Structure-activity relationship of an ozonide carboxylic acid (oz78) against fasciola hepatica

Article abstract of DOI:10.1021/jm100226t

In this paper, we describe the SAR of ozonide carboxylic acid OZ78 (1) as the first part of our search for a trematocidal synthetic peroxide drug development candidate. We found that relatively small structural changes to 1 resulted most commonly in loss of activity against Fasciola hepatica in vivo. A spiroadamantane substructure and acidic functional group (or ester prodrug) were required for activity. Of 26 new compounds administered at single 100 mg/kg oral doses to F. hepatica infected rats, 8 had statistically significant worm burden reductions, 7 were partially curative, and 1 (acylsulfonamide 6) was completely curative and comparable to 1 in flukicidal efficacy. This study also showed that the activity of 1 is peroxide-bond-dependent, suggesting that its flukicidal efficacy depends upon hemoglobin digestion in F. hepatica.

Full text of DOI:10.1021/jm100226t

J. Med. Chem. 2010, 53, 4223–4233 4223
DOI: 10.1021/jm100226t
Structure-Activity Relationship of an Ozonide Carboxylic Acid (OZ78) against Fasciola hepatica
Qingjie Zhao,† Mireille Vargas,‡,§ Yuxiang Dong,† Lin Zhou,† Xiaofang Wang,† Kamaraj Sriraghavan,† Jennifer Keiser,‡,§ and
Jonathan L. Vennerstrom*,†
†College of Pharmacy, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, Nebraska, ‡Swiss Tropical and Public
Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland, and §University of Basel, Petersplatz 1, CH-4003 Basel, Switzerland
Received February 20, 2010
In this paper, we describe the SAR of ozonide carboxylic acid OZ78 (1) as the first part of our search for a
trematocidal synthetic peroxide drug development candidate. We found that relatively small structural
changes to 1 resulted most commonly in loss of activity against Fasciola hepatica in vivo. A spiro-
adamantane substructure and acidic functional group (or ester prodrug) were required for activity. Of
26 new compounds administered at single 100 mg/kg oral doses to F. hepatica infected rats, 8 had
statistically significant worm burden reductions, 7 were partially curative, and 1 (acylsulfonamide 6) was
completely curative and comparable to 1 in flukicidal efficacy. This study also showed that the activity
of 1 is peroxide-bond-dependent, suggesting that its flukicidal efficacy depends upon hemoglobin
digestion in F. hepatica.
Some 250 million people are infected with parasitic trema-
tode worms. Of these, the most widespread are blood flukes of
the genus Schistosoma.1 However, the liver flukes Fasciola
hepatica and F. gigantica are also important pathogenic
trematodes infecting an estimated 2.4-17 million people.2
Fascioliasis is of considerable public health and great veter-
inary significance and occurs worldwide, with the highest
number of infected people in the Andean countries, Cuba,
Western Europe, Egypt, and Iran.2,3 Triclabendazole, which
has been routinely used since the early 1980s in veterinary
medicine, is currently the sole drug used to treat human
fascioliasis and is registered in only four countries.4 Evidence
of drug resistance to triclabendazole in veterinary medicine5
provides animpetus for the discovery anddevelopment ofnew
drugs against fascioliasis. In this respect, data from a recent
clinical trial in Vietnam6 demonstrated that artesunate, a
semisynthetic artemisinin derivative, had good efficacy in
the treatment of human fascioliasis, although it was less
effective than triclabendazole.
they, as well as other peroxidic compounds, possess both
antiplasmodial and trematocidal7-9 activities, since both
plasmodia and several trematodes including Fasciola spp.
degrade hemoglobin to generate free heme, a possible target10
for bioactive peroxides. That the artemisinins and ozonide
OZ78 (1) are so much less effective against the nonhemoglobin-
degrading intestinal fluke Echinostoma caproni than against
hemoglobin-degrading flukes F. hepatica, Clonorchis sinensis,
and various schistosome species9,11-13 supports this hypo-
thesis.14 Thus, the artemisinins and synthetic peroxides offer
excellent starting points15,16 for the discovery of broad-spectrum,
orally active trematocidal agents that would minimize drug
resistance and lead to superior treatment and control options.
In this paper, we describe the SAR of ozonide 1 as the first part
of our search for a flukicidal synthetic peroxide drug develop-
ment candidate.
Chemistry
Following the method of Tsandi et al.,17 acylsulfonamide 6
(Scheme 1) was prepared by reaction of 1 with methanesulfo-
namide in the presence of DMAP and DCC.a Hydrazide 10
was readily prepared by reaction of the corresponding methyl
ester 2 with hydrazine. Ozonides 7 and 8, the glycine and
taurine conjugates of 1, were synthesized by reaction of HOBt
active ester 2818 with glycine ethyl ester (subsequent ester
hydrolysis) and taurine, respectively.
Ozonide dicarboxylic acid 14 was synthesized in a five-step
sequence (Scheme 2) starting from 29,19 the Knoevenagel
condensation product of 1,4-cyclohexanedione monoethy-
lene ketal and isopropylidene malonate (Meldrum’s acid).20
Although the semisynthetic artemisinins are best known for
their powerful antimalarial properties, it is not surprising that
a Abbreviations: AS, artesunate; DIPEA, diisopropylethylamine; DMAP,
dimethylaminopyridine; DCC, dicyclohexylcarbodiimide; DMF, dimethyl-
formamide; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehy-
drochloride; HOBt, 1-hydroxybenzotriazole; p-TSA, p-toluenesulfonic
acid; sg, silica gel.
*To whom correspondence should be addressed. Phone: 402-559-
5362. Fax: 402-559-9543. E-mail: jvenners@unmc.edu.
r
2010 American Chemical Society
Published on Web 04/27/2010
pubs.acs.org/jmc

4224 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Zhao et al.
Scheme 1a
Scheme 3a
a Reagents and conditions: (a) MeSO2NH2, DMAP, DCC, CH2Cl2,
room temp, 24 h, then 1 M HCl; (b) hydrazine hydrate, MeOH/THF,
60 °C, 24 h; (c) glycine ethyl ester HCl, DIPEA, CH2Cl2, room temp,
24 h; (d) aqueous NaOH, EtOH, then 1 M HCl to pH 5; (e) taurine,
THF/aqueous NaOH.
Scheme 2a
a
˚
Reagents and conditions: (a) 4 A molecular sieves, pyridine, room
temp, 2 days; (b) H2, 10% Pd-C, CH2Cl2, room temp, 24 h; (c) MeOH/
Et2O, H2SO4, reflux, 12 h; (d) O3, CH2Cl2/cyclohexane, 0 °C, solid
NaHCO3, 2 h; (e) 1 N KOH/THF, room temp, 12 h, then 1 M HCl.
a Reagents and conditions: (a) O3, CH2Cl2/cyclohexane, 0 °C;
(b) Me3SiOK, THF, 50 °C, 4 h; (c) MSA, CH3CN, room temp, 24 h;
(d) BrCH2COOEt, aqueous K2CO3/THF, room temp, 6 h; (e) aqueous
Reduction (99%), deketalization (27%), and esterification
(37%) afforded diester ketone 30 which underwent Gries-
baum coozonolysis21 with oxime ether 3122 to afford diester
ozonide 32 in 75% yield. The latter was hydrolyzed to afford
14 in high yield.
NaOH/EtOH, room temp, 2 h; (f) MeONH2 HCl, EtOH, pyridine,
3
room temp, 2-6 h; (g) 2-adamantanone, O3, CH2Cl2/cyclohexane, 0 °C;
(h) 1 N NaOH, aqueous EtOH, 25-60 °C, 3 h, then 1 M HCl, 0 °C.
The synthesis of ozonide 16, the monoethylphosphonic acid
isostere of 1, began with the synthesis of ketophosphonate
diester 33 (26% yield) (Scheme 3) from 4-(bromomethyl)-
cyclohexanone in an Arbuzov reaction following the method
of Yamagishi et al.23 4-(Bromomethyl)cyclohexanone, in turn,
was obtained by HCl deprotection of the corresponding
ethylene ketal24 in 74% yield. Ozonide diethyl phosphonate
34, obtained in good yield by Griesbaum coozonolysis21
between oxime ether 3122 and 33, was treated with potassium
trimethylsilanolate25 to afford 16. Ozonide piperidine carboxy-
late 17 was obtained by hydrolysis of its corresponding ester
36 in 84% yield; the latter was obtained by deprotection of
BOC ozonide 3526 with methanesulfonic acid followed by
alkylation with ethyl bromoacetate in 90% overall yield.
Ozonide ester 39, the precursor of ozonide carboxylic acid
18, the trans isomer of 1, was obtained in 21% yield by
Griesbaum coozonolysis21 of oxime ether 38 and 2-adaman-
tanone. With this combination of reaction partners, cis (2) and
trans (39) ester ozonides were produced in a ratio of 1:2.5 and
39 was purified by flash column chromatography. In contrast,
2 is the major reaction product in a Griesbaum coozonolysis
of oxime ether 31 and keto ester 37.27 Ozonide carboxylic
acids 19 and 20, regioisomers of 1, were both obtained in a
straightforward two-step Griesbaum coozonolysis/ester hy-
drolysis sequence starting from oxime ether 31 and the
corresponding keto esters 40 and 42. Unexpectedly, both 41
and 43 were formed with high diastereoselectivity and were
isolated as single isomers. Assuming the peroxide bond is
axial and the alkyl ester substituent is equatorial,18,27-29 we
Scheme 4a
a Reagents and conditions: (a) O3, CH2Cl2/cyclohexane, 0 °C; (b) 1 N
NaOH, aqueous EtOH, 25-60 °C, 3 h, then 1 M HCl, 0 °C.
assigned structures for 19 and 20 as indicated in Scheme 3,
although X-ray crystallographic analysis would be required
to substantiate this.
Ozonide acids 21 (2.3:1 mixture of isomers) and 22 (single
isomer) (Scheme 4) were obtained by hydrolysis of ozonide
esters 45 (4:1 mixture of isomers) and 47 (9:1 mixture of
isomers), which in turn were obtained by Greisbaum
coozonolysis21 of keto ester 3718 and oxime ethers 4430 and
46,28 respectively. On thebasis of the axial peroxide bonds and
equatorial cyclohexyl substituents in ozonide products from
similar coozonolysis reactions,18,27-29 we assigned the struc-
tures for 47 and 22 as indicated in Scheme 4.
