N1-arylsulfonyl-N2-(1-aryl)ethyl-3-phenylpropane-1,2-diamines as novel calcimimetics acting on the calcium sensing receptor

Article abstract of DOI:10.1016/S0960-894X(00)00382-6

The synthesis and calcimimetic properties of N¹-arylsulfonyl-N²-(1-aryl)ethyl-3-phenylpropane-1,2-diamines are described. The most active compound of the series (3n, used at 10 μM) produced 974±11% of the maximal stimulation of [³H]IP production obtained by 10 mM Ca²⁺ in CHO cells expressing the calcium sensing receptor (CaSR). This calcimimetic activity was due to a specific interaction of this compound with the CaSR. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00382-6

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2001±2004
N1-Arylsulfonyl-N2-(1-aryl)ethyl-3-phenylpropane-1,2-diamines as
Novel Calcimimetics Acting on the Calcium Sensing Receptor
Philippe Dauban,a Sandrine Ferry,b Helene Faure,b Martial Ruatb
and Robert H. Dodda,*
aInstitut de Chimie des Substances Naturelles, Centre National de la Recherche Scienti®que, 91198 Gif-sur-Yvette Cedex, France
Â
Laboratoire de Neurobiologie Cellulaire et Moleculaire, Junior Group ATIPE, UPR 9040 du CNRS, 91198 Gif-sur-Yvette Cedex, France
b
Received 17 April 2000; revised 26 June 2000; accepted 30 June 2000
AbstractÐThe synthesis and calcimimetic properties of N1-arylsulfonyl-N2-(1-aryl)ethyl-3-phenylpropane-1,2-diamines are
described. The most active compound of the series (3n, used at 10 mM) produced 97Æ11% of the maximal stimulation of [3H]IP
production obtained by 10 mM Ca2+ in CHO cells expressing the calcium sensing receptor (CaSR). This calcimimetic activity was
due to a speci®c interaction of this compound with the CaSR. # 2000 Elsevier Science Ltd. All rights reserved.
The extracellular calcium sensing receptor (CaSR) is a
G-protein coupled receptor which senses extracellular
calcium [Ca2+]e as demonstrated recently by the cloning
of its cDNA from various tissues including parathyroid
and brain.1 3 This protein belongs to the heptahelical
family of receptors that includes metabotropic glutamate
receptors (mGluR)4 and the g-aminobutyric acid receptor
(GABAB-R)5 as well as certain pheromone and taste
receptors.6,7
inositol phosphates, intracellular calcium mobilization
and release of arachidonates.
Besides calcium, cloned CaSR is activated by magne-
sium and other divalent cations, by charged molecules
such as spermine, spermidine and aminoglycosides and
possibly by b-amyloid peptides.1,14,15,17,18 Recently, two
small, non-cationic organic molecules (NPS 568 and
NPS 467, 1 and 2, respectively) were reported to interact
with cloned CaSR and to mimic or potentiate the e€ects
of [Ca2+]e.15,16,19 These compounds, referred to as cal-
cimimetics, were shown to increase the concentration of
cytoplasmic calcium ([Ca2+]i) in bovine parathyroid
cells and inhibit PTH secretion. In the thyroid, CaSR
presumably regulates calcitonin secretion and might be
preferentially targetted by NPS 467 compared to para-
thyroid CaSR. This selectivity is possibly related to minor
biochemical di€erences in the core protein, suggesting that
tissue-speci®c calcimimetics can be designed.8 Since these
compounds only produce e€ects in the presence of
[Ca2+]e, it has been hypothesized that they interact
allosterically with the CaSR to increase its sensitivity to
[Ca2+]e.
