Substituted pyrazolopyridopyridazines as orally bioavailable potent and selective PDE5 inhibitors: Potential agents for treatment of erectile dysfunction

Article abstract of DOI:10.1021/jm0256068

Novel pyrazolopyridopyridazine derivatives have been prepared as potent and selective PDE5 inhibitors. Compound 6 has been identified as a more potent and selective PDE5 inhibitor than sildenafil (1). It is as efficacious as sildenafil in in vitro and in

Full text of DOI:10.1021/jm0256068

J . Med. Chem. 2003, 46, 457-460
457
hydrolyzing enzyme present in the corpus cavernosum,
the smooth muscle tissue in the penis that is engorged
with blood during an erection. Upon sexual stimulation,
nitric oxide (NO) is released from nonadrenergic, non-
cholinergic neurons in the penis. NO activates guanylyl
cyclase, which in turn produces cGMP. cGMP initiates
a protein phosphorylation cascade, which causes a
decrease in the intracellular calcium concentration
within corpus cavernosal smooth muscle cells, resulting
in vasorelaxation, inflow of arterial blood, and ulti-
mately an erection.7 Inhibition of PDE5 increases the
effective concentration of cGMP in the corpus caverno-
sum, potentiating the above-described effects, leading
to enhanced erectile function.
Su bstitu ted P yr a zolop yr id op yr id a zin es
a s Or a lly Bioa va ila ble P oten t a n d
Selective P DE5 In h ibitor s: P oten tia l
Agen ts for Tr ea tm en t of Er ectile
Dysfu n ction
Guixue Yu,*,† Helen Mason,† Ximao Wu,†
J ian Wang,‡ Saeho Chong,‡ Bruce Beyer,§
Andrew Henwood,§ Ronald Pongrac,# Laurie Seliger,#
Bin He,# Diane Normandin,# Pam Ferrer,#
Rongan Zhang,# Leonard Adam,#
William G. Humphrey,‡ J ohn Krupinski,# and
J ohn E. Macor†
The therapeutic benefit of sildenafil in ED results
from its potent inhibition of PDE5. However, it is only
modestly selective toward some other PDE isozymes,
most notably PDE1 and PDE6 (Table 1). At relevant
therapeutic doses of sildenafil, it is likely that measur-
able inhibition of PDE1 and PDE6 occurs. As noted
earlier, this may be the cause of some of the side effects
associated with sildenafil use. Consistent with this
hypothesis is the fact that the incidence of side effects
associated with sildenafil use is dose-proportional. The
visual disturbances associated with sildenafil treatment
can be likely linked to inhibition of PDE6, the sole cGMP
PDE in the retina. Other side effects may be due, at
least in part, to nonspecific inhibition of PDE1, which
is abundant throughout most of the vasculature.8a Thus,
more selective PDE5 inhibitors should be of substantial
clinical interest as already exemplified by 2 and 3.2,8b
This manuscript details the discovery of a novel series
of pyrazolopyridopyridazine derivatives that are potent,
selective, efficacious, and orally bioavailable PDE5
inhibitors.
We have recently disclosed 5 as a potent and selective
PDE5 inhibitor.9a Upon examination of the X-ray struc-
ture of 5, it became apparent that the hydrogen from
the benzylic amine (-NH-) formed a hydrogen bond
with the amide carbonyl. This observation, coupled with
the structure of Eisai’s potent PDE5 inhibitor 4,10 led
us to hypothesize that constraining the conformation
of 5 should provide a novel and potent PDE5 scaffold.
Thus, the structural combination of 4 and 5 led to the
discovery of the pyrazolopyridopyridazine (PPP) scaffold,
typified by compounds 6-9, which were determined to
be potent and selective PDE5 inhibitors.
Discovery Chemistry, Drug Metabolism and
Pharmacokinetics, Drug Safety, and Metabolic and
Cardiovascular Drug Discovery, Bristol-Myers Squibb
Pharmaceutical Research Institute, P.O. Box 5400,
Princeton, New J ersey 08543-5400
Received November 6, 2002
Abstr a ct: Novel pyrazolopyridopyridazine derivatives have
been prepared as potent and selective PDE5 inhibitors.
