6-Methyl-2,4-disubstituted pyridazin-3(2H)-ones: A novel class of small-molecule agonists for formyl peptide receptors

Article abstract of DOI:10.1021/jm900592h

Following a ligand-based drug design approach, a potent mixed formyl peptide receptor 1 (FPR1) and formyl peptide receptor-like 1 (FPRL1) agonist (14a) and a potent and specific FPRL1 agonist (14x) were identified. These compounds belong to a large series of pyridazin-3(2H)-one derivatives substituted with a methyl group at position 6 and a methoxy benzyl at position 4. At position 2, an acetamide side chain is essential for activity. Likewise, the presence of lipophilic and/or electronegative substituents in the position para to the aryl group at the end of the chain plays a critical role for activity. Affinity forFPR1 receptors was evaluated by measuring intracellular calcium flux in HL-60 cells transfected with FPR1, FPRL1, and FPRL2. Agonists were able to activate intracellular calcium mobilization and chemotaxis in human neutrophils. Themost potent chemotactic agent (EC₅₀ =0.6 μM) was the mixed FPR/FPRL1 agonist 14h.

Full text of DOI:10.1021/jm900592h

5044 J. Med. Chem. 2009, 52, 5044–5057
DOI: 10.1021/jm900592h
6-Methyl-2,4-Disubstituted Pyridazin-3(2H)-ones: A Novel Class of Small-Molecule Agonists for
Formyl Peptide Receptors
Agostino Cilibrizzi,† Mark T. Quinn,‡ Liliya N. Kirpotina,‡ Igor A. Schepetkin,‡ Jeff Holderness,‡ Richard D. Ye,§
Marie-Josephe Rabiet, Claudio Biancalani,† Nicoletta Cesari,† Alessia Graziano,† Claudia Vergelli,† Stefano Pieretti,^
Vittorio Dal Piaz,† and Maria Paola Giovannoni*,†
†
‡
Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Firenze, Via Ugo Schiff 6, Sesto Fiorentino 50019 Firenze, Italy, Department of
ꢀ
Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717, §Department of Pharmacology, University of Illinois,
ꢀ ꢁ ꢁ
Chicago, Illinois 60612, CEA, DSV, iRTSV, Laboratoire Biochimie et Biophysique des Systemes Integres, 17 rue des Martyrs, Grenoble F-38054,
France, and ^Department of Therapeutic Research and Medicine Evaluation, Viale Regina Elena 299, 00161 Roma, Italy
Received May 7, 2009
Following a ligand-based drug design approach, a potent mixed formyl peptide receptor 1 (FPR1) and
formyl peptide receptor-like 1 (FPRL1) agonist (14a) and a potent and specific FPRL1 agonist (14x) were
identified. These compounds belong to a large series of pyridazin-3(2H)-one derivatives substituted with a
methyl group at position 6 and a methoxy benzyl at position 4. At position 2, an acetamide side chain is
essential for activity. Likewise, the presence of lipophilic and/or electronegative substituents in the position
para to the aryl group at the end of the chain plays a critical role for activity. Affinity for FPR1 receptors was
evaluated by measuring intracellular calcium flux in HL-60 cells transfected with FPR1, FPRL1, and
FPRL2. Agonists were able to activate intracellular calcium mobilization and chemotaxis in human
neutrophils. The most potent chemotactic agent (EC50 =0.6 μM) was the mixed FPR/FPRL1 agonist 14h.
Introduction
(i.e., N-formylmethionine-leucine-phenylalanine, fMLF),
which are high affinity agonists for FPR1, strongly supports
the hypothesis that these receptors work as antibacterial
receptors.7 According to this idea, Fpr1-knockout mice show
reduced resistance to infections provoked by some types of
bacteria.8 Moreover, a key role of FPRL1 has been proposed
in host response to HIV-1 infection, and synthetic peptide
domains of HIV-1 envelope proteins are able to activate
FPRL1 leading to attenuation of cell response to chemokines
through cross-desensitization and down-regulation of the
monocyte receptors CCR5 and CXCR4.9 The potent in vitro
anti-HIV-1 activity of the synthetic gp41 peptide T2 (DP178),
which is an agonist of FPR, strongly supports this view.10
Evidence for a pro-inflammatory role of FPRL1 in various
neurodegenerative diseases was also found. In fact, the 42
amino acid form of amyloid β (AB42), which is believed to
playa key role in Alzheimer disease-induced neuronal damage
by recruitment and activation of mononuclear phagocytes, is
a chemotactic agonist of FPRL1.11 Likewise, the neurotoxic
protein fragment PrP106-126, which behaves as the patho-
logic isoform of prion protein, was chemotactic and indepen-
dently induced cytokine secretion in human monocytes
through interaction with FPRL1.12 On the other hand, anti-
inflammatory properties are displayed by different types of
FPRL1 ligands. For example, lipoxin A4 (LXA4) is an
endogenous arachidonic acid metabolite that has been shown
to promote resolution of inflammation through FPRL1
agonism.13 Likewise, annexin A1, a glucocorticoid-regulated
protein that is particularly abundant in neutrophils, is able to
inhibit leucocytes migration/activation through hFPRL1
agonism.14 Taken together, these data clearly support a
key role of FPRL1 in a variety of acute inflammatory and
infective pathologies. Thus, this receptor has recently become
Innate immunity is a very important mechanism in defend-
ing humans against infectious microbes.1 Thus, immune dis-
regulation due to viral infections (i.e., AIDS) and immuno-
suppression treatments following transplantation expose pa-
tients to life-threatening risks. Moreover, in the context of
increasing aging of the population in more developed coun-
tries, it was observed that older people exhibit a natural
immune function disregulation, which may be exacerbated
in chronic stress conditions.2 In addition, the ongoing emer-
gence of bacterial and viral strains resistant to multiple classes
of chemotherapeutics increases morbidity, mortality, and
costs associated with nosocomial infections.3 Thus, identifica-
tion and development of bioactive molecules that selectively
stimulate the innate immune response is an important chal-
lenge both for biologists and for chemists.4
The human formyl peptide receptor (FPR) family belongs
to a group of G-protein coupled receptors that are predomi-
nantly expressed on neutrophils and monocytes.5 Three FPR
subtypes (FPR1a, FPRL1, and FPRL2) have been identified
in humans, while eight FPR-related receptors have been
characterized in mice.6 Evidence for an important role of
FPRs both in infective and inflammatory processes, has been
well documented in the literature. The finding that bacteria
are the major natural source of chemotactic formyl peptides
*To whom correspondence should be addressed. Phone: þ30-055-
4573682. Fax: þ39 55 4573671. E-mail mariapaola.giovannoni@
unifi.it.
a Abbreviations: FPRs, formyl peptide receptors; FPR1, formyl
peptide receptor 1; FPRL1, formyl peptide receptor-like 1; FPRL2,
formyl peptide receptor-like 2; fMLF, N-formylmethionine-leucine-
phenylalanine; LXA4, lipoxin A4; WT, wild-type; HBD, hydrogen bond
donor; HBA, hydrogen bond acceptor.
r
pubs.acs.org/jmc
Published on Web 07/29/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5045
Figure 1. Structures of FPRL1 agonists.
a prospective target for therapeutic intervention.15 In addi-
tion, the identification of novel agonists and antagonists may
be a useful tool to clarify the FPR-mediated (patho)-
physiological effects.
the new 11a by condensation with the appropriate aromatic
aldehyde in the presence of KOH. Compounds 11, in turn,
werealkylated with ethyl bromoacetateto give the esters 12a-
c, whose 12b,c were previously reported.24,25 Alkaline hydro-
lysis of compounds 12 gave the known 13c23 and the new
carboxylic acids 13a,b. These compounds were treated with
ethyl chloroformate in THF in presence of triethylamine,
affording the intermediate mixed anhydrides, which, in turn,
were transformed into the final amides 14a-s and 14u-x by
treatment with the appropriate aryl or cycloalkyl amine. To
obtain the ester 14t, the mixed anhydride was treated with
4-bromophenol.
Scheme 3 outlines the synthetic procedure for compounds
15-19: the precursor 11a was alkylated with the appropriate
bromo ester to give compounds 15a,b, which, in turn, were
converted into the corresponding acids 16a,b. The final step
was the transformation of these compounds to the final
amides 17a,b using the same procedure described for com-
pounds 14. Compounds 18a,b were obtained by alkylation of
11a with the appropriate halide in standard conditions.
Moreover the precursor 11a was converted into the final
19a,b through a Mannich reaction (CH2O þ NH3). The
intermediate amine was not isolated and was converted into
the urea 19a by treatment with 4-bromophenyl isocyanate and
into the amide 19b with 4-bromobenzoyl chloride.
The final compounds 21, 23, and 25 were synthesized as
shown in Scheme 4. The intermediate 12a was reduced with
sodium borohydride in THF/MeOH to generate the primary
alcohol 20. This compound was the starting material for the
synthesis both of the ether 21, through a coupling reaction
with the 4-bromophenylboronic acid in presence of Cu(Ac)2,
and of compound 23, through the mesylate 22, which was
converted into the final compound 23 by nucleophilic replace-
ment with 4-bromo aniline. Treatment of 22 with ammonia
gave the intermediate 24 from which the amide 25b and
the urea 25a were obtained using 4-bromobenzoyl chloride
or 4-bromophenyl isocyanate, respectively, using the same
In addition to natural peptides like the bacterial fMLF,16
synthetic peptides17 and small molecule nonpeptide ligands of
FPRL1 have been recently reported. Quin-C118,19 (compound
1, Figure 1) was the first small-molecule FPRL1-selective
agonist shown to induce chemotaxis in peripheral blood
neutrophils, with a relative potency 1000-fold lower than the
synthetic peptide agonist WKYMVm. More recently, re-
searchers from Amgen reported a series of pyrazolone deri-
vatives displaying nanomolar selective affinity for FPRL1.20
These types of agonists, of which 2 is an example, are
characterized by the presence of an arylurea substructure
linked to the heterocyclic backbone and served us as starting
point to develop novel chemotypes of FPR ligands. Thus we
report here the synthesis and evaluation of two series of
functionalized pyridazinones of general structure 3 and 4.
Chemistry
The synthetic pathways employed to prepare the final
compounds are depicted in Schemes 1-5.
Compound 521 in anhydrous toluene was refluxed with
the appropriate aryl isocyanate to afford the urea derivatives
6a-c (Scheme 1). The 4-butoxy analogue 7 was synthesized
with an alternative procedure starting from the same precur-
sor and using triphosgene in anhydrous THF, followed by
treatment with the appropriate aniline. The aromatic amide 8
was obtained from 5 by treatment with the opportune aroyl
chloride in toluene at reflux. Finally, coupling of the amine 5
with 4-butoxyphenylboronic acid in CH2Cl2 in presence of
Cu(Ac)2 gave the aromatic amine 9.
In Scheme 2 is depicted the synthesis of compounds 14a-x
(Table 2). The dihydropyridazinone 1022 was converted into
the previously described 4-benzyl derivatives 11b,c23,24 and

5046 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Cilibrizzi et al.
Scheme 1a
a Reagents and conditions: (a) anhydrous toluene, aryl isocyanate, reflux, 3-7 h; (b) THF, anhydrous sodium acetate, triphosgene 70 ꢀC, 2 h,
4-butoxyaniline, 12 h, rt; (c) Et3N, RCOCl, toluene, 110 ꢀC, 4 h; (d) Cu(Ac)2, 4-butoxyphenylboronic acid, Et3N, rt, 12 h.
reaction conditions described for the analogues 7 and 8 in
Scheme 1.
