Synthesis and pharmacological evaluations of sildenafil analogues for treatment of erectile dysfunction

Article abstract of DOI:10.1021/jm701400r

The 5-[2-ethoxy-5-(4-methylpiperazin-1-ylsulfonyl)phenyl]-1-methyl-3- propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, sildenafil, is a cGMP-specific phosphodiesterase-5 (PDE5) inhibitor used for penile erectile dysfunction. In the search for more potent and selective PDE5 inhibitors, new sildenafil analogues (6a-v), characterized by the presence on the sulfonyl group in the 5′ position of novel N-4-substituted piperazines or ethylenediamine moiety, were prepared by traditional and microwave-assisted synthesis and tested in rabbit isolated aorta and corpus cavernosum. Similarly to sildenafil, several analogues showed IC₅₀ values in the nanomolar range. In the in vitro studies, all the tested compounds caused concentration-dependent relaxations in both rabbit isolated aorta and corpus cavernosum. All sildenafil analogues potentiated the nitric oxide-dependent vasodilation in endothelium-intact rabbit aorta. Compound 6f exhibited great pEC₅₀ value in corpus cavernosum, and compounds 6r and 6u in isolated aorta were found as potent as sildenafil for inhibiting PDE5. Because several analogues were significantly more lipophilic than sildenafil, these compounds may offer a new lead for development of new sildenafil analogues.

Full text of DOI:10.1021/jm701400r

J. Med. Chem. 2008, 51, 2807–2815
2807
Synthesis and Pharmacological Evaluations of Sildenafil Analogues for Treatment of Erectile
Dysfunction
Haroldo A. Flores Toque,† Fernanda B. M. Priviero,† Cleber E. Teixeira,† Elisa Perissutti,‡ Ferdinando Fiorino,‡
Beatrice Severino,‡ Francesco Frecentese,‡ Raquel Lorenzetti,† Juliana S. Baracat,† Vincenzo Santagada,‡ Giuseppe Caliendo,‡
Edson Antunes,† and Gilberto De Nucci*,†
Department of Pharmacology, Faculty of Medical Sciences, State UniVersity of Campinas (UNICAMP), Campinas, Brazil, Dipartimento di
Chimica Farmaceutica e Tossicologica, UniVersità di Napoli “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy
ReceiVed NoVember 7, 2007
The 5-[2-ethoxy-5-(4-methylpiperazin-1-ylsulfonyl)phenyl]-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-
d]pyrimidin-7-one, sildenafil, is a cGMP-specific phosphodiesterase-5 (PDE5) inhibitor used for penile erectile
dysfunction. In the search for more potent and selective PDE5 inhibitors, new sildenafil analogues (6a-v),
characterized by the presence on the sulfonyl group in the 5′ position of novel N-4-substituted piperazines
or ethylenediamine moiety, were prepared by traditional and microwave-assisted synthesis and tested in
rabbit isolated aorta and corpus cavernosum. Similarly to sildenafil, several analogues showed IC50 values
in the nanomolar range. In the in vitro studies, all the tested compounds caused concentration-dependent
relaxations in both rabbit isolated aorta and corpus cavernosum. All sildenafil analogues potentiated the
nitric oxide-dependent vasodilation in endothelium-intact rabbit aorta. Compound 6f exhibited great pEC50
value in corpus cavernosum, and compounds 6r and 6u in isolated aorta were found as potent as sildenafil
for inhibiting PDE5. Because several analogues were significantly more lipophilic than sildenafil, these
compounds may offer a new lead for development of new sildenafil analogues.
Introduction
The compound 5-[2-ethoxy-5-(4-methylpiperazin-1-ylsulfo-
nyl)phenyl]-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-
d]pyrimidin-7-one (sildenafil), is a selective and potent inhibitor
of PDE5, which elevates intracellular cGMP levels, enhances
NO-dependent corpus cavernosum relaxation in vitro,7–11 and
augments the penile erection in vivo.11,12 Elevation of cGMP
levels causes a decrease in intracellular calcium concentration,
leading to enhanced relaxation of smooth muscle, increased
arterial inflow, venous congestion, thus ameliorating the erectile
dysfunction. PDE5 is a therapeutic target of considerable
research interest as can be seen by numerous publications
regarding the design, synthesis, and optimization of new PDE5
inhibitors such as 1-[[3-(1,4-dihydro-5-methyl-4-oxo-7-propyl-
imidazo[5,1-f][1,2,4]triazin-2-yl)-4-ethoxyphenyl]sulfonyl]-4-
ethyl-piperazine (vardenafil), (6R-trans)-6-(1,3-benzodioxol-5-
yl)-2,3,6,7,12,12a-hexahydro-2-methyl-pyrazino[1,2:1,6]pyrido[3,4-
b]indole-1,4-dione (tadalafil) and other compounds under clinical
trials (see Chart 1).13–16
Cyclic nucleotide phosphodiesterases (PDEs) are enzymes
that catalyze the hydrolysis of cyclic nucleotides, cAMP and
cGMP, to their respective 5′-nucleoside monophosphate by
cleavage of the phosphodiester bond at the 3′-position.1 To date,
11 families of PDEs have been classified according to the
sequence homology and biochemical properties.2,3 Phosphodi-
esterase 5 (PDE5)a, a cGMP-binding cGMP-specific PDE, is
widely distributed in vascular and visceral smooth muscle cells,
platelets, kidney, lung, and corpus cavernosum and plays a key
role in the regulation of the cellular level of cGMP. The main
therapeutic indications for PDE5 inhibitors are the treatment
of erectile dysfunction and idiopathic pulmonary hypertension,
although several other potentials have also been identified such
as systemic hypertension and prosthate hyperplasia.4 Common
side-effects include headache, facial flushing, nasal congestion,
dyspepsia, and transient visual impairment.5 Although some of
these secondary effects such as visual impairment were at-
tributed to unspecific inhibition of PDE6, recent data demon-
strate that human retina expresses both PDE5 and PDE6
isoforms.6
To develop novel PDE5 inhibitors with improved therapeutic
efficacy based on the structure of sildenafil, we have explored
a series of sildenafil analogues in which the methyl-piperazine
moiety was replaced with novel N-4-substituted-piperazines. A
large number of aryl-, alkyl-, and cycloalkyl- substituents were
selected with a large variety of hydrophobic, electronic, and
steric properties. Moreover, to investigate the importance of the
piperazine moiety, we have designed a novel series of deriva-
tives in which an ethylenediamine structure has been introduced
in place of the piperazine ring. These modifications led to a
large set of sildenafil analogues (6a-v), which were prepared
by traditional and microwave-assisted synthesis. Because sildena-
fil is being used not only for treatment of male erectile
dysfunction but also for pulmonary hypertension, we have
evaluated the effect of the newly synthesized compounds as
PDE5 inhibitors in both corpus cavernosum and aorta. The aim
* To whom correspondence should be addressed. Phone: +55-19-
35219555. Fax: +55-19-32892968. E-mail: denucci@gdenucci.com. Ad-
dress: Gilberto De Nucci, Department of Pharmacology, Faculty of Medical
Sciences, State University of Campinas-UNICAMP, PO Box 6111, Campi-
nas, SP 13084-971, Brazil.
†
Department of Pharmacology, Faculty of Medical Sciences, State
University of Campinas.
‡
Dipartimento di Chimica Farmaceutica e Tossicologica, Università di
Napoli “Federico II”.
a Abbreviations: PDE5, phosphodiesterase type-5; DMSO, dimethyl
sulfoxide; WP, washed platelets; EDTA, diethylene (2-aminoethylether)-
N,N,N′,N′-tetra-acetic acid; NO, nitric oxide; sGC, soluble guanylate cyclase;
PE, phenylephrine; ACh, acetylcholine; PRP, platelets-rich plasma; ACD
buffer, acid-citrate dextrose buffer; TEA, triethylamine; DMAP, dimethyl-
aminopyridine; ESI/MS, electrospray ionization mass spectrometry; 1H
NMR, nuclear magnetic resonance spectra for proton.
