8-(3-Chloro-4-methoxybenzyl)-8H-pyrido[2,3-d]pyrimidin-7-one derivatives as potent and selective phosphodiesterase 5 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.02.041

A novel series of highly selective phosphodiesterase 5 (PDE5) inhibitors was found. 8H-Pyrido[2,3-d]pyrimidin-7-one derivatives bearing an (S)-2-(hydroxymethyl)pyrrolidin-1-yl group at the 2-position and a 3-chloro-4-methoxybenzyl group at the 8-position

Full text of DOI:10.1016/j.bmcl.2015.02.041

Bioorganic & Medicinal Chemistry Letters 25 (2015) 1431–1435
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
8-(3-Chloro-4-methoxybenzyl)-8H-pyrido[2,3-d]pyrimidin-7-one
derivatives as potent and selective phosphodiesterase 5 inhibitors
Toshiaki Sakamoto a, Yuichi Koga a, Masataka Hikota a, Kenji Matsuki a, Hideki Mochida b, Kohei Kikkawa b,
Kotomi Fujishige c, Jun Kotera c, Kenji Omori c,d, Hiroshi Morimoto a, , Koichiro Yamada a
⇑
a Medicinal Chemistry Research Laboratories II, Mitsubishi Tanabe Pharma Corporation, 2-2-50 Kawagishi, Toda, Saitama 335-8505, Japan
b Pharmacology Research Laboratories II, Mitsubishi Tanabe Pharma Corporation, 2-2-50 Kawagishi, Toda, Saitama 335-8505, Japan
c Advanced Medical Research Laboratories, Mitsubishi Tanabe Pharma Corporation, 2-2-50 Kawagishi, Toda, Saitama 335-8505, Japan
d Industry and Academia Cooperation Research Project, Laboratory of Target and Drug Discovery, Graduate School of Pharmaceutical Sciences, Nagoya University, Furo-cho,
Chikusa-ku, Nagoya 464-8601, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of highly selective phosphodiesterase 5 (PDE5) inhibitors was found. 8H-Pyrido[2,3-
d]pyrimidin-7-one derivatives bearing an (S)-2-(hydroxymethyl)pyrrolidin-1-yl group at the 2-position
and a 3-chloro-4-methoxybenzyl group at the 8-position exhibited potent PDE5 inhibitory activities
and high PDE5 selectivity over PDE6. Among the synthesized compounds, the 5-methyl analogue (5b)
showed the most potent relaxant effect on isolated rabbit corpus cavernosum with an EC30 value of
0.85 nM.
Received 16 January 2015
Revised 10 February 2015
Accepted 16 February 2015
Available online 21 February 2015
Keywords:
PDE5
Ó 2015 Elsevier Ltd. All rights reserved.
PDE6
Erectile dysfunction
Phosphodiesterase 5 (PDE5) is a cyclic guanosine monophos-
phate (cGMP)-specific hydrolytic enzyme, and the inhibitors of
PDE5 are used for the treatment of erectile dysfunction (ED)1,2
and pulmonary arterial hypertension (PAH).3 The first approved
orally active PDE5 inhibitor, sildenafil showed high efficacy, but
visual side effects such as cyanopsia and visual disturbance were
reported in some cases.4,5 These adverse effects are indicated to
be attributed to inhibition of PDE6.6 After the launch of sildenafil,
PDE5-selective inhibitors having various frameworks, such as
PDE6 (PDE6/PDE5 = 5700), but its relaxant effect on isolated rabbit
corpus cavernosum was insufficient (EC30 >100 nM) owing to its
high lipophilicity (cLogP = 4.01). Aiming to potentiate the relaxant
effect, further synthetic study was carried out using 2a as a lead
compound. First, the trimethoxybenzene moiety was modified to
reduce the lipophilicity (compounds 4). As a next step, the
substituents at the 4- and 5-positions of the pyrimidine ring were
bound to potentiate PDE5 inhibitory activity (compounds 5).
