A novel series of potent and selective PDE5 inhibitors with potential for high and dose-independent oral bioavailability

Article abstract of DOI:10.1021/jm060113e

Sildenafil (5-[2-ethoxy-5-(4-methyl-1-piperazinylsulfonyl)phenyl]-1-methyl- 3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one), a potent and selective phosphodiesterase type 5 (PDE5) inhibitor, provided the first oral treatment for male erectile dy

Full text of DOI:10.1021/jm060113e

J. Med. Chem. 2006, 49, 3581-3594
3581
A Novel Series of Potent and Selective PDE5 Inhibitors with Potential for High and
Dose-Independent Oral Bioavailability†
Charlotte M. N. Allerton,*,‡ Christopher G. Barber,‡ Kevin C. Beaumont,§ David G. Brown,| Susan M. Cole,§ David Ellis,‡
Charlotte A. L. Lane,‡ Graham N. Maw,‡ Natalie M. Mount, David J. Rawson,‡ Colin M. Robinson,|
Stephen D. A. Street,‡ and Nicholas W. Summerhill‡
DiscoVery Chemistry, Pharmacokinetics, Dynamics and Metabolism, Lead DiscoVery, and DiscoVery Biology, Pfizer Global Research &
DeVelopment, Ramsgate Road, Sandwich, Kent CT13 9NJ, United Kingdom
ReceiVed February 2, 2006
Sildenafil (5-[2-ethoxy-5-(4-methyl-1-piperazinylsulfonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7H-pyra-
zolo[4,3-d]pyrimidin-7-one), a potent and selective phosphodiesterase type 5 (PDE5) inhibitor, provided
the first oral treatment for male erectile dysfunction. The objective of the work reported in this paper was
to combine high levels of PDE5 potency and selectivity with high and dose-independent oral bioavailability,
to minimize the impact on the Cmax of any interactions with coadministered drugs in the clinic. This goal
was achieved through identification of a lower clearance series with a high absorption profile, by replacing
the 5′-piperazine sulfonamide in the sildenafil template with a 5′-methyl ketone. This novel series provided
compounds with low metabolism in human hepatocytes, excellent caco-2 flux, and the potential for good
aqueous solubility. The in vivo oral and iv pharmacokinetic profiles of example compounds confirmed the
high oral bioavailability predicted from these in vitro screens. 5-(5-Acetyl-2-butoxy-3-pyridinyl)-3-ethyl-
2-(1-ethyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (2) was selected for progression into
the clinic.
Introduction
impact on Cmax of any interactions with coadministered drugs.
In general, good solubility, high absorption across the intestinal
wall, and low first-pass clearance are required for high oral
bioavailability.4 We focused on reducing the first-pass clearance
of soluble, rule-of-five compliant5 PDE5 inhibitors to achieve
this objective.
The launch of sildenafil (Figure 1) as the first oral treatment
for male erectile dysfunction revolutionized the treatment of
this disease.1 Sildenafil is a potent and selective inhibitor of
phosphodiesterase type 5 (PDE5), the predominant isozyme
responsible for the metabolism of cGMP in the corpus caver-
nosum of the penis. Following sexual stimulation, nitric oxide
is released from the nonadrenergic, noncholinergic nerves within
the penis, which activates guanylate cyclase to form cGMP via
cyclization of GTP. This increase in intracellular cGMP leads
to smooth muscle relaxation, which enables blood to flow into
the penis and hence erection to occur. Hydrolysis of cGMP to
GMP by PDE5 reverses this process, restoring muscle tone with
the consequential loss of erection.2 Administration of sildenafil
inhibits PDE5 activity and therefore slows cGMP breakdown.
This enhances the action of nitric oxide and cGMP, and
facilitates penile erection in individuals suffering from male
erectile dysfunction (MED).
Following the development of sildenafil, we sought PDE5
inhibitors with greater selectivity over phosphodiesterase type
6 (PDE6), since this enzyme is believed to be responsible for
the low incidence of adverse visual events, such as abnormalities
in color vision, associated with high doses of sildenafil. SAR
describing how this selectivity was achieved through the
discovery of the pyridyl methyl analogue 1 (Figure 1) has
already been published.3 The objective for the current program
was to identify a potent and selective PDE5 inhibitor with high
and dose-independent oral bioavailability, to minimize the
The piperazine sulfonamide group is the primary site of
metabolism in the sildenafil series.6 Consequently, we concen-
trated on finding a more metabolically stable replacement for
this functionality. This led to a novel series of potent and
selective PDE5 inhibitors, with a methyl ketone at the 5′-position
of the 5-(2-alkoxy-3-pyridinyl)-2,6-dihydro-7H-pyrazolo[4,3-
d]pyrimidin-7-one template. These compounds are relatively
small (MW < 500) with moderate lipophilicity, and this results
in good oral absorption. In addition, they have low clearance
(the predominant metabolite in human hepatocytes being the
secondary alcohol) and a basic group attached at the N2 position,
which provides good aqueous solubility via salt formation. The
combination of these factors leads to high oral bioavailability
in vivo. In particular, compound 2 was shown to have the
potential for high and dose-independent oral absorption in man
and was therefore progressed to the clinic to determine its
potential as an oral agent for the treatment of MED. This paper
explains the key results that led to its selection.
Chemistry
Initially, the 5′-methyl ketone functionality was introduced
into the pyrazolopyrimidinone template via mercury-mediated
hydrolysis of an acetylene (Scheme 1). The key intermediate,
5-iodo-2-propoxynicotinic acid 4, synthesized via iodination of
2-propoxynicotinic acid 3,7 was converted to the acid chloride
and then coupled with 4-amino-5-ethyl-1-(2-pyridinylmethyl)-
1H-pyrazole-3-carboxamide.8 Subsequent cyclization with po-
tassium bis(trimethylsilyl)amide in n-propanol yielded the
pyrazolopyrimidinone template 6. The acetylene functionality
was then introduced via a Sonagashira reaction with (trimethyl-
† Coordinates have been deposited for the structure of the complex of
compound 2 with the catalytic domain of PDE5. PDB ID: 2CHM.
* To whom correspondence should be addressed. Telephone: +44 1304
616161. Fax: +44 1304 651817. E-mail: charlotte.allerton@pfizer.com.
‡ Discovery Chemistry.
§ Pharmacokinetics, Dynamics and Metabolism.
| Lead Discovery.
Discovery Biology.
10.1021/jm060113e CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/24/2006

3582 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Allerton et al.
Figure 1. Discovery of 5′-ketone series.
silyl)acetylene,9 followed by deprotection with potassium
fluoride. The acetylene intermediate 7 was hydrolyzed under
acidic conditions in the presence of mercury sulfate to yield
the methyl ketone 8. Finally, the 2′-butoxy analogue 9 was
synthesized by heating compound 8 in butanol in the presence
of potassium bis(trimethylsilyl)amide. The reaction was low
yielding due to competing Meerwein reduction10 of the methyl
ketone.
pyrazole-3-carboxamide8 was coupled with 2-butoxy-5-iodoni-
cotinic acid 11 and then the N2-substituent was introduced
regioselectively via alkylation with N-(2-chloroethyl)morpholine.
Some of the N2-azetidinyl and N2-piperidinyl analogues were
also synthesized by a similar route (Scheme 3). Acidic hydroly-
sis of the acetylene functionalities in compounds 23/24 led to
partial BOC deprotection, which was then completed by stirring
the crude product in trifluoroacetic acid. Alkyl substituents were
then introduced onto the azetidine or piperidine nitrogens via
reductive amination. This worked well with formaldehyde and
The N2-morpholinoethyl analogue 16 was synthesized by a
similar route (Scheme 2). In this case, 4-amino-5-ethyl-1H-
Scheme 1. Synthesis of 5′-Ketones Using Sonagashira Methodology
Reagents and conditions: (a) NIS, TFA, TFAA, reflux; (b) (COCl)2, CH2Cl2, DMF, 0 °C to rt, then 4-amino-5-ethyl-1-(2-pyridinylmethyl)-1H-pyrazole-
3-carboxamide, pyridine, DCM, rt; (c) KHMDS, n-propanol, reflux; (d) (i) trimethylsilylacetylene, Pd(PPh3)2Cl2, CuI, triethylamine, MeCN, 60 °C; (ii) KF,
DMF, H2O, rt; (e) H2SO4, HgSO4, Me2CO, reflux; (f) KHMDS, n-BuOH, 90 °C.
Scheme 2. Synthesis of N2-Morpholinoethyl Analogues
Reagents and conditions: (a) KHMDS, n-BuOH, THF 0 °C to rt; (b) (COCl)2, CH2Cl2, DMF, 0 °C to rt, then 4-amino-5-ethyl-1H-pyrazole-3-carboxamide,
pyridine, DCM, rt; (c) N-(2-chloroethyl)morpholine hydrochloride, Cs2CO3, DMF, 80 °C; (d) KHMDS, n-BuOH, reflux; (e) (i) trimethylsilylacetylene,
Pd(PPh3)2Cl2, CuI, triethylamine, MeCN, 60 °C; (ii) KF, DMF, H2O, RT; (f) H2SO4, HgSO4, Me2CO, reflux; (g) KHMDS, 2-methoxyethanol, 120 °C.

