Synthesis, HPLC enantioresolution, and X-ray analysis of a new series of C5-methyl pyridazines as N-formyl peptide receptor (FPR) Agonists

Article abstract of DOI:10.1002/chir.22162

The synthesis of three racemates and the corresponding non-chiral analogues of a C5-methyl pyridazine series is described here, as well as the isolation of pure enantiomers and their absolute configuration assignment. In order to obtain optically active compounds, direct chromatographic methods of separation by HPLC-UV were investigated using four chiral stationary phases (CSPs: Lux Amylose-2, Lux Cellulose-1, Lux Cellulose-2 and Lux Cellulose-3). The best resolution was achieved using amylose tris(5-chloro-2-methylphenylcarbamate) (Lux Amylose-2), and single enantiomers were isolated on a semipreparative scale with high enantiomeric excess, suitable for biological assays. The absolute configuration of optically active compounds was unequivocally established by X-ray crystallographic analysis and comparative chiral HPLC-UV profile. All compounds of the series were tested for formyl peptide receptor (FPR) agonist activity, and four were found to be active, with EC₅₀ values in the micromolar range. Chirality 25:400-408, 2013.

Full text of DOI:10.1002/chir.22162

CHIRALITY 25:400–408 (2013)
Synthesis, HPLC Enantioresolution, and X-ray Analysis of a
New Series of C5-methyl Pyridazines as N-Formyl Peptide
Receptor (FPR) Agonists
1
1
1
1
1
*
AGOSTINO CILIBRIZZI, LETIZIA CROCETTI, MARIA PAOLA GIOVANNONI, ALESSIA GRAZIANO, CLAUDIA VERGELLI,
GIANLUCA BARTOLUCCI,1 GIACOMO SOLDANI,1 MARK T. QUINN,2 IGOR A. SCHEPETKIN,2 AND CRISTINA FAGGI3
1Università degli Studi di Firenze, Dipartimento di Scienze Farmaceutiche, Via Ugo Schiff 6, Sesto Fiorentino, 50019 Firenze, Italy
2Montana State University, Department of Immunology and Infectious Diseases, Bozeman, MT 59717 USA
3Università degli Studi di Firenze, Dipartimento di Chimica, Via della Lastruccia 13, Sesto Fiorentino, 50019 Firenze, Italy
ABSTRACT
The synthesis of three racemates and the corresponding non-chiral analogues of
a C5-methyl pyridazine series is described here, as well as the isolation of pure enantiomers and
their absolute configuration assignment. In order to obtain optically active compounds, direct
chromatographic methods of separation by HPLC-UV were investigated using four chiral station-
ary phases (CSPs: Lux Amylose-2, Lux Cellulose-1, Lux Cellulose-2 and Lux Cellulose-3). The
best resolution was achieved using amylose tris(5-chloro-2-methylphenylcarbamate) (Lux Amy-
lose-2), and single enantiomers were isolated on a semipreparative scale with high enantiomeric
excess, suitable for biological assays. The absolute configuration of optically active compounds
was unequivocally established by X-ray crystallographic analysis and comparative chiral HPLC-
UV profile. All compounds of the series were tested for formyl peptide receptor (FPR) agonist
activity, and four were found to be active, with EC50 values in the micromolar range. Chirality
25:400-408, 2013. © 2013 Wiley Periodicals, Inc.
KEY WORDS: liquid chromatography; chiral stationary phase; Lux Cellulose; Lux Amylose;
absolute configuration; pyridazinones; FPRs; inflammation
INTRODUCTION
new series of chiral C5-methyl pyridazine derivatives
(compounds (Æ)-4a-c, Fig. 1) and the corresponding aromatic
non-chiral analogues (8a-c) in order to further investigate the
role of stereochemistry in their pharmacological activity. Due
to the hypothetical stereoselectivity of the drug–receptor
interaction, we developed a chiral stationary phases (CSPs)/
eluent system suitable for a semi-preparative scale and
performed chiral HPLC-UV separation of optically active
enantiomers from each racemates (Æ)-4a-c. Analytical methods
on four different CSPs (Lux Amylose-2, Lux Cellulose-1, Lux
Cellulose-2, and Lux Cellulose-3) were initially investigated to
determine the best resolution conditions, and changes in
mobile-phase composition were studied to determine the effect
of solvents on the column selectivity. On the basis of the prelim-
inary chromatographic results, the resolution of the racemates
was subseqently transferred in the semi-preparative system.
The absolute configuration of collected optically active com-
pounds was univocally established by X-ray crystallographic
analysis of one of the enantiomers in each pair and by compar-
ative elution order during chiral HPLC-UV analysis. Finally, we
investigated the pharmacological properties of all racemates,
enantiomers and non-chiral anologues as FPRs agonists using
in vitro functional assays.
Inflammation is the first response of the immune system to
infection, irritation, and trauma, and it plays a central role in
the pathogenesis of many human diseases, such as asthma,
rheumatoid arthritis, multiple sclerosis, cancer, and
Alzheimer’s disease.1 The inflammatory response requires
tight regulation and control to avoid overshooting, prolonga-
tion, and self-induced damage.2 Considering that most cur-
rently used anti-inflammatory drugs have important
limitations,3 the extensive search to find immunomodulatory
agents that regulate innate immune responses represents a
promising strategy for treating inflammation and infectious
diseases.
The development of bioactive molecules that selectively
stimulate the immune system represents an important chal-
lenge in medicinal chemistry.4 To this end, the modulation
of formyl peptide receptors (FPRs), mediators of key events
in the regulation of endogenous inflammation and resolution
of inflammation, represents a distinctive approach to control
inflammatory events.5 FPRs belong to the seven transmem-
brane domain G protein-coupled receptor (GPCR) family,
and three subtypes have been identified in humans (FPR1,
FPR2/ALX, FPR3) which exhibit a high level of amino acidic
homology.6 Being expressed in the majority of white blood
cells, FPRs interact with a wide range of structurally different
pro- and anti-inflammatory ligands associated with different
diseases.
