Development of AC265347-Inspired Calcium-Sensing Receptor Ago-Positive Allosteric Modulators

Article abstract of DOI:10.1002/cmdc.202100368

The calcium-sensing receptor (CaSR) is a clinical target in the treatment of hyperparathyroidism and related diseases. However, clinical use of approved CaSR-targeting drugs such as cinacalcet is limited due to adverse side effects including hypocalcaemia

Full text of DOI:10.1002/cmdc.202100368

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Development of AC265347-Inspired Calcium-Sensing
Receptor Ago-Positive Allosteric Modulators
+
[a, b]
+ [b]
[b]
[b]
Le Vi Dinh ,
Aaron DeBono , Andrew N. Keller, Tracy M. Josephs,
[b, c]
[b, c]
[a]
Karen J. Gregory,
Katie Leach,*
and Ben Capuano*
The calcium-sensing receptor (CaSR) is a clinical target in the
treatment of hyperparathyroidism and related diseases. How-
ever, clinical use of approved CaSR-targeting drugs such as
cinacalcet is limited due to adverse side effects including
hypocalcaemia, nausea and vomiting, and in some instances, a
lack of efficacy. The CaSR agonist and positive allosteric
modulator (ago-PAM), AC265347, is chemically distinct from
clinically-approved CaSR PAMs. AC265347 potently suppressed
parathyroid hormone (PTH) release in rats with a lower
propensity to cause hypocalcaemia compared to cinacalcet and
may therefore offer benefits over current CaSR PAMs. Here we
report a structure activity relationship (SAR) study seeking to
optimise AC265347 as a drug candidate and disclose the
discovery of AC265347-like compounds with diverse pharmacol-
ogy and improved physicochemical and drug-like properties.
Introduction
The human CaSR is a class C G protein-coupled receptor (GPCR).
2
+
The CaSR plays a pivotal role in extracellular calcium (Ca o)
homeostasis via negative regulation of parathyroid hormone
(
PTH) synthesis and secretion from chief cells of the parathyroid
2
+
glands. When serum Ca
kidneys, intestines and bone to restore Ca
a physiologically normal range. For patients suffering from
hyperparathyroidism such as that which occurs secondary to
chronic kidney disease, hyper-secretion of PTH leads to health
concentrations fall, PTH acts on the
+
o
2
Figure 1. CaSR positive allosteric modulators; cinacalcet (1) and AC265347
concentrations to
o
(2).
[1]
[2]
complications including bone and cardiovascular disease.
ing the need for improved therapies to correct the signalling or
expression of naturally occurring mutations.
Potentiation of CaSR-mediated suppression of PTH release by
PAMs such as cinacalcet (1, Figure 1) is therefore an important
strategy to normalise serum PTH concentrations in
The PAMs evocalcet and etelcalcetide have recently entered
the market for secondary hyperparathyroidism. However, some
[2b,3]
[2b]
hyperparathyroidism.
Further, cinacalcet has been success-
patients experience adverse side effects from cinacalcet,
fully used in some patients that harbour naturally occurring
loss-of-function mutations in the CASR gene, or in genes
encoding the CaSR interacting proteins, Gα (encoded by
GNA11) and adaptor protein 2 (AP2). However, some patients
with these mutations do not respond to cinacalcet, demonstrat-
evocalcet or etelcalcetide, including gastrointestinal issues and
[5]
hypocalcaemia, which limit clinical use. There is subsequently
interest in the discovery of improved CaSR PAMs devoid of
detrimental adverse complications. In 2011, ACADIA Pharma-
ceuticals identified AC265347 (2, Figure 1) as a CaSR PAM with
1
1
[4]
[6,7]
reduced adverse effect potential.
In rodents, the (S)-enan-
+
[
a] L. V. Dinh, Prof. B. Capuano
tiomer of AC265347 suppressed PTH release at concentrations
that were 300-fold lower than those that suppressed blood
Medicinal Chemistry
Monash Institute of Pharmaceutical Sciences (Monash University)
2+
Ca
concentrations, suggesting (S)-AC265347 may have a
3
81 Royal Parade, Monash University
[6]
Parkville, VIC 3052 (Australia)
lower propensity to induce hypocalcaemia. Further, (S)-
AC265347 was more effective than cinacalcet at restoring the
signalling of select naturally occurring loss-of-function CaSR
E-mail: ben.capuano@monash.edu
b] L. V. Dinh, Dr. A. DeBono, Dr. A. N. Keller, Dr. T. M. Josephs,
Dr. K. J. Gregory, Dr. K. Leach
+
+
[
[6]
Drug Discovery Biology
Monash Institute of Pharmaceutical Sciences (Monash University)
mutants. AC265347 may therefore offer a novel scaffold from
which to develop more efficacious therapies for disorders of
3
81 Royal Parade, Monash University
2+
PTH or Ca
homeostasis.
o
Parkville, VIC 3052 (Australia)
E-mail: katie.leach@monash.edu
c] Dr. K. J. Gregory, Dr. K. Leach
Department of Pharmacology, Monash University
A previous SAR study demonstrated core characteristics of
the benzothiazole scaffold that were required to retain
AC265347-like PAM activity (Figure 2). However, this study
and others focusing on target- and mechanism-based chemical
optimization of CaSR PAMs have relied on modulator titration
curves to quantify PAM SAR. In such a paradigm, the response
[
[
[7]
9
Ancora Imparo Way, Clayton, VIC 3800 (Australia)
+
] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202100368
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Results and Discussion
SAR rationale
In previous SAR work, AC265347-like compounds were function-
alized at the 2-position and attached to a number of substituted
[7]
phenyl rings via a stereogenic centre. Furthermore, substitu-
1
ents at R were explored including hydrogen, methyl, cyclo-
propyl and phenyl. The replacement of the methyl group at the
stereogenic centre for either a hydrogen or phenyl at R
Figure 2. Gustafsson’s first generation 1-(benzothiazol-2-yl)-1-phenylethanol
derivatives suggested the hydroxyl and small non-hydrogen substituents at
the stereogenic centre were mandatory for activity, while the phenyl ring
1
abolished activity, whilst the presence of a cyclopropyl group
significantly reduced potency by ~20-fold compared to a
[7]
tolerated small or rigid substituents.
1
methyl substituent. Derivatives with a methyl at R and hydroxyl
groups retained activity when coupled with smaller substituents
2
around the aromatic ring (R ). These substituents included
mono- and di-substituted aromatics encompassing either meth-
yl substituents, small halogen atoms or both. Bulky substituents
attached to the phenyl were not tolerated, and replacement of
the phenyl group with a β-naphthyl group also resulted in a
significant loss of activity. The necessity of the hydroxyl group
at the stereogenic centre was also examined with the replace-
ment by an exo-methylene group resulting in a complete loss
Figure 3. Proposed structural modifications on the scaffold of 2 detailed in
this work.
[7]
of activity, thereby demonstrating its importance for potency.
Since prior work demonstrated that only small substituents
on the phenyl moiety were tolerated, we expanded upon the
initial series by generating a library of compounds encompass-
ing small substituents with differing electronic and steric
properties (Figure 3). Along with the exploration of various
substituents on the phenyl moiety, we also explored: (i) isosteric
replacement of the CH for CF at the stereogenic centre; (ii)
substituents on the benzothiazole moiety; (iii) a scaffold hop
from the benzothiazole core to the unsubstituted and sub-
stituted thiazole core, as analogues of this nature have not
to a fixed concentration (e.g. EC ) of orthosteric agonist is
2
0
measured in the presence of increasing concentrations of PAM
[8]
to determine modulator potency.
A limitation of this approach is that it may yield apparently
flat” structure–activity relationship (SAR) profiles if a single
3
3
“
potency parameter is used as the sole determinant of
compound activity. This is because, in an experimental para-
digm exemplified by the titration curve, PAM potency and
maximal effect are amalgams of four parameters: affinity (KB);
cooperativity, which defines the magnitude and direction of the
PAM effect on orthosteric ligand affinity (α) or efficacy (β); and
been described thus far; and (iv)
a multi-functionalised
approach that incorporated the ‘best’ previously mentioned
modifications into a single molecular entity. All analogues
reported by Gustafsson et al., were synthesized as racemates,
and subsequently resolved by chiral HPLC to give the individual
enantiomers. The S-enantiomer of AC265347 demonstrated the
highest potency, but it was only 2.5-fold more potent than the
[9]
PAM intrinsic efficacy (τB). Thus, changes in one of these four
parameters as a consequence of chemical modification, may be
offset by changes in another of the parameters in the opposite
direction, accounting for flat SAR based on potency alone. With
this in mind, and in consideration of the potential of AC265347
as a novel CaSR PAM, here we sought to further optimize the
AC265347 scaffold (Figure 3), and quantified PAM pharmacol-
[7]
racemate mixture. Herein, we therefore characterised only the
racemate mixtures of AC265347-like compounds.
