Synthesis and pharmacological evaluation of new pyridazin-based thioderivatives as formyl peptide receptor (FPR) agonists

Article abstract of DOI:10.1002/ddr.21076

Preclinical Research A new series of pyridazinone-based thioderivatives and pyridazine analogs was synthesized and tested for their ability to bind to the three human formyl peptide receptor (FPR) isoforms (FPR1, FPR2, and FPR3) and to activate intracellular calcium mobilization and chemotaxis in human neutrophils. Among the pyridazin-3(2H)-one derivatives tested, analogs 8b and 8c were mixed FPR1/FPR2 agonists, with median effective concentration values in the micromolar range, and were able to activate chemotaxis and Ca ²⁺ flux in human neutrophils in the low micromolar range. Molecular docking studies showed that interaction of a ligand with Arg205 of FPR1 is important for FPR1 agonist activity. For FPR2, differences in activity between oxygen-containing compounds and their thio-analogs were due to steric bulkiness of sulfur-containing groups.

Full text of DOI:10.1002/ddr.21076

DRUG DEVELOPMENT RESEARCH 74 : 259–271 (2013)
Research Article
Synthesis and Pharmacological Evaluation of New
Pyridazin-Based Thioderivatives as Formyl Peptide
Receptor (FPR) Agonists
Letizia Crocetti,1 Claudia Vergelli,1 Agostino Cilibrizzi,1 Alessia Graziano,1
Andrei I. Khlebnikov,2 Liliya N. Kirpotina,3 Igor A. Schepetkin,3 Mark T. Quinn,3 and
Maria Paola Giovannoni1*
1Dipartimento di Scienze Farmaceutiche, Sesto Fiorentino 50019, Firenze, Italy
2Department of Chemistry, Altai State Technical University, Barnaul, Russia
3Department of Immunology and Infectious Diseases, Montana State University, Bozeman,
Montana 59717, USA
Strategy, Management and Health Policy
Enabling
Preclinical Development Clinical Development
Technology,
Genomics,
Proteomics
Preclinical
Research
Toxicology, Formulation
Drug Delivery,
Pharmacokinetics
Phases I-III
Regulatory, Quality,
Manufacturing
Postmarketing
Phase IV
ABSTRACT
A new series of pyridazinone-based thioderivatives and pyridazine analogs was synthe-
sized and tested for their ability to bind to the three human formyl peptide receptor (FPR) isoforms (FPR1,
FPR2, and FPR3) and to activate intracellular calcium mobilization and chemotaxis in human neutrophils.
Among the pyridazin-3(2H)-one derivatives tested, analogs 8b and 8c were mixed FPR1/FPR2 agonists,
with median effective concentration values in the micromolar range, and were able to activate chemotaxis
and Ca2+ flux in human neutrophils in the low micromolar range. Molecular docking studies showed that
interaction of a ligand with Arg205 of FPR1 is important for FPR1 agonist activity. For FPR2, differences in
activity between oxygen-containing compounds and their thio-analogs were due to steric bulkiness of
sulfur-containing groups. Drug Dev Res 74 : 259–271, 2013.
© 2013 Wiley Periodicals, Inc.
Key words: pyridazine; pyridazin-3(2H)-one; formyl peptide receptors; neutrophil; chemotaxis; Ca2+ mobilization
INTRODUCTION
responses, e.g. directional movement of neutrophils,
lysosomal enzyme release [Schiffmann et al., 1975],
degranulation, and production of superoxide anion
[Prossnitz and Ye, 1997; Mills et al., 1999; Le et al.,
2002]. Three FPR subtypes have been identified in
humans (FPR1, FPR2, and FPR3) [Ye et al., 2009].
FPR1 was the first that was biochemically defined
and was a high-affinity binding site on the surface of
Formyl peptide receptors (FPRs) are a family of
chemoattractant receptors that play an essential role in
host defense against infection and trauma. In addition,
they are involved at different levels in the regulation of
inflammatory reactions and sensing cellular dysfunction
[Migeotte et al., 2006]. FPRs belong to the seven trans-
membrane domain G-protein-coupled receptor family,
are expressed in the majority of white blood cells,
and are important in host defense and inflammation
[Ye et al., 2009]. All major neutrophil functions accom-
plished through FPRs can be inhibited by treatment
with pertussis toxin [Bokoch and Gilman, 1984], indi-
cating that the G proteins coupled to FPRs belong
to the Gi family of heterotrimeric proteins [Simon
et al., 1991]. Activation of FPRs induces a variety of
*Correspondence to: Maria Paola Giovannoni, Diparti-
mento di Scienze Farmaceutiche, Via Ugo Schiff 6, Sesto
Fiorentino 50019, Firenze, Italy.
E-mail: mariapaola.giovannoni@unifi.it
Received 23 January 2013; Accepted 4 February 2013
Published online in Wiley Online Library (wileyonlinelibrary.
com). DOI: 10.1002/ddr.21076
© 2013 Wiley Periodicals, Inc.

