Dual Antitubercular and Antileishmanial Profiles of Quinoxaline Di-N-Oxides Containing an Amino Acidic Side Chain

We present a new category of quinoxaline di-N-oxides (QdNOs) containing amino acid side chains with dual antituberculosis and antileishmanial activity. These compounds were synthesized by combining a regioselective 2,5-piperazinedione opening and a Beirut reaction and were screened for their activity against Mycobacterium tuberculosis and the promastigote and amastigote forms of representative species of the Leishmania genus. Most QdNOs exhibited promising antitubercular activity with IC50 values ranging from 4.28 to 49.95 μM, comparable to clinically established drugs. Structure–activity relationship analysis emphasized the importance of substituents on the aromatic ring and the side chain. Antileishmanial tests showed that some selected compounds exhibited activity comparable to the positive control miltefosine against promastigotes of Leishmania amazonensis and Leishmania donovani. Notably, some compounds were found to be also more potent and less toxic than miltefosine in intracellular amastigote assays against Leishmania amazonensis. The compound showing the best dual antitubercular and leishmanicidal profile and a good selectivity index, 4h, can be regarded as a hit compound that opens up new opportunities for the development of integrated therapies against co-infections.


Introduction
Worldwide mortality and disability caused by infectious agents is a serious global health problem and one of the most important scientific challenges of our time [1].Neglected tropical diseases (NTDs) are a group of protozoan, viral, bacterial or parasitic diseases that are particularly prevalent among the less favored population, mostly in tropical areas.Changes in migration patterns and environmental factors [2] may have complex effects on the distribution of disease vectors of NTDs such as malaria, Chagas disease and leishmaniasis, which may alter their geographical distribution.In particular, leishmaniasis (LH) and tuberculosis (TB) are endemic NTDs that affect a considerable fraction of the population in tropical and subtropical regions.
Worldwide, an estimated 1.6 million new cases of leishmaniasis occur annually, with approximately 12 million patients being currently infected in about 90 countries and about 350 million people being at risk.These cases include per year 700,000 to 1.2 million new cases of cutaneous leishmaniasis, a disfiguring disease, and 200,000 to 400,000 patients with visceral leishmaniasis (kala-azar), which proves fatal when left untreated [3,4].
Currently, there are no approved vaccines for human leishmaniasis [5], and the primary approach to combating the disease is focused on chemotherapy.The present arsenal of drugs available against leishmaniasis is rather small, and moreover, these drugs are limited by disadvantages such as the requirement for prolonged treatments, the need for parenteral routes of administration, high toxicity in many cases and the fact that they are becoming increasingly ineffective due to the appearance of resistant forms of the parasite [6].Although the global leishmaniasis incidence has remained relatively stable over the past decade, a major problem exists in the immunocompromised population, often co-infected with other NTSs or with the human immunodeficiency virus (HIV) [7][8][9], and thus, due to the increasing numbers of HIV patients, leishmaniasis chemotherapy is further complicated by variations in efficacy associated with immune status.In this context, tuberculosis is also an immunosuppressive condition that affects the treatment of leishmaniasis, which depends on an effective immune response that activates macrophages.Furthermore, tuberculosis may cause progression of latent leishmaniasis to the clinical stage, while visceral leishmaniasis can also reactivate latent tuberculosis.Thus, pulmonary tuberculosis and leishmaniasis (as well as other parasitic diseases) have been shown to be risk factors for each other [10] and due to their geographical overlap, co-infections are very common [11][12][13][14].The low level of development of the countries with the highest prevalence of both infections makes the simultaneous treatment of these diseases difficult.Therefore, there is an urgent need for affordable new therapies with integrated dual activity against both infections.If successful, these compounds can be regarded as a particular case of multi-target directed ligands, one of the most promising current paradigms in drug discovery [15].
In this context, we present a method for synthesizing a previously unexplored category of quinoxaline-1,4-dioxides that incorporate a glycine side chain, starting from the widely acknowledged 2,5-piperazinedione template [31].The compounds thus generated were screened for their activity against Mycobacterium tuberculosis and were also studied as antileishmanial agents in an effort to obtain a hit compound that can be used as a starting point toward addressing the above-mentioned co-infection problem.The inclusion of amino acid structural fragments is a useful technique in drug design for the modulation of physicochemical properties and exploiting protein transporters to improve membrane permeation [32][33][34].Furthermore, it provides opportunities for specific drug uptake by bacteria or parasite-specific proteins, which may be useful to achieve selective toxicity.Mycobacteria are characterized by the very low permeability of their wall and are therefore highly dependent on the uptake of nutrients by transporter proteins, although the identity of many of these transporters is still unknown [35].