[10]
ogy using an operational model of allosterism providing a
more “enriched” SAR for describing CaSR allostery. The synthetic
strategies used herein included exploration of small substitu-
ents at both the stereogenic center and phenyl moiety, while
implementing bioisosteric and scaffold hopping strategies to
define new chemical space.
Homology modelling
To predict the likelihood that our novel compounds would bind
to the CaSR prior to chemical synthesis, AC265347 and
analogues were docked into a CaSR 7TM homology model
based on the crystal structure of metabotropic glutamate
[11]
receptor 5 (mGlu5) (Figure 4A, B). Furthermore, mutagenesis
studies were performed to validate the homology model of
[12]
AC265347. AC265347 is predicted to bind deep within the
CaSR 7TM with the benzothiazole moiety occupying a small
hydrophobic pocket pointing towards the intracellular cavity
[12]
(Figure 4).
Computational docking suggested substituents
attached at the para-position would project towards the
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Scheme 1. Synthesis of AC265347 derivatives with varying substituents on
the phenyl ring: a) nBuLi, substituted acetophenone, THF, À 78°C, 27–61%;
b) acetic acid, RT, quantitative yield.
dropwise addition of nBuLi to promote lithiation of the scaffold
at the 2-position. Each reaction was stirred for 30 min, allowing
sufficient time to effect complete lithiation followed by the
dropwise addition of the selected acetophenone (4-isopropyl, 4;
Figure 4. A) The representative S-enantiomer of the parent compound 2 was
modelled at the CaSR 7TM; B) sample compounds 5, 21 and 26 highlight the
structural differences to AC265347 - green (methylenedioxy bridge on
phenyl ring), pink (4,5-dimethyl substitution on thiazole core) and blue (6-
methyl substitution of the benzothiazole scaffold), respectively. The S-
enantiomers were docked and overlayed with 2 (beige) showing the relative
binding poses to the parent compound.
3
7
,4-dioxolyl, 5; 4-methoxy-2-methyl, 6; 2,4-bis(trifluoromethyl),
; 3,5-bis(trifluoromethyl), 8; tetrahydropyranyl (THP)-protected
4-hydroxy-2-methyl, 9a) in THF to deliver the desired com-
pound (9b). These reactions generally proceeded in accordance
with the literature without much concern affording the desired
products in yields ranging from 27–61%. However, instability of
the lithiated benzothiazole species often led to lower yields.
Compounds 16 and 17 (Scheme 2) were synthesized using
the corresponding substituted benzoic acids. Modification at
the stereogenic centre of AC265347 employed an isosteric
replacement strategy to examine the electronic effect of the
extracellular loops and not impede the binding mode. Docking
poses of representative analogues from each type of modifica-
tion illustrated in Figure 3 are depicted in Figure 4 with all
compounds predicted to bind to a similar site in a similar
orientation to AC265347, thus validating the subsequent library
of compounds that were synthesised.
electron-withdrawing group, CF , on the functional activity of
3
analogues. The initial single modification of 16 was followed by
the global isosteric replacement of the available methyl groups
for CF represented in 17.
3
Chemical synthesis
The
2,4-dimethylbenzoic
acid
(10)
and
2,4-bis
(trifluoromethyl)benzoic acid (11) were reacted with oxalyl
Our first series of compounds focused on varying substituents
expected to exert different steric and electronic effects within
the binding site. A branched isopropyl group at the para-
position of 4 was installed to explore the tolerability of a
medium-sized hydrophobic group in the binding pocket, and
identify additional ligand-receptor interactions, e.g. van der
Waals. 5 contained a methylenedioxy group comprising H-bond
accepting ability to test for possible hydrophilic interactions in
the binding cleft. Single modifications made to the scaffold of
AC265347 with 4-methoxy and 4-hydroxy substituents were
represented by 6 and 9b, respectively. Both analogues con-
tained ring activating electron-donating groups and H-bond
acceptors, while a H-bond donor is exclusive to 9b. The
replacement of the weakly electron-donating methyl group for
chloride in the presence of catalytic DMF to deliver the acid
chlorides. The resulting product was reacted without further
purification with N,O-dimethylhydroxylamine hydrochloride to
give the corresponding Weinreb amides: N-methoxy-N,2,4-
trimethylbenzamide (12) and N-methoxy-N-methyl-2,4-bis
(trifluoromethyl)benzamide (13). Following silica column purifi-
cation, the Weinreb amide intermediates were subsequently
a CF group at the ortho- and para- positions of 7 allowed
3
examination of the electron-withdrawing effect on the activity
of analogues whilst maintaining a similar size. This type of
replacement is commonly seen in drug design and develop-
ment settings as fluorine displays a similar size to hydrogen yet
[13]
is significantly more electronegative and metabolically stable.
Scheme 2. Synthesis of AC265347 derivatives exhibiting isosteric replace-
Subsequently, we examined the effect of shifting both CF3
groups in 7 to meta- positions as exemplified in compound 8.
Analogues 4–9 (Scheme 1) were synthesized by dissolving
benzothiazole in anhydrous THF at À 78°C, followed by the
ment of the –CH
and 17): a) i) (COCl)
3
group at the stereogenic centre for -CF
3
(compounds 16
2
, DMF(cat), CH
2
2
Cl , rt, ii) N,O-dimethylhydroxylamine·HCl,
Et N, CH Cl , rt, 59–64%, b) benzothiazole, nBuLi, THF,À 78°C, 76%, c)
3
2
2
(
3 4 3 3 3
CH ) NF, CF Si(CH ) , THF, rt, 32–45%.
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reacted with the lithiated benzothiazole species generated
using the aforementioned method to give the methanone
intermediates 14 and 15. These diarylketones were reacted with
Ruppert-Prakash reagent (CF Si(CH ) ) in the presence of
tetramethylammonium fluoride (TMAF), to give the desired
compounds 16 and 17 in 45% and 32% yield, respectively,
replacing the CH3 group at the stereogenic centre of the
for ease of comparison of pharmacological activity. The
incorporation of fluorine to give 27 was rationalized by the
comparative size to hydrogen (an isostere) yet different
electronic properties as aforementioned. Additionally, the
isosteric replacement of the aromatic carbon for nitrogen on
the fused ring of 28 introduced a hydrogen-bond donor to
capture new interactions within the binding cleft. The reagents:
6-methylbenzothiazole (23), 6-fluorobenzothiazole (24) and
thiazolo[5,4-c]pyridine (25) were independently lithiated using
the previously described standard procedure and subsequently
reacted with 2,4-dimethylacetophenone to give the desired
compounds 26, 27 and 28, respectively.
3
3 3
AC265347 scaffold with an isosteric CF group.
3
Furthermore, we generated a series of analogues focused
on the modification of the unexplored benzothiazole motif on
AC265347. We utilized truncation, substitution and expansion
strategies on the benzothiazole moiety to provide further
insight into the chemical space and tolerability surrounding the
cavity deep within the binding pocket. First, we examined the
importance of the fused ring by scaffold hopping from the
traditional benzothiazole to the truncated thiazole moiety. The
Pharmacology & SAR
2
analogues generated include deletion of the fused sp -
AC265347 and novel analogues synthesized in this study were
tested in calcium mobilization assays in FlplnHEK-Trex-CaSR
3
hybridized benzo ring system and replacement with a sp -
2
+
hybridized methyl group. Compounds 20–22 (Scheme 3) were
synthesized using either 2-bromothiazole (18) or 2-bromo-4,5-
dimethylthiazole (19). The respective bromothiazole starting
materials underwent lithium-halogen exchange using nBuLi.
Lithiation of 18 and subsequent reaction with 2,4-dimeth-
ylacetophenone gave the desired compound 20, albeit in low
yield due to the relative instability of the lithiated thiazole
intermediate. Lithiation of 19 and the subsequent reaction with
cells for the ability to potentiate the response to Ca o.
2
+
Concentration-response curves to Ca
were generated in the
o
absence and presence of multiple concentrations of test
compounds to quantify PAM affinity (pK ), cooperativity (αβ)
B
and efficacy (τ ) using an operational model of allosterism
B
(Eq 1) (Table 1), thus determining both allosteric agonism and
2
+
allosteric potentiation of Ca o. Of note, some modulators
possess genuine agonist activity (τ modulator response in the
B,
2
,4-dimethylacetophenone or 2,4-trifluoromethylacetophenone
presence of vehicle) determined using operational model of
allosterism, which differentiates the effect of the modulator on
led to the desired analogues 21 and 22, respectively, in good
yield.
Accordingly, we explored the introduction of substituents
on the benzothiazole motif (Scheme 4). Modifications made on
the benzothiazole ring were kept consistent at the 6-position
Table 1. Functional affinity (pK
B
), positive cooperativity (αβ) and efficacy
2+
(τ ) for AC265347 analogues, determined using Ca mobilization assay in
B
i
HEK293 cells. The data presented were extrapolated from the number of
independently repeated experiments stated (n) performed in duplicate.