Contract grant sponsor: National Institutes of Health; Contract grant number:
GM103500.
*Correspondence to: Maria Paola Giovannoni, Dipartimento di Scienze
Farmaceutiche, Via Ugo Schiff 6, Sesto Fiorentino 50019 Firenze. E-mail
mariapaola.giovannoni@unifi.it
Received for publication 23 July 2012; Accepted 11 January 2013
DOI: 10.1002/chir.22162
Published online 6 June 2013 in Wiley Online Library
(wileyonlinelibrary.com).
Previously, we identified a large number of potent pyridazine-
based FPR1/FPR2 agonists.7 Starting from the literature
evidence that chiral C4- or C5-substituted pyridazines are gen-
erally of particular interest to search for biological activity,8–10
as well as reports that stereoselective interaction may enhance
the binding with FPRs,11–14 we report herein the synthesis of a
© 2013 Wiley Periodicals, Inc.

C5-METHYL PYRIDAZINES AS FPR AGONISTS
401
Synthesis
General procedure for preparation of racemate (Æ)-2 and non-
chiral analogue 6. A mixture of the appropriate compound (Æ)-1 or
515 (7.41 mmol), K2CO3 (14.82 mmol), and ethyl bromoacetate
(11.12 mmol) in CH3CN (5 mL) was refluxed under stirring for 2–3 h.
The mixture was then concentrated in vacuo, diluted with cold water,
and extracted with CH2Cl2 (3 Â 15 mL). The organic layer was evaporated
in vacuo, and the final compounds (Æ)-2 and 616 were purified by column
chromatography using cyclohexane/ethyl acetate 1:1 as eluent.
Fig. 1. Structures of chiral FPR ligands designed.
(Æ)-ethyl-2-[5-methyl-6-oxo-3-phenyl-5,6-dihydropyridazin-1(4H)-
yl]acetate [(Æ)-2]. Yield = 99.9%, yellow oil.1H nmR (CDCl3) d 1.28
(m, 6H, CHCH3 + CH2CH3), 2.67-2.79 (m, 2H, CHCH2), 3.07-3.14
(m, 1H, CH3CH), 4.24 (q, 2H, CH3CH2, J = 6.9 Hz), 4.59 (s, 2H, NCH2),
7.40-7.43 (m, 3H, Ar), 7.72-7.75 (m, 2H, Ar).
MATERIAL AND METHODS
Reagents, starting materials, and HPLC-grade solvents were obtained
from commercial sources and used without further purification. Extracts
were dried over Na2SO4, and the solvents were removed under reduced
pressure. All reactions were monitored by thin layer chromatography
(TLC) using commercial plates precoated with Merck silica gel 60 F-254.
Visualization was performed by UV fluorescence (lmax = 254 nm) or by
staining with iodine or potassium permanganate. Chromatographic separa-
tions were performed on a silica gel column by gravity chromatography
(Kieselgel 40, 0.063–0.200 mm; Merck) or flash chromatography (Kieselgel
40, 0.040-0.063 mm; Merck). Yields refer to chromatographically and spec-
troscopically pure compounds, unless otherwise stated. Compounds were
named following IUPAC rules, as applied by Beilstein-Institut AutoNom
2000 (4.01.305) or CA Index Name. The identity and purity of intermediates
and final compounds was ascertained through nmR, TLC, and analytical
HPLC-UV. All melting points were determined on a microscope hot stage
Büchi apparatus and are uncorrected.1H nmR spectra were recorded with
Avance 400 instruments (Bruker Biospin Version 002 with SGU). Chemical
shifts (d) are reported in ppm to the nearest 0.01 ppm, using the solvent as
an internal standard. Coupling constants (J values) are given in Hz and were
calculated using ‘TopSpin 1.3’ software rounded to the nearest 0.1 Hz. Mass
spectra (m/z) were recorded on a ESI-MS triple quadrupole (Varian 1200L)
system, in positive ion mode, by infusing a 10 mg/L solution of each analyte
dissolved in a mixture of mQ H2O:acetonitrile 1:1 v/v. Microanalyses were
performed with a Perkin-Elmer 260 elemental analyzer for C, H, N, and the
results were within Æ 0.4% of the theoretical values, unless otherwise stated.
Analytical HPLC-UV was performed on an Agilent 1200 Series with an
autosampler, column oven, and diode array detector (DAD) using chiral
Lux Amylose-2, Lux Cellulose-1, Lux Cellulose-2, and Lux Cellulose-3
(50 mm  4.6 mm I.D., 3 mm particle size; Phenomenex, Bologna, Italy)
columns. For analytical enantioseparations, the sample solutions were
prepared by diluting stock solutions of each racemate at a concentration
of 0.1 mg/mL in the same mixture of solvents used as mobile phase.
The injection volume was 10 mL, the flow rate was 1.0 mL/min, the tem-
perature of column was 40ꢀC, and the detector wavelength was fixed at
250 nm. The signal was acquired and processed by Chemstation revision
B.03.03-SR2 software. HPLC-grade solvents were supplied by Sigma-
Aldrich (Milan, Italy). The mobile phases tested were mixtures of aceto-
nitrile (MeCN) or n-hexane (n-Hex), both with isopropanol (IPA) as polar
modifier. The values of retention time (tR) are given in minutes.
Semi-preparative HPLC-UV enantioseparations were performed using a
Lux Amylose-2 (250 mm  4.6 mm I.D., 5 mm particle size; Phenomenex,
Bologna, Italy) column. The HPLC apparatus consisted of a Perkin-Elmer
(Norwalk, CT, USA) series 200 with a quaternary pump, autosampler, col-
umn oven, UV–VIS detector,and Biologic BioFrac fraction collector (from
Bio-Rad, Milan, Italy). The column temperature was 40ꢀC, and the UV de-
tector wavelength was fixed at 250 nm. The signal was acquired and
processed by Totalchrom 6.3.1.0504 software. The enantiomeric excess
(ee) values were calculated from relative peak areas under analytical
conditions.
