Metabolism-guided discovery of a potent and orally bioavailable urea-based calcimimetic for the treatment of secondary hyperparathyroidism

Article abstract of DOI:10.1016/j.bmcl.2013.10.050

A series of urea based calcimimetics was optimized for potency and oral bioavailability. Crucial to this process was overcoming the poor pharmacokinetic properties of lead thiazole 1. Metabolism-guided modifications, characterized by the use of metabolite identification (ID) and measurement of time dependent inhibition (TDI) of CYP3A4, were essential to finding a compound suitable for oral dosing. Calcimimetic 18 exhibited excellent in vivo potency in a 5/6 nephrectomized rat model and cross-species pharmacokinetics.

Full text of DOI:10.1016/j.bmcl.2013.10.050

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6625–6628
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Metabolism-guided discovery of a potent and orally bioavailable
urea-based calcimimetic for the treatment of secondary
hyperparathyroidism
c
d
e
Paul M. Wehn a, Paul E. Harrington b, , Timothy J. Carlson , James Davis , Pierre Deprez ,
Christopher H. Fotsch b, Mark P. Grillo c, Jenny Ying-Lin Lu f, Sean Morony d, Kanaka Pattabiraman a,
Steve F. Poon b, Jeff D. Reagan f, David J. St. Jean Jr. b, Taoues Temal e, Minghan Wang d, Yuhua Yang f,
Charles Henley III d, Sarah E. Lively a
⇑
a Department of Medicinal Chemistry, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, USA
b Department of Medicinal Chemistry, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
c Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, USA
d Department of Metabolic Disorders, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
e Galapagos SASU, 102 Avenue Gaston Roussel, 93230 Romainville, France
f Department of Metabolic Disorders, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of urea based calcimimetics was optimized for potency and oral bioavailability. Crucial to this
process was overcoming the poor pharmacokinetic properties of lead thiazole 1. Metabolism-guided
modifications, characterized by the use of metabolite identification (ID) and measurement of time depen-
dent inhibition (TDI) of CYP3A4, were essential to finding a compound suitable for oral dosing. Calcimi-
metic 18 exhibited excellent in vivo potency in a 5/6 nephrectomized rat model and cross-species
pharmacokinetics.
Received 3 September 2013
Revised 22 October 2013
Accepted 23 October 2013
Available online 31 October 2013
Keywords:
Calcimimetics
Ó 2013 Elsevier Ltd. All rights reserved.
Positive allosteric modulators
1,2,4-Thiadiazoles
Metabolite ID
Time dependent inhibition
Secondary hyperparathyroidism (secondary HPT) is a condition
characterized by the chronic elevation of parathyroid hormone
(PTH) that often develops in patients with compromised kidney
function.1 The regulation of PTH levels is governed by the cal-
cium-sensing receptor (CaSR), a class 3 G protein-coupled receptor
(GPCR) primarily expressed on the parathyroid gland.2 Increases in
serum Ca2+ concentration raise the level of activation of the CaSR
and inhibit PTH secretion, whereas decreases in serum Ca2+ concen-
tration reduce CaSR activation and enhance PTH secretion.3 Left
untreated, secondary HPT can lead to limb deformities as well as
bone and joint pain. The discovery of positive allosteric modulators
of the CaSR, type II calcimimetics, represents a novel therapy for the
treatment of secondary HPT.4 Such agents increase the sensitivity of
the CaSR to serum Ca2+ and thereby decrease secretion of PTH.
II calcimimetic with the disclosure of R-568 and related analogues.5
Subsequent work to identify new calcimimetic agents has focused
almost entirely on derivatives that retain a basic
a-methylbenzyl
amine moiety.6 A novel series of benzothiazole urea based calcim-
imetics arising from R-568 and fendiline was recently disclosed.7
Further development of this series led to 1.8 In vivo studies with
1, using a 5/6 nephrectomized rat model of kidney failure wherein
O
N
H
NHSO2Me
Ph
N
N
N
In
a groundbreaking publication, Nemeth and co-workers
1
described the in vitro and in vivo pharmacology of the first type
Ph
O
S
hCaSR EC50 = 16 nM
Rat CL = 3.8 L/h/kg
⇑
Corresponding author. Tel.: +1 805 313 5564; fax: +1 805 480 1337.
E-mail address: pharring@amgen.com (P.E. Harrington).
Figure 1. Thiazole 1, a urea-based calcimimetic.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.10.050