260
CROCETTI ET AL.
neutrophils for the prototypic peptide formyl- 3.4 and 3.8 mM for FPR1 and FPR2, respectively),
methionine-leucine-phenylalanine (fMLF) [Ye et al., whereas compound 2 is a FPR2-specific agonist (EC50 =
2009]. FPR2 shares 69% amino acid identity with 2.4 mM). Structural analysis of this series of compounds
human FPR1, but despite the relatively high level of indicated that an acetamide spacer at N-2 of the pyridazi-
sequence homology, is a low-affinity receptor for fMLF none ring is an essential requirement for specificity and
[Quehenberger et al., 1993]. FPR3 shares 56% and potency of these compounds, and the role of both C = O
83% sequence identity with human FPR1 and FPR2, (H-bond acceptor) and amidic NH (H-bond donor) in
respectively, but does not bind N-formyl peptides, e.g. the side chain was crucial for binding to FPRs. More-
fMLF. Instead, FPR3 responds to some nonformylated over, the presence of a lipophilic and/or electronegative
chemotactic peptides identified as FPR2 agonists.
para substituent in the aromatic system of the side chain
FPRs interact with a wide range of structurally at position 2 was an important element required for
different pro- and anti-inflammatory ligands associated potency and selectivity [Cilibrizzi et al., 2009].
with different diseases, including amyloidosis and
Based on previous reports demonstrating benefi-
Alzheimer’s disease [Cui et al., 2002], some types of cial substitutions of C = S for C = O and methylthio
cancers and related alopecia induced by most antican- (SCH3) for methoxy (OCH3) [Wrobel et al., 1989;
cer agents [Edwards et al., 2005; Tsuruky et al., 2007], Güngör et al., 2006], we designed a series of thio-analogs
prion diseases [Zhou et al., 2009], HIV [Kilby et al., characterized by properties similar to those of previously
1998], stomach ulcers [De Paulis et al., 2004], and reported parent compounds [Cilibrizzi et al., 2009].
nociception associated with inflammatory processes Furthermore, a series of pyridazine analogs where the
[Pieretti et al., 2004]. Many studies indicate that FPRs phenylacetamide moiety was moved to position 3 of the
are important mediators of key events in endogenous ring was also designed. For all new compounds, we
anti-inflammation, and it has been proposed that FPR report herein the synthesis and biological evaluation for
modulation is an attractive approach for the targeting FPR agonist activity, selectivity versus FPR1/FPR2/
biochemical processes for resolving inflammation FPR3, and the ability to activate intracellular calcium
[Gilroy et al., 2004]. Likewise, FPRs may represent mobilization and chemotaxis in human neutrophils.
therapeutic targets for selectively stimulating innate
immune responses [Serhan et al., 2007; Zhang and
MATERIALS AND METHODS
Falla, 2009; Dufton and Perretti, 2010].
Chemistry
Previous studies led to the identification of a wide
range of structurally unrelated nonpeptide and peptide
Syntheses of the new pyridazin-3(2H)-one and
FPR agonists, including synthetic molecules and both pyridazine thioderivatives are depicted in Figures
host-derived and pathogen-derived agents [Bokoch 2 and 3. In Figure 2, procedures to obtain the
et al., 1984; Gierschik et al., 1989], and more recently, pyridazinone-3(2H)-one thioderivatives 8a-c, 9 and
we identified a large number of potent FPR agonists with the thiopyridazine analog 11 are reported. In both
a pyridazin-3(2H)-one scaffold that contained a meth- cases, the starting material was the 6-methyl-4,5-
oxybenzyl group at position 4 [Cilibrizzi et al., 2009, dihydropyridazin-3(2H)-one compound 4 [Meng and
2012]. Two of the most interesting compounds of the Hesse, 1990], which was directly converted in the new
series are shown in Figure 1: compound 1 is a mixed benzyl derivative 5a and the previously described 5b,c
FPR1/FPR2 agonist with activity in the low micromolar [Cilibrizzi et al., 2009] by Knoevenagel condensa-
range (median effective concentration values [EC50] = tion using the appropriate aromatic aldehyde in the
O
O
O
H
N
H
N
H
N
O
Br
Br
N
N
N
N
N
N
O
O
O
O
O
O
1
3
3.4 μM (FPR1)
7.7 μM (FPR1)
2
EC50
=
EC50
=
μ
μ
14.4 M (FPR2)
3.8 M (FPR2)
μ
EC50 = 2.4 M (FPR2)
Fig. 1. Structures of selected FPRs agonists 1–3.
Drug Dev. Res.

FPR MIXED AGONISTS
261
O
O
O
OH
N
R1
N
HN
N
HN
N
N N
A
C
B
O
O
O
O
O
D
NH
N
4
N
O
R
R
R
R
5
5a-c
6a,b
7a,b
a
b
c
SCH3 (p)
OCH3 (m)
OCH3 (p)
E
R
R
6, 7
1, 8a-c
5c
for
a
b
SCH3 (p)
OCH3 (m)
1
for
E
R1
HN
N
N
N
comp
R
Br
HS
S
1
Br
Br
I
OCH3 (m)
SCH3 (p)
SCH3 (p)
8a
8b
S
8c OCH3 (m)
NH
N
SCH3
N
O
O
O
Br
10
F
O
O
NH
N
N
S
9
O
11
Fig. 2. Synthesis of pyridazin-3(2H)-one-based thioderivatives 8a-c, 9 and pyridazine analog 11.
Reagents and conditions: (A) substituted benzaldehyde (1 equiv), KOH 5% (w/v) in anhydrous EtOH, 1–5 h, reflux; (B) ethyl bromoacetate (1.5
equiv), K2CO3 (2 equiv), anhydrous CH3CN, 2–4 h, reflux; (C) NaOH 6 N, 1–2 h, 80°C; (D) ethyl chloroformate (1.1 equiv), Et3N (3.5 equiv),
substituted aniline (2 equiv), anhydrous THF, 12 h, -5°C → rt; (E) Lawesson’s reagent (2 equiv), anhydrous toluene, 2–3 h, 80°C; (F)
N-(4-bromophenyl)-2-chloroacetamide (1.5 equiv), K2CO3 (2 equiv), anhydrous CH3CN, 3 h, reflux.
presence of KOH in absolute EtOH (5% w/v). Alkyla- was present only and univocally on the side chain.
tion of intermediates 5a, 5b with ethyl bromoacetate Figure 2 also depicts the synthetic procedure for
resulted in the esters 6a, 6b, [Cilibrizzi et al., 2009], obtaining the pyridazine analog 11. Using Lawesson’s
which after alkaline hydrolysis gave the corresponding reagent, as described above, the precursor 5c [Cilibrizzi
acids 7a,b [Cilibrizzi et al., 2009]. Subsequent reaction et al., 2009] was transformed into the intermediate 10
of 7a,b with ethyl chloroformate in THF in presence of which, in turn, was alkylated under standard conditions
triethylamine resulted in the intermediate mixed anhy- with N-(4-bromophenyl)-2-chloroacetamide [Baraldi
drides, which in turn were transformed in good yields et al., 2007] to obtain the final compound 11, contain-
into the final amide 1 [Cilibrizzi et al., 2009] and the ing the phenylacetamide moiety at C-3 of the ring.
new compounds 8a-c. Moreover, the thioamide analog
The synthetic procedure to obtain pyridazine
9 was directly generated from compound 1 by treat- analogs 15a,b and 18 is depicted in Figure 3. The
ment with Lawesson’s reagent in toluene at 80°C previously described 3-chloro-6-phenylpyridazin-4-ol,
[Jesberger et al., 2003]. Considering that compound 1 compound 12 [Sircar, 1983] was converted into
has two carbonyl groups susceptible to thionation, both intermediate 13 by performing a cross-coupling reac-
1H NMR and MS(ESI) analyses were performed to tion with the appropriate aryl boronic acids, using
confirm structure 9. MS analysis clearly demonstrated copper acetate as catalyst and a weak base (triethy-
the presence of a fragment of compound 9 containing lamine) in CH2Cl2 [Quach and Batey, 2003; Chiang
1
the pyridazinone nucleous, and H NMR experiments and Olsson, 2004]. Compound 13 was then treated
showed a clear difference in the chemical shift of the with saturated ethanolic ammonia at 180°C to obtain
signal related to N-CH2-CO in 1 and the corresponding the corresponding 3-amino derivative 14, which
N-CH2-CS in compound 9. These findings suggest that was directly reacted under standard conditions with
the pyridazinone ring was unchanged, and the sulfur the 4-bromophenylisocyanate or the 4-bromobenzoyl
Drug Dev. Res.

