Characterization of the two major CYP450 metabolites of ozonide (1,2,4-trioxolane) OZ277

Article abstract of DOI:10.1016/j.bmcl.2008.01.087

The antimalarial synthetic ozonide OZ277 (RBx11160) was hydroxylated by human liver microsomes at the distal bridgehead carbon atoms of the spiroadamantane substructure to form two carbinol metabolites devoid of antimalarial activity.

Full text of DOI:10.1016/j.bmcl.2008.01.087

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1555–1558
Characterization of the two major CYP450 metabolites
of ozonide (1,2,4-trioxolane) OZ277
a
b
b
a
c
´
Lin Zhou, Andre Alker, Armin Ruf, Xiaofang Wang, Francis C. K. Chiu,
Julia Morizzi,c Susan A. Charman,c William N. Charman,c Christian Scheurer,d
Sergio Wittlin,d Yuxiang Dong,a Daniel Hunzikerb and Jonathan L. Vennerstroma,*
aCollege of Pharmacy, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198, USA
bF. Hoffmann-La Roche Ltd, Grenzacherstrasse 124, CH-4070 Basel, Switzerland
cVictorian College of Pharmacy, Monash University, 381 Royal Parade, Parkville, Vic. 3052, Australia
dSwiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
Received 19 December 2007; revised 23 January 2008; accepted 25 January 2008
Available online 30 January 2008
Abstract—The antimalarial synthetic ozonide OZ277 (RBx11160) was hydroxylated by human liver microsomes at the distal bridge-
head carbon atoms of the spiroadamantane substructure to form two carbinol metabolites devoid of antimalarial activity.
ꢀ 2008 Elsevier Ltd. All rights reserved.
The antimalarial sesquiterpene lactone artemisinin con-
tains a peroxide bond in the form of a 1,2,4-trioxane
heterocycle.1 The peroxide bond in artemisinin, semisyn-
thetic artemisinins, and synthetic peroxides is essential,
O
O
Y
X
O
NH NH2
O
but not sufficient, for high antimalarial efficacy.1,2
A
1 (OZ277) X, Y = H
2 (OZ397) X = OH, Y = H
synthetic peroxide antimalarial drug has yet to be iden-
tified,3,4 although a synthetic ozonide (OZ277 or
RBx11160)5 is now in Phase II clinical trials. Under-
standing the stability of the pharmacophoric peroxide
bond in OZ277 (1)6 to cytochrome P450 (CYP450)
metabolism is key to elucidating its pharmacokinetics
and pharmacodynamics. To this end, we studied the
reaction profile of 1 with human liver microsomes, and
we now report the structural identification, synthesis,7
and antimalarial activity of two major OZ277 hydroxyl-
ated metabolites 2 (OZ397) and 3 (OZ381) (Fig. 1).
3 (OZ381) X = H, Y = OH
Figure 1. Ozonide 1 (OZ277) and its two hydroxylated metabolites 2
(OZ397) and 3 (OZ381).
tation of spiroadamantane ozonides (such as 1) in ESI–
MS is characterized by peroxide bond scission and sub-
sequent rearrangement resulting in the elimination of an
adamantane lactone fragment with the loss of 166 mass
units (Fig. 2C). Peaks b and c exhibited a loss of 182
mass units in the MS/MS spectra indicating incorpora-
tion of an oxygen atom in the adamantane substructure
(Fig. 2D). The minor hydroxylated metabolite (peak d)
showed a loss of 166 mass units indicating that the ada-
mantane moiety had not been modified and that incor-
poration of the oxygen atom had occurred at the
cyclohexyl side of the molecule (Fig. 2E). Although
the sites of hydroxylation could not be deduced from
the MS/MS fragmentation spectra alone, it was specu-
lated that the sites of hydroxylation of the two major
metabolites (peaks b and c) were at the two distal
bridgehead positions. This is consistent with the
observed preference for bridgehead oxidation in
Incubation of 1 (Fig. 2A) with human liver microsomes
in vitro produced three new peaks on LC/MS (Fig. 2B),
each with an increase in molecular weight of 16 suggest-
ing the presence of hydroxylated metabolites.8 The
hydroxylated metabolite peaks were further character-
ized by MS/MS (Fig. 2D and E). The MS/MS fragmen-
Keywords: 1,2,4-Trioxolane; Ozonide; Antimalarial; Synthetic perox-
ide; CYP450.
