Antileishmanial Effect of 1,5- and 1,8-Substituted Fused Naphthyridines

Citation: Melcón-Fernandez, E.; Martín-Encinas, E.; Palacios, F.; Galli, G.; Reguera, R.M.; Martínez-Valladares, M.; Balaña-Fouce, R.; Alonso, C.; Pérez-Pertejo, Y. Antileishmanial Effect of 1,5and 1,8-Substituted Fused Naphthyridines. Molecules 2024, 29, 74. https://doi.org/10.3390/ molecules29010074 Academic Editors: Maria Isabel L. Soares and Susana M. M. Lopes Received: 4 November 2023 Revised: 18 December 2023 Accepted: 20 December 2023 Published: 22 December 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). molecules Article Antileishmanial Effect of 1,5and 1,8-Substituted Fused Naphthyridines Estela Melcón-Fernandez 1, Endika Martín-Encinas 2, Francisco Palacios 2, Gulio Galli 1, Rosa M. Reguera 1, María Martínez-Valladares 1, Rafael Balaña-Fouce 1, Concepción Alonso 2,* and Yolanda Pérez-Pertejo 1,* 1 Departamento de Ciencias Biomédicas, Facultad de Veterinaria, Universidad de León, Campus de Vegazana s/n, 24071 León, Spain; [email protected] (G.G.) 2 Departamento de Química Orgánica I, Facultad de Farmacia, Lascaray Research Center, Universidad del País Vasco/Euskal Herriko Unibertsitatea (UPV/EHU), Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain *Correspondence: [email protected] (C.A.); [email protected] (Y.P.-P.) Abstract: In the absence of a vaccine, there is a need to find new drugs for the treatment of neglected tropical diseases, such as leishmaniasis, that can overcome the many drawbacks of those currently used. These disadvantages include cost, the need to maintain a cold chain, the route of administration, the associated adverse effects and the generation of resistance. In this work we have evaluated the antileishmanial effect of 1,5and 1,8-substituted fused naphthyridines through in vitro and ex vivo assays, using genetically modified axenic and intramacrophagic Leishmania infantum amastigotes. The toxicity of these compounds has been tested in the mammalian host cell using murine splenic macrophages, as well as in murine intestinal organoids (miniguts) in order to assess their potential for oral administration. The 1,8derivatives showed greater leishmanicidal activity and the presence of a nitrogen atom in the fused ring to the naphthyridine was important to increase the activity of both types of molecules. The aromatization of the pyridine ring also had marked differences in the activity of the compounds. Keywords: visceral leishmaniasis; fused 1,5-naphthyridines; fused 1,8-naphthyridines; intramacrophagic Leishmania parasites; mouse intestinal organoids 1. Introduction Current treatment of leishmaniasis, a complex of vector-borne diseases caused by parasitic protists of the genus Leishmania, remains a challenge [ 1 – 3 ]. The lack of a vaccine makes chemotherapy the only way to manage the infection when the first symptoms appear. However, the ability of the parasite to evade the host immune system and the intracellular localization of the parasite within host macrophages are pitfalls that hinder and delay the discovery and development of new drugs to treat these diseases [ 4 ]. Last but not least, leishmaniasis is classified by the WHO as a neglected tropical disease widely spread in the tropical and subtropical areas of the planet [ 5 ], where the drugs currently in use are scarce, outdated and expensive. In addition, current drugs exhibit toxicity, adverse side effects, poor oral bioavailability and loss of efficacy due to the increasing emergence of resistant strains [1,6,7]. Despite their significant poor tolerability, pentavalent antimonials are still the firstline treatment for different types of leishmaniasis. In addition to their cardiotoxicity, they require prolonged and painful parenteral administration, which makes adherence difficult [ 8 ]. Formulations of the polyene fungicide amphotericin B are strongly effective against the visceral form of the disease, but they must be administered by intravenous infusions and require a cold chain to reach the point of care [ 4 , 9 ]. Finally, miltefosine is the only approved oral antileishmanial drug, but unfortunately, it cannot be administered in pregnant women due to its teratogenicity and easily promotes resistance [ 9 ]. Combinations of these drugs and with the antibiotic paromomycin represent an interesting scenario until Molecules 2024,29, 74. https://doi.org/10.3390/molecules29010074 https://www.mdpi.com/journal/molecules Molecules 2024,29, 74 2 of 16 more effective and safer molecules are registered [ 10 , 11 ]. Given the limited number of drugs currently used against leishmaniasis and their drawbacks, it is urgently needed to search for new compounds that allow short, safe, inexpensive treatments and whose requirements are in line with the needs of developing countries (target product profile), where the disease is most prevalent [12]. There is a broad consensus among medicinal chemists that the presence of nitrogen atoms in the heterocyclic structure is relevant to improve interactions in biological systems as nitrogenated heterocycles are part of the chemical structure of many natural products and agents with significant biological activity, such as antiviral, antibiotic and antitumor drugs [ 13 – 20 ]. In fact, the FDA database reveals that a large number of accepted drugs contain a nitrogen heterocycle in their structure [ 21 ]. Therefore, in the drug discovery process, the development of practical synthetic routes to access these structural motifs in the simplest way possible is an important goal for synthetic and medicinal chemists. One of the most straightforward and versatile processes for the preparation of heterocyclic nitrogen compounds is the Povarov reaction [ 22 ]. With this in mind, our group has developed a suitable, efficient, fast and versatile methodology for the synthesis of nitrogenated heterocycles and good results were observed as topoisomerase IB (TopIB) inhibitors of these derivatives, with antileishmanial activity [23,24]. In addition, if the traditional Povarov reaction represents an excellent tool for the preparation of bicyclic nitrogenated heterocycles, such as the aforementioned quinoline or naphthyridine derivatives, compounds with a greater number of fused cycles can also be prepared with this