At the surface of the parathyroid cell, the CaSR detects
and responds to small changes of circulating [Ca2+
]
e
leading to regulation of parathyroid hormone (PTH)
secretion.2,8 Elevated concentrations of [Ca2+]e decrease
PTH secretion such that the CaSR provides the essential
component of calcium homeostasis in the mammalian
organism. In humans, point mutations in the CaSR
structure produce modi®cations in [Ca2+
] set point
e
that can result in dramatic physiological e€ects.9 11
Expression of CaSR occurs also in other tissues impli-
cated in calcium homeostasis including thyroid and
kidney, and in brain where its expression has been
observed in neurons and oligodendrocytes.3,12,13
Fibroblasts transfected with cloned rat CaSR have
allowed initiation of detailed studies of CaSR pharma-
cology and its associated transduction signals.14 16
CaSR is positively coupled to phospholipases C and A2
and its activity can be evaluated by the accumulation of
In rats, NPS 568 reduced or eliminated osteitis ®brosa20
and allowed 20±50% reduction of cell proliferation
*Corresponding author. Tel.: +33-1-69-82-45-04; fax: +33-1-69-07-
72-47; e-mail: robert.dodd@icsn.cnrs-gif.fr
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00382-6

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P. Dauban et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2001±2004
observed in the parathyroid gland in a model reprodu-
cing chronic renal de®ciency.21 In humans, NPS 568
reduced PTH concentrations in patients su€ering from
the same disorder22 and was, moreover, shown to
decrease PTH and [Ca2+]e levels in menopaused women
exhibiting primary hyperthyroidism.23 Thus, while the
clinical value of calcimimetics acting at the CaSR has
been clearly demonstrated in syndromes where calcium
homeostasis and PTH secretion are perturbed, there is
an obvious need for new compounds showing improved
activity, tissue speci®city and/or pharmacokinetic pro-
®les. As a ®rst step in this direction, we wish to report
herein the calcimimetic activities of N-arylsulfonyl-1,2-
diamines of general structure 3, a new class of CaSR
ligands.
corresponding mono-N-arylsulfonyl derivatives 3c±e,
3g±r. The hydrochloride salts of these amines were pre-
pared for biological evaluation.
The calcimimetic-like properties of the synthesized com-
pounds 3a±r were evaluated in a biochemical assay per-
formed on Chinese hamster ovarian (CHO) cells
transfected with an expression vector containing cloned
CaSR from rat brain [CHO(CaSR)] or not [CHO(WT*)].14
CHO(CaSR) has been shown to stimulate tritiated ino-
sitol phosphate ([3H]IP) accumulation upon activation
of the CaSR by Ca2+ and other divalent cations and by
NPS 568.14 Thus, [3H]IP accumulation produced by
10 mM of each compound or NPS 568 in the presence of
2 mM [Ca2+]e was measured and compared to the e€ect
produced by 10 mM [Ca2+]e (a concentration eliciting
maximal CaSR activation). Results are shown in Table 1.
For several representative compounds, a comparison of
their potency in stimulating [3H]IP accumulation in
CHO(CaSR) and CHO(WT*) cells was made in the pre-
sence of 2 mM [Ca2+]e. Results are shown in Figure 1.