10.1021/jm701400r CCC: $40.75
2008 American Chemical Society
Published on Web 04/05/2008

2808 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Chart 1. Commercially Available PDE5 Inhibitors
Flores Toque et al.
Table 1. Conventional Heating vs Microwave Irradiation for Intermediates 3-5 and Final Compounds 6a-va
conventional heatingb
microwave irradiation
compd
yield (%)
time (h)
temp (°C)
yield (%)
time (min)
power (W)
temp (°C)
3
4
5
40
72
97
38–70
65
2
2.5
2
2
2
25
90
25
25
70
25
25
75
60
5
25
200
300
25
100
6a-o
6p
6q
6r-v
65–90c
96
5
15
200
200
30
90
70
30–60
24
2
60–92c
5
200
30
a The experimental conditions used on microwave irradiation were similar to those used by conventional heating, with the same amount of starting
reagent and solvent. More details on the experimental conditions of the reactions are reported in the Experimental Section. b Oil-bath. c Ranging between the
reported percentage.
of the present study is also to investigate the effects of
compounds 6a-v on PDE5 in human platelets.
7(6H)-one (4). Reaction of 4 with chlorosulphonic acid yielded
the desired key intermediate 4-ethoxy-3-(6,7-dihydro-1-methyl-
7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)benzene-1-
sulfonyl chloride (5a). Alkylation of intermediate 5a with the
appropriate N-4-substituted-piperazine gave the desired final
compounds 5-{2-ethoxy-5-[(4-R)piperazinylsulphonyl]phenyl)-
1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-
7-ones (6a-o). Alternatively, treatment of intermediate 5a with
the appropriate N-N′-substituted ethylenediamine yielded final
compounds 5-(2-ethoxy-5-R′,R′-ethylenediamine-sulfonyl)phe-
nyl)-1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo-[4,3d]-py-
rimidin-7-ones (6r-t). It is worth noting that, from the reaction
of intermediate 5a with N-ꢀ-aminoethyl glycine, also the dimer
6u was isolated. The synthesis of compounds 6r and 6s did not
provide dimers because of the lack of nucleophilicity of
nitrogens linked to the aromatic rings of N-phenyl- and
N-naphthyl-ethylenediamine, respectively. On the other hand,
when 5a was treated with 2-(2-amino-ethylamino)-ethanol only,
dimer 6v was isolated without the formation of the monosub-
stituted derivative and this could be addressed to the high
nucleophilicity of the starting reagent. Scheme 2 represents the
synthesis of the final compounds 6p and 6q. The key intermedi-
ate 5-{2-ethoxy-5-[(4-hydroxyethyl)piperazinylsulphonyl]phenyl}-
1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-
7-one (5b) was prepared according to the procedure reported
in Scheme 1 starting from 5a and N-hydroxyethyl-piperazine.
Acylation of 5b with decanoyl chloride, in anhydrous CHCl3,
Chemistry
The preparation of some intermediates and final compounds
(6a-v) was performed with traditional and microwave-assisted
synthesis using a microwave oven especially designed for
organic chemistry. The experimental conditions used in the
microwave-assisted synthesis were similar to those used in the
conventional heating with the same concentration of starting
materials and volume of solvent.
All reactions were performed in sealed vessels using an
appropriate microwave program, composed by ramping and
holding steps. The optimum profile power/time and temperature
for each considered compound is reported in Table 1.
The final compounds 6a-v were prepared following the
synthetic routes reported in Schemes 1, 2. The intermediate
compounds 3, 4, 5a, and 5b were synthesized following
procedures reported in literature with some modifications.17
Scheme 1 shows the acylation of the commercially available
4-amino-4,5-dihydro-1-methyl-3-propyl-1H-pyrazole-5-carbox-
amide (1) with 2-ethoxy-benzoyl chloride (2) in presence of
triethylamine (TEA)/4-(dimethylamino) pyridine (DMAP), yield-
ing the desired 4-(2-ethoxybenzamido)-1-methyl-3-propyl-1H-
pyrazole-5-carboxamide (3). Cyclization of intermediate 3, in
the presence of 35% H2O2 and NaOH pellets, furnished 5-(2-
ethoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-

Sildenafil Analogues for Treatment of Erectile Dysfunction
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2809
Scheme 1a
a Reagents and conditions: (i) TEA, DMAP/anhydrous CH2Cl2, µν; (ii) NaOH pellets, H2O2 (35%), H2O/ETOH, µν; (iii) ClSO3H, 0 °C, N2; (iv) appropriate
4-R-substituted piperazine, TEA/absolute ETOH, µν; (v) N,N′-substituted-ethylenediamine TEA/ absolute ETOH, µν.
Scheme 2a
yielding 5c, which was converted in the desired compound 6q
by treatment with AgNO3 in CH3CN.
Analytical purification of each product was obtained by
chromatography on silica gel column and further crystallization
from diethyl ether. All new compounds gave satisfactory
1
elemental analyses (C, H, N) and were characterized by H
1
NMR and ESI mass spectrometry. H NMR and MS data for
all final compounds were consistent with the proposed structures.
Pharmacology
The newly synthesized compounds were tested in vitro in
order to evaluate the effects of PDE5 inhibition in human
platelets. Phosphodiesterase (PDE) activity was determined as
the concentration of cGMP or cAMP measured as a percentage
of the inactive PDE (where the control with inactive PDE
indicates maximal concentration of cGMP or cAMP present in
the sample under the assay conditions). The measurement was
performed by LC-MS/MS analysis on a triple quadrupole mass
spectrometer (MS) API 4000. IC50 is defined as the concentra-
tion of inhibitor where 50% of the cGMP or cAMP is present
in the sample when compared to the inactive PDE.
Functional studies in rabbit corpus cavernosum and in both
endothelium-intact and -denuded rabbit isolated aorta were
determined to evaluate smooth muscle relaxation induced by
sildenafil analogues (6a-v). In phenylephrine-contracted prepa-
rations, cumulative concentration–response curves for 6a-v
were obtained in aortic rings (0.0001–10 µM) and corpus
cavernosum (0.001–10 µM). Experimental values were calcu-
lated relative to the maximal changes from the contraction
produced by phenylephrine in each tissue, which was taken as
100%. Data represent the mean ( SEM of 3–5 experiments.
a Reagents and conditions: (i) 4-hydroxyethyl piperazine, TEA/ absolute
ETOH, µν; (ii) decanoyl chloride, anhydrous CHCl3, µν; (iii) chlorobutyryl
chloride, anhydrous CHCl3/NaHCO3, 0 °C; (iv) AgNO3, CH3CN, at reflux
under light exclusion.
Results and Discussion
gave the final compound 6p. Intermediate 5b was also reacted
with chlorobutyryl chloride in anhydrous CHCl3/NaHCO3,
The application of microwave energy to organic compounds
for conducting synthetic reactions at highly accelerated rates

2810 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Flores Toque et al.
has become a well-known technique.18 In later years, our
interests were directed to the use of microwave irradiation in
the field of heterocyclic synthesis;19 starting from the acquired
experience in this area, we performed the preparation of all the
final compounds (6a-v) by microwave flash-heating. The
obtained yields were higher (60–96%) than those obtained by
conventional heating (Table 1) and the overall reaction times
were dramatically reduced from 2-2.5 h to 5-25 min. Similar
results were obtained for intermediates 3–5 (Table 1). The
obtained yields and time reaction reduction confirm the ef-
ficiency of microwave flash-heating chemistry and place this
technology as a powerful alternative to conventional heating.