methodology in its intramolecular version [ 25 ]. In these cases, the aza-Diels–Alder process (a [4+2] cycloaddition reaction) involves an aldehyde functionalized with a dienophile and an aromatic amine. Moreover, if the aromatic amine used is heterocyclic such as 2-aminopyridine or 3-aminopyridine, this process allows the direct preparation of fused heterocycles with several heteroatoms in their structure. Under this context, the objective of the present study was to screen a series of fused 1,5and 1,8-naphthyridines to evaluate their antileishmanial activity by using in vitro and ex vivo tests. The antiparasitic activity of these compounds was assessed in both axenic and intramacrophagic forms of Leishmania infantum using an in-house improved bioimaging system. In addition, the potential toxicity of the assayed compounds was evaluated in mouse splenic macrophages and murine intestinal organoids in order to assess their tolerability as oral drugs. 2. Results 2.1. Chemistry The synthesis of fused 1,5-naphthyridines and 1,8-naphthyridines is depicted in Schemes 1and 2. The synthesis of fused 1,5-naphthyridines (Scheme 1) was performed by an intramolecular Povarov type [4+2] cycloaddition reaction. Thus, when 3-aminopyridine 1a was reacted with functionalized aldehydes 2in refluxing chloroform, in the presence of 2 equivalents of BF 3· Et 2 O, tetrahydro[1,5]naphthyridine derivatives 4,6,8(Scheme 1, Figure 1) were regioand diastereoselectively obtained as previously reported by us [ 26 , 27 ]. Molecules 2023, 28, x FOR PEER REVIEW 3 of 17 Scheme 1. Intramolecular Povarov reaction of 3-aminopyridine 1a with functionalized aldehydes 2. Figure 1. Structures of fused 1,5-naphthyridine derivatives 4–9 tested as antileishmanial agents. When 2-aminopyridine 1b was used tetrahydro[1,8]naphthyridines derivatives 10, 12, 14 (Scheme 2, Figure 2) were isolated as well in a regioand diastereoselective way [28]. Scheme 2. Intramolecular Povarov reaction of 2-aminopyridine 1b and functionalized aldehydes 2. Figure 2. Structures of fused 1,8-naphthyridine derivatives 10–15 tested as antileishmanial agents. In addition, the subsequent dehydrogenation of tetrahydronaphthyridine derivatives with MnO2 in refluxing toluene yielded the corresponding 1,5-naphthyridines 5, 7 and 9 (Scheme 1, Figure 1) and 1,8-naphthyridines 11, 13 and 15 (Scheme 2, Figure 2), respectively, as previously reported [26–28]. The described methodologies represent an easy and efficient strategy for the preparation of these compounds containing a wide range of electron-releasing and electron-withdrawing substituents. Scheme 1. Intramolecular Povarov reaction of 3-aminopyridine 1a with functionalized aldehydes 2. Molecules 2024,29, 74 3 of 16 Molecules 2023, 28, x FOR PEER REVIEW 3 of 17 Scheme 1. Intramolecular Povarov reaction of 3-aminopyridine 1a with functionalized aldehydes 2. Figure 1. Structures of fused 1,5-naphthyridine derivatives 4–9 tested as antileishmanial agents. When 2-aminopyridine 1b was used tetrahydro[1,8]naphthyridines derivatives 10, 12, 14 (Scheme 2, Figure 2) were isolated as well in a regioand diastereoselective way [28]. Scheme 2. Intramolecular Povarov reaction of 2-aminopyridine 1b and functionalized aldehydes 2. Figure 2. Structures of fused 1,8-naphthyridine derivatives 10–15 tested as antileishmanial agents. In addition, the subsequent dehydrogenation of tetrahydronaphthyridine derivatives with MnO2 in refluxing toluene yielded the corresponding 1,5-naphthyridines 5, 7 and 9 (Scheme 1, Figure 1) and 1,8-naphthyridines 11, 13 and 15 (Scheme 2, Figure 2), respectively, as previously reported [26–28]. The described methodologies represent an easy and efficient strategy for the preparation of these compounds containing a wide range of electron-releasing and electron-withdrawing substituents. Scheme 2. Intramolecular Povarov reaction of 2-aminopyridine 1b and functionalized aldehydes 2. Molecules 2023, 28, x FOR PEER REVIEW 3 of 17 N X Y R2 N BF3稥t2O N HN Y X R2 O X Y R2 NH2 N + 34 X = O; Y = CH2 6X = O; Y = CO 8 X = NTs; Y = CH2 MnO2 N NY X R2 HH 5X = O; Y = CH2 7X = O; Y = CO 9X = NTs; Y = CH2 1a 2a X = O; Y = CH2 2b X = O; Y = CO 2c X = NTs; Y = CH2 R1 MS 4 Å R1R1R1 R1 = H, 4-Br, 6-Br, 6-OMe Scheme 1. Intramolecular Povarov reaction of 3-aminopyridine 1a with functionalized aldehydes 2. N HN O R1 4a (R1 = H) 4c (R1 = OMe) N N O R1 N HN O R1 6a (R1 = H) 6b (R1 = Br) 6c (R1 = OMe) O N N O R1 O N HN NTs 8a (R1 = H) 8b (R1 = 4-Br) 8c (R1 = 6-OMe) 8d (R1 = 6-Br) R1N N NTs 5a (R1 = H) 5b (R1 = Br) 5c (R1 = OMe) 7a (R1 = H) 7b (R1 = Br) 7c (R1 = OMe) R1 9a (R1 = H) 9b (R1 = 4-Br) 9c (R1 = 6-OMe) 9d (R1 = 6-Br) Figure 1. Structures of fused 1,5-naphthyridine derivatives 4–9 tested as antileishmanial agents. When 2-aminopyridine 1b was used tetrahydro[1,8]naphthyridines derivatives 10, 12, 14 (Scheme 2, Figure 2) were isolated as well in a regioand diastereoselective way [28]. N X Y R2 N R1 BF3稥t2O N HN Y X R2 O X Y R2 NH2 N+ 310 X = O; Y = CH2 12 X = O; Y = CO 14 X = NTs; Y = CH2 MnO2 N NY X R2 HH 11 X = O; Y = CH2 13 X = O; Y = CO 15 X = NTs; Y = CH2 R3 1b 2a X = O; Y = CH2 2b X = O; Y = CO 2c X = NTs; Y = CH2 R1 R1 R3R3 R3 MS 4 Å R1 R1= H, 5-Br Scheme 2. Intramolecular Povarov reaction of 2-aminopyridine 1b and functionalized aldehydes 2. N HN O 10a (R2 = H) 10b (R2 = F) 10c (R2 = Me) N N O N HN O R1 12a (R1 = H, R2 = H) 12b (R1 = Br, R2 = H) O N N O R1 O N HN N 14a N N N R1 11a (R2 = H, R3 = H) 11b (R2 = F, R3 = H) 11c (R2 = H, R3 = OMe) 13a (R1 = H, R2 = H) 13b (R1 = Br, R2 = H) 13c (R1 = H, R2 = F) 15a (R1 = H) 15b (R1 = Br) R2R2 R3 R2R2 Ts Ts Figure 2. Structures of fused 1,8-naphthyridine derivatives 10–15 tested as antileishmanial agents. In addition, the subsequent dehydrogenation of tetrahydronaphthyridine derivatives with MnO2 in refluxing toluene yielded the corresponding 1,5-naphthyridines 5, 7 and 9 (Scheme 1, Figure 1) and 1,8-naphthyridines 11, 13 and 15 (Scheme 2, Figure 2), respectively, as previously reported [26–28]. The described methodologies represent an easy and efficient strategy for the preparation of these compounds containing a wide range of electron-releasing and electron-withdrawing substituents. Figure 1. Structures of fused 1,5-naphthyridine derivatives 4–9tested as antileishmanial agents. When 