Functionalized pyrazoles and pyrazolo[3,4-d]pyridazinones: Synthesis and evaluation of their phosphodiesterase 4 inhibitory activity

Article abstract of DOI:10.1016/j.bmc.2010.03.066

A series of pyrazoles and pyrazolo[3,4-d]pyridazinones were synthesized and evaluated for their PDE4 inhibitory activity. All the pyrazoles were found devoid of activity, whereas some of the novel pyrazolo[3,4-d]pyridazinones showed good activity as PDE4 inhibitors. The most potent compounds in this series showed an IC₅₀ in the nanomolar range. The ability to inhibit TNF-α release in human PBMCs was determined for two representative compounds, finding values in the sub-micromolar range. SARs studies demonstrated that the best arranged groups around the heterocyclic core are 2-chloro-, 2-methyl- and 3-nitrophenyl at position 2, an ethyl ester at position 4 and a small alkyl group at position 6. Molecular modeling studies performed on a representative compound allowed to define its binding mode to the PDE4B isoform.

Full text of DOI:10.1016/j.bmc.2010.03.066

Bioorganic & Medicinal Chemistry 18 (2010) 3506–3517
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Functionalized pyrazoles and pyrazolo[3,4-d]pyridazinones: Synthesis and
evaluation of their phosphodiesterase 4 inhibitory activity
Pierfrancesco Biagini a, Claudio Biancalani a, Alessia Graziano a, Nicoletta Cesari a, Maria Paola Giovannoni a,
,
*
Agostino Cilibrizzi a, Vittorio Dal Piaz a, Claudia Vergelli a, Letizia Crocetti a, Maurizio Delcanale b,
Elisabetta Armani b, Andrea Rizzi b, Paola Puccini b, Paola Maria Gallo b, Daniele Spinabelli b, Paola Caruso b
a Dipartimento di Scienze Farmaceutiche, Università degli Studi di Firenze, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Italy
b Chiesi Farmaceutici S.p.A., Via Palermo 26/A, 43100 Parma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyrazoles and pyrazolo[3,4-d]pyridazinones were synthesized and evaluated for their PDE4
inhibitory activity. All the pyrazoles were found devoid of activity, whereas some of the novel pyrazol-
o[3,4-d]pyridazinones showed good activity as PDE4 inhibitors. The most potent compounds in this series
Received 2 February 2010
Revised 23 March 2010
Accepted 25 March 2010
Available online 31 March 2010
showed an IC50 in the nanomolar range. The ability to inhibit TNF-a release in human PBMCs was deter-
mined for two representative compounds, finding values in the sub-micromolar range. SARs studies dem-
onstrated that the best arranged groups around the heterocyclic core are 2-chloro-, 2-methyl- and 3-
nitrophenyl at position 2, an ethyl ester at position 4 and a small alkyl group at position 6. Molecular
modeling studies performed on a representative compound allowed to define its binding mode to the
PDE4B isoform.
Keywords:
Pyrazoles
Pyrazolo[3,4-d]pyridazinones
PDE4 inhibitors
Structure–activity relationship
Molecular modeling
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Under this point of view, c-AMP elevation mediated by PDE4
inhibitors represents a promising single treatment both for COPD
In recent years the interest for novel chemotypes of phosphodi-
esterase 4 (PDE4) inhibitors increased considerably due to their
possible therapeutic applications in a wide range of inflammatory
and immune disorders1 like asthma2, chronic obstructive pulmon-
ary disease (COPD)3–5, reumathoid arthritis6, inflammatory bowel
disease7, multiple sclerosis8 and so on. Moreover, very recently fur-
ther therapeutic targets emerged since several studies have shown
that selective PDE4 inhibitors are able to improve cognitive perfor-
mances in a variety of assays performed in animal models.9,10 The
main part of PDE4 inhibitors is targeted to asthma and COPD, being
these pulmonary diseases among the most common chronic disor-
ders in advanced western countries. Both the pathologies are
increasing in incidence and, namely for COPD, which is at the pres-
ent the fifth cause of death in USA, there is an urgent need for ther-
apeutic treatments.11 None of the existing medications for COPD
demonstrated to modify the long-term decline in lung function
which is the hallmark of this disease. Therefore, the goal of COPD
pharmacotherapy is to provide relief of symptoms, prevent compli-
cations and/or progression of the disease with a minimum of side
effects.
and asthma due to the antiinflammatory and bronchodilatory
effect exerted by these agents. Interestingly in COPD patients
Cilomilast A12, which together Roflumilast B13 (Fig. 1) is the most
advanced PDE4 inhibitor in clinical trials (Phase III), showed evi-
dence for an antiinflammatory and potentially disease modifying
effect, which is completely absent in subjects treated with steroids,
b2 agonists and antimuscarinics.14
The potential of the approach based on PDE inhibition is clearly
demonstrated by the recent success of several chemical entities
targeting different PDE subtypes. Thus Cilostazol, which is a
O
OH
Cl
N
O
O
O
O
O
N
F
H
CN
Cl
F
b
a
O
O
N
N
NO2
c
* Corresponding author. Tel.: +39 055 4573682.
E-mail address: mariapaola.giovannoni@unifi.it (M.P. Giovannoni).
Figure 1. PDE4 inhibitors.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.066

P. Biagini et al. / Bioorg. Med. Chem. 18 (2010) 3506–3517
3507
selective PDE3 inhibitor, is used as peripheral vasodilator15 and the
O
EtOOC COCH2OMe
N
EtOOC
Cl
C
N
O
selective PDE5 inhibitors Sildenafil, Tadalafil and Vardenafil are
extensively prescribed for male erectile disfunction.16 Among
PDE4 inhibitors Ibudilast was approved in Japan for the treatment
of asthma and stroke.17
On these grounds is not surprising the great interest of the
pharmaceutical community for identifying novel chemical entities
endowed with potent and selective PDE4 inhibitory activity.
As continuation of our previous synthetic efforts in PDE inhibi-
tors area18–22, we report here the synthesis and evaluation of a ser-
ies of pyrazoles and pyrazolo[3,4-d]pyridazinones some of which
are structurally related to compound C, belonging to a series of po-
tent PDE4 inhibitors described by researchers from Plexxikon.23
Me
Cl
CH
H
N
a
HC
+
Cl
NH
NMe2
5
6
7
Scheme 2. Synthesis of pyrazole 7. Reagents and conditions: (a) anhydrous toluene,
Et3N, reflux.
R3
N N
EtOOC COR
O
R
N
a
R2
N
N
R2
N
2. Chemistry
R1
R1
b
c
7,8
10a-e
All compounds were synthesized as reported in Schemes 1–6.
Scheme 1 shows the synthetic procedure affording pyrazoles
HN N
O
R
3a–e and 4a–e. The isoxazolo[3,4-d]pyridazinones 2a–e (2c24
)
N
N
were prepared by condensing the precursors 1a–d (1a18, 1b,c25
and 1d26) with the opportune arylaldehydes under standard
conditions. Treatment of 2a–e with 2 N NaOH in ethanol at 80 °C
afforded pyrazoles 3a–e27 which were transformed into the corre-
sponding pyrazoles 4a–e by reduction with ammonium formate
and Pd/C.
R2
R1
9a,b
R2
R1
Compd
R
Compound 7 was obtained by reacting derivative 528 and 629 in
anhydrous toluene under reflux (Scheme 2).
H
2-Cl-Ph
7, 9a
8, 9b
CH2OMe
Me
Me
3-NO2-Ph
The pyrazolo[3,4-d]pyridazinones 10a–e were obtained as de-
picted in Scheme 3. Pyrazoles 7 and 830 were treated with hydra-
zine hydrate in ethanol affording the intermediates 9a,b, which,
were alkylated under standard conditions to give the final products
10a–d. Compound 10e was obtained by treatment of the precursor
8 with PPA and ethylhydrazine oxalate.
The synthesis of derivatives 14a–n and 15a–c (Scheme 4) was
performed starting from the isoxazolo[3,4-d]pyridazinones
11a,b25,26, which were alkylated under standard conditions (com-
pounds 1a and 12a30, 12b, 12c26 and 12d,e) and in turn were trans-
formed into the 4-nitro-5-acetyl derivatives 13a–f (13a18 and
13b31) by oxidative cleavage of the isoxazole ring.32 Treatment
with opportune hydrazines in ethanol at room temperature affor-
ded the final products 14a–n. Further elaboration of 14d by alkyl-
ation with appropriate arylalkyl halides in anhydrous acetone at
60 °C afforded the final pyrazolo[3,4-d]pyridazinones 15a–c.
Modifications of COOEt group at position 4 of the pyridazine
ring (Scheme 5) were performed starting from compounds
14e–g,j. The intermediates carboxylic acids 16a–d, obtained by
10
R2
R
R3
Et
R1
H
H
a
b
c
d
e
CH2OMe
CH2OMe
Me
2-Cl-Ph
cyclopropylmethyl
2-Cl-Ph
Me CH2COOEt
Me CH2CH2COOEt
Me Et
3-NO2-Ph
3-NO2-Ph
3-NO2-Ph
Me
Me
Scheme 3. Synthesis of 2H-pyrazolo[3,4-d]pyridazin-7(6H)-ones 10a–e. Reagents
and conditions: (a) ethylhydrazine oxalate, PPA, 80–90 °C; (b) hydrazine hydrate,
EtOH, rt; (c) R3X, anhydrous DMF or MeCOMe, K2CO3, reflux.
hydrolysis of the corresponding esters, were converted into the
secondary and tertiary amides 17a–f through the intermediate acyl
chloride. The synthesis of 18a,b was carried out through the inter-
mediates 17a,b, which were transformed into the final 5-cyano
derivatives 18a,b, using POCl3 as reagent. Finally, esterification in
R1
N N
R1
N N
R
CN
CH=CHR2
R
CN
CH2CH2R2
c
b
a
N
N
O
R
O
R
N
R1
N
R1
N
N
CH=CHR2
2a-e
Me
O
O
1a-d
3a-e
4a-e
2-4
R
R1 R2
1
R
R1
a
b
c
d
e
Ph
Ph
Me
Ph
Et α-naphthyl
a
b
c
d
Ph
Me
Ph
Et
Et 2-COOH-Ph
Me Ph
Me
Me
Et
Me 3,4-OMe-Ph
4-pyridyl Et α-naphthyl
4-pyridyl
Scheme 1. Synthesis of pyrazoles 3a–e and 4a–e. Reagents and conditions: (a) R2CHO, MeONa, abs MeOH, reflux; (b) NaOH 2 N, EtOH, 80 °C; (c) HCOONH4, Pd/C, EtOH, reflux.

