Synthesis and Evaluation of Marine-Inspired Compounds Result in Hybrids with Antitrypanosomal and Antileishmanial Activities

Natural products are a very rich source for obtaining new compounds with therapeutic potential. In the search for new antiparasitic and antimicrobial agents, molecular hybrids were designed based on the structures of antimicrobial marine quinazolinones and eugenol, a natural phenolic compound. Following reports of the therapeutic potential of quinazolinones and eugenol derivatives, it was expected that the union of these pharmacophores could generate biologically relevant substances. The designed compounds were obtained by classical synthetic procedures and were characterized by routine spectrometric techniques. Nine intermediates and final products were then evaluated in vitro against Trypanosoma brucei and Leishmania infantum. Antifungal and antibacterial activity were also evaluated. Six compounds (9b, 9c, 9d, 10b, 10c, and 14) showed mild activity against T. brucei with IC50 in the range of 11.17–31.68 μM. Additionally, intermediate 9c showed anti-Leishmania activity (IC50 7.54 μM) and was six times less cytotoxic against THP-1 cells. In conclusion, novel derivatives with a simple quinazolinone scaffold showing selectivity against parasites without antibacterial and antifungal activities were disclosed, paving the way for new antitrypanosomal agents.


Introduction
Parasites are responsible for a high rate of mortality each year and represent a global health burden worldwide, with 11 parasitic infections identified as neglected tropical diseases by the World Health Organization (WHO) [1,2].Parasitic infections are rapidly spreading and becoming a major cause of chronic diseases due to climate change and environmental pollution, as well as increased resistance to the used drug arsenal [3].
Human African trypanosomiasis (HAT), also known as sleeping sickness, is a parasitecaused neglected disease that greatly affects sub-Saharan Africa, putting about 55 million people at risk (according to 2020 data) [4].This potentially fatal disease is caused by two Trypanosoma brucei species, namely Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, and the parasites are transmitted to the human host by the bite of the tsetse fly [5,6].WHO, in collaboration with governmental and nongovernmental organizations, has been able to significantly reduce the incidence of HAT to fewer 1000 cases per year, attributable to access to novel drugs [4,6].
Leishmaniasis is a parasitic neglected disease caused by Leishmania spp., which are transmitted to mammal hosts by the bite of infected female sandflies.Visceral leishmaniasis is the most severe and life-threatening form and is caused by Leishmania donovani on the Asian and African continents and by Leishmania infantum in the Mediterranean Basin (Portugal, Spain, Greece, and Italy), the Middle East, Central Asia, and Central/North America [2,7].Annually, there are reports of up to 1 million new cases although infection by parasites does not always correlate with the development of disease [8].
The research on novel antiparasitic agents has been limited and represents a low interest investment for large pharmaceutical companies due to their incidence occurring mainly in secluded, poor, and disadvantaged populations with limited resources [9].A large proportion of the approved and new-drug applicants is occupied by natural products or derivatives, furthers suggesting their impact in medicine over the years [10,11].
Marine natural products have shown a variety of biological activities, such as antimicrobial, antioxidant, anticancer, and antiparasitic.In terms of antiparasitic activity, various compounds from marine sources have been reported, and reviews of their antiprotozoal potential have shown several examples of novel compounds for this biological activity [9,12].In particular, quinazolinones have rendered derivatives with several pharmacological and biological activities, such as antibacterial, antifungal, and antiparasitic [13].Examples of naturally occurring quinazolinones from Bacillus cereus present in sea mud with antifungal activity were reported (Figure 1) [14].Recently, our research group disclosed for the first time new marine-derived indolylmethylpyrazinoquinazolines active against Plasmodium and Trypanosomatids [2].55 million people at risk (according to 2020 data) [4].This potentially fatal disease is caused by two Trypanosoma brucei species, namely Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, and the parasites are transmitted to the human host by the bite of the tsetse fly [5,6].WHO, in collaboration with governmental and nongovernmental organizations, has been able to significantly reduce the incidence of HAT to fewer 1000 cases per year, attributable to access to novel drugs [4,6].
Leishmaniasis is a parasitic neglected disease caused by Leishmania spp., which are transmitted to mammal hosts by the bite of infected female sandflies.Visceral leishmaniasis is the most severe and life-threatening form and is caused by Leishmania donovani on the Asian and African continents and by Leishmania infantum in the Mediterranean Basin (Portugal, Spain, Greece, and Italy), the Middle East, Central Asia, and Central/North America [2,7].Annually, there are reports of up to 1 million new cases although infection by parasites does not always correlate with the development of disease [8].