2-aminopyridine 1b was used tetrahydro[1,8]naphthyridines derivatives 10,12, 14 (Scheme 2, Figure 2) were isolated as well in a regioand diastereoselective way [28]. Molecules 2023, 28, x FOR PEER REVIEW 3 of 17 Scheme 1. Intramolecular Povarov reaction of 3-aminopyridine 1a with functionalized aldehydes 2. Figure 1. Structures of fused 1,5-naphthyridine derivatives 4–9 tested as antileishmanial agents. When 2-aminopyridine 1b was used tetrahydro[1,8]naphthyridines derivatives 10, 12, 14 (Scheme 2, Figure 2) were isolated as well in a regioand diastereoselective way [28]. Scheme 2. Intramolecular Povarov reaction of 2-aminopyridine 1b and functionalized aldehydes 2. Figure 2. Structures of fused 1,8-naphthyridine derivatives 10–15 tested as antileishmanial agents. In addition, the subsequent dehydrogenation of tetrahydronaphthyridine derivatives with MnO2 in refluxing toluene yielded the corresponding 1,5-naphthyridines 5, 7 and 9 (Scheme 1, Figure 1) and 1,8-naphthyridines 11, 13 and 15 (Scheme 2, Figure 2), respectively, as previously reported [26–28]. The described methodologies represent an easy and efficient strategy for the preparation of these compounds containing a wide range of electron-releasing and electron-withdrawing substituents. Figure 2. Structures of fused 1,8-naphthyridine derivatives 10–15 tested as antileishmanial agents. In addition, the subsequent dehydrogenation of tetrahydronaphthyridine derivatives with MnO 2 in refluxing toluene yielded the corresponding 1,5-naphthyridines 5,7and 9 (Scheme 1, Figure 1) and 1,8-naphthyridines 11,13 and 15 (Scheme 2, Figure 2), respectively, as previously reported [ 26 – 28 ]. The described methodologies represent an easy and efficient strategy for the preparation of these compounds containing a wide range of electronreleasing and electron-withdrawing substituents. 2.2. In Silico ADME In silico ADME studies, also known as computer-based studies, [ 29 – 32 ] play a crucial role in the drug discovery and development process. ADME stands for absorption, distribution, metabolism and excretion, which are key factors that determine the pharmacokinetics and efficacy of a drug. In silico ADME studies involve the use of computational models and algorithms to predict the ADME properties of a drug which not only saves time and resources, but also helps in the identification of potential safety concerns. Molecular properties such as the partition coefficient (Log P o/w), molecular weight, hydrogen bond donors and acceptors, topological polar surface area (TPSA) and violation of Lipinski’s rule of five were assessed (Table 1). Molecules 2024,29, 74 4 of 16 Table 1. Physicochemical properties and drug-likeness for chromeno[4,3-b][1,5]naphthyridine and chromeno[4,3-b][1,8]naphthyridine derivatives. #-number. Compound LogP Mol. Wt. H-Donor H-Acceptor TPSA Lipinski #Violations 4a 2.71 314.38 1 2 34.15 0 4c 2.76 344.41 1 3 43.38 0 6a 2.51 328.36 1 3 51.22 0 6b 2.69 407.26 1 3 51.22 0 6c 3.02 358.39 1 4 60.45 0 8a 3.11 467.58 1 3 70.68 0 8b 3.73 546.48 1 3 70.68 2 8c 3.81 497.61 1 4 79.91 0 8d 3.81 546.48 1 3 70.68 2 5a 3.26 310.35 0 3 35.01 0 5b 3.60 389.24 0 3 35.01 0 5c 3.60 340.37 0 4 44.24 0 7a 2.80 324.33 0 4 55.99 0 7b 3.12 403.23 0 4 55.99 0 7c 3.19 354.36 0 5 65.22 0 9a 3.70 463.55 0 4 71.54 0 9b 3.87 542.45 0 4 71.54 2 9c 3.97 493.58 0 5 80.77 0 9d 3.52 542.45 0 4 71.54 2 10a 3.05 314.38 1 2 34.15 0 10b 2.76 332.37 1 3 34.15 0 10c 2.92 328.41 1 2 34.15 0 12a 2.56 328.36 1 3 51.22 0 12b 2.94 407.26 1 3 51.22 0 14a 3.38 467.58 1 3 70.68 0 11a 2.99 310.35 0 3 35.01 0 11b 3.08 328.34 0 4 35.01 0 11c 3.27 340.37 0 4 44.24 0 13a 2.61 324.33 0 4 55.99 0 13b 3.00 403.23 0 4 55.99 1 13c 2.69 342.32 0 5 55.99 0 15a 3.48 463.55 0 4 71.54 0 15b 3.70 542.45 0 4 71.54 2 In general, a drug candidate with a LogP value between 0 and 5 is considered to have favorable ADME properties. A LogP value that is too low may indicate poor lipid solubility, which can affect the absorption and distribution of the drug in the body. A LogP value that is too high may indicate poor aqueous solubility, which can lead to poor bioavailability and potential toxicity due to the accumulation of the drug in fatty tissues. In the case of our compounds, they have a LogP value between 2.5 and 4 (with a mean of 3.12), so they would fall within the favorable range mentioned above. TPSA, or the topological polar surface area, is another commonly used parameter in drug discovery and development to predict the ADME properties of a drug candidate. TPSA is a measure of the polar surface area of a compound, which is important for its interaction with biological targets and its ability to cross biological membranes. In general, a drug candidate with a TPSA value between 20 and 140 Å 2 is considered to have favorable ADME properties. In Table 1, all the compounds tested in this work fall within the favorable range for good oral bioavailability. Finally, Lipinski’s Rule of Five [ 33 ], a widely used rule in drug discovery to assess the drug-like properties of a compound, was performed to study our compounds. It is recommended that the orally active drug candidate should not have more than one violation of Lipinski’s rule. All compounds tested except compounds 8b,8d,9b,9d and 15b meet the established criteria (Table 1). The absorption of a drug is a critical factor in determining its efficacy and safety. It refers to the process by which a drug enters the bloodstream and reaches its target site of action. Understanding the factors that influence drug absorption is essential for optimizing drug therapy and minimizing adverse effects. Because of this, the absorption of Molecules 2024,29, 74 5 of 16 drug was evaluated based on aqueous solubility, intestinal absorption and permeability (Table 2). Thus, the aqueous solubility (Log S) of all compounds ranges from − 7.48 to −4.40 log mol/L , which shows the moderate solubility in water of the synthesized compounds. In addition, all of them show intestinal absorption above 93%, which is interesting because most orally administered drugs are absorbed mainly through the small intestine due to its large surface area. Finally, the Caco-2 permeability, the logarithm of the apparent permeability coefficient (log Papp > 8 × 10 −6 cm/s), was predicted. Since the compound is