Synthesis
2,5-Diketopiperazines (DKPs) are the simplest cyclic peptides and are readily accessible from amino acids, representing highly versatile scaffolds in synthesis [31,36,37].In this context, we [38] and others [39,40] have been actively involved in the development of new DKP-based synthetic methodologies, with a specific focus on the regioselective cleavage of suitably functionalized DKP derivatives.The synthetic strategy to obtain the QdNO compounds 4 and 5 involved a three-step sequence starting from 3-arylmethylene-2,5-piperazinediones 1.The required compounds 1 were readily available by aldol condensation of 1,4-diacetyl-2,5-piperazinedione with substituted benzaldehydes in the presence of potassium tert-butoxide [41], and they were opened by acid-promoted alcoholysis of the 2,5-piperazinedione ring under microwave irradiation or reflux conditions [42] to give the N-(3-arylpyruvylamino) esters 2. In order to study the effect on the bioactivity of substituents with varied electronic effects, chloro, fluoro and trifluoromethyl were used as electron-withdrawing groups, and methyl and methoxy as electron-donating groups.Yields were generally good, independently of the nature of these substituents at the aromatic ring.For the second step, methanol and benzyl alcohol were employed as nucleophiles for the regioselective opening of the DKP ring to furnish compounds 2 (Scheme 1).The scope and yields of these reactions are summarized in Table 1.
The target quinoxaline-1,4-dioxide framework was synthesized using a modification of the Beirut reaction [43][44][45] by treating 2a-i with the suitable benzofuraxan derivatives 3. Some of the compounds 4 thus obtained were used as starting materials for the synthesis of the acids 5.After screening several reaction conditions, lithium hydroxide in H2O/dioxane ultimately provided the most satisfactory results for the saponification of 4. As shown in Scheme 2 and Table 2, this short multi-step sequence demonstrated good tolerance for both electron-withdrawing and electron-donating groups.

Synthesis
2,5-Diketopiperazines (DKPs) are the simplest cyclic peptides and are readily accessible from amino acids, representing highly versatile scaffolds in synthesis [31,36,37].In this context, we [38] and others [39,40] have been actively involved in the development of new DKP-based synthetic methodologies, with a specific focus on the regioselective cleavage of suitably functionalized DKP derivatives.The synthetic strategy to obtain the QdNO compounds 4 and 5 involved a three-step sequence starting from 3-arylmethylene-2,5-piperazinediones 1.The required compounds 1 were readily available by aldol condensation of 1,4-diacetyl-2,5-piperazinedione with substituted benzaldehydes in the presence of potassium tert-butoxide [41], and they were opened by acid-promoted alcoholysis of the 2,5-piperazinedione ring under microwave irradiation or reflux conditions [42] to give the N-(3-arylpyruvylamino) esters 2. In order to study the effect on the bioactivity of substituents with varied electronic effects, chloro, fluoro and trifluoromethyl were used as electron-withdrawing groups, and methyl and methoxy as electron-donating groups.Yields were generally good, independently of the nature of these substituents at the aromatic ring.For the second step, methanol and benzyl alcohol were employed as nucleophiles for the regioselective opening of the DKP ring to furnish compounds 2 (Scheme 1).The scope and yields of these reactions are summarized in Table 1.The target quinoxaline-1,4-dioxide framework was synthesized using a modification of the Beirut reaction [43][44][45] by treating 2a-i with the suitable benzofuraxan derivatives 3. Some of the compounds 4 thus obtained were used as starting materials for the synthesis of the acids 5.After screening several reaction conditions, lithium hydroxide in H 2 O/dioxane ultimately provided the most satisfactory results for the saponification of 4. As shown in Scheme 2 and Table 2, this short multi-step sequence demonstrated good tolerance for both electron-withdrawing and electron-donating groups.Scheme 1. Synthesis of tricarbonyl compounds 2 from 2,5-diketopiperazine.In summary, our method represents a new entry into functionalized quinoxaline-1,4di-N-oxides, generating five bonds over three steps, as summarized in Figure 2.For reference purposes, we added to this study compound 6, an analogue of 4j lacking the glycine side chain.This compound was prepared by hydrolysis of the amide bond in compound 4j by treatment with lithium hydroxide in dioxane-water (Scheme 3).Scheme 3. Preparation of the additional compound 6 used as a reference for the biological studies.

Antituberculosis Activity
We first examined our compounds for activity against Mycobacterium tuberculosis by conducting high-throughput screening of their in vitro activity against the H37Rv strain, using an assay adapted from the microdilution AlamarBlue (AB) broth assay reported by Collins and Franzblau [46].The results obtained, expressed as IC50, IC90 and MIC, are shown in Figure 3 and Table S1.Our compounds showed IC50 values in the 4.28-49.95µM range, with compounds 4c, 4h, 4l, 4m, 4n, 5f and 5m showing the best inhibition results, which are comparable to those found for two antitubercular drugs in clinical use, namely, pyrimetamine (IC50: 37.35 µM) and cycloserine (IC50: 12.47 µM).Importantly, all compounds showed low cytotoxicity values against Vero cells.S1 (Supporting Information).None of the compounds showed cytotoxicity in Vero cells at concentrations up to 6.25 μg/mL.

