The structure - Activity relationship of the antimalarial ozonide arterolane (OZ277)

Article abstract of DOI:10.1021/jm901473s

The structure and stereochemistry of the cyclohexane substituents of analogues of arterolane (OZ277) had little effect on potency against Plasmodium falciparum in vitro.Weak base functional groups were not required for high antimalarial potency, but they were essential for high antimalarial efficacy in P. berghei-infected mice. Five new ozonides with antimalarial efficacy and ADME profiles superior or equal to that of arterolane were identified.

Full text of DOI:10.1021/jm901473s

J. Med. Chem. 2010, 53, 481–491 481
DOI: 10.1021/jm901473s
The Structure-Activity Relationship of the Antimalarial Ozonide Arterolane (OZ277)
Yuxiang Dong,† Sergio Wittlin,‡ Kamaraj Sriraghavan,† Jacques Chollet,‡ Susan A. Charman,§ William N. Charman,§
Christian Scheurer,‡ Heinrich Urwyler,^ Josefina Santo Tomas,§ Christopher Snyder,‡ Darren J. Creek,§ Julia Morizzi,§
Maria Koltun,§ Hugues Matile, Xiaofang Wang,† Maniyan Padmanilayam,† Yuanqing Tang,† Arnulf Dorn, Reto Brun,‡ and
Jonathan L. Vennerstrom*,†
†College of Pharmacy, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE, ‡Swiss Tropical Institute,
Socinstrasse 57, CH-4002 Basel, Switzerland, §Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash
University (Parkville Campus), 381 Royal Parade, Parkville, Victoria 3052, Australia, ^Basilea Pharmaceutica Ltd., Grenzacherstrasse 487,
CH-4058 Basel, Switzerland, and F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, CH-4070 Basel, Switzerland
Received October 5, 2009
The structure and stereochemistry of the cyclohexane substituents of analogues of arterolane (OZ277)
had little effect on potency against Plasmodium falciparum in vitro. Weak base functional groups were
not required for high antimalarial potency, but they were essential for high antimalarial efficacy in
P. berghei-infected mice. Five new ozonides with antimalarial efficacy and ADME profiles superior or
equal to that of arterolane were identified.
The semisynthetic artemisinins artemether and artesunate
(AS)a are important first-line antimalarial drugs.1 They are
employed with partner drugs in artemisinin combination
treatments (ACT) to allow for treatment regimens of 3 days2
and to protect against potential artemisinin drug resistance.3
In efforts to improve upon the biopharmaceutical properties
of the artemisinins, newer semisynthetic artemisinins such as
artelinic acid and artemisone have been investigated.4-6 In
addition, work to identify synthetic peroxides6-8 that com-
bine the powerful antimalarial action of the artemisinins with
good biopharmaceutical properties is ongoing. With this goal
in mind, we identified the ozonide arterolane (OZ277, 1), a
synthetic peroxide drug development candidate9 thatisnow in
phase III clinical trials in the form of an arterolane maleate/
piperaquine phosphate combination.6,10
good antimalarial profiles.12,14,15 In this paper, we describe
new insightsfromthe SARofthe ozonidesrelatedto1 and our
efforts to identify analogs of 1 with superior biopharma-
ceutical and antimalarial properties.
Chemistry
We identified HOBt active ester 2 as a key common inter-
mediate suitable for the preparation of numerous analogs
of 1 using parallel chemistry. HOBt active ester 2 was obtained
(Scheme 1) starting with Griesbaum coozonolysis16 of O-methyl-
2-adamantanone oxime (3)17 and methyl 4-oxocyclohexyl
acetate (4)18 to form ozonide ester 5 (78%), which was
isolated by solvent removal followed by crystallization from
80% aq EtOH. Ester hydrolysis of 5 afforded ozonide acid 6
in 96% yield. Treatment of 6 with HOBt and EDCI in DMF
afforded, after precipitation with water, HOBt active ester 2
in 95% yield. For these ozonides, X-ray crystallographic
data19,20 reveals that the peroxide bond is axial and
the cyclohexane substituent is equatorial, establishing the
stereochemistry as cis.
A wide range of ozonide amides and amino amides was
obtained by stirring the corresponding amine or diamine with
2 in CHCl3 or in a mixture of CHCl3/EtOH at rt, typically for
1-4 h. For example, under these conditions, treatment of 2
with 1,2-diamino-2-methylpropane, guanidine, glycinamide,
1,2-diaminoethane, 1-amino-1-(aminomethyl)cyclopropane,
1,3-diaminopropane, 2,2-dimethyl-1,3-diaminopropane, trans-
1,4-diaminocyclohexane, 1-piperazinecarboxaldehyde, pipera-
zine, N-(2-hydroxyethyl)piperazine, piperazin-2-one, 4-hydro-
xypiperidine, isonipecotic acid, and isonipecotamide afforded 1
(84%), 17 (94%), 18 (75%), 19 (83%), 20 (60%), 21 (76%), 22
(35%), 23 (47%), 26 (58%), 28 (75%), 29 (65%), 30 (51%), 32
(37%), 34 (82%), and 35 (76%), respectively (Scheme 2).
Similarly, treatment of 2 with 4-(tert-butoxycarbonyl)piperi-
dine followed by deprotection with PTSA afforded 33 (31%
overall) or with thiomorpholine and subsequent oxidation with
m-CPBA afforded 25 (79% overall). Ozonides 15 (65%), 16
The structure-activity relationship (SAR) for this class of
dispiro synthetic ozonides can be summarized as follows: (1)
the spiroadamantane ring system11,12 and peroxide bond11,13
are essential for activity; (2) more lipophilic ozonides tend to
have better oral activities than their more polar counter
parts,11,12 an outcome consistent with that seen for other
classes of synthetic peroxides;7 (3) ozonides with a wide range
of neutral and basic, but not acidic, functional groups have
*To whom correspondence should be addressed. E-mail: jvenners@
unmc.edu. Tel: 402-559-5362. Fax: 402-559-9543.
a Abbreviations: ACT, artemisinin combination treatment; AS,
artesunate; CQ, chloroquine; DHA, dihydroartemisinin; ER, hepatic
extraction ratios; MF, mefloquine; PfATP6, Plasmodium falciparum
sarcoendoplasmic reticulum Ca2þ ATPase; SSV, standard suspension
vehicle; TPP, triphenylphosphine.
r
2009 American Chemical Society
Published on Web 11/19/2009
pubs.acs.org/jmc

482 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Dong et al.
Scheme 1
Table 1. Metabolic Stability and Activity of Hydroxamic Acid, Acyl-
guanidine, and Neutral Amide Derivatives of Ozonide Carboxylic
Acid 78 against P. falciparum in Vitro and P. berghei in Vivo (1 ꢀ
10 mg/kg po)
IC50 (ng/mL),a
K1/NF54
compd
R
activity (%)b
ERc
6d
OH
NH2
34/45
0.91/1.1
1.4/2.0
2.4/2.0
3.9/3.6
1.3/1.6
95
97
0.28
0.49
0.88
e
Scheme 2
15
16
17
18
ASd
NHOH
NHCdNH(NH2)
NHCH2CONH2
97
97
99.6
67
e
0.43f
a Mean from n = 2-3. Individual measurements differed by less than
50%. bGroups of five P. berghei-infected NMRI mice were treated orally
1 day postinfection with ozonides dissolved or suspended in SSV.
Antimalarial activity was measured by percent reduction in parasitemia
on day 3 postinfection. Individual measurements differed by less than
10%. c Predicted hepatic extraction ratios (ER) using human micro-
somes.28 d Data from Vennerstrom et al.9 e Not determined. f Value for
dihydroartemisinin (DHA), the primary metabolite of artesunate (AS).
Table 2. Metabolic Stability and Activity of Ozonide Amino Amides
against P. falciparum in Vitro and P. berghei in Vivo (1 ꢀ 10 mg/kg po)
(28%), 24 (74%), and 31 (62%) were obtained from the mixed
anhydride of 6, which was formed by treatment of 6 with ethyl
chloroformate and Et3N at 0 ꢀC, followed by exposure to
ammonia (7 N in MeOH), hydroxylamine, morpholine, and
piperidine, respectively. Ozonide 27 (87%) was obtained by
treatment of 28 with methanesulfonyl chloride/pyridine in
CH2Cl2 at 0 ꢀC. Ozonide tetrazole 43 was obtained by treat-
ment of 1 with TPP, trimethylsilyl azide, and DIAD according
to the method of Duncia et al.21
IC50 (ng/mL),
K1/NF54
compd
X
activity (%)
ER
1a,b
19a
20a
21a
22c
23a
CH2C(CH3)2
CH2CH2
1.0/0.91
1.8/1.9
99.9
99.9
0.32
0.42
Ozonide 36 was obtained by reaction of ozonide acid 720
with HOBt and EDCI in DMF followed by reaction of the
thus formed HOBt active ester (95%) with 1,2-diamino-
2-methylpropane in CHCl3 (85%) (Scheme 2). Ozonide 37
was obtained by coozonolysis of oxime ether 317 with methyl
3-(4-oxocyclohexyl)propionate (8)18 to form ozonide ester 9
(59%) followed by ester hydrolysis (95%), HOSu active ester
formation (100%), and reaction with 1,2-diamino-2-methyl-
propane (74%). Ozonide ureas 38 (31%) and 39 (25%) were
obtained by treatment of the 4-nitrophenylcarbamate22 deri-
vatives of ozonide amines 10 and 1115 with 1,2-diamino-
2-methylpropane. Ozonide carbamate 40 (26%) was obtained
by methylation of imidazole carbamate 1212 with methyl
trifluoromethylsulfonate followed by reaction with 1,2-dia-
mino-2-methylpropane. trans-Ozonide 41 was obtained by
reaction of ozonide acid 1323 with HOBt and EDCI in DMF
followed by reaction of the thus-formed HOBt active ester
(64%) with 1,2-diamino-2-methylpropane in CHCl3 (82%).
trans-Ozonide 42 was obtained starting with repeated crystal-
lizations of 5 from the reaction mother liquor to enrich the
proportion of the minor trans-ozonide ester isomer of 5; using
this approach, we obtained a 1:1 mixture of the cis- and trans-
ozonide esters. Subsequent hydrolysis and formation of the
4-nitrophenyl ozonide esters afforded after repeated crystal-
lizations from EtOH, the pure trans isomer 14 (5%). Treat-
ment of the latter with 1,2-diamino-2-methylpropane affor-
ded 42 (68%). As indicated in Tables 1-4, most of the weak
base ozonides were isolated and tested as their tosylate
salts.
CH2C(CH2CH2)
CH2CH2CH2
CH2C(CH3)2CH2
trans-1,4-C6H10
0.40/0.52
3.5/2.3
99.9
99.9
0.50
d
0.46/0.49
2.8/3.0
99.9
>99.9
0.43
0.54
a Tosylate salt. b Data from Vennerstrom et al.9 c Mesylate salt. d Not
determined.
Table 3. Metabolic Stability and Activity of Ozonide Piperidine, Mor-
pholine, Thiomorpholine, and Piperazine Amides against P. falciparum
in Vitro and P. berghei in Vivo (1 ꢀ 10 mg/kg po)
IC50 (ng/mL),
K1/NF54
compd
X
activity (%)
ER
b
24
25
26
27
28a
29
30
31
32
33a
34
35
O
SO2
1.7/3.0
1.8/1.2
99.8
<40
99.8
<40
>99.9
96
b
b
b
NCOH
0.39/0.63
1.2/2.1
NSO2CH3
NH
NCH2CH2OH
0.21/0.24
1.0/0.90
0.60/0.89
1.6/1.6
0.50
b
99.2
99.3
99.7
>99.9
43
0.72
b
H
OH
0.49/0.76
0.35/0.39
19/27
0.75
0.45
NH2
COOH
CONH2
b
b
1.2/1.7
86
a Tosylate salt. b Not determined.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 483
Table 4. Metabolic Stability and Activity of Homologs, Isosteres, and
Stereoisomers of Ozonide Amino Amide 277 against P. falciparum
in Vitro and P. berghei in Vivo (1 ꢀ 10 mg/kg po)
Table 5. In Vivo Activity in P. berghei Infected Mice Following Three
Consecutive Daily Oral Doses of 3 mg/kga
compd
activity (%)
survival (days)
cureb (%)
control
1c
23
0
6-7
11.4
22.0
14.8
27.0
24.4
9.8
0
0
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
70
0
28
33
0
60
0
37
40
0
ASc
CQc
MFc
9.2
9.4
0
99.7
99
0
15.8
0
IC50 (ng/mL),
K1/NF54
a Groups of five P. berghei-infected NMRI mice were treated orally
on days þ1, þ2, and þ 3 postinfection with ozonides dissolved or
suspended in SSV. Antimalarial activity was measured by percent
reduction in parasitemia on day 4 postinfection. b No detectable para-
sites at 30 days postinfection. c Data from Vennerstrom et al.9
compd
X
activity (%)
ER
36a
37a
38a
39a
40a
41a
42a
43a
none
CH2CH2
NH
0.66/0.25
0.55/0.82
8.4/6.2
96
>99.9
63
b
0.37
b
b
CH2NH
CH2O
none
3.2/3.5
97
methylene R to the carboxamide of 1 with an NH (38) or with
a CH2NH (39) decreased overall antimalarial efficacy; in
contrast, replacement with a CH2O endowed 40 with an
antimalarial profile comparable to that of 1. The relatively
low in vivo activities of ureas 38 and 39 indicate that for
reasonably polar ozonides such as 1 with an experimental log
DpH7.4 valueof 2.9, adding H-bond donating groups decreases
overall antimalarial efficacy in vivo. Tetrazole isostere 43 was
as potent as 1 in vitro but was more than 20-fold less effective
in vivo. The two cis/trans isomer pairs 36/41 and 1/42
exhibited little differences in in vitro potency but considerable
differences in in vivo activity; for example, 1 was more than
40-foldmore effective than 42. The latter phenomenonmay be
explained in part by the 3.5-fold higher aqueous degradation
rate26 and 2.2-fold higher iron(II) reactivity27 of the trans-42
vs the cis-1.
