The discovery of UK-369003, a novel PDE5 inhibitor with the potential for oral bioavailability and dose-proportional pharmacokinetics

Article abstract of DOI:10.1016/j.bmc.2011.10.022

This paper describes our recent efforts to design and synthesise potent and selective PDE5 inhibitors and the use of in vitro predictors of clearance, absorption and permeability to maximise the potential for dose-proportional pharmacokinetics and good oral bioavailability in man. Optimisation of the preclinical profile resulted in the identification of UK-369003 (19a) and its nomination as a clinical candidate. The clinical pharmacokinetic and safety profile has enabled us to progress the compound to test its efficacy in patients with lower urinary tract symptoms (LUTS) associated with benign prostatic hyperplasia (BPH) and a paper describing its efficacy has recently been published.^2,3.

Full text of DOI:10.1016/j.bmc.2011.10.022

Bioorganic & Medicinal Chemistry 20 (2012) 498–509
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The discovery of UK-369003, a novel PDE5 inhibitor with the potential
for oral bioavailability and dose-proportional pharmacokinetics
David J. Rawson a, , Stephen Ballard c, Christopher Barber a, Laura Barker c, Kevin Beaumont b,
⇑
Mark Bunnage a, Susan Cole b, Martin Corless a, Stephen Denton a, David Ellis a, Marion Floc’h a,
Laura Foster c, James Gosset b, Frances Holmwood a, Charlotte Lane a, David Leahy c, John Mathias a,
Graham Maw a, William Million a, Cedric Poinsard a, Jenny Price a, Rachel Russel c, Stephen Street a,
Lesa Watson a
a Worldwide Medicinal Chemistry, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
b Department of Pharmacokinetics, Dynamics and Metabolism, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
c Biology Department, Ramsgate Road, Pfizer Global Research and Development, Sandwich, Kent CT13 9NJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper describes our recent efforts to design and synthesise potent and selective PDE5 inhibitors and
the use of in vitro predictors of clearance, absorption and permeability to maximise the potential for
dose-proportional pharmacokinetics and good oral bioavailability in man. Optimisation of the preclinical
profile resulted in the identification of UK-369003 (19a) and its nomination as a clinical candidate. The
clinical pharmacokinetic and safety profile has enabled us to progress the compound to test its efficacy in
patients with lower urinary tract symptoms (LUTS) associated with benign prostatic hyperplasia (BPH)
and a paper describing its efficacy has recently been published.2,3
Received 1 August 2011
Revised 2 October 2011
Accepted 7 October 2011
Available online 24 October 2011
Keywords:
PDE5
Caco-2
Ó 2011 Elsevier Ltd. All rights reserved.
Phosphodiesterase
Oral bioavailability
1. Introduction
P-glycoprotein was considered to be the main source of the non-pro-
portional pharmacokinetic profile observed in man.4
The launch of ViagraÒ (Sildenafil citrate, 1) in 1998 as the first
oral treatment of male erectile dysfunction (MED) generated a
huge amount of interest in the potential of PDE5 inhibitors to treat
MED and other diseases associated with lowering cGMP levels.
Kidney, heart and lung are all tissues rich in PDE5 and PDE5 inhib-
itors have been used extensively in clinical trials to assess their po-
tential to treat diseases associated with vascular tone in these
organs.1 RevatioÒ was successfully launched for the treatment of
pulmonary hypertension in 2005.
The aim of the current work was to maintain the PDE selectivity
profile of UK-343664 whilst maximising the potential for oral bio-
availability and dose-proportional pharmacokinetics through min-
imisation of Pgp affinity and optimisation of physicochemical
parameters (such as molecular weight, lipophilicity, solubility and
H-bonding capacity).5 We have been able to explore these changes
through modulation of substituents at the N2, C3 and 20-positions
O
N
O
N
N
N
N
O
HN
O
HN
2. Chemistry
N
N
UK-343664 (2) (Fig. 1) was designed as a follow-up to Sildenafil
with improved selectivity for PDE5 over PDE6 and was progressed
into development. It was shown to have dose-dependent pharmaco-
kinetics in man with supraproportional increases in both AUC and
Cmax with increasing oral dose. The high affinity of UK-343664 for
O
S
N
O
O
S
N
O
1
2
N
N
Sildenafil
IC50 PDE5 = 3.5nM
UK-343664
IC50 PDE5 = 1.1nM
PDE6/PDE5 = 11
MW = 474
PDE6/PDE5 = 103
MW = 565
⇑
Corresponding author.
E-mail address: davidrawson1@gmail.com (D.J. Rawson).
Figure 1.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.022

D. J. Rawson et al. / Bioorg. Med. Chem. 20 (2012) 498–509
499
N2
O
N
O
N
O
R1
R1
H2N
O
O
N
N
N
O
HN
O
HN
O
2'
N
R
N
N
N
O
HN
N
N
N
SO2Me
N
N
i, ii, iii
N
N
H
C3
R3
N
O
NBOC
O
S
N
O
O
S
N
O
O
S
N
O
S
N
O
8
6i
N
N
R2
R2
N
N
ii
R = Alkyl, alkoxyalkyl or heteroarylalkyl
R1 = Alkyl, alkoxyalkyl or heteroarylalkyl
R2 = Me or Et
R3 = H or Me
O
O
N
H2N
O
N
O
HN
O
Figure 2.
N
v
N
N
N
N
N
N
H
(seeFig. 2). This has enabled us to discover novel compounds with a
significantly improved balance of potency, selectivity, metabolic
stability and oral absorption characteristics commensurate with
progression to clinical development.
The two classes of compounds which we are described in this
paper are shown in Figure 2.
NR
O
S
N
O
O
S
N
O
9
R = H
iv
N
N
11
10 R = Me
The synthetic route described in Scheme 1 proved to be a versa-
tile and regioselective route to N2 alkylated compounds (7).
Acid 3 was coupled with aminocarboxamide 46 using standard
procedures (either addition of the acid chloride of 3 to amine 4
or via carbodiimide-mediated coupling). A completely regioselec-
tive N2 alkylation of 5 could be achieved using an appropriate
alkyl-, alkoxyalky- or aminoalkyl-mesylate or chloride to give
6a–6k. This was followed by a one-pot cyclisation/displacement
procedure which facilitated conversion of 6 directly into 7 through
use of the appropriate alcoholic solvent (R1OH) and 4 equiv
KHMDS at reflux which resulted in pyrimidinone formation with
Scheme 2. Reagents and conditions: (i) KHMDS, nBuOH, 120–130 °C, pressure
vessel (ii) TFA, CH2Cl2; (iii) methanesulphonyl chloride, NEt3, CH2Cl2; (iv) HOAc,
NaCNBH3, CH2O (v) KHMDS, nBuOH, reflux.
concomitant alkoxypyridyl exchange (7a–7k). The regiochemistry
of the initial alkylation was proved by correlation with compounds
made using Scheme 3 (vide infra).
Cyclisation followed by deprotection of 6j with TFA and sulpho-
nylation furnished 8. The methylpiperidine analogue (11) was
formed by deprotection of 6j with TFA and reductive methylation
prior to the cyclisation (Scheme 2).
An alternative route to the compounds described in Scheme 1 is
shown in Scheme 3. Alkylation of 12 resulted in a separable mix-
ture of regioisomers, 13 and 14.7 The desired N2 isomer 15 was
made through hydrogenation of 13 and used in the synthesis of
19a–d and 20a–i.
For the novel fused tricyclic inhibitors (26 and 27 in Scheme 4)
the key step was formation of the pyrazole ring from the interme-
diate 22.8 N-nitrosolation of pipecolinic acid (21a) (or 3-methyl-
pipecolinic acid (21b)) using nitrous acid, followed by reaction of
the intermediate with TFAA in ether, gave the betaine 22. A mix-
ture of isomeric pyrazoles was obtained on heating the betaine
in xylene in the presence of ethylpropiolate which were readily
separated by chromatography. Nitration and concomitant ester
hydrolysis of 23 followed by amide formation and nitro reduction
furnished 24, which was converted to 26 and 27 by the methods
described in Schemes 1 and 2. Preparative chiral HPLC was used
to separate enantiomeric products.9
O
H2N
O
O
O
O
N
N
N
N
N
OH
H
O
H
i
N
H2N
H2N
O
S
N
O
O
S
N
O
NH
4
5
3
N
R2
N
R2
3a R1 = Et
3b R2 = Me
O
N
O
R1
H2N
O
N
O
HN
O
N
N
R
ii
iii
N
N
N
N
R
H
O
S
N
O
O
S O
7
6
N
N
R2
N
R2
3. Results and discussion
R2 = Et
R2 = Me
Phosphodiesterase inhibitory activity was assessed on human
corpus cavernosum-derived PDE5 with IC50 values determined
from concentration–response curves using concentrations from
7a R= nPr, R1= -(CH2)2OMe
7b R= iPr, R1= -(CH2)2OMe
7c R= cBu, R1= -(CH2)2OMe
7d R= iBu, R1= -(CH2)2OMe
7j R = nPr, R1 = -(CH2)2OMe
7k R = iBu, R1 = -(CH2)2OMe
1 nM to 10 lM. Our primary PDE selectivity assay was IC50 against
7e R= cPrCH2, R1= -(CH2)2OMe
PDE6cone derived from dog retina. PDE6cone was chosen for the pri-
mary selectivity screen as it has been associated with the low inci-
dence of adverse visual effects associated with high doses of
Sildenafil.10
7f R= 4-tetrahydropyran, R1= -(CH2)2OMe
7g R= -(CH2)2NMe2, R1=iBu
7h R= -(CH2)3NMe2, R1=nBu
7i R= 4-N-BOCpiperidine, R1=nBu
We have previously described how improved selectivity for
PDE5/6 could be achieved through introduction of a 20-methoxy-
ethyloxy pyridyl and large N2 pyrazole motifs (2). The series of
compounds shown in Table 1 combine the 20-methoxyethoxy func-
tionality with smaller N2 substituents targeting compounds with a
Scheme 1. Reagents and conditions: (i) 1-(3-dimethylaminopropyl)-3-ethyl-car-
bodiimide hydrochloride, hydroxybenzotriazole, di-isopropylethylamine, THF,
20 °C, 20 h; (ii) caesium carbonate, alkyl mesylate or alkyl chloride, DMF, 20 °C,
20 h; (iii) KHMDS, R1OH, 120 °C, 20 h.

