Synthesis and Evaluation of the First Fluorescent Antagonists of the Human P2Y2 Receptor Based on AR-C118925

Article abstract of DOI:10.1021/acs.jmedchem.8b00139

The human P2Y₂ receptor (hP2Y₂R) is a G-protein-coupled receptor that shows promise as a therapeutic target for many important conditions, including for antimetastatic cancer and more recently for idiopathic pulmonary fibrosis. As such, there is a need for new hP2Y₂R antagonists and molecular probes to study this receptor. Herein, we report the development of a new series of non-nucleotide hP2Y₂R antagonists, based on the known, non-nucleotide hP2Y₂R antagonist AR-C118925 (1), leading to the discovery of a series of fluorescent ligands containing different linkers and fluorophores. One of these conjugates, 98, displayed micromolar affinity for hP2Y₂R (pK_d = 6.32 ± 0.10, n = 17) in a bioluminescence-energy-transfer (BRET) assay. Confocal microscopy with this ligand revealed displaceable membrane labeling of astrocytoma cells expressing untagged hP2Y₂R. These properties make 98 one of the first tools for studying hP2Y₂R distribution and organization.

Full text of DOI:10.1021/acs.jmedchem.8b00139

Subscriber access provided by - Access paid by the | UCSB Libraries
Synthesis and Evaluation of the first Fluorescent Antagonists
2
of the Human P2Y Receptor based on AR-C118925
Sean Conroy, Nicholas D. Kindon, Jacqueline Glenn, Leigh A. Stoddart,
Richard James Lewis, Stephen J. Hill, Barrie Kellam, and Michael John Stocks
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00139 • Publication Date (Web): 20 Mar 2018
Downloaded from http://pubs.acs.org on March 21, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Synthesis and Evaluation of the first Fluorescent
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Antagonists of the Human P2Y Receptor based on
2
ARꢀC118925.
1,#
1,#
2,3
2,3
Sean Conroy , Nicholas D. Kindon , Jacqueline Glenn , Leigh A. Stoddart , Richard J.
4
2,3
1,3
1*
Lewis , Stephen J. Hill , Barrie Kellam , Michael J. Stocks
1
School of Pharmacy, Centre for Biomolecular Sciences, University Park Nottingham,
Nottingham, NG7 2RD, UK.
2
Division of Physiology, Pharmacology & Neuroscience, Medical School, University of
Nottingham, Nottingham, NG7 2UH, UK.
3
Centre of Membrane Proteins and Receptors, University of Birmingham and University of
Nottingham, the Midlands, NG7 2UH, UK.
4
Medicinal Chemistry, Respiratory, Inflammation and Autoimmunity, IMED Biotech Unit,
AstraZeneca, 431 83 Mölndal, Gothenburg, Sweden
#
These authors contributed equally to this work.
KEYWORDS: P2Y R, antagonists, fluorescence, BRET, G ProteinꢀCoupled Receptor, GPCR
2
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT
The human P2Y receptor (hP2Y R) is a G proteinꢀcoupled receptor that shows promise as a
2
2
therapeutic target for many important conditions including antiꢀmetastatic cancer therapy and
more recently for the treatment of idiopathic pulmonary fibrosis. As such, there is a need for new
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
hP2Y R antagonists and molecular probes to study this receptor. Herein, we report the
2
development of a new series of nonꢀnucleotide hP2Y R antagonists leading to the discovery of a
2
series of fluorescent ligands containing different linkers and fluorophores based on the known,
nonꢀnucleotide hP2Y R antagonist ARꢀC118925 (1). One of these conjugates 98 displayed
2
micromolar affinity for the hP2Y R (pK = 6.32 ± 0.10; n=17) using a bioluminescence energy
2
d
transfer (BRET) assay. Confocal microscopy with this ligand revealed displaceable membrane
labeling of astrocytoma cells expressing unꢀtagged hP2Y R. These properties, make 98 one of
2
the first tools for studying hP2Y R distribution and organization.
2
INTRODUCTION
P2Y receptors (P2YRs) are G proteinꢀcoupled receptors (GPCRs) that are activated by
extracellular nucleotides. The P2Y family is composed of eight members encoded by distinct
genes that can be subdivided into two groups based on their primary signaling through specific G
1
proteins and sequence homology. The first subꢀgroup includes the P2Y1
receptors which
,2,4,6,11
primarily signal though G , with the second subꢀgroup signaling through G encompassing the
q
i
2
P2Y12,13,14 receptor subtypes. Notably, the P2Y receptor (P2Y R) is activated by the
2
2
endogenous agonists uridineꢀ5′ꢀtriphosphate (UTP hP2Y , EC = 140 nM) and adenosineꢀ5′ꢀ
2
50
3
triphosphate (ATP, hP2Y , EC = 230 nM). As the P2Y R is predominately G ꢀcoupled,
2
50
2
q
receptor activation leads to stimulation of phospholipase C, IP release and elevation of
3
ACS Paragon Plus Environment
2