Fluorinated ozonide acids 23-25 (Scheme 5) were obtained
inparallelfive-tosix-stepsequencesforwhich thekeyreaction

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4225
Scheme 5a
a Reagents and conditions: (a) bis(2-methoxyethyl)aminosulfur trifluoride, CH2Cl2, 0 °C, 1 h; (b) concentrated HCl, aqueous acetone, room temp, 1 h;
(c) MeONH2 HCl, EtOH, pyridine, room temp, 2-6 h; (d) O3, CH2Cl2/cyclohexane, 0 °C; (e) 2-adamantanone, O3, CH2Cl2/cyclohexane, 0 °C;
3
(f) NaBH4, MeOH, 0-10 °C to room temp, 24 h; (g) bis(2-methoxyethyl)aminosulfur trifluoride, CH2Cl2, room temp, 24 h.
Scheme 6a
Table 1. Worm Burden Reductions in Adult F. hepatica Harbored in
Rats after the Administration of Ozonides 1-11 at Single Oral Doses of
100 mg/kg
compd
R
worm burden reduction (%)
cures
control
0/12
2/5
10/10
2/3
2/4
0/3
0/4
4/4
2/4
0/4
0/3
0/3
0/4
ASa,b
1c
2
30
100e
99e
36
34
0
100e
86e
0
OH
OMe
OEt
NHOH
3d
4
5
NHCdNH(NH2)
NHSO2CH3
NHCH2COOH
NH(CH2)2SO3Na
NH2
a Reagents and conditions: (a) 50% H2O2, I2, MeOH, room temp,
24 h; (b) Et3SiOTf, Et3N, DMF, 0 °C to room temp, 24 h; (c) 1 N SnCl4,
CH2Cl2,-78 to -30 °C, 12 h; (d) 15% aqueous KOH, 60 °C,20h;(e) p-TSA,
CH2Cl2, room temp, 4 h.
6
7
8
9
7
10
11
NHNH2
N(CH2CH2)2NH
12
0
was a Griesbaum coozonolysis21 between fluorinated oxime
ethers 51, 57, and 61 and keto ester 3718 to form the corre-
sponding ozonide esters 52, 58, and 62 in low to moderate
yields; hydrolysis of the latter yielded 23-25 (62-99%).
Ozonide esters 58 and 62 were obtained as single diastereo-
mers and were assigned as cis based on the previously
observed27 diastereoselectivityof the Griesbaum coozonolysis
reaction. Ozonide ester 52 was obtained as a 3:1 ratio of
diastereomers; as depicted in Scheme 5, the major isomer was
assigned as trans, cis based on the diastereoselectivity of
similar coozonolysis reactions with other 5-substituted-2-
adamantanones.31 The fluorinated oxime ethers 51, 57, and
61 were obtained in high overall yields by successive treatment
of 5-hydroxy-2-adamantanone ethylene ketal (48),32 6-hydroxy-
2-adamantanone ethylene ketal (54), and 2,6-adamantanedione
monoethylene ketal (53)33 with bis(2-methoxyethyl)aminosulfur
trifluoride followed by deprotection and condensation with
methoxylamine.
a AS = artesunate. b Data from Keiser et al.13 c Data from Keiser
et al.12 d Tested at 50 mg/kg. e p < 0.05 from the Kruskal-Wallis test
comparing the median values of the responses between the treatment
and control groups.
1,2-Dioxolane ester 65 was obtained as a single diastereomer
and was assigned a cis configuration based on the stereochemis-
try observed in similar reactions.36 Ketal 27, the nonperoxidic
isostere of 1, was obtained in 79% yield as a 1.3:1 mixture of
isomers by p-TSA catalyzed condensation14 of 2-hydroxy-
methyl-2-adamantanol (66)37 and 2-(4-oxocyclohexyl)acetic
acid (67). Ozonides 1-5, 9, 11-13, and 15 were obtained as
previously described.18,27,38
Activity against F. hepatica
Efficacy data for the target compoundsadministered at oral
doses of 100 mg/kg to F. hepatica infected rats39 are shown in
Tables 1-3. At 8 weeks postinfection, rats were treated with
single 50-100 mg/kg oral doses of target compounds pre-
pared as suspensions in 7% (v/v) Tween-80 and 3% (v/v)
EtOH. At day 6 after treatment, rats were sacrificed and adult
flukes were recovered from the bile ducts and livers. Target
compound efficacies were evaluated by comparing the mean
Triethylsilylperoxyketalester63, the key intermediate inthe
synthesis of 1,2-dioxolane 26, was obtained in a two-step
sequence34 from keto ester 37 (Scheme 6). Formation of the
peroxycarbenium intermediate35 by treatment of 63 with SnCl4
in the presence of 2-methyleneadamantane afforded 1,2-dioxo-
lane ester 65 (40%) that was hydrolyzed to form 26 (93%).

4226 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Zhao et al.
Table 2. Worm Burden Reductions in Adult F. hepatica Harbored in Rats after the Administration of Ozonides 12-20 at Single Oral Doses of 100 mg/kg
compd
R
worm burden reduction (%)
cures
12
13
14
15
16
17
18
19
20
COOH
CH2CH2COOH
CH(COOH)2
52a
14
0
1/4
0/5
0/4
0/3
0/3
0/3
2/3
0/3
2/3
CH2NHCOCOOH
CH2PO(OEt)OK
14
16
0
95a
46
90a
a p < 0.05 from the Kruskal-Wallis test comparing the median values of the responses between the treatment and control groups.
Table 3. Worm Burden Reductions in Adult F. hepatica Harbored in
Rats after the Administration of Ozonides 21-27 at Single Oral Doses of
100 mg/kg
total worm burdens of treated and untreated control rats. Statis-
tical significance was calculated using the Kruskal-Wallis test.
Table 1 shows efficacy data for a range of acidic, neutral,
and weak base amide derivatives of 1. Of these compounds,
the only one with efficacy equal to 1 was acylsulfonamide 6,
although methyl ester 2 and glycine conjugate 7 achieved
statistically significant worm burden reductions and partial
cures; a 50 mg/kg dose of ethyl ester 3 also produced partial
cures. The presence of an acidic functional group was no
guarantee of good activity for these ozonides. For example,
hydroxamic acid 4, amphoteric acylguanidine 5, and taurine
conjugate 8 either were completely inactive or produced
marginal decreases in total worm burdens and cured no
infected animals. Unlike 2 and 3, the alkyl ester prodrugs of 1,
the primary amide (9), hydrazide (10), and piperazinamide (11)
derivatives were inactive or only weakly active and cured no
infected rats.
Table 2 shows efficacy data for compounds that probe the
effect of changing the position (12, 13, 19, 20) and stereo-
chemistry (18) of the carboxylic acid functional group of 1.
The position and stereochemistry of the carboxylic acid func-
tional group in 1 seem to be optimal, since removing (12) or
extending (13) the connecting alkyl link reduced efficacy, as
did changing the stereochemistry from cis (1) to trans (18) and
changing the position of attachment of the carboxymethyl
substituent on the cyclohexyl substructure (19, 20). Even
though the preceding five compounds were less effective than
1, ozonides 12, 18, and 20 were partially curative and achieved
statistically significant worm burden reductions. In addition,
the effects of an additional carboxylic acid functional group
(14), replacement of the carboxylic acid with a monoethylpho-
sphonic acid (16) or carboxyoxamide (15), and substitution of
the spirocyclohexyl substructure of 1 with a spiropiperidinyl
in 17 were examined. Not one of compounds 14-17 signifi-
cantly reduced worm burden reductions nor were they cura-
tive; the lack of efficacy of 17 is consistent with SAR trends of
antimalarial ozonides.26
compd
worm burden reduction (%)
cures
21
22
23
24
25
26
27
0
0/3
0/3
2/3
1/3
0/3
0/3
0/3
51
80a
76b
80a
7
0
a p < 0.05 from the Kruskal-Wallis test comparing the median values
of the responses between the treatment and control groups. b p = 0.051.
inactivating CYP450 metabolism of the distal bridgehead
carbon atoms of 1 based on the known inactive CYP450
metabolites31 of arterolane (OZ277).40 Although 23-25 had
moderate efficacies against F. hepatica, none was more active
than 1.
The complete loss of efficacy for 27 (Table 3), the nonper-
oxidic isostere of 1, shows that the activity of 1 is peroxide-
bond-dependent. The lack of efficacy for the peroxidic 1,2-
dioxolane 26, consistent with our previous data35 showing
that 1,2-dioxolanes have very low to no antimalarial activity,
provides additional mechanistic insight. 1,2-Dioxolanes react
with Fe(II) primarily by two-electron vs one-electron reduc-
tion to form inactive diol reaction products rather than
carbon-centered radicals,35 the latter of which are formed
by β-scission reactions of the initially formed Fe(III) com-
plexed oxy radicals. These β-scission reactions are accele-
rated by the adjacent oxygen atom41 present in ozonides
(1,2,4-trioxolanes) but absent in 1,2-dioxolanes.
Table 3 shows efficacy data for compounds that probe the
effect of replacing the spiroadamantyl substructure of 1 with a
spirocyclohexyl (21) or bicyclo[3.3.1]nonane (22) or of fluorine
substitution (23-25) of the spiroadamantyl substructure of 1.
