Process for obtaining Anti-inflammatory compounds, said compounds, pharmaceutical composition and use of said compounds and of the pharmaceutical composition

Abstract

The present invention relates to a process for synthesising non-steroidal anti-inflammatory compounds derived from spilanthol. The present invention provides a highly desirable optimised process for the development of new anti-inflammatory agents, being an alternative to corticosteroids which present serious side effects with long-term use. The compounds disclosed in the present invention have applications in the pharmaceutical industry, since they have the potential to be used in the treatment of diseases that involve the development of inflammatory processes, such as migraines, rheumatism, osteoarthritis, infections or orthopaedic injuries.

Description

[0001] FIELD OF THE INVENTION

[0001] The present invention relates to a process for producing non-steroidal anti-inflammatory compounds that inhibit the production of Prostaglandin E2 (PGE2) and Leukotriene B4 (LTB4) derived from spilanthol. Furthermore, said compounds that present high inhibitory potency of the production of both relevant inflammatory mediators, PGE2 and LTB4, and a pharmaceutical composition containing said compounds are disclosed.

[0002] The present invention falls within the field of Pharmacotechnology, more precisely in the area of ​​Medicinal Chemistry. FUNDAMENTS OF THE INVENTION TRPV1 ION CHANNELS

[0003] Transient receptor potential vanilloid type 1 (TRPV1) ion channels are targets of interest in the search for new substances with analgesic and anti-inflammatory activity. These channels are a family of non-selective ion channels found primarily in the central and peripheral nervous system. They detect mechanical, physical, or chemical stimuli in the environment, causing pain and inflammation (BUJAK et al., 2019; JARDÍN et al., 2017).

[0004] In this case, activation of TRPV1 channels increases the permeability of Na+ and Ca2+ cations across the neuronal cell membrane, causing an influx of these ions into the cells. This effect generates a mild, momentary sensation of pain and keeps the neurons in a depolarized state for longer. Thus, these cells become less excitable, reducing pain transmission and intensity (JARA-OSEGUERA; SIMON; ROSENBAUM, 2008).

[0005] Inhibition of TRPV1 channels also reduces pain sensation. Reducing neuronal membrane permeability to Na+ and Ca2+ cations causes the threshold voltage for depolarization to take longer to be reached (PREMKUMAR; SIKAND, 2008). Therefore, both inhibition and activation of these channels cause the action potential to take longer to occur, reducing the release of neuropeptides and neurotransmitters responsible for pain transmission (LI et al., 2016). The same occurs with the release of pro-inflammatory substances and chemotactic agents in other cell types. Overall, these effects decrease sensitivity to pain and inflammatory stimuli (BUJAK et al., 2019; LI et al., 2016).

[0006] Studies have been conducted on the importance and participation of TRPV1 ion channels in inflammatory processes, but contradictory results have led to uncertain conclusions. A summary of the results obtained can be seen in Figure 1 (BUJAK et al., 2019). When TRPV1 channel antagonists, such as AMG 9810, SB 366791, capsazepine (CPZ), or BCTC, were used, the inflammatory process was inhibited. Effects such as decreased expression of pro-inflammatory substances, including neuropeptides, substance P, epinephrine, norepinephrine, and cytokines from the interleukin (IL) group, were observed after LPS administration (Figure 1, A) (WANG et al., 2008). Paradoxically, genetic deletion of TRPV1 channel expression, or chemical ablation, caused elevated inflammatory responses in mice. Increased expression of TNF-α, interleukins and infiltration of neutrophils and macrophages were observed (Figure 1, B) (CLARK et al., 2007; FENG et al., 2017; MONEY; MONEY, 2013). .

[0007] In turn, the use of TRPV1 channel agonists, such as capsaicin (CAP), also reduced the inflammatory process. Inhibitions of prostaglandin E, NF-κβ, and cytokine production, as well as decreased tissue inflammation, were observed in mice injected with LPS that had been treated with capsaicin (Figure 1, C) (KOBAYASHI et al., 2012; WANG et al., 2017). However, also contradictorily, when analyzing the effects of natural activation of TRPV1 channels, such as under conditions of low pH or high temperatures, an increase in the inflammatory process was observed. Hyperalgesia, allodynia, leukocyte infiltration, and increased release of substance P and cytokines were observed (Figure 1, D). Results such as these, and the fact that these channels are involved with several types of inflammatory mediators, in addition to other physiological processes, do not allow final conclusions about the function of TRPV1 in inflammatory processes.Furthermore, these ion channels are present in more than one type of cell, with different functions throughout the body, from neurons to immune system cells (BUJAK et al., 2019).

[0008] Natural products of plant origin that are TRPV1 channel activators (agonists) are known. Some of these are fatty acid alkylamides, such as capsaicin and spilanthol (HE-1) (DE LA ROSA-LUGO et al., 2017; VENNEKENS; VRIENS; NILIUS, 2008). There is also an enyne analogue of spilanthol, which exhibits more potent and long-lasting antinociceptive activity, here designated as HE-3. This derivative proved to be more potent in the tailflick test, but further studies are still needed to confirm whether its activity also involves the TRPV1 channel (ALONSO; PASTRE, 2016), as illustrated in the chemical structures below: capsaicin, spilanthol, eugenol, and the enyne analogue of spilanthol.

[0009] Capsaicin was one of the first substances to be reported with this type of activity, but its potency and selectivity are limited, so its prolonged use causes extensive itching and neuronal damage. Therefore, its therapeutic application as an analgesic and anti-inflammatory agent is disregarded (BASITH et al., 2016; HUANG et al., 2013).

[0010] Regarding spilanthol, it is still unclear whether it is a TRPV1 channel agonist or antagonist, but it has shown minor adverse effects such as pruritus and neuronal damage by decreasing nociception. Prior art studies indicate that the hexane extract of Acmella oleracea (jambu), consisting of approximately 85% spilanthol, has TRPV1 antagonist activity at low concentrations (0.1 µg / mL) and agonist activity at high concentrations (30 µg / mL) (DALLAZEN et al., 2018). This dual effect may occur because different concentrations of the ligand can alter the conformation of the ion channel, altering the mechanism of interaction between conformational selection and induced fit. In this case, high concentrations of spilanthol may cause an agonist interaction, similar to a natural ligand (GREIVES; ZHOU, 2014). There is also the possibility that higher concentrations of the other constituents of the jambu extract may alter the conformation of the active site.

[0011] Eugenol is another antinociceptive natural product that is a TRPV1 channel agonist, and its structural similarity to part of capsaicin suggests a similar mechanism of action. Eugenol also exhibits activity against TRPA1 ion channels (YANG et al., 2003). These channels act similarly to TRPV1 but are more specific for nociception from other types of mechanical or chemical stimuli, such as low temperature or various endogenous agonists. The distribution ratio among different cell types also differs from that of TRPV1, when neurons are disregarded (FERNANDES; FERNANDES; KEEBLE, 2012). Like spilanthol, eugenol does not present a high incidence of pruritus and neuronal destruction with prolonged use (CHUNG et al., 2014). INFLAMMATORY PROCESS

[0012] Regarding anti-inflammatory agents, the most investigated biological targets for substances with this activity are the enzymes cyclooxygenases 1 and 2 (COX-1 and COX-2, Figure 2), which are the targets of most anti-inflammatory drugs currently used (TILO GROSSER; EMER SMYTH; GARRET A. FITZGERALD, 2015). However, COX-2 was previously considered an inducible isoform, being more expressed in inflammatory processes and therefore responsible for the production of prostaglandins that act as mediators of pain and inflammation in tissues. The anti-inflammatory effects of drugs that inhibit both isoforms are due to the inhibition of COXs (BERGQVIST; MORGENSTERN; JAKOBSSON, 2020). COX-1 inhibition is the isoform of the enzyme initially called constitutional, being present throughout the organism in processes responsible for homeostasis, in addition to inflammatory processes (SIMON, 1999).It is due to the inhibition of COX-1 that chronic use and high doses of anti-inflammatory drugs can cause adverse gastrointestinal effects and bleeding, because this isoform is responsible for the synthesis of prostaglandins that regulate these physiological processes related to these effects (CHAKRABORTI et al., 2010).

[0013] However, although these data suggest that drugs capable of selectively inhibiting the inducible isoform, COX-2, are more desirable and would not present significant adverse effects, the continuous use of celecoxib has shown this to be incorrect. When using this drug for prolonged periods or at high doses, patients began to experience heart and kidney problems. Studies after the launch of celecoxib found that COX-2 acts as a constitutional enzyme in cardiac and kidney tissues, maintaining homeostasis in these organs (HARRIS, 2006; SETA et al., 2009). Because of these factors, treatments with selective COX-2 inhibitors should be considered for each patient, taking into account pre-existing conditions. Because selectivity reduced, but did not eliminate, gastrointestinal effects, both selective and non-selective anti-inflammatory drugs are valid drug options (MENDES et al., 2012).

[0014] The main strategy used in the development of selective COX-2 inhibitors takes advantage of a single amino acid difference in the catalytic site of these enzymes. COX-1 has an isoleucine amino acid residue at its site (Ile523), close to the arachidonic acid interaction site, as can be seen (Figure 3, A), but with ibuprofen in the same location as the acid. This isoleucine residue also interacts with the substrate, but makes the catalytic site narrower (MALKOWSKI et al., 2000; TAVARES et al., 2017). In turn, the COX-2 enzyme has a valine residue (Val509) instead of isoleucine, which also interacts with the natural substrate. However, the fact that valine is less bulky than isoleucine allows ligands in the COX-2 catalytic site to reach a pocket with hydrophilic amino acid residues, including Arg499, His75 and Gln178 (Figure 3, B).This characteristic of COX-2 is the most explored in the search for selective inhibitors of this isoform (DUGGAN et al., 2010; MEEK; VAN DE LAAR; E. VONKEMAN, 2010).

[0015] Another enzyme important in inflammatory processes is 5-lipoxygenase (5-LOX), which participates in the conversion of arachidonic acid into leukotrienes, such as LTB4 (leukotriene B4). Increased neutrophil recruitment in the inflammatory process is primarily caused by LTB4 (RUBBO; WOOD, 2020). Leukotrienes are important inflammatory mediators, especially in diseases such as asthma and allergic rhinitis. Despite this, compounds that inhibit 5-LOX alone are not very effective as anti-inflammatories and are not widely sought after, except for the treatment of these diseases. This specific inhibition contributes to a decrease in inflammation but leaves out mediators with more significant effects, such as PGE2 (BRUNE, 2004; MARTEL-PELLETIER, 2003).

[0016] One drawback of inhibiting only COX or 5-LOX enzymes is that both use the same substrate, arachidonic acid. Prolonged inhibition of one of these enzymes results in an imbalance in substrate utilization (FIORUCCI et al., 2001). For example, patients taking COX-2 inhibitors may develop allergic rhinitis and asthma. This inflammation of the nasal mucosa and bronchi is largely mediated by leukotrienes, which are then overproduced due to the excess substrate for the 5-LOX enzyme. Thus, a similar problem could occur with specific 5-LOX inhibition, leading to excess synthesis of mediators synthesized by COX enzymes (MARTEL-PELLETIER, 2003). Because of these issues, dual inhibitors of COX and 5-LOX enzymes are currently being investigated as anti-inflammatory agents.Inhibitors with these activities exhibit a synergistic effect in reducing inflammation because they are capable of addressing a wider range of mediators. With this effect profile, they can achieve greater efficacy with lower doses (CELOTTI; DURAND, 2003). Currently, corticosteroids are the most potent class of anti-inflammatory drugs used in therapy, exhibiting this dual activity profile.

[0017] However, because they are synthetic substances derived from hormones produced by the adrenal glands, patients begin to experience adverse effects such as excessive fatigue, anxiety, and immunosuppression when using them for prolonged periods or in high doses (CHARLIER; MICHAUX, 2003). In turn, some nonsteroidal anti-inflammatory drugs (NSAIDs), such as indomethacin and acetylsalicylic acid, also present this dual activity profile, but with considerably less potency compared to 5-LOX (PAAJANEN; MÄNNISTÖ; UOTILA, 1982; UOTILA et al., 1981). Because of these issues, the search and demand for NSAIDs capable of acting with high potency on both COX and 5-LOX enzymes is growing.

[0018] Below, we present the chemical structures of some known compounds that exhibit dual inhibition of COX and 5-LOX enzymes. Plant extracts that exhibit this dual activity are known, such as the lipid extract of Acrocomia crispa fruits and the methanolic extract of Polyalthia longifolia seeds (NGUYEN et al., 2020; PÉREZ et al., 2017). One of the diterpenes found in the Polyalthia longifolia extract had an IC50 in the nanomolar range (NGUYEN et al., 2020). Synthetic derivatives of chalcones or benzothiophenes also exhibit this dual activity against COX and 5-LOX, with IC50 values ​​in the micromolar range (P et al., 2018).

[0019] In addition to the aforementioned antinociceptive activity, spilanthol and eugenol also exhibit anti-inflammatory activity reported in the literature. Spilanthol has been shown to inhibit the inflammatory process through several pathways. Extracts from A. oleracea flowers inhibited nitric oxide (NO) production and the activity of the enzymes nitric oxide synthase (iNOS) and COX-2 in activated macrophages. Nitric oxide is also an inflammatory mediator with potent vasodilatory activity (BAKONDI et al., 2019). Additionally, spilanthol also reduced the concentration of other mediators, such as interleukins 6 and 1β, and TNF-α in RAW2 cells64,7. Interleukins 6 and 1β are mediators involved in lymphocyte recruitment and pyrogenesis during inflammation. TNF-α is important in regulating the inflammatory response and production of other mediators and is also a pyrogen (ABDUL RAHIM et al., 2021; WU et al., 2008).Finally, it was demonstrated that spilanthol could suppress NF-κB and MAPK signaling in lung epithelial cells, decreasing the production of PGE2 and the inflammatory mediator MCP-1, responsible for monocyte recruitment (HUANG et al., 2018b).

[0020] In turn, eugenol also demonstrates anti-inflammatory activity. This natural product decreased the concentration of inflammatory mediators such as interleukin 6, TNF-α, NO, and PGE2, in addition to decreasing the production of the enzymes iNOS and COX-2 (BARBOZA et al., 2018; DE ARAÚJO LOPES et al., 2018). Eugenol also demonstrated antinociceptive and anti-inflammatory activities in in vivo tests with carrageenan and acetic acid performed in mice (DANIEL et al., 2009). MOLECULAR HYBRIDIZATION STRATEGY

[0021] Molecular hybridization is a drug development strategy that involves combining two or more pharmacophoric groups of distinct substances into a single compound. These groups generally belong to different classes of substances that promote the same type of activity or desired physiological effect. Thus, the principle is that, by combining these groups, the pharmacological effect of the resulting hybrid will be superior (BARREIRO; ALBERTO MANSSOUR FRAGA, 2015). Possible results obtained from molecular hybridization can be divided into two categories. In one, the resulting hybrid has affinity for, and interactions with, more than one type of receptor or enzyme therapeutically relevant to a specific disease. In this case, the objective is to obtain a synergistic therapeutic effect as a result of this type of combination and the affinity with different active sites (OLIVEIRA PEDROSA et al., 2017).Another possibility of hybridization is that its interaction with a single biological site is increased. Instead of promoting interaction in more than one site at the same time, this hybridization adds the intermolecular interactions of the groups used against a specific biological target (BÉRUBÉ, 2016).

[0022] New substances that act on different therapeutic targets, seeking a synergistic pharmacological effect, are known as multitarget drugs (MAKHOBA et al., 2020). Some examples of well-known and widely used multitarget drugs are acetaminophen, ibuprofen, acetylsalicylic acid, and dipyrone. The mechanisms of action of these drugs are not yet fully understood, but they all have at least one pharmacological effect, such as analgesia, anti-inflammatory, and antipyretic effects (STRAUBE et al., 2011). Recently, synergistic hybridization with affinities for different targets has shown improved pharmacodynamic and therapeutic results, especially in more complex diseases. Diseases such as cancer (WANG et al., 2019), diabetes (CEFALO et al., 2019), Alzheimer's disease (LI et al., 2014b) and resistant bacterial infections (KLAHN; BRÖNSTRUP, 2017) show good therapeutic responses to multi-target substances.These results break the paradigm that the more specific and selective a candidate is, the better and easier its approval and adoption as a drug. However, to be well-planned, this approach has the limitation of knowing more than one therapeutic target relevant to the pathology, in addition to substances that show affinity for these targets (CATTO et al., 2018).

[0023] Hybrids can be developed from two possibilities. The first combines pharmacophores using chemical structures called connectors, or linkers, without drastically modifying the initial structures. The second type of hybridization involves the fusion of pharmacophoric groups from distinct substances into a structure that bears little resemblance to the initial ones. As a result, the union of these pharmacophoric groups can transform the pharmacophore into a new chemical entity (PROSCHAK; STARK; MERK, 2019).

[0024] Examples of drug candidates developed using molecular hybridization strategies, combining two structures using linkers, will be presented below. The first, as shown in the diagram below, involves the development of a multifunctional drug for the treatment of Alzheimer's disease, using a linker structure to unite both pharmacophores.

[0025] Suggested causes for this disease include abnormal deposits of β-amyloid proteins, oxidative stress, unwanted metal accumulation, and low levels of acetylcholine, all in the brain. Because crystallographic studies of the enzyme acetylcholinesterase indicate that it has a peripheral anionic site near its catalytic site, the development of a ligand that could interact simultaneously in these two regions was rationalized. Another justification for this approach is that biochemical studies indicate that interactions with this anionic site are responsible for some of the accumulation of β-amyloid proteins in the brain. Therefore, the authors utilized the inhibitory properties of tacrine, a drug already approved for the treatment of Alzheimer's disease, which has a high affinity for the acetylcholinesterase catalytic site.The other pharmacophore used was rhein, which interacts with the aforementioned peripheral site and exhibits activities relevant to the treatment of this disease, such as reducing β-amyloid formation in brain tissue, as well as antioxidant and metal chelating properties. This strategy led to the preparation of a hybrid compound that is five times more potent (IC50 = 58.7 nM) than tacrine in inhibiting acetylcholinesterase, likely due to its co-interaction with the enzyme's peripheral site. Furthermore, the same hybrid reduced β-amyloid accumulation in brain tissue by 70.2% at a concentration of 100 µM (LI et al., 2014).

[0026] Aiming at the development of antibacterials against strains resistant to current drugs, multitarget inhibitors of DNA gyrase enzymes were developed, as shown in the diagram below.

[0027] One class of drugs that effectively inhibit this enzyme is fluoroquinolones, such as ciprofloxacin. However, several bacterial species have strains resistant to this class of antibacterials, with the most common resistance mechanism being the expression of efflux pumps. Thus, the state of the art takes advantage of the fact that plant extracts containing high concentrations of flavonoids have been shown to be effective in inhibiting efflux pumps similar to those of fluoroquinolones. By hybridizing various types of nuclei belonging to these two classes, a hybrid was developed between ciprofloxacin and naringenin, substances from each class that showed the greatest effectiveness when combined. The activity of the resulting hybrid, IC50 in µg / mL, against several drug-resistant bacterial species was 8 to 88 times greater than the control of pure ciprofloxacin.Confirmation of the product's multitarget activity was reinforced because higher concentrations of the hybrid accumulated in the bacterial cytoplasm. Furthermore, there was a prevalence of twisted DNA in the cell nucleus, which is consistent with DNA gyrase inhibition (XIAO et al., 2014).

[0028] There are also drug candidates developed through molecular hybridization strategies that fuse pharmacophoric groups into a new pharmacophore. Many azo compounds, such as benzoxazoles and benzothiazoles, and more specifically, 4'-amino-3'-methyl-2-benzothiazoles, have reported antitumor activity, both in vitro and in vivo, against various cancer cell lines. However, their activity declines rapidly due to metabolism by the CYP 450 enzyme. Other compounds relevant for antineoplastic treatments are those containing the imide group, such as thalidomide, due to their angiogenesis inhibitory activity.