Page 3 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2+
intracellular Ca concentration, as well as the initiation of protein kinase C and activation of the
mitogen activated protein kinase cascade.
Defining the clinical role for P2Y R antagonism has been hampered by the lack of high affinity
2
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and drugꢀlike receptor antagonists. However, it has been reported that ATP released from tumor
cellꢀactivated platelets induces the opening of the endothelial barrier, leading to migration of
tumor cells and hence cancer proliferation. More importantly, the P2Y R was identified as the
2
primary mediator of this effect; a strong reduction of tumor cell metastasis was observed in
P2Y R deficient mice revealing a therapeutic potential of P2Y R antagonists as antiꢀmetastatic
2
2
5
, 6
agents.
Recently, it has been reported that both inflammation and fibrosis were reduced in
P2Y Rꢀdeficient mice compared to wild type animals. In addition mechanistic studies have
2
demonstrated that recruitment of neutrophils into the lungs, proliferation and migration of lung
fibroblasts and ILꢀ6 production are all key P2Y Rꢀmediated processes. These studies clearly
2
demonstrate the involvement of P2Y R subtypes in the pathogenesis of fibrotic lung diseases in
2
humans and mice and support the development of selective P2Y R antagonists for the treatment
2
7
of idiopathic pulmonary fibrosis (IPF). To date, the only reported high affinity P2Y R
2
antagonists were those developed by scientists from AstraZeneca resulting in the nonꢀnucleotide
8
, 9
P2Y R antagonist ARꢀC118925 (1).
2
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Several in vivo and ex vivo studies using 1 have been reported further validating the therapeutic
benefit of P2Y R antagonists. Importantly, it has been shown that 1, which was reported to be
2
inactive at 10 ꢁM against a panel of 37 other receptors, was able to concentrationꢀdependently
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
antagonize ATPγSꢀinduced mucin secretion in an ex vivo model of human bronchial epithelial
10
cells. In addition, Müller et al recently demonstrated that 1 was a selective, high affinity
11
reversible antagonist of the P2Y R.
2
We were drawn to the exciting possibility of using 1 as a chemical template to design new
12
P2Y R antagonists and synthesize fluorescently labeled chemical tools to further probe P2Y R
2
2
1
3
function. Using fluorescence as a means to study GPCRs allows access to a large range of
1
4
pharmacological techniques that can capture dynamic processes in living cells. In particular,
fluorescently labeled receptor antagonists have been developed to target GPCRs allowing
1
5–17
visualization of GPCR function at the cellular level.
In addition, fluorescent ligands can be
used in resonance energy transfer (RET) techniques, in particular those that utilize the luciferase
NanoLuc, to quantify ligandꢀreceptor interactions and determine the affinity of unlabeled
18
ligands. This offers advantages for receptors such as P2Y R for which there are currently no
2
commercially available radio ligands. In addition, as the reported antagonists for P2Y R have
2
mid to high affinity it is proposed that fluorescent ligands designed on these ligands might also
have affinity in this range. This may prove problematic for techniques which directly monitor
fluorescent ligand binding but NanoBRET has been shown to display low nonꢀspecific binding
1
8, 19
at high fluorescent ligand concentrations.
RESULTS AND DISCUSSION
Synthesis and evaluation of hP2Y R antagonists. The medicinal chemistry strategy involved an
2
initial exploration of the structure activity relationship (SAR) around 1, to enable the design of
ACS Paragon Plus Environment
4

Page 5 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
structural analogues with improved predicted physicochemical properties and to guide our design
strategy through highlighting suitable linking sites to attach the fluorophore groups (Figure 1 and
Table 1).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. SAR for P2Y R antagonists showing changes from the furan in 1, to thiazole (reduced
2
lipophilicity) and change from 2,8ꢀdimethylꢀ5Hꢀdibenzo[a,d][7]annulene to 7ꢀchloroꢀ4Hꢀ
benzo[5,6]cyclohepta[1,2ꢀd]thiazole tricylic ring system.
Table 1 The estimated affinity value for P2Y R antagonists 1ꢀ6 obtained from the calcium
2
mobilization assay
a
a
Example
hP2Y pKb
Example
hP2Y pKb
2
2
1
7.51 ± 0.09 (12)
6.43 ± 0.08 (3)
7.11 ± 0.14 (7)
4
5
6
i.a.
b
2
6.48 ± 0.10 (3)
5.99 ± 0.03 (7)
3
a
The estimated affinity value for each antagonist (pK ) was calculated from the shift of UTPγS
b
concentration response curve brought about by addition of a single concentration of antagonist
using the Gaddum equation. i.a. = less than 50% inhibition of the response to 0.1ꢀM UTPγS in
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
b
8
the presence of 10 ꢀM compound. UTPγS EC = 7.9 ± 1.3 nM (n=25). literature value pA 5.7
5
0
2
Data shown is mean ± SEM, number of separate experiments given in parentheses.
20
1
has previously been shown to have high lipophilicity (cLogP 4.2)
and poor
8
physicochemical properties for oral delivery. We therefore synthesized the thiazole analogue 3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
cLogP 3.8), with little loss of affinity for the P2Y R. Replacement of the 2,8ꢀdimethylꢀ5Hꢀ
2
dibenzo[a,d][7]annulene to 7ꢀchloroꢀ4Hꢀbenzo[5,6]cyclohepta[1,2ꢀd]thiazole tricylic ring gave 5
2
1
(
pK 6.5, clogP 3.4). In addition, nonꢀparallel SAR was observed in the replacement of the Nꢀ1
b
thiouracil substituent with a methyl group to give a compound 6 with P2Y R affinity, whereas
2
the corresponding analogue 4 proved inactive. In order to explore this intriguing finding we
synthesized a range of analogues of 6 to study this SAR (Table 2).
Table 2 Calcium mobilization activity for P2Y R antagonists 6ꢀ19
2
a
Example
R3
hP2Y pKb
2
7
8
9
NH
2
6.56 ± 0.14 (4)
i.a. (3)
PhNH
PhCH
NH
i.a. (3)
2
10
11
12
13
14
15
16
PhCH
2
CH
2
NH
i.a. (3)
1ꢀmethyl piperazinꢀ4ꢀyl
morpholinyl
i.a. (3)
i.a. (3)
piperidinyl
i.a. (3)
2ꢀmethoxyethanꢀ1ꢀaminyl 6.60 ± 0.21 (4)
2ꢀmethoxypropanꢀ1ꢀaminyl 6.56 ± 0.09 (3)
2ꢀethoxyethanꢀ1ꢀaminyl
6.73 ± 0.05 (3)
ACS Paragon Plus Environment
6

