The discovery of an orally efficacious positive allosteric modulator of the calcium sensing receptor containing a dibenzylamine core

Article abstract of DOI:10.1016/j.bmcl.2010.07.060

The discovery of a series of novel and orally efficacious type II calcimimetics, developed from the lead compound 1, is described herein. Compound 22 suppressed plasma PTH levels relative to vehicle when dosed orally in a rat pharmacodynamic model.

Full text of DOI:10.1016/j.bmcl.2010.07.060

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5544–5547
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery of an orally efficacious positive allosteric modulator of the
calcium sensing receptor containing a dibenzylamine core
a,
d
e
a
Paul E. Harrington a, , David J. St. Jean Jr. , Jeffrey Clarine , Thomas S. Coulter , Michael Croghan ,
Adam Davenport e, James Davis b, Chiara Ghiron e, Jonathan Hutchinson e, Michael G. Kelly a, Fred Lott b,
Jenny Ying-Lin Lu c, David Martin b,à, Sean Morony b, Steve F. Poon a, Elena Portero-Larragueta e,
Jeff D. Reagan c, Kelly A. Regal d,§, Andrew Tasker a, Minghan Wang b, Yuhua Yang c, Guomin Yao a,
Qingping Zeng a, Charles Henley III b, Christopher Fotsch a
*
*
,
a Chemistry Research and Discovery, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
b Metabolic Disorders, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
c Metabolic Disorders, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
d Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
e Evotec (UK) Ltd, 114 Milton Park, Abingdon, Oxfordshire OX14 4SA, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery of a series of novel and orally efficacious type II calcimimetics, developed from the lead
compound 1, is described herein. Compound 22 suppressed plasma PTH levels relative to vehicle when
dosed orally in a rat pharmacodynamic model.
Received 9 June 2010
Revised 13 July 2010
Accepted 15 July 2010
Available online 12 August 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Calcimimetic
Allosteric modulator
The regulation of serum calcium ions is vital to a wide range of
physiological processes. The mechanism by which the concentra-
tion of serum ionized calcium is controlled is complex. The para-
thyroid and thyroid glands, bone, kidney, and intestine all act in
concert to tightly regulate the concentration of ionized calcium.1
The calcium sensing receptor (CaSR) plays a fundamental role in
the regulation of extracellular calcium within the physiological
range of 1.1–1.3 mM.2 The CaSR is a cell surface receptor that is
part of the superfamily of G protein-coupled receptors (GPCR)
and is comprised of 1078 amino acids with seven transmembrane
domains. On parathyroid chief cells, the CaSR controls the secre-
tion of parathyroid hormone (PTH), a hormone that is responsible
for raising serum ionized calcium levels.
vesicles. PTH then raises extracellular Ca2+ by stimulating calcium
resorption from the bone and enhancing calcium reabsorption
from the kidney and gut. However, when extracellular Ca2+ con-
centrations are increased, the CaSR is activated, and secretion of
PTH is suppressed. Thus, the CaSR acts as a biological brake, being
activated during times of high extracellular Ca2+, which keeps the
parathyroid from secreting excess PTH. Ligands that mimic the ef-
fect Ca2+ has on the CaSR are called calcimimetics.3
Type I calcimimetics such as inorganic4 and organic polycations
directly activate the CaSR in the form of classic agonists. Type II
calcimimetics function as positive allosteric modulators of the
CaSR and thus increase the sensitivity of the CaSR to extracellular
calcium. Cinacalcet HCl (SensiparÒ, MimparaÒ), a type II calcimi-
metic, is on the market in certain territories for diseases related
to hypercalcemia;5 which include primary and secondary hyper-
parathyroidism (1HPT and 2HPT) and parathyroid carcinoma.
In vitro screening for novel type II calcimimetics was performed
using a cell-based assay in the presence and absence of extracellu-
The concentration of extracellular ionized calcium is regulated
via a feedback loop mechanism involving Ca2+ ions, the CaSR, and
PTH. In response to low extracellular calcium levels (low Ca2+),
the chief cells of the parathyroid secrete PTH from intracellular
lar Ca2+ 6
presence of extracellular Ca2+ were positive allosteric activators
of the CaSR. Benzyl amine 1 (CaSR EC50 = 1.2 M, Fig. 1) was iden-
. Compounds which activated the receptor only in the
* Corresponding authors.
