Antagonists of the calcium receptor. 2. Amino alcohol-based parathyroid hormone secretagogues

Article abstract of DOI:10.1021/jm900563e

When administered as a single agent to rats, the previously reported calcium receptor antagonist 3 elicited a sustained elevation of plasma PTH resulting in no increase in overall bone mineral density. The lack of a bone building effect for analogue 3 was

Full text of DOI:10.1021/jm900563e

J. Med. Chem. 2009, 52, 6599–6605 6599
DOI: 10.1021/jm900563e
Antagonists of the Calcium Receptor. 2. Amino Alcohol-Based Parathyroid
Hormone Secretagogues
Robert W. Marquis,*,† Amparo M. Lago,† James F. Callahan,† Attiq Rahman,† Xiaoyang Dong,† George B. Stroup,‡
Sandra Hoffman,‡ Maxine Gowen,‡ Eric G. DelMar,^ Bradford C. Van Wagenen,^ Sarah Logan,^ Scott Shimizu,^
John Fox,^ Edward F. Nemeth,^ Theresa Roethke, Brian R. Smith, Keith W. Ward, and Pradip Bhatnagar†
†Departments of Medicinal Chemistry and ‡Bone and Cartilage Biology, Drug Metabolism and Pharmacokinetics,
GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, and
^NPS Pharmaceuticals, 550 Hills Drive, Bedminster, New Jersey 07921
Received May 6, 2009
When administered as a single agent to rats, the previously reported calcium receptor antagonist 3 elicited a
sustained elevation of plasma PTH resulting in no increase in overall bone mineral density. The lack of a
bone building effect for analogue 3 was attributed to the large volume of distribution (Vdss(rat)=11 L/kg),
producing a protracted plasma PTH profile. Incorporation of a carboxylic acid functionality into the
amino alcohol template led to the identification of 12 with a lower volume of distribution (Vdss(12) =
1.18 L/kg) and a shorter half-life. The zwitterionic nature of antagonist 12 necessitated the utility of an
ester prodrug approach to increase overall permeability. Antagonist 12 elicited a rapid and transient
increase in circulating levels of PTH following oral dosing of the ester prodrug 11 in the dog. The
magnitude and duration of the increases in plasma levels of PTH would be expected to stimulate new bone
formation.
Introduction
An alternative approach to the development of an orally
active bone forming agent, based upon the clinically validated
mechanism of subcutaneously administered osteogenic PTH
peptides, would be to identify an agent that would elicit the
secretion of endogenously stored parathyroid hormone from
the parathyroid glands. Here, the secretion of parathyroid
hormone is under the control of the calcium receptor (CaR).3
This family C G-protein-coupled receptor is capable of
sensing minute fluctuations in circulating levels of serum
calcium such that a drop in the concentration of calcium levels
(hypocalcaemia) elicits the release of PTH from the parathy-
roid glands while an increase in calcium levels (hypercalcimia)
suppresses the release of this hormone. The effects of hyper-
calcemia can be mimicked by calcimimetic compounds that
inhibit the secretion of PTH. One such compound is cinacalcet
(1, Figure 1),4 an allosteric activator of the CaR that is used to
treathyperparathyroidism. Calcimimetic compoundsvalidate
the view that circulating levels of PTH can be pharmacologi-
cally controlled by a small molecule acting at the parathyroid
gland CaR. On the other hand, small molecule antagonists of
the CaR, known as calcilytics, serve to mimic a hypocalcemic
state and elicit the secretion of PTH.5 If the profile of PTH
secretion following antagonism of the CaR is rapid and
transient then this should promote the formation of new
bone. If however the profile of PTH secretion following
antagonism of the receptor is protracted, then this will have
an overall catabolic effect and lead ultimately to an undesi-
rable excess resorption of bone. As such, the design of a CaR
antagonist capable of promoting overall bone growth is
predicated upon this compound’s ability to briefly antagonize
the receptor, thereby eliciting a transient release of endogen-
ous PTH.6
Osteoporosis is a disease afflicting more than 10 million
women and men in the U.S. alone.1 The overall weakening of
bone that results in osteoporosis is caused by a shift in the
equilibrium of bone remodeling in which bone formation by
osteoblasts is overtaken by the resorption of old bone by
osteoclasts. Current treatments to correct this imbalance may
be divided into two distinct mechanistic classes. The first class
is the antiresorptives which directly inhibit osteoclast activity,
thereby slowing the resorption side of the bone remodeling
equilibrium. This class is exemplified by bisphosphonates,
selective estrogen receptor modulators (SERMsa), hormone
replacement therapy (HRT), and calcitonin. All of these
antiresorptive agents have proven efficacy in humans for the
treatment of osteoporosis, slowing the excess resorption of
bonebut offering littleintermsof stimulatingthe formation of
new bone. The second mechanistic class for the treatment of
osteoporosis is the bone forming agents which increase osteo-
blast activity allowing these cells to keep pace with or out-
perform bone resorbing osteoclasts. Both parathyroid
hormone (PTH) (Nycomed) and its N-terminal 1-34 amino
acid fragment teriparatide (Lilly),2 when administered daily
by subcutaneous injection, stimulate the formation of new
bone, increase bone mineral density, and decrease the risk of
fractures.
*To whom correspondence should be addressed. Phone: 1-610-917-
7368. Fax: 1-610-917-4206. E-mail Robert.W.Marquis@gsk.com.
a Abbreviations: CaR, calcium receptor; PTH, parathyroid hormone;
HEK, human embryonic kidney; OVX, ovariectomized; SAR, struc-
ture-activity relationship; DAT, dopamine transporter; SERT, seroto-
nin transporter; NET, norepinephrine transporter; CYP2D6, cyto-
chrome P450 2D6; hERG, human ether-a-go-go related gene; FLIPR,
fluorimetric imaging plate reader; SERM, selective estrogen receptor
modulator; HRT, hormone replacement therapy.