considered to have high Caco-2 permeability if the predicted value is >0.90, in our case, all the synthesized compounds have high Caco-2 permeability. Finally, the volume of distribution (VDss), blood–brain barrier permeability (BBB permeability) [ 34 , 35 ] and the fraction of unbound of the synthesized compounds were further assessed. Considering that a Log VDss > 0.45 L/kg indicates a high volume of drug distribution, we can say that all synthesized compounds have low to moderate volume of distribution in tissues, except for compound 5a. On the other hand, a compound is said to be readily permeable across the BBB if the predicted value of log BBB is >0.3 and poorly distributed if the value is < − 1. In our estimations it can be seen that chromeno[4,3-b][1,5]naphthyridines (compounds 4,5,6and 7) and chromeno[4,3-b][1,8] naphthyridines (compounds 10,11,12 and 13) show excellent BBB parameters but those fused to quinolines (compounds 8,9,14 and 15) are not easily permeable through the BBB. Additional factors to consider in drug development include metabolite formation mediated by cytochrome P450 (CYP) enzyme activities [ 36 ] and drug clearance (CL) [ 37 ]. CYP450 enzymes are a family of heme-containing enzymes that are involved in the metabolism of a wide range of endogenous compounds and xenobiotics. These enzymes play a crucial role in drug metabolism, and their activity can influence the efficacy and safety of many therapeutic agents. Compounds were studied as possible CYP2D6, CYP3A4, CYP1A2, CYP2C19 and CYP2C9 enzyme inhibitors. Noteworthy, all compounds show the ability to inhibit the CYP2C19 enzyme. Compounds with a methoxy group in their structure (5c,9c and 11c) show inhibition of CYP2C9, which is primarily expressed in the liver and plays a critical role in the metabolism of numerous drugs. Conversely, the total clearance is primarily a combination of hepatic as well as renal clearance and is measured by the proportionality constant CLtot in log(mL/min/kg). The predicted value of all the synthesized compounds shows low CLtot ranging from − 0.154 to 1.91. These results clearly indicate that all these compounds show appropriate pharmacokinetic properties and can be considered antileishmanial agents. The boiled egg model [ 38 ] visually represents some of the ADME parameters of the evaluated 1,5and 1,8-naphthyridines (Figure 3). Compounds that would cross both the blood–brain barrier (BBB) and human gastrointestinal tract (HIA) are placed in the yolk and those with only HIA-absorption in the white. Most of the tested compounds have positive BBB and HIA permeability, except compounds 8,9a,14a and 15a which only show positive HIA and compounds 9b–dand 15b which do not show results for either parameter. P-glycoprotein (PGP) is a transporter protein found in the membrane of cells in various tissues, such as the gastrointestinal tract, liver and kidneys [ 39 , 40 ]. Its main function is to transport foreign substances, such as drugs, out of cells. Therefore, it may be desirable for a drug to be a PGP substrate, as it can help protect against toxicity or unwanted side effects. Dots colored blue correspond to molecules that are predicted to be P-glycoprotein substrates (PGP+) and are therefore actively pumped from the brain or into the gastrointestinal lumen (Figure 3). If they are predicted not to be P-glycoprotein substrates (PGP − ), the corresponding dot appears in red. In the case of our tested compounds, it should be noted that quinolino naphthyridine derivatives 8,9,14 and 15 and chromeno naphthyridine derivatives 7and 13 do not qualify as PGP-substrate candidates. In contrast, all the other compounds, derivatives 4,5,6,10,11 and 12, are suitable as PGP substrate candidates. Molecules 2024,29, 74 6 of 16 Table 2. Results of the ADME predictive study for chromeno[4,3-b][1,5]naphthyridine and chromeno[4,3-b][1,8]naphthyridine derivatives. Compound Log S (Log mol/L) Caco-2 Perm. (Log Paap in 10−6cm/s) Int. Abs. (% abs) VDss (L/kg) Fract. Unb. (Fu.) BBB Permeability (log BB) CYP1A2 Inhibitor CYP2C19 Inhibitor CYP2C9 Inhibitor CYP2D6 Inhibitor CYP3A4 Inhibitor Total Clearence Numeric (Log mL/min/kg) 4a −4.73 1.779 96.038 0.312 0.027 Yes No Yes No Yes Yes 0.180 4c −5.00 1.195 94.748 −0.121 0.109 Yes No Yes No Yes Yes 0.493 6a −4.47 1.268 98,408 0.190 0.003 Yes No Yes No Yes No 0.003 6b −5.59 1.210 93.336 −0.277 0.122 Yes No Yes No No No −0.024 6c −4.75 1.182 95.066 −0.303 0.112 Yes No Yes No Yes Yes 0.379 8a −6.21 1.153 98.895 −0.719 0.081 No No Yes No Yes Yes 0.286 8b −7.32 1.136 97.026 −0.669 0.073 No No Yes No No No −0.154 8c −6.49 1.063 95.572 −0.961 0.238 No No Yes No Yes Yes 0.392 8d −7.12 1.140 97.414 −0.810 0.087 No No Yes No Yes No −0.103 5a −4.92 1.660 100 0.525 0.26 Yes Yes Yes No Yes Yes 0.834 5b −6.03 1.143 96.994 0.099 0.354 Yes Yes Yes No No Yes 0.135 5c −5.18 1.122 98.725 0.025 0.343 Yes Yes Yes Yes Yes Yes 0.881 7a −5.00 1.410 100 0.403 0.368 Yes Yes Yes No No No 0.921 7b −6.11 1.137 98.66 0.193 0.363 Yes Yes Yes No No No 0.363 7c −5.27 1.098 100 0.108 0.352 Yes Yes Yes Yes No Yes 1.091 9a −6.36 1.119 100 −0.312 0.334 No Yes Yes No No Yes 0.819 9b −7.48 1.114 100 −0.293 0.333 No Yes Yes No No No 0.137 9c −6.64 1.167 99.946 −0.574 0.393 No No Yes Yes No Yes 0.949 9d −7.27 1.106 100 −0.386 0.338 No Yes Yes No No No 0.123 10a −4.92 1.765 96.706 0.142 0.035 Yes No Yes No Yes No 0.577 10b −5.07 1.803 94.633 −0.151 0.031 Yes No Yes No Yes No 0.268 10c −5.21 1.054 95.189 0.014 0.027 Yes No Yes No Yes No 0.601 12a −4.66 1.247 97.879 −0.184 0 Yes No Yes No Yes No 0.467 12b −5.56 1.044 94.333 −0.230 0.007 Yes No Yes No No No −0.092 14a −6.40 0.431 98.124 −0.935 0.086 No No Yes No Yes Yes 0.384 11a −5.07 1.550 100 0.297 0.285 Yes Yes Yes No Yes Yes 0.928 11b −5.23 1.659 98.699 −0.046 0.306 Yes Yes Yes No No Yes 0.777 11c −5.13 1.121 100 0.131 0.294 Yes Yes Yes Yes Yes Yes 0.905 13a −5.16 1.393 100 0.273 0.344 Yes Yes Yes No No No 1.068 13b −6.06 1.101 99.395 0.046 0.349 Yes Yes Yes No No No 0.046 13c −5.31 1.430 100 0.028 0.346 Yes Yes Yes No No No 1.006 15a −6.51 1.000 100 −0.464 0.335 No Yes Yes No No Yes 0.851 15b −7.43 0.974 100 −0.596 0.341 No No Yes No No No −0.026 Molecules 2024,29, 74 7 of 16 Molecules 2023, 28, x FOR PEER REVIEW 7 of 17 Additional factors to consider in drug development