*
Corresponding author. Tel.: +1 402 559 5362; fax: +1 402 559
9543; e-mail: jvenners@unmc.edu
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.087

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L. Zhou et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1555–1558
100
was obtained by treatment of 5-hydroxy-2-adamanta-
3.85
A
B
none with methoxylamine HCl and pyridine (97%) fol-
lowed by acetylation with Ac2O, pyridine, and DMAP
(cat.) (91%). Ozonolysis of unsymmetrical oxime ethers
such as 4 produce enantiomeric carbonyl oxide interme-
diates, which in cycloaddition reactions with 4-substi-
tuted cyclohexanones (such as 5) could form four
isomeric ozonides.13 Based on our previous data,14,15
we expected to observe the formation of two major
ozonide isomers with substituent and peroxo groups at
the equatorial and axial positions in the cyclohexane
ring. Indeed, proton and carbon NMR of the purified
(sg, 10% ether in hexanes) reaction mixture (30%) indi-
cated formation of two predominant ozonide diester iso-
mers16 from which we were able to crystallize the major
isomer 6 in 17% yield. Conversion of 6 to minor metab-
olite 3 was relatively straightforward beginning with es-
ter hydrolysis (96%) to hydroxy acid 7 followed by
conversion to active ester 8 (89%). Amide bond (80%)
and salt formation with TsOH (94%) completed the
reaction sequence. The trans,cis configuration of 6,
and therefore of 3, was assigned based on the conversion
of trans,cis ozonide diester 10 (vide infra) to 7 (86%).
a
%
%
50
0
1
2
3
4
5
6
2.75
100
b
50
0
2.88
d
c
4.36
1
2
3
4
5
6
Retention Time (min)
100
50
227.18
C
%
%
%
210.16
243.18
393.29
400
0
100
150
150
150
200
250
300
300
300
350
350
350
100
227.17
D
50
Similarly, major metabolite 217 was obtained in a four-
step sequence (Scheme 2) starting with a Griesbaum
coozonolysis11,12 reaction between 5-(4-methylbenz-
oxy)-2-adamantanone O-methyl oxime (9) and keto es-
ter 5 to afford, after chromatography (sg, 0–20% ether
in hexanes), a mixture of four ozonide diester diastereo-
mers (54%).13,16 Oxime ether 9 was obtained by acyla-
210.15
200
243.17
250
409.26
400
0
100
100
E
243.17
50
0
tion
of
5-hydroxy-2-adamantanone
with
4-
210.15
200
243.22
250
methylbenzoyl chloride in pyridine (90%) followed by
treatment with methoxylamine HCl and pyridine
(94%). The 4-methylbenzoate (rather than acetate) was
chosen to increase molecular weight to allow for more
convenient fractional crystallization of the minor ozon-
ide diester isomers, and to provide convenient benzylic
singlet proton NMR signals to distinguish between iso-
mers. Repeated chromatography (sg, 8% ether in hex-
anes) gave four fractions (isomers A + B), isomer B,
isomers B + C, and isomers C + D. Crystallization of
isomer B from acetone gave 10 as colorless crystals,
established as the trans,cis diastereomer by X-ray crys-
tallographic analysis (Fig. 3). Repeated chromatography
(sg, 8% ether in hexanes) of isomers B + C gave isomer
C which was crystallized from acetone to give 11 as col-
orless crystals, established as the cis,cis diastereomer by
X-ray crystallographic analysis (Fig. 3). Diester ozonide
11 was converted to major metabolite 2 following a sim-
ilar sequence to that described for minor metabolite 3:
ester hydrolysis to hydroxy acid 12 (81%), conversion
to active ester 13 (83%) followed by amide bond forma-
tion and conversion to the tosylate salt (71% combined
yield).
409.24
400
100
m/z
Figure 2. Mass chromatograms of (A) ozonide 1 (OZ277) and (B)
three hydroxylated metabolites formed upon incubation with human
liver microsomes. Peak a, ozonide 1 (OZ277); peak b, adamantane
hydroxylated metabolite 2 (OZ397); peak c, adamantane hydroxylated
metabolite 3 (OZ381) and peak d, cyclohexyl hydroxylated metabolite.
