Discovery and SAR of a novel series of Natriuretic Peptide Receptor-A (NPR-A) agonists

Article abstract of DOI:10.1016/j.bmcl.2017.09.028

Novel thienopyrimidine compounds 2 and 3 were discovered from high-throughput screening as Natriuretic Peptide Receptor A (NPR-A) agonists. Scaffold hopping of a thienopyrimidine ring to a quinazoline ring, introduction of the basic functional group and optimization of the substituent on the 6-position of the benzene ring of quinazoline led to improved agonistic activity. We discovered compound 48, which showed potent agonistic activity for NPR-A with an EC₅₀ value of 0.073 μM, indicating 350-fold potency compared to the hit compound 3.

Full text of DOI:10.1016/j.bmcl.2017.09.028

Accepted Manuscript
Discovery and SAR of a novel series of Natriuretic Peptide Receptor–A
(NPR–A) agonists
Takehiko Iwaki, Yuji Nakamura, Taisaku Tanaka, Yasuyuki Ogawa, Osamu
Iwamoto, Yoshihiko Okamura, Yumi Kawase, Mayumi Furuya, Yoshiaki
Oyama, Takahiro Nagayama
PII:
S0960-894X(17)30923-X
http://dx.doi.org/10.1016/j.bmcl.2017.09.028
BMCL 25292
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 August 2017
5 September 2017
13 September 2017
Please cite this article as: Iwaki, T., Nakamura, Y., Tanaka, T., Ogawa, Y., Iwamoto, O., Okamura, Y., Kawase, Y.,
Furuya, M., Oyama, Y., Nagayama, T., Discovery and SAR of a novel series of Natriuretic Peptide Receptor–A
(NPR–A) agonists, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.
2017.09.028
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Discovery and SAR of a novel series of Natriuretic Peptide Receptor–A (NPR–A)
agonists
Takehiko Iwakia∗, Yuji Nakamurab, Taisaku Tanakaa, Yasuyuki Ogawab, Osamu Iwamotob, Yoshihiko
Okamuraa, Yumi Kawaseb, Mayumi Furuyaa, Yoshiaki Oyamaa, Takahiro Nagayamab
aAsubio Pharma Co., Ltd, 6-4-3, Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan
bDaiichi Sankyo Co., Ltd, 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Novel thienopyrimidine compounds 2 and 3 were discovered from high-throughput screening as
Natriuretic Peptide Receptor A (NPR-A) agonists. Scaffold hopping of a thienopyrimidine ring
to a quinazoline ring, introduction of the basic functional group and optimization of the
substituent on the 6-position of the benzene ring of quinazoline led to improved agonistic
activity. We discovered compound 48, which showed potent agonistic activity for NPR-A with
an EC50 value of 0.073 µM, indicating 350-fold potency compared to the hit compound 3.
Revised
Accepted
Available online
Keywords:
Natriuretic Peptide Receptor A
Agonist
2017 Elsevier Ltd. All rights reserved.
Scaffold hopping
Thienopyrimidine derivatives
Quinazoline derivatives
Atrial natriuretic peptide (ANP) is a cardiovascular hormone
that consists of 28 amino acids. ANP is mainly secreted from the
cardiac atria and binds to the natriuretic peptide receptor-A
(NPR-A), which exerts diuresis vasodilation.1,2 Human ANP
(HANP®) is already being used clinically for the treatment of
acute heart failure in Japan.3,4 Recent studies have shown that
ANP has a potent inhibitory effect on cardiac fibrosis and
hypertrophy, and exhibits anti-inflammatory effects.4,5 These
results suggest that the non-peptidic NPR-A agonists that are
suitable for chronic setting might also be useful for the treatment
of cardiocirculatory diseases such as chronic heart failure,
chronic kidney failure and arteriosclerosis obliterans. The clinical
utility of ANP is limited to the acute setting because of its rapid
clearance from the body, as is often the case with other peptide
drugs.5
unsuitable lead compound for clinical candidate. Based on the
above consideration, we re-conducted high-throughput
screening campaign of the Daiichi Sankyo compound library to
a
It has been reported that ANP binds to the dimeric NPR-A and
causes a twist motion of the two extracellular domains, which
lead to the conformational changes in the intracellular guanylyl
cyclase domain and the activation of eyzymatic activity.6 It is a
big challenge to develop small molecules which mimic the action
of ANP. We have previously reported non-peptidic small
molecules with a novel triazine scaffold that showed NPR-A
agonistic effects like ANP. We succeeded in the discovery of the
potent NPR-A agonist
1 via a dimerization approach (Figure
1).7 Compound 1 has a high molecular weight (~700) and 20
nitrogen atoms in its structure, and these attributes make it an
———
Figure 1. Triazine compound 1 and hit compounds from high throughput
screening.
∗ Corresponding author. Tel.: +81-78-306-5307; fax: +81-78-306-5971; e-mail: iwaki.takehiko.v7@asubio.co.jp

