Synthesis of Polycyclic Ether-Benzopyrans and In Vitro Inhibitory Activity against Leishmania tarentolae

Construction of a focused library of polycyclic ether-benzopyrans was undertaken in order to discover new therapeutic compounds that affect Leishmania growth and infectivity. This is especially of interest since there are few drug therapies for leishmaniasis that do not have serious drawbacks such high cost, side effects, and emerging drug resistance. The construction of these polycyclic ether-benzopyrans utilized an acetoxypyranone-alkene [5+2] cycloaddition and the Suzuki-Miyaura cross-coupling. The multi-gram quantity of the requisite aryl bromide was obtained followed by effective Pd-catalyzed coupling with boronic acid derivatives. Compounds were tested in vitro using the parasitic protozoan, Leishmania tarentolae. Effects of concentration, time, and exposure to light were evaluated. In addition, the effects on secreted acid phosphatase activity and nitric oxide production were investigated, since both have been implicated in parasite infectivity. The data presented herein are indicative of disruption of the Leishmania tarentolae and thus provide impetus for the development and testing of a more extensive library.


Introduction
According to the Centers for Disease Control and Prevention (CDC), leishmaniasis is a neglected tropical disease caused by parasitic protozoans in the genus Leishmania [1] that affects humans and animals in nearly 100 countries [2] with cases reported on every continent except Australia and Antarctica [3]. The exact number of leishmaniasis cases is not known but is reported to be between 1.5 and 2.0 million new cases per year with more than 350 million people at risk worldwide [4]. Although less common in the United States than many other parts of the western hemisphere [5], cases of cutaneous leishmaniasis have been documented in Oklahoma and Texas [6,7], raising concerns that this neglected disease will become a US public health issue sooner rather than later [8]. Leishmaniasis is caused by 20 different species of Leishmania and is spread by the bite of approximately 30 different species of phlebotomine sandflies [2]. Leishmaniasis is presented clinically in three different forms: cutaneous, visceral, or mucosal [2,9]. Although several treatments exist, they are not without liabilities [10,11]. For example, amphotericin B is cost-prohibitive, antimony compounds are toxic, and both therapies have significant side-effects. There are three main modes of therapy, including systemic parenteral, systemic oral, and local (i.e., topical application). Examples of systemic parenteral therapies include various drug formulations of pentavalent antimony (Sb +5 ) compounds. Systemic oral therapy may include the phosphocholine derivative miltefosine or a triazole-containing small molecule such as fluconazole. Examples of local therapies range from cryotherapy with liquid nitrogen to thermotherapy with localized current field radiofrequency heat, in addition to the intralesional administration of antimony-based compounds or topical application of paromomycin [12]. These treatments can be expensive and may result in harsh side effects with reports of drug resistance becoming a serious problem, so there is a critical need for new drugs to treat this neglected tropical disease [1,2]. Recently, a polycyclic ether-containing natural product, englerin A, was reported to be toxic in renal cancer cells and has become a very popular synthetic target (Figure 1) [13][14][15][16]. One hypothesis regarding the mechanism of action is a complex array of metabolic pathways involving PKCθ and HSF1, leading to an inhibition of glucose transporter (Glut1) activity [17]. In addition, Feng et al. reported that glucose transporters are critical for Leishmania mexicana viability and infectivity [18]. Furthermore, benzopyrans [19][20][21] are classified as privileged structures toward medicinal chemistry efforts [22] and benzopyran-chalcones have been shown to inhibit Leishmania major [23,24]. Thus, we hypothesized that hybrid oxabicyclo[3.2.1]-octane-benzopyrans 1 offered a unique scaffold to pursue inhibitory activity against the protozoan parasite Leishmania tarentolae [25]. This strain of Leishmania is generally regarded as non-pathogenic to humans, and it therefore serves as a model system in vitro [26]. We observed that these compounds negatively affect parasite growth and viability in vitro with mechanistic targets such as nitric oxide production and secreted acid phosphatase activity, both of which are proposed to be involved in parasite-host interactions [27,28]. We also included tests against mammalian glial cells, in culture, to evaluate toxicity for another cell type.

Proposed Suzuki-Miyaura Pathway toward Construction of Ether-Benzopyran Analogues
Toward construction of a library of polycyclic ether-benzopyran analogues 1, we envisioned the synthesis of aryl bromide 6 that would be suitable for Suzuki cross-coupling [89,90] with biologically relevant [91] boronic acid derivatives 5 (Scheme 2). The alcohol 6 would be accessible via Luche reduction [92] of enone 7, which would arise from the key [5+2] cycloaddition. The cycloadduct 7 would be accessible by the aforementioned sequence (cf. Scheme 1) utilizing aryl bromide 8, which is readily accessible upon the allylation of commercially available 5-bromo-salicyladehyde (not shown).

