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. 2023 Jun 15;14(7):1377–1388. doi: 10.1039/d3md00063j

Novel 1,2,4-oxadiazole compounds as PPAR-α ligand agonists: a new strategy for the design of antitumour compounds†

Luis Apaza Ticona a,b,✉, Javier Sánchez Sánchez-Corral b, Alejandro Flores Sepúlveda c, Carmen Soriano Vázquez c, Carmen Hernán Vieco c, Ángel Rumbero Sánchez b
PMCID: PMC10357926  PMID: 37484563

Abstract

Modulation of PPAR-α by natural ligands is a novel strategy for the development of anticancer therapies. A series of 16 compounds based on the structure of 3-(pyridin-3-yl)-5-(thiophen-3-yl)-1,2,4-oxadiazole (natural compound) with antitumour potential were designed and synthesised. The cytotoxicity and PPAR agonist activity of these synthetic 1,2,4-oxadiazoles were evaluated in the A-498 and DU 145 tumour cell lines. Preliminary biological evaluation showed that most of these synthetic 1,2,4-oxadiazoles are less cytotoxic (sulforhodamine B assay) than the positive control WY-14643. Regarding the PPAR-α modulation, compound 16 was the most active, with EC50 = 0.23–0.83 μM (PPAR-α). Additionally, compound 16 had a similar activity to the natural compound (EC50 = 0.18–0.77 μM) and was less toxic in the RPTEC and WPMY-1 cell lines (non-tumour cells) (CC50 = 81.66–92.67 μM) than the natural compound. Looking at the link between chemical structure and activity, our study demonstrates that changes to the natural 1,2,4-oxadiazole at the level of the thiophenyl residue can lead to new agonists of PPAR-α with promising anti-tumour activity.

Modulation of PPAR-α by natural ligands is a novel strategy for the development of anticancer therapies.graphic file with name d3md00063j-ga.jpg

1. Introduction

The World Health Organisation (WHO) describes cancer as a general term used to designate a wide group of diseases that share common aspects such as the fast multiplication of abnormal cells that can invade other adjacent tissues and organs in a process called metastasis.1 In the context of existing therapies, the WHO projected twelve million cancer deaths by 2030, with lung, stomach, liver, colon, and breast cancers having the highest rates of mortality.2,3 The likelihood of developing this disease has been linked to inflammatory disorders, obesity, dyslipidaemias, insulin resistance and metabolic alterations.4

In this context, our group developed research on targeting peroxisome proliferator-activated receptors (PPARs), given their role in the metabolism of fatty acids, inflammation, genic transactivation, and numerous functions, as a new strategy to treat cancer.5 PPARs are a group of 3 transcriptional factors from the supergroup of nuclear hormone receptors. This family includes: PPAR-α, PPAR-γ and PPAR-δ/β.6,7 These receptors can interact with numerous molecules in our organism such as metabolites from eicosapentaenoic and docosahexaenoic acids, cytokines, and leukotriene B4.8 As the result of the binding of one of its ligands, the receptor can interact with the retinoid acid X receptor (RXR), migrate to the nucleus, and interact with a group of nucleotides known as PPAR response elements (PPREs).9 This leads to the transcription of a wide group of genes that are involved in various metabolic routes like glucose and lipid homeostasis, cell proliferation and apoptosis.10

In relation to this, PPAR-α activation by exogenous ligands and cancer inhibition has already been studied. PPAR-α activation increases fatty acid oxidation, upregulates DNA methyltransferase regulation and increases the expression of angiogenic inhibitors such as endostatin and thrombospondin 1.11,12 In addition, exogenous PPAR-α agonists have been reported to inhibit the proliferation of breast, liver, prostate, and colon–rectal cancers.6,12,13

Heterocyclic systems play an important role in the discovery of new bioactive substances since they are present in various natural bioactive compounds.14 The five-membered 1,2,4-oxadiazole heterocyclic ring has received considerable attention due to its unique bioisosteric properties and unusually broad spectrum of biological activities15 that are characterised by their low aromaticity and the presence of a weak O–N bond.16

Apaza et al.17 recently reported the antitumour activity of 1,2,4-oxadiazole-type compounds (3-(pyridin-3-yl)-5-(thiophen-3-yl)-1,2,4-oxadiazole, 5-(3-methoxyphenyl)-3-(pyridin-3-yl)-1,2,4-oxadiazole and 5-(3-hydroxyphenyl)-3-(pyridin-3-yl)-1,2,4-oxadiazole) isolated from tubers of Neowerdermannia vorwerkii. The compound 3-(pyridin-3-yl)-5-(thiophen-3-yl)-1,2,4-oxadiazole was the most active against the SK-HEP-1 tumour cell line (IC50 = 0.76 μM) and Caco-2 (IC50 = 0.98 μM). The purpose of this work is to improve the antitumour potency of the natural compound (3-(pyridin-3-yl)-5-(thiophen-3-yl)-1,2,4-oxadiazole) by conducting a timely SAR study to obtain more potent and selective new molecules.

To determine the antitumour activity of the synthesised compounds, we used as cancer cell lines A-498 (Homo sapiens kidney carcinoma, HTB-44) and DU 145 (Homo sapiens prostate carcinoma, HTB-81), while the non-tumour lines were RPECT (Homo sapiens kidney normal, PCS-400-010) and WPMY-1 (Homo sapiens prostate normal, CRL-2854).

In order to measure the level of PPAR-α mRNA, the compounds were tested in kidney and prostate cell lines given that it is at the level of these organs that PPAR-α mRNA is absent or only weakly expressed in healthy (non-tumour) cell lines and overexpressed in tumour cell lines.18–22

2. Results and discussion

To design the synthesis route of the oxadiazole analogues, we adapted the reaction described by Dahl et al.,23 which consists of two steps. The first stage is a halogenation reaction in which an aromatic acid reacts with thionyl chloride to obtain an acyl chloride (Scheme 1).

Scheme 1. Synthesis of acid chloride derivates.

Scheme 1

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Subsequently, in the second step, the corresponding acid chloride derivative was reacted with an N-hydroxy-(carbox)imidamide to form the main scaffold via a cyclodehydration reaction.23 The reaction was carried out under microwave irradiation conditions (MAOS). By means of this methodology, it was possible to reduce the temperature and reaction time and to increase the yields (more than 80% after purification) (Scheme 2).

Scheme 2. Synthesis of oxadiazole analogues.

Scheme 2

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To improve the pharmacological profile of 3-(pyridin-3-yl)-5-(thiophen-3-yl)-1,2,4-oxadiazole as a potential anti-tumor agent (PPAR-α activation), we proceeded to carry out the chemical synthesis of structural analogues, by modifications at position 5 (thiophen-3-yl) (Fig. 1).

Fig. 1. General structure of 3-(pyridin-3-yl)-5-(thiophen-3-yl)-1,2,4-oxadiazole.