Scheme 1. Reagents and conditions: (a) trichloroacetyl isocyanate, THF, r.t., 96%; (b) 7 N NH3/MeOH, r.t.; then 4 N NaOH, 100 °C, 80%; (c) phosphorus
oxychloride, N,N-diethylaniline, 110 °C, 93%; (d) Ar-CH2NH2, DIPEA, THF, r.t., 60–93%; (e) R1-NH2, H2O, microwave, 160 °C, 69–99%; (f) AcCl, DIPEA,
THF r.t., 91–94%; (g) 1-methyl-3-piperidinecarboxylic acid, HATU, DIPEA, CH2Cl2, r.t., 72%.
discover new small scaffolds using human NPR-A expressing
CHO cells.5 As a result, we identified the thienopyrimidine
derivatives 2 and 3 which enhanced the production of cyclic
guanosine monophosphate (cGMP) in the assay. These
compounds did not act on NRR-B, another natriuretic peptide
receptor subtype, expressing and mock CHO cells in a counter
assay at 20 µg/mL, and we considered compounds 2 and 3 to
selectively activate NPR-A (Figure 1). Since these compounds
showed only weak agonistic activity but had lower molecular
weights and a fewer number of nitrogen atoms than those of the
triazine compound 1, we decided to optimize their structures to
improve their agonistic activity.
the thienopyrimidine core were prepared, as shown in Scheme 1.
The urea 5 was prepared by reacting commercially available
methyl 3-amino-5-phenylthiophene-2-carboxylate
4
with
trichloroacetyl isocyanate. After deprotection of the
trichloroacetyl group with ammonia, cyclization under alkaline
conditions afforded the bicyclic core 6, which was then
chlorinated with phosphorus oxychloride to give the intermediate
7. Aminomethylpyridines and aminobenzylamines were
introduced to the 4-position of 7, and subsequent introduction of
alkylamines to the 2-position assisted microwave irradiation
yielded compounds 13b–17b. Acetylation of the compounds 16b
and 17b afforded 18 and 19, respectively, and the condensation
of 17b with 1-methyl-3-piperidinecarboxylic acid8 led to the
formation of compound 20.
In order to elucidate the structure-activity relationship,
compounds that could be modified at the 2- and 4-positions of
Table 1. EC50 values of thienopyrimidine NPR-A agonists. The concentration of each compound required for half-maximum accumulation of cGMP (EC50) was
determined from an analysis of the plots of cGMP contents versus log concentration of the compound in human NPR-A expressing CHO cells. Maximum cGMP
concentration induced by human ANP was regarded as 100% (EC50 = 0.1 nM). a Racemate.

