Optimized Synthesis and Antimalarial Activity of 1,2-Dioxane-4-carboxamides

FULL PAPER

DOI: 10.1002/ejoc.201301394

Optimized Synthesis and Antimalarial Activity of 1,2-Dioxane-4-carboxamides

Marco Lombardo,*[a,b] Dhiraj P. Sonawane,[c] Arianna Quintavalla,[a,b] Claudio Trombini,[a,b]

Dilip D. Dhavale,[c] Donatella Taramelli,[b,d] Yolanda Corbett,[b,d] Francesca Rondinelli,[b,e]

Caterina Fattorusso,*[b,e] Marco Persico,[b,e][‡] and Orazio Taglialatela-Scafati[b,e][‡]

Keywords: Manganese / Redox chemistry / Amides / Cycloaddition / Radicals / O–O activation / Medicinal chemistry /Antimalarial activity

We recently proposed 3-methoxy-4-methoxycarbonyl-1,2-di-oxanes 3 as promising scaffolds enabling access to potentialantimalarial drugs. We present here an optimized two-stepsynthesis of 3 characterized by high yields, simple work-upprocedures and high diastereoselectivity allowing us to read-ily prepare 3 on multigram scale. The versatility of the 1,2-dioxane scaffold was demonstrated by our generation of anew family of 1,2-dioxane-4-carboxamides 8a–h and the re-alization of their in vitro activities against chloroquine-sensi-

Introduction

Artemisinin-based combination therapies (ACTs) are themost effective strategies by which to treat uncomplicatedmalaria, although they are too expensive for the poorestpeople of malaria endemic regions.[1] Consequently, reliablelow-cost supplies of artemisinin (1, Figure 1) are urgentlyneeded. The presence of the endoperoxide bridge is essentialto antimalarial activity.[2,3] Consequently, many syntheticendoperoxides have been proposed as potential antimalarialdrugs in the last few years.[4] For example, 1,2,4-trioxolaneOZ439[5] and 1,2,4-trioxane CDRI-97/78[6] have recentlyreached phase II clinical trials (Figure 1). Structurally sim-pler endoperoxide-containing polyketides isolated from ma-rine sources, such as plakortin (2, Figure 1) obtained from

[a] Dipartimento di Chimica “Giacomo Ciamician”, Alma MaterStudiorum, Università di Bologna,Via Selmi 2, 40126 Bologna, ItalyE-mail: [email protected]://www.unibo.it/faculty/marco.lombardo

[b] Italian Malaria Network - Centro Interuniversitario di Ricerchesulla Malaria (CIRM), Dipartimento di Medicina Sperimentalee Scienze Biochimiche,Via del Giochetto, 06126 Perugia, Italy

[c] Garware Research Centre, Department of Chemistry,University of Pune,411007 Pune, India

[d] Dipartimento di Scienze Farmacologiche e BiomolecolariUniversità di Milano,Via Pascal 36, 20133 Milano, Italy

[e] Dipartimento di Farmacia, Università di Napoli “Federico II”,Via D. Montesano 49, 80131 Napoli, ItalyE-mail: [email protected]

[‡] These authors equally contributed to the workSupporting information for this article is available on theWWW under http://dx.doi.org/10.1002/ejoc.201301394.

Eur. J. Org. Chem. 2014, 1607–1614 © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1607

tive (D10) and chloroquine-resistant (W2) P. falciparumstrains. In particular, one of these amides (8e), displayed anti-malarial activity on par with the natural product lead plakor-tin and was 5-fold more active than its methyl ester analogue3. Structure–activity relationship (SAR) analysis supported byDFT calculations revealed that, for this family of compounds,alkyl substituents at C6 dictate, in large part, the degree ofantimalarial activity. This finding contrasts those previouslyobserved for the ester series.

the Caribbean sponge Plakortis simplex, also display inter-esting in vitro antimalarial activities against chloroquine re-sistant (CQ-R) Plasmodium falciparum (Pf) strains and,more importantly, fail to display cellular toxicity.[7] Anexhaustive computational and experimental study of thesemolecules provided useful insights into the mechanism ofaction and the pharmacologic requirements needed for anti-malarial activity.[8] These efforts revealed that antimalarialbioactivity results from a concerted mechanism, where thefollowing events occur in a single step as in the case ofplakortin: (i) electron uptake, (ii) O1–O2 bond breakingwith consequent O1 radical formation, (iii) O1–C9 bond

Figure 1. Structures of natural and synthetic antimalarial endo-peroxides.

M. Lombardo, C. Fattorusso et al.FULL PAPERformation with consequent radical shift onto C10 (Fig-ure 1). This latter species represents the key toxic intermedi-ate responsible for plakortin antimalarial activity and is in-volved in subsequent intermolecular reactions. Thus, thestructure must simultaneously orient all intramolecular re-action partners in order to trigger production of the toxiccarbon radical. This realization inspired us to design aseries of simple differently substituted endoperoxides char-acterized by a 3-methoxy-1,2-dioxane scaffold (3, Figure 1).En route to achieving this goal we also developed an inex-pensive two-pot MnIII-mediated synthesis and determinedthe antimalarial activities of the synthetic endoperoxidesagainst CQ-R Pf strains to be in the low micromolarrange.[9]

Although the bioactivities identified are not sufficientlypotent to be directly exploitable, the ester group at C4 andalkyl groups R1–R3 offer a number of opportunities for in-troduction of chemical diversity within scaffold 3 in searchof improved activity levels. Here we present an optimizedsynthetic route to 3, as well as the synthesis of a novel fam-ily of 1,2-dioxane-4-carboxamides 8a–h, one of which wasas active as the natural product lead plakortin.