SelectiVe PDE5 Inhibitors with Oral BioaVailability
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3583
Scheme 3. Synthesis of N2-Azetidinyl and N2-Piperidinyl Analogues
Reagents and conditions: (a) (COCl)2, CH2Cl2, DMF, 0 °C to rt, then 4-amino-5-ethyl-1H-pyrazole-3-carboxamide, pyridine, DCM, rt; (b) tert-butyl-3-
iodo-1-azetidinecarboxylate (19) or tert-butyl 4-[(methylsulfonyl)oxy]-1-piperidinecarboxylate (20), Cs2CO3, DMF, 100 °C; (c) KHMDS, n-PrOH, reflux;
(d) (i) trimethylsilylacetylene, Pd(PPh3)2Cl2, CuI, triethylamine, MeCN, 60 °C; (ii) KF, DMF, H2O, rt; (e) H2SO4, HgSO4, Me2CO, reflux, then trifluoracetic
acid, CH2Cl2, rt; (f) HCHO (27/32) or Me2CO (29), Na(OAc)3BH, CH2Cl2, rt or MeCHO, NaCNBH3, NaOAc, MeOH, rt (28/33); (g) Cs2CO3, n-BuOH,
reflux; (h) Cs2CO3, i-BuOH , reflux.
acetone but led to low-yielding reactions with acetaldehyde. As
with the synthesis of compound 9, the 2′-alkoxy substituent
could then be varied by performing an exchange reaction in
the presence of the required alcohol and cesium carbonate.
Cesium carbonate was preferable to potassium bis(trimethylsi-
lyl)amide for the exchange reactions, since it minimized
Meerwein reduction of the ketone.
The routes outlined above allowed rapid exploration of SAR,
since the versatile 5′-iodo group enabled the introduction of a
wide range of functionality at this position (although only 5′-
ketones are discussed in this paper). Once the 5′-methyl ketone
substituent had been identified as yielding potent, selective
PDE5 inhibitors with high bioavailability, we investigated
alternative routes directly to this series. Therefore, an alternative
route was designed that involved earlier introduction of the 5′-
methyl ketone functionality.
3611 (Scheme 4). This was coupled with 4-amino-3-ethyl-1H-
pyrazole-5-carboxamide,8 and the resulting amide 37 was
alkylated to introduce the desired N2 substituents 38/39.
Subsequent cyclization with cesium carbonate in n-butanol at
reflux was accompanied by concomitant 2′-ether exchange to
yield the pyrazolopyrimidinones 40/41. BOC deprotection with
trifluoroacetic acid followed by either reductive amination or
alkylation gave the target compounds 2/44. An identical route,
starting from 4-amino-3-propyl-1H-pyrazole-5-carboxamide,12
was followed to synthesize the C3-propyl N2-azetidine ana-
logues 45/46 discussed in Table 3 (see Experimental Section
for synthetic details).
The N2-methylimidazole analogues 48/49 were synthesized
by a similar procedure, in which the 5-acetyl-2-ethoxynicotinic
acid 36 was coupled with 4-amino-5-ethyl-1-[(1-methyl-1H-
imidazol-2-yl)methyl]-1H-pyrazole-3-carboxamide.7 The cy-
clization reaction of intermediate 47 with simultaneous 2′-
A Heck reaction between 5-bromo-2-ethoxynicotinic acid 35
and butyl vinyl ether yielded the key methyl ketone intermediate
Scheme 4. Synthesis of 5′-Ketones Using Heck Methodology
Reagents and conditions: (a) butyl vinyl ether, Pd(OAc)2, tri-o-tolylphosphine, Et3N, Na2CO3, MeCN, reflux, (65%); (b) 4-amino-3-ethyl-1H-pyrazole-
5-carboxamide, HATU, CH2Cl2, rt; (c) tert-butyl-3-iodo-1-azetidinecarboxylate (38) or 1-(tert-butoxycarbonyl)-4-piperidinyl methanesulfonate (39), Cs2CO3,
DMF, 100 °C; (d) Cs2CO3, n-BuOH, molecular sieves, reflux; (e) trifluoroacetic acid, CH2Cl2, rt; (f) EtI, K2CO3, MeCN, 45 to 50 °C.

3584 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Scheme 5. Synthesis of N2-Methylimidazole Analogues
Allerton et al.
Reagents and conditions: (a) 4-amino-5-ethyl-1-[(1-methyl-1H-imidazol-2-yl)methyl]-1H-pyrazole-3-carboxamide, HATU, CH2Cl2, rt; (b) Cs2CO3, n-PrOH,
molecular sieves, reflux; (c) Cs2CO3, n-BuOH, molecular sieves, reflux.
Scheme 6. Optimized Synthetic Route to N2-Azetidinyl Metabolite
Reagents and conditions: (a) 1-benzhydryl-3-azetidinyl methanesulfonate, Na2CO3, NaI, THF/H2O, reflux; (b) 1-chloroethyl chloroformate, CH2Cl2, then
MeOH, reflux; (c) (i) MeCHO, Na(OAc)3BH, triethylamine, DCM, MeOH, 0 °C to rt; (ii) H2 (60 psi), 10% Pd/C, EtOH, rt; (d) 36, 1,1′-carbonyldiimidazole,
EtOAc, reflux; (e) Cs2CO3, n-BuOH, reflux.
Scheme 7. Synthesis of Alcohol Metabolite
The alcohol metabolite of 2 was synthesized by reduction of
the ketone with sodium borohydride, to yield compound 55
(Scheme 7).
Results and Discussion14
Background. Since the development of sildenafil, extensive
effort has been focused on identifying PDE5 inhibitors with
greater selectivity over PDE6, since this isozyme is responsible
for the low incidence of visual disturbances reported with high
doses of sildenafil. Previous publications have outlined how
greater selectivity was introduced into the sildenafil template
by replacing the phenylsulfonamide with pyridylsulfonamide,
varying the 2′-substituent, and introducing N-1 or N-2 pyrazole
substituents. This led to the development of compound 1 (Figure
1), which is a potent PDE5 inhibitor displaying approximately
300-fold selectivity over canine cone PDE6.3 In clinical trials,
compound 1 showed dose-dependent increases in Cmax and AUC
over the dose range 1-800 mg and increases in these parameters
when coadministered with ketoconazole (a potent inhibitor of
P-glycoprotein and CYP3A4).15 Further studies showed that
compound 1 is a substrate for human P-glycoprotein and
CYP3A4, which explains the variations in pharmacokinetic data
that arose when these species were either saturated or inhibited.
As a consequence of these clinical data, we desired a PDE5
inhibitor with high and dose-independent oral bioavailability,
since this would minimize the impact of any interactions with
coadministered drugs in the clinic.
Reagents and conditions: (a) NaBH4, MeOH, 0 °C to rt.
exchange was performed in n-propanol to give 48 and in
n-butanol to give 49 (Scheme 5).
To supply bulk quantities of 2 to support our development
program, we were keen to investigate a more convergent route,
which avoided the use of the potential carcinogen ethyl iodide
(Scheme 6). Therefore, the nitro pyrazole intermediate 508 was
alkylated with 1-(diphenylmethyl)-3-azetidinylmethanesulfonate
to give a 2:3 ratio of N1:N2 regioisomers.13 The N2 regioisomer
51 was separated by trituration and deprotected with 1-chloro-
ethyl chloroformate to yield compound 52. The ethyl group was
then cleanly introduced by reductive amination with acetalde-
hyde, and the nitro group was reduced to yield amine 53.
Addition of amine 53 to the acyl imidazole derivative of
5-acetyl-2-ethoxynicotinic acid 36 yielded amide 54, which then
underwent cyclization and simultaneous 2′-exchange to yield
the target compound 2.

SelectiVe PDE5 Inhibitors with Oral BioaVailability
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3585
Figure 2. Discovery of 5′-methyl ketone series.
The high oral bioavailability of a compound is generally a
result of good solubility, high absorption across the intestinal
wall, and low first-pass hepatic extraction.4 Measured solubilities
were used as a filter for our compound selection. Caco-2
monolayers were used to predict human intestinal absorption
potential.16 A high absorptive flux (Papp > 10 × 10-6 cm/s)17
and an efflux ratio close to unity across the concentration range
10-100 µM should ensure that concentration dependence is
avoided in man.15 The metabolism of compounds by human
hepatocytes was used as an in vitro prediction of first-pass
clearance.18 These in vitro assays were used to select compounds
to profile in vivo. In addition, we only synthesized rule-of-five-
compliant compounds to maximize our chances of achieving
good oral absorption.5 With these measures in place, we then
focused on reducing first-pass clearance. Since the predominant
route of clearance for most sildenafil analogues is cytochrome
P-450-mediated metabolic oxidation of the piperazine ring by
CYP3A4,6 we decided to remove this functionality from the
template and replace it with a less metabolically vulnerable
group.
enhancing potency and selectivity, while maintaining good
Caco-2 flux.
First, we replaced the phenyl ring for a pyridyl and introduced
a N-2 pyridylmethyl group. Both of these changes had previ-
ously led to higher in vitro selectivity in the sildenafil series
(1, Figure 1).3 In addition a 2′-butoxy substituent was introduced,
since SAR predicted that this would lead to an increase in
potency and selectivity.3 The resulting compound 9 displayed
excellent levels of PDE5 potency and PDE6 selectivity, with
Caco-2 cell flux indicative of complete oral absorption (Figure
2). In vitro human hepatocyte stability data predicted this
compound would have low first-pass clearance. As expected,
compound 9 displayed low total clearance in the dog iv PK
study, predictive of 84% bioavailability (Table 1).
Compound 9 showed that low first-pass clearance was
achievable within this methyl ketone series. However, the
subsequent oral PK study in dog showed the compound to have
variable oral bioavailability, due to its poor aqueous solubility
(3 µg/mL at pH 7.2). Hence, we decided to introduce more basic
substituents at the N-2 position to enable the synthesis of more
soluble salt forms (Table 2).
Discovery of Selective 5′-Ketone Series. An early analogue
of sildenafil, compound 56, contained a ketone at the 5′-position
(Figure 2). This compound was a potent PDE5 inhibitor, with
an excellent pharmacokinetic profile in man (oral bioavailability
> 80%, clearance ) 5 mL/min/kg), but since it displayed no in
vitro selectivity advantage over sildenafil, it was not progressed
further.19 On the basis of the pharmacokinetic profile of this
compound in man, we felt that low clearance and high
bioavailablity was achievable in other 5′-ketone series. To
introduce PDE6 selectivity into this template, we planned to
use the SAR developed in the sildenafil series (outlined above),
including the introduction of N-2 substituents. However, before
undertaking this work, we were keen to find the minimal
pharmacophore required for PDE5 potency in the 5′-ketone
series, to ensure that the molecular weight of compounds did
not exceed 500.5 Therefore, we profiled compound 57, the 5′-
methyl ketone analogue of compound 56, and found that it
retained moderate PDE5 potency.19 This compound provided a
low molecular weight, potent template onto which N-2 substit-
uents and larger 2′-groups could be introduced with the aim of
The SAR at the 2′-position (R1) follows that seen in the
sildenafil series, with the butoxy substituent giving rise to more
potent and selective compounds than the lower alkoxy groups.
A small subset of analogues in the C3 propyl series were also
synthesized, but since these offered little potency or selectivity
advantages over the C3 ethyl series and were more lipophilic,
no further analogues were made (Table 3).
The most potent and selective analogues from Table 2 were
profiled through the in vitro pharmacokinetic assays described
earlier (Table 4). The morpholine-containing analogue 16
displayed low solubility, and its weakly basic center did not
lend itself to salt formation; hence, this compound did not meet
our criteria. The imidazole containing analogue 49 showed an
unacceptably high rate of metabolism in human hepatocytes,
and a low Papp value (which may have been due to its low
solubility); therefore, this compound was not progressed for
future studies. The more basic amine derivatives 29, 2, and 44
all displayed low rates of metabolism in human hepatocytes,
Papp values predictive of complete oral absorption, and adequate
Table 1. Pharmacokinetic Parameters for Compound 9 Administered Intravenously to Dog (mean values for n ) 2)
a PPB ) plasma protein binding.