The research on novel antiparasitic agents has been limited and represents a low interest investment for large pharmaceutical companies due to their incidence occurring mainly in secluded, poor, and disadvantaged populations with limited resources [9].A large proportion of the approved and new-drug applicants is occupied by natural products or derivatives, furthers suggesting their impact in medicine over the years [10,11].
Marine natural products have shown a variety of biological activities, such as antimicrobial, antioxidant, anticancer, and antiparasitic.In terms of antiparasitic activity, various compounds from marine sources have been reported, and reviews of their antiprotozoal potential have shown several examples of novel compounds for this biological activity [9,12].In particular, quinazolinones have rendered derivatives with several pharmacological and biological activities, such as antibacterial, antifungal, and antiparasitic [13].Examples of naturally occurring quinazolinones from Bacillus cereus present in sea mud with antifungal activity were reported (Figure 1) [14]  On the other hand, eugenol, a phenolic natural compound (Figure 2), has presented various biological activities, including antiparasitic and antimicrobial, among others [15][16][17].Chemical modifications of the eugenol structure or integration of this unit into a given privileged structure is an approach often taken by medicinal chemists to obtain derivatives with improved biological profiles.On the other hand, eugenol, a phenolic natural compound (Figure 2), has presented various biological activities, including antiparasitic and antimicrobial, among others [15][16][17].Chemical modifications of the eugenol structure or integration of this unit into a given privileged structure is an approach often taken by medicinal chemists to obtain derivatives with improved biological profiles.55 million people at risk (according to 2020 data) [4].This potentially fatal disease is caused by two Trypanosoma brucei species, namely Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, and the parasites are transmitted to the human host by the bite of the tsetse fly [5,6].WHO, in collaboration with governmental and nongovernmental organizations, has been able to significantly reduce the incidence of HAT to fewer 1000 cases per year, attributable to access to novel drugs [4,6].
Leishmaniasis is a parasitic neglected disease caused by Leishmania spp., which are transmitted to mammal hosts by the bite of infected female sandflies.Visceral leishmaniasis is the most severe and life-threatening form and is caused by Leishmania donovani on the Asian and African continents and by Leishmania infantum in the Mediterranean Basin (Portugal, Spain, Greece, and Italy), the Middle East, Central Asia, and Central/North America [2,7].Annually, there are reports of up to 1 million new cases although infection by parasites does not always correlate with the development of disease [8].
The research on novel antiparasitic agents has been limited and represents a low interest investment for large pharmaceutical companies due to their incidence occurring mainly in secluded, poor, and disadvantaged populations with limited resources [9].A large proportion of the approved and new-drug applicants is occupied by natural products or derivatives, furthers suggesting their impact in medicine over the years [10,11].
Marine natural products have shown a variety of biological activities, such as antimicrobial, antioxidant, anticancer, and antiparasitic.In terms of antiparasitic activity, various compounds from marine sources have been reported, and reviews of their antiprotozoal potential have shown several examples of novel compounds for this biological activity [9,12].In particular, quinazolinones have rendered derivatives with several pharmacological and biological activities, such as antibacterial, antifungal, and antiparasitic [13].Examples of naturally occurring quinazolinones from Bacillus cereus present in sea mud with antifungal activity were reported (Figure 1) [14]  On the other hand, eugenol, a phenolic natural compound (Figure 2), has presented various biological activities, including antiparasitic and antimicrobial, among others [15][16][17].Chemical modifications of the eugenol structure or integration of this unit into a given privileged structure is an approach often taken by medicinal chemists to obtain derivatives with improved biological profiles.Therefore, keeping in mind the diversity and potential of marine compounds, particularly of the marine quinazolinones, a series of derivatives of novel quinazolinones associated with the eugenol moiety was designed via a well-known click chemistry Mar.Drugs 2023, 21, 551 3 of 15 reaction [18].We hypothesized that this new structure scaffold could be promising against parasitic diseases since it brings together essential moieties from bioactive compounds.The synthesis of these quinazolinone-eugenol and related compounds will be herein discussed, as well as their antiparasitic activity and further screenings for antimicrobial activities.

