Chemical Stability of the Peroxide Bond Enables Diversified Synthesis of Potent Tetraoxane Antimalarials(1)

Abstract

The development of widespread drug resistance to chloroquine (CQ(a)) has resulted in severe health issues for countries in malaria endemic regions. The antimalarial properties of artemisinin 2 and of other peroxides, such as 1,2,4,5-tetraoxacy-cloalkanes (tetraoxanes), have recently begun to be exploited in the development of new approaches to fighting CQ-resistant strains of malaria. New tetraoxanes employing a steroidal backbone have now been prepared that are highly active, are inexpensive, and demonstrate low toXieity.(5,6) A part of our research in this field is focused on the development of a new type of tetraoxane with nonidentical substituents(6) that utilize a steroid and small cyclohexylidene carriers possessing secondary amide bonds. Also, during our work in this field we discovered that tetraoxanes are unusually stable, even. at pH 1.6,(6c) a characteristic that subsequently allowed the synthesis of many interesting derivatives. This communication encompasses the synthesis of various amino-functionalized antimalarials based on the appreciable stability of the tetraoxane moiety to reaction conditions such as reductive amination and LiAlH4 reduction. Their respective antimalarial activities and the pronounced antiproliferative activity of certain products are reported along with in vitro metabolism studies.

Full text

Chemical Stability of the Peroxide Bond Enables Diversified Synthesis of Potent Tetraoxane
Antimalarials
1
Igor Opsenica,
†
Dejan Opsenica,
†
Kirsten S. Smith,
¶
Wilbur K. Milhous,
¶
and Bogdan A. Šolaja*
,§
Institute of Chemistry, Technology and Metallurgy, Belgrade, Serbia, DiVision of Experimental Therapeutics, Walter Reed Army Institute of
Research, Washington, D.C. 20307-5100, and Faculty of Chemistry, UniVersity of Belgrade, Belgrade, Serbia
ReceiVed NoVember 10, 2007
Of 17 prepared 1,2,4,5-tetraoxacyclohexanes stable to reductive and acidic conditions, 3 of them were more
active than artemisinin against CQ and MFQ resistant strain TM91C235 and all compounds were more
active in vitro against W2 than against D6 strain. In vivo, amines 10 and 11a cured all mice at higher doses
with MCD e37.5 (mg/kg)/day. Triol 13 was exceptionally active against melanoma (LOX IMVI) and
ovarian cancer (IGROV1), both with LC50 )60 nM.
Introduction
The development of widespread drug resistance to chloro-
quine (CQ
a
) has resulted in severe health issues for countries
in malaria endemic regions. The antimalarial properties of
artemisinin
2
and of other peroxides, such as 1,2,4,5-tetraoxacy-
cloalkanes (tetraoxanes),
3,4
have recently begun to be exploited
in the development of new approaches to fighting CQ-resistant
strains of malaria. New tetraoxanes employing a steroidal
backbone have now been prepared that are highly active, are
inexpensive, and demonstrate low toxicity.
5,6
A part of our research in this field is focused on the
development of a new type of tetraoxane with nonidentical
substituents
6
that utilize a steroid and small cyclohexylidene
carriers possessing secondary amide bonds. Also, during our
work in this field we discovered that tetraoxanes are unusually
stable, even at pH 1.6,
6c
a characteristic that subsequently
allowed the synthesis of many interesting derivatives.
This communication encompasses the synthesis of various
amino-functionalized antimalarials based on the appreciable
stability of the tetraoxane moiety to reaction conditions such
as reductive amination and LiAlH4reduction. Their respective
antimalarial activities and the pronounced antiproliferative
activity of certain products are reported along with in vitro
metabolism studies.
Results and Discussion
The discovery of the appreciable stability of tetraoxanes to
basic (pH 12, NaOH/i-PrOH/H2O, room temp f80 °C)
5a
and
acidic (pH 1.6, CH3OH/HCl, 37 °C)
6c
conditions initiated our
research into the application of classical reagents for reductive
amination conditions (NaBH3CN, NaBH(OAc)3), reduction
(NaBH4, LiAlH4), and acetylation (cat. TMSOTf/Ac2O).
7
As noted previously,
5,6
our approach to functionalized tet-
raoxanes consists of an ester facid famide sequence. Thus,
we prepared 1,1-dihydroperoxycyclohexane (1) in 50% yield,
8,9
which was subsequently coupled to methyl 4-oxocyclohexane-
carboxylate, affording 2in 28–35% yield and the side product
hexaoxonane 4(Scheme 1).
10
Upon transformations furnishing
tetraoxane amides (2f3f5-7) in 65–79% yield, we explored
the stability of the tetraoxane moiety under reducing conditions.
We discovered that ester 2was reduced in very high yield to
alcohol 8(Scheme 1) with no appreciable cleavage of the
tetraoxane moiety observed with use of LiAlH4.
