Structure-Activity Relationship of the Antimalarial Ozonide Artefenomel (OZ439)

Article abstract of DOI:10.1021/acs.jmedchem.6b01586

Building on insights gained from the discovery of the antimalarial ozonide arterolane (OZ277), we now describe the structure-activity relationship (SAR) of the antimalarial ozonide artefenomel (OZ439). Primary and secondary amino ozonides had higher metab

Full text of DOI:10.1021/acs.jmedchem.6b01586

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Article
SAR of the Antimalarial Ozonide Artefenomel (OZ439)
Yuxiang Dong, Xiaofang Wang, Sriraghavan Kamaraj, Vivek J. Bulbule, Francis C. K. Chiu,
Jacques Chollet, Dhanasekaran Manickam, Christopher D. Hein, Petros Papastogiannidis,
Julia Morizzi, David M. Shackleford, Helena Barker, Eileen Ryan, Christian Scheurer,
Yuanqing Tang, Qingjie Zhao, Lin Zhou, Karen L. White, Heinrich Urwyler, William N.
Charman, Hugues Matile, Sergio Wittlin, Susan A. Charman, and Jonathan L. Vennerstrom
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01586 • Publication Date (Web): 04 Jan 2017
Downloaded from http://pubs.acs.org on January 4, 2017
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SAR of the Antimalarial Ozonide Artefenomel (OZ439)
†
†
†
†
§
Yuxiang Dong , Xiaofang Wang , Sriraghavan Kamaraj , Vivek J. Bulbule , Francis C. K. Chiu ,
‡,£
†
†
‡,£
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Jacques Chollet , Manickam Dhanasekaran , Christopher D. Hein , Petros Papastogiannidis ,
§
§
§
§
‡,£
Julia Morizzi , David M. Shackleford , Helena Barker , Eileen Ryan , Christian Scheurer ,
†
†
†
§
∞
Yuanqing Tang , Qingjie Zhao , Lin Zhou , Karen L. White , Heinrich Urwyler , William N.
§
¶
‡,£
§
Charman , Hugues Matile , Sergio Wittlin , Susan A. Charman *, and Jonathan L.
†
Vennerstrom *
†
College of Pharmacy, University of Nebraska Medical Center, 986125 Nebraska Medical
Center, Omaha, NE, United States
§
Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash
University (Parkville Campus), 381 Royal Parade, Parkville, Victoria 3052, Australia
‡
Swiss Tropical and Public Health Institute, Socinstrasse 57, CHꢀ4002 Basel, Switzerland
£
University of Basel, CHꢀ4003 Basel, Switzerland
∞
Basilea Pharmaceutica Ltd., Grenzacherstrasse 487, CHꢀ4058 Basel, Switzerland
¶
F. HoffmannꢀLa Roche Ltd., Grenzacherstrasse 124, CHꢀ4070 Basel, Switzerland
*S.A.C.: Phone: 61 3 9903 9626. Eꢀmail: susan.charman@monash.edu
*
J.L.V.: Phone: 402 559 5362. Eꢀmail: jvenners@unmc.edu
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Abstract
Building on insights gained from the discovery of the antimalarial ozonide arterolane (OZ277),
we now describe the structureꢀactivity relationship (SAR) of the antimalarial ozonide
artefenomel (OZ439). Primary and secondary amino ozonides had higher metabolic stabilities
than tertiary amino ozonides, consistent with their higher pK and lower Log D values. For
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a
7.4
primary amino ozonides, addition of polar functional groups decreased in vivo antimalarial
efficacy. For secondary amino ozonides, additional functional groups had variable effects on
metabolic stability and efficacy, but the most effective members of this series also had the
highest Log D values. For tertiary amino ozonides, addition of polar functional groups with Hꢀ
7
.4
bond donors increased metabolic stability, but decreased in vivo antimalarial efficacy. Primary
and tertiary amino ozonides with cycloalkyl and heterocycle substructures were superior to their
acyclic counterparts. The high curative efficacy of these ozonides was most often associated
with high and prolonged plasma exposure, but exposure on its own did not explain the presence
or absence of either curative efficacy or in vivo toxicity.
Keywords: 1,2,4ꢀtrioxolane, ozonide, antimalarial, artemisinin, OZ439, artefenomel
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The semisynthetic artemisinins (Figure 1) are vitally important firstꢀline antimalarial drugs that
1
,2
comprise the fastꢀacting component of artemisinin combination therapies (ACT). Recently,
delayed parasite clearance times in patients treated with ACTs have become increasingly
prevalent in parts of Southeast Asia and are now known to be associated with polymorphisms in
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the kelch domain–carrying protein K13. The discovery of artemisinin led to extensive
investigation of structurally diverse synthetic peroxides containing many different types of
peroxide heterocycles including 1,2ꢀdioxanes, 1,2,4ꢀtrioxanes, 1,2,4ꢀtrioxolanes, and 1,2,4,5ꢀ
7
ꢀ10
tetraoxanes.
Although the precise mechanism of action of antimalarial peroxides is still not
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fully understood, it is hypothesized that the pharmacophoric peroxide bond undergoes
1
2ꢀ14
reductive activation by heme released during parasite hemoglobin digestion
to produce
1
2,15ꢀ21
carbonꢀcentered radicals that alkylate heme and parasite proteins
leading to downstream
17,22,23
peroxidative damage and oxidative stress.
Figure 1. Artemisinin (ART) and its semisynthetic derivatives dihydroartemisinin (DHA),
artemether (AM), and artesunate (AS) and ozonides 1 and 2.
2
4
The first synthetic peroxide to be registered was ozonide 1 (OZ277), also known as
2
5ꢀ27
arterolane,
which was approved for the Indian market as a threeꢀdose combination product
with piperaquine (Synriam) for the treatment of uncomplicated malaria. More recently, nextꢀ
2
8
generation ozonide 2 (OZ439, artefenomel) has progressed through to phase IIb clinical trials
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and is being developed as a single dose combination treatment, although the partner drug has not
2
9ꢀ32
yet been determined (Figure 1).
Compared to 8’ꢀalkyl substituted ozonides such as 1, 8′ꢀaryl
substituted ozonides such as 2 are much more stable to premature bloodꢀmediated degradation,
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yet maintain sufficient ferrous ironꢀreactivity to rapidly kill parasites.
The 8’ꢀaryl versus 8’ꢀ
alkyl substituent shifts the conformer equilibrium to the less reactive axialꢀperoxide
3
3ꢀ35
conformer.
It is likely that the high in vivo antimalarial efficacy of 2 and its analogs is a
direct consequence of the significantly prolonged in vivo exposure profile which in turn is at
2
8
least partially linked to an optimized ferrous iron reactivity profile. For example, the halfꢀlives
25,27
29,31
in humans for 1 and 2 are approximately 3 h
and 46ꢀ60 h,
respectively.