General procedure for preparation of racemate (Æ)-3 and non-
chiral analogue 7. A suspension of the appropriate compound (Æ)-2 or
6 (7.29 mmol) in 6 N NaOH (10 mL) was stirred at 80ꢀC for 3–5 h. The mix-
ture was then diluted with cold water and acidified with 6 N HCl. Products
(Æ)-3 and 7 were filtered off by suction and recrystallized from ethanol.
(Æ)-2-[5-Methyl-6-oxo-3-phenyl-5,6-dihydropyridazin-1(4H)-yl]acetic
acid [(Æ)-3]. Yield = 99.9%, mp = 87-89ꢀC (EtOH).1H nmR (CDCl3) d 1.31
(d, 3H, CHCH3, J= 6.4 Hz), 2.68–2.78 (m, 2H, CHCH2), 3.04–3.16
(m, 1H, CH3CH), 4.64 (s, 2H, NCH2), 6.98 (exch br s, 1H, OH), 7.41–7.43
(m, 3H, Ar), 7.73–7.75 (m, 2H, Ar).
2-[5-Methyl-6-oxo-3-phenylpyridazin-1(6H)-yl]acetic acid (7).
Yield = 90%, mp = 92–94ꢀC (EtOH).1H nmR (CDCl3) d 2.34 (s, 3H, CH3),
3.48 (exch br s, 1H, OH), 5.05 (s, 2H, NCH2), 7.45-7.50 (m, 3H, Ar), 7.63
(s, 1H, Ar), 7.78 (d, 2H, Ar, J = 4.5 Hz).
General procedure for preparation of racemates (Æ)-4a-c and non-
chiral analogues 8a-c. To a cooled (À5ꢀC) and stirred solution of the
appropriate derivative (Æ)-3 or 7 (2.06 mmol) in anhydrous THF
(6 mL), Et3N (7.21 mmol) was added. After 30 min, the mixture was
allowed to warm up to 0ꢀC, and ethyl chloroformate (2.27 mmol) was
added. After 1 h, the appropriate arylamine (4.12 mmol) was added.
The reaction was carried out at room temperature for 12 h. The mixture
was then concentrated in vacuo, diluted with cold water (20–30 mL),
and extracted with CH2Cl2 (3 Â 15 mL). The solvent was evaporated to
obtain the final compounds, which were purified by column chromatogra-
phy using cyclohexane/ethyl acetate 2:1 (for compound (Æ)-4a), n-
hexane/ethyl acetate 3:2 (for compounds (Æ)-4b,c), and cyclohexane/
ethyl acetate 1:1 (for compounds 8a-c) as eluents.
(Æ)-N-(4-Bromophenyl)-2-[5-methyl-6-oxo-3-phenyl-5,6-dihydropyridazin-
1(4H)-yl]acetamide [(Æ)-4a]. Yield= 23%, mp= 148–149ꢀC (EtOH).1H
nmR (CDCl3) d 1.37 (d, 3H, CHCH3, J = 6.4 Hz), 2.74–2.81 (m, 2H, CHCH2),
3.14–3.22 (m, 1H, CH3CH), 4.68 (s, 2H, NCH2), 7.23 (d, 2H, Ar, J = 8.8 Hz),
7.38–7.42 (m, 3H, Ar), 7.57 (d, 2H, Ar, J = 8.8 Hz), 7.77–7.79 (m, 2H, Ar),
8.15 (exch br s, 1H, NH). MS (ESI) calcd. for C19H18BrN3O2, averaged
400.27. Found: m/z 400/402 with a correct isotopic ratio 1:1 of ions species
[M+ H]+. Anal. calcd. for C19H18BrN3O2: C, 57.01; H, 4.53; N, 10.50. Found:
C, 56.95; H, 4.51; N, 10.47.
(Æ)-N-(4-Fluorophenyl)-2-[5-methyl-6-oxo-3-phenyl-5,6-dihydropyridazin-
1(4H)-yl]acetamide [(Æ)-4b]. Yield= 61%, mp = 164–165ꢀC (EtOH).
1 nmR (CDCl3) d 1,35 (d, 3H, CHCH3, J = 6.4 Hz), 2,69–2.80 (m, 2H, CHCH2),
3,11–3,20 (m, 1H, CH3CH), 4,68 (s, 2H, NCH2), 6.98 (t, 2H, Ar, J = 8.8 Hz),
7,42–7.49 (m, 5H, Ar), 7,76–7.78 (m, 2H, Ar), 8.25 (exch br s, 1H, NH).
MS (ESI) calcd. for C19H18FN3O2, 339.36. Found: m/z 340 [M + H]+. Anal.
calcd. for C19H18FN3O2: C, 67.24; H, 5.35; N, 12.38. Found: C, 67.17;
H, 5.36; N, 12.33.
Specific rotations of enantiomers were measured at 589 nm with a
Perkin-Elmer polarimeter model 241 equipped with a Na lamp. The
volume of the cell was 2 mL and the optical path was 10 cm. A standard
solution was prepared by dissolving 20 mg of the compounds into 2 mL
of CHCl3 (c = 1). The system was set at a temperature of 20ꢀC using a
Neslab RTE 740 cryostat.
(Æ)-N-(1,3-Benzo-dioxol-5-yl)-2-[5-methyl-6-oxo-3-phenyl-5,6-
dihydropyridazin-1(4H)-yl] acetamide [(Æ)-4c].. Yield= 88%, mp=
170–171ꢀC (EtOH).1H nmR (CDCl3) d 1.35 (d, 3H, CHCH3, J = 6.4 Hz),
2.69–2.80 (m, 2H, CHCH2), 3.10–3.18 (m, 1H, CH3CH), 4.66
Chirality DOI 10.1002/chir

402
CILIBRIZZI ET AL.
equation k’ = (tr
(s, 2H, NCH2), 5.94 (s, 2H, OCH2O), 6.72 (d, 1H, Ar, J = 8.3 Hz), 6.80 (dd, 1H,
Ar, J = 6.2 Hz, J = 2.1 Hz), 7.24 (s, 1H, Ar), 7.44 (t, 3H, Ar, J= 2.8 Hz), 7.77-7.79
(m, 2H, Ar), 8.01 (exch br s, 1H, NH). MS (ESI) calcd. for C20H19N3O4,
365.38. Found: m/z 366 [M + H]+. Anal. calcd. for C20H19N3O4: C, 65.74; H,
5.24; N, 11.50. Found: C, 65.61; H, 5.22; N, 11.56.