3508
P. Biagini et al. / Bioorg. Med. Chem. 18 (2010) 3506–3517
Et
R3
R3
R3
N N
N N
N N
N N
HN N
O
COOEt
Me
N
O
R
d
c
b
O
R
O
R
O
R
a
for 14d
N
N
N
R1
O2N COMe
N
Me
N
O
Me
Me
O
R1
13a-f
11a,b
1a, 12a-e
14a-n
15a-c
R3
Et
R
Compd.
11
R
15
R1
Ph
Ph
Ph
COOEt
COOEt
COOEt
1a, 13a
a
b
Ph
COOEt
CH2COOEt
CH2CH2COOEt
Et
n-propyl
cyclopropylmethyl
12a, 13b
12b, 13c
12c, 13d
12d, 13e
12e, 13f
a
b
c
4-CN
4-NO2
4-COOMe
14
R3
Et
R1
R
a
b
c
d
e
f
g
h
i
3-NO2-Ph
Ph
Ph
Ph
CH2COOEt
(CH2)2COOEt
Et
Et
Et
Et
Et
Et
3-NO2-Ph
3-NO2-Ph
H
3-NO2-Ph
Ph
2-Cl-Ph
3-CH2OH-Ph
2-CH3-Ph
2,6-Cl-Ph
3-NO2-Ph
2-Cl-Ph
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
Et
n-propyl
cyclopropylmethyl
j
k
l
H3C
Et
COOEt
m
COOCH3
Cl
Et
COOEt
n
CONH2
Scheme 4. Synthesis of 3-methyl-2H-pyrazolo[3,4-d]pyridazin-7(6H)-ones 14a–n and 15a–c. Reagents and conditions: (a) R3X, anhydrous DMF or MeCOMe, K2CO3, reflux;
(b) CAN, HNO3, AcOH, 50/55 °C; (c) R1NHNH2, EtOH, rt; (d) arylalkyl halides, MeCOMe, K2CO3, 60 °C.
standard condition of the carboxylic acid 16a afforded the final
19a–c.
ence pyrazole discovered by Plexxikon (compound C). Among
them the most potent compound (IC50 = 32 nM) was the ethyl ester
14g (R1 = 2-Cl, R2 = Me, R3 = Et). The analogue 14e with 3-NO2–
C6H4 at position 2 was about twofold less potent. The correspond-
ing methyl ester 19a was slightly more potent (IC50 = 46 nM) sug-
gesting that a minimum size for the ester function is an important
requirement for the activity. Support for this hypothesis was done
by the results obtained with the corresponding n-propyl and iso-
propyl esters (19b and 19c, respectively) which proved completely
devoid of activity. Replacement of COOEt in compound 14e with
the metabolically stable oxadiazole bioisoster (compound 23)
was associated with a strongly reduced activity (48% inhibition
Finally, in Scheme 6 is reported a further elaboration of the
function at position 4. The precursor 16c was converted into the
primary alcohol 20, which in turn, was transformed into the ether
21 using NaH and MeI in DMF at room temperature. The final prod-
uct 23, bearing a oxadiazole ring at position 4, was synthesized
through a three step procedure: treatment of the precursor 16a
with SOCl2 followed by treatment of the crude chloride with ace-
thydrazide (22) and cyclization with POCl3. Elemental analyses of
all new compounds are reported as Supplementary data.
at 1 lM). Any other modification of the ester function provoked
3. Biological results and discussion
loss of activity: thus the carboxylic acid 16a and the amides 17c–
f, as well as the nitriles 18a,b, the primary alcohol 20 and the
methyl ether 21 showed absence of activity or reduced activity
(21). In any case the presence of polar and hydrogen-bond donor
groups (COOH, CH2OH, CONH-Me) in this part of the molecule
was very detrimental for the activity. Moreover, the importance
of a hydrogen-bond acceptor substituent (COOEt) was suggested
by the present molecular modeling studies. In fact, the obtained
data demonstrated that the replacement of ester function (14g)
with the methyl ether (21) is associated with loss activity
All compounds were evaluated for their ability to inhibit PDE4
from U-937 cells at 1 lM concentration. For the most part of com-
pounds showing more then 50% inhibition at this concentration,
dose–response curves were constructed to calculate IC50 and the
results are reported in Tables 1 and 2. As showed in Table 1, all
the pyrazole derivatives resulted inactive at the tested concentra-
tion, being only 35% at 1 lM the higher value of inhibition (com-
pound 3a). With the synthesis of pyrazolopyridazinones (Table 2)
we realized bicyclic structures more closely related to the refer-
(IC50 = 32 nM and 41% inhibition at 1 lM, respectively). Replace-

P. Biagini et al. / Bioorg. Med. Chem. 18 (2010) 3506–3517
3509
Et
Et
Et
Et
N N
N N
N N
N N
O
CN
Me
O
CONRR'
Me
O
COOH
Me
O
COOEt
Me
b
c
a
N
N
N
N
N
N
R1
for 17a,b
N
R1
N
R1
R1
18a,b
17a-f
16a-d
14e,f,g,j
d for 16a
17
R
R'
Et
R1
N N
Compd.
14e, 16a
14f, 16b Ph
14g, 16c 2-Cl-Ph
R1
3-NO2-Ph
a
b
c
d
e
H
H
H
Me
Me
H
H
Me
Et
Me
2-Cl-Ph
O
COOR
Me
2,6-Cl-Ph
3-NO2-Ph
3-NO2-Ph
3-NO2-Ph
N
N
Cl
2,6-Cl-Ph
14j, 16d
NO2
H
N
f
Ph
19a-c
Cl
19
R
18
R1
a
b
c
Me
CH2CH2Me
CHMe2
a
b
2-Cl-Ph
2,6-Cl-Ph
Scheme 5. Synthesis of 3-methyl-2H-pyrazolo[3,4-d]pyridazin-7(6H)-ones 17a–f, 18a,b and 19a–c. Reagents and conditions: (a) NaOH, EtOH, rt; (b) SOCl2, Et3N, RR0NH, THF;
(c) POCl3; (d) RX, anhydrous DMF or MeCOMe, K2CO3, reflux.
Table 1
Et
N N
Et
N N
PDE4 inhibitory activity of pyrazoles
R
CN
CH2
O
CH2OMe
O
CH2OH
R
CN
CH CH R2
N
N
b
CH2 R2
N
N
N
N
Me
Cl
a
Me
N
N
R1
Cl
Et
for 16c
COOH
R1
N N
3a-e
4a-e
O
21
20
PDE4a
N
R
R1
R2
-Naphtyl
Me
Et
N
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
Ph
Ph
Me
Ph
Et
Et
Me
Me
Et
Et
Et
Me
Me
Et
a
35
6
1.3
9
19
-3
11
2.1
14
2.8
Et
N N
R1
16a,c
2-(COOH)Ph
Ph
3,4-OCH3-Ph
Me
O
N
Me
N N
O
O
CONHNHCOMe
N
d
c
4-pyridyl
Ph
a
a
-Naphtyl
-Naphtyl
N
N
N
N
Me
for 16a
Ph
Me
Ph
2-(COOH)Ph
Ph
3,4-OCH3-Ph
NO2
NO2
22
23
4-Pyridyl
a-Naphtyl
a
Data are indicated as inhibition percentage at 1
lM concentration.
Scheme 6. Synthesis of 3-methyl-2H-pyrazolo[3,4-d]pyridazin-7(6H)-ones 20, 21
and 23. Reagents and conditions: (a) NaBH4, anhydrous THF, MeOH, 70 °C; (b) NaH,
MeI, DMF, rt; (c) SOCl2, Et3N, acethydrazide; (d) POCl3, 60 °C.
activity, suggesting that a small alkyl group is the best arranged
in position 6. For compounds substituted at position 4 with Me,
the presence of ester function at R3 (10c,d) gave products showing
moderate activity (compound 10c, R3 = CH2COOEt, 64% inhibition
ment of COOEt with a lipophilic methyl group resulted in com-
pound 10e (IC50 = 385 nM). Despite an order of magnitude reduc-
tion of activity, this finding suggests that the introduction of a
small alkyl group at position 4 may be an useful approach to over-
come the possible metabolic instability of the corresponding
esters. Moreover, this result could be interpreted to indicate a
different binding mode of these compounds with catalytic site.
at 1 lM) or inactivity (10d, R3 = (CH2)2COOEt). A similar trend
was also observed with compounds bearing Ph at position 4, where
14a, bearing Et at R3 was more active that 14b (R3 = CH2COOEt)
and 14c (R3 = (CH2)2COOEt).
The best substituents in the phenyl group appended at position
2 were 2-chloro and 3-nitro groups. Isosteric replacement of Cl in
compound 14g (IC50 = 32 nM) with Me gave a compound (14i)
showing a slightly reduced activity (IC50 = 59 nM). Introduction
of a second Cl in position 6 of the phenyl (14j) was associated with
a strongly reduced activity (IC50 = 237 nM). A similar effect was ob-
served for compounds 14m and 14n where a second substituent
Moderate activity (47% inhibition at 1 lM) was also found for the
analogue 14a with a phenyl group at position 4.
The best substituents at R3 were ethyl and n-propyl. In fact
compounds 14e and 14k proved equipotent (IC50 = 75 and 72 nM,
respectively). By comparing the 2-(2-chlorophenyl) derivatives
14g and 14l it emerged that replacement of ethyl with cyclopro-
pylmethyl was associated with 1 order of magnitude reduction of

