Discovery and structure-activity relationships of 2-benzylpyrrolidine- substituted aryloxypropanols as calcium-sensing receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2004.11.071

A structure-activity relationship study of the amine portion of the calcilytic compound NPS-2143 resulted in the discovery of substituted 2-benzylpyrrolidines as replacements for the 1,1-dimethyl-2-naphthalen-2-yl- ethylamine. When compared to NPS-2143, a

Full text of DOI:10.1016/j.bmcl.2004.11.071

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1225–1228
Discovery and structure–activity relationships of
2-benzylpyrrolidine-substituted aryloxypropanols as
calcium-sensing receptor antagonists
Wu Yang,a,* Yufeng Wang,a Jacques Y. Roberge,a Zhengping Ma,b Yalei Liu,a
R. Michael Lawrence,a David P. Rotella,a Ramakrishna Seethala,b
Jean H. M. Feyenb and John K. Dickson, Jr.a
aDiscovery Chemistry, Bristol–Myers Squibb Pharmaceutical Research Institute, P.O. Box 5400, Princeton,
NJ 08543-5400, USA
bMetabolic and Cardiovascular Drug Discovery, Bristol–Myers Squibb Pharmaceutical Research Institute, P.O. Box 5400,
Princeton, NJ 08543-5400, USA
Received 4 October 2004; revised 22 November 2004; accepted 29 November 2004
Available online 23 December 2004
Abstract—A structure–activity relationship study of the amine portion of the calcilytic compound NPS-2143 resulted in the discov-
ery of substituted 2-benzylpyrrolidines as replacements for the 1,1-dimethyl-2-naphthalen-2-yl-ethylamine. When compared to NPS-
2143, a newly discovered compound, 3h, exhibited similar potency as a calcium-sensing receptor (CaR) antagonist and a superior
human ether-a-go-go related gene (hERG) profile.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Osteoporosis is a major and growing disease that affects
over 28 million people, especially postmenopausal
women. Currently, available therapies include anti-
resorptive agents such as estrogen, bisphosphonates,
calcitonin and selective estrogen receptor modulators
(SERMs). These anti-resorptive agents can prevent bone
loss but have limited effects on new bone formation and
each has been associated with efficacy, safety, tolerabil-
ity and/or cost limitations. The only FDA-approved
anabolic agent, teriparatide, a synthetic 1-34 amino acid
peptide fragment of human parathyroid hormone
(PTH), has been used to treat severe osteoporosis as a
daily subcutaneous injection.1 As an alternative to daily
PTH injections, one could envision stimulating the re-
lease of endogenous PTH from the parathyroid glands.
Evidence suggests that physiologically relevant plasma
levels of endogenous PTH are sufficientto elicita bone
anabolic response.2 It is well established that PTH secre-
tion is inversely related to plasma calcium concentra-
tion.3 The cloning of a calcium-sensing receptor (CaR)
from parathyroid tissue has recently elucidated the
molecular basis of this control mechanism.4 A small
molecule CaR antagonist and/or a negative allosteric
modulator could act as an oral anabolic agent. NPS-
2143 is the first reported orally active low molecular
weight calcilytic that stimulates the secretion of endoge-
nous PTH by antagonizing the CaR on the surface of
parathyroid cells.5 The resulting increase in endogenous
circulating PTH results in the stimulation of new bone
growth. In animal studies, when co-administered with
the anti-resorptive agent 17b-estradiol, NPS-2143 in-
creased trabecular bone volume and bone mineral den-
sity, and the results were comparable to those
obtained by daily PTH injection.
In vitro profiling shows that NPS-2143 has potential lia-
bilities such as potent hERG inhibition. Cardiac hERG
channel blockage is an important mechanism for drug-
induced QT prolongation associated with torsades de
pointes and sudden death6 and in a patch-clamp assay,
NPS-2143 inhibited the hERG K+ channel with an
IC50 of 194 nM. In an attempt to modulate this activity,
we embarked on a search for a replacementof hte
secondary gem-dimethylamine in NPS-2143, a common
pharmacophore in mostof ht e compounds disclosed in
Keywords: Osteoporosis; Calcium-sensing receptor antagonist.