Chemistry
The synthetic route used to obtain the hybrids consisted initially of the functionalization of eugenol (1) to obtain the propargyl ether 2 and the azide 4 (see Scheme 1).To obtain 2, eugenol (1) was subjected to O-alkylation with propargyl bromide in basic medium, according to a procedure described before [19].The azide (4) was synthesized in three steps, namely by a hydroxymethylation reaction of eugenol (1) with formaldehyde in basic medium [20], followed by chlorination of the alcoholic intermediate ( 3) with thionyl chloride and one-pot nucleophilic substitution with sodium azide.Intermediate 4 formation was confirmed by NMR, in which a singlet relative to methylene protons alpha to the azido group could be observed at 4.37 ppm.In the 13 C NMR spectrum, the signal relative to this group was found in the region expected for this type of carbon at 49.2 ppm.Therefore, keeping in mind the diversity and potential of marine compounds, particularly of the marine quinazolinones, a series of derivatives of novel quinazolinones associated with the eugenol moiety was designed via a well-known click chemistry reaction [18].We hypothesized that this new structure scaffold could be promising against parasitic diseases since it brings together essential moieties from bioactive compounds.The synthesis of these quinazolinone-eugenol and related compounds will be herein discussed, as well as their antiparasitic activity and further screenings for antimicrobial activities.

Chemistry
The synthetic route used to obtain the hybrids consisted initially of the functionalization of eugenol (1) to obtain the propargyl ether 2 and the azide 4 (see Scheme 1).To obtain 2, eugenol (1) was subjected to O-alkylation with propargyl bromide in basic medium, according to a procedure described before [19].The azide (4) was synthesized in three steps, namely by a hydroxymethylation reaction of eugenol (1) with formaldehyde in basic medium [20], followed by chlorination of the alcoholic intermediate (3) with thionyl chloride and one-pot nucleophilic substitution with sodium azide.Intermediate 4 formation was confirmed by NMR, in which a singlet relative to methylene protons alpha to the azido group could be observed at 4.37 ppm.In the 13 C NMR spectrum, the signal relative to this group was found in the region expected for this type of carbon at 49.2 ppm.The synthetic route used to obtain the quinazolinone counterpart is shown in Scheme 2. First, anthranilic acids 5a-5d were subjected to an N-acylation reaction with propynoyl chloride, prepared from propiolic acid and thionyl chloride, as indicated elsewhere [21].Anthranilic acid (5a) was further N-acylated with chloroacetyl chloride, and the intermediate 11 was converted into the azide 12, following the procedures described before [22].Amide intermediates 6a-6d were treated with acetic anhydride under reflux to form benzoxazinone intermediates 7a-7d, according to the general procedure reported [23].Our attempts to convert these benzoxazinones directly into the respective quinazolinones by reaction with ammonia did not result in success, contrary to what was reported in another work [24].In this case, instead of quinazolinones, 2-acylaminobenzamides 8a-8d were obtained as described by Kelleher and coworkers [18], who used this ammonia-based method for the synthesis of this type of benzamide.Intermediates 6a and 7a were easy to handle and could be purified and properly characterized.The main findings in the characterization of 6a by 1 H NMR were the characteristic signal of the acetylenic proton at 4.51 ppm and the signals for both acetylenic carbons at 79.48 and 78.84 ppm in 13 C NMR spectra.Still, in the 13 C NMR spectrum, the signals at 170.71 and 150.84 ppm, relative to the amide and acid carbonyls, established this characterization.For the intermediate benzoxazolone 7a, the main evidence was the presence of a single carbonyl signal at 159.47 ppm, in addition to the azomethine carbon at 146.79 ppm.
The amides 6b-6d, benzoxazinones 7b-7d, and 2-acylamino-benzamides 8a-8d were found to be quite unstable, decomposing rapidly during purification and characterization attempts.Thus, intermediates 8a-8d were used in crude form for the synthesis of triazole intermediates 9a-9d, while intermediates 12 and 7a could generate the additional triazoles The synthetic route used to obtain the quinazolinone counterpart is shown in Scheme 2. First, anthranilic acids 5a-5d were subjected to an N-acylation reaction with propynoyl chloride, prepared from propiolic acid and thionyl chloride, as indicated elsewhere [21].Anthranilic acid (5a) was further N-acylated with chloroacetyl chloride, and the intermediate 11 was converted into the azide 12, following the procedures described before [22].Amide intermediates 6a-6d were treated with acetic anhydride under reflux to form benzoxazinone intermediates 7a-7d, according to the general procedure reported [23].Our attempts to convert these benzoxazinones directly into the respective quinazolinones by reaction with ammonia did not result in success, contrary to what was reported in another work [24].In this case, instead of quinazolinones, 2-acylamino-benzamides 8a-8d were obtained as described by Kelleher and coworkers [18], who used this ammonia-based method for the synthesis of this type of benzamide.Intermediates 6a and 7a were easy to handle and could be purified and properly characterized.The main findings in the characterization of 6a by 1 H NMR were the characteristic signal of the acetylenic proton at 4.51 ppm and the signals for both acetylenic carbons at 79.48 and 78.84 ppm in 13 C NMR spectra.Still, in the 13 C NMR spectrum, the signals at 170.71 and 150.84 ppm, relative to the amide and acid carbonyls, established this characterization.For the intermediate benzoxazolone 7a, the main evidence was the presence of a single carbonyl signal at 159.47 ppm, in addition to the azomethine carbon at 146.79 ppm.
The amides 6b-6d, benzoxazinones 7b-7d, and 2-acylamino-benzamides 8a-8d were found to be quite unstable, decomposing rapidly during purification and characterization attempts.Thus, intermediates 8a-8d were used in crude form for the synthesis of triazole intermediates 9a-9d, while intermediates 12 and 7a could generate the additional triazoles 13 and 14, respectively.These and the other triazoles (9a-9d) were prepared by the copper catalyzed azide-alkyne cycloaddition reaction, following the classical click procedure described elsewhere [25].The signal relative to the triazole hydrogen of products 9a-9d, 10a-10c, 13, and 14 was clearly observed in the range of 8.25-8.62ppm.
Quinazolinone compounds 10a-10c were obtained from 9a-9c, following the method of cyclocondensation of 2-acylamino anthranilamides in hot aqueous NaOH, as described before [26].Despite our efforts, attempts at cyclization with the chlorinated intermediate (9d) were unsuccessful, so it was not possible to evaluate the activity of the respective quinazolinone.The structure characterization was performed by NMR and HRMS techniques (experimental).