0.22/0.36
1.4/0.74
1.7/1.5
>99.9
84
96
0.32
b
b
b
CH2
2.3/1.5
98
a Tosylate salt. b Not determined.
Antimalarial Activity
In vitro and in vivo antimalarial activities9 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 1 ꢀ 10 and 3 ꢀ
3 mg/kg oral doses of the ozonides administered in a non-
solubilizing, standard suspension vehicle (SSV) formulation
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.
To assess whether the five (23, 28, 33, 37, and 40) most
promising new ozonides (activity >99.9% at 1 ꢀ 10 mg/kg
po) could cure P. berghei-infected mice, we administered
a 3 mg/kg daily dose on days þ1, þ2, and þ3 postinfection
(Table 5). In this experiment, only 33 cured any (3/5) of the
infected mice; however the other ozonides all increased sur-
vival time compared with the untreated control and were as or
more effective than AS and chloroquine (CQ) in this respect.
Of these, 23 and 37 were as or more effective than mefloquine
(MF) at increasing survival time.
The data in Tables 1-4 reveal that with the exception of the
ozonide urea 38 with IC50’s between 6 and 9 ng/mL, each of
the neutral and weak base ozonides had IC50 values in the
relatively narrow range of 0.2-3.9 ng/mL, similar to that of
the control artesunate. Further, there was no significant
difference in in vitro potency between neutral and weak base
ozonides. The weakly acidic hydroxamic acid 16 and ampho-
teric24 acylguanidine 17 were similarly potent. The only
compounds for which the nature of the cyclohexane substi-
tuent affected potency significantly were the acidic ozonides 6
and 34, which, in contrast to the acidic semisynthetic arte-
sunate, were an order of magnitude less potent against
P. falciparum in vitro. Studies to examine the erythrocyte
partitioning characteristics for a range of ozonides indicated
that in contrast to the weak base compounds, 6 did not
partition well into erythrocytes, a factor that could contribute
in part to its poor antimalarial activity (data not shown).
Whereas the structure of the neutral and weak base ozonides
had little to no effect on potency against P. falciparum in vitro,
it significantly affected potency against P. berghei in vivo.
Thus, all six ozonides (Table 1-4) with activities >99.9%
were weak bases; of these, five were primary amines and one
(28) was a secondary amine. None of the neutral (or acidic)
ozonides had an in vivo activity that exceeded 99.8%.
Metabolism and Pharmacokinetics
Where measured, the predicted hepatic extraction ratios
(ER)28 for nine weak base and two neutral ozonides indicated
that the former were more metabolically stable than the latter.
The most metabolically stable compound was carboxylic acid
6 with an ER of 0.28; in contrast, the corresponding hydro-
xamic acid 16 was relatively unstable (ER=0.88).
A subset of the most promising ozonides (28, 33, 37) along
with 42, the trans isomer of 1, and the neutral compound 32
(Table 5) were administered intravenously (iv) and orally (po)
to rats, and the pharmacokinetic data are shown in Table 6.
The data indicated that half-lives for the four active ozonides
were similar, and all were longer than that for dihydroarte-
misinin(DHA). Ozonide33, whichhadthe highest cure rate in
vivo, also had the highest oral bioavailability in rats being
approximately 2-fold higher than the other more active com-
pounds tested. In comparison, 42, the trans isomer of 1, which
had reduced efficacy when tested in vivo, had a very high
plasma clearance, short half-life, and low oral bioavailability.
The data in Table 4 focus more precisely on the SAR of the
amino amide substructure of 1. Removing (36) or adding (37)
a methylene between the amide functional group and the
cyclohexane ring had little effect on in vitro potency but did
influence in vivo efficacy; 37 was as effective whereas 36 was
more than 40-fold25 less effective than 1. Replacing the

484 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Dong et al.
Table 6. Pharmacokinetic Parametersa after Intravenous and Oral Administration to Rats
intravenous administration
oral administration
bioavailability (%)
compd
half-life (min)
vol of distribution (L/kg)
plasma (blood) clearance (mL/(min kg))
3
1
28
76
102
660
83
16
25
36
19
12
16
3.0
141 (62)
168 (56.3)
37d
26
37
5
32
33
157 (39.1)
134d
298d
72.0 (72)
78
32
37
42
DHAb,c
60
37
15
e
26
a Values represent the average of two to three determinations. b Dihydroartemisinin (DHA), the primary metabolite of artesunate (AS). c Data from
Dong et al.11 d Not determined. e Not dosed PO.
Ozonide 32 had a long half-life after IV administration due to
its large volume of distribution and moderate plasma clear-
ance; however, it exhibited very low oral bioavailability in
comparison to the more active ozonides, most likely as a result
of its very poor aqueous solubility. Inhibition assays29 with
the 3A4, 2C9, and 2D6 CYP450isoforms revealed that33, like
1, had no inhibitory effect on enzyme activities at concentra-
tions up to 50 μM.
in vitro potency of antimalarial ozonides (1,2,4-trioxolanes),
similar to what has previously been found31,32 for enantio-
meric synthetic 1,2,4-trioxanes. However data for 42, the trans
isomer of 1, indicated that it was more rapidly degraded in
aqueous solution26 and in the presence of Fe(II).27 The
pharmacokinetic properties of 42 also indicated that the
plasma clearance was approximately 2-fold higher than that
for 1 and its oral bioavailability was relatively low. The
preceding observations together with the twin findings that
1 alkylates heme33 but only weakly interacts with the putative
artemisinin target PfATP634 suggests that hemoglobin diges-
tion35 in the parasite food vacuole36-38 is the parasite bio-
chemistry13 that accounts for the antimalarial properties of
synthetic peroxides and probably semisynthetic artemisinins
as well. Sixth, we identified five new ozonides (23, 28, 33,
37, 40) with antimalarial efficacies and ADME profiles super-
ior or equal to that of 1. Ongoing studies will determine the
potential of these and other weak base ozonides as antimalar-
ial drug development candidates.
Toxicology
Preliminary toxicological experiments were performed in
male rats given daily oral doses of 100 and 300 mg/kg of 33 for
five days. On day 5, maximum plasma levels of approximately
1000 and 3000 ng/mL were measured for 33 at the 100 and
300 mg/(kg day) doses. Even at the high dose, this ozonide,
3
like 1 and AS, was minimally toxic with the liver, lymphatic
organs, and possibly the kidneys, as target organs. No signs of
neurotoxicity were seen. Findings tended to be reversible at
the end of a 1-week recovery period. The overall toxicity of 33
was quite similar to that of 1.9
Experimental Section
Summary
General. Melting points are uncorrected. Using CDCl3,
1
CD3OD, or DMSO-d6 as solvents, H and 13C NMR spectra
Data for these ozonides provide several insights for the
ongoing identification of more effective4,6-8 synthetic perox-
ide antimalarials. First, it is evident that in vitro potency was
not a reliable predictor of in vivo activity, and determination
of IC50’s against P. falciparum was insufficient to select
compounds for further metabolic and pharmacokinetic pro-
filing; rather, data from experiments in P. berghei infected
mice were essential for compound differentiation. Second,
acidic functional groups decrease antimalarial potency, con-
sistent with our previous observations.12 Third, the relatively
narrow IC50 range of the neutral and weak base ozonides
indicates that the cyclohexane substituent played only a minor
role in intrinsic potency against P. falciparum. This suggests
that once an active synthetic peroxide structural framework is
identified, there is considerable latitude in the range of per-
oxide heterocycles and functional groups that can be em-
ployed to improve both antimalarial activity and ADME
properties. One such peroxide heterocycle is the 1,2,4,5-tetra-
oxane analog of ozonide (1,2,4-trioxolane) 24; this 1,2,4,
5-tetraoxane has excellent antimalarial properties including
an IC50 of 2.1 ng/mL against the 3D7 strain of P. falciparum
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 and HPLC analysis confirmed that all target
compounds possessed purities g95%.
Starting Material Synthesis. Methyl 4-oxocyclohexyl ace-
tate39,40 and methyl 3-(4-oxocyclohexyl)propionate18 were obtained
in a modified two-step sequence18 by reduction (H2, 900 psi,
Rh-C, 100 ꢀC, 5 h, isopropanol) of the corresponding phenol
esters to the diastereomeric cyclohexanol esters (97-99%)
followed by Jones oxidation (83-89%). In each case, some
(ca. 5-8%) over-reduction occurred to form the correspon-
ding cyclohexyl esters. 1-Amino-1-(aminomethyl)cyclopropane
dihydrochloride was obtained from N-(diphenylmethylene)-
aminoacetonitrile in a three-step sequence according to the
method of Vergne et al.41 As indicated below, the remaining
starting materials were commercially available or were prepared
according to known procedures.
cis-Adamantane-2-spiro-30-80-[[[(20-amino-20-methylpropyl)-
amino]carbonyl]methyl]-10,20,40-trioxaspiro[4.5]decane p-Tosy-
late (1). To a solution of 2 (13.19 g, 30 mmol, 1 equiv) in CHCl3
(300 mL) was added rapidly a solution of 1,2-diamino-2-methyl-
propane (5.29 g, 60 mmol, 2 equiv) in CHCl3 (50 mL). The
resulting mixture was stirred at rt for 1 h before being quenched
with water (500 mL). After separation of the organic layer, the
aqueous layer was extracted with CHCl3 (2 ꢀ 100 mL). The
combined extracts were washed with water (3 ꢀ 500 mL) and
brine (300 mL), dried over MgSO4, and filtered. To the filtrate
was added a solution of p-toluenesulfonic acid monohydrate
and an ED50 of 3.2 mg/(kg day) against P. berghei in the
3
4-day Peters test.30 Fourth, although weak base functional
groups were not required for high antimalarial potency
against P. falciparum in vitro, they were essential for high
antimalarial efficacy in P. berghei-infected mice; this may be a
function of the superior ADME properties of weak base vs
neutral ozonides. Fifth, stereochemistry had little effect on the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 485
(5.71 g, 30 mmol, 1 equiv) in EtOH (30 mL). After evaporation
of the solvents, the residue was treated with EtOH (100 mL),
filtered, and washed with hexanes (200 mL) to afford 1 (14.25 g,
84%) as a colorless solid. Mp 160-162 ꢀC; 1H NMR (DMSO-
d6) δ 1.01-1.17 (m, 2H), 1.16 (s, 6H), 1.58-1.99 (m, 21H), 2.05
(d, J=7.1 Hz, 2H), 2.28 (s, 3H), 3.19 (d, J=6.3 Hz, 2H), 7.11 (d,
J=8.0 Hz, 2H), 7.48 (d, J=8.0 Hz, 2H), 7.70 (s, 3H), 8.02 (t, J=
6.2 Hz, 1H); 13C NMR (DMSO-d6) δ 20.92, 23.47, 25.96, 26.36,
29.68, 32.71, 33.56, 34.40, 35.91, 36.23, 41.97, 46.02, 54.52,
108.52, 110.63, 125.55, 125.73, 128.20, 137.80, 145.82, 172.49.
