Comparative antimalarial activities and ADME profiles of ozonides (1,2,4-trioxolanes) OZ277, OZ439, and their 1,2-dioxolane, 1,2,4-trioxane, and 1,2,4,5-tetraoxane isosteres

Article abstract of DOI:10.1021/jm400004u

To ascertain the structure-activity relationship of the core 1,2,4-trioxolane substructure of dispiro ozonides OZ277 and OZ439, we compared the antimalarial activities and ADME profiles of the 1,2-dioxolane, 1,2,4-trioxane, and 1,2,4,5-tetraoxane isosteres. Consistent with previous data, both dioxolanes had very weak antimalarial properties. For the OZ277 series, the trioxane isostere had the best ADME profile, but its overall antimalarial efficacy was not superior to that of the trioxolane or tetraoxane isosteres. For the OZ439 series, there was a good correlation between the antimalarial efficacy and ADME profiles in the rank order trioxolane > trioxane > tetraoxane. As we have previously observed for OZ439 versus OZ277, the OZ439 series peroxides had superior exposure and efficacy in mice compared to the corresponding OZ277 series peroxides.

Full text of DOI:10.1021/jm400004u

Article
pubs.acs.org/jmc
Comparative Antimalarial Activities and ADME Profiles of Ozonides
(1,2,4-trioxolanes) OZ277, OZ439, and Their 1,2-Dioxolane, 1,2,4-
Trioxane, and 1,2,4,5-Tetraoxane Isosteres
Xiaofang Wang,† Yuxiang Dong,† Sergio Wittlin,‡,§ Susan A. Charman,∥ Francis C. K. Chiu,∥
Jacques Chollet,‡,§ Kasiram Katneni,∥ Janne Mannila,∥ Julia Morizzi,∥ Eileen Ryan,∥ Christian Scheurer,‡,§
Jessica Steuten,∥ Josefina Santo Tomas,‡,§ Christopher Snyder,‡,§ and Jonathan L. Vennerstrom*,†
†College of Pharmacy, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, Nebraska, United States
‡Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
§University of Basel, CH-4003 Basel, Switzerland
∥Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381
Royal Parade, Parkville, Victoria 3052, Australia
ABSTRACT: To ascertain the structure−activity relationship
of the core 1,2,4-trioxolane substructure of dispiro ozonides
OZ277 and OZ439, we compared the antimalarial activities
and ADME profiles of the 1,2-dioxolane, 1,2,4-trioxane, and
1,2,4,5-tetraoxane isosteres. Consistent with previous data,
both dioxolanes had very weak antimalarial properties. For the OZ277 series, the trioxane isostere had the best ADME profile,
but its overall antimalarial efficacy was not superior to that of the trioxolane or tetraoxane isosteres. For the OZ439 series, there
was a good correlation between the antimalarial efficacy and ADME profiles in the rank order trioxolane > trioxane > tetraoxane.
As we have previously observed for OZ439 versus OZ277, the OZ439 series peroxides had superior exposure and efficacy in mice
compared to the corresponding OZ277 series peroxides.
he discovery of artemisinin from Artemisia annua in the
1,2,4-trioxane PA1103/SAR11624212 and 1,2,4,5-tetraoxane
RKA 18213 were identified as synthetic peroxide drug
development candidates.8 To ascertain the structure−activity
relationship of the core 1,2,4-trioxolane substructure in dispiro
8′-alkyl (1a) and 8′-aryl (1b) 1,2,4-trioxolanes, we now
describe the synthesis and comparative antimalarial activities
and ADME profiles of the 1,2-dioxolane (2), 1,2,4-trioxane (3),
and 1,2,4,5-tetraoxane (4) isosteres (Figure 2). This study is
the first to provide a head-to-head comparison of these four
peroxide heterocycles within a common structural framework.
1
Tearly 1970s gave rise to the semisynthetic artemisinins
and, more recently, to structurally diverse synthetic peroxide2
antimalarials. Although the precise mechanism of action of
these drugs is still not fully understood, it is hypothesized that
the pharmacophoric peroxide bond3 undergoes reductive
activation by heme released during parasite hemoglobin
digestion4 to produce carbon-centered radicals that alkylate
heme and parasite proteins.2a,5 The first synthetic peroxide drug
development candidates were fenozan B076 and arteflene,7 of
which the latter progressed to a phase II clinical trial before
further development was discontinued8 (Figure 1).
In 2012, ozonide OZ277 (1a),9 also known as arterolane
maleate,10 was approved for the Indian market as a combination
product with piperaquine phosphate (Synriam) for the
treatment of malaria. More recently, the next-generation
ozonide OZ439 (1b)11 has completed phase IIa clinical trials
(Figure 2). In addition to these ozonides (1,2,4-trioxolanes),
■
CHEMISTRY
Ozonides 1a and 1b were obtained via Griesbaum coozonol-
ysis14 as previously described.2c,9,11a The key step in the
synthesis of 1,2-dioxolanes 2a and 2b was fragmentation of
15
triethylsilylperoxy ketals 7 and 8 with SnCl4 to form the
corresponding peroxycarbenium ions that underwent annula-
tion16 with 2-methyleneadamantane17 to form dioxolane esters
9 and 10 (Scheme 1). Subsequent hydrolysis followed by either
amide or ether bond formation afforded 2a and 2b,
respectively. Dioxolanes 2a and 2b were obtained as single
diastereomers and assigned a cis configuration based on the
stereochemistry observed in similar reactions.16
Received: January 2, 2013
Figure 1. Artemisinin and first generation synthetic peroxides.
© XXXX American Chemical Society
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Figure 2. Next generation synthetic peroxides and OZ277 and OZ439 isosteres.
a
Scheme 1
a
Reagents and conditions: (a) 30% H2O2, I2, MeOH, rt, 24 h; (b) Et3SiOTf, Et3N, DMF, 0 °C to rt, 24 h; (c) 2-methyleneadamantane, 1 N SnCl4 in
CH2Cl2, −78 to −30 °C, 12 h; (d) 15% aq KOH, EtOH, 60 °C, 20 h, then AcOH; (e) (1) HOSu, EDCI, DMF, rt, 24 h, (2) 1,2-diamino-2-
methylpropane, CHCl3, rt, 4 h, (3) p-TsOH, ether; (f) powdered NaOH, (Bu)4NHSO4, N-(2-chloroethyl)morpholine HCl, CH3CN, rt to 60 °C, 12
h, then MSA, CH2Cl2/ether, rt.