Antileishmanial Activity
The QdNO derivatives were also tested against promastigote forms of two Leishmania parasites, namely, L. amazonensis, which causes cutaneous and mucocutaneous For reference purposes, we added to this study compound 6, an analogue of 4j lacking the glycine side chain.This compound was prepared by hydrolysis of the amide bond in compound 4j by treatment with lithium hydroxide in dioxane-water (Scheme 3).For reference purposes, we added to this study compound 6, an analogue of 4j lacking the glycine side chain.This compound was prepared by hydrolysis of the amide bond in compound 4j by treatment with lithium hydroxide in dioxane-water (Scheme 3).Scheme 3. Preparation of the additional compound 6 used as a reference for the biological studies.

Antituberculosis Activity
We first examined our compounds for activity against Mycobacterium tuberculosis by conducting high-throughput screening of their in vitro activity against the H37Rv strain, using an assay adapted from the microdilution AlamarBlue (AB) broth assay reported by Collins and Franzblau [46].The results obtained, expressed as IC50, IC90 and MIC, are shown in Figure 3 and Table S1.Our compounds showed IC50 values in the 4.28-49.95µM range, with compounds 4c, 4h, 4l, 4m, 4n, 5f and 5m showing the best inhibition results, which are comparable to those found for two antitubercular drugs in clinical use, namely, pyrimetamine (IC50: 37.35 µM) and cycloserine (IC50: 12.47 µM).Importantly, all compounds showed low cytotoxicity values against Vero cells.S1 (Supporting Information).None of the compounds showed cytotoxicity in Vero cells at concentrations up to 6.25 μg/mL.

Antileishmanial Activity
The QdNO derivatives were also tested against promastigote forms of two Leishmania parasites, namely, L. amazonensis, which causes cutaneous and mucocutaneous Scheme 3. Preparation of the additional compound 6 used as a reference for the biological studies.

Biology 2.2.1. Antituberculosis Activity
We first examined our compounds for activity against Mycobacterium tuberculosis by conducting high-throughput screening of their in vitro activity against the H37Rv strain, using an assay adapted from the microdilution AlamarBlue (AB) broth assay reported by Collins and Franzblau [46].The results obtained, expressed as IC 50 , IC 90 and MIC, are shown in Figure 3 and Table S1.Our compounds showed IC 50 values in the 4.28-49.95µM range, with compounds 4c, 4h, 4l, 4m, 4n, 5f and 5m showing the best inhibition results, which are comparable to those found for two antitubercular drugs in clinical use, namely, pyrimetamine (IC 50 : 37.35 µM) and cycloserine (IC 50 : 12.47 µM).Importantly, all compounds showed low cytotoxicity values against Vero cells.For reference purposes, we added to this study compound 6, an analogue of 4j lacking the glycine side chain.This compound was prepared by hydrolysis of the amide bond in compound 4j by treatment with lithium hydroxide in dioxane-water (Scheme 3).Scheme 3. Preparation of the additional compound 6 used as a reference for the biological studies.

Antituberculosis Activity
We first examined our compounds for activity against Mycobacterium tuberculosis by conducting high-throughput screening of their in vitro activity against the H37Rv strain, using an assay adapted from the microdilution AlamarBlue (AB) broth assay reported by Collins and Franzblau [46].The results obtained, expressed as IC50, IC90 and MIC, are shown in Figure 3 and Table S1.Our compounds showed IC50 values in the 4.28-49.95µM range, with compounds 4c, 4h, 4l, 4m, 4n, 5f and 5m showing the best inhibition results, which are comparable to those found for two antitubercular drugs in clinical use, namely, pyrimetamine (IC50: 37.35 µM) and cycloserine (IC50: 12.47 µM).Importantly, all compounds showed low cytotoxicity values against Vero cells.S1 (Supporting Information).None of the compounds showed cytotoxicity in Vero cells at concentrations up to 6.25 μg/mL.

Antileishmanial Activity
The QdNO derivatives were also tested against promastigote forms of two Leishmania parasites, namely, L. amazonensis, which causes cutaneous and mucocutaneous  S1 (Supporting Information).None of the compounds showed cytotoxicity in Vero cells at concentrations up to 6.25 µg/mL.

