Synthesis of novel (benzimidazolyl)isoquinolinols and evaluation as adenosine A1 receptor tools

Article abstract of DOI:10.1039/c7ra13148h

G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane receptors in eukaryotes. The adenosine A₁ receptor (A₁AR) is a class A GPCR that is of interest as a therapeutic target particularly in the treatment of cardiovascular disease and neuropathic pain. Increased knowledge of the role A₁AR plays in mediating these pathophysiological processes will help realise the therapeutic potential of this receptor. There is a lack of enabling tools such as selective fluorescent probes to study A₁AR, therefore we designed a series of (benzimidazolyl)isoquinolinols conjugated to a fluorescent dye (31-35, 42-43). An improved procedure for the synthesis of isoquinolinols from tetrahydroisoquinolinols via oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and atmospheric oxygen is reported. This synthetic method offers advantages over previous metal-based methods for the preparation of isoquinolinols and isoquinolines, which are important scaffolds found in many biologically active compounds and natural products. We report the first synthesis of the (benzimidazolyl)isoquinolinol compound class, however the fluorescent conjugates were not successful as A₁AR fluorescent ligands.

Full text of DOI:10.1039/c7ra13148h

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Synthesis of novel (benzimidazolyl)isoquinolinols
and evaluation as adenosine A1 receptor tools†
Cite this: RSC Adv., 2018, 8, 16362
Sameek Singh,a Samantha L. Cooper,b Jacqueline R. Glenn,b Jessica Beresford,b
a
Lydia R. Percival,b Joel D. A. Tyndall,
Stephen J. Hill,b Laura E. Kilpatrickb
a
*
and Andrea J. Vernall
G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane receptors in
eukaryotes. The adenosine A1 receptor (A1AR) is a class A GPCR that is of interest as a therapeutic target
particularly in the treatment of cardiovascular disease and neuropathic pain. Increased knowledge of the
role A1AR plays in mediating these pathophysiological processes will help realise the therapeutic
potential of this receptor. There is a lack of enabling tools such as selective fluorescent probes to study
A1AR, therefore we designed a series of (benzimidazolyl)isoquinolinols conjugated to a fluorescent dye
(31–35, 42–43). An improved procedure for the synthesis of isoquinolinols from tetrahydroisoquinolinols
via oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and atmospheric oxygen is
reported. This synthetic method offers advantages over previous metal-based methods for the
preparation of isoquinolinols and isoquinolines, which are important scaffolds found in many biologically
active compounds and natural products. We report the first synthesis of the (benzimidazolyl)isoquinolinol
compound class, however the fluorescent conjugates were not successful as A1AR fluorescent ligands.
Received 8th December 2017
Accepted 21st April 2018
DOI: 10.1039/c7ra13148h
rsc.li/rsc-advances
remains a need to develop uorescent ligands with high
subtype selectivity for A1AR.
Introduction
One approach to the design of a uorescent ligand is to
identify an existing pharmacophore with the requisite selec-
tivity and affinity for the target receptor, and then identify
a location that a linker and uorophore can be attached.5 With
this design in mind, it is then necessary to synthesise and test
a range of pharmacophore, pharmacophore-linker and uo-
rescent ligands as modications have the potential to alter the
affinity and efficacy of the parent pharmacophore. In an effort to
develop a selective A1AR uorescent ligand we chose to avoid
Adenosine receptors (ARs) are class A G protein-coupled
receptors (GPCRs), classied in four distinct subtypes – A1,
A
2A, A2B and A3. Adenosine A1 receptor (A1AR) is expressed
throughout the human body with predominant distribution in
brain, heart, kidney and adipose tissue and is an attractive
therapeutic target.1 To this end, A1AR ligands are of interest and
are being developed to treat various pathological conditions
such as atrial brillation, angina pectoris, congestive heart
failure, diabetes, neuropathic pain and renal disorders.2 Fluo-
rescent ligands have emerged as useful and powerful tools to
study real-time and live cell dynamic processes of GPCRs.3
Fluorescent ligands have been developed for ARs,4 including
some with high AR subtype selectivity for A3AR.5 There is one
report of uorescent dansyl-linked N6 NECA derivatives with
subtype selectivity for rat A1AR,6 however the short excitation
wavelength of the dansyl uorophore is not ideal for many
applications. When the same research group conjugated the
lead pharmacophore-linker to longer excitation wavelength
uorophores A3AR selective probes ensued.6 Therefore there
a
xanthine amine congener (XAC)-based pharmacophore
because of subtype selectivity concerns as a range of XAC-
uorescent conjugates have been reported as either non AR
subtype selective or A3AR selective.7 Cosimelli et al. recently
reported a series of (benzimidazolyl)isoquinolines as selective
antagonists for human A1AR (hA1AR), among them ligands 1–3
(Fig. 1),8 which were used as the inspiration for the uorescent
ligand design reported herein.
