2-Arylacetamido-4-phenylamino-5-substituted pyridazinones as formyl peptide receptors agonists

Article abstract of DOI:10.1016/j.bmc.2016.04.019

N-Formyl peptide receptors (FPRs: FPR1, FPR2, and FPR3) are G protein-coupled receptors that play key roles in modulating immune cells. FPRs represent potentially important therapeutic targets for the development of drugs that could enhance endogenous ant

Full text of DOI:10.1016/j.bmc.2016.04.019

Accepted Manuscript
2-Arylacetamido-4-Phenylamino-5-Substituted Pyridazinones as Formyl Pep-
tide Receptors Agonists
Claudia Vergelli, Igor A. Schepetkin, Giovanna Ciciani, Agostino Cilibrizzi,
Letizia Crocetti, Maria Paola Giovannoni, Gabriella Guerrini, Antonella
Iacovone, Liliya N. Kirpotina, Andrei I. Khlebnikov, Richard D. Ye, Mark T.
Quinn
PII:
S0968-0896(16)30253-X
http://dx.doi.org/10.1016/j.bmc.2016.04.019
BMC 12934
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 January 2016
1 April 2016
8 April 2016
Please cite this article as: Vergelli, C., Schepetkin, I.A., Ciciani, G., Cilibrizzi, A., Crocetti, L., Giovannoni, M.P.,
Guerrini, G., Iacovone, A., Kirpotina, L.N., Khlebnikov, A.I., Ye, R.D., Quinn, M.T., 2-Arylacetamido-4-
Phenylamino-5-Substituted Pyridazinones as Formyl Peptide Receptors Agonists, Bioorganic & Medicinal
Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.04.019
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2-Arylacetamido-4-Phenylamino-5-Substituted Pyridazinones as Formyl Peptide
Receptors Agonists
Claudia Vergelli,[a] Igor A. Schepetkin,[b] Giovanna Ciciani,[a] Agostino Cilibrizzi,[c] Letizia
Crocetti,[a] Maria Paola Giovannoni,[a]* Gabriella Guerrini,[a] Antonella Iacovone,[a] Liliya N.
Kirpotina,[b] Andrei I. Khlebnikov,[d] Richard D. Ye,[e] and Mark T. Quinn,[b]
aNEUROFARBA, Sezione di Farmaceutica e Nutraceutica, Università degli Studi di Firenze, Via
Ugo Schiff 6, 50019 Sesto Fiorentino, Italy.
bDepartment of Microbiology and Immunology, Montana State University, Bozeman, MT 59717,
USA.
cDepartment of Chemistry, Imperial College London, South Kensington, London SW7 2AZ, UK.
dDepartment of Biotechnology and Organic Chemistry, Tomsk Polytechnic University, Tomsk
634050, Russia & Department of Chemistry, Altai State Technical University, Barnaul, Russia.
eInstitute of Chinese Medical Sciences, University of Macau, Macau SAR, China
* corresponding authors:
Maria Paola Giovannoni
Dipartimento di NEUROFARBA
Via Ugo Schiff 6
Sesto Fiorentino 50019 Firenze
Tel +39-055-4573682
E-mail mariapaola.giovannoni@unifi.it
Key-words: formyl peptide receptor (FPR), agonist, pyridazin-3(2H)-one, neutrophil, Ca2+
mobilization
1

Abstract
N-Formyl peptide receptors (FPRs: FPR1, FPR2, and FPR3) are G protein-coupled receptors that
play key roles in modulating immune cells. FPRs represent potentially important therapeutic targets
for the development of drugs that could enhance endogenous anti-inflammation systems associated
with various pathologies, thereby reducing the progression of inflammatory conditions. Previously,
we identified 2-arylacetamide pyridazin-3(2H)-ones as FPR1- or FPR2-selective agonists, as well as
a large number of FPR1/FPR2-dual agonists and several mixed-agonists for the three FPR isoforms.
Here, we report a new series of 2-arylacetamido-4-aniline pyridazin-3(2H)-ones substituted in
position 5 as a further development of these FPR agonists. Chemical manipulation presented in this
work resulted in mixed FPR agonists 8a, 13a and 27b, which had EC50 values in nanomolar range.
In particular, compound 8a showed a preference for FPR1 (EC50 = 45 nM), while 13a and 27b
showed a moderate preference for FPR2 (EC50 = 35 and 61 nM, respectively). Thus, these
compounds may represent valuable tools for studying FPR activation and signaling.
2

1. Introduction
Human formyl peptide receptors (FPR1, FPR2, and FPR3) are a family of versatile G-
protein-coupled receptors (GPCRs) that represent attractive therapeutic targets because of their
involvement in a wide range of normal physiological processes, as well as pathological events
associated with inflammatory conditions [1-4]. Originally identified in phagocytic leucocytes, FPRs
mediates chemotaxis and activation of the majority of immune system cells in response to bacterial
products and various inflammatory stimuli [3]. However, recent studies indicate that FPRs are also
expressed in a variety of nonhematopoietic cells, such as lung epithelial cells, platelets, osteoblasts,
and hepatocytes, suggesting a wider role for FPRs beyond inflammation and host defense [5].
FPR activation can induce pro- or anti-inflammatory responses, depending on the nature of
the ligand and cell types involved. For example, FPRs have been reported to contribute to
inflammation associated with amyloidosis and Alzheimer's disease, prion disease, human
immunodeficiency virus, stomach ulcer, some cancers, nociception associated with inflammatory
processes, chronic obstructive pulmonary disease (COPD), stroke and ischemia-reperfusion injury
[6-13]. Conversely, stimulation of FPRs with certain agonists can also induce pro-resolving
responses or endogenous anti-inflammatory systems [3]. Indeed, screening of commercial libraries
and new synthetic compounds has resulted in the discovery of a number of small-molecule non-
peptide FPR agonists and antagonists with a wide range of chemical diversity and activities [14-16].
In previous studies, we identified several pyridazin-3(2H)-one-based derivatives that
showed an interesting profile as FPR agonists, combining an appreciable potency and differential
selectivity toward the three human FPR isoforms [17-21]. Key requirements for agonist activity of
this class of compounds were the presence of a 4-bromophenylacetamide side chain at the N-2
position of the scaffold [17, 19] and the presence of a methyl group at C-6 [20]. Position 4 could be
substituted with a benzyl or phenylamino group, resulting in compounds with micromolar activity
(Figure 1, general structure A).
3

Figure 1
O
X
NH
N
Br
N
O
X = NH, CH2
FPR1/FPR2 = 2.3-10.8M
(m, p)
O
A
In the present study, we further investigated pyridazinone derivatives belonging to the series
of 4-phenylamino derivatives (structure A, X = NH) which until now was only poorly studied [20].
In particular, we inserted at position 5 of the pyridazinone scaffold a variety of substituents, such as
alkyl or acyl groups, ester, unsaturated chains, and pyrazole rings, in order to evaluate how such
modifications affected target specificity and compound potency.
2. Chemistry
All compounds were synthesized as reported in Schemes 1-5, and the structures were
confirmed using analytical and spectral data.
The synthetic pathway leading to the final compounds 6a-f bearing an acyl group at position
5 is outlined in Scheme 1. Previously described isoxazolo-pyridazinones of type 1 [22-25] were
transformed into the corresponding 4-amino-5-acetyl derivatives 2a-f (2a-d [25]) by reductive
cleavage with 10% Pd/C and HCOONH4 in ethanol. The products were then converted to the 4-
bromophenylacetamide derivatives 5a-f as follows. Intermediates 2a and 2d,e were alkylated with
ethylbromoacetate under standard conditions to generate 3a [20] and 3d,e, which were transformed
into the corresponding carboxylic acids 4a [22] and 4d,e through alkaline hydrolysis. These were
transformed into the final amides of type 5 by treatment with 4-bromoaniline, ethyl chloroformate
4

and triethylamine in THF. Compounds 5b,c and 5f were obtained starting from their respective
intermediates by a direct alkylation with N-(4-bromophenyl)-2-chloroacetamide [26] under standard
conditions. Finally, a coupling reaction between 5a-f and 3-methoxybenzenboronic acid was
carried out in the presence of copper(II)acetate and Et3N to generate the final 5-alkyl pyridazinones
6a-f.
Scheme 1
O
O
HN N
N N
HN N
i
ii
O
O
O
for 2a,d,e
N
O
H2N
R
H2N
R
R
O
O
3a,d,e
2a-f
1a-f
v
for 2b,c,f
iii
Br
O
OH
1-6
R
O
NH
N N
a
b
c
d
e
f
CH3
iv
O
C2H5
C3H7
C4H9
cC5H9
cC6H11
N N
O
H2N
R
O
H2N
R
4a,d,e
O
5a-f
Br
vi
O
NH
N N
O
O
NH
R
O
6a-f
Reagents and conditions: i) 10% Pd/C, HCOONH4, anhydrous EtOH, reflux, 1 h; ii) ethyl bromoacetate, K2CO3,
anhydrous CH3CN, reflux, 2 h; iii) 6N NaOH, EtOH, 60 °C, 1 h; iv) Et3N, anhydrous THF, ethyl chloroformate, 4-
bromoaniline, -5 °C, rt and then 17.5 h; v) N-(4-bromophenyl)-2-chloroacetamide, K2CO3, anhydrous CH3CN,
reflux, 2 h; vi) 3-methoxybenzenboronic acid, (CH3COO)2Cu, Et3N, anhydrous CH2Cl2, rt, 16 h.
Synthetic routes used to obtain the 5-alkyl (11a-d) and 5-vinyl (13) derivatives are shown in
Scheme 2. Intermediates 2a-d [25] were converted to the desired final compounds 11a-d through
5

