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

Abstract

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.

Keywords: Leishmania; Suzuki-Miyaura; benzopyran; cycloaddition; nitric oxide; oxabicyclo[3.2.1]-octane; oxidopyrylium; secreted acid phosphatase.

Figure 1

Hybrid polycyclic ether-benzopyran (1).

Figure 2

Diels–Alder [4+2] cycloaddition versus oxidopyrylium-alkene [5+2] cycloaddition.

Scheme 1

Previous synthesis of a polycyclic ether-benzopyran 3.

Scheme 2

Proposed Suzuki-Miyaura pathway for the construction of ether-benzopyran analogues 1.

Scheme 3

Synthesis of Suzuki-Miyaura cross-coupling partner 6.

Figure 3

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) absorbance values from cells exposed to a single dose of 30 µM polycyclic ether-benzopyrans in DMSO were obtained at each interval (0 h and every 24 h subsequently). Values are the mean for n = 4 replicates and all SD were smaller than 2% of the mean. DMSO cells are considered to be the appropriate control.

Figure 4

MTT values at 24 h intervals post addition of a single dose of a polycyclic ether-benzopyran. The compounds (30 µM) were added directly after first reading. Arrow indicates time that cells were given fresh medium with no added compound (recovery phase). Values are the mean values from n = 4 replicates.

Figure 5

Absorbance (A 405 nm) after 24 h incubation with pNPP. Values are mean ± SD for n = 4 replicates.

Figure 6

Normalized secreted acid phosphatase (SAP) assay absorbance to MTT absorbance per condition.

Figure 7

Absorbance values (A 405 nm) resulting from differing pre-incubation with polycyclic ether-benzopyrans. Values are the mean ± SD for n = 4 replicates. Blue: 15 min; orange: 18 h; gray: 46 h.

Figure 8

The dose-response curve at 24 and 48 h after a single addition of test compound 1e at concentrations ranging from 4.5 to 200 µM.