Construction of the Suzuki-Miyaura Aryl Bromide Precursor
En route to cross-coupling partner 6, commercially available 5-bromosalicylaldehyde 9 was heated in acetonitrile with allyl bromide and K2CO3 to give allyl ether 8 in 97% yield (Scheme 3). The treatment of furan with nBuLi in THF provided the 2-furyllithium, which underwent addition to aldehyde 8 to afford the alcohol 10. Next, mCBPA-mediated Achmatowicz rearrangement [85][86][87][88] delivered the hydroxypyranone (not shown), which was acylated to give the acetoxypyranone 11 in 71% yield over two steps. NMP was utilized to promote the [5+2] cycloaddition [83] to afford the ketone 7 in 95% yield as a single diastereomer. Luche reduction [92] gave the allylic alcohol 6, which was included to provide a hydrogen-bond donor and remove the reactive enone functionality [93].

Proposed Suzuki-Miyaura Pathway toward Construction of Ether-Benzopyran Analogues
Toward construction of a library of polycyclic ether-benzopyran analogues 1, we envisioned the synthesis of aryl bromide 6 that would be suitable for Suzuki cross-coupling [89,90] with biologically relevant [91] boronic acid derivatives 5 (Scheme 2). The alcohol 6 would be accessible via Luche reduction [92] of enone 7, which would arise from the key [5+2] cycloaddition. The cycloadduct 7 would be accessible by the aforementioned sequence (cf. Scheme 1) utilizing aryl bromide 8, which is readily accessible upon the allylation of commercially available 5-bromo-salicyladehyde (not shown).

Proposed Suzuki-Miyaura Pathway toward Construction of Ether-Benzopyran Analogues
Toward construction of a library of polycyclic ether-benzopyran analogues 1, we envisioned the synthesis of aryl bromide 6 that would be suitable for Suzuki cross-coupling [89,90] with biologically relevant [91] boronic acid derivatives 5 (Scheme 2). The alcohol 6 would be accessible via Luche reduction [92] of enone 7, which would arise from the key [5+2] cycloaddition. The cycloadduct 7 would be accessible by the aforementioned sequence (cf. Scheme 1) utilizing aryl bromide 8, which is readily accessible upon the allylation of commercially available 5-bromo-salicyladehyde (not shown). Scheme 2. Proposed Suzuki-Miyaura pathway for the construction of ether-benzopyran analogues 1.

Construction of the Suzuki-Miyaura Aryl Bromide Precursor
En route to cross-coupling partner 6, commercially available 5-bromosalicylaldehyde 9 was heated in acetonitrile with allyl bromide and K2CO3 to give allyl ether 8 in 97% yield (Scheme 3). The treatment of furan with nBuLi in THF provided the 2-furyllithium, which underwent addition to aldehyde 8 to afford the alcohol 10. Next, mCBPA-mediated Achmatowicz rearrangement [85][86][87][88] delivered the hydroxypyranone (not shown), which was acylated to give the acetoxypyranone 11 in 71% yield over two steps. NMP was utilized to promote the [5+2] cycloaddition [83] to afford the ketone 7 in 95% yield as a single diastereomer. Luche reduction [92] gave the allylic alcohol 6, which was included to provide a hydrogen-bond donor and remove the reactive enone functionality [93].

Construction of the Suzuki-Miyaura Aryl Bromide Precursor
En route to cross-coupling partner 6, commercially available 5-bromosalicylaldehyde 9 was heated in acetonitrile with allyl bromide and K 2 CO 3 to give allyl ether 8 in 97% yield (Scheme 3). The treatment of furan with nBuLi in THF provided the 2-furyllithium, which underwent addition to aldehyde 8 to afford the alcohol 10. Next, mCBPA-mediated Achmatowicz rearrangement [85][86][87][88] delivered the hydroxypyranone (not shown), which was acylated to give the acetoxypyranone 11 in 71% yield over two steps. NMP was utilized to promote the [5+2] cycloaddition [83] to afford the ketone 7 in 95% yield as a single diastereomer. Luche reduction [92] gave the allylic alcohol 6, which was included to provide a hydrogen-bond donor and remove the reactive enone functionality [93].

Palladium Catalyzed Suzuki-Miyaura Cross-Coupling
Suzuki-Miyaura cross-coupling [89,90] is one of the most widely used reactions to construct C-C bonds and affords an effective delivery of relevant heterocycles [91]. Aryl bromide 6 provides the necessary functional group handle to react with boronic acid derivatives 5a-e with catalytic palladium to deliver products 1a-e (Table 1). In general, Pd(Ph3)4 was utilized as the catalyst with Cs2CO3 in a mixed solvent system of toluene, DME, and water to afford the desired cross-coupling products 1a,c-e (entries 1, [3][4][5]. However, in the case of 2-fluoropyridine-4-boronic acid 5b (entry 2), Pd(dppf)Cl2 and K2CO3 in toluene:EtOH provided the desired product 1b [94]. In all cases, the starting material 6 was consumed according to 1 H NMR analyses of the crude reaction mixtures.

