Evaluation of the Pharmacophoric Role of the O-O Bond in Synthetic Antileishmanial Compounds: Comparison between 1,2-Dioxanes and Tetrahydropyrans

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Evaluation of the Pharmacophoric Role of the O−O Bond in

Synthetic Antileishmanial Compounds: Comparison between 1,2-

Dioxanes and Tetrahydropyrans

Margherita Ortalli,^⊥Stefania Varani,^⊥Giorgia Cimato, Ruben Veronesi, Arianna Quintavalla,*

Marco Lombardo,* Magda Monari, and Claudio Trombini

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ABSTRACT: Leishmaniases are neglected diseases that can be

treated with a limited drug arsenal; the development of new

molecules is therefore a priority. Recent evidence indicates that

endoperoxides, including artemisinin and its derivatives, possess

antileishmanial activity. Here, 1,2-dioxanes were synthesized with

their corresponding tetrahydropyrans lacking the peroxide bridge,

to ascertain if this group is a key pharmacophoric requirement for

the antileishmanial bioactivity. Newly synthesized compounds

were examined in vitro, and their mechanism of action was

preliminarily investigated. Three endoperoxides and their corre-

sponding tetrahydropyrans eﬀectively inhibited the growth of

Leishmania donovani promastigotes and amastigotes, and iron did

not play a signiﬁcant role in their activation. Further, reactive

oxygen species were produced in both endoperoxide- and tetrahydropyran-treated promastigotes. In conclusion, the peroxide group

proved not to be crucial for the antileishmanial bioactivity of endoperoxides, under the tested conditions. Our ﬁndings reveal the

potential of both 1,2-dioxanes and tetrahydropyrans as lead compounds for novel therapies against Leishmania.

formulation of more eﬀective combination therapies.²⁹

INTRODUCTION

■

However, the cost increase³⁰⁻³²and the rapid recurrence of

Leishmania protozoa are endemic in most tropical and

subtropical areas worldwide and in the Mediterranean

resistance phenomena³³⁻³⁷prompted the scientiﬁc community

Europe.¹⁻⁴Due to global warming and climate change, these

to turn its attention toward the development of new

antileishmanial drugs,³⁸⁻⁵¹which should be eﬀective, safe,

parasites and their sand ﬂy vectors are at risk to spread into

countries previously considered nonendemic, including central

and western Europe.⁵⁻¹⁰The protozoa of the genus

Leishmania are responsible for various forms of human

leishmaniasis. While cutaneous leishmaniasis can be self-

limiting, mucocutaneous infection can lead to profoundly

disﬁguring lesions, and visceral leishmaniasis is fatal if left

untreated.¹¹Collectively, the parasites belonging to the genus

Leishmania cause one of the most burdensome neglected

tropical diseases, aﬀecting predominantly poor populations,

providing annually 1.5−2 million new cases and roughly

70,000 deaths.¹²Currently, the pharmacological treatment for

human leishmaniasis is based on few drugs¹³(Figure 1), which

lead to unsatisfactory results due to their toxicity, complex

administration protocols (slow and painful intravenous

infusion or intramuscular injection), variable eﬀectiveness

(depending on the disease clinical form or the infecting

Leishmania species), and, more recently, growing drug

resistance.¹⁴⁻²⁶

and not expensive. Concerning the research and development

of new improved drugs, four main approaches are exploited:

(i) drug repurposing, as in the case of fexinidazole⁴⁰(Figure

2); (ii) diversity-oriented screening of collections of chemicals,

such as oxaborole AN-4169^39,52and aminopyrazole amide 1³⁹

(Figure 2); (iii) phenotypic screening, leading to the

identiﬁcation of new biological targets and eﬀective inhib-

itors,⁵³⁻⁵⁶such as 17-AAG (HSP90 inhibitor)⁵⁷(Figure 2);

and (iv) use of known or new natural products with limited

side eﬀects,⁵⁸⁻⁶¹especially those deriving from plants, such as

fucoidan⁵⁸and 11,13-dehydrocompressanolide⁶¹(Figure 2).

Received: September 10, 2020

The early eﬀorts to overcome these limitations were directed

to the improvement of the drug delivery systems^27,28and the

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Figure 1. Drugs currently employed for the treatment of human leishmaniases (the reported IC₅₀values are relative to bioassays on promastigotes

of Leishmania donovani strains).

Figure 2. Some potential new leads for the treatment of leishmaniasis.

Among the plant-derived antiparasitic compounds, some

natural endoperoxides have shown a certain antileishmanial

activity.⁶²⁻⁶⁵In particular, artemisinin and some of its

semisynthetic derivatives (Figure 3) have shown remarkable

biological activities,^66,67especially as antimalarials; these

compounds are currently recommended by World Health

Organization as ﬁrst-line treatment for Plasmodium falciparum

malaria.^68,69Artemisinin and its derivatives also exhibited a

promising bioactivity against other pathogenic protozoa,

including Trypanosoma spp. and Leishmania spp.^{66,67,70−76}

Focusing on the antileishmanial properties, it is worth

mentioning that the in vitro potency of some semisynthetic

artemisinin derivatives (BB201 and BB241; Figure 3) is

comparable to or higher than those of some currently

employed drugs (amphotericin B and miltefosine).^66,67More-

over, artemisinin-derived endoperoxides are able to inhibit the

parasite metabolism with limited adverse eﬀects on the host

cells.^66,77

recently proposed. In 2015, Cortes and co-workers reported on

the fully synthetic trioxolanes LC50 and LC95 (Figure 3),

showing a promising bioactivity when tested in vitro against

promastigotes (IC₅₀range, 3.5−9.4 μM) and intracellular

amastigotes (IC₅₀range, 79.8−107.9 μM) of L. infantum.⁷⁹In

2019, Iwanaga et al. described the oral activity of the fully

synthetic tetraoxaspiro N-251 (Figure 3) against amastigotes

of L. donovani (IC₅₀= 6.7 μM).⁸⁰

We recently proposed new structurally simple 3-methoxy-

1,2-dioxanes 2 (Figure 3), obtained through an eﬃcient and

cheap synthetic approach.⁸¹⁻⁸⁷The simple and ﬂexible

protocol developed for their synthesis allowed us to collect a

small library of 1,2-dioxanes 2, variably substituted on C3, C4,

and C6. When tested for in vitro antileishmanial activity on

promastigotes of L. donovani, some of these compounds

exhibited good inhibitory activities (IC₅₀range, 4.0−16.3 μM)

and low toxicity (selectivity index range, 12.2−35).⁸⁷The most

interesting compounds in terms of activity and selectivity were

further tested in vitro on L. donovani amastigotes and L. tropica,

L. major, and L. infantum promastigotes, showing good

performance. A preliminary investigation of the structure−

activity relationships (SARs) allowed us to identify some of the

key pharmacophoric requirements for this class of antileishma-

One of the limits to the development of drugs based on

natural products is the low availability of the desired

compound in the natural source, which renders the large-

scale production diﬃcult and expensive. For this reason, the

total synthesis of structurally simple cyclic peroxides has been

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reductive decomposition of the peroxide function generates

radical oxygen species, which trigger alkylation processes,

leading to the parasite death.⁸⁸⁻¹⁰⁰A pharmacophoric role of

the peroxide bond in dioxanes has also been suggested against

Leishmania parasites,^{62,64,65,79,80,101}but it has not yet entirely

proved. Few studies investigated the mechanism of action of

endoperoxides on Leishmania parasites, focusing only on

natural scaﬀolds. In fact, Chatterjee et al. established that

artemisinin exerts its activity on L. donovani promastigotes by

an iron-dependent generation of free radicals.^102,103Gille and

co-workers stated that the activation by Fe(II) of the peroxide

bond in ascaridole and artemisinin is an essential part of their

pharmacological mechanism in vitro on L. tarentolae

promastigotes.¹⁰⁴Accordingly, the objectives of the present

study are as follows: (i) the synthesis of tetrahydropyrans 3,

bearing the same substitution pattern of the already studied

endoperoxides 2 and lacking the peroxide bridge (Figure 4);

(ii) the evaluation of their antileishmanial properties to

establish the role played by the O−O bond; and (iii) the

preliminary investigation of the mechanism of action of the

most promising bioactive compounds.

Figure 3. Antileishmanial activity of some recently studied natural and

synthetic cyclic peroxides (IC₅₀values are relative to bioassays on

promastigotes for all compounds, except for N-251, where the value is

relative to amastigotes).⁷⁸

RESULTS

■

Synthesis of Tetrahydropyrans 3. Our retrosynthetic

analysis for the construction of tetrahydropyrans 3 is sketched

in Scheme 1. The amino side chain is installed on C3

exploiting the ester group of intermediate 9, while the six-

membered ring is built by a spontaneous intramolecular

ketalization of intermediate A. The unsaturated β-ketoester 8

could in turn be obtained via a Knoevenagel condensation,

starting from the protected 2,2-disubstituted 3-hydroxypropa-

nal 7.

Considering the good antileishmanial properties of C6-

diphenyl and C6-dibutyl endoperoxides bearing the amino-

propyl imidazole side chain on C4 (2b and 2a, respectively;

Table 1),⁸⁷we initially decided to prepare the analogous

tetrahydropyrans 3b (R²= Ph and R¹= Me) and 3a (R²= Bu

and R¹= Me) as well as the tetrahydropyran 3c (R²= R¹=

Me), analogue of the inactive endoperoxide 2c (Table 1), to

conﬁrm the pharmacophoric requirements of the lipophilic

side chains on C6. The synthesis of 3b (Scheme 2) started

nial endoperoxides (Figure 4): (i) lipophilic side chains must

be present on C6; (ii) a long lipophilic side chain on C3

increases the cytotoxicity; (iii) the C3−C4 relative stereo-

chemistry weakly aﬀects the antileishmanial activity; and (iv) a

heteroaromatic ring must be present on the C4 side chain and

its nature signiﬁcantly aﬀects both activity and toxicity of 1,2-

dioxane.

In the present study, we further investigate the structure−

activity relationship of our synthetic 1,2-dioxanes, in order to

establish whether the peroxide group is a crucial pharmaco-

phoric requirement for the antileishmanial bioactivity (Figure

4). Concerning P. falciparum malaria, it has been demonstrated

that the antimalarial peroxides act via intraerythrocytic

activation of the O−O bond by Fe(II). The homolytic

̈

Figure 4. Previously identiﬁed pharmacophoric requirements of 3-methoxy-1,2-dioxanes 2 as antileishmanial agents⁸⁷and aim of the present work.

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three simple and high-yielding synthetic steps, consisting of a

trans-acetalization (step a), a DIBALH-promoted acetal

reduction (step b), and a Dess−Martin periodinane-promoted

oxidation (step c).

Scheme 1. Retrosynthetic Analysis of Tetrahydropyrans 3

(PG = Protective Group)

The β-ketoester moiety was inserted on the intermediate

̈

aldehyde 7b via a Knoevenagel reaction. The high steric

hindrance close to the reactive site of the α,α-disubstituted

aldehyde and the generation of a congested trisubstituted

oleﬁn particularly hampered this process, which required harsh

reaction conditions and excess of reagents.¹⁰⁶Despite the

optimization attempts by varying the reaction temperature and

reagent ratio, the yield of 8b could not be signiﬁcantly

improved. A single stereoisomer of the diphenyl oleﬁn 8b was

observed and isolated from the crude mixtures. The benzyl O-

protecting group was selected in order to integrate in a single

synthetic step the alcohol deprotection and the double bond

hydrogenation, leading to the desired intermediate A (Scheme

1). Unexpectedly, 2-methoxy tetrahydropyran 9b (Scheme 2)

was directly obtained in good yield after the hydrogenation

step, proving that four diﬀerent transformations took place in a

with the commercially available diphenylacetaldehyde, which

was converted to the desired 2,2-disubstituted propanediol 4b

using a reported literature procedure.¹⁰⁵The protected

aldehyde 7b was thus obtained exploiting a sequence of

Table 1. Inhibitory Activity of Tetrahydropyrans 3 and Endoperoxides 2 against Promastigotes of L. donovani, Cytotoxicity in

Mammalian Kidney Epithelial Cells (Vero), and Selectivity Index (SI)

a

b

Compounds tested as racemates. IC₅₀represents the concentration of a compound that causes 50% growth inhibition. Results represent the

c

mean ( standard deviation, SD) of three independent experiments performed in duplicate. Additional susceptibility tests for selected compounds

were performed on L. donovani cultures using 10% FBS in the culture medium, in order to compare the variation of IC₅₀with a less nutrient- and

d

antioxidant-rich environment (see the Supporting Information, Table S2). CC₅₀represents 50% cytotoxic concentration on Vero cells. Results

e

represent the mean ( standard deviation, SD) of three independent experiments performed in duplicate. Selectivity index (SI) = CC₅₀/IC₅₀; nd =

not determined due to the low antileishmanial potency.

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a

Scheme 2. Synthetic Route Developed for the Construction of Tetrahydropyrans 3 (See Experimental Section for Details)

a

Reagents and conditions: (a) CSA (1 mol %), DCM, 0 °C to rt, and 2−12 h; (b) DIBALH (2 equiv), DCM, −78 °C to rt, and 12 h; (c) Dess−

Martin periodinane (1.05 equiv), DCM, 0 °C to rt, and overnight; (d) methyl acetoacetate (2 equiv), pyridine (4 equiv), TiCl₄·2THF (2 equiv),

THF, 0 °C to reﬂux, and 18 h; (e) Pd/C (20% w/w), H₂(ﬁlled balloon), MeOH, rt, and 12−16 h; (f) LiAlH₄(1 equiv), THF, 0 °C, and 1−3 h;

(g) Dess−Martin periodinane (1.05 equiv), DCM, 0 °C to rt, and overnight; (h) 1-(3-aminopropyl)imidazole (1 equiv), MeOH, rt, and overnight;

and (i) NaBH₄(1.5 equiv), 0 °C to rt, and 1 h.

one-pot fashion: O-debenzylation, double bond reduction,

cyclization, and methyl ketal formation.¹⁰⁷Moreover, this

process was not only very eﬃcient but also highly cis-

stereoselective, as established by the X-ray crystallographic

analysis of the isolated product 9b (Figure 5, see the

Supporting Information for details).¹⁰⁸

At last, the replacement of the ester group on C3 with the

aminopropyl imidazole side chain was accomplished following

the protocol developed for the corresponding endoperoxides

2,⁸⁷consisting in the reduction to an alcohol, followed by

oxidation to the corresponding aldehyde and reductive

amination (steps f−i, Scheme 2).

The synthetic approach proposed for tetrahydropyran 3b

was applied also to the construction of 3a and 3c (Scheme 2).

The dibutyl (4a) and dimethyl (4c) diols are both

commercially available, and they were eﬃciently converted

into the corresponding α,α-disubstituted aldehydes 7a and 7c,

̈

respectively. The Knoevenagel reaction proceeded, as

expected, with a moderate yield on the hindered α,α-dibutyl

aldehyde 7a but with an excellent yield on the α,α-dimethyl

aldehyde 7c, proving that the performance of this process is

governed by the steric hindrance. In addition, it is worth

mentioning that unlike the diphenyl derivative 8b (Scheme 2),

the dialkyl-substituted oleﬁns 8a and 8c were obtained as

mixtures of two isomers (8a: Z/E = 65:35, 8c: Z/E =

60:40).¹⁰⁹To investigate the impact of the double bond

geometry on the outcome of the following one-pot trans-

formation, providing the tetrahydropyran system 9, we carried

out the reaction on the two isomers Z-8a and E-8a, separately

(Scheme 3). Invariably, we obtained only the 2,3-cis

tetrahydropyran 9a from both oleﬁns, demonstrating not

only the cis-stereoconvergence¹¹⁰proper of the process but

also the comparable reaction rates for the two isomers. On this

Scheme 3. Reagents and Conditions: Pd/C (20% w/w), H₂

(ﬁlled balloon), MeOH, rt, and Overnight

Figure 5. Determination of the relative stereochemistry of 2-methoxy

tetrahydropyran 9b through X-ray crystallographic analysis (thermal

ellipsoids are drawn at 30% of the probability level).

