Dispiro-1,2,4-trioxane analogues of a prototype dispiro-1,2,4-trioxolane: Mechanistic comparators for artemisinin in the context of reaction pathways with iron(II)

Article abstract of DOI:10.1021/jo050385+

Single electron reduction of the 1,2,4-trioxane heterocycle of artemisinin (1) forms primary and secondary carbon-centered radicals. The complex structure of 1 does not lend itself to a satisfactory dissection of the electronic and steric effects that influence the formation and subsequent reaction of these carbon-centered free radicals. To help demarcate these effects, we characterized the reactions of achiral dispiro-1,2,4-trioxolane 4 and dispiro-1,2,4-trioxanes 5-7 with ferrous bromide and 4-oxo-TEMPO. Our results suggest a small preference for attack of Fe(II) on the nonketal peroxide oxygen atom of 1. For 4, but not for 5 and 6, there was a strong preference for attack of Fe(II) on the less hindered peroxide bond oxygen atom. The steric hindrance afforded by a spiroadamantane in a five-membered trioxolane is evidently much greater than that for a corresponding six-membered trioxane. Unlike 1, 5-7 fragment by entropically favored β-scission pathways forming relatively stable α-oxa carbon-centered radicals. These data suggest that formation of either primary or secondary carbon-centered radicals is a necessary but insufficient criterion for antimalarial activity of 1 and synthetic peroxides.

Full text of DOI:10.1021/jo050385+

Dispiro-1,2,4-trioxane Analogues of a Prototype
Dispiro-1,2,4-trioxolane: Mechanistic Comparators for Artemisinin
in the Context of Reaction Pathways with Iron(II)
Yuanqing Tang,† Yuxiang Dong,† Xiaofang Wang,† Kamaraj Sriraghavan,†
James K. Wood,‡ and Jonathan L. Vennerstrom*,†
College of Pharmacy, University of Nebraska Medical Center, 986025 Nebraska Medical Center,
Omaha, Nebraska 68198-6025, and Department of Chemistry, University of Nebraska at Omaha,
60th and Dodge Street, Omaha, Nebraska 68192-0109
jvenners@unmc.edu
Received March 1, 2005
Single electron reduction of the 1,2,4-trioxane heterocycle of artemisinin (1) forms primary and
secondary carbon-centered radicals. The complex structure of 1 does not lend itself to a satisfactory
dissection of the electronic and steric effects that influence the formation and subsequent reaction
of these carbon-centered free radicals. To help demarcate these effects, we characterized the reactions
of achiral dispiro-1,2,4-trioxolane 4 and dispiro-1,2,4-trioxanes 5-7 with ferrous bromide and 4-oxo-
TEMPO. Our results suggest a small preference for attack of Fe(II) on the nonketal peroxide oxygen
atom of 1. For 4, but not for 5 and 6, there was a strong preference for attack of Fe(II) on the less
hindered peroxide bond oxygen atom. The steric hindrance afforded by a spiroadamantane in a
five-membered trioxolane is evidently much greater than that for a corresponding six-membered
trioxane. Unlike 1, 5-7 fragment by entropically favored â-scission pathways forming relatively
stable R-oxa carbon-centered radicals. These data suggest that formation of either primary or
secondary carbon-centered radicals is a necessary but insufficient criterion for antimalarial activity
of 1 and synthetic peroxides.
Introduction
carbon-centered free radicals3 or carbocations4 that may
convey the parasiticidal effects and unique antimalarial
specificity of the artemisinins by alkylation of heme5 or
proteins,6 including the putative target SERCA PfATP6.7
The two major pathways (Scheme 1) for reductive cleav-
age of 18 are initiated by delivery of an electron from
Artemisinin and Iron(II). The discovery of artemisi-
nin (1, qinghaosu) and its semisynthetic derivatives
provided a new class of antimalarial drugs. As sum-
marized by Klayman,1 early work demonstrated that the
pharmacophoric peroxide bond in the 1,2,4-trioxane
heterocycle of 1 is essential for activity. Considerable
evidence2 suggests that the peroxide bond in 1 undergoes
reductive activation by heme released by parasite hemo-
globin digestion. This irreversible redox reaction produces
(3) Butler, A. R.; Gilbert, B. C.; Hulme, P.; Irvine, L. R.; Renton, L.;
Whitwood, A. C. Free Rad. Res. 1998, 28, 471-476.
(4) Szpilman, A. M.; Korshin, E. E.; Hoos, R.; Posner, G. H.; Bachi,
M. D. J. Org. Chem. 2001, 66, 6531-6540.
(5) Robert, A.; Coppel, Y.; Meunier, B. Chem. Commun. 2002, 414-
415.
(6) Meshnick, S. R. Artemisinin: Mechanisms of Action, Resistance
and Toxicity. Int. J. Parasitol. 2002, 32, 1655-1660.
(7) 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, 957-961.
(8) (a) Posner, G. H.; Oh, C. H. J. Am. Chem. Soc. 1992, 114, 8328-
8329. (b) Jefford, C. W.; Vicente, M. G. H.; Jacquier, Y.; Favarger, F.;
Mareda, J.; Millasson-Schmidt, P.; Brunner, G.; Burger, U. Helv. Chim.
Acta 1996, 79, 1475-1487. (c) Gu, J.; Chen, K.; Jiang, H.; Leszczynski,
J. J. Phys. Chem. A 1999, 103, 9364-9369.
* To whom correspondence should be addressed. Tel: 402.559.5362.
