New 2-arylpyrazolo[4,3-c]quinoline derivatives as potent and selective human A3 adenosine receptor antagonists

Article abstract of DOI:10.1021/jm050125k

In this paper we report the synthesis and biological evaluation of a new class of 2-phenyl-2,5-dihydro-pyrazolo[4,3-c]quinolin-4-ones as A₃ adenosine receptor antagonists. We designed a new route based on the Kira-Vilsmeier reaction for the syn

Full text of DOI:10.1021/jm050125k

J. Med. Chem. 2005, 48, 5001-5008
5001
New 2-Arylpyrazolo[4,3-c]quinoline Derivatives as Potent and Selective Human
A3 Adenosine Receptor Antagonists
Pier Giovanni Baraldi,*,† Mojgan Aghazadeh Tabrizi,† Delia Preti,† Andrea Bovero,† Francesca Fruttarolo,†
Romeo Romagnoli,† Naser Abdel Zaid,§ Allan R. Moorman,| Katia Varani,‡ and Pier Andrea Borea‡
Dipartimento di Scienze Farmaceutiche, Dipartimento di Medicina Clinica e Sperimentale-Sezione di Farmacologia,
Universita` di Ferrara, 44100 Ferrara Italy, College of Pharmacy, An-Najah National University, Nablus, and
King Pharmaceutical R & D, 4000 CentreGreen Way, Suite 300 Cary, North Carolina 27513
Received February 9, 2005
In this paper we report the synthesis and biological evaluation of a new class of 2-phenyl-2,5-
dihydro-pyrazolo[4,3-c]quinolin-4-ones as A3 adenosine receptor antagonists. We designed a
new route based on the Kira-Vilsmeier reaction for the synthesis of this class of compounds.
Some of the synthesized compounds showed A3 adenosine receptor affinity in the nanomolar
range and good selectivity as evaluated in radioligand binding assays at human (h) A1, A2A,
A2B, and A3 adenosine receptor subtypes. We introduced several substituents on the 2-phenyl
ring. In particular substitution at the 4-position by methyl, methoxy, and chlorine gave optimal
activity and selectivity 6c (KihA1, A2A>1000 nM, EC50hA2B>1000 nM, KihA3 ) 9 nM), 6d
(KihA1, A2A>1000 nM, EC50hA2B>1000 nM, KihA3 ) 16 nM), 6b (KihA1, A2A >1000 nM, EC50-
hA2B>1000 nM, KihA3 ) 19 nM). In conclusion, the 2-phenyl-2,5-dihydro-pyrazolo[4,3-c]quinolin-
4-one derivatives described herein represent a new family of in vitro selective antagonists for
the adenosine A3 receptor.
Introduction
the study of adenosine receptor antagonists17-21 and
they focused their attention on 2-arylpyrazolo[3,4-c]-
quinoline derivatives. More recently, they have focused
on 2-aryl-1,2,4-triazolo[4,3-a]quinoxaline-1,4-diones, 2-ar-
yl-1,2,4-triazolo[4,3-a]quinoxalin-4-amino-1-ones, find-
ing potent and/or selective A3 adenosine receptor an-
tagonists.22,23 In the past few years, different classes of
nonxanthinic structures have been reported to be A3
adenosine receptor antagonists.24 In particular the
pyrazolo-triazolo-pyrimidine structure has been exten-
sively investigated by our research group.25-28 Consid-
ering the activity profile of the 2-arylpyrazolo[3,4-
c]quinoline derivatives synthesized by Colotta et al., we
decided to synthesize and biologically evaluate the
structural isomers of this class of compounds, a new
series of 2-arylpyrazolo[4,3-c]quinoline. In this account
we describe a new synthetic route for the synthesis of
2-arylpyrazolo[4,3-c]quinolines as potent and selective
A3 adenosine receptor antagonists. Our goal was to
synthesize a series of 2-arylpyrazolo[4,3-c]quinolines
variously substituted to evaluate its affinity toward A1,
A2A, A2B and A3 adenosine receptor subtypes.
Initially we investigated the nature and the position
of the substituents on the 2-phenyl ring. Para-substitu-
tion provided the most potent hA3 compounds. From our
studies it also emerged the relevance of a 4-oxo func-
tional group for A3 antagonist activity. In fact, substitu-
tion of the 4-oxo moiety with different functionalities,
like amines, ureas, hydrazides, ethers, or thioethers
induces a loss of affinity. Replacement of the 4-oxo
functional group with a thione moiety also leads to a
decrease in affinity. The N5-methylated compound (20)
shows no affinity at any of the adenosine receptor
subtypes. Enlargement of the ring from a quinoline
structure to a benzazocine structure (23) causes a loss
of affinity.
