StructureâActivity Relationship of the Antimalarial Ozonide

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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§, 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, CH-4002 Basel, Switzerland

£

University of Basel, CH-4003 Basel, Switzerland

∞

¶

Basilea Pharmaceutica Ltd., Grenzacherstrasse 487, CH-4058 Basel, Switzerland

F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, CH-4070 Basel, Switzerland

*S.A.C.: Phone: 61 3 9903 9626. E-mail: [email protected] *J.L.V.: Phone: 402 559 5362. E-mail: [email protected]

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Abstract 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 metabolic stabilities than tertiary amino ozonides, consistent with their higher pKa and lower Log D7.4 values. For 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 D7.4 values. For tertiary amino ozonides, addition of polar functional groups with Hbond 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: 1,2,4-trioxolane, ozonide, antimalarial, artemisinin, OZ439, artefenomel

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The semisynthetic artemisinins (Figure 1) are vitally important first-line antimalarial drugs that comprise the fast-acting component of artemisinin combination therapies (ACT).1,2 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 the kelch domain–carrying protein K13.3,4 The discovery of artemisinin5,6 led to extensive investigation of structurally diverse synthetic peroxides containing many different types of peroxide heterocycles including 1,2-dioxanes, 1,2,4-trioxanes, 1,2,4-trioxolanes, and 1,2,4,5tetraoxanes.7-10 Although the precise mechanism of action of antimalarial peroxides is still not fully understood, it is hypothesized that the pharmacophoric peroxide bond11 undergoes reductive activation by heme released during parasite hemoglobin digestion12-14 to produce carbon-centered radicals that alkylate heme and parasite proteins12,15-21 leading to downstream peroxidative damage and oxidative stress.17,22,23

Figure 1. Artemisinin (ART) and its semisynthetic derivatives dihydroartemisinin (DHA), artemether (AM), and artesunate (AS) and ozonides 1 and 2.

The first synthetic peroxide to be registered was ozonide 1 (OZ277),24 also known as arterolane,25-27 which was approved for the Indian market as a three-dose combination product with piperaquine (Synriam) for the treatment of uncomplicated malaria. More recently, nextgeneration ozonide 2 (OZ439, artefenomel)28 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 yet been determined (Figure 1).29-32 Compared to 8’-alkyl substituted ozonides such as 1, 8′-aryl substituted ozonides such as 2 are much more stable to premature blood-mediated degradation, yet maintain sufficient ferrous iron-reactivity to rapidly kill parasites.28,33 The 8’-aryl versus 8’alkyl substituent shifts the conformer equilibrium to the less reactive axial-peroxide conformer.33-35 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 least partially linked to an optimized ferrous iron reactivity profile.28 For example, the half-lives in humans for 1 and 2 are approximately 3 h25,27 and 46-60 h,29,31 respectively.

In this paper, we profile ozonides 3-49 to describe the structure-activity 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 1,2,4-trioxolane is superior to the corresponding 1,2,4-trioxane, 1,2,4,5-tetraoxane, and 1,2-dioxolane peroxy heterocycles, the latter of which are nearly inactive;36 2) the spiroadamantane ring system37,38 and peroxide bond11,37 are essential for activity; 3) more lipophilic ozonides tend to have better oral antimalarial efficacies than their more polar counterparts, 37,38 an outcome consistent with that seen for other classes of synthetic peroxides;7 4) ozonides with a wide range of neutral and basic, but not acidic, functional groups have good antimalarial activity;38-41 5) weak base ozonides possess the best overall ADME properties;40,42 and 6) hydroxylated ozonide CYP450 metabolites have relatively low antimalarial activities.37,43 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 N-oxidation 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 D7.4 5.4 for 2 vs 0.9 for 1) that well illustrates one important hurdle in the optimization of these next generation ozonides.

Chemistry

As shown in Schemes 1-7, ozonide target compounds were prepared as their mesylate or tosylate salts; structures depicted are the free base forms. We identified ozonide phenol 5044 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 epoxide45 followed by tosylate salt formation afforded 16 in 48% overall yield; similarly, alkylation of the phenoxide of 50 with corresponding Boc-protected epoxide gave Boc ozonide 51 which underwent one-pot deprotection and mesylate salt formation to afford 15 in an overall yield of 78%. Alkylation of 50 with the requisite Boc-protected piperidine mesylate46 produced Boc ozonide 52 in 81% yield; a subsequent one-pot deprotection and mesylate salt formation afforded 17 in 78% yield.

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Scheme 1 a

a

Reagents and conditions: (a) 1-oxa-6-azaspiro[2,5]octane-6-carboxylic acid t-butyl ester,

isopropanol, 1 M aq. KOH 60 °C, 20 h; (b) 5,5,7,7-tetramethyl-1-oxa-6-azaspiro[2,5]octane, isopropanol, 1 M aq. KOH 60 °C, 20 h, then TsOH, EtOAc, rt; (c) powdered NaOH, tetrabutylammonium hydrogensulfate, CH3CN, rt, 0.5 h, then t-butyl 4-(2methylsulfonyloxyethyl)piperidine-1-carboxylate, 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 one-pot Boc deprotection and mesylate salt formation (57%) (Scheme 2). Intermediate 55 was obtained by reduction of Boc-protected ester 5347 to alcohol 54 (94%) followed by mesylate formation (82%).

Scheme 2a

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, CH2Cl2, 0 °C

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for 1 h, then rt for 12 h; (c) powdered NaOH, tetrabutylammonium hydrogensulfate, CH3CN, rt, 0.5 h, then 55 (t-butyl 4-[2-(methanesulfonyloxy)ethoxy]piperidine-1-carboxylate), 60 °C, 12 h; (d) 1.5 M MsOH in THF, rt, 4 h.

Reductive amination of amino ozonide 428 followed by mesylate salt formation afforded 21 in 50% yield (Scheme 3). Alkylation of bromoethoxy ozonide 5748 with azetidine or 2-oxa-6azaspiro [3,3]heptane49 followed by tosylate salt formation afforded 22 (24%) and 39 (18%), respectively.

Scheme 3a

a

Reagents and conditions: (a) tetrahydro-4H-pyran-4-one, AcOH, sodium triacetoxyborohydride,

CH2Cl2, rt, 12 h, then MsOH in EtOAc, rt, 0.5 h; (b) azetidine HCl, K2CO3, CH3CN, 60 °C, 24 h, then TsOH, EtOAc/CH2Cl2, rt; (c) 2-oxa-6-azaspiro [3,3]heptane tosylate, K2CO3, CH3CN, 60 °C, 24 h, then TsOH, EtOAc, rt.

Following a modified method of Erhardt et al.,50 keto phenol 58 was converted into keto epoxide 59 in 87% yield (Scheme 4). Griesbaum coozonolysis51 of 59 and O-methyl-2-adamantanone

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oxime (60)52 formed epoxy ozonide 61 (54%), which after exposure to morpholine and mesylate salt formation gave 36 in 60% yield.

Scheme 4a

a

Reagents and conditions: (a) epichlorohydrin, K2CO3, CH3CN, reflux, 24 h; (b) O3, 8:2

cyclohexane:CH2Cl2, 0 °C, 5 min; (c) morpholine, MeOH, 60 °C, 12 h, then MsOH, ether, rt, 0.5 h.

Griesbaum coozonolysis51 of keto acetate 6244 and fluorinated O-methyl-2-adamantanone oximes 63 and 6453 afforded ozonide esters 65 (54%) and 66 (44%), respectively (Scheme 5). These intermediates underwent one-pot acetate hydrolysis/alkylation with N-(2chloroethyl)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 5a

a

Reagents and conditions: (a) O3, 5-7:1 cyclohexane:CH2Cl2, 0 °C, 5 min; (b) N-(2-

chloroethyl)morpholine HCl, NaOH, tetrabutylammonium hydrogensulfate, CH3CN, 25 to 60 °C, 12 h, then MsOH, ether, rt, 0.5 h.

Alkylation of morpholine or isonipecotamide with ozonide benzyl chloride 6748 followed by salt formation afforded the mesylate of 45 (69%) and tosylate of 46 (61%) (Scheme 6). Conversion of ozonide acid 6838 to its N-hydroxysuccinimide active ester 69 (91%) followed by reaction of the latter with piperazine and salt formation afforded 47 (75%) as its mesylate.

Scheme 6a

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a

Reagents and conditions: (a) morpholine, Et3N, CH3CN, rt to 60 °C, 48 h, then MsOH in ether,

0 °C, 0.5 h; (b) isonipecotamide, K2CO3, CH3CN, rt to 60 °C, 48 h, then TsOH in EtOAc/ether, 0 °C, 0.5 h; (c) N-hydroxysuccinimide, EDCI, DMF, 0 ºC to rt, 12 h; (d) piperazine, CH2Cl2, rt, 12 h, then MsOH, EtOAc/ether, rt, 0.5 h.

Chlorosulfonylation of ketone 70 afforded crude 71 (10%) which was converted into pentafluorosulfonate ester 72 (70%) (Scheme 7). Griesbaum coozonolysis51 of 72 and 6052 formed ozonide sulfonate ester 73 (61%). Treatment of 73 with piperazine or 4-aminopiperidine followed by mesylate salt formation afforded sulfonamides 48 (52%) and 49 (42%), respectively.

Scheme 7a

a

Reagents and conditions: (a) chlorosulfonic acid, CH2Cl2, -10 °C, 6 h; (b) pentafluorophenol,

Et3N, CH2Cl2, rt, 2 h; (c) O3, 9:1 cyclohexane:CH2Cl2, 0 °C, 5 min; (d) piperazine, Et3N, CH3CN, rt to 60 °C, 12 h, then MsOH, EtOAc/CH2Cl2, 0 °C; (e) 4-aminopiperidine, Et3N, CH3CN, rt to 70 °C, 12 h, then MsOH, EtOAc/CH2Cl2, 0 °C.