1,2,4-Trioxane 3a was obtained by conversion of trioxane
acid 1118 into the corresponding HOSu active ester followed by
amide bond formation (Scheme 2). The synthesis of trioxane
3b (Scheme 2) began with conversion of ketophenol 12 into
alkylidene acetate 13 followed by epoxidation and regioselective
perhydrolysis19 to form β-hydroperoxy alcohol 14, predom-
inately as its trans isomer.18 Acid-catalyzed condensation of 14
with 2-adamantanone followed by acetate hydrolysis (Scheme
2) yielded trioxane phenol 15, which was alkylated with N-(2-
chloroethyl)morpholine to afford 3b. 1,2,4,5-Tetraoxanes 4a
and 4b (Scheme 3) were obtained by condensation of
a
a
Scheme 2
Scheme 3
a
Reagents and conditions: (a) HOSu, EDCI, 1,2-diamino-2-methyl-
propane, CH3CN/CH2Cl2, rt, 16 h, then MSA, EtOAc; (b) see (f) for
Scheme 1.
tetraoxane acid 1613 with the corresponding diamine and O-
alkylation of tetraoxane phenol 1718 with N-(2-chloroethyl)-
morpholine, respectively. Peroxides 1a−3a were isolated and
tested as their tosylate salts; the remaining peroxides were
isolated and tested as their mesylate salts.
ANTIMALARIAL ACTIVITY
■
a
Reagents and conditions: (a) HOSu, EDCI, DMF, rt, 24 h; (b) 1,2-
The activity data shown in Table 1 reveals several SAR trends.
Consistent with our previous data17 demonstrating that 1,2-
dioxolanes react with iron(II) primarily by two-electron
reduction to form inactive diol reaction products rather than
one-electron reduction to form carbon-centered radicals, 2a
and 2b were 2 orders of magnitude less potent than the
corresponding ozonides 1a and 1b. Erhardt et al.20 demon-
diamino-2-methylpropane, CHCl3, rt, 5 h, then p-TsOH, EtOAc; (c)
Ph3PCH2, 4-(4-hydroxyphenyl)cyclohexanone, rt for 1 h, then
reflux for 12 h; (d) Ac2O, pyridine, CH2Cl2, 0 °C to rt, 12 h; (e)
peracetic acid, CHCl3, NaOAc, 0 °C to rt, 12 h; (f) 50% H2O2,
MoO2(acac)2, Et2O, MgSO4, rt, 12 h; (g) 2-adamantanone, CSA,
CH2Cl2, rt, 12 h; (h) 15% aq KOH, EtOH/THF, 50 °C, 4 h, then
AcOH; (i) see (f) for Scheme 1.
B
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provided little insight to account for the divergent antimalarial
activities and ADME properties.
Table 1. Activity of Dispiro Peroxides against P. falciparum
in Vitro and P. berghei in Vivo (1 × 30 mg/kg po)
Metabolic stability studies in mouse liver microsomes
indicated that with the exception of trioxane 3a, which was
quite stable, the remaining OZ277 isosteres (1a, 2a, and 4a)
exhibited similar metabolic stabilities (Table 3). For OZ439
and its isosteres, trioxolane 1b was marginally more
metabolically stable than the remaining isosteres (2b, 3b, and
4b), and similar to that of OZ277 (1a).
a
IC50 (nM) K1/ in vivo act.
survival
(days)
b
c
compd
control
X
NF54
(%)
cures
−
O
−
0
6−7
11
−
d
1a
1.8/1.6
170/440
19/29
99.9
0/5
ND
0/3
0/3
5/5
ND
3/5
0/3
0/5
0/5
0/5
Table 3. ADME Properties of Dispiro Peroxides
d
2a
3a
4a
CH2
OCH2
OO
O
ND
99.1
99.5
>99.9
ND
99.2
99.3
92
ND
15
in vitro CLint
(μL/min/mg)/EH
in vitro blood T1/2 in vivo AUC
b
c0−24h
a
compd
(h)
(nM·h)
0.90/1.7
2.8/3.4
370/1,200
2.2/2.4
2.9/4.0
3.4/4.2
120/9.9
7.2/14
9
d
d
1b
>30
ND
27
1a
2a
3a
4a
1b
2b
3b
4b
17/0.43
12/0.35
<7/<0.2
14/0.37
14/0.38
28/0.55
38/0.63
31/0.58
0.93
1.0
2.3
0.91
10.4
f
5130
e
2b
3b
4b
CH2
OCH2
OO
−
ND
22377
13511
52726
ND
15
d
AS
9
d
CQ
−
−
99.9
99.6
10
d
MF
22
8.6
2.6
34419
18448
a
Mean from n = 2−3. Individual measurements differed by less than
50%. Individual measurements differed by less than 10%. No
detectable parasites at 30 days postinfection. Artesunate (AS),
b
c
a
b
c
Mouse liver microsomes. Mouse blood. 30 mg/kg oral dose to
d
d
mice. AUC to 8 h only as concentrations were below the limit of
quantitation at 24 h. ND = not determined. No measurable
degradation.
e
f
chloroquine (CQ), and mefloquine (MF) data from Vennerstrom et
al.9 and Charman et al.11a ND = not determined.
d
strated that β-scission reactions that form carbon-centered
radicals are accelerated by the adjacent oxygen atom present in
ozonides (1,2,4-trioxolanes), but absent in 1,2-dioxolanes.
Although the trioxane (3a) and tetraoxane (4a) isosteres of
OZ277 (1a) had good potency against Plasmodium falciparum
in vitro, neither compound was curative in the P. berghei model.
Similarly, trioxane 3b and tetraoxane 4b were no less potent
than OZ439 (1b) in vitro, but of these, only 3b was partially
curative in the Plasmodium berghei model.
For the OZ277 series, the stability in mouse blood showed
the same trend as seen with the microsomes, with trioxane 3a
being about 2-fold more stable than 1a, 2a, and 4a (Table 3).
Consistent with previous results in human and rat blood,11a
OZ439 (1b) was about 10-fold more stable in mouse blood
compared to OZ277 (1a). No degradation was detected over
the 4 h incubation period for dioxolane 2b, whereas 3b
exhibited similar stability to 1b, and 4b was about 4-fold less
stable compared to 1b.
PHYSICOCHEMICAL AND ADME PROPERTIES
■
Additional studies were conducted to explain why the ozonide
heterocycle in this particular dispiro structural framework (1a
and 1b) appears to have the best overall antimalarial profile.
The predicted physicochemical parameters (Table 2)
indicated that OZ277 and its isosteres were more basic and
In vivo, 3a had the highest area under the curve (AUC) of
the OZ277 isosteres, consistent with its better metabolic
stability and marginally better blood stability; this trioxane also
produced the longest average survival time for the OZ277
series, despite having somewhat weaker activity against P.
falciparum in vitro (Table 3). As shown in Figure 3, panel A, 3a
was also the only compound from the OZ277 series that had
plasma concentrations above about 10 ng/mL for more than 24
h. In contrast, concentrations of both 1a and 4a dropped below
1 ng/mL by the 24 h time point. Interestingly, the 2-fold
increase in the AUC of 4a vs 1a did not translate into markedly
superior in vivo antimalarial efficacy of the former. For the
OZ439 series, the rank ordering of AUC values mirrored the in
vitro blood stability data with 1b and 3b having similar AUC
values, whereas 4b had an AUC that was approximately 2- to 3-
fold lower. The rank order of exposure (i.e., 1b > 3b > 4b) was
also consistent with the in vivo efficacy results where 1b
resulted in the longest survival (>30 days), 3b resulted in an
average survival of 27 days, and animals treated with 4b
survived for an average of only 15 days. Each of the compounds
from the OZ439 series maintained plasma concentrations
above about 30 ng/mL for more than 24 h, with concentrations
of 1b and 3b being above about 200 ng/mL at the 24 h time
point (Figure 3, panel B).