Recently we have reported on the amino alcohol analogue 2
(Figure 1 and Table 1) which was identified as a weak
r
2009 American Chemical Society
Published on Web 10/12/2009
pubs.acs.org/jmc

6600 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Marquis et al.
antagonist of the CaR through a screening effort of the former
SmithKline Beecham compound collection.7 Compound 2
had originally been profiled in a legacy program focused on
the discovery of β-adrenergic receptor agonists and antago-
nists where it was shown to be a potent antagonist of the
β2-adrenergic receptor and an agonist of the β3-adrenergic
receptor. Additionally, 2, being a lipophilic basic amine and a
common pharmacophore for a variety of targets, was also
a potent inhibitor of several monoamine transporters, the
human ether a-go-go related gene (hERG) ion channel as well
as the 2D6 cytochrome P450 (Table 1).8 Structure-activity
studies to optimize the antagonist potency of 2 and to reduce
off-target interactions led to the identification of amino
alcohol 3 (Figure 1 and Table 1), which is a potent antagonist
of the CaR with improved selectivity over the β-adrenergic
receptor family. Here, the key selectivity determinant for the
CaR compared to β-adrenergic receptors was shown to be the
chirality of the secondary hydroxyl group. Although this
modification resulted in a significant reduction in affinity
for the β-receptor, it did not greatly improve selectivity
against the other off-target interactions mentioned above
(see Table 1). Despite concerns regarding these off-target
interactions, amino alcohol 3 was progressed into an ovar-
iectomized(OVX)ratstudytoassessitsabilitytostimulate the
formation of new bone.9 When dosed orally for 8 weeks,
analogue 3 elicited a robust but sustained elevation of plasma
PTH in the OVX rat. This protracted rise in PTH levels
ultimately led to increased bone turnover with no increases
in overall bone mineral density. The undesirable PTH profile
elicited by analogue 3 was attributed to its prolonged phar-
macokinetic profile in the rat where it was shown to be rapidly
cleared (Cl = 73.8 (mL/min)/kg) with a large volume of
distribution (Vdss=11 L/kg). On the basis of these data, an
ideal follow-up to analogue 3 would have a significantly
reduced volume of distribution while maintaining the rapid
rate of clearance. A molecule with these two characteristics
would be expected to have an improved pharmacokinetic
profile with a short-lasting antagonism of the CaR, resulting
in a transient plasma PTH exposure. In this account we detail
structure-activity and pharmacokinetic studies that further
address the off-target interactions associated with the amino
alcohol class of antagonists as well as the protracted pharma-
cokinetic profile of analogue 3.
Synthesis Chemistry
The analogues detailed in this account were synthesized
according to the reactions provided in Schemes 1-3. Com-
pound 5 of Scheme 1 was synthesized starting with the
coupling of 2-cyanophenol (4) with (2R)-2-oxiranylmethyl
4-nitrobenzenesulfonate to provide the intermediate chiral
epoxide (not shown). Opening of this epoxide with [1,1-
dimethyl-2-(2-naphthalenyl)ethyl]amine in refluxing ethanol
provided 5. In a similar fashion, as outlined in Scheme 2,
alkylation of ethyl 4-hydroxyhydrocinnamate (6) with (2S)-2-
oxiranylmethanol provided the epoxide 7 which was opened
with [1,1-dimethyl-2-(2-naphthalenyl)ethyl]aminein refluxing
ethanol to provide the ethyl ester 8. Hydrolysis of 8 under
basic conditions gave the acid 9. Analogues 11 and 12 of
Scheme 3 were synthesized beginning with bromination of
4-hydroxyhydrocinnamate (6) followed by cyanation and
alkylation with (2S)-2-oxiranylmethanol to provide epoxide
10. Opening of epoxide 10 with [1,1-dimethyl-2-(2-naphtha-
lenyl)ethyl]amine provided the ester 11 which was hydrolyzed
under basic conditions to give the acid 12.
Results and Discussion
To assess the ability of synthesized analogues to antagonize
the CaR, two in vitro assays were used. The first was a
fluorimetric imaging plate reader (FLIPR) based assay mea-
suring the ability of test compounds to block increases in the
concentration of cytoplasmic Ca2þ in HEK293 cells expres-
sing the human parathyroid CaR. The second was a binding
assay where analogues were tested for their ability to displace
the tritiated calcimimetic radioligand (2-(2-hydroxyphenyl)-
6-methyl-5-(2-methylpropyl)-3-(2-phenylethyl)-4(3H)-pyri-
midinone) from membrane preparations of HEK293 cells
expressing the human CaR. The hydrochloride salts of the
synthesized compounds were prepared in order to facilitate
both compound solubility and handling.
As detailed previously analogue 3 is a potent antagonist of
the CaR in both the binding assay and the FLIPR assay with
Scheme 1. Synthesis of Antagonist 5a
a Reagents and conditions: (a) NaH, DMF, (2R)-2-oxiranylmethyl
4-nitrobenzenesulfonate, 0 ꢀC; (b) [1,1-dimethyl-2-(2-naphthalenyl)ethyl]-
amine, EtOH, 70 ꢀC.
Figure 1. Calcimimetic and calcilytic compounds.
Table 1. Potency and Selectivity Dataa
IC50 (μM)
compd
CaR FLIPR
CaR binding
SERT binding
NET binding
DAT binding
CYP2D6
hERG Bbinding
β2 binding
2
11.0
31.8
0.061
0.002
0.003
0.068
0.078
0.023
0.60
0.20
0.003
16.0
0.037
5.40
1.20
0.20
0.002
3.7
1.26
11.8
3.00
3.20
38.5
1.7
0.0013
4.3
ND
3
0.058
0.018
>25
0.034
0.200
0.003
0.003
4.8
0.045
0.100
52.0
5
9
>30.0
14.0
>30.0
11
12
0.003
0.022
0.059
2.80
3.0
>50.0
32.7
a IC50 values represent the average of at least three individual titrations. ND=not determined.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6601
Scheme 2. Synthesis of Antagonists 8 and 9a
on this aromatic group was found to be required for potent
antagonism of the CaR. Despite the lack of CaR antagonist
potency for analogue 9, what was most encouraging was the
substantial increase in selectivity for the majority of the off-
target interactions characteristic of this amino alcohol series.