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P. M. Wehn et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6625–6628
H
N
NHSO2Me
N
N
H
NHSO2Me
N
N
N
O
S
S
O
N
O
S
O
NHAc
Reactive epoxide metabolite
HO2C
1a
, probable N-acetylcysteine adduct
RLM
NHSO2Me
(revealed by tandem MS)
H
N
HO
Ph
N
N
H
O
N
N
Ph
O
S
1
H
N
S
N
O
HO2C
Ph
O
NHAc
Reactive quinone methide
OH
1b
, probable N-acetylcysteine adduct
(revealed by tandem MS)
Figure 2. Metabolites of 1 formed from incubation with rat liver microsomes (RLM) and elucidated by tandem mass spectrometry.
Table 1
Table 2
Heterocyclic replacements of thiazole
Impact of electron withdrawing groups on TDI potential
O
N
O
N
H
N
H
Ph
Ph
N
Ph
N
N
N
Ar
Ph
O
S
Ph
O
X
Compound
Ar
hCaSR EC50 (nM)a
300
Compound
X
%3A4 Activity Remaininga
hCaSR EC50 (nM)b
Ph
N
O
7
8
9
10
11
H
F
22
28
59
76
91
36
53
17
157
18
2
SO2Me
CN
Cl
Ph
Ph
Ph
Ph
3
4
5
6
1030
286
175
23
N
N
S
N
S
a
CYP3A4 experiments were performed as previously described Ref. 11
Values are an average of at least two determinations.
b
N
N
Table 3
Chlorothiazole and thiadiazole analogues of 1
N
N
O
N
N
S
a
Values are an average of at least two determinations.
H
R
Ph
N
N
N
Ph
O
S
X
one kidney and 2/3 of the second kidney have been surgically re-
moved, showed promise demonstrating a modest reduction in ser-
Compound
X
R
hCaSR EC50 (nM)a PK Datab CL (L/h/kg)/%F
um PTH levels. Despite these positive features,
1 exhibited
12
13
14
15
CCl NHSO2Me
CCl SO2NH2
3
2
6
3
1.4/37
0.42/16
4.0/23
3.8/23
substantial liabilities including poor pharmacokinetics (PK). This
account describes our systematic efforts to address the metabolism
of 1 (Fig. 1).
N
N
NHSO2Me
SO2NH2
a
To understand the high clearance of 1, a series of metabolite ID
studies was performed. Incubation of 1 in the presence of rat liver
microsomes (RLM) indicated sites of oxidation at the thiazole, mor-
pholine, and gem-diphenyl moieties. Further studies to elaborate
the specific nature of the oxidations were performed by incubating
1 in RLM containing N-acetylcysteine (Fig. 2). These studies sug-
gested the intermediacy of a reactive epoxide in the formation of
metabolite 1a and implicated oxidation of the phenyl ring to a qui-
none methide in the formation of metabolite 1b. The intermediacy
of specific oxidations in the formation of metabolites 1a and 1b
suggested a potential strategy to improve their stability.
Values are an average of at least two determinations.
Pharmacokinetic studies were conducted in male Sprague–Dawley rats and
were administered at 0.5 mg/kg IV and 2 mg/kg PO.
b
Numerous reports indicate thiazoles as elements of oxidative
susceptibility.9 Our strategy to prevent epoxidation was to replace
the thiazole nucleus with other heterocycles lacking the potential
for epoxidation (Table 1). Despite considerable effort, only 1,2,4-
thiadiazole 6 (hCaSR EC50 = 23 nM)10 was identified as a competent
bioisostere of 1.