As previously described,28,48,54,55 the remaining ozonides (5-14, 19, 20, 24-35, and 37-42) were obtained from ozonide phenol 50 or its acetate ester 6244 using parallel chemistry.

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Antimalarial Activity and ADME Properties

In vitro and in vivo antimalarial activities were measured using the chloroquine-resistant K1 and chloroquine-sensitive NF54 strains of P. falciparum, and P. berghei-infected mice, respectively, as previously described.24 In vivo efficacy data were obtained using single 30 mg/kg oral doses of the ozonides administered one day post-infection in an aqueous suspension vehicle. Calculated pKa and Log D7.4 values were generated using JChem for Excel and in vitro intrinsic clearance (CLint) values were determined for all compounds using human liver microsomes. For 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 1-6. First, the in vitro antimalarial potency for ozonides 2-49 fell in the narrow range of approximately 0.8 to 12 nM. Second, all of the ozonides reduced parasitemia by >99% on day 3 post-infection 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 pKa and lower Log D7.4 values, but neither of these parameters 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 (CLint, µL/min/mg microsomal protein) in human liver microsomes as a function of calculated pKa (left) and Log D7.4 (right). Dashed line represents the nominal cutoff for more stable compounds (nominally defined as CLint < 15 µL/min/mg protein).

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 single-dose curative efficacy in the P. berghei mouse model, increasing the average survival to approximately 30 days resulting in 3 of the 5 animals having no detectable parasites. As shown in Figure 3A and discussed previously,28 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 (4-6) led to a marginal reduction in overall survival (22-25 days), but a similar number of cures (2-3 out of 5 mice). Incorporation of gem-dimethyl 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 4). Interestingly, 2, 34, and 38 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 6,6difluoro analogue 43 exhibited comparable stability across species. The corresponding thiomorpholine sulfones had no curative efficacy (data not shown). Homomorpholine 35, the ring-expanded 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 latter. As anticipated,56 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 stability or solubility (41). Ozonides 42 and 43 were designed to block the metabolic oxidation29 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 (44-46), or carboxamide (47) and sulfonamide (48, 49) functional groups. Compared to 2, each of these compounds had improved metabolic stability. The poorly-soluble morpholinecontaining 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 H-bond 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. berghei-infected mice. As we have shown before,28 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 pre-infection (Table 8). Again, AS and 1 afforded no protection in this experiment, whereas mefloquine (MFQ) protected 5/5 of the infected animals at 48 h, but only extended survival time 3-fold 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 pre-infection dosing times. Most notably, 2 and 38 were fully protective when administered 96 h prior to infection.

Mouse Exposure

A subset of the most promising ozonides were administered to non-infected 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 non-infected 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 (1/5-3/5 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.

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 single-dose curative efficacy is most often associated with high and prolonged exposure, consistent with the improved efficacy of the cis-8’aryl ozonides relative to their cis-8’-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 2-4, 10, 11, 19, 33, and 38 were administered by oral gavage at 3-day intervals to male rats. The spectrum of toxicities were comparable between the tested ozonides and included dosedependent 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).

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 1- to 4-fold 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 (1-2 µg/mL at 300 mg/kg), only minor signs of toxicity were seen. Finally, administration of 2 resulted in significantly (4 to 25-fold) 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 pKa or Log D7.4 values and toxicity for these ozonides, it is evident that the three ozonides with the lowest toxicities were tertiary amines.

Summary

As we have previously observed42 in the SAR of the cis-8’-alkyl ozonide 1, the in vitro antimalarial potency for these cis-8’-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 pKa nor Log D7.4 correlated with in vivo antimalarial efficacy, although we note an overall trend that metabolically stable ozonides tended to have higher pKa and lower Log D7.4 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 D7.4 values. For the tertiary amino ozonides, addition of polar functional groups with H-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. 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 6-difluoro-substituted 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 single-dose curative efficacy in this series of cis-8’-aryl ozonides seemed to self-select 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 General. Melting points are uncorrected. 1D 1H and 13C NMR spectra were recorded on a 500 MHz spectrometer using CDCl3 or DMSO-d6 as solvents. All chemical shifts are reported in parts per million (ppm) and are relative to internal (CH3)4Si (0 ppm) for 1H and CDCl3 (77.2 ppm) or DMSO-d6 (39.5 ppm) for 13C NMR. Silica gel (sg) particle size 32−63 µm was used for 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 cis-adamantane-2-spiro-3'-8'-(4'hydroxyphenyl)-1',2',4'-trioxaspiro[4.5]decane (50)44 (0.50 g, 1.4 mmol) and 1-oxa-6azaspiro[2,5]octane-6-carboxylic acid t-butyl 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 cis-adamantane-2-spiro-3'-8'-[4'-[(1'-tertbutyloxycarbonyl-4'-hydroxy-4'-piperidinyl)methoxy]phenyl]-1',2',4'-trioxaspiro[4.5]decane (51) as a colorless solid (0.71 g, 89%). 1H NMR (CDCl3) δ 1.47 (s, 9H), 1.52-1.64 (m, 4H), 1.65-2.08 (m, 22H), 2.19 (s, 1H), 2.44-2.56 (m, 1H), 3.14-3.30 (m, 2H), 3.78 (s, 2H), 3.78-4.00 (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 solid (0.44 g, 88%). Mp 154-155 °C; 1H NMR (CDCl3) δ 1.60-2.10 (m, 26H), 2.46-2.54 (m, 1H),

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2.79 (s, 3H), 3.29-3.45 (m, 4H), 3.84 (s, 2H), 6.82 (d, J = 8.5 Hz, 2H), 7.13 (d, J = 8.5 Hz, 2H), 8.52 (brs, 1H), 8.86 (brs, 1H); 13C NMR (CDCl3) δ 26.45, 26.84, 30.35, 31.60, 34.66, 34.77, 36.37, 36.77, 39.30, 40.09, 41.98, 67.32, 74.84, 108.35, 111.36, 114.46, 127.76, 139.25, 156.54. Anal. Calcd for C29H43NO8S 2.5 H2O: C, 57.03; H, 7.92; N, 2.29. Found: C, 56.78; H, 7.66; N, 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 2,2,6,6-tetramethyl-4-piperidinone monohydrate (3.46 g, 20 mmol), trimethylsulfoxonium iodide (4.4 g, 20 mmol), potassium t-butoxide (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 MgSO4, filtered, and concentrated to afford 5,5,7,7tetramethyl-1-oxa-6-azaspiro[2,5]octane45 as a colorless oil (3.0 g, 89%). 1H NMR (CDCl3) δ 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 5044 (0.50 g, 1.40 mmol) and 5,5,7,7-tetramethyl-1-oxa-6-azaspiro[2,5]octane45 (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 ptoluenesulfonic 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 153-154 °C; 1H NMR (CDCl3) δ 1.54-2.08 (m, 38H), 2.26 (s, 3H), 2.45-2.54 (m, 1H), 2.49 (s, 1H), 3.52 (s, 2H), 6.73 (d, J = 8.5 Hz, 2H), 7.07-7.16 (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); 13C NMR (CDCl3) δ 21.24, 25.84, 26.45, 26.85, 31.21, 31.62, 34.69, 34.78, 36.37, 36.77, 40.93, 42.03, 56.29, 70.39, 108.37, 111.40, 114.46, 125.84, 127.70, 128.88, 139.27, 140.26, 142.31, 156.41. Anal. Calcd for C39H55NO8S: C, 67.12; H, 7.94; N, 2.01. 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'trioxaspiro[4.5]decane mesylate (17). Step 1. To a solution of 5044 (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 tert-butyl 4(2-methylsulfonyloxyethyl)piperidine-1-carboxylate (52)46 (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 CH2Cl2. After solvent removal in vacuo, the crude product was purified by crystallization from 1:1 EtOH:H2O to afford cis-adamantane-2-spiro-3'-8'-[4'-[2'-(1'-tert-butyloxycarbonyl -4'piperidinyl)ethoxy]phenyl]-1',2',4'-trioxaspiro[4.5]decane (52) as a colorless solid (0.90 g, 81%). 1

H NMR (CDCl3) δ 1.10-1.22 (m, 2H), 1.45 (s, 9H), 1.64-2.08 (m, 29H), 2.45-2.54 (m, 1H),

2.60-2.76 (m, 2H), 3.98 (t, J = 6.0 Hz, 2H), 4.00-4.20 (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), and dried in vacuo to afford 17 as a colorless solid (0.70 g, 78%). Mp 139-140 °C; 1H NMR (CDCl3) δ 1.56-2.08 (m, 29H), 2.45-2.54 (m, 1H), 2.78 (s, 3H), 2.82-2.94 (m, 2H), 3.44-3.53 (m, 2H), 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), 8.76 (brs, 1H); 13C NMR (CDCl3) δ 16.94, 26.46, 26.86, 28.62, 31.05, 31.63, 34.72, 34.79, 35.11, 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 C30H45NO7S: C, 63.92; H, 8.05; N, 2.48. Found: C, 63.73; H, 7.91; N, 2.56.