500
D. J. Rawson et al. / Bioorg. Med. Chem. 20 (2012) 498–509
O
R
R
R
iii
N
R
i, ii
H2N
N+
N
O
O
O
N
R
N
N
CO2H
N
H
O
O2N
O
H2N
N
H2N
N
22
NH
i
N
R
21
ii
+
CO2Et
23
13
O2N
H2N
21a R = H
21b R = Me
(Racemic)
N
H2N
O2N
12
O
N
15
H2N
O
N
O
R
N
iv, v, vi
vii, viii
ix
14
N
N
O
H
O
NH2
N
R
H2N
N
O
S O
N
O
O
N
N
CONH2
16d R =
O
N
24
N
N
25
v
R
iii
H
N
N
vi
N
H
17 R =
18 R =
x
O
S
N
O
xi
16
Me
O
O
R1
N
N
N
HN
O
HN
O
N
N
N
N
O
O
O
N
N
O
R1
O
N
N
R
R
O
HN
HN
N
R
N R
S
N
O
S
N
O
iv
vii
27
N
N
N
N
26
N
N
26a R = H
26b R = Me (+)
26c R = Me (-)
27a R = H, R1 = Pr
O
S
N
O
O
S
N
O
27b R = H, R1 = MeO(CH2)2
27c R = H, R1 = MeO(CH2)2 (+)
27d R = H, R1 = MeO(CH2)2 (-)
19
20
N
N
Scheme 4. Reagents and conditions: (i) NaNO2, HCl, H2O; (ii) TFAA, Et2O; (iii) ethyl
propynoate, xylene, reflux, 2 h; (iv) NaOH, H2O, dioxan; (v) HNO3/H2SO4, 40–55 °C;
(vi) (COCl)2, CH2Cl2, DMF; (vii) NH3, THF; (viii) 10% Pd/C, EtOH, 60 psi H2, 20 °C,
14 h; (ix) acid chloride of 3, NEt3, CH2Cl2; (x) KHMDS, EtOH, 130 °C, 14 h, pressure
vessel; (xi) methoxyethanol, KHMDS, reflux, 14 h.
20a R = -(CH2)2OMe, R1 = nPr
20b R = -(CH2)2OMe, R1 = iBu
19a R = -(CH2)2OMe
19b R = -(CH2)2morpholine
19c R = Et
20c R = -(CH2)2OMe, R1 = 2-pyridylmethyl
20d R = -(CH2)2OMe, R1 = -(CH2)2OMe
20e R = -(CH2)2morpholine, R1=cPrCH2
20f R = -(CH2)2morpholine, R1=MeO(CH2)2
20g R = Et, R1 = MeO(CH2)2-
19d R =
N
Me
20h R =
R1 = nBu
N
Me
Table 1
Scheme 3. Reagents and conditions: (i) caesium carbonate, RCl, DMF; (ii) 50 psi H2,
10% Pd/C (iii) 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride,
HOBT, di-isopropylethylamine, THF, 20 °C, 20 h; (iv) KHMDS, ethanol, 120 °C,
pressure vessel; (v) TFA, CH2Cl2; (vi) CH2O, HOAc, NaCNBH3; (vii) R1OH, KHMDS,
120 °C.
O
O
N
O
HN
N
R
N
O
N
logD of 1–3 to maximise the potential for metabolic stability and
oral absorption. Replacement of the pyridyl group of 2 with alkyl
groups of varying lengths maintained good PDE5 potency and
selectivity over PDE6.
Replacement of the N2 pyridyl substituent of 2 with a methoxy-
ethyl substituent enabled us to explore substituents at the R1 posi-
tion with increased lipophilicity whilst maintaining our desired
lipophilicity range (Table 2).
Basic functionalities were also tolerated at the N2 position (Ta-
ble 3) with alkyloxy groups required at the 20 position to achieve
the levels of lipophilicity required for oral absorption. Excellent
levels of PDE5 potency could be achieved and PDE5/6 selectivity
maintained for the more lipophilic analogues.
A small series of inhibitors was synthesised by cyclising the N2
alkyl and C-3 substituents to form a third ring appended to the pyr-
azolopyrimidinone system (Table 4). A methyl group was intro-
duced into the fused ring (R2) to improve PDE5 potency by
accessing the lipophilic pocket exploited by Sildenafil.11 Significant
improvements in PDE5 potency were observed (26b and 26c, 27c
and 27d) with 27d showing good PDE5 potency combined with im-
proved PDE5/6 selectivity.
S
N
O
N
R2
Example
R
R2
MW
IC50 PDE5 (nM)
PDE5/6 selectivity
7a
7b
7c
7d
nPr
iPr
Et
Et
Et
Et
534
534
544
548
1.83
1.36
1.76
1.04
170
354
219
331
cBu
iBu
7e
7f
Et
Et
546
576
1.01
1.66
189
241
O
7i
7j
nPr
iBu
Me
Me
520
534
4.35
2.89
165
237
N
2
Et
597
1.1
842
PDE5 potency and PDE5/6 selectivity over a narrow lipophilicity
range. The selectivity and MW of the compounds described have
been plotted in Figure 3 and a general trend for increased selectiv-
ity with higher MW can be seen. The desired ‘target zone’ to max-
The compounds shown in Tables 1–4 demonstrate that both the
R and R1 positions on the pyrazolopyrimidinone template are very
tolerant to a wide range of substituents, with excellent levels of

D. J. Rawson et al. / Bioorg. Med. Chem. 20 (2012) 498–509
501
Table 2
Table 4
O
N
O
N
R1
R1
N
N
O
HN
O
HN
N
R
N
*
N
N
R2
O
S
N
O
O
S
N
O
N
N
Example
R
R1
MW IC50 PDE5
(nM)
PDE5/6
selectivity
Example R2
R1
MW IC50 PDE5 (nM) PDE5/6 selectivity
26a
27a
H
H
Et
488
502
10.8
2.47
70
55
nPr
nPr
iBu
534
546
0.69
0.69
51
OMe
OMe
OMe
20a
20b
27b
26b
26c
H
518
502
502
8.5
3.35
3.70
392
125
55
MeO
204
Me Et
Me Et
N
20c
583
0.75
309
27c
27d
Me
Me
532
532
2.76
1.96
149
490
MeO
MeO
OMe
OMe
20d
19a
550
534
1.53
1.23
306
117
MeO
Et
With the predominant mode of clearance being hepatic P450-
mediated metabolic oxidation of the piperazine ring and N-2 posi-
tion by CYP3A4, we have used human liver microsome incubations
as a surrogate for first-pass metabolism.
Table 3
O
N
R1
N
Caco-2 monolayers have been used extensively as a high
throughput screen to assess human intestinal absorption potential
and have been very useful in predicting potential for good bioavail-
ability in man from an in vitro system.13 A high absorptive flux
(Papp > 10 ꢀ 10ꢁ6 cm/s)14 and an efflux ratio close to unity across
N
O
HN
N
R
O
S
N
O
the concentration range 10–100 lM increases the potential for
concentration independent absorption in man.4
N
Using a panel of in vitro screens we have been able to screen
many compounds and progress only those which combine PDE5
potency and PDE5/6 selectivity with good stability in human liver
microsomes (T1/2 >20 min), good Caco-2 flux (Papps >10 ꢀ
10ꢁ6 cm/s) and low efflux ratio (A–B/B–A <2) to further PK testing
in rat and dog. Tables 5 and 6 summarise some of the SAR trends. A
decrease in metabolic stability (associated with increased CYP
turnover) and an increase in membrane permeability are both
associated with increased lipophilicity as would be expected for
compounds metabolised through CYPs.
Example
R
R1
MW IC50
PDE5/6
selectivity
PDE5
(nM)
7g
7h
–(CH2)2NMe2
–(CH2)3NMe2
nBu
iBu
561
575
1.00
2.44
171
404
nBu
nBu
Et
651
587
517
531
559
0.31
0.74
15.8
8.2
100
184
28
N
N
SO2Me
8
11
17
18
20h
Within a relatively narrow logD range (1.1–2.9) clear differenti-
ation between similar analogues has been achieved facilitating
effective selection of inhibitors to be taken forward and 19a and
27d were progressed to rat and dog PK studies.
NH
N
N
Et
52
nBu
0.78
209
O
O
O
19b
20e
20f
Et
575
601
605
0.86
0.26
1.27
184
153
210
N
N
N
cPrCH2
MeO
imise the potential for dose-proportional pharmacokinetics (PK)
and minimise the incidence of visual side-effects has been shaded.
Further in vitro tests were required to differentiate between
inhibitors and select compounds for further development.
The oral bioavailability of most drugs is determined by solubil-
ity, permeability across the intestinal wall and hepatic first pass
extraction.12 As solubility is not limiting in this series we have
made extensive use of in vitro models of absorption and first pass
extraction to select compounds for progression with potential for
oral bioavailability.
Figure 3. Effect of MW on PDE5/6 selectivity. Red spots represent the dibasic
examples in Table 3. Blue spots are the monobasic examples in Tables 1–4.

502
D. J. Rawson et al. / Bioorg. Med. Chem. 20 (2012) 498–509
Table 5
O
R1
N
O
HN
N
R
N
N
O
S
N
O
N
R2
Example
R
R1
R2
LogD (pH7.4)
HLMa (min)
Caco-2b Papp ꢀ 106
A-B/B-A efflux ratioc
20d
19a
7j
Et
1.4
2.1
2.3
2.8
2.9
70
28
28
11
7
9
4.2
1.5
2.2
2.5
1.1
MeO
MeO
iBu
MeO
Et
Et
16
19
24
40
Me
Et
MeO
MeO
iBu
7d
iBu
20b
Et
MeO
7g
nBu
Et
Et
3
—
2.8
6
7
4
N
N
20h
nBu
2.8
74
MeO
O
O
20f
Et
Et
1.1
1.6
48
18
2.8
2.5
8.0
N
N
19b
Et
11.2
a
b
c
Half-life of parent compound upon incubation with human liver microsomes.
Apical to basolateral flux across Caco-2 cell monolayer at 25 M.
Ratio of flux in the apical:basalateral direction to flux in the basolateral:apical direction.
l
Table 6
Table 8
In vitro parameters for 27d
Pharmacokinetic parameters of 19a in man
Example
LogD
(pH 7.4)
HLM
(min)
Caco-2
A-B/B-A
efflux ratio
Dose (mg/kg)
0.43 (iv), 1.43 (oral)
Papp ꢀ 106
Clearance (ml/min/kg)
Vd (L/kg) (iv)
Elimination T1/2 (h) (iv)
Cmax (ng/ml)(oral)
Tmax (h) (oral)
12.8 2.0
2.8 0.05
2.6 0.43
204.8 117.5
1.0 0.54
95.2
27d
1.6
11.2
24
1.5
AUC (ng h/ml)(oral)
Oral bioavailability (%)
Table 7
Pharmacokinetic parameters for 19a and 27d
34 10.6
Species
Rat 19a
Dog 19a
Dog 27d
Dose (mg/kg)
i.v. Clearance (ml/min/
kg)
1.0 i.v., 2.0 p.o. 0.5 i.v., 1.0 p.o. 0.5 i.v., 1.0 p.o.
4. Conclusions
26.6
16.0
20.0
Vd (L/kg)
Elimination T1/2 (h)
Oral bioavailability (%)
2.2
0.95
61
2.8
2.1
55
2.6
1.5
18
In summary, we report the discovery of several PDE5 inhibitors
which show excellent in vitro potency for corpus cavernosum de-
rived PDE5 and selectivity for dog retinal PDE6cone. High through-
put in vitro assays have been used to optimise bioavailability
through determination of oral absorption potential and metabolic
stability. Based on in vitro data collected, 19a and 27d were se-
lected for progression to dog pharmacokinetics and, based on the
results from these studies, 19a was progressed further to clinical
studies.
A paper describing the efficacy of UK-369003 (19a) in patients
with lower urinary tract symptoms associated with clinical benign
prostatic hyperplasia has already been published3 and a full paper
describing the dose dependence of the PK and a full analysis of the
metabolites of UK-369003 in man is in preparation.
The PK data for both 19a and 27d show clearance values of
approximately half hepatic blood flow. The oral bioavailability of
19a is close to 50%, indicating that the compound is essentially
completely absorbed from the g.i. tract. This would be expected
from the aqueous solubility (100 mg/ml @ pH 6.7, >14 mg/ml in
0.2 M lactate buffer @pH 4.3) and the Caco-2 cell flux data (Table 7).
The improved bioavailability and reduced synthetic complexity
of 19a compared to 27d clearly differentiated the two lead com-
pounds 19a has been progressed to clinical development as a selec-
tive, low dose, orally active agent for the treatment of diseases
associated with PDE5-modulated levels of cGMP. The PK parame-
ters of 19a in man are summarised in Table 8 with both rat and
dog predicting well for both clearance and volume of distribution.
The compound was also well tolerated in healthy volunteers. The
preclinical and Phase 1 profile of 19a encouraged us to progress
to further clinical efficacy studies.
5. Experimental
1H NMR spectra in deuterochloroform (CDCl3) or dimethyl
sulphoxide (DMSO-d6) were recorded on a Varian Unity Inova-
300, a Varian Unity Inova-400, or a Varian Mercury-400 spectrom-
eter. Room temperature means 20–25 °C. Unless otherwise stated,

