Research on heterocyclic compounds, XLI. 2-phenylimidazo[1,2- b]pyridazine-3-acetic derivatives: Synthesis and anti-inflammatory activity

Article abstract of DOI:10.1016/S0223-5234(99)00112-9

The synthesis of a group of 2-phenylimidazo[1,2-b]pyridazine-3-acetic esters and acids is described. The structures of the new compounds are supported by ¹H-NMR spectra. These compounds were tested in vivo for their anti-inflammatory, analgesic and ulcerogenic activity. All new compounds showed remarkable anti-inflammatory action in the carrageenan rat paw oedema (one third of that for indomethacin) but no significant analgesic activity in the acetic acid writhing test together with negligible ulcerogenic action, and were also found to be lacking inhibitory activity on cyclooxygenase in vitro.

Full text of DOI:10.1016/S0223-5234(99)00112-9

Eur. J. Med. Chem. 34 (1999) 1003−1008
© 1999 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
1003
Short communication
Research on heterocyclic compounds, XLI.
2-Phenylimidazo[1,2-b]pyridazine-3-acetic derivatives: synthesis
and anti-inflammatory activity
Antonia Sacchia, Sonia Laneria*, Francesca Arenaa, Enrico Abignentea,
Marina Gallitellia, Michele D’amicob, Walter Filippellib, Francesco Rossib
aDipartimento di Chimica Farmaceutica e Tossicologica,
Facoltà di Farmacia, Università di Napoli Federico II, Via Domenico Montesano 49, I-80131 Napoli, Italy
bIstituto di Farmacologia e Tossicologia, Facoltà di Medicina e Chirurgia,
II Università di Napoli, Via Costantinopoli 16, I-80138 Napoli, Italy
(Received 21 January 1999; accepted 21 April 1999)
Abstract – The synthesis of a group of 2-phenylimidazo[1,2-b]pyridazine-3-acetic esters and acids is described. The structures of the new
1
compounds are supported by H-NMR spectra. These compounds were tested in vivo for their anti-inflammatory, analgesic and ulcerogenic
activity. All new compounds showed remarkable anti-inflammatory action in the carrageenan rat paw oedema (one third of that for
indomethacin) but no significant analgesic activity in the acetic acid writhing test together with negligible ulcerogenic action, and were also
found to be lacking inhibitory activity on cyclooxygenase in vitro. © 1999 Éditions scientifiques et médicales Elsevier SAS
imidazo[1,2-b]pyridazines / anti-inflammatory activity / analgesic activity / ulcerogenic activity / cyclooxygenase inhibition
1. Introduction
In the context of our research on the structure-activity
relationships and mode of action of bicyclic imidazo-
derivatives with anti-inflammatory and analgesic activity,
we synthesized a series of 2-phenylimidazo[1,2-b]pyri-
dazine-3-carboxylic acids 1 (figure 1) which showed high
analgesic activity in the acetic acid writhing test in mice,
low or no anti-inflammatory activity in the carrageenan-
induced rat paw oedema and low ulcerogenic action on
the rat gastric mucosa [1, 2]. We then synthesized three
series of analogues, namely 2-methylimidazo[1,2-b]-
pyridazine-3-carboxylic acids 2 [3], imidazo[1,2-b]-
pyridazine-2-acetic acids 3 [4] and imidazo[1,2-b]-
pyridazine-2-carboxylic acids 4 [5].
In comparison with the first series of acids 1, com-
pounds 2 and 3 showed a lower analgesic activity,
whereas the same activity reached the lowest level in the
last series (acids 4). However, the pharmacological pro-
file was the same in all four groups of compounds, since
Figure 1. Imidazo[1,2-b]pyridazine acidic derivatives.
the analgesic activity was always coupled with low or no
anti-inflammatory and ulcerogenic action.
In consideration of the fact that the highest analgesic
activity was shown by the first type of compounds, i.e.
2-phenylimidazo[1,2-b]pyridazine-3-carboxylic acid 1,
and in order to extend the study of the structure-activity
relationships, we have synthesized a new series of
2-phenyl derivatives, with only one structural change, i.e.
the replacement of the carboxylic moiety in position 3 by
an acetic group.
*Correspondence and reprints

