Penta-substituted benzimidazoles as potent antagonists of the calcium-sensing receptor (CaSR-antagonists)

Article abstract of DOI:10.1016/j.bmcl.2010.07.016

A series of novel benzimidazole derivatives has been designed via a scaffold morphing approach based on known calcilytics chemotypes. Subsequent lead optimisation led to the discovery of penta-substituted benzimidazoles that exhibit attractive in vitro and in vivo calcium-sensing receptor (CaSR) inhibitory profiles. In addition, synthesis and structure-activity relationship data are provided.

Full text of DOI:10.1016/j.bmcl.2010.07.016

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5161–5164
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Penta-substituted benzimidazoles as potent antagonists of the calcium-sensing
receptor (CaSR-antagonists)
*
Marc Gerspacher , Eva Altmann, René Beerli, Thomas Buhl, Ralf Endres, Rainer Gamse,
Jacques Kameni-Tcheudji, Michaela Kneissel, Karl Heinz Krawinkler, Martin Missbach, Alfred Schmidt,
Klaus Seuwen, Sven Weiler, Leo Widler
Novartis Institutes for BioMedical Research, Novartis Pharma AG, WKL-136.4.84, CH-4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel benzimidazole derivatives has been designed via a scaffold morphing approach based on
known calcilytics chemotypes. Subsequent lead optimisation led to the discovery of penta-substituted
benzimidazoles that exhibit attractive in vitro and in vivo calcium-sensing receptor (CaSR) inhibitory
profiles. In addition, synthesis and structure–activity relationship data are provided.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 9 June 2010
Revised 2 July 2010
Accepted 3 July 2010
Available online 3 August 2010
Keywords:
Calcium-sensing receptor (CaSR)
Parathyroid hormone (PTH)
Bone formation
Osteoporosis
CaSR-antagonist
Benzimidazoles
Osteoporosis is a disease characterised by low bone mass and
microarchitectural deterioration of bone tissue that leads to bone
fragility and increased risk of fractures.1 Most therapies for the
treatment of osteoporosis inhibit bone resorption and prevent fur-
ther bone loss. However, as many osteoporosis patients have al-
ready lost a substantial amount of bone at the time of diagnosis
there is a need for agents that stimulate new bone formation.2
The only currently commercially available anabolic treatment for
osteoporosis are PTH (parathyroid hormone) or PTH fragments
such as ForteoÒ (teriparatide, the 1–34 fragment of PTH), which
causes a significant increase in bone mass and reduces vertebral
fracture risk substantially.3 However, this peptide must be admin-
istered by sc injections. An orally active low molecular weight
compound with the same efficacy would be a highly attractive
alternative for the patient. Instead of applying exogenous PTH,
mobilisation of endogenous stores of the hormone can be envis-
aged. PTH is stored in relatively large amounts in parathyroid cells
and its secretion is controlled by a calcium-sensing receptor (CaSR)
located on the cell surface. As the release of PTH is negatively cou-
pled to CaSR activation, CaSR-antagonists (calcilytics) mimic a
state of hypocalcemia and thus stimulate PTH release into the
blood stream.4
It is well documented that elevated levels of PTH only result in
higher bone mass if they are transient, that is, they do not persist
for more than about 2–4 h. Elevated PTH levels sustained for sev-
eral hours activate not only osteoblasts but also osteoclasts leading
to an increase in bone turnover instead of higher bone mass.5
Therefore, the drug discovery goal of this approach is the identifi-
cation of a CaSR-antagonist, that is, rapidly absorbed (short Tmax
)
and fairly rapidly eliminated. At Novartis the starting point for lead
optimisation towards a compound exhibiting such properties was
the HTS hit 1 (Fig. 1), a close analogue of Proquazone (an analgesic).
Lead optimisation led to compounds such as 2 and 3 that showed
O
N
O
O
N
X
N
OH
N
N
N
MeO
O
Proquazone
1
X = O
2
3
S
* Corresponding author. Tel.: +41 61 696 76 36; fax: +41 61 696 62 46.
E-mail address: marc.gerspacher@novartis.com (M. Gerspacher).
Figure 1. Proquazone and proquazone type calcilytics.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.016

