Preparation of bivalent agonists for targeting the mu opioid and cannabinoid receptors

Article abstract of DOI:10.1016/j.ejmech.2019.05.037

In order to obtain novel pharmacological tools and to investigate a multitargeting analgesic strategy, the CB₁ and CB₂ cannabinoid receptor agonist JWH-018 was conjugated with the opiate analgesic oxycodone or with an enkephalin related tetrapeptide. The opioid and cannabinoid pharmacophores were coupled via spacers of different length and chemical structure. In vitro radioligand binding experiments confirmed that the resulting bivalent compounds bound both to the opioid and to the cannabinoid receptors with moderate to high affinity. The highest affinity bivalent derivatives 11 and 19 exhibited agonist properties in [³⁵S]GTPγS binding assays. These compounds activated MOR and CB (11 mainly CB₂, whereas 19 mainly CB₁) receptor-mediated signaling, as it was revealed by experiments using receptor specific antagonists. In rats both 11 and 19 exhibited antiallodynic effect similar to the parent drugs in 20 μg dose at spinal level. These results support the strategy of multitargeting G-protein coupled receptors to develop lead compounds with antinociceptive properties.

Full text of DOI:10.1016/j.ejmech.2019.05.037

Accepted Manuscript
Preparation of bivalent agonists for targeting the mu opioid and cannabinoid receptors
Szabolcs Dvorácskó, Attila Keresztes, Adriano Mollica, Azzurra Stefanucci, Giorgia
Macedonio, Stefano Pieretti, Ferenc Zádor, Fruzsina Walter, Mária Deli, Gabriella
Kékesi, László Bánki, Gábor Tuboly, Gyöngyi Horváth, Csaba Tömböly
PII:
S0223-5234(19)30447-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.037
EJMECH 11347
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 March 2019
Revised Date: 30 April 2019
Accepted Date: 12 May 2019
Please cite this article as: S. Dvorácskó, A. Keresztes, A. Mollica, A. Stefanucci, G. Macedonio, S.
Pieretti, F. Zádor, F. Walter, Má. Deli, G. Kékesi, Láó. Bánki, Gá. Tuboly, Gyö. Horváth, C. Tömböly,
Preparation of bivalent agonists for targeting the mu opioid and cannabinoid receptors, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.05.037.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Preparation of bivalent agonists for targeting the mu opioid and cannabinoid
receptors
a
a,#
b
b
Szabolcs Dvorácskó, Attila Keresztes, Adriano Mollica, Azzurra Stefanucci, Giorgia
b
c
d
e
e
f
Macedonio, Stefano Pieretti, Ferenc Zádor, Fruzsina Walter, Mária Deli, Gabriella Kékesi,
g
h
f
a,
László Bánki, Gábor Tuboly, Gyöngyi Horváth, Csaba Tömböly *
a
d
Laboratory of Chemical Biology, and Laboratory of Opioid Research, Institute of
e
Biochemistry, and Biological Barriers Research Group, Institute of Biophysics, Biological
Research Centre of the Hungarian Academy of Sciences, Temesvári krt. 62., 6726 Szeged,
b
Hungary; Dipartimento di Farmacia, Università di Chieti-Pescara “G. d’Annunzio”, Via dei
c
Vestini 31, 66100 Chieti, Italy; Istituto Superiore di Sanità, Centro Nazionale Ricerca e
Valutazione Preclinica e Clinica dei Farmaci, Viale Regina Elena 299, 00161 Rome, Italy;
f
Department of Physiology, Faculty of Medicine, University of Szeged, 6720 Szeged, Dóm tér
g
h
1
0., Hungary; Department of Traumatology and Department of Neurology, Faculty of
Medicine, University of Szeged, 6725 Szeged, Semmelweis u. 6., Hungary.
*
Corresponding author:
Csaba Tömböly, PhD
Laboratory of Chemical Biology
Biological Research Centre of the Hungarian Academy of Sciences
Temesvári krt. 62., H-6726 Szeged, Hungary
E-mail: tomboly@brc.hu
#
Present Address:
The University of Arizona, College of Medicine, Department of Pharmacology, P.O. Box
2
45050, 1501 N. Campbell Ave., Tucson, AZ 85724-5050
1

ACCEPTED MANUSCRIPT
Abstract
In order to obtain novel pharmacological tools and to investigate a multitargeting analgesic
strategy, the CB and CB cannabinoid receptor agonist JWH-018 was conjugated with the opiate
1
2
analgesic oxycodone or with an enkephalin related tetrapeptide. The opioid and cannabinoid
pharmacophores were coupled via spacers of different length and chemical structure. In vitro
radioligand binding experiments confirmed that the resulting bivalent compounds bound both to
the opioid and to the cannabinoid receptors with moderate to high affinity. The highest affinity
3
5
bivalent derivatives 11 and 19 exhibited agonist properties in [ S]GTPγS binding assays. These
compounds activated MOR and CB (11 mainly CB , whereas 19 mainly CB ) receptor-mediated
2
1
signaling, as it was revealed by experiments using receptor specific antagonists. In rats both 11
and 19 exhibited antiallodynic effect similar to the parent drugs in 20 µg dose at spinal level.
These results support the strategy of multitargeting G-protein coupled receptors to develop lead
compounds with antinociceptive properties.
Graphical abstract
2

ACCEPTED MANUSCRIPT
Highlights:
2
Mu opioid (oxycodone or Tyr-D-Ala-Gly-Phe-NH ) and cannabinoid (JWH-018) receptor
-
agonists were covalently coupled via short spacers.
3
-
JWH-018 was labeled with tritium and [ H]JWH-018 was validated as a novel radioligand for
cannabinoid receptors.
-
In vitro studies revealed that the bivalent compounds 11 and 19 could bind both to the mu
opioid and to the cannabinoid receptors.
-
The bivalent compounds 11 and 19 were found to be agonists both for the mu opioid and for
3
5
the cannabinoid receptors in [ S]GTPγS binding experiments.
-
At spinal level the bivalent compounds 11 and 19 were equieffective with the parent drugs at
0 µg dose in a chronic osteoarthritis pain model in rats.
2
Keywords: cannabinoid receptor agonist, mu opioid receptor agonist, multi-targeting, bivalent
ligand, radioligand
3

ACCEPTED MANUSCRIPT
Abbreviations
ACN, acetonitrile; AM 251, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazole-3-carboxamide; AM 630, 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-
indol-3-yl](4-methoxyphenyl)methanone; BBB, blood-brain barrier; Boc, tert-butyloxycarbonyl;
BSA, bovine serum albumin; CB, cannabinoid; DAMGO, H-Tyr-
DCM, dichloromethane; DIC, N,N’-diisopropylcarbodiimide; DIEA, diisopropylethylamine;
DMF, dimethylformamide; DOR, delta opioid receptor; EDC, 1-ethyl-3-(3-
D-Ala-Gly-N-MePhe-Gly-ol;
dimethylaminopropyl)carbodiimide; EtOAc, ethylacetate; EtOH, ethanol; GPCR, G-protein-
coupled receptor; GTPγS, guanosine 5'-O-(3-thiotriphosphate); HOBt, 1H-benzotriazol-1-ol;
HPLC,
high-performance
liquid
chromatography;
HS-665,
3-(2-
((cyclobutylmethyl)(phenethyl)amino)ethyl)phenol; i.t., intrathecal; JWH-018 (or AM 678),
naphthalen-1-yl(1-pentyl-1H-indol-3-yl)methanone; k’, retention factor (HPLC); KOR, kappa
opioid receptor; MOR, mu opioid receptor; MsCl, methanesulfonyl chloride; NMM, 4-
methylmorpholine; NMR, nuclear magnetic resonance (spectroscopy); R
f
, retention factor
(TLC); RVD-hemopressin, H-Arg-Val-Asp-Pro-Val-Asn-Phe-Lys-Leu-Leu-Ser-His-OH; SEM,
9
9
standard error of mean; TEA, triethylamine; TFA, trifluoroacetic acid; ∆ -THC, (–)-trans-∆ -
tetrahydrocannabinol; THF, tetrahydrofuran; TLC, thin layer chromatography; WIN-55,212-2,
(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-
1
-naphthalenylmethanone mesylate.
4

ACCEPTED MANUSCRIPT
Introduction
Mu opioid receptor (MOR) agonists are the most common therapeutics in clinic to alleviate
severe pain. However, their dose-limiting adverse effects inspire the development of novel
1
analgesics. Cannabinoid (CB) receptor agonists can modulate hyperalgesia and show effective
2
therapeutic value against inflammatory and chronic pain including neuropathic pain. The co-
administration of MOR and CB receptor agonists has been shown to enhance the antinociceptive
effect with decreased opiate-related side-effects, and the synergism of opioid and cannabinoid
3
4–11
12–15
16–19
ligands has been extensively studied in mice,
in rats,
in rhesus monkeys
and in an
2
0
experimental pain model applied to volunteers.
2
1–23
Initiated by the possible dimerization interaction of the opioid and cannabinoid receptors
bivalent compounds, i.e. spacer linked pharmacophores, were also considered to decrease the
opioid side-effects. Conjugating the MOR agonist fentanyl to the CB antagonist/inverse agonist
1
2
4
rimonabant resulted in MOR-CB antagonists. Coupling of an enkephalin-related peptide to
2
5
rimonabant led to the loss of analgesic effects in hot plate and tail flick tests. In contrast,
bivalent compounds of the MOR agonist α-oxymorphamine and a rimonabant analogue with
oxydiacetic acid-based spacers were found to exhibit antinociception in tail flick test without
2
6
producing tolerance in 24 h. Another important goal of the combination treatments is to
decrease the effective dose of opioids, especially in the treatment of severe chronic pains. It
3
,27–30
could be potentially achieved by combining opioid agonists with cannabinoid agonists.
In a
case study of a patient with familial Mediterranean fever it was reported that the administration
9
9
of ∆ -tetrahydrocannabinol (∆ -THC) reduced the morphine consumption by about 50% to
2
7
alleviate chronic pain.
In order to target the MOR and CB receptors with a single compound, bivalent ligands
consisting of a MOR and a CB agonist were designed. In one set the MOR agonist
1
,31–33
34
oxycodone,
that is widely used in the treatment of severe pain was applied. The other set
3
5–37
contained the enkephalin-related tetrapeptide Tyr-
D
-Ala-Gly-Phe
as the opioid
pharmacophore. Both opioid agonists were coupled with naphthalen-1-yl(1-pentyl-1H-indol-3-
yl)methanone (JWH-018 or AM 678), a full CB agonist. JWH-018 is an indole-type synthetic
CB receptor agonist that structurally relates to WIN-55,212-2. It exhibits typical cannabinoid
pharmacology in vivo and has high affinity for both CB receptors (K
i
(CB
1 i 2
)= 9.00 nM, K (CB )=
3
8–41
2
.94 nM).
The receptor binding and signaling properties of the resulting bivalent compounds
5