D. J. Rawson et al. / Bioorg. Med. Chem. 20 (2012) 498–509
503
all reactions were carried out using commercially available anhy-
drous solvents. Flash column chromatography refers to column
chromatography on silica gel (Kieselgel 60, 230–400 mesh). Thin-
layer chromatography was performed on either precoated Merck
silica gel (60 F254) plates or Macherey–Nagel Polygram Si G/UV
(0.2 mm silica gel) plates. Mass spectra were recorded using a Fi-
sons Instruments Trio mass spectrometer in the thermospray ion-
ization 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 ob-
served ions quoted refer to the isotopic composition of lowest
mass. Combustion analyses were conducted by Exeter Analytical
UK Ltd, Uxbridge, Middlesex.
mo-2-ethoxy-5-(4-ethylpiperazine-1-ylsulphonyl) pyridine as a
brown solid (6.41 g). 1H NMR (CDCl3) d: 1.06 (t, 3H), 1.48 (t, 3H),
2.42 (q, 2H), 2.56 (m, 4H), 3.09 (m, 4H), 4.54 (q, 2H), 8.10 (s, 1H),
8.46 (s, 1H). LRMS (Thermospray): m/z 378, 380 (M+1)+.
(vi) A mixture of the bromide (6.40 g, 16.92 mmol), triethyl-
amine (12 mL, 86.1 mmol), and catalytic palladium(0) tris(tri-
phenylphosphine) in ethanol (60 mL) was heated at 100 °C and
200 psi, under a carbon monoxide atmosphere, for 18 h, then
cooled. The reaction mixture was evaporated under reduced
pressure and the residue purified by column chromatography
(dichloromethane and methanol) to afford pyridine 2-ethoxy-5-
(4-ethylpiperazin-1-ylsulphonyl)-3-carboxylic acid ethyl ester as
an orange oil (6.2 g). 1H NMR (CDCl3) d: 1.02 (t, 3H), 1.39 (t,
3H), 1.45 (t, 3H), 2.40 (q, 2H), 2.54 (m, 4H), 3.08 (m, 4H), 4.38
(q, 2H), 4.55 (q, 2H), 8.37 (s, 1H), 8.62 (s, 1H). LRMS (Thermo-
spray): m/z 372 (M+1)+.
5.1. Pyridine 2-ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)-3-
carboxylic acid (3a)
(vii) A mixture of the ethyl ester (4.96 g, 13.35 mmol) and 2 N
NaOH solution (25 mL, 50.0 mmol) in ethanol (25 mL) was stirred
at room temperature for 2 h. The reaction mixture was concen-
trated under reduced pressure to half it is volume, washed with
ether and acidified to pH 5 using 4 N HCl. The aqueous solution
was extracted with dichloromethane (3 ꢀ 30 mL), the combined
organic extracts dried (MgSO4) and evaporated under reduced
pressure to afford the title compound, pyridine 2-ethoxy-5-(4-eth-
ylpiperazin-1-ylsulphonyl)-3-carboxylic acid (3a) as a tan coloured
solid (4.02 g). Melting point 206–207 °C. 1H NMR (Dmso-d6) d: 1.18
(t, 3H), 1.37 (t, 3H), 3.08 (q, 2H), 3.17-3.35 (m, 8H), 4.52 (q, 2H),
8.30 (s, 1H), 8.70 (s, 1H). LRMS (Thermospray): m/z 344 (M+1)+.
(i) 2-Aminopyridine (80 g, 0.85 mol) was added portionwise
over 30 min to oleum (320 g) and the resulting solution heated
at 140 °C for 4 h. On cooling, the reaction was poured onto ice
(200 g) and the mixture stirred in an ice/salt bath for a further
2 h. The resulting suspension was filtered, the solid washed with
ice water (200 mL) and cold IMS (200 mL) and dried under suction
to afford pyridine-2-amino-5-sulphonic acid as a solid (111.3 g).
LRMS (Thermospray): m/z 175 (M+1)+.
(ii) Bromine (99 g, 0.62 mol) was added dropwise over 1 h, to a
hot solution of the sulphonic acid (108 g, 0.62 mol) in water
(600 mL) so as to maintain a steady reflux. Once the addition
was complete the reaction was cooled and the resulting mixture
filtered. The solid was washed with water and dried under suction
to afford pyridine-2-amino-3-bromo-5-sulphonic acid (53.4 g). 1H
NMR (DMSO-d6) d: 8.08 (s, 1H), 8.14 (s, 1H).
5.2. 4-[2-Ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-
ylcarboxamido]-1H-3-ethylpyrazole-5-carboxamide (5a)
(iii) A solution of sodium nitrite (7.6 g, 110.0 mmol) in water
(30 mL) was added dropwise to an ice-cooled solution of the bro-
mo sulphonic acid (25.3 g, 100.0 mmol) in 20% aqueous hydrochlo-
ric acid (115 mL), so as to maintain the temperature below 6 °C.
The reaction was stirred for 30 min at 0 °C and for a further 1 h
at room temperature. The reaction mixture was evaporated under
reduced pressure and the residue dried under vacuum at 70 °C for
72 h. A mixture of this solid, phosphorus pentachloride (30.0 g,
144.0 mmol) and phosphorus oxychloride (1 mL, 10.8 mmol) was
heated at 125 °C for 3 h, and then cooled. The reaction mixture
was poured onto ice (100 g) and the resulting solid filtered, and
washed with water. The product was dissolved in dichlorometh-
ane, dried (MgSO4), and evaporated under reduced pressure to af-
ford pyridine-3-bromo-2-chloro-5-sulphonyl chloride as a yellow
solid (26.58 g). 1H NMR (CDCl3) d: 8.46 (s, 1H), 8.92 (s, 1H).
(iv) A solution of 1-ethylpiperazine (11.3 mL, 89.0 mmol) and
triethylamine (12.5 mL, 89.0 mmol) in dichloromethane (150 mL)
was added dropwise to an ice-cooled solution of the sulphonyl
chloride (23.0 g, 79.0 mmol) in dichloromethane (150 mL) and
the reaction stirred at 0 °C for 1 h. The reaction mixture was con-
centrated under reduced pressure and the residual brown oil was
purified by column chromatography (dichloromethane and metha-
nol) to afford 3-bromo-2-chloro-5-(4-ethylpiperazine -1-ylsulpho-
nyl)pyridine as an orange solid (14.5 g). 1H NMR (CDCl3) d: 1.05 (t,
3H), 2.42 (q, 2H), 2.55 (m, 4H), 3.12 (m, 4H), 8.24 (s, 1H), 8.67 (s,
1H).
A
solution of 3-ethyl-1H-pyrazole-5-carboxamide (9.2 g,
59.8 mmol) in DMF (60 mL) was added to a solution of 3a
(21.7 g, 62.9 mmol), 1-hydroxybenzotriazole hydrate (10.1 g,
66.0 mmol) and triethylamine (13.15 mL, 94.3 mmol) in dichloro-
methane (240 mL). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiim-
ide hydrochloride (13.26 g, 69.2 mmol) was added and the reaction
stirred at room temperature for 6 h. The dichloromethane was re-
moved under reduced pressure, the remaining solution poured into
ethyl acetate (400 mL), and this mixture washed with aqueous
NaHCO3 (400 mL). The resulting crystalline precipitate was filtered,
washed with ethyl acetate and dried under vacuum, to afford the
title compound, as a white powder (22 g, 77%). 1H NMR (CDCl3 + 1
drop DMSO-d6) d: 0.96 (t, 3H), 1.18 (t, 3H), 1.50 (t, 3H), 2.25-2.56
(m, 6H), 2.84 (q, 2H), 3.00 (m, 4H), 4.70 (q, 2H), 5.60 (br s, 1H),
6.78 (br s, 1H), 8.56 (d, 1H), 8.76 (d, 1H), 10.59 (s, 1H), 12.10–
12.30 (br s, 1H). LRMS (Thermospray) 480 (M+1)+.
5.3. 4-[2-Ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-
ylcarboxamido]-3-ethyl-2-n-propylpyrazole-5-carboxamide
(6a)
A
mixture of 5a (1.0 g, 2.09 mmol), n-propyl bromide
(0.23 mL, 2.5 mmol) and caesium carbonate (374 mg, 1.15 mmol)
in DMF (5 mL), was heated at 60 °C for 18 h. The solvent was re-
moved under reduced pressure, the residue suspended in water
and extracted with dichloromethane. The combined organic ex-
tracts were washed with brine, dried (MgSO4), and evaporated
under reduced pressure. This product was triturated with ether
and the resulting white solid filtered and dried to give the title
compound (205 mg, 19%). 1H NMR (CDCl3) d: 0.97 (t, 3H), 1.00
(t, 3H), 1.20 (t, 3H), 1.60 (t, 3H), 1.90 (m, 1H), 2.40 (q, 2H),
2.55 (m, 4H), 3.90 (q, 2H), 3.08 (m, 4H), 4.00 (t, 2H), 4.80 (q,
2H), 5.25 (br s, 1H), 6.65 (br s, 1H), 8.60 (s, 1H), 8.80 (s, 1H),
10.55 (s, 1H).
(v) A mixture of the sulphonyl chloride (6.60 g, 17.9 mmol) and
sodium ethoxide (6.09 g, 89.6 mmol) in ethanol (100 mL) was
heated under reflux for 18 h, then cooled. The reaction mixture
was concentrated under reduced pressure, the residue partitioned
between water (100 mL) and ethyl acetate (100 mL), and the layers
separated. The aqueous phase was extracted with ethyl acetate
(2 ꢀ 100 mL) and the combined organic solutions were dried
(MgSO4) and evaporated under reduced pressure to afford 3-bro-