Similar to previous analogues, 9 remained a potent inhibitor
of the serotonin transporter with IC50=0.068 μM, however,
a substantive reduction in potency versus the norepinephrine
and dopamine transporters was seen relative to analogues 3
and 5. Additionally, incorporation of the propionic acid
moiety also led to a reduction in activity versus the 2D6
cytochrome P450 isozyme (IC50 = 52 μM), the hERG ion
channel (IC50=38.5 μM), and the β2-adrenoreceptor (IC50>
30 μM). Pharmacokinetic analysis in the rat revealed that 9
had a high rate of clearance (Cl=117.5 (mL/min)/kg) with a
low volume of distribution (Vdss=2.1 L/kg), a short half-life
(T1/2 =0.4 h), and mean residence time (MRT=0.3 h). As
discussed above, this type of pharmacokinetic profile was
desirable for eliciting a rapid and transient release of PTH.
Unfortunately the oral bioavailbilty of 9 was low (appro-
ximately 4%), which was likely a reflection of both the high
clearance and the low intestinal absorption attributable to the
zwitterionic nature of this compound.
a Reagents and conditions: (a) DIAD, PPh3, (2S)-2-oxiranylmetha-
nol, THF, 0 ꢀC to room temp; (b) [1,1-dimethyl-2-(2-naphthalenyl)ethyl]-
amine, EtOH, 70 ꢀC; (c) NaOH, EtOH, H2O.
Scheme 3. Synthesis of Antagonists 11 and 12a
Although analogue 9 was not a potent antagonist of the
CaR its improved receptor selectivity and desirable intrave-
nous pharmacokinetic profile made this analogue the focus of
additional studies aimed at improving both potency and
bioavailability. As discussed above, structure-activity studies
had revealed that the incorporation of an electron withdraw-
ing moiety on this aromatic group was deemed essential for
antagonist potency. Incorporation of the propionic acid side
chain of 9 on the aromatic group of the potent antagonist5 led
to analogue 12, which was a potent antagonist of the CaR
with IC50=0.022 μM in the receptor binding assay and IC50=
0.20 μM in the FLIPR assay. Despite the improved potency
versus the CaR, no significant improvement in selectivity for
12 against the serotonin transporter (SERT IC50=0.023 μM)
was observed while improved selectivity over the norepine-
pherine (NET IC50 =5.40 μM) and dopamine transporters
(DAT IC50=2.80 μM) was observed relative to analogue 3
(SERT IC50=0.002 μM; NET IC50=0.002 μM, DAT IC50=
0.20 μM). Incorporation of the propionic acid moiety also led
to a significant increase in selectivity of 12 versus CYP2D6
(IC50>50.0 μM), thehERGion channel (IC50=32.7 μM), and
the β2-adrenoreceptor (IC50>30.0 μM).
Pharmacokinetic analysis of 12 in the rat showed this
compound to have a desirable profile with a short half-life
(T1/2=0.56 h), lower mean residence time (MRT=0.32 h),
rapid clearance (Cl = 63.6 mL/min/kg), and low volume of
distribution (Vdss =1.18 L/kg). Analogue 12 had a low oral
bioavailability (approximately 0.3%) when administered or-
ally as a suspension (48 mg/kg). Dosing into the portal vein of
the rat revealed that the first pass hepatic extraction of 12 was
moderate to high at 68 ( 21%, suggesting that it is likely a
combination of poor intestinal absorption and high first pass
hepatic extraction that is limiting the systemic exposure of 12
in the rat following oral administration. Evaluation of the
pharmacokinetics of 12 in the dog revealed that this compound
had a short half-life (T1/2=1.23 h) and mean residence time
(MRT = 0.85 h) with a small volume of distribution
(Vdss = 0.78 L/kg) and a moderate rate of clearance (Cl =
15.4 (mL/min)/kg). As was observed in the rat, the rate of first
pass hepatic extraction of 12 was high in the dog (90 ( 6%)
following intraportal administration. The oral bioavailability
a Reagents and conditions: (a) Br2, CHCl3, 0 ꢀC; (b) CuCN, NMP,
reflux; (c) DIAD, PPh3, (2S)-2-oxiranylmethanol, THF, 0 ꢀC to room
temp; (d) [1,1-dimethyl-2-(2-naphthalenyl)ethyl]amine, EtOH, 70 ꢀC;
(e) CH3OH, 3 N NaOH.
IC50 values of 0.003 and 0.058 μM, respectively (Table 1). This
potency represents a greater than 10000-fold increase in
potency in the CaR binding assay and a 190-fold increase in
potency in the functional assay over the original lead 2. In a
binding assay format, analogue 3 is also a potent inhibitor of
the serotonin, norepinepherine, and dopamine transporters
withIC50 valuesof0.002, 0.200, and 0.200μM, respectively, as
well as both the 2D6 cytochrome P450 (IC50=0.045 μM) and
the hERG ion channel (IC50=3.00 μM). Importantly, the
affinity of 3 for the β2-adrenergic receptor was reduced almost
3300-fold relative to 2. Analogue 5, in which the chlorine atom
of 3 was removed, was also a potent antagonist of the CaR
with IC50 values of 0.003 and 0.018 μM in the binding and
functional assays, respectively. Not unexpectedly, this modi-
fication did not greatly affect the off-target interactions ob-
served within analogues of this series of antagonists, and as
such, this analogue was not further profiled in pharmacoki-
netic studies. However, this modification did indicate that
potent antagonism of the CaR was achievable with the
incorporation of a benzonitrile moiety. Incorporation of a
propionic acid on the aromatic group to provide analogue 9
resulted in a significant loss in antagonist potency. This result
is consistent with structure-activity trends defined earlier
within this series in which an electron withdrawing moiety

6602 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Marquis et al.
