Synthesis, enantioresolution, and activity profile of chiral 6-methyl-2,4-disubstituted pyridazin-3(2H)-ones as potent N-formyl peptide receptor agonists

Article abstract of DOI:10.1016/j.bmc.2012.04.043

A series of chiral pyridazin-3(2H)-ones was synthesized, separated as pure enantiomers, and evaluated for N-formyl peptide receptor (FPR) agonist activity. Characterization of the purified enantiomers using combined chiral HPLC and chiroptical studies (circular dichroism, allowed unambiguous assignment of the absolute configuration for each pair of enantiomers). Evaluation of the ability of racemic mixtures and purified enantiomers to stimulate intracellular Ca ²⁺ flux in FPR-transfected HL-60 cells and human neutrophils and to induce β-arrestin recruitment in FPR-transfected CHO-K1 cells showed that many enantiomers were potent agonists, inducing responses in the sub-micromolar to nanomolar range. Furthermore, FPRs exhibited enantiomer selectivity, generally preferring the R-(-)-forms over the S-(+)-enantiomers. Finally, we found that elongation of the carbon chain in the chiral center of the active compounds generally increased biological activity. Thus, these studies provide important new information regarding molecular features involved in FPR ligand preference and report the identification of a novel series of FPR agonists.

Full text of DOI:10.1016/j.bmc.2012.04.043

Bioorganic & Medicinal Chemistry 20 (2012) 3781–3792
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, enantioresolution, and activity profile of chiral 6-methyl-2,4-disubs-
tituted pyridazin-3(2H)-ones as potent N-formyl peptide receptor agonists
Agostino Cilibrizzi a, Igor A. Schepetkin b, Gianluca Bartolucci a, Letizia Crocetti a, Vittorio Dal Piaz a,
a
b
b
a
Maria Paola Giovannoni a, , Alessia Graziano , Liliya N. Kirpotina , Mark T. Quinn , Claudia Vergelli
⇑
a Dipartimento di Scienze Farmaceutiche, Università degli Studi di Firenze, Via Ugo Schiff 6, Sesto Fiorentino 50019, Italy
b Department of Immunology and Infectious Diseases, Montana State University, Bozeman, MT 59717, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of chiral pyridazin-3(2H)-ones was synthesized, separated as pure enantiomers, and evaluated
for N-formyl peptide receptor (FPR) agonist activity. Characterization of the purified enantiomers using
combined chiral HPLC and chiroptical studies (circular dichroism, allowed unambiguous assignment of
the absolute configuration for each pair of enantiomers). Evaluation of the ability of racemic mixtures
and purified enantiomers to stimulate intracellular Ca2+ flux in FPR-transfected HL-60 cells and human
neutrophils and to induce b-arrestin recruitment in FPR-transfected CHO-K1 cells showed that many
enantiomers were potent agonists, inducing responses in the sub-micromolar to nanomolar range. Fur-
thermore, FPRs exhibited enantiomer selectivity, generally preferring the R-(ꢀ)-forms over the S-(+)-
enantiomers. Finally, we found that elongation of the carbon chain in the chiral center of the active com-
pounds generally increased biological activity. Thus, these studies provide important new information
regarding molecular features involved in FPR ligand preference and report the identification of a novel
series of FPR agonists.
Received 2 February 2012
Revised 18 April 2012
Accepted 21 April 2012
Available online 27 April 2012
Keywords:
Formyl peptide receptors
Inflammation
Neutrophils
Enantiomers
Pyridazin-3(2H)-ones
Chiral agonists
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
events in inflammation and endogenous anti-inflammation and
represents an attractive target for the discovery of new therapeu-
Inflammation represents a primary response of the immune
system to infection, irritation, and trauma, and is mediated
through the action of many cell types with distinct but overlapping
roles.1 However, if an injurious agent persists or disrupts leukocyte
recruitment, both acute and chronic inflammatory disorders may
ensue.2 Indeed, dysregulated inflammatory processes play an
important role in many diseases, such as asthma,3 rheumatoid
arthritis, multiple sclerosis, periodontal disease, cancer, cardiovas-
cular and Alzheimer’s diseases.4 Thus, the host inflammatory re-
sponse requires stringent regulation to avoid self-induced
damage,5 and anti-inflammatory drugs are used to treat such
inflammatory syndromes.
The majority of current anti-inflammatory drugs have limita-
tions due to their interference with pro-inflammatory mediators
and potential to interfere with recently described biochemical pro-
cesses required for resolving inflammation.6 Thus, the develop-
ment of bioactive molecules that selectively stimulate innate
immune responses without causing excessive inflammation has
been a significant challenge.7 In this context, the human N-formyl
peptide receptor (FPR) family has emerged as mediator of key
tics.8 FPRs are seven transmembrane domain G protein-coupled
receptors (GPCR), and the three subtypes identified in humans
(FPR1, FPR2/ALX, and FPR3) exhibit a high level of amino acidic
homology.9 FPR1 is relatively abundant on phagocytic cells, and
it was first defined biochemically as a high-affinity binding site
on the surface of neutrophils for the prototypic peptide
N-formyl-methionine-leucine-phenylalanine (fMLF).10 FPR2/ALX
shares 69% amino acidic identity with human FPR1 but, despite
the relatively high level of sequence homology, is a low-affinity
receptor for fMLF.11 FPR2/ALX has a tissue distribution similar to
that of FPR1, but it is also expressed in other cell types, including,
hepatocytes, epithelial cells, T lymphocytes, neuroblastoma cells,
astrocytoma cells, and microvascular endothelial cells.10 This pat-
tern of tissue expression suggests that FPR2/ALX may participate
in a number of functions in addition to host defense. FPR3 shares
56% and 83% sequence identity with human FPR1 and FPR2/ALX,
respectively. It does not bind N-formyl peptides, such as fMLF,
but it responds to some non-formylated chemotactic peptides
identified for FPR2/ALX. Unlike FPR1 and FPR2/ALX, FPR3 is not
expressed in neutrophils but is specifically expressed on mono-
cytes and dendritic cells.12
FPRs interact with a range of structurally different pro- and
anti-inflammatory ligands associated with different diseases,
⇑
Corresponding author. Tel.: +39 055 4573682; fax +39 055 4573780.
E-mail address: mariapaola.giovannoni@unifi.it (M.P. Giovannoni).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.043