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Scheme 4. Synthesis of Tetrahydropyran 3d Starting from Intermediate Alcohol 10b (see Experimental Section for Details)

Table 2. Inhibitory Activity of Tetrahydropyrans 3 and Endoperoxides 2 against Amastigotes of L. donovani, Cytotoxicity in

Mammalian Kidney Epithelial Cells (Vero), and Selectivity Indexes (SIs)

a

b

Compounds tested as racemates. IC₅₀represents the concentration of a compound that causes 50% growth inhibition. Results represent the

c

mean ( standard deviation, SD) of three independent experiments performed in duplicate. CC₅₀represents 50% cytotoxic concentration on Vero

cells. Results represent the mean ( standard deviation, SD) of three independent experiments performed in duplicate. Additional cytotoxicity tests

d

were performed employing the THP-1 cell line (see the Supporting Information, Table S3). Selectivity index (SI) = CC₅₀/IC₅₀.

basis, the mixtures of Z and E isomers were directly used

without separation.

The subsequent usual manipulations of the C3 side chain

provided the desired aminopropyl imidazole tetrahydropyrans

3a and 3c (Scheme 2).

Vero cells (Table 1) and on THP-1 cells (see the Supporting

Information, Table S3) and the corresponding selectivity

indexes (SIs) were calculated. The performance of each

tetrahydropyran 3 was compared with that of the correspond-

ing endoperoxide 2 (Table 1).

Finally, the intermediate alcohol 10b (Scheme 2) was used

as the starting material for the synthesis of the tetrahydropyran

3d, analogue of the highly active antileishmanial endoperoxide

2d (Table 1). This molecule, characterized by the presence of

a triazole ring and a phosphonium salt in the C3 side chain,

was prepared through an alkylation/click cycloaddition

sequence developed for the construction of the corresponding

endoperoxide 2d (Scheme 4).⁸⁷

In Vitro Growth Inhibition of L. donovani Promasti-

gotes and Amastigotes. The bioactivity of the new

tetrahydropyrans 3 was initially evaluated against the

extracellular promastigote forms of L. donovani (MHOM/

NP/02/BPK282/0cl4) as the reference strain, indicative of the

leishmaniasis in the Old World. The results were expressed as

IC₅₀, i.e., the concentration of the product required to inhibit

the parasite growth by 50%. When a compound could inhibit

the promastigote growth, its cytotoxicity was also evaluated on

We observed a similar bioactivity proﬁle for endoperoxides 2

and the corresponding tetrahydropyrans 3; the aminopropyl

imidazole derivatives 3a/2a and 3b/2b showed IC₅₀in the low

micromolar range (Table 1). The parallel behavior was

maintained also for methyl derivatives 3c and 2c, which were

both inactive under our bioassay conditions (40 μM

compound). The same set of tetrahydropyrans 3 and

endoperoxides 2 was also tested on intramacrophage

amastigote forms of L. donovani (Table 2).

The good antileishmanial potency in the low micromolar

range observed on promastigotes was preserved on amastigotes

for both classes of compounds. Moreover, it is interesting to

note that our synthetic structurally simple compounds,

endoperoxides 2 and tetrahydropyrans 3, are signiﬁcantly

more potent against L. donovani parasites (promastigote and

amastigote forms) than artemisinin and artesunate (Tables 1

and 2).

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Table 3. Inhibitory Activity of Tetrahydropyran 3a and Endoperoxide 2a against Promastigotes of L. donovani in the Presence

or Absence of the Iron Chelator DFO

a

b

Compounds tested as racemates. IC₅₀represents the concentration of a compound that causes 50% growth inhibition. Results represent the

mean ( standard deviation, SD) of three independent experiments performed in duplicate. We also tested these compounds in the presence of

deferiprone (DFP), which is a more lipophilic iron chelator than DFO (see the Supporting Information, Table S5 and Figure S3). DFO =

desferrioxamine.

Figure 6. (A, B) Isobolograms depicting the interaction of (A) tetrahydropyran 3a with the iron chelator DFO and (B) endoperoxide 2a with the

iron chelator DFO. A dark gray line is line of additivity. X axes depict the fractional inhibitory concentration (FIC = IC₅₀of the drug in the

combination/IC₅₀of the drug when tested alone). Y axes depict the fractional of DFO. Square in the ﬁgure indicates ΣFIC values from each drug

combination.

In particular, tetrahydropyrans 3 revealed to be slightly more

active against amastigotes than the corresponding endoper-

oxides 2.

DFO at diﬀerent concentrations. The variation of IC₅₀of the

compounds in relation to the diﬀerent doses of DFO was

determined by the alamarBlue assay (Table 3), and the isobole

technique was employed to depict the interactions of 2a and

3a with DFO (Figure 6).

Investigation on the Mechanism of Action of 1,2-

Dioxanes 2 and Tetrahydropyrans 3 against L.

donovani. Study on the Iron Role in the Activation of

Synthetic Antileishmanial Compounds 2 and 3. Little is

known about the mechanism of action of endoperoxides on

Leishmania parasites. To investigate whether iron plays a role

in triggering the antileishmanial bioactivity of our synthetic

compounds, we studied whether the inhibition caused by 2a

and 3a on L. donovani promastigotes was inﬂuenced by the

presence of an iron chelator (desferrioxamine, DFO);^111,112

the results are presented in Table 3.

As shown in Table 3, the inhibitory activity of 3a and 2a

against Leishmania parasites did not undergo signiﬁcant

variation in the presence of various concentrations of DFO,

proving a scarce inﬂuence of the iron chelator on the

bioactivity of the compounds. The isobole technique was

used for depicting synergistic or antagonistic interaction

between the iron chelator and the two compounds on the

parasite growth (Figure 6). Combinations are expressed as the

sum of the fractional inhibitory concentrations (ΣFIC), and

FIC values are deﬁned as synergism (<0.5), antagonism

(>4.0), and additivity (no interaction). The strength of

synergism (or antagonism) is indicated by the degree of

deviation from the line of additivity. As shown in Figure 6,

At ﬁrst, we determined IC₅₀and CC₅₀values of the iron

chelator DFO (187 and >400 μM, respectively). Then, the

eﬀect of the iron chelator presence on the bioactivity of the

two compounds was studied by coincubating 2a and 3a with

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combination of DFO with 2a or 3a resulted in additive eﬀects

as no antagonism or synergism was observed. These ﬁndings

suggest that low-molecular-weight iron species do not play a

crucial role in triggering the antileishmanial activity of the

tested compounds.

The eﬀect of the iron chelator DFO was investigated also on

the pair of compounds 2b and 3b, conﬁrming the same

behavior observed for 2a and 3a, respectively (see the

Supporting Information, Table S4 and Figure S2).

Reactive Oxygen Species (ROS) Production Induced by

Antileishmanial Compounds 2 and 3 in L. donovani

Promastigotes. We evaluated the ability of our synthetic

compounds to generate reactive oxygen species (ROS) in L.

donovani promastigotes. We selected the endoperoxide 2a and

the tetrahydropyran 3a as model substrates, which were

administered to the parasites in the presence of 2,7-

dichlorodihydroﬂuorescein diacetate (H₂DCFDA), a live-cell-

permeable dye, which is oxidized by ROS, providing a

ﬂuorescent compound (DCF) (see Experimental Section for

details).¹⁰³Therefore, the resultant ﬂuorescence is a direct

measure of the amount of ROS generated.

Figure 8. Quantiﬁcation of ROS generated upon treatment of L.

donovani promastigotes with tetrahydropyran 3a (IC₅₀), endoperoxide

2a (IC₅₀), miltefosine (22 μM), and H₂O₂(20 μM) or without

treatment (negative control, NC) for 1−60 min. Results represent the

mean ( standard deviation, SD) of three independent experiments

performed in triplicate.

As shown in Figure 7, endoperoxide 2a and tetrahydropyran

3a were both capable of inducing an increase in the

control H₂O₂(Figure 8). The data obtained from the ROS

levels evaluation led us to two main observations: (i) the

tetrahydropyran 3a induced a higher increase in intracellular

ROS levels than the endoperoxide 2a, and (ii) the amount of

ROS generated by 3a and 2a was in accordance with the IC₅₀

proﬁle of the compounds as a higher inhibitory activity was

found for 3a (IC₅₀3.4 μM) as compared to 2a (IC₅₀, 7.5 μM).

Localization Studies of Tetrahydropyrans 3 and 1,2-

Dioxanes 2 in Parasitic Cells. In order to acquire

information on the ability of the tested compounds to enter

the L. donovani promastigote cell and on their biodistribution

within the parasite, we accomplished a confocal microscopy

investigation. For this purpose, we synthesized new com-

pounds carrying a ﬂuorescent probe (Schemes 5 and 6). In

particular, the most active butyl derivatives were labeled with

an acridine-based ﬂuorescent dye (2e and 3e, Scheme 5),

which was introduced exploiting the ﬁnal reductive amination

step of the previously developed synthetic route. The isolation

yields of the ﬂuorescent endoperoxide 2e and tetrahydropyran

Figure 7. Measurement of ROS in promastigotes of L. donovani in the

presence of endoperoxide 2a and tetrahydropyran 3a tested at their

IC₅₀. Generation of ROS was measured using 2,7-dichlorodihydro-

ﬂuorescein diacetate (H₂DCFDA). Miltefosine (22 μM) and H₂O₂

(20 μM) were used as positive controls. NC: negative control

(untreated parasites). The parasites were analyzed by ﬂuorimetry.

Results represent the mean of three independent experiments

performed in triplicate.

Scheme 5. Reagents and Conditions: (a) N¹-(6-Chloro-2-

methoxyacridin-9-yl)butane-1,4-diamine (1 equiv), MeOH,

rt, and Overnight and (b) NaBH₄(1.5 equiv), 0 °C to rt,

a

and 1 h (see Experimental Section for Details)

intracellular ROS levels at their IC₅₀as compared to the

negative control (untreated promastigotes). In details, 3a

showed a higher ROS production when compared to H₂O₂(20

μM, positive control) but a slightly lower ROS production

when compared to miltefosine (22 μM, positive control).

Compound 2a showed a lower ﬂuorescence intensity when

compared to H₂O₂and miltefosine; however, a higher amount

of ROS was detected in 2a-treated promastigotes in

comparison with untreated cells. Quantiﬁcation of the

generated ROS was monitored for 1−60 min. As shown in

Figure 8, intracellular ROS levels increased over time in

untreated and treated promastigotes, but the increase was

higher in parasites that were treated with 3a and 2a than in

untreated cells. These results indicate that 3a and 2a induced

oxidative stress in L. donovani promastigotes, with ROS

production induced by 3a overlapping with the positive

a

For the synthesis of 13, see ref 87.

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Scheme 6. Reagents and Conditions: (a) tert-Butyl (4-Aminobutyl)carbamate (1 equiv), MeOH, rt, and Overnight; (b) NaBH₄

(1.5 equiv), 0 °C to rt, and 1 h; (c) HCl in Et₂O (1 M, 20 equiv), Et₂O, rt, and Overnight; and (d) NBD-Cl (1.1 equiv), TEA

a

(3 equiv), DCM, 0 °C to rt, and 4 h (see Experimental Section for Details)

a

For the synthesis of 13, see ref 87.

Table 4. Inhibitory Activity of Fluorescent Compounds 2e, 2f, and 3e against Promastigotes of L. donovani, Cytotoxicity in

Mammalian Kidney Epithelial Cells (Vero), and Selectivity Index (SI)

a

b

c

Compounds tested as racemates. IC₅₀represents the concentration of a compound that causes 50% growth inhibition. CC₅₀represents 50%

d

cytotoxic concentration on Vero cells. Selectivity index (SI) = CC₅₀/IC₅₀.

Figure 9. Cell localization of labeled endoperoxide 2f and TO-PRO-3 in promastigotes of L. donovani. (A−C) Confocal microscopy images show

(A) uptake of 2f with green ﬂuorescence, (B) overlay of uptake of 2f with green ﬂuorescence and uptake of TO-PRO-3 with blue ﬂuorescence, and

(C) uptake of TO-PRO-3, staining nuclei and kinetoplasts with blue ﬂuorescence. Red arrows indicate nuclei, and yellow arrows indicate

kinetoplasts.

3e were moderate due to their poor solubility in organic

solvents, which made the puriﬁcation diﬃcult.

We were aware that the ﬂuorescence of acridine-derived

labels can be quenched under certain conditions (e.g., acidic

pH value, presence of hematin, etc.) and that this kind of

ﬂuorophore can easily bind to DNA, leading to altered

results.¹¹³To validate the biodistribution data obtained from

acridine-containing compounds 2e and 3e, we synthesized also

the endoperoxide 2f (Scheme 6) bearing the nitrobenzyldia-

zole (NBD) ﬂuorophore, to be used as a comparison since it

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Figure 10. Cell localization of labeled tetrahydropyran 3e and TO-PRO-3 in promastigotes of L. donovani. (A−C) Confocal microscopy images

show (A) uptake of 3e with green ﬂuorescence, (B) overlay of uptake of 3e with green ﬂuorescence and uptake of TO-PRO-3 with blue

ﬂuorescence, and (C) uptake of TO-PRO-3 with blue ﬂuorescence. Red arrows indicate nuclei, and yellow arrows indicate kinetoplasts.

Figure 11. Cell localization of labeled endoperoxide 2e and TO-PRO-3 in promastigotes of L. donovani. (A−C) Confocal microscopy images show

(A) uptake of 2e with green ﬂuorescence, (B) overlay of uptake of 2e with green ﬂuorescence and uptake of TO-PRO-3 with blue ﬂuorescence, and

(C) uptake of TO-PRO-3 with blue ﬂuorescence. Red arrows indicate nuclei, and yellow arrows indicate kinetoplasts.

does not have the abovementioned limitations. The con-

struction of 2f (Scheme 6) was diﬃcult as the nitro-

benzyldiazole system revealed to be not stable under reductive

amination conditions. In particular, the introduction of the

diamino side chain on C4 proceeded smoothly (14, 79%

yield). Conversely, the N-Boc deprotection employing

anhydrous HCl and the isolation of the corresponding product

as the hydrochloride salt revealed to be critical (step c, Scheme

6). For this reason, the coupling with NBD-Cl was carried out

on the crude intermediate. However, the isolated unoptimized

yield of the ﬂuorescent endoperoxide 2f was modest.

At ﬁrst, the bioactivity of the new ﬂuorescent compounds

was evaluated on promastigotes of L. donovani and the

corresponding cytotoxicity on Vero cells was also assayed

(Table 4). The acridin-containing derivatives 3e and 2e

revealed to be more potent against L. donovani promastigotes

than the corresponding imidazole-containing products 3a and

2a, respectively (Table 1). This eﬀect could be due to a toxicity

contribution of the acridin system, as suggested by the low

selectivity index (SI) of these compounds, which were more

toxic on Vero cells than the corresponding nonlabeled

compounds. However, it is important to emphasize that the

behavior of endoperoxide 2e and tetrahydropyran 3e remains

comparable also for this set of labeled molecules.

(Table 1). However, its SI was low, conﬁrming a signiﬁcant

toxicity of these ﬂuorescent products on Vero cells.

The labeled compounds were incubated with L. donovani

promastigotes, and then confocal microscopy images were

acquired (see Experimental Section for details). Figure 9 shows

data obtained with the NBD-labeled endoperoxide 2f. We

noticed a marked accumulation of the ﬂuorescent probe in the

parasite cytoplasm, whereas a dark area was evident inside the

parasite (Figure 9A), likely corresponding to the nucleus. To

conﬁrm this interpretation, we marked the nucleus and

kinetoplast of the parasite with TO-PRO-3 (Figure 9C), a

speciﬁc dye for nucleic acids.^114,115The overlapped color

image (Figure 9B) clearly shows the dark blue nucleus (red

arrows, Figure 9) and the cyan kinetoplast (yellow arrows,

Figure 9), whose color derived from a colocalization of green

and blue ﬂuorescence. This ﬁnding suggested that endoper-

oxide 2f spread throughout the parasite cytoplasm and it was

able to enter the kinetoplast but not the nucleus.

A similar intraparasitic distribution was observed for

acridine-labeled tetrahydropyran 3e by confocal microscopy

(Figure 10).

Concerning acridine-labeled endoperoxide 2e, we observed

a colocalization of green and blue ﬂuorescence not only in the

kinetoplast but also in the nucleus (Figure 11B). This ﬁnding

suggests that this peculiar acridine-containing compound can

enter the nucleus core.