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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 t-butyl 4-(2-ethoxy-2oxoethoxy)piperidine-1-carboxylate (53)47 (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 5 mL), water (2 x 5 mL), and brine (5 mL), dried over MgSO4, filtered and concentrated to afforded t-butyl 4-(2-hydroxyethoxy)piperidine-1-carboxylate (54) as a colorless oil (0.80 g, 94%). 1H NMR (CDCl3) δ 1.46 (s, 9H), 1.44-1.68 (m, 2H), 1.80-1.90 (m, 2H), 3.03-3.12 (m, 2H), 3.46-3.53 (m, 1H), 3.56-3.61 (m, 2H), 3.70-3.80 (m, 4H). Step 2. To a solution of 54 (0.80 g, 3.30 mmol) and triethylamine (1.00 ml, 8.00 mmol) in CH2Cl2 (20 mL) at 0 °C was added 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 MgSO4, filtered, and concentrated to afford t-butyl 4-[2-(methanesulfonyloxy)ethoxy]piperidine1-carboxylate (55) as a pale yellow oil (0.86 g, 82%). 1H NMR (CDCl3) δ 1.46 (s, 9H), 1.36-1.60 (m, 2H), 1.76-1.90 (m, 2H), 3.06 (s, 3H), 3.00-3.14 (m, 2H), 3.48-3.56 (m, 1H), 3.66-3.82 (m, 4H), 4.37 (t, J = 4.0 Hz, 2H). Step 3. To a solution of 5044 (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, 2.67 mmol) was added. The reaction mixture was stirred at 60 °C overnight, cooled to rt, filtered, and washed with CH2Cl2. After the filtrate was concentrated, the crude product was purified by crystallization from 5:1 EtOH:H2O to afford cis-adamantane-2-spiro-3'-8'-[4'-[2'-(4'piperidinyloxy)ethoxy]phenyl]-1',2',4'-trioxaspiro[4.5]decane (56) as a colorless solid (0.70 g,

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85%). 1H NMR (CDCl3) δ 1.45 (s, 9H), 1.64-2.08 (m, 26H), 2.44-2.54 (m, 1H), 3.04-3.14 (m, 2H), 3.48-3.60 (m, 1H), 3.66-3.82 (m, 4H), 4.09 (t, J = 5.0 Hz, 2H), 6.84 (d, J = 8.5 Hz, 2H), 7.11 (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 (14 mL) was stirred at rt for 4 h. The resulting precipitate was filtered off, washed with ether (30 mL), and dried in vacuo to afford 18 as a colorless solid (0.40 g, 57%). Mp 136-137 °C; 1H NMR (CDCl3) δ 1.64-2.16 (m, 26H), 2.44-2.54 (m, 1H), 2.79 (s, 3H), 3.14-3.24 (m, 2H), 3.263.36 (m, 2H), 3.70-3.80 (m, 1H), 3.78 (t, J = 4.5 Hz, 2H), 4.08 (t, J = 4.5 Hz, 2H), 6.82 (d, J = 8.5 Hz, 2H), 7.12 (d, J = 8.5 Hz, 2H), 8.48 (brs, 1H), 8.59 (brs, 1H); 13C NMR (CDCl3) δ 26.46, 26.85, 27.07, 31.62, 34.71, 34.79, 36.37, 36.78, 40.20, 42.02, 67.01, 67.49, 70.34, 108.41, 111.37, 114.41, 127.68, 138.76, 156.92. Anal. Calcd for C30H45NO8S 1.5 H2O: C, 59.38; H, 7.97; 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]1',2',4'-trioxaspiro[4.5]decane mesylate (21). A mixture of cis-adamantane-2-spiro-3'-8'-[4'-(3'aminopropoxy)phenyl]-1',2',4'-trioxaspiro[4.5]decane (4)28 (0.395 g, 0.955 mmol), tetrahydro4H-pyran-4-one (0.098 g, 0.97 mmol), acetic acid (0.7 mL, 12 mmol), and sodium triacetoxyborohydride (0.342 g, 1.53 mmol) in CH2Cl2 (10 mL) was stirred overnight at rt. The 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 MgSO4, filtered, and concentrated to afford the free base of 21. To a solution of the free base of 21 in 1:1 CH2Cl2:EtOAc (18 mL) was 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%). Mp 138–140 °C dec; 1H NMR (CDCl3) δ 1.64-2.10 (m, 26H), 2.31-2.35 (m, 2H), 2.46-2.51 (m,

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1H), 2.73 (s, 3H), 3.17-3.27 (m, 3H), 3.35-3.39 (m, 2H), 4.02-4.06 (m, 4H), 6.79 (d, J = 8.8 Hz, 2H), 7.09 (d, J = 8.8 Hz, 2H), 8.82-8.87 (brs, 2H); 13C NMR (CDCl3) δ 25.83, 26.40, 26.80, 28.92, 31.56, 34.64, 34.73, 36.32, 36.72, 39.54, 41.84, 41.92, 54.43, 64.67, 65.99, 108.32, 111.30, 114.17, 127.63, 138.79, 156.61. Anal. Calcd for C31H47NO8S: C, 62.71; H, 7.98; N, 2.36. 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 cis-adamantane-2-spiro-3'-8'-[4'-(2'bromoethoxy)phenyl]-1',2',4'-trioxaspiro[4.5]decane (57)48 (0.50 g, 1.08 mmol), azetidine hydrochloride (0.20 g, 2.14 mmol), and K2CO3 (2.0 g) in dry acetonitrile (80 mL) was heated at 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 CH2Cl2 (5 mL) to which a solution of p-toluenesulfonic 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 solid (0.16 g, 24%). Mp 125-126 °C; 1H NMR (CDCl3) δ 1.60-2.10 (m, 23H), 2.34 (s, 3H), 2.442.55 (m, 1H), 2.79-2.91 (m, 1H), 3.48-3.58 (m, 2H), 4.04-4.16 (m, 2H), 4.25-4.32 (m, 2H), 4.474.57 (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, J = 8.5 Hz, 2H), 11.68 (brs, 1H); 13C NMR (CDCl3) δ 17.09, 21.32, 26.43, 26.83, 31.57, 34.65, 34.76, 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 (ESI-TOF) m/z: [M + H]+ Calcd for C27H38NO4 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 4-(4-hydroxyphenyl)cyclohexanone (58) (5.00 g, 26.3 mmol), epichlorohydrin (10.0 g, 108 mmol), and potassium carbonate (7.26 g,

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52.6 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 4-[4-(oxiran-2-ylmethoxy)phenyl]cyclohexanone (59) as a colorless solid (5.60 g, 87%). Mp 76-77 °C; 1H NMR (CDCl3) δ 1.84-1.98 (m, 2H), 2.15-2.25 (m, 2H), 2.45-2.55 (m, 4H), 2.76 (dd, J = 5.0, 2.5 Hz, 1H), 2.91 (t, J = 4.0 Hz, 1H), 3.34-3.40 (m, 1H), 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), 7.16 (d, J = 9.0 Hz, 2H); 13C NMR (CDCl3) δ 34.14, 41.35, 41.88, 44.70, 50.16, 68.78, 114.68, 127.61, 137.47, 157.09, 211.29. Step 2. A solution of 59 (1.00 g, 4.10 mmol) and O-methyl 2adamantanone oxime (60)52 (1.09 g, 6.10 mmol) in cyclohexane (80 mL) and CH2Cl2 (20 mL) was treated with ozone according to the method of Dong et al.37 After removal of solvents, the crude product was purified by sg flash chromatography (10% - 20% ether in hexane) to afford cis-adamantane-2-spiro-3'-8'-[4'-(oxiran-2-ylmethoxy)phenyl]-1',2',4'-trioxaspiro[4.5]decane (61) as a colorless solid (0.90 g, 54%). Mp 102-103 °C; 1H NMR (CDCl3) δ 1.64-2.08 (m, 22H), 2.45-2.55 (m, 1H), 2.75 (dd, J = 5.5, 3.0 Hz, 1H), 2.90 (t, J = 4.5 Hz, 1H), 3.32-3.38 (m, 1H), 3.95 (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, J = 9.0 Hz, 2H); 13C NMR (CDCl3) δ 26.44, 26.84, 31.60, 34.70, 34.77, 36.36, 36.76, 42.01, 44.75, 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:H2O to afford the pure free base of 36. To a solution of 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, 60%). Mp 167-168 °C; 1H NMR (DMSO-d6) δ 1.48-1.58 (m, 2H), 1.62-1.96 (m, 20H), 2.30 (s,