- t0)/t0, where tr is the retention time. The
enantioselectivity (a) and the resolution factors (R) were calculated as
follows: a = k2’/k1’ = 2 (tr,2 - tr,1)/(w1 + w2), where tr,2 and tr,1 are the
retention times of the second and the first enantiomers respectively, while
w1 and w2 are the corresponding base peak widths. Table 1 reports the
analytical parameters for the resolution of racemates (Æ)-4a-c carried out
on the four amylose- and cellulose-derived CSPs, using mixtures of MeCN
or n-Hex containing 10% IPA.
From analysis of the data obtained in analytical enantioseparations
(Table 1), we found the best chromatographic profile for the resolution
of all racemic mixtures (Æ)-4a-c was achieved with a Lux Amylose-2
column using MeCN/IPA 90:10 as the mobile phase (Fig. 2). These
conditions were then chosen for semipreparative purification, which
was found in analytical experiments to represent the best compromise
between high resolution of the two enantiomers, high loading capacity
of analytes, and short separation time. Quantitative isolation of the
optically active isomers (À)-4a-c and (+)-4a-c was then performed at
40ꢀC on a semipreparative apparatus, using a Lux Amylose-2 column
(250 mm  4.6 mm I.D., 5 mm) and MeCN/IPA 90:10 as the isocratic
mobile phase. The flow rate was 1 mL/min and the sample injection
was in the range of 150 to 200 mg of analyte for each run. Wavelength
used for the detection was 250 nm. Finally, pure enantiomers were
properly recovered by partition of the eluate according to the profile of
the chromatograms, as shown in Figure 3.
N-(4-Bromophenyl)-2-[5-methyl-6-oxo-3-phenylpyridazin-1(6H)-
yl]acetamide (8a). Yield = 38%, mp = 149–151ꢀC (EtOH).1H nmR
(CDCl3) d 2.37 (s, 3H, CH3), 5.08 (s, 2H, CH2CO), 7.40–7.49 (m, 7H,
Ar), 7.68 (s, 1H, Ar), 7.82 (dd, 2H, Ar, J = 5.8 Hz, J = 1.4 Hz), 9.02 (exch
br s, 1H, NH). MS (ESI) calcd. for C19H16BrN3O2, averaged 398.25.
Found: m/z 398/400 with a correct isotopic ratio 1:1 of ions species
[M + H]+. Anal. calcd. for C19H16BrN3O2: C, 57.30; H, 4.05; N, 10.55.
Found: C, 57.24; H, 4.07; N, 10.52.
N-(4-Fluoro phenyl)-2-[5-methyl-6-oxo-3-phenylpyridazin-1(6H)-
yl]acetamide (8b). Yield = 45%, mp = 117–119ꢀC (EtOH).1H nmR
(CDCl3) d 2.34 (s, 3H, CH3), 5.11 (s, 2H, CH2CO), 6.89-6.93 (td, 2H, Ar,
J = 3.3 Hz, J = 2.0 Hz), 7.44-7.49 (m, 5H, Ar), 7.65 (s, 1H, Ar), 7.80 (dd,
2H, Ar, J = 3.3 Hz, J = 2.0 Hz), 9.21 (exch br s, 1H, NH). MS (ESI) calcd.
for C19H16FN3O2, 337.35. Found: m/z 338 [M + H]+. Anal. calcd. for
C19H16FN3O2: C, 67.65; H, 4.78; N, 12.46. Found: C, 67.72; H, 4.80; N,
12.50.
N-(1,3-Benzodioxol À5-yl)-2-[5-methyl-6-oxo-3-phenylpyridazin-1
(6H)-yl]acetamide (8c). Yield = 25%, mp = 215–217ꢀC (EtOH).1H nmR
(DMSO-d6) d 2.19 (s, 3H, CH3), 4.95 (s, 2H, OCH2O), 5.99 (s, 2H,
CH2CO), 6.87 (d, 1H, Ar, J = 8.4 Hz), 6.96 (d, 1H, Ar, J = 8.4 Hz), 7.29
(s, 1H, Ar), 7.49 (q, 3H, Ar, J = 7.3 Hz), 7.89 (d, 2H, Ar, J = 7.3 Hz), 8.04
(s, 1H, Ar), 10.26 (exch br s, 1H, NH). MS (ESI) calcd. for C20H17N3O4,
363.37. Found: m/z 364 [M + H]+. Anal. calcd for C19H16FN3O2:
C, 66.11; H, 4.72; N, 11.56. Found: C, 66.04; H, 4.70; N, 11.58.
S-(À)-N-(4-Bromophenyl)-2-[5-methyl-6-oxo-3-phenyl-5,6-
dihydropyridazin-1(4H)-yl] acetamide [S-(À)-4a]. mp= 148–149ꢀC
(EtOH). [a]20D = À117.8ꢀ (c 1, CHCl3). ee= 99.9%.1H nmR (CDCl3) d 1.33
(d, 3H, CH2CHCH3, J = 6.4 Hz), 2.69–2.79 (m, 2H, CH2CHCH3), 3.11–3.19
(m, 1H, CH2CHCH3), 4.64 (s, 2H, NCH2CO), 7.40–7.43 (m, 7H, Ar), 7.74–
7.77 (m, 2H, Ar), 8.07 (exch br s, 1H, NH). MS (ESI) calcd. for
C
19H18BrN3O2, averaged 400.27. Found: m/z 400/402 with a correct
isotopic ratio 1:1 of ions species [M + H]+. Anal. calcd. for C19H18BrN3O2:
C, 57.01; H, 4.53; N, 10.50. Found: C, 57.07; H, 4.55; N, 10.53.