262
CROCETTI ET AL.
Br
N
X
N
N
N
N
N
B
C
H2N
HN
Cl
O
O
O
13
14
15a,b
A
O
O
O
15
X
N
N
Cl
a
b
CONH
CO
D
Br
HO
12
O
O
HN
O
O
OH
N
O
O
N
N
N
N
N
E
F
HN
HN
HN
O
18
17
16
O
O
O
Fig. 3. Synthesis of pyridazine-based analogs 15a,b and 18.
Reagents and conditions: (A) 4-methoxyphenylboronic acid (2 equiv), Cu(OAc)2 (1.5 equiv), Et3N (2 equiv), CH2Cl2, 12 h, rt; (B) NH3/anhydrous
EtOH, 7 h, 180°C; (C) for 15a: 4-bromophenylisocyanate (1.1 equiv) anhydrous toluene, 6 h, reflux; for 15b: 4-bromobenzoyl chloride (4 equiv),
Et3N (catalytic), anhydrous CH2Cl2, 24 h, 0°C/rt; (D) ethyl bromoacetate (3 equiv), K2CO3 (2 equiv), anhydrous CH3CN, 8 h, reflux; (E) NaOH
6 N, 2 h, 80°C; (F) ethyl chloroformate (1.1 equiv), Et3N (3.5 equiv), 4-bromoaniline (2 equiv), anhydrous THF, 12 h, -5°C/rt.
1
1
chloride to obtain the urea 15a and the amide 15b, through H NMR and TLC. H NMR spectra were
respectively. Alternatively, final compound 18 was pre- recorded with Avance 400 instruments (Bruker Biospin
pared from intermediate 14 by performing the same Version 002, Bruker AXS Inc., Madison, WI USA with
reaction sequence (alkylation with ethyl bromoacetate, full digital signal generation [SGU]). Chemical shifts (d)
alkaline hydrolysis, and finally, through the intermedi- were reported in ppm to the nearest 0.01 ppm, using
ate mixed anhydride, coupling with 4-bromoaniline) the solvent as the internal standard. Coupling constants
shown in Figure 2 for compounds 1 and 8a-c.
(J-values) are given in Hz and were calculated using
TopSpin 1.3 software (Bruker) rounded to the nearest
0.1 Hz. Mass spectra (m/z) were recorded on an
ESI-MS triple quadrupole mass spectrometer (Varian
1200L, Sunnyvale, CA USA). Microanalyses were per-
formed with a Perkin-Elmer 260 elemental analyzer
(Waltham, MA USA) for C, H, and N, and the results
were within Ϯ 0.4% of the theoretical values, unless
otherwise stated.
Experimental
All chemicals were purchased from Sigma-Aldrich
(St. Louis, MO, USA). 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 (Merck, Darms-
tadt, Germany). Visualization was performed by UV
fluorescence (lmax = 254 nm) or by staining with iodine
or potassium permanganate. Chromatographic separa-
tions were performed on a silica gel column by gra-
vity chromatography (Kieselgel 40, 0.063–0.200 mm;
Merck) or flash chromatography (Kieselgel 40, 0.040–
0.063 mm; Merck). All melting points were determined
on a microscope hot stage Büchi apparatus (Assago,
Milano, Italy) and are uncorrected. Yields refer to
chromatographically and spectroscopically pure com-
pounds, unless otherwise stated. The identity and purity
of intermediates and final compounds were ascertained
General procedure for compounds (5a-c)
To 12 ml of a solution of KOH in absolute EtOH
(5%, w/v), compound 4 (4.46 mmol) and the appropri-
ate aromatic aldehyde (4.46 mmol) were added, and the
solution was refluxed under stirring for 1–5 h. After
cooling, the mixture was concentrated in vacuo, diluted
with ice-cold water (20–25 ml) and acidified with 2 N
HCl. The suspension was extracted with CH2Cl2
(3 ¥ 25 ml), and removal of the solvent resulted in com-
pounds 5a-c, which were purified by crystallization in
ethanol.
Drug Dev. Res.

FPR MIXED AGONISTS
263
6-Methyl-4-[4-(methylthio)benzyl]pyridazin-3(2H)-
one (5a)
carried out at room temperature (rt) for 12 h. The
mixture was then concentrated in vacuo, diluted with
Mp = 151–153°C; crystallization solvent = EtOH; cold water (10–15 ml) and extracted with CH2Cl2
1
yield = 46%. H NMR (CDCl3), d: 2.26 (3H, s), 2.51 (3 ¥ 15 ml). The solvent was evaporated to obtain final
(3H, s), 3.87 (2H, s), 6.71 (1H, s), 7.18 (2H, d, compounds 1 and 8a-c, which were purified by column
J = 8.2 Hz), 7.27 (3H, dd, J = 9.3, 1.8 Hz).
chromatography using cyclohexane/ethyl acetate 1:1 for
compounds 1 and 8a,c and cyclohexane/ethyl acetate
2:1 for 8b as eluents.
General procedure for compounds (6a,b)
A mixture of the suitable intermediate 5a,b
(4.50 mmol), K2CO3 (9.00 mmol), and ethyl bromoac- N-(4-Bromophenyl)-2-{3-methyl-5-[4-(methylthio)
etate (6.75 mmol) in CH3CN (10 ml) was refluxed benzyl]-6-oxo-pyridazin-1(6H)-yl}acetamide (8a)
under stirring for 2–4 h. The mixture was then concen-
Mp = 97–99°C; purified by column chromatogra-
1
trated in vacuo, diluted with cold water, and extracted phy (cyclohexane/ethyl acetate 1:1); yield = 10%. H
with CH2Cl2 (3 ¥ 15 ml). The solvent was evaporated in NMR (CDCl3) d 2.30 (3H, s), 2.50 (3H, s), 3.89 (2H, s),
vacuo, and compounds 6a and b were purified by crys- 4.94 (2H, s), 6.80 (1H, s), 7.17 (2H, d, J = 8.2 Hz), 7.24
tallization from ethanol.
(2H, d, J = 8.2 Hz), 7.39 (4H, dd, J = 2.6, 5.7 Hz), 9.01
(1H, exch br s). MS (ESI) Calcd. for C21H20BrN3O2S,
458.37. Found: m/z 458.17 [M + H]+. Anal. Calcd for
C21H20BrN3O2S: C, 55.03; H, 4.40; N, 9.17. Found: C,
54.92; H, 4.39; N, 9.20.
Ethyl-2-{3-methyl-5-[4-(methylthio)benzyl]-6-
oxopyridazin-1(6H)-yl}acetate (6a)
Mp = 129–131°C; crystallization solvent = EtOH;
1
yield = 99.9%. H NMR (CDCl3), d: 1.31 (3H, t, J =
6.8 Hz), 2.24 (3H, s), 2.51 (3H, s), 3.87 (2H, s), 4.27 N-(4-Iodophenyl)-2-{3-methyl-5-[4-(methylthio)
(2H, q, J = 6.8 Hz), 4.86 (2H, s), 6.68 (1H, s), 7.17 (2H, benzyl]-6-oxo-pyridazin-1(6H)-yl}acetamide (8b)
d, J = 7.3 Hz), 7.25–7.29 (2H, m).
Mp = 68–70°C; purified by column chromatogra-
1
phy (cyclohexane/ethyl acetate 2:1); yield = 46%. H
NMR (CDCl3) d 2.30 (3H, s), 2.51 (3H, s), 3.89 (2H, s),
4.93 (2H, s), 6.80 (1H, s), 7.18 (2H, s, J = 8.4 Hz), 7.24-
General procedure for compounds (7a,b)
A suspension of the intermediate 6a and 6b 7.28 (4H, m), 7.61 (2H, d, J = 8.8 Hz), 8.92 (1H, exch br
(4.40 mmol) in 6 N NaOH (10 ml) was stirred at 80°C s). MS (ESI) Calcd. for C21H20IN3O2S, 505.37. Found:
for 1–2 h. The mixture was first diluted with ice-cold m/z 506.11 [M + H]+. Anal. Calcd for C21H20IN3O2S:
water and then acidified with 6 N HCl. Products 7a C, 49.91; H, 3.99; N, 8.31. Found: C, 49.86; H, 4.01;
and 7b were filtered by suction and recrystallized from N, 8.34.
ethanol.
2-[5-(3-Methoxybenzyl)-3-methyl-6-oxopyridazin-1
2-{3-Methyl-5-[4-(methylthio)benzyl]-6-
oxopyridazin-1(6H)-yl}acetic acid (7a)
(6H)-yl]-N-[4-(methylthio)phenyl]acetamide (8c)
Mp = 166–167°C; purified by column chromatog-
Mp = 87–89°C; crystallization solvent = EtOH; raphy (cyclohexane/ethyl acetate 1:1); yield = 99.9%. 1H
1
yield = 99.9%. H NMR (CDCl3), d 2.25 (3H, s), 2.51 NMR (CDCl3), d 2.28 (3H, s), 2.45 (3H, s), 3.80 (3H, s),
(3H, s), 3.87 (2H, s), 4.91 (2H, s), 6.69 (1H, s), 7.16 (2H, 3.89 (2H, s), 4.95 (2H, s), 6.79–6.85 (4H, m), 7.12–
d, J = 7.6 Hz), 7.25 (2H, d, J = 7.6 Hz).
7.15 (1H, m), 7.27 (1H, t, J = 7.8 Hz), 7.39 (2H, d,
J = 5.0 Hz), 9.13 (1H, exch br s). MS (ESI) Calcd. for
C22H23N3O3S, 409.50. Found: m/z 410.11 [M + H]+.
Anal. Calcd for C22H23N3O3S: C, 64.53; H, 5.66; N,
10.26. Found: C, 64.66; H, 5.64; N, 10.22.
General procedure for compounds (1) [Cilibrizzi
et al., 2009] and (8a-c)
To a cooled (-5°C) and stirred solution of the
appropriate carboxylic acid 7a,b (0.90 mmol), in anhy-
drous tetrahydrofuran (6 ml), Et3N (3.15 mmol) was N-(4-Bromophenyl)-2-[5-(3-methoxybenzyl)-
added. After 30 min, the mixture was allowed to warm 3-methyl-6-oxo-pyridazin-1(6H)-
up to 0°C, and ethyl chloroformate (0.99 mmol) was yl]ethanethioamide (9)
added. After 1 h, the appropriately substituted ary-
Compound 9 was obtained by slow addition of
lamine (1.80 mmol) was added, and the reaction was Lawesson’s reagent (0.28 mmol) to a stirred solution of
Drug Dev. Res.