Fax: 402.559.9543.
† University of Nebraska Medical Center.
‡ University of Nebraska at Omaha.
(1) Klayman, D. L. Science 1985, 228, 1049-1055.
(2) (a) Robert, A.; Meunier, B. Chem. Soc. Rev. 1998, 27, 273-279.
(b) Jefford, C. W. Curr. Med. Chem. 2001, 8, 1803-1826. (c) Dong, Y.;
Vennerstrom, J. L. Redox Rep. 2003, 8, 284-288. (d) Posner, G. H.;
O’Neill, P. H. Acc. Chem. Res. 2004, 37, 397-404. (e) O’Neill, P. M.;
Posner, G. H. J. Med. Chem. 2004, 47, 2945-2964.
10.1021/jo050385+ CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/20/2005
J. Org. Chem. 2005, 70, 5103-5110
5103

Tang et al.
SCHEME 1
SCHEME 2. Synthesis of 5-7
Fe(II) to the antibonding σ* orbital of the peroxide bond
to form a pair of oxy radicals. The first involves the attack
of Fe(II) on O1 of the peroxide bond, producing an O2 free
radical. This oxygen free radical is short-lived and
rearranges via â-scission to a C4-centered primary radi-
cal thermodynamically facilitated by concomitant forma-
tion of an ester functionality; apparently, no methyl
radical is produced by the alternate â-scission pathway.
In the second, initial attack of Fe(II) on O2 of the peroxide
linkage results in formation of an O1 free radical. Rather
than fragment by any of the three possible â-scission
pathways a-c, the O1 free radical undergoes a 1,5
H-shift, giving rise to a C4-centered secondary radical.
Both artemisinin-derived C4-centered radicals are ir-
reversibly formed kinetic intermediates2b that self-
quench9 in intramolecular reactions to produce 2 and 3,
both of which are devoid of antimalarial activity. In
summary, available evidence suggests that electron
transfer leading to formation of carbon-centered radicals
may constitute the required activation step of the arte-
misinins and other peroxidic antimalarials.
Results and Discussion
Trioxane Synthesis. Preparation of target trioxanes
5-7 (Scheme 2) required precursor â-hydroperoxy alco-
hols 10 and 11. Although â-hydroperoxy alcohols have
been prepared by treatment of epoxides with anhydrous
H2O2 with12,13 or without14 an acid catalyst, we elected
to use the commercially available 50% aqueous H2O2 as
a safer alternative. Hydroperoxidation of spiro epoxide
815 with 50% aqueous H2O2 (10 equiv) pretreated with
anhydrous MgSO4 (ca. 1 g of anhydrous MgSO4 per 1 mL
of 50% H2O2 in 15 mL of ether) and molybdenyl acety-
lacetonate (5%)16 as catalyst afforded 1012 in modest yield.
Whether the primary or secondary carbon-centered
radicals or a combination of the two contribute to the
antimalarial activity of 1 has been a subject of debate.
The difficulty lies in the fact that both carbon-centered
radicals can be observed in iron(II) models, largely
depending on the particular reaction conditions, including
solvents and counterions as discussed in detail by Wu et
al.10 The complex structure of 1 does not lend itself to
satisfactory dissection of the electronic and steric effects
that influence the formation and subsequent reaction of
these carbon-centered free radicals. On the basis of the
achiral prototype dispiro-1,2,4-trioxolane 4,11 we envi-
sioned that achiral dispiro-1,2,4-trioxanes 5-7 could
provide tools to help demarcate these effects.
(10) Wu, W. M.; Wu, Y.; Wu, Y.-L.; Yao, Z.-J.; Zhou, C.-M.; Li, Y.;
Shan, F. J. Am. Chem. Soc. 1998, 120, 3316-3325.
(11) Vennerstrom, J. L.; Arbe-Barnes, S.; Brun R.; Charman, S. A.;
Chiu, F. C. K.; Chollet, J.; Dong, Y.; Dorn, A.; Hunziker, D.; Matile,
H.; McIntosh, K.; Padmanilayam, M.; Santo Tomas, J.; Scheurer, C.;
Scorneaux, B.; Tang, Y.; Urwyler, H.; Wittlin, S.; Charman, W. N.
Nature 2004, 430, 900-904.
(12) Kerr, B.; McCullough, K. J. J. Chem. Soc., Chem. Commun.
1985, 590-592.
(13) Subramanyam, V.; Brizuela, C. L.; Soloway, A. H. J. Chem. Soc.
Chem. Commun. 1976, 508-509.
(14) Adam, W.; Peters, K.; Renz, M. J. Org. Chem. 1997, 62, 3183-
3189.
(15) (a) Corey, E. J.; Chaykovsky, M. J. Am. Chem. Soc. 1962, 84,
3782-3783. (b) Johnson, C. R.; Mori, K.; Nakanishi, A. J. Org. Chem.
1979, 44, 2065-2067.
(16) Mattucci, A. M.; Perrotti, E.; Santambrogio, A. J. Chem. Soc.,
Chem. Commun. 1970, 1198-1199.
(9) Wu, Y. Acc. Chem. Res. 2002, 35, 255-259.