Table 2. Pharmacokinetic Data for Antagonists 3, 9, 11, and 12a
pharmacokinetics
Cl
((mL/min)/
kg)
MRT
(h)
T1/2
(h)
Vdss
(L/kg)
compd species
F (%)
3
rat
rat
2.5
0.3
2.1
0.4
73.8
117.5
11.03
2.1
11.4
4.0
9
11
11
12
12
rat
0.7
0.88
1.23
0.56
1.23
9.5b
9.1b
0.29
ND
dog
rat
1.37
0.32
0.85
63.6
15.4
1.18
0.78
dog
a Three rats or dogs were employed for the po and iv arms of these
studies. ND=not determined. b Oral bioavailability of acid 12 following
oral dosing of ester 11.
Figure 2. Mean pharmacokinetics and pharmacodynamics of ana-
logue 12 following the oral dosing of a 23.4 mg/kg suspension dose
(1% methylcellulose) of 11 in the dog (n=3).
of 12 in the dog was not determined in these studies because of
the low levels of intestinal absorption and high hepatic extrac-
tion, similar to what had been previously observed in the rat.
The systemic exposure of 12 in the rat and the dog was
limited by two factors: high hepatic extraction and poor
intestinal absorption following oral administration. The high
hepatic extraction of 12 is likely a required characteristic of a
compound that by necessity has to be cleared rapidly in order to
limit systemic exposure and antagonize briefly the CaR. Alter-
natively, in order to address the poor intestinal absorption of
12, a classic ester prodrug strategy was employed. The ethyl
ester prodrug 11 of the parent acid 12 was a potent antagonist
of the CaR with an IC50 of 0.003 and 0.034 μM in binding and
functional assays, respectively (Table 1). Not unexpectedly, the
gains in receptor selectivity realized with the parent acid 12 were
virtually abolished with the ester prodrug 11, a likely reflection
of this compound’s greater lipophilicity (clogP(11)=5.1 versus
clogP(12) = 2.0) and cell permeability. This loss of target
selectivity was of nominal concern because of the well pre-
cedented rates of ester prodrug hydrolysis which would lead to
the generation of the significantly more selective parent acid 12
in the systemic circulation. Data were therefore gathered in an
effort to assess the rates of this transformation in vivo.
In the rat, the oral bioavailability of the parent acid 12
following dosing of a suspension of the ester prodrug 11
(4.7 mg/kg in 1% methylcellulose) was 9.5%, representing a
32-fold increase in exposure when compared to the dosing of a
solution of the parent acid 12 (48 mg/kg) (Table 2). None of
the ester 11 was detectable in the systemic circulation, indicat-
ing complete hydrolysis to the parent acid 12 at this dose.
Following intravenous administration, the fractional conver-
sion of the ester 11 to the acid 12 had an apparent half-life of
approximately 7 min. Additionally, none of the ester prodrug
11 was observed in the systemic circulation following intra-
portal infusion (1.8 mg/kg), indicating rapid hydrolysis and/
or high hepatic extraction of this compound. A high dose
study in the rat (47.5 mg/kg po) conducted to support safety
assessment studies revealed that systemic exposure to the
ester 11 was very low with maximum blood concentrations
of 15-20 ng/mL, again indicative of rapid ester hydrolysis. In
the dog, the oral bioavailability of the acid 12 was 9.1%
following oral suspension dosing of the ester 11. In this species
the parent acid had a desired short half-life (T1/2=1.23 h) and
mean residence time (MRT = 1.37 h) following the oral
administration of a suspension dose of the ester 11, suggesting
rapid dissolution and absorption. The half-life of the ester 11
was estimated to be approximately 7 min with rapid conver-
sion to the parent acid 12 following intravenous administra-
tion. Portal vein concentrations indicated that the ester 11 was
well absorbed across the intestinal wall and that the acid 12
comprised the majority of the total portal vein AUC (73%).
Combined, these data indicate that it is again high first pass
hepatic extraction, not poor absorption, limiting the bioavail-
ability of the parent acid 12 following the oral administration
of the ester 11 in these two species.
The promising pharmacokinetic profile of the potent acid
12 following oral dosing of the ester prodrug 11 prompted
evaluation of this acid/ester prodrug pair for its ability to elicit
an increase in plasma levels of PTH in the dog. As shown in
Figure 2, oral administration of the ester 11 (23.4 mg/kg)
produced a rapid increase of the parent acid 12 in the systemic
circulation with an oral Tmax of 0.5 h and an oral Cmax of
597 ng/mL. This profile suggests that the ester 11 rapidly
dissolved in intestinal fluid, was well absorbed across the
intestinal wall, and was completely converted to the parent
acid 12 via ester hydrolysis. Levels of the acid 12 returned to
baseline in approximately 2 h, consistent with the desired
profile for brief antagonism of the CaR (Figure 2). A con-
comitant rise in plasma PTH levels was also observed follow-
ing the oral dosing of the ester 11. Plasma PTH levels reached
Tmax at 0.4 h with a Cmax of approximately 180 ng/mL, a phar-
macokinetic profile essentially overlaying that of the acid 12.
Conclusions
In this account we have detailed structure-activity studies
focused on the improvement of both the in vitro selectivity and
in vivo pharmacokinetic profiles of the first generation amino
alcohol-based CaR antagonist 3. The incorporation of a polar
carboxylic acid moiety into this template to provide analogue
12 served the dual purposes of improving both the CaR selec-
tivity and pharmacokinetic profiles of 3. However, because of
the high first pass hepatic extraction and low intestinal absorp-
tion, the oral bioavailability of 12 was low, prompting the use
of an acid/ester prodrug strategy. The ester 11 was able to
deliver significant levels of the acid following oral administra-
tion in both the dog and the rat and maintained a desired short
exposure profile. Oral administration of the ester 11 in the dog
elicited a rapid and transient increase in plasma levels of PTH.
The magnitude and duration of the increases in plasma levels
of PTH would be expected to stimulate new bone formation.
