Allosteric agonists of the calcium receptor (CaR): Fluorine and SF 5 analogues of cinacalcet

Article abstract of DOI:10.1039/c2ob26402a

Three selectively fluorinated cinacalcet analogues are prepared and their activity as calcium-sensing receptor (CaR) agonists is assessed. Individual (2R,1′R)-2 and (2S,1′R)-3 fluorocinacalcet diastereoisomers were prepared using the MacMillan asymmetric fluorination reaction. Assays with the recombinant human CaR revealed that both diastereoisomers have a similar potency to each other although slightly lower (75-80%) than that of cinacalcet 1. The SF₅-cinacalcet analogue 4 was prepared from meta-pentafluorosulfanyl benzyl alcohol and has ～75% agonist activity relative to cinacalcet 1 indicating that the SF₅ group can replace the CF₃ group and retain significant bioactivity.

Full text of DOI:10.1039/c2ob26402a

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PAPER
Allosteric agonists of the calcium receptor (CaR): fluorine and SF5 analogues
of cinacalcet†
Poh Wai Chia,a Sarah C. Brennan,b Alexandra M. Z. Slawin,a Daniela Riccardib and David O’Hagan*a
Received 18th July 2012, Accepted 21st August 2012
DOI: 10.1039/c2ob26402a
Three selectively fluorinated cinacalcet analogues are prepared and their activity as calcium-sensing
receptor (CaR) agonists is assessed. Individual (2R,1′R)-2 and (2S,1′R)-3 fluorocinacalcet
diastereoisomers were prepared using the MacMillan asymmetric fluorination reaction. Assays with
the recombinant human CaR revealed that both diastereoisomers have a similar potency to each
other although slightly lower (75–80%) than that of cinacalcet 1. The SF5-cinacalcet analogue 4 was
prepared from meta-pentafluorosulfanyl benzyl alcohol and has ∼75% agonist activity relative to
cinacalcet 1 indicating that the SF5 group can replace the CF3 group and retain significant bioactivity.
as agonists of the glutamate receptor, and found again a very
different level of response for the two diastereoisomers, attribu-
Introduction
Cinacalcet 1 is an allosteric activator of the calcium receptor
(CaR) and in that respect it mimics the role of Ca2+ ions. Such
calcimimetic drugs are used for the treatment of hyperpara-
thyroidism in the clinic.1–5 The CaR has other roles too, such as
a regulator of gastrointestinal epithelia function, fluid secretion
in the colon and stimulation of gastrin in the stomach.6–11 Also
the CaR is triggered in certain osteoporosis treatments.12 Thus
improved calcimimetic drugs are of value. In this paper we
report the synthesis and evaluation of the three fluorinated
cinacalcet analogues 2–4.
We have been interested in influencing the conformation of
biologically significant amines by the strategic incorporation of a
C–F bond.13–15 When fluorine is placed beta to a protonated
amine then the electrostatic attraction between the opposing C–F
and N+–H dipoles favours a gauche rather than anti relationship
between these bonds.16–21 In this context we have prepared the
enantiomers of 3-fluoro-γ-aminobutyric acid (3F-GABA) and
shown that they have very different agonist activities with the
GABAc receptor,15 but similar efficacies with the GABAA
receptor,14 indicating that GABA has different binding modes
with each receptor. In a recent study we have explored the rela-
tive efficacy of 3-fluoro-N-methyl-D-aspartic acid (3F-NMDA)
ted to the C–F bond dictating different conformational prefer-
ences for each stereoisomer.22 In this paper, we describe a
synthesis of the two β-fluoroamine diastereoisomers 2 and 3. It
is anticipated that the C–F bond will have a strong preference to
lie gauche rather than anti to the C–N+ bond. The relative
efficacy of these diastereoisomers with the CaR receptor should
give some insight into the favoured binding mode to the receptor.
