The discovery of novel calcium sensing receptor negative allosteric modulators

Article abstract of DOI:10.1016/j.bmcl.2009.04.044

The design and profile of a series of zwitterionic calcium sensing receptor negative allosteric modulators is described. Evaluation of key analogues using a rat model demonstrate a robust response, significantly improved potency over ronacaleret and have

Full text of DOI:10.1016/j.bmcl.2009.04.044

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3328–3332
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery of novel calcium sensing receptor negative allosteric modulators
Gayatri Balan, Jonathan Bauman, Samit Bhattacharya, Mayda Castrodad, David R. Healy, Michael Herr,
Paul Humphries, Sandra Jennings, Amit S. Kalgutkar, Brendon Kapinos, Vishal Khot, Kimberly Lazarra,
*
*
Mei Li, Yan Li, Constantin Neagu, Robert Oliver, David W. Piotrowski , David Price , Hong Qi,
Holly A. Simmons, James Southers, Liuqing Wei, Yan Zhang, Vishwas M. Paralkar
Pfizer Global Research and Development, Groton/New London Laboratories, Pfizer Inc., Eastern Point Road, Groton, CT 06340, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The design and profile of a series of zwitterionic calcium sensing receptor negative allosteric modulators
is described. Evaluation of key analogues using a rat model demonstrate a robust response, significantly
improved potency over ronacaleret and have the potential as an oral, anabolic treatment for osteoporosis.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 5 March 2009
Revised 6 April 2009
Accepted 8 April 2009
Available online 18 April 2009
Keywords:
Calcium sensing receptor
Negative allosteric modulators
Osteoporosis
Parathyroid hormone
From 2000 to 2005 in the United States the number of patients
greater than 55 years old seeking treatment for osteoporosis has
increased by 10%. This increase is clearly linked to the improved
longevity of the population.1 Currently the majority of patients
are treated with antiresorptive therapy of which the bisphospho-
nate class are the most commonly prescribed. Alendronic acid
agent that can be used more widely in the treatment and preven-
tion of osteoporosis.
Rather than focusing resource into delivering an orally available
form of the peptide PTH an alternative approach is to deliver a tra-
ditional small molecule that could stimulate the release of PTH
endogenously. The calcium-sensing receptor (CaSR) is a class C
G-protein coupled receptor primarily located on the surface of
parathyroid cells that senses extracellular levels of calcium ion lev-
els and increases release of PTH when calcium levels are low.5 Neg-
ative allosteric modulators of the calcium receptor (also known as
calcilytics) are therefore able to stimulate the release of PTH.6 It is
important to note that these CaSR negative allosteric modulators
must be short acting to stimulate a pulsatile release of PTH. In or-
der to mimic the short half life of endogenous PTH or teriparatide,
these agents should have a pharmacokinetic half life of approxi-
mately one hour. This knowledge has been key to the medicinal
chemistry design of the calcilytics throughout all research groups,
in particular the pharmacokinetic properties of the small molecule.
Assuming that the free concentration of the negative allosteric
modulator drives the release of the PTH, this necessitates that
the compounds have a rapid onset of action (short Tmax after oral
administration) and a sufficiently high clearance and/or low vol-
ume of distribution so that the drug is cleared rapidly. It is also
important to deliver a compound that is not susceptible to interpa-
tient variability through drug-drug interactions or has a risk of
accumulation because these effects could lead to safety concerns
due to hypercalcemia. This is a difficult pharmacokinetic profile
to predict preclinically and so a multiple clinical candidate strategy
TM
(marketed as Fosamax ) is the market leader and is now available
through generic manufacturers.2
Patients who either fail or are intolerant to antiresorptive ther-
apy used to have limited alternatives. However, when teriparatide
TM
(marketed as Forteo ), a recombinant form of parathyroid hormone
(PTH) was introduced in 2002, anabolic therapy became an option
for those patients with severe osteoporosis. Teriparatide is the
1–34 amino acid portion of the complete human parathyroid
hormone molecule that contains amino acid sequence 1–84.3
Endogenous PTH is the primary regulator of calcium and phos-
phate metabolism in bone and kidney. Daily subcutaneous injec-
tions of teriparatide stimulate new bone formation leading to
increased bone mineral density. However, it is known that sus-
tained increase of PTH levels can lead to increased bone resorption
and so have an opposite effect to that needed within the osteopo-
rosis arena.4 Although, sales of teriparatide have increased since its
launch its use is limited due to a restricted label and the necessity
for an administration route via injection. Therefore there is still a
large commercial opportunity for an orally available bone anabolic
* Corresponding authors.
E-mail address: david.a.price@pfizer.com (D. Price).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.044