Structure–activity relationships (SAR) from the Cosimelli
et al. study indicated small, lipophilic benzimidazole substitu-
ents such as methyl and ethylthio groups in some positions
improved A1AR affinity and in others abolished it.8 This sensi-
tivity to slight positional change, along with potential buried,
lipophilic interactions these groups could be making with the
A1AR to enhance affinity led us to discount linker substitution
from the benzimidazole ring. Since 1,3-substituted isoquinoline
analogues such as VUF5455 (ref. 9) and LUF6096 (ref. 10) have
aSchool of Pharmacy, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
E-mail: andrea.vernall@otago.ac.nz; Tel: +64 3 479 4518
bPhysiology, Pharmacology and Neuroscience Division, School of Life Sciences,
University of Nottingham, Nottingham, NG7 2UH, UK
† Electronic supplementary information (ESI) available. See \DOI:
10.1039/c7ra13148h
16362 | RSC Adv., 2018, 8, 16362–16369
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
tetrahydroisoquinols to isoquinolinols under mild conditions
as an alternate to heavy metal-based or harsh reaction
conditions.
Results and discussion
The synthesis of 1-substituted 6-hydroxyl tetrahydroisoquino-
line 5 began with attempts to carry out a Pictet–Spengler reac-
tion of 3-(2-aminoethyl)phenol hydrobromide 4 with glyoxylic
acid monohydrate following the procedure Li et al.28 (no spec-
troscopic data provided for 5, ref. 29), however these were
unsuccessful. Pleasingly, following the synthesis of 5 reported
by Maillard et al.29 tetrahydroisoquinoline 5 was obtained in
good yield, the point of difference between the Li et al.28 and
Maillard et al.29 procedures being addition of triethylamine to
the amine hydrobromide salt prior to addition of glyoxylic acid
monohydrate (Scheme 1). The neutralisation presumably meant
the imine could form more readily and hence the Pictet–Spen-
gler reaction could proceed. Attempts to aromatise carboxylic
acid 5 using DDQ and isolate the hydroxyisoquinoline carbox-
ylic acid (using optimised conditions described below) were
problematic, in part due to difficulty analysing and purifying
the polar carboxylic acid. Therefore 5 was esteried to give
methyl ester 6 (compound 6 was previously reported by Ma
et al.30 but with no spectroscopic data). Synthesis of the 3-
substituted tetrahydroisoquinolinol series began with Pictet–
Spengler condensation of (+/ꢀ)-m-tyrosine 8 with formaldehyde
to give 9 according to a literature procedure reporting the
synthesis of 9,31 followed by esterication to give 10. The
synthesis of methyl ester 10 from 9 has previously been re-
ported18,25,32–34 however none of these reports include spectro-
scopic data for 10. The rst attempt to synthesise the 7-hydroxyl
tetrahydroisoquinoline series via condensation of tyramine
with glyoxylic acid monohydrate according to the procedure for
5 (except without trimethylamine as the amine was not a salt)
was not successful. Failure of this reaction was attributed to the
lack of an activating group in tyramine, compared to the pres-
ence of an activating para hydroxyl relative to the point of
condensation for 4 and 8. Instead, Fmoc-protected tyramine 12
was reacted with glyoxylic acid monohydrate to give a carboxylic
acid according to the synthesis of this carboxylic acid reported
by Maillard et al.29 which was esteried to give 13 in low yield
over 2 steps. NMR spectra of 13 showed a mixture of isomers
that were conrmed as rotamers rather than regioisomers via
variable temperature NMR experiments.
Fig.
1 A1AR subtype selective (benzimidazolyl)isoquinolines with
previously reported AR affinities.8
been reported as allosteric ligands for A3AR, albeit with quite
different substituents, we chose to instead explore the previously
unexplored isoquinoline C5–8 positions for linker attachment.
A1AR ligands such as 1–3 lack a reactive functional group for
linker and subsequently uorophore attachment in these posi-
tions, therefore we designed a series of (benzimidazolyl)iso-
quinolinols. To the best of our knowledge, there are no previous
literature reports on the synthesis of (benzimidazolyl)iso-
quinolinols. The hydroxyl group offers accessible chemistry for
linker attachment, e.g. via an ether bond that remains unionised
at physiological pH. Succinimidyl-6-[2-(p-{(E)-2-[4,4-diuoro-5-
(2-thienyl)-3a,4a-diaza-4-bora-s-indacen-3-yl]-ethenyl}phenoxy)
acetylamino]hexanoate (BODIPY630/650-SE) was chosen as the
uorophore for coupling because of its success in developing
uorophores for studying GPCRs in previous studies4,5,11 and its
suitable spectroscopic properties as an acceptor in biolumines-
cence resonance energy transfer (BRET) assays.12
We envisaged using the Pictet–Spengler reaction13 in our
synthesis to form a hydroxytetrahydroisoquinoline carboxylic
acid followed by aromatisation to give a hydroxyisoquinoline
carboxylic acid. This required an aromatisation reaction
tolerant of functional groups such as a phenol, amine and
carboxylic acid. Although isoquinoline is an important phar-
macophore in various natural products, metal co-ordination
ligands and marketed drugs such as papaverine, ripasudil,
quinisocaine and moxaverine, a general method for the
construction of isoquinolines with broad functional group
tolerance, including the synthesis of isoquinolinols, is lacking.