5104 J. Org. Chem., Vol. 70, No. 13, 2005

Dispiro-1,2,4-trioxane Analogues
SCHEME 3
Use of alternative solvents and catalysts such as THF
and sulfuric acid, respectively, led to decreased ratios of
10 and diol 1217 and lower reaction yields. Under nearly
identical conditions, hydroperoxidation of spiro epoxide
on the mechanism of action of 1 but also to understand
the striking loss of antimalarial activity of 5 and 6
compared to that of 1 and 4.
Reaction of 4 with iron(II) produced lactone 14, bro-
moacid 15 and unsaturated acid 1623 in a combined
isolated yield of 72% along with a 3% yield of 6-bromo-
hexanoic acid 1724 (Scheme 3). This result was confirmed
by the 22:1 ratio of cyclohexanone and 2-adamantanone
trapped as their oxime ethers25 by post-reaction treat-
ment with O-benzylhydroxylamine and pyridine and
quantitated by 1H NMR.26 The 22-24:1 ratio of reaction
products resulting from â-scission of the spiroadaman-
tane and spirocyclohexanone, respectively, indicates re-
gioselective formation of the oxy radical resulting from
preferential attack of Fe(II) on the less hindered peroxide
bond oxygen atom (O1) of 4. Similar regioselective iron-
mediated decompositions of other unsymmetical trioxo-
lanes due to steric hindrance were noted by Abe et al.27
Reaction of 5 with iron(II) produced bromoester
carbinols 18 and 19, unsaturated ester carbinol 20,
2-adamantanone, cyclohexanone, and formaldehyde
(Scheme 4). A 2:1 ratio of the O1:O2 pathways was evident
in the 2:1:1:0.43 ratio of (18 + 20)/2-adamantanone/
cyclohexanone/formaldehyde, the latter three of which
were quantitated26 as their O-benzyl oximes.25,28 The less
than theoretical quantity of formaldehyde observed was
probably due to the loss of formaldehyde during the
reaction and of its O-benzyl oxime during the reaction
workup.29 Bromoester carbinol 18 was initially formed
as a 2:1 mixture of two isomers that isomerized both in
the crude reaction mixture and in CDCl3 solution to form
a mixture of four isomers. These four 3,7-disubstituted
bicyclo[3.3.1]nonane isomers could be purified to 80-95%
by RP-HPLC, but their exo/endo stereochemistry was not
9
18 afforded 11 in low yield accompanied by nearly equal
quantities of diol 13.19 Little or no conversion of 9 to 11
took place using other solvents (ether, acetonitrile, 2-pro-
panol) or catalysts.
In the presence of catalytic p-toluenesufonic acid
(PTSA), target trioxanes 5 and 720,21 were readily formed
by reaction of 10 with 2-adamantanone and cyclohex-
anone, respectively. Similarly, reaction of 11 with cyclo-
hexanone in the presence of catalytic camphorsulfonic
acid (CSA) afforded target trioxane 6. Trioxane 5 was also
obtained (77% yield) using the triethylsilylperoxy deriva-
tive of 10, although the latter was obtained in yields of
less than 30% from 1-cyclohexene-1-methanol using the
method of O′Neill et al.21a (data not shown).
Antimalarial Activity. The antimalarial properties
of 5-7 compared to that of 1 and 4 were measured
against P. falciparum in vitro and P. berghei in vivo.22
The results were unexpected in that 5 and 7 with IC50’s
of 49 and 120 ng/mL were an order of magnitude less
potent than 1 and 4 (IC50’s of 1.6 and 1.2 ng/mL).
Trioxane 6 (IC50 > 1000 ng/mL) was inactive against P.
falciparum in vitro. Furthermore, each of the trioxanes
was completely inactive against P. berghei when they
were administered at 100 mg/kg oral and subcutaneous
doses on day 1 postinfection; in contrast 1 and 4 had oral
ED50/ED90’s of 9.1/14 and 4.0/8.8 mg/kg.
Reactions with Iron(II). Next, we turned our atten-
tion to investigating the reaction pathways of 4-7 with
FeBr2 (1 equiv) in THF8a not only to shed further light
(17) Itami, K.; Kamei, T.; Mitsudo, K.; Nokami, T.; Yoshida, J.-I. J.
Org. Chem. 2001, 66, 3970-3976.
(18) Olah, G. A.; Wu, A.-H. Synthesis 1990, 887-889.
(23) Bridges, A. J.; Raman, P. S.; Ng, G. S. Y.; Jones, J. B. J. Am.
Chem. Soc. 1984, 106, 1461-1467.
(19) Farcasiu, D.; Schleyer, P. V. R.; Ledlie, D. B. J. Org. Chem.
1973, 38, 3455-3459.
(24) McGrady, J.; Laughlin, R. G. Synthesis 1984, 426-428.
(25) Bhat, J. I.; Clegg, W.; Maskill, H.; Elsegood, M. R. J.; Menneer,
I. D.; Miatt, P. C. J. Chem. Soc., Perkin Trans. 2 2000, 1435-1446.
(26) Because of the volatility of 2-adamantanone, cyclohexanone, and
formaldehyde, their less volatile O-benzyl oximes (refs 25 and 28) were
independently prepared to identify diagnostic 1H NMR signals ap-
propriate for quantitation: δ 3.57 (s), 2.20 (t), and 7.08 (d), respectively.
(27) Abe, M.; Inakazu, T.; Munakata, J.; Nojima, M. J. Am. Chem.