Thus 14a was selected as lead and extensive structure-
activity relationship (SAR) studies on this prototype were
performed by modifying the nature and the length of the
functionalized side chain and by replacing Br with a variety of
substituents. Elongation of carbon chain from one to two
methylene groups gave compound 17a, which resulted less
potent at FPR1 and FPRL1 of 14a (Table 1). Branching the
carbon chain resulted in compound 17b, which did not
activate FPRL1 but retained activity for FPR1 similar to that
of 14a. Thus, 17b is an FPR1-specific agonist. Modifications
more substantial of the functionalized chain were obtained
with compounds 18a,b and were completely detrimental.
Further inactive compounds were the urea derivative 19a,
the inverse amide 19b, and their superior homologues 25a and
25b. Replacement of CONH with an ether group and a
secondary amine gaves compounds 21 and 23, respectively,
which resulted also inactive. Moreover, compounds 29 and
30, in which the substituents at position 2 and 4 were inter-
changed, were inactive.
Other modifications of the functionalized chain were per-
formed with compounds 14r-u (Table 2). When the NH was
converted toNCH3 (compound 14r), the activity disappeared.
Compound 14s, where the CONH is in the middle of the
spacer, was also inactive. Replacement of CONH with COO
(14t) or inclusion of amidic nitrogen in piperazine nucleus
(14u) gave the same effect.
The nature and the position of the substituent on the phenyl
group at the end of the chain also played a crucial role in
ligand activity. Moving Br to positions meta (14b) and ortho
(14c) resulted in a complete loss of FPR1/FPRL1 activity.
Among halo-derivatives, the 4-chloro analogue 14d exhibited
In Scheme 5 is depicted the synthesis of compounds 29 and
30. Treatment of the commercially available 6-methylpyrida-
zinone 26 with m-methoxybenzyl chloride in acetonitrile
resulted in compound 27, which, in turn, gave the correspond-
ing 4-amino derivative 28 by heating with N2H4 under hard
conditions. The amide 29 and the urea 30 were obtained from
28 by treatment with the proper aroyl chloride or the aryl
isocyanate by following the same conditions reported for 25a
and 25b.
Results and Discussion
All synthesized compounds were evaluated for their ability to
induce intracellular Ca2þ flux in HL-60 cells transfected with
FPR1,FPRL1,orFPRL2,andresultswerereportedinTables1
and 2. All compounds were also evaluated in WT (wild-type
nontransfected HL-60 cells) and were inactive. Moreover, both
EC50 values and relative efficacy compared to the peptide
agonists, fMLF and WKYMVm, were determined.
In the early phase of our project, we synthesized pyridazin-
3(2H)-ones bearing a phenyl group at position 2 and a
functionalized side chain at position 4 (compounds 6a-c, 7,
8, and 9), which may be regarded as enlarged analogues of 2
(Figure 1). Because these compounds failed to show activity
(see Table 1), we turned our attention to analogues with a
substituted benzyl at position 4 and a similar functionalized
chain at N-2 (compounds 4, Figure 1). In this series, we were
able to identify compound 14a, a potent mixed FPR1 and
FPRL1 agonist.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5047
Scheme 2a
a Reagents and conditions: (a) aromatic aldehyde, 5% KOH, EtOH, reflux, 2-5 h; (b) ethyl bromoacetate, K2CO3, anhydrous CH3CN, reflux, 1-6 h;
(c) NaOH, 60-80 ꢀC, 1-4 h; (d) ethyl chloroformate, anhydrous THF, Et3N, appropriate substituted aryl(cycloalkyl)amine (or 4-bromophenol for
compound 14t), 12 h, rt.
the same profile as 14a. The corresponding 4-iodo derivative
14e was 2 times less potent at FPRL1. For this compound, a
weak effect at FPRL2 was also observed. The 4-fluoro
analogue 14f was less potent compared to 14a, but specificity
for FPR1 was evident. The elimination of Br (compound 14g)
was associated with loss of activity. Replacement of Br in 14a
with substituents having similar steric properties, such as
t-But (14i), OCF3 (14o), and CN (14q), led to loss of activity
at both FPR1 and FPRL1. The 4-trifluoromethyl and the
4-nitro analogues (14n and 14p, respectively) as well as
compound 14h, bearing a methyl group at position 4, had
relatively low activity. Introduction of alkoxy groups gave
interesting results: the 4-methoxy derivative 14j and the 3,4-
dimethoxy derivative 14l had low activity at both receptors,
whereas the 3,4-methylendioxy derivative14m was specific for
FPR1. It is worth noting that compounds 14v, 14w, and 14k,
where Br in the para position was substituted with the typical
buthoxy group characteristic of Quin-C1, were found to be
completely devoid of activity.
Only a few substitutions were performed at the level of the
aromatic system in the benzyl group. Moving OCH3 from the
meta to the para position (compound 14x) resulted in high
activity (EC50 = 2.4 μM) and selectivity for FPRL1. This
exciting result prompted us to design several additional
analogues, which are currently under investigation. Taken
together, these data suggest that regarding the aromatic
system at the end of the functionalized chain in position 2,
the presence of a lipophilic and/or electronegative substituent,
such as F, Br, or CH3, in position para is an essential
requirement for potency and/or selectivity. Likewise, the
presence of an acetamide spacer at pyridazine N-2 also plays
a crucial role in specificity and potency. The role of both CO
and NH in the side chain seems to indicate that a hydrogen
bond donor (HBD) neighboring an acceptor (HBA) system is
also an essential requirement for binding at FPRs, according
to the Quin-C1 and pyrazolone structures 2, where the pre-
sence of an urea is a common structural element. Moreover,
this HBD\HBA system must be placed at an appro-
priate distance from both the aromatic and the heterocyclic
scaffold.
For compound 14a were also calculated dose-response
curves (Figure 2). Note that no response was observed in
control, undifferentiated HL-60 cells, which do not express
FPRs. Furthermore, the effect of selected agonists on Ca2þ
flux in human neutrophils was also determined to verify the
HL-60 results in primary phagocytes (Table 3). We found that
both selective and nonselective agonists identified in HL-60
cell assays also induced Ca2þ flux in human neutrophils, with
EC50 values in the range 0.8-21.7 μM.
The most potent chemotactic compound was 14h (EC50 =
0.6 μM), followed by 14j (EC50 =0.9 μM), which were both
nonselective agonists. The FPRL1-selective agonist (14x)
showed lower potency (EC50 =13.1 μM) as a chemotactic
agent.
In conclusion, we have identified a novel chemotype of
interesting and selective FPR1 or FPRL1 agonists and mixed
FPR/FPRL1 agonists active in the low micromolar range in
human neutrophils. Moreover, some of these compounds
proved to be able to stimulate chemotaxis at submicromolar
concentrations.

5048 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Cilibrizzi et al.
Scheme 3a
a Reagents and conditions: (a) appropriate alkyl halide, K2CO3, anhydrous CH3CN, reflux, 3 h; (b) NaOH, 60 ꢀC, rt; (c) ethyl chloroformate,
anhydrous THF, Et3N, 4-bromoaniline, 12 h, rt; (d) appropriate alkyl halide, K2CO3, anhydrous CH3CN, reflux, 2-4 h; (e) 40% CH2O, 33% NH3,
dioxane, CH2Cl2, 4-bromophenyl isocyanate (for compound 19a), or 4-bromobenzoyl chloride (for compound 19b), 0 ꢀC to rt, 6-12 h.
Experimental Section
6c). In the case of 6a, a second batch of product was obtained
from evaporation of the filtrate. The residue, after treatment
with cold water, was extracted with CH2Cl2 (3 ꢀ 20 mL).
Removal of the solvent gave a residue that was purified
by column chromatography using CH2Cl2/CH3OH 9.9:0.1 as
eluent.
1-(4-Iodophenyl)-3-(6-methyl-3-oxo-2-phenyl-2,3-dihydropyri-
dazin-4-yl)urea (6a). Yield =45%; mp =264-265 ꢀC (EtOH/
toluene). IR (cm-1) 3270 (NH), 3256 (NH), 1708 (CO), 1627
(CO). 1H NMR (CDCl3) δ 2.44 (s, 3H, CH3), 6.50 (d, 2H, Ar, J=
8.7 Hz), 7.21 (t, 1H, Ar, J =7.5 Hz), 7.36-7.41 (m, 4H, Ar), 7.68
(d, 2H, Ar, J=7.7 Hz), 8.15 (s, 1H, Ar), 8.41 (exch br s, 1H, NH),
9.38 (exch br s, 1H, NH). MS (ESI) m/z 447.03 [M þ H]þ. Anal.
(C18H15IN4O2) C, H, N.
1-(4-Bromophenyl)-3-(6-methyl-3-oxo-2-phenyl-2,3-dihydro-
pyridazin-4-yl)urea (6b). Yield = 50%; mp = 263-265 ꢀC
(toluene). IR (cm-1) 3271 (NH), 3250 (NH), 1704 (CO), 1625
(CO). 1H NMR (CDCl3) δ 2.43 (s, 3H, CH3), 6.60 (d, 2H, Ar, J=
8.6 Hz), 7.19-7.22 (m, 3H, Ar), 7.38 (t, 2H, Ar, J=7.8 Hz), 7.69
(d, 2H, Ar, J =7.9 Hz), 8.16 (s, 1H, Ar), 8.44 (exch, br, s, 1H,
NH), 9.40 (exch br s, 1H, NH). MS (ESI) m/z 399.05 [M þ H]þ.
Anal. (C18H15BrN4O2) C, H, N.
Chemistry. All melting points were determined on a Buchi
apparatus and are uncorrected. IR spectra were measured as
Nujol mulls with a PerkinElmer spectrometer (FT-IR, Spectrum
1000). 1H NMR spectra were recorded with Avance 400 instru-
ments (Bruker Biospin Version 002 with SGU). Chemical shifts
are reported in ppm, using the solvent as internal standard.
Mass spectra (m/z) were recorded on a ESI-TOF mass spectro-
meter (Bruker Micro TOF). Extracts were dried over Na2SO4,
and the solvents were removed under reduced pressure. Merck
F-254 commercial plates were used for analytical TLC to follow
the course of reaction. Silica gel 60 (Merck 70-230 mesh) was
used for column chromatography. Microanalyses were per-
formed with a Perkin-Elmer 260 elemental analyzer for C, H,
N, and the results were within (0.4% of the theoretical values
unless otherwise stated. Reagents and starting materials were
commercially available.
General Procedure for 6a-c. To a stirred solution of com-
pound 521(0.35 mmol) in anhydrous toluene (2 mL), the proper
aryl isocyanate (0.40 mmol) was added. The mixture was
refluxed for 4-7 h. After cooling, the precipitate was collected
by suction and purified by crystallization from toluene (6b and

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5049
Scheme 4a
a Reagents and conditions: (a) anhydrous THF, NaBH4, CH3OH, 60 ꢀC, 1 h; (b) Cu(Ac)2, 4-bromophenylboronic acid, anhydrous CH2Cl2, Et3N, rt,
20 h; (c) pyridine, CH2Cl2, methanesulfonyl chloride, 0 ꢀC to rt, 4 h; (d) 2-propanol, 4-bromoaniline, 60 ꢀC, 6 h; (e) 33% NH3, isopropanol, 60 ꢀC, 3 h; (f)
anhydrous toluene, 4-bromophenyl isocyanate, 110 ꢀC, 5 h (for compound 25a); anhydrous CH2Cl2, Et3N, 4-bromobenzoyl chloride, 0 ꢀC, 6 h (for
compound 25b).