E-mail address: pharring@amgen.com (P.E. Harrington).
l
Present address: Sundance Ranch, 12955 Back Canyon Road, Caliente, CA 93518,
USA.
tified as a lead candidate for further optimization.
à
Initial SAR focused on the B-ring analogs 1–4. The synthesis of
Present address: South San Francisco, CA 94080, USA.
Present address: Array BioPharma Inc., Boulder, CO 80301, USA.
the benzyl amine compounds 1–9 is shown in Scheme 1.7 Reductive
§
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.060

P. E. Harrington et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5544–5547
5545
CF3
H
N
OMe
Br
MeO
(HO)2B
Pd(PPh3)4
H
Me
N
R1
1
K2CO3
µM
CaR EC50 = 1.2 0.3
Me
10
Figure 1. Benzyl amine 1.
OMe
H
N
R1
Me
11-14
CF3
OHC
R
NaBH3CN or
NaBH(OAc)3
H
N
A
B
Scheme 2. Synthesis of biphenyl amines 11–14.
NH2
Me
MeO
MeO
R
Me 1–9
Scheme 1. Synthesis of benzyl amines 1–9.
Cl
Cl
Br
HO2C
Br
H
N
Table 1
SAR of B-ring analogs 1–4
NH2
EDCI, HOBt
86% yield
MeO
MeO
15
O
Me
Me
R3
R2
H
N
CF3
MeO
Me
R1
Cl
(HO)2B
BH3·THF
H
N
a
Compound
R1
R2
R3
CaSR EC50
(
l
M)
CaSR maxa,b (%)
Pd(PPh3)4, Na2CO3
23% yield (from 15)
MeO
Br
1
2
3
4
H
Cl
H
H
H
H
H
H
H
1.2 0.3c
3.7 1.5
0.35 0.20
0.37 0.02
24 4c
110 69
97 11
16
Me
o-ClPh
H
Cl
OMe
88
2
H
N
a
Mean SD with n P 3.
Max (%) is the ratio of the compound induced FLIPR response divided by the
MeO
b
17
Me
maximum FLIPR response induced by a saturating dose of CaCl2 (10 mM) multiplied
by 100.
CF3
c
n = 2.
Scheme 3. Synthesis of chlorobiphenyl analog 17.
Table 2
Table 2 highlights the results from the optimization of the C-
ring phenyl substituent. Compounds 6–7 were prepared to further
examine the SAR at the ortho position (R1) of the C-ring. Within this
limited set of compounds, no improvement in potency relative to 3
was achieved. However, the C-ring para substituted analogs, meth-
SAR of C-ring biphenyl analogs 5–9
R1
H
N
A
B
MeO
C
Me
R2
oxy 8 and trifluoromethyl 9, had an EC50 <1 lM.
Having established the importance of the C-ring phenyl for
in vitro potency, we sought to further improve these compounds
by adding a B-ring methoxy group, a functional group that im-
proved the activity of analog 4 relative to 1. The para-methoxy ana-
logs 11–14 were prepared as shown in Scheme 2. Bromide 10 was
prepared through a reductive amination (Scheme 1). Palladium
catalyzed Suzuki–Miyaura coupling of 10 with the corresponding
boronic acid yielded biphenyls 11–14. The B-ring para-chloro ana-
log 17 was prepared by the route highlighted in Scheme 3. Amide
coupling of (R)-1-(3-methoxyphenyl)ethanamine with 3-bromo-4-
chlorobenzoic acid gave 15 in 86% yield. Amide reduction with
BH3ÁTHF followed by Suzuki–Miyaura coupling provided 17.