ACCEPTED MANUSCRIPT
were investigated and the in vitro active compounds were tested in vivo after spinal
administration for antinociception in a chronic pain model, which might be clinically relevant.
Materials and Methods
General. The purity of all reagents and solvents were analytical or the highest commercially
available grade. Starting materials, buffer components, GDP, GTPγS were purchased from
Sigma-Aldrich Kft. (Budapest, Hungary), fatty acid free bovine serum albumin (BSA) was from
Serva (Heidelberg, Germany), DAMGO was obtained from Bachem AG (Bubendorf,
5
,6
Switzerland), Ile -deltorphin-2 was prepared in the Laboratory of Chemical Biology (BRC,
Hungary), naloxone was kindly provided by Endo Laboratories (Wilmington, DE, USA), WIN-
3
5
5
5,212-2 was purchased from Tocris Inc. (Bristol, UK), [ S]GTPγS (s.a. >37 TBq/mmol) was
3
purchased from Hartmann Analytic (Braunschweig, Germany). The radioligands [ H]JWH-018
3
3
(
s.a. 1.48 TBq/mmol), [ H]WIN-55,212-2 (s.a. 485 GBq/mmol), [ H]DAMGO (s.a. 1.43
3
5,6
3
TBq/mmol), [ H]Ile -deltorphin-2 (s.a. 725 GBq/mmol) and [ H]HS-665 (s.a. 1.13 TBq/mmol)
were prepared in the Laboratory of Chemical Biology (BRC, Hungary). Tritium labeling was
4
2
carried out in a self-designed vacuum manifold and radioactivity was measured with a Packard
Tri-Carb 2100 TR liquid scintillation analyser using Insta Gel scintillation cocktail of
PerkinElmer. Analytical thin layer chromatography (TLC) was performed on 5×10 cm glass
plates precoated with silica gel 60 F254 (Merck, Darmstadt, Germany), spots were visualized with
UV light. Flash chromatography was carried out on silica gel 60 (Sigma Ltd., St. Louis, MO,
USA) using the indicated solvents. Analytical HPLC separations were performed with a Merck-
Hitachi LaChrom system on an Alltech Altima HP C18 (150 × 4.6 mm, 5 µm) or on a Vydac
2
18TP54 (250 × 4.6 mm, 5 µm) column using the indicated gradients of ACN (0.08% (v/v)
TFA) (eluent B) in H O (0.1%(v/v) TFA) (eluent A) at a flow rate of 1 mL/min, and UV
2
detection at λ= 216 nm was applied. Radio-HPLC was performed on a Phenomenex Luna C18(2)
(150 × 4.6 mm, 5 µm) column using a Jasco HPLC system equipped with a Packard Radiomatic
1
13
5
5
05 TR Flow Scintillation Analyser. H and C NMR spectra were recorded on a Bruker Avance
00 MHz or on a Varian Mercury 300 MHz spectrometer and chemical shifts (δ) are reported in
1
13
ppm after calibration to the solvent signals. The assignments are based on H, C(DEPT),
HSQC, HMBC, GQ-COSY and 2D-TOCSY experiments, and on the reported assignment of
6

ACCEPTED MANUSCRIPT
4
3
JWH-018. Molecular weight of the compounds was determined by ESI-MS analysis on a
Finnigan Mat LCQ spectrometer.
Oxycodone O-carboxymethyloxime (1). Oxycodone (1 g, 3.17 mmol) was dissolved in 250
mL of EtOH then 365 mg of 2-(aminooxy)acetic acid hemihydrochloride (3.32 mmol) and 400
µL of pyridine were added. The solution was stirred at 80 °C for 75 min then the precipitate was
filtered and dried under vacuum. The crude product was purified by HPLC on a Vydac
2
18TP1010 column (250 × 10 mm, 10 µm) using a linear gradient of 10→50% B in A over 25
min at a flow rate of 4 mL/min (λ= 216 nm) to give 1.14 g (93%) of pure 1 as a white solid. Rf
.26 (CHCl –MeOH–NH3(aq) 9:1:0.1); HPLC k’= 4.90 (t = 12.4 min, linear gradient of 5→30%
B in A over 25 min, flow rate: 1 mL/min, λ= 216 nm); H NMR (500 MHz, MeOD) δ 6.88 (d,
H, J= 8.2 Hz, 2-H), 6.79 (d, 1H, J= 8.2 Hz, 1-H), 5.03 (s, 1H, 5-H), 4.54 and 4.53 (2×s, 2×1H,
CH -COOH), 3.85 (s, 3H, OCH ), 3.59 (d, 1H, J= 6.4 Hz, 9-H), 3.47 (d, 1H, J= 19.9 Hz, 10-H),
0
3
R
1
1
2
3
3
2
.19 (dd, 1H, J= 13.0, 4.6 Hz, 16-H), 3.11 (dd, 1H, J= 19.9, 6.4 Hz, 10-H’), 2.93 (s, 3H, NCH3),
.87 (dd, 1H, J= 13.0, 3.9 Hz, 16-H’), 2.72 (ddd, 1H, J= 17.3, 7.0, 2.2 Hz, 15-H), 2.62 (m, 2H, 7-
H, 15-H’), 1.75 (m, 1H, 7-H’), 1.71 (dd, 1H, J= 7.0, 2.6 Hz, 8-H), 1.46 (ddd, 1H, J= 14.1, 11.5,
1
3
7
.0 Hz, 8-H’); C NMR (126 MHz, MeOD) δ 175.6 (COOH), 156.7 (C-6), 146.7 (C-4), 144.8
C-3), 130.1 (C-12), 124.2 (C-11), 121.1 (C-1), 117.7 (C-2), 87.5 (C-5), 72.9 (O-CH -COOH),
1.2 (C-14), 68.3 (C-9), 57.7 (OCH ), 48.3 (C-16), 47.2 (C-13), 41.7 (NCH ), 30.0 (C-7), 28.9
(
2
7
3
3
+
(
24 2 6
C-8), 24.7 (C-10), 18.6 (C-15); ESI-MS calcd for C20H N O 388.16, found 388.59 [M+H] .
Oxycodone O-(N-(2-(N-Boc-amino)ethyl)carboxamidomethyl)oxime (2). Oxime 1 (20 mg,
.
5
1.5 µmol) and HOBt H O (7.9 mg, 51.5 µmol) were dissolved in 1.5 mL of DMF and DIC (8
2
µL, 51.5 µmol) was added. It was stirred for 5 min, then tert-butyl 2-aminoethylcarbamate
hydrochloride (20 mg, 102 µmol) and DIEA (18 µL, 102 µmol) were added to the solution. The
mixture was stirred at 50 °C for 16 h then it was evaporated in vacuo. The crude product was
purified by column chromatography on silica gel 60 with CHCl –MeOH (8:2) to give 22.2 mg
3
(
81%) of 2 as yellowish oil. R 0.45 (CHCl –MeOH 9:1); HPLC k’= 4.65 (t = 11.9 min, linear
f
3
R
1
gradient of 10→60% B in A over 25 min); H NMR (500 MHz, CDCl
) δ 6.83 (brs, 1H, 2-H),
3
6
5
.74 (d, 1H, J= 8.2 Hz, 1-H), 6.58 (brs, CONH), 5.88 (brs, 1H, CONH), 5.07 and 5.01 (2×s, 1H,
-H), [4.68 and 4.58 (2×d, J= 16.9 Hz), 4.53 (d, J= 16.0 Hz)] (2H, O-CH -CO), 3.90 (s, 3H,
2
OCH ), 3.71 (m, 1H, 9-H), 3.28 (overlapping m, 6H, 1”-H, 2”-H, 10-H, 16-H), 3.08 (d, 1H, J=
3
1
3
8.5 Hz, 10-H’), 2.90 (brs, 4H, NCH , 15-H), 2.77 (m, 2H, 7-H, 16-H’), 2.33 (m, 1H, 15-H’),
7