3510
P. Biagini et al. / Bioorg. Med. Chem. 18 (2010) 3506–3517
Table 2
PDE4 inhibitory activity of pyrazolopyridazinones
O
R
O
R
N
N
R1
R3
R3
N
N
N
N
N
N
R2
R2
R1
10a-e, 14a-n, 16-19, 20, 21, 23
15a-c
Compd
R
R1
R2
R3
PDE4a
10a
10b
10c
10d
10e
14a
14b
14c
14e
14f
14g
14h
14i
CH2OCH3
CH2OCH3
CH3
CH3
CH3
Ph
Ph
Ph
2-Cl
2-Cl
H
H
Et
65 7%
19 7%
64.1 7.5%
Eb
3-NO2
3-NO2
3-NO2
3-NO2
3-NO2
3-NO2
3-NO2
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH2COOEt
(CH2)2COOEt
Et
Et
CH2COOEt
(CH2)2COOEt
Et
Et
Et
Et
Et
Et
n-Propyl
Eb
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
4
3%
385
47 12%
1
1
4%
0%
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOH
CONHCH3
CON(CH3)CH2CH3
CON(CH3)2
Ab
CN
CN
75
300
32
39 9%
59
237
72
374
876
757
2-Cl
3-CH2OH
2-CH3
2,6-Cl
3-NO2
2-Cl
14j
14k
14l
14m
14n
15a
15b
15c
16a
17c
17d
17e
17f
18a
18b
19a
19b
19c
20
Cb
Db
4-CN
4-NO2
4-COOCH3
3-NO2
3-NO2
3-NO2
3-NO2
H
5% (10
1140
lM)
1200
2
5%
14 4%
ꢀ2 3%
4
3
4%
3%
2-Cl
ꢀ2 9%
2,6-Cl
3-NO2
3-NO2
3-NO2
2-Cl
2 13%
COOCH3
COO(CH2)2CH3
COOCH(CH3)2
CH2OH
CH2OCH3
Bb
46 M
14 4%
6
6%
Et
Et
Et
ꢀ4 1%
41 1%
48 5%
21
23
2-Cl
3-NO2
a
Data are indicated as IC50 (nM) or inhibition percentage at 1
l
M concentration or at indicated concentration (
lM).
b
Cl
Cl
H3C
CH3
O
CONH
N
A=
B=
C=
D=
E=
N
N
Cl
CONH2
COOCH3
was inserted in the aromatic ring. Finally, elimination of the substi-
tuent (compound 14f) provoked a 10-fold reduction of potency.
Thus keeping Me at R2, COOEt at R and Et at R3 for the substituent
R1 the following order of efficacy can be established: 2-Cl > 2-
CH3 > 3-NO2 > 2,6-Cl > H.
Introduction of a methylenic spacer between the aromatic por-
tion and the pyrazole was detrimental for the activity. In fact all
the synthesized benzyl derivatives (15a–c) were significantly less
potent with respect the phenyl analogues, being their IC50, at the
best, in the low micromolar range.
tors such as TNF-a 33
(14e and 14i) for their ability to inhibit TNF-
,
we decided to test two selected compounds
release in human
a
peripheral blood mononuclear cells (PBMCs). Both compounds
exhibited high activity being their IC50 = 383 (170–861) and
127 nM (38–424), respectively (confidence limits in brackets).
Finally, in view of a possible development of these compounds,
we examined their drug-like properties following the Lipinski’s
rule of five.34 We found that as 14e and 14i meet the requirements
of this rule being HBD <5, HBA <10, MW <500 and log P <5. Evalu-
ation of lipophilicity was performed by measuring log P with Mol-
inspiration Free log P calculator.35 The following values were
found: 2.263 for 14e and 2.729 for 14i.
As regards R2 there is only a couple of compounds that may fur-
nish an indication on the role played by the methyl group: thus by
comparing compound 21 (41% inhibition at 1
lM) and its nor
derivative 10a (62.9% inhibition at 1 M) it seems that in this part
of the molecule the presence of an hydrogen is better that a methyl
l
4. Molecular modeling studies
group.
In order to explain the high potency of the ethyl ester 14g
(IC50 = 32 nM) compared to the other less active inhibitors bearing
different groups in position 4, the binding mode of 14g was stud-
Since the inhibition of PDE4 leads to an increase in intracellular
cAMP which, in turn, inhibits the release of inflammatory media-

P. Biagini et al. / Bioorg. Med. Chem. 18 (2010) 3506–3517
3511
ied, using as catalytic site the 1XM4 complex structure of Piclam-
ilast bound to the PDE4B isoform.
0.7 mmol), and sodium methoxide (0.5–1 mmol) in anhydrous
methanol was heated for 5–10 min at 60–70 °C. After the mixture
was cooled, the precipitate was recovered by suction. For com-
pound 2b, after cooling, 6 N HCl was added, the precipitate recov-
ered by suction and purified by column chromatography using
dichlomethane/methanol/acetic acid 9:1:0.1. For compound 2e,
to a mixture of 1d26 in absolute EtOH (1 mL), 1-naphthaldehyde
(1 mmol), and piperidine (0.5 mL) was added. The mixture was
stirred in a sealed tube at 110 °C for 6 h. After evaporation of sol-
vent, ice-water was added and compound 2e was recovered by
suction.
Besides the six water molecules directly linked to the metals, an
additional water molecule was retained in the hydrophobic clamp,
where the selective Gln443 side chain forms important hydrogen
bonds with the inhibitors.36
Several poses of 14g were generated in Glide,37 using the extra
precision mode and imposing as docking constraint the hydrogen
bond with the Gln443 residue.
In the proposed binding mode of 14g (Fig. 2) the ethyl ester
group sits in a small binding pocket and forms two hydrogen bonds
with the Gln443 side chain and with a water molecule. Another
hydrogen bond is formed between the nitrogen atom in position
5 of the core structure and the Gln443 side chain. This particular
pose of 14g explains the loss of activity when the ethyl ester is re-
placed by bulkier groups.
Additional binding energy is found by the 2-chlorophenyl group
in position 2, which interacts with the His234, Ile410 and Phe414
residues through significant Van der Waals contacts.
6.1.1.1. 6-Ethyl-3-((E)-2-(naphthalen-1-yl)vinyl)-4-phenylisox-
azolo[3,4-d]pyridazin-7(6H)-one 2a. Yield = 52%; mp = 195–
196 °C (EtOH); 1H NMR (CDCl3) d 1.45 (t, 3H, NCH2CH3), 4.35
(q, 2H, NCH2CH3), 6.90 (d, 1H, CH@CHAr), 7.40–7.70 (m, 9H,
Ar), 7.90 (m, 2H, Ar), 8.20 (d, 1H, Ar), 8.50 (d, 1H, CH@CHAr).
6.1.1.2. 2-((1E)-2-(6-ethyl-6,7-dihydro-7-oxo-4-phenylisoxazolo
[3,4-d]pyridazin-3-yl)vinyl)benzoic acid 2b. Yield = 40%; mp =
189–192 °C; 1H NMR (CDCl3) d 1.43 (t, 3H, NCH2CH3), 4.31 (q, 2H,
NCH2CH3), 6.70 (d, 1H, CH@CHAr), 7.32 (d, 1H, Ar), 7.45–7.68 (m,
7H, Ar), 8.10 (d, 1H, Ar), 8.58 (d, 1H, CH@CHAr).
5. Conclusions
In conclusion only the series of pyrazolopyridazinones showed
interesting activity; the best arranged groups around the heterocy-
clic core are 2-chloro-, 2-methyl- and 3-nitrophenyl at position 2, a
ethyl ester at position 4 and a small alkyl group (ethyl, n-propyl) at
position 6. As regards position 3 a more in depth exploration of this
part of the molecule is necessary and studies in this sense are in
progress.
6.1.1.3. 3-(3,4-dimethoxystyryl)-6-methyl-4-phenylisoxazolo-
[3,4-d]pyridazin-7(6H)-one 2d. Yield = 63%; mp = 234–235 °C
(EtOH); 1H NMR (CDCl3) d 3.82 (s, 3H, OCH3), 3.84 (s, 3H, OCH3),
3.95 (s, 3H, NCH3), 6.60 (d, 1H, CH@CHAr), 6.78 (s, 1H, Ar), 6.87
(d, 1H, Ar), 6.99 (d, 1H, Ar), 7.53–7.67 (m, 6H, (5H, Ar; 1H,
CH@CHAr)).
6. Experimental section
6.1. Chemistry
6.1.1.4. 6-Ethyl-3-((E)-2-(naphthalen-1-yl)vinyl)-4-(pyridin-4-
yl)isoxazolo[3,4-d]pyridazin-7(6H)-one 2e. Yield = 61%; mp =
222–224 °C (EtOH); 1H NMR (CDCl3) d 1.48 (t, 3H, NCH2CH3),
4.35 (q, 2H, NCH2CH3), 6.94 (d, 1H, CH@CHAr), 7.45–7.69 (m,
6H, (4H, Ar; 2H, Py)), 7.93 (t, 2H, Ar), 8.21 (d, 1H, Ar), 8.59 (d,
1H, CH@CHAr), 8.89 (d, 2H, Py).
All melting points were determined on a Büchi apparatus and
are uncorrected. 1H NMR spectra were recorded with Avance 400
instruments (Bruker Biospin Version 002 with SGU). Chemical
shifts are reported in ppm, using the solvent as internal standard.
Infrared spectra (IR) were recorded as Nujol mulls with a Perkin–
Elmer spectrometer. Mass spectra (m/z) were recorded on a LTQ
mass spectrometer (ThermoFisher, San Jose, CA). Extracts were
dried over Na2SO4 and the solvents were removed under reduced
pressure. Merck F-254 commercial plates were used for analytical
TLC to follow the course of reaction. Silica Gel 60 (Merck 70–
230 mesh) was used for column chromatography.
6.1.2. General procedure for 3a–e
A mixture of appropriate isoxazolo[3,4-d]pyridazinones 2a–e
(2c24) (0.25–0.45 mmol), 2 N NaOH (2–4 mL) in ethanol (1.5–
5 mL) was refluxed under stirring for 2–3 h. Then the mixture
was concentrated in vacuo and diluted with cold water, and the
precipitate was recovered by suction. For compound 3b, after dilu-
tion with cold water, 6 N HCl was added to the mixture and the
precipitate was purified by column chromatography using dichlo-
methane/methanol/acetic acid 9:1:0.1.
6.1.1. General Procedure for 2a,b,d,e
A mixture of isoxazolopyridazinones 1a–c18,25,26 (1a18, 1b25
,
1c26
) (0.4–0.6 mmol), the appropriate aryl aldehyde (0.4–
6.1.2.1. 1-Ethyl-5-((E)-2-(naphthalen-1-yl)vinyl)-3-phenyl-1H-
pyrazole-4-carbonitrile 3a. Yield = 34%; mp = 139–140 °C (EtOH);
1H NMR (CDCl3) d 1.58 (t, 3H, NCH2CH3), 4.38 (q, 2H, NCH2CH3),
7.05 (d, 1H, CH@CHAr), 7.45–7.60 (m, 5H, Ar), 7.65 (t, 1H, Ar),
7.82 (d, 1H, Ar), 7.92 (d, 2H, Ar), 8.05 (d, 2H, Ar), 8.28 (d, 1H, Ar),
8.60 (d, 1H, CH@CHAr).
6.1.2.2.
vinyl)benzoic acid 3b. Yield = 20%; mp = 178–181 °C; IR (Nujol):
1740 (CO), 2200 (CN), 960 (trans CH@CH) cmꢀ1 1H NMR (CDCl3)
2-((E)-2-(4-Cyano-1-ethyl-3-phenyl-1H-pyrazol-5-yl)
;
d 1.55 (t, 3H, NCH2CH3), 4.37 (q, 2H, NCH2CH3), 6.85 (d, 1H,
CH@CHAr), 7.40–7.51 (m, 4H, Ar), 7.66 (t, 1H, Ar), 7.72 (d, 1H,
Ar), 8.01 (d, 2H, Ar), 8.18 (d, 1H, Ar), 8.52 (d, 1H, CH@CHAr); MS
(ESI): m/z 343.38 [M+H]+.
6.1.2.3. 1,3-Dimethyl-5-styryl-1H-pyrazole-4-carbonitrile 3c.
Yield = 24%; mp = 115 °C (EtOH); IR (Nujol): 2225 (CN), 960
Figure 2. Docking of the potent inhibitor 14g (IC50 = 32 nM) into the PDE4B
catalytic binding pocket.
(trans CH@CH) cmꢀ1 1H NMR (CDCl3) d 2.40 (s, 3H, CCH3), 3.90
;

