Synthesis, Biological Evaluation, and Utility of Fluorescent Ligands Targeting the μ-Opioid Receptor

Article abstract of DOI:10.1021/acs.jmedchem.5b01664

Fluorescently labeled ligands are useful pharmacological research tools for studying receptor localization, trafficking, and signaling processes via fluorescence imaging. They are also employed in fluorescent binding assays. This study is centered on the design, synthesis, and pharmacological evaluation of fluorescent probes for the opioid receptors, for which relatively few non-peptidic fluorescent probes currently exist. The known μ-opioid receptor (MOR) partial agonist, buprenorphine, was structurally elaborated to include an amidoalkylamine linker moiety that was coupled with a range of fluorophores to afford new fluorescent probes. All compounds proved to be selective MOR antagonists. Confocal fluorescence microscopy studies revealed that the probe incorporating a sulfonated cyanine-5 fluorophore was the most appropriate for imaging studies. This ligand was subsequently employed in an automated fluorescence-based competition binding assay, allowing the pK_i values of several well-known opioid ligands to be determined. Thus, this new probe will prove useful in future studies of MOR receptor pharmacology.

Full text of DOI:10.1021/acs.jmedchem.5b01664

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Article
Synthesis, Biological Evaluation and Utility of
Fluorescent Ligands Targeting the µ-Opioid Receptor
Luke S. Schembri, Leigh A. Stoddart, Stephen J. Briddon, Barrie
Kellam, Meritxell Canals, Bim Graham, and Peter J. Scammells
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01664 • Publication Date (Web): 03 Dec 2015
Downloaded from http://pubs.acs.org on December 6, 2015
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Synthesis, Biological Evaluation and Utility of Fluorescent
Ligands Targeting the µꢀOpioid Receptor
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†
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Luke S. Schembri, Leigh A. Stoddart, Stephen J. Briddon, Barrie Kellam,
‡
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,†
Meritxell Canals, Bim Graham,* and Peter J. Scammells*
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‡
Medicinal Chemistry and Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences,
Monash University, Parkville, Victoria, 3052, Australia.
Cell Signaling Research Group, School of Life Sciences, Queen’s Medical Centre, University of
Nottingham, Nottingham NG7 2UH, UK
School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7
2RD, UK
KEYWORDS: opioid receptor, fluorescent imaging, opioid antagonist, fluorescent ligand
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ABSTRACT: Fluorescentlyꢀlabeled ligands are useful pharmacological research tools for studying
receptor localization, trafficking and signaling processes via fluorescence imaging. They are also
employed in fluorescent binding assays. This study is centered on the design, synthesis and
pharmacological evaluation of fluorescent probes for the opioid receptors, for which relatively few
nonꢀpeptidic fluorescent probes currently exist. The known µꢀopioid receptor (MOR) partial agonist,
buprenorphine, was structurally elaborated to include an amidoalkylamine linker moiety that was
coupled with a range of fluorophores to afford new fluorescent probes. All compounds proved to be
selective MOR antagonists. Confocal fluorescence microscopy studies revealed that the probe
incorporating a sulfonated cyanineꢀ5 fluorophore (3) was the most appropriate for imaging studies.
This ligand was subsequently employed in an automated fluorescenceꢀbased competition binding assay,
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allowing the pK values of several wellꢀknown opioid ligands to be determined. Thus, this new probe
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will prove useful in future studies of MOR receptor pharmacology.
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ꢀ
INTRODUCTION
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Opiates, such as morphine, are one of the most efficacious classes of analgesics in use today for
the treatment of acute and chronic pain. They elicit their effects by binding to the three “classical”
opioid receptors (ORs) – the ꢁ, κ and δ receptors (MOR, KOR and DOR) – and some to the more
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recently discovered nociceptin receptor (NOR), all of which are classified as class A, peptideꢀ
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binding, γꢀsubtype G proteinꢀcoupled receptors (GPCRs). The four ORs couple to the same major
intracellular signaling pathways, interacting with G proteins of the G family. Upon GPCR activation,
i/o
the β/γ subunit of the heterotrimeric G protein is released and acts to inhibit voltageꢀgated calcium
channels and activate inwardlyꢀrectifying potassium channels.
This in turn leads to cell
4
hyperpolarization and inhibition of neurotransmitter release, thus inhibiting neuronal signaling.
Additionally, the α subunit of the G protein inhibits adenylyl cyclase and hence decreases intracellular
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,6
cyclic adenosine monophosphate (cAMP) production.
Notwithstanding their effectiveness as analgesics, opiates suffer from a myriad of doseꢀlimiting
sideꢀeffects, including constipation, hallucinations, sedation, respiratory depression and, perhaps most
7
significantly, tolerance and physical dependence. In order to develop opiates with an improved sideꢀ
effect profile, more fundamental biological research is needed to determine how such adverse effects
arise. Additionally, more structureꢀactivity relationships (SARs) must be established so as to be able to
enhance or maintain potency whilst reducing or eliminating undesirable sideꢀeffects.
Fluorescentlyꢀlabeled GPCR ligands are useful for the above endeavors as they can be used in an
array of pharmacological experiments, most notably, realꢀtime monitoring of cellular processes such as
8–13
receptor trafficking and ligand binding.
Due to their high sensitivity, such probes can generate
meaningful pharmacological data from a few cells (such as a biopsy) and thus may be useful in
determining pharmacological differences between diseased and healthy tissue. Moreover, they may be
used to determine the binding affinity of drug candidates via fluorescence resonance energy transfer
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(
FRET)ꢀbased assays or assays involving displacement of a probe containing an environmentallyꢀ
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sensitive fluorophore.
Traditionally, receptor binding assays have been performed using competitive radioligands, with
the binding affinity of a drug candidate determined from its ability to displace a highꢀaffinity
radiolabeled ligand. Such experiments require a large population of cells, whereas information can be
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obtained from just a few cells, or even a single cell, using fluorescent probeꢀbased experiments.
Fluorescent probes are generally easier to handle, provide high sensitivity, and can be used to visually
differentiate between specific and nonspecific binding if used in conjunction with fluorescently labeled
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receptors. Moreover, from a safety point of view, the waste generated from the use of such probes is
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far less dangerous to the environment and the user than that produced by radiometric assays, making
them more amenable to highꢀthroughput screening (HTS) applications. A fluorescent ligand must,
however, be carefully designed to ensure that it retains (or improves upon) the pharmacological
properties of the ligand upon which it is based. Due consideration must be given to not only the choice
of ligand, but also linker length, linker chemistry, site of linker attachment, as well as the
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fluorophore.