Palladium Catalyzed Suzuki-Miyaura Cross-Coupling
Suzuki-Miyaura cross-coupling [89,90] is one of the most widely used reactions to construct C-C bonds and affords an effective delivery of relevant heterocycles [91]. Aryl bromide 6 provides the necessary functional group handle to react with boronic acid derivatives 5a-e with catalytic palladium to deliver products 1a-e (Table 1). In general, Pd(Ph 3 ) 4 was utilized as the catalyst with Cs 2 CO 3 in a mixed solvent system of toluene, DME, and water to afford the desired cross-coupling products 1a,c-e (entries 1, [3][4][5]. However, in the case of 2-fluoropyridine-4-boronic acid 5b (entry 2), Pd(dppf)Cl 2 and K 2 CO 3 in toluene:EtOH provided the desired product 1b [94]. In all cases, the starting material 6 was consumed according to 1 H NMR analyses of the crude reaction mixtures.

Palladium Catalyzed Suzuki-Miyaura Cross-Coupling
Suzuki-Miyaura cross-coupling [89,90] is one of the most widely used reactions to construct C-C bonds and affords an effective delivery of relevant heterocycles [91]. Aryl bromide 6 provides the necessary functional group handle to react with boronic acid derivatives 5a-e with catalytic palladium to deliver products 1a-e (Table 1). In general, Pd(Ph3)4 was utilized as the catalyst with Cs2CO3 in a mixed solvent system of toluene, DME, and water to afford the desired cross-coupling products 1a,c-e (entries 1, [3][4][5]. However, in the case of 2-fluoropyridine-4-boronic acid 5b (entry 2), Pd(dppf)Cl2 and K2CO3 in toluene:EtOH provided the desired product 1b [94]. In all cases, the starting material 6 was consumed according to 1 H NMR analyses of the crude reaction mixtures.

Palladium Catalyzed Suzuki-Miyaura Cross-Coupling
Suzuki-Miyaura cross-coupling [89,90] is one of the most widely used reactions to construct C-C bonds and affords an effective delivery of relevant heterocycles [91]. Aryl bromide 6 provides the necessary functional group handle to react with boronic acid derivatives 5a-e with catalytic palladium to deliver products 1a-e (Table 1). In general, Pd(Ph3)4 was utilized as the catalyst with Cs2CO3 in a mixed solvent system of toluene, DME, and water to afford the desired cross-coupling products 1a,c-e (entries 1, [3][4][5]. However, in the case of 2-fluoropyridine-4-boronic acid 5b (entry 2), Pd(dppf)Cl2 and K2CO3 in toluene:EtOH provided the desired product 1b [94]. In all cases, the starting material 6 was consumed according to 1 H NMR analyses of the crude reaction mixtures.

Palladium Catalyzed Suzuki-Miyaura Cross-Coupling
Suzuki-Miyaura cross-coupling [89,90] is one of the most widely used reactions to construct C-C bonds and affords an effective delivery of relevant heterocycles [91]. Aryl bromide 6 provides the necessary functional group handle to react with boronic acid derivatives 5a-e with catalytic palladium to deliver products 1a-e (Table 1). In general, Pd(Ph3)4 was utilized as the catalyst with Cs2CO3 in a mixed solvent system of toluene, DME, and water to afford the desired cross-coupling products 1a,c-e (entries 1, [3][4][5]. However, in the case of 2-fluoropyridine-4-boronic acid 5b (entry 2), Pd(dppf)Cl2 and K2CO3 in toluene:EtOH provided the desired product 1b [94]. In all cases, the starting material 6 was consumed according to 1 H NMR analyses of the crude reaction mixtures.

Palladium Catalyzed Suzuki-Miyaura Cross-Coupling
Suzuki-Miyaura cross-coupling [89,90] is one of the most widely used reactions to construct C-C bonds and affords an effective delivery of relevant heterocycles [91]. Aryl bromide 6 provides the necessary functional group handle to react with boronic acid derivatives 5a-e with catalytic palladium to deliver products 1a-e (Table 1). In general, Pd(Ph3)4 was utilized as the catalyst with Cs2CO3 in a mixed solvent system of toluene, DME, and water to afford the desired cross-coupling products 1a,c-e (entries 1, [3][4][5]. However, in the case of 2-fluoropyridine-4-boronic acid 5b (entry 2), Pd(dppf)Cl2 and K2CO3 in toluene:EtOH provided the desired product 1b [94]. In all cases, the starting material 6 was consumed according to 1 H NMR analyses of the crude reaction mixtures.