In this paper, we profile ozonides 3ꢀ49 to describe the structureꢀactivity relationship (SAR) of 2,
beginning with the discovery of 3. Previous work on the SAR for this class of dispiro synthetic
ozonides has demonstrated that: 1) the core 1,2,4ꢀtrioxolane is superior to the corresponding
1
,2,4ꢀtrioxane, 1,2,4,5ꢀtetraoxane, and 1,2ꢀdioxolane peroxy heterocycles, the latter of which are
36
37,38
11,37
nearly inactive; 2) the spiroadamantane ring system
and peroxide bond
are essential for
activity; 3) more lipophilic ozonides tend to have better oral antimalarial efficacies than their
3
7,38
more polar counterparts,
an outcome consistent with that seen for other classes of synthetic
7
peroxides; 4) ozonides with a wide range of neutral and basic, but not acidic, functional groups
3
8ꢀ41
have good antimalarial activity;
5) weak base ozonides possess the best overall ADME
40,42
properties;
and 6) hydroxylated ozonide CYP450 metabolites have relatively low antimalarial
3
7,43
activities.
In this respect, 1 is hydroxylated at the distal bridgehead carbon atoms of the
spiroadamantane substructure to form two isomeric carbinol metabolites, whereas 2 is
metabolized principally by hydroxylation at the three distal carbon atoms of the
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spiroadamantane, with minor contributions from Nꢀoxidation of the morpholine nitrogen and
cleavage of the morpholine ring to form a total of five primary and six secondary Phase I
metabolites, the most potent of which was an order of magnitude less active than 2. The lower
metabolic stability of 2 versus 1 is consistent with the higher Log D7.4 of the former (calculated
Log D 5.4 for 2 vs 0.9 for 1) that well illustrates one important hurdle in the optimization of
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these next generation ozonides.
Chemistry
As shown in Schemes 1ꢀ7, ozonide target compounds were prepared as their mesylate or tosylate
4
4
salts; structures depicted are the free base forms. We identified ozonide phenol 50 as a key
common intermediate suitable for the preparation of numerous target compounds using parallel
chemistry (Scheme 1). For example, alkylation of the phenoxide of 50 with the corresponding
45
epoxide followed by tosylate salt formation afforded 16 in 48% overall yield; similarly,
alkylation of the phenoxide of 50 with corresponding Bocꢀprotected epoxide gave Boc ozonide
51 which underwent oneꢀpot deprotection and mesylate salt formation to afford 15 in an overall
4
6
yield of 78%. Alkylation of 50 with the requisite Bocꢀprotected piperidine mesylate produced
Boc ozonide 52 in 81% yield; a subsequent oneꢀpot deprotection and mesylate salt formation
afforded 17 in 78% yield.
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a
Scheme 1
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a
Reagents and conditions: (a) 1ꢀoxaꢀ6ꢀazaspiro[2,5]octaneꢀ6ꢀcarboxylic acid tꢀbutyl ester,
isopropanol, 1 M aq. KOH 60 °C, 20 h; (b) 5,5,7,7ꢀtetramethylꢀ1ꢀoxaꢀ6ꢀazaspiro[2,5]octane,
isopropanol, 1 M aq. KOH 60 °C, 20 h, then TsOH, EtOAc, rt; (c) powdered NaOH,
tetrabutylammonium hydrogensulfate, CH CN, rt, 0.5 h, then tꢀbutyl 4ꢀ(2ꢀ
3
methylsulfonyloxyethyl)piperidineꢀ1ꢀcarboxylate, 60 °C, 12 h; (d) 1.5 M MsOH in THF, rt, 3–4
h.
Ozonide 18 was obtained by alkylation of the phenoxide of 50 with mesylate 55 to afford Boc
ozonide 56 (85%) which underwent subsequent oneꢀpot Boc deprotection and mesylate salt
formation (57%) (Scheme 2). Intermediate 55 was obtained by reduction of Bocꢀprotected ester
4
7
53 to alcohol 54 (94%) followed by mesylate formation (82%).
a
Scheme 2
a
Reagents and conditions: (a) 2 M lithium borohydride in THF followed by 1 M lithium
triethylborohydride in THF, rt, 12 h; (b) methanesulfonyl chloride, triethylamine, CH Cl , 0 °C
2
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for 1 h, then rt for 12 h; (c) powdered NaOH, tetrabutylammonium hydrogensulfate, CH CN, rt,
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0.5 h, then 55 (tꢀbutyl 4ꢀ[2ꢀ(methanesulfonyloxy)ethoxy]piperidineꢀ1ꢀcarboxylate), 60 °C, 12 h;
(d) 1.5 M MsOH in THF, rt, 4 h.
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Reductive amination of amino ozonide 4 followed by mesylate salt formation afforded 21 in
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0% yield (Scheme 3). Alkylation of bromoethoxy ozonide 57 with azetidine or 2ꢀoxaꢀ6ꢀ
49
azaspiro [3,3]heptane followed by tosylate salt formation afforded 22 (24%) and 39 (18%),
respectively.
a
Scheme 3
a
Reagents and conditions: (a) tetrahydroꢀ4Hꢀpyranꢀ4ꢀone, AcOH, sodium triacetoxyborohydride,
CH Cl , rt, 12 h, then MsOH in EtOAc, rt, 0.5 h; (b) azetidine HCl, K CO , CH CN, 60 °C, 24 h,
2
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then TsOH, EtOAc/CH Cl , rt; (c) 2ꢀoxaꢀ6ꢀazaspiro [3,3]heptane tosylate, K CO , CH CN, 60
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°
C, 24 h, then TsOH, EtOAc, rt.
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Following a modified method of Erhardt et al., keto phenol 58 was converted into keto epoxide
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9 in 87% yield (Scheme 4). Griesbaum coozonolysis of 59 and Oꢀmethylꢀ2ꢀadamantanone
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oxime (60) formed epoxy ozonide 61 (54%), which after exposure to morpholine and mesylate
salt formation gave 36 in 60% yield.
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a
Scheme 4
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Reagents and conditions: (a) epichlorohydrin, K CO , CH CN, reflux, 24 h; (b) O , 8:2
2
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cyclohexane:CH Cl , 0 °C, 5 min; (c) morpholine, MeOH, 60 °C, 12 h, then MsOH, ether, rt, 0.5
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h.
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44
Griesbaum coozonolysis of keto acetate 62 and fluorinated Oꢀmethylꢀ2ꢀadamantanone
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oximes 63 and 64 afforded ozonide esters 65 (54%) and 66 (44%), respectively (Scheme 5).
These intermediates underwent oneꢀpot acetate hydrolysis/alkylation with Nꢀ(2ꢀ
chloroethyl)morpholine followed by mesylate salt formation to afford fluorinated ozonides 42
and 43 in overall yields of 77 and 72%, respectively. Ozonide 42 was isolated as a 1:1 mixture
of diastereomers (cis, cis isomer shown).
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Scheme 5
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Reagents and conditions: (a) O , 5ꢀ7:1 cyclohexane:CH Cl , 0 °C, 5 min; (b) Nꢀ(2ꢀ
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chloroethyl)morpholine HCl, NaOH, tetrabutylammonium hydrogensulfate, CH CN, 25 to 60
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°
C, 12 h, then MsOH, ether, rt, 0.5 h.