include metabolite formation mediated by cytochrome P450 (CYP) enzyme activities [36] and drug clearance (CL) [37]. CYP450 enzymes are a family of heme-containing enzymes that are involved in the metabolism of a wide range of endogenous compounds and xenobiotics. These enzymes play a crucial role in drug metabolism, and their activity can influence the efficacy and safety of many therapeutic agents. Compounds were studied as possible CYP2D6, CYP3A4, CYP1A2, CYP2C19 and CYP2C9 enzyme inhibitors. Noteworthy, all compounds show the ability to inhibit the CYP2C19 enzyme. Compounds with a methoxy group in their structure (5c, 9c and 11c) show inhibition of CYP2C9, which is primarily expressed in the liver and plays a critical role in the metabolism of numerous drugs. Conversely, the total clearance is primarily a combination of hepatic as well as renal clearance and is measured by the proportionality constant CLtot in log(mL/min/kg). The predicted value of all the synthesized compounds shows low CLtot ranging from −0.154 to 1.91. These results clearly indicate that all these compounds show appropriate pharmacokinetic properties and can be considered antileishmanial agents. The boiled egg model [38] visually represents some of the ADME parameters of the evaluated 1,5and 1,8-naphthyridines (Figure 3). Compounds that would cross both the blood–brain barrier (BBB) and human gastrointestinal tract (HIA) are placed in the yolk and those with only HIA-absorption in the white. Most of the tested compounds have positive BBB and HIA permeability, except compounds 8, 9a, 14a and 15a which only show positive HIA and compounds 9b–d and 15b which do not show results for either parameter. P-glycoprotein (PGP) is a transporter protein found in the membrane of cells in various tissues, such as the gastrointestinal tract, liver and kidneys [39,40]. Its main function is to transport foreign substances, such as drugs, out of cells. Therefore, it may be desirable for a drug to be a PGP substrate, as it can help protect against toxicity or unwanted side effects. Dots colored blue correspond to molecules that are predicted to be P-glycoprotein substrates (PGP+) and are therefore actively pumped from the brain or into the gastrointestinal lumen (Figure 3). If they are predicted not to be P-glycoprotein substrates (PGP−), the corresponding dot appears in red. In the case of our tested compounds, it should be noted that quinolino naphthyridine derivatives 8, 9, 14 and 15 and chromeno naphthyridine derivatives 7 and 13 do not qualify as PGP-substrate candidates. In contrast, all the other compounds, derivatives 4, 5, 6, 10, 11 and 12, are suitable as PGP substrate candidates. Figure 3. Boiled egg illustration of 1,5-naphthyridines (A) and 1,8-naphthyridines (B). The white region is the physicochemical space of molecules with the highest probability of being absorbed by the gastrointestinal tract, and the yellow region (yolk) is the physicochemical space of molecules with the highest probability of permeating into the brain. In the illustration, several compounds are represented with the same spot. Figure 3. Boiled egg illustration of 1,5-naphthyridines (A) and 1,8-naphthyridines (B). The white region is the physicochemical space of molecules with the highest probability of being absorbed by the gastrointestinal tract, and the yellow region (yolk) is the physicochemical space of molecules with the highest probability of permeating into the brain. In the illustration, several compounds are represented with the same spot. 2.3. Antileishmanial Effect The 1,5and 1,8-fused naphthyridine derivatives were evaluated as antileishmanial agents in assays using two forms of L. infantum amastigotes: axenic amastigotes recovered from bone marrow cells of infected BALB/c mice and intramacrophagic amastigotes obtained from splenic explants of the same subjects. The L. infantum strain used is a genetically modified strain previously engineered by our group to constitutively express the infrared protein iRFP from Rhodopseudomonas palustris bacteriophytochrome (iRFP L. infantum) [ 41 , 42 ]. This strain allows monitoring the viability of both forms of the parasite by quantifying the fluorescence emitted at 700 nm by the iRFP protein produced by living amastigotes [42]. Amastigotes obtained from mouse bone marrow cells were used to perform a first screening with the complete series of fused 1,5and 1,8-naphthyridines. Molecules that did not inhibit amastigote growth at a concentration of 25 µ M were considered to be poorly active and were discarded for subsequent assays on intramacrophagic amastigotes ex vivo. In the case of the tests carried out with intramacrophage amastigotes, the compounds that showed activity at a concentration lower than 20 µ M were selected to perform dose–response curves, obtaining their corresponding EC 50 values. The toxicity of these selected compounds was assessed in non-infected splenic cells and the cytotoxic concentration (CC 50 ) value obtained was used to calculate the selective index (SI). The oral tolerability of these same compounds was tested by calculating the viability of murine intestinal organoids exposed to two concentrations (50 and 25 µM). Table 3shows the results obtained with the chromeno[4,3-b][1,5]naphthyridines and chromeno[4,3-b][1,5]naphthyridines-6-one derivatives. Of these compounds, only two were candidates to be tested in intramacrophagic amastigotes (6b and 7c), obtaining EC 50 values of 12.86 ± 0.82 µ M and 36.99 ± 2.87 µ M, respectively. However, the SI of both molecules was not very high (4.3 and 2.6), and the toxicity in murine intestinal organoids was similar to that observed in mouse splenic macrophages cells. The replacement of the oxygen atom by nitrogen in these types of molecules produced more active compounds (Table 4). In this group, we found the more active and selective compounds such as molecules 8b and 8c, with EC 50 values of 5.53 ± 0.26 µ M and 4.93 ±0.35 µM , respectively, and SI values of >18.1 and >20.2. In the case of compound 8c the viability of murine intestinal organoids was of 100% at 50 µ M. Compound 9b has the peculiarity of being less toxic in murine intestinal organoids than in splenic cells, obtaining an SI of only 4.6 while the viability in organoids was of 100% at 