Table 2. Calculated Physicochemical Properties of Dispiro
Peroxides
compd log P/log DpH 7.4
pKa
PSA (Å2) H-bond donor/acceptor
1a
2a
3a
4a
1b
2b
3b
4b
2.07/0.69
3.21/1.83
2.44/1.05
2.26/0.87
4.63/4.53
5.66/5.56
4.88/4.78
4.81/4.71
9.05
9.05
9.05
9.05
6.81
6.82
6.82
6.81
82.8
73.6
82.8
92.0
49.4
40.2
49.4
58.6
3/6
3/5
3/6
3/7
0/6
0/5
0/6
0/7
less lipophilic than OZ439 and its isosteres. While the OZ277
series would be expected to have better aqueous solubility at
physiological pH, the OZ439 series would likely have better
permeability properties due to a higher log D and lower polar
surface area (PSA). However, within the two peroxide series,
the differences in these calculated physicochemical properties
C
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Figure 3. Plasma concentration versus time profiles for (A) OZ277 isosteres and (B) OZ439 isosteres following oral administration of 30 mg/kg to
noninfected Swiss outbred mice. Open symbols represent individual concentrations for n = 2 mice per time point, and filled symbols represent the
average concentrations. Symbols: 1a and 1b (circles), 3a and 3b (squares), and 4a and 4b (triangles). For 1a, concentrations were below the limit of
quantitation of 0.5 ng/mL by the 24 h time point.
mmol) and 50% H2O2 (4.5 mL, 40 mmol) in MeOH15b (50 mL) was
added methyl 2-(4-oxocyclohexyl)acetate (5) (1.70 g, 10 mmol). The
mixture was stirred at rt for 24 h and concentrated to a residue that
was partitioned between CH2Cl2 (30 mL) and water (30 mL). The
SUMMARY AND CONCLUSIONS
In summary, this work confirms that dioxolanes such as 2a and
2b have very weak antimalarial properties, which we attribute to
■
their reaction with ferrous iron to form inactive diol reaction
products.17 In the OZ277 series, trioxane 3a had the best
ADME profile, but this was compensated by a somewhat lower
antimalarial potency, which may be due in part to subtle
differences in the iron(II) and heme reaction profiles of
trioxanes, trioxolanes, and tetraoxanes.19b,21 For the OZ439
series, there was a good correlation between antimalarial
efficacy and in vivo exposure (AUC) with the rank order being
trioxolane 1b > trioxane 3b > tetraoxane 4b. As we have noted
before,11a the superior exposure profile of 8′-aryl (OZ439) vs
8′-alkyl (OZ277) ozonides translates into superior antimalarial
efficacy (as evidenced by increased survival time) of the former.
Our data suggest that this same trend also holds true for
trioxanes (e.g., 3b versus 3a) and tetraoxanes (e.g., 4b versus
4a), which is consistent with recent data22 indicating that aryl
substituted tetraoxanes may have superior antimalarial efficacies
compared to alkyl substituted tetraoxanes such as RKA182.13
Finally, we suggest that the inferior blood stability and PK
profiles of tetraoxanes 4a and 4b versus the corresponding
trioxanes 3a and 3b results from the second peroxide bond of
the former. For example, each of the degenerate conformers of
the tetraoxanes has an equatorial peroxide bond with a more
accessible LUMO4e,19b,21c that would be expected to react more
readily with iron(II) than the corresponding trioxanes which
have only one conformer with an equatorial peroxide bond.
aqueous layer was extracted with CH2Cl2 (2 × 30 mL). The combined
extracts were washed with water and brine, dried over MgSO4, filtered,
and concentrated to afford methyl 2-(4-hydroperoxy-4-
methoxycyclohexyl)acetate as a 1:1 mixture of diastereomers (2.15 g,
99%), which was used immediately in the next step: 1H NMR
(CDCl3) δ 0.92−2.46 (m, 11H), 3.30 (s, 1.5H), 3.34 (s, 1.5H), 3.70
(s, 3H), 7.42 (s, 0.5H), 7.52 (s, 0.5H). Step 2. The unpurified methyl
2-(4-hydroperoxy-4-methoxycyclohexyl)acetate (2.15 g, 9.86 mmol) in
DMF (100 mL) was treated with Et3N (4.5 mL, 32 mmol) and
Et3SiOTf (2.54 mL, 12 mmol) at 0 °C. The reaction mixture was
stirred at rt for 24 h and then diluted with ice-cold hexane (100 mL)
and ice−water (100 mL). The organic layer was separated, and the
aqueous layer was extracted with hexane (3 × 100 mL). The extracts
were combined, dried over MgSO4, and concentrated to afford methyl
2-[4-methoxy-4-(triethylsilylperoxy)cyclohexyl]acetate (7) as a 1:1
mixture of diastereomers (3.02 g, 92%), which was used immediately
1
in the next step: H NMR (CDCl3) δ 0.68−0.80 (m, 6H), 0.94−1.08
(m, 9H), 0.84−2.44 (m, 11H), 3.26 (s, 1.5H), 3.29 (s, 1.5H), 3.67 (s,
3H). Step 3. To a −78 °C solution of 7 (3.02 g, 9.10 mmol) in
CH2Cl2 (50 mL) was added 2-methyleneadamantane (0.67 g, 4.53
mmol) followed by 1 M SnCl4 in CH2Cl2 (10 mL, 10 mmol). The
resulting mixture was stirred at −78 °C for 30 min and then kept at
−30 °C overnight. The reaction mixture was allowed to warm to −3
°C and quenched with ice−water (50 mL). After separation of the
organic layer, the aqueous layer was extracted with CH2Cl2 (2 × 50
mL). The combined extracts were washed with water (50 mL) and
brine (50 mL), dried over MgSO4, filtered, and concentrated.
Purification by silica gel (sg) chromatography (0−10% ether in
hexane) afforded cis-adamantane-2-spiro-3′-8′-[(methoxycarbonyl)-
methyl]-1′,2′-dioxaspiro[4.5]decane (9) as a colorless solid (0.60 g,
EXPERIMENTAL SECTION
■
General. Melting points are uncorrected. 1H and 13C NMR spectra
were recorded on a 500 MHz spectrometer. All chemical shifts are
reported in parts per million (ppm) and are relative to internal
(CH3)4Si (0 ppm) for 1H and CDCl3 (77.0 ppm) or DMSO-d6 (39.7
ppm) for 13C NMR. Combustion and HPLC analysis confirmed that
all target compounds possessed purities ≥95%. As indicated below,
starting materials were commercially available or were prepared
according to known procedures.
