pharmaceuticals Communication Sevanol and Its Analogues: Chemical Synthesis, Biological Effects and Molecular Docking 1, , 1,2, 1 1 Olga A. Belozerova * y, Dmitry I. Osmakov y , Andrey Vladimirov , Sergey G. Koshelev , Anton O. Chugunov 1,3,4 , Yaroslav A. Andreev 1,2 , Victor A. Palikov 5, Yulia A. Palikova 5, Elvira R. Shaykhutdinova 5, Artem N. Gvozd 6, Igor A. Dyachenko 5, Roman G. Efremov 1,3,4, Vadim S. Kublitski 1 and Sergey A. Kozlov 1,* 1 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia; [email protected] (D.I.O.); [email protected] (A.V.); [email protected] (S.G.K.); [email protected] (A.O.C.); [email protected] (Y.A.A.); [email protected] (R.G.E.); [email protected] (V.S.K.) 2 Institute of Molecular Medicine, Sechenov First Moscow State Medical University, 119991 Moscow, Russia 3 National Research University Higher School of Economics, 101000 Moscow, Russia 4 Moscow Institute of Physics and Technology (State University), Dolgoprudny, 141701 Moscow Oblast, Russia 5 Branch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 6 Nauki Avenue, 142290 Pushchino, Russia; [email protected] (V.A.P.); [email protected] (Y.A.P.); [email protected] (E.R.S.); [email protected] (I.A.D.) 6 Federal State Budgetary Institution «Scientific Center of Biomedical Technologies of Federal Medical and Biological Agency» (FSBI SCBT FMBA of Russia), 1 Svetlye Gory, Moscovskaya Oblast, 143442 Krasnogorskiy Rayon, Russia; [email protected] * Correspondence: [email protected] (O.A.B.); [email protected] (S.A.K.) These authors contributed equally to this work. y Received: 10 June 2020; Accepted: 21 July 2020; Published: 24 July 2020 Abstract: Among acid-sensing ion channels (ASICs), ASIC1a and ASIC3 subunits are the most widespread and prevalent in physiological and pathophysiological conditions. They participate in synaptic plasticity, learning and memory, as well as the perception of inflammatory and neurological pain, making these channels attractive pharmacological targets. Sevanol, a natural lignan isolated from Thymus armeniacus, inhibits the activity of ASIC1a and ASIC3 isoforms, and has a significant analgesic and anti-inflammatory effect. In this work, we described the efficient chemical synthesis scheme of sevanol and its analogues, which allows us to analyze the structure–activity relationships of the different parts of this molecule. We found that the inhibitory activity of sevanol and its analogues on ASIC1a and ASIC3 channels depends on the number and availability of the carboxyl groups of the molecule. At the structural level, we predicted the presence of a sevanol binding site based on the presence of molecular docking in the central vestibule of the ASIC1a channel. We predicted that this site could also be occupied in part by the FRRF-amide peptide, and the competition assay of sevanol with this peptide confirmed this prediction. The intravenous (i.v.), intranasal (i.n.) and, especially, oral (p.o.) administration of synthetic sevanol in animal models produced significant analgesic and anti-inflammatory effects. Both non-invasive methods of sevanol administration (i.n. and p.o.) showed greater efficacy than the invasive (i.v.) method, thus opening new horizons for medicinal uses of sevanol. Keywords: acid-sensing ion channel; sevanol; electrophysiology; molecular docking; nociception; analgesia; lignan; total synthesis Pharmaceuticals 2020, 13, 163; doi:10.3390/ph13080163 www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2020, 13, 163 2 of 20 1. Introduction Pathological conditions, such as tissue damage, cancer and inflammation, are often accompanied by the acidification of the extracellular environment, which is a primary cause of pain in the sufferer. Acid-sensing ion channels (ASICs), members of the degenerin/epithelial Na+-channels family, are sensitive to even a slight decrease of the extracellular pH [1,2]. In mammals, four genes encode at least six ASIC isoforms: ASIC1a and ASIC3 isoforms are the most studied channels, and they contribute substantially to different physiological processes and pathological conditions. ASIC1a is expressed throughout the central and peripheral nervous systems, and plays an important role in synaptic plasticity, learning and memory, as well as in ischemic processes and anxiety disorders [3–5]. ASIC3 is widely distributed in the peripheral nervous system and non-neuronal tissues, and is involved in the perception of acid-mediated inflammatory pain (both acute and chronic) [6,7], as well as pain from various mechanical stimuli [8,9]. As a result, these channels are regarded as promising targets for the development of analgesic and anti-inflammatory drugs. Indeed, ASIC channels were shown to be inhibited for low-molecular