50 µ M. This result could be related to the negative HIA permeability (Figure 3) of this compound. Another point to highlight among the compounds of this group is the greater activity of compounds that lack the aromatic naphthyridine ring versus those that have the aromaticity. Molecules 2024,29, 74 8 of 16 Table 3. Biological activity of chromeno[4,3-b][1,5]naphthyridines and chromeno[4,3-b][1,5]naphthyridine6-ones. The antileishmanial effect was evaluated in axenic IRFP-L.infantum amastigotes from bone marrow cells and subsequently in intramacrophagic IRFP-L.infantum from murine spleens. Cytotoxicity was assessed in uninfected splenic macrophages and intestinal tolerance in murine gut organoids. Molecules 2023, 28, x FOR PEER REVIEW 8 of 17 2.3. Antileishmanial Effect The 1,5and 1,8-fused naphthyridine derivatives were evaluated as antileishmanial agents in assays using two forms of L. infantum amastigotes: axenic amastigotes recovered from bone marrow cells of infected BALB/c mice and intramacrophagic amastigotes obtained from splenic explants of the same subjects. The L. infantum strain used is a genetically modified strain previously engineered by our group to constitutively express the infrared protein iRFP from Rhodopseudomonas palustris bacteriophytochrome (iRFP L. infantum) [41,42]. This strain allows monitoring the viability of both forms of the parasite by quantifying the fluorescence emitted at 700 nm by the iRFP protein produced by living amastigotes [42]. Amastigotes obtained from mouse bone marrow cells were used to perform a first screening with the complete series of fused 1,5and 1,8-naphthyridines. Molecules that did not inhibit amastigote growth at a concentration of 25 µM were considered to be poorly active and were discarded for subsequent assays on intramacrophagic amastigotes ex vivo. In the case of the tests carried out with intramacrophage amastigotes, the compounds that showed activity at a concentration lower than 20 µM were selected to perform dose–response curves, obtaining their corresponding EC50 values. The toxicity of these selected compounds was assessed in non-infected splenic cells and the cytotoxic concentration (CC50) value obtained was used to calculate the selective index (SI). The oral tolerability of these same compounds was tested by calculating the viability of murine intestinal organoids exposed to two concentrations (50 and 25 µM). Table 3 shows the results obtained with the chromeno[4,3-b][1,5]naphthyridines and chromeno[4,3-b][1,5]naphthyridines-6-one derivatives. Table 3. Biological activity of chromeno[4,3-b][1,5]naphthyridines and chromeno[4,3-b][1,5]naphthyridine-6-ones. The antileishmanial effect was evaluated in axenic IRFP-L.infantum amastigotes from bone marrow cells and subsequently in intramacrophagic IRFP-L.infantum from murine spleens. Cytotoxicity was assessed in uninfected splenic macrophages and intestinal tolerance in murine gut organoids. Compound R1 Axenic Amastigotes a Intracellular Amastigotes EC50 (µM) Mouse Splenic Macrophages CC50 (µM) SI Intestinal Organoids 50 µM (%Viability) 25 µM (%Viability) 4a H >25 n.d.* n.d - 4c OMe >25 n.d. n.d. - 5a H >25 n.d. n.d. - 5b Br >25 n.d. n.d. - 5c OMe >25 n.d. n.d. - 6a H >25 n.d. n.d. - 6b Br <25 12.86 ± 0.82 55.66 ± 6.42 4.3 60 ± 7 72 ± 5 6c OMe >25 n.d. n.d. - 7a H >25 n.d. n.d. - 7b Br >25 n.d. n.d. - 7c OMe <25 36.99 ± 2.87 97.68 ± 1.77 2.6 85 ± 13 99 ± 4 * n.d. not determined. a Compounds with inhibition at concentrations higher than 25 µM were considered not very active and were discarded for subsequent assays. Compound R1Axenic Amastigotes a Intracellular Amastigotes EC50 (µM) Mouse Splenic Macrophages CC50 (µM) SI Intestinal Organoids 50 µM (%Viability) 25 µM (%Viability) 4a H >25 n.d.* n.d - 4c OMe >25 n.d. n.d. - 5a H >25 n.d. n.d. - 5b Br >25 n.d. n.d. - 5c OMe >25 n.d. n.d. - 6a H >25 n.d. n.d. - 6b Br <25 12.86 ±0.82 55.66 ±6.42 4.3 60 ±7 72 ±5 6c OMe >25 n.d. n.d. - 7a H >25 n.d. n.d. - 7b Br >25 n.d. n.d. - 7c OMe <25 36.99 ±2.87 97.68 ±1.77 2.6 85 ±13 99 ±4 * n.d. not determined. a Compounds with inhibition at concentrations higher than 25 µ M were considered not very active and were discarded for subsequent assays. Table 4. Biological activity of quinolino[4,3-b][1,5]naphthyridines. The antileishmanial effect was evaluated in axenic IRFP-L.infantum amastigotes from bone marrow cells and subsequently in intramacrophagic IRFP-L. infantum from murine spleens. Cytotoxicity was assessed in uninfected splenic macrophages and intestinal tolerance in murine gut organoids. Molecules 2023, 28, x FOR PEER REVIEW 9 of 17 Of these compounds, only two were candidates to be tested in intramacrophagic amastigotes (6b and 7c), obtaining EC50 values of 12.86 ± 0.82 µM and 36.99 ± 2.87 µM, respectively. However, the SI of both molecules was not very high (4.3 and 2.6), and the toxicity in murine intestinal organoids was similar to that observed in mouse splenic macrophages cells. The replacement of the oxygen atom by nitrogen in these types of molecules produced more active compounds (Table 4). In this group, we found the more active and selective compounds such as molecules 8b and 8c, with EC50 values of 5.53 ± 0.26 µM and 4.93 ± 0.35 µM, respectively, and SI values of >18.1 and >20.2. In the case of compound 8c the viability of murine intestinal organoids was of 100% at 50 µM. Compound 9b has the peculiarity of being less toxic in murine intestinal organoids than in splenic cells, obtaining an SI of only 4.6 while the viability in organoids was of 100% at 50 µM. This result could be related to the negative HIA permeability (Figure 3) of this compound. Another point to highlight among the compounds of this group is the greater activity of compounds that lack the aromatic naphthyridine ring versus those that have the aromaticity. Table 4. Biological activity of quinolino[4,3-b][1,5]naphthyridines. The antileishmanial effect was evaluated in axenic IRFP-L.infantum amastigotes from bone marrow cells and subsequently in intramacrophagic IRFP-L. infantum from murine spleens. Cytotoxicity was assessed in uninfected splenic macrophages and intestinal tolerance in murine gut organoids. Compound R1 Axenic Amastigotes a Intracelullar Amastigotes EC50 (µM) Mouse Splenic Macrophages CC50 (µM) * SI