Soc. 1999, 121, 6556-6562.
(20) Haq, A.; Kerr, B.; McCullough, K. J. J. Chem. Soc., Chem.
Commun. 1993, 1076-1078.
(21) (a) O’Neill, P. M.; Hindley, S.; Pugh, M. D.; Davies, J.; Bray, P.
G.; Park, B. K.; Kapu, D. S.; Ward, S. A.; Stocks, P. A. Tetrahedron
Lett. 2003, 44, 8135-8138. (b) Ahmed, A.; Dussault, P. H. Org. Lett.
2004, 6, 3609-3611. In this paper, the same Co(thd)2 catalyst is
employed as the method of choice for hydroperoxysilylation.
(22) In vitro activity was measured against the chloroquine-sensitive
NF54 (airport, unknown origin) and chloroquine-resistant KI (Thai-
land) strains of Plasmodium falciparum. The IC50’s are average values
(n ) 2 to 3) against the NF54 and K1 strains. In vivo activity was
measured against P. berghei as previously described (ref 11). Against
the K1 strain, O’Neill et al. (ref 21) report an IC50 of 33 ng/mL for 7;
our measured IC50 of 130 ng/mL for 7 was 4-fold higher.
(28) Hart, D. J.; Seely, F. L. J. Am. Chem. Soc. 1988, 110, 1631-
1633.
(29) For 5-7, we also observed small quantities of benzaldehyde
O-benzyl oxime (δ 8.14) (ref 25) that may have arisen from Fe(III)
oxidation of O-benzylhydroxylamine to form benzaldehyde that sub-
sequently reacted with O-benzylhydroxylamine.
J. Org. Chem, Vol. 70, No. 13, 2005 5105

Tang et al.
SCHEME 4
SCHEME 5
defined30 because of difficulty in calculating coupling
constants, although exo 7-bromo and 3-ester substituents
in one (18d) of the isomers were deduced by a strong
correlation between H-3 (2.82-2.91 ppm) and H-7 (4.68-
4.76 ppm) in the NOESY spectrum. The only way we
could account for the formation of bromoester carbinol
19 (vide infra; Scheme 6) was via iron(II) decomposition
of trioxane 7 formed from cyclohexanone and â-hydro-
peroxy alcohol 10 liberated during the course of the
reaction of 5 with iron(II). An analogous Lewis acid
catalyzed heterolytic fragmentation of the 1,2,4-trioxane
ring of artemisinin has been previously proposed.31
This 2:1 proportion of reaction products indicates a
modest preference for attack of Fe(II) on the less hindered
peroxide bond oxygen atom (O1) of 5. Interestingly, attack
of Fe(II) on the more hindered peroxide bond oxygen atom
(O2) generates an Fe(III)-complexed oxy radical that can
undergo two â-scission pathways, only one of which is
observed. In this pathway, an R-oxa primary carbon-
centered radical and cyclohexanone are initially formed,
and the former then unravels to 2-adamantanone and
formaldehyde. We observed no R-hydroxy ketone reaction
product that would have arisen from the â-scission
pathway to form the primary carbon-centered radical.
Reaction of 6 with iron(II) produced bromoester carbinol
21, 2-adamantanone, cyclohexanone, and formaldehyde
(Scheme 5). A 1:3 ratio of the O1:O2 pathways was evident
in the 1:3.1:3.3:1.5 ratio of 21/2-adamantanone/cyclohex-
anone/formaldehyde, the latter three of which were
quantitated26 as their O-benzyl oximes.25,28 As was the
case for 5, the less than theoretical quantity of formal-
dehyde observed was probably due to the loss of formal-
dehyde during the reaction and of its O-benzyl oxime
during the reaction workup. This 1:3 proportion of
reaction products indicates a modest preference for attack
of Fe(II) on the less hindered peroxide bond oxygen atom
(O2) of 6. Attack of Fe(II) on O2 generates an Fe(III)
complexed oxy radical that can undergo two â-scission
pathways, only one of which is observed. In this pathway,
2-adamantanone and an R-oxa primary carbon-centered
radical are initially formed, and the latter then unravels
(30) (a) Peters, J. A., Van Der Toorn, J. M.; Van Bekkum, H.
Tetrahedron 1975, 31, 2273-2281. (b) Peters, J. A.; van Ballegoyen-
Eekhout, G. W. M.; van de Graaf, B., Bove´e, W. M. M. J.; Baas, J. M.
A.; van Bekkum H. Tetrahedron 1983, 39, 1649-1654.
(31) Haynes, R. K.; Pai, H. H. O.; Voerste, A. Tetrahedron Lett. 1999,
40, 4715-4718.
5106 J. Org. Chem., Vol. 70, No. 13, 2005

Dispiro-1,2,4-trioxane Analogues
SCHEME 6
to cyclohexanone and formaldehyde. Similar to 5, we
observed no R-hydroxy ketone reaction product that
would have arisen from the â-scission pathway to form
the secondary carbon-centered radical.
intra- or intermolecular electron transfer between Fe(III)
and the carbon-centered radicals to simultaneously re-
generate Fe(II) and produce the corresponding carbo-
cations,2b,4 the latter of which could react with bromide
or undergo elimination.