G. Balan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3328–3332
3329
was adopted. A flurry of recent publications, patents and disclo-
sures on clinical compounds has prompted us to report our
research into this highly competitive arena.7
by staying within the amino-acid expression we would have an
opportunity to deliver a safe and efficacious agent providing we
could improve the potency against the human and rat CaSR by
approximately an order of magnitude.
The first orally bioavailable small molecule calcilytic reported
was NPS-2143, however, this compound was hampered by hERG
and P450 inhibition and inappropriate pharmacokinetics due to a
long half life.8 The most advanced compound in the clinic was rona-
caleret which had reached Phase 2 by the start of 2008, Figure 1.9 The
structural similarities between NPS-2143 and ronacaleret are clear
and the addition of the carboxylic acid moiety could improve both
the off target pharmacology issues associated with NPS-2143 and
also modulate the pharmacokinetic half life. Ronacaleret has shown
insufficient efficacy in clinical trials and development has been ter-
minated. Clearly this lack of efficacy could be due to a variety of fac-
tors such as poor exposure, limited ability to dose escalate due to
safety concerns or simply due to a lack of confidence in the mecha-
nism. The confidence in the mechanism within the clinical setting
was reestablished shortlyafter with the disclosurethat JTT-305(also
known as MK-5442) had shown a positive proof of concept in a 12
week Phase 2a study. The structure of JTT-305 is unknown but is be-
lieved to be in a similar chemotype to that of ronacaleret.
Taking ronacaleret as the project lead the ability of ronacaleret to
increase endogenous PTH secretion was evaluated in Sprague–Daw-
ley male rats following oral administration. The plasma concentra-
tion of PTH1-84 was measured by using a commercially available
PTH ELISA kit which detects the full length of rat PTH1-84 specifi-
cally. No PTH spike was discernible upon oral administration of
ronacaleret at 10 mg/kg (suspension in 0.5% methylcellulose)
despite achieving a reasonably high total systemic exposures
(Cmax ꢀ 300 nM); the free concentration of ronacaleret was there-
fore <3 nM. In a parallel study the oral bioavailability of ronacaleret
in rats was determined to be 12%. The IC50 of ronacaleret against the
rat CaSR receptor expressed in HEK cells was determined to be
320 nM. The conclusion was that for compounds with equivalent
pharmacokinetic properties a significant increase in potency would
be required to deliver an anticipated in vivo efficacy signal of PTH
elevation. Therefore, it was decided that an initial goal of identifying
significantly more potent compounds than ronacaleret was an
appropriate project starting point. In our hands the IC50 of ronacaler-
et against the human CaSR receptor expressed in HEK cells was
determined to be 150 nM, which was within threefold of the rat po-
tency. Previous experience of working within the amino-alcohol ser-
ies had shown a consistent relationship between rat and human
potencies (<threefold variation). We did not foresee species differ-
ences in potency to be a major concern in project prosecution and
this was indeed the case. For the sake of expediency only rat CaSR
data was generated for compounds that we were going to progress
to in vivo studies to measure the PTH response.
A review of the available literature suggested that the carbox-
ylic acid was tolerated in a variety of vectors attached to the phen-
oxy motif. Indeed, using in silico structural overlays, it was
impossible to have the carboxylic acid overlap from the differing
expressions within this chemotype while maintaining the posi-
tions of the basic center and hydroxyl groups. When NPS-2143
and ronacaleret were profiled in our laboratories it appeared that
NPS-2143 was significantly more potent as a negative allosteric
modulator of the CaSR. This confirmed that the acid expression is
not delivering a key interaction with the receptor, rather it is mod-
ifying the overall physio-chemistry of the molecule. Our initial
strategy was to retain the acid expression and then deliver novel,
inventive methods of attaching the carboxylic acid to the amino-
alcohol template. Comparing the human microsomal stability of
NPS-2143 and ronacaleret it was clear that the acid expression
has also significantly reduced the lipophilicity of the series and
delivered a significant improvement in human microsomal stabil-
ity, Table 1. Both NPS-2143 and ronacaleret have an equivalent free
fraction in the microsomal preparation so this is not driving the
difference in stability. The disconnect between rat and human liver
microsomal stability for ronacaleret was concerning and difficult to
find a strong rationale for. This data gave us the opportunity to
moderately increase the lipophilicity of the series while retaining
the microsomal stability and potentially improving potency
through hydrophobic interactions with the receptor. We were also
aware that there were many other non P450 mediated clearance
pathways available to lipophilic amino-acids, and it was decided
to progress compounds aggressively to in vivo studies as decision
making experiments.
In order to meet these design criteria, the team was intrigued at
the utility of palladium mediated, sequential reactions on norbor-
nadiene as illustrated in Scheme 1. Not only is the starting material
readily available, but the hydroarylation reaction is tolerant of a
variety of differing aryl functionalities giving us synthetic flexibil-
ity in the preparation of analogues.10 Indeed, both the cyclopropa-
nation and hydroarylation reactions can be safely performed on
multigram quantities. It was decided that for the sake of speed
the project would screen the final mixture of diastereoisomers
and then for compounds of interest separate the diastereoisomers
for in vivo profiling.
We were delighted to find that the compounds prepared com-
pared favorably in terms of potency with compound 1 being only
twofold less potent than NPS-2143. Progression of 3 to microsomal
preparations also suggested that in this molecular expression we
had similar clearance rates between rat and human microsomes
which gave us hope that we would be able to predict our clinical
dose with an improved degree of confidence. We were aware that
within this series we working at the higher end of accepted phys-
iochemical space for orally bioavailable drugs. There have been
numerous analysis’ investigating molecular properties that influ-
ence oral bioavailability and it is generally agreed that molecular
Profiling of ronacaleret through a battery of binding assays
showed no significant activity against a range of pharmacologically
relevant enzymes, ion channels and receptors up to 10 lM, as mea-
sured in binding competition and functional assays. Further profil-
ing for cardiac effects and in particular those mediated via hERG
showed only very weak affinity using whole cell voltage clamp
techniques with <10% inhibition at 10
lM. We were confident that
F
OH
H
N
CN
OH
H
F
O
Cl
O
N
CO2H
Figure 1. Structure of NPS-2143 and ronacaleret.