Palladium Catalyzed Suzuki-Miyaura Cross-Coupling
Suzuki-Miyaura cross-coupling [89,90] is one of the most widely used reactions to construct C-C bonds and affords an effective delivery of relevant heterocycles [91]. Aryl bromide 6 provides the necessary functional group handle to react with boronic acid derivatives 5a-e with catalytic palladium to deliver products 1a-e (Table 1). In general, Pd(Ph3)4 was utilized as the catalyst with Cs2CO3 in a mixed solvent system of toluene, DME, and water to afford the desired cross-coupling products 1a,c-e (entries 1, [3][4][5]. However, in the case of 2-fluoropyridine-4-boronic acid 5b (entry 2), Pd(dppf)Cl2 and K2CO3 in toluene:EtOH provided the desired product 1b [94]. In all cases, the starting material 6 was consumed according to 1 H NMR analyses of the crude reaction mixtures.

Palladium Catalyzed Suzuki-Miyaura Cross-Coupling
Suzuki-Miyaura cross-coupling [89,90] is one of the most widely used reactions to construct C-C bonds and affords an effective delivery of relevant heterocycles [91]. Aryl bromide 6 provides the necessary functional group handle to react with boronic acid derivatives 5a-e with catalytic palladium to deliver products 1a-e (Table 1). In general, Pd(Ph3)4 was utilized as the catalyst with Cs2CO3 in a mixed solvent system of toluene, DME, and water to afford the desired cross-coupling products 1a,c-e (entries 1, [3][4][5]. However, in the case of 2-fluoropyridine-4-boronic acid 5b (entry 2), Pd(dppf)Cl2 and K2CO3 in toluene:EtOH provided the desired product 1b [94]. In all cases, the starting material 6 was consumed according to 1 H NMR analyses of the crude reaction mixtures.

Incubation Effects on L. tarentolae Growth
No major differences were observed via light microscopy (data not shown) in the morphology or motility of L. tarentolae following 96-h incubations. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay [95] was used to evaluate cell viability with and without the test compounds. Initial MTT values were essentially the same, suggesting that the same number of viable cells were present in each of the separate flasks prior to the addition of the polycyclic ether-benzopyrans. Subsequent MTT values were altered in the presence of some of the test compounds in comparison to the DMSO control ( Figure 3) and this is especially evident on day 2 post addition of the 1e compound. On day 1 post addition of compounds 1a,1c, 1d, and 1e, values were significantly different (p, 0.05) from DMSO control cells. One day 2, all additions resulted in significant differences relative to DMSO control cells. On day 3, all additions resulted in significant differences except compound 1d. On day 4, all additions resulted in significant differences except compound 1a.
It is of interest that compound 1e has an apparent early inhibitory effect on cells, relative to control cells, but that, with time, the compound is not stable, so the inhibition is relieved, and the cells recover.
Molecules 2020, 25, x 5 of 20 No major differences were observed via light microscopy (data not shown) in the morphology or motility of L. tarentolae following 96-h incubations. The 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) viability assay [95] was used to evaluate cell viability with and without the test compounds. Initial MTT values were essentially the same, suggesting that the same number of viable cells were present in each of the separate flasks prior to the addition of the polycyclic ether-benzopyrans. Subsequent MTT values were altered in the presence of some of the test compounds in comparison to the DMSO control ( Figure 3) and this is especially evident on day 2 post addition of the 1e compound. On day 1 post addition of compounds 1a,1c, 1d, and 1e, values were significantly different (p, 0.05) from DMSO control cells. One day 2, all additions resulted in significant differences relative to DMSO control cells. On day 3, all additions resulted in significant differences except compound 1d. On day 4, all additions resulted in significant differences except compound 1a. It is of interest that compound 1e has an apparent early inhibitory effect on cells, relative to control cells, but that, with time, the compound is not stable, so the inhibition is relieved, and the cells recover.

Test Compound Inhibition and Apparent Recovery by Leishmania after Transfer to New Medium
Inhibition of L. tarentolae growth was seen by several compounds (Figure 4) with the most effective inhibitory compounds being compound 1c and co-incubation of compounds 1c + 1e. Slopes obtained for the original growth period and the secondary recovery phase indicate some level of recovering capabilities after exposure to different compounds (Table 2), since a greater ratio of the slope corresponds to an increased ability to recover after compound exposure. For all days post