MS/MS spectra of (C) ozonide 1 (OZ277), (D) adamantane hydrox-
ylated metabolites 2 (OZ397) and 3 (OZ381), and (E) the cyclohexyl
hydroxylated metabolite.
adamantane-containing structures.9 We also reasoned
that hydroxylation at the two proximal bridgehead car-
bon atoms was less likely to occur due to an inductive
effect of the ozonide heterocycle. Based on this hypoth-
esis, we synthesized putative hydroxylated metabolites 2
and 3 (vide infra) and found that they had identical
chromatographic retention characteristics and MS frag-
mentation patterns to the two major in vitro metabolites
formed in human liver microsomes. A repeat of the
in vitro metabolism studies quantitating metabolite for-
mation indicated that 2 was the major, and 3 was the
minor, metabolite, in an approximate 4:1 ratio.
The crystallization conditions described above delivered
suitable crystals for X-ray analysis of 10 and 11.18 In
both cases, single crystals were mounted in a loop and
data were collected on a STOE Imaging Plate Diffrac-
tion System (STOE, Darmstadt) with Mo-radiation
Minor metabolite 310 was obtained in a four-step
sequence (Scheme 1) starting with a Griesbaum coozon-
olysis11,12 reaction between 5-acetoxy-2-adamantanone
O-methyl oxime (4) and keto ester 5. Oxime ether 4
˚
(0.71 A) at room temperature. Data were processed with
STOE IPDS-software and the crystal structures were

L. Zhou et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1555–1558
1557
OCH3
N
AcO
O
O
O
O
O
HO
a
AcO
b
4
5
+
O
O
R
OMe
O
O
6
7, R = OH
8, R = OSu
OMe
c
major trans,cis isomer
O
d
3
Scheme 1. Reagents and conditions: (a) O3, cyclohexane:CH2Cl2 3:1, 0 ꢁC; (b) 1.25 M aq NaOH:EtOH:THF 0.8:1:1, 50 ꢁC, 4 h, then acidification to
pH 3 with 1 M HCl; (c) HOSu, EDCI, DMF, rt, 24 h; (d) 1,2-diamino-2-methylpropane, CHCl3, rt, 1 h, then PTSA, EtOH, rt.
H3C
O
O
O
O
O
O
OMe
O
10
OCH3
N
O
a
major trans,cis isomer
+
5
O
+
9
H3C
O
O
O
O
b
O
O
O
HO
OMe
R
O
O
H3C
11
minor cis,cis isomer
12, R = OH
13, R = OSu
c
d
2
Scheme 2. Reagents and conditions: (a) O3, cyclohexane:CH2Cl2 4:1, 0 ꢁC; (b) 1 M aq NaOH:EtOH:THF 1:1:1, 50 ꢁC, 18 h, then acidification to pH
4 with 0.2 M HCl; (c) HOSu, EDCI, DMF, rt, 24 h; (d) 1,2-diamino-2-methylpropane, CHCl3, rt, 3 h, then PTSA, ether:CH2Cl2 5:1, rt.
„trans“
„cis“
„cis“
„cis“
10
11
major trans,cis isomer
minor cis,cis isomer
Figure 3. Ellipsoid plots of diester ozonides 10 and 11; displacement ellipsoids are shown at the 50% probability level. In both structures, the
methoxycarbonylmethyl and epoxide substituents are in equatorial positions on the cyclohexane ring, and the peroxide substituent is in the axial
position. In 10 and 11, the 4-methylbenzoate is trans and cis, respectively, to the peroxide substituent.
solved and refined with ShelXTL (Bruker AXS, Kar-
lsruhe). The relative configuration of 10 was confirmed
by an independent X-ray analysis of a second crystal.
amantane ring system to the antimalarial properties of
1. It is conceivable that the steric hindrance provided
by the bridgehead carbinols in 2 and 3 prevents effi-
cient alkylation reactions of the spiroadamantane-de-
rived secondary carbon-centered radicals19 from
occurring when the ozonide reacts with iron(II) in
the parasite. Consistent with a general SAR trend
for this class of antimalarial peroxides,20 it is also
likely that the greater polarity of 2 and 3 (LogD
1.8) vs. 1 (LogD 3.2) may, in part, account for the
inactivity of the former.