Relatively few fluorescent probes have been developed thus far for the ORs. Fluorescentlyꢀ
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labeled enkephalin peptides (endogenous OR ligands) were the first such compounds to be reported.
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.
Artamangkul, Macey and coworkers appended the Alexa 488 and 594 fluorophores to dermorphin
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0,11,13
and employed these conjugates in various imaging and receptor trafficking studies.
Most
recently, Ghosh and coꢀworkers decorated fluorescent quantum dots with multiple copies of a
DeltorphinꢀII analog peptide, permitting singleꢀmolecule imaging of the human DOR expressed in
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CHO cells. In terms of nonꢀpeptidic designs, Kolb et al. have labeled oxymorphone, naltrexone and
naloxone at the Cꢀ6 position with fluorescein (and rhodamineꢀB in the case of naloxone). Although the
resulting probes broadly retain the ORꢀmediated activities of their parent compounds, their receptor
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binding affinities were significantly diminished, and they were not employed for fluorescent
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visualization/detection of ORs.
Medzihradsky, Emmerson and coꢀworkers have attached the
environmentallyꢀsensitive (solvatochromic) fluorophore, 7ꢀnitrobenzoꢀ2ꢀoxaꢀ1,3ꢀdiazole (NBD), to a
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tetrahydrothebaine derivative, and both NBD and the 4,4ꢀdifluoroꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindacene
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(BODIPY) fluorophore to βꢀnaltrexamine.
The NBDꢀbearing compounds lacked significant
selectivity, and the absorbance and emission wavelengths of the NBD and greenꢀemitting BODIPY
fluorophores are not optimal for biological imaging applications. The tetrahydrothebaine derivative
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was also too hydrophobic, leading to nonꢀspecific binding to cellular membranes.
The BODIPY
derivatives exhibited MOR affinities in the nanomolarꢀtoꢀsubꢀnanomolar range, with one showing a
selectivity ratio of > 40 for MOR over DOR and KOR. Portoghese and coꢀworkers have reported the
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development of fluoresceinꢀlabeled KORꢀselective arylacetamide agonists, however fluorescein
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suffers from a high photoꢀbleaching rate and its photophysical properties are generally not optimal for
receptor imaging studies.
It is clear from the above that there still remains considerable scope for the development of
fluorescent OR probes of practical utility, i.e., probes possessing both binding and photophysical
properties suitable for imaging of OR localization and trafficking, and/or fluorescentꢀbased screening
assays. Herein, we present the synthesis, biological evaluation and application of a series of fluorescent
OR antagonists with improved MOR selectivity (Figure 1), based on the highꢀaffinity opioid,
buprenorphine. The latter is indicated in Europe for the relief of pain, whilst globally it is used in the
treatment of opiate abuse. It is a partial MOR and NOR agonist, KOR and DOR antagonist, is more
potent than morphine, and has less unwanted sideꢀeffects owing to its ceiling effect on tolerance and
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respiratory depression. It provides an attractive scaffold for generating fluorescent probes for ORs
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because of its strong binding affinity [pK (MOR) = 8.8] and the possibility to couple it to certain
i
chemical moieties, such as a spacer or linker. Indeed, some related compounds already have alkyl
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chains appended at the Cꢀ20 position and this has resulted in extremely high affinity ligands. Inspired
by this work, probes were designed featuring four different fluorophores: sulfonated cyanineꢀ5 (sulfoꢀ
TM
Cy5), boron dipyrromethene difluoride (BODIPY ) 630/650, 4ꢀ((6ꢀmethoxyꢀ1,2,4,5ꢀtetrazinꢀ3ꢀ
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yl)oxy)butanoic acid (tetrazine) and 3ꢀ(6ꢀaminoꢀ1,3ꢀdioxoꢀ1Hꢀbenzo[de]isoquinolinꢀ2(3H)ꢀ
yl)propanoic acid (naphthalimide). Through measurement of their respective receptor pharmacology,
alongside fluorescence confocal microscopy, this study clearly demonstrates the suitability of the sulfoꢀ
Cy5ꢀbearing fluorescent ligand, in particular, for receptor localization studies, as well as in vitro
binding assays. This probe possesses the advantage of exhibiting increased fluorescence in the MORꢀ
bound state, obviating the need for a wash step included in many competitive binding assays.
NH2
R =
O
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HO
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SO3-
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S
MeO
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OMe
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Figure 1. Structures of fluorescent OR ligands synthesized in this study.
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ꢀ
RESULTS AND DISCUSSION
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Fluorescent ligand design and synthesis. A range of ligand design options were considered in
which the linker and fluorophore could be attached to different positions of a morphinan core. Linking
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from the tertiary amine seemed chemically feasible (e.g. via Nꢀdemethylation and alkylation), however
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the OR crystal structures
suggested that this amine is buried deep within the active site and would
therefore have limited tolerance to groups appended at this position. Some substitutions at this position
can convert agonists to antagonists, as evidenced by naltrexone. Linking from the 3ꢀposition of the
phenol moiety was another potential strategy. However, even simple methylation at this position
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(codeine) reduces binding affinity by approximately 200ꢀfold.
A more interesting possibility for
linker attachment was the 7ꢀposition of the morphinan scaffold, via a DielsꢀAlder reaction, since this
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could lead to an increase in affinity, as demonstrated by ligands such as etorphine and buprenorphine.
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Previous work conducted on the development of fluorescently labeled OR ligands
as well as
indicates that the Cꢀring of the morphinan scaffold (which incorporates the 6ꢀ and
ꢀpositions) is generally reasonably tolerant of substitution. With these factors in mind, the synthesis
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bivalent ligands
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and biological evaluation of three different congeners (Figure 2) featuring a linker attached via the 7ꢀ
position were pursued. In two of these structures (9 and 11) the linker was attached via a DielsꢀAlder
reaction involving the Cꢀring diene of oripavine and methyl acrylate. Subsequent saponification and
amide coupling incorporated the full linker unit (amide congeners). The third congener was derived
from the DielsꢀAlder reaction of oripavine with a fully formed linker unit that possessed a terminal Nꢀ
substituted maleimide group (imide congener). Literature precedence suggests that conjugation of a
maleimide group retains affinity when connected in this position, and alkyl chains are also
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tolerated.
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Figure 2. Structures of buprenorphineꢀlike congeners prepared in this study.