Aromatisation of tetrahydroisoquinoline to isoquinoline is
usually carried out using heavy metals such as palladium with
harsh conditions with poor functional group tolerance.14–21
Protection/deprotection of functional groups is another
common strategy employed for successful aromatisation to
prepare functionalised isoquinolines.22,23 Use of 2,3-dichloro-
5,6-dicyano-1,4-benzoquinone (DDQ) has been reported for
the aromatisation of tetrahydroisoquinoline22,24,25 and dihy-
droisoquinoline26,27 to isoquinolines at high temperature. In
this study, we explored the use of DDQ to aromatise
There is one report of the aromatisation of an unprotected
hydroxytetrahydroisoquinoline (10) with DDQ in patent literature
(conversion of 10 to 11, no spectroscopic data for 11 provided25)
however in our hands all initial attempts to aromatise 6 or 10 using
DDQ with varying temperatures and solvents and with either
oxygen excluded or in a closed reaction vessel failed. When 6 and
ꢁ
DDQ were dissolved in THF and dioxane and heated to 100 C
under a N2 atmosphere the partially oxidised methyl 6-hydroxy-3,4-
dihydroisoquinoline-1-carboxylate was isolated as the major
product. However, at moderate temperature and with vigorous
stirring in a ask open to the atmosphere, 6 and DDQ were reacted
to give the desired product 7 in moderate yield. To our knowledge,
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 16362–16369 | 16363

View Article Online
RSC Advances
Paper
Scheme 1 Synthesis of isoquinolines. Reagents and conditions: (i) Et3N, glyoxylic acid monohydrate, EtOH, 71%; (ii) SOCl2, MeOH, 65 ꢁC,
quantitative; (iii) DDQ, THF, 1,4 dioxane, 45 ꢁC, 49–52%; (iv) HCl, H2O, formaldehyde (37 wt% in H2O) 100 ꢁC, 48%; (v) H2SO4, MeOH, 96%; (vi)
glyoxylic acid monohydrate, H2SO4, CH3CO2H; (vii) SOCl2, MeOH, 18% over two steps; (viii) DMSO, MeOH, 60 ꢁC; (ix) DDQ, THF, 1,4 dioxane,
45 ꢁC, 61% over two steps.
isoquinoline 7 has only been reported once before in the literature a product with low organic solvent solubility from the viscous
in a Japanese patent35 but no spectroscopic data was provided. The reaction mixture. NMR spectra of 17–19 showed a mixture of
procedure to convert 6 to 7 was also used to successfully aromatise conformers, which was elucidated to be tautomers using vari-
10 to 11. Since the aromatised product did not form without able solvent and high temperature NMR experiments and high
vigorous stirring open to the atmosphere it is likely that DDQ performance liquid chromatography (HPLC) (discussed in
mediates formation of a dihydroisoquinoline, which is then aro- ESI†). The phenolic group of 17–19 was reacted with tert-butyl
matised by oxygen as the active oxidant. Oxygen has previously bromoacetate to afford corresponding tert-butyl esters 20–22,
been reported as an oxidant in aromatisation reactions36,37 and which were reacted with triuoroacetic acid to give carboxylic
indeed Dong et al.38 used a base in dimethyl sulfoxide (DMSO) acids 23–25. Carboxylic acid 24 was coupled to three different
along with oxygen from air for conversion of N-tosyltetrahy- amino linkers – a C8 alkyl, short polyethylene glycol (PEG) or L-
droisoquinolines into isoquinolines. Dong et al.38 also isolated an Ala-L-Ala short peptide to give 27, 28 or 29 respectively.