1004
prepared using as starting material a mixture (nearly 1:1)
of 3-amino-6-chloro-5-methylpyridazine (5f) and
3-amino-6-chloro-4-methylpyridazine (5g): this mixture
was obtained from 3,6-dichloro-4-methylpyridazine [8]
following the Mori procedure [9]. After reaction with 6,
the products 7f and 7g were separated by column chro-
matography. The above mixture of the amines 5f and 5g
afforded the corresponding dehalogenated mixture of
3-amino-5-methylpyridazine (5d) and 3-amino-4-methyl-
pyridazine (5e) by catalytic hydrogenation. The reaction
of 6 with the mixture 5d–5e afforded the esters 7d and 7e.
The correct structural assignments to these products
were performed by 1H-NMR spectra (table I), and are in
accordance with the literature data, in particular with the
chemical shifts found by Kobe et al. [10] for H-6, H-7,
H-8 in the imidazo[1,2-b]pyridazine.
3. Pharmacology
The new esters 7a–g and acids 8a–g were tested in
vivo using the acetic acid writhing test in mice and
carrageenan induced rat paw oedema to study analgesic
and anti-inflammatory activity. Higher doses were admin-
istered to rats to study the irritative and ulcerogenic
action on the mucosa of the stomach and small intestine.
Indomethacin was used in all tests as reference drug.
These three tests should allow us not only to extend the
SAR study to another series of imidazopyridazines, but
also to obtain some information about their mechanism of
action: they should display a similar level of activity in all
three tests if they are inhibitors of prostaglandin biosyn-
thesis. In order to unequivocally resolve this question,
some new compounds (the more and less active ones)
were also subjected to two different cyclooxygenase
activity assays in vitro [11, 12].
Figure 2. Synthetic method for 2-phenylimidazo[1,2-b]pyri-
dazine-3-acetic derivatives.
2. Chemistry
The required compounds were prepared using the
synthetic method depicted in figure 2. The reaction in dry
ethanol of 3-aminopyridazines 5a–g with ethyl
3-benzoyl-3-bromopropionate 6, prepared ad hoc, af-
forded the corresponding ethyl 2-phenylimidazo[1,2-
b]pyridazine-3-acetates 7a–g. These esters were con-
verted into the respective acids 8a–g by acid hydrolysis.
Esters 7a, 7b and 7c were prepared by reacting 6 with
3-aminopyridazine 5a, 3-amino-6-chloropyridazine 5b,
and 3-amino-6-methoxypyridazine 5c, respectively, pre-
pared ad hoc following the methods described by Wer-
muth et al. [6] and Steck et al. [7].
4. Results and discussion
The anti-inflammatory activity displayed by the com-
pounds under examination is reported in table II. The
esters 7c, 7d and 7g, and the acids 8a, 8c, 8f and 8g are
the most active compounds with comparable levels of
activity, as can be seen on the basis of reported values of
ED50 (approximately one third of that for indomethacin).
The results obtained in the acetic acid writhing test are
quite different (table III), in fact all compounds show
weak or no activity. The gastrointestinal irritative and
ulcerogenic action (table IV) was almost completely ab-
sent in all compounds.
The couples of isomers 7d–e and 7f–g were obtained
with the same method previously employed to obtain the
corresponding isomeric couples in the preceding series of
imidazo[1,2-b]pyridazines 1–4 [5]. The couple 7f–g was
There is no parallelism between the results of all three
in vivo tests. This is certainly not the pharmacological