264
CROCETTI ET AL.
compound 1 (0.14 mmol) in toluene (3 ml), and the (2.62 mmol), copper acetate (1.96 mmol), and Et3N
reaction was carried out at reflux for 3 h. The solvent (2.62 mmol) in CH2Cl2 (10 ml) was stirred for 12 h at rt.
was removed in vacuo, and the mixture was diluted with The copper salts were filtered off, and the organic
ice-cold water and extracted with CH2Cl2 (3 ¥ 10 ml). layer was extracted with 50% aqueous ammonia
The crude product was finally purified by flash column (3 ¥ 10 ml), washed with water (10 ml), and dried over
chromatography using cyclohexane/ethyl acetate 1:1 as Na2SO4. After removal of the solvent in vacuo, the
eluent, to yield 9 as an amorphous solid. Mp = 68– residue was purified by flash column chromato-
1
70°C; crystallization solvent = EtOH; yield = 30%. H graphy using cyclohexane/ethyl acetate 1:1 as eluent.
NMR (CDCl3), d 2.32 (3H, s), 3.81 (3H, s), 3.92 (2H, s), Mp = 124–126°C; purified by column chromatography
1
5.37 (2H, s), 6.81 (1H, s), 6.83-6.87 (3H, m), 7.28 (1H, (cyclohexane/ethyl acetate 1:1); yield = 48%. H-NMR
t, J = 5.0 Hz), 7.48 (2H, d, J = 8.8 Hz), 7.74 (2H, d, (CDCl3), d: 3.86 (3H, s), 6.99 (2H, d, J = 9.2 Hz), 7.45-
J = 8.8 Hz), 11.46 (1H, exch br s). MS (ESI) Calcd. for 7.47 (3H, m), 7.61 (2H, d, J = 9.2 Hz), 7.78-7.80 (2H,
C21H20BrN3O2S, 458.37. Found: m/z 458.17 [M + H]+, m), 7.93 (s, 1H).
482.36 [M + Na]+, 378.45 [M – Br]+, 231.17 [M –
C8H7BrNS]+. Anal. Calcd for C21H20BrN3O2S: C, 55.03; 4-(4-Methoxyphenoxy)-6-phenylpyridazin-3-
H, 4.40; N, 9.17. Found: C, 55.08; H, 4.39; N, 9.19.
amine (14)
A solution of compound 13 in saturated ethanolic
ammonia was heated in a sealed stainless steel tube
at 180°C for 7 h. The mixture was then concen-
trated in vacuo, and the residue was treated with cold
diethyl ether to obtain a crude solid that was recovered
by suction and recrystallized by ethanol. Mp = 160–
162°C; crystallization solvent = EtOH; yield = 99.9%.
1H-NMR (DMSO), d: 3.79 (3H, s), 6.66 (2H, exch br s),
6.78 (1H, s), 7.01 (2H, d, J = 8.4 Hz), 7.38-7.43 (3H, m),
7.53 (2H, d, J = 8.4 Hz), 7.73 (2H, d, J = 7.2 Hz).
4-(4-Methoxybenzyl)-6-methylpyridazine-3(2H)-
thione (10)
Lawesson’s reagent (0.87 mmol) was slowly added
to a stirred solution of intermediate 5c (0.87 mmol) in
toluene (3 ml), and the reaction was carried out at reflux
for 2 h. The mixture was cooled and after 1 h stirring
in ice-bath, the precipitate was filtered and purified
by recrystallization from ethanol. Mp = 191–193°C;
1
crystallization solvent = EtOH; yield = 47%. H NMR
(CDCl3), d 2.30 (3H, s), 3.85 (3H, s), 4.11 (2H, s), 6.57
(1H, s), 6.93 (2H, d, J = 8.5 Hz), 7.17 (2H, d,
J = 8.5 Hz), 12.12 (1H, exch br s).
1-(4-Bromophenyl)-3-[4-(4-methoxyphenoxy)-6-
phenylpyridazin-3-yl]urea (15a)
To a stirred solution of compound 14 (0.23 mmol)
in anhydrous toluene (3 ml), 4-bromophenyl isocyanate
(0.26 mmol) was added. The mixture was refluxed for
6 h. After cooling, the solvent was removed under
reduced pressure, and the residue was dissolved in
CH2Cl2 and washed, in turn, with 2 N HCl (3 ¥ 15 ml),
2 N NaOH (3 ¥ 15 ml), and with H2O (15 ml). The
organic layer was dried over Na2SO4 and evaporated in
vacuo to obtain the crude products, which were purified
by flash column chromatography using CH2Cl2/CH3OH
(gradient 100:0 to 98:2) as eluent. Mp = 267–269°C;
purified by column chromatography (CH2Cl2/CH3OH,
gradient 100:0 to 98:2); yield = 25%. 1H-NMR (CDCl3),
d: 3.65 (3H, s), 6.82 (4H, d, J = 8.8 Hz), 7.42–7.48 (5H,
m), 7.64 (2H, d, J = 8.8 Hz), 7.89–7.92 (2H, m), 8.55
(1H, exch br s), 8.80 (1H, s), 9.42 (1H, exch br s). MS
(ESI) Calcd. for C24H19BrN4O3, 491.34. Found: m/z
491.11 [M + H]+. Anal. Calcd for C24H19BrN4O3: C,
58.67; H, 3.90; N, 11.40. Found: C, 58.55; H, 3.89;
N, 11.35.
N-(4-Bromophenyl)-2-[4-(4-methoxybenzyl)-6-
methylpyridazin-3-ylthio]acetamide (11)
A mixture of compound 10 (0.41 mmol), K2CO3
(0.82 mmol), and N-(4-bromophenyl)-2-chloro aceta-
mide (0.61 mmol) in CH3CN (4 ml) was refluxed under
stirring for 1.5 h. After cooling, the solvent was evapo-
rated, and the mixture was diluted with cold water. The
precipitate was filtered and purified by flash chroma-
tography using cyclohexane/ethyl acetate 1:2 as eluent.
Mp = 116–118°C; purified by column chromatography
1
(cyclohexane/ethyl acetate 1:2); yield = 96%. H NMR
(CDCl3), d 2.62 (3H, s), 3.84 (3H, s), 3.87 (2H, s), 4.08
(2H, s), 6.84 (1H, s), 6.92 (2H, d, J = 8.5 Hz), 7.11 (2H,
d, J = 8.5 Hz), 7.38 (2H, d, J = 8.8 Hz), 7.48 (2H, d,
J = 8.8 Hz), 10.26 (1H, exch br s). MS (ESI) Calcd. for
C21H20BrN3O2S, 458.37. Found: m/z 458.11 [M + H]+.
Anal. Calcd for C21H20BrN3O2S: C, 55.03; H, 4.40; N,
9.17. Found: C, 54.81; H, 4.38; N, 9.19.
3-Chloro-4-(4-methoxyphenoxy)-6-
phenylpyridazine (13)
4-Bromo-N-[4-(4-methoxyphenoxy)-6-
phenylpyridazin-3-yl]benzamide (15b)
Et3N (0.025 ml) and 4-bromobenzoyl chloride
A
suspension of 3-chloro-6-phenylpyridazin-
4-ol 12 (1.31 mmol), 4-methoxyphenylboronic acid (0.67 mmol) were added to a cooled (0°C) and stirred
Drug Dev. Res.