Antileishmanial Activity
The QdNO derivatives were also tested against promastigote forms of two Leishmania parasites, namely, L. amazonensis, which causes cutaneous and mucocutaneous leishmaniasis, and L. donovani, which is associated with visceral leishmaniasis.While intracellular amastigote models, which have the advantage of including the effects of the host cell, are normally regarded as the gold standard for in vitro drug discovery against Leishmania [47], axenic systems such as promastigotes provide a fast and convenient way to perform the initial screening of a compound library.The results obtained in the study of our compounds against L. amazonensis and L. donovani promastigotes are summarized in Figures 4 and 5 and Table S2 (Supporting Information).
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 6 of 19 leishmaniasis, and L. donovani, which is associated with visceral leishmaniasis.While intracellular amastigote models, which have the advantage of including the effects of the host cell, are normally regarded as the gold standard for in vitro drug discovery against Leishmania [47], axenic systems such as promastigotes provide a fast and convenient way to perform the initial screening of a compound library.The results obtained in the study of our compounds against L. amazonensis and L. donovani promastigotes are summarized in Figures 4 and 5 and Table S2 (Supporting Information).The numerical data corresponding to this graphic can be found in Table S2 (Supporting Information).
Figure 5. Selectivity indexes, obtained as the ratio between the toxicity against J774 macrophages (CC50, µM) and the IC50 values against Leishmania amazonensis and Leishmania donovani promastigotes (IC50, µM).The numerical data corresponding to this graphic can be found in Table S2 (Supporting Information).
Based on these results and in order to complete the characterization of our library, we selected compounds 4a, 4d, 4g, 4h, 4i, 4k, 4m and 6 to be assayed in the intracellular amastigote model against both L. amazonensis and L. donovani, with the results shown in Figures 6 and 7 and Table S3 (Supporting Information).S2 (Supporting Information).
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 6 of 1 leishmaniasis, and L. donovani, which is associated with visceral leishmaniasis.While in tracellular amastigote models, which have the advantage of including the effects of th host cell, are normally regarded as the gold standard for in vitro drug discovery agains Leishmania [47], axenic systems such as promastigotes provide a fast and convenient way to perform the initial screening of a compound library.The results obtained in the study of our compounds against L. amazonensis and L. donovani promastigotes are summarized in Figures 4 and 5 and Table S2 (Supporting Information).Based on these results and in order to complete the characterization of our library we selected compounds 4a, 4d, 4g, 4h, 4i, 4k, 4m and 6 to be assayed in the intracellula amastigote model against both L. amazonensis and L. donovani, with the results shown in Figures 6 and 7 and Table S3 (Supporting Information).Based on these results and in order to complete the characterization of our library, we selected compounds 4a, 4d, 4g, 4h, 4i, 4k, 4m and 6 to be assayed in the intracellular amastigote model against both L. amazonensis and L. donovani, with the results shown in Figures 6 and 7 and Table S3 (Supporting Information).Figure 6.Antileishmanial activity against Leishmania amazonensis and Leishmania donovani amastigotes (IC50, µM).The numerical data corresponding to this graphic can be found in Table S3 (Supporting Information).
Figure 7. Selectivity indexes, obtained as the ratio between the toxicity against J774 macrophages (CC50, µM) and the IC50 values against Leishmania amazonensis and Leishmania donovani amastigotes (IC50, µM).The numerical data corresponding to this graphic can be found in Table S3 (Supporting Information).

Discussion
Regarding antitubercular structure-activity relationships, the presence of substituents on the aromatic ring in the C-3 position of the quinazoline skeleton increases activity (compare 4a/4c with 4d and 4f/4h with 4f).In all cases, compounds bearing this substituent in the para position showed better results than their ortho or meta analogues (compare 4j/4k and 4b with 4g).Additionally, the presence of a substituent at the C-6 position of the quinoxaline ring led to an increase in the activity (compare 4a/4f and 4n; 4l/4c and 4d/4m), and similar results were observed for methyl and methoxy groups (compare 4i/4m and 4h/4l).The presence of the trifluoromethyl group in any of the rings improved the inhibition results (4c, 4h and 4l).Regarding the glycine side chain, the presence of the carboxylic acid seemed to increase activity compared to the methyl ester (compare compounds 4f/5f and 4i/5i).Compound 6, with a carboxylic group attached directly to the C-2 position of the heterocyclic skeleton, showed the lowest activity of the whole series, showcasing the relevance of the glycine side chain for antitubercular activity.
Compounds 4c, 4f, 4l and their derivatives 5, containing a carboxylic acid group at the side chain, were inactive against both Leishmania species.An analysis of the results obtained against L. amazonensis showed that four of the compounds (4e, 4m, 4n and 6) exhibited similar in vitro activity to the drug miltefosine, used as a positive control, with IC50 values in the 20-97 µM range (miltefosine gave 47.9 µM).Unfortunately, compound 4e was highly toxic to macrophages, and therefore, it showed very little selectivity (SI = 0.7).The presence of a para substituted phenyl at C-3 increased activity compared to unsubstituted phenyl (compare 4a/4d/4b with 4f/4h/4i/4j).Electronic effects were not relevant for the activity values (compare 4a/4b/4d, 4g/4h/4i/4k and 4n/4m/4l).The presence Figure 6.Antileishmanial activity against Leishmania amazonensis and Leishmania donovani amastigotes (IC 50 , µM).The numerical data corresponding to this graphic can be found in Table S3 (Supporting Information).The numerical data corresponding to this graphic can be found in Table S3 (Supporting Information).
Figure 7. Selectivity indexes, obtained as the ratio between the toxicity against J774 macrophages (CC50, µM) and the IC50 values against Leishmania amazonensis and Leishmania donovani amastigotes (IC50, µM).The numerical data corresponding to this graphic can be found in Table S3 (Supporting Information).