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3H), 2.50-2.60 (m, 1H), 3.08-3.30 (m, 4H), 3.40-3.50 (m, 2H), 3.70 (t, J = 12.0 Hz, 1H), 3.79 (t, J = 12.0 Hz, 1H), 3.88-4.04 (m, 4H), 4.26-4.34 (m, 1H), 5.95 (brs, 1H), 6.88 (d, J = 9.0 Hz, 2H), 7.14 (d, J = 9.0 Hz, 2H), 9.68 (brs, 1H); 13C NMR (DMSO-d6) δ 25.98, 26.40, 31.48, 34.27, 34.44, 35.96, 36.26, 40.86, 50.59, 53.05, 58.59, 63.26, 70.11, 108.31, 110.74, 114.68, 127.71, 138.67, 156.70. Anal. Calcd for C30H45NO9S: C, 60.48; H, 7.61; N, 2.35. Found: C, 60.30; H, 7.40; N, 2.37. cis-Adamantane-2-spiro-3'-8'-[4'-[2'-(2'-oxa-6'-azaspiro[3.3]hept-6'-yl)ethoxy]phenyl]1',2',4'-trioxaspiro[4.5]decane p-tosylate (39). A mixture of 5748 (0.50 g, 1.08 mmol), 2-oxa-6azaspiro [3,3]heptane tosylate49 (0.25 g, 0.98 mmol) and K2CO3 (2 g) in dry acetonitrile (80 mL) was heated at 60 °C for 24 h. The reaction mixture was cooled to rt, filtered, and concentrated. The residue was washed with water (50 mL) and dried in vacuo to afford the crude free base of 39. To a solution of the crude free base of 39 in ethyl acetate (5 mL) was added a solution of ptoluenesulfonic acid monohydrate (0.13 g, 0.68 mmol) in ethyl acetate (30 mL). The precipitate was collected by filtration to afford 39 as a colorless solid (0.13 g, 18%). Mp 136-137 °C; 1H NMR (CDCl3) δ 1.50-2.08 (m, 22H), 2.35 (s, 3H), 2.45-2.56 (m, 1H), 3.44-3.52 (m, 2H), 4.124.20 (m, 2H), 4.25-4.32 (m, 2H), 4.72 (s, 2H), 4.76-4.84 (m, 2H), 4.91 (s, 2H), 6.76 (d, J = 8.5 Hz, 2H), 7.07-7.20 (m, 4H), 7.72 (d, J = 8.0 Hz, 2H), 12.01 (brs, 1H); 13C NMR (CDCl3) δ 21.35, 26.45, 26.85, 31.59, 34.66, 34.79, 36.38, 36.77, 39.11, 42.02, 54.24, 63.92, 108.32, 111.44, 114.36, 125.82, 127.95, 128.91, 140.00, 140.35, 141.59, 155.43. HRMS (ESI-TOF) m/z: [M + H3O]+ Calcd for C29H42NO6 500.3012; Found 500.3005. cis-5-Fluoroadamantane-2-spiro-3'-8'-[4'-[2'-(4'-morpholinyl)ethoxy]phenyl]-1',2',4'trioxaspiro[4.5]decane mesylate (42). Step 1. A solution of O-methyl 5-fluoro-2-adamantanone oxime (63)53 (760 mg, 3.85 mmol) and 4-(4-acetoxyphenyl)cyclohexanone (62) (1.34 g, 5.78

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mmol) in cyclohexane (150 mL) and CH2Cl2 (30 mL) was treated with ozone according to the method of Dong et al.37 After removal of the solvents, the crude product was purified by sg flash chromatography (20:1 hexane:ethyl acetate) to afford cis-adamantane-5-fluoro-2-spiro-3'-8'-(4'acetoxyphenyl)-1',2',4'-trioxaspiro[4.5]decane (65) (900 mg, 54%, 2:1 mixture of diastereomers). 1

H NMR (CDCl3) δ 1.57–2.36 (m, 24H), 2.53–2.58 (m, 1H), 6.99 (d, J = 8.3 Hz, 2H), 7.20 (d, J

= 7.8 Hz, 2H); 13C NMR (CDCl3) δ 21.09, 29.40 (d, J = 10.1 Hz), 31.35, 33.14 (d, J = 1.9 Hz), 34.47, 38.78 (d, J = 10.6 Hz), 39.21 (d, J = 19.7 Hz), 41.96 (d, J = 17.3 Hz), 42.19, 91.20 (d, J = 184.7 Hz), 108.85, 109.52, 121.34, 127.65, 143.47, 148.84, 169.64. Step 2. To a solution of 65 (620 mg, 1.43 mmol) in dry acetonitrile (20 mL) were added powdered NaOH (343 mg, 8.56 mmol) and tetrabutylammonium hydrogensulfate (97 mg, 0.29 mmol). After the mixture was stirred at 25 ºC for 30 min, N-(2-chloroethyl)morpholine hydrochloride (531 mg, 2.85 mmol) was added. The reaction mixture was stirred at 60 ºC overnight and cooled to rt. The inorganic solid was filtered off and washed with CH2Cl2 (10 mL). After removal of the solvents in vacuo, the residue was dissolved in EtOAc (60 mL). The organic layer was washed with water (50 mL), brine (50 mL), and dried over MgSO4. Filtration and solvent removal in vacuo afforded the free base of 42. To a solution of the above free base in EtOAc (10 mL) was added dropwise a solution of MsOH (133 mg, 1.38 mmol) in ether (5 mL) at 0 ºC. The resulting solid was filtered, washed with ether (5 mL), and dried in vacuo at 40 ºC to afford 42 (642 mg, 77%, 1:1 mixture of diastereomers) as a colorless solid. Mp 146–148 °C; 1H NMR (CDCl3) δ 1.57–2.23 (m, 21H), 2.50–2.55 (m, 1H), 2.82 (s, 3H), 3.08 (brs, 2H), 3.54 (brs, 2H), 3.65–3.68 (m, 2H), 4.00–4.13 (m, 4H), 4.48 (t, J = 4.2 Hz, 2H), 6.84 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 8.8 Hz, 2H), 11.74 (brs, 1H); 13

C NMR (CDCl3) δ 29.44 (d, J = 10.1 Hz), 31.49, 33.18 (d, J = 1.9 Hz), 34.51, 38.51 (d, J =

10.6 Hz), 39.24 (d, J = 19.7 Hz), 39.36, 41.89, 41.99 (d, J = 17.3 Hz), 52.93, 56.84, 62.86, 63.83,

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91.25 (d, J = 184.3 Hz), 108.91, 109.56, 109.57, 114.43, 127.98, 139.81, 155.36. Anal. Calcd for C29H42FNO8S: C, 59.67; H, 7.25; N, 2.40. Found: C, 59.42; H, 7.23; N, 2.60. cis-6,6-Difluoroadamantane-2-spiro-3'-8'-[4'-[2'-(4'-morpholinyl)ethoxy]phenyl]-1',2',4'trioxaspiro[4.5]decane mesylate (43). Step 1. A solution of O-methyl 6,6-difluoro-2adamantanone oxime (64)53 (300 mg, 1.4 mmol) and 4-(4-acetoxyphenyl)cyclohexanone (62) (486 mg, 2.1 mmol) in cyclohexane (30 mL) and CH2Cl2 (4 mL) was treated with ozone according to the method of Dong et al.37 After removal of the solvents in vacuo, the crude product was purified by flash chromatography (sg, 30:1 hexane:ethyl acetate) to afford an cis6,6-difluoroadamantane-2-spiro-3'-8'-(4'-acetoxyphenyl)-1',2',4'-trioxaspiro[4.5]decane (66) (269 mg, 44%) as a white solid. 1H NMR (CDCl3) δ 1.69–2.15 (m, 24H), 2.29 (s, 3H), 2.52-2.58 (m, 1H), 7.00 (d, J = 8.3 Hz, 2H), 7.20 (d, J = 8.8 Hz, 2H); 13C NMR (CDCl3) δ 21.13, 30.72 (3J = 3.8 Hz), 30.75 (3J = 3.8 Hz), 31.38, 34.17 (2J = 21.6 Hz), 34.51, 34.59 (2J = 21.6 Hz), 34.65, 42.27, 108.85, 109.21, 121.39, 124.00 (1J = 247.1 Hz), 127.68, 143.43, 148.90, 169.69. Step 2. To a solution of 66 (182 mg, 0.46 mmol) in dry acetonitrile (5 mL) were added powdered NaOH (110 mg, 2.7 mmol) and Bu4NHSO4 (31 mg, 0.1 mmol). After stirring for 30 min at 25 ºC, N-(2chloroethyl)morpholine hydrochloride (170 mg, 0.76 mmol) was added. The reaction mixture was stirred at 60 ºC for overnight and cooled to rt. The inorganic solid was filtered off and washed with EtOAc (2 x 10 mL). After removal of the solvents in vacuo, the residue was dissolved in EtOAc (50 mL). The organic layer was washed with water (50 mL), brine (50 mL) and dried over MgSO4. Filtration and solvent removal in vacuo afforded the free base of 43. To a solution of the above crude free base of 43 in EtOAc (4 mL) was added dropwise a solution of MsOH (41 mg, 0.43 mmol) in ether (2 mL) at 0 ºC to produce a solid that was filtered, washed with ether (5 mL) and dried in vacuo at 40 ºC to afford 43 as a white solid (200 mg, 72%). Mp