P. M. Wehn et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6625–6628
6627
Table 4
Stabilization of the diphenyl motif by fluorination
O
N
H
N
F
R
N
N
O
S
X
F
Compound
F
X
R
hCaSR EC50 (nM)a
PK Datab CL (L/h/kg)/%F
16
17
18
19
20
21
2-F
3-F
4-F
4-F
4-F
4-F
CCl
CCl
CCl
CCl
N
SO2NH2
SO2NH2
SO2NH2
NHSO2Me
SO2NH2
NHSO2Me
4
2.3/20
—
0.32/36
0.81/1.0
1.6/39
1.3/48
19
10
9
9
20
N
a
Values are an average of at least two determinations.
Pharmacokinetic studies were conducted in male Sprague–Dawley rats and were administered at 0.5 mg/kg IV and 2 mg/kg PO.
b
formation. Using 78 as a baseline, substitution with –F, –SO2Me,
–CN, and –Cl was explored (Table 2 and 8–11). Reduced TDI poten-
tial was observed upon substitution with chloro analog 11 proving
most effective. Fluorine did not significantly decrease bioactivation
of the thiazole ring (7 vs 8).
Table 5
PK summary for compound 18
Species
CL (L/h/kg)
Vss (L/kg)
MRT IV (h)
t1/2 (h)
%F
Rat
0.32
0.024
0.098
2.0
0.21
1.2
6.3
8.9
13
7.0
8.0
11
36
15
47
The chloro substituted thiazole analogue 128 demonstrated a
nearly 3-fold improvement in clearance relative to 1 (Table 3).
Interestingly, the reverse sulfonamide analogue 138 resulted in a
further reduction in clearance. Unfortunately, both thiadiazole12
analogues 14 and 15 were rapidly cleared in vivo. Further improve-
ments to PK were realized by modification of the gem-diphenyl
group.
Nonhuman Primate
Beagle Dog
An alternate strategy to reduce the oxidative susceptibility of
the thiazole in 1 was to modulate reactivity by substitution at
the 5-position of the thiazole. We measured time-dependent inac-
tivation (TDI) of CYP3A411 as an estimate of reactive metabolite
Figure 3. Sustained reduction of PTH and ionized calcium after oral administration of 18 to 5/6 nephrectomized rats.
O
O
N
O
O
H
Ar1
O
Ar2
Ar1
Ar2
a, b
c, d
e
H2N
+
N
OEt
Ar2
O
Ar1
NH2
X
N
Ar1
NH
23
P(OEt)2
EtO
22
O
Ar2
O
N
H
f
Ar1
N
N
Ar3
Ar2
O
Scheme 1. (a) NaH, PhMe, 0 °C for 1 h, then 50 °C, 3 d; (b) Pd/C, 1 atm H2, EtOH, 25 °C, 16 h; (c) LiAlH4, THF/PhMe, –78 °C to 0 °C; (d) SO3Ápyridine, Et3N, DMSO, 25 °C; (e)
NaBH(OAc)3, ClCH2CH2Cl, 25 °C; (f) Ar3NH2, DMAP, CDI, 40 °C, 2.5 d.