Adenosine, an endogenous modulator of a wide range
of biological functions in the nervous, cardiovascular,
renal, and immune systems, interacts with at least four
cell surface receptor subtypes classified as A1, A2A, A2B
and A3.1,2 All four adenosine receptor subtypes are
coupled via G-protein to the enzyme adenylate cyclase
in either an inhibitory (A1 and A3 subtypes) or stimu-
latory manner (A2A and A2B subtypes).3 In addition, the
A3 adenosine receptor subtype, that is distributed in
different organs (lung, liver, heart, kidney, and, in low
density, in the brain)4 also exerts its action through the
stimulation of phospholipases C4 and D5.5 The potential
therapeutic applications of activating or antagonizing
this receptor subtype have been investigated in recent
years6,7 and in particular, antagonists for the A3 receptor
may prove to be useful for the treatment of inflamma-
tion and in the regulation of cell growth.8 Activation of
A3 adenosine receptors in the rat results in hypotension
by promotion of release or inflammatory mediators from
mast cells.9 It has also been suggested that the A3
receptor plays an important role in brain ischemia,10,11
immunosuppression,12 and bronchospasm in several
animal models.13 From these pharmacological observa-
tions, highly selective A3 adenosine receptor antagonists
are being sought as potential antiasthmatic,14 antiin-
flammatory,15 and cerebroprotective agents.16 In the
past few years, Colotta et al. directed much effort toward
* To whom correspondence should be addressed: Prof: Pier Gio-
vanni Baraldi, Dipartimento di Scienze Farmaceutiche, Universita` di
Ferrara, Via Fossato di Mortara 17-19, 44100 Ferrara Italy. Tel:
+39-0532-291293. Fax: +39-0532-291296. E-mail: pgb@dns.unife.it.
† Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara.
‡ Dipartimento di Medicina Clinica
Farmacologia, Universita` di Ferrara.
§ An-Najah National University.
| King Pharmaceutical R & D.
e Sperimentale-Sezione di
10.1021/jm050125k CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/06/2005

5002 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Baraldi et al.
Scheme 1
Reagents: a: Ethanol, acetic acid, reflux; b: 1) POCl3, 0 °C, DMF 2) reflux; c: KMnO4, KOH/H2O, rt; d: ethanol, H2SO4, reflux; e: 1)
H2/C-Pd, ethanol, 2) reflux or 2-methoxy-ethanol/water, Fe/HCl conc, reflux; f: ethanol, H2/Pd-C, reflux.
Chemistry
120 °C with 2-furoic acid hydrazide or 4-chlorobenzoic
acid hydrazide, the corresponding tetracyclic compounds
15 and 16 respectively, were produced. Treating 6a with
Lawesson’s reagent afforded 18, which was alkylated
with methyl iodide to yield 19. (Scheme 3) Compound
20 was obtained by alkylation of 6a in the presence of
methyl iodide and K2CO3.
The synthesis of the benzazocine derivative 23 is
displayed in Scheme 4. Aldehyde 3a was reacted with
triphenyl-λ5-phosphanylidene-acetic acid ethyl ester
(Wittig reaction) to obtain 21, which was hydrogenated
to obtain 22 and subsequently treated with NaH to
afford 23.
The synthetic pathways employed to obtain the pyra-
zolo[4,3-c]quinoline nucleus are illustrated in Scheme
1: phenyl-hydrazones (2a-k) were obtained according
to Werber et al.29 by reacting the corresponding phe-
nylhydrazines with o-nitro-acetophenone. The pyrazole-
4-carboxaldehydes (3a-k) were obtained by the Vils-
meier reaction, adding the corresponding phenyl-
hydrazone to POCl3 and DMF. The oxidation of the
pyrazole-4-carboxaldehydes with KMnO4 yielded the
pyrazole-4-carboxylic acids (4a-k) that were trans-
formed to the respective esters (5a-k) in a mixture of
ethanol and sulfuric acid. Hydrogenation of 3a and
subsequently boiling the amine intermediate in ethanol
affords compound 7.30 Reduction of 5a, 5c, 5d, 5i-k
with hydrogen/Pd-C followed by refluxing of the amine
intermediate in ethanol afforded 6a, 6c, 6d, 6i-k, while
the reduction of 5b, e, f, g, h containing the halogen
atoms on the 2-phenyl ring, was realized with an
alternative method31 using iron powder and concen-
trated solution of HCl to yield 6b, e, f, g, h.
Schemes 2 and 3 show the synthetic routes adopted
to functionalize the 4-position of the pyrazoloquinoline
nucleus. Refluxing 6a in a mixture of POCl3/PCl5 gave
8 which was treated with the corresponding amines to
obtain 9-12. The urea derivative 14 was prepared by
adding phenylisocyanate to amine 9. Treatment of 8
with sodium methoxide provided 13. Reaction of 8 with
requisite 4-chlorobenzoic acid hydrazide at 70 °C af-
forded 17. When the same reaction was performed at
4-substituted-phenylhydrazines 1a-h are available
commercially, the 4-alkyl-phenylhydrazines 1i-k were
prepared according to the procedure reported in the
literature.32
Results and Discussion
Affinities of the pyrazolo[4,3-c]quinoline derivatives
in radioligand binding assays at hA1, hA2A, and hA3
receptors are reported in Table 1. Receptor binding
assays were performed using Chinese Hamster Ovary
cells (CHO) transfected with human A1, A2A, or A3
adenosine receptors. Potency of the compounds versus
hA2B adenosine receptors was studied by evaluating
their capability to inhibit (100 nM) NECA stimulated
cAMP levels. Moreover, the three compounds that
showed high affinity to hA3 adenosine receptors were
also studied to calculate their potencies. cAMP experi-

2-Arylpyrazolo[4,3-c]quinoline Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 5003
Scheme 2
Reagents: a: POCl3, PCl5, reflux; b: NH3 liq, ethanol/ethyl ether, 120 °C (for compound 9), corresponding amines, 120 °C, sealed tube,
sodium methylate, methanol, reflux (for compound 13); c: 4-chlorobenzoic acid hydrazide, ethanol, TEA, 70 °C; d: 2-furoic acid hydrazide
or 4-chlorobenzoic acid hydrazide, 120 °C, sealed tube, TEA, ethanol; e: phenylisocyanate, dioxane, reflux.