Additionally we have prepared the cinacalcet analogue 4 where
the meta aryl-CF3 group has been replaced by a meta aryl-SF5
group. The SF5 group is gaining in prominence in the medicinal
chemistry literature due to its high chemical stability, electron
withdrawing nature and hydrophobicity.23 It has potential in
structure activity programmes as an alternative to CF3 as a
stable lipophilic substituent for modifying pharmacokinetic
properties.24–26 There are a few examples in the literature com-
paring the relative efficacy of SF5 and CF3 analogues directly in
medicinal products. For example the antimalarial mefloquine
showed improved bioactivity and selectivity when its CF3-group
was replaced by the SF5-group.26
aUniversity of St Andrews, School of Chemistry and Centre for
Biomolecular Sciences, North Haugh, St Andrews, Fife, KY16 9ST, UK.
E-mail: do1@st-andrews.ac.uk; Fax: +44 (0)1334 463800;
Tel: +44 (0)1334 467171
bCardiff University, Biomedical Sciences Building, Museum Avenue,
Cardiff CF10 3AX, UK. E-mail: Riccardi@Cardiff.ac.uk;
Fax: +44 (0)29 208 74116; Tel: +44 (0)29 208 79132
†Electronic supplementary information (ESI) available: Experimental
methods and crystallographic data. CCDC 892697. For ESI and crystal-
lographic data in CIF or other electronic format see DOI:
10.1039/c2ob26402a
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Results and discussion
Synthesis of the calcimimetic β-fluoro diastereoisomers 2 and 3
The synthesis of (2R,1′R) 2 commenced with the preparation of
aldehyde 5 (Scheme 1), an established intermediate in cinacalcet
synthesis.27 Aldehyde 5, was then subjected to a MacMillan
asymmetric fluorination protocol28 where it was treated with
NFSI (2.5 eq) and either organocatalyst (R)- or (S)-6 (10% mol)
in THF–isopropanol (9 : 1). Most of the substrate was converted
to the desired mono fluorinated aldehyde 7, however about
5–10% of the corresponding difluoro aldehyde 8 was apparent
by GC-MS. The enantiomeric excess of (R)- or (S)-7 in each
case was at least 97% ee as determined by chiral phase GC-MS
analysis (see ESI†). A reduction of each enantiomer of aldehyde
7 was carried out with LiAlH4. Any contaminating difluoroalco-
hol 10 could be separated at this stage by careful chromato-
graphy to afford 9 in good yield (80%). Each enantiomer was
then separately progressed to the corresponding triflate 11 and
then a nucleophilic displacement with (R)-1-(1-naphthyl)ethyl-
amine 12 gave cinacalcet analogues 2 and 3.
Fig. 1 Comparison of the X-ray crystal structure of cinacalcet29 1 and
the fluorinated stereoisomer (2R,1′R) 2. Both show an extended alkyl
chain.
Table 1 1H-NMR derived JHH and JHF coupling constants (Hz)
for (2R,1′R) 2 and (2S,1′R) 3 indicating an extended (as shown)
conformation in solution. R = (R)-1-(1-naphthyl)ethyl
The high diastereomeric purity of the 2-fluorocinacalcet
stereoisomers was confirmed by 19F-NMR (see ESI†). Also a
suitable crystal of the (2R,1′R)-2 was obtained for X-ray structure
analysis. The resultant structure shown in Fig. 1 confirmed the
relative and absolute configuration as (2R,1′R)-2. The F–C–C–
N+ dihedral angle in this structure is 53.0° consistent with a tight
gauche alignment of the C–F and C–N+ bonds in the solid state.
The analogous Hpro-R–C–C–N+ is 57.6° for cinacalcet. Also the
alkyl chain of 2 adopts an extended anti zig–zag conformation
similar to the structure of (non-fluorinated) cinacalcet29 1 also
shown in Fig. 1. Thus the stereoelectronics associated with the
C–F bond appear to reinforce the conformation adopted by cina-
calcet itself.
Modelling of the coupling constants (Bruker-DAISY pro-
gramme) from 1H- and 19F-NMR indicate that this extended con-
formation is also the relevant one in solution for both of the
fluorinated cinacalcet diastereoisomers 2 and 3 (see ESI†).