[0029] This type of activity can be exploited to prevent and control tumor growth, since blood flow to solid tumors is a key factor for their growth. In this context, the hybrid shown in the diagram below was developed, a benzothiazole-phthalimide that has an IC50 value of 6 nM against MCF7 tumor cells.

[0030] This hybrid was developed by combining pharmacophoric groups from distinct substances into a pharmacophore of a new chemical entity. Cell cycle analyses show that apoptosis induction was the primary factor responsible for antineoplastic activity, rather than kinase enzyme inhibition (PHILOPPES; LAMIE, 2019).

[0031] Inhibiting the interaction of the Eukaryotic Translation Initiator Factors (EIF4E) and eIF4G has become a therapeutic target of interest in recent years for the treatment of cancers that exhibit normal or exaggerated expression of these factors. This approach has shown promise in sensitizing cells to antitumor treatments to which they have acquired resistance, since the complex formed between the factors is important for mRNA translation. Therefore, it is possible to control the overexpression of proteins and structures linked to the control of apoptosis, proliferation, and cell metastasis. Based on these data, the hybridization of one of the few known inhibitors of this complex, 4EGI-1, was rationalized. It was hybridized with WLJ18, another inhibitor developed in previous research and illustrated in the diagram below.

[0032] After initial hybridization, an REA study optimized the initially obtained hybrid, resulting in another, more potent derivative, EGPI-1. This compound exhibits activities 2- to 23-fold greater than its predecessors against different tumor cell lines, in addition to a satisfactory pharmacokinetic profile (WANG et al., 2019).

[0033] In view of all the above, the present invention investigates the synthesis and development of spilanthol derivatives with more potent anti-inflammatory and antinociceptive activities, for systemic or local use. In this sense, the present invention proposes the use of a structural modification strategy, called molecular hybridization, to develop new and surprising hybrid compounds of spilanthol or its analogue enyne, such that the new compounds are more effective and potent than the compounds described and saturated in the prior art.

[0034] Some prior art documents describe the synthesis of hybrid derivatives of substances with anti-inflammatory and antinociceptive activities, such as spilanthol and eugenol, such as:

[0035] Patent document EP 2636666 B1 entitled “TRANS-2-DECENOIC ACID DERIVATIVE AND MEDICAMENT CONTAINING SAME” discloses a list of pharmaceutical compounds as active ingredients that have highly safe neurotrophic activity or alleviating action for side effects induced by the administration of anticancer agents. Compound 28, selected from the listing of said document, has a similar structure to the active compounds of the present invention.

[0036] However, the aforementioned document does not present the anti-inflammatory activity of the compounds to be protected, in terms of inhibiting the production of PGE2 and LTB4. In this case, the effect presented is neurotrophic, which is not related to the anti-inflammatory activity in question. Although compound 28 in the aforementioned document has a similar structure, there are thousands of possible structural derivatives of this compound until reaching the structures of the derivatives revealed in the present invention. There are modifications in at least seven atoms of the structure, which would not be obvious, even if it referred to the same type of activity. It is noteworthy that the aforementioned document presents another type of biological activity, which is not directly related to the anti-inflammatory effect.The molecules are considerably simpler than those developed in the present invention, and the anti-inflammatory activity presented in the present invention is not directly related to the neurotrophic effect presented in the aforementioned document. Furthermore, the compounds of the present invention exhibit anti-inflammatory activity through a dual mechanism, exhibiting high inhibitory potency on the production of both relevant inflammatory mediators, PGE2 and LTB4. This effect is highly desirable for the development of new anti-inflammatory agents, being an alternative to corticosteroids, which have serious side effects with chronic use.

[0037] Patent document BR 10 2016 017871 1 A2 entitled “PROCESS FOR OBTAINING SPILANTHOL AND ANALOGUES, SPILANTHOL AND ANALOGUES OBTAINED” discloses a process for obtaining spilanthol and analogues. The analogue compounds mentioned have a structure similar to the active compounds of the present invention.

[0038] However, it should be noted that the concatenation of the steps, with regard to the reactions and their ordering for the synthesis of the compounds of the present invention, is not obvious given the state of the art. All the steps performed underwent optimizations, including the need for the use of protecting groups, which could not have been anticipated in the aforementioned document.

[0039] It is noted that the aforementioned document describes the synthesis of compounds simpler than the compounds disclosed in the present invention that exhibited anti-inflammatory activity. The compounds were also evaluated in the ex vivo study and were found to be less active. The present invention describes the synthesis of more complex compounds, for example with a different substitution pattern, that exhibit anti-inflammatory activity ex vivo and in vivo.

[0040] The scientific papers entitled “A NEW APPROACH FOR THE TOTAL SYNTHESIS OF SPILANTHOL AND ANALOGUE WITH IMPROVED ANESTHETIC ACTIVITY” and “NEW APPROACH FOR THE TOTAL SYNTHESIS OF SPILANTHOL AND EVALUATION OF ANTINOCICEPTIVE ACTIVITY” refer, respectively, to a simplified process for obtaining spilanthol and its analogues. The analogue compounds cited showed superior effects when compared to spilanthol and the commercial standard EMLA. ® , the main topical anesthetic currently used. These analogs have a chemical structure similar to the compound of the present invention.

[0041] However, although the substances of the present invention are, initially, derivatives of the compounds disclosed in said document, the anti-inflammatory activity of the present invention is not obvious in light of the aforementioned prior art. The document in question presents the antinociceptive activity of the initial compounds (spilanthol and enine analogue). This anti-inflammatory activity of said derivative was then surprisingly enhanced by the present invention, arriving at the derivatives described herein HE-8, 13, 16 and 17.

[0042] It is also considered that the antinociceptive activity observed for the compounds in this document occurred after topical application. The results of the present invention demonstrate that the compounds can act systemically, as they exhibited activity in a much more complex environment. The present invention optimized the anti-inflammatory activity of the enyne derivative, which was also described during the development of the present invention. The mechanisms for antinociceptive and analgesic activities are different from those for anti-inflammatory activities. The compounds exhibit anti-inflammatory activity through a dual mechanism, exhibiting high inhibitory potency on the production of both relevant inflammatory mediators, PGE2 and LTB4.

[0043] The scientific document entitled "EXPERIMENTAL AND THEORETICAL SPECTROSCOPIC CHARACTERIZATION, NLO RESPONSE, AND REACTIVITY OF THE PHARMACOLOGICAL AGENT SPILANTHOL AND ANALOGUES" reveals an evaluation of spilanthol analogs with great potential for use as therapeutic agents. Furthermore, the structure of the analog S2 in the aforementioned document is similar to the compounds of the present invention.

[0044] The aforementioned document presents an experimental and theoretical (DFT) investigation of various electronic properties related to the molecular structure of spilanthol (S1) and the other two analogs (S2 and S3). The document focuses on optical-electronic materials, which seeks to predict whether certain chemical structures might exhibit biological activity. However, the document concludes that in vitro and in vivo studies are still needed to confirm and identify such biological activities. The present invention, in turn, reveals that the anti-inflammatory activity exhibited by the developed compounds is unrelated to the optical properties revealed in the aforementioned document.

[0045] The scientific paper, titled "IDENTIFICATION OF A PARTIAL AND SELECTIVE TRPV1 AGONIST CPIPC FOR RELIEF OF INFLAMMATORY PAIN," reveals the TRPV1 partial agonist, MDR-652, and discusses its antinociceptive activity in mouse models of inflammatory and neuropathic pain. Additionally, it reveals that it has less toxicity compared to capsaicin, suggesting that the development of subpotent TRPV1 partial agonists may be an effective strategy for pain relief. Furthermore, the TRPV1 partial agonist MDR-652 has been shown to be an effective strategy for treating inflammatory and neuropathic pain with less toxicity.

[0046] The aforementioned document presents the analgesic effect on inflammatory pain of another precursor of the substances of the present invention, MDR-652. In this case, MDR-652 presents this analgesic effect because it is a partial agonist of TRPV1 ion channels, which are closely related to pain transmission, and which studies indicate is sensitized in inflammatory processes by PGE2. Therefore, MDR-652 does not exhibit the same activity as the compounds in the present invention, which is to inhibit the production of the inflammatory mediators PGE2 and LTB4, possibly by inhibiting the enzymes COX and 5-LOX.

[0047] The initial planning of the compounds of the present invention was TRPV1 channel agonists, however, due to the lack of a specific test for this activity, a surprising and unexpected anti-inflammatory activity was demonstrated, which was much more potent and effective.

[0048] Furthermore, as previously noted, the relationship between TRPV1 channel activity and inflammation remains controversial, and it cannot be stated that a TRPV1 agonist or antagonist compound will exhibit anti-inflammatory activity. MDR-652 and the compounds presented in this document reduce the sensation of inflammatory pain, but do not reduce the inflammation itself, an activity demonstrated by the compounds of the present invention. Thus, the derivatives disclosed in this invention exhibit anti-inflammatory activity, which is not observed for MDR-652, and reduce the pain caused by this inflammation.

[0049] It should also be noted that the structures of the compounds of the present invention are quite distinct from MDR-652.

[0050] The scientific document entitled “ANTI-INFLAMMATORY AND QUINONE REDUCTASE-INDUCING COMPOUNDS FROM BEILSCHMIEDIA MANNII” discloses compounds with a chemical structure similar to the active compounds of the present invention, such as compound 3 or 2 of the aforementioned document. Additionally, the document highlights the anti-inflammatory characteristics of the compounds.

[0051] It is well-known that fagaramide, a natural product, exhibits anti-inflammatory activity. Therefore, its molecule is also used as a precursor for the substances in this invention. However, the specific mechanism of fagaramide's anti-inflammatory action, much less its pharmacophoric group, is not known. The molecular docking assay performed in this invention suggests greater inhibitory activity of the COX and 5-LOX enzymes obtained by the more active derivatives, compared to the compounds used as prototypes. Furthermore, it is noteworthy that the modifications made to the chemical structure of fagaramide yielded a significantly more active compound. Such modifications are not obvious given the large number of possibilities for structural modification of fagaramide. Therefore, the present proposal presents a derivative of this natural product that is more potent against these enzymes (HE-17).The most active molecules in the ex vivo assay are much more complex and potent than fagaramide.

[0052] Furthermore, the aforementioned document reveals that the anti-inflammatory activity of fagaramide is not related to the inhibition of NF-kB and is not capable of suggesting a mechanism.

[0053] The scientific document entitled “ANTI-INFLAMMATORY DRUG DESIGN USING A MOLECULAR HYBRIDIZATION APPROACH” describes the use of molecular hybridization for the analysis and development of new anti-inflammatory drugs with the aim of obtaining drugs with multiple ligands, acting on one or multiple targets, allowing synergistic action and minimizing toxicity.

[0054] This document is a review that presents a collection of anti-inflammatory substances obtained through the use of molecular hybridization in the development of new drugs. The definition of molecular hybridization presupposes knowledge of the pharmacophoric groups of the molecules to be combined in the new hybrid. In the case of the molecules chosen as prototypes for this invention, the pharmacophoric groups of spilanthol (or its enyne derivative) or eugenol are unknown in terms of anti-inflammatory activity. Therefore, it would not be possible to determine which parts of the molecules should be combined in a new chemical structure to potentially generate a new, more active chemical entity. The same reasoning applies to the compound MDR-652.

[0055] This strategy can often work to obtain compounds more active than their precursors, for any type of biological activity of interest, but it has the same probability of failure. In the present invention, for example, of all the compounds designed with this strategy, only three actually showed improved activity. This was verified in our study, with HE-16 losing its PGE2 production inhibition activity. It can be seen that hybridization from portions of the more active derivatives HE-8 and 13 was not as efficient in improving, or even maintaining, the PGE2 production inhibition activity of the HE-16 derivative. A significant loss of activity occurred for this derivative.

[0056] Molecular hybridization can serve as a guide, but without adequate knowledge and rationalization, success is unlikely. Therefore, using this strategy is far from making new drug development an obvious process.

[0057] The present invention discloses compounds that exhibit anti-inflammatory activity through a dual mechanism, exhibiting high inhibitory potency on the production of both relevant inflammatory mediators, PGE2 and LTB4. This effect is highly desirable for the development of new anti-inflammatory agents, representing an alternative to corticosteroids, which have serious side effects with chronic use.

[0058] It is noteworthy that knowledge about which modifications would be made to the molecules, and how the prototypes would be joined, is not obvious given the state of the art due to the lack of knowledge about the groups responsible for the biological activities of each molecule individually (eugenol and spilanthol / enyne analog or MDR-652). Therefore, the synthesis of these compounds would not be obvious. For example, the results suggest that the presence of the eugenol structure (HE-2) in the alkyl chain of HE-3 contributes to the inhibitory activity of PGE2 production. However, this only occurs when the number of carbons between the α,β-unsaturated amide and the alkyne of the alkyl chain is maintained (HE-8 and 9: 84 and 86 nM [PGE2], respectively).

[0059] The prior art, taken together, could reveal a process for synthesizing compounds, but anti-inflammatory activity through inhibition of the production of PGE2 and LTB4 mediators is unknown for any of the compounds presented in this document or in any other prior art document. Spilanthol's anti-inflammatory activity was known, but its mechanism of action was unknown. In the case of MDR-652, only its partial agonist activity of TRPV1 channels is known, which reduces pain caused by inflammatory processes, but not the inflammation itself. However, the more potent anti-inflammatory activity of the derivatives developed in this invention was described based on a development of the initial studies.

[0060] Furthermore, the use of molecular hybridization strategies may facilitate and shorten the process of developing new drugs, reducing the number of derivative substances that need to be synthesized and evaluated to achieve a more active compound. However, this is far from making the process obvious. Considerations such as which parts of each compound's chemical structures will be used and fused, the biological target, and the desired synergistic effect are not obvious to someone skilled in the art. Furthermore, the anti-inflammatory activity of precursors against the inhibition of PGE2 and LTB4 production was unknown.

[0061] It should be noted that the anti-inflammatory activity presupposes different mechanisms, and no prior art document teaches how to obtain the proposed derivatives or the specific mechanism for inhibiting the formation of the PGE2 and LTB4 mediators, making it not trivial to use the teachings described for the proposed invention.

[0062] The interaction of capsaicin and other compounds (such as MDR-652) with the TRPV1 receptor occurs in a head-to-tail fashion. In other words, there are well-known interactions between the polar (head) and nonpolar (tail) parts of capsaicin and the receptors. On the other hand, the interaction mechanisms with COX and LOX enzymes are different, as demonstrated by the molecular docking performed for the compounds prepared with this technology. Therefore, it is not considered trivial to know which parts of the molecules (spilanthol / enine analog and MDR-652) should be combined to develop anti-inflammatory drugs with superior or improved activity. Furthermore, the pharmacophoric groups of the parent molecules are unknown. Therefore, which parts of the molecules should be combined to achieve molecules with superior anti-inflammatory activity would not be considered trivial.In fact, seventeen molecules were prepared and it was possible to verify which substitution patterns are important for activity and which are not. The compound HE-11, also resulting from the hybridization between spilanthol (enyne derivative) and eugenol, was shown to be inactive in the assay that evaluated the inhibition of PGE2 production. .

[0063] Furthermore, the insights presented in this invention are surprising because choosing the portion of the compounds disclosed herein is not trivial. The MDR-652 molecule is much more complex (polyfunctionalized). In addition to a substituted aromatic ring, it has a thiazole ring on the left side of the molecule that bears no resemblance to the nonpolar side chains of spilanthol (and other N-alkylamides). Therefore, the prior art cannot guide the choice of the thiazole ring to combine with spilanthol. This thiazole ring allows for several intermolecular interactions (since it has hydrogen bond acceptors) with the receptor that cannot be achieved with the nonpolar chain of spilanthol. Even the synthesis of MDR-652 from capsaicin (or spilanthol) would not be obvious, since capsaicin has an sp3 carbon separating the aromatic ring from the amide nitrogen.Thus, this change completely alters the electronics of the amide system, which in turn could impact the interaction with amino acid residues in the molecular target.

[0064] It should be added that the uses are distinct, and it is not obvious that a combination of established knowledge would lead to the compounds with anti-inflammatory activity and a dual mechanism of inhibition of the formation of the inflammatory mediators PGE2 and LTB4 of the present invention. The molecules, which were developed based on the analgesic activities of the precursors spilanthol and its analogue enyne, eugenol, and MDR-652, exhibit an unexpected and surprising activity, dually inhibiting the production of the inflammatory mediators PGE2 and LTB4, thus exhibiting anti-inflammatory activity. This occurs because no established prior art knowledge, used alone or in combination, explicitly mentions this biological effect. This anti-inflammatory mechanism is not mentioned, so predicting this effect is neither trivial nor obvious.Having knowledge of which modifications could be carried out on molecules, which molecular modification strategies could be considered, as well as how and where to use them, without any teaching in any of the prior art knowledge, does not arise in an evident manner and, therefore, cannot be considered obvious.

[0065] Initially, the compounds in this invention were designed for potential interaction with TRPV1 ion channels, so MDR-652 was considered a prototype. However, biological activity was explored, and the compounds were evaluated for other types of therapeutic activity / targets, such as antitumor, acaricidal, and anti-inflammatory. In the new analyses, none of the compounds demonstrated antitumor activity, one demonstrated acaricidal activity, and four demonstrated anti-inflammatory activity through dual inhibition of the mediators PGE2 and LTB4. It is noteworthy that almost all of the molecules exhibit some PGE2 inhibition activity, thus all have potential for anti-inflammatory use, except HE-10, HE-11, and HE-7, which were inactive in the experimental models used.Eugenol and spilanthol are compounds with acaricidal activity reported in the literature, yet molecular hybridization between these substances did not improve their acaricidal effect, as can be seen in patent BR1020220170754.

[0066] There are at least six types of strategies widely used in the development of new drugs, such as hybridization, simplification, homologation, or molecular rigidification, as well as bioisosterism and latency. These strategies aim to reduce the difficulty, time, and money spent on drug research, but they are far from always effective. If the use of such strategies were obvious or infallible, the number of drugs available would be much greater; therefore, their use does not detract from the merit of the present invention. Knowledge and experience regarding the best strategy to use, and which active substances, or parts thereof, should be modified or studied, are not obvious. This involves intellectual effort and creativity that derive from the knowledge and results obtained by the present invention throughout its development.

[0067] No prior art document reveals that the molecule, or part thereof, has the mechanism of action proposed by the present invention, nor would the combination of documents be able to guide a person skilled in the art to propose such molecules for the development of a superior anti-inflammatory drug. As mentioned previously, anti-inflammatory activity through inhibition of the production of PGE2 and LTB4 mediators was not explicitly known for any of the compounds presented in these articles / patents. In the case of MDR-652, only its partial agonist activity of TRPV1 channels is known, which reduces pain caused by inflammatory processes, but not the inflammation itself. The prior art reveals compounds that exhibit (topical) anesthetic activity, and these compounds act by blocking ion channels to cause desensitization and consequent pain relief.Further research reveals a TRPV1 channel partial agonist compound, thus exhibiting analgesic activity. The anti-inflammatory activity, via decreased PGE2 and LTB4 levels, has no direct or well-established relationship with activity at TRPV1 receptors. The molecular docking results of the present invention point to the mechanism of action of the substances via inhibition of the COX and 5-LOX enzymes, which participate in the biosynthesis of PGE2 and LTB4, respectively. Therefore, it is completely independent of any action at the TRPV1 receptor.

[0068] Disregarding the significant structural differences between the present invention and the prior art, activity via NFkB could indeed confer anti-inflammatory activity, considering that NFkB is a central regulator of inflammation. However, it is well established that action via NFkB, i.e., central action, is associated with significant adverse effects. A classic example of this is corticosteroid anti-inflammatory drugs, which act on NFkB. Inhibition of PGE2 and LTB4 production by pathways other than NFkB has been associated with greater efficacy and fewer adverse effects than current drugs, reinforcing the importance of protecting the results obtained. Again, the combination of the prior art does not demonstrate that combining some of the molecules known in the prior art would lead to the synthesis of new molecules with anti-inflammatory activity with a dual inhibition mechanism.