These efforts led to the finding of a novel chemical series of highly
selective PDE5 inhibitors which exhibit potent relaxant effect on
isolated rabbit corpus cavernosum. In this Letter, we report the
detail of the synthesis and biological activities of novel 8H-pyri-
do[2,3-d]pyrimidin-7-one derivatives 5.
In the exploration of the optimal substituent at the 2-position of
the pyrimidine derivatives 2, we found that incorporation of polar
substituents to reduce the lipophilicity of the compounds led to
an improvement of the relaxant effect.15 Therefore, we focused on
modification of the 3,4,5-trimethoxybenzene moiety at the
5-position, to which high lipophilicity of 2a (cLogP = 4.01) is
attributed, and pyrimidine-5-carbaldehyde 4a (cLogP = 2.78) and
5-acetylpyrimidine 4b (cLogP = 3.25) were designed by deletion
of the 3,4,5-trimethoxyphenyl group. In addition, 4c
(cLogP = 3.79), 4e (cLogP = 3.07), and 4f (cLogP = 3.48) were
designed by replacement of the 3,4,5-trimethoxybenzene with N-
containing heterocycles.
tetrahydro-b-carboline,7,8
phthaladine,9
quinazoline,10
quinolone,11 pyrazolopyrimidine,12 pyridopyrazinone,13 and
pyrimidin-4(3H)-one14 have been reported to date.
In previous Letters, we reported findings of 5-(3,4,5-trimethoxy-
benzoyl)-pyrimidine derivatives 2 as a novel chemical class of high-
ly PDE5-selective inhibitors by a scaffold hopping strategy using
isoquinolin-1-one derivative T-1032 (1) as a lead compound.15
Furthermore, transformation to pyrimidine-5-carboxamide deriva-
tives and subsequent optimization of the substituents led to the
discovery of avanafil (3), which has been approved by the FDA for
the treatment of ED (Fig. 1).16 In the course of this study, 5-ben-
zoylpyrimidine derivative 2a was found to show a potent PDE5
inhibitory activity (IC50 = 0.21 nM) and a high PDE5 selectivity over
⇑
Corresponding author. Tel.: +81 045 963 7164; fax: +81 045 963 7165.
E-mail address: morimoto.hiroshi@md.mt-pharma.co.jp (H. Morimoto).
http://dx.doi.org/10.1016/j.bmcl.2015.02.041
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

1432
T. Sakamoto et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1431–1435
OMe
Cl
OH
N
N
NH
N
O
NH2
HN
O
R
NH
O
Y
N
N
N
N
N
N
O
CO2Me
OMe
3
avanafil
MeO
MeO
OMe
OMe
Cl
OMe
OMe
OH
N
1
2
T-1032
N
NH
N
O
R5
acyl(formyl)pyrimidine
OMe
Cl
4
OH
N
N
NH
OMe
2a
N
O
IC50 = 0.21 nM
OH
N
Cl
Selectivity
N
N
O
PDE6 / PDE5 = 5700
EC30 > 100 nM
cLogP = 4.01
MeO
OMe
N
OMe
R5
5 pyridopyrimidinone
Figure 1. Structures of pyrimidine derivatives (2, 3, 4) and pyridopyrimidinone derivatives 5.
The synthesis of the pyrimidine derivatives (4a–c, 4e, 4f) was
illustrated in Scheme 1. 4-Aminopyrimidine derivative 7 was
obtained by condensation of commercially available 6 with 3-
chloro-4-methoxybenzylamine. Reduction of the ester group in 7
with LiAlH4 and subsequent oxidation of the resulting alcohol with
MnO2 gave pyrimidine-5-carbaldehyde 8. The 2-position of the
pyrimidine ring was substituted with (S)-prolinol after oxidation
of the methylsulfanyl group with mCPBA to give 4a. 5-
Acylpyrimidine derivatives 4b–d were obtained by addition of
the corresponding R5-metal reagents to the formyl group of 4a
and the following oxidation with MnO2. The 1-methylimidazol-2-
yl analogue 4e was synthesized via 9 which was prepared from 8
in the same manner as that described above. 4f was obtained by
1,4-addition of morpholine to 5-acryloylpyrimidine 4d.