This pharmacokinetic profile represents a significant improve-
ment over the first generation antagonist 3. The ability of
analogues such as 12 to promote a bone building effect in vivo
will be the subject of a future publication.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6603
3
Experimental Procedures
and 25 μL of 40 nM H-compound in 100% EtOH for a final
concentration of 2 nM. The binding reaction is initiated by the
addition of 50 μL of 80-200 μg/mL HEK 293 4.0-7 membrane
diluted in incubation buffer, and the mixture is allowed to
incubate at 4 ꢀC for 30 min. Wash buffer is 50 mM Tris-HCl
containing 0.1% PEI. Nonspecific binding is determined by the
addition of 100-fold excess of unlabeled homologous ligand and
is generally 20% of total binding. The binding reaction is
terminated by rapid filtration onto 1% PEI pretreated GF/C
filters using a Brandel harvestor. Filters are placed in scintilla-
tion fluid, and radioactivity is assessed by liquid scintillation
counting.
Calcium Receptor Inhibitor Assay. HEK 293 4.0-7 cells were
constructed as described by Rogers et al. (J. Bone Miner. Res.
1995, 10 (Suppl. 1), S483). At 48 h prior to running the CaR
assay, frozen HEK293 CaRec4.0-c17 cells are thawed, counted,
and diluted to 3ꢀ105/mL (15K/50 μL). The cell medium used
to dilute the cells consists of 1:1 DMEM/F12 (HAM’S) with
L-glutamine, 15 mM HEPES, phenol red, 10% fetal bovine
serum, and 1% penicillin-streptomycin solution. Cell solution
is seeded at (15K cells/50 μL)/well in Greiner poly-D-lysine
coated 38-well, black, clear-bottom tissue culture plates and left
at room temperature for 1 h to reduce the edge effect. After the
first hour at room temperature, cell plates are then placed into a
37 ꢀC, 5% CO2 incubator for 48 h.
On the day of the experiment, plate confluency is first checked
via microscope. Wells should be ∼100% confluent in an even
cell monolayer. Assay reagents are prepared fresh. Dye load
buffer consists of Hank’s buffered saline solution with 0.75 mM
calcium, without magnesium, without sodium bicarbonate, with
20 mM Hepes, probenecid (2.5 mM final concentration), Fluo4
(2 μM final concentration), and Brilliant Black (500 μM final
concentration). Compound dilution buffer consists of Hank’s
buffered saline solution without calcium, without magnesium
(without sodium bicarbonate), and with CHAPS (0.01% final
concentration). A ligand curve plate is also prepared fresh. A 16
point curve is dispensed into a Greiner 384-well polypropylene
plate. The top concentration of the ligand, CaCl, is 2.875 mM
(final concentration), and the lowest concentration is 0.375 mM
(final concentration). An EC80 value is generated from the curve
data.
The CaR assay begins when the cell medium is aspirated
from the cell plate using a Tecan plate washer, leaving nothing
but the cell monolayer. Dye load buffer is added to the cell plate
at 20 μL/well using a Multidrop, and the loaded plate is
incubated for 45 min at 37 ꢀC, 5% CO2. Compound plates are
received with 1 μL of compound stamped at 5 mM top con-
centration (25 μM final concentration in cell plate). Compound
dilution buffer is added to columns 1-24 in the compound plate
at 65 μL/well using a Multidrop. Column 6 is prestamped with
1 μL of DMSO to represent the high control, and column 18
receives 65 μL of buffer as the low control. The compound
addition takes place on a Cybi Well dispenser when 10 μL of
diluted compound is added to the dye loaded cell plate. The cell
plate with compound is then incubated at room temperature for
5 min. The antagonist addition takes place on the FLIPR when
10 μL of EC80 challenge is added to the cell plate and fluores-
cence imaging proceeds for 65 s. Column 18 of the EC80 challenge
plate contains only buffer to represent a low control or tool
antagonist.
Calcium Receptor Binding Assay. HEK 293 4.0-7 cells stably
transfected with the human parathyroid calcium receptor
(“HuPCaR”) were scaled up in T180 tissue culture flasks. Plasma
membrane is obtained by Polytron homogenization or glass
douncing in buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 3 mM
MgCl2) in the presence of a protease inhibitor cocktail contain-
ing 1 μM leupeptin, 0.04 μM pepstatin, and 1 mM PMSF.
Aliquoted membrane was snap frozen and stored at -80 ꢀC. 3H
labeled compound was radiolabeled to a radiospecific activity of
44 Ci/mmol and was aliquoted and stored in liquid nitrogen for
radiochemical stability.
The purities of all tested compounds that were not analyzed
via elemental analysis were determined by high performance
liquid chromatography. Method A was performed on a Agilent
1100 series, with a UV detector monitoring at 254 nm, a Zorbax
SB-C8 column (4.6 mm ꢀ 150 mm, 5 μm), 5-100% CH3CN/
H2O (with 0.02% TFA) over 12.5 min and hold for 2.5 min, and
flow rate of 1.5 mL/min at 40ꢀC. Method B was performed on a
Agilent 1200 series, with a UV detector monitoring at 254 nm, a
Phenomenex C18 column (4.6 mm ꢀ 150 mm, 3 μm), 5-95%
CH3CN/H2O (with 0.1% TFA) over 15 min, and flow rate of
1 mL/min at 40 ꢀC. Method C was performed on an Agilent 1200
series, with a UV detector monitoring at 254/214 nm, a Zorbax-
XDB-C18 column (4.6 mmꢀ75 mm, 3.5 μm), 5-95% CH3CN/
H2O (each with 0.10 % TFA) over 4 min and hold for 1 minute
at 95 % of CH3CN (0.10% TFA), and flow rate of 2.0 mL/min.
All compounds analyzed by these methods possessed purities
equal to or greater than 95% with the exception of analogue 5,
which was 95.5% pure by method A and 94.9% pure by method B.