Anal. (C29H44N2O7S) C, H, N.
cis-Adamantane-2-spiro-30-80-[[(10H-benzotriazol-10-yloxy)-
carbonyl]methyl]-10,20,40-trioxaspiro[4.5]decane (2). A solution
of 6 (12.92 g, 40 mmol, 1 equiv), HOBt (6.49 g, 48 mmol, 1.2
equiv), and EDCI (9.20 g, 48 mmol, 1.2 equiv) in DMF (300 mL)
under Ar was stirred at rt for 24 h. Under ice cooling, the
reaction was quenched with water (150 mL). The precipitate was
collected by filtration, washed with 95% EtOH (150 mL), and
dried to afford 2 (16.61 g, 95%) as a colorless solid. Mp 154-
156 ꢀC; 1H NMR (CDCl3) δ 1.37-1.51 (m, 2H), 1.63-2.17 (m,
21H), 2.72 (d, J=7.1 Hz, 2H), 7.39 (d, J=78.5 Hz, 1H), 7.43 (dd,
J=8.2, 7.2 Hz, 1H), 7.56 (dd, J=8.0, 7.4 Hz, 1H), 8.07 (d, J=8.5
Hz, 1H).
recrystallization from EtOH to afford 16 (95 mg, 28%) as a
colorless solid. Mp 138-140 ꢀC (EtOH); 1H NMR (DMSO-d6)
δ 0.81-1.27 (m, 3H), 1.40-2.19 (m, 22H), 8.65 (s, 1H), 10.33 (s,
1H); 13C NMR (DMSO-d6) δ 25.84, 26.25, 29.40, 32.60, 33.35,
34.26, 35.81, 36.13, 38.68, 108.33, 110.44, 167.91. Anal.
(C18H27NO5) C, H, N.
cis-Adamantane-2-spiro-30-80-(20-oxo-20-guanidinoethyl)-10,20,
40-trioxaspiro[4.5]decane (17). A solution of potassium tert-but-
oxide (0.56 g, 5.0 mmol) and guanidine hydrochloride (0.48 g,
5.0 mmol) in dioxane (20 mL) was heated under Ar at 50 ꢀC for
20 min. After the mixture was cooled to rt, a solution of 2 (0.46 g,
1.05 mmol) in CHCl3 (20 mL) was added dropwise. The mixture
was stirred at rt for 4 h and then concentrated. After addition of
water (30 mL), the resulting precipitate was collected, washed
with water, and dried to give 17 (0.36 g, 94%) as a colorless solid.
Mp 146-147 ꢀC; 1H NMR (CDCl3) δ 1.19-1.35 (m, 2H),
1.61-2.07 (m, 21H), 2.21 (d, J = 7.3 Hz, 2H); 13C NMR
(CDCl3) δ 26.49, 26.89, 30.13, 33.71, 34.16, 34.80, 36.39,
36.82, 47.35, 108.87, 111.21, 161.10. Anal. (C19H29N3O4) C,
H, N.
cis-Adamantane-2-spiro-30-80-[[[(20-amino-20-oxoethyl)amino]-
carbonyl]methyl]-10,20,40-trioxaspiro[4.5]decane (18). To a solu-
tion of glycinamide hydrochloride (221 mg, 2.0 mmol), triethy-
lamine (304 mg, 3 mmol), EtOH (5 mL), and water (1 mL) in
CHCl3 (10 mL) was added 2 (440 mg, 1.0 mmol). The resulting
mixture was stirred at rt for 17 h before being diluted with
CHCl3 (20 mL) and water (40 mL). After separation of the
organic layer, the aqueous layer was extracted with CHCl3 (3 ꢀ
10 mL). The combined extracts were washed with water (2 ꢀ
20 mL) and brine (2 ꢀ 20 mL), dried over MgSO4, filtered, and
concentrated. The residue was crystallized from 30% aq EtOH
to afford 18 (284 mg, 75%) as a colorless solid. Mp 130-132 ꢀC;
1H NMR (CDCl3) δ 1.11-1.38 (m, 2H), 1.42-2.07 (m, 21H),
2.15 (d, J=5.4 Hz, 2H), 3.95 (s, 2H), 5.84 (br s, 1H), 6.52 (br s,
1H), 6.73 (br s, 1H); 13C NMR (CDCl3) δ 26.45, 26.84, 29.94,
33.47, 33.88, 34.75, 36.34, 36.77, 42.77, 42.88, 108.50, 111.33,
171.40, 172.88. Anal. (C20H30N2O5) C, H, N.
cis-Adamantane-2-spiro-30-80-[[[(20-aminoethyl)amino]carbonyl]-
methyl]-10,20,40-trioxaspiro[4.5]decane p-Tosylate (19). To a so-
lution of 2 (440 mg, 1.0 mmol) in CHCl3 (15 mL) was added
rapidly a solution of ethylenediamine (601 mg, 10 mmol) in
EtOH (5 mL). The resulting mixture was stirred at rt for 2 h
before being 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 (3 ꢀ
20 mL) and brine (20 mL), dried over MgSO4, filtered, and
concentrated. The residue was dissolved in CH2Cl2 (20 mL) and
treated with a solution of p-toluenesulfonic acid monohydrate
(191 mg, 1.0 mmol) in EtOH (2 mL). After evaporation of the
solvents, the residue was treated with ether (20 mL), filtered, and
washed with ether (20 mL) to afford 19 (448 mg, 83%) as a
colorless solid. Mp 140-142 ꢀC; 1H NMR (CDCl3) δ 0.91-1.11
(m, 2H), 1.59-2.07 (m, 23H), 2.37 (s, 3H), 2.99-3.05 (m, 2H),
3.39-3.44 (m, 2H), 7.16 (d, J=7.8 Hz, 2H), 7.47 (s, 1H), 7.69 (d,
J=7.8 Hz, 2H), 7.73 (s, 3H); 13C NMR (CDCl3) δ 21.33, 26.48,
26.86, 29.73, 33.08, 33.84, 34.77, 36.37, 36.80, 36.98, 40.16,
42.46, 108.44, 111.15, 111.31, 125.73, 129.32, 140.71, 141.33,
173.94. Anal. (C27H40N2O7S) C, H, N.
cis-Adamantane-2-spiro-30-80-methoxycarbonylmethyl-10,20,
40-trioxaspiro[4.5]decane (5). A solution of O-methyl 2-adaman-
tanone oxime (3)17 (11.06 g, 61.7 mmol, 1.5 equiv) and methyl
4-oxocyclohexyl acetate (4)39,40 (7.0 g, 41.1 mmol, 1 equiv) in
cyclohexane (200 mL) and CH2Cl2 (40 mL) was treated with
ozone according to the method of Dong et al.12 After removal of
solvents, the crude product was purified by crystallization from
80% aq EtOH (200 mL) to afford 5 (10.83 g, 78%) as a colorless
1
solid. Mp 96-98 ꢀC; H NMR (CDCl3) δ 1.20-1.33 (m, 2H),
1.61-2.09 (m, 21H), 2.22 (d, J=6.8 Hz, 2H), 3.67 (s, 3H).
cis-Adamantane-2-spiro-30-80-carboxymethyl-10,20,40-trioxas-
piro[4.5]decane (6). To a solution of 5 (10.83 g, 32.19 mmol, 1
equiv) in 95% EtOH (150 mL) was added NaOH (3.86 g, 96.57
mmol, 3 equiv) solution in water (80 mL). The mixture was
stirred at 50 ꢀC for 4 h, cooled to 0 ꢀC, and treated with 1 M HCl
(129 mL, 4 equiv). The precipitate was collected by filtration,
washed with 50% aq EtOH (150 mL), and dried in a vacuum
oven at 40 ꢀC to give 6 (9.952 g, 96%) as a colorless solid. Mp
146-148 ꢀC (95% EtOH); 1H NMR (CDCl3) δ 1.19-1.41 (m,
2H), 1.60-2.05 (m, 21H), 2.27 (d, J=6.8 Hz, 2H); 13C NMR
(CDCl3) δ 26.58, 26.97, 29.91, 33.00, 33.95, 34.86, 36.49, 36.89,
40.39, 108.38, 111.40, 177.75. Anal. (C18H26O5) C, H.
cis-Adamantane-2-spiro-30-80-(20-amino-20-oxoethyl)-10,20,
40-trioxaspiro[4.5]decane (15). To a solution of 6 (322 mg, 1
mmol) in CH2Cl2 (10 mL) at 0 ꢀC was added triethylamine (202
mg, 2 mmol) followed by ethyl chloroformate (217 mg, 2 mmol).
After 15 min, ammonia (7 N in MeOH, 3 mL) was added, and
the stirring was continued for 12 h. The precipitate was filtered
and dried to afford 15 (210 mg, 65%) as a colorless solid. Mp
140-142 ꢀC; 1H NMR (CDCl3) δ 1.09-1.43 (m, 3H), 1.45-2.15
(m, 20H), 2.11 (d, J=7.1 Hz, 2H), 5.48 (s, 1H), 5.66 (s, 1H); 13
C
NMR (CDCl3) δ 26.47, 26.85, 29.97, 33.32, 33.95, 34.77,
36.38, 36.78, 42.55, 108.50, 111.35, 174.39. Anal. (C18H27NO4)
C, H, N.
cis-Adamantane-2-spiro-30-80-(20-hydroxyamino-20-oxoethyl)-
10,20,40-trioxaspiro[4.5]decane (16). To a solution of 6 (322 mg,
1.0 mmol) in ether (10 mL) at 0 ꢀC were added triethylamine (202
mg, 2 mmol) and ethyl chloroformate (217 mg, 2 mmol). The
mixture was stirred at 0 ꢀC for 15 min before a freshly prepared
solution of hydroxylamine was added. The latter was prepared
as follows: a suspension of hydroxylamine hydrochloride (170
mg, 2.48 mmol) and sodium bicarbonate (203 mg, 2.48 mmol) in
MeOH (5 mL) was stirred at rt for 15 min and then filtered. The
resulting mixture was stirred at rt for 12 h, diluted with ether
(10 mL), washed with water (10 mL), dried over MgSO4,
and concentrated. The crude product was purified by flash
chromatography (sg, 5% MeOH in CH2Cl2) and by subsequent
cis-Adamantane-2-spiro-30-80-[[[[(10-aminocyclopropyl)methyl]-
amino]carbonyl]methyl]-10,20,40-trioxaspiro[4.5]decane p-Tosylate
(20). To a solution of 1-amino-1-(aminomethyl)cyclopropane
dihydrochloride41 (0.162 g, 1.02 mmol) in EtOH (5 mL) and
CHCl3 (10 mL) was added triethylamine (0.5 mL) followed by a
solution of 2 (0.22 g, 0.50 mmol) in CHCl3 (15 mL). The resulting
mixture was stirred at rt for 1 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 (3 ꢀ 20 mL) and brine (20 mL), dried over
MgSO4, and filtered. To the filtrate was added a solution of
p-toluenesulfonic acid monohydrate (0.10 g, 0.53 mmol) in EtOH

486 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Dong et al.
(10 mL). After evaporation of the solvents, the residue was
purified by crystallization from EtOH to afford 20 (0.17 g,
60%) as a colorless solid. Mp 175-176 ꢀC; 1H NMR (DMSO-
d6) δ 0.72-0.77 (m, 2H), 0.78-0.85 (m, 2H), 1.02-1.17 (m, 2H),
1.56-1.94 (m, 21H), 2.02 (d, J=6.9 Hz, 2H), 2.28 (s, 3H), 3.30 (d,
J=6.9 Hz, 2H), 7.11 (d, J=7.8 Hz, 2H), 7.47 (d, J=7.8 Hz, 2H),
8.03-8.07 (m, 4H); 13C NMR (DMSO-d6) δ 10.66, 21.22, 26.23,
26.40, 26.63, 29.90, 33.15, 33.88, 34.69, 34.84, 36.20, 36.49, 42.35,
44.95, 108.88, 111.02, 125.94, 128.64, 138.52, 145.52, 172.71.
Anal. (C29H42N2O7S) C, H, N.
cis-Adamantane-2-spiro-30-80-[[[(30-aminopropyl)amino]carbo-
nyl]methyl]-10,20,40-trioxaspiro[4.5]decane p-Tosylate (21). To a
solution of 1,3-propanediamine (0.50 g, 6.74 mmol) in CHCl3
(10 mL) was added dropwise the solution of 2 (0.4 g, 0.91 mmol)
in CHCl3 (20 mL). The resulting mixture was stirred at rt for 1 h
and then quenched with water (30 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. The residue was dissolved in CHCl3 (20 mL), and
then a solution of p-toluenesulfonic acid monohydrate (0.18 g,
0.95 mmol) in EtOH (10 mL) was added. After evaporation of
the solvent, the crude product was purified by crystallization
from EtOH to afford 21 (0.38 g, 76%) as a colorless solid. Mp
154-156 ꢀC; 1H NMR (DMSO-d6) δ 1.01-1.18 (m, 2H),
1.54-1.94 (m, 23H), 1.97 (d, J = 6.8 Hz, 2H), 2.28 (s, 3H),
2.69-2.81 (m, 2H), 3.08 (td, J=6.8, 5.9 Hz, 2H), 7.11 (d, J=7.8
Hz, 2H), 7.47 (d, J=8.3 Hz, 2H), 7.63 (s, 3H), 7.95 (t, J=5.6 Hz,
1H); 13C NMR (DMSO-d6) δ 20.95, 25.98, 26.38, 27.70, 29.65,
32.94, 33.59, 34.42, 35.61, 35.94, 36.26, 37.05, 42.06, 108.56,
110.67, 125.67, 128.23, 137.80, 145.88, 171.82. Anal.