3
For example in (2R,1′R) 2, the JHF coupling constant F/H4 is
3
17.5 Hz consistent with a gauche relationship, whereas the JHF
coupling constant F/H3 is larger at 30.8 Hz, consistent with an
anti relationship.30 Similarly on the other side of the fluorine the
3JHF coupling constant for F/H2 is 20.9 Hz, whereas that for
F/H1 is 31.0 Hz consistent with gauche and anti relationships
respectively. A similar analysis was carried out for (2S,1′R) 3. In
each case the data reinforce an extended alkyl chain confor-
mation in solution, similar to that observed for the solid state
structure of 2. The JHH and JHF coupling constants (Hz) around
fluorine for both isomers are shown in Table 1.
Synthesis of SF5-cinacalcet analogue 4
The synthesis of SF5-cinacalcet analogue 4 started with a nucleo-
philic displacement on benzyl bromide 1331 with diethyl
malonate and sodium hydride at −78 °C (Scheme 2). Decarbox-
ylation was mediated after saponification (6 M NaOH/EtOH)
and then treatment with 4 M HCl. Acidic hydrolysis gave car-
boxylic acid 14 which was purified by careful chromatography.
Carboxylic acid 14 was then treated with LiAlH4 to generate
alcohol 15 and then a Dess–Martin oxidation gave aldehyde 16
in good yield (90%). This aldehyde was then subject to a
Scheme 1 Asymmetric fluorination approach to the preparation of the
cinacalcet diastereoisomers 2 and 3.
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(∼20–25%) in efficacy relative to cinacalcet 1, possibly due to
fluorine lowering the amine pKa. The observation that both
isomers are active suggests, to a first approximation, that confor-
mer A of A–C shown in Fig. 3, is most consistent with the pre-
dicted binding to the CaR. Only conformer A enables both
stereoisomers to adopt a common conformation orientating R2
similarly and simultaneously having a C–N+ to C–F gauche
relationship. The SF5-cinacalcet 4 had a 25% lower maximum
response but a comparable EC50 to that of cinacalcet 1
suggesting that the SF5 group can replace the CF3 without a sig-
nificant change in agonist binding.
Scheme 2 Synthetic route to the SF5 analogue 4 of cinacalcet.
reductive amination with (R)-1-(1-naphthyl)ethylamine 12 and
NaBH(OAc)3 to give 4 as a free base, which was then treated
with HCl/MeOH in diethyl ether to afford 4 as its HCl salt.
Conclusions
Calcium receptor (CaR) assays
In this study we have prepared three novel cinacalcet analogues
2–4. Two of these (2 and 3) are selectively fluorinated diastereo-
isomers with the fluorine placed beta to the amine on the alkyl
chain. This design was explored as the C–F bond is known to
influence the molecular conformation such that a gauche
relationship is favoured between the C–F and C–N+ bonds, and
conformations where these bonds are anti to each other are disfa-
voured. Both fluorinated calcimimetics stereoisomers 2 and 3
were similarly active suggesting that they bind the CaR allosteric
site with the alkyl group anti-periplanar to the amine (Conformer
A, Fig. 3). The third analogue directly replaced the CF3 group of
cinacalcet 1 with the SF5 group. This analogue also retained
good bioactivity.
The three fluorinated calcimimetics 2–4 were evaluated by
intracellular [Ca2+] mobilization using microfluorimetry.
HEK293 cells, which stably expressed the human CaR (HEK-
hCaR), were loaded with the calcium sensitive fluorophore,
fura-2. HEK-hCaR cells were then excited using alternate wave-
lengths of 340 and 380 nm and imaged at 510 nm. The fura-2
excitation ratio (F340/F380) was used to detect changes in
intracellular [Ca2+]. Stimulation of the CaR was achieved at a
concentrations ≥100 nM in the presence of 1.5 mM extra-
cellular [Ca2+]o for each of the calcimimetics 2–4 against a cina-
calcet 1 control.32 Fura-2 excitation ratios were then integrated to
determine the approximate area under the curve, and concen-
tration-response curves (Fig. 2) were fitted using the Hill
equation to determine maximum response and the EC50
(Table 2).