Scheme 3
such as methyl (6c) or methoxy (6d) at the 4-position
of the phenyl ring enhanced affinity. The presence of
an electron-withdrawing chlorine (6b) or fluorine (6g)
atom at the p-position of the phenyl ring produced
comparable affinity at the adenosine receptor subtypes.
The presence of a chlorine atom at the m-position of the
phenyl ring induced a loss of affinity, such as was seen
with the 2-(3-chlorophenyl) (6e) and 2-(3,4-dichlorophe-
nyl) (6f) derivatives. The presence of a chlorine atom
at the o-position (6h) did not affect affinity relative to
the parent (6a).
Considering the activity of compound 6c, we thought
to explore the effect of chain elongation at the p-position
of the 2-phenyl ring, creating an homologous series of
derivatives (p-ethyl, 6j; p-isopropyl, 6k; p-n-butyl, 6i).
We observed that these bulky groups decrease the
affinity. The p-ethyl derivative maintains a slight activ-
ity, but further increases in the number of carbon atoms
affords a loss of activity.
Investigation of the amide function on the pyrazolo-
quinoline nucleus provided interesting information about
the importance of this moiety for the activity of this class
of compounds. Transformation of the amide function of
the quinoline nucleus into an imine moiety (compound
7) led to a significant decrease of affinity for A3 adenos-
ine receptor. Replacement of the 4-oxo functional with
the 4-amino group (9) yielded compounds with no
significant affinity for A3 adenosine receptor. In fact the
pyrazoloquinoline-4-amine (9), pyrazoloquinoline-4-cy-
clohexylamine (10) and pyrazoloquinoline-4-(4-methyl)-
piperazine (12) were completely inactive. Only the
pyrazoloquinoline-4-benzylamine (11) showed a slight
affinity toward A3 adenosine receptor. The presence of
an urea moiety at the 4-position (14) determined a loss
of affinity. Substitution of the 4-oxo functional group
with the corresponding thio-derivative (18) or methoxy
Reagents: a: Lowesson’s reagent, toluene, reflux; b: sodium
acetate, ethanol, CH3I, reflux; c: K2CO3, CH3I, DMF, rt.
ments were performed evaluating their capability to
block the inhibitory effect mediated by Cl-IB-MECA
(100 nM). (Figure 1).
All the examined compounds proved to be almost
inactive in rA3CHO membranes showing different per-
centage of inhibition in the range 1-22% of specific
binding at a concentration of 10 µM. These results are
in agreement with low degree of sequence homology
(72%) of the A3 adenosine receptor subtypes.
The synthesis of these pyrazolo[4,3-c]quinolines has
produced some potent and selective A3 antagonists
(6a-d, 6g). No compound showed affinity at A1, A2A and
A2B adenosine receptor subtypes, while the structurally
isomeric pyrazolo[3,4-c]quinoline derivatives reported in
the literature showed good affinity toward both the A1
and A3 adenosine receptors.
The parent unsubstituted 2-phenyl (compound 6a)
showed good affinity and selectivity for the adenosine
A3 receptor. Introduction of an electron-donating group,

5004 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Baraldi et al.
Scheme 4
Reagents: triphenyl-λ5-phosphanylidene-acetic acid ethyl ester, CHCl3, 70 °C; b: ethanol, C-Pd, H2, c: NaH 60%, toluene, reflux.
Table 1. Adenosine Receptor Affinities of Derivatives 6a-d,
6g, h, j, 7, 11, 13
In conclusion, to delineate the SAR of the 2-aryl-
pyrazolo[4,3-c]quinolin-4-one class as A3 adenosine re-
ceptor antagonist, we have investigated different por-
tions of this structure, modifying the substituents. It is
emerged from this study that within the pyrazolo-
quinolone nucleus certain functionalities, such as the
amide linkage, are important for the activity of these
compounds. Modification of the 2-phenyl ring at the
para-position with sterically small groups is permitted,
and may improve the A3 adenosine receptor affinity.
a
b
c
d
hA1
Ki (nM)
hA2A
Ki (nM)
hA2B
IC50 (nM)
hA3
Ki (nM)
Compd
6a
6b
6c
6d
6g
6h
6j
>1000 (5%) >1000 (2%) >1000 (1%) 27 (23-32)
>1000 (4%) >1000 (1%) >1000 (1%) 19 (14-25)
>1000 (3%) >1000 (1%) >1000 (2%) 9 (7-11)
>1000 (1%) >1000(1%) >1000 (2%) 16 (12-23)
>1000 (10%) >1000 (10%) >1000 (10%) 27 (24-31)
>1000 (3%) >1000 (1%) >1000 (8%) 44 (36-54)
>1000 (12%) >1000 (1%) >1000 (13%) 157 ( 18
>1000 (5%) >1000 (3%) >1000 (1%) 212 (188-240)
>1000 (4%) >1000 (2%) >1000 (1%) 125 (98-159)
>1000 (16%) >1000 (5%) >1000 (10%) 166 (140-197)
>1000 (14%) >1000(8%) >1000 (11%) 220 (202-239)
7
11
13
18
Experimental Section
Chemistry General. Reactions were routinely monitored
by thin-layer chromatography (TLC) on silica gel (precoated
F245 Merck plates). Products were visualized with iodine or
potassium permanganate solution. 1H NMR spectra were
determined in CDCl3 or DMSO-d6 solutions with a Bruker AC
200 spectrometer. Peaks positions are given in parts per
million (δ) downfield from tetramethylsilane as internal
standard, and J values are given in Hz. Light petroleum ether
refers to the fractions boiling at 40-60 °C. Melting points were
determined on a Buchi-Tottoli instrument and are uncorrected.