Antiparasitic Activity
The compounds were screened for their antiparasitic activity against Leishmania infantum promastigotes and Trypanosoma brucei parasites, and the cytotoxicity was also evaluated using THP-1 cells.The results obtained allowed us to calculate the half maximal inhibitory concentration (IC50), which is the concentration of compounds that inhibits parasite growth by 50%, and the cytotoxic concentration 50 (CC50), which corresponds to the concentration of compounds required to reduce cell viability by 50%.Both measures, IC50  Quinazolinone compounds 10a-10c were obtained from 9a-9c, following the method of cyclocondensation of 2-acylamino anthranilamides in hot aqueous NaOH, as described before [26].Despite our efforts, attempts at cyclization with the chlorinated intermediate (9d) were unsuccessful, so it was not possible to evaluate the activity of the respective quinazolinone.The structure characterization was performed by NMR and HRMS techniques (experimental).

Biological Tests 2.2.1. Antiparasitic Activity
The compounds were screened for their antiparasitic activity against Leishmania infantum promastigotes and Trypanosoma brucei parasites, and the cytotoxicity was also evaluated using THP-1 cells.The results obtained allowed us to calculate the half maximal inhibitory concentration (IC 50 ), which is the concentration of compounds that inhibits parasite growth by 50%, and the cytotoxic concentration 50 (CC 50 ), which corresponds to the concentration of compounds required to reduce cell viability by 50%.Both measures, IC 50 and CC 50 , were calculated in µM with a 95% confidence interval and were used to calculate the selectivity index (SI).The antiparasitic activity, cytotoxicity, and SI are depicted in Table 1.
Table 1.Antiparasitic potential of 9a, 9b, 9c, 9d, 10a, 10b, 10c, 13, 14, eugenol (1), pentamidine, and miltefosine.The antiparasitic activity was evaluated by the IC 50 against L. infantum promastigotes and T. brucei bloodstream parasites, and the cytotoxicity was evaluated by the CC 50 in the THP-1 cell line.The selectivity index (SI) for each parasite is also presented.The SI for the compounds was calculated by the ratio of the antiparasitic activity to the CC 50 in THP-1.Except for 9c, the synthesized hybrids presented no detectable antiparasitic activity against L. infantum promastigotes until 40 µM (the highest concentration tested).This compound (9c) presented an IC 50 of 7.54 µM (5% CI of 5.85-9.58).Regarding T. brucei, six compounds (9b, 9c, 9d, 10b, 10c, and 14) presented a quantifiable IC 50 .The most potent IC 50 values were between 20 and 10 µM.Concerning the cytotoxicity against PMA-differentiated THP-1 cells, the tested series did not induce viability loss in the tested concentrations except for 9c, which presented a CC 50 value of 45.82 µM (95% CI of 38.04-55.30).All active compounds presented SI values superior to 2.