The NBD derivative 2f showed lower activity against L.

donovani promastigotes than the corresponding acridin-

derivative 2e (Table 4), and its IC₅₀value was similar to

that of the corresponding imidazole-containing compound 2a

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cytoplasm and in the kinetoplast but not in the nucleus,

while the acridine-labeled endoperoxide 2e was also found in

the nucleus. Although these ﬁndings are preliminary, they

allow us to make some considerations. It is known that the

localization of a labeled species in a cell depends on the

structure of both the bioactive compound and the ﬂuorescent

tag. By tagging our products with diﬀerent probes, we observed

a common feature, i.e., a marked accumulation of ﬂuorescent

compounds in the parasitic cytoplasm and kinetoplast.

Compound 2f, bearing the nitrobenzyldiazole (NBD)

ﬂuorophore, appears as the most similar to the corresponding

not labeled bioactive product 2a, considering its IC₅₀value

(Table 4) and calculated log P (see the Supporting

Information). Therefore, considering the cell localization of

the most representative labeled derivative 2f, we speculate that

cytoplasm and kinetoplast are the main accumulation sites of

the newly synthesized molecules, where the biological target/s

of this family of compounds might be localized.

In conclusion, we synthesized a small library of tetrahy-

dropyrans 3, bearing the same substitution pattern of the

corresponding endoperoxides 2 but lacking the peroxide

bridge. Both classes of compounds were tested for their

antileishmanial activity, and we observed a similar bioactivity

proﬁle for 1,2-dioxanes 2 and tetrahydropyrans 3. We also

found that in our biological systems: (i) iron appeared not to

play a crucial role in triggering the activity against Leishmania

of the selected molecules; (ii) both 1,2-dioxanes 2 and

tetrahydropyrans 3 induced a signiﬁcant oxidative stress in L.

donovani promastigotes, implying that the cleavage of the O−

O bond is not necessary for the generation of free radicals in

treated promastigotes; and ﬁnally, (iii) ﬂuorescent-tagged 1,2-

dioxanes and tetrahydropyrans mainly accumulated in the

Leishmania cytoplasm and kinetoplast, suggesting that their

biological target/s might be localized in these parasitic

compartments. Our ﬁndings reveal the potential role of both

1,2-dioxanes and tetrahydropyrans as lead compounds for

novel therapies against Leishmania and provide new insights

into their mechanism of action; the peroxide group proved not

to be a crucial pharmacophoric requirement for the

antileishmanial bioactivity of the new synthesized endoper-

oxides in our biological system. Additional studies are needed

to corroborate our ﬁndings.

DISCUSSION AND CONCLUSIONS

■

Little is known on the mechanism of action of endoperoxides

against Leishmania. Previous mechanistic studies on natural

endoperoxides, such as artemisinin, suggested that they act

through an iron(II)-mediated homolytic cleavage of the

peroxide function, leading to the formation of cytotoxic

radicals.^102−104Based on these studies, we expected that

endoperoxides 2 would exhibit higher antileishmanial activity

than the corresponding tetrahydropyrans 3, the latter lacking

the O−O bond. Surprisingly, we observed a similar bioactivity

proﬁle for the two classes of compounds (Table 1) and

tetrahydropyrans 3 revealed to be slightly more active against

intracellular amastigotes than the corresponding endoperoxides

2 (Table 2). The latter ﬁnding is particularly meaningful taking

into account that Leishmania amastigotes obtain the iron

necessary for its metabolism from the host macrophage.^102,116

Considering the greater iron availability for amastigotes than

promastigotes, the compounds activated by iron are normally

more active against amastigotes than promastigotes,¹⁰²while

we observed an opposite behavior for our products. These

ﬁndings suggest that in the tested conditions, the primary

mechanism of action of endoperoxides 2 and tetrahydropyrans

3 on L. donovani parasites does not involve an iron-mediated

activation. Thus, the peroxide group appears not to be a crucial

pharmacophoric requirement for the tested 1,2-dioxanes,

which is divergent from previous ﬁndings obtained with the

natural endoperoxide artemisinin and ascaridole.^102−104

Iron is an essential nutrient for all the organisms, including

Leishmania, and its supply plays a crucial role in the parasite

survival.^117,118However, the parasitic cells are also vulnerable

to the toxicity of iron and iron-induced ROS. Based on the

abovementioned results, we investigated the role of iron in the

activation of our synthetic compounds 2 and 3. In contrast

with the mentioned previous studies, we showed that the

inhibitory activity of 3a and 2a against Leishmania did not

undergo signiﬁcant variation in the presence of various

concentrations of the iron chelators DFO and DFP, suggesting

that low-molecular-weight iron species do not play a crucial

role in triggering the antileishmanial bioactivity of our

synthetic endoperoxides. This result is particularly meaningful

for endoperoxide 2a, and it is supported by the previous

observations that tetrahydropyrans 3 and endoperoxides 2

show a similar antileishmanial potency (Table 1) and that they

have a comparable activity against both promastigote and

amastigote forms of L. donovani (Tables 1 and 2).

EXPERIMENTAL SECTION

Chemistry. General Information. All the commercial chemicals

■

were purchased from Sigma-Aldrich, VWR, Alfa Aesar, or TCI

Chemicals and used without additional puriﬁcations. The ¹H and ¹³

C

To further investigate the role of the peroxide bridge on

bioactivity, we analyzed the levels of free radicals in Leishmania

cultures treated with our synthetic compounds 2 and 3. We

observed that the tetrahydropyran 3a induced a higher increase

in intracellular ROS levels than the endoperoxide 2a in L.

donovani promastigotes. This ﬁnding corroborates our

hypothesis that the generation of free radicals by cleaving the

O−O bond is not the main mechanism of action of the

synthetic peroxides tested in our biological model. Indeed, the

peroxide bond in our compounds could be very stable, thus

not contributing to the pharmacological activity.

At last, in order to acquire information on the ability of the

tested molecules to enter the L. donovani promastigote cell and

on the compound distribution within the parasite, we carried

out confocal microscopy investigations on new synthesized

ﬂuorescent endoperoxides and tetrahydropyrans. We detected

endoperoxide 2f and tetrahydropyran 3e in the parasite

NMR spectra were recorded on a Varian INOVA 400 NMR

instrument with a 5 mm probe. All chemical shifts have been quoted

relative to deuterated solvent signals; chemical shifts (δ) are reported

in ppm, and coupling constants (J) are reported in hertz (Hz). Low-

resolution MS (LRMS) ESI analyses were performed on an Agilent

Technologies MSD1100 single-quadrupole mass spectrometer. Mass

spectrometric detection was performed in the full-scan mode from m/

z 50 to 2500, with a scan time of 0.1 s in the positive ion mode, ESI

spray voltage of 4500 V, nitrogen gas pressure of 35 psi, drying gas

ﬂow rate of 11.5 mL min⁻¹, and fragmentor voltage of 30 V. LRMS EI

analyses were performed on a Hewlett-Packard 5971 with EI

ionization at 70 eV. High-resolution MS (HRMS) ESI analyses

were performed on an LTQ Orbitrap XL (Thermo Scientiﬁc) mass

spectrometer. Melting point (mp) measurements were performed on

Bibby Stuart Scientiﬁc SMP3 apparatus. Flash chromatography

puriﬁcations were carried out using VWR silica gel (40−63 μm

particle size). Thin-layer chromatography was performed on Merck 60

F254 plates.

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The purity of bioactive target compounds was ≥95% established by

HPLC analyses performed on an Agilent Technologies HP1100

instrument. A Phenomenex Gemini C18 3 μm (100 × 3 mm) column

was employed for the chromatographic separation, and two diﬀerent

analytical methods were used: method A: mobile phase, H₂O/

CH₃CN; gradient from 30 to 80% of CH₃CN in 8 min, 80% of

CH₃CN until 22 min, then up to 90% of CH₃CN in 2 min, and stop

time at 25 min; and ﬂow rate, 0.4 mL min⁻¹; method B: gradient

analogous to method A employing a mobile phase (H₂O/CH₃CN)

containing 0.2% of formic acid. The peak identity was conﬁrmed by

LRMS ESI analyses.

Endoperoxides 2a−2d are known, and they were prepared

according to our previous work.⁸⁷Compound 4b is known, and it

was synthesized according to the literature procedure.¹¹⁹Its physical

and spectroscopic data matched the reported ones. N¹-(6-Chloro-2-

methoxyacridin-9-yl)butane-1,4-diamine is a known compound, and it

was prepared according to the literature procedure.¹²⁰Its physical and

spectroscopic data matched the reported ones. Benzaldehyde diethyl

acetal is known, and it was prepared according to the literature

procedure.¹²¹After being synthesized, it was used in the following

reaction without puriﬁcation.

137.6, 128.6, 128.2, 128.1, 127.8, 127.7, 126.5, 76.2, 73.6, 68.8, 52.6.

LRMS (ESI⁺) m/z: 301.2 [M − OH]⁺, 319.2 [M + H]⁺, 341.2 [M +

Na]⁺.

3-(Benzyloxy)-2,2-dimethylpropan-1-ol (6c). The compound is

known, and its physical and spectroscopic data matched the reported

ones.¹²²

Synthesis of 3-(Benzyloxy)-2,2-disubstituted-propanals (7).

Compounds 7 were synthesized from the corresponding starting

materials 6 according to the literature procedure.¹²²

2-((Benzyloxy)methyl)-2-butylhexanal (7a). Yield: 86%. Colorless

liquid. Mobile phase for the chromatographic puriﬁcation: CyH/

1

Et₂O, 9:1. H NMR (400 MHz, CDCl₃): δ 9.47 (s, 1H), 7.31−7.18

(m, 5H), 4.43 (s, 2H), 3.46 (s, 2H), 1.52 (ddd, J = 13.8, 9.0, 5.7 Hz,

4H), 1.27 (h, J = 7.3 Hz, 4H), 1.11 (dq, J = 7.9, 4.4 Hz, 4H), 0.87 (t, J

= 7.3 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃): δ 205.2, 137.9, 128.0,

127.2, 127.2, 73.0, 70.3, 52.9, 29.5, 25.2, 23.1, 13.6. LRMS (EI⁺) m/z

(%): 276 (<1) [M]⁺, 113 (15), 91 (100).

3-(Benzyloxy)-2,2-diphenylpropanal (7b). Yield: 95%. White waxy

solid. Mobile phase for the chromatographic puriﬁcation: CyH/

1

AcOEt, 95:5. H NMR (400 MHz, CDCl₃): δ 9.91 (s, 1H), 7.41−

7.27 (m, 8H), 7.25−7.11 (m, 7H), 4.52 (s, 2H), 4.25 (s, 2H). ¹³C

NMR (100 MHz, CDCl₃): δ 199.0, 138.8, 137.8, 129.2, 128.5, 128.3,

127.5, 127.4, 73.6, 72.5, 64.1. LRMS (ESI⁺) m/z: 299.2 [M − OH]⁺,

317.2 [M + H]⁺, 334.2 [M + NH₄]⁺, 339.2 [M + Na]⁺.

3-(Benzyloxy)-2,2-dimethylpropanal (7c). The compound is

known, and its physical and spectroscopic data matched the reported

ones.¹²²

Synthetic Procedures and Compound Characterizations. Syn-

thesis of 2-Phenyl-5,5-disubstituted-1,3-dioxanes (5). Compounds

5 were prepared according to the literature procedure¹²²modiﬁed as

follows. (1S)-(+)-Camphorsulfonic acid (CSA, 0.01 equiv) was added

at 0 °C to a solution of the starting diol 4 and benzaldehyde diethyl

acetal (1.05 equiv) in anhydrous CH₂Cl₂(2 mL mmol⁻¹) under a N₂

atmosphere. The reaction mixture was warmed to room temperature

and stirred for 2−12 h, before being quenched with a saturated

aqueous solution of NaHCO₃. The mixture was extracted with

CH₂Cl₂(3 × 10 mL), and the organic phase was dried over Na₂SO₄

and ﬁltered. The solvent was removed under reduced pressure, and

the product was puriﬁed by ﬂash chromatography on silica gel.

5,5-Dibutyl-2-phenyl-1,3-dioxane (5a). Yield: 95%. Colorless

liquid. Mobile phase for chromatographic puriﬁcation: CyH/Et₂O,

̈

Synthesis of the Knoevenagel Adducts (8). Compounds 8 were

synthesized according to the literature procedure¹⁰⁶from the

corresponding starting materials 7 using the commercially available

preformed titanium complex (2 equiv) and by stirring the reaction

mixture at reﬂux for 18 h.

Methyl (E)-2-Acetyl-4-((benzyloxy)methyl)-4-butyloct-2-enoate

(E-8a). Total (Z and E isomers) yield: 47%. Yellowish liquid. Mobile

1

phase for the chromatographic puriﬁcation: CyH/AcOEt, 95:5. H

1

9:1. H NMR (400 MHz, CDCl₃): δ 7.49 (dd, J = 7.9, 1.8 Hz, 2H),

NMR (400 MHz, CDCl₃): δ 7.37−7.25 (m, 5H), 6.69 (s, 1H), 4.40

(s, 2H), 3.77 (s, 3H), 3.31 (s, 2H), 2.30 (s, 3H), 1.49−1.41 (m, 4H),

1.26 (q, J = 7.3 Hz, 6H), 1.19−1.07 (m, 2H), 0.87 (t, J = 7.2 Hz, 6H).

¹³C NMR (100 MHz, CDCl₃): δ 202.5, 165.4, 151.3, 138.2, 128.2,

7.40−7.31 (m, 3H), 5.39 (s, 1H), 3.95 (dd, J = 10.3, 1.2 Hz, 2H),

3.60 (dd, J = 10.3, 1.2 Hz, 2H), 1.84−1.68 (m, 2H), 1.42−1.32 (m,

2H), 1.32−1.23 (m, 4H), 1.21−1.12 (m, 2H), 1.11−1.03 (m, 2H),

0.94 (t, J = 7.2 Hz, 3H), 0.92 (t, J = 7.2 Hz, 3H). ¹³C NMR (100

MHz, CDCl₃): δ 138.5, 128.3, 127.7, 125.8, 101.5, 75.0, 34.3, 32.1,

30.4, 25.1, 24.0, 23.2, 23.2, 13.8, 13.6. LRMS (EI⁺) m/z (%): 276

(53) [M]⁺, 275 (92), 245 (5), 140 (49), 105 (65), 56 (100).

2,5,5-Triphenyl-1,3-dioxane (5b). Yield: 94%. White solid. Puriﬁed

by recrystallization from cold CH₂Cl₂. Melting point, 97−101 °C. ¹H

NMR (400 MHz, CDCl₃): δ 7.58−7.50 (m, 2H), 7.47−7.39 (m, 2H),

7.38−7.29 (m, 7H), 7.25−7.19 (m, 2H), 7.17−7.08 (m, 2H), 5.64 (s,

1H), 4.87 (d, J = 11.6 Hz, 2H), 4.42 (d, J = 11.6 Hz, 2H). ¹³C NMR

(100 MHz, CDCl₃): δ 144.6, 143.6, 138.2, 129.2, 129.0, 128.8, 128.4,

128.2, 127.0, 126.6, 126.3, 102.3, 75.4, 44.7. LRMS (ESI⁺) m/z: 339.2

[M + Na]⁺.

5,5-Dimethyl-2-phenyl-1,3-dioxane (5c). Yield: 87%. The com-

pound is known, and its physical and spectroscopic data matched the

reported ones.¹²²

Synthesis of 3-(Benzyloxy)-2,2-disubstituted-propan-1-ols (6).

Compounds 6 were synthesized from the corresponding starting

materials 5 according to the literature procedure.¹¹⁹

2-((Benzyloxy)methyl)-2-butylhexan-1-ol (6a). Yield: 99%. Color-

less liquid. Mobile phase for the chromatographic puriﬁcation: CyH/

AcOEt, 1:1. ¹H NMR (400 MHz, CDCl₃): δ 7.40−7.27 (m, 5H), 4.50

(s, 2H), 3.51 (d, J = 6.1 Hz, 2H), 3.37 (s, 2H), 2.67 (t, J = 5.8 Hz,

1H), 1.35−1.07 (m, 12H), 0.89 (t, J = 7.1 Hz, 6H). ¹³C NMR (100

MHz, CDCl₃): δ 138.0, 127.9, 127.1, 127.0, 75.6, 75.6, 73.0, 67.5,

67.5, 40.6, 30.6, 24.7, 23.3, 13.7. LRMS (EI⁺) m/z (%): 278 (2) [M]⁺,

187 (1), 91 (100).