the following steps: reduction of the acetyl at C-5 with NaBH4 (compounds 7a-d), dehydratation
with polyphosphoric acid (PPA) (8a-d), reduction of the vinyl group with a Parr instrument (9a-d),
insertion of the fragment at N-2 (10a-d), and coupling with 3-methoxybenzeneboronic acid (11a-d).
To obtain the final compound 13, direct alkylation with N-(4-bromophenyl)-2-chloroacetamide [26]
on intermediate 8a was performed (compound 12), followed by a coupling reaction with 3-
methoxybenzenboronic acid.
Scheme 2
HN N
HN N
HN N
i
ii
O
O
O
H2N
R
H2N
H2N
R
R'
O
O
HO
8a-d
2a-d
7a-d
iii
for 8a
iv
O
Br
NH
2, 7
R
HN N
iv
N N
O
a
b
c
CH3
NH
Br
O
C2H5
C3H7
C4H9
H2N
N N
H2N
R'
d
O
R'
9a-d
10a-d
H2N
v
12
v
8-11
R'
O
Br
O
NH
a
b
c
H
NH
Br
CH3
C2H5
C3H7
N N
N N
O
d
O
HN
HN
R'
O
O
11a-d
13
Reagents and conditions: i) NaBH4, CH3OH, rt, 1 h; ii) PPA, reflux, 5 h; iii)10% Pd/C, anhydrous EtOH, H2, Parr, 30
PSI, 3 h; iv) N-(4-bromophenyl)-2-chloroacetamide, K2CO3, anhydrous CH3CN, reflux, 2-6 h; v) 3-
methoxybenzenboronic acid, (CH3COO)2Cu, Et3N, anhydrous CH2Cl2, rt, 16 h.
6

Scheme 3 shows the synthetic pathway for compounds 17a-b and 18: intermediate 14 [27]
was converted into the pyridazinone 15 through isoxazole ring cleavage using the appropriate
alcohol and a catalytic amount of Et3N. Compounds of type 15 were then transformed into the final
17a,b following the same procedure reported in Schemes 1 and 2. Moreover, basic hydrolysis of
the ester led to the final carboxylic derivative 18.
Scheme 3
O
NH
Br
N N
HN N
ii
HN N
i
O
O
O
R
R
H2N
O
H2N
O
N
O
O
O
16a,b
15a,b
14
iii
O
O
H
NH
Br
N
Br
N N
N N
O
O
iv
O
O
R
NH
O
NH
OH
O
O
17a,b
18
15-17
R
a
CH3
b
C2H5
Reagents and conditions: i) Et3N, MeOH or EtOH, 60 °C, 4 h; ii) N-(4-bromophenyl)-2-chloroacetamide, K2CO3,
anhydrous CH3CN, reflux, 2-4 h; iii) 3-methoxybenzenboronic acid, (CH3COO)2Cu, Et3N, anhydrous CH2Cl2, rt, 16 h;
iv) 2N NaOH, EtOH, rt, 1 h.
The pyrazolyl derivatives 25a,b were obtained following the procedures outlined in
Scheme 4. Compound 19 [22] was transformed into the intermediated 20, as previously reported,
and then was condensed with N,N-dimethylformamide dimethyl acetal to obtain derivative 21.
7

Treatment with Mo(CO)6 [28] led to 22. Condensation with hydrazine hydrate gave the C-5
pyrazolyl pyridazinone 23 which, in turn, was alkylated with iodomethane (compound 24) and
coupled with 3-methoxybenzenboronic acid (25a,b).
Scheme 4
O
O
O
OH
NH
Br
ii
NH
Br
N N
N N
N N
i
O
O
O
N
N
N
N
O
O
O
21
19
20
iii
O
O
O
NH
Br
NH
Br
iv
NH
Br
v
N N
N N
N N
O
O
O
H2N
H2N
N
H2N
N
N
O
N
N
22
23
H
24
vi
O
NH
Br
N N
O
25 OCH3
m
p
a
b
NH
O
N
N
25a,b
Reagents and conditions: i) Et3N, anhydrous THF, ethyl chloroformate, 4-bromoaniline, -5 °C and then rt, 17.5 h; ii)
DMF/DMA, 90°C, 3 h; iii) Mo(CO)6, H2O, CH3CN, reflux, 2h; iv) N2H4 . H2O, EtOH, reflux, 3 h; v) CH3I, K2CO3,
anhydrous DMF, 90°C, 3 h; vi) 3- or 4-methozybenzenboronic acid, (CH3COO)2Cu, Et3N, anhydrous CH2Cl2, rt, 16 h.
8

Finally Scheme 5 describes the synthetic pathway leading to the final 5-unsubstituted
pyridinone and pyridazinone derivatives 28a-b, which were obtained starting from appropriate
intermediates 26a [29] and 26b [30] following the usual synthetic procedures described in previous
schemes.
Scheme 5
O
O
NH
Br
NH
N X
Br
HN X
NH2
N X
i
ii
O
O
O
NH
NH2
OCH3
27a,b
26a,b
28a,b
26-28
X
a
b
C
N
Reagents and conditions: i) N-(4-bromophenyl)-2-chloroacetamide, K2CO3, anhydrous CH3CN, reflux, 6 h; ii) 3-
methoxybenzenboronic acid, (CH3COO)2Cu, Et3N, anhydrous CH2Cl2, rt, 16 h.
3. Results and discussion
3.1. Biological results
All new compounds were evaluated for their ability to induce intracellular Ca2+ flux in
human HL-60 cells transfected with FPR1, FPR2, or FPR3, and the results are reported as EC50
values in Tables 1-2.
Analysis of compounds substituted with various groups at position 5 (Table 1) demonstrated
that several were potent mixed FPR agonists. Among them, the acetyl derivative 8a was active in
the nanomolar range and preferred FPR1 (EC50 = 45 nM). On the other hand, elongation of the
9

aliphatic chain of keto(alkyl) derivatives (compounds 8b-d) was detrimental for FPR agonist
activity. Although the butyl analogue 8d was selective for FPR1, it had only moderate activity
(EC50 = 15.6 µM). Further modification at position 5, such as the introduction of cyclopentyl and
cyclohexyl rings (8e-f), also led to decreased activity.
Replacement of the ketone at C-5 (Table 1) with a pyrazole resulted in two mixed FPR
agonists with reasonable agonist activity (27a-b). These two agonists differ only in the position of
the methoxy group of the aniline at C-4 (meta for 27a versus para for 27b). Compound 27b was a
potent FPR2 agonist (EC50 = 35 nM), although it did have some activity at the other two FPR
subtypes (FPR2>FPR3>>FPR1). Elimination of the methoxyphenylamino group at C-4 of the
pyridazinone (compound 26) led to decreased activity that was comparable to that of the other two
4-amino derivatives 24 and 25. Likewise, introduction of methyl or ethyl esters at C-5 of
pyridazinone (19a-b) also led to compounds with micromolar activity. On the other hand,
introduction of a carboxylic function in the same position (20) led to a slight increase of selectivity
toward FPR1 (EC50 = 0.6 M). Finally, the vinyl derivative 15 exhibited mixed agonist activity for
the three FPR isoforms but had a higher preference for FPR1 and FPR2 (EC50 = 0.23 and 0.11 M,
respectively).
Activities of the 5-alkyl derivatives 13a-d are presented in Table 2. Compound 13a, in
which R = C2H5, was the most potent of this series. It was active in the nanomolar range toward the
three FPR subtypes but had a preference for FPR2 (EC50 = 61 nM). Elongation of the aliphatic
chain was detrimental for activity, resulting in compounds with micromolar EC50 values for FPR1
and FPR2 and no activity at FPR3. Surprisingly, the 5-propyl derivative 13b was completely
devoid of activity. This is likely due to the loss of H-bonding between this compound and the
receptor (see molecular modeling details below). Furthermore, elimination of the C-5 substituent
led to a compound 29a with high nanomolar agonist activity for FPR1 (EC50 = 0.24 M). Finally,
compound 29b exhibited moderate mixed agonist activity for FPR1 and FPR2, but was one order of
10