Table 2. IC50 (µM) Values for Compounds 6a-q as PDE Inhibitors in
Human Platelets and Relaxant Effects (pEC50 and Emax) of the PDE5
Inhibitor in Rabbit Corpus Cavernosum Preparations Contracted by
Phenylephrine
First, the sildenafil analogues were tested in vitro to evaluate
the inhibition of PDE5 activity in human platelets. In this assay,
the inhibitory potencies (IC50 values) of 6c, 6e, 6f, 6 h, 6i, 6l,
and 6o were similar to sildenafil (IC50 ) 0.053 µΜ), ranging
between 0.05 and 0.15 µΜ. Compounds 6m, 6n, and 6q showed
higher IC50 values (0.37, 0.32, and 0.53 µΜ, respectively), while
compounds 6a, 6b, 6d, 6g, and 6p presented less than 50% of
enzymatic inhibition (Tables 2 and 3). Derivatives embodying
ethylenediamine moiety (6r, 6s, 6t, and 6v) showed a good
activity, with IC50 values ranging between 0.20 and 0.51 µΜ.
Interestingly, the analogue 6u showed an IC50 value of 0.038
µΜ, which represents the lowest obtained value.
Second, we examined the potency of 6a-q analogues in
inducing relaxation on rabbit corpus cavernosum. Our data
showed that 6a, 6c, 6e, 6f, 6n, 6o, and 6p were as potent as
sildenafil in relaxing the corpus cavernosum, with values of
pEC50 between 6.36 and 7.08 µΜ. Particularly, compound 6f
showed the best pEC50 value for relaxing corpus cavernosum
(7.08 ( 0.09 µΜ). In contrast, compounds 6b, 6d, 6g, 6 h, 6i,
6l, and 6q were found to be significantly less potent than
sildenafil in this assay. The maximal responses for these
compounds (Emax values between 100 and 119%) were similar
to sildenafil (Emax 105 ( 3%). Table 2 shows the pEC50 and
Emax values for these compounds. The compounds containing
an ethylenediamine moiety (6r-v) exhibited activity lower than
the corresponding N-4-substituted-piperazine analogues in cor-
pus cavernosum. In fact, it should be noted that all compounds
exhibited values of pEC50 and Emax lower than 6.26 ( 0.15 µΜ
and 69 ( 4%, respectively. Overall, our results demonstrated
that a set of different N-4-substituted piperazine with a large
variety of hydrophobic, electronic, and steric properties are able
to bind PDE5 enzyme, and several compounds yielded an
excellent combination of both enzymatic inhibition and corpus
cavernosum relaxation.
In the last set of experiments, functional studies in rabbit
isolated aortic rings were performed to evaluate smooth muscle
relaxation. Cumulative addition of 6a-v analogues (0.0001–10
µM) evoked sustained relaxations in endothelium-intact and
-denuded aortic rings precontracted with phenylephrine (PE, 1
µΜ) in a concentration-dependent manner. The removal of the
endothelium caused a significant rightward shift of the concen-
tration–response curve to 6a, 6c, 6d, 6e, 6f, 6g, 6 h, 6i, 6l, 6n,
6o, 6p, and 6q (Table 4). Sildenafil showed similar values in
both endothelium-intact and -denuded preparations (pEC50: 7.25
( 0.07 µΜ, 6.57 ( 0.11 µΜ, respectively). These results
indicate that the tested compounds potentiate the basal NO
release from endothelium. In contrast, analogues 6b and 6m
were less potent than sildenafil in endothelium-intact aortic rings
(pEC50: 6.77 ( 0.19 µΜ, 6.81 ( 0.19 µΜ, respectively). Table
4 summarizes the pEC50 values and maximal responses for
sildenafil analogues in both endothelium-denuded and endot-
helium-intact aorta.
* Experimental values were calculated relative to the maximal changes
from the contraction produced by phenylephrine and represented as -log
of the molar concentration to produce 50% of the maximal relaxation elicited
by sildenafil and analogues in contracted tissues. Data represent the mean
( SEM of n experiments. ** Were not obtained values at 50% of enzymatic
inhibition. ClogP ) octanol-water Partition Coefficient.
The derivatives embodying ethylenediamine moiety (ana-
logues 6r-v), presented pEC50 values similar to sildenafil in
both endothelium-intact and -denuded preparations (Table 4).

Sildenafil Analogues for Treatment of Erectile Dysfunction
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2811
Table 3. IC50 (µM) Values for Compounds 6r-v as PDE Inhibitors in
Human Platelets and Relaxant Effects (pEC50 and Emax) of the PDE5
Inhibitor in Rabbit Corpus Cavernosum Preparations Contracted by
Phenylephrine
the active site pocket mainly by various hydrophobic interactions
and two hydrogen bonds.21
In conclusion, we have synthesized several compounds that
exhibited a good PDE5 inhibitory activity that was as potent as
sildenafil. In particular, compound 6f in rabbit isolated corpus
cavernosum, and compounds 6r and 6u in aorta, showed the
best pharmacological profile and may offer a new lead for
development of novel sildenafil analogues. Because these
compounds are more lipophilic than sildenafil, they may have
better bioavailability and prolonged action in vivo. Further
studies on the pharmacokinetic profile of these compounds are
required to reveal their therapeutic potential.
Experimental Section
General Chemistry. All starting materials were commercially
available and used without further purification. Synthesis was
performed using a microwave oven ETHOS 1600, Milestone. All
reactions were performed in standard Pyrex glassware with a reflux
condenser fitted through the roof of the microwave cavity and were
performed by a microwave program that was composed by
appropriate temperature ramping and holding steps. Optimum profile
power/time and temperature employed for the synthesis is reported
in Table 1. Conventional heating (oil bath) and microwave
irradiation of the reactions were compared (Table 1). The temper-
ature of the stirred reaction mixture was monitored directly by a
microwave-transparent fluoroptic probe inserted into the solution.
Organic solutions were dried over anhydrous sodium sulfate.
Melting points were determined using a Kofler hot-stage apparatus
and are uncorrected. Thin-layer chromatography was performed on
precoated silica gel Kieselgel 60F254 (E. Merck, AG, Darmstadt,
Germany) plates. Column chromatography was performed on
200–400 mesh silica gel. Molecular weights of final compounds
were assessed by electrospray ionization mass spectrometry (ESI/
MS) on a ThermoFinnigan LCQ Ion-Trap. Nuclear magnetic
resonance spectra for proton (1H NMR) were recorded on a Bruker
AM-500 spectrometer. The chemical shift values are expressed in
ppm (part per million) relative to tetramethylsilane as internal
standard: s ) singlet, d ) doublet, t ) triplet, q ) quartet, m )
multiplet, and br ) broad singlet. The relative integrals of peak
areas agreed with those expected for the assigned structures.
Elemental compositions are within (0.4% of the calculate values.
The ClogP values reported in table 2 have been determined using
the software “Octanol-Water Partition Coefficients, ClogP for
Windows, version 2.0” (BioByte Corp., Claremont, CA).
4-(2-Ethoxybenzamido)-1-methyl-3-propyl-1H-pyrazole-5-
carboxamide (3). A mixture of 4-amino-4,5-dihydro-1-methyl-3-
propyl-1H-pyrazole-5-carboxamide (1) (3.0 g, 16.4 mmol), 2-ethoxy-
benzoyl chloride (2) (6.1 g, 33.0 mmol), DMAP (0.02 g, 0.164
mmol), and TEA (3.34 g, 33.0 mmol) in anhydrous dichloromethane
(50 mL) was introduced into the reaction vessel and the desired
parameters (microwave, power, temperature, and time) were set as
reported in Table 1. The solvent was evaporated and the residue
dissolved in a mixture (250 mL) of dichloromethane/methanol 19/1
(v/v); the obtained solution was washed with 1 N hydrochloric acid
(100 mL). The organic layers were dried over anhydrous Na2SO4
and concentrated in vacuo. The obtained residue was purified by
column chromatography on silica gel, eluting with a mixture of
dichloromethane/methanol 19.4/0.6 (v/v). The combined fractions
were evaporated providing the crude product 3 as a pale-pink solid
(75%); mp: 153–155 °C.