3-(Benzyloxy)-2,2-diphenylpropan-1-ol (6b). Yield: 85%. White

waxy solid. Mobile phase for chromatographic puriﬁcation: CyH/

Et₂O, 8:2. ¹H NMR (400 MHz, CDCl₃): δ 7.39−7.26 (m, 7H), 7.25−

7.16 (m, 8H), 4.58 (s, 2H), 4.36 (d, J = 6.4 Hz, 2H), 4.14 (s, 2H),

2.50 (t, J = 6.5 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃): δ 143.8,

127.6, 127.5, 72.6, 72.4, 52.2, 44.9, 35.8, 31.3, 25.9, 23.3, 13.9. LRMS

(ESI⁺) m/z: 343.2 [M − OMe]⁺, 392.4 [M + NH₄]⁺.

Methyl (Z)-2-Acetyl-4-((benzyloxy)methyl)-4-butyloct-2-enoate

(Z-8a). Total (Z and E isomers) yield: 47%. Yellowish liquid. Mobile

1

phase for the chromatographic puriﬁcation: CyH/AcOEt, 95:5. H

NMR (400 MHz, CDCl₃): δ 7.39−7.27 (m, 5H), 6.65 (s, 1H), 4.48

(s, 2H), 3.74 (s, 3H), 3.38 (s, 2H), 2.28 (s, 3H), 1.52−1.42 (m, 3H),

1.26 (h, J = 7.3 Hz, 5H), 1.20−1.07 (m, 4H), 0.87 (t, J = 7.2 Hz, 6H).

¹³C NMR (100 MHz, CDCl₃): δ 195.3, 168.1, 151.8, 138.2, 135.9,

128.2, 127.5, 73.0, 72.6, 51.9, 44.7, 34.9, 25.9, 25.8, 23.2, 13.9. LRMS

(ESI⁺) m/z: 343.2 [M − OMe]⁺, 392.4 [M + NH₄]⁺.

Methyl 2-Acetyl-5-(benzyloxy)-4,4-diphenylpent-2-enoate (8b).

Yield: 31%. Yellowish oil. Mobile phase for the chromatographic

1

puriﬁcation: CyH/AcOEt, 9:1. H NMR (400 MHz, CDCl₃): δ 7.52

(s, 1H), 7.33−7.26 (m, 8H), 7.25−7.18 (m, 7H), 4.52 (s, 2H), 4.12

(s, 2H), 3.11(s, 3H), 2.35 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ

195.4, 166.3, 149.0, 141.0, 137.5, 136.9, 128.9, 128.2, 128.0, 127.6,

127.6, 127.0, 75.5, 73.3, 55.3, 51.4, 26.5. LRMS (ESI⁺) m/z: 415.2 [M

+ H]⁺, 432.2 [M + NH₄]⁺, 437.2 [M + Na]⁺.

Methyl (E)-2-Acetyl-5-(benzyloxy)-4,4-dimethylpent-2-enoate (E-

8c). Total (Z and E isomers) yield: 92%. Yellowish oil. Mobile phase

1

for the chromatographic puriﬁcation: CyH/AcOEt, 95:5. H NMR

(400 MHz, CDCl₃): δ 7.36−7.25 (m, 5H), 6.82 (s, 1H), 4.47 (s, 2H),

3.75 (s, 3H), 3.24 (s, 2H), 2.35 (d, J = 1.2 Hz, 3H), 1.10 (s, 6H). ¹³C

NMR (100 MHz, CDCl₃): δ 203.1, 165.2, 151.3, 138.1, 133.8, 128.2,

127.5, 127.4, 78.0, 72.9, 52.2, 38.6, 31.7, 24.6. LRMS (ESI⁺) m/z:

291.2 [M + H]⁺, 308.2 [M + NH₄]⁺, 313.2 [M + Na]⁺.

Methyl (Z)-2-Acetyl-5-(benzyloxy)-4,4-dimethylpent-2-enoate

(Z-8c). Total (Z and E isomers) yield: 92%. Yellowish oil. Mobile

1

phase for the chromatographic puriﬁcation: CyH/AcOEt, 95:5. H

NMR (400 MHz, CDCl₃): δ 7.36−7.26 (m, 5H), 6.82 (s, 1H), 4.51

L

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Article

(s, 2H), 3.78 (s, 3H), 3.27 (s, 2H), 2.27 (s, 3H), 1.13 (s, 6H). ¹³C

NMR (100 MHz, CDCl₃): δ 195.6, 168.1, 151.7, 138.2, 135.6, 128.3,

127.6, 127.5, 78.6, 73.2, 52.1, 38.5, 25.9, 23.6. LRMS (ESI⁺) m/z:

291.2 [M + H]⁺, 308.2 [M + NH₄]⁺, 313.2 [M + Na]⁺.

Synthesis of Methyl Tetrahydropyran-3-carboxylates (9). The

appropriate starting materials 8 were added to a suspension of 10%

Pd/C (20% w/w with respect to the substrate) in MeOH (10 mL

mmol⁻¹), and the mixture was vigorously stirred at room temperature

under a H₂atmosphere (1 atm) for 12−16 h. The reaction mixture

was then ﬁltered, and the solvent was removed under reduced

pressure. The product was isolated from the crude by ﬂash

chromatography on silica gel.

Methyl 5,5-Dibutyl-2-methoxy-2-methyltetrahydro-2H-pyran-3-

carboxylate (9a). Yield: 56%. Colorless oil. Mobile phase for the

chromatographic puriﬁcation: CyH/AcOEt, 95:5. ¹H NMR (400

MHz, CDCl₃): δ 3.68 (d, J = 1.0 Hz, 3H), 3.32 (d, J = 11.1 Hz, 1H),

3.24 (dd, J = 11.1, 2.5 Hz, 1H), 3.19 (s, 3H), 2.70 (dd, J = 13.3, 4.3

Hz, 1H), 1.88 (t, J = 13.4 Hz, 1H), 1.54 (ddd, J = 13.6, 4.3, 2.4 Hz,

1H), 1.46 (s, 3H), 1.40 (ddd, J = 9.9, 5.6, 3.5 Hz, 2H), 1.35−1.04 (m,

10H), 0.90 (dt, J = 8.4, 7.1 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃): δ

172.6, 97.3, 67.8, 51.6, 48.0, 47.0, 36.3, 34.1, 31.5, 30.9, 25.4, 24.7,

23.5, 23.5, 22.8, 14.1, 14.0. LRMS (ESI⁺) m/z: 269.2 [M − OMe]⁺,

301.2 [M + H]⁺, 323.2 [M + Na]⁺, 623.4 [2 M + Na]⁺.

Methyl 2-Methoxy-2-methyl-5,5-diphenyltetrahydro-2H-pyran-

3-carboxylate (9b). Yield: 72%. White solid. Mobile phase for the

chromatographic puriﬁcation: CyH/AcOEt, 95:5. ¹H NMR (400

MHz, CDCl₃): δ 7.41−7.39 (m, 2H), 7.31−7.25 (m, 3H), 7.22−7.18

(m, 5H), 4.28 (dd, J = 11.9, 2.9 Hz, 1H), 3.71 (d, J = 11.8 Hz, 1H),

3.68 (s, 3H), 3.23 (s, 3H), 3.09 (t, J = 12.9 Hz, 1H), 2.44 (dd, J =

12.8, 3.4 Hz, 1H), 2.37 (dt, J = 12.9, 3.2 Hz, 1H), 1.43 (s, 3H). ¹³C

NMR (100 MHz, CDCl₃): δ 172.2, 145.7, 144.7, 128.3, 127.0, 126.4,

126.0, 97.3, 67.2, 51.7, 48.1, 47.5, 45.7, 32.4, 22.6. LRMS (ESI⁺) m/z:

309.2 [M − OMe]⁺, 363.2 [M + Na]⁺.

1H), 2.87 (bd, J = 9.6 Hz, 1H), 1.87 (t, J = 12.9 Hz, 1H), 1.83−1.75

(m, 1H), 1.41 (s, 3H), 1.21 (dt, J = 12.5, 3.1 Hz, 1H), 1.04 (s, 3H),

0.86 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 100.0, 70.3, 63.6, 47.4,

42.0, 35.2, 30.2, 27.1, 23.5, 21.8. LRMS (ESI⁺) m/z: 189.1 [M + H]⁺.

Synthesis of Tetrahydropyran-3-carboxyaldehydes (11). Com-

pounds 11 were synthesized from the corresponding starting materials

10 according to our previously reported procedure,⁸⁷and they were

immediately used in the following step.

Synthesis of 3-Aminopropyltetrahydropyrans (3). Compounds 3

were synthesized from the corresponding starting materials 11

according to our previously reported procedure.⁸⁷

N-((5,5-Dibutyl-2-methoxy-2-methyltetrahydro-2H-pyran-3-yl)-

methyl)-3-(1H-imidazol-1-yl)propan-1-amine (3a). Yield: 45%.

Colorless oil. Mobile phase for the chromatographic puriﬁcation:

1

AcOEt/MeOH, 6:4. H NMR (400 MHz, CDCl₃): δ 7.46 (s, 1H),

7.04 (s, 1H), 6.90 (s, 1H), 4.02 (t, J = 6.9 Hz, 2H), 3.28−3.18 (m,

2H), 3.15 (s, 3H), 2.64 (dd, J = 11.8, 3.8 Hz, 1H), 2.54 (t, J = 6.8 Hz,

2H), 2.42 (dd, J = 11.8, 7.8 Hz, 1H), 1.91 (p, J = 6.8 Hz, 2H), 1.74

(ddt, J = 12.1, 7.8, 4.0 Hz, 1H), 1.52−1.39 (m, 4H), 1.32 (s, 3H),

1.38−1.02 (m, 10H), 0.89 (q, J = 7.3 Hz, 6H). ¹³C NMR (100 MHz,

CDCl₃): δ 137.2, 129.4, 118.8, 99.1, 67.8, 51.4, 47.6, 46.6, 44.7, 40.8,

36.6, 34.8, 33.8, 31.2, 31.2, 25.5, 24.7, 23.6, 23.5, 22.0, 14.2, 14.0.

LRMS (ESI⁺) m/z: 348.4 [M − OMe]⁺, 402.4 [M + Na]⁺. HRMS

(ESI⁺) m/z: [M + Na]⁺calcd for C₂₂H₄₁N₃NaO₂, 402.3096; found,

402.3104.

3-(1H-Imidazol-1-yl)-N-((2-methoxy-2-methyl-5,5-diphenyltetra-

hydro-2H-pyran-3-yl)methyl)propan-1-amine (3b). Yield: 85%.

White waxy solid. Mobile phase for the chromatographic puriﬁcation:

AcOEt/MeOH, 8:2. ¹H NMR (400 MHz, CD₃OD): δ 7.61 (d, J = 1.3

Hz, 1H), 7.47−7.39 (m, 2H), 7.34−7.11 (m, 8H), 7.10 (s, 1H), 6.96

(s, 1H), 4.33 (dd, J = 11.9, 2.8 Hz, 1H), 4.03 (td, J = 7.0, 1.6 Hz, 2H),

3.63 (d, J = 11.8 Hz, 1H), 3.21 (s, 3H), 2.66 (dd, J = 12.4, 3.5 Hz,

1H), 2.61−2.52 (m, 2H), 2.49 (d, J = 12.3 Hz, 1H), 2.47−2.38 (m,

2H), 1.99−1.87 (m, 2H), 1.56 (ddd, J = 12.1, 8.2, 4.0 Hz, 1H), 1.27

(s, 3H). ¹³C NMR (100 MHz, CD₃OD): δ 147.9, 147.0, 138.4, 129.5,

129.3, 129.1, 129.1, 127.9, 127.3, 126.9, 120.5, 100.2, 68.3, 51.7, 48.1,

47.5, 47.4, 45.9, 42.1, 35.3, 31.5, 22.1. LRMS (ESI⁺) m/z: 388.2 [M −

OMe]⁺, 420.2 [M + H]⁺. HRMS (ESI⁺) m/z: [M + H]⁺calcd for

C₂₆H₃₄N₃O₂, 420.2651; found, 420.2657.

Methyl 2-Methoxy-2,5,5-trimethyltetrahydro-2H-pyran-3-car-

boxylate (9c). Yield: 63%. Colorless oil. Mobile phase for the

chromatographic puriﬁcation: CyH/AcOEt, 95:5. ¹H NMR (400

MHz, CDCl₃): δ 3.69 (s, 3H), 3.38 (d, J = 10.9 Hz, 1H), 3.20 (s,

3H), 3.09 (dd, J = 10.9, 2.6 Hz, 1H), 2.74 (dd, J = 13.3, 4.3 Hz, 1H),

2.00 (t, J = 13.3 Hz, 1H), 1.48 (s, 3H), 1.41 (ddd, J = 13.4, 4.4, 2.8

Hz, 1H), 1.02 (s, 3H), 0.87 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ

172.0, 96.8, 69.8, 51.3, 47.7, 47.3, 34.7, 29.3, 26.6, 23.0, 22.5. LRMS

(ESI⁺) m/z: 185.2 [M − OMe]⁺, 239.2 [M + Na]⁺, 455.2 [2 M +

Na]⁺.

3-(1H-Imidazol-1-yl)-N-((2-methoxy-2,5,5-trimethyltetrahydro-

2H-pyran-3-yl)methyl)propan-1-amine (3c). Yield: 99%. Colorless

oil. Mobile phase for the chromatographic puriﬁcation: CH₂Cl₂/

1

MeOH/NH₃, 9:1:0.1. H NMR (400 MHz, CDCl₃): δ 7.47 (s, 1H),

7.05 (d, J = 1.2 Hz, 1H), 6.91 (d, J = 1.3 Hz, 1H), 4.03 (t, J = 7.0 Hz,

2H), 3.30 (d, J = 10.8 Hz, 1H), 3.17 (s, 3H), 3.06 (dd, J = 10.8, 2.4

Hz, 1H), 2.66 (dd, J = 11.8, 3.8 Hz, 1H), 2.55 (t, J = 6.8 Hz, 2H),

2.46 (dd, J = 11.9, 7.8 Hz, 1H), 1.92 (p, J = 6.9 Hz, 2H), 1.79 (ddt, J

= 12.3, 8.1, 4.3 Hz, 1H), 1.51−1.40 (m, 1H), 1.41−1.34 (m, 1H),

1.34 (s, 3H), 1.03 (s, 3H), 0.83 (s, 3H). ¹³C NMR (100 MHz,

CDCl₃): δ 137.0, 129.1, 118.7, 98.8, 70.1, 51.1, 47.5, 46.5, 44.6, 41.4,

37.2, 31.1, 30.1, 27.1, 23.6, 21.8. LRMS (ESI⁺) m/z: 296.2 [M + H]⁺,

559.8 [2M − OMe]⁺. HRMS (ESI⁺) m/z: [M + H]⁺calcd for

C₁₆H₃₀N₃O₂, 296.2338; found, 296.2332.

Synthesis of 3-Hydroxymethyltetrahydropyrans (10). Com-

pounds 10 were synthesized from the corresponding starting materials

9 according to our previously reported procedure.⁸⁷

(5,5-Dibutyl-2-methoxy-2-methyltetrahydro-2H-pyran-3-yl)-

methanol (10a). Yield: 81%. Colorless oil. Mobile phase for the

chromatographic puriﬁcation: CyH/AcOEt, 9:1. ¹H NMR (400 MHz,

CDCl₃): δ 3.90 (d, J = 11.4 Hz, 1H), 3.43 (t, J = 10.4 Hz, 1H), 3.26

(d, J = 1.2 Hz, 2H), 3.22 (s, 3H), 2.86 (d, J = 9.8 Hz, 1H), 1.81−1.70

(m, 2H), 1.45 (dd, J = 12.2, 5.0 Hz, 2H), 1.40 (s, 3H), 1.38−1.04 (m,

10H), 0.90 (q, J = 7.3 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃): δ

100.6, 68.1, 63.9, 47.5, 41.3, 36.6, 34.9, 31.8, 31.1, 25.5, 24.6, 23.6,

23.5, 21.9, 14.1, 14.0. LRMS (ESI⁺) m/z: 273.2 [M + H]⁺.