imine under an argon atmosphere instead of the isoquinoline Carboxylic acid 23 underwent amide coupling to give PEG-
product, analogous to the partially oxidised imine intermediate linked 26 while the 7-substituted carboxylic acid 25 was con-
(methyl 6-hydroxy-3,4-dihydroisoquinoline-1-carboxylate) isolated verted to PEG-linked 30. These linkers were selected to explore
in our study with exclusion of air. Formation of an iminium ion variations in the physicochemical properties of the resulting
intermediate (3,4-dihydroisoquinoline) has also been reported for uorescent probes, in particular in light of lipophilicity and
DDQ-based cross-dehydrogenative coupling reactions of non-specic membrane binding. Boc deprotection of 26–30
tetrahydroisoquinolines.39
provided amines that were reacted with BODIPY630/650-SE to
Diethylamine was initially used for Fmoc deprotection of 13 give uorescent conjugates 31–35. 3-(Benzimidazolyl)
however subsequent aromatisation with DDQ provided poor isoquinolinol-based uorescent conjugates 42 and 43 were
yield of 14, most likely due to residual diethylamine. High synthesised using analogous methodology (Scheme 3).
temperature NMR spectra in DMSO-d6, carried out to study Absorption and emission spectra of uorescent compounds 31–
rotamers of 13 revealed cleavage of Fmoc, and indeed there is 35 and 42–43 showed excitation maxima at 624 nm and emis-
precedent in the literature that Fmoc cleavage can be conducted sion maxima at either 641 nm or 642 nm (detailed in ESI†).
in neat DMSO at high temperature.40 Thus, Fmoc cleavage of 13
in DMSO, followed by DDQ and air mediated aromatisation
afforded 14 in comparable yield to 7 and 11. The moderate yield
of DDQ-air mediated aromatisation is unclear but may be due to
low solubility in organic solvent of these compounds and
susceptibility to methyl ester hydrolysis during basic work up.
Hydrolysis of 7 and 14 under basic conditions provided 15
and 16, which were condensed with o-phenylenediamine or 2,4-
diaminotoluene in hot polyphosphoric acid (PPA) to afford 1-
(benzimidazolyl)isoquinolinols 17–19 in low yield (Scheme 2).
This low yield was likely due to a number of factors, including
poor double dehydration yield and a difficult isolation of
Pharmacology
Fluorescent ligands 31–35 and 42–43 were analysed for their
ability to bind to A1AR according to a previously reported
bioluminescence resonance energy transfer (NanoBRET) satu-
ration binding assay using the novel luciferase NanoLuc (NLuc;
Promega Corporation, USA).12 HEK293 cell lines stably trans-
fected with N-terminal NLuc labelled A1AR were treated with
increasing concentrations of the (benzimidazolyl)isoquinolinol
uorescent ligands, in the presence and absence of 1 mM
DPCPX (a high affinity, non-uorescent, selective A1AR antag-
onist) to determine levels of non-specic binding.
16364 | RSC Adv., 2018, 8, 16362–16369
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Scheme 2 Synthesis of 1-(1H-benzimidazol-2-yl)isoquinolinols. Reagents and conditions: (i) LiOH, THF, H2O, 70–96%; (ii) PPA, 250 ꢁC, 21–38%;
(iii) tert-butyl bromoacetate, K2CO3, THF, 60 ꢁC, 66–96%; (iv) TFA, DCM, 60% – quantitative; (v) HATU, DIPEA, DMF, 29–69%; (vi) BODIPY630/
650-SE, DIPEA, DMF, 33–84%.
Unfortunately, all ligands 31–35 and 42–43 did not show any Fluorescent ligands 31–34 were then analysed for their ability to
indication of binding to A1AR, for example data shown for 31 bind to NLuc-labelled A3AR in analogous NanoBRET experi-
and 33 (Fig. 2a and b) (other compound data in ESI Fig. S16†). ments, using A3AR selective MRS1220 to establish non-specic
Scheme 3 Synthesis of 3-(1H-benzimidazol-2-yl)isoquinolinols. Reagents and conditions: (i) LiOH, THF, H2O, 90%; (ii) PPA, 250 ꢁC, 26%; (iii) tert-
butyl bromoacetate, K2CO3, THF, 60 ꢁC, 94%; (iv) TFA, DCM, quantitative; (v) HATU, DIPEA, DMF, 37–39%; (vi) BODIPY630/650-SE, DIPEA, DMF,
73–86%.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 16362–16369 | 16365

View Article Online
RSC Advances
Paper
Fig. 2 HEK293 cells stably transfected with N-terminally NLuc-tagged A1AR or A3AR were treated with increasing concentrations of fluorescent
ligand and the BRET ratio measured after direct addition of the NLuc substrate furimazine (10 mM).12 Non-specific binding was assessed in the
absence and presence of 1 mM DPCPX (NLuc-A1AR) or 1 mM MRS1220 (NLuc-A3AR). Pooled data of raw BRET ratio's were baseline corrected
(minus vehicle + furimazine BRET ratios) so that data are expressed as fold increase in BRET ratios over basal. (a) NLuc-A1AR and 31, (b) NLuc-
A1AR and 33, (c) NLuc-A3AR and 31 and (d) NLuc-A3AR and 33. Data represents four–seven independent experiments (in triplicate).
binding. Ligands 32 and 34 did not show any specic binding to an additional region of A3AR distinct from the orthosteric
NLuc-A3AR (data not shown), while 31 and to a lesser extent 33 binding site of MRS 1220.