Chromatography was performed using Merck 60-200 mesh
silica gel. All products reported showed 1H NMR spectra in
agreement with the assigned structures. Organic solutions
were dried over anhydrous magnesium sulfate. Elemental
analyses were performed by the microanalytical laboratory of
Dipartimento di Chimica, University of Ferrara, and were
within 0.4% of the calculated values.
a Displacement of specific [3H]DPCPX binding at human A1
receptors expressed in CHO cells (n ) 3-6). b Displacement of
specific [3H]ZM241385 binding at human A2A receptors expressed
in CHO cells (n ) 3-6). c Potency (IC50) of examined compounds
to inhibit 100 nM-NECA stimulation cAMP levels in hA2B CHO
cells. In parentheses are reported the % of inhibition to hA1, A2A
and A2B CHO cells. d Displacement of specific [3H]MRE3008-F20
binding at human A3 receptors expressed in CHO cells (n ) 3-6).
Data are expressed as geometric means with 95% confidence
limits. For compounds 6e, f, i, k, 9, 10, 12, 14-17, 19, 20,
23: hA1, A2A, A2B, A3 > 1000 nM.
General Procedure for the Preparation of Phenyl-
hydrazones (2a-k). Classic method for the synthesis of
phenyl-hydrazones was applied: To a solution of o-nitro-
acetophenone (50 mmol) in ethanol (50 mL), substituted
phenylhydrazines (50 mmol) and acetic acid (2 mL) were
added. The reaction mixture was refluxed for 8 h. The solvent
was removed and the residue was taken up with chloroform
and washed with a saturated solution of NaHCO3. Organic
phases were dried over anhydrous MgSO4, filtered and con-
centrated at reduced pressure to afford a residue that was used
without purification in the next reaction.
General Procedure for the Preparation of 3-
(2-Nitrophenyl)-1-phenyl-1H-pyrazole-4-carbaldehydes
(3a-k). A variant of the Vilsmeyer reaction was used: POCl3
(100 mmol) was added dropwise to anhydrous DMF (100 mmol)
at 0 °C and stirring was continued for 20 min untill the
formation of Vilsmeyer complex. The corresponding phenyl-
hydrazone (50 mmol) dissolved in DMF (20 mL) was added
and the reaction mixture was stirred for 30 min at room
temperature and then refluxed for 10 h. The reaction mixture
was portioned in water/ice (100 mL) and was allowed to cool
to 0 °C, 2 N NaOH was added and stirred for 30 min. The
precipitated product was filtered and the solid obtained was
purified by crystallization from ethanol (yield: 65-80%).
General Procedure for the Preparation of 3-(2-Nitro-
phenyl)-1-phenyl-1H-pyrazole-4-carboxylic acids (4a-k).
To a stirred solution of the corresponding 1H-pyrazole-4-
carboxaldehydes 3a-k (5 mmol) in dioxane (20 mL) and water
(5 mL), was added KOH (7.5 mmol) in water (3 mL), followed
by KMnO4 (7.5 mmol). The reaction mixture was stirred at
Figure 1. Inhibitory curves of cAMP accumulation in human
A3 adenosine receptors by adenosine antagonists blocking the
effect of 100 nM Cl-IB-MECA.
group (13), caused a decrease of A3 adenosine receptor
affinity, while the presence of a methyl-thio moiety in
this position (19) led to a complete loss of affinity.
Replacement of the 4-oxo moiety with an hydrazide (17)
or the corresponding tetracyclic compounds obtained by
cyclization (15 and 16), afforded inactive ligands at the
adenosine receptors.
Alkylation of the nitrogen at the 5-position (20) caused
a loss of affinity. Indeed the presence of an hydrogen
atom in this position appears essential for the activity
of this class of compounds.
Finally, we investigated the effects of the cycle
enlargement, switching from a quinoline nucleus to a
benzazocine nucleus (23). This compound was inactive.

2-Arylpyrazolo[4,3-c]quinoline Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 5005
room temperature and monitored by TLC. The excess of
KMnO4 was decomposed by addition of diluted H2O2. The
reaction mixture was filtered through a Celite pad, the filtrate
was evaporated under reduced pressure, and the residue was
acidified with HCl solution 10% (100 mL). The white solid
precipitate that formed was filtered and washed with water.