Compd
pK
(K , μM)
B
B
�SD
Logαβ�SD
(αβ)
Logτ
B
�SD (τ
B
)
n
AC265347 (2)
5.1�0.09 (7.9)
4.3�0.8 (50)*
4.9�0.3 (13)
5.2�0.3 (6.3)
5.8�0.3 (1.6)*
2.5�0.9 (320)
2.3�0.8 (200)
1.6�0.6 (40)
2.2�0.4 (160)
1.3�0.4 (20)*
0.5�0.2 (3.2)
5
4
4
4
6
5
4
3
6
4
7
3
3
6
6
[
a]
4
5
6
7
8
ND
Scheme 3. Thiazole-containing derivatives employing a scaffold-hopping
strategy: a) nBuLi, substituted acetophenone, THF, À 78°C, 17–40%.
-0.7�0.5 (0.2)
1.0�0.3 (10)
0.7�0.2 (5.0)
[
b]
[b]
[b]
ND
ND
ND
9
1
1
2
2
2
2
2
2
b
6
7
0
1
2
6
7
8
4.6�0.2 (25)
5.2�0.2 (6.3)
5.2�0.4 (4.0)
4.7�0.2 (20)
5.2�0.2 (6.3)
5.2�0.4 (6.3)
2.2�0.4 (160)
1.6�0.9 (40)
1.7�0.5 (50)
1.4�0.2 (25)*
2.1�0.1 (130)
1.6�0.9 (40)
ND[a]
0.7�0.2 (5.0)
0.4�0.2 (2.5)
[
a]
ND
0.8�0.4 (6.3)
0.4�0.1 (2.5)
[
a]
[a]
[a]
ND
ND
ND
4.9�0.4 (13)
1.7�0.4 (50)
0.3�0.6 (0.5)
[
b]
[b]
[b]
ND
ND
ND
[
a]
[b]
Scheme 4. Modifications of the benzothiazole ring including isosteric
replacement with thiazolo[5,4-c]pyridine, and replacement of H with CH
and F substituents at the 6-position: a) nBuLi, 2,4-dimethylacetophenone,
THF, À 78°C, 5–49%.
ND: negligible or no response detected; not determined due to lack
of saturable allosteric effect. *p<0.05 where statistical significance
compared to 2 was determined by one-way ANOVA with Dunnett’s post-
test.
3
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2
+
2+
ambient Ca
versus agonism. Figure 5 depicts the Ca
methoxy and p-hydroxy substituents; compounds 5, 6 and 9b
o
concentration-response curves in the presence of selected
analogues of AC265347 demonstrating the effects of represen-
tative structural modifications, including varying substituents,
isosteric replacement and scaffold hopping, on PAM activity
compared to the parent compound, 2.
The first analogue of our series, 4, was designed to explore
the biochemical impact of introducing the simplest branched
hydrocarbon. Introduction of the isopropyl group (4) resulted in
a modest but significant loss in affinity whilst maintaining
comparable cooperativity to AC265347 (Table 1) suggesting
that small, less hindered substituents at the para position are
preferable for binding affinity.
respectively. These compounds were anticipated to donate
electron density into the aromatic ring creating an electron-rich
environment and encourage improved π-π and potential π-
cation interactions, and promote hydrogen-bonding interac-
tions. 5 and 6 retained activity (affinity, cooperativity, efficacy)
compared to AC265347 (2). Introducing a hydroxy substituent
(9b) resulted in a modest reduction in affinity and negligible
agonism. The trio set of analogues demonstrated no significant
improvement in activity when compared to 2.
Compounds 7, 8, 16 and 17 explored the introduction of
trifluoromethyl group(s) as isosteric replacements of the methyl
groups of parent compound 2. Replacement of the 2- and 4-
methyl groups of 2 with trifluoromethyl groups to generate 7,
significantly improved affinity with maintenance of efficacy,
while cooperativity reduced significantly comparable to parent
compound 2 (Table 1). Unfortunately altering the substitution
However, the incorporation of such a branched hydro-
carbon group was detrimental to τB with a loss of agonist
activity (Table 1). We then explored the introduction of
electron-donating groups in the form of 3,4-methylenedioxy, p-
2
+
Figure 5. Pharmacological evaluation of AC265347 derivatives for PAM activity in FlpIn HEK TRex cells stably expressing the CaSR using intracellular Ca
mobilization assays. The representative series of concentration-response curves shown above denotes the effect of structural changes to pharmacological
activity. Dose-response curves are shown for compounds 2, 5, 21 and 26 as representative examples of compounds where modulator activity was altered. (a)
2
+
AC265347 (2) demonstrated a left-ward shift in the Ca
o
concentration-response curve as the modulator concentration increases, which is typical of a PAM, in
2+
addition to an increase in the response to vehicle, indicative of agonist activity as well as potentiation of ambient Ca
o
in the vehicle. (b) replacement of the
xylyl motif for benzodioxole (5) resulted in a reduction in agonist efficacy. (c) compound 21 demonstrated similar activity to that of the parent compound
with the truncation of the benzothiazole moiety to the corresponding 4,5-dimethylthiazole. (d) the incorporation of a methyl substituent on the benzothiazole
core, 26, resulted in the virtually complete absence of any PAM or agonist activity at the CaSR. Data are mean � SEM determined from nꢀ3 performed in
duplicate. Curves through the data points are the best fit of Eq.1 to the data.
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pattern of the trifluoromethyl groups from the 2- and 4-
positions (7) to the 3- and 5-positions (8) did not improve
activity, where the affinity, cooperativity and efficacy could not
be quantified due to the unsaturable allosteric effect (Table 1).
We then explored the impact of exchange of the stereogenic
methyl of AC265347 for the similarly sized but electron-
withdrawing trifluoromethyl group while retaining the 2,4-
dimethyl substitution on the phenyl ring. Compound 16
retained affinity, cooperativity and efficacy compared to 2. Since
both bioisosteric replacement strategies led to compounds that
retained/improved CaSR affinity and agonist activity, we
implemented a global bioisosteric strategy replacing the
stereogenic À CH and the À CH substituents of the phenyl ring
fluoro substituent. Accordingly, we examined the effect of
substituting the benzothiazole ring for a thiazolo-pyridine ring
because the introduction of nitrogen maintains a similar
molecular size relative to 2. 28 resulted in a loss of activity
compared to 2. Although isosteric replacement of carbon for
nitrogen would enable H-bond accepting ability and reduce
electron density of the aromatic ring, the introduction signifi-
cantly diminished compound activity. In addition, the methyl
substituent at the 6-position of the benzothiazole core is not
well tolerated, whilst isosteric replacement of H for F at the
same position maintained activity comparable to 2. Therefore,
further substitution on the benzothiazole core may be
detrimental to activity and hence maintaining the unsubstituted
pharmacophore could prove best for the desired ago-allosteric
profile.
3
3
with À CF groups leading to 17. The aforementioned modifica-
3
tions (17) gratifyingly retained affinity, cooperativity and
agonism at the CaSR when compared to 2.
Ligand efficiency (LE, (kcal/mol)/heavy atom) and lipophillic
ligand efficiency (LLE) are metrics used in drug discovery
programs to evaluate a ligand’s binding affinity to a biological
protein relative to the compound‘s heavy atom count (molec-
As modifications conducted on the benzothiazole ring of 2
have yet to be reported in the literature, we next exchanged
the benzothiazole scaffold for its simplest unit (an unsubsti-
tuted thiazole) yielding compound 20. Unfortunately, replacing
the benzothiazole fused ring system for a five-membered
heterocyclic ring resulted in a significant reduction in coopera-
tivity and loss of agonist activity, although affinity was retained
compared to 2.
To address the previous significant structural change in
scaffold, we explored the replacement of the benzothiazole
system for a 4,5-dimethylthiazole core (21) to foster additional
hydrophobic interactions, while retaining the stereogenic meth-
yl, hydroxyl group and 2,4-dimethylphenyl motif of 2. Impor-
tantly 21 retained affinity, cooperativity and efficacy compared
to 2, highlighting a new chemotype with notable activity for
the CaSR. We subsequently explored a multifunctional strategy
incorporating the 4,5-dimethylthiazole core with the 2,4-bis
[14]
ular size) and lipophilicity (logP), respectively.
In general,
structural modifications promoting an increase in LE and LLE
are typically associated with mutually improved potential for
lead optimization and drug-like properties. Through successive
iterations of compound refinement, both the LE and LLE of the
parent scaffold have been enhanced compared to that of 2,
with the most efficient compounds being 21 (Figure 6, LLE=
2.11, LE=0.41, clogP=3.09) and 20 (Figure 6, LLE=2.56, LE=
0.42, clogP=2.56). Compound 21 has emerged as a genuine
lead optimisation candidate with improved physicochemical
and drug-like properties compared to 2, whilst importantly
retaining receptor affinity and cooperativity, and intrinsic
efficacy. This study also reveals the versatility of the thiazole
scaffold for further structural interrogation and development of
2
+
(trifluoromethyl)phenyl motif (22). These modifications demon-
ligands that allosterically modulate Ca
the CaSR.
affinity and efficacy at
o
strated retention of affinity, efficacy and cooperativity com-
pared with the parent compound 2, thus highlighting the
success of implementing the multifuctionalised scaffold hop-
ping and bioisosteric replacement strategies.