Compound 6 has been identified as a more potent and selective
PDE5 inhibitor than sildenafil (1). It is as efficacious as
sildenafil in in vitro and in vivo PDE5 inhibition models, and
it is orally bioavailable in rats and dogs. The superior isozyme
selectivity of 6 is expected to exert less adverse effects in
humans when used for erectile dysfunction treatment.
Prior to the introduction of sildenafil [Viagra (1),
Figure 1] in 1998, erectile dysfunction (ED) was largely
an untreated medical disorder.1,2 Studied originally as
a treatment for angina, sildenafil is a potent inhibitor
of phosphodiesterase type 5 (PDE5).3 Its effectiveness
in treating ED was discovered serendipitously in phase
II angina trials. Despite its success in treating ED,
sildenafil has several notable side effects such as
headache, nausea, cutaneous flushing, and visual
disturbances.3a,4 These side effects may be attributed
to the limited selectivity of 1 against other PDE
isozymes, most notably PDE1 and PDE6 (Table 1).3
Thus, the need exists for improved PDE5 inhibitors
possessing greater PDE isozyme selectivity, which
should potentially lead to drugs with fewer side effects.2
Recently, compounds 2 (Vardenafil)5 and 3 (Tadalafil)6
have been studied in human clinical trials and have
demonstrated reduced adverse effects when compared
with sildenafil, probably due to their improved isozyme
selectivities verses PDE1 and PDE6. Here, we report a
new series of potent PDE5 inhibitors represented by the
clinical entity 6 with in vivo efficacy comparable to that
of 1 but with a much-improved PDE isozyme selectivity
profile.
The chemistry leading to 611 is shown in Scheme 1.
Pyrazolopyridine 10 was prepared using methodology
that we had previously published.9b Reaction of 10 with
hydrazine expanded the fused maleimide ring, forming
the pyridazine dione 11. Exposure of 11 to phosphorus
oxychloride afforded the dichloropyridazine derivative
12, which served as the key PPP scaffold allowing
independent examination of both the R- and â-positions
on the pyridazine ring (Scheme 1).
PDE5 is a member of the phosphodiesterase family
of enzymes that are responsible for the hydrolysis of
cGMP and/or cAMP.4 PDE5 is the primary cGMP
Using the chemistry sequence described in Scheme
1, we have examined substitutions at both positions. As
we previously observed,9a the 3-chloro-4-methoxy-
phenylmethylamine9c group constituted the best sub-
stituent at the R-position (Scheme 1). When the R-sub-
* To whom correspondence should be addressed. Phone: 609-818-
6528. Fax: 609-818-3450. E-mail: guixue.yu@bms.com.
† Discovery Chemistry.
‡ Drug Metabolism and Pharmacokinetics.
§ Drug Safety.
#
Metabolic and Cardiovascular Drug Discovery.
10.1021/jm0256068 CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/08/2003

458 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Letters
F igu r e 1. PDE 5 inhibitors.
Ta ble 1. PDE5 Inhibition and Isozyme Selectivities8
compd
IC50 PDE5 (nM)
IC50 ratio of 1/5
IC50 ratio of 2/5
IC50 ratio of 3/5
IC50 ratio of 4/5
IC50 ratio of 6/5
1a
25
3b
4c
5a
6a
7
1.6 ( 0.5
0.7
1.3
160
400
>104
>104
>104
1600
9200
>104
4000
>104
>104
4100
>104
>104
>104
3800
>104
7500
>104
>104
2900
>104
>104
1900
600
7
49
450
d
47
150
80
>104
>104
6400
>104
5700
>104
>104
0.6
0.8 ( 0.5
0.3 ( 0.1
0.7 ( 0.3
0.03 ( 0.01
0.10 ( 0.04
>104
300
8a
9a
7500
4000
30
210
a
Determined in house. Enzyme sources: PDE1, bovine heart; PDE2, rat kidney; PDE3, human platelet; PDE4, rat kidney; PDE5,
human platelet; PDE6, bovine retina. All IC50 determinations are averages based on three determinations. Taken from ref 5. c Taken
from ref 7. Not available.
b
d
stituent as the 3-chloro-4-methoxyphenylmethylamine
group was held constant, varied substitution at the
â-position provided numerous low nanomolar inhibitors,
typified by compounds 6-9. The broad SAR at this
position (Table 1, Figure 1) provided us with a unique
opportunity to modulate the pharmacokinetic properties
of this series of compounds. Eventually, 6 was chosen
to be evaluated further.12
The in vitro potency and selectivity of 6 were much
improved compared to sildenafil (1), particularly the
significantly improved selectivity for PDE5 versus PDE6
and PDE1 (Table 1).13
The ability of a compound to potentiate relaxation of
corpus cavernosal tissue strips in vitro has been used
as a functional measure of the PDE5 inhibition.9a This
model requires that the drug penetrates corpus caver-
nosal cells, since PDE5 is an intracellular enzyme.