1-(4-Chlorophenyl)-3-(6-methyl-3-oxo-2-phenyl-2,3-dihydro-
pyridazin-4-yl)urea (6c). Yield = 85%; mp = 274-276 ꢀC
(toluene). IR (cm-1) 3269 (NH), 3255 (NH), 1705 (CO), 1626
(CO). 1H NMR (CDCl3) δ 2.44 (s, 3H, CH3), 6.65 (d, 2H, Ar, J=
8.8 Hz), 7.07 (d, 2H, Ar, J=8.8 Hz), 7.21 (t, 1H, Ar, J=7.5 Hz),
7.38 (t, 2H, Ar, J =7.9 Hz), 7.69 (d, 2H, Ar, J =7.6 Hz), 8.16 (s,
1H, Ar), 8.43 (exch br s, 1H, NH), 9.40 (exch br s, 1H, NH). MS
(ESI) m/z 355.10 [M þ H]þ. Anal. (C18H15ClN4O2) C, H, N.
1-(4-Butoxyphenyl)-3-(6-methyl-3-oxo-2-phenyl-2,3-dihydro-
pyridazin-4-yl)urea (7). To a cooled and stirred suspension of 5
(0.5 mmol) and anhydrous sodium acetate (1.19 mmol) in
anhydrous THF (5 mL), triphosgene (1.18 mmol) was added.
The mixture was stirred for an additional 10 min at 0 ꢀC and
refluxed for 2 h. The solvent was removed in vacuo, and the
residue was dissolved in anhydrous THF (2 mL). 4-Butoxyani-
line (1.25 mmol) was added, and the mixture was stirred at room
temperature for 12 h. After dilution with cold water, the
suspension was extracted with CH2Cl2 (3 ꢀ 20 mL). After
removal of the solvent, the residue was purified by column
chromatography using absolute EtOH/CH2Cl2/petroleum
ether/toluene/33% ammonia 3.3:19.7:6.5:70:0.5 as eluent. Yield
=31%; mp =189-191 ꢀC (EtOH). IR (cm-1) 3270 (NH), 3255
(NH), 1705 (CO), 1625 (CO). 1H NMR (CDCl3) δ 1.01 (t, 3H,
CH2CH3, J=7.4 Hz), 1.52 (sext, 2H, CH2CH3, J=7.4 Hz), 1.79
(quint, 2H, CH2CH2CH2, J =7.0 Hz), 2.40 (s, 3H, 6-CH3), 3.95
(t, 2H, OCH2, J =6.6 Hz), 6.70 (s, 4H, Ar), 7.17 (t, 1H, Ar, J =
7.5 Hz), 7.32 (t, 2H, Ar, J=7.8 Hz), 7.61 (d, 2H, Ar, J=7.9 Hz),
8.11 (exch br s, 1H, NH), 8.14 (s, 1H, Ar), 9.33 (exch br s, 1H,
NH). MS (ESI) m/z 393.19 [M þ H]þ. Anal. (C22H24N4O3) C, H,
N.
4-Butoxy-N-(6-methyl-3-oxo-2-phenyl-2,3-dihydropyridazin-
4-yl)benzamide (8). 4-Butoxybenzoic acid (0.36 mmol) was
converted to the corresponding chloride as follows: SOCl2
(20.7 mmol) was added and the stirred suspension was cooled
to 0 ꢀC and treated with Et3N (0.99 mmol). After 4 h of reflux,
the mixture was evaporated in vacuo and the residue was washed

5050 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Cilibrizzi et al.
Scheme 5a
a Reagents and conditions: (a) 3-methoxybenzyl chloride, anhydrous CH3CN, anhydrous K2CO3, 80 ꢀC, 2 h; (b) hydrazine hydrate, 180 ꢀC, 11 h;
(c) 4-bromobenzoyl chloride, Et3N, 0 ꢀC, 4 h; (d) 4-bromophenyl isocyanate, anhydrous toluene, reflux, 7 h.
with cyclohexane (3 ꢀ 5 mL). The residue was dissolved in
anhydrous toluene (5 mL), and the solution was treated with a
solution of compound 5 (0.40 mmol) in toluene (1 mL) and Et3N
(1.48 mmol). After 4 h of reflux, the solvent was removed in
vacuo and the residue was treated with cold water and extracted
with CH2Cl2 (3 ꢀ 20 mL). After removal of the solvent, the
residue was purified by column chromatography (eluent: cyclo-
hexane/ethyl acetate 3:1). Yield=37%; mp=95-97 ꢀC (EtOH).
IR (cm-1) 3269 (NH), 1682 (CO), 1644 (CO). 1H NMR (CDCl3)
δ 1.01 (t, 3H, CH2CH3, J=7.4 Hz), 1.53 (sext, 2H, CH2CH3, J=
7.5 Hz), 1.82 (quint, 2H, CH2CH2CH2, J =7.0 Hz), 2.45 (s, 3H,
6-CH3), 4.06 (t, 2H, CH2O, J =6.5 Hz), 6.99 (d, 2H, Ar, J =8.8
Hz), 7.43 (t, 1H, Ar, J=7.4 Hz), 7.52 (t, 2H, Ar, J=7.8 Hz), 7.65
(d, 2H, Ar, J =7.7 Hz), 7.91 (d, 2H, Ar, J =8.8 Hz), 8.24 (s, 1H,
Ar), 9.46 (exch br s, 1H, NH). MS (ESI) m/z 378.18 [M þ H]þ.
Anal. (C22H23N3O3) C, H, N.
4-(4-Butoxyphenylamino)-6-methyl-2-phenylpyridazin-3(2H)-
one (9). To a suspension of 5 (0.55 mmol), copper acetate (0.82
mmol) and 4-butoxyphenylboronic acid (1.08 mmol) in CH2Cl2
(2 mL), Et3N (1.08 mmol) was added and the mixture was stirred
at room temperature for 12 h. The suspension was extracted
with 15% aqueous ammonia (10 mL), and the organic layer was
washed with 10 mL of water and dried over sodium sulfate.
After removal of the solvent, the residue was purified by column
chromatography using as eluent cyclohexane/ethyl acetate 3:1.
The analytical sample was obtained from a further purification
performed on a silica gel preparative column (eluent: cyclohex-
ane/ethyl acetate 3:1). Yield =16%; mp =137-139 ꢀC (EtOH).
IR (cm-1) 3275 (NH), 1633 (CO). 1H NMR (CDCl3) δ 1.02 (t,
3H, CH2CH3, J =7.4 Hz), 1.54 (sext, 2H, CH2CH3, J =7.0 Hz),
1.81 (quint, 2H, CH2CH2CH2), 2.31 (s, 3H, 6-CH3), 4.01 (t, 2H,
CH2O, J=6.5 Hz), 6.40 (s, 1H, Ar), 6.97 (d, 2H, Ar, J=8.9 Hz),
7.20 (d, 2H, Ar, J =8.8 Hz), 7.41 (t, 1H, Ar, J =7.4 Hz), 7.51
(t, 3H, Ar, J=7.7 Hz), 7.64 (d, 2H, Ar, J=8.0 Hz). MS (ESI) m/
z 350.19 [M þ H]þ. Anal. (C21H23N3O2) C, H, N.
4-(3-Methoxybenzyl)-6-methylpyridazin-3(2H)-one (11a). To
a solution of 10 (1.79 mmol) in 7 mL of a solution of KOH 5%
(w/v) in absolute EtOH, 3-methoxybenzaldehyde (1.79 mmol)
was added and the mixture was refluxed under stirring for 3 h.
After cooling, the sample was concentrated in vacuo, diluted
with cold water (10-15 mL), and acidified with 2 N HCl. The
suspension was extracted with CH2Cl2 (3 ꢀ 15 mL). Removal of
the solvent afforded compound 11a, which was purified by flash
chromatography using cyclohexane/ethyl acetate 1:1 as eluent.
Yield =61%; mp =133-134 ꢀC (EtOH). 1H NMR (CDCl3) δ
2.26 (s, 3H, 6-CH3), 3.82 (s, 3H, OCH3), 3.90 (s, 2H, CH2-Ar),
6.73 (s, 1H, Ar), 6.82-6.86 (m, 3H, Ar), 7.276-7.28 (m, 1H, Ar).
[5-(3-Methoxybenzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-
acetic acid ethyl ester (12a). A mixture of 11a (1.13 mmol),
K2CO3 (2.26 mmol), and ethyl bromoacetate (3.40 mmol) in
CH3CN (3 mL) was refluxed under stirring for 6 h. The mixture
was then concentrated in vacuo, diluted with cold water, and
extracted with CH2Cl2 (3 ꢀ 15 mL). The solvent was evaporated
in vacuo, and compound 12a was purified by column chroma-
tography using cyclohexane/ethyl acetate 1:1 as eluent. Yield =
98%; oil. 1H NMR (CDCl3) δ 1.29t, 3H, CH2CH3, J =7.1 Hz),
2.22 (s, 3H, 3-CH3), 3.80 (s, 3H, OCH3), 3.86 (s, 2H, CH2-Ar),
4.22-4.25 (m, 2H, OCH2CH3), 4.85 (s, 2H, NCH2CO), 6.67 (s,
1H, Ar), 6.78-6.82 (m, 3H, Ar), 7.26 (t, 1H, Ar, J =7.8 Hz).
General Procedure for 13a,b. A suspension of the appropriate
derivatives 12a or 12b,24 respectively (0.5-1.11 mmol), and 6 N
NaOH (6-8 mL) in ethanol (2 mL) was stirred at rt to 80 ꢀC for
1-4 h. The mixtures were then concentrated in vacuo, diluted
with cold water, and acidified with 6 N HCl, and the final
products were filtered off with suction and recrystallized from
ethanol.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5051
Table 1. Activity of the Compounds in Human HL-60 Cells Expressing Human FPR1, FPRL1, or FPRL2
Ca2þ mobilization EC50 (μM) and efficacy (%)a
FPR1 FPRL1 FPRL2
NA
compd
X
Y
R1
R2
6a
6b
6c
7
NHCONH
NHCONH
NHCONH
NHCONH
NHCO
NH
I
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Br
Cl
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
1.9
OC4H9
OC4H9
OC4H9
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
4-Br
8
9
14a
CH2
CH2
CH2CONH
(CH2)2CONH
CH(CH3)CONH
CH2
4-Br
4-Br
3.4 ( 1.6 (75)
9.7 ( 2.7 (30)
3.2 ( 1.5 (90)
NA
NA
3.8 ( 1.5 (70)
5.4 ( 1.2 (25)
NA
17a
17b
CH2
CH2
4-Br
4-Br
18a
18b
NA
NA
CH2
CH2
CH2CO
4-Br
4-Br
19a
19b
CH2NHCONH
CH2NHCO
(CH2)2O
NA
NA
NA
NA
CH2
CH2
4-Br
4-Br
21
23
NA
NA
NA
NA
CH2
CH2
(CH2)2NH
(CH2)2NHCONH
(CH2)2NHCO
CH2
4-Br
4-Br
25a
25b
NA
NA
NA
NA
CH2
NHCO
NHCONH
4-Br
3-OCH3
3-OCH3
29
30
NA
NA
NA
NA
CH2
4-Br
fMLF
WKYMVm
0.01
0.5
20.4
0.001
0.01
a NA, no activity was observed (no response was observed during first 2 min after addition of compounds under investigation). The EC50 values are
presented as the mean ( SD 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
bracket) is expressed as percent of the response induced by 5 nM fMLF (FPR1) or 5 nM WKYMVm (FPRL1 and FPRL2).