Results and Discussion

Optimization of the Synthesis of 1,2-Dioxanes 3

Among the different synthetic routes to 1,2-dioxanes, themanganese(III)-promoted [2 + 2 + 2] cycloaddition ofactive methylene compounds (i.e., β-keto esters 4), 1,1-di-substituted alkenes 5 and molecular oxygen (Scheme 1) pro-posed by Nishino and co-workers in the 1990s[10a] and ex-tensively applied by the same author in a variety of success-ful transformations,[10b] is particularly appealing. This 3-component cycloaddition is formally described by a cata-lytic cycle driven by the MnIII/MnII redox couple.[9] How-ever, even though the process is catalytic in MnIII, the[2 + 2 + 2] cycloaddition is often run in acetic acid in anoxygen or air flow, using a stoichiometric amount ofMn(OAc)2 and a catalytic amount of Mn(OAc)3.[10c,10d]

Scheme 1. Synthesis of 3-methoxy-1,2-dioxanes 3. R1 = Me, nBu;R2/R3 = Me/Me, Me/nBu, nBu/Me, nBu/nBu.

The large amount of manganese salts requires a corre-sponding amount of acetic acid (i.e. 25 mL/mmol of thelimiting reagent), and the reaction work-up is biased by

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both the metal and solvent quantities. Alkene component5, with very few exceptions, is a gem-diaryl or monoarylethylene. These olefins produce a stabilized intermediatebenzyl or benzhydryl free-radical benefitting both final re-action yields and overall reaction rates. The choice of"which olefin to use" was the first question to answer sinceour strategy inspired by plakortin 2 required the use of gem-dialkyl olefins. Thus, for the preparation of a small libraryof 3-hydroxy-1,2-dioxanes 6 we adopted the reaction condi-tions reported by Nishino (Scheme 1). Desired hemiketals6 were obtained in the 40–90% yield range following atroublesome work-up procedure.[9] As for the stereoselecti-vity, compounds displaying the –COOMe (equatorial) at C4and the –OH (axial) at C3 in a cis orientation were theonly products attained (hereafter named 3,4-cis) albeit inracemic form. However, compounds with structure 6 werefound to be completely inactive against Pf strains. Alterna-tively, corresponding 3-methoxy-1,2-dioxanes 3, obtainedby a methanol ketalization reaction promoted by (1S)-(+)-10-camphorsulfonic acid (CSA), displayed promising bioac-tivities. Thus, we carried out an optimization study of thereaction conditions for the overall process depicted inScheme 1.

We first modified the original procedure for synthesis of6[10c] by (i) decreasing the amount of acetic acid employedfrom 25 mL/mmol to 5 mL/mmol, (ii) using a catalyticamount of MnIII acetate (10 mol-%) with a stoichiometricamount of MnII acetate, and (iii) using a 3:1 molar ratio ofβ-keto ester 4 and alkene 5 in the presence of O2 at atmo-spheric pressure. The resulting heterogeneous reactionworked satisfactorily on the mmol scale (Table 1, Entry 1).On scale up however, several problems became apparent,related primarily to the use of stoichiometric amounts ofmanganese salts that made the final reaction work-up ut-terly impractical. Using methyl acetoacetate (4a) and 2-but-yl-2-hexene (5a) as components of the benchmark reaction,we then ran the reaction using catalytic amounts of manga-nese salts and further reduced the acetic acid employed as

Table 1. Optimization runs for synthesis of endoperoxide 6a.[a]

Entry 4a Mn(OAc)2 Mn(OAc)3 t Yield[equiv.] [equiv.] [equiv.] [h] [%][b]

1 3 1 0.1 18 71[c]

2 3 0.1 0.1 4 683 3 0.05 0.05 4 744 3 0.01 0.01 4 645 2 0.05 0.05 4 766 1.2 0.05 0.05 4 707[d] 2 0.05 0.05 4 82

[a] Unless otherwise stated, reactions run on 1 mmol of 5a.[b] Yields after purification by flash-chromatography on silica.[c] Data from ref.[9] [d] Reaction run on 20 mmol of 5a.

Synthesis and Biological Activity of 1,2-Dioxane-4-carboxamides

the solvent from 5 mL/mmol to 2 mL/mmol. The results ob-tained in the optimization runs are collected in Table 1.

Using a catalytic amount of MnIII acetate and of MnII

acetate (both 10 mol-%, Table 1, Entry 2), we succeeded inisolating desired endoperoxide 6a in almost the same yieldas when using stoichiometric conditions (Table 1, Entry 1)thus highlighting higher conversions in shorter reactiontimes. These encouraging results prompted us to further re-duce the amount of employed manganese salts to 5 mol-%(Table 1, Entry 3). In this latter case, a completely homo-geneous reaction mixture ensued and manganese salts werecompletely soluble during the basic aqueous work-up, thusallowing product isolation by simple extraction. The reac-tion was carried out also using 1 mol-% of both manganesesalts (Table 1, Entry 4), and only a slight reduction in yieldwas noted. Finally, using 5 mol-% of manganese salts, wetried to also reduce the excess of β-keto esters commonlyemployed in these transformations. Very good yields wereobtained using only 2 equiv. of 4a (Table 1, Entry 5), inplace of the described 3 equiv., and notable results were alsoobtained using only a 20 % excess of β-keto ester (Table 1,Entry 6). The counterintuitive observation that reductionsin catalyst loading and solvent improve isolated productyields is perhaps best explained by a coordinative reductionin undesired side-reactions.[10d] Reactions are much cleanerand more efficient when smaller quantities of 4a and aceticacid are used. Having realized that the use of 5 mol-% ofboth manganese salts and of 2 equiv. of 4a represents agood trade-off between practicality and overall processeconomy, we scaled up the reaction to 20 mmol scale(Table 1, Entry 7), obtaining 6a in 82 % isolated yield. In allcases the reaction proved to be completely diastereoselec-tive, exclusively affording 3,4-cis adduct 6. Correspondingly,the relative C3–C4 and C4–C6 configurations were con-firmed by 2D NMR ROESY, as previously reported.[9]