[0069] Regarding the strategy used in the synthesis of the compounds (a series of 17 molecules), it is more of a SAR (structure-activity relationship assessment) approach. Several compounds were prepared to verify the structural requirements for expression of activity. The anti-inflammatory activity of the derivatives developed in this invention was described based on an extension of the initial studies that had a different focus. Furthermore, it is emphasized that knowing what modifications would be made to the molecules, and how the prototypes would be assembled, is not obvious.

[0070] The surprising effect is the anti-inflammatory activity through the dual mechanism of inhibition of the production of the inflammatory mediators PGE2 and LTB4. It should also be considered that the activity (mechanism of action and therapeutic target) is different from those reported for the molecules most closely related to the present invention (analgesic or antinociceptive activities). The effect is surprising, as molecules that exhibit this dual inhibition mechanism, highly desirable for the development of anti-inflammatory drugs, are rare. Corticosteroids exhibit this dual mechanism, but their use in clinical medicine is very limited due to their collateral effects, especially with prolonged use. The molecules of the present invention exhibited such dual inhibition, but they are not steroidal. Indeed, the mechanism of action involved does not differ from that already disclosed in the prior art for compounds that share some structural similarity.

[0071] Again, it is worth mentioning that the molecules developed in the present invention present distinct activity (use) (anti-inflammatory activity) and also have a dual mechanism, thus constituting a difference and advantage in relation to what exists in the state of the art.

[0072] Having highlighted the differences and relevance of this proposal in relation to the prior art, it is clear that this proposal is an optimized synthesis process for obtaining compounds that exhibit anti-inflammatory activity through a dual mechanism, exhibiting high inhibitory potency on the production of both relevant inflammatory mediators, PGE2 and LTB4. This effect is highly desirable for the development of new anti-inflammatory agents, representing an alternative to corticosteroids, which have serious side effects with chronic use. Therefore, they have potential for use in the treatment of diseases involving the development of inflammatory processes, such as migraines, rheumatism, osteoarthritis, infections, or orthopedic injuries. BRIEF DESCRIPTION OF THE INVENTION

[0073] The present invention aims to propose an optimized process for the production of nonsteroidal anti-inflammatory compounds. Furthermore, the present invention proposes four chemical structures derived from Spilanthol, namely HE-8, 13, 16, and 17, capable of acting as anti-inflammatory inhibitors of the production of Prostaglandin E2 (PGE2) and Leukotriene B4 (LTB4). The anti-inflammatory activity of these structures occurs through a dual mechanism highly desirable for the development of new alternative agents to corticosteroids, which have serious side effects after chronic use.

[0074] Furthermore, the present invention also proposes a pharmaceutical composition comprising said compounds and at least one pharmaceutically acceptable excipient, as well as the use of said compounds or composition to prepare a medicament for the treatment of diseases involving the development of inflammatory processes, such as: migraines, rheumatism, osteoarthritis, infections or orthopedic injuries. BRIEF DESCRIPTION OF THE FIGURES

[0075] In order to obtain a complete and total view of the objective of this invention, the Figures to which reference is made are presented, as follows.

[0076] Figure 1 illustrates the results obtained when investigating the role of TRPV1 channels in inflammation. Item A corresponds to the results obtained with the use of TRPV1 receptor antagonists, such as capsazepine (CPZ). Item B corresponds to the results of genetic deletion or chemical ablation of these receptors. Item C corresponds to the effects observed when using TRPV1 channel agonists, such as capsaicin. Finally, Item D corresponds to the effects of natural activation of these receptors, such as high temperatures or low pH.

[0077] Figure 2 graphically illustrates prostaglandin biosynthesis.

[0078] Figure 3 illustrates the inhibitors at the catalytic sites of COX-1 and COX-2. In (A) Ibuprofen is shown at the COX-1 site (non-selective); in (B) Celecoxib is shown at the COX-2 site (selective).

[0079] Figure 4 graphically illustrates the anti-inflammatory results of the first ex vivo test. In which the parameters: arepresents values ​​in ng / mL; b represents values ​​in (%) calculated in comparison with vehicle; * represents P<0.05 different compared with vehicle; ** represents P<0.05 different compared with dexamethasone and indomethacin.

[0080] Figure 5 graphically illustrates the anti-inflammatory results of the second ex vivo test. In which the parameters: a represents values ​​in ng / mL; b represents values ​​in (%) calculated in comparison with vehicle; * represents P<0.05 different compared with vehicle; ** represents P<0.05 different compared with dexamethasone and indomethacin.

[0081] Figure 6 graphically illustrates the anti-inflammatory results of the third ex vivo test. In which the parameters: a represents values ​​in ng / mL; b represents values ​​in (%) calculated in comparison with vehicle; * represents P<0.05 different compared with vehicle; ** represents P<0.05 different compared with dexamethasone and indomethacin.

[0082] Figure 7 illustrates a comparison of the poses assumed by the studied ligands with the 5-LOX inhibitors. White carbons: Studied ligands; Cyan: Zileuton; Pink: Meclofenamic acid. In (A) the poses assumed by HE-1, 2, 3, 13, and 14 are presented; in (B) the pose assumed by HE-8 is presented; and in (C) the poses assumed by HE-16 and 17 are presented.

[0083] Figure 8 graphically illustrates the results of the in vivo ear edema assays. Data were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test. All samples were tested at a concentration of 1.5 µM / ear. * p value ≤0.05 compared with vehicle (negative control) and # p value ≤0.05 compared with dexamethasone (positive control).

[0084] Figure 9 graphically illustrates the quantification of myeloperoxidase (MPO) enzymatic activity of the samples. The results were analyzed by analysis of variance (one-way ANOVA) followed by Dunnett's multiple comparison test. * p value ≤0.05 compared with vehicle (negative control) and # p value ≤0.05 compared with dexamethasone (positive control).

[0085] Figure 10 illustrates the evaluation of PGE2 concentration in ear fragments obtained in the in vivo ear edema assay. Samples were tested at a concentration of 1.5 µM / ear. The results were analyzed by univariate analysis of variance (one-way ANOVA) followed by Dunnett's multiple comparison test. * p value < 0.0001 compared with vehicle (negative control) and # p value < 0.0001 compared with dexamethasone (positive control).

[0086] Figure 11 illustrates the evaluation of LTB4 concentration in ear fragments obtained in the in vivo ear edema assay. Samples were tested at a concentration of 1.5 µM / ear. The results were analyzed by univariate analysis of variance (one-way ANOVA) followed by Dunnett's multiple comparison test. * p value ≤ 0.0002 compared with vehicle (negative control) and # p value ≤ 0.0002 compared with dexamethasone (positive control).

[0087] Figure 12 illustrates the molecular docking of the conformations assumed by the ligands in the COX-1 active site. A) Superposition of the conformations related to all ligands. B) Pose pattern of the HE-1 and HE-3 ligands. C) Pose pattern of the HE-2 ligand. D) Pose pattern of the HE-8 ligand. E) Pose pattern of the HE-13 ligand. F) Pose pattern of the HE-14 and HE-17 ligands. G) Pose pattern of the HE-16 ligand.

[0088] Figure 13 illustrates the molecular docking of the ligand interaction modes in the COX-1 active site. HE-1 (A), HE-2 (B), HE-3 (C), HE-8 (D), HE-13 (E), HE-14 (F), HE-16 (G), and HE-17 (H) (white carbons). COX-1 active site (green carbons). Hydrogen bonds are plotted in green dashed lines, and halogen bonds are plotted in blue dashed lines.

[0089] Figure 14 illustrates the molecular docking of the conformations assumed by the ligands in the active site of COX-2. HE-1 (A), HE-2 (B), HE-3 (C), HE-8 (D), HE-13 (E), HE-14 (F), HE-16 (G) and HE-17 (H).

[0090] Figure 15 illustrates the molecular docking of the ligand interaction modes in the COX-2 active site. HE-1 (A), HE-2 (B), HE-3 (C), HE-8 (D), HE-13 (E), HE-14 (F), HE-16 (G) and HE-17 (H) (white carbons). COX-2 active site (green carbons). Hydrogen bonds are represented by green dashed lines, halogen bonds are represented by blue dashed lines and T-stacking interactions are represented by pink dashed lines.

[0091] Figure 16 illustrates the molecular docking of the conformations assumed by the ligands in the 5-LOX active site. A) Superposition of the conformations related to all ligands. B) Pose pattern of the ligands HE-1 and HE-13. C) Pose pattern of the ligand HE-2 and HE-8. D) Pose pattern of the ligand HE-3. E) Pose pattern of the ligands HE-14, HE-16 and HE-17.

[0092] Figure 17 illustrates the molecular docking of the interaction modes of the ligands in the active site of 5-LOX. HE-1 (A), HE-2 (B), HE-3 (C), HE-8 (D), HE-13 (E), HE-14 (F), HE-16 (G), and HE-17 (H) (white carbons) in the active site of 5-LOX (green carbons). Hydrogen bonds are represented by green dashed lines and halogen bonds are represented by blue dashed lines. DETAILED DESCRIPTION OF THE INVENTION

[0093] The present invention relates, in a first embodiment, to a process for producing non-steroidal anti-inflammatory compounds that inhibit the production of Prostaglandin E2 (PGE2) and Leukotriene B4 (LTB4) derived from Spilanthol.

[0094] The process for synthesizing the most potent derivatives of the present invention, namely HE-8, 13, 16 and 17, comprises the following steps: I. Procedure for synthesizing isobutylphosphonatoacetamide (iBuPA); II. Procedure for obtaining intermediates 13.a, 13.b, 13.c, 13.d and 13.e; III. Procedure for obtaining intermediate 8.a; IV. Procedure for Sonogashira reaction; V. Procedure for Swern oxidation reaction; VI. Procedure for Horner-Wadsworth-Emmons (HWE) reaction; and VII. Procedure for removing the t-butyldimethylsilyl group.

[0095] Therefore, to demonstrate the potential of the aforementioned synthesis process, the present invention will be described in more detail in terms of the steps performed and their respective parameters for each potent derivative obtained by the present invention.

[0096] In this sense, the synthesis to obtain the derivatives consists of a convergent process. The phosphonatoacetamide intermediate that will be used specifically for the synthesis of each derivative (intermediates iBuPA or 13.e) is prepared. At the same time, the aldehyde intermediate specific for the synthesis of each derivative is also prepared from a Swern oxidation of a previously prepared alcohol (intermediates 1.b, 8.c, or piperonal). Both intermediates are then combined in a Horner-Wadsworth-Emmons (HWE) reaction.

[0097] Intermediate 17.a is obtained using piperonal as the aldehyde. Also in the case of intermediates 13.f, 16.a, and 17.a, a deprotection reaction is performed to remove the TBDMS (tert-butyl dimethyl silane) group using n-Bu4NF (tetra-n-butylammonium fluoride):

[0098] For the synthesis of intermediate 1.a, a Sonogashira reaction is carried out between 4-pentyn-1-ol and 1-bromo-1-propene, using a base diisopropylamine, a palladium catalyst, preferably Pd(Ph3P)4, a copper(I) salt, preferably CuI, in a polar aprotic solvent, preferably dimethylformamide. Intermediate 8.b is prepared in a similar manner, but intermediate 8.a is used instead of 1-bromo-1-propene. Intermediate 8.a was prepared from the reaction of eugenol with phenyltriflimide using an inorganic base, preferably K2CO3, in a polar aprotic solvent, preferably tetrahydrofuran:

[0099] In turn, the intermediate IBuPA is synthesized from direct amidation with the diethyl ester ethyl phosphonoacetate, using lanthanum(III) triflate as a catalyst. Phosphonatoacetamide 13.e is prepared in a few steps. 2-Fluoro-4-nitrobenzoic acid is esterified with methanol and sulfuric acid to form intermediate 13.a, which is then reduced to intermediate 13.b with sodium borohydride. The alcohol of intermediate 13.b is transformed into a silyl ether group by reaction with a silyl chloride, preferably t-butyldimethylsilyl chloride, to form intermediate 13.c. The nitro group in this intermediate is reduced with Pd / C and H2, forming intermediate 13.d. From the amidation of intermediate 13.d with diethylphosphonoacetic acid, in the presence of EDC and DMAP, phosphonatoacetamide 13.e is formed:

[0100] In a second embodiment, the present invention describes the compounds obtained by the aforementioned synthesis process using the following Markush formula: where: R1 can be selected from 3-fluorophenyl-(4-hydroxymethylene) and isobutyl: R2 can be selected from 4-allyl-1-(but-1-yn-1-yl)-2-methoxybenzene, (E)-hept-2-en-4-yne benzo[d][1,3]dioxol: from the above, it can be inferred that the following compounds are obtained by means of Markush: - Compound HE-8 (E)-7-(4-allyl-2-methoxyphenyl)-N-isobutylhept-2-en-6-ynamide, with formula:

[0102] Obtained as a white solid and with a final yield for the synthesis reaction of 70%. - Compound HE-13 (2E,8E)-N-(3-fluoro-4-(hydroxymethyl)phenyl)deca-2,8-dien-6-ynamide, with formula:

[0103] Obtained as a white solid and with a final yield for the synthesis reaction of 88%. - Compound HE-16 (E)-7-(4-allyl-2-methoxyphenyl)-N-(3-fluoro-4-(hydroxymethyl)phenyl)hept-2-en-6-ynamide of formula:

[0104] Obtained as a white solid and with a final yield for the synthesis reaction of 91%. - Compound HE-17 (E)-3-(benzo[d][1,3]dioxol-5-yl)-N-(3-fluoro-4-(hydroxymethyl)phenyl)acrylamide of formula:

[0105] Obtained as a white solid and with a final yield for the synthesis reaction of 93%.

[0106] Additionally, in order to prove the effectiveness of the present invention, the following characterization and activity verification tests were performed in relation to the most potent compounds HE-8, 13, 16 and 17: a. Molecular docking; b. Ex vivo assay; and c. Ear edema assay.

[0107] The molecular docking results suggest that the synthesized derivatives interact with the COX-1 and COX-2 enzymes, therefore being capable of inhibiting the production of the inflammatory mediator PGE2. This analysis also suggests that the compounds interact with the 5-LOX enzyme, thus being capable of inhibiting the production of the inflammatory mediator LTB4.

[0108] Ex vivo assays performed at a concentration of 1µg / mL showed that the developed compounds have the ability to dually inhibit the production of the inflammatory mediators PGE2 and LTB4, thus acting on two pathways responsible for the inflammatory process, simultaneously and at a low concentration. Since this assay was performed on human blood, without an isolated target, it would not be possible to conclude the compounds' mechanism of action, only that they can inhibit both pathways of inflammatory mediator production. The other assays help to corroborate the activity of the compounds and highlight the mechanism of action, such as molecular docking.

[0109] In in vivo assays of mouse ear edema induced by Croton oil, it was possible to observe that the compounds presented anti-inflammatory activity by reducing the size of the edema, which was observed in mice treated with the derivatives.PGE2 is the main inflammatory mediator responsible for edema, therefore, the anti-edematogenic effect indicates that the COX pathway that produces PGE2 was inhibited. All derivatives showed statistically different activity from the vehicle, the blank used in the test. Among the derivatives, HE-8 demonstrated the most potent activity in this in vivo analysis of topical anti-edematogenic activity (Figure 8).

[0110] The inflammatory response involves the recruitment of immune cells, such as neutrophils, to the site of injury or infection. Neutrophils migrate to the site of inflammation and play a fundamental role in the body's natural defense. However, excessive neutrophil infiltration can cause tissue damage and contribute to the pathogenesis of inflammatory diseases, thus neutrophil recruitment is an interesting therapeutic target for the development of anti-inflammatory drugs.

[0111] This study investigated the inhibitory effects of spilanthol derivatives on neutrophil migration in the same experimental model of ear edema used to verify anti-edematogenic activity. The drug dexamethasone, a corticosteroid known to inhibit not only edema but also neutrophil recruitment, as it is a central inhibitor of the inflammatory cascade, was used as a positive control [5]. Topical applications of HE-8, HE-13, HE-16, and HE-17 significantly inhibited neutrophil migration compared to the negative control (p-value < 0.001). Dexamethasone inhibited neutrophil migration by 56.7%, while the HE-8 derivative inhibited neutrophil migration by 43.5% (Figure 9).

[0112] Neutrophil migration in a tissue can be estimated by the activity of MPO, an enzyme mainly present in neutrophils.Leukotrienes B4 (LTB4), produced through the LOX pathway, are one of the main compounds that act in the recruitment of these polymorphonuclear cells to the inflamed site. Therefore, lower levels of myeloperoxidase (MPO) also indicate potential inhibition of the LOX pathway. Dexamethasone, a steroid drug, indirectly inhibits neutrophil recruitment, whereas current nonsteroidal anti-inflammatory drugs (NSAIDs) act primarily on the COX pathway without necessarily inhibiting neutrophil recruitment. Therefore, the new spilanthol derivatives have potential anti-inflammatory activity through a novel mechanism of action, with potential dual inhibition of the COX and LOX pathways.

[0113] The chemical mediators of inflammation PGE2 and LTB4 are produced by two main pathways of the inflammatory cascade: COX and LOX, respectively. These mediators can be evaluated in the experimental ear edema model mentioned above.All derivatives evaluated demonstrated a significant reduction in PGE2 and LTB4 levels, comparable to the reference drug dexamethasone, and different from the negative control (vehicle; p < 0.0002, Figures 10 and 11). Derivative HE-8 showed the greatest reduction, 60.0% and 14.2%, in PGE2 and LTB4 levels, respectively. The same derivative HE-8 also showed greater inhibition of ear edema, corroborating the result of edema and COX pathway inhibition (Figure 8). The other derivatives HE-13, HE-16, and HE-17 also showed significant inhibition of PGE2 production of 53.2%, 54.0%, and 56.6% and LTB4 of 13.0%, 13.8%, and 14.0%, respectively, compared with the vehicle. All of them demonstrated similarity of effect when statistically compared with the steroidal reference drug, dexamethasone (p < 0.0001 and p ≤ 0.0002) (Figures 10 and 11).The results demonstrated that substances HE-8, HE-13, HE-16 and HE-17 presented dual inhibition of the COX and LOX pathways, and consequently the inhibition of the synthesis of PGE2 and LTB4 production.

[0114] In a third embodiment, the invention provides a pharmaceutical composition comprising said compounds of the present invention and at least one pharmaceutically acceptable excipient. The pharmaceutical composition of the present invention may be in the form of tablets, coated tablets, capsules, dragees or lozenges. Alternatively, the pharmaceutical composition may be in the form of a liquid as a solution for intravenous administration or in topical form, as a cream or ointment. Pharmaceutically acceptable excipients may be selected from carriers, fillers, binders, disintegrants, glidants, lubricants, humectants, buffers, preservatives, colorants, flavorings, sweeteners, thickeners, emulsifiers and antioxidants.

[0115] In a fourth embodiment, the present invention provides for the use of said compounds HE-8, 13, 16 and 17 or pharmaceutical composition for the preparation of a medicament for the treatment of inflammatory diseases, or diseases that present inflammatory pathways involved, such as: migraines, rheumatism, osteoarthritis, infections or orthopedic injuries and asthma or allergic rhinitis, in addition to inflammatory skin diseases.

[0116] To demonstrate its potential, the present invention will also be described in more detail under the aspect of the implemented examples. It should be emphasized that the following description is only for the purpose of elucidating the understanding of the proposed invention and revealing, in more detail, the embodiment of the invention without limiting it to the same. Thus, variables similar to the example are also within the scope of the invention.Examples of Realization DERIVATIVE DESIGN

[0117] The first compounds to be designed, synthesized, and evaluated were derivatives HE-8, 9, 10, and 11. The objective of these derivatives was the structural combination of derivative HE-3 with eugenol, employing the molecular hybridization strategy. The enyne analogue of spilanthol (HE-3) was chosen since it had shown greater antinociceptive and anti-inflammatory activity than spilanthol in the formalin test.