Page 7 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
7
8
9
2ꢀisopropoxyethanꢀ1ꢀaminyl 6.49 ± 0.09 (3)
2ꢀphenoxyethanꢀ1ꢀaminyl
phenyl
i.a. (3)
i.a. (3)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
The estimated affinity value for each antagonist (pK ) was calculated from the shift of the
b
UTPγS concentration response curve brought about by addition of a single concentration of
antagonist using the Gaddum equation. i.a. = less than 50% inhibition of 0.1ꢀM UTPγS response
in the presence of 10 ꢀM compound (n=3). n.b. the corresponding uracil derivatives were
inactive at 10 ꢀM compound. Data shown is mean ± SEM, number of separate experiments
given in parentheses.
From the SAR study of 6, it was shown that replacing thiazole 2ꢀmethyl substituent with an
amino group increased hP2Y R affinity (compare 6 with 7) whereas a sterically demanding
2
substituent, such as 19 showed complete loss of affinity for the hP2Y R. We therefore explored
2
substitution of the amino group and showed that both sterically demanding amino groups (8, 9
and 10) and cyclic tertiary amines (11, 12 and 13) were inactive. However the linear, less
sterically demanding alkyl amino groups (14, 15, 16 and 17) increased hP2Y R affinity, although
2
the bulkier 2ꢀphenoxyethanꢀ1ꢀamino substituent 18 proved inactive.
Thus far, all the compounds synthesized were tested as racemic mixtures. To try and determine
whether the activity resided in one enantiomer, resolution of 14 and 16 was achieved through
semiꢀpreparative chiral HPLC and the biology of the resolved enantiomers independently
assessed (Table 3).
Table 3 Calcium mobilization activity for resolved enantiomers of 14 and 15
a
b
Example Racemic compound Enantiomeric excess (ee)
hP2Y Pkb
2
2
2
2
0
1
2
99%
78%
95%
6.63 ± 0.11 (6)
5.82 ± 0.05 (3)
i.a. (3)
1
4
16
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
3
96%
6.78 ± 0.05 (6)
a
b
Compounds separated using Phenomenex’s Lux 5 ꢁm amyloseꢀ2 stationary phase. The
estimated affinity value for each antagonist (pK ) was calculated from the shift of UTPγS
concentration response curve brought about by addition of a single concentration of antagonist
using the Gaddum equation. i.a. = less than 50% inhibition of 0.1ꢀM UTPγS response in the
presence of 10 ꢀM compound (n=3). Data shown is mean ± SEM, number of separate
experiments given in parentheses.
b
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
From these results it is possible to see that the hP2Y R antagonist affinity observed for the
2
racemic compounds (14 and 16) resides largely in just one enantiomer 20 and 23 respectively.
Although some antagonist activity is observed for 21, this may be attributed to residual active
enantiomer 20 which constitutes 11% of the sample. Unfortunately, the resolved enantiomers
proved to be amorphous powders and so single crystal Xꢀray determination of the absolute
chirality could not be used for structural determination. However, vibrational circular dichroism
of 22 and 23 was used, where spectra were acquired on both samples and fitted to the calculated
2
2–24
spectra.
with the calculated spectrum for the (S)- enantiomer and therefore 23 was assigned as the active
R)- enantiomer (Figure 2).
The results (supplementary information) showed that there was a good match for 22
(
ACS Paragon Plus Environment
8

Page 9 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 2. Chiral resolution of 16 and structural assignment made by vibrational circular
dichroism.
In an attempt to increase affinity within the new series of compounds we incorporated key
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
structural features of 14 and 5 to generate compound 24.
However, 24 did not demonstrate the expected increase in affinity from combining the features
of 14 and 5 and showed a similar level of hP2Y R affinity (pK 7.02 ± 0.05, n=4) to both 1 and
2
d
2
5
3, demonstrating nonꢀadditive SAR within the series of compounds.
The synthesis of compounds 2ꢀ4 is illustrated in Scheme 1. Alkylation of 5ꢀ(2,8ꢀdimethylꢀ5Hꢀ
8
dibenzo[a,d][7]annulenꢀ5ꢀyl)pyrimidineꢀ2,4(1H,3H)ꢀdione with ethyl 2ꢀ(bromomethyl)thiazoleꢀ
8
4
ꢀcarboxylate followed by treatment with Lawesson’s reagent and saponification gave 2 which
was reacted with 2ꢀamino tetrazole using benzotriazolꢀ1ꢀylꢀoxytripyrrolidinophosphonium
hexafluorophosphate activation to afford 3. In a similar manner, alkylation with methyl iodide
followed by conversion to the thiouracil gave 4, in good over all yield.
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 1. (a) (i) N,Oꢀbis(trimethylsilyl)trifluoroacetamide, DCM, reflux, 18 h; (ii) ethyl 2ꢀ
o
(
1
bromomethyl)thiazoleꢀ4ꢀcarboxylate, 50 C, 24 h (55%). (b) Lawesson’s reagent, 1, 4ꢀdioxane,
o
00 C, 18 h (85%). (c) NaOH, methanol/H O, reflux, 1 h (91%). (d) 5ꢀaminotetrazole,
2
benzotriazolꢀ1ꢀylꢀoxytripyrrolidinophosphonium hexafluorophosphate, DMF, DIPEA, rt, 1 h
(49%). (e) (i) N,Oꢀbis(trimethylsilyl)trifluoroacetamide, DCM, reflux, 18 h; (ii). iodomethane,
o
50 C, 24 h (61%). (f) Lawesson’s reagent, 1,4ꢀdioxane, reflux, 18 h (40%).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cl
O
S
Cl
I
Cl
N
Cl
a
b
d
CO R
2
O
S
R = Et
R = H
c
N
t
Cl
N
O Bu
Br
Br
e
N
N
f
tBuO
Cl
N
Cl
Cl
Cl
Cl
tBuO
N
i
O
N
h
O
g
S
N
OH
N
HN
O
HN
HN
S
S
S
S
N
N
tBuO
N
N
O
N
H
O
j
Cl
Cl
Cl
S
N
O
N
S
k
l
HN
HN
HN
S
N
S
S
O
N
N
O
O
N
N
O
N
O
O
NH
N
N
N
NH
S
EtO
S
EtO
S
N
35
36
5
Scheme 2. Reagents and conditions: (a) Allyl alcohol, tetrabutylammonium chloride, DMF,
o
3
% mol. palladium(II)acetate, NaHCO , 50 C, 18 h (92%). (b) (i) ethyl dichloroacetate, sodium
3
o
ethoxide, diethyl ether, 30 min, 0ꢀ40 C; (ii) thioacetamide, ethanol, reflux, 3 h (30% over 2
steps). (c) NaOH, H O/THF (1:1), rt, 18 h (99%). (d) (i) oxalyl chloride, cat. DMF, DCM, rt, 3 h;
2
ACS Paragon Plus Environment
10