With an improved method for the synthesis of 3-hydroxy-1,2-dioxanes 6 on multigram scale in hand, we then turnedour attention to the optimization of the subsequent ketal-ization step (Scheme 1). We originally used a large excess ofacid to promote ketalization and this brought about partialepimerization at C3, affording mixtures of 3,4-cis and 3,4-trans isomers 3 with ratios ranging from 50:50 to 70:30 infavor of the 3,4-cis adducts. This diastereoisomer equilibra-tion initially provided us the opportunity to evaluate theeffect of relative stereochemistry on antimalarial activityand to better model their mechanism of action.[9] Throughthese studies we found that, generally speaking, the 3,4-cisadducts possessed greater antimalarial activity than their3,4-trans congeners. Thus, we investigated ketalization of 6ausing different loadings of CSA in refluxing anhydrousmethanol (0.5 m) for 24 h in efforts to optimize cis stereo-isomer generation. Diastereoselectivity was found to relyheavily on the amount of acid employed, favouring 3,4-cis-3a when catalytic amounts of CSA were used. The resultsgenerated during these trials are depicted in Table 2.

The catalytic conditions identified herein enabled us toeasily perform ketalization on 20 mmol scale thereby af-fording desired methyl ketal 3a in very good isolated yield.

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Table 2. Synthesis of methyl ketal 3a.[a]

Entry CSA [equiv.] Yield [%][b] 3,4-cis-3a/3,4-trans-3a

1 5 75 50:502 1 78 60:403 0.15 87 90:104[c] 0.15 85 87:13

[a] Unless otherwise stated, reactions were run on 1 mmol of 6ausing CSA in refluxing anhydrous methanol (0.5 m) for 24 h.[b] Yields after purification by flash-chromatography on silica.[c] Reaction run on 20 mmol of 6a.

Moreover the 3,4-cis adduct was preferentially generatedwith a diastereoselectivity of 87:13 (Table 2, Entry 4).

Synthesis and Biological Activity of Endoperoxide Amides 8

Having optimized a scalable synthetic procedure, we thenmoved to the second goal of this study, namely the prepara-tion of a small library of 1,2-dioxane derivatives carryingan amide functionality at C4. The amide functionality wasselected on the basis of its lower susceptibility to both enzy-matic and non-enzymatic hydrolysis compared to the corre-sponding ester. The most straightforward route to amides 8was to replace 4 in Scheme 1 with desired β-ketoamides.[11]

However, all attempts to remodel to simple β-ketoamidesusing the previously discussed process, invariably failed inour hands.

Thus, having identified suitable reaction conditions forthe medium scale synthesis of 3-methoxy-1,2-dioxanes 3, wedecided to adopt a two-step protocol consisting of the basichydrolysis of the ester moiety to give carboxylic acids 7,followed by O-(benzotriazol-1-yl)-N,N,N�,N�-tetrameth-yluronium tetrafluoroborate (TBTU)-promoted couplingwith the appropriate amine to give a new family of endo-peroxides of general structure 8 (Scheme 2). In this prelimi-nary investigation we used either butylamine or 1-(3-amino-propyl)-imidazole as amines of choice.

Scheme 2. Synthesis of 1,2-dioxane-4-carboxamides 8. R1 = Me,nBu; R2/R3 = Me/Me, Me/nBu, nBu/Me, nBu/nBu; R4 = nBu, N-[3-(1H-imidazol-1-yl)propyl].

Results obtained using this two-step procedure, togetherwith the in vitro antimalarial activities against both CQ-S(D10) and CQ-R (W2) Pf strains, are collected in Table 3.

M. Lombardo, C. Fattorusso et al.FULL PAPERTable 3. Structures, overall synthesis yields and antimalarial ac-tivity of 1,2-dioxane-4-carboxamides (8) against chloroquine-sensi-tive (D10) and chloroquine-resistant (W2) P. falciparum strains.

[a] Overall yields of the two-step sequence after purification byflash-chromatography on silica. [b] Data are the mean�SD ofthree different experiments in duplicate. [c] Chloroquine.

Initially we prepared a series of amides using butylamine8a–c. In agreement with our previous pharmacophoremodel and SAR studies,[9] compound 8a, presenting only


methyl substituents on the endoperoxide ring, is not able toform the carbon centered radical on the alkyl side-chains,and was thus completely inactive (Table 3, Entry 1). Theintroduction of a butyl side-chain at C3 (Table 3, Entry 2)or of two butyl side-chains on C6 (Table 3, Entry 3) wasfound to restore some antimalarial activity; the potenciesof these analogs were comparable to those of their parentmethyl esters (W2 IC50 2.2�0.8 μm for 8b vs. 1.5 �0.5 forthe corresponding methyl ester, 1.95� 0.05 μm for 8c vs.2.5�0.7 for the corresponding methyl ester).[9]

Our previous DFT studies[9] indicated that, in the ab-sence of iron, there is no definite preference between O1and O2 for the acquisition of the radical after O–O re-ductive scission. Thus, iron coordination is a determinantfor formation of the radical on one oxygen rather than theother. In agreement with their observed antimalarial activi-ties, optimization of a low energy conformer of 8b in com-plex with FeII at the DFT level afforded the same resultsthat had previously been obtained for its ester analogue.[9]

In particular, (i) the methoxy group in the axial position ledto the formation of the FeII–O1/O2/O7 pre-reactive com-plex, able to form the oxygen radical on both O1 and O2,and to evolve either on the C3 or C6 butyl chain, and (ii)the FeII–O1/O2 starting complex led to the inactive O2–C3heterolytic scission product (Figure 1, Supporting Infor-mation).