11
Stability of
this moiety to LAH was confirmed by azide-to-amine reduction
(Scheme 1) and the reduction of steroidal tetraoxane 12 to triol
13 (Scheme 2). Established stability
6c
of a tetraoxacyclohexane
at pH 1.6 enabled us to use an acidic workup procedure (see
Experimental Section). Additionally, we successfully applied a
TMSOTf/Ac2O esterification method en route to mononalcohol
15, which was further oxidized in 83% yield under aprotic
conditions. Finally, NaBH(OAc)3and NaBH4were applied for
reductive ammination and the reduction of mixed anhydride to
alcohol, respectively. Thus, we have shown that the tetraoxane
moiety is stable to reducing conditions (LiAlH4, NaBH(OAc)3,
and NaBH4) and mild acidic conditions (protic and aprotic).
Biological Screening. Antimalarial Activity. All synthesized
compounds were screened in vitro against CQ-susceptible, CQ-
resistant, and multidrug resistant strains, D6, W2, and TM91C235
(Thailand), respectively.
12
The least active compounds were
hexaoxonane 4, a type of peroxide much less active than
tetraoxanes, trioxanes, or trioxolanes,
4c
and the most polar
compounds 3and 13. The significantly lower in vitro activity
* To whom correspondence should be addressed. Phone: +381-11-263-
86-06. Fax: +381-11-263-60-61. E-mail: bsolaja@chem.bg.ac.yu.
†
Institute of Chemistry, Technology and Metallurgy.
¶
Walter Reed Army Institute of Research.
§
University of Belgrade.
a
Abbreviations: CQ, chloroquine; MFQ, mefloquine; ART, artemisinin;
CA, cholic acid; DCA, deoxycholic acid; MCD, mimimal curative dose;
MAD, minimal active dose; MG_MID, mean graph midpoint.
Scheme 1
a
a
(a) 30% H2O2/HCl, CH3CN/CH2Cl2; (b) methyl 4-oxocyclohexanecar-
boxylate, CH2Cl2,H
2SO4/CH3CN; (c) NaOH, i-PrOH/H2O/∆; (d) ClCO2Et/
Et3N, R1NH2, (e) LiAlH4,Et
2O; (f) (1) MsCl, Py, (2) NaN3, DMF; (g)
LiAlH4,Et
2O; (h) carbonyl, NaBH(OAc)3,CH
2Cl2.
J. Med. Chem. 2008, 51, 2261–2266 2261
10.1021/jm701417a CCC: $40.75 2008 American Chemical Society
Published on Web 03/11/2008

of acid 3, in comparison to corresponding methyl ester 2, was
expected on the basis of previous results.
4a,6c
However, one can
observe that the differences in activities of the tetraoxane acids
and corresponding esters diminish with a decrease of polarity
of the molecule. An example is in Table 1, 12 and 19, with
further examples in refs 6a and 6b. Steroidal triol 13 is much
less active than its diacetoxy derivative 15, and this information
clearly indicates that the protected hydroxy groups at C(7) and
C(12) of cholic acid are needed for good activity. In addition,
no effect on activity is excerted by the C(24)-O functionality;
triacetate 14, monoalcohol 15, and starting ester 12 have very
similar activities, and this trend is similar to that seen with
trioxolanes.
13
The analysis given above possibly points to the
high importance of the substitution pattern at C(7) and C(12)
of the steroidal tetraoxanes.
The in vitro antimalarial potency of dicyclohexylidene
carboxylic amides 5–7, prepared via mixed anhydrides, is higher
than that of the ester 3; however, they are less active than most
amino derivatives. Thus, when directly compared (on the same
plate), the activity of primary amide 5appears to be one-half
that of the corresponding amine 10. Of the amides, the most
active was the N,N-dimethylethan-1,2-diamino derivative 7,
projected to possess a weak base structural subunit. Resitance
of the tetraoxane moiety to LAH and applied reductive
amination conditions enabled easy approach to amines (8f9
f10 f11). For the first time we tested tetraoxane azides as
possible antimalarial candidates. Interestingly, dicyclohexylidene
azide 9and its steroidal analogue 18 (19 f15 f18) are
equipotent antimalarials with activities very similar to that of
artemisinin.
In vitro metabolism studies were performed on compounds
7,11a-c,17a,bto assess the bioavailability of possible drug
candidates after oral administration. Metabolic stability assays
were done using human and mouse liver microsomes.
6c
Stable
compounds were defined as having half-lives of >60 min, and
the relevant data are given in Table 1. The data showed that 7,
17a, and 17b were metabolically stable. However, 11a,11b,
and 11c were metabolically less stable, with half-lives of 43.9,
9.9, and 3.5 min in mouse, and the lesser in vivo activity of
11b compared to 11a might be ascribed to the shorter half-life.
Five achiral dicyclohexylidene tetraoxanes were chosen for
further evaluation in vivo against P. berghei infected mice using
a modified Thompson test.