3330
G. Balan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3328–3332
Table 1
Comparison of potencies and microsomal stability of phenoxy analogues
R2
OH
R2
OH
H
H
N
R1
R1
O
N
O
R3
R3
CO2H
HO2C
Compoundsa
R1
R2
R3
h-CaSR (IC50, nM)b
HLM (clint)c
RLM (clint)d
MWt
clog P (log D)
NPS-2143
Ronacaleret
23
146
157
<8
>100
90
409
448
5.1
2.1 (1.5)
*
*
1
2
3
4
F
F
56
19
15
17
23
35
50
25
38
526
478
515
470
3.7 (2.6)
3.5
H
H
H
H
900
6
*
*
CN
H
3.3 (2.0)
3.3
F
3500
Me
F
*
*
*
5
6
H
H
H
F
300
23
20
15
9
Not done
499
507
530
3.4
Me
F
CN
CN
27
3.0 (2.3)
Me
N
7
350
<10
1.5
O
a
All compounds are a 50:50 mixture of two diastereoisomers as drawn.
b
c
Potency at human recombinant CaSR recpetore expressed in HEK cells. All assay determinations n = 3.
Human liver microsome stability expressed as intrinsic clearance ( L/min/mg of microsomal protein).
L/min/mg of microsomal protein).
l
d
Rat liver microsome stability expressed as intrinsic clearance (
l
weight and hydrogen bonding propensity are key components.11
We were keen that any further manipulations within this series
did not further compromise these characteristics. Incorporating
the 2-cyano group generated compound 3 where we had achieved
high potency, acceptable microsomal stabilities and physiochemi-
cal profile. Attempts to improve upon compound 3 through alter-
native expressions to the indane were unsuccessful with
compound 6 being the most interesting profiled.
Comparing compounds 3 and 6 from their in vitro profile, they
were difficult to differentiate except that compound 3 had a repro-
ducibly improved potency against the CaSR. It was decided to sep-
arate the diastereoisomers by careful chromatography and perform
rat in vivo pharmacokinetic studies on the individual diastereoiso-
mers. Interestingly, the separated diastereoisomers were equipo-
tent at the CaSR receptor again confirming our hypothesis that
the carboxylic acid is not undergoing a key interaction with the
a
CO2H
b
Ar
Ar
CO2H
CO2H
c
CO2H
Scheme 1. Synthesis of cyclopropyl acids; Reagents and conditions: (a) ethyldi-
azoacetate, PdCl2, hexanes, 60 °C; (b) NaOMe, IMS, rt then conc. H2SO4, EtOH, rt; (c)
ArI, Pd(OAc)2, DMSO, Et3N, P(o-tol)3, HCO2H, 75 °C.