[5-(3-Methoxybenzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-
acetic Acid (13a). Yield =97%; mp =194-195 ꢀC (EtOH). 1H
NMR (DMSO) δ 2.21 (s, 3H, 3-CH3), 3.72 (s, 3H, OCH3), 3.76
(s, 2H, CH2-Ar), 4.69 (s, 2H, NCH2CO), 6.79-6.85 (m, 3H, Ar),
7.07 (s, 1H, Ar), 7.22 (t, 1H, Ar, J =7.8 Hz).
[5-(4-Methoxybenzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-
acetic Acid (13b). Yield =96%; mp =144-145 ꢀC (EtOH). 1H
NMR (CDCl3) δ 2.26 (s, 3H, 3-CH3), 3.84 (s, 3H, OCH3), 3.87
(s, 2H, CH2-Ar), 4.93 (s, 2H, NCH2CO), 6.69 (s, 1H, Ar), 6.91
(d, 2H, Ar, J =8.5 Hz), 7.16 (d, 2H, Ar, J =8.5 Hz).
General Procedure for 14a-x. To a cooled (-5 ꢀC) and stirred
solution of compound 13a (0.35 mmol) in anhydrous tetrahydro-
furan (3-5 mL), Et3N (1.22 mmol) was added. After 30 min, the
mixture was allowed to warm up to 0 ꢀC, and ethyl chloroformate
(0.38 mmol) was added. After 1 h, the appropriate substituted
aryl(cycloalkyl)amine (for compounds 14a-s and 14u-x) or
4-bromophenol (for compound 14t), commercially available
(0.7 mmol), was added. The reactions were carried out at room
temperature for 12 h. The mixtures were then concentrated in
vacuo, diluted with cold water (20-30 mL), and extracted with
CH2Cl2 (3 ꢀ 15 mL). The solvent was evaporated to afford final
compounds 14a-x, which were purified by column chromatogra-
phy using cyclohexane/ethyl acetate 1:1 as eluent for compounds
14a, 14d, 14f-m, 14q, 14w; toluene/NH4OH/EtOH/CH2Cl2/
petroleum ether 7:0.05:0.30:2:0.65 for compounds 14b,c; 14x;
cyclohexane/ethyl acetate 1:2 for compounds 14e, 14n-p, 14s,
14v; cyclohexane/ethyl acetate 2:1 for compounds 14r,t and
CH2Cl2/CH3OH 9.5:0.5 for compound 14u.
N-(4-Bromophenyl)-2-[5-(3-methoxybenzyl)-3-methyl-6-oxo-
6H-pyridazin-1-yl]-acetamide (14a). Yield =86%; mp =171-
172 ꢀC (EtOH). IR (cm-1) 3300 (NH), 1708 (CO), 1644 (CO). 1H
NMR (CDCl3) δ 2.20 (s, 3H, 3-CH3), 3.70 (s, 3H, OCH3), 3.73
(s, 2H, CH2-Ar), 4.80 (s, 2H, NCH2CO), 6.77-6.82 (m, 3H, Ar),
7.13 (s, 1H, Ar), 7.20 (t, 1H, Ar, J=8.0 Hz), 7.47 (s, 4H, Ar). MS
(ESI) m/z 442.08 [M þ H]þ. Anal. (C21H20BrN3O3) C, H, N.
N-(3-Bromophenyl)-2-[5-(3-methoxybenzyl)-3-methyl-6-oxo-
6H-pyridazin-1-yl]-acetamide (14b). Yield =85%; mp =73-75
1
ꢀC (EtOH). IR (cm-1) 3295 (NH), 1708 (CO), 1642 (CO). H
NMR (CDCl3) δ 2.30 (s, 3H, 3-CH3), 3.80 (s, 3H, OCH3), 3.92
(s, 2H, CH2-Ar), 4.95 (s, 2H, NCH2CO), 6.80-6.86 (m, 4H, Ar),
7.14 (t, 1H, Ar, J =8.0 Hz), 7.22 (m, 1H, Ar, J =8.2 Hz), 7.27-
7.31 (m, 1H, Ar), 7.39 (d, 1H, Ar, J =8.0 Hz), 7.77 (s, 1H, Ar),
9.05 (exch br s, 1H, NH). MS (ESI) m/z 271.11 [M þ H]þ. Anal.
(C21H20BrN3O3) C, H, N.
N-(2-Bromophenyl)-2-[5-(3-methoxybenzyl)-3-methyl-6-oxo-
6H-pyridazin-1-yl]-acetamide (14c). Yield =80%; oil. IR (cm-1
)
3300 (NH), 1708 (CO), 1644 (CO). 1H NMR (CDCl3) δ 2.30 (s,
3H, 3-CH3), 3.82 (s, 3H, OCH3), 3.92 (s, 2H, CH2-Ar), 5.00 (s,
2H, NCH2CO), 6.76 (s, 1H, Ar), 6.80-6.86 (m, 3H, Ar), 7.00 (t,
1H, Ar, J =7.7 Hz), 7.27-7.34 (m, 2H, Ar), 7.52 (d, 1H, Ar, J =
8.0 Hz), 8.36 (d, 1H, Ar, J =8.2 Hz), 8.54 (exch br s, 1H, NH).
MS (ESI) m/z 442.08 [M þ H]þ. Anal. (C21H20BrN3O3) C, H, N.

5052 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Cilibrizzi et al.
Table 2. Activity of the Compounds in Human HL-60 Cells Expressing Human FPR1, FPRL1, or FPRL2
Ca2þ mobilization EC50 (μM) and efficacy (%)a
compd
14a
14b
X
Y
R1
FPR1
FPRL1
FPRL2
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NH-C6H4-Br (p)
NH-C6H4-Br (m)
NH-C6H4-Br (o)
NH-C6H4-Cl (p)
NH-C6H4-I (p)
3.4 ( 1.6 (75)
NA
3.8 ( 1.5 (70)
NA
NA
NA
NA
NA
14c
14d
NA
2.6 ( 0.3 (110)
2.8 ( 0.2 (90)
7.6 ( 0.2 (40)
NA
NA
4.0 ( 1.6 (35)
6.8 ( 2.2 (40)
NA
NA
14e
14f
13.0 ( 3.1 (30)
NA
NA
NH-C6H4-F (p)
NH-C6H5
14g
14h
NH-C6H4-CH3 (p)
NH-C6H4-tC4H9 (p)
NH-C6H4-OCH3 (p)
NH-C6H4-OC4H9 (p)
NH-C6H4-(OCH3)2 (m, p)
Ab
7.2 ( 2.2 (120)
NA
10.9 ( 3.4 (50)
NA
NA
NA
14i
14j
7.7 ( 2.5 (65)
NA
14.4 ( 2.0 (35)
NA
NA
NA
14k
14 L
14m
14n
15.5 ( 2.9 (25)
2.3 ( 1.1 (50)
5.7 ( 1.8 (50)
NA
16.8 ( 3.2 (25)
NA
NA
NA
NH-C6H4-CF3 (p)
NH-C6H4-OCF3 (p)
NH-C6H4-NO2 (p)
NH-C6H4-CN (p)
N(CH3)-C6H4-Br (p)
NHCH2-C6H4-Br (p)
O-C6H4-Br (p)
8.8 ( 2.3 (95)
NA
NA
NA
14o
14p
10.5 ( 2.9 (60)
12.3 ( 2.5 (55)
NA
NA
NA
14q
14r
NA
NA
NA
NA
NA
NA
NA
NA
0.01
0.5
NA
NA
NA
NA
14s
14t
NA
NA
NA
NA
14u
14v
Bb
OCH3
Cl
OCH3
NH-C6H4-OC4H9 (p)
NH-C6H4-OC4H9 (p)
NH-C6H4-Br (p)
NA
NA
NA
NA
14w
14x
fMLF
WKYMVm
H
H
2.4 ( 0.9 (70)
20.4
0.001
NA
1.9
0.01
a NA, no activity was observed (no response was observed during first 2 min after addition of compounds under investigation). The EC50 values are
presented as the mean ( SD 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
b
bracket) is expressed as percent of the response induced by 5 nM fMLF (FPR1) or 5 nM WKYMVm (FPRL1 and FPRL2).
N-(4-Chlorophenyl)-2-[5-(3-methoxybenzyl)-3-methyl-6-oxo-
6H-pyridazin-1-yl]-acetamide (14d). Yield =86%; mp =170-
171 ꢀC (EtOH). IR (cm-1) 3296 (NH), 1705 (CO), 1644 (CO). 1H
NMR (CDCl3) δ 2.30 (s, 3H, 3-CH3), 3.80 (s, 3H, OCH3), 3.90
(s, 2H, CH2-Ar), 4.95 (s, 2H, NCH2CO), 6.79 (s, 1H, Ar), 6.80-
6.85 (m, 3H, Ar), 7.16 (d, 2H, Ar, J=8.8 Hz), 7.25-7.29 (m, 1H,
Ar), 7.38 (d, 2H, Ar, J =8.8 Hz), 9.28 (exch br s, 1H, NH). MS
(ESI) m/z 398.13 [M þ H]þ. Anal. (C21H20ClN3O3) C, H, N.
N-(4-Iodophenyl)-2-[5-(3-methoxybenzyl)-3-methyl-6-oxo-
6H-pyridazin-1-yl]-acetamide (14e). Yield =99%; mp =179-
180 ꢀC (EtOH). IR (cm-1) 3300 (NH), 1707 (CO), 1645 (CO). 1H
NMR (CDCl3) δ 2.30 (s, 3H, 3-CH3), 3.79 (s, 3H, OCH3), 3.88
(s, 2H, CH2-Ar), 4.95 (s, 2H, NCH2CO), 6.78-6.84 (m, 4H, Ar),
7.17 (d, 2H, Ar, J =8.7 Hz), 7.24-7.28 (m, 1H, Ar), 7.45 (d, 2H,
Ar, J =8.7 Hz), 9.41 (exch br s, 1H, NH). MS (ESI) m/z 490.06
[M þ H]þ. Anal. (C21H20IN3O3) C, H, N.
(EtOH). IR (cm-1) 3300 (NH), 1707 (CO), 1643 (CO). 1H NMR
(CDCl3) δ 2.30 (s, 3H, 3-CH3), 3.80 (s, 3H, OCH3), 3.90 (s, 2H,
CH2-Ar), 4.95 (s, 2H, NCH2CO), 6.80-6.84 (m, 4H, Ar), 6.90
(t, 2H, Ar, J = 8.7 Hz), 7.25-7.29 (m, 1H, Ar), 7.39-7.42
(m, 2H, Ar), 9.21 (exch br s, 1H, NH). MS (ESI) m/z 382.16
[M þ H]þ. Anal. (C21H20FN3O3) C, H, N.
2-[5-(3-Methoxybenzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-N-
phenyl-acetamide (14g). Yield= 97%; oil. IR (cm-1) 3295 (NH),
1708 (CO), 1644 (CO). 1H NMR (CDCl3) δ 2.28 (s, 3H, 3-CH3),
3.81 (s, 3H, OCH3), 3.91 (s, 2H, CH2-Ar), 4.96 (s, 2H, NCH2CO),
6.78-6.86 (m, 4H, Ar), 7.08 (t, 1H, Ar, J =7.4 Hz), 7.26-7.30 (m,
3H, Ar), 7.50 (d, 2H, Ar, J =8.0 Hz), 8.93 (exch br s, 1H, NH). MS
(ESI) m/z 364.16 [M þ H]þ. Anal. (C21H21N3O3) C, H, N.