6c
The amide 7was tested orally,
while tetraoxane azide 9and amines 10,11a, and 11b were
administered subcutaneously. In both tests the mice were
infected on day 0, and the tested compounds were administered
accordingly on days 3-5 postinfection. To our surprise,
tetraoxane 7, despite being a metabolically stable compound
(t1/2 >60 min, no metabolite produced upon incubation with
human, mouse, rat, and rhesus monkey microsomes), was
inactive in the in vivo test even at a dose of 320 (mg/kg)/day
(MTD >960 mg/kg, Table 2). However, peroxide azide 9cured
4 of 5 mice at a dose of 300 (mg/kg)/day, with a mean survival
time of 30.6 days versus 7–9 days in the control mice. Cure of
2 of 5 mice and increased survival were also seen in mice dosed
with 150 (mg/kg)/day, with a group mean survival time of >26
days. In the present set of compounds, the most active were
tetraoxane amines 10,11a, and 11b, with a minimum curative
dose (MCD) of e37.5 (mg/kg)/day. Primary amine 10 cured
all test animals at doses of 300 and 150 (mg/kg)/day and 2 of
5 at 37.5 (mg/kg)/day, with a minimum active dose (MAD) of
9.3 (mg/kg)/day. Secondary amines 11a and 11b were less active
than 10, both with minimal curative dose of 37.5 (mg/kg)/day.
Toxicity was not observed at any dose; all animals not cured in
the above tests died of malaria.
Antiproliferative Activity. The antiproliferative activity of
five compounds in Table 3 was tested against a diverse panel
of 60 human cancer cell lines at NIH-NCI, starting at 10
-4
M.
14
Compounds 2,5,7, and 17b showed low to moderate activity
as exemplified by low MG_MID values (Table 3). However,
the most polar, and one of the least active compounds in
antimalarial screen, tetraoxane 13, was found to be a very
effective antiproliferative agent against a broad spectrum of
cancer cells. The results of the activity against 19 cancer cell
lines, shown in Table 4, indicate that triol 13 totally inhibits
the cancer growth (TGI) at submicromolar levels, with an
average concentration of 0.40 µM. This pronounced antitumor
activity is further accented by very high and selective toxicity
of 13 against melanoma (LOX IMVI, LC50 )60 nM) and
ovarian cancer (IGROV1, LC50 )60 nM).
Conclusion
The stability of the tetraoxane moiety to hydride reduction
and to acidic conditions (up to pH 1.6) enabled the synthesis
of a series of mixed dicyclohexylidene tetraoxanes and a new
type of steroidal mixed tetraoxane. In contrast to the steroidal
tetraoxanes, the amines of the dicyclohexylidene series were
more active in vitro and in vivo than the corresponding
carboxylic amides. In vitro, all compounds were more active
against the CQ-resistant W2 strain than against the CQ-
susceptible D6. Compounds 7,11a,11b were more active than
ART against the CQ and MFQ-resistant strain TM91C235
(Thailand). In vivo, amines 10 and 11a cured all mice at higher
doses and exhibited MCD e37.5 (mg/kg)/day. As in our earlier
studies, no peroxide bond scission was observed in any in vitro
ADME studies. Of the tested compounds, triol 13 is exception-
Scheme 2
a
a
(a) LiAlH4,Et
2O; (b) Ac2O, TMSOTf, CH2Cl2; (c) K2CO3, MeOH;
(d) PCC, CH2Cl2; (e) RNH2, NaBH(OAc)3,CH
2Cl2; (f) (i) ClCO2Et/Et3N/
THF; (ii) NaBH4; (g) (i) MsCl/Py; (ii) NaN3/DMF.
2262 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 Opsenica et al.

ally active in an in vitro antiproliferative screen against a panel
of 60 cell lines.
Experimental Section
For general remarks see ref 6c.
1,1-Dihydroperoxycyclohexane (1). Cyclohexanone (1 mL, 10
mmol) was dissolved at room temperature in a CH2Cl2/CH3CN
mixture (20 mL, 1:3 v/v) followed by 30% H2O2(10.4 mL, 0.1
mol) and 6 drops of concentrated HCl. The reaction mixture was
stirred for2hatroom temperature and quenched with saturated
NaHCO3and CH2Cl2. The organic layer was separated, and the
water layer was additionally extracted with EtOAc (3 ×50 mL).
The combined organic layers were dried over anhydrous MgSO4
and evaporated to dryness. The obtained crude product (740 mg,
50%) was used in the following step.
Methyl 7,8,15,16-Tetraoxadispiro[5.2.5.2]hexadecane-3-car-
boxylate and Methyl 7,8,15,16,23,24-Hexaoxatrispiro[5.2.5.2.