The nonexistent to low efficacies for 21 and 22 is an outcome
consistent with SAR trends for antimalarial ozonides.28 Target
ozonides 23-25 were designed to slow or block potentially
Of 26 new compounds tested (Tables 1-3), 8 had statisti-
cally significant worm burden reductions, 7 were partially
curative, and 1 (6) was completely curative. Compounds that

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4227
were more effective in reducing worm burdens in the F.
hepatica infected rats also tended to result in partial cures,
although ozonide ester 3 (36% worm burden reduction at
50 mg/kg, 2/4 cures) and artesunate (30% worm burden
reduction, 2/5 cures) were exceptions. Given that acylsulfo-
namide 6 was the onlycompoundwithefficacy equal tothatof
1 at the 100 mg/kg dose, it was tested at the lower dose of
50 mg/kg and compared to existing data12 for 1. At 50 mg/kg,
1 and 6 reduced worm burdens by 53% and 17%, respectively,
but only 1 cured (2/4) the infected rats. Interestingly, previous
SAR18,26,29,42 reveals thatacidicozonides have relatively weak
antimalarial activities so that it may be possible to identify an
ozonidewithselectivityfor inhibition of F. hepatica. However,
1and 6are clearly less effective than triclabendazole; at 10 mg/kg,
triclabendazole reduced burden reduction by 95% and cured
3/4 infected rats.16
resulting mixture was stirred at room temperature for 2 h before
quenching with water (30 mL). After separation of the water
layer, the organic layer was washed with water (4ꢀ30 mL), dried
over MgSO4, filtered, and concentrated in vacuo. The residue
was crystallized from 1:5 EtOH/H2O to afford cis-adamantane-
2-spiro-30-80-[[[(ethoxycarbonylmethyl)amino]carbonyl]methyl]-
10,20,40-trioxaspiro[4.5]decane (1.50 g, 81%) as a white solid. Mp
157-159 °C; 1H NMR (CDCl3) δ 1.23-1.30 (m, 5H), 1.68-1.99
(m, 21H), 2.13 (d, J=6.8 Hz, 2H), 4.03 (d, J=4.9 Hz, 2H), 4.22
(q, J=7.3 Hz, 2H), 5.97 (brs, 1H); 13C NMR (CDCl3) δ 14.10,
26.42, 26.79, 29.94, 33.47, 33.92, 34.73, 36.31, 36.74, 41.29,
43.10, 61.53, 108.50, 111.30, 169.95, 172.11.
Step 2. To a solution of cis-adamantane-2-spiro-30-80-
[[[(ethoxycarbonylmethyl)amino]carbonyl]methyl]-10,20,40-triox-
aspiro[4.5]decane (1.01 g, 2.5 mmol) in EtOH (80 mL) was added
a solution of NaOH (198 mg, 5.0 mmol) in water (15 mL). The
mixture was stirred for 16 h at room temperature, evaporated
to give an oil, and acidified with 1 M aqueous HCl to pH 5. After
the aqueous phase was extracted with ethyl acetate (4ꢀ50 mL),
the EtOAc extracts were combined, dried over MgSO4, filtered,
and evaporated to give an oil that was crystallized from CHCl3
to afford 7 (752 mg, 80%) as a white solid. Mp 146-148 °C; 1H
NMR (DMSO-d6) δ 1.04-1.12 (m, 2H), 1.62-1.89 (m, 21H),
2.02 (d, J=6.8 Hz, 2H), 3.71 (d, J=5.9 Hz, 2H), 8.15 (t, J=5.9
Hz, 1H), 12.44 (brs, 1H); 13C NMR (DMSO-d6) δ 26.00, 26.41,
29.65, 33.07, 33.64, 34.45, 35.94, 36.28, 40.72, 41.82, 108.62,
110.66, 171.60, 171.79. Anal. (C20H29NO6) C, H, N.
Summary
These data indicate that relatively small structural changes
to 1 led, in most cases, to substantial, if not complete, loss of
activity against F. hepatica in vivo. A peroxide bond, spiroa-
damantane substructure, and acidic functional group (or
prodrug) were required for activity. Although 1 seems to be
an “optimized” ozonide structure for efficacy against F. hepatica,
its peroxide-dependent activity suggests that, like antimala-
rial peroxides,14 its efficacy depends upon hemoglobin digestion
in F. hepatica. The mechanistic basis for the superior efficacy
of ozonide acids is unclear, but it may be derived from a
favorable distribution of the un-ionized forms to the low pH
mileau43 of the trematode gut, the site of hemoglobin diges-
tion. Indeed, in an electron microscopy study44 of ex vivo
F. hepatica, 1 caused extensive damage to the gut. Investigation
of the flukicidal properties of other peroxy heterocycles is in
progress.
cis-Adamantane-2-spiro-30-80-[[[(20-sulfoethyl)amino]carbonyl]-
methyl]-10,20,40-trioxaspiro[4.5]decane Sodium Salt (8). To a
solution of 2818 (1.50 g, 3.4 mmol) and taurine (387 mg, 3.1 mmol)
in THF (200 mL) was added a solution of NaOH (247 mg,
6.2 mmol) in water (15 mL). After the mixture was stirred for
24 h at room temperature, an additional portion of 28 (44 mg,
0.1 mmol) was added. After the mixture was further stirred for
24 h at room temperature, the solvents were removed in vacuo to
produce an oil that was purified by reverse phase chromatography
(C18, 1:1 MeOH/H2O) to afford 8 (1.23 g, 80%) as a white solid.
1
Mp 154-156 °C; H NMR (DMSO-d6) δ 1.02-1.10 (m, 2H),
1.61-1.93 (m, 23H), 2.53 (t, J=7.9 Hz, 2H), 3.28 (td, J=7.9,
6.9 Hz, 2H), 7.73 (s, 1H); 13C NMR (DMSO-d6) δ 26.02, 26.42,
29.71, 33.02, 33.66, 34.46, 35.64, 35.95, 36.30, 42.38, 50.88, 108.63,
110.63, 170.91. Anal. (C20H30NNaO7S) C, H, N.
Experimental Section
General. Melting points are uncorrected. H and 13C NMR
1
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),
CD3OD (49.0 ppm), or DMSO-d6 (39.7 ppm) for 13C NMR.
Combustion analysis confirmed that all target compounds
possessed purities of g95%.
cis-Adamantane-2-spiro-30-80-(20-oxo-20-hydrazinoethyl)-10,20,
40-trioxaspiro[4.5]decane (10). To a stirred solution of 2 (0.68 g,
2 mmol) in MeOH (10 mL) and THF (5 mL) was added
hydrazine monohydrate (3.0 g, 60 mmol). The resulting mixture
was heated at 50-60 °C for 24 h, then cooled to room temperature
and concentrated. The residue was dissolved in EtOAc (100 mL),
washedwithwater (50mL) andbrine(50mL), dried over MgSO4,
and filtered. After removal of the solvent, the crude product was
purified by crystallization from 5:1 CH2Cl2/EtOH to afford 10
cis-Adamantane-2-spiro-30-80-[[[(methylsulfonyl)amino]carbonyl]-
methyl]-10,20,40-trioxaspiro[4.5]decane (6). Ozonide 6 was synthe-
sized following the method of Tsandi et al.17 To a solution of 1
(1.61 g, 5.0 mmol), methanesulfonamide (951 mg, 10.0 mmol), and
DMAP (611 mg, 5.0 mmol) in CH2Cl2 (40 mL) was added DCC
(1.14 g, 5.5 mmol). After 24 h, the reaction mixture was filtered and
1 M aqueous HCl was added to the filtrate. After the aqueous layer
was extracted with CH2Cl2 (2ꢀ 30 mL), the combined CH2Cl2
extracts were dried over MgSO4, filtered, and evaporated in vacuo
to produce a residue that was purified by chromatography (sg, 10:1
CH2Cl2/MeOH) to afford 6 (1.05 g, 72%) as a white solid. Mp
131-133 °C; 1H NMR (CDCl3) δ 1.26-1.30 (m, 3H), 1.68-1.96
(m, 20H), 2.22 (d, J = 6.4 Hz, 2H), 3.32 (s, 3H), 8.26 (s, 1H);
13C NMR (CDCl3) δ 26.40, 26.79, 29.78, 32.82, 33.77, 34.74,
36.32, 36.72, 41.63, 42.88, 108.16, 111.50, 170.96. Anal. (C19H29-
NO6S) C, H, N.
1
(0.56 g, 83%) as a colorless solid. Mp 124-126 °C; H NMR
(CDCl3) δ 1.15-1.35 (m, 2H), 1.61-2.02 (m, 21H), 2.03 (d, J=
6.8 Hz, 2H), 3.55-4.09 (m, 2H), 6.76 (s, 1H); 13C NMR (CDCl3)
δ 26.46, 26.85, 29.99, 33.36, 33.91, 34.77, 36.37, 36.77, 41.23,
108.43, 111.40, 172.77. Anal. (C18H28N2O4) C, H, N.
cis-Adamantane-2-spiro-30-80-dicarboxymethyl-10,20,40-triox-
aspiro[4.5]decane (14). Step 1. A mixture of 1,4-cyclohexane-
dione monoethylene ketal (31.2 g, 200 mmol), isopropylidene
˚
malonate (32.4 g, 220 mmol), molecular sieves, 4 A (9 g), and
pyridine (300 mL) was stirred at room temperature for 2 days
under Ar. The reaction suspension was filtered, and the filtrate
was concentrated in vacuo. The residue was dissolved in CH2Cl2
(500 mL) and washed with 1 M HCl (2ꢀ150 mL) and water (3ꢀ
150 mL). The CH2Cl2 layer was dried over MgSO4 and evapo-
rated in vacuo to give 5-(1,4-dioxaspiro[4.5]dec-8-ylidene)-2,2-
dimethyl-1,3-dioxane-4,6-dione (29)19 (39.1 g, 69%) as a red
cis-Adamantane-2-spiro-30-80-[[[(carboxymethyl)amino]carbonyl]-
methyl]-10,20,40-trioxaspiro[4.5]decane (7). Step 1. To a solution
of cis-adamantane-2-spiro-30-80-[[(10H-benzotriazol-10-yloxy)-
carbonyl]methyl]-10,20,40-trioxaspiro[4.5]decane (28)18 (2.00 g,
4.6 mmol) and glycine ethyl ester hydrochloride (762 mg, 5.4 mmol)
in CH2Cl2 (60 mL) was added DIPEA (1.29 g, 10 mmol). The
1
powder. H NMR (CDCl3) δ 1.75 (s, 6H), 1.91 (t, J=6.6 Hz,
4H), 3.15 (t, J=6.6 Hz, 4H), 4.00 (s, 4H); 13C NMR (CDCl3) δ
26.9, 30.5, 35.4, 64.6, 103.8, 106.9, 115.2, 160.8, 178.6.

4228 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Zhao et al.