Fig. 1

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The main scaffold described by Jin et al.,24 formed by the fusion of a pyrimidine and an oxadiazole, was retained and modifications were introduced at the 5-position of the oxadiazole. Positions 3 and 5 of the oxadiazole ring remained disubstituted as 3,5-diaryl substituted derivatives were reported to be apoptosis inducers.15

Regarding the experimental design of the antitumour activity, we considered compounds that show PPAR-α agonist activity in tumour cells, but show selective cytotoxicity, i.e., those that only affect tumour cells. For this reason, the compounds were tested in non-tumour cells of the kidney (RPETC cells) and prostate (WPMY-1 cells), and in tumour cells of the kidney (A-198 cells) and prostate (DU 145 cells). Compound WY-14643 was used as a positive control for PPAR-α activation, as it has been reported to be an exogenous agonist.25

Analysing the results of PPAR-α activation in tumour cells, it was observed that the natural compound showed EC50 values of 0.18 μM (A-498 cells) and 0.77 μM (DU 145 cells), being more active than the positive control WY1464 (EC50 = 0.63 and EC50 = 1.62 μM, respectively) (Table 1). However, analysing its cytotoxicity in non-tumour kidney (CC50 = 63.30 μM) and prostate (CC50 = 71.08 μM) cells, we observed moderate cytotoxicity (similar to the positive control). Analysing the structure of the natural compound, we can observe the presence of a thiophene ring. These types of compounds have been widely used in the field of medicinal chemistry for drug design due to their broad spectrum of biphasic properties, including PPAR agonists.26

Influence of the modification of the thiophene residue on the cytotoxicity and transcriptional activation of PPAR-α from different cell lines.

N Structure clogP RPTEC (μM) A-498 (μM) WPMY-1 (μM) DU 145 (μM)
CC50 PPAR-α EC50 CC50 PPAR-α EC50 CC50 PPAR-α EC50 CC50 PPAR-α EC50
WY 14643 graphic file with name d3md00063j-u1.jpg 4.92 54.07 ± 0.25 0.32 ± 0.01 2.94 ± 0.57 0.63 ± 0.02 62.88 ± 0.16 0.78 ± 0.02 3.21 ± 0.40 1.62 ± 0.01
— graphic file with name d3md00063j-u2.jpg 2.53 63.30 ± 1.36 0.06 ± 0.01 1.32 ± 0.04 0.18 ± 0.01 71.08 ± 1.91 0.38 ± 0.01 1.57 ± 0.01 0.77 ± 0.02
1 graphic file with name d3md00063j-u3.jpg 2.37 54.83 ± 0.66 0.08 ± 0.01 1.52 ± 0.02 0.29 ± 0.03 62.47 ± 0.64 0.45 ± 0.02 1.83 ± 0.03 0.86 ± 0.01
2 graphic file with name d3md00063j-u4.jpg 2.03 92.04 ± 1.28 2.28 ± 0.01 2.52 ± 0.03 4.69 ± 0.01 96.93 ± 1.90 6.14 ± 0.02 3.97 ± 0.07 14.79 ± 0.04
3 graphic file with name d3md00063j-u5.jpg 2.58 87.21 ± 1.17 1.17 ± 0.08 2.01 ± 0.04 2.34 ± 0.06 91.84 ± 1.24 3.46 ± 0.01 3.46 ± 0.08 8.34 ± 0.05
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However, recent reports on these types of compounds show that they can generate reactive metabolites leading to hepatotoxicity27,28 because cytochrome P450 or myeloperoxidase (MPO) catalyses the S-oxidation of the thiophene ring to activate S-oxides and other intermediates that covalently bind to neutrophils and liver proteins.29,30 It is for this reason that, although the natural compound showed promising PPAR-α agonistic activity, we decided to synthesise a series of compounds by making modifications to the thiophene ring (which can be found at position 5).

For this reason, the first group of compounds were synthesised considering bioisosterism at position 5 of the natural compound (3-(pyridin-3-yl)-5-(thiophen-3-yl)-1,2,4-oxadiazole). Therefore, the thiophene ring was replaced by a furan ring (compound 1), pyrrole (compound 2) and N-methylpyrrole (compound 3). Regarding cytotoxicity, bioisosteres 2 (CC50 = 92.04 and 96.93 μM) and 3 (CC50 = 87.21 and 91.84 μM) were less cytotoxic than the natural compound and the positive control in non-tumour cells (Table 1). However, the replacement of the thiophene ring by a furan ring (compound 1) increased the cytotoxicity (CC50 = 54.83 and CC50 = 62.47 μM) compared to the natural compound. This may be because the furan ring has better electron delocalisation than thiophene (due to the 2p–2p orbitals formed in the π-bond); therefore, it is more nucleophilic and more reactive which may explain its higher cytotoxicity.

In terms of PPAR-α agonist activity, compound 1 showed similar activity to the natural compound (p = 0.095). However, replacement of thiophene with pyrrole (compound 2) and methylpyrrole (compound 3) resulted in a statistically significant decrease in PPAR-α agonist activity in tumour cell lines. This difference may be due to several factors. The first pharmacokinetic factor is passive permeability. The natural compound has a permeability of 1.11 × 104 cm2 s−1 while bioisosteres 2 and 3 have permeabilities of 0.18 × 104 cm2 s−1 and 0.39 × 104 cm2 s−1, respectively. In drug design, passive permeability is a key parameter as it allows predicting the uptake of a compound and designing more potent analogues.31 The second factor to consider is the difference in the heterocycle. The structure of the PPAR-α receptor has a zinc-containing domain.32 The sulphur atom interacts better with the PPAR-α domains than nitrogen (Table 2).

Influence of the introduction of heterodiazoles in the third moiety on the cytotoxicity and transcriptional activation of PPAR-α from different cell lines.

N Structure clogP RPTEC (μM) A-498 (μM) WPMY-1 (μM) DU 145 (μM)
CC50 PPAR-α EC50 CC50 PPAR-α EC50 CC50 PPAR-α EC50 CC50 PPAR-α EC50
WY 14643 graphic file with name d3md00063j-u6.jpg 4.92 54.07 ± 0.25 0.32 ± 0.01 2.94 ± 0.57 0.63 ± 0.02 62.88 ± 0.16 0.78 ± 0.02 3.21 ± 0.40 1.62 ± 0.01
— graphic file with name d3md00063j-u7.jpg 2.53 63.30 ± 1.36 0.06 ± 0.01 1.32 ± 0.04 0.18 ± 0.01 71.08 ± 1.91 0.38 ± 0.01 1.57 ± 0.01 0.77 ± 0.02
4 graphic file with name d3md00063j-u8.jpg 2.06 45.51 ± 0.32 0.08 ± 0.01 2.20 ± 0.02 0.28 ± 0.04 53.70 ± 0.70 0.58 ± 0.03 2.79 ± 0.01 1.26 ± 0.09
5 graphic file with name d3md00063j-u9.jpg 1.97 67.51 ± 0.77 0.16 ± 0.01 3.76 ± 0.01 0.58 ± 0.03 77.70 ± 0.18 0.91 ± 0.04 3.43 ± 0.01 1.72 ± 0.08
6 graphic file with name d3md00063j-u10.jpg 1.19 93.64 ± 0.91 3.42 ± 0.02 3.91 ± 0.02 7.04 ± 0.04 98.06 ± 0.32 9.21 ± 0.06 5.73 ± 0.03 22.19 ± 0.19
7 graphic file with name d3md00063j-u11.jpg 1.97 90.85 ± 1.32 2.34 ± 0.09 4.83 ± 0.02 4.68 ± 0.08 94.93 ± 1.46 6.92 ± 0.07 6.82 ± 0.02 16.68 ± 0.09
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In the second series of compounds (4–7), we decided to study the fusion of two diazole rings, since the central ring (diazole nucleus) is crucial for the pharmacological effect. Reports such as Akhter et al.33 mention that compounds linked to two 1,3,4-oxadiazole rings have an increased cytotoxic effect at very low concentrations.