5162
M. Gerspacher et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5161–5164
Table 1
IC50 values in the nanomolar range in the in vitro receptor assay
and also displayed the appropriate PK- and PTH-release properties
as demonstrated in in vivo experiments in rats and dogs (short
Tmax, sharp PTH plasma peak).6
In vitro activity of compounds 7–24
R3
4
N
In an attempt to identify another template, three structures 4, 5
and 6 of the proquazone (or structurally related) series were super-
imposed as shown in Figure 2. Based on this superimposition a
new template 7, a benzimidazole derivative, displaying the struc-
tural features thought to be necessary for adequate receptor affin-
ity, was designed (morphed) and subsequently prepared. Testing of
1N
7
R1
R2
Compd R1
R2
R3
CaSR FLIPR IC50 (lM)
7
8
9
n-Bu
Methoxy-ethyl
n-Bu
H
H
H
H
i-PrO
i-PrO
MeO
H
H
H
2.55
1.95
4.69
17
7 revealed an IC50 of 2.5
lM in the in vitro assay.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Methoxy-ethyl
Methoxy-ethyl
Methoxy-ethyl
Methoxy-ethyl
Methoxy-ethyl
Methoxy-ethyl
Methoxy-ethyl
Methoxy-ethyl
Methoxy-ethyl
Methoxy-ethyl
Methoxy-ethyl
Methoxy-ethyl
A fluorimetric assay was used as the primary screen to deter-
mine inhibitory potency of the compounds against the CaSR. The
assay (FLIPR assay) measures calcium mobilisation in hamster
fibroblasts transfected with the human receptor.6
MeO
Me
Me
MeO Me
MeO Cl
MeO Br
MeO
MeO CF3
MeO MeO
Cl
1.90
2.10
1.90
0.21
0.05
0.022
0.013
0.051
1.82
0.77
1.00
0.19
0.79
>10
MeO
At the initial stages of lead optimisation (Table 1), derivatives
7–9 showed that methoxy-ethyl as the R1 residue (leading to
slightly more soluble compounds, compared to the n-butyl deriva-
tives) can easily replace n-butyl and that the isopropyloxy residue
(R3) can be replaced by the smaller methoxy without any signifi-
cant loss in potency. The unsubstituted derivative 10 (R1, R2 = H)
was less active. It could also be demonstrated that moving the
methoxy residue from the 4 to the 7 position (11) is tolerated.
Replacement of the 7-OMe residue with a methyl-group was also
tolerated (12). Introduction of an additional methyl residue at po-
sition 7 of 9 leading to 13 did not have much impact on potency of
the resulting compound. Surprisingly, however, was the 10-fold in-
crease in potency of 14 where the positions of the 4- and the 7-res-
idues have been switched. Further increases in activity could be
achieved with the introduction of halogens or trifluoromethyl
residues at position 4, with bromo- and iodo-residues being most
preferred (15, 16, 17 and 18). The 4,7-dimethoxy- and the 4-meth-
oxy-7-chloro-derivatives 19 and 20 proved to be significantly less
I
MeO
Br
Br
N,N-Dimethyl-amino-ethyl MeO Br
n-Pr
MeO Me
MeO Br
N,N-Dimethyl-acetamido
potent than the 4-chloro-7-methoxy derivative 15. It was ob-
served, that fairly lipophilic R1 residues were needed for good po-
tency. The N-dimethylamino-ethyl derivative 22 showed a ca. 10-
fold lower activity in comparison with the methoxy-ethyl com-
pound 16 and the propyl derivative 23 showed a fourfold decrease
in potency. An amide moiety as present in 24 led to a substantial
loss of activity.
In order to further increase the potency of these benzimidazole
derivatives certain additional residues in position 5 can be benefi-
cial as shown in Table 2. At position 5, halogens and especially a
benzyl residue in combination with a halogen or trifluoro moiety
at position 4 led to a slight increase in potency, while a cyano res-
idue was just tolerated (compounds 25–29 and 33). However, an
ethyl or phenyl residue led to loss in potency (compounds 30 and
32). The reduced activity of 31 showed that substitution of the 5 po-
sition alone is not sufficient for producing potent compounds and
that a residue (e.g., halogen or trifluoromethyl) at position 4 was
needed for good activity. Introduction of additional substituents
in the meta- and para-positions of the 5-benzyl residues led to a de-
crease in activity (compounds 34–36), while introduction of an
ortho SO-Me moiety (37) resulted in a highly potent compound.
Replacement of 5-benzyl with a 3-pyridine-methyl- (38) group
was tolerated as well. Introduction of an additional substitutent
such as 2-methoxy, 2-methyl-sufanyl and 2-methyl-sulfinyl into
the pyridine moiety was not only well tolerated but can lead to a
further slight increase in potency as shown specifically for com-
pounds 39–42. The thiazolyl-methyl-moiety (43) led to another
highly potent compound.
The synthesis scheme (Scheme 1) illustrates the synthetic prep-
aration of the compounds 40, 41 and 42. The commercially avail-
able aniline 44 was converted into 45 via O-methylation and
Sandmeyer reaction. Introduction of the methoxyethyl-amino resi-
due via nucleophilic substitution of the chlorine by treatment with
methoxy-ethylamine, followed by selective bromination at position
5 led to 47 in a reasonable overall yield. Reduction of the nitro group
with Raney-Ni, acylation of the aniline nitrogen with 4-isopropyl
benzoic acid followed by cyclisation to the benzimidazole deriva-
tive 48 proceeded in almost quantitative overall yield. Palladium
assisted replacement of the bromo moiety by a cyano residue fol-
lowed by Raney-Ni reduction afforded the aldehyde 49 in moderate
yield. Reaction of 49 with the lithium derivative obtained from 50
Figure 2. Overlay of structures and scaffold morphing.