weight compounds of various natures and may, therefore, be promising objects for the study of channels and the design of drugs. Amiloride (a potassium-sparing diuretic agent) inhibits all of the functional isoforms of ASICs (IC50 of 10–60 µM) [10]. A-317567 non-selectively inhibits the ASIC currents (IC50 of 2–30 µM) [11]. Nonsteroidal anti-inflammatory drugs act on different isoforms of ASIC: ibuprofen and flurbiprofen inhibit the ASIC1a-containing channels (IC50 of 350 µM), whereas aspirin and diclofenac inhibit the ASIC3-containing channels (IC50 of 260 and 92 µM, respectively) [12]. Diarylamidines cause the non-specific inhibition of neuronal ASIC currents (IC50 of 0.3–40 µM) [13,14]. NMDA receptor channel blockers, such as memantine, IEM-2117 and 9-aminoacridine, inhibit ASIC1a in submillimolar concentrations [15]. Most of these compounds are applied in clinics, and they have all demonstrated analgesic, anti-inflammatory or neuroprotective effects in tests in vivo [4,7,11]. More effective inhibitory activity on ASIC1a and/or ASIC3 isoforms has been identified in peptide molecules from spider [16,17], snake [18,19] and sea anemone [20–23] venoms, but such animal toxins have not thus far been successfully rendered as medicinal drugs. Sevanol, a lignan isolated from Thymus armeniacus, inhibited human ASIC3 channels with IC50 of 0.3 mM and possessed pain relief activity [24]. The parenteral administration of sevanol (0.01–10 mg/kg) in mice significantly reversed Complete Freund’s adjuvant (CFA) -induced thermal hyperalgesia and reduced the number of writhes in an acetic acid-induced writhing test [25]. An effective scheme of chemical synthesis for sevanol and the fragment making up half of its ((1S,2S)-1-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}propane-1,2,3-tricarboxylic acid, according to IUPAC) was developed, and the biological activity of the synthetic compounds was confirmed in vitro as well as in vivo [26,27]. According to previous work [26], the biological activity of Sevanol’s diastereomer decreased on 20% of the natural Sevanol activity in electrophysiological experiments. The investigation of structure–activity relationships (SAR) is the one of the effective tools for understanding the ligand–receptor interaction and, consequently, for drug design. Here, we present an improved scheme for the synthesis of sevanol and its analogues, and demonstrate a correlation between the activity of the compounds and the number of free carboxyl groups, as well as other physico-chemical descriptors. The competition of sevanol with the RF-amide peptide for ASIC1a modulation, together with molecular modeling, allowed us to predict the presence of the sevanol molecule’s binding site in the central vestibule of the channel. We also demonstrate for the first time that sevanol is effective as an analgesic and anti-inflammatory agent by non-invasive intranasal and, to a greater extent, oral administration. This opens up promising prospects for its use in medicine. Pharmaceuticals 2020, 13, 163 3 of 20 2.Pharmaceuticals Results and 2020 Discussion, 13, x FOR PEER REVIEW 3 of 21 2.1.2.1. Chemistry Chemistry 2.1.1.2.1.1. Sevanol Sevanol Production Production InIn the the present present study, study, we triedwe tried to trace to thetrace correlation the correlation between between the structure the structure of the sevanol of the molecule sevanol (Imolecule) and its biological(I) and its activity. biological We activity. modified We the modified carboxylic the part carboxylic of I and part obtained of I and sevanol obtained derivatives: sevanol s788derivatives: (II), s590 s788 (III) ( andII), s590 epiphyllic (III) and acid epiphyllic EA (IV) (Figureacid EA1 ).(IV) (Figure 1). FigureFigure 1. 1.Sevanol Sevanol andand its its analogues: analogues:I —sevanol;I—sevanol; IIII——sevanolsevanol withwith methylatedmethylated carboxyliccarboxylic groups;groups; IIIIII—sevanol—sevanol shortened shortened by by two two acetic acetic acid acid groups; groups;IV IV—epiphyllic—epiphyllic acid. acid. TheThe synthesissynthesis ofof sevanolsevanol (I()I) waswas completedcompleted basedbased onon thethe methodmethod describeddescribed previouslypreviously [[27].27]. TheThe synthetic synthetic path path included included the the parallel parallel synthesis synthesis of of two two molecules: molecules: the the t-butyl t-butyl isocitrate isocitrate1 1and and the the suitablysuitably protected protected ca caffeicffeic acid acid2 2(Scheme (Scheme1). I). The The method method of of obtaining obtaining the the intermediate intermediate4 4included included removingremoving protecting protecting groups groups (PG) (PG) from from the phenolicthe phenolic hydroxyls hydroxyls of 3. Ourof 3 careful.