Step 2. A solution of crude 29 (39.1 g, 138.5 mmol) and 10%
Pd-C (1.0 g) in CH2Cl2 (450 mL) was hydrogenated at 500 psi at
room temperature for 1 day. After filtration, the filtrate was
concentrated in vacuo to give crude 5-(1,4-dioxaspiro[4.5]dec-8-
yl)-2,2-dimethyl-1,3-dioxane-4,6-dione (38.8 g, 99%) which was
used for the next step. An analytical sample was obtained by
flash chromatography (sg, 50% ether in hexane) as a yellow
42.5 mmol) in EtOH (70 mL) and 6 M aqueous HCl (15 mL,
90 mmol) was stirred overnight at room temperature. After
addition of saturated aqueous NaHCO3 to bring the pH to 8,
most of the solvent was removed in vacuo and water (100 mL)
and ether (100 mL) were added. After separation of the ether
layer, the aqueous phase was extracted with ether (3ꢀ100 mL). The
combined organic phases were dried and evaporated to give
an oil that was purified by sg chromatography (sg, 10% EtOAc
in hexane) to afford 4-(bromomethyl)cyclohexanone (6.0 g,
74%) as a colorless oil. 1H NMR (CDCl3) δ 1.51-1.59 (m, 2H),
2.11-2.18 (m, 1H), 2.20-2.24 (m, 3H), 2.34-2.45 (m, 4H), 3.39
(d, J=6.3 Hz, 2H); 13C NMR (CDCl3) δ 31.00, 37.83, 38.29, 40.02,
210.74.
1
powder. Mp 145-148 °C dec; H NMR (CDCl3) δ 1.55-1.65
(m, 4H), 1.75 (s, 3H), 1.77 (s, 3H), 1.80 (s, 1H), 1.82 (s, 1H),
1.97-2.05 (m, 2H), 2.41-2.46 (m, 1H), 3.45 (d, J=2.9 Hz, 1H),
3.95 (s, 4H); 13C NMR (CDCl3) δ 26.4, 27.4, 28.2, 34.7, 37.6,
50.2, 64.2, 64.3, 104.8, 107.8, 164.8.
Step 3. A solution of 5-(1,4-dioxaspiro[4.5]dec-8-yl)-2,2-
dimethyl 1,3-dioxane-4,6-dione (38.8 g, 136.6 mmol) and pyr-
idinium p-toluenesulfonate (5.0 g, 20 mmol) in acetone (400 mL)
was refluxed for 20 h. The reaction solution was concentrated
and then dissolved in CH2Cl2 (300 mL), washed with water (3ꢀ
100 mL), dried over MgSO4, and evaporated to dryness. Be-
cause of the poor yield of the deprotection and the difficulty of
separation, the above process was repeated four times to
produce crude 5-(4-oxocyclohexyl)-2,2-dimethyl-1,3-dioxane-
4,6-dione (9.0 g, 27%) which was used for the next step. An
analytical sample was obtained by flash chromatography (sg,
60% ether in hexane) as a white powder. Mp 152-154 °C dec.;
1H NMR (CDCl3) δ 1.77 (s, 3H), 1.81 (s, 3H), 1.94-1.99 (m,
2H), 2.18-2.27 (m, 2H), 2.37-2.48 (m, 4H), 2.88-2.95 (m, 1H),
3.61 (d, J=2.9 Hz, 1H); 13C NMR (CDCl3) δ 27.0, 28.1, 28.2,
36.2, 40.7, 49.8, 105.0, 164.3, and 209.9.
Step 2. According to the method of Yamagishi et al.,23
a
mixture of 4-(bromomethyl)cyclohexanone (3.0 g, 15.7 mmol)
and triethyl phosphite (10.4 g, 62.8 mmol) was heated to 170 °C
for 8 h. After the mixture was cooled, saturated aqueous
NaHCO3 was added to raise the pH to 8. The aqueous phase
was extracted with CH2Cl2 (3ꢀ100 mL). The combined organic
extracts were dried with MgSO4, filtered, and evaporated to give
an oil that was purified with chromatography (sg, 50% EtOAc
and hexane to 100% EtOAc) to afford diethyl [(4-oxocyclohexyl)-
methyl]phosphonate (33) (1.0 g, 26%). 1H NMR (CDCl3) δ
1.33-1.36 (t, J=7.3 Hz, 6H), 1.52-1.56 (m, 2H), 1.75-1.80 (dd,
J=18.6, 7.4 Hz, 1H), 2.23-2.26 (m, 3H), 2.37-2.40 (m, 4H),
4.08-4.16 (m, 4H); 13C NMR (CDCl3) δ 16.46 (d, J=5.8 Hz),
31.21 (d, J=3.8 Hz), 31.54 (d, J=140.6 Hz), 33.79 (d, J=11.0
Hz), 40.54, 61.51 (d, J=6.7 Hz).
Step 3. A solution of 3122 (11.06 g, 61.7 mmol) and 33 (7.0 g,
41.1 mmol) in cyclohexane (200 mL) and CH2Cl2 (40 mL)
was treated with ozone following the method of Dong et al.28
After removal of solvents in vacuo, the crude product was
purified by chromatography (sg, 50% EtOAc in hexane) to
afford cis-adamantane-2-spiro-30-80-[(diethoxyphosphinyl)-
methyl]-10,20,40-trioxaspiro[4.5]decane (34) (10.83 g, 78%) as
an oil. 1H NMR (CDCl3) δ 1.29-1.34 (m, 8H), 1.65-1.99
(m, 23H), 4.04-4.13 (m, 4H); 13C NMR (CDCl3) δ 16.44 (d,
J=5.0 Hz), 26.47, 26.86, 31.15, 31.36 (d, J=11.4 Hz), 32.10 (d,
J=133.2 Hz), 33.96, 34.77, 36.37, 36.79, 61.37 (d, J=6.4 Hz),
108.27, 111.35.
Step 4. A solution of 5-(4-oxocyclohexyl)-2,2-dimethyl-1,3-
dioxane-4,6-dione (9.0 g, 37.5 mmol), MeOH (300 mL), Et2O
(300 mL), and concentrated H2SO4 (2.0 mL, 37.6 mmol) was
refluxed overnight. The reaction mixture was cooled to room
temperature and concentrated in vacuo. The residue was dis-
solved in CH2Cl2 (150 mL) and washed with water (3ꢀ100 mL).
The CH2Cl2 layer was dried over MgSO4, filtered, and evapo-
rated to dryness. The residue was purified by flash chromatography
(sg, 50% ether in hexane) to give 4-[bis(methoxycarbonyl)methyl]-
cyclohexanone (30) (3.2 g, 37%) as a white solid. Mp 59-60 °C;
1H NMR (CDCl3) δ 1.53-1.61 (m, 2H), 2.06-2.09 (m, 2H),
2.39-2.42 (m, 4H), 2.54-2.60 (m, 1H), 3.33 (d, J=8.8 Hz,1H),
3.76 (s, 6H); 13C NMR (CDCl3) δ 29.9, 35.8, 40.1, 52.4, 56.1,
168.4, 210.2.
Step 5. A suspension of 30 (3.2 g, 14 mmol), O-methyl
2-adamantanone oxime (31)22 (5.0 g, 28 mmol), NaHCO3 (no
product was formed without the addition of solid NaHCO3) (1.7 g,
20 mmol) in CH2Cl2 (80 mL), and cyclohexane (240 mL) was
treated with ozone at 0 °C for 2 h. The reaction solution was
concentrated and the residue was purified by flash chromatog-
raphy (sg, 6% ether in hexane) to give cis-adamantane-2-spiro-
30-80-[bis(methoxycarbonyl)methyl]-10,20,40-trioxaspiro[4.5]decane
(32) (4.2 g, 75%) as colorless crystals. Mp 94-95 °C (ethanol);
1H NMR (CDCl3) δ 1.29-1.37 (m, 2H), 1.68-1.99 (m, 20H),
2.09-2.16 (m, 1H), 3.19 (d, J=8.8 Hz, 1H), 3.73 (s, 6H); 13C
NMR (CDCl3) δ 26.4, 26.8, 27.7, 33.7, 34.7, 36.2, 36.3, 36.7,
52.4, 56.9, 108.0, 111.4, and 168.9.
Step 4. According to the method of Dziemidowicz et al.,25
a
mixture of 34 (333 mg, 0.8 mmol) and potassium trimethylsila-
nolate (412 mg, 3.2 mmol) in anhydrous THF (12 mL) was
stirred at 50 °C for 4 h. Removal of the solvent gave a residue
that was purified by reverse phase chromatography (C18, 1:1
CH3OH/H2O) to afford 16 (264 mg, 85%) as a white solid.
Mp 160-162 °C; 1H NMR (CDCl3) δ 1.21-1.23 (m, 5H), 1.44-
1.48 (m, 2H), 1.61-1.99 (m, 21H), 3.81 (m, 2H); 13C NMR
(CDCl3) δ 16.66 (d, J=6.7 Hz), 26.45, 26.84, 31.49 (d, J=9.6
Hz), 32.10 (d, J=2.2 Hz), 33.00 (d, J=130.0 Hz), 34.10, 34.73,
36.35, 36.77, 60.28 (d, J=4.6 Hz), 108.60, 111.11. Anal. (C19H30-
KO6P) C, H.
