Literature DB >> 33091297

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

Margherita Ortalli¹, Stefania Varani^1,2, Giorgia Cimato¹, Ruben Veronesi³, Arianna Quintavalla^3,4, Marco Lombardo^3,4, Magda Monari³, Claudio Trombini^3,4.

Abstract

Leishmanian>ses 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 corresponding 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.

Entities: CellLine Chemical Disease Gene Species

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Year: 2020 PMID： 33091297 PMCID： PMC8018184 DOI： 10.1021/acs.jmedchem.0c01589

Source DB: PubMed Journal: J Med Chem ISSN： 0022-2623 Impact factor: 7.446

Introduction

Leishmania protozoan> are endemic in most tropical and subtropical areas worldwide and in the Mediterranean Europe.[1−4] Due to global warming and climate change, these parasites and their sand fly vectors are at risk to spread into countries previously considered nonendemic, including central and western Europe.[5−10] The 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.[11] Collectively, 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.[12] Currently, the pharmacological treatment for human leishmaniasis is based on few drugs[13] (Figure ), 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]

Figure 1

Drugs currently employed for the treatment of human leishmaniases (the reported IC50 values are relative to bioassays on promastigotes of Leishmania donovani strains).

Drugs currently employed for the treatment of humann> leishmaniases (the reported IC50 values are relative to bioassays on promastigotes of Leishmania donovani strains). The early efforts to overcome these limitations were directed to the improvement of the drug delivery systems[27,28] and the formulation of more effective combination therapies.[29] However, the cost increase[30−32] an class="Chemical">nd the rapid recurrence of resistance phenomena[33−37] prompted the scientific community to turn its attention toward the development of new antileishmanial drugs,[38−51] which should be effective, safe, 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 fexinidazole[40] (Figure ); (ii) diversity-oriented screening of collections of chemicals, such as oxaborole AN-4169(39,52) and aminopyrazole amide 1(39) (Figure ); (iii) phenotypic screening, leading to the identification of new biological targets and effective inhibitors,[53−56] such as 17-AAG (HSP90 inhibitor)[57] (Figure ); and (iv) use of known or new natural products with limited side effects,[58−61] especially those deriving from plants, such as fucoidan[58] and 11,13-dehydrocompressanolide[61] (Figure ).

Figure 2

Some potential new leads for the treatment of leishmaniasis.

Some potential new leads for the treatment of leishmanian class="Chemical">sis. Among the plant-derived antiparasitic compoun class="Chemical">nds, some natural endoperoxides have shown a certain antileishmanial activity.[62−65] In particular, artemisinin and some of its semisynthetic derivatives (Figure ) have shown remarkable biological activities,[66,67] especially as antimalarials; these compounds are currently recommended by World Health Organization as first-line treatment for Plasmodium falciparum malaria.[68,69] Artemisinin 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 ) is comparable to or higher than those of some currently employed drugs (amphotericin B and miltefosine).[66,67] Moreover, artemisinin-derived endoperoxides are able to inhibit the parasite metabolism with limited adverse effects on the host cells.[66,77]

Figure 3

Antileishmanial activity of some recently studied natural and synthetic cyclic peroxides (IC50 values are relative to bioassays on promastigotes for all compounds, except for N-251, where the value is relative to amastigotes).[78]

Antileishmanian>l activity of some recently studied natural and synthetic cyclic peroxides (IC50 values are relative to bioassays on promastigotes for all compounds, except for N-251, where the value is relative to amastigotes).[78] One of the limits to the development of drugs based on natural products is the low availability of the desired compoun class="Chemical">nd 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 recently proposed. In 2015, Cortes and co-workers reported on the fully synthetic trioxolanes LC50 and LC95 (Figure ), showing a promising bioactivity when tested in vitro against promastigotes (IC50 range, 3.5–9.4 μM) and intracellular amastigotes (IC50 range, 79.8–107.9 μM) of L. infantum.[79] In 2019, Iwanaga et al. described the oral activity of the fully synthetic tetraoxaspiro N-251 (Figure ) against amastigotes of L. donovani (IC50 = 6.7 μM).[80] We recently proposed new structurally simple <n class="Gene">span class="Chemical">3-methoxy-1,2-dioxanes 2 (Figure ), obtained through an efficient and cheap synthetic approach.[81−87] The simple and flexible protocol developed for their synthesis allowed us to collect a small library of n class="Chemical">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 (IC50 range, 4.0–16.3 μM) and low toxicity (selectivity index range, 12.2–35).[87] The 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 antileishmanial endoperoxides (Figure ): (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 stereochemistry 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.