(Fig. 2c and d) showed a small degree of binding to NLuc-A3AR.
A possible explanation for the lack of binding of uorescent
However, the specic binding window was small and the stan- ligands 31–35 and 42–43 to NLuc A1AR, is steric occlusion of
dard error's of the calculated Kd values for specic binding to binding due to the presence of the N-terminal NanoLuc tag. To
NLuc-A3AR were large (31 Kd ¼ 162 ꢂ 65.5 nM, n ¼ 5; 33 Kd ¼ investigate this, NanoBRET was used to quantify the specic
534 ꢂ 254 nM, n ¼ 4).
binding of CA200645, a known uorescent non selective AR
This NanoBRET assay is an excellent way to assess new ligand at NLuc A1AR. Saturable binding was observed, with
uorescent ligands41 since it measures the interaction of the minimal non-specic binding and a large observation window
uorescent ligand and receptor directly without relying on the (Fig. 3A; Kd ¼ 110 nM + 18.46 n ¼ 5) consistent with previous
ability of the test ligand to compete with another tracer such as NanoBRET observations of CA200645 binding to NLuc A1AR or
a radioligand. In this study the level of uorescent ligand non- NLuc A3AR.12 NanoBRET assay plates were then washed and
specic/membrane-binding was dened by measuring BRET in imaged using a IX Ultra confocal plate reader (MetaXpress,
the presence, for A3AR, of a competing high concentration of Molecular Devices) with uorescence intensity quantied per
unlabelled MRS 1220. Therefore this measure of specicity is well using a modied multiple wavelength cell scoring algo-
dened by the ability of MRS 1220 (a[1,2,4]triazolo[1,5-c]qui- rithm.42 Saturable binding of CA200645 was observed at NLuc
nazolinyl pharmacophore) to prevent binding of the uorescent A1AR (Kd ¼ 189.3 nM ꢂ 69.6; n ¼ 4, data not shown), with a Kd
compounds, which implies each would be interacting with the value consistent with that measured by NanoBRET (Fig. 3A).
same or an overlapping region of the ligand binding pocket of The binding of CA200645 to untagged A1AR stably expressed in
the receptor. It is clear from Fig. 2c and d, however, that the HEK293, was also quantied using this uorescent imaging-
non-specic binding component (obtained in the presence of based technique with the resultant estimated binding affinity
MRS 1220) is not linear and is better t with a saturable binding comparable to that seen for NLuc tagged A1AR (144.8 nM ꢂ 63.6;
curve. It therefore remains a possibility (along with non-specic n ¼ 3; data not shown). From these results it can be concluded
membrane interactions) that 31 and 33 may be interacting with that the NanoLuc tag does not occlude binding of CA200645 to
16366 | RSC Adv., 2018, 8, 16362–16369
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Fig. 3 HEK293 cells stably expressing N-terminal NLuc tagged A1AR were treated with increasing concentrations of CA200645 (5–500 nM; 1 h at
37 ꢁC; A). Non specific binding was defined using 1 mM DPCPX, an A1AR selective antagonist. The NanoLuc substrate furimazine was added (10
mM), with luminescence and fluorescence emissions recorded using a Pherastar FS. Data was pooled from independent experiments (n ¼ 5) and
baseline corrected (minus vehicle + furimazine BRET ratios) so that data are expressed as fold increase in BRET ratios over basal and where
appropriate fit using one site saturation binding (mean ꢂ SEM). For competition experiments (B), NLuc A1AR were co-incubated with a fixed
concentration of CA200645 and increasing concentrations of unlabelled ligand (1 h at 37 ꢁC). Total CA200645 binding and vehicle are shown by
the black and white bars respectively. Data were pooled from five independent experiments and are expressed as mean ꢂ SEM.
the receptor, therefore it seems unlikely this tag is the reason for aromatisation reaction, conversion of 6 to 7 was as follows: to
the poor binding of uorescent ligands 31–35 and 42–43.