Amide congeners. Scheme 1 details the synthetic pathway used to access the amide derivatives,
with oripavine as the starting molecule. Following protection of phenol 5 to its corresponding tertꢀ
butyldimethylsilyl (TBS) ether (6), a DielsꢀAlder reaction between the latter and methyl acrylate,
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7
afforded ester 7. Compared to dienophiles such as methyl vinyl ketone, the ester presents a weaker
electronꢀwithdrawing group compared to the ketone, therefore requiring more forcing conditions to
achieve complete conversion to the DielsꢀAlder product. This was achieved through irradiating the
o
sample for three hours at 130 C in a microwave in neat methyl acrylate. Compound 7 was isolated in
good yield (69%) following recrystallization from minimal hot methanol, which was necessary to
remove a small amount of the 8ꢀregioisomer byproduct.
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a
Scheme 1. Synthesis of amide congeners.
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Reagents and conditions: (a) TBSCl, imidazole, DMF, RT, 1 min, 94%; (b) methyl acrylate, µW, 130
o
C, 3 h, 69%; (c) i. 1
M LiOH, THF (1:1), RT, 14 h; ii. EDC.HCl, HOBt, DIPEA, DMF, N RT, 14 h,
2,
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4%; (d) 10% Pd/C, H , RT, 10 bar, 2 h, 86%; (e) 4 M HCl, RT, 30 min, 39% for 9, 29% (over two step
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from 8) for 11.
Saponification of methyl ester 7 with lithium hydroxide and subsequent amide coupling with tertꢀ
butyl (6ꢀaminohexyl)carbamate using 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide hydrochloride
(EDC.HCl) and Nꢀhydroxybenzotriazole (HOBt) afforded Bocꢀprotected congener 8 in a respectable
yield (44%) with > 95% purity according to HPLC. An “Hꢀcube” hydrogenation system was then
employed to reduce the olefin in the next step (small scales of 500 mg or less). This occurred cleanly
within two hours to produce 10 in high yield (86%). Acidolytic Boc group removal was performed on
both the unreduced (8) and reduced (10) versions of the protected congeners to give compounds 9 and
11 respectively, as their bisꢀHCl salts following lyophilization.
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Imide congener. The synthetic route for these derivatives commenced with synthesis of a
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maleimideꢀbearing linker group though reaction of Nꢀ(ethoxycarbonyl)maleimide with monoꢀBocꢀ
protected diaminohexane (Scheme 2). After one hour, 12 was obtained as a pure white solid in high
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0
yield (89%). This high yield was only achieved if fresh (no more than a week old) Nꢀ
o
(
ethoxycarbonyl)maleimide was used and if the amine was stirred in saturated NaHCO at 0 C before
3
adding in the maleimide.
a
Scheme 2. Synthesis of the imide congener.
a
o
Reagents and conditions: (a) i. 3:5 THF/sat. NaHCO3 (aq), 0 C, 15 min; ii. 3:17 (v/v) THF/sat.
NaHCO3 (aq), RT, 45 min, 89%; (b) EtOH, reflux, 5 h, 52%; (c) 4 M HCl, RT, 30 min, 86%.
Compound 13 was synthesized via a DielsꢀAlder reaction between 12 and oripavine (Scheme 2).
This reaction required less forcing conditions compared to methyl acrylate as the maleimide group is a
stronger dienophile. Additionally, as the maleimide is connected through both the 7 and 8 positions, no
minor regioisomer byproducts were produced and hence purification was performed via silica
chromatography.
Unfortunately, reducing the alkene moiety of these compounds proved problematic. Reaction
conditions trialed in the Hꢀcube included pressures as high as 20 bar (280 psi), yet no reduction took
place, even after two hours. One possible explanation for this difficulty is that the bulkiness of the
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3
maleimide group hinders the olefin’s approach to the palladium catalyst. Only the unreduced version
of this congener was therefore tested, and it was assumed that enough information about the effect of
the olefin on potency and affinity could be garnered from the amide congeners. Finally, acidolytic
removal of the Boc protecting group of 13, followed by column chromatography to remove minor
impurities, afforded congener 14 as its bisꢀHCl salt.
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
The different congeners were compared pharmacologically to assess which modifications were
tolerated by the MOR and which, if any, retained the partial MOR selectivity of the parent molecule
(vide infra). In all cases, the corresponding tert-butyloxycarbonyl (BOC) protected congeners were
also evaluated in order to help assess if there was potential receptor tolerance for further chemical
elaboration at the end of the linker unit.
Fluorescent ligands synthesis. The four fluorophores chosen for incorporation into our probes
TM
were BODIPY 630/650, naphthalimide, sulfoꢀCy5 and tetrazine. The necessary precursors were
TM
commercially available (BODIPY 630/650 SE) or readily accessed by literature procedures (sulfoꢀ
4
9
50
Cy5 with a hexanoic acid pendant and 3ꢀ(4ꢀaminoꢀ1,8ꢀnaphthalimido)propanoic acid ) for all but the
tetrazine probe. Preparation of the later required the synthesis of a novel conjugatable tetrazine
5
1
derivative (19) from 3,6ꢀdichlorotetrazine (15) according to Scheme 3. Compound 18 features a
butanoic acid linker at one end and a methoxy “cap” at the other. Such alkoxy groups render tetrazines
51–54
fluorescent.
Additionally, these groups ensure that no other group reacts with the tetrazine via
5
1
nucleophilic substitution, in particular thiols and amines, which cause loss of tetrazine fluorescence.
Compound 15 was first reacted with excess methanol to replace one of the chloro groups with a
5
4
methoxy substituent. Following this, the remaining chloro group was substituted with 1,4ꢀbutanediol
5
3
in low yield (19%) by adapting the method of Gong et al. and employing NaH to deprotonate the
alcohol reactant (substitution with 4ꢀhydroxybutanoic acid was also trialed in an attempt to directly
access 18, however this led to degradation of the tetrazine core). The low yield of 17 is thought to be
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1
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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
due to partial degradation of the tetrazine by the reactive alkoxide, combined with the formation of
byproducts resulting from 1,4ꢀbutanediol displacing both the methoxy and chloro groups of 16 or
forming crosslinked species (an extra equivalent of the diol was added in an attempt to minimize
doubleꢀdeprotonation of the diol). The penultimate step involved oxidation of the terminal alcohol
under mild conditions, employing a catalytic amount of chromium trioxide (1.2 mol%) and periodic
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
5
5
acid as per the method of Zhao et al. Finally, the carboxylic acid group of 18 was converted to the Nꢀ
1
hydroxysuccinimide (NHS) ester using dicyclohexylcarbodiimide (DCC) as the coupling reagent. H
NMR analysis showed that the isolated sample of 19 contained dicyclohexylurea (DCU, 25% by mass
1
according to H NMR), however it was considered adequate for the subsequent coupling step.
a
Scheme 3. Synthesis of the conjugatable tetrazine fluorophore.
a
Reagents and conditions: (a) MeOH, RT, 1 h, 82%; (b) i. NaH, 1,4ꢀbutanediol, dry THF, N , RT, 30 min;
2
o
o
ii. N , dry THF, ꢀ78 C, 1 h; iii. RT, 1 h, 19%; (c) i. CrO , H IO , ACN, 0 C, 30 min; ii. RT, 30 min,
2
3
5
6
o
100%; (d) i. DCC, NHS, DCM, 0 C, 1 h; ii. RT, 1 h, 97% recovery (75% 19, 25% DCU by mass).