Somewhat unexpectedly, we found that both 2 and 3
had IC50 values >100 ng/mL against the chloroquine-
resistant K1 strain of Plasmodium falciparum
in vitro; in comparison, 1 has an IC50 of 1.0 ng/mL
against this same parasite strain.5 The complete lack
of antiplasmodial activity of 2 and 3 demonstrates
the essential contribution of an unsubstituted spiroad-

1558
L. Zhou et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1555–1558
1.46–2.02 (m, 20H), 2.06 (d, J = 6.8 Hz, 2H), 2.29 (s, 3H),
Acknowledgment
3.19 (d, J = 5.8 Hz, 2H), 4.47 (s, 1H), 7.12 (d, J = 7.8 Hz,
2H), 7.49 (d, J = 7.8 Hz, 2H), 7.71 (br s, 3H), 8.04 (t,
J = 6.0 Hz, 1H); 13C NMR (125.7 MHz, DMSO-d6) d
20.95, 23.49, 28.24, 29.69, 32.70, 33.35, 33.53, 37.80, 41.90,
41.97, 44.44, 46.03, 54.53, 65.49, 108.69, 110.08, 125.66,
128.24, 137.84, 145.80, 172.51. Anal. Calcd for
C29H44N2O8S: C, 59.98; H, 7.64; N, 4.82. Found: C,
59.71; H, 7.48; N, 5.02.
This investigation received financial support from the
Medicines for Malaria Venture (MMV).
References and notes
1. Klayman, D. L. Science 1985, 228, 1049.
2. O’Neill, P. M.; Posner, G. H. J. Med. Chem. 2004, 47,
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3. Jefford, C. W. Drug Discovery Today 2007, 12, 487.
4. Tang, Y.; Dong, Y.; Vennerstrom, J. L. Med. Res. Rev.
2004, 24, 425.
5. 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.
6. Kaiser, M.; Wittlin, S.; Nehrbass-Stuedli, A.; Dong, Y.;
Wang, X.; Hemphill, A.; Matile, H.; Brun, R.; Venner-
strom, J. L. Antimicrob. Agents Chemother. 2007, 51,
2991.
11. Griesbaum, K.; Liu, X.; Kassiaris, A.; Scherer, M. Libigs
Ann./Recueil 1997, 1381.
12. Griesbaum, K. Trends Org. Chem. 1997, 6, 145.
13. Although the mechanism of the Griesbaum coozonolysis
is not completely understood, the facial selectivity in
reactions of the putative enantiomeric carbonyl oxide
intermediates parallels the facial selectivity in reactions of
5-substituted 2-adamantyl derivatives. This requires con-
sideration of both electrostatic and hyperconjugative
effects, as, for example, discussed and debated in the
following reviews: (a) Adcock, W.; Trout, N. A. Chem.
Rev. 1999, 99, 1415; (b) Gung, B. W. Chem. Rev. 1999, 99,
1377.
14. Tang, Y.; Dong, Y.; Karle, J. M.; DiTusa, C. A.;
Vennerstrom, J. L. J. Org. Chem. 2004, 69, 6470.
15. Dong, Y.; Chollet, J.; Matile, H.; Charman, S. A.; Chiu,
F. C. K.; Charman, W. N.; Scorneaux, B.; Urwyler, H.;
Santo Tomas, J.; Scheurer, C.; Snyder, C.; Dorn, A.;
Wang, X.; Karle, J. M.; Tang, Y.; Wittlin, S.; Brun, R.;
Vennerstrom, J. L. J. Med. Chem. 2005, 48, 4953.
16. Two major isomers were formed in an approximate ratio
of 3:1; the two minor isomers could not be quantified due
to overlapping low intensity signals.
7. All new compounds provided satisfactory 1H and 13C
NMR and elemental analysis data. Full experimental
details can be found in: Vennerstrom, J. L.; Dong, Y.;
Chollet, J.; Matile, H.; Wang, X.; Sriraghavan, K.;
Charman, W. N. Spiro and Dispiro 1,2,4-trioxolane
Antimalarials, and their Preparation, Pharmaceutical
Compositions, and Use in the Treatment of Malaria,
Cancer, and Schistosomiasis. U.S. Pat. Appl. Publ. 2005,
23p, Cont.-in-part of U.S. Ser. No. 742,010. Although we
encountered no difficulties in working with these second-
ary ozonides (1,2,4-trioxolanes), routine precautions such
as the use of shields, fume hoods, and avoidance of metal
salts should be observed whenever possible.