Table 3 summarizes the SAR for the substituted biphenyl analogs
11–14 and 17. Significant in vitro potency gains were achieved by
combining the C-ring para-trifluoromethylphenyl with the B-
ring para-methoxy substituents. For example, compound 11
a
Compound
R1
R2
CaSR EC50 (lM)
CaSR maxa (%)
109 40
5
6
7
8
9
F
H
H
H
OMe
CF3
2.7 2.0
2.2 0.3
1.4 0.2
Me
OMe
H
93
92
95
86
7
6
4
9
0.87 0.05
0.62 0.08
H
a
Mean SD with n P 3.
amination8 of the commercially available aldehydes with (R)-1-
(3-methoxyphenyl)ethanamine yielded 1–9 in a single step.
The potency and percent activation in the CaSR assay of the B-
ring analogs 1–4 is shown in Table 1. Both the unsubstituted phe-
nyl analog 1 and the chloro analog 2 had EC50 >1 lM. In addition, 1
had a lower maximal activation. However, compound 3, with the
B-ring ortho-chlorophenyl substituent, was significantly more po-
(EC50 = 0.023 lM) was significantly more potent than either 4 or 9
tent than either 1 or 2 with an EC50 = 0.35
l
M. Furthermore, com-
thus confirming the additive effect of the C-ring para-trifluorometh-
ylphenyl and B-ring para-methoxy groups. However, the B-ring
para-chloro analog 17 was approximately 10-fold less potent than
the corresponding methoxy compound 11. Optimization of the A-
pound 4 (EC50 = 0.37 M) was also more potent than the parent
l
compound 1. These results provided two avenues, B-ring meta aryl
and B-ring para substitution, for further SAR studies and opportu-
nities to further improve the dibenzylamine scaffold.
ring revealed that phenyl (12, EC50 = 0.027 lM) and meta-fluoro

5546
P. E. Harrington et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5544–5547
Table 3
CF3
SAR of substituted biphenyl analogs 11–14 and 17
OMe
OMe
(HO)2B
Pd(PPh3)4, K2CO3
83-100% yield
R2
R
H
R
H
H
Br
N
R1
O
O
O
O
CF3
Me
18, R = H
19
CF3
M)
, R = Me
CF3
Compound
R1
R2
CaSR EC50
(l
CaSR maxa (%)
a
OCF3
OCF3
Cl
11
12
13
14b
17
OMe
H
F
Me
OMe
OMe
OMe
OMe
OMe
Cl
0.023 0.009
0.027 0.017
0.037 0.046
0.16 0.10
87
92
1
9
(HO)2B
99 10
99 13
93 11
Pd2(dba)3, Cy3P
96% yield
20
CF3
0.28 0.02
a
Mean SD with n P 3.
Racemic.
b
F
Table 4
Rat pharmacokinetic parameters for compounds 11 and 12 administered iv
NH2
Me
F
OMe
H
N
Compound
Dosea
CL
Vss
t1/2
AUC0–inf
18
NaBH3CN, MeOH
AcOH
(mg/kg)
(L/h/kg)
(L/kg)
(h)
(ng h/mL)
Me
21
CF3
11
12
3.5
3.0
4.0
3.3
28
22
4.9
6.1
886
905
99% yield
a
Pharmacokinetic studies were conducted in male Sprague–Dawley rats and
were administered in 20% Captisol/water.
NH2
1.
Me
TiCl4, DCM
OMe
H
N
(13, EC50 = 0.037
analog (14, EC50 = 0.16
l
M) were both more active than the meta-methyl
M).
19
2. NaBH4, MeOH
l
R1 R2
Me
54% yield
The pharmacokinetic profiles of the two most promising ana-
logs, 11 and 12, are shown in Table 4. Unfortunately, both com-
pounds exhibited clearance at or above liver blood flow in male
Sprague–Dawley rats when dosed intravenously. Despite the high
clearance, the high volume of distribution for both 11 and 12 re-
sulted in half-lives of 4.9 and 6.1 h, respectively. With these results,
we sought to decrease the in vivo clearance of 12 by blocking po-
tential sites of metabolism. These positions included one of the
benzylic carbons, the para-position on the A-ring, and the B-ring
methoxy, which were predicted as metabolically labile positions
using MetaSite software.9 Based on this theory, fluorinated and
substituted derivatives were synthesized via the routes described
in Scheme 4. Palladium catalyzed Suzuki–Miyaura coupling10 pro-
duced the functionalized aldehydes or acetophenone (18–20) in
high yields. Reductive amination using standard conditions deliv-
ered analogs 21 and 24. Methylated derivatives 22 and 23 were
synthesized in an analogous manner; although the reductive ami-
nation required pre-formation of the acetophenone imine with
TiCl4.11 The diastereomeric ratio was 14:1 favoring the R,R-diaste-
reomer (22). The stereochemistry of the major diastereomer was
confirmed by single crystal X-ray analysis.