The final step was to conjugate the fluorophores as per Scheme 4. The amide congener 11 was
selected over the imide congener due to its higher binding affinity (vide infra) (note that the bisꢀTFA
salt form of 11 (11b), rather than the bisꢀHCl salt (11a), was used for three of the probes). While
TM
BODIPY 630/650 and the tetrazine derivative were reacted as Nꢀhydroxysuccinimide esters, 2ꢀ(6ꢀ
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3
chloroꢀ1Hꢀbenzotriazoleꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetramethylaminium hexafluorophosphate (HCTU) was used to
couple the unactivated naphthalimide and sulfoꢀCy5 derivatives to the congener. The naphthalimide
probe 1 was isolated in reasonable yield (48%) via this method, however a guanidiunium byproduct
was detected in significant amounts for the sulfoꢀCy5 probe 3, formed as a result of the amine reacting
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
5
6
directly with the coupling reagent.
The use of benzotriazolꢀ1ꢀylꢀoxytripyrrolidinophosphonium
hexafluorophosphate (PyBOP) as a coupling agent eliminated this problem, although 3 could still only
be isolated in low yield (18%).
TM
Conjugation of the BODIPY 630/650 and tetrazine fluorophores was performed using the
5
7
reaction conditions set out by Middleton et al. Purification of 2 required only preparative HPLC
(
32% yield), however for 4, an extra extraction step (1:3 IPA:CHCl ) was required to remove the DCU
3
carried over from the previous step (18% yield).
a
Scheme 4. Synthesis of fluorescent OR ligands.
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4
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
a
Reagents and conditions: (1) HCTU, DIPEA, N , dry DMF, 2 h, RT, 48%; (2) DIPEA, N , dry DMF,
2
2
4
h, RT, 32%; (3) PyBOP, DIPEA, N , dry DMF, 1 h, RT, 18%; (4) DIPEA, N , dry DMF, 1 h, RT,
2 2
18%.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Pharmacology. Initially, the OR binding affinities of the six congeners were measured via a
3
competitive radioligand binding assay using [ H]diprenorphine in membrane preparations of CHOꢀ
FlpIn cells stably expressing the human MOR. The results (Table 1) revealed some clear trends.
Firstly, the amide linker is generally better tolerated than the imide linker; three of the four compounds
(
8, 10 and 11) with amide linkers have a higher binding affinity than the imide congeners. The amide
equivalent (8) of the protected, unreduced imide congener (13) displayed approximately 35ꢀfold higher
affinity for the ORs. One possible explanation for this is that the amide group is more flexible than the
imide group and is thus able to rotate itself into a position where it can make more favorable hydrogenꢀ
bonding interactions with surrounding residues, or minimize unfavorable steric interactions. The
reduction of the olefin is associated with a 2–3ꢀfold increase in OR affinity (10 vs. 8 and 11 vs. 9),
which may again be due to increased flexibility. Finally, the Bocꢀprotected congeners, 8, 10 and 13,
exhibited approximately 26ꢀ, 49ꢀ and 8ꢀfold higher OR affinity compared to the respective deprotected
analogs, 9, 11 and 14. This indicates not only that the linker is well tolerated, but that it is sufficiently
long that reasonably bulky groups can be attached to its terminus without disrupting binding.
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5
Table 1. OR affinities (pK ) of congeners, measured using a radioligand binding assay in membrane
i
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
preparations of CHO FlpIn cells stably expressing human MOR.
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
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1
2
3
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2
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0
Compound
R
Single/Double Bond
pKi
DAMGO
ꢀꢀ
ꢀꢀ
ꢀꢀ
5.66 ± 0.08
†
Buprenorphine
ꢀꢀ
8.8
§
8
9
ꢀ(CH
2
)
6
NHBoc
Double
Double
Single
Single
Double
Double
8.13 ± 0.08
6.71 ± 0.08
8.66 ± 0.08
6.97 ± 0.08
6.58 ± 0.07
5.69 ± 0.07
ꢀ(CH
)
NH .2HCl
2
2
6
10
11
13
14
ꢀ(CH
2
)
6
NHBoc
NH .2HCl
NHBoc
ꢀ(CH
)
2
6
2
ꢀ(CH )
2 6
ꢀ(CH ) NH .2HCl
2 6 2
†
§
58
from IUPHAR database.
Errors represent ± SEM of 3 independent experiments
The efficacy of congeners 8–14 to stimulate ERK phosphorylation via the MOR was also tested
in CHO FlpIn cells stably expressing human MOR. Timeꢀcourse experiments involving incubation of
the congeners (10 µM) with MORꢀoverexpressing cells for a 30ꢀmin period showed no ERK
phosphorylation in the presence of any of the congeners (data not shown), indicating that all congeners
have no detectable agonist activity at this signaling pathway at this time point.
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1
1
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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
Incubation of increasing concentrations of each of the fluorescent probes with an EC50
2
4
concentration of a MOR, DOR or KOR agonist ([DꢀAla , NꢀMePhe , Glyꢀol]ꢀenkephalin (DAMGO),
SNC80 and ICI199441, respectively) in CHO FlpIn cells stably expressing human MOR, DOR or
KOR, resulted in an inhibition of agonistꢀinduced ERK phosphorylation in all cases (Figure S21) (in
the case of the DOR and KOR, time course experiments akin to those performed with the congeners
and MORꢀoverexpressing cells were also conducted, indicating that all congeners have no detectable
agonist activity at this signaling pathway after 30 min (data not shown)). Assuming competitive
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
binding, the affinities (pA ) of the fluorescent probes for the three receptor subtypes (Table 2) were
2
5
9
determined using the GaddumꢀSchild equation as per Lane et al. Each of the probes showed high
affinity for the MOR, together with a strong preference for this OR subtype over KOR and DOR
(selectivity ratios of 9–45 and > 200, respectively). The selectivity ratios for 2 and 4 indicate that they
25–27
are amongst the most MORꢀselective fluorescent opiate antagonists reported to date.