ACCEPTED MANUSCRIPT
1
3 3
.80 (m, 2H, 7-H’, 8-H), 1.48 (m, 1H, 8-H’), 1.40 (s, 9H, C(CH ) ); ESI-MS calcd for
+
C H N O 530.27, found 531.30 [M+H] .
27
38
4
7
Oxycodone O-(N-(6-(N-Boc-amino)hexyl)carboxamidomethyl)oxime (3). Prepared as
described for 2 but tert-butyl 6-aminohexylcarbamate (22 mg, 102 µmol) was used. The crude
product was purified by column chromatography on silica gel 60 with CHCl –MeOH (8:2) to
3
give 23.1 mg (77%) of 3 as pale yellow oil. R
f
0.44 (CHCl
3
1
–MeOH 9:1); HPLC k’= 6.76 (t
R
=
1
6.3 min, linear gradient of 10→60% B in A over 25 min); H NMR (500 MHz, CDCl ) δ 6.83
3
(d, 1H, J= 8.2 Hz, 2-H), 6.74 (d, 1H, J= 8.2 Hz, 1-H), 6.09 (t, 1H, J= 5.1 Hz, CONH), 5.05 (s,
1
9
H, 5-H), [4.60 and 4.52 (2×d, 2×1H, J= 15.7 Hz, O-CH
2 3
-CO), 3.90 (s, 3H, OCH ), 3.85 (m, 1H,
-H), 3.27 (overlapping m, 4H, 1”-H, 6”-H), 3.08 (m, 3H, 10-H, 10-H’, 16-H), 2.91 (s, 3H,
NCH ), 2.82 (m, 2H, 7-H, 16-H’), 2.71 (brs, 1H, 15-H), 2.33 (m, 1H, 15-H’), 1.85 (brs, 1H, 8-
3
3 3
H), 1.77 (d, 1H, J= 9.7 Hz, 7-H’), 1.47 (m, 5H, 2”-H, 5”-H, 8-H’), 1.43 (s, 9H, C(CH ) ), 1.31
+
(
m, 4H, 3”-H, 4”-H); ESI-MS calcd for C H N O 586.34, found 587.40 [M+H] .
3
1
46
4
7
Oxycodone O-(N-(13-(N-Boc-amino)-4,7,10-trioxatridecyl)carboxamidomethyl)oxime (4).
Prepared as described for 2 but N-Boc-4,7,10-trioxa-1,13-tridecanediamine (33 mg, 102 µmol)
was used. The crude product was purified by column chromatography on silica gel 60 with
3 f 3
CHCl –MeOH (8:2) to give 23.6 mg (66%) of 4 as yellowish oil. R 0.52 (CHCl –MeOH 9:1);
1
HPLC k’= 4.94 (t = 12.5 min, linear gradient of 5→95% B in A over 25 min); H NMR (500
R
3
MHz, CDCl ) δ 6.82 (d, 1H, J= 8.2 Hz, 2-H), 6.73 (d, 1H, J= 8.2 Hz, 1-H), 6.43 (brs, CONH),
5
.06 (s, 1H, 5-H), 5.01 (brs, CONH), 4.58 and 4.51 (2×d, 2×1H, J= 15.8 Hz, O-CH -CO), 3.90
2
(
s, 3H, OCH ), 3.84 (m, 1H, 9-H), (3.61, 3.57, 3.52) (3×m, 12H, 3”-H, 5”-H, 6”-H, 8”-H, 9”-H,
3
1
1
8
1”-H), 3.43-3.19 (overlapping m, 6H, 1”-H, 13”-H, 10-H, 16-H), 3.08 (dd, 1H, J= 19.6, 6.0 Hz,
0-H’) 2.90 (s, 3H, NCH ), 2.81 (m, 3H, 7-H, 15-H, 16-H’), 2.69 (m, 1H, 15-H’), 1.83 (m, 1H,
3
3 3
-H), 1.74 (m, 5H, 7-H’, 2”-H, 12”-H), 1.44 (m, 1H, 8-H’), 1.43 (s, 9H, C(CH ) ); ESI-MS calcd
+
for C H N O 690.38, found 691.15 [M+H] .
3
5
54
4
10
Oxycodone O-(N-(2-aminoethyl)carboxamidomethyl)oxime (5). The N-protected oxime 2
22 mg, 41.5 µmol) was dissolved in 2 mL of DCM containing 50% (v/v) TFA and it was stirred
(
for 30 min at rt. The solution was evaporated in vacuo that yielded the TFA salt of 5. 21 mg
(95%); R
f
0.27 (CHCl
3
–MeOH 9:1); HPLC k’= 3.64 (t
R
= 10.2 min, linear gradient of 5→30% B
+
in A over 25 min); ESI-MS calcd for C H N O 430.22, found 431.30 [M+H] .
2
2
30
4
5
8

ACCEPTED MANUSCRIPT
Oxycodone O-(N-(6-aminohexyl)carboxamidomethyl)oxime (6). Prepared as described for
5
. Yield 22 mg (96%); R 0.26 (CHCl –MeOH 9:1); HPLC k’= 4.90 (t = 12.4 min, linear
f
3
R
gradient of 5→95% B in A over 25 min); ESI-MS calcd for C H N O 486.28, found 487.11
2
6
38
4
5
+
[
M+H] .
Oxycodone O-(N-(13-amino-4,7,10-trioxatridecyl)carboxamidomethyl)oxime (7). Prepared
as described for 5. Yield 22.5 mg (95%); R 0.33 (CHCl –MeOH 9:1); HPLC k’= 4.82 (t = 12.8
min, linear gradient of 5→30% B in A over 25 min); ESI-MS calcd for C H N O 590.33,
f
3
R
3
0
46
4
8
+
found 591.09 [M+H] .
-(1H-indol-1-yl)hexanoic acid (8). To a stirred solution of indole (1.17 g, 10 mmol) in ACN
10 mL) were added triethylamine (1.39 mL, 10 mmol) and 6-bromohexanoic acid (1.94 g, 10
6
(
mmol), then the solution was stirred at 80 °C for 16 h. The solvent was evaporated in vacuo and
the residue was extracted with water and CHCl (3×20 mL). The combined organic phase was
3
washed with brine, and dried over Na SO . After evaporation the crude product was purified by
2
4
column chromatography on silica gel 60 with EtOAc−n-hexane 2:1 to give 1.76 g (77%) of pure
8
as yellow oil. R 0.38 (EtOAc–n-hexane 2:1); HPLC k’= 4.36 (t = 15.0 min, linear gradient of
f
R
1
5
→60% B in A over 25 min); H NMR (500 MHz, CDCl
3
) δ 7.63 (d, 1H, J= 7.9 Hz, 4-H), 7.33
(d, 1H, J= 8.2 Hz, 7-H), 7.20 (t, 1H, J= 7.6 Hz, 5-H), 7.10 (t, 1H, J= 7.6 Hz, 6-H), 7.09 (d, 1H,
J= 3.2 Hz, 2-H), 6.48 (d, 1H, J= 3.1 Hz, 3-H), 4.13 (t, 2H, J= 7.1 Hz, 1’-H), 2.33 (t, 2H, J= 7.4
Hz, 5’-H), 1.87 (quin, 2H, J= 7.3 Hz, 2’-H), 1.67 (quin, 2H, J= 7.5 Hz, 4’-H), 1.38 (quin, 2H, J=
1
3
7
.7 Hz, 3’-H); C NMR (126 MHz, CDCl ) δ 178.2 (COOH), 136.0 (C-7a), 128.7 (C-3a), 127.9
3
(C-2), 121.5 (C-6), 121.1 (C-4), 119.4 (C-5), 109.4 (C-7), 101.1 (C-3), 46.3 (C-1’), 33.8 (C-5’),
3
0.1 (C-2’), 26.6 (C-3’), 24.4 (C-4’); ESI-MS calcd for C H NO 231.13, found 231.93
1
4
17
2
+
[
M+H] .
6-(3-(1-Naphthoyl)-1H-indol-1-yl)hexanoic acid (9). To a stirred solution of 8 (1.5 g, 6.49
mmol) in 5 mL of dry DCM 6.5 mL of 1M Et AlCl in hexane (6.49 mmol) was added dropwise.
2
It was stirred at 0 °C for 1 h then 1.2 g of 1-naphthoyl chloride (6.49 mmol) dissolved in 3 mL of
DCM was added dropwise. The reaction mixture was stirred at 0 °C for 16 h then it was carefully
poured into a mixture of ice and 0.1 M HCl and it was extracted with DCM. The combined
organic phase was washed with brine and dried over Na
and the crude product was purified by column chromatography on silica gel 60 with (EtOAc−n-
hexane 1:1) to give 1.05 g (42%) of pure 9 as yellow oil that became crystalline in a day. R 0.26
2 4
SO . The organic phase was evaporated
f
9