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A. Cilibrizzi et al. / Bioorg. Med. Chem. 20 (2012) 3781–3792
including amyloidosis and Alzheimer’s disease,13 some kinds of
cancers and related alopecia induced by most anticancer
agents,14,15 prion disease,16 HIV,17 stomach ulcer,18 and nocicep-
tion associated with inflammatory processes.19 In addition to
endogenous agonists and natural peptides like the bacterial fMLF,
synthetic peptides and small non-peptide FPR ligands have re-
cently been reported.10,20 Additionally, interactions of FPRs with
some ligands have been found to be stereoselective. For example,
lipoxin A4 (LXA4; Fig. 1), the first identified endogenous ligand
for FPR2/ALX (EC50 = 1.0 nM),21 displays stereoselective and highly
potent anti-inflammatory and pro-resolving activity in vivo in
many mammalian systems,3 and changes in potency were associ-
ated with double bond isomerization and alcohol chirality (R or
S).22–24 More recently, researchers from Amgen developed a series
of N-substituted benzimidazoles as potent FPR2 ligands,25 also
displaying stereospecific receptor binding. Among these ligands,
difference in activity between two enantiomers, designated as
compounds A and B (Fig. 1), was noted, being the R-isomer (A) sig-
nificantly more potent than the S-enantiomer (B).
Previously, we identified a large number of potent mixed FPR1/
FPR2 agonists belonging to a series of pyridazin-3(2H)-one deriva-
tives bearing a methyl group at position 6, a methoxybenzyl at
O
H
N
O
HO OH
N
OH
∗
N
N
O
OH
LXA4 (EC50 = 1 nM FPR2)
A [(
B [(
R
S
)-isomer, EC50 = 0.034 μM FPR2]
)-isomer, EC50 = 3.5 μM FPR2]
O
H
O
H
N
Br
N
Br
R
R1
∗
∗
N N
O
N N
O
O
O
( )-5a (EC50 = 3.2 μM FPR1)
5
Figure 1. Structures of selected chiral FPR ligands and the newly designed series.
O
R
O
R
H
N
Br
NH2
HN
R1
O
R1
R1
O
Br
N
N
a
b
R
OH
+
HN
N
Br
O
Br
Br
O
3a-h
1a-h
2
O
4
5a-h
1, 3, 5
R
R1
compd
a
A
CH3
H
( )-b
( )-c
( )-d
( )-e
( )-f
( )-g
h
H
H
H
H
H
C2H5
chiral HPLC
pure enantiomers
(+)-5a-g (-)-5a-g
n
-C3H7
i-C3H7
n-C4H9
C6H5
and
C2H5 CH3
CH3 CH3
a A: 1 and 3a = pure enantiomers; 5a = racemate.
Scheme 1. Synthesis of chiral pyridazin-3(2H)-one derivatives ( )-5a–g and non-chiral analog 5h.

A. Cilibrizzi et al. / Bioorg. Med. Chem. 20 (2012) 3781–3792
3783
Table 1
Conditions for chiral HPLC separations of the racemates ( )-5a–g
Enantiomer
Stationary phasea
Chiralcel OD
Eluent (n-hexane/IPA)
Flow rate (mL/min)
1.2
Temperature (°C)
Retention time (tR, min)b
S-(+)-5a
R-(ꢀ)-5a
S-(+)-5b
R-(ꢀ)-5b
S-(+)-5c
R-(ꢀ)-5c
S-(+)-5d
R-(ꢀ)-5d
S-(+)-5e
R-(ꢀ)-5e
S-(+)-5f
R-(ꢀ)-5f
S-(+)-5g
R-(ꢀ)-5g
95:5
25
19.2
23.2
15.9
17.9
5.1
9.4
3.9
10.1
5.4
9.7
Chiralcel OD
Amylose-2
Amylose-2
Amylose-2
Amylose-2
Amylose-2
95:5
1.2
1.5
1.5
1.5
1.5
1.5
25
40
40
40
40
40
60:40
60:40
60:40
60:40
60:40
12.3
7.6
8.6
6.7
a
Column dimensions were all 250 mm ꢁ 4.6 mm I.D.
b
Elution was monitored by UV absorbance at 250 nm.
Figure 2. Chiral semi-preparative HPLC resolution of racemates ( )-5a–c. Conditions were as follows: Chiralcel OD column (10
95:5 (v/v) as eluent, flow rate 1.2 mL/min, detection by UV at 250 nm, temperature 25 °C. At the bottom it is shown the lack of selectivity when using Chiralcel OD column
under the same conditions to separate racemate ( )-5c.
l
m, 4.6 mm I.D. ꢁ 250 mm), n-hexane/IPA
position 4, and an acetamidic side chain at position 2.26 One of the
most interesting compound in this series was compound ( )-5a26
Fig. 1), which is a FPR1-specific agonist (EC50 = 3.2 M) that
contains a chiral center at the level of the side chain in position
2. Considering the potential importance of stereoselectivity in
FPR-ligand interactions, we evaluated this issue using pure enanti-
omers of racemate ( )-5a (Fig. 1) and a newly synthesized series of
chiral and branched pyridazin-3(2H)-one derivatives (general
structure 5, Fig. 1). These studies led to the identification of novel
mixed FPR1/FPR2 agonists with potent activity for human neutro-
phils. We also found that FPRs exhibited a preference for the R-(ꢀ)-
enantiomers of these compounds and that elongation of the carbon
chain in their chiral center generally increased biological activity.
Thus, in addition to the identification of novel FPR agonists, these
studies provide important new information about molecular
features involved in FPR ligand preference.
(
l

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A. Cilibrizzi et al. / Bioorg. Med. Chem. 20 (2012) 3781–3792
Figure 3. Chiral semi-preparative HPLC resolution of racemates ( )-5c–g. Conditions were as follows: Lux Amylose-2 column (5
60:40 (v/v) as eluent, flow rate 1.5 mL/min, detection by UV at 250 nm, temperature 40 °C.
lm, 4.6 mm I.D. ꢁ 250 mm), n-hexane/IPA
Table 2
relative configuration of the starting carboxylic acids, which was
due to lack of participation of the chiral center in the reaction
and the non-basic reaction conditions. The amides R-(+)-3a and S
-(ꢀ)-3a were then used to alkylate the intermediate 426 (Scheme
1). Previous studies have described alkylation of N-2 on the pyrid-
azinone ring as a classical SN2 reaction,28,29 so this methodology
could be theoretically applied to the synthesis of both enantiomers
(+)-5a and (ꢀ)-5a, with complete inversion of the configuration of
starting amides. Nevertheless, inversion of the configuration was
not complete, and racemization occurred. Thus, we synthesized
the other planned compounds as racemates using the same syn-
thetic procedure reported in Scheme 1 and separated them, to-
gether with the already reported racemate ( )-5a,26 through
semi-preparative chiral HPLC. The synthesis was performed using
the commercially available 4-bromophenyl amides ( )-3b,d, the al-
ready described ( )-3f–h,30–32 and the new ( )-3c,e as intermedi-
ates. Synthesis of ( )-3g required preparation of the starting
bromo-derivative ( )-1g,33 obtained as reported previously by
treating 2-methylbutanoic acid with N-bromosuccinimide in CCl4
at reflux.
Specific rotation and enantiomeric excess determined for purified enantiomers
½ ꢂ (CHCl3, c = 1)
a 2D0
Enantiomer
Enantiomeric excess (%)
S-(+)-5a
R-(ꢀ)-5a
S-(+)-5b
R-(ꢀ)-5b
S-(+)-5c
R-(ꢀ)-5c
S-(+)-5d
R-(ꢀ)-5d
S-(+)-5e
R-(ꢀ)-5e
S-(+)-5f
R-(ꢀ)-5f
S-(+)-5g
R-(ꢀ)-5g
+80°
ꢀ78°
>99.9
94.8
>99.9
96.0
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
98.6
+129°
ꢀ129°
+139°
ꢀ137°
+99°
ꢀ97°
+111°
ꢀ110°
+16°
ꢀ17°
+17°
ꢀ18°
>99.9
2. Chemistry
Initially, we attempted to obtain pure enantiomers (+)-5a and
(ꢀ)-5a of the previously described racemic ( )-5a26 by asymmetric
synthesis (Scheme 1). Commercially available bromopropionic
acids R-(+)-1a and S-(ꢀ)-1a were first treated with SOCl2, resulting
in the intermediate acid chlorides, which in turn were transformed
into amides R-(+)-3a and S-(ꢀ)-3a27 using 4-bromoaniline 2
(Scheme 1). Amide bond formation occurred by retention of the
2.1. Chiral chromatographic resolution of racemates ( )-5a–g
and absolute configuration assignment
Compounds ( )-5a–g (Scheme 1) were separated as pure
enantiomers [enantiomeric excess (ee) = 94.8–99.9%] by semi-