For 4-6, confirmation of the proposed carbon-centered
free radical intermediates was provided by spin-trapping
experiments with the nitroxide free radical, 4-oxo-2,2,6,6-
tetramethyl-1-piperidinyloxy (4-oxo-TEMPO). Treatment
of 4 with 1.5 equiv each of FeBr2 and 4-oxo-TEMPO in
THF did form the expected aminoxy acid 22 in 7% yield,
but the major product was 14 (70%) accompanied by 15
(8%), 16 (3%), and 17 (6%). However, using conditions
(1.5 equiv of Fe(OAc)2 and 2 equiv of 4-oxo-TEMPO in
1:1 CH2Cl2/CH3CN) that we had used earlier in a similar
experiment11 with a trioxolane ketone, 22 was produced
in 56% yield along with 14 (28%) and 16 (6%). For 5 and
6, reactions were conducted with 2.5 equiv each of FeBr2
and 4-oxo-TEMPO in THF. Trioxane 5 formed aminoxy
ester alcohol 23 (57%), 2-adamantanone (3%), and un-
saturated ester carbinol 20 (4%). Trioxane 6 formed
aminoxy ester alcohol 24 (22%) and 2-adamantanone
(39%). Aminoxy reaction products 22-24 correspond to
the proposed carbon-centered free radical intermediates
invoked to account for the observed reaction products in
Schemes 3-5. However, this does not necessarily imply
that bromoacids 15 and 17, bromoester carbinols 18, 19
(Scheme 6; vide infra), and 21, the unsaturated acid 16
and ester carbinol 20 were formed exclusively by carbon-
centered radical pathways. For example, an alternative
mechanism to account for the formation of 15-21 is
Reaction of 7 with iron(II) produced bromoester carbinol
19, cyclohexanone, and formaldehyde (Scheme 6). A 1:1.4
ratio of 19:cyclohexanone was observed; the latter was
quantitated26 as its O-benzyl oxime.25 Since the O2
pathway produces 2 equiv of cyclohexanone, this result
indicates a 3:2 ratio of the O1:O2 pathways. Since steric
effects do not play a role in reaction of 7 with iron(II),
this proportion of reaction products indicates a small
preference for attack of Fe(II) on the nonketal peroxide
oxygen atom (O1) of the peroxide bond. In this pathway,
subsequent â-scission simultaneously forms a primary
carbon-centered radical and ester. Attack of Fe(II) on O2
generates an Fe(III) complexed oxy radical that can
undergo two â-scission pathways, only one of which is
observed. In this pathway, cyclohexanone and an R-oxa
primary carbon-centered radical are initially formed, and
the latter then unravels to formaldehyde and a second
molecule of cyclohexanone. As was observed for 5 and 6,
such entropically favored O2 â-scission reaction pathways
that form relatively stable32 R-oxa primary carbon-
centered radicals are apparently much too rapid to
observe the competing â-scission pathways33 leading to
the unobserved R-hydroxy ketone reaction products.
Similar R-oxa carbon-centered radicals may be invoked
to account for the carbonyl end products produced from
single-electron reductions of other synthetic trioxanes.34
Discussion. We suggest that the observed reaction
product distributions in the reactions of 4-7 with Fe(II)
(32) Henry, D. J.; Parkinson, C. J.; Mayer, P. M.; Radom, L. J. Phys.
Chem. A 2001, 105, 6750-6756.
(33) Bietti, M.; Lanzalunga, O.; Salamone, M. J. Org. Chem. 2005,
70, 1417-1422.
(34) (a) Fujisaka, T.; Miura, M.; Nojima, M.; Kusabayashi, S. J.
Chem. Soc., Perkin Trans. 1 1989, 1031-1039. (b) Posner, G. H.; Park,
S. B.; Gonza´lez, L.; Wang, D.; Cumming, J. N.; Klinedinst, D.; Shapiro,
T. A.; Bachi, M. D. J. Am. Chem. Soc. 1996, 118, 3537-3538. (c) O’Neill,
P. M.; Mukhtar, A.; Ward, S. A.; Bickley, J. F.; Davies, J.; Bachi, M.
D.; Stocks, P. A. Org. Lett. 2004, 6, 3035-3038. (d) Provot, O.;
Camuzat-Dedenis, B.; Hamzaoui, M.; Moskowitz, H.; Mayrargue, J.;
Robert, A.; Cazelles, J.; Meunier, B.; Zouhiri, F.; Desmae¨le, D.;
d’Angelo, J.; Mahuteau, J.; Gay, F.; Cice´ron, L. Eur. J. Org. Chem.
1999, 1935-1938.
J. Org. Chem, Vol. 70, No. 13, 2005 5107

Tang et al.
are determined by regioselective formation of one of two
possible oxy radicals generated by delivery of an electron
from Fe(II) to the peroxide bond antibonding σ* orbitals27
rather than the relative stability of the two oxy radical
species per se. As is observed for 1,2b the ensuing con-
versions of the oxy radicals to the more stable8c carbon-
centered radical intermediates by â-scission pathways35
are irreversible and strongly exothermic. For 5-7, the
observed reaction products also confirm12,20 that ring-
opening â-scissions arising from oxy radicals at ketal (O2)
positions occur much faster than competing ring-opening
â-scissions arising from oxy radicals at nonketal (O1)
positions.