In light of the poor binding of uorescent ligands 31–35 and 1 : 1, v/v) at 45 C was added DDQ (0.38 g, 1.67 mmol) and the
42–43 to A1AR as measured using NanoBRET competition reaction was stirred vigorously at 45 ꢁC for 5 h with the mouth of
a solution of 6 (0.17 g, 0.83 mmol) in 1,4-dioxane : THF (10 mL
ꢁ
binding experiments,
a
subset of (benzimidazolyl)iso- the ask open to the atmosphere to allow mixing of air. 1,4-
quinolinols described in Schemes 2 and 3 (1, 17–19, 28, 30, 37 Dioxane (10 mL) was added and the reaction mixture ltered,
and 40) and literature reference compound 1 were tested using the ltrate was diluted with EtOAc and washed three times with
NanoBRET competition assays. HEK293 NLuc A1AR cells were sat aq. NaHCO3 solution. The organic washings were combined,
treated with a xed concentration of CA200645 (25 nM) and washed once with water, brine solution, then dried over MgSO4,
increasing concentrations of unlabelled ligand. The A1AR concentrated under reduced pressure and puried by silica gel
selective antagonist DPCPX was used as a positive control. No ash column chromatography (30–50% EtOAc/hexane) to
displacement of CA200645 binding was observed with benzi- provide 7 (84 mg, 0.413 mmol, yield 49%) as an off-white solid.
midazolyl)isoquinolinols 1, 17–19, 28, 30, 37 and 40 until ligand 1H NMR (400 MHz, MeOD-d4) d 4.04 (s, 3H, OCH3), 7.16 (d, J ¼
concentrations were in excess of 10ꢀ6 M (Fig. 3B; representative 2.5 Hz, 1H, ArH), 7.28 (dd, 1H, J ¼ 2.5, 9.3 Hz, ArH), 7.73 (d, 1H, J
(benzimidazolyl)isoquinolinols shown). With the exception of ¼ 5.7 Hz, ArH), 8.30 (d, 1H, J ¼ 5.7 Hz, ArH), 8.52 (d, 1H, J ¼
DPCPX, it was not possible to calculate a pKi value for all 9.3 Hz, ArH). 13C NMR (101 MHz, MeOD-d4) d 53.24, 108.72,
compounds studied (DPCPX pKi; 8.82 ꢂ 0.16). To our surprise, 122.70, 122.89, 124.25, 129.49, 141.07, 141.83, 149.28, 161.18,
no displacement of CA200645 was observed when using 1, 167.61. HRMS calculated for C11H10NO3 (M + H)+, 204.0655;
which was previously reported as a high affinity and selective found, 204.0647.
A1AR ligand (hA1AR Ki ¼ 14.1 ꢂ 1.6 nM, Fig. 1) in a [3H]DPCPX
binding assay in membranes obtained from CHO cells stably
expressing hA1AR.8 Interestingly both 1-(1H-1,3-benzodiazol-2- Conclusions
yl)isoquinolin-6-ol 17 and 3-(6-methyl-1H-1,3-benzodiazol-2-yl)
Our overall goal was to design new A1AR selective uorescent
isoquinolin-6-ol 37 both showed better (although still limited)
ligands, however it can be concluded that a (benzimidazolyl)iso-
quinolinol scaffold linked via the C5 or C6 isoquinoline position to
a uorophore is not suitable for this purpose. Although explora-
binding to NLuc A1AR than 1 (Fig. 3B).
tion of other regions of the (benzimidazolyl)isoquinoline scaffold
Experimental
(such as from the benzimidazole) for linker attachment are
Full details of the synthesis of 1, 4–43, including all method- possible, in light of the poor A1AR binding observed for 1, the
ology, chemical synthesis, pharmacology, equipment and selection of a different A1AR selective pharmacophore may be
compound characterisation can be found in the ESI,† along optimal. Although the end biological aim of A1AR uorescent
with a comprehensive tautomer study of 18. All compounds ligands was not achieved, a method for aromatisation of tetrahy-
tested in the BRET assay were shown by analytical HPLC to droisoquinolinols to give isoquinolinols and conformational
possess >95% purity. As a representative examples of a DDQ (tautomer) studies of (benzimidazolyl)isoquinolinols are reported.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 16362–16369 | 16367

View Article Online
RSC Advances
Paper
Since there are only two reports25,43 for non-metal-mediated aro- 12 L. A. Stoddart, E. K. M. Johnstone, A. J. Wheal, J. Goulding,
matisation of unprotected tetrahydroisoquinolinol to iso-
quinolinol, for which experimental details are unclear and no
M. B. Robers, T. Machleidt, K. V. Wood, S. J. Hill and
K. D. G. Peger, Nat. Methods, 2015, 12, 661–663.
yields are provided, the method described herein using DDQ and 13 E. D. Cox and J. M. Cook, Chem. Rev., 1995, 95, 1797–1842.
atmospheric oxygen in mild conditions will prove useful for the 14 H.-P. Buchstaller, L. Burgdorf, D. Finsinger, C. Amendt,
preparation of biologically important isoquinolines and
isoquinolinols.
M. Grell, C. Sirrenberg, F. Zenkeand Preparation of
isoquinoline derivatives as kinase inhibitors,
WO2005082858A2, Merck Patent Gmbh, 2005.
15 H.-P. Buchstaller, L. Burgdorf, D. Finsinger, F. Stieber,
C. Sirrenberg, C. Amendt, M. Grell, F. Zenke and M. Krier,
Bioorg. Med. Chem. Lett., 2011, 21, 2264–2269.