Table 2. pA values of the fluorescent probes at the three ORs (n = 3). Agonists used were DAMGO
2
(MOR), SNC80 (DOR) and ICI199441 (KOR).
Compound
pA (MOR)
pA (DOR)
pA (KOR)
MOR/KOR
selectivity
2
2
2
§
Agonist (pEC )
7.67 ± 0.01
8.18 ± 0.07
10.32 ± 0.04
ꢀ
50
#
1
2
3
4
7.87 ± 0.08
8.37 ± 0.07
7.31 ± 0.22
8.01 ± 0.27
< 5
6.91 ± 0.18
6.72 ± 0.25
6.30 ± 0.04
6.37 ± 0.04
9
< 5
< 5
< 5
45
10
44
§
#
Errors represent ± SEM of three independent experiments performed in triplicate. < 5 indicates that
no fitting could be performed within the concentrations tested, indicating very low affinity.
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Imaging studies. The absorption and emission profiles of the synthesized probes (Figure S23)
were recorded using a FlexStation 3 plate reader in both hydrophilic (HEPES buffered saline solution,
HBSS) and hydrophobic (dimethylsulfoxide, DMSO) environments at a concentration of 10 ꢁM.
These solvents were used to represent the polar aqueous and comparatively nonꢀpolar ligand binding
site environment within an OR, respectively. Such measurements were necessary to ensure that
appropriate excitation wavelength and dichroic emission filters were used in the subsequent imaging
experiments.
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
In general, all of the observed excitation and emission maxima (Table S1) closely matched those
5
0,60,61
reported in the literature for the corresponding “free” fluorophore.
All probes experienced a
significant decrease in emission intensity and, in most cases, a hypsochromic shift in maxima upon
moving from DMSO to HBSS, which is particularly useful from the perspective of developing
fluorescent displacementꢀstyle binding assays. Unfortunately, no signal could be detected for the
tetrazine probe, 4, even at 100 ꢁM, despite the fact that yellow fluorescence could be observed under
longꢀwavelength UV irradiation (TLC lamp).
TM
It was encouraging that the BODIPY
630/650 derivative (2) exhibited the highest MOR
affinity (vide supra) as it has particularly favorable photophysical properties for imaging and assay
applications. Most BODIPY fluorophores have high quantum yields, often approaching unity, and a
ꢀ1 ꢀ1 62
high molar extinction coefficient (101,000 cm M ).
Probe 2 displayed bright fluorescence in a
relatively nonꢀpolar environment (DMSO), but almost full quenching of fluorescence in an aqueous
environment (Figure S23), which would obviate the need for a washing step in a fluorescent
displacementꢀstyle binding assay.
Singleꢀtime point confocal images of human embryonic kidney cells (HEK) stably expressing the
human MORꢀGFP fusion protein were captured after incubation with each of the probes (Figure 3).
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1
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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
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5
5
5
5
5
5
5
6
The MOR was expressed with green fluorescent protein at its Cꢀterminus so that the binding specificity
of each probe could be assessed from the degree of coꢀlocalization of probe and GFP fluorescence.
TM
The strong binding affinity and favorable photophysical properties of BODIPY
630/650
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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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
allowed probe 2 to be clearly imaged at relatively low concentration (5 nM). There was a high degree
of colocalization of the probe with MORꢀGFP and significant displacement of binding by the nonꢀ
fluorescent antagonist, naloxone, providing strong evidence that binding is specifically to the MOR.
However, the fact that not all the fluorescent signal was lost upon incubation with a large excess of the
unlabeled MORꢀspecific ligand indicates some degree of nonꢀspecific interaction of 2 with the cellular
membrane (Figure 3).
The concentration required to image binding of probe 3 (50 nM) was higher than that required for
2, consistent with its almost 10ꢀfold lower affinity for the MOR. Unlike 2, this probe could be almost
completely displaced using excess naloxone (Figure 3), again indicating that it binds specifically to the
MOR, but also that the use of a more hydrophilic sulfoꢀCy5 fluorophore reduces nonꢀspecific
interaction with the cellular membrane.
Displacement of 3 with naloxone was also accompanied by a large decrease in fluorescence
intensity, consistent with the reduced fluorescence of this probe in an aqueous environment. It should
be noted that no wash steps were performed in these imaging experiments, so the observed reductions
in fluorescence were not simply due to a concentration gradientꢀdriven reꢀequilibration process.
In the case of 1 and 4, no fluorescence was observed after incubating cells with the probes; hence
their quantum yields appear to be inadequate for imaging purposes under the conditions employed here.
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9
MORꢀGFP
Ligand
Merge
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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51
52
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54
55
56
57
58
59
60
2
(5 nM)
2
(5 nM)
+
1 ꢀM Naloxone
3
(50 nM)
3
(50 nM)
+
1 ꢀM Naloxone
Figure 3. Live cell confocal imaging of 2 and 3 in cells expressing MORꢀGFP. Confocal images of
o
HEKꢀMORꢀGFP cells incubated with probes 2 or 3 (30 min, 37 C) at the concentrations shown, with
o
and without prior incubation with naloxone (1 µM, 30 min, 37 C). All images are of a 102 µm × 102
µm area and are representative images of those obtained in three separate experiments.
Fluorescent binding assays. The imaging studies suggested that probes 2 and 3 may be suitable
6
3
for use in a fluorescenceꢀbased competition binding assay as per Stoddart et al. As a prelude to this,
experiments were performed to establish an appropriate preꢀincubation time (Figure 4).
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2
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5
6
The results for the sulfoꢀCy5ꢀbearing probe 3 (Figure 4, top panels) showed that equilibrium was
rapidly reached (within 5 min), whilst for the BODIPYꢀbearing probe 2 (Figure 4, bottom panels), the
system was still equilibrating after 30 minutes. Experiments performed in the presence of an excess of
naloxone again confirmed that there was minimal nonꢀspecific interaction in the case of 3, and a single
wash step removed essentially all of the bound probe, reflective of fast dissociation kinetics. In
contrast, significant amounts of 2 bound to the cells in the presence of naloxone, and washing produced
very little change in fluorescence, indicating a high degree of nonꢀspecific interaction and/or slower
dissociation kinetics. Given these results, 3 was considered the most appropriate probe for use in a
fluorescent binding assay.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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00 nM 3
100 nM 3
+ 1 ꢀM
Naloxone
1
0 nM 2
10 nM 2
1 ꢀM
Naloxone
+
Figure 4. Determining the suitability of 2 and 3 for a live cell fluorescenceꢀbased competition
binding assay. Confocal images obtained on the IX Ultra of HEKꢀMOR cells incubated with 2 (10
o
nM) and 3 (100 nM) for 5–30min at 37 C. Cells in the bottom three rows of the plate were coincubated
with naloxone (1 µM) for their respective time periods, and those in the five rightꢀhand side columns
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were washed once after incubation. The image is a montage of four images taken per well and is
representative of images taken in three separate experiments.
