Synthesis and Biological Evaluations of Granulatamide B and its Structural Analogues

Background: While granulatamides A and B have been previously isolated, their biological activities have been only partially examined. The aim of this study was to synthesize granulatamide B (4b), a tryptamine-derivative naturally occurring in Eunicella coral species, using the well-known procedure of Sun and Fürstner and its 12 structural analogues by modifying the side chain, which differs in length, degree of saturation as well as number and conjugation of double bonds. background: The extreme physical and chemical conditions in the marine environment have led macro- and micro-organisms to adapt by synthesizing secondary metabolites for purposes of defense, attack, or signaling [1]. These compounds are often characterized by unique chemical structures, higher oxygen content, and the presence of halogen atoms, and thus differ significantly from compounds isolated from terrestrial organisms[2]. Because marine metabolites exhibit a wide range of biological activities, including anticancer [3], anti-inflammatory [4], antimicrobial [5], antioxidant [6] and others, they have attracted the attention of the scientific community, particularly with regard to the discovery of new compounds as potential drugs that are more potent and less toxic. Although the upsurge of marine (blue) biotechnology did not begin until the middle of the last century, more than 30,000 compounds have been isolated to date, largely due to the implementation of the extensive marine bioprospecting programs [7–9]. As a result, a large number of steroids, terpenes, phenols, polypeptides, glycosides and alkaloids have been extracted, characterized, synthesized and subjected on numerous studies of their biological effects, with a focus on anticancer activity [10]. Marine indole alkaloids include a group of different heterocyclic products that exhibit various pharmacological activities, as already described in the recent literature [11, 12]. So far, they have been isolated mainly from marine microorganisms (fungi and bacteria), sponges, bryozoans, tunicates, molluscs, red algae, mangroves and corals [11]. 2 / 21 However, the latter, are not a prolific source of these chemicals, as alkaloids account for only 4% of all metabolites studied that have been isolated from cnidarians, according to Rocha et al. [13]. Moreover, the coral microbiome may be the actual producer of indole compounds, as the indole synthesis pathway from tryptophan is well-known in bacteria [14] and 3-hydroxy-L-kynurenine, a tryptophan metabolite, has been detected in the coral genus Eunicella [15]. Nevertheless, there are more than 100 indole alkaloids obtained from the coral extracts that exhibit remarkable biological properties such as antibacterial, insecticidal and cytotoxic activities [16]. Notable among them are two N-acyl tryptamines, granulatamides A and B, isolated in 2006 from the soft coral Eunicella granulata (Grasshoff, 1992) and previously studied only on few cell lines for their antiproliferative activities [17]. Such N-fatty acyl tryptamines are a rare group of natural products that have been isolated mainly from terrestrial organisms [18–20] and for which few biological studies were conducted [21–24]. In addition to isolation from natural organisms, total synthetic approaches to the preparation of granulatamides A [25] and B [26] have also been published. Therefore, both granulatamides are important because of their interesting chemical structure consisting of 3 main components: indole nucleus, amide bond and long fatty acyl chain. The indole nucleus is a planar and aromatic heterocyclic motif that is considered a privileged structure in organic and medicinal chemistry [27–29]. Moreover, it is a pharmacophore of numerous natural and synthetic compounds that exhibit various biological activities with the potential to treat different diseases as observed in preclinical and clinical studies [30]. The versatility of pharmacological activities is the result of the chemical nature of indoles, particularly the hydrogen bond donation, high π-electron density and high HOMO (highest occupied molecular orbital) energy. Consequently, indole compounds interact with various target proteins and nucleobases [12, 24]. Due to the delocalization of the nitrogen lone-pair to the π-electron system, the majority of organic reactions occur at the C-3 position (protonation, oxidation, electrophilic substitutions, cycloaddition), whereas the substitution of the N-H bond in the ring occurs under basic conditions [29, 30]. An example of C-3 substituted indoles, are indolamines such as the neurotransmitters tryptamine and its 5-hydroxy derivative (serotonin) or the neurohormone melatonin (5-methoxy-N-acetyltryptamine) [28]. Their potential is shown by their ability to form an amide bond when conjugated, for example, with certain organic acids, leading to more effective biological properties and binding to various biological targets [31]. In addition, such compounds tend to be more stable due to the resonance stability of the amide bond and lower reactivity and susceptibility to nucleophilic attack [32]. Among fatty acids, polyunsaturated fatty acids (PUFAs) play an important role because the presence of a long hydrocarbon chain and a reactive carboxylic group enables the preparation of pharmacologically heterogeneous compounds. In 2020, Jóźwiak et al. extensively investigated the advantages of saturated and unsaturated fatty acids and their condensation with heterocyclic compounds leading to increased tumour selectivity and lower toxicity. Interestingly, ω-3 PUFAs have been shown to modulate inflammatory processes, and act as antibacterial, neuroprotective or anticancer agents. The latter results from the fact that tumour cells use fatty acids more extensively due to increased metabolism, indicating the possibility of increasing the selectivity of compounds conjugated to such acids [33]. Furthermore, the anticancer effect of PUFAs may be related to modulation of membrane integrity and interference with ion channels in the membranes of cancer cells, as has been widely discussed by Mukerjee et al. [34]. Adding the assumption that ω-3 PUFAs may be antioxidant rather than prooxidant, the potential of these acids becomes even clearer [35, 36]. Method: The prepared library of compounds underwent comprehensive assessment for their biological activities, encompassing antioxidative, antiproliferative, and antibacterial properties, in addition to in vivo toxicity evaluation using a Zebrafish model. Compound 4i, which consists of a retinoic acid moiety, exhibited the strongest scavenging activity against ABTS radicals (IC50 = 36 ± 2 μM). In addition, 4b and some of the analogues (4a, 4c and 4i), mostly containing an unsaturated chain and conjugated double bonds, showed moderate but non-selective activity with certain IC50 values in the range of 20-40 μM. objective: Considering the above-mentioned properties of indole-containing compounds, the insufficient data on biological properties and as part of our project Bioprospecting of the Adriatic Sea in relation to the soft corals of genus Eunicella [37, 38], the marine natural product granulatamide B was synthesized alongside with twelve structural analogues. Furthermore, the compounds were screened for their antioxidant, antiproliferative and antibacterial activities and complemented with in vivo toxicity data. Our strategy was based on modulating the biological properties by modifying the side chain using fatty acids that differ in length, degree of saturation, number and position of double bonds and their conjugation. Result: In contrast, the analogue 4l, a derivative of alpha-linolenic acid, was the least toxic towards normal cell lines. Moreover, 4b was also highly active against Gram-positive Bacillus subtilis with an MIC of 125 μM. Nevertheless, both 4b and 4i, known for the best-observed effects, caused remarkable developmental abnormalities in the zebrafish model Danio rerio. Conclusion: Since modification of the side chain did not significantly alter the change in biological activities compared to the parent compound, granulatamide B (4b), the substitution of the indole ring needs to be considered. Our group is currently carrying out new syntheses focusing on the functionalization of the indole core.