General. Except where indicated materials and reagents were
used as supplied. Nuclear magnetic resonance spectra were
recorded at either 250 or 400 MHz using, respectively, a Bruker
AM 250 or Bruker AC 400 spectrometer. Mass spectra were
taken on a PE Syx API III instrument using electrospray (ES)
ionization techniques. Elemental analyses were obtained using a
Perkin-Elmer 240C elemental analyzer. Reactions were moni-
tored by TLC analysis using Analtech silica gel GF or E. Merck
silica gel 60 F-254 thin layer plates. Flash chromatography was
carried out on E. Merck Kieselgel 60 (230-400 mesh) silica gel.
The purity of all tested compounds, which were not analyzed via
elemental analysis, were determined by high performance liquid
chromatography. Method A was performed on an Agilent 1100
series, with a UV detector monitoring at 254 nm, a Zorbax SB-
C8 column (4.6 mm ꢀ 150 mm, 5 μm), 5-100% CH3CN/H2O
(with 0.02% TFA) over 12.5 min and hold for 2.5 min, and
flow rate of 1.5 mL/min at 40 ꢀC. Method B was performed on
a Agilent 1200 series, with a UV detector monitoring at 254 nm,
a Phenomenex C18 column (4.6 mmꢀ150 mm, 3 μm), 5-95%
CH3CN/H2O (with 0.1% TFA) over 15 min, and flow rate of
1 mL/min at 40 ꢀC.
2-[((2R)-3-{[1,1-Dimethyl-2-(2-naphthalenyl)ethyl]amino}-2-
hydroxypropyl)oxy]benzonitrile (5). To a solution of (2S)-glyci-
dol (2 g, 27 mmol) in dichloromethane (25 mL) at 0 ꢀC was
slowly added triethylamine (3.75 mL). Then a solution of 4-nitro-
benzenesulfonyl chloride (6 g, 27 mmol) in dichloromethane
(13 mL) was added to the reaction mixture at such a rate that the
temperature did not rise above 15 ꢀC. After 1 h at 0 ꢀC the
solution was allowed to warm to room temperature and main-
tained at this temperature overnight. The reaction mixture was
concentrated and the crude material purified by flash column
chromatography (0-60% EtOAc/hexane) to afford 4.2 g of the
desired product (58%). MS (m/z): 260.1 (MþH).
A typical reaction mixture contains 2 nM 3H compound ((R,
R)-N-40-methoxy-t-3-30-methyl-10-ethylphenyl-1-(1-naphthyl)-
ethylamine) or 3H compound ((R)-N-[2-hydroxy-3-(3-chloro-2-
cyanophenoxy)propyl]-1,1-dimethyl-2-(4-methoxyphenyl)ethyl-
amine) and 4-10 μg of membrane in homogenization buffer
containing 0.1% gelatin and 10% EtOH in a reaction volume of
0.5 mL. Incubation is performed in 12ꢀ75 polyethylene tubes in
an ice-water bath. To each tube 25 μL of test sample in 100%
EtOH is added, followed by 400 μL of cold incubation buffer
Sodium hydride (70 mg, 1.76 mmol, 60% oil suspension) was
washed twice with hexane and then placed in a two-necked
round-bottom flask under N2 and cooled to 0 ꢀC with an ice-
water bath. Then DMF (5 mL), 2-hydroxybenzonitrile (190 mg,
1.6 mmol), and (2R)-2-oxiranylmethyl 4-nitrobenzenesulfonate
(500 mg, 1.92 mmol) were added to this mixture. The solution
was maintained at 0 ꢀC for 6 h, whereupon it was concentrated

6604 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Marquis et al.
and the crude material was purified by flash column chroma-
tography (0-80% EtOAc/hexane) to afford 240 mg of the
2-{[(2R)-2-oxiranylmethyl]oxy}benzonitrile (86%).
was slowly added bromine (2.85 g, 17.86 mmol) over 25 min. The
solution was stirred for 60 min at room temperature and then
quenched with water and washed with brine. The organic layer
was dried over Na2SO4, filtered, concentrated in vacuo, and
purified by flash chromatography (Biotage, 0-20% EtOAc/
hexanes) to yield ethyl 3-(3-bromo-4-hydroxyphenyl)propano-
Asolutionof2-{[(2R)-2-oxiranylmethyl]oxy}benzonitrile(239mg,
1.37 mmol) and 2-methyl-1-(2-naphthalenyl)-2-propanamine
(327 mg, 1.64 mmol) in ethanol (10 mL) were combined and
heated at 70 ꢀC for 16 h. The mixture was concentrated and
purified by column chromatography (0-10% CH2Cl2/MeOH)
and afforded the desired product (272 mg, 53.2%). This material
was dissolved in CH2Cl2, and HCl in ether was added. This
material was concentrated and azeotropically dried with toluene
to provide the hydrochloride salt 5. 1H NMR (DMSO, 400 MHz):
δ 8.95 (m, 1H), 8.72 (m, 1H), 7.08-7.90 (m, 11H), 6.05 (m, 1H),
4.15-4.40 (m, 3H), 3.31-3.46 (m, 1H), 3.15-3.20 (m, 3H), 1.30
(s, 6H). MS (m/z): 375.3 (M þ H). HPLC purities: method A,
95.5%, tR=7.2 min; method B, 94.9%, tR=8.9 min.
1
ate (3.54 g, 76%). H NMR (400 MHz, CDCl3): δ 1.26 (t, J=
7.07 Hz, 3 H), 2.56-2.63 (t, J=7.6 Hz, 2 H), 2.88 (t, J=7.71 Hz,
2 H), 4.11-4.19 (q, J=7.2 Hz, 2 H), 6.95 (d, J=8.34 Hz, 1 H),
7.07 (dd, J=8.34, 2.02 Hz, 1 H), 7.33 (d, J=2.27 Hz, 1 H).
To a solution of ethyl 3-(3-bromo-4-hydroxyphenyl)pro-
panoate (3.54 g, 12.97 mmol) in NMP (20 mL) was added CuCN
(1.22 g, 13.62 mmol), and the mixture was refluxed for 2 h. The
reaction mixture was cooled to room temperature, diluted with
ethyl acetate (70 mL), and then washed with 1 N HCl (60 mLꢀ2)
and brine (60 mL). The organic layer was dried over Na2SO4,
filtered, concentrated in vacuo, and purified by flash chroma-
tography (Biotage, 10-28%, EtOAc/hexanes) to yield ethyl
3-(3-cyano-4-hydroxyphenyl)propanoate (2.1 g, 73.8%). LCMS
(m/z): 220.0 [MþH]þ.