Test Compound Inhibition and Apparent Recovery by Leishmania after Transfer to New Medium
Inhibition of L. tarentolae growth was seen by several compounds (Figure 4) with the most effective inhibitory compounds being compound 1c and co-incubation of compounds 1c + 1e. Slopes obtained for the original growth period and the secondary recovery phase indicate some level of recovering capabilities after exposure to different compounds (Table 2), since a greater ratio of the slope corresponds to an increased ability to recover after compound exposure. For all days post addition of compounds 1a, 1c, 1d, and co-incubation with 1c + 1e, there was a statistically significant change relative to control cells (p < 0.05).
Molecules 2020, 25, x 6 of 20 addition of compounds 1a, 1c, 1d, and co-incubation with 1c + 1e, there was a statistically significant change relative to control cells (p < 0.05).  Significant differences were observed in the differential growth patterns of experimental cultures compared to DMSO control. Complexes appear to be affecting various pathways involved in cell maturation and division. Growth curve plots show severe growth inhibition in the presence of multiple compounds. Although indole 1c shows the highest inhibitory activity, this compound also reveals synergistic activity in the presence of pyrimidine 1e. The difference in recovery is minimal between each sample when comparing the ratio of the slopes. No condition appeared to bring the cellular viability to 0. However, this was a one-dose study (addition at 0 h) and it is likely that a multidose study would further affect the cellular viability of the parasite.

Incubation Effects of Secreted Acid Phosphatase (SAP) Activity
Secreted acid phosphatase (SAP) activity on the fourth day (96 h post-addition) showed a marked difference between control and experimental conditions ( Figure 5). Normalizing the absorbance obtained for SAP activity to the day 4 MTT absorbance suggests that SAP activity is directly or indirectly affected in the presence of the test compounds ( Figure 6). For all compounds   Significant differences were observed in the differential growth patterns of experimental cultures compared to DMSO control. Complexes appear to be affecting various pathways involved in cell maturation and division. Growth curve plots show severe growth inhibition in the presence of multiple compounds. Although indole 1c shows the highest inhibitory activity, this compound also reveals synergistic activity in the presence of pyrimidine 1e. The difference in recovery is minimal between each sample when comparing the ratio of the slopes. No condition appeared to bring the cellular viability to 0. However, this was a one-dose study (addition at 0 h) and it is likely that a multidose study would further affect the cellular viability of the parasite.

Incubation Effects of Secreted Acid Phosphatase (SAP) Activity
Secreted acid phosphatase (SAP) activity on the fourth day (96 h post-addition) showed a marked difference between control and experimental conditions ( Figure 5). Normalizing the absorbance obtained for SAP activity to the day 4 MTT absorbance suggests that SAP activity is directly or indirectly affected in the presence of the test compounds ( Figure 6). For all compounds tested, there was a significant increase (p < 0.05) in secreted acid phosphatase activity relative to DMSO control cells.
Molecules 2020, 25, x 7 of 20 tested, there was a significant increase (p < 0.05) in secreted acid phosphatase activity relative to DMSO control cells.

Standard SAP Direct Enzymic Effect Assay
A 15-min pre-incubation period before substrate addition showed a decrease in SAP activity with compounds 1b and 1e being the most effective (Figure 7). The 18-h and 46-h pre-incubation periods showed little difference to control in terms of SAP activity, suggesting that enzymatic inhibition may have occurred during the 15-min pre-incubation period, resulting in a lower enzyme activity and less product formed. However, with time, the enzyme activity appears to recover, suggesting that the test compounds may not be stable under these incubation conditions. Normalized SAP Activity to Viability: Ratio of A405 nm to A595 nm tested, there was a significant increase (p < 0.05) in secreted acid phosphatase activity relative to DMSO control cells.

Standard SAP Direct Enzymic Effect Assay
A 15-min pre-incubation period before substrate addition showed a decrease in SAP activity with compounds 1b and 1e being the most effective (Figure 7). The 18-h and 46-h pre-incubation periods showed little difference to control in terms of SAP activity, suggesting that enzymatic inhibition may have occurred during the 15-min pre-incubation period, resulting in a lower enzyme activity and less product formed. However, with time, the enzyme activity appears to recover, suggesting that the test compounds may not be stable under these incubation conditions. Normalized SAP Activity to Viability: Ratio of A405 nm to A595 nm

Standard SAP Direct Enzymic Effect Assay
A 15-min pre-incubation period before substrate addition showed a decrease in SAP activity with compounds 1b and 1e being the most effective (Figure 7). The 18-h and 46-h pre-incubation periods showed little difference to control in terms of SAP activity, suggesting that enzymatic inhibition may have occurred during the 15-min pre-incubation period, resulting in a lower enzyme activity and less product formed. However, with time, the enzyme activity appears to recover, suggesting that the test compounds may not be stable under these incubation conditions.