* Experimental values were calculated relative to the maximal changes
from the contraction produced by phenylephrine, and represented as -log
of the molar concentration to produce 50% of the maximal relaxation elicited
by sildenafil and analogues in contracted tissues. Data represent the mean
( SEM of n experiments. ClogP ) octanol-water Partition Coefficient.
In contrast to the other compounds, the relaxations evoked by
the compounds 6r and 6u were not affected by the endothelium
denudation, whereas those evoked by 6s, 6t, and 6v were greatly
attenuated by endothelium removal, producing a marked
rightward shift, thus making 6r and 6u potential compounds to
treat erectile dysfunction. The maximal responses to 6r, 6s, 6t,
6u, and 6v were not affected by endothelium denudation.
Thus, our findings suggest that piperazine moiety is not
crucial in order to obtain PDE inhibitory activity. The ethyl-
enediamine derivative 6u, for example, showed an IC50 value
on PDE5 in human platelets lower than sildenafil (0.038 and
0.053 µM, respectively). The pharmacological results obtained
with compounds 6r-v are very interesting because, associated
with the absence of the piperazine structure, the newly
synthesized derivatives support one or two acidic functions that
make the molecules anphoter (6r, 6s, and 6t) or just acid (6u
and 6v). This improved inhibitory activity could be explained
assuming that the acidic function mimick the phosphate group
of cGMP. These findings are in accordance with recently
reported 3D-QSAR studies on sildenafil analogues that indicate
that compounds with substituents charged or larger than a methyl
group, present on the sildenafil piperazine moiety, are essential
for high inhibitory activity.20 Opening the piperazine ring
represents a crucial alteration that gives more flexibility to the
molecules allowing different conformations of the supported
functional groups.
5-(2-Ethoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]py-
rimidin-7(6H)-one (4). 4-(2-Ethoxybenzamido)-1-methyl-3-propyl-
1H-pyrazole-5-carboxamide (3) (2.23 g, 6.76 mmol) was added
portion wise to a solution of sodium hydroxide (0.54 g, 13.5 mmol)
and 30% hydrogen peroxide solution (2.24 mL) in water (20 mL).
Ethanol (70 mL) was added, the reaction mixture was introduced
into the reaction vessel, and the desired parameters (microwave,
power, temperature, and time) were set as reported in Table 1. The
mixture was cooled and evaporated in vacuo. The resulting solid
Therefore, the obtained results show that our molecules,
which have been designed by introducing different substituents
on piperazine or N,N′-ethylendiamine moiety, have a good
inhibitory activity on PDE-5. The biological profile is due to a
combination of the inserted structural modifications and the
unmodified sildenafil pharmacophoric portion that binds into

2812 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Flores Toque et al.
Table 4. Potency (pEC50) and Maximal Response (Emax) Values Derived from Concentration-Response Curves to the Sildenafil Analogues (0.0001-10
µM each) in Endothelium-Intact (E+) and -Denuded Aortic Rings (E-) Contracted with Phenylephrine (1 µM). Data Represent the Mean ( SEM of 5
Experiments
compd
6a
pEC50 values
Emax values
compd
pEC50 values
Emax values
E+ 7.18 ( 0.17
E- 6.74 ( 0.22a
E+ 6.77 ( 0.19b
E- 6.48 ( 0.20a
E+ 7.16 ( 0.17
E- 6.54 ( 0.31a
E+ 7.22 ( 0.08
E- 6.78 ( 0.11a
E+ 7.28 ( 0.10
E- 6.68 ( 0.09a
E+ 7.28 ( 0.10
E- 6.71 ( 0.10a
E+ 7.40 ( 0.06
E- 6.76 ( 0.10a
E+ 7.02 ( 0.14
E- 6.49 ( 0.14a
E+ 7.08 ( 0.13
E- 6.61 ( 0.12a
E+ 7.18 ( 0.14
E- 6.48 ( 0.22a
E+ 7.25 ( 0.07
E- 6.57 ( 0.11a
59 ( 3
41 ( 3a
69 ( 9
49 ( 4a
63 ( 8
40 ( 4a
86 ( 1
70 ( 3a
90 ( 4
75 ( 5a
89 ( 3
67 ( 4a
91 ( 4
73 ( 4a
66 ( 6
47 ( 3a
74 ( 6
52 ( 6a
77 ( 3
46 ( 4a
76 ( 6
56 ( 1
6m
E+ 6.81 ( 0.19b
E- 6.42 ( 0.26a
E+ 7.18 ( 0.14
E- 6.69 ( 0.11a
E+ 7.32 ( 0.09
E- 6.64 ( 0.10a
E+ 7.46 ( 0.06
E- 6.68 ( 0.10a
E+ 7.04 ( 0.09
E- 6.54 ( 0.07a
E+ 7.21 ( 0.10
E- 7.01 ( 0.11
E+ 7.28 ( 0.11
E- 6.75 ( 0.11a
E+ 7.21 ( 0.10
E- 6.73 ( 0.11a
E+ 6.93 ( 0.12
E- 6.81 ( 0.09
E+ 7.07 ( 0.15
E- 6.47 ( 0.19a
66 ( 2
42 ( 2a
72 ( 4
61 ( 4
91 ( 5
60 ( 4a
93 ( 2
72 ( 6a
87 ( 3
88 ( 3
82 ( 4
82 ( 3
70 ( 5
66 ( 7
73 ( 3
69 ( 5
75 ( 5
68 ( 7
69 ( 8
51 ( 5
6b
6n
6o
6p
6q
6r
6s
6c
6d
6e
6f
6g
6h
6t
6i
6u
6v
6l
sildenafil
a p < 0.05 compared with the respective control value. b p < 0.05 compared to sildenafil E+ value.
was treated with 2 N hydrochloric acid (3.8 mL), with external
cooling, and the mixture was extracted with dichloromethane. The
combined organic extracts were then washed with saturated aqueous
sodium carbonate solution and brine and then dried over anhydrous
Na2SO4 and concentrated in vacuo. The resulting crude residue was
purified on silica gel column eluting with a mixture of dichlo-
romethane/ethanol 19.4/0.6 (v/v), and the final compound 4 was
obtained as a colorless solid (45% yield); mp: 143–146 °C.
4-Ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyra-
zolo[4,3-d]pyrimidin-5-yl)benzene-1-sulfonyl chloride (5a). Car-
boxamide compound (4) (10.0 g, 32.1 mM) was added portion wise
to stirred and cooled chlorosulphonic acid (20 mL) in an ice bath
under nitrogen atmosphere; the reaction mixture was then warmed
to room temperature gradually for 2 h after the addition. The
reaction solution was cautiously added to ice–water (150 mL), and
the aqueous mixture extracted with a mixture of dichloromethane/
methanol 9/1 (v/v). The combined extracts were dried over
anhydrous Na2SO4 and evaporated under vacuum to give the
required sulfonyl chloride 5 as a white solid (97% yield); mp:
179–181 °C.
General Procedure for Preparation of Derivatives 6a-o by
Condensation of 4-Substituted Piperazines with Intermediate
5a. A mixture of chlorosulphonyl derivative (5a) (1 mmol), an
appropriate 4-substituted piperazine (1 mmol), and triethylamine
(0.20 g, 2 mmol) in anhydrous ethanol (20 mL) was added; the
reaction was introduced into the reaction vessel, and the desired
parameters (microwave, power, temperature, and time) were set as
reported in Table 1. The reaction mixture was dried under reduced
pressure, and the resulting crude residue was purified on silica gel
column using dichloromethane/methanol 9.7/0.3 (v/v) as eluent,
unless otherwise indicated. The crude products 6a-o were crystal-
lized by diethyl ether. The intermediate compound 5b was obtained
by reaction of 5a with 4-hydroxyethyl piperazine under the same
reaction conditions.17
5-{2-Ethoxy-5-[(4-o-tolyl)piperazinylsulphonyl]phenyl)-1-meth-
yl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one
(6b). It was synthesized from 5a and o-tolylpiperazine. Yield 90%;
mp: 233–236 °C. 1H NMR (500 MHz, DMSO) δ: 0.94 (t, 3H,
CH2CH2CH3, J ) 7.5 Hz); 1.32 (t, 3H, OCH2CH3, J ) 6.9 Hz);
1.69–1.81 (m, 2H, CH2CH2CH3); 2.49 (t, 2H, CH2CH2CH3, J )
7.5 Hz); 2.76 (s, 3H, CH3); 3.20–3.60 (m, 8H, 4 NCH2); 4.14 (s,
3H, NCH3); 4.20 (q, 2H, OCH2CH3, J ) 6.9 Hz); 6.92 (d, 1H,
H-3′, J ) 9.0 Hz); 7.12 (t, 1H, J ) 6.9); 7.39 (d, 2H, J ) 7.2);
7.86–7.92 (m, 3H); 12.30 (br s, 1H, NH); ESI/MS: 551.44 [M +
H]+. Anal. (C28H34N6O4S) C, H, N.