3586 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Table 2. SAR for 5′-Methyl Ketones with Basic N2 Substituents
Allerton et al.
a Platelet PDE5 enzyme used.
Table 3. Comparison of SAR for C3 Ethyl and C3 Propyl Methyl
Ketones
greater than 75%, with half-life values commensurate with oral,
pro re nata dosing.
On the basis of the dog pharmacokinetic studies and the
resulting predicted human pharmacokinetic parameters, it was
felt that both compounds 29 and 2 were suitable for progression
into the clinic. On the basis of clinical data from previous PDE5
inhibitor development candidates, the level of selectivity seen
with these compounds will prevent visual disturbances from
occurring at all clinically relevant doses. Due to the greater
selectivity of compound 2 over canine retinal cone PDE6, this
compound was progressed at highest priority. In human hepa-
tocytes, the predominant metabolite from compound 2 was the
secondary alcohol 55,21 formed by ketone reductase. This
secondary alcohol is oxidized back to the 5′-ketone in hepato-
cytes, so the parent ketone and its alcohol metabolite exist in
equilibrium.22 Some CYP3A4 metabolism was also observed,
giving rise to the des-alkyl compound 42 as a minor metabolite
(Table 2). Ketone reductase is present in the cytosol in the liver;
therefore, the human hepatocyte assay provided us with a good
prediction of rate of clearance via this route as well as via
cytochrome P-450-mediated metabolism. The alcohol metabolite
55 and the des-alkyl metabolite 42 both showed significant
decreases in PDE5 and PDE6 potency, which will prevent any
problems associated with active circulating metabolites (Figure
3 and Table 2).
solubility (with the potential to prepare more soluble salt forms).
Therefore, since these compounds also displayed low rates of
metabolism in dog liver microsomes, they were progressed for
dog iv and oral pharmacokinetic studies.
Compound 44 displayed a higher total clearance in dog than
the other compounds, which was surprising considering its lower
lipophilicity and stability in human hepatocytes (Table 5).
Therefore compound 44 was less likely to achieve an oral
bioavailability of greater than 75% in man. Both compounds
29 and 2 displayed low total clearances and oral bioavailabilities
Absorption Profile of Compound 2. To determine whether
compound 2 would display the dose-independent oral absorption
desired in man, its Caco-2 flux was measured across the

SelectiVe PDE5 Inhibitors with Oral BioaVailability
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3587
Table 4. In Vitro Pharmacokinetic Data on Key 5′-Methyl Ketones
a Predicted from hepatocytes. b e.r. ) efflux ratio.
concentration range 10-100 µM (Figure 4). For the Pgp
substrate 1, the absorptive flux increased, and hence the efflux
ratio decreased, both as the compound concentration increased
(due to saturation of the P-glycoprotein efflux mechanism) and
in the presence of the P-glycoprotein inhibitor ketoconazole.
Hence, when compound 1 was coadministered with ketocona-
zole in phase II clinical trials, this variability in absorption
combined with the expected inhibition of CYP3A4 led to
significant increases in Cmax and AUC.15 For compound 2, the
absorptive flux in the Caco-2 cell model remained consistently
high across the concentration range, and also in the presence
of ketoconazole. This predicts that compound 2 will display
high and dose independent oral absorption in man. These data
are consistent with Lipinski’s predictions for good oral absorp-
tion, since compound 2 meets all of the rule-of-five criteria.
Compound 1, on the other hand, exceeds the MW rule, and the
corresponding increase in molecular size may be the cause of
variability in its absorption profile.
Rationale for PDE6 Selectivity of Compound 2. The SAR
shows that potent and selective PDE5 inhibitors can be achieved
with either basic alkyl or heteroaryl N-2 pyrazole substituents,
suggesting that these groups may be pointing out toward bulk
solvent rather than making any specific interactions with the
PDE5 enzyme as detailed in the X-ray crystal structures of PDE5
Table 5. Dog Pharmacokinetic Data on Key 5′-Methyl Ketones (n ) 2)

3588 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Allerton et al.
Figure 5. Crystallographic overlay of structures of sildenafil (green
C atoms) and compound 2 (purple C atoms).
Figure 3. Biological activity of alcohol metabolite.
catalytic domain in complex with inhibitors of this class.23 The
simple addition of a methyl at the N2 position of the series
causes a complete “flip” in binding mode compared to sildenafil
and helps explain the change in PDE6 selectivity for this N2
substituted series (Figure 5).
The new binding mode adopted by compound 2 clearly makes
different interactions with the catalytic site of PDE5 (Figure
6). The major potency-gaining hydrophobic interactions with
the Phe820 and Phe376 are maintained, but the hydrogen-
bonding pattern with the conserved glutamine Gln817 is
changed, and access to a deep hydrophobic pocket (the 2′
substituent pocket) occurs from a different angle. In addition,
the 5′-ketone makes an additional hydrogen bond to Gln775,
which stabilizes interactions with the absolutely conserved
Gln817. As a result of the flipped binding mode adopted by
compound 2, there is an associated shift in the side chain of
the Leu804 residue flanking the deep 2′-alkoxy pocket, which
enables formation of significant interactions between the inhibi-
tor and Leu804 in PDE5. This interaction does not occur in
PDE6, since the Leu804 present in PDE5 is replaced by
methionine in PDE6, explaining the increased PDE6 selectivity
displayed by compounds adopting the “flipped” binding mode.
Figure 6. Crystal structure of compound 2 in the active site of the
PDE5 catalytic domain.
Experimental Section
Chemistry. Room temperature means 20-25 °C. Unless oth-
erwise stated, all reactions were carried out using commercially
available anhydrous solvents. Flash column chromatography refers
to column chromatography on silica gel (Kieselgel 60, 230-400
mesh). Thin-layer chromatography was performed on either pre-
coated Merck silica gel (60 F254) plates or Macherey-Nagel
Polygram Si G/UV (0.2 mm silica gel) plates. Melting points are
Conclusions
1
In this paper we have reported the discovery of a novel series
of PDE5 inhibitors, with excellent in vitro potency for corpus
cavernosum PDE5 and selectivity over canine retinal cone
PDE6. Replacement of the piperazine sulfonamide functionality
in sildenafil with a metabolically more stable methyl ketone
led to a series of PDE5 inhibitors with low clearance and dose-
independent oral absorption. The predicted high and dose-
independent bioavailability for compound 2 should ensure that
the impact on Cmax of any interactions with coadministered drugs
in the clinic are minimized. Further data on this compound will
be reported in due course.
uncorrected. H nuclear magnetic resonance (NMR) spectra were
recorded using a Varian Unity Inova-300, a Varian Unity Inova-
400, or a Varian Mercury-400 spectrometer and were in all cases
consistent with the proposed structures. Mass spectra were recorded
using a Fisons Instruments Trio mass spectrometer in the thermo-
spray ionization mode (TSP) or using a Finnigan navigator with
electrospray ionization (ES) in positive and/or negative ionization
mode. LRMS means low-resolution mass spectrum and the
calculated and observed ions quoted refer to the isotopic composi-
tion of lowest mass. High-resolution mass spectra were obtained
with a Bruker Apex II FTICR-MS with a 4.7 T magnet using Xmass
software. Combustion analyses were conducted by Exeter Analytical
Figure 4. Caco-2 flux of compounds 1 and 2 at increasing concentrations.