The reference compound, NPS 568 (1), produced
106Æ15% of the maximal response and thus acts as a
calcimimetic in CHO(CaSR) cells. New compounds
having an unsubstituted a-methylbenzylamino group
were ®rst studied. While both the p-nitrophenylsulfona-
mide 3a and its reduced amino form 3b (obtained by
catalytic hydrogenation of 3a) were practically inactive,
the p-toluenesulfonamide 3c showed some weak calcimi-
metic character (16Æ2%). By analogy with the structure
of NPS 568, an m-methoxy group was then included on
the a-methylbenzyl moiety. This led, in the case of com-
pound 3d, to a small increase in activity (36Æ8% stimula-
tion) compared to the de-OMe derivative 3c. The
simultaneous presence of a p-methoxy group on the
phenylsulfonamide moiety (compound 3e) had no e€ect
on this activity (34Æ4% stimulation). The a-methylben-
zyl group was then replaced by the 1-(1-naphthyl)ethyl
The compounds developed in this study were synthe-
sized as shown in Scheme 1. Thus, commercially-avail-
able aryl-substituted allylbenzenes 4 in acetonitrile were
treated at 0 ꢀC with 1 equiv of [(N-p-nitrophenylsulfo-
nyl)imino]phenyliodinane in the presence of catalytic
copper (I or II) tri¯ate and molecular sieves to give the
corresponding N-sulfonylaziridines 5.24,25 The latter,
upon reaction with a 1-arylethylamine at room tem-
perature, a€orded the p-nitro derivatives 3a, 3f, 6 in 90±
95% yields. Removal of the N-p-nitrophenylsulfonyl
group could be e€ected by heating the latter compounds
at 50 ꢀC in acetonitrile containing 2% DMSO, 3 equiv of
thiophenol and an excess potassium carbonate.26 The
resulting primary amines were then reacted in dichloro-
methane with 1.1 equiv of various substituted arylsulfonyl
chlorides in the presence of triethylamine to provide the
Scheme 1.

P. Dauban et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2001±2004
2003
Table 1. Accumulation of [3H]IP induced by the test compounds of
general structure 3 in CHO(CaSR)a cells expressing rat cloned CaSR.
Compound
(10 mM)
Ar
R1
R2
% [3H]IP
Accumulation
NPS 568
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
106Æ15
8Æ3
Ph
Ph
Ph
4-NO2
4-NH2
4-CH3
4-CH3
4-CH3O
4-NO2
4-CH3
4-Cl
4-CF3
H
4-CH3O
2-Cl
3-Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
2Æ2
16Æ2
36Æ8
34Æ4
28Æ10
68Æ8
38Æ15
27Æ2
78Æ7
80Æ8
79Æ7
83Æ3
97Æ11
60Æ3
65Æ1
45Æ1
47Æ2
3-CH3OPh
3-CH3OPh
1-Naphthyl
1-Naphthyl
1-Naphthyl
1-Naphthyl
1-Naphthyl
1-Naphthyl
1-Naphthyl
1-Naphthyl
Figure 1. E€ects of test compounds of general structure 3 and NPS
568 in CHO(CaSR) cells expressing rat cloned CaSR or in CHO(WT*)
cells. CHO(CaSR) or CHO(WT*) cells were prelabeled with myo-
[3H]inositol in basal Ham's F-12 medium as described in legend to
Table 1. [3H]IP accumulation was determined for each test compound
and for NPS 568 at 10 mM and in presence of 2 mM [Ca2+]e. Results
are expressed as % of [3H]IP basal level and are means ÆS.E.M. of
three to ®ve independent experiments performed in triplicate. In
CHO(WT*) cells, test compounds and NPS 568 have limited e€ects on
[3H]IP basal level (less than 40% of basal value) whereas they pro-
duced more robust accumulation of [3H]IP in CHO(CaSR) cells sug-
gesting that these compounds stimulate phospholipase C activity by
interacting speci®cally with CaSR.
1-Naphthyl 3,4-di-CH3O
H
2-Cl
2-CH3O
4-CH3O
3,4-OCH2O
3o
3p
3q
3r
1-Naphthyl
1-Naphthyl
1-Naphthyl
1-Naphthyl
4-CH3O
4-CH3O
4-CH3O
4-CH3O
aCHO(CaSR) cells expressed rat Ca2+ sensing receptor cloned from rat
brain.3 These cells were cultured in basal Ham's F-12 medium (0.3 mM
Ca2+, 0.6 mM Mg2+) as previously described.14 Cells, in 24-well plates,
were cultured overnight in their growth medium containing 0.5 mCi/mL
myo-[3H]inositol (Amersham Corp.). Drugs were dissolved at 10 mM in
ethanol and then diluted in basal medium. Cells were incubated with
drugs (10 mM) at 37 ꢀC and activation of the phospholipase C enzyme
was estimated after quanti®cation of [3H]inositol phosphates ([3H]IP),
accumulation that was performed as described.14 Results are expressed
as percentage of the maximal response obtained by 10 mM Ca2+ and
are means ÆS.E.M. of two to ®ve independent experiments performed
in triplicate. Results varied by less than 5% in any given experiment.
modi®cation was shown to be detrimental to calcimimetic
activity (compare to compound 3k). Other substituents [o-
methoxy (3p), p-methoxy (3q), 3,4-methylenedioxy (3r)]
also led to diminished activities.