Discussion
Regarding antitubercular structure-activity relationships, the presence of substituents on the aromatic ring in the C-3 position of the quinazoline skeleton increases activity (compare 4a/4c with 4d and 4f/4h with 4f).In all cases, compounds bearing this substituent in the para position showed better results than their ortho or meta analogues (compare 4j/4k and 4b with 4g).Additionally, the presence of a substituent at the C-6 position of the quinoxaline ring led to an increase in the activity (compare 4a/4f and 4n; 4l/4c and 4d/4m), and similar results were observed for methyl and methoxy groups (compare 4i/4m and 4h/4l).The presence of the trifluoromethyl group in any of the rings improved the inhibition results (4c, 4h and 4l).Regarding the glycine side chain, the presence of the carboxylic acid seemed to increase activity compared to the methyl ester (compare compounds 4f/5f and 4i/5i).Compound 6, with a carboxylic group attached directly to the C-2 position of the heterocyclic skeleton, showed the lowest activity of the whole series, showcasing the relevance of the glycine side chain for antitubercular activity.

Discussion
Regarding antitubercular structure-activity relationships, the presence of substituents on the aromatic ring in the C-3 position of the quinazoline skeleton increases activity (compare 4a/4c with 4d and 4f/4h with 4f).In all cases, compounds bearing this substituent in the para position showed better results than their ortho or meta analogues (compare 4j/4k and 4b with 4g).Additionally, the presence of a substituent at the C-6 position of the quinoxaline ring led to an increase in the activity (compare 4a/4f and 4n; 4l/4c and 4d/4m), and similar results were observed for methyl and methoxy groups (compare 4i/4m and 4h/4l).The presence of the trifluoromethyl group in any of the rings improved the inhibition results (4c, 4h and 4l).Regarding the glycine side chain, the presence of the carboxylic acid seemed to increase activity compared to the methyl ester (compare compounds 4f/5f and 4i/5i).Compound 6, with a carboxylic group attached directly to the C-2 position of the heterocyclic skeleton, showed the lowest activity of the whole series, showcasing the relevance of the glycine side chain for antitubercular activity.
Compounds 4c, 4f, 4l and their derivatives 5, containing a carboxylic acid group at the side chain, were inactive against both Leishmania species.An analysis of the results obtained against L. amazonensis showed that four of the compounds (4e, 4m, 4n and 6) exhibited similar in vitro activity to the drug miltefosine, used as a positive control, with IC 50 values in the 20-97 µM range (miltefosine gave 47.9 µM).Unfortunately, compound 4e was highly toxic to macrophages, and therefore, it showed very little selectivity (SI = 0.7).The presence of a para substituted phenyl at C-3 increased activity compared to unsubstituted phenyl (compare 4a/4d/4b with 4f/4h/4i/4j).Electronic effects were not relevant for the activity values (compare 4a/4b/4d, 4g/4h/4i/4k and 4n/4m/4l).The presence of a substituent at C-6 increases the activity, and electron-withdrawing groups are preferred, since CF 3 leads to a higher activity than CH 3 and the latter than MeO (compare 4n/4f/4a with 4i/4m).The ester group in the glycine side chain increased activity compared to the corresponding carboxylic acid.Finally, compound 6, bearing a carboxylic group at C-2, showed a slightly increased activity, suggesting that the glycine side chain is counterproductive in terms of antileishmanial activity in the promastigote stage.Nevertheless, its key role in terms of antitubercular properties makes it an essential structural feature for the desired dual activity.
An analysis of the results obtained against L. donovani revealed lower inhibition activity in comparison to miltefosine.However, eleven of the tested compounds exhibited interesting inhibition and low toxicity results.An analysis of the structure-activity relationships led to the same conclusions as those of the L. amazonensis study.The esters showed better results than their carboxylic acid analogues and the C-3-phenyl-substituted phenyls compounds performed better than their unsubstituted analogues.On the subject of the substituents in the C-6 position, although the electronic effect of the substituent was not relevant for the activity, the presence of a methoxy group increased the selectivity index (compounds 4g, 4h, 4i, 4j and 4k).
Compound 4g showed the best SI value (310.6) and the presence of the methoxy group in compound 4d (IC 50 = 9.8 µM; SI = 54) improved the potency and the selectivity index compared to methyl analogue 4m (IC 50 = 74.4µM; SI = 6.1).The QNOs 4k and 4d (in this order) were the two most potent compounds in these experiments.Furthermore, 4k (IC 50 = 7.2 µM; SI = 330) and 4d (IC 50 = 9.8 µM; SI = 539) were considerably more potent and less toxic than the reference antileishmanial drug miltefosine (IC 50 = 136.1 µM; SI = 2.87).A similar study was performed with L. donovani amastigotes, showing lower activities for all compounds and suggesting that they are more promising for cutaneous and mucocutaneous leishmaniasis.Nevertheless, 4h maintained a respectable activity against Leishmania donovani amastigotes and can thus be regarded as the compound with a better-rounded antileishmanial profile.
As an overall conclusion, the best-balanced dual antitubercular/antileishmanial profile corresponds to compound 4h.It is relevant to note that this compound shows activities in the same order of magnitude for all organisms assayed, a very important feature for a drug aimed at several targets, and can therefore be proposed as a hit for further development.