3512
P. Biagini et al. / Bioorg. Med. Chem. 18 (2010) 3506–3517
(s, 2H, NCH3), 6.88 (d, 1H, CH@CHAr), 7.35–7.45 (m, 3H, Ar), 7.54–
7.63 (m, 3H, (2H, Ar; 1H, CH@CHAr)); MS (ESI): m/z 223.27 [M+H]+.
which was purified by column chromatography using cyclohex-
ane/ethyl acetate, 1:1, as eluent. Yield = 21%; mp = 66–68 °C
(EtOH); 1H NMR (CDCl3) d 1.44 (t, 3H, OCH2CH3), 3.46 (s, 3H,
OCH3), 4.47 (q, 2H, OCH2CH3), 4.54 (s, 2H, COCH2OCH3), 7.43 (m,
2H, Ar), 7.56 (m, 1H, Ar), 7.62 (m, 1H, Ar), 8.40 (s, 1H, Ar).
6.1.2.4.
5-(3,4-Dimethoxystyryl)-1-methyl-3-phenyl-1H-pyra-
zole-4-carbonitrile 3d. Yield = 50%; mp = 157–158 °C (EtOH); 1H
NMR (CDCl3) d 3.87 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 4.03 (s, 3H,
NCH3), 6.80 (d, 1H, CH@CHAr), 6.92 (d, 1H, Ar), 7.09 (s, 1H, Ar),
7.20 (d, 1H, Ar), 7.40–7.55 (m, 3H, Ar), 7.63 (d, 1H, CH@CHAr),
8.00 (d, 2H, Ar).
6.1.5. General procedure for 9a,b
A suspension of 7 or 829 (0.35–0.37 mmol) in absolute ethanol
(3–4 mL) and hydrazine hydrate (3–6 mmol) was stirred at room
temperature for 1–2 h. Then the mixture was cooled for 1 h and
the precipitate was recovered by suction.
6.1.2.5. 1-Ethyl-5-((E)-2-(naphthalen-1-yl)vinyl)-3-(pyridin-4-
yl)-1H-pyrazole-4-carbonitrile 3e. Yield = 38%; mp = 238–240 °C
(EtOH); 1H NMR (CDCl3) d 1.60 (t, 3H, NCH2CH3), 4.42 (q, 2H,
NCH2CH3), 7.03 (d, 1H, CH@CHAr), 7.58 (q, 2H, Ar), 7.65 (t, 1H,
Ar), 7.83 (d, 1H, Ar), 7.92 (t, 2H, Ar), 8.00 (d, 2H, Py), 8.28 (d, 1H,
Ar), 8.60 (d, 1H, CH@CHAr), 8.78 (d, 2H, Py).
6.1.5.1. 2-(2-Chlorophenyl)-4-(methoxymethyl)-2H-pyrazolo-
[3,4-d]pyridazin-7(6H)-one 9a. Yield = 74%; mp = 193–194 °C
(EtOH); 1H NMR (DMSO-d6) 3.35 (s, 3H, CH2OCH3), 4.53 (s, 2H,
CH2OCH3), 7.58–7.69 (m, 2H, Ar), 7.79 (t, 2H, Ar), 8.92 (s, 1H, Ar),
12.39 (exch br s, 1H, NH).
6.1.3. General procedure for 4a–e
A mixture of appropriate pyrazoles 3a–e (0.26–0.50 mmol), 10%
Pd/C (30–50 mg), and ammonium formate (0.6–1.2 mmol) in EtOH
(2–3 mL) was refluxed for 1–2 h. After the mixture was cooled,
CH2Cl2 (5 mL) was added, the catalyst was filtered off, and the sol-
vent was evaporated in vacuo to afford 4a–e.
6.1.5.2. 3,4-Dimethyl-2-(3-nitrophenyl)-2H-pyrazolo[3,4-d]pyri-
dazin-7(6H)-one 9b. Yield = 81%; mp = >300 °C (EtOH); 1H NMR
(DMSO-d6) 2.50 (s, 3H, 4-CH3), 2.70 (s, 3H, 3-CH3), 7.90 (t, 1H,
Ar), 8.15 (d, 1H, Ar), 8.46 (m, 2H, Ar), 12.10 (exch br s, 1H, NH).
6.1.6. General procedure for 10a–d
6.1.3.1. 1-Ethyl-5-(2-(naphthalen-1-yl)ethyl)-3-phenyl-1H-pyra-
zole-4-carbonitrile 4a. Yield = 44%; mp = 95–97 °C (EtOH); 1H
NMR (CDCl3) d 1.23 (t, 3H, NCH2CH3), 3.28 (t, 2H, CH2CH2Ar),
3.55 (t, 2H, CH2CH2Ar), 3.76 (q, 2H, NCH2CH3), 7.38–7.60 (m, 7H,
Ar), 7.80 (d, 1H, Ar), 7.90 (d, 1H, Ar), 8.00 (d, 2H, Ar), 8.10 (d, 1H,
Ar).
A mixture of 9a or 9b (0.14–0.28 mmol), K2CO3 (0.29–
0.56 mmol), and the appropriate (cyclo)alkyl halide (0.2–
0.48 mmol) in anhydrous acetone (4 mL) for 10a,b or anhydrous
DMF (1.5 mL) for 10c,d was refluxed under stirring for 2–8 h. For
compounds 10a,b, the reaction mixture was concentrated in vacuo
and diluted with cold water, and the precipitate 10a was recovered
by suction. For compound 10b, the suspension was extracted with
ethyl acetate (3 ꢁ 15 mL) and evaporation of the solvent afforded
an oil as final product. For compounds 10c,d, after cooling, the sus-
pension was diluted with cold water and the precipitates were
recovered by suction. Compound 10d was purified by column chro-
matography using CHCl3/MeOH, 9.5:0.5, as eluent.
6.1.3.2. 2-(-2-(4-Cyano-1-ethyl-3-phenyl-1H-pyrazol-5-yl)ethyl)
benzoic acid 4b. Yield = 33%; mp = 94–98 °C (EtOH); IR (Nujol):
1740 (CO), 2210 (CN) cmꢀ1 1H NMR (CDCl3) d 1.45 (t, 3H,
;
NCH2CH3), 3.20 (t, 2H, CH2CH2Ar), 3.40 (t, 2H, CH2CH2Ar), 4.13 (q,
2H, NCH2CH3), 7.30–7.60 (m, 6H, Ar), 7.96 (d, 2H, Ar), 8.12 (d, 1H,
Ar); MS (ESI): m/z 345.39 [M+H]+.
6.1.6.1. 2-(2-Chlorophenyl)-6-ethyl-4-(methoxymethyl)-2H-pyr-
azolo[3,4-d]pyridazin-7(6H)-one 10a. Yield = 73%; mp = 58–60 °C
(EtOH); 1H NMR (CDCl3) d 1.41 (t, 3H, NCH2CH3), 3.44 (s, 3H,
CH2OCH3), 4.29 (q, 2H, NCH2CH3), 4.62 (s, 2H, CH2OCH3), 7.46 (m,
2H, Ar), 7.59 (m, 1H, Ar), 7.71 (m, 1H, Ar), 8.46 (s, 1H, Ar).
6.1.3.3. 1,3-Dimethyl-5-phenethyl-1H-pyrazole-4-carbonitrile
4c. Yield = 41%; oil; IR (Nujol): 2220 (CN) cmꢀ1 1H NMR (CDCl3)
;
d 2.33 (s, 3H, CCH3), 3.00 (m, 4H, (2H, CH2CH2Ar; 2H, CH2CH2Ar)),
3.40 (s, 3H, NCH3), 7.09 (m, 2H, Ar), 7.28 (m, 3H, Ar); MS (ESI):
m/z 225.29 [M+H]+.
6.1.6.2. 2-(2-Chlorophenyl)-6-(cyclopropylmethyl)-4-(methoxy-
methyl)-2H-pyrazolo[3,4-d]pyridazin-7(6H)-one
6.1.3.4.
pyrazole-4-carbonitrile 4d. Yield = 30%; mp = 62–64 °C (EtOH);
2220 (CN) cmꢀ1 1H NMR (CDCl3) d 3.00 (t, 2H, CH2CH2Ar), 3.12
(t, 2H, CH2CH2Ar), 3.52 (s, 3H, NCH3), 3.82 (s, 3H, OCH3), 3.89 (s,
3H, OCH3), 6.58 (s, 1H, Ar), 6.68 (d, 1H, Ar), 6.83 (d, 1H, Ar),
7.40–7.50 (m, 3H, Ar), 7.97 (d, 2H, Ar).
5-(3,4-Dimethoxyphenethyl)-1-methyl-3-phenyl-1H-
10b. Yield = 42%; oil; 1H NMR (CDCl3) d 0.45–0.55 (m, 4H, cC3H5),
1.38–1.46 (m, 1H, cC3H5), 3.46 (s, 3H, CH2OCH3), 4.12 (d, 2H,
NCH2), 4.63 (s, 2H, CH2OCH3), 7.48 (m, 2H, Ar), 7.60 (m, 1H, Ar),
7.74 (m, 1H, Ar), 8.47 (s, 1H, Ar).
;
6.1.6.3. Ethyl 2-(3,4-dimethyl-2-(3-nitrophenyl)-7-oxo-2H-pyr-
azolo[3,4-d]pyridazin-6(7H)-yl)acetate 10c. Yield = 58%; mp =
189–190 °C (EtOH); IR (Nujol): 1755 (CO), 1690 (CO), 1520 and
6.1.3.5. 1-Ethyl-5-(2-(naphthalen-1-yl)ethyl)-3-(pyridin-4-yl)-
1H-pyrazole-4-carbonitrile 4e. Yield = 78%; mp = 135–138 °C
(EtOH); 1H NMR (CDCl3) d 1.22 (t, 3H, NCH2CH3), 3.30 (t, 2H,
CH2CH2Ar), 3.57 (t, 2H, CH2CH2Ar), 3.73 (q, 2H, NCH2CH3), 7.21
(d, 1H, Ar), 7.39 (1H, Ar), 7.50–7.62 (m, 2H, Ar), 7.82 (d, 1H, Ar),
7.93 (d, 1H, Ar), 8.06 (d, 3H, (1H, Ar; 2H, Py)), 8.77 (d, 2H, Py).
1350 (NO2) cmꢀ1 1H NMR (CDCl3) d 1.32 (t, 3H, COOCH2CH3),
;
2.60 (s, 3H, 4-CH3), 2.78 (s, 3H, 3-CH3), 4.25 (q, 2H, COOCH2CH3),
4.95 (s, 2H, NCH2), 7.80 (t, 1H, Ar), 7.96 (m, 1H, Ar), 8.43 (m, 2H,
Ar); MS (ESI): m/z 371,35 [M+H]+.
6.1.4. Ethyl-1-(2-chlorophenyl)-4-(2-methoxyacetyl)-1H-
pyrazole-3-carboxylate 7
6.1.6.4. Ethyl 3-(3,4-dimethyl-2-(3-nitrophenyl)-7-oxo-2H-pyr-
azolo[3,4-d]pyridazin-6(7H)-yl)propanoate 10d. Yield = 33%;
mp = 175–176 °C (EtOH); IR (Nujol): 1750 (CO), 1690 (CO), 1530
A mixture of the compound 628 (2.94 mmol) in anhydrous tolu-
ene (6 mL), Et3N (3.6 mmol) and compound 527 (2.95 mmol) was
refluxed under stirring for 4 h. The reaction mixture was concen-
trated in vacuo, diluted with cold water (10 mL) and neutralized
with 6 N HCl (10 mL). The suspension was extracted with ethyl
acetate (3 ꢁ 15 mL) and the solvent was evaporated to afford 7,
and 1360 (NO2) cmꢀ1 1H NMR (CDCl3) d 1.28 (t, 3H, COOCH2CH3),
;
2.60 (s, 3H, 4-CH3), 2.75 (s, 3H, 3-CH3), 2.85 (t, 2H, NCH2CH2), 4.20
(q, 2H, COOCH2CH3), 4.53 (t, 2H, NCH2CH2), 7.80 (t, 1H, Ar), 7.95 (m,
1H, Ar), 8.43 (m, 2H, Ar); MS (ESI): m/z 385.37 [M+H]+.