M. Gerspacher et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5161–5164
5163
Table 2
NO2
Cl
NO2
NO2
NH2
a, b
c
In vitro activity of compounds 25–37
O
R3
N
H
(70%)
R4
4
O
O
OH
d
N
N
5
44
45
46
O
O
NO2
Br
Br
e, f, g
N
N
Compd
R4
R3
CaSR FLIPR IC50 (lM)
O
N
H
(>90%)
25
26
27
28
29
30
31
32
33
34
35
36
Br
Br
CF3
Br
CN
Et
CF3
Ph
Br
I
0.014
0.006
0.011
0.016
0.025
0.19
0.36
0.06
0.005
0.026
0.043
0.34
O
O
O
48
47
Br
CF3
Br
Br
H
Br
Br
CF3
Br
Br
(62% over 2 steps)
N
50
HOC
N
N
h, i
S
Br
Bn
2-Methoxy-benzyl
3,4-Dimethoxy-benzyl
3,4-Dimethyl-benzyl
j, k
(36%)
O
O
49
(52-65%)
S(O)Me
37
38
39
Br
0.005
0.015
0.004
F
F
F
R
N
N
Br
N
N
OMe
N
N
l, m
S
CF3
O
(ca. 40%)
N
N
O
SMe
N
n
41 R = SMe
o
40
41
Br
0.004
O
42 R = S(O)Me
51
O
S
Br
SMe
N
N
N
N
CF3
0.0016
O
S(O)Me
N
42
43
CF3
CF3
0.0026
0.002
O
40 (60%)
Scheme 1. Preparation of 40, 41 and 42. Reagents and conditions: (a) NaH, MeI,
DMF; (b) NaNO2, CuCl, HCl; (c) 2-methoxyethylamine; Hünig’s base, sealed tube, 4;
(d) Br2, HOAc; (e) Raney-Ni, THF, H2; (f) isopropylbenzoic acid, EDC, DMAP, CH2Cl2;
(g) HOAc, 100 °C; (h) Zn(CN)2, DMF, tetrakis(triphenylphosphine)palladium; (i)
Raney-Ni, sodiumhypophosphite, water, pyridine, HOAc; (j) n-BuLi; (k) Zn, HCOOH;
(l) I2, Ag2CO3, AcOH, 80 °C; (m) CuI, FSO2-CF2COOMe, DMF, 120 °C; (n) Br2, HOAc;
(o) H2O2, H2O, AcOH.
2-Thiazolyl-2-methyl
by Br–Li exchange with n-butyl-lithium and subsequent reduction
of the resulting secondary alcohol led to the 5-benzyl-benzimid-
azole derivative 51. Iodination of 51 at position 4, and conversion
of the iodo-substituent into
a trifluoromethyl residue using
methyl-2,2-difluoro-2-(fluorosulfonyl) acetate7 led to 41 in moder-
ate yields, while bromination of 51 with bromine in acetic acid
afforded 40 in better yields. Hydrogen peroxide mediated oxidation
of the sulphur in 41 afforded the sulfinyl derivative 42.
350
300
250
200
150
100
50
0.20
0.15
0.10
0.05
0.00
40: 3.0 mg/kg p.o.
hPTH equiv.
All other compounds described in this letter can be prepared
starting from the appropriately substituted di-aniline derivatives
as described above for the preparation of 40–42 (Scheme 1) or
by using previously published synthetic procedures.8
As pointed out in the introductory part of the letter, a useful cal-
cilytic compound (CaSR-antagonist) must be rapidly absorbed
(short Tmax) upon po dosing and fairly rapidly eliminated, thus
inhibiting the target only for few hours per day (leading to elevated
PTH levels for 2–4 h).
Based on its overall in vitro and physicochemical properties, 40
was selected for in vivo PK/PD assessment.6 As shown in Figure 3,
in dogs 40, dosed at 3 mg/kg po (in a microemulsion formulation)
reached its maximum plasma concentration of 155 nM within 2 h
post administration, with a significant decrease at the 3 h time
point. Six hours post dosing the plasma levels of 40 had decreased
to a level of ca. 14% of Cmax. An excellent PK/PD relationship was
observed, with maximal PTH concentration/drug levels at ca. 2 h
post dosing. The overall shape of the PTH curve followed nicely
0
0
1
2
3
6
10
24
time (h)
Figure 3. Plasma levels of PTH as determined by ELISA and of 40 as determined by
LC/MS after oral administration of 30 mg/dog (ca. 3 mg/kg) in a microemulsion
formulation.
the shape of the compound plasma concentration curve. The PTH
peak levels are considered sufficient for achieving a bone anabolic
effect.