When 6 was evaluated in rabbit corpus cavernosum
tissue strips, it demonstrated efficacy equivalent to that
of sildenafil (1, Table 2). Thus, in addition to being a
more potent and selective PDE5 inhibitor in vitro
(compared with sildenafil), compound 6 was at least
equivalent to sildenafil in its in vitro functional PDE5
activity in rabbit corpus cavernosal tissue.
the intracavernosal blood pressure in the penis of an
anesthetized rabbit as our in vivo measure of erectile
function.14 Accordingly, compound 6 was then examined
in vivo in anesthetized male rabbits, using sildenafil as
a control. Compound 6 demonstrated equal efficacy with
sildenafil in its ability to elevate mean blood pressure
in the corpus cavernosum (Table 2). This animal model
suggested that 6 and sildenafil were equivalent in vivo
in rabbits.
Further profiling of 6 was pursued given the excellent
in vitro and in vivo results obtained. Thus, examination
of the pharmacokinetic profile of 6 in rats (2.0 µmol/kg)
and dogs (5.1 µmol/kg) demonstrated equal or better
exposure upon oral dosing of the drug in rats and dogs
when compared with 1 (Table 2). The relatively short
terminal half-life of 6 in both species was consistent
with the on-demand dosing that is likely best suited for
a medication for this quality-of-life disorder (Table 2).
These results gave confidence to the expectation that 6
would be orally active in humans.
Appropriate toxicity studies of 6 necessarily followed
the pharmacokinetic studies. Accordingly, the safety
profile of 6 in rats was evaluated at doses of 10, 50, and
250 mg/kg, and there were no drug-related adverse
effects at 10 and 50 mg/kg. The only adverse effect
observed at 250 mg/kg was limited to lower body weight
in male rats (6% lower than control). There were no
drug-related gross-pathologic lesions observed at any
Since sildenafil was identified in human clinical trials
and not as a result of a preclinical studies, we needed
to develop our own preclinical model of erectile function.
To accomplish this, we used drug-induced increases in

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 459
Sch em e 1a
Ewing and Carl Decicco for helpful comments during
the manuscript preparation. We thank Dr. Jack Gougou-
tas, Mr. J ohn DiMarco, and Ms. Mary Malley for the
X-ray structure determination.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails, X-ray structure of 6, and PDE assay protocol. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Refer en ces
(1) (a) Martel, A. M.; Graul, A.; Rabasseda, X.; Castan˜er, R.
Treatment of erectile dysfunction, Phosphodiesterase V inhibitor.
Drugs Future 1997, 22, 138-143. (b) Terrett, N. K.; Bell, A. S.;
Brown, D.; Ellis, P. Sildenafil (Viagra), a potent and selective
inhibitor of type 5 cGMP phosphodiesterase with utility for the
treatment of male erectile dysfunction. Bioorg. Med. Chem. Lett.
1996, 6, 1819-1824. (c) Boolell, M.; Allen, M. J .; Ballard, S. A.;
Gepi-Attee, S.; Muirhead, G. J .; Naylor, A. M.; Osterloh, I. H.;
Gingell, C. Sildenafil: an orally active type 5 cyclic GMP-specific
phosphodiesterase inhibitor for the treatment of penile erectile
dysfunction. Int. J . Urol. Res. 1996, 8, 47-52. (d) Sorbera, L.