2-[5-(3-Methoxybenzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-
N-4-tolyl-acetamide (14h). Yield = 78%; mp = 142-144 ꢀC
(EtOH). IR (cm-1) 3300 (NH), 1708 (CO), 1644 (CO). 1H NMR
(CDCl3) δ 2.28 (s, 3H, 3-CH3), 2.30 (s, 3H, CH3-Ar), 3.81 (s, 3H,
OCH3), 3.90 (s, 2H, CH2-Ar), 4.94 (s, 2H, NCH2CO), 6.76
N-(4-Fluorophenyl)-2-[5-(3-methoxybenzyl)-3-methyl-6-oxo-6H-
pyridazin-1-yl]-acetamide (14f). Yield =97%; mp=145-147 ꢀC

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5053
Figure 2. Analysis of Ca2þ mobilization in phagocytes treated with compound 14a. HL-60-FPR1 and HL-60-FPRL1 cells (A) or human
neutrophils (B) were loaded with FLIPR calcium 3 dye, and Ca2þ flux was analyzed, as described. Responses were normalized to the response
induced by 5 nM fMLF for HL-60-FPR1 cells and neutrophils, or 5 nM WKYMVm for HL-60-FPRL1 cells, which were assigned a value of
100%. (C) Representative kinetics of Ca2þ mobilization after treatment with compound 14a or fMLF. Human neutrophils were treated with
the compound 14a (1.5 and 3 μM), 5 nM fMLF (positive control), or 1% DMSO (negative control), and Ca2þ flux was monitored for the
indicated times. The data are from one experiment that is representative of three independent experiments.
Table 3. Ca2þ Mobilization and Chemotactic Activity in Human
(EtOH). IR (cm-1) 3296 (NH), 1705 (CO), 1644 (CO). 1H NMR
(CDCl3) δ 1.30 (s, 9H, C-(CH3)3), 2.27 (s, 3H, 3-CH3), 3.81 (s, 3H,
OCH3), 3.90 (s, 2H, CH2-Ar), 4.96 (s, 2H, NCH2CO), 6.76 (s, 1H,
Ar), 6.80 (s, 1H, Ar), 6.81-6.86 (m, 2H, Ar), 7.26-7.30 (m, 3H,
Ar), 7.43 (d, 2H, Ar, J = 8.6 Hz), 8.88 (exch br s, 1H, NH).
MS (ESI) m/z 420.22 [M þ H]þ. Anal. (C25H29N3O3) C, H, N.
2-[5-(3-Methoxybenzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-
N-(4-methoxyphenyl)-acetamide (14j). Yield =75%; mp =65-
67 ꢀC (EtOH). IR (cm-1) 3300 (NH), 1705 (CO), 1644 (CO).
1H NMR (CDCl3) δ 2.28 (s, 3H, 3-CH3), 3.80 (s, 3H, OCH3),
3.81 (s, 3H, OCH3), 3.91 (s, 2H, CH2-Ar), 4.94 (s, 2H,
NCH2CO), 6.77 (s, 1H, Ar), 6.80 (s, 1H, Ar), 6.81-6.86 (m,
4H, Ar), 7.27-7.30 (m, 1H, Ar), 7.42 (d, 2H, Ar, J =8.9 Hz),
8.71 (exch br s, 1H, NH). MS (ESI) m/z 394.18 [M þ H]þ. Anal.
(C22H23N3O4) C, H, N.
Nneutrophils Treated with Selected FPR1/FPRL1 Agonistsa
EC50 (μM)
compd
Ca2þ mobilization
chemotaxis
14a
14d
14e
14f
2.6 ( 0.3
6.7 ( 1.1
3.2 ( 1.2
3.9 ( 0.6
3.2 ( 0.3
1.6 ( 0.8
1.1 ( 0.6
3.6 ( 1.0
3.6 ( 0.8
21.7 ( 4.2
4.3 ( 1.1
11.3 ( 2.8
0.8 ( 0.2
2.1 ( 0.8
1.6 ( 0.2
1.8 ( 0.3
8.2 ( 1.4
0.6 ( 0.3
0.9 ( 0.2
1.1 ( 0.6
1.2 ( 0.6
4.5 ( 2.5
1.9 ( 0.6
13.1 ( 2.3
11.8 ( 2.6
0.6 ( 0.4
14h
14j
14l
14m
14n
14p
14x
17a
17b
N-(4-Butoxyphenyl)-2-[5-(3-methoxybenzyl)-3-methyl-6-oxo-
6H-pyridazin-1-yl]-acetamide (14k). Yield=73%; oil. IR (cm-1
)
3300 (NH), 1708 (CO), 1645 (CO). 1H NMR (CDCl3) δ 0.99 (t,
3H, O(CH2)3CH3), 1.50 (sext, 2H, OCH2CH2CH2, J =7.5 Hz),
1.76 (quint, 2H, OCH2CH2CH2, J =7.0 Hz), 2.28 (s, 3H, 3-
CH3), 3.81 (s, 3H, OCH3), 3.90 (s, 2H, CH2-Ar), 3.93 (t, 2H,
OCH2CH2CH2, J=6.5 Hz), 4.94 (s, 2H, NCH2CO), 6.76 (s, 1H,
Ar), 6.80-6.86 (m, 5H, Ar), 7.26-7.30 (m, 1H, Ar), 7.40 (d, 2H,
Ar, J =8.9 Hz), 8.76 (exch br s, 1H, NH). MS (ESI) m/z 436.23
[M þ H]þ. Anal. (C25H29N3O4) C, H, N.
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 dose-response curves (5-6 points) generated using
GraphPad Prism 5 with 95% confidential interval ( p < 0.05).
(s, 1H, Ar), 6.80-6.86 (m, 3H, Ar), 7.08 (d, 2H, Ar, J =8.3 Hz),
7.26-7.30 (m, 1H, Ar), 7.38 (d, 2H, Ar, J =8.4 Hz), 8.82 (exch
br s, 1H, NH). MS (ESI) m/z 378.18 [M þ H]þ. Anal.
(C22H23N3O3) C, H, N.
N-(4-tert-Butylphenyl)-2-[5-(3-methoxybenzyl)-3-methyl-6-oxo-
6H-pyridazin-1-yl]-acetamide (14i). Yield=95%; mp=54-56 ꢀC
N-(3,4-Dimethoxyphenyl)-2-[5-(3-methoxybenzyl)-3-methyl-
6-oxo-6H-pyridazin-1-yl]-acetamide (14l). Yield =74%; mp =
57-59 ꢀC (EtOH). IR (cm-1) 3298 (NH), 1708 (CO), 1640 (CO).
1H NMR (CDCl3) δ 2.29 (s, 3H, 3-CH3), 3.80 (s, 3H, OCH3),

5054 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Cilibrizzi et al.
3.83 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.90 (s, 2H, CH2-Ar),
4.95 (s, 2H, NCH2CO), 6.72 (d, 1H, Ar, J =8.6 Hz), 6.78 (s, 2H,
Ar), 6.82-6.88 (m, 3H, Ar), 7.25-7.29 (m, 1H, Ar), 7.32 (d, 1H,
Ar, J =2.2 Hz), 8.93 (exch br s, 1H, NH). MS (ESI) m/z 424.19
[M þ H]þ. Anal. (C23H25N3O5) C, H, N.
J =8.7 Hz), 7.26-7.30 (m, 1H, Ar), 7.50 (d, 2H, Ar, J =8.7 Hz).
MS (ESI) m/z 443.06 [M þ H]þ. Anal. (C21H19BrN2O4) C, H, N.
4-(3-Methoxybenzyl)-6-methyl-2-[2-(4-methylpiperazin-1-yl)-
2-oxo-ethyl]-pyridazin-3(2H)-one (14u). Yield = 62%; oil. IR
(cm-1) 1673 (CO), 1644 (CO). 1H NMR (CDCl3) δ 2.25 (s, 3H,
3-CH3), 2.55 (s, 3H, CH3N), 2.74-2.81 (m, 4H, Ar), 3.77-3.82
(m, 7H (4H, Ar; 3H, OCH3)), 3.87 (s, 2H, CH2-Ar), 4.96 (s, 2H,
NCH2CO), 6.69 (s, 1H, Ar), 6.80 (s, 1H, Ar), 6.82-6.85 (m, 2H,
Ar), 7.26-7.30 (m, 1H, Ar). MS (ESI) m/z 371.21 [M þ H]þ.
Anal. (C20H26N4O3) C, H, N.
N-(4-Butoxyphenyl)-2-[5-(4-methoxybenzyl)-3-methyl-6-oxo-
6H-pyridazin-1-yl]-acetamide (14v). Yield =60%; mp =160-
161 ꢀC (EtOH). IR (cm-1) 3300 (NH), 1707 (CO), 1644 (CO).
1H NMR (CDCl3) δ 0.98 (t, 3H, O(CH2)3CH3, J =7.4 Hz),
1.45-1.54 (m, 2H, OCH2CH2CH2CH3), 1.73-1.80 (m, 2H,
OCH2CH2CH2CH3), 2.28 (s, 3H, 3-CH3), 3.83 (s, 3H, OCH3),
3.88 (s, 2H, CH2-Ar), 3.94 (t, 2H, OCH2CH2CH2CH3, J =6.5
Hz), 4.93 (s, 2H, NCH2CO), 6.74 (s, 1H, Ar), 6.82 (d, 2H, Ar, J=
9.0 Hz), 6.90 (d, 2H, Ar, J=8.6 Hz), 7.17 (d, 2H, Ar, J=8.5 Hz),
7.39 (d, 2H, Ar, J =9.0 Hz), 8.67 (exch br s, 1H, NH). MS (ESI)
m/z 436.23 [M þ H]þ. Anal. (C25H29N3O4) C, H, N.
N-Benzo[1,3]dioxol-5-yl-2-[5-(3-methoxybenzyl)-3-methyl-6-
oxo-6H-pyridazin-1-yl]-acetamide (14m). Yield =98%; oil. IR
1
(cm-1) 3300 (NH), 1707 (CO), 1643 (CO). H NMR (CDCl3)
δ 2.28 (s, 3H, 3-CH3), 3.81 (s, 3H, OCH3), 3.89 (s, 2H, CH2-Ar),
4.93 (s, 2H, NCH2CO), 5.92 (s, 2H, O-CH2-O), 6.65 (d, 1H, Ar,
J =8.3 Hz), 6.77-6.85 (m, 5H, Ar), 7.21 (d, 1H, Ar, J =2.0 Hz),
7.25-7.29 (m, 1H, Ar), 9.04 (exch br s, 1H, NH). MS (ESI) m/z
408.16 [M þ H]þ. Anal. (C22H21N3O5) C, H, N.
2-[5-(3-Methoxybenzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-
N-(4-trifluoromethylphenyl)-acetamide (14n). Yield = 80%;
mp =175-176 ꢀC (EtOH). IR (cm-1) 3297(NH), 1708 (CO),
1646 (CO). 1H NMR (CDCl3) δ 2.33 (s, 3H, 3-CH3), 3.79 (s, 3H,
OCH3), 3.91 (s, 2H, CH2-Ar), 5.00 (s, 2H, NCH2CO), 6.81-6.85
(m, 3H, Ar), 6.89 (s, 1H, Ar), 7.25-7.29 (m, 1H, Ar), 7.38 (d, 2H,
Ar, J =8.7 Hz), 7.47 (d, 2H, Ar, J =8.7 Hz), 9.62 (exch br s, 1H,
NH). MS (ESI) m/z 432.16 [M þ H]þ. Anal. (C22H20F3N3O3)
C, H, N.