Malaria parasites catabolize haemoglobin in an acidic(pH ≈ 5.5) food vacuole, producing toxic free heme, whichis detoxified by conversion into insoluble hemozoin.[12] Thisprocess is crucial for the activity of many antimalarialdrugs. Indeed, chloroquine and related drugs are believedto interfere with this detoxification process,[13] whereas arte-misinin and, generally, endoperoxides are probably bio-acti-vated by interaction with heme–FeII, leading to the homo-lytic endoperoxide bond cleavage and production of toxicradical species.[12,14] To exploit its antimalarial activity, theendoperoxide must be able to reach the parasite cytoplasmand enter the ferrous-rich acidic food vacuole. On the basisof this pathway, we reasoned that the presence in the endo-peroxide core scaffold of a basic side chain containing animidazole ring might both enhance iron interaction andcause drug accumulation in the acidic food vacuole. Thus,we prepared a series of amides 8d–h using commerciallyavailable 1-(3-aminopropyl)imidazole. Calculated logD andprevalent ionic forms of the designed compounds at cyto-plasmic and parasite food vacuole pH for compounds 8a–h are reported in Table 4.

Some interesting and unexpected results were obtainedfor amides 8d–h. Indeed, the introduction of an imidazolering within the C4 chain abolished any contribution of theC3 butyl chain to antimalarial activity (8d vs. 8b, Table 3).In this new series, antimalarial activity seems to be solelydependent on the number of butyl chains at C6 (8e and 8hvs. 8f and 8g, Table 3). It is noteworthy that 8e is 4-foldmore active than analogue 8c (Table 3) and 5-fold moreactive than its methyl ester analogue (W2 IC50 2.5 �0.7μm).[9] Thus, in the case of 8e, introduction of the imidazolering produced the expected increase in antimalarial activity.


Table 4. Occurrence rate of ionic forms and logD of 1,2-dioxane-4-carboxamides (8).

Ionic form [%][a,b] logD[b]

Entry 8 pH 7.2 pH 5.5 pH 7.2 pH 5.5

1 8a N (100) N(100) 1.98 1.982 8b N (100) N (100) 3.36 3.363 8c N (100) N (100) 4.66 4.664 8d P (38), N (62) P (97), N (3) 2.06 0.85 8e P (38), N (62) P (97), N (3) 3.35 2.096 8f P (38), N (62) P (97), N (3) 3.35 2.097 8g P (38), N (62) P (97), N (3) 3.35 2.098 8h P (38), N (62) P (97), N (3) 4.85 3.59

[a] Percentage of ionic form in brackets; P: protonated form; N:neutral form. [b] Calculated using ACD/Percepta software, version14.0.0 (Advanced Chemistry Development, Inc., Toronto, ON,Canada).

When the most active compound of the series, 8e, wasallowed to react with FeCl2 (6 equiv.) in a 4:1 mixture ofCH3CN/H2O, lactone 9 (Scheme 3) was obtained as themajor product. Formation of 9 was not surprising and isconsistent with the cleavage mechanism already describedfor other members of this family.[9] As previously discussedin our previous manuscript, this model reaction with FeCl2proved unable to predict the in vivo outcome of the reactionwith the FeII center for the class of endoperoxoketal deriva-tives.

Scheme 3. Reaction of compounds 8e with FeCl2. R = N-[3-(1H-imidazol-1-yl)propyl].

In order to properly analyze structure–activity relation-ships (SARs) of this new series of antimalarial endoperox-ides, a computational analysis (see Exp. Sect. for details) ofthe possible ionic forms of 8b, 8d and 8e was performed,taking into account the radical shift mechanism already re-ported for plakortins[8] and related synthetic derivatives.[9,15]

Possible conformers were ranked by their potential energyvalues and grouped into families on the basis of their 1,2-dioxane ring conformation (Table S1, Supporting Infor-mation). Due to the electronic and steric repulsions amongC3, C4, and C6 substituents, the most populated ring con-former was the chair form characterized by the equatorialposition of the C4 substituent, named chair A (Table S1,Supporting Information), as reported for plakortins[8] andsynthetic analogues.[9,15] All conformers within 5 kcal/molof the global minimum of 8b, 8d and 8e were further ana-lyzed. It was determined that the presence of the imidazoleside-chain at C4 (8d and 8e), both, in neutral and proton-ated forms (Table 4), enables formation of intramolecularhydrogen bonds between O7 and/or the amide group(Table S2, Supporting Information and Figure S2, Support-ing Information). Consequently, the C4 chain folds towardsitself and/or C3 (Figure S2, Supporting Information). Con-sidering the rotation of the methoxy group upon iron bind-


ing (Figure 2, a), it is likely that steric crowding betweenC3 and C4 substituents (Figure 2, b) prevents the correctorientation of all reaction partners required for radical shiftonto the C3 butyl chain.

Figure 2. (a) DFT structure of 8b pre-reactive complex. van derWaals volume of iron is shown (scaled by 60% for clarity of presen-tation). (b) PM7 conformer of 8d presenting an intramolecular dis-tance suitable for the 1,4-H shift. The hydrogens involved in the1,4-H shift are evidenced as balls. Hydrogen bonds are highlightedby a green dashed line. O1, O2, O7, C3, C4, and FeII atoms arelabeled. Atoms are colored by atom type (O = red; N = blue; H =white; FeII = cyan).