504
D. J. Rawson et al. / Bioorg. Med. Chem. 20 (2012) 498–509
5.4. 3-Ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-(2-
methoxyethoxy)pyridin-3-yl]-2-n-propyl-2,6-dihydro-7H-
pyrazolo[4,3-d]pyrimidin-7-one (7a)
(m, 2H), 2.40 (q, 2H), 2.45 (m, 2H), 2.54 (m, 4H), 2.95 (m, 2H),
3.15 (m, 4H), 3.55 (s, 3H), 3.80 (m, 2H), 4.74 (m, 2H), 4.95 (m,
1H), 8.60 (s, 1H), 8.98 (s, 1H), 10.75 (s, 1H).
A mixture of 6a (200 mg, 0.38 mmol) and potassium bis(tri-
methylsilyl)amide (381 mg, 1.91 mmol) in 2-methoxyethanol
(3 mL) was heated under reflux for 18 h. The solvent was removed
under reduced pressure, the residue partitioned between water
and dichloromethane and the mixture neutralised using 2 N HCl.
The phases were separated, and the organic layer dried (MgSO4)
and evaporated under reduced pressure. The crude product was
purified by column chromatography (dichloromethane and metha-
nol), and the product triturated with ether to give the title com-
pound as a white solid (48 mg, 23%).
5.8. 2-iso-Butyl-3-ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-
2-(2-methoxyethoxy)pyridin-3-yl]-2,6-dihydro-7H-
pyrazolo[4,3-d]pyrimidin-7-one (7d)
The title compound was obtained as a pale yellow solid in 21%
yield, from 6d, and 2-methoxyethanol, according to the method of
7a. Anal. (C25H37N7O5S.0.2H2O): C, 54.22; H, 6.73; N, 17.61. Found:
C, 54.47; H, 6.84; N, 17.79. 1H NMR (CDCl3) d: 0.95 (d, 6H), 1.05 (t,
3H), 1.40 (d, 3H), 2.40 (m, 3H), 2.55 (m, 4H), 3.00 (q, 2H), 3.10 (m,
4H), 3.55 (s, 3H), 3.85 (t, 2H), 5.05 (d, 2H), 4.80 (t, 2H), 8.60 (s,
1H), 8.95 (s, 1H), 10.80 (s, 1H). LRMS (Thermospray): m/z 549
(M+1)+.
1H NMR (CDCl3) d: 0.95 (t, 3H), 1.05 (t, 3H), 1.4 (t, 3H), 2.00 (q,
2H), 2.42 (q, 2H), 2.55 (m, 4H), 3.02 (q, 2H), 3.10 (m, 4H), 3.55 (s,
3H), 3.86 (t, 2H), 4.25 (t, 2H), 4.75 (t, 2H), 8.62 (s, 1H), 8.98 (s,
1H), 10.75 (br s, 1H). Anal. calcd for C24H35N7O5S.H2O: C, 52.25;
H, 6.69; N, 17.54. Found: C, 52.25; H, 6.76; N, 17.77.
5.9. 2-Cyclopropylmethyl-3-ethyl-5-[5-(4-ethylpiperazin-1-
ylsulphonyl)-2-(2-methoxyethoxy)pyridin-3-yl]-2,6-dihydro-
7H-pyrazolo[4,3-d]pyrimidin-7-one (7e)
5.5. 5-[2-iso-Butoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-
3-yl]-2-[2-(dimethylamino)ethyl]-3-ethyl-2,6-dihydro-7H-
pyrazolo[4,3-d]pyrimidin-7-one (7h)
The title compound was obtained as a white solid in 15% yield,
from 6e, and 2-methoxyethanol, according to the method of 7a.
Anal. (C25H35N7O5S.0.5H2O): C, 54.13; H, 6.54; N, 17.68. Found:
C, 53.82; H, 6.34; N, 17.61. 1H NMR (CDCl3) d: 0.43 (m, 2H),
0.60 (m, 2H), 0.80 (m, 1H), 1.00 (t, 3H), 1.40 (t, 3H), 2.40 (q,
2H), 2.54 (m, 4H), 3.00 (q, 2H), 3.07 (m, 4H), 3.50 (s, 3H), 3.80
(m, 2H), 4.20 (d, 2H), 4.78 (m, 2H), 8.60 (s, 1H), 8.97 (s, 1H),
10.75 (br s, 1H).
A mixture of 6g (127 mg, 0.23 mmol) potassium bis(trimeth-
ylsilyl)amide (230 mg, 1.15 mmol) and ethyl acetate (20 mg,
0.23 mmol) in iso-butanol (30 mL) was heated at 130 °C in a
sealed vessel for 6 h. The solvent was removed under reduced
pressure from the cooled mixture, and the residue partitioned
between ethyl acetate and NaHCO3 solution. The layers were
separated, the aqueous phase extracted with ethyl acetate
(3ꢀ), and the combined organic extracts dried (Na2SO4) and
evaporated under reduced pressure. The residual gum was puri-
fied by column chromatography (dichloromethane and metha-
nol) to give the title compound, (30 mg, 23%). 1H NMR (CDCl3)
d: 1.02 (t, 3H), 1.12 (d, 6H), 1.42 (t, 3H), 2.31 (m, 7H), 2.42 (q,
2H), 2.57 (m, 4H), 2.90 (t, 2H), 3.06 (q, 2H), 3.16 (m, 4H),
4.38–4.47 (m, 4H), 8.61 (d, 1H), 9.01 (d, 1H), 10.60 (s, 1H). Anal.
calcd for C26H40N8O4S.0.6H2O: C, 54.85; H, 7.25; N, 19.16. Found
C, 54.64; H, 7.27; 19.61.
5.10. 3-Ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-(2-
methoxyethoxy)pyridin-3-yl]-2-(tetrahydropyran-4-yl)-2,6-
dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (7f)
The title compound was obtained as a pale yellow solid in 21%
yield, from 6f, and 2-methoxyethanol, according to the method of
7a. Anal. (C26H37N7O6S.1.8H2O) C, H, N. 1H NMR (CDCl3) d: 1.00 (t,
3H), 1.40 (t, 3H), 1.83 (m, 2H), 2.40 (q, 2H), 2.55 (m, 6H), 3.06 (q,
2H), 3.10 (m, 4H), 3.55 (s, 3H), 3.60 (t, 2H), 3.80 (t, 2H), 4.20 (m,
2H), 4.48 (m, 1H), 4.80 (t, 2H), 8.60 (s, 1H), 9.00 (s, 1H), 10.80 (br
s, 1H). LRMS (Electrospray): m/z 576.6 (M+1)+.
5.6. 3-Ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-(2-
methoxyethoxy)pyridin-3-yl]-2-iso-propyl-2,6-dihydro-7H-
pyrazolo[4,3-d]pyrimidin-7-one (7b)
5.11. 5-[2-n-Butoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-
3-yl]-2-[3-(dimethylamino)-n-propyl]-3-ethyl-2,6-dihydro-7H-
pyrazolo[4,3-d]pyrimidin-7-one (7g)
The title compound was obtained as a white solid in 21% yield,
from 6b, and 2-methoxyethanol, according to the method of 7a.
Anal. (C24H35N7O5S.0.4H2O): C, 53.30; H, 6.67; N, 18.13. Found: C,
53.53; H, 6.64; N, 17.89. 1H NMR (CDCl3) d: 1.00 (t, 3H), 1.40 (t,
3H), 1.60 (d, 6H), 2.40 (q, 2H), 2.55 (m, 4H), 3.02 (q, 2H), 3.15
(m, 4H), 3.55 (s, 3H), 3.80 (m, 2H), 4.70 (m, 1H), 4.78 (m, 2H),
8.60 (s, 1H), 9.00 (s, 1H), 10.75 (s, 1H). LRMS (Thermospray): m/z
534.8 (M+1)+.
The title compound was obtained in 44% yield, from 6h and n-
butanol, by the method of 7g. Anal. (C27H42N8O4S.H2O): C, 55.76; H,
7.53; N, 18.88. Found: C, 55.55; H, 7.42; N, 19.20. 1H NMR (CDCl3)
d: 1.01 (t, 6H), 1.40 (t, 3H), 1.56 (m, 2H), 1.95 (m, 2H), 2.17 (m, 2H),
2.21 (s, 6H), 2.24 (t, 2H), 2.40 (q, 2H), 2.57 (m, 4H), 3.06 (q, 2H),
3.17 (m, 4H), 4.37 (t, 2H), 4.65 (t, 2H), 8.61 (d, 1H), 9.02 (d, 1H),
10.59 (s, 1H).
5.7. 2-Cyclobutyl-3-ethyl-5-[5-(4-ethylpiperazin-1-
ylsulphonyl)-2-(2-methoxyethoxy)pyridin-3-yl]-2,6-dihydro-
7H-pyrazolo[4,3-d]pyrimidin-7-one (7c)
5.12. 3-Ethyl-5-[2-(2-methoxyethoxy)-5-(4-methylpiperazin-1-
ylsulphonyl)pyridin-3-yl]-2-n-propyl-2,6-dihydro-7H-
pyrazolo[4,3-d]pyrimidin-7-one (7i)
A mixture of 6c (450 mg, 0.85 mmol), potassium bis(trimethyl-
silyl)amide (505 mg, 2.55 mmol) and ethyl acetate (100 lL,
The title compound was synthesised from 6j according to the
method of 7a.
1.28 mmol) in 2-methoxyethanol (8 ml) was heated under reflux
for 18 h. The solvent was removed under reduced pressure, and
the residue purified by column chromatography (dichlorometh-
ane/methanol) to afford the title compound, (133 mg, 29%). Anal.
(C24H33N7O4S): C, 55.90; H, 6.45; N, 19.01. Found: C, 55.98; H,
6.45; N, 18.88. 1H NMR (CDCl3) d: 1.00 (t, 3H), 1.38 (t, 3H), 1.90
Anal. (C23H33N7O5S.0.5H2O): C, 53.13; H, 6.69; N, 18.07.
Found: C, 53.15; H, 6.56; N, 18.03. 1H NMR (CDCl3) d: 0.97 (t,
3H), 1.40 (t, 3H), 2.00 (q, 2H), 2.25 (s, 3H), 2.50 (m, 4H), 3.00
(q, 2H), 3.18 (m, 4H), 3.82 (m, 2H), 4.22 (t, 2H), 4.80 (m, 2H),
8.60 (s, 1H), 9.00 (s, 1H), 10.75 (br s, 1H). LRMS (Thermospray):
m/z 520.2 (M+1)+.