Logically, we focused on the introduction of substituents on
the benzothiazole core since to date analogues with substitu-
ents on the benzothiazole have not been disclosed. Our
preliminary docking studies predicted the 6-methyl or 6-fluoro
benzothiazole derivatives would bind within the 7TM in a
similar manner to AC265347. We therefore coupled 6-substi-
tuted benzothiazoles with the 2,4-dimethylphenyl substituent
present in 2. The 6-methyl and 6-fluoro substituted variants
were chosen since the methyl represented the smallest alkyl
substituent, whilst fluorine is an isostere of hydrogen with their
atomic sizes being remarkably similar but possessing strikingly
different electronegativities. Given the size constraints of the
binding cleft, it was hypothesized that the fluorine derivative
Figure 6. Lipophillic ligand efficiencies (LLE), and ligand efficiencies (LE) of 2,
, 21, and 20 calculated according to equilibrium dissociation constants
6
2
+
derived from functional interaction experiments measuring Ca mobiliza-
tion. Multiple compound iterations have yielded improvements in the
scaffold’s efficiency through decreased molecular weight and comparable
functional binding affinity. This is particularly evident with compounds 20
and 21 as they exhibit a marked improvement in molecular weight (MW),
lipophilicity (clogP), LLE and LE compared to that of the parent compound,
2, resulting in promising new starting scaffolds that can be elaborated
through new vectors. (ACD Percepta 2019.1.1).
2
7 would retain affinity, and the methylated analogue 26 would
demonstrate a modest drop in affinity. Analogue 26 (6-CH3
benzothiazole core) lost activity, whereas the fluorinated
analogue 27 (6-F) maintained affinity, cooperativity and efficacy
compared to 2. These data suggest the methyl group at the 6-
position was sterically disfavoured compared to the smaller
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Conclusion
100% B [1 min]; 0.5 mL/min). Low-resolution mass spectra (MS)
were run on a Agilent 6100 series single quad LC–MS coupled with
an Agilent 1200 series HPLC, G1311 A quaternary pump, G1329 A
thermostated autosampler, and G1314B variable wavelength
detector (214 and 254 nm). LC conditions were as follows:
Phenomenex Luna C8(2) column (100 Å, 5 μm, 50×4.6 mm), 30°C;
We have expanded on a class of PAMs previously described by
Gustafsson et al. by generating a focused library of compounds,
providing further insight into the SAR of these benzothiazole
derived analogues. Analogues in our series explored three key
regions of the AC265347 lead core: the benzothiazole motif, the
substituent effects on the phenyl ring and the stereogenic
centre, after which a multifunctionalised approach was em-
ployed incorporating the ‘best’ modifications. The above data
suggests modifications to the parent compound afford novel
PAMs that retain affinity, positive cooperativity and efficacy at
the CaSR. Our work highlights bioisosteric replacement and
scaffold hopping strategies can deliver compounds with
comparable affinity and efficacy to the lead compound
AC265347 (2). Minor alterations to the compound structure
suggested detrimental effects on activity, e.g. changing the
position of isosteres from 2,4-diCF (7) to 3,5-diCF , (8) resulted
in the loss of activity. Our exploration of 6-position substituted
benzothiazole analogues resulted in compounds with reduced
activity (26 and 28), highlighting the possible tight and hydro-
phobic nature of the 7TM allosteric binding pocket and limited
scope for further functionalization. Importantly we have dem-
onstrated compounds with a favourable mix of affinity,
cooperativity and efficacy profiles and improved drug-like
properties that could find application in CaSR-related disorders
where AC265347 may be effective in rescuing loss-of-function
the sample (5 μL) was eluted using a binary gradient (solvent A:
0.1% aq. HCO
10 min], 100% B [10 min]; 0.5 mL/ min). MS conditions were as
H; solvent B: 0.1% HCO H in CH CN; 5–100% B
2
2 3
[
follows: quadrupole ion source with multimode ESI; drying gas
temperature, 300°C; vaporizer temperature, 200°C; capillary volt-
age, 2000 V (positive mode) or 4000 V (negative mode); scan range,
100–1000 m/z; and step size, 0.1 s over 10 min. High-resolution MS
was performed on an Agilent 6224 TOF LCMS coupled to an Agilent
1290 Infinity LC. All data were acquired and reference mass
corrected via a dual-spray electrospray ionization (ESI) source. Each
scan or data point on the total ion chromatogram (TIC) is an
average of 13700 transients, producing a spectrum every second.
Mass spectra were created by averaging the scans across each peak
and subtracting the background from first 10 s of the TIC.
Acquisition was performed using the Agilent Mass Hunter Data
Acquisition software (ver. B.05.00, build 5.0.5042.2), and analysis
was performed using Mass Hunter Qualitative Analysis (ver. B.05.00
build 5.0.519.13). Acquisition parameters were as follows: mode,
ESI; drying gas flow, 11 L/min; nebulizer pressure, 45 psi; drying gas
temperature, 325°C; voltages: capillary, 4000 V; fragmentor, 160 V;
3
3
skimmer, 65 V; octapole RF, 750 V; scan range, 100–1500 m/z; and
positive ion mode internal reference ions, m/z 121.050873 and
9
22.009798. LC conditions were as follows: Agilent Zorbax SBÀ C18
rapid resolution HT (2.1×50 mm, 1.8 μm column), 30°C; the sample
(
5 μL) was eluted using a binary gradient (solvent A: 0.1% aq.
HCO H; solvent B: 0.1% HCO H in CH CN; 5–100% B [3.5 min],
2
2
3
[6]
CaSR mutants, with potentially reduced side-effects. Of
particular note was that truncation of the benzothiazole scaffold
of 2 led to the discovery of new chemotypes: 4,5-dimeth-
ylthiazole (21) and the unsubstituted thiazole (20), with
improved LE and LLE, and drug-like properties that could be
further optimized to discover ‘next generation’ analogues with
a spectrum of allosteric profiles for potential therapeutic
application devoid of clinically-limiting adverse effects.
°
0
.5 mL/min). All glassware was dried in an oven at 110 C and
cooled under nitrogen gas prior to use.
Syntheses
[15]
General procedure A
Under anhydrous conditions, benzothiazole or substituted benzo-
thiazole (0.4 mmol, 1.2 equiv.) was dissolved in anhydrous THF
(
2 mL) then cooled to À 78°C. nBuLi (1.4 equiv., 2.5 M in THF) was
subsequently added dropwise and after complete addition the
reaction was allowed to stir for 30 min. Substituted acetophenone
Experimental Section
(1 equiv) was then added to the cooled solution in anhydrous THF
General Procedures
(1 mL). The resulting orange reaction mixture was subsequently left
stirring for 1.5 h at À 78°C before allowing the reaction to warm to
Thin layer chromatography (TLC) was performed using TLC silica
gel 60 F254 aluminium sheets (0.25 mm, Merck). Flash column
chromatography was carried out with silica gel 60, 0.6–0.20 mm
room temperature and stirred for an additional 2 h. The reaction
was quenched with NH Cl
(5 mL) and extracted with EtOAc (3×
4
(sat’d)
7
mL). Organic layer was collected and subsequently washed with
1
13
(
70–230 mesh, Merck). H (400.13 MHz) and C NMR (100.62 MHz)
brine then collected, dried over anhydrous Na SO and concen-
2
4
spectra obtained on a Bruker Avance III Nanobay spectrometer with
a BACS 60 sample changer using solvents from Cambridge Isotope
Laboratories. Chemical shifts (δ, ppm) are reported relative to the
trated under reduced pressure.
1
13
solvent peak (CDCl : 7.26 [ H] or 77.16 [ C]. Coupling constants (J)
[16]
3
General procedure B
are given in hertz (Hz), and the following abbreviations were
assigned for signal multiplicities: brs=broad singlet, s=singlet, d=
doublet, t=triplet, and m=multiplet. The purity of the compounds
The appropriately substituted benzoic acid (2 mmol, 1 equiv.) was
stirred in CH Cl (5 mL) followed by the drop-wise addition of oxalyl
2
2
(>95%) was established analytical HPLC. Analytical HPLC was
chloride (1.25 equiv.) and DMF (2-3 drops). The reaction was stirred
for 1.5 h, and then reduced under vacuum. The clear oil was re-
acquired on an Agilent 1260 Infinity analytical HPLC coupled with a
G1322 A degasser, G1312B binary pump, G1367E high-performance
autosampler, and G4212B diode array detector. Conditions were as
follows: Zorbax Eclipse Plus C18 rapid resolution column (4.6×
suspended in CH
ylhydroxylamine HCl salt (1.4 equiv.) and the drop-wise addition of
Et N (3 equiv.). The resultant reaction was stirred for 18 h after
which the reaction was quenched with Na CO3(satd), extracted with
CH Cl and washed with brine. The organic layer was dried over
anhydrous Na SO and concentrated under reduced pressure.