1
40%): mp 119−120 °C; H NMR (CDCl3) δ 1.24−1.36 (m, 2H),
1.44−1.96 (m, 17H), 1.95−2.02 (m, 2H), 2.06−2.14 (m, 2H), 2.13 (s,
2H), 2.20 (d, J = 7.5 Hz, 2H), 3.66 (s, 3H); 13C NMR (CDCl3) δ
26.44, 26.99, 29.07, 33.45, 34.93, 35.64, 36.24, 37.21, 41.17, 51.42,
55.47, 84.02, 88.71, 173.47. Step 4. To a solution of 9 (0.45 g, 1.35
mmol) in EtOH (20 mL) was added 15% aq KOH solution (2 mL).
The resulting mixture was stirred at 60 °C for 20 h, concentrated to
∼5 mL, then diluted with water (10 mL), and acidified with acetic acid
(5 mL). The precipitate was collected by filtration, washed with cold
water, and dried in vacuo at 40 °C to afford cis-adamantane-2-spiro-3′-
cis-Adamantane-2-spiro-3′-8′-[[[(2′-amino-2′-
methylpropyl)amino]carbonyl]methyl]-1′,2′-dioxaspiro[4.5]-
decane p-Tosylate (2a). Step 1. To a solution of I2 (0.254 g, 1.0
D
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8′-(carboxymethyl)-1′,2′-dioxaspiro[4.5]decane as a colorless solid
mixture was allowed to warm up to −3 °C and quenched with ice−
water (100 mL). After separation of the organic layer, the aqueous
layer was extracted with CH2Cl2 (2 × 100 mL). The combined extracts
were washed with water (100 mL) and brine (100 mL), dried over
MgSO4, filtered, and concentrated. Crystallization from EtOH afforded
cis-adamantane-2-spiro-3′-8′-(4′-acetoxyphenyl)-1′,2′-dioxaspiro[4.5]-
1
(0.40 g, 93%): mp 184−185 °C; H NMR (CDCl3) δ 1.27−1.40 (m,
2H), 1.42−1.96 (m, 17H), 1.95−2.04 (m, 2H), 2.06−2.14 (m, 2H),
2.13 (s, 2H), 2.24 (d, J = 7.5 Hz, 2H), 11.14 (brs, 1H); 13C NMR
(125.7 MHz, CDCl3) δ 26.44, 26.99, 29.00, 33.26, 33.45, 34.90, 35.64,
36.24, 37.20, 41.04, 55.46, 83.98, 88.75, 178.74. Step 5. A solution of
cis-adamantane-2-spiro-3′-8′-(carboxymethyl)-1′,2′-dioxaspiro[4.5]-
decane (0.22 g, 0.69 mmol), HOSu (0.09 g, 0.78 mmol), and EDCI
(0.15 g, 0.78 mmol) in DMF (10 mL) was stirred at rt for 24 h. Under
ice cooling, the reaction was quenched with water (30 mL). The
precipitate was collected by filtration, washed with cold water, and
dried in a vacuum oven at 40 °C to afford the dioxolane active ester as
a colorless solid (0.28 g, 99%): mp 188−189 °C; 1H NMR (CDCl3) δ
1.33−1.98 (m, 19H), 1.98−2.06 (m, 2H), 2.06−2.14 (m, 2H), 2.13 (s,
2H), 2.27 (d, J = 7.0 Hz, 2H), 2.96 (s, 4H). Step 6. To a solution of
1,2-diamino-2-methylpropane (0.30 g, 3.41 mmol) in CHCl3 (10 mL)
was added dropwise to the solution of the dioxolane active ester (0.28
g, 0.68 mmol) in CHCl3 (10 mL). The resulting mixture was stirred at
rt for 4 h and then quenched with water (10 mL). After separation of
the organic layer, the aqueous layer was extracted with CHCl3 (2 × 20
mL). The combined extracts were washed with water (2 × 10 mL) and
brine (10 mL), dried over MgSO4, filtered, and concentrated. The
residue was dissolved in ether (10 mL), and then the solution of p-
toluenesulfonic acid monohydrate (0.14 g) in ether (10 mL) was
added. The precipitate was collected by filtration to afford 2a as a
1
decane (10) as a colorless solid (5.60 g, 70%): mp 149−150 °C; H
NMR (CDCl3) δ 1.52−1.88 (m, 16H), 1.93−1.98 (m, 2H), 2.07−2.16
(m, 4H), 2.18 (s, 2H), 2.28 (s, 3H), 2.45−2.53 (m, 1H), 6.98 (d, J =
8.5 Hz, 2H), 7.19 (d, J = 8.5 Hz, 2H); 13C NMR (CDCl3) δ 21.14,
26.46, 27.02, 30.52, 33.47, 35.63, 35.67, 36.28, 37.22, 42.83, 55.61,
83.89, 88.79, 121.25, 127.75, 144.49, 148.70, 169.73. Step 4. To a
solution of 10 (5.60 g, 14.0 mmol) in THF (80 mL) and EtOH (160
mL) was added 15% aq KOH solution (5 mL) The resulting mixture
was stirred at 60 °C for 20 h. The solution was concentrated to ∼5
mL, and the residue was diluted with water (10 mL) and acidified with
acetic acid (5 mL). The precipitate was collected by filtration, washed
with cold water, and dried in a vacuum oven at 40 °C to afford cis-
adamantane-2-spiro-3′-8′-(4′-hydroxyphenyl)-1′,2′-dioxaspiro[4.5]-
1
decane as a colorless solid (4.50 g, 90%): mp 210−211 °C; H NMR
(500 MHz, CDCl3) δ 1.52−1.90 (m, 16H), 1.93−2.00 (m, 2H), 2.06−
2.17 (m, 4H), 2.18 (s, 2H), 2.38−2.48 (m, 1H), 4.72 (brs, 1H), 6.75
(d, J = 8.5 Hz, 2H), 7.07 (d, J = 8.5 Hz, 2H); 13C NMR (125.7 MHz,
CDCl3) δ 26.45, 27.01, 30.71, 33.48, 35.67, 35.72, 36.28, 37.22, 42.47,
55.63, 84.03, 88.83, 115.10, 127.83, 139.25, 153.71. Step 5. To a
solution of cis-adamantane-2-spiro-3′-8′-(4′-hydroxyphenyl)-1′,2′-
dioxaspiro[4.5]decane (0.50 g, 1.40 mmol) in dry acetonitrile (80
mL) were added powered NaOH (0.30 g, 7.50 mmol) and
tetrabutylammonium hydrogen sulfate (0.06 g, 0.20 mmol). The
mixture was stirred at rt for 30 min before 3-chloropropylamine
hydrochloride (0.70 g, 3.75 mmol) was added. The reaction mixture
was stirred at 60 °C overnight, cooled to rt, filtered, and washed with
CH2Cl2. After the filtrate was concentrated, the residue was dissolved
in CH2Cl2, washed with water and brine, dried over MgSO4, filtered,
and concentrated. The residue was dissolved in CH2Cl2 (5 mL), and
then a solution of methanesulfonic acid (MSA) (0.11 g) in ether (20
mL) was added. The precipitate was collected by filtration to afford 2b
as a colorless solid (0.41 g, 51%): mp 175−176 °C; 1H NMR (CDCl3)
δ 1.52−1.87 (m, 16H), 1.92−1.97 (m, 2H), 2.07−2.16 (m, 4H), 2.18
(s, 2H), 2.40−2.50 (m, 1H), 2.83 (s, 3H), 3.02−3.12 (m, 2H), 3.50−
3.57 (m, 2H), 3.62−3.70 (m, 2H), 3.97−4.04 (m, 2H), 4.06−4.16 (m,
2H), 4.44−4.51 (m, 2H), 6.82 (d, J = 8.5 Hz, 2H), 7.14 (d, J = 8.5 Hz,
2H), 11.84 (brs, 1H); 13C NMR (CDCl3) δ 26.45, 27.00, 30.63, 33.47,
35.63, 35.66, 36.28, 37.20, 39.36, 42.45, 52.88, 55.59, 56.79, 62.84,
63.86, 83.90, 88.81, 114.36, 128.02, 140.85, 155.20. Anal. Calcd for
C30H45NO7S: C, 63.92; H, 8.05; N, 2.48. Found: C, 63.69; H, 7.89; N,
2.52.