Scheme 2. Reagents and conditions: (a) trichloroacetyl isocyanate, THF, r.t., 80–89%; (b) 7 N NH3/MeOH, r.t.; then reflux, 88–97%; (c) phosphorus
oxychloride, N,N-diethylaniline, reflux, 75–77%; (d) aminobenzylamines or trans-1,4-cyclohexanediamine, DIPEA, THF, r.t., 63–84%; (e) isopropylamine,
H2O, microwave, 160°C, 68–98%; (f) 1-methyl-3-piperidinecarboxylic acid or nicotinic acid, HATU, DIPEA, CH2Cl2, r.t., 64–99%; (g) phenylboronic acids,
Pd(PPh3)4, K3PO4, 1,4-dioxane-H2O (2:1), microwave, 130 °C, 37–98%; (h) H2, 10% Pd/C, EtOH, r.t., 86%.
We evaluated the NPR-A agonistic activity9 of compounds
13b–15b and 18–20 using human NPR-A expressing CHO cells
(Table 1). Our initial efforts focused on varying the size of the
alkylamino group at the 2-position of compound 2. It was found
that the agonistic activity was slightly improved as the size of the
alkylamino group increased (14a and 14b), although compound
14c with the larger isobutylamino group showed decreased
agonistic activity. Therefore, the substituent at the 2-position was
fixed as isopropylamino group for further study.
Compounds 13b and 15b with different positions of the
nitrogen atom on the pyridine ring did not improve the agonistic
activity. However, changing the position of the acetylamino
group of compound 3 decreased NPR-A agonistic activity (18
and 19). Compound 20 was designed with the basic functional
group 1-methyl-3-piperidine on the acetylamino group of
compound 19 because the negatively charged amino acids
typically cluster at the ANP binding site of NPR-A receptor10. In
fact, compound 20 showed improved agonistic activity with an
EC50 value of 5.0 µM. Therefore, it seems that the introduction of
a basic functional group at this position is effective in improving
agonistic activity.
Subsequently, we designed quinazoline derivatives based on
the reports that a benzene ring could be treated as an equivalent
of
a
thiophene ring11. The synthesis of the quinazoline
compounds shown in Table 2 and Table 3 is described in Scheme
2. Intermediates 27 and 28 were prepared from commercially
available 21 and 22 via a synthetic method similar to that used to
obtain 7. Compounds 29–33 were prepared by the introduction of
aminobenzylamines and trans-1,4-cyclohexanediamine at the 4-
Table 2. NPR-A agonist EC50 values of quinazoline compounds. Effect of
amide groups on benzylamine, conversion of linker to trans-1,4-
cyclohexanediamine and importance of 7-positioned phenyl group. a
Racemate, except for compound 43.