A. Cilibrizzi et al. / Bioorg. Med. Chem. 20 (2012) 3781–3792
3785
Figure 4. Chiral analytical HPLC analysis of the chromatographically resolved enantiomers (+)-5a–g/(ꢀ)-5a–g. Conditions were as follows: Lux Amylose-2 column (3
lm,
4.6 mm I.D. ꢁ 50 mm), n-hexane/IPA 60:40 (v/v) as eluent, flow rate 1.0 mL/min, detection by UV at 250 nm, temperature 40 °C, injection volume of 10
l
L. For analytical
enantioseparations, a standard solution was prepared by dissolving 0.1 mg of each enantiomer into 1 mL of ethanol. The enantiomer excess values (Table 2) were calculated
from relative peak areas. Arrows indicate the percentage of the enantiomeric impurities for (ꢀ)-5a, (+)-5b, and (+)-5g, whereas all the other enantiomers were obtained with a
purity >99.9%.
preparative chiral HPLC using two distinct polysaccharide-based
chiral stationary phases (CSPs) (Table 1). A Chiralcel ODÒ column
[cellulose tris(3,5-dimethylphenylcarbamate)] was used to sepa-
rate the racemates ( )-5a,b, according to a procedure reported in
literature for analogue compounds.34 Since the increasing sterical
hindrance of the chiral center led to a progressive lack of effective-
ness of the CSP (Fig. 2), the racemates ( )-5c–g were separated
using a Lux Amylose-2Ò column [amylose tris(5-chloro-2-methyl-
phenyl-carbamate)] ( Fig. 3). The polarimetric analysis was per-
hindrance of the phenyl group in ( )-5f and the absence of H on
the chiral center in ( )-5g, were the key determinants in this inver-
sion of chromatographic elution.
Since all attempts to obtain crystals suitable for X-ray analysis
to assign the absolute configurations of the pure enantiomers of
( )-5a–g failed, we utilized an approach extensively reported in
the literature, which is based on comparison of the circular dichro-
ism (CD) spectra of the unknown enantiomers with the CD profile
of reference compounds whose absolute configuration has already
been established.36,37 Commercially available N-acetyl-S-(ꢀ)-ala-
formed to establish the specific rotations (½a D20
ꢂ
) of enantiomers
(Table 2), and the enantiomeric excess was determined and re-
ported in Figure 4.
nine [S-(ꢀ)-6], R-(ꢀ)-2-phenylglycine [R-(ꢀ)-7], and S-(ꢀ)-
a-
methylvaline [S-(ꢀ)-9] were chosen as reference molecules, con-
sidering that the substituents at their stereogenic centers are sim-
ilar to those of our compounds (highlighted in red in Figure 5). In
fact, N-acetyl-S-(ꢀ)-alanine [S-(ꢀ)-6], showing a similar sequence
of groups/atoms, can be used as reference for (+)-5a/(ꢀ)-5a in CD
analysis, and the results can be extended to the enantiomeric pairs
(+)-5b–e/(ꢀ)-5b–e. Analogously, we considered R-(ꢀ)-838 and S-
(ꢀ)-10, which are the acetyl derivatives of R-(ꢀ)-2-phenylglycine
Chiral HPLC and polarimetric analysis indicated that for race-
mates ( )-5a–e, the first eluted enantiomers using both columns
rotated polarized light in the positive direction, accordingly to
the similar nature of substituents on the stereogenic center (R = al-
kyl, R1 = H, Scheme 1). Conversely, the elution sequence was re-
versed in the case of racemates ( )-5f,g, where the enantiomers
(ꢀ)-5f,g eluted faster than the corresponding (+)-forms. Clearly,
enantiomer elution order is an important aspect in the study of
enantiorecognition mechanisms.35 In our case, structural differ-
ences on the chiral center between analytes, such as the greater
[R-(ꢀ)-7] and S-(ꢀ)- -methylvaline [S-(ꢀ)-9] (Fig. 5) respectively,
a
as reference compounds for (+)-5f/(ꢀ)-5f and (+)-5g/(ꢀ)-5g. The
acetylation reaction, performed in H2O using acetic anhydride at

3786
A. Cilibrizzi et al. / Bioorg. Med. Chem. 20 (2012) 3781–3792
Br
Br
O
O
O
NH
NH
OH
similar
CD profile
R
HPLC :
same-sense
N N
N N
NH
O
O
O
chiral recognition
mechanism
N-Acetyl-S-(-)-alanine,
S-(-)-6
O
O
(+)-5b, (-)-5b (R = CH2CH3)
(+)-5a/(-)-5a
(+)-5c, (-)-5c (R = CH2CH2CH3)
(+)-5d, (-)-5d (R = CH(CH3)2)
(+)-5e, (-)-5e (R = CH2CH2CH3CH3)
Br
O
NH
O
O
similar
CD profile
OH
OH
N N
a
O
NH
NH2
O
R-(-)-2-Phenylglycine,
R-(-)-7
R-(-)-8
O
(+)-5f/(-)-5f
Br
O
NH
O
O
similar
CD profile
OH
OH
a
N N
O
NH
NH2
O
S-(-)-α-Methylvaline,
S-(-)-9
S-(-)-10
O
(+)-5g/(-)-5g
Figure 5. N-Acetyl-S-(ꢀ)-alanine [S-(ꢀ)-6] and acetyl derivatives of R-(ꢀ)-2-phenylglycine [R-(ꢀ)-7] and S-(ꢀ)-a-methylvaline [S-(ꢀ)-9] used as reference compounds in CD
experiments in comparison to enantiomeric pairs (+)-5a/(ꢀ)-5a, (+)-5f/(ꢀ)-5f, (+)-5g/(ꢀ)-5g due to their comparable substituents (groups/atoms) on the stereogenic center
(highlighted in red). Reagents and conditions: (a) acetic anhydride (7.5 equiv), H2O, 0.5 h, 70 °C.
Figure 6. ORTEP view of R-(ꢀ)-8 and S-(ꢀ)-10 used as reference compounds in CD experiments.