N-(4-Butoxyphenyl)-2-[5-(4-chlorobenzyl)-3-methyl-6-oxo-
6H-pyridazin-1-yl]-acetamide (14w). Yield =70% mp =146-
147 ꢀC (EtOH). IR (cm-1) 3298 (NH), 1708 (CO), 1642 (CO). 1H
NMR (CDCl3) δ 0.99 (t, 3H, O(CH2)3CH3, J =7.4 Hz), 1.49
(sext, 2H, OCH2CH2CH2CH3, J =7.5 Hz), 1.76 (quint, 2H,
OCH2CH2CH2CH3, J =7.0 Hz), 2.29 (s, 3H, 3-CH3), 3.88 (s,
2H, CH2-Ar), 3.93 (t, 2H, CH2-O, J = 6.5 Hz), 4.94 (s, 2H,
NCH2CO), 6.76 (s, 1H, Ar), 6.81 (d, 2H, Ar, J=9.0 Hz), 7.18 (d,
2H, Ar, J=8.4 Hz), 7.32 (d, 2H, Ar, J=8.4 Hz), 7.38 (d, 2H, Ar,
J=9.0 Hz), 8.69 (exch br s, 1H, NH). MS (ESI) m/z 440.17 [M þ
H]þ. Anal. (C24H26ClN3O3) C, H, N.
N-(4-Bromophenyl)-2-[5-(4-methoxybenzyl)-3-methyl-6-oxo-
6H-pyridazin-1-yl]-acetamide (14x). Yield=73%; mp =139-
141 ꢀC (EtOH). IR (cm-1) 3300 (NH), 1709 (CO), 1644 (CO). 1H
NMR (CDCl3) δ 2.29 (s, 3H, 3-CH3), 3.83 (s, 3H, OCH3), 3.88
(s, 2H, CH2-Ar), 4.94 (s, 2H, NCH2CO), 6.78 (s, 1H, Ar), 6.90
(d, 2H, Ar, J =8.6 Hz), 7.17 (d, 2H, Ar, J =8.6 Hz), 7.40 (s, 4H,
Ar), 9.01 (exch br s, 1H, NH). MS (ESI) m/z 442.08 [M þ H]þ.
Anal. (C21H20BrN3O3) C, H, N.
2-[5-(3-Methoxybenzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-
N-(4-trifluoromethoxyphenyl)-acetamide (14o). Yield = 87%;
mp =168-169 ꢀC (EtOH). IR (cm-1) 3300 (NH), 1708 (CO),
1644 (CO). 1H NMR (CDCl3) δ 2.31 (s, 3H, 3-CH3), 3.78 (s, 3H,
OCH3), 3.89 (s, 2H, CH2-Ar), 5.00 (s, 2H, NCH2CO), 6.79-6.83
(m, 3H, Ar), 6.87 (s, 1H, Ar), 7.00 (d, 2H, Ar, J =8.6 Hz), 7.24-
7.28 (m, 1H, Ar), 7.41 (d, 2H, Ar, J=9.0 Hz), 9.54 (exch br s, 1H,
NH). MS (ESI) m/z 448.15 [M þ H]þ. Anal. (C22H20F3N3O4)
C, H, N.
2-[5-(3-Methoxybenzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-
N-(4-nitrophenyl)-acetamide (14p). Yield=49%; mp=165-166
ꢀC (EtOH). IR (cm-1) 3298 (NH), 1708 (CO), 1644 (CO).
1H NMR (CDCl3) δ 2.34 (s, 3H, 3-CH3), 3.79 (s, 3H, OCH3),
3.92 (s, 2H, CH2-Ar), 5.01 (s, 2H, NCH2CO), 6.80-6.84 (m, 3H,
Ar), 6.94 (s, 1H, Ar), 7.25-7.28 (m, 1H, Ar), 7.49 (d, 2H, Ar, J=
9.2 Hz), 7.99 (d, 2H, Ar, J =9.2 Hz), 9.92 (exch br s, 1H, NH).
MS (ESI) m/z 409.15 [M þ H]þ. Anal. (C21H20N4O5) C, H, N.
N-(4-Cyanophenyl)-2-[5-(3-methoxybenzyl)-3-methyl-6-oxo-
6H-pyridazin-1-yl]-acetamide (14q). Yield =55%; mp =156-
158 ꢀC (EtOH). IR (cm-1) 3285 (NH), 2221 (CN), 1716 (CO),
1644 (CO). 1H NMR (CDCl3) δ 2.32 (s, 3H, 3-CH3), 3.80 (s, 3H,
OCH3), 3.91 (s, 2H, CH2-Ar), 4.98 (s, 2H, NCH2CO), 6.80-6.88
(m, 4H, Ar), 7.26-7.30 (m, 1H, Ar), 7.50-7.57 (m, 4H, Ar), 9.60
(exch br s, 1H, NH). MS (ESI) m/z 389.16 [M þ H]þ. Anal.
(C22H20N4O3) C, H, N.
2-[5-(3-Methoxybenzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-
N-(4-methoxyphenyl)-N-methyl-acetamide (14r). Yield =45%;
mp =125-127 ꢀC (EtOH). IR (cm-1) 1709 (CO), 1644 (CO).
1H NMR (CDCl3) δ 2.21 (s, 3H, 3-CH3), 3.32 (s, 3H, CH3N),
3.82 (s, 3H, OCH3), 3.86 (s, 2H, CH2-Ar), 4.64 (s, 2H,
NCH2CO), 6.62 (s, 1H, Ar), 6.77 (s, 1H, Ar), 6.80-6.85 (m,
2H, Ar), 7.26-7.30 (m, 3H, Ar), 7.60 (d, 2H, Ar, J=8.4 Hz). MS
(ESI) m/z 456.09 [M þ H]þ. Anal. (C22H22BrN3O3) C, H, N.
N-(4-Bromobenzyl)-2-[5-(3-methoxybenzyl)-3-methyl-6-oxo-
6H-pyridazin-1-yl]-acetamide (14s). Yield =97%; mp =184-
185 ꢀC (EtOH). IR (cm-1) 3300 (NH), 1708 (CO), 1644 (CO).
1H NMR (DMSO-d6) δ 2.21 (s, 3H, 3-CH3), 3.73 (s, 3H, OCH3),
3.76 (s, 2H, CH2-Ar), 4.27 (d, 2H, CH2Ar), 4.68 (s, 2H,
NCH2CO), 6.80-6.86 (m, 3H, Ar), 7.05 (s, 1H, Ar), 7.23-
7.26 (m, 3H, Ar), 7.51 (d, 2H, Ar, J=8.3 Hz), 8.63 (exch br t, 1H,
NH, J = 5.8 Hz). MS (ESI) m/z 456.09 [M þ H]þ. Anal.
(C22H22BrN3O3) C, H, N.
General Procedure for 15a,b. Compounds 15a,b were ob-
tained starting from 11a following the general procedure de-
scribed for 12a, using the appropriate alkyl halide in the place of
ethyl bromoacetate.
3-[5-(3-Methoxybenzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-
propionic Acid Ethyl Ester (15a). Yield=86%; oil. H NMR
1
(CDCl3) δ 1.26 (t, 3H,OCH2CH3, J =7.1 Hz), 2.22 (s, 3H, 3-
CH3), 2.84 (t, 2H, NCH2CH2COO, J =7.2 Hz), 3.83 (s, 3H,
OCH3), 3.87 (s, 2H, CH2-Ar), 4.17 (q, 2H, OCH2CH3, J =7.1
Hz), 4.45 (t, 2H, NCH2CH2COO, J =7.3 Hz), 6.64 (s, 1H, Ar),
6.80 (s, 1H, Ar), 6.83 (d, 2H, Ar, J =7.9 Hz), 7.26-7.30 (m,
1H, Ar).
2-[5-(3-Methoxybenzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-
propionic Acid Ethyl Ester (15b). Yield=85%; oil. H NMR
1
(CDCl3) δ 1.22 (t, 3H,OCH2CH3, J = 7.1 Hz), 1.65 (d, 3H,
CH3CHN, J =7.2 Hz), 2.21 (s, 3H, 3-CH3), 3.78 (s, 3H, OCH3),
3.84 (s, 2H, CH2-Ar), 4.19 (q, 2H, OCH2CH3, J =6.7 Hz), 5.51
(q, 1H, CH3CHN, J =7.2 Hz), 6.64 (s, 1H, Ar), 6.77-6.81 (m,
3H, Ar), 7.22-7.26 (m, 1H, Ar).
General Procedure for 16a,b. Compounds 16a,b were ob-
tained starting from 15a,b following the same general procedure
described for 13a,d. After dilution with cold water and acidifica-
tion with 6N HCl, the mixtures were extracted with CH2Cl2 (3 ꢀ
15 mL) and the solvent evaporated in vacuo.
3-[3-(3-Methoxybenzyl)-5-methyl-2-oxo-2H-pyridin-1-yl]-
propionic Acid (16a). Yield =96%; mp =86-88 ꢀC (EtOH). 1H
NMR (CDCl3) δ 2.22 (s, 3H, 3-CH3), 2.89 (t, 2H, NCH2-
CH2COO, J =7.2 Hz), 3.81 (s, 3H, OCH3), 3.87 (s, 2H, CH2-
Ar), 4.46 (t, 2H, NCH2CH2COO, J =7.2 Hz), 6.66 (s, 1H, Ar),
[5-(3-Methoxybenzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-
acetic Acid 4-Bromo-phenyl Ester (14t). Yield = 97%; mp =
111-112 ꢀC (EtOH). IR (cm-1) 3300 (NH), 1745 (CO), 1644
1
(CO). H NMR (CDCl3) δ 2.27 (s, 3H, 3-CH3), 3.81 (s, 3H,
OCH3), 3.90 (s, 2H, CH2-Ar), 5.10 (s, 2H, NCH2COO), 6.73 (s,
1H, Ar), 6.80 (s, 1H, Ar), 6.83-6.86 (m, 2H, Ar), 7.05 (d, 2H, Ar,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5055
6.78 (s, 1H, Ar), 6.81-6.85 (m, 2H, Ar), 7.25-7.29 (m, 1H, Ar),
9.99 (exch br s, 1H, OH).
purified by flash chromatography using cyclohexane/ethyl
acetate 3:1 as eluent.
1-(4-Bromophenyl)-3-[5-(3-methoxybenzyl)-3-methyl-6-oxo-
6H-pyridazin-1-ylmethyl]-urea (19a). Yield =27%; mp =112-
114 ꢀC (EtOH). IR (cm-1) 3270 (NH), 3265 (NH), 1708 (CO),
1630 (CO). 1H NMR (CDCl3) δ 2.25 (s, 3H, 3-CH3), 3.82 (s, 3H,
OCH3), 3.88 (s, 2H, CH2-Ar), 6.13 (s, 2H, NCH2N), 6.68 (s, 1H,
Ar), 6.78-6.81 (m, 1H, Ar), 6.83-6.86 (m, 2H, Ar), 6.99 (exch
br s, 1H, NH), 7.27-7.31 (m, 3H, Ar), 7.41 (m, 2H, Ar, J =8.8
Hz). MS (ESI) m/z 458.07 [M þ H]þ. Anal. (C21H21BrN4O3)
C, H, N.
2-[5-(3-Methoxybenzyl)-3-methyl-6-oxo-6H-pyridazin-1-yl]-
propionic Acid (16b). Yield =85%; oil. 1H NMR (CDCl3) δ 1.71
(d, 3H, CH3CHN, J =7.2 Hz), 2.25 (s, 3H, 3-CH3), 3.81 (s, 3H,
OCH3), 3.88 (s, 2H, CH2-Ar), 5.54 (q, 1H, CH3CHN, J =7.2
Hz), 6.67 (s, 1H, Ar), 6.79-6.85 (m, 3H, Ar), 7.26-7.29 (m,
1H, Ar).