P. Biagini et al. / Bioorg. Med. Chem. 18 (2010) 3506–3517
3513
6.1.6.5.
6-Ethyl-3,4-dimethyl-2-(3-nitrophenyl)-2H-pyrazol-
6.1.9. General procedure for 14a–n
o[3,4-d]pyridazin-7(6H)-one 10e. Compound 10e was obtained
starting from 8 treating with PPA and ethylhydrazine oxalate at
80 °C for 1 h. After cooling, cold water was added and the precipi-
tate was recovered by suction. Yield = 61%; mp = 215–216 °C
(EtOH); 1H NMR (CDCl3) d 1.40 (t, 3H, NCH2CH3), 2.60 (s, 3H, 4-
CH3), 2.77 (s, 3H, 3-CH3), 4.28 (q, 2H, NCH2CH3), 7.80 (t, 1H, Ar),
7.98 (d, 1H, Ar), 8.43 (m, 2H, Ar).
A suspension of 13a–f (13a18 and 13b30) (0.15–0.57 mmol) and
the appropriate hydrazines, which are all commercially available
with exception of 1-m-methylhydroxyphenylhydrazine38, (0.39–
1.96 mmol) in absolute ethanol (3–10 mL), was stirred for 5 min–
12 h at a temperature ranging from 20 to 80 °C. Then the reaction
mixture was concentrated in vacuo and diluted with cold water.
Compounds 14a–e,k were recovered by suction and recrystallized
by ethanol. For compounds 14f–j,l–n, the suspension was ex-
tracted with CH2Cl2 (2 ꢁ 15 mL), the organic layer was washed
with 6 N HCl (10 mL), and then evaporated in vacuo. Compounds
14f–j,l–n were purified by column chromatography, using cyclo-
hexane/ethyl acetate, 1:1 for compounds 14f,i,j,l; cyclohexane/
ethyl acetate, 1:3, for compound 14h and cyclohexane/ethyl ace-
tate, 2:1, for compounds 14g,m.
6.1.7. General procedure for 12b,d,e
Compounds 12b,d,e were obtained starting from 11a25,b26 fol-
lowing the same procedure described for 10a–d. For compound
12b, the reaction was carried out in anhydrous DMF and the sus-
pension was diluted with cold water and extracted with CH2Cl2
(3 ꢁ 15 mL) to afford the final product. For compounds 12d,e anhy-
drous acetone was used as solvent and the precipitate 12d was
recovered by suction. Compound 12e was obtained after extraction
with ethyl acetate (3 ꢁ 15 mL) and evaporation in vacuo.
6.1.9.1. 6-Ethyl-3-methyl-2-(3-nitrophenyl)-4-phenyl-2H-pyraz-
olo[3,4-d]pyridazin-7(6H)-one
14a. Yield = 57%;
mp = 163–
164 °C (EtOH); 1H NMR (CDCl3) d 1.48 (t, 3H, NCH2CH3), 2.30 (s,
3H, 3-CH3), 4.38 (q, 2H, NCH2CH3), 7.52–7.63 (m, 5H, Ar), 7.80 (t,
1H, Ar), 8.00 (d, 1H, Ar), 8.43 (m, 2H, Ar).
6.1.7.1. Ethyl 3-(3-methyl-7-oxo-4-phenylisoxazolo[3,4-d]pyri-
dazin-6(7H)-yl)propanoate
12b. Yield = 75%;
mp = 94–96 °C
(EtOH); 1H NMR (CDCl3) d 1.20 (t, 3H, COOCH2CH3), 2.60 (s, 3H,
3-CH3), 2.90 (t, 2H, NCH2CH2), 4.10 (q, 2H, COOCH2CH3), 4.55 (t,
2H, NCH2CH2), 7.55 (s, 5H, Ar).
6.1.9.2. Ethyl 2-(3-methyl-2-(3-nitrophenyl)-7-oxo-4-phenyl-
2H-pyrazolo[3,4-d]pyridazin-6(7H)-yl)acetate 14b. Yield = 40%;
mp = 207–209 °C (EtOH); IR (Nujol): 1755 (CO), 1690 (CO), 1535
6.1.7.2. Ethyl 6,7-dihydro-3-methyl-7-oxo-6-propylisoxazol-
o[3,4-d]pyridazine-4-carboxylate 12d. Yield = 45%; mp = 66–
67 °C (EtOH); 1H NMR (CDCl3) d 1.00 (t, 3H, NCH2CH2CH3), 1.46
(t, 3H, COOCH2CH3), 1.86 (m, 2H, NCH2CH2CH3), 3.00 (s, 3H, 3-
CH3), 4.20 (t, 2H, NCH2CH2CH3), 4.48 (q, 2H, COOCH2CH3).
and 1350 (NO2) cmꢀ1 1H NMR (CDCl3) d 1.32 (t, 3H, COOCH2CH3),
;
2.30 (s, 3H, 3-CH3), 4.27 (q, 2H, COOCH2CH3), 5.07 (s, 2H, NCH2),
7.55 (m, 3H, Ar), 7.61 (m, 2H, Ar), 7.80 (t, 1H, Ar), 8.00 (d, 1H,
Ar), 8.44 (m, 2H, Ar); MS (ESI): m/z 433.42 [M+H]+.
6.1.7.3. Ethyl 6-(cyclopropylmethyl)-6,7-dihydro-3-methyl-7-
oxoisoxazolo[3,4-d]pyridazine-4-carboxylate 12e. Yield = 48%;
mp = 86–87 °C (EtOH); 1H NMR (CDCl3) d 0.61 (m, 4H, cC3H5),
1.36 (m, 1H, cC3H5), 1.47 (t, 3H, COOCH2CH3), 3.04 (s, 3H, 3-CH3),
4.12 (t, 2H, NCH2), 4.49 (q, 2H, COOCH2CH3).
6.1.9.3. Ethyl 3-(3-methyl-2-(3-nitrophenyl)-7-oxo-4-phenyl-
2H-pyrazolo[3,4-d]pyridazin-6(7H)-yl)propanoate
14c. Yield = 38%; mp = 152–154 °C (EtOH); 1H NMR (CDCl3) d 1.24
(t, 3H, COOCH2CH3), 2.30 (s, 3H, 3-CH3), 2.92 (t, 2H, NCH2CH2), 4.15
(q, 2H, COOCH2CH3), 4.66 (t, 2H, NCH2CH2), 7.57 (m, 5H, Ar), 7.80 (t,
1H, Ar), 8.00 (d, 1H, Ar), 8.43 (m, 2H, Ar).
6.1.8. General Procedure for 13c–f
To a suspension of 12b–e (12c26) (0.8–1.08 mmol), AcOH 50%
(7 mL) and HNO3 65% (1.5–2 mL) heated at 50–60 °C, CAN
(6.38 mmol) was added portionwise. The mixture was heated for
1–2 h at 60 °C. After dilution with cold water (10–15 mL) the sus-
pension was extracted with CH2Cl2 (2 ꢁ 15 mL); evaporation of the
solvent afforded compounds 13c,e,f. Compound 13d was recovered
by suction.
6.1.9.4. Ethyl 6-ethyl-6,7-dihydro-3-methyl-7-oxo-2H-pyrazol-
o[3,4-d]pyridazine-4-carboxylate 14d. Yield = 80%; mp = 40 °C
(EtOH); 1H NMR (CDCl3) d 1.42 (t, 3H, COOCH2CH3), 1.51 (t, 3H,
NCH2CH3), 2.63 (s, 3H, 3-CH3), 4.39–4.49 (m, 4H, (2H, COOCH2CH3;
2H, NCH2CH3)), 6.13 (exch br s, 1H, NH).
6.1.9.5. Ethyl 6-ethyl-6,7-dihydro-3-methyl-2-(3-nitrophenyl)-
7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-carboxylate
14e. Yield = 49%; mp = 219–221 °C (EtOH); 1H NMR (CDCl3) d 1.48
(m, 6H, (3H, COOCH2CH3; 3H, NCH2CH3)), 2.82 (s, 3H, 3-CH3), 4.40
(q, 2H, COOCH2CH3), 4.52 (q, 2H, NCH2CH3), 7.81 (t, 1H, Ar), 7.95 (d,
1H, Ar), 8.45 (m, 2H, Ar).
6.1.8.1. Ethyl 3-(4-acetyl–5-nitro-6-oxo-3-phenylpyridazin-1(6H)-
yl)propanoate 13c. Yield = 67%; oil; 1H NMR (CDCl3) d 1.15 (t, 3H,
COOCH2CH3), 2.15 (s, 3H, COCH3), 2.95 (t, 2H, NCH2CH2), 4.15 (q,
2H, COOCH2CH3), 4.65 (t, 2H, NCH2CH2), 7.45 (m, 5H, Ar).
6.1.8.2. Ethyl 4-acetyl-1-ethyl-1,6-dihydro-5-nitro-6-oxopyrid-
azine-3-carboxylate 13d. Yield = 53%; mp = 90–91 °C (EtOH); 1H
NMR (CDCl3) d 1.40 (m, 6H, (3H, COOCH2CH3; 3H, NCH2CH3)),
2.61 (s, 3H, COCH3), 4.41 (m, 4H, (2H COOCH2CH3; 2H, NCH2CH3)).
6.1.9.6. Ethyl 6-ethyl-6,7-dihydro-3-methyl-7-oxo-2-phenyl-2H-
pyrazolo[3,4-d]pyridazine-4-carboxylate 14f. Yield = 53%; mp =
119–121 °C (EtOH); 1H NMR (CDCl3) d 1.46 (m, 6H, (3H, COO-
CH2CH3; 3H, NCH2CH3)), 2.70 (s, 3H, 3-CH3), 4.38 (q, 2H, COOCH2-
CH3), 4.50 (q, 2H, NCH2CH3),7.54 (m, 5H, Ar).
6.1.8.3. Ethyl 4-acetyl-1,6-dihydro-5-nitro-6-oxo-1-propylpyrid-
azine-3-carboxylate 13e. Yield = 47%; mp = 58–60 °C (EtOH); 1H
NMR (CDCl3) d 1.00 (t, 3H, NCH2CH2CH3), 1.40 (t, 3H, COOCH2CH3),
1.80–1.95 (m, 2H, NCH2CH2CH3), 2.60 (s, 3H, COCH3), 4.30 (t, 2H,
NCH2CH2CH3), 4.42 (q, 2H, COOCH2CH3).
6.1.9.7. Ethyl 2-(2-chlorophenyl)-6-ethyl-6,7-dihydro-3-methyl-
7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-carboxylate
14g. Yield = 39%; mp = 113–115 °C (EtOH); 1H NMR (CDCl3) d 1.48
(m, 6H, (3H, COOCH2CH3; 3H, NCH2CH3)), 2.62 (s, 3H, 3-CH3), 4.40
(q, 2H, COOCH2CH3), 4.51 (q, 2H, NCH2CH3), 7.49 (m, 2H, Ar), 7.50–
7.60 (m, 1H, Ar), 7.63 (m, 1H, Ar).
6.1.8.4. Ethyl 4-acetyl-1-(cyclopropylmethyl)-1,6-dihydro-5-
nitro-6-oxopyridazine-3-carboxylate 13f. Yield = 45%; mp = 59–
61 °C (EtOH); 1H NMR (CDCl3) d 0.59–0.69 (m, 4H, cC3H5), 1.30–
1.40 (m, 1H, cC3H5), 1.42 (t, 3H, COOCH2CH3), 3.16 (s, 3H, COCH3),
4.23 (t, 2H, NCH2), 4.44 (q, 2H, COOCH2CH3).
6.1.9.8. Ethyl 6-ethyl-6,7-dihydro-2-(3-(hydroxymethyl) phe-
nyl)-3-methyl-7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-carboxyl-
ate 14h. Yield = 30%; mp = 152–154 °C (EtOH); IR (Nujol): 1750