5-{2-Ethoxy-5-[(4-(1-(2-methoxyphenyl))piperazinylsul-
phonyl]phenyl)-1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-
d]pyrimidin-7-one (6c). It was synthesized from 5a and 1-(2-
methoxyphenyl) piperazine. Yield 90%; mp: 205–207 °C. 1H NMR
(500 MHz, DMSO) δ: 0.94 (t, 3H, CH2CH2CH3, J ) 7.5 Hz); 1.32
(t, 3H, OCH2CH3, J ) 6.9 Hz); 1.69–1.81 (m, 2H, CH2CH2CH3);
2.49 (t, 2H, CH2CH2CH3, J ) 7.5 Hz); 3.19 (s, 3H, OCH3);
3.54–3.66 (m, 8H, 4NCH2); 4.14 (s, 3H, NCH3); 4.20 (q, 2H,
OCH2CH3, J ) 6.9 Hz); 6.60 (m, 2H, H-2′, H-4′, and H-6′); 6.92
(d, 1H, H-3′, J ) 9.0 Hz); 7.08 (t, 2H, H-3′, and H-5′, J ) 7.2);
7.86–7.92 (m, 2H, H-4′, and H-6′); 12.30 (br s, 1H, NH); ESI/MS:
567.44 [M + H]+. Anal. (C28H34N6O5S) C, H, N.
5-{2-Ethoxy-5-[(4-(3-(trifluoromethyl)phenyl)piperazinyl-
sulphonyl]phenyl)-1-methyl-3-N-propyl-1,6-dihydro-7H-pyra-
zolo[4,3-d]pyrimidin-7-one (6d). It was synthesized from 5a and
3-(trifluoromethyl)phenylpiperazine. Yield 85%; mp: 184–186 °C.
1H NMR (500 MHz, DMSO) δ: 0.94 (t, 3H, CH2CH2CH3, J ) 7.5
Hz); 1.32 (t, 3H, OCH2CH3, J ) 6.9 Hz); 1.69–1.81 (m, 2H,
CH2CH2CH3); 2.49 (t, 2H, CH2CH2CH3, J ) 7.5 Hz); 3.20–3.60
(m, 8H, 4NCH2); 4.14 (s, 3H, NCH3); 4,20 (q, 2H, OCH2CH3, J )
6.9 Hz); 6.92 (d, 2H, J ) 9.0 Hz); 7.09–7.19 (m, 2H); 7.86–7.92
(m, 4H, H-4′, and H-6′); 12.30 (br s, 1H, NH); ESI/MS: 606.44
[M + H]+. Anal. (C28H31F3N6O4S) C, H, N.
5-{2-Ethoxy-5-[(4-phenyl)piperazinylsulphonyl]phenyl)-1-
methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-
one (6a). It was synthesized from 5a and phenylpiperazine. Yield
5-{2-Ethoxy-5-[(4-(2-fluorophenyl)piperazinylsulphonyl]phenyl)-
1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-
7-one (6e). It was synthesized from 5a and 2-(fluorophenyl)
piperazine. Yield 90%; mp: 208–210 °C. 1H NMR (500 MHz,
DMSO) δ: 0.94 (t, 3H, CH2CH2CH3, J ) 7.5 Hz); 1.32 (t, 3 H,
OCH2CH3, J ) 6.9 Hz); 1.69–1.81 (m, 2 H, CH2CH2CH3); 2.49 (t,
2 H, CH2CH2CH3, J ) 7.5 Hz); 3.20–3.60 (m, 8 H, 4NCH2); 4.14
(s, 3H, NCH3); 4.20 (q, 2H, OCH2CH3, J ) 6.9 Hz); 6.60 (m, 2H,
H-4′, and H-6′); 6.92 (d, 1H, H-3′, J ) 9.0 Hz); 7.08 (t, 2H, H-3′,
1
80%; mp: 203–205 °C. H NMR (500 MHz, DMSO) δ: 0.94 (t,
3H, CH2CH2CH3, J ) 7.5 Hz); 1.32 (t, 3 H, OCH2CH3, J ) 6.9
Hz); 1.69–1.81 (m, 2 H, CH2CH2CH3); 2.49 (t, 2 H, CH2CH2CH3,
J ) 7.5 Hz); 3.20–3.60 (m, 8 H, 4 NCH2); 4.14 (s, 3H, NCH3);
4.20 (q, 2H, OCH2CH3, J ) 6.9 Hz); 6.60 (m, 3H, H-2′, H-4′, and
H-6′); 6.92 (d, 1H, H-3′, J ) 9.0 Hz); 7.08 (t, 2H, H-3′, and H-5′,
J ) 7.2); 7.86–7.92 (m, 2H, H-4′, and H-6′); 12.30 (br s, 1H, NH);
ESI/MS: 537.44 [M + H]+. Anal. (C27H32N6O4S) C, H, N.

Sildenafil Analogues for Treatment of Erectile Dysfunction
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2813
and H-5′, J ) 7.2); 7.86–7.92 (m, 2H, H-4′, and H-6′); 12.21 (br
s, 1H, NH); ESI/MS: 555.44 [M + H]+. Anal. (C27H31FN6O4S) C,
H, N.
6.62–7.47 (m, 9H); 12.30 (br s, 1H, NH); ESI/MS: 587.44 [M +
H]+. Anal. (C31H34N6O4S) C, H, N.
5-{2-Ethoxy-5-[(4-(1-piperonyl)piperazinylsulphonyl]phenyl)-
1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-
7-one (6n). It was synthesized from 5a and 1-piperonylpiperazine.
Yield 85%; mp: 193–195 °C. 1H NMR (500 MHz, DMSO) δ: 0.94
(t, 3H, CH2CH2CH3, J ) 7.5 Hz); 1.32 (t, 3H, OCH2CH3, J ) 6.9
Hz); 1.69–1.81 (m, 2H, CH2CH2CH3); 2.49 (t, 2H, CH2CH2CH3, J
) 7.5 Hz); 3.28–3.34 (m, 10H, 4NCH2, CH2); 4.14 (s, 3H, NCH3);
4.20 (q, 2H, OCH2CH3, J ) 6.9 Hz); 5.94 (s, 2H); 6.66–6.79 (m,
3H); 7.36 (d, 1H, H-3′, J ) 9.0 Hz); 7.79–7.81 (m, 2H, H-4′, and
H-6′); 12.30 (br s, 1H, NH); ESI/MS: 595.44 [M + H]+. Anal.
(C28H32N6O6S) C, H, N.