The fluorinated calcimimetics (2R,1′R) 2 and (2S,1′R) (3)
were both found to be active although both displayed a loss
Fig. 2 Concentration response curves for all four calcimimetics in the
Fig. 3 The preferred binding conformation of Cinacalcet HCl (1) in
solution as extended conformation as implied by conformational study.
presence of 1.5 mM [Ca2+]o.
Table 2 The maximum response and EC50 of the fluorinated and non-fluorinated calcimimetics obtained from three individual experiments
Cinacalcet HCl (1)
(2R,1′R)-2F cinacalcet HCl (2)
(2S,1′R)-2F cinacalcet HCl (3)
SF5-cinacalcet HCl (4)
Maximum response
EC50 (μm)
4.8 0.4
0.1252 0.0360
4.0 0.3
0.1357 0.0327
3.6 0.2
0.1604 0.0334
3.6 0.4
0.1234 0.0316
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extracted into EtOAc (3 × 50 mL). The combined organic layers
were concentrated under reduced pressure. This product (R)-11
was used for the next reaction without purification. (R)-1-(1-
Naphthyl)ethylamine (0.11 ml, 0.7 mmol) 12 was added to a
solution of (R)-11 (200.0 mg, 0.6 mmol) in toluene (15 mL) and
the reaction was heated at reflux for 16 h. The mixture was then
concentrated and the product purified over silica gel (hexane–
EtOAc; 8 : 2) to afford (2R,1′R)-2 (199.5 mg, 95%) as a pale
yellow oil. HCl in diethyl ether (0.1 mL, 1 M) was added into
(2R,1′R)-2 to afford as the hydrochloride salt.
[α]D +4.4 (c 1.0, CHCl3); δH (400 MHz, CDCl3): 8.02–7.22
(11H, m, Ar-H), 5.07 (dm, J = 47.9 Hz, CHF), 4.92–4.85 (1H,
q, J = 6.4 Hz, CHMe), 2.85–2.72 (4H, m, 2 × CH2) and 1.64
(3H, d, J = 5.8 Hz, CH3); δc (100 MHz, CDCl3) 133.9 (Ar C),
132.6 (Ar CH), 131.0 (Ar C), 129.1 (Ar CH), 128.9 (Ar CH),
128.5 (Ar CH), 126.5 (Ar CH), 126.0 (Ar CH), 125.8 (Ar CH),
123.8 (Ar CH), 122.3 (Ar CH), 92.9 (d, J = 172.8 Hz, CF), 54.1
(CH), 49.8 (d, J = 22.2 Hz, CH2), 39.0 (d, J = 20.9 Hz, CH2)
and 22.5 (CH3); δF {1H} (376 MHz, CDCl3): −63.0 (3F, s,
CF3), −184.2 (1F, s, CF); IR (nujol mull) νmax/cm−1 2985,
1453, 1329, 1207, 1074, 1026, 801 and 750. m/z (ESI+) 398
[M + Na+] (30). HRMS (ES+): Found 376.1676. Calcd For
[M + H]+ C10H10OF3, 376.1683.
Experimental
General
All reactions were conducted in oven-dried glassware under a
nitrogen atmosphere. Reactions were monitored by thin-layer
chromatography using Merck Kieselgel 60 plates; visualisation
was achieved by short-wave UV and or staining with phospho-
molybdic acid. NMR spectra were recorded a Bruker AV-400 or
a Bruker AV-300 instrument in solutions of CDCl3. Where
necessary, molecular connectivities were assigned using two-
dimensional (COSY, HSQC, homonuclear J-resolved) experi-
ments, and coupling constants for 2 and 3 were determined by
simulation/iteration sequences using the Daisy module of the
Bruker TopSpin software.