Chiral Chromatographic Resolution
Analytical chiral HPLC separations of racemates (Æ)-4a-c were
performed by using Lux Amylose-2, Lux Cellulose-1, Lux Cellulose-2 and
Lux Cellulose-3 columns (50 mm  4.6 mm I.D., 3 mm) from Phenomenex
(Bologna, Italy) having amylose tris(5-chloro-2-methylphenylcarbamate),
cellulose tris(3,5-dimethylphenylcarbamate), cellulose tris(3-chloro-4-
methylphenylcarbamate), and cellulose tris(4-methylbenzoate) as chiral
stationary phases respectively. All chromatographic resolutions were
performed at 40ꢀC using isocratic elutionand alternatively mixtures of
MeCN or n-Hex and different concentrations of IPA as the polar modifier.
The dead times (t0) of the tested columns, estimated at flow rate of
1.0 mL/min, were comparable in the range of 0.7–0.8 min. The values of
t0 found for each column and for each mobile phase employed were used
for the retention factor (k’) determination. The k’ was calculated using the
R-(+)-N-(4-Bromophenyl)-2-[5-methyl-6-oxo-3-phenyl-5,6-
dihydropyridazin-1(4H)-yl] acetamide [R-(+)-4a]. mp = 148–149ꢀC
(EtOH). [a]20D = +114.2ꢀ (c 1, CHCl3). ee= 98.1%.1H nmR (CDCl3) and MS
(ESI) spectra are identical to that of the enantiomer S-(À)-4a. Anal. calcd
for C19H18BrN3O2: C, 57.01; H, 4.53; N, 10.50. Found: C, 57.12; H, 4.52; N,
10.52.
S-(À)-N-(4-Fluorophenyl)-2-[5-methyl-6-oxo-3-phenyl-5,6-
dihydropyridazin-1(4H)-yl] acetamide [S-(À)-4b]. mp = 164–165ꢀC
(EtOH). [a]20D = À133.8ꢀ (c 1, CHCl3). ee= 98.1%.1H nmR (CDCl3) d 1.34
(d, 3H, CH2CHCH3, J = 6.4 Hz), 2.68–2.79 (m, 2H, CH2CHCH3), 3.11-3.19
(m, 1H, CH2CHCH3), 4.65 (s, 2H, NCH2CO), 6.98 (t, 2H, Ar, J= 8.8 Hz),
TABLE 1. Analytical chromatographic parameters of compounds (Æ)-4a-c calculated on cellulose and amylose CSPs testeda
(Æ)-4a
k’2
(Æ)-4b
k’2
(Æ)-4c
k’2
Column and
Mobile Phase
k’1
0.85
a
R
k’1
a
R
k’1
a
R
Lux Amylose-2
MeCN:IPA 9:1
Hex:IPA 9:1
2.50
29.41
2.95
1.35
3.9
3.2
0.40
27.58
1.00
31.40
2.52
1.14
2.5
1.4
1.14
NRb
3.02
NR
2.65
NR
3.5
NR
21.73
Lux Cellulose-1
MeCN:IPA 9:1
Hex:IPA 9:1
0.28
6.55
0.41
10.28
1.44
1.57
0.6
3.3
0.19
5.99
0.24
8.46
1.27
1.41
0.3
2.6
NR
8.95
NR
11.47
NR
1.28
NR
1.8
Lux Cellulose-2
MeCN:IPA 9:1
Hex:IPA 9:1
NR
13.07
NR
14.01
NR
1.07
NR
0.5
NR
11.87
NR
13.33
NR
1.12
NR
0.6
NR
NR
NR
NR
NR
NR
NR
NR
Lux Cellulose-3
MeCN:IPA 9:1
Hex:IPA 9:1
NR
4.62
NR
5.25
NR
1.14
NR
1.0
NR
3.53
NR
3.85
NR
1.09
NR
0.7
NR
11.95
NR
12.59
NR
1.05
NR
0.5
aColumns, 50 Â 4.6 mm I.D, 3 mm particle size; isocratic elution; flow rate, 1 mL/min; oven, 40ꢀC; UV detection, 250 nm.
bNR, no resolution due to the lack of selectivity or strong retention on the stationary phase.
Chirality DOI 10.1002/chir

C5-METHYL PYRIDAZINES AS FPR AGONISTS
403
Ar), 7.40–7.42 (m, 3H, Ar), 7.74-7.77 (m, 2H, Ar), 7.88 (exch br s, 1H,
NH). MS (ESI) calcd. for C20H19N3O4, 365.38. Found: m/z 366 [M + H]+.
Anal. calcd for C20H19N3O4: C, 65.74; H, 5.24; N, 11.50. Found: C, 65.81;
H, 5.26; N, 11.53.
R-(+)-N-(1,3-Benzo-dioxol-5-yl)-2-[5-methyl-6-oxo-3-phenyl-5,6-
dihydropyridazin-1(4H)-yl] acetamide [R-(+)-4c]. mp = 170–171ꢀC
(EtOH). [a]20D = +136.6ꢀ (c 1, CHCl3). ee= 99.2%.1H nmR and MS (ESI) spec-
tra are identical to that of the enantiomer S-(À)-4c. Anal. calcd. for
C20H19N3O4: C, 65.74; H, 5.24; N, 11.50. Found: C, 65.87; H, 5.23; N, 11.46.
X-ray Crystal Structure Analysis
X-ray diffraction analysis of compounds S-(À)-4a,c and R-(+)-
4b. X-ray analyses were carried out with a Goniometer Oxford Diffraction
KM4 Xcalibur2. Graphite-monochromated Mo/Ka radiation (40 mA/–40
KV) and a KM4 CCD/SAPPHIRE detector were used for cell parameter
determination and data collection of S-(À)-4a and S-(À)-4c. Cu/Ka radiation
(40 mA/–40 KV), monochromated by an Oxford Diffraction Enhance UL-
TRA assembly, and an Oxford Diffraction Excalibur PX Ultra CCD were
used for cell parameter determination and data collection of R-(+)-4b. The
integrated intensities, measured using the o scan mode, were corrected
for Lorentz and polarization effects.17 The substantial redundancy in data
allowed empirical absorption corrections (SADABS18) to be applied using
multiple measurements of symmetry-equivalent reflections. The structure
was solved by direct methods of SIR200419 and refined using the full-
matrix least squares on F2 provided by SHELXL97.20 In all three com-
pounds, the non-hydrogen atoms were refined anisotropically. Aromatic,
methylic, methylenic, and methinic hydrogens were assigned in calculated
positions, only hydrogen on N-3 in S-(À)-4c was found in the Fourier synthe-
sis; all hydrogen atoms were refined as isotropic.