16 K. Iwasa, M. Moriyasu, Y. Tachibana, H.-S. Kim, Y. Wataya,
W. Wiegrebe, K. F. Bastow, L. M. Cosentino, M. Kozuka
and K.-H. Lee, Bioorg. Med. Chem., 2001, 9, 2871–2884.
17 F. Navas and P. K. Spearing, Preparation of isoxazole
compounds as therapeutic farnesoid X receptor agonists,
US pat., 20080096921A1, Smithkline Beecham Corporation,
2008.
18 E. F. Calderwood, M. Duffey, A. E. Gould, P. D. Greenspan,
B. Kulkarni, M. J. Lamarche, R. S. Rowland, M. Tregay, and
T. J. Vos, Bicyclic compounds with kinase inhibitory
activity, WO2007067444A1, Millennium Pharmaceuticals,
Inc., 2007.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
This work was supported by a University of Otago Research
Grant and the School of Pharmacy, University of Otago and the
University of Nottingham. S. S. is supported by a Doctoral
Scholarship from the School of Pharmacy, University of Otago.
The authors thank Dr Leigh Stoddart of the Physiology, Phar-
macology and Neuroscience group at the University of Not-
tingham for making the NLuc A3AR construct and Promega
Corporation for making the NLuc A1AR construct.
19 S. Chakraborty, W. W. Brennessel and W. D. Jones, J. Am.
Chem. Soc., 2014, 136, 8564–8567.
20 W. Yao, Y. Zhang, X. Jia and Z. Huang, Angew. Chem., Int. Ed.,
2014, 53, 1390–1394.
References
1 J.-F. Chen, H. K. Eltzschig and B. B. Fredholm, Nat. Rev. Drug 21 R. Chen, J. Qi, Z. Mao and S. Cui, Org. Biomol. Chem., 2016,
Discovery, 2013, 12, 265–286. 14, 6201–6204.
2 C. E. Muller and K. A. Jacobson, Biochim. Biophys. Acta, 2011, 22 A. M. M. Mjalli, D. Jones, D. R. Gohimmukkula, G. Huang,
¨
1808, 1290–1308.
J. Zhu, M. Rao, R. C. Andrews, and T. Ren, Benzazole
derivatives, compositions, and methods of use as b-
secretase inhibitors, WO2006099379A2, Transtech Pharma,
Inc., 2006.
3 L. A. Stoddart, C. W. White, K. Nguyen, S. J. Hill and
K. D. Peger, Br. J. Pharmacol., 2016, 173, 3028–3037.
4 E. Kozma, P. S. Jayasekara, L. Squarcialupi, S. Paoletta,
S. Moro, S. Federico, G. Spalluto and K. A. Jacobson, 23 T. R. Burke Jr, P. L. Russ and V. E. Marquez, Heterocycles,
Bioorg. Med. Chem. Lett., 2013, 23, 26–36.
1992, 34, 757–764.
5 A. J. Vernall, S. J. Hill and B. Kellam, Br. J. Pharmacol., 2014, 24 A. Jirgensons, E. Loza, M. Charlton, P. W. Finn,
171, 1073–1084.
D. P. L. Ribas, and A. Saint-Leger, Novel N-acyl-
arylsulfonamide derivatives as aminoacyl-tRNA synthetase
inhibitors, WO2016129983A1, Latvian Institute Of Organic
Synthesis, 2016.
6 M. Macchia, F. Salvetti, S. Barontini, F. Calvani, M. Gesi,
M. Hamdan, A. Lucacchini, A. Pellegrini, P. Soldam and
C. Martini, Bioorg. Med. Chem. Lett., 1998, 8, 3223–3228.
7 A. J. Vernall, L. A. Stoddart, S. J. Briddon, H. W. Ng, 25 A. Abeywardane, S. R. Brunette, M. J. Burke, T. M. Kirrane Jr,
C. A. Laughton, S. W. Doughty, S. J. Hill and B. Kellam,
Org. Biomol. Chem., 2013, 11, 5673–5682.
C. C. Man, D. R. Marshall, A. K. Padyana, H. Razavi, R. Sibley,
K. L. L. Smith, R. J. Snow, R. J. Sorcek, H. Takahashi,
S. J. Taylor, M. R. Turner, E. R. R. Young, Q. Zhang,
Y. Zhang, and R. M. Zindell, Pyrazole derivatives which
8 B. Cosimelli, S. Taliani, G. Greco, E. Novellino, A. Sala,
E. Severi, F. Da Settimo, C. La Motta, I. Pugliesi,
L. Antonioli, M. Fornai, R. Colucci, C. Blandizzi, S. Daniele,
M. L. Trincavelli and C. Martini, ChemMedChem, 2011, 6,
1909–1918.
inhibit
leukotriene
production,
WO2014014874A1,
Boehringer Ingelheim International Gmbh, 2014.