Page 11 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
0
ii) aluminium(III)chloride, DCM, rt, 18 h (39% over two steps). (e) Sodium tert-butoxide, THF,
Cꢀrt, 18 h (61%) (f) (i) nꢀbutyllithium, THF, ꢀ78 C, 30 mins; (ii) 29, ꢀ78 C to rt, 1 h (65%). (g)
o
o
o
trifluoroacetic acid, reflux, 72 h (36%). (h) (i) N,Oꢀbis(trimethylsilyl)trifluoroacetamide, DCM,
reflux, 18 h; (ii) iodomethane, 50 C, 24 h (61%). (i) Lawesson’s reagent, 1,4ꢀdioxane, reflux, 18
h (40%). (j) (i) N,Oꢀbis(trimethylsilyl)trifluoroacetamide, DCM, reflux, 18 h; (ii) ethyl 2ꢀ
(bromomethyl)thiazoleꢀ4ꢀcarboxylate, 50 C, 24 h (57%). (k) Lawesson’s reagent, 1, 4ꢀdioxane,
o
o
o
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
00 C, 18 h (89%). (l) (i) NaOH, methanol/H O, reflux, 1 h (91%). (ii) 5ꢀaminotetrazole,
2
benzotriazolꢀ1ꢀylꢀoxytripyrrolidinophosphonium hexafluorophosphate, DMF, DIPEA, rt, 1 h
(30%).
Scheme 2 shows the synthetic route to 5 and 6. The first step involved performing a Heck
reaction, coupling 3ꢀchloroiodobeneze 25 to allyl alcohol and this successfully isomerized in situ
yielding the desired aldehyde 26. This was reacted with ethyl dichloroacetate in a Darzens
25
condensation to generate a αꢀchloro epoxide which was reacted directly with thioacetamide to
afford desired 2ꢀmethylthiazole 27 with moderate yields achieved over two steps. Freshly
prepared sodium ethoxide, generated from sodium metal in dried ethanol was found to be the
optimal base for the Darzens condensation. Saponification gave carboxylic acid 28 and treatment
with oxalyl chloride generated the acyl chloride which was immediately cyclized to give the
tricyclic ketone 29 as a single regioisomer. Lithiation of the di tertꢀbutyl ether-protected uracil
25
3
0, was readily achieved with nꢀbutyllithium and this underwent a 1, 2ꢀaddition to ketone 29 to
give tertiary alcohol 32. Concomitant deprotection and dehydration gave uracil 33 through
heating in trifluoroacetic acid. Although the yield for this reaction was poor, other acidic
conditions were ineffective. Alkylation at the N1ꢀposition of the uracil was achieved in a oneꢀpot
process of silylation with N,Oꢀbis(trimethylsilyl)trifluoroacetamide (BSTFA), alkylation with
iodomethane and subsequent desilylation to give 34. Finally, reaction with Lawesson’s reagent
gave 6. From the uracil intermediate 33 alkylation with ethyl 2ꢀ(bromomethyl) thiazoleꢀ4ꢀ
26
carboxylate gave 35 which was subsequently reacted with Lawesson’s reagent to give 36.
Hydrolysis was followed by reaction of the resulting carboxylic acid with 5ꢀaminotetrazole and
benzotriazolꢀ1ꢀylꢀoxytripyrrolidinophosphonium hexafluorophosphate to give 5.
ACS Paragon Plus Environment
11

Journal of Medicinal Chemistry
Page 12 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Reagents and conditions: (a) (i) Ethyl dichloroacetate, sodium ethoxide, diethyl
ether, 30 min, 0ꢀ40 C; (ii) thiourea, ethanol, reflux, 2.5 h (46% over 2 steps). (b)
o
copper(II)chloride, tertꢀbutyl nitrite, acetonitrile, rt, 2 h (65%) (c) NaOH, H O/THF (1:1), rt, 18
2
h (quant.). (d) (i) Oxalyl chloride, cat. DMF, DCM, rt, 3 h; (ii) AlCl , DCM, rt, 18 h (83% over
3
o
o
two steps). (e) (i). 31, nꢀbutyllithium, THF, ꢀ78 C, 30 mins; (ii). 82, ꢀ78 C to rt, 1 h (79%). (f)
o
1
:1
acetic
acid/1,4ꢀdioxane
140 C,10
min
(58%).
(g)
(i)
N,Oꢀ
o
bis(trimethylsilyl)trifluoroacetamide, DCM, reflux, 18 h; (ii). iodomethane, 50 C, 24 h (61%) (h)
Phenylboronic acid, Na CO , 1% mol. bis(triphenylphosphine) palladium(II)chloride, 1,4ꢀ
dioxane, water, MW, 150 C, 5 min (68%). (i) Lawesson’s reagent, 1,4ꢀdioxane, reflux, 18 h
2
3
0
o
(
76%). (j) R R NH, 1, 4ꢀdioxane, MW, 100 C, 4h (51ꢀ92 %). (k) Lawesson’s reagent, 1,4ꢀ
1 2
dioxane, reflux, 18 h (44ꢀ88%). (l) (a) (i) N,Oꢀbis(trimethylsilyl)trifluoroacetamide, 1,2ꢀ
dichloroethane, reflux, 18 h; (ii) ethyl 2ꢀ(bromomethyl)thiazoleꢀ4ꢀcarboxylate, reflux, 48 h
(91%). (m) 2ꢀMethoxyethyl amine, triethylamine, 1, 4ꢀdioxane, reflux, 72 h (59%). (n)
Lawesson’s reagent, 1, 4ꢀdioxane, reflux, 18 h (81%). (o) (i) NaOH, methanol/H O, reflux, 1 h
2
(91%).
(ii)
5ꢀAminotetrazole,
benzotriazolꢀ1ꢀylꢀoxytripyrrolidinophosphonium
hexafluorophosphate, DMF, DIPEA, rt, 2 h (34%).
In a similar sequence to Scheme 2, aldehyde 26 was reacted with ethyl dichloroacetate and the
resulting crude ꢀchloro epoxide reacted with thiourea to afford the 2ꢀaminothiazole 37 which
α
was converted to the 2ꢀchlorothiazole 38 (Scheme 3). The ethyl ester was hydrolyzed to afford
ACS Paragon Plus Environment
12