5-{2-Ethoxy-5-[(4-(1-(cyclohexyl))piperazinylsulphonyl]phenyl)-
1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-
7-one (6o). It was synthesized from 5a and 1-(cyclohexyl)
piperazine. Yield 90%; mp: 209–211 °C. 1H NMR (500 MHz,
DMSO) δ: 0.92 (t, 3H, CH2CH2CH3, J ) 7.5 Hz); 1.12 (t, 3H,
OCH2CH3, J ) 6.9 Hz); 1.29–1.32 (m, 10H); 1.65–1.74 (m, 2H,
CH2CH2CH3); 2.49 (t, 2H, CH2CH2CH3, J ) 7.5 Hz); 2.70–2.83
(m, 1H); 3.20–3.60 (m, 8H, 4NCH2); 4.14 (s, 3H, NCH3); 4.20 (q,
2H, OCH2CH3, J ) 6.9 Hz); 7.36 (d, 1H, H-3′, J ) 9.0 Hz);
7.79–7.80 (m, 2H, H-4′, and H-6′); 12.30 (br s, 1H, NH); ESI/MS:
543.44 [M + H]+. Anal. (C27H38N6O4S) C, H, N.
5-{2-Ethoxy-5-[(4-(4-fluorophenyl)piperazinylsulphonyl]phenyl)-
1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-
7-one (6f). It was synthesized from 5a and 4-(fluorophenyl)
piperazine. Yield 85%; mp: 195–197 °C. 1H NMR (500 MHz,
DMSO) δ: 0.94 (t, 3H, CH2CH2CH3, J ) 7.5 Hz); 1.28 (t, 3H,
OCH2CH3, J ) 6.9 Hz); 1.62–1.73 (m, 2H, CH2CH2CH3); 2.49 (t,
2H, CH2CH2CH3, J ) 7.5 Hz); 3.20–3.60 (m, 8H, 4NCH2); 4.14
(s, 3H, NCH3); 4.20 (q, 2H, OCH2CH3, J ) 6.9 Hz); 6.60 (m, 2H,
H-4′, and H-6′); 6.92 (d, 1H, H-3′, J ) 9.0 Hz); 7.08 (t, 2H, H-3′,
and H-5′, J ) 7.2); 7.86–7.92 (m, 2H, H-4′, and H-6′); 12.20 (br
s, 1H, NH); ESI/MS: 555.44 [M + H]+. Anal. (C27H31FN6O4S) C,
H, N.
5-{2-Ethoxy-5-[(4-(2-chlorophenyl))piperazinylsulpho-
nyl]phenyl)-1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-
d]pyrimidin-7-one (6g). It was synthesized from 5a and (2-
1
chlorophenyl) piperazine. Yield 65%; mp: 213–215 °C. H NMR
(500 MHz, DMSO) δ: 0.92 (t, 3H, CH2CH2CH3, J ) 7.5 Hz); 1.32
(t, 3H, OCH2CH3, J ) 6.9 Hz); 1.70–1.74 (m, 2H, CH2CH2CH3);
2.49 (t, 2H, CH2CH2CH3, J ) 7.5 Hz); 2.74–3.05 (m, 8H, 4NCH2);
4.14 (s, 3H, NCH3); 4.20 (q, 2H, OCH2CH3, J ) 6.9 Hz); 6.60 (m,
2H, H-4′, and H-6′); 6.92 (d, 1H, H-3′, J ) 9.0 Hz); 7.08 (t, 2H,
H-3′, and H-5′, J ) 7.2); 7.86–7.88 (m, 2H, H-4′, and H-6′); 12.21
(br s, 1H, NH); ESI/MS: 572.44 [M + H]+. Anal. (C27H31ClN6O4S)
C, H, N.
5-{2-Ethoxy-5-[(4-decanoic acid ethyl ester)piperazinylsul-
phonyl]phenyl)-1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-
d]pyrimidin-7-one (6p). A mixture of 5-{2-ethoxy-5-[(4-hydroxy-
ethyl)piperazinylsulphonyl]phenyl}-1-methyl-3-N-propyl-1,6-dihydro-
7H-pyrazolo[4,3-d]pyrimidin-7-one (5b) (0.5 g, 1 mmol) and
decanoyl chloride (0.19 g, 1 mmol) in anhydrous chloroform (20
mL) was added; the reaction mixture was introduced into the
reaction vessel, and the desired parameters (microwave, power,
temperature, and time) were set as reported in Table 1. The solvent
was evaporated in vacuo, the residue was dissolved in H2O (50
mL) and brine, and then the solution was washed with ethyl acetate
(70 mL), dried with Na2SO4, and evaporated under vacuum. The
resulting crude residue was purified on silica gel column using
dichloromethane/methanol 15/1 (v/v) as eluent. The product was
6p crystallized as white solid from diethyl ether. Yield (90%); mp:
5-{2-Ethoxy-5-[(4-(3-chlorophenyl))piperazinylsulpho-
nyl]phenyl)-1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-
d]pyrimidin-7-one (6h). It was synthesized from 5a and (3-
1
chlorophenyl) piperazine. Yield 77%; mp: 200–202 °C. H NMR
(500 MHz, DMSO) δ: 0.92 (t, 3H, CH2CH2CH3, J ) 7.5 Hz); 1.30
(t, 3H, OCH2CH3, J ) 6.9 Hz); 1.70–1.74 (m, 2H, CH2CH2CH3);
2.49 (t, 2H, CH2CH2CH3, J ) 7.5 Hz); 2.74–3.05 (m, 8H, 4NCH2);
4.14 (s, 3H, NCH3); 4.20 (q, 2H, OCH2CH3, J ) 6.9 Hz); 6.78 (m,
2H, H-4′, and H-6′); 6.91 (d, 1H, H-3′, J ) 9.0 Hz); 7.16 (t, 2H,
H-3′, and H-5′, J ) 7.2); 7.86–7.88 (m, 2H, H-4′, and H-6′); 12.18
(br s, 1H, NH); ESI/MS: 572.44 [M + H]+. Anal. (C27H31ClN6O4S)
C, H, N.
1
5-{2-Ethoxy-5-[(4-(4-chlorophenyl))piperazinylsulpho-
nyl]phenyl)-1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-
d]pyrimidin-7-one (6i). It was synthesized from 5a and (4-
149–151 °C. H NMR (500 MHz, DMSO) δ: 0.81–0.93 (m, 6H);
1.16–1.43 (m, 15H); 1.72–1.73 (m, 4H); 2.74–2.86 (m, 6H);
3.20–3.60 (m, 8H, 4NCH2); 4.03–4.20 (m, 5H); 4.20 (q, 2H,
OCH2CH3, J ) 6.9 Hz); 6.92 (d, 1H, H-3′, J ) 9.0 Hz); 7.86–7.92
(m, 2H, H-4′, and H-6′); 12.30 (br s, 1H, NH); ESI/MS: 659.85
[M + H]+. Anal. (C33H50N6O6S) C, H, N.
1
chlorophenyl) piperazine. Yield 66%; mp: 212–214 °C. H NMR
(500 MHz, DMSO) δ: 0.92 (t, 3H, CH2CH2CH3, J ) 7.5 Hz); 1.32
(t, 3H, OCH2CH3, J ) 6.9 Hz); 1.70–1.74 (m, 2H, CH2CH2CH3);
2.49 (t, 2H, CH2CH2CH3, J ) 7.5 Hz); 2,74–3,02 (m, 8H, 4NCH2);
4,10 (s, 3H, NCH3); 4,21 (q, 2H, OCH2CH3, J ) 6.9 Hz); 6.89 (m,
2H, H-4′, and H-6′); 6.92 (d, 1H, H-3′, J ) 9.0 Hz); 7.37 (t, 2H,
H-3′, and H-5′, J ) 7.2); 7.83–7.86 (m, 2H, H-4′, and H-6′); 12.21
(br s, 1H, NH); ESI/MS: 572.44 [M + H]+. Anal. (C27H31ClN6O4S)
C, H, N.