A. Cilibrizzi et al. / Bioorg. Med. Chem. 20 (2012) 3781–3792
3787
Figure 7. Circular dichroism (CD) spectra of the pure enantiomeric pairs (+)-5a/(ꢀ)-5a, (+)-5f/(ꢀ)-5f, (+)-5g/(ꢀ)-5g and reference compounds S-(ꢀ)-6, R-(ꢀ)-8 and S-(ꢀ)-10
(recorded in methanol at 25 °C). In each spectra, the black trace represents the reference compound, whereas solid and dashed red traces correspond to the analyzed (S)- and
(R)-enantiomers, respectively.
Table 3
Biological activity of racemates ( )-5a–g, enantiomers S-(+)-5a–g/R-(ꢀ)-5a–g, and non-chiral analogue 5h
Br
O
NH
N
N
∗
O
R
R1
O
Compound
R
R1
Ca2+ mobilization
FPR1
b-Arrestin recruitment
hPMN
FPR2
FPR1
FPR2
EC50
(
l
M) and efficacya (%)
( )-5a26
S-(+)-5a
R-(ꢀ)-5a
( )-5b
S-(+)-5b
R-(ꢀ)-5b
( )-5c
S-(+)-5c
R-(ꢀ)-5c
( )-5d
S-(+)-5d
R-(ꢀ)-5d
( )-5e
S-(+)-5e
R-(ꢀ)-5e
( )-5f
S-(+)-5f
R-(ꢀ)-5f
( )-5g
S-(+)-5g
R-(ꢀ)-5g
5h
H
CH3
0.8 (95)
4.9 (65)
1.4 (110)
1.8 (145)
2.8 (120)
0.57 (125)
0.3 (120)
NA
0.93 (145)
3.0 (90)
NA
2.3 (105)
0.06 (120)
4.7 (45)
0.073 (135)
4.3 (140)
NA
3.2 (90)
3.2 (70)
8.5 (95)
1.3 (95)
13.4 (115)
2.8 (120)
2.8 (75)
NA
3.0 (85)
2.0 (75)
NA
9.4 (75)
1.1 (110)
20.8 (35)
0.5 (110)
3.1 (80)
NA
NA
16.1 (60)
10.2 (80)
2.2 (95)
22.2 (100)
2.3 (150)
3.6 (85)
NA
0.84 (120)
6.5 (40)
NA
5.4 (105)
0.1 (105)
7.0 (65)
0.089 (120)
1.8 (90)
NA
0.54 (140)
2.1 (80)
NA
12.6 (25)
11.4 (85)
NA
21.2 (20)
H
C2H5
13.8 (80)
3.1 (90)
NA
8.3 (70)
H
n-C3H7
i-C3H7
n-C4H9
C6H5
NA
3.8 (120)
NA
6.8 (100)
H
NA
12.4 (65)
NA
NA
H
15.1 (25)
3.5 (155)
6.1 (60)
3.4 (40)
H
NA
4.0 (95)
NA
2.3 (60)
0.062 (190)
0.5 (100)
1.4 (85)
1.2 (90)
4.7 (70)
0.005
0.94 (110)
1.5 (70)
7.0 (65)
4.5 (70)
6.3 (50)
0.01
CH3
CH3
C2H5
11.7 (65)
11.3 (70)
20.1 (20)
0.007
NA
11.2 (60)
NA
13.7 (110)
20.4 (60)
20.4
CH3
fMLF
12.5
WKYMVm
0.001
0.50
0.001
0.015
0.009
a
NA, no activity (no response was observed during the first 2 min after addition of compounds under investigation) considering the limits of efficacy >20% and EC50
<50 lM. EC50 values are presented as the average of four 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 a 95% confidential interval (p <0.05). For Ca2+ flux assays, efficacy (in
parentheses) is expressed as % of the response induced by 5 nM fMLF (FPR1) or 5 nM WKYMVm (FPR2). For b-arrestin recruitment assays, efficacy is expressed as % of the
response induced by 5 nM WKYMVm (FPR1/FPR2).
70 °C, occurred by complete retention of the absolute configuration
configuration to (+)-5a and the (R)-absolute configuration to (ꢀ)-
5a. The differences in curve profile (Fig. 7) may depend on the dif-
ferent types of chromophores between (+)-5a/(ꢀ)-5a and S-(ꢀ)-6,
which does not contain any aromatic systems. Moreover, due to
the similarity on the chiral centers, the result obtained for S-(+)-
5a/R-(ꢀ)-5a may efficiently be transferred to enantiomeric pairs
(+)-5b–e/(ꢀ)-5b–e (Fig. 5), allowing us to conclude that enantio-
mers of the same sign of optical rotation have the same absolute
configuration.
Likewise, we compared CD spectra of reference compounds R-
(ꢀ)-838 and S-(ꢀ)-10 with the enantiomeric pairs (ꢀ)-5f/(+)-5f
and (ꢀ)-5g/(+)-5g, respectively, and assigned to enantiomers (ꢀ)-
5f/(ꢀ)-5g the (R)-absolute configuration, while to (+)-5f/(+)-5g
as reported in literature39 and confirmed by both polarimatric
(½a 2D0
ꢂ
) and X-ray analysis of R-(ꢀ)-838 and S-(ꢀ)-10 (Fig. 6). Crystals
of R-(ꢀ)-838 and S-(ꢀ)-10 suitable for X-ray experiments were
grown by crystallization from acetonitrile solution, and their spa-
tial coordinates are provided as Supplementary data.
Compounds (+)-5a and (ꢀ)-5a were analyzed in comparison
with N-acetyl-S-(ꢀ)-alanine [S-(ꢀ)-6] ( Fig. 7). The CD spectrum
of the (+)-5a enantiomer displayed a broad positive Cotton effect
around 300 nm and, oppositely, the (ꢀ)-5a enantiomer exhibited
the corresponding mirror-image CD. With the positive Cotton
effect of the N-acetyl-S-(ꢀ)-alanine in the wavelength range be-
tween 215 and 250 nm, we can reasonably assign the (S)-absolute

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A. Cilibrizzi et al. / Bioorg. Med. Chem. 20 (2012) 3781–3792
Figure 9. Analysis of b-arrestin recruitment in Cho-K1 cells treated with enanti-
omers S-(+)-5f and R-(+)-5f. CHO-K1 cells transfected with FPR1 (upper panel) or
FPR2 (lower panel) were treated with the indicated compounds (500 nM) or DMSO
vehicle (negative control) and analyzed for b-arrestin translocation, as described
under Materials and Methods. Representative experiment from three independent
experiments.
Figure 8. Analysis of Ca2+ mobilization in phagocytes treated with enantiomers S-
(+)-5f and R-(+)-5f. HL-60-FPR2 cells (upper panel) or human neutrophils (PMN,
lower panel) were loaded with Fluo-4 AM dye, and Ca2+ flux in response to the
indicated compounds (500 nM) or DMSO vehicle (negative control) was analyzed,
(arrow indicates when treatment was added). Representative experiment from
three independent experiments.
FPR2, and a loss of selectivity for FPR1. Likewise, the R-(ꢀ)-enantio-
mer [R-(ꢀ)-5b] was found to be more potent than S-(+)-5b and
exhibited comparable activity to the racemate ( )-5b. Interest-
ingly, the S-(+)-5c enantiomer was completely devoid of activity,
whereas its enantiomer R-(ꢀ)-5c had high activity in the
was assigned the (S)-form (Fig. 7). In both cases, the bathochromic
shift of the CD curves of (ꢀ)-5f/(+)-5f and (ꢀ)-5g/(+)-5g in compar-
ison with the profiles of R-(ꢀ)-8 and S-(ꢀ)(ꢀ)-10, seemed to de-
pend on the different chromophores of the molecules.
On the basis of combined chromatographic and chiroptical
studies, the absolute configuration assignment was unambigu-
ously completed, and configurations were assigned to the seven
enantiomeric pairs synthesized: S-(+)-5a–g/R-(ꢀ)-5a–g. Due to
the comparable chiral centers in this group of molecules, the re-
sults clearly assigned the (S)-absolute configuration to all dextro-
rotatory enantiomers of the series and the (R)-absolute
configuration to the corresponding levorotatory isomers.
sub-micromolar range (EC50 = 0.84 lM at FPR2) and was even
more active than the racemic mixture of enantiomers [( )-5c].
Similarly, S-(+)-5d was completely inactive, while enantiomer
R-(ꢀ)-5d had an EC50 in the micromolar range for both FPR1 and
FPR2. Elongation of carbon chain on the chiral center resulted in
the n-butyl derivatives ( )-5e which exhibited decreased enantio-
mer preference by the FPR subtypes compared to the 5c and 5d
analogues. On the other hand, the enantiomer R-(ꢀ)-5e was the
most potent FPR1/FPR2 mixed agonist of the series (EC50 ꢃ500
and 90 nM for FPR1 and FPR2, respectively). Moreover, racemic
( )-5e and both enanantiomers R-(ꢀ)-5e and S-(+)-5e were the
only compounds in this series exhibiting preferential activity for
FPR2. Enantiomers of ( )-5f, which have a phenyl group at the
chiral center, again exhibited distinct enantiomer preference by
the receptor, with R-(ꢀ)-5f being highly active, while isomer
S-(+)-5f was completely devoid of activity at either FPR1 or FPR2
(Fig. 8). The introduction of an additional alkyl group to the chiral
center, forming derivatives ( )-5g, resulted in a loss of receptor
affinity, but for S-(+)-5g a FPR1 selectivity was found. Finally, the
non-chiral analogue 5h was found to be a mixed FPR1/FPR2 agonist
with somewhat higher activity for FPR1. Thus, in general for most
compounds, the R-enantiomers were relatively more active than
the S-enantiomers and had similar activities to the racemic
3. Biological results and discussion
3.1. Structure–activity relationship analysis
All newly synthesized compounds were tested as racemates
and pure enantiomers for their ability to induce intracellular
Ca2+ flux in HL-60 cells transfected with FPR1 or FPR2, and the
responses were compared with the activity of fMLF and
WKYMVm (Table 3). To confirm the selectivity for FPRs, all final
compounds were also tested in wild-type non-transfected HL-60
cells and were found to be inactive (data not shown).
Starting from racemic ( )-5a, which we reported previously to
be an FPR1-specific agonist (EC50 = 3.2
isomers R-(ꢀ)-5a and S-(+)-5a showed a similar activity on FPR1/
l
M),26 we found that