Recrystallization of the crude product from ethanol provided
the title compounds. (yield: 70-80%)
General Procedure for the Preparation of Ethyl 3-(2-
nitrophenyl)-1-phenyl-1H-pyrazole-4-carboxylates (5a-
k). To a solution of the corresponding 3-(2-nitrophenyl)-1-
phenyl-1H-pyrazole-4-carboxylic acid (5 mmol) in absolute
ethanol (30 mL), H2SO4 (2 mL) was added and refluxed for 8
h. The reaction mixture was concentrated in vacuo, the residue
was neutralized with a saturated solution of NaHCO3. The
white solid precipitated was filtered and purified by crystal-
lization from ethanol. (yield: 90-95%)
General Procedure for the Preparation of 2-
Phenyl-2H-pyrazolo[4,3-c]quinolin-4(5H)-ones (6a, 6c,
6d, 6i-k). To a solution of the corresponding ethyl 3-(2-
nitrophenyl)-1-phenyl-1H-pyrazole-4-carboxylate (5 mmol) in
absolute ethanol (100 mL), 10% Pd-C (0.5 g) was added
portion wise. The reaction mixture was hydrogenated (50 psi)
at room temperature for 2 h, then filtered through a Celite
pad and the filtrate was stirred at reflux for 8 h. Ethanol was
evaporated under reduced pressure and the oily residue was
purified by crystallization from ethanol to give a white solid.
(yield: 60-65%)
refluxed for 4 h under stirring. Water (10 mL), was added and
the reaction mixture was stirred for 10 min at room temper-
ature and filtered. (yield 86%)
1-Phenyl-3-(2-phenyl-2H-pyrazolo[4,3-c]quinolin-4-yl)-
urea (14). To a solution of compound 9 (0.17 mmol) in
anhydrous dioxane (50 mL) was added phenyisocyanate (0.1
mL). The reaction mixture was refluxed for 10 h with stirring,
then allowed to cool to room temperature. The solvent was
evaporated in vacuo to yield a solid. Recrystallization of the
crude product from dioxane provided the title compound as a
white solid. (yield: 75%).
2-Phenyl-6-(furan-2-yl)-2H-pyrazolo[4,3-c][1,2,4]triazolo-
[4,3-a]quinoline (15) and 2-Phenyl-6-(4-chlorophenyl)-
2H-pyrazolo[4,3-c][1,2,4]triazolo[4,3-a]quinoline (16). A
solution of 8 (0.36 mmol), furan-2-carbohydrazide or 4-chlo-
robenzohydrazide (0.54 mmol) and triethylamine (0.36 mmol)
in ethanol (5 mL) was heated in a sealed tube at 120 °C for 10
h. The reaction mixture was concentrated in vacuo, the residue
was suspended in ethyl acetate and filtered. The product was
purified by column chromatography (ethyl acetate/petroleum
ether 7:3) to afford a white solid. (yield 70%).
4-Chloro-N′-(2-phenyl-2H-pyrazolo[4,3-c]quinolin-4-yl)-
benzohydrazide (17). A solution of 8 (0.50 mmol), 4-chloro-
benzohydrazide (0.75 mmol) and triethylamine (0.5 mmol) in
ethanol (10 mL) was heated at 70 °C for 1 h. The precipitate
was filtered, washed with water and purified by crystallization
from DMF/water to provide compound 17 as a yellow solid.
(yield: 60%).
General Procedure for the Preparation of 2-
Phenyl-2H-pyrazolo[4,3-c]quinolin-4(5H)-ones (6b, 6e,
6f-h). To a solution of the appropriate 3-(2-nitrophenyl)-1-
phenyl-1H-pyrazole-4-carboxylate (0.2 mmol) in 2-methoxy-
ethanol (10 mL) and water (2.5 mL), iron powder (100 mg)
was added followed by concentrated HCl (0.1 mL). The mixture
was stirred at reflux for 1 h, then cooled and filtered through
a Celite pad. The filter cake was washed well with hot
2-methoxyethanol and the filtrate was diluted with water to
precipitate the product that was purified by crystallization
from ethanol. (yield: 65-70%)
2-Phenyl-2H-pyrazolo[4,3-c]quinoline (7). To a solution
of 3a (5 mmol) in absolute ethanol (100 mL) was added Pd-C
(0.5 g) portion wise. The reaction mixture was hydrogenated
(50 psi) at room temperature for 2 h, then was heated to reflux
with stirring for 30 min. After filtration through a Celite pad,
the filtrate was evaporated under reduced pressure and the
oily residue was purified by crystallization from ethanol/water
to give a white solid. (yield: 90%)
4-Chloro-2-phenyl-2H-pyrazolo[4,3-c]quinoline (8). A
mixture of 6a (1.15 mmol), POCl3 (10.5 mL) and PCl5 (0.35
mmol) was refluxed with stirring for 2 h. The solvent was
evaporated and the crude solid obtained was suspended in
water/ice (30 mL) and filtered to afford the product as a white
solid. (Yield: 90%).
2-Phenyl-2H-pyrazolo[4,3-c]quinolin-4-amine (9). A so-
lution of 8 (1 mmol) and liquid NH3 (5 mL) in 20 mL of ethanol/
ethyl ether 9:1 was heated in a sealed tube at 120 °C for 7 h.
The reaction mixture was evaporated in vacuo and the solid
residue was dissolved in ethyl ether, heated and filtered to
afford a white solid. (yield: 83%)
2-Phenyl-2H-pyrazolo[4,3-c]quinoline-4(5H)-thione (18).