3514
P. Biagini et al. / Bioorg. Med. Chem. 18 (2010) 3506–3517
(CO), 1680 (CO) cmꢀ1
;
1H NMR (DMSO-d6)
d
1.30 (t, 3H,
(m, 6H, (3H, COOCH2CH3; 3H, NCH2CH3)), 2.64 (s, 3H, 3-CH3),
4.35 (q, 2H, COOCH2CH3), 4.51 (q, 2H, NCH2CH3), 5.94 (s, 2H,
CH2Ar), 7.48 (d, 2H, Ar), 7.63 (d, 2H, Ar); MS (ESI): m/z 351.36
[M+H]+.
COOCH2CH3), 1.35 (t, 3H, NCH2CH3), 2.60 (s, 3H, 3-CH3), 4.20 (q,
2H, COOCH2CH3), 4.40 (q, 2H, NCH2CH3), 4.62 (d, 2H, CH2OH),
5.41 (exch br t, 1H, CH2OH), 7.48–7.61 (m, 4H, Ar); MS (ESI): m/z
356.38 [M+H]+.
6.1.10.2. Ethyl 2-(4-nitrobenzyl)-6-ethyl-6,7-dihydro-3-methyl-
7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-carboxylate
6.1.9.9. Ethyl 6-ethyl-6,7-dihydro-3-methyl-7-oxo-2-o-tolyl-2H-
pyrazolo[3,4-d]pyridazine-4-carboxylate 14i. Yield = 33%; mp =
94–96 °C (EtOH); 1H NMR (CDCl3) d 1.48 (m, 6H, (3H, COOCH2CH3;
3H, NCH2CH3)), 2.05 (s, 3H, 2-CH3Ar), 2.56 (s, 3H, 3-CH3), 4.40(q,
2H, COOCH2CH3), 4.50 (q, 2H, NCH2CH3), 7.27 (m, 1H, Ar), 7.36–
7.48 (m, 3H, Ar).
15b. Yield = 33%; mp = 131–132 °C (cyclohexane); IR (Nujol): 1745
(CO), 1690 (CO), 1530 and 1350 (NO2) cmꢀ1 1H NMR (CDCl3) d
;
1.40–1.50 (m, 6H, (3H, COOCH2CH3; 3H, NCH2CH3)), 2.65 (s, 3H,
3-CH3), 4.36 (q, 2H, COOCH2CH3), 4.50 (q, 2H, NCH2CH3), 6.00 (s,
2H, CH2Ar), 7.55 (d, 2H, Ar), 8.20 (d, 2H, Ar); MS (ESI): m/z
371.35 [M+H]+.
6.1.9.10. Ethyl
2-(2,6-dichlorophenyl)-6-ethyl-6,7-dihydro-3-
methyl-7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-carboxylate 14j.
Yield = 43%; mp = 216–218 °C (EtOH); 1H NMR (CDCl3) d 1.48 (m,
6H, (3H, COOCH2CH3; 3H, NCH2CH3)), 2.60 (s, 3H, 3-CH3), 4.40 (q,
2H, COOCH2CH3), 4.51 (q, 2H, NCH2CH3), 7.47–7.57 (m, 3H, Ar).
6.1.10.3. Ethyl 2-(3-(methoxycarbonyl)benzyl)-6-ethyl-6,7-dihy-
dro-3-methyl-7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-carboxyl-
ate 15c. Yield = 33%; mp = 131–132 °C (EtOH); 1H NMR (CDCl3) d
1.40–1.50 (m, 6H, (3H, COOCH2CH3; 3H, NCH2CH3)), 2.63 (s, 3H,
3-CH3), 3.92 (s, 3H, COOCH3), 4.38 (q, 2H, COOCH2CH3), 4.50 (q,
2H, NCH2CH3), 5.95 (s, 2H, CH2Ar), 7.42 (t, 1H, Ar), 7.58 (d, 1H,
Ar), 7.98 (d, 1H, Ar), 8.07 (s, 1H, Ar).
6.1.9.11. Ethyl 6,7-dihydro-3-methyl-2-(3-nitrophenyl)-7-oxo-
6-propyl-2H-pyrazolo[3,4-d]pyridazine-4-carboxylate
14k.
Yield = 35%; mp = 178–179 °C (EtOH); 1H NMR (CDCl3) d 1.00 (t,
3H, NCH2CH2CH3),1.48 (t, 3H, COOCH2CH3), 1.90 (m, 2H,
NCH2CH2CH3), 2.80 (s, 3H, 3-CH3), 4.30 (t, 2H, NCH2CH2CH3), 4.50
(q, 2H, COOCH2CH3), 7.80 (t, 1H, Ar), 7.93 (d, 1H, Ar), 8.43 (m,
2H, Ar).
6.1.11. General procedure for 16a–d
A suspension of 14e–g and 14j (0.15–0.7 mmol) in 2 N NaOH
(2–5 mL) was stirred at 25–80 °C for 40 min–2 h. After cooling,
the mixture was acidified with 6 N HCl and the precipitate was
recovered by suction.
6.1.9.12. Ethyl 2-(2-chlorophenyl)-6-(cyclopropylmethyl)-6,7-
dhydro-3-methyl-7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-car-
boxylate 14l. Yield = 21%; mp = 132–134 °C (EtOH); 1H NMR
(CDCl3) d 0.54 (m, 4H, cC3H5), 1.22–1.35 (m, 1H, cC3H5),1.48 (t,
3H, COOCH2CH3), 2.62 (s, 3H, 3-CH3), 4.21 (d, 2H, NCH2), 4.50 (q,
2H, COOCH2CH3), 7.49 (d, 2H, Ar), 7.56 (m, 1H, Ar), 7.63 (d, 1H, Ar).
6.1.11.1. 6-Ethyl-6,7-dihydro-3-methyl-2-(3-nitrophenyl)-7-oxo
-2H-pyrazolo[3,4-d]pyridazine-4-carboxylic acid 16a. Yield =
68%; mp = 227–229 °C dec (EtOH); IR (Nujol): 1730 (CO), 1680
(CO), 1530 and 1360 (NO2) cmꢀ1 1H NMR (DMSO-d6) d 1.32 (t,
;
3H, NCH2CH3), 2.60 (s, 3H, 3-CH3), 4.21 (q, 2H, NCH2CH3), 7.95 (t,
1H, Ar), 8.18 (d, 1H, Ar), 8.48 (d, 1H, Ar), 8.53 (s, 1H, Ar), 13.70
(exch br s, 1H, COOH); MS (ESI): m/z 343.29 [M+H]+.
6.1.9.13. Ethyl 2-(5-(methoxycarbonyl)-2-methylphenyl)-6-
ethyl-6,7-dihydro-3-methyl-7-oxo-2H-pyrazolo[3,4-d]pyrida-
zine-4-carboxylate 14m. Yield = 60%; mp = 192–193 °C (EtOH);
1H NMR (CDCl3) d 1.42–1.51 (m, 6H, (3H, COOCH2CH3; 3H,
NCH2CH3)), 2.12 (s, 3H, 2-CH3Ar), 2.58 (s, 3H, 3-CH3), 3.94 (s, 3H,
COOCH3), 4.41 (q, 2H, COOCH2CH3), 4.51 (m, 2H, NCH2CH3), 7.50
(d, 1H, Ar), 7.97 (dd, 1H, Ar), 8,16 (dd, 1H, Ar).
6.1.11.2. 6-Ethyl-6,7-dihydro-3-methyl-7-oxo-2-phenyl-2H-pyr-
azolo[3,4-d]pyridazine-4-carboxylic acid 16b. Yield = 90%; mp =
230–232 °C dec (EtOH); 1H NMR (DMSO-d6)
d 1.29 (t, 3H,
NCH2CH3), 2.61 (s, 3H, 3-CH3), 4.18 (q, 2H, NCH2CH3), 7.62 (m,
5H, Ar).
6.1.9.14. Ethyl 2-(5-carbomoyl-2-chlorophenyl)-6-ethyl-6,7-
dihydro-3-methyl-7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-car-
boxylate 14n. Yield = 60%; mp = 171–172 °C (EtOH); 1H NMR
(CDCl3) d 1.42–1.51 (m, 6H, (3H, COOCH2CH3; 3H, NCH2CH3)),
2.60 (s, 3H, 3-CH3), 4.30–4.48 (q, 2H, COOCH2CH3), 4.51 (m, 2H,
NCH2CH3), 6.06 (exch br s, 1H, NH2), 7.00 (exch br s, 1H, NH2),
7.69 (d, 1H, Ar), 8.07 (m, 2H, Ar).
6.1.11.3. 2-(2-Chlorophenyl)-6-ethyl-6,7-dihydro-3-methyl-7-oxo-
2H-pyrazolo[3,4-d]pyridazine-4-carboxylic acid 16c. Yield = 83%;
mp = 202–204 °C (EtOH); 1H NMR (DMSO-d6)
d 1.32 (t, 3H,
NCH2CH3), 2.48 (s, 3H, 3-CH3), 4.20 (q, 2H, NCH2CH3), 7.65 (t, 1H,
Ar), 7.72 (t, 2H, Ar), 7.84 (d, 1H, Ar).
6.1.11.4. 2-(2,6-Dichlorophenyl)-6-ethyl-6,7-dihydro-3-methyl-
7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-carboxylic acid 16d.
Yield = 72%; mp = 222–224 °C (EtOH); 1H NMR (DMSO-d6) d
1.33 (t, 3H, NCH2CH3), 2.45 (s, 3H, 3-CH3), 4.20 (q, 2H, NCH2CH3),
7.76 (m, 1H, Ar), 7.87 (m, 2H, Ar), 13.70 (exch br s, 1H, COOH).
6.1.10. General procedure for 15a–c
Compounds 15a–c were obtained starting from 14d and appro-
priate arylalkyl halides, following the same procedure described
for 10a–d. For these compounds, the reaction was carried out in
anhydrous acetone at 60 °C for 1–2 h. Compound 15a was recov-
ered by suction and purified by column chromatography using
cyclohexane/ethyl acetate, 1:1, as eluent. Compounds 15b,c were
obtained after extraction with CH2Cl2 (3 ꢁ 20 mL) and purification
by column chromatography using cyclohexane/ethyl acetate, 2:1,
as eluent for compound 15b and cyclohexane/ethyl acetate, 3:1,
as eluent for compound 15c.
6.1.12. General Procedure for 17a–f
A mixture of 16a–d (0.1–0.6 mmol), anhydrous Et3N (0.15 mL)
and SOCl2 (3–27.5 mmol) was stirred at 60 °C for 1–2 h. The excess
of SOCl2 was removed in vacuo, and then appropriate amine (0.5–
2 mmol) was added to the residue oil dissolved in cold anhydrous
THF. For compound 17f NaH (60% dispersion in mineral oil)
(0.6 mmol) was added. The mixture was stirred at room tempera-
ture for 20 min–2 h, diluted with cold water (2–5 mL), and the pre-
cipitate was recovered by suction for compounds 17a,d. For
compounds 17b,c,e,f, the suspension was neutralized with 2 N
NaOH, extracted with ethyl acetate (3 ꢁ 15 mL) and the solvent
was evaporated in vacuo. The compounds 17b,c,e were recrystal-
6.1.10.1. Ethyl 2-(4-cyanobenzyl)-6-ethyl-6,7-dihydro-3-meth-
yl-7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-carboxylate 15a.
Yield = 14%; mp = 130–131 °C (cyclohexane); IR (Nujol): 2220
(CN), 1745 (CO), 1680 (CO) cmꢀ1 1H NMR (CDCl3) d 1.40–1.50
;