To confirm that 3 was suitable for use in determining the affinity of unlabeled ligands, MOR
receptorꢀexpressing cells were coꢀincubated with 3 (50 nM) and increasing concentrations of either the
MOR agonists, DAMGO and dermorphin, or the MOR antagonists, diprenorphine, naloxone,
naltrexone and naloxonazine, with xanthine amine congener (XAC), an adenosine receptor antagonist
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included as a negative control. The affinity of each test compound (pK , Table 3) was determined from
i
the IC50 value obtained from the competition curve constructed by measuring the decrease in
fluorescence intensity caused by the compound competing with 3 for the MOR (Figure 5).
Figure 5. Competition binding curves. Competition curves for displacement of 3 (50 nM) by MORꢀ
binding test compounds and XAC, constructed by measuring the decrease in fluorescence intensity
with increasing concentrations of test compound. Each data point represents mean ± SEM of three
experiments each performed in duplicate.
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For the antagonists, excellent agreement was found between the pK values obtained from the
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automated confocal imaging and literature values derived from radioligand binding assays (Table 3).
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In contrast, the pK values obtained for the agonists were significantly lower than the literature
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64–67
values.
However, it is of note that the literature values were obtained from radioligand binding
3
assays using a titrated agonist ([ H]DAMGO). As such, the affinities reported in literature represent the
highꢀaffinity G proteinꢀcoupled state of the receptor. As our fluorescent ligand competition binding
assays were performed in whole cells, where the intracellular concentration of GDP is significantly
higher than GTP, the receptor will exist predominantly in the lowꢀaffinity G protein uncoupled state
state, which is reflected in the lower agonist affinities obtained. As expected, no decrease in
fluorescence intensity was observed in the presence of XAC, meaning that there is no decrease in
binding of the probe over time and its binding is specific to the MOR.
Table 3. MOR affinity (pK ) of test compounds, as determined by ChengꢀPrusoff analysis of data
i
presented in Figure 5 (n = 3). Literature values derived from radioligand binding assays are included
6
4–67
for comparison.
Compound
DAMGO
pK from fluorescent binding assay
Literature pKi
i
§
7.06 ± 0.13
8.9
Dermorphin
Diprenorphine
Naloxonazine
Naloxone
7.71 ± 0.08
9.91 ± 0.08
8.84 ± 0.05
8.94 ± 0.11
9.56 ± 0.04
8.8
9.7
8.5
8.9
9.7
Naltrexone
§
Errors represent ± SEM of three independent experiments performed in duplicate.
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ꢀ
CONCLUSION
In conclusion, we have successfully synthesized four new nonꢀpeptidic fluorescent OR ligands
based on the buprenorphine scaffold. All four compounds acted as antagonists at the MOR, DOR and
KOR, exhibiting highest affinity for the MOR. Although we have not yet explored the NORꢀbinding
properties of the compounds, the fact that buprenorphine has affinity for this fourth OR subtype
suggests that this would be a worthwhile subject for future studies. The ligand linked to the sulfoꢀCy5
fluorophore (3) was found to display the best properties for use in imaging and fluorescent binding
assays, exhibiting rapid and reversible MOR binding, characterized by increased fluorescence in the
bound state. It was successfully used to image the MOR in HEK cells overexpressing this receptor, as
well as to measure the MOR affinities of a series of ligands via a fluorescent competitive binding assay.
We envisage that this compound will prove useful as a research tool for further investigating the
pharmacology of the MOR in in vitro studies, as well as in developing new OR agonists/antagonists
with improved efficacy and sideꢀeffect profiles.
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ꢀ
EXPERIMENTAL SECTION
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Chemistry. All chemicals were purchased from SigmaꢀAldrich Pty, Invitrogen, Matrix Scientific,
Tocris Bioscience or Merck Group Ltd and used without purification, unless otherwise indicated. tertꢀ
Butylꢀ(6ꢀaminohexyl)carbamate, triaminoguanidine hydrochloride, 3,6ꢀbis(3,5ꢀdimethyl1Hꢀpyrazolꢀ1ꢀ
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yl)ꢀ1,2ꢀdihydroꢀsꢀtetrazine,
3,6ꢀbis(3,5ꢀdimethyl1Hꢀpyrazolꢀ1ꢀyl)ꢀsꢀtetrazine,
3,6ꢀdihydrazinylꢀsꢀ
51,54,68
tetrazine, 15 and 16 were synthesized as per previous literature.