(R)-2-Fluoro-3-(meta-trifluoromethyl)phenyl)propan-1-ol (R)-
9. N-Fluorobenzenesulfonimide (7.80 g, 0.02 mol) and (5R)-
(+)-2,2,3-trimethyl-5-benzyl-4-imidazolidinone
dichloroacetic
acid salt (516 mg, 15% mol) was added to a solution of alde-
hyde 527 (2.0 g, 9.9 mmol) in THF–isopropanol (9 : 1) at
−15 °C. The reaction was monitored by chiral phase GC-MS
until all the starting material was consumed. Aldehydes (R)-7
and 8 (∼20 : 1) could be assayed by GC-MS (see ESI†) and
were judged to be >97% ee, but for preparative purposes they
were directly reduced. Thus after 16 h, lithium aluminium
hydride (564 mg, 0.01 mol) was added to the reaction and stir-
ring was continued for 1 h. The reaction was quenched with
NaHCO3 solution (30 ml) at −15 °C. The aqueous layer was
washed with (NH4)2SO4 and extracted into EtOAc (3 × 50 mL).
The combined organics were concentrated under reduced
pressure and purified over silica gel (hexane–EtOAc; 7 : 3) to
give (R)-9 (2.4 g, 80%) as a pale yellow oil.
(2S,1′R)-2-Fluorocinacalcet HCl 3. Trifluoromethanesulfonic
anhydride (0.3 mL, 1.8 mmol) was added to a solution of Et3N
(0.33 mL, 4.5 mmol) and DMAP (15 mg, 0.1 mmol) in dry
DCM (30 mL) at 0 °C. (S)-9 (258 mg, 1.2 mmol) was added
into the reaction mixture via cannula and left to stir for 2 h at
0 °C. The reaction mixture was subsequently warmed to RT and
stirring continued for 16 h. The reaction was quenched with satu-
rated NaHCO3 solution (30 ml) and the aqueous layer extracted
into EtOAc (3 × 50 mL). The combined organics were concen-
trated under reduced pressure to afford (S)-11. The product was
used for the next reaction without purification. (R)-1-(1-
Naphthyl)ethylamine 12 (0.2 ml, 1.8 mmol) was added into the
solution of (S)-11 (245 mg, 0.7 mmol) in toluene (15 mL) and
the reaction was heated at reflux for 16 h. After 16 h, the reaction
was concentrated. Purification over silica gel (hexane–EtOAc;
8 : 2) gave (2S,1′R)-3 (207 mg, 78%) as a pale yellow oil. HCl
in diethyl ether (0.1 mL, 1 M) was added to afford the hydro-
chloride salt.
[α]D +9.55 (c 2.0, CHCl3); δH (300 MHz, CDCl3): 7.43 (5H,
m, Ar-H), 4.71 (dm, JHF = 48.5 Hz, CHF), 3.67 (2H, m, CH2)