Adamantane-2-spiro-30-80-carboxymethyl-10,20,40-trioxa-80-
azaspiro[4.5]decane Sodium Salt (17). Step 1. To a suspension of
adamantane-2-spiro-30-80-tert-butoxycarbonyl-10,20,40-trioxa-80-
azaspiro[4.5]decane (35)26 (1.462 g, 4.0 mmol) in THF (10 mL)
was added dropwise a solution of methanesulfonic acid (1.54 g,
16.0 mmol) in CH3CN (2 mL) at room temperature, and the
mixture was stirred at room temperature for 24 h. A solution of
K2CO3 (2.211 g, 16.0 mmol) in H2O (6 mL) and a solution of
ethyl bromoacetate (802 mg, 4.8 mmol) in THF (12 mL) were
added. After the mixture was stirred for 6 h, the solvents were
removed and the residue was diluted with EtOAc (40 mL) and
H2O (30 mL). After phase separation, the aqueous phase was
extracted with EtOAc (3ꢀ30 mL). The combined organic layers
were dried over MgSO4, filtered, and concentrated in vacuo. The
crude product was purified by sg chromatography (sg, 20%
EtOAc in hexane) to afford adamantane-2-spiro-30-80-ethoxy-
carbonylmethyl-10,20,40-trioxa-80-azaspiro[4.5]decane (36) (1.26 g,
Step 6. To a solution of 32 (1.0 g, 2.5 mmol) in THF (50 mL)
was added a solution of KOH (1.12 g, 20 mmol) in H2O
(3.0 mL). After being stirred at room temperature for 12 h, the
reaction solution was concentrated. The residue was dissolved in
EtOAc (100 mL) and H2O (50 mL) and acidified with 1 M HCl
to pH 2. The EtOAc layer was separated, washed with H2O (3ꢀ
50 mL), dried over MgSO4, and evaporated in vacuo to give 14
(0.81 g, 87%) as a white powder. Mp 152-153 °C dec; 1H NMR
(DMSO-d6) δ 1.16-1.25 (m, 2H), 1.65-1.93 (m, 21H), 3.02
(d, J=8.8 Hz, 1H), 12.7 (s, 2H); 13C NMR (DMSO-d6) δ 26.0,
26.5, 27.5, 33.6, 34.5, 35.3, 36.0, 36.3, 57.1, 108.4, 110.8, 170.2.
Anal. (C19H26O7) C, H.
cis-Adamantane-2-spiro-30-80-[[(ethoxy)hydroxyphosphinyl]-
methyl]-10,20,40-trioxaspiro[4.5]decane Potassium Salt (16). Step 1.
A solution of 8-(bromomethyl)-1,4-dioxaspiro[4.5]decane24 (10 g,
1
90%) as an oil. H NMR (CDCl3) δ 1.28 (t, J=6.8 Hz, 3H),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4229
1.67-2.03 (m, 18H), 2.58-2.62 (m, 2H), 2.71-2.75 (m, 2H), 3.23
(s, 2H), 4.19 (q, J=6.8 Hz, 2H); 13C NMR (CDCl3) δ 14.07, 26.26,
26.66, 34.08, 34.54, 34.60, 36.17, 36.56, 50.96, 58.94, 60.43, 106.44,
111.47, 170.21.
and treated with 1 M HCl to pH 3. The resulting solid was
filtered, washed with water, and dried in vacuo to afford
19 (320 mg, 93%) as a white solid. Mp 66-69 °C; H NMR
(CDCl3) δ 1.29-1.39 (m, 2H), 1.44-2.37 (m, 23H), 2.84 (dd, J=
6.1, 3.9 Hz, 1H); 13C NMR (CDCl3) δ 23.61, 24.30, 26.29, 26.72,
29.66, 33.80, 34.07, 34.72, 34.90, 35.34, 35.83, 36.63, 36.89,
39.24, 109.65, 111.62, 178.90. Anal. (C18H26O5) C, H.
trans-Adamantane-2-spiro-30-70-carboxymethyl-10,20,40-triox-
aspiro[4.5]decane (20). Step 1. A solution of 3122 (1.44 g, 8.0 mmol)
and methyl 2-(3-oxocyclohexyl)acetate (42)45 (1.14 g, 6.7mmol) in
cyclohexane (30 mL) and CH2Cl2 (10 mL) was treated with ozone
according to the method of Dong et al.28 After removal of the
solvents in vacuo, the crude product was purified by column
chromatography (sg, 50:1 hexane/ethyl acetate) to afford trans-
adamantane-2-spiro-30-70-methoxycarbonylmethyl-10,20,40-triox-
aspiro[4.5]decane (43) (998 mg, 45%) as an oil. 1H NMR (CDCl3)
δ 0.93-1.00 (m, 1H), 1.40-2.09 (m, 22H), 2.20-2.29 (m, 2H),
3.66 (s, 3H); 13C NMR (CDCl3) δ22.44, 26.30, 26.70, 30.76, 32.32,
34.02, 34.56, 34.59, 36.20, 36.58, 40.38, 40.70, 51.16, 108.44,
110.99, 172.39.
1
Step 2. To a solution of 36 (517 mg, 1.47 mmol) in EtOH
(50 mL) was added a solution of NaOH (117 mg, 2.94 mmol) in
water (10 mL). After the mixture was stirred at room tempera-
ture for 2 h, the resulting precipitate was collected by filtration,
washed with H2O (5 mL), and dried in vacuo at 40 °C to give 17
1
(305 mg, 84%) as a white solid. Mp 140-142 °C; H NMR
(CD3OD) δ 1.71-2.05 (m, 18H), 2.56 (m, 2H), 2.67 (m, 2H),
2.98 (s, 2H); 13C NMR (CD3OD) δ 27.96, 28.36, 35.06, 35.75,
35.83, 37.81, 37.83, 52.38, 63.29, 108.06, 112.57, 177.62. Anal.
(C17H24NNaO5) C, H, N.
trans-Adamantane-2-spiro-30-80-carboxymethyl-10,20,40-triox-
aspiro[4.5]decane (18). Step 1. To a solution of methyl 2-(4-
oxocyclohexyl)acetate (37) (5.106 g, 30 mmol) in EtOH (100 mL)
was added pyridine (3.559 g, 45 mmol) followed by methoxyla-
mine hydrochloride (2.756 g, 33 mmol). The reaction mixture was
stirred at room temperature for 4.5 h, concentrated in vacuo, and
diluted with CH2Cl2 (50 mL) and water (50 mL). The organic
phase was separated, and the aqueous layer was extracted with
CH2Cl2 (3ꢀ50 mL). The combined organic layers were washed
with 1 M HCl (40 mL), saturated aqueous NaHCO3 (40 mL), and
brine (40 mL) and dried over MgSO4. Removal of the solvents in
vacuo afforded methyl 2-[4-(methoxyimino)cyclohexyl]acetate
(38) (5.02 g, 84%) as an oil. 1H NMR (CDCl3) δ 1.12-1.28 (m,
2H), 1.68-2.20 (m, 6H), 2.26 (d, J=7.3 Hz, 2H), 2.37-2.41 (m,
1H), 3.16-3.21 (m, 1H), 3.68 (s, 3H), 3.81 (s, 3H); 13C NMR
(CDCl3) δ 23.96, 31.09, 31.35, 32.58, 33.85, 40.50, 51.47, 60.96,
158.88, 172.92.
Step 2. To a solution of 43 (627 mg, 1.8 mmol) in EtOH
(16 mL) was added a solution of NaOH (224 mg, 5.6 mmol) in
water (4 mL), and the mixture was stirred at 60 °C for 3 h.
Removal of the solvents afforded an oil that was cooled to 0 °C
and treated with 1 M HCl to pH 3. The resulting solid was
filtered, washed with water, and dried in vacuo to afford 20 (567
mg, 94%) as a white solid. Mp 66-68 °C; 1H NMR (CDCl3) δ
0.95-1.02 (m, 1H), 1.42-2.08 (m, 22H), 2.22-2.33 (m, 2H); 13
C
NMR (CDCl3) δ 22.61, 26.44, 26.83, 30.86, 32.29, 34.18, 34.76,
34.78, 36.35, 36.76, 40.53, 40.96, 108.65, 111.35, 178.46. Anal.
(C18H26O5) C, H.
3-Carboxymethyl-7,14,15-trioxadispiro[5.1.5.2]pentadecane (21).
Step 1. A solution of O-methylcyclohexanone oxime (44)30 (2.16 g,
17 mmol) and 37 (3.47 g, 20.4 mmol) in cyclohexane (120 mL) and
CH2Cl2 (30 mL) was treated with ozone following the method of
Dong et al.28 After removal of solvents in vacuo, the crude product
was purified by chromatography (sg, 4% EtOAc in hexane)
followed by crystallization from cold MeOH to afford 3-methoxy-
carbonylmethyl-7,14,15-trioxadispiro[5.1.5.2]pentadecane (45)
(2.4 g, 50%, 4:1 mixture of two isomers based on the 1H NMR
doublets at 2.22 and 2.26) as a white solid. Mp 110-112 °C; 1H
NMR (CDCl3) δ 1.27 (m, 2H), 1.36 (m, 1H), 1.43 (m, 1H),
1.56-1.82 (m, 13H), 1.94 (d, J = 15.6 Hz, 2H), 2.22 (d, J =
7.5 Hz, 1.6H), 2.26 (d, J=7.0 Hz, 0.4H), 3.67 (s, 3H); 13C NMR
(CDCl3) δ 22.46, 22.58, 23.72, 23.77, 24.82, 29.55, 29.84, 33.08,
33.22, 33.50, 33.76, 34.54, 40.36, 40.59, 51.46, 108.32, 108.86,
109.24, 171.11, 173.21.
Step 2. To a solution of 45 (678 mg, 2.24 mmol) in EtOH (50 mL)
was added a solution of NaOH (180 mg, 4.48 mmol) in water
(10 mL). The mixture was stirred at room temperature for 12 h,
cooled to 0 °C, and treated with 1 M aqueous HCl (5 mL) and H2O
(50 mL). The precipitate was collected by filtration, washed with
50% aqueous EtOH (10 mL), and dried in vacuo at 40 °C to give 21
(450 mg, 74%, 2.3:1 mixture of two isomers based on the 1H NMR
doublets at 2.27 and 2.30) as a colorless solid. Mp 144-146 °C; 1H
NMR (CDCl3) δ 1.24-1.47 (m, 4H), 1.54-1.95 (m, 15H), 2.27 (d,
J=6.8 Hz, 1.4H), 2.30 (d, J=6.8 Hz, 0.6H); 13C NMR (CDCl3) δ
23.74, 23.79, 24.84, 24.87, 29.52, 29.80, 32.88, 33.01, 33.51, 33.76,
34.56, 40.28, 40.41, 40.51, 108.27, 108.28, 108.96, 109.33, 178.76.
Anal. (C14H22O5) C, H.