Figure 4

Previously identified pharmacophoric requirements of 3-methoxy-1,2-dioxanes 2 as antileishmanial agents[87] and aim of the present work.

Previously identified pharmacophoric requirements of 3-methoxy-1,2-dioxanesn> 2 as antileishmanial agents[87] and aim of the present work. In the present study, we further investigate the structure–activity relationship of our synthetic 1,2-dioxanesn>, in order to establish whether the peroxide group is a crucial pharmacophoric requirement for the antileishmanial bioactivity (Figure ). 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 reductive decomposition of the peroxide function generates radical oxygen species, which trigger alkylation processes, leading to the parasite death.[88−100] A 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.[102,103] Gille 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.[104] Accordingly, 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 ); (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.

Results

Synthesis of Tetrahydropyrans 3

Our retrosn>ynthetic analysis for the construction of tetrahydropyrans 3 is sketched in Scheme . 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 Knöevenagel condensation, starting from the protected 2,2-disubstituted 3-hydroxypropanal 7.

Scheme 1

Retrosynthetic Analysis of Tetrahydropyrans 3 (PG = Protective Group)

Considering the good anti<n class="Gene">span class="Disease">leishmanial properties of C6-diphenyl and C6-dibutyl <ne">span>n class="Chemical">endoperoxides bearing the aminopropyl imidazole side chain on C4 (2b and 2a, respectively; Table ),[87] we 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 ), to confirm the pharmacophoric requirements of the lipophilic side chains on C6. The synthesis of 3b (Scheme ) started with the commercially available diphenylacetaldehyde, which was converted to the desired 2,2-disubstituted propanediol 4b using a reported literature procedure.[105] The protected aldehyde 7b was thus obtained exploiting a sequence of 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).

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)

Compounds tested as racemates.

IC50 represents the concentration of a compound that causes 50% growth inhibition. Results represent the 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 IC50 with a less nutrient- and antioxidant-rich environment (see the Supporting Information, Table S2).

CC50 represents 50% cytotoxic concentration on Vero cells. Results 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.

Scheme 2

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

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.

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

Reagents and con class="Chemical">nditions: (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. Compounds tested as racemates. IC50 represents the concentration of a compound that causes 50% growth inhibition. Results represent the mean (± stan class="Chemical">ndard deviation, SD) of three independent experiments performed in duplicate. Additional susceptibility tests for selected compounds were performed on L. donovani cultures un class="Chemical">sing 10% FBS in the culture medium, in order to compare the variation of IC50 with a less nutrient- and antioxidant-rich environment (see the Supporting Information, Table S2). CC50 represents 50% cytotoxic concentration on Vero cells. Results represent the mean (± standard deviation, SD) of three independent experiments performed in duplicate. Selectivity index (n class="Chemical">SI) = CC50/IC50; nd = not determined due to the low antileishmanial potency. The β-ketoestern> moiety was inserted on the intermediate aldehyde 7bvia a Knöevenagel 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.[106] Despite 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 ). Unexpectedly, 2-methoxy tetrahydropyran 9b (Scheme ) was directly obtained in good yield after the hydrogenation step, proving that four different transformations took place in a one-pot fashion: O-debenzylation, double bond reduction, cyclization, and methyl ketal formation.[107] Moreover, 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 , see the Supporting Information for details).[108]

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).