1005
1
Table I. Yields, m.p. and H-NMR spectral data of 2-phenylimidazo[1,2-b]pyridazine-3-acetic derivatives.
Compounds1 yield m.p.
1H-NMR in ppm2
%
°C
δ: H-6
H-7
H-8
J6, 7
J6, 8
Hz
J7, 8
Ph
CH2 Ethyl
(s)
Substituents
7a
7b
7c
7d
7e
7f
7g
8a
8b
8c
8d
8e
8f
32
20
40
40
40
30
30
55
55
50
55
60
60
60
103–105 8.50 (dd)
108–110
104–105
110–112 8.25 (d)
121–123 8.15 (d)
113–115
7.20 (dd)
7.00 (d)
6.65 (d)
7.85 (dd)
7.90 (d)
8.70 (d)
7.95 (d)
4.5
2.0
2.4
10
10
10
7.70 (m) 7.60 (m)
7.70 (m) 7.40 (m)
8.60 (m) 8.40 (m)
7.50 (m) 7.40 (m)
7.60 (m) 7.50 (m)
7.65 (m) 7.50 (m)
4.10 4.00 (q) 1.00 (t)
4.10 4.05 (q) 1.20 (t)
4.10 4.05 (q) 1.10 (t) 6-OCH3: 3.90 (s)
4.10 4.08 (q) 1.20 (t) 7-CH3: 2.60 (s)
4.10 4.05 (q) 1.30 (t) 8-CH3: 2.80 (s)
4.20 4.10 (q) 1.30 (t) 7-CH3: 2.50 (s)
7.19 (d)
4.3
4.2
7.70 (s)
125–127
6.85 (s)
7.20 (dd)
7.21 (d)
6.75 (d)
7.80 (m) 7.50 (m) 7.30 (m) 4.20 4.10 (q) 1.30 (t) 8-CH3: 2.70 (s)
> 200
> 200
> 200
> 200
> 200
> 200
> 200
8.49 (dd)
7.80 (dd)
7.95 (d)
8.77 (d)
8.04 (d)
2.0
2.3
10
10
10
7.60 (m) 7.55 (m)
7.90 (m) 7.68 (m)
8.67 (m) 8.42 (m)
7.60 (m) 7.45 (m)
7.65 (m) 7.50 (m)
7.90 (m) 7.50 (m)
7.82 (m) 7.45 (m)
4.05
4.10
4.10
4.10
4.10
4.10
4.20
6-OCH3: 3.90 (s)
7-CH3: 2.65 (s)
8-CH3: 2.55 (s)
7-CH3: 2.55 (s)
8-CH3: 2.68
8.35 (d)
8.25 (d)
7.20 (d)
6.80 (s)
4.3
7.75 (s)
8g
1All compounds were analysed for C, H, N (also for Cl when present): found values were within ± 0.4% compared with theoretical values.
2Solvents: CDCl3 for 7a–g, CD3OD for 8a–g.
Table II. Anti-inflammatory activity by the carrageenan rat paw oedema test.
Compound
Dose mg/kg
p.o.
% Oedema inhibition relative to control at the:
ED50, mg/kg (fiducial limits)
1st h
2nd h
3rd h
4th h
3rd h
4th h
7a
7b
7c
40
40
20
40
80
20
40
80
40
40
20
40
80
–17
–7
–26
–14
–53
–26
–72
–14
–56
–78
–26
–20
–17
–34
–75
–29
–19
–34
–47
–80
–24
–53
–84
–47
–43
–20
–37
–82
–44
–21
–37
–59
–87
–42
–51
–90
–44
–44
–37
–51
–77
–
–
–
–
–62
–17
–52
–28
–63
–62
–17
0
35.1
(29.4–41.8)
28.9
(24.0–34.8)
7d
36.5
(32.1–41.7)
–
7e
7f
7g
–
–
–
–
–
–5
–27
–57
33.2
(27.0–40.7)
8a
10
20
40
40
10
20
40
40
40
10
20
40
10
20
40
–30
–47
–53
–14
–30
–58
–83
–67
–33
–22
–66
–86
–22
–58
–86
–31
–42
–68
–31
–20
–42
–66
–51
–51
–33
–65
–81
–28
–42
–72
–22
–33
–73
–33
–22
–41
–55
–43
–43
–33
–51
–76
–22
–41
–76
–32
–47
–66
–24
–24
–34
–64
–25
–38
–22
–55
–75
–25
–34
–75
–
21.4
(16.7–27.5)
8b
8c
–
31.4
(24.5–40.1)
–
27.8
(22.6–34.2)
8d
8e
8f
–
–
–
–
17.8
(14.7–21.5)
19.8
(17.0–23.1)
8g
21.9
(18.9–25.4)
23.0
(1.6–33.7)
IMA
5
7.5
10
–3
–16
–39
–40
–33
–55
–38
–49
–67
–35
–55
–79
7.0
(4.5–10.8)
6.7
(4.8–8.7)