Step 2. A solution of 2-adamantanone (2.25 g, 14.4 mmol) and
38 (1.91 g, 9.59 mmol) in cyclohexane (50 mL) and CH2Cl2
(10 mL) was treated with ozone according to the method of
Dong et al.28 After removal of the solvents in vacuo, the crude
product was purified by flash chromatography (sg, 100:1 hex-
ane/ethyl acetate) to afford trans-adamantane-2-spiro-30-80-
methoxycarbonylmethyl-10,20,40-trioxaspiro[4.5]decane (39) (680 mg,
1
21%) as an oil. H NMR (CDCl3) δ 1.35-1.43 (m, 2H), 1.58-
2.03 (m, 21H), 2.27 (d, J= 6.8 Hz, 2H), 3.67 (s, 3H); 13C NMR
(125.7 MHz, CDCl3) δ 26.38, 26.81, 29.65, 33.27, 33.61, 34.64, 34.83,
36.28, 36.70, 40.41, 51.43, 108.35, 111.51, 173.19.
Step3. Toa solutionof39(402 mg, 1.2 mmol) inEtOH(10mL)
was added a solution of NaOH (143 mg, 3.6 mmol) in water
(3 mL). The mixture was stirred at 60°C for 3 h and evaporated to
give an oil. The residue was cooled to 0 °C and treated with 1 M
HCl to pH 3. The resulting solid was filtered, washed with water,
and dried in vacuo to afford 18 (346 mg, 90%) as a white solid.
Mp 144-146 °C; 1H NMR (CDCl3) δ 1.38-1.45 (m, 2H), 1.60-
2.03 (m, 21H), 2.31 (d, J=6.8 Hz, 2H); 13C NMR (CDCl3) δ
26.43, 26.85, 29.66, 33.10, 33.65, 34.70, 34.88, 36.32, 36.74, 40.31,
108.33, 111.65, 178.66. Anal. (C18H26O5) C, H.
cis-Adamantane-2-spiro-30-60-carboxymethyl-10,20,40-trioxaspiro-
[4.5]decane (19). Step 1. A solution of O-methyl 2-adamanta-
none oxime (31)22 (896 mg, 5.0 mmol) and ethyl 2-(2-oxocyclo-
hexyl)acetate (40) (1.38 g, 7.5 mmol) in cyclohexane (30 mL) and
CH2Cl2 (6 mL) was treated with ozone according to the method
of Dong et al.28 After removal of the solvents in vacuo, the crude
product was purified by flash chromatography (sg, 40:1 hexane/
ethyl acetate) to afford cis-adamantane-2-spiro-30-60-ethoxycar-
bonylmethyl-10,20,40-trioxaspiro[4.5]decane (41) (380 mg, 22%)
as an oil. 1H NMR (CDCl3) δ 1.25-1.31 (m, 5H), 1.40-2.40 (m,
23H), 2.76 (dd, J=15.6, 2.4 Hz, 1H), 4.12-4.18 (m, 2H); 13C
NMR (CDCl3) δ 23.50, 26.18, 26.59, 27.27, 33.80, 33.94, 34.57,
35.16, 35.65, 36.10, 36.52, 36.73, 39.07, 39.26, 46.77, 60.18,
109.58, 111.26, 172.98.
Bicyclo[3.3.1]nonane-9-spiro-30-80-carboxymethyl-10,20,40-triox-
aspiro[4.5]decane (22). Step 1. A solution of O-methyl bicyclo-
[3.3.1]nonan-9-one oxime (46)28 (792 mg, 4.7 mmol) and 37 (1.21g,
7.1 mmol) in cyclohexane (30 mL) and CH2Cl2 (50 mL) was
treated with ozone following the method of Dong et al.28 After
removal of the solvents, the crude product was purified by column
chromatography (sg, 50:1 hexane/ethyl acetate) to afford bicyclo-
[3.3.1]nonane-9-spiro-30-80-methoxycarbonylmethyl-10,20,40-triox-
aspiro[4.5]decane (47) (807 mg, 53%) as a white solid. Mp 82-
84 °C; 1HNMR(CDCl3) δ1.22-2.06 (m, 23H), 2.22 (d, J=7.3 Hz,
Step 2. To a solution of 41 (350 mg, 1.0 mmol) in EtOH (8 mL)
was added a solution of NaOH (120 mg, 3.0 mmol) in water
(3 mL). The mixture was stirred at room temperature for 3 h and
concentrated in vacuo to afford an oil that was cooled to 0 °C

4230 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Zhao et al.
2H), 3.67 (s, 3H); 13C NMR (CDCl3) δ 20.41, 20.82, 29.33, 29.52,
29.87, 33.10, 33.94, 36.20, 40.60, 51.40, 108.27, 111.22, 173.15.
Step 2. To a solution of 47 (785 mg, 2.4 mmol) in EtOH (20
mL) was added a solution of NaOH (290 mg, 7.3 mmol) in water
(3 mL). The mixture was stirred at 60 °C for 3 h, evaporated to
give an oil, cooled to 0 °C, and treated with 1 M HCl to pH 3.
The resulting solid was filtered, washed with water, and dried in
vacuo to afford 22 (651 mg, 87%) as a white solid. Mp 146-148
°C; 1H NMR (CDCl3) δ 1.25-1.33 (m, 2H), 1.44-1.52 (m, 2H),
1.64-2.07 (m, 19H), 2.27 (d, J=7.3 Hz, 2H); 13C NMR (CDCl3)
δ 20.44, 20.84, 29.36, 29.54, 29.84, 32.91, 33.95, 36.21, 40.55,
108.24, 111.35, 178.79. Anal. (C17H26O5) C, H.
give an oil. The residue was cooled to 0 °C and treated with 1 M
aqueous HCl (4 mL) and CH2Cl2 (30 mL). After separation of the
organic layer, the aqueous phase was extracted with CH2Cl2 (2ꢀ
20 mL). The combined organic phase was washed with saturated
aqueous NaHCO3 (2ꢀ20 mL) and brine (20 mL), dried over MgSO4,
filtered, and evaporated to give 23 (266 mg, 62%, 5:1 mixture of
diastereomers) as a white solid. Mp 147-149 °C; 1H NMR
(CDCl3) δ 1.24-1.32 (m, 2H), 1.55-1.93 (m, 15H), 2.19-2.28
(m, 7H); 13C NMR (CDCl3) δ 29.40 (d, J=9.6 Hz), 29.74, 32.78,
33.15 (d, J=1.9 Hz), 33.73, 38.77 (d, J=10.6 Hz), 39.21 (d, J=19.7
Hz), 40.47, 41.97 (d, J=17.3 Hz), 91.23 (d, J=184.7 Hz), 108.93,
109.53, 178.80. Anal. (C18H25FO5) C, H.
cis-6-Fluoroadamantane-2-spiro-30-80-carboxymethyl-10,20,40-
trioxaspiro[4.5]decane (24). Step 1. Sodium borohydride (1.11 g,
29.2 mmol) was added portionwise to a ice-cold solution of 2,6-
adamantandione monoethylene ketal (53)33 (1.74 g, 8.35 mmol)
in MeOH (100 mL) at such a rate as to keep the internal
temperature below 10 °C. The reaction mixture was allowed to
warm to room temperature and stirred for 24 h. After the
reaction was quenched with water (10 mL), the solvents were
removed in vacuo and the residue was partitioned between
CH2Cl2 (80 mL) and water (60 mL). After phase separation,
the aqueous phase was extracted with CH2Cl2 (3ꢀ40 mL). The
combined organic phases were dried over MgSO4, filtered, and
concentrated in vacuo to afford 6-hydroxy-2-adamantanone
monoethylene ketal (54) (1.6 g, 91%) as a white solid. Mp
128-130 °C; 1H NMR (CDCl3) δ 1.62-2.08 (m, 13H), 3.80 (s,
1H), 3.95 (s, 4H); 13C NMR (CDCl3) δ 28.21, 33.29, 33.39,
35.24, 35.83, 64.19, 73.55, 110.95.
trans,cis-5-Fluoroadamantane-2-spiro-30-80-carboxymethyl-
10,20,40-trioxaspiro[4.5]decane (23). Step 1. To a stirred solution
of 5-hydroxy-2-adamantanone ethylene ketal (48)32 (100 mg,
0.5 mmol) in CH2Cl2 (6 mL) at 0 °C was added bis(2-methoxy-
ethyl)aminosulfur trifluoride (158 mg, 0.7 mmol). After 1 h of
stirring, the reaction was quenched with water (2 mL) and the
aqueous phase was extracted with CH2Cl2 (2 ꢀ 15 mL). The
combined organic layers were dried with MgSO4, filtered, and
concentrated in vacuo to afford a residue that was purified by
crystallization from 1:1 MeOH/H2O to afford 5-fluoro-2-ada-
mantanone ethylene ketal (49) (81 mg, 80%) as a white solid. Mp
1
44-46 °C; H NMR (CDCl3) δ 1.55-1.57 (d, 2H), 1.73-1.75
(m, 2H), 1.89-1.92 (m, 4H), 2.01 (s, 2H), 2.18-2.22 (m, 3H),
3.92-3.98 (m, 4H); 13C NMR (CDCl3) δ 29.80 (d, J=10.1 Hz),
33.23 (d, J=1.9 Hz), 38.76 (d, J=10.6 Hz), 39.39 (d, J=19.2 Hz),
42.30 (d, J = 16.8 Hz), 64.32, 64.48, 91.56 (d, J = 183.8 Hz),
109.74.
Step 2. To a solution of 49 in 2:1 acetone/water (12 mL) was
added concentrated HCl (4 mL), and the reaction mixture was
stirred for 1 h at room temperature before removal of the
solvents in vacuo. CH2Cl2 (6 mL) and water (6 mL) were added,
and the two phases were separated followed by extraction of the
aqueous phase with CH2Cl2 (2ꢀ4 mL). The combined organic
layers were dried over MgSO4 and the solvent was removed in
vacuo to afford 5-fluoro-2-adamantanone (50) (55 mg, 86%) as
Step 2. To a solution of 54 (808 mg, 3.8 mmol) in CH2Cl2 (40mL)
was added bis(2-methoxyethyl)aminosulfur trifluoride (850 mg,
3.8 mmol), and the reaction mixture was stirred at room tempera-
ture for 24 h before quenching with water (30 mL). The aqueous
phase was extracted with CH2Cl2 (2ꢀ40 mL) and the combined
organic phases were washed with saturated aqueous NaHCO3 (2ꢀ
30 mL) and brine (30 mL), dried over MgSO4, and concentrated in
vacuo to afford crude 6-fluoro-2-adamantanone monoethylene
1
1
a colorless solid. Mp 269-271 °C; H NMR (CDCl3) δ 1.66-
ketal (55) (540 mg, 66%) as a white solid. Mp 45-47 °C; H
2.44 (m, 11H), 2.68 (s, 2H); 13C NMR (125.7 MHz, CDCl3) δ
30.43 (d, J=10.1 Hz), 37.94 (d, J=2.4 Hz), 41.55 (d, J=17.8 Hz),
42.05 (d, J=20.2 Hz), 47.05 (d, J=10.1 Hz), 90.18 (d, J=185.7 Hz),
214.92 (d, J=1.9 Hz).
