3H-Quinazolin-4-ones as a new calcilytic template for the potential treatment of osteoporosis

Article abstract of DOI:10.1016/j.bmcl.2005.01.078

Structure-activity relationship studies, focused on identification of the active pharmacophore fragments in a single high-throughput screening calcilytic hit, resulted in the discovery of potent calcium receptor antagonists, substituted 3H-quinazolin-4-on

Full text of DOI:10.1016/j.bmcl.2005.01.078

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1557–1560
3H-Quinazolin-4-ones as a new calcilytic template for the
potential treatment of osteoporosis
Irina Shcherbakova,* Manuel F. Balandrin, John Fox, Anjan Ghatak,z
William L. Heaton and Rebecca L. Conklin
Drug Discovery, NPS Pharmaceuticals, Inc., 383 Colorow Drive, Salt Lake City, UT 84108, USA
Received 2 December 2004; revised 28 January 2005; accepted 31 January 2005
Abstract—Structure–activity relationship studies, focused on identification of the active pharmacophore fragments in a single high-
throughput screening calcilytic hit, resulted in the discovery of potent calcium receptor antagonists, substituted 3H-quinazolin-4-
ones.
Ó 2005 Elsevier Ltd. All rights reserved.
Daily injections of parathyroid hormone (PTH) that in-
duce transient increases in PTH levels stimulate the
growth of new bone and might be a new anabolic ther-
apy for osteoporosis.1 An alternative therapeutic ap-
proach might involve stimulation of the secretion of
endogenous PTH from the parathyroid glands that is
regulated by the calcium receptor (CaR).2,3 Compounds
that block CaR activity (calcilytics) stimulate transient
PTH release producing anabolic effects in bone and thus
are implicated as a viable treatment for osteoporosis.3,4
As demonstrated in animal studies, the first reported
small molecule calcilytic, NPS 2143 (Fig. 1) induced
the secretion of PTH and stimulated the growth of
new bone.4,5 Recently, a novel class of calcium receptor
antagonists, N1-arylsulfonyl-N2-(1-(1-naphthyl)ethyl)-
1,2-diamino-cyclohexanes have been discovered.6 The
ligand binding site model for the in vitro potent com-
pound, Calhex 231 has been proposed,7 although no in
vivo effect has been reported for this pharmacophore
type.
O
N
CN
OH
C
H
N
O
Cl
N
B
A
O
NPS 53574 (IC50 = 3.5 µM)
NPS 2143 (IC50 = 0.043 µM)
Figure 1.
4-one, NPS 53574 (Fig. 1) via high-throughput screen-
ing. The biological activities of substituted 3H-quinazo-
lin-4-ones are well documented; examples include
sedative–hypnotic (methaqualone),8 antitussive (chloro-
qualone),9 and analgesic (diproqualone).10 Herein, we
succinct structure–activity relationship
(SAR) study around NPS 53574 that led to the discov-
ery of novel potent CaR antagonists.11,12
describe
a
We first compared the effect on calcilytic activity of
replacing the 2-furan-2-yl function (ring A) in NPS
53574 with an unsubstituted and substituted phenyl
ring. Evaluation of the substitution in the annelated
benzene ring B and in the phenyl ring C of the 3-phen-
ethyl substituent of 3H-quinazolin-4-ones targeted iden-
tification of the active pharmacophoric regions. The
general method for synthesis of 2-aryl-substituted 3H-
quinazolin-4-ones is the modification of literature meth-
ods13,14 and shown in Scheme 1. The cyclization of
substituted anthranilic acids 1 with aroyl chlorides 2
was accomplished in pyridine at room temperature to
give benzoxazinones 3 in 60–90% yield. Heating
As part of an ongoing search for novel drug-like calcilyt-
ics with a good potential for optimization into potent,
selective, nontoxic compounds with acceptable pharma-
cokinetic properties, we have identified a structurally
new single hit, 3-phenethyl-2-furan-2-yl-3H-quinazolin-
Keywords: Calcium receptor antagonists; Calcilytics; Osteoporosis;
3H-Quinazolin-4-ones.
*
Corresponding author. Tel.: +1 801 583 4939; fax: +1 801 583
4961; e-mail: ishcherbakova@npsp.com
z Deceased.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.078