D. J. Rawson et al. / Bioorg. Med. Chem. 20 (2012) 498–509
505
5.13. 2-iso-Butyl-3-ethyl-5-[2-(2-methoxyethoxy)-5-(4-methylpi
perazin-1-ylsulphonyl)pyridin-3-yl]-2,6-dihydro-7H-pyrazolo
[4,3-d]pyrimidin-7-one (7j)
vigorously for 30 min. Acetic acid (88
l
L, 1.69 mmol) was added,
the solution stirred for a further 30 min, then sodium triacetoxy-
borohydride (169 mg, 0.80 mmol) was added and the reaction stir-
red at room temperature for 16 h. The reaction mixture was poured
into aqueous NaHCO3 solution, and extracted with ethyl acetate.
The combined organic extracts were dried (MgSO4) and evaporated
under reduced pressure. The residue was purified by column chro-
matography (dichloromethane and methanol and 0.88 ammonia)
to afford the title compound (70 mg, 35%). 1H NMR (CDCl3) d:
1.02 (t, 3H), 1.22 (t, 3H), 1.58 (t, 3H), 1.92 (m, 2H), 2.14 (m, 2H),
2.25–2.45 (m, 7H), 2.54 (m, 4H), 2.91 (q, 2H), 2.99–3.16 (m, 6H),
4.08 (m, 1H), 4.78 (q, 2H), 5.11 (br s, 1H), 6.65 (br s, 1H), 8.63 (d,
1H), 8.83 (d, 1H), 10.53 (s, 1H). LRMS (Thermospray): m/z 344
(M+1)+.
The title compound was obtained as a white solid in 5% yield,
from 6k, and 2-methoxyethanol, according to the method of 7a.
Anal. (C24H35N7O5S.2.5H2O): C, 54.02; H, 6.61; N, 18.37. Found: C,
49.70; H, 6.99; N, 16.88. 1H NMR (CDCl3) d: 0.97 (d, 6H), 1.40 (t,
3H), 2.28 (m, 4H), 2.52 (m, 4H), 3.02 (q, 2H), 3.16 (m, 4H), 3.57
(s, 3H), 3.86 (t, 2H), 4.10 (d, 2H), 4.78 (t, 2H), 8.61 (d, 1H), 8.98
(d, 1H), 10.79 (s, 1H). LRMS (Thermospray): m/z 534 (M+1)+.
5.14. 5-[2-n-Butoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-
3-yl]-3-ethyl-2-[1-(methylsulphonyl)piperidin-4-yl]-2,6-
dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (8)
5.17. 5-[2-n-Butoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-
3-yl]-3-ethyl-2-(1-methylpiperidin-4-yl)-2,6-dihydro-7H-
pyrazolo[4,3-d]pyrimidin-7-one (11)
(i) 5-[2-n-Butoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-
yl]-2-(1-tert-butoxycarbonylpiperidin-4-yl)-3-ethyl-2,6-dihydro-
7H-pyrazolo[4,3-d]pyrimidin-7-one (7i) was obtained in 69% yield,
from 6i and n-butanol, by the method of 7g. 1H NMR (CDCl3): 1.01
(t, 6H), 1.34-1.60 (m, 14H), 1.93 (m, 4H), 2.41 (m, 4H), 2.57 (m, 4H),
2.90 (m, 2H), 3.00–3.20 (m, 6H), 4.38 (m, 3H), 4.66 (t, 2H), 8.61 (d,
1H), 9.00 (s, 1H), 10.58 (s, 1H).
(ii) Trifluoroacetic acid (1.5 mL) was added to a solution 7i
(150 mg, 0.22 mmol) in dichloromethane (1.5 mL), and the solu-
tion stirred for 2½ h at room temperature. The mixture was evap-
orated under reduced pressure, the residue triturated well with
ether, and the resulting precipitate, filtered and dried to give a
white powder (156 mg).
The title compound was obtained in 50% yield, from 10 by the
method of 7g using n-butanol as the solvent. 1H NMR (CDCl3) d:
1.02 (m, 6H), 1.40 (t, 3H), 1.57 (m, 2H), 1.94 (m, 4H), 2.16 (m,
2H), 2.37 (s, 3H), 2.41 (q, 2H), 2.56 (m, 6H), 3.03 (m, 4H), 3.15
(m, 4H), 4.22 (m, 1H), 4.66 (t, 2H), 8.62 (d, 1H), 9.01 (d, 1H),
10.55 (s, 1H). Anal. calcd for C28H42N8O4S.0.75H2O: C, 56.01; H,
7.27; N, 18.51. Found C, 56.03; H, 7.30; N, 18.67.
5.18. 3-Ethyl-1-(2-methoxyethyl)-4-nitropyrazole-5-
carboxamide (14a) and 3-ethyl-2-(2-methoxyethyl)-4-
nitropyrazole-5-carboxamide (13a)
(iii) Methanesulphonyl chloride (23
to a solution of this intermediate in dichloromethane (4 mL) and
triethylamine (110 L, 0.79 mmol), and the reaction stirred at
lL, 0.30 mmol) was added
l
A mixture of 12 (1.7 g, 8.8 mmol), 2-bromoethyl methyl ether
(0.85 mL, 8.85 mmol) and caesium carbonate (2.9 g, 9.0 mmol) in
DMF (20 mL) was stirred at room temperature for 20 h. The reac-
tion mixture was concentrated under reduced pressure and the
residue was partitioned between ethyl acetate (125 mL) and brine
(100 mL). The phases were separated, and the organic layer was
dried (Na2SO4), and evaporated under reduced pressure. The crude
product was purified by column chromatography (ethyl acetate/
methanol) to afford the title compound 14a (831 mg, 39%).
1H NMR (DMSO-d6) d: 1.19 (t, 3H), 2.82 (q, 2H), 3.20 (s, 3H), 3.68
(t, 2H), 4.22 (t, 2H), 8.18 (s, 1H), 8.38 (s, 1H). LRMS (Thermospray):
m/z 260 (M+18)+ and the title compound of 13a, (793 mg, 37%).
1H NMR (CDCl3) d: 1.18 (t, 3H), 2.98 (q, 2H), 3.22 (s, 3H), 3.70 (t,
2H), 4.28 (t, 2H), 7.65 (s, 1H), 7.94 (s, 1H). LRMS (Thermospray): m/
z 243 (M+1)+.
room temperature for 1½ h. The mixture was treated with satu-
rated aqueous NaHCO3 solution (10 mL), and extracted with ethyl
acetate (2 ꢀ 10 mL). The combined organic extracts were dried
(MgSO4) and evaporated under reduced pressure to give a gum.
The crude product was purified by column chromatography
(dichloromethane and methanol) to afford the title compound as
a white solid (50 mg, 34%). 1H NMR (CDCl3) d: 1.01 (t, 6H), 1.40
(t, 3H), 1.55 (m, 2H), 1.95 (m, 2H), 2.08 (m, 2H), 2.42 (q, 2H),
2.57 (m, 6H), 2.90 (s, 3H), 3.01-3.18 (m, 8H), 4.01 (m, 2H), 4.42
(m, 1H), 4.66 (t, 2H), 8.62 (d, 1H), 9.01 (d, 1H), 10.60 (s, 1H). Anal.
calcd for C28H42N8O6S2: C, 51.67; H, 6.50; N, 17.22. Found C, 51.39;
H, 6.62; N, 17.05.
5.15. 4-[2-Ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-
ylcarboxamido]-3-ethyl-2-(piperidin-4-yl)pyrazole-5-
carboxamide ditrifluoroacetate (9)
5.19. 4-Amino-3-ethyl-2-(2-methoxyethoxy)pyrazole-5-
carboxamide (15a)
Trifluoroacetic acid (3 mL) was added to a solution of the piper-
idine 6i (309 mg, 0.47 mmol) in dichloromethane (4 mL), and the
solution stirred for 2½ h. The reaction was evaporated under re-
duced pressure and the residue triturated well with ether. The
resulting solid was sonicated in ether for 1 minute, the resulting
precipitate filtered and dried to afford the title compound as a
white solid (278 mg, 75%). 1H NMR (DMSO-d6) d: 1.15 (m, 6H),
1.46 (t, 3H), 2.04 (m, 2H), 2.20 (m, 2H), 2.40–2.84 (m, 6H), 3.00-
3.22 (m, 6H), 3.25–3.60 (m, 4H), 3.76 (m, 1H), 4.62 (m, 4H), 7.27
(s, 1H), 7.40 (s, 1H), 8.41 (m, 2H), 8.70 (m, 2H), 10.24 (s, 1H).
A mixture of the title compound from 14a (500 mg, 2.07 mmol)
and 10% palladium on charcoal (50 mg) in ethanol (20 mL) was
hydrogenated at 50 psi and room temperature for 18 h. The reac-
tion mixture was filtered through ArbocelÒ, and the filtrate evapo-
rated under reduced pressure to afford the title compound as a
white solid. 1H NMR (DMSO-d6) d: 1.03 (t, 3H), 2.57 (q, 2H), 3.20
(s, 3H), 3.63 (t, 2H), 4.09 (t, 2H), 4.39 (s, 2H), 6.90 (s, 1H), 7.01 (s,
1H). LRMS (Thermospray): m/z 213 (M+1)+.
5.20. 4-[2-Ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-
ylcarboxamido]-3-ethyl-2-(2-methoxyethyl)pyrazole-5-
carboxamide (16a)
5.16. 4-[2-Ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-
ylcarboxamido]-3-ethyl-2-(1-methylpiperidin-4-yl)pyrazole-5-
carboxamide (10)
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-
ride (5.26 g, 27.4 mmol) was added to a solution of 3a (7.25 g,
21.1 mmol), and 15a (4.45 g, 20.9 mmol), 1-hydroxybenzotriazole
Formaldehyde (217
a solution of 9, in dichloromethane (8 mL), and the solution stirred
lL, 37% aqueous, 2.90 mmol) was added to