Synthesis 4.1.1. General Synthetic Experimental Information
NMR spectra were registered on a Bruker Avance 250 spectrometer (250 MHz for 1 H, 63 MHz for 13 C) (Bruker, Rivas-Vaciamadrid, Spain) and maintained by the Unidad de Resonancia Magnética Nuclear, UCM.Infrared spectra were obtained by preparing KBr disks for each compound and registering their spectra on a Perkin Elmer Paragon 1000 FT-IR spectrophotometer (Tres Cantos, Spain).Melting points were taken on a Reichert 723 hot stage microscope (Vienna, Austria).CHN combustion elemental analyses were performed by the Unidad de Microanálisis Elemental, UCM, using a Leco CHNS-932 combustion microanalyzer (Tres Cantos, Spain).

General Procedure for the Synthesis of 3-Arylmethylene-2,5-Piperazinediones (1)
A solution of 1,4-diacetyl-2,5-piperazinedione (4.41 mmol) and the corresponding aldehyde (4.41 mmol) in DCM (12 mL) was treated dropwise with a 1M solution of potassium tert-butoxide in tert-butyl alcohol (4.41 mL).The solution was stirred at room temperature for 5 h.Then, 10 mL of a saturated aqueous solution of NH 4 Cl was added to the reacting mixture, and the formed solid was collected by filtration and dried to obtain compounds 1.Their characterization data are given below.

General Procedure for Preparation of Benzofurazane Oxides 3a-c
Benzofurazan oxide 3a and its derivatives were synthesized according to a modified literature procedure [51].Derivatives of o-nitroaniline (43.5 mmol) were dissolved in 20% ethanol/KOH (250 mL).The deep red solution was cooled in an ice bath (0-5 • C).Commercial aqueous NaOCl (Clorox) (200 mL) was added with stirring until no o-nitroaniline was present in the solution, that is, when the solution no longer changed color from yellow to red upon the addition of NaOCl.The bright yellow precipitate was collected by suction filtration and was washed with cold water.The solid was dried under vacuum to yield yellow solid benzofurazan oxides 3a-3c (65-76% yield).

General Procedure for Preparation of 3-arylquinoxaline-1,4-di-N-oxides 4
The suitable arylpiruvyl ester 2 (0.42 mmol) was added to a solution of the suitable benzofuranoxane 3 (0.5 mmol) and KOH (0.5 mmol) in dry THF (10 mL).The reaction mixture was stirred at room temperature for 3-16 h, and when no starting material was evident by TLC, 2 mL of a 1M HCl solution was added.The reaction mixture was extracted with AcOEt (2 × 10 mL).The organic layer was washed with brine and dried over anhydrous sodium sulfate and the solvent was evaporated under reduced pressure.The residue was crystallized from chloroform-hexane to give the pure quinoxaline-1,4-di-N-oxides 4.

In Vitro Activity against Mycobacterium tuberculosis 4.2.1. First Protocol for Activity Measurement
The test samples were analyzed in vitro against Mycobacterium tuberculosis H37Rv (Mtb H37Rv) in a high-throughput screen using an assay adapted from the microdilution AlamarBlue (AB) broth assay as reported by Collins and Franzblau [47].All manipulations of Mtb H37Rv were conducted in accordance with the Biosafety in Microbiological and Biomedical Laboratories (BMBL) in Biosafety Level 3 containment laboratories.In brief, our assay uses Mtb H37Rv in a 384-well plate format with in-plate DMSO carrier controls, 3.198 µM amikacin, 0.17 µM amikacin controls and 320 compounds.The Mtb H37Rv plus DMSO carrier control provides a 100% growth control for each plate.The 3.198 µM amikacin completely inhibits the growth of the bacteria and is used in place of uninoculated media as the background control; meanwhile, the 0.17 µM amikacin control approximates the MIC of amikacin ranging from 30 to 80% inhibition, indicative of a positive growth inhibition and proper assay performance.The medium used for both compound preparation and Mtb H37Rv plating was assessed for contamination by plating two 384-well plates with the medium alone.The plates were checked for contamination by visual inspection and endpoint detection.The compounds were evaluated in a 10-point stacked plate dose-response method.The compounds were serially diluted at 1:2 from 100 µM to 0.195 µM.The plates were read fluorometrically after incubation with the compounds and the addition of AB.