SelectiVe PDE5 Inhibitors with Oral BioaVailability
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3589
U.K. Ltd., Uxbridge, Middlesex. NOE refers to nuclear Overhauser
effect, which was recorded using a Varian Inova 500 MHz
spectrometer. Other abbreviations are used in conjunction with
standard chemical practice.
scopic foam (1.65 g), which was taken onto the next stage crude:
LRMS (m/z) (TSP+) 397.5 (MH+). Anal. (C20H24O3N6·2.0H2O·TFA)
C, H, N.
5-(5-Acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(1-methyl-3-aze-
tidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (27). Form-
aldehyde (400 µL of a 37-41% aq solution, 5.04 mmol) was added
to a stirring solution of amine 25 (500 mg, 1.26 mmol) in DCM
(30 mL). Sodium triacetoxyborohydride (670 mg, 3.15 mmol) was
added after 15min. After a further 30 min the reaction mixture was
diluted with DCM (15 mL) and washed with saturated aqueous
sodium bicarbonate solution (15 mL). The layers were separated,
and the aqueous layer was extracted further with DCM (15 mL).
The combined organic layers were washed with brine (10 mL),
dried (MgSO4), and concentrated in vacuo. The crude product was
purified by flash column chromatography (eluting with 95:5:1 CH2-
Cl2:MeOH:0.88 N NH3) to yield the title compound as a white,
hygroscopic solid (288 mg, 0.70 mmol, 56%): mp 175.9-177.0
°C; LRMS (m/z) (TSP+) 411.6 (MH+). Anal. (C21H26O3N6·0.7H2O)
C, H, N.
5-(5-Acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(1-ethyl-3-aze-
tidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (28). So-
dium cyanoborohydride (92 mg, 1.47 mmol) was added to a stirring
solution of amine 25 (500 mg, 0.98 mmol), acetaldehyde (64 µL,
1.18 mmol), and sodium acetate (161 mg, 1.96 mmol) in MeOH
(10 mL) under nitrogen at room temperature. After 1 h the mixture
was poured into saturated aqueous sodium bicarbonate solution (20
mL), and extracted with DCM (3 × 15 mL). The combined organic
layers were dried (MgSO4) and concentrated in vacuo. The crude
product was purified by flash column chromatography (eluting with
95:5:0.5-80:20:1 EtOAc:MeOH:0.88 N NH3) to yield the title
compound as a white solid (140 mg, 0.33 mmol, 34%): LRMS
(m/z) (TSP+) 425.3 (MH+).
5-(5-Acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(2-pyridinylmethyl)-
2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (9). To a solution
of compound 8 (210 mg, 0.49 mmol) in n-butanol (20 mL) was
added KHMDS (291 mg, 1.49 mmol). The reaction mixture was
heated at 90 °C for 48 h and then concentrated in vacuo. The
reaction mixture was then dissolved in ethyl acetate (50 mL),
washed with saturated sodium bicarbonate solution (20 mL) and
brine (20 mL), dried (MgSO4), and concentrated in vacuo. Tritu-
ration from ether, followed by flash column chromatography
(eluting with 99:1 EtOAc:MeOH), yielded the target compound as
a white solid (88 mg, 0.20 mmol, 41%): LRMS (m/z) (TSP+) 447.9
(MH+). (C24H26O3N6‚0.25H2O·0.1EtOAc) C, H, N.
5-[5-Acetyl-2-(2-methoxyethoxy)-3-pyridinyl]-3-ethyl-2-[2-(4-
morpholinyl)ethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-
one (17) was prepared in 46% yield from the butoxy analogue 16
using 2-methoxyethanol as solvent and heating to 120 °C, following
a procedure similar to that described for compound 9: LRMS (m/
z) (TSP+) 471.1 (MH+). Anal. (C23H30O5N6·0.25H2O) C, H, N.
Irradiation of the pyrazolopyrimidinone C3-CH2CH3 and pyrazole
NCH2CH2NR2 showed mutual NOE enhancements, confirming the
regiochemistry.
5-(5-Acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-[2-(4-morpholinyl)-
ethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (16). Sul-
furic acid (1 N, 0.25 mL) was added to a stirred solution of 15
(78.0 mg, 0.17 mmol) in acetone (5 mL) at room temperature.
Mercury sulfate (8 mg, 0.03 mmol) was added and the mixture
heated at reflux for 4 h. Further aliquots of mercury sulfate (8 mg,
0.03 mmol), 1 N sulfuric acid (0.25 mL), and acetone (5 mL) were
then added, and the reaction mixture was heated at reflux for a
further 4 h before being left at room temperature overnight. The
reaction mixture was concentrated in vacuo, and the residue
partitioned between ethyl acetate (10 mL) and saturated sodium
bicarbonate solution (10 mL). The organic layer was separated and
the aqueous layer was extracted further with ethyl acetate (3 × 10
mL). The combined organic layers were washed with brine (10
mL), dried (MgSO4), and concentrated in vacuo. Purification by
flash column chromatography (eluting with 100:0 to 99:1 EtOAc:
MeOH) yielded the target compound as a white solid (48 mg, 0.10
mmol, 59%): LRMS (m/z) (ES+) 469 (MH+). Anal. (C24H32O4N6·
0.25H2O·0.2Et2O) C, H, N.
5-(5-Acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-[(1-methyl-1H-
imidazol-2-yl)methyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-
7-one (48) was prepared in 31% yield from the amide 47 using
n-propanol as solvent, following a procedure similar to that
described for compound 2 (route B): mp 243.1-245.0 °C; LRMS
(m/z) (TSP+) 436.1 (MH+). Anal. (C22H25O3N7·0.7H2O) C, H, N.
5-(5-Acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-[(1-methyl-1H-
imidazol-2-yl)methyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-
7-one (49) was prepared in 25% yield from the amide 47 using
n-butanol as solvent, following a similar procedure to that described
for compound 2 (route B) : mp 251.7-253.0 °C; LRMS (m/z)
(TSP+) 450.2 (MH+). Anal. (C23H27O3N7·0.3H2O) C, H, N.
5-(5-Acetyl-2-propoxy-3-pyridinyl)-2-(3-azetidinyl)-3-ethyl-
2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (25). Sulfuric
acid (1 N, 6 mL) was added to a stirred solution of acetylene 23
(1.44 g, 3.01 mmol) in acetone (50 mL) at room temperature.
Mercury sulfate (268 mg, 0.90 mmol) was added and the mixture
heated at reflux for 6 h. The reaction mixture was poured into
saturated aqueous sodium bicarbonate solution (20 mL). The
aqueous was then extracted with DCM (6 × 20 mL). The combined
organic layers were washed with brine (20 mL), dried (MgSO4),
and concentrated in vacuo to yield a brown oil. The oil was
dissolved in 40% TFA in DCM and stirred at room temperature
for 1 h. The reaction mixture was then concentrated in vacuo, and
the crude product was purified by flash column chromatography
(eluting with 95:5:1-80:20:1 CH2Cl2:MeOH:0.88 N NH3) to yield
the trifluoroacetate salt of the title compound as a white, hygro-
5-(5-Acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(1-isopropyl-3-
azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (29)
was prepared in 31% yield from the secondary amine 25 and
acetone, following a similar procedure to that described for
compound 27: mp 162.8-163.6 °C; LRMS (m/z) (TSP+) 439.6
(MH+). Anal. (C23H30O3N6) C, H, N.
5-(5-Acetyl-2-isobutoxy-3-pyridinyl)-3-ethyl-2-(1-methyl-3-
azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (30).
The 2′-propoxy analogue 27 (70 mg, 0.17 mmol) and cesium
carbonate (167 mg, 0.51 mmol) were dissolved in isobutyl alcohol
(2 mL) and heated in a reactivial at 90 °C with 4 Å molecular
sieves for 24 h. A further aliquot of cesium carbonate (56 mg, 0.17
mmol) and isobutyl alcohol (0.5 mL) were added, and heating was
continued for a further 24 h. The reaction mixture was then poured
into DCM (10 mL) and a saturated aqueous sodium bicarbonate
solution (5 mL). The organic layer was separated and the aqueous
layer extracted further with DCM (3 × 10 mL). The combined
organics were dried (MgSO4) and concentrated in vacuo. Purifica-
tion by flash column chromatography (eluting with 97.5:2.5:0.5
CH2Cl2:MeOH:0.88 N NH3) yielded an inseparable mixture of title
compound and starting material. The mixture was dissolved in
isobutyl alcohol (2 mL), cesium carbonate (167 mg, 0.51 mmol)
was added, and the reaction mixture was heated at 90 °C for 48 h.
The reaction was then worked up as described previously and
purified by flash column chromatography (eluting with 95:5:0.5-
85:15:0.5 EtOAc:MeOH:0.88 N NH3 and then repeated eluting with
99:1:0.1-95:5:0.5 CH2Cl2:MeOH:0.88 N NH3) to yield the title
compound as a white solid (32 mg, 0.08 mmol, 44%): LRMS (m/
z) (TSP+) 425.5 (MH+); HRMS (C22H28N6O3) calcd for (M + 1)+
425.2296, found 425.2287.
5-(5-Acetyl-2-butoxy-3-pyridinyl)-2-(3-azetidinyl)-3-ethyl-2,6-
dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (42). TFA (25 mL,
31% vol) was added to a solution of the carbamate 40 (13.4 g, 262
mmol) in DCM (80 mL) at 0 °C, and the mixture was then stirred
at room temperature for 1 h. The reaction mixture was poured into
toluene (100 mL) and concentrated in vacuo to yield an oil. The
oil was azeotroped again with toluene (50 mL) and the residue taken
up in isopropyl acetate. The resulting precipitate was removed by
filtration and dried in vacuo to yield the trifluoroacetate salt of the

3590 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Allerton et al.
title compound as a white solid (11.2 g, 17.5 mmol, 67%): LRMS
(m/z) (ES+) 411.0 (MH+), (ES-) 409.0 (MH-). Anal. (C21H26O3N6·
2.0H2O·0.05DIPE) C, H, N.
5-(5-Acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(1-methyl-3-aze-
tidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (31) was
prepared in 33% yield from 2′ propoxy analogue 27 and n-butanol,
following a similar procedure to that described for compound 30:
LRMS (m/z) (ES+) 425.0 (MH+), (ES-) 423.0 (MH-); HRMS
(C22H28N6O3) calcd for (M + 1)+ 425.2296, found 425.2294.
sample was crystallized from ethyl acetate for analysis: mp 182.0-
183.0 °C; LRMS (m/z) (TSP+) 467.3 (MH+). Anal. (C25H34O3N6)
C, H, N.
5-(5-Acetyl-2-propoxy-3-pyridinyl)-2-(3-azetidinyl)-3-propyl-
2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (45) was pre-
pared in 67% yield from the carbamate 58, using a similar procedure
to that described for compound 42. An analytical sample was
prepared by crystallization from diethyl ether: mp 185 °C (dec);
LRMS (m/z) (ES+) 411 (MH)+; (ES-) 409 (M - H)-. Anal.
(C21H26N6O3·0.3H2O) C, H, N.
5-(5-Acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(1-ethyl-3-azetidi-
nyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (2). Route
A. Potassium carbonate (4.80 g, 34.7 mmol) and ethyl iodide (1.4
mL, 17.5 mmol) were added to a cloudy solution of the secondary
amine 42 (11.1 g, 17.4 mmol) in MeCN (600 mL), and then the
reaction mixture was heated to 45-50 °C for 2.5 h. The solvent
was then removed in vacuo and the residue dissolved in 95:5:0.5
CH2Cl2:MeOH:0.88 N NH3 (50 mL). The resulting solution was
filtered and then purified by flash column chromatography (eluting
with 95:5:0.5-92:8:1 CH2Cl2:MeOH:0.88 N NH3). The product
was crystallized from diisopropyl ether to yield the title compound
as white crystals (4.90 g, 11.2 mmol, 64%): mp 143.0-144.0 °C.
Route B. Cesium carbonate (38.6 g, 119 mmol) was added to a
solution of the amide 54 (25.4 g, 59.3 mmol) in n-butanol (400
mL) in the presence of powdered 4-Å molecular sieves (10 g). The
reaction mixture was then heated to reflux and 20 mL of solvent
removed via distillation into a splash trap. Refluxing was then
continued for 4 h, after which the reaction mixture was cooled and
filtered. The filtrate was concentrated in vacuo and then purified
by flash column chromatography (eluting with 95:5:0.5 CH2Cl2:
MeOH:0.88 N NH3) to yield a green oil. The crude product was
then purified by crystallization from ethyl acetate, to yield the title
compound as a white solid (9.00 g, 20.5 mmol, 35%): mp 143.0-
144.0 °C; LRMS (m/z) (TSP+) 439.2 (MH+). Anal. (C23H30O3N6)
C, H, N. Inversion of the pyrazolopyrimidinone C3-CH2CH3 and
azetidine-C3-H showed mutual NOE enhancements, confirming the
regiochemistry.
5-(5-Acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(4-piperidinyl)-
2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (26) was pre-
pared in 65% yield from the acetylene 24, following a similar
procedure to that described for compound 25: LRMS (m/z) (TSP+)
425.5 (MH+). Anal. (C22H28O3N6·1.45DCM) C; H, N.
5-(5-Acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(1-methyl-4-pi-
peridinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (32)
was prepared in 66% yield from the secondary amine 26, following
a similar procedure to that described for compound 27: mp 219.0-
220.0 °C; LRMS (m/z) (TSP+) 439.5 (MH+). Anal. (C23H30O3N6·
0.2H2O·0.1DCM) C, H, N.
5-(5-Acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(1-ethyl-4-pi-
peridinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (33)
was prepared in 28% yield from the secondary amine 26, following
a similar procedure to that described for compound 28: LRMS-
(m/z) (TSP+) 453.8 (MH+). Anal. (C24H32O3N6·0.2H2O·0.1DCM·
0.1MeOH) C, H, N.
5-(5-Acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(4-piperidinyl)-
2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (43) was pre-
pared in 90% crude yield (contained ∼10% impurity) from the
carbamate 41, following a similar procedure to that described
for compound 42: LRMS (m/z) (TSP+) 439 (MH+). HRMS
(C23H30N6O3) calcd for (M + 1)+ 439.2452, found 439.2446.
5-(5-Acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(1-methyl-4-pi-
peridinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (34)
was prepared in 18% yield from the 2′-propoxy analogue 32 using
n-butanol as solvent, following a similar procedure to that described
for compound 30: mp 198.0-199.0 °C; LRMS (m/z) (TSP+) 453-
(MH+). Anal. (C24H32O3N6·0.25H2O·0.1DIPE) C, H, N.
5-(5-Acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(1-ethyl-4-pi-
peridinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (44)
was prepared in 65% yield from the secondary amine 43, following
a similar procedure to that described for compound 2 (route A). A
5-(5-Acetyl-2-propoxy-3-pyridinyl)-2-(1-methyl-3-azetidinyl)-
3-propyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (46)
was prepared in 20% yield from the secondary amine 45, using a
similar procedure to that described for compound 27: mp 118-
122 °C; LRMS (m/z) (ES+) 447 (MNa)+, 425 (MH)+; (ES-) 423
(M - H)-. Anal. (C22H28N6O3·0.3H2O·0.5EtOAc) C, H, N.
5-[2-Butoxy-5-(1-hydroxyethyl)-3-pyridinyl]-3-ethyl-2-(1-eth-
yl-3-azetidinyl) -2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one
(55). Sodium borohydride (17 mg, 0.46 mmol) was added to an
ice-cooled suspension of ketone 2 (400 mg, 0.91 mmol) in MeOH
(10 mL) under a nitrogen atmosphere, and the solution was allowed
to warm to room temperature, with stirring, over 1 h. Additional
sodium borohydride (17 mg, 0.46 mmol) was then added, and the
reaction stirred at room temperature for 30 min. The mixture was
concentrated under reduced pressure, the residue was partitioned
between ethyl acetate (30 mL) and water (20 mL), and the layers
were separated. The aqueous phase was extracted with ethyl acetate
(2 × 20 mL), and the combined organic solutions were dried
(MgSO4) and concentrated in vacuo. The residual yellow foam was
purified by flash column chromatography (eluting with DCM:
MeOH:0.88 N NH3 95:5:0.5). The resulting foam was crystallized
from Et2O to afford the title compound as a white solid (285 mg,
0.65 mmol, 71%): mp 117-119 °C; LRMS (m/z) (TSP+) 441.2
(MH+). Anal. (C23H32N6O3) C, H, N.
5-(5-Acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(2-pyridinyl-
methyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (8) was
prepared in 12% yield from the acetylene 7, using a similar
procedure to that described for compound 16: LRMS (m/z) (TSP+)
433.4 (MH+). Anal. (C23H24O3N6·0.5H2O·0.5DCM) C, H, N.
3-Ethyl-5-(5-ethynyl-2-propoxy-3-pyridinyl)-2-(2-pyridinyl-
methyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (7). Po-
tassium fluoride (239 mg, 4.12 mmol) was added to a stirring
solution of trimethylsilylacetylene 59 (1.00 g, 2.06 mmol) in DMF
(20 mL) and water (2 mL) at 0 °C. The reaction mixture was then
stirred at room temperature for 4 h. The reaction mixture was then
concentrated in vacuo, and the residue was taken up in water (50
mL) and ethyl acetate (50 mL) and filtered through Arbocel. The
organic layer was separated, and the aqueous layer was extracted
further with ethyl acetate (3 × 50 mL) and DCM (50 mL). The
combined organic layers were dried (MgSO4) and concentrated in
vacuo to give the title compound as a beige solid (700 mg, 1.69
mmol, 82%): mp 189 °C; LRMS (m/z) (TSP+) 415.6 (MH+). Anal.
(C23H22O2N6·0.7H2O) C, H, N.
3-Ethyl-5-(2-propoxy-5-[(trimethylsilyl)ethynyl]-3-pyridinyl)-
2-(2-pyridinylmethyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-
7-one (59). Pd(PPh3)2Cl2 (60.0 mg, 0.08 mmol), trimethylsilylacet-
ylene (615 µL, 4.44 mmol), and cuprous iodide (15 mg, 0.08 mmol)
were added to a stirred slurry of iodo analogue 6 (1.50 g, 2.90
mmol) in triethylamine (23 mL) and MeCN (1 mL) at room
temperature under nitrogen. The mixture was stirred at room
temperature for 3 h, after which a further aliquot of trimethylsily-
lacetylene (300 µL, 2.17 mmol) was added. After stirring for a
further 1 h, another aliquot of trimethylsilylacetylene (300 µL, 2.17
mmol) was added. After 1 h the reaction was concentrated in vacuo
and then partitioned between ethyl acetate (50 mL) and water (50
mL). The organic layers were separated, and the aqueous layer was
extracted further with ethyl actetate (2 × 50 mL). The combined
organic layers were washed with brine (75 mL), dried (MgSO4),
and concentrated in vacuo to give a yellow solid. Trituration from
ether yielded the title compound as an off-white solid (873 mg,
1.80 mmol, 62%): LRMS (m/z) (TSP+) 487.5 (MH+).