A.; Martin, L.; Leeson, P. A.; Castaner, J . IC-351. Drug Future
2001, 26, 15-19.
a
Reagents: (a) hydrazine hydrate (2.0 equiv), EtOH/water,
(2) Rotella, D. P. Phosphodiesterase 5 Inhibitors: Current Status
and Potential Applications. Nat. Rev. Drug Discovery 2002, 1,
674-682.
room temperature, 2 h (100%); (b) POCl3 (3.0 equiv, neat), reflux
3 h (85%); (c) 15 (1.05 equiv), (i-Pr)2NEt (3.0 equiv), NMP, at 80
°C for 3 h (40%); (d) 3-Cl-4-MeO-phenylmethylamine‚HCl (1.1
equiv), (i-Pr)2NEt (3.0 equiv), NMP, at 110 °C for 3 h (85%); (e)
SOCl2 (3.0 equiv), DMF (cat.), toluene, reflux 2 h (100%); (f)
CH3NH2 (5.0 equiv), THF at room temperature (90%).
(3) (a) Eardley, I. The role of phosphodiesterase inhibitors in
impotence. Expert Opin. Invest. Drugs 1997, 6, 1803-1810. (b)
Garcia-Reboll, L.; Mulhall, J . P.; Goldstein, I. Drugs for the
treatment of impotence. Drugs Aging 1997, 11, 140-151. (c)
Truss, M. C.; Stief, C. G. Phosphodiesterase inhibitors in the
treatment of erectile dysfunction. Drugs Today 1998, 34, 805-
812.
Ta ble 2. Characteristics of 6 Compared to Sildenafil (1)a
(4) (a) Beavo, J . A. Cyclic nucleotide phosphodiesterases: functional
implications of multiple isoforms. Physiol. Rev. 1995, 75, 725-
748. (b) Scrip 1997, October 9.
(5) Ormrod, D.; Easthope, S. E.; Figgitt, D. P. Vardenafil. Drugs
Aging 2002, 19, 217-227.
(6) Eardley, I.; Cartledge, J . Int. J . Clin. Pract. 2002, 56, 300-304.
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Physiol. Rev. 1995, 75, 191-236.
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Cyclic GMP phosphodiesterase-5: target of sildenafil. J . Biol.
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EC50
in vitro
strip
ED50 rabbit
in vivo
half-life (h)
in male
F (%) in male
rats (R)
model
rats (R) or
dogs (D)
compd
assay (nM)
(nmol/kg)
or dogs (D)
1 (sildenafil)
19
13
110
100
15 (R)
54 (D)
29 (R)
42 (D)
0.3 (R)
5.2 (D)
1.2 (R)
2.0 (D)
6
a
Oral bioavailability (% F) and half-life of sildenafil taken from
the Viagra NDA.
(9) (a) Yu, Guixue, Mason, H. J .; Wu, X.; Wang, J .; Chong, S.;
Dorough, G.; Henwood, A.; Pongrac, R.; Seliger, L.; He, B.;
Normandin, D.; Adam, L.; Krupinski, J .; Macor, J . E. Substituted
pyrazolopyridine as potent and selective PDE5 inhibitors: potent
agents for treatment of erectile dysfunction. J . Med. Chem. 2001,
44, 1025-1027. (b) Mason, H. J .; Wu, X.; Schmitt, R.; Yu, G.;
Macor, J . E. Synthesis of fused pyridopyrrolidine dione deriva-
tives using hetero Diels-Alder reactions Tetrahedron Lett. 2001,
42, 8931-8934. (c) Yu, G.; Mason, H. J .; Wu, X.; Endo, M.;
Douglas, J .; Macor, J . E. A mild and convenient method for
aromatic chlorination of electron-rich arylalkyl amines. Tetra-
hedron Lett. 2001, 42, 3247-3249. (d) Yu, G.; Macor, J . E.;
Chung, H.-J .; Humora, M.; Katipally, K.; Wang, Y.; Kim, S.
Patent WO 0056719, 2000.
(10) Watanabe, N.; Adachi, H.; Takase, Y.; Ozaki, H.; Matsukura,
M.; Miyazaki, K.; Ishibashi, K.; Ishihara, H.; Kodama, K.;
Nishino, M.; Kakiki, M.; Kabasawa, Y. 4-(3-Chloro-4-methoxy-
benzyl)aminophthalazines: synthesis and inhibitory activity
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2529.