On the contrary, low energy PM7 conformers of 8b pre-sented an extended conformation of the C4 chain, pointingaway from the C3 butyl chain, thus allowing the hypothe-sized radical shift, in agreement with the observed antima-larial activity (8b vs. 8d, Table 3). The positioning of theC4 imidazole side-chain does not interfere with the putativeradical shift onto C6 butyl chains (Figure S3, SupportingInformation). Accordingly, 8e displayed the expected im-provement in antimalarial activity relative to 8c (Table 3).Further study, including the rational synthesis of new ana-logues, will be performed to investigate a possible role ofthe imidazole moiety in iron coordination and/or radicalevolution. Such structural features may readily enable thedevelopment of new potential antimalarial drugs.

Conclusions

An optimized catalytic protocol is reported for the two-step synthesis of 3-methoxy-1,2-dioxanes 3 and is charac-terized by higher yields, simpler product isolation pro-cedures, higher diastereoselectivity and overall increasedsustainability. This family of endoperoxides represents aninteresting chemically flexible scaffold for diversity orientedidentification of new antimalarial drug candidates. Here wedemonstrated the possibility of easily introducing new sidearms at C4 by converting the original ester group intoamides. The new series of 1,2-dioxane-4-carboxamides 8was tested for in vitro activity against chloroquine-sensitive(D10) and chloroquine-resistant (W2) P. falciparum strains.It was found that, contrary to what we previously observedfor the ester series, alkyl substituents at C6 largely dictateantimalarial potencies and the introduction of an imid-azole-containing side chain at C4 improves antimalarial ac-

M. Lombardo, C. Fattorusso et al.FULL PAPERtivity. In particular, compound 8e was 5-fold more activethan its methyl ester analogue, thus achieving antimalarialactivity comparable to the natural product lead plakortin(2). These results pave the way for the development of new,potent and affordable antimalarial endoperoxides througha simple and inexpensive synthesis. An in-depth theoreticalinvestigation into the role played by the imidazole moietyin the expression of antimalarial activity, as well as thepreparation of new compounds characterized by this mo-lecular framework but possessing different C4 substituents,are currently underway and will be reported in due course.

Experimental SectionOptimized Synthesis of 3-Hydroxy-1,2-dioxanes 6. General Pro-cedure: The appropriate alkene (1–20 mmol) was added at roomtemperature to a mixture of the desired β-keto ester (2–3 equiv.),Mn(OAc)3·2H2O (5 mol-%), and Mn(OAc)2·4H2O (5 mol-%) inacetic acid (2 mL/mmol). The resulting homogeneous solution wasstirred at room temperature for 4 h under oxygen at atmosphericpressure (O2 filled balloon) and the conversion was monitored byTLC. The reaction mixture was neutralized with stoichiometricNaOH (3 m aqueous solution) and then made slightly basic with asaturated NaHCO3 solution. The aqueous phase was extractedwith CH2Cl2 and the combined organic phases were dried(Na2SO4) and the solvents evaporated to dryness. Intermediate 3-hydroxy-1,2-dioxanes 6 were purified by flash chromatography onsilica gel, eluting with cyclohexane/ethyl acetate mixtures. The spec-troscopic data and physical properties of compounds 6 were iden-tical to the previously reported ones.[9]

Optimized Synthesis of 3-Methoxy-1,2-dioxanes 3. General Pro-cedure: (1S)-(+)-Camphorsulfonic acid (15 mol-%) was added atroom temperature to a solution of the desired 3-hydroxy-1,2-diox-ane 6 (1–20 mmol) in anhydrous methanol (2 mL/mmol) and thesolution was stirred at 65 °C for 24 h. The reaction was quenchedat 0 °C with saturated NaHCO3 solution, and the aqueous phasewas extracted with CH2Cl2. The combined organic phases weredried (Na2SO4) and solvents evaporated to dryness. 3-Methoxy-1,2-dioxanes 3 were isolated after flash chromatography on silica gelby elution with cyclohexane/ethyl acetate mixtures. The spectro-scopic data and physical properties of compounds 3 were identicalto the previously reported ones.[9]

Synthesis of 1,2-Dioxane-4-carboxamide 8. General Procedure: So-dium hydroxide (2 m aqueous solution, 1.5 equiv.) was added to asolution of 3 in methanol (0.5 m) and the reaction mixture washeated to reflux until no more starting was detected by TLC (4–12 h). The solution was cooled and evaporated under reduced pres-sure. The residue was diluted with water and acidified with hydro-chloric acid (1 m aqueous solution). The aqueous mixture was ex-tracted with ethyl acetate and the combined organic extracts weredried with Na2SO4, filtered, and evaporated under reduced pres-sure. The crude residue was purified by silica-gel columnchromatography (cyclohexane/ethyl acetate mixtures) to give de-sired carboxylic acid 7. N,N-Diisopropylethylamine (DIPEA,2 equiv.) and O-(benzotriazol-1-yl)-N,N,N�,N�-tetramethyluroniumtetrafluoroborate (TBTU, 1.2 equiv.) were added at room tempera-ture under inert atmosphere to a solution of acid 7 in DCM. Themixture was stirred for 1.5 h at room temperature and then desiredamine compound [butylamine or 1-(3-aminopropyl)-imidazole,1 equiv.] was added. The reaction mixture was stirred at room tem-perature overnight (≈ 12–16 h). The crude reaction mixture was


washed with water, the DCM was dried with Na2SO4, concentratedunder vacuum and the crude residue was purified by silica-gel col-umn chromatography (cyclohexane/ethyl acetate, 90:10 for 8a–cand DCM/methanol, 95:5 for 8d–h).