To a solution of 6a (0.20 mmol) in anhydrous toluene (5 mL)
was added Lawesson’s reagent (0.10 mmol), and the reaction
mixture was heated at reflux under a nitrogen atmosphere
for 15 min. The solvent was evaporated under reduced pres-
sure, the residue was dissolved in ethanol, heated and filtered
to remove Lawesson’s reagent. The filtrate was evaporated
under reduced pressure and the residue was crystallized from
ethanol. (yield: 90%)
4-(Methylthio)-2-phenyl-2H-pyrazolo[4,3-c]quinoline
(19). To a solution of 18 (0.35 mmol) in ethanol (10 mL) was
added anhydrous sodium acetate (0.70 mmol) and methyl
iodide (0.70 mmol). The reaction mixture was heated at reflux
for 2 h. Then the solvent was evaporated under reduced
pressure, to the residue was added 10 mL of water, and the
precipitate was collected by filtration to afford the title
compound. (yield: 65%)
5-Methyl-2-phenyl-2H-pyrazolo[4,3-c]quinolin-4(5H)-
one (20). A solution of 6a (0.35 mmol), K2CO3 (0.40 mmol)
and iodomethane (0.50 mmol) in DMF (5 mL) was stirred for
24 h at room temperature. The solvent was evaporated and
the reaction mixture was suspended in water, filtered and
purified by crystallization from DMF/water. (yield: 75%)
(E)-ethyl 3-(3-(2-nitrophenyl)-1-phenyl-1H-pyrazol-4-
yl)acrylate (21). To a solution of 3a (30 mmol) in chloroform
(20 mL) triphenyl-λ5-phosphanylidene)-acetic acid ethyl ester
(30 mmol) was added, and the reaction mixture was heated
at 70 °C with stirring for 3 h. The solvent was evaporated and
the residue was purified by crystallization from ethyl ether/
petroleum ether to afford a yellow solid. (yield: 80%).
Ethyl 3-(3-(2-aminophenyl)-1-phenyl-1H-pyrazol-4-yl)-
propanoate (22). To a solution of 21 (0.50 mmol) in absolute
ethanol (10 mL), Pd-C (0.26 g) was added portion wise. The
reaction mixture was hydrogenated at room temperature at
50 psi for 3 h. The suspension was filtered through a Celite
pad and the filtrate was evaporated under reduced pressure.
The residue was purified by column chromatography (ethyl
ether/petroleum ether 1:1), to afford an oil (yield: 90%).
4-Phenyl-3,4,10-triaza-tricyclo[9.4.0.2,6]pentadeca-
1(15),2,5,11,13-pentaen-9-one (23). A solution of 22 (0.30
mmol) and NaH 60% (300 mg) in anhydrous toluene (10 mL)
was heated at reflux for 6 h and allowed to cool to room
temperature. The mixture was diluted with water, the organic
phase was separated, washed with water (10 mL), and
evaporated. Compound 23 was purified by crystallization from
ethyl acetate/petroleum ether. (yield: 90%)
N-Cyclohexyl-2-phenyl-2H-pyrazolo[4,3-c]quinolin-4-
amine (10), N-benzyl-2-phenyl-2H-pyrazolo[4,3-c]quino-
lin-4-amine (11) and 4-(4-methylpiperazin-1-yl)-2-phenyl-
2H-pyrazolo[4,3-c]quinoline (12). A solution of 8 (0.3 mmol)
in 1 mL of cyclohexylamine, benzylamine or N-methyl-pipera-
zine was heated in a sealed tube at 120 °C for 8 h. The reaction
mixture was cooled, ethyl ether (5 mL) was added and the
product was filtered. The title compounds 10 and 12 were
purified by crystallization respectively from ethyl-ether/hexane
and ethanol/water, while the compound 11 was purified by
column chromatography (ethyl acetate/petroleum ether 1:1).
(yield: 70-80%)
4-Methoxy-2-phenyl-2H-pyrazolo[4,3-c]quinoline (13).
To a solution of compound 8 in methanol (10 mL) was added
sodium methoxide (1.75 mmol). The reaction mixture was

5006 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Baraldi et al.
Biology Experiments
recombinant A3 adenosine receptors was previously
performed.37 Competition experiments were carried out
in duplicate in a final volume 250 µl in test tubes
containing 1 nM [125I]-AB-MECA, 50 mM Tris HCl
buffer, 10 mM MgCl2, pH 7.4 and 20 µl of diluted
membranes (12 µg of protein/assay) and at least 6-8
different concentration of examined ligands for 60 min
at 37 °C. Nonspecific binding was defined as binding in
the presence of 50 µM R-PIA and was about 30% of
total binding. Bound and free radioactivity were sepa-
rated by filtering the assay mixture through Whatman
GF/B glass fiber which were washed three times with
ice-cold buffer. Filter bound radioactivity was measured
by scintillation spectrometry (SL-1800 Beckman) after
addition of Aquensure liquid.