5-{2-Ethoxy-5-[(4-chlorobutyric acid ethyl ester)piperazinyl-
sulphonyl] phenyl)-1-methyl-3-N-propyl-1,6-dihydro-7H-pyra-
zolo[4,3-d]pyrimidin-7-one (5c). A mixture of 5-{2-ethoxy-5-[(4-
hydroxyethyl)piperazinylsulphonyl]phenyl}-1-methyl-3-N-propyl-
1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (5b) (0.5 g, 1
mmol), chlorobutyryl chloride (0.1 g mmol) in anhydrous chloro-
form (20 mL), and a solution of saturated NaHCO3 (10 mL) was
stirred at 0 °C for 2 h. The solvent was evaporated under vacuum,
the residue dissolved in H2O (50 mL) and brine, and then the
organic phase was separated, dried over anhydrous Na2SO4, and
evaporated under vacuum. (90%); mp: 169–170 °C.
5-{2-Ethoxy-5-[(4-(4-nitrophenyl))piperazinylsulphonyl]phenyl)-
1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-
7-one (6l). It was synthesized from 5a and (4-nitrophenyl)
piperazine and eluted with dichloromethane/methanol 9/1 (v/v).
Yield 90%; mp: 221–223 °C. 1H NMR (500 MHz, DMSO) δ: 0.91
(t, 3H, CH2CH2CH3, J ) 7.5 Hz); 1.27 (t, 3H, OCH2CH3, J ) 6.9
Hz); 1.68–1.73 (m, 2H, CH2CH2CH3); 2.49 (t, 2H, CH2CH2CH3, J
) 7.5 Hz); 2.71–2.74 (m, 8H, 4NCH2); 3.32 (s, 3H, NCH3); 4.14
(q, 2H, OCH2CH3, J ) 6.9 Hz); 6.98 (m, 2H, H-4′, and H-6′);
7.31 (d, 1H, H-3′, J ) 9.0 Hz); 7.81 (t, 2H, H-3′, and H-5′, J )
7.2); 8.01–8.02 (m, 2H, H-4′, and H-6′); 12.21 (br s, 1H, NH);
ESI/MS: 572.44 [M + H]+. Anal. (C27H31N7O6S) C, H, N.
5-{2-Ethoxy-5-[4-(1-naphthyl)piperazinylsulphonyl]phenyl)-
1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-
7-one (6m). It was synthesized from 5a and 1-naphthylpiperazine.
Yield 89%; mp: 260–262 °C. 1H NMR (500 MHz, DMSO) δ: 0.94
(t, 3H, CH2CH2CH3, J ) 7.5 Hz); 1.32 (t, 3H, OCH2CH3, J ) 6.9
Hz); 1.69–1.81 (m, 2H, CH2CH2CH3); 2.49 (t, 2H, CH2CH2CH3, J
) 7.5 Hz); 3.20–3.60 (m, 8H, 4NCH2); 4.14 (s, 3H, NCH3); 4.20
(q, 2H, OCH2CH3, J ) 6.9 Hz); 6.92 (d, 1H, H-3′, J ) 9.0 Hz);
5-{2-Ethoxy-5-[(4-nitro-butyric acid ethyl ester)piperazinyl-
sulphonyl] phenyl)-1-methyl-3-N-propyl-1,6-dihydro-7H-pyra-
zolo[4,3-d] pyrimidin-7-one (6q). A mixture of 5c (0.6 g, 1 mmol)
with AgNO3 (0.34 g, 2 mmol) in acetonitrile (30 mL) was stirred
at reflux under light exclusion. The mixture was filtered on Celite,
the solvent was evaporated in vacuo, the residue was dissolved in
H2O (50 mL) and brine, and then the organic phase was separated,
dried with Na2SO4, and evaporated in vacuo. The resulting crude
residue was purified on silica gel column using diethyl ether/ethanol,
1
8:2 (v/v) as eluent. Yield 90%; mp: 153–155 °C. H NMR (500
MHz, DMSO) δ: 0.92 (t, 3H, CH2CH2CH3, J ) 7.5 Hz); 1.32 (t,
3H, OCH2CH3, J ) 6.9 Hz); 1.72–1.73 (m, 2H, CH2CH2CH3);
1.85–1.87 (m, 2H); 2.35–2.37 (m, 4H); 2.49–2.52 (m, 8H, 4NCH2);
2.74–2.77 (m, 2H); 3.30–3.31 (m, 2H); 4.05–4.19 (m, 5H); 4.20
(q, 2H, OCH2CH3, J ) 6.9 Hz); 6.92 (d, 1H, H-3′, J ) 9.0 Hz);

2814 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Flores Toque et al.
7.86–7.92 (m, 2H, H-4′, and H-6′); 12.30 (br s, 1H, NH); ESI/MS:
636.44 [M + H]+. Anal. (C27H37N7O9S) C, H, N.
In Vitro Studies: Preparation of PDE Crude Extracts from
Human Platelets. Blood samples from healthy male volunteers
were collected in 0.1 vol of 3.13% sodium citrate and centrifuged
at 200g for 15 min at 10 °C. Platelet-rich plasma (PRP) was
removed and followed by addition of 10 mM EDTA. Next, platelet-
rich plasma (PRP) was centrifuged at 900g for 15 min at 10 °C.
The pellets were then washed in calcium-free Krebs buffer
containing 10 mM EDTA. The pellets were resuspended in Krebs
buffer containing 0.32 M sucrose to a final concentration of 1 ×
109 cells mL-1 and subsequently sonicated for 10 s. The protein
concentration was determined by a Protein Assay kit (BioRad) using
bovine serum albumin as the standard. The enzyme sample was
frozen and stored at -80 °C until experimentation.
General Procedure for Synthesis of Ethylenediamine Deriva-
tives 6r-v. A mixture of chlorosulphonyl derivative (5a) (0.410
g, 1 mmol), an appropriate N,N′-substituted ethylendiamine (1
mmol), and TEA (0.202 g, 2 mmol) in anhydrous ethanol (25 mL)
was added; the mixture was introduced into the reaction vessel,
and the desired parameters (microwave, power, temperature, and
time) were set as reported in Table 1. The reaction mixture was
dried under reduced pressure, and the resulting crude residue was
purified on a silica gel column using dichloromethane/ethanol, 19.4/
0.6 (v/v), as eluent, unless otherwise indicated. The final compounds
6r-v, obtained as solids, were crystallized by diethyl ether.
Determination of PDE Activity by LC-MS/MS Analysis. The
standard enzymatic reaction mixture (total volume of 200 µL)
contained 50 mM Tris-HCl (pH 8.0), 100 mM MgCl2, and PDE
enzyme sample (final protein concentration 0.5 mg/mL).22,27
Increasing concentrations (0.005–1 µM) of the agents under study
(sildenafil analogues) were prepared in dimethylsulfoxide (DMSO)
and preincubated in the enzymatic mixture for 5 min at room
temperature. Reaction was initiated by the addition of the substrate
cGMP (5 µM) at 35 °C for 30 min. The reaction was stopped by
immersing the sample in boiling water for 2 min. Samples were
stored at -20 °C. DMSO alone was added in the samples used to
measure 100% of enzyme activity. The chromatographic system
consisted of an analytical column (Genesis C18, 4 µmol, 100 mm
× 2.1 mm, i.d., reversed phase). The mobile phase was a mixture
of eluent A (water containing 10 mM formic acid) and eluent B
(acetonitrile containing 10 mM formic acid).27 The flow rate was
350 µL/min and the injection volume was 40 µL of each sample,
previously diluted with water. The total run time was 10 min. The
triple quadrupole mass spectrometer (MS) API 4000, AB/MDS
Sciex Instruments (Toronto, Canada), was equipped with an
electrospray source. To optimize all of the MS parameters, analysis
ionization was carried out using standard solutions of GMP and
cGMP infused directly into the mass spectrometer in positive ion
mode, specific monitoring compounds were identified by multiple
reaction monitoring (MRM) of ion GMP m/z 363.9 and its fragment
m/z 152.1 and ion cGMP m/z 346.0 and its fragment m/z 152.2. A
six-point calibration curve was constructed.