position of intermediates 27 and 28. The introduction of the
isopropylamino group at the 2-position of compounds 29–33 with
microwave irradiation followed by the condensation of 1-methyl-
3-piperidinecarboxylic acid or nicotinic acid afforded compounds
34–38 and 40, which were further coupled with phenylboronic
acids to give compounds 41–51. Compound 38 was
dehalogenated using Pd/C and H2 to afford compound 39.
activity with an EC50 value of 0.073 µM (Table 3). Fluorine was
more suitable as the substituent at the meta position of the phenyl
group because compounds 50 and 51 with chloro and methoxy
substituents, respectively, did not show any improvement in the
agonistic activity as compared to compound 45.
In conclusion, novel thienopyrimidine compounds 2 and 3
were found to show weak agonistic activity via high throughput
screening. The scaffold hopping of thienopyrimidine to
quinazoline was examined, and it was revealed that the
quinazoline derivatives showed more potent agonistic activity
than the thienopyrimidine derivatives. From structural
optimization of the quinazoline compounds, the basic moiety and
the phenyl group at 6-position on the quinazoline ring were found
to be important for agonistic activity. Finally, we discovered the
novel quinazoline compound 48 which showed potent agonistic
activity toward NPR-A with an EC50 value of 0.073 µM. This
value was 350-fold higher than that of the hit compound 3.
Compared to triazine 1, compound 48 has a smaller molecular
weight (518 g/mol) and considerably fewer nitrogen atoms. This
highly active compound 48 is expected to be useful tool for
studying cardiovascular diseases such as cardiac fibrosis,
hypertrophy and chronic heart failure.
The NPR-A agonistic activities of the quinazoline derivatives
are listed in Table 2. Overall, quinazoline derivatives showed
more potent agonistic activity than that of the thienopyrimidine
derivatives. The agonistic activity of 41 was 3.5-fold higher than
that of the thienopyrimidine compound 20 with an EC50 value of
1.4 µM. Compound 42 which had the piperidine amide group at
the 3-position of benzylamine showed further improved agonistic
activity with an EC50 value of 0.72 µM in contrast to compounds
41 and 44. It was evident from the loss of the activity in
compound 43 that the basic 1-methyl-3-piperidinylamide moiety
was important for agonistic activity. Compound 45 with a trans-
1,4-cyclohexanediamino linker for adjusting the position of the
1-methyl-3-piperidinylamide moiety showed improved agonistic
activity with an EC50 value of 0.17 µM. The importance of the
phenyl group on the quinazoline ring for agonistic activity was
indicated by the reduction of the activity in compound 46 and the
loss of the activity in case of compound 39.
References and notes
The optimization of the phenyl group at 6-position on the
quinazoline ring was performed in order to further improve the
1. Nakao, K.; Ogawa, Y.; Suga, S.; Imura, H. J. Hypertens. 1992, 10,
907-912.
agonistic activity. Compounds 47 and 49 with
a fluoro
2. Levin, E. R.; D. G. Gardner.; W. K. Samson. N. Engl. J. Med.
1998, 339-321.
substituent at the ortho and para positions did not show any
remarkable change in agonistic activity, but compound 48 with a
fluoro substituent at the meta position had an enhanced agonistic
3. Suwa, M.; Seino, Y.; Nomachi, Y.; Matsuki, S.; Funahashi, K.
Circ. J. 2005, 69, 283-290.
4. Kasama, S.; Furuya, M.; Toyama, T.; Ichikawa, S.; Kurabayashi,
M. Eur. Heart J. 2008, 29, 1485–1494.
5. Kitano, K.; Fukuda, Y.; Nagahira, K.; Nasu, T.; Izumi, R.;
Kawashima, K.; Nakanishi, T. Immunology Letters. 1995, 41, 215-
222.
6. Misono, K. S. Ogawa, H. Qiu, Y. Ogata, C. M. Peptides, 2005, 26,
957-968.
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Sugiyama, M.; Yamaki, A.; Furuya, M. Bioorg. Med. Chem. 2017,
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Brusco, D. Chuchani, G. International Journal of Chemical
Kinetics. 2006, 38, 106 –114.
9. Evaluation of human NPR-A agonist activity; Human NPR-A
agonist activities of the compounds were evaluated in CHO cells
expressing human NPR-A5. The NPR-A expressing CHO cells
were seeded in 96-well plate at 1 × 104 cells/well and cultured for
1 day under 5% carbon dioxide gas at 37°C. The cells were
washed with Hanks buffer containing 20 mM HEPES and 0.1%
bovine serum albumin, pH 7.4 (Hanks-HEPES-BSA buffer) two
times, and incubated with human ANP or the compounds in the
presence of 0.5 mM 1-methyl-3-isobutylxanthine (Sigma-Aldrich
Japan, Co. LLC, Tokyo, Japan) for 30 min at 37°C. The
incubation was terminated by aspirating the medium and washed
once by Hanks-HEPES-BSA buffer. The cells were disrupted by
the addition of 0.1N HCl, and intracellular cGMP was released to
the solution. Cellular cGMP concentration was determined using a
cGMP radioimmunoassay kit (Yamasa Co., Chiba, Japan).
10. van den Akker, F. Zhang, X. Miyagi, M. Huo, X. Misono, K. S.
Yee, V. C. Nature, 2000, 406, 101-104.
Table 3. NPR-A agonist EC50 values of quinazoline compounds.
Optimization of phenyl group at 7-position on the quinazoline ring for
improvement of agonistic activity. a Racemate.
11. (a) El-Ansary, A. K.; Kamal, A. M.; Al-Ghorafi, M. A. Eur. J.
Med. Chem. 2014, 86, 202-210. (b) El-Ansary, A. K.; Kamal, A.
M.; Al-Ghorafi, M. Chem. Pharm. Bull. 2016, 64, 1172–1180.

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Discovery and SAR of a novel series of
Natriuretic Peptide Receptor-A (NPR-A)
agonists
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Takehiko Iwakia∗, Yuji Nakamurab, Taisaku Tanakaa, Yasuyuki Ogawab, Osamu Iwamotob, Yoshihiko
Okamuraa, Yumi Kawaseb, Yoshiaki Oyamaa, Mayumi Furuyaa and Takahiro Nagayamab
aAsubio Pharma Co., Ltd, 6-4-3, Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan
bDaiichi Sankyo Co., Ltd, 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
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