1
colorless solid (0.35 g, 90%): mp 224−225 °C; H NMR (CDCl3) δ
0.96−1.08 (m, 2H), 1.33 (s, 6H), 1.24−1.94 (m, 21H), 2.06 (s, 2H),
2.02−2.16 (m, 2H), 2.39 (s, 3H), 3.39 (d, J = 6.5 Hz, 2H), 7.25 (d, J =
9.0 Hz, 2H), 7.30 (brs, 3H), 7.66 (d, J = 9.0 Hz, 2H); 13C NMR
(CDCl3) δ 21.35, 23.98, 26.47, 27.02, 28.92, 32.08, 33.21, 33.48, 34.93,
35.65, 36.24, 37.24, 42.97, 46.23, 55.52, 56.07, 84.00, 88.49, 125.53,
129.44, 140.34, 141.54, 173.26. Anal. Calcd for C30H46N2O6S: C,
64.03; H, 8.24; N, 4.98. Found: C, 64.08; H, 7.99; N, 4.71.
cis-Adamantane-2-spiro-3′-8′-[4′-[2′-(4′-morpholinyl)-
ethoxy]phenyl]-1′,2′-dioxaspiro[4.5]decane Mesylate (2b).
Step 1. To a solution of I2 (0.254 g, 1.0 mmol) and 30% H2O2 (4.5
mL, 40 mmol) in MeOH15b (50 mL) was added 4-(4-acetoxyphenyl)-
cyclohexanone (6) (2.32 g, 10 mmol). The mixture was stirred at rt for
24 h and concentrated to a residue that was partitioned between
CH2Cl2 (30 mL) and water (30 mL). The aqueous layer was extracted
with CH2Cl2 (2 × 30 mL). The combined extracts were washed with
water and brine, dried over MgSO4, filtered, and concentrated to afford
4-(4-hydroperoxy-4-methoxycyclohexyl)phenyl acetate as a 3:2
mixture of diastereomers (2.8 g, 100%), which was used immediately
1
in the next step: H NMR (CDCl3) δ 1.52−2.00 (m, 6H), 2.17−2.34
(m, 5H), 2.48−2.62 (m, 1H), 3.34 (s, 1.2H), 3.36 (s, 1.8H), 6.97−7.04
(m, 2H), 7.20−7.26 (m, 2H), 7.58 (s, 0.4H), 7.62 (s, 0.6H); 13C NMR
(CDCl3) δ 21.12, 30.06, 30.30, 30.78, 31.30, 33.96, 41.28, 43.01, 48.31,
48.56, 104.90, 105.12, 121.31, 121.33, 127.75, 127.76, 143.65, 143.75,
148.86, 169.73. Step 2. The unpurified 4-(4-hydroperoxy-4-
methoxycyclohexyl)phenyl acetate (2.8 g, 10 mmol) in DMF (100
mL) was treated with Et3N (4.5 mL, 32 mmol) and Et3SiOTf (2.54
mL, 12 mmol) at 0 °C. The reaction mixture was stirred at rt for 24 h
and then diluted with ice-cold hexane (100 mL) and ice−water (100
mL). The organic layer was separated, and the aqueous layer was
extracted with hexane (3 × 100 mL). The extracts were combined,
dried over MgSO4, and concentrated. Purification by sg chromatog-
raphy (0−10% ether in hexane) afforded 4-[4-methoxy-4-
(triethylsilylperoxy)cyclohexyl]phenyl acetate (8) as a 2:3 mixture of
diastereomers (2.18 g, 55%): 1H NMR (CDCl3) δ 0.68−0.82 (m, 6H),
0.94−1.07 (m, 9H), 1.42−1.84 (m, 6H), 2.18−2.25 (m, 1.2H), 2.29 (s,
1.8H), 2.29 (s, 1.2H), 2.31−2.38 (m, 0.8H), 2.50−2.59 (m, 1H), 3.32
(s, 1.8H), 3.34 (s, 1.2H), 6.97−7.03 (m, 2H), 7.17−7.25 (m, 2H); 13C
NMR (CDCl3) δ 3.79, 3.80, 5.79, 6.57, 6.75, 6.79, 21.13, 30.21, 30.51,
31.32, 31.71, 43.01, 43.26, 48.15, 48.40, 104.20, 104.35, 121.26,
121.32, 127.68, 127.79, 143.99, 144.17, 148.82, 169.68. Step 3. To a
−78 °C solution of 8 (8.0 g, 20 mmol) in CH2Cl2 (200 mL) was
added 2-methyleneadamantane (3.2 g, 21.6 mmol) followed by 1 M
SnCl4 in CH2Cl2 (30 mL, 30 mmol). The resulting mixture was stirred
at −78 °C for 30 min and then kept at −30 °C overnight. The reaction
Adamantane-2-spiro-3′-9′-[[[(2′-amino-2′-methylpropyl)-
amino]carbonyl]methyl]-1′,2′,4′-trioxaspiro[5.5]undecane p-
Tosylate (3a). Step 1. A solution of adamantane-2-spiro-3′-9′-
(carboxymethyl)-1′,2′,5′-trioxaspiro[5.5]undecane (11)18 (0.20 g, 0.60
mmol), HOSu (0.08 g, 0.70 mmol), and EDCI (0.14 g, 0.73 mmol) in
DMF (10 mL) was stirred at rt for 24 h. The reaction was quenched
with water (30 mL) at 0 °C, and the precipitate was collected by
filtration, washed with cold water, and dried in vacuo at 40 °C to give
the trioxane active ester (0.24 g, 96%) as a colorless solid, which was
1
used directly in the next step: H NMR (CDCl3) δ 1.00−2.10 (m,
21H), 2.48−2.64 (m, 2H), 2.76−2.90 (m, 4H), 2.95 (brs, 1H), 3.18
(brs, 1H), 3.76 (s, 2H). Step 2. To a solution of 1,2-diamino-2-
methylpropane (0.20 g, 2.30 mmol) in CHCl3 (5 mL) was added
dropwise to the solution of the trioxane active ester (0.24 g, 0.58
mmol) in CHCl3 (15 mL). The resulting mixture was stirred at rt for 5
h and then quenched with water (20 mL). After separation of the
organic layer, the aqueous layer was extracted with CHCl3 (2 × 20
mL). The combined extracts were washed with water (2 × 20 mL) and
brine (20 mL), dried over MgSO4, filtered, and concentrated to afford
the free base of 3a (0.18 g) as a colorless solid. The free base of 3a
(0.18 g) was dissolved in EtOAc (20 mL) to which was added a
solution of p-toluenesulfonic acid monohydrate (0.09 g) in EtOAc (10
mL). The solid was collected and dried in vacuo at 40 °C to afford 3a
E
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as a colorless solid (0.24 g, 69%): mp 166−167 °C; 1H NMR (CDCl3)
δ 1.34 (s, 6H), 0.50−2.15 (m, 23H), 2.41 (s, 3H), 2.41 (brs, 1H), 2.90
(brs, 1H), 3.40 (s, 2H), 3.54 (s, 2H), 7.25 (d, J = 8.0 Hz, 2H), 7.33 (t,
J = 7.0 Hz, 1H), 7.65 (d, J = 8.0 Hz, 2H), 7.76 (brs, 3H); 13C NMR
(CDCl3): δ 21.39, 23.90, 27.12, 28.51 (br), 31.44 (br), 33.33, 33.58,
36.05 (br), 37.14, 41.97, 46.15, 56.27, 62.45, 77.58, 104.23, 125.50,
129.41, 140.34, 141.78, 172.99. Anal. Calcd for C30H46N2O7S: C,
62.26; H, 8.01; N, 4.84. Found: C, 62.49; H, 7.86; N, 4.77.