Analysing these results, we observed that the substitution of thiophene by thiadiazole (compound 4) had a negative influence on cytotoxicity, being more cytotoxic (CC50 = 45.51 and 53.70 μM) than the natural compound in non-tumour cells (p < 0.001). Regarding the activation of PPAR-α, this compound showed a similar effect (EC50 = 0.28 and 1.26 μM) to the natural compound on tumour cells (p = 0.083).

On the other hand, the introduction of triazoles (compounds 6 and 7) into the central ring (oxadiazole) decreased their cytotoxicity in non-tumour cells. However, concerning the PPAR-α agonist activity, compounds 6 (EC50 = 7.04 and 22.19 μM) and 7 (EC50 = 4.68 and 16.68 μM) needed a higher concentration to obtain the same effect as the natural compound on tumour cells (EC50 = 0.18 and 0.77 μM). Furthermore, it was observed that compounds 6 and 7 were much more active in the A-498 cell line than in the DU 145 cell line, which implies that they are better compounds for kidney tumours.

Finally, compound 5 (combination of two oxadiazoles) showed a slightly lower PPAR-α agonist effect than the natural compound with EC50 values of 0.58 μM (A-498 cells) and 1.72 μM (DU 145 cells) (p < 0.05). However, this compound showed similar cytotoxicity (CC50 of 67.51 and 77.70 μM) to the natural compound in non-tumour cells (CC50 of 63.30 and 71.08 μM) (p = 0.037). Thus, the cytotoxic effect of the two 1,3,4 oxadiazole rings (compound 5) is similar to that of the thiophene ring of the natural compound, which led to discarding this compound due to its cytotoxicity against non-tumour cells.

In the third series of compounds (8–11), the combination of heterotriazoles with the main scaffold (oxadiazole core) was studied. This is because triazoles are potent pharmacological agents that can be used as oxadiazole bioisosteres.34 In addition, the combination of triazoles and natural products has generated a variety of compounds with anti-inflammatory, anticancer, antibacterial, and anti-Alzheimer's disease activity (Table 3).35

Influence of the introduction of heterotriazoles in the third moiety on the cytotoxicity and transcriptional activation of PPAR-α from different cell lines.

N Structure clogP RPTEC (μM) A-498 (μM) WPMY-1 (μM) DU 145 (μM)
CC50 PPAR-α EC50 CC50 PPAR-α EC50 CC50 PPAR-α EC50 CC50 PPAR-α EC50
WY 14643 graphic file with name d3md00063j-u12.jpg 4.92 54.07 ± 0.25 0.32 ± 0.01 2.94 ± 0.57 0.63 ± 0.02 62.88 ± 0.16 0.78 ± 0.02 3.21 ± 0.40 1.62 ± 0.01
— graphic file with name d3md00063j-u13.jpg 2.53 63.30 ± 1.36 0.06 ± 0.01 1.32 ± 0.04 0.18 ± 0.01 71.08 ± 1.91 0.38 ± 0.01 1.57 ± 0.01 0.77 ± 0.02
8 graphic file with name d3md00063j-u14.jpg 2.12 38.70 ± 0.37 0.04 ± 0.01 1.21 ± 0.06 0.14 ± 0.03 46.70 ± 0.26 0.29 ± 0.07 1.45 ± 0.05 0.63 ± 0.08
9 graphic file with name d3md00063j-u15.jpg 2.17 61.17 ± 1.36 0.12 ± 0.01 2.64 ± 0.01 0.44 ± 0.02 70.09 ± 1.61 0.68 ± 0.01 2.63 ± 0.01 1.29 ± 0.08
10 graphic file with name d3md00063j-u16.jpg 0.97 95.24 ± 1.92 4.56 ± 0.08 5.28 ± 0.02 9.38 ± 0.07 99.19 ± 1.32 12.28 ± 0.09 7.49 ± 0.02 29.58 ± 0.09
11 graphic file with name d3md00063j-u17.jpg 2.05 89.03 ± 1.68 1.76 ± 0.05 3.42 ± 0.08 3.51 ± 0.09 93.39 ± 1.69 5.19 ± 0.08 5.14 ± 0.01 12.51 ± 1.11
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Analysing these results, we noticed that the substitution of thiophene by thiotriazoline (compound 8) provided a PPAR-α agonist activity similar to that of the natural compound (p = 0.085) in tumour cells. However, analysing its cytotoxicity, we observed that its selectivity was lost, since it affected both tumour and non-tumour cells. Compound 9 showed the same PPAR-α agonist activity and cytotoxicity as the natural compound. In the case of compounds 10 (CC50 = 95.24 and 99.19 μM) and 11 (CC50 = 89.03 and 93.39 μM), although they showed lower cytotoxicity compared to the natural compound (p < 0.001), both needed higher concentrations to obtain the same effect as the natural compound.

Thus, our results showed that the new compounds were more active than the natural compound and more selective, but not simultaneously. The literature suggests that, to obtain more effective and less toxic ligands, the ligand must have a balanced affinity for the PPAR-α receptor.36 This could be achieved by attaching a lipophilic moiety to the pharmacophore. Therefore, a series of non-heterocyclic modifications were designed in series 4 (Table 4).

Influence of the introduction of cycloalkanes and alkanes and degree of unsaturation in the cycloalkanes in the third moity on the cytotoxicity and transcriptional activation of PPAR-α from different cell lines.

N Structure clogP RPTEC (μM) A-498 (μM) WPMY-1 (μM) DU 145 (μM)
CC50 PPAR-α EC50 CC50 PPAR-α EC50 CC50 PPAR-α EC50 CC50 PPAR-α EC50
WY 14643 graphic file with name d3md00063j-u18.jpg 4.92 54.07 ± 0.25 0.32 ± 0.01 2.94 ± 0.57 0.63 ± 0.02 62.88 ± 0.16 0.78 ± 0.02 3.21 ± 0.40 1.62 ± 0.01
— graphic file with name d3md00063j-u19.jpg 2.53 63.30 ± 1.36 0.06 ± 0.01 1.32 ± 0.04 0.18 ± 0.01 71.08 ± 1.91 0.38 ± 0.01 1.57 ± 0.01 0.77 ± 0.02
12 graphic file with name d3md00063j-u20.jpg 2.56 66.65 ± 0.15 0.16 ± 0.02 1.37 ± 0.05 0.31 ± 0.01 78.60 ± 0.16 0.60 ± 0.01 1.63 ± 0.06 1.19 ± 0.04
13 graphic file with name d3md00063j-u21.jpg 2.65 68.39 ± 1.11 0.19 ± 0.01 1.39 ± 0.08 0.36 ± 0.02 81.31 ± 1.79 0.67 ± 0.02 1.68 ± 0.05 1.38 ± 0.05
14 graphic file with name d3md00063j-u22.jpg 2.54 74.82 ± 1.96 0.21 ± 0.02 1.53 ± 0.05 0.47 ± 0.02 87.99 ± 1.28 0.73 ± 0.01 1.81 ± 0.08 1.50 ± 0.03
15 graphic file with name d3md00063j-u23.jpg 2.66 84.43 ± 1.24 0.27 ± 0.02 1.79 ± 0.06 0.58 ± 0.01 96.38 ± 1.38 0.84 ± 0.02 2.81 ± 0.01 1.77 ± 0.04
16 graphic file with name d3md00063j-u24.jpg 2.89 81.66 ± 1.93 0.10 ± 0.01 1.66 ± 0.04 0.23 ± 0.02 92.67 ± 1.25 0.43 ± 0.02 1.93 ± 0.07 0.83 ± 0.02
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In terms of the cytotoxicity results in non-tumour cells, all compounds in the series (12–16) showed better results than the natural compound and the control. In terms of activity, compound 16 showed similar activity (EC50 = 0.23 and 0.83 μM) to the natural compound in prostate tumour cells and slightly lower activity in kidney tumour cells, but showed a lower cytotoxicity in healthy cells, making it a more promising anti-tumour candidate than the natural compound.