magnitude lower in activity than 29a at FPR1, suggesting that the pyridonic scaffold was less
appropriate compared with the pyridazinone scaffold.
Table 1. Activity of C-4 and C-5 substituted pyridazinones 8a-f, 15, 19a-b, 20, 24-26, 27a-b.
O
O
NH
N
Br
NH
N
Br
N
N
O
O
NH
R
H2N
R
O
24-26
8a-f, 15, 19a,b, 20, 27a,b
OCH3
m
R
hPMN[a]
FPR1-HL60[a]
FPR2-HL60[a]
FPR3-HL60[a]
Compd
8a
0.036 ± 0.007
(150)
0.045 ± 0.016
(185)
0.17 ± 0.038
(60)
0.21 ± 0.044
(150)
COCH3
m
m
m
m
m
m
m
m
m
-
COCH2CH3
CO(CH2)2CH3
CO(CH2)3CH3
COcC5H9
5.5 ± 1.6 (110)
24.0 ± 4.2 (60)
8.0 ± 2.6 (90)
1.7 ± 0.4 (110)
28.9 ± 6.6 (120)
2.7 ± 0.8 (130)
11.8 ± 3.5 (70)
15.6 ± 2.6 (45)
1.6 ± 0.4 (110)
10.0 ± 3.2 (110)
0.78 ± 0.1 (190)
8.1 ± 2.1 (90)
N.A. a
N.A. a
N.A. a
N.A. a
N.A. a
N.A. a
8b
8c
8d
8e
10.5 ± 2.1 (60)
8.7 ± 0.47 (115)
0.11 ±0.014 (105)
2.3 ± 0.49 (60)
1.9 ± 0.03 (110)
3.1 ± 0.78 (115)
5.7 ± 2.3 (50)
13.5 ± 1.8 (60)
1.9 ± 0.71 (40)
0.59 ± 0.18 (90)
0.035 ± 0.1 (120)
COcC6H11
8f
CH=CH2
0.84 ± 0.18 (130)
22.1 ± 5.3 (25)
6.1 ± 1.7 (95)
0.23 ± 0.07 (120)
4.4 ± 0.6 (25)
6.0 ± 2.1 (75)
N.A. a
15
COOCH3
19a
19b
20
COOCH2CH3
COOH
2.5 ± 0.7 (90)
N.A. a
1.5 ± 0.5 (155)
9.3 ± 2.3 (105)
11.8 ± 2.4 (140)
3.5 ± 0.32 (60)
0.59 ± 0.21 (135)
0.6 ± 0.1 (130)
5.1 ± 1.7 (120)
8.4 ± 1.5 (135)
2.9 ± 0.14 (75)
3.6 ± 0.28 (130)
4.0 ± 0.89 (115)
17.1 ± 4.3 (75)
N.A. a
COCHCHN(CH3)2
pyrazole
24
-
N.A. a
25
-
1-methylpyrazole
1-methylpyrazole
N.A.
N.A. a
26
m
27a
p
1-methylpyrazole 0.30 ± 0.044 (130)
0.67 ± 0.22 (110)
27b
[a]Values, expressed as EC50 (µM) and Efficacy (% in brackets) were evaluated in a Ca2+ flux assay. EC50 values
represent the average mean of three independent experiments and were determined by nonlinear regression analysis of
the concentration-response curves (5-6 points) generated using GraphPad Prism 5 with 95% confidential interval (p <
0.05). Efficacy (in brackets) is expressed as % of the response induced by 5 nM fMLF in human polymorphonuclear
neutrophils (hPMN) and FPR1-HL60 cells or by 5 nM WKYMVm in FPR2-HL60 and FPR3-HL60 cells. [b]N.A., no
activity (no response was observed during first 2 min after addition of compounds under investigation) considering the
limits of efficacy < 20% and EC50 < 50 μM.
11

Table 2. Activity of C-4 and C-5 substituted pyridazinones 13a-d and 29a-b.
O
O
NH
N
Br
NH
Br
N
N
H3CO
H3CO
O
O
NH
R
NH
29b
13a-d, 29a
R
hPMN[a]
FPR1-HL60[a]
0.18 ± 0.004 (185)
N.A.
FPR2-HL60[a]
FPR3-HL60[a]
0.46 ± 0.014 (35)
N.A.
Compd
13a
CH2CH3
CH2CH2CH3
(CH2)3CH3
(CH2)4CH3
H
0.34 ± 0.11 (115)
N.A.
0.061 ± 0.022 (35)
N.A.
13b
1.40 ± 0.8 (45)
5.70 ± 1.2 (55)
0.56 ± 0.12 (85)
4.31 ± 0.4 (60)
3.6 ± 1.1 (65)
1.4 ± 0.34 (95)
0.24 ± 0.09 (120)
2.50 ± 0.7 (110)
4.5 ± 1.3 (30)
0.19 ± 0.018 (115)
9.6 ± 2.0 (65)
8.10 ± 1.7 (75)
N.A.
13c
N.A.
13d
N.A.
29a
-
N.A.
29b
a]Values, expressed as EC50 (µM) and Efficacy (% in brackets) were evaluated in Ca2+ flux assay. EC50
[
values represent the average mean of three independent experiments and were determined by nonlinear
regression analysis of the concentration-response curves (5-6 points) generated using GraphPad Prism 5 with
95% confidential interval (p < 0.05). Efficacy (in brackets) is expressed as % of the response induced by 5
nM fMLF in human polymorphonuclear neutrophils (hPMN) and FPR1-HL60 cells or by 5 nM WKYMVm
in FPR2-HL60 and FPR3-HL60 cells. [b]N.A., no activity (no response was observed during first 2 min after
addition of compounds under investigation) considering the limits of efficacy < 20% and EC50 < 50 μM.
The most active derivatives (8a, 15 and 27a-b) were also evaluated for chemoattractant
activity in human neutrophils. As expected for FPR agonists, all four compounds had
chemoattractant activity and induced this response in the micromolar range (Table 3).
Table 3. Chemoattractant activity of selected pyridazinones in human neutrophils
EC50 (µM)[a] in
Compd
migration assay
2.2 ± 0.51
1.2 ± 0.24
1.1 ± 0.22
0.45 ± 0.17
8a
15
27a
27b
[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 concentration-
response curves (5-6 points) generated using GraphPad Prism 5 with 95% confidential interval (p < 0.05).
Some of the synthesized compounds were also evaluated for their ability to induce
intracellular Ca2+ flux in mouse neutrophils and RBL cells transfected with Fpr1 or Fpr2 (Table 4).
12

Although all compounds tested were active in human neutrophils, only eight of these compounds
activated Ca2+ flux in mouse neutrophils. Of these, three compounds were also agonists for mouse
Fpr1, mouse Fpr2, or both. The reason for this species-specific difference in activity is currently not
understood; however, we and other group have observed quite different response patterns to some
agonists and/or antagonists in human and mouse neutrophils [14].
Table 4. Activity of pyridazinones in mouse neutrophils and mouse Fpr-transfected RBL cells.
mPMN[a]
mFpr1-RBL[a]
mFpr2-RBL[a]
Compd
N.A.[b]
8.4 ± 2.6 (75)
N.A.[b]
24.1 ± 6.1 (105)
15.9 ± 4.9 (120)
N.A.[b]
N.A.[b]
N.A.[b]
N.A.[b]
N.A.[b]
N.A.[b]
N.A.[b]
N.A.[b]
N.A.[b]
N.A.[b]
N.A.[b]
N.A.[b]
8a
8b
8c
8d
8e
8f
15
19a
19b
20
N.A.[b]
N.A.[b]
N.A.[b]
N.A.[b]
22.8 ± 6.4 (30)
N.A.[b]
N.A.[b]
N.A.[b]
N.A.[b]
N.A.[b]
1.0 ± 0.34 (100) 4.7 ± 1.3 (50)
N.A.[b]
13.5 ± 3.7 (50)
6.7 ± 2.1 (55)
9.4 ± 2.7 (65)
24
25
26
27a
27b
N.A.[b]
N.A.[b]
N.A.[b]
5.6 ± 2.4 (45)
N.A.[b]
6.0 ± 1.9 (80)
N.A.[b]
N.A.[b]
N.A.[b]
N.A.[b]
18.5 ± 4.1 (25) 18.3 ± 3.8 (35)
[a]The EC50 (µM) and Efficacy (% in brackets) were evaluated in Ca2+ flux assay. Values are expressed EC50 presented
as the average mean of three independent experiments, in which EC50 values were determined by nonlinear regression
analysis of the concentration-response curves (5-6 points) generated using GraphPad Prism 5 with 95% confidential
interval (p < 0.05). Efficacy (in brackets) is expressed as % of the response induced by 5 nM WKYMVm in mouse
polymorphonuclear neutrophils (mPMN) or RBL cells transfected with mouse Fpr1 (mFpr1-RBL) or Fpr2 (mFpr2-
RBL). [b]N.A., no activity (no response was observed during first 2 min after addition of compounds under
investigation) considering the limits of efficacy < 20% and EC50 < 50 µM.
3.2. Molecular docking studies
Our data suggest that larger substituents at position 5 may cause steric hindrance and that the
optimal length of this group was two carbon atoms. To evaluate the role of steric hindrance from
bulky acyl groups, we performed molecular docking of compounds 8a and 8e into the binding site
of an FPR1 homology model and compounds 13a-c into the binding site of an FPR2 homology
model. With each of these pairs, the compounds differ in size of acyl or alkyl groups at position 5
of the pyridazine heterocycle.
13

As shown in Figure 2A, the best docking pose of 8a occupies areas of FPR1 characteristic of
other FPR1 agonists [31]. For example, the p-bromo substituted aromatic ring of 8a is positioned
near channel A, and the acetyl group enters cavity B (receptor regions designated as previously
reported [31]). Additionally, strong H-bonds form between the carbonyl oxygen and anilide
nitrogen atoms of 8a and Thr265 of FPR1, whereas a weaker H-bond is formed between the
methoxy substituent and Arg205 (Figure 2B). These interactions may contribute to the agonist
activity of compound 8a. Close contact of the acetyl group in 8a and FPR1 Leu198 was observed
for the calculated ligand-receptor complex (Figure 2B), and the shortest interatomic distance
between the acetyl oxygen and hydrogen atom of Leu198 located at the wall of cavity C was ~2.7
Å, indicating close proximity of the agonist 8a and the receptor. Thus, a larger acyl group at
position 5 of the heterocycle would be expected to hinder ligand orientation similar to 8a. Indeed,
the lowest-energy pose of compound 8e had a very different arrangement within the FPR1 binding
site. The bulky cyclopentyl substituent prevented positioning the molecule near cavity B, such that
8e leans toward hole C of FPR1 (Figure 2A). This difference in binding explains the decreased
agonist activity for 8e and its analogs with large acyl groups.
14