SelectiVe PDE5 Inhibitors with Oral BioaVailability
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3591
3-Ethyl-5-(5-iodo-2-propoxy-3-pyridinyl)-2-(2-pyridinylmethyl)-
2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (6). KHMDS
(3.65 g, 18.3 mmol) was added to a stirring solution of the amide
5 (6.50 g, 12.2 mmol) in 3-methyl-3-pentanol (50 mL) at room
temperature under nitrogen. The reaction mixture was then heated
at 120 °C for 42 h, after which a further aliquot of KHMDS (3.65
g, 18.3 mmol) was added. Heating at 120 °C was continued for a
further 24 h, followed by another addition of KHMDS (3.65 g,
18.3 mmol). After a further 3 h of heating at 120 °C, the reaction
mixture was concentrated in vacuo. The residue was partitioned
between ethyl acetate (200 mL) and saturated aqueous sodium
bicarbonate solution (200 mL). The organic layer was separated
and the aqueous layer extracted further with ethyl acetate (3 × 100
mL). The combined organic layers were dried (MgSO4) and
concentrated in vacuo. The crude product was triturated in ether to
yield some of the target compound. The residue was purified by
flash column chromatography (eluting with 99.8:0.2 to 99:1 CH2-
Cl2:MeOH) to yield the title compound (which was combined with
that obtained earlier) as a solid (1.40 g, 2.71 mmol, 22%):
mp 228.9-233.8 °C; LRMS (m/z) (ES+) 517 (MH+).
Anal. (C21H21O2N6I) C, H, N.
N-{3-(Aminocarbonyl)-5-ethyl-1-[2-(4-morpholinyl)ethyl]-1H-
pyrazol-4-yl}-2-butoxy-5-iodonicotinamide (13). Cesium carbon-
ate (880 mg, 2.70 mmol) was added to a stirring solution of amide
12 (750 mg, 1.60 mmol) and N-(2-chloroethyl)morpholine hydro-
chloride (370 mg, 1.99 mmol) in DMF (5 mL) under a nitrogen
atmosphere. The mixture was stirred at room temperature for 16 h
and then heated at 60 °C for 24 h. Further aliquots of N-(2-
chloroethyl)morpholine hydrochloride (185 mg, 0.99 mmol) and
cesium carbonate (486 mg, 1.49 mmol) were then added and heating
continued for a further 16 h. The reaction mixture was cooled and
poured into water (50 mL), and the aqueous layer was extracted
with ether (3 × 50 mL). The combined organic layers were washed
with brine (50 mL), dried (MgSO4), and concentrated in vacuo.
Purification by flash column chromatography (eluting with 99:1 to
98:2 CH2Cl2:MeOH) yielded the title compound (310 mg, 0.54
mmol, 34%): LRMS (m/z) (ES+) 571 (MH+).
N-[3-(Aminocarbonyl)-5-ethyl-1H-pyrazol-4-yl]-2-butoxy-5-
iodonicotinamide (12) was prepared in 98% yield from acid 11
and 4-amino-3-ethyl-1H-pyrazole-5-carboxamide,8 following a
similar procedure to that described for compound 5: LRMS (m/z)
(TSP+) 457.9 (MH+).
N-[3-(Aminocarbonyl)-5-ethyl-1-(2-pyridinylmethyl)-1H-pyra-
zol-4-yl]-5-iodo-2-propoxynicotinamide (5). Oxalyl chloride (3.93
mL, 45 mmol) was added dropwise to a solution of acid 4 (4.61 g,
15 mmol) in DCM (100 mL) and DMF (100 µL) under nitrogen at
0 °C. The reaction mixture was allowed to warm to room
temperature and then stirred for 16 h. The reaction mixture was
then concentrated in vacuo and azeotroped three times with toluene,
to give a yellow/brown solid. This solid was then dissolved in DCM
(20 mL) and added to a solution of 4-amino-5-ethyl-1-(2-pyridi-
nylmethyl)-1H-pyrazole-3-carboxamide8 (3.68 g, 15 mmol) in
pyridine (70 mL) stirring under nitrogen. After 4 h the reaction
mixture was concentrated in vacuo, and the product was partitioned
between ethyl acetate (200 mL) and saturated aqueous sodium
bicarbonate solution (200 mL). The organic layer was separated
and the aqueous layer extracted with ethyl acetate (3 × 50 mL).
The combined organic layers were washed with brine, dried
(MgSO4), and concentrated in vacuo to yield the target compound
as a peach-colored compound (7.70 g, 14.4 mmol, 96%): LRMS
(m/z) (ES+) 535 (MH+), (ES-) 533 (MH-). Anal. (C21H23O3N6I)
C, H, N.
2-n-Butoxy-5-iodonicotinic Acid (11). n-Butanol (2.96 g, 40.0
mmol) was added dropwise to a stirring suspension of KHMDS
(7.96 g, 40.0 mmol) in THF (30 mL), with cooling. The reaction
mixture was then stirred at room temperature for 15 min. A solution
of methyl-2-chloro-5-iodonicotinate 1024 (1.98 g, 6.60 mmol) in
THF (10 mL) was then added dropwise over 10 min. After 2 h a
solution of sodium hydroxide (532 mg, 13.2 mmol) in water (3
mL) was added dropwise over 5 min. The reaction was then
neutralized to pH 8-9 with 2 M HCl, and the bulk of solvent was
removed in vacuo. The pH of the remaining solution was adjusted
to pH 8-9 again and then partitioned with ethyl acetate (100 mL).
The resulting precipitate was separated by filtration and shown to
be the title compound (640 mg, 1.99 mmol, 30%). The organic
and aqueous layers were separated, and the aqueous layer was re-
extracted with ethyl acetate (100 mL). The combined organic layers
were washed with 2 M HCl (50 mL), dried (MgSO4), and
concentrated in vacuo to give a brown solid. This solid was
combined with those separated earlier to yield the title compound
(730 mg, 2.27 mmol, 34%): LRMS (m/z) (TSP) 321.9 (MH+).
2-Propoxy-5-iodonicotinic Acid (4). 2-Propoxynicotinic acid 3
(9.30 g, 51.3 mmol) was dissolved slowly in TFA (75 mL) and
trifluoroacetic anhydride (19 mL). NIS (18.6 g, 82.7 mmol) was
then added portionwise, and the red/brown solution was heated at
reflux for 6 h followed by 16 h at room temperature. The reaction
mixture was then concentrated in vacuo and water (150 mL) added.
The aqueous mixture was extracted with DCM (3 × 150 mL). The
combined organic layers were extracted with aqueous sodium
hydroxide solution (1 N, 200 mL), and then the aqueous layer was
acidified with concentrated hydrochloric acid. The acidic aqueous
layer was then extracted with DCM (4 × 150 mL), and the
combined organic layers were washed with brine (150 mL), dried
(MgSO4), and concentrated in vacuo. Trituration from pentane
yielded the target compound as an off-white solid (11.3 g, 36.8
mmol, 72%): LRMS (m/z) (ES-) 306 (MH-).
5-(2-Butoxy-5-ethynyl-3-pyridinyl)-3-ethyl-2-[2-(4-morpholin-
yl)ethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (15)
was prepared in 78% yield from the trimethylsilyl analogue 60,
following a similar procedure to that described for compound 7:
LRMS (m/z) (ES+) 451 (MH+).
5-{2-Butoxy-5-[(trimethylsilyl)ethynyl]-3-pyridinyl}-3-ethyl-
2-[2-(4-morpholinyl)ethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyri-
midin-7-one (60) was prepared in 68% yield from the iodo analogue
14, following a similar procedure to that described for compound
59: LRMS (m/z) (ES+) 523 (MH+).
5-Acetyl-N-{3-(aminocarbonyl)-5-ethyl-1-[(1-methyl-1H-imi-
dazol-2-yl)methyl]-1H-pyrazol-4-yl}-2-ethoxynicotinamide (47)
was prepared in 70% yield from acid 36 and 4-amino-5-ethyl-1-
[(1-methyl-1H-imidazol-2-yl)methyl]-1H-pyrazole-3-carboxam-
ide,7 following a similar procedure to that described for compound
37: LRMS (m/z) (TSP+) 440.7 (MH+).
5-Acetyl-2-ethoxynicotinic Acid (36). Triethylamine (354 mL,
2.54 M) was added to a slurry of 5-bromo-2-ethoxynicotinic acid25
(250 g, 1.02 M) in MeCN (1 L). To this reaction mixture was added
palladium(II) acetate (4.56 g, 20.3 mmol), butyl vinyl ether (305
g, 3.05 M), and tri-o-tolylphosphine (12.4 g, 40.6 mmol), each
addition being washed in with MeCN. The remaining solvent was
then added and the reaction mixture refluxed under nitrogen for
22 h. The reaction mixture was left at room temperature for 16 h,
and then the precipitate was removed by filtration and discarded.
The filtrate was concentrated in vacuo to give a brown gum, which
was then stirred for 1 h in water (1 L) and concentrated HCl (1 L).
The reaction mixture was diluted with water (6.25 L) and extracted
with DCM (6 × 500 mL). The combined organic layers were
extracted with 5% aqueous sodium bicarbonate solution (1.2 L, 2
× 400 mL). The basic aqueous extracts were washed with DCM
(250 mL) and then acidified to pH 3. After stirring for 30 min the
precipitated product was removed by filtration, washed with water
(250 mL), and dried at 50 °C in vacuo to yield the target compound
as a white solid (134 g, 64.1 mmol, 63%): LRMS (m/z) (ES-)
208 (MH-).
5-(2-Butoxy-5-iodo-3-pyridinyl)-3-ethyl-2-[2-(4-morpholinyl)-
ethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (14) was
prepared in 67% yield from the amide 13 using n-butanol as solvent,
following a similar procedure to that described for compound 6:
LRMS (m/z) (ES+) 553 (MH+).
tert-Butyl 3-[3-ethyl-5-(5-ethynyl-2-propoxy-3-pyridinyl)-7-
oxo-6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-2-yl]-1-azetidin-
ecarboxylate (23) was prepared in 85% yield from the trimethylsilyl