A. Cilibrizzi et al. / Bioorg. Med. Chem. 20 (2012) 3781–3792
3789
mixtures. This pattern was further supported by analysis of the
racemates and enantiomers in human neutrophils, which also
exhibited relative selectivity for the R-enantiomers and had similar
enantiomer selectivity as the transfected HL-60 cells (Table 3).
1H NMR spectra were recorded using Avance 400 instruments
(Bruker Biospin Version 002 with SGU). Chemical shifts (d) are re-
ported in ppm to the nearest 0.01 ppm, using the solvent as inter-
nal standard. Coupling constants (J values) are given in Hz and
were calculated using ‘TopSpin 1.3’ software rounded to the near-
est 0.1 Hz. 13C NMR spectra were recorded using Avance 400
instruments (Bruker Biospin Version 002 with SGU). Chemical
shifts (d) are reported in ppm to the nearest 0.1 ppm, using the sol-
vent as internal standard. Mass spectra (m/z) were recorded on an
ESI-TOF mass spectrometer (Bruker Micro TOF), and reported mass
values are within the error limits of 5 ppm mass units. Microanal-
yses were performed with a Perkin-Elmer 260 elemental analyzer
for C, H, N, and the results were within 0.4% of the theoretical val-
ues, unless otherwise stated.
3.2. b-Arrestin recruitment
To confirm the FPR1/FPR2 agonist activity of the enantiomers,
we evaluated the ability of the individual enantiomers to stimulate
b-arrestin recruitment to the activated receptor.40 Because b-arres-
tin recruitment occurs independently of G-protein coupling, this
assay provides a complementary approach to monitor FPR activa-
tion.41 As shown in Table 3, we observed a relatively strong corre-
lation between EC50 values obtained for Ca2+ mobilization and b-
arrestin recruitment. Importantly, no b-arrestin recruitment was
observed in FPR1/FPR2 CHO-K1 cells treated with the S-(+)-enanti-
omers of 5b, 5c, 5d, and 5f, confirming enantiomer preference ob-
served in Ca2+ flux assays. Representative dose–response curves for
S-(+)-5f and R-(ꢀ)-5f are shown in Fig. 9. As positive controls, fMLF
or WKYMVm treatment of FPR1 CHO-K1 cells resulted in dose-
dependent recruitment of b-arrestin, with EC50 values of 5 and
19 nM, respectively, whereas no recruitment was induced by fMLF
in FPR2 CHO-K1 cells (Table 3). Receptor specificity of this cell sys-
tem has also been demonstrated previously with the FPR2-specific
agonist WKYMVM and selective FPR2 inhibitor PBP10.42
Semi-preparative HPLC enantioseparations were performed
using stainless-steel Chiralcel ODÒ (250 mm ꢁ 4.6 mm I.D.,
10 lm particle size, Chiral Technologies Europe, Illkirch, France)
and Lux Amylose-2Ò (250 mm ꢁ 4.6 mm I.D., 5
lm particle size,
Phenomenex, Bologna, Italy) columns. The HPLC consisted of a Per-
kin-Elmer (Norwalk, CT) series 200 comprised of a quaternary
pump, autosampler, Peltier column oven, and UV–VIS detector
coupled with a Biologic BioFrac fraction collector (Bio-Rad, Milan,
Italy). HPLC-grade solvents were supplied by Sigma–Aldrich (Mi-
lan, Italy). The mobile phase mixtures of n-hexane/IPA were de-
gassed by sonication just before use. The signal was acquired and
processed by Totalchrom 6.3.1.0504 software. The values of reten-
tion time (tR) are given in minutes. The analytical HPLC to deter-
mine the enantiomeric excess of the optically pure compounds
was performed on the same instrument using chiral Lux Amy-
4. Conclusions
lose-2Ò (50 mm ꢁ 4.6 mm I.D., 3
lm particle size, Phenomenex,
Overall, we synthesized and characterized a series of chiral
compounds that exhibited high agonist activity for FPR1/FPR2.
Among these compounds, we identified potent mixed FPR1/FPR2
agonists R-(ꢀ)-5b, R-(ꢀ)-5c, R-(ꢀ)-5e, and R-(ꢀ)-5f, which had
agonist activity in the sub-micromolar to nanomolar range in hu-
man neutrophils. The higher selectivity of FPRs for the R-(ꢀ)-enan-
tiomers may be related to the ability of these compounds to
establish a better interaction with the receptors, as compared to
the respective S-(+)-forms. Elongation of the carbon chain at the
chiral center (in R-enantiomers) generally increased biological
activity (CH3 < C2H5 = n-C3H7 < n-C4H9 = C6H5), suggesting the
importance of considering this molecular feature in further
development of this project.
Bologna, Italy) column and mobile phase of n-hexane/IPA 60:40.
In analytical enantioseparations, the sample solutions were pre-
pared from stock solutions of analytes (1 mg/1 mL) by diluting
with ethanol to reach a concentration of 0.1 mg/1 mL. The injection
volume was 10 lL, the flow rate was 1.0 mL/min, the column tem-
perature was 40 °C, and the UV detector wavelength was fixed at
250 nm. Enantiomeric excess values were calculate from peak
areas using the formula (A1ꢀA2)/(A1+A2), where A1 is the major
enantiomer.
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
compound into 2 mL of CHCl3 (c = 1). The system was set at a tem-
perature of 20 °C using a Neslab RTE 740 cryostat.
The CD spectra of enantiomers (dissolved in methanol at con-
centrations ꢃ0.25 mg/mL for analytes and 1 mg/mL for reference
molecules) were measured in a quartz cell (0.1 cm-path length)
under nitrogen atmosphere at 25 °C using a Jasco model J-810
spectropolarimeter (Jasco, Ishikawa-cho, Hachioji City, Tokyo, Ja-
pan). The spectra were averaged over five instrumental scans from
350 to 215 nm at 50 nm/min scanning speed with a spectral band
width of 1 nm and data resolution of 0.2 nm. The spectra were ac-
quired and processed with Spectra Analysis software. The intensi-
ties are presented in terms of ellipticity values (mdeg).
5. Experimental section
5.1. Chemistry
Reagents and starting materials were obtained from commer-
cial sources. Extracts were dried over Na2SO4, and the solvents
were removed under reduced pressure. All reactions were moni-
tored by thin layer chromatography (TLC) using commercial plates
precoated with Merck silica gel 60 F-254. Visualization was
performed by UV fluorescence (kmax = 254 nm) or by staining with
iodine or potassium permanganate. Chromatographic separations
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 chromato-
graphically and spectroscopically pure compounds, unless other-
wise stated. When reactions were performed in anhydrous
conditions, the mixtures were maintained under nitrogen atmo-
sphere. 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. All
melting points were determined on a microscope hot stage Büchi
apparatus and are uncorrected.
5.1.1. General procedure for enantiomers S-(ꢀ)-3a27/R-(+)-3a,
racemates ( )-3b–g and 3h30–32
To a stirred solution of the suitable carboxylic acid 1a–h
(1.31 mmol), in anhydrous CH2Cl2 (5 mL), SOCl2 (7.86 mmol) was
added drop-wise, and the reaction was carried out at 40 °C for
4 h. The mixtures were allowed to cool down, the solvent and
the excess SOCl2 were removed in vacuo, the residues were dis-
solved in anhydrous THF (5 mL), 4-bromoaniline (2.62 mmol)
was added, and the reactions were stirred for an additional 4 h at
room temperature. After removal of the solvent under reduced