Cl (5 mL) followed by addition of N,O-dimeth-
2
2
3
1
00 mm) with UV detection at 254 and 214 nm, 30°C; the sample
2
was eluted using a gradient system, where solvent A was 0.1% aq.
TFA and solvent B was 0.1% TFA in CH CN (5–100% B [9 min],
2
2
3
2
4
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[16]
General procedure C
1-(Benzo[d]thiazol-2-yl)-1-(4-methoxy-2-methylphenyl)
ethan-1-ol (6)
To a solution of benzothiazole (0.3 mmol, 1.1 equiv.) in dry THF
(
3 mL) under anhydrous conditions at À 78°C was added nBuLi
Synthesis as described in general procedure A
solution (2.5 M, 1.3 equiv.) in a dropwise fashion and stirred for 1 h.
The Weinreb amide (12 or 13) in dry THF solution was subsequently
added dropwise, then the reaction mixture was allowed to reach r.t
and stirred for 24 h. The resulting mixture was quenched by
The resulting crude was purified via silica gel column chromatog-
raphy (5:1 PET spirits:EtOAc) to afford the product as a white solid
1
(
48 mg, 58%). H NMR (401 MHz, Acetone-d ) δ 7.99 (ddd, J=7.8,
6
saturated NH Cl (5 mL) and extracted with EtOAc (3×7 mL) and
4
1
.4, 0.6 Hz, 1H), 7.88 (ddd, J=8.1, 1.2, 0.6 Hz, 1H), 7.62 (d, J=8.6 Hz,
then washed with brine. The combined organic layer was dried
over anhydrous Na SO then concentrated under reduced pressure.
1H), 7.49–7.44 (m, 1H), 7.39 (ddd, J=7.9, 7.2, 1.3 Hz, 1H), 6.77 (ddd,
2
4
J=8.7, 2.8, 0.6 Hz, 1H), 6.70 (d, J=2.8 Hz, 1H), 5.40 (s, OH), 3.78 (s,
1
3
3
H), 2.17 (s, 3H), 2.08 (s, 3H). C NMR (101 MHz, Acetone) δ 181.64
(C), 160.02 (C), 154.13 (C), 139.47 (C), 136.63 (C), 136.47 (C), 128.33
CH), 126.59 (CH), 125.70 (CH), 123.63 (CH), 122.70 (CH), 118.42 (CH),
110.70 (CH), 77.19 (C), 55.37 (CH ), 31.00 (CH ), 21.74 (CH ). HRMS
(m/z): C17 S requires 300.1053 [M+H] ; found 300.1061.
HPLC: t 5.452 min, >95% purity (214 & 254 nm)
[17]
General procedure D
(
A solution of benzo[d]thiazol-2-yl(phenyl)methanone derivative (14
or 15) (0.2 mmol, 1 equiv.) in THF solution was cooled to 0°C
3
3
3
+
H
17NO
2
followed by the addition of CF SiMe (4.7 equiv.) and Me NF
R
3
3
4
(
0.25 equiv.). The reaction mixture was stirred at 0°C for 30 min
before warming to room temperature and stirring for an additional
4
1
-(Benzo[d]thiazol-2-yl)-1-(2,4-bis(trifluoromethyl)phenyl)
h. The resulting reaction mixture was quenched by NaHCO
3(satd)
ethan-1-ol (7)
(
5 mL), extracted with EtOAc (3×7 mL), dried over anhydrous
Na SO and concentrated under reduced pressure.
2
4
Synthesis as described in general procedure A
1
-(Benzo[d]thiazol-2-yl)-1-(4-isopropylphenyl)ethan-1-ol (4)
The resulting crude mixture was purified using silica gel column
chromatography (8:1 PET spirits:EtOAc) to afford the product as
1
yellow paste (52%, 32 mg). R =0.4. H NMR (401 MHz, CDCl ) δ 8.03
f
3
Synthesis as described in general procedure A
(s, 1H, H3”), 7.98 (ddd, J=8.2, 1.1, 0.6 Hz, 1H, H4’), 7.92 (d, J=8.4 Hz,
The resulting crude mixture was purified using silica gel column
1H, H6”), 7.84 (ddd, J=8.0, 1.2, 0.6 Hz, 1H, H7’), 7.79 (dd, J=8.4,
chromatography (8:1 PET spirits:EtOAc) to afford the product as a
1.3 Hz, 1H, H5”), 7.48 (ddd, J=8.3, 5.9, 1.3 Hz, 1H, H5’), 7.39 (ddd,
1
13
white solid (31 mg, 34%). R (8:1 PET spirits:EtOAc)=0.18. H NMR
J=8.0, 6.8, 1.2 Hz, 1H, H6’), 3.89 (bs, 1H, OH), 2.21 (s, 3H, H2).
NMR (101 MHz, CDCl
C
f
(
7
401 MHz, CDCl ) δ 8.00 (d, J=8.1 Hz, 1H), 7.84 (d, J=7.9 Hz, 1H),
.54 (d, J=8.4 Hz, 2H), 7.46 (ddd, J=8.3, 7.3, 1.2 Hz, 1H), 7.35 (ddd,
3
) δ 177.31 (C), 152.47 (C), 147.40 (C), 135.59
3
(C), 130.00 (CH), 129.28 (m, 2 C), 128.56 (m, CH), 126.52 (CH), 126.39
J=8.1, 7.2, 1.0 Hz, 1H), 7.21 (d, J=8.2 Hz, 2H), 3.81 (bs, OH), 2.88
(q, J=253 Hz, CF), 125.85 (m, CH), 125.68 (CH), 123.40 (CH), 122.16
1
3
19
(
hept, J=6.9 Hz, 1H), 2.13 (s, 3H), 1.24 (s, 3H), 1.23 (s, 3H). C NMR
(q, J=247 Hz, CF), 121.94 (CH), 79.13 (C), 32.18 (CH
(377 MHz, CDCl ), À 63.04 (CF
) δ À 55.50 (CF
+H] . HRMS (m/z): C17 NOS requires 392.0538 [M+H] ; found
392.0555. HPLC: t 7.131 min, >95% purity (214 & 254 nm)
). F NMR
3
(
1
1
101 MHz, CDCl ) δ 179.24 (C), 152.90 (C), 148.58 (C), 142.45 (C),
35.77 (C), 126.64 (2CH), 126.14 (CH), 125.52 (2CH), 125.14 (CH),
3
3
). LCMS (m/z): 391.9 [M
3
+
3
+
H F
11 6
23.21 (CH), 121.82 (CH), 76.61 (C), 33.83 (CH), 30.36 (CH ), 24.00
R
3
+
(
2CH ). LCMS (m/z): 320.0 [M+Na] . HRMS (m/z): C H NOS
3
18 19
+
requires [M+H] 298.1260; found 298.1271. HPLC: tR 6.998 min,
1
-(Benzo[d]thiazol-2-yl)-1-(3,5-bis(trifluoromethyl)phenyl)
>
95% purity (214 & 254 nm)
ethan-1-ol (8)
1
-(Benzo[d][1,3]dioxol-5-yl)-1-(benzo[d]thiazol-2-yl)ethan-1-ol
Synthesis as described in general procedure A
(5)
The resulting crude mixture was purified via silica gel column
chromatography (12:1 PET spirits:EtOAc) to afford the product as a
Synthesis as described in general procedure A
1
light-yellow oil (16 mg, 27%). R =0.16. H NMR (401 MHz, CDCl ) δ
f
3
The resulting crude was purified via silica gel column chromatog-
raphy (12:1 toluene: acetone) to afford the product as a light-
8.17 (s, 1H, H2” and H6”), 8.02 (d, J=8.1 Hz, 1H, H4’), 7.87 (d, J=
8.0 Hz, 1H, H7’), 7.81 (s, 1H, H4”), 7.50 (ddd, J=8.3, 7.3, 1.2 Hz, 1H,
H5’), 7.40 (ddd, J=8.1, 7.2, 0.9 Hz, 1H, H6’), 3.85 (bs, OH), 2.18 (s, 3H,
1
yellow paste (45 mg, 61%). R =0.36. H NMR (401 MHz, CDCl ) δ
f
3
1
3
7
.99 (d, J=8.1 Hz, 1H, H4’), 7.84 (d, J=7.9 Hz, 1H, H7’), 7.46 (ddd,
H2). C NMR (101 MHz, CDCl ) δ 176.57 (C), 152.83 (C), 147.61 (C),
3
J=8.3, 7.3, 1.2 Hz, 1H, H5’), 7.35 (ddd, J=8.1, 7.3, 0.9 Hz, 1H), 7.12
131.87 (q, J=66.7, 2 C), 126.60 (CH), 126.04 (m, 2CH), 125.73 (CH),
(
1
d, J=1.7 Hz, 1H), 7.09 (dd, J=8.2, 1.8 Hz, 1H), 6.77 (d, J=8.1 Hz,
H), 5.93 (s, 2H), 3.84 (bs, OH), 2.10 (s, 3H, H2). C NMR (101 MHz,
124.76 (C),124.35, (q, J =247.2 Hz, 2 C), 123.55 (CH), 122.02 (CH),
13
19
121.93 (m, CH), 76.21 (C), 31.12 (CH
). HRMS (m/z): C17
). F NMR (377 MHz, CDCl
) δ
3
3
+
CDCl ) δ 178.87 (C), 152.74 (C), 147.77 (C), 146.98 (C), 139.10 (C),
À 62.76 (2CF
3
H
11
F
6
NOS requires 392.0538 [M+H] ;
7.492 min, >95% purity (214 & 254 nm)
3
1
1
35.60 (C), 126.09 (CH), 125.12 (CH), 123.15 (CH), 121.75 (CH),
found 392.0553. HPLC: t
R
18.88 (CH), 107.98 (CH), 106.63 (CH), 101.20 (CH ), 76.37 (C), 30.45
2
+
(CH ). LCMS (m/z): 281.9 [M-OH] . HRMS (m/z): C H NO S requires
3
16 13
3
+
3
00.0689 [M+H] ; found 300.0699. HPLC: tR 5.837 min, >95%
purity (214 & 254 nm)
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1
-(benzo[d]
J=8.0 Hz, 1H, H6), 7.57 (d, J=8.0 Hz, 1H, H7), 3.89 (bs, 0.4H), 3.43 (s,
2.6H), 3.37 (s, 2.6H), 3.05 (bs, 0.4H). LCMS (m/z): 302.0 [M+H] .