[0118] The scheme below shows the design of the four initial derivatives. The hybridizations to form derivatives HE-8 and HE-9 were performed while maintaining the structures of eugenol and the analogue HE-3. Derivative HE-10 was designed by decreasing the number of atoms in the alkyl chain of HE-3, but with the insertion of an aromatic ring with the eugenol substitution pattern.In the HE-11 derivative, the isobutylamide moiety was replaced by an aromatic amide that also has the eugenol substitution pattern.

[0002]

[0119] After the initial anti-inflammatory results, another 11 compounds were planned. In this case, the derivatives HE-4 and HE-5 evaluate the simplification of the alkyl chain of HE-1 and the analogue HE-3 as shown in the scheme below.

[0120] These analogs allow us to observe the importance of the diene group and the α, β unsaturation conjugated to the amide for the anti-inflammatory effect. The compound HE-15 was evaluated because it is a structural simplification of the derivative HE-8, the most active in the first test, and because it replaces the diene group of HE-1 with a more stable ring, see, for example, the scheme below.

[0121] Fagaramide (HE-14) is a natural product of the N-alkyl amide class, similar to spilanthol, and has a known anti-inflammatory activity, with results that suggest inhibition of prostaglandin production, see the diagram below. Its preparation allows evaluating a simplification of the unsaturated alkyl chain of spilanthol, in addition to confirming its PGE2 inhibitory activity (PEREZ G., 2001).

[0122] The HE-6 derivative was designed from the modification of the substituents of the aromatic ring of the HE-8 derivative, aiming to analyze the effect of inversion of the electron density in this ring, see the example in the scheme below.

[0123] This substitution pattern was chosen because the reagent was available for immediate use. With the HE-7 derivative, the aim was to analyze the effect of replacing the isobutylamide of HE-3 with an aromatic moiety with a similar size to that of capsaicin, but without the same substitution pattern, which is known to be responsible for the pungency of capsaicin, see the scheme below (HUANG et al., 2013; JANUSZ et al., 1993).

[0124] The HE-12 derivative was designed from molecular hybridization with lidocaine, which is widely used as a topical or injectable anesthetic (CARACAS et al., 2009; VAN DER WAL et al., 2015) see the scheme below

[0125] It is known that oral applications of lidocaine are limited due to its low bioavailability, since the tertiary amine and aromatic amide portion of its chemical structure are susceptible to metabolic reactions. Thus, the effect of first-pass metabolism drastically reduces the concentrations of lidocaine that are ingested, hindering and drastically increasing the required dosage (MAO; CHEN, 2000). Despite this, as there is also interest in developing a new analgesic and anti-inflammatory agent for topical or local use, a lidocaine derivative with the HE-3 analogue was prepared. In turn, the HE-13 derivative was designed as a molecular hybrid of MDR-652, a very potent TRPV1 channel agonist recently described in the literature (see the diagram below). This substance was developed by Ann et al. in 2020 and is currently sold as a standard non-capsinoid agonist for this type of receptor (ANN et al., 2020).

[0126] Finally, the HE-16 and HE-17 derivatives were planned based on the results obtained in the first two tests, with the intention of uniting the substitutions that presented greater activity in each of the investigated regions of the HE-3 prototype as defined in the scheme below. METHODOLOGY APPLIED IN THE PRESENT INVENTION

[0127] All reagents used in the present invention were of analytical standard. Palladium tetrakis triphenylphosphine was purchased from Oakwood Chemical (Estill, South Carolina, USA), 4-pentyn-1-ol was purchased from Tokyo Chemical Industry (Kashiai, Fukaya City, Saitama, Japan). Anhydrous dimethyl sulfoxide (DMSO) and the remaining reagents were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Triethylamine and diisopropylamine were treated with CaH2 and distilled before use. Tetrahydrofuran (THF) was treated with metallic sodium, benzophenone and distilled before use. The anhydrous reactions were carried out under an atmosphere of piped anhydrous N2 gas. The progress of the reactions was monitored with thin layer chromatography (TLC) on aluminum plates obtained from Merck (silica gel 60 F254). Purifications by silica column chromatography (SCC) were performed with flash silica (silica gel 60, 220-440 mesh, Sigma-Aldrich).When applicable, GC-MS chromatographic analyses were performed on an Agilent model 7890 GC gas chromatograph coupled to a mass spectrometer. The chromatographic column used in the analyses was an Agilent HP-5MS. The heating ramp started at 60°C, followed by heating to 180°C at a rate of 15°C / min, and then a second heating to 280°C at a rate of 10°C / min. The injector and ion source temperatures were set at 280°C and 230°C, respectively. The electron impact energy was set at 70 eV, and the mass spectrometer operated over a mass / charge range of 30–400 m / z. NMR analyses of. 1 H and of 13 C were obtained on a Bruker 250 MHz spectrometer, chemical shifts (δ) were reported in parts per million (ppm) relative to the residual deuterated solvent as internal standard (CDCl3: 7.26 ppm for C NMR 1 H and 77.16 ppm for NMR 13 W; DMSO-d6: 2.5 ppm for NMR1 H e 39.52 ppm para NMR of 13 C; Acetone-d6: 2.05 ppm para NMR de 1 H e 29.84 ppm para NMR of 13C). The following abbreviations were used to represent the coupling multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, non = nonet, dt = doublet of triplets, and m = multiplet. The unit of coupling constants (J) is hertz (Hz). NMR spectra were processed using MestReNova 6.0.2-5475 software. Chemical structure names were generated using ChemDraw Ultra 12.0 software. Melting points were obtained with a Mettler Toledo MP50 Melting Point System and are uncorrected. HRMS analyses were obtained on an Orbitrap Thermo-Q Exactive direct injection mass spectrometer. SYNTHESIS PROCEDURE OF ISOBUTYL PHOSPHONATEACETAMIDE (iBuPA)

[0128] To an oven-dried 10 mL reaction flask were added 1 eq. of triethyl phosphonoacetate (4 mmol), 1.2 eq. of isobutylamine and 10 mol% of anhydrous lanthanum triflate. The reaction was kept under stirring for 6 hours and then diluted in THF.The resulting solution was filtered through a silica filter and the recovered solvent was evaporated. The product was already adequately pure by NMR analysis and was used in the next step without further purification.

[0129] Compound (2-(isobutylamino)-2-oxoethyl)phosphonate. It was obtained as a colorless oil in 91% yield. GENERAL PROCEDURE FOR AMIDATION REACTIONS WITH EDC

[0130] To a previously oven-dried 25 mL reaction flask filled with 10 mL of anhydrous dichloromethane were added 1.2 eq. of a specific amine, 1 eq. of diethylphosphonoacetic acid or decanoic acid (1.5 mmol), 10 mol% DMAP, and 1 eq. of EDC. The atmosphere in the flask was replaced by N2 and the reaction was kept under stirring for 18 h at 25°C. Then, the reaction was extracted with distilled water (3x10 mL), saturated NaHCO3 solution (3x10 mL), 1M HCl (3x10 mL), 10 mL of saturated NaCl solution and dried with anhydrous Na2SO4.Dichloromethane was evaporated under reduced pressure and the product already showed adequate purity.

[0131] Compound N-isobutyldecanamide (HE-5). Obtained using decanoic acid instead of diethylphosphonoacetic acid and isobutylamine as amine.

[0132] Compound diethyl (2-((2-methoxybenzyl)amino)-2-oxoethyl)phosphonate (7.a). 85% yield, appearance of white solid. Obtained according to the general procedure for amidation reactions using (2-methoxyphenyl)methanamine as amine. 1H NMR (CDCl3, 250 MHz): δ 1.27 (t, J = 7.1 Hz, 6H); 2.85 (d, JH-P = 20.6 Hz, 2H); 3.85 (s, 3H); 4.06 (dq, J = 14.2, 7.1 Hz, 4H); 4.45 (d, J = 5.9 Hz, 2H); 6.88 (dd, J = 12.1, 7.8 Hz, 2H); 7.08 - 7.35 (m, 3H). NMR of. 13 C (62.5 MHz, CDCl3): δ 16.2; 16.3; 35.28 (d, J C-P = 130.6 Hz); 39.4; 55.3; 55.3; 62.5; 62.6; 110.1; 120.5; 126.0; 128.7; 129.4; 157.4; 163.71 (d, JC-P = 3.7 Hz). HRMS (ESI+): m / z calculated for C 14 H 22 NO5P [M+H +]: 316.13084; located at 316,13012.

[0133] Compound diethyl (2-((2,6-dimethylphenyl)amino)-2- oxoethyl)phosphonate (12.a). 82% efficiency, solid pack aspect. MRI de 1 H (CDCl3, 250 MHz): δ 1.36 (t, J = 7.4 Hz, 6H); 2.24 (s, 6H); 3.04 (d, J = 20.6 Hz, 2H); 4.18 (quint, J = 7.4 Hz, 4H); 7.02 – 7.08 (m, 3H); 8.18 (sl, 1H). MRI de 13 C (62.5 MHz, CDCl3): δ 16.4 (d, J = 6.2 Hz); 18.5; 35.09 (d, J = 130.1 Hz); 62.94 (d, J = 6.6 Hz); 127.4; 128.2; 133.8; 135.4; 162.44 (d, J = 3.5 Hz). MRI de 31 P: (CDCl3, 250 MHz): δ 23.0. HRMS (ESI+): m / z calculated for C14H22NO4P [M+Na +]: 322.11787; found 322.11708. GENERAL PROCEDURE FOR THE SONOGASHIRA REACTION

[0134] To an oven-dried 10 mL flask were added 10 mol% Pd(PPh3)4, 10 mol% CuI, 10 mol% PPh3, and 5 mL of anhydrous DMF. Then, the atmosphere in the flask was exchanged for N2 and after 5 min of stirring, 1.1 eq. of the aryl or vinyl triflate or halide, 1 eq. of 4-pentyn-1-ol (2 mmol), and 2 eq. of anhydrous triethylamine or diisopropylamine were added to the flask. The reaction was stirred at a specific temperature for each substrate for 2 to 16 h. Once the reaction was complete, as assessed by TLC, it was diluted with 15 mL of ethyl ether and filtered through a silica filter. The remaining solvent and base were evaporated. The product was purified by a silica column, with a hexane / ethyl acetate solution 95:5 to 70:30 being used as eluent.

[0135] Compound (E)-oct-6-en-4-yn-1-ol (1.a). 56% yield, appearance of yellow oil.Obtained according to the general procedure for the Sonogashira reaction using 3 eq. of 1-bromo-1-propene 60:40 Z:E as the vinyl halide, at 25°C for 2h.

[0136] Compound 5-(3,5-bis(trifluoromethyl)phenyl)pent-4-yn-1-ol (6.a) obtained according to the general procedure for the Sonogashira reaction using 1-bromo-3,5-bis(trifluoromethyl)benzene as the aryl bromide and temperature of 80°C for 16h. 73% yield, appearance of yellow oil. 5H NMR. 1 H (CDCl3, 250 MHz): δ 1.72 (s, 1H); 1.88 (quint, J = 6.6 Hz, 2H); 2.57 (t, J = 6.6 Hz, 2H); 3.81 (t, J = 6.6 Hz, 2H); 7.75 (s, 1H); 7.80 (s, 2H). NMR of 13 C (62.5 MHz, CDCl3): δ 15.9; 31.1; 61.5; 78.6; 93.6; 121.17 (hep, JC-F = 3.8 Hz); 123.1 (q, JC-F = 271.0 Hz); 126.2; 131.6; 131.8 (q, JC-F = 33.6 Hz). NMR of 19 F (235 MHz, CDCl3): δ -63. HRMS (ESI+): m / z calculated for C13H10F6O [M+H +]: 297.07086; found 297.07011.

[0137] Compound 5-(4-allyl-2-methoxyphenyl)pent-4-yn-1-ol (8.b), obtained according to the general procedure for the Sonogashira reaction, using compound 1.a as aryl triflate and temperature of 70°C for 16h. 70% yield, appearance of yellowish oil. NMR of 1 H (250 MHz, CDCl3): δ 1.87 (quint, J = 6.5 Hz, 2H); 2.58 (t, J = 6.5 Hz, 2H); 3.36 (d, J = 6.6 Hz, 2H); 3.85 (sl, 5H); 4.97 – 5.23 (m, 2H); 5.72 – 6.13 (m, 1H); 6.67 (s, 1H); 6.71 (d, J = 7.8 Hz, 1H); 7.27 (d, J = 7.8 Hz, 1H). NMR of 13 C (62.5 MHz, CDCl3): δ 16.8; 31.3; 40.4; 55.7; 62.3; 77.5; 93.2; 110.5; 110.9; 116.3; 120.7; 133.2; 136.8; 141.6; 160.0. HRMS (ESI+): m / z calculated for C15H18O2[M+H +]: 231.13796; found 231.13761.

[0138] Compound 4-allyl-2-(5-hydroxypent-1-yn-1-yl)-6-methoxyphenol (9.b). Obtained according to the general procedure for the Sonogashira reaction using intermediate 9.a as the aryl halide, at 80°C for 16 h. 48% yield, appearance of yellowish oil. NMR 1 H (CDCl3, 250 MHz): δ 2.01 (quint, J = 6.8 Hz, 2H); 2.88 (t, J = 7.3 Hz, 2H); 3.43 (d, J = 6.7 Hz, 2H); 3.72 (t, J = 6.8 Hz, 2H); 3.98 (s, 3H); 4.94 – 5.22 (m, 2H); 6.00 (ddt, J = 16.8, 10.0, 6.7 Hz, 1H); 6.35 (s, 1H); 6.57 (s, 1H); 6.90 (s, 1H). NMR of 13C (62.5 MHz, CDCl3): δ 24.9; 30.8; 40.6; 56.0; 62.0; 102.6; 106.6; 112.3; 115.6; 130.6; 135.4; 138.1; 142.6; 144.6; 159.1. GENERAL PROCEDURE FOR THE SWERN REACTION

[0139] 5 mL of anhydrous dichloromethane was added to an oven-dried 10 mL reaction flask. The atmosphere in the flask was exchanged for N2 and 3 eq. of oxalyl chloride was added. Then, the flask was cooled to -78°C and 2 eq. of anhydrous DMSO was added dropwise. After gas formation ceased, 1 eq. of the specific alcohol (1.5 mmol). The reaction was stirred under cooling for 1 h and then 2 eq. of anhydrous triethylamine were added. After 20 min, cooling was stopped and stirring continued for another 2 h at room temperature. Once the reaction was complete by TLC, 20 mL of distilled water were added to the flask and the solution extracted with dichloromethane (5x10 mL).The resulting organic phase was washed with 20 mL of saturated NaCl solution and the remaining water was removed with anhydrous Na2SO4. The solvent was then evaporated on a rotary evaporator. All aldehydes obtained were used in the following reactions without further purification and as they were prepared.

[0140] Compound (E)-oct-6-en-4-ynal (1.b). Obtained according to the general procedure for the Swern reaction, using substance 1.a as alcohol. The product was used immediately in the following reactions, without being completely purified and isolated. Appearance of orange oil.

[0141] Compound 5-(3,5-bis(trifluoromethyl)phenyl)pent-4-ynal (6.b). Obtained according to the general procedure for the Swern reaction, using intermediate 6.a as alcohol. Appearance of yellow oil. This intermediate was used in the following reaction without being completely purified and isolated.

[0142] 5-(4-allyl-2-methoxyphenyl)pent-4-inal compound (8.c).Obtained according to the general procedure for the Swern reaction using intermediate 8.b as alcohol. Appearance of orange oil. This intermediate was used in the following reaction without being completely purified and isolated.

[0143] Compound 5-(5-allyl-2-hydroxy-3-methoxyphenyl)pent-4-ynal (9.c). Obtained according to the general procedure for the Swern reaction using intermediate 9.b as alcohol. Appearance of orange oil. This intermediate was used in the following reaction without being completely purified and isolated. GENERAL PROCEDURE FOR THE HORNER-WADSWORTH-EMMONS REACTION

[0144] 5 eq. of 60% NaH in mineral oil were added to an oven-dried 10 mL reaction flask, followed by 5 mL of anhydrous THF. This suspension was cooled to 0°C, and then 1.1 eq. of the specific phosphonatoacetamide was added. After 5 minutes, 1 eq. of the specific aldehyde (1 mmol) dissolved in anhydrous THF was added dropwise.The ice bath was removed and the reaction stirred for 1 to 2 h at room temperature. After the reaction was completed, as verified by TLC, it was acidified with saturated NH4Cl solution and the resulting aqueous solution was extracted with ethyl acetate (5x10 mL). After extraction, the combined organic phase was washed with 20 mL of saturated NaCl solution, dried with anhydrous Na2SO4 and the solvent evaporated on a rotary evaporator. The final product was purified by column chromatography on silica using hexane / ethyl acetate 90:10 to 70:30 as eluent.

[0145] Compound (E)-N-isobutyldec-2-enamide (HE-4). Obtained using n-octanal as aldehyde and iBuPA as phosphonatoacetamide. 81% yield, appearance of a white pasty solid (ALONSO; PASTRE, 2016).

[0146] Compound (E)-7-(3,5-bis(trifluoromethyl)phenyl)-N-isobutylhept-2-en-6-enamide (HE-6). Obtained using 1.5 eq. of tBuOK and 2 eq. of LiCl instead of sodium hydride. Intermediate 6 was used.b as aldehyde and iBuPA as phosphonatoacetamide. 43% yield, appearance of white solid. NMR. 1 H (CDCl3, 250 MHz): δ 0.92 (d, J = 6.7 Hz, 6H); 1.79 (no, J = 6.7 Hz, 1H); 2.38 – 2.68 (m, 4H); 3.17 (t, J = 6.5 Hz, 2H); 5.58 (s, 1H); 5.90 (d, J = 15.2 Hz, 1H); 6.90 (dt, J = 15.2; 6.5 Hz, 1H); 7.76 (s, 1H); 7.80 (s, 2H). NMR of 13 C (CDCl3, 62.5 MHz): δ 18.7; 20.2; 28.7; 30.9; 47.0; 79.0; 92.8; 121.29 (quint, J C-F = 3.8 Hz); 123.1 (q, JC-F = 270.6 Hz); 125.2; 126.0; 131.6; 131.9 (q, J = 33.6 Hz); 141.7; 165.6. NMR 19 F (235 MHz, CDCl3) -63. HRMS (ESI+): m / z calculated for C 19 H 19 F6NO [M+H + ]: 392.1444; found: 392.14553.

[0147] Compound (2E,8E)-N-(2-methoxybenzyl)deca-2,8-dien-6-enamide (HE-7). Obtained using intermediate 1.b as aldehyde and 7.a as phosphonatoacetamide. 71% yield in two steps, appearance of white solid. δ NMR 1H (CDCl3, 250 MHz): δ 1.74 (dd, J = 6.8, 1.8 Hz, 3H); 2.40 (s, 4H); 3.86 (s, 3H); 4.50 (d, J = 5.9 Hz, 2H); 5.44 (dd, J = 15.5, 1.8 Hz, 1H); 5.82 (d, J = 15.5 Hz, 1H); 5.94 – 6.14 (m, 2H); 6.80 – 6.94 (m, 3H); 7.22 – 7.29 (m, 2H). NMR of 13 C (62.5 MHz, CDCl3): δ 18.5; 18.6; 31.4; 39.4; 55.4; 80.1; 86.8; 110.4; 110.9; 120.8. 124.7; 126.3; 126.9; 130.0; 138.6; 142.3; 157.6; 165.5. HRMS (ESI+): m / z calculated for C18H21NO [M+Na + ]: 306.14645; found: 306.14664.