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A. Cilibrizzi et al. / Bioorg. Med. Chem. 20 (2012) 3781–3792
pressure, the mixtures were dissolved in CH2Cl2 and washed
sequentially with 2 N HCl (3 ꢁ 15 mL), 2 N NaOH (3 ꢁ 15 mL),
and H2O (15 mL). The organic layer was dried over Na2SO4 and
evaporated under vacuum to give the crude products, which were
purified by flash column chromatography using toluene/ethyl ace-
tate 6:1 as eluent.
J = 7.8 Hz), 7.40 (d, 4H, Ar, J = 4.9 Hz), 8.99 (exch br s, 1H, NH).
MS (ESI) calcd. for C22H22BrN3O3, 456.33; found: m/z 457.11
[M+H]+. Anal. (C22H22BrN3O3) C, H, N.
5.1.3.2.
methyl-6-oxopyridazin-1(6H)-yl]propanamide,
5a. Light-yellow oil.
R-(ꢀ)-N-(4-Bromophenyl)-2-[5-(3-methoxybenzyl)-3-
R-(ꢀ)-
ꢀ 78° (c = 1, CHCl3). ee = 94.8%
½ ꢂ
a 2D0
5.1.1.1. R-(+)-2-Bromo-N-(4-bromophenyl)propanamide, R-(+)-
(determined by analytical chiral HPLC). 1H NMR (CDCl3) d 1.71
(d, 3H, CHCH3, J = 7.0 Hz), 2.30 (s, 3H, 3-CH3), 3.81 (s, 3H, OCH3),
3.91 (s, 2H, CH2-Ar), 5.71 (q, 1H, CHCH3, J = 7.0 Hz), 6.78 (s, 1H,
Ar), 6.81(s, 1H, Ar), 6.84 (d, 2H, Ar, J = 8.3 Hz), 7.28 (t, 1H, Ar,
J = 7.8 Hz), 7.40 (d, 4H, Ar, J = 3.0 Hz), 9.01 (exch br s, 1H, NH).
MS (ESI) calcd. for C22H22BrN3O3, 456.33; found: m/z 457.11
[M+H]+. Anal. (C22H22BrN3O3) C, H, N.
3a.
Yield = 12%. mp = 150–151 °C (EtOH). ½a D20
þ 25 deg (c = 1,
ꢂ
CHCl3). ee >99.9%. 1H NMR (CDCl3) d 1.98 (d, 3H, CHCH3, J = 7.2 Hz),
4.56 (q, 1H, CHCH3, J = 7.0 Hz), 7.45–7.50 (dd, 4H, Ar, J = 6.3 Hz,
J = 2.9 Hz), 8.09 (exch br s, 1H, NH). Anal. (C9H9Br2NO) C, H, N.
5.1.2. General procedure for racemates ( )-5b–g and 5h
Mixtures of compound 4 (0.13 mmol), K2CO3 (0.26 mmol), and
the suitable intermediate ( )-3b–g or 3h (0.14 mmol), in CH3CN
(3 mL) were refluxed under stirring for 4–5 h. After cooling, the sol-
vent was evaporated, and ice-cold water was added to the mix-
tures. For racemate ( )-5b, after 1 h stirring in an ice-bath, the
precipitate was filtered off and purified by crystallization in cyclo-
hexane. For racemates ( )-5c–g and compound 5h, the mixtures
were extracted using CH2Cl2 (3 ꢁ 10 mL). The organic layer was
collected, dried over Na2SO4 and evaporated in vacuo. The crude
residue was purified by flash column chromatography using cyclo-
hexane/ethyl acetate 2:1 as eluent for racemates ( )-5c–e, cyclo-
hexane/ethyl acetate 1:3 as eluent for racemate ( )-5f, and
cyclohexane/ethyl acetate 1:1 as eluent for compounds ( )-5g
and 5h.
5.1.4. HPLC resolution of ( )-5c–g by Chiral HPLC
Racemates were separated by chiral HPLC with a Lux Amylose-
2Ò (250 mm ꢁ 4.6 mm I.D., 5
lm particle size) column. The eluent
mixture n-hexane/IPA 60:40 was used in isocratic mode (1.5 mL/
min at 40 °C), and the UV signal was monitored at k = 250 nm.
Racemates were dissolved in ethanol (2 mg/mL solution), and
100 lL of ( )-5c–f and 80 lL of ( )-5g were injected each time.
In order to separate 20 mg of racemic mixtures, about 100 injec-
tions were needed, requiring about 25 h to obtain the resolved
enantiomeric pairs S-(+)5c/R-(ꢀ)-5c (tR = 5.1, tR = 9.4), S-(+)-5d/R-
(ꢀ)-5d (tR = 3.9, tR = 10.1), S-(+)-5e/R-(ꢀ)-5e (tR = 5.4, tR = 9.7), R-
(ꢀ)-5f/S-(+)-5f (tR = 7.6, tR = 12.3) and R-(ꢀ)-5g/S-(+)-5g (tR = 6.7,
tR = 8.6) (see Sections 2 and 5.1 for assignment of the absolute
configuration).
5.1.2.1.
methyl-6-oxopyridazin-1(6H)-yl]butanamide,
5b.
Yield = 98%. Mp = 79–81 °C (cyclohexane). 1H NMR
( )-N-(4-Bromophenyl)-2-[5-(3-methoxybenzyl)-3-
( )-
5.1.4.1.
S-(+)-N-(4-Bromophenyl)-2-[5-(3-methoxybenzyl)-3-
methyl-6-oxopyridazin-1(6H)-yl]pentanamide,
5c.
S-(+)-
(CDCl3) d 0.95 (t, 3H, CHCH3, J = 7.2 Hz), 2.19–2.38 (m, 5H, 3-CH3
and CH2CH3), 3.81 (s, 3H, OCH3), 3.91 (s, 2H, CH2-Ar), 5.46 (dd,
1H, NCHCH2, J = 1.4 Hz, J = 6.9 Hz), 6.77–6.85 (m, 4H, Ar), 7.27 (t,
1H, Ar, J = 7.9 Hz), 7.30–7.44 (m, 4H, Ar), 9.13 (exch br s, 1H, NH).
13C NMR (CDCl3) d 168.1 (C), 161.4 (C), 159.9 (C), 145.3 (C), 142.1
(C), 138.5 (C), 137.2 (C), 131.5 (2 CH), 130.7 (CH), 129.9 (CH),
121.6 (CH), 121.1 (2 CH), 116.3 (C), 115.3 (CH), 112.0 (CH), 64.5
(CH), 55.2 (CH3), 36.1 (CH2), 22.9 (CH2), 21.2 (CH3), 10.8 (CH3).
MS (ESI) calcd. for C23H24BrN3O3, 470.36; found: m/z 471.12
[M+H]+. Anal. (C23H24BrN3O3) C, H, N.
Light-yellow oil. ½a D20
þ 139° (c = 1, CHCl3). ee >99.9%
ꢂ
(determined by analytical chiral HPLC). 1H NMR (CDCl3) d 0.98 (t,
3H, CHCH3, J = 7.4 Hz), 1.27–1.42 (m, 2H, CH2CH2CH3), 2.15–2.28
(m, 2H, CHCH2CH2), 2.30 (s, 3H, 3-CH3), 3.81 (s, 3H, OCH3), 3.90
(s, 2H, CH2-Ar), 5.56 (t, 1H, NCHCH2, J = 7.4 Hz), 6.75 (s, 1H, Ar),
6.79 (d, 1H, Ar, J = 1.9 Hz), 6.82–6.86 (m, 2H, Ar), 7.28 (t, 1H, Ar,
J = 7.8 Hz), 7.39 (s, 4H, Ar), 9.02 (exch br s, 1H, NH). MS (ESI) calcd.
for
(C24H26BrN3O3) C, H, N.
C
24H26BrN3O3, 484.39; found: m/z 485.14 [M+H]+. Anal.
5.1.4.2.
R-(ꢀ)-N-(4-Bromophenyl)-2-[5-(3-methoxybenzyl)-3-
5.1.3. HPLC resolution of ( )-5a,b by chiral phase HPLC
methyl-6-oxopyridazin-1(6H)-yl]pentanamide,
R-(ꢀ)-
Both racemates were separated by chiral HPLC using a Chiralcel
5c.
Light-yellow oil.
½
a 2D0
ꢂ
ꢀ 137 (c = 1, CHCl3). ee = 99.9%
ODÒ (250 mm ꢁ 4.6 mm I.D., 10
l
m particle size) column. The
(determined by analytical chiral HPLC). 1H NMR (CDCl3) d 0.98 (t,
3H, CHCH3, J = 7.4 Hz), 1.28–1.40 (m, 2H, CH2CH2CH3), 2.17–2.28
(m, 2H, CHCH2CH2), 2.30 (s, 3H, 3-CH3), 3.81 (s, 3H, OCH3), 3.90
(s, 2H, CH2-Ar), 5.56 (t, 1H, NCHCH2, J = 7.4 Hz), 6.75 (s, 1H, Ar),
6.79 (d, 1H, Ar, J = 1.5 Hz), 6.82–6.86 (m, 2H, Ar), 7.28 (t, 1H, Ar,
J = 7.8 Hz), 7.39 (s, 4H, Ar), 9.14 (exch br s, 1H, NH). MS (ESI) calcd.
eluent mixture n-hexane/IPA 95:5 was used in isocratic mode
(1.2 mL/min at 25 °C), and the UV signal was monitored at
k = 250 nm. Racemates were dissolved in ethanol (1 mg/mL solu-
tion), and 50 lL were injected each time. Under the described
conditions, the resolution of ( )-5a,b was lower that 1.5 in both
cases (Fig. 2); therefore, it was necessary to collect the co-eluting
fractions, dry them by evaporation, and reprocess the samples. In
order to separate about 20 mg of racemic mixtures, 600 injections
were needed, requiring ꢃ200 h to obtain the resolved enantiomeric
pairs S-(+)-5a/R-(ꢀ)-5a (tR = 19.2, tR = 23.2) and S-(+)-5b/R-(ꢀ)-5b
(tR = 15.9, tR = 17.9) (see Sections 2 and 5.1 for assignment of the
absolute configuration).
for
C
24H26BrN3O3, 484.39; found: m/z 485.14 [M+H]+. Anal.
(C24H26BrN3O3) C, H, N.
5.1.5. General procedure for R-(ꢀ)-838 and S-(ꢀ)-10
To an aqueous (75 mL) solution of R-(ꢀ)-2-phenylglycine or S-
(ꢀ)-
a-methylvaline (99.33 mmol) respectively, acetic anhydride
(0.75 mol) was added, and the mixture was stirred for 0.5 h at
70 °C until all the amino acid was dissolved. When the reaction
mixture was cooled to 5 °C, the crystallized amide was separated
by filtration, and the final products were purified by recrystalliza-
tion from CH3CN.
5.1.3.1.
methyl-6-oxopyridazin-1(6H)-yl]propanamide,
5a. Light-yellow oil.
S-(+)-N-(4-Bromophenyl)-2-[5-(3-methoxybenzyl)-3-
S-(+)-
½
a 2D0
ꢂ
þ 80° (c = 1, CHCl3). ee >99.9%
(determined by analytical chiral HPLC). 1H NMR (CDCl3) d 1.71
(d, 3H, CHCH3, J = 7.0 Hz), 2.29 (s, 3H, 3-CH3), 3.82 (s, 3H, OCH3),
3.91 (s, 2H, CH2-Ar), 5.71 (q, 1H, CHCH3, J = 7.0 Hz), 6.78 (s, 1H,
Ar), 6.81(s, 1H, Ar), 6.84 (d, 2H, Ar, J = 7.4 Hz), 7.28 (t, 1H, Ar,
5.1.5.1. S -(ꢀ)-2-Acetamido-2,3-dimethylbutanoic acid, S-(ꢀ)-
10.
Yield = 36%. mp = 213–214 °C (EtOH). ½a D20
ꢀ 1:4° (c = 1,
ꢂ
EtOH). ee >99.9%. 1H NMR (DMSO-d6) d 0.84 (d, 3H, CHCH3,