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151-153 ºC; 1H NMR (CDCl3) δ 1.63-1.73 (m, 2H), 1.80-2.16 (m, 18H), 2.49-2.54 (m, 1H), 2.81 (s, 3H), 3.09 (br, 2H), 3.53-3.55 (m, 2H), 3.65 (br, 2H), 4.04-4.11 (m, 4H), 4.48 (t, J = 4.4 Hz, 2H), 6.84 (d, J = 8.3 Hz, 2H), 7.14 (d, J = 8.3 Hz, 2H); 13C NMR (CDCl3) δ 30.69 (3J = 3.8 Hz), 30.72 (3J = 3.8 Hz), 31.45, 34.14 (2J = 21.6 Hz), 34.49, 34.55 (2J = 21.6 Hz), 34.62, 39.37, 41.88, 52.88, 56.78, 62.83, 63.81, 108.84, 109.18, 114.43, 123.97 (1J = 247.1 Hz), 127.93, 139.71, 155.39. Anal. Calcd for C29H41F2NO8S: C, 57.89; H, 6.87; N, 2.33. Found: C, 57.73; H, 7.08; N, 2.38. cis-Adamantane-2-spiro-3'-8'-[4'-[(4'-morpholinyl)methyl]phenyl]-1',2',4'trioxaspiro[4.5]decane mesylate (45). To a stirred mixture of morpholine (1.12 g, 12.8 mmol) and Et3N (1.30 g, 12.8 mmol) in acetonitrile (60 mL) at rt was added cis-adamantane-2-spiro-3'8'-[(4'-chloromethyl)phenyl]-1',2',4'-trioxaspiro[4.5]decane (67)48 (0.50 g, 1.28 mmol). The reaction mixture was stirred at 60 °C for 48 h. After cooling to rt, the resulting solid product was filtered and washed with EtOAc. The filtrate was evaporated to dryness in vacuo. The residue was dissolved with EtOAc (60 mL), washed with H2O (2 x 50 mL), brine (50 mL), and dried over MgSO4. Filtration and solvent removal in vacuo afforded the free base of 45. To a solution of the free base of 45 in EtOAc (10 mL) was added dropwise a solution of MsOH (0.098 g, 1.02 mmol) in ether (10 mL) at 0 °C and stirred for 30 min. The resulting solid obtained was filtered, washed with ether (50 mL) and dried in vacuo at 50 °C to afford 45 as a colorless solid (0.47 g, 69%). Mp 134-137 °C; 1H NMR (CDCl3) δ 1.70-2.07 (m, 22H), 2.55-2.60 (m, 1H), 2.86 (s, 3H), 3.05 (brs, 4H), 3.99 (brs, 4H), 4.13 (s, 2H), 7.27 (d, J = 7.5 Hz, 2H), 7.43 (d, J = 7.5 Hz, 2H); 13C NMR (CDCl3) δ 26.36, 26.76, 31.18, 34.49, 34.70, 36.30, 36.67, 39.47, 42.54, 51.51, 61.11, 64.00, 108.07, 111.42, 126.32, 127.61, 131.20, 148.17. Anal. Calcd for C28H41NO7S: C, 62.78; H, 7.71; N, 2.61. Found: C, 62.90; H, 7.63; N, 2.80.

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cis-Adamantane-2-spiro-3'-8'-[4'-[[4'-(aminocarbonyl)-1'-piperidinyl]methyl]phenyl]1',2',4'-trioxaspiro[4.5]decane p-tosylate (46). To a stirred mixture of isonipecotamide (0.39 g, 3.08 mmol) and K2CO3 (2.13 g, 15.4 mmol) in acetonitrile (50 mL) at rt was added 6748 (0.60 g, 1.54 mmol). The reaction mixture was stirred at 60 °C for 48 h. After cooling to rt, the resulting solid material was filtered off and washed with EtOAc. The filtrate was evaporated to dryness in vacuo. The residue was dissolved with EtOAc (60 mL), washed with H2O (2 x 50 mL), brine (50 mL), and dried over MgSO4. Filtration and solvent removal in vacuo afforded the free base of 46. To a solution of the free base of 46 in EtOAc (10 mL) was added dropwise a solution of ptoluenesulfonic acid monohydrate (0.19 g, 1.0 mmol) in ether (10 mL) at 0 °C. The resulting precipitate was filtered, washed with ether (50 mL) and dried in vacuo at 50 °C to afford 46 as a colorless solid (0.61 g, 61%). Mp 140-142 °C; 1H NMR (CDCl3) δ 1.62-2.20 (m, 26H), 2.37 (s, 3H), 2.50-2.65 (m, 2H), 2.88 (brs, 2H), 3.32-3.40 (m, 2H), 4.05 (s, 2H), 5.53 (s, 0.3H), 6.53 (s, 0.7H), 7.05 (d, J = 7.5 Hz, 2H), 7.19 (d, J = 7.5 Hz, 2H), 7.25 (d, J = 8.0 Hz, 2H), 7.48 (s, 0.3H), 7.62 (s, 0.7H), 7.74 (d, J = 8.0 Hz, 2H), 9.71 (s, 0.7H), 9.83 (s, 0.3H); 13C NMR (CDCl3) δ 21.34, 25.93, 26.43, 26.83, 31.19, 34.55, 34.76, 36.36, 36.74, 42.56, 49.38, 51.73, 60.48, 108.13, 111.43, 125.83, 126.12, 127.48, 128.96, 131.26, 140.26, 142.35, 148.01, 176.38. Anal. Calcd for C36H48N2O7S: C, 66.23; H, 7.41; N, 4.29. Found: C, 66.20; H, 7.18; N, 4.25. cis-Adamantane-2-spiro-3'-8'-[4'-(1'-piperazinylcarbonyl)phenyl]-1',2',4'trioxaspiro[4.5]decane mesylate (47). Step 1. To a solution of cis-adamantane-2-spiro-3'-8'-(4'carboxyphenyl)-1',2',4'-trioxaspiro[4.5]decane (68)38 (2.5 g, 6.5 mmol) and Nhydroxysuccinimide (0.96 g, 8.35 mmol) in DMF (30 mL) was added EDCI (1.6 g, 8.3 mmol) at 0 ºC. After the addition, the reaction mixture was stirred at rt for overnight. After the reaction mixture was poured onto ice, the resulting precipitate was filtered, washed with water (3 x 25

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mL) and dried to afford cis-adamantane-2-spiro-3'-8'-[4'-[[(2',5'-dioxopyrrolidin-1'yl)oxy]carbonyl]phenyl]-1',2',4'-trioxaspiro[4.5]decane (69) (2.8 g, 91%). Mp 124-126 °C; 1H NMR (CDCl3) δ 1.70-2.08 (m, 22H), 2.61-2.67 (m, 1H), 2.89 (brs, 4H), 7.35 (d, J = 8.3 Hz, 2H), 8.06 (d, J = 8.3 Hz, 2H). Step 2. To a solution of 69 (0.7 g, 1.46 mmol) in dichloromethane (25 mL) was added dropwise a solution of piperazine (1.25 g, 14.6 mmol) in dichloromethane (5 mL) at rt. After stirring for overnight, the reaction was quenched by addition of water (25 mL) and dichloromethane (10 mL). The organic layer was separated, washed with water (3 x 25 mL), and dried over MgSO4. After filtration, the solvent was removed in vacuo to afford the free base of 47. To the solution of the free base of 47 in ethyl acetate (50 mL) was added a solution of MsOH (0.14 g, 1.46 mmol) in ether (10 mL) at rt and the reaction mixture was stirred for 0.5 h. The solid obtained was filtered, washed with ether (2 x 25 mL), and dried to afford 47 (0.6 g, 75%). Mp 148-150 ºC; 1H NMR (DMSO-d6) δ 1.58-1.94 (m, 22H), 2.38 (s, 3H), 2.64-2.72 (m, 1H), 3.12-3.20 (brs, 4H), 3.50-3.84 (brs, 4H), 7.31 (d, J = 7.8 Hz, 2H), 7.40 (d, J = 7.8 Hz, 2H), 8.91 (brs, 2H); 13C NMR (DMSO-d6) δ 26.03, 26.43, 31.10, 34.21, 34.47, 35.99, 36.29, 39.96, 41.59, 42.87, 108.25, 110.82, 126.90, 127.70, 132.82, 148.17, 169.44. Anal. Calcd for C28H40N2O7S: C, 61.29; H, 7.35; N, 5.11. Found: C, 61.31; H, 7.18; N, 5.33. cis-Adamantane-2-spiro-3'-8'-[4'-(1'-piperazinylsulfonyl)phenyl]-1',2',4' trioxaspiro[4.5]decane mesylate (48). Step 1. To a precooled solution of 4phenylcyclohexanone (70) (20.0 g, 0.114 mol) in dichloromethane (120 mL) at -10 °C was added dropwise (45 min) a solution of chlorosulfonic acid (80.3 g, 46.0 mL, 0.689 mole) in dichloromethane (120 mL). After the mixture was stirred at -10 °C for an additional 5 h, the reaction was quenched by pouring onto ice water (100 mL). After the aqueous layer was separated, organic layer was washed with cold water (100 mL), brine (50 mL), and dried over

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MgSO4, filtered, and concentrated to provide a crude product of 4-(4oxocyclohexyl)benzenesulfonyl chloride (71) as a viscous black-brown oil (3.10 g, 10 %). 1H NMR (CDCl3) δ 1.92-2.03 (m, 2H), 2.23-2.31 (m, 2H), 2.53-2.58 (m, 4H), 3.14-3.23 (m, 1H), 7.50 (d, J = 8.0 Hz, 2H), 8.00 (d, J = 8.0 Hz, 2H). Step 2. To a solution of pentafluorophenol (2.30 g, 0.0124 mol) in dichloromethane (25.0 mL) was added TEA (4.03 g, 5.60 mL, 0.0398 mol). After the mixture was stirred at rt for 30 min, a solution of crude 71 (3.09 g, 0.011 mol) in dichloromethane (30 mL) was added and the reaction mixture was stirred at rt for 2 h. The solvent was evaporated in vacuo and the residue was purified by crystallization from 1:1 EtOAc:hexane to give pentafluorophenyl 4-(4-oxocyclohexyl)benzenesulfonate (72) as a white solid (3.33 g, 70%). Mp 133-135 °C; 1H NMR (CDCl3) δ 1.93-2.04 (m, 2H), 2.23-2.31 (m, 2H), 2.53-2.58 (m, 4H), 3.14-3.23 (m, 1H), 7.50 (d, J = 8.5 Hz, 2H), 7.94 (d, J = 8.5 Hz, 2H). Step 3. A solution of 60 (1.437 g, 8.01 mmol) and 72 (2.33 g, 5.34 mmol) in 9:1 cyclohexane:DCM (100 mL) was treated with ozone according to the method of Dong et al.37 After removal of the reaction solvents in vacuo, the residue was purified by crystallization from ethanol to give cisadamantane-2-spiro-3'-8'-[4'-[(2',3',4',5',6'-pentafluorophenoxy)sulfonyl]phenyl]-1',2',4'trioxaspiro[4.5]decane (73) (1.90 g, 61%) as a colorless solid. Mp 138-140 °C; 1H NMR (CDCl3) δ 1.68-2.12 (m, 22H), 2.66-2.74 (m, 1H), 7.44 (d, J = 8.5 Hz, 2H), 7.89 (d, J = 8.5 Hz, 2H). Step 4. To a solution of 73 (0.5 g, 0.85 mmol) in dry acetonitrile (30 mL) were added piperazine (0.98 g, 0.011 mol) and triethylamine (0.50 g, 0.80 mL, 4.74 mmol). The mixture was stirred at rt for 30 min and then heated at 60 °C for 12 h. After cooling to rt, the organic solvents were evaporated to give a residue. The residue was dissolved in EtOAc (50 mL), washed with water (20 mL), brine (20 mL), and dried over MgSO4. The filtrate was concentrated and dried in vacuo to afford the free base of 48. To the solution of the free base of 48 in 5:1 EtOAc:CH2Cl2 was