In this regard, it was observed that the introduction of a lipophilic residue (16) significantly enhances the selective apoptosis of tumour cells vs. healthy cells without a notable loss of PPAR-α agonist activity. To confirm the interactions of compound 16 with the PPAR-α (PDB: 4BCR) protein, we decided to perform in silico docking studies using the GOLD software,37 which uses a genetic algorithm (GA) to generate different ligand conformations. The binding site was defined using the reference ligand WY-14643 and restricted to atoms within 10 Å.25

Using the GA, 300 docking poses were generated, and then ranked using ChemPLP as a scoring function38 and GoldScore as a re-scoring function. Docked solutions were clustered based on how similar their poses were in terms of RMSD (distance between clusters = 0.8 Å). The top ranked solution (PLPFitness = 53.62; GoldScore = 37.05; RMSd = 0.45 Å) of the largest cluster was selected for further discussion and its interactions were analysed using the LigandScout software.39

Compound 16 exhibited a mode of binding characterised by hydrogen bonding that had been previously observed for other agonists. These unions occur between the polar head of the compound and the different chains of arm I, on the one hand, and the hydrophobic interactions between its alkyl chain and arm II35, on the other hand.40

Our compound's polar component (pyridine) has formed interactions with arm I residues. The polar nature of arm I helps retain the AF2-helix, promoting co-activator binding.32 The residues with which it forms hydrogen bonds have been previously described as relevant for agonist activation of PPAR-α.40–43

Likewise, three hydrogen bonds were found: TYR-464 (1.79 Å), TYR-314 (2.50 Å) and TYR-280 (2.39 Å), coloured red in Fig. 2. The hydrogen bond with TYR464, located in the AF-12 helix, is especially relevant for the reasons mentioned above. The pyridine ring was also observed to have hydrophobic interactions with PHE-273, VAL-444 and LEU-460.

Fig. 2. Compound 16 at the binding site, forming hydrogen bonds with residues (highlighted in red) previously identified as crucial for PPAR agonist interactions.

Fig. 2

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On the other hand, the alkyl tail of compound 16 was observed to have hydrophobic interactions with THR-279, MET-330, LEU-321 and VAL-332, as shown in Fig. 3.

Fig. 3. 2D diagram displaying hydrophobic interactions (highlighted in yellow) and hydrogen bond acceptors (indicated by red arrows).

Fig. 3

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In conclusion, the molecular docking experiments carried out in this study show that compound 16 binds directly to the PPAR-α protein, similar to the agonist WY 14643, exerting an agonist effect on PPAR-α. Once PPAR-α is stimulated, the exact mechanism by which PPAR-α regulates apoptosis can be hypothesised to involve several pathways. One proposed mechanism might involve the regulation of the expression of pro- and anti-apoptotic genes.12 PPAR-α has been shown to induce the expression of pro-apoptotic genes, such as Bax, while inhibiting the expression of anti-apoptotic genes, such as Bcl-2.44 In this sense, direct activation of PPAR-α may trigger its binding to the Bcl2 protein and its subsequent polyubiquitination, thus generating an apoptotic effect.

A second mechanism might involve the regulation of the expression of several key proteins involved in apoptosis, including caspases, which are proteases that cleave and activate other proteins involved in the apoptotic pathway.45 PPAR-α has also been shown to activate the p53 tumour suppressor protein, which can induce apoptosis in response to DNA damage or other stressors.4

Overall, the exact mechanism by which PPAR-α regulates apoptosis is complex and likely to involve multiple pathways and factors. Therefore, additional assays such as real-time PCR could confirm our results. Likewise, once the interaction with PPAR-α is confirmed, in vivo studies (animal models) could provide additional evidence that compound 16 is a promising anticancer compound.

In the present study, a series of 1,2,4-oxadiazole derivatives have been proposed as potential antitumor agents by activating PPAR-α. For this, the natural product 3-(pyridin-3-yl)-5-(thiophen-3-yl)-1,2,4-oxadiazole has been used as a starting point and structural modifications have been made. Pharmacologically, all compounds were more active (PPAR-α agonist effect) than the positive control (WY-14643). As a result of molecular coupling, it has been observed that compound 16 showed hydrophobic interactions and hydrogen bonds with residues such as SER-280 and TYR-464, important for the maintenance of the closed conformation of PPAR-α.

Moreover, future trials could evaluate the action of compound 16 against PPAR-γ/δ to study if its mechanism is selective, or if it exerts dual activation mechanisms.

3. Materials and methods

3.1. General experimental procedures

First-grade organic solvents were used for isolating the compounds. The solvents were purchased from Sigma-Aldrich. Thin-layer chromatography (TLC, Merck Silica gel 60-F254 plates) was used for compound identification. TLC plates thus obtained were visualised with UV light (Spectroline® E-Series UV lamp with one longwave (365 nm) and one shortwave (254 nm) tube, 230 V, New York, USA) and through heating a plate stained with a 5% phosphomolybdic acid solution (12Mo12O3·H3PO4 ≥99.99% Sigma-Aldrich, CAS number 51429-74-4) in 95% ethanol (EtOH), followed by heat application. Manual column chromatography was performed with silica gel (40–63 μm and 20–45 μm, Merck) and the eluent was used in accordance with standard techniques.

1D and 2D NMR spectra were recorded on a Bruker BioSpin GmbH spectrometer operated at 300 MHz (1H) and 75 MHz (13C), with tetramethylsilane (TMS ≥99.9% Sigma-Aldrich, CAS number 75-76-3) as an internal standard. The deuterated solvent was CDCl3-d1 (99.8 atom% D Sigma-Aldrich, CAS number 865-49-6). Spectra were calibrated through the assignment of the residual solvent peak at δH 7.26/δC 77.16 for CDCl3-d1.

HRESIMS analyses were performed using a mass spectrometer with a hybrid quadrupole time-of-flight (QTOF) MAXIS II analyser model from the Bruker commercial house. Samples were analysed through the electrospray ionisation technique by direct infusion at a flow of 3 μL min−1, using methanol (MeOH anhydrous 99.8% Sigma-Aldrich, CAS number 67-56-1) with 0.1% formic acid (97.5–98.5% Sigma-Aldrich, CAS number 64-18-6) as the ionising phase. The source parameters are the following: end plate offset: 500 V; capillary: 3500; nebuliser: 0.2 bar; dry gas: 2.0 L min−1; dry temperature: 250 °C; and mass range of 50–3000 Da.

3.2. General procedure for acyl chloride derivative synthesis

A mixture of the corresponding acid (1.0 eq.) and thionyl chloride (4.0 eq.) in toluene (0.100 eq.) was stirred at room temperature (25 °C) for 3 h. The organic phase was washed with water (15 mL), NaHCO3 sat. solution (15 mL) and brine solution (15 mL). The solvent was removed in vacuo. The product was used directly in the next step.

3.3. General procedure for the synthesis of oxadiazole analogues

To a mixture of (Z)-N′-hydroxynicotinimidamide (1.0 eq.) in DMF (5 mL) was acyl chloride (2.0 eq.) added under a nitrogen atmosphere and the mixture was heated at 120 °C for 40 min under MW irradiation. After 40 min, the reaction was completed (no starting material in TLC), DMF was removed, and water was added. The aqueous phase was extracted with dichloromethane (DCM, 3 × 15 mL). The organic phase was dried over anhydrous Na2SO4 and filtered, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography using an ethyl acetate (AcOEt) gradient (10–80%) in cyclohexane (cHex) as the eluent.