FPR MIXED AGONISTS
265
solution of 14 (0.33 mmol) in anhydrous CH2Cl2 (4 ml), N-(4-Bromophenyl)-2-[4-(4-methoxyphenoxy)-6-
and the reaction was mantained under stirring at phenylpyridazin-3-ylamino]acetamide (18)
0°C for 3 h. Additional 4-bromobenzoyl chloride
To a cooled (-5°C) and stirred solution of the
(0.67 mmol) was added, and the mixture was stirred at carboxylic acid 17 (0.14 mmol) in anhydrous tetrahy-
rt for 24 h. The solid residue was removed by filtration, drofuran (5 ml), Et3N (0.49 mmol) was added. After
and the organic layer was washed with 6 N NaOH 30 min, the mixture was allowed to warm up to 0°C, and
(3 ¥ 10 ml), 6 N HCl (3 ¥ 10 ml) and, finally, with cold ethyl chloroformate (0.15 mmol) was added. After 1 h,
water (2 ¥ 10 ml). Drying with Na2SO4 and evaporation 4-bromoaniline (0.28 mmol) was added. The reaction
of the solvent in vacuo resulted in crude compound was carried out at room temperature for 5 h. The
15b, which was purified by flash chromatography mixture was then concentrated in vacuo, diluted with
using CH2Cl2 as eluent. Mp = 203–204°C; purified cold water (10–15 ml), and extracted with CH2Cl2
by column chromatography (CH2Cl2); yield = 63%. (3 ¥ 15 ml). The solvent was evaporated to obtain final
1H-NMR (CDCl3), d: 3.87 (3H, s), 7.02 (2H, d, compound 18, which was purified by column chroma-
J = 8.4 Hz), 7.44–7.49 (3H, m), 7.66 (4H, t, J = 8.4 Hz), tography using cyclohexane/ethyl acetate 1:1 as eluent.
7.83 (2H, s. J = 8.4 Hz), 7.91 (2H, dd, J = 6.0, 2.0 Hz), Mp = 238–239°C; purified by column chromatography
1
8.84 (1H, s), 9.52 (1H, exch br s). MS (ESI) Calcd. for (cyclohexane/ethyl acetate 1:1); yield = 28%. H-NMR
C24H18BrN3O3, 476.32. Found: m/z 476.11 [M + H]+. (CDCl3), d: 3.83 (3H, s), 4.09 (2H, s), 6.60 (1H, s),
Anal. Calcd for C24H18BrN3O3: C, 60.52; H, 3.81; N, 6.96 (2H, d, J = 9.2 Hz), 7.41-7.49 (7H, m), 7.58 (2H,
8.82. Found: C, 60.46; H, 3.82; N, 8.85.
d, J = 9.2 Hz), 7.76 (2H, dd, J = 5.2, 2.8 Hz), 8.25 (1H,
exch br s). MS (ESI) Calcd. for C25H21BrN4O3,
505.36. Found: m/z 505.10 [M + H]+. Anal. Calcd for
C25H21BrN4O3: C, 59.42; H, 4.19; N, 11.09. Found: C,
59.53; H, 4.17; N, 11.11.
Ethyl 2-[4-(4-methoxyphenoxy)-6-phenylpyridazin-
3-ylamino]acetate (16)
A mixture of the intermediate 14 (0.33 mmol),
K2CO3 (0.66 mmol), and ethyl bromoacetate (1.00
mmol) in CH3CN (4 ml) was refluxed with stirring for
8 h. The mixture was then concentrated, diluted with
cold water, and extracted with CH2Cl2 (3 ¥ 15 ml). The
organic layer was dried over Na2SO4 and evaporated
in vacuo to obtain the crude product 16, which was
purified by flash chromatography using cyclohexane/
ethyl acetate 2:1 as eluent. Mp = 130–132°C; purified
by column chromatography (cyclohexane/ethyl acetate
Pharmacology
Cell culture
Human promyelocytic leukemia HL-60 cells
stably transfected with FPR1 (HL-60-FPR1), FPR2
(HL-60-FPR2), or FPR3 (HL-60-FPR3) were cultured
in RPMI 1640 medium supplemented with 10% heat-
inactivated fetal calf serum, 10 mM HEPES, 100 mg/ml
streptomycin, 100 U/ml penicillin, and G418 (1 mg/ml),
as previously described [Christophe et al., 2002]. Wild-
type HL-60 cells were cultured under the same condi-
tions but without G418.
1
2:1); yield = 79%. H NMR (CDCl3), d: 1.32 (3H, t,
J = 7.2 Hz), 3.83 (3H, s), 4.04 (2H, d, J = 5.2 Hz), 4.26
(2H, q, J = 7.2 Hz), 6.39 (1H, s), 6.98 (2H, d, J = 8.8 Hz),
7.39-7.43 (3H, m), 7.62 (2H, d, J = 8.8 Hz), 7.78 (2H, d,
J = 7.6 Hz).
Isolation of human neutrophils
Blood was collected from healthy donors in accor-
dance with a protocol approved by the Institutional
Review Board at Montana State University. Neutro-
2-[4-(4-Methoxyphenoxy)-6-phenylpyridazin-3-
ylamino]acetic acid (17)
A suspension of compound 16 (0.30 mmol) in 6 N phils were purified from the blood using dextran
NaOH (4 ml) was stirred at rt to 80°C for 2 h. The sedimentation, followed by Histopaque 1077 (Sigma
mixture was first diluted with ice-cold water, acidified Aldrich, St. Louis, MO USA) gradient separation and
with 6 N HCl, and then extracted with CH2Cl2. The hypotonic lysis of red blood cells, as previously
organic layer was dried over Na2SO4 and evaporated in described [Schepetkin et al., 2007]. Isolated neutro-
vacuo to obtain the crude product 17, which was puri- phils were washed twice and resuspended in HBSS
fied by recrystallization from ethanol. Mp = 203–205°C; without Ca2+ and Mg2+ (Hank’s balanced salt solution
1
crystallization solvent = EtOH; yield = 54%. H NMR [HBSS-]; Mediatech, Inc., Herndon, VA). Neutrophil
(CDCl3), d: 3.83 (3H, s), 3.95 (2H, s), 6.42 (1H, s), preparations were routinely >95% pure, as determined
6.94–6.97 (2H, m), 7.40–7.48 (3H, m), 7.57 (2H, d, by light microscopy, and >98% viable, as determined by
J = 8.8 Hz), 7.76–7.79 (2H, m).
trypan blue exclusion.
Drug Dev. Res.