Compounds
It is important to note that, among the synthesized compounds, only 9c presented activity in both parasites.However, it is worth noting that 9c was also the most toxic molecule from the series, with a CC 50 value of 45.82 µM.This finding might be suggestive of a non-specific mechanism of action.
The structure-activity relationship revealed that the different substituents used in intermediate 9 and final derivative 10 influenced the antitrypanosomal activity.Compounds 9b and 9c (R=OMe and R=F, respectively) presented a quantifiable IC 50 , while 9a (R=H) did not show any detectable IC 50 .Among derivatives 10, compounds 10b (R=OMe) and 10c (R=F) had similar IC 50 values to 9b and 9c, respectively.The unsubstituted compounds 10a and 9a did not show detectable values of IC 50 .It is important to note that 10c had the most potent IC 50 value against T. brucei, but its IC 50 value against L. infantum promastigote was greater than 40 µM.On the other hand, 9c was not the most potent anti-trypanosome compound but presented activity against Leishmania.This outcome is suggestive of parasite-specific activity.In conclusion, new hybrid compounds were disclosed as promising scaffolds for the synthesis of new antiparasitic molecules.

Antimicrobial Activity
The compounds were additionally evaluated for antimicrobial activity using the broth microdilution method against three different fungal strains and against two different bacteria, and the results were demonstrated as the minimum inhibitory concentration (MIC), which is the minimal concentration to cause inhibition of fungal or bacteria growth according to the Clinical and Laboratory Standards Institute (CLSI) protocols.Voriconazole and gentamicin were used as quality control drugs for the antifungal and antibacterial assays, respectively, and the obtained results were according to the followed norms.The obtained results for the synthesized compounds revealed poor to no antimicrobial activity for the marine eugenol hybrids (Supplementary Material, Table S1).
The tested series presented MIC values of >512 µg/mL against C. albicans and A. fumigatus, meaning that the synthesized substances are not promising antifungal agents against these strains.The results against T. rubrum were slightly improved for compounds 9a, 9d, 10a, 10c, and 14, with values ranging from 256 to 512 µg/mL.Nevertheless, the MIC values are still viewed as high; therefore, the compounds were considered not promising, and no further fungal strains were tested.
The MLCs were tested for compounds 9a, 9d, 10a, 10c, and 14 for T. rubrum, and all compounds presented values of >512 µg/mL.For eugenol, the MLC results were between 256 and 512 µg/mL for T. rubrum and were >512 µg/mL for C. albicans and A. fumigatus.
In terms of the antibacterial activity, 9c demonstrated a minor improvement in MIC values in comparison to the remaining compounds (MIC between 256 and 512 µg/mL, versus >512 µg/mL) when tested against E. coli and S. aureus.For E. coli and S. aureus, the only MIC evaluated was for 9c, which presented a value of >512 µg/mL and ≤512 µg/mL, respectively.Once again, the hybrids were not considered promising compounds for this biological activity.