Page 13 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
carboxylic acid 39, converted to the acid chloride and cyclized to tricyclic ketone 40. Lithiation
of the tertꢀbutyl ether-protected uracil 31 and reaction with 40 gave the tertiary alcohol 41. After
o
screening a range of milder acidic conditions, heating to 140 C with MW irradiation in 1:1 acetic
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
acid/1,4ꢀdioxane for 10 minutes was found to be optimal for the formation of the desired uracil
intermediate 42, which was subsequently methylated to give 43. The chlorine atom in compound
4
3 was readily displaced through nucleophilic aromatic substitution with a range of primary and
secondary amines on heating under basic conditions. These conditions were unsuccessful when
using aniline and, in this instance, heating in the microwave with hydrochloric acid (catalytic)
proved successful to give 46. Microwaveꢀbased conditions were employed for the substitution
with ammonia in the synthesis of 45. A Suzuki reaction with phenylboronic acid gave 44. Using
Lawesson’s reagent, it was then possible to convert these uracil derivatives (44ꢀ54) to the
respective 4ꢀthiouracil derivatives (6ꢀ19). Employing a route analogous to the synthesis of
compound 7, it was possible to generate the desired tetrazole analogue 24. Uracil intermediate 42
was alkylated at the N1ꢀposition with ethyl 2ꢀ(bromomethyl)thiazoleꢀ4ꢀcarboxylate to give 57.
This was reacted with 2ꢀmethoxyethylamine to afford 58, which was subsequently reacted with
Lawesson’s reagent to give the 4ꢀthiouracil 59. Hydrolysis, followed by benzotriazolꢀ1ꢀylꢀ
oxytripyrrolidinophosphonium hexafluorophosphate activation and reaction with 5ꢀ
aminotetrazole, afforded 24.
Synthesis of hP2Y R fluorescent ligands. With a view to developing a series of fluorescent
2
conjugates suitable for both a bioluminescence resonance energy transfer (BRET) ligand binding
18,27–29
assay
and imaging through confocal microscopy, we embarked on a strategy to synthesize
bodipy conjugates, specifically dyes bodipy A (absorption max 628 nm, emission max 642 nm)
ACS Paragon Plus Environment
13

Journal of Medicinal Chemistry
Page 14 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
or bodipy B (absorption max 503 nm, emission max 509 nm) as this would allow us the
opportunity of ligand choice in future imaging work.
Two positions on to the P2Y R antagonist core structure were considered for attachment of the
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
linker and fluorophore (Figure 3). In order to simplify the synthetic chemistry and increase SAR
within the series, we examined replacement of the furan ring of 1 and the thiazole ring of 2 with
a 1,3,5 triꢀsubstituted phenyl ring. This would allow attachment of the acyl tetrazole group in
addition to providing a second free carboxylic acid group to attach the fluorescent conjugates.
Fortunately, the 3,5ꢀdicarboxylic acid 60 (hP2Y R pK 6.53 ± 0.04, n= 7) was well tolerated with
2
d
no loss of affinity for the P2Y R compared with compound 2. Therefore, the first series of
2
compounds have the linkerꢀfluorophore attached via the phenyl ring of 60.
Having established that alkoxyalkyl amines are tolerated for activity in the 2ꢀposition of the
thiazole in the 4Hꢀbenzo[5,6]cyclohepta[1,2ꢀd]thiazolꢀ4ꢀyl) tricyclic ring of compounds of the
type shown in Figure 2, this position was chosen as the second point of attachment of the linkerꢀ
fluorophore. Finally, a third generation of fluorescent ligands would be explored containing the
optimal second generation fluorescent ligand with incorporation of the acyl tetrazole functional
group.
ACS Paragon Plus Environment
14

Page 15 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Design of fluorescent P2Y R antagonists based from SAR on 1. The figure shows
2
potential attachment points from which to attach either bodipy A or bodipy B via a suitable
linking group selected. * Represents attachment points for fluorophore linkage via a suitable
linking group.
First generation hP2Y R fluorescent ligands. The general synthetic route to the first generation
2
P2Y R fluorescent antagonists is shown in Scheme 4.
2
ACS Paragon Plus Environment
15

Journal of Medicinal Chemistry
Page 16 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 4. a) Dimethyl 5ꢀ(bromomethyl)isophthalate, BSTFA, 1,2ꢀdichloroethane, reflux, 16 h.
b) Lawesson’s reagent, 1,4ꢀdioxane, reflux, 16 h. c) 2eq NaOH, MeOH, toluene, H O, 4h. 65%
2
(3 steps) d) tert-butyl (2ꢀ(2ꢀaminoethoxy)ethyl)carbamate (53%) or tert-butyl (2ꢀ
aminoethyl)carbamate (63%), NEt3, benzotriazolꢀ1ꢀylꢀoxytripyrrolidinophosphonium
hexafluorophosphate, DMF, rt, 0.5 h. e) LiOH, MeOH, H O, 16 h, rt. 14ꢀ30%. f) 4ꢀ
2
aminotetrazole, NEt , Benzotriazolꢀ1ꢀylꢀoxytripyrrolidinophosphonium hexafluorophosphate,
3
DMF, 1h, rt. g) 4M HCl in 1,4ꢀdioxane, 1h. h) bodipyꢀSE, DIPEA, DMF, rt, 1ꢀ4 h.15ꢀ45%.
8
Alkylation of 5ꢀ(2,8ꢀdimethylꢀ5Hꢀdibenzo[a,d][7]annulenꢀ5ꢀyl)pyrimidineꢀ2,4(1H,3H)ꢀdione
with dimethyl 5ꢀ(bromomethyl)isophthalate, followed by treatment with Lawesson’s reagent and
selective hydrolysis of one of the methyl esters gave 61. The carboxylic acid 61 was activated
using benzotriazolꢀ1ꢀylꢀoxytripyrrolidinophosphonium hexafluorophosphate and coupled to form
the appropriate amide (62ꢀ63). The second ester was then hydrolyzed and converted to the
amidotetrazoles (64ꢀ65). Finally, the Boc protecting group was removed and the resulting amine
ACS Paragon Plus Environment
16

Page 17 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
coupled to the fluorophore using the appropriate commercially available BODIPY succinimidyl
ester to give a small library of 4 fluorescent conjugates (66ꢀ69).
To determine whether any of these conjugates had affinity at the P2Y R and consequently if
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
they could be used in NanoBRET binding assays, a 1321N1 astrocytoma cellꢀline expressing
recombinant P2Y R tagged on its Nꢀterminus with NanoLuc (NanoLucꢀP2Y R) was generated.
2
2
The NanoLucꢀtagged P2Y receptors exhibited normal calcium signals (EC for UTPγS of 91 ±
2
50
1
2 nM; n=3). These NanoLucꢀP2Y cells were treated with increasing concentrations of 66ꢀ69
2
and then treated with the NanoLuc substrate furimazine, before the resulting BRET signal was
monitored. All four compounds showed moderate to low affinity for the NanoLucꢀP2Y R (Table
2
4
), with the conjugates 68 and 69 having the higher affinities. However, this did illustrate the
power of using NanoBRET to monitor ligand binding to low affinity receptors.
Table 4: Affinity of 66, 67, 68 and 69 determined in 1321N1 astrocytoma cells expressing
recombinant NLucꢀP2Y R
2
b
c
a
Example n
R
NanoBRET pKd
5.56 ± 0.1 (3)
5.78 ± 0.18 (3)
6.12 ± 0.07 (3)
6.07 ± 0.15 (3)
6
6
6
6
6
7
8
9
0
0
1
1
A
B
A
B
a
b, c
pK values derived from saturation binding curves.
n and R previously defined from
d
Scheme 4. Data shown is mean ± SEM, number of separate experiments given in parentheses.
Second generation hP2Y R fluorescent ligands. The general synthetic route to the second
2
generation P2Y R fluorescent antagonists, in which the linkerꢀfluorophore is attached to the
2
thiazole ring, is illustrated in Scheme 5.
ACS Paragon Plus Environment
17