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added dropwise a solution of MsOH (71 mg, 0.749 mmol) in EtOAc (5 mL) at 0 °C. The resulting precipitate was filtered, washed with ether (25 mL) and dried in vacuo at 40 °C to afford 48 as a colorless solid (0.260 g, 52%). Mp 165-167 °C; 1H NMR (DMSO-d6) δ 1.56-1.99 (m, 22H), 2.30 (s, 3H), 2.74-2.82 (m, 1H), 3.10 (brs, 4H), 3.20 (brs, 4H), 7.55 (d, J = 8.0 Hz, 2H), 7.70 (d, J = 8.0 Hz, 2H), 8.58 (brs, 2H); 13C NMR (DMSO-d6) δ 25.81, 26.22, 30.73, 33.90, 34.27, 35.79, 36.08, 41.39, 42.30, 42.86, 107.94, 110.70, 127.95, 128.06, 131.74, 152.17. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C26H37N2O5S 489.2423; Found 489.2446. cis-Adamantane-2-spiro-3'-8'-[4'-[(4'-amino-1'-piperidinyl)sulfonyl]phenyl]-1',2',4'trioxaspiro[4.5]decane mesylate (49). To a solution of 73 (0.6 g, 1.02 mmol) in dry acetonitrile (35 mL) were added 4-aminopiperidine (0.684 g, 6.833 mol) and triethylamine (1.37 g, 1.90 mL, 0.013 mol). The mixture was stirred at rt for 30 min and then heated at 70 °C for 12 h. After cooling to rt, the organic solvents were evaporated to give a residue that was dissolved in EtOAc (50 mL), washed with water (20 mL), brine (20 mL), and dried over MgSO4. The filtrate was concentrated and dried in vacuo to afford the free base of 49. To a solution of the free base of 49 in 5:1 EtOAc:CH2Cl2 was added dropwise a solution of MsOH (60 mg, 0.616 mmol) in EtOAc (5 mL) at 0 °C. The resulting precipitate was filtered, washed with ether (25 mL), and dried in vacuo at 40 °C to afford 49 as a colorless solid (0.255 g, 42%). Mp 162-165 °C; 1H NMR (CDCl3) δ 1.68-2.10 (m, 26H), 2.29-2.35 (m, 2H), 2.60-2.69 (m, 2H), 2.70 (s, 3H), 3.02 (brs, 1H), 3.80-3.86 (m, 2H), 7.36 (d, J = 8.5 Hz, 2H), 7.64 (d, J = 8.5 Hz, 2H), 7.68 (brs, 3H); 13C NMR (CDCl3) δ 26.43, 26.83, 29.25, 31.09, 34.47, 34.77, 36.37, 36.74, 39.58, 42.84, 44.58, 48.01, 107.97, 111.57, 127.66, 127.85, 133.30, 151.84. Anal. Calcd for C28H42N2O8S2•H2O: C, 54.52; H, 7.19; N, 4.54. Found: C, 54.75; H, 7.23; N, 4.63.

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Antimalarial Screens. In vitro and in vivo antimalarial data was obtained as previously described. 57,78

Ionization Constants and Partition Coefficients. pKa and Log D7.4 values were calculated using JChem for Excel (ChemAxon, Budapest, Hungary, ver 16.4).

Kinetic Solubility. Solubility was assessed by dilution of a concentration stock solution prepared in DMSO into Milli-Q water (final DMSO concentration of 1%) and allowing the sample to stand at ambient temperature for 30 min. Solubility ranges were determined by nephelometry as described previously.59

In vitro metabolism. As fully described in Coteron et al.,59 compounds were incubated with human liver microsomes (either BD Gentest, Discovery Labware Inc., Woburn, MA or XenoTech LLC, Lenexa, KS) and appropriate co-factors at a substrate concentration of 1 µM and a microsomal protein concentration of 0.4 mg/mL. Loss of parent compound was monitored by LC/MS and intrinsic clearance values were calculated using the depletion rate constant.

Mouse Exposure. All animal studies were conducted using established procedures in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, and the study protocols were reviewed and approved by the Monash Institute of Pharmaceutical Sciences Animal Ethics Committee. Mouse exposure studies were conducted in non-fasted female FVB mice (20-30 g). Oral suspension formulations (containing 0.5% w/v hydroxypropylmethyl cellulose, 0.4% v/v Tween 80, 0.5% benzyl alcohol, 0.9% w/v sodium

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chloride in water) were administered by gavage. Blood samples were collected under isoflurane anesthesia via terminal cardiac puncture. Blood samples were immediately centrifuged at 4ºC and plasma was separated, stored at -20 ºC. Plasma samples were processed by precipitation of the plasma proteins with acetonitrile (2:1 or 3:1 volume ratio) and the supernatant assayed by LC/MS using either a Waters Micromass Quattro Ultima PT triple quadrupole instrument coupled to a Waters 2795 HPLC or a Waters Micromass Quattro Premier triple quadrupole instrument coupled to a Waters Acquity UPLC. Concentrations were obtained by comparison of the peak area ratio (using diazepam as the internal standard) to that for a calibration curve prepared in blank mouse plasma. Plasma pharmacokinetic parameters were calculated using noncompartmental methods (WinNonlin ver. 5.2, Pharsight Corporation, Cary, North Carolina, USA).

Exploratory Toxicology. Toxicology studies were conducted by Basilea Pharmaceutica Ltd (Basel, Switzerland). Ozonides or vehicle (same formulation vehicle as that used for the mouse efficacy and oral exposure studies) were administered orally to male rats (100 or 300 mg/kg) every third day for 5 doses. Satellite groups were included for the collection of blood samples for kinetic examinations and for a 2-week recovery phase. Toxicity was assessed by clinical observations, body weight development, and clinical laboratory investigations (hematology, clinical chemistry and urine analysis). At the end of the study periods, all rats were sacrificed, necropsied and selected organs examined histopathologically.

Supporting Information: Supporting information [CSV Smiles data] is available free of charge on the ACS Publications

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website. Corresponding Authors: J.L.V.: Phone: 402.559.5362. E-mail: [email protected] S.A.C.: Phone: 61 3 9903 9626. E-mail: [email protected] Present Addresses: †

For V.J.B.: Allexcell, Inc., 135 Wood Street, West Haven, CT 06516, United States.

†

For C.D.H.: Allergan, 2525 DuPont Drive, Bldg. RD-3, #3170, Irvine, CA 92612, United States.

†

For S.K.: Organic Chemistry Division, School of Advanced Sciences, Vellore Institute

of Technology University, Vellore-632 014, India. †

For M.D.: Syngene International Ltd., Plot No.2 & 3, Bommasandra IV Phase, Jigani Link

Road, Bangalore 560 099, India. †

For Y.T.: Melinta Therapeutics, Inc., 300 George Street, Suite 301, New Haven, Connecticut

06511, United States. †

For Q.Z.: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, No. 501, Haike

Rd., Pudong New District, Shanghai 201210, China. Notes: The authors declare no competing interests.

Acknowledgments This investigation received financial support from Medicines for Malaria Venture (MMV). We acknowledge the support and advice of J. Carl Craft, Arnulf Dorn, Alan T. Hudson, Daniel Hunziker, Marcel Tanner, Timothy N. C. Wells, and the late Ian C. Bathurst and the expert scientific and technical assistance of Josefina Santo Tomas, Christopher Snyder, and Tien Nguyen.

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Abbreviations Used: ACT, artemisinin combination therapies; AS, artesunate; CLint, in vitro intrinsic clearance; MFQ, mefloquine; MsOH, methanesulfonic acid.

References (1) White, N. J. Qinghaosu (Artemisinin): The price of success. Science 2008, 320, 330-334. (2) Eastman, R. T.; Fidock, D. A. Artemisinin-based combination therapies: a vital tool in efforts to eliminate malaria. Nat. Rev. Microbiol. 2009, 7, 864-874. (3) Sibley, C. H. Understanding artemisinin resistance. Science 2015, 347, 373-374. (4) Paloque, L.; Ramadani, A. P.; Mercereau-Puijalon, O.; Augereau, J.-M.; Benoit-Vical, F. Plasmodium falciparum: Multifaceted resistance to artemisinins. Malar. J. 2016, 15, 149. (5) Tu, Y. The discovery of artemisinin (qinghaosu) and gifts from Chinese medicine. Nat. Med. 2011, 17, 1217-1220. (6) Haynes, R. K. From artemisinin to new artemisinin antimalarials: Biosynthesis, extraction, old and new derivatives, stereochemistry and medicinal chemistry requirements. Curr. Topics Med. Chem. 2006, 6, 509-537. (7) Tang, Y.; Dong, Y.; Vennerstrom, J. L. Synthetic peroxides as antimalarials. Med. Res. Rev. 2004, 24, 425-448. (8) Jefford, C. W. Synthetic peroxides as potent antimalarials. News and views. Curr. Top. Med. Chem. 2012, 12, 373−399. (9) Tilley, L.; Charman, S. A.; Vennerstrom, J. L. Semisynthetic artemisinin and synthetic peroxide antimalarials. In Neglected Diseases and Drug Discovery; Palmer, M. J., Wells, T. N. C., Eds.; Royal Society of Chemistry: Cambridge, U.K., 2012; pp 33−64. (10) Slack, R. D.; Jacobine, A. M.; Posner, G. H. Antimalarial peroxides: Advances in drug discovery and design. Med. Chem. Commun. 2012, 3, 281-297.