The X-ray CIF file for each of the three structures has been deposited
at the Cambridge Crystallographic Data Center and allocated with the
deposition numbers CCDC 833484 [S-(À)-4a], CCDC 833486 [R-(+)-4b]
and CCDC 833485 [S-(À)-4c] respectively. Copies of the data can be
obtained, free of charge, from CCDC, 12 Union Road, Cambridge,
CB2 1EZ UK (e-mail: deposit@ccdc.cam.ac.uk; internet: //www.ccdc.
cam.ac.uk).
Fig. 2. Analytical separation of racemates (Æ)-4a-c obtained by simulta-
neous on-line UV detection. Column, Lux Amylose-2 (50 mm  4.6 mm I.D.,
3 mm); eluent, MeCN/IPA 90:10; flow rate, 1.0 mL/min; temperature, 40ꢀC;
detection, 250 nm; for other experimental conditions, see Table 1.
Compound S-(À)-4a. 4x(C19H18N3O2Br), M = 1601.10, Triclinic, space
group P1, a = 10.070(1), b = 12.244(1), c = 15.328(1)Å, V = 1832.0(3)Å,3
Z = 1 Dc = 1.451, m = 2.261 mm-1, F(000) = 816, experiment T = 293ꢀK.
Single crystals of S-(À)-4a were obtained by slow crystallization from
EtOH. The asymmetric unit contained four independent molecules, as
indicated in the molecular formula. 13926 reflections were collected with
a 4.27 < y < 29.41 range; 9827 were independent; the parameters were
902 and the final R index was 0.0518 for reflections having I > 2sI. There
are four non equivalent molecules in the asymmetric unit. In PARST
analysis, we found three significant intermolecular hydrogen bonds.
They have the following parameters and show interactions along the a
and b axes:
7.41-7.47 (m, 5H, Ar), 7.74–7.80 (m, 2H, Ar), 8.01 (exch br s, 1H, NH).
MS (ESI) calcd. for C19H18FN3O2, 339.36. Found: m/z 340 [M + H]+. Anal.
calcd. for C19H18FN3O2: C, 67.24; H, 5.35; N, 12.38. Found: C, 67.31; H,
5.34; N, 12.43.
R-(+)-N-(4-Fluorophenyl)-2-[5-methyl-6-oxo-3-phenyl-5,6-
dihydropyridazin-1(4H)-yl]acetamide
[R-(+)-4b]. mp= 164–165ꢀC
(EtOH). [a]20D = +128.3ꢀ (c 1, CHCl3). ee= 99.1%.1H nmR (CDCl3) and
N3A -H3A
0.86 (.01)
N3B -H3B
0.860 (0.008)
N3C -H3C
0.860 (0.009)
N3A . . .O1B
2.94(.01)
N3B . . .O1D
2.89 (0.1)
N3C . . .O1A
2.87 (0.1)
H3A ..O1B
2.10 (.01)
H3B ..O1D
2.045 (0.09)
H3C ..O1A
2.013 (0.08)
N3A -H3A. .O1B
164.2 (0.6)
N3B -H3B. .O1D
166.9 (0.6)
N3C -H3C. .O1A
173.6 (0.7)
Operation symmetry
x + 1,+y,+z
x,+y + 1,+z
x,+y-1,+z
MS (ESI) spectra are identical to that of the enantiomer S-(À)-4b. Anal.
calcd for C19H18FN3O2: C, 67.24; H, 5.35; N, 12.38. Found: C, 67.10;
H, 5.37; N, 12.40.
Compound R-(+)-4b. 2x(C19H18FN3O2)
,
M = 678.73, Tetragonal,
space group P À4 21 c, a = 19.982(1), b = 19.982(1), c = 17.455(1)Å,
V = 6969.4(6)Å,3 Z = 8 Dc = 1.294, 0.768 mm-1, F(000) = 2848,
m
=
experiment T = 293ꢀK. Single crystals of R-(+)-4b were obtained by
slow crystallization from MeCN. The asymmetric unit contains two
independent molecules, as indicated in the molecular formula.
48 444 reflections were collected with a 4.43 < y < 65.20 range;
5917 were independent; the parameters were 452 and the final R
index was 0.0682 for reflections having I > 2sI. In PARST analysis,
we found two significant hydrogen bonds. The first is
S-(À)-N-(1,3-Benzo-dioxol-5-yl)-2-[5-methyl-6-oxo-3-phenyl-5,6-
dihydropyridazin-1(4H)-yl] acetamide [S-(À)-4c]. mp= 170–171ꢀC
(EtOH). [a]20D = À132.3ꢀ (c 1, CHCl3). ee= 99.9%.1H nmR (CDCl3) d 1.35
(d, 3H, CH2CHCH3, J= 6.4 Hz), 2.67–2.78 (m, 2H, CH2CHCH3), 3.10-3.18
(m, 1H, CH2CHCH3), 4.63 (s, 2H, NCH2CO), 5.92 (s, 2H, OCH2O), 6.71
(d, 1H, Ar, J= 8.3 Hz), 6.80 (dd, 1H, Ar, J= 6.4 Hz, J = 1.9 Hz), 7.21 (s, 1H,
Chirality DOI 10.1002/chir

404
CILIBRIZZI ET AL.