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Alkylation of morpholine or isonipecotamide with ozonide benzyl chloride 67 followed by salt
formation afforded the mesylate of 45 (69%) and tosylate of 46 (61%) (Scheme 6). Conversion
3
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of ozonide acid 68 to its Nꢀhydroxysuccinimide active ester 69 (91%) followed by reaction of
the latter with piperazine and salt formation afforded 47 (75%) as its mesylate.
a
Scheme 6
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a
Reagents and conditions: (a) morpholine, Et N, CH CN, rt to 60 °C, 48 h, then MsOH in ether,
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0
°C, 0.5 h; (b) isonipecotamide, K CO , CH CN, rt to 60 °C, 48 h, then TsOH in EtOAc/ether, 0
2 3 3
°
C, 0.5 h; (c) Nꢀhydroxysuccinimide, EDCI, DMF, 0 ºC to rt, 12 h; (d) piperazine, CH Cl , rt, 12
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h, then MsOH, EtOAc/ether, rt, 0.5 h.
Chlorosulfonylation of ketone 70 afforded crude 71 (10%) which was converted into
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pentafluorosulfonate ester 72 (70%) (Scheme 7). Griesbaum coozonolysis of 72 and 60
formed ozonide sulfonate ester 73 (61%). Treatment of 73 with piperazine or 4ꢀaminopiperidine
followed by mesylate salt formation afforded sulfonamides 48 (52%) and 49 (42%), respectively.
a
Scheme 7
a
Reagents and conditions: (a) chlorosulfonic acid, CH Cl , ꢀ10 °C, 6 h; (b) pentafluorophenol,
2
2
Et N, CH Cl , rt, 2 h; (c) O , 9:1 cyclohexane:CH Cl , 0 °C, 5 min; (d) piperazine, Et N,
3
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CH CN, rt to 60 °C, 12 h, then MsOH, EtOAc/CH Cl , 0 °C; (e) 4ꢀaminopiperidine, Et N,
3
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CH CN, rt to 70 °C, 12 h, then MsOH, EtOAc/CH Cl , 0 °C.
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8,48,54,55
As previously described,
the remaining ozonides (5ꢀ14, 19, 20, 24ꢀ35, and 37ꢀ42) were
44
obtained from ozonide phenol 50 or its acetate ester 62 using parallel chemistry.
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Antimalarial Activity and ADME Properties
In vitro and in vivo antimalarial activities were measured using the chloroquineꢀresistant K1 and
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chloroquineꢀsensitive NF54 strains of P. falciparum, and P. bergheiꢀinfected mice, respectively,
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as previously described. In vivo efficacy data were obtained using single 30 mg/kg oral doses
of the ozonides administered one day postꢀinfection in an aqueous suspension vehicle.
Calculated pK and Log D values were generated using JChem for Excel and in vitro intrinsic
a
7.4
clearance (CL ) values were determined for all compounds using human liver microsomes. For
int
selected compounds, intrinsic clearance was also measured in mouse, rat and dog liver
microsomes to assess potential species differences. For a subset of ozonides, kinetic solubility in
water and exposure in mice following oral dosing (30 mg/kg) were also assessed.
We note several overarching trends from the data in Tables 1ꢀ6. First, the in vitro antimalarial
potency for ozonides 2ꢀ49 fell in the narrow range of approximately 0.8 to 12 nM. Second, all of
the ozonides reduced parasitemia by >99% on day 3 postꢀinfection compared to an untreated
control group, however there was no correlation between in vitro antimalarial activity in vivo
curative efficacy. Third, there was an overall trend (Figure 2) that the more metabolically stable
ozonides tended to have higher pK and lower Log D values, but neither of these parameters
a
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directly correlated with oral exposure or efficacy. Finally, the more effective compounds tended
to have high and prolonged exposure in mice; however exposure on its own did not explain the
presence or absence of in vivo efficacy.
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Figure 2. In vitro intrinsic clearance (CL µL/min/mg microsomal protein) in human liver
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microsomes as a function of calculated pKa (left) and Log D (right). Dashed line represents the
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.4
nominal cutoff for more stable compounds (nominally defined as CL < 15 µL/min/mg protein).
int
As we noted in the introduction, the data in Table 1 begins with the discovery that aminoethyl
ether ozonide 3, in contrast to 1 and AS, had singleꢀdose curative efficacy in the P. berghei
mouse model, increasing the average survival to approximately 30 days resulting in 3 of the 5
2
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animals having no detectable parasites. As shown in Figure 3A and discussed previously, this
improved efficacy most likely results from the substantially prolonged exposure profile of the 8’ꢀ
aryl versus the 8’ꢀalkyl ozonides. Extending the length of the alkyl chain between the primary
amino and ether functional groups (4ꢀ6) led to a marginal reduction in overall survival (22ꢀ25
days), but a similar number of cures (2ꢀ3 out of 5 mice). Incorporation of gemꢀdimethyl groups
in the alkyl chain maintained (9) or increased (10) curative efficacy. However, substitution of
one of the side chain methylene carbons with an ether (7) or sulfone (8) decreased in vivo
antimalarial efficacy (average survival <15 days and 0/5 cures). Similarly, substitution of one or
more of the side chain methylene carbons with other polar functional groups including alcohols,
carboxamides, oxamides, and ureas decreased in vivo antimalarial efficacy (data not shown).
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Lastly, replacing the alkyl chain between the primary amino and ether functional groups with a
cyclohexyl substructure in 11 and 12 resulted in high antimalarial activity and metabolic
stability. For 1, 4 and 11, the in vitro metabolic stability was also generally comparable in
mouse, rat and dog liver microsomes compared to that for human microsomes. Primary amino
ozonides 10 and 11 exhibited high and extended exposure and high efficacy, consistent with their
metabolic stability and reasonably good aqueous solubility (Tables 1ꢀ2, Figure 3A).
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Figure 3. Plasma concentration versus time profiles for selected ozonides following oral
administration of 30 mg/kg to nonꢀinfected mice. Panel A: primary amino ether ozonides, Panel
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B: secondary amino ether ozonides, Panel C: piperidine, piperazine and morpholine ozonides.
Each point represents the average concentration for two mice.
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The data in Table 3 show that secondary amineꢀcontaining ozonides 13ꢀ21 had generally good
metabolic stability, high aqueous solubility (with the exception of 14), high and prolonged
exposure (Figure 3B and Table 2) and high antimalarial efficacy with greater than 24 days
average survival and cures ranging from 2/5 to 5/5. For 13, 16, and 17, there was also no notable
difference in metabolic stability in the other species. As expected, 13ꢀ21 had higher calculated
pK values than the corresponding primary (Tables 1 and 6) and tertiary (Tables 4 and 5) amineꢀ
a
containing ozonides. Additional polar functional groups including ethers (18, 21), and secondary
(20) and tertiary (15, 16, 19) alcohols seemed to have minimal impact on metabolic stability,
solubility or antimalarial efficacy and for the compounds tested, exposure remained high (Figure
3B and Table 2). Of these, 16 and 17 had the highest in vivo antimalarial efficacy and also had
the highest Log D values (Log D ~ 3.3) and exposure of the compounds tested.