General Procedure for 17a,b. Compounds 17a,b were ob-
tained starting from 16a,b following the general procedure
described for 14a-x. Purification of the final compounds was
performed by column chromatography using cyclohexane/ethyl
acetate 1:2 as eluent for compound 17a, cyclohexane/ethyl
acetate 2:1 for compound 17b.
4-Bromo-N-[5-(3-methoxybenzyl)-3-methyl-6-oxo-6H-pyrida-
zin-1-ylmethyl]-benzamide (19b). Yield =30%; oil. IR (cm-1
)
3290 (NH), 1708 (CO), 1640 (CO). 1H NMR (CDCl3) δ 2.26 (s,
3H, 3-CH3), 3.83 (s, 3H, OCH3), 3.90 (s, 2H, CH2-Ar), 6.29 (s,
2H, NCH2N), 6.69 (s, 1H, Ar), 6.81-6.87 (m, 3H, Ar), 7.28-
7.32 (m, 1H, Ar), 7.58 (d, 2H, Ar, J =8.6 Hz), 7.94 (d, 2H, Ar,
J =8.6 Hz). MS (ESI) m/z 443.06 [M þ H]þ. Anal. (C21H20-
BrN3O3) C, H, N.
N-(4-Bromophenyl)-3-[3-(3-methoxybenzyl)-5-methyl-2-oxo-
2H-pyridazin-1-yl]-propionamide (17a). Yield = 82%; mp =
123-125 ꢀC (EtOH). IR (cm-1) 3297 (NH), 1710 (CO), 1644
1
(CO). H NMR (CDCl3) δ 2.27 (s, 3H, 3-CH3), 3.00 (t, 2H,
NCH2CH2COO, J =6.3 Hz), 3.80 (s, 3H, OCH3), 3.88 (s, 2H,
CH2-Ar), 4.54 (t, 2H, NCH2CH2COO, J =6.3 Hz), 6.76-6.84
(m, 4H, Ar), 7.25 (t, 1H, Ar, J =7.9 Hz), 7.40 (d, 2H, Ar, J =8.8
Hz), 7.50 (d, 2H, Ar, J =8.8 Hz), 9.29 (exch br s, 1H, NH). MS
(ESI) m/z 456.09 [M þ H]þ. Anal. (C22H22BrN3O3) C, H, N.
N-(4-Bromophenyl)-2-[5-(3-methoxybenzyl)-3-methyl-6-oxo-
6H-pyridazin-1-yl]-propionamide (17b). Yield = 53%; oil; IR
2-(2-Hydroxyethyl)-4-(3-methoxybenzyl)-6-methyl-pyridazin-
3(2H)-one (20). To a refluxed mixture of compound 12a (0.47
mmol) and NaBH4 (2.64 mmol) in anhydrous THF (6 mL),
CH3OH (1.45 mL) was slowly added. After stirring for 1 h at
60 ꢀC, the mixture was concentrated in vacuo, diluted with cold
water (10-15 mL), and extracted with CH2Cl2 (3 ꢀ 15 mL).
Evaporation of the solvent afforded the final compound (20).
Yield =95%; oil. 1H NMR (CDCl3) δ 2.23 (s, 3H, 6-CH3), 3.80
(s, 3H, OCH3), 3.86 (s, 2H, CH2-Ar), 4.00 (t, 2H, NCH2CH2OH,
J =5.1 Hz), 4.35 (t, 2H, NCH2CH2OH, J =5.0 Hz), 4.66 (exch
br s, 1H, CH2OH), 6.68 (s, 1H, Ar), 6.78-6.84 (m, 3H, Ar),
7.25-7.29 (m, 1H, Ar).
2-[2-(4-Bromophenoxy)-ethyl]-4-(3-methoxybenzyl)-6-meth-
yl-pyridazin-3(2H)-one (21). Compound 21 was obtained start-
ing from 20 following the general procedure described for 9.
Compound 21 was purified by flash chromatography using
toluene/ethyl acetate 8:2 as eluent. Yield =26%; mp =82-
83 ꢀC (EtOH). IR (cm-1) 1643 (CO). 1H NMR (CDCl3) δ 2.24
(s, 3H, 6-CH3), 3.82 (s, 3H, OCH3), 3.87 (s, 2H, CH2-Ar), 4.36 (t,
2H, NCH2CH2O, J =5.9 Hz), 4.54 (t, 2H, NCH2CH2O, J =
5.8 Hz), 6.66 (s, 1H, Ar), 6.79-6.86 (m, 5H, Ar), 7.27-7.35 (m,
1H, Ar), 7.35-7.37 (m, 2H, Ar). MS (ESI) m/z 431.08 [M þ H]þ.
Anal. (C21H21BrN2O3) C, H, N.
Methanesulfonic Acid 2-[5-(3-Methoxybenzyl)-3-methyl-6-
oxo-6H-pyridazin-1-yl]-ethyl Ester (22). To a cooled (0 ꢀC) and
stirred solution of 20 (0.47 mmol) and pyridine (0.5 mmol) in
anhydrous CH2Cl2 (2 mL), methanesulfonyl chloride (0.61
mmol) was added dropwise, and the mixture was stirred at room
temperature for 4 h. Then ice cold water was added and the
mixture was extracted with CH2Cl2 (3 ꢀ 15 mL): evaporation of
the solvent afforded the desired compound. Yield=85%; oil. 1H
NMR (CDCl3) δ 2.22 (s, 3H, 3-CH3), 2.98 (s, 3H, CH3SO3), 3.80
(s, 3H, OCH3), 3.86 (s, 2H, CH2-Ar), 3.87-3.90 (m, 2H,
NCH2CH2O), 4.45 (t, 2H, NCH2CH2O, J =6.6 Hz), 6.66 (s,
1H, Ar), 6.78 (s, 1H, Ar), 6.82 (d, 2H, Ar, J=8.1 Hz), 7.24-7.28
(m, 1H, Ar).
2-[2-(4-Bromophenylamino)-ethyl]-4-(3-methoxybenzyl)-6-
methyl-pyridazin-3(2H)-one (23). A solution of 22 (0.4 mmol)
and 4-bromoaniline (0.8 mmol) in 2-propanol (2 mL) was heated
under stirring for 6 h at 60 ꢀC. After the mixture was concen-
trated in vacuo and cold water (30 mL) was added, the suspen-
sion was extracted with CH2Cl2 (3 ꢀ 15 mL). Evaporation of the
solvent afforded the final compound 23, which was purified by
column chromatography using toluene/ethyl acetate 8:2 as
1
(cm-1) 3300 (NH), 1709 (CO), 1643 (CO). H NMR (CDCl3)
δ 1.71 (d, 3H, CH3CHN, J =7.1 Hz), 2.31 (s, 3H, 3-CH3), 3.80
(s, 3H, OCH3), 3.90 (s, 2H, CH2-Ar), 5.71 (q, 1H, CH3CHN, J=
7.0 Hz), 6.79-6.85 (m, 4H, Ar), 7.25-7.29 (m, 1H, Ar), 7.35-
7.36 (m, 4H, Ar), 9.18 (exch br s, 1H, NH). MS (ESI) m/z 456.09
[M þ H]þ. Anal. (C22H22BrN3O3) C, H, N.
General Procedure for 18a,b. Compounds 18a,b were ob-
tained starting from 11 following the procedure described for
12a. The final compounds were purified by column chromatog-
raphy using cyclohexane/ethyl acetate 2:1 as eluent for com-
pound 18a and cyclohexane/ethyl acetate 1:1 for compound 18b.
2-(4-Bromobenzyl)-4-(3-methoxybenzyl)-6-methyl-pyridazin-
3(2H)-one (18a). Yield =68%; mp =112-114 ꢀC (EtOH). IR
(cm-1) 1638 (CO). 1H NMR (CDCl3) δ 2.23 (s, 3H, 6-CH3), 3.81
(s, 3H, OCH3), 3.86 (s, 2H, CH2-Ar), 5.25 (s, 2H, NCH2Ar), 6.64
(s, 1H, Ar), 6.78-6.85 (m, 3H, Ar), 7.26-7.30 (m, 1H, Ar), 7.35
(d, 2H, Ar, J =8.4 Hz), 7.45-7.47 (m, 2H, Ar). MS (ESI) m/z
399.07 [M þ H]þ. Anal. (C20H19BrN2O2) C, H, N.
2-[2-(4-Bromophenyl)-2-oxo-ethyl]-4-(3-methoxybenzyl)-6-
methyl-pyridazin-3(2H)-one (18b). Yield =95%; oil. IR (cm-1
)
1715 (CO), 1644 (CO). 1H NMR (CDCl3) δ 2.25 (s, 3H, 6-CH3),
3.83 (s, 3H, OCH3), 3.89 (s, 2H, CH2-Ar), 5.52 (s, 2H,
NCH2CO), 6.72 (s, 1H, Ar), 6.80 (s, 1H, Ar), 6.84 (d, 2H, Ar,
J =7.9 Hz), 7.27-7.31 (m, 1H, Ar), 7.66 (d, 2H, Ar, J =8.6 Hz),
7.88 (d, 2H, Ar, J =8.5 Hz). MS (ESI) m/z 427.07 [M þ H]þ.
Anal. (C21H19BrN2O3) C, H, N.
General Procedure for 19a,b. A mixture of 11a (0.32 mmol),
40% formaldehyde (3 mL), and 33% NH3 (1.5 mL) in dioxane
(1.5-2 mL) was heated at 50 ꢀC for 1 h. The solvent was then
evaporated in vacuo, and the residue was extracted with CH2Cl2
(3 ꢀ 15 mL). The organic layer was dried with Na2SO4 and
evaporated to afford an oil.
For compound 19a, the residual oil was dissolved in 2 mL of
anhydrous CH2Cl2 and 4-bromophenyl isocyanate (0.35 mmol)
was added. The mixture was stirred at room temperature for
12 h and then the solid residue was filtered off and the solution
was evaporated in vacuo to afford compound 19a, which was
purified by flash chromatography using cyclohexane/ethyl acet-
ate 3:1 as eluent.
For compound 19b, the residual oil was dissolved in 3 mL of
anhydrous CH2Cl2 and, after cooling (0 ꢀC), 4-bromobenzoyl
chloride (0.44 mmol) was added and the mixture was stirred at
0 ꢀC for 6 h. Finally, the residue was washed with cold 0.5 N
NaOH (3 ꢀ 10 mL) and with cold water (2 ꢀ 10 mL). Evapora-
tion of the organic layer afforded compound 19b, which was
eluent. Yield = 58%; mp = 86-88 ꢀC (EtOH). IR (cm-1
)
3350 (NH), 1643 (CO). 1H NMR (CDCl3) δ 2.24 (s, 3H, 6-CH3),
3.58 (t, 2H, NCH2CH2NHAr, J =5.7 Hz), 3.82 (s, 3H, OCH3),
3.86 (s, 2H, CH2-Ar), 4.46 (t, 2H, NCH2CH2NHAr, J=5.8 Hz),
6.65-6.68 (m, 3H, Ar), 6.79-6.86 (m, 3H, Ar), 7.27-7.31 (m,
3H, Ar). MS (ESI) m/z 428.10 [M þ H]þ. Anal. (C21H22BrN3O2)
C, H, N.

5056 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Cilibrizzi et al.