Journal of Medicinal Chemistry
Page 18 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 5. Reagents and conditions: (a) amine, NEt , 1,4ꢀdioxane, reflux, 72 h. 96ꢀ97% (b)
Lawesson’s reagent, 1,4ꢀdioxane, 100 C, 18 h. 39ꢀ74% (c) TFA, DCM, rt, 30 min. (d) amine,
Benzotriazolꢀ1ꢀylꢀoxytripyrrolidinophosphonium hexafluorophosphate, DMF, DIPEA, rt, 1 h.
36ꢀ58% (2 steps) (e) TFA, DCM, rt, 30 min; f) bodipyꢀSE, DIPEA, DMF, rt, 1ꢀ4 h. 10ꢀ54% (2
steps).
3
o
Nucleophilic displacement of the 2ꢀchloro substituent of 43 by either tertꢀbutyl 3ꢀ
aminopropanoate or tertꢀbutyl 6ꢀaminohexanoate gave the corresponding tertꢀbutyl esters (70
o
and 71). Treatment with Lawesson’s reagent in 1,4ꢀdioxane at 100 C resulted in formation of the
4
ꢀthiouracils (72 and 73). The carboxylic acids were generated from the tertꢀbutyl esters using
TFAꢀmediated hydrolysis and subsequently coupled with tertꢀbutyl (2ꢀaminoethyl)carbamate or
tertꢀbutyl (2ꢀ(2ꢀaminoethoxy)ethyl)carbamate to generate the corresponding amides (74ꢀ77). The
Boc protecting group was removed using TFA and the resulting amines coupled with the
appropriate commercially available bodipyꢀsuccinimidyl ester (SE), generating a small library of
ACS Paragon Plus Environment
18

Page 19 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
4 fluorescent conjugates (78ꢀ91). These fluorescent conjugates were initially tested using the
aforementioned NanoBRET binding assay at fixed concentrations of 10 µM in the presence and
absence of 10 µM 1 (see figure S1 in the supplementary material). It was found that 80, 85, 86
and 87 generated the largest specific NanoBRET signal, therefore their affinity was determined
from saturation binding assays demonstrating the excellent signal to noise ratios observed from
the NanoBRET assay even for low affinity conjugates (Figure 4; Table 5).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. NanoBRET saturation binding isotherm determined in 1321N1 astrocytoma cells
expressing recombinant NLucꢀP2Y R. This has been performed for (a) 80, (b) 85, (c) 86 and (d)
2
8
7 in the absence and presence of 10 µM 1. Data points are mean values ± SEM (n= 3 or 4).
ACS Paragon Plus Environment
19

Journal of Medicinal Chemistry
Page 20 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 5. Affinity of 85, 86, 87 and 80 determined in 1321N1 astrocytoma cells expressing
recombinant NLucꢀP2Y R
2
Example pKd
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
0
5.29 ± 0.17 (4)
4.91 ± 0.14 (4)
5.67 ± 0.10 (4)
5.38 ± 0.19 (3)
85
86
87
pK values derived from saturation binding curves. Data shown is mean ± SEM, number of
d
separate experiments given in parentheses.
Reassuringly, the affinities determined for the nonꢀfluorescent P2Y R antagonist 1, with either
2
86 (pK 7.45 ± 0.13, n = 4) or 87 (pK 7.32 ± 0.13, n = 4) where consistent with the affinity
i
i
determined using the P2Y R functional assay (pK 7.51 ± 0.09); demonstrating that the P2Y R
2
b
2
fluorescent ligands could be used in a NanoBRET assay for determining the affinity of nonꢀ
fluorescent P2Y R antagonists. The clear demonstration of saturable specific binding with these
2
low affinity fluorescent ligands confirmed the utility of the NanoBRET binding format and the
ability to exploit the good signal to noise ratio of this proximityꢀbased assay. To explore the
opportunity to develop higher affinity fluorescent conjugates, we embarked on a synthetic
strategy to incorporate the affinityꢀenhancing acylꢀtetrazole functional group into the fluorescent
compounds.
Third generation P2Y R fluorescent ligands. The general synthetic route to the third generation
2
P2Y R fluorescent antagonists is illustrated in Scheme 6. Displacement of the chlorine atom in
2
compound 57 with tertꢀbutyl 3ꢀaminopropanoate gave tertꢀbutyl ester 92. Treatment with TFA
afforded conversion of the tertꢀbutyl ester to the corresponding acid which was immediately
ACS Paragon Plus Environment
20

Page 21 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
activated and coupled to tertꢀbutyl (2ꢀaminoethyl)carbamate 93 using HATU. Hydrolysis of the
ethyl
ester,
activation
of
the
carboxylic
acid
using
benzotriazolꢀ1ꢀylꢀ
oxytripyrrolidinophosphonium hexafluorophosphate and coupling with 5ꢀaminotetrazole gave
the amidotetrazole 94. The Boc protecting group was removed and the resulting amine attached
to the fluorophore using the appropriate bodipy succinimidyl ester (Scheme 6).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 6. Reagents and conditions: (a) amine, NEt , 1,4ꢀdioxane, reflux, 72 h. (89%). (b)
3
Lawesson’s reagent, 1,4ꢀdioxane, reflux, 18 h (48%). (c) TFA, DCM, rt, 30 min (55%). (d) tertꢀ
butyl (2ꢀaminoethyl)carbamate, HATU, DCM, DIPEA, rt, 24 h (63%). (e) 1. NaOH, MeOH,
reflux,
4
h;
2.
5ꢀaminotetrazole,
Benzotriazolꢀ1ꢀylꢀoxytripyrrolidinophosphonium
hexafluorophosphate, DIPEA, DMF, rt 4 h (28% over 2 steps). (f) 1. TFA, DCM, rt, 30 min; 2.
bodipyꢀSE, DIPEA, DMF, rt, 1ꢀ4 h. (11ꢀ18%, 2 steps). The structures of compounds 95ꢀ98 are
assigned in table 6.
In contrast to nonꢀfluorophore compounds, 14 and 24 where there was little change in affinity
in changing the uracil N1ꢀsubstituent, in three of the four compounds (95ꢀ98) there was a
ACS Paragon Plus Environment
21