5.2]tetracosane-3-carboxylate (2 and 4). To a cooled solution (ice
bath) of dihydroperoxide 1(0.34 g, 2.3 mmol) in CH2Cl2(20 mL)
was added ketone 3(0.36 g, 2.3 mmol). After the mixture was
stirred for 30 min at the same temperature, a cooled H2SO4/CH3CN
mixture (1.66 mL, 1:10, v/v) was added dropwise. After an
additional 50 min of stirring, the mixture was worked up in the
usual manner and was purified by SiO2column chromatography
(Lobar B, LichroPrep Si 60, eluent heptane/EtOAc )95/5)
affording 185 mg (28%) 2and 37 mg (8%) 4.2: colorless foam,
softness 75–80 °C. Anal. (C14H22O6·
1
/4H2O) C, H. 4: solid oil. Anal.
(C20H32O8·H2O) C, H.
7,8,15,16-Tetraoxadispiro[5.2.5.2]hexadecane-3-carboxylic Acid
(3). Methyl ester 2(142 mg, 0.5mmol) was hydrolyzed at 80 °C
with NaOH (29.5 mg, 0.7mmol) in an i-PrOH/H2O mixture (12
mL, 3:1 v/v). After 15 min, the mixture was cooled and diluted
with H2O (20 mL) and CH2Cl2(50 mL). The water layer was
acidified to pH 2 with diluted HCl, and the layers were separated.
The water layer was further extracted with CH2Cl2(3 ×20 mL).
Then the combined organic layers were washed with water and
brine, dried over anhydrous MgSO4, and evaporated to dryness.
Trituration with Et2O afforded 120 mg (88%) of the product. With
heating at 144–156 °C, the amorphous powder transforms into
rhombic crystals, which melt at 168 °C. Anal. (C13H20O6·
1
/3H2O)
C, H.
General Procedure for Preparation of Amides 5–7. A solution
of 4(250 mg, 0.92 mmol) in dry CH2Cl2(25 mL) with added Et3N
(130µL, 0.92mmol) and ClCO2Et (90 µL, 0.92mmol) was stirred
Table 1. In Vitro Antimalarial Activities of Tetraoxanes 2–19 against P. falciparum D6,
a
W2,
b
and TM91C235
c
Strains
IC50 (nM) IC90 (nM) met. stab. t1/2 (min)
compd D6 W2 TM91C235 D6 W2 TM91C235 human mouse
229.20
d
40.41
d
26.96 83.92 62.48 110.22
3429.27
d
413.18
d
410.58 467.17 519.94 522.55
4>499.41 >499.41 >499.41
523.96 20.16 27.24 42.75 40.36 55.62
619.27 21.98 25.34 49.17 44.54 53.89
711.24 9.72 7.24 15.10 17.87 12.79 >60 >60
815.18 6.54 12.62 22.52 11.57 23.66
99.87 6.07 11.29 20.34 11.04 22.24
10 12.84 7.79 19.75 24.14 16.00 28.98
11a 11.18 6.17 10.78 14.02 17.68 13.93 15.9 43.9
11b 9.40 7.54 9.27 14.54 22.91 16.73 38.7 9.9
11c 13.82 8.19 13.89 34.52 20.60 39.51 42.0 3.5
12 21.47
e
16.96
e
13 129.72 108.88 165.77 623.30 297.96 339.88
14 15.92 11.61 19.24 28.40 26.34 29.31
15 15.64 9.77 18.15 28.67 24.21 27.19
17a 24.12 11.10 20.77 49.29 26.85 73.88 >60 >60
17b 46.77 18.51 49.67 109.81 65.44 88.09 >60 >60
18 8.60 6.30 13.93 13.31 21.60 20.56
19 30.57
e
19.22
e
MFQ
f
7.34 4.89 22.45 19.49 9.45 50.14
CQ
f
13.17 616.94 244.76 17.58 1019.71 345.08
ART
g
9.0 6.7 13.04 12.8 11.5 17.40
a
P. falciparum African D6 clone.
b
P. falciparum Indochina W2 clone.
c
P. falciparum multidrug resistant TM91C23 strain (Thailand).
d
Taken from ref
3b for comparison.
e
Taken from ref 6a for comparison.
f
Control drugs.
g
Average of greater than eight replicates.