(2-Methoxy-2-methyl-5,5-diphenyltetrahydro-2H-pyran-3-yl)-

methanol (10b). Yield: 95%. White waxy solid. Mobile phase for the

chromatographic puriﬁcation: CyH/AcOEt, 75:25. ¹H NMR (400

MHz, CDCl₃): δ 7.48−7.35 (m, 2H), 7.34−7.12 (m, 8H), 4.32 (dd, J

= 11.9, 3.0 Hz, 1H), 3.90 (dd, J = 11.6, 2.8 Hz, 1H), 3.66 (d, J = 11.9

Hz, 1H), 3.43 (dd, J = 11.7, 3.4 Hz, 1H), 3.26 (s, 3H), 3.00 (t, J =

13.0 Hz, 1H), 2.17 (dt, J = 13.0, 3.2 Hz, 1H), 1.50 (dt, J = 13.0, 3.2

Hz, 1H), 1.37 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 146.2, 145.5,

128.3, 128.3, 128.1, 126.9, 126.4, 125.8, 100.4, 67.5, 63.9, 47.6, 46.6,

41.8, 32.9, 21.7. LRMS (ESI⁺) m/z: 313.2 [M + H]⁺.

Synthesis of Tetrahydropyran-3-propargyl Ether (12). Com-

pound 12 was synthesized from the corresponding starting material

10b according to our previously reported procedure.⁸⁷

2-Methoxy-2-methyl-5,5-diphenyl-3-((prop-2-yn-1-yloxy)-

methyl)tetrahydro-2H-pyran (12). Yield: 77%. Colorless waxy solid.

Mobile phase for the chromatographic puriﬁcation: CyH/AcOEt,

1

95:5 to 8:2. H NMR (400 MHz, CDCl₃): δ 7.46−7.40 (m, 2H),

7.32−7.22 (m, 4H), 7.21−7.14 (m, 4H), 4.30 (dd, J = 11.8, 2.9 Hz,

1H), 4.06 (d, J = 2.4 Hz, 2H), 3.71−3.65 (m, 2H), 3.35 (dd, J = 9.4,

7.9 Hz, 1H), 3.20 (s, 3H), 2.48 (t, J = 12.5 Hz, 2H), 2.38 (t, J = 2.4

Hz, 1H), 1.76 (ddt, J = 12.2, 8.5, 4.2 Hz, 1H), 1.31 (s, 3H). ¹³C NMR

(100 MHz, CDCl₃): δ 146.5, 145.4, 128.3, 128.2, 128.1, 126.9, 126.3,

125.8, 98.3, 79.9, 74.2, 71.5, 67.2, 58.1, 47.7, 46.1, 41.2, 33.7, 22.0.

LRMS (ESI⁺) m/z: 319.2 [M − OMe]⁺, 373.2 [M + Na]⁺.

Synthesis of Tetrahydropyran-3-triazolyl Ether (3d). Compound

3d was synthesized from the corresponding starting material 12

according to our previously reported procedure.⁸⁷

(2-Methoxy-2,5,5-trimethyltetrahydro-2H-pyran-3-yl)methanol

(10c). Yield: 47%. Colorless oil. Mobile phase for the chromato-

graphic puriﬁcation: CyH/AcOEt, 75:25. ¹H NMR (400 MHz,

CDCl₃): δ 3.91 (dd, J = 11.5, 2.7 Hz, 1H), 3.44 (bt, J = 10.4 Hz, 1H),

3.32 (d, J = 10.9 Hz, 1H), 3.23 (s, 3H), 3.10 (dd, J = 10.9, 2.6 Hz,

M

https://dx.doi.org/10.1021/acs.jmedchem.0c01589

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(3-(4-(((2-Methoxy-2-methyl-5,5-diphenyltetrahydro-2H-pyran-

3-yl)methoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)-

triphenylphosphonium bromide (3d). Yield: 51%. Pinkish waxy

solid. Mobile phase for the chromatographic puriﬁcation: CH₂Cl₂/

atmosphere. The reaction mixture was warmed at room temperature

and stirred for 4 h. The solvent was removed under reduced pressure,

and the product was isolated by ﬂash chromatography on silica gel.

N¹-((6,6-Dibutyl-3-methoxy-3-methyl-1,2-dioxan-4-yl)methyl)-

N⁴-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)butane-1,4-diamine (2f).

Yield: 21%. Dark waxy solid. Mobile phase for the chromatographic

puriﬁcation: CH₂Cl₂/MeOH, 9:1 to 8:2. ¹H NMR (400 MHz,

CD₃OD): δ 8.50 (d, J = 8.9 Hz, 1H), 6.36 (d, J = 8.9 Hz, 1H), 3.75−

3.49 (m, 2H), 3.27 (s, 3H), 3.07−2.93 (m, 1H), 2.89 (t, J = 7.4 Hz,

2H), 2.79 (dd, J = 12.5, 8.5 Hz, 1H), 2.28−2.07 (m, 1H), 1.96−1.71

(m, 5H), 1.57−1.46 (m, 1H), 1.37−1.30 (m, 2H), 1.31 (s, 3H),

1.31−1.19 (m, 10H), 0.97−0.82 (m, 6H). LRMS (ESI⁺) m/z: 508.6

[M + H]⁺, 1015.0 [2 M + H]⁺.

Parasitology. Parasites. Promastigotes of a L. donovani reference

strain (MHOM/NP/02/BPK282/0cl4) were maintained at 26 °C in

a liquid custom-made medium, HOMEM Cat. no: 1140-082 (Gibco

Thermo Fisher Scientiﬁc Inc., Waltham, USA), complete composi-

tion: S-MEM (Eagle) 1 × 1 liter pack, glucose (1−2 g), sodium

bicarbonate (0.3 g), sodium pyruvate (0.11 g), p-aminobenzoic acid

(1 mg), biotin (0.1 mg), HEPES (4.77−5.96 g), MEM amino acids

(10 mL), MEM nonessential amino acids (10 mL), pH 7.5−7.6,

supplemented with 20% fetal bovine serum (FBS, EuroClone SpA,

Milan, Italy)¹²³and 1% penicillin−streptomycin (EuroClone SpA).

Cell Cultures. Vero cells (kidney of African green monkey epithelial

cell line) were cultured at 37 °C in the MEM liquid medium

supplemented with 10% FBS (EuroClone SpA), 1% levoglutamine

(EuroClone SpA), and 1% penicillin−streptomycin (EuroClone

SpA). THP-1 cells (human leukemia monocytic cell line) were

cultured at 37 °C in the RPMI 1640 (EuroClone SpA) liquid medium

supplemented with 10% FBS (EuroClone SpA), 1% levoglutamine

(EuroClone SpA), 1% penicillin−streptomycin, and mercaptoethanol

(Gibco) 50 μM.

Promastigote Growth Inhibition Assay. To evaluate the eﬃcacy of

the compounds on the extracellular forms of L. donovani,

promastigotes in their late log/stationary phase were seeded with

the complete HOMEM medium at 10⁶cells mL⁻¹in 96-well plates

and incubated with tested compounds at a range concentration of

600−1.6 μM in a 26 °C incubator for 72 h. The antileishmanial drug

amphotericin B was used as the positive control, and untreated

promastigotes were the negative control. Each experiment was

performed in duplicate. Stock solution of the compounds was 8

mM in DMSO. To estimate the concentration at which the

compounds caused 50% inhibition of growth (IC₅₀), the alamarBlue

assay was employed (Life Technologies, Thermo Fisher Scientiﬁc

Inc., Waltham, USA). The alamarBlue assay includes a colorimetric

growth indicator based on detection of metabolic activity. Speciﬁcally,

the system incorporates an oxidation−reduction (REDOX) indicator

that changes color in response to chemical reduction of the growth

medium resulting from cell growth. The method monitors the

reducing environment of proliferating cells; the cell-permeable

resazurin is added (nonﬂuorescent form, blue color) and, upon

entering cells, is reduced to resoruﬁn (ﬂuorescent form, red color) as

a result of cellular metabolic activity. Evaluation was performed by

adding 20 μL of alamarBlue and incubating at 26 °C for 24 h. The

reducing environment was evaluated after 24 h by absorbance

measurement at the Multiskan ascent plate reader (Thermo Fisher

Scientiﬁc Inc.) at 550 and 630 nm.

Cytotoxicity Test. The cytotoxicity of the compounds was

determined using mammalian kidney epithelial cells (Vero cell line)

and human acute monocytic leukemia cell line (THP-1). Cells were

seeded (10⁵cells mL⁻¹) on 96-well plates with the complete MEM

medium and incubated with test compounds at 37 °C in a 5% CO₂

incubator. After 72 h of incubation, 20 μL of the alamarBlue reagent

was added to each well and incubated at 37 °C for 24 h. Reduction of

resazurin to resoruﬁn was evaluated after 24 h by absorbance

measurement at the Multiskan ascent plate reader (Thermo Fisher

Scientiﬁc Inc.,) at 550 and 630 nm. DMSO used for compound

dilution was also tested to control the employed concentration, which

was not toxic and did not inﬂuence the toxicity of the compounds.

Each experiment was performed in duplicate. The selectivity index

1

MeOH, 9:1. H NMR (400 MHz, CD₃OD): δ 8.13 (bs, 1H), 7.95−

7.79 (m, 4H), 7.81−7.69 (m, 11H), 7.38 (d, J = 7.7 Hz, 2H), 7.22

(td, J = 7.5, 4.7 Hz, 4H), 7.13 (tt, J = 9.0, 5.3 Hz, 4H), 4.63 (t, J = 5.7

Hz, 2H), 4.49 (s, 2H), 4.31 (d, J = 11.8 Hz, 1H), 3.71 (bs, 1H), 3.60

(d, J = 11.8 Hz, 1H), 3.53−3.34 (m, 3H), 3.17 (s, 3H), 2.45−2.36

(m, 2H), 2.35−2.22 (m, 2H), 1.78−1.59 (bs, 1H), 1.23 (s, 3H). ¹³C

NMR (100 MHz, CD₃OD): δ 146.5, 145.4, 135.2 (d, J = 3.0 Hz),

133.8 (d, J = 10.0 Hz), 130.6 (d, J = 12.6 Hz), 128.3, 128.2, 128.1,

126.9, 126.1, 125.7, 117.8 (d, J = 86.4 Hz), 98.3, 72.2, 67.1, 60.3, 46.9

(d, J = 166.7 Hz), 41.2, 33.7, 29.6, 23.4 (d, J = 2.3 Hz), 22.1, 21.0,

20.0 (d, J = 52.9 Hz), 14.1. LRMS (ESI⁺) m/z: 332.8 [M − OMe]²⁺,

696.6 [M]⁺. HRMS (ESI⁺) m/z: [M − Br⁻]⁺calcd for

C₄₄H₄₇N₃O₃P⁺, 696.3350; found, 696.3360.

Synthesis of Acridine-Labeled Compounds. Compounds 2e and

3e were synthesized from the corresponding starting materials (13⁸⁷

and 11a, respectively) and N¹-(6-chloro-2-methoxyacridin-9-yl)-

butane-1,4-diamine following our previously reported procedure.⁸⁷

N¹-(6-Chloro-2-methoxyacridin-9-yl)-N⁴-((6,6-dibutyl-3-me-

thoxy-3-methyl-1,2-dioxan-4-yl)methyl)butane-1,4-diamine (2e).

Yield: 45%. Yellow waxy solid. Mobile phase for the chromatographic

puriﬁcation: CH₂Cl₂/MeOH/NH₃, 9:1:0.1. ¹H NMR (400 MHz,

CDCl₃): δ 8.04 (d, J = 2.1 Hz, 1H), 8.01 (d, J = 9.3 Hz, 1H), 7.97 (d,

J = 9.4 Hz, 1H), 7.39 (dd, J = 9.5, 2.6 Hz, 1H), 7.28 (dd, J = 9.2, 2.1

Hz, 1H), 7.23 (d, J = 2.7 Hz, 1H), 3.95 (s, 3H), 3.73 (t, J = 6.9 Hz,

2H), 3.26 (s, 3H), 2.74 (dd, J = 11.9, 4.0 Hz, 1H), 2.64 (td, J = 6.7,

1.5 Hz, 2H), 2.50 (dd, J = 11.9, 8.1 Hz, 1H), 1.94 (ddt, J = 12.4, 8.4,

4.4 Hz, 1H), 1.87−1.74 (m, 3H), 1.69−1.55 (m, 4H), 1.50 (dd, J =

13.2, 5.0 Hz, 1H), 1.47−1.26 (m, 4H), 1.25 (s, 3H), 1.24−1.08 (m,

6H), 0.86 (dt, J = 16.4, 7.1 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃): δ

155.8, 149.9, 148.0, 134.9, 131.0, 124.3, 124.2, 117.7, 115.5, 102.2,

99.6, 81.8, 55.5, 50.8, 50.5, 49.7, 48.6, 39.2, 36.4, 32.2, 31.5, 29.5,

27.5, 25.5, 24.8, 23.2, 23.2, 18.9, 14.1, 13.9. LRMS (ESI⁺) m/z: 277.8

[M − OMe + H]²⁺, 586.6 [M + H]⁺.

N¹-(6-Chloro-2-methoxyacridin-9-yl)-N⁴-((5,5-dibutyl-2-me-

thoxy-2-methyltetrahydro-2H-pyran-3-yl)methyl)butane-1,4-dia-

mine (3e). Yield: 32%. Yellow waxy solid. Mobile phase for the

chromatographic puriﬁcation: CH₂Cl₂/MeOH/NH₃, 9:1:0.1. ¹H

NMR (400 MHz, CDCl₃): δ 8.05 (d, J = 9.3 Hz, 1H), 8.02 (d, J =

2.0 Hz, 1H), 7.93 (d, J = 9.3 Hz, 1H), 7.36 (d, J = 2.5 Hz, 1H), 7.31

(d, J = 9.4 Hz, 1H), 7.22 (dd, J = 9.3, 2.1 Hz, 1H), 3.97 (s, 3H), 3.84

(t, J = 6.8 Hz, 2H), 3.16 (s, 3H), 2.87−2.67 (m, 3H), 2.58 (dd, J =

12.1, 7.9 Hz, 1H), 2.00−1.85 (m, 2H), 1.85−1.72 (m, 2H), 1.57 (d, J

= 13.0 Hz, 1H), 1.48−1.36 (m, 2H), 1.34 (s, 3H), 1.32−0.98 (m,

14H), 0.86 (dt, J = 10.3, 7.1 Hz, 6H). LRMS (ESI⁺) m/z: 276.2 [M +

H − OMe]²⁺, 584.6 [M + H]⁺.

Synthesis of NBD-Labeled Endoperoxide (2f). Compound 14 was

prepared from 13⁸⁷and the commercially available N-Boc-1,4-

butanediamine following our previously reported procedure.⁸⁷

tert-Butyl (4-(((6,6-Dibutyl-3-methoxy-3-methyl-1,2-dioxan-4-

yl)methyl)amino)butyl)carbamate (14). Yield: 79%. Colorless waxy

solid. Mobile phase for the chromatographic puriﬁcation: CH₂Cl₂/

1

MeOH, 9:1. H NMR (400 MHz, CDCl₃): δ 4.76 (bs, 1H), 3.30 (s,

3H), 3.14 (d, J = 6.6 Hz, 2H), 2.87 (bs, 1H), 2.68 (bs, 2H), 2.07 (bs,

1H), 1.95−1.83 (m, 1H), 1.74−1.46 (m, 14H), 1.44 (s, 9H), 1.32 (s,

3H), 1.37−1.23 (m, 4H), 0.91 (dt, J = 9.1, 6.8 Hz, 6H). ¹³C NMR

(100 MHz, CDCl₃): δ 156.0, 102.0, 81.7, 50.5, 49.5, 48.5, 40.2, 38.8,

36.3, 32.1, 31.4, 28.3, 27.7, 26.6, 25.4, 24.7, 23.1, 18.7, 14.0, 13.8.

LRMS (ESI⁺) m/z: 445.6 [M + H]⁺, 711.8 [2M − Boc]⁺.