[0148] Compound (E)-7-(4-allyl-2-methoxyphenyl)-N-isobutylhept-2-en-6-ynamide (HE-8). Obtained according to the general procedure for the HWE reaction using intermediate 8.c as aldehyde and iBuPA as phosphonatoacetamide. 70% yield in two steps, appearance of white solid. δ NMR 1H (250 MHz, CDCl3): δ 0.91 (d, 6.5 Hz, 6H); 1.78 (no, J = 6.5 Hz, 1H); 2.5 (q, J = 6.4 Hz, 2H); 2.59 (d, J = 6.5 Hz, 2H); 3.14 (t, J = 6.4 Hz, 2H); 3.35 (d, J = 6.6 Hz, 2H); 3.8 (s, 3H); 5.07 (m, 2H); 5.62 (s, 1H); 5.89 (d, J = 15.2 Hz, 1H); 5.92 (m, 1H); 6.67 (s, 1H); 6.7 (d, J = 7.6 Hz, 1H); 6.89 (dt, J = 15.3 Hz, 6.4 Hz, 1H); 7.26 (d, J = 7.6 Hz, 1H). NMR of 13 C (62.5 MHz, CDCl3): δ 18.9; 20.0; 28.5; 31.3; 40.3; 46.8; 55.74; 92.2; 110.4; 110.9; 116.1; 120.6; 124.7; 133.4; 136.7; 141.6; 142.0; 159.8; 165.8. HRMS (ESI+): m / z calculated for C21H27NO2 [M+H + ]: 326.2115; found: 326.2118.

[0149] Compound (E)-7-(5-allyl-2-hydroxy-3-methoxyphenyl)-N-isobutylhept-2-en-6-ynamide (HE-9). Obtained according to the general procedure for the HWE reaction using intermediate 2.c as the aldehyde and iBuPA as the phosphonatoacetamide. 62% yield in 2 steps, appearance of a slightly yellowish solid. δ NMR 1H (250 MHz, CDCl3): δ 0.90 (d, 6.6 Hz, 6H); 1.77 (no, J = 6.7 Hz, 1H); 2.62 (q, J = 6.9 Hz, 2H); 2.91 (t, J = 7.8 Hz, 2H); 3.12 (t, J = 6.4 Hz, 2H); 3.42 (d, J = 6.6 Hz, 2H); 3.9 (s, 3H); 5.08 (m, 2H); 5.54 (s, 1H); 5.81 (d, J = 15.2 Hz, 1H); 5.99 (m, 1H); 6.33 (s, 1H); 6.57 (s, 1H); 6.88 (s, 1H); 6.85 (dt, J = 15.2 Hz, 6.7 Hz, 1H). NMR of 13 C (62.5 MHz, CDCl3): δ 20.0; 27.2; 28.5; 30.0; 40.4; 46.8; 55.9; 102.7; 106.5; 112.3; 115.5; 124.6; 130.3; 135.3; 137.9; 142.3; 142.4; 144.5; 157.9; 165.7. HRMS (ESI+): m / z calculated for C21H27NO3 [M+H + ]: 342.2064; found: 342.2065.

[0150] Compound (E)-3-(5-allyl-2-hydroxy-3-methoxyphenyl)-N-isobutylacrylamide (HE-10). Obtained according to the general procedure for the HWE reaction, using intermediate 10.a as the aldehyde, iBuPA as the phosphonatoacetamide and a hexane:ethyl acetate 7:3 solution as eluent in the chromatographic column. 89% yield, appearance of a slightly yellowish solid. NMR 1H (CDCl3, 250 MHz): δ 0.94 (d, J = 6.7 Hz, 6H); 1.83 (no, J = 6.7 Hz, 1H); 3.20 (t, J = 6.4 Hz, 2H); 3.29 (d, J = 6.7 Hz, 2H); 3.88 (s, 3H); 5.07 (m, 2H); 5.76 (s, 1H); 5.92 (m, 1H); 6.12 (s, 1H); 6.64 (d, J = 15.7 Hz, 1H); 6.65 (d, J = 1.4 Hz, 1H); 6.85 (d, J = 1.4 Hz, 1H); 7.75 (d, J = 15.7 Hz, 1H). NMR of 13 C (CDCl362.5 MHz): δ 20.1; 28.6; 39.7; 47.0; 56.1; 111.6; 115.8; 120.8; 121.2; 122.3; 131.1; 136.0; 137.3; 143.36; 146.6; 166.6. HRMS (ESI+): m / z calculated for C 17 H 23 NO3[M+H + ]: 290.1751; found: 290.1746.

[0151] Compound (2E,8E)-N-(5-allyl-2-hydroxy-3-methoxyphenyl)deca-2,8-dien-6-ynamide (HE-11). Obtained according to the general procedure for the HWE reaction using compounds 1.b as the aldehyde (prepared just before the reaction) and 11.c as the phosphonatoacetamide. 63% yield for 2 steps, appearance of a reddish oil. NH-NMR 1H (CDCl3, 250 MHz): δ 1.74 (dd, J = 6.7; 1.3 Hz, 3H); 2.24 (s, 4H); 3.29 (d, J = 6.6 Hz, 2H); 3.85 (s, 3H); 5.01- 5.09 (m, 2H); 5.44 (dd, J = 15.7; 1.6 Hz, 1H); 5.83-5.97 (m, 1H); 6.01-6.07 (m, 1H); 6.0-6.11 (m, 1H); 6.50 (d, J = 1.3 Hz); 6.83 (s, 1H); 6.99 (dt, J = 15.2; 6.5 Hz, 1H); 7.40 (s; 1H); 7.69 (s, 1H). NMR of 13 C (CDCl3; 62.5 MHz): δ 18.4; 31.3; 40.0; 56.0; 80.1; 86.4; 107.5; 110.7; 113.5; 115.7; 124.4; 125.6; 131.7; 134.1; 137.4; 138.6; 144.5; 147.3; 164.1. HRMS (ESI+): m / z calculated for C20H23NO3 [M+H + ]: 326.1751; found: 326.1752.

[0152] Compound (2E,8E)-N-(2,6-dimethylphenyl)deca-2,8-dien-6-enamide (HE-12). Obtained using intermediate 1.b as aldehyde and 12.a as phosphonatoacetamide. 49% yield in two steps, appearance of white solid. NH4 NMR 1H (Acetone-d6, 500 MHz): δ 1.73 (d, J = 6.3 Hz, 3H); 2.19 (s, 6H); 2.39 - 2.51 (m, 4H); 5.51 (d, J = 15.7 Hz, 1H); 6.04 (dq, J = 13.6; 6.7 Hz, 1H); 6.28 (d, J = 15.3 Hz, 1H); 6.89 (dt, J = 14.8; 6.4 Hz, 1H); 7.06 (s, 3H); 8.55 (s, 1H). NMR is available 13 C (100 MHz, Acetone-d6): δ 18.4; 18.6; 19.0; 32.0; 80.8; 87.8; 112.0; 125.9; 127.4; 128.5; 136.2; 136.4; 138.9; 142.9; 164.2. HRMS (ESI+): m / z calculated for C18H21NO [M+Na +]: 290.15154; found: 290.15225.

[0153] Compound (E)-3-(benzo[d][1,3]dioxol-5-yl)-N-isopropylacrylamide (HE-14). Obtained through the general procedure for HWE reaction using piperonal as aldehyde and iBuPA as phosphonatoacetamide. 86% yield, appearance of white solid (ALONSO; PASTRE, 2016).

[0154] Compound (E)-N-isobutyl-5-phenylpent-2-enamide (HE-15). Obtained through the general procedure for HWE reaction using hydrocinnamaldehyde as aldehyde and iBuPA as phosphonatoacetamide. 88% yield, appearance of white solid (ALONSO; PASTRE, 2016).

[0155] Compound (2E,8E)-N-(4-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluorophenyl)deca-2,8-dien-6-enamide (13.f). Obtained using 1.5 eq. of tBuOK and 2 eq. of LiCl instead of sodium hydride. Intermediate 1.b was used as aldehyde and 13.e as phosphonatoacetamide. 62% yield in 2 steps, appearance of colorless oil. NH4 NMR 1H (CDCl3, 250 MHz): δ 0,09 (s, 6H); 0,93 (s, 9H); 1,74 (dd, J = 6,8; 1,6 Hz, 3H); 2,43 (s, 4H); 4,74 (s, 2H); 5,44 (dd, J = 15,7; 1,7 Hz, 1H); 5,90 – 6,14 (m, 2H); 6,99 (ddd, J = 13,2; 8,3; 4,4 Hz, 1H); 7,15 (dd, J = 8,3; 1,7 Hz, 1H); 7,38 (t, J = 8,2 Hz, 1H); 7,54 (d, J = 12,0 Hz, 1H); 7,68 (s, 1H). RMN de 13 C (62,5 MHz, CDCl3): δ -5,2; 18,5; 18,5; 18,6; 26,0; 31,5; 58,8 (d, J = 4,0 Hz); 80,4; 86,6; 107,2 (d, J = 26,9 Hz); 110,9; 115,2 (d, J = 2,4 Hz); 124,3 (d, J = 15,0 Hz); 124,8; 128,7 (d, J = 6,1 Hz); 138,3 (d, J = 11,1 Hz); 138,9; 144,6; 159,8 (d, J = 244,7 Hz); 164,1. RMN de 19 F (235 MHz, CDCl3): δ -117,9. HRMS (ESI+): m / z calculada para C 23 H 32 FNO2Si [M+H +]: 402.22591; found 402.22538.

[0156] Compound (E)-7-(4-allyl-2-methoxyphenyl)-N-(4-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluorophenyl)hept-2-en-6-enamide (16.a). Obtained using 1.5 eq. of tBuOK and 2 eq. of LiCl instead of sodium hydride. Intermediate 8.c was used as aldehyde and 13.e as phosphonatoacetamide. 70% yield in two steps, appearance of white solid. NH4 NMR 1 H (CDCl3, 250 MHz): δ 0.10 (s, 6H); 0.93 (s, 9H); 2.38 - 2.68 (m, 4H); 3.36 (d, J = 6.6 Hz, 2H), 4.74 (s, 2H); 3.83 (s, 3H); 5.08 (dd, J = 13.4; 1.6 Hz, 2H); 5.91 (ddd, J = 13.4; 7.9; 4.8 Hz, 1H); 6.01 (dd, J = 12.3; 7.9 Hz, 1H); 6.71 (d, J = 7.9 Hz, 1H); 6.68 (s, 1H); 7.84 (s, 1H); 7.03 (dt, J = 15.1; 6.4 Hz, 1H); 7.18 (d, J = 8.3 Hz, 1H); 7.28 (d, J = 7.8 Hz, 1H); 7.37 (t, J = 8.2 Hz, 1H); 7.55 (d, J = 12.1 Hz, 1H); 7.84 (s, 1H). NMR of 13C (62.5 MHz, CDCl3): δ -5.2; 18.4; 18.9; 26.0; 31.3; 40.4; 55.8; 58.8 (d, JC-F = 4.0 Hz); 78.0; 92.2; 107.0 (d, JC-F = 26.2 Hz); 110.4; 111.2; 115.1; 116.4; 120.9; 124.1 (d, JC-F = 14.9 Hz); 124.9; 128.7 (d, JC-F = 6.0 Hz); 133.6; 136.8; 138.4 (d, JC-F = 10.9 Hz), 141.9, 144.3; 160.0; 159.8 (d, JC- F = 244.6 Hz); 164.2. NMR of 19 F (235 MHz, CDCl3): δ -117.9. HRMS (ESI+): m / z calculated for C30H38FNO3Si [M+K + ]: 546.22366; found 546.22361.

[0157] Compound (E)-3-(benzo[d][1,3]dioxol-5-yl)-N-(3- fluoro-4-(hydroxymethyl)phenyl)acrylamide (17.a). Obtained using piperonal as aldehyde and intermediate 13.e as phosphonateacetamide. 82% yield in two stages, white solid appearance. NMR of 1 H (CDCl3, 250 MHz): δ 0.10 (s, 6H); 0.93 (s, 9H); 4.75 (s, 2H); 5.99 (s, 2H); 6.37 (d, J = 15.4 Hz, 1H); 6.78 (d, J = 8.4 Hz, 1H); 6.97 (d, J = 7.3 Hz, 1H); 6.98 (s, 1H); 7.20 (d, J = 8.3 Hz, 1H); 7.40 (t, J = 8.2 Hz, 1H); 7.56 – 7.71 (m, 3H). NMR of 13C (CDCl3, 62.5 MHz): δ -5.2; 18.5; 26.0; 58.8 (d, J C-F = 4.1 Hz); 101.7; 106.5; 107.2 (d, JC-F = 28.7 Hz); 108.7; 115.2 (d, JC-F = 4.7 Hz); 118.6; 124.3 (d, JC-F = 18.5 Hz); 124.5; 128.7 (d, JC-F = 6.1 Hz); 129.0; 138.4 (d, J C-F = 10.9 Hz); 142.7; 148.4; 149.6; 159.8 (d, JC-F = 244.8 Hz); 164.5. MRI de 19 F (235 MHz, CDCl3): δ -117.8. HRMS (ESI+): m / z calculated for C23H28FNO4Si [M+K +]: 468.14032; found 468.13967. GENERAL PROCEDURE FOR REMOVAL OF THE t-BUTYLDIMETHYLSILYL GROUP

[0158] To a 25 mL reaction flask containing 1 mmol of the silicon ether to be deprotected, 10 mL of THF was added. The resulting solution was cooled in an ice bath and kept under magnetic stirring. Then, 1.1 eq. of a THF solution containing 1 M n-Bu4NF was added slowly via syringe. After the addition, the ice bath was removed and the reaction kept under stirring at 25ºC for 1 h. After checking the completion of the reaction by TLC, 10 mL of saturated NH4Cl solution was added and the mixture was extracted with 3x10 mL of ethyl acetate. The combined organic phase was washed with 2x10 mL of saturated NaCl solution and dried over anhydrous Na2SO4. After evaporating the solvent, the oil obtained was purified through a silica column, with a hexane:ethyl acetate solution 90:10 to 70:30 as eluent.

[0159] Compound (2E,8E)-N-(3-fluoro-4-(hydroxymethyl)phenyl)deca-2,8-dien-6-ynamide (HE-13). Compound obtained by the general procedure for removal of the t-butyldimethylsilyl group using intermediate 13.f as substrate. 88% yield, obtained as a white solid. 1 H NMR. 1 H (250 MHz, Acetone-d6): δ 1.72 (dd, J = 6.7; 1.6 Hz, 3H); 2.33-2.54 (m, 4H); 4.21 (t, J = 5.8 Hz, 1H); 4.63 (d, J = 5.5 Hz, 2H); 5.49 (dd, J = 15.8; 1.8 Hz, 1H); 6.02 (dq, J = 15.7; 6.8 Hz, 1H); 6.17 (dd, J = 15.3; 5.7 Hz, 1H); 6.96 (dt, J = 15.2; 6.6 Hz, 1H); 7.32 (dd, J = 8.4; 1.9 Hz, 1H); 7.41 (t, J = 8.3 Hz, 1H); 7.73 (dd, J = 12.7; 1.9 Hz, 1H); 9.39 (s, 1H). NMR of 13 C (Acetone-d6; 62.5 MHz): δ 18.42; 18.8; 31.9; 58.1 (d, J = 3.9 Hz); 80.7; 87.6; 106.9 (d, J = 27.3 Hz), 111.9; 115.4 (d, J = 3.0 Hz); 124.6 (d, J = 15.5 Hz); 126.0; 130.1 (d, J = 6.2 Hz); 138.9; 140.9 (d, J = 11.2 Hz); 144.3; 160.9 (d, J = 242.2 Hz); 164.4. NMR of 19F (235 MHz, Acetone-d6): δ -120. HRMS (ESI+): m / z calculated for C17H18FNO2 [M+Na + ]: 310.12138; found: 310.12127.

[0160] Compound (E)-7-(4-allyl-2-methoxyphenyl)-N-(3-fluoro-4-(hydroxymethyl)phenyl)hept-2-en-6-ynamide (HE-16). Compound obtained by the general procedure for removal of the t-butyldimethylsilyl group using intermediate 16.a as substrate. 91% yield, appearance of white solid. δ NMR 1 H (Acetone-d6, 250 MHz): δ 2.46 – 2.63 (m, 4H); 3.37 (d, J = 6.6 Hz, 2H); 3.83 (s, 3H); 4.21 (t, J = 5.5 Hz, 1H); 4.64 (d, J = 5.5 Hz, 2H); 5.02 – 5.12 (m, 2H); 5.96 (ddt, J = 16.8; 9.9; 6.7 Hz, 1H); 6.22 (d, J = 15.3 Hz, 1H); 6.73 (d, J = 7.7 Hz, 1H); 6.84 (s, 1H); 7.07 (dt, J = 14.9; 6.5 Hz, 1H); 7.25 (d, J = 7.7 Hz, 1H); 7.29 – 7.48 (m, 2H); 7.75 (d, J = 12.8 Hz, 1H); 9.40 (s, 1H). NMR of 13 C (Acetone-d6, 250 MHz): δ 19.3; 32.1; 40.9; 55.9; 58.1 (d, J C-F= 3.9 Hz); 78.7; 92.8; 106.9 (d, JC-F = 27.2 Hz); 111.6; 112.1; 115.4 (d, JC-F = 2.8 Hz); 116.2; 121.2; 124.6 (d, JC-F = 15.5 Hz); 126.1; 130.1 (d, J C-F = 6.2 Hz); 133.9; 138.1; 140.9 (d, J C-F = 11.3 Hz); 142.7; 144.4; 160.9 (d, JC-F = 242.4 Hz); 161.2; 164.5. NMR of 19 F (235 MHz, Acetone-d6): δ -119.9. HRMS (ESI+): m / z calculated for C 24 H 24 FNO3[M+H + ]: 394.18130; found 394.18076.

[0161] Compound (E)-3-(benzo[d][1,3]dioxol-5-yl)-N-(3-fluoro-4-(hydroxymethyl)phenyl)acrylamide (HE-17). Compound obtained by the general procedure for removal of the t-butyldimethylsilyl group using intermediate 17.a as substrate. 93% yield, appearance of white solid. δ NMR 1H (DMSO-d6, 250 MHz): δ 4,48 (d, J = 5,5 Hz, 2H); 5,17 (t, J = 5,5 Hz, 1H); 6,09 (s, 2H); 6,63 (d, J = 15,6 Hz, 1H); 6,98 (d, J = 8,0 Hz, 1H); 7,07 – 7,24 (m, 2H); 7,26 – 7,45 (m, 2H); 7,52 (d, J = 15,6 Hz, 1H); 7,67 (d, J = 12,0 Hz, 1H); 10,28 (s, 1H). RMN de 13 C (DMSO-d6, 62,5 MHz): δ 56,5 (d, J = 3,3 Hz); 101,6; 105,7 (d, J = 26,8 Hz); 106,4; 108,7; 114,6 (d, J = 2,3 Hz); 119,9; 123,5 (d, J = 15,7 Hz); 123,8; 129,0; 129,4 (d, J = 6,4 Hz); 139,7 (d, J = 11,3 Hz); 140,5; 148,0; 148,9; 159,4 (d, JC-F = 242,1 Hz); 163,9. RMN de 19 F (235 MHz, DMSO-d6): δ -118,4. HRMS (ESI+): m / z calculada para C17H14FNO4 [M+H +]: 316.09729; found 316.09796. SYNTHETIC PROCEDURE FOR OBTAINING INTERMEDIATE 8.a.

[0162] To a 5 mL microwave vial were added 1 eq. of eugenol (3 mmol), 1.3 eq. of K2CO3, 3 mL of THF and 0.6 eq. of N-phenyl-bis(trifluoromethanesulfonimide). The reaction was stirred at 120ºC for 10 min in a microwave reactor and after completion, confirmed by TLC, it was diluted in 10 mL of ethyl ether and filtered through a silica filter. The filtered solvent was evaporated and the brownish oil obtained was purified on a silica column (CCS), being eluted with a hexane:ethyl acetate solution 100:0 to 90:10.

[0163] Compound 4-allyl-2-methoxyphenyl trifluoromethanesulfonate (8.a). 72% yield, obtained as a colorless oil (YOUNG; WHITE, 2008). SYNTHETIC PROCEDURES TO OBTAIN INTERMEDIATE 9.a.