P. Biagini et al. / Bioorg. Med. Chem. 18 (2010) 3506–3517
3515
lized by ethanol, compound 17f was purified by column chroma-
NCH2CH3), 2.58 (s, 3H, 3-CH3), 4.37 (q, 2H, NCH2CH3), 7.56 (m,
tography, using cyclohexane/ethyl acetate, 1:1, as eluent.
3H, Ar).
6.1.12.1. 2-(2-Chlorophenyl)-6-ethyl-6,7-dihydro-3-methyl-7-
oxo-2H-pyrazolo[3,4-d]pyridazine-4-carboxamide 17a. Yield =
78%; mp = 203–205 °C (EtOH); 1H NMR (DMSO-d6) d 1.34 (t, 3H,
NCH2CH3), 2.52 (s, 3H, 3-CH3), 4.19 (q, 2H, NCH2CH3), 7.65 (t, 1H,
Ar), 7.70 (exch br s, 1H, NH2), 7.72 (s, 1H, Ar), 7.83 (d, 1H, Ar),
8.00 (exch br s, 1H, NH2).
6.1.14. General procedure for 19a–c
Compounds 19a–c were obtained starting from 16a following
the same procedure described for 10a–d. For compound 19a, the
reaction was carried out in anhydrous acetone and the suspen-
sion was diluted with cold water and extracted with ethyl ace-
tate (3 ꢁ 15 mL) to afford the final product. For compounds
19b,c, the reaction was carried out in anhydrous DMF and after
dilution with cold water the final products were recovered by
suction.
6.1.12.2. 2-(2,6-Dichlorophenyl)-6-ethyl-6,7-dihydro-3-methyl-
7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-carboxamide 17b. Yield =
75%; mp = 243–245 °C (EtOH); 1H NMR (DMSO-d6) d 1.36 (t, 3H,
NCH2CH3), 2.52 (s, 3H, 3-CH3), 4.19 (q, 2H, NCH2CH3), 7.74 (exch
br s, 1H, NH2), 7.77 (m, 1H, Ar), 7.85 (s, 1H, Ar), 7.87 (s, 1H, Ar),
8.02 (exch br s, 1H, NH2).
6.1.14.1. Methyl 6-ethyl-6,7-dihydro-3-methyl-2-(3-nitrophen-
yl)-7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-carboxylate 19a. Yield =
96%; mp = 185–188 °C dec (EtOH); IR (Nujol): 1745 (CO), 1690
6.1.12.3. 6-Ethyl-6,7-dihydro-N,3-dimethyl-2-(3-nitrophenyl)-
7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-carboxamide 17c. Yield =
64%; mp = 250–252 °C (EtOH); IR (Nujol): 3430 (NH), 1680 (2
(CO), 1525 and 1350 (NO2) cmꢀ1 1H NMR (CDCl3) d 1.47 (t, 3H,
;
NCH2CH3), 2.84 (s, 3H, 3-CH3), 4.05 (s, 3H, COOCH3), 4.41 (q, 2H,
NCH2CH3), 7.82 (t, 1H, Ar), 7.96 (d, 1H, Ar), 8.46 (m, 2H, Ar); MS
(ESI): m/z 357.32 [M+H]+.
CO), 1525 and 1350 (NO2) cmꢀ1 1H NMR (DMSO-d6) d 1.33 (t,
;
3H, NCH2CH3), 2.70 (s, 3H, 3-CH3), 2.80 (s, 3H, NHCH3), 4.18 (q,
2H, NCH2CH3), 7.92 (t, 1H, Ar), 8.15 (d, 1H, Ar), 8.45 (d, 1H, Ar),
8.50 (s, 1H, Ar), 8.55 (exch br s, 1H, NHCH3); MS (ESI): m/z
356.34 [M+H]+.
6.1.14.2. Propyl 6-ethyl-6,7-dihydro-3-methyl-2-(3-nitrophen-
yl)-7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-carboxylate 19b. Yield =
96%; mp = 184–186 °C dec (EtOH); IR (Nujol): 1745 (CO), 1690
6.1.12.4. N,6-Diethyl-6,7-dihydro-N,3-dimethyl-2-(3-nitrophen-
yl)-7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-carboxamide 17d.
Yield = 64%; mp = 250–252 °C (EtOH); IR (Nujol): 1690 (CO),
(CO), 1530 and 1350 (NO2) cmꢀ1 1H NMR (CDCl3) d 1.08 (t, 3H,
;
CJJCH2CH2CH3), 1.47 (t, 3H, NCH2CH3), 1.89 (m, 2H, CJJCH2-
CH2CH3), 2.82 (s, 3H, 3-CH3), 4.40 (m, 4H, (2H, CJJCH2CH2CH3;
2H, NCH2CH3)), 7.81 (t, 1H, Ar), 7.95 (d, 1H, Ar), 8.45 (m, 2H, Ar);
MS (ESI): m/z 385.37 [M+H]+.
1670 (CO), 1515 and 1350 (NO2) cmꢀ1 1H NMR (CDCl3) d 1.30
;
(t, 3H, NCH2CH3),1.40 (t, 3H, N(CH3)CH2CH3), 2.60 (d, 3H, 3-
CH3), 3.1 5 (d, 3H, N(CH3)CH2CH3), 3.48 (q, 1H, N(CH3)CH2CH3),
3.65 (q, 1H, N(CH3)CH2CH3), 4.30 (q, 2H, NCH2CH3), 7.78 (t, 1H,
Ar), 7.94 (d, 1H, Ar), 8.41 (m, 2H, Ar); MS (ESI): m/z 384.39
[M+H]+.
6.1.14.3. Isopropyl 6-ethyl-6,7-dihydro-3-methyl-2-(3-nitro-
phenyl)-7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-carboxylate 19c.
Yield = 96%; mp = 164–166 °C dec (EtOH); 1H NMR (CDCl3) d
1.48 (m, 9H, (6H, CJJCH(CH3)2; 3H, NCH2CH3), 2.80 (s, 3H, 3-
CH3), 4.40 (m, 2H, NCH2CH3), 5.35 (m, 1H CJJCH(CH3)2), 7.80 (t,
1H, Ar), 7.95 (d, 1H, Ar), 8.41 (m, 2H, Ar).
6.1.12.5. 6-Ethyl-6,7-dihydro-N,N,3-trimethyl-2-(3-nitrophenyl)
-7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-carboxamide 17e. Yield =
41%; mp = 196–198 °C (EtOH); 1H NMR (CDCl3) d 1.41 (t, 3H,
NCH2CH3), 2.61 (s, 3H, 3-CH3), 3.1 9 (s, 3H, N(CH3)2), 3.2 2 (s, 3H,
N(CH3)2), 4.31 (q, 2H, NCH2CH3), 7.80 (t, 1H, Ar), 7.94 (d, 1H, Ar),
8.42 (m, 2H, Ar).
6.1.15. 2-(2-Chlorophenyl)-6-ethyl-4-(hydroxymethyl)-3-methyl-
2H-pyrazolo[3,4-d]pyridazin-7(6H)-one 20
A mixture of 16c (0.33 mmol), NaBH4 (1.98 mmol) in anhydrous
THF was heated at 70 °C for 20 min, then methanol (1 mL) was
slowly added. The mixture was stirred for 1 h, and then concen-
trated in vacuo. After dilution with cold water (10 mL), the suspen-
sion was extracted with ethyl acetate (3 ꢁ 15 mL) and the solvent
was evaporated to afford 20, which was recrystallized by ethanol.
Yield = 85%; mp = 169–171 °C dec (EtOH); IR (Nujol): 1690 (CO)
6.1.12.6. N,2,6-Dichloro(4-pyridin)-6-ethyl-6,7-dihydro-2-phenyl-
7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-carboxamide 17f. Yield =
8%; mp = 246–248 °C (EtOH); 1H NMR (CDCl3) d1.19 (t, 3H,
NCH2CH3), 2.71 (s, 3H, 3-CH3), 4.10 (m, 2H, NCH2CH3), 7.49–7.59
(m, 7H, Ar), 8.67 (exch br s, 1H, NHAr).
cmꢀ1 1H NMR (CDCl3) d 1.27 (exch br s, 1H, CH2OH), 1.43 (t, 3H,
;
NCH2CH3), 2.52 (s, 3H, 3-CH3), 4.30 (m, 2H, NCH2CH3), 4.90 (s,
2H, CH2OH), 7.49 (m, 2H, Ar), 7.55 (m, 1H, Ar), 7.61 (m, 1H, Ar);
MS (ESI): m/z 318.76 [M+H]+.
6.1.13. General procedure for 18a,b
A suspension of 17a or 17b (0.21 mmol) in POCl3 (5.81 mmol)
was stirred at 60 °C for 1–2 h. Then the reaction mixture in the cold
water (15 mL) was slowly added. The suspension was extracted
with ethyl acetate (3 ꢁ 15 mL) and the organic layer evaporated
affording a crude precipitate which was recrystallized by ethanol.
6.1.16. 2-(2-Chlorophenyl)-6-ethyl-4-(methoxymethyl)-3-methyl-
2H-pyrazolo[3,4-d]pyridazin-7(6H)-one 21
A mixture of 20 (0.22 mmol) in anhydrous DMF (3 mL) was stir-
red at 0 °C, NaH (60% dispersion in mineral oil) (0.44 mmol) was
added during 10 min and then CH3I (0.22 mmol) in anhydrous
DMF (1 mL) was added dropwise. The suspension was stirred at
room temperature for 2 h. After dilution with cold water, the mix-
ture was extracted with CH2Cl2 (3 ꢁ 15 mL) and the solvent was
evaporated in vacuo to afford 21. Yield = 85%; oil; 1H NMR (CDCl3)
d 1.42 (t, 3H, NCH2CH3), 2.52 (s, 3H, 3-CH3), 3.45 (s, 3H, CH2OCH3),
4.30 (m, 2H, NCH2CH3), 4.52–4.69 (m, 2H, CH2OCH3), 7.49 (m, 2H,
Ar), 7.54 (m, 1H, Ar), 7.61 (m, 1H, Ar).
6.1.13.1. 2-(2-Chlorophenyl)-6-ethyl-6,7-dihydro-3-methyl-7-
oxo-2H-pyrazolo[3,4-d]pyridazine-4-carbonitrile 18a. Yield =
91%; mp = 155–158 °C (EtOH); IR (Nujol): 2225 (CN), 1690 (CO)
cmꢀ1 1H NMR (CDCl3) d 1.46 (t, 3H, NCH2CH3), 2.62 (s, 3H, 3-
;
CH3), 4.37 (q, 2H, NCH2CH3), 7.51 (m, 4H, Ar); MS (ESI): m/z
313.74 [M+H]+.
6.1.13.2. 2-(2,6-Dichlorophenyl)-6-ethyl-6,7-dihydro-3-methyl-
7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-carbonitrile 18b. Yield =
70%; mp = 206–209 °C (EtOH); 1H NMR (CDCl3) d 1.47 (t, 3H,