266
CROCETTI ET AL.
Ca2+ mobilization assay
compounds/spontaneous cell migration in response to
Changes in intracellular Ca2+ were measured with control medium) ¥ 100. EC50 values were determined
a FlexStation II scanning fluorometer using a FLIPR 3 by nonlinear regression analysis of the dose–response
calcium assay kit (Molecular Devices, Sunnyvale, CA) curves generated using Prism 5 software.
for human neutrophils and HL-60 cells. All active com-
pounds were evaluated in parent (wild-type) HL-60
cells to verify that agonists are inactive in nontrans- Molecular modeling
fected cells (data not shown). Human neutrophils or
The FPR1 homology model was created using the
HL-60 cells, suspended in HBSS- containing 10 mM crystal structure of bovine rhodopsin, as reported pre-
HEPES, were loaded with Fluo-4 AM dye (Invitrogen, viously [Movitz et al., 2010]. A Protein Data Bank
Eugene, OR) (1.25 mg/ml final concentration) and incu- (PDB) file of the homology model was loaded into the
bated for 30 min in the dark at 37°C. After dye loading, Molegro Virtual Docker (MVD) program (MVD
the cells were washed with HBSS- containing 10 mM 2010.4.2, Molegro ApS, Katrinebjerg, Denmark). The
HEPES, resuspended in HBSS containing 10 mM position of the ligand binding site in the model was
HEPES and Ca2+ and Mg2+ (HBSS+), and aliquotted recently localized by docking studies of different FPR1
into the wells of a flat-bottomed, half-area-well black agonists [Movitz et al., 2010; Khlebnikov et al., 2012].
microtiter plates (2 ¥ 105 cells/well). The compound Docking search space was defined as a sphere centered
source plate contained dilutions of test compounds in at the carbonyl carbon of the Ala residue in the FPR1
HBSS+. Changes in fluorescence were monitored peptide agonist Ac-QAWF in its docking pose obtained
(lex = 485 nm, lem = 538 nm) every 5 s for 240 s at room by Movitz et al. [2010]. The radius of the sphere was
temperature after automated addition of compounds. adopted to be equal to 11 Å. This search space encom-
Maximum change in fluorescence, expressed in arbi- passed the whole Ac-QAWF molecule and included, at
trary units over baseline, was used to determine agonist least partially, 36 residues of FPR1. Among these, side
response. Responses were normalized to the response chains of 23 residues closest to the center of the search
induced by 5 nM fMLF (Sigma Chemical Co., St. space were set flexible during the docking simulation.
Louis, MO) for HL-60-FPR1 and neutrophils, or 5 nM
For FPR2 homology modeling, the primary amino
Trp-Lys-Tyr-Met-Val-D-Met-NH2 (WKYMVm) (Cal- acid sequence of FPR2 was submitted to the Phyre2
biochem, San Diego, CA) for HL-60-FPR2 and HL-60- (Protein Homology/analogy Recognition Engine V2.0)
FPR3 cells, which were assigned a value of 100%. protein fold recognition server (Structural Bioinformat-
Curve fitting (5–6 points) and calculation of EC50 were ics Group, Imperial College, London; http://www.sbg.
performed by nonlinear regression analysis of the dose– bio.ic.ac.uk/phyre2) [Kelley and Sternberg, 2009], and
response curves generated using Prism 5 (GraphPad the homology model, created using the crystal structure
Software, Inc., San Diego, CA).
of bovine rhodopsin, was optimized, as described
recently [Schepetkin et al., 2013].
Taking into account a lack of structural informa-
tion about any ligand-receptor complex with FPR2, we
Chemotaxis assay
Neutrophils were suspended in HBSS+ containing tried to locate cavities in the macromolecule obtained
2% (v/v) fetal bovine serum (FBS) (2 ¥ 106 cells/ml), by homology modeling in order to identify the search
and chemotaxis was analyzed in 96-well ChemoTx space for docking. Use of the MVD “Detect cavity”
chemotaxis chambers (Neuroprobe, Gaithersburg, module with probe size 1.2 Å gave two cavities with
MD), as previously described [Schepetkin et al., 2007]. volumes of 241 and 25 Å3 in the region of the binding
In brief, lower wells were loaded with 30 ml of HBSS+ site. Positions of these two cavities reflect an asymmet-
containing 2% (v/v) FBS and the indicated concentra- ric dumb-bell shape of the binding site. Hence, for
tions of test compound, DMSO (negative control), and FPR2, we also chose a spherical search space with a
1 nM fMLE as a positive control. The number of default radius 15 Å centered at the terminus of the
migrated cells was determined by measuring ATP in larger cavity directed to the smaller one. This sphere
lysates of transmigrated cells using a luminescence- embraced two cavities and eight residues reported by
based assay (CellTiter-Glo; Promega, Madison, WI), Fujita et al. [Fujita et al., 2011]. Side chains of 45 resi-
and luminescence measurements were converted to dues closest to the center of the sphere were considered
absolute cell numbers by comparison of the values flexible in the docking study.
with standard curves obtained with known numbers of
Before docking, structures of the compounds
neutrophils. The results are expressed as percentage were preoptimized using HyperChem software (Hyper-
of negative control and were calculated as follows: cube, Inc., Gainesville, FL) with MM+ force field and
(number of cells migrating in response to test saved in Tripos MOL2 format. The ligand structures
Drug Dev. Res.