*
Corresponding author. Tel.: +1 609 818 6493; fax: +1 609 818 3460;
e-mail: wu.yang@bms.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.071

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W. Yang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1225–1228
CN
OH
CN
OH
Ph
H
N
Cl
O
Cl
O
N
NPS-2143
3a CaR FLIPR IC50 = 2.2 µM
Figure 2. Hitfrom library.
CN
OH R1
Cl
O
N
R2
The 2,5-difluoro-substituted compound 3d showed sim-
ilar activity to 3a. Moving the benzyl group from the
4-position to the 3- or 2-positions on the piperidine ring
(3e and 3f) led to greatly reduced activity. However,
when the ring size was reduced from the six-membered
ring piperidine to a five-membered ring pyrrolidine,
an improvement in activity was observed. While the
3-benzylpyrrolidine 3g showed an IC50 of 10 lM, the
Figure 1.
the patent literature (Fig. 1).7 A solution phase library
approach was undertaken where a structurally diverse
setof amines was employed.
The synthesis of the solution phase library was carried
outin a 96-well mini-reacotr ( Scheme 1). 3-Chloro-2-
cyanophenol 1 was treated with an excess of (R)-epichlo-
rohydrin in the presence of Cs2CO3 in refluxing CH3CN
overnight. Upon completion of the reaction, the solids
were filtered and the excess reagent was removed by ro-
tary evaporation. The resulting crude epoxide 2 was
then redissolved in i-PrOH and aliquoted into a set of
93 primary and secondary amines. The reaction mix-
tures were heated in capped vials at 80 ꢁC overnight.
The excess primary amines were trapped with formyl-
ated polystyrene resin and the excess secondary amines
were removed with an isocyanate resin. The crude prod-
ucts were purified by loading onto a cation exchange re-
sin (SCX) column, which was firstwashed wiht MeOH
to remove the nonbasic impurities and then with NH3/
MeOH to afford the final products 3 in 8–100% yields
(average 80%) after preparative HPLC purification.
Table 1. Inhibition of intracelluar Ca2+ release by substituted piper-
idines and pyrrolidines as measured by FLIPRTM
CN
OH R1
N
Cl
O
R2
Compd
NR1R2
FLIPR IC50 (lM)a
H
N
NPS-2143
0.08 ( 0.03)
Ph
3a
3b
2.2 ( 0.04)
>30
N
N
OMe
OMe
The products from the above library were screened in a
fluorescence based intracellular Ca2+ mobilization assay
in HEK cells containing over-expressed recombinant
human calcium-sensing receptor. Inhibition by test com-
pounds of intracellular Ca2+ release stimulated by extra-
cellular Ca2+ was measured using FLIPR technology as
previously described.8 4-Benzylpiperidine 3a was identi-
fied from this assay as a lead CaR antagonist with an
IC50 of 2.2 lM (Fig. 2).
3c
0.64 ( 0.04)
N
F
F
3d
3e
3f
2.1 ( 0.34)
N
N
>30
Ph
A brief SAR study of compound 3a was carried out
focusing on different substituted cyclic amines. Table 1
summarizes the antagonist activities of exemplary piper-
idine and pyrrolidine analogues of compound 3a. meta-
Substitution of the 4-benzyl group with a methoxy
group (3c) improved the potency by 3-fold, while para-
substitution (3b) resulted in a dramatic loss of activity.
Ph
N
>30
Ph
3g
3h
10 ( 0.37)
0.35 ( 0.004)
N
OH R1
N
Ph
N
CN
CN
CN
O
Cl
Cl
Cl
OH
O
O
a
b
R2
1
2
3
(S)-3h
(R)-3h
0.12 ( 0.006)
>30
Scheme 1. Reagents and conditions: (a) (R)-epichlorohydrin, Cs2CO3,
CH3CN, reflux overnight, 84%; (b) i-PrOH, NHR1R2, 80 ꢁC, over-
night, 8–100%.
a Values are means of two experiments, standard deviation is given in
parentheses.