CF3
22, R1 = Me, R2 = H
(d.r. = 14:1)
23, R1 = H, R2 = Me
NH2
Me
OCF3
H
N
20
NaBH(OAc)3, DCE
AcOH
Me
24
CF3
74% yield
Scheme 4. Synthesis of analogs 21–24.
Table 5
SAR of fluorinated and substituted analogs of 12
R1
R4
H
N
R2 R3
Me
CF3
M)
a
Table 5 summarizes the SAR for analogs 21–24. An attempt to
incorporate a fluorine at the para position of the A-ring resulted
in decreased activity relative to 12. In addition, the B-ring trifluo-
romethyl compound 24 was less active than 12. However, incorpo-
ration of a methyl group at the unsubstituted benzylic carbon (R3)
resulted in a compound equipotent to 12. There was a strong ste-
reochemical preference for the R,R-diastereomer (cf. analog 22 vs
Compound
R1
R2
R3
R4
CaSR EC50
(
l
CaSR maxa (%)
87 13
21
22
23
24
F
H
H
H
Me
H
OMe
OMe
OMe
OCF3
0.058 0.042
0.017 0.022
3.0 2.9b
H
H
H
Me
H
H
94
8
163 103b
74c
0.11c
a
b
c
Mean SD with n P 3.
n = 2.
n = 1.
23, which had EC50 values of 0.017 and 3.0 lM, respectively).
The pharmacokinetic profile of 22 is shown in Table 6. Relative
to analog 12, the clearance was significantly improved. The high Vss
and the moderate clearance led to a significantly higher AUC rela-
tive to compound 12. Given the promising in vitro and in vivo pro-
file of 22, along with good rat oral bioavailability12 (%F = 60,
Cmax = 90 ng/mL), this molecule was progressed to a rat pharmaco-
dynamic model to access its ability to lower PTH levels.
In this study, compound 22 was dosed orally to normal male
Sprague–Dawley rats to determine the effect on circulating PTH
at varying doses (0.3, 1, 3 mg/kg).13 After a single 0.3 mg/kg (green
line) dose, plasma PTH levels were reduced relative to vehicle
(black line) out to 8 h (Fig. 2). At the highest dose (3 mg/kg, red
line) PTH levels were significantly suppressed after 1 day.

P. E. Harrington et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5544–5547
5547
Table 6
Rat pharmacokinetic parameters for compound 22 administered iva
Acknowledgments
Compound
22
CL (L/h/kg)
1.6
Vss (L/kg)
t1/2 (h)
AUC0–inf (ng h/mL)
2020
We acknowledge Roger Zanon and Yiping Wu for their assis-
tance with formulations and rat pharmacokinetics, respectively.
The authors also thank Rashid Syed for providing X-ray crystallo-
graphic support.
18
9.3
a
Pharmacokinetic study was conducted in male Sprague–Dawley rats and was
administered in 100% DMSO at 3 mg/kg.
References and notes
Compound 22 (3 mg/kg)
1. (a) Brown, E. M. Physiol. Rev. 1991, 71, 371; (b) Mihai, R.; Farndon, J. R. Br. J.
Anaesth. 2000, 85, 29; (c) Silver, J.; Kilav, R.; Naveh-Many, T. Am. J. Physiol. Renal
Physiol. 2002, 283, F367; (d) Kurokawa, K. Kidney Int. 1994, 44, S97; (e)
Dempster, D. W.; Cosman, F.; Parisien, M.; Shen, V.; Lindsay, R. Endocr. Rev.
1993, 14, 690.