ACCEPTED MANUSCRIPT
(EtOAc–n-hexane 2:1); HPLC k’= 5.07 (t
R
= 17.0 min, linear gradient of 20→100% B in A over
1
2
5 min); H NMR (500 MHz, CDCl ) δ 8.50 (m, 1H, 4-H), 8.19 (d, 1H, J= 8.3 Hz, 15’-H), 7.98
3
(d, 1H, J= 8.2 Hz, 11’-H), 7.92 (d, 1H, J= 8.1 Hz, 12’-H), 7.67 (d, 1H, J= 7.0 Hz, 9’-H), [7.54 (t,
1
7
7
3
1
H, J= 8.2 Hz) and 7.52 (t, 1H, J= 8.2 Hz)] (10’-H and 13’-H), 7.47 (t, 1H, J= 7.1 Hz, 14’-H),
.41-7.34 (overlapping m, 4H, 2-H, 5-H, 6-H, 7-H), 4.08 (t, 2H, J= 7.3 Hz, 1’-H), 2.26 (t, 2H, J=
.4 Hz, 5’-H), 1.83 (quin, 2H, J= 7.4 Hz, 2’-H), 1.62 (quin, 2H, J= 7.6 Hz, 4’-H), 1.31 (m, 2H,
1
3
’-H); C NMR (126 MHz, CDCl ) δ 192.5 (3-CO), 181.6 (COOH), 138.9 (C-8’), 137.6 (C-2),
3
36.9 (C-7a), 133.8 (C-11a’), 130.6 (C-15a’), 130.4 (C-11’), 128.4 (C-12’), 127.0 (C-14’), 126.8
(C-3a), 126.5 (C-13’), 126.0 (C-9’), 125.7 (C-15’), 124.7 (C-10’), 123.3 (C-6), 122.7 (C-5),
1
4
22.2 (C-4), 117.4 (C-3), 110.1 (C-7), 46.4 (C-1’), 37.3 (C-5’), 29.9 (C-2’), 26.3 (C-3’), 25.3 (C-
+
’); ESI-MS calcd for C25
H
23NO
3
385.17, found 386.03 [M+H] .
Bivalent compound 10. The carboxylic acid 9 (7.4 mg, 19 µmol) and HOBt.H O (2.9 mg, 19
2
µmol) were dissolved in 1.5 mL of DMF and DIC (2.9 µL, 19 µmol) was added. It was stirred
for 5 min, then 5 (20.7 mg, 38 µmol) and DIEA (6.6 µL, 38 µmol) were added and the solution
was stirred at 50 °C for 16 h. Then it was evaporated in vacuo and the crude product was purified
by semipreparative HPLC on a Vydac 218TP1010 column that yielded 12.1 mg of 10 (79%) as
yellow oil. R 0.63 (CHCl –MeOH 9:1); HPLC k’= 5.82 (t = 14.3 min, linear gradient of
f
3
R
1
1
1
0→100% B in A over 25 min); H NMR (500 MHz, CDCl ) δ H NMR (CDCl ) δ 8.40 (d, 1H,
3 3
J= 6.8 Hz, 4’-H), 8.15 (d, 1H, J= 8.4 Hz, 15’-H), 7.96 (d, 1H, J= 8.1 Hz, 11’-H), 7.90 (d, 1H, J=
.1 Hz, 12’-H), 7.65 (d, 1H, J= 6.8 Hz, 9’-H), 7.52 (t, 1H, J= 7.6 Hz, 10’-H), 7.50 (t, 1H, J= 7.5
Hz, 13’-H), 7.45 (t, 1H, J= 7.6 Hz, 14’-H), 7.41 (s, 1H, 2’-H), 7.39 (s, 1H, 7’-H), 7.32 (m, 2H,
’-H, 6’-H), 6.81 (d, 1H, J= 8.2 Hz, 2-H), 6.74 (brs, CONH), 6.72 (d, 1H, J= 8.2 Hz, 1-H), 6.43
brs, CONH), 4.99 (s, 1H, 5-H), 4.57 and 4.47 (2×d, 2×1H, J= 16.1 Hz, O-CH -CO), 4.08 (t, 2H,
8
5
(
2
J= 6.9 Hz, 16’-H), 3.85 (s, 3H, OCH
3
), 3.73 (brs, 1H, 9-H), 3.26 (2×brs, 5H, 1”-H, 2”-H, 16-H),
3
.21 (d, 1H, J= 19.0 Hz, 10-H), 3.00 (d, 1H, J= 19.0 Hz, 10-H’), 2.84 (s, 4H, NCH , 15-H), 2.73
3
(brs, 2H, 7-H, 16-H’), 2.40 (d, 1H, J= 8.2 Hz, 15-H’), 2.09 (t, 2H, J= 5.7 Hz, 20’-H), 1.81 (quin,
2
1
1
1
H, J= 7.1 Hz, 17’-H), 1.76 (m, 1H, 8-H), 1.65 (d, 1H, J= 8.0 Hz, 7-H’), 1.56 (quin, 2H, 6.9 Hz,
1
3
9’-H), 1.35 (m, 1H, 8-H’), 1.27 (m, 2H, 18’-H); C NMR (126 MHz, CDCl ) δ 192.3 (Ar-CO),
3
74.3 (20’-CONH), 170.9 (O-CH
2
-CONH), 156.7 (C-6), 145.9 (C-4), 143.8 (C-3), 139.0 (C-8’),
38.2 (C-2’), 137.2 (C-7a’), 133.9 (C-11a’), 130.9 (C-15a’), 130.2 (C-11’), 128.6 (C-12), 128.4
(C-12’), 127.1 (C-3a’), 126.9 (C-14’), 126.5 (C-13’), 126.1 (C-9’), 126.0 (C-15’), 124.8 (C-10’),
10

ACCEPTED MANUSCRIPT
1
1
4
2
23.8 (C-6’), 123.0 (C-5’), 122.9 (C-4’), 121.6 (C-11), 120.0 (C-1), 117.6 (C-3’), 115.8 (C-2),
10.3 (C-7’), 86.9 (C-5), 73.2 (O-CH -CO), 70.4 (C-14), 65.7 (C-9), 56.8 (OCH ), 47.3 (C-16),
2
3
7.1 (C-16’), 46.2 (C-13), 42.1 (NCH ), 39.8 and 39.6 (C-1”, C-2”), 36.1 (C-20’), 29.6 (C-17’),
3
9.3 (C-7), 28.6 (C-8), 26.4 (C-18’), 25.1 (C-19’), 24.1 (C-10), 17.3 (C-15); MALDI-MS calcd
+
for C H N O 797.38, found 798.34 [M+H] .
4
7
51
5
7
Bivalent compound 11. Prepared as described for 10, but 6 (23 mg, 38 µmol) was used. Yield
1.6 mg of 11 (71%) as brown oil. R 0.60 (CHCl –MeOH 9:1); HPLC k’= 6.18 (t = 15.1 min,
1
f
3
1
R
linear gradient of 10→100% B in A over 25 min); H NMR (500 MHz, CDCl ) δ 8.41 (d, 1H, J=
3
7
.5 Hz, 4’-H), 8.14 (d, 1H, J= 8.3 Hz, 15’-H), 7.96 (d, 1H, J= 8.2 Hz, 11’-H), 7.90 (d, 1H, J= 8.1
Hz, 12’-H), 7.63 (d, 1H, J= 6.6 Hz, 9’-H), 7.52 (t, 1H, J= 7.9 Hz, 10’-H), 7.50 (t, 1H, J= 8.2 Hz,
3’-H), 7.44 (t, 1H, J= 7.6 Hz, 14’-H), 7.38 (overlapping d, 1H, 7’-H), 7.37 (s, 1H, 2’-H), 7.33
m, 2H, 5’-H, 6’-H), 6.81 (d, 1H, J= 8.2 Hz, 2-H), 6.72 (d, 1H, J= 8.3 Hz, 1-H), 6.26 (brs, 1H,
”-NH), 6.06 (brs, 6”-NH), 5.00 (s, 1H, 5-H), 4.58 and 4.50 (2×d, 2×1H, J= 15.9 Hz, O-CH2-
CO), 4.07 (t, 2H, J= 6.8 Hz, 16’-H), 3.85 (s, 3H, OCH ), 3.78 (brs, 1H, 9-H), 3.30 (q, 1H, J= 6.3
Hz, 1”-H), 3.24 (brs, 1H, 16-H), 3.23 (d, 1H, J= 19.4 Hz, 10-H), 3.14 (m, 2H, 1”-H’, 6”-H), 3.03
d, 1H, J= 19.3 Hz, 10-H’), 2.91 (d, 1H, J= 18.7 Hz, 6”-H’), 2.84 (s, 3H, NCH ), 2.73 (brs, 2H,
-H, 16-H’), 2.69 (m, 1H, 15-H), 2.60 (m, 1H, 15-H’), 2.11 (t, 2H, J= 6.4 Hz, 20’-H), 1.80 (quin,
H, J= 7.0 Hz, 17’-H, 8-H), 1.68 (d, 1H, J= 8.5 Hz, 7-H’), 1.57 (quin, 2H, 6.3 Hz, 19’-H), [1.42
1
(
1
3
(
3
7
3
(m, 4H) and 1.26 (brs, 4H)] (2”-H, 3”-H, 4”-H, 5”-H), 1.34 (m, 1H, 8-H’), 1.26 (brs, 2H, 18’-H);
1
3
C NMR (126 MHz, CDCl ) δ 192.4 (Ar-CO), 173.8 (20’-CONH), 170.0 (O-CH -CONH),
3
2
1
1
56.8 (C-6), 145.6 (C-4), 143.9 (C-3), 139.0 (C-8’), 138.3 (C-2’), 137.2 (C-7a’), 133.9 (C-11a’),
30.8 (C-15a’), 130.2 (C-11’), 128.4 (C-12’), 128.3 (C-12), 127.1 (C-3a’), 126.9 (C-14’), 126.5
(
C-13’), 126.1 (C-9’), 126.0 (C-15’), 124.8 (C-10’), 123.9 (C-6’), 123.1 (C-5’), 123.0 (C-4’),
21.3 (C-11), 120.0 (C-1), 117.6 (C-3’), 116.0 (C-2), 110.2 (C-7’), 86.4 (C-5), 73.3 (O-CH
CO), 70.4 (C-14), 66.0 (C-9), 56.9 (OCH ), 47.6 (C-16), 47.1 (C-16’), 46.0 (C-13), 42.0 (NCH ),
1
2
-
3
3
3
9.4 (C-6”), 38.7 (C-1”), 36.2 (C-20’), 29.5 (C-17’), (29.2, 28.3, 26.1, 26.0) (C-2”, C-3”, C-4”,
C-5”), 29.0 (C-7), 28.4 (C-8), 26.4 (C-18’), 25.3 (C-19’), 24.0 (C-10), 17.9 (C-15); MALDI-MS
+
calcd for C H N O 853.44, found 854.49 [M+H] .
5
1
59
5
7
Bivalent compound 12. Prepared as described for 10, but 7 (26.8 mg, 38 µmol) was used.
Yield 11.1 mg of 12 (61%) as yellow oil. R 0.70 (CHCl –MeOH 9:1); HPLC k’= 6.06 (t = 14.8
f
3
R
1
min, linear gradient of 10→100% B in A over 25 min); H NMR (500 MHz, CDCl
3
) δ 8.42 (d,
11