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D. J. Rawson et al. / Bioorg. Med. Chem. 20 (2012) 498–509
hydrate (3.71 g, 27.4 mmol), and N-diisopropylethylamine
(10.96 mL, 63.3 mmol) in dichloromethane (70 mL), and the reac-
tion stirred for 18 h. The reaction mixture was diluted with dichlo-
romethane (100 mL), washed with water (100 mL), saturated
aqueous NaHCO3 solution (100 mL), and brine (100 mL), dried
(MgSO4), and evaporated under reduced pressure. The crude prod-
uct was purified by column chromatography (dichloromethane
and methanol) to give the title compound as a foam, (10.1 g,
94%). 1H NMR (CDCl3) d: 1.03 (t, 3H), 1.20 (t, 3H), 1.58 (t, 3H),
2.40 (q, 2H), 2.54 (m, 4H), 2.95 (q, 2H), 3.10 (m, 4H), 3.37 (s, 3H),
3.80 (t, 2H), 4.26 (t, 2H), 4.78 (q, 2H), 5.27 (s, 1H), 6.66 (s, 1H),
8.65 (s, 1H), 8.85 (s, 1H), 10.51 (s, 1H). LRMS (Thermospray): m/z
538 (M+1)+.
8.62 (d, 1H), 9.04 (d, 1H), 10.61 (s, 1H). LRMS (Thermospray):
m/z 574 (M+1)+.
5.24. 5-[2-Ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-
yl]-2-ethyl-3-ethyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-
one (19c)
The title compound was obtained as a solid, from 16c and eth-
anol, by the method of 7g, in the absence of ethyl acetate, and after
trituration from ether. Anal. (C23H33N7O5S.0.2Et2O): C, 53.29; H,
6.26; N, 18.19. Found C, 53.49; H, 6.60; N, 18.35. 1H NMR (CDCl3)
d: 1.02 (t, 3H), 1.40 (t, 3H), 1.58 (m, 6H), 2.41 (q, 2H), 2.55 (m,
4H), 3.00–3.18 (m, 6H), 4.38 (q, 2H), 4.75 (q, 2H), 8.63 (s, 1H),
9.04 (s, 1H), 10.63 (s, 1H). LRMS: m/z 490 (M+1)+.
5.21. 4-[2-Ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-
ylcarboxamido]-3-ethyl-2-(1-methylazetidin-3-yl)pyrazole-5-
carboxamide (17)
5.25. 5-[2-Ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-
yl]-3-ethyl-2-(1-methylazetidin-3-yl)-2,6-dihydro-7H-
pyrazolo[4,3-d]pyrimidin-7-one (19d)
Trifluoroacetic acid (2.5 mL) was added to a solution of 16d
(700 mg, 1.1 mmol) in dichloromethane (3.5 mL) and the solution
stirred at room temperature for 2½ h. The reaction mixture was
evaporated under reduced pressure and the residue triturated well
with ether and dried under vacuum. The solid was suspended in
saturated aqueous NaHCO3 solution, extracted with ethyl acetate,
and the combined organic extracts evaporated under reduced
pressure.
A mixture of 18 (470 mg, 0.86 mmol) and potassium bis(tri-
methylsilyl)amide (600 mg, 3.0 mmol) in ethanol (45 mL) was
heated at 130 °C for 16 h. The cooled mixture was concentrated un-
der reduced pressure, the solution diluted with aqueous NaHCO3
solution to give pH 8, and extracted with ethyl acetate
(3 ꢀ 100 mL). The combined organic extracts were dried (MgSO4)
and evaporated under reduced pressure. The crude product was
purified by column chromatography (dichloromethane:metha-
nol:0.88 ammonia) to give the title compound, (170 mg, 37%). 1H
NMR (CDCl3) d: 1.02 (t, 3H), 1.38 (t, 3H), 1.58 (m, 3H), 2.40 (q,
2H), 2.50 (s, 3H), 2.57 (m, 4H), 3.01 (q, 2H), 3.16 (m, 4H), 3.79 (t,
2H), 3.90 (t, 2H), 4.78 (q, 2H), 5.12 (m, 1H), 8.62 (d, 1H), 9.01 (d,
1H), 10.62 (s, 1H). Anal. calcd for C24H34N8O4S.1.33 H2O: C,
52.31; H, 6.53; N, 19.80. Found C, 51.97; H, 6.66; N,20.20.
Formaldehyde (280
solution of the intermediate amine in dichloromethane (8 mL), and
the solution stirred vigorously for 30 min. Acetic acid (53 L,
lL, 37% aqueous, 4.4 mmol) was added to a
l
1.1 mmol) was added, the solution stirred for a further 30 min,
then sodium triacetoxyborohydride (238 mg, 1.12 mmol) was
added and the reaction stirred at room temperature for 16 h. The
reaction mixture was poured into aqueous NaHCO3 solution
(30 mL), and extracted with ethyl acetate (2 ꢀ 30 mL). The com-
bined organic extracts were dried (MgSO4) and evaporated under
reduced pressure. The residue was purified by column chromatog-
raphy (dichloromethane/methanol/0.88 ammonia) to afford the ti-
tle compound (470 mg, 78%). 1H NMR (CDCl3) d: 1.01 (t, 3H), 1.18
(t, 3H), 1.58 (t, 3H), 2.40 (q, 2H), 2.48 (s, 3H), 2.54 (m, 4H), 2.85 (q,
2H), 3.10 (m, 4H), 3.59 (t, 2H), 3.82 (t, 2H), 4.79 (q, 2H), 4.96 (m,
1H), 5.32 (br s, 1H), 6.79 (br s, 1H), 8.64 (d, 1H), 8.82 (d, 1H),
10.52 (s, 1H).
5.26. 3-Ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-n-
propoxypyridin-3-yl]-2-(2-methoxyethyl)-2,6-dihydro-7H-
pyrazolo[4,3-d]pyrimidin-7-one (20a)
Potassium bis(trimethylsilyl)amide (153 mg, 0.77 mmol) was
added to a solution of 19a (100 mg, 0.19 mmol) in n-propanol
(5 mL), and the reaction heated under reflux for 18 h. The solvent
was removed under reduced pressure, the residue partitioned be-
tween water and dichloromethane and the mixture neutralised
using solid CO2. The layers were separated, the aqueous phase ex-
tracted with dichloromethane, and the combined organic extracts
dried (Na2SO4) and evaporated under reduced pressure. The crude
product was purified by column chromatography (dichlorometh-
ane and methanol) to give the title compound (68 mg, 66%). 1H
NMR (CDCl3) d: 1.02 (t, 3H), 1.40 (t, 3H), 1.52 (t, 3H), 1.98 (m,
2H), 2.40 (q, 2H), 2.57 (m, 4H), 3.14 (m, 6H), 3.32 (s, 3H), 3.94 (t,
2H), 4.46 (t, 2H), 4.62 (t, 2H), 8.61 (s, 1H), 9.02 (s, 1H), 10.62 (s,
1H). Anal. calcd for C24H35N7O5S.H2O: C, 53.81; H, 6.61; N, 18.27.
Found C, 54.02; H, 6.61; N,18.27. LRMS (Thermospray): m/z 534
(M+1)+.
5.22. 5-[2-Ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-
yl]-3-ethyl-2-(2-methoxyethyl)-2,6-dihydro-7H-pyrazolo[4,3-
d]pyrimidin-7-one (19a)
The title compound was obtained in 27% yield, after recrystalli-
sation from ethyl acetate, from 16a and ethanol, by the method of
7g.
mp 161–162 °C. 1H NMR (CDCl3) d: 1.04 (t, 3H), 1.40 (t, 3H), 1.58
(t, 3H), 2.41 (q, 2H), 2.57 (m, 4H), 3.08 (q, 2H), 3.14 (m, 4H), 3.30 (s,
3H), 3.92 (t, 2H), 4.46 (t, 2H), 4.75 (q, 2H), 8.62 (d, 1H), 9.04 (d, 1H),
10.61 (s, 1H). Anal. calcd for C24H35N7O6S.0.2EtOAc: C, 53.12; H,
6.47; N, 18.15. Found C, 53.21; H, 6.49; N,18.25. LRMS (Thermo-
spray): m/z 520 (M+1)+.
5.27. 5-[2-iso-Butoxy-5-(4-ethylpiperazin-1-ylsulphonyl)
pyridin-3-yl]-3-ethyl-2-(2-methoxyethyl)-2,6-dihydro-7H-
pyrazolo[4,3-d]pyrimidin-7-one (20b)
5.23. 5-[2-Ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-
yl]-3-ethyl-[2-(morpholin-4-yl)ethyl]-2,6-dihydro-7H-
pyrazolo[4,3-d]pyrimidin-7-one (19b)
The title compound was obtained as a solid, from 19a and 2-
methyl-n-propanol, by the method of 20a. Anal. (C25H37N7O5S):
C, 54.84; H, 6.84; N, 17.65. Found C, 54.83; H, 6.81; N,17.90. 1H
NMR (CDCl3) d: 1.02 (t, 3H), 1.12 (d, 6H), 1.40 (t, 3H), 2.30 (m,
1H), 2.42 (q, 2H), 2.57 (m, 4H), 3.08 (q, 2H), 3.13 (m, 4H), 3.30 (s,
3H), 3.90 (t, 2H), 4.46 (m, 4H), 8.62 (s, 1H), 9.02 (s, 1H), 10.60 (s,
1H). LRMS (Thermospray): m/z 548 (M+1)+.
The title compound was obtained as a solid, from 16b and eth-
anol, by the method of 7g. Anal (C26H38N8O5S): C, 54.16; H, 6.66; N,
19.34. Found C, 54.33; H, 6.66; N,19.50. 1H NMR (CDCl3) d: 1.02 (t,
3H), 1.42 (t, 3H), 1.57 (t, 3H), 2.40 (q, 2H), 2.54 (m, 8H), 2.98 (t, 2H),
3.04 (q, 2H), 3.15 (m, 4H), 3.66 (m, 4H), 4.40 (t, 2H), 4.76 (q, 2H),

D. J. Rawson et al. / Bioorg. Med. Chem. 20 (2012) 498–509
507
5.28. 3-Ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-
5.33. 5-[2-iso-Butoxy-5-(4-ethylpiperazin-1-
[(pyridin-2-yl)methyl]pyridin-3-yl]-2-(2-methoxyethyl)-2,6-
dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (20c)
ylsulphonyl)pyridin-3-yl]-3-ethyl-2-(1-methylazetidin-3-yl)-
2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (20h)
The title compound was obtained as a solid, from 20a and
pyridine-2-methanol, by the method of 20a. Anal. (C27H34N8
O5S.0.5H2O): C, 55.20; H, 5.88; N, 18.97. Found C, 55.31; H,
5.91; N, 19.11. 1H NMR (CDCl3) d: 1.02 (t, 3H), 1.42 (t, 3H), 2.42
(q, 2H), 2.57 (m, 4H), 3.12 (m, 6H), 3.30 (s, 3H), 3.94 (t, 2H),
4.46 (t, 2H), 5.90 (s, 2H), 7.35 (m, 2H), 7.78 (m, 1H), 8.59 (s,
1H), 8.84 (m, 2H), 12.70 (s, 1H). LRMS (Thermospray): m/z 583
(M+1)+.
The title compound was obtained in 45% yield, from 19d and n-
butanol, by the method of 20a. 1H NMR (CDCl3) d: 1.02 (t, 6H), 1.38
(t, 3H), 1.57 (m, 2H), 1.96 (m, 2H), 2.41 (q, 2H), 2.50 (s, 3H), 2.56
(m, 4H), 3.00 (q, 2H), 3.15 (m, 4H), 3.79 (t, 2H), 3.94 (t, 2H), 4.68
(t, 2H), 5.12 (m, 1H), 8.62 (d, 1H), 9.01 (d, 1H), 10.61 (s, 1H). Anal.
(C26H38N8O4S.1.25H2O): C, 54.13; H, 6.75; N, 18.86. Found C, 53.73;
H, 7.02; N, 19.28.
5.34. cis-3-Methyl-2-piperidinecarboxylic acid (21b)
5.29. 3-Ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-(2-
methoxyethoxy)pyridin-3-yl]-2-(2-methoxyethyl)-2,6-dihydro-
7H-pyrazolo[4,3-d]pyrimidin-7-one (20d)
A mixture of 2-cyano-3-methylpyridine (20.0 g, 169 mmol) and
conc. HCl (50 mL) in water (50 mL) was heated under reflux for 8 h.
The reaction was evaporated under reduced pressure, the residue
azeotroped with water, then triturated with acetone. The resulting
pink solid was recrystallised from acetonitrile/water (50:50 by vol-
ume) to give an off-white solid (13.8 g).
A mixture of this intermediate acid, and platinum oxide (5.0 g)
in ethanol (70 mL) and water (70 mL) was hydrogenated at 60 °C
and 60 psi for 24 h. The cooled mixture was filtered through Arbo-
celÒ and the filtrate concentrated under reduced pressure to a vol-
ume of 50 mL. This solution was cooled in ice, and the resulting
precipitate filtered and dried to afford the title compound,
(8.15 g, 34%). LRMS (Thermospray) m/z: 144.1 (M+1)+.
The title compound was obtained as a solid, from 19a and 2-
methoxyethanol, by the method of 20a. Anal. (C25H37N7O6S): C,
52.45; H, 6.42; N, 17.84. Found C, 52.26; H, 6.46; N,17.56. 1H
NMR (CDCl3) d: 1.02 (m, 6H), 1.84 (m, 2H), 2.42 (q, 2H), 2.56 (m,
4H), 3.01 (t, 2H), 3.15 (m, 4H), 3.29 (s, 3H), 3.57 (s, 3H), 3.88 (m,
4H), 4.44 (t, 2H), 4.78 (t, 2H), 8.61 (s, 1H), 8.98 (s, 1H), 10.76 (s,
1H). LRMS (Thermospray): m/z 564 (M+1)+.
5.30. 5-[2-Cyclopropylmethoxy-5-(4-ethylpiperazin-1-
ylsulphonyl)pyridin-3-yl]-3-ethyl-[2-(morpholin-4-yl)ethyl]-
2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (20e)
5.35. 4,5,6,7-Tetrahydro[1,2,3]oxadiazolo[3,4-a]pyridin-8-ium-
3-olate (22a)
The title compound was obtained as a white solid in 38%
yield, after recrystallisation from ethyl acetate, from 19b and
cyclopropanemethanol, by the method of 20a. Anal. (C28H40N8
O5S.0.5H2O): C, 55.36; H, 6.78; N, 18.24. Found C, 52.26; H,
6.78; N, 17.56. 1H NMR (CDCl3) d: 0.46 (m, 2H), 0.77 (m, 2H),
1.01 (t, 3H), 1.42 (t, 3H), 2.40 (q, 2H), 2.54 (m, 8H), 2.97 (t,
2H), 3.01–3.20 (m, 4H), 3.68 (m, 4H), 4.40 (t, 2H), 4.50 (d, 2H),
8.60 (d, 1H), 9.05 (d, 1H), 10.76 (s, 1H). LRMS (Thermospray)
601 (M+1)+.
A solution of sodium nitrite (14.0 g, 203 mmol) in water (30 mL)
was added dropwise over 1 h, to a solution of pipecolinic acid
(25.0 g, 193 mmol) in water (90 mL) and conc HCl (16.5 mL,
198 mmol), cooled in an ice/acetone bath. Once addition was com-
plete, the solution was stirred at 0 °C for 1 h, then allowed to warm
to room temperature. The reaction was extracted with dichloro-
methane (2 ꢀ 200 ml), the combined organic extracts dried
(Na2SO4), and evaporated under reduced pressure. The residual
oil was dissolved in ether (400 mL), this solution cooled in ice, tri-
fluoroacetic anhydride (28 mL, 200 mmol) was added dropwise
over 1 h, and the reaction stirred at room temperature for 18 h.
The reaction mixture was concentrated under reduced pressure
and the residual oil was purified by column chromatography (ethyl
acetate), to give the title compound as a white solid (13.0 g, 43%).
1H NMR (CDCl3) d: 1.95 (m, 2H), 2.10 (m, H), 2.62 (t, 2H), 4.43 (t,
2H). LRMS (Thermospray) m/z: 158.1 (M+1)+.
5.31. 3-Ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-(2-
methoxyethoxy)pyridin-3-yl]-[2-(morpholin-4-yl)ethyl]-2,6-
dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (20f)
The title compound was obtained as a white solid in 48% yield,
from 19b and 2-methoxyethanol, by the method of 20a. Anal.
(C27H40N8O6S.0.5H2O): C, 52.72; H, 6.76; N, 18.11. Found C,
52.84; H, 6.73; N,18.25. 1H NMR (CDCl3) d: 1.02 (t, 3H), 1.42 (t,
3H), 2.41 (q, 2H), 2.53 (m, 8H), 2.96 (t, 2H), 3.05 (q, 2H), 3.16 (m,
4H), 3.57 (s, 3H), 3.66 (m, 4H), 4.40 (t, 2H), 4.78 (t, 2H), 8.62 (d,
1H), 8.99 (d, 1H), 10.78 (s, 1H).
5.36. Ethyl 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-2-
carboxylate (23a)
A mixture of 22a (13.0 g, 93 mmol), and ethyl propiolate
(28.5 mL, 278 mmol), in xylene (250 mL) was heated under reflux
for 2 h. The cooled mixture was evaporated under reduced pres-
sure. The residual oil was purified by column chromatography
(diethylether), to afford the title compound as an oil, (5.82 g,
32%). 1H NMR (CDCl3) d: 1.39 (t, 3H), 1.88 (m, 2H), 2.06 (m, 2H),
2.82 (t, 2H), 4.20 (t, 2H), 4.39 (q, 2H), 6.55 (s, 1H). LRMS (Thermo-
spray) m/z: 195.2 (M+1)+.
5.32. 2-Ethyl-3-ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-
(2-methoxyethoxy)pyridin-3-yl]-2,6-dihydro-7H-pyrazolo[4,3-
d]pyrimidin-7-one (20g)
The title compound was obtained as a solid, from 19c and 2-
methoxyethanol, by the method of 20a, using ethyl acetate/dieth-
ylamine for column chromatography. Anal. (C23H33N7O5S): C,
53.29; H, 6.26; N, 18.19. Found C, 53.49; H, 6.60; N, 18.35. 1H
NMR (CDCl3) d: 1.02 (t, 3H), 1.42 (t, 3H), 1.59 (t, 3H), 2.44 (q,
2H), 2.57 (m, 4H), 3.05 (q, 2H), 3.16 (m, 4H), 3.58 (s, 3H), 3.86 (t,
2H), 4.28 (q, 2H), 4.79 (t, 2H), 8.62 (s, 1H), 8.99 (s, 1H). LRMS (Ther-
mospray): m/z 520 (M+1)+.
5.37 3-Amino-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-2-
carboxamide (24a)
(i) Sodium hydroxide solution (20 mL, 2 M, 40 mmol) was
added to a solution of 23a (5.8 g, 30 mmol) in dioxan (100 mL),