26 V. K. Tiwari, N. Kamal and M. Kapur, Org. Lett., 2017, 19,
262–265.
¨
9 Z.-G. Gao, J. E. Van Muijlwijk-Koezen, A. Chen, C. E. Muller,
A. P. Ijzerman and K. A. Jacobson, Mol. Pharmacol., 2001, 60, 27 A. G. Schultz, W. G. McMahon, R. R. Staib and R. K. Kullnig,
1057–1063.
Tetrahedron Lett., 1987, 28, 4929–4932.
10 L. H. Heitman, A. Goblyos, A. M. Zweemer, R. Bakker, 28 J. J. Li and J. A. Tino, Tetrahydroisoquinoline analogs useful
T. Mulder-Krieger, J. P. van Veldhoven, H. de Vries,
J. Brussee and A. P. IJzerman, J. Med. Chem., 2009, 52, 926–
931.
as growth hormone secretagogues, WO2001085695A1,
Bristol-Myers Squibb Co., 2001.
29 M. C. Maillard, F. A. Brookeld, S. M. Courtney,
F. M. Eustache, M. J. Gemkow, R. K. Handel,
11 Z. Ma, L. Du and M. Li, J. Med. Chem., 2014, 57, 8187–8203.
16368 | RSC Adv., 2018, 8, 16362–16369
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
L. C. Johnson, P. D. Johnson, M. A. Kerry, F. Krieger,
RSC Advances
inhibit (block) bitter taste in compositions and use
thereof, WO2011106114A1, Senomyx, Inc., USA, 2011.
˜
´
M. Meniconi, I. Munoz-Sanjuan, J. J. Palfrey, H. Park,
S. Schaertl, M. G. Taylor, D. Weddell and C. Dominguez, 35 S. Ishiguro, N. Kawaguchi, M. Nakakoshi, S. Shimada,
Biorg. Med. Chem., 2011, 19, 5833–5851.
30 D. Ma, W. Wu, G. Yang, J. Li, J. Li and Q. Ye, Bioorg. Med.
Chem. Lett., 2004, 14, 47–50.
31 P. L. Ornstein, M. B. Arnold, N. K. Augenstein and
J. W. Paschal, J. Org. Chem., 1991, 56, 4388–4392.
32 A. M. M. Mjalli, D. Jones, D. R. Gohimmukkula, G. Huang,
M. Seya, M. Nomoto, M. Okue, and H. Tomizuka,
Preparation of isoquinoline derivatives as protease
inhibitors, JP07304744A, Snow Brand Milk Prod Co Ltd,
Japan, 1995.
36 S. Chen, Y. Li, P. Ni, B. Yang, H. Huang and G.-J. Deng, J. Org.
Chem., 2017, 82, 2935–2942.
J. Zhu, M. Rao, R. C. Andrews, and T. Ren, Benzazole 37 T. Shamim, M. Gupta and S. Paul, J. Mol. Catal. A: Chem.,
derivatives, compositions, and methods of use as b- 2009, 302, 15–19.
secretase inhibitors, WO2006099379A2, Transtech Pharma, 38 J. Dong, X.-X. Shi, J.-J. Yan, J. Xing, Q. Zhang and S. Xiao, Eur.
Inc., 2006. J. Org. Chem., 2010, (36), 6987–6992.
33 S. Seiwert, L. Beigelman, B. Buckman, V. Serebryany, and 39 A. S.-K. Tsang, P. Jensen, J. M. Hook, A. S. K. Hashmi and
A. D. Stoycheva, Preparation of proline tripeptides and M. H. Todd, Pure Appl. Chem., 2011, 83, 655–665.
analogs as inhibitors of hepatitis C virus replication for 40 S. Hock, R. Marti, R. Riedl and M. Simeunovic, CHIMIA
¨
treating hepatitis
WO2010045266A1, InterMune, Inc., USA, 2010.
34 D. S. Karanewsky, J. Fotsing, C. Tachdjian, and M. Arellano,
C
infection and liver brosis,
International Journal for Chemistry, 2010, 64, 200–202.
41 L. A. Stoddart, L. E. Kilpatrick and S. J. Hill, Trends
Pharmacol. Sci., 2018, 39, 136–147.
Identication of human T2R receptors that respond to bitter 42 M. A. Arruda, L. A. Stoddart, K. Gherbi, S. J. Briddon,
compounds that elicit the bitter taste in compositions, and B. Kellam and S. J. Hill, Front. Pharmacol., 2017, 8, 908.
the use thereof in assays to identify compounds that 43 B. Franck, G. Blaschke and G. Schlingloff, Angew. Chem.,
1963, 75, 957–965.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 16362–16369 | 16369