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I. Shcherbakova et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1557–1560
O
NH2
O
N
O
Cl
O
R3
N
O
R3
R2
OH
NH2
4
2
R1
R1
N
R1
pyridine
200 oC, 2-3 h
R4
5 a,b,d-k,m-t
0 oC to rt
R2
3 a,b,d-k,m-t
1
Scheme 1. Compounds 3, 5: R2 = R4 if R2 5 OAc or 2-OMe; R4 = OH if R2 = OAc or 2-OMe. For other substituents, see Tables 1 and 2.
compounds 3 with a 10-fold excess of the amines 4 pro-
duced 3H-quinazolin-4-ones 5 in good to excellent
yields, while the earlier method reported the formation
of the mixture of quinazolinones and the ring-opened
byproducts, bisamides.14
The 2-acetoxy group in the benzoxazinones 3 was
cleaved under the reaction conditions to form the phenol
moiety in quinazolinones 5, even if a reduced excess
of amines 4 (2-fold instead of 10-fold) was used as
exemplified in Scheme 2. Surprisingly, regioselective
demethylation was observed on interaction of 2-(meth-
oxy-phenyl)-substituted benzoxazinones 3d–f with phen-
ethylamine: whereas a methoxy group at the meta- or
para-position of the 2-phenyl ring remained unchanged,
the ortho-methoxy group was cleaved yielding the
hydroxy-substituted quinazolinone 5j (Scheme 4). A
similar regioselective demethylation of substituted
2-methoxybenzoic acids and their amides was recently
reported as the first and so far only example of nucleo-
philic O-dealkylation in the presence of aliphatic
amines.15 The use of 1.1 equiv of phenethylamine in
the reaction with benzoxazinone 3f resulted in a complex
mixture. The 2-(2-methoxy-phenyl)-substituted quinaz-
olinone 5f was obtained by methylation of the phenol
5j (Scheme 4).
During the course of the latter reaction, undesired nucleo-
philic displacement of the fluorine substituent at posi-
tion 7, 8, or 5 in benzoxazinones 3l,u,v led to the
amino-substituted quinazolinones 6l,u,v, respectively,
as the major products (Scheme 2), probably due to the
reaction conditions (elevated temperature and the ab-
sence of solvent). The application of microwave irradia-
tion resulted in the preparation of 7-fluoro-substituted
quinazolinone 5l, whereas 8- and 5-fluoro-substituted
benzoxazinones 3u,v still produced products of nucleo-
philic displacement 6u,v.
Similarly to the formation of the amino-substituted qui-
nazolinones 6l,u,v, fusion of 2-(2-fluoro-phenyl)-substi-
tuted benzoxazinone 3c and phenethylamine (1.1
equiv) at 200 °C resulted in the dominant nucleo-
philic displacement of the fluorine substituent with the
amino moiety. For preservation of the 2-(2-fluoro-phe-
nyl) fragment, synthesis of the intermediate bisamide 7
was carried out in pyridine at 120 °C followed by cycli-
zation to 2-(2-fluoro-phenyl)-3-phenethyl-3H-quinazo-
lin-4-one 5c on fusion (Scheme 3) similarly to the
previously reported procedure.13
The quinazolinone compounds 5a–t were evaluated in
vitro for their calcilytic activity. Calcilytic activity was
determined by a compoundÕs ability to block, in a con-
centration-dependent manner, increases in the concen-
tration of intracellular Ca2+ elicited by increases in
extracellular Ca2+ in HEK 293 4.0–7 cells stably express-
ing the human CaR. Increases in intracellular Ca2+
,
measured using fluo-3, a fluorescent calcium indicator
O
O
O
H
N
5
5
Ph
Ph
F
Ph
N
N
O
b
a
7
F
N
7
98%
N
40-45%
N
8
8
HO
HO
AcO
6 l,u,v
5l
3 l,u,v
Scheme 2. Reagents and conditions: (a) PhCH2CH2NH2 (2 equiv), 200 °C, 4 h; (b) PhCH2CH2NH2 (2 equiv), DMF, microwave irradiation, 240 °C,
10 min. Compounds 3l: 7-F; 3u: 8-F; 3v: 5-F; 6l: 7-NHCH2CH2Ph; 6u: 8-NHCH2CH2Ph; 6v: 5-NHCH2CH2Ph.
O
O
N
O
N
Ph
Ph
N
H
PhCH2CH2NH2 (1.1 equiv)
N
F
O
F
neat
200 oC, 48 h
NH
pyridine, ∆, 4 h
O
F
5c
3c
7
Scheme 3.