5164
M. Gerspacher et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5161–5164
2. Deal, C. Nat. Clin. Pract. Rheumatol. 2009, 5, 20.
In summary, a novel series of benzimidazoles has been designed
3. (a) Neer, R. M.; Arnaud, C. D.; Zanchetta, J. R.; Prince, R.; Gaich, G. A.; Reginster,
J.-Y.; Hodsman, A. B.; Eriksen, E. F.; Ish-Shalom, S.; Genant, H. K.; Wang, O.;
Mitlak, B. H. N. Engl. J. Med. 2001, 344, 1434; (b) Gallagher, J. C.; Genant, H. K.;
Crans, G. G.; Vargas, S. J.; Krege, J. H. J. Clin. Endocrinol. Metab. 2005, 90, 1583; (c)
Hamann, K. L.; Lane, N. E. Rheum. Dis. Clin. North Am. 2006, 32, 703; (d)
Bilezikian, J. P. Women Health 2007, 3, 243.
4. (a) Trivedi, R.; Mithal, A.; Chattopadhyay, N. Curr. Med. Chem. 2008, 15, 178; (b)
Brown, E. M. Subcell. Biochem. 2007, 45, 139; (c) Jensen, A. A.; Brauner-Osborne,
H. Curr. Neuropharmacol. 2007, 5, 180.
5. (a) Dobnig, H.; Turner, R. T. Endocrinology 1997, 138, 4607; (b) Frolik, C. A.; Black,
E. C.; Cain, R. L.; Satterwhite, J. H.; Brown-Augsburger, P. L.; Sato, M.; Hock, J. M.
Bone 2003, 33, 372; (c) Onyia, J. E.; Helvering, L. M.; Gelbert, L.; Wei, T.; Huang,
S.; Chen, P.; Dow, E. R.; Maran, A.; Zhang, M.; Lotinun, S.; Lin, X.; Halladay, D. L.;
Miles, R. R.; Kulkarni, N. H.; Ambrose, E. M.; Ma, Y. L.; Frolik, C. A.; Sato, M.;
Bryant, H. U.; Turner, R. T. J. Cell. Biochem. 2005, 95, 403.
6. Widler, L.; Altmann, E.; Beerli, R.; Breitenstein, W.; Bouhelal, R.; Buhl, T.; Gamse,
R.; Gerspacher, M.; Halleux, C.; John, M. R.; Lehmann, H.; Kalb, O.; Kneissel, M.;
Missbach, M.; Müller, I. R.; Reidemeister, S.; Renaud, J.; Taillardat, A.; Tommasi,
R.; Weiler, S.; Wolf, R. M.; Seuwen, K. J. Med. Chem. 2010, 53, 2250.
7. Chen, Q.; Wu, S. J. Chem. Soc., Chem. Commun. 1989, 705.
and optimised as inhibitors of the calcium-sensing receptor
(calcilytics). A selected example has been shown to be a potent
calcilytic with in vivo activity. Compound 40 showed a sharp
PTH-release peak in dogs after oral administration in a microemul-
sion formulation.
Acknowledgements
The authors would like to thank Esther Roehn, Karin Ryffel, Ger-
ard Schreiber, Nina Svennum, Sandra Ammon Vetter, Jean-Louis
Runser, Heinz Anklin, Valérie Cordier, Esther Scheidegger and Sven
Guldbransen for excellent technical assistance.
References and notes
1. (a) Kanis, J. A.; McCloskey, E. V.; Johansson, H.; Oden, A.; Melton, L. J.; Khaltaev,
N. Bone 2008, 42, 467; (b) Poole, K. E. S.; Compston, J. E. BMJ 2006, 333, 1251; (c)
Sambrook, P.; Cooper, C. Lancet 2006, 367, 2010.
8. (a) Gerspacher, M.; Weiler, S. PCT Int. Appl., 2005, WO2005068433.; (b)
Gerspacher, M., Krawinkler, K. H. PCT Int. Appl., 2007, WO2007112913.