[0164] To a 25 mL reaction flask were added 1 eq. of eugenol (3 mmol), 1.1 eq.lithium bromide, 10 mL glacial acetic acid, and 6.5 mol% anhydrous copper (II) acetate were added. After adding these reagents, the flask was coupled to a condenser, and the atmosphere in both was exchanged for O . Two flasks filled with the same gas were left in the condenser, and the reaction was heated at 80°C for 16 h. After completion of the reaction, as observed by TLC, the solvent was evaporated, and the resulting pasty solid was neutralized with 20 mL of saturated NaHCO . This aqueous solution was extracted with dichloromethane (5 × 10 mL), and the resulting organic phase was washed with 50 mL of saturated NaCl solution. The organic phase was then dried with anhydrous Na 2 SO 4 and evaporated under reduced pressure. The final product was purified by TLC, eluting with a 100:0 to 80:20 hexane:ethyl acetate solution.

[0165] Compound 4-allyl-2-bromo-6-methoxyphenol (9.a). 55% yield, obtained as a brownish oil. SYNTHETIC PROCEDURES FOR OBTAINING INTERMEDIATE 10.a.

[0166] Initially, 1 eq. of eugenol (3 mmol) was solubilized in 15 mL of chloroform inside a 25 mL reaction flask. Then, 3 eq. of KOH were added and the reaction mixture was stirred under reflux for 5 h. After this time, 5 mL of a 1 M aqueous HCl solution were added and stirring was continued for another 20 minutes at rt. The reaction was then extracted with dichloromethane (5x10 mL), the combined organic phase was washed with 20 mL of saturated NaCl solution, dried with anhydrous Na2SO4 and the solvent evaporated on a rotary evaporator. NMR spectrum. 1H indicated that the product was already pure.

[0167] Compound 5-allyl-2-hydroxy-3-methoxybenzaldehyde (10.a). 84% yield, appearance of a greenish solid. SYNTHETIC PROCEDURES TO OBTAIN INTERMEDIATE 11.a; 11.b and 11.c.

[0168] 10 mL of dichloromethane and 1 eq. of eugenol (4 mmol) were added to a 25 mL reaction flask. Then, 3 eq. of flash silica previously wetted with distilled water (1 g SiO2 / 1 mL H2O), 1 eq. of NaNO3 and 1 eq. of KHSO4 were added to the reaction flask. The resulting suspension was stirred at rt for 24 h. Once the reaction was complete by TLC, it was gravity filtered through a funnel containing anhydrous Na2SO4, and the dichloromethane was evaporated. The product was removed from the crude reaction by hexane extraction of the viscous oil obtained after evaporation of the reaction solvent. Hexane was added to the oil, stirred for 2 minutes, and the supernatant was removed and transferred to a flask (5x10 mL).The supernatant accumulated in the flask was evaporated on a rotary evaporator to provide the desired product with adequate purity.

[0169] Compound 4-allyl-2-methoxy-6-nitrophenol (11.a). 92% yield, orange oil appearance (AZEVEDO-BARBOSA et al., 2021).

[0170] In the second reaction of the phosphonatoacetamide synthesis, 1 eq. of intermediate 11.a (3 mmol) and 5 eq. of SnCl2.2H2O were added to a 25 mL reaction flask containing 10 mL of ethanol. This reaction was stirred under reflux for 3 h, cooled and then neutralized with 20 mL of saturated NaHCO3 solution. The reaction was then extracted with ethyl ether (5x10 mL) and the combined organic phase was washed with 20 mL of saturated NaCl solution, dried with anhydrous Na2SO4 and the solvent evaporated on a rotary evaporator. The aminoeugenol formed was used immediately in the following reaction as it degrades easily.

[0171] Compound 4-allyl-2-methoxy-6-aminophenol (11.b). 69% yield, appearance of a yellow solid.

[0172] In the third reaction to form phosphonatoacetamide, 1 eq. of intermediate 11.b (2 mmol) was added to a 10 mL reaction flask containing 5 mL of anhydrous dichloromethane. Then, 1.2 eq. of diethylphosphonoacetic acid and 1 eq. of EDC were added to the flask. The reaction was stirred at 25ºC for 16 h and then extracted with distilled water (2x10 mL), 1M HCl (2x10 mL), saturated NaHCO3 solution (2x10 mL), washed with 20 mL of saturated NaCl solution and finally dried with anhydrous Na2SO4. The dichloromethane was evaporated under reduced pressure. The NMR spectrum. 1 H of the crude obtained showed that the product was already pure.

[0173] Compound (2-((5-allyl-2-hydroxy-3-methoxyphenyl)amino)-2-oxoethyl)phosphonate diethyl (11.c). 78% yield, appearance of brownish solid. NMR 1H (CDCl3, 250 MHz): δ 1,13 (t, J = 7,0 Hz, 6H); 3,07 (d, J = 20,9 Hz, 2H); 3,26 (d, J = 6,72 Hz, 2H); 3,82 (s, 1H); 4,16 (quint, J = 7,4 Hz, 4H); 4,99-5,07 (m, 3H); 5,81-5,97 (m, 1H); 6,47 (d, J = 1,4 Hz, 1H); 7,30 (d, J = 1,4 Hz, 1H); 8,97 (s, 1H). RMN de 13 C (CDCl3; 62,5 MHz): δ 16,2 (d, J C-P = 6,0 Hz); 36,1 (d, JC-P = 129,8 Hz); 39,9; 56,0; 63,0 (d, JC-P = 6,5 Hz); 107,8; 113,6; 115,6; 125,5; 131,5; 134,3; 137,4; 147,4; 162,6 (d, J C-P= 4.2 Hz). SYNTHETIC PROCEDURES TO OBTAIN INTERMEDIATE 13.a, 13.b, 13.c, 13.d and 13.e.

[0174] Initially, 1 eq. of 2-fluoro-4-nitrobenzoic acid (5 mmol), 30 mL of methanol and 25 mol% of sulfuric acid were added to a 100 mL round-bottom flask. The flask was then connected to a condenser and the reaction was stirred for 20 h under reflux. Then, the reaction was cooled and 30 mL of saturated NaHCO3 solution was added to the flask. The methanol was evaporated and the suspension formed was filtered under vacuum, being washed with distilled water (5x10 mL). After being dried in a desiccator, the solid obtained already presented adequate purity.

[0175] Compound methyl 2-fluoro-4-nitrobenzoate (13.a). Quantitative yield, appearance of white solid.

[0176] Then, 1 eq. of intermediate 13.a (5 mmol) was added to a 100 mL flask together with 30 mL of methanol. The solution was cooled to 0ºC with an ice bath and then 4 eq. of NaBH4 was slowly added to the reaction, over 5 min.The reaction was kept in an ice bath for 1 h and then the bath was removed. The reaction was then kept for another 4 h at 25°C until completion as verified by TLC. The reaction was cooled again and 15 mL of distilled water was added to the flask and the final solution stirred for 5 min. The methanol was then evaporated and the suspension formed was vacuum filtered, being washed with distilled water (5x10 mL). After being dried in a desiccator, the solid obtained already presented adequate purity.

[0177] Compound 2-fluoro-4-nitrophenyl methanol (13.b). 78% yield, appearance of a slightly orange solid.

[0178] Then, 1 eq. of intermediate 13.b (3.5 mmol) was added to a 25 mL oven-dried flask and solubilized with 10 mL of anhydrous DCM and 1 mL of anhydrous DMF. Then, 2 eq. of imidazole to the balloon and it was closed with a septum and its atmosphere exchanged for N2. The balloon was cooled in an ice bath and kept under rigorous stirring. 1.5 eq. were added.of t-butyldimethylsilyl chloride was slowly added to the flask via syringe. The ice bath was removed, and the reaction was kept under rigorous stirring at 25°C for 24 h. Upon verifying the completion of the reaction by TLC, it was vacuum filtered, and the organic solvent obtained was extracted with 3x10 mL of saturated NH4Cl solution, 3x10 mL of distilled water, and 2x10 mL of saturated NaCl solution. The final organic phase was dried over anhydrous Na2SO4 and evaporated. The oil obtained was purified through a silica column, with a hexane:ethyl acetate solution 100:0 to 80:20 being used as eluent.

[0179] Compound tert-butyl((2-fluoro-4-nitrobenzyl)oxy)dimethylsilane (13.c) 71% yield, appearance of colorless oil. 1 H NMR. 1 H (CDCl3, 250 MHz): δ 0.14 (s, 6H); 0.96 (s, 9H); 4.86 (s, 2H); 7.72 (t, J = 8.1 Hz, 1H); 7.87 (dd, J = 9.7; 2.0 Hz, 1H); 8.06 (dd, J = 8.1; 2.0 Hz, 1H). NMR of 13C (CDCl3; 62.5 MHz): δ -5.3; 18.5; 26.0; 58.7 (d, JC-F = 4.7 Hz); 110.6 (d, JC-F = 26.2 Hz); 119.4 (d, J C-F = 3.5 Hz); 128.4 (d, J C-F = 5.2 Hz); 136.6 (d, J C-F = 14.6 Hz); 147.7 (d, JC-F = 8.9 Hz); 158.8 (d, JC-F = 249.9 Hz). MRI de 19 F (235 MHz, CDCl3): δ -116. HRMS (ESI+): m / z calculated for C 13 H 20 FNO3Si [M+H +]: 286.12692; found 286.12619.

[0180] Next, 1 eq. of intermediate 13.c (2.4 mmol) was added to a 25 mL flask together with 10 mL of methanol and 10 mol% Pd / C 10%. After the flask was capped with a septum, the atmosphere inside it was replaced by H2 using a bladder. The reaction was then kept for 2 h under rigorous stirring at 25°C. After this time, the hydrogen was removed from the flask with a N2 flow and the suspension was filtered on a silica pad. The filtered solvent was evaporated and the crude obtained was purified through a silica column, with a hexane:ethyl acetate solution 90:10 to 60:40 as eluent.

[0181] Compound 4-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoroaniline (13.d). 89% yield, appearance of a reddish solid. NMR 1 H (CDCl3, 250 MHz) δ 0.09 (s, 6H); 0.92 (s, 9H); 3.70 (s, 2H); 4.67 (s, 2H); 6.34 (dd, J = 11.8; 2.2 Hz, 1H); 6.44 (dd, J = 8.2, 2.2 Hz, 1H); 7.19 (t, J = 8.2 Hz, 1H). NMR of 13C (CDCl3; 62.5 MHz) δ -5.1; 18.6; 26.1; 59.0 (d, JC-F = 4.1 Hz); 101.9 (d, JC-F = 24.8 Hz); 110.7 (d, JC-F = 2.7 Hz); 118.1 (d, JC-F = 15.2 Hz); 129.9 (d, JC-F = 6.5 Hz); 147.3 (d, JC-F = 10.7 Hz); 161.0 (d, JC-F = 244.2 Hz). MRI de 19 F (235 MHz, CDCl3): δ -119.4. HRMS (ESI+): m / z calculated for C13H22FNOSi [M+H +]: 256.15275; found 256.15215.

[0182] In the next step, 1 eq. of intermediate 13.d (2.1 mmol) was added to a previously oven-dried 25 mL reaction flask, followed by 15 mL of anhydrous DCM, 1.1 eq. of diethylphosphonoacetic acid, 0.1 eq. of DMAP, and 1 eq. of EDC. The atmosphere in the flask was replaced by N2 and the reaction was kept under stirring for 24 h at 25ºC. Then, the reaction was extracted with distilled water (3x10 mL), 1M HCl (3x10 mL), saturated NaHCO3 solution (3x10 mL), washed with 2x10 mL of saturated NaCl solution and finally dried over anhydrous Na2SO4. The dichloromethane was evaporated under reduced pressure. The product did not require further purification.

[0183] Compound diethyl (2-((4-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluorophenyl)amino)-2-oxoethyl)phosphonate (13.e). 92% yield, obtained as a colorless oil that solidifies in the refrigerator. NMR 1H (CDCl3, 250 MHz): δ 0.09 (s, 6H); 0.93 (s, 9H); 1.37 (t, J = 7.1 Hz, 6H); 3.09 (d, JH-P = 21.4 Hz, 2H); 4.21 (dq, JP-HH = 14.3; 7.1 Hz, 4H); 4.69 (s, 2H); 7.08 (dd, J = 8.3; 1.8 Hz, 1H); 7.26 (t, J = 8.3 Hz, 1H); 7.39 (dd, J = 12.2, 1.8 Hz, 1H); 9.60 (s, 1H). NMR of 13 C (CDCl3; 62.5 MHz): δ −5.3; 16.4 (d, JC-P = 6.2 Hz); 18.5; 26.0; 36.4 (d, JC-P = 129.9 Hz); 58.7 (d, JC-F = 4.2 Hz); 63.2 (d, JC-P = 6.6 Hz); 106.7 (d, JC-F = 26.6 Hz); 114.8 (d, JC-F = 3.1 Hz); 123.9 (d, JC-F = 14.9 Hz); 128.3 (d, JC-F = 6.0 Hz); 138.3 (d, JC-F = 11.0 Hz); 159.5 (d, JC-F = 244.1 Hz); 162.3 (d, JC-P = 4.6 Hz). NMR of 19 F (235 MHz, CDCl3): δ −118.5. NMR of 31 P (101.25 MHz, CDCl3): δ 22.6 (tp, J = 24.5; 8.1 Hz). HRMS (ESI+): m / z calculated for C19H33FNO5Psi [M+H +]: 434.19224; found 434.19168. Z-SELECTIVE CATALYTIC HYDROGENATION PROCEDURE

[0184] To a 10 mL round-bottomed flask was added 0.25 mmol of the substrate to be hydrogenated, followed by 2.5 mL of methanol. Then, 5 or 10 mol% of Lindlar's catalyst (5% Pd in ​​CaCO3) and 10 or 20 mol% of quinoline were added. After adding a magnetic stirrer to the flask, it was inserted into a pressure reactor. The reactor was hermetically sealed and connected to a hydrogen cylinder with a regulating valve. The reactor outlet valve was kept open and the atmosphere inside it was replaced by H2. After the atmosphere was replaced, the reactor outlet valve was closed and the internal reactor pressure was increased to 5 or 10 bar. The reactor inlet valve was then closed and the reactor was disconnected from the H2 cylinder. The reactor was left on a magnetic plate, under rigorous stirring.After the reaction time, the reactor was purged with N2 gas and the lid removed. After filtration through a silica pad, substrate conversion and product formation were qualitatively evaluated by GC / MS. Yields were quantitatively evaluated by NMR, using acetanilide as an internal standard, for product 1.c. For product HE-1, yields were evaluated by the mass isolated after CCS.

[0185] Compound (2E,6Z,8E)-N-isobutyldeca-2,6,8-trienamide (HE-1). Obtained using compound HE-13 as substrate. 76% yield, appearance of colorless oil.

[0186] Compound (4Z,6E)-octa-4,6-dien-1-ol (1.c). Obtained using compound 1.a as substrate. 59% yield, appearance of colorless oil. MOLECULAR DOCKING

[0187] Compounds HE-1, 2, 3, 8, 13, 14, 16 and 17 were designed and optimized using the BIOVIA Discovery Studio v16.1.0.15350 program. For optimization, a Dreiding type force field was used.The file containing the coordinates for the crystallographic structure of cyclooxygenase-1 (COX-1) complexed with indomethacin-alpha-ethylethanolamide (PDB ID 2OYU), cyclooxygenase-2 (COX-2) complexed with naproxen (PDB ID 3NT1), and 5-lipoxygenase (5-LOX) complexed with arachidonic acid (PDB ID 3V99) were obtained from the Protein Data Bank. Water molecules, ligands, and extra chains were removed from the crystallographic structures of both COX and LOX isoforms, keeping only chain A of the base crystallographic structure for study. For lipoxygenase, the catalytic iron atom was maintained. Regarding the PDB 4YK5 structure, the three-dimensional trimer structure obtained with the aid of the PISA software was obtained from the PDB. From this structure, in a first analysis, the third monomer, water molecules and other ligands were excluded, keeping only two monomers.In a second analysis, the glutathione (GSH) molecule was maintained in its binding site between the two monomers used to assess whether the interaction of the test substances was interfered with by the presence of this substance in the enzyme's active site. AutoDockTools 1.5.7 was used to prepare the macromolecules (addition of Gasteiger charges and AD4 atomic type) and ligands (definition of torsional aspects), allowing the respective PDBQT files for the aforementioned structures to be obtained. Docking studies were performed using AutoDock Vina 1.1.1. To define the conformational search area, the previously indicated inhibitor interaction regions were considered in each case. For COX isoforms, the conformational search area was defined as a box with dimensions 18 Å x 18 Å x 18 Å, centered at the coordinate point 20.524 x 50.052 x 11.247 for COX-1 and -40.957 x -51.293 x -22.318 for COX-2.For the 5-LOX structure, the conformational search area was 30 Å x 20 Å x 30 Å, centered at the coordinate point 19.367 x -77.797 x -34.355. EX VIVO ASSAY

[0188] Human blood was collected from volunteer donors using a Falcon tube and a sterile heparinized syringe. All materials used in pipetting and plating of blood samples were previously autoclaved. All these materials were also kept inside a laminar flow hood prior to testing, being sterilized by UV for 15 minutes.

[0189] The LPS E. coli O26:B6 was used as an inflammatory inducer. The samples and LPS were diluted in PBS 1x and, when necessary, dilutions were made inside the laminar flow hood, in previously autoclaved Eppendorf bottles. Samples should be at a final concentration of 1µg / mL in the plate well.

[0190] Samples were added to the wells of a 96-well plate containing 200 µL of blood.After addition of LPS, the plates were incubated. The final volume in each well was always 250 µL. For the negative control, 25 µL of PBS was used. For the positive control, the commercial anti-inflammatory drugs dexamethasone (steroidal) and indomethacin (non-steroidal) were used.

[0191] Then, the plates were incubated for 24 h in an incubator with a 5% CO2 atmosphere, at 37°C. After incubation, the plate was centrifuged, the plasma was removed and, when necessary, frozen until subsequent measurements.

[0192] For PGE2 quantification, an ACN:MeOH solution was added to the previously obtained plasma to cause precipitation of plasma proteins. Then, the plasma was centrifuged, the supernatant was removed and transferred to a 15 mL Falcon tube containing MilliQ water.

[0193] Using solid phase extraction, Supelclean cartridges (LC-18 SPE 500mg, #57012) are initially conditioned by the addition of MeOH, followed by MilliQ water with 0.1% acetic acid.The sample is then added to the cartridge, followed by another fraction of MilliQ water with 0.1% acetic acid. The sample containing PGE2 to be quantified is collected in eppendorf tubes by elution with MeOH acidified with 0.1% acetic acid. The samples are then stored in a freezer, if necessary. Each sample was dried using Speed ​​Vaccum.

[0194] In the final step, the samples are resuspended in chloramphenicol PI for injection into the LC-MS / MS. The injection volume was 20 µL. Samples should always be transported on ice to avoid PGE2 degradation.

[0195] The chromatographic quantification method was validated according to ANVISA (2012) standards. Concentrations of 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, and 0.195 ng / mL were used to construct the calibration curve. A gradient method was used for the mobile phase. The mobile phase consisted of MilliQ water with 0.1% formic acid as phase A and acetonitrile as phase B.The total run time was 8.5 min for each sample. The column oven operated at 30°C. The vial should be kept on ice and protected from light until injection, and returned after injection. Run data for chloramphenicol: 320.90>152.10 m / z; Q1: 16; CE: 20; Q3: 29. Run data for PGE2: 351.10>271.30 m / z; Q1: 13; CE: 18; Q3: 28. EAR EDEMA ASSAY ANIMALS

[0196] Adult male Swiss mice weighing 20-30 g (n=60) were used in this study. The animals were obtained from the Central Animal Facility of the Federal University of Alfenas – Minas Gerais, Brazil. The mice were randomly divided into treatment and control groups, and the researchers were not informed of the group assignment. The animals were housed under controlled light conditions (12-hour light / dark cycle; lights on at 6:00 AM), with a room temperature of 22°C, and access to food and water ad libitum.Mice were acclimated in groups to housing conditions for 3 days prior to the experiments, in their respective treatment boxes. All experiments were conducted under the approval of the Ethics Committee of the Federal University of Alfenas (protocol number 013 / 2022). CROTON OIL-INDUCED EAR EDEMA ASSAY

[0197] The ear edema assay was performed to evaluate the topical anti-inflammatory activity of four new synthetic spilanthol derivatives. This in vivo model can assess edema inhibition and thus access the cyclooxygenase (COX) pathway, as well as measure neutrophil recruitment in the same experiment for indirect assessment of lipoxygenase (LOX) pathway inhibition. Therefore, this in vivo assay allows the evaluation of anti-inflammatory activity in the COX and LOX pathways, with the potential to indicate compounds with dual mechanisms of action.