7
.4
As we had determined that the N,NꢀdiMe and other acyclic tertiary amine analogs of 3 and 4 had
good potency against P. falciparum, but had no in vivo curative efficacy and were metabolically
unstable (data not shown), we turned our attention to tertiary amineꢀcontaining heterocycles
(Table 4). Although azetidine 22 was metabolically stable and had good solubility, it had no
curative efficacy, similar to the ring expanded pyrrolidine (23) and piperidine (24) analogs (the
latter also had significantly reduced solubility). Ozonide 25, the gemꢀdifluoro isostere of 24, also
had no curative efficacy in vivo and was metabolically unstable. However, substitution of the
piperidine distal carbon in 24 with carboxamide (26) and alcohol (27) functional groups had
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minimal impact on metabolic stability and somewhat improved in vivo efficacy for the latter (21
days survival and 1/5 cures). The diprotic piperazine 28 and piperazine urea 29 had no curative
efficacy in vivo, but they were metabolically stable and 28 exhibited good solubility and
exposure (Figure 3C and Table 2). Carboxamide 30 was metabolically unstable and afforded no
cures, whereas sulfonamide 31 cured 2/5 mice, but was also metabolically unstable. For 31,
extending the ethyl to a propyl linker (32) significantly improved curative efficacy (5/5 cures),
and marginally improved metabolic stability, but 32 had very low solubility. Finally, piperidine
tertiary carbinol 33 had good antimalarial efficacy (30 days survival and 4/5 cures), intermediate
metabolic stability and reasonably good exposure in spite of its very poor aqueous solubility.
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Expanding the SAR of the piperidine and piperazine ozonides (Table 4), the profiles of the
corresponding morpholino and thiomorpholine 1ꢀoxide (thiomorpholine sulfoxide) ozonides are
described in Table 5. In addition, 42 and 43 illustrate the effect of adding fluorine atoms to the
distal carbon atoms of the spiroadamantane substructure of 2. Morpholines 2 and 34 and
thiomorpholine sulfoxides 37 and 38 afforded complete cures (5/5 mice), but they had marginal
metabolic stability in human liver microsomes and generally poor aqueous solubility, not
surprising due to their relatively high lipophilicity (Log D > 4). Interestingly, 2, 34, and 38
7
.4
were considerably more stable in microsomes from mice, rats and dogs compared to human
microsomes, a characteristic not seen for the other ozonides tested. In comparison, the 6,6ꢀ
difluoro analogue 43 exhibited comparable stability across species. The corresponding
thiomorpholine sulfones had no curative efficacy (data not shown). Homomorpholine 35, the
ringꢀexpanded analog of 2, was completely curative, and despite its high lipophilicity, it had
better metabolic stability than the latter although its in vivo exposure was somewhat lower than
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the other highly efficacious compounds (Table 2 and Figure 3C). Ozonide 36, the secondary
alcohol derivative of 34, had lower antimalarial efficacy and lower metabolic stability than the
5
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latter. As anticipated, 39, the spirocyclic derivative of 2, had better metabolic stability and
solubility than the latter, although it had no in vivo curative efficacy. The ortho (41) and meta
(40) regioisomers of 2 were less effective in vivo than the latter and they had no better metabolic
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stability or solubility (41). Ozonides 42 and 43 were designed to block the metabolic oxidation
of the spiroadamantane substructure of 2 with fluorine substitution. Both 42 and 43 had high
antimalarial efficacy; 42 had similar metabolic stability compared to 2, whereas 43 was
marginally more metabolically stable and had similarly poor aqueous solubility.
Data in Table 6 illustrate the outcome of replacing the aryl ether oxygen atom with a methylene
carbon (44ꢀ46), or carboxamide (47) and sulfonamide (48, 49) functional groups. Compared to 2,
each of these compounds had improved metabolic stability. The poorlyꢀsoluble morpholineꢀ
containing 45, which was also the most lipophilic of the series, was as effective as 2 in the P.
berghei model whereas the primary carboxamide 46 and primary amine 49, with Hꢀbond
donating functional groups, had only moderate curative efficacy (1/5 to 2/5 cures). Both 47 and
48 had poor in vivo antimalarial efficacy.
To assess whether some of the most promising ozonides (> 3/5 cures) had prophylactic potential,
they were each administered as a single 30 mg/kg oral dose 24 h prior to infection (Table 7). In
this experiment, only 35 was not at least partially protective, although it did increase survival
time. The remaining ozonides protected from 1/5 to 5/5 of the P. bergheiꢀinfected mice. As we
28
have shown before, AS and 1 afforded no protection in this experiment whereas mefloquine
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(
MFQ) protected 3/5 of the infected animals. Extending the prophylaxis data in Table 7, selected
ozonides were administered as single 100 mg/kg oral doses 48, 72, and 96 h preꢀinfection (Table
). Again, AS and 1 afforded no protection in this experiment, whereas mefloquine (MFQ)
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protected 5/5 of the infected animals at 48 h, but only extended survival time 3ꢀfold when given
72 h before infection. This is in contrast to the other ozonides, all of which (with the exception of
4
) were partially to fully protective at all of the preꢀinfection dosing times. Most notably, 2 and
8 were fully protective when administered 96 h prior to infection.
3
Mouse Exposure
A subset of the most promising ozonides were administered to nonꢀinfected mice at a single oral
dose of 30 mg/kg in an aqueous suspension vehicle to see if there was a correlation between
curative efficacy, prophylactic efficacy and exposure. From a practical standpoint it was
necessary to assume that exposure profiles generated in nonꢀinfected mice provided a reasonable
estimation of exposure in P. berghei infected mice.
Compared to 8’ꢀalkyl ozonide 1, the 8’ꢀaryl ozonides all had significantly higher and more
prolonged oral exposures as illustrated in Figure 3 and Table 2. Even in the absence of data for
many of the less efficacious compounds, the results suggest that compounds curing 4/5 or 5/5
mice (2, 10, 11, 16, 17, 19, 33) generally had higher exposure compared to ozonides that cured
no mice or had only limited curative efficacy (1/5ꢀ3/5 cures; 5, 9, 13, 14, 18, 20, 28). The
exceptions to this included 35 where the exposure profile was somewhat lower than for the other
highly curative compounds yet it was still highly effective, and 18 which had very good exposure
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but more limited efficacy. In terms of prophylaxis, the high efficacy of 2 was consistent with its
high and extended exposure profile. Ozonides 33 and 10 were also consistent with this trend as
both compounds protected 4/5 mice and had high and extended exposure profiles. Ozonide 19
was an exception as it would have been expected to be more protective than it was based on its
exposure profile.
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One factor not taken into account in these exposure studies is the potential difference in plasma
protein binding between compounds and the impact this would have on unbound concentrations
and efficacy. The other interesting factor is that some of the tested ozonides exhibiting very poor
aqueous solubility (i.e. 2, 33, 35) still had good exposure and high efficacy. This could be a
reflection of the short term nature of the kinetic solubility experiment or may indicate that
components of the aqueous suspension vehicle used for dosing (i.e. hydroxypropylmethyl
cellulose and/or Tween 80) have a positive impact on compound solubility. Based on their
physicochemical properties, all of these compounds would also be expected to have good
membrane permeability which could also partially mitigate the impact of poor solubility. Taken
together, the exposure results suggest that high singleꢀdose curative efficacy is most often
associated with high and prolonged exposure, consistent with the improved efficacy of the cisꢀ8’ꢀ
aryl ozonides relative to their cisꢀ8’ꢀalkyl counterparts such as 1. However, exposure on its own
does not explain the presence or absence of curative efficacy for this series of highly potent
ozonides indicating that other yet to be identified factors also contribute to the in vivo efficacy.