dose in both male and female rats. Because of its
excellent in vitro and in vivo potency, selectivity, and
safety profile, compound 6 was progressed into human
clinical trials and was found to be well tolerated at the
highest dose tested (50 mg) in healthy human volun-
teers.15
In summary, we have identified a series of novel and
potent PDE5 inhibitors based on a pyrazolopyridopy-
ridazine template (i.e., 6).12 These compounds were
characterized by potent PDE5 inhibition in vitro and
potent efficacy in vivo. Compound 6 distinguished itself
by its selectivity profile and in vivo efficacy. Its PDE1
and PDE6 isozyme selectivities were superior to that
of 1 (sildenafil). Additional studies demonstrated that
6 had a desirable pharmacokinetic profile in two animal
species with no safety concerns. Accordingly, studies in
healthy volunteers were conducted, and no safety
concerns were revealed. Thus, because of its improved
PDE isozyme selectivity profile compared with that of
sildenafil, compound 6 demonstrated fewer PDE-related
side effects, such as visual disturbances, in healthy
volunteers. A detailed SAR examination of this series
and the effects of 6 in human subjects will be reported
in due course.
(11) The characterization of compound 6 was as follows. Compound
6: 1H NMR (400 MHz, CD3Cl) δ 1.56 (t, 3H), 3.03 (d, 3H), 3.87
(s, 3H), 4.75 (q, 2H), 4.97 (d, 2H), 5.79 (bs, 1H, NH), 6.90 (d,
2H), 7.25 (m, 1H), 7.37 (m, 1H), 7.48 (s, 1H), 8.02 (d, 2H), 8.47
(s, 1H), 9.01 (s, 1H); 13C NMR (400 MHz, CDCl3) δ 15.0, 25.8,
43.3, 45.6, 56.2, 99.9, 104.1, 112.3, 113.8, 118.1, 122.5, 122.8,
127.6, 129.9, 130.6, 131.1, 137.3, 138.4, 143.0, 146.2, 149.3, 154.2,
162.7. Anal. Calcd for C23H22ClN9O2: C, 56.16; H, 4.51; N, 25.63;
Cl, 7.21. Found: C, 55.72; H, 4.32; N, 25.94; Cl, 7.23. Mp 181-
183 °C and 206-207 °C.
(12) A more comprehensive examination of the SAR within this series
will be presented separately.
(13) Enzyme inhibition assays were carried out as described in the
following. Rotella, D.; Sun, Z.; Zhu, Y.; Krupinski, J .; Pongrac,
R.; Seliger, L.; Normandin, D.; Macor, J . E. N-3-Substituted
imidazoquinazolinones: potent and selective PDE5 inhibitors as
potential agents for treatment of erectile dysfunction. J . Med.
Chem. 2000, 43 (7), 1257-1263.
Ack n ow led gm en t. We thank Drs. David Rotella,
Yingzhi Bi, and J acques Roberge for their helpful
discussions throughout this project. We thank Drs. Rick

460 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Letters
(14) Rabbits were anesthetized with a combination of ketamine (10
mg/kg, im) followed by urethane (0.5 g/kg, iv). Arterial and
venous accesses were obtained via the femoral artery and vein
for the measurement of blood pressure and the administration
of compounds, respectively. Intracavernosal pressure (ICP) was
monitored continuously during the course of the experiment.
After a baseline ICP was measured in an animal, SNAP was
injected intracavernosally (ic). The total amount of SNAP
injected ranged from 1 to 5 µg/kg in a final volume of 100 µL of
Dulbecco’s phosphate-buffered saline (DPBS). The catheter was
flushed with 200 mL of DPBS containing heparin (5 units/mL)
to ensure that all of the SNAP was injected into the corpus. ICP
responses to SNAP after the initial transient injection artifact
were characterized by the peak pressure that developed (in
mmHg), the duration of the response (in seconds (s)), and most
importantly the area under the curve of the response (AUC in
mmHg s). Control responses to SNAP in each animal were
measured in triplicate before the iv administration of 6 or
sildenafil. After determination of control SNAP-dependent ICP
responses, increasing doses of either 6 or sildenafil were given
followed by SNAP to determine the effect of the PDE5 inhibitors
on the NO-dependent changes in ICP. Increasing doses of the
compound (10, 30, 100, 300, and 1000 nmol/kg) were given 30
min apart, and SNAP was injected 10 min after the administra-
tion of each dose. Standard dose-dependent curves were gener-
ated, and the ED50 values were determined using these curves.
(15) The results of the human clinical trials of 6 will be reported
elsewhere in due course.
J M0256068