(C28H42N2O7S) C, H, N.
2.84-3.06 (m, 1H), 3.38-3.52 (m, 1H), 7.12 (d, J = 7.8 Hz,
2H), 7.48 (d, J=7.8 Hz, 2H), 7.71 (s, 1H), 7.73 (s, 3H); 13C NMR
(DMSO-d6) δ 20.95, 25.97, 26.37, 29.26, 29.57, 30.16, 33.03,
33.58, 34.42, 35.92, 36.25, 42.10, 46.60, 48.75, 108.57, 110.63,
125.65, 128.25, 137.87, 145.76, 170.48. Anal. (C31H46N2O7S) C,
H, N.
cis-Adamantane-2-spiro-30-80-[20-(40-morpholinyl)-20-oxoethyl]-
10,20,40-trioxaspiro[4.5]decane (24). To a solution of 6 (322 mg,
1.0 mmol) in ether (10 mL) at 0 ꢀC were added triethylamine (202
mg, 2 mmol) and ethyl chloroformate (217 mg, 2 mmol). The
mixture was stirred at 0 ꢀC for 15 min before morpholine (175
mg, 2 mmol) was added. The resulting mixture was stirred at rt
for 12 h, diluted with ether (10 mL), washed with water (10 mL),
dried over MgSO4, and concentrated. The crude product was
purified by crystallization from EtOH to afford 24 (290 mg,
74%) as a colorless solid. Mp 118-120 ꢀC (EtOH); 1 H NMR
(CDCl3) δ 1.16-1.35 (m, 2H), 1.60-2.16 (m, 21H), 2.21 (d, J=
6.9 Hz, 2H), 3.38-3.54 (m, 2H), 3.55-3.82 (m, 6H); 13C NMR
(125.7 MHz, CDCl3) δ 26.48, 26.86, 30.28, 33.26, 34.05, 34.79,
36.39, 36.79, 39.01, 41.93, 46.18, 66.66, 66.97, 108.58, 111.35,
170.67. Anal. (C22H33NO5) C, H, N.
cis-Adamantane-2-spiro-30-80-[[(10,10-dioxido-40-thiomorpholinyl)-
carbonyl]methyl]-10,20,40-trioxaspiro[4.5]decane (25). Step 1. To
a solution of 2 (0.5 g, 1.14 mmol) in CHCl3 (15 mL) was added
dropwise a solution of thiomorpholine (0.15 g, 1.45 mmol) in
CHCl3 (10 mL). The resulting mixture was stirred at rt for 1.5 h
and then quenched with water (30 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, and filtered. After
removal of the solvent, the crude product was purified by
crystallization from ether to afford the thioether intermediate
1
cis-Adamantane-2-spiro-30-80-[[[(30-amino-20,20-dimethylpropyl)-
amino]carbonyl]methyl]-10,20,40-trioxaspiro[4.5]decane Mesylate
(22). To a solution of 2,2-dimethyl-1,3-propanediamine (0.35 g,
3.43 mmol) in CHCl3 (10 mL) was added dropwise a solution of
2 (0.5 g, 1.14 mmol) in CHCl3 (20 mL). The resulting mixture
was stirred at rt for 1 h and then quenched with water (30 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 crude product
(0.41 g, 89%) as a white solid. After being washed with ether, the
crude product (0.25 g) was dissolved in CHCl3 (20 mL), and then
a solution of methanesulfonic acid (60 mg, 0.62 mmol) in CHCl3
(10 mL) was added. After evaporation of the solvent, the crude
product was purified by crystallization from EtOH to afford 22
(0.45 g, 97%) as a colorless solid. Mp 126-127 ꢀC; H NMR
(CDCl3) δ 1.15-1.31 (m, 2H), 1.59-2.05 (m, 21H), 2.20 (d, J=
6.8 Hz, 2H), 2.57-2.63 (m, 4H), 3.69-3.78 (m, 2H), 3.85-3.92
(m, 2H); 13C NMR (125.7 MHz, CDCl3) δ 26.48, 26.86, 27.46,
27.94, 30.31, 33.21, 34.05, 34.79, 36.39, 36.79, 39.36, 44.29,
48.41, 108.59, 111.35, 170.40. Step 2. To a solution of the above
thioether intermediate (0.39 g, 0.96 mmol) in CH2Cl2 (10 mL) at
0 ꢀC was added dropwise a solution of m-CPBA (0.52 g, 2.1
mmol) in CH2Cl2 (15 mL). The resulting mixture was stirred at rt
for 24 h and then partitioned between CH2Cl2 (20 mL) and
saturated aq NaHCO3 (20 mL). The organic layer was washed
with water (20 mL) and brine (20 mL), dried over MgSO4, and
filtered. After removal of the solvent, the crude product was
purified by crystallization from CH2Cl2/EtOH to afford 25 (0.34
g, 81%) as a colorless solid. Mp 159-160 ꢀC; 1H NMR (CDCl3)
δ 1.15-1.33 (m, 2H), 1.59-2.03 (m, 21H), 2.26 (d, J=6.8 Hz,
2H), 3.02 (s, 4H), 3.96 (s, 2H), 4.11 (s, 2H); 13C NMR (CDCl3) δ
26.46, 26.85, 30.25, 33.07, 33.96, 34.78, 36.38, 36.77, 39.04,
40.22, 43.90, 52.14, 52.28, 108.35, 111.48, 170.54. Anal.
(C22H33NO6S) C, H, N.
1
(0.20 g, 35%) as a colorless solid. Mp 162-164 ꢀC; H NMR
(DMSO-d6) δ 0.88 (s, 6H), 1.02-1.18 (m, 2H), 1.54-1.95 (m,
21H), 2.07 (d, J=6.8 Hz, 2H), 2.29 (s, 3H), 2.51 (s, 2H), 2.95 (d,
J=6.4 Hz, 2H), 7.64 (s, 3H), 8.20 (t, J=6.3 Hz, 1H); 13C NMR
(DMSO-d6) δ 23.22, 25.96, 26.36, 29.62, 32.87, 33.53, 34.41,
34.83, 35.92, 36.24, 41.78, 45.53, 46.01, 108.50, 110.69, 173.23.
Anal. (C24H42N2O7S) C, H, N.
cis-Adamantane-2-spiro-30-80-[[(40-formyl-10-piperazinyl)carbo-
nyl]methyl]-10,20,40-trioxaspiro[4.5]decane (26). To a solution of
2 (440 mg, 1.0 mmol) in CHCl3 (20 mL) was added a solution of
1-piperazinecarboxaldehyde (137 mg, 1.2 mmol) in CHCl3
(1 mL). The resulting mixture was stirred at rt for 2 h before
removal of the solvent. The residue was crystallized from 1:2
EtOH/water to afford 26 (242 mg, 58%, 2:1 mixture of
rotamers) as a colorless solid. Mp 142-144 ꢀC; 1H NMR
(CDCl3) δ 1.15-1.32 (m, 2H), 1.45-2.09 (m, 21H), 2.20-2.28
(m, 2H), 3.32-3.78 (m, 8H), 8.10 (s, 1H); 13C NMR (CDCl3) δ
26.46, 26.84, 30.27, 33.18, 34.01, 34.77, 36.38, 36.77, 39.26,
39.99, 40.22, 41.02, 42.15, 45.15, 45.33, 45.70, 46.25, 108.49,
111.39, 160.74, 160.92, 170.55, 170.72. Anal. (C23H34N2O5) C,
H, N.
cis-Adamantane-2-spiro-30-80-[[[(trans-40-aminocyclohexyl)-
amino]carbonyl]methyl]-10,20,40-trioxaspiro[4.5]decane p-Tosy-
late (23). To a solution of 2 (220 mg, 0.5 mmol) in CHCl3
(10 mL) was added rapidly a solution of trans-1,4-diaminocy-
clohexane (343 mg, 3.0 mmol) in CHCl3 (10 mL). The resulting
mixture was stirred at rt for 1 h and filtered. The filtrate was
diluted with water (30 mL). After separation of the organic
layer, the aqueous layer was extracted with CHCl3 (2 ꢀ 10 mL).
The combined extracts were washed with water (2 ꢀ 20 mL) and
brine (20 mL), dried over MgSO4, and filtered. To the filtrate
was added a solution of p-toluenesulfonic acid monohydrate
(50 mg, 0.26 mmol) in EtOH (1 mL). After evaporation of the
solvents, the residue was treated with ether (20 mL), filtered, and
washed with ether (20 mL) to afford 23 (139 mg, 47%) as a
colorless solid. Mp 140 ꢀC dec; 1H NMR (DMSO-d6) δ
0.97-1.43 (m, 6H), 1.54-2.02 (m, 27H), 2.29 (s, 3H),
cis-Adamantane-2-spiro-30-80-[[[40-(methylsulfonyl)-10-pipera-
zinyl]carbonyl]methyl]-10,20,40-trioxaspiro[4.5]decane (27). To a
solution of the free base of 28 (0.43 g, 1.10 mmol) in CH2Cl2
(10mL) at 0 ꢀC under Ar was added pyridine (0.3 mL, 3.79 mmol),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 487
followed by methanesulfonyl chloride (0.12 mL, 1.57 mmol).
The resulting mixture was stirred at 0 ꢀC for 1 h and then
quenched with saturated aq K2CO3 (5 mL). After separation of
the organic layer, the aqueous layer was extracted with CH2Cl2
(3 ꢀ 10 mL). The combined extracts were washed successively
with water (10 mL), 1 M aq HCl (5 mL), and brine (10 mL), dried
over MgSO4, and filtered. The filtrate was concentrated to
about 3 mL and diluted with ether (20 mL). The precipitate
was collected and washed with hexanes (10 mL) to afford 27
36.38, 36.78, 38.31, 39.34, 39.48, 40.83, 41.34, 42.40, 46.06,
49.01, 108.46, 108.50, 111.35, 111.43, 166.60, 167.91, 170.37,
170.62. Anal. (C22H32N2O5) C, H, N.
cis-Adamantane-2-spiro-30-80-[(10-
piperidinylcarbonyl)methyl]-10,20,40-trioxaspiro[4.5]decane (31).
To a solution of 6 (322 mg, 1.0 mmol) in CH2Cl2 (10 mL) at
0 ꢀC were added triethylamine (202 mg, 2 mmol) and ethyl
chloroformate (217 mg, 2 mmol). The mixture was stirred at 0 ꢀC
for 15 min before piperidine (100 mg, 1.2 mmol) was added. The
resulting mixture was stirred at rt for 12 h, concentrated, and
triturated with water. The crude product was purified by crystal-
lization from EtOH to afford 31 (0.24 g, 62%) as a colorless
solid. Mp 98-100 ꢀC (EtOH); 1H NMR (CDCl3) δ 1.15-1.39
(m, 2H), 1.43-2.17 (m, 27H), 2.21 (d, J=6.8 Hz, 2H), 3.30-3.49
(m, 2H), 3.50-3.65 (m, 2H); 13C NMR (CDCl3) δ 24.57, 25.66,
26.49, 26.60, 26.87, 30.31, 33.43, 34.11, 34.79, 36.39, 36.81,
39.26, 42.69, 46.87, 108.72, 111.29, 170.28. Anal. (C23H35NO4)
C, H, N.
cis-Adamantane-2-spiro-30-80-[[(40-hydroxy-10-piperidinyl)-
carbonyl]methyl]-10,20,40-trioxaspiro[4.5]decane (32). To a solu-
tion of 2 (440 mg, 1.0 mmol) in CHCl3 (20 mL) was added 4-
hydroxypiperidine (121 mg, 1.2 mmol). The resulting mixture
was stirred at rt for 1 h before being 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 (3 ꢀ 30 mL) and brine (20 mL), dried
over MgSO4, filtered, and concentrated. The residue was crys-
tallized from 10:1 ether/CHCl3 to afford 32 (152 mg, 37%) as a
colorless solid. Mp 154-156 ꢀC; 1H NMR (CDCl3) δ 1.15-1.35
(m, 2H), 1.41-1.59 (m, 2H), 1.60-2.09 (m, 23H), 2.23 (d, J=7.1
Hz, 2H), 3.09-3.31 (m, 2H), 3.68-3.82 (m, 1H), 3.87-4.03 (m,
1H), 4.05-4.21 (m, 1H); 13C NMR (CDCl3) δ 26.49, 26.86,
30.28, 33.39, 34.01, 34.07, 34.66, 34.78, 36.39, 36.80, 38.92,
39.22, 42.97, 67.20, 108.64, 111.32, 170.39. Anal. (C23H35NO5)
C, H, N.