3592 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Allerton et al.
analogue 61, following a similar procedure to that described for
compound 7: LRMS (m/z) (TSP+) 496.6 (MNH4+), 379.7 (MH+
- BOC).
tert-Butyl 3-(3-ethyl-7-oxo-5-{2-propoxy-5-[(trimethylsilyl)-
ethynyl]-3-pyridinyl}-6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-
2-yl)-1-azetidinecarboxylate (61) was prepared in 83% yield from
the iodo analogue 21, following a similar procedure to that described
for compound 59: LRMS (m/z) (TSP+) 568.6 (MNH4+), 452.0
(MH+). Anal. (C28H38O4N6Si) C, H, N.
tert-Butyl 3-[3-ethyl-5-(5-iodo-2-propoxy-3-pyridinyl)-7-oxo-
6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-2-yl]-1-azetidinecar-
boxylate (21) was prepared in 63% yield from the amide 19 using
n-propanol as solvent, following a similar procedure to that
described for compound 6: LRMS (m/z) (TSP+) 598.1 (MNH4+).
Anal. (C23H29O4N6I) C, H, N. Irradiation of the pyrazolopyrimi-
dinone C3-CH2CH3 and azetidine-C3-H showed mutual NOE
enhancements, confirming regiochemistry.
tert-Butyl 3-(3-(aminocarbonyl)-5-ethyl-4-{[(5-iodo-2-propoxy-
3-pyridinyl)carbonyl]amino}-1H-pyrazol-1-yl)-1-azetidinecar-
boxylate (19) was prepared in 65% yield from unsubstituted
pyrazole 18 and tert-butyl-3-iodo-1-azetidinecarboxylate,26 follow-
ing a similar procedure to that described for compound 13: LRMS
(m/z) (TSP+) 373.2 (MH+ - BOC and I). Anal. (C23H31O5N6I·
0.2DCM) C, H, N.
N-[3-(Aminocarbonyl)-5-ethyl-1H-pyrazol-4-yl]-5-iodo-2-pro-
poxynicotinamide (18) was prepared in 74% yield from acid 4
and 4-amino-3-ethyl-1H-pyrazole-5-carboxamide,8 following a
similar procedure to that described for compound 5: LRMS (m/z)
(TSP+) 444.3 (MH+). Anal. (C15H18O3N5I) C, H, N.
tert-Butyl 3-[5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-7-oxo-
6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-2-yl]-1-azetidinecar-
boxylate (40) was prepared in 46% crude yield from amide 38
using n-butanol as solvent, following a similar procedure to that
described for compound 2 (route B). Crystallization from diiso-
propyl ether yielded the title compound, containing a 10% impurity,
as white crystals: LRMS (m/z) (ES+) 433 (MNa+), (ES-) 509
(MH-).
tert-Butyl 3-[4-{[(5-acetyl-2-ethoxy-3-pyridinyl)carbonyl]-
amino}-3-(aminocarbonyl)-5-ethyl-1H-pyrazol-1-yl]-1-azetidin-
ecarboxylate (38) was prepared in 63% yield from unsubstituted
pyrazole 37 and tert-butyl-3-iodo-1-azetidinecarboxylate,26 follow-
ing a similar procedure to that described for compound 13: mp
220-223 °C; LRMS (m/z) (ES+) 523.0 (MNa+), (ES-) 499.0
(MH-). Anal. (C24H32O6N6) C, H, N.
5-Acetyl-N-[3-(aminocarbonyl)-5-ethyl-1H-pyrazol-4-yl]-2-
ethoxynicotinamide (37). A solution of acid 36 (5.70 g, 27.3 mmol)
and HATU (10.9 g, 28.6 mmol) in DCM (100 mL) was added to
a solution of 4-amino-3-ethyl-1H-pyrazole-5-carboxamide8 (4.20
g, 27.3 mmol) and N,N-diisopropylethylamine (23.7 mL, 136.2
mmol) in DCM (115 mL) under nitrogen. After 1 h the mixture
was diluted with brine (100 mL) and washed with saturated aqueous
sodium bicarbonate solution (100 mL) and 2 N HCl (100 mL).
Each aqueous layer was extracted with DCM (100 mL), and the
combined organics were washed with brine (100 mL), dried
(MgSO4), and concentrated in vacuo. An analytical sample of the
title compound was obtained by trituration with ethyl acetate,
followed by crystallization from ethanol, while the remainder was
purified by flash column chromatography (eluting with 95:5 CH2-
Cl2:MeOH) to yield the title compound as a white solid (total weight
) 7.8 g, 22.5 mmol, 83%): mp 217-219 °C; LRMS (m/z) (TSP+)
346.2 (MH+). Anal. (C16H19O4N5) C, H, N.
The solid was washed with water:ethanol 90:10 and then dried in
vacuo to yield the title compound as a white solid (24.0 g, 56.0
mmol, 69%): mp 230-233 °C; LRMS (m/z) (TSP+) 429.2 (MH+).
Anal. (C21H28O4N6) C, H, N.
4-Amino-5-ethyl-1-(1-ethyl-3-azetidinyl)-1H-pyrazole-3-car-
boxamide (53). A mixture of nitropyrazole 62 (22.0 g, 82.3 mmol)
and 10% palladium on charcoal (2.0 g) in ethanol (500 mL) was
hydrogenated at 60 psi and room temperature for 4 h. The reaction
mixture was then filtered through Arbocel under nitrogen, and the
filtrate was concentrated in vacuo to yield the title compound as a
cream solid (19.6 g, 82.6 mmol, 100%): mp 155-157 °C; LRMS
(m/z) (TSP+) 238.2 (MH+). Anal. (C11H19O1N5) C, H, N.
5-Ethyl-1-(1-ethyl-3-azetidinyl)-4-nitro-1H-pyrazole-3-car-
boxamide (62). To a stirring solution of the secondary amine 52
(31.1 g, 113 mmol) and triethylamine (14.1 mL, 102 mmol) in DCM
(400 mL) and MeOH (400 mL) at 0 °C was added sodium
triacetoxyborohydride (60 g, 282 mmol) in one portion. Acetalde-
hyde (19 mL, 339 mmol) was then added dropwise over 2 min.
The reaction mixture was then allowed to warm to room temperature
over 30 min. The solvent was then removed in vacuo and the residue
partitioned between DCM (500 mL) and water (300 mL). The
organic layer was separated, and the aqueous layer was basified
with solid sodium bicarbonate and extracted with DCM (500 mL)
and DCM:MeOH (95:5, 500 mL; 90:10, 500 mL). The combined
organic layers were dried (MgSO4) and concentrated in vacuo. The
residue was triturated from hot ethyl acetate and a white solid
separated by filtration. The filtrate was concentrated in vacuo and
purified by flash column chromatography (eluting with 95:5:0.5
CH2Cl2:MeOH:0.88 N NH3) to give a white solid which was
combined with the previous batch to yield the title compound (23.3
g, 86.8 mmol, 77%): mp 177-179 °C; LRMS (m/z) (TSP+) 268.3
(MH+). Anal. (C11H17O3N5) C, H, N.
1-(3-Azetidinyl)-5-ethyl-4-nitro-1H-pyrazole-3-carboxamide
(52). To a suspension of the benzhydryl compound 51 (35.3 g, 87.1
mmol) in DCM (700 mL) at 0 °C under nitrogen was added
1-chloroethyl chloroformate (10.4 mL, 95.8 mmol) dropwise. The
reaction mixture was stirred at 0 °C for 30 min and at room
temperature for 18 h. The reaction mixture was then concentrated
in vacuo, and the oily residue dissolved in MeOH (700 mL) and
refluxed for 1 h. The solvent was then removed in vacuo, and the
crude product triturated from ethyl acetate (200 mL) and acetone
(200 mL) to yield the dihydrochloride salt of the title compound
as a beige solid (21.3 g, 77.3 mmol, 89%): mp 164-167 °C; LRMS
(m/z) (TSP+) 240.3 (MH+). Anal. (C9H13O3N5·2HCl·0.2Me2CO) C,
H, N.
1-(1-Benzhydryl-3-azetidinyl)-5-ethyl-4-nitro-1H-pyrazole-3-
carboxamide (51). 5-Ethyl-4-nitro-1H-pyrazole-3-carboxamide 508
(25.0 g, 136 mmol), sodium carbonate (57.6 g, 543 mmol), sodium
iodide (40.7 g, 272 mmol), and 1-benzhydryl-3-azetidinyl meth-
anesulfonate27 (86.2 g, 272 mmol) were dissolved in THF (338
mL) and water (38 mL) and refluxed for 5 days. The reaction
mixture was then concentrated in vacuo and taken up in ethyl acetate
(500 mL) and water (300 mL). The resulting precipitate was filtered
and washed with ethyl acetate and water to yield the title compound
as a white solid (17.0 g, 41.9 mmol, 31%): mp 257-260 °C; LRMS
(m/z) (TSP+) 406.2 (MH+). Irradiation of pyrazole-CH2CH3 and
azetidine-C3-H showed mutual NOE enhancements, confirming the
regiochemistry.
tert-Butyl 4-[3-ethyl-5-(5-ethynyl-2-propoxy-3-pyridinyl)-7-
oxo-6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-2-yl]-1-piperi-
dinecarboxylate (24) was prepared in 100% yield from the
trimethylsilyl analogue 63, following a similar procedure to that
described for compound 7: mp 221 °C; LRMS (m/z) (TSP+) 507.7
(MH+), 524.3 (MNH4+). Anal. (C27H34O4N6·0.5H2O) C, H, N.
5-Acetyl-N-[3-(aminocarbonyl)-5-ethyl-1-(1-ethyl-3-azetidinyl)-
1H-pyrazol-4-yl]-2-ethoxynicotinamide (54). 1,1-Carbonyldiimi-
dazole (13.9 g, 85.8 mmol) was added to a suspension of acid 36
(17.1 g, 81.8 mmol) in ethyl acetate (140 mL) under nitrogen, and
the reaction mixture was stirred at 45 °C for 45 min and refluxed
for 90 min. The reaction mixture was then cooled to room
temperature and a slurry of amine 53 (19.4 g, 81.8 mmol) in ethyl
acetate (70 mL) was added. The reaction mixture was then refluxed
for 16 h, after which a precipitate had formed. The suspension was
cooled to room temperature and the solid removed by filtration.
tert-Butyl 4-(3-ethyl-7-oxo-5-{2-propoxy-5-[(trimethylsilyl)-
ethynyl]-3-pyridinyl}-6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-
2-yl)-1-piperidinecarboxylate (63) was prepared in 75% yield from
the iodo analogue 22, following a similar procedure to that described
for compound 59: LRMS (m/z) (TSP+) 580 (MH+), 479.7 (MH+
- BOC). Anal. (C30H42O4N6Si·0.2H2O) C, H, N.