1006
Table III. Analgesic activity by the acetic acid writhing test in
The second test was carried out by measuring the rate
of conversion of exogenous arachidonic acid into PGE2
in the rat medullary and cortical kidney microsomes.
Inhibition of microsomal PGE2 production was measured
by radioimmunoassay as reported previously [13]. This
test confirmed the above results: no compound showed
significant activity (< 12% relative to control), except
indomethacin (90–94%) at the same concentration
(10 µM).
Therefore in the present case the remarkable anti-
inflammatory activity shown by these compounds was
found to be independent of the cyclooxygenase inhibi-
tion. It should be noted that we recently came to the same
conclusion for a group of imidazo[1,2-a]pyrimidine ana-
logues [14].
From the point of view of our studies on structure-
activity relationships of imidazo[1,2-b]pyridazine acidic
derivatives 1–4 (figure 1), the most significant finding is
that the 2-phenylimidazo[1,2-b]pyridazine-3-acetic de-
rivatives (esters 7 and acids 8) showed weak analgesic
activity and proved to be completely lacking ulcerogenic
action, whereas showed a significant anti-inflammatory
activity. The pharmacological profile resulting from the
above data for these 2-phenylimidazo[1,2-b]pyridazine-
3-acetic derivatives is different from that previously
observed for 1–4 series in which the analgesic activity
was clearly prevailing over the anti-inflammatory action,
so further investigation will be necessary in order to
explain the activity of these imidazo-derivatives, which is
probably due to the multiple mechanism of action differ-
ently influenced by the structural changes.
mice.
Compound Dose mg/kg p.o.
% Decrease of mean no. of
writhes in 25 min after treat-
ment relative to control
7a
7b
7c
7d
7e
40
40
40
40
40
40
40
40
40
40
40
40
40
40
5
–22.5
–4.5
–34.4
–8.8
–32.3
–4.0
–25.1
–17.2
–8.9
–26.5
–10.8
–9.3
–29.6
–15.8
–51.2
7f
7g
8a
8b
8c
8d
8e
8f
8g
IMA
profile expected from compounds acting as inhibitors of
the prostaglandin biosynthesis. In order to investigate this
aspect of the question, our compounds were tested in
vitro for their cyclooxygenase-inhibiting activity.
The most active compounds 7c, 7d, 8c, 8f and 8g were
tested for their cyclooxygenase-inhibiting activity by
measuring the rate of conversion of [1-14C]arachidonic
acid into PGE2 in the microsomal fraction of mucosa
preparation of rabbit distal colon after incubation with
test compounds, following the method previously re-
ported [13].
All compounds were found to be devoid of inhibitory
activity, i.e. 0.7–9.0% relative to control, compared with
90–92% of indomethacin at the same concentration
(10 µM).
5. Experimental protocols
5.1. Chemistry
Thin layer chromatography by precoated silica gel
plates (Merck 60 F254) was used to control the course of
reactions and purity of products: all compounds were
designated as pure when they showed a single spot after
elution with chloroform/methanol mixture (95:5); detec-
tion of components was made by UV light and/or
treatment with iodine vapors. Preparative separations
were performed in columns packed with silica gel from
Farmitalia Carlo Erba (RS, J mm 0.05:0.20). Melting
points were determined with a Kofler hot stage micro-
scope and are uncorrected. Elemental analyses indicated
by the symbols of the elements were within ± 0.4% of the
Table IV. Incidence of gastrointestinal lesions in rats.
Compound Dose mg/kg
p.o.
Remarks at 6 h after treatment:
% animal with:
hyperaemia
ulcers
7a
7b
7c
100
100
100
100
100
100
100
100
100
100
5
20
10
30
30
20
30
40
20
30
30
80
0
0
0
0
0
0
0
0
0
0
50
7f
7g
8a
8b
8c
8f
8g
IMA
1
theoretical values. The H-NMR spectra were recorded
using a Bruker AMX- 500 spectrometer equipped with a
Bruker X-32 computer; chemical shift values are reported