Preparation of Rabbit Isolated Corpus Cavernosum and
Aorta. Male New Zealand white rabbits (2–3 kg) were anaesthe-
tized with sodium pentobarbital (Hypnol, 50 mg/kg, i.v.), exsan-
guinated via the carotid artery, and aorta and penis removed. The
protocol was approved by the University Ethics Committee. The
aorta was cleaned of all loosely adherent tissue. Corpus cavernosum
preparations were obtained, following dissection of the tunica
albuginea and surrounding connective tissues. Tissues were im-
mediately placed in chilled Krebs solution of the following
composition (mM): NaCl, 118; NaHCO3, 25; glucose, 5.6; KCl,
4.7; KH2PO4, 1.2; MgSO4 ·7H2O, 1.17; CaCl2 ·2H2O, 2.5. In some
aortic rings, the endothelium was removed mechanically by rubbing
the lumen of the aorta with a closed pair of fine-tipped forceps.
Endothelium removal was confirmed by the lack of a relaxant response
to acetylcholine (ACh, 1 µM) at the start of the experiments. Aortic
rings and cavernosal strips were mounted under tension 10 mN in 10
mL organ chambers, as previously described.23–25 Isometric force was
recorded using a PowerLab 400 data acquisition system (software
chart, version 4.2, AD Instruments, MA). The tissues were allowed
to equilibrate for 1 h before the start of the experiments, and
phenylephrine (PE, 1 and 10 µM) was added to increase the basal
tone. Thereafter, when reaching the submaximal contraction,
sildenafil analogues (6a-v) and sildenafil itself were tested in the
concentration range of 0.0001–10 µM (aortic rings) or 0.001–10
µM (corpus cavernosum). One concentration–response curve to
6a-v was obtained for each preparation owing to the lower
contractile effect of PE observed in the second curve.
5-(2-Ethoxy-5-(N-phenyl-ethylenediamine-sulfonyl)phenyl)-
1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-
7-one (6r). It was synthesized from 5a and N-phenylethylenedi-
amine. Yield 92%; mp: 165–167 °C. 1H NMR (500 MHz, DMSO)
δ: 0.96 (t, 3H, CH2CH2CH3, J ) 7.5 Hz); 1.31 (t, 3H, OCH2CH3,
J ) 6.9 Hz); 1.70–1.73 (m, 2H, CH2CH2CH3); 2.08 (d, 1H);
2.74–2.77 (m, 2H); 2.87–2.88 (m, 2H); 3.08–3.41 (m, 6H);
4.15–4.17 (m, 3H); 5.52 (t, 2H); 6.47–6.48 (d, 1H); 6.99–7.02 (t,
1H); 7.29–7.31 (d, 1H); 7.74–7.96 (m, 2H); 12.13 (br s, 1H, NH).
ESI/MS: 510.64 [M + H]+. Anal. (C25H30N6O4S) C, H, N.
5-(2-Ethoxy-5-(N-naphthyl-ethylenediamine-sulfonyl)phenyl)-
1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-
7-one (6s). It was synthesized from 5a (0.410 g, 1 mmol) and
N-naphthylethylenediamine (0.259 g, 1 mmol). Yield 55%; mp:
192–194 °C. 1H NMR (500 MHz, DMSO) δ: 0.96 (t, 3H,
CH2CH2CH3, J ) 7.5 Hz); 1.31 (t, 3H, OCH2CH3, J ) 6.9 Hz);
1.70–1.73 (m, 2H, CH2CH2CH3); 2.08 (d, 1H); 2.74–2.77 (m, 2H);
2.87–2.88 (m, 2H); 3.08–3.41 (m, 6H); 4.15–4.17 (s, 3H); 5.52 (t,
2H); 6.42 (d, 1H); 7.07–7.39 (m, 3H); 7.70 (d, 1H); 7.87 (d, 1H);
8.00 (d, 1H); 12.13 (br s, 1H, NH); ESI/MS: 560.44 [M + H]+.
Anal. (C29H32N6O4S) C, H, N.
5-(2-Ethoxy-5-(N-acetic acid ethylenediamine-sulfonyl)phe-
nyl)-1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyri-
midin-7-one (6t), 5-(2-Ethoxy-5-(N-acetic acid,N′-5-(2-ethoxy-
5-sulfonyl)phenyl)-1-methyl-3-N-propyl-1,6-dihydro-7H-
pyrazolo[4,3-d]pyrimidin-7-one-ethylenediamine-sulfonyl)phenyl)-
1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo [4,3-d]pyrimidin-
7-one (6u). Compounds 6t and 6u were synthesized in one step
from 5a (0.410 g, 1 mmol) and N-ꢀ-aminoethyl glycine (0.118 g,
1 mmol).
Compound 6t. Yield 60%; mp: 198–200 °C. 1H NMR (500
MHz, DMSO) δ: 0.96 (t, 3H, CH2CH2CH3, J ) 7.5 Hz); 1.31 (t,
3H, OCH2CH3, J ) 6.9 Hz); 1.70–1.73 (m, 2H, CH2CH2CH3); 2.08
(d, 1H); 2.74–2.77 (m, 2H); 2.87–2.88 (m, 2H); 3.08–3.41 (m, 6H);
3.31–3.39 (s, 2H); 4.15–4.17 (m, 3H); 5.52 (t, 2H); 12.13 (br s,
1H, NH); ESI/MS: 493.6 [M + H]+. Anal. (C21H28N6O6S) C,
H, N.
Compound 6u. Yield 20%; mp: 167–169 °C. 1H NMR (500
MHz, DMSO) δ: 0.96 (t, 6H, CH2CH2CH3, J ) 7.5 Hz); 1.31 (t,
6H, OCH2CH3, J ) 6.9 Hz); 1.70–1.73 (m, 4H, CH2CH2CH3); 2.08
(d, 2H); 2.74–2.77 (m, 4H); 2.87–2.88 (m, 4H); 3.08–3.41 (m, 6H);
3.31–3.39 (s, 2H); 4.15–4.17 (m, 3H); 5.52 (t, 4H); 7.07–7.28 (m,
1H); 7.83–7.94 (m, 1H); 12.13 (br s, 2H, NH); ESI/MS: 868.20
[M + H]+. Anal. (C38H46N10O10S2) C, H, N.
5-(2-Ethoxy-5-(N-ethanol,N′-5-(2-ethoxy-5-sulfonyl)phenyl)-
1-methyl-3-N-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-
7-one-ethylenediamine-sulfonyl)phenyl)-1-methyl-3-N-propyl-
1,6-dihydro-7H-pyrazolo [4,3-d]pyrimidin-7-one (6v). It was
synthesized from 5a (0.410 g, 1 mmol) and 2-(2-amino-ethylamino)-
ethanol (0,104 g, 1 mmol). Yield 45%; mp: 211–213 °C. 1H NMR
(500 MHz, DMSO) δ: 0.96 (t, 6H, CH2CH2CH3, J ) 7.5 Hz); 1.31
(t, 6H, OCH2CH3, J ) 6.9 Hz); 1.70–1.73 (m, 4H, CH2CH2CH3);
2.08 (t, 2H); 2.48 (t, 2H); 2.74–2.77 (m, 4H); 2.87–2.88 (m, 4H);
3.08–3.41 (m, 6H); 3.31–3.39 (s, 2H); 4.15–4.17 (m, 3H); 5.52 (t,
4H); 7.07–7.28 (m, 1H); 7.83–7.94 (m, 1H); 12.13 (br s, 2H, NH);
ESI/MS: 854.20 [M + H]+. Anal. (C38H48N10O9S2) C, H, N.
Acknowledgment. Haroldo Flores Toque is thankful to
Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP) for financial support. The NMR spectral data were

Sildenafil Analogues for Treatment of Erectile Dysfunction
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2815
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provided by Centro di Ricerca Interdipartimentale di Analisi
Strumentale, Universita degli Studi di Napoli “Federico II”. The
assistance of the staff is gratefully appreciated.
´
Supporting Information Available: Elemental analysis data for
compounds 6a–v. This material is available free of charge via the
Internet at http://pubs.acs.org.
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