Measurement of cyclic AMP levels in CHO cells
transfected with human A2B and A3 adenosine
receptors. CHO cells transfected with human A2B or
A3 adenosine receptors were washed with phosphate-
buffered saline, diluted trypsin and centrifuged for 10
min at 200 g. The pellet containing the CHO cells (1 ×
106 cells/assay) was suspended in 0.5 mL of incubation
mixture (mM): NaCl 15, KCl 0.27, NaH2PO4 0.037,
MgSO4 0.1, CaCl2 0.1, Hepes 0.01, MgCl2 1, glucose 0.5,
pH 7.4 at 37 °C, 2 IU/ml adenosine deaminase and 4-(3-
butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-
1724) as phosphodiesterase inhibitor and preincubated
for 10 min in a shaking bath at 37 °C. The potencies of
antagonists studied versus hA2B adenosine receptors
were determined by antagonism of NECA (100 nM)-
induced stimulation of cyclic AMP levels. The potencies
of antagonists examined versus hA3 adenosine receptors
were determined by antagonism of Cl-IB-MECA (100
nM)-induced inhibition of cyclic AMP levels. The reac-
tion was terminated by the addition of cold 6% trichlo-
roacetic acid (TCA). The TCA suspension was centri-
fuged at 2000 g for 10 min at 4 °C and the supernatant
was extracted four times with water-saturated diethyl
ether. The final aqueous solution was tested for cyclic
AMP levels by a competition protein binding assay.
Samples of cyclic AMP standard (0-10 pmoles) were
added to each test tube containing the incubation buffer
(trizma base 0.1 M, aminophylline 8.0 mM, 2 mercap-
toethanol 6.0 mM, pH 7.4) and [3H] cyclic AMP in a total
volume of 0.5 mL. The binding protein previously
prepared from beef adrenals, was added to the samples
previously incubated at 4 °C for 150 min, and after the
addition of charcoal were centrifuged at 2000 g for 10
min. The clear supernatant was counted in a LS-1800
Beckman scintillation counter.
CHO membranes preparation. The human A1, A2A,
2B and A3 receptors has been transfected in CHO cells
A
according to the method previously described.33 The cells
were grown adherently and maintained in Dulbecco’s
modified Eagles medium with nutrient mixture F12
(DMEM/F12) without nucleosides, containing 10% fetal
calf serum, penicillin (100 U/ml), streptomycin (100 µg/
mL), L-glutamine (2 mM) and Geneticin (G418, 0.2 mg/
mL) at 37 °C in 5% CO2/95% air. Cells were split 2 or 3
times weekly at a ratio between 1:5 and 1:20. For
membrane preparation the culture medium was re-
moved and the cells were washed with PBS and scraped
off T75 flasks in ice-cold hypotonic buffer (5 mM Tris
HCl, 2 mM EDTA, pH 7.4). The cell suspension was
homogenized with Polytron and the homogenate was
spun for 10 min at 1000 × g. The supernatant was then
centrifuged for 30 min at 100000 × g. The membrane
pellet was suspended in 50 mM Tris HCl buffer pH 7.4
(for A3 adenosine receptors: 50 mM Tris HCl, 10 mM
MgCl2, 1 mM EDTA) and incubated with 3 IU/ml of
adenosine deaminase for 30 min at 37 °C. Then the cell
suspension was frozen at -80 °C.
Human cloned A1, A2A and A3 Adenosine Recep-
tor Binding Assay. All synthesized compounds have
been tested for their affinity at human A1, A2A and A3
adenosine receptors. Displacement experiments of [3H]-
DPCPX to CHO cells transfected with the human
recombinant A1 adenosine receptor were performed for
120 min at 25 °C in 0.2 mL of 50 mM Tris HCl buffer
pH 7.4 containing 1 nM [3H]-DPCPX, diluted mem-
branes (50 µg of protein/assay) and at least 6-8 different
concentrations of antagonists studied. Non specific
binding was determined in the presence of 10 µM of
CHA and this was always e 10% of the total binding.34
Binding of [3H]-ZM 241385 to CHO cells transfected
with the human recombinant A2A adenosine receptors
(50 µg of protein/assay) was performed using 0.2 mL 50
mM Tris HCl buffer, 10 mM MgCl2 pH 7.4 and at least
6-8 different concentrations of antagonists studied for
an incubation time of 60 min at 4 °C. Nonspecific
binding was determined in the presence of 1 µM ZM
241385 and was about 20% of total binding.35
Binding of [3H]-MRE 3008F20 to CHO cells trans-
fected with the human recombinant A3 adenosine recep-
tors was previously performed.36 Competition experi-
ments were carried out in duplicate in a final volume
of 250 µL in test tubes containing 1 nM [3H]-MRE
3008F20, 50 mM Tris HCl buffer, 10 mM MgCl2, 1 mM
EDTA, pH 7.4 and 100 µL of diluted membranes (50 µg
of protein/assay) and at least 6-8 different concentra-
tions of examined ligands for 120 min at 4 °C. Non-
specific binding was defined as binding in the presence
of 1 µM MRE 3008F20 and was about 25% of total
binding. Bound and free radioactivity were separated
by filtering the assay mixture through Whatman GF/B
glass filters which were washed three times with ice-
cold buffer. Filter bound radioactivity was measured by
scintillation spectrometry (LS-1800 Beckman) after
addition of Aquensure liquid.
