Ligand Growing Experiments Suggested 4-amino and 4-ureido pyridazin-3(2H)-one as Novel Scaffold for FABP4 Inhibition

Fatty acid binding protein (FABP4) inhibitors are of synthetic and therapeutic interest and ongoing clinical studies indicate that they may be a promise for the treatment of cancer, as well as other diseases. As part of a broader research effort to develop more effective FABP4 inhibitors, we sought to identify new structures through a two-step computing assisted molecular design based on the established scaffold of a co-crystallized ligand. Novel and potent FABP4 inhibitors have been developed using this approach and herein we report the synthesis, biological evaluation and molecular docking of the 4-amino and 4-ureido pyridazinone-based series.


Introduction
Fatty acids (FAs) are long carbon chain organic carboxylic acids responsible for different actions in the human organism [1,2]. Their chronic high concentration in circulation leads to various disorders [3,4], including atherosclerosis [5], diabetes [6] and obesity [7]. Considering that their chemical structure is characterized by high lipophilicity, FAs are insoluble in water, and their trafficking into the body requires specific carriers such as the fatty acid-binding proteins (FABPs). [8]. Since their discovery, FABPs have been classified into different families based on their localization in the human body, such as 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 expressed in adipocytes [9], and the research into small molecule inhibitors for such protein initially started when it was reported that knockout animal models of FABP4 produced protective effects against the development of insulin resistance [10], as well as several pathological events linked to the metabolic syndrome and atherosclerosis [11][12][13]. Interestingly, pharmacological approaches with small molecules that inhibit the normal function of the protein are also valid in this regard, demonstrating similar results as the genetic procedures by mimicking the phenotype of FABP4-deficient mice [14]. This family of transporter proteins also has a role in cancer progression [15], and it was discovered that non-physiological expressions of FABPs are present in some of the most common cancers such as renal cell carcinoma, bladder and prostate, as well as other types of cancer cells [16][17][18]. It was recently discovered that FABP4 promotes the metastasis and invasion of colon cancer and that the treatment with a classical small molecule inhibitor (BMS309403) weakened the migration and invasion of colon cancer cells [19]. FABP4 leads also to abnormal metastasis patterns in ovarian cancer, and recent findings demonstrate that the protein is responsible for the disease's aggressivity, contributing to poor prognosis in this tumor [20]. Moreover, the transporter has also been shown to play a role in accelerating glioblastoma cell growth [21]. All these recent findings related to cancer research proved that FABP4 targeting may represent an effective and promising therapeutic strategy against oncological conditions, in addition to the established effects on metabolic and cardiovascular diseases.
Recently, a variety of effective FABP4 inhibitors (FABP4i) have been developed, but unfortunately, none of them is currently in the clinical research phases [14,22]. Computeraided drug design represents a promising and effective tool for the identification of molecular hits as FABP4i [23][24][25][26][27]. In line with our recent interest in the development of new antitumor compounds and the identification of novel bioactive heterocycles [28][29][30][31][32], herein we report the design, synthesis and in vitro characterization of 4-amino and 4-ureido pyridazinone-based series of FABP4i inspired by the scaffold hopping of an established ligand co-crystallized within the protein.

Heterocyclic Small-Molecule Design
To generate a novel series of FABP4 inhibitors we have exploited a two-step computing assisted molecular design. As shown in Figure 1, in the first step of the drug-design process we focused on the search for bioisosteric-replacements/scaffold hopping of the pyrimidine scaffold of the co-crystallyzed ligand (2-[(2-oxo-2-piperidin-1 -ylethyl)sulfanyl]-6-(trifluoromethyl)pyrimidin-4-ol; pdbID: 1TOU). Our bioisosteric replacement analysis led to the selection of three nitrogen-containing heterocyclic frameworks, i.e., pyridazinones, pyridines and benzo [d]thiazole (see Supplementary Materials). Considering the synthetic accessibility of pyridazinone-based molecules and that pyridazinone was not investigated earlier as a scaffold to access FABP4 inhibitors, we envisaged to use this heterocycle to carry out automated ligand growing experiments inside the FABP4 cavity, as described in the Section 3, leading to 52 target molecules. The compounds were then synthesized and screened against FABP4 and the chemical structures are reported in Tables 1 and 2. Both the scaffold hopping and the ligand growing experiments were conducted using Spark (https://www.cresset-group.com/products/spark/ accessed on 15 June 2022) [33]. well as other types of cancer cells [16][17][18]. It was recently discovered that FABP4 promotes the metastasis and invasion of colon cancer and that the treatment with a classical small molecule inhibitor (BMS309403) weakened the migration and invasion of colon cancer cells [19]. FABP4 leads also to abnormal metastasis patterns in ovarian cancer, and recent findings demonstrate that the protein is responsible for the disease's aggressivity, contributing to poor prognosis in this tumor [20]. Moreover, the transporter has also been shown to play a role in accelerating glioblastoma cell growth [21]. All these recent findings related to cancer research proved that FABP4 targeting may represent an effective and promising therapeutic strategy against oncological conditions, in addition to the established effects on metabolic and cardiovascular diseases.
Recently, a variety of effective FABP4 inhibitors (FABP4i) have been developed, but unfortunately, none of them is currently in the clinical research phases [14,22]. Computeraided drug design represents a promising and effective tool for the identification of molecular hits as FABP4i [23][24][25][26][27]. In line with our recent interest in the development of new antitumor compounds and the identification of novel bioactive heterocycles [28][29][30][31][32], herein we report the design, synthesis and in vitro characterization of 4-amino and 4ureido pyridazinone-based series of FABP4i inspired by the scaffold hopping of an established ligand co-crystallized within the protein.

Heterocyclic Small-Molecule Design
To generate a novel series of FABP4 inhibitors we have exploited a two-step computing assisted molecular design. As shown in Figure 1, in the first step of the drug-design process we focused on the search for bioisosteric-replacements/scaffold hopping of the pyrimidine scaffold of the co-crystallyzed ligand (2-[(2-oxo-2-piperidin-1 -ylethyl)sulfanyl]-6-(trifluoromethyl)pyrimidin-4-ol; pdbID: 1TOU). Our bioisosteric replacement analysis led to the selection of three nitrogen-containing heterocyclic frameworks, i.e., pyridazinones, pyridines and benzo[d]thiazole (see Supplementary Materials). Considering the synthetic accessibility of pyridazinone-based molecules and that pyridazinone was not investigated earlier as a scaffold to access FABP4 inhibitors, we envisaged to use this heterocycle to carry out automated ligand growing experiments inside the FABP4 cavity, as described in the Experimental section, leading to 52 target molecules. The compounds were then synthesized and screened against FABP4 and the chemical structures are reported in Tables 1 and 2. Both the scaffold hopping and the ligand growing experiments were conducted using Spark (https://www.cresset-group.com/products/spark/ accessed on 15 June 2022) [33].

Chemistry
The synthetic procedures carried out to obtain the target compounds containing the pyridazinone scaffold are reported in Schemes 1-9. The structures were confirmed on th basis of analytical and spectral data. Scheme 1 shows the synthetic pathway affording th final compounds 4a,b, 5a,b, 6 and 7. Intermediate 2 [34] was obtained starting from isox azole-pyridazinone 1, synthesized by adopting previously reported protocols [29][30][31][32] and using methanol and triethylamine for opening the isoxazole nucleus. The subsequent hy drolysis (acid 3 [35]) and acylation with thionyl chloride, triethylamine and appropriate amine led to final compounds 4a,b. Products 5a,b were obtained from alkylation reaction of 4a,b with ethyl bromide in standard conditions. The opening of isoxazole core of th starting material 1 with 33% NH4OH afforded to amide 6 [28] which, by subsequent de hydration with POCl3, led to compound 7. The synthesis of final compounds 16-18 is re ported in Scheme 2. The reaction between sodium salt of diketone 8 with the commercially available ethyl chloro(hydroximino)acetate 9 in ethanol led to a mixture of isomers 10 and 11 [36] that were cyclized to isoxazole-pyridazinone 12 and 13 using phenylhydrazine and PPA. After chromatographic separation, the latter were subjected to a series of reaction

Chemistry
The synthetic procedures carried out to obtain the target compounds containing the pyridazinone scaffold are reported in Schemes 1-9. The structures were confirmed on the basis of analytical and spectral data. Scheme 1 shows the synthetic pathway affording the final compounds 4a,b, 5a,b, 6 and 7. Intermediate 2 [34] was obtained starting from isoxazole-pyridazinone 1, synthesized by adopting previously reported protocols [29][30][31][32] and using methanol and triethylamine for opening the isoxazole nucleus. The subsequent hydrolysis (acid 3 [35]) and acylation with thionyl chloride, triethylamine and appropriate amine led to final compounds 4a,b. Products 5a,b were obtained from alkylation reaction of 4a,b with ethyl bromide in standard conditions. The opening of isoxazole core of the starting material 1 with 33% NH 4 OH afforded to amide 6 [28] which, by subsequent dehydration with POCl 3 , led to compound 7. The synthesis of final compounds 16-18 is reported in Scheme 2. The reaction between sodium salt of diketone 8 with the commercially available ethyl chloro(hydroximino)acetate 9 in ethanol led to a mixture of isomers 10 and 11 [36] that were cyclized to isoxazole-pyridazinone 12 and 13 using phenylhydrazine and PPA. After chromatographic separation, the latter were subjected to a series of reactions to obtain the compounds 16-18. The treatment of intermediate 13 with ammonium formate and Pd/C provided compound 18, while the treatment of 12 with methanol and triethylamine led to pyridazinone 14. Intermediate 14 was first hydrolyzed to acid (15), then converted to amide (16) and finally treated with POCl 3 to obtain the cyano derivative 17. The final compounds 21 and 22 were obtained through a procedure similar to that shown in Scheme 1 for amide derivative 6 and cyano derivative 7, using intermediate 20 as the starting material, which was obtained by reaction of cyclohexyl hydrazine and PPA with isoxazole 19 [34] (see Scheme 3). Scheme 4 reports the synthesis of the pyridazinone-based derivatives of type 24 and 25 (unsubstituted at position 5), compound 28 and the thio-derivative 27. Intermediate 23 [37] was reacted with the appropriate brominated alkylating agent in presence of potassium carbonate and dry DMF to afford 24a-f derivatives (24a, [38]; 24c, [34]). The formation of urea derivatives of type 25 was carried out using sodium acetate and triphosgene in dry THF at reflux, and then treated with ammonia. The urea 28 was directly obtained from intermediate 23 using the same conditions used for compound type 25. The transformation of the carbonyl (C=O) in thiocarbonyl group (C=S) was carried out using the Lawesson's reagent in toluene (26) and the subsequently alkylation with methyl iodide in standard condition led to the thio derivative 27. In Schemes 5 and 6 are reported the synthetic procedures of other un-substituted pyridazinones at position 5, but bearing different groups/functions at position 4 and 6. In particular, Scheme 5 depicts the synthetic pathways for compounds with a phenyl ring at position 6 and a methyl group at N-2, while different substituents are introduced at position 4. Starting from compound 24a [38] (Scheme 4), the amino group at position 4 was acylated using the suitable anhydride in pyridine in a sealed/pressure vessel to obtain the final compounds 29a-d. Moreover, the same amino group was also subjected to a coupling reaction using the appropriate R-phenylboronic acid in presence of copper (II) acetate and triethylamine to furnish the derivatives 30a,b and 33. The substituent R on the phenyl at position 4 was further elaborated. The m/o-CN group of compounds 30a,b was converted into m/o-CONH 2 (compounds 31a,b, respectively) with 80% sulfuric acid under reflux. The 4-carbethoxy function in product 33 was firstly hydrolyzed to acid 34, converted into the corresponding acid chloride with thionyl chloride and then acylated with 1-acetylpiperazine (compound 35). Lastly, the carbonyl group of intermediate 24a was converted in thiocarbonyl (32) using the same procedure discussed in Scheme 4. In Scheme 6 are depicted pyridazinone-based derivatives with a methyl group or a hydrogen at N-2, an amino group or urea functionality at position 4, but bearing different groups e/o functions (e.g., R-phenyl, alkyl, cycloalkyl) at position 6. Starting from commercially available intermediates 36a-f, the introduction of an amino group at position 4 with hydrazine hydrate at high temperature led to compounds 37a-e (37e, [38]) and the subsequent alkylation with methyl iodide provided products 38ad. The derivatives 39a,b and 40 were obtained from reaction with triphosgene and ammonia in the same conditions reported in Scheme 4, starting from 38b,c and 37e, respectively. The direct alkylation of the intermediates 36e and 36f afforded the corresponding N-methyl derivatives 41a,b (41a, [39]), which were subsequently converted into compounds 42a,b through the same reaction used to obtain 37a-e. In particular, the reaction conditions used to introduce an amino group at position 4 led also to the reduction in the nitro group in compound 42b. The latter was subjected to acylation reaction with acetyl chloride to obtain product 44. Instead, intermediate 42a was subjected to triphosgene treatment to obtain the urea derivative 43. Scheme 7 reports the synthesis of final compounds 48 and 49. Intermediate 47 was obtained starting from isoxazole 45, previously synthesized by us [34] by cyclization reaction with methyl hydrazine (46) and subsequent opening of the isoxazole ring with ammonium formate and palladium on carbon. The deacetylation (on 47) with 48% bromic acid at high temperature led to compound 48, which was subsequently treated with triphosgene and ammonia to obtain the urea derivative 49. Compound 51 was obtained through alkylation reaction using standard conditions [40], but starting from product 50 [41] (Scheme 8). Lastly, the final compound 55 (Scheme 9) was obtained starting from intermediate 52 [42], through the same reactions of isoxazole nucleus opening (53 [42]), deacetylation (54 [43]) and formation of the urea function. In Scheme 9 is also illustrated the treatment of 52 with dimethylformamide dimethyl acetal to generate intermediate 56, which was subsequently converted into compound 57 using hydrazine hydrate. the same reactions of isoxazole nucleus opening (53 [42]), deacetylation (54 [43]) and formation of the urea function. In Scheme 9 is also illustrated the treatment of 52 with dimethylformamide dimethyl acetal to generate intermediate 56, which was subsequently converted into compound 57 using hydrazine hydrate. Based on the analytical and spectral data (proton and carbon NMR) and mass spectrometry (MS), all the new compounds confirmed the predicted chemical structures, as well as satisfactory results in terms of formulation and purity (See Experimental section; in SI are reported representative examples of analytical characterization data of the compounds processed to FABP4 inhibition assay in vitro). Reversed phase liquid chromatography was used to perform a qualitative analysis of the dataset's purity. The formation of the products was monitored by UV absorbance at wavelengths of 281 nm and 254 nm. The retention times range was from 6 to 17 min (See Experimental section and SI). The overall feature of mass spectra (LC-MS) of this series of pyridazinone-derivatives is the presence of a predominant peak corresponding to the molecular ion [  Based on the analytical and spectral data (proton and carbon NMR) and mass spectrometry (MS), all the new compounds confirmed the predicted chemical structures, as well as satisfactory results in terms of formulation and purity (See Section 3; in Supporting Information are reported representative examples of analytical characterization data of the compounds processed to FABP4 inhibition assay in vitro). Reversed phase liquid chromatography was used to perform a qualitative analysis of the dataset's purity. The formation of the products was monitored by UV absorbance at wavelengths of 281 nm and 254 nm. The retention times range was from 6 to 17 min (See Section 3 and Supporting Information). The overall feature of mass spectra (LC-MS) of this series of pyridazinonederivatives is the presence of a predominant peak corresponding to the molecular ion [M + H] + (See Section 3 and Supporting Information).

FABP4 Inhibition Evaluation
FABP4 inhibitory activity was assessed by measuring the decrease in fluorescent signal of a detection reagent (DR) when displaced by a strong FABP4 ligand. Specifically, the DR exhibits an increased fluorescence intensity when bound to FABP4. Therefore, any effective ligand of the protein, which binds to the same binding pocket and can displace the DR, determines a reduction in the fluorescence read-out. The new molecular series was screened in a two-step procedure. Firstly, a single concentration of 5 µM was used to gain an estimation of the overall inhibitory effect of all the molecules. Subsequently, only the compounds that were able to reduce the fluorescence reading of at least 95% were further evaluated by measuring the IC50 values (µM), which were lastly compared with the activity of the arachidonic acid (i.e., FABP4 established ligand). The single point displacement results are reported in Figure 2. Based on the data of the first screening, 10 molecules were selected as most effective compounds-i.e., able to reduce the fluorescence of the DR to at least 95%, for which the IC50 (µM) was calculated. Arachidonic acid was used as a positive control, resulting with an IC50 of 3.42 µM. The IC50 values of our set of compounds are reported in Table 3. Compound 25a demonstrated a potent inhibitory activity, with an IC50 value (i.e., 2.97 µM) lower than the reference arachidonic acid.

FABP4 Inhibition Evaluation
FABP4 inhibitory activity was assessed by measuring the decrease in fluorescent signal of a detection reagent (DR) when displaced by a strong FABP4 ligand. Specifically, the DR exhibits an increased fluorescence intensity when bound to FABP4. Therefore, any effective ligand of the protein, which binds to the same binding pocket and can displace the DR, determines a reduction in the fluorescence read-out. The new molecular series was screened in a two-step procedure. Firstly, a single concentration of 5 µM was used to gain an estimation of the overall inhibitory effect of all the molecules. Subsequently, only the compounds that were able to reduce the fluorescence reading of at least 95% were further evaluated by measuring the IC 50 values (µM), which were lastly compared with the activity of the arachidonic acid (i.e., FABP4 established ligand). The single point displacement results are reported in Figure 2. Based on the data of the first screening, 10 molecules were selected as most effective compounds-i.e., able to reduce the fluorescence of the DR to at least 95%, for which the IC 50 (µM) was calculated. Arachidonic acid was used as a positive control, resulting with an IC 50 of 3.42 µM. The IC 50 values of our set of compounds are reported in Table 3. Compound 25a demonstrated a potent inhibitory activity, with an IC 50 value (i.e., 2.97 µM) lower than the reference arachidonic acid.

Molecular Modelling Studies
Since the first apo-FABP crystal structure was published in 1992, many other holo-FABP structures with a variety of ligands have been solved. The hydrophobic pocket side chains engage a hydrogen bond to the carboxylate of FAs toward several amino acids. Moreover, a network of water molecules may be involved in mediating these interactions. The docking experiments of the molecular series compounds were conducted on the most active compounds 4b, 25a, 30b, and 22. Figure 3 shows the 2D binding interactions for the molecules, while Figure 4 displays the predicted poses inside the binding pocket of FABP4. All the compounds are able to engage several interactions with relevant residues in the binding pocket, such as R126 and Y128, as well as R106. R126 can interact with both the carbonyls of the most potent compound 25a, that also interacts directly with Y128 and, through the network of water molecules, with S53. The 4b is well allocated inside the binding pocket and is engaging a strong H-bond interaction with R126. Differently, compound 22 is not suitably allocated inside the pocket to generate appropriate binding with R126 and Y128 and most of the stabilizing interactions are due to pi-pi stacking with A75, F16 and M20. Lastly, the -CN group of 30b results responsible of the stabilizing interaction with R126 and Y128, that are likely to account for the lower activity of the compound, as determined by the lower binding interaction for this group with the residues.

Molecular Modelling Studies
Since the first apo-FABP crystal structure was published in 1992, many other holo-FABP structures with a variety of ligands have been solved. The hydrophobic pocket side chains engage a hydrogen bond to the carboxylate of FAs toward several amino acids. Moreover, a network of water molecules may be involved in mediating these interactions. The docking experiments of the molecular series compounds were conducted on the most active compounds 4b, 25a, 30b, and 22. Figure 3 shows the 2D binding interactions for the molecules, while Figure 4 displays the predicted poses inside the binding pocket of FABP4. All the compounds are able to engage several interactions with relevant residues in the binding pocket, such as R126 and Y128, as well as R106. R126 can interact with both the carbonyls of the most potent compound 25a, that also interacts directly with Y128 and, through the network of water molecules, with S53. The 4b is well allocated inside the binding pocket and is engaging a strong H-bond interaction with R126. Differently, compound 22 is not suitably allocated inside the pocket to generate appropriate binding with R126 and Y128 and most of the stabilizing interactions are due to pi-pi stacking with A75, F16 and M20. Lastly, the -CN group of 30b results responsible of the stabilizing interaction with R126 and Y128, that are likely to account for the lower activity of the compound, as determined by the lower binding interaction for this group with the residues.

General Remarks
All 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 (TLC) using commercial plates (Merck) pre-coated with silica gel 60 F-254. Visualization was performed by UV fluorescence (λmax = 254 nm) or by staining with iodine or potassium permanganate. Chromatographic separations were performed on silica gel columns by gravity ( The instrument used consisted of a Thermo Accela LC system interfaced to a Thermo TSQ Access triple quadrupole mass spectrometer with a HESI source. The data were processed with Xcalibur software (version 2.0). An amount of 10 µL of sample was analyzed in flow injection, with a flow rate of 0.2 mL/min of mobile phase 0.1% HCOOC in MeOH/H 2 O (50:50). Parameters used for the analysis in positive ion mode were: spray voltage 3500 V; vaporizer temperature 300 • C; sheath gas pressure 50 au; capillary temperature 350 • C; capillary offset 35.

General Procedure for Compounds 4a,b
A mixture of 3 (0.35 mmol) [35], a catalytic amount of Et 3 N (0.1 mL) and SOCl 2 (9.35 mmol) was stirred at room temperature for 30 min. Then the excess of SOCl 2 was removed in vacuo and the residue oil was dissolved in cold anhydrous THF (1 mL). To this suspension, the appropriate amine (0.75 mmol) was added and the mixture was stirred at room temperature for 2 h. After cooling, cold water was added (2-5 mL) and the suspension was extracted with CH 2 Cl 2 (3 × 15 mL); the solvent was evaporated under vacuum to afford the desired final compounds, which were purified by flash column chromatography using cyclohexane/ethyl acetate 1:2 as eluent (4a), or by crystallization from ethanol (4b).

General Procedure for Compounds 5a,b
A mixture of 4a,b (0.43 mmol), K 2 CO 3 (0.86 mmol) and 0.50 mmol of ethyl bromide in anhydrous DMF (2 mL) was refluxed for 30-90 min. After cooling, the mixture was diluted with cold water (15 mL) and compound 5a was recovered by filtration under vacuum. For compound 5b the suspension was extracted with CH 2 Cl 2 (3 × 15 mL) and the solvent was evaporated in vacuo. The crude products were purified by crystallization from ethanol.

5-Acetyl-4-amino-2-phenylpyridazin-3(2H)-one (18)
Intermediate 13 (1.01 mmol) was suspended in 3.5 mL of EtOH, then 6.08 mmol of HCOONH 4 and 40 mg of 10% Pd/C were added. The mixture was refluxed for 2 h and after cooling, CH 2 Cl 2 (5 mL) was added. The solution was stirred for 5 min, then the catalyst was filtered off and the solvent was evaporated in vacuo to furnish desiderd compound 18.

General Procedure for Compounds 25a-f
To a cooled (0 • C) and stirred suspension of the appropriate pyridazinone 24a-f (0.65 mmol) in anhydrous THF (1-3 mL), anhydrous sodium acetate (1.55 mmol) and triphosgene (2.26 mmol) were added. The mixture was stirred for 10 min at room temperature and refluxed for 2 h. Then, the suspension was cooled to 0 • C and 1 mL of 33% NH 3 was added and the mixture was stirred for 30-90 min at room temperature. After evaporation of the solvent, ice/cold water was added (15 mL) and the precipitate obtained was recovered by filtration under vacuum and purified by crystallization from ethanol to obtain the pure samples of 25a-f.       (26) A mixture of 23 [37] (0.86 mmol) and Lawesson's reagent (1.71 mmol) in anhydrous toluene (2-3 mL) was heated at 90 • C for 5 h. After cooling the solvent was evaporated under vacuum, cold water was added (10 mL) and the mixture was extracted with CH 2 Cl 2 (3 × 15 mL). Evaporation of the solvent afforded 26 which was purified by flash column chromatography using CH 2 Cl 2 /CH 3 OH 10:1 as eluent. Yield = 63%; mp = 175-178 • C (Cyclohexane). 1 (27) Compound 27 was obtained, starting from compound 26, through the general procedure described for 24b and 24d-f. After dilution with cold water, the precipitate was recovered by suction and purified by crystallization. Yield

General procedure for compounds 30a,b and 33
A mixture of compound 24a [38] (0.79 mmol), the appropriate R-phenylboronic acid (0.79 mmol), copper acetate (1.19 mmol) and triethylamine (1.59 mmol) in CH 2 Cl 2 (5 mL) was stirred at room temperature for 3-12 h. After evaporation of the solvent, ethyl acetate was added (15-20 mL) and the solution was extracted first with 33% NH 3 (3 × 5 mL) and then with water (2 × 5 mL). The organic layer was evaporated under vacuum and the residue was purified by crystallization from ethanol.  13 (32) Compound 32 was obtained, starting from compound 24a [38], through the same procedure described for 26. In this case, the mixture was refluxed for 10 h. After cooling, ice/cold water was added. The precipitate was recovered by suction and purified by flash column chromatography using cyclohexane/ethyl acetate 1:1 as eluent. Yield = 85%; mp = 134-135 • C (EtOH). 1   Compound 35 was obtained starting from 34 through the same procedure described for 4a,b. In this case the mixture was stirred at room temperature for 40 min. After cooling, THF was removed in vacuo and cold water was added (10 mL). The crude precipitate was recovered by filtration under vacuum and purified by crystallization. Yield

General Procedure for Compounds 38a-d
A mixture of the appropriate pyridazinone 37a-d (0.67 mmol), K 2 CO 3 (1.34 mmol) and CH 3 I (1.01 mmol) in anhydrous DMF (1.5 mL) was stirred at 80 • C for 1-4 h. After cooling, the mixture was diluted with cold water (15 mL) and compound 38a was recovered by suction and crystallized from ethanol. For compounds 38b-d the suspension was extracted with CH 2 Cl 2 (3 × 15 mL) and the solvent was evaporated in vacuo. The final compounds were purified by flash column chromatography using cyclohexane/ethyl acetate 1:2 (for 38b,d) or CH 2 Cl 2 /CH 3 OH 9.5:0.5 (for 38c) as eluents.

Molecular Modeling and Biological Data
The 2D chemical structures were built using Marvin Sketch and all the structures were subjected to molecular mechanics energy minimization using the MMFF94 force field present in the same software [45]. The 3D geometry of all compounds was then optimized using the PM3 Hamiltonian [46], as implemented in MOPAC 2016 package assuming a pH of 7.0 [47]. Once built and optimized, all structures were used in the bioisostere replacement tool Spark 10.4.0. Five hundred compounds were generated for the substitution (50 best compounds reported in the Supplementary Materials). The isosteric replacement was performed using the same 178,558 fragments for each part; in particular, the fragments derive from ChEMBL and Zinc databases with a protocol already reported and validated [27,48,49]. Ligand growing experiments were performed in the selected pyridazinone structure using an already reported protocol [50]. Docking calculations were made using AutoDock with the default docking parameters and a validated protocol [51,52]. The setup was done with YASARA [47]. 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. The X-ray crystal structures of the co-crystal FABP4/(2-[(2-oxo-2-piperidin-1ylethyl)sulfanyl]-6-(trifluoromethyl)pyrimidin-4-ol) (PDBid: 1TOU) was downloaded from the Protein Data Bank (www.rcsb.org accessed on 15 June 2022).

FABP Inhibitory Activity Assays
To analyze the inhibitory activity of FABP4 ligands, a displacement assay was utilized as described by the Cayman's instruction, FABP4 Inhibitor/Ligand Screening Assay Kit, Item 10,010,231 (see Supplementary Materials for additional details). The samples of compounds for activity determination were prepared as a stock solution (1 mM) in DMSO. On the day of activity assay, the compounds were all diluted in phosphate buffer solution (PBS, pH 7.4) to different concentrations (100, 50, 10, 5, 2, 1, and 0 µM). Appropriate concentrations of DMSO in PBS were used as control. The detection reagent (FABP Assay Detection Reagent, Item 10010376) was used as provided by the Cayman's kit. The diluted Detection Reagent probe was mixed with FABP4 protein present in the kit and incubated for 10 min at room temperature. Compounds were then added and equilibrated for another 10 min. Lastly, the fluorescence signal was recorded at 470 nm (i.e., emission, with the excitation fixed at 370 nm) with a CytoFluor ® Series 4000 Fluorescence Multi-Well Plate Reader. The IC 50 was calculated as indicated in the kit booklet of FABP4 Inhibitor/Ligand Screening Assay Kit (Item No. 10010231) Cayman chemicals, as follows: 1) calculate the average fluorescence of each sample; 2) calculate the background corrected fluorescence (BCF) by subtracting the blank; 3) divide the BCF of each sample by the maximum BCF and multiply by 100% (this is the value in percent fluorescence units, i.e., % FU); 4) plot the % FU values against the concentration of inhibitor/ligand used; 5) find the concentration of inhibitor/ligand that corresponds to 50% FU, to determine IC 50 values.

Conclusions
We have identified novel 4-amino and 4-ureido pyridazinone-based FABP4 inhibitors whose design was directed by computing assisted molecular design of bioisosteric-replacements/ scaffold hopping of the pyrimidine skeleton of the co-crystallyzed ligand 1TOU. Selected compounds have been synthesized and tested for their ability to inhibit FABP4. Among the new series, ten compounds were further evaluated on the basis of their inhibitory activity on FABP4 established via a single point displacement assay. In particular, 4b, 25a, 30b and 22 exhibited high FABP4 inhibitory activity with IC 50 in the low micromolar range. The results demonstrated that compound 25a was the most potent analogue in terms of displacement of the arachidonic acid, with an IC 50 value of 2.97 µM, which is lower than the IC 50 of the positive control (3.42 µM). Docking experiments, conducted with the most active compounds 4b, 25a, 30b, 22, confirmed the ability of these molecules to interact with several amino acid residues present inside the FABP4 binding pocket, with the stronger interaction exhibited by compound 25a. This result is in agreement with the higher activity recorded in vitro for 25a, in comparison to the other 4-amino and 4-ureido pyridazinone-based analogues developed in this study.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ph15111335/s1, 1 H NMRs of selected compounds; 13 C NMRs of selected compounds; Mass spectra of selected compounds; HPLC/UV chromatograms of selected compounds; 50 'best-fit' compounds generated with scaffold hopping replacement; Info on the FABP4 inhibitor assay kit; Averaged data as Background corrected fluorescence for IC 50 measured compounds.