Compound 14 was subsequently converted into the corresponding

N-deprotected hydrochloride salt according to a literature proce-

dure,¹⁰⁰and it was immediately used in the following synthetic step

without any puriﬁcation.

Compound 2f was prepared according to a literature procedure¹⁰⁰

modiﬁed as follows: 4-chloro-7-nitrobenzofurazan (1.1 equiv) was

added to a solution of the hydrochloride salt and triethylamine (3

equiv) in anhydrous CH₂Cl₂(30 mL mmol⁻¹) at 0 °C under a N₂

N

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Article

(SI) for each compound was calculated as the ratio between

cytotoxicity (CC₅₀/72 h) and activity (IC₅₀/72 h) against Leishmania

promastigotes.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01589.

■

sı

Amastigote Growth Inhibition Assay. To evaluate the eﬃcacy of

the compounds on the intracellular form of L. donovani, human acute

monocytic leukemia cell line (THP-1) was infected with L. donovani

promastigotes. Cells were harvested and seeded in a 96-well plate (10⁵

cells mL⁻¹) in the complete RPMI 1640 medium, and PMA (0.1 μM,

Cayman Chemical Company, Ann Arbor, Michigan, USA) was added

for obtaining maturation and cell adherence. Cells were incubated at

37 °C in a 5% CO₂incubator. After 48 h, the medium was replaced

with the fresh medium containing stationary-phase promastigotes that

were then phagocyted by monocytic cells and transformed into

intracellular amastigotes. After 24 h of incubation, newly synthesized

compounds were added and the plates were incubated at 37 °C in a

5% CO₂incubator for 72 h. After incubation, wells were washed,

ﬁxed, and stained with Giemsa. Staining was detected using a Nikon

Eclipse E200 light microscope (Nikon, Tokyo, Japan). The infectivity

index (% of infected macrophages × average number of amastigotes

per macrophage) was determined by counting at least 100 cells in

duplicate cultures.

X-ray crystallographic analysis, hypothesis on cis-stereo-

preference in tetrahydropyran 9 construction, suscepti-

bility tests on Leishmania promastigotes with the

modiﬁed assay medium, cytotoxicity evaluation in

THP-1 cell line, study on the iron chelator (DFO)

eﬀect on 2b and 3b bioactivity, study on the iron

chelator (DFP) eﬀect on 2a and 3a bioactivity,

calculated log P, NMR spectra, HLPC analyses (PDF)

Compound number, SMILES formula, IC₅₀against L.

donovani promastigotes and amastigotes, CC₅₀against

Vero cells and THP-1 cells of compounds 2a−2f and

3a−3e (CSV)

AUTHOR INFORMATION

Corresponding Authors

■

Role of an Iron Chelator in the Activation of Compounds.^111,112

To analyze the eﬀect of an iron chelator (DFO or DFP) on the

compounds, the late log/stationary phase of promastigotes was

seeded with the complete HOMEM medium at 10⁶cells mL⁻¹in 96-

well plates and incubated with the iron chelator for 24 h at 26 °C.

Four nontoxic doses of the iron chelator were prepared, in 1:2

dilutions. Each compound was added using a concentration range

including the IC₅₀of each compound. Each experiment was

performed in duplicate. The viability of the parasites was determined

by the alamarBlue assay. The fractional inhibitory concentration

(FIC; FIC = IC₅₀of the drug in the combination/IC₅₀of the drug

when tested alone) of each compound was calculated and plotted as

an isobologram.

Arianna Quintavalla − Department of Chemistry “G.

Ciamician”, Alma Mater Studiorum - University of Bologna,

40126 Bologna, Italy; Centro Interuniversitario di Ricerca sulla

Malaria (CIRM) - Italian Malaria Network (IMN), University

of Milan, 20100 Milan, Italy; orcid.org/0000-0002-0993-

6855; Email: arianna.quintavalla@unibo.it

Marco Lombardo − Department of Chemistry “G. Ciamician”,

Alma Mater Studiorum - University of Bologna, 40126

Bologna, Italy; Centro Interuniversitario di Ricerca sulla

Malaria (CIRM) - Italian Malaria Network (IMN), University

of Milan, 20100 Milan, Italy; orcid.org/0000-0001-8415-

8363; Email: marco.lombardo@unibo.it

Evaluation of Intracellular ROS Levels. To monitor the eﬀect of

the compounds on the production of ROS in promastigotes of L.

donovani, an oxidant-sensitive green ﬂuorescent dye (2,7-dichloro-

ﬂuorescein diacetate, H₂DCFDA) was used. Promastigotes in their

late log/stationary phase were seeded with the complete HOMEM

medium at 10⁶cells mL⁻¹in 96-well plates and incubated with tested

compounds at their IC₅₀in a 26 °C incubator for 24 h. Subsequently,

parasitic cells were washed in PBS (pH 7.4), loaded with 10 μM

permeant probe H₂DCFDA (Sigma, St. Louis, MO, USA), and

incubated in the dark for 35 min at 26 °C.¹⁰³H₂O₂(20 μM)¹²⁴and

miltefosine (22 μM)¹²⁵were used as positive controls. Each

experiment was performed in triplicate. Reactive oxygen species

(ROS) production was measured as an increase in ﬂuorescence

caused by the conversion of nonﬂuorescent dye to highly ﬂuorescent

2,7-dichloroﬂuorescein, with an excitation wavelength of 488 nm and

an emission wavelength of 530 nm. Plates were read by employing a

ﬂuorescence microplate reader Varioskan Flash (Thermo Fisher

Scientiﬁc Inc.).

Confocal Microscopy. For imaging, promastigotes of L. donovani

were seeded (10⁶cells mL⁻¹) on 96-well plates at 26 °C for 72 h.

Then, the labeled compounds, at their IC₅₀concentrations, were

added and the plate was incubated at 26 °C for 45/50 min.

Subsequently, cells were collected, washed with PBS, and immobilized

on a slide. TO-PRO-3 (1 μM) (Thermo Fisher Scientiﬁc Inc.), a

speciﬁc dye for nucleic acids,^114,115was also added after cell

permeabilization, made by incubating promastigotes in methanol/

acetone for 10 min at −20 °C. Fluorescence microscopy analysis was

performed using a TCS SP2 scanning confocal microscope (Leica

Microsystems, Wetzlar, Germany) with a 63× oil objective, the

excitation wavelength utilized for the labeled compounds was 488 nm,

and emission was detected at 505 nm. The excitation wavelength for

TO-PRO-3 was 642 nm, and emission was detected at 661 nm. An

antifade agent to reduce photo bleaching was not used.

Authors

Margherita Ortalli − Unit of Clinical Microbiology, Regional

Reference Centre for Microbiological Emergencies (CRREM), St.

Orsola-Malpighi University Hospital, 40138 Bologna, Italy

Stefania Varani − Unit of Clinical Microbiology, Regional

Reference Centre for Microbiological Emergencies (CRREM), St.

Orsola-Malpighi University Hospital, 40138 Bologna, Italy;

Department of Experimental, Diagnostic and Specialty

Medicine, Alma Mater Studiorum - University of Bologna,

40138 Bologna, Italy

Giorgia Cimato − Unit of Clinical Microbiology, Regional

Reference Centre for Microbiological Emergencies (CRREM), St.

Orsola-Malpighi University Hospital, 40138 Bologna, Italy

Ruben Veronesi − Department of Chemistry “G. Ciamician”,

Alma Mater Studiorum - University of Bologna, 40126

Bologna, Italy

Magda Monari − Department of Chemistry “G. Ciamician”,

Alma Mater Studiorum - University of Bologna, 40126

Bologna, Italy

Claudio Trombini − Department of Chemistry “G. Ciamician”,

Alma Mater Studiorum - University of Bologna, 40126

Bologna, Italy; Centro Interuniversitario di Ricerca sulla

Malaria (CIRM) - Italian Malaria Network (IMN), University

of Milan, 20100 Milan, Italy

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01589

Author Contributions

^⊥M.O. and S.V. contributed equally to this work. A.Q. and

M.O. conceived the study. M.O. and G.C. performed the

O

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J. Med. Chem. XXXX, XXX, XXX−XXX

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Article

(8) Horrillo, L.; Castro, A.; Matía, B.; Molina, L.; García-Martínez,

J.; Jaqueti, J.; García-Arata, I.; Carrillo, E.; Moreno, J.; Ruiz-Giardin, J.

M.; San Martín, J. Clinical aspects of visceral Leishmaniasis caused by

L. infantum in adults. Ten years of experience of the largest outbreak

in Europe: what have we learned? Parasites Vectors 2019, 12, 359.

(9) Koch, L. K.; Kochmann, J.; Klimpel, S.; Cunze, S. Modeling the

climatic suitability of Leishmaniasis vector species in Europe. Sci. Rep.

2017, 7, 13325.

promastigote and amastigote growth inhibition assays, the

cytotoxicity tests, the evaluation of iron role and intracellular

ROS levels, and the confocal microscopy experiments. M.O.

and S.V. supervised the bioassays and analyzed the biological

results. R.V. and A.Q. synthesized and characterized 1,2-

dioxanes and tetrahydropyrans. A.Q., M.L., and C.T. designed

the compound synthesis and analyzed the synthetic and

biological results. A.Q. and M.L. supervised the compound

synthesis. M.M. performed the X-ray crystallographic analysis.

A.Q. and M.O. prepared the original manuscript draft. All

authors contributed to writing and reviewing the ﬁnal

manuscript. C.T., M.L., and S.V. provided ﬁnancial resources.

(10) Chalghaf, B.; Chemkhi, J.; Mayala, B.; Harrabi, M.; Benie, G.

B.; Michael, E.; Salah, A. B. Ecological niche modeling predicting the

potential distribution of leishmanial vectors in the Mediterranean

basin: impact of climate change. Parasites Vectors 2018, 11, 461.

(11) Saliba, M.; Shalhoub, A.; Taraif, S.; Loya, A.; Houreih, M. A.; El

Hajj, R.; Khalifeh, I. Cutaneous leishmaniasis: an evolving disease

with ancient roots. Int. J. Dermatol. 2019, 58, 834−843.

Funding

̀

This research was supported by Ministero dell’Universita e

(12) World Health Organization Leishmaniasis; https://www.who.

int/leishmaniasis/en/ (accessed Dec 15, 2019).

della Ricerca (PRIN2015 - 20154JRJPP), Fondazione Carisbo

(project 2018/0356), and University of Bologna (RFO).

(13) Gradoni, L.; Rogelio, L. V.; Mourad, M. Manual on case

management and surveillance of the leishmaniases in the WHO European

Region, 2017; World Health Oragnization: Web site: http://www.

euro.who.int/en/publications/abstracts/manual-on-case-

management-and-surveillance-of-the-leishmaniases-in-the-who-

european-region-2017 (accessed Dec 20, 2019).

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

We thank Ms. G. Di Donatantonio and Mr. G. Ulivi for the

execution of some synthetic steps.

■

(14) Barrett, M. P.; Croft, S. L. Management of trypanosomiasis and

leishmaniasis. Br. Med. Bull. 2012, 104, 175−196.

(15) Singh, N.; Kumar, M.; Singh, R. K. Leishmaniasis: current

status of available drugs and new potential drug targets. Asian Pac. J.

Trop. Med. 2012, 5, 485−497.

ABBREVIATIONS

■

CC₅₀, half-maximum toxic concentration; SI, selectivity index

= CC₅₀/IC₅₀; R&D, research and development; HSP90, 90

kDa heat shock protein; PG, protective group; CSA,

camphorsulfonic acid; equiv, equivalents; nd, not determined;

L., Leishmania; DFO, desferrioxamine; DFP, deferiprone; FIC,

fractional inhibitory concentration = IC₅₀of drug in

combination/IC₅₀of drug alone; H₂DCFDA, 2,7-dichlorodi-

hydroﬂuorescein diacetate; DCF, dichloroﬂuorescein; NC,

negative control; NBD, nitrobenzyldiazole; TEA, triethyl-

amine; TO-PRO-3, a carbocyanine monomer nucleic acid

stain; LRMS, low-resolution mass spectrometry; CyH, cyclo-

hexane; bs, broad singlet; bt, broad triplet; dq, double quartet;

FBS, fetal bovine serum; THP-1 cells, human leukemia

monocytic cells

(16) Uliana, S. R. B.; Trinconi, C. T.; Coelho, A. C. Chemotherapy

of leishmaniasis: present challenges. Parasitology 2018, 145, 464−480.

́

(17) Vermeersch, M.; da Luz, R. I.; Tote, K.; Timmermans, J.-P.;

Cos, P.; Maes, L. In vitro susceptibilities of Leishmania donovani

promastigote and amastigote stages to antileishmanial reference

drugs: practical relevance of stage-specific differences. Antimicrob.

Agents Chemother. 2009, 53, 3855−3859.

(18) Lachaud, L.; Bourgeois, N.; Plourd, M.; Leproho, P.; Bastien,

P.; Ouellette, M. Parasite susceptibility to amphotericin B in failures

of treatment for visceral leishmaniasis in patients coinfected with HIV

type 1 and Leishmania infantum . Clin. Infect. Dis. 2009, 48, No. e16.

(19) den Boer, M.; Davidson, R. N. Treatment options for visceral

leishmaniasis. Exp. Rev. Anti-Infect. Ther. 2014, 4, 187−197.

(20) Rodrigues, I. A.; Mazotto, A. M.; Cardoso, V.; Alves, R. L.;

Amaral, A. C. F.; de Andrade Silva, J. R.; Pinheiro, A. S.; Vermelho, A.

B. Natural products: insights into leishmaniasis inflammatory

response. Mediators Inflammation 2015, 2015, 1.

REFERENCES

■

(21) Croft, S. L.; Olliaro, P. Leishmaniasis chemotherapy −

challenges and opportunities. Clin. Microbiol. Infect. 2011, 17,

1478−1483.

(1) World Health Organization Leishmaniasis; https://www.who.

int/news-room/fact-sheets/detail/leishmaniasis (accessed Dec 20,

2019).

(22) Ponte-Sucre, A.; Gamarro, F.; Dujardin, J.-C.; Barrett, M. P.;

(2) Torres-Guerrero, E.; Quintanilla-Cedillo, M. R.; Ruiz-

Esmenjaud, J.; Arenas, R. Leishmaniasis: a review. F1000Res. 2017,

6, 750.

(3) Inceboz, T. Epidemiology and Ecology of Leishmaniasis. In

Current Topics in Neglected Tropical Diseases; Rodriguez-Morales, A. J.,

Ed.; IntechOpen: London, 2019, 1−15, DOI: 10.5772/inte-

chopen.86359.

́

Lopez-Velez, R.; García-Hernandez, R.; Pountain, A. W.;

Mwenechanya, R.; Papadopoulou, B. Drug resistance and treatment

failure in leishmaniasis: A 21st century challenge. PLoS Neglected Trop.

Dis. 2017, 11, No. e0006052.

(23) Vanaerschot, M.; Huijben, S.; Van den Broeck, F.; Dujardin, J.

C. Drug resistance in vectorborne parasites: multiple actors and

scenarios for an evolutionary arms race. FEMS Microbiol. Rev. 2014,

38, 41−55.

(24) Hendrickx, S.; Boulet, G.; Mondelaers, A.; Dujardin, J. C.; Rijal,

S.; Lachaud, L.; Cos, P.; Delputte, P.; Maes, L. Experimental selection

of paromomycin and miltefosine resistance in intracellular amastigotes

of Leishmania donovani and L. infantum . Parasitol. Res. 2014, 113,

1875−1881.

(25) Hendrickx, S.; Mondelaers, A.; Eberhardt, E.; Delputte, P.; Cos,

P.; Maes, L. In vivo selection of paromomycin and miltefosine

resistance in Leishmania donovani and L. infantum in a syrian hamster

model. Antimicrob. Agents Chemother. 2015, 59, 4714−4718.

(26) Mandal, S.; Maharjan, M.; Singh, S.; Chatterjee, M.;

Madhubala, R. Assessing aquaglyceroporin gene status and expression

profile in antimony-susceptible and -resistant clinical isolates of

(4) Aronson, N. E.; Magill, A. J. Leishmaniasis. In Hunter’s Tropical

Medicine and Emerging Infectious Diseases; Ryan, E. T.; Hill, D. R.;

Solomon, T.; Aronson, N.; Endy, T. P., Eds.; Elsevier: Amsterdam,

2020; 776−798.