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D. J. Rawson et al. / Bioorg. Med. Chem. 20 (2012) 498–509
and the reaction stirred at room temperature for 18 h. The mixture
was evaporated under reduced pressure and HCl (2 M, 21 mL) was
added. The mixture was evaporated under reduced pressure, the
residue azeotroped with toluene, and the product dried under
vacuum.
5.39. 2-{2-Ethoxy-5-[(4-ethyl-1-piperazinyl)sulfonyl]-3-
pyridinyl}-7,8,9,10-tetrahydropyrido[20,10:5,1]pyrazolo[4,3-
d]pyrimidin-4(3H)-one (26a)
A mixture of 25a (1.0 g, 1.98 mmol) and potassium bis(trimeth-
(ii) Fuming nitric acid (5 mL) was added to concentrated sul-
phuric acid (30 mL) with ice cooling. The resulting mixture was
warmed to 40 °C and the intermediate acid (5.0 g) was added por-
tionwise over 1 h so as to keep the internal temperature between
50 and 60 °C. The reaction was stirred at 60 °C for 1 h followed
by 4 h at 50 °C. The mixture was allowed to cool to room temper-
ature and stirred overnight. The mixture was poured onto ice and
the resulting precipitate filtered off, washed with water, and dried
to afford the acid intermediate, 3-nitro-4,5,6,7-tetrahydropyrazol-
o[1,5-a] pyridine-2-carboxylic acid (4.6 g). 1H NMR (CDCl3) d: 2.00
(m, 2H), 2.15 (m, 2H), 3.20 (t, 2H), 4.28 (t, 2H). LRMS (Thermo-
spray) m/z: 229.3 (M+18)+.
ylsilyl)amide (1.20 g, 6.0 mmol) and ethyl acetate (100 lL,
1.0 mmol) in ethanol (80 mL) was heated in a sealed vessel at
130 °C for 18 h. The solvent was removed under reduced pressure,
the residue partitioned between dichloromethane and water and
the mixture neutralised by the addition of solid CO2. The layers
were separated, the aqueous phase extracted with dichlorometh-
ane (2 ꢀ 100 mL), and the combined organic extracts dried
(Na2SO4) and evaporated under reduced pressure. The crude prod-
uct was purified by column chromatography (dichloromethane
and methanol) to afford the title compound (490 mg, 51%). 1H
NMR (CDCl3) d: 1.01 (t, 3H), 1.57 (t, 3H), 2.01 (m, 2H), 2.18 (m,
2H), 2.40 (q, 2H), 2.55 (m, 4H), 3.14 (m, 6H), 4.40 (m, 2H), 4.75
(q, 2H), 8.61 (s, 1H), 9.04 (s, 1H), 10.66 (s, 1H). Anal. calcd for
(iii) Oxalyl chloride (5.7 mL, 65 mol) was added over 1 min to an
ice-cooled solution of this acid in dichloromethane (100 mL) and
C22H29N7O4S.H2O: C, 52.65; H, 6.02; N19.46. Found C, 52.25; H,
DMF (100
lL) and the reaction was allowed to warm to room tem-
6.18; N,19.39.
perature over 2.5 h. The mixture was evaporated under reduced
pressure, dried under vacuum and the resulting solid was dissolved
in tetrahydrofuran (100 mL) and cooled in an ice bath. Ammonia
gas was bubbled through for 10 min and the reaction stirred a
room temperature for 5 h. The mixture was evaporated under re-
duced pressure and the resulting solid was treated with water
(20 mL), washed with water (5 mL) and dried to give 3-nitro-
5.40. 2-{2-Ethoxy-5-[(4-ethyl-1-piperazinyl)sulfonyl]-3-
pyridinyl}-10-methyl-7,8,9,10-tetrahydropyrido[20,10:5,1]-
pyrazolo[4,3-d]pyrimidin-4(3H)-one (26b)
The title compound was made by the method of 26a and the
enantiomers were separated by chiral preparative HPLC Chiralpak
IA, 50/50 EtOH:MeOH. Anal. calcd for (C23H31N7O4S.H2O): C, 53.16;
H, 6.40; N, 18.87. Found C, 53.48; H, 6.28; N, 18.76. 1H NMR (CDCl3)
d: 1.02 (t, 3H), 1.59 (m, 7H), 2.02–2.28 (m, 3H), 2.41 (q, 2H), 2.57
(m, 4H), 3.15 (m, 4H), 3.38 (m, 1H), 4.34 (m, 1H), 4.43 (m, 1H),
4.77 (q, 2H), 8.62 (d, 1H), 9.03 (d, 1H), 10.65 (s, 1H).
4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-2-carboxamide, as
a
beige solid (4.2 g). 1H NMR (CDCl3) d: 1.96 (m, 2H), 2.10 (m, 2H),
3.17 (t, 2H), 4.21 (m, 2H), 6.04 (br s, 1H), 7.76 (br s, 1H). LRMS
(Thermospray) m/z: 211.1 (MH)+.
(iv) A mixture of the carboxamide, and 10% Pd/C (500 mg) in
methanol (200 mL) was hydrogenated at 60 psi and room temper-
ature for 5 h. The mixture was diluted with methanol (200 mL)
with warming, in order to dissolve the residual solid then filtered
through Arbocel and the filtrate evaporated under reduced pres-
sure to afford the title compound as a brown solid (3.15 g, 58%).
1H NMR (DMSO-d6) d: 1.75 (m, 2H), 1.90 (m, 2H), 2.55 (t, 2H),
3.96 (t, 2H), 4.40 (br s, 2H), 6.86 (br s, 1H), 7.04 (br s, 1H). LRMS
(Thermospray) m/z: 181.5 (M+1)+.
5.41. 2-{2-Ethoxy-5-[(4-ethyl-1-piperazinyl)sulfonyl]-3-
pyridinyl}-10-methyl-7,8,9,10-tetrahydropyrido[20,10:5,1]-
pyrazolo[4,3-d]pyrimidin-4(3H)-one (26c)
The title compound was made by the method of 26a and the
enantiomers were separated by chiral preparative HPLC Chiralpak
IA, 50/50 EtOH:MeOH. Anal. calcd for (C23H31N7O4S.H2O): C, 53.16;
H, 6.40; N, 18.87. Found C, 53.37; H, 6.24; N, 18.87. 1H NMR (CDCl3)
d: 1.02 (t, 3H), 1.59 (m, 7H), 2.02–2.28 (m, 3H), 2.41 (q, 2H), 2.57
(m, 4H), 3.15 (m, 4H), 3.38 (m, 1H), 4.34 (m, 1H), 4.43 (m, 1H),
4.77 (q, 2H), 8.62 (d, 1H), 9.03 (d, 1H), 10.65 (s, 1H).
5.38. 3-[({2-Ethoxy-5-[(4-ethyl-1-piperazinyl) sulfonyl]-3-
pyridinyl}carbonyl)amino]-4-4,5,6,7-tetrahydropyrazolo[1,5-
a]pyridine-2-carboxamide (25a)
Oxalyl chloride (4.4 mL, 50.0 mmol) was added to an ice-
cooled solution of 3a (5.0 g, 13.0 mmol) in dichloromethane
(100 mL) and DMF (0.2 mL), and the reaction stirred for 1 h at
0 °C, then for 4 h at room temperature. The solvent was removed
under reduced pressure, and the residue azeotroped with tolu-
ene, and dried under vacuum. The resulting acid chloride was
suspended in dichloromethane (200 mL), cooled in an ice-bath,
24a (2.37 g, 13.0 mmol) and Et3N (5.0 mL, 36.0 mmol) added,
and the reaction stirred at room temperature for 18 h. The reac-
tion was diluted with dichloromethane, washed with saturated
NaHCO3 solution, dried (Na2SO4) and evaporated under reduced
pressure. The crude was purified by column chromatography
(dichloromethane and methanol) to give the title compound as
a white powder (6.0 g, 91%). 1H NMR (CDCl3) d: 1.01 (t, 3H),
1.58 (t, 3H), 1.90 (m, 2H), 2.27 (m, 2H), 2.39 (q, 2H), 2.53 (m,
4H), 3.00 (t, 2H), 3.10 (m, 4H), 4.17 (t, 2H), 4.79 (q, 2H), 5.30
(br s, 1H), 6.63 (br s, 1H), 8.62 (s, 1H), 8.81 (s, 1H), 10.74 (s,
1H). LRMS (Thermospray) m/z: 506.9 (M+1)+.
5.42. 2-{5-[(4-Ethyl-1-piperazinyl)sulfonyl]-2-n-propoxy-3-
pyridinyl}-7,8,9,10-tetrahydropyrido[20,10:5,1]pyrazolo[4,3-
d]pyrimidin-4(3H)-one (27a)
A solution of 25 (250 mg, 0.51 mmol) in n-propanol (20 mL)
was heated to reflux, and some solvent allowed to evaporate off.
The solution was cooled to room temperature, potassium bis(tri-
methylsilyl)amide (510 mg, 2.6 mmol) was added, and the reaction
heated under reflux for 18 h. The cooled mixture was concentrated
under reduced pressure and the residue partitioned between
dichloromethane and water. This mixture was neutralised using
CO2 pellets, the layers separated, and the aqueous phase extracted
with dichloromethane (3ꢀ). The combined organic extracts were
dried (Na2SO4) and evaporated under reduced pressure. The crude
product was purified by column chromatography (dichlorometh-
ane/methanol) to afford the title compound as a solid, (130 mg,
51%). Anal. (C23H31N7O4S.0.5H2O): C, 54.10; H, 6.31; N, 19.20.
Found C, 54.09; H, 6.27; N, 19.14. 1H NMR (CD3OD) d: 1.02 (m,