1
(0.45 g, 87%) as a colorless solid. Mp 143-145 ꢀC; H NMR
(CDCl3) δ 1.15-1.33 (m, 2H), 1.61-2.07 (m, 21H), 2.23 (d, J=
6.8 Hz, 2H), 2.80 (s, 3H), 3.16-3.28 (m, 4H), 3.55-3.63 (m, 2H),
3.70-3.79 (m, 2H); 13C NMR (CDCl3) δ 26.46, 26.84, 30.26,
33.15, 34.01, 34.77, 34.85, 36.38, 36.76, 39.21, 41.14, 45.34,
45.69, 45.93, 108.48, 111.40, 170.53. Anal. (C23H36N2O6S)
C, H, N.
cis-Adamantane-2-spiro-30-80-[(10-piperazinylcarbonyl)methyl]-
10,20,40-trioxaspiro[4.5]decane p-Tosylate (28). To a solution of 2
(13.19 g, 30 mmol, 1 equiv) in CHCl3 (300 mL) was added
rapidly a solution of piperazine (12.92 g, 150 mmol, 5 equiv) in
CHCl3 (50 mL). The resulting mixture was stirred at rt for 1.5 h
before being quenched with water (500 mL). After separation of
the organic layer, the aqueous layer was extracted with CHCl3
(2 ꢀ 100 mL). The combined extracts were washed with water
(3 ꢀ 500 mL) and brine (300 mL), dried over MgSO4, and
filtered. To the filtrate was added a solution of p-toluenesulfonic
acid monohydrate (5.71 g, 30 mmol, 1 equiv) in EtOH (30 mL).
After evaporation of the solvents, the residue was dissolved in
CHCl3 (70 mL), and the product was precipitated by adding
isopropanol (420 mL), filtered, and washed with 6:1 isopropa-
nol/CHCl3 (210 mL) and hexanes (300 mL). The solid was
redissolved in CHCl3 (60 mL), precipitated by adding hexanes
(600 mL), filtered, and washed with hexanes (200 mL) to afford
28 (12.58 g, 75%) as a colorless solid. Mp 148-150 ꢀC; 1H NMR
(CDCl3) δ 1.03-1.29 (m, 2H), 1.49-2.05 (m, 21H), 2.15 (d, J=
6.3 Hz, 2H), 2.39 (s, 3H), 3.18 (s, 2H), 3.25 (s, 2H), 3.71 (s, 2H),
3.83 (s, 2H), 7.22 (d, J=7.7 Hz, 2H), 7.71 (d, J=7.4 Hz, 2H), 9.25
(s, 2H); 13C NMR (CDCl3) δ 21.35, 26.47, 26.85, 30.19, 33.00,
33.96, 34.77, 36.38, 36.78, 38.19, 38.90, 42.36, 43.68, 43.78,
108.41, 111.37, 125.66, 129.22, 140.94, 141.11, 170.45. Anal.
(C29H42N2O7S) C, H, N.
cis-Adamantane-2-spiro-30-80-[[[40-(20-hydroxyethyl)-10-pipera-
zinyl]carbonyl]methyl]-10,20,40-trioxaspiro[4.5]decane (29). To a
solution of N-(2-hydroxyethyl)piperazine (325 mg, 2.5 mmol) in
CHCl3 (10 mL) was added 2 (440 mg, 1.0 mmol) The resulting
mixture was stirred at rt for 2 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. Crystallization of the
residue from EtOAc gave 29 (281 mg, 65%) as a colorless solid.
Mp 143-145 ꢀC; 1H NMR (CDCl3) δ 1.26-1.31 (m, 2H),
1.62-2.04 (m, 21H), 2.21 (d, J=6.3 Hz, 2H), 2.44-2.54 (m,
4H), 2.56 (t, J=4.4 Hz, 2H), 2.62 (brs, 1H), 3.49 (t, J=4.0 Hz,
2H), 3.64 (t, J=4.4 Hz, 4H); 13C NMR (CDCl3) δ 26.43, 26.81,
30.25, 33.29, 34.02, 34.74, 36.34, 36.74, 39.10, 41.54, 45.71,
52.62, 53.15, 57.76, 59.25, 108.56, 111.28, 170.41. Anal.
(C24H38N2O5) C, H, N.
cis-Adamantane-2-spiro-30-80-[[(40-amino-10-piperidinyl)carbo-
nyl]methyl]-10,20,40-trioxaspiro[4.5]decane p-Tosylate (33). Step
1. To a solution of 2 (880 mg, 2 mmol) in CHCl3 (40 mL) was
added 4-(tert-butoxycarbonylamino)piperidine (481 mg, 2.4
mmol). The resulting mixture was stirred at rt for 1 h before
solvent removal. The residue was crystallized from 50% aq
EtOH (80 mL) to give the BOC ozonide (995 mg, 99%). Mp
146-148 ꢀC; 1H NMR (DMSO-d6) δ 1.02-1.29 (m, 4H), 1.38 (s,
9H), 1.59-1.94 (m, 23H), 2.19 (d, J=6.8 Hz, 2H), 2.63 (dd, J=
11.8, 11.8 Hz, 1H), 3.02 (dd, J=12.2, 12.2 Hz, 1H), 3.39-3.51
(m, 1H), 3.80 (d, J=13.7 Hz, 1H), 4.23 (d, J=13.2 Hz, 1H), 6.84
(d, J=7.8 Hz, 1H). Step 2. A mixture of the above BOC ozonide
(505 mg, 1.0 mmol) and p-toluenesulfonic acid monohydrate
(951 mg, 5.0 mmol) in EtOAc/isopropanol (9:1, 50 mL) was
stirred at rt for 48 h. The precipitate was filtered, washed with
EtOAc (20 mL), dissolved in 20% aq EtOH (90 mL), and
basified with 14% aq KOH (10 mL). The solid was filtered,
dissolved in CHCl3, dried over MgSO4, and concentrated. To a
solution of the above free base (170 mg) in EtOAc (10 mL) was
added a solution of p-toluenesulfonic acid monohydrate (80 mg,
0.42 mmol) in EtOAc (10 mL). The precipitate was collected by
filtration, washed with EtOAc (10 mL), and dried to give 33 (180
mg, 31%) as a colorless solid. Mp 154-156 ꢀC; 1H NMR
cis-Adamantane-2-spiro-30-80-[[(30-oxo-10-piperazinyl)carbo-
nyl]methyl]-10,20,40-trioxaspiro[4.5]decane (30). To a solution of
2 (440 mg, 1.0 mmol) in CHCl3 (20 mL) was added piperazin-
2-one (120 mg, 1.2 mmol). The resulting mixture was stirred at rt
for 3 h before removal of the solvent. The residue was crystal-
lized from 1:1 EtOH/water to afford 30 (207 mg, 51%, 3:2
(DMSO-d6) δ 1.05-1.18 (m, 2H), 1.20-1.29 (m, 1H),
1.30-1.42 (m, 1H), 1.59-1.97 (m, 23H), 2.22 (d, J=5.4 Hz,
2H), 2.29 (s, 3H), 2.57 (dd, J=11.8, 11.8 Hz, 1H), 3.02 (dd, J=
12.2, 12.2 Hz, 1H), 3.24 (br s, 1H), 3.92 (d, J=13.7 Hz, 1H), 4.39
(d, J=13.2 Hz, 1H), 7.12 (d, J=7.8 Hz, 2H), 7.48 (d, J=7.8 Hz,
2H), 7.82 (s, 3H); 13C NMR (DMSO-d6) δ 20.94, 25.97, 26.37,
29.61, 29.77, 30.48, 32.73, 33.67, 34.41, 35.93, 36.25, 38.37,
43.32, 47.66, 47.75, 108.58, 110.62, 125.64, 128.23, 137.81,
145.84, 169.78. Anal. (C30H44N2O7S) C, H, N.
1
mixture of rotamers) as a colorless solid. Mp 150-152 ꢀC; H
NMR (CDCl3) δ 1.17-1.34 (m, 2H), 1.57-2.09 (m, 21H), 2.21
(d, J=6.8 Hz, 1.2H), 2.24 (d, J=6.8 Hz, 0.8H), 3.39 (s, 1.2H),
3.42 (s, 0.8H), 3.67 (t, J=5.0 Hz, 0.8H), 3.82 (t, J=5.4 Hz, 1.2H),
cis-Adamantane-2-spiro-30-80-[[(40-carboxy-10-piperidinyl)-
carbonyl]methyl]-10,20,40-trioxaspiro[4.5]decane (34). To a solu-
tion of 2 (220 mg, 0.5 mmol), water (5 mL), and EtOH (10 mL) in
4.12 (s, 1.2H), 4.25 (s, 0.8H), 6.42 (s, 0.6H), 6.58 (s, 0.4H); 13
C
NMR (CDCl3) δ 26.47, 26.85, 30.20, 30.26, 32.98, 34.00, 34.78,

488 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Dong et al.
CHCl3 (10 mL) was added isonipecotic acid (129 mg, 1.0 mmol).
The resulting mixture was stirred at rt for 16.5 h before removal
of the solvents. The residue was crystallized from 1:1 EtOH/
water to afford 34 (179 mg, 82%) as a colorless solid. Mp
159-161 ꢀC; 1H NMR (CDCl3) δ 1.18-1.35 (m, 2H),
1.58-2.06 (m, 25H), 2.24 (d, J=6.8 Hz, 2H), 2.60 (tt, J=10.7,
3.9 Hz, 1H), 2.86 (t, J=11.2 Hz, 1H), 3.14 (t, J=11.2 Hz, 1H),
3.85 (d, J=13.7 Hz, 1H), 4.44 (d, J=13.6 Hz, 1H); 13C NMR
(CDCl3) δ 26.48, 26.86, 27.72, 28.33, 30.27, 33.37, 34.06, 34.79,
36.39, 36.80, 39.23, 40.46, 40.93, 44.96, 108.62, 111.34, 170.53,
178.19. Anal. (C24H35NO6) C, H, N.
cis-Adamantane-2-spiro-30-80-[[[40-(aminocarbonyl)-10-piperi-
dinyl]carbonyl]methyl]-10,20,40-trioxaspiro[4.5]decane (35). To a
solution of 2 (220 mg, 0.5 mmol) in CHCl3 (10 mL) and EtOH
(10 mL) was added isonipecotamide (128 mg, 1.0 mmol). The
resulting mixture was stirred at rt for 4 h before removal of the
solvents. The residue was crystallized from 2:1 EtOH/water to
afford 35 (164 mg, 76%) as a colorless solid. Mp 149-151 ꢀC; 1H
NMR (CDCl3) δ 1.16-1.32 (m, 2H), 1.58-2.06 (m, 25H), 2.22
(d, J=6.8 Hz, 2H), 2.39 (tt, J=11.2, 3.9 Hz, 1H), 2.69 (t, J=11.5
Hz, 1H), 3.06 (t, J=11.7 Hz, 1H), 3.92 (d, J=13.7 Hz, 1H), 4.60
(d, J=13.2 Hz, 1H), 5.54 (s, 2H); 13C NMR (CDCl3) δ 26.46,
26.84, 28.60, 29.02, 30.25, 30.30, 33.31, 34.05, 34.77, 36.37,
36.78, 39.24, 41.04, 42.37, 45.15, 108.61, 111.31, 170.39,
176.16. Anal. (C24H36N2O5) C, H, N.
cis-adamantane-2-spiro-30-80-(20-carboxyethyl)-10,20,40-trioxas-
piro[4.5]decane (13.2 g, 95%) as a colorless solid. Mp 144-
146 ꢀC; 1H NMR (CDCl3) δ 1.12-1.26 (m, 2H), 1.27-1.38 (m,
1H), 1.57 (dt, J=7.8, 7.1 Hz, 2H), 1.62-2.04 (m, 20H), 2.36 (t,
J=7.6 Hz, 2H), 11.58 (br s, 1H); 13C NMR (CDCl3) δ 26.47,
26.86, 29.72, 30.80, 31.71, 34.02, 34.76, 34.78, 35.44, 36.37,
36.79, 108.76, 111.25, 180.06. Step 3. A solution of the above
ozonide acid (0.5 g, 1.5 mmol), HOSu (0.21 g, 1.79 mmol), and
EDCI (0.34 g, 1.79 mmol) in DMF (20 mL) under Ar was stirred
at rt for 24 h before being quenched with water (25 mL) at 0 ꢀC.
