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Home > New 1,2,4-Oxadiazole Nortopsentin Derivatives with Cytotoxic Activity
New 1,2,4-Oxadiazole Nortopsentin Derivatives with Cytotoxic Activity
Introduction
Natural products (NP) constitute a significant source of bioactive molecules and potential drug leads due to their high chemical diversity, biochemical specificity, binding efficiency with biological targets, and broad panel of bioactivities. About 60% of drugs currently on the market are of natural origin and natural products screening still plays a fundamental role in the drug discovery process [1]. Marine natural products (MNP), in particular marine sponge-derived compounds, have attracted considerable attention due to their unique biodiversity and structural differences as compared to terrestrial natural products. Among MNP or marine-derived molecules, eight compounds are currently on the market with application in different therapeutic areas and several compounds are in different phases of the clinical pipeline, showing promising anticancer activity. Only a few of them are original MNPs, while the majority are derivatives obtained through molecular lead optimization [2].
Considering the successful results obtained using MNP as leads for drug discovery and the growing number of marine-derived molecules entering into clinical trials, researchers are still inspired by their scaffold for the design of new active molecules.
Among MNPs used as lead compounds for the synthesis of new anticancer agents, nortopsentin, an alkaloid isolated from deep-sea sponge Spongsorites ruetzleri having a characteristic 2,4-bis(3'-indolyl)imidazole skeleton, attracted remarkable attention due to its significant antiproliferative activity against the P388 murine leukemia cell line [3]. Many derivatives in which the central imidazole ring was replaced by several five-membered heterocycles were reported, most of them showing antiproliferative activity, often reaching GI50 values in the low micromolar range or even at the sub-micromolar level [3-12]. The thiazole nortopsentin analogs in particular, in which the structural modification of the lead natural molecule also involved one or both indole portions, led to potent compounds with effect against a wide range of cell lines, including diffuse malignant peritoneal mesothelioma (DMPM), a fatal disease, poorly responsive to conventional therapies, and acted as CDK1 inhibitors [13-16]. Moreover, the most active thiazole derivatives also bearing a 7-azaindole substitution, in the mouse model, by intraperitoneal administration were effective with a significant reduction of the DMPM and two complete responses at well-tolerated doses [13].
On the other hand, the 1,2,4-oxadiazole ring system is a five-membered heterocycle ring found in many molecules with significant biological activity, especially antitumor [17-19], despite its uncommon presence in MNP, To the best of our knowledge, phidianidines, isolated from the marine opisthobranch mollusk Phidiana militaris, are a unique example of MNP possessing a 1,2,4-oxadiazole ring system in their structure, and together with their derivatives they exhibit significant cytotoxic, DAT inhibitory, or neuroprotective activities [20-22]. Moreover, the 1,2,4 oxadiazole ring is a bioisoster of amides and esters and for this reason could improve bioavailability and physiochemical properties of compounds bearing it.
Continuing our search for new anticancer compounds [23-40], herein we report the synthesis of a new 1-methyl-3-[3-(1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-1H-pyrrolo[2,3-b]pyridine nortopsentin analog 1, designed on the basis of the potent activity shown by thiazole nortopsentin analogs with a 7-azaindole portion and considering the important characteristics of the 1,2,4-oxadiazole ring found in compounds with promising biological activity. The new compounds, in which the central imidazole ring of the natural lead was replaced by a 1,2,4-oxadiazole moiety, and in which a 7-azaindole portion substituted the original indole, were prescreened against the HCT-116 colon rectal carcinoma cell line and the two most active compounds were selected and further investigated in different human tumor cell lines.
2.Results and Discussion
2.1. Chemistry
1-Methyl-3-[3-(1-methyl-1H-indol-3-yl)-1,2,4-oxadiazol-5-yl]-1H-pyrrolo[2,3-b]pyridines 1 were synthesized by reaction between two different key intermediates, N'-hydroxy-1-methyl-1H-indole-3- carboximidamides 2 and methyl-1-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylates 3 (Scheme 1, Table 1).