I. Shcherbakova et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1557–1560
1559
O
O
O
N
O
N
a
a
N
O
N
N
b
N
H
N
OMe
p-
OMe
p-
o-
O
MeO
m-
m-
5f
5j
5d,e
3d-f
Scheme 4. Reagents and conditions: (a) PhCH2CH2NH2 (2 equiv), 200 °C, 3 h; (b) MeI, K2CO3, THF, D, 24 h. Compounds 3, 5: d m-OMe; e p-OMe;
f o-OMe.
(Biotium) were elicited by increasing extracellular Ca2+
from 1.0 to 1.3 mM. Fluorescence signals were measured
Table 2. B and C ring SAR
O
C
as the peak height of the response and normalized to the
response elicited by extracellular Ca2+ in the absence of
test compound. All compounds were tested in duplicate
at eight concentrations with the highest concentration
being 30 lM. IC50 values of new compounds were nor-
malized to that of a standard (internal reference) com-
pound tested in parallel in each assay.
N
B
R3
R1
N
H
O
Compound 5
R1
R3
IC50 (lM)
k
l
8-Me
7-F
H
0.7
0.6
H
It was found that replacement of the 2-furan-2-yl frag-
ment A in NPS 53574 (Fig. 1) with the phenyl ring de-
creased but did not erase calcilytic activity (5a, Table
1). Table 1 summarizes the SAR with respect to substi-
tution in the 2-phenyl ring for quinazolinones 5a–j.
Introduction of the hydroxy substituents at the phenyl
ring showed a trend toward increased calcilytic activity
(5g,i), and the ortho-hydroxy-substituted compound 5j
demonstrated as much as a 50-fold potency increase
when compared to the parent compound 5a.
m
n
o
p
q
r
7-Cl
6-Cl
6-Me
H
3-F
H
1.6
0.3
3-F
3-Cl
H
0.52
0.8
5-Me
5-Me
6-F
0.25
0.25
0.21
0.19
3-F
H
s
t
6-F
3-F
Further optimization of the compound 5j via mono- and
di-substitution in the rings B and C improved potency
modestly and yielded several compounds with attractive
profiles (Table 2).
Thus, a single ortho-hydroxy substituent in the 2-phenyl
ring of 3H-quinazolin-4-ones is beneficial to the calci-
lytic activity. The effect of such a substitution might be
the result of an intramolecular hydrogen bond in a bio-
active conformation as illustrated for 2-(2-hydroxy-phe-
nyl)-3H-quinazolin-4-one 5j in (Scheme 4). The effect of
a single intramolecular hydrogen bond on increasing
permeability is known to be significant and can easily
amount to a factor of 10 or more.16 The contribution
of this effect could be favorable to the overall pharmaco-
logical profile of the optimized compounds.
In vivo, the quinazolinones 5 with IC50 values <0.5 lM
were very effective at transiently increasing plasma PTH
levels following bolus intravenous injection in rats.
Thus, compound 5j (3 or 10 lmol/kg) or vehicle was
administered by i.v. injection over about 15 s to normal
conscious male Sprague–Dawley rats with chronic
indwelling arterial and venous catheters. Arterial blood
samples were collected immediately before, and at 1, 5,
10, and 30 min after the start of the injection for
Table 1. 2-Phenyl ring SAR
3 µmol/kg
10 µmol/kg
O
C
N
B
8000
6000
4000
2000
0
N
R4
Compound 5
R4
IC50 (lM)
a
b
c
d
e
f
H
3-F
14
>30
>30
>30
>30
>30
2.8
2-F
3-OMe
4-OMe
2-OMe
3-OH
4-OH
2,5-Di-OH
2-OH
0
10
20
30
g
h
i
Time, min
>30
1.6
0.3
Figure 2. Plasma levels of compound 5j after bolus i.v. injection in
normal rats.
j

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I. Shcherbakova et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1557–1560
References and notes
Vehicle
3 µmol/kg
10 µmol/kg
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Time, min
Figure 3. Effect of bolus i.v. injection of compound 5j on plasma PTH
levels in normal rats.
measurement of the levels of PTH, Ca2+, and compound 5j
in plasma. PTH was measured using specific rat PTH(1-
84) ELISA (Immutopics, San Clemente, CA) and com-
pound 5j was quantified by an LC–MS/MS method.
Plasma levels of compound 5j were maximal at 1 min
after injection and declined rapidly during the next 10–
30 min (Fig. 2). Injection of compound 5j induced a
rapid, but transient dose-related increase in plasma PTH
levels that were maximal at 1 min after the injection.
Plasma PTH levels had returned to pre-dose levels by
10 min (Fig. 3). There were no changes in plasma Ca2+
levels, probably because PTH levels were elevated for
such a short time and/or because the study was only
of 30 min duration. For example, when NPS 2143
(Fig. 1) was infused intravenously, plasma Ca2+ levels
did not start to increase for 60 min.5
In summary, optimization of the substituted 3H-quinaz-
olin-4-ones resulted in the generation of novel potent
calcilytics, which stimulated PTH secretion in vivo.
The series offers a new perspective for the development
of clinically relevant calcium receptor antagonists.