PDE5 inhibitory activities of the synthesized compounds were
evaluated using the enzyme isolated from canine lung.17 As shown
in Table 1, the pyrimidine-5-carbaldehyde derivative 4a showed a
moderate PDE5 inhibitory activity (IC50 = 33 nM), and the modifi-
cation of the formyl group into an acetyl group led to the improve-
ment of the potency (4b, IC50 = 5.2 nM). Although the N-containing
heterocycles were tolerated on the acyl groups, all of the synthe-
sized 5-position variants were more than ten times less potent
than 2a.
d]pyrimidin-7-one derivatives were designed and synthesized as
described in Scheme 2.
Pyrimidine-5-carbaldehyde
8 was converted into a,b-un-
saturated ester 10 by Horner–Wadsworth–Emmons reaction, and
10 was cyclized in the presence of NaH to give pyridopyrimidinone
derivative 11a. Alternatively, 11e was prepared from 5-
acetylpyrimidine 9 by treatment with trimethylphosphonoacetate
and excess NaH in toluene at 100 °C.20 The 2-methylsulfanyl
groups of these compounds were substituted with (S)-prolinol in
the same manner as that described above to give 5a and 5e. 1,4-
Addition of a cuprate, which was prepared from MeLi and CuCN,
to 10 and concomitant cyclization gave 12. After substitution of
the 2-position, resulting 13 was treated with DDQ to give 5b.
Compound 5c was obtained from 4c in 17% yield, accompanied
by recovery of 48% of starting material. In the synthesis of 5f,
methyl trimethylsilylacetate was used instead of the Horner–
Wadsworth–Emmons reagent,21 since 5f was not obtained by the
same procedure as used for preparation of 5c.
PDE5 inhibitory activities of these compounds were evaluated,
and the light-activated bovine retina PDE6 was used for evaluation
of PDE6 inhibitory activity. As summarized in Table 2, pyridopy-
rimidinone derivatives (5a–c, 5e, 5f) showed high potency, which
were superior to the corresponding cyclization precursors, pyrim-
idine derivatives (4a–c, 4e, 4f), respectively. These results may be
attributed to the restriction of the molecule into a bioactive confor-
mation. Among the synthesized compounds, the 5-methyl ana-
logue (5b) showed the most potent PDE5 inhibitory activity
(IC50 = 0.86 nM) and the highest selectivity over PDE6 (PDE6/
PDE5 = 2300).
Aiming to improve their PDE5 inhibitory activity, we planned
the construction of a bicyclic framework by cyclization between
the substituents at the 4- and 5-positions of the pyrimidine ring.
Ring closure modifications reduce the flexibility of the molecules
and sometimes achieve dramatic changes in potency, selectivity
permeability, and so on.18,19 Based on this idea, 8H-pyrido[2,3-

T. Sakamoto et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1431–1435
1433
OMe
Cl
OMe
Cl
MeS
N
Cl
MeS
N
NH
MeS
N
NH
d
a
b, c
N
N
N
CO2Et
CO2Et
CHO
6
7
8
OMe
Cl
OMe
Cl
4b (R5 = Me)
4c (R5 = pyridin-2-yl)
4d (R5 = vinyl)
OH
OH
N
N
NH
N
N
NH
e
N
N
O
CHO
R5
4a
4b-d
OMe
Cl
OMe
OH
Cl
MeS
N
NH
N
N
NH
f
g
8
N
O
N
N
O
Me
Me
N
N
N
9
4e
OMe
Cl
OH
N
N
NH
h
4d
N
O
O
N
4f
Scheme 1. Reagents and conditions: (a) 3-chloro-4-methoxybenzylamine, Et3N, THF, rt (quant); (b) LiAlH4, THF, rt (83%); (c) MnO2, CH2Cl2, rt (91%); (d) (i) mCPBA, CHCl3,
0 °C, (ii) (S)-prolinol, Et3N, CHCl3, 0 °C (83%); (e) (i) MeLi, Et2O, THF, À78 °C or n-BuLi, 2-bromopyridine, Et2O, THF, À78 °C or vinylmagnesium bromide, THF, 0 °C (ii) MnO2,
CHCl3, rt (4b 60%, 4c 57%, 4d 43%, 2 steps); (f) (i) 1-methylimidazole, n-BuLi, THF, À78 °C, (ii) MnO2, CHCl3, rt (61%, 2 steps); (g) (i) mCPBA, CHCl3, 0 °C, (ii) (S)-prolinol, Et3N,
CHCl3, rt (86%, 2 steps); (h) morpholine, EtOH, rt (98%).