2
and 2.97 (2H, m, CH2); δc (75 MHz, CDCl3) 132.7 (Ar CH),
129.0 (Ar CH), 125.9 (Ar CH), 123.7 (Ar CH), 94.8 (d, J =
172.8 Hz, CHF), 64.1 (d, J = 22.2 Hz, COH) and 37.2 (d, J =
21.6 Hz, CH2); δF {1H} (376 MHz, CDCl3): −63.1 (3F, s, CF3),
−189.0 (1F, s, CHF). m/z (ESI−) 203 (M − F−, 40%); IR (nujol
mull) νmax/cm−1 3353, 2934, 1461, 1328, 1199, 1072, 908, 835
and 752. m/z (CI+) 203.07 [M − F−] (100). HRMS (CI+): Found
203.0684. Calcd For (M − F)− C10H10OF3, 203.0668.
[α]D −8.0 (c 0.6, CHCl3); δH (400 MHz, CDCl3): 8.22–7.39
(11H, m, Ar-H), 5.39 (dm, J = 49.2 Hz, CHF), 5.20–5.14 (1H,
q, J = 6.7 Hz, CHMe), 3.17–2.79 (4H, m, 2 × CH2) and 1.61
(3H, d, J = 6.7 Hz, CH3); δc (100 MHz, CDCl3) 138.2 (Ar C),
134.4 (Ar CH), 133.1 (Ar CH), 131.6 (Ar CH), 129.4 (Ar CH),
129.2 (Ar CH), 128.0 (Ar CH), 126.4 (Ar CH), 126.1 (Ar CH),
125.9 (Ar CH), 123.9 (Ar CH), 123.9 (Ar CH), 123.4 (Ar CH),
123.1 (Ar CH), 94.6 (d, J = 171.6 Hz, CHF), 54.1 (CH), 51.1
(d, J = 21.5 Hz, CH2) and 39.5 (d, J = 21.6 Hz, CH2) and 23.9
(CH3); δF (376 MHz, CDCl3): −63.1 (3F, s, CF3), −183.6 (1F, s,
CF). m/z (ESI+) 398 [M + Na+] (30). HRMS (ES+): Found
376.1688. Calcd For [M + H]+ C10H10OF3, 376.1683.
(S)-2-Fluoro-3-(meta-trifluoromethyl)phenyl)propan-1-ol (S)-
9. Enantiomer (S)-9 was prepared following the procedure for
(R)-9, starting with aldehyde 5. The product was obtained as a
pale yellow oil (2.4 g, 80%). [α]D −14.4 (c 2.1, CHCl3); spectra
data were identical to (R)-9. m/z (CI+) 203.07 [M − F−] (100).
HRMS (CI+): Found 203.0684. Calcd For [M − F]− C10H10OF3,
203.0668.
(2R,1′R)-2-Fluorocinacalcet HCl 2. Trifluoromethanesulfonic
anhydride (0.71 mL, 3.4 mmol) was added to a solution of Et3N
(0.33 mL, 4.5 mmol) and DMAP (27.9 mg, 0.2 mmol) in dry
DCM (30 mL) at 0 °C. (R)-9 (500 mg, 2.2 mmol) was added
into the reaction mixture via cannula and was left to stir for 2 h
at 0 °C. The reaction mixture was subsequently warmed to RT
and continued stirring for 16 h. The reaction was quenched with
saturated NaHCO3 solution (30 ml) and the aqueous layer was
3-(Meta-pentafluorosulfur)phenyl)propanoic acid 14. Diethyl
malonic (2.5 mL, 0.01 mol) and sodium hydride (404.0 mg,
0.01 mol) were added to a solution of 13 (5.0 g, 0.01 mol) in a
dry THF (50 mL) at −78 °C. After 1 h of stirring, the reaction
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mixture was warmed to RT and stirring continued for 16 h. After
all the starting material was consumed, as confirmed by TLC
analysis, the reaction was quenched with hydrochloric acid
(10 mL) in an ice-cooled water bath. The aqueous layer was
extracted into ethyl acetate (3 × 50 mL) and the combined organ-
ics concentrated. This product was used directly. NaOH (6 M,
30 mL) and EtOH (5 mL) were added to the reaction mixture,
which was then heated at reflux. After 16 h, the reaction was
worked-up by addition of cHCl to pH 2 at 0 °C and the mixture
was extracted into ethyl acetate (3 × 50 mL). The organic layers
were combined and the product was concentrated under reduced
pressure. Concentrated HCl (4 M, 30 mL) was added and the
reaction was heated at reflux. After 16 h, the reaction was
quenched with saturated NaHCO3 solution at 0 °C and acidified
with cHCl until pH 2. The reaction was extracted into ethyl
acetate (3 × 100 mL) and the combined organics were concen-
trated and purified over silica gel (hexane–ethyl acetate; 8 : 2) to
give 14 (2.3 g, 50%) as a colourless oil.
δH (300 MHz, CDCl3) 7.65–7.41 (4H, m, Ar-H) 3.06 (2H, t,
J = 7.7 Hz, CH2) and 2.76 (2H, t, J = 7.7 Hz, CH2); δc
(75 MHz, CDCl3) 141.0 (Ar C), 131.8 (Ar CH), 129.0 (Ar CH),
125.0 (Ar CH), 123.3 (Ar CH), 35.5 (CH2) and 30.2 (CH2); δF
(376 MHz, CDCl3): 62.4 (1F, d, J = 149.5 Hz, Feq) and 83.9
(4F, q, Faxial); IR (nujol mull) νmax/cm−1 3353, 3006, 1484,
1313, 1272, 1109, 886, 827 and 764. m/z (ESI+) 299 [M + Na+]
(50). HRMS (ESI+): Found 299.0141. Calcd For [M + Na]+
C9H9SF5O2, 299.0136.