In consideration of the small preference for attack of
Fe(II) on the nonketal peroxide oxygen atom (O1) of the
peroxide bond in 7, we observed a less than expected 2:1
preference for attack of Fe(II) on the nonketal and
sterically less-hindered oxygen atom (O1) of the peroxide
bond in 5 keeping in mind the 3:1 preference for attack
of Fe(II) on the ketal and sterically less-hindered oxygen
atom (O2) of the peroxide bond in 6. For 4 on the other
hand, there is a strong preference for attack of Fe(II) on
the less hindered peroxide bond oxygen atom (O1). Since
there is little difference in the electronic character of the
peroxide oxygen atoms in 4, apparently steric hindrance
to attack of Fe(II) on the more hindered peroxide bond
oxygen atom (O2) determines the observed regioselectiv-
ity. In the subsequent reaction pathways, both â-scission
pathways are equally facilitated by concomitant ester
formation. The regioselectivity of attack of Fe(II) on the
peroxide bond afforded by the spiroadamantane in the
five-membered 1,2,4-trioxolane 4 is evidently much greater
than that for the six-membered 1,2,4-trioxanes 5 and 6.
This is illustrated by the greater steric crowding on the
adamantane side of the peroxide bond antibonding σ*
orbital in 4 vs 5 and 6 (Figure 1).
FIGURE 1. LUMO (blue) mapped on isodensity surfaces of
4, 5, and 6 viewed down the axis of the equatorial peroxide
bond from the adamantane (a) and cyclohexane (b) sides.
FIGURE 2. LUMO (blue) mapped on the isodensity surface
of 1 viewed down the axis of the peroxide bond from the O1 (a)
and O2 (b) direction.
Although conformers of 4-636 with the axial peroxide
are more stable (∆E ) Eeq - Eax ) 0.280, 1.45, and 0.145
kcal/mol for 4, 5, and 6), conformers with the equatorial
peroxide (depicted in Figure 1) provide less steric hin-
drance to the peroxide antibonding σ* orbital. As viewed
down the axis of the O-O bond of 4, the σ* (LUMO) is
not visible to the incoming Fe(II) from the adamantane
side but is clearly visible and presumably readily acces-
sible to the incoming Fe(II) from the cyclohexane side.
For 5 and 6, the LUMO is not as accessible from either
direction as it is from the cyclohexane side of 4. The
reduced accessibility of the LUMO in 5 and 6 is due to
the additional methylene group of the trioxane hetero-
cycle increasing the steric hindrance of the peroxide bond
by both ring systems. This increased steric effect is
manifested by a decrease in the angle between the
adamantane and cyclohexane rings as measured through
the axis of the peroxide bond. By defining planes of the
spiro and vicinal carbon atoms, the angle between the
planes of the adamantane and cyclohexane rings can be
calculated; for 4 it is 214°, whereas for 5 and 6 it is 167°.
The less exposed and presumably more biologically
stable peroxide bonds in 5 and 6 vs 1 and 4 (Figures 1
and 2) may explain why the two trioxanes are so much
less active than 1 or their trioxolane chemical cousin 4,
although the release of ring strain with peroxide bond
cleavage in the six-membered 1,2,4-trioxane 1 is less than
that for the five-membered 1,2,4-trioxolane 4. Another
contributing factor could be that 5 and 6, unlike 1 and
4, undergo reaction pathways to form relatively stable
R-oxa primary carbon-centered radicals that unravel to
carbonyl-containing end products. The latter, unlike
carbon-centered radicals, presumably contribute little to
their antimalarial properties. In this regard, it is notable
that 6, the trioxane completely devoid of antimalarial
activity, also produced the largest proportion of carbonyl-
containing end products and the lowest proportion of
carbon-centered radicals. Consistent with this, 1 does not
undergo â-scission pathway c (Scheme 1) that would form
the stabilized R-dioxa (methylenedioxy) carbon-centered
radical, as evidenced by the absence of the expected
diketoacid end product 25 (Scheme 7). A complementary
example is provided by synthetic trioxane 2637 that
undergoes exclusive â-scission to form an R-oxa primary
(35) Walling, C.; Padwa, A. J. Am. Chem. Soc. 1963, 85, 1593-1597.
(36) Conformational analysis of 4-6 using MMFF94 with the Monte
Carlo method revealed that the chair conformations of the cyclohexane
rings in 4-6 are the most stable conformations. Also, in both the
trioxolane and trioxane rings there exist two stable conformations of
the same energy. The four stable conformations of each compound were
subjected to ab initio geometry optimizations via HF/6-31G*. Again,
the trioxolane and trioxane rings were found to have two equal energy
conformations. Consequently, only two conformations of the cyclohex-
ane ring where the peroxide linkage was in either the axial or
equatorial positions were considered.
5108 J. Org. Chem., Vol. 70, No. 13, 2005

Dispiro-1,2,4-trioxane Analogues
SCHEME 7
carbon-centered radical; as predicted, this trioxane is
inactive. The foregoing, however, does not provide a
satisfactory explanation for the 4-fold greater potency of
deoxoartemisinin (27) vs that of 1,38 as the major iron-
(II) reaction product of the former is diketo formate ester
2839 produced by a putative â-scission pathway2b that we
suggest proceeds by way of an R-dioxa (methylenedioxy)
carbon-centered radical.40 Derivatives of 27 seem to
fragment by this same pathway to afford carbonyl-
containing end products.41
Although this study did not provide definitive answers
to whether the primary or secondary carbon-centered
radicals or a combination of the two contribute to the
antimalarial activity of 1, the results did suggest a small
preference for attack of Fe(II) on the nonketal peroxide
oxygen atom (O1) of 1.42 In addition, the iron(II) frag-
mentation of 4 clearly demonstrates that a peroxide need
not fragment to form a primary carbon-centered radical
to be active. Nevertheless, 1 alkylates free heme5 or
hemoglobin heme43 by way of the primary carbon-
centered radical (O1 pathway), possibly the only malaria-
parasite-relevant fully characterized alkylation reactions
so far reported for 1.
