2-Arylacetamido-4-Phenylamino-5-Substituted Pyridazinones as Formyl Peptide Receptors Agonists

Abstract

N-Formyl peptide receptors (FPRs: FPR1, FPR2, and FPR3) are G protein-coupled receptors that play key roles in modulating immune cells. FPRs represent potentially important therapeutic targets for the development of drugs that could enhance endogenous anti-inflammation systems associated with various pathologies, thereby reducing the progression of inflammatory conditions. Previously, we identified 2-arylacetamide pyridazin-3(2H)-ones as FPR1- or FPR2-selective agonists, as well as a large number of FPR1/FPR2-dual agonists and several mixed-agonists for the three FPR isoforms. Here, we report a new series of 2-arylacetamido-4-aniline pyridazin-3(2H)-ones substituted in position 5 as a further development of these FPR agonists. Chemical manipulation presented in this work resulted in mixed FPR agonists 8a, 13a and 27b, which had EC₅₀ values in nanomolar range. In particular, compound 8a showed a preference for FPR1 (EC₅₀ = 45 nM), while 13a and 27b showed a moderate preference for FPR2 (EC₅₀ = 35 and 61 nM, respectively). Thus, these compounds may represent valuable tools for studying FPR activation and signaling.

Graphical abstract

1. Introduction

Human formyl peptide receptors (FPR1, FPR2, and FPR3) are a family of versatile G-protein-coupled receptors (GPCRs) that represent attractive therapeutic targets because of their involvement in a wide range of normal physiological processes, as well as pathological events associated with inflammatory conditions [1–4]. Originally identified in phagocytic leucocytes, FPRs mediates chemotaxis and activation of the majority of immune system cells in response to bacterial products and various inflammatory stimuli [3]. However, recent studies indicate that FPRs are also expressed in a variety of nonhematopoietic cells, such as lung epithelial cells, platelets, osteoblasts, and hepatocytes, suggesting a wider role for FPRs beyond inflammation and host defense [5].

FPR activation can induce pro- or anti-inflammatory responses, depending on the nature of the ligand and cell types involved. For example, FPRs have been reported to contribute to inflammation associated with amyloidosis and Alzheimer's disease, prion disease, human immunodeficiency virus, stomach ulcer, some cancers, nociception associated with inflammatory processes, chronic obstructive pulmonary disease (COPD), stroke and ischemia-reperfusion injury [6–13]. Conversely, stimulation of FPRs with certain agonists can also induce pro-resolving responses or endogenous anti-inflammatory systems [3]. Indeed, screening of commercial libraries and new synthetic compounds has resulted in the discovery of a number of small-molecule non-peptide FPR agonists and antagonists with a wide range of chemical diversity and activities [14–16].

In previous studies, we identified several pyridazin-3(2H)-one-based derivatives that showed an interesting profile as FPR agonists, combining an appreciable potency and differential selectivity toward the three human FPR isoforms [17–21]. Key requirements for agonist activity of this class of compounds were the presence of a 4-bromophenylacetamide side chain at the N-2 position of the scaffold [17, 19] and the presence of a methyl group at C-6 [20]. Position 4 could be substituted with a benzyl or phenylamino group, resulting in compounds with micromolar activity (Figure 1, general structure A).

Figure 1.

General structure of FPR1/FPR2 agonist, based on pyridazin-3(2H)-one scaffold.

In the present study, we further investigated pyridazinone derivatives belonging to the series of 4-phenylamino derivatives (structure A, X = NH) which until now was only poorly studied [20]. In particular, we inserted at position 5 of the pyridazinone scaffold a variety of substituents, such as alkyl or acyl groups, ester, unsaturated chains, and pyrazole rings, in order to evaluate how such modifications affected target specificity and compound potency.

2. Chemistry

All compounds were synthesized as reported in Schemes 1–5, and the structures were confirmed using analytical and spectral data.

Scheme 1.

Reagents and conditions: i) 10% Pd/C, HCOONH₄, anhydrous EtOH, reflux, 1 h; ii) ethyl bromoacetate, K₂CO₃, anhydrous CH₃CN, reflux, 2 h; iii) 6N NaOH, EtOH, 60 °C, 1 h; iv) Et₃N, anhydrous THF, ethyl chloroformate, 4-bromoaniline, −5 °C, rt and then 17.5 h; v) N-(4-bromophenyl)-2-chloroacetamide, K₂CO₃, anhydrous CH₃CN, reflux, 2 h; vi) 3-methoxybenzenboronic acid, (CH₃COO)₂Cu, Et₃N, anhydrous CH₂Cl₂, rt, 16 h.

Scheme 5.

Reagents and conditions: i) N-(4-bromophenyl)-2-chloroacetamide, K₂CO₃, anhydrous CH₃CN, reflux, 6 h; ii) 3-methoxybenzenboronic acid, (CH₃COO)₂Cu, Et₃N, anhydrous CH₂Cl₂, rt, 16 h.

The synthetic pathway leading to the final compounds 6a–f bearing an acyl group at position 5 is outlined in Scheme 1. Previously described isoxazolo-pyridazinones of type 1 [22–25] were transformed into the corresponding 4-amino-5-acetyl derivatives 2a–f (2a–d [25]) by reductive cleavage with 10% Pd/C and HCOONH₄ in ethanol. The products were then converted to the 4-bromophenylacetamide derivatives 5a–f as follows. Intermediates 2a and 2d,e were alkylated with ethylbromoacetate under standard conditions to generate 3a [26] and 3d,e, which were transformed into the corresponding carboxylic acids 4a [22] and 4d,e through alkaline hydrolysis. These were transformed into the final amides of type 5 by treatment with 4-bromoaniline, ethyl chloroformate and triethylamine in THF. Compounds 5b,c and 5f were obtained starting from their respective intermediates by a direct alkylation with N-(4-bromophenyl)-2-chloroacetamide [27] under standard conditions. Finally, a coupling reaction between 5a–f and 3-methoxybenzenboronic acid was carried out in the presence of copper(II)acetate and Et₃N to generate the final 5-alkyl pyridazinones 6a–f.

Synthetic routes used to obtain the 5-alkyl (11a–d) and 5-vinyl (13) derivatives are shown in Scheme 2. Intermediates 2a–d [25] were converted to the desired final compounds 11a–d through the following steps: reduction of the acetyl at C-5 with NaBH₄ (compounds 7a–d), dehydratation with polyphosphoric acid (PPA) (8a–d), reduction of the vinyl group with a Parr instrument (9a–d), insertion of the fragment at N-2 (10a–d), and coupling with 3-methoxybenzeneboronic acid (11a–d). To obtain the final compound 13, direct alkylation with N-(4-bromophenyl)-2-chloroacetamide [27] on intermediate 8a was performed (compound 12), followed by a coupling reaction with 3-methoxybenzenboronic acid.

Scheme 2.

Reagents and conditions: i) NaBH₄, CH₃OH, rt, 1 h; ii) PPA, reflux, 5 h; iii)10% Pd/C, anhydrous EtOH, H₂, Parr, 30 PSI, 3 h; iv) N-(4-bromophenyl)-2-chloroacetamide, K₂CO₃, anhydrous CH₃CN, reflux, 2–6 h; v) 3-methoxybenzenboronic acid, (CH₃COO)₂Cu, Et₃N, anhydrous CH₂Cl₂, rt, 16 h.

Scheme 3 shows the synthetic pathway for compounds 17a–b and 18: intermediate 14 [28] was converted into the pyridazinone 15 through isoxazole ring cleavage using the appropriate alcohol and a catalytic amount of Et₃N. Compounds of type 15 were then transformed into the final 17a,b following the same procedure reported in Schemes 1 and 2. Moreover, basic hydrolysis of the ester led to the final carboxylic derivative 18.

Scheme 3.

Reagents and conditions: i) Et₃N, MeOH or EtOH, 60 °C, 4 h; ii) N-(4-bromophenyl)-2-chloroacetamide, K₂CO₃, anhydrous CH₃CN, reflux, 2–4 h; iii) 3-methoxybenzenboronic acid, (CH₃COO)₂Cu, Et₃N, anhydrous CH₂Cl₂, rt, 16 h; iv) 2N NaOH, EtOH, rt, 1 h.

The pyrazolyl derivatives 25a,b were obtained following the procedures outlined in Scheme 4. Compound 19 [22] was transformed into the intermediated 20, as previously reported, and then was condensed with N,N-dimethylformamide dimethyl acetal to obtain derivative 21. Treatment with Mo(CO)₆ [29] led to 22. Condensation with hydrazine hydrate gave the C-5 pyrazolyl pyridazinone 23 which, in turn, was alkylated with iodomethane (compound 24) and coupled with 3-methoxybenzenboronic acid (25a,b).

Scheme 4.

Reagents and conditions: i) Et₃N, anhydrous THF, ethyl chloroformate, 4-bromoaniline, −5 °C and then rt, 17.5 h; ii) DMF/DMA, 90°C, 3 h; iii) Mo(CO)₆, H₂O, CH₃CN, reflux, 2h; iv) N₂H₄. H₂O, EtOH, reflux, 3 h; v) CH₃I, K₂CO₃, anhydrous DMF, 90°C, 3 h; vi) 3- or 4-methozybenzenboronic acid, (CH₃COO)₂Cu, Et₃N, anhydrous CH₂Cl₂, rt, 16 h.

Finally Scheme 5 describes the synthetic pathway leading to the final 5-unsubstituted pyridinone and pyridazinone derivatives 28a–b, which were obtained starting from appropriate intermediates 26a [30] and 26b [31] following the usual synthetic procedures described in previous schemes.

3. Results and discussion

3.1. Biological results

All new compounds were evaluated for their ability to induce intracellular Ca²⁺ flux in human HL-60 cells transfected with FPR1, FPR2, or FPR3, and the results are reported as EC₅₀ values in Tables 1–2.

Table 1.

Activity of C-4 and C-5 substituted pyridazinones 8a–f, 15, 19a–b, 20, 24–26, 27a–b.

Compd	OCH3	R	hPMN[a]	FPR1-HL60[a]	FPR2-HL60[a]	FPR3-HL60[a]
8a	m	COCH3	0.036 ± 0.007 (150)	0.045 ± 0.016 (185)	0.17 ± 0.038 (60)	0.21 ± 0.044 (150)
8b	m	COCH2CH3	5.5 ± 1.6 (110)	2.7 ± 0.8 (130)	0.78 ± 0.1 (190)	N.A.a
8c	m	CO(CH2)2CH3	24.0 ± 4.2 (60)	11.8 ± 3.5 (70)	8.1 ± 2.1 (90)	N.A.a
8d	m	CO(CH2)3CH3	8.0 ± 2.6 (90)	15.6 ± 2.6 (45)	N.A.a	N.A.a
8e	m	COcC5H9	1.7 ± 0.4 (110)	1.6 ± 0.4 (110)	10.5 ± 2.1 (60)	N.A.a
8f	m	COcC6H11	28.9 ± 6.6 (120)	10.0 ± 3.2 (110)	8.7 ± 0.47 (115)	N.A.a
15	m	CH=CH2	0.84 ± 0.18 (130)	0.23 ± 0.07 (120)	0.11 ±0.014 (105)	6.0 ± 2.1 (75)
19a	m	COOCH3	22.1 ± 5.3 (25)	4.4 ± 0.6 (25)	2.3 ± 0.49 (60)	N.A.a
19b	m	COOCH2CH3	6.1 ± 1.7 (95)	2.5 ± 0.7 (90)	1.9 ± 0.03 (110)	N.A.a
20	m	COOH	1.5 ± 0.5 (155)	0.6 ± 0.1 (130)	3.1 ± 0.78 (115)	17.1 ± 4.3 (75)
24	-	COCHCHN(CH3)2	9.3 ± 2.3 (105)	5.1 ± 1.7 (120)	5.7 ± 2.3 (50)	N.A.a
25	-	pyrazole	11.8 ± 2.4 (140)	8.4 ± 1.5 (135)	13.5 ± 1.8 (60)	N.A.a
26	-	1-methylpyrazole	3.5 ± 0.32 (60)	2.9 ± 0.14 (75)	1.9 ± 0.71 (40)	N.A.
27a	m	1-methylpyrazole	0.59 ± 0.21 (135)	3.6 ± 0.28 (130)	0.59 ± 0.18 (90)	N.A.a
27b	p	1-methylpyrazole	0.30 ± 0.044 (130)	4.0 ± 0.89 (115)	0.035 ± 0.1 (120)	0.67 ± 0.22 (110)

^[a]Values, expressed as EC₅₀ (µM) and Efficacy (% in brackets) were evaluated in a Ca²⁺ flux assay. EC₅₀ values represent the average mean of three independent experiments and were determined by nonlinear regression analysis of the concentration-response curves (5–6 points) generated using GraphPad Prism 5 with 95% confidential interval (p < 0.05). Efficacy (in brackets) is expressed as % of the response induced by 5 nM fMLF in human polymorphonuclear neutrophils (hPMN) and FPR1-HL60 cells or by 5 nM WKYMVm in FPR2-HL60 and FPR3-HL60 cells.

^[b]N.A., no activity (no response was observed during first 2 min after addition of compounds under investigation) considering the limits of efficacy < 20% and EC₅₀ < 50 µM.

Table 2.

Activity of C-4 and C-5 substituted pyridazinones 13a–d and 29a–b.

Compd	R	hPMN[a]	FPR1-HL60[a]	FPR2-HL60[a]	FPR3-HL60[a]
13a	CH2CH3	0.34 ± 0.11 (115)	0.18 ± 0.004 (185)	0.061 ± 0.022 (35)	0.46 ± 0.014 (35)
13b	CH2CH2CH3	N.A.	N.A.	N.A.	N.A.
13c	(CH2)3CH3	1.40 ± 0.8 (45)	3.6 ± 1.1 (65)	4.5 ± 1.3 (30)	N.A.
13d	(CH2)4CH3	5.70 ± 1.2 (55)	1.4 ± 0.34 (95)	0.19 ± 0.018 (115)	N.A.
29a	H	0.56 ± 0.12 (85)	0.24 ± 0.09 (120)	9.6 ± 2.0 (65)	N.A.
29b	-	4.31 ± 0.4 (60)	2.50 ± 0.7 (110)	8.10 ± 1.7 (75)	N.A.

^[a]Values, expressed as EC₅₀ (µM) and Efficacy (% in brackets) were evaluated in Ca²⁺ flux assay. EC₅₀ values represent the average mean of three independent experiments and were determined by nonlinear regression analysis of the concentration-response curves (5–6 points) generated using GraphPad Prism 5 with 95% confidential interval (p < 0.05). Efficacy (in brackets) is expressed as % of the response induced by 5 nM fMLF in human polymorphonuclear neutrophils (hPMN) and FPR1-HL60 cells or by 5 nM WKYMVm in FPR2-HL60 and FPR3-HL60 cells.

^[b]N.A., no activity (no response was observed during first 2 min after addition of compounds under investigation) considering the limits of efficacy < 20% and EC₅₀ < 50 µM.

Analysis of compounds substituted with various groups at position 5 (Table 1) demonstrated that several were potent mixed FPR agonists. Among them, the acetyl derivative 8a was active in the nanomolar range and preferred FPR1 (EC₅₀ = 45 nM). On the other hand, elongation of the aliphatic chain of keto(alkyl) derivatives (compounds 8b–d) was detrimental for FPR agonist activity. Although the butyl analogue 8d was selective for FPR1, it had only moderate activity (EC₅₀ = 15.6 µM). Further modification at position 5, such as the introduction of cyclopentyl and cyclohexyl rings (8e–f), also led to decreased activity.

Replacement of the ketone at C-5 (Table 1) with a pyrazole resulted in two mixed FPR agonists with reasonable agonist activity (27a–b). These two agonists differ only in the position of the methoxy group of the aniline at C-4 (meta for 27a versus para for 27b). Compound 27b was a potent FPR2 agonist (EC₅₀ = 35 nM), although it did have some activity at the other two FPR subtypes (FPR2>FPR3>>FPR1). Elimination of the methoxyphenylamino group at C-4 of the pyridazinone (compound 26) led to decreased activity that was comparable to that of the other two 4-amino derivatives 24 and 25. Likewise, introduction of methyl or ethyl esters at C-5 of pyridazinone (19a–b) also led to compounds with micromolar activity. On the other hand, introduction of a carboxylic function in the same position (20) led to a slight increase of selectivity toward FPR1 (EC₅₀ = 0.6 µM). Finally, the vinyl derivative 15 exhibited mixed agonist activity for the three FPR isoforms but had a higher preference for FPR1 and FPR2 (EC₅₀ = 0.23 and 0.11 µM, respectively).

Activities of the 5-alkyl derivatives 13a–d are presented in Table 2. Compound 13a, in which R = C₂H₅, was the most potent of this series. It was active in the nanomolar range toward the three FPR subtypes but had a preference for FPR2 (EC₅₀ = 61 nM). Elongation of the aliphatic chain was detrimental for activity, resulting in compounds with micromolar EC₅₀ values for FPR1 and FPR2 and no activity at FPR3. Surprisingly, the 5-propyl derivative 13b was completely devoid of activity. This is likely due to the loss of H-bonding between this compound and the receptor (see molecular modeling details below). Furthermore, elimination of the C-5 substituent led to a compound 29a with high nanomolar agonist activity for FPR1 (EC₅₀ = 0.24 µM). Finally, compound 29b exhibited moderate mixed agonist activity for FPR1 and FPR2, but was one order of magnitude lower in activity than 29a at FPR1, suggesting that the pyridonic scaffold was less appropriate compared with the pyridazinone scaffold.

The most active derivatives (8a, 15 and 27a–b) were also evaluated for chemoattractant activity in human neutrophils. As expected for FPR agonists, all four compounds had chemoattractant activity and induced this response in the micromolar range (Table 3).

Table 3.

Chemoattractant activity of selected pyridazinones in human neutrophils

Compd	EC50 (µM)[a] in migration assay
8a	2.2 ± 0.51
15	1.2 ± 0.24
27a	1.1 ± 0.22
27b	0.45 ± 0.17

^[a]The data are presented as the mean ± SD of three independent experiments with cells from different donors, in which median effective concentration values (EC₅₀) were determined by nonlinear regression analysis of the concentration-response curves (5–6 points) generated using GraphPad Prism 5 with 95% confidential interval (p < 0.05).

Some of the synthesized compounds were also evaluated for their ability to induce intracellular Ca²⁺ flux in mouse neutrophils and RBL cells transfected with Fpr1 or Fpr2 (Table 4). Although all compounds tested were active in human neutrophils, only eight of these compounds activated Ca²⁺ flux in mouse neutrophils. Of these, three compounds were also agonists for mouse Fpr1, mouse Fpr2, or both. The reason for this species-specific difference in activity is currently not understood; however, we and other group have observed quite different response patterns to some agonists and/or antagonists in human and mouse neutrophils [14].

Table 4.

Activity of pyridazinones in mouse neutrophils and mouse Fpr-transfected RBL cells.

Compd	mPMN[a]	mFpr1-RBL[a]	mFpr2-RBL[a]
8a	N.A.[b]	N.A.[b]	N.A.[b]
8b	8.4 ± 2.6 (75)	N.A.[b]	N.A.[b]
8c	N.A.[b]	N.A.[b]	N.A.[b]
8d	24.1 ± 6.1 (105)	N.A.[b]	N.A.[b]
8e	15.9 ± 4.9 (120)	N.A.[b]	N.A.[b]
8f	N.A.[b]	N.A.[b]	N.A.[b]
15	22.8 ± 6.4 (30)	N.A.[b]	N.A.[b]
19a	N.A.[b]	N.A.[b]	N.A.[b]
19b	N.A.[b]	N.A.[b]	N.A.[b]
20	1.0 ± 0.34 (100)	4.7 ± 1.3 (50)	N.A.[b]
24	13.5 ± 3.7 (50)	6.7 ± 2.1 (55)	9.4 ± 2.7 (65)
25	N.A.[b]	N.A.[b]	N.A.[b]
26	5.6 ± 2.4 (45)	N.A.[b]	N.A.[b]
27a	N.A.[b]	N.A.[b]	N.A.[b]
27b	6.0 ± 1.9 (80)	18.5 ± 4.1 (25)	18.3 ± 3.8 (35)

^[a]The EC₅₀ (µM) and Efficacy (% in brackets) were evaluated in Ca²⁺ flux assay. Values are expressed EC₅₀ presented as the average mean of three independent experiments, in which EC₅₀ values were determined by nonlinear regression analysis of the concentration-response curves (5–6 points) generated using GraphPad Prism 5 with 95% confidential interval (p < 0.05). Efficacy (in brackets) is expressed as % of the response induced by 5 nM WKYMVm in mouse polymorphonuclear neutrophils (mPMN) or RBL cells transfected with mouse Fpr1 (mFpr1-RBL) or Fpr2 (mFpr2-RBL).

^[b]N.A., no activity (no response was observed during first 2 min after addition of compounds under investigation) considering the limits of efficacy < 20% and EC₅₀ < 50 µM.

3.2. Molecular docking studies

Our data suggest that larger substituents at position 5 may cause steric hindrance and that the optimal length of this group was two carbon atoms. To evaluate the role of steric hindrance from bulky acyl groups, we performed molecular docking of compounds 8a and 8e into the binding site of an FPR1 homology model and compounds 13a–c into the binding site of an FPR2 homology model. With each of these pairs, the compounds differ in size of acyl or alkyl groups at position 5 of the pyridazine heterocycle.

As shown in Figure 2A, the best docking pose of 8a occupies areas of FPR1 characteristic of other FPR1 agonists [32]. For example, the p-bromo substituted aromatic ring of 8a is positioned near channel A, and the acetyl group enters cavity B (receptor regions designated as previously reported [32]). Additionally, strong H-bonds form between the carbonyl oxygen and anilide nitrogen atoms of 8a and Thr265 of FPR1, whereas a weaker H-bond is formed between the methoxy substituent and Arg205 (Figure 2B). These interactions may contribute to the agonist activity of compound 8a. Close contact of the acetyl group in 8a and FPR1 Leu198 was observed for the calculated ligand-receptor complex (Figure 2B), and the shortest interatomic distance between the acetyl oxygen and hydrogen atom of Leu198 located at the wall of cavity C was ~2.7 Å, indicating close proximity of the agonist 8a and the receptor. Thus, a larger acyl group at position 5 of the heterocycle would be expected to hinder ligand orientation similar to 8a. Indeed, the lowest-energy pose of compound 8e had a very different arrangement within the FPR1 binding site. The bulky cyclopentyl substituent prevented positioning the molecule near cavity B, such that 8e leans toward hole C of FPR1 (Figure 2A). This difference in binding explains the decreased agonist activity for 8e and its analogs with large acyl groups.

Figure 2.

Docking of compounds 8a and 8e into the FPR1 homology model. Panel A. Docking poses of compounds 8a (violet) and 8e (light-blue) into the FPR1 binding site of FPR1. The ligand binding site is represented by a surface colored according to electrostatic properties (red – negatively charged areas, blue – positively charged areas). Panel B. Docking pose of compound 8a and residues of FPR1 within 4 Å from the pose. H-bonds are shown as light-blue dashed lines.

A homology model of FPR2 was similarly used to perform molecular docking with 13a–c. These molecules have quite subtle differences in structure and are oriented in the binding site with brominated benzene rings directed deep into the binding site (Figure 3A). This orientation of the p-bromophenyl moiety is analogous to that observed previously for parent compounds [20]. Agonist 13a with an ethyl substituent at position 5 of the pyridazine ring is H-bonded with Thr177, while the m-methoxyphenyl substituent occupies a hydrophobic subpocket surrounded by Ala181, Gly264, Leu268, Tyr277, and Ile279 (Figure 3B). Changing the alkyl substituent from ethyl to n-butyl in compound 13c led to flipping of the molecule so that the butyl chain of this ligand was now located in the hydrophobic subpocket, while the m-methoxyphenyl group formed an H-bond with Asn171 (Figure 3C). For 13b, a flipped pose similar to 13c was obtained. However, the substituted pyridazine 13b did not form H-bonds with the receptor, which likely led to decreased affinity of the propyl derivative and complete lack of biological activity (see Table 2).

Figure 3.

Docking of compounds 13a–c into the FPR2 homology model. Panel A. Docking poses of compounds 13a (blue), 13b (green), and 13c (yellow) with a fragment of the FPR2 surface (cut for clarity). Panel B. Docking pose of compound 13a and residues of FPR2 within 4 Å from the pose. H-bonds are shown as light-blue dashed lines. Panel C. Docking pose of compound 13c and residues of FPR2 within 4 Å from the pose. H-bonds are shown as light-blue dashed lines.

Thus, larger substituents in position 5 of the pyridazine heterocycle cause steric effects on the binding modes of the ligands in the FPR1 and FPR2 binding sites, which is consistent with their reduced or lost agonist activity.

4. Conclusions

We describe the synthesis of new series of C-5 substituted 2-arylacetamide pyridazin-3(2H)-ones that exhibit improved potency and selectivity toward FPR isoforms. Biological analysis of these compounds confirmed the suitability of pyridazinone-based compounds as a relevant system to develop novel human FPR agonists. Indeed, the majority of compounds described herein were mixed FPR agonists, with compounds 8a and 27b being the most potent (EC₅₀ values in the nanomolar range). Overall, we show that modification of position C-5 of the pyridazinone ring in 2-arylacetamide pyridazin-3(2H)-ones represents an effective approach for the development of active FPR agonists. These compounds represent valuable tools for studying FPR activation and signaling in inflammatory conditions.

5. Experimental section

5.1. Chemistry

Reagents and starting materials were obtained from commercial sources. Extracts were dried over Na₂SO₄, and the solvents were removed under reduced pressure. All reactions were monitored by thin layer chromatography (TLC) using commercial plates precoated with Merck silica gel 60 F-254. Visualization was performed by UV fluorescence (λ_max = 254 nm) or by staining with iodine or potassium permanganate. Chromatographic separations were performed on a silica gel column using gravity chromatography (Kieselgel 40, 0.063–0.200 mm; Merck), flash chromatography (Kieselgel 40, 0.040–0.063 mm; Merck), or silica gel preparative TLC (Kieselgel 60 F₂₅₄, 20 × 20 cm, 2 mm). Yields refer to chromatographically and spectroscopically pure compounds, unless otherwise stated. Compounds were named following IUPAC rules, as applied by Beilstein-Institut AutoNom 2000 (4.01.305) or CA Index Name. All melting points were determined on a microscope hot stage Büchi apparatus and are uncorrected. The identity and purity of intermediates and final compounds were determined through NMR analysis and TLC chromatography. ¹H NMR, ¹³C NMR and NOESY spectra were recorded with Avance 400 instruments (Bruker Biospin Version 002 with SGU). Chemical shifts (δ) are reported in ppm to the nearest 0.01 ppm (for ¹H NMR) or 0.1 ppm (for ¹³C NMR), using the solvent as an internal standard. Coupling constants (J values) of ¹H NMR are given in Hz and were calculated using ‘TopSpin 1.3’ software rounded to the nearest 0.1 Hz. Microanalyses were performed with a Perkin-Elmer 260 elemental analyzer for C, H, and N, and the results were within ± 0.4 % of the theoretical values, unless otherwise stated.

5.1.1. General procedures for 2e and 2f

Ammonium formate (3.11 mmol) and 10% Pd/C (0.05 mmol) were added to a solution of the appropriate intermediate 1e and 1f [22–24] (1.04 mmol) in 5 mL of anhydrous EtOH, and the reaction was refluxed for 1 h. After cooling, 20 mL of CH₂Cl₂ were added to the mixture, and the precipitate was removed by filtration under vacuum. The organic layer was then evaporated, and the desired products 2e,f were obtained pure after recrystallization from EtOH.

5.1.1.1. 4-Amino-5-cyclopentanecarbonyl-6-methylpyridazin-3(2H)-one, 2e

Yield = 98%; mp = 225–227 °C (EtOH). ¹H-NMR (CDCl₃) δ 1.62-1.72 (m, 2H, cC₅H₉); 1.73-1.81 (m, 2H, cC₅H₉); 1.83-1.89 (m, 4H, cC₅H₉); 2.45 (s, 3H, CH₃); 3.44 (quin, 1H, cC₅H₉, J = 7.6 Hz); 6.76 (exch br s, 2H, NH₂); 7.98 (exch br s, 1H, NH). Anal. Calcd for C₁₁H₁₅N₃O₂ (221.26): C, 59.71; H, 6.83; N, 18.99; Found: C, 59.52; H, 6.84; N, 18.95.

5.1.1.2. 4-Amino-5-cyclohexanecarbonyl-6-methylpyridazin-3(2H)-one, 2f

Yield = 96%; mp = 232–234 °C (EtOH). ¹H-NMR (CDCl₃) δ 1.20-1.40 (m, 2H, cC₆H₁₁); 1.40-1.60 (m, 2H, cC₆H₁₁); 1.60-1.80 (m, 2H, cC₆H₁₁); 1.80-1.90 (m, 4H, cC₆H₁₁); 2.53 (s, 3H, CH₃); 2.90-2.93 (m, 1H, cC₆H₁₁);); 7.21 (exch br s, 2H, NH₂); 8.56 (exch br s, 1H, NH). Anal. Calcd for C₁₂H₁₇N₃O₂ (235.28): C, 61.26; H, 7.28; N, 17.86; Found: C, 61.11; H, 7.27; N, 17.89.

5.1.2. General procedures for 3d and 3e

To a solution of the appropriate substrate 2d [25] and 2e (1.60 mmol) in anhydrous CH₃CN (5 mL), K₂CO₃ (3.20 mmol) and ethyl bromoacetate (2.40 mol) were added. The solution was stirred for 2–3 h at reflux, and the solvent was evaporated. The residue was mixed with ice-cold water (10 mL) and the precipitate was recovered by suction and recrystallized from ethanol (compound 3d); alternatively, for compound 3e, the suspension was extracted with CH₂Cl₂ (3 × 15 mL) and the organic layer was dried over Na₂SO₄ and evaporated in vacuo. Finally, 3e was purified by column flash chromatography using cyclohexane/ethyl acetate 1:1 as eluent.

5.1.2.1. (5-Amino-3-methyl-6-oxo-4-pentanoylpyridazin-1(6H)-yl)acetic acid ethyl ester, 3d

Yield = 97%; mp = 92–94 °C (EtOH). ¹H-NMR (CDCl₃) δ 0.94 (t, 3H, CH₂CH₂CH₃, J = 7.2 Hz); 1.29 (t, 3H, OCH₂CH₃, J = 7.2 Hz); 1.36 (sext, 2H, CH₂CH₂CH₃, J = 7.2 Hz); 1.68 (quin, 2H, COCH₂CH₂, J = 7.2 Hz); 2.48 (s, 3H, N=CCH₃); 2.82 (t, 2H, COCH₂CH₂, J = 7.2 Hz); 4.24 (q, 2H, OCH₂CH₃, J = 7.2 Hz); 4.81 (s, 2H, NCH₂); 8.89 (exch br s, 2H, NH₂). Anal. Calcd for C₁₄H₂₁N₃O₄ (295.33): C, 56.94; H, 7.17; N, 14.23; Found: C, 57.08; H, 7.16; N, 14.27.

5.1.2.2. (5-Amino-4-cyclopentanecarbonyl-3-methyl-6-oxopyridazin-1(6H)-yl)acetic acid ethyl ester, 3e

Yield = 64%; mp = 83–85 °C (EtOH). ¹H-NMR (CDCl₃) δ 1.29 (t, 3H, CH₂CH₃, J = 7.2 Hz); 1.61-1.65 (m, 2H, cC₅H₉); 1.74-1.79 (m, 2H, cC₅H₉); 1.80-1.89 (m, 4H, cC₅H₉); 2.43 (s, 3H, N=CCH₃); 3.45 (m, 1H, cC₅H₉); 4.24 (q, 2H, CH₂CH₃, J = 7.2 Hz); 4.81 (s, 2H, NCH₂); 6.87 (exch br s, 2H, NH₂). Anal. Calcd for C₁₅H₂₁N₃O₄ (307.34): C, 58.62; H, 6.89; N, 13.67; Found: C, 58.47; H, 6.88; N, 13.71.

5.1.3. General procedures for 4d and 4e

To a solution of the suitable ester 3d and 3e (0.49–0.58 mmol) in 96% EtOH (5 mL), 6N NaOH (3 mL) was added. The reaction was carried out at 60°C for 1–2 h. After evaporation of the solvent, the mixture was diluted with ice-cold water, acidified with 6N HCl, and extracted with CH₂Cl₂ (3 × 15 mL). The organic layer was dried over Na₂SO₄ and evaporated in vacuo to give desired final compounds, which were purified by crystallization from ethanol.

5.1.3.1. (5-Amino-3-methyl-6-oxo-4-pentanoylpyridazin-1(6H)-yl)acetic acid, 4d

Yield = 98%; mp = 97–99 °C (EtOH). ¹H-NMR (CDCl₃) δ 0.93 (t, 3H, CH₂CH₃, J = 7.2 Hz); 1.36 (sext, 2H, CH₂CH₃, J = 7.2 Hz); 1.66 (quin, 2H, COCH₂CH₂, J = 7.2 Hz); 2.48 (s, 3H, N=CCH₃); 2.81 (t, 2H, COCH₂CH₂, J = 7.2 Hz); 4.87 (s, 2H, NCH₂); 5.32 (exch br s, 1H, OH); 7.45 (exch s br, 2H, NH₂). Anal. Calcd for C₁₂H₁₇N₃O₄ (267.28): C, 53.92; H, 6.41; N, 15.72; Found: C, 54.08; H, 6.40; N, 15.68.

5.1.3.2. (5-Amino-4-cyclopentanecarbonyl-3-methyl-6-oxopyridazin-1(6H)-yl)acetic acid, 4e

Yield = 73%; mp = 168–169 °C (EtOH). ¹H-NMR (CDCl₃) δ 1.60-1.70 (m, 2H, cC₅H₉); 1.71-1.80 (m, 2H, cC₅H₉); 1.81-1.91 (m, 4H, cC₅H₉); 2.45 (s, 3H, CH₃); 3.45 (m, 1H, cC₅H₉); 4.88 (s, 2H, NCH₂); 5.03 (exch br s, 1H, OH); 6.89 (exch br s, 2H, NH₂). Anal. Calcd for C₁₃H₁₇N₃O₄ (279.29): C, 55.91; H, 6.14; N, 15.05; Found: C, 55.76; H, 6.13; N, 15.09.

5.1.4. General procedures for 5a, and 5d–e

To a cooled (−5 °C) and stirred solution of the appropriate substrate 4a, 4d–e (4a [22]) (0.98 mmol) in anhydrous tetrahydrofuran (5 mL), Et₃N (3.43 mmol) was added. After 0.5 h, the mixture was allowed to warm up to 0 °C, and ethyl chloroformate (1.02 mmol) was added. After 1 h, 4-bromoaniline (1.96 mmol) was added. The reaction was carried out at room temperature for 16 h. The mixture was then concentrated in vacuo, diluted with cold water (20–30 mL), and extracted with CH₂Cl₂ (3 × 15 mL). The organic layer was dried over Na₂SO₄ and evaporated to obtain crude final compounds, which were purified by crystallization from ethanol for compound 5a and by column flash chromatography using cyclohexane/ethyl acetate 1:1 as eluent, followed by recrystallization from EtOH, for compounds 5d and 5e.

5.1.4.1. 2-(4-Acetyl-5-amino-3-methyl-6-oxopyridazin-1(6H)-yl)-N-(4-bromophenyl) acetamide, 5a

Yield = 90%; mp = 240–242 °C (EtOH). ¹H-NMR (CDCl₃) δ 2.57 (s, 3H, COCH₃); 2.60 (s, 3H, N=CCH₃); 4.90 (s, 2H, NCH₂); 7.54 (s, 4H, Ar); 8.50 (exch br s, 1H, NH); 6.32 (exch br s, 2H, NH₂). Anal. Calcd for C₁₅H₁₅BrN₄O₃ (379.21): C, 47.51; H, 3.99; N, 14.77; Found: C, 47.64; H, 3.98; N, 14.80.

5.1.4.2. 2-(5-Amino-3-methyl-6-oxo-4-pentanoylpyridazin-1(6H)-yl)-N-(4-bromophenyl) acetamide, 5d

Yield = 42%; mp = 164–165 °C (EtOH). ¹H-NMR (CDCl₃) δ 0.93 (t, 3H, CH₂CH₃, J = 7.2 Hz); 1.35 (sext, 2H, CH₂CH₃, J =7.2 Hz); 1.67 (quin, 2H, COCH₂CH₂, J = 7.2 Hz); 2.51 (s, 3H, N=CCH₃); 2.82 (t, 2H, COCH₂CH₂, J = 7.2 Hz); 4.88 (s, 2H, NCH₂); 7.38 (s, 4H, Ar); 8.63 (exch br s, 1H, NH); 8.56 (exch br s, 2H, NH₂). Anal. Calcd for C₁₈H₂₁BrN₄O₃ (421.29): C, 51.32; H, 5.02; N, 13.30; Found: C, 51.46; H, 5.02; N, 13.27.

5.1.4.3. 2-(5-Amino-4-cyclopentanecarbonyl-3-methyl-6-oxopyridazin-1(6H)-yl)-N-(4-bromophenyl)acetamide, 5e

Yield = 19%; mp = 179–181 °C (EtOH). ¹H-NMR (CDCl₃) δ 1.61-1.71 (m, 2H, cC₅H₉); 1.72-1.82 (m, 2H, cC₅H₉); 1.83-1.92 (m, 4H, cC₅H₉); 2.48 (s, 3H, CH₃); 3.45 (m, 1H, cC₅H₉); 4.89 (s, 2H, NCH₂); 6.89 (exch br s, 2H, NH₂); 7.40 (s, 4H, Ar); 8.66 (exch br s, 1H, NH). Anal. Calcd for C₁₉H₂₁BrN₄O₃ (433.30): C, 52.67; H, 4.89; N, 12.93; Found: C, 52.49; H, 4.88; N, 12.91.

5.1.5. General procedures for 5b–c, 5f

A mixture of the appropriate intermediate 2b–c or 2f (0.96 mmol), K₂CO₃ (1.93 mmol) and N-(4-bromophenyl)-2-chloroacetamide [27] (0.96–1.20 mmol) in CH₃CN (10–15 mL) was refluxed under stirring for 2–6 h. The mixture was then concentrated in vacuo, and ice cold water was added. After 1 h stirring in an ice-bath, the precipitate was recovered by suction to obtain pure compound 5b–c. For compound 5f further purification was performed by column chromatography using cyclohexane/ethyl acetate 1:2 as eluent.

5.1.5.1. 2-(5-Amino-3-methyl-6-oxo-4-propionylpyridazin-1(6H)-yl)-N-(4-bromophenyl) acetamide, 5b

Yield = 98%; mp = 174–175 °C (EtOH). ¹H-NMR (CDCl₃) δ 1.22 (t, 3H, CH₂CH₃, J = 7.2 Hz); 2.56 (s, 3H, N=CCH₃); 2.88 (q, 2H, CH₂CH₃, J = 7.2 Hz); 4.90 (s, 2H, NCH₂); 7.43 (s, 4H, Ar); 8.55 (exch br s, 1H, NH); 8.76 (exch br s, 2H, NH₂). Anal. Calcd for C₁₆H₁₇BrN₄O₃ (393.24): C, 48.87; H, 4.36; N, 14.25; Found: C, 48.99; H, 4.35; N, 14.21.

5.1.5.2. 2-(5-Amino-4-butyryl-3-methyl-6-oxopyridazin-1(6H)-yl)-N-(4-bromophenyl) acetamide, 5c

Yield = 96%; mp = 161–163 °C (EtOH). ¹H-NMR (CDCl₃) δ 0.99 (t, 3H, CH₂CH₃, J = 7.2 Hz); 1.75 (sext, 2H, CH₂CH₃, J = 7.2 Hz); 2.54 (s, 3H, N=CCH₃); 2.83 (t, 2H, COCH₂, J = 7.2 Hz); 4.90 (s, 2H, NCH₂); 7.43 (s, 4H, Ar); 8.57 (exch br s, 1H, NH); 9.04 (exch br s, 2H, NH₂). Anal. Calcd for C₁₇H₁₉BrN₄O₃ (407.26): C, 50.14; H, 4.70; N, 13.76; Found: C, 50.05; H, 4.69; N, 13.73.

5.1.5.3. 2-(5-Amino-4-cyclohexanecarbonyl-3-methyl-6-oxopyridazin-1(6H)-yl)-N-(4-bromophenyl) acetamide, 5f

Yield = 53%; mp = 97–99 °C (EtOH). ¹H-NMR (CDCl₃) δ 1.20-1.40 (m, 2H, cC₆H₁₁); 1.40-1.60 (m, 2H, cC₆H₁₁); 1.60-1.80 (m, 2H, cC₆H₁₁); 1.80-1.90 (m, 4H, cC₆H₁₁); 2.44 (s, 3H, CH₃); 2.91-2.98 (m, 1H, cC₆H₁₁); 4.89 (s, 2H, NCH₂); 6.73 (exch br s, 2H, NH₂); 7.38 (s, 4H, Ar); 8.70 (exch br s, 1H, NH). Anal. Calcd for C₂₀H₂₃BrN₄O₃ (447.33): C, 53.70; H, 5.18; N, 12.52; Found: C, 53.85; H, 5.17; N, 12.56.

5.1.6. General procedures for 6a–f

To a suspension of the appropriate intermediate 5a–f (0.91 mmol), copper(II) acetate (1.36 mmol), 3-methoxyphenylboronic acid (0.91–1.82 mmol) in CH₂Cl₂ (10 mL), and Et₃N (1.82 mmol) were added, and the mixture was stirred at room temperature for 16 h. The suspension was extracted with 15% aqueous ammonia (3 × 10 mL), and the organic layer was washed with water (10 mL) and dried over Na₂SO₄. After removal of the solvent in vacuo, the final desired compounds were purified by column flash chromatography using as eluent CH₂Cl₂/MeOH 98:2, for compounds 6b–f and cycloexane/ethyl acetate 1:3 for compound 6a. Compounds 6d and 6e were further purified by recrystallization from cyclohexane.

5.1.6.1. 2-[4-Acetyl-5-(3-methoxyphenylamino)-3-methyl-6-oxopyridazin-1(6H)-yl]-N-(4-bromophenyl)acetamide, 6a

Yield = 41%; mp = 115–116 °C (EtOH). ¹H-NMR (CDCl₃) δ 1.92 (s, 3H, COCH₃); 2.24 (s, 3H, N=C-CH₃); 3.80 (s, 3H, OCH₃); 4.97 (s, 2H, NCH₂); 6.61 (s, 1H, Ar); 6.68 (d, 1H, Ar, J = 8.4 Hz); 6.74 (d, 1H, Ar, J = 8.0 Hz); 7.24 (m, 1H, Ar); 7.42-7.48 (m, 4H, Ar); 8.5 (exch br s, 1H, NH); 9.21 (exch br s, 1H, NH). ¹³C-NMR (CDCl₃) δ 19.6 (CH₃); 31.2 (CH₃); 55.3 (CH₃); 57.0 (CH₂); 108.1 (CH); 112.0 (CH); 115.9 (CH); 116.6 (C); 117.0 (C); 121.4 (2CH); 130.5 (CH); 131.8 (2CH); 136.7 (C); 137.7 (C); 139.6 (C); 144.3 (C); 146.6 (C); 157.6 (C); 160.4 (C); 164.9 (C). Anal. Calcd for C₂₂H₂₁BrN₄O₄ (485.33): C, 54.44; H, 4.36; N, 11.54; Found: C, 54.57; H, 4.35; N, 11.57.

5.1.6.2. N-(4-Bromophenyl)-2-[5-(3-methoxyphenylamino)-3-methyl-6-oxo-4-propionyl pyridazin-1(6H)-yl]acetamide, 6b

Yield = 10%; mp = 87–89 °C (EtOH). ¹H-NMR (CDCl₃) δ 0.57 (t, 3H, CH₂CH₃, J = 7.2 Hz); 2.19 (s, 3H, N=CCH₃); 2.28 (q, 2H, CH₂CH₃, J = 7.2 Hz); 3.81 (s, 3H, OCH₃); 4.97 (s, 2H, NCH₂); 6.60 (s, 1H, Ar); 6.67 (d, 1H, Ar, J = 8.4 Hz); 6.73 (d, 1H, Ar, J = 8.4 Hz); 7.24 (t, 1H, Ar, J = 8.4 Hz); 7.45 (s, 4H, Ar); 7.69 (exch br s, 1H, NH); 8.61 (exch br s, 1H, NH). ¹³C-NMR (CDCl₃) δ 14.1 (CH₃); 20.3 (CH₃); 37.6 (CH₂); 55.4 (CH₃); 57.5 (CH₂); 108.5 (CH); 112.3 (CH); 115.3 (CH); 116.5 (C); 117.0 (C); 121.5 (2 × CH); 130.5 (CH); 132.0 (2 × CH); 136.6 (C); 137.4 (C); 139.7 (C); 144.6 (C); 146.5 (C); 157.6 (C); 160.6 (C); 165.1 (C). Anal. Calcd for C₂₃H₂₃BrN₄O₄ (499.36): C, 55.32; H, 4.64; N, 11.22; Found: C, 55.47; H, 4.63; N, 11.18.

5.1.6.3. N-(4-Bromophenyl)-2-[4-butyryl-5-(3-methoxyphenylamino)-3-methyl-6-oxo pyridazin-1(6H)-yl]acetamide, 6c

Yield = 11%; mp = 113–115 °C (EtOH). ¹H-NMR (CDCl₃) δ 0.67 (t, 3H, CH₂CH₃, J = 7.2 Hz); 1.09 (sext, 2H, CH₂CH₃, J = 7.2 Hz); 2.19 (s, 3H, N=CCH₃); 2.22 (t, 2H, COCH₂, J = 7.2 Hz); 3.80 (s, 3H, OCH₃); 4.97 (s, 2H, NCH₂); 6.60 (s, 1H, Ar); 6.67 (d, 1H, Ar, J = 8.0 Hz); 6.73 (d, 1H, Ar, J = 8.0 Hz); 7.23 (t, 1H, Ar, J = 8.0 Hz); 7.42-7.47 (m, 4H, Ar); 7.69 (exch br s, 1H, NH); 8.57 (exch br s, 1H, NH). ¹³C-NMR (CDCl₃) δ 13.5 (CH₃); 16.3 (CH₂); 20.3 (CH₃); 46.2 (CH₂); 55.4 (CH₃); 57.5 (CH₂); 108.3 (CH); 112.3 (CH); 115.1 (CH); 116.6 (C); 117.0 (C); 121.5 (2 × CH); 130.5 (CH); 132.0 (2 × CH); 136.6 (C); 137.3 (C); 139.7 (2 × C); 144.6 (C); 157.6 (C); 160.5 (C); 165.0 (C). Anal. Calcd for C₂₄H₂₅BrN₄O₄ (513.38): C, 56.15; H, 4.91; N, 10.91; Found: C, 56.63; H, 4.90; N, 10.88.

5.1.6.4. N-(4-Bromophenyl)-2-[5–(3-methoxyphenylamino)-3-methyl-6-oxo-4-pentanoyl pyridazin-1(6H)-yl]acetamide, 6d

Yield = 13%; mp = 96–98 °C (cyclohexane). ¹H-NMR (CDCl₃) δ 0.73 (t, 3H, CH₂CH₃, J = 7.2 Hz); 0.93-1.06 (m, 4H, (CH₂)₂CH₃); 2.17 (s, 3H, N=CCH₃); 2.22 (t, 2H, COCH₂CH₂, J = 7.2 Hz); 3.78 (s, 3H, OCH₃); 4.95 (s, 2H, NCH₂); 6.58 (s,1H, Ar); 6.64 (dd, 1H, Ar, J = 8.4 Hz, J = 2.4 Hz); 6.71 (dd, 1H, Ar, J = 8.4 Hz, J = 2.4 Hz); 7.12 (t, 1H, Ar, J = 8.4 Hz); 7.44 (s, 4H, Ar); 7.66 (exch br s, 1H, NH); 8.56 (exch br s, 1H, NH). ¹³C-NMR (CDCl₃) δ 13.0 (CH₃); 17.5 (CH₂); 20.1 (CH₃); 24.7 (CH₂); 44.1 (CH₂); 55.4 (CH₃); 57.5 (CH₂); 108.3 (CH); 112.3 (CH); 115.1 (CH); 116.6 (C); 117.1 (C); 121.4 (2 × CH); 129.8 (CH); 132.0 (2 × CH); 136.5 (C); 137.3 (C); 139.6 (C); 144.6 (C); 146.5 (C); 154.6 (C); 160.6 (C); 164.1 (C). Anal. Calcd for C₂₅H₂₇BrN₄O₄ (527.41): C, 56.93; H, 5.16; N, 10.62; Found: C, 57.03; H, 5.15; N, 10.57.

5.1.6.5. N-(4-Bromophenyl)-2-[4–cyclopentanecarbonyl-5-(3-methoxyphenylamino)-3-methyl-6-oxopyridazin-1(6H)-yl]acetamide, 6e

Yield = 11%; mp = 140–142 °C (cyclohexane). ¹H-NMR (CDCl₃) δ 1.13-1.22 (m, 2H, cC₅H₉); 1.29-1.36 (m, 2H, cC₅H₉); 1.37-1.43 (m, 2H, cC₅H₉); 1.53 (m, 2H, cC₅H₉); 2.19 (s, 3H, N=CCH₃); 3.04 (quin, 1H, cC₅H₉, J = 7.6 Hz); 3.87 (s, 3H, OCH₃); 4.97 (s, 2H, NCH₂); 6.56 (s, 1H, Ar); 6.67 (dd, 1H, Ar, J = 8.4 Hz, J = 2.4 Hz); 6.71 (dd, 1H, Ar, J = 8.4 Hz, J = 2.4 Hz); 7.22 (t, 1H, Ar, J = 8.4 Hz); 7.44-7.47 (m, 4H, Ar); 8.54 (exch br s, 1H, NH); 9.98 (exch br s, 1H, NH). ¹³C-NMR (CDCl₃) δ 20.9 (CH₃); 22.7 (CH₂); 25.8 (CH₂); 28.3 (CH₂); 31.9 (CH₂); 52.2 (CH); 55.4 (CH₃); 57.6 (CH₂); 107.5 (CH); 112.0 (CH); 114.6 (CH); 116.6 (C); 119.0 (C); 121.5 (2 × CH); 130.5 (CH); 132.0 (2 × CH); 136.6 (C); 137.5 (C); 138.5 (C); 145.2 (C); 147.3 (C); 157.8 (C); 160.4 (C); 165.3 (C). Anal. Calcd for C₂₆H₂₇BrN₄O₄ (539.42): C, 57.89; H, 5.05; N, 10.39; Found: C, 57.70; H, 5.05; N, 10.42.

5.1.6.6. N-(4-Bromophenyl)-2-[4-cyclohexanecarbonyl-5-(3-methoxyphenylamino)-3-methyl-6-oxopyridazin-1(6)-yl]acetamide, 6f

Yield = 34%; mp = 181–183 °C (EtOH). ¹H-NMR (CDCl₃) δ 0.80-1.00 (m, 8H, cC₆H₁₁); 1.40-1.60 (m, 2H, cC₆H₁₁); 2.00 (s, 3H, N=CCH₃); 2.46-2.50 (m, 1H, cC₆H₁₁); 3.68 (s, 3H, OCH₃); 4.87 (s, 2H, NCH₂); 6.60-6.62 (m, 3H, Ar); 7.12-7.17 (m, 1H, Ar); 7.56-7.47 (m, 4H, Ar); 8.82 (exch br s, 1H, NH); 10.44 (exch br s, 1H, NH). ¹³C-NMR (CDCl₃) δ 20.6 (CH₃); 25.8 (2 × CH₂); 27.6 (2 × CH₂); 29.5 (CH₂); 51.3 (CH); 55.5 (CH₃); 55.6 (CH₂); 108.2 (CH); 111.1 (CH); 115.2 (CH); 115.6 (C); 115.8 (C); 121.6 (2 × CH); 130.3 (CH); 132.1 (2 × CH); 138.1 (C); 138.5 (C); 141.6 (2 × C); 142.6 (C); 157.1 (C); 160.3 (C); 165.7 (C). Anal. Calcd for C₂₇H₂₉BrN₄O₄ (553.45): C, 58.59; H, 5.28; N, 10.12; Found: C, 58.40; H, 5.27; N, 10.09.

5.1.7. General procedures for 7a–d

To a cooled (0 °C) suspension of compounds 2a–d (1.20 mmol) in 5 mL of MeOH, NaBH₄ (3.59–7.18 mmol) was slowly added, and the mixture was stirred for 1 h at room temperature. After cooling, cold water was added (10 mL) and compound 7a was recovered by filtration under vacuum. For compounds 7b–d the suspension was extracted with ethyl acetate (3 × 10 mL) and the organic layer was dried over Na₂SO₄ and evaporated to obtain crude final compounds, which were purified by crystallization from ethanol.

5.1.7.1. 4-Amino-5-(1-hydroxyethyl)-6-methylpyridazin-3(2H)-one, 7a

Yield = 49%; mp = >300 °C (MeOH). ¹H-NMR (DMSO-d₆) δ 1.28 (d, 3H, CHCH₃, J = 6.4 Hz); 2.09 (s, 3H, N=CCH₃); 4.78 (q, 1H, CH, J = 6.4 Hz); 5.98 (exch br s, 1H, NH); 8.56 (exch br s, 2H, NH₂). Anal. Calcd for C₇H₁₁N₃O₂ (169.18): C, 49.70; H, 6.55; N, 24.84; Found: C, 49.85; H, 6.56; N, 24.79.

5.1.7.2. 4-Amino-5-(1-hydroxypropyl)-6-methylpyridazin-3(2H)-one, 7b

Yield = 74%; mp = 184–189 °C (EtOH). ¹H-NMR (DMSO-d₆) δ 0.86 (t, 3H, CH₂CH₃, J = 7.2 Hz); 1.56-1.61 (m, 1H, CH₂CH₃); 1.65-1.70 (m, 1H, CH₂CH₃); 2.09 (s, 3H, N=CCH₃); 4.49-4.54 (m, 1H, CHOH); 8.56 (exch br s, 2H, NH₂); 12.26 (exch br s, 1H, NH). Anal. Calcd for C₈H₁₃N₃O₂ (183.21): C, 52.45; H, 7.15; N, 22.94; Found: C, 52.57; H, 7.17; N, 22.89.

5.1.7.3. 4-Amino-5-(1-hydroxybutyl)-6-methylpyridazin-3(2H)-one, 7c

Yield = 72%; mp = 212–215 °C (EtOH). ¹H-NMR (DMSO-d₆) δ 0.86 (t, 3H, CH₂CH₃, J = 7.2 Hz); 1.24-1.30 (m, 2H, CH₂CH₂CH₃); 1.40-1.49 (m, 2H, CH₂CH₂CH₃); 2.07 (s, 3H, N=CCH₃); 4.60 (m, 1H, CHOH); 8.92 (exch br s, 2H, NH₂); 12.26 (exch br s, 1H, NH). Anal. Calcd for C₉H₁₅N₃O₂ (197.23): C, 54.81; H, 7.67; N, 21.30; Found: C, 54.66; H, 7.69; N, 21.36.

5.1.7.4. 4-Amino-5-(1-hydroxypentyl)-6-methylpyridazin-3(2H)-one, 7d

Yield = 74%; mp = 206–208 °C (EtOH). ¹H-NMR (DMSO-d₆) δ 0.85 (t, 3H, CH₂CH₃, J = 7.2 Hz); 1.18-1.30 (m, 4H, CH₂CH₂CH₂CH₃); 1.40-1.71 (m, 2H, CH₂CH₂CH₂CH₃); 2.08 (s, 3H, N=CCH₃); 4.58 (m, 1H, CHOH); 5.52 (exch br d, 1H, OH); 5.91 (exch br s, 2H, NH₂); 12.59 (exch br s, 1H, NH). Anal. Calcd for C₁₀H₁₇N₃O₂ (211.26): C, 56.85; H, 8.11; N, 19.89; Found: C, 57.01; H, 8.12; N, 19.83.

5.1.8. General procedures for 8a–d

A stirred suspension of appropriate intermediates 7a–d (1.20 mmol) in PPA (29.5–55.1 mmol) was heated at 90–110 °C for 2–5 h. After cooling, ice-cold water was added, and the mixture was neutralized by slow addition of 6N NaOH. The resulting suspension was extracted with ethyl acetate, and the organic phase was dried over Na₂SO₄. Evaporation of the solvent under vacuum resulted in final compounds 8a–b. Instead, for compounds 8c–d, after neutralization, was observed the formation of a precipitate, which was recovered by suction.

5.1.8.1. 4-Amino-6-methyl-5-vinylpyridazin-3(2H)-one, 8a

Yield = 90%; mp = 270–272 °C (EtOH). ¹H-NMR (CDCl₃) δ 2.22 (s, 3H, CH₃); 5.19 (exch br s, 2H, NH₂); 5.61 (d, 1H, CH-H, J = 18 Hz); 5.74 (d, 1H, CH-H, J = 12 Hz); 6.46 (dd, 1H, CH, J = 18 Hz, J = 12 Hz); 8.03 (exch br s, 1H, NH). Anal. Calcd for C₇H₉N₃O (151.17): C, 55.62; H, 6.00; N, 27.80; Found: C, 55.75; H, 5.99; N, 27.74.

5.1.8.2. 4-Amino-6-methyl-5-propenylpyridazin-3(2H)-one, 8b

Yield = 62%; mp = 264–266 °C (EtOH). ¹H-NMR (CDCl₃) δ 1.84 (d, 3H, CH₃CH, J = 6.8 Hz); 2.10 (s, 3H, CH₃); 5.92-5.97 (m, 1H, CH=CH-CH₃); 6.01-6.13 (m, 1H, CH=CH-CH₃); 6.89 (exch br s, 2H, NH₂); 12.30 (exch br s, 1H, NH). Anal. Calcd for C₈H₁₁N₃O (165.19): C, 58.17; H, 6.71; N, 25.44; Found: C, 58.03; H, 6.70; N, 25.51.

5.1.8.3. 4-Amino-5-but-1-enyl-6-methylpyridazin-3(2H)-one, 8c

Yield = 42%; mp = 257–259 °C (EtOH). ¹H-NMR (CDCl₃) δ 1.11-1.16 (m, 3H, CH₂CH₃, J = 7.2 Hz); 1.82-1.91 (m, 2H, CHCH₂CH₃); 2.38 (s, 3H, N=CCH₃); 4.57-4.63 (m, 1H, CH=CH-CH₂); 5.03 (m, 1H, CH=CH-CH₃); 5.55 (exch br s, 1H, NH); 6.59 (exch br s, 2H, NH₂). Anal. Calcd for C₉H₁₃N₃O (179.22): C, 60.32; H, 7.31; N, 23.45; Found: C, 60.48; H, 6.58; N, 23.39.

5.1.8.4. 4-Amino-6-methyl-5-pent-1-enylpyridazin-3(2H)-one, 8d

Yield = 90%; mp = 216–218 °C dec. (EtOH). ¹H-NMR (CDCl₃) δ 0.96 (t, 3H, CH₂CH₃, J = 7.4 Hz); 1.48-1.53 (m, 2H, CH₂CH₂CH₃); 2.19 (s, 3H, N=CCH₃); 2.21-2.26 (m, 2H, CH₂CH₂CH₃); 5.06 (exch br s, 2H, NH₂); 6.02-6.09 (m, 2H, CH=CH-CH₂); 8.03 (exch br s, 1H, NH). Anal. Calcd for C₁₀H₁₅N₃O (193.25): C, 62.15; H, 7.82; N, 21.74; Found: C, 62.31; H, 7.83; N, 21.78.

5.1.9. General procedures for 9a–d

Compound 8a–d (0.46 mmol) was subjected to catalytic reduction with 10% Pd/C (0.23 mmol) in EtOH (20 mL) for 3 h in a Parr instrument at 30 PSI. The catalyst was filtered off, and the solvent was evaporated under vacuum, affording in the final compounds.

5.1.9.1. 4-Amino-5-ethyl-6-methylpyridazin-3(2H)-one, 9a

Yield = 56%; mp = 260–262 °C (EtOH). ¹H-NMR (CDCl₃) δ 1.13 (t, 3H, CH₂CH₃, J = 7.6 Hz); 2.25 (s, 3H, N=CCH₃); 2.39 (q, 2H, CH₂CH₃, J = 7.6 Hz); 6.81 (exch br s, 2H, NH₂); 8.03 (exch br s, 1H, NH). Anal. Calcd for C₇H₁₁N₃O (153.18): C, 54.89; H, 7.24; N, 27.43; Found: C, 54.76; H, 7.23; N, 27.51.

5.1.9.2. 4-Amino-6-methyl-5-propylpyridazin-3(2H)-one, 9b

Yield = 85%; mp = 242–244 °C dec. (EtOH). ¹H-NMR (CDCl₃) δ 0.91 (t, 3H, CH₂CH₃, J = 7.2 Hz); 1.34-1.40 (m, 2H, CH₂CH₂CH₃); 2.09 (s, 3H, N=CCH₃); 2.28-2.33 (m, 2H, CH₂CH₂CH₃); 5.89 (exch br s, 2H, NH₂); 12.18 (exch br s, 1H, NH). Anal. Calcd for C₈H₁₃N₃O (167.21): C, 57.46; H, 7.84; N, 25.13; Found: C, 57.59; H, 7.83; N, 25.06.

5.1.9.3. 4-Amino-5-butyl-6-methylpyridazin-3(2H)-one, 9c

Yield = 82%; mp = 205–206 °C (EtOH). ¹H-NMR (CDCl₃) δ 0.96 (t, 3H, CH₂CH₃, J = 7.2 Hz); 1.43-1.50 (m, 4H, CH₂CH₂CH₂CH₃); 2.34 (s, 3H, N=CCH₃); 2.38 (t, 2H, CH₂CH₂CH₂CH₃, J = 7.2 Hz); 6.03 (exch br s, 2H, NH₂); 11.89 (exch br s, 1H, NH). Anal. Calcd for C₉H₁₅N₃O (181.23): C, 59.64; H, 8.34; N, 23.19; Found: C, 59.77; H, 8.33; N, 23.12.

5.1.9.4. 4-Amino-6-methyl-5-pentylpyridazin-3(2H)-one, 9d

Yield = 70%; mp = 230–232 °C (EtOH). ¹H-NMR (CDCl₃) δ 0.90 (t, 3H, CH₂CH₃, J = 7.2 Hz); 1.35-1.40 (m, 4H, CH₂CH₂CH₂CH₃); 1.45-1.53 (m, 2H, CH₂CH₂CH₂CH₃); 2.26 (s, 3H, N=CCH₃); 2.34-2.40 (m, 2H, CH₂C₄H₉); 4.86 (exch br s, 1H, NH); 6.88 (exch br s, 2H, NH₂). Anal. Calcd for C₁₀H₁₇N₃O (195.26): C, 61.51; H, 8.78; N, 21.52; Found: C, 61.70; H, 8.77; N, 21.56.

5.1.10. General procedures for 10a–d

Compounds 10a–d were obtained starting from 9a–d following the same procedure described for 5b– c and 5f. For compounds 10b–d the suspension was extracted with ethyl acetate (3 × 15 mL); the organic layer was dried over Na₂SO₄ and evaporated to give desired final compounds which were purified by column chromatography using cyclohexane/ethyl acetate 1:2 (for 10b and 10d) or cyclohexane/ethyl acetate 1:3 (for 10c) as eluents.

5.1.10.1. 2-(5-Amino-4-ethyl-3-methyl-6-oxopyridazin-1(6H)-yl)-N-(4-bromophenyl) acetamide, 10a

Yield = 74%; mp = 206–208 °C (EtOH). ¹H-NMR (CDCl₃) δ 1.14 (t, 3H, CH₂CH₃, J = 7.6 Hz); 2.28 (s, 3H, N=CCH₃); 2.41 (q, 2H, CH₂CH₃, J = 7.6 Hz); 4.89 (s, 2H, NCH₂); 6.88 (exch br s, 2H, NH₂); 7.36-7.40 (m, 4H, Ar); 9.00 (exch br s, 1H, NH). Anal. Calcd for C₁₅H₁₇BrN₄O₂ (365.23): C, 49.33; H, 4.69; N, 15.34; Found: C, 49.21; H, 4.68; N, 15.38.

5.1.10.2. 2-(5-Amino-3-methyl-6-oxo-4-propylpyridazin-1(6H)-yl)-N-(4-bromophenyl) acetamide, 10b

Yield = 19%; mp = 197–199 °C dec. (EtOH). ¹H-NMR (CDCl₃) δ 1.02 (t, 3H, CH₂CH₃, J = 7.2 Hz); 1.51-1.59 (m, 2H, CH₂CH₂CH₃); 2.27 (s, 3H, N=CCH₃); 2.37 (t, 2H, CH₂CH₂CH₃, J = 7.2 Hz); 4.89 (s, 2H, NCH₂); 6.84 (exch br s, 2H, NH₂); 7.35-7.41 (m, 4H, Ar); 9.04 (exch br s, 1H, NH). Anal. Calcd for C₁₆H₁₉BrN₄O₂ (379.25): C, 50.67; H, 5.05; N, 14.77; Found: C, 50.53; H, 5.06; N, 14.74.

5.1.10.3. 2-(5-Amino-4-butyl-3-methyl-6-oxopyridazin-1(6H)-yl)-N-(4-bromophenyl) acetamide, 10c

Yield = 26%; oil. ¹H-NMR (CDCl₃) δ 0.96 (t, 3H, (CH₂)₃CH₃, J = 7.2 Hz); 1.43-1.50 (m, 4H, CH₂CH₂CH₂CH₃); 2.34 (s, 3H, N=CCH₃); 2.38 (t, 2H, CH₂(CH₂)₂CH₃, J = 6.4 Hz); 4.90 (s, 2H, NCH₂); 6.59 (exch br s, 2H, NH₂); 7.34-7.40 (m, 4H, Ar); 8.87 (exch br s, 1H, NH). Anal. Calcd for C₁₇H₂₁BrN₄O₂ (393.28): C, 51.92; H, 5.38; N, 14.25; Found: C, 51.76; H, 5.07; N, 14.22.

5.1.10.4. 2-(5-Amino-3-methyl-6-oxo-4-pentylpyridazin-1(6H)-yl)-N-(4-bromophenyl) acetamide, 10d

Yield = 75%; mp = 167–171 °C (EtOH). ¹H-NMR (CDCl₃) δ 0.90 (t, 3H, (CH₂)₄CH₃, J = 7.2 Hz); 1.34-1.39 (m, 4H, (CH₂)₂CH₂CH₂CH₃); 1.47-1.52 (m, 2H, CH₂CH₂(CH₂)₂CH₃); 2.26 (s, 3H, N=CCH₃); 2.33-2.38 (m, 2H, CH₂(CH₂)₃CH₃); 4.88 (s, 2H, NCH₂); 6.59 (exch br s, 2H, NH₂); 7.36-7.41 (m, 4H, Ar); 9.04 (exch br s, 1H, NH). Anal. Calcd for C₁₈H₂₃BrN₄O₂ (407.30): C, 53.08; H, 5.69; N, 13.76; Found: C, 53.26; H, 5.68; N, 13.79.

5.1.11. General procedures for 11a–d

Compounds 11a–d were obtained starting from 10a–d following the same procedure described for 6a–f. The final desired compounds were purified by column chromatography using as eluent CH₂Cl₂/MeOH 98:2 for compounds 11a, CH₂Cl₂/MeOH 99:1 for 11c and cyclohexane/ethyl acetate 1:2 for compounds 11b and 11d.

5.1.11.1. N-(4-Bromophenyl)-2-[4-ethyl-5-(3-methoxyphenylamino)-3-methyl-6-oxopyridazin-1(6H)-yl]acetamide, 11a

Yield = 18%; mp = 81–84 °C (EtOH). ¹H-NMR (CDCl₃) δ 0.88 (t, 3H, CH₂CH₃, J = 7.2 Hz); 2.28 (s, 3H, N=CCH₃); 2.30-2.36 (m, 2H, CH₂CH₃); 3.78 (s, 3H, CH₃O); 4.93 (s, 2H, NCH₂); 6.51-6.66 (m, 3H, Ar); 7.01 (exch br s, 1H, NH); 7.19 (t, 1H, Ar, J = 8.2 Hz); 7.32-7.39 (m, 4H, Ar); 9.11 (exch br s, 1H, NH). Anal. Calcd for C₂₂H₂₃BrN₄O₃ (471.35): C, 56.06; H, 4.92; N, 11.89; Found: C, 56.21; H, 4.91; N, 11.92.

5.1.11.2. N-(4-Bromophenyl)-2-[5-(3-methoxyphenylamino)-3-methyl-6-oxo-4-propyl pyridazin-1(6H)-yl]acetamide, 11b

Yield = 18%; oil. ¹H-NMR (CDCl₃) δ 0.66 (t, 3H, (CH₂)₂CH₃, J = 7.2 Hz); 1.26-1.32 (m, 2H, CH₂CH₂CH₃); 2.23-2.31(m, 2H, CH₂CH₂CH₃); 2.35 (s, 3H, N=CCH₃); 3.78 (s, 3H, OCH₃); 4.92 (s, 2H, NCH₂); 6.52-6.68 (m, 4H, Ar); 6.84 (exch br s, 1H, NH); 7.35-7.42 (m, 4H, Ar); 9.04 (exch br s, 1H, NH). Anal. Calcd for C₂₃H₂₅BrN₄O₃ (485.37): C, 56.91; H, 5.19; N, 11.54; Found: C, 56.73; H, 5.20; N, 11.51.

5.1.11.3. N-(4-Bromophenyl)-2-[4-butyl-5-(3-methoxyphenylamino)-3-methyl-6-oxopyridazin-1(6H)-yl]acetamide, 11c

Yield = 15%; oil. ¹H-NMR (CDCl₃) δ 0.68 (t, 3H, (CH₂)₃CH₃, J = 7.2 Hz); 1.00-1.08 (m, 4H, CH₂CH₂CH₂CH₃); 2.24-2.30 (m, 2H, CH₂CH₂CH₂CH₃); 2.33 (s, 3H, N=CCH₃); 3.78 (s, 3H, OCH₃); 4.93 (s, 2H, NCH₂); 6.60-6.69 (m, 4H, Ar); 6.84 (exch br s, 1H, NH); 7.35-7.42 (m, 4H, Ar); 9.01 (exch br s, 1H, NH). Anal. Calcd for C₂₄H₂₇BrN₄O₃ (499.40): C, 57.72; H, 5.45; N, 11.22; Found: C, 57.85; H, 5.46; N, 11.25.

5.1.11.4. N-(4-Bromophenyl)-2-[5-(3-methoxyphenylamino)-3-methyl-6-oxo-4-pentyldazin-1(6H)-yl]acetamide, 11d

Yield = 16%; oil. ¹H-NMR (CDCl₃) δ 0.68 (t, 3H, (CH₂)₄CH₃, J = 7.2 Hz); 0.98-1.01 (m, 2H, (CH₂)₃CH₂CH₃); 1.08-1.14 (m, 2H, (CH₂)₂CH₂CH₂CH₃); 1.24-1.29 (m, 4H, CH₂CH₂CH₂CH₃); 2.31 (s, 3H, N=CCH₃); 3.77 (s, 3H, OCH₃); 4.92 (s, 2H, NCH₂); 6.50-6.69 (m, 4H, Ar); 6.91 (exch br s, 1H, NH); 7.33-7.39 (m, 4H, Ar); 9.06 (exch br s, 1H, NH). Anal. Calcd for C₂₅H₂₉BrN₄O₃ (513.43): C, 58.48; H, 5.69; N, 10.91; Found: C, 58.31; H, 5.70; N, 10.97.

5.1.12. 2-(5-Amino-3-methyl-6-oxo-4-vinylpyridazin-1(6H)-yl)-N-(4-bromophenyl)acetamide, 12

Compound 12 was obtained following the same procedure described for 5b–c, 5f starting from 8a, followed by purification with column flash chromatography using cyclohexane/ethyl acetate 1:4 as eluent. Yield = 52%; mp = 194–196 °C (EtOH). ¹H-NMR (CDCl₃) δ 2.26 (s, 3H, N=CCH₃); 4.96 (s, 2H, NCH₂); 5.26 (exch br s, 2H, NH₂); 5.64 (d, 1H, CHCH-H, J = 18 Hz); 5.78 (d, 1H, CHCH-H, J = 12 Hz); 6.47 (dd, 1H, CHCH₂, J =18 Hz, J =12 Hz); 7.38 (m, 4H, Ar); 9.00 (exch br s, 1H, NH). Anal. Calcd for C₁₅H₁₅BrN₄O₂ (363.21): C, 49.60; H, 4.16; N, 15.43; Found: C, 49.76; H, 4.17; N, 15.39.

5.1.13. N-(4-Bromophenyl)-2-[5-(3-methoxyphenylamino)-3-methyl-6-oxo-4-vinylpyridazin-1(6H)-yl]acetamide, 13

Compound 13 was obtained starting from 12 following the general procedure described for 6a–f. The residue was purified by crystallization from ethanol, followed by column flash chromatography using CH₂Cl₂/MeOH 98:2 as eluent. Yield = 39%; mp = 182–184 °C (EtOH). ¹H-NMR (CDCl₃) δ 2.32 (s, 3H, N=CCH₃); 3.79 (s, 3H, OCH₃); 5.00 (s, 2H, NCH₂); 5.07 (d, 1H, CHCH-H, J = 18 Hz); 5.29 (d, 1H, CHCH-H, J = 12 Hz); 6.24 (dd, 1H, CHCH₂, J = 18 Hz, J = 12 Hz); 6.41 (s, 1H, Ar); 6.48 (d, 1H, Ar, J = 8.0 Hz); 6.63 (d, 1H, Ar, J = 8.0 Hz); 7.18 (t, 1H, Ar, J = 8.0 Hz); 7.38 (m, 4H, Ar); 7.51 (exch br s, 1H, NH); 9.09 (exch br s, 1H, NH). ¹³C-NMR (CDCl₃) δ 21.0 (CH₃); 55.3 (CH₃); 57.5 (CH₂); 108.4 (CH); 109.4 (CH); 114.8 (CH); 116.8 (C); 117.0 (C); 121.4 (2 × CH); 121.9 (CH₂); 129.2 (2 × CH); 131.8 (2 × CH); 135.9 (C); 136.8 (C); 139.9 (C); 147.3 (C); 158.2 (C); 159.9 (C); 165.3 (C). Anal. Calcd for C₂₂H₂₁BrN₄O₃ (469.33): C, 56.30; H, 4.51; N, 11.94; Found: C, 56.48; H, 4.50; N, 11.91.

5.1.14. General procedures for 15a and 15b

A catalytic amount of Et₃N (0.20 mL) was added to a solution of 14 [28] (1.32 mmol) in 3 mL of appropriate solvent (MeOH for 15a or EtOH for 15b), and the reaction was carried out at 60°C for 4 h. After cooling, the precipitate was recovered by suction to obtain pure 15a–b.

5.1.14.1. Methyl 5-amino-1,6-dihydro-3-methyl-6-oxopyridazine-4-carboxylate, 15a

Yield = 99%; mp = 253–254 °C (MeOH). ¹H-NMR (CDCl₃) δ 2.49 (s, 3H, N=CCH₃); 3.94 (s, 3H, COOCH₃); 5.43 (exch br s, 1H, NH); 8.23 (exch br s, 2H, NH₂). Anal. Calcd for C₇H₉N₃O₃ (183.16): C, 45.90; H, 4.95; N, 22.94; Found: C, 45.76; H, 4.94; N, 22.99.

5.1.14.2. Ethyl 5-amino-1,6-dihydro-3-methyl-6-oxopyridazine-4-carboxylate, 15b

Yield = 73%; mp = 226–228 °C (EtOH). ¹H-NMR (CDCl₃) δ 1.43 (t, 3H, CH₂CH₃, J = 7.2 Hz); 2.50 (s, 3H, N=CCH₃); 4.40 (q, 2H, CH₂CH₃, J = 7.2 Hz); 5.86 (exch br s, 1H, NH); 8.87 (exch br s, 2H, NH₂). Anal. Calcd for C₈H₁₁N₃O₃ (197.19): C, 48.73; H, 5.62; N, 21.31; Found: C, 48.86; H, 5.61; N, 21.36.

5.1.15. General procedures for 16a and 16b

Compounds 16a–b were obtained starting from 15a–b following the same procedure described for 5b–c, and 5f.

5.1.15.1. Methyl 1-[(4-bromophenylcarbamoyl)methyl]-5-amino-1,6-dihydro-3-methyl-6-oxopyridazine-4-carboxylate, 16a

Yield = 98%; mp = 197–199 °C (EtOH). ¹H-NMR (CDCl₃) δ 2.51 (s, 3H, N=CCH₃); 3.94 (s, 3H, OCH₃); 4.90 (s, 2H, NCH₂); 7.43 (s, 4H, Ar); 8.50 (exch br s, 1H, NH); 8.90 (exch br s, 1H, NH₂). Anal. Calcd for C₁₅H₁₅BrN₄O₄ (395.21): C, 45.59; H, 3.83; N, 14.18; Found: C, 45.68; H, 3.82; N, 14.21.

5.1.15.2. Ethyl 1-[(4-bromophenylcarbamoyl)methyl]-5-amino-1,6-dihydro-3-methyl-6-oxopyridazine-4-carboxylate, 16b

Yield = 94%; mp = 217–219 °C (EtOH). ¹H-NMR (CDCl₃) δ 1.43 (t, 3H, CH₂CH₃, J = 7.2 Hz); 2.52 (s, 3H, N=CCH₃); 4.40 (q, 2H, CH₂CH₃, J = 7.2 Hz); 4.91 (s, 2H, NCH₂); 7.43 (s, 4H, Ar); 8.52 (exch br s, 1H, NH); 9.21 (exch br s, 2H, NH₂). Anal. Calcd for C₁₆H₁₇BrN₄O₄ (409.23): C, 46.96; H, 4.19; N, 13.69; Found: C, 47.09; H, 4.18; N, 13.65.

5.1.16. General procedures for 17a and 17b

Compounds 17a–b were obtained starting from 16a–b following the same procedure described for 6a–f. The final desired compounds were purified by crystallization from ethanol, followed by column flash chromatography using CH₂Cl₂/MeOH 98:2 as eluent.

5.1.16.1. Methyl 1-[(4-bromophenylcarbamoyl)methyl]-5-(3-methoxyphenylamino)-1,6-dihydro-3-methyl-6-oxopyridazine-4-carboxylate, 17a

Yield = 12%; mp = 194–195 °C (EtOH). ¹H-NMR (CDCl₃) δ 2.33 (s, 3H, CCH₃); 3.20 (s, 3H, COOCH₃); 3.82 (s, 3H, OCH₃); 4.97 (s, 2H, NCH₂); 6.64 (s, 1H, Ar); 6.70 (d, 1H, Ar, J = 8.4 Hz); 6.77 (d, 1H, Ar, J = 8.4 Hz); 7.26 (t, 1H, Ar, J = 8.4 Hz); 7.42 (s, 4H, Ar); 7.90 (exch br s, 1H, NH); 8.59 (exch br s, 1H, NH). ¹³C-NMR (CDCl₃) δ 20.6 (CH₃); 51.6 (CH₃); 55.4 (CH₃); 57.3 (CH₂); 108.0 (CH); 110.0 (C); 112.3 (CH); 114.0 (C); 115.0 (CH); 117.0 (C); 121.5 (2 × CH); 130.2 (CH); 131.9 (2 × CH); 136.5 (C); 138.8 (C); 139.4 (C); 145.5 (C); 157.0 (C); 161.3 (C); 165.4 (C). Anal. Calcd for C₂₂H₂₁BrN₄O₅ (501.33): C, 52.71; H, 4.22; N, 11.18; Found: C, 52.87; H, 4.23; N, 11.16.

5.1.16.2. Ethyl 1-[(4-bromophenylcarbamoyl)methyl]-5-(3-methoxyphenylamino)-1,6-dihydro-3-methyl-6-oxopyridazine-4-carboxylate, 17b

Yield = 17%; mp = 185–187 °C (EtOH). ¹H-NMR (CDCl₃) δ 1.02 (t, 3H, CH₂CH₃, J = 7.2 Hz); 2.33 (s, 3H, N=CCH₃); 3.58 (q, 2H, CH₂CH₃, J = 7.2 Hz); 3.80 (s, 3H, OCH₃); 4.98 (s, 2H, NCH₂); 6.65 (s, 1H, Ar); 6.70 (d, 1H, Ar, J = 8.4 Hz); 6.75 (d, 1H, Ar, J = 8.4 Hz); 7.25 (t, 1H, Ar, J = 8.4 Hz); 7.41 (s, 4H, Ar); 7.92 (exch br s, 1H, NH); 8.80 (exch br s, 1H, NH). ¹³C-NMR (CDCl₃) δ 13.7 (CH₃); 20.7 (CH₃); 55.4 (CH₃); 57.2 (CH₂); 61.5 (CH₂); 107.9 (CH); 108.1 (C); 112.1 (CH); 114.9 (CH); 117.0 (C); 121.5 (2 × CH); 130.1 (CH); 131.9 (2 × CH); 136.7 (C); 138.5 (C); 139.6 (C); 145.4 (C); 157.1 (C); 160.3 (C); 164.8 (C); 165.2 (C). Anal. Calcd for C₂₃H₂₃BrN₄O₅ (515.36): C, 53.60; H, 4.50; N, 10.87; Found: C, 53.47; H, 4.49; N, 10.90.

5.1.17. 1-[(4-Bromophenylcarbamoyl)-methyl]-5-(3-methoxyphenylamino)-1,6-dihydro-3-methyl-6-oxopyridazine-4-carboxylate, 18

To prepare 18, 0.12 mmol of 17a were suspended in 2N NaOH (2 mL), and the reaction was stirred for 1 h at room temperature. The mixture was then diluted with ice-cold water and acidified with 6N HCl. The resulting precipitate was recovered by suction and purified by column flash chromatography using CH₂Cl₂/MeOH 9:1 as eluent. Yield = 39%; mp = 211–213 °C (EtOH). ¹H-NMR (CD₃OD-d₄) δ 2.39 (s, 3H, N=CCH₃); 3.76 (s, 3H, OCH₃); 4.93 (s, 2H, NCH₂); 6.57-6.69 (m, 3H, Ar); 7.14 (t, 1H, Ar, J = 8.4 Hz); 7.53-7.54 (m, 4H, Ar). ¹³C-NMR (MeOD-d₄) δ 19.7 (CH₃); 54.3 (CH₃); 55.0 (CH₂); 107.2 (CH); 109.9 (CH); 114.1 (CH); 116.2 (C); 117.0 (C); 119.5 (C); 121.5 (2 × CH); 128.8 (CH); 131.4 (2 × CH); 136.9 (C); 137.5 (C); 141.1 (C); 145.0 (C); 157.5 (C); 159.9(C); 166.0 (C). Anal. Calcd for C₂₁H₁₉BrN₄O₅ (487.30): C, 51.76; H, 3.93; N, 11.50; Found: C, 51.90; H, 3.92; N, 11.47.

5.1.18. N-(4-Bromophenyl)-2-(3,4-dimethyl-7-oxoisoxazolo[3,4-d]pyridazin-6(7H)-yl) acetamide, 20

Compound 20 was obtained starting from 19 [22] following the same procedure described for 5a, d, e. Finally, compound 20 was purified by crystallization from EtOH. Yield = 46%; mp = 227–228 °C (EtOH). ¹H-NMR (CDCl₃) δ 2.52 (s, 3H, N=CCH₃); 2.90 (s, 3H, C=CCH₃); 4.93 (s, 2H, NCH₂); 7.40-7.45 (m, 4H, Ar); 8.25 (exch br s, 1H, NH). Anal. Calcd for C₁₅H₁₃BrN₄O₃ (377.19): C, 47.76; H, 3.47; N, 14.85; Found: C, 47.82; H, 3.46; N, 14.88.

5.1.19. N-(4-Bromophenyl)-2-[3-(2-dimethylaminovinyl)-4-methyl-7-oxoisoxazolo[3,4-d] pyridazin-6(7H)-yl]acetamide, 21

A suspension of intermediate 20 (0.79 mmol) in DMF/DMA (4.5 mL) was heated at 90 °C for 3 h in the dark. After cooling, ice-cold water was added to the mixture, and the precipitate formed was recovered through filtration under vacuum. Yield = 88%; mp = 228–229 °C (EtOH). ¹H-NMR (CDCl₃) δ 2.47 (s, 3H, N=CCH₃); 2.60 (s, 3H, NCH₃); 2.89 (s, 3H, NCH₃); 4.91 (s, 2H, NCH₂); 5.22 (d, 1H, CH=CH, J = 12.4 Hz); 7.38-7.46 (m, 4H, Ar); 7.62 (d, 1H, CH=CH, J = 12.4 Hz); 8.65 (exch br s, 1H, NH). Anal. Calcd for C₁₈H₁₈BrN₅O₃ (432.27): C, 50.01; H, 4.20; N, 16.20; Found: C, 50.15; H, 4.19; N, 16.17.

5.1.20. 2-[5-Amino-4-(3-dimethylaminoacryloyl)-3-methyl-6-oxo-5,6-dihydro-4H-pyridazin-1-yl]-N-(4-bromo-phenyl)acetamide, 22

To a stirred solution of 21 (0.19 mmol) in anhydrous CH₃CN (5 mL), Mo(CO)₆ (0.21 mmol) and a catalytic amount of H₂O (0.1 mL) were added at 50 °C. The reaction was then carried out at reflux for 1.5 h [29]. The solvent was removed under vacuum, and ice-cold water was added to the mixture. After 1 h stirring in an ice-bath, the precipitate was recovered by suction and suspended in CH₂Cl₂. The resulting precipitate was filtered, and the pure 22 was obtained through evaporation of the organic layer under vacuum and purification by crystallization from EtOH. Yield = 46%; mp = 208–209 °C (EtOH). ¹H-NMR (DMSO) δ 2.08 (s, 3H, N=CCH₃); 2.57 (s, 3H, NCH₃); 2.60 (s, 3H, NCH₃); 4.91 (s, 2H, NCH₂); 6.41 (d, 1H, CH=CH, J = 7.2 Hz); 7.48-7.54 (m, 4H, Ar); 7.82 (d, 1H, CH=CH, J = 5.6 Hz); 8.78 (exch br s, 2H, NH₂); 10.44 (exch br s, 1H, NH). Anal. Calcd for C₁₈H₂₀BrN₅O₃ (434.29): C, 49.78; H, 4.64; N, 16.13; Found: C, 49.81; H, 4.63; N, 16.11.

5.1.21. 2-[5-Amino-3-methyl-6-oxo-4-(1H-pyrazol-3-yl)-6H-pyridazin-1-yl]-N-(4-bromo--phenyl)acetamide, 23

Hydrazine hydrate (1.38 mmol) was slowly added drop wise to a solution of intermediate 22 (0.69 mmol) in 4 mL of 96% EtOH, and the reaction was refluxed for 3 h. After cooling, the solvent was removed under vacuum. Ice-cold water was added, and the precipitate was recovered by filtration under vacuum. A second batch of compound 23 was obtained through extraction of the aqueous phase with CH₂Cl₂ (3 × 15 mL), drying over Na₂SO₄, and evaporation under vacuum. Yield = 67%; mp = 229–230 °C (EtOH). 1H-NMR (CDCl₃) δ 2.44 (s, 3H, N=CCH₃); 4.98 (s, 2H, NCH₂); 6.60 (d, 1H, Ar, J = 1.6 Hz); 6.80 (exch br s, 1H, NH); 7.40-7.45 (m, 4H, Ar); 7.75 (d, 1H, Ar, J = 2.0 Hz); 8.98 (exch br s, 1H, NH); 9.03 (exch br s, 2H, NH₂). Anal. Calcd for C₁₆H₁₅BrN₆O₂ (403.23): C, 47.66; H, 3.75; N, 20.84; Found: C, 47.52; H, 3.76; N, 20.88.

5.1.22. 2-[5-Amino-3-methyl-4-(1-methyl-1H-pyrazol-3-yl)-6-oxopyridazin-1(6H)-yl]-N-(4-bromophenyl)acetamide, 24

K₂CO₃ (0.87 mmol) was added to a solution of intermediate 23 (0.43 mmol) in 4 mL of anhydrous DMF. After 2 h stirring, 1.19 mmol of CH₃I were added, and the reaction was carried out for additional 3 h at 90°C. After cooling, ice-cold water was added, and the mixture was extracted with CH₂Cl₂. The organic phase was dried over Na₂SO₄ and evaporated. Yield = 46%; mp = 232–233 °C (EtOH). ¹H-NMR (CDCl₃) δ 2.44 (s, 3H, N=CCH₃); 4.01 (s, 3H, NCH₃); 4.97 (s, 2H, NCH₂); 6.50 (d, 1H, Ar, J = 2.0 Hz); 7.38-7.42 (m, 4H, Ar); 7.44 (exch br s, 2H, NH₂,); 7.45-7.50 (m, 1H, Ar); 9.02 (exch br s, 1H, NH). Anal. Calcd for C₁₇H₁₇BrN₆O₂ (417.26): C, 48.93; H, 4.11; N, 20.14; Found: C, 48.76; H, 4.10; N, 20.18.

5.1.23. General procedures for 25a and 25b

Compounds 25a–b were obtained starting from 24 following the same general procedure described for 6a–f. After removal of the solvent in vacuo, compound 25a was obtained by crystallization from ethanol and preparative TLC using ethyl acetate as eluent.

5.1.24. N-(4-Bromophenyl)-2-[5-(3-methoxyphenylamino)-3-methyl-4-(1-methyl-1H-pyrazol-3-yl)-6-oxo-6H-pyridazin-1-yl]acetamide, 25a

Yield = 15%; mp = 112–113 °C (EtOH). ¹H-NMR (CDCl₃) δ 2.29 (s, 3H, N= CCH₃); 3.67 (s, 3H, NCH₃); 3.72 (s, 3H, OCH₃); 5.01 (s, 2H, NCH₂); 5.99 (d, 1H, Ar, J = 2.4 Hz); 6.30 (s, 1H, Ar); 6.42 (d, 1H, Ar, J = 8.0 Hz); 6.5 (d, 1H, Ar, J = 8.4 Hz); 6.97 (t, 1H, Ar, J = 8.0 Hz); 7.07 (exch br s, 1H, NH); 7.42-7.48 (m, 4H, Ar); 7.81 (d, 1H, Ar, J = 2.4 Hz); 8.89 (exch br s, 1H, NH). ¹³C-NMR (CDCl₃) δ 21.4 (CH₃); 38.7 (CH₃); 55.1 (CH₃); 57.7 (CH₂); 107.6 (CH); 108.0 (CH); 110.2 (CH); 115.0 (CH); 116.9 (C); 121.6 (2CH); 125.0 (CH); 126.5 (CH); 131.9 (2CH); 135.2 (C); 136.8 (C); 137.5 (C); 139.8 (C); 145.0 (C); 146.3 (C); 157.0 (C); 160.2 (C); 165.4 (C). Anal. Calcd for C₂₄H₂₃BrN₆O₃ (523.38): C, 55.08; H, 4.43; N, 16.06; Found: C, 55.23; H, 4.42; N, 16.11.

5.1.25. N-(4-Bromophenyl)-2-[5-(4-methoxyphenylamino)-3-methyl-4-(1-methyl-1H-pyrazol-3-yl)-6-oxo-6H-pyridazin-1-yl]acetamide, 25b

Yield = 64%; mp = 103–104 °C (EtOH). ¹H-NMR (CDCl₃) δ 2.21 (s, 3H, N=CCH₃); 3.67 (s, 3H, NCH₃); 3.75 (s, 3H, OCH₃); 5.00 (s, 2H, CH₂CO); 5.93 (s, 1H, Ar); 6.58-6.59 (m, 2H, Ar); 6.73-6.75 (m, 2H, Ar); 7.40-7.46 (m, 4H, Ar); 7.71 (s, 1H, Ar); 8.91 (exch br s, 1H, NH). ¹³C-NMR (CDCl₃) δ 21.2 (CH₃); 38.6 (CH₃); 55.5 (CH₃); 57.5 (CH₂); 107.8 (CH); 112.9 (CH); 113.6 (2CH); 116.9 (C); 121.5 (2CH); 125.0 (CH); 126.0 (CH); 130.1 (C); 131.0 (CH); 131.5 (C); 132.0 (CH); 136.8 (C); 138.2 (C); 144.2 (C); 148.5 (C); 156.7 (C); 157.0 (C); 165.4 (C). Anal. Calcd for C₂₄H₂₃BrN₆O₃ (523.38): C, 55.08; H, 4.43; N, 16.06; Found: C, 55.17; H, 4.43; N, 16.10.

5.1.26. General procedures for 27a and 27b

Compounds 27a–b were obtaining starting from appropriate substrate 26a [30] and 26b [31] following the same procedure described for 5b–c, 5f. The desired final compounds were purified by column flash chromatography using, as eluent, cyclohexane/ethyl acetate 1:2 for compound 27a and CH₂Cl₂/MeOH 9.5:0.5 for compound 27b.

5.1.26.1. 2-(3-Amino-5-methyl-2-oxopyridin-1(2H)-yl)-N-(4-bromophenyl)acetamide, 27a

Yield = 34%; mp = 151–153 °C dec. (EtOH). ¹H-NMR (CDCl₃) δ 2.06 (s, 3H, N=CCH₃); 4.64 (s, 2H, NCH₂); 6.51 (exch br s, 1H, NH); 6.66 (s, 1H, Ar); 7.36-7.47 (m, 4H, Ar); 9.64 (exch br s, 2H, NH₂). Anal. Calcd for C₁₄H₁₄BrN₃O₂ (336.18): C, 50.02; H, 4.20; N, 12.50; Found: C, 50.16; H, 4.21; N, 12.47.

5.1.26.2. 2-(5-Amino-3-methyl-6-oxopyridazin-1(6H)-yl)-N-(4-bromophenyl)acetamide, 27b

Yield = 29%; mp = 241–244 °C dec. (EtOH). ¹H-NMR (CDCl₃) δ 2.22 (s, 3H, N=CCH₃); 4.90 (s, 2H, NCH₂); 5.88 (exch br s, 2H, NH₂); 6.19 (s, 1H, Ar); 7.33-7.39 (m, 4H, Ar); 8.91 (exch br s, 2H, NH₂). Anal. Calcd for C₁₃H₁₃BrN₄O₂ (337.17): C, 46.31; H, 3.89; N, 16.62; Found: C, 46.44; H, 3.90; N, 16.67.

5.1.27. General procedures for 28a and 28b

Compounds 28a–b were obtaining starting from appropriate substrate 27a–b following the same procedure described for 6a–f. The desired final compounds were purified by column flash chromatography using, as eluent, cyclohexane/ethyl acetate 1:2 for compound 28a and CH₂Cl₂/MeOH 9.5:0.5 for compound 28b.

5.1.27.1. N-(4-Bromophenyl)-2-[3-(3-methoxyphenylamino)-5-methyl-2-oxopyridin-1(2H)-yl]acetamide, 28a

Yield = 11%; mp = 210–213 °C (EtOH). ¹H-NMR (CDCl₃) δ 2.08 (s, 3H, N=CCH₃); 3.80 (s, 3H, OCH₃); 4.71 (s, 2H, NCH₂); 6.51 (exch br s, 1H, NH); 6.59 (d, 1H, Ar, J = 7.3 Hz); 6.73 (m, 2H, Ar); 6.77 (d, 1H, Ar, J = 7.2 Hz); 7.05 (s, 1H, Ar); 7.25 (s, 1H, Ar); 7.36-7.41 (m, 4H, Ar); 9.51 (exch br s, 1H, NH). Anal. Calcd for C₂₁H₂₀BrN₃O₂ (442.31): C, 57.02; H, 4.56; N, 9.50; Found: C, 57.19; H, 4.57; N, 9.48.

5.1.27.2. N-(4-Bromophenyl)-2-[5-(3-methoxyphenylamino)-3-methyl-6-oxopyridazin-1(6H)-yl]-acetamide, 28b

Yield = 16%; oil. ¹H-NMR (CDCl₃) δ 2.27 (s, 3H, N=CCH₃); 3.82 (s, 3H, OCH₃); 4.94 (s, 2H, NCH₂); 6.43 (exch br s, 1H, NH); 6.64 (s, 1H, Ar); 6.71-6.76 (m, 2H, Ar); 6.81 (m, 1H, Ar); 7.35 (m, 1H, Ar); 7.38-7.43 (m, 4H, Ar); 8.68 (exch br s, 1H, NH). Anal. Calcd for C₂₀H₁₉BrN₃O₂ (443.29): C, 54.19; H, 4.32; N, 12.64; Found: C, 54.31; H, 4.31; N, 12.61.

5.2. Biology

5.2.1. Cell culture

Human promyelocytic leukemia HL-60 cells stably transfected with FPR1 (FPR1-HL60), FPR2 (FPR2-HL60), or FPR3 (FPR3-HL60) (kind gift from Dr. Marie-Josephe Rabiet, INSERM, Grenoble, France) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 10 mM HEPES, 100 µg/ml streptomycin, 100 U/ml penicillin, and G418 (1 mg/mL), as previously described [33]. were a Wild-type HL-60 cells were cultured under the same conditions, but without G418. Rat basophilic leukemia (RBL-2H3) cells transfected with mouse Fpr1 (Fpr1-RBL) or mouse Fpr2 (Fpr2-RBL) were cultured in DMEM supplemented with 20% (v/v) FBS, 10 mM HEPES, 100 µg/ml streptomycin, 100 U/ml penicillin, and G418 (250 µg/ml). Wild-type HL-60 and RBL-2H3 cells were cultured under the same conditions, but without G418.

5.2.2. Isolation of human neutrophils

Blood was collected from healthy donors in accordance with a protocol approved by the Institutional Review Board at Montana State University. Neutrophils were purified from the blood using dextran sedimentation, followed by Histopaque 1077 gradient separation and hypotonic lysis of red blood cells, as previously described [34]. Isolated neutrophils were washed twice and resuspended in Hank's balanced salt solution (HBSS) without Ca²⁺ and Mg²⁺ (HBSS⁻). Neutrophil preparations were routinely > 95% pure, as determined by light microscopy, and > 98% viable, as determined by trypan blue exclusion.

5.2.3. Isolation of mouse neutrophils

Mouse bone marrow neutrophils were isolated from bone marrow leukocyte preparations, as described previously [35]. In brief, bone marrow leukocytes were flushed from tibias and femurs of BALB/c mice with HBSS⁻, filtered through a 70 µm nylon cell strainer (BD Biosciences, Franklin Lakes, NJ) to remove cell clumps and bone particles, and resuspended in HBSS⁻ at 10⁶ cells/ml. Bone marrow leukocytes were resuspended in 3 ml of 45% Percoll solution and layered on top of a Percoll gradient consisting of 2 ml each of 50, 55, 62, and 81% Percoll solutions in a conical 15-ml polypropylene tube. The gradient was centrifuged at 1600g for 30 min at 10°C, and the cell band located between the 61 and 81% Percoll layers was collected. The cells were washed, layered on top of 3 ml of Histopaque 1119, and centrifuged at 1600g for 30 min at 10°C to remove contaminating red blood cells. The purified neutrophils were collected, washed, and resuspended in HBSS⁻. All animal use was conducted in accordance with a protocol approved by the Institutional Animal Care and Use Committee at Montana State University.

5.2.4. Ca²⁺ mobilization assay

Changes in intracellular Ca²⁺ were measured with a FlexStation II scanning fluorometer using a FLIPR 3 calcium assay kit (Molecular Devices, Sunnyvale, CA) for human neutrophils and HL-60 cells, as described previously [36]. All active compounds were evaluated in wild-type HL-60 and RBL cells to verify that the agonists are inactive in non-transfected cells. Human neutrophils, HL-60 or RBL cells, suspended in HBSS⁻ containing 10 mM HEPES, were loaded with Fluo-4 AM dye (Invitrogen; 1.25 µg/mL final concentration) and incubated for 30 min in the dark at 37 °C. After dye loading, the cells were washed with HBSS⁻ containing 10 mM HEPES, resuspended in HBSS containing 10 mM HEPES, and aliquotted into the wells of a flat-bottomed, half-area-well black microtiter plates (2 × 10⁵ cells/well). The compound of interest was added from a source plate containing dilutions of test compounds in HBSS with 10% dimethyl sulfoxide (DMSO), and changes in fluorescence were monitored (λ_ex = 485 nm, λ_em = 538 nm) every 5 s for 240 s at room temperature after automated addition of compounds. Maximum change in fluorescence, expressed in arbitrary units over baseline, was used to determine agonist response. Responses were normalized to the response induced by 5 nM fMLF (Sigma Chemical Co., St. Louis, MO) for FPR1-HL60 cells and human neutrophils, or 5 nM WKYMVm (Calbiochem, San Diego, CA) for murine neutrophils, FPR2-HL60, FPR3-HL60, Fpr1-RBL, and Fpr2-RBL cells, which were assigned a value of 100%. Curve fitting (5–6 points) and calculation of median effective concentration values (EC₅₀) were performed by nonlinear regression analysis of the concentration-response curves generated using Prism 5 (GraphPad Software, Inc., San Diego, CA).

5.2.5. Cell migration assay

Human neutrophils were suspended in HBSS containing 2% (v/v) fetal bovine serum (FBS) (2×10⁶ cells/mL), and cell migration was analyzed in 96-well ChemoTx chemotaxis chambers (Neuroprobe, Gaithersburg, MD), as previously described [20]. Briefly, lower wells were loaded with 30 µL of HBSS containing 2% (v/v) FBS and the indicated concentrations of test compound, DMSO (negative control), or 1 nM fMLF as a positive control. Neutrophils were added to the upper wells and allowed to migrate through the 5.0 µm pore polycarbonate membrane filter for 60 min at 37 °C and 5% CO₂. The number of migrated cells was determined by measuring ATP in lysates of transmigrated cells using a luminescence-based assay (CellTiter-Glo; Promega, Madison, WI), and luminescence measurements were converted to absolute cell numbers by comparison of the values with standard curves obtained with known numbers of neutrophils. The results are expressed as percentage of negative control and were calculated as follows: (number of cells migrating in response to test compounds/spontaneous migration in response to control medium)×100. EC₅₀ values were determined by nonlinear regression analysis of the concentration-response curves generated using Prism 5 software.

5.3. Molecular modeling

We used the FPR1 and FPR2 homology models created previously [26, 37]. Both models are based on the crystal structure of the bovine rhodopsin receptor. Before docking, structures of compounds 8a, 8e, and 13a–c were built and optimized using HyperChem 7.0 software with the MM+ force field and saved in Tripos MOL2 format. The ligand structures were then imported into MVD with the options “Create explicit hydrogens”, “Assign charges (calculated by MVD)”, and “Detect flexible torsions in ligands” enabled. The molecules were docked into FPR1 and FPR2 using the search spaces as applied in our previous publications [20, 32] and with a rigid receptor structure. MolDock score functions were applied with 0.3 Å grid resolution. Ligand flexibility was accounted for with respect to torsion angles auto-detected in MVD. The “Internal HBond” option was activated in the “Ligand evaluation” menu of Docking Wizard. Thirty docking runs were performed for each molecule. The option “Return multiple poses for each run” was enabled, and the post-processing options “Energy minimization” and “Optimize H-bonds” were applied after docking. Similar poses were clustered at a RMSD threshold of 1 Å.