Standard Leishmania SAP Enzyme Kinetic Assay
A 15-min pre-incubation period was allotted for the enzyme to interact with each test compound before starting the enzymatic reaction with the addition of substrate. V vs. [S] curves between conditions show no difference in enzyme activity depending on either test compound present or substrate concentration. Lineweaver-Burk plots for each condition yield very similar KM and Vmax values, suggesting no direct modulation as either an activator or inhibitor of Leishmania SAP activity (Table 3). In all cases (cf. Figure 5), the detectable SAP activity was similar to or larger than the DMSO control cells, suggesting some effect on either the cells' ability to synthesize or release this important parasite enzyme. It was of interest that incubation with compound 1d resulted in the highest value of detectable SAP activity. When normalizing the absorbance obtained for SAP activity to the day 4 MTT absorbance, it appears that the SAP activity is directly or indirectly affected in the presence of the test compounds ( Figure 6). Comparable values for both KM and Vmax for all conditions (Table 3) suggests no direct enzymic effect of activation or inhibition on SAP by 6, 1c, and 1d. These data suggest that some test compounds affect the Leishmania cells' ability to synthesize or release the secreted acid phosphatase (SAP) enzyme, but do not directly affect the enzyme activity.
2.5.6. Detection of Leishmania Nitric Oxide Using a Specific Fluorescent Probe, DAF-FM Aliquots (equal volumes from each condition) of cells were centrifuged, resuspended and incubated, washed, and plated for microscopy. All compounds showed a modest decrease in NO production relative to the 1% DMSO control (Table 4) . However, 1b, 1e, and co-incubation with 1b + 1e show a more substantial decrease in NO levels than the other compounds. These trends are shown in the microscope images ( Figures S15-S20) at 40x magnification in which green indicates cells responding to the probe for nitric oxide. Note that there were no observable differences in cell

Standard Leishmania SAP Enzyme Kinetic Assay
A 15-min pre-incubation period was allotted for the enzyme to interact with each test compound before starting the enzymatic reaction with the addition of substrate. V vs. [S] curves between conditions show no difference in enzyme activity depending on either test compound present or substrate concentration. Lineweaver-Burk plots for each condition yield very similar K M and V max values, suggesting no direct modulation as either an activator or inhibitor of Leishmania SAP activity (Table 3). In all cases (cf. Figure 5), the detectable SAP activity was similar to or larger than the DMSO control cells, suggesting some effect on either the cells' ability to synthesize or release this important parasite enzyme. It was of interest that incubation with compound 1d resulted in the highest value of detectable SAP activity. When normalizing the absorbance obtained for SAP activity to the day 4 MTT absorbance, it appears that the SAP activity is directly or indirectly affected in the presence of the test compounds ( Figure 6). Comparable values for both K M and V max for all conditions (Table 3) suggests no direct enzymic effect of activation or inhibition on SAP by 6, 1c, and 1d. These data suggest that some test compounds affect the Leishmania cells' ability to synthesize or release the secreted acid phosphatase (SAP) enzyme, but do not directly affect the enzyme activity.

Detection of Leishmania Nitric Oxide Using a Specific Fluorescent Probe, DAF-FM
Aliquots (equal volumes from each condition) of cells were centrifuged, resuspended and incubated, washed, and plated for microscopy. All compounds showed a modest decrease in NO production relative to the 1% DMSO control (Table 4). However, 1b, 1e, and co-incubation with 1b + 1e show a more substantial decrease in NO levels than the other compounds. These trends are shown in the microscope images ( Figures S15-S20) at 40x magnification in which green indicates cells responding to the probe for nitric oxide. Note that there were no observable differences in cell morphology when comparing control (with DMSO) to experimental cells. This is of interest since the production of nitric oxide by Leishmania has been proposed as one mechanism by which these parasites protect themselves from oxidative stress [27]. Then a reduction in nitric oxide production may render these parasites less infective to host cells. Further studies should be conducted using standard inducible nitric oxide synthase (iNOS) to test if either of these two compounds acts as a direct enzyme inhibitor. No acute toxicity was seen in glial cells following a 2-h incubation with the various test compounds relative to DMSO control (Table 5). In contrast to the viability assays with L tarentolae, our investigations of all test compounds were not found to be acutely toxic to glial cells in vitro. This suggests that these compounds may not be as hazardous to mammalian cells. Short term incubation, with other compounds, has been shown previously to elicit acute cytotoxicity in glial cells [96]. A therapeutic use of these compounds targeted toward inhibiting Leishmania based infection should be further investigated.

ED-50 (Effective Dose) Study on Leishmania
The toxicity assay was replicative of earlier incubation results, suggesting no toxicity from 6, and a cytotoxic effect from 1c and 1e. The formation of a trendline was used on the linear portion for both 1c and 1e to estimate an ED-50 concentration for both days after addition (Table 6). Compound 6 had no apparent effect on the cell viability at levels up to 200 µM. Compounds 1c and 1e clearly show some level of toxicity to L. tarentolae, whereas 6 does not. The second day MTT measurement of 1c incubated L. tarentolae decreased from the first day suggesting that the L. tarentolae may be unable to recover from exposure, especially at higher concentrations. This ability to recover is seen at lower (less than 40 µM) but not higher concentrations with 1e, as shown in Figure 8.