G. Balan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3328–3332
3331
Table 2
Comparison of potencies and microsomal stability of benzyloxy analogues
R
R
O
N
H
N
H
O
OH
OH
CO2H
CO2H
Compoundsa
R
h-CaSR (IC50, nM)b
HLM (clint)c
35
RLM (clint)d
MWt
510
clog P (log D)
F
*
8
25
45
3.5 (2.4)
Me
*
9
50
16
14
530
3.7 (2.8)
F
*
Cl
Cl
10
18
33
23
Not done
38
526
470
4.1 (2.6)
3.7
Me
*
11
3500
a
All compounds are a 50:50 mixture of two diastereoisomers as drawn.
b
c
Potency at human recombinant CaSR recpetore expressed in HEK cells. All assay determinations n = 3.
Human liver microsome stability expressed as intrinsic clearance ( L/min/mg of microsomal protein).
L/min/mg of microsomal protein).
l
d
Rat liver microsome stability expressed as intrinsic clearance (
l
receptor in terms of binding energy. Following iv administration
(1 mg/kg) for each diastereoisomer the volume of distribution
was moderate for both (Vd = 5 L/kg), however, with a clearance of
approximately liver blood flow (70 and 83 mL/min/kg) the mea-
sured half life of the diastereoisomers was 1.5 h. An oral leg was
performed (10 mg/kg), and the oral bioavailability was poor for
both diastereoisomers (<10%) due to the high clearance. The exper-
iment was valuable as it directed the team to move away from the
phenoxy template and seek alternative expressions for the acid
moiety where our in vitro assays were more predictive of in vivo
outcomes. The in vitro/in vivo clearance disconnect is not entirely
surprising as the microsomal preparation will only significantly
predict for the P450 mediated oxidation component of the
in vivo clearance. Within this series the opportunities for a range
of other in vivo clearance mechanisms such as glucuronidation
and organic anion/cation transporters is high and with our current
knowledge of these systems an accurate prediction of in vivo clear-
ance is impossible.12 This confirmed our desire within the project
to drive compounds as rapidly as possible to an in vivo assessment
of pharmacokinetics rather than relying on in vitro assays alone.
A reassessment of the literature suggested that the phenoxy
expression could be replaced by a benzyloxy motif.13 This was
highly attractive as we could again use the hydroarylation method-
ology and reuse phenethyl intermediates previously prepared. This
enabled the rapid identification of a number of interesting com-
pounds, Table 2. Again these compounds are of equivalent potency
to ronacaleret and the correlation between human and rat micro-
somal stability was good except for one outlier, compound 11.
We have no clear rationale as to why there should be a significant
difference between the microsomal stabilities in human and rat for
compound 11. From these compounds it was decided to progress
compound 8 as the strongest representative of the series into
in vivo rat pharmacokinetic studies. After separation of the diaste-
reoisomers we were delighted to find that after oral administration
(10 mg/kg) a diastereoisomer showed high oral bioavailability
(>60%) and a total plasma Cmax of 1 M. With this drug concentra-
l
tion we were confident that this was a compound of quality to pro-
gress to in vivo efficacy studies. Advancing 8 to a PTH secretion
experiment in Sprague–Dawley male rats using oral administration
(10 mg/kg) the compound displayed a robust PTH response compa-
rable to literature agents. Further progression through a panel of
screens to assess off target pharmacology showed no significant
activity <10 lM and no activity at the hERG channel or P450 en-
zymes. With this improvement over both NPS-2143 and ronacaler-
et in terms of both efficacy and off target pharmacology the
compound was profiled in an in vivo toleration study. Pleasingly
it showed no significant adverse effects at the 500 mg/kg dose used
in the 4 day rat study and was accepted for progression into pre-
clinical studies.
In conclusion we have described the project strategy and design
criteria we used for the discovery of a calcylitic using a simple yet
powerful synthetic methodology. The compound displays robust
efficacy in a standard rodent model that is routinely used in the
osteoporosis arena. It also has appropriate pharmacokinetics in
rat with good oral bioavailability and is well tolerated in prelimin-
ary in vitro and in vivo safety studies. Further work in this highly
attractive mechanism will be described in following publications.
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