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P. M. Wehn et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6625–6628
Metabolite ID studies with N-acetylcysteine had indicated the
References and notes
formation of covalent adducts at the phenyl rings suggesting the
intermediacy of a quinone methide (Fig. 2, 1b). As with the thia-
zole, the installation of electron withdrawing groups on the unsub-
stituted phenyl ring was hypothesized to reduce metabolism.
Efforts focused on the incorporation of electron withdrawing ele-
ments. As such, a series of fluorinated derivatives were prepared
(Table 4 and 16–21). Ortho-fluorination resulted in a 2-fold
decrease in potency relative to 13. Meta- and para-fluorination
compromised activity by 10- and 5-fold, respectively, relative to
13. Unfortunately, the ortho-fluorinated derivative 16 was cleared
more rapidly in rats than the parent analogue 13.13 In all cases,
para-fluorination had lower clearance than their unsubstituted
congeners, especially in the case of the thiadiazoles 20 and 21.
Additional pharmacokinetic profiling of 18 in higher species
confirmed the good cross-species pharmacokinetics (Table 5).
These results demonstrate the pharmacokinetic improvements
realized by a metabolism-guided approach to analogue design. A
screen of off-target activities using a functional GPCR platform at
CEREP indicated that 18 showed no confounding off-target activ-
ity.14 Moreover, 18 exhibited robust lowering of PTH and ionized
calcium (Ca2+), relative to vehicle, in a 5/6 nephrectomized rat
model of kidney failure when dosed orally at 6 mg/kg (Fig. 3).15
The modular route by which analogues were prepared is de-
scribed in Scheme 1. The gem-diphenyl ester 22 formed from the
corresponding benzophenone via a Horner–Wadsworth–Emmons
reaction. Amine 23 was formed in three steps from 22 by a reduc-
tion–oxidation-reductive amination sequence. The urea function-
ality was formed by coupling ArNH2 with carbonyldiimidazole
(CDI) in the presence of 4-dimethylaminopyridine (DMAP).
1. (a) Cunningham, J.; Locatelli, F.; Rodriguez, M. Clin. J. Am. Soc. Nephrol. 2011, 6,
913; (b) Rodriguez, M.; Nemeth, E.; Martin, D. Am. J. Physiol. Renal Physiol. 2005,
288, F253; (c) Skoreckim, K.; Green, J.; Brenner, B. M. In Harrison’s Principles of
Internal Medicine; Kasper, D. L., Braunwald, E., Fauci, A. S., Hauser, S. L., Longo, D.
L., Jameson, J. L. Eda., Eds., 16th ed.; ; McGraw-Hill: New York, NY, 2005; Vol.
261, pp 1653–1663.
2. 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.
3. (a) Ward, B. K.; Magno, A. L.; Walsh, J. P.; Ratajczak, T. Clin. Biochem. 2012, 45,
943; (b) Nemeth, E. F. In Principles of Bone Biology; Bilezikian, J. P., Raisz, L. G.,
Rodan, G. A., Eds., 2nd ed.; Academic Press: San Diego, CA, 2002; pp 1339–
1359.
4. (a) Nemeth, E. F.; Shoback, D. Best Pract. Res. Clin. Endocrinol. Metab. 2013, 27,
373; (b) Conn, P. J.; Christopoulos, A.; Lindsley, C. W. Nat. Rev. Drug Discov.
2009, 8, 41; (c) Harrington, P. E.; Fotsch, C. Curr. Med. Chem. 2007, 14, 3027; (d)
Sorbera, L. A.; Castaner, R. M.; Bayes, M. Drugs of the Future 2002, 27, 831.
5. Nemeth, E. F.; Steffey, M. E.; Hammerland, L. G.; Hung, B. C. P.; Van Wagenen, B.
C.; DelMar, E. G.; Balandrin, M. F. Proc. Natl. Acad. Sci. USA 1998, 95, 4040.
6. Kiefer, L.; Leiris, S.; Dodd, R. H. Expert Opin. Ther. Patents 2011, 21, 681.
7. Deprez, P.; Temal, T.; Jary, H.; Auberval, M.; Lively, S.; Guédin, D.; Vevert, J. P.
Bioorg. Med. Chem. Lett. 2013, 23, 2455.
8. The discovery and optimization of 1 will be described in a future publication.
9. (a) Dalvie, D. K.; Kalgutkar, A. S.; Khojasteh-Bakht, S. C.; Obach, R. S.; O’Donnell,
J. P. Chem. Res. Toxicol. 2002, 15, 269; (b) St. Jean, D. J.; Fotsch, C. J. Med. Chem.
2012, 55, 6002; For a similar strategy that successfully addressed the metabolic
liabilities of a thiazole, see: (c) Kalgutkar, A. S.; Driscoll, J.; Zhao, S. X.; Walker,
G. S.; Shephard, R. M.; Soglia, J. R.; Atherton, J.; Yu, L.; Mutlib, A. E.; Munchhof,
M. J.; Reiter, L. A.; Jones, C. S.; Doty, J. L.; Trevena, K. A.; Shaffer, C. L.; Ripp, S. L.
Chem. Res. Toxicol. 2007, 20, 1954.
10. The hCaSR assay was performed in human embryonic kidney 293 cells
expressing the human parathyroid CaSR as previously described, see: 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, 308, 627.
11. Wong, S. G.; Fan, P. W.; Subramanian, R.; Tonn, G. R.; Henne, K. R.; Johnson, M.
G.; Tadano Lohr, M.; Wong, B. K. Drug Metab. Dispos. 2010, 38, 841.
12. For the preparation of substituted thiadiazoles, see: Wehn, P. M.; Harrington, P.
E.; Eksterowicz, J. E. Org. Lett. 2009, 11, 5666.
In summary, we identified 18, a novel calcimimetic with the po-
tential to lower elevated parathyroid hormone levels associated
13. A similar finding has been made at Pfizer using a systematic and pairwise
analysis of aromatic halogenation on microsomal clearance. Sun, H.; Keefer, C.
E.; Scott, D. O. Drug Metab. Lett. 2011, 5, 232.
with secondary hyperparathyroidism.
A metabolism guided
approach to analogue design, highlighted by the use of metabolite
ID experiments and measurement of TDI of CYP3A4, addressed PK
issues and enabled the identification of 18. Most consequential was
the use of thiazole chlorination in combination with fluorination of
the gem-diphenyl moiety. Lastly, 1,2,4-thiadiazoles appear to be
equipotent replacements of the chlorothiazole although the chlo-
rothiazoles had lower rat IV clearance.
14. Compound 18 was tested in a CEREP panel of 34 enzymes, 19 receptors, 5
transporters, and 5 channels at 10 lM. Three assays with POC < 50 (adenosine
transporter, guanylyl cyclase, and carbonic anhydrase II) were not expected to
interfere with the observed in vivo pharmacology. Solubility data for 18: 0.01 N
HCl = 0.017 mg/mL, Simulated Intestinal Fluid (SIF) = 0.040 mg/mL, Phosphate
Buffered Saline (PBS) = 0.001 mg/mL.
15. Compound 18 was dosed orally to 5/6 nephrectomized male Sprague–Dawley
rats as a suspension in 2% HPMC/1% Pluronic F68/5% Captisol in water. A
statistically significant (repeated measures ANOVA, p < 0.05) decrease in PTH
relative to vehicle was measured at 4 h for both doses and at 8 h for the 6 mg/
kg dose. The decrease in blood ionized calcium was statistically significant
(repeated measures ANOVA, p < 0.05) relative to vehicle at 1, 4, and 8 h for the
6 mg/kg dose and 4 and 8 h for the 2 mg/kg dose.
Acknowledgments
We acknowledge Roger Zanon and Yiping Wu for their assis-
tance with formulations and rat pharmacokinetics, respectively.