3-{4-[((2R)-3-{[1,1-Dimethyl-2-(2-naphthalenyl)ethyl]amino}-
2-hydroxypropyl)oxy]phenyl}propanoic Acid (8). To a solution
of ethyl 3-(4-hydroxyphenyl)propanoate (6, 5.0 g, 25.8 mmol) in
100 mL of THF was added (2S)-2-oxiranylmethanol (2.48 g,
33.5 mmol) and triphenylphosphine (10.2 g, 38.7 mmol). The reac-
tion mixture was cooled to 0 ꢀC, and DIAD (8.10 g, 38.7 mmol)
was added dropwise. The mixture was allowed to warm to room
temperature and maintained at this temperature overnight. A
white precipitate formed upon addition with Et2O and was fil-
tered off through a fine fritted funnel. The filtrate was concen-
trated and purified by column chromatography to give 4.8 g (75%)
of ethyl 3-(4-{[(2R)-2-oxiranylmethyl]oxy}phenyl)propanoate (7).
A solution of ethyl 3-(4-{[(2R)-2-oxiranylmethyl]oxy}phenyl)-
propanoate (1.2 g, 4.8 mmol) and [1,1-dimethyl-2-(2-naphtha-
lenyl)ethyl]amine (1.0 g, 5.0 mmol) in ethanol (10 mL) was com-
binedand heated at 70 ꢀC for 15 h. The mixture was concentrated
and purified by column chromatography (0-90% ethyl acetate/
hexane) and afforded ethyl 3-{4-[((2R)-3-{[1,1-dimethyl-2-(2-
naphthalenyl)ethyl]amino}-2-hydroxypropyl)oxy]phenyl}pro-
panoate (8, 1.4 g, 78%). 1H NMR (400 MHz, CDCl3): δ 9.41 (br,
1 1 H), 8.82 (br, 1 H), 7.91 (m, 3 H), 7.52 (s, 1 H), 7.50 (m, 2 H),
7.40 (d, 1 H, J=8.4 Hz), 7.15 (d, 2 H, J=8.4 Hz), 6.89 (d, 2 H, J=
8.8 Hz), 5.95 (d, 1 H, J=4.8 Hz), 4.28 (m, 1 H), 4.03 (m, 4 H),
3.21-3.09 (m, 4 H), 2.79 (t, 2 H, J=7.6 Hz), 2.57 (t, 2 H, J=
7.6 Hz), 1.30 (s, 6 H), 1.15(t, J= 7.2 Hz). MS(m/z): 450.2 (MþH).
To a solution of ethyl 3-(3-cyano-4-hydroxyphenyl)propano-
ate (1.95 g, 8.90 mmol) in dry THF (15 mL) was added in
sequence (S)-glycidol (1.32 g, 17.81 mmol) and PPh3 (4.67 g,
17.81). The mixture was cooled in an ice bath, and DIAD (4.67 g,
17.81 mmol) was added dropwise. After 20 h, an additional
0.65 equiv of (S)-glycidol, PPh3, and DIAD was added and the
mixture was stirred for 3 h at room temperature. The solvent was
then evaporated and the residue was purified by flash chroma-
tography (Biotage, 0-1.2% THF/CH2Cl2) to yield ethyl 3-(3-
cyano-4-{[(2R)-2-oxiranylmethyl]oxy}phenyl)propanoate (1.40 g,
57%). MS (m/z): 294.2 [Mþ18]þ.
To a solution of ethyl 3-(3-cyano-4-{[(2R)-2-oxiranylmethyl]-
oxy}phenyl) propanoate (0.627 g, 2.3 mmol) in ethanol (10 mL)
was added [1,1-dimethyl-2-(2-naphthalenyl)ethyl]amine (0.571 g,
2.87 mmol), and the mixture was stirred at 70 ꢀC for 20 h. The
solvent was evaporated, and the residue was purified by flash
chromatography (Biotage, 0-3.2% MeOH/CH2Cl2) to yield
ethyl 3-{3-cyano-4-[((2R)-3-{[1,1-dimethyl-2-(2-naphthalenyl)-
ethyl]amino}-2-hydroxypropyl)oxy]phenyl}propanoate (0.89 g,
82%). To a solution of ethyl 3-{3-cyano-4-[((2R)-3-{[1,1-di-
methyl-2-(2-naphthalenyl)ethyl]amino}-2-hydroxypropyl)oxy]-
phenyl}propanoate (202 mg, 0.426 mmol) in acetonitrile (4.0 mL)
was added 1 N HCl (solution in diethyl ether) (2.13 mL, 2.13 mmol).
After the mixture was stirred for 20 min the solvent was
evaporated and the residue was azeotroped with toluene to yield
ethyl 3-{3-cyano-4-[((2R)-3-{[1,1-dimethyl-2-(2-naphthalenyl)-
ethyl]amino}-2-hydroxypropyl)oxy]phenyl}propanoate hydro-
Anal. Calcd for C28H35NO4 HCl: C 69.19; H 7.46; N 2.88.
3
Found: C 69.14; H 7.44; N 2.83.
3-{4-[((2R)-3-{[1,1-Dimethyl-2-(2-naphthalenyl)ethyl]amino}-
2-hydroxypropyl)oxy]phenyl}propanoic Acid (9). Sodium hydro-
xide (3 N, 1.0 mL, 3.0 mmol) was added to a solution of ethyl
3-{4-[((2R)-3-{[1,1-dimethyl-2-(2-naphthalenyl)ethyl]amino}-
2-hydroxypropyl)oxy]phenyl}propanoate (8, 0.50 g, 1.0 mmol)
in methanol (15 mL). The mixture was maintained at room
temperature for 15 h. The methanol was removed in vacuo, and
the aqueous layer was diluted with water. The pH of the aqueous
layer was adjusted to pH ∼6.0 with 6 N HCl while stirring.