1007
in δ units (ppm) relative to tetramethylsilane used as
internal standard.
5.1.4. 2-Phenylimidazo[1,2-b]pyridazine-3-acetic acids
8a–g
General procedure: a mixture of 10 mmol of each ethyl
ester and 40 mL of 10% aqueous HCl was refluxed for
2–3 h. After cooling, the solution was adjusted to pH 4–5
with NaHCO3 to obtain the precipitation of the acid
which was recrystallized from ethanol.
5.1.1. Ethyl 3-benzoyl-3-bromopropionate 6
A solution of 3-benzoylpropionic acid (19.6 g,
0.1 mol) in 100 mL of dry ethanol with concentrated
H2SO4 (5 mL) was refluxed for 7 h. After cooling,
ethanol was removed in vacuo, and the residue dissolved
in diethyl ether and extracted with NaHCO3 saturated
solution. The organic extract was washed with water,
dried on Na2SO4 and evaporated in vacuo to obtain ethyl
3-benzoylpropionate as an oil in 75% yield.
A solution of ethyl 3-benzoylpropionate in 100 mL of
diethyl ether was added slowly with an equimolar amount
of bromine at 0 °C. The solution was stirred at room
temperature for 1 h. The organic solution was washed
three times with NaHCO3 saturated solution, dried on
Na2SO4 and evaporated in vacuo to obtain the required
compound 6 as an oil in 65% yield.
5.2. Pharmacology
As regards the experiments carried out in vivo, test
compounds were administered orally by gavage in 1%
methylcellulose suspension. In the oedema and writhing
test each compound was first tested at 40 mg/kg. If a
significant activity was observed, lower and/or higher
doses were administered in order both to study the
dose-dependence of the pharmacological activity and to
calculate ED50 values, when possible. Gastric ulcero-
genic action was studied in rats which were treated orally
with higher doses (100 mg/kg).
Indomethacin was included in all tests for comparison
purposes (IMA in tables II–IV).
5.1.2. Ethyl-2-phenylimidazo[1,2-b]pyridazine-3-aceta-
tes 7a and 7c–g
General procedure: a mixture of the starting 3-amino-
pyridazine and ethyl 3-benzoyl-3-bromopropionate 6
(molar ratio 1:1.5) in ligroine was refluxed for 3 h. After
cooling, the mixture was filtered and the filtrate was
extracted with 10% aqueous HCl. The aqueous layer was
separated and adjusted to pH 7–8 with NaHCO3 to obtain
the precipitation of the ester which was then recrystal-
lized from n-hexane. This procedure allowed us to obtain
the esters 7a and 7c.
In the case of the isomeric mixtures 7d–e and 7f–g,
obtained starting from the mixtures of the isomeric
amines 5d–e and 5f–g, respectively, the crude product
precipitated was subjected to column chromatographic
separation, eluting with n-hexane/diethyl ether mixtures
with increasing percentage of ether. The single products
obtained from the columns were then recrystallized from
n-hexane.
5.2.1. Anti-inflammatory activity
The paw oedema inhibition test [15] was used on rats.
Groups of 5 rats of both sexes (body weight 150–200 g),
pregnant females excluded, were given a dose of a test
compound. After 30 min, 0.2 mL of 1% carrageenan
suspension in 0.9% NaCl solution was injected subcuta-
neously into the plantar aponeurosis of the hind paw and
the paw volume was measured by a water plethysmom-
eter Socrel and then measured again 1, 2, 3 and 4 h later.
The mean increase of paw volume at each time interval
was compared with that of a control group (5 rats treated
with carrageenan, but not treated with test compounds) at
the same time intervals and percentage inhibition values
were calculated. The experimental results are listed in
table II.
5.2.2. Analgesic activity
5.1.3. Ethyl 6-chloro-2-phenylimidazo[1,2-b]pyridazine-
3-acetate, 7b
Acetic acid writhing test [16] was used on mice.
Groups of 5 mice of both sexes (body weight 20–25 g),
pregnant females excluded, were given a dose of a test
compound. After 30 min the animals were injected intra-
peritoneally with 0.25 mL/mouse of 0.5% acetic acid
solution and writhes were counted during the following
25 min. The mean number of writhes for each experimen-
tal group and percentage decrease compared with the
control group (5 mice not treated with test compounds)
were calculated. The experimental results are listed in
table III.
A mixture of a 3-amino-6-chloropyridazine 5b and
ethyl 3-benzoyl-3-bromopropionate 6 (molar ratio 1:1.5)
in anhydrous ethanol was refluxed for 10 h. After cooling,
ethanol was removed in vacuo, and the residue treated
with NaHCO3 saturated solution and extracted with
CHCl3. The organic extract was washed with water, dried
on Na2SO4 and evaporated in vacuo to obtain the
required product 7b, which was recrystallized from
n-hexane.

1008
[2] Abignente E., Arena F., Luraschi E., Saturnino C., Marmo E.,
Berrino L., Donnoli D., Farmaco 45 (1990) 1075–1087.
5.2.3. Ulcerogenic action
Groups of 10 rats of both sexes (body weight
200–220 g), pregnant females excluded, fasted for 24 h,
were given an oral dose of a test compound, except the
control group. All animals were sacrificed 6 h after
dosing and their stomachs and small intestine were
macroscopically examined to assess the incidence of
hyperaemia and ulcers. The experimental results are
listed in table IV.
[3] Abignente E., Arena F., Luraschi E., Saturnino C., Rossi F., Berrino
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Acknowledgements
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The NMR spectra were performed at the “Centro di
Ricerca Interdipartimentale di Analisi Srtumentale,” Uni-
versità di Napoli “Federico II,” Naples. The assistance of
staff is much appreciated. This research has been finan-
cially supported by the Italian Ministry of University and
Scientific and Technological Research (MURST, Rome).
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