As shown in Figure 1, NPS 568 had limited e€ect on
[3H]IP accumulation in CHO(WT*) cells (i.e., not
expressing CaSR) indicating that the large stimulation
in [3H]IP accumulation produced by this compound in
CHO(CaSR) cells (460% of basal values) is due to its
speci®c interaction with the CaSR. A similar observation
was made with the test compounds. Thus, even the
weakly stimulating compounds 3c, 3d and 3f, did so
only in the presence of the CaSR, with only negligible
e€ects in CHO(WT*) cells. The selective action was
even more evident for the more active compounds 3g, 3l
and 3m. For the most active compound 3n, a pro®le
similar to that of NPS 568 was observed, with a stimu-
lation of [3H]IP accumulation of 446% in CHO(CaSR)
cells and again, less than 40% in CHO(WT*) cells. It
thus appears that these arylsulfonyl derivatives are sti-
mulating IP production by interacting speci®cally with
the CaSR.
group. In the case of the p-nitrophenylsulfonamide deri-
vative 3f, this led to a substantial increase in activity
(28Æ10% stimulation) compared to 3a, while replacement
of the nitro function of 3f by a methyl group (com-
pound 3g) led to a further 2.5-fold increase in activity
(68Æ8% stimulation). Whereas replacement of the
methyl group of 3g by chloride (compound 3h) or tri-
¯uoromethyl (3i) led to signi®cant loss of activity
(38Æ15% and 27Æ2% stimulation respectively), both
the absence of a para substituent (3j) or the presence of
a p-methoxy group (3k) improved activity (78±80% sti-
mulation). This activity was maintained in the case of
the o-Cl and m-Cl derivatives 3l and 3m, respectively.
The 3,4-dimethoxy derivative 3n was subsequently
observed to be the most active calcimimetic of this series,
providing 97Æ11% of the stimulation of [3H]IP accumu-
lation produced by 10mM Ca2+, and thus comparable to
NPS 568 in activity.
In conclusion, the new N1-arylsulfonyl-N2-arylethyl dia-
mines described herein represent the second family of
compounds displaying calcimimetic activity via interac-
tion with the CaSR. While these compounds incorporate
an NPS-like backbone, the presence of the additional
arylsulfonamide moiety allows for a great deal of further
structural modi®cations. Such modi®cations should
Attention was also given to substituents on the phenyl
ring separated from the secondary amine function by
three carbon atoms, as in NPS 568. By analogy with the
latter, the o-chloro derivative was prepared (3o) but this

2004
P. Dauban et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2001±2004
permit improvement of potencies and development of
tissue-speci®c calcimimetics. Studies aimed at further
characterizing the in vitro and in vivo calcimimetic
properties of these compounds are currently underway.
11. Pollak, M. R.; Brown, E. M.; Estep, H. L.; McLaine, P.
N.; Kifor, O.; Park, J.; Hebert, S. C.; Seidman, C. E.; Seid-
man, J. G. Nat. Genet. 1994, 8, 303.
12. Ferry, S.; Trai€ort, E.; Stinnakre, J.; Ruat, M. Eur. J.
Neurosci. 2000, 12, 1.
13. Rogers, K. V.; Dunn, C. K.; Hebert, S. C.; Brown, E. M.
Brain Res. 1997, 744, 47.
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The ®nancial support of the Association pour la
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gratefully acknowledged. S.F. was the recipient of a
DRET fellowship.
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