Figure 2
A homology model of FPR2 was similarly used to perform molecular docking with 13a-c.
These molecules have quite subtle differences in structure and are oriented in the binding site with
brominated benzene rings directed deep into the binding site (Figure 3A). This orientation of the p-
bromophenyl moiety is analogous to that observed previously for parent compounds [20]. Agonist
13a with an ethyl substituent at position 5 of the pyridazine ring is H-bonded with Thr177, while
the m-methoxyphenyl substituent occupies a hydrophobic subpocket surrounded by Ala181,
Gly264, Leu268, Tyr277, and Ile279 (Figure 3B). Changing the alkyl substituent from ethyl to n-
butyl in compound 13c led to flipping of the molecule so that the butyl chain of this ligand was now
located in the hydrophobic subpocket, while the m-methoxyphenyl group formed an H-bond with
Asn171 (Figure 3C). For 13b, a flipped pose similar to 13c was obtained. However, the substituted
15

pyridazine 13b did not form H-bonds with the receptor, which likely led to decreased affinity of the
propyl derivative and complete lack of biological activity (see Table 2).
Figure 3
16

Thus, larger substituents in position 5 of the pyridazine heterocycle cause steric effects on
the binding modes of the ligands in the FPR1 and FPR2 binding sites, which is consistent with their
reduced or lost agonist activity.
4. Conclusions
We describe the synthesis of new series of C-5 substituted 2-arylacetamide pyridazin-3(2H)-
ones that exhibit improved potency and selectivity toward FPR isoforms. Biological analysis of
these compounds confirmed the suitability of pyridazinone-based compounds as a relevant system
to develop novel human FPR agonists. Indeed, the majority of compounds described herein were
mixed FPR agonists, with compounds 8a and 27b being the most potent (EC50 values in the
nanomolar range). Overall, we show that modification of position C-5 of the pyridazinone ring in
2-arylacetamide pyridazin-3(2H)-ones represents an effective approach for the development of
active FPR agonists. These compounds represent valuable tools for studying FPR activation and
signaling in inflammatory conditions.
5. Experimental section
5.1. Chemistry
Reagents and starting materials were obtained from commercial sources. Extracts were dried
over Na2SO4, and the solvents were removed under reduced pressure. All reactions were monitored
by thin layer chromatography (TLC) using commercial plates precoated with Merck silica gel 60 F-
254. Visualization was performed by UV fluorescence (λmax = 254 nm) or by staining with iodine or
potassium permanganate. Chromatographic separations were performed on a silica gel column using
gravity chromatography (Kieselgel 40, 0.063-0.200 mm; Merck), flash chromatography (Kieselgel
40, 0.040-0.063 mm; Merck), or silica gel preparative TLC (Kieselgel 60 F254, 20 x 20 cm, 2 mm).
Yields refer to chromatographically and spectroscopically pure compounds, unless otherwise stated.
17

Compounds were named following IUPAC rules, as applied by Beilstein-Institut AutoNom 2000
(4.01.305) or CA Index Name. All melting points were determined on a microscope hot stage Büchi
apparatus and are uncorrected. The identity and purity of intermediates and final compounds were
1
determined through NMR analysis and TLC chromatography. H NMR, 13C NMR and NOESY
spectra were recorded with Avance 400 instruments (Bruker Biospin Version 002 with SGU).
1
13
Chemical shifts (δ) are reported in ppm to the nearest 0.01 ppm (for H NMR) or 0.1 ppm (for C
1
NMR), using the solvent as an internal standard. Coupling constants (J values) of H NMR are
given in Hz and were calculated using ‘TopSpin 1.3’ software rounded to the nearest 0.1 Hz.
Microanalyses were performed with a Perkin-Elmer 260 elemental analyzer for C, H, and N, and the
results were within ± 0.4 % of the theoretical values, unless otherwise stated.
5.1.1. General procedures for 2e and 2f
Ammonium formate (3.11 mmol) and 10% Pd/C (0.05 mmol) were added to a solution of the
appropriate intermediate 1e and 1f [22-24] (1.04 mmol) in 5 mL of anhydrous EtOH, and the
reaction was refluxed for 1 h. After cooling, 20 mL of CH2Cl2 were added to the mixture, and the
precipitate was removed by filtration under vacuum. The organic layer was then evaporated, and the
desired products 2e,f were obtained pure after recrystallization from EtOH.
5.1.1.1. 4-Amino-5-cyclopentanecarbonyl-6-methylpyridazin-3(2H)-one, 2e. Yield = 98%; mp =
225-227 °C (EtOH). 1H-NMR (CDCl3) δ 1.62-1.72 (m, 2H, cC5H9); 1.73-1.81 (m, 2H, cC5H9); 1.83-
1.89 (m, 4H, cC5H9); 2.45 (s, 3H, CH3); 3.44 (quin, 1H, cC5H9, J = 7.6 Hz); 6.76 (exch br s, 2H,
NH2); 7.98 (exch br s, 1H, NH). Anal. Calcd for C11H15N3O2 (221.26): C, 59.71; H, 6.83; N, 18.99;
Found: C, 59.52; H, 6.84; N, 18.95.
5.1.1.2. 4-Amino-5-cyclohexanecarbonyl-6-methylpyridazin-3(2H)-one, 2f. Yield = 96%; mp =
1
232-234 °C (EtOH). H-NMR (CDCl3) δ 1.20-1.40 (m, 2H, cC6H11); 1.40-1.60 (m, 2H, cC6H11);
1.60-1.80 (m, 2H, cC6H11); 1.80-1.90 (m, 4H, cC6H11); 2.53 (s, 3H, CH3); 2.90-2.93 (m, 1H,
18

cC6H11); ); 7.21 (exch br s, 2H, NH2); 8.56 (exch br s, 1H, NH). Anal. Calcd for C12H17N3O2
(235.28): C, 61.26; H, 7.28; N, 17.86; Found: C, 61.11; H, 7.27; N, 17.89.
5.1.2. General procedures for 3d and 3e
To a solution of the appropriate substrate 2d [25] and 2e (1.60 mmol) in anhydrous CH3CN
(5 mL), K2CO3 (3.20 mmol) and ethyl bromoacetate (2.40 mol) were added. The solution was
stirred for 2-3 h at reflux, and the solvent was evaporated. The residue was mixed with ice-cold
water (10 mL) and the precipitate was recovered by suction and recrystallized from ethanol
(compound 3d); alternatively, for compound 3e, the suspension was extracted with CH2Cl2 (3 x 15
mL) and the organic layer was dried over Na2SO4 and evaporated in vacuo. Finally, 3e was purified
by column flash chromatography using cyclohexane/ethyl acetate 1:1 as eluent.
5.1.2.1. (5-Amino-3-methyl-6-oxo-4-pentanoylpyridazin-1(6H)-yl)acetic acid ethyl ester, 3d.
1
Yield = 97%; mp = 92-94 °C (EtOH). H-NMR (CDCl3) δ 0.94 (t, 3H, CH2CH2CH3, J = 7.2 Hz);
1.29 (t, 3H, OCH2CH3, J = 7.2 Hz); 1.36 (sext, 2H, CH2CH2CH3, J = 7.2 Hz); 1.68 (quin, 2H,
COCH2CH2, J = 7.2 Hz); 2.48 (s, 3H, N=CCH3); 2.82 (t, 2H, COCH2CH2, J = 7.2 Hz); 4.24 (q, 2H,
OCH2CH3, J = 7.2 Hz); 4.81 (s, 2H, NCH2); 8.89 (exch br s, 2H, NH2). Anal. Calcd for C14H21N3O4
(295.33): C, 56.94; H, 7.17; N, 14.23; Found: C, 57.08; H, 7.16; N, 14.27.
5.1.2.2. (5-Amino-4-cyclopentanecarbonyl-3-methyl-6-oxopyridazin-1(6H)-yl)acetic acid ethyl
1
ester, 3e. Yield = 64%; mp = 83-85 °C (EtOH). H-NMR (CDCl3) δ 1.29 (t, 3H, CH2CH3, J = 7.2
Hz); 1.61-1.65 (m, 2H, cC5H9); 1.74-1.79 (m, 2H, cC5H9); 1.80-1.89 (m, 4H, cC5H9); 2.43 (s, 3H,
N=CCH3); 3.45 (m, 1H, cC5H9); 4.24 (q, 2H, CH2CH3, J = 7.2 Hz); 4.81 (s, 2H, NCH2); 6.87 (exch
br s, 2H, NH2). Anal. Calcd for C15H21N3O4 (307.34): C, 58.62; H, 6.89; N, 13.67; Found: C, 58.47;
H, 6.88; N, 13.71.
5.1.3. General procedures for 4d and 4e
To a solution of the suitable ester 3d and 3e (0.49-0.58 mmol) in 96% EtOH (5 mL), 6N
NaOH (3 mL) was added. The reaction was carried out at 60°C for 1-2 h. After evaporation of the
solvent, the mixture was diluted with ice-cold water, acidified with 6N HCl, and extracted with
19