All solvents were reagent grade
(chloroform, dichloromethane, ethyl acetate, methanol, acetonitrile). Microwave reactions were
conducted in a Biotage Initiator™, in 0.5–2.0 mL vials unless otherwise indicated, according to the
manufacturer’s instructions. Where indicated, reduction reactions were performed using a Thalesnano
TM
TM
Hydrogenation Reactor (HꢀCube ) continuous flow apparatus system using a 10% PD/C Catcart
cartridge with parameters as indicated. Thin layer chromatography (TLC) was performed on Merck
Silica Gel 60 F254 plates. TLC plates were visualized using a UV lamp at 254 nm or through the use
1
13
19
of KMnO staining agent. H, C and F nuclear magnetic resonance (NMR) spectra were recorded at
4
4
00.13 MHz, 100.61 and 376 MHz, respectively, using an Avance III Nanobay 400 MHz Bruker
spectrometer coupled to the BACS 60 automatic sample changer. Data acquisition and processing was
managed using Topspin software package version 3. Chemical shifts (δ) were measured in parts per
million (ppm) referenced to an internal standard of residual solvent; 3.31 ppm and 49.00 ppm for
deuterated methanol, 7.26 ppm and 77.16 ppm for deuterated chloroform, 2.50 ppm and 39.52 ppm for
deuterated dimethylsulfoxide (d ꢀDMSO), for proton and carbon spectra, respectively. Spectroscopic
6
data are given using the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; br, broad; J, coupling constant; app, apparent pentet. Analytical highꢀperformance liquid
chromatography (HPLC) was carried out on a Waters 2690 Separation Module coupled with a Waters
9
96 Photodiode Array Detector with a Phenomenex Luna 5 micron C8 150 × 4.6 mm column using the
following elution protocol: 0–10 min, gradient from 0% to 80% buffer B/20% buffer A, 10–11 min,
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0% buffer B/20% buffer A, 11–12 min, gradient from 80% buffer B/20% buffer A to 100% buffer B;
2–22 min, 100% B (buffer A = 99.9% H O, 0.1% TFA; buffer B = 80% ACN, 19.9% H O, 0.1%
1
2
2
TFA). Compound 3 was analyzed using an Agilent 1260 Infinity Analytical HPLC using a Zorbax
Eclipse Plus Rapid Resolution 3.5 micron C18 4.6 x 100 mm, column with a 1260 infinity diode array
detector, and the following elution protocol: 0–9 min, gradient from 5% buffer D/ 95% buffer C to
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00% buffer D; 9–10 min, 100% buffer D (buffer C = 99.9% H O, 0.1% TFA; buffer D = 99.9% ACN,
2
0
.1% TFA). The purities of all compounds submitted for biological testing were hence determined to
be ≥ 95%. Preparatory HPLC purification was carried out on an Agilent 1260 Prep HPLC with an
Alltima 5 micron C8 250 x 22 mm column and a 1260 Infinity diode array detector VL, and the
following elution protocol: 0–9 min, gradient from 5% buffer D/ 95% buffer C to 100% buffer D; 9–10
min, 100% D (buffer C = 99.9% H O, 0.1% TFA; buffer D 99.9% ACN, 0.1% TFA). Highꢀresolution
2
mass spectrometric (HRMS) analyses were performed on a Waters LCT TOF LC/MS Mass
Spectrometer coupled to a 2795 Alliance Separations module. All data were acquired and mass
corrected via a dualꢀspray Leucine Enkephaline reference sample. Mass spectra were created by
averaging the scans across each peak and background subtracted of the TIC. Acquisition and analysis
were performed using the MassLynx software version 4.1. The mass spectrometer conditions were as
ꢀ1
follows: electrospray ionization mode, desolvation gas flow of 550 L h , desolvation temperature of
50 °C, source temperature of 110 °C, capillary voltage of 2400 V, sample cone voltage of 60 V, scan
2
range acquired between 100–1500 m/z, one sec scan times and internal reference ions for positive ion
mode (Leucine Enkephaline) of 556.2771. Reverseꢀphase chromatography was performed using
Davasil C18ꢀmodified reverseꢀphase silica using buffers C and D. Liquid chromatographyꢀmass
spectrometry (LCꢀMS) was performed using an Agilent 6100 Series Single Quad LC/MS coupled to an
Agilent 1200 Series HPLC with the following conditions and equipment for MS: 1200 Series G1311A
Quaternary pump, 1200 Series G1329A Thermostatted Autosampler, 1200 Series G1314B Variable
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Wavelength Detector. MS conditions: Quadrupole ion source, multimodeꢀES ion mode, 300 °C drying
gas temperature, 200 °C vaporizing temperature, capillary voltage of 2000 V (positive), capillary
voltage of 4000 V (negative), scan range between 100–1000 m/z with an 0.1 sec step size and a 10 min
acquisition time. LC equipment and conditions were as follows: reverseꢀphase HPLC analysis on a
Luna 5 micron C8(2) 50 x 4.6 mm column using a column temperature of 30 °C, an injection volume
of 5 µL, and the following elution protocol: 0–4 min, gradient from 5% buffer F/95 % buffer E to
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00% buffer F; 4–7 min, 100% buffer F; 4–7 min, gradient from 100% buffer F to 5% F/95 % buffer E
(buffer E = 99.9% H O, 0.1% formic acid; buffer F: 99.9% ACN, 0.1% formic acid). Detection was at
2
2
54 nm. Emission and excitation spectra were obtained using a FlexStation 3. All measurements were
made on 10 µM solutions of compounds in HBSS or DMSO, with the solvent signals subtracted from
the relevant spectra. The following parameters were used. Compound 1: Emission spectra: excitation
at 400 nm, cutoff at 420 nm, scan emission from 430–750 nm at 2 nm steps. Excitation spectra:
emission at 580 nm, cutoff at 570 nm, scan excitation from 300–550 nm at 2 nm steps. Compound 2:
Emission spectra: excitation at 360 nm, cutoff at 420 nm, scan emission from 430–710 nm at 2 nm
steps. Excitation spectra: emission at 700 nm, cutoff at 695 nm, scan excitation from 300–680 nm at 2
nm steps. Compound 3: Emission spectra: excitation at 590 nm, cutoff at 610 nm, scan emission from
6
20–800 nm at 2 nm steps. Excitation spectra: emission at 700 nm, cutoff at 695 nm, scan excitation
from 300–680 nm at 2 nm steps. All measurements were normalized and plotted using GraphPad
Prism 6 (GraphPad Software, San Diego, CA).
(3R,6S,7R,7aR,12bS)-6-((6-(3-(6-Amino-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-
yl)propanamido)hexyl)carbamoyl)-9-hydroxy-7-methoxy-3-methyl-1,2,3,4,5,6,7,7a-octahydro-4a,7-
ethano-4,12-methanobenzofuro[3,2-e]isoquinolin-3-ium 2,2,2-trifluoroacetate (1). Compound 11a
(351 mg, 0.649 mmol) and DIPEA (340 µl, 1.95 mmol) were dissolved in dry DMF (2 mL). 3ꢀ(4ꢀ
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Aminoꢀ1,8ꢀnaphthalimido)propanoic acid (140 mg, 0.492 mmol) and HCTU (247 mg, 0.597 mmol)
were added to this solution, preꢀdissolved in dry DMF (2 mL). The reaction mixture was left to stir
under a nitrogen atmosphere for 2 h. Compound 1 was obtained as a greenꢀyellow solid after reverseꢀ
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phase column chromatography (0–40% buffer D in C) and lyophilization (201 mg, 48%). H NMR
(
MeOD) δHH 8.49–8.45 (m, 2H), 8.25 (d, J = 8.4 Hz, 1H), 7.62 (dd, J = 8.4, 7.4 Hz, 1H), 6.86 (d, J =
8
.4 Hz, 1H), 6.74 (d, J = 8.1 Hz, 1H), 6.65 (d, J = 8.1 Hz, 1H), 4.56 (d, J = 1.9 Hz, 1H), 4.40 (t, J = 7.0
Hz, 2H), 3.70 (d, J = 6.9 Hz, 1H), 3.41 (s, 3H), 3.29–3.28 (m, 1H), 3.17 – 3.06 (m, 5H), 2.99 (s, 3H),
2.90 (dd, J = 19.9, 7.1 Hz, 1H), 2.73–2.69 (m, 1H), 2.60–2.52 (m, 3H), 2.30 (td, J = 13.7, 5.2 Hz, 1H),
1
3
2
1
.07–1.87 (m, 3H), 1.55–1.41 (m, 7H), 1,25–1.23 (m, 4H), 0.84–0.78 (m, 1H); C NMR (MeOD) δ
CH
74.0, 173.5, 166.0, 165.4, 154.4, 147.5, 140.8, 135.5, 132.5, 131.3, 131.1, 130.2, 125.2, 124.3, 123.1,
121.2, 121.0, 119.4, 107.7, 109.4, 92.6, 77.1, 64.8, 52.2, 47.7, 45.4, 44.4, 42.7, 40.3, 40.2, 37.8, 37.1,
19
3
5.8, 33.1, 30.7, 30.1, 30.0, 29.7, 27.3, 27.2, 25.2, 19.0; F NMR (MeOD) δ ꢀ76.8. HRMS (ESIꢀTOF)
+
m/z C H N O [MH] calcd, 735.3632; found, 735.3622.