3.4. General procedure for acyl chloride derivative synthesis

A mixture of the corresponding acid (1.0 eq.) and thionyl chloride (11.0 eq.) in toluene (0.2 eq.) was stirred at reflux for 2 h. The solvent was removed in vacuo. The product was used directly in the next step.

3.5. Spectroscopic data

5-(Furan-3-yl)-3-(pyridin-3-yl)-1,2,4-oxadiazole (1)

5-(Furan-3-yl)-3-(pyridin-3-yl)-1,2,4-oxadiazole (1) was prepared from furan-3-carbonyl chloride (48.3 mg, 0.37 mmol) and (Z)-N′-hydroxynicotinimidamide (25.0 mg, 0.185 mmol), in DMF for 40 min at 120 °C. The crude product was purified by column chromatography in cHex/AcOEt (10% to 70% AcOEt) to obtain the required product as a yellowish oil (25.9 mg). 1H NMR (300 MHz, CDCl3) δH 9.26 (d, J = 1.4 Hz, 1H), 8.70–8.61 (m, 1H), 8.38 (dt, J = 7.5, 1.5 Hz, 1H), 8.12 (t, J = 1.5 Hz, 1H), 7.59 (dd, J = 7.5, 1.5 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 7.25 (dd, J = 7.5, 1.5 Hz, 1H).13C NMR (75 MHz, CDCl3) δC 169.9, 167.8, 150.8, 149.7, 145.3, 139.5, 135.5, 123.7, 122.9, 114.3, 113.7. HRESIMS m/z [M + H]+m/z = 214.0251 and [M + Na]+m/z = 236.0489 (calculated for C11H8N3O2+, 214.0617 and C11H7N3NaO2+, 236.0436).

3-(Pyridin-3-yl)-5-(1H-pyrrol-3-yl)-1,2,4-oxadiazole (2)

3-(Pyridin-3-yl)-5-(1H-pyrrol-3-yl)-1,2,4-oxadiazole (2) was prepared from 1H-pyrrole-3-carbonyl chloride (48.0 mg, 0.37 mmol) and (Z)-N′-hydroxynicotinimidamide (25.0 mg, 0.185 mmol), in DMF for 40 min at 120 °C. The crude product was purified by column chromatography in cHex/AcOEt (10% to 70% AcOEt) to obtain the required product as a yellowish oil (30.2 mg). 1H NMR (300 MHz, CDCl3) δH 9.75 (dd, J = 9.5, 7.8 Hz, 2H), 9.26 (d, J = 1.4 Hz, 2H), 8.70–8.61 (m, 2H), 8.37 (dt, J = 7.5, 1.5 Hz, 2H), 7.90 (ddd, J = 9.5, 2.1, 1.5 Hz, 2H), 7.43 (t, J = 7.5 Hz, 2H), 6.70 (td, J = 7.7, 2.1 Hz, 2H), 6.25 (dd, J = 7.5, 1.5 Hz, 2H). 13C NMR (75 MHz, CDCl3) δC 170.1, 167.3, 150.8, 149.7, 135.6, 123.7, 122.9, 122.7, 120.0, 108.8, 107.9. HRESIMS m/z [M + H]+m/z = 213.0852 and [M + Na]+m/z = 235.0782 (calculated for C11H9N4O+, 213.0776 and C11H8N4NaO+, 235.0596).

5-(1-Methyl-1H-pyrrol-3-yl)-3-(pyridin-3-yl)-1,2,4-oxadiazole (3)

5-(1-Methyl-1H-pyrrol-3-yl)-3-(pyridin-3-yl)-1,2,4-oxadiazole (3) was prepared from 1-methyl-1H-pyrrole-3-carbonyl chloride (53.1 mg, 0.37 mmol) and (Z)-N′-hydroxynicotinimidamide (25.0 mg, 0.185 mmol), in DMF for 40 min at 120 °C. The crude product was purified by column chromatography in cHex/AcOEt (10% to 70% AcOEt) to obtain the required product as a yellowish oil (35.0 mg). 1H NMR (300 MHz, CDCl3) δH 9.26 (d, J = 1.5 Hz, 1H), 8.65 (dd, J = 7.5, 1.5 Hz, 1H), 8.37 (dt, J = 7.5, 1.5 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H), 7.17–7.08 (m, 2H), 7.08–7.00 (m, 1H), 3.72 (d, J = 0.6 Hz, 3H).13C NMR (75 MHz, CDCl3) δC 168.5, 167.2, 150.8, 149.7, 135.55, 124.6, 123.7, 123.4, 122.9, 109.6, 109.0, 36.6. HRESIMS m/z [M + H]+m/z = 227.0357 and [M + Na]+m/z = 249.0433 (calculated for C12H11N4O+, 227.0933 and C12H10N4NaO+, 249.0752).

3-(Pyridin-3-yl)-5-(1,2,4-thiadiazol-3-yl)-1,2,4-oxadiazole (4)

3-(Pyridin-3-yl)-5-(1,2,4-thiadiazol-3-yl)-1,2,4-oxadiazole (4) was prepared from 1,2,4-thiadiazole-3-carbonyl chloride (55.0 mg, 0.37 mmol) and (Z)-N′-hydroxynicotinimidamide (25.0 mg, 0.185 mmol), in DMF for 40 min at 120 °C. The crude product was purified by column chromatography in cHex/AcOEt (10% to 70% AcOEt) to obtain the required product as a yellowish oil (33.2 mg). 1H NMR (300 MHz, CDCl3) δH 10.60 (s, 1H), 9.43 (d, J = 1.5 Hz, 1H), 8.66 (ddd, J = 7.5, 1.5, 0.4 Hz, 1H), 8.42 (dt, J = 7.5, 1.5 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H). 13C NMR (75 MHz, CDCl3) δC 173.3, 169.4, 166.5, 159.7, 150.4, 149.7, 135.5, 123.7, 123.4. HRESIMS m/z [M + H]+m/z = 232.0915 and [M + Na]+m/z = 254.0804 (calculated for C9H6N5OS+, 232.0293 and C9H5N5NaOS+, 254.0113).

3′-(Pyridin-3-yl)-3,5′-bi(1,2,4-oxadiazole) (5)

3′-(Pyridin-3-yl)-3,5′-bi(1,2,4-oxadiazole) (5) was prepared from 1,2,4-oxadiazole-3-carbonyl chloride (49.0 mg, 0.37 mmol) and (Z)-N′-hydroxynicotinimidamide (25.0 mg, 0.185 mmol), in DMF for 40 min at 120 °C. The crude product was purified by column chromatography in cHex/AcOEt (10% to 70% AcOEt) to obtain the required product as a yellowish oil (33.0 mg). 1H NMR (300 MHz, CDCl3) δH 9.43 (d, J = 1.5 Hz, 1H), 9.25 (s, 1H), 8.70–8.61 (m, 1H), 8.43 (dt, J = 7.5, 1.5 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H).13C NMR (75 MHz, CDCl3) δC 167.1, 165.8, 163.2, 162.3, 150.3, 149.7, 135.5, 123.7, 123.6. HRESIMS m/z [M + H]+m/z = 216.0074 and [M + Na]+m/z = 238.0749 (calculated for C9H6N5O2+, 216.0521 and C9H5N5NaO2+, 238.0341).