+
thiazol-2-yl)-1-(2-methyl-4-((tetrahydro-2H-pyran-2-yl)oxy)
phenyl)ethan-1-ol (9a)
HPLC: t 6.058 min, >95% purity (214 & 254 nm).
R
Synthesis as described in general procedure A
Benzo[d]thiazol-2-yl(2,4-dimethylphenyl)methanone (14)
The resulting crude was purified via silica gel column chromatog-
raphy (4:1 PET spirit:EtOAc) to afford the product as a solid (83 mg,
Synthesis as described in general procedure C
35%).
The resulting crude mixture was purified using silica gel column
chromatography (20:1 PET spirits:acetone) to afford the product as
1
H NMR (401 MHz, CDCl ) δ 7.97 (d, J=8.2 Hz, 1H), 7.79 (t, J=7.7 Hz,
3
1
1
H), 7.54 (d, J=8.7 Hz, 1H), 7.47–7.40 (m, 1H), 7.37–7.30 (m, 1H),
.91 (dt, J=8.7, 2.1 Hz, 1H), 6.83 (t, J=1.9 Hz, 1H), 5.44 (t, J=2.9 Hz,
H), 3.96–3.86 (m, 2H), 3.64–3.56 (m, 1H), 2.14 (s, 3H), 2.09 (s, 3H),
white crystal (164 mg, 76%,). R =0.17. H NMR (401 MHz, CDCl ) δ
f
3
6
1
1
1
1
1
1
7
2
8
.20 (m, 1H, H4’), 8.01 (m, 2H, H7’and H6’’), 7.54 (m, 2H, H5’’ and
H6’’), 7.17 (m, 2H, H3’’ and H5’’), 2.53 (s, 3H, 2’’-CH ), 2.41 (s, 3H, 4’’-
CH3). C NMR (101 MHz, CDCl ) δ 188.73 (C), 168.16 (C), 153.91 (C),
3
3
13
1
3
.88–1.82 (m, 2H), 1.75–1.52 (m, 3H). C NMR (101 MHz, CDCl ) δ
3
80.08 (C), 157.11 (C), 152.46 (C), 139.55 (d, J=2.3 Hz, C), 136.03 (C),
34.95 (d, J=2.1 Hz, C), 127.60 (d, J=1.5 Hz, CH), 126.12 (CH),
25.17 (CH), 123.16 (CH), 121.83 (CH), 120.45 (d, J=11.1 Hz, CH),
1
43.01 (C), 139.77 (C), 137.32 (C), 132.64 (CH), 132.30 (C), 132.17
(CH), 127.64 (CH), 126.97 (CH), 126.19 (CH), 125.87 (CH), 122.32 (CH),
+
2
1.70 (CH ), 20.92 (CH ). HRMS (m/z): C H NOS requires [M+H]
3 3 16 13
12.88 (d, J=13.7 Hz, CH), 96.22 (d, J=2.8 Hz, CH), 76.66 (C or CH2),
268.0797; found 268.0791. HPLC: t 7.499 min, >95% purity (214 &
R
6.60 (C or CH ), 62.09 (d, J=2.0 Hz, CH ), 31.51 (CH ), 30.46 (CH ),
2
2
3
2
254 nm)
5.33 (CH ), 21.57 (CH ), 18.83 (CH ). HRMS (m/z): C H NO S
2
3
2
21 23
3
+
requires 370.1471 [M+H] ; found 370.1481. HPLC: tR 5.965 min,
95% purity (214 & 254 nm)
>
Benzo[d]thiazol-2-yl(2,4-bis(trifluoromethyl)phenyl)
methanone (15)
4
-(1-(Benzo[d]thiazol-2-yl)-1-hydroxyethyl)-3-methylphenol
Synthesis as described in general procedure C
(9b)
The resulting crude mixture was purified using silica gel column
chromatography (12:1 PET spirits:acetone) to afford the product as
9
a was stirred for in 2 mL of 50% acetic acid at 50°C for 2 h with a
few drops of EtOAc to help dissolve the solid. The crude mixture
was allowed to cool to room temperature followed by extraction
with EtOAC, wash with water, dried over Na SO and reduced under
1
white solid (65 mg, 76%). R =0.23. H NMR (401 MHz, CDCl ) δ 8.14
f
3
(
m, 1H, H4’), 8.08 (s, 1H, H3’’), 8.04 (m, 1H, H7’’), 7.98 (d, J=8.1 Hz,
2
3
13
1
1H, H5’’), 7.87 (d, J=8.1 Hz, 1H, H6’’), 7.59 (m, 2H, H5’ and H6’).
C
pressure to furnish solid product in quantitative yield. H NMR
401 MHz, Acetone-d ) δ 8.23 (s, 1H), 7.99 (ddd, J=7.9, 1.3, 0.6 Hz,
NMR (101 MHz, CDCl ) δ 187.48 (C), 165.10 (C), 153.77 (C), 139.38
3
(
1
(
(
1
(
(
6
(
C), 137.74 (C), 133.20 (q, J=33.8 Hz, C), 130.31 (CH), 129.88 (q, J=
3.6 Hz, C), 128.66 (CH), 128.54 (m, C), 127.55 (C), 126.22 (CH),
H), 7.87 (ddd, J=8.1, 1.2, 0.6 Hz, 1H), 7.51 (d, J=8.5 Hz, 1H), 7.46
3
1
ddd, J=8.2, 7.2, 1.3 Hz, 1H), 7.38 (ddd, J=7.9, 7.2, 1.3 Hz, 1H), 6.67
ddd, J=8.5, 2.7, 0.6 Hz, 1H), 6.62 (dd, J=2.7, 0.8 Hz, 1H), 5.32 (s,
24.30 (m, C), 123.11 (q, J=273.0 Hz, CF), 122.94 (q, J=279.3 Hz,
1
9
13
CF) 122.61 (CH). F NMR (377 MHz, CDCl
3
) δ À 58.18, À 63.14. HRMS
H), 2.12 (s, 3H), 2.06 (s, 3H). C NMR (101 MHz, Acetone) δ 206.20
+
(
m/z): C H F NOS requires [M+H] 375.0098; found 375.0147.