Photosensitivity study on Leishmania
No difference in light exposure sensitivity was seen immediately after uptake period and light exposure between experimental and control cultures (Table 7). However, by 24 h, cells with test compound 1c exhibited a reduction in cell viability but independent of light treatment. In contrast, 1e or 6 treated cells exhibited no toxicity throughout the entire trial under any light condition. In general, L. tarentolae appeared to be resistant to exposure to both UV and fluorescent light under these experimental conditions. Compound 1e MTT levels are in line with DMSO control, which was unexpected, as all other studies showed some level of toxicity. One possible explanation is that this molecule is not as stable as the others and thus stock preparations undergoing multiple freeze-thaw cycles in DMSO from −20 °C to tepid water rendered this inactive. Compound 1c continued to exhibit the same toxic effect to L. tarentolae, as it was not dependent on the light exposure received implying that light exposure has no effect on uptake and/or toxicity of 1c. Table 7. MTT absorbances relative to 1% DMSO cell control, 6, 1c, or 1e at 2, 24, and 48 h post addition. D = DMSO, N = normal conditions (dark), F = fluorescent bulb, UV = ultraviolet bulb.

Conclusions
Due to the pressing need for new small molecules to treat Leishmaniasis, hybrid polycyclic ether-benzopyrans were constructed in order to combine the potential glucose transporter inhibition with the privileged benzopyran structure. The construction of this new scaffold was undertaken by utilizing the complexity-generating [5+2] cycloaddition in conjunction with the Suzuki-Miyaura cross-coupling to deliver biologically relevant heterocycles. Leishmania tarentolae growth was shown to be affected in the presence of these organic molecules to differing extents with no noticeable change in cell morphology, motility, or clumping. Although the biochemical mechanisms are not fully understood, two different enzymes were evaluated in this study, namely secreted acid phosphatase and (indirectly) nitric oxide synthase. SAP secretion or transcriptional regulation may be affected by the presence of these polycyclic ether-benzopyrans, since we found that there is differing MTT

Photosensitivity study on Leishmania
No difference in light exposure sensitivity was seen immediately after uptake period and light exposure between experimental and control cultures (Table 7). However, by 24 h, cells with test compound 1c exhibited a reduction in cell viability but independent of light treatment. In contrast, 1e or 6 treated cells exhibited no toxicity throughout the entire trial under any light condition. In general, L. tarentolae appeared to be resistant to exposure to both UV and fluorescent light under these experimental conditions. Compound 1e MTT levels are in line with DMSO control, which was unexpected, as all other studies showed some level of toxicity. One possible explanation is that this molecule is not as stable as the others and thus stock preparations undergoing multiple freeze-thaw cycles in DMSO from −20 • C to tepid water rendered this inactive. Compound 1c continued to exhibit the same toxic effect to L. tarentolae, as it was not dependent on the light exposure received implying that light exposure has no effect on uptake and/or toxicity of 1c.

Conclusions
Due to the pressing need for new small molecules to treat Leishmaniasis, hybrid polycyclic ether-benzopyrans were constructed in order to combine the potential glucose transporter inhibition with the privileged benzopyran structure. The construction of this new scaffold was undertaken by utilizing the complexity-generating [5+2] cycloaddition in conjunction with the Suzuki-Miyaura cross-coupling to deliver biologically relevant heterocycles. Leishmania tarentolae growth was shown to be affected in the presence of these organic molecules to differing extents with no noticeable change in cell morphology, motility, or clumping. Although the biochemical mechanisms are not fully understood, two different enzymes were evaluated in this study, namely secreted acid phosphatase and (indirectly) nitric oxide synthase. SAP secretion or transcriptional regulation may be affected by the presence of these polycyclic ether-benzopyrans, since we found that there is differing MTT normalized SAP readings from in vitro incubation. However, none of the compounds tested appear to have a direct, modulatory effect on SAP as an activator or inhibitor. The reduction in detectable nitric oxide by some of the test compounds was substantial and may reduce the ability of these cells to detoxify reactive oxygen species of host cells. Acute toxicity does not seem to occur in cultured glial cells indicating a differential effect relative to cell type. Polycyclic ether-benzopyrans 1c and 1e appear to have the most inhibitory effects on Leishmania tarentolae viability through a concentration and time-dependent manner. Exposure to different wavelengths of light does not seem to affect toxicity levels of these two complexes. Under some conditions, the compounds do not appear to be stable with time, suggesting that potential therapy will involve multiple dosing to bring about successful reduction of the parasitic load. Future work will focus on evaluation of the mechanism of action especially involving the GLUT transporter by which these polycyclic ether-benzopyrans affect Leishmania in vitro.