The precipitate was filtered, washed with 95% aq EtOH
(10 mL), and dried to afford the active ester (0.64 g, 100%) as
a colorless solid. Mp 144-146 ꢀC; 1H NMR (500 MHz, CDCl3)
δ 1.16-1.29 (m, 2H), 1.35-1.44 (m, 1H), 1.63-2.02 (m, 22H),
2.62 (t, J=7.6 Hz, 2H), 2.83 (br s, 4H). Step 4. To a solution of
the ozonide active ester (0.5 g, 1.15 mmol) in CHCl3 (25 mL) was
added in one portion a solution of 1,2-diamino-2-methylpro-
pane (0.20 g, 2.3 mmol) in CHCl3 (15 mL). The reaction mixture
was stirred at rt for 1 h before being quenched with water
(50 mL). After separation of the organic layer, the aqueous
layer was extracted with CHCl3 (2 ꢀ 25 mL). The combined
extracts were washed with water (3 ꢀ 50 mL), dried over MgSO4,
and filtered. To the filtrate was added a solution of p-toluene-
sulfonic acid monohydrate (0.22 g, 1.16 mmol) in EtOH
(10 mL). After evaporation of the solvents, the residue was treated
with ether (15 mL). The solid was collected by filtration, washed
with ether (30 mL), and dried to afford 37 (0.49 g, 74%) as a
colorless solid. Mp 114-118 ꢀC; 1H NMR (CDCl3) δ 0.86-1.02
(m, 3H), 1.33 (s, 6H), 1.34-1.58 (m, 6H), 1.62-2.06 (m, 18H), 2.39
(s, 3H), 3.42 (d, J=5.9 Hz, 2H), 7.23 (d, J=7.8 Hz, 2H), 7.64 (br s,
1H), 7.67 (d, J=7.8 Hz, 2H), 7.78 (s, 3H); 13C NMR (CDCl3) δ
21.36, 23.85, 26.45, 26.85, 29.52, 31.53, 33.33, 33.98, 34.76, 35.58,
36.36, 36.77, 46.54, 56.17, 108.61, 111.11, 125.64, 129.32, 140.03,
cis-Adamantane-2-spiro-30-80-[[(20-amino-20-methylpropyl)-
amino]carbonyl]-10,20,40-trioxaspiro[4.5]decane p-Tosylate (36).
Step 1. A mixture of 712 (8.7 g, 28.3 mmol), HOBt (4.59 g,
34.0 mmol), and EDCI (6.52 g, 34.0 mmol) in DMF (70 mL) was
stirred at rt overnight before addition of water (70 mL). The
precipitate was collected by filtration and dried to afford the
ozonide active ester (11.0 g, 91%) as a colorless solid. Mp
153-154 ꢀC; 1H NMR (CDCl3) δ 1.64-2.16 (m, 20 H),
2.20-2.30 (m, 2H), 2.88-2.98 (m, 1H), 7.35 (d, J = 8.3 Hz,
1H), 7.43 (t, J=7.8 Hz, 1H), 7.55 (t, J=7.8 Hz, 1H), 8.07 (d, J=
8.3 Hz, 1H). Step 2. A mixture of the above active ester (425 mg,
1 mmol) and 1,2-diamino-2-methylpropane (229 mg, 2.5 mmol)
in CHCl3 (10 mL) was stirred at rt for 4 h. The reaction mixture
was washed with water (10 mL) and brine (10 mL) and dried
over MgSO4. After filtration, a solution of p-toluenesulfonic
acid monohydrate (190 mg, 1 mmol) in MeOH (2 mL) was
added. The resulting mixture was concentrated and triturated
with EtOAc to afford 36 (360 mg, 65%) as a colorless solid. Mp
141.70, 175.48. Anal. (C30H46N2O7S 1.2H2O) C, H, N.
3
cis-Adamantane-2-spiro-30-80-[[[(20-amino-20-methylpropyl)-
amino]carbonyl]amino]-10,20,40-trioxaspiro[4.5]decane p-Tosy-
late (38). Step 1. To a solution of the free base of 1015 (0.70 g,
2.51 mmol) in CH2Cl2 (100 mL) was added pyridine (0.3 mL)
followed by a solution of the 4-nitrophenyl chloroformate (0.6 g,
2.98 mmol) in CH2Cl2 (50 mL) at 0 ꢀC. The resulting mixture
was stirred at rt for 24 h, then diluted with CH2Cl2 (100 mL),
washed with 1 M aq NaHCO3 (2 ꢀ 100 mL), water (2 ꢀ 100 mL),
and brine (100 mL), dried over MgSO4, and filtered. After
removal of solvent, the crude product was purified by crystal-
lization from EtOH to afford the cis-adamantane-2-spiro-
30-80-[[(40-nitrophenoxy)carbonyl]amino]-10,20,40-trioxaspiro-
[4.5]decane (1.0 g, 90%). Step 2. To a solution of 1,2-diamino-
2-methylpropane (0.40 g, 4.64 mmol) in CHCl3 (10 mL) was
added dropwise a solution of the above 4-nitrophenyl carba-
mate (0.4 g, 0.90 mmol) in CHCl3 (10 mL) and MeOH (20 mL).
The resulting mixture was stirred at rt for 4 h and then quenched
with water (30 mL). After separation of the organic layer, the
aqueous layer was extracted with CHCl3 (2 ꢀ 30 mL). The
combined extracts were washed with 1 M aq NaHCO3 (2 ꢀ
20 mL), water (3 ꢀ 20 mL), and brine (20 mL), dried over
MgSO4, filtered, and concentrated. The residue was dissolved in
CHCl3 (20 mL) and then treated with a solution of p-toluene-
sulfonic acid monohydrate (0.17 g, 0.89 mmol) in EtOH
(10 mL). After evaporation of the solvent, the crude product
was purified by crystallization from 1:4 EtOH/CH2Cl2 to afford
38 (0.16 g, 31%) as a colorless solid. Mp 172-173 ꢀC; 1H NMR
(DMSO-d6) δ 1.13 (s, 6H), 1.27-1.39 (m, 2H), 1.59-1.93 (m,
20H), 2.28 (s, 3H), 3.10 (d, J=5.8 Hz, 2H), 3.44-3.54 (m, 1H),
6.01-6.11 (m, 2H), 7.10 (d, J=7.8 Hz, 2H), 7.47 (d, J=7.8 Hz,
2H), 7.65 (s, 3H); 13C NMR (DMSO-d6) δ 20.95, 23.31, 23.36,
25.95, 26.35, 30.04, 32.16, 34.41, 34.43, 35.87, 36.22, 46.37,
47.26, 54.51, 108.08, 110.87, 125.66, 128.20, 137.72, 145.97,
158.14. Anal. (C28H43N3O7S) C, H, N.
1
154-156 ꢀC; H NMR (CDCl3) δ 1.19-1.31 (m, 2H), 1.36 (s,
6H), 1.48-2.03 (m, 21H), 2.40 (s, 3H), 3.45 (d, J=6.3 Hz, 2H),
7.24 (d, J=8.3 Hz, 2H), 7.33 (d, J=6.6 Hz, 1H), 7.67 (d, J=8.3
Hz, 2H), 7.75 (s, 3H); 13C NMR (CDCl3) δ 21.33, 23.89, 26.43,
26.52, 26.85, 33.34, 34.74, 34.76, 36.36, 36.75, 43.04, 46.13,
56.44, 107.69, 111.15, 125.50, 129.39, 140.40, 141.67, 175.98.
Anal. (C28H42N2O7S) C, H, N.
cis-Adamantane-2-spiro-30-80-[20-[[(20-amino-20-methylpropyl)-
amino]carbonyl]ethyl]-10,20,40-trioxaspiro[4.5]decane p-Tosylate
(37). Step 1. A solution of 317 (22.35 g, 124.7 mmol) and methyl
3-(4-oxocyclohexyl)propionate18 (15.3 g, 83.1 mmol) in cyclo-
hexane (400 mL) and CH2Cl2 (80 mL) was treated with ozone
according to the method of Dong et al.12 After removal of
solvents, the crude product was purified by crystallization from
80% aq EtOH (150 mL) to afford adamantane-2-spiro-30-80-(20-
methoxycarbonylethyl)-10,20,40-trioxaspiro[4.5]decane (17.18 g,
59%) as a colorless solid. Mp 102-104 ꢀC; 1H NMR (CDCl3) δ
1.15-1.23 (m, 2H), 1.24-1.37 (m, 1H), 1.54-1.61 (m, 2H),
1.62-2.05 (m, 20H), 2.32 (t, J=7.8 Hz, 2H), 3.66 (s, 3H); 13
C
NMR (CDCl3) d 26.46, 26.86, 29.72, 31.10, 31.74, 34.04, 34.75,
34.77, 35.53, 36.36, 36.78, 51.49, 108.79, 111.20, 174.17. Step 2.
To a solution of the above ozonide ester (14.43 g, 41.2 mmol) in
95% aq EtOH (200 mL) was added NaOH (4.90 g, 123.7 mmol)
solution in water (100 mL). The mixture was stirred at 50 ꢀC for
4 h, cooled to 0 ꢀC, and treated with 1 M HCl (162 mL). The
precipitate was collected by filtration, washed with 50% aq
EtOH (150 mL), and dried in a vacuum oven at 40 ꢀC to give
cis-Adamantane-2-spiro-30-80-[[[[(20-amino-20-methylpropyl)-
amino]carbonyl]amino]methyl]-10,20,40-trioxaspiro[4.5]decane

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 489
p-Tosylate (39). A mixture of cis-adamantane-2-spiro-30-80-
[[[(40-nitrophenoxy)carbonyl]amino]methyl]-10,20,40-trioxaspiro-
[4.5]decane23 (0.32 g, 0.7 mmol) and 1,2-diamino-2-methylpro-
pane (154 mg, 3.5 mmol) in CHCl3 (10 mL) was stirred at rt
overnight. The mixture was then diluted with CHCl3 (10 mL),
washed with water and brine, dried over MgSO4, filtered, and
concentrated. The crude product was dissolved in CH2Cl2
(10 mL) and added to a solution of p-toluenesulfonic acid
monohydrate (100 mg, 0.53 mmol) in MeOH (1 mL). After
concentration, the solid was crystallized from 1:4 CH2Cl2/ether
to afford 39 (100 mg, 25%) as a colorless solid. Mp 184 ꢀC dec.;
1H NMR (CDCl3) δ 0.85-1.03 (m, 2H), 1.27 (s, 6H), 1.41-2.05
(m, 21H), 2.40 (s, 3H), 2.72 (d, J=5.9 Hz, 2H), 3.26 (d, J=7.3
Hz, 2H), 5.23-5.39 (m, 1H), 5.91-6.07 (m, 1H), 7.24 (d, J=8.3
Hz, 2H), 7.56 (s, 3H), 7.69 (d, J = 8.3 Hz, 2H); 13C NMR
(CDCl3) δ 21.37, 23.57, 26.48, 26.88, 27.56, 33.71, 34.79, 36.38,
36.57, 36.81, 45.34, 47.00, 56.55, 108.68, 111.16, 125.62, 129.42,
140.42, 141.69, 158.63. Anal. (C29H45N3O7S) C, H, N.
the above mixture of ozonide ester isomers (4.1 g, 12.20 mmol) in
95% EtOH (50 mL) was added a solution of NaOH (1.46 g,
36.60 mmol) in water (10 mL). The mixture was stirred at 50 ꢀC
for 4 h, cooled to 0 ꢀC, and neutralized with 1.0 M aq HCl. The
precipitate was collected by filtration, washed with 50% aq
EtOH (50 mL), and dried in vacuo at 40 ꢀC to give a 1:1 mixture
of cis (6) and trans ozonide acids (3.10 g, 77%) as a colorless
solid. Step3. A solution of the above ozonide acid isomers (3.10 g,
9.62 mmol), HOBt (1.56 g, 11.55 mmol), and EDCI (2.28 g,
11.55 mmol) in DMF (50 mL) was stirred at 0 ꢀC for 2 h before
4-nitrophenol (1.6 g, 11.55 mmol) was added. After being stirred
for an additional 2 h at 0 ꢀC, the reaction was stirred at rt
overnight. The reaction was quenched with ice-water (50 mL)
at 0 ꢀC and extracted with EtOAc (3 ꢀ 50 mL). The organic phase
was washed with water (3 ꢀ 100 mL) and brine and dried over
MgSO4. After removal of the solvent, the residue was purified by
chromatography followed by repeated crystallizations from
EtOH to give pure trans-4-nitrophenyl ozonide ester (0.210 g,
5%). 1H NMR (CDCl3) δ 1.51-2.05 (m, 23 H), 2.57 (d, J=6.84
Hz, 2H), 7.27 (d, J=9.3 Hz, 2H)), 8.27 (d, J=9.3 Hz, 2H). Step 4.
To a solution of trans-4-nitrophenyl ozonide ester (0.180 g, 0.42
mmol) in CHCl3 (5 mL) was rapidly added a solution of 1,2-
diamino-2-methylpropane (0.184 g, 2.10 mmol) in CHCl3 (5 mL).
The reaction mixture was stirred at rt overnight and then
quenched with water (15 mL). After separation of the organic
layer, the aqueous layer was extracted with CHCl3 (2 ꢀ 10 mL).