Compound 8a: ESIMS: m/z = 282 [M + Na]+. HREIMS: m/z =282.1683, calcd. for C13H25NO4Na m/z 282.1681. 1H NMR(400 MHz, CDCl3): δ = 6.94 (broad s, 1 H), 3.41 (s, 3 H), 3.33–3.13 (m, 2 H), 2.80 (dd, J = 13.5, 4.4 Hz, 1 H), 1.94 (t, J = 13.2 Hz,1 H), 1.75 (dd, J = 12.9, 4.5 Hz, 1 H), 1.48 (ddd, J = 14.3, 7.6,4.6 Hz, 2 H), 1.39 (s, 3 H), 1.38–1.29 (m, 2 H), 1.34 (s, 3 H), 1.19(s, 3 H), 0.94 (t, J = 7.3 Hz, 4 H) ppm. 13C NMR (100 MHz,CDCl3): δ = 171.6, 100.3, 77.8, 49.0, 48.9, 38.9, 35.4, 31.5, 27.1,22.2, 20.0, 18.9, 13.7 ppm.

Compound 8b: ESIMS: m/z = 324 [M + Na]+. HREIMS: m/z =324.2155, calcd. for C16H31NO4Na m/z 324.2151. 1H NMR(400 MHz, CDCl3): δ = 7.00 (broad s, 1 H), 3.40 (s, 3 H), 3.26 (dq,J = 13.2, 6.8 Hz, 1 H), 3.17 (dq, J = 13.2, 7.0 Hz, 1 H), 2.94 (dd,J = 13.3, 4.6 Hz, 1 H), 1.94 (t, J = 13.1 Hz, 1 H), 1.76 (dd, J =12.9, 4.5 Hz, 1 H), 1.73–1.58 (m, 4 H), 1.56–1.42 (m, 2 H), 1.37 (s,3 H), 1.38–1.21 (m, 4 H), 1.18 (s, 3 H), 0.93 (t, J = 7.3 Hz, 3 H),0.88 (t, J = 7.1 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ =172.0, 101.7, 77.7, 48.6, 46.1, 38.9, 36.0, 32.8, 31.5, 27.1, 25.6, 23.1,22.3, 20.1, 13.8, 13.7 ppm.

Compound 8c: ESIMS: m/z = 366 [M + Na]+. HREIMS: m/z =366.2627, calcd. for C19H37NO4Na m/z 366.2620. 1H NMR(400 MHz, CDCl3): δ = 6.95 (broad t, J = 5.9 Hz, 1 H), 3.40 (s, 3H), 3.35–3.24 (m, 1 H), 3.23–3.11 (m, 1 H), 2.78 (dd, J = 13.3,4.6 Hz, 1 H), 1.88 (t, J = 13.2 Hz, 1 H), 1.77 (dd, J = 13.0, 4.6 Hz,1 H), 1.55–1.44 (m, 4 H), 1.39–1.18 (m, 12 H), 1.32 (s, 3 H), 0.96–0.87 (m, 9 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 171.9, 100.4,81.7, 48.9, 48.4, 38.9, 36.1, 32.6, 31.5, 31.1, 25.4, 24.8, 23.2, 23.1,20.1, 18.9, 14.0, 13.9, 13.7 ppm.

Compound 8d: ESIMS: m/z = 376 [M + Na]+. HREIMS: m/z =376.2220, calcd. for C18H31N3O4Na m/z 376.2212. 1H NMR(400 MHz, CDCl3): δ = 7.51 (s, 1 H), 7.07 (s, 1 H), 7.07–7.05 (m,1 H), 6.94 (s, 1 H), 3.97 (t, J = 7.0 Hz, 2 H), 3.40 (s, 3 H), 3.28–3.14 (m, 2 H), 2.95 (dd, J = 13.3, 4.5 Hz, 1 H), 2.02–1.94 (m, 2 H),1.92 (t, J = 13.2 Hz, 1 H), 1.74 (dd, J = 12.8, 4.6 Hz, 1 H), 1.71–1.54 (m, 2 H), 1.37 (s, 3 H), 1.35–1.24 (m, 4 H), 1.18 (s, 3 H), 0.88(t, J = 7.0 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 172.6,137.0, 129.8, 118.8, 101.6, 77.6, 48.8, 46.0, 44.5, 36.3, 36.0, 32.9,31.2, 27.1, 25.7, 23.0, 22.3, 13.8 ppm.

Compound 8e: ESIMS: m/z = 418 [M + Na]+. HREIMS:: m/z =418.2688, calcd. for C21H37N3O4Na m/z 418.2682. 1H NMR(400 MHz, CDCl3): δ = 7.49 (s, 1 H), 7.07 (s, 1 H), 7.02 (broad, J

= 6.1 Hz, 1 H), 6.94 (s, 1 H), 3.97 (t, J = 7.0 Hz, 2 H), 3.41 (s, 3H), 3.23 (q, J = 6.7 Hz, 2 H), 2.80 (dd, J = 13.3, 4.6 Hz, 1 H), 2.00(p, J = 7.0 Hz, 2 H), 1.87 (t, J = 13.1 Hz, 1 H), 1.75 (dd, J = 13.0,4.6 Hz, 1 H), 1.59–1.45 (m, 2 H), 1.31 (s, 3 H), 1.37–1.18 (m, 10H), 0.92 (t, J = 7.1 Hz, 3 H), 0.89 (t, J = 6.9 Hz, 3 H) ppm. 13CNMR (100 MHz, CDCl3): δ = 172.5, 137.0, 129.8, 118.8, 110.0,100.2, 81.7, 49.0, 48.3, 44.5, 36.4, 36.1, 32.5, 31.2, 31.0, 25.3, 24.8,23.2, 23.1, 19.1, 14.0, 13.9 ppm.