2. (a) Brown, E. M.; Gamba, G.; Riccardi, D.; Lombardi, M.; Butters, R.; Kifor, O.;
Sun, A.; Hediger, M. A.; Lytton, J.; Hebert, S. C. Nature 1993, 366, 575; (b) Brown,
E. M.; MacLeod, R. J. Physiol. Rev. 2001, 81, 239; (c) Hofer, A. M.; Brown, E. M.
Nat. Rev. Mol. Cell Biol. 2003, 4, 530.
3. (a) Nagano, N. Pharmacol. Ther. 2006, 109, 339; (b) Urena, P.; Frazao, J. M. Kidney
Int. 2003, 63, S91; (c) Steddon, S. J.; Cunningham, J. Lancet 2005, 365, 2237; (d)
Nemeth, E. F. In Principles of Bone Biology; Bilezikian, J. P., Raisz, L. G., Rodan, G.
A., Eds.; Academic Press: San Diego, 2002; pp 1339–1359; (e) Hebert, S. C. Annu.
Rev. Med. 2006, 57, 349; (f) Harrington, P. E.; Fotsch, C. Curr. Med. Chem. 2007,
14, 3027.
400
300
200
100
0
Compound 22 (1 mg/kg)
Compound 22 (0.3 mg/kg)
Vehicle
4. Nemeth, E. F.; Scarpa, A. J. Biol. Chem. 1987, 262, 5188.
5. Balfour, J. A. B.; Scott, L. J. Drugs 2005, 65, 271.
6. (a) Nemeth, E. F.; Heaton, W. H.; Miller, M.; Fox, J.; Balandrin, M. F.; Van
Wagenen, B. C.; Colloton, M.; Karbon, W.; Scherrer, J.; Shatzen, E.; Rishton, G.;
Scully, S.; Qi, M.; Harris, R.; Lacey, D.; Martin, D. J. Pharmacol. Exp. Ther. 2004,
0
4
8
12
16
20
24
Time (h)
Figure 2. Dose–response effects of 22 (0.3, 1, and 3 mg/kg) or vehicle (2% HPMC/1%
Tween 80/5% Captisol) on plasma parathyroid hormone (PTH) following oral
administration to normal male Sprague–Dawley rats (n = 4/group).
308, 627; (b) The following compounds had CaSR EC50 values <5
absence of extracellular Ca2+
(1.7 M), 11 (0.72 M), 13 (1.3
(0.43 M), 21 (1.7 M), 22 (2.1 M). All remaining compounds had CaSR EC50
values >5
l
M in the
:
3
l
l
lM), 14
l
l
l
l
M in the absence of extracellular Ca2.
7. All compounds were characterized by 1H NMR and LC/MS and their purity was
determined to be >95% by reverse phase HPLC.
In conclusion, a novel series of type II calcimimetics has been
discovered. Although analog 12 possessed excellent in vitro po-
8. Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.; Shah, R. D. J.
Org. Chem. 1996, 61, 3849.
9. Cruciani, G.; Carosati, E.; De Boeck, B.; Ethirajulu, K.; Mackie, C.; Howe, T.;
Vianello, R. J. Med. Chem. 2005, 48, 6970.
10. Kudo, N.; Perseghini, M.; Fu, G. C. Angew. Chem., Int. Ed. 2006, 45, 1282.
11. Bringmann, G.; Geisler, J.-P. Tetrahedron Lett. 1989, 30, 317.
12. Oral pharmacokinetic study was conducted in male Sprague–Dawley rats and
was administered in 2% HPMC/1% Tween 80/5% Captisol in water at 3 mg/kg.
tency relative to 1 (EC50 = 0.027 vs 1.2 lM), it displayed high
in vivo clearance in rats (CL = 3.3 L/h/kg). Systematic attempts to
improve clearance or slow metabolism led to the discovery of
analog 22, which maintained the in vitro potency of 12 while sig-
nificantly improving the in vivo clearance. In a rat pharmacody-
namic model, compound 22 showed a decrease in circulating
PTH levels in a dose dependent manner.
13. The concentration of 22 at 1 h was:
7
1 ng/mL (0.3 mpk), 17 8 ng/mL
(1 mpk), and 37 12 ng/mL (3 mpk).