40



1 2 3 4 5 6 7 8 9 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

Page 42 of 60

(11) 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. (12) Meshnick, S. R. Artemisinin: mechanisms of action, resistance and toxicity. Int. J. Parasitol. 2002, 32, 1655−1660. (13) Klonis, N.; Crespo-Ortiz, M. P.; Bottova, I.; Abu-Bakar, N.; Kenny, S.; Rosenthal, P. J.; Tilley, L. Artemisinin activity against Plasmodium falciparum requires hemoglobin uptake and digestion. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 11405−11410. (14) Klonis, N.; Creek, D. J.; Tilley L. Iron and heme metabolism in Plasmodium falciparum and the mechanism of action of artemisinins. Curr. Opin. Microbiol. 2013, 16, 722-727. (15) Meunier, B.; Robert, A. Heme as trigger and target for trioxane-containing antimalarial drugs. Acc. Chem. Res. 2010, 43, 1444-1451. (16) 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 antimalarials. Antimicrob. Agents Chemother. 2008, 52, 1291-1296. (17) Bousejra-El Garah, F.; Wong, M. H. L.; Amewu, R. K.; Muangnoicharoen, S.; Maggs, J. L.; Stigliani, J. L.; Park, B. K.; Chadwick, J.; Ward, S. A.; O’Neill, P. M. Comparison of the reactivity of antimalarial 1,2,4,5-tetraoxanes with 1,2,4-trioxolanes in the presence of ferrous iron salts, heme, and ferrous iron salts/ phosphatidylcholine. J. Med. Chem. 2011, 54, 6443−6455. (18) Wang, J.; Zhang, C. J.; Chia, W. N.; Loh, C. C.; Li, Z.; Lee, Y. M.; He, Y.; Yuan, L. X.; Lim, T. K.; Liu, M.; Liew, C. X.; Lee, Y. Q.; Zhang, J.; Lu, N.; Lim, C. T.; Hua, Z.-C.; Liu, B.;

41


Page 43 of 60

1 2 3 4 5 6 7 8 9 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


Shen, H. M.; Tan, K. S.; Lin, Q. Haem-activated promiscuous targeting of artemisinin in Plasmodium falciparum. Nat. Commun. 2015, 6, 10111. (19) Ismail, H. M.; Barton, V.; Phanchana, M.; Charoensutthivarakul, S.; Wong, M. H.; Hemingway, J.; Biagini, G. A.; O'Neill, P. M.; Ward, S. A. Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 2080-2085. (20) Ismail, H. M.; Barton, V. E.; Panchana, M.; Charoensutthivarakul, S.; Biagini, G. A.; Ward, S. A.; O'Neill, P. M. A click chemistry-based proteomic approach reveals that 1,2,4-trioxolane and artemisinin antimalarials share a common protein alkylation profile. Angew. Chem. Int. Ed. Engl. 2016, 55, 6401-6405. (21) Jourdan, J.; Matile, H.; Reift, E.; Biehlmaier, O.; Dong, Y.; Wang, X.; Mäser, P.; Vennerstrom, J. L.; Wittlin, S. Monoclonal antibodies that recognize the alkylation sSignature of antimalarial ozonides OZ277 (arterolane) and OZ439 (artefenomel). ACS Infect. Dis. 2016, 2, 54-61. (22) Hartwig, C. L.; Lauterwasser, E. M.W.; Mahajan, S. S.; Hoke, J. M.; Cooper, R. A.; Renslo, A. R. Investigating the antimalarial action of 1,2,4-trioxolanes with fluorescent chemical probes. J. Med. Chem. 2011, 54, 8207−8213. (23) Antoine T.; Fisher, N.; Amewu, R.; O'Neill, P. M.; Ward, S. A.; Biagini, G. A. Rapid kill of malaria parasites by artemisinin and semi-synthetic endoperoxides involves ROS-dependent depolarization of the membrane potential. J. Antimicrob. Chemother. 2014, 69, 1005-1016. (24) 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.

42



1 2 3 4 5 6 7 8 9 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

Identification of an antimalarial synthetic trioxolane drug development candidate. Nature 2004, 430, 900-904. (25) Gautam, A.; Ahmed, T.; Sharma, P.; Varshney, B.; Kothari, M.; Saha, N.; Roy, A.; Moehrle, J. J.; Paliwal, J., Pharmacokinetics and pharmacodynamics of arterolane maleate following multiple oral doses in adult patients with P. falciparum malaria. J. Clin. Pharmacol. 2011, 51, 1519-1528. (26) Valecha, N.; Looareesuwan, S.; Martensson, A.; Abdulla, S. M.; Krudsood, S.; Tangpukdee, N.; Mohanty, S.; Mishra, S. K.; Tyagi, P. K.; Sharma, S. K.; Moehrle, J.; Gautam, A.; Roy, A.; Paliwal, J. K.; Kothari, M.; Saha, N.; Dash, A. P.; Bjorkman, A., Arterolane, a new synthetic trioxolane for treatment of uncomplicated Plasmodium falciparum malaria: A phase II, multicenter, randomized, dose-finding clinical trial. Clin. Infect. Dis. 2010, 51, 684-691. (27) Valecha, N.; Krudsood, S.; Tangpukdee, N.; Mohanty, S.; Sharma, S. K.; Tyagi, P. K.; Anvikar, A.; Mohanty, R.; Rao, B. S.; Jha, A. C.; Shahi, B.; Singh, J. P. N.; Roy, A.; Kaur, P.; Kothari, M.; Mehta, S.; Gautam, A.; Paliwal, J. K.; Arora, S.; Saha, N., Arterolane maleate plus piperaquine phosphate for treatment of uncomplicated Plasmodium falciparum malaria: A comparative, multicenter, randomized clinical trial. Clin. Infect. Dis. 2012, 55, 663-671. (28) Charman, S. A.; Arbe-Barnes, S.; Bathurst, I. C.; Brun, R.; Campbell, M.; Charman, W. N.; Chiu, F. C. K.; Chollet, J.; Craft, J. C.; Creek, D. J.; Dong, Y.; Matile, H.; Maurer, M.; Morizzi, J.; Nguyen, T.; Papastogiannidis, P.; Scheurer, C.; Shackleford, D. M.; Sriraghavan, K.; Stingelin, L.; Tang, Y.; Urwyler, H.; Wang, X.; White, K. L.; Wittlin, S.; Zhou, L.; Vennerstrom, J. L., Synthetic ozonide drug candidate OZ439 offers new hope for a single-dose cure of uncomplicated malaria. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 4400-4405.

43


Page 44 of 60

Page 45 of 60

1 2 3 4 5 6 7 8 9 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


(29) Moehrle, J. J.; Duparc, S.; Siethoff, C.; van Giersbergen, P. L. M.; Craft, J. C.; Arbe-Barnes, S.; Charman, S. A.; Gutierrez, M.; Wittlin, S.; Vennerstrom, J. L., First-in-man safety and pharmacokinetics of synthetic ozonide OZ439 demonstrates an improved exposure profile relative to other peroxide antimalarials. Br. J. Clin. Pharmacol. 2013, 75, 524-537. (30) Wells, T. N, Hooft van Huijsduijnen, R.; Van Voorhis, W. C. Malaria medicines: A glass half full? Nat. Rev. Drug Discovery 2015, 14, 424-442. (31) Phyo, A. P.; Jittamala, P.; Nosten, F. H.; Pukrittayakamee, S.; Imwong, M.; White, N. J.; Duparc, S.; Macintyre, F.; Baker, M.; Möhrle, J. J. Antimalarial activity of artefenomel (OZ439), a novel synthetic antimalarial endoperoxide, in patients with Plasmodium falciparum and Plasmodium vivax malaria: An open-label phase 2 trial. Lancet Infect. Dis. 2016, 16, 61-69. (32) McCarthy, J. S.; Baker, M.; O'Rourke, P.; Marquart, L.; Griffin, P.; Hooft van Huijsduijnen R.; Möhrle, J. J. Efficacy of OZ439 (artefenomel) against early Plasmodium falciparum bloodstage malaria infection in healthy volunteers. J. Antimicrob. Chemother. 2016, 71, 2620-2627. (33) 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 dispiro1,2,4-trioxolane antimalarials. J. Pharm. Sci. 2007, 96, 2945-2956. (34) Tang, Y.; Dong, Y.; Wang, X.; Sriraghavan, K.; Wood, J. K.; Vennerstrom, J. L. Dispiro1,2,4-trioxane analogs of a prototype dispiro-1,2,4-trioxolane: Mechanistic comparators for artemisinin in the context of reaction pathways with iron (II). J. Org. Chem. 2005, 70, 51035110. (35) Creek, D. J.; Chalmers, D. K.; Charman, W. N.; Duke, B. J. K. Quantum chemical study of the intermediate complex required for iron-mediated reactivity and antimalarial activity of dispiro-1,2,4-trioxolanes. J. Mol. Graphics Modell. 2008, 27, 394-400.