Table 2. In Vivo Antimalarial Activities of Tested Tetraoxanes against
P. berghei
a
mg ·kg
-1
·
day
-1
mice dead/day died
mice alive
day 31/total
survival time
(day)
b
Compound 7
c
320 1/14, 1/16, 1/17, 1/19, 1/21 0/5 17.4
80 3/7, 1/8, 1/9 0/5 7.6
20 1/6, 3/7, 1/9 0/5 7.2
Compound 9
d
300 1/29 4/5 30.6
150 1/15, 1/27, 1/28 2/5 26.4
37.5 1/10, 1/17, 1/20, 1/24, 1/25 0/5 19.2
9.3 1/8, 3/10, 1/12 0/5 10
Compound 10
d
300 5/5 >31
150 5/5 >31
37.5 1/17, 1/20, 1/24 2/5 24.6
9.3 1/8, 1/10, 1/14, 1/20, 1/31 0/5 16.6
Compound 11a
d
300 5/5 >31
150 1/21 4/5 29
37.5 2/17, 2/24 1/5 22.6
9.3 1/8, 2/11, 1/17, 1/20 0/5 13.4
Compound 11b
d
300 1/6, 1/19, 1/29 2/5 23.2
150 1/17, 1/18, 1/25, 1/27 1/5 23.6
37.5 1/8, 1/10, 1/16, 1/26 1/5 18.2
9.3 2/8, 2/9, 1/25 0/5 11.8
Infected Controls
e
0 7–9 0/5
a
Groups of five P. berghei (KBG 173 strain) infected CD-1 mice were
treated on days 3-5 postinfection with tetraoxanes suspended in 0.5%
hydroxyethylcellulose/0.1% Tween-80 (po) or sesame oil (sc). Mice alive
on day 31 with no parasites in a blood film are considered cured.
b
Including
cured mice.
c
Compound administered orally.
d
Compounds administered
subcutaneously.
e
All noninfected age controls survived (5/5).
Synthesis of Tetraoxane Antimalarials Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2263

for 90 min at 0 °C. Amine was added, and after 30 min of stirring
the mixture was warmed to room temperature. After 90 min it was
diluted with H2O, the layers were separated, and the organic layer
was washed with brine, dried over anhydrous MgSO4, and
evaporated to dryness.
7,8,15,16-Tetraoxadispiro[5.2.5.2]hexadecane-3-carboxam-
ide (5). By use of the above procedure, 3was reacted with 10 equiv
of NH4Cl and 10 equiv of Et3N in dry CH2Cl2(20 mL) to afford
the primary amide 5(200 mg, 80%), which was then triturated with
Et2O. 5: mp 168–172 °C. Anal. (C13H21NO5·H2O) C, H.
N-Propyl-7,8,15,16-tetraoxadispiro[5.2.5.2]hexadecane-3-car-
boxamide (6). Acid 3(250 mg, 0.92 mmol) was transformed into
amide 6(220 mg, 77%) using 10 equiv of n-PrNH2in dry CH2Cl2
(45 mL). Column chromatography: Lobar B, LichroPrep RP-18,
eluent MeOH/H2O)8/2. Colorless foam, softness at 200–203 °C.
Anal. (C16H27NO5)C,H.
N-[2-(Dimethylamino)ethyl]-7,8,15,16-tetraoxadispiro[5.2.5.2]-
hexadecane-3-carboxamide (7). Acid 4(250 mg, 0.92 mmol) was
transformed into amide 7(280 mg, 90%) using 10 equiv of
Me2NCH2CH2NH2in dry CH2Cl2(45 mL). Column chromatogra-
phy: Lobar B, LichroPrep RP-18, eluent MeOH. Colorless foam,
softness at 179–182 °C. Anal. (C17H30N2O5·H2O) C, H, N.
7,8,15,16-Tetraoxadispiro[5.2.5.2]hexadec-3-ylmethanol (8). A
solution of methyl ester 2(1 g, 3.5 mmol) in dry ether (5 mL) was
added in portions to a suspension of LiAlH4(177 mg, 4.7 mmol)
in dry ether (5 mL) at room temperature. After 50 min it was diluted
with H2O and EtOAc. The water layer was acidified to pH 2 with
diluted HCl, the layers were separated, and the water layer was
further extracted with EtOAc (3 ×50 mL). The combined organic
layers were dried over anhydrous Na2SO4and evaporated to dryness.
The crude product was purified using dry flash chromatography,
with an eluent of heptane/EtOAc (8/2). Yield 813 mg (90%).
Colorless foam, softness 116–118 °C. Anal. (C13H22O5)C,H.
3-(Azidomethyl)-7,8,15,16-tetraoxadispiro[5.2.5.2]hexadecane
(9). To a solution of alcohol 8(650 mg, 2.5 mmol) in pyridine (5
mL) at room temperature was added methanesulfonyl chloride (250
µL, 3.0 mmol). The mixture was stirred at room temperature for
2 h, then diluted with H2O and EtOAc. The water layer was acidified
with diluted HCl, and the layers were separated. The water layer
was further extracted with EtOAc (3 ×50 mL). The combined
organic layers were dried over anhydrous Na2SO4and evaporated
to dryness. The obtained crude product was used in the following
step. To a solution of mesylate (1.4 g, 4.2 mmol) in DMF (15 mL)
was added NaN3(2.7g, 42 mmol). The mixture was stirred at 50
°C for 16 h before being quenched with water and EtOAc, and the
layers were separated. The water layer was further extracted with
EtOAc (3 ×75 mL). The combined organic layers were washed
with brine, dried over anhydrous Na2SO4, and evaporated to dryness.