General Methods-Organic Synthesis
All reactions were performed under Ar atmosphere in oven-dried or flame-dried glassware unless otherwise noted. Diethyl ether (Et 2 O) was dried over pressed Na metal. All other commercially available anhydrous solvents and reagents were used as received. Sodium hydride (NaH) was a 60% dispersion in mineral oil. Thin layer chromatography was performed with glass or aluminum plates (silica gel F 254 , Art 5715, 0.25 mm), visualized by fluorescence quenching under UV light, and stained with potassium permanganate. Flash column chromatography (FCC) was performed with silica gel 60A 40-63 µm (200-400 mesh). Mass spectral data were acquired using positive mode Electrospray Ionization (ESI+) and a high-resolution Time of Flight (TOF) mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA). 1 H NMR spectra were acquired at 400 or 500 MHz and 13 C{ 1 H} NMR spectra were acquired at 100 MHz or 125 MHz as noted (Bruker, Billerica, MA, USA). 1 H and 13 C{ 1 H} NMR chemical shifts are reported in ppm (δ) relative to the residual solvent peaks. 1 H NMR coupling constants (J) are reported in Hertz (Hz), and multiplicities are indicated as follows: s (singlet), br. s (broad singlet), d (doublet), t (triplet), q (quartet), quint (quintet), sext (sextet), sept (septet), dd (doublet of doublets), ddd (doublet of doublet of doublets), dt (doublet of triplets), td (triplet of doublets), ddt (doublet of doublet of triplets), dq (doublet of quartets), qq (quartet of quartets), ovlp (overlapping), m (multiplet), app. (apparent). Based on intensity in the 13 C{ 1 H} spectra, both magnetic and chemical shift equivalent peaks are noted in parentheses. Compounds 6 and 1a-1e were solubilized with DMSO to 30 mM for stock concentration (stored at −20 • C).
Evaluation of Direct Enzymic Effect of Test Compounds on SAP: A stock culture of L. tarentolae in the late stationary phase was centrifuged (3000 rpm, 15 min @ 7 • C, Labnet HERMLE Z 400K centrifuge), and the cell-free supernatant was collected. This standard pool of enzyme was separated into equal volumes and allowed to incubate with each of the 6 compounds at 30 µM or 1% DMSO for 15 min, 18 h, or 46 h before starting the SAP assay.
SAP Enzyme Kinetic Assay using Selected Test Compounds (following the method of Dorsey et al. [28]): A stock culture of L. tarentolae in the late stationary phase, was pelleted, retaining the supernatant. This standard pool of SAP enzyme was separated into equal volumes and allowed to incubate with 30 µM compounds 6, 1c, and 1e. Pre-incubated samples were aliquoted with 13 varying substrate concentrations ranging from 4.5 µM to 1787 µM.
Evaluation of Test Compound Acute Effects on Glial Viability: Test compounds prepared in DMSO and diluted into incomplete DMEM were introduced, at a concentration of 30 µM, when the glial cells in the 96-well plates reached confluency. MTT assays were conducted in incomplete DMEM diluted 1:10 with sterile saline following a two-hour incubation with the compounds.
Effective Dose (ED-50) Study on L. tarentolae: Equal volumes of late log-phase L. tarentolae were aliquoted into separate flasks with compounds 6, 1c, or 1e at 8 varying final concentrations ranging from 3 to 200 µM. MTT assays were performed 24 h and 48 h post-addition of compounds.
Evaluation of Photosensitivity of L. tarentolae in the Presence of Test Compounds: Equal volumes of late log-phase L. tarentolae were aliquoted into separate flasks with 30 µM of compounds 6, 1c, or 1e. Cultures were incubated 1 h in the dark to allow uptake of compounds before exposing to a fluorescent bulb (following the method of Morgenthaler et al. [97]), or a UV bulb for 1 h. Cells kept in the dark were considered the control cells. MTT assays were performed directly after compound addition as well as 2 h, 24 h, and 48 h after light exposure.
Evaluation of production of nitric oxide by Leishmania in the presence of test compounds: Nitric oxide (NO) assay was performed using DAF-FM diacetate (a compound that fluoresces after reaction with nitric oxide; from Millipore Sigma, Darmstadt, Germany) at a final concentration of 20 µM and 1% DMSO in saline. Cells were centrifuged (10,000 rpm, 60 s, Eppendorf Centrifuge 5415C) and resuspended in this DAF-FM diacetate solution for 1 h before re-centrifuging, which was followed by washing the cell pellet with 500 µL saline. Cells were resuspended in a final total volume of saline such that there were a similar number of cells per sample. Prior to imaging, 10 µL of each sample was loaded onto a glass slide within a silicon grease ring and then covered with a cover slip. Sample slides were placed in a BZX-810 Keyence Fluorescent Microscope for imaging using a green fluorescent protein filter (GFP; 470 nm: 525 nm excitation-emission coupling). Images were captured at a 1.5 s exposure time and overlays of fluorescent cells with the brightfield images were produced using the analyzer software. Cells were then counted for number of green fluorescent cells (indicating production of nitric oxide) and number of non-green cells.

Conflicts of Interest:
The authors declare no conflict of interest.