(5) Dujardin, J.-C.; Campino, L.; Canavate, C.; Dedet, J.-P.;

̃

Gradoni, L.; Soteriadou, K.; Mazeris, A.; Ozbel, Y.; Boelaert, M.

Spread of vector-borne diseases and neglect of Leishmaniasis, Europe.

Emerg. Infect. Dis. 2008, 14, 1013−1018.

́

(6) Alvar, J.; Velez, I. D.; Bern, C.; Herrero, M.; Desjeux, P.; Cano,

J.; Jannin, J.; den Boer, M.; The WHO Leishmaniasis control team.

Leishmaniasis worldwide and global estimates of its incidence.

PLoSOne 2012, 7, No. e35671.

(7) Steverding, D. The history of Leishmaniasis. Parasites Vectors

2017, 10, 82.

P

https://dx.doi.org/10.1021/acs.jmedchem.0c01589

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Leishmania donovani from India. J. Antimicrob. Chemother. 2010, 65,

496−507.

E. R.; da Silva Rodrigues, J. H.; Simao

̃

, A. N. C.; Yamauchi, L. M.;

Nakamura, C. V.; Verri, W. A., Jr.; Costa, I. N.; Conchon-Costa, I.;

Pavanelli, W. R. Trans -chalcone modulates Leishmania amazonensis

infection in vitro by Nrf2 overexpression affecting iron availability.

Eur. J. Pharmacol. 2019, 853, 275−288.

(27) Mostafavi, M.; Sharifi, I.; Farajzadeh, S.; Khazaeli, P.; Sharifi,

H.; Pourseyedi, E.; Kakooei, S.; Bamorovat, M.; Keyhani, A.; Parizi,

M. H.; Khosravi, A.; Khamesipour, A. Niosomal formulation of

amphotericin B alone and in combination with glucantime: in vitro

and in vivo leishmanicidal effects. Biomed. Pharmacother. 2019, 116,

108942.

(43) Upadhyay, A.; Chandrakar, P.; Gupta, S.; Parmar, N.; Singh, S.

K.; Rashid, M.; Kushwaha, P.; Wahajuddin, M.; Sashidhara, K. V.;

Kar, S. Synthesis, biological evaluation, structure −activity relationship,

and mechanism of action studies of quinoline −metronidazole

derivatives against experimental visceral leishmaniasis. J. Med. Chem.

2019, 62, 5655−5671.

(44) Otero, E.; García, E.; Palacios, G.; Yepes, L. M.; Carda, M.;

Agut, R.; Velez, I. D.; Cardona, W. I.; Robledo, S. M. Triclosan-caffeic

acid hybrids: synthesis, leishmanicidal, trypanocidal and cytotoxic

activities. Eur. J. Med. Chem. 2017, 141, 73−83.

(45) Baquedano, Y.; Moreno, E.; Espuelas, S.; Nguewa, P.; Font, M.;

(28) Scariot, D. B.; Volpato, H.; Fernandes, N. d. S.; Soares, E. F. P.;

Ueda-Nakamura, T.; Dias-Filho, B. P.; Din, Z. U.; Rodrigues-Filho, E.;

Rubira, A. F.; Borges, O.; Sousa, M. D. C.; Nakamura, C. V. Activity

and cell-death pathway in Leishmania infantum induced by sugiol:

vectorization using yeast cell wall particles obtained from Saccha-

romyces cerevisiae . Front. Cell. Infect. Microbiol. 2019, 9, 208.

(29) Rebello, K. M.; Andrade-Neto, V. V.; Gomes, C. R. B.; de

Souza, M. V. N.; Branquinha, M. H.; Santos, A. L. S.; Torres-Santos,

E. C.; d’Avila-Levy, C. M. Miltefosine-lopinavir combination therapy

against Leishmania infantum infection: in vitro and in vivo approaches.

Front. Cell. Infect. Microbiol. 2019, 9, 229.

(30) World Health Organization − Access to essential antileishmanial

medicines and treatment; http://www.who.int/leishmaniasis/

research/en/ (accessed Dec 15, 2019). In particular, see: World

Health Organization − Costs of medicines in current usefor the treatment

of leishmaniasis. http://www.who.int/leishmaniasis/research/978_

92_4_12_949_6_Annex6.pdf?ua=1 (accessed Dec 15, 2020).

(31) Seifert, K. Structures, targets and recent approaches in anti-

leishmanial drug discovery and development. Open Med. Chem. J.

2011, 5, 31−39.

́

Gutierrez, K. J.; Jimenez-Ruiz, A.; Palop, J. A.; Sanmartín, C. Novel

hybrid selenosulfonamides as potent antileishmanial agents. Eur. J.

Med. Chem. 2014, 74, 116−123.

(46) Yousuf, M.; Mukherjee, D.; Dey, S.; Pal, C.; Adhikari, S.

Antileishmanial ferrocenylquinoline derivatives: synthesis and bio-

logical evaluation against Leishmania donovani . Eur. J. Med. Chem.

2016, 124, 468−479.

́

(47) Felix, M. B.; de Souza, E. R.; de Lima, M. d. C. A.; Frade, D. K.

G.; Serafim, V. d. L.; Rodrigues, K. A. d. F.; Neris, P. L. d. N.; Ribeiro,

F. F.; Scotti, L.; Scotti, M. T.; de Aquino, T. M.; Mendoca Junior, F. J.

́

̧

B.; de Oliveira, M. R. Antileishmanial activity of new thiophene-indole

hybrids: design, synthesis, biological and cytotoxic evaluation, and

chemometric studies. Bioorg. Med. Chem. 2016, 24, 3972−3977.

(48) Ramu, D.; Garg, S.; Ayana, R.; Keerthana, A. K.; Sharma, V.;

Saini, C. P.; Sen, S.; Pati, S.; Singh, S. Novel β -carbolinequinazolinone

hybrids disrupt Leishmania donovani redox homeostasis and show

promising antileishmanial activity. Biochem. Pharmacol. 2017, 129,

26−42.

(49) Barea, P.; Barbosa, V. A.; Bidoia, D. L.; de Paula, J. C.;

Stefanello, T. F.; da Costa, W. F.; Nakamura, C. V.; Sarragiotto, M. H.

Synthesis, antileishmanial activity and mechanism of action studies of

novel β -carboline-1,3,5-triazine hybrids. Eur. J. Med. Chem. 2018, 150,

579−590.

(50) Tiuman, T. S.; Santos, A. O.; Ueda-Nakamura, T.; Filho, B. P.

D.; Nakamura, C. V. Recent advances in leishmaniasis treatment. Int.

J. Infect. Dis. 2011, 15, No. e525.

(32) Olliaro, P.; Darley, S.; Laxminarayan, R.; Sundar, S. Cost-

effectiveness projections of single and combination therapies for

visceral leishmaniasis in Bihar, India. Trop. Med. Int. Health. 2009, 14,

918−925.

(33) Srivastava, S.; Mishra, J.; Gupta, A. K.; Singh, A.; Shankar, P.;

Singh, S. Laboratory confirmed miltefosine resistant cases of visceral

leishmaniasis from India. Parasites Vectors 2017, 10, 49.

́

(34) Garcia-Hernandez, R.; Manzano, J. I.; Castanys, S.; Gamarro, F.

Leishmania donovani develops resistance to drug combinations. PLoS

Neglected Trop. Dis. 2012, 6, No. e1974.

(35) Hendrickx, S.; da Luz, R. A. I.; Bhandari, V.; Kuypers, K.; Shaw,

C. D.; Lonchamp, J.; Salotra, P.; Carter, K.; Sundar, S.; Rijal, S.;

Dujardin, J.-C.; Cos, P.; Maes, L. Experimental induction of

paromomycin resistance in antimony-resistant strains of L. donovani :

outcome dependent on in vitro selection protocol. PLoS Neglected

Trop. Dis. 2012, 6, No. e1664.

(36) Hendrickx, S.; Guerin, P. J.; Caljon, G.; Croft, S. L.; Maes, L.

Evaluating drug resistance in visceral leishmaniasis: the challenges.

Parasitology 2018, 145, 453−463.

(37) Collett, C. F.; Kitson, C.; Baker, N.; Steele-Stallard, H. B.;

Santrot, M.-V.; Hutchinson, S.; Horn, D.; Alsford, S. Chemogenomic

profiling of anti-leishmanial efficacy and resistance in the related

kinetoplastid parasite Trypanosoma brucei . Antimicrob. Agents Chemo-

ther. 2019, 63, No. e00795. See also ref 22.

(38) World Health Organization Accelerating work to overcome the

global impact of neglected tropical diseases: a roadmap for

implementation; http://www.who.int/neglected_diseases/NTD_

RoadMap_2012_Fullversion.pdf (accessed Dec 20, 2019).

(39) Burrows, J. N.; Elliott, R. L.; Kaneko, T.; Mowbray, C. E.;

Waterson, D. The role of modern drug discovery in the fight against

neglected and tropical diseases. Med. Chem. Commun. 2014, 5, 688−

700.

(51) Gil, C. Drug Discovery for Leishmaniasis; The Royal Society of

Chemistry: London, 2017.

(52) Elliott, R. L. Third World Diseases; Springer-Verlag: Berlin

Heidelberg, 2011.

(53) Vijayakumar, S.; Das, P. Recent progress in drug targets and

inhibitors towards combating leishmaniasis. Acta Trop. 2018, 181,

95−104.

(54) Negrao, F.; Eberlin, M. N.; Giorgio, S. Proteomic approaches

̃

for drug discovery against tegumentary leishmaniasis. Biomed.

Pharmacother. 2017, 95, 577−582.

(55) Sharma, M.; Shaikh, N.; Yadav, S.; Singh, S.; Garg, P. A

systematic reconstruction and constraint-based analysis of Leishmania

donovani metabolic network: identification of potential antileishma-

nial drug targets. Mol. BioSyst. 2017, 13, 955−969.

(56) Rossi, M.; Fasel, N. How to master the host immune system?

Leishmania parasites have the solutions! Int. Immunol. 2018, 30, 103−

111.

(57) de Oliveira Almeida Petersen, A. L.; Campos, T. A.; dos Santos

Dantas, D. A.; de Souza Rebouca̧ s, J.; da Silva, J. C.; de Menezes, J. P.

B.; Formiga, F. R.; de Melo, J. V.; Machado, G.; Veras, P. S. T.

Encapsulation of the HSP-90 chaperone inhibitor 17-AAG in stable

liposome allow increasing the therapeutic index as assessed, in vitro ,

on Leishmania (L) amazonensis amastigotes-hosted in mouse CBA

macrophages. Front. Cell. Infect. Microbiol. 2018, 8, 303.

(40) Wyllie, S.; Patterson, S.; Stojanovski, L.; Simeons, F. R. C.;

Norval, S.; Kime, R.; Read, K. D.; Fairlamb, A. H. The anti-

trypanosome drug fexinidazole shows potential for treating visceral

leishmaniasis. Sci. Transl. Med. 2012, 4, 119re1.

(41) de Melo Mendes, V.; Tempone, A. G.; Borborema, S. E. T.

Antileishmanial activity of H1-antihistamine drugs and cellular

alterations in Leishmania (L.) infantum . Acta Trop. 2019, 195, 6−14.

(42) Miranda-Sapla, M. M.; Tomiotto-Pellissier, F.; Assolini, J. P.;

(58) Sharma, G.; Kar, S.; Ball, W. B.; Ghosh, K.; Das, P. K. The

Carloto, A. C. M.; da Silva Bortoleti, B. T.; Gonca̧ lves, M. D.; Tavares,

curative effect of fucoidan on visceral leishmaniasis is mediated by

Q

https://dx.doi.org/10.1021/acs.jmedchem.0c01589

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

activation of map kinases through specific protein kinase c isoforms.

Cell. Mol. Immunol. 2014, 11, 263−274.

artemisinin for the treatment of murine visceral leishmaniasis. Int. J.

Nanomed. 2017, 12, 2189−2204.

(77) Krishna, S.; Bustamante, L.; Haynes, R. K.; Staines, H. M.

Artemisinins: their growing importance in medicine. Trends

Pharmacol. Sci. 2008, 29, 520−527. References cited therein.

(78) Leishmania parasites are transmitted into a mammalian host by

the bite of phlebotomine sand ﬂies (genus Phlebotomus in the Old

World and Lutzomyia in the New World) as promastigotes

(ﬂagellates), which transform into aﬂagellated amastigotes in host

macrophages.

(59) Kyriazis, I. D.; Koutsoni, O. S.; Aligiannis, N.; Karampetsou, K.;

Skaltsounis, A.-L.; Dotsika, E. The leishmanicidal activity of

oleuropein is selectively regulated through inflammation- and

oxidative stress-related genes. Parasites Vectors 2016, 9, 441.

(60) Nirma, C.; Rangel, G. T.; Alves, M. A.; Casanova, L. M.;

Moreira, M. M.; Rodrigues, L. M.; Hamerski, L.; Tinoco, L. W. New

Leishmania donovani nucleoside hydrolase inhibitors from Brazilian

flora. RSC Adv. 2019, 9, 18663−18669.

(79) Cortes, S.; Albuquerque, A.; Cabral, L. I. L.; Lopes, L.;

Campino, L.; Cristiano, M. L. S. In vitro susceptibility of Leishmania

infantum to artemisinin derivatives and selected trioxolanes.

Antimicrob. Agents Chemother. 2015, 59, 5032−5035.

(80) Kwofie, K. D.; Sato, K.; Sanjoba, C.; Hino, A.; Shimogawara,

R.; Amoa-Bosompem, M.; Ayi, I.; Boakye, D. A.; Anang, A. K.; Chang,

K.-S.; Ohashi, M.; Kim, H.-S.; Ohta, N.; Matsumoto, Y.; Iwanaga, S.

Oral activity of the antimalarial endoperoxide 6-(1,2,6,7-

tetraoxaspiro[7.11]nonadec-4-yl) hexan-1-ol (N-251) against Leish-

mania donovani complex. PLoS Neglected Trop. Dis. 2019, 13,

No. e0007235.

(81) Persico, M.; Quintavalla, A.; Rondinelli, F.; Trombini, C.;

Lombardo, M.; Fattorusso, C.; Azzarito, V.; Taramelli, D.; Parapini,

S.; Corbett, Y.; Chianese, G.; Fattorusso, E.; Taglialatela-Scafati, O. A

new class of antimalarial dioxanes obtained through a simple two-step

synthetic approach: rational design and structure −activity relationship

studies. J. Med. Chem. 2011, 54, 8526−8540.

(82) Persico, M.; Parapini, S.; Chianese, G.; Fattorusso, C.;

Lombardo, M.; Petrizza, L.; Quintavalla, A.; Rondinelli, F.; Basilico,

N.; Taramelli, D.; Trombini, C.; Fattorusso, E.; Taglialatela-Scafati, O.

Further optimization of plakortin pharmacophore: structurally simple

4-oxymethyl-1,2-dioxanes with promising antimalarial activity. Eur. J.

Med. Chem. 2013, 70, 875−886.

(83) Lombardo, M.; Sonawane, D. P.; Quintavalla, A.; Trombini, C.;

Dhavale, D. D.; Taramelli, D.; Corbett, Y.; Rondinelli, F.; Fattorusso,

C.; Persico, M.; Taglialatela-Scafati, O. Optimized synthesis and

antimalarial activity of 1,2-dioxane-4-carboxamides. Eur. J. Org. Chem.

2014, 1607−1614.

(84) Sonawane, D. P.; Persico, M.; Corbett, Y.; Chianese, G.; Di

Dato, A.; Fattorusso, C.; Taglialatela-Scafati, O.; Taramelli, D.;

Trombini, C.; Dhavale, D. D.; Quintavalla, A.; Lombardo, M. New

antimalarial 3-methoxy-1,2-dioxanes: optimization of cellular pharma-

cokinetics and pharmacodynamics properties by incorporation of

amino and N -heterocyclic moieties at C4. RSC Adv. 2015, 5, 72995−

73010.