CH2Cl2 (3 x 15 mL). The organic layer was dried over Na2SO4 and evaporated in vacuo to give
desired final compounds, which were purified by crystallization from ethanol.
5.1.3.1. (5-Amino-3-methyl-6-oxo-4-pentanoylpyridazin-1(6H)-yl)acetic acid, 4d. Yield = 98%;
1
mp = 97-99 °C (EtOH). H-NMR (CDCl3) δ 0.93 (t, 3H, CH2CH3, J = 7.2 Hz); 1.36 (sext, 2H,
CH2CH3, J = 7.2 Hz); 1.66 (quin, 2H, COCH2CH2, J = 7.2 Hz); 2.48 (s, 3H, N=CCH3); 2.81 (t, 2H,
COCH2CH2, J = 7.2 Hz); 4.87 (s, 2H, NCH2); 5.32 (exch br s, 1H, OH); 7.45 (exch s br, 2H, NH2).
Anal. Calcd for C12H17N3O4 (267.28): C, 53.92; H, 6.41; N, 15.72; Found: C, 54.08; H, 6.40; N,
15.68.
5.1.3.2. (5-Amino-4-cyclopentanecarbonyl-3-methyl-6-oxopyridazin-1(6H)-yl)acetic acid, 4e.
1
Yield = 73%; mp = 168-169 °C (EtOH). H-NMR (CDCl3) δ 1.60-1.70 (m, 2H, cC5H9); 1.71-1.80
(m, 2H, cC5H9); 1.81-1.91 (m, 4H, cC5H9); 2.45 (s, 3H, CH3); 3.45 (m, 1H, cC5H9); 4.88 (s, 2H,
NCH2); 5.03 (exch br s, 1H, OH); 6.89 (exch br s, 2H, NH2). Anal. Calcd for C13H17N3O4 (279.29):
C, 55.91; H, 6.14; N, 15.05; Found: C, 55.76; H, 6.13; N, 15.09.
5.1.4. General procedures for 5a, and 5d-e
To a cooled (-5 °C) and stirred solution of the appropriate substrate 4a, 4d-e (4a [22]) (0.98
mmol) in anhydrous tetrahydrofuran (5 mL), Et3N (3.43 mmol) was added. After 0.5 h, the mixture
was allowed to warm up to 0 °C, and ethyl chloroformate (1.02 mmol) was added. After 1 h, 4-
bromoaniline (1.96 mmol) was added. The reaction was carried out at room temperature for 16 h.
The mixture was then concentrated in vacuo, diluted with cold water (20-30 mL), and extracted with
CH2Cl2 (3 x 15 mL). The organic layer was dried over Na2SO4 and evaporated to obtain crude final
compounds, which were purified by crystallization from ethanol for compound 5a and by column
flash chromatography using cyclohexane/ethyl acetate 1:1 as eluent, followed by recrystallization
from EtOH, for compounds 5d and 5e.
5.1.4.1.
2-(4-Acetyl-5-amino-3-methyl-6-oxopyridazin-1(6H)-yl)-N-(4-bromophenyl)
acetamide, 5a. Yield = 90%; mp = 240-242 °C (EtOH). 1H-NMR (CDCl3) δ 2.57 (s, 3H, COCH3);
2.60 (s, 3H, N=CCH3); 4.90 (s, 2H, NCH2); 7.54 (s, 4H, Ar); 8.50 (exch br s, 1H, NH); 6.32 (exch
20

br s, 2H, NH2). Anal. Calcd for C15H15BrN4O3 (379.21): C, 47.51; H, 3.99; N, 14.77; Found: C,
47.64; H, 3.98; N, 14.80.
5.1.4.2.
2-(5-Amino-3-methyl-6-oxo-4-pentanoylpyridazin-1(6H)-yl)-N-(4-bromophenyl)
acetamide, 5d. Yield = 42%; mp = 164-165 °C (EtOH). 1H-NMR (CDCl3) δ 0.93 (t, 3H, CH2CH3, J
= 7.2 Hz); 1.35 (sext, 2H, CH2CH3, J =7.2 Hz); 1.67 (quin, 2H, COCH2CH2, J = 7.2 Hz); 2.51 (s,
3H, N=CCH3); 2.82 (t, 2H, COCH2CH2, J = 7.2 Hz); 4.88 (s, 2H, NCH2); 7.38 (s, 4H, Ar); 8.63
(exch br s, 1H, NH); 8.56 (exch br s, 2H, NH2). Anal. Calcd for C18H21BrN4O3 (421.29): C, 51.32;
H, 5.02; N, 13.30; Found: C, 51.46; H, 5.02; N, 13.27.
5.1.4.3.
2-(5-Amino-4-cyclopentanecarbonyl-3-methyl-6-oxopyridazin-1(6H)-yl)-N-(4-
1
bromophenyl)acetamide, 5e. Yield = 19%; mp = 179-181 °C (EtOH). H-NMR (CDCl3) δ 1.61-
1.71 (m, 2H, cC5H9); 1.72-1.82 (m, 2H, cC5H9); 1.83-1.92 (m, 4H, cC5H9); 2.48 (s, 3H, CH3); 3.45
(m, 1H, cC5H9); 4.89 (s, 2H, NCH2); 6.89 (exch br s, 2H, NH2); 7.40 (s, 4H, Ar); 8.66 (exch br s,
1H, NH). Anal. Calcd for C19H21BrN4O3 (433.30): C, 52.67; H, 4.89; N, 12.93; Found: C, 52.49; H,
4.88; N, 12.91.
5.1.5. General procedures for 5b,c, 5f
A mixture of the appropriate intermediate 2b-c or 2f (0.96 mmol), K2CO3 (1.93 mmol) and
N-(4-bromophenyl)-2-chloroacetamide [26] (0.96-1.20 mmol) in CH3CN (10-15 mL) was refluxed
under stirring for 2-6 h. The mixture was then concentrated in vacuo, and ice cold water was added.
After 1 h stirring in an ice-bath, the precipitate was recovered by suction to obtain pure compound
5b-c. For compound 5f further purification was performed by column chromatography using
cyclohexane/ethyl acetate 1:2 as eluent.
5.1.5.1.
2-(5-Amino-3-methyl-6-oxo-4-propionylpyridazin-1(6H)-yl)-N-(4-bromophenyl)
acetamide, 5b. Yield = 98%; mp = 174-175 °C (EtOH). 1H-NMR (CDCl3) δ 1.22 (t, 3H, CH2CH3, J
= 7.2 Hz); 2.56 (s, 3H, N=CCH3); 2.88 (q, 2H, CH2CH3, J = 7.2 Hz); 4.90 (s, 2H, NCH2); 7.43 (s,
4H, Ar); 8.55 (exch br s, 1H, NH); 8.76 (exch br s, 2H, NH2). Anal. Calcd for C16H17BrN4O3
(393.24): C, 48.87; H, 4.36; N, 14.25; Found: C, 48.99; H, 4.35; N, 14.21.
21

5.1.5.2.
2-(5-Amino-4-butyryl-3-methyl-6-oxopyridazin-1(6H)-yl)-N-(4-bromophenyl)
acetamide, 5c. Yield = 96%; mp = 161-163 °C (EtOH). 1H-NMR (CDCl3) δ 0.99 (t, 3H, CH2CH3, J
= 7.2 Hz); 1.75 (sext, 2H, CH2CH3, J = 7.2 Hz); 2.54 (s, 3H, N=CCH3); 2.83 (t, 2H, COCH2, J = 7.2
Hz); 4.90 (s, 2H, NCH2); 7.43 (s, 4H, Ar); 8.57 (exch br s, 1H, NH); 9.04 (exch br s, 2H, NH2).
Anal. Calcd for C17H19BrN4O3 (407.26): C, 50.14; H, 4.70; N, 13.76; Found: C, 50.05; H, 4.69; N,
13.73.
5.1.5.3.
2-(5-Amino-4-cyclohexanecarbonyl-3-methyl-6-oxopyridazin-1(6H)-yl)-N-(4-
bromophenyl) acetamide, 5f. Yield = 53%; mp = 97-99 °C (EtOH). 1H-NMR (CDCl3) δ 1.20-1.40
(m, 2H, cC6H11); 1.40-1.60 (m, 2H, cC6H11); 1.60-1.80 (m, 2H, cC6H11); 1.80-1.90 (m, 4H, cC6H11);
2.44 (s, 3H, CH3); 2.91-2.98 (m, 1H, cC6H11); 4.89 (s, 2H, NCH2); 6.73 (exch br s, 2H, NH2); 7.38
(s, 4H, Ar); 8.70 (exch br s, 1H, NH). Anal. Calcd for C20H23BrN4O3 (447.33): C, 53.70; H, 5.18;
N, 12.52; Found: C, 53.85; H, 5.17; N, 12.56.
5.1.6. General procedures for 6a-f
To a suspension of the appropriate intermediate 5a-f (0.91 mmol), copper(II) acetate (1.36
mmol), 3-methoxyphenylboronic acid (0.91-1.82 mmol) in CH2Cl2 (10 mL), and Et3N (1.82 mmol)
were added, and the mixture was stirred at room temperature for 16 h. The suspension was extracted
with 15% aqueous ammonia (3 x 10 mL), and the organic layer was washed with water (10 mL) and
dried over Na2SO4. After removal of the solvent in vacuo, the final desired compounds were
purified by column flash chromatography using as eluent CH2Cl2/MeOH 98:2, for compounds 6b-f
and cycloexane/ethyl acetate 1:3 for compound 6a. Compounds 6d and 6e were further purified by
recrystallization from cyclohexane.
5.1.6.1.
2-[4-Acetyl-5-(3-methoxyphenylamino)-3-methyl-6-oxopyridazin-1(6H)-yl]-N-(4-
bromophenyl)acetamide, 6a. Yield = 41%; mp = 115-116 °C (EtOH). 1H-NMR (CDCl3) δ 1.92 (s,
3H, COCH3); 2.24 (s, 3H, N=C-CH3); 3.80 (s, 3H, OCH3); 4.97 (s, 2H, NCH2); 6.61 (s, 1H, Ar);
6.68 (d, 1H, Ar, J = 8.4 Hz); 6.74 (d, 1H, Ar, J = 8.0 Hz); 7.24 (m, 1H, Ar); 7.42-7.48 (m, 4H, Ar);
8.5 (exch br s, 1H, NH); 9.21 (exch br s, 1H, NH). 13C-NMR (CDCl3) δ 19.6 (CH3); 31.2 (CH3);
22