3H), 2.56−2.66 (m, 1H), 2.61 (brs, 2H), 3.87 (brs, 2H), 7.00 (d, J =
8.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H); 13C NMR (CDCl3) δ 21.13,
27.12, 28.60 (br), 29.66 (br), 30.55 (br), 33.18 (br), 33.36, 36.00 (br),
37.14, 39.23, 42.96, 62.28, 77.65, 104.40, 121.40, 127.63, 143.37,
148.87, 169.70. Step 6. To a solution of adamantane-2-spiro-3′-9′-(4′-
acetoxyphenyl)-1′,2′,4′-trioxaspiro[5.5]undecane (0.30 g, 0.73 mmol)
in THF (10 mL) and EtOH (20 mL) was added 15% aq KOH (1
mL). The resulting mixture was stirred at 50 °C for 4 h, concentrated
to ∼5 mL, diluted with water (30 mL), and acidified with acetic acid (2
mL). The precipitate was collected by filtration, washed with cold
water, and dried in vacuo at 40 °C to afford adamantane-2-spiro-3′-9′-
(4′-hydroxyphenyl)-1′,2′,4′-trioxaspiro[5.5]undecane (15) as a color-
less solid (0.24 g, 89%): mp 178−179 °C; 1H NMR (CDCl3) δ 1.30−
2.20 (m, 20H), 2.51−2.60 (m, 1H), 2.82 (brs, 1H), 2.98 (brs, 1H),
3.89 (brs, 2H), 6.77 (d, J = 8.5 Hz, 2H), 7.06 (d, J = 8.5 Hz, 2H); 13C
NMR (CDCl3) δ 27.13, 30.69 (br), 32.26 (br), 33.18, 33.36, 34.18
(br), 37.15, 42.67, 62.30, 77.75, 104.40, 115.17, 127.74, 138.19,
153.78. Step 7. To a solution of 15 (0.10 g, 0.27 mmol) in dry
acetonitrile (20 mL) were added powered NaOH (0.20 g, 5.0 mmol)
and tetrabutylammonium hydrogen sulfate (0.01 g, 0.03 mmol). The
mixture was stirred at rt for 30 min before N-(2-chloroethyl)-
morpholine hydrochloride (0.14 g, 0.75 mmol) was added. The
reaction mixture was stirred at 60 °C overnight, cooled to rt, filtered,
and washed with CH2Cl2. After the filtrate was concentrated, the
residue was dissolved in CH2Cl2, washed with water and brine, dried
over MgSO4, filtered, and concentrated. The residue (0.12 g) was
dissolved in EtOAc (10 mL) to which was added a solution of MSA
(0.02 g) in ether (20 mL). The precipitate was collected by filtration
Adamantane-2-spiro-3′-9′-[4′-[2′-(4′-morpholinyl)ethoxy]-
phenyl]-1′,2′,4′-trioxaspiro[5.5]undecane Mesylate (3b). Step
1. To a solution of n-BuLi (2.5 M in hexane, 6.3 mL, 15.8 mmol) in
dry ether (50 mL) was added methyltriphenylphosphonium bromide
(5.7 g, 16.0 mmol). The resulting mixture was stirred at rt for 1 h, and
then a solution of 4-(4-hydroxyphenyl)cyclohexanone (12) (1.5 g, 7.9
mmol) in dry ether (30 mL) was added dropwise. The mixture was
then refluxed overnight. After removal of ether, the residue was
partitioned between ether (30 mL) and water (30 mL). The aqueous
layer was extracted with ether (2 × 30 mL). The combined extracts
were washed with water and brine, dried over MgSO4, filtered, and
concentrated. Purification by sg chromatography (0−10% ether in
hexane) afforded 4-(4-methylenecyclohexyl)phenol as a colorless solid
1
(0.92 g, 49%): mp 89−90 °C; H NMR (CDCl3) δ 1.43−1.55 (m,
2H), 1.91−1.99 (m, 2H), 2.12−2.22 (m, 2H), 2.37−2.44 (m, 2H),
2.56−2.66 (m, 1H), 4.63 (s, 1H), 4.67 (m, 2H), 6.73−6.78 (m, 2H),
7.03−7.10 (m, 2H); 13C NMR (CDCl3) δ 35.15, 35.75, 43.24, 107.28,
115.10, 127.87, 139.28, 148.91, 153.61. Step 2. To a solution of 4-(4-
methylenecyclohexyl)phenol (0.92 g, 4.65 mmol) in CH2Cl2 (10 mL)
at 0 °C was added dry pyridine (5 mL) followed by acetic anhydride
(2 mL). The resulting mixture was stirred at rt overnight. After
removal of the solvents, the residue was partitioned between CH2Cl2
(30 mL) and water (30 mL). The aqueous layer was extracted with
CH2Cl2 (2 × 30 mL). The combined extracts were washed with 1 M
HCl (2 × 10 mL) and water (10 mL), dried over MgSO4, filtered, and
concentrated to afford 4-(4-methylenecyclohexyl)phenyl acetate (13)
as a colorless solid (1.06 g, 99%): 1H NMR (CDCl3) δ 1.43−1.60 (m,
2H), 1.91−1.99 (m, 2H), 2.12−2.22 (m, 2H), 2.29 (s, 3H), 2.37−2.44
(m, 2H), 2.56−2.66 (m, 1H), 4.68 (m, 2H), 6.99 (d, J = 8.5 Hz, 2H),
7.20 (d, J = 8.5 Hz, 2H). Step 3. Peracetic acid (5.7 g, 32 wt % in aq
acetic acid) was mixed with CHCl3 (25 mL), the aqueous layer was
discarded, and the organic layer was dried over MgSO4 to yield an
anhydrous peracetic acid solution, which was added dropwise to an ice
cold solution of 13 (1.80 g, 7.83 mmol) in CHCl3 (15 mL) containing
sodium acetate (0.5 g). The reaction mixture was then warmed to rt
and stirred overnight. The reaction mixture was washed with water (3
× 20 mL) and saturated NaHCO3 (3 × 20 mL), dried over MgSO4,
filtered, and concentrated to afford 4-(1-oxaspiro[2.5]octan-6-yl)-
phenyl acetate as a pale yellow oil (1.80 g, 93%): 1H NMR (CDCl3) δ
1.32−1.50 (m, 2H), 1.76−2.00 (m, 4H), 2.00−2.16 (m, 2H), 2.30 (s,
3H), 2.56−2.66 (m, 1H), 2.70 (s, 2H), 7.01 (d, J = 8.5 Hz, 2H), 7.24
(d, J = 8.5 Hz, 2H). Step 4. Anhydrous MgSO4 (10 g) was added to a
mixture of 50% H2O2 (10.0 g, 114 mmol) and ether (150 mL) at 0 °C.