42 49
5
7
5,5-Difluoro-3-((Z)-4-(2-((6-((6-((3R,6S,7R,7aR,12bS)-9-hydroxy-7-methoxy-3-methyl-
,2,3,4,5,6,7,7a-octahydro-4a,7-ethano-4,12-methanobenzofuro[3,2-e]isoquinolin-3-ium-6-
1
carboxamido)hexyl)amino)-6-oxohexyl)amino)-2-oxoethoxy)styryl)-7-(thiophen-2-yl)-5H-
dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide 2,2,2-trifluoroacetate (2). Compound 11b
(
13.8 mg, 19.8 µmol), DIPEA (10 µl, 57.4 µmol) and 6ꢀ(((4,4ꢀdifluoroꢀ5ꢀ(2ꢀthienyl)ꢀ4ꢀboraꢀ3a,4aꢀ
diazaꢀsꢀindaceneꢀ3ꢀyl)styryloxy)acetyl)aminohexanoic acid, succinimidyl ester (6.70 mg, 10.1 µmol)
was dissolved in dry DMF (500 µl). The reaction was left to stir under a nitrogen atmosphere for 4 h
with the exclusion of light. The resulting solution was added to MeOH and purified by reverseꢀphase
prep HPLC (60–100% buffer D in C) to give compound 2 as a blue solid after lyophilization (3.60 mg,
1
3
2
2%). H NMR (MeOD) δ 8.12 (dd, J = 3.8, 1.0 Hz, 1H), 7.65–7.60 (m, 3H), 7.55 (d, J = 4.0 Hz,
HH
H), 7.37 (s, 1H), 7.22–7.20 (m, 2H), 7.14 (d, J = 4.4 Hz, 2H), 7.06 (d, J = 8.9 Hz, 2H), 6.86 (d, J = 4.3
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Hz, 1H), 6.74 (d, J = 8.1 Hz, 1H), 6.63 (d, J = 8.1 Hz, 1H), 4.57 (s, 2H), 4.51 (d, J = 1.9 Hz, 1H), 3.62
d, J = 6.9 Hz, 1H), 3.38 (s, 3H), 3.29–3.20 (m, 3H), 3.16–3.02 (m, 4H), 2.98–2.78 (m, 4H), 2.71–2.66
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(
(m, 1H), 2.53–2.40 (m, 1H), 2.23 (td, J = 14.0, 5.2 Hz, 1H), 2.15 (t, J = 7.4 Hz, 2H), 2.07–1.96 (m,
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
19
1
H), 1.96–1.77 (m, 2H), 1.68–1.21 (m, 18H), 0.82–0.74 (m, 1H); F NMR (MeOD) δ ꢀ77.0. HRMS
+
(ESIꢀTOF) m/z C H BF N O S [MH] calcd, 1013.4733; found, 1013.4731.
56 65
2
6
7
2
-((1E,3E,5E)-5-(1-Ethyl-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-1-(6-((6-
(6S,7R,7aR,12bS)-9-hydroxy-7-methoxy-3-methyl-1,2,3,4,5,6,7,7a-octahydro-4a,7-ethano-4,12-
(
methanobenzofuro[3,2-e]isoquinoline-6-carboxamido)hexyl)amino)-6-oxohexyl)-3,3-dimethyl-3H-
indol-1-ium-5-sulfonate, 2,2,2-trifluoroacetate salt (3). Compound 11b (19.3 mg, 27.7 µmol) was
dissolved in dry DMF (200 µl). DIPEA (40 µl, 230 µmol), Cy5 (15 mg, 22.8 µmol) and PyBOP (14.7
mg, 28.3 µmol) were added to this solution, preꢀdissolved in dry DMF (200 µL). The reaction mixture
was left to stir under a nitrogen atmosphere for 1 h with the exclusion of light. The resulting solution
was added to MeOH (1 mL) and purified by reverseꢀphase prep HPLC (4–37% buffer D in C) to give
1
compound 3 as a blue solid after lypholization (5.00 mg, 18%). H NMR (MeOD) δ 8.53–8.16 (m,
HH
2
H), 8.02–7.76 (m, 4H), 7.34 (dd, J = 15.1, 8.5 Hz, 2H), 6.85–6.58 (m, 3H), 6.44–6.30 (m, 2H), 4.58
(
s, 1H), 4.22–4.14 (m, 4H), 3.68 (d, J = 6.9 Hz, 1H), 3.40 (s, 3H), 3.22–3.14 (m, 6H), 3.00 (s, 3H),
2
.97–2.71 (m, 2H), 2.67–2.50 (m, 1H), 2.47–2.28 (m, 1H), 2.18 (t, J = 6.8 Hz, 2H), 1.99–1.64 (m,
19
2
0H), 1.51–1.26 (m, 16H), 0.83–0.74 (m, 1H); F NMR (MeOD) δ ꢀ77.3. HRMS (ESIꢀTOF) m/z
+
C H N O S [MH] calcd, 1108.5134; found, 1108.5113.
60
77
5
11 2
(
3R,6S,7R,7aR,12bS)-9-hydroxy-7-methoxy-6-((6-(4-((6-methoxy-1,2,4,5-tetrazin-3-
yl)oxy)butanamido)hexyl)carbamoyl)-3-methyl-1,2,3,4,5,6,7,7a-octahydro-4a,7-ethano-4,12-
methanobenzofuro[3,2-e]isoquinolin-3-ium 2,2,2-trifluoroacetate (4). Compound 11b (132 mg, 189
µmol) was dissolved in dry DMF (1 mL). DIPEA (150 µl, 861 µmol) and 19 (91.3 mg, 293 µmol)
were added and the reaction mixture left to stir under a nitrogen atmosphere for 1 h. 1:3 (v/v)
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