Determination of the relative stereochemistry of 2-methoxy tetrahydropyrann> 9b through X-ray crystallographic analysis (thermal ellipsoids are drawn at 30% of the probability level). At last, the replacement of the estern> group on C3 with the aminopropyl imidazole side chain was accomplished following the protocol developed for the corresponding endoperoxides 2,[87] consisting in the reduction to an alcohol, followed by oxidation to the corresponding aldehyde and reductive amination (steps f–i, Scheme ). The synthetic approach proposed for tetrahydropyrann> 3b was applied also to the construction of 3a and 3c (Scheme ). 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 Knöevenagel 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 ), the dialkyl-substituted olefins 8a and 8c were obtained as mixtures of two isomers (8a: Z/E = 65:35, 8c: Z/E = 60:40).[109] To investigate the impact of the double bond geometry on the outcome of the following one-pot transformation, providing the tetrahydropyran system 9, we carried out the reaction on the two isomers Z-8a and E-8a, separately (Scheme ). Invariably, we obtained only the 2,3-cis tetrahydropyran 9a from both olefins, demonstrating not only the cis-stereoconvergence[110] proper of the process but also the comparable reaction rates for the two isomers. On this basis, the mixtures of Z and E isomers were directly used without separation.

Scheme 3

Reagents and Conditions: Pd/C (20% w/w), H2 (filled balloon), MeOH, rt, and Overnight

The subsequent usual manipulations of the C3 side chain provided the den class="Chemical">sired aminopropyl imidazole tetrahydropyrans 3a and 3c (Scheme ). Finally, the intermediate alcohol 10bn> (Scheme ) was used as the starting material for the synthesis of the tetrahydropyran 3d, analogue of the highly active antileishmanial endoperoxide 2d (Table ). 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 ).[87]

Scheme 4

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

In Vitro Growth Inhibition of L. donovani Promastigotes and Amastigotes

The bioactivity of the new tetrahydropyransn> 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 Vero cells (Table ) 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 corresponding endoperoxide 2 (Table ). We observed a similar bioactivity profile for <n class="Gene">span class="Chemical">endoperoxides 2 and the corresponding tetrahydropyrans 3; the aminopropyl imidazole derivatives 3a/2a and 3b/2b showed IC50 in the low micromolar range (Table ). 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 ).

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)

Compounds tested as racemates.

IC50 represents the concentration of a compound that causes 50% growth inhibition. Results represent the mean (± standard deviation, SD) of three independent experiments performed in duplicate.

CC50 represents 50% cytotoxic concentration on Vero cells. Results represent the mean (± standard deviation, SD) of three independent experiments performed in duplicate. Additional cytotoxicity tests were performed employing the THP-1 cell line (see the Supporting Information, Table S3).

Selectivity index (SI) = CC50/IC50.

Compounds tested as racemates. IC50 represents the concentration of a compound that causes 50% growth inhibition. Results represent the mean (± stan class="Chemical">ndard deviation, SD) of three independent experiments performed in duplicate. CC50 represents 50% cytotoxic concentration on Vero cells. Results represent the mean (± standard deviation, SD) of three in class="Chemical">ndependent experiments performed in duplicate. Additional cytotoxicity tests were performed employing the THP-1 cell line (see the Supporting Information, Table S3). Selectivity index (n class="Chemical">SI) = CC50/IC50. The good antileishmanian>l 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 and 2). In particular, tetrahydropyransn> 3 revealed to be slightly more active against amastigotes than the corresponding endoperoxides 2.

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 en class="Chemical">ndoperoxides on <ne">span>n class="Disease">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 .

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

Compounds tested as racemates.

IC50 represents 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.

Compounds tested as racemates. IC50 represents the concentration of a compound that causes 50% growth inhibition. Results represent the mean (± stan class="Chemical">ndard 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. At first, we determined IC50 and CC50 values of the <n class="Gene">span class="Chemical">iron chelator DFO (187 and >400 μM, respectively). Then, the effect of the <ne">span>n class="Chemical">iron chelator presence on the bioactivity of the two compounds was studied by coincubating 2a and 3a with DFO at different concentrations. The variation of IC50 of the compounds in relation to the different doses of DFO was determined by the alamarBlue assay (Table ), and the isobole technique was employed to depict the interactions of 2a and 3a with DFO (Figure ).

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 = IC50 of the drug in the combination/IC50 of the drug when tested alone). Y axes depict the fractional of DFO. Square in the figure indicates ΣFIC values from each drug combination.