νmax/cm−1 3006, 1710, 1484, 1435, 1275, 1100, 828, 805 and
783. m/z (CI+) 241 [M − F−] (100). HRMS (CI+): Found
241.0310. Calcd For [M − F]+ C9H9OF3S, 243.0310.
SF5-Cinacalcet HCl analogue 4. (R)-1-(1-Naphthyl)ethyl-
amine 12 (0.15 mL, 0.9 mmol) was added to a solution of 16
(0.2 g, 0.7 mmol) in THF and stirred at RT for 5 h. Sodium tri-
acetoxyborohydride (0.19 mg, 0.9 mmol) was then added into
the reaction mixture and stirring continued for 16 h at RT. The
reaction was concentrated under reduced pressure. Purification
over silica gel (hexane–EtOAc; 8 : 2) followed by the addition of
HCl in diethyl ether (0.1 mL, 1 M) gave the title compound 4
(0.28 g, 90%) as a colourless oil.
[α]D +14.0 (c 0.4, CHCl3); δH (400 MHz, CDCl3): 8.15–7.38
(11H, m, Ar-H), 5.11 (1H, brm, CH), 2.69 (2H, brm, CH2), 2.47
(2H, brm, CH2), 2.19 (2H, brm, CH2) and 1.91 (3H, d, J =
6.0 Hz, CH3); δc (100 MHz, CDCl3) 141.0 (Ar C), 133.8
(Ar C), 132.0 (Ar C), 131.3 (Ar C), 130.6 (Ar CH), 129.6
(Ar CH), 129.5 (Ar CH), 128.7 (Ar CH), 127.4 (Ar CH), 126.3
(Ar CH), 126.1 (Ar CH), 125.6 (Ar CH), 125.0 (Ar CH), 123.8
(Ar CH), 121.2 (Ar CH), 53.5 (C), 45.4 (CH2), 32.6 (CH2), 27.3
(CH2) and 21.3 (CH2); δF (376 MHz, CDCl3): 62.4 (1F, d, J =
150.09 Hz, Feq) and 84.0 (4F, q, Faxial); IR (nujol mull)
ν
max/cm−1 2925, 2848, 1275, 1261, 845, 805 and 750. m/z
(ESI+) 416 [M + H+] (100). HRMS (ES+): Found 416.1469.
Calcd For [M + H]+ C21H23NF5S, 416.1471.
Cinacalcet HCl 1. Cinacacet HCl was synthesized following a
published method.27 The product was obtained as a pale yellow
oil (2.4 g, 80%) and the analytical data was in accordance with
the literature.
[α]D +8.7 (c 1, CHCl3) lit. [α]D +10.0 (c 1, CHCl3); δH
(400 MHz, CDCl3): 8.12–7.21 (11H, m, Ar-H), 4.57 (1H, q,
CH), 2.71–2.46 (4H, m, 2 × CH2) and 1.75 (3H, d, J = 6.44 Hz,
CH3); δF {1H} (376 MHz, CDCl3): −63.0 (3F, s, CF3). m/z
(ESI+) 416 [M + H+] (100).
3-(Meta-pentafluorosulfur)phenyl)propan-1-ol
15. Lithium
aluminium hydride (206 mg, 5.4 mmol) was added to a solution
of 14 (1.0 g, 3.6 mmol) in THF (30 mL) and the reaction heated
under reflux for 1 h. Saturated NaHCO3 (15 mL) was added and
the reaction was extracted into EtOAc (3 × 50 mL). Concen-
tration gave 15 (848.8 mg, 90%) as a colourless oil.