Data Analysis. The protein concentration was de-
termined according to a Bio-Rad method38 with bovine
albumin as a standard reference. Inhibitory binding
constants, Ki values, were calculated from those of IC50
according to Cheng & Prusoff equation39 Ki ) IC50/(1+-
[C*]/KD*), where [C*] is the concentration of the radio-
ligand and KD* its dissociation constant. A weighted
nonlinear least-squares curve fitting program LIGAND40
was also used for computer analysis of inhibition
experiments. Potency values (IC50) obtained in cyclic
AMP assays were calculated by nonlinear regression
analysis using the equation for a sigmoid concentra-
tion-response curve (Graph PAD Prism, San Diego,
CA). Affinity values are expressed as geometric mean
Rat cloned A3 Adenosine Receptor Binding As-
say. All synthesized compounds have been tested for
their affinity to rat A3 adenosine receptors. Binding of
[
125I]-AB-MECA to CHO cells transfected with the rat

2-Arylpyrazolo[4,3-c]quinoline Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 5007
1-aryl-1,4-dihydro-3-methyl[1]benzopyrano[2,3-c]pyrazol-4-
ones, 1-aryl- 4,9-dihydro-3-methyl-1H-pyrazolo[3,4-b]quinolin-
4-ones, and 1-aryl-1H-imidazo[4,5-b]quinoxalines. J. Med. Chem.
1995, 38, 1330-1336.
with 95% or 99% confidence limits in parentheses and
IC50 values are expressed as the arithmetic mean (
s.e.mean.
(19) Colotta, V.; Catarzi, D.; Varano, F.; Cecchi, L.; Filacchioni, G.;
Martini, C.; Trincavelli, L.; Lucacchini, A. 4-Amino-6-benzyl-
amino-1,2-dihydro-2-phenyl-1,2,4-triazolo[4,3a] quinoxalin-1-
Acknowledgment. We thank King Pharmaceutical
Research and Development, Inc., 4000 CentreGreen
Way, Suite 300 Cary, NC 27513, for financial support.
We also thank Prof. Karl-Norbert Klotz for cDNA
encoding the human adenosine receptors.
one:
a new A2A adenosine receptor antagonist with high
selectivity versus A1 receptors. Arch. Pharm. (Weinheim, Ger.
1999, 332, 39-41.
(20) Colotta, V.; Catarzi, D.; Varano, F.; Cecchi, L.; Filacchioni, G.;
Martini, C.; Trincavelli, L.; Lucacchini, A. Synthesis and struc-
ture-activity relationships of a new set of 2-arylpyrazolo[3,4-
c]quinoline derivatives as adenosine receptor antagonists. J.
Med. Chem. 2000, 43, 3118-3124.
(21) Colotta, V.; Catarzi, D.; Varano, F.; Calabri, F. R.; Lenzi, O.;
Filacchioni, G.; Martini, C.; Trincavelli, L.; Delforian, F.; Moro,
S. 1,2,4-triazolo[4,3-a]quinoxalin-1-one moiety as an attractive
scaffold to develop new potent and selective human A3 adenosine
receptor antagonists: synthesis, pharmacological, and ligand-
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quinoxalin-1-one: a versatile tool for the synthesis of potent and
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(23) Colotta, V.; Catarzi, D.; Varano, F.; Filacchioni, G.; Martini, C.;
Trincavelli, L.; Lucacchini, A. Synthesis and structure-activity
relationships of a new set of 1,2,4-Triazolo[4,3-a]quinoxalin-1-
one Derivatives as Adenosine Receptor Antagonists. Bioorg. Med.
Chem. 2003, 11, 3541-3550.
(24) Baraldi, P. G.; Aghazadeh Tabrizi, M.; Fruttarolo, F.; Bovero,
A.; Avitabile, B.; Preti, D.; Romagnoli, R.; Merighi, S.; Gessi, S.;
Varani, K.; Borea, P. A. Recent Developments in the Field of A3
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(25) Baraldi, P. G.; Cacciari, B.; Romagnoli, R.; Klotz, K. N.; Leung,
E.; Varani, K.; Gessi, S.; Meighi, S.; Borea, P. A. Pyrazolo[4,3-
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and Selective Human A3 Adenosine Receptor Antagonists. J.
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(26) Baraldi, P. G.; Cacciari, B.; Romagnoli, R.; Spalluto, S.; Moro,
S.; Klotz, K. N.; Leung, E.; Varani, K.; Gessi, S.; Merighi, S.;
Borea, P. A. Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine De-
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Receptor Antagonists: Influence of the Chain at the N8 Pyrazole
Nitrogen. J. Med. Chem. 2000, 43, 4768-4780.
(27) Baraldi, P. G.; Cacciari, B.; Moro, S.; Romagnoli, R.; Ji, X. D.;
Jacobson, K. A.; Gessi, S.; Borea, P. A.; Spalluto, G. Fluorosul-
fonyl- and Bis-(â-chloroethyl)amino-phenylamino Functionalized
Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine Derivatives: Ir-
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Molecular Modelling Studies. J. Med. Chem. 2001, 44, 2735-
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(28) Baraldi, P. G.; Cacciari, B.; Moro, S.; Spalluto, G.; Pastorin, G.;
Da Ros, T.; Klotz, K. N.; Varani, K.; Gessi, S.; Borea, P. A.
Synthesis, Biological Activity, and Molecular Modeling Inves-
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Supporting Information Available: Spectroscopic and
elemental analysis data. This material is available free of
charge via the Internet at http://pubs.acs.org.
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