1
6
7 6
C), 157.71 (C), 154.14 (C), 139.46 (C), 136.67 (C), 135.36 (C), 128.42
CH), 126.57 (CH), 125.67 (CH), 123.62 (CH), 122.69 (CH), 119.61 (CH),
HPLC: t 7.655 min, >95% purity (214 & 254 nm)
R
1
12.52 (CH), 77.22 (C), 31.07 (CH ), 21.65 (CH ). HRMS (m/z):
3 3
+
C H F NO S requires 286.0896 [M+H] ; found 286.0904. HPLC: t
1
6
15
3
2
R
1
-(Benzo[d]
thiazol-2-yl)-1-(2,4-dimethylphenyl)-2,2,2-trifluoroethan-1-ol
16)
4.640 min, >95% purity (214 & 254 nm)
(
N-Methoxy-N,2,4-trimethylbenzamide (12)
Synthesis as described in general procedure D
Synthesis as described in general procedure B
Purification of resulting crude mixture was performed using silica
gel column chromatography (20:1 PET spirits:EtOAc) to afford the
Purification using silica gel column chromatography (4:1 PET spirits:
1
product as a white solid (28 mg, 45%). R =0.25. H NMR (401 MHz,
f
EtOAc) was performed to afford product as a clear oil (260 mg,
1
CDCl
.2, 0.6 Hz, 1H, H7’), 7.63 (dq, J=8.0, 2.3 Hz, 1H, H5’’), 7.55 (ddd, J=
8.3, 7.3, 1.2 Hz, 1H, H5’), 7.44 (ddd, J=8.2, 7.3, 1.1 Hz, 1H, H6’), 7.11
3
) δ 8.12 (ddd, J=8.3, 1.1, 0.7 Hz, 1H, H4’), 7.83 (ddd, J=8.0,
6
7%,). R =0.37. H NMR (401 MHz, CDCl ) δ 7.14 (d, J=7.6 Hz, 1H,
f
3
1
H6), 7.00 (s, 1H, H3), 6.98 (d, J=0.5 Hz, 1H, H5), 3.55 (s, 3H, O-CH ),
3
1
3
3
.27 (s, 3H, N-CH ), 2.31 (s, 3H, 4-CH ), 2.28 (s, 3H, 5-CH ). C NMR
3
3
3
(
(
m, 1H, H6’’), 7.00 (m, 1H, H3’’), 5.17 (s, 1H), 2.35 (s, 3H, 2’’-CH ), 2.06
3
(101 MHz, CDCl ) δ 139.09 (C), 134.81 (C), 132.32 (C), 130.92 (CH),
3
13
s, 3H, 4’’-CH3). C NMR (101 MHz, CDCl ) δ 169.29 (C), 151.23 (C),
3
1
26.31 (CH), 126.06 (CH), 61.01 (CH ), 23.86 (CH ), 21.25 (CH ), 19.06
3 R
3 3 3
1
39.78 (C), 139.28 (C), 137.13 (C), 134.14 (CH), 131.03 (C), 126.87 (d,
J=3.9 Hz, CH), 126.71 (CH), 126.41 (CH), 126.21 (CH), 124.29 (q, J=
88 Hz, CF) 123.95 (CH), 121.94 (CH), 79.95 (q, J=29.0 Hz, C), 21.23
(CH ). HPLC: t 9.934 min, >95% purity (214 nm). Does not ionize in
LCMS.
2
1
9
(CH ), 21.07 (CH ). F NMR (377 MHz, CDCl ) δ À 73.21 (s). HRMS (m/
3
3
3
+
z): C H F NOS requires 338.0821 [M+H] ; found 338.0834. HPLC:
N-Methoxy-N-methyl-2,4-bis(trifluoromethyl)benzamide (13)
17 14 3
tR 7.012 min, >95% purity (214 & 254 nm)
Synthesis as described in general procedure B
Purification using silica gel column chromatography (4:1 PET spirits:
EtOAc) was performed to afford the product as a clear oil (69 mg,
1
5
9%). R =0.17. H NMR (401 MHz, CDCl ) δ 7.95 (s, 1H, 4H), 7.86 (d,
f
3
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1
-(Benzo[d]thiazol-2-yl)-1-(2,4-bis(trifluoromethyl)
H6’’), 3.81 (s, 1H, OH), 2.32 (d, J=0.7 Hz, 3H, 4’-CH
3
), 2.28 (d, J=
), 2.05 (s, 3H, H2). C NMR (101 MHz, CDCl ) δ
71.56 (C), 148.75 (C), 147.80 (C), 130.41 (q, J=67.2, C), 129.99 (CH),
29.03 (q, J=65.3, C), 128.32 (d, J=3.2 Hz, C), 127.59 (CH), 125.64
1
3
phenyl)-2,2,2-trifluoroethan-1-ol (17)
0.7 Hz, 3H, 5’-CH
3
3
1
1
Synthesis as described in general procedure D
(m, CH), 123.78 (q, J=274.0, CF), 123.44 (q, J=272.4 Hz, CF), 76.44
19
The resulting crude mixture was purified using silica gel column
chromatography (12:1 PET spirits:acetone) to afford the product as
white solid (15 mg, 32%). R =0.2. H NMR (401 MHz, CDCl ) δ 8.21
(C), 31.83 (CH
3
), 14.73 (CH
3
), 11.44 (CH
). F NMR (377 MHz, CDCl ) δ
3 3
+
À 55.32 (C HF ) and À 63.01 (CF
(m/z): C15 13F NOS requires 370.0695 [M+H] ; found 370.0708.
6
). LCMS (m/z): 369.9 [M+H] . HRMS
3
3
1
+
f
3
(
d, J=8.4 Hz, 1H, H6”), 8.13 (m, 1H, H3”), 8.12 (m, 1H, H4’), 7.96 (dd,
J=8.5, 1.5 Hz, 1H, H5”), 7.84 (ddd, J=8.1, 1.2, 0.7 Hz, 1H, H7’), 7.57
ddd, J=8.3, 6.4, 1.2 Hz, 1H, H5’), 7.46 (ddd, J=8.3, 7.3, 1.2 Hz, 1H,
HPLC: t 6.619 min, >95% purity (214 & 254 nm)
R
(
13
1
-(2,4-Dimethylphenyl)-1-(6-methylbenzo[d]thiazol-2-yl)
H6’), 5.50 (s, OH). C NMR (101 MHz, CDCl ) δ 166.55 (C), 151.32 (C),
3
1
3
1
1
7
38.85 (C), 136.59 (C), 132.45 (q, J=34.8 Hz, C), 131.20 (q, J=
3.4 Hz, C), 129.13 (dd, J=9.5, 4.8 Hz, CH), 128.73 (CH), 127.14 (CH),
26.80 (m, CH), 126.62 (CH), 124.17 (CH), 123.83 (q, J=289 Hz, CF),
23.13 (q, J=274.2 Hz, CF), 123.04 (q, J=272.8 Hz, CF) 121.96 (CH),
ethan-1-ol (26)
Synthesis as described in general procedure A
19
9.51 (q, J=29.8 Hz, C). F NMR (377 MHz, CDCl ) δ À 55.48 (CF ),
The resulting crude mixture was purified using silica gel column
3
3
+
À 63.34 (CF ), À 73.04 (CF ). LCMS (m/z): 445.8 [M+H] . HRMS (m/z):
chromatography (10:1 PET spirits:EtOAc) to afford the product as a
3
3
+
1
C H F NOS requires 446.0256 [M+H] ; found 446.0269. HPLC: t
R
white solid (49 mg, 49%). R
f
=0.19. H NMR (401 MHz, CDCl
3
) δ 7.88
1
7
8
9
7.390 min, >95% purity (214 & 254 nm).
(d, J=8.3 Hz, 1H, H4’), 7.60 (s, 1H, H7’), 7.54 (d, J=8.0 Hz, 1H, H6’’),
7.28 (dd, J=8.3, 1.3 Hz, 1H, H5’), 7.07 (d, J=8.0 Hz, 1H, H5’’), 6.98 (s,
1
H, H3’’), 3.83 (bs, 1H, OH), 2.47 (s, 3H, 6’-CH ), 2.34 (s, 3H, 2’’-CH ),
3
3
1
3
1
-(2,4-Dimethylphenyl)-1-(thiazol-2-yl)ethan-1-ol (20)
2.14 (s, 3H, 4’’-CH
), 2.10 (s, 3H, H2). C NMR (101 MHz, CDCl
) δ
3
3
178.56 (C), 150.58 (C), 138.98 (C), 138.37 (C), 137.62 (C), 136.23 (C),
1
35.33 (C), 133.41 (CH), 127.70 (CH), 126.40 (CH), 126.29 (CH),
Synthesis as described in general procedure A
122.73 (CH), 121.57 (CH), 76.72 (C), 31.20 (CH ), 21.61 (CH ), 21.29
3
3
+
The resulting crude mixture was purified using silica gel column
chromatography (6:1 PET spirits:EtOAc) to afford the product as a
(CH
C
3
), 21.04 (CH
19NOS requires 298.1260 [M+H] ; found 298.1273. HPLC: t
R
). LCMS (m/z): 280.0 [M-OH] . HRMS (m/z):
3
+
H
18
1
white solid (16 mg, 25%). R =0.24. H NMR (401 MHz, CDCl ) δ 7.70
6.884 min, >95% purity (214 & 254 nm)
f
3
(
1
2
d, J=2.5 Hz, 1H, H4’), 7.50 (d, J=8.0 1H, H6”), 7.29 (d, J=2.9 Hz,
H, H5’), 7.04 (d, J=8.5 Hz, 1H, H5”), 6.96 (s, 1H, H3”), 3.4 (bs, OH),
13
.31 (s, 3H, 2”-CH ), 2.08 (s, 3H, 4”-CH ), 2.05 (s, 3H, H2). C NMR
1-(2,4-Dimethylphenyl)-1-(6-fluorobenzo[d]thiazol-2-yl)
3
3
101 MHz, CDCl ) δ 179.14 (C), 141.58 (CH), 139.44 (C), 138.10 (C),
ethan-1-ol (27)
3
36.99 (C), 133.35 (CH), 126.28 (CH), 126.02 (CH), 119.63 (CH), 76.39
+
Synthesis as described in general procedure A
13
15
HPLC: t 5.262 min, >95% purity (214 & 254 nm)
The resulting crude mixture was purified using silica gel column
chromatography (10:1 PET spirits:EtOAc) to afford the product as a
R
1
yellow solid (12 mg, 12%). R =0.25. H NMR (401 MHz, CDCl ) δ
f
3
1
-(2,4-Dimethylphenyl)-1-(4,5-dimethylthiazol-2-yl)ethan-1-ol
(21)
Synthesis as described in general procedure A
The resulting crude mixture was purified using silica gel column
chromatography (8:1 PET spirits:EtOAc) to afford the product as a
(C), 137.51 (C), 133.51 (CH), 126.53 (CH), 126.31 (C), 124.22 (d, J=
9.4 Hz, CH), 114.79 (d, J=24.7 Hz, CH), 108.05 (d, J=26.6 Hz, CH),
1
19
yellow paste (28 mg, 40%). R =0.29. H NMR (401 MHz, CDCl ) δ
79.60 (C), 31.19 (CH
3
), 21.32 (CH
3
), 21.05 (CH
3
). F NMR (377 MHz,
f
3
.45 (d, J=8.0 Hz, 1H, H6’’), 7.01 (d, J=8.0 Hz 1H, H5’’), 6.96 (d, J=
CDCl
3
) δ À 116.08 (CF). HRMS (m/z): C17
H16FNOS requires 302.1009
6.755 min, >95% purity (214 &
+
[M+H] ; found 302.1016. HPLC t
254 nm).