SelectiVe PDE5 Inhibitors with Oral BioaVailability
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3593
tert-Butyl 4-[3-ethyl-5-(5-iodo-2-propoxy-3-pyridinyl)-7-oxo-
6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-2-yl]-1-piperidinecar-
boxylate (22) was prepared in 16% yield from the amide 20 using
n-propanol as solvent, following a similar procedure to that
described for compound 6: LRMS (m/z) (TSP+) 609.7 (MH+),
509.0 (MH+ - BOC).
tert-Butyl 4-(3-(aminocarbonyl)-5-ethyl-4-{[(5-iodo-2-propoxy-
3-pyridinyl)carbonyl]amino}-1H-pyrazol-1-yl)-1-piperidinecar-
boxylate (20) was prepared in 52% yield from the unsubstituted
pyrazole 18 and tert-butyl 4-[(methylsulfonyl)oxy]-1-piperidin-
ecarboxylate,28 following a similar procedure to that described
for compound 13: LRMS (m/z) (TSP+) 627.4 (MH+). Anal.
(C25H35O5N6I·0.3H2O) C, H, N.
tert-Butyl 4-[5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-7-oxo-
6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-2-yl]-1-piperidinecar-
boxylate (41) was prepared in 40% yield from amide 39 using
n-butanol as solvent, following a similar procedure to that described
for compound 2 (route B). Trituration from diethyl ether gave the
title compound as a white powder: mp 194-195 °C; LRMS (m/z)
(TSP+) 539 (MH+), 439 (MH+ - BOC). Anal. (C28H38O5N6) C,
H, N.
the highest level of cGMP-hydrolytic activity (determined as below)
were pooled, aliquotted, and stored in liquid nitrogen until use.
PDE activity was measured using a scintillation proximity assay
(SPA)-based method as previously described.30 The effect of PDE
inhibitors was investigated by assaying a fixed amount of enzyme
in the presence of varying inhibitor concentrations and low substrate
3
(cGMP in a 3:1 ratio unlabeled to H-labeled at a concentration
∼1/3Km) such that IC50 = Ki. The final assay volume was made
up to 102 µL with assay buffer (20 mM Tris-HCl pH 7.4 at 30 °C,
5 mM MgCl2, 1 mg/mL bovine serum albumin). Reactions were
initiated with substrate, incubated for 30-60 min at 30 °C to give
<30% substrate turnover, and terminated with 50 µL of yttrium
silicate SPA beads (Amersham Pharmacia Biotech Ltd, Little
Chalfont, UK). Plates were resealed and shaken for 20 min, after
which the beads were allowed to settle for 20 min in the dark and
then counted on a TopCount plate reader (Packard, Meridien, CT).
Radioactivity units were converted to percent activity of an
uninhibited control (100%) and plotted against inhibitor concentra-
tion, and inhibitor IC50 values were obtained using the ‘Fit Curve’
Microsoft Excel extension.
Structural Biology. The structure of the Escherichia coli
engineered PDE5 was solved by molecular replacement (MR) using
a model of PDE5.23 X-ray diffraction data were collected with a
RaxisIV image plate detector on an in-house RU200HB rotating
anode (Rigaku), with Blue Osmic mirrors (MSC). All data were
processed using the HKL package.31 Data collection statistics for
compound 2 are available in the Supporting Information. The
crystals belong to the monoclinic space group C2, with cell
dimensions a ) 55.84 Å, b ) 76.66 Å, c ) 81.27 Å, â ) 102.584°.
There is one molecule per asymmetric unit (Mw ) 37 562.41 kDa)
and a calculated solvent content of 45.14% (VM ) 2.26).32
Molecular replacement was performed using AMORE (CCP4).33
The resulting map was of good quality and the structure was refitted
using QUANTA.34 Refinement was carried out in the resolution
range 35-1.6 Å using CNX35 with the “mlf” maximum likelihood
target function. Partial structure factors from a flat bulk-solvent
model and anisotropic B-factor correction were supplied throughout
the refinement. The R-factor for the current model is 0.31 (free
R-factor, 5% of data, 0.32) for all data in the resolution range 35-
1.6 Å. The refinement statistics for compound 2 are available in
the Supporting Information.
tert-Butyl 4-[4-{[(5-acetyl-2-ethoxy-3-pyridinyl)carbonyl]-
amino}-3-(aminocarbonyl)-5-ethyl-1H-pyrazol-1-yl]-1-piperidi-
necarboxylate (39) was prepared in 57% yield from unsubstituted
pyrazole 37 and 1-(tert-butoxycarbonyl)-4-piperidinyl methane-
sulfonate,28 following a similar procedure to that described for
compound 13: mp 197-202 °C; LRMS (m/z) (ES+) 529 (MH+).
Anal. (C26H36O6N6·0.5H2O) C, H, N.
tert-Butyl 3-[5-(5-acetyl-2-propoxy-3-pyridinyl)-7-oxo-3-pro-
pyl-6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-2-yl]-1-azetidine-
carboxylate (58) was prepared in 16% yield from amide 64 using
n-propanol as solvent, following a similar procedure to that
described for compound 2 (route B): mp 195-198 °C; LRMS (m/
z) (ES+) 533 (MNa)+; (ES-) 509 (M - H)-. Anal. (C26H34N6O5)
C, H, N. Irradiation of pyrazole-CH2CH2CH3 and azetidine-C3-H
showed mutual NOE enhancements, confirming the regiochemistry.
tert-Butyl 3-[4-{[(5-acetyl-2-ethoxy-3-pyridinyl)carbonyl]-
amino}-3-(aminocarbonyl)-5-propyl-1H-pyrazol-1-yl]-1-azeti-
dinecarboxylate (64) was prepared as an indistinguishable mixture
of regioisomers in 83% yield from the unsubstituted pyrazole 65
and 3-iodo-1-azetidinecarboxylic tbutyl ester,26 following a similar
procedure to that described for compound 13: mp 200-203 °C;
LRMS (m/z) (EI+) 537 (MNa)+, 515 (MH)+. Anal.(C25H34N6O6)
C, H, N.
5-Acetyl-N-[3-(aminocarbonyl)-5-propyl-1H-pyrazol-4-yl]-2-
ethoxynicotinamide (65) was prepared in 48% yield from acid 36
and 4-amino-5-propyl-1H-pyrazole-3-carboxamide,8 following a
similar procedure to that described for compound 37: mp 196-
199 °C; LRMS (m/z) (ES+) 382 (MNa)+, 360 (MH)+; (ES-) 358
(MH-). Anal. (C17H21N5O4·0.9H2O) C, H, N.
PDE5 Enzyme Inhibitor Assays. Phosphodiesterase type 5 was
prepared from human corpus cavernosum tissue as previously
described.29 Phosphodiesterase type 6 (cone) was prepared from
canine retina. Following dissection, retinas were suspended in cold
20 mM Hepes, 1 mM EDTA, 250 mM sucrose, pH 7.8 buffer, to
which a Complete Inhibitor Cocktail tablet (Roche Molecular
Biologicals) had been added prior to use. Retinas were homogenized
on ice using 5 × 5-s bursts of an Ultra-Turrax hand-held
homogenizer. The homogenate was filtered through two layers of
surgical gauze and centrifuged at 100 000g for 60 min at 4 °C.
The supernatant was filtered through a 0.22 µm filter and applied
to a 1 mL Resource Q anion exchange column equilibrated in 20
mM HEPES, 5 mM MgCl2, 0.2 mM EGTA, pH 7.4, using a
Pharmacia FPLC system (Amersham Pharmacia Biotech Ltd, Little
Chalfont, UK). The bound protein was washed with 5 column
volumes and eluted using a 0-300 mM NaCl linear gradient over
65 mL, followed by a steeper gradient of 300-500 mM NaCl over
10 mL, and a final 1 M NaCl wash for 10 mL. The flow rate was
5 mL/min, and the fractions were 2 mL. Cone PDE6 eluted at
approximately 150 mM NaCl. The column fractions comprising
Acknowledgment. We would like to thank Steve Denton,
Edel Evrard, Mark Ness, Gemma Parsons, Jenny Price, Nick
Smith, and Lesa Watson for compound synthesis and route
optimization; Sue Bethell, Eugene Gbekor, Julie Gupta, and
Shirley Jones for biological screening; James Gosset and Rhys
Jones for pharmacokinetic data generation; Janet Williams and
Mike Cram for solubility data generation; Kevin Dack and Mark
Kemp for useful discussions; and Heather Ringrose for crys-
talliztion work.
Supporting Information Available: Tables of 1H NMR data,
elemental analysis data for target compounds, X-ray data collection
statistics for compound 2, and crystallographic refinement statistics
of deposited model for compound 2. This material is available free
of charge via the Internet at http://pubs.acs.org.
References
(1) For recent reviews on the pharmacological therapy for the treatment
of erectile dysfunction, see: (a) Maw, G. N. Pharmacological therapy
for the treatment of erectile dysfunction. Annu. Rep. Med. Chem.
1999, 34, 71-79. (b) Cartledge, J.; Eardley, I. Viagra and beyond.
Curr. Opin. CPNS InVest. Drugs 1999, 2, 240-245.
(2) Eardley, I. The role of phosphodiesterase inhibitors in impotence.
Expert Opin. InVest. Drugs 1997, 12 (6), 1803-1810.
(3) Bunnage, M. E. Design and synthesis of selective PDE5 inhibitors
for the treatment of MED. 224th ACS National Meeting, Boston, USA,
August 18-22, 2002; Bunnage, M. E. Highly selective chiral PDE5
inhibitors for treatment of MED. 227th ACS National Meeting,