The crude product was purified using dry flash chromatography
with a heptane/EtOAc eluent (8/2). Yield 1.57 g (>99%). Colorless
foam, softness 86–87 °C. Anal. (C13H21N3O4)C,H,N.
1-(7,8,15,16-Tetraoxadispiro[5.2.5.2]hexadec-3-yl)metha-
namine (10). A solution of azide 9(900 mg, 3.18 mmol) in dry
ether (5 mL) was added in one portion to a suspension of LiAlH4
(165 mg, 4.35 mmol) in dry ether (5 mL) at room temperature.
After 50 min it was diluted with H2O and NaOH (10%). The
solution was filtered, and the residue was washed with a small
portion of ether. The filtrate was extracted with ether (2 ×50 mL),
and the combined organic layers were dried over anhydrous Na2SO4
and evaporated to dryness. The crude product was purified using
dry flash chromatography with an EtOAc/MeOH/NH3eluent (8/
1/1). Yield 475 mg (60%). Colorless foam, softness 75–77 °C. Anal.
(C13H23NO4·
1
/2H2O) C, H, N.
7,8,15,16-Tetraoxadispiro[5.2.5.2]hexadecane-3-metha-
namine, N-cyclohexyl- (11a). To a mixture of amine 10 (145 mg,
0.56 mmol) and cyclohexanone (59 µL, 0.56 mmol) in CH2Cl2(10
mL) was added sodium triacetoxyborohydride (286 mg, 1.35 mmol).
After the mixture was stirred at room temperature for 18 h, it was
poured into water and extracted with CH2Cl2(2 ×50 mL). The
combined organic layers were dried over anhydrous Na2SO4and
evaporated to dryness. The crude product was purified by dry flash
chromatography with an eluent of EtOAc/MeOH/NH3aq )27/0.5/
0.5. Yield 97 mg (48%). Colorless foam, softness 104–106 °C. Anal.
(C19H33NO4·
1
/3H2O) C, H, N.
N-(7,8,15,16-Tetraoxadispiro[5.2.5.2]hexadec-3-ylmethyl)pro-
pan-2-amine (11b). Amine 10 (220 mg, 0.85 mmol) was trans-
formed into amine 11b (172 mg, 67%) using acetone (69 µL, 0.94
mmol) and NaBH(OAc)3(473 mg, 2.23 mmol). The crude product
was purified using dry flash chromatography with an eluent of
EtOAc/MeOH (8/2). Colorless foam, softness 87–89 °C. Anal.
(C16H29NO4)C,H,N.
N-(Phenylmethyl)-7,8,15,16-tetraoxadispiro[5.2.5.2]hexadecane-
3-methanamine (11c). Amine 10 (100 mg, 0.39 mmol) was
transformed into amine 11c (45 mg, 50%) using PhCHO (40 µL,
0.39 mmol) and NaBH(OAc)3(200 mg, 0.94 mmol). The crude
product was purified using dry flash chromatography with an eluent
of EtOAc/MeOH (8/2) and repeated dry flash chromatography using
EtOAc. Colorless foam, softness 61–62 °C. Anal. (C20H29NO4·
1
/
2H2O) C, H, N.
5β-Cholan-7r,12r,24-triol-3-spiro-6′-(1′,2′,4′,5′-tetraoxacyclo-
hexane)-3′-spirocyclohexane (13). A solution of methyl ester 12
(100 mg, 0.16 mmol) in dry ether (5 mL) was added in one portion
to a suspension of LiAlH4(21 mg, 0.57 mmol) in dry ether (5 mL)
at room temperature. After 50 min the reaction was quenched with
H2O and EtOAc. The water layer was acidified to pH 2 with diluted
HCl, and layers were separated. The water layer was further
extracted with EtOAc (3 ×50 mL), and the combined organic layers
were dried over anhydrous Na2SO4and evaporated to dryness.
Crude triol 13 was purified using dry flash chromatography using
a heptane/EtOAc eluent (2/8). Yield 81 mg (98%). Colorless foam,
softness119–121°C.[R]
20
D+24.0(c0.2,CHCl3).Anal.(C30H50O7·
1
/
2H2O) C, H.
7r,12r,24-Triacetoxy-5β-cholan-3-spiro-6′-(1′,2′,4′,5′-tetraox-
acyclohexane)-3′-spirocyclohexane (14). Alcohol 13 (1.67 g, 3.19
mmol) was dissolved in a previously prepared solution of Ac2O
(1.7 mL) and TMSOTf (35 µL, 0.19 mmol) in dry CH2Cl2(30 mL)
at room temperature. After stirring for 15 min, the reaction was
quenched with saturated NaHCO3and the layers were separated.