ACCEPTED MANUSCRIPT
1
1
H, J= 6.8 Hz, 4’-H), 8.16 (d, 1H, J= 8.3 Hz, 15’-H), 7.97 (d, 1H, J= 8.1 Hz, 11’-H), 7.90 (d,
H, J= 8.1 Hz, 12’-H), 7.65 (d, 1H, J= 6.6 Hz, 9’-H), 7.53 (t, 1H, J= 7.6 Hz, 10’-H), 7.51 (t, 1H,
J= 7.5 Hz, 13’-H), 7.45 (t, 1H, J= 7.3 Hz, 14’-H), 7.40 and 7.39 (2×s, 2×1H, 2’-H, 7’-H), 7.34
m, 2H, 5’-H, 6’-H), 6.80 (d, 1H, J= 8.2 Hz, 2-H), 6.71 (d, 1H, J= 8.3 Hz, 1-H), 6.68 (brs,
CONH), 6.55 (brs, CONH), 5.01 (s, 1H, 5-H), 4.59 and 4.50 (2×d, 2×1H, J= 15.9 Hz, O-CH2-
CO), 4.09 (t, 2H, J= 6.9 Hz, 16’-H), 3.87 (s, 3H, OCH ), 3.77 (brs, 1H, 9-H), 3.58-3.41 (m, 12H,
”-H, 5”-H, 6”-H, 8”-H, 9”-H, 11”-H), 3.27 (brs, 4H, 1”-H, 13”-H), 3.26 (brs, 1H, 16-H), 3.22
d, 1H, J= 19.1 Hz, 10-H), 3.03 (d, 1H, J= 18.6 Hz, 10-H’), 2.86 (s, 3H, NCH ), 2.74 (brs, 2H, 7-
(
3
3
(
3
H, 16-H’), 2.70 (brs, 1H, 15-H), 2.60 (d, 1H, J= 12.0 Hz, 15-H’), 2.12 (t, 2H, J= 6.5 Hz, 20’-H),
.81 (m, 3H, 8-H, 17’-H), 1.70 (m, 5H, 7-H’, 2”-H, 12”-H), 1.59 (quin, 2H, 6.7 Hz, 19’-H), 1.37
1
1
3
(
brs, 1H, 8-H’), 1.27 (m, 2H, 18’-H); C NMR (126 MHz, CDCl
3
) δ 192.3 (Ar-CO), 173.8 (20’-
CONH), 169.9 (O-CH -CONH), 156.7 (C-6), 145.7 (C-4), 143.9 (C-3), 139.0 (C-8’), 138.2 (C-
2
2
’), 137.2 (C-7a’), 133.9 (C-11a’), 130.9 (C-15a’), 130.2 (C-11’), 128.4 (2C, C-12, C-12’), 127.1
(C-3a’), 126.9 (C-14’), 126.5 (C-13’), 126.1 (C-9’), 126.0 (C-15’), 124.8 (C-10’), 123.8 (C-6’),
1
8
23.05 (C-5’), 122.97 (C-4’), 121.3 (C-11), 119.9 (C-1), 117.6 (C-3’), 116.0 (C-2), 110.2 (C-7’),
6.5 (C-5), 73.3 (O-CH -CO), (70.3, 70.1, 70.0, 69.9, 69.4) (7C, C-14, C-3”, C-5”, C-6”, C-8”,
2
C-9”, C-11”), 66.0 (C-9), 57.0 (OCH ), 47.6 (C-16), 47.1 (C-16’), 46.1 (C-13), 42.0 (NCH ),
3
3
3
1
9
8.1 and 37.1 (C-1”, C-13”), 36.1 (C-20’), 30.6 (C-8), 29.6 (C-17’), 29.2 (C-7), 28.9 (C-2”, C-
67 5 10
2”), 26.4 (C-18’), 25.2 (C-19’), 24.1 (C-10), 17.8 (C-15); MALDI-MS calcd for C55H N O
+
57.49, found 958.23 [M+H] .
(1H-Indol-3-yl)(naphthalen-1-yl)methanone (13). Indole (250 mg, 2.13 mmol) was
dissolved in 5 mL of DCM and 1.74 mL of Et AlCl (25% (w/w) in toluene (3.2 mmol) was
2
added at 0 °C. The mixture was stirred at 0 °C for 30 min and 1-naphthoyl chloride (609 mg, 3.2
mmol dissolved in 8 mL of DCM) was added dropwise to the solution at 0 °C, and it was stirred
for 16 h. Then the reaction mixture was quenched with 100 mM NaHCO . The precipitate was
3
filtered and the filtrate was evaporated in vacuo. The crude product was purified by column
chromatography on silica gel 60 (n-hexane–EtOAc 2:1) to give 13 (406 mg, 70%) as yellow
1
solid. R 0.44 (n-hexane–EtOAc 2:1); H NMR (300 MHz, CDCl ) δ 8.73 (brs, 1H, NH indole),
f
3
8
7
1
.50 (d, 1H, J= 6.6 Hz, 4-H), 8.17 (d, 1H, J= 8.1 Hz, 15’-H), 7.96 (d, 1H, J= 8.4 Hz, 11’-H),
.89 (d, 1H, J= 7.5 Hz, 12’-H), 7.66 (d, 1H, J= 6.8 Hz, 9’-H), 7.53-7.36 (m, 7H, 10’-H, 13’-H,
1
3
4’-H, 2-H, 5-H, 6-H, 7-H); C NMR (300 MHz, CDCl ) δ 138.7, 136.5, 134.9, 133.7, 130.7,
3
12

ACCEPTED MANUSCRIPT
1
30.1, 128.1, 126.8, 126.3, 125.9, 125.8, 124.5, 124.1, 123.0, 122.7, 119.2, 111.4; ESI-MS calcd
+
for C H NO 271.10, found 272.24 [M+H] .
1
9
13
tert-Butyl 5-bromopentylcarbamate (14). To a stirred solution of tert-butyl 5-hydroxypentyl-
carbamate (500 mg, 2.46 mmol) and TEA (498 mg, 4.92 mmol) in 5 mL DCM at –10 °C was
added MsCl (338 mg, 2.95 mmol) dropwise and the solution was stirred at the same temperature
for 5 h. The reaction was then quenched with water. The organic layer was washed with water,
brine, dried over MgSO , filtered and evaporated under reduced pressure to give the desired
4
product as yellow oil (552 mg, 80%, R 0.5 (EtOAc)) The mesylate was used in the next step
f
2
without any further purification. Under N atmosphere 5-(tert-butoxycarbonylamino)pentyl
methanesulfonate (350 mg, 1.2 mmol) was dissolved in 5 mL THF followed by the addition of
LiBr (313 mg, 3.6 mmol) to the solution. The reaction mixture was stirred for 16 h under reflux,
then THF was removed under vacuum. The mixture was diluted with 10 mL water and it was
extracted with DCM (3 × 10 mL). The combined organic phase was washed with water (3 × 10
4
mL) and brine (3 × 10 mL), dried over MgSO and evaporated in vacuo. The product was
purified by column chromatography on silica gel 60 (n-hexane–EtOAc 9:1) to give white
1
crystalline product (230 mg, 72%). R
f
0.6 (n-hexane–EtOAc 4:1); H NMR (300 MHz, CDCl
3
) δ
4
7
1
.59 (brs, 1H, NH), 3.37 (t, 2H, J= 8.4 Hz, 5-H), 3.09 (q, 2H, J= 7.8 Hz, 1-H), 1.84 (quin, 2H, J=
1
3
.3 Hz, 4-H), 1.49-1.36 (m, 4H, 2-H, 3-H), 1.40 (s, 9H, CH ); C NMR (300 MHz, CDCl ) δ
3
3
55.9 (CONH), 79.0 (C(CH
3
)
3
), 40.2 (C-1), 33.6 (C-5), 32.2 (C-4), 29.2 (CH
3
), 28.3 (C-2), 25.3
+
(
C-3); ESI-MS calcd for C H BrNO 265.07, found 266.12 [M+H] .
10
20
2
tert-Butyl (5-(3-(1-naphthoyl)-1H-indol-1-yl)pentyl)carbamate (15). To a stirred solution
of NaH (60% dispersion in mineral oil, 15.4 mg, 0.44 mmol) in 5 mL of DMF at 0 °C was added
3 (100 mg, 0.368 mmol) in 10 mL DMF dropwise and the mixture was stirred at 80 °C for 1 h.
1
The reaction mixture was cooled to 0 °C and a solution of 14 (108 mg, 0.41 mmol) in 5 mL
DMF was added dropwise and stirred at 0 °C for 30 min, and then stirred for 18 h at rt. Then it
was evaporated and the oily residue was dissolved in EtOAc (50 mL). The organic layer was
2 4
washed with water (3 × 50 mL) and brine (3 × 50 mL), dried over Na SO and evaporated in
vacuo. The crude residue was purified by column chromatography on silica gel 60 (ethyl acetate
/
hexane 1:2) to yield 15 (142 mg, 85%) as orange-red oil. R
f
0.59 (n-hexane–EtOAc 2:1); HPLC
1
k’= 6.36 (t = 20.6 min, linear gradient of 5→95% B in A over 25 min); H NMR (300 MHz,
R
DMSO-d ) δ 8.29 (d, 1H, J=8.4 Hz, 15’-H), 8.07 (d, 1H, J= 8.1 Hz, 11’-H), 8.00 (t, 1H, 14’-H),
6
13