The combined extracts were washed with water (3 ꢀ 25 mL),
dried over MgSO4, and concentrated to afford 42 free base (0.130
cis-Adamantane-2-spiro-30-80-[[[[(20-amino-20-methylpropyl)-
amino]carbonyl]oxy]methyl]-10,20,40-trioxaspiro[4.5]decane p-Tosy-
late (40). To a solution of imidazole carbamate 1212 (358 mg,
1 mmol) in THF (10 mL) at 0 ꢀC was added methyl trifluor-
omethanesulfonate (164 mg, 1 mmol). The mixture was stirred
at 0 ꢀC for 30 min before 1,2-diamino-2-methylpropane (440 mg,
5 mmol) was added. The mixture was stirred at 0 ꢀC for
additional 2 h and then stirred at rt for 18 h. After removal of
the solvent, the residue was dissolved in CH2Cl2 (30 mL),
washed with water and brine, dried over MgSO4, filtered, and
concentrated. The residue was triturated with 60% aq EtOH
(20 mL) and filtered. The solid was dissolved in CHCl3 (10 mL)
and added to a solution of p-toluenesulfonic acid monohydrate
(65 mg, 0.34 mmol) in MeOH (2 mL). After concentration, the
crude product was triturated with ether to afford 40 (150 mg, 26%)
as a colorless solid. Mp 66-70 ꢀC; 1H NMR (CDCl3) δ 0.99-1.14
(m, 2H), 1.31 (s, 6H), 1.36-2.05 (m, 21H), 2.39 (s, 3H), 3.30 (d,
J=6.3 Hz, 2H), 3.71 (d, J=6.8 Hz, 2H), 6.51 (t, J=6.3 Hz, 1H),
1
g, 80%) as a colorless solid. H NMR (CDCl3) δ 1.11 (s, 6H),
1.20 (brs, 2H), 1.34-1.44 (m, 2H), 1.58-2.03 (m, 21H), 2.15
(d, J=6.8 Hz, 2H), 3.14 (d, J=5.8 Hz, 2H), 6.02 (brs, 1H); 13
C
NMR (MHz, CDCl3) δ 26.43, 26.88, 28.74, 29.79, 33.75,
33.79, 34.69, 34.90, 36.32, 36.75, 43.46, 49.74, 50.22, 108.53,
111.52, 172.13. To a solution of the free base of 42 in CHCl3
(5 mL) was added a solution of p-toluenesulfonic acid mono-
hydrate (0.072 g, 0.38 mmol) in EtOH (1 mL). After removal of
the solvent, the residue was treated with ether (15 mL). The
precipitate was filtered, washed with ether (20 mL), and dried to
afford 42 (0.156 g, 85%) as a colorless solid. Mp 150-152 ꢀC; 1H
NMR (CDCl3) δ 1.17 (s, 6H), 1.21-1.34 (m, 2H), 1.51-1.98 (m,
21H), 2.10 (d, J=7.3 Hz, 2H), 2.29 (s, 3H), 3.20 (d, J=6.4 Hz,
2H), 7.12 (d, J = 7.8 Hz, 2H), 7.48 (d, J = 7.8 Hz, 2H), 7.71
(brs, 3H), 8.05 (t, J=6.3 Hz, 1H); 13C NMR (CDCl3) δ 20.95,
23.48, 25.96, 26.37, 29.45, 32.85, 32.26, 34.38, 34.56, 35.93,
36.23, 41.71, 46.02, 54.53, 108.59, 110.92, 125.66, 128.24,
137.83, 145.81, 172.54.
cis-Adamantane-2-spiro-30-80-[[10-(20-amino-20-methylpropyl)-
10H-tetrazol-50-yl]methyl]-10,20,40-trioxaspiro[4.5]decane (43).
To a mixture of the free base of 1 (0.46 g, 1.17 mmol),
triphenylphosphine (0.77 g, 2.94 mmol), and trimethylsilyl azide
(0.34 g, 2.95 mmol) in THF (20 mL) at 0 ꢀC under Ar was added
dropwise diisopropyl azodicarboxylate (0.71 g, 3.51 mmol). The
mixture was slowly warmed to rt and stirred for 72 h. The
reaction mixture was diluted with water (50 mL) and extracted
with EtOAc (2 ꢀ 50 mL). The combined extracts were washed
with saturated aqueous NaHCO3 (2 ꢀ 50 mL) and brine (50 mL),
dried over MgSO4, filtered, and concentrated. To a solution of
the residue CHCl3 (20 mL) was added a solution of p-toluene-
sulfonic acid monohydrate (0.22 g) in EtOH (10 mL). After
evaporation of the solvents, the crude product was purified by
crystallization from 1:4 MeOH/CH2Cl2 to afford 43 (0.12 g,
17%) as a colorless solid. Mp 220 ꢀC dec; 1H NMR (CD3OD) δ
1.28-1.38 (m, 2H), 1.38 (s, 6H), 1.65-2.09 (m, 21H), 2.37 (s,
3H), 2.86 (d, J=6.8 Hz, 2H), 4.58 (s, 2H), 7.23 (d, J=7.8 Hz,
2H), 7.70 (d, J=7.8 Hz, 2H); 13C NMR (CD3OD) δ 21.30, 23.79,
27.97, 28.37, 30.04, 30.87, 34.87, 35.77, 35.78, 36.04, 37.80,
37.88, 54.02, 55.39, 109.53, 112.40, 126.97, 129.81, 141.66,
157.09.
7.20 (d, J=7.8 Hz, 2H), 7.72 (d, J=7.8 Hz, 2H), 7.79 (s, 3H); 13
C
NMR (CDCl3) δ 21.38, 23.59, 26.48, 26.54, 26.88, 33.57, 34.80,
35.66, 36.39, 36.80, 48.40, 56.08, 68.98, 108.56, 111.26, 125.82,
129.06, 140.81, 141.02, 157.56. Anal. (C29H44N2O8S) C, H, N.
trans-Adamantane-2-spiro-30-80-[[(20-amino-20-methylpropyl)-
amino]carbonyl]-10,20,40-trioxaspiro[4.5]decane p-Tosylate (41).
Step 1. A mixture of 1323 (8.0 g, 26 mmol), HOBt (5.3 g, 39
mmol), and EDCI (6.9 g, 36.4 mmol) in DMF (70 mL) was
stirred at rt overnight. The precipitate was collected by filtration
and dried to afford the desired active ester (7.0 g, 64%) as a
colorless solid. Mp 145-147 ꢀC; 1H NMR (CDCl3) d 1.64-2.10
(m, 18H), 2.11-2.20 (m, 2H), 2.22-2.30 (m, 2H), 2.90-2.98 (m,
1H), 7.38 (d, J=8.3 Hz, 1H), 7.43 (t, J=7.8 Hz, 1H), 7.55 (t, J=
7.8 Hz, 1H), 8.07 (d, J=8.3 Hz, 1H). Step 2. A mixture of the
above active ester (425 mg, 1 mmol) and 1,2-diamino-2-methyl-
propane (229 mg, 2.5 mmol) in CHCl3 (10 mL) was stirred at rt
for 4 h. The reaction mixture was washed with water (10 mL)
and brine (10 mL) and dried over MgSO4. After filtration, a
solution of p-toluenesulfonic acid monohydrate (190 mg, 1
mmol) in MeOH (2 mL) was added. The resulting mixture was
concentrated and triturated with EtOAc to afford 41 (450 mg,
82%) as a colorless solid. Mp 162-165 ꢀC; 1H NMR (CDCl3) δ
1.04-1.17 (m, 2H), 1.34 (s, 6H), 1.58-2.03 (m, 21H), 2.39 (s,
3H), 3.42 (d, J=6.8 Hz, 2H), 7.23 (d, J=7.8 Hz, 2H), 7.31 (d, J=
6.3 Hz, 1H), 7.65 (d, J=7.8 Hz, 2H), 7.76 (s, 3H); 13C NMR
(CDCl3) δ 21.58, 24.12, 26.64, 26.68, 27.06, 33.38, 34.95, 35.03,
36.56, 36.99, 43.04, 46.33, 56.65, 107.98, 111.94, 125.68, 129.65,
140.62, 142.11, 176.22. Anal. (C28H42N2O7S) C, H, N.
trans-Adamantane-2-spiro-30-80-[[[(20-amino-20-methylpropyl)-
amino]carbonyl]methyl]-10,20,40-trioxaspiro[4.5]decane p-Tosy-
late (42). Step 1. A 1:1 mixture of the cis (5) and trans ozonide
esters (4.14 g, 21%) was obtained from a 59 mmol scale
preparation of ozonide ester 5, after chromatography and
repeated crystallizations from EtOH to remove 5. Step 2. To
Antimalarial Screens. In vitro and in vivo antimalarial data
was obtained as previously described.42,43

490 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Dong et al.
Partition Coefficients. Log DpH7.4 values, calculated as pre-
viously described,9,11 denote the octanol/buffer partition coeffi-
cients at pH 7.4 that are relevant for the ionizable ozonides.
Experimental log DpH7.4 values (elog D) for ozonides 277 and
339 were determined using a modification of a previously
reported chromatographic method.44
Functional Group Polarity on the Antimalarial Activity of Spiro
and Dispiro-1,2,4-Trioxolanes. Bioorg. Med. Chem. 2006, 14,
6368–6382.
(13) Kaiser, M.; Wittlin, S.; Nehrbass-Stuedli, A.; Dong, Y.; Wang, X.;
Hemphill, A.; Matile, H.; Brun, R.; Vennerstrom, J. L. Peroxide
Bond-Dependent Antiplasmodial Specificity of Artemisinin and
OZ277 (RBx11160). Antimicrob. Agents Chemother. 2007, 51,
2991–2993.
Pharmacokinetic Experiments. In vivo pharmacokinetic stu-
dies were conducted in conscious male Sprague-Dawley rats.
Compounds were dosed by intravenous infusion over 5-10 min
through a cannula previously implanted in the jugular vein at
doses of 2.5-3.5 mg/kg. The IV formulations consisted of either
DMSO (4-10% v/v)/aqueous buffer or propylene glycol
(10-40% v/v)/ethanol (10-15% v/v)/water. Oral doses were
administered by gavage as aqueous suspensions prepared in
0.5% w/v hydroxypropylmethyl cellulose or carboxymethyl
cellulose, 0.4% Tween 80, and 0.5% benzyl alcohol. Blood
samples were collected via a cannula previously implanted in
the carotid artery, and plasma was immediately obtained by
centrifugation after which samples were stored at -20 ꢀC. For
analysis, samples were thawed, plasma proteins were precipi-
tated with acetonitrile, and aliquots of the supernatant were
analyzed by LC/MS. Quantitation was conducted by compar-
ison to the response obtained for calibration standards also
prepared in plasma using the same methods.
(14) Padmanilayam, M.; Scorneaux, B.; Dong, Y.; Chollet, J.; Matile,
H.; Charman, S. A.; Creek, D. J.; Charman, W. N.; Santo Tomas,
J.; Scheurer, C.; Wittlin, S.; Brun, R.; Vennerstrom, J. L. Anti-
malarial Activity of N-Alkyl Amine, Carboxamide, Sulfonamide,
and Urea Derivatives of a Dispiro-1,2,4-Trioxolane Piperidine.
Bioorg. Med. Chem. Lett. 2006, 16, 5542–5545.
(15) Tang, Y.; Dong, Y.; Wittlin, S.; Charman, S. A.; Chollet, J.; Chiu,
F. C. K.; Charman, W. N.; Matile, H.; Urwyler, H.; Dorn, A.;
Bajpai, S.; Wang, X.; Padmanilayam, M.; Karle, J. M.; Brun, R.;
Vennerstrom, J. L. Weak Base Dispiro-1,2,4-Trioxolanes: Potent
Antimalarial Ozonides. Bioorg. Med. Chem. Lett. 2007, 17, 1260–
1265.
(16) Griesbaum, K.; Liu, X.; Kassiaris, A.; Scherer, M. Ozonolyses of
O-Alkylated Ketoximes in the Presence of Carbonyl Groups: A
Facile Access to Ozonides. Liebigs Ann./Recl. 1997, 1381–1390.
(17) Dong, Y.; Vennerstrom, J. L. Dispiro-1,2,4,5-Tetraoxanes via
Ozonolysis of Cycloalkanone O-Methyl Oximes: A Comparison
with the Peroxidation of Cycloalkanones in Acetonitrile Sulfuric
Acid Media. J. Org. Chem. 1998, 63, 8582–8585.
(18) Marvell, E. N.; Sturmer, D.; Rowell, C. Bicyclo[3.3.1]nonanes. A
New Synthesis of 2-Bicyclo[3.3.1]-nonanone. Tetrahedron 1966, 22,
861–866.
(19) Zhou, L.; Alker, A.; Ruf, A.; Wang, X.; Chiu, F. C. K.; Morizzi, J.;
Charman, S. A.; Charman, W. N.; Scheurer, C.; Wittlin, S.; Dong,
Y.; Hunziker, D.; Vennerstrom, J. L. Characterization of the Two
Major CYP450 Metabolites of Ozonide (1,2,4-trioxolane) OZ277.
Bioorg. Med. Chem. Lett. 2008, 18, 1555–1558.