W. Yang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1225–1228
1227
Table 2. Inhibition of intracelluar Ca2+ release by substituted 2-
benzylpyrrolidines as measured by FLIPRTM
Ph
Ph
c
O
a,b
R3
P
O
CO2H
N
H
N
N
CN
OH
ArMgBr
O
O
R5
Cl
O
N
O
CF3
O
CF3
4
5
R4
R3
CN
O
OH
Cl
d or e
N
f
Compd
R3
H
R4
R5
FLIPR IC50 (lM)a
R3
R3
2
N
H
R4
(S)-3h
7a
7b
7c
H
H
H
H
H
H
H
H
H
H
0.12 ( 0.006)
>30
R4
2-MeO
3-MeO
4-MeO
H
6
7
H
0.13 ( 0.006)
0.20 ( 0.006)
0.41 ( 0.02)
0.10 ( 0.007)
>30
R
3 = OMe
R3 = OH
H
g
7d
7e
3,4-Di-MeO
2-OH
4-OH
3-F
H
H
Scheme 2. Reagents and conditions: (a) CF3CO2Et, 1,1,3,3-tetramethyl-
guanidine, 82%; (b) ClP(O)Ph2, NEt3, 79%; (c) THF, À70 ꢁC t o rt ,
17–60%; (d) H2/Pd/C, EtOH, HCl, 48 h, 100%; (e) (i) BF3ÆOEt2,
Et3SiH, 41–55%; (ii) 12.5 N HCl, i-PrOH reflux, 100%; (f) EtOH,
110 ꢁC, 16 h, 47–55%; (g) BBr3, CH2Cl2, À70 ꢁC to rt, 80%.
7f
H
7g
7h
7i
H
0.15 ( 0.005)
1.9 ( 0.16)
>30
4-Me2N
3-MeO
3-F
H
OH
OH
H
H
7j
H
2.3 ( 0.19)
1.4 ( 0.22)
2.8 ( 0.29)
>30
7k
7l
H
OH(R)
F(S)
Di-F
H
H
2-benzylpyrrolidine 3h was over 6-fold more potent than
3a, wit h an IC of 0.35 lM.
50
7m
H
H
a Values are means of two experiments, standard deviation is given in
parentheses.
The two diastereomers composing 3h were separated by
chiral preparative HPLC.9 The fasteluting diastereomer
A was found to have an IC50 of 120 nM while the slow
eluting diastereomer B was inactive at concentrations up
t o 30lM. The absolute stereochemistry at the 2-benzyl
position of diastereomer A was subsequently confirmed
t o beS after an independent asymmetric synthesis of
diastereomer A starting from chiral (S)-2-benzylpyrrol-
idine prepared by the procedure of Nordlander et al.10
in these compounds were demethylated, a surprising
pattern was observed. The 4-hydroxy compound 7f
was inactive, while the 2-hydroxyl compound 7e showed
potent inhibition with an IC50 of 100 nM. A basic dim-
ethylamino group (7h) led to a >10-fold decrease in
activity. Hydroxyl substitution at the benzylic position
(7i and 7j), as well as hydroxylation on the pyrrolidine
ring (7k), proved unfavourable for the antagonistic
activity. Attempts to reduce the basicity of the pyrrol-
idine nitrogen through the addition of b-fluorines (7l
and 7m) led to a stepwise reduction of activity as the
number of fluorines increased.
An alternate route was utilized for the synthesis of other
substituted 2-benzylpyrrolidine analogues 7a–j as illus-
trated in Scheme 2.11 D-proline was protected with a tri-
fluoroacetyl group and converted to the mixed
anhydride 4. Treatment of 4 with various Grignard re-
agents provided the ketones 5, which were converted
to benzylpyrrolidines 6 (R4 = H) either in one step by
hydrogenolysis in acidic EtOH, or in a stepwise fashion
by reduction with BF3ÆOEt2/Et3SiH followed by depro-
tection with refluxing HCl in i-PrOH. In some cases,
The synthesis of the fluorinated analogues is shown in
Scheme 3 starting from the known compounds 2-benzyl-
pyrrolidin-3-ol or 2-benzylpyrrolidin-3-one.12 Fluori-
nation of the alcohol or ketone with bis(2-meth-
oxyethyl)aminosulfur trifluoride (BAST)13 followed by
TFA deprotection provided the mono- or di-fluorinated
pyrrolidines, which were coupled with epoxide 2 in the
absence of solvent to provide the final compounds 7l
and 7m.