NMR (CDCl3) δ 1.59 (d, J=12.7 Hz, 2H), 1.74 (s, 2H), 1.79 (d, J=
12.7 Hz, 2H), 2.01-2.11 (m, 6H), 3.94 (s, 4H), 4.59 (d, J=50.8 Hz,
1H); 13C NMR (CDCl3) δ 28.40 (J=1.0 Hz), 31.34 (J=18.2 Hz),
32.37 (J= 8.7 Hz), 34.92 (J= 1.4 Hz), 35.45, 64.11, 94.17 (J=
179.5 Hz), 110.29.
Step 3. To a solution of 50 (800 mg, 4.8 mmol) in EtOH (40 mL)
was added pyridine (564 mg, 7.1 mmol) followed by methoxyla-
mine hydrochloride (477 mg, 5.7 mmol). The reaction mixture was
stirred at room temperature for 2 h, concentrated in vacuo, and
diluted with water (10 mL). After filtration, O-methyl-5-fluoro-2-
adamantanone oxime (51) was obtained as a white solid (773 mg,
82%). Mp 70-72 °C; 1H NMR (CDCl3) δ 1.70-2.06 (m, 10H),
Step 3. To a solution of 55 in 2:1 acetone/water (12 mL) was
added concentrated HCl (4 mL), and the reaction mixture was
stirred at 70 °C for 1 h. After removal of the solvents in vacuo,
CH2Cl2 (6 mL) and water (6 mL) were added. After phase
separation, the aqueous phase was extracted with CH2Cl2 (2ꢀ
4 mL). The combined organic phases were dried over MgSO4 and
concentrated in vacuo to afford 6-fluoro-2-adamantanone (56)
(55 mg, 86%) as a white solid. Mp 230-232 °C; 1H NMR
(CDCl3) δ 1.81-2.49 (m, 12H), 4.85 (dt, J=49.8, 3.4 Hz, 1H);
13C NMR (CDCl3) δ 31.73 (J=18.7 Hz), 32.68 (J=1.4 Hz), 36.42
(J=8.6Hz), 44.67 (J=1.9Hz), 45.48, 92.72 (J=180.9 Hz), 215.95.
Step 4. To a solution of 56 (336 mg, 2.0 mmol) in EtOH (25 mL)
was added pyridine (316 mg, 3.0 mmol) followed by methoxyla-
mine hydrochloride (200 mg, 2.4 mmol). The reaction mixture was
stirred at room temperature for 6 h, concentrated in vacuo, and
diluted with CH2Cl2 (50 mL) and water (50 mL). The organic
phase was separated, and the aqueous layer was extracted with
CH2Cl2 (3ꢀ30 mL). The combined organic phases were washed
with 1 M HCl (30 mL), saturated aqueous NaHCO3 (30 mL), and
brine (30 mL) and dried over MgSO4. Removal of the solvents in
vacuo afforded O-methyl-6-fluoro-2-adamantanone oxime (57)
(370 mg, 94%, 1:1 mixture of diastereomers) as a white solid.
Mp 70-72 °C; 1H NMR (CDCl3) δ 1.61-2.49 (m, 11H), 3.41-
3.44 (m, 1H), 3.81 (s, 1.5H), 3.82 (s, 1.5H), 4.71-4.82 (m, 1H); 13C
NMR (CDCl3) δ 31.24 (J=1.0 Hz), 32.30 (J=18.2 Hz), 32.36 (J=
18.2 Hz), 34.36, 35.00, 35.28 (J=8.6 Hz), 36.53 (J=8.6 Hz), 60.96,
44.67 (J=1.9 Hz), 45.48, 92.72 (J=180.9 Hz), 215.95.
2.37 (d, J=2.0 Hz, 1H), 2.78 (s, 1H), 3.69 (s, 1H), 3.82 (s, 3H); 13
C
NMR (CDCl3) δ 30.78 (d, J=10.0 Hz), 30.96 (d, J=9.6 Hz), 36.08
(d, J=2.4 Hz), 37.50 (d, J=1.9 Hz), 37.90 (d, J=10.6 Hz), 41.45 (d,
J=18.7 Hz), 41.74 (d, J=17.3 Hz), 42.69 (d, J=18.7 Hz), 61.12,
91.23 (d, J=185.2 Hz), 162.81 (d, J=2.4 Hz).
Step 4. A solution of 51 (918 mg, 4.66 mmol) and 37 (1.19 g,
6.99 mmol) in cyclohexane (40 mL) and CH2Cl2 (20 mL) was
treated with ozone according to the method of Dong et al.28
After removal of solvents, the crude product was purified by
crystallization from EtOH to afford trans,cis-5-fluoroadaman-
tane-2-spiro-30-80-methoxycarbonylmethyl- 10,20,40-trioxaspiro-
[4.5]decane (52) (450 mg, 27%, 3:1 mixture of diastereomers) as
a white solid. Mp 126-128 °C; 1H NMR (CDCl3) δ 1.23-1.28
(m, 2H), 1.55-1.95 (m, 15H), 2.18-2.23 (m, 7H), 3.67 (s, 3H);
13C NMR (CDCl3) δ 29.40 (d, J=10.1 Hz), 29.80, 32.99, 33.14
(d, J=1.9 Hz), 33.75, 38.76 (d, J=11.4 Hz), 39.20 (d, J=19.7
Hz), 40.54, 41.97 (d, J=17.3 Hz), 51.49, 91.20 (d, J=184.3 Hz),
109.01, 109.46, 173.12.
Step 5. To a solution of 52 (450 mg, 1.27 mmol) in EtOH (10 mL)
was added a solution of NaOH (152 mg, 3.81 mmol) in water
(4 mL). The mixture was stirred for 4 h at 55 °C and evaporated to

Article
Step 5. A solution of 57 (370 mg, 1.9 mmol) and 37 (479 mg,
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4231
33.59 (J=3.4 Hz), 34.06, 34.87 (J=3.4 Hz), 35.53 (J=3.4 Hz),
61.10, 123.90 (J=247.1 Hz), 162.97.
2.8 mmol) in cyclohexane (30 mL) and CH2Cl2 (60 mL) was
treated with ozone according to the method of Dong et al.28
After removal of solvents in vacuo, the crude product was
purified by crystallization from cold EtOH to afford cis-6-
fluoroadamantane-2-spiro-30-80-methoxycarbonylmethyl-10,20,40-
trioxaspiro[4.5]decane (58) (250 mg, 38%) as a white solid. Mp
114-116 °C; 1H NMR (CDCl3) δ 1.23-1.30 (m, 2H), 1.58-2.13
(m, 19H), 2.22 (d, J=7.3 Hz, 1H), 3.67 (s, 3H), 4.59 (d, J=
50.8 Hz); 13C NMR (CDCl3) δ 28.51 (J=1.0 Hz), 28.57, 29.84
(J=3.8 Hz), 31.02 (J=18.2 Hz), 31.43 (J=18.2 Hz), 32.38 (J=
4.3 Hz), 32.45 (J = 4.3 Hz), 33.07, 33.83 (J = 2.9 Hz), 34.86
(J=1.4 Hz), 35.43, 40.57, 51.46, 93.90 (J=179.5 Hz), 108.74,
110.32, 173.15.
Step 4. A solution of 61 (460 mg, 2.1 mmol) and 37 (546 mg,
3.2 mmol) in cyclohexane (30 mL) and CH2Cl2 (6 mL) was
treated with ozone according to the method of Dong et al.28
After removal of solvents in vacuo, the crude product was
purified by crystallization from cold EtOH to afford cis-6,6-
difluoroadamantane-2-spiro-30-80- methoxycarbonylmethyl-
10,20,40-trioxaspiro[4.5]decane (62) (322 mg, 41%) as a white
solid. Mp 129-131 °C; 1H NMR (CDCl3) δ 1.20-1.29 (m, 2H),
1.69-2.14 (m, 19H), 2.23 (d, J=6.8 Hz, 2H), 3.70 (s, 3H); 13
C
NMR (CDCl3) δ 30.65 (J=3.8 Hz), 30.68 (J=3.4 Hz), 32.99,
34.11 (J =21.6 Hz), 34.52 (J =21.6 Hz), 34.57, 40.49, 51.47,
108.94, 109.08, 123.94 (J=247.1 Hz), 173.11.
Step 6. To a solution of 58 (250 mg, 0.7 mmol) in EtOH (12 mL)
was added a solution of NaOH (85 mg, 2.1 mmol) in water (3 mL).
The mixture was stirred at 60 °C for 3 h and concentrated in vacuo.
The residue was cooled to 0 °C and treated with 1 M aqueous HCl
(5 mL) and H2O (10 mL). The precipitate was collected by
filtration and dried in vacuo at 40 °C to afford 24 (237 mg, 99%)
as a white solid. Mp 144-146 °C; 1H NMR (CDCl3) δ 1.24-1.32
(m, 2H), 1.59-2.10 (m, 19H), 2.27 (d, J=6.8 Hz, 1H), 4.60 (d, J=
50.8 Hz); 13C NMR (CDCl3) δ 28.52, 28.57, 29.78 (J=3.4 Hz),
30.99 (J=18.2 Hz), 31.41 (J=18.2 Hz), 32.37 (J=3.8 Hz), 32.44
(J=3.8 Hz), 32.85, 33.80 (J=2.9 Hz), 34.83, 35.40, 40.44, 93.93 (J=
179.0 Hz), 108.66, 110.39, 178.46. Anal. (C18H25FO5) C, H.
cis-6,6-Difluoroadamantane-2-spiro-30-80-carboxymethyl-10,20,
40-trioxaspiro[4.5]decane (25). Step 1. To a solution of 2,6-
adamantanedione monoethylene ketal (53)33 (1.17 g, 5.6 mmol)
in CH2Cl2 (85 mL) was added bis(2-methoxyethyl)aminosulfur
trifluoride (2.27 g, 10.3 mmol), and the reaction mixture was
stirred at room temperature for 24 h before quenching with
water (50 mL). The aqueous phase was separated and extracted
with CH2Cl2 (2 ꢀ 40 mL). The combined organic layers were
washed with saturated aqueous NaHCO3 (2ꢀ40 mL) and brine
(40 mL), dried over MgSO4, filtered, and concentrated in vacuo
to give a residue that was purified by flash chromatography (sg,
40:1 hexane/ethyl acetate) to afford 6,6-difluoro-2-adamanta-
none monoethylene ketal (59) (1.10 g, 85%) as a colorless solid.