ACCEPTED MANUSCRIPT
7
.75 (s, 1H, 2’-H), 7.68-7.49 (m, 5H, 9’-H, 7’-H, 13’-H, 10’-H, 12’-H), 7.30 (m, 2H, 5’-H, 6’-
H), 6.72 (t, 1H, NH-Boc), 4.17 (t, 2H, J= 7.3 Hz, 1’-H), 2.81 (q, 2H, 5’-H), 1.68 (quin, 2H, J=
1
3
7
.3 Hz, 2’-H), 1.29-1.15 (m, 13H, 4’-H, 3’-H, 3×CH ); C NMR (300 MHz, DMSO-d ) δ 191.3
3 6
(3-CO), 155.9 (CONH), 139.8 (C-8’), 138.9 (C-2), 137.2 (C-7a), 133.7 (C-11a’), 130.5 (C-15a’),
1
1
30.1 (C-11’), 128.7 (C-12’), 127.1 (C-14’), 126.8 (C-3a), 126.7 (C-13’), 126.2 (C-9’), 125.7 (C-
5’), 125.4 (C-10’), 123.7 (C-6), 122.9 (C-4), 122.1 (C-5), 116.4 (C-3), 111.5 (C-7), 77.7
(
C(CH
3
)
3
3
), 46.6 (C-1’), 29.5 (C-4’), 29.3 (C-2’), 28.6 (CH ), 23.7 (C-3’); ESI-MS calcd for
+
C H N O 456.24, found 457.12 [M+H] .
29
32
2
3
(1-(5-Aminopentyl)-1H-indol-3-yl)(naphthalen-1-yl)methanone (16). The Boc-protected
amine 15 (137 mg, 0.3 mmol) was dissolved in 2 mL DCM containing 50% (v/v) TFA and it was
stirred for 30 min at rt. The solution was evaporated and the product was washed with DCM and
evaporated in vacuo to give 16 (135 mg, 97%); R
f
0.56 (MeOH–AcOH 95:5); HPLC k’= 4.22
(
t = 11.0 min, linear gradient of 10→100% B in A over 25 min); ESI-MS calcd for C H N O
R
24 24
2
+
3
56.19, found 357.08 [M+H] .
N-(5-(3-(1-Naphthoyl)-1H-indol-1-yl)pentyl)acetamide (17). The amine 16 (17mg, 36 µmol)
dissolved in 1 mL of DCM followed by the addition of 0.3 mL TEA and 0.3 mL acetic
anhydride. The mixture was then stirred at rt for 16 h, then it was evaporated in vacuo. The crude
1
7 was purified by column chromatography on silica gel 60 (EtOAc–DCM 9:1) to give 17 (13
mg, 91%); R
f
0.54 (n-hexane–EtOAc 2:1); HPLC k’=4.70 (t
R
= 16.0 min, linear gradient of
1
2
1
0→100% B in A over 25 min); H NMR (300 MHz, DMSO-d
6
) δ 8.31 (d, 1H, 15’-H), 8.06 (d,
H, J= 8.1 Hz, 11’-H), 8.00 (d, 1H, J= 8.2 Hz, 14’-H), 7.76 (s, 1H, 2’-H), 7.74-7.48 (m, 6H, 9’-
H, 7’-H, 13’-H, 10’-H, 12’-H, NH), 7.30 (m, 2H, 5’-H, 6’-H), 4.17 (t, 2H, J= 7.3 Hz, 1’-H), 2.93
(q, 2H, J= 5.7 Hz, 5’-H), 1.71-1.65 (m, 5H, CH
3
and 2’-H), 1.32 (quin, 2H, J= 7.3 Hz, 4’-H),
1
3
1
.17 (quin, 2H, J= 7.4 Hz, 3’-H); C NMR (300 MHz, DMSO-d ) δ 191.3 (3-CO), 169.3
6
(CONH), 139.8 (C-8’), 138.9 (C-2), 137.2 (C-7a), 133.7 (C-11a’), 130.5 (C-15a’), 130.1 (C-11’),
1
1
2
3
28.7 (C-12’), 127.1 (C-14’), 126.8 (C-3a), 126.7 (C-13’), 126.2 (C-9’), 125.7 (C-15’), 125.4 (C-
0’), 123.7 (C-6), 122.9 (C-4), 122.2 (C-5), 116.4 (C-3), 111.5 (C-7), 46.5 (C-1’), 38.6 (C-5’),
3 26 2 2
9.6 (C-4’), 29.0 (C-2’), 23.9 (CH ), 23.01 (C-3’); ESI-MS calcd for C26H N O 398.20, found
+
99.02 [M+H] .
Peptide synthesis, general procedure. To an ice-cooled mixture containing N-protected
amino acid or peptide (0.28 mmol) in DCM (5 mL), EDC.HCl (1.1 equiv., 0.28 mmol), HOBt
14

ACCEPTED MANUSCRIPT
(1.1 equiv., 0.28 mmol), NMM (3.3 equiv., 0.85 mmol), the required protected amino acid (1
equiv., 0.25 mmol) dissolved in DMF (2.5 mL) was added. The reaction mixture was allowed to
warm at rt for 16 h and evaporated under reduced pressure. The residue was then dissolved in
3
EtOAc and washed with three portions of 5% citric acid, NaHCO and brine. The organic phase
was dried over Na SO , and the solvent evaporated under reduced pressure to give the desired
2
4
product. All final Boc-protected intermediates have been purified by flash chromatography on
silica gel 60 and then treated with a mixture of TFA/DCM (1:1) for 30 min at ambient
temperature. The final products as TFA salts were lyophilised and then characterized as follows.
4
4
Tyr-
2
D-Ala-Gly-Phe-NH . It was prepared as described.
Bivalent compound 18. Overall isolated yield 21%; R 0.71 (ACN–MeOH–H O 4:1:1); HPLC
f
2
1
k’= 4.43 (t
R
= 15.2 min, linear gradient of 20→100% B in A over 25 min); H NMR (300 MHz,
-Ala NH), 8.28 (d, 1H, J= 7.2 Hz,
5’-H), 8.20 (t, 1H, Gly NH), 8.08-7.94 (m, 8H, Phe ArH, Tyr NH, Phe NH, 5’-NH), 7.74 (s, 1H,
-H), 7.66-7.44 (m, 5H, 9’-H, 10’-H, 11’-H, 12’-H, 13’-H), 7.33 (quin, 1H, 14’-H), 7.21-7.09
DMSO-d ) δ 9.34 (s, 1H, Tyr OH), 8.57 (d, 1H, J= 6.9 Hz,
D
6
1
2
(m, 4H, 4-H, 5-H, 6-H, 7-H), 7.01 (d, 2H, J= 8.7 Hz, Tyr ArH), 6.68 (d, 2H, J= 8.7 Hz, Tyr
α
α
α
ArH), 4.41 (q, 1H, Phe H ), 4.28 (quin, 1H, D-Ala H ), 4.15 (t, 2H, 1’-H), 3.95 (q, 1H, Tyr H ),
α
β
β
3
.61 (dd, 2H, Gly H ), 2.97-2.67 (m, 6H, Phe H , Tyr H , 5’-H), 1.65 (quin, 2H, 2’-H), 1.25
β
(
50 6 6
quin, 2H, 4’-H) 1.10-1.02 (m, 5H, 3’-H, D-Ala H ); ESI-MS calcd for C47H N O 794.38,
+
found 795.63 [M+H] .
Bivalent compound 19. Overall isolated yield 14%; R 0.73 (ACN–MeOH–H O 4:1:1); HPLC
f
2
1
k’= 4.24 (t
R
= 14.7 min, linear gradient of 20→100% B in A over 25 min); H NMR (300 MHz,
-Ala NH), 8.27 (m, 2H, Gly NH,
5’-H), 8.18 (t, 1H, Gly NH), 8.07-7.96 (m, 6H, 9’-H, 10’-H, 11’-H, 12’-H, 13’-H, Tyr NH, Phe
NH), 7.87 (brs, 1H, Tyr NH), 7.75 (s, 1H, 2-H), 7.65-7.48 (m, 5H, 4-H, 5-H, 6-H, 7-H, 5’-NH),
.29 (m, 1H, 14’-H), 7.19-7.13 (m, 5H, Phe ArH), 6.98 (d, 2H, J= 8.7 Hz, Tyr ArH), 6.66 (d, 2H,
DMSO-d ) δ 9.30 (s, 1H, Tyr OH), 8.49 (d, 1H, J= 6.9 Hz,
D
6
1
7
α
α
J= 8.7 Hz, Tyr ArH), 4.47 (q, 1H, Phe H ), 4.27 (quin, 1H, D-Ala H ), 4.16 (t, 2H, 1’-H), 3.91
α
α
β
β
(
q, 1H, Tyr H ), 3.56 (d, 4H, Gly H ), 2.96-2.70 (m, 6H, 5’-H, Tyr H , Phe H ), 1.68 (quin, 2H,
β
2
’-H), 1.36 (quin, 2H, 4’-H), 1.18 (quin, 2H, 3’-H), 1.01 (d, 3H,
D
-Ala H ); ESI-MS calcd for
+
C H N O 851.40, found 852.63 [M+H] .
49
53
7
7
Bivalent compound 20. Overall isolated yield 25%; R 0.68 (ACN–MeOH–H O 4:1:1); HPLC
f
2
1
k’= 6.59 (t
R
= 15.9 min, linear gradient of 5→95% B in A over 25 min); H NMR (300 MHz,
15