FPR MIXED AGONISTS
267
were then imported into the MVD program with the and WKYMVm) and previously described pyrida-
following options enabled: “Create explicit hydrogens,” zinones 1–3 (Fig. 1 and [Cilibrizzi et al., 2009]), were
“Assign charges (calculated by MVD),” and “Detect determined. All compounds were also evaluated in
flexible torsions in ligands.” Selected molecules were wild-type, nontransfected HL-60 cells to verify re-
docked into FPR1 and FPR2 using the search spaces sponse specificity. Moreover, the compounds that
indicated above with a rigid receptor structure. Ligand showed the best profile were selected to evaluate their
flexibility was accounted for with respect to torsion chemotactic activity and capacity to mobilize Ca2+ in
angles autodetected in MVD. MolDock score functions human neutrophils.
were used with 0.3 Å grid resolution. The “Internal
Analysis of pyridazinone derivatives containing a
H-bond” option was activated in the “Ligand evalua- methylthio group in the phenyl ring at position C-4
tion” menu of Docking Wizard. Fifteen docking runs (Table 1) showed that substitution of OCH3 at this posi-
were performed for each molecule with side chain flex- tion of the previously published FPR2-specific agonist 2
ibility enabled for the residues mentioned above. The (Fig. 1 and [Cilibrizzi et al., 2009]) with SCH3 resulted
postprocessing options “Energy minimization” and in compound 8a, which was inactive for all FPRs. Thus,
“Optimize H-bonds” were applied after docking.
differences in steric hindrance between oxygen and
sulfur appear to result in loss of agonist activity. Sur-
prisingly, the iodine analog, compound 8b, had agonist
activity in the micromolar range for both FPR1 and
RESULTS AND DISCUSSION
We synthesized a series of new pyridazin-3(2H)- FPR2. Similarly, substitution of OCH3 in the para posi-
one-based thioderivatives and pyridazine analogs and tion of the phenylacetamide linker of reference com-
screened them in order to identify novel molecules able pound 3 (Fig. 1 and [Cilibrizzi et al., 2009]) with SCH3
to activate human neutrophils through FPRs. The final (compound 8c) did not change FPR agonist activity,
compounds were evaluated for their ability to induce keeping the potency at micromolar levels. These data
intracellular Ca2+ flux in HL-60 cells transfected with suggest that it is possible to modify the para position of
FPR1, FPR2, or FPR3, as it is possible to estimate FPR phenylacetamide side chain, resulting in compounds
binding using this assay system [Prossnitz et al., 1991; with similar activity. In contrast, transformation of our
Didsbury et al., 1992]. Both EC50 values and relative lead compound 1 (Fig. 1 and [Cilibrizzi et al., 2009]) by
efficacy, compared with the peptide agonists (fMLF substitution of the oxygen atom in the amide bridge
TABLE 1. Functional Activity of New Pyridazin-3(2H)-One Thioderivatives 8a-c and 9 in HL-60 Cells Expressing Human FPR1, FPR2, or
FPR3, in Comparison with Reference Compounds 1–3*
O
X
N
N
NH
R
R1
Ca2+ mobilization EC50 (mM) and efficacy (%)†
Compd.
R
X
R1
Br
Br
Br
FPR1
FPR2
FPR3
1*
9
2*
8a
3*
8c
8b
OCH3 (m)
OCH3 (m)
OCH3 (p)
SCH3 (p)
OCH3 (m)
OCH3 (m)
SCH3 (p)
O
S
3.4 Ϯ 1.6 (75)
N.A.
3.8 Ϯ 1.5 (70)
N.A.
2.4 Ϯ 0.9 (70)
N.A.
14.4 Ϯ 2.0 (35)
8.2 Ϯ 2.5 (40)
9.4 Ϯ 2.7 (60)
20.4
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
1.9
O
O
O
O
O
N.A.
N.A.
Br
OCH3
SCH3
I
7.7 Ϯ 2.5 (65)
2.2 Ϯ 0.69 (70)
2.3 Ϯ 0.72 (80)
0.01
fMLF
WKYMVm
0.5
0.001
0.01
*Values of activity and efficacy are from Cilibrizzi et al. [2009].
†N.A., no activity was observed (no response was observed during first 2 min after addition of compounds under investigation) considering the
limits of efficacy >20% and EC50 <50 mM. EC50 values are presented as the mean Ϯ standard deviation of three independent experiments, in
which median effective concentration values (EC50) were determined by nonlinear regression analysis of the dose–response curves (5–6 points)
generated using GraphPad Prism 5 with 95% confidential interval (P < 0.05). Efficacy (in parentheses) is expressed as % of the response induced
by 5 nM fMLF (FPR1) or 5 nM WKYMVm (FPR2 and FPR3).
Drug Dev. Res.

268
CROCETTI ET AL.
A
B
Fig. 5. Docking poses of selected pyridazine derivatives within the
FPR1 binding site. (A) Poses of active compound 8b (blue) and inactive
compound 8a (brown). (B) Poses of active compound 1 (yellow) and
inactive compound 9 (sage). [Color figure can be viewed in the online
issue which is available at wileyonlinelibrary.com]
Fig. 4. Activation of human neutrophils by selected compounds. (A)
Representative kinetics of neutrophil intracellular Ca2+ flux after treat-
ment with compound 8b or fMLF. Human neutrophils were treated
with different concentrations of the compound 8b, 5 nM fMLF, or 1%
DMSO (negative control), and Ca2+ flux was monitored for the indi-
cated times. The data are from one experiment that is representative of
three independent experiments. (B) Human neutrophil chemotaxis
toward the indicated concentrations of compounds 8b (᭿) and 8c (᭺)
was determined, as described under Materials and Methods. The
data are presented as the mean Ϯ standard deviation of duplicate
samples from one experiment that is representative of two indepen-
dent experiments.
Molecular docking of selected thioderivatives into
the FPR1 and FPR2 binding sites was performed to
assess how the presence of the sulfur atom might influ-
ence the binding modes of the compounds as compared
with their oxygen-containing analogs. Taking into
account that FPR1 has a more open and broader
binding site than FPR2, the docking results were ana-
lyzed in terms of partial interactions of ligands with
FPR1 residues. As shown in Figure 5, compounds 8a,
8b, 1, and 9 occupy different locations within the
receptor. Arg205 is one of the key residues contributing
with a sulfur atom led to thioamide 9, which was inac- to FPR1 agonist activity [Mills et al., 2000; Khlebnikov
tive. Lastly, none of the pyridazine analogs (11, 15a,b, et al., 2012], and the highly active peptide fMLE and
and 18) exhibited FPR agonist activity.
2-(benzimidazol-2-ylthio)-N-phenylacetamide-derived
Both FPR1/FPR2 agonists 8b and 8c activated FPR1 agonists strongly interacted with Agr205, includ-
human neutrophil Ca2+ flux (EC50 = 2.2 and 2.1 mM, ing formation of H-bonds [Khlebnikov et al., 2012].
respectively) and chemotaxis (EC50 = 2.9 and 2.4 mM, Likewise, our docking studies indicated that compound
respectively). Representative dose–response curves for 8b had interaction energy of -20.02 kcal/mol with
these activities are shown in Figure 4.
Arg205 and was H-bonded to this residue, whereas the
Drug Dev. Res.