3516
P. Biagini et al. / Bioorg. Med. Chem. 18 (2010) 3506–3517
6.1.17. N0-Acetyl-6-ethyl-6,7-dihydro-3-methyl-2-(3-nitro-
phenyl)-7-oxo-2H-pyrazolo[3,4-d]pyridazine-4-carbohydrazide
22
ployed as the stimulus (3 ng/mL, corresponding to EC84) was se-
lected on the basis of preliminary experiments, which showed
this dose producing nearly maximal stimulation of TNF-a synthe-
Compound 22 was obtained from 16a and acethydrazide, fol-
lowing the procedure described for 17a–f. The mixture was stirred
at room temperature for 10 min, diluted with cold water (3 mL),
and the precipitate was recovered by suction. Yield = 35%;
mp = 146–148 °C dec (EtOH); 1H NMR (CDCl3) d 1.43 (t, 3H,
NCH2CH3), 2.20 (s, 3H, NHCOCH3), 2.87 (s, 3H, 3-CH3), 4.31 (m,
2H, NCH2CH3), 7.41 (exch br s, 1H, NH), 7.49 (m, 2H, Ar), 7.80 (t,
1H, Ar), 7.97 (d, 1H, Ar), 8.42 (d, 1H, Ar), 8.50 (s, 1H, Ar), 9.87 (exch
br s 1H, NH).
sis. In each experimental session, the effect produced by roflumi-
last (3 nM, corresponding to its EC50) was estimated as control.
The plates were incubated for 18 h at 37 °C in a humidified
incubator under an atmosphere of 95% air and 5% CO2. Afterwards,
the culture supernatant was collected and stored at ꢀ80 °C until
determination of TNF-a contents.
6.2.3. TNF-
a
measurement
was measured using a commercially available
Human TNF-
a
ELISA kit (Biosource International, Camarillo, CA; Arcus Biologicals,
6.1.18. 6-Ethyl-3-methyl-4-(5-methyl-1,3,4-oxadiazol-2-yl)-2-
(3-nitrophenyl)-2H-pyrazolo[3,4-d]pyridazin-7(6H)-one 23
Compound 23 was obtained as a precipitate starting from 22
following the same general procedure described for 18a,b.
Yield = 40%; mp = 194–196 °C (EtOH); IR (Nujol): 1690 (CO), 1530
Modena, Italy; detection limit: 1.7 pg/mL); the assay was per-
formed according to the manufacturer’s instructions. TNF-
tent was expressed as pg/mL.
a con-
and 1350 (NO2) cmꢀ1 1H NMR (CDCl3) d 1.50 (t, 3H, NCH2CH3),
;
Supplementary data
2.73 (s, 3H, 5-CH3 oxadiazole), 3.03 (s, 3H, 3-CH3), 4.44 (m, 2H,
NCH2CH3), 7.83 (t, 1H, Ar), 7.96 (d, 1H, Ar), 8.46 (d, 1H, Ar), 8.49
(t, 1H, Ar); MS (ESI): m/z 381.35 [M+H]+.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.03.066.
6.2. Biological assays
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to stimulate TNF-
served to measure basal TNF-
a
synthesis, except that in n = 3 wells/plate which
production. LPS concentration em-
a

P. Biagini et al. / Bioorg. Med. Chem. 18 (2010) 3506–3517
3517
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