Experimental Section
Adamantane-2-spiro-3′-1′,2′,4′-trioxaspiro[5.5]undec-
ane (5). p-Toluenesulfonic acid monohydrate (40 mg, 0.2
mmol) was added to a mixture of 1012 (280 mg, 1.9 mmol),
2-adamantanone (450 mg, 3 mmol), CH2Cl2 (10 mL) and 1,2-
dichloroethane (10 mL). The reaction mixture was stirred at
room temperature overnight, washed with saturated NaHCO3
(15 mL), water (15 mL) and brine (15 mL), dried over MgSO4,
filtered and concentrated. The crude product was purified by
flash chromatography using gradient elution (sg, 2-4% ether
in hexane) to afford 5 as a colorless solid (500 mg, 95%). Mp
1
45-48 °C (ethanol/H2O 2:1); H NMR δ 1.22-2.05 (m, 22H),
2.37 (brs, 1H), 2.95 (brs, 1H), 3.51 (brs, 1H), 3.69 (brs, 1H);
13C NMR δ 21.4, 25.9, 27.17, 27.19, 28.5, 30.2, 32.3, 33.3, 33.4,
36.2, 37.2, 65.3, 77.3, 104.2. Anal. Calcd for C17H26O3: C, 73.34;
H, 9.41. Found: C, 73.50; H, 9.21.
Adamantane-2-spiro-3′-1′,2′,5′-trioxaspiro[5.5]undec-
ane (6). 10-Camphorsulfonic acid (80 mg, 0.35 mmol) was
added to a mixture of 11 (680 mg, 3.5 mmol), cyclohexanone
(686 mg, 7.0 mmol) and CH2Cl2 (30 mL). The reaction mixture
was stirred at room temperature overnight, washed with
saturated NaHCO3 (30 mL), water (30 mL), and brine (30 mL),
dried over MgSO4, filtered and concentrated. The crude
product was crystallized from 80% aqueous ethanol to afford
6 as a colorless solid (740 mg, 76%). Mp 85-87 °C (80%
(37) Cazelles, J.; Camuzet-Dedenis, B.; Provot, O.; Robert, A.;
Mayrargue, J.; Meunier, B. J. Chem. Soc., Perkin Trans. 1 2000, 1265-
1270.
(38) Jung, M.; Li, X.; Bustos, D. A.; ElSohly, H. N.; McChesney, J.
D.; Milhous, W. K. J. Med. Chem. 1990, 33, 1516-1518.
(39) Avery, M. A.; Fan P.; Karle, J. M.; Bonk, J. D.; Miller, R.; Goins,
D. K. J. Med. Chem. 1996, 39, 1885-1897.
(40) Alternate pathways (refs 10 and 39) to account for the formation
of 28 from 27 have been proposed.
1
aqueous ethanol); H NMR δ 1.40-2.36 (m, 23H), 2.73 (brs,
1H), 3.71 (brs, 1H), 4.05 (brs, 1H); 13C NMR δ 22.4, 25.6, 27.4,
27.5, 28.6, 29.3, 32.0, 33.6, 34.2, 37.8, 63.6, 81.1, 101.9. Anal.
Calcd for C17H26O3: C, 73.34; H, 9.41. Found: C, 73.49; H,
9.48.
(41) O’Neill, P. M.; Pugh, M.; Stachulski, A. V.; Ward, S. A.; Davies,
J.; Park, B. K. J. Chem. Soc., Perkin Trans. 1 2001, 2682-2689.
(42) As one reviewer pointed out, it is interesting to note that
epiartemisinin (C9 R-epimer), with its more sterically hindered inner
peroxide oxygen atom (O1), is 1.5- to 7-fold less potent than 1. This
activity difference was rationalized in terms of a preferential attack
of Fe(II) (heme) at the outer peroxide oxygen atom (O2) of epiartemisi-
nin to give mainly the secondary carbon-centered radical. (a) Acton,
N.; Klayman, D. L. Planta Med. 1987, 266-268. (b) Avery, M. A.; Gao,
F.; Chong, W. K. M.; Mehrotra, S.; Milhous, W. K. J. Med. Chem. 1993,
36, 4264-4215. (c) Jefford, C. W.; Burger, U.; Millasson-Schmidt, P.;
Bernardinelli, G.; Robinson, B. L.; Peters, W. Helv. Chim. Acta 2000,
83, 1239-1246.
7-(4-Oxo-2,2,6,6-tetramethyl-1-piperidinyloxy)bicyclo-
[3.3.1]nonane-3-carboxylic acid (22). To a solution of 4 (264
mg, 1.0 mmol), 4-oxo-TEMPO (340 mg, 2.0 mmol) in CH2Cl2
(10 mL) and CH3CN (10 mL) was added Fe(OAc)2 (261 mg,
1.5 mmol). The resulting mixture was stirred under N2 and
35 °C for 24 h before being quenched with water (50 mL) and
acetic acid (3 mL). After separation of the organic layer, the
aqueous layer was extracted with CH2Cl2 (2 × 20 mL). The
combined extracts were washed with brine (2 × 30 mL), dried
over MgSO4, filtered and concentrated. The crude product was
purified by flash chromatography using gradient elution (sg,
(43) Selmeczi, K.; Robert, A.; Claparols, C.; Meunier, B. FEBS Lett.