Intestinal Organoids 50 µM (%Viability) 25 µM (%Viability) 8a H <25 5.30 ± 0.71 45.03 ± 3.91 8.5 89 ± 10 93 ± 7 8b 4-Br <25 5.53 ± 0.26 >100 >18.1 98 ± 13 >100 8c 6-OMe <25 4.93 ± 0.35 >100 >20.28 >100 >100 8d 6-Br <25 34.36 ± 9.70 >50 1.5 3 ± 1 4 ± 1 9a H >25 n.d. n.d. - 9b 4-Br <25 3.63 ± 0.15 16.89 ± 0.86 4.6 >100 >100 9c 6-OMe >25 >20 n.d. - 9d 6-Br >25 >20 n.d. - * n.d. not determined. a Compounds with inhibition at concentrations higher than 25 µM were considered not very active and were discarded for subsequent assays. Four chromeno[4,3-b][1,8]naphthyridines and chromeno[4,3-b][1,8]naphthyridine-6one derivatives were tested in intramacrophagic amastigotes (10b, 11a, 13a and 13b, Table 5). Of these four, the highest SI was obtained with 11a (EC50 = 7.70 ± 0.29 µM; SI ≥ 13). Compound 10b was less toxic in intestinal organoids than in mouse splenic macrophages, showing 90.04% of viability in organoids at 50 µM against of a CC50 value of 50.72 ± 0.99 µM in mouse splenic macrophages. The in silico predictions for this compound have not identified problems relative to its HIA absorption. Contrary to 1,5-naphthyridines (Table 4) compounds in this group were more active with the aromatic naphthyridine ring. Compound R1Axenic Amastigotes a Intracelullar Amastigotes EC50 (µM) Mouse Splenic Macrophages CC50 (µM) * SI Intestinal Organoids 50 µM (%Viability) 25 µM (%Viability) 8a H <25 5.30 ±0.71 45.03 ±3.91 8.5 89 ±10 93 ±7 8b 4-Br <25 5.53 ±0.26 >100 >18.1 98 ±13 >100 8c 6-OMe <25 4.93 ±0.35 >100 >20.28 >100 >100 8d 6-Br <25 34.36 ±9.70 >50 1.5 3 ±1 4 ±1 9a H >25 n.d. n.d. - 9b 4-Br <25 3.63 ±0.15 16.89 ±0.86 4.6 >100 >100 9c 6-OMe >25 >20 n.d. - 9d 6-Br >25 >20 n.d. - * n.d. not determined. a Compounds with inhibition at concentrations higher than 25 µ M were considered not very active and were discarded for subsequent assays. Four chromeno[4,3-b][1,8]naphthyridines and chromeno[4,3-b][1,8]naphthyridine-6one derivatives were tested in intramacrophagic amastigotes (10b,11a,13a and 13b, Table 5). Of these four, the highest SI was obtained with 11a (EC 50 = 7.70 ± 0.29 µ M; SI ≥ 13). Compound 10b was less toxic in intestinal organoids than in mouse splenic macrophages, show- Molecules 2024,29, 74 9 of 16 ing 90.04% of viability in organoids at 50 µ M against of a CC 50 value of 50.72 ±0.99 µM in mouse splenic macrophages. The in silico predictions for this compound have not identified problems relative to its HIA absorption. Contrary to 1,5-naphthyridines (Table 4) compounds in this group were more active with the aromatic naphthyridine ring. Table 5. Biological activity of chromeno[4,3-b][1,8]naphthyridines and chromeno[4,3-b][1,8]naphthyridine6-ones. The antileishmanial effect was evaluated in axenic IRFP-L.infantum amastigotes from bone marrow cells and subsequently in intramacrophagic IRFP-L.infantum from murine spleens. Cytotoxicity was assessed in uninfected splenic macrophages and intestinal tolerance in murine gut organoids. Molecules 2023, 28, x FOR PEER REVIEW 10 of 17 Table 5. Biological activity of chromeno[4,3-b][1,8]naphthyridines and chromeno[4,3-b][1,8]naphthyridine-6-ones. The antileishmanial effect was evaluated in axenic IRFP-L.infantum amastigotes from bone marrow cells and subsequently in intramacrophagic IRFP-L.infantum from murine spleens. Cytotoxicity was assessed in uninfected splenic macrophages and intestinal tolerance in murine gut organoids. Compound R1 R2 R3 Axenic Amastigo tes a Intracelullar Amastigotes EC50 (µM) Mouse splenic Macrophages CC50 (µM) * SI Intestinal Organoids 50 µM (%Viability) 25 µM (%Viability) 10a - H H >25 >20 n.d. - 10b - F H <25 7.57 ± 0.44 50.72 ± 0.99 6.7 90 ± 22 99 ± 8 10c - Me H >25 >20 n.d. - 11a - H H <25 7.70 ± 0.29 >100 >13 96 ± 1 92 ± 1 11b - F H >25 >20 n.d. - 11c - H OMe >25 >20 n.d. - 12a H H - >25 >20 n.d. - 12b Br H - >25 >20 n.d. - 13a H H - <25 10.58 ± 1.21 34.65 ± 1.79 3.3 53 ± 5 87 ± 15 13b Br H - <25 7.53 ± 0.97 >50 >6.6 61 ± 19 90 ± 5 13c H F - >25 >20 n.d. - * n.d. not determined. a Compounds with inhibition at concentrations higher than 25 µM were considered not very active and were discarded for subsequent assays. The three available 1,8-derivatives with nitrogen atoms instead of oxygen were tested in intramacrophagic amastigotes (Table 6). However, their SI was low and the toxicity in organoids was similar than obtained with splenic cells. Table 6. Biological activity of quinolino[4,3-b][1,8] naphthyridines. The antileishmanial effect was evaluated in axenic IRFP-L.infantum amastigotes from bone marrow cells and subsequently in intramacrophagic IRFP-L.infantum from murine spleens. Cytotoxicity was assessed in uninfected splenic macrophages and intestinal tolerance in murine gut organoids. Compound R1 Axenic Amastigotes a Intracelullar Amastigotes EC50 (µM) Mouse Splenic Macrophages CC50 (µM) SI Intestinal Organoids 50 µM (%Viability) 25 µM (%Viability) 14a H <25 9.34 ± 0.77 27.19 ± 1.55 2.9 56 ± 14 90 ± 9 15a H <25 67.43 ± 9.78 8.78 ± 2.21 0.1 15b Br <25 6.16 ± 0.54 24.64 ± 3.59 4.0 13 ± 3 58 ± 16 a Compounds with inhibition at concentrations higher than 25 µM were considered not very active and were discarded for subsequent assays. Compound R1R2R3Axenic Amastigotes a Intracelullar Amastigotes EC50 (µM) Mouse Splenic Macrophages CC50 (µM) * SI Intestinal Organoids 50 µM (%Viability) 25 µM (%Viability) 10a - H H >25 >20 n.d. - 10b - F H <25 7.57 ±0.44 50.72 ±0.99 6.7 90 ±22 99 ±8 10c - Me H >25 >20 n.d. - 11a - H H <25 7.70 ±0.29 >100 >13 96 ±1 92 ±1 11b - F H >25 >20 n.d. - 11c - H OMe >25 >20 n.d. - 12a H H - >25 >20 n.d. - 12b Br H - >25 >20 n.d. - 13a H H - <25 10.58 ±1.21 34.65 ±1.79 3.3 53 ±5 87 ±15 13b Br H - <25 7.53 ±0.97 >50 >6.6 61 ±19 90 ±5 13c H F - >25 >20 n.d. - * n.d. not determined. a Compounds with inhibition at concentrations higher than 25 µ M were considered not very active and were discarded for subsequent assays. The three available 1,8-derivatives with nitrogen atoms instead of oxygen were tested in intramacrophagic amastigotes (Table 6). However, their SI was low and the toxicity in organoids was similar than obtained with splenic cells. Table 6. Biological activity of quinolino[4,3-b][1,8] naphthyridines. The antileishmanial effect was evaluated in axenic IRFP-L.infantum amastigotes from bone marrow cells