General Methods
Reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA), Acros Organics (Thermo Fisher Scientific, Geel, Belgium), or Fisher Scientific (Thermo Fisher Scientific, Loughborough, UK) and were used without further purification.Thin layer chromatography (TLC) using Merck silica gel 60 (GF254)-precoated plates (0.2 mm of thickness) with appropriate mobile phases were used to follow reaction progressions.Ultraviolet light (254 and 365 nm) and 3% aqueous FeCl 3 (for phenolic derivatives) were used to visually detect compounds on chromatograms.When necessary, purifications of the synthesized compounds were performed by flash column chromatography using silica gel 60 (0.040-0.063 mm, Merck, Darmstadt, Germany) or preparative thin layer chromatography (PTLC) using Merck silica gel 60 (GF254) plates.The 1 H and 13 C NMR spectra were taken at the Centro de Materiais (CEMUP)-University of Porto on a Bruker Avance III 400 spectrometer (400 MHz for 1 H and 100 MHz for 13 C) or at the University of Aveiro on a Bruker Avance 300 spectrometer (300.13MHz for 1 H and 75.47 MHz for 13 C) in DMSO-d 6 or CDCl 3 (Deutero GmbH, Ely, UK) at room temperature.Chemical shifts are expressed in δ (ppm) values relative to tetramethylsilane (TMS) as an internal reference.Coupling constants are reported in hertz (Hz). 13C NMR assignments were made by comparison with the assignments of similar molecules.High-resolution mass spectrometry (HRMS) was performed on an LTQ OrbitrapTM XL hybrid mass spectrometer (Thermo Fischer Scientific, Bremen, Germany) controlled by LTQ Tune Plus 2.5.5 and Xcalibur 2.1.0 in positive mode at CEMUP-University of Porto.The capillary voltage of the electrospray ionization source (ESI) was set to 3.1 kV.The capillary temperature was 275 • C. The sheath gas was at 6 (arbitrary unit, as provided by the software settings).The capillary voltage was 46 V, and the tube lens voltage was 120 V.The synthesis and purification of compounds were undertaken as described in the following sections.[22], respectively.The 1 H and 13 C NMR spectra of 2, 3, and 12 were in accordance with the reported data.
Synthesis of 4-allyl-2-azido-6-methoxyphenol (4) Compound 3 (0.5 g, 2.6 mmol) was dissolved in anhydrous N,N-dimethylformamide (5.0 mL) and stirring solution cooled by an ice water bath.Then, thionyl chloride (0.62 g, 0.38 mL, 5.2 mmol) was added dropwise to this solution, followed by anhydrous potassium carbonate (1.1 7.8 mmol).The mixture was left under stirring at room temperature for 2 h.Subsequently, sodium azide (0.17 g, 2.6 mmol) was added as a solution in anhydrous dimethylsulfoxide (1.0 mL).The mixture was kept under the same conditions for 24 h when TLC (hexane/ethyl acetate, 7:3) showed the end of the reaction.Water (10 mL) was added to the mixture, and the crude product was extracted with ethyl acetate (5 × 10 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated until dry.The pure product was obtained after flash column chromatography (hexane/ethyl acetate, 7:3).

Synthesis of Acetylenic Intermediates 6a and 7a
Anthranilic acid 5a (1 eq) and triethylamine (2 eq) were dissolved in dry dichloromethane (10 mL), and the mixture was stirred and cooled to 0 • C in an ice bath.Then, propynoyl chloride (1.2 eq) was added dropwise.The reaction mixture was kept under the same conditions until the consumption of the starting material, which was visualized by TLC (hexane/ethyl acetate, 1:1).Then, the reaction mixture was poured into crushed ice and stirred vigorously to precipitate the product 6a, which was filtered off under reduced pressure.The product was used in the next step without further purification.Intermediate 6a (1 eq) was then dissolved in acetic anhydride (20 mL), and the mixture was heated at 130 • C for 2 h.After this time, the reaction mixture was cooled to room temperature and then poured into crushed ice.After vigorous stirring, the solid that precipitated was filtered off under reduced pressure and washed copiously with water.The obtained product (7a) was pure enough to be used in the next step.

Synthesis of 2-(propioloylamino)benzamides 8a-8d
Compounds 8a-8d were prepared in three steps from the respective anthranilic acids, following the classical methods reported before [18,23].As the intermediates 6b-6d, 7b-7d and 8a-8d were quite unstable, they were readily used in the subsequent reactions without purification.However, their identities were confirmed indirectly by the success in obtaining the triazole products.
Synthesis of Triazoles 9a-9d, 13, and 14 The corresponding alkyne (2 or 8a-8d, 1 eq) and azide (4 or 12, 1 eq) intermediates were dissolved in a mixture of tetrahydrofuran-water (9:1), and to this solution was added sodium ascorbate (0.1 eq) and copper II sulfate (0.01 eq).The mixture was left under vigorous magnetic stirring at room temperature for 4-24 h.The progress of the reaction was monitored by TLC (100% ethyl acetate), and once the reaction was complete, the solvent was evaporated using a rotary evaporator, and the resulting solid was pre-purified by liquidliquid extraction using water and ethyl acetate.The product isolated from the organic phase was purified by crystallization with diethyl ether or flash column chromatography (ethyl acetate/hexane, 9:1), which led to the desired triazoles.