2004, 556, 245-248.
J. Org. Chem, Vol. 70, No. 13, 2005 5109

Tang et al.
10-90% EtOAc in hexane) to give 2-adamantanone (8 mg, 5%),
14 (46 mg, 28%), 1623 (10 mg, 6%), and 22 (190 mg, 56%) as a
colorless solid. For 22: mp 179-180 °C (ether/hexane 1:3); 1H
NMR δ 1.14 (s, 6H), 1.14-1.18 (m, 1H), 1.24-1.35 (m, 3H),
1.30 (s, 6H), 1.50-1.61 (m, 3H), 2.08-2.30 (m, 7H), 2.48-2.62
(m, 3H), 4.04-4.13 (m, 1H); 13C NMR δ 22.7, 26.0, 28.7, 29.2,
33.8, 35.4, 39.5, 53.5, 62.8, 76.1, 181.7, 208.6. HRMS-FAB for
C19H31NO4 [M + H]+: calcd 338.2253, found 338.2321.
(1-Hydroxycyclohexyl)methyl 7-(4-Oxo-2,2,6,6-tetra-
methyl-1-piperidinyloxy)-bicyclo[3.3.1]nonane-3-carbox-
ylate (23). A mixture of 5 (240 mg, 0.86 mmol), FeBr2 (370
mg, 1.72 mmol), 4-oxo-TEMPO (294 mg, 1.72 mmol) and THF
(10 mL) was stirred at ambient temperature under a N2
atmosphere for 16 h and concentrated. The crude product was
dissolved in EtOAc, washed with water and brine, dried over
MgSO4, filtered and concentrated. The crude product was
purified by flash chromatography using gradient elution (sg,
10-60% EtOAc in hexane) to afford 23 (220 mg, 57%) as a
colorless oil (single isomer). 1H NMR δ 1.12 (s, 6H), 1.10-1.18
(m, 1H), 1.28 (s, 6H), 1.24-1.34 (m, 4H), 1.40-1.68 (m, 12H),
1.85 (brs, 1H, OH), 2.08-2.30 (m, 8H), 2.52-2.60 (m, 2H), 3.98
(s, 2H), 4.04-4.11 (m, 1H); 13C NMR δ 21.5, 22.6, 25.6, 25.9,
28.4, 29.4, 33.9, 34.3, 35.6, 39.5, 53.5, 62.6, 70.5, 71.3, 76.1,
176.5, 208.6. HRMS-FAB for C26H44NO5 [M + H]+: calcd
450.3219, found 450.3210.
washed with water and brine, dried over MgSO4, filtered and
concentrated. The crude product was purified by flash chro-
matography using gradient elution (sg, 10-50% EtOAc in
hexane) to give 2-adamantanone (50 mg, 39%) and 24 (85 mg,
1
22%) as a colorless oil. H NMR δ 1.15 (s, 6H), 1.28 (s, 6H),
1.40-1.46 (m, 2H), 1.52-1.87 (m, 16H), 2.05 (brs, 1H, OH),
2.20 (d, J ) 12.7 Hz, 2H), 2.23 (d, J ) 15.6 Hz, 2H), 2.39 (t,
J ) 7.4 Hz, 2H), 2.55 (d, J ) 12.7 Hz, 2H), 3.82 (t, J ) 6.4 Hz,
2H), 4.29 (s, 2H); 13C NMR δ 22.5, 25.1, 26.0, 27.0, 27.4, 28.3,
32.4, 32.5, 34.2, 34.4, 34.6, 38.0, 53.5, 62.9, 68.9, 74.0, 76.7,
173.8, 208.3. HRMS-FAB for C26H44NO5 [M + H]+: calcd
450.3219, found 450.3221.
Acknowledgment. We thank the Medicines for
Malaria Venture (MMV) for generous support of this
research and Reto Brun and Sergio Wittlin of the Swiss
Tropical Institute for the antimalarial data of 1, and
4-7. We acknowledge the Nebraska Center for Mass
Spectrometry for the HRMS data.
Supporting Information Available: Alternate synthesis
of 5 and synthesis and characterization data for 7-21,
O-benzyl oxime synthesis and quantitation, antimalarial as-
says, computational chemistry methods, 13C NMR spectra for
compounds 18a-d and 19-24, and total energies and atomic
coordinates to support the M.M. and M.O. calculations. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(2-Hydroxy-2-adamantyl)methyl 6-(4-Oxo-2,2,6,6-tet-
ramethyl-1-piperidinyloxy)hexanoate (24). A mixture of
6 (240 mg, 0.86 mmol), FeBr2 (370 mg, 1.72 mmol), 4-oxo-
TEMPO (294 mg, 1.72 mmol) and THF (10 mL) was stirred at
ambient temperature under a N2 atmosphere for 30 h and
concentrated. The crude product was dissolved in EtOAc,
JO050385+
5110 J. Org. Chem., Vol. 70, No. 13, 2005