[0198] The method used in this study to evaluate ear edema in mice involved the application of croton oil and was performed in groups of ten animals, in accordance with other works in the literature. The compounds (1.5 µM / ear) and the vehicle (control animals) were applied topically 30 minutes after the application of 20 µL of croton oil (5% v / v in acetone) to the inner surface of each left ear of the mice. Dexamethasone (1.5 µM / ear) was used as a positive control. Edema was measured 6 hours after application of the compounds / vehicle by the difference in weight between the 6 mm ear fragments removed from the left and right ears of the mice. Data were expressed as mean ± SEM and analyzed by one-way ANOVA followed by Dunnett's multiple comparison test. The inhibition percentages were calculated according to the formula: [(mean values ​​of the vehicle group - mean values ​​of the treated group) / mean values ​​of the vehicle group] × 100.QUANTIFICATION OF THE CHEMICAL MEDIATORS OF INFLAMMATION PGE2 AND LTB4 AND DETERMINATION OF NEUTROPHIL RECRUITMENT

[0199] The left ear fragments obtained in the ear edema evaluation were stored in 400 µL of NaEDTA / NaCl buffer (pH 4.7) and frozen at -20°C until analysis to evaluate the quantification of the chemical mediators of inflammation PGE2 and LTB4. The ear fragments were homogenized and ground using the Turrax tissue macerator (13,000 rpm), in the absence of light, on crushed ice, and were centrifuged at 3000 rpm for 15 min at 4°C. The precipitate was reserved for the following assay to quantify neutrophil recruitment. The supernatant obtained was lyophilized until dry and evaluated for its concentration of the eicosanoid PGE2 and the leukotriene LTB4, important mediators of the inflammatory process

[0013] .The dried supernatants were resuspended in a volume of 100 µL of a 25 ng / mL solution of chloramphenicol (CAP), an internal standard solubilized in acetonitrile. The samples were homogenized in an ultrasonic bath for 2 min and vortexed to solubilize the chemical mediators of inflammation PGE2 and LTB4. After that, the samples were centrifuged for 10 min at 10,000 rpm at 4°C, and 80 µL of the sample supernatant was analyzed in UHLPC-MS / MS.

[0200] The mediators, PGE2 and LTB4, were quantified in an ultra-performance liquid chromatograph (UHPLC) coupled to an MS / MS mass spectrometer with a triple quadrupole (QqQ) analyzer and electrospray ionizer (ESI). An injection volume of 20 µL and the gradient chromatographic method of 60 to 90% acetonitrile (MeCN) in acidified water (0.1% formic acid) from 0 to 4.5 min, 90 to 100% from 4.5 to 5.5 min were used., maintained at 100% from 5.5 to 6.5 min, reconditioned in the initial mobile phase from 100% to 60% from 6.5 to 7.5 min, maintaining at 60% until a total of 10 min of analysis. A flow rate of 0.3 mL / min, compatible pre-column and C18 column (Agilent Poroshell 120 EC-C18) 2.7 µm, 3 mm x 100 mm were used. Multiple Reaction Monitoring (MRM) of the ions of the mediadotes PGE2, LTB4 and the internal standard CAP was performed at 351.1>271.2; 335.1>195.1; 321.1>152.5 m / z, respectively.

[0201] Neutrophil recruitment and myeloperoxidase (MPO) activity are important markers of inflammation, as MPO is found abundantly in neutrophils. To determine neutrophil recruitment, fragments of the left ear obtained in the previous experiments. Before using the kit, the precipitate that was reserved as previously described was homogenized and centrifuged and the supernatant was used to measure MPO activity.MPO activity was determined using an enzymatic reaction with substrate reagent (tetramethylbenzidine and hydrogen peroxide) following the instructions of the BD-OptEIA™ kit from BD Biosciences (San Diego, CA, USA). After 10 minutes, the reaction was stopped with 2.5 M H2SO4 and the absorbance was measured at 450 nm. The results were expressed as MPO activity. Statistical analysis was performed using the same methods as in the previous evaluation. RESULTS OBTAINED BY THE PRESENT INVENTION CHEMICAL SYNTHESIS OF THE DERIVATIVES

[0202] In general, spilanthol and HE-3 analogs were obtained through a methodology useful for performing structural modifications on spilanthol.

[0203] Isobutylphosphonatocetamide (iBuPA) used in the synthesis of the derivatives HE-1, 3, 4, 5, 6, 8, 9, 10, 14 and 15 was prepared using a direct amidation reaction as described in Scheme 1, using the Lewis acid lanthanum triflate as catalyst:.

[0204] The spilanthol-eugenol hybrid HE-8 was synthesized according to the synthetic route shown in Scheme 2 below. The synthesis of this derivative differs from HE-9 in step initial. In this case, a triflate is formed from the phenol of the eugenol structure (intermediate 8.a), using phenyl triflimide. The preparation of the eugenol triflate showed a 72% yield.

[0205] For the derivative HE-9, an o-selective bromination is carried out with respect to the phenol group of eugenol, shown in Scheme 3, intermediate 9.a, below. The method used is the only one that showed selectivity in the bromination position, with a yield of 55%. The subsequent synthetic steps were carried out similarly for both derivatives, starting from each of the initial products obtained. The Sonogashira reaction used the bromide (9.a) or the eugenol triflate aryl bromides (8.a) that were obtained as substrates, together with 4-pentyn-1-ol. Both reactions required heating due to steric hindrance in each of the substrates. Aryl bromides also exhibit lower reactivity in coupling reactions, when compared to slightly hindered vinyl bromides (1-bromo-1-propene). The yields were 70% for 8.b and 48% for 9.b (Schemes 2 and 3, respectively).

[0206] The Swern oxidation and Horner–Wadsworth–Emmons (HWE) olefination reactions were accessed to complete the hybrids HE-8 and 9. The final yields for the two reactions in the synthesis of each compound were 70% and 52%, respectively.

[0207] The derivative HE-10 was obtained according to Scheme 4 below. The Reimer–Tiemann reaction for the formylation of eugenol showed 84% yield, with selectivity for the ortho to hydroxyl position. The HWE reaction used in the synthesis of HE-10 showed 89% yield, which is possibly due to the fact that aldehyde 10.a can be isolated and stored, so this yield is relative to only one reaction step.

[0208] Some derivatives used aldehyde 1.b to be synthesized, as shown in Scheme 5, A below. The reactions The Sonogashira reactions performed for the formation of the enyne alcohol 1.a were performed according to the methodology reported for the synthesis of spilanthol, with 56% yield and selectivity greater than 9:1 for the trans isomer. Also similar to the reported methodology, the aldehyde 1.b was then obtained from the oxidation of the alcohol 1.a, through the Swern reaction (ALONSO et al., 2018). The aldehyde 1.b is volatile and difficult to isolate, in addition to being unstable. Therefore, the aldehydes obtained as products of the Swern reactions were not completely isolated. The crude products of these reactions were used in the next step, after undergoing only a liquid-liquid extraction.

[0209] For the synthesis of the hybrid HE-11, a specific phosphonatoacetamide was prepared to converge in the HWE reaction, as shown in Scheme 5, B above.The nitration reaction of this route was selective, with no evidence of nitration in other regions, possibly due to the substitution pattern of the eugenol benzene ring, resulting in 92% yield. SnCl2.2H2O was used to reduce the aromatic nitro group because it exhibits high selectivity in reducing this type of group compared to Pd / C, which could reduce the allylic chain. The desired amine was obtained in 69% yield. Finally, in the reaction to form phosphonatoacetamide, DMAP was not used as a catalyst to increase the selectivity of amide formation over esters, resulting in the formation of the product in 78% yield. The Swern oxidation reaction, using alcohol 1.a, followed by the olefination of HWE, resulted in 63% yield, similar to that obtained with isobutylphosphonatoacetamide.

[0210] For the synthesis of the HE-11 hybrid, a specific phosphonatoacetamide was prepared to converge in the HWE reaction, as shown in Scheme 5, B above. The nitration reaction of this route occurred selectively, with no evidence of nitrations in other regions, possibly due to the substitution pattern of the benzene ring of eugenol, presenting 92% yield. SnCl2.2H2O was used to reduce the aromatic nitro group because it presents high selectivity in the reduction of this type of group, when compared to Pd / C, which could reduce the allylic chain. The desired amine was obtained in 69% yield. Finally, in the reaction to form phosphonatoacetamide, DMAP was not used as a catalyst in order to increase the selectivity of amide formation over esters, leading to the formation of the product in 78% yield. The Swern oxidation reaction, using alcohol 1.a, followed by olefination of HWE, showed 63% yield, similar to that when using isobutylphosphonatoacetamide.

[0211] For the synthesis of HE-4, an HWE reaction between commercially obtained n-octanal and iBuPA is carried out, with 81% yield, as shown in Scheme 6, A below. The HE-5 derivative is obtained in 94% yield from a direct amidation between decanoic acid and isobutylamine, using EDC and DMAP (Scheme 6, B).

[0212] It was not possible to obtain the desired product when using the HWE reaction with sodium hydride to synthesize the HE-6 derivative, as shown in Scheme 7 below. It is possible to assume that the hydrogen alpha to the alkyne group, conjugated with a highly electron-withdrawing ring, may have sufficient acidity to be abstracted by the hydride present in the medium. Thus, several undesired reactions may have occurred. Because of this, a methodology that is useful when dealing with substrates sensitive to strong bases was evaluated. This method uses tBuOK and LiCl and showed selectivity above 95% in the formation of the trans product. The selectivity of the reactions of HWE were confirmed by two peaks common to the NMR spectra of 1H of the products. One of them refers to a doublet with a chemical shift between 6 and 6.4 ppm and a J value around 15 Hz. The other refers to a doublet triplet between 6.7 and 7.1 ppm, with J values ​​around 15 and 6.5 Hz. The yield of this methodology for the Swern reaction followed by the HWE reaction was 43%.

[0213] Like derivative HE-11, derivatives HE-7, 12, and 13 were synthesized from aldehyde 1.b (Scheme 5, A), but using different phosphonatoacetamides. Intermediates 7.a and 12.a were prepared in 94 and 82% yields, respectively, according to Schemes 8, A and B below. The yields of the HWE reactions for compounds HE-7 and HE-12 were in 71% and 49% yield, respectively.

[0214] In turn, the synthesis of the HE-13 derivative started from the commercial compound 2-fluoro-4-nitrobenzoic acid, with an initial sequence of reactions similar to those carried out by Ann et al., 2020, to obtain intermediate 13.b, as shown in Scheme 9 below. The subsequent reactions led to the formation of phosphonatoacetamide 13.e, which was used in the HWE reaction for the synthesis of the HE-13 derivative. Initially, this derivative was synthesized without protecting the hydroxyl group of intermediate 13.b with the TBDMS group. However, the amidation reactions with EDC, even without DMAP, and HWE showed yields of less than 29% and formed byproducts that were difficult to remove. More than one chromatographic column and one recrystallization were required to adequately purify the final product. Thus, hydroxyl protection resulted in easier purifications and, consequently, improvements in yields.The HWE reaction conditions were similar to those of the HE-6 derivative, as they were milder and required simpler purification. All reactions were successful. yields, including the final reaction for hydroxyl deprotection with nBu4NF. This allowed the preparation of larger quantities of the HE-13 and HE-17 derivatives for in vivo analyses.

[0215] The HE-14 derivatives shown in Scheme 10A below and HE-15, shown in Scheme 10B below, were synthesized from commercially available aldehydes. The synthesis of these compounds required only two steps, which showed high yields. The analogues HE-4, 5, 14, and 15 had previously been synthesized by Isabella Gonçalves Alonso, but had not been evaluated as potential anti-inflammatory agents.

[0216] Scheme 11, shown below, shows the synthetic route used for derivatives HE-16 and HE-17. Since these two derivatives were designed from the previous ones, the same intermediates and starting materials were reused. In order to maintain structural fragments of derivatives HE-8 and 13 in derivative HE-16, aldehyde 8.c and phosphonatoacetamide 13.e were used in the HWE reaction (Scheme 11, A). This reaction showed 53% yield for this derivative, also considering the Swern reaction from the previous step. The hydroxyl deprotection step of intermediate 16.a occurred in 91% yield. Finally, the last compound to be synthesized was HE-17, which maintains fragments of derivative HE-14 (fagaramide) and HE-13. As for the synthesis of fagaramide, piperonal was also used, in addition to phosphonatoacetamide 13.e, in the HWE reaction (scheme 11, B).The HWE reaction showed a 70% yield, which is probably higher because it is only one step. reaction, starting from a commercially available aldehyde, which can be isolated and stored. The deprotection of the hydroxyl group of intermediate 17.a also showed a high yield of 93%. RESULTS OBTAINED FOR THE PRESENT INVENTION IN RELATION TO EX VIVO ASSESSMENTS AND EAR EDEMA ASSAY Ex vivo assays

[0217] This assay performs a quantitative analysis of inflammatory chemical mediators present in human blood plasma, such as PGE2. The chemical samples are added to the wells containing blood and, after inducing an inflammatory process with LPS (E. coli O26:B6), the mediators are evaluated by LC-MS.

[0218] The results of the initial ex vivo analysis are shown in Figure 4 and the derivatives are shown in the structures below. It can be observed that the derivatives HE-8 and HE-9 have activities statistically similar to indomethacin and dexamethasone in inhibiting the production of PGE2.The results suggest that the presence of the eugenol HE-2 structure in the alkyl chain of HE-3 contributes to the inhibitory activity of PGE2 production. However, this only occurs when the number of carbons between the α,β-unsaturated amide and the alkyne of the alkyl chain is maintained (HE-8 and 9: 84 and 86 nM [PGE2], respectively). It is observed that the presence of the phenolic hydroxyl of eugenol and the position of the alkyne in the aromatic ring, para or meta to the allylic chain, do not affect the activity. The removal of the alkyne and the decrease of 4 carbons in the alkyl chain between the amide and the eugenol ring abolished the activity (HE-10, 139 nM [PGE2]). The same occurred with the insertion of eugenol in the moiety. amide of HE-3 (HE-11, 141 nM [PGE2]).

[0219] The results presented in Figure 5 refer to the second ex vivo test performed; the other derivatives are shown in the diagram below. It can be seen that the replacement of the isobutylamide of HE-3 by an amide similar to that in compound MDR-652 was the modification that showed the best anti-inflammatory activity, statistically similar to the standards.

[0220] When comparing the activity of derivatives HE-12 and 13 (more active) with derivatives HE-7 and HE-11 (less active), it can be expected that the substituents of the amide moiety are interacting with a very narrow hydrophobic region in the site of action. This can be suggested because the greater distance between the amide nitrogen and the aromatic ring in that moiety (HE-7: 48 nM [PGE2]) reduced the activity. Furthermore, the trisubstituted aromatic ring with bulky substituents in compound HE-11 also reduced the activity. In turn, less bulky substituents such as isobutylamine and rings present in derivatives HE-12 (19 nM [PGE2]) and HE-13 (8 nM [PGE2]) showed greater activity.Additionally, the alcoholic hydroxyl group of HE-13 may be interacting with some hydrophilic amino acid residue close to this hydrophobic region at the site of action, but which the phenolic hydroxyl group of HE-11 should not reach. The results obtained from the molecular modifications performed suggest an interaction with the active sites of COX enzymes, which is corroborated by the docking assays.

[0221] The removal of the diene or enyne systems caused a slight decrease in activity (HE-4: (27 nM [PGE2]) compared to HE-1. The removal of the double bond conjugated with the amide in the HE-5 derivative (23 nM [PGE2]) does not appear to have a significant influence on the biological activity, when compared to the HE-4 derivative.

[0222] Inverting the electron density of the aromatic ring inserted in the alkyl chain significantly reduced the activity (HE-6: 23 nM [PGE2]).The same occurred when removing the allylic chain, the methoxyl and the alkyne from the eugenol ring inserted in the same chain (HE-15: 21 nM [PGE2]).

[0223] Although fagaramide (HE-14, 15 nM [PGE2]) was not comparable to indomethacin and dexamethasone, it showed significant inhibition of PGE2, being the second most potent substance in the second test. Thus, as the benzodioxol group simplifies the structure and synthesis of derivatives,. It is justifiable to maintain it in the design of a new analogue.

[0224] Considering the inhibition of PGE2 and LTB4, observed in the in vivo assay, all substances H8, H13, H16, and H17 showed significant activity, all of which were statistically different from the vehicle (Figures 10 and 11). All of them showed both inhibition of PGE2 and LTB4 production, making it a very promising derivative. As mentioned, compounds capable of dually inhibiting the synthesis of these mediators, and which are not corticosteroids, have been highly sought after. The molecular docking results presented below corroborate the anti-inflammatory activity of the evaluated compounds. They are possibly interacting with the COX-1 and COX-2 enzymes, which participate in PGE2 biosynthesis. Docking studies also suggest that the inhibition of LTB4 biosynthesis results from an interaction with the 5-LOX enzyme. Molecular docking in COX-1, COX-2 and 5-LOX enzymes.

[0225] Table 1 presents the predicted energies for the most stable complex formed by the ligand under study and the crystallographic structures of COX-1, COX-2, and 5-LOX. The derivatives evaluated were HE-1, 2, 3, 8, 13, 14, 16, and 17 (Table 1). The redocking energy presented in the tables was calculated by Autodock Vina from the structure of the crystallographic complex of a known inhibitor or ligand with the specific enzyme. The 2OYU complex refers to indomethacin bound to the COX-1 enzyme. 3NT1 is the naproxen complex bound to the COX-2 enzyme. Finally, 3V99 is the arachidonic acid complex with the 5-LOX enzyme. A docking assay was also performed on the mpges-1 enzyme, but since the energy values ​​were higher, they were not. will be addressed. Table 1: Summary of binding energy results obtained in molecular docking studies between the evaluated compounds and the COX-1, COX-2 and 5-LOX enzymes.

[0226] From the results in Table 1, considering only the energy values ​​presented in general for all enzymes, it is possible to see that the evaluated derivatives have a greater tendency to interact with the active site of the COX-1 and 2 enzyme isoforms. There is also a tendency to interact with the 5-LOX enzyme, but less than with the COX enzymes. It is worth noting that the lower the energy value of the complex formed (more negative), the greater interaction the ligand evaluated in the docking had with the enzyme. This result may indicate that the greater the compound's capacity to interact and inhibit the evaluated enzyme would be. When comparing only the hybrids (derivatives HE-8 to HE-17), HE-13 is the one that shows the lowest affinity for the COX-1 active site, and higher affinities for the active sites of COX-2 and 5-LOX.Despite the lower activity against COX-1, this result may corroborate the high inhibition value (82%) that this derivative presented in the ex vivo test (Figure 5), because one of the enzymes most expressed in inflammation caused by LPS is COX-2. Regarding the HE-8 derivative, it presents interaction values ​​similar to those of HE-14, being slightly higher only in relation to 5-LOX. Both compounds showed high inhibition capacity of prostaglandin production in the ex vivo tests, being considerably more active than the other precursor compounds (HE-1 to HE-3). Derivative HE-16, on the other hand, shows the lowest affinity for the COX-2 active site, with a slightly better affinity for COX-1 than HE-13. As mentioned, the increased surface size of the structure compared to HE-13 may have hindered this derivative's accommodation in the active sites of COX enzymes.It is important to consider that in silico analyses evaluate the affinity of compounds in specific conformations with the active site, in isolation. Factors such as physicochemical characteristics that may interfere with the diffusion of these compounds through biological membranes, solubility in biological media, or metabolism, as in the case of prodrugs, are not considered in the analysis. These factors can significantly impact the results of ex vivo, in vivo, and in vitro biological assays. Comparing the energy values ​​of the hybrid derivatives, HE-8, 13, 16, and 17, with their precursors, HE-1, 2, 3, and 14, reveals an increase in the ligands' affinity for the enzymes in general. Therefore, the results of the ex vivo and docking assays performed are consistent, indicating the relative success of the adopted molecular modification strategy.