Synthesis of Quinazolinones 10a-10c
A solution of 10 M aqueous NaOH (2.0 mL) was added to compounds 9a-9c (0.2 mmol) solubilized in ethanol (18.0 mL).The mixture was then heated under reflux for 1 h.After that time, the resulting solution was cooled, and the pH was adjusted to 5 with HCl 1 M.The product was obtained by extraction with ethyl acetate, followed by solvent evaporation, needing no further purification.

Fungal and Bacterial Strains
Fungal strains, including reference strains and clinical isolates, were used for the study of the antifungal activity: a yeast reference strain from American Type Culture Collection (ATCC), Candida albicans ATCC 10231; filamentous fungi reference strain Aspergillus fumigatus ATCC 240305; and a clinical isolate of dermatophytes Trichophyton rubrum FF5.Candida krusei ATCC 6258 was used as a quality control.All fungal strains were stored in Sabouraud dextrose broth (SDB-Bio-Mèrieux, Marcy L'Etoile, France) with glycerol (20%) at −80 • C and were subcultured in Sabouraud dextrose agar (SDA-Bio-Mèrieux, Marcy L'Etoile, France) for 24-72 h (yeasts and A. fumigatus) or 5-7 days (T.rubrum) before each assay to obtain optimal growth and purity conditions.Two bacteria strains were used, one Gram positive and one Gram negative: Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922, respectively.The bacterial strains were stored in trypticase soy broth (TSB-Biolife, Milan, Italy) with glycerol (10%) at −80 • C and subcultured in Mueller-Hinton agar (MHA-Bio-Mèrieux, Marcy L'Etoile, France) for 24 h before each assay.

Antifungal Activity
For the purpose of quantitatively measuring the antifungal activity in vitro, a broth microdilution method was used to determine the MICs using the CLSI reference protocols M27-A3 [27] for yeasts and M38-A2 [28] for filamentous fungi.In short, two-fold serial dilutions of each stock solution were prepared using RPMI medium (pH 7) to obtain a range of test concentrations from 16 to 512 µg/mL.The yeasts or spore suspensions were prepared from 24 to 72-h cultures (yeasts and Aspergillus) or from 5-7 days of cultures (dermatophyte) in saline solution (with a drop of Tween 20 for the filamentous fungi).The transmittance of the cell density of yeasts was adjusted to 0.5 McFarland standard, and for filamentous fungi, the spores were counted using a Neubauer camera.Dilutions were performed using RPMI to obtain cell final concentrations in the plate of 0.5-2.5 × 10 3 colony forming units (CFU)/mL for yeasts, 0.4-5 × 10 4 CFU/mL for Aspergillus, and 1-3 × 10 3 CFU/mL for dermatophytes.37 • C in a 5% CO 2 environment with subculture every 7 days at 1 × 10 6 /mL in 5-mL T25 ventilated flasks.

Anti-T. brucei Activity
The compounds' efficacy against bloodstream-stage trypomastigotes was evaluated using a resazurin-based assay.Parasites were added to 100 µL of serial dilutions of compounds in supplemented complete medium at a cell density of 5 × 10 3 /mL.As a quality control, a dose-response curve for the antitrypanosomal pentamidine was included in all the assays.The final volume of the assay was 200 µL/well.Each condition was carried out in duplicate.Following 72 h of incubation at the specific conditions for parasites, 20 samples of a 0.5 mM resazurin solution was added, and the plates were incubated for a further 4 h under the same conditions.Fluorescence was measured at 544 nm and 590 nm excitation and emission wavelength, respectively, using a Synergy 2 Multi-Mode Reader (Biotek, Winooski, VT, USA).The results are shown as % of parasite growth inhibition compared to control (untreated parasites) and represent the average of at least three independent experiments.The effect was evaluated by the determination of the IC 50 value (concentration required to inhibit growth in 50%) and calculated by non-linear regression curves using GraphPad Prism software, version 8.1.1 for Windows (GraphPad Software, San Diego, CA, USA).