8. Ozonide 1 was incubated for 2 h with human liver
microsomes (BD Gentest, Discovery Labware Inc.,
Woburn, MA) at a substrate concentration of 1 lM and
a microsomal protein concentration of 0.4 mg/mL as
previously described.5 Loss of parent compound and
appearance of metabolites were monitored by LC/MS.
LC/MS analysis was conducted on a Waters (Milford,
MA) Micromass Q-TOF Micro quadrupole-time-of-flight
mass spectrometer coupled to a Waters Alliance 2795
HPLC. Chromatographic separation was achieved using
an acetonitrile–water gradient (containing 0.05% formic
acid) at a flow rate of 0.4 mL/min and a Phenomenex
(Torrance, CA) Luna C8(2) column (50 · 2.1 mm, 5 lm
particle size) equipped with a precolumn of the same
packing material. Formation of metabolites was moni-
tored using MS full scans and confirmed using collision-
induced dissociation experiments.
17. cis,cis-5-Hydroxyadamantane-2-spiro-30-80-[[[(20-amino-
20-methylpropyl)-amino]carbonyl]methyl]-10,20,40-trioxa-
1
spiro[4.5]decane p-tosylate (2): mp 150–152 ꢁC; H NMR
(500 MHz, DMSO-d6) d 1.04–1.20 (m, 2H), 1.16 (s, 6H),
1.44–1.82 (m, 17H), 1.94–2.02 (m, 3H), 2.07 (d, J = 7.3 Hz,
2H), 2.29 (s, 3H), 3.19 (d, J = 5.8 Hz, 2H), 4.47 (br s, 1H),
7.11 (d, J = 8.3 Hz, 2H), 7.47 (d, J = 7.8 Hz, 2H), 7.67 (br
s, 3H), 8.05 (t, J = 6.0 Hz, 1H); 13C NMR (125.7 MHz,
DMSO-d6) d 20.94, 23.50, 28.61, 29.70, 33.35, 33.43, 37.58,
41.97, 44.50, 46.04, 54.54, 65.09, 108.65, 110.11, 125.66,
128.18, 137.66, 146.06, 172.55. Anal. Calcd for
C29H44N2O8S: C, 59.98; H, 7.64; N, 4.82. Found: C,
59.97; H, 7.40; N, 4.93.
18. Crystallographic data (excluding structural factors) for
structures 10 and 11 in this paper have been deposited with
the Cambridge Crystallographic Data Centre (CCDC) as
supplementary publication numbers CCDC 671176 and
671177. Copies of the data can be obtained, free of charge,
on application to CCDC, 12 Union Road, Cambridge,
CB2 1EZ, UK [fax: +44 (0)1223 336033 or e-mail:
deposit@ccdc.cam.ac.uk].
19. Tang, Y.; Dong, Y.; Wang, X.; Sriraghavan, K.; Wood, J.
K.; Vennerstrom, J. L. J. Org. Chem. 2005, 70, 5103.
20. Dong, Y.; Tang, Y.; Chollet, J.; Matile, H.; Wittlin, S.;
Charman, S. A.; Charman, W. N.; Santo Tomas, J.;
Scheurer, C.; Snyder, C.; Scorneaux, B.; Bajpai, S.;
Alexander, S. A.; Wang, X.; Padmanilayam, M.; Cheruku,
S. R.; Brun, R.; Vennerstrom, J. L. Bioorg. Med. Chem.
2006, 14, 6368.
9. White, R. E.; McCarthy, M.-B.; Egeberg, K. D.; Sligar, S.
G. Arch. Biochem. Biophys. 1984, 228, 493.
10. trans,cis-5-Hydroxyadamantane-2-spiro-30-80-[[[(20-amino-
20-methylpropyl)-amino]carbonyl]methyl]-10,20,40-trioxa-
1
spiro[4.5]decane p-tosylate (3): mp 200 ꢁC dec; H NMR
(500 MHz, DMSO-d6) d 1.02–1.15 (m, 2H), 1.16 (s, 6H),