Synthesis and biological profiling of novel isocoumarin derivatives and related compounds

J. Serb. Chem. Soc. 86 (7–8) 639–649 (2021) JSCS–5450 Original scientific paper Synthesis and biological profiling of novel isocoumarin derivatives and related compounds MILENA R. SIMIĆ1*#, SLAVICA ERIĆ2, IVAN BORIĆ3, ANNAMARIA LUBELSKA4, GNIEWOMIR LATACZ4, KATARZYNA KIEC-KONONOWICZ4, SANDRA VOJNOVIĆ5, JASMINA NIKODINOVIĆ-RUNIĆ5 and VLADIMIR M. SAVIC1# 1University of Belgrade, Faculty of Pharmacy, Department of Organic Chemistry, Vojvode Stepe 450, 11221 Belgrade, Serbia; 2University of Belgrade, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Vojvode Stepe 450, 11221 Belgrade, Serbia, 3IQVIA, Clinical Department, Rudnička 2, 11000 Belgrade, Serbia, 4Jagiellonian University, Medical College, Department of Technology and Biotechnology of Drugs, Medyczna 9, 30-688 Kraków, Poland and 5University of Belgrade, Institute for Molecular Genetics and Genetic Engineering, Laboratory for Microbial Molecular Genetics and Ecology, Vojvode Stepe 444a, 11042 Belgrade, Serbia (Received 1 December 2020, revised 20 March, accepted 23 March 2021) Abstract: In the continuation of our study of substituted isocoumarins a series of novel 3-azolyl isocoumarin and their thio derivatives, including some related lactone compounds was prepared and biologically profiled against C. albicans showing moderate activity with MIC values in range of 4–60 μg mL-1, in general. The additional characterisation of selected compounds was carried out by exploring their activity on CYP3A4 and CYP2D6 enzymes, while experiments on mutagenicity were performed by AMES test. The representative isocoumarins 3b, 4a and 4b showed lower inhibitory activity on CYP enzymes, when compared to the reference inhibitors, ketoconazole and quinidine. Compound 4a showed a higher mutagenic potential than the other two compounds. Further characterization included cytotoxicity profiling against normal MRC5 cells. Keywords: antifungal compounds; isocoumarins; Candida albicans; CYP enzymes; AMES. INTRODUCTION Isocoumarin derivatives have been intensively studied in recent decades.1–3 In synthetic chemistry, they have been widely explored as starting compounds for the preparation of other heterocyclic derivatives.4,5 Furthermore, these compounds have been subject of detailed research in medicinal chemistry due to their promising general biological potential.6–8 Isocoumarin skeleton can be con* Corresponding author. Е-mail: # Serbian Chemical Society member. https://doi.org/10.2298/JSC201201025S 639 Available on line at www.shd.org.rs/JSCS/ ________________________________________________________________________________________________________________________ (CC) 2021 SCS. 640 SIMIĆ et al. sidered as a privileged structure, as it is frequently encountered in naturally occurring compounds and also in plethora of synthetic derivatives exhibiting whole array of physiological properties. Natural isocoumarins may or may not have C(3) double bond but are rarely unsubstituted. They possess various substituents diversely positioned around the core, although most frequent substitution pattern, represented by structures in Fig. 1, is characterized by the oxygenated aromatic ring and functionality at C(3). Fig. 1. Structures of some natural isocoumarins. The C(3)-derived isocoumarins, either natural or synthetic, are known to have broad range of biological activities, such as anti-inflammatory, antimalarial, antimicrobial, antifungal, cytotoxic, antiangiogenic.6–8 We have disclosed earlier a novel class of these derivatives possessing a heterocyclic ring at C(3).9 Those compounds showed excellent antifungal properties, in some instances even better than voriconazole, which was used as a standard. Some derivatives demonstrated activity against azole resistant strains such as Candida krusei and Candida parapsilosis. Brief exploration of the mechanism of action suggested that the selected compounds did not cause any degradation or any interactions with Candida albicans DNA, implying that the origin of the biological profile of studied isocoumarins is not related to structural modifications of DNA molecules. In order to explore further the structure–activity relationship (SAR) for this class of compounds and perform wider biological characterisation, a small series of additional isocoumarin derivatives and related compounds were synthesised and tested for antifungal activity on C. albicans. Further biological profiling was carried out by exploring cytotoxicity on healthy human MRC5 cells, inhibitory potential on cytochrome P450 (CYP) enzymes, and mutagenicity using AMES assay. EXPERIMENTAL General The NMR spectra were recorded on Bruker Ascend 400 (400 MHz) and Bruker Avance III (500 MHz) spectrometers. Chemical shifts are given in parts per million (δ) downfield from tetramethylsilane as the internal standard. Deuterochloroform was used as a solvent, unless otherwise stated. Mass spectral data were recorded using Agilent Techologies 6520 Q-TOF spectrometer coupled with Agilent 1200 HPLC or Agilent Technologies 5975C MS coupled with Agilent Technologies 6890N GC. IR spectra were recorded on IR Termo Scientific Nicolet iS10 (4950) spectrometer. Melting points were determined using Boetius PHMK 05 apparatus without correction. Flash chromatography was carried out using silica gel 60 Available on line at www.shd.org.rs/JSCS/ ________________________________________________________________________________________________________________________ (CC) 2021 SCS. BIOLOGICAL STUDY OF AZOLYL-ISOCOUMARINS 641 (230–400 mesh) while thin layer chromatography was carried out using alumina plates with 0.25 mm silica layer (Kieselgel 60 F254, Merck). Compounds were seen by staining with potassium permanganate solution and Dragendorff reagent. The solvents were purified by distillation before use. Analytical and spectral data, as well as additional experimental details are given in the Supplementary material to this paper. General procedure for synthesis of thioisocoumarins Thioisocoumarins were synthesised following literature procedure.10 To a solution of isocoumarin (0.12 mmol) in dry toluene (2 mL) Lawesson’s reagent (48.5 mg, 0.12 mmol) was added under nitrogen atmosphere and the mixture was refluxed for 12 h. After the reaction was complete, as indicated by TLC, the mixture was cooled to room temperature and solvent was removed under reduced pressure. The residue was purified by flash chromatography in order to get the clean the product. General procedure for synthesis of azolyl-methylisocoumarins Azolyl-methylisocoumarins were synthesised from 3-bromomethylisocoumarin and corresponding azoles following literature procedure.11,12 General procedure for synthesis of 3-azolyl-phthalides Starting compound, 3-bromophthalide (7) was prepared from phthalide and NBS.13 3-Azolyl-phthalides 8a–c were synthesised from 3-bromophthalide and corresponding azoles following the procedure from literature.14 Biology Antifungal activity. Standard dis (...truncated)