3
R
1
3
C
3
3
3
NMR (101 MHz, CDCl ) δ 173.73 (C2’), 146.87 (C), 139.81 (C), 137.91
3
C), 137.38 (C), 133.33 (CH), 127.17 (C), 126.23 (CH), 126.21 (CH),
6.01 (C), 31.27 (C), 21.41 (CH ), 21.00 (CH ), 14.81 (CH ), 11.46 (CH ).
1-(2,4-Dimethylphenyl)-1-(thiazolo[5,4-c]pyridin-2-yl)
ethan-1-ol (28)
3
+
3
3
3
1
5
19
+
62.1260 [M+H] ; found 262.1270. HPLC: tR 5.136 min, >95%
purity (214 & 254 nm).
Synthesis as described in general procedure A.
The resulting crude was purified via silica gel column chromatog-
1
-(2,4-Bis(trifluoromethyl)phenyl)-1-(4,5-dimethylthiazol-2-yl)
raphy (8:1 toluene:acetone) to afford the product as a clear paste
1
ethan-1-ol (22)
(5 mg, 5%). H NMR (401 MHz, CDCl
3
) δ 9.09 (s, 1H), 8.59 (s, 1H),
7
1
.85 (d, J=5.4 Hz, 1H), 7.54 (d, J=8.0 Hz, 1H), 7.07 (d, J=8.0 Hz,
13
H), 6.98 (s, 1H), 4.09 (s, 1H), 2.32 (s, 3H), 2.13 (s, 3H), 2.12 (s, 3H).
C
Synthesis as described in general procedure A
NMR (101 MHz, CDCl ) δ 186.12 (C), 159.09 (C), 157.91 (C), 145.59
3
The resulting crude mixture was purified using silica gel column
chromatography (8:1 PET spirits:EtOAc) to afford the product as
(CH), 144.88 (CH), 144.36 (CH), 138.82 (C), 138.44 (C), 137.42 (C),
133.58 (CH), 126.62 (CH), 126.35 (CH), 77.29 (C), 31.39 (CH
), 21.37
). LCMS (m/z): 285.3 [M+H] . HRMS? HPLC: t
7.390 min, >95% purity (214 & 254 nm).
3
1
+
yellow solid (24 mg, 17%). R =0.2. H NMR (401 MHz, CDCl ) δ 8.01
(CH
3
), 21.04 (CH
3
R
f
3
(s, 1H, H3’’), 7.77 (d, J=8.6 Hz, 1H, H5’’), 7.74 (dd, J=8.5, 1.6 Hz, 1H,
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ChemMedChem
General Pharmacology
Homology modelling
The homology model of the CaSR 7TM domain was constructed
and validated as described previously. Briefly, the ICM software
Materials
[12]
TM
TM
package (Molsoft; San Diego, CA, USA) was used to thread the CaSR
protein sequence onto the mGlu5 template coordinates (PDB:
Flp-In TREX Human Embryonic Kidney (HEK) 293 cells were
purchased from Invitrogen (Carlsbad, USA). Fluo-8-AM
(
[11]
4OO9). Incomplete regions in the template structure, or regions
acetoxymethyl ester) was purchased from Abcam. AC265347,
with poor sequence alignment, were modelled using Monte Carlo
optimization in internal coordinates using ICM.
Dulbecco’s Modified Eagle’s Medium (high glucose), poly-D-lysine,
hygromycin B, blasticidin HCl, tetracycline and all other reagents
were purchased from Sigma Aldrich (St Louis, USA). Ionomycin was
Docking was performed using ICM, where the SAR candidate was
placed in the 7TM binding pocket of the CaSR homology model
[
18]
purchased from Cayman Chemicals (Michigan, USA).
[
12]
identified in.
The pharmacophore binding poses were then
[22]
refined as described previously.
Briefly, a flexible receptor
Cell lines
approach was employed where biased probability Monte Carlo
optimisation of receptor residue sidechains was undertaken while
simultaneously sampling PAM shape and position using Monte
FlpIn TRex HEK293 cells stably expressing the cmyc-tagged WT
[
18b,19]
CaSR have been described previously
DMEM supplemented with 5% FBS and antibiotic selection
200 μg/mL hygromycin; 5 μg/mL blasticidin).
and were maintained in
[
23]
Carlo randomisation.
Binding poses were assessed using ICM
[24]
docking scores and calculated binding energies. After sampling,
the binding pose with the lowest calculated binding energy for
each analogue was taken as the hypothesized binding mode.
(
2
+
Ca mobilisation assays
i
2
+
Ca
mobilisation assays were performed as previously
[20]
i
described. Specifically, FlpIn TRex HEK293 stable cell lines were
Acknowledgements
seeded at 80 000 cells/well in poly-D-lysine (50 μg/mL) coated clear
9
6 well plates and incubated overnight at 37°C, 5% CO , in the
2
This work was supported by the Australian Research Council
(Discovery project grant DP170104228) and the National Health
and Medical Research Council of Australia (Project grant
presence of 100 ng/mL tetracycline. Cells were washed in assay
buffer (150 mM NaCl, 2.6 mM KCl, 1.18 mM MgCl , 10 mM D-
2
glucose, 10 mM HEPES, 0.1 mM CaCl , 0.5% BSA, 4 mM probenecid,
2
pH 7.4) and loaded with 1 μM Fluo-8 AM in assay buffer for 1 hour.
Agonist and PAMs (0, 0.03, 0.1, 0.3, 1, 3 or 10 μM) were co-added
1
138891). KL and KJG are Australian Research Council Future
2
+
Fellows (fellowships FT160100075 and FT170100392, respectively).
and measurements of Ca
mobilisation were performed in
i
duplicate at 37°C using a Flexstation 1 or 3 (Molecular Devices;
Sunnyvale, CA, USA), using 490 nm excitation and 520 nm emission.
Data were normalised to the % of ionomycin response. 1 μM Conflict of Interest
ionomycin was used as an internal assay control.
The authors declare no conflict of interest.
Data and Statistical analysis
2
+
Keywords: calcium-sensing receptor · AC265347 · positive
Data describing the interaction between Ca
and the PAMs were
[10]
o
allosteric modulators
relationship
· class C GPCR · structure-activity
fitted to the following operational model of allosterism:
Effect ¼
À
�
nB
nB nT
Em ½A þ Cꢁ ðK þab½BꢁÞþt ½Bꢁ½EC ꢁ
(1)
B
B
50
nB
nB
nB nT
½
EC ꢁ ðK þ½BꢁÞþð½A þ Cꢁ ðK þab ½BꢁÞþt ½Bꢁ½EC ꢁ Þ
5
0
B
B
B
50
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ChemMedChem 2021, 16, 1–13
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Manuscript received: May 25, 2021
Accepted manuscript online: July 2, 2021
Version of record online: ■■■, ■■■■
Scammells, A. D. Conigrave, A. Christopoulos, K. Leach, Br. J. Pharmacol.
ChemMedChem 2021, 16, 1–13
www.chemmedchem.org
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FULL PAPERS
L. V. Dinh, Dr. A. DeBono, Dr. A. N.
Keller, Dr. T. M. Josephs, Dr. K. J.
Gregory, Dr. K. Leach*, Prof. B.
Capuano*
1
– 13
Development of AC265347-
Inspired Calcium-Sensing
Receptor Ago-Positive Allosteric
Modulators
Positive cooperativity: The CaSR is a
clinical target in the treatment of hy-
perparathyroidism and related
diseases. AC265347 is an ago-PAM
chemically distinct from clinically
approved CaSR PAMs. AC265347
potently suppressed PTH release in
rats and is virtually devoid of hypocal-
caemia and may therefore offer
unique clinical benefits. Herein we
report an SAR study disclosing
AC265347-like compounds with
diverse pharmacology and improved
physicochemical characteristics.