The relaxant effects on isolated rabbit corpus cavernosum were
evaluated for selected compounds, and the results were summa-
rized in Table 3.22 5a (cLogP = 1.91), 5b (cLogP = 2.41), and 5f
(cLogP = 2.03), which were less lipophilic compared to 2a, showed
improved relaxant effects despite of the slightly reduced PDE5
inhibitory activity. Among them, 5b showed the most potent relax-
ant effect on isolated rabbit corpus cavernosum with an EC30 value
of 0.85 nM. These values were superior to those of sildenafil
(EC30 = 8.7 nM, PDE6/PDE5 = 500).
In conclusion, we found 8H-pyrido[2,3-d]pyrimidin-7-one
derivatives as a novel series of highly selective PDE5 inhibitors,
which were designed by the modification to reduce the lipophili-
city and subsequent cyclization to constrain the conformation of
the pyrimidine derivatives. A series of pyridopyrimidinone deriva-
tives showed superior potency in comparison to the corresponding
pyrimidine derivatives, respectively; therefore, a methyl group and
basic functionalities were tolerated at the 5-position of the pyri-
dopyrimidinone ring with potent PDE5 inhibitory activities. By
reducing lipophilicity while maintaining potent PDE5 inhibitory
activity, pyridopyrimidinone derivatives showed improved relax-
ant effects compared to that of 2a. Among the synthesized com-
pounds, 5b bearing a methyl group at the 5-position of the
pyridopyrimidinone ring showed the most potent relaxant effect
on isolated rabbit corpus cavernosum (EC30 = 0.85 nM) and the
highest PDE5 selectivity over PDE6 (PDE6/PDE5 = 2300). These
findings encouraged us to carry out further optimization of 5b.
Table 1
PDE5 inhibitory activity of pyrimidine derivatives
OMe
Cl
OH
N
N
NH
N
O
R5
4a-c, e, f
–R5
Compound
IC50 (nM)
a
4a
4b
33
H
5.2
Me
N
4c
4e
4f
12
12
2.9
Me
N
N
N
O
a
IC50 values of these compounds were calculated by an equation of the first
degree using two data; just above and below 50% inhibition. Some of the IC50 values
were calculated using PrismÒ, version 3.0.