Mp 76-78 °C; 1H NMR (CDCl3) δ 1.77 (s, 2H), 1.87-1.90 (d,
J=12.7 Hz, 4H), 2.00-2.02 (d, J=12.2 Hz, 4H), 2.11 (s, 2H),
3.95 (s, 4H); 13C NMR (CDCl3) δ 30.76 (J=3.8 Hz), 34.58 (J=
21.6 Hz), 34.87, 64.37, 109.31, 124.43 (J=246.6 Hz).
Step 2. To a solution of 59 (1.1 g, 4.78 mmol) in 5:1 acetone/
water (96 mL) was added concentrated HCl (40 mL), and the
reaction mixture was heated to 70 °C gradually. After the dis-
appearance of the starting material as determined by GC-MS
(0.5 h), the mixture was cooled to 0 °C and NaHCO3 was added
to adjust the pH to 7. After CH2Cl2 (80 mL) was added to the
mixture, the organic layer was separated. The aqueous phase
was extracted with CH2Cl2 (3ꢀ50 mL). The combined organic
layers were dried over MgSO4, filtered, and concentrated in
vacuo to afford 6,6-difluoro-2-adamantanone (60) as a white
solid (760 mg, 85%). Mp 252-255 °C; 1H NMR (CDCl3) δ 2.01
(d, J=12.7 Hz, 4H), 2.29 (d, J=12.7 Hz, 4H), 2.35 (s, 2H), 2.53
(s, 2H); 13C NMR (CDCl3) δ 34.86 (J=3.8 Hz), 35.06 (J=22.1
Hz), 44.47, 123.15 (J=247.6 Hz), 214.28.
Step 5. To a solution of 62 (322 mg, 0.87 mmol) in EtOH
(8 mL) was added a solution of NaOH (104 mg, 2.6 mmol) in water
(3 mL). The mixture was stirred at room temperature for 4 h and
evaporated to give an oil. After the residue was treated with 1 M
HCl to lower the pH to 3, the aqueous phase was extracted with
CH2Cl2 (3 ꢀ 50 mL). The combined extracts were dried over
MgSO4, filtered, and evaporated to afford 25 (277 mg, 89%) as a
white solid. Mp 148-150 °C; 1H NMR (CDCl3) δ 1.29-1.37 (m,
2H), 1.75-2.20 (m, 19H), 2.23 (d, J=6.8 Hz, 2H); 13C NMR
(125.7 MHz, CDCl3) δ 29.72, 30.67 (J=3.8 Hz), 30.70 (J=3.8
Hz), 32.79, 33.73, 34.12 (J=21.6 Hz), 34.54 (J=21.6 Hz), 34.58,
40.53, 108.87, 109.16, 123.96 (J = 247.1 Hz), 179.20. Anal.
(C18H24F2O5) C, H.
Adamantane-2-spiro-30-80-carboxymethyl-10,20-dioxaspiro[4.5]-
decane (26). Step 1. To a solution of I2 (0.254 g, 1.0 mmol) and
50% H2O2 (4.5 mL, 40 mmol) in MeOH (50 mL) was added 37
(1.70 g, 10 mmol). After the mixture was stirred at room
temperature for 24 h, it was concentrated in vacuo and the
residue was partitioned between CH2Cl2 (30 mL) and water
(30 mL). The aqueous layer was extracted with CH2Cl2 (2ꢀ30 mL).
The combined extracts were washed with water, 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
1
next step. H 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. To a solution of the unpurified methyl 2-(4-hydro-
peroxy-4-methoxycyclohexyl)acetate (2.15 g, 9.86 mmol) in
DMF (100 mL) at 0 °C was added Et3N (4.5 mL, 32 mmol)
followed by Et3SiOTf (2.54 mL, 12 mmol). The reaction mixture
was stirred at room temperature 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 (3ꢀ100 mL). The organic extracts were combined, dried
over MgSO4, and concentrated to afford methyl 2-[4-methoxy-
4-(triethylsilylperoxy)cyclohexyl]acetate (63) as a 1:1 mixture of
diastereomers (3.02 g, 92%) which was used immediately in the
1
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 solution of 63 (3.02 g, 9.10 mmol) in CH2Cl2
(50 mL) at -78 °C was added 2-methyleneadamantane46 (64)
(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 al-
lowed 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 (2ꢀ50 mL). The combined extracts were
washed with water (50 mL) and brine (50 mL), dried over
MgSO4, filtered, and concentrated in vacuo. Purification by
chromatography (sg, 0-10% ether in hexane) afforded ada-
mantane-2-spiro-30-80-methoxycarbonylmethyl-10,20-dioxaspiro-
[4.5]decane (65) as a colorless solid (0.60 g, 40%). Mp 119-120 °C;
1H 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,
Step 3. To a solution of 60 (760 mg, 4.1 mmol) in EtOH (50 mL)
was added pyridine (483 mg, 6.1 mmol) followed by methoxyla-
mine hydrochloride (409 mg, 4.9 mmol). The reaction mixture was
stirred at room temperature for 6 h, concentrated in vacuo, and
diluted with CH2Cl2 (50 mL) and water (50 mL). The organic
phase was separated, and the aqueous layer was extracted with
CH2Cl2 (3ꢀ50 mL). The combined organic phases were washed
with 1 M HCl (40 mL), saturated aqueous NaHCO3 (40 mL) and
brine (40 mL), and dried over MgSO4. Removal of the solvent in
vacuo afforded O-methyl-6,6-difluoro-2-adamantanone oxime
(61) (760 mg, 87%) as a white solid. Mp 114-116 °C; 1H NMR
(CDCl3) δ 1.80-1.89 (m, 4H), 2.09-2.18 (m, 4H), 2.30 (s, 1H),
2.52 (s, 1H), 3.46 (s, 1H), 3.82 (s, 1H); 13C NMR (CDCl3) δ 27.22,

4232 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Zhao et al.
33.45, 34.93, 35.64, 36.24, 37.21, 41.17, 51.42, 55.47, 84.02, 88.71,
173.47.
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Step 4. To a solution of 65 (0.45 g, 1.35 mmol) in EtOH (20 mL)
was added 15% aqueous KOH (2 mL), and 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 vacuo at 40 °C to
afford 26 as a colorless solid (0.40 g, 93%). Mp 184-185 °C; 1H
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 (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. Anal. (C19H28O4) C, H.
Adamantane-2-spiro-20-80-carboxymethyl-10,40-dioxaspiro[4.5]-
decane (27). p-Toluenesulfonic acid monohydrate (260 mg,
1.37 mmol) was added to a solution of 2-hydroxymethyl-2-ada-
mantanol (66)37 (1.22 g, 6.69 mmol) and 2-(4-oxocyclohexyl)-
acetic acid (67) (1.14 g, 7.29 mmol) in CH2Cl2 (200 mL). The
reaction mixture was stirred at room temperature for 4 h, washed
with water (100 mL) and brine (100 mL), dried over MgSO4,
filtered, and concentrated in vacuo. The crude product was purified
by crystallization from aqueous MeOH to afford 27 as a white solid
1
(1.69 g, 79%, 1.3:1 mixture of isomers based on the H NMR
singlets at 3.84 and 3.88). Mp 133-135 °C; 1H NMR (CDCl3) δ
1.25-1.85 (m, 21H), 2.18 (q, 2H), 2.26 (d, J=6.8 Hz, 0.78H), 2.29
(d, J=6.8 Hz, 1.11H), 3.84 (s, 0.87H), 3.88 (s, 1.13H); 13C NMR
(CDCl3) δ 26.67, 26.70, 26.79, 26.93, 30.01, 30.05, 33.08, 33.46,
33.64, 35.82, 35.91, 35.96, 36.33, 37.25, 37.34, 37.41, 40.66, 40.74,
72.14, 72.32, 84.33, 84.48, 108.35, 108.46, 178.79, 178.89. Anal.
(C19H28O4) C, H.
Evaluation of Activity against F. hepatica. All animal studies39
were approved by regulatory authorities following Swiss
National regulations. Metacercariae of F. hepatica were pur-
chased from Baldwin Aquatics (Monmouth, OR). Female
Wistar rats (weight, ∼100 g) were purchased from Harlan
(Horst, The Netherlands). Animals were kept in groups of four
in Makrolon cages in environmentally controlled conditions
(temperature, ∼25 °C; humidity, ∼70%; 12 h light/dark cycle)
and acclimatized for 1 week. Rats were infected with ∼20
metacercariae each. They had free access to water and rodent
diet. At 8 weeks after infection, rats were treated with single
50-100 mg/kg oral doses of target compounds prepared as
suspensions in 7% (v/v) Tween-80 and 3% (v/v) EtOH. At day 6
after treatment, rats were sacrificed by CO2 and adult flukes
were recovered from the bile ducts and livers. Target compound
efficacies were evaluated by comparing the mean total worm
burdens of treated and untreated control rats. Statistical signific-
ance (at a significance level of 5%) was calculated using the
Kruskal-Wallis test (Statsdirect, version 2.4.5; Cheshire, U.K.).
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monoethylene ketal and dimethylmalonate with TiCl4 and pyridine
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Acknowledgment. This investigation received financial
support from NIH (Grant R21AI076783), the Nebraska
Research Initiative, the Swiss National Science Foundation
(Project No. PPOOA-114941), and the Medicines for Malaria
Venture.
Supporting Information Available: Elemental analysis results
for 6-8, 10, 14, and 16-27. This material is available free of
charge via the Internet at http://pubs.acs.org.
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