FPR MIXED AGONISTS
269
inactive derivative 8a had a very weak, nonbonded
attraction to Arg205. Additionally, molecule 8b formed
H-bonds with Thr265, whereas 8a was not H-bonded
to any FPR1 residue (Fig. 5A). A similar situation
occurred for the active and inactive compounds 1 and
9, respectively (Fig. 5B). Active pyridazine 1 formed an
H-bond with Arg205 and had total interaction energy of
32.9 kcal/mol with this residue. On the other hand,
compound 9 had a much weaker, nonbonded interac-
tion with Arg205 (-8.82 kcal/mol).
Pairs of sulfur- and oxygen-containing compounds
were also compared for their ability to interact with
FPR2. As an example, a detailed comparison of docking
poses for the active oxygen-containing compound 2 and
its inactive methylthio analog 8a showed that com-
pound 8a did not have any satisfactory binding mode
within the FPR2 binding site and that its best docking
pose had significant repulsive interactions with Tyr277
and Ile280 of FPR2. These steric conflicts made it
impossible for the sulfur-containing ligand to effectively
penetrate into the receptor cavity. In spite of H-
bonding with Asn171 and Asp173, this docking pose
was energetically unfavorable, and its MolDock score
was about 76 kcal/mol higher than that of the active
methoxy-derivative 2. In comparison, the best pose of
agonist 2 was well incorporated into the cavity of the
241 Å3 binding site obtained with the MVD “Detect
Cavity” feature (see Materials and Methods). The
closest nonvalent contact of 2.48 Å occurred between
the oxygen atom of the ligand 2 methoxy group and
Thr168. An analogous pose would be impossible for
compound 8a whose methylthio group is far more bulky
than the methoxy substituent in molecule 2.
Fig. 6. Docking poses of selected pyridazine derivatives within the
FPR2 binding site. The cavity of FPR2 found by the MVD program is
shown by a green grid, and residues closest to the cavity are shown.
(A) Poses of active compounds 1 (yellow), 2 (light-blue), 3 (green), and
8c (blue) fit the receptor cavity well. (B) Poses of inactive compounds
8a (magenta) and 9 (red) have molecular fragments outside of the
cavity (p-bromophenyl groups of compounds 8a and 9; methyl and
methythio groups of compound 8a). [Color figure can be viewed in the
online issue which is available at wileyonlinelibrary.com]
The docking poses of active oxygen-containing
derivatives 1–3 and 8c fit the FPR2 cavity well
(Fig. 6A). In contrast, the inactive sulfur-containing
pyridazine derivatives 8a and 9 had molecular frag-
ments outside the cavity, which caused steric hin-
drances and prevented an effective binding of the to a receptor was shown here for some active FPR1 and
ligands with FPR2 (Fig. 6B). Such positions of com- FPR2 agonists, the docking studies indicated that
pounds 8a and 9 within the binding site were restricted replacement of the sulfur atom by oxygen did not lead
by the bulkiness of sulfur-containing molecular frag- to emergence of an H-bond with participation of this
ments. Hence, these molecules cannot adopt more oxygen atom in docking. We also confirmed that most
suitable conformations that are possible for the corre- active FPR1 agonists strongly interact with Arg205 of
sponding oxygen-containing derivatives.
the receptor, as described previously [Mills et al., 2000;
Overall, our docking studies showed that binding Khlebnikov et al., 2012].
modes of oxygen-containing pyridazine derivatives are
In conclusion, we synthesized several novel FPR
quite different from their thio-analogs. In the case of agonists with pyridazinone or pyridazine scaffolds as
FPR2, this is caused mainly by bulkiness of sulfur- modified analogs of the previously reported series
containing groups, i.e. by a higher van der Waals radius of FPR agonists [Cilibrizzi et al., 2009]. Among the
and longer valence bonds formed by sulfur atoms with pyridazinone analogs tested, thioderivatives 8b and 8c
respect to oxygen atoms. It is also known that sulfur were mixed FPR1/FPR2 agonists with micromolar
forms much weaker H-bonds than oxygen [Wood et al., activity, whereas all pyridazine analogs and thioderiva-
2008]. Although the importance of H-bonds for binding tives 8a and 9 were completely inactive. Furthermore,
Drug Dev. Res.

270
CROCETTI ET AL.
Dufton N, Perretti M. 2010. Therapeutic anti-inflammatory poten-
tial of formyl-peptide receptor agonists. Pharmacol Ther 127:175–
188.
molecular docking studies suggested that the inactivity
of these thioderivatives was due to a weak H-bonding
acceptor and bulkiness of sulfur-containing molecular
fragments. These findings confirmed the crucial impor-
tance of the pyridazinone scaffold for the activity of this
class of compounds.
Edwards BS, Bologa C, Young SM, Balakin KV, Prossnitz ER,
Savchuck NP, Sklar LA, Oprea TI. 2005. Integration of virtual
screening with high-throughput flow cytometry to identify novel
small molecule formylpeptide receptor antagonists. Mol Pharma-
col 68:1301–1310.
Fujita H, Kato T, Watanabe N, Takahashi T, Kitagawa S. 2011.
Stimulation of human formyl peptide receptors by calpain inhibi-
tors: homology modeling of receptors and ligand docking simula-
tion. Arch Biochem Biophys 516:121–127.
ACKNOWLEDGMENTS
This work was supported in part by National Insti-
tutes of Health grant GM103500 and the Montana
State University Agricultural Experimental Station.
Gierschik P, Sidiropoulos D, Jakobs KH. 1989. Two distinct
Gi-proteins mediate formyl peptide receptor signal transduction in
human leukemia (HL-60) cells. J Biol Chem 264:21470–21473.
Gilroy DW, Lawrence T, Perretti P, Rossi AG. 2004. Inflammatory
resolution: new opportunities for drug discovery. Nat Rev Drug
Discov 3:401–416.
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