55.3 (CH3); 57.0 (CH2); 108.1 (CH); 112.0 (CH); 115.9 (CH); 116.6 (C); 117.0 (C); 121.4 (2CH);
130.5 (CH); 131.8 (2CH); 136.7 (C); 137.7 (C); 139.6 (C); 144.3 (C); 146.6 (C); 157.6 (C); 160.4
(C); 164.9 (C). Anal. Calcd for C22H21BrN4O4 (485.33): C, 54.44; H, 4.36; N, 11.54; Found: C,
54.57; H, 4.35; N, 11.57.
5.1.6.2.
N-(4-Bromophenyl)-2-[5-(3-methoxyphenylamino)-3-methyl-6-oxo-4-propionyl
pyridazin-1(6H)-yl]acetamide, 6b. Yield = 10%; mp = 87-89 °C (EtOH). 1H-NMR (CDCl3) δ 0.57
(t, 3H, CH2CH3, J = 7.2 Hz); 2.19 (s, 3H, N=CCH3); 2.28 (q, 2H, CH2CH3, J = 7.2 Hz); 3.81 (s, 3H,
OCH3); 4.97 (s, 2H, NCH2); 6.60 (s, 1H, Ar); 6.67 (d, 1H, Ar, J = 8.4 Hz); 6.73 (d, 1H, Ar, J = 8.4
Hz); 7.24 (t, 1H, Ar, J = 8.4 Hz); 7.45 (s, 4H, Ar); 7.69 (exch br s, 1H, NH); 8.61 (exch br s, 1H,
NH). 13C-NMR (CDCl3) δ 14.1 (CH3); 20.3 (CH3); 37.6 (CH2); 55.4 (CH3); 57.5 (CH2); 108.5 (CH);
112.3 (CH); 115.3 (CH); 116.5 (C); 117.0 (C); 121.5 (2 x CH); 130.5 (CH); 132.0 (2 x CH); 136.6
(C); 137.4 (C); 139.7 (C); 144.6 (C); 146.5 (C); 157.6 (C); 160.6 (C); 165.1 (C). Anal. Calcd for
C23H23BrN4O4 (499.36): C, 55.32; H, 4.64; N, 11.22; Found: C, 55.47; H, 4.63; N, 11.18.
5.1.6.3.
N-(4-Bromophenyl)-2-[4-butyryl-5-(3-methoxyphenylamino)-3-methyl-6-oxo
1
pyridazin-1(6H)-yl]acetamide, 6c. Yield = 11%; mp = 113-115 °C (EtOH). H-NMR (CDCl3) δ
0.67 (t, 3H, CH2CH3, J = 7.2 Hz); 1.09 (sext, 2H, CH2CH3, J = 7.2 Hz); 2.19 (s, 3H, N=CCH3); 2.22
(t, 2H, COCH2, J = 7.2 Hz); 3.80 (s, 3H, OCH3); 4.97 (s, 2H, NCH2); 6.60 (s, 1H, Ar); 6.67 (d, 1H,
Ar, J = 8.0 Hz); 6.73 (d, 1H, Ar, J = 8.0 Hz); 7.23 (t, 1H, Ar, J = 8.0 Hz); 7.42-7.47 (m, 4H, Ar);
7.69 (exch br s, 1H, NH); 8.57 (exch br s, 1H, NH). 13C-NMR (CDCl3) δ 13.5 (CH3); 16.3 (CH2);
20.3 (CH3); 46.2 (CH2); 55.4 (CH3); 57.5 (CH2); 108.3 (CH); 112.3 (CH); 115.1 (CH); 116.6 (C);
117.0 (C); 121.5 (2 x CH); 130.5 (CH); 132.0 (2 x CH); 136.6 (C); 137.3 (C); 139.7 (2 x C); 144.6
(C); 157.6 (C); 160.5 (C); 165.0 (C). Anal. Calcd for C24H25BrN4O4 (513.38): C, 56.15; H, 4.91; N,
10.91; Found: C, 56.63; H, 4.90; N, 10.88.
5.1.6.4.
N-(4-Bromophenyl)-2-[5-(3-methoxyphenylamino)-3-methyl-6-oxo-4-pentanoyl
1
pyridazin-1(6H)-yl]acetamide, 6d. Yield = 13%; mp = 96-98 °C (cyclohexane). H-NMR (CDCl3)
δ 0.73 (t, 3H, CH2CH3, J = 7.2 Hz); 0.93-1.06 (m, 4H, (CH2)2CH3); 2.17 (s, 3H, N=CCH3); 2.22 (t,
23

2H, COCH2CH2, J = 7.2 Hz); 3.78 (s, 3H, OCH3); 4.95 (s, 2H, NCH2); 6.58 (s,1H, Ar); 6.64 (dd,
1H, Ar, J = 8.4 Hz, J = 2.4 Hz); 6.71 (dd, 1H, Ar, J = 8.4 Hz, J = 2.4 Hz); 7.12 (t, 1H, Ar, J = 8.4
Hz); 7.44 (s, 4H, Ar); 7.66 (exch br s, 1H, NH); 8.56 (exch br s, 1H, NH). 13C-NMR (CDCl3) δ 13.0
(CH3); 17.5 (CH2); 20.1 (CH3); 24.7 (CH2); 44.1 (CH2); 55.4 (CH3); 57.5 (CH2); 108.3 (CH); 112.3
(CH); 115.1 (CH); 116.6 (C); 117.1 (C); 121.4 (2 x CH); 129.8 (CH); 132.0 (2 x CH); 136.5 (C);
137.3 (C); 139.6 (C); 144.6 (C); 146.5 (C); 154.6 (C); 160.6 (C); 164.1 (C). Anal. Calcd for
C25H27BrN4O4 (527.41): C, 56.93; H, 5.16; N, 10.62; Found: C, 57.03; H, 5.15; N, 10.57.
5.1.6.5. N-(4-Bromophenyl)-2-[4-cyclopentanecarbonyl-5-(3-methoxyphenylamino)-3-methyl-
1
6-oxopyridazin-1(6H)-yl]acetamide, 6e. Yield = 11%; mp = 140-142 °C (cyclohexane). H-NMR
(CDCl3) δ 1.13-1.22 (m, 2H, cC5H9); 1.29-1.36 (m, 2H, cC5H9); 1.37-1.43 (m, 2H, cC5H9); 1.53 (m,
2H, cC5H9); 2.19 (s, 3H, N=CCH3); 3.04 (quin, 1H, cC5H9, J = 7.6 Hz); 3.87 (s, 3H, OCH3); 4.97 (s,
2H, NCH2); 6.56 (s, 1H, Ar); 6.67 (dd, 1H, Ar, J = 8.4 Hz, J = 2.4 Hz); 6.71 (dd, 1H, Ar, J = 8.4 Hz,
J = 2.4 Hz); 7.22 (t, 1H, Ar, J = 8.4 Hz); 7.44-7.47 (m, 4H, Ar); 8.54 (exch br s, 1H, NH); 9.98
13
(exch br s, 1H, NH). C-NMR (CDCl3) δ 20.9 (CH3); 22.7 (CH2); 25.8 (CH2); 28.3 (CH2); 31.9
(CH2); 52.2 (CH); 55.4 (CH3); 57.6 (CH2); 107.5 (CH); 112.0 (CH); 114.6 (CH); 116.6 (C); 119.0
(C); 121.5 (2 x CH); 130.5 (CH); 132.0 (2 x CH); 136.6 (C); 137.5 (C); 138.5 (C); 145.2 (C); 147.3
(C); 157.8 (C); 160.4 (C); 165.3 (C). Anal. Calcd for C26H27BrN4O4 (539.42): C, 57.89; H, 5.05; N,
10.39; Found: C, 57.70; H, 5.05; N, 10.42.
5.1.6.6. N-(4-Bromophenyl)-2-[4-cyclohexanecarbonyl-5-(3-methoxyphenylamino)-3-methyl-
1
6-oxopyridazin-1(6)-yl]acetamide, 6f. Yield = 34%; mp = 181-183 °C (EtOH). H-NMR (CDCl3)
δ 0.80-1.00 (m, 8H, cC6H11); 1.40-1.60 (m, 2H, cC6H11); 2.00 (s, 3H, N=CCH3); 2.46-2.50 (m, 1H,
cC6H11); 3.68 (s, 3H, OCH3); 4.87 (s, 2H, NCH2); 6.60-6.62 (m, 3H, Ar); 7.12-7.17 (m, 1H, Ar);
7.56-7.47 (m, 4H, Ar); 8.82 (exch br s, 1H, NH); 10.44 (exch br s, 1H, NH). 13C-NMR (CDCl3) δ
20.6 (CH3); 25.8 (2 x CH2); 27.6 (2 x CH2); 29.5 (CH2); 51.3 (CH); 55.5 (CH3); 55.6 (CH2); 108.2
(CH); 111.1 (CH); 115.2 (CH); 115.6 (C); 115.8 (C); 121.6 (2 x CH); 130.3 (CH); 132.1 (2 x CH);
24