3-(Pyridin-3-yl)-5-(1H-1,2,4-triazol-3-yl)-1,2,4-oxadiazole (6)

3-(Pyridin-3-yl)-5-(1H-1,2,4-triazol-3-yl)-1,2,4-oxadiazole (6) was prepared from 1H-1,2,4-triazole-3-carbonyl chloride (48.7 mg, 0.37 mmol) and (Z)-N′-hydroxynicotinimidamide (25.0 mg, 0.185 mmol), in DMF for 40 min at 120 °C. The crude product was purified by column chromatography in cHex/AcOEt (10% to 70% AcOEt) to obtain the required product as a yellowish oil (29.0 mg). 1H NMR (300 MHz, CDCl3) δH 12.90 (d, J = 4.3 Hz, 1H), 9.43 (d, J = 1.5 Hz, 1H), 8.70–8.61 (m, 1H), 8.43 (dt, J = 7.6, 1.5 Hz, 1H), 8.31 (d, J = 4.3 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H). 13C NMR (75 MHz, CDCl3) δC 166.4, 165.4, 150.3, 149.7, 148.7, 144.5, 135.5, 123.7, 123.5. HRESIMS m/z [M + H]+m/z = 215.0999 and [M + Na]+m/z = 237.0034 (calculated for C9H7N6O+, 215.0681 and C9H6N6NaO+, 237.0501).

5-(1-Methyl-1H-1,2,4-triazol-3-yl)-3-(pyridin-3-yl)-1,2,4-oxadiazole (7)

5-(1-Methyl-1H-1,2,4-triazol-3-yl)-3-(pyridin-3-yl)-1,2,4-oxadiazole (7) was prepared from 1-methyl-1H-1,2,4-triazole-3-carbonyl chloride (53.8 mg, 0.37 mmol) and (Z)-N′-hydroxynicotinimidamide (25.0 mg, 0.185 mmol), in DMF for 40 min at 120 °C. The crude product was purified by column chromatography in cHex/AcOEt (10% to 70% AcOEt) to obtain the required product as a yellowish oil (29.4 mg). 1H NMR (300 MHz, CDCl3) δH 9.43 (d, J = 1.5 Hz, 1H), 8.70–8.61 (m, 1H), 8.54 (q, J = 0.7 Hz, 1H), 8.42 (dt, J = 7.5, 1.5 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H), 4.02 (d, J = 0.7 Hz, 3H). 13C NMR (75 MHz, CDCl3) δC 167.4, 166.2, 151.7, 150.3, 149.7, 144.7, 135.5, 123.7, 123.5, 36.4. HRESIMS m/z [M + H]+m/z = 229.0019 and [M + Na]+m/z = 251.0054 (calculated for C10H9N6O+, 229.0838 and C10H8N6NaO+, 251.0657).

3-(Pyridin-3-yl)-5-(1,2,3,5-thiatriazol-4-yl)-1,2,4-oxadiazole (8)

3-(Pyridin-3-yl)-5-(1,2,3,5-thiatriazol-4-yl)-1,2,4-oxadiazole (8) was prepared from 1,2,3,5-thiatriazole-4-carbonyl chloride (55.3 mg, 0.37 mmol) and (Z)-N′-hydroxynicotinimidamide (25.0 mg, 0.185 mmol), in DMF for 40 min at 120 °C. The crude product was purified by column chromatography in cHex/AcOEt (10% to 70% AcOEt) to obtain the required product as a yellowish oil (32.4 mg). 1H NMR (300 MHz, CDCl3) δH 9.43 (d, J = 1.5 Hz, 1H), 8.70–8.61 (m, 1H), 8.42 (dt, J = 7.5, 1.5 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H). 13C NMR (75 MHz, CDCl3) δC 167.2, 165.1, 161.7, 150.3, 149.7, 135.5, 123.7, 123.5. HRESIMS m/z [M + H]+m/z = 233.0548 and [M + Na]+m/z = 255.0063 (calculated for C8H5N6OS+, 233.0246 and C8H4N6NaOS+, 255.0065).

3-(Pyridin-3-yl)-5-(1,2,3,5-oxatriazol-4-yl)-1,2,4-oxadiazole (9)

3-(Pyridin-3-yl)-5-(1,2,3,5-oxatriazol-4-yl)-1,2,4-oxadiazole (9) was prepared from 1,2,3,5-oxatriazole-4-carbonyl chloride (49.4 mg, 0.37 mmol) and (Z)-N′-hydroxynicotinimidamide (25.0 mg, 0.185 mmol), in DMF for 40 min at 120 °C. The crude product was purified by column chromatography in cHex/AcOEt (10% to 70% AcOEt) to obtain the required product as a yellowish oil (26.8 mg). 1H NMR (300 MHz, CDCl3) δH 9.43 (d, J = 1.5 Hz, 1H), 8.70–8.61 (m, 1H), 8.42 (dt, J = 7.5, 1.5 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H). 13C NMR (75 MHz, CDCl3) δC 167.3, 163.6, 162.7, 150.3, 149.7, 135.5, 123.7, 123.6. HRESIMS m/z [M + H]+m/z = 217.0767 and [M + Na]+m/z = 239.0612 (calculated for C8H5N6O2+, 217.0474 and C8H4N6NaO2+, 239.0293).

3-(Pyridin-3-yl)-5-(2H-tetrazol-5-yl)-1,2,4-oxadiazole (10)

3-(Pyridin-3-yl)-5-(2H-tetrazol-5-yl)-1,2,4-oxadiazole (10) was prepared from 2H-tetrazole-5-carbonyl chloride (49.0 mg, 0.37 mmol) and (Z)-N′-hydroxynicotinimidamide (25.0 mg, 0.185 mmol), in DMF for 40 min at 120 °C. The crude product was purified by column chromatography in cHex/AcOEt (10% to 70% AcOEt) to obtain the required product as a yellowish oil (29.4 mg). 1H NMR (300 MHz, CDCl3) δH 12.51 (s, 1H), 9.43 (d, J = 1.5 Hz, 1H), 8.70–8.61 (m, 1H), 8.43 (dt, J = 7.5, 1.5 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H). 13C NMR (75 MHz, CDCl3) δC 167.0, 162.9, 150.3, 150.3, 149.7, 135.5, 123.7. HRESIMS m/z [M + H]+m/z = 216.0953 and [M + Na]+m/z = 238.0224 (calculated for C8H6N7O+, 216.0434 and C8H5N7NaO+, 238.0453).

5-(2-Methyl-2H-tetrazol-5-yl)-3-(pyridin-3-yl)-1,2,4-oxadiazole (11)

5-(2-Methyl-2H-tetrazol-5-yl)-3-(pyridin-3-yl)-1,2,4-oxadiazole (11) was prepared from 2-methyl-2H-tetrazole-5-carbonyl chloride (54.2 mg, 0.37 mmol) and (Z)-N′-hydroxynicotinimidamide (25.0 mg, 0.185 mmol), in DMF for 40 min at 120 °C. The crude product was purified by column chromatography in cHex/AcOEt (10% to 70% AcOEt) to obtain the required product as a yellowish oil (33.1 mg). 1H NMR (300 MHz, CDCl3) δH 9.43 (d, J = 1.5 Hz, 1H), 8.70–8.61 (m, 1H), 8.43 (dt, J = 7.5, 1.5 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H), 4.43 (s, 3H). 13C NMR (75 MHz, CDCl3) δC 166.9, 160.4, 156.8, 150.3, 149.7, 135.5, 123.7, 38.7. HRESIMS m/z [M + H]+m/z = 230.0269 and [M + Na]+m/z = 252.0264 (calculated for C9H8N7O+, 230.0790 and C9H7N7NaO+, 252.0610).