3594 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Allerton et al.
Anaheim, CA, March 28-April 1, 2004. Bunnage, M. E. Viagra &
Beyond: Selective PDE5 Inhibitors for Treatment of MED Inter-
national Symposium on AdVances in Synthetic, Combinatorial &
Medicinal Chemistry, Moscow, Russia, May 5-8, 2004.
tion. The preferred method is filtration accompanied with an
assessment of filter binding. Following separation, the pH of the
soluble fraction is determined and the soluble material diluted in
mobile phase for analysis by HPLC or MS. Bulk form is assessed
by microscopy.
(4) Chan, O. H.; Stewart, B. H. Physicochemical and drug-delivery
considerations for oral drug bioavailability. Drug DiscoVery Today
1996, 1, 461-473.
(21) The stereoselectivity of the reduction of the 5′-ketone to the alcohol
by ketone reductase was never determined. In addition, it was never
identified whether reoxidation to the ketone occurred with the racemic
alcohol or was specific to one of the enantiomers.
(22) Chu-Moyer, M. Y.; Ballinger, W. E.; Beebe, D. A.; Coutcher, J. B.;
Day, W. W.; Li, J.; Oates, P. J.; Weekly, R. M. SAR and species/
stereo-selective metabolism of the sorbitol dehydrogenase inhibitor,
CP-470,711. Bioorg. Med. Chem. Lett. 2002, 12 (11), 1477-1480.
Felsted, R. L.; Bachur, N. R. Ketone reductases. Enzym. Basis
Detoxicat. 1980, 1, 281-293.
(23) Brown, D. G.; Groom, C. R.; Hopkins, A. L.; Jenkins, T. M.; Kamp,
S. H.; O’Gara, M. M.; Ringrose, H. J.; Robinson, C. M.; Taylor, W.
E. Crystal structures of human phosphodiesterase 5 and its ligand
complex and their use in the design of site-directed mutants and
design or screening of inhibitor compounds. PCT Int. Appl. WO
2003038080 A1. Brown, D. G.; Groom, C. R.; Hopkins, A. L.;
Jenkins, T. M.; Kamp, S. H.; O’Gara, M. M.; Ringrose, H. J.;
Robinson, C. M.; Taylor, W. E. Crystal structures of human
phosphodiesterase 5 and its ligand complex and their use in screening
for inhibitor. PCT Int. Appl. WO 2004097010 A1. Sung, B. J.;
Hwang, K. Y.; Jeon, Y. H.; Lee, J. I., Heo, Y. S.; Kim, J. H.; Moon,
J.; Yoon, J. M.; Hyun, Y. L. Structure of the catalytic domain of
human phosphodiesterase 5 with bound drug molecules. Nature 2003,
425 (6953), 98-102.
(24) Taylor, E. C.; Wong, G. S. K. Convergent and efficient palladium-
effected synthesis of 5,10-dideaza-5,6,7,8-tetrahydrofolic acid
(DDATHF) J. Org. Chem. 1989, 54, 4 (15), 3618-3624.
(25) Supplied by Key Organics.
(26) Fox, D. N. A. Preparation of 1-benzoyl-3-piperidinylethylpiperidines
and analogs as tachykinin receptor antagonists. Eur. Pat. Appl. EP
992493.
(27) Jung, M. E.; Choi, Y. M. New Synthesis of 2-azetidines and
1-azabutadienes and the use of the latter in Diels-Alder reactions:
total synthesis of (()-γ-coniceine. J. Org. Chem. 1991, 56(24),
6729-30.
(28) Greengrass, C. W.; Street, S. D. A.; Whittle, P. J. Preparation of
antiviral peptide analogs PCT Int. Appl. WO 9319059.
(29) 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. Impotence Res.
1996, 8, 7-52.
(5) Lipinski, C. A.; Lombardo, F.; Dominy, B.l W.; Feeney, P. J.
Experimental and computational approaches to estimate solubility
and permeability in drug discovery and development settings. AdV.
Drug DeliVery ReV. 1997, 23, 3-25.
(6) Walker, D. K.; Ackland, M. J.; James, G. C.; Muirhead, G. J.; Rance,
D. J.; Wastall, P.; Wright, P. A. Pharmacokinetics and metabolism
of sildenafil in mouse, rat, rabbit, dog and man. Xenobiotica 1999,
29, 297-310.
(7) Bunnage, M. E.; Mathias, J. P.; Street, S. D. A.; Wood, A. Preparation
of pyrazolopyrimidinone cGMP PDE5 inhibitors for the treatment
of sexual dysfunction. PCT Int. Appl. WO 9954333.
(8) Bunnage, M. E.; Mathias, J. P.; Street, S. D. A.; Wood, A. Preparation
of pyrazolopyrimidinones as inhibitors of type 5 cyclic guanosine
3′,5′-monophosphate phosphodiesterase (cGMP PDE5) for the treat-
ment of sexual dysfunction. PCT Int. Appl. WO 9849166.
(9) Thomas, R. J.; Campbell, K. N.; Hennion, G. F. Catalytic hydration
of alkylacetylenes. J. Am. Chem. Soc. 1938, 60, 718-720.
(10) Wilds, A. L. Reduction with aluminium alkoxides (the Merwein-
Pondorf-Verley reduction). Org. React. 1944, 2, 178-223.
(11) Wright, S. W.; Hageman, D. L.; McClure, L. D. Synthesis of
functionalised 3-pyridyl methyl ketones. J. Heterocycl. Chem. 1998,
35, 719-723.
(12) Bell, A. S.; Brown, D.; Terrett, N. K. Preparation of pyrazolo[4,3-
d]pyrimidin-7-ones as cardiovascular agents. Eur. Pat. Appl. EP
463756.
(13) Confirmed by nuclear Overhauser effect (see Experimental Section).
(14) For all the compounds reported in this paper, IC50 values were
determined from concentration-response curves across the range
from 1 nM to 10 µM (unless otherwise stated) using human corpus
cavenosum-derived PDE5. For our primary selectivity assay, IC50
values were determined using cone PDE6 derived from canine retina
(unless otherwise stated), since this is the isozyme against which
sildenafil shows least selectivity. Ballard, S. A.; Gingell, C. J.; Tang,
K.; Turner, L. A.; Price, M. E.; Naylor, A. M. Effects of sildenafil
on the relaxation of human corpus cavernosum tissue in vitro and
on the activities of cyclic nucleotide phosphodiesterase isozymes. J.
Urol. 1998, 159, 2164-2171.
(15) Abel, S.; Beaumont, K. C.; Crespi, C. L.; Eve, M. D.; Fox, L.; Hyland,
R.; Jones, B. C.; Muirhead, G. J.; Smith, D. A.; Venn, R. F.; Walker,
D. K. Potential role for P-glycoprotein in the non-proportional
pharmacokinetics of UK-343,664 in man. Xenobiotica 2001, 31 (8/
9), 665-676; Walker, D. K.; Beaumont, K. C.; Comby, P.; Evans,
K. M.; Gedge, J. I.; Halliday, R. C.; Roffey, S. J.; Wright, P. A.
Pharmacokinetics and metabolism of a selective PDE5 inhibitor (UK-
343,664) in rat and dog. Xenobiotica 2001, 31 (8/9), 651-664.
(16) (a) Artursson, P. Epithelial transport of drugs in cell culture. I: A
model for studying the passive diffusion of drugs over intestinal
absorptive (Caco-2) cells. J. Pharm. Sci. 1990, 79, 476-482. (b)
Stewart, B. H.; Chan, O. H.; Lu, R. H.; Reyner, E. L.; Schmid, H.
L.; Hamilton, H. W.; Steinbaugh, B. A.; Taylor, M. D. Comparison
of intestinal permeabilities determined in multiple in vitro and in
situ models: Relationship to absorption in humans Pharm. Res. 1995,
12, 693-699. (c) Bailey, C. A.; Bryla, P.; Malick, A. W. The use of
the intestinal epithelial cell culture model, Caco-2, in pharmaceutical
development AdV. Drug. DeliV. ReV. 1996, 22, 85-103.
(30) Fawcett, L.; Baxendale R.; Stacey, P.; McGrouther, C.; Harrow I.;
Soderling S.; Hetman, J.; Beavo, J. A.; Phillips, S. C. Molecular
cloning and characterisation of a distinct human phosphodiesterase
gene family: PDE11A. PNAS 2000, 97, 3702-3707.
(31) Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data
collected in oscillation mode. Methods Enzymol. 1997, 276, 307-
326.
(32) Matthews, B. W. Solvent content of protein crystals. J. Mol. Biol.
1968, 33, 491-7.
(33) Collaborative Computational Project Number 4. The CCP4 suite:
Programs for protein crystallography. Acta Crystallogr., Sect. D: Biol.
Crystallogr. D 1994, 50 (5), 760-763.
(34) Quanta98, 1998, version 98.1111; Molecular Simulations Inc., San
Diego, CA 92121-3752.
(17) Papp (cm/s) ) amount transported/time × surface area × initial
concentration; all data was measured at 25 µM unless otherwise
stated; efflux ratio is the ratio of secretory flux to absorptive flux.
(18) Carlille, D.; Zomorodi, K.; Houston, B. Scaling factors to relate drug
metabolic clearance in hepatic microsomes isolated hepatocytes, and
the intact liver Drug Met. Dispos. 1997, 25, 903-911.
(19) Bell, A. S.; Terrett, N. K. Preparation of pyrazolopyrimidinones as
cGMP phosphodiesterase inhibitors. Eur. Pat. Appl. EP 526004.
(20) Aliquots of compounds are mixed with either phosphate buffer (pH
7.4) or water for >16 h at room temperature or 37 °C. Soluble and
insoluble compounds are separated by filtration or double centrifuga-
(35) Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros,
P.; Grosse-Kunstleve, R. W.; Jiang, J.-S.; Kuszewski, J.; Nilges, M.;
Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L.
Crystallography & NMR system: A new software suite for macro-
molecular structure determination. Acta Crystallogr., Sect. D: Biol.
Crystallogr. D 1998, 54 (5), 905-921.
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