(85) Sonawane, D. P.; Corbett, Y.; Dhavale, D. D.; Taramelli, D.;

Trombini, C.; Quintavalla, A.; Lombardo, M. D-glucose-derived 1,2,4-

trioxepanes: synthesis, conformational study, and antimalarial activity.

Org. Lett. 2015, 17, 4074−4077.

(86) Persico, M.; Fattorusso, R.; Taglialatela-Scafati, O.; Chianese,

G.; de Paola, I.; Zaccaro, Z.; Rondinelli, F.; Lombardo, M.;

Quintavalla, A.; Trombini, C.; Fattorusso, E.; Fattorusso, C.; Farina,

B. The interaction of heme with plakortin and a synthetic

endoperoxide analogue: new insights into the heme-activated

antimalarial mechanism. Sci. Rep. 2017, 7, 45485.

(61) da Silva, B. P.; Cortez, D. A.; Violin, T. V.; Filho, B. P. D.;

Nakamura, C. V.; Ueda-Nakamura, T.; Ferreira, I. C. P. Anti-

leishmanial activity of a guaianolide from Tanacetum parthenium (L.)

Schultz Bip. Parasitol. Int. 2010, 59, 643−646.

(62) Correa, E.; Cardona, D.; Quinones, D.; Torres, F.; Franco, A.

̃

́

E.; Velez, I. D.; Robledo, S.; Echeverri, F. Leishmanicidal activity of

Pycnoporus sanguineus . Phytother. Res. 2006, 20, 497−499.

(63) Chianese, G.; Fattorusso, E.; Scala, F.; Teta, R.; Calcinai, B.;

Bavestrello, G.; Dien, H. A.; Kaiser, M.; Tasdemir, D.; Taglialatela-

Scafati, O. Manadoperoxides, a new class of potent antitrypanosomal

agents of marine origin. Org. Biomol. Chem. 2012, 10, 7197−7207.

(64) Monzote, L.; Geroldinger, G.; Tonner, M.; Scull, R.; De Sarkar,

S.; Bergmann, S.; Bacher, M.; Staniek, K.; Chatterjee, M.; Rosenau,

T.; Gille, L. Interaction of ascaridole, carvacrol, and caryophyllene

oxide from essential oil of Chenopodium ambrosioides L. with

mitochondria in Leishmania and other eukaryotes. Phytother. Res.

2018, 32, 1729−1740. References cited therein.

(65) Monzote, L.; García, M.; Pastor, J.; Gil, L.; Scull, R.; Maes, L.;

Cos, P.; Gille, L. Essential oil from Chenopodium ambrosioides and

main components: activity against Leishmania , their mitochondria and

other microorganisms. Exp. Parasitol. 2014, 136, 20−26.

(66) Loo, C. S. N.; Lam, N. S. K.; Yu, D.; Su, X.-z.; Lu, F.

Artemisinin and its derivatives in treating protozoan infections

beyond malaria. Pharmacol. Res. 2017, 117, 192−217.

(67) Mishina, Y. V.; Krishna, S.; Haynes, R. K.; Meade, J. C.

Artemisinins inhibit Trypanosoma cruzi and Trypanosoma brucei

rhodesiense in vitro growth. Antimicrob. Agents Chemother. 2007, 51,

1852−1854.

(68) WHO World Malaria Report; 2013, ISBN: 9 789241 56469 4.

(69) World Health Organization Guidelines for the treatment of

malaria;

http://whqlibdoc.who.int/publications/2010/

9789241547925_eng.pdf.

(70) Sen, R.; Bandyopadhyay, S.; Dutta, A.; Mandal, G.; Ganguly,

G.; Saha, P.; Chatterjee, M. Artemisinin triggers induction of cell-

cycle arrest and apoptosis in Leishmania donovani promastigotes. J.

Med. Microbiol. 2007, 56, 1213−1218.

(71) Want, M. Y.; Islamuddin, M.; Chouhan, G.; Ozbak, H. A.;

Hemeg, H. A.; Dasgupta, A. K.; Chattopadhyay, A. P.; Afrin, F.

Therapeutic efficacy of artemisinin-loaded nanoparticles in exper-

imental visceral leishmaniasis. Colloids Surf., B 2015, 130, 215−221.

(72) Ghaffarifar, F.; Heydari, F. E.; Dalimi, A.; Hassan, Z. M.;

Delavari, M.; Mikaeiloo, H. Evaluation of apoptotic and antileishma-

nial activities of artemisinin on promastigotes and BALB/C mice

infected with leishmania major. Iran J. Parasitol. 2015, 10, 258−267.

(73) Mutiso, J. M.; Macharia, J. C.; Barasa, M.; Taracha, E.;

Bourdichon, A. J.; Gicheru, M. M. In vitro and in vivo antileishmanial

efficacy of a combination therapy of diminazene and artesunate

against Leishmania donovani in BALB/c mice. Rev. Inst. Med. Trop. Sao

Paulo 2011, 53, 129−132.

(74) Chollet, C.; Crousse, B.; Bories, C.; Bonnet-Delpon, D.;

Loiseau, P. M. In vitro antileishmanial activity of fluoro-artemisinin

derivatives against Leishmania donovani . Biomed. Pharmacother. 2008,

62, 462−465.

(75) Sen, R.; Ganguly, S.; Saha, P.; Chatterjee, M. Efficacy of

artemisinin in experimental visceral leishmaniasis. Int. J. Antimicrob.

Agents 2010, 36, 43−49.

(76) Want, M. Y.; Islammudin, M.; Chouhan, G.; Ozbak, H. A.;

Hemeg, H. A.; Chattopadhyay, A. P.; Afrin, F. Nanoliposomal

(87) Ortalli, M.; Varani, S.; Rosso, C.; Quintavalla, A.; Lombardo,

M.; Trombini, C. Evaluation of synthetic substituted 1,2-dioxanes as

novel agents against human leishmaniasis. Eur. J. Med. Chem. 2019,

170, 126−140.

(88) Robert, A.; Dechy-Cabaret, O.; Cazelles, J.; Meunier, B. From

mechanistic studies on artemisinin derivatives to new modular

antimalarial drugs. Acc. Chem. Res. 2002, 35, 167−174.

(89) Jefford, C. W. Why artemisinin and certain synthetic peroxides

are potent antimalarials. Implications for the mode of action. Curr.

Med. Chem. 2001, 8, 1803−1826.

(90) Posner, G. H.; Oh, C. H.; Wang, D.; Gerena, L.; Milhous, W.

K.; Meshnick, S. R.; Asawamahasadka, W. Mechanism-based design,

synthesis, and in vitro antimalarial testing of new 4-methylated

trioxanes structurally related to artemisinin: the importance of a

R

https://dx.doi.org/10.1021/acs.jmedchem.0c01589

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

carbon-centered radical for antimalarial activity. J. Med. Chem. 1994,

37, 1256−1258.

(91) Posner, G. H.; Wang, D.; Cumming, J. N.; Oh, C. H.; French,

A. N.; Bodley, A. L.; Shapiro, T. A. Further evidence supporting the

importance of and the restrictions on a carbon-centered radical for

high antimalarial activity of 1,2,4-trioxanes like artemisinin. J. Med.

Chem. 1995, 38, 2273−2275.

brevicomin, and (−)-isolaurepan. Tetrahedron: Asymmetry 2007, 18,

1419−1427.

(108) Access Structures; Crystallographic data of compound 9b have

been deposited with the Cambridge Crystallographic Data Centre

(CCDC) as supplementary publication number CCDC 1972697.

Copies of the data can be obtained free of charge via www.ccdc.cam.

ac.uk/getstructures.

(109) NMR experiments (nOe) on the two isomers of oleﬁn 8a

were used to establish the double bond geometry (see the Supporting

Information for details). The results were extended to oleﬁn 8c.

(110) The favored cis relative stereochemistry established by X-ray

crystallographic analysis for product 9b was extended to the other

tetrahydropyrans 9a and 9c obtained using the same stereoselective

synthetic approach. For a hypothesis justifying the cis-stereo-

preference in the construction of tetrahydropyrans 9, see the

Supporting Information.

(92) Wang, J.; Zhang, C.-J.; Chia, W. N.; Loh, C. C. Y.; 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.; Shen, H.-M.; Tan,

K. S. W.; Lin, Q. Haem-activated promiscuous targeting of artemisinin

in Plasmodium falciparum . Nat. Commun. 2015, 6, 10111.

(93) Robert, A.; Cazelles, J.; Meunier, B. Characterization of the

alkylation product of heme by the antimalarial drug artemisinin.

Angew. Chem., Int. Ed. 2001, 40, 1954−1957.

(94) Cazelles, J.; Robert, A.; Meunier, B. Alkylating capacity and

reaction products of antimalarial trioxanes after activation by a heme

model. J. Org. Chem. 2002, 67, 609−619.

(111) Bell, A. Antimalarial drug synergism and antagonism:

mechanistic and clinical significance. FEMS Microbiol. Lett. 2005,

253, 171−184.

(95) Robert, A.; Benoit-Vical, F.; Meunier, B. The key role of heme

to trigger the antimalarial activity of trioxanes. Coord. Chem. Rev.

2005, 249, 1927−1936.

(96) 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 heme alkylation for spiro- and dispiro-1,2,4-

trioxolane antimalarials. Antimicrob. Agents Chemother. 2008, 52,

1291−1296.

(97) Taglialatela-Scafati, O.; Fattorusso, E.; Romano, A.; Scala, F.;

Barone, V.; Cimino, P.; Stendardo, E.; Catalanotti, B.; Persico, M.;

Fattorusso, C. Insight into the mechanism of action of plakortins,

simple 1,2-dioxane antimalarials. Org. Biomol. Chem. 2010, 8, 846−

856.

(112) Benoit-Vical, F.; Robert, A.; Meunier, B. In vitro and in vivo

potentiation of artemisinin and synthetic endoperoxide antimalarial

drugs by metalloporphyrins. Antimicrob. Agents Chemother. 2000,

2836−2841.

(113) See ref 100 and references cited therein.

(114) Milani, L.; Maurizii, M. G. Vasa expression in spermatogenic

cells during the reproductive-cycle phases of Podarcis sicula (Reptilia,

Lacertidae). J. Exp. Zool., Part B 2015, 324, 424−434.

(115) Milani, L.; Pecci, A.; Ghiselli, F.; Passamonti, M.; Bettini, S.;

Franceschini, V.; Maurizii, M. G. VASA expression suggests shared

germ line dynamics in bivalve molluscs. Histochem. Cell Biol. 2017,

148, 157−171.

(116) Das, N. K.; Biswas, S.; Solanki, S.; Mukhopadhyay, C. K.

Leishmania donovani depletes labile iron pool to exploit iron uptake

capacity of macrophage for its intracellular growth. Cell. Microbiol.

2009, 11, 83−94.

(117) Zaidi, A.; Singh, K. P.; Ali, V. Leishmania and its quest for

iron: an update and overview. Mol. Biochem. Parasitol. 2017, 211, 15−

25.

(98) O’Neill, P. M.; Barton, V. E.; Ward, S. A. The molecular

mechanism of action of artemisinin - the debate continues. Molecules

2010, 15, 1705−1721.

̈

(99) Tilley, L.; Straimer, J.; Gnadig, N. F.; Ralph, S. A.; Fidock, D. A.

Artemisinin action and resistance in Plasmodium falciparum . Trends

Parasitol. 2016, 32, 682−696.

(100) Stocks, P. A.; Bray, P. G.; Barton, V. E.; Al-Helal, M.; Jones,

M.; Araujo, N. C.; Gibbons, P.; Ward, S. A.; Hughes, R. H.; Biagini,

G. A.; Davies, J.; Amewu, R.; Mercer, A. E.; Ellis, G.; O’Neill, P. M.

Evidence for a common non-heme chelatable-iron-dependent

activation mechanism for semisynthetic and synthetic endoperoxide

antimalarial drugs. Angew. Chem., Int. Ed. 2007, 46, 6278−6283.

(101) Geroldinger, G.; Tonner, M.; Fudickar, W.; De Sarkar, S.;

Dighal, A.; Monzote, L.; Staniek, K.; Linker, T.; Chatterjee, M.; Gille,

L. Activation of anthracene endoperoxides in Leishmania and

impairment of mitochondrial functions. Molecules 2018, 23, 1680.

(102) Sen, R.; Saha, P.; Sarkar, A.; Ganguly, S.; Chatterjee, M. Iron

enhances generation of free radicals by artemisinin causing a caspase-

independent, apoptotic death in Leishmania donovani promastigotes.

Free Radical Res. 2010, 44, 1289−1295.

(118) Sarkar, A.; Andrews, N. W.; Laranjeira-Silva, M. F.

Intracellular iron availability modulates the requirement for

Leishmania Iron Regulator 1 (LIR1) during macrophage infections.

Int. J. Parasitol. 2019, 49, 423−427.

(119) Sato, T.; Onuma, T.; Nakamura, I.; Terada, M. Platinum-

catalyzed cycloisomerization of 1,4-enynes via 1,2-alkenyl rearrange-

ment. Org. Lett. 2011, 13, 4992−4995.

(120) Ishikawa, Y.; Yamashita, A.; Uno, T. Efficient photocleavage of

DNA by cationic porphyrin-acridine hybrids with the effective length

of diamino alkyl linkage. Chem. Pharm. Bull. 2001, 49, 287−293.

(121) Shi, M.; Shen, Y.-M. Transition-metal-catalyzed reactions of

propargylamine with carbon dioxide and carbon disulfide. J. Org.

Chem. 2002, 67, 16−21.

(122) Yang, Y.; Wang, J.; Kayser, M. Approaches to the synthesis of

enantiopure α -hydroxy-β -lactams with functionalized side chains.

Tetrahedron: Asymmetry 2007, 18, 2021−2029.

(123) Silva, A. M.; Cordeiro-da-Silva, A.; Coombs, G. H. Metabolic

variation during development in culture of Leishmania donovani

promastigotes. PLoS Neglected Trop. Dis. 2011, 5, No. e1451.

(124) Ahmad, B.; Islam, A.; Khan, A.; Khan, M. A.; ul Haq, I.; Jafri,

L.; Ahmad, M.; Mehwish, S.; Khan, A.; Ullah, N. Comprehensive

investigations on anti-leishmanial potentials of Euphorbia wallichii

root extract and its effects on membrane permeability and apoptosis.

Comp. Immunol., Microbiol. Infect. Dis. 2019, 64, 138−145.

(125) Stroppa, P. H. F.; Antinarelli, L. M. R.; Carmo, A. M. L.;

Gameiro, J.; Coimbra, E. S.; da Silva, A. D. Effect of 1,2,3-triazole

salts, non-classical bioisosteres of miltefosine, on Leishmania

amazonensis . Bioorg. Med. Chem. 2017, 25, 3034−3045.

(103) De Sarkar, S.; Sarkar, D.; Sarkar, A.; Dighal, A.; Chakrabarti,

S.; Staniek, K.; Gille, L.; Chatterjee, M. The leishmanicidal activity of

artemisinin is mediated by cleavage of the endoperoxide bridge and

mitochondrial dysfunction. Parasitology 2019, 146, 511−520.

(104) Geroldinger, G.; Tonner, M.; Hettegger, H.; Bacher, M.;

Monzote, L.; Walter, M.; Staniek, K.; Rosenau, T.; Gille, L.

Mechanism of ascaridole activation in Leishmania . Biochem.

Pharmacol. 2017, 132, 48−62.

̈

(105) Rideau, E.; Masing, F.; Fletcher, S. P. Asymmetric conjugate

addition of alkylzirconocenes to cyclopent-4-ene-1,3-dione mono-

acetals. Synthesis 2015, 47, 2217−2222.

(106) Hong, R.; Chen, Y.; Deng, L. Catalytic enantioselective total

syntheses of bisorbicillinolide, bisorbicillinol, and bisorbibutenolide.

Angew. Chem. 2005, 44, 3478−3481.

(107) The ketalization involving the solvent is probably favored by

acidic traces present in the catalyst. See for example: Prasad, K. R.;

Anbarasan, P. Enantioselective synthesis of α -benzyloxy-ω -alkenals:

application to the synthesis of (+)-exo -brevicomin, (+)-iso-exo -

S

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Evaluation of the Pharmacophoric Role of the O-O Bond in Synthetic Antileishmanial Compounds: Comparison between 1,2-Dioxanes and Tetrahydropyrans

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