D. J. Rawson et al. / Bioorg. Med. Chem. 20 (2012) 498–509
509
6H), 1.83 (m, 2H), 2.00 (m, 2H), 2.18 (m, 2H), 2.41 (q, 2H), 2.57 (m,
4H), 3.10 (m, 4H), 4.38 (t, 2H), 4.54 (t, 2H), 8.44 (d, 1H), 8.64 (d,
1H). LRMS (Thermospray): m/z 502 (M+1)+.
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 approxi-
mately 150 mM NaCl. The column fractions comprising the highest
level of cGMP-hydrolytic activity (determined as below) were
pooled, aliquotted, and stored in liquid nitrogen until use. PDE
5.43. 2-{5-[(4-Ethyl-1-piperazinyl)sulfonyl]-2-(2-methoxy-
ethoxy)-3-pyridinyl}-7,8,9,10-tetrahydropyrido[20,10:5,1]-
pyrazolo[4,3-d]pyrimidin-4(3H)-one (27b)
activity was measured using
a scintillation proximity assay
(SPA)-based method as previously described. The effect of PDE
inhibitors was investigated by assaying a fixed amount of enzyme
in the presence of varying inhibitor concentrations and low sub-
strate (cGMP in a 3:1 ratio unlabeled to 3H-labeled at a concentra-
tion_1/3Km) such that IC50 = Ki. The final assay volume was made
up to 102 íL with assay buffer (20 mM Tris–HCl pH 7.4 at 30 °C,
5 mM MgCl2, 1 mg/mL bovine serum albumin). Reactions were ini-
tiated with substrate, incubated for 30–60 min at 30 °C to give
<30% substrate turnover, and terminated with 50 íL of yttrium sil-
icate 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). Radio-
activity units were converted to percent activity of an uninhibited
control (100%) and plotted against inhibitor concentration, and
inhibitor IC50 values were obtained using the ‘Fit Curve’ Microsoft
Excel extension.
The title compound was obtained as a white solid in 59% yield,
from 29a, by the method of 31a. Anal. (C23H31N7O5S;0.5H2O): C,
52.45; H, 6.12; N, 18.62. Found C, 52.32; H, 6.11; N, 18.54. 1H
NMR (CDCl3, 300 MHz) d: 1.00 (t, 3H), 2.00 (m, 2H), 2.18 (m, 2H),
2.40 (q, 2H), 2.56 (m, 4H), 3.12 (6H, m), 3.57 (s, 3H), 3.84 (t, 2H),
4.40 (t, 2H), 4.78 (t, 2H), 8.60 (d, 1H), 8.98 (d, 1H), 10.80 (s, 1H).
LRMS (Thermospray): m/z 518.1 (MH)+.
5.44. 2-{5-[(4-Ethyl-1-piperazinyl)sulfonyl]-2-(2-metho-
xyethoxy)-3-pyridinyl}-10-methyl-7,8,9,10-tetrahydro-
pyrido[20,10:5,1]pyrazolo[4,3-d]pyrimidin-4(3H)-one (27c)
The title compound was made by the method and the enantio-
mers were separated by chiral preparative HPLC Chiralpak IA, 50/
50 EtOH:MeOH. Anal. calcd for (C24H33N7O5S.0.5H2O): C, 53.32;
H, 6.34; N, 18.14. Found C, 53.23; H, 6.31; N, 18.03. 1H NMR (CDCl3)
d: 1.02 (t, 3H), 1.58 (d, 6H), 2.07 (m, 1H), 2.20 (m, 2H), 2.40 (q, 2H),
2.55 (m, 4H), 3.15 (m, 4H), 3.38 (m, 1H), 3.57 (s, 3H), 3.86 (t, 2H),
4.32 (m, 1H), 4.42 (m, 1H), 4.78 (t, 2H), 8.61 (d, 1H), 8.98 (d, 1H),
10.77 (s, 1H). LRMS (Thermospray): m/z: 532 (M+1)+.
References and notes
1. For recent reviews on The Pharmacological Therapy for the Treatment of
Erectile Dysfunction and other disease associated with PDE5 inhibition see: (a)
Maw, G. N. Annu. Rep. Med. Chem. 1999, 34, 71; (b) Cartledge, J.; Eardley, I. Curr.
Opin. CPNS Investig. Drugs 1999, 1, 240; (c) Konstantinos, G.; Petros, P. Curr.
Pharm. Design 2009, 15, 3540; (d) Kouvelas, D.; Goulas, A.; Papazisis, G.; Sardeli,
C.; Porzitaki, C. Curr. Pharm. Design 2009, 15, 3464.
2. Cole, S. Beaumont, K.C. in preparation.
3. Tamimi, N. A. M.; Mincik, I.; Haughie, S.; Lamb, J.; Crossland, A.; Ellis, P. Br. J.
Urol. Int. 2010, 106(5), 674.
5.45. 2-{5-[(4-Ethyl-1-piperazinyl)sulfonyl]-2-(2-
methoxyethoxy)-3-pyridinyl}-10-methyl-7,8,9,10-
tetrahydropyrido[20,10:5,1]pyrazolo[4,3-d]pyrimidin-4(3H)-one
(27d)
4. 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. W. Xenobiotica 2001, 31,
665.
5. (a) Allerton, C. M. N.; Barber, C. G.; Beaumont, K. C.; Brown, D. G.; Cole, S. M.;
Ellis, D.; Lane, C. A. L.; Maw, G. N.; Mount, N. M.; Rawson, D. J.; Robinson, C. M.;
Street, S. D. A.; Summerhill, N. W. J. Med. Chem. 2006, 49, 3581; (b) Bunnage, M.
E.; Mathias, J. P.; Wood, A.; Miller, D.; Street, S. D. A. Biorg. Med. Chem. Lett.
2008, 18, 6033.
6. Bunnage, M. E.; Mathias, J. P.; Street, S. D. A.; Wood, A. PCT Int. Appl. WO
9849166.
7. The regiochemistry of the N-ethylation was confirmed by NMR experiments.
Irradiation of either methylene afforded an NOE enhancement of the opposite
The title compound was made by the method and the enantio-
mers were separated by chiral preparative HPLC on Chiralpak IA,
50/50 EtOH:MeOH. Anal. calcd for (C24H33N7O5S.H2O): C, 52.44;
H, 6.42; N, 17.84. Found C, 52.51; H, 6.36; N, 17.93. 1H NMR (CDCl3)
d: 1.02 (t, 3H), 1.58 (d, 6H), 2.07 (m, 1H), 2.20 (m, 2H), 2.40 (q, 2H),
2.55 (m, 4H), 3.15 (m, 4H), 3.38 (m, 1H), 3.57 (s, 3H), 3.86 (t, 2H),
4.32 (m, 1H), 4.42 (m, 1H), 4.78 (t, 2H), 8.61 (d, 1H), 8.98 (d, 1H),
10.77 (s, 1H). LRMS (Thermospray): m/z: 532 (M+1)+.
ethyl methylene for 13a but not for 14a.
O
6. PDE5 and PDE6 enzyme inhibitor assays
N
H2N
O2N
N
H
Phosphodiesterase type 5 was prepared from human corpus
cavernosum tissue as previously described. Phosphodiesterase
type 6 (cone) was prepared from canine retina. Following dissec-
tion, 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 fil-
tered 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 ím filter and ap-
plied 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 Phar-
macia FPLC system (Amersham Pharmacia Biotech Ltd, Little Chal-
font, UK). The bound protein was washed with 5 column volumes
and eluted using a 0–300 mM NaCl linear gradient over 65 mL,
H
H
H
13a
8. Ranganathan, D.; Bamezai, S. Tetrahedron Letters 1983, 24, 1067.
9. Enantiomers were separated on Chiralpak IA, 50/50 EtOH/MeOH.
10. Ballard, S. A.; Gingell, C. J.; Tang, K.; Turner, L. A.; Price, M. E.; Naylor, A. M. J.
Urol. 1998, 159, 2164.
11. 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. Nature 2003, 425, 98.
12. Chan, O. H.; Stewart, B. H. Drug Discovery Today 1996, 1, 461.
13. (a) Artursson, P. J. Pharm. Sci 1990, 79, 476; (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.
Pharm. Sci. 1995, 12, 693; (c) Bailey, C. A.; Bryla, P.; Malick, A. W. Adv. Drug.
Delivery Rev. 1996, 22, 85; (d) Yee, S. Pharm. Res. 1997, 14, 763.
14. Papp (cm/s) is apparent permeability coefficient and is calculated using the
following equation:
Papp = (F ꢀ VD)/SA ꢀ MD)
where SA is the surface area for transport, VD is the donor volume and MD is the
donor amount at t = 0. Efflux ratio is the ratio of secretory flux to absorptive
flux.