(20) Tang, Y.; Dong, Y.; Karle, J. M.; DiTusa, C. A.; Vennerstrom, J. L.
Synthesis of Tetrasubstituted Ozonides by the Griesbaum Coozo-
nolysis Reaction: Diastereoselectivity and Functional Group
Transformations by Post-Ozonolysis Reactions. J. Org. Chem.
2004, 69, 6470–6473.
Acknowledgment. This investigation received financial
support from Medicines for Malaria Venture (MMV).
Supporting Information Available: Elemental analysis and
HRMS data for 1, 6, and 15-43. This material is available free
of charge via the Internet at http://pubs.acs.org.
References
(1) Winstanley, P.; Ward, S. Malaria Chemotherapy. Adv. Parasitol.
2006, 61, 47–76.
(2) White, N. J. Qinghaosu (Artemisinin): The Price of Success.
(21) Duncia, J. V.; Pierce, M. E.; Santella, J. B., III Three Synthetic
Routes to a Sterically Hindered Tetrazole. A New One-Step Mild
Conversion of an Amide into a Tetrazole. J. Org. Chem. 1991, 56,
2395–2400.
Science 2008, 320, 330–334.
(3) Breman, J. G.; Alilio, M. S.; White, N. J. Defining and Defeating
the Intolerable Burden of Malaria. III. Progress and Perspectives.
Am. J. Trop. Med. Hyg. 2007, 77 (S6), vi–xi.
(4) O’Neill, P. M.; Posner, G. H. A Medicinal Chemistry Perspective
on Artemisinin and Related Endoperoxides. J. Med. Chem. 2004,
47, 2945–2964.
(22) Liu, Q.; Luedtke, N. W.; Tor, Y. A Simple Conversion of Amines
into Monosubstituted Ureas in Organic and Aqueous Solvents.
Tetrahedron Lett. 2001, 42, 1445–1447.
(23) Vennerstrom, J. L.; Dong, Y.; Chollet, J.; Matile, H. Spiro and
Dispiro 1,2,4-Trioxolane Antimalarials. U. S. Patent 6,486,199,
November 26, 2002.
(5) Haynes, R. K. From Artemisinin to New Artemisinin Antimalar-
ials: Biosynthesis, Extraction, Old and New Derivatives, Stereo-
chemistry and Medicinal Chemistry Requirements. Curr. Top.
Med. Chem. 2006, 6, 509–537.
(6) Muraleedharan, K. M.; Avery, M. A. Progress in the Development
of Peroxide-based Anti-parasitic Agents. Drug Discovery Today
2009, 14, 793–803.
(24) Adang, A. E. P.; Lucas, H.; Adrianus, P. A.; de, M.; Engh, R. A.;
Grootenhuis, P. D. G. Novel Acylguanidine Containing Thrombin
Inhibitors With Reduced Basicity at the P1 Moiety. Bioorg. Med.
Chem. Lett. 1998, 8, 3603–3608.
(25) Activity is defined as percent reduction on day 3 postinfection
compared with an untreated control group. For example, a com-
pound 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%.
(7) Tang, Y.; Dong, Y.; Vennerstrom, J. L. Synthetic Peroxides as
Antimalarials. Med. Res. Rev. 2004, 24, 425–448.
(8) Jefford, C. W. New Developments in Synthetic Peroxidic Drugs as
Artemisinin Mimics. Drug Discovery Today 2007, 12, 487–495.
(9) Vennerstrom, J. L.; Arbe-Barnes, S.; Brun, R.; Charman, S. A.;
Chiu, F. C. K.; Chollet, J.; Dong, Y.; Dorn, A.; Hunziker, D.;
Matile, H.; McIntosh, K.; Padmanilayam, M.; Santo Tomas, J.;
Scheurer, C.; Scorneaux, B.; Tang, Y.; Urwyler, H.; Wittlin, S.;
Charman, W. N. Identification of an Antimalarial Synthetic
Trioxolane Drug Development Candidate. Nature 2004, 430,
900–904.
(10) Olliaro, P.; Wells, T. N. C. The Global Portfolio of New Anti-
malarial Medicines under Development. Clin. Pharmacol. Ther.
2009, 85, 584–595.
(11) Dong, Y.; Chollet, J.; Matile, H.; Charman, S. A.; Chiu, F. C. K.;
Charman, W. N.; Scorneaux, B.; Urwyler, H.; Santo Tomas, J.;
Scheurer, C.; Snyder, C.; Dorn, A.; Wang, X.; Karle, J. M.; Tang,
Y.; Wittlin, S.; Brun, R.; Vennerstrom, J. L. Spiro and Dispiro-
1,2,4-Trioxolanes as Antimalarial Peroxides: Charting a Workable
Structure-Activity Relationship Using Simple Prototypes. J. Med.
Chem. 2005, 48, 4953–4961.
(26) Perry, C. S.; Charman, S. A.; Prankerd, R. J.; Chiu, F. C. K.; Dong,
Y.; Vennerstrom, J. L.; Charman, W. N. Chemical Kinetics and
Aqueous Degradation Pathways of a New Class of Synthetic
Ozonide Antimalarials. J. Pharm. Sci. 2006, 95, 737–747.
(27) Creek, D. J.; Charman, W. N.; Chiu, F. C. K.; Prankerd, R. J.;
McCullough, K. J.; Dong, Y.; Vennerstrom, J. L.; Charman,
S. A. Iron Mediated Degradation Kinetics of Substituted Dispiro-
1,2,4-Trioxolane Antimalarials. J. Pharm. Sci. 2007, 96, 2945–
2956.
(28) As fully described in Vennerstrom et al.,9 compounds were incu-
bated with human liver microsomes and appropriate cofactors (BD
Gentest, Discovery Labware Inc., Woburn, MA) at a substrate
concentration of 1-10 μM and a microsomal protein concentra-
tion of 0.4 mg/mL. Loss of parent compound was monitored by
LC/MS. Extraction ratios were calculated using appropriate scal-
ing factors as previously described by Obach, R. S. Prediction of
Human Clearance of Twenty-Nine Drugs From Hepatic Micro-
somal Intrinsic Clearance Data: An Examination of In Vitro Half-
Life Approach and Nonspecific Finding to Microsomes. Drug
Metab. Dispos. 1999, 27, 1350–1359.
(12) Dong, Y.; Tang, Y.; Chollet, J.; Matile, H.; Wittlin, S.; Charman, S.
A.; Charman, W. N.; Santo Tomas, J.; Scheurer, C.; Snyder, C.;
Scorneaux, B.; Bajpai, S.; Alexander, S. A.; Wang, X.; Padmani-
layam, M.; Cheruku, S. R.; Brun, R.; Vennerstrom, J. L. Effect of
(29) Crespi, C. L.; Miller, V. P.; Penman, B. W. Microtiter Plate Assays
for Inhibition of Human, Drug-Metabolizing Cytochromes P450.
Anal. Biochem. 1998, 248, 188–190.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 491
(30) Amewu, R.; Stachulski, A. V.; Ward, S. A.; Berry, N. G.; Bray, P.
G.; Davies, J.; Labat, G.; Vivas, L.; O’Neill, P. M. Design and
Synthesis of Orally Active Dispiro 1,2,4,5-Tetraoxanes; Synthetic
Antimalarials With Superior Activity to Artemisinin. Org. Biomol.
Chem. 2006, 4, 4431–4436.
(37) Crespo, M. D. P.; Avery, T. D.; Hanssen, E.; Fox, E.; Robinson, T.
V.; Valente, P.; Taylor, D. K.; Tilley, L. Artemisinin and a Series of
Novel Endoperoxide Antimalarials Exert Early Effects on Diges-
tive Vacuole Morphology. Antimicrob. Agents Chemother. 2008,
52, 98–109.
(38) Hartwig, C. L.; Rosenthal, A. S.; D’Angelo, J.; Griffin, C. E.;
Posner, G. H.; Cooper, R. A. Accumulation of Artemisinin
Trioxane Derivatives Within Neutral Lipids of Plasmodium falci-
parum Malaria Parasites is Endoperoxide-dependent. Biochem.
Pharmacol. 2009, 77, 322–336.
(39) Yadav, G. C.; Dorwal, H. N.; Valavala, S.; Sharma, V. K. A
Process for the Preparation of Known Antimalarials Spiro and
Dispiro 1,2,4-Trioxolanes and Salts Thereof. PCT Int. Appl. WO
2007138435 2007,.
(40) Seo, J. W.; Comninos, J. S.; Chi, D. Y.; Kim; Dong, W.; Carlson,
K. E.; Katzenellenbogen, J. A. Fluorine-Substituted Cyclofenil
Derivatives as Estrogen Receptor Ligands: Synthesis and Struc-
ture-Affinity Relationship Study of Potential Positron Emission
Tomography Agents for Imaging Estrogen Receptors in Breast
Cancer. J. Med. Chem. 2006, 49, 2496–2511.
(31) Jefford, C. W.; Kohmoto, S.; Jaggi, D.; Timari, G.; Rossier, J.-C.;
ꢀ
Rudaz, M.; Barbuzzi, O.; Gerard, D.; Burger, U.; Kamalaprija, P.;
Mareda, J.; Bernardinelli, G.; Manzanares, I.; Canfield, C. J.;
Fleck, S. L.; Robinson, B. L.; Peters, W. 51. Synthesis, Structure,
and Antimalarial Activity of Some Enantiomerically Pure, cis-
Fused Cyclopenteno-1,2,4-trioxanes. Helv. Chim. Acta 1995, 78,
647–662.
(32) O’Neill, P. M.; Rawe, S. L.; Borstnik, K.; Miller, A.; Ward, S. A.;
Bray, P. G.; Davies, J.; Oh, C. H.; Posner, G. H. Enantiomeric
1,2,4-Trioxanes Display Equivalent in Vitro Antimalarial Activity
versus Plasmodium falciparum Malaria Parasites: Implications for
the Molecular Mechanism of Action of the Artemisinins. Chem-
BioChem 2005, 6, 2048–2054.
(33) Creek, D. J.; Charman, W. N.; Chiu, F. C. K.; Prankerd, R. J.;
Dong, Y.; Vennerstrom, J. L.; Charman, S. A. Relationship
between Antimalarial Activity and Haem Alkylation for Spiro-
and Dispiro-1,2,4-trioxolane Antimalarial. Antimicrob. Agents
Chemother. 2008, 52, 1291–1296.
(41) Vergne, F.; Aitken, D. J.; Husson, H.-P. Synthesis of 1-Amino-
1-(aminomethyl)cyclopropane and its Monobenzamides. J. Org.
Chem. 1992, 57, 6071–6075.
(34) Uhlemann, A.-C.; Wittlin, S.; Matile, H.; Bustamante, L. L.;
Krishna, S. Mechanism of Antimalarial Action of Synthetic Triox-
olane RBX11160 (OZ277). Antimicrob. Agents Chemother. 2007,
51, 667–672.
(42) Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D.
Quantitative Assessment of Antimalarial Activity in Vitro by a
Semiautomated Microdilution Technique. Antimicrob. Agents
Chemother. 1979, 16, 710–718.
(43) Ridley, R. G.; Matile, H.; Jaquet, C.; Dorn, A.; Hofheinz, W.;
Leupin, W.; Masciadri, R.; Theil, F. P.; Richter, W. F.; Girometta,
M. A.; Guenzi, A.; Urwyler, H.; Gocke, E.; Potthast, J. M.; Csato,
M.; Thomas, A.; Peters, W. Antimalarial Activity of the Bisquino-
line trans-N1,N2-Bis-(7-chloroquinolin-4-yl)cyclohexane-1,2-diamine:
Comparison of Two Stereoisomers and Detailed Evaluation of the
S,S Enantiomer, Ro 47-7737. Antimicrob. Agents Chemother. 1997, 41,
677–686.
(35) Elliott, D. A.; McIntosh, M. T.; Hosgood, H. D., III; Chen, S.;
Zhang, G.; Baevova, P.; Joiner, K. A. Four Distinct Pathways of
Hemoglobin Uptake in the Malaria Parasite Plasmodium falci-
parum. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2463–2468.
(36) Stocks, P. A.; Bray, P. G.; Barton, V. E.; Al-Helal, M.; Jones, M.;
Araujo, N. C.; Gibbons, P.; Ward, S. A.; Hughes, R. H.; Biagini, G.
A.; Davies, J.; Amewu, R.; Mercer, A. E.; Ellis, G.; O’Neill, P. M.
Evidence for a Common Non-Heme Chelatable-Iron-Dependent
Activation Mechanism for Semisynthetic and Synthetic Endoper-
oxide Antimalarial Drugs. Angew. Chem., Int. Ed. 2007, 46, 6278–
6283.
(44) Lombardo, F.; Shalaeva, M. Y.; Tupper, K. A.; Gao, F.; Abraham,
M. H. ElogPoct: A Tool for Lipophilicity Determination in Drug
Discovery. J. Med. Chem. 2000, 43, 2922–2928.