The water layer was further extracted with CH2Cl2(3 ×15 mL),
and the combined organic layers were washed with brine, dried
over anhydrous Na2SO4, and evaporated to dryness. Crude triacetate
14 was purified using dry flash chromatography using a heptane/
Table 3. MG_MID (TGI) Values for Compounds 2,5,7,13, and 17b
2 5 7 13 17b
-4.34 -4.13 -4.00 -5.92 -4.64
Table 4. In Vitro Antiproliferative Activities of Tetraoxane 13 (µM,
after 48 h, Selected Data)
cell line GI50
a
TGI
b
LC50
c
leukemia CCRF-CEM 0.10 0.24 0.56
HL-60(TB) 0.15 0.31 0.62
MOLT-4 0.18 0.41 0.96
SR 0.16 0.34 0.72
non-small-cell lung cancer EKVX 0.14 0.31 0.65
HOP-92 0.14 0.32 0.73
NCI-H460 0.24 0.62 2.68
CNS cancer SF-295 0.17 0.33 0.66
SF-539 0.17 0.32 0.59
U251 0.21 0.46 0.98
melanoma LOX IMVI 0.02 0.03 0.06
SK-MEL-2 0.20 0.39 0.73
ovarian cancer IGROV1 <0.01 0.01 0.06
renal cancer 786-0 0.23 0.54 2.01
UO-31 0.04 0.17 0.61
prostate cancer PC-3 0.29 0.99 3.41
DU-145 0.11 0.23 0.48
breast cancer NCI/ADR-RES 0.25 0.59 4.27
T-47D 0.33 0.93 5.30
a
50% growth inhibitory activity.
b
Total growth inhibition.
c
Concentration
of the compound at which 50% of the cells are killed.
2264 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 Opsenica et al.

EtOAc eluent (7/3). Yield 1.92 g (93%). Colorless foam, softness
136–137 °C. [R]
20
D+55.5 (c0.2, CHCl3). Anal. (C36H56O10)C,H.
7r,12r-Diacetoxy-5β-cholan-24-ol-3-spiro-6′-(1′,2′,4′,5′-tet-
raoxacyclohexane)-3′-spirocyclohexane (15). Hydrolysis of Tri-
acetate 14. Triacetate 14 (1.67 g, 2.57 mmol) was dissolved in
dry methanol (50 mL), followed by addition of anhydrous K2CO3
(640 mg, 4.63 mmol). The suspension was stirred at room
temperature for 5 h. The mixture was evaporated to dryness,
dissolved in CH2Cl2and H2O, and the layers were separated. The
organic layer was washed with brine, dried over anhydrous Na2SO4,
and evaporated to dryness. The crude monoalcohol 15 was purified
using dry flash chromatography using a heptane/EtOAc eluent (4/
6). Yield 1.50 g (96%). Colorless foam, softness 207–210 °C. [R]
20
D
+57.0 (c0.2, CHCl3). Anal. (C34H54O9)C,H.
Via Mixed Anhydride. Acid 19 (50 mg, 0.08 mmol) was
dissolved in dry THF (5 mL) and treated with Et3N (23 µL, 0.16
mmol) and ClCO2Et (15.34 µL, 0.16 mmol). After3hofstirring
at 0 °C, NaBH4(30.5 mg, 0.8 mmol) was added. After an additional
24 h of stirring at room temperature, the mixture was diluted with
H2O and CH2Cl2, and the layers were separated. The water layer
was further extracted with CH2Cl2(2 ×50 mL) and the combined
organic layers were dried over anh. Na2SO4and evaporated to
dryness. The crude alcohol 15 was purified using dry flash
chromatography using a heptane/EtOAc eluent (1/1). Yield 37 mg
(76%).
7r,12r-Diacetoxy-5β-cholan-24-al-3-spiro-6′-(1′,2′,4′,5′-tet-
raoxacyclohexane)-3′-spirocyclohexane (16). Alcohol 15 (100 mg,
0.16 mmol) was dissolved in dichloromethane (20 mL) followed
by the addition of pyridinium chlorochromate (53 mg, 0.25 mmol).
After 2 h the mixture was transferred to a silica gel column and
eluted with CH2Cl2to afford 83 mg (83%) of 16 as a colorless
solid.
N-(n-Propyl)-7r,12r-diacetoxy-5β-cholan-24-amine-3-spiro-
6′-(1′,2′,4′,5′-tetraoxacyclohexane)-3′-spirocyclohexane (17a). To
a mixture of crude aldehyde 16 (83 mg, 0.14 mmol) and n-PrNH2
(23 µL, 0.28 mmol) in dichloromethane (20 mL), sodium triac-
etoxyborohydride (58 mg, 0.28 mmol) was added. The mixture was
stirred at room temperature for 18 h. The mixture was then poured
onto water and extracted with CH2Cl2(2 ×50 mL). The combined
organic layers were dried over anhydrous Na2SO4and evaporated
to dryness. The crude amine 17a was purified by dry flash
chromatography using an eluent of EtOAc/MeOH/NH3aq )8/1/1.
Yield 64 mg (72%). Colorless foam, softness 76–78 °C. [R]
20
D
+43.0 (c0.2, CHCl3). Anal. (C37H61NO8)C,H,N.