After stirring for 20 min, the mixture was filtered and the filtrate was
added to a mixture of 4-(1-oxaspiro[2.5]octan-6-yl)phenyl acetate
(1.80 g, 7.32 mmol) and molybdenyl acetylacetonate (228 mg, 0.7
mmol) in ether (10 mL). After the reaction mixture was stirred
overnight at rt, it was washed with water (100 mL) and brine (100
mL), dried over MgSO4, filtered, and concentrated to afford 4-[(4-
hydroperoxy-4-(hydroxymethyl)cyclohexyl)]phenyl acetate (14) as a
1
to afford 3b as a colorless solid (0.13 g, 83%): mp 188−189 °C; H
NMR (CDCl3) δ 1.30−2.14 (m, 20H), 2.53−2.62 (m, 1H), 2.83 (s,
3H), 2.74−2.90 (m, 1H), 2.96 (brs, 1H), 3.02−3.13 (m, 2H), 3.51−
3.58 (m, 2H), 3.64−3.70 (m, 2H), 3.89 (brs, 2H), 3.98−4.05 (m, 2H),
4.06−4.16 (m, 2H), 4.47−4.53 (m, 2H), 6.84 (d, J = 8.5 Hz, 2H), 7.13
(d, J = 8.5 Hz, 2H), 11.81 (brs, 1H); 13C NMR (CDCl3) δ 27.12,
28.46 (br), 29.68 (br), 30.62 (br), 33.36, 34.77 (br), 36.01 (br), 37.13,
39.36, 42.66, 52.94, 56.86, 62.28, 62.88, 63.83, 77.65, 104.40, 114.45,
127.92, 139.70, 155.35. Anal. Calcd for C30H45NO8S·0.5H2O: C,
61.20; H, 7.88; N, 2.38. Found: C, 61.15; H, 7.95; N, 2.36.
Adamantane-2-spiro-3′-9′-[[[(2′-amino-2′-methylpropyl)-
amino]carbonyl]methyl]-1′,2′,4′,5′-tetraoxaspiro[5.5]-
undecane Mesylate (4a). To a solution of adamantane-2-spiro-3′-9′-
(carboxymethyl)-1′,2′,4′,5′-tetraoxaspiro[5.5]undecane (16)13 (339
mg, 1 mmol) in CH3CN (10 mL) and CH2Cl2 (10 mL) was added
HOSu (127 mg, 1.1 mmol) followed by EDCI (211 mg, 1.1 mmol).
After the mixture was stirred at rt for 16 h, 1,2-diamino-2-
methylpropane (264 mg, 3 mmol) was added. After stirring for 2 h,
the mixture was concentrated, diluted with water (50 mL), and
extracted with CH2Cl2 (2 × 20 mL). The combined organic layers
were washed with water (30 mL) and brine (20 mL), dried over
MgSO4, and concentrated. To a solution of the residue in EtOAc (5
mL) was added a solution of MSA (96 mg, 1 mmol) in EtOAc (5 mL).
The precipitate was collected by filtration and purified by
crystallization from CH3CN to give 4a as a colorless solid (243 mg,
48%): mp 150 °C dec; 1H NMR (CDCl3) δ 1.18−1.37 (m, 2H), 1.36
(s, 6H), 1.42−2.08 (m, 19H), 2.17 (d, J = 7.3 Hz, 2H), 3.00−3.26 (m,
2H), 11.15 (brs, 1H); 13C NMR (CDCl3) δ 23.93, 26.97, 26.99, 27.75
(br), 28.36 (br), 28.78 (br), 30.04 (br), 31.07 (br), 33.07, 33.11, 33.44,
34.24 (br), 36.87, 39.86, 42.63, 46.25, 56.14, 107.62, 110.37, 172.80.
Anal. Calcd for C23H40N2O8S: C, 54.74; H, 7.99; N, 5.55. Found: C,
54.79; H, 8.01; N, 5.42.
Adamantane-2-spiro-3′-9′-[4′-[2′-(4′-morpholinyl)ethoxy]-
phenyl]-1′,2′,4′,5′-tetraoxaspiro[5.5]undecane Mesylate (4b).
To a solution of adamantane-2-spiro-3′-9′-(4′-hydroxyphenyl)-
1′,2′,4′,5′-tetraoxaspiro[5.5]undecane (17)18 (0.5 g, 1.34 mmol) in
dry acetonitrile (50 mL) were added powered NaOH (0.30 g, 7.50
mmol) and tetrabutylammonium hydrogen sulfate (0.06 g, 0.20
mmol). The mixture was stirred at rt for 30 min before N-(2-
chloroethyl) morpholine hydrochloride (0.75 g, 4.03 mmol) was
added. The reaction mixture was stirred at 60 °C overnight, cooled to
rt, filtered, and washed with CH2Cl2. After the filtrate was
1
pale yellow oil (1.46 g, 71%): H NMR (CDCl3) δ 1.35−2.20 (m,
8H), 2.30 (s, 3H), 2.46−2.56 (m, 1H), 3.91 (s, 2H), 7.01 (d, J = 8.5
Hz, 2H), 7.21 (d, J = 8.5 Hz, 2H), 7.63 (brs, 1H). Step 5. 10-
Camphorsulfonic acid (CSA) (0.20 g, 0.88 mmol) was added to a
mixture of 14 (1.46 g, 5.20 mmol) and 2-adamantanone (1.20 g, 8.00
mmol) in CH2Cl2 (50 mL). The reaction mixture was stirred
overnight at rt, washed with saturated NaHCO3 (20 mL), water (20
mL), and brine (20 mL), dried over MgSO4, filtered, and
concentrated. The crude product was purified by crystallization from
5:1 EtOH:H2O to afford adamantane-2-spiro-3′-9′-(4′-acetoxyphen-
yl)-1′,2′,4′-trioxaspiro[5.5]undecane as a colorless solid (0.46 g, 21%):
1
mp 155−156 °C; H NMR (CDCl3) δ 1.30−2.10 (m, 20H), 2.28 (s,
F
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Journal of Medicinal Chemistry
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concentrated, the residue was dissolved in CH2Cl2, washed with water
and brine, dried over MgSO4, filtered, and concentrated. The residue
(0.60 g) was dissolved in EtOAc (10 mL) to which was added a
solution of MSA (0.11 g) in ether (20 mL). The precipitate was
collected by filtration to afford 4b as a colorless solid (0.45 g, 58%):
calibration standards prepared in blank mouse whole blood processed
in the same manner.