Fig. 3. Semipreparative resolution of racemates (Æ)-4a-c. Column, Lux Amylose-2 (250 mm  4.6 mm I.D., 5 mm); eluent, MeCN/IPA 90:10; flow-rate, 1.0 mL/
min; temperature, 40ꢀC; detection, 250 nm.
intramolecular, whereas the second is intermolecular. They have
the following parameters:
analysis, we found a significant intermolecular hydrogen bond that has
the following parameters and shows an interaction along the a axis:
N3B -H3B
0.860 (.003)
N3A -H3A
N3B . . .O1A
2.768(.004)
N3A . . .O1B
2.812 (0.004)
H3B ..O1A
1.988 (.003)
H3A ..O1B
2.035 (0.003)
N3B –H3B. .O1A
150.3 ( 0.2)
N3A -H3A. .O1B
149.8 (0.2)
Operation symmetry
y + 1/2,+x-1/2,+z-1/2
0.860 (0.003)
N3 -HN3
0.74 (.06)
N3 . . .O2
2.81(.01)
HN3 ..O2
2.10 (.06)
N3 –HN3. .O2
165 ( 2)
Operation symmetry
x-1,+y,+z
Compound S-(À)-4c.
C20H19N3O4 , M = 365.38, Orthorhombic, space
Biological Assays
group P 21 21 21, a = 4.721(1), b = 16.863(3), c = 21.597(3)Å, V = 1719.3(5)
Å,3 Z = 4 Dc = 1.412, m = 0.100 mm-1, F(000) = 768, experiment T = 150ꢀK.
Single crystals of S-(À)-4c were obtained by vapour diffusion from
the EtOH/n-hexane mixture. 5351 reflections were collected with a
4.42 < y < 28.02 range; 3243 were independent; the parameters were 248
and the final R index was 0.0981 for reflections having I > 2sI. In PARST
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 strepto-
mycin, 100 U/ml penicillin, and G418 (1 mg/mL), as previously
Chirality DOI 10.1002/chir

C5-METHYL PYRIDAZINES AS FPR AGONISTS
405
described.21 Wild-type HL-60 cells were cultured under the same condi-
tions, but without G418.
curves generated using Prism 5 (GraphPad Software, Inc., San Diego,
CA, USA).
RESULTS AND DISCUSSION
Isolation of Human Neutrophils. Blood was collected from healthy
donors in accordance with a protocol approved by the Institutional Review
Board at Montana State University. Neutrophils were purified from the
blood using dextran sedimentation, followed by Histopaque 1077 gradi-
ent separation and hypotonic lysis of red blood cells, as previously
described.22 Isolated neutrophils were washed twice and resuspended
in Hank’s balanced-salt solution without Ca2+ and Mg2+ (HBSS-).
Neutrophil preparations were routinely >95% pure, as determined by
light microscopy, and >98% viable, as determined by trypan blue
exclusion.
Synthesis
The already described (Æ)-1 and aromatic analogue 5,15
which was obtained from (Æ)-1 with bromine in acetic acid as
the oxidizing agent23 (Scheme 1), were the key building blocks
for synthesis of the final compounds (Æ)-4a-c and 8a-c. The
alkylation under standard conditions with ethyl bromoacetate
resulted in the racemate (Æ)-2 and the known non-chiral 6,16
from which the corresponding intermediates (Æ)-3 and 7 were
obtained after alkaline hydrolysis. The final racemates (Æ)-4a-c
and the non-chiral analogues 8a-c were prepared in good
yields by treating the corresponding carboxylic acids (Æ)-3
and 7 with ethyl chloroformate in THF in the presence of
triethylamine; the intermediate mixed anhydrides were not
isolated but were directly transformed into the final amides
using the appropriate aryl amine (Scheme 1). The pure
compounds (Æ)-4a-c and 8a-c were crystallized from ethanol,
and the results of1H nmR, MS(ESI), and elemental analysis
agreed with the assigned structure.
Ca2+ Mobilization Assay. Changes in intracellular Ca2+ were mea-
sured with a FlexStation II scanning fluorometer using a FLIPR 3 calcium
assay kit (Molecular Devices, Sunnyvale, CA, USA) for human neutro-
phils and HL-60 cells. All active compounds were evaluated in parent
(wild-type) HL-60 cells for supporting that the agonists are inactive in
non-transfected cells. Human neutrophils or HL-60 cells, suspended in
HBSS- containing 10 mm HEPES, were loaded with Fluo-4 AM dye
(Invitrogen) (1.25 mg/mL final concentration) and incubated for 30 min
in the dark at 37ꢀC. After dye loading, the cells were washed with HBSS-
containing 10 mm HEPES, resuspended in HBSS containing 10 mm
HEPES and Ca2+ and Mg2+ (HBSS+), and aliquotted into the wells of a
flat-bottomed, half-area-well black microtiter plates (2 Â 105 cells/well).
The compound source plate contained dilutions of test compounds in
HBSS+. Changes in fluorescence were monitored (lex = 485 nm, lem = 538
nm) every 5 sec for 240 sec 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 Chemi-
cal Co., St. Louis, MO, USA) for HL-60-FPR1 and neutrophils, or 5 nm
WKYMVm (Calbiochem, San Diego, CA, USA) for HL-60-FPR2 and HL-
60-FPR3 cells, which were assigned a value of 100%. Curve fitting (5–6
Chiral Resolution
In order to obtain complete resolution of the racemates (Æ)-
4a-c, we performed an analytical chromatographic approach
based on the screening of four different chiral stationary
phases: Lux Amylose-2, Lux Cellulose-1, Lux Cellulose-2 and
Lux Cellulose-3 (50 mm 4.6 mm I.D., 3 mm particle size).
Different mobile phases were tested to determine the best
combination of mobile and stationary phases for the resolution
of racemates. The chromatographic results of the screening are
reported in Table 1 as retention factor (k’), enantioselectivity
(a) and resolution factors (R). Experimental data indicated that
points) and calculation of median effective concentration values (EC50
)
were performed by nonlinear regression analysis of the dose–response
Scheme 1. Synthesis of chiral compounds (Æ)-4a-c and non-chiral analogues 8a c.
Chirality DOI 10.1002/chir

406
CILIBRIZZI ET AL.