(A, B) Isobolograms depicting the interaction of (A) tetrahydropyrann> 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 = IC50 of the drug in the combination/IC50 of the drug when tested alone). Y axes depict the fractional of DFO. Square in the figure indicates ΣFIC values from each drug combination. As shown in Table , the inhibitory activity of 3a and 2a against <n class="Gene">span class="Disease">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 ). 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 , 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 ironn> 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 <n class="Gene">span class="Chemical">reactive oxygen species (<ne">span>n class="Chemical">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).[103] Therefore, the resultant fluorescence is a direct measure of the amount of ROS generated. As shown in Figure , en class="Chemical">ndoperoxide 2a and <ne">span>n class="Chemical">tetrahydropyran 3a were both capable of inducing an increase in the intracellular ROS levels at their IC50 as 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 H2O2 and 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 , 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 control H2O2 (Figure ). 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 (IC50 3.4 μM) as compared to 2a (IC50, 7.5 μM).

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-dichlorodihydrofluorescein 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.

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.

Measurement of ROSn> 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-dichlorodihydrofluorescein 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. Quantification of ROSn> 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.

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 <n class="Gene">span class="Species">L. donovani promastigote cell and on their biodistribution within the parasite, we accomplished a confocal mic<ne">span>n class="Chemical">roscopy investigation. For this purpose, we synthesized new compounds carrying a fluorescent probe (Schemes and 6). In particular, the most active butyl derivatives were labeled with an acridine-based fluorescent dye (2e and 3e, Scheme ), 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 3e were moderate due to their poor solubility in organic solvents, which made the purification difficult.

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, and 1 h (see Experimental Section for Details)

For the synthesis of 13, see ref (87).

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

For the synthesis of 13, see ref (87).

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, and 1 h (see Experimental Section for Details)

For the synthesis of 13, see ref (87).

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

For the synthesis of 13, see ref (87). We were aware that the fluorescence of acridinen>-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.[113] To validate the biodistribution data obtained from acridine-containing compounds 2e and 3e, we synthesized also the endoperoxide 2f (Scheme ) bearing the nitrobenzyldiazole (NBD) fluorophore, to be used as a comparison since it does not have the abovementioned limitations. The construction of 2f (Scheme ) was difficult as the nitrobenzyldiazole 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 ). 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 <n class="Gene">span class="Species">L. donovani and the corresponding n class="Disease">cytotoxicity on Vero cells was also assayed (Table ). 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 ). 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 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)

Compounds tested as racemates.

IC50 represents the concentration of a compound that causes 50% growth inhibition.

CC50 represents 50% cytotoxic concentration on Vero cells.

Selectivity index (SI) = CC50/IC50.

Compounds tested as racemates. IC50 represents the concentration of a compound that causes 50% growth inhibition. CC50 represents 50% cytotoxic concentration on Vero cells. Selectivity index (n class="Chemical">SI) = CC50/IC50. The n class="Chemical">NBD derivative 2f showed lower activity against L. donovani promastigotes than the corresponding <ne">span>n class="Chemical">acridin-derivative 2e (Table ), and its IC50 value was similar to that of the corresponding imidazole-containing compound 2a (Table ). However, its SI was low, confirming a significant toxicity of these fluorescent products on Vero cells. The labeled compounds were incubated with <n class="Gene">span class="Species">L. donovani promastigotes, and then confocal mic<ne">span>n class="Chemical">roscopy images were acquired (see Experimental Section for details). Figure 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 A), likely corresponding to the nucleus. To confirm this interpretation, we marked the nucleus and kinetoplast of the parasite with TO-PRO-3 (Figure C), a specific dye for nucleic acids.[114,115] The overlapped color image (Figure B) clearly shows the dark blue nucleus (red arrows, Figure ) and the cyan kinetoplast (yellow arrows, Figure 9), whose color derived from a colocalization of green and blue fluorescence. This finding suggested that endoperoxide 2f spread throughout the parasite cytoplasm and it was able to enter the kinetoplast but not the nucleus.

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.