5-(Cyclopenta-1,4-dien-1-yl)-3-(pyridin-3-yl)-1,2,4-oxadiazole (12)

5-(Cyclopenta-1,4-dien-1-yl)-3-(pyridin-3-yl)-1,2,4-oxadiazole (12) was prepared from cyclopenta-1,4-diene-1-carbonyl chloride (47.6 mg, 0.37 mmol) and (Z)-N′-hydroxynicotinimidamide (25.0 mg, 0.185 mmol), in DMF for 40 min at 120 °C. The crude product was purified by column chromatography in cHex/AcOEt (0% to 40% AcOEt) to obtain the required product as a yellowish oil (30.2 mg). 1H NMR (300 MHz, CDCl3) δH 9.24 (d, J = 1.4 Hz, 1H), 8.65 (ddd, J = 7.5, 1.5, 0.4 Hz, 1H), 8.38 (dt, J = 7.6, 1.5 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H), 7.11 (dd, J = 10.9, 1.0 Hz, 1H), 6.56 (tt, J = 6.2, 1.1 Hz, 1H), 6.07 (dtd, J = 10.9, 6.2, 1.3 Hz, 1H), 2.54 (t, J = 6.2 Hz, 2H). 13C NMR (75 MHz, CDCl3) δC 171.2, 166.3, 150.8, 149.7, 148.4, 139.5, 135.7, 134.3, 128.4, 123.7, 122.5, 40.0. HRESIMS m/z [M + H]+m/z = 212.0805 and [M + Na]+m/z = 234.0829 (calculated for C12H10N3O+, 212.0824 and C11H9N3NaO+, 234.0643).

5-(Cyclopent-1-en-1-yl)-3-(pyridin-3-yl)-1,2,4-oxadiazole (13)

5-(Cyclopent-1-en-1-yl)-3-(pyridin-3-yl)-1,2,4-oxadiazole (13) was prepared from cyclopent-1-ene-1-carbonyl chloride (48.3 mg, 0.37 mmol) and (Z)-N′-hydroxynicotinimidamide (25.0 mg, 0.185 mmol), in DMF for 40 min at 120 °C. The crude product was purified by column chromatography in cHex/AcOEt (0% to 40% AcOEt) to obtain the required product as a yellowish oil (26.6 mg). 1H NMR (300 MHz, CDCl3) δH 9.27 (d, J = 1.5 Hz, 1H), 8.69–8.60 (m, 1H), 8.39 (dt, J = 7.5, 1.5 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H), 6.07 (tt, J = 6.2, 1.0 Hz, 1H), 3.08 (tdt, J = 7.2, 1.1, 0.5 Hz, 2H), 2.97–2.84 (m, 2H), 1.69 (p, J = 7.1 Hz, 2H). 13C NMR (75 MHz, CDCl3) δC 168.7, 167.3, 150.8, 149.7, 135.7, 130.6, 130.2, 123.7, 122.7, 30.9, 28.2, 24.8. HRESIMS m/z [M + H]+m/z = 214.0959 and [M + Na]+m/z = 236.0539 (calculated for C12H12N3O+, 214.0980 and C12H11N3NaO+, 236.0800).

5-(Cyclopent-2-en-1-yl)-3-(pyridin-3-yl)-1,2,4-oxadiazole (14)

5-(Cyclopent-2-en-1-yl)-3-(pyridin-3-yl)-1,2,4-oxadiazole (14) was prepared from cyclopent-2-ene-1-carbonyl chloride (48.3 mg, 0.37 mmol) and (Z)-N′-hydroxynicotinimidamide (25.0 mg, 0.185 mmol), in DMF for 40 min at 120 °C. The crude was purified by column chromatography in cHex/AcOEt (0% to 40% AcOEt) to obtain the required product as a yellowish oil (29.0 mg). 1H NMR (300 MHz, CDCl3) δH 9.23 (d, J = 1.5 Hz, 1H), 8.69–8.60 (m, 1H), 8.37 (dt, J = 7.5, 1.5 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 6.57 (dd, J = 10.9, 6.2 Hz, 1H), 5.80 (dtd, J = 10.9, 6.1, 1.8 Hz, 1H), 4.29–4.15 (m, 1H), 2.49–2.30 (m, 2H), 2.35–2.07 (m, 2H). 13C NMR (75 MHz, CDCl3) δC 179.1, 165.6, 150.4, 149.7, 135.3, 133.4, 131.8, 123.6, 123.1, 44.7, 39.5, 30.0. HRESIMS m/z [M + H]+m/z = 214.0723 and [M + Na]+m/z = 236.0733 (calculated for C12H12N3O+, 214.0980 and C12H11N3NaO+, 236.0800).

5-Cyclopentyl-3-(pyridin-3-yl)-1,2,4-oxadiazole (15)

5-Cyclopentyl-3-(pyridin-3-yl)-1,2,4-oxadiazole (15) was prepared from cyclopentanecarbonyl chloride (49.1 mg, 0.37 mmol) and (Z)-N′-hydroxynicotinimidamide (25.0 mg, 0.185 mmol), in DMF for 40 min at 130 °C. The crude product was purified by column chromatography in cHex/AcOEt (0% to 40% AcOEt) to obtain the required product as a yellowish oil (33.6 mg). 1H NMR (300 MHz, CDCl3) δH 9.23 (d, J = 1.5 Hz, 1H), 8.65 (dd, J = 7.5, 1.5 Hz, 1H), 8.38 (dt, J = 7.6, 1.5 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 3.52 (p, J = 7.0 Hz, 1H), 2.17-1.99 (m, 4H), 1.82–1.58 (m, 4H). 13C NMR (75 MHz, CDCl3) δC 182.8, 165.5, 150.4, 149.7, 135.3, 123.6, 123.1, 38.7, 30.9, 25.8. HRESIMS m/z [M + H]+m/z = 216.0518 and [M + Na]+m/z = 238.0556 (calculated for C12H14N3O+, 216.1137 and C12H13N3NaO+, 238.0956).

5-Pentyl-3-(pyridin-3-yl)-1,2,4-oxadiazole (16)

5-Pentyl-3-(pyridin-3-yl)-1,2,4-oxadiazole (16) was prepared from hexanoyl chloride (49.8 mg, 0.37 mmol) and (Z)-N′-hydroxynicotinimidamide (25.0 mg, 0.185 mmol), in DMF for 40 min at 120 °C. The crude product was purified by column chromatography in cHex/AcOEt (0% to 40% of AcOEt) to obtain the required product as a yellowish oil (31.2 mg). 1H NMR (300 MHz, CDCl3) δH 9.23 (d, J = 1.5 Hz, 1H), 8.69–8.60 (m, 1H), 8.35 (dt, J = 7.5, 1.5 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 2.93 (t, J = 7.1 Hz, 2H), 1.89 (p, J = 7.1 Hz, 2H), 1.50–1.36 (m, 1H), 1.42–1.26 (m, 3H), 0.98–0.82 (m, 3H). 13C NMR (75 MHz, CDCl3) δC 177.3, 165.8, 150.3, 149.7, 135.1, 123.6, 123.0, 30.9, 27.2, 25.0, 22.6, 14.1. HRESIMS m/z [M + H]+m/z = 218.0826 and [M + Na]+m/z = 240.0965 (calculated for C12H16N3O+, 218.1293 and C12H15N3NaO+, 240.1113).