1434
T. Sakamoto et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1431–1435
c (R5 = 1-methylimidazol-2-yl)
OMe
OMe
Cl
OMe
Cl
Cl
MeS
N
NH
MeS
N
NH
MeS
N
N
O
a (R5 = H)
b
N
O
N
N
CO2Me
R5
R5
d
8, 9
10
11a, e
e
OMe
OMe
Cl
OMe
Cl
OH
OH
N
Cl
f
MeS
N
N
N
O
N
N
N
O
N
N
O
g
N
N
Me
Me
R5
12
13
5a, b, e
OMe
OMe
Cl
OH
OH
N
Cl
h
(R5 = H)
N
N
NH
N
N
O
8, 11a, 5a
5b
4c, 5c
9, 11e, 5e
4f, 5f
(R5 = Me)
N
O
N
5
(R = pyridin-2-yl)
R5
R5
(R5 = 1-methylimidazol-2-yl)
5
(R = 2-morpholin-4-yl-ethyl)
4c, f
5c, f
Scheme 2. Reagents and conditions: (a) (MeO)2P(O)CH2CO2Me, NaH, THF, rt (59%); (b) NaH, MeOH, rt (50%); (c) (MeO)2P(O)CH2CO2Me, NaH, toluene 100 °C (13%); (d) (i)
mCPBA, CHCl3, 0 °C, (ii) (S)-prolinol, Et3N, CHCl3, 0 °C (5a 77%, 5e 96%, 2 steps); (e) MeLi, CuCN, EtO2, THF, 0 °C, (67%); (f) (i) mCPBA, CHCl3, 0 °C, (ii) (S)-prolinol, Et3N, CHCl3,
60 °C (75%, 2 steps); (g) DDQ, dioxane, 60 °C (30%).; (h) (MeO)2P(O)CH2CO2Me, NaH, toluene, 100 °C (5c 17%) or TMSCH2CO2Me, dicyclohexylamine, n-BuLi, THF, rt (5f 8%).
Table 2
Table 3
PDE5 inhibitory activity and selectivity against PDE6 of pyridopyrimidinone deriva-
tives
Relaxant effect on isolated rabbit corpus cavernosum
a
Compound
Relaxant effect EC30 (nM)
OMe
5a
2.2
5b
5f
0.85
8.6
OH
Cl
N
N
N
O
Sildenafil
8.7
a
EC30 values were determined from the logarithmic concentration–inhibition
curve. The value is given as the average of at least two experiments, where the
variation from the mean value is 30% or less.
N
R5
5a-c, e, f
–R5
Compound
IC50 (nM)
Selectivity (PDE6/5)
a
References and notes
5a
5b
3.3
850
H
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Me
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N
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5f
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N
N
0.98
1600
O
a
IC50 values of these compounds were calculated by an equation of the first
degree using two data; just above and below 50% inhibition. Some of IC50 values
were calculated using PrismÒ, version 3.0.

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20. In this reaction, a,b-unsaturated ester was obtained in 9% yield, and 66% of
starting material 9 was recovered.
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22. The initial resting isomeric tension of each tissue strip was adjusted to 1.5 g by
gradual incremental stretching in 10 mL of organ bath chambers containing
physiological salt solution (PSS) at 37 0.5 °C, continuously aerated with 95%
O2 and 5% CO2. The contractile response to high-KCl (120 mM) PSS was
checked twice. Phenylephrine (PE) (5 Â 10À6 M) was added into each organ
bath in order to obtain a tonic contraction. After the PE contractile response
was stabilized, test compound (10À10–10À6 M) or vehicle was added to the
preparation at an interval of 30 min. Papaverine hydrochloride was added into
each organ bath chamber (final concentration, 10À4 M) to confirm the maximal
relaxation of the tissue strips at the end of experiment. The composition of PSS
was as follows (mM): NaCl 118, KCl 4.7, MgSO4 1.2, CaCl2 1.5, KH2PO4 1.2,
NaHCO3 25.0, dextrose 11.0, EDTA-2Na 0.023 (pH 7.3 or 7.4).
17. PDE assay was done by the radiolabeled nucleotide method. PDE enzyme
(100 ll) in 50 mM Tris–HCl (pH 8.0) was added to 200 ll assay buffer
composed of 50 mM Tris–HCl (pH 8.0), 12.5 mM MgCl2, 10 mM 2-
mercaptoethanol and 0.825 mg/ml bovine serum albumin. The enzyme
reaction was started by adding 200 ll substrate solution containing
[3H]cGMP plus unlabeled cGMP or in 50 mM Tris–HCl (pH 8.0). Reaction
mixtures were incubated at 37 °C for 30 min and then boiled for 1.5 min.
Subsequently 100 ll of 1 mg/ml solution of Crotalus atrox snake venom was