the partially reduced hydroxyl intermediates
6
(R4 = OH) were also isolated. Coupling of pyrrolidines
6 with epoxide 2 provided the final compounds 7. In
cases where the R3 substituent was a methoxy group,
the compound could be demethylated to the correspond-
ing phenol by BBr3. The diastereomeric purity of 7 was
determined by chiral analytical HPLC and was found to
be generally over 95% exceptfor 7a, where epimeriza-
tion had occurred at the 2-position of the pyrrolidine
during the synthesis and a 2:3 mixture of diastereomers
was obtained.
Compound (S)-3h was tested in a K+ patch-clamp assay
for the hERG inhibitory activity and was found to
show reduced potency (IC50 = 1.24 lM) compared with
NPS-2143 (IC50 = 0.19 lM). The decreased hERG
Y
F
X
CN
OH
Y
F
Y
a
b,c
Cl
O
N
Ph
Ph
Table 2 summarizes the SAR around the 2-benzylpyrr-
olidine of (S)-3h. Substitution of a methoxy group at
the meta- or para-position of the phenyl ring, as well
as an electron-withdrawing 3-F substitution was toler-
ated (compounds 7b, 7c and 7g). Moving the methoxy
group to the ortho-position (7a) resulted in a dramatic
loss of activity. Disubstitution in compound 7d led to
a 3-fold decrease in potency. When the methoxy groups
N
N
Ph
Boc
Boc
9a (Y = H)
9b (Y = F)
7l (Y = H)
7m (Y = F)
8a (X = OH, Y = H)
8b (X = Y = O)
Scheme 3. Reagents and conditions: (a) BAST, CH2Cl2, À78 ꢁC t o rt ,
overnight, 23% (Y = H), 72% (Y = F); (b) TFA, CH2Cl2; (c) 2, 90 ꢁC,
overnight, two steps 37% (Y = H), 74% (Y = F).

1228
W. Yang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1225–1228
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Technol. Today 1999, 2, 270; (b) Vandenberg, J. I.;
Walker, B. D.; Campbell, T. J. Trends Pharmacol. Sci.
2001, 22, 240.
7. (a) Lago, A. M.; Callahan, J. F.; Bhatnagar, P. K.; Del
Mar, E. G.; Bryan, W. M; Burgess, J. L. WO 0153254,
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activity of (S)-3h suggests it is possible to maintain the
CaR antagonist activity while reducing the hERG liabil-
ity for this series of compounds.
In conclusion, a solution phase library approach was
undertaken to find a novel series of calcium-sensing
receptor antagonists as exemplified by (S)-3h. This new
compound showed comparable potency to NPS-2143
as a calcium-sensing receptor antagonist but possessed
reduced hERG blocking activity.
8. Schroeder, K. S.; Neagle, B. D. J. Biomol. Screen. 1996, 1,
75.
9. Chiral preparative HPLC conditions: CHIRALPAKꢂ
OD column, 5 · 50 cm, with 20% isopropanol/hexane
containing 0.1% triethylamine as the eluent at a flow rate
of 50 mL/min.
Acknowledgements
We would like to thank Mary Lee Conder for perform-
ing the hERG patch-clamp assays on NPS-2143 and
compound (S )-3h. We are also thankful to Drs. Robert
Zahler and Ruth Wexler for valuable suggestions.
10. Nordlander, J. E.; Njoroge, F. G.; Payne, M. J.; Warman,
D. J. Org. Chem. 1985, 50, 3481.
11. (a) Fisher, L. E.; Rosenkranz, R. P.; Clark, R. D.;
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Caroon, J. M.; Galeazzi, E.; Eglen, R.; Whiting, R. L.
Bioorg. Med. Chem. Lett. 1995, 5, 2371; (b) Fisher, L. E.;
Muchowski, J. M.; Galeazzi, E.; Rosenkranz, R. P.;
McClelland, D. L. US Patent 5100912, 1992.
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