ACCEPTED MANUSCRIPT
DMSO-d
NH), 8.09-7.97 (m, 9H, Phe ArH, Phe NH, β-Ala NH, Tyr NH, 5’-NH), 7.77-7.49 (m, 7H, 2-H,
’-H, 10’-H, 11’-H, 12’-H, 13’-H, 14’-H), 7.34-7.12 (m, 4H, 4-H, 5-H, 6-H, 7-H), 7.01 (d, 2H,
6
) δ 9.33 (s, 1H, OH Tyr), 8.55 (d, 1H, D-Ala NH), 8.28 (d, 1H, 15’-H), 8.20 (t, 1H, Gly
9
α
α
Tyr ArH), 6.68 (d, 2H, Tyr ArH), 4.39 (q, 1H, Phe H ), 4.29 (quin, 1H, D-Ala H ), 4.16 (t, 2H,
α
α
β
β
1
’-H), 3.95 (q, 1H, Tyr H ), 3.58 (d, 2H, Gly H ), 2.93-2.71 (m, 6H, 5’-H, Tyr H , Phe H ), 2.13
α
β
(
t, 2H, βAla H ), 1.67 (quin, 2H, 2’-H), 1.33 (quin, 2H, 4’-H), 1.21-1.15 (m, 4H, β-Ala H , 3’-
β
+
55 7 7
H), 1.05 (d, 3H, D-Ala H ); ESI-MS calcd for C50H N O 865.42, found 866.14 [M+H] .
Bivalent compound 21. Overall isolated yield 12%; R 0.67 (ACN–MeOH–H O 4:1:1); HPLC
f
2
1
k’= 4.27 (t = 14.8 min, linear gradient of 20→100% B in A over 25 min); H NMR (300 MHz,
R
DMSO-d
NH), 8.06-8.00 (m, 8H, Phe ArH, Phe NH, Tyr NH, 5’-NH), 7.75 (s, 1H, 2-H), 7.63-7.52 (m, 6H,
2’-H, 9’-H, 10’-H, 11’-H, 14’-H, 13’-H), 7.30-7.11 (m, 4H, 4-H, 5-H, 6-H, 7-H), 6.96 (d, 2H,
6
) δ 9.32 (s, 1H, Tyr OH), 8.54 (d, 1H, D-Ala NH), 8.29 (d, 1H, 15’-H), 8.20 (t, 1H, Gly
1
α
α
Tyr ArH), 6.69 (d, 2H, Tyr ArH), 4.40 (q, 1H, Phe H ), 4.29 (quin, 1H, D-Ala H ), 4.18 (t, 2H,
α
α
β
β
1
’-H), 3.96 (q, 1H, Tyr H ), 3.64 (d, 2H, Gly H ), 2.94-2.70 (m, 7H, Tyr H , Phe H , Gaba
α
β
NH, 5’-H), 1.93 (t, 2H, Gaba H ), 1.69 (quin, 2H, J= 7.3 Hz, 2’-H), 1.49 (quin, 2H, Gaba H ),
γ
β
1
.33 (quin, 2H, 4’-H), 1.21 (m, 4H, 3’-H, Gaba H ), 1.06 (d, 3H, D-Ala H ); ESI-MS calcd for
+
C H N O 879.43, found 880.23 [M+H] .
51
57
7
7
1-Pentyl-1H-indole (22). To a stirred solution of indole (1.17 g, 10 mmol) in ACN (10 mL)
were added TEA (1.01 g, 10 mmol) and 1-iodopentane (1.98 g, 10 mmol), then the solution was
stirred at 80 °C for 16 h. The solvent was evaporated in vacuo and the residue was extracted with
water and CHCl (3×20 mL). The combined organic phase was washed with brine, and dried
3
over Na SO . After evaporation the crude product was purified by column chromatography (n-
2
4
hexane−EtOAc 95:5) to give 1.40 g (75%) of pure 22 as an oil. R
0.70 (n-hexane−EtOAc 95:5);
f
1
HPLC k’= 4.30 (t = 11.7 min, linear gradient of 50→100% B in A over 25 min); H NMR (500
R
3
MHz, CDCl ) δ 7.63 (d, 1H, J= 7.9 Hz, 4-H), 7.35 (d, 1H, J= 8.1 Hz, 7-H), 7.20 (t, 1H, J= 7.6
Hz, 5-H), 7.10 (d, 1H, J= 3.1 Hz, 2-H), 7.09 (t, 1H, J= 8.0 Hz, 6-H), 6.49 (d, 1H, J= 3.1 Hz, 3-
H), 4.12 (t, 2H, J= 7.2 Hz, 1’-H), 1.85 (quin, 2H, J= 7.2 Hz, 2’-H), 1.33 (m, 4H, 3’-H, 4’-H),
1
3
0
3 3
.89 (t, 3H, J= 7.0 Hz, CH ); C NMR (126 MHz, CDCl ) δ 136.1 (C-7a), 128.7 (C-3a), 127.9
(C-2), 121.4 (C-6), 121.1 (C-4), 119.3 (C-5), 109.5 (C-7), 100.9 (C-3), 46.6 (C-1’), 30.1 (C-2’),
+
2
9.3 (C-3’), 22.5 (C-4’), 14.1 (CH ); ESI-MS calcd for C H N 187.14, found 188.02 [M+H] .
3
13 17
16

ACCEPTED MANUSCRIPT
5-Bromo-1-pentyl-1H-indole (23). 1.96 g of 5-bromo-1H-indole (10 mmol) was dissolved in
2
0 mL of DMF containing 1.6 g of powdered NaOH, then 1-iodopentane (1.98 g, 10 mmol) was
added dropwise. After 4 h stirring at ambient temperature the mixture was filtered and the filtrate
was evaporated in vacuo. The resulting oil was dissolved in CHCl and extracted with water. The
organic phase was washed with brine and dried over Na SO . The crude product was purified by
3
2
4
column chromatography (n-hexane–EtOAc 95:5) to give 1.75 g (66%) of pure 23 as an oil. R
f
0
.62 (n-hexane–EtOAc 95:5); HPLC k’= 6.18 (t = 15.8 min, linear gradient of 50→100% B in A
R
1
1
over 25 min); H NMR (CDCl ) H NMR (500 MHz, CDCl ) δ 7.74 (d, 1H, J= 1.6 Hz, 4-H),
3
3
7
.27 (dd, 1H, J= 8.8 Hz, 1.6 Hz, 7-H), 7.21 (d, 1H, J= 8.8 Hz, 6-H), 7.09 (d, 1H, J= 3.0 Hz, 2-
H), 6.42 (d, 1H, J= 2.9 Hz, 3-H), 4.08 (t, 2H, J= 7.2 Hz, 1’-H), 1.82 (quin, 2H, J= 7.3 Hz, 2’-H),
1
3
1
7
4
2
3 3
.31 (m, 4H, 3’-H, 4’-H), 0.88 (t, 3H, J= 7.1 Hz, CH ); C NMR (126 MHz, CDCl ) δ 134.8 (C-
a), 130.3 (C-3a), 129.1 (C-2), 124.3 (C-6), 123.5 (C-4), 112.6 (C-5), 111.0 (C-7), 100.6 (C-3),
6.7 (C-1’), 30.1 (C-2’), 29.2 (C-3’), 22.4 (C-4’), 14.1 (CH ); ESI-MS calcd for C H BrN
3
13 16
+
65.05, found 266.18 [M+H] .
Naphthalen-1-yl(1-pentyl-1H-indol-3-yl)methanone (24). To a stirred solution of 22 (281
mg, 1.5 mmol) in 10 mL of dry DCM at 0 °C was added dropwise 1.5 mL of 1M Et AlCl in
hexane (1.5 mmol). The solution was stirred at 0 °C for 1 h followed by the dropwise addition of
86 mg of 1-naphthoyl chloride (1.5 mmol) in 3 mL DCM. The reaction mixture was stirred at 0
C for 16 h then the solution was poured carefully into a mixture of ice and 0.1 M HCl and it was
extracted with DCM. The combined organic phase was evaporated and the residue was dissolved
in diethyl ether that was washed with 15% K CO . The organic phase was evaporated and the
crude product was purified by column chromatography (n-hexane−EtOAc 4:1) to give 368 mg
2
2
°
2
3
(
72%) of pure 24 as an oil. R 0.44 (n-hexane−EtOAc 4:1); HPLC k’= 8.08 (t = 19.1 min, linear
f
R
1
gradient of 50→95% B in A over 25 min); H NMR (500 MHz, CDCl
3
) δ 8.49 (m, 1H, 4-H),
8
7
1
.19 (d, 1H, J= 8.4 Hz, 15’-H), 7.97 (d, 1H, J= 8.2 Hz, 11’-H), 7.91 (d, 1H, J= 8.1 Hz, 12’-H),
.66 (d, 1H, J= 6.9 Hz, 9’-H), [7.53 (t, 1H, J= 7.5 Hz) and 7.52 (t, 1H, J= 7.1 Hz)] (10’-H and
3’-H), 7.47 (t, 1H, J= 7.6 Hz, 14’-H), 7.41-7.35 (overlapping m, 4H, 2-H, 5-H, 6-H, 7-H), 4.07
(t, 2H, J= 7.3 Hz, 1’-H), 1.81 (quin, 2H, J= 7.4 Hz, 2’-H), 1.28 (m, 4H, 3’-H, 4’-H), 0.85 (t, 3H,
1
3
3 3
J= 7.0 Hz, CH ); C NMR (126 MHz, CDCl ) δ 192.2 (CO), 139.3 (C-8’), 138.1 (C-2), 137.2
(C-7a), 133.9 (C-11a’), 131.0 (C-15a’), 130.1 (C-11’), 128.3 (C-12’), 127.2 (C-3a), 126.9 (C-
1
4’), 126.4 (C-13’), 126.2 (C-9’), 126.0 (C-15’), 124.7 (C-10’), 123.7 (C-6), 123.1 (C-5), 123.0
17