A. Cilibrizzi et al. / Bioorg. Med. Chem. 20 (2012) 3781–3792
3791
J = 6.8 Hz), 0.91 (d, 3H, CHCH3, J = 6.8 Hz), 1.25 (s, 3H, CHCCH3),
O1–HO1
0.883(0.00)
O1...O3
2.610(0.00)
HO1...O3
1.772(0.00)
O1–HO1...O3
157.57(0.00)
1.81 (s, 3H, COCH3), 1.91–1.99 (m, 1H, CCH(CH3)2), 7.07 (exch
br s, 1H, NH), 12.08 (exch br s, 1H, OH). Anal. (C8H15NO3) C,
H, N.
5.1.5.2. Crystallographic data for compounds R-(ꢀ)-838 and S-
5.2. Biological assays
5.2.1. Cell culture
Human promyelocytic leukemia HL-60 cells stably transfected
with FPR1 (HL-60-FPR1) or FPR2 (HL-60-FPR2) were cultured in
RPMI 1640 medium supplemented with 10% heat-inactivated fetal
(ꢀ)-10.
RX-analysis was carried out with a Goniometer Ox-
ford Diffraction KM4 Xcalibur2 at room temperature. Cu/Ka radia-
tion (40 mA/ꢀ40 KV), monochromated by an Oxford Diffraction
Enhance ULTRA assembly, and an Oxford Diffraction Excalibur PX
Ultra CCD were used for cell parameter determination and data
collection. The integrated intensities, measured using the
x scan
calf serum, 10 mM HEPES, 100 lg/mL streptomycin, 100 U/mL
mode, were corrected for Lorentz and polarization effects.43 Direct
methods of SIR200444 were used in solving the structure, which
was refined using the full-matrix least squares on F2 provided by
SHELXL97.45 Multi-scan symmetry-related measurement was used
as the experimental absorption correction type. The non-hydrogen
atoms were refined anisotropically, whereas hydrogen atoms were
refined as isotropic.
penicillin, and G418 (1 mg/mL), as previously described.46 Wild-
type HL-60 cells were cultured under the same conditions, but
without G418.
5.2.2. Isolation of human neutrophils
Blood was collected from healthy donors in accordance with a
protocol approved by the Institutional Review Board at Montana
State University. Neutrophils were purified from the blood using
dextran sedimentation, followed by Histopaque 1077 gradient sep-
aration and hypotonic lysis of red blood cells, as previously de-
scribed.47 Isolated neutrophils were washed twice and
resuspended in HBSS without Ca2+ and Mg2+ (HBSSꢀ). Neutrophil
preparations were routinely >95% pure, as determined by light
microscopy, and >98% viable, as determined by trypan blue
exclusion.
The X-ray CIF files for these structures have been deposited at
the Cambridge Crystallographic Data Centre and allocated with
deposition numbers CCDC 833453 [compound R-(ꢀ)-8] and CCDC
830537 [compound S-(ꢀ)-10]. Copies of the crystallographic data
for this article 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].
5.1.5.3. Crystallographic details for R-(ꢀ)-8.
C10H11NO3,
5.2.3. Ca2+ mobilization assay
M = 193.20, Orthorhombic, space group P 21 21 21, a = 9.196(1),
b = 9.850(1), c = 11.323(1)Å, V = 1025.6(2)Å3, Z = 4 Dc = 1.251,
Changes in intracellular Ca2+ were measured with a FlexStation
II scanning fluorometer using a FLIPR 3 calcium assay kit (Molecu-
lar Devices, Sunnyvale, CA) for human neutrophils and HL-60 cells.
All active compounds were evaluated in parent (wild-type) HL-60
cells to verify that the agonists were inactive in non-transfected
cells. Human neutrophils or HL-60 cells, suspended in HBSSꢀ con-
taining 10 mM HEPES, were loaded with Fluo-4 AM dye (Invitro-
l
= 0.777 mmꢀ1, F(000) = 408. 3364 reflections were collected
with a 5.95 < h < 70.80 range with a completeness to theta 97.3%;
1663 were independent, the parameters were 140 and the final R
index was 0.0415 for reflections having I >2rI, and 0.0632 for all
data. Flack parameter is ꢀ0.1(3) for the exact configuration.
Hydrogen atoms were assigned in calculated positions except
HN1 on N1, HO1 on O1, and H1 on asymmetric carbon C1, which
were found by Fourier analysis. Colorless prismatic shaped crystal
(0.12 ꢁ 0.07 ꢁ 0.06), suitable for collection, was obtained from
CH3CN. In PARST analysis, we found a strong intermolecular hydro-
gen bond between O1—HO1 and O3 in the molecule reported for
(ꢀx+1/2, ꢀy+2, +z+1/2) operation symmetry. It has the following
parameters:
gen) (1.25 lg/mL final concentration) and incubated for 30 min
in the dark at 37 °C. After dye loading, the cells were washed with
HBSSꢀ containing 10 mM HEPES, resuspended in HBSS containing
10 mM HEPES and Ca2+ and Mg2+ (HBSS+), and aliquoted into the
wells of a flat-bottomed, half-area-well black microtiter plates
(2 ꢁ 105 cells/well). The compound source plate contained dilu-
tions of test compounds in HBSS+. Changes in fluorescence were
monitored (kex = 485 nm, kem = 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 normal-
ized 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-FPR2 cells, which were as-
signed a value of 100%. Curve fitting (5–6 points) and calculation
of median effective concentration values (EC50) were performed
by nonlinear regression analysis of the dose–response curves gen-
erated using Prism 5 (GraphPad Software, Inc., San Diego, CA).
O1–H3
0.932(0.04)
O1...O3
2.550(0.00)
HO1...O3
1.622(0.04)
O1–HO1...O3
173.32(3.94)
5.1.5.4. Crystallographic details for S-(ꢀ)-10.
C8H15NO3,
M = 173.21, orthorhombic, space group P 21 21 21, a = 5.934(1),
b = 10.731(1), c = 15.001(1)Å, V = 955.2(2)Å3, Z = 4 Dc = 1.204,
l
= 0.760 mmꢀ1, F(000) = 376. 2985 reflections were collected with
a 5.07 < h < 69.93 range with a completeness to theta 94.6%;
1517 were independent, the parameters were 121, and the final
R index was 0.0352 for reflections having I >2rI, and 0.0402 for
5.2.4. b-Arrestin recruitment assay
The PathHunterÒ eXpress b-arrestin recruitment assay was per-
formed according to the manufacturer’s protocol using CHO-K1
FPR1 and CHO-K1 FPR2 cells (DiscoveRx Corporation, Fremont,
CA). These cell lines monitor GPCR activity by detecting the inter-
action of b-arrestin with the activated GPCR using b-galactosidase
(b-gal) enzyme fragment complementation. Assay plates [96-well
half area plates with clear bottom (Greiner Bio-One, Monroe,
all data. Flack parameter is 0.02(3) for the exact configuration.
Hydrogen atoms were assigned in calculated positions except
HN1 on N1, HO1 on O1, and H4 on C4, which were found by Fourier
analysis. Colorless cubic shaped crystals (0.08 ꢁ 0.07 ꢁ 0.05), suit-
able for collection, were obtained from CH3CN. Also in this case we
found, in PARST analysis, a strong intermolecular hydrogen bond
between O1—HO1 and O3 in the molecule reported for (ꢀx+1,
+yꢀ1/2, ꢀz+1/2) operation symmetry. It has the following
parameters:
NC)] were prepared with 5000 cells/well in 50
Optimized Cell Culture Medium. After incubation at 37 °C (5%
lL of DiscoveRx