Cell localization of labeled en class="Chemical">ndoperoxide 2f and <ne">span>n class="Chemical">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. A similar intraparan class="Chemical">sitic distribution was observed for acridine-labeled tetrahydropyran 3e by confocal microscopy (Figure ).

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.

Cell localization of labeled tetrahydropyran n class="Chemical">3e and <ne">span>n class="Chemical">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. Concerning acridinen>-labeled endoperoxide 2e, we observed a colocalization of green and blue fluorescence not only in the kinetoplast but also in the nucleus (Figure B). This finding suggests that this peculiar acridine-containing compound can enter the nucleus core.

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.

Cell localization of labeled en class="Chemical">ndoperoxide 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.

Discussion and Conclusions

Little is known on the mechanism of action of en class="Chemical">ndoperoxides against <ne">span>n class="Disease">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.[102−104] Based 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 ) and tetrahydropyrans 3 revealed to be slightly more active against intracellular amastigotes than the corresponding endoperoxides 2 (Table ). 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,[102] while 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] Ironn> is an essential nutrient for all the organisms, including Leishmania, and its supply plays a crucial role in the parasite survival.[117,118] However, 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 ) and that they have a comparable activity against both promastigote and amastigote forms of L. donovani (Tables and 2). To further investigate the role of the peroxiden> 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. donovanin> 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 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 IC50 value (Table ) 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 synthen class="Chemical">sized a small library of tetrahydropyrans 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 endoperoxides in our biological system. Additional studies are needed to corroborate our findings.

Experimental Section

Chemistry

General Information

All the commercial chemicals were purchased from Sigma-Aldrich, VWR, Alfa Aesar, or TCI Chemicals an class="Chemical">nd used without additional purifications. The 1H and 13C 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. 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, an class="Chemical">nd 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. En class="Chemical">ndoperoxides 2a–2d are known, and they were prepared according to our previous work.[87] Compound 4b is known, and it was synthesized according to the literature procedure.[119] Its physical and spect<ne">span>n class="Chemical">roscopic 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.[120] Its physical and spectroscopic data matched the reported ones. Benzaldehyde diethyl acetal is known, and it was prepared according to the literature procedure.[121] After being synthesized, it was used in the following reaction without purification.

Synthetic Procedures and Compound Characterizations

Synthesis of 2-Phenyl-5,5-disubstituted-1,3-dioxanes (5)

Compounds 5 were prepared according to the literature procedure[122] modified as follows. (1S)-(+)-<n class="Gene">span class="Chemical">Camphorsulfonic acid (CSA, 0.01 equiv) was added at 0 °C to a solution of the starting <ne">span>n class="Chemical">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: CyHn>/Et2O, 9:1. 1H NMR (400 MHz, CDCl3): δ 7.49 (dd, J = 7.9, 1.8 Hz, 2H), 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 Cn class="Chemical">H2Cl2. Melting point, 97–101 °C. 1H NMR (400 MHz, <ne">span>n class="Chemical">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 compound is known, an class="Chemical">nd its physical and spectroscopic data matched the reported ones.[122]

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

Compounds 6 were synthen class="Chemical">sized from the corresponding starting materials 5 according to the literature procedure.[119]

2-((Benzyloxy)methyl)-2-butylhexan-1-ol (6a)

Yield: 99%. Colorless liquid. Mobile phase for the chromatographic purification: CyHn>/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: CyHn>/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, 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, an class="Chemical">nd its physical and spectroscopic data matched the reported ones.[122]

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

Compounds 7 were synthen class="Chemical">sized 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: CyHn>/Et2O, 9:1. 1H 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: CyHn>/AcOEt, 95:5. 1H 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, an class="Chemical">nd its physical and spectroscopic data matched the reported ones.[122]

Synthesis of the Knöevenagel Adducts (8)