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Exploratory Toxicology
In multiple dose exploratory toxicology studies, five consecutive doses of 100 and 300 mg/kg of
ozonides 2ꢀ4, 10, 11, 19, 33, and 38 were administered by oral gavage at 3ꢀday intervals to male
rats. The spectrum of toxicities were comparable between the tested ozonides and included doseꢀ
dependent signs of impaired general condition, inhibition of body weight development, changes
of biochemistry parameters (albumin, triglycerides, transaminases, bilirubin), adrenal cortical
vacuolation, atrophic changes of lymphoid organs, and indication of gastric irritation (glandular
vacuolation and focal ulceration in the forestomach).
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After daily oral dosing of primary amino ozonide 3, the only significant finding was slight
lymphoid atrophy and adrenal cortical vacuolation. For 3 and the other primary amino ozonides
4
, 10, and 11, there was a 1ꢀ to 4ꢀfold accumulation of concentrations over the dosing period.
The spectrum and severity of toxicological findings and plasma concentrations after
administration of 4 and 11 were comparable to that of 3. Plasma levels of 10 were in the range
observed for the other primary amino ozonides, but the signs of toxicity were more severe
(mortality in 9/12 rats at 300 mg/kg). Administration of secondary amino ozonide 19 led to
relatively high plasma concentrations (> 1.9 µg/mL at 300 mg/kg) and resulted in an overall
moderate toxicity (mortality in 3/12 rats at 300 mg/kg). Although plasma concentrations of 33
and 38 were also relatively high (1ꢀ2 µg/mL at 300 mg/kg), only minor signs of toxicity were
seen. Finally, administration of 2 resulted in significantly (4 to 25ꢀfold) higher peak plasma
concentrations (4.6 and 6.3 µg/mL at 100 and 300 mg/kg) than the other ozonides, but this was
associated with only minor signs of toxicity.
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In conclusion, the spectrum of toxicities was comparable among these ozonides. In contrast,
plasma exposure was variable, but this was not generally paralleled by the severity of toxicity. In
consideration of variable exposure, the toxicity of the ozonides after oral administration to male
rats can be ranked as follows: 2 < 33 < 38 < 11 < 4 < 3 < 19 < 10. Although there was no
correlation between pK or Log D values and toxicity for these ozonides, it is evident that the
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a
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three ozonides with the lowest toxicities were tertiary amines.
Summary
4
2
As we have previously observed in the SAR of the cisꢀ8’ꢀalkyl ozonide 1, the in vitro
antimalarial potency for these cisꢀ8’ꢀaryl ozonide analogs of 2 differed by less than one order of
magnitude and there was no direct correlation between in vitro and in vivo antimalarial activity.
Neither pK nor Log D correlated with in vivo antimalarial efficacy, although we note an
a
7.4
overall trend that metabolically stable ozonides tended to have higher pK and lower Log D7.4
a
values (Figure 2) and the primary and secondary amino ozonides had better metabolic stabilities
and aqueous solubility than the tertiary amino ozonides. For the primary amino ozonides,
addition of polar functional groups decreased in vivo antimalarial efficacy. For the secondary
amino ozonides, additional functional groups had variable effects on metabolic stability and
efficacy, but the most effective members of this series also had the highest Log D values. For
7
.4
the tertiary amino ozonides, addition of polar functional groups with Hꢀbond donors increased
metabolic stability, but decreased in vivo antimalarial efficacy. Primary and tertiary amino
ozonides with cycloalkyl and heterocycle substructures were superior to their acyclic
counterparts. Replacing the ether oxygen with a methylene carbon, carboxamide, or sulfonamide
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significantly improved metabolic stability, but only the most lipophilic compound had high
curative efficacy. Ozonides with fluorine substitution in the spiroadamantane substructure had
high efficacy and the 6ꢀdifluoroꢀsubstituted analog also had improved metabolic stability. The
high curative efficacy of these ozonides was most often associated with high and prolonged
plasma exposure, but exposure on its own did not explain the presence or absence of either
curative efficacy or in vivo toxicity. It is evident that our focus on singleꢀdose curative efficacy
in this series of cisꢀ8’ꢀaryl ozonides seemed to selfꢀselect for relatively lipophilic compounds in
spite of the negative impact this has on aqueous solubility. Of the ozonides tested, the three that
were the least toxic were tertiary amines.
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Experimental Section
1
13
General. Melting points are uncorrected. 1D H and C NMR spectra were recorded on a 500
MHz spectrometer using CDCl or DMSOꢀd as solvents. All chemical shifts are reported in
3
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parts per million (ppm) and are relative to internal (CH ) Si (0 ppm) for H and CDCl (77.2
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3
1
3
ppm) or DMSOꢀd (39.5 ppm) for C NMR. Silica gel (sg) particle size 32−63 ꢁm was used for
6
all flash column chromatography. Reported reaction temperatures are those of the oil bath.
HPLC and combustion analysis confirmed that all target compounds have a purity of at least
95%
cis-Adamantane-2-spiro-3'-8'-[4'-[(4'-hydroxy-4'-piperidinyl)methoxy]phenyl]-1',2',4'-
trioxaspiro[4.5]decane mesylate (15). Step 1. To a solution of cisꢀadamantaneꢀ2ꢀspiroꢀ3'ꢀ8'ꢀ(4'ꢀ
44
hydroxyphenyl)ꢀ1',2',4'ꢀtrioxaspiro[4.5]decane (50) (0.50 g, 1.4 mmol) and 1ꢀoxaꢀ6ꢀ
azaspiro[2,5]octaneꢀ6ꢀcarboxylic acid tꢀbutyl ester (0.36 g, 1.69 mmol) in isopropanol (15 mL)
was added 1 M aqueous KOH solution (1 mL). The resulting mixture was stirred at 60 °C for 20
h and then quenched with water (20 mL). The precipitate was collected by filtration, washed
with water, and dried in vacuo at 40 °C to afford cisꢀadamantaneꢀ2ꢀspiroꢀ3'ꢀ8'ꢀ[4'ꢀ[(1'ꢀtertꢀ
butyloxycarbonylꢀ4'ꢀhydroxyꢀ4'ꢀpiperidinyl)methoxy]phenyl]ꢀ1',2',4'ꢀtrioxaspiro[4.5]decane (51)
1
as a colorless solid (0.71 g, 89%). H NMR (CDCl ) δ 1.47 (s, 9H), 1.52ꢀ1.64 (m, 4H), 1.65ꢀ2.08
3
(m, 22H), 2.19 (s, 1H), 2.44ꢀ2.56 (m, 1H), 3.14ꢀ3.30 (m, 2H), 3.78 (s, 2H), 3.78ꢀ4.00 (m, 2H),
6
.83 (d, J = 8.5 Hz, 2H), 7.13 (d, J = 8.5 Hz, 2H). Step 2. A mixture of 51 (0.50 g, 0.88 mmol)
and 1.5 M MsOH in THF (28 mL) was stirred at rt for 3 h. The resulting precipitate was
collected by filtration, washed with ether (30 mL), and dried in vacuo to afford 15 as a colorless
1
solid (0.44 g, 88%). Mp 154ꢀ155 °C; H NMR (CDCl ) δ 1.60ꢀ2.10 (m, 26H), 2.46ꢀ2.54 (m, 1H),
3
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.79 (s, 3H), 3.29ꢀ3.45 (m, 4H), 3.84 (s, 2H), 6.82 (d, J = 8.5 Hz, 2H), 7.13 (d, J = 8.5 Hz, 2H),
1
3
.52 (brs, 1H), 8.86 (brs, 1H); C NMR (CDCl ) δ 26.45, 26.84, 30.35, 31.60, 34.66, 34.77,
3
6.37, 36.77, 39.30, 40.09, 41.98, 67.32, 74.84, 108.35, 111.36, 114.46, 127.76, 139.25, 156.54.