3792
A. Cilibrizzi et al. / Bioorg. Med. Chem. 20 (2012) 3781–3792
12. Migeotte, I.; Riboldi, E.; Franssen, J. D.; Grégoire, F.; Loison, C.; Wittamer, V.;
CO2, 95% relative humidity) for 48 h, 5.5 lL of test compound was
added, and the incubation was continued at 37 °C for 90 min.
Detection reagent (25 L) was added, and the incubation was con-
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monitored using a Fluoroskan Ascent FL microtiter plate reader
(Thermo Fisher Scientific, Waltham, MA). Maximum change in
luminescence, expressed in arbitrary units over baseline, was used
to determine agonist response. Responses were normalized to the
response induced by 5 nM WKYMVm for both CHO-FPR1 and CHO-
FPR2, which was assigned a value of 100%. Curve fitting (5–6
points) and calculation of median effective concentration values
(EC50) were performed by nonlinear regression analysis of the
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The authors wish to thank Dr. Cristina Faggi for X-ray structure
analysis of compounds R-(ꢀ)-8 and S-(ꢀ)-10 and Dr. Leonardo
Gonnelli (CERM, Magnetic Resonance Center, University of Flor-
ence) for providing the spectropolarimeter for CD analysis. This
work was supported in part by National Institutes of Health grants
RR020185 and GM103500, an equipment grant from the M.J. Mur-
dock Charitable Trust, and the Montana State University Agricul-
tural Experimental Station.
Supplementary data
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Chemical and physical characteristics and spectral data for all
the remaining new intermediates ( )-3c,e, racemates ( )-5c–g,
enantiomers S-(+)-5b–g/R-(ꢀ)-5b–g and for non-chiral analogue
5h. Elemental analysis for all new compounds. Experimental de-
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