and subsequently in intramacrophagic IRFP-L.infantum from murine spleens. Cytotoxicity was assessed in uninfected splenic macrophages and intestinal tolerance in murine gut organoids. Molecules 2023, 28, x FOR PEER REVIEW 10 of 17 Table 5. Biological activity of chromeno[4,3-b][1,8]naphthyridines and chromeno[4,3-b][1,8]naphthyridine-6-ones. The antileishmanial effect was evaluated in axenic IRFP-L.infantum amastigotes from bone marrow cells and subsequently in intramacrophagic IRFP-L.infantum from murine spleens. Cytotoxicity was assessed in uninfected splenic macrophages and intestinal tolerance in murine gut organoids. Compound R1 R2 R3 Axenic Amastigo tes a Intracelullar Amastigotes EC50 (µM) Mouse splenic Macrophages CC50 (µM) * SI Intestinal Organoids 50 µM (%Viability) 25 µM (%Viability) 10a - H H >25 >20 n.d. - 10b - F H <25 7.57 ± 0.44 50.72 ± 0.99 6.7 90 ± 22 99 ± 8 10c - Me H >25 >20 n.d. - 11a - H H <25 7.70 ± 0.29 >100 >13 96 ± 1 92 ± 1 11b - F H >25 >20 n.d. - 11c - H OMe >25 >20 n.d. - 12a H H - >25 >20 n.d. - 12b Br H - >25 >20 n.d. - 13a H H - <25 10.58 ± 1.21 34.65 ± 1.79 3.3 53 ± 5 87 ± 15 13b Br H - <25 7.53 ± 0.97 >50 >6.6 61 ± 19 90 ± 5 13c H F - >25 >20 n.d. - * n.d. not determined. a Compounds with inhibition at concentrations higher than 25 µM were considered not very active and were discarded for subsequent assays. The three available 1,8-derivatives with nitrogen atoms instead of oxygen were tested in intramacrophagic amastigotes (Table 6). However, their SI was low and the toxicity in organoids was similar than obtained with splenic cells. Table 6. Biological activity of quinolino[4,3-b][1,8] naphthyridines. The antileishmanial effect was evaluated in axenic IRFP-L.infantum amastigotes from bone marrow cells and subsequently in intramacrophagic IRFP-L.infantum from murine spleens. Cytotoxicity was assessed in uninfected splenic macrophages and intestinal tolerance in murine gut organoids. Compound R1 Axenic Amastigotes a Intracelullar Amastigotes EC50 (µM) Mouse Splenic Macrophages CC50 (µM) SI Intestinal Organoids 50 µM (%Viability) 25 µM (%Viability) 14a H <25 9.34 ± 0.77 27.19 ± 1.55 2.9 56 ± 14 90 ± 9 15a H <25 67.43 ± 9.78 8.78 ± 2.21 0.1 15b Br <25 6.16 ± 0.54 24.64 ± 3.59 4.0 13 ± 3 58 ± 16 a Compounds with inhibition at concentrations higher than 25 µM were considered not very active and were discarded for subsequent assays. Compound R1Axenic Amastigotes a Intracelullar Amastigotes EC50 (µM) Mouse Splenic Macrophages CC50 (µM) SI Intestinal Organoids 50 µM (%Viability) 25 µM (%Viability) 14a H <25 9.34 ±0.77 27.19 ±1.55 2.9 56 ±14 90 ±9 15a H <25 67.43 ±9.78 8.78 ±2.21 0.1 15b Br <25 6.16 ±0.54 24.64 ±3.59 4.0 13 ±3 58 ±16 a Compounds with inhibition at concentrations higher than 25 µ M were considered not very active and were discarded for subsequent assays. Molecules 2024,29, 74 16 of 16 42. Calvo-Álvarez, E.; Stamatakis, K.; Punzón, C.; Álvarez-Velilla, R.; Tejería, A.; Escudero-Martínez, J.M.; Pérez-Pertejo, Y.; Fresno, M.; Balaña-Fouce, R.; Reguera, R.M. Infrared fluorescent imaging as a potent tool for in vitro , ex vivo and in vivo models of visceral leishmaniasis. PLoS Negl. Trop. Dis. 2015,9, e0003666. [CrossRef] 43. Kandepedu, N.; Gonzàlez Cabrera, D.; Eedubilli, S.; Taylor, D.; Brunschwig, C.; Gibhard, L.; Njoroge, M.; Lawrence, N.; Paquet, T.; Eyermann, C.J.; et al. Identification, Characterization, and optimization of 2,8-disubstituted-1,5-naphthyridines as novel Plasmodium falciparum phosphatidylinositol-4-kinase inhibitors with in vivo efficacy in a humanized mouse model of malaria. J. Med. Chem. 2018,12, 5692–5703. [CrossRef] [PubMed] 44. Araújo-Neto, J.B.; Silva, M.; Oliveira-Tintino, C.D.; Begnini, I.M.; Rebelo, R.A.; Silva, L.E.; Mireski, S.L.; Nasato, M.C.; Krautler, M.I.; Ribeiro-Filho, J.; et al. Enhancement of antibiotic activity by 1,8-naphthyridine derivatives against multi-resistant bacterial strains. Molecules 2021,26, 7400. [CrossRef] [PubMed] 45. Mithula, S.; Nandikolla, A.; Murugesan, S.; Kondapalli, V.G. 1,8-naphthyridine derivatives: An updated review on recent advancements of their myriad biological activities. Future Med. Chem. 2021,13, 1591–1618. [CrossRef] [PubMed] 46. Madaan, A.; Verma, R.; Kumar, V.; Singh, A.T.; Jain, S.K.; Jaggi, M. 1,8-Naphthyridine Derivatives: A Review of Multiple Biological Activities. Arch. Pharm. 2015,348, 837–860. [CrossRef] [PubMed] 47. Sriram, D.; Senthilkumar, P.; Dinakaran, M.; Yogeeswari, P.; China, A.; Nagaraja, V. Antimycobacterial activities of novel 1-(cyclopropyl/tert-butyl/4-fluorophenyl)-1,4-dihydro6-nitro-4-oxo-7-(substituted secondary amino)-1,8-naphthyridine-3carboxylic acid. J. Med. Chem. 2007,50, 6232–6239. [CrossRef] [PubMed] 48. Tsuzuki, Y.; Tomita, K.; Sato, Y.; Kashimoto, S.; Chiba, K. Synthesis and structure-activity relationships of 3-substituted 1,4dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridines as novel antitumor agents. Bioorg. Med. Chem. Lett. 2004,14, 3189–3193. [CrossRef] 49. Eweas, A.F.; Khalifa, N.M.; Ismail, N.S.; Al-Omar, M.A.; Soliman, A.M.M. Synthesis, molecular docking of novel 1,8-naphthyridine derivatives and their cytotoxic activity against HepG2 cell lines. Med. Chem. Res. 2014,23, 76–86. [CrossRef] 50. Abuzahra, M.M.; Ahmed, N.S.; Sarhan, M.O.; Mahgoub, S.; Abdelhameed, A.; Zaghary, W.A. Novel substituted 1,8naphthyridines: Design, synthesis, radiolabeling, and evaluation of apoptosis and topoisomerase II inhibition. Arch. Pharm. 2023, 356, e2300035. [CrossRef] 51. Nandikolla, A.; Srinivasarao, S.; Karan Kumar, B.; Murugesan, S.; Aggarwal, H.; Balaña-Fouce, R.; Melcón-Fernández, E.; Pérez-Pertejo, Y.; Chandra Sekhar, K.V.G. Novel phenanthridine amide analogs as potential anti-leishmanial agents: In vitro and in silico insights. Bioorg. Chem. 2021,117, 105414. [CrossRef] 52. Du, Y.; Li, X.; Niu, Q.; Mo, X.; Qui, M.; Ma, T.; Kuo, C.J.; Fu, H. Development of a miniaturized 3D organoid culture platform for ultra-high-throughput screening. J. Mol. Cell. Biol. 2020,12, 630–643. [CrossRef] 53. SwissADME Webpage. Available online: http://www.swissadme.ch/ (accessed on 25 October 2023). 54. pkCSM—Pharmacokinetics Webpage. Available online: http://biosig.unimelb.edu.au/pkcsm/ (accessed on 25 October 2023). Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.