δH (400 MHz, CDCl3) 7.61–7.38 (4H, m, Ar-H) 3.7 (2H, t,
J = 6.3 Hz, CH2) 2.82 (2H, t, J = 7.8 Hz, CH2) and 1.93 (2H, m,
CH2); δc (100 MHz, CDCl3) 131.6 (Ar CH), 128.6 (Ar CH),
125.7 (Ar CH), 123.5 (Ar CH), 61.8 (COH), 33.9 (CH2) and
31.9 (CH2); δF (376 MHz, CDCl3): 62.4 (1F, d, J = 149.8 Hz,
Feq) and 84.4 (4F, q, Faxial); IR (nujol mull) νmax/cm−1 3351,
2937, 1434, 1328, 1274, 1110, 918, 824 and 744. m/z (CI+)
243 [M − F−] (100). HRMS (CI+): Found 243.0464. Calcd For
[M − F]+ C9H11OF3S, 243.0467.
Intracellular Ca2+ mobilisation assays. HEK-hCaSR cells
were sub-cultured on to sterile 13 mm diameter glass coverslips
in DMEM/10% fetal bovine serum 48–72 h prior to experiment.
Cells were loaded with 3 μM fura-2 AM in loading solution,
made up of extracellular solution (ECS; 135 mM NaCl, 5.0 mM
KCl, 5.0 mM HEPES, 10 mM D-glucose, 1.2 mM MgCl2,
pH 7.4), with the addition of 1 mg mL−1 BSA and 1.0 mM
CaCl2. Once cells were incubated for 1.5 h at 37 °C, the fura-2
containing solution was removed and the cells were washed to
remove any remaining extracellular fura-2.
3-(Meta-pentafluorosulfur)phenyl)propanal 16. Dess–Martin
periodinane (1.9 g, 4.58 mmol) was added to a solution of 15
(0.8 g, 3.0 mmol) in dry DCM (20 mL) at 0 °C and the reaction
was slowly warmed to RT. After 1 h of stirring, the reaction was
quenched with saturated NaHCO3 solution (30 mL). The
aqueous layer was washed with EtOAc (3 × 50 mL) and the
combined organics were concentrated under reduced pressure.
Purification over silica gel (hexane–EtOAc; 9 : 1) gave 16 (0.7 g,
90%) as a colourless oil.
δH (400 MHz, CDCl3) 9.76 (1H, s, CHO), 7.52–7.29 (4H, m,
Ar-H) 2.95 (2H, t, J = 7.4 Hz, CH2) and 2.76 (2H, t, J = 7.4 Hz,
CH2); δc (100 MHz, CDCl3) 200.3 (CvO), 141.6 (Ar C), 131.6
(Ar CH), 128.9 (Ar CH), 125.8 (Ar CH), 124.0 (Ar CH),
44.9 (CH2) and 27.8 (CH2); δF (376 MHz, CDCl3): 62.4 (1F, d,
J = 150.09 Hz, Feq) and 84.1 (4F, q, Faxial); IR (nujol mull)
Fura-2 loaded cells were then transferred to a custom made
perfusion chamber mounted on an Olympus IX71 microscope
equipped with
a Cairn monochromotor-base fluorescence
system. Cells were then perifused with ECS containing 1.5 mM
CaCl2 and increasing concentrations of either cinalcalcet 1 or the
other calcimimetics 2–4 (0.03–3 μM). Fura-2 was alternatively
excited at 340 and 380 nm, and images at 510 nm were acquired
by a slow scan CCD camera.
Intracellular mobilization data for single cells consisted of
fluorescence excitation ratios (F340/F380) plotted against time
(min). These data were then integrated determine the approxi-
mate area under the curve/minute for each calcimimetic concen-
tration – termed the Integrated Response – and normalised with
7926 | Org. Biomol. Chem., 2012, 10, 7922–7927
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respect to the IRU for the control Ca2+o concentration (ECS with
1.5 mM CaCl2).
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Integrated responses were then expressed as concentration-
response curves and then fitted using the following form of the
Hill Equation: d + [(a − d) × Xb]/[cb × Xb]) where a = maximum
response, b = the Hill co-efficient, c = EC50, d = the minimum
response and X = the ratiometric response. Experimental data
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Acknowledgements
We are grateful to the University of Malaysia Terengganu, and
the Ministry of Higher Education of Malaysia for a PhD Student-
ship. DO’H acknowledges the ERC for an Advanced Investi-
gator Grant. DR acknowledges the Marie Curie ITN
“Multifaceted CaSR” (grant 264663) for the financial support.
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