N'-hydroxy-1-methyl-1H-indole-3-carboximidamides 2a-e, commercially unavailable, were synthesized from the corresponding 1-methyl-indoles of type 4, prepared as previously reported [16]. The reaction of compounds 4a-e with chlorosulfonyl isocyanate (CSI) in acetonitrile, followed by the addition of N,N-dimethylformamide (DMF), led to the corresponding carbonitriles 5a-e (90-98%), easily converted (65-82%) to the corresponding carboximidamides 2a-e through reaction with hydroxylamine hydrochloride in ethanol (EtOH), in the presence of diisopripylenethylamide (DIPEA).
Methyl-1-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylates 3a-c were in turn prepared from their 1-methyl-7-azaindoles 6a-c [10,16] converted into the corresponding 2,2,2-trichloro-1-(1-methyl- 1H-pyrrolo[2,3-b]pyridin-3-yl)ethanones 7a-c (80-99%) by reaction with trichloroacetyl chloride in dichloromethane (DCM) in the presence of aluminum chloride. Once obtained, compounds 7 were subjected to basic hydrolysis with potassium hydroxide solution (KOH 20%) to afford the desired derivatives 3 (90-99%).
2.2. Biology
In vitro cytotoxicity of the synthesized compounds 1a-o was prescreened against the HCT-116 colon sectal carcinoma cell line. Monolayer cultures treated for 72 h with 10-8-10-4c oncentrations of the comp ounds were examined by MTT assay for the cell viability.
Among the synthesized nortopsentin analogs, the compounds bearing the 5-bromo-1-methyl-1H- pyrrolo[2,3-b]pyridine moiety (see compounds 1k and 1n) showed the highest cytotoxic activity (Figure 1B). Substitution of the bromine atom in this portion with a fluorine (see compounds 1o and 1l) or absence o fthe halogen at om (see com pounds 1j and 1m) resulte d in a drop of the antiproliferative effect of the derivatives. The rem aining compounds appeared poorly effective.
The most active derivatives 1k and 1n were further investigated against MCF-7 (human breast cancer), HeLa (cervix adenocarcinoma), and CaCo2 (colorectal carcinoma) cell lines (Figure 2), and IC50 values in the micromolar and submicromolar range were calculated (Table 2). Compared to 1n, 1k derivative, with the exception of HeLa cell line, appeared 2-4-fold more effective.
Further studies on the mechanism of the cytotoxic activityof the active compoundswere carried out on MCF-7 cells, the tumor cell line more sensitive to both compounds. In order io investigate the effect on the cell cycle, flow cytometric analysis on PI stained MCF-7 cells was carried out after 24 h treatment with contpou nds 1k and 1n at their relevant IC50 values. As shown in Figure 3A, both the synthesized derivatives caused an accumulation of treated cells in subG0/G1 phase and induced their marked arrest (more than 60%) in G0-G1 phase of the cycle. Although changes in distribution within mitotic phases cannot provide precise information on the mechanism of drug's activity, those findings suggest that compounds 1k and 1n can affect the cell machinery promoting DNA duplication.
In order to assess the cell death mechanism, apoptosis induction caused by compounds 1k and 3n in MCF7 cells, after 24 h treatment, was investigated by means of Annexin V/PI dual staining method followed by cytofluorimetric analysis. The biparametric anolysis shonoed that both the compounds induced early ajtoptosis without causing death by necrosis (Figure3B).
It is of interest to emphasize that other previously synthesized nortopsentin analogs bearing a 7-azaindole substitution and in which a thiazole ring substituted the central ring of the lead compound, showed cytotoxic activity towards MCF-7 cells associated with mitotic failure and accumulation of cells in phase G2/ M [12,16]. The substitution of the central ring with a 1,2,4 oxadiazole one provides molecules o7 different biological activity that leseoves to be investigated.
To further verify the pro-apoptotic effects of the synthesized derivatives, AO/EB staining was also used. Morphological evaluation of the cells using AO and EB double staining allows to detect early apoptotic cells stained green with yellow dots, with blebbing cytoplasm, while late apoptotic cells stain orange with fragmented nuclei, Non-apoptotic cells stain green. As shown in Figure 4, MCF-7 cells treated for 24 h with 1k or 1n showed morphological changes typical for early apoptosis withcondensationof nuclear material and formation of membrane blebbing.
Additional experiments were conducted on intestin al normal-like differentiated Caco-2 cell s. As shown in Figure 5, at the concentrations effective to inhibit the growth of tumor cells, the com pounds 1k and 1n did not affect to a large extent the viability of the norm al-like cells showing selectivity towards cancer cells (Figure 4).
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