138.1 (C); 138.5 (C); 141.6 (2 x C); 142.6 (C); 157.1 (C); 160.3 (C); 165.7 (C). Anal. Calcd for
C27H29BrN4O4 (553.45): C, 58.59; H, 5.28; N, 10.12; Found: C, 58.40; H, 5.27; N, 10.09.
5.1.7. General procedures for 7a-d
To a cooled (0 °C) suspension of compounds 2a-d (1.20 mmol) in 5 mL of MeOH, NaBH4
(3.59-7.18 mmol) was slowly added, and the mixture was stirred for 1 h at room temperature. After
cooling, cold water was added (10 mL) and compound 7a was recovered by filtration under vacuum.
For compounds 7b-d the suspension was extracted with ethyl acetate (3 x 10 mL) and the organic
layer was dried over Na2SO4 and evaporated to obtain crude final compounds, which were purified
by crystallization from ethanol.
5.1.7.1. 4-Amino-5-(1-hydroxyethyl)-6-methylpyridazin-3(2H)-one, 7a. Yield = 49%; mp = >300
°C (MeOH). 1H-NMR (DMSO-d6) δ 1.28 (d, 3H, CHCH3, J = 6.4 Hz); 2.09 (s, 3H, N=CCH3); 4.78
(q, 1H, CH, J = 6.4 Hz); 5.98 (exch br s, 1H, NH); 8.56 (exch br s, 2H, NH2). Anal. Calcd for
C7H11N3O2 (169.18): C, 49.70; H, 6.55; N, 24.84; Found: C, 49.85; H, 6.56; N, 24.79.
5.1.7.2. 4-Amino-5-(1-hydroxypropyl)-6-methylpyridazin-3(2H)-one, 7b. Yield = 74%; mp =
1
184-189 °C (EtOH). H-NMR (DMSO-d6) δ 0.86 (t, 3H, CH2CH3, J = 7.2 Hz); 1.56-1.61 (m, 1H,
CH2CH3); 1.65-1.70 (m, 1H, CH2CH3); 2.09 (s, 3H, N=CCH3); 4.49-4.54 (m, 1H, CHOH); 8.56
(exch br s, 2H, NH2); 12.26 (exch br s, 1H, NH). Anal. Calcd for C8H13N3O2 (183.21): C, 52.45; H,
7.15; N, 22.94; Found: C, 52.57; H, 7.17; N, 22.89.
5.1.7.3. 4-Amino-5-(1-hydroxybutyl)-6-methylpyridazin-3(2H)-one, 7c. Yield = 72%; mp = 212-
1
215 °C (EtOH). H-NMR (DMSO-d6) δ 0.86 (t, 3H, CH2CH3, J = 7.2 Hz); 1.24-1.30 (m, 2H,
CH2CH2CH3); 1.40-1.49 (m, 2H, CH2CH2CH3); 2.07 (s, 3H, N=CCH3); 4.60 (m, 1H, CHOH); 8.92
(exch br s, 2H, NH2); 12.26 (exch br s, 1H, NH). Anal. Calcd for C9H15N3O2 (197.23): C, 54.81; H,
7.67; N, 21.30; Found: C, 54.66; H, 7.69; N, 21.36.
5.1.7.4. 4-Amino-5-(1-hydroxypentyl)-6-methylpyridazin-3(2H)-one, 7d. Yield = 74%; mp =
1
206-208 °C (EtOH). H-NMR (DMSO-d6) δ 0.85 (t, 3H, CH2CH3, J = 7.2 Hz); 1.18-1.30 (m, 4H,
CH2CH2CH2CH3); 1.40-1.71 (m, 2H, CH2CH2CH2CH3); 2.08 (s, 3H, N=CCH3); 4.58 (m, 1H,
25

CHOH); 5.52 (exch br d, 1H, OH); 5.91 (exch br s, 2H, NH2); 12.59 (exch br s, 1H, NH). Anal.
Calcd for C10H17N3O2 (211.26): C, 56.85; H, 8.11; N, 19.89; Found: C, 57.01; H, 8.12; N, 19.83.
5.1.8. General procedures for 8a-d
A stirred suspension of appropriate intermediates 7a-d (1.20 mmol) in PPA (29.5-55.1
mmol) was heated at 90-110 °C for 2-5 h. After cooling, ice-cold water was added, and the mixture
was neutralized by slow addition of 6N NaOH. The resulting suspension was extracted with ethyl
acetate, and the organic phase was dried over Na2SO4. Evaporation of the solvent under vacuum
resulted in final compounds 8a-b. Instead, for compounds 8c-d, after neutralization, was observed
the formation of a precipitate, which was recovered by suction.
5.1.8.1. 4-Amino-6-methyl-5-vinylpyridazin-3(2H)-one, 8a. Yield = 90%; mp = 270-272 °C
1
(EtOH). H-NMR (CDCl3) δ 2.22 (s, 3H, CH3); 5.19 (exch br s, 2H, NH2); 5.61 (d, 1H, CH-H, J =
18 Hz); 5.74 (d, 1H, CH-H, J = 12 Hz); 6.46 (dd, 1H, CH, J = 18 Hz, J = 12 Hz); 8.03 (exch br s,
1H, NH). Anal. Calcd for C7H9N3O (151.17): C, 55.62; H, 6.00; N, 27.80; Found: C, 55.75; H,
5.99; N, 27.74.
5.1.8.2. 4-Amino-6-methyl-5-propenylpyridazin-3(2H)-one, 8b. Yield = 62%; mp = 264-266 °C
(EtOH). 1H-NMR (CDCl3) δ 1.84 (d, 3H, CH3CH, J = 6.8 Hz); 2.10 (s, 3H, CH3); 5.92-5.97 (m, 1H,
CH=CH-CH3); 6.01-6.13 (m, 1H, CH=CH-CH3); 6.89 (exch br s, 2H, NH2); 12.30 (exch br s, 1H,
NH). Anal. Calcd for C8H11N3O (165.19): C, 58.17; H, 6.71; N, 25.44; Found: C, 58.03; H, 6.70; N,
25.51.
5.1.8.3. 4-Amino-5-but-1-enyl-6-methylpyridazin-3(2H)-one, 8c. Yield = 42%; mp = 257-259 °C
1
(EtOH). H-NMR (CDCl3) δ 1.11-1.16 (m, 3H, CH2CH3, J = 7.2 Hz); 1.82-1.91 (m, 2H,
CHCH2CH3); 2.38 (s, 3H, N=CCH3); 4.57-4.63 (m, 1H, CH=CH-CH2); 5.03 (m, 1H, CH=CH-CH3);
5.55 (exch br s, 1H, NH); 6.59 (exch br s, 2H, NH2). Anal. Calcd for C9H13N3O (179.22): C, 60.32;
H, 7.31; N, 23.45; Found: C, 60.48; H, 6.58; N, 23.39.
5.1.8.4. 4-Amino-6-methyl-5-pent-1-enylpyridazin-3(2H)-one, 8d. Yield = 90%; mp = 216-218
1
°C dec. (EtOH). H-NMR (CDCl3) δ 0.96 (t, 3H, CH2CH3, J = 7.4 Hz); 1.48-1.53 (m, 2H,
26