Journal of Medicinal Chemistry
Page 22 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
significant increase in affinity relative to the nonꢀamidotetrazole series ꢀ compare 98 & 86, 96 &
8
5 and 97 & 80 (Table 5 and Table 6).
Table 6. Affinity of 95, 96, 97 and 98 determined in 1321N1 astrocytoma cells expressing
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
recombinant NLucꢀP2Y R
2
b
c
a
Example m
R
NanoBRET pKd
6.05 ± 0.12 (3)
7.05 ± 0.05 (3)
6.89 ± 0.06 (7)
6.32 ± 0.10 (17)
9
9
9
9
5
6
7
8
0
0
1
1
A
B
A
B
a
b, c
pK values derived from saturation binding curves
m and R previously defined from
d
Scheme 6. Data shown is mean ± SEM, number of separate experiments given in parentheses.
Pharmacological evaluation of third generation P2Y R fluorescent ligands. To further evaluate
2
the utility of fluorescent ligands to study the pharmacology of the P2Y R, one bodipy A (97) and
2
one bodipy B (98) linked fluorescent ligand was chosen for further studies. Initially, we
confirmed that 97 and 98 still retained the ability to functionally antagonize the P2Y R (Figure
2
2
+
5
). In the Ca mobilization assay, a modest rightwards shift of the agonist doseꢀresponse curve
was observed for both 10ꢀM 97 (pK 5.69 ± 0.05, n=8) and 10ꢀM 98 (pK 5.87 ± 0.05, n=7).
b
b
ACS Paragon Plus Environment
22

Page 23 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
+
Figure 5. Pharmacological evaluation of: 97 and 98 showing the effect on Ca mobilization in
hP2Y Rꢀ1321N1 cells induced by 0.1 ꢀM UTPγS (n=7 or 8).
2
Compounds 97 and 98 bought about a clear concentrationꢀdependent increase in the BRET
ratio in the NLUCꢀtagged hP2Y R assay (Figure 6). This was antagonized by 1 (Figures 6 c &
2
d) yielding pKi values of 7.66 ± 0.11 (n = 9) and 7.38 ± 0.04 (n = 6) for antagonism of 97 and 98
binding respectively.
ACS Paragon Plus Environment
23

Journal of Medicinal Chemistry
Page 24 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. Pharmacological evaluation of: a) 97 and b) 98 showing BRET saturation binding
(n=7); c) and d) Displacement of 97 (c) (100nM, n=9) and 98 (d) (1µM, n=6) binding in NLucꢀ
P2Y 1321N1 cells by 1. Values show Mean ± SEM.
2
To further evaluate the utility of the fluorescent conjugate 98 in the NanoBRETꢀligand binding
assay the affinity of a selection of P2Y R antagonists (1, 3, 6, 22, 23, 60, 86) and the previously
2
8
reported stabilized triphosphate P2Y R antagonist 99 over a range of P2Y R affinity were
2
2
determined in competition binding experiments.
ACS Paragon Plus Environment
24

Page 25 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
All eight compounds induced a concentration dependent decrease in specific binding of 98
that enabled their affinity to be determined. There was a good correlation in the values obtained
2
+
in the NanoBRET assay to those determined in the Ca mobilization assay (Table 7). In
addition to antagonists, the NanoBRET assay was also used to estimate the affinity of UTPγS.
As there has been no reports of radioligands for the P2Y R, this measurement has not previously
2
been possible.
2
+
Table 7. Comparison of affinity estimates obtained in Ca mobilization and NanoBRET assays
2
+
a
b
Example pK Ca mobilization pK NanoBRET (98)
b
i
1
3
6
2
2
6
8
9
7.51 ± 0.09 (12)
7.11± 0.14 (7)
5.99 ± 0.03 (7)
<5.0 (6)
7.38 ± 0.04 (6)
6.89 ± 0.05 (6)
5.81 ± 0.10 (6)
5.28 ± 0.05 (3)
6.56 ± 0.16 (3)
6.84 ± 0.02 (6)
6.40 ± 0.01 (6)
7.08 ± 0.03 (3)
5.46 ± 0.04 (6)
2
3
0
6
9
6.74 ± 0.10 (6)
6.53 ± 0.04 (7)
6.30 ± 0.05 (6)
c
6.59 ± 0.12 (3)
UTPγS
ND
a
The estimated affinity value for each antagonist (pK ) was calculated from the shift of the
b
agonist dose response curve brought about by addition of a single concentration of antagonist
ACS Paragon Plus Environment
25

Journal of Medicinal Chemistry
Page 26 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
b
using the Gaddum equation. Measured in competition binding experiment using fluorescent
c
8
ligand 98. reported pA 8.0 . ND = not determined due to agonist activity. Data shown is mean
SEM, number of separate experiments given in parentheses.
2
±
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The availability of both a green (98) and a red (97) fluorescent P2Y R ligand with reasonable
2
affinity for the hP2Y R suggested that they may both have utility for imaging of the receptor in
2
living cells. Confocal microscope images of fluorescent ligands 97 and 98 with astrocytoma
1
321N1 cells expressing hP2Y R (Figures 7a and c) showed localized membrane fluorescence
2
and very little intracellular fluorescence. When cells were pretreated with 1, the specificꢀ
membrane fluorescence of 97 and 98 was substantially reduced (Figures 7b and d), indicating
that the majority of the membrane fluorescence observed was specific labeling of the P2Y R.
2
ACS Paragon Plus Environment
26