Anti-Leishmania Activity
The compounds' efficacy against L. infantum promastigotes was evaluated using a resazurin-based assay.Parasites were added to 100 µL of serial dilutions of compounds in supplemented complete medium at a cell density of 5 × 10 5 /mL.As a quality control, a dose-response curve to the antileishmanial drug miltefosine was included in all the assays.The final volume of the assay was 200 µL/well.Each condition was carried out in duplicate.Following 72 h of incubation at the specific conditions for parasites, 20 µL of a 0.5 mM resazurin solution was added, and the plates were incubated for a further 4 h under the same conditions.Fluorescence was measured at 544 nm and 590 nm excitation and emission wavelengths, respectively, using a Synergy 2 Multi-Mode Reader (Biotek, Winooski, VT, USA).Results are shown as % of parasite growth inhibition compared to control (untreated parasites) and represent the average of at least three independent experiments.The effect was evaluated by the determination of the IC 50 value (concentration required to inhibit growth in 50%) and was calculated by non-linear regression curves using GraphPad Prism software, version 8.1.1 for Windows (GraphPad Software, San Diego, CA, USA).

Cytotoxicity Assay
A human leukemia cell line, THP-1 (ATCC ® TIB-202™), was cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/mL penicillin/streptomycin, and 20 mM HEPES.The cell line was maintained in a humidified incubator at 37 • C and 5% CO 2 by subculture every 3 days in 20 mL of media at a concentration of 2 × 10 5 /mL in a T75 flask.All cell culture reagents were purchased from Lonza-Bioscience (Morrisville, NC, USA).
The cytotoxicity effect of compounds on THP-1-derived macrophages was assessed by colorimetric MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide).Briefly, THP-1 cells were suspended in RPMI complete medium at a density of 1 × 10 6 cells/mL and 100 µL/well and were seeded in a 96-well plate and differentiated into macrophages by addition of 40 ng/mL of phorbol-myristate 13-acetate (PMA, Sigma, Saint Louis, MI, USA) for 24 h, followed by replacement with fresh medium for 24 more h.Subsequently, the cells were incubated with 100 µL of compounds ranging from 100 to 12.5 µM after dilution in the RPMI complete medium.Each condition was carried out in quadruplicate.After 72 h of incubation at 37 • C and 5% CO 2 , the medium was removed, and 200 µL of 0.5 mg/mL MTT solution diluted in RPMI was added.The plates were incubated for an additional 4 h.Then, 160 µL of media was removed, and the same volume of 2-propanol was added.Absorbance was read at 570 nm using a Synergy 2 Multi-Mode Reader (Biotek, Winooski, VT, USA).Cytotoxicity was evaluated by the determination of the CC 50 value (drug concentration that reduced the percentage of viable cells by 50%) and calculated by non-linear regression analysis using GraphPad Prism software, version 8.1.1 for Windows (GraphPad Software, San Diego, CA, USA).The results represent the average of at least three independent experiments.For each compound, the selectivity index (SI) was calculated as the ratio between cytotoxicity in THP-1 (CC 50 , 72 h) and activity against parasites (IC 50 , 72 h).

Conclusions
New compounds, designed by molecular hybridization from a marine quinazolinone and eugenol, were synthesized and evaluated against protozoan species involved in neglected parasitic diseases and as possible antifungal and antibacterial agents.Initially we hypothesized that the marine quinazolinone described as having antifungal activity (a, Figure 1) could benefit from hybridization with eugenol.Moreover, the conjugation through a triazole moiety, critical for azole antifungal drugs, was hypothesized to increase the potential antifungal activity.In contrast to our expectations, no antifungal activity was detected for the series.Although no hybrid showed relevant antimicrobial action, inspired in our previous studies with indolylmethylpyrazinoquinazolines (b, Figure 1), six of the substances tested (intermediates 9b, 9c, and 9d and final products 10b, 10c, and 14) presented mild antitrypanosomal activity.One of them, the fluorinated intermediate 9c, additionally showed a relevant leishmanicidal effect.Natural products, including marine specialized metabolites, are one potential source from which novel trypanocidal compounds have been disclosed.Most of these compounds have activity against multiple (micro)organisms, which could limit their application.The breakthrough of this work was the discovery of derivatives with a simple quinazolinone scaffold selective against parasites without antibacterial and antifungal activities, which are synthetically accessible and without chiral centers, in contrast to previously reported antiparasitic alkaloids, such as the indolylmethylpyrazinoquinazolines.The structural pattern explored constitutes a relevant starting point for future optimization in an attempt to find marine-inspired candidates for leishmanicidal and trypanosomicidal drugs.