Compounds 8 were synthen class="Chemical">sized according to the literature procedure[106] from 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 phase for the chromatographic purification: <n class="Gene">span class="Chemical">CyH/<ne">span>n class="Chemical">AcOEt, 95:5. 1H 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, 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 phase for the chromatographic purification: <n class="Gene">span class="Chemical">CyH/<ne">span>n class="Chemical">AcOEt, 95:5. 1H 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 oiln>. Mobile phase for the chromatographic purification: CyH/AcOEt, 9:1. 1H 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%. <n class="Gene">span class="Chemical">Yellowish oil. Mobile phase for the chromatographic purification: <ne">span>n class="Chemical">CyH/AcOEt, 95:5. 1H 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%. <n class="Gene">span class="Chemical">Yellowish oil. Mobile phase for the chromatographic purification: <ne">span>n class="Chemical">CyH/AcOEt, 95:5. 1H NMR (400 MHz, CDCl3): δ 7.36–7.26 (m, 5H), 6.82 (s, 1H), 4.51 (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% n class="Chemical">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 H2 atmosphere (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 oiln>. 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: CyHn>/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]+.

Methyl 2-Methoxy-2,5,5-trimethyltetrahydro-2H-pyran-3-carboxylate (9c)

Yield: 63%. Colorless oiln>. 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]+.

Synthesis of 3-Hydroxymethyltetrahydropyrans (10)

Compounds 10 were synthen class="Chemical">sized 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 oiln>. 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: CyHn>/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]+.

(2-Methoxy-2,5,5-trimethyltetrahydro-2H-pyran-3-yl)methanol (10c)

Yield: 47%. Colorless oiln>. Mobile phase for the chromatographic 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, 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)

Compounds 11 were synthen class="Chemical">sized from the corresponding starting materials 10 according to our previously reported procedure,[87] and they were immediately used in the following step.

Synthesis of 3-Aminopropyltetrahydropyrans (3)

Compounds 3 were synthen class="Chemical">sized 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 oiln>. Mobile phase for the chromatographic purification: AcOEt/MeOH, 6:4. 1H 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-diphenyltetrahydro-2H-pyran-3-yl)methyl)propan-1-amine (3b)

Yield: 85%. White waxy solid. Mobile phase for the chromatographic purification: AcOEtn>/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.

3-(1H-Imidazol-1-yl)-N-((2-methoxy-2,5,5-trimethyltetrahydro-2H-pyran-3-yl)methyl)propan-1-amine (3c)

Yield: 99%. Colorless oiln>. Mobile phase for the chromatographic purification: CH2Cl2/MeOH/NH3, 9:1:0.1. 1H 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 Tetrahydropyran-3-propargyl Ether (12)

Compound 12 was synthen class="Chemical">sized 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: CyHn>/AcOEt, 95:5 to 8:2. 1H 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 synthen class="Chemical">sized from the corresponding starting material 12 according to our previously reported procedure.[87]

(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: Cn class="Chemical">H2Cl2/<ne">span>n class="Chemical">MeOH, 9:1. 1H 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 n class="Chemical">2e and 3e were synthesized from the corresponding starting materials (13[87] 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-methoxy-3-methyl-1,2-dioxan-4-yl)methyl)butane-1,4-diamine (2e)

Yield: 45%. Yellow waxy solid. Mobile phase for the chromatographic purification: Cn class="Chemical">H2Cl2/<ne">span>n class="Chemical">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-methoxy-2-methyltetrahydro-2H-pyran-3-yl)methyl)butane-1,4-diamine (3e)

Yield: 32%. Yellow waxy solid. Mobile phase for the chromatographic purification: Cn class="Chemical">H2Cl2/<ne">span>n class="Chemical">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 13(87) an class="Chemical">nd 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: Cn class="Chemical">H2Cl2/<ne">span>n class="Chemical">MeOH, 9:1. 1H 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 correspon class="Chemical">nding N-deprotected hydrochloride salt according to a literature procedure,[100] and it was immediately used in the following synthetic step without any purification. Compound 2f was prepared according to a literature procedure[100] modified as follows: <n class="Gene">span class="Chemical">4-chloro-7-nitrobenzofurazan (1.1 equiv) was added to a solution of the <ne">span>n class="Chemical">hydrochloride salt and triethylamine (3 equiv) in anhydrous CH2Cl2 (30 mL mmol–1) at 0 °C under a N2 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: Cn class="Chemical">H2Cl2/<ne">span>n class="Chemical">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. donovanin> 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 composition: 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)[123] and 1% penicillin–streptomycin (EuroClone SpA).