Considering both systems (Lux Cellulose-1 with n-Hex/
IPA 90:10 and Lux Amylose-2 with MeCN/IPA 90:10) showed
a comparable chiral resolving ability, the criteria used to dis-
criminate between methods was the solubility of the analytes
in the respective mobile phases. Indeed, since the solubility
of the racemates (Æ)-4a-c was poorer in n-Hex/IPA 90:10 than
in MeCN/IPA 90:10, the latter method was chosen in order to
increase the amount of analyte injected for each separation cy-
cle and to reduce the times of separations. Once optimized,
these analytical conditions were applied to the semipreparative
system. Separations were performed on Lux Amylose-2 column
(250 mm 4.6 mm I.D., 5 mm) (Fig. 3) and were suitable to
collect the required amounts of pure enantiomers (À)-4a-c
and (+)-4a-c for biological studies. Finally, the fractions
collected were analyzed in the analytical system using Lux
Amylose-2 column and the aforementioned conditions to deter-
mine the enantiomeric excess (ee) of pure isomers, which
resulted in ee values between 98.1% and 99.9% (Fig. 4).
Configurational Assignment
In order to establish absolute configuration of optically ac-
tive compounds in the series, X-ray analysis was performed.
Considering the difficulties in obtaining suitable crystals of
all enantiomers, one isomer for each pair was processed for
X-ray analysis, as reported for (À)-4a, (+)-4b and (À)-4c
(Fig. 5) in the experimental section. Assignment of the
absolute configurations at C5 was then unambiguously
established for all of the enantiomers by a combination of
crystallographic data, polarimetric analysis, and comparative
elution order determined by chiral HPLC separations.
Among the three analyzed enantiomers, compound (À)-4a
contains a bromine as a heavy atom that was useful in deter-
mining the exact configuration using the Flack parameter
derived from X-ray analysis.24 For enantiomers (+)-4b and
(À)-4c, the absolute configurations at C5 of the pyridazine ring
could not be determined positively by using only Flack param-
eters, as these compounds don’t contain heavy atoms in their
structures. Thus, these crystallographic results needed to be
supported by a correlation with both polarimetric and chiral
chromatographic analysis. Chiral HPLC and polarimetric
analysis indicated that racemates (Æ)-4a-c have same-sense
chiral recognition mechanism and, in all cases, the first eluted
enantiomer on Lux Amylose-2 column rotated the polarized
light in the negative direction (Figs. 3 and 4). Since the chiral
stationary phases are given just in one configuration, enantio-
mer elution order is a very important aspect in the study of
enantiorecognition mechanisms.25 In a series of analogue com-
pounds, such as in our case, the similarity of both skeleton and
Fig. 4. Chiral chromatographic analysis of isolated enantiomers (À)-4a-c
and (+)-4a-c to determine enantiomeric excess (ee). Column, Lux Amylose-2
(50 mm  4.6 mm I.D., 3 mm); eluent, MeCN/IPA 90:10; flow rate, 1.0 mL/min;
temperature, 40ꢀC; detection, 250 nm.
the best resolutions (R> 1.5) were achieved using cellulose tris
(3,5-dimethylphenylcarbamate) (Lux Cellulose-1) and n-Hex/
IPA 90:10 or amylose tris (3,5-dimethylphenylcarbamate)
(Lux Amylose-2) and MeCN/IPA 90:10. The other combina-
tions of mobile/stationary phases tested were found to be
unsuitable for resolving the samples tested.
Fig. 5. An ORTEP view of the molecular structure of S-(À)-4a, R-(+)-4b, and S-(À)-4c.
Chirality DOI 10.1002/chir

C5-METHYL PYRIDAZINES AS FPR AGONISTS
407
TABLE 2. Analysis of FPR activation by racemates (Æ)-4a-c, pure
CONCLUSIONS
enantiomers S-(À)-4a-c/R-(+)-4a-c, and non-chiral analogues 8a-c
Taken together, our data confirmed that the stereogenic cen-
ter at C5 of the pyridazine ring plays only a marginal role in the
activity and selectivity of the FPR agonists. On the other hand,
the N-bromophenylacetamide moiety on the lactam nitrogen of
the heterocyclic scaffold seems to play a major role in agonist
activity, which is in agreement with previously reported
agonists.7,11 Considering that pyridazine-based compounds
represent good candidates for FPR-specific agonists, further
studies are in progress to evaluate the influence of stereochem-
istry in other experimental in vitro models such as chemotaxis
and neutrophils activation.
in HL-60 cells expressing human FPR1, FPR2, or FPR3
Ca2+ Mobilization EC50 (mM) and Efficacy (%)a
compd
FPR1
FPR2
FPR3
(Æ)-4a
S-(À)-4a
R-(+)-4a
8a
23.5 (55)
19.5 (75)
24.4 (80)
21.5 (50)
7.0 (65)
10.7 (95)
10.0 (85)
10.1 (45)
N.A.
8.3 (40)
7.1 (40)
NA
(Æ)-4b
S-(À)-4b
R-(+)-4b
8b
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
ACKNOWLEDGMENTS
This work was supported in part by National Institutes of
Health grant GM103500, an equipment grant from the M.J.
Murdock Charitable Trust, and the Montana State University
Agricultural Experimental Station.
(Æ)-4c
S-(À)-4c
R-(+)-4c
8c
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
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for their ability to induce intracellular Ca2+ flux in HL-60 cells
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their ability to act as FPR agonists.26,27 As shown in Table 2,
biological assays revealed that the bromo derivatives (Æ)-4a,
S-(À)-4a, R-(+)-4a and 8a activated FPR1 and FPR2 with
EC50 values in a similar micromolar range, whereas they were
slightly more potent FPR2 agonists, with EC50 values be-
tween 7.0 and 10.7 mm. Moreover, enantiomers S-(À)-4a and
R-(+)-4a also exhibited an appreciable agonistic activity for
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=
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were inactive. To confirm specificity of the observed FPR ag-
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WT cells (wild-type non transfected HL-60 cells) and were
found to be inactive.
Chirality DOI 10.1002/chir

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Chirality DOI 10.1002/chir