3.6. Cell culture

Two human tumour cell lines were used in this study: A-498 (Homo sapiens kidney carcinoma, HTB-44) and DU 145 (Homo sapiens prostate carcinoma, HTB-81), and two human non-tumour cell lines RPECT (Homo sapiens kidney normal, PCS-400-010) and WPMY-1 (Homo sapiens prostate normal, CRL-2854). They were obtained from the American Type Culture Collection (ATCC). The cells were maintained in Dulbecco's modified Eagle medium (DMEM, Sigma-Aldrich St. Louis, USA) containing 2 mM l-glutamine (AppliChem, Denmark), supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, USA), 100 units per mL penicillin (Fisher Scientific, Pittsburgh, USA) and 100 μg mL−1 streptomycin (Fisher Scientific, Pittsburgh, USA) in culture flasks, in an incubator with a humidified atmosphere with 5% CO2, at 37 °C under normoxic conditions (20–21% O2) for non-tumour cells and under hypoxic conditions (1% O2) for tumour cells (thus mimicking the tumour in vivo microenvironment).

Solutions of compounds (100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 and 0.20 μM, prepared in culture medium with 0.5% DMSO) were prepared from stock solutions at a concentration of 1 mM by dissolving them in dimethyl sulfoxide (DMSO ≥99.9% Merck, cat. no. 67-68-5). WY 14643 (PPAR-α agonist modulator) (≥98% Sigma-Aldrich, CAS number 50892-23-4) was used as a positive control at ten concentrations (100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 and 0.20 μM, prepared in culture medium with 0.5% DMSO). To determine the cytotoxicity of the vehicle (DMSO), culture medium with 0.5% DMSO was added to tumour and non-tumour cells, at the corresponding proportions of the compound solutions. Finally, as a negative control, only culture medium was added to the tumour and non-tumour cells, at the corresponding proportions of the compound solutions.

3.7. Cytotoxicity assay

Cells were seeded in 96-well plates (200 μL, 2 × 104 cells per well) and were grown for 12 h at 37 °C under normoxic (for non-tumour cells) and hypoxic (for tumour cells) conditions. Subsequently, the cells were treated in parallel with different samples (solutions of compounds, positive control, DMSO and negative control) for 72 h under normoxic (for non-tumour cells) and hypoxic (for tumour cells) conditions. The cells were then fixed with cold 10% trichloroacetic acid (TCA ≥99.0% Sigma-Aldrich, CAS number 76-03-9) for 1 h at 4 °C. After washing with water, 100 μL of 0.4% sulforhodamine B (SRB dye content 75% Sigma-Aldrich, CAS number 3520-42-1) in 1% acetic acid (AcOH ≥99.0% Sigma-Aldrich, CAS number 64-19-7) were added to each well and incubated for 20 min. After washing them three times with 1% TCA, the plates were air-dried, and stained cells were dissolved with 100 μL per well of 10 mM unbuffered tris(hydroxymethyl)aminomethane (Tris base ≥99.9% Sigma-Aldrich, CAS number 77-86-1). The optical density was measured at 540 nm, using a microplate reader (ELISA plate reader, SpectraMax® i3, Molecular Devices).

3.8. PPAR-α transcriptional assays

To determine the effects of the tested compounds on PPAR-α transcriptional activity, cells were transiently co-transfected with the expression vector GAL4-PPAR-α and the luciferase reporter vector GAL4-luc using Roti©-Fect (Carl Roth, Karlsruhe, Germany), following the manufacturer's instructions. 24 h after the transfection under normoxic (for non-tumour cells) and hypoxic (for tumour cells) conditions, the cells were treated in parallel with different samples (solutions of compounds, positive control, DMSO and negative control) for 72 h under normoxic (for non-tumour cells) and hypoxic (for tumour cells) conditions. Protein lysates were prepared in a buffer containing 25 mM Tris-phosphate (pH = 7.8), 8 mM magnesium chloride (MgCl2 anhydrous ≥98% Sigma-Aldrich, CAS number 7786-30-3), 1 mM dl-dithiothreitol (DTT ≥99.0% Sigma-Aldrich, CAS number 3483-12-3), 1% polyethylene glycol tert-octylphenyl ether (Triton X-100 ≥99.0% Sigma-Aldrich, CAS number 9036-19-5) and 7% 1,2,3-propanetriol (glycerol ≥99.5% Sigma-Aldrich, CAS number 56-81-5). Luciferase activity was determined using a luciferase assay kit (Promega, Madison, WI, USA), in an Autolumat LB 9510 (Berthold) following the instructions of the luciferase assay kit (Promega, Madison, WI, USA). The protein concentration in the cell extracts was measured by the Bradford method (BioRad). The RLU μg−1 was calculated and the results were expressed as the percentage of induction of GAL4-Luc activity. Specific transactivation was expressed as fold induction over the control (untreated cells).

3.9. Statistical analysis

50% cytotoxicity concentration (CC50) and 50% effective concentration (EC50) values were determined by non-linear regression. All the experiments were performed in triplicate. One-way ANOVA statistical analysis (Tukey's multiple comparison test, **p < 0.05; ***p < 0.001) was performed to evaluate if the differences among values are statistically significant. All the analyses were performed using the 1994–2020 GraphPad Prism Software LLC version 9.0.0. (86) for Mac (producer Dennis Radushev, San Diego, California USA, https://www.graphpad.com).

3.10. Molecular docking

In silico docking studies were carried out using GOLD software (Hermes 2022.2.0 (Build 353591)).37 The protocol was validated by removing the co-crystallised ligand of the receptor and re-docking it into the active site using RMSD for evaluation of the result. The energy-minimised ligand (compound 16) was docked using 300 GA runs and 125 000 operations.

3.11. Protein and ligand preparation

The crystal structure of PPAR-α in complex with WY14643 was obtained from the RCSB Protein Data Bank (PDB ID: 4BCR). Hydrogen atoms were added, the co-crystallised ligand was extracted, and water molecules removed. The binding site was defined using the reference ligand WY-14643 and restricted to atoms within 10 Å.

The structure of compound 16 was drawn using ChemDraw version 21.0.0.28 (1998–2022 PerkinElmer Informatics, Inc.) and then converted to a 3D structure using Avogadro version 1.93.0 (AvogadroLibs version 1.93.1., Qt version 5.15.3).43 Subsequently, the resulting ligand was optimised, and energy-minimisation was carried out through the MMFF94s force field implemented in Ligand Scout.

3.12. Visualisation

Protein–ligand interactions were analysed using Ligand Scout version 4.4.9 build 20220817 [i1_10] © 1999–2023 by Intel:Ligand GmH.39

Author contributions

LAT and JSSC performed the experimental design of the chemistry study; AFS performed the computational modelling; LAT and ARS contributed to the analysis of the spectral data; CSV and CHV contributed to the conception and experimental design of the pharmacological study and analysis of the measurements of the clogP values; LAT contributed to the writing and review of the manuscript.

Conflicts of interest

None.

Supplementary Material

MD-014-D3MD00063J-s001
†

Electronic supplementary information (ESI) available: 1H- and 13C-NMR spectra of the synthetic 1,2,4-oxadiazoles analysed in this study are provided (Fig. S1–S36). See DOI: https://doi.org/10.1039/d3md00063j

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Associated Data

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Supplementary Materials

MD-014-D3MD00063J-s001
MD-014-D3MD00063J-s001.pdf (1.4MB, pdf)

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