Mouse Exposure Studies. Animal studies were conducted using
established procedures9,11a in accordance with the Australian Code of
Practice for the Care and Use of Animals for Scientific Purposes, and
the study protocols were reviewed and approved by the Monash
Institute of Pharmaceutical Sciences Animal Ethics Committee.
Exposure following oral administration to noninfected mice was
assessed at a dose of 30 mg/kg using an aqueous formulation vehicle
containing 0.5% (w/v) hydroxypropylmethylcellulose, 0.5% (v/v)
benzyl alcohol, and 0.4% (v/v) Tween 80. Compounds were
administered by oral gavage (3 mL/kg) to 6−8 week old male Swiss
outbred mice. An abbreviated blood sampling protocol was used to
collect samples at 1, 4, 8, and 24 h postdose (two mice at each time
point) with blood sampling either by cheek-bleed or terminal cardiac
puncture with a maximum of two samples per mouse. Plasma was
immediately separated from erythrocytes and frozen at −20 °C prior
to analysis by LC−MS. Chromatography, MS, and sample processing
were as described for the metabolic stability, and concentrations were
determined by comparison to a calibration curve prepared in mouse
plasma. Limits of quantitation were between 0.5 and 1 ng/mL for all
analytes in plasma. Area under the plasma concentration−time profiles
(AUC) were approximated using WinNonLin version 5.2.1 (Pharsight
Corporation, Mountain View, CA).
1
mp 182−183 °C; H NMR (CDCl3) δ 1.56−2.10 (m, 20H), 2.53−
2.63 (m, 1H), 2.83 (s, 3H), 3.01−3.13 (m, 2H), 3.16−3.36 (m, 2H),
3.48−3.58 (m, 2H), 3.67 (d, J = 12.5 Hz, 2H), 3.95−4.16 (m, 4H),
4.45−4.53 (m, 2H), 6.84 (d, J = 8.5 Hz, 2H), 7.16 (d, J = 7.0 Hz, 2H),
11.83 (brs, 1H); 13C NMR (CDCl3) δ 27.03, 29.84 (br), 31.90 (br),
33.14, 34.28 (br), 36.92, 39.36, 42.76, 52.93, 56.85, 62.87, 63.84,
107.45, 110.52, 114.43, 128.08, 139.74, 155.39. Anal. Calcd for
C29H43NO9S: C, 59.88; H, 7.45; N, 2.41. Found: C, 59.97; H, 7.67; N,
2.33.
In Vitro and in Vivo Antimalarial Activity. In vitro and in vivo
antimalarial activities9,11a,23 were measured using the chloroquine-
resistant K1 and chloroquine-sensitive NF54 strains of P. falciparum,
and P. berghei-infected mice, respectively. In vivo data were obtained
using single 30 mg/kg oral doses (relative to the free base of each
compound) of 1−4 administered in a nonsolubilizing, standard
suspension vehicle (SSV) comprising 0.5% w/v carboxymethyl
cellulose, 0.5% v/v benzyl alcohol, 0.4% v/v Tween 80, and 0.9%
w/v sodium chloride in water, except for 3a, which was dosed in 3% v/
v ethanol and 7% v/v Tween 80 in water. Activity is defined as percent
reduction in parasitemia on day 3 postinfection compared to an
untreated control group. For example, a compound with an activity of
99.9% is 10-fold more active than one with an activity of 99.0% and
100-fold more active than one with an activity of 90%. Cures in the P.
berghei model are defined as having no detectable parasites on day 30
postinfection.
AUTHOR INFORMATION
Corresponding Author
*Phone: 402.559.5362. E-mail: jvenners@unmc.edu.
■
Notes
The authors declare no competing financial interest.
Physicochemical Properties. Physicochemical properties (log
DpH 7.4, pKa and PSA) were calculated using ACD/Laboratories
Release 9.0 software (Advanced Chemistry Laboratories, Toronto).
Metabolic Stability. Metabolic stability was assessed in vitro by
incubating compounds (1 μM) at 37 °C in mouse liver microsomes
(Xenotech, Lenexa, KS) suspended in 0.1 M phosphate buffer (pH
7.4) at a final protein concentration of 0.4 mg/mL. Metabolic
reactions were initiated by the addition of an NADPH-regenerating
system (1 mg/mL NADP, 1 mg/mL glucose-6-phosphate, 1 U/mL
glucose-6-phosphate dehydrogenase) and MgCl2 (0.67 mg/mL) and
were quenched at various time points up to 60 min by the addition of
ice-cold acetonitrile. Quenched samples were centrifuged, and the
relative loss of parent compound was monitored by LC−MS using a
Waters/Micromass Xevo TQ triple quadrupole mass spectrometer
coupled to a Waters Acquity UPLC (Waters Corporation, Milford,
MA). Chromatography was conducted using a Supelco Ascentis
Express RP Amide column (50 × 2.1 mm, 2.7 μm particle size,
Supelco, Bellefonte, PA) operated at 40 °C and compounds were
eluted with a methanol−water gradient buffered with 0.05% formic
acid and delivered at a flow rate of 0.4 mL/min. Mass spectrometry
was conducted in positive electrospray ionization mode with multiple-
reaction monitoring using MS/MS parameters optimized for each
compound. Concentration versus time data were fitted to an
exponential decay function to determine the first order rate constant
for substrate depletion, which was then used to calculate the
degradation half-life, an in vitro intrinsic clearance value (mL/min/
mg microsomal protein), and subsequently a predicted in vivo intrinsic
clearance value.24 Predicted in vivo CLint values were converted to
predicted in vivo hepatic extraction ratios (EH) using the following
equation: EH = CLint/Q + CLint where Q is liver blood flow, which was
assumed to be 90 mL/min/kg for mice.25
ACKNOWLEDGMENTS
■
This investigation received financial and scientific support from
Medicines for Malaria Venture (MMV) and the Nebraska
Research Initiative (NRI). We thank the manuscript reviewers
for their many useful comments and suggestions.
ABBREVIATIONS USED
■
CSA, 10-camphorsulfonic acid; EDCI, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride; HOSu, N-
hydroxysuccinimide; MSA, methanesulfonic acid; SSV, stand-
ard suspending vehicle
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