[0227] The results of the molecular docking assays for each specific enzyme are presented below.Table 2 presents the results of molecular docking studies involving the COX-1 enzyme. This table provides an overview of the interactions performed by each compound in the COX-1 active site. The precursors HE-1 and HE-3 exhibit very similar conformations and points of interaction in the active site, likely due to their high structural similarity. The alkyl chain of the HE-13 derivative, on the other hand, exhibited a significantly different conformation and spatial arrangement than these two precursors. Changing the amide portion of this derivative altered one type of interaction and many of the amino acid residues involved, in addition to adding a new point of interaction. Considering the derivatives HE-8, 13, and 16, it can be observed that the conformations and spatial arrangements between these derivatives were quite different. Despite having the fewest points of interaction, the HE-8 derivative had the lowest energy.The HE-16 derivative exhibits the highest number of interactions, including hydrogen and halogen bonds, but an energy 0.3 kcal / mol higher than that of HE-8. Compared to compounds HE-14 and HE-17, the insertion of the amide moiety of HE-13 did not cause significant changes in their conformation and spatial arrangement in the active site. The total number of interactions remained the same in both compounds, but HE-14 exhibits two additional hydrogen bonds, presenting a lower energy. Additionally, some additional peculiarities can be observed regarding the positioning of some compounds. It is worth mentioning that HE-13, HE-14, HE-16, and HE-17 have at least one of their aromatic rings with equivalent poses, maintaining van der Waals interactions with aromatic amino acid residues, such as PHE381 and TRP387.Furthermore, they also show proximity to the SER530 residue, important for the irreversible inhibition mechanism of acetylsalicylic acid (MALKOWSKI et al., 2000). An important point to highlight regarding the analysis of the interaction of the precursors HE-1 and HE-3 is that the amide group of both ligands is positioned close to the amino acid residues TYR355 and ARG120, essential amino acids for interaction with the carboxyl group of arachidonic acid. This interaction is reproduced by compounds HE-14 and HE-16, also via their amide groups, which may suggest that this group acts as an "anchor" group for these compounds (MALKOWSKI et al., 2000). Finally, there is evidence of a possible halogen bond between the fluorine atoms in the structures of HE-13 and HE-16 and the residues MET522 and LEU352, respectively.Even considering that fluorine is a weak halogen bond donor, it is possible that this atom contributes at least to the modulation of polar interactions with the enzyme. Table 2: Binding energy and interaction points presented in the complexes formed by the compounds under study with the COX-1 enzyme. a Energy value of the lowest-energy complex formed, in kcal / mol. (LH): Hydrogen bonding, (VdW): Van der Waals forces, (LHal): Halogen bonding. Images of the in silico COX-1 assay are shown in Figures 12 and 13.

[0228] Table 3 presents the energy values ​​obtained during the docking assay with the COX-2 enzyme, in addition to an overview of the interaction points between the compounds evaluated and the active site. In this study, it is observed that the interactions between the COX-2 enzyme and the compounds evaluated remain similar to those found in the studies with COX-1. The largest variations in terms of binding energy occur for the ligands HE-13 (-7.3 to 8.0 kcal / mol) and HE-16 (-7.8 to -6.6 kcal / mol). For HE-13, the maintenance of the possible halogen bond between this compound and the MET522 residue in the active sites of both COX isoforms is observed.However, the alkyl chain of this derivative appears to fit better into the COX-2 enzyme, extending along the catalytic site of this enzyme, when compared with the pose assumed by this portion of the HE-13 structure in COX-1. This difference may have optimized hydrophobic interactions with the binding site, which is narrower and shorter in COX-1 than in COX-2 due to the Ile523 residue (KURUMBAIL et al., 1996). This characteristic appears to increase the stability of the COX-2:HE-13 enzyme-ligand complex. In the case of the HE-16 derivative, the effect appears to be the opposite. This derivative inverted its position in the COX-2 binding site, which appears to hinder the possibility of interactions with residues ARG120 and TYR355, which are observed for the complex formed with COX-1.Although this inversion maintains the halogen bond with residue LEU352, in addition to ensuring other polar interactions with residues GLN192 and PHE518, this change may explain the increase in the complex's energy. This positional inversion at the COX enzyme sites can also be observed for compounds HE-8, 14, and 17, but in these cases, there were no significant changes in the predicted binding energies. However, it is important to note that the changes primarily impact the possibility of hydrogen bond formation with residues ARG120 and TYR355. Assays have suggested that these residues should be considered an important interaction site to be explored in these ligands for affinity enhancement. Table 3: Binding energies and interaction sites presented in the complexes formed by the compounds under study with the COX-2 enzyme. aEnergy value of the lowest-energy complex formed, in kcal / mol. (LH): Hydrogen bonding, (VdW): Van der Waals forces, (LHal): Halogen bonding. Images of the in silico COX-2 assay are shown in Figures 14 and 15.

[0229] The next docking to be presented is that of the 5-LOX enzyme; this enzyme is significantly different from the previously evaluated COX enzymes. Despite this, it also has arachidonic acid as a substrate, participating in the biosynthesis of LTB4, another important inflammatory mediator. Table 4 presents the main results of the molecular docking studies involving the 5-LOX enzyme. For comparative purposes, the results of the molecular docking study with the 5-LOX inhibitor drugs zileuton and meclofenamic acid will be mentioned. Table 4: Binding energy and interaction points presented in the complexes formed by the compounds under study with the 5-LOX enzyme. a Energy value of the lowest-energy complex formed, in kcal / mol. (LH): Hydrogen bonding, (VdW): Van der Waals forces, (LHal): Halogen bonding. Images of the in silico assay on 5-LOX are shown in Figures 16 and 17.

[0230] In general, the interaction potential analyzed solely in relation to the binding energy of the compounds with the 5-LOX active site does not stand out in relation to the other enzymes evaluated. Compound HE-17 appears to show the greatest affinity for this enzyme, followed by HE-13, while HE-1 appears to have the lowest affinity for it, excluding HE-2 (eugenol). Because this is a very broad active site for conformational searching, diverse interaction patterns between the ligands evaluated and the enzyme can be observed.HE-1 and HE-13 maintain their fatty acid chains in similar positions, exploiting interactions with similar amino acid residues, differing in the interactions performed by the substituent in the amide portion of each compound's structure. These interactions are possibly responsible for increasing the stability of the 5-LOX:HE-13 complex compared to 5-LOX:HE-1 (Table 4). The same observation can be made when comparing HE-2 with HE-8, in which the interactions involving the aromatic moiety and its substituents are similar. However, in the case of HE-8, this is compounded by the possibility of interaction of the isobutylamide moiety, derived from HE-1 and HE-3, with additional hydrophobic residues in the active site. The sum of the interactions of these two portions of HE-8 allows for a reduction in the interaction energy compared to the precursors HE-1, 2, and 3. Considering the ligands HE-14, 16, and 17, the amino acid residue ASN554 appears to be a common interaction point.The larger surface area of ​​HE-16 and HE-17 allows for a greater number of interactions with the enzyme, which may explain the greater stability of the complexes formed with these compounds when compared to the complex formed by HE-14.

[0231] Figure 7 shows a comparison of the poses assumed in the docking assays by the ligands under study (white carbons) with the poses assumed by two drugs with 5-LOX inhibitory action in its active site. The inhibitory drugs are zileuton (cyan carbons) and meclofenamic acid (pink carbons). It can be seen that the ligands HE-1, 2, 3, 13, and 14 assume conformations and poses that make it difficult to achieve and reproduce the same interactions performed by the inhibitors. However, the amide portion of the HE-13 group may allow additional hydrogen and halogen bonds in the active site of 5-LOX, as well as in the HE-16 derivative.For the HE-8 derivative, it is possible to observe that its lowest energy conformation corresponds to a pose very similar to that assumed by the drug meclofenamic acid. The HE-16 and 17 derivatives assume poses that allow them to interact with common interaction points of both meclofenamic acid and zileuton. The high similarity of some of the interactions presented by these last four compounds suggests a potential for interaction and inhibition of 5-LOX. Ear edema assay ANTI-INFLAMMATORY ACTIVITY BY EAR EDEMA ASSAY

[0232] All spilanthol derivatives tested in the in vivo assay (HE-8, HE-13, HE-16, and HE-17) demonstrated anti-edematogenic activity compared to the vehicle (negative control), according to statistical analysis by ANOVA followed by Dunnett's multiple comparison test (p < 0.001).The HE-8 derivative showed a substantial reduction in inflammation with a 69.02% reduction (2.375 mg / ear ± 1) compared to the negative control (7.667 mg / ear ± 0.8). Furthermore, HE-8 showed a statistically comparable reduction in inflammation to the positive control dexamethasone (82.61% inhibition, 1.333 mg / ear ± 1), a classic anti-inflammatory steroid drug. This result is relevant since the samples were evaluated at the same concentration of 1.5 µmol / ear, and HE-8 is not a corticosteroid derivative, which are drugs known for a number of adverse reactions. The results demonstrate that HE-8 has comparable potency to the positive standard (FIGURE 8).

[0233] Although HE-13, HE-16, and HE-17 are less potent, they still exhibit significant anti-inflammatory activity.The results corroborate those obtained in the ex vivo assay, since prostaglandin E2 (PGE2) is produced via COX, the same pathway that is capable of reducing edema associated with the inflammatory response and increased local vascular permeability. Low PGE2 levels indicate anti-inflammatory and anti-edematogenic activity. ANTI-INFLAMMATORY ACTIVITY BY ASSESSMENT OF NEUTROPHIL RECRUITMENT

[0234] The inflammatory response involves the recruitment of immune cells, such as neutrophils, to the site of injury or infection. Neutrophils migrate to the site of inflammation and play a fundamental role in the body's natural defense. However, excessive neutrophil infiltration can cause tissue damage and contribute to the pathogenesis of inflammatory diseases, thus neutrophil recruitment is an interesting therapeutic target for the development of anti-inflammatory drugs.

[0235] This study investigated the inhibitory effects of spilanthol derivatives on neutrophil migration in a mouse ear edema model induced by croton oil. The drug dexamethasone, a corticosteroid known to inhibit not only COX-mediated edema but also neutrophil recruitment by inhibiting the initiation of the inflammatory cascade, was used as a positive control. Topical applications of HE-8, HE-13, HE-16, and HE-17 inhibited neutrophil migration compared to the negative control, with relevant percentage inhibition values ​​and statistical significance (p-value < 0.001). Dexamethasone inhibited neutrophil migration by 56.7%, while the HE-8 derivative inhibited neutrophil migration by 43.5% (FIGURE 19). Neutrophil migration in a tissue can be estimated by the activity of MPO, an enzyme mainly present in neutrophils.Leukotrienes B4 (LTB4), produced through the LOX pathway, are one of the main compounds involved in recruiting these polymorphonuclear cells to the inflamed site. Therefore, lower MPO levels also indicate potential inhibition of the LOX pathway. Dexamethasone, a steroid drug, indirectly inhibits neutrophil recruitment, while current nonsteroidal anti-inflammatory drugs (NSAIDs) act primarily on the COX pathway without necessarily inhibiting neutrophil recruitment. Therefore, the new spilanthol derivatives have potential anti-inflammatory activity through a novel mechanism of action, with potential dual inhibition of the COX and LOX pathways.

[0236] The chemical mediators of inflammation, PGE2 and LTB4, are produced by two main pathways of the inflammatory cascade: COX and LOX, respectively. These mediators can be evaluated in the experimental model of ear edema.All derivatives evaluated demonstrated a significant reduction in PGE2 and LTB4 levels, comparable to the reference drug dexamethasone, and different from the negative control (vehicle; p < 0.0002, Figures 10 and 11). Derivative H-8 showed the greatest reduction, 60.0% and 14.2%, in PGE2 and LTB4 levels, respectively. The same derivative H-8 also showed greater inhibition of ear edema, corroborating the result of edema and COX pathway inhibition (Figures 9, 10 and 11). The other derivatives HE-13, HE-16 and HE-17 also showed inhibition of PGE2 production of 53.2%, 54.0% and 56.6% and LTB4 of 13.0%, 13.8%, 14.0% respectively, unlike the vehicle and demonstrated to be comparable with the reference drug dexamethasone (p < 0.0001 and p ≤ 0.0002) (Figures 10 and 11). The results demonstrated that substances HE-8, HE-13, HE-16 and HE-17 showed dual inhibition of the COX and LOX pathways, and consequently inhibition of the synthesis of PGE2 and LTB4 production.

[0237] In view of the above, the synthetic route for obtaining spilanthol developed can be used in the synthesis of various spilanthol derivatives, as demonstrated. Of the 17 derivatives synthesized and evaluated, 4 were more active than spilanthol and eugenol in inhibiting PGE2 production. This shows that the design based on the molecular hybridization strategy presented good results in relation to the improvement of anti-inflammatory activity.

[0238] A molecular docking analysis on the COX-1, COX-2, and 5-LOX enzymes allowed corroborating the results presented by the compounds in the ex vivo tests, suggesting that their activity in inhibiting PGE2 production is related to the inhibition of the COX-1 and COX-2 enzymes. These in silico assays also suggest that the inhibition of LTB4 production may be due to the interaction with the 5-LOX enzyme.Because ex vivo analysis evaluates anti-inflammatory activity in an environment that simulates human blood, this activity is not evaluated on a specific target, but rather as a whole. Therefore, docking analyses of these enzymes help to elucidate the mechanism of action of these compounds. Similarly, quantification of the mediators PGE2 and LTB4 in ear fragments obtained in the in vivo anti-inflammatory assay. Of all the compounds designed and evaluated, the HE-8 derivative showed the best anti-inflammatory activity in both the ex vivo and in vivo assays. This compound significantly inhibited both the inflammatory mediators PGE2 and LTB4, in addition to reducing neutrophil recruitment. Mice treated with HE-8 had the lowest ear edema burden, indicating reduced inflammation.

[0240] The other derivatives presented a similar edema mass, larger than that of the HE-8 derivative, but significantly smaller than that of the vehicle used in the study. The activities against PGE2 and LTB4 inhibition of the HE-13, HE-16 and HE-17 derivatives in the in vivo assays were not significantly different, all showed high activity, the HE-17 derivative was the second greatest PGE2 inhibitor. In the case of inhibition of neutrophil recruitment, the HE-17 derivative was as active as the HE-8 derivative.

Claims

1 / 6 CLAIMS 1. Process for obtaining non-steroidal anti-inflammatory compounds, characterized by comprising the steps: I. Procedure for synthesizing isobutylphosphonatoacetamide (iBuPA); II. Procedure for synthesizing intermediates 13.a, 13.b, 13.c, 13.d and 13.e; III. Procedure for synthesizing intermediate 8.a; IV. Procedure for the Sonogashira reaction; V. Procedure for the Swern oxidation reaction; VI. Procedure for the Horner-Wadsworth-Emmons (HWE) reaction; and VII. Procedure for removing the t-butyldimethylsilyl group.

2. Process according to claim 1, characterized by the fact that it is a convergent process, in which steps I to IV, obtaining the phosphonatoacetamide intermediate, occur at the same time as step V, which obtains the aldehyde intermediate specific for each derivative. 3.Process according to claim 1 or 2, characterized in that the phosphonatoacetamide intermediate and the specific aldehyde intermediate are joined in the Horner-Wadsworth-Emmons (HWE) reaction of step VI so as to obtain the intermediates:. . 2 / 6 4. Process according to claim 1, characterized in that in step IV, for the synthesis of intermediate 1.a, a Sonogashira reaction is carried out between 4-pentyn-1-ol and 1-bromo-1-propene, using an organic base, preferably diisopropylamine, a palladium catalyst, preferably Pd(PhP)4, a copper (I) salt, preferably CuI, in a polar aprotic solvent, preferably dimethylformamide.

5. Process according to claim 1, characterized in that in step III, intermediate 8.a is prepared from a reaction of eugenol with phenyltriflimide, using an inorganic base, preferably K2CO3, in a polar aprotic solvent, preferably tetrahydrofuran.

6. Process according to claim 1, characterized in that in step IV, for the synthesis of intermediate 8.b, intermediate 8.a is used instead of 1-bromo-1-propene. 7.Process according to claim 3, characterized in that intermediate 17.a is obtained using piperonal as aldehyde.

8. Process according to claim 1, characterized in that in step VII, to obtain the derivatives HE-13, HE-16 and HE-17, a deprotection reaction is carried out to remove the TBDMS (tert-butyl dimethylsilane) group, using n-Bu4NF (tetra-n-butylammonium fluoride).

9. Process according to claim 1, characterized in that in step I the intermediate IBuPA is synthesized from a direct amidation with o. 3 / 6 ethyl phosphonoacetate diethyl ester using lanthanum (III) triflate as catalyst.

10. Process according to claim 1, characterized in that in step II the phosphonatoacetamide 13.e is prepared.

11. Process according to claim 1, characterized in that in step II the 2-fluoro-4-nitrobenzoic acid is esterified with methanol and sulfuric acid to form intermediate 13.a which is then reduced to intermediate 13.b with sodium borohydride, in which the alcohol of intermediate 13.b is transformed into a silyl ether group, from the reaction with a silyl chloride, preferably t-butyldimethylsilyl chloride, to form intermediate 13.c, in which the nitro group in this intermediate is reduced with Pd / C and H2, forming intermediate 13.d.

12. Process, according to claim 11, characterized in that it starts from the amidation of intermediate 13.d with diethylphosphonoacetic acid, in the presence of EDC and DMAP, phosphonatoacetamide 13 is formed.

13. Non-steroidal anti-inflammatory compounds, characterized by the fact that they are obtained by the process, as defined in any one of claims 1 to 12, and have the following Markush formula:. where R1 can be selected from (3-fluorophenyl)-4-metanyl and isobutyl: 4 / 6 R2 can be selected from 4-allyl-1-(but-1-yn-1-yl)-2-methoxybenzene, (E)-hept-2-en-4-yne and benzo[d][1,3]dioxol:

14. Compound HE-8 (E)-7-(4-allyl-2-methoxyphenyl)-N-isobutylhept-2-en-6-ynamide characterized by the fact that it has the following formula: .

15. Compound HE-8, according to claim 14, characterized by the fact that it is obtained as a white solid and a final synthesis yield of 70%.

16. Compound HE-13 (2E,8E)-N-(3-fluoro-4-(hydroxymethyl)phenyl)deca-2,8-dien-6-ynamide, characterized by the fact that it has the following formula: 5 / 6 .

17. Compound HE-13, according to claim 16, characterized by the fact that it is obtained as a white solid and a final synthesis yield of 88%.

18. Compound HE-16 (E)-7-(4-allyl-2-methoxyphenyl)-N-(3-fluoro-4-(hydroxymethyl)phenyl)hept-2-en-6-ynamide, characterized by the fact that it has the following formula: .

19. Compound HE-16, according to claim 18, characterized by the fact that it is obtained as a white solid and a final synthesis yield of 91%.

20. Compound HE-17 (E)-3-(benzo[d][1,3]dioxol-5-yl)-N-(3-fluoro-4-(hydroxymethyl)phenyl)acrylamide, characterized by the fact that it has the following formula: . 6 / 6 21. Compound HE-17, according to claim 20, characterized by the fact that it is obtained as a white solid and a final synthesis yield of 93%.

22. Pharmaceutical composition, characterized by the fact that it comprises the compounds, as defined in claim 13, and a pharmaceutically acceptable excipient.

23. Use of the compounds, as defined in claim 13, and a pharmaceutically acceptable excipient, or of a pharmaceutical composition, as defined in claim 22, characterized by the fact that it is in the preparation of a medicament for the treatment of inflammatory diseases, or that involve an inflammatory process in the pathophysiology, such as: migraines, rheumatism, osteoarthritis, orthopedic infections or injuries and asthma or allergic rhinitis, in addition to inflammatory skin or systemic diseases.

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