2-(2-Aminoethyl)-4-(3-methoxybenzyl)-6-methyl-pyridazin-
3(2H)-one (24). A mixture of 22 (0.43 mmol) and 33% NH3
(3 mL) in isopropanol (2 mL) was stirred at 60 ꢀC for 3 h. After
concentration of the solvent and dilution with cold water
(20 mL), the mixture was extracted with CH2Cl2 (3 ꢀ 15 mL).
Evaporation of the solvent afforded desired compound 24.
2H, Ar), 7.26-7.30 (m, 1H, Ar), 7.66 (d, 1H, Ar, J =8.7 Hz),
7.80 (d, 2H, Ar, J =8.6 Hz), 8.14 (s, 1H, Ar), 9.37 (exch br s, 1H,
NH). MS (ESI) m/z 428.06 [M þ H]þ. Anal. (C20H18BrN3O3)
C, H, N.
1-(4-Bromophenyl)-3-[2-(3-methoxybenzyl)-6-methyl-3-oxo-
2,3-dihydropyridazin-4-yl]urea (30). Compound 30 was obtained
from compound 28 following the general procedure reported for
6a-c. After dilution with cold water, the mixture was extracted
with CH2Cl2 (3 ꢀ 15 mL), and the solvent was evaporated in
vacuo. Compound 30 was purified by column chromatography
using first CH2Cl2 to remove the 4-bromophenyl urea, followed
by cyclohexane/ethyl acetate 2:1 as eluent. Yield =56%; mp =
207-209 ꢀC (EtOH). IR (cm-1) 3270 (NH), 3265 (NH), 1705
(CO), 1630 (CO). 1H NMR (CDCl3) δ 2.12 (s, 3H, CH3), 3.57 (s,
3H, OCH3), 5.08 (s, 2H, CH2), 6.51-6.58 (m, 3H, Ar), 6.97-
7.04 (m, 3H, Ar), 7.23 (d, 2H, Ar, J =8.7 Hz), 7.86 (s, 1H, Ar),
8.70 (exch br s, 1H, NH), 8,94 (exch, br,s, 1H, NH). MS (ESI) m/
z 443.07 [M þ H]þ. Anal. (C20H19BrN4O3) C, H, N.
1
Yield = 68%; oil. H NMR (CDCl3) δ 2.25 (s, 3H, 6-CH3),
3.82 (s, 3H, OCH3), 3.88 (s, 2H, CH2-Ar), 4.02-4.04 (m, 2H,
NCH2CH2NH2), 4.37 (t, 2H, NCH2CH2NH2, J =4.9 Hz), 5.38
(exch br s, 2H, NH2), 6.69 (s, 1H, Ar), 6.79-6.85 (m, 3H, Ar),
7.26-7.30 (m, 1H, Ar).
General Procedure for 25a,b. Compounds 25a,b were ob-
tained by starting from 24. For compound 25a, the general
procedure reported for 6a-c was followed, and the final com-
pound was purified by flash chromatography using cyclohex-
ane/ethyl acetate 1:1 as eluent. For compound 25b, Et3N
(1.8 mmol) and 4-bromobenzoyl chloride (1.43 mmol) were
added to a cooled (0 ꢀC) and stirred solution of 24 (0.73 mmol)
in anhydrous CH2Cl2 (2 mL), and the mixture was stirred at 0 ꢀC
for 6 h. The solid residue was filtered off, and the solution was
washed with 6 N NaOH (3 ꢀ 10 mL) and then with cold water
(2 ꢀ 10 mL). The organic layer was dried with Na2SO4 and
evaporated in vacuo to afford compound 25b, which was
purified by flash chromatography using CH2Cl2/MeOH 99:1
as eluent.
Biological Assays. Cell Culture. Human HL-60 cells stably
transfected with FPR1 (HL-60-FPR1), FPRL1 (HL-60-
FPRL1), or FPRL2 (HL-60-FPRL2) were as previously de-
scribed.25 The transfected cells were cultured in RPMI supple-
mented with 10% heat inactivated fetal calf serum, 10 mM
HEPES, 100 μg/mL streptomycin, 100 U/mL penicillin, and
G418 (1 mg/mL), as described previously.26 Parent (wild-type)
HL-60 cells were cultured under the same conditions but with-
out G418.
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. Neu-
trophils were purified from the blood using dextran sedimenta-
tion, followed by Histopaque 1077 gradient separation and
hypotonic lysis of red blood cells, as described previously.27
Isolated neutrophils were washed twice and resuspended in
HBSS without Ca2þ and Mg2þ (HBSS-). Neutrophil prepara-
tions were routinely >95% pure, as determined by light micro-
scopy, and >98% viable, as determined by trypan blue
exclusion.
1-(4-Bromophenyl)-3-{2-[5-(3-methoxybenzyl)-3-methyl-6-
oxo-6H-pyridazin-1-yl]-ethyl}-urea (25a). Yield =15%; mp =
114-115 ꢀC (EtOH). IR (cm-1) 3270 (NH), 3265 (NH), 1705
(CO), 1630 (CO). 1H NMR (CDCl3) δ 2.23 (s, 3H, 3-CH3), 3.81
(s, 3H, OCH3), 3.87 (s, 2H, CH2-Ar), 4.47 (t, 2H,
NCH2CH2NH, J =5.2 Hz), 4.57 (t, 2H, NCH2CH2NH, J =
5.1 Hz), 6.69 (s, 1H, Ar), 6.78-6.84 (m, 3H, Ar), 7.04 (exch br s,
1H, NH), 7.25-7.28 (m, 3H, Ar), 7.41 (d, 2H, Ar, J =8.8 Hz).
MS (ESI) m/z 472.08 [M þ H]þ. Anal. (C22H23BrN4O3) C, H, N.
4-Bromo-N-{2-[5-(3-methoxybenzyl)-3-methyl-6-oxo-6H-pyr-
idazin-1-yl]-ethyl}-benzamide (25b). Yield =13%; oil. IR (cm-1
)
3300 (NH), 1707 (CO), 1643 (CO). 1H NMR (CDCl3) δ 2.18 (s,
3H, 3-CH3), 3.82 (s, 3H, OCH3), 3.87 (s, 2H, CH2-Ar), 4.55 (t,
2H, NCH2CH2NH, J=5.4 Hz), 4.70 (t, 2H, NCH2CH2NH, J=
5.4 Hz), 6.67 (s, 1H, Ar), 6.68-6.85 (m, 3H, Ar), 7.26 (d, 1H, Ar,
J =7.8 Hz), 7.56 (d, 2H, Ar, J =8.5 Hz), 7.86 (d, 2H, Ar, J =8.5
Hz). MS (ESI) m/z 457.08 [M þ H]þ. Anal. (C22H22BrN3O3)
C, H, N.
Ca2þ Mobilization Assay. Changes in intracellular Ca2þ 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. All active compounds
were evaluated in parent (wild-type) HL-60 cells for supporting
that the agonists are inactive in nontransfected cells. Human
neutrophils or HL-60 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 Ca2þ and Mg2þ (HBSSþ), and
aliquotted into the wells of a flat-bottomed, half-area-well black
microtiter plates (2 ꢀ 105 cells/well). The compound source plate
contained dilutions of test compounds in HBSSþ. Changes in
fluorescence were monitored (λex =485 nm, λem =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 HL-
60-FPR1 and neutrophils, or 5 nM WKYMVm (Calbiochem,
San Diego, CA) for HL-60-FPRL1, which were assigned a value
of 100%. Curve fitting (5-6 points) and calculation of median
effective concentration values (EC50) were performed by non-
linear regression analysis of the dose-response curves generated
using Prism 5 (GraphPad Software, Inc., San Diego, CA).
Chemotaxis Assay. Neutrophils were suspended in HBSSþ
containing 2% (v/v) fetal bovine serum (FBS) (2 ꢀ 106 cells/
mL), and chemotaxis was analyzed in 96-well ChemoTx che-
motaxis chambers (Neuroprobe, Gaithersburg, MD), as de-
scribed previously.27 In brief, lower wells were loaded with
2-(3-Methoxybenzyl)-6-methylpyridazin-3(2H)-one (27). Com-
pound 27 was obtained starting from 26 following the general
procedure described for 12a. Compound 27 was purified by flash
chromatography using CH2Cl2/MeOH 9.9:0.1 as eluent. Yield =
86%; mp =53-55 ꢀC (cyclohexane). 1H NMR (CDCl3) δ 2.32 (s,
3H, CH3), 3.80 (s, 3H, OCH3), 5.26 (s, 2H, CH2), 6.81-6.84 (m,
1H, Ar), 6.87 (d,1H, Ar, J =9.4 Hz), 6.97-7.05 (m, 2H, Ar), 7.07
(d, 1H, Ar, J =9.4 Hz), 7.22-7.26 (m, 1H, Ar).
4-Amino-2-(3-methoxybenzyl)-6-methylpyridazin-3(2H)-one
(28). A suspension of 27 (0.78 mmol) and hydrazine hydrate (322
mmol) was stirred in a sealed tube at 180 ꢀC for 12 h. After
cooling, ice-cold water was added, the suspension was kept at
0 ꢀC for 2 h, and the precipitate was filtered off. The solution was
saturated with NH4Cl and extracted with CH2Cl2 (3 ꢀ 25 mL).
Removal of the solvent afforded a second batch of crude
1
product. Yield = 89%; mp = 96-98 ꢀC (EtOH). H NMR
(CDCl3) δ 2.23 (s, 3H, CH3), 3.81 (s, 3H, OCH3), 4.85 (exch br s,
2H, NH2), 5.27 (s, 2H, CH2), 6.16 (s, 1H, Ar), 6.81-6.85 (m, 1H,
Ar), 6.97-7.02 (m, 2H, Ar), 7.23-7.28 (m, 1H, Ar).
4-Bromo-N-[2-(3-methoxybenzyl)-6-methyl-3-oxo-2,3-dihy-
dropyridazin-4-yl]benzamide (29). Compound 29 was obtained
from compound 28 following the procedure described for 25b.
The final compound was purified by flash chromatography
using cyclohexane/ethyl acetate 3:1 as eluent. Yield = 28%;
mp =162-164 ꢀC (EtOH). IR (cm-1) 3300 (NH), 1709 (CO),
1644 (CO). 1H NMR (CDCl3) δ 2.40 (s, 3H, CH3), 3.82 (s, 3H,
OCH3), 5.32 (s, 2H, CH2), 6.84-6.87 (m, 1H, Ar), 6.98-7.02 (m,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5057
30 μL of HBSSþ containing 2% (v/v) FBS and the indicated
concentrations of test compound, DMSO (negative control),
and 1 nM fMLF as a positive control. The number of migrated
cells was determined by measuring ATP in lysates of transmi-
grated 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 neutro-
phils. The results are expressed as percentage of negative control
and were calculated as follows: (number of cells migrating in
response to test compounds/spontaneous cell migration in
response to control medium) ꢀ 100. EC50 values were deter-
mined by nonlinear regression analysis of the dose-response
curves generated using Prism 5 software.
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Acknowledgment. We thank Dr. Francois Boulay
ꢀ
(Laboratoire de Biochimie et Biophysique des Systemes In-
tegres, CEA-Grenoble, France) for help in obtaining HL-60
ꢁ ꢁ
cells transfected with FPR subtypes. This work was supported
in part by NIH contract HHSN266200400009C and P20
RR020185. We also thank Dr. Brian Bothner and Navid
Mohaved (Department of Chemistry & Biochemistry, Mon-
tana State University, Bozeman, MT) for their help in mass
spectrometry analysis.
Supporting Information Available: Elemental analysis results
for all target compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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