Cell Cultures

Vero cells (kidney of African green monkeyn> 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 <n class="Gene">span class="Chemical">HOMEM medium at 106 cells mL–1 in 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 anti<ne">span>n class="Disease">leishmanial 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 cytotoxicityn> 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 (105 cells 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 (SI) for each compound was calculated as the ratio between cytotoxicity (CC50/72 h) and activity (IC50/72 h) against Leishmania promastigotes.

Amastigote Growth Inhibition Assay

To evaluate the efficacy of the compounds on the intracellular form of L. donovani, <n class="Gene">span class="Species">human acute monocytic <ne">span>n class="Disease">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% CO2 incubator. 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% CO2 incubator 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.

Role of an Iron Chelator in the Activation of Compounds[111,112]

To analyze the effect of an ironn> chelator (DFO or DFP) on the compounds, the late log/stationary phase of promastigotes was seeded with the complete HOMEM medium at 106 cells mL–1 in 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 IC50 of 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 = IC50 of the drug in the combination/IC50 of the drug when tested alone) of each compound was calculated and plotted as an isobologram.

Evaluation of Intracellular ROS Levels

To monitor the effect of the compounds on the production of <n class="Gene">span class="Chemical">ROS in promastigotes of <ne">span>n class="Species">L. donovani, an oxidant-sensitive green fluorescent dye (2,7-dichlorofluorescein diacetate, H2DCFDA) was used. Promastigotes in their late log/stationary phase were seeded with the complete HOMEM medium at 106 cells mL–1 in 96-well plates and incubated with tested compounds at their IC50 in 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.[103] H2O2 (20 μM)[124] and miltefosine (22 μM)[125] were 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. donovanin> were seeded (106 cells mL–1) on 96-well plates at 26 °C for 72 h. Then, the labeled compounds, at their IC50 concentrations, 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,[114,115] was 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.

101 in total

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Review 2. Chemotherapy of leishmaniasis: present challenges.

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Journal: Mol Biosyst Date: 2017-05-02

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Journal: Biomed Pharmacother Date: 2019-05-30 Impact factor: 6.529

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Authors: Paula Baréa; Valéria Aquilino Barbosa; Danielle Lazarin Bidóia; Jéssica Carreira de Paula; Talitha Fernandes Stefanello; Willian Ferreira da Costa; Celso Vataru Nakamura; Maria Helena Sarragiotto
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6. Vasa expression in spermatogenic cells during the reproductive-cycle phases of Podarcis sicula (Reptilia, Lacertidae).

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Journal: J Exp Zool B Mol Dev Evol Date: 2015-05-06 Impact factor: 2.656

7. Triclosan-caffeic acid hybrids: Synthesis, leishmanicidal, trypanocidal and cytotoxic activities.

Authors: Elver Otero; Elisa García; Genesis Palacios; Lina M Yepes; Miguel Carda; Raúl Agut; Iván D Vélez; Wilson I Cardona; Sara M Robledo
Journal: Eur J Med Chem Date: 2017-09-29 Impact factor: 6.514

Review 8. Leishmaniasis: a review.

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Journal: F1000Res Date: 2017-05-26

9. 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.

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Journal: Front Cell Infect Microbiol Date: 2018-08-30 Impact factor: 5.293

10. Miltefosine-Lopinavir Combination Therapy Against Leishmania infantum Infection: In vitro and in vivo Approaches.

Authors: Karina M Rebello; Valter V Andrade-Neto; Claudia Regina B Gomes; Marcos Vinícius N de Souza; Marta H Branquinha; André L S Santos; Eduardo Caio Torres-Santos; Claudia M d'Avila-Levy
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1. Influence of N-Methylation and Conformation on Almiramide Anti-Leishmanial Activity.

Authors: Anh Minh Thao Nguyen; Skye Brettell; Noélie Douanne; Claudia Duquette; Audrey Corbeil; Emanuella F Fajardo; Martin Olivier; Christopher Fernandez-Prada; William D Lubell
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1 in total