Compound 8f: ESIMS: m/z = 418 [M + Na]+. HREIMS: m/z =418.2679, calcd. for C21H37N3O4Na m/z 418.2682. 1H NMR(400 MHz, CDCl3): δ = 7.70 (s, 1 H), 7.16–7.07 (m, 2 H), 7.00 (s,1 H), 4.01 (t, J = 6.9 Hz, 2 H), 3.41 (s, 3 H), 3.23 (dq, J = 6.8,2.6 Hz, 2 H), 2.96 (dd, J = 13.2, 4.5 Hz, 1 H), 2.01 (p, J = 7.1 Hz,2 H), 1.89 (t, J = 12.9 Hz, 1 H), 1.76–1.54 (m, 3 H), 1.45–1.39 (m,2 H), 1.34 (s, 3 H), 1.33–1.23 (m, 8 H), 0.95–0.84 (m, 6 H) ppm.13C NMR (100 MHz, CDCl3): δ = 172.7, 137.3, 129.7, 118.9, 101.8,


79.7, 48.7, 45.7, 44.5, 40.1, 36.3, 34.4, 33.0, 31.2, 25.7, 25.0, 23.1,23.0, 20.1, 13.9, 13.8 ppm.

Compound 8g: ESIMS: m/z = 418 [M + Na]+. HREIMS: m/z =418.2680, calcd. for C21H37N3O4Na m/z 418.2682. 1H NMR(400 MHz, CDCl3): δ = 7.53 (s, 1 H), 7.12–7.02 (m, 2 H), 6.95 (s,1 H), 3.98 (t, J = 7.0 Hz, 2 H), 3.40 (s, 3 H), 3.31–3.13 (m, 2 H),2.94 (dd, J = 12.7, 5.2 Hz, 1 H), 1.99 (p, J = 7.0 Hz, 2 H), 1.93–1.77 (m, 2 H), 1.76–1.49 (m, 2 H), 1.39–1.23 (m, 10 H), 1.11 (s, 3H), 0.92 (t, J = 6.8 Hz, 3 H), 0.88 (t, J = 6.9 Hz, 3 H) ppm. 13CNMR (100 MHz, CDCl3): δ = 172.7, 137.1, 129.7, 118.8, 101.5,79.6, 48.7, 45.6, 44.5, 36.3, 34.8, 33.7, 32.9, 31.2, 25.8, 25.6, 24.1,23.1, 23.0, 14.0, 13.8 ppm.

Compound 8h: ESIMS: m/z = 460 [M + Na]+. HREIMS: m/z =460.3155, calcd. for C24H43N3O4Na m/z 460.3151. 1H NMR(400 MHz, CDCl3): δ = 7.57 (s, 1 H), 7.13–7.05 (m, 2 H), 6.96 (s,1 H), 3.99 (t, J = 7.0 Hz, 2 H), 3.40 (s, 3 H), 3.22 (q, J = 6.7 Hz,2 H), 2.94 (dd, J = 13.0, 4.8 Hz, 1 H), 2.00 (p, J = 7.1 Hz, 2 H),1.86 (t, J = 13.0 Hz, 2 H), 1.77 (dd, J = 12.9, 4.8 Hz, 1 H), 1.73–1.56 (m, 2 H), 1.56–1.41 (m, 2 H), 1.38–1.17 (m, 14 H), 0.97–0.83(m, 9 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 172.8, 137.0,129.7, 118.8, 101.7, 81.5, 48.7, 45.3, 44.5, 36.3, 36.1, 33.1, 33.0,31.2, 31.1, 25.7, 25.3, 24.8, 23.2, 23.1, 23.0, 14.0, 13.9, 13.8 ppm.

Reaction of Compound 8e with FeCl2: Compound 8e, (6.0 mg,0.015 mmol) was dissolved in CH3CN/H2O, 4:1 (4 mL) and freshlypurchased FeCl2·4H2O (17.5 mg, 0.09 mmol) was added. The reac-tion mixture was stirred at room temperature for 2 h and then theobtained mixture was partitioned between water and EtOAc. Theorganic phase, dried with Na2SO4, was purified by HPLC (SI60 n-hexane/EtOAc, 8:2) affording compound 9 (2.1 mg, 0.006 mmol,40%).

Compound 9: ESIMS: m/z = 372 [M + Na]+. HRESIMS: m/z =372.2266, calcd. for C19H31N3O3Na m/z 372.2263. 1H NMR(400 MHz, CDCl3): δ = 7.50 (s, 1 H), 7.05 (s, 1 H), 6.92 (s, 1 H),4.39 (t, J = 7.0 Hz, 2 H), 3.25 (overlapped, 3 H), 2.00 (p, J = 7.0 Hz,2 H), 1.75 (overlapped, 4 H), 1.55–1.35 (m, 8 H), 0.90 (t, J =7.1 Hz, 6 H) ppm.

Parasite Growth and Drug Susceptibility Assay: The CQ-S (D10)and the CQ-R (W2) strains of P. falciparum were cultured in vitroas described by Trager and Jensen.[16] Parasites were maintained at5% hematocrit (human type A-positive red blood cells) in RPMI1640 (EuroClone, Celbio) medium with the addition of 1% Al-buMax (Invitrogen, Milan, Italy), 0.01% hypoxanthine, 20 mm

HEPES and 2 mm glutamine, at 37 °C in a standard gas mixtureconsisting of 1 % O2, 5% CO2, and 94% N2. Compounds weredissolved in DMSO and then diluted with medium to achieve therequired concentrations (final DMSO concentration �1%, which isnontoxic to the parasite). Drugs were placed in 96 well flat-bottommicroplates (COSTAR) and serial dilutions made. Asynchronouscultures with parasitemia of 1–1.5% and 1% final hematocrit werealiquoted into the plates and incubated for 72 h at 37 °C. Parasitegrowth was determined spectrophotometrically (OD650) by measur-ing the activity of the parasite lactate dehydrogenase (pLDH), ac-cording to a modified version of Makler’s method in control anddrug-treated cultures.[17] Antiplasmodial activity is expressed as the50% inhibitory concentrations (IC50). Each IC50 value is themean� standard deviation of at least three separate experimentsperformed in duplicate.[18]

Computational Studies: Molecular modeling calculations were per-formed on SGI Origin 200 8XR12000 and E4 Server Twin 2�DualXeon-5520, equipped with two nodes. Each node: 2� Intel® Xeon®

QuadCore E5520-2.26 Ghz, 36 GB RAM. The molecular modeling


graphics were carried out on SGI Octane 2 workstations. Estima-tion of apparent pKa and logD values of the newly designed com-pounds were calculated by using ACD/Percepta software.[19] Ac-cordingly, the percentage of neutral/ionized forms were computedat pH 7.2 (cytoplasmic pH value) and 5.5 (parasite vacuole pH)using the Handerson-Hasselbach equation.