CH2CH2CH3); 2.19 (s, 3H, N=CCH3); 2.21-2.26 (m, 2H, CH2CH2CH3); 5.06 (exch br s, 2H, NH2);
6.02-6.09 (m, 2H, CH=CH-CH2); 8.03 (exch br s, 1H, NH). Anal. Calcd for C10H15N3O (193.25):
C, 62.15; H, 7.82; N, 21.74; Found: C, 62.31; H, 7.83; N, 21.78.
5.1.9. General procedures for 9a-d
Compound 8a-d (0.46 mmol) was subjected to catalytic reduction with 10% Pd/C (0.23
mmol) in EtOH (20 mL) for 3 h in a Parr instrument at 30 PSI. The catalyst was filtered off, and the
solvent was evaporated under vacuum, affording in the final compounds.
5.1.9.1. 4-Amino-5-ethyl-6-methylpyridazin-3(2H)-one, 9a. Yield = 56%; mp = 260-262 °C
(EtOH). 1H-NMR (CDCl3) δ 1.13 (t, 3H, CH2CH3, J = 7.6 Hz); 2.25 (s, 3H, N=CCH3); 2.39 (q, 2H,
CH2CH3, J = 7.6 Hz); 6.81 (exch br s, 2H, NH2); 8.03 (exch br s, 1H, NH). Anal. Calcd for
C7H11N3O (153.18): C, 54.89; H, 7.24; N, 27.43; Found: C, 54.76; H, 7.23; N, 27.51.
5.1.9.2. 4-Amino-6-methyl-5-propylpyridazin-3(2H)-one, 9b. Yield = 85%; mp = 242-244 °C
1
dec. (EtOH). H-NMR (CDCl3) δ 0.91 (t, 3H, CH2CH3, J = 7.2 Hz); 1.34-1.40 (m, 2H,
CH2CH2CH3); 2.09 (s, 3H, N=CCH3); 2.28-2.33 (m, 2H, CH2CH2CH3); 5.89 (exch br s, 2H, NH2);
12.18 (exch br s, 1H, NH). Anal. Calcd for C8H13N3O (167.21): C, 57.46; H, 7.84; N, 25.13; Found:
C, 57.59; H, 7.83; N, 25.06.
5.1.9.3. 4-Amino-5-butyl-6-methylpyridazin-3(2H)-one, 9c. Yield = 82%; mp = 205-206 °C
(EtOH). 1H-NMR (CDCl3) δ 0.96 (t, 3H, CH2CH3, J = 7.2 Hz); 1.43-1.50 (m, 4H,
CH2CH2CH2CH3); 2.34 (s, 3H, N=CCH3); 2.38 (t, 2H, CH2CH2CH2CH3, J = 7.2 Hz); 6.03 (exch br
s, 2H, NH2); 11.89 (exch br s, 1H, NH). Anal. Calcd for C9H15N3O (181.23): C, 59.64; H, 8.34; N,
23.19; Found: C, 59.77; H, 8.33; N, 23.12.
5.1.9.4. 4-Amino-6-methyl-5-pentylpyridazin-3(2H)-one, 9d. Yield = 70%; mp = 230-232 °C
(EtOH). 1H-NMR (CDCl3) δ 0.90 (t, 3H, CH2CH3, J = 7.2 Hz); 1.35-1.40 (m, 4H,
CH2CH2CH2CH3); 1.45-1.53 (m, 2H, CH2CH2CH2CH3); 2.26 (s, 3H, N=CCH3); 2.34-2.40 (m, 2H,
CH2C4H9); 4.86 (exch br s, 1H, NH); 6.88 (exch br s, 2H, NH2). Anal. Calcd for C10H17N3O
(195.26): C, 61.51; H, 8.78; N, 21.52; Found: C, 61.70; H, 8.77; N, 21.56.
27

5.1.10. General procedures for 10a-d
Compounds 10a-d were obtained starting from 9a-d following the same procedure described
for 5b- c and 5f. For compounds 10b-d the suspension was extracted with ethyl acetate (3x 15 mL);
the organic layer was dried over Na2SO4 and evaporated to give desired final compounds which
were purified by column chromatography using cyclohexane/ethyl acetate 1:2 (for 10b and 10d) or
cyclohexane/ethyl acetate 1:3 (for 10c) as eluents.
5.1.10.1.
2-(5-Amino-4-ethyl-3-methyl-6-oxopyridazin-1(6H)-yl)-N-(4-bromophenyl)
acetamide, 10a. Yield = 74%; mp = 206-208 °C (EtOH). 1H-NMR (CDCl3) δ 1.14 (t, 3H, CH2CH3,
J = 7.6 Hz); 2.28 (s, 3H, N=CCH3); 2.41 (q, 2H, CH2CH3, J = 7.6 Hz); 4.89 (s, 2H, NCH2); 6.88
(exch br s, 2H, NH2); 7.36-7.40 (m, 4H, Ar); 9.00 (exch br s, 1H, NH). Anal. Calcd for
C15H17BrN4O2 (365.23): C, 49.33; H, 4.69; N, 15.34; Found: C, 49.21; H, 4.68; N, 15.38.
5.1.10.2.
2-(5-Amino-3-methyl-6-oxo-4-propylpyridazin-1(6H)-yl)-N-(4-bromophenyl)
1
acetamide, 10b. Yield = 19%; mp = 197-199 °C dec. (EtOH). H-NMR (CDCl3) δ 1.02 (t, 3H,
CH2CH3, J = 7.2 Hz); 1.51-1.59 (m, 2H, CH2CH2CH3); 2.27 (s, 3H, N=CCH3); 2.37 (t, 2H,
CH2CH2CH3, J = 7.2 Hz); 4.89 (s, 2H, NCH2); 6.84 (exch br s, 2H, NH2); 7.35-7.41 (m, 4H, Ar);
9.04 (exch br s, 1H, NH). Anal. Calcd for C16H19BrN4O2 (379.25): C, 50.67; H, 5.05; N, 14.77;
Found: C, 50.53; H, 5.06; N, 14.74.
5.1.10.3.
2-(5-Amino-4-butyl-3-methyl-6-oxopyridazin-1(6H)-yl)-N-(4-bromophenyl)
1
acetamide, 10c. Yield = 26%; oil. H-NMR (CDCl3) δ 0.96 (t, 3H, (CH2)3CH3, J = 7.2 Hz); 1.43-
1.50 (m, 4H, CH2CH2CH2CH3); 2.34 (s, 3H, N=CCH3); 2.38 (t, 2H, CH2(CH2)2CH3, J = 6.4 Hz);
4.90 (s, 2H, NCH2); 6.59 (exch br s, 2H, NH2); 7.34-7.40 (m, 4H, Ar); 8.87 (exch br s, 1H, NH).
Anal. Calcd for C17H21BrN4O2 (393.28): C, 51.92; H, 5.38; N, 14.25; Found: C, 51.76; H, 5.07; N,
14.22.
5.1.10.4.
2-(5-Amino-3-methyl-6-oxo-4-pentylpyridazin-1(6H)-yl)-N-(4-bromophenyl)
acetamide, 10d. Yield = 75%; mp = 167-171 °C (EtOH). 1H-NMR (CDCl3) δ 0.90 (t, 3H,
(CH2)4CH3, J = 7.2 Hz); 1.34-1.39 (m, 4H, (CH2)2CH2CH2CH3); 1.47-1.52 (m, 2H,
28

CH2CH2(CH2)2CH3); 2.26 (s, 3H, N=CCH3); 2.33-2.38 (m, 2H, CH2(CH2)3CH3); 4.88 (s, 2H,
NCH2); 6.59 (exch br s, 2H, NH2); 7.36-7.41 (m, 4H, Ar); 9.04 (exch br s, 1H, NH). Anal. Calcd for
C18H23BrN4O2 (407.30): C, 53.08; H, 5.69; N, 13.76; Found: C, 53.26; H, 5.68; N, 13.79.
5.1.11. General procedures for 11a-d
Compounds 11a-d were obtained starting from 10a-d following the same procedure
described for 6a-f. The final desired compounds were purified by column chromatography using as
eluent CH2Cl2/MeOH 98:2 for compounds 11a, CH2Cl2/MeOH 99:1 for 11c and cyclohexane/ethyl
acetate 1:2 for compounds 11b and 11d.
5.1.11.1. N-(4-Bromophenyl)-2-[4-ethyl-5-(3-methoxyphenylamino)-3-methyl-6-oxopyridazin-
1
1(6H)-yl]acetamide, 11a. Yield = 18%; mp = 81-84 °C (EtOH). H-NMR (CDCl3) δ 0.88 (t, 3H,
CH2CH3, J = 7.2 Hz); 2.28 (s, 3H, N=CCH3); 2.30-2.36 (m, 2H, CH2CH3); 3.78 (s, 3H, CH3O); 4.93
(s, 2H, NCH2); 6.51-6.66 (m, 3H, Ar); 7.01 (exch br s, 1H, NH); 7.19 (t, 1H, Ar, J = 8.2 Hz); 7.32-
7.39 (m, 4H, Ar); 9.11 (exch br s, 1H, NH). Anal. Calcd for C22H23BrN4O3 (471.35): C, 56.06; H,
4.92; N, 11.89; Found: C, 56.21; H, 4.91; N, 11.92.
5.1.11.2.
N-(4-Bromophenyl)-2-[5-(3-methoxyphenylamino)-3-methyl-6-oxo-4-propyl
pyridazin-1(6H)-yl]acetamide, 11b. Yield = 18%; oil. 1H-NMR (CDCl3) δ 0.66 (t, 3H, (CH2)2CH3,
J = 7.2 Hz); 1.26-1.32 (m, 2H, CH2CH2CH3); 2.23-2.31(m, 2H, CH2CH2CH3); 2.35 (s, 3H,
N=CCH3); 3.78 (s, 3H, OCH3); 4.92 (s, 2H, NCH2); 6.52-6.68 (m, 4H, Ar); 6.84 (exch br s, 1H,
NH); 7.35-7.42 (m, 4H, Ar); 9.04 (exch br s, 1H, NH). Anal. Calcd for C23H25BrN4O3 (485.37): C,
56.91; H, 5.19; N, 11.54; Found: C, 56.73; H, 5.20; N, 11.51.
5.1.11.3. N-(4-Bromophenyl)-2-[4-butyl-5-(3-methoxyphenylamino)-3-methyl-6-oxopyridazin-
1
1(6H)-yl]acetamide, 11c. Yield = 15%; oil. H-NMR (CDCl3) δ 0.68 (t, 3H, (CH2)3CH3, J = 7.2
Hz); 1.00-1.08 (m, 4H, CH2CH2CH2CH3); 2.24-2.30 (m, 2H, CH2CH2CH2CH3); 2.33 (s, 3H,
N=CCH3); 3.78 ( s, 3H, OCH3); 4.93 (s, 2H, NCH2); 6.60-6.69 (m, 4H, Ar); 6.84 (exch br s, 1H,
NH); 7.35-7.42 (m, 4H, Ar); 9.01 (exch br s, 1H, NH). Anal. Calcd for C24H27BrN4O3 (499.40): C,
57.72; H, 5.45; N, 11.22; Found: C, 57.85; H, 5.46; N, 11.25.
29