ACCEPTED MANUSCRIPT
3
(C-4), 117.7 (C-3), 110.1 (C-7), 47.3 (C-1’), 29.6 (C-2’), 29.1 (C-3’), 22.3 (C-4’), 14.0 (CH );
+
ESI-MS calcd for C H NO 341.18, found 341.95 [M+H] .
2
4
23
Naphthalen-1-yl(5-bromo-1-pentyl-1H-indol-3-yl)methanone (25). Prepared as described
for 24, but starting from 23 (400 mg, 1.5 mmol). The crude product was purified by column
chromatography (n-hexane–EtOAc 4:1) to give 517 mg (82%) of pure 25 as an oil. R 0.40 (n-
f
R
hexane–EtOAc 4:1); HPLC k’= 7.62 (t = 18.1 min, linear gradient of 50→100% B in A over 25
1
min); H NMR (500 MHz, CDCl ) δ 8.71 (d, 1H, J= 1.6 Hz, 4-H), 8.17 (d, 1H, J= 8.3 Hz, 15’-
3
H), 7.98 (d, 1H, J= 8.2 Hz, 11’-H), 7.92 (d, 1H, J= 8.0 Hz, 12’-H), 7.65 (dd, 1H, J= 6.9 Hz, 0.7
Hz, 9’-H), [7.53 (t, 1H, J= 7.6 Hz) and 7.52 (t, 1H, J= 6.7 Hz)] (10’-H and 13’-H), 7.48 (dt, 1H,
J= 7.7 Hz, 1.2 Hz, 14’-H), 7.45 (dd, 1H, J= 8.7 Hz, 1.8 Hz, 6-H), 7.32 (s, 1H, 2-H), 7.26 (d, 1H,
J= 8.4 Hz, 7-H), 4.04 (t, 2H, J= 7.2 Hz, 1’-H), 1.79 (quin, 2H, J= 7.4 Hz, 2’-H), 1.26 (m, 4H, 3’-
1
3
H, 4’-H), 0.85 (t, 3H, J= 7.1 Hz, CH ); C NMR (126 MHz, CDCl ) δ 191.9 (CO), 138.8 (C-8’),
3
3
1
1
1
1
38.5 (C-2), 135.9 (C-7a), 133.9 (C-11a), 130.9 (C-15a), 130.4 (C-11’), 128.7 (C-3a), 128.4 (C-
2’), 127.0 (C-14’), 126.8 (C-13’), 126.5 (C-9’), 126.0 (2C, C-15’, C-6), 125.8 (C-4), 124.7 (C-
0’), 117.2 (C-3), 116.8 (C-5), 111.5 (C-7), 47.5 (C-1’), 29.6 (C-2’), 29.0 (C-3’), 22.3 (C-4’),
+
4.0 (CH ); ESI-MS calcd for C H BrNO 419.09, found 420.14 [M+H] .
3
24 22
3
[
H]Naphthalen-1-yl(1-pentyl-1H-indol-3-yl)methanone (26). Tritium labeling was
performed with 3.6 mg of 25 (8.5 µmol) dissolved in 0.6 mL of EtOAc in the presence of 3 mg
of Pd/C (10% Pd) catalyst and triethylamine (1.5 µL, 10.7 µmol). The reaction mixture was
degassed prior to tritium reduction by two freeze-thaw cycles, and then it was stirred under 0.25
bar of tritium gas for 4 h at rt. The unreacted tritium gas was then adsorbed onto pyrophoric
uranium and the catalyst was filtered off with a syringe filter. The filtrate was evaporated in
vacuo and the labile tritium was removed by repeated evaporations from EtOH solution. Finally
3
7
.03 GBq of [ H]JWH-018 was isolated as a white solid that was purified by HPLC on a
Phenomenex Luna C18(2) column (k’= 8.08 (t = 19.1 min), linear gradient of 50→95% B in A
R
over 25 min). The specific activity was determined by using an HPLC peak area calibration
curve recorded with 24 and it was found to be 1.48 TBq/mmol. The tritium labeled JWH-018
was dissolved in EtOH (37 MBq/mL) and stored under liquid nitrogen.
Tritium labeling of 11. 2 mL 1.15 mg/mL MeOH solution of 9 (6 µmol) was mixed with 250
µL 3 % (v/v) ICl in MeOH (14.2 µmol) and the solution was stirred at ambient temperature for
6
2 2 5
0 min. Then 50 mg/mL Na S O in water was added until decolorization, and the iodo
18

ACCEPTED MANUSCRIPT
derivative of 9 was purified by semipreparative HPLC on a Phenomenex Luna C18(2) stationary
phase. The resulting 1.6 mg (55%) of iodo-9 was dissolved in 400 µL DMF and 3 mg of
Pd/BaSO (10% Pd) catalyst and triethylamine (1.4 µL, 10 µmol) were added and tritium
4
3
3
labeling was performed as described for [ H]JWH-018 to give 64 MBq of [ H]9 with a specific
3
activity of 64 GBq/mmol. Finally, 37 MBq of [ H]9 and HOBt.H O (0.3 mg, 1.9 µmol) were
2
dissolved in 150 µL of DMF and DIC (0.3 µL, 1.9 µmol) was added. It was stirred for 5 min,
then 6 (2.1 mg, 2.9 µmol) and DIEA (1.4 µL, 8 µmol) were added and the solution was stirred at
rt for 16 h. It was then evaporated in vacuo and the crude product was purified by HPLC on a
3
Phenomenex Luna C18(2) column that yielded 5.5 MBq [ H]11 (15%). S.a. 64 GBq/mmol;
R
HPLC k’= 5.48 (t = 13.6 min, linear gradient of 20→100% B in A over 25 min).
Tritium labeling of 19. To a solution of 19 (970 µL 1 mg/mL MeOH, 1 µmol) 1.8 mg of
IPy BF (4.8 µmol) and 4.4 µL of HBF in Et O were added and the reaction mixture was stirred
2
4
4
2
for 1 h at rt under nitrogen. The reaction was quenched with a solution of Na S O in water and
2
2
5
the iodo derivative of 19 was purified by HPLC on a Phenomenex Luna C18(2) stationary phase
yielding 0.8 mg (60%) of diiodo-19. It was dissolved in 400 µL DMF and 2.5 mg of Pd/BaSO
10% Pd) catalyst and triethylamine (0.8 µL, 5.6 µmol) were added and tritium labeling was
4
(
3
3
performed as described for [ H]JWH-018 to give 80 MBq of [ H]19 with a specific activity of
1
85 GBq/mmol. HPLC k’= 6.78 (t = 16.3 min, linear gradient of 5→95% B in A over 25 min).
R
Preparation of brain membrane homogenates. Wistar rats and guinea pigs were locally bred
and handled according to the EU Directive 2010/63/EU and to the Regulations on Animal
Protection (40/2013. (II. 14.) Korm) of Hungary. Crude membrane fractions were prepared from
the brain without cerebellum. Brains were quickly removed from the euthanized animals and
directly put in ice-cold 50 mM Tris-HCl (pH 7.4) buffer. The collected tissue was then
homogenized in 30 volumes (v/w) of ice-cold buffer with a Braun Teflon-glass homogenizer at
the highest rpm. The homogenate was centrifuged at 20 000 × g for 25 min and the resulting
pellet was suspended in the same volume of cold buffer followed by incubation at 37 °C for 30
min to remove endogenous ligands. After centrifugation the pellets were taken up in five
volumes of 50 mM Tris-HCl (pH 7.4) buffer containing 0.32 M sucrose and stored in aliquots at
–
80 °C. Prior to the experiment, aliquots were thawed and centrifuged at 20 000 × g for 25 min
and the pellets were resuspended in 50 mM Tris-HCl (pH 7.4), homogenized with a Dounce
followed by the determination of the protein content by the method of Bradford. The membrane
19

ACCEPTED MANUSCRIPT
3
5
suspensions were immediately used either in radioligand binding experiments or in [ S]GTPγS
functional assays.
3
Radioligand binding assays. Binding experiments of [ H]JWH-018 were performed at 30 °C
for 60 min in 50 mM Tris-HCl binding buffer (pH 7.4) containing 2.5 mM EGTA, 5 mM MgCl
2
and 0.5 mg/mL fatty acid free BSA in plastic tubes in a total assay volume of 1 mL that
3
contained 0.3–0.5 mg/mL membrane protein. Association time course of [ H]JWH-018 binding
3
was obtained by incubating 0.6 nM [ H]JWH-018 with rat brain membrane (0.45 mg/mL
protein) at 30 °C for various periods of time (0–90 min) in the absence or presence of 10 µM
3
JWH-018 to assess specific binding. Dissociation time course of [ H]JWH-018 was obtained by
3
incubating 0.6 nM [ H]JWH-018 with rat brain membrane (0.45 mg/mL protein) at 30 °C for 60
min, then dissