Synthesis of Five- and Six-Membered N-Phenylacetamido Substituted Heterocycles as Formyl Peptide Receptor Agonists

Article abstract of DOI:10.1002/ddr.21370

(Table presented.). Formyl peptide receptors (FPRs) are G-protein-coupled receptors that play an important role in the regulation of inflammatory process and cellular dysfunction. In humans, three different isoforms are expressed (FPR1, FPR2, and FPR3). FPR2 appears to be directly involved in the resolution of inflammation, an active process carried out by specific pro-resolving mediators that modulate specific receptors. Previously, we identified 2-arylacetamido pyridazin-3(2H)-ones as FPR1- or FPR2-selective agonists, as well as a large number of mixed-agonists for the three isoforms. Here, we report a new series of 2-arylacetamido pyridazinones substituted at position 5 and their development as FPR agonists. We also synthesized a new series of 2-oxothiazolones bearing a 4-bromophenylacetamido fragment, which was fundamental for activity in the pyridazinone series. The compounds of most interest were 4a, a potent, mixed FPR agonist recognized by all three isotypes (FPR1 EC₅₀ = 19 nM, FPR2 EC₅₀ = 43 nM, FPR3 EC₅₀ = 40 nM), and 4b, which had potent activity and a preference for FPR2 (EC₅₀ = 13 nM). These novel compounds may represent valuable tools for studying FPR activation and signaling. Drug Dev Res 78 : 49–62, 2017.

Full text of DOI:10.1002/ddr.21370

DRUG DEVELOPMENT RESEARCH 00 : 00–00 (2016)
Research Article
Synthesis of Five- and Six-Membered N-Phenylacetamido
Substituted Heterocycles as Formyl Peptide Receptor
Agonists
Claudia Vergelli,1 Igor A. Schepetkin,2 Giovanna Ciciani,1 Agostino Cilibrizzi,3
Letizia Crocetti,1 Maria Paola Giovannoni,1* Gabriella Guerrini,1 Antonella Iacovone,1
Liliya N. Kirpotina,2 Richard D. Ye,4 and Mark T. Quinn2
1
ꢀ
Department of NEUROFARBA, Sezione di Farmaceutica e Nutraceutica, Universita degli Studi
di Firenze, Via Ugo Schiff 6, Sesto, Fiorentino 50019, Italy
2Department of Microbiology and Immunology, Montana State University, Bozeman,
Montana 59717
3Department of Chemistry, Imperial College London, South Kensington, London SW7 2AZ,
United Kingdom
4Institute of Chinese Medical Sciences, University of Macau, Macau, China
Strategy, Management and Health Policy
Enabling
Preclinical Development
Toxicology, Formulation
Drug Delivery,
Clinical Development
Phases I-III
Preclinical
Research
Postmarketing
Phase IV
Technology,
Genomics,
Proteomics
Regulatory, Quality,
Manufacturing
Pharmacokinetics
ABSTRACT
Formyl peptide receptors (FPRs) are G-protein-coupled receptors that play an important
role in the regulation of inflammatory process and cellular dysfunction. In humans, three different iso-
forms are expressed (FPR1, FPR2, and FPR3). FPR2 appears to be directly involved in the resolution of
inflammation, an active process carried out by specific pro-resolving mediators that modulate specific
receptors. Previously, we identified 2-arylacetamido pyridazin-3(2H)-ones as FPR1- or FPR2-selective
agonists, as well as a large number of mixed-agonists for the three isoforms. Here, we report a new
series of 2-arylacetamido pyridazinones substituted at position 5 and their development as FPR ago-
nists. We also synthesized a new series of 2-oxothiazolones bearing a 4-bromophenylacetamido frag-
ment, which was fundamental for activity in the pyridazinone series. The compounds of most interest
were 4a, a potent, mixed FPR agonist recognized by all three isotypes (FPR1 EC50 5 19 nM, FPR2
EC50 5 43 nM, FPR3 EC50 5 40 nM), and 4b, which had potent activity and a preference for FPR2
(EC50 5 13 nM). These novel compounds may represent valuable tools for studying FPR activation and
C
V
signaling. Drug Dev Res 00 : 000–000, 2016.
2016 Wiley Periodicals, Inc.
Key words: formyl peptide receptor; agonist; pyridazin-3(2H)-one; neutrophil; Ca21 flux
Conflict of Interest: The authors declare to have no financial/commercial conflict of interest.
Contract grant sponsor: National Institutes of Health IDeA Program COBRE; contract grant numbers: GM110732 (to M.T.Q.) and
AI033503 (to R.D.Y.), 1009546; Contract grant sponsors: M.J. Murdock Charitable Trust (to M.T.Q.), USDA National Institute of Food and
Agriculture Hatch project (to M.T.Q.), Montana University System Research Initiative 51040-MUSRI2015-03, and the Montana State Univer-
sity Agricultural Experiment Station (to M.T.Q.)
*Correspondence to: Maria Paola Giovannoni, Dipartimento di NEUROFARBA, Via Ugo Schiff 6, Sesto Fiorentino 50019
Firenze. E-mail mariapaola.giovannoni@unifi.it
Received 20 September 2016; Accepted 12 October 2016
Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/ddr.21370
C
V
2016 Wiley Periodicals, Inc.

2
VERGELLI ET AL.
INTRODUCTION
Inflammation is a primary response of the
immune system to injury or infection by invading
microbial pathogens and is mediated by the action of
many cell types with distinct functions [Beutler,
2004]. The critical role of the inflammatory process
in health and disease is essential; however, the mech-
anisms leading to the restoration of homeostasis and
resolution of inflammation (ROI) have only recently
been elucidated. These studies have demonstrated
that the ROI is an active process carried out by
specific pro-resolving mediators, such as lipoxin,
resolvins, protectins, and maresins, which have anti-
inflammatory and pro-resolving activities that are
mediated via interactions with specific receptors
[Serhan et al., 2002; Serhan and Levy, 2003; Serhan
et al., 2011; Serhan et al., 2015]. Among the recep-
tors activated by pro-resolving mediators is formyl
peptide receptor 2 (FPR2), which belongs to a group
of G-protein coupled receptors [Corminboeuf and
Fig. 1. FPR1/FPR2 mixed agonists.
4-bromophenylacetamide moiety at N-2, and a benzyl/
aniline group at position 4 of the scaffold. In the series
of 4-anilino derivatives, we identified some compounds
with good potency and with a moderate preference for
Leroy, 2015]. In addition to FPR2, two other iso- FPR2 subtype [Vergelli et al., 2016]. In the present
forms, FPR1 and FPR3, have been identified in article, we further investigated 4-benzylpyridazinones
humans and exhibit a high level of amino acid homol-
ogy with FPR2 [Ye et al., 2009]. Despite their high
level of sequence homology, the FPRs differ in their
ability to bind the prototypic N-formyl peptide N-for-
myl-methionine-leucine-phenylalanine (fMLF). FPR1
has high affinity receptor for this ligand, FPR2 low-
affinity receptor, while FPR3 does not bind fMLF.
FPR1 and FPR2 have a similar distribution in a vari-
ety of tissues and cells involved in inflammation,
including endothelial cells, platelets and immature
dendritic cells, monocytes, neutrophils, macrophages,
T lymphocytes, and epithelial cells [Migeotte et al.,
2006; Ye et al., 2009], whereas FPR3 is expressed in
monocytes and dendritic cells [Migeotte et al., 2005,
2006].
The involvement of FPR2 in the ROI makes
this receptor an attractive target for treating a variety
of pathologies, including rheumatoid arthritis, asth-
ma, chronic obstructive pulmonary disease, cancer,
cardiovascular, and Alzheimer’s diseases [Libby,
2002, 2015; Mantovani et al., 2008; Bozinovski et al.,
2013]. There are only a few reported examples of
FPR2-selective ligands [Schepetkin et al., 2014a].
Our research in the field of FPR ligands led to
the identification of a large number of pyridazin-
3(2H)-one derivatives that were mixed FPR1/FPR2
agonists (Fig. 1, general structure A) [Cilibrizzi et al.,
2009, 2012; Cilibrizzi et al., 2013; Crocetti et al.,
2013; Giovannoni et al., 2013, Vergelli et al., 2016].
derivatives as isosteres of the 4-anilino derivatives and
examine their structure-activity relationships [Vergelli
et al., 2016], as well as several derivatives lacking a
benzyl fragment at position 4. We also synthesized and
evaluated a series of 2-oxothiazolones by maintaining
the 4-bromophenylacetamide side fragment.
MATERIALS AND METHODS
Chemistry
The synthetic pathways followed to obtain the
final compounds are shown in Figures 2 and 3, and
the structures were confirmed by analytical and spec-
tral data.
In Figure 2, synthesis of the 4-benzylpyridazinones
4a-f and the 4-unsubstituted derivatives of type 6 and 7
is presented. Previously described oxoacids 1a-e [Metz
and Schwenker, 1980; Xu et al., 2005; Holloway et al.,
2010] were reacted with hydrazine hydrate to obtain the
corresponding 4,5-dihydropyridazinones (6)2a-e (com-
pounds (6)2a,b,d [Pinna et al., 1988; Haider and
Holzer, 2004; Xu et al., 2005]), which were converted
into derivatives 3a-f by treatment with the appropriate
arylaldehyde in the presence of KOH. Alkylation of 3a-f
with commercially available N-(4-bromophenyl)22-
chloroacetamide in anhydrous CH3CN resulted in the
final compounds 4a-f. Compounds (6)2a,b [Pinna
et al., 1988; Haider and Holzer, 2004] were also key
intermediates for the synthesis of 4-unsubstituted deriva-
Key requirements for activity were the presence of a tives
6 and 7 by direct alkylation with N-(4-
methyl group at position 6 of the pyridazinone ring, a bromophenyl)22-chloroacetamide (compounds 6a,b) or
Drug Dev. Res.

PYRIDAZINONES AS FPR AGONISTS
3
Fig. 2. Synthesis of the final compounds 4a-f, (6)6a,b, 7a,b. Reagents and conditions: (i) N2H4 H2O, EtOH, reflux, 3h; (ii) 3 or 4-
methoxybenzaldehyde, KOH 5% (w/v) in anhydrous EtOH, reflux, 7–20 h; (iii) N-(4-bromophenyl)-2-chloroacetamide, K2CO3, anhydrous
CH3CN, reflux, 5–7 h; (iv) Br2/AcOH, reflux, 8h.
by oxidation with Br2 and acetic acid (compounds 5a,b)
In Figure 3, synthesis of the final oxothiazolones
[Coates and McKillop, 1993; Haider and Holzer, 2004] 9a-h and 10 is presented. The previously described
and further alkylation (7a,b).
compounds 8a-h [Isomura et al., 1988; Pihlaja et al.,
Drug Dev. Res.

4
VERGELLI ET AL.
Fig. 3. Synthesis of the final compounds 9a-h and 10. Reagents and conditions: (i) N-(4-bromophenyl)-2- chloroacetamide, K2CO3,anhy-
drou CH3CN, reflux, 5–7 h; (ii) 10% Pd/C, anhydrous EtOH, H2, Parr, 30 PSI, 2 h.
2002; Wang et al., 2005; Yarligan et al., 2005; Zhao to chromatographically and spectroscopically pure
et al., 2013] were alkylated as shown to obtain the
desired 9a-h. The final 9h was then subject to cata-
lytic reduction with Pd/C in a Parr instrument to
obtain compound 10.
compounds, unless otherwise stated. Compounds are
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 Bu€chi 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 (d) are reported in ppm
Experimentals
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 pre-coated with Merck silica gel 60 F-254, and
visualization was performed by UV fluorescence
(kmax 5 254 nm) or by staining with iodine or potassi-
um 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
1
to the nearest 0.01 ppm (for H NMR) or 0.1 ppm
(for 13C NMR) using the solvent as an internal stan-
1
dard. Coupling constants (J values) of H NMR are
given in Hz and were calculated using “TopSpin 1.3”
software and rounded to the nearest 0.1 Hz. Micro-
analyses were performed with a Perkin-Elmer 260
elemental analyzer for C, H, and N, and the results
were within 6 0.4% of the theoretical values, unless
(Kieselgel 60 F254, 20 3 20 cm, 2 mm). Yields refer otherwise stated.
Drug Dev. Res.

PYRIDAZINONES AS FPR AGONISTS
5
General Procedures for (6)2c and (6)2e
4-(3-methoxybenzyl)-5,6-Dimethylpyridazin-3(2H)-
One, 3a
To a solution of appropriate acid 1c and 1e
(1.52 mmol) [Holloway et al., 2010] in EtOH (5 mL),
hydrazine hydrate (3.04 mmol) was added. The mix-
ture was refluxed for 3 h. After cooling, the solvent
was evaporated under vacuum and cold water
(10 mL) was added. The suspension was extracted
with CH2Cl2 (3 3 15 mL), the organic phase was
dried over Na2SO4 and evaporated to give the final
compounds (6)2c and (6)2e.
Yield 5 58%; mp 5 185–1868C (EtOH). 1H-
NMR (CDCl3) d 2.18 (s, 3H, N 5 CCH3); 2.28 (s,
3H, CH3); 3.79 (s, 3H, OCH3); 4.03 (s, 2H, CH2);
6.76 (d, 1H, Ar, J 5 10.8 Hz); 6.81–6.83 (m, 2H, Ar);
7.20 (t, 1H, Ar, J 5 8.0 Hz); 9.98 (exch br s, 1H,
NH). Anal. Calcd for C14H16N2O2 (244.29): C, 68.83;
H, 6.60; N, 11.47; Found: C, 69.01; H, 6.59; N,
11.50.
4-(4-methoxybenzyl)-5,6-Dimethylpyridazin-3(2H)-
(6)5-Ethyl-6-Methyl-4,5-Dihydropyridazin-3(2H)-
One, (6)2c
One, 3b
Yield 5 44%; mp 5 150–1518C (EtOH). 1H-
NMR (CDCl3) d 2.19 (s, 3H, N 5 CCH3); 2.28 (s,
3H, CH3); 3.79 (s, 3H, OCH3); 3.98 (s, 2H, CH2);
6.82 (d, 2H, Ar, J 5 8.4 Hz); 7.21 (d, 2H, Ar, J 5 8.4
Hz); 10.33 (exch br s, 1H, NH). Anal. Calcd for
C14H16N2O2 (244.29): C, 68.83; H, 6.60; N, 11.47;
Found: C, 68.99; H, 6.59; N, 11.49.
1
Yield 5 26%; oil. H-NMR (CDCl3) d 0.94 (t,
3H, CH3CH2, J 5 7.2 Hz); 1.50–1.65 (m, 2H,
CH2CO); 2.00 (s, 3H, CH3C 5 N); 2.30–2.38 (m, 2H,
CH3CH2); 2.47–2.53 (m, 1H, CHCH2CH3); 9.17
(exch br s, 1H, NH). Anal. Calcd for C7H12N2O
(140.18): C, 59.98; H, 8.63; N, 19.98; Found: C,
59.85; H, 8.65; N, 19.92.
5-Ethyl-4-(3-methoxybenzyl)-6-Methylpyridazin-
(6)5-Butyl-6-Methyl-4,5-Dihydropyridazin-3(2H)-
One, (6)2e
3(2H)-One, 3c
Yield 5 44%; mp 5 168–1708C (EtOH). 1H-
NMR (CDCl3) d 1.08 (t, 3H, CH3CH2, J 5 7.6 Hz);
2.37 (s, 3H, N 5 CCH3); 2.62 (q, 2H, CH3CH2,
J 5 7.6 Hz); 3.79 (s, 3H, OCH3); 4.03 (s, 2H,
CH2Ph); 6.76 (m, 1H, Ar); 6.82 (m, 2H, Ar); 7.20 (m,
1H, Ar). Anal. Calcd for C15H18N2O2 (258.32): C,
69.74; H, 7.02; N, 10.84; Found: C, 69.91; H, 7.01;
N, 10.87.
1
Yield 5 34%; mp 5 63–678C (Cyclohexane). H-
NMR (CDCl3) d 0.91–0.95 (m, 3H, CH3(CH2)3);
1.32–1.37 (m, 2H, CH3CH2(CH2)2); 1.44–1.50 (m,
2H,
CH3CH2CH2CH2);
1.87–1.89
(m,
2H,
CH3(CH2)2CH2); 2.07 (s, 3H, CH3C 5 N); 2.37–2.42
(m, 1H, CH(CH2)3CH3); 2.48–2.60 (m, 2H, CH2CO);
8.24 (exch br s, 1H, NH). Anal. Calcd for C9H16N2O
(168.24): C, 64.25; H, 9.59; N, 16.65; Found: C,
64.41; H, 9.58; N, 16.59.
4-(3-methoxybenzyl)-6-Methyl-5-Propylpyridazin-
3(2H)-One, 3d
Yield 5 23%; mp 5 134–1378C (EtOH). 1H-
NMR (CDCl3) d 1.00 (t, 3H, CH3(CH2)2, J 5 7.6
Hz); 1.38–1.44 (m, 2H, CH3CH2CH2); 2.33 (s, 3H,
N 5 CCH3); 2.49–2.54 (m, 2H, CH3CH2CH2); 3.79
(s, 3H, OCH3); 4.03 (s, 2H, CH2Ph); 6.75 (m, 1H,
Ar); 6.82–6.85 (m, 2H, Ar); 7.19 (t, 1H, Ar, J 5 7.2
Hz). Anal. Calcd for C16H20N2O2 (272.34): C, 70.56;
H, 7.40; N, 10.29; Found: C, 70.39; H, 7.41; N,
10.23.
General Procedures for 3a-f
To a solution of the suitable intermediate
(6)2a-e ((6)2a,b and (6)2d [Pinna et al., 1988;
Haider and Holzer, 2004; Xu et al., 2005]) (1.44
mmol) in 5–9 mL of KOH/absolute EtOH (5% w/v),
3- or 4-methoxybenzaldehyde (1.44–2.16 mmol) was
added and the mixture was refluxed under stirring
for 7–20 h. After cooling, the mixture was concentrat-
ed in vacuo, diluted with ice-cold water (5–10 mL),
neutralized with N HCl and extracted with CH2Cl2
(3 3x 20 mL). Removal of the solvent resulted in
crude compounds 3a,b,f, which were purified by
flash chromatography using cyclohexane/ethyl acetate
1:3 (for 3a,b) and cyclohexane/ethyl acetate 2:1 (for
3f) as eluents. Compounds 3c-e were recovered as
crude precipitate after dilution and neutralization.
These final compounds were purified by crystalliza-
tion from EtOH.
5-Butyl-4-(3-methoxybenzyl)-6-Methylpyridazin-
3(2H)-One, 3e
Yield 5 22%; mp 5 144–1468C (EtOH/H2O 1:1).
1H-NMR (CDCl3) d 0.95 (t, 3H, CH3(CH2)3, J 5 7.2
Hz); 1.32–1.46 (m, 4H, CH3CH2CH2CH2); 2.35 (s, 3H,
N 5 CCH3); 2.52–2.57 (m, 2H, CH3CH2CH2CH2);
3.79 (s, 3H, OCH3); 4.02 (s, 2H, CH2Ph); 6.76 (m, 1H,
Ar); 6.82 (m, 2H, Ar); 7.19 (t, 1H, Ar, J 5 7.6 Hz). Anal.
Drug Dev. Res.

6
VERGELLI ET AL.
Calcd for C17H22N2O2 (286.37): C, 71.30; H, 7.74; N, 16.0 (CH3); 20.3 (CH3); 31.3 (CH2); 55.2 (CH3); 58.1
9.78; Found: C, 71.49; H, 7.75; N, 9.81.
(CH2); 114.1 (2 CH); 116.4 (C); 121.2 (2CH); 129.4
(2 CH); 129.6 (C); 131.5 (2 CH); 137.0 (C); 138.5
(C); 140.3 (C); 146.8 (C); 158.3 (C); 161.2 (C); 165.6
(C). Anal. Calcd for C22H22N3O3 (456.33): C, 57.90;
H, 4.86; N, 9.21; Found: C, 57.69; H, 4.85; N, 9.18.
4-(3-methoxybenzyl)-6-Methyl-5-Phenylpyridazin-
3(2H)-One, 3f
1
Yield 5 32%; oil. H-NMR (CDCl3) d 2.14 (s,
3H, N 5 CCH3); 3.73 (s, 3H, OCH3); 3.80 (s, 2H,
CH2); 6.42 (s, 1H, Ar); 6.53 (d, 1H, Ar, J 5 10.8 Hz);
6.71 (dd, 1H, Ar, J 5 2.4 Hz, J 5 5.6 Hz); 7.02–7.17
(m, 3H, Ar); 7.41–7.51 (m, 3H, Ar). Anal. Calcd for
C19H18N2O2 (306.36): C, 74.49; H, 5.92; N, 9.14;
Found: C,74.68; H, 5.91; N, 9.16.
N-(4-bromophenyl)-2-[4-ethyl-5-(3-
methoxybenzyl)-3-methyl-6-oxopyridazin-1(6H)-
Yl]Acetamide, 4c
Yield 5 39%; mp5 86–888C (EtOH). 1H-NMR
(CDCl3) d 1.10 (t, 3H, CH3CH2, J 5 7.6 Hz); 2.39 (s,
3H, N 5 CCH3); 2.63 (q, 2H, CH3CH2, J 5 7.6 Hz);
3.75 (s, 3H, OCH3); 4.05 (s, 2H, CH2Ar); 4.93 (s, 2H,
CH2CO); 6.77 (m, 3H, Ar); 7.18 (t, 1H, Ar, J 5 8.8
Hz); 7.33–7.38 (m, 4H, Ar); 9.31 (exch br s, 1H, NH).
Anal. Calcd for C23H24BrN3O3 (470.36): C, 58.73; H,
5.14; N, 8.93; Found: C, 58.89; H, 5.13; N, 8.90.
General Procedures for 4a-f
To a mixture of the appropriate intermediate
3a-f (0.40 mmol) and K2CO3 (0.80–1.20 mmol) in
CH3CN (5–10 mL), N-(4-bromophenyl)22-chloroa-
cetamide (0.40–0.72 mmol) was added and the sus-
pension was refluxed under stirring for 5–7 h. After
cooling, the mixture was concentrated in vacuo, ice
cold water was (5–10 mL) added and then further
stirred for 1 h. The precipitate was recovered by suc-
tion and purified by flash chromatography using
cyclohexane/ethyl acetate 1:1 (compounds 4a,b,e),
cyclohexane/ethyl acetate 1:2 (compounds 4c,d) and
cyclohexane/ethyl acetate 2:1 for 4f as eluents.
N-(4-bromophenyl)-2-[5-(3-methoxybenzyl)-3-
methyl-6-oxo-4-propylpyridazin-1(6H)-
yl]Acetamide, 4d
Yield 5 30%; mp5 81–838C (EtOH). 1H-NMR
(CDCl3) d 1.04 (t, 3H, CH3(CH2)2, J 5 7.2 Hz); 1.42–
1.49 (m, 2H, CH3CH2CH2); 2.38 (s, 3H, N 5 CCH3);
2.54–2.58 (m, 2H, CH3CH2CH2); 3.74 (s, 3H, OCH3);
4.03 (s, 2H, CH2Ar); 4.88 (s, 2H, CH2CO); 6.74–6.78
(m, 3H, Ar); 7.17 (t, 1H, Ar, J 5 7.6 Hz); 7.25 (m, 4H,
Ar); 9.50 (exch br s, 1H, NH). Anal. Calcd for
C24H26BrN3O3 (484.39): C, 59.51; H, 5.41; N, 8.67;
Found: C, 59.67; H, 5.40; N, 8.64.
N-(4-bromophenyl)-2-[5-(3-methoxybenzyl)-3,4-
dimethyl-6-oxopyridazin-1(6H)-Yl]Acetamide, 4a
Yield 5 48%; mp 5 123–1248C (EtOH). 1H-
NMR (CDCl3) d 2.23 (s, 3H, N 5 CCH3); 2.34 (s,
3H, C 5 CCH3); 3.76 (s, 3H, OCH3); 4.06 (s, 2H,
CH2Ar); 4.94 (s, 2H, NCH2); 6.76 (dd, 1H, Ar,
J 5 6.4 Hz, J 5 1.6 Hz); 6.79–6.82 (m, 2H, Ar); 7.19
(t, 1H, Ar, J 5 8.0 Hz); 7.30–7.35 (m, 4H, Ar); 9.31
(exch br s, 1H, NH). 13C-NMR (CDCl3) d 16.0
(CH3); 20.4 (CH3); 32.1 (CH2); 55.1 (CH3); 57.9
(CH2); 111.3 (CH); 114.7 (CH); 116.3 (C); 120.7
(CH); 121.1 (2 CH); 129.5 (CH); 131.5 (2CH); 137.1
(C); 137.9 (C); 139.4 (C); 140.7 (C); 146.6 (C); 159.8
(C); 161.2 (C); 165.6 (C). Anal. Calcd for
C22H22N3O3 (456.33): C, 57.90; H, 4.86; N, 9.21;
Found: C, 57.74; H, 4.85; N, 9.24.
N-(4-bromophenyl)-2-[4-butyl-5-(3-
methoxybenzyl)-3-methyl-6-oxopyridazin-1(6H)-
yl]Acetamide, 4e
1
Yield 5 57%; mp 5 75–778C (Cyclohexane). H-
NMR (CDCl3) d 0.93 (t, 3H, CH3(CH2)3, J 5 7.2 Hz);
1.28–1.45 (m, 4H, CH3CH2CH2CH2); 2.38 (s, 3H,
N 5 CCH3); 2.54–2.58 (m, 2H, CH3CH2CH2CH2);
3.73 (s, 3H, OCH3); 3.99 (s, 2H, CH2Ar); 4.88 (s, 2H,
CH2CO); 6.75–6.78 (m, 3H, Ar); 7.17 (t, 1H, Ar,
J 5 7.6 Hz). 7.25–7.27 (m, 4H, Ar); 9.53 (exch br s,
1H, NH). Anal. Calcd for C25H28BrN3O3 (498.41): C,
60.24; H, 5.66; N, 8.43; Found: C, 60.08; H, 5.66; N,
8.46.
N-(4-bromophenyl)-2-[5-(4-methoxybenzyl)-3,4-
dimethyl-6-oxopyridazin-1(6H)-Yl]Acetamide, 4b
Yield 5 58%; mp 5 177–1788C (EtOH). 1H-
NMR (CDCl3) d 2.24 (s, 3H, N 5 CCH3); 2.33 (s,
3H, C 5 CCH3); 3.78 (s, 3H, OCH3); 4.02 (s, 2H,
CH2Ar); 4.94 (s, 2H, NCH2); 6.80 (d, 2H, Ar, J 5 8.4
N-(4-bromophenyl)-2-[5-(3-methoxybenzyl)-3-
methyl-6-oxo-4-phenylpyridazin-1(6H)-
yl]Acetamide, 4f
1
Yield 5 24%; oil. H-NMR (CDCl3) d 2.09 (s,
Hz); 7.16 (d, 2H, Ar, J 5 8.4 Hz); 7.30–7.38 (m, 4H, 3H, CCH3,); 3.65 (s, 3H, OCH3); 3.87 (s, 2H,
Ar); 9.26 (exch br s, 1H, NH). 13C-NMR (CDCl3) d CH2Ar); 5.00 (s, 2H, NCH2); 6.38 (s, 1H, Ar); 6.64
Drug Dev. Res.

PYRIDAZINONES AS FPR AGONISTS
7
(d, 1H, Ar, J 5 8.8 Hz); 7.02–7.06 (m, 2H, Ar); 7.19
N-(4-bromophenyl)-2-[3,4-dimethyl-6-
oxopyridazin-1(6H)-yl]Acetamide, 7a
(d, 1H, Ar, J5 7.6 Hz); 7.42 (m, 8H, Ar); 9.22 (exch
br s, 1H, NH). 13C-NMR (CDCl3) d 21.1 (CH3);
31.1 (CH2); 55.2 (CH3); 58.3 (CH2); 111.4 (CH);
112.1 (CH); 114.7 (CH); 115.3 (CH); 116.4 (C);
120.9 (CH); 121.1 (CH); 125.4 (C); 127.5 (CH);
128.0 (CH); 128.8 (CH); 129.0 (CH); 129.3 (CH);
129.8 (CH); 131.5 (CH); 134.7 (C); 137.0 (C); 138.5
(C); 139.7 (C); 145.2 (C); 145.8 (C); 161.2 (C); 165.5
(C). Anal. Calcd for C27H24N3O3 (518.40): C, 62.56;
H, 4.67; N, 8.11; Found: C, 62.74; H, 4.68; N, 8.13.
Yield 5 81%; mp 5 205–2068C (EtOH). 1H-
NMR (CDCl3) d 2.23 (s, 3H, N 5 CCH3); 2.33 (s,
3H, 5CCH3); 4.92 (s, 2H, CH2); 6.81 (s, 1H, Ar);
7.39–7.44 (m, 4H, Ar); 9.11 (exch br s, 1H, NH).
13C-NMR (CDCl3) d 19.0 (CH3); 19.2 (CH3); 57.5
(CH2); 116.0 (C); 121.0 (2CH); 127.4 (CH); 132.0
(2CH); 137.0 (C); 144.8 (C); 161.1 (C); 165.4 (C).
Anal. Calcd for C14H14BrN3O2 (336.18): C, 50.02; H,
4.20; N, 12.50; Found: C, 50.16; H, 4.19; N, 12.53.
General Procedures for (6)6a,b and 7a,b
N-(4-bromophenyl)-2-[3-methyl-6-oxo-4-
phenylpyridazin-1(6H)-yl]Acetamide, 7b
Compounds (6)6a,b and 7a,b were obtained
starting from appropriate substrates (6)2a,b [Pinna
et al., 1988; Haider and Holzer, 2004] and 5a,b
[Coates and McKillop, 1993; Haider and Holzer,
2004] following the same procedure described for
4a-f. The final compounds were purified by column
chromatography using cyclohexane/ethyl acetate 1:1
as eluent.
Yield 5 64%; mp 5 199–2008C (EtOH). 1H-
NMR (CDCl3) d 2.30 (s, 3H, CH3); 5.01 (s, 2H,
CH2); 6.91 (s, 1H, Ar); 7.40–7.42 (m, 2H, Ar); 7.43–
7.48 (m, 4H, Ar); 7.49–7.5 (m, 3H, Ar); 9.22 (exch br
s, 1H, NH). 13C-NMR (CDCl3) d 20.4 (CH3); 58.0
(CH2); 116.9 (C); 121.4 (CH); 127.9 (CH); 128.0
(2CH); 128.9 (2CH); 129.4 (2CH); 131.8 (2CH);
135.5 (C); 137.0 (C); 145.5 (C); 148.3 (C); 161.0 (C);
165.2 (C). Anal. Calcd for C19H16BrN3O2 (398.25):
C, 57.30; H, 4.05; N, 10.55; Found: C, 57.43; H,
4.06; N, 10.58.
(6)N-(4-bromophenyl)-2-[3,4-dimethyl-6-oxo-5,6-
dihydropyridazin-1(4H)-yl]Acetamide, (6)6a
1
Yield 5 36%; mp 5 84–858C (EtOH). H-NMR
(CDCl3) d 1.22 (d, 3H, CHCH3, J 5 6.8 Hz); 2.08 (s,
3H, N 5 CCH3); 2.33–2.40 (m, 1H, CH3CH); 2.64–
2.70 (m, 2H, CHCH2); 4.49 (d, 1H, NCH-H, J 5 15.6
General Procedures for 9a-h
Compounds 9a-h were obtained starting from
Hz); 4.58 (d, 1H, NCH-H, J 5 15.6 Hz); 7.30–7.40 appropriate substrate of type 8 [Isomura et al., 1988;
(m, 4H, Ar); 8.19 (exch br s, 1H, NH). 13C-NMR
Pihlaja et al., 2002; Wang et al., 2005; Yarligan et al.,
2005; Zhao et al., 2013] following the same proce-
dure described for 4a-f. The final compounds were
purified by column chromatography using cyclohex-
ane/ethyl acetate 1:1 (for 9a,f,g), cyclohexane/ethyl
acetate 1:2 (for 9b), cyclohexane/ethyl acetate 2:1
(for 9d), and CH2Cl2/CH3OH 98:2 (9c,e) as eluents.
(CDCl3) d 15.7 (CH3); 21.1 (CH3); 31.9 (CH); 34.1
(CH2); 53.1 (CH2); 116.8 (C); 121.4 (2 CH); 131.8 (2
CH); 136.8 (C); 158.6 (C); 166.3 (C); 166.5 (C). Anal.
Calcd for C14H16BrN3O2 (338.20): C, 49,72; H, 4.77;
N, 12.42; Found: C, 49.59; H, 4.75; N, 12.47.
(6)N-(4-bromophenyl)-2-[3-methyl-6-oxo-4-phenyl-
5,6-dihydropyridazin-1(4H)-yl]Acetamide, (6)6b
2-(5-acetyl-4-methyl-2-oxo-thiazol-3-yl)-N-(4-
bromophenyl)Acetamide, 9a
1
Yield 5 62%; oil. H-NMR (CDCl3) d 2.06 (s,
Yield 5 19%; mp 5 228–2308C (EtOH). 1H-
NMR (CDCl3) d 2.39 (s, 3H, COCH3); 2.65 (s, 3H,
CH3); 4.55 (s, 2H, CH2CO); 7.34–7.44 (m, 4H, Ar);
8.19 (exch br s, 1H, NH). Anal. Calcd for
C14H13BrN2O3S (369.23): C, 45.54; H, 3.55; N, 7.59;
Found: C, 45.65; H, 3.54; N, 7.60.
3H, CH3); 2.82 (dd, 1H, CHCH-H, J 5 4.8 Hz,
J 5 11.6 Hz); 2.96 (dd, 1H, CHCH-H, J 5 7.6 Hz,
J 5 9.2 Hz); 3.84 (dd, 1H, CH-Ph, J 5 5.2 Hz, J 5 2.0
Hz); 4.46 (d, 1H, NCH-H, J 5 15.6 Hz); 4.72 (d, 1H,
NCH-H, J 5 15.6 Hz); 7.17–7.19 (m, 2H, Ar); 7.28–
7.34 (m, 3H, Ar); 7.42 (d, 2H, Ar, J 5 8.8 Hz) 7.90
(exch br s, 1H, NH). 13C-NMR (CDCl3) d 22.3
(CH3); 34.4 (CH2); 43.5 (CH); 53.7 (CH2); 116.9 (C);
121.4 (CH); 127.2 (2 CH); 128.1 (CH); 129.2 (CH);
129.5 (2 CH); 131.8 (2 CH); 136.6 (C); 137.1 (C);
155.9 (C); 165.8 (C); 166.4 (C). Anal. Calcd for
C19H19BrN3O2 (400.27): C, 57.01; H, 4.53; N, 10.50;
Found: C, 57.17; H, 4.52; N, 10.47.
N-(4-bromophenyl)-2-(4-methyl-2-oxo-thiazol-3-
yl)Acetamide, 9b
Yield 5 35%; mp 5 204–2058C (EtOH). 1H-
NMR (CDCl3) d 2.40 (s, 3H, CH3); 4.45 (s, 2H,
CH2CO); 5.85 (s, 1H, H5-thiazole); 7.36–7.43 (m,
4H, Ar); 8.43 (exch br s, 1H, NH). Anal. Calcd for
Drug Dev. Res.

8
VERGELLI ET AL.
C12H11BrN2O2S (327.20): C, 44.05; H, 3.39; N, 8.56;
Found: C, 44.17; H, 3.39; N, 8.54.
N-(4-bromophenyl)-2-[4-(4-chlorophenyl)-2-
oxothiazol-3-yl]Acetamide, 9g
Yield 5 18%; mp 5 230–2328C (EtOH). 1H-
NMR (CDCl3) d 4.36 (s, 2H, CH2CO); 6.14 (s, 1H,
H5-thiazole); 7.37–7.45 (m, 8H, Ar); 8.73 (exch br s,
1H, NH). Anal. Calcd for C17H12BrClN2O2S
(423.71): C, 48.19; H, 2.85; N, 6.61; Found: C, 48.03;
H, 2.85; N, 6.63.
N-(4-bromophenyl)-2-(2-oxo-4-phenyl-thiazol-3-
yl)Acetamide, 9c
Yield 5 18%; mp 5 181–1838C (EtOH). 1H-
NMR (CDCl3) d 4.41 (s, 2H, CH2CO); 6.14 (s, 1H,
H5-thiazole); 7.32 (s, 4H, Ar); 7.45 (s, 5H, Ar); 8.45
(exch br s, 1H, NH). 13C-NMR (CDCl3) d 48.8
(CH2); 99.4 (CH); 117.1 (C); 121.3 (CH); 121.4
(CH); 129.9 (CH); 130.3 (CH); 130.8 (C); 131.8
(CH); 136.6 (C); 138.3 (C); 165.1 (2 CO). Anal.
Calcd for C17H13BrN2O2S (389.27): C, 52.45; H,
3.37; N, 7.20; Found: C, 52.29; H, 3.36; N, 7.22.
N-(4-bromophenyl)-2-[4-(4-nitrophenyl)-2-
oxothiazol-3-yl]Acetamide, 9h
Yield 5 21%; mp 5 162–1648C (EtOH). 1H-
NMR (CDCl3) d 4.38 (s, 2H, CH2CO); 6.30 (s, 1H,
H5-thiazole); 7.41 (s, 4H, Ar); 7.72 (d, 2H, Ar,
J 5 8.8 Hz); 8.83 (d, 2H, Ar, J 5 8.8); 8.68 (exch br s,
1H, NH). Anal. Calcd for C17H12BrN3O4S (434.26):
C, 47.02; H, 2.79; N, 9.68; Found: C, 47.16; H, 2.78;
N, 9.71.
N-(4-bromophenyl)-2-[4-(3-methoxyphenyl)-2-
oxothiazol-3-yl]-Acetamide, 9d
Yield 5 25%; mp 5 162–1658C (EtOH). 1H-
NMR (CDCl3) d 3.83 (s, 3H, CH3); 4.39 (s, 2H,
CH2CO); 6.15 (s, 1H, H5-thiazole); 7.01 (s, 3H, Ar);
7.42 (s, 5H, Ar); 8.63 (exch br s, 1H, NH). 13C-NMR
(CDCl3) d 48.8 (CH2); 55.5 (CH3); 99.3 (CH); 114.3
(CH); 116.0 (CH); 116.9 (CH); 121.2 (CH); 121.4
(CH); 130.2 (CH); 131.5 (C); 131.7 (CH); 136.7 (C);
138.3 (C); 159.9 (C); 165.2 (CO); 174.2 (CO). Anal.
Calcd for C18H15BrN2O3S (419.29): C, 51.56; H,
3.61; N, 6.68; Found: C, 51.43; H, 3.60; N, 6.72.
2-[4-(4-Aminophenyl)-2-Oxothiazol-3-Yl]-N-(4-
bromophenyl)Acetamide, 10
Compound 9h (0.11 mmol) was subject to cata-
lytic reduction with 10% Pd/C (0.05 mmol) in EtOH
(20 mL) for 2 h in a Parr instrument (30 PSI). The
catalyst was filtered off, and the solvent was evaporat-
ed under vacuum, affording the final compound,
which was purified by column chromatography using
cyclohexane/ethyl acetate 1:1 as eluent. Yield 5 27%;
1
mp 5 188–1898C (EtOH). H-NMR (CDCl3) d 4.32
(s, 2H, CH2CO); 5.41 (exch br s, 2H, NH2); 6.21 (s,
1H, H5-thyazole); 6.53 (m, 2H, Ar); 7.01 (m, 2H,
Ar); 7.45 (s, 4H, Ar); 10.28 (exch br s, 1H, NH).
Anal. Calcd for C17H14BrN3O2S (404.28): C, 50.50;
H, 3.49; N, 10.39; Found: C, 50.61; H, 3.48; N,
10.37.
N-(4-bromophenyl)-2-[4-(4-methoxyphenyl)-2-
oxothiazol-3-yl]Acetamide, 9e
Yield 5 15%; mp 5 210–2138C (EtOH). 1H-
NMR (CDCl3) d 3.84 (s, 3H, CH3); 4.37 (s, 2H,
CH2CO); 6.07 (s, 1H, H5-thiazole); 6.96 (d, 2H, Ar,
J 5 8.8 Hz); 7.38 (m, 6H, Ar); 8.79 (exch br s, 1H,
NH). 13C-NMR (CDCl3) d 48.7 (CH2); 55.3 (CH3);
100.1 (CH); 114.4 (CH); 115.8 (CH); 117.1 (CH);
121.7 (CH); 121.3 (CH); 129.8 (CH); 131.2 (C);
132.1 (CH); 135.9 (C); 138.4 (C); 160.1 (C); 164.2
(CO); 173. (CO). Anal. Calcd for C18H15BrN2O3S
(419.29): C, 51.56; H, 3.61; N, 6.68; Found: C, 51.61;
H, 3.61; N, 6.65.
Cell Culture
Human promyelocytic leukemia HL60 cells sta-
bly transfected with FPR1 (FPR1-HL60), FPR2
(FPR2-HL60), or FPR3 (FPR3-HL60) were cultured
in RPMI 1640 medium supplemented with 10%
heat-inactivated fetal calf serum, 10 mM HEPES,
100 lg/ml streptomycin, 100 U/ml penicillin, and
G418 (1 mg/mL), as previously described [Giovan-
noni et al., 2013]. 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) fetal bovine serum
(FBS), 10 mM HEPES, 100 lg/ml streptomycin, 100
U/ml penicillin, and G418 (250 lg/ml). Wild-type
N-(4-bromophenyl)-2-[4-(3-chlorophenyl)-2-
oxothiazol-3-yl]Acetamide, 9f
Yield 5 16%; mp 5 203–2048C (EtOH). 1H-
NMR (CDCl3) d 4.37 (s, 2H, CH2CO); 6.18 (s, 1H,
H5-thiazole); 7.36–7.46 (m, 8H, Ar); 8.59 (exch br s,
1H, NH). Anal. Calcd for C17H12BrClN2O2S
(423.71): C, 48.19; H, 2.85; N, 6.61; Found: C, 48.35; HL60 and RBL-2H3 cells were cultured under the
H, 2.84; N, 6.62.
same conditions, but without G418.
Drug Dev. Res.

PYRIDAZINONES AS FPR AGONISTS
9
TABLE 1. Activity of 4-Benzyl-5-Substituted Pyridazinones 4a-f in Human Neutrophils and FPR-Transfected HL60 Cell
Ca21 flux
Human neutrophils
FPR1-HL60
FPR2-HL60
FPR3-HL60
Compd
R5
OCH3
EC50 (mM) and efficacy (%)a
4a
4b
4c
4d
4e
4f
CH3
CH3
C2H5
C3H5
C4H9
C6H5
m
p
m
m
m
m
0.006 6 0.002 (150)
0.27 6 0.025 (140)
1.2 6 0.3 (100)
3.2 6 1.2 (70)
2.7 6 0.7 (25)
5.4 6 0.26 (100)
0.019 6 0.005 (85)
0.9 6 0.033 (60)
3.2 6 0.6 (120)
2.2 6 0.5 (115)
N.A.b
0.043 6 0.016 (80)
0.013 6 0.003 (95)
1.9 6 0.41 (100)
4.6 6 1.3 (90)
15.7 6 4.2 (90)
N.A.
0.040 6 0.011 (165)
0.13 6 0.032 (100)
4.1 6 1.7 (100)
3.4 6 0.76 (65)
N.A.
2.2 6 0.62 (160)
N.A.
aEC50 values represent the average of means from 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 is
expressed as % of the response induced by 5 nM fMLF (FPR1), 5 nM WKYMVm (FPR2), or 10 nM WKYMVM (FPR3).
bN.A., no activity (no response was observed during the first 2 min after addition of compounds under investigation) considering the limits
of efficacy > 20% and EC50 < 50 mM.
Briefly, bone marrow leukocytes were flushed from
tibias and femurs of BALB/c mice with HBSS, fil-
tered through a 70 lm nylon cell strainer (BD Bio-
sciences, 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 poly-
propylene tube. The gradient was centrifuged at
1600g for 30 min at 108C, 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 108C 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 Insti-
Isolation of Human Neutrophils
Blood was collected from healthy donors in
accordance with a protocol approved by the Institu-
tional Review Board at Montana State University.
Neutrophils were purified from the blood using dex-
tran sedimentation, followed by Histopaque 1077 gra-
dient separation and hypotonic lysis of red blood
cells, as previously described [Schepetkin et al.,
2014b]. Isolated neutrophils were washed twice and
resuspended in HBSS without Ca21 and Mg21
(HBSS2). Neutrophil preparations were routinely >
95% pure, as determined by light microscopy, and
greater than 98% viable, as determined by trypan blue
exclusion.
Isolation of Murine Neutrophils
Murine bone marrow neutrophils were isolated
from bone marrow leukocyte preparations, as tutional Animal Care and Use Committee at Montana
described previously [Schepetkin et al., 2014a]. State University.
Drug Dev. Res.

10
VERGELLI ET AL.
TABLE 2. Activity of C-5 Substituted Pyridazinones (6)26a-b and 7a-b in Human Neutrophils and FPR-Transfected HL60 Cells
Ca21 flux
Human neutrophils
FPR1-HL60
FPR2-HL60
FPR3-HL60
Compd
R5
EC50 (mM) and efficacy (%)a
(6)-6a
(6)-6b
7a
CH3
C6H5
CH3
13.0 6 1.8 (115)
1.4 6 0.38 (75)
2.0 6 0.29 (130)
0.20 6 0.056 (110)
13.0 6 4.6 (60)
4.1 6 1.6 (90)
5.7 6 2.1 (110)
N.A.
2.6 6 0.89 (120)
0.63 6 0.17 (110)
0.51 6 0.19 (90)
0.15 6 0.052 (115)
11.8 6 3.0 (150)
0.49 6 0.14 (120)
N.A.b
7b
C6H5
0.97 6 0.23 (95)
aEC50 values represent the average of means from 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 is
expressed as % of the response induced by 5 nM fMLF (FPR1-HL60), 5 nM WKYMVm (FPR2-HL60), or 10 nM WKYMVM (FPR3-HL60).
bN.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 mM.
Ca21 Mobilization Assay
temperature after automated addition of compounds.
Changes in intracellular Ca21 were measured
Maximum change in fluorescence, expressed in arbi-
trary units over baseline, was used to determine ago-
with a FlexStation II scanning fluorometer using a
nist response. Responses were normalized to the
FLIPR 3 calcium assay kit (Molecular Devices, Sun-
nyvale, CA) for human and murine neutrophils, as
well as HL60 and RBL cells, as described previously
response induced by 5 nM fMLF (Sigma Chemical
Co., St. Louis, MO) for FPR1 HL60 cells and human
neutrophils, 5 nM WKYMVm (Calbiochem, San
[Schepetkin et al., 2013]. All active compounds were
Diego, CA) for FPR2 HL60 cells, 10 nM WKYMVM
evaluated in wild-type HL60 and RBL cells to verify
(Tocris Bioscience) for FPR3-HL60 cells, and 10 nM
that the agonists are inactive in non-transfected cells.
WKYMVm for mouse neutrophils, mFpr1-RBL, and
Neutrophils or HL60 and RBL cells, suspended in
HBSS2 containing 10 mM HEPES, were loaded
mFpr2-RBL cells, which were assigned a value of
100%. Curve fitting (5–6 points) and calculation of
with Fluo-4 AM dye (Invitrogen; 1.25 lg/mL final
concentration) and incubated for 30 min in the dark
median effective concentration values (EC50 values)
were performed using nonlinear regression analysis
of the concentration-response curves generated using
at 378C. After dye loading, the cells were washed
with HBSS2 containing 10 mM HEPES, resus-
Prism 5 (GraphPad Software, Inc., San Diego, CA).
pended in HBSS containing 10 mM HEPES and
Ca21 and Mg21 (HBSS1), and aliquotted into the
Cell Migration Assay
Neutrophils were suspended in HBSS1 contain-
wells of a flat-bottomed, half-area-well black microti-
ter plates (2 3 105 cells/well). The compound of
interest was added from a source plate containing ing 2% (v/v) FBS (2 3 106 cells/mL), and cell migra-
dilutions of test compounds in HBSS1, and changes tion was analyzed in 96-well ChemoTx chemotaxis
in fluorescence were monitored (kex 5 485 nm, chambers (Neuroprobe, Gaithersburg, MD), as previ-
kem 5 538 nm) every 5 s for 240 s at room ously described [Schepetkin et al., 2014b]. Briefly,
Drug Dev. Res.

PYRIDAZINONES AS FPR AGONISTS
11
TABLE 3. Activity of 9a-h, 10 in Human Neutrophils and FPR-Transfected HL60 Cells
Ca21 flux
Human neutrophils
FPR1-HL60
FPR2-HL60
FPR3-HL60
Compd
R4
R5
EC50 (mM) and efficacy (%)a
9a
9b
9c
9d
9e
9f
9g
9h
10
CH3
CH3
Ph
3-OCH3Ph
4-OCH3Ph
3-ClPh
4-ClPh
4-NO2Ph
4-NH2Ph
COCH3
12.2 6 2.5(55)
10.7 6 2.3 (55)
6.0 6 1.5 (95)
1.3 6 0.3 (125)
7.4 6 2.3 (110)
11.1 6 2.8 (140)
12.2 6 3.1 (125)
1.5 6 0.4 (95)
36.1 6 3.9 (65)
8.9 6 1.9 (75)
5.8 6 1.4 (60)
4.1 6 1.1 (80)
2.1 6 0.6 (95)
0.23 6 0.04 (120)
1.8 6 0.16 (100)
3.0 6 0.7 (110)
2.4 6 0.5 (75)
N.A.
N.A.b
27.8 6 3.2 (85)
19.7 6 2.4 (135)
5.1 6 1.7 (100)
36.1 6 3.3 (85)
32.2 6 3.6 (75)
N.A.
H
H
H
H
H
H
H
H
12.4 6 2.6 (70)
1.8 6 0.6 (100)
0.28 6 0.08 (90)
2.6 6 0.6 (110)
6.0 6 1.7 (75)
2.8 6 0.7 (75)
9.1 6 1.4 (60)
34.9 6 4.2 (80)
N.A.
N.A.
14.7 6 3.7 (65)
aEC50 values represent the average of means from 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 is
expressed as % of the response induced by 5 nM fMLF (FPR1-HL60), 5 nM WKYMVm (FPR2-HL60), or 10 nM WKYMVM (FPR3-HL60).
bN.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 mM.
based assay (CellTiter-Glo; Promega, Madison, WI),
TABLE 4. Chemoattractant Activity of Selected Pyridazinones in
and luminescence measurements were converted to
Human Neutrophils
absolute cell numbers by comparison of the values
Compd
EC50 (mM)a
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 com-
pounds/spontaneous migration in response to control
medium)3100. EC50 values were determined by non-
linear regression analysis of the concentration-
response curves generated using Prism 5 software.
4a
4b
7b
0.27 6 0.06
0.51 6 0.11
2.1 6 0.53
aThe data are presented as the mean 6 SD of three independent
experiments with cells from different donors, in which median effec-
tive 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).
RESULTS AND DISCUSSION
lower wells were loaded with 30 mL of HBSS1 con-
taining 2% (v/v) FBS and the indicated concentra-
tions 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 mm pore polycarbonate membrane
filter for 60 min at 378C and 5% CO2. The number
of migrated cells was determined by measuring ATP
The newly synthesized compounds were evalu-
ated for their ability to induce intracellular Ca21 flux
in human neutrophils and HL60 cells transfected
with FPR1, FPR2, and FPR3 (Tables 1–3). Compounds
were also evaluated in mouse neutrophils and RBL-2H3
cells transfected with mouse FPR1 and FPR2. To verify
receptor specificity, these compounds were also evaluat-
ed, as applicable, in wild-type non-transfected HL60
in lysates of transmigrated cells using a luminescence- and RBL cells and were found to be inactive.
Drug Dev. Res.

12
VERGELLI ET AL.
mixtures of isomers, it is possible that one of the iso-
mers could be more active than the other, reducing
overall specific activity or even interfering with bind-
ing of the active isomer.
TABLE 5. Activity of Selected Pyridazinones in Mouse Neutrophils
and Mouse Fpr-Transfected RBL Cells
Ca21 flux
Table
3 reports the activities of the 2-
Mouse neutrophils
mFpr1-RBL
mFpr2-RBL
oxothiazole derivatives, 9a-h and 10 that exhibited
EC50 values in the micromolar range for the three
FPR isoforms, with the exception of compound 9d,
which had EC50 values of 0.28 mM and 0.23 mM for
FPR1 and FPR2, respectively. No appreciable FPR2
selectivity was observed for this series, although they
did have a lower affinity for FPR3 (EC50 ꢀ 20-fold
higher than for FPR1 and FPR2).
Compd
EC50 (mM) and efficacy (%)a
4a
4b
4f
(6)-6a
(6)-6b
7a
N.A.b
N.A.
N.A.
N.A.
N.A.
N.A.
24.3 6 4.8 (95)
N.A.
14.2 6 3.4 (160)
21.7 6 5.2 (70)
15.7 6 4.1 (75)
N.A.
28.8 6 5.2 (180)
N.A
3.5 6 1.1 (220)
N.A.
25.1 6 4.5 (70)
2.3 6 0.7 (60)
N.A.
7b
N.A.
N.A
The most active derivatives (4a,b and 7b) were
also evaluated for their chemoattractant activity
toward human neutrophils (Table 4). As expected for
FPR agonists, they stimulated neutrophil migrations
with EC50 values that correlated well with their ability
to induce human neutrophil intracellular Ca21 flux.
All of the synthesized compounds were also
evaluated in mouse neutrophils and RBL cells trans-
fected with mouse Fpr1 or Fpr2 (Table 5). The
majority of tested compounds had no agonist effects
at mouse neutrophils or RBL cells transfected with
mouse Fpr; however, four compounds were active in
mouse neutrophils and three had activity in either
Fpr1 or Fpr2 RBL cells. As shown in Table 5, com-
pounds 4f, (6)6a,b, and 7a,b all activated Ca21 flux
in mouse neutrophils with low micromolar activity.
From the literature, it is well known that there are
differences in affinity between FPR1 and Fpr1 for
fMLF and other FPR agonists/antagonists [He et al.,
2000] and these receptors have only 72% sequence
similarities [Dahlgren et al., 2016]. However, it is dif-
ficult to explain the complete inactivity of the most
potent human FPR agonists of this series (4a and
4b) toward mouse neutrophils or mouse Fpr-
transfected RBL cells. Comparison of the results
between human FPR-transfected HL60 cells and
mouse Fpr1/Fpr2-transfected RBL cells was also dif-
ficult to interpret. For example, (6)6a was active for
all human FPR and mouse Fpr subtypes tested,
whereas 4f was inactive in FPR2-HL60 cells but had
an EC50 value of 28.8 mM for mouse Fpr2. Likewise,
(6)6a was active for all human FPR subtypes but
only activated mFpr1 and not mouse neutrophils or
mFpr2. Thus, the difference in human and mouse
FPR responses to various agonists are clearly evident
and will need to be considered when using agonists
in mouse models involving Fpr function.
aEC50 values represent the average of means from 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
is expressed as % of the response induced by 10 nM WKYMVm.
bN.A., no activity (no response was observed during first 2 min after
addition of the compounds under investigation).
In the 4-benzylpyridazinone series (Table 1),
the introduction of a methyl at C-5 was associated
with high FPR agonist activity (compounds 4a,b)
leading to some of themost potent mixed agonists.
For example, 4a activated all three FPR subtypes
with good potency (FPR1 EC50 5 19 nM, FPR2
EC50 5 43 nM, FPR3 EC50 5 40 nM). Although 4b
was an FPR1 and FPR3 agonist with submicromolar
activity (EC50 5 0.9 and 0.13 mM, respectively), it exhib-
ited a preference for FPR2 (EC50 5 13 nM).These
results are consistent with our previous studies [Cili-
brizzi et al., 2009], which showed that when OCH3 was
shifted from the meta to the para position of the benzyl
at C-4, the selectivity of a given ligand shifted from
mixed FPR1/FPR2 to FPR2-specific.
Elongation of the aliphatic chain (compounds
4c-e) was detrimental for activity and led to mixed
agonists with EC50 values in the micromolar range,
with the exception of the 3-butyl derivative 4e, which
was a weak but selective FPR2 agonist (EC50 5 15.7
mM). Surprisingly, the 5-phenyl derivative 4f exhib-
ited FPR1 selectivity (EC50 5 2.2 mM, Table 1).
As shown in Table 2 elimination of the substitu-
ent at position 4 and introduction of CH3 or C6H5 at
C-5 (products of type 6 and 7) led to compounds
endowed with micromolar activity and a slight prefer-
ence for FPR2. The unsaturation at C-4/C-5 of the
heterocyclic ring (7a,b) appeared to be associated
In conclusion, we have identified a new series
with increased agonist activity and selectivity (gener- of FPRs agonists with EC50 values in the nanomolar
ally submicromolar range) relative to the 4,5-dihydro to low micromolar range. The majority of compounds
analogues, (6)6a,b. As compounds (6)6a,b were were mixed FPRs agonists, but with
a slight
Drug Dev. Res.

PYRIDAZINONES AS FPR AGONISTS
13
chemotactic agonist for mouse formyl peptide receptor.
Immunol 165:4598–4605.
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of 19 nM (FPR1), 43 nM (FPR2), and 40 nM
(FPR3). Similarly interesting is compound 4b, which
had considerable potency, but exhibited a preference
for FPR2 (EC50 5 13 nM) and could represent a nov-
el lead compound for further chemical manipulation
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