Optimization of 4‐amino‐pyridazin‐3(2H)‐one as a valid core scaffold for FABP4 inhibitors

Current clinical research suggests that fatty acid‐binding protein 4 inhibitors (FABP4is), which are of biological and therapeutic interest, may show potential in treating cancer and other illnesses. We sought to uncover new structures through the optimization of the previously reported 4‐amino and 4‐ureido pyridazinone‐based series of FABP4is as part of a larger research effort to create more potent FABP4 inhibitors. This led to the identification of 14e as the most potent analog with IC50 = 1.57 μM, which is lower than the IC50 of the positive control. Advanced modeling investigations and in silico absorption, distribution, metabolism, and excretion ‐ toxicity calculations suggested that 14e represents a potential candidate for in vivo studies such as FABP4i.


| INTRODUCTION
Organic carboxylic acids with a long carbon chain known as fatty acids (FAs) have several roles in the body. [1,2]Their persistently high quantity in the bloodstream causes a variety of diseases, [3,4] including atherosclerosis, diabetes, and obesity.FAs are insoluble in water, due to the high lipophilicity of their chemical structure, and they must be transported into biological fluids by specific carriers so-called fatty acid-binding proteins (FABPs). [5]Based on the location where they are found in the human body, FABPs have been classified into various families: A-FABP (adipocyte), B-FABP (brain), E-FABP (epidermal), H-FABP (muscle and heart), I-FABP (intestinal), Il-FABP (ileal), L-FABP (liver), M-FABP (myelin), and T-FABP (testis).FABP4 (aP2 or A-FABP) is the subtype predominantly expressed in adipocytes.When it was revealed that FABP4 knockout animal models showed protective effects against the development of insulin resistance as well as several pathological events linked to metabolic syndrome and atherosclerosis, research into small molecule inhibitors for this protein was first launched.Significantly, pharmacological therapies based on compounds that block the FABP4's normal activity are equally effective in this regard, showing comparable outcomes to genetic methods by simulating the phenotype of FABP4-deficient animals. [6]10] Moreover, it was recently reported that FABP4 facilitates colon cancer metastasis and invasion and that administration of a conventional FABP4 small molecule inhibitor (BMS309403) reduced colon cancer cells' ability to migrate and invade. [11]Additionally, FABP4 causes irregular metastatic patterns in ovarian cancer, and new studies show that the protein plays a role in the aggressiveness of this type of tumor, generating complications for its prognosis and treatment. [12]Overall, this new body of evidence in the context of cancer research suggests that FABP4 targeting could offer new opportunities in the treatment of oncological conditions, in addition to the recognized effects on metabolic and cardiovascular conditions.
Nevertheless, there are no FABP4 inhibitors (FABP4is) under clinical investigation at the present time. [6,13]6] In line with our research focus in the discovery of novel bioactive heterocycles and the development of new anticancer agents, [17,18] in this article, we describe the design, synthesis, and in vitro testing of novel heterocyclic small-molecule series as FABP4is, derived from the structural optimization of our recently reported 4-amino and 4-ureido pyridazinonebased inhibitors. [19]| RESULTS AND DISCUSSION 2.1 | Chemistry

| Heterocyclic small-molecule design
As shown in Figure 1, recently we took use of a two-step computationally assisted molecular design to find novel FABP4i scaffolds starting from the bioisosteric-replacement/scaffold-hopping of the pyrimidine core of the cocrystallized ligand (i.e., 2-[(2-oxo-2-piperidin-1-ylethyl)sulfanyl]-6-(trifluoromethyl)pyrimidin-4-ol; PBDID: 1TOU).Our examination of bioisosteric replacement resulted in the choice of 4-amino and 4-ureido pyridazinone as a suitable scaffold to develop a series of FABP4is with low micromolar activities. [19]In this work we have further optimized the structure of the identified scaffold, leading to new inhibitors with increased potency.We have carried out a scaffold hopping analysis as reported in Figure 1 and then scored the compounds with Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) calculations, as described in the section (see Supporting Information Material for details of the selection criteria for the in vitro evaluation of the compounds).This approach has enabled the selection of 25 target molecules.The compounds were then synthesized and the 13 predicted as most active (see Supporting Information: Table S1) were screened against FABP4 in vitro.The chemical structures of the new series of compounds are reported in Tables 1 and 2.

| Chemistry
All intermediates and final compounds were synthesized as reported in Schemes 1-3 and the structure were confirmed on the basis of analytical and spectral data.
Scheme 1 shows the synthetic pathways affording the final products of type 3, 6, and 7, in which position 2, 4, 5, and 6 of pyridazinone were suitably modified to introduce selected residues of interest.For the synthesis of compounds 3a-h, intermediates 1a-c [20][21][22] were reacted with cerium ammonium nitrate (CAN) in a mixture of 50% AcOH and 65% HNO 3 , adopting a previously established procedure, [23] affording intermediates 2a,b [22,24] and the new 2c.The displacement of the nitro group with suitable alkyl-or heteroarylamine lead to the desired final compounds of type 3.For the synthesis of final compounds 7a,b, the intermediate 1c [22] was converted into compound 4 adopting previously established reduction conditions with ammonium formate and 10% Pd/C in EtOH. [25]bsequently, the treatment of 4 with bromine and 47% HBr in glacial acetic acid afforded intermediate 5, which was reacted with thioacetamide in ethanol to give the final compound 6.Compounds Intermediate 8 [26] was treated with the suitable alcoholate to give the isoxazolopyridazines 9a,b (for 9b [26] ).The reductive cleavage with 10% Pd/C in Parr instrument at 60 Psi gave 4-amino-5-acetyl derivatives 10a,b, which were in turn coupled with 3-chlorophenylboronic acid following the same conditions previously described for 7a,b.Scheme 3 depicts the synthetic procedures adopted to obtain the final products 14a-h, 15, 16a,b, and 17, showing various functional groups in position 4 of the pyridazinone.Through condensation with the appropriate aromatic aldehyde in the presence of KOH, dihydropyridazinones 12a,b [27] were firstly converted into the previously described 4-benzyl derivatives 13a-g [28][29][30][31][32] and in the new analogs 13h-j.Subsequently, compounds of type 13 were finally alkylated with ethyl bromide in anhydrous dimethylformamide (DMF) under standard conditions to give final compounds 14a-h and 16a,b.
For 16a,b, these reaction conditions produced also the esterification of the carboxylic group in the para (13i) or ortho (13j) position of the benzyl group, simultaneously to the alkylation of N-1 on the pyridazinone.Lastly, compound 15 was obtained from intermediate 14c through treatment with Lawesson's reagent in anhydrous toluene, while the hydrolysis of 16a with 6 N NaOH in EtOH afforded the final compound 17.

| FABP4 inhibition evaluation
The reduction in fluorescence signal of when a powerful FABP4 ligand displaces a detection reagent (DR), was used to measure the FABP4 inhibitory activity.When bound to FABP4, the DR displays a higher fluorescence intensity.Therefore, a decrease in the fluorescence read-out is caused by any ligand that can successfully bind to the same binding pocket and has the ability to displace the DR.The new molecular series was examined in two steps.First, an assessment of the inhibitory effect of all the compounds was obtained using a single dose of 5 µM.Then, only the substances that could lower the fluorescence reading by at least 95% were assessed further, by measuring the IC 50 values (µM), which were then contrasted with the arachidonic acid's activity (i.e., FABP4 established ligand).Figure 2 reports the single-point displacement results.Based on the data of the first screening, four molecules were selected as the analogs with higher efficacy-that is, able to reduce the fluorescence of the DR to at least 95%, worth to be progressed to IC 50 calculation.In IC 50 experiments, using arachidonic acid as a positive control, IC 50 of 3.30 µM was obtained.Table 3 lists the IC 50 values for the compounds in our set.Molecule 14e revealed a strong inhibitory effect, with an IC 50 value (i.e., 1.57 µM) value lower than the standard arachidonic acid and lower than the previous FABP4i that we had found (compound A, [19] Figure 1).Furthermore, these interactions may be mediated by a network of water molecules.Figure 3 shows the interactions for the most active compounds identified in this study-that is, 3e, 11a, 14c, and 14e (two-dimensional [2D] interactions are reported in the Supporting Information: Figures S1-S4).Each compound has many interactions with the appropriate residues in the binding pocket, for instance, Y128 and R106.As shown in the Figure 3, compounds 3e, 14c, and 14e prefer a binding pose where the carbonyl of the central core interacts with R126 and the other aromatic rings are allocated in the additional hydrophobic pockets, in a binding pose similar to that of the starting reference compound (Figure 1).In contrast, analog 11a does not interact with R126, rather a hydrogen bond is formed between this analog and S53 (See Supporting Information: Figure S2).
This missing interaction with R126 may explain the compound's decreased activity, as seen by the lower binding energy interaction.
The stability over time of the most potent molecule was further investigated by molecular dynamic (MD) experiments.One hundred nanoseconds of simulation were run in explicit water at neutral pH (7.4)   as described in the experimental part.The results of the simulation confirmed that molecule 14e is able to maintain the binding poses over time, as determined by the calculated root mean square deviation (RMSD) that maintain a constant level (Figure 4), after the initial stabilization of the starting structure (0 ns).As expected, the protein structure is also not influenced by the binding of molecule 14e, confirming a certain degree of stability of the protein-14e complex (see Supporting Information: Figure S5).Using FABP3 crystallized protein, [33] the possible activity of our compounds against FABP3 was investigated through docking studies.
To validate the calculation of the binding affinity we used five compounds as reference for which the experimental K i toward FABP3 was reported. [34]The results have been reported in Supporting Information: Table S4, from which it is possible to deduce that our compounds 3e, 11a, 14c, and 14e have very low activity against FABP3.
By examining pharmacokinetic profiles and potential negative side effects of 14e, the in silico assessment has been enhanced.
green), 14c (orange), and 14e (yellow) poses inside the binding pocket of fatty acid-binding protein 4 (FABP4) compared with the reference compound (blue; see Figure 1 for molecular structure).
Table S3.From these experiments, the compound results orally available and moderately soluble in water.and is a substrate of the renal organic cation transporter 2. Lastly, the compound did not pass the AMED toxicity test (and it could be hepatotoxic), whereas no skin senitization is recorded.The max tolerated human dose is 0.328 (log mg/kg/day).
After neutralization with acetic acid, the mixture was concentrated to a reduced volume.After cooling, the precipitate was recovered by suction and recrystallized from ethanol.Yield = 85%; mp > 300°C (EtOH).

| General procedure for the synthesis of compounds 13h-j
The suitable 2-or 4-substituted benzaldehyde (0.90 mmol) was added to a solution of 12a,b [27] (0.90 mmol) in 3.5 mL of KOH 5% (w/v) in absolute EtOH and the mixture was refluxed under stirring for 1-2 h.After cooling, the sample was concentrated in vacuo, diluted with ice-cold water (10-15 mL) and acidified with 2 N HCl.
For compounds 14a, 14d, and 14 g, the suspension was extracted with CH 2 Cl 2 (3 × 10 mL) and, after evaporation of the solvent, the final compounds were crystallized from ethanol. the PM3 Hamiltonian, [36] as implemented in MOPAC 2016 package assuming a pH of 7.0. [37]Once built and optimized, all structures were used in the bioisostere replacement tool Spark 10.4.0. [36,38]][41][42] Using AutoDock's default docking parameters and a validated protocol, docking calculations were performed. [40]The setup was done with YASARA.The Lamarckian genetic algorithm implemented in AutoDock was used for the calculations.The ligand-centered maps were generated by AutoGrid with a spacing of 0.375 Å and dimensions that encompass all atoms extending 5 Å from the surface of the ligand.All of the parameters were inserted at their default settings.PDBid: 1TOU and 3WBG were downloaded from the Protein Data Bank (www.rcsb.org),and used for the calculations.MM/PBSA rescoring procedures were obtained by using fastDRH as open access web server (http://cadd.zju.edu.cn/fastdrh/overview, accessed on December 05, 2022). [43]The molecular dynamics simulations of the complexes were performed with the YASARA.A periodic simulation cell extending 10 Å from the surface of the protein was employed.The cell was filled with water, with a maximum sum of all water bumps of 1.0 Å and a density of 0.997 g/mL.
The setup included optimizing the hydrogen bonding network [44] to increase the solute stability and a pK a prediction to fine-tune the protonation states of protein residues at the chosen pH of 7.4. [45]With an excess of either Na or Cl to neutralize the cell, NaCl ions were supplied at a physiological concentration of 0.9%.The simulation was run using the ff14SB force field [46] for the solute, GAFF2, [47] AM1BCC [48] for ligands, and TIP3P for water.The cutoff was 10 Å for Van der Waals forces (the default used by AMBER), [49] and no cutoff was applied to electrostatic forces (using the Particle Mesh Ewald algorithm). [50]The equations of motions were integrated with multiple time steps of 2.5 fs for bonded interactions and 5.0 fs for nonbonded interactions at a temperature of 298 K and a pressure of 1 atm using algorithms described in detail previously. [51]Short MD simulation was run on the solvent only to remove clashes.The entire system was then energy minimized using a steepest descent minimization to remove conformational stress, followed by a simulated annealing minimization until convergence (<0.01 kcal/ mol Å).Finally, 100 ns MD simulation without any restrictions was conducted, and the conformations were recorded every 200 ps.
7a,b were then obtained from 6 through coupling reaction with the appropriate aryl boronic acid in the presence of dry cupric acetate and triethylamine in anhydrous CH 2 Cl 2 .In Scheme 2 is reported the synthesis of the final compounds 11a,b featuring a pyridazine scaffold in the place of the pyridazinone.F I G U R E 1 Computer-aided design of the new scaffolds shown schematically.Y = O, S. X = NH, CH 2 .

FLORESTA ET AL. | 5 of 14 2. 3 |
Molecular docking and ADMET predictionSince the first apo-FABP crystal structure was published in 1992, numerous different holo-FABP structures with a variety of ligands have been discovered.It is now established, that the side chains of the FABP hydrophobic pocket form a hydrogen bond with the carboxylate of the FAs toward various amino acidic residues.
14e resulted as a Pgp substrate and no violation to the Lipinski rule of 5 is reported.Other four drug-likeness rules named Ghose, Egan, Veber, and Muegee, were simultaneously satisfied.The result of the PAINS interference structures model assay, that was designed to exclude molecules that are most likely to show false positives in biological assays, did not point out any inconsistency.The calculated absorption and distribution have been graphically represented by the Edan-Egg model reported in Supporting Information: FigureS10(Brain or IntestinaL EstimateD, BOILED-Egg).The visual analysis of the Edan-Egg model highlights that 14e was predicted to passively permeate the blood-brain barrier.Regarding the absorption parameters, the compound presents a promising oral availability due to the optimal Caco-2 cell permeability and intestinal absorption.Additionally, the molecule is anticipated to be available to interact with the pharmaceutical target because of the plasma's high unbound proportion.The calculated value of total clearance indicates that 14e has a good renal elimination was directed by bioisosteric-replacements/scaffold hopping and evaluated by MM/GBSA calculations.Twenty-five new best-scoring molecules have been synthesized and the most active hits were further biologically evaluated for their FABP4 inhibitory activity.The structural optimization of our previous series led to the identification of several new analogs (e.g., 3e, 11a, 14c, 14e) with higher FABP4 inhibitory activity (i.e., IC 50 in the low micromolar range) than the former compounds.In particular, 14e resulted in the most potent analog, with IC 50 = 1.57μM, which is lower than the IC 50 of the positive control (arachidonic acid, IC 50 = 3.30 µM).MD experiments confirmed the capability of 14e to establish interactions with several amino acids into FABP4 binding pocket, being this in agreement with the higher activity recorded in vitro for 14e, in comparison to the other analogs developed in this study.Lastly, in silico absorption, distribution, metabolism, and excretiontoxicity (ADMET) calculations suggested that 14e would be optimally absorbed, distributed, metabolized, and excreted, as well as well adsorbed by the skin and intestine, having the potential to enable topical and oral route of administration during future in vivo studies as FABP4i.Chemistry4.1.1 | GeneralAll the chemical reagents were purchased from Merk and Sigma Aldrich of reagent grade and were used without any further purification.Extracts were dried over Na 2 SO 4 and the solvents were removed under reduced pressure.All reactions were monitored by thin layer chromatography F I G U R E 4 Solute RMSD from the starting structure as a function of simulation time.Fatty acid-binding protein 4 (FABP4) in blue.14e in red.All the simulated system in green.