N-(2-Dimethylamino)ethyl)-7r,12r-diacetoxy-5β-cholan-24-
amine-3-spiro-6′-(1′,2′,4′,5′-tetraoxacyclohexane)-3′-spirocyclo-
hexane (17b). Aldehyde 16 (200 mg, 0.33 mmol) was transformed
into amine 17b (168 mg, 75%) using Me2NCH2CH2NH2(72.5 µL,
0.66 mmol) and NaBH(OAc)3(140 mg, 0.66 mmol). The crude
product was purified using dry flash chromatography with an
EtOAc/MeOH/NH3aq (8/1/1) eluent. Solid. [R]
20
D+45.0 (c0.2,
CHCl3). Anal. (C38H64N2O8·5H2O) C, H, N.
7r,12r-Diacetoxy-5β-cholan-24-azido-3-spiro-6′-(1′,2′,4′,5′-
tetraoxacyclohexane)-3′-spirocyclohexane (18). To a solution of
alcohol 15 (200 mg, 0.33 mmol) in pyridine (4 mL) at room
temperature was added methanesulfonyl chloride (31 µL, 0.4 mmol).
The mixture was stirred at room temperature for 2 h, then diluted
with H2O and EtOAc. The water layer was acidified with diluted
HCl, and layers were separated. The water layer was further
extracted with EtOAc (3 ×50 mL), and the combined organic layers
were dried over anhydrous Na2SO4and evaporated to dryness. The
obtained crude product was used in the following step. To a solution
of mesylate (226 mg, 0.33 mmol) in DMF (5 mL) was added NaN3
(214 mg, 3.3 mmol). The mixture was stirred at 50 °C for 16 h
before being quenched with water and EtOAc, and layers were
separated. The water layer was further extracted with EtOAc (3 ×
75 mL), and the combined organic layers were washed with brine,
dried over anhydrous Na2SO4, and evaporated to dryness. The crude
product was purified using dry flash chromatography using a
heptane/EtOAc (7/3) eluent. Yield 198,5 mg (95%). Solid oil. [R]
20
D
+44.5 (c0.2, CHCl3). Anal. (C34H53N3O8·2H2O) C, H, N.
In Vitro Antimalarial Activity. The in vitro antimalarial drug
susceptibility screen is a modification of the procedures first
published by Desjardins et al.,
15
with modifications developed by
Milhous et al.,
16
and the details are given in ref 5a.
In Vivo Antimalarial Activity. The P. berghei mouse efficacy
tests were conducted using a modified version of the Thompson
test. Groups of five mice were inoculated intraperitoneally with
erythrocytes infected with a drugsensitive strain of P. berghei on
day 0. Drugs were suspended in 0.5% hydroxyethylcellulose/0.1%
Tween-80 (for po administration) or in sesame oil (for sc
administration). Drugs were administered orally once a day
beginning on day 3 postinfection. Dosings are given in Table 2.
Cure was defined as survival until day 31 posttreatment. Untreated
control mice die on day 7–9 postinfection.
In Vitro Metabolism Studies. The metabolic stability assay
sample preparation was performed in a 96-well plate on a TECAN
Genesis robotic sample processor. All incubations were carried out
in 0.1 M sodium phosphate buffer (pH 7.4) in the presence of an
NADPH-regenerating system (NADP
+
sodium salt, MgCl2·6H2O,
and glucose 6-phosphate). Test drug (10 µM), microsomes (1 mg/
mL total protein), buffer, and NADPH-regenerating system were
warmed to 37 °C, and the reaction was initiated by the addition of
glucose 6-phosphate dehydrogenase (G6PD). Samples were quenched
using an equal volume of cold methanol. Samples were centrifuged
to pellet the proteins, and the supernatant was analyzed by LC-MS/
MS using fast LC gradient or isocratic methods. Percentages of
parent drug remaining at each time point were calculated using the
ratio of the peak area at each time point to the area of the time
zero point. To calculate the half-life, a first-order rate of decay was
assumed. A plot of the natural log (ln) of the drug concentration
versus time was generated, where the slope of that line was -k.
The half-life was calculated as 0.693/k.
Acknowledgment. This work was supported by the Ministry
of Science of Serbia (Grant No. 142022) and the Serbian
Academy of Sciences and Arts. Research was conducted in
compliance with the Animal Welfare Act and other federal
statutes and regulations relating to animals and experiments
involving animals and adheres to principles stated in the Guide
for the Care and Use of Laboratory Animals, NRC Publication,
1996 edition. Material has been reviewed by the Walter Reed
Army Institute of Research. There is no objection to its
presentation or publication. The opinions or assertions contained
herein are the private views of the author and are not to be
construed as official or as reflecting the true views of the
Department of the Army or the Department of Defense.
Supporting Information Available: Analytical data of synthe-
sized/isolated compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
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2266 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 Opsenica et al.