Second Protocol for Activity Measurement
An alternative method for end-point detection was assessed using the Promega reagent BacTiter-Glo™ Microbial Cell Viability (BTG).The BTG plates were briefly incubated for 20 min at room temperature, sealed with Perkin Elmer clear TopSeal A and read from the top using luminescence on a Perkin Elmer Envision instrument (Tres Cantos, Spain).

Autofluorescence
The compounds in media were pre-read from the high concentration plate with no AlamarBlue or bacteria added.Fold increase was calculated using the median of the positive control wells from the AlamarBlue Mtb H37Rv assay for the Mtb H37Rv control wells.The criterion for a compound being considered autofluorescent was defined as having >50% fluorescence of the Mtb H37Rv control wells.None of the analyzed compounds were found to be autofluorescent.The following species of Leishmania were used: L. donovani (MHOM/IN/80/DD8), which was purchased (ATCC, Manassas, VA, USA), and L. amazonensis (MHOM/Br/79/Maria), which was kindly provided by Prof. Alfredo Toraño (Instituto de Salud Carlos III, Madrid, Spain).Promastigotes were cultured in Schneider's Insect Medium supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) (SIGMA-Merck, Madrid, Spain) and 1000 U/L of penicillin plus 100 mg/L of streptomycin in 25 mL culture flasks at 26 • C.

Promastigote Susceptibility Assay
The activity of the compounds on Leishmania promastigotes was performed according to the method previously reported by us [52].Briefly, promastigotes (2.5 × 10 5 parasites/well) were cultured in 96-well plastic plates.The compounds were dissolved in dimethylsulfoxide (DMSO).Serial dilutions at 1:2 of the test compounds in fresh culture medium were performed (100, 50, 25, 12.5, 6.25, 3.12, 1.56 and 0.78 µg/mL) in a final volume of 200 µL; the solutions were then added to the parasite suspension.After an incubation period of 48 h at 26 • C, a volume of 20 µL of 2.5 mM resazurin solution in PBS was added and the plates were incubated for 3 h under the same conditions.Finally, the Relative Fluorescence Unit (RFU) (535 ex -590 em nm wavelength) was determined in a fluorimeter (Infinite 200 Tecan i-Control).Growth inhibition (%) was calculated by 100-[(RFU treated wells-RFU signal-to-noise)/(RFU untreated-RFU signal-to-noise) × 100].All tests were carried out in triplicate and the IC50 was calculated by probit analysis using SPSS 17.0 Statistics Software.Miltefosine (Sigma-Merck, Madrid, Spain) was used as the reference drug.

Intracellular Amastigote Assay
We performed an intracellular amastigote assay on L. amazonensis and L. donovani according to a fluorometric method reported by us [53].To carry out the in vitro macrophage infections, 5 × 10 4 cells were cultured in RPMI-1640 medium and then were infected with 5 × 10 5 promastigotes.In the L. donovani assays, the plates were incubated for 48 h at 37 • C, while in the L. amazonensis assays, they were incubated at 33 • C for the first 24 h and then the temperature was increased to 37 • C for the next 24 h.After, the culture medium was removed and the cells were washed with buffered RPMI-HEPES at pH 7.4 several times in order to eliminate the non-internalized promastigotes.A total of 100 µL of fresh medium tempered at 37 • C was added to the infected cells, which then were exposed to 100 µL of the test compounds in RPMI-1640 at different concentrations (100, 50, 25, 12.5, 6.25, 3.12, 1.56 and 0.78 µg/mL in a final volume of 200 µL) for 48 h at 37 • C. The culture medium was removed by centrifugation at 3500 rpm for 5 min (Centrifuge 5403 Eppendorf) and a lysis solution (0.02% sodium dodecyl sulfate in RPMI-HEPES) was added.After 20 min, the treated cells were harvested (centrifugation at 3500 rpm for 5 min at 4 • C) and the supernatants were replaced by 200 µL of fresh Schneider's culture medium.The plates were then incubated at 26 • C for another 3 days to allow for the transformation of viable amastigotes into promastigotes and proliferation.Afterwards, 20 µL/well of 2.5 mM resazurin was added and incubated for another 3 h.Finally, URF was measured and IC50 was estimated as described above.All tests were carried out in triplicate.Miltefosine was used as the reference drug.

Cytotoxicity Assay on Macrophages
Cell viability was evaluated using a modification with resazurin of the colorimetric method described previously [47].J774 macrophages were seeded (5 × 10 4 cells/well) in 96-well flat-bottom microplates with 100 µL of RPMI-1640 medium.The cells were allowed to adhere for 4 h at 37 • C in 5% CO 2 , then different concentrations of the test compounds in 100 µL of the medium were added and exposed for 24 h.Growth controls were also included.To evaluate cell viability, 20 µL of a 2.5 mM resazurin solution was added and the plates were returned to the incubator for another 3 h.The reduction in resazurin was determined by measuring the fluorescence intensity (excitation wavelength, 535 nm; emission wavelength, 590 nm).Each concentration was assayed three times.The medium and controls were used in each test as blanks.The cytotoxic effect of the compounds was defined as the 50% reduction in cell viability of treated culture cells with respect to the untreated culture (CC 50 ).

Selectivity Index Calculations
The selectivity index (SI) was calculated as SI = CC 50 /IC 50 , as described by Ferreira et al. [54].

Conclusions
We describe the design of a novel synthetic route and biological evaluation of new QdNOs with amino acid side chains as bifunctional therapeutic agents against tuberculosis and leishmaniasis.The basic structure of quinoxaline 1,4-dioxide was modified with a peptide side chain and aryl side arm at position 2 and 3, respectively.The synthetic route toward these compounds involves a selective diketopiperazine ring opening followed by a modified Beirut reaction.The strategic design of molecules possessing dual properties represents a promising paradigm in drug discovery, effectively tackling the specific challenges associated with infectious diseases and potentially leading to more efficient and less costly treatments.The main conclusions that can be drawn from this SAR study are as follows: 1.
The presence of a peptide chain at C-2 improves the antitubercular activity compared to the presence of a carboxylic acid.

2.
A higher state of oxidation of the nitrogen atoms of the heterocyclic skeleton increases its activity.

3.
The presence of substituents in the benzene rings improves the activity of the compounds.4.
Substituents at the para position of the aromatic side arm are more favorable than their ortho and meta analogues.
The identification of compound 4h, showing good activity and selectivity both as an antitubercular and antileishmanial agent, establishes a foundation for future investigations for the desired dual profile, with a good balance between the activities found against all of the organisms studied.This compound can be considered an initial candidate for further development.

Figure 1 .
Figure 1.Representative examples of QdNOs with diverse biological activities.

Figure 1 .
Figure 1.Representative examples of QdNOs with diverse biological activities.

Pharmaceuticals 2024 , 19 Figure 2 .
Figure 2. A summary of the bonds generated in our synthetic strategy.

Figure 3 .
Figure 3. Antibacterial activity against Mycobacterium tuberculosis Mtb H37Rv.The numerical data corresponding to this graphic can be found in TableS1(Supporting Information).None of the compounds showed cytotoxicity in Vero cells at concentrations up to 6.25 μg/mL.

Figure 2 .
Figure 2. A summary of the bonds generated in our synthetic strategy.

Pharmaceuticals 2024 , 19 Figure 2 .
Figure 2. A summary of the bonds generated in our synthetic strategy.

Figure 3 .
Figure 3. Antibacterial activity against Mycobacterium tuberculosis Mtb H37Rv.The numerical data corresponding to this graphic can be found in TableS1(Supporting Information).None of the compounds showed cytotoxicity in Vero cells at concentrations up to 6.25 μg/mL.

Figure 2 .
Figure 2. A summary of the bonds generated in our synthetic strategy.

Figure 3 .
Figure 3. Antibacterial activity against Mycobacterium tuberculosis Mtb H37Rv.The numerical data corresponding to this graphic can be found in TableS1(Supporting Information).None of the compounds showed cytotoxicity in Vero cells at concentrations up to 6.25 μg/mL.

Figure 3 .
Figure 3. Antibacterial activity against Mycobacterium tuberculosis Mtb H37Rv.The numerical data corresponding to this graphic can be found in TableS1(Supporting Information).None of the compounds showed cytotoxicity in Vero cells at concentrations up to 6.25 µg/mL.

Figure 4 .
Figure 4. Antileishmanial activity against Leishmania amazonensis and Leishmania donovani pro mastigotes (IC50, µM).The numerical data corresponding to this graphic can be found in Table S (Supporting Information).

Figure 5 .
Figure5.Selectivity indexes, obtained as the ratio between the toxicity against J774 macrophage (CC50, µM) and the IC50 values against Leishmania amazonensis and Leishmania donovani promastigote (IC50, µM).The numerical data corresponding to this graphic can be found in TableS2(Supporting Information).

Figure 5 .
Figure5.Selectivity indexes, obtained as the ratio between the toxicity against J774 macrophages (CC 50 , µM) and the IC 50 values against Leishmania amazonensis and Leishmania donovani promastigotes (IC 50 , µM).The numerical data corresponding to this graphic can be found in TableS2(Supporting Information).

Figure 7 .
Figure7.Selectivity indexes, obtained as the ratio between the toxicity against J774 macrophages (CC 50 , µM) and the IC 50 values against Leishmania amazonensis and Leishmania donovani amastigotes (IC 50 , µM).The numerical data corresponding to this graphic can be found in TableS3(Supporting Information).

Table 1 .
Scope and yields obtained in two-step synthesis of compounds 2.

Table 1 .
Scope and yields obtained in two-step synthesis of compounds 2.
Scheme 2. Application of the Beirut reaction to the synthesis of compounds 4 and their hydrolysis to 5.

Table 2 .
Scope and yields obtained in synthesis of compounds 4 and 5. -oxides, generating five bonds over three steps, as summarized in Figure2.