Conformational Analysis: Newly designed compounds 8b, 8d, and8e were built taking into account the prevalent ionic forms at theconsidered different pH values using the Insight 2005 Builder mod-ule (Accelrys Software Inc., San Diego). Atomic potentials andcharges were assigned using the CFF91 force field.[20] Partialcharges of the compounds, considered protonated at the imidazolemoiety as consequence of the estimation of apparent pKa values,were assigned by comparing partial charges assigned by CFF91force field[20] with those calculated by MNDO[21] semiempirical 1SCF calculations performed on the neutral and the ionized com-pounds. In particular, CFF91 force field partial charges were addedto the algebraic difference between MNDO partial charges of theprotonated form and MNDO partial charge of the neutral form.

The conformational space of the compounds was sampled through200 cycles of Simulated Annealing (ε = 1). In simulated annealing,the temperature is altered in time increments from an initial tem-perature to a final temperature by adjusting the kinetic energy ofthe structure (by rescaling the velocities of the atoms). The follow-ing protocol was applied: the system was heated up to 1000 K over2000 fs (time step: 3.0 fs); the temperature of 1000 K was appliedto the system for 2000 fs (time step: 3.0 fs) with the aim of sur-mounting torsional barriers; successively, temperature was linearlyreduced to 300 K in 1000 fs (time step: 1.0 fs). Resulting conforma-tions were then subjected to molecular mechanic (MM) energy mi-nimization within Insight 2005 Discover module (CFF91 forcefield; ε = 1) until the maximum RMS derivative was less than0.001 kcal/Å, using Conjugate Gradient[22] as minimization algo-rithm. All MM conformers were then subjected to a full geometryoptimization by semiempirical calculations, using the quantum me-chanical method PM7 in the Mopac2012 package[23] and EF[24]

(Eigenvector Following routine) as geometry optimization algo-rithm. GNORM value was set to 0.01. To reach a full geometryoptimization the criteria for terminating all optimizations was in-creased by a factor of 100, using the keyword PRECISE.

Resulting conformers were ranked by their potential energy values(i.e., ΔE from the global energy minimum) and grouped into fami-lies on the basis of their 1,2-dioxane ring conformation (i.e. chairA, chair B; skew boat A and skew boat B).

All PM7 conformers within 5 kcal/mol from the global minimumcharacterized by chair A 1,2-dioxane ring conformation were fur-ther classified on the basis of (i) distance between endoperoxideoxygens (O1 and O2) and possible partners for a “through space”(1,4 and 1,5) intramolecular radical shift (� 3 Å); (ii) steric accessi-bility of endoperoxide oxygen lone pairs and (iii) intramolecularinteractions among C3, C4 and C6 substituents. The accessible sur-face area of endoperoxide oxygens lone pairs has been evaluatedby calculating Connolly surfaces (Insight 2005, Accelrys SoftwareInc., San Diego).

A low energy conformer of 8b meeting the assumed pharmacoph-oric requirements, in complex with FeII, was subjected to DFT cal-culations. All possible FeII coordination geometries (i.e. O1, O1/O2, O2/O7 and O1/O2/O7) were used as starting structures for theDFT study.

Density Functional Calculations: All DFT calculations were carriedout using the Gaussian09 suite of programs.[25] The theoretical pro-

M. Lombardo, C. Fattorusso et al.FULL PAPERtocol chosen for these calculations included B3LYP hybrid ex-change-correlation functional in connection with 6-311++G** andDZVP (opt) basis sets for non-metal atoms and iron,[26–28] respec-tively. Optimized contracted Gaussian DZVP (opt) basis set, set upad hoc for B3LYP, ensures a reliable representation of metal proper-ties in opposition to the pseudopotential behaviour.[29] The DFTminimum structure was characterized by vibrational analysis at thesame level of theory. APT charges and both Mulliken and NBOspin densities were evaluated for the metal compound.[30]

Supporting Information (see footnote on the first page of this arti-cle): 1H and 13C NMR spectra for 8a–h. DFT structures of 8bbioactive conformer in complex with FeII. PM7 conformers of 8din neutral and in protonated form. PM7 conformer of 8e presentingan intramolecular distance suitable for the 1,4-H shift. Table ofoccurrence rate of 1,2-dioxane ring conformations of 8b, 8d and 8e.Table of hydrogen bonds of 8b, 8d and 8e.

Acknowledgments

This work was supported by the Ministero degli Affari Esteri(MAE), Rome contribution PGR00124 (Design and developmentof new antimalarial leads for chloroquine-resistant plasmodiumstrains) to CT and EU project Bluegenics (grant number 311848)to O. T. S. and C. F. The authors are particularly thankful to AVISComunale Milano for collecting blood samples for parasite culture.

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2469–2660.Received: September 13, 2013

Published Online: January 8, 2014

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Optimized Synthesis and Antimalarial Activity of 1,2-Dioxane-4-carboxamides