The precipitated white solid was collected by filtration and
dried under high vacuum in the oven (∼55 ꢀC) for ∼ 20 h. The
zwitterion was taken up in acetonitrile, and 1 N HCl in ether
was added and stirred for ∼2 h and then concentrated in vacuo
to afford 3-{4-[((2R)-3-{[1,1-dimethyl-2-(2-naphthalenyl)ethyl]-
amino}-2-hydroxypropyl)oxy]phenyl}propanoic acid (9, 0.4 g,
84%) as white foam solid. 1H NMR (400 MHz, CDCl3): δ 11.8
(s, 1 H), 9.41 (br, 1H), 8.85 (br, 1 H), 7.91 (m, 3 H), 7.78 (s, 1 H),
7.51 (m, 2 H), 7.40 (m, 1 H), 7.15 (d, 2 H, J=8.4 Hz), 6.89 (d, 2 H,
J=8.4 Hz), 5.96 (d, 1 H, J=4.4 Hz), 4.30 (m, 1 H), 4.02 (d, 2 H,
J=5.2 Hz), 3.23-3.03 (m, 4 H), 2.76 (t, 2 H, J=7.6 Hz), 2.50 (t,
2 H, J=7.6 Hz), 1.30 (s, 6 H). MS (m/z): 422.2 (MþH). Anal.
1
chloride as a white solid (quantitative). H NMR (400 MHz,
DMSO-d6): δ 1.16 (s, 3 H) 1.29 (s, 6 H) 2.59-2.65 (m, 2 H),
2.79-2.86 (m, 2 H), 3.13-3.24 (m, 3 H), 3.34-3.44 (m, 1 H),
4.00-4.08 (m, 2 H), 4.15-4.31 (m, 3 H), 5.96-6.01 (m, 1 H),
7.21-7.25 (m, 1 H), 7.37-7.42 (m, 1 H), 7.48-7.59 (m, 3 H),
7.63 (s, 1 H), 7.77-7.80 (m, 1 H), 7.87-7.94 (m, 3 H), 8.60-8.81
(m, 2 H). 13C NMR: δ 14.07, 22.33, 22.41, 28.78, 34.74, 42.68,
43.88, 59.70, 59.84, 65.30, 70.64, 100.55, 113.30, 116.42, 125.86,
126.17, 127.44, 127.59, 129.00,129.22, 131.94, 132.84, 132.88,
133.06, 133.82, 135.10, 158.41, 171.96. LCMS (m/z): 475.2 [Mþ
H]þ. [R]2D0.4 þ15.43 (c 0.77, MeOH). Anal. Calcd for C29H35-
N2O4Cl 0.25H2O: C, 67.56%, H 6.94%, N 5.43%. Found: C
3
67.16%, H 6.99%, N 5.33%.
3-{3-Cyano-4-[((2R)-3-{[1,1-dimethyl-2-(2-naphthalenyl)ethyl]-
amino}-2-hydroxypropyl)oxy]phenyl}propanoic Acid Hydrochloride
(12). To a solution ethyl 3-{3-cyano-4-[((2R)-3-{[1,1-dimethyl-
2-(2-naphthalenyl)ethyl]amino}-2-hydroxypropyl)oxy]phenyl}-
propanoate (11, 468. 0 mg, 0.987 mmol) in methanol (4.0 mL)
was added aqueous 3 N NaOH (0.823 mL, 2.47 mmol). After
overnight stirring at room temperature, the solvent was evapo-
rated and the residue was diluted with water (15 mL). The
mixture was acidified to pH ∼6 to obtain a solid that was filtered
and washed with ethyl ether and acetonitrile. The solid was then
Calcd for C26H31NO4 HCl: C 68.18; H 6.61; N 2.98. Found: C
3
68.06; H 7.04; N 3.06
Ethyl 3-{3-Cyano-4-[((2R)-3-{[1,1-dimethyl-2-(2-naphthalenyl)-
ethyl]amino}-2-hydroxypropyl)oxy]phenyl}propanoate Hydrochlo-
ride (11). To a solution of ethyl 3-(4-hydroxyphenyl)propanoate
(3.30 g, 17.01 mmol) in chloroform (30 mL) cooled in an ice bath

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6605
azeotroped with toluene, suspended in acetonitrile, and then
treated with 1 N HCl (in ethyl ether) (2.71 mL, 2.71 mmol). The
solid product was filtered and washed with a small amount of
development of calindol as a new calcimimetic acting at the calcium-
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1
acetonitrile to yield the title compound (245 mg, 38.4%). H
NMR (400 MHz, DMSO-d6): δ 1.29 (s, 6 H), 2.54-2.59 (m, 2 H),
2.77-2.84 (m, 2 H), 3.18 (s, 3 H), 3.36-3.43 (m, 1 H), 4.15-4.30
(m, 3 H), 5.97-6.04 (m, 1 H), 7.24 (d, J=8.59 Hz, 1 H), 7.39-
7.43 (m, 1 H), 7.48-7.59 (m, 3 H), 7.63 (s, 1 H), 7.78 (s, 1 H),
7.87-7.94 (m, 3 H), 8.65-8.72 (m, 1 H), 8.76-8.85 (m, 1 H),
12.15 (s, 1 H). 13C NMR: δ 22.42, 28.86, 34.94, 42.75, 43.89,
54.89, 65.38, 70.64, 100.54, 113.30, 116.46, 125.85, 126.16,
127.44, 127.57, 129.01, 129.21, 131.93, 132.82, 133.01, 134.19,
135.08, 158.35, 173.52. HRMS: 447.2282 (calcd 447.2284).
[R]2D3.6 þ11.75 (c 0.74, DMSO). HPLC purities: method A,
98.5%, tR=6.95 min; method C, 100%, tR=2.76 min.
Acknowledgment. The authors thank Drs. John Gleason
and Brian Metcalf for their support of this work.
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