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1
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0
Anal. Calcd for C H NO S 2.5 H O: C, 57.03; H, 7.92; N, 2.29. Found: C, 56.78; H, 7.66; N,
2
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2
2.05.
cis-Adamantane-2-spiro-3'-8'-[4'-[(4'-hydroxy-2',2',6',6'-tetramethyl-4'-
piperidinyl)methoxy]phenyl]-1',2',4'-trioxaspiro[4.5]decane p-tosylate (16). Step 1. A
mixture of 2,2,6,6ꢀtetramethylꢀ4ꢀpiperidinone monohydrate (3.46 g, 20 mmol),
trimethylsulfoxonium iodide (4.4 g, 20 mmol), potassium tꢀbutoxide (4.4 g, 40 mmol) in
ethylene glycol dimethyl ether (80 mL) was refluxed for 8 h with under Ar. After cooling to rt,
the reaction mixture was concentrated to 50 mL, diluted with ether (300 mL), washed with water
(300 mL) and brine (300 mL), dried over MgSO , filtered, and concentrated to afford 5,5,7,7ꢀ
4
45
1
tetramethylꢀ1ꢀoxaꢀ6ꢀazaspiro[2,5]octane as a colorless oil (3.0 g, 89%). H NMR (CDCl ) δ
3
4
4
1
.21 (s, 6H), 1.25 (s, 6H), 1.48 (q, J = 7.5 Hz, 4H), 2.69 (s, 2H). Step 2. To a solution of 50
45
(0.50 g, 1.40 mmol) and 5,5,7,7ꢀtetramethylꢀ1ꢀoxaꢀ6ꢀazaspiro[2,5]octane (0.50 g, 2.96 mmol)
in isopropanol (25 mL) was added 1 M aq. KOH (4 mL). The resulting mixture was stirred at 60
C for 20 h, cooled to rt, and quenched with water (20 mL). The precipitate was collected by
°
filtration and washed with water to afford a 1:1 mixture of the unreacted 50 and the desired free
base of 16. After the crude product was dissolved in ether (10 mL), a solution of pꢀ
toluenesulfonic acid monohydrate (0.15 g, 0.79 mmol) in ethyl acetate (30 mL) was added. The
precipitate was collected by filtration to afford 16 as a colorless solid (0.47 g, 48%). Mp 153ꢀ154
1
°
C; H NMR (CDCl ) δ 1.54ꢀ2.08 (m, 38H), 2.26 (s, 3H), 2.45ꢀ2.54 (m, 1H), 2.49 (s, 1H), 3.52
3
(s, 2H), 6.73 (d, J = 8.5 Hz, 2H), 7.07ꢀ7.16 (m, 4H), 7.55 (d, J = 12.0 Hz, 1H), 7.73 (d, J = 8.0
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Hz, 2H), 8.79 (d, J = 12.0 Hz, 1H); C NMR (CDCl ) δ 21.24, 25.84, 26.45, 26.85, 31.21, 31.62,
3
3
4.69, 34.78, 36.37, 36.77, 40.93, 42.03, 56.29, 70.39, 108.37, 111.40, 114.46, 125.84, 127.70,
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28.88, 139.27, 140.26, 142.31, 156.41. Anal. Calcd for C H NO S: C, 67.12; H, 7.94; N, 2.01.
3
9
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59
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Found: C, 66.89; H, 7.78; N, 2.12.
cis-Adamantane-2-spiro-3'-8'-[4'-[2'-(4'-piperidinyl)ethoxy]phenyl]-1',2',4'-
4
4
trioxaspiro[4.5]decane mesylate (17). Step 1. To a solution of 50 (0.70 g, 1.97 mmol) in dry
acetonitrile (80 mL) were added powered NaOH (0.50 g, 12.5 mmol) and tetrabutylammonium
hydrogensulfate (0.05 g, 0.15 mmol). The mixture was stirred at rt for 30 min before tertꢀbutyl 4ꢀ
46
(2ꢀmethylsulfonyloxyethyl)piperidineꢀ1ꢀcarboxylate (52) (0.61 g, 1.98 mmol) was added. The
reaction mixture was stirred at 60 °C for 12 h, cooled to rt, filtered, and washed with CH Cl .
2
2
After solvent removal in vacuo, the crude product was purified by crystallization from 1:1
EtOH:H O to afford cisꢀadamantaneꢀ2ꢀspiroꢀ3'ꢀ8'ꢀ[4'ꢀ[2'ꢀ(1'ꢀtertꢀbutyloxycarbonyl ꢀ4'ꢀ
2
piperidinyl)ethoxy]phenyl]ꢀ1',2',4'ꢀtrioxaspiro[4.5]decane (52) as a colorless solid (0.90 g, 81%).
1
H NMR (CDCl ) δ 1.10ꢀ1.22 (m, 2H), 1.45 (s, 9H), 1.64ꢀ2.08 (m, 29H), 2.45ꢀ2.54 (m, 1H),
3
2.60ꢀ2.76 (m, 2H), 3.98 (t, J = 6.0 Hz, 2H), 4.00ꢀ4.20 (m, 2H), 6.81 (d, J = 8.5 Hz, 2H), 7.11 (d,
J = 8.5 Hz, 2H). Step 2. A mixture of 52 (0.90 g, 1.59 mmol) and 1.5 M MsOH in THF (30 mL)
was stirred at rt for 4 h. The resulting precipitate was filtered off, washed with ether (30 mL),
1
and dried in vacuo to afford 17 as a colorless solid (0.70 g, 78%). Mp 139ꢀ140 °C; H NMR
(CDCl ) δ 1.56ꢀ2.08 (m, 29H), 2.45ꢀ2.54 (m, 1H), 2.78 (s, 3H), 2.82ꢀ2.94 (m, 2H), 3.44ꢀ3.53 (m,
3
2
8
3
H), 3.97 (t, J = 6.0Hz, 2H), 6.80 (d, J = 8.5 Hz, 2H), 7.11 (d, J = 8.5 Hz, 2H), 8.41 (brs, 1H),
1
3
.76 (brs, 1H); C NMR (CDCl ) δ 16.94, 26.46, 26.86, 28.62, 31.05, 31.63, 34.72, 34.79,
3
5.11, 36.38, 36.78, 39.29, 42.02, 44.19, 64.59, 108.41, 111.38, 114.23, 127.67, 138.59, 157.00.
Anal. Calcd for C H NO S: C, 63.92; H, 8.05; N, 2.48. Found: C, 63.73; H, 7.91; N, 2.56.
3
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cis-Adamantane-2-spiro-3'-8'-[4'-[2'-(4'-piperidinyloxy)ethoxy]phenyl]-1',2',4'-
trioxaspiro[4.5]decane mesylate (18). Step 1. To a solution of tꢀbutyl 4ꢀ(2ꢀethoxyꢀ2ꢀ
4
7
oxoethoxy)piperidineꢀ1ꢀcarboxylate (53) (1.00 g, 3.48 mmol) in ether (10 mL) and THF (2 mL)
was added dropwise 2 M lithium borohydride in THF (1.74 ml, 3.48 mmol) followed by 1 M
lithium triethylborohydride in THF (0.35 mL, 0.35 mmol). The resulting mixture was stirred at rt
overnight and then diluted with ether (30 mL). The mixture was washed with 2 M aq. NaOH (2 x
0
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7
8
9
0
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5
mL), water (2 x 5 mL), and brine (5 mL), dried over MgSO , filtered and concentrated to
4
afforded tꢀbutyl 4ꢀ(2ꢀhydroxyethoxy)piperidineꢀ1ꢀcarboxylate (54) as a colorless oil (0.80 g,
1
9
4%). H NMR (CDCl ) δ 1.46 (s, 9H), 1.44ꢀ1.68 (m, 2H), 1.80ꢀ1.90 (m, 2H), 3.03ꢀ3.12 (m,
3
2H), 3.46ꢀ3.53 (m, 1H), 3.56ꢀ3.61 (m, 2H), 3.70ꢀ3.80 (m, 4H). Step 2. To a solution of 54 (0.80
g, 3.30 mmol) and triethylamine (1.00 ml, 8.00 mmol) in CH Cl (20 mL) at 0 °C was added
2
2
dropwise methanesulfonyl chloride (0.27 ml, 3.50 mmol). After stirring at 0 °C for 1 h and at rt
for 12 h, the reaction mixture was washed with water (2 x 10 mL) and brine (10 mL), dried over
MgSO , filtered, and concentrated to afford tꢀbutyl 4ꢀ[2ꢀ(methanesulfonyloxy)ethoxy]piperidineꢀ
4
1
1
ꢀcarboxylate (55) as a pale yellow oil (0.86 g, 82%). H NMR (CDCl ) δ 1.46 (s, 9H), 1.36ꢀ1.60
3
(m, 2H), 1.76ꢀ1.90 (m, 2H), 3.06 (s, 3H), 3.00ꢀ3.14 (m, 2H), 3.48ꢀ3.56 (m, 1H), 3.66ꢀ3.82 (m,
4
4
4
H), 4.37 (t, J = 4.0 Hz, 2H). Step 3. To a solution of 50 (0.50 g, 1.40 mmol) in dry acetonitrile
60 mL) were added powered NaOH (0.10 g, 2.5 mmol) and tetrabutylammonium
hydrogensulfate (0.50 g, 1.47 mmol). The mixture was stirred at rt for 30 min before 55 (0.86 g,
.67 mmol) was added. The reaction mixture was stirred at 60 °C overnight, cooled to rt, filtered,
and washed with CH Cl After the filtrate was concentrated, the crude product was purified by
(
2
2
2.
crystallization from 5:1 EtOH:H O to afford cisꢀadamantaneꢀ2ꢀspiroꢀ3'ꢀ8'ꢀ[4'ꢀ[2'ꢀ(4'ꢀ
2
piperidinyloxy)ethoxy]phenyl]ꢀ1',2',4'ꢀtrioxaspiro[4.5]decane (56) as a colorless solid (0.70 g,
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5%). H NMR (CDCl ) δ 1.45 (s, 9H), 1.64ꢀ2.08 (m, 26H), 2.44ꢀ2.54 (m, 1H), 3.04ꢀ3.14 (m,
3
H), 3.48ꢀ3.60 (m, 1H), 3.66ꢀ3.82 (m, 4H), 4.09 (t, J = 5.0 Hz, 2H), 6.84 (d, J = 8.5 Hz, 2H),
.11 (d, J = 8.5 Hz, 2H). Step 4. A mixture of 56 (0.70 g, 1.20 mmol) and 1.5 M MsOH in THF
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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36
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
14 mL) was stirred at rt for 4 h. The resulting precipitate was filtered off, washed with ether (30
1
mL), and dried in vacuo to afford 18 as a colorless solid (0.40 g, 57%). Mp 136ꢀ137 °C; H
NMR (CDCl ) δ 1.64ꢀ2.16 (m, 26H), 2.44ꢀ2.54 (m, 1H), 2.79 (s, 3H), 3.14ꢀ3.24 (m, 2H), 3.26ꢀ
3
3
.36 (m, 2H), 3.70ꢀ3.80 (m, 1H), 3.78 (t, J = 4.5 Hz, 2H), 4.08 (t, J = 4.5 Hz, 2H), 6.82 (d, J =
1
3
8.5 Hz, 2H), 7.12 (d, J = 8.5 Hz, 2H), 8.48 (brs, 1H), 8.59 (brs, 1H); C NMR (CDCl ) δ 26.46,
3
2
6.85, 27.07, 31.62, 34.71, 34.79, 36.37, 36.78, 40.20, 42.02, 67.01, 67.49, 70.34, 108.41,
1
11.37, 114.41, 127.68, 138.76, 156.92. Anal. Calcd for C H NO S 1.5 H O: C, 59.38; H, 7.97;
30 45
8
2
N, 2.31. Found: C, 59.26; H, 7.95; N, 2.27.
cis-Adamantane-2-spiro-3'-8'-[4'-[3'-[(4'-tetrahydropyranyl)amino]propoxy]phenyl]-
',2',4'-trioxaspiro[4.5]decane mesylate (21). A mixture of cisꢀadamantaneꢀ2ꢀspiroꢀ3'ꢀ8'ꢀ[4'ꢀ(3'ꢀ
1
2
8
aminopropoxy)phenyl]ꢀ1',2',4'ꢀtrioxaspiro[4.5]decane (4) (0.395 g, 0.955 mmol), tetrahydroꢀ
Hꢀpyranꢀ4ꢀone (0.098 g, 0.97 mmol), acetic acid (0.7 mL, 12 mmol), and sodium
triacetoxyborohydride (0.342 g, 1.53 mmol) in CH Cl (10 mL) was stirred overnight at rt. The
4
2
2
solution was then sequentially washed with 1 M aq. NaOH (30 mL), water (50 mL), and brine
30 mL). The organic layer was dried over anhydrous MgSO , filtered, and concentrated to
(
4
afford the free base of 21. To a solution of the free base of 21 in 1:1 CH Cl :EtOAc (18 mL) was
2
2
added a solution of MsOH (0.064 g, 0.67 mmol) in ethyl acetate (2 mL). After stirring for 30
min, the solution was concentrated and triturated with ethyl acetate (20 mL). The precipitate was
collected by filtration, washed with ethyl acetate (10 mL), and dried to afford 21 (0.281 g, 50%).
1
Mp 138–140 °C dec; H NMR (CDCl ) δ 1.64ꢀ2.10 (m, 26H), 2.31ꢀ2.35 (m, 2H), 2.46ꢀ2.51 (m,
3
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2
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H), 2.73 (s, 3H), 3.17ꢀ3.27 (m, 3H), 3.35ꢀ3.39 (m, 2H), 4.02ꢀ4.06 (m, 4H), 6.79 (d, J = 8.8 Hz,
1
3
2H), 7.09 (d, J = 8.8 Hz, 2H), 8.82ꢀ8.87 (brs, 2H); C NMR (CDCl ) δ 25.83, 26.40, 26.80,
3
2
8.92, 31.56, 34.64, 34.73, 36.32, 36.72, 39.54, 41.84, 41.92, 54.43, 64.67, 65.99, 108.32,
0
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0
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9
0
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8
9
0
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
11.30, 114.17, 127.63, 138.79, 156.61. Anal. Calcd for C H NO S: C, 62.71; H, 7.98; N, 2.36.
3
1
47
8
Found: C, 63.07; H, 8.03; N, 2.51.
cis-Adamantane-2-spiro-3'-8'-[4'-[2'-(1'-azetidinyl)ethoxy]phenyl]-1',2',4'-
trioxaspiro[4.5]decane p-tosylate (22). A mixture of cisꢀadamantaneꢀ2ꢀspiroꢀ3'ꢀ8'ꢀ[4'ꢀ(2'ꢀ
4
8
bromoethoxy)phenyl]ꢀ1',2',4'ꢀtrioxaspiro[4.5]decane (57) (0.50 g, 1.08 mmol), azetidine
hydrochloride (0.20 g, 2.14 mmol), and K CO (2.0 g) in dry acetonitrile (80 mL) was heated at
2
3
60 °C for 24 h. The reaction mixture was cooled to rt, filtered, and concentrated. After being
washed with water (50 mL) and dried in vacuo, the free base of 22 was dissolved in CH Cl (5
2
2
mL) to which a solution of pꢀtoluenesulfonic acid monohydrate (0.09 g, 0.47 mmol) in ethyl
acetate (30 mL) was added. The precipitate was collected by filtration to afford 22 as a colorless
1
solid (0.16 g, 24%). Mp 125ꢀ126 °C; H NMR (CDCl ) δ 1.60ꢀ2.10 (m, 23H), 2.34 (s, 3H), 2.44ꢀ
3
2.55 (m, 1H), 2.79ꢀ2.91 (m, 1H), 3.48ꢀ3.58 (m, 2H), 4.04ꢀ4.16 (m, 2H), 4.25ꢀ4.32 (m, 2H), 4.47ꢀ
4.57 (m, 2H), 6.74 (d, J = 8.5 Hz, 2H), 7.10 (d, J = 8.5 Hz, 2H), 7.13 (d, J = 8.5 Hz, 2H), 7.75 (d,
1
3
J = 8.5 Hz, 2H), 11.68 (brs, 1H); C NMR (CDCl ) δ 17.09, 21.32, 26.43, 26.83, 31.57, 34.65,
3
3
4.76, 36.36, 36.75, 41.99, 54.25, 56.02, 63.82, 108.32, 111.40, 114.35, 125.88, 127.83, 128.91,
+
139.69, 140.34, 141.60, 155.59. HRMS (ESIꢀTOF) m/z: [M + H] Calcd for C H NO
2
7
38
4
440.2801; Found 440.2803.
cis-Adamantane-2-spiro-3'-8'-[4'-[2'-hydroxy-3'-(4'-morpholinyl)propoxy]phenyl]-1',2',4'-
trioxaspiro[4.5]decane mesylate (36). Step 1. A mixture of 4ꢀ(4ꢀhydroxyphenyl)cyclohexanone
(58) (5.00 g, 26.3 mmol), epichlorohydrin (10.0 g, 108 mmol), and potassium carbonate (7.26 g,
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2.6 mmol) in acetonitrile (100 mL) was heated to reflux for 24 h. After cooling, the reaction
mixture was filtered and concentrated in vacuo to afford a residue that was purified by
crystallization from ether to afford 4ꢀ[4ꢀ(oxiranꢀ2ꢀylmethoxy)phenyl]cyclohexanone (59) as a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
colorless solid (5.60 g, 87%). Mp 76ꢀ77 °C; H NMR (CDCl ) δ 1.84ꢀ1.98 (m, 2H), 2.15ꢀ2.25
3
(m, 2H), 2.45ꢀ2.55 (m, 4H), 2.76 (dd, J = 5.0, 2.5 Hz, 1H), 2.91 (t, J = 4.0 Hz, 1H), 3.34ꢀ3.40 (m,
1
H), 3.95 (dd, J = 11.0, 6.0 Hz, 1H), 4.21 (dd, J = 11.0, 3.0 Hz, 1H), 6.89 (d, J = 9.0 Hz, 2H),
1
3
7
.16 (d, J = 9.0 Hz, 2H); C NMR (CDCl ) δ 34.14, 41.35, 41.88, 44.70, 50.16, 68.78, 114.68,
3
127.61, 137.47, 157.09, 211.29. Step 2. A solution of 59 (1.00 g, 4.10 mmol) and Oꢀmethyl 2ꢀ
52
adamantanone oxime (60) (1.09 g, 6.10 mmol) in cyclohexane (80 mL) and CH Cl (20 mL)
2
2
3
7
was treated with ozone according to the method of Dong et al. After removal of solvents, the
crude product was purified by sg flash chromatography (10% ꢀ 20% ether in hexane) to afford
cisꢀadamantaneꢀ2ꢀspiroꢀ3'ꢀ8'ꢀ[4'ꢀ(oxiranꢀ2ꢀylmethoxy)phenyl]ꢀ1',2',4'ꢀtrioxaspiro[4.5]decane (61)
1
as a colorless solid (0.90 g, 54%). Mp 102ꢀ103 °C; H NMR (CDCl ) δ 1.64ꢀ2.08 (m, 22H),
3
2
.45ꢀ2.55 (m, 1H), 2.75 (dd, J = 5.5, 3.0 Hz, 1H), 2.90 (t, J = 4.5 Hz, 1H), 3.32ꢀ3.38 (m, 1H),
.95 (dd, J = 11.0, 6.0 Hz, 1H), 4.18 (dd, J = 11.0, 3.0 Hz, 1H), 6.85 (d, J = 9.0 Hz, 2H), 7.12 (d,
3
1
3
J = 9.0 Hz, 2H); C NMR (CDCl ) δ 26.44, 26.84, 31.60, 34.70, 34.77, 36.36, 36.76, 42.01,
3
4
4.75, 50.18, 68.74, 108.39, 111.35, 114.48, 127.66, 138.92, 156.79. Step 3. To a solution of 61
(
0.30 g, 0.73 mmol) in methanol (20 mL) was added morpholine (1.0 mL). The reaction mixture
was stirred at 60 °C overnight and cooled to rt. After removal of the solvents, the residue was
purified by crystallization from 1:1 EtOH:H O to afford the pure free base of 36. To a solution of
2
the free base of 36 in ether (10 mL) was added a solution of MsOH (0.05 g, 0.52 mmol) in ether
(
10 mL). The precipitate was collected by filtration to afford 36 as a colorless solid (0.26 g,
1
6
0%). Mp 167ꢀ168 °C; H NMR (DMSOꢀd ) δ 1.48ꢀ1.58 (m, 2H), 1.62ꢀ1.96 (m, 20H), 2.30 (s,
6
28
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