Page 27 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 7. Visualization of the binding of 97 (a) and 98 (c) on astrocytoma cells expressing
hP2Y R. In each case, the images in the left hand column shows a confocal image with 100 nM
2
of the fluorescent ligand, while the right hand column shows the images (b) and (d) with the
addition of 10 ꢀM of 1. In all conditions, cells were incubated for 30 min at 37°C in the presence
or absence of 10 ꢀM of 1. Single equatorial confocal images were then obtained in the continued
presence of the fluorescent ligand (97 or 98) and/or unlabeled antagonist. Images shown are from
a single experiment representative of 4 performed.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CONCLUSION
We have described the synthesis and evaluation of new examples of acidic hP2Y R antagonists
2
based on the known hP2Y R antagonist 1. In addition, we have shown the discovery of a new
2
series of neutral hP2Y R antagonists and demonstrated SAR leading to the identification of
2
potent hP2Y R antagonists (such as 20 and 23). In addition, we have shown a stereochemical
2
preference for biological activity within this series as typified by the resolved examples 20 & 21
and 22 and 23. Vibrational circular dichroism has suggested that, in the case of 16 all hP2Y R
2
biological activity resides in the (R)-enantiomer 23 – although single crystal Xꢀray work will be
required to confirm this initial stereochemical assignment. The SAR studies led to the
identification of suitable linking sites for attachment of the fluorescent ligand to generate three
distinct series of fluorescentlyꢀlabelled hP2Y R antagonists. From this extensive synthetic work,
2
2
+
two examples (97 and 98) were identified demonstrating both functional antagonist activity (Ca
mobilization assay) and sufficient affinity for the hP2Y R through a new bioluminescence
2
resonance energy transfer (BRET) assay. In addition, confocal microscopy revealed clear,
displaceable membrane labeling of astrocytoma cells expressing the hP2Y R. The excellent
2
imaging properties, make 97 and 98 ideal tools for studying hP2Y R distribution and
2
ACS Paragon Plus Environment
27

Journal of Medicinal Chemistry
Page 28 of 96
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
organization. Finally, the discovery of the new hP2Y R antagonist fluorescent ligands (97 and
2
9
8) became realized as a result of an extensive program of synthetic chemistry where it proved
essential to explore the parallel changes of linker attachment points, fluorophores and linking
3
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
group. From this study, only a very few fluorescent conjugates were shown to possess
sufficient affinity to enable the establishment of a new NanoBRETꢀbased fluorescent assay for
the identification of new hP2Y R fragments and ligands.
2
EXPERIMENTAL SECTION
CHEMISTRY ꢀ GENERAL METHODS
Chemicals and solvents were provided by Fisher Scientific UK, Acros Organics, Sigmaꢀ
®
Aldrich, Merck Millipore or Fluorochem. BODIPY FLꢀXꢀNHS (D6102) and
®
BODIPY 630/650ꢀXꢀNHS (D10000) were purchased from Molecular Probes (Invitrogen, UK).
All reactions were monitored by TLC using Merck Silica Gel 60Å F254 TLC plates or by LCꢀ
MS. Unless otherwise stated, all compounds were dried under high vacuum either at rt or within
o
an oven at 40 C. LCꢀMS data was collected on a Shimadzu UFLCXR HPLC system coupled to
an Applied Biosystems API 2000 LC/MS/MS electrospray ionization (ESI). The column used
was a Phenomenex GeminiꢀNX 3 ꢁmꢀ110Â C18, 50x2mm at 40ºC. The flow rate was 0.5
mL/min, the UV detection was at 220 nm and 254 nm. Method 1 for the LCꢀMS ran for 1 min at
5
% B; 5 to 98% B over 2 min, 98% B for 2 min, 98 to 5% B over 0.5 min and then 5% for 1 min.
Method 2 for the LCꢀMS ran for 1.5 min at 10% B; 10 to 98% B over 8 min; 98% B for 2 min;
8 to 10% B over 0.5 min and then 10% B for 1 min. Where solvent A: 0.1% formic acid in
9
water; solvent B: acetonitrile. Unless otherwise stated compounds reported had a purity >95% at
the wavelength and method quoted. HRMS data was collected on a Bruker microTOF II mass
spectrometer using electrospray ionization (ESIꢀTOF). Adducts within error of ±10 ppm are
ACS Paragon Plus Environment
28

Page 29 of 96
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
reported. Preparative RPꢀHPLC was performed on a Waters 2767 sample manager coupled to
Waters 2525 binary gradient module and a Waters 2457 dual wavelength absorbance detector.
The column used was a Phenomenex Gemini® 5 ꢁm NX C18 110 Å, 150x21 mm at ambient
temperature. The flow rate was 40 mL/min, the UV detection was at 254 nm. Method 3 for the
preparative RPꢀHPLC ran for 1 min at 10% B; then 10 to 35% B over 4 min; then 35 to 40% B
over 20 min; then 40 to 90% B over 2 min; then 90 to 10% B over 2 min and then for 1 min at
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
10% B. Method 4 for the preparative RPꢀHPLC ran for 1 min at 10% B; 10 to 45% B over 4 min;
45 to 50% B over 20 min; 50 to 90% B over 2 min; 90 to 10% B over 2 min and then for 1 min at
10% B. Where solvent A was 0.1% trifluoroacetic acid in water and solvent B was 0.1%
trifluoroacetic acid in acetonitrile. ChiralꢀHPLC was performed on a Dionex ICSꢀ3000 SP single
pump coupled to a Rheodyne 9725i PEEKinjector coupled to a Dionex UltiMate 3000 Variable
Wavelength Detector. The columns used were Phenomenex Lux® 5 ꢁm Amyloseꢀ2, 250 x 4.6
mm and 250 x 10 mm; for analytical and semiꢀpreparative runs respectively. These operated at
ambient temperature and, unless otherwise stated, the mobile phase was 25% ethanol/hexane
with a flow rate of 1 mL/min for analytical runs and 5 mL/min for semiꢀpreparative runs. The
UV detection was at 254 nm. NMR spectroscopy was performed using a Bruker AV(III) HD 400
1
13
NMR spectrometer equipped with a 5 mm BBFO+ probe, recording H and C NMR at 400.25
MHz and 100.66 MHz respectively; or a Bruker AV(III) 500 NMR spectrometer equipped with a
1
13
1
13
5
mm dual H/ C heliumꢀcooled cryoprobe, recording H and C NMR at 500.13 MHz and
125.77 MHz respectively. NMR data was processed using iNMR (version 5.5.7) referencing
spectra to residual solvents. Chemical shifts are quoted as δ: values in ppm; coupling constants J
are given in Hz and multiplicities are described as follows: s ꢀ singlet, d ꢀ doublet, t ꢀ triplet, q ꢀ
quartet, qi ꢀ quintet, sep – septet, m – multiplet, app – apparent, br ꢀ broad.
ACS Paragon Plus Environment
29