44



1 2 3 4 5 6 7 8 9 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

Page 46 of 60

(36) Wang, X.; Dong, Y.; Wittlin, S.; Charman, S.; Chiu, F.; Chollet, J.; Katneni, K.; Mannila, J.; Morizzi, J.; Ryan, E.; Scheurer, C.; Steuten, J.; Santo Tomas, J.; Snyder, C.; Vennerstrom, J. L. Comparative antimalarial activities and ADME profiles of ozonides (1,2,4-trioxolanes) OZ277, OZ439 and their 1,2-dioxolane, 1,2,4-trioxane, and 1,2,4,5-tetraoxane isosteres. J. Med. Chem. 2013, 56, 2547−2555. (37) 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-1,2,4trioxolanes as antimalarial peroxides: Charting a workable SAR using simple prototypes. J. Med. Chem. 2005, 48, 4953-4961. (38) 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.; Padmanilayam, M.; Cheruku, S. R.; Brun, R.; Vennerstrom, J. L. Effect of functional group polarity on the antimalarial activity of spiro and dispiro-1,2,4-trioxolanes. Bioorg. Med. Chem. 2006, 14, 6368-6382. (39) 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. Antimalarial 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. (40) 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.

45


Page 47 of 60

1 2 3 4 5 6 7 8 9 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


(41) Tang, Y.; Wittlin, S.; Charman, S. A.; Chollet, J.; Chiu, F. C. K.; Morizzi, J.; Johnson, L. M.; Santo Tomas, J.; Scheurer, C.; Snyder, C.; Zhou, L.; Dong, Y.; Charman, W. N.; Matile, H.; Urwyler, H.; Dorn, A.; Vennerstrom, J. L. The comparative antimalarial properties of weak base and neutral synthetic ozonides. Bioorg. Med. Chem. Lett. 2010, 20, 563-566. (42) Dong, Y.; Wittlin, S.; Sriraghavan, K.; Chollet, J.; Charman, S. A.; Charman, W. N.; Scheurer, C.; Urwyler, H.; Santo Tomas, J.; Snyder, C.; Creek, D. J.; Morizzi, J.; Koltun, M.; Matile, H.; Wang. X.; Padmanilayam, M.; Tang, Y.; Dorn, A.; Brun, R.; Vennerstrom, J. L. The structure-activity relationship of the antimalarial ozonide arterolane (OZ277). J. Med. Chem. 2010, 53, 481-491. (43) 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. (44) Tang, Y.; Dong, Y.; Karle, J. M.; DiTusa, C. A.; Vennerstrom, J. L. Synthesis of tetrasubstituted ozonides by the Griesbaum coozonolysis reaction: Diastereoselectivity and functional group transformations by post-ozonolysis reactions. J. Org. Chem. 2004, 69, 64706473. (45) Cygler, M.; Grabowski, M. J.; Strzyzewski, W.; Turala, L.; Skowronski, R. The synthesis and structure of some 4-(aryloxymethyl)-2,2,6,6-tetramethyl-4-piperidinol and 4(aryloxymethyl)-4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy derivatives. Can. J. Chem. 1986, 64, 670-680. (46) Alliot, J.; Gravel, E.; Buisson, D.-A.; Larquetoux, L.; Nicolas, M.; Doris, E. A straightforward enantioselective synthesis of F17807. Tetrahedron 2015, 71, 9383–9387.

46



1 2 3 4 5 6 7 8 9 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

(47) Kaneko, T.; Clark, R. S. J.; Ohi, N.; Ozaki, F.; Kawahara, T.; Kamada, A.; Okano, K.; Yokohama, H.; Ohkuro, M.; Muramoto, K.; Takenaka, O.; Kobayashi, S. Piperidine carboxylic acid derivatives of 10H-pyrazino[2,3-b][1,4]benzothiazine as orally active adhesion molecule inhibitors. Chem. Pharm. Bull. 2004, 52, 675-687. (48) Vennerstrom, J. L.; Dong, Y.; Charman, S. A.; Wittlin, S.; Chollet, J.; Wang, X.; Sriraghavan, K.; Zhou, L.; Matile, H.; Charman, W. N. Spiro and dispiro 1,2,4-trioxolane antimalarials. PCT WO 2009/091433 A2, July 23, 2009. (49) Wuitschik, G.; Rogers-Evans, M.; Buckl, A.; Bernasconi, M.; Marki, M.; Godel, T.; Fischer, H.; Wagner, B.; Parrilla, I.; Schuler, F.; Schneider, J.; Alker, A.; Schweizer, W. B.; Muller, K.; Carreira, E. M. Spirocyclic oxetanes: Synthesis and properties. Angew. Chem. Int. Ed. 2008, 47, 4512-4515. (50) Erhardt, P. W.; Woo, C. M.; Gorczynski, R. J.; Anderson, W. G. Ultra-short-acting βadrenergic receptor blocking agents. 1. (Aryloxy)propanolamines containing esters in the nitrogen substituent. J. Med. Chem. 1982, 25, 1402–1407. (51) Griesbaum, K.; Liu, X.; Kassiaris, A.; Scherer, M. Ozonolyses of O-alkylated ketoximes in the presence of carbonyl groups: A facile access to ozonides. Libigs Ann./Recueil 1997, 1381– 1390. (52) 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. (53) Zhao, Q.; Vargas, M.; Dong, Y.; Zhou, L.; Wang, X.; Sriraghavan, K.; Keiser, J.; Vennerstrom, J. L. Structure-activity relationship of an ozonide carboxylic acid (OZ78) against Fasciola hepatica. J. Med. Chem. 2010, 53, 4223-4233

47


Page 48 of 60

Page 49 of 60

1 2 3 4 5 6 7 8 9 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


(54) Vennerstrom, J. L.; Dong, Y.; Charman, S. A.; Wittlin, S.; Chollet, J.; Creek, D. J.; Wang, X.; Sriraghavan, K.; Zhou, L.; Matile, H.; Charman, W. N. Dispiro 1,2,4-trioxolane antimalarials. PCT WO 2009/058859 A2, May 7, 2009. (55) Vennerstrom, J. L.; Dong, Y.; Charman, S. A.; Wittlin, S.; Chollet, J.; Creek, D. J.; Wang, X.; Sriraghavan, K.; Zhou, L.; Matile, H.; Charman, W. N. Dispiro 1,2,4–trioxolane antimalarials. U. S. Patent 8,067,620, November 29, 2011. (56) Wuitschik, G.; Carreira, E. M.; Wagner, B.; Fischer, H.; Parrilla, I.; Schuler, F.; RogersEvans, M.; Müller, K. Oxetanes in drug discovery: Structural and synthetic insights. J. Med. Chem. 2010, 53, 3227-3246. (57) 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. (58) 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 bisquinoline 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, 677686. (59) Coteron, J. M.; Marco, M.; Esquivias, J.; Deng, X.; White, K. L.; White, J.; Koltun, M.; El Mazouni, F.; Kokkonda, S.; Katneni, K.; Bhamidipati, R.; Shackleford, D. M.; Angulo-Barturen, I.; Ferrer, S. B.; Jimenez-Diaz, M. B.; Gamo, F. J.; Goldsmith, E. J.; Charman, W. N.; Bathurst, I.; Floyd, D.; Matthews, D.; Burrows, J. N.; Rathod, P. K.; Charman, S. A.; Phillips, M. A., Structure-guided lead optimization of triazolopyrimidine-ring substituents identifies potent

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Plasmodium falciparum dihydroorotate dehydrogenase inhibitors with clinical candidate potential. J. Med. Chem. 2011, 54, 5540-5561. (60) Morgenthaler, M.; Schweizer, E.; Hoffmann-Röder, A.; Benini, F.; Martin, R. E.; Jaeschke, G.; Wagner, B.; Fischer, H.; Bendels, S.; Zimmerli, D.; Schneider, J.; Diederich, F.; Kansy, M.; Müller, K. Predicting and tuning physicochemical properties in lead optimization: Amine basicities. ChemMedChem 2007, 2, 1100-1115.

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In vitro ADME and antimalarial activity of primary amino ether ozonides 3-12

Table 1.

against P. falciparum in vitro and P. berghei in vivo

O O O R= ∗ R

O 3-10

NH2 ∗ 11

NH2 12

IC50 (nM)b K1/NF54

Cures (mean survival in days) c

CLintd (µL/min/ mg protein)

Sol watere (µg/mL)

Compd

R

pKaa

Log D7.4a

3

(CH2)2NH2

9.3

3.0

1.7/2.3 f

3/5 (29)f

100

4

(CH2)3NH2

9.9

2.5

3.7/3.3f

3/5 (29)f

30

5/5

4

>30

5/5

24

3/5

14

0/5

11

>30

5/5

>30

5/5

24

3/5

32

>30

5/5

27

4/5

24

3/5

34

>30

5/5

>30

5/5

27

4/5

38

>30

5/5

>30

5/5

>30

5/5

1

7

0/5

ND

ND

ND

ND

AS

7

0/5

ND

ND

ND

ND

MFQ

>30

5/5

18

0/5

ND

ND

ND = not determined

59


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Table of Contents Graphic

O O O O

O O O

N

Artefenomel Focus on single-dose curative efficacy self-selects for lipophilic compounds.

A

B

C

D

O

A = H, F B = O, CH 2, CONR, SO 2NR C = alkyl, cycloalkyl D = weak base ± polar functional groups

60


StructureâActivity Relationship of the Antimalarial Ozonide

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StructureâActivity Relationship of the Antimalarial Ozonide