
calculated: C 77.77%, H 6.77%, N 3.48%; found C
77.83%, H 6.46%, N 3.25%. Compound 9a: FT-IR (KBr,
cmÀ1) 1602.0, 3408.4; 1H NMR (200 MHz, CDCl3) d
1.49–2.04 (m, 7H), 2.61–2.85 (bm, 1H), 3.40–3.64 (bm,
2H), 3.83 (dd, 1H, J=11.96, 2.97 Hz), 3.90–4.09 (m, 1H),
5.31 (dd, 1H, J=9.98, 2.97 Hz), 5.35 and 5.57 (2Âs,
2Â1H), 6.57–6.71 (m, 2H), 7.12–7.20 (m, 2H), 7.25–7.60
(m, 10H); 13C NMR (50 MHz, CDCl3) d 27.03and 27.60
(t), 28.48 and 28.74 (t), 28.98 and 29.09 (t), 32.62 and
33.52 (t), 50.81 and 51.14 (d), 63.23 and 63.52 (t), 80.69
(d), 102.35 and 102.43 (s), 113.69 (2Âd), 116.76 and
116.84 (t), 117.69 (d), 127.20 (2Âd), 127.43(2 Âd), 127.70
(2Âd), 127.93(d), 129.25 (2 Âd), 129.75 (2Âd), 137.81 and
137.85 (s), 140.84 (s), 141.51 (s), 143.33 and 143.37 (s),
147.60 (s); FABMS (m/z) 428 (M++1). Anal. calculated:
C 78.65%, H 6.93%, N 3.27%; found C 78.25%, H
Heetika Malik is grateful to the Council of Scientific
and Industrial Research (CSIR), New Delhi for award
of Senior Research Fellowship.
References and notes
1. For reviews on artemisinin and its analogues see: (a)
Klayman, D. L. Science 1985, 228, 1049. (b) Luo, X. D.;
Shen, C. C. Med. Res. Rev. 1987, 7, 29. (c) Zaman, S. S.;
Sharma, R. P. Heterocycles 1991, 32, 1593. (d) Cumming,
J. N.; Ploypradith, P.; Posner, G. H. Adv. Pharmacol.
1997, 37, 253. (e) Zhou, W. S.; Xu, X. X. Acc. Chem. Res.
1994, 27, 211. (f) Bhattacharya, A. K.; Sharma, R. P.
Heterocycles 1999, 51, 1681.
2. For current status of artemisinin derivatives see: Asthana,
O. P.; Srivastava, J. S.; Valecha, N. J. Parasitic Diseases
1997, 21, 1.
3. (a) Singh, C.; Misra, D.; Saxena, G.; Chandra, S. Bioorg.
Med. Chem. Lett. 1992, 2, 497. (b) Singh, C.; Misra, D.;
Saxena, G.; Chandra, S. Bioorg. Med. Chem. Lett. 1995,
5, 1913. (c) Singh, C.; Gupta, N.; Puri, S. K. Bioorg. Med.
7.26%, N 3.61%. Compound 9d: FT-IR (KBr, cmÀ1
)

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C. Singh et al. / Bioorg. Med. Chem. Lett. 14 (2004) 459–462
1
1612.0, 3305.5; H NMR (200 MHz, CDCl3) d 1.33–2.07
(m, 7H), 2.12 (s, 3H), 2.59–2.85 (bm, 1H), 3.31–3.41 (bm,
2H), 3.83 (dd, 1H, J=11.89, 3.06 Hz), 3.90–4.08 (m, 1H),
5.30 (dd, 1H, J=10.01, 3.06 Hz), 5.34 and 5.57 (2Âs,
2Â1H), 6.55 (d, 2H, J=8.62 Hz), 7.02 (s, 1H, NH), 7.24
(d, 2H, J=8.62 Hz) 7.31–7.60 (m, 9H); 13C NMR
(50 MHz, CDCl3) d 24.52 (q), 27.02 and 27.56 (t), 28.42
and 28.66 (t), 29.00 (t), 32.59 and 33.48 (t), 51.18 and
51.49 (d), 63.20 and 63.49 (t), 80.68 (d), 102.36 and 102.44
(s), 113.99 (2Âd), 116.86 (t), 122.98 (d), 127.19 (2Âd),
127.41 (2Âd), 127.69 (2Âd), 127.95 (2Âd), 129.26 (2Âd),
137.82 (s), 140.79 (2Âs), 141.47 (s), 143.27 (s), 144.74 (s),
168.90 (s); FABMS (m/z) 485 (M++1). Anal. calculated:
C 74.44%, H 6.66%, N 5.78%; found C 74.57%, H
6.67%, N 5.38%.
10. The in vivo efficacy of compounds was evaluated against
Plasmodium yoelii (MDR) in Swiss mice model. The mice
were inoculated with 1Â106 parasitised RBC on day zero
and treatment was administered to a group of five mice at
each dose, from day 0 to 3, in two divided doses daily.
The required drug dilutions were prepared in groundnut
oil and 0.1 mL volume was administered intramuscularly
and orally for each dose. Parasitaemia level were recorded
from thin blood smears between day 4–28.11 Mice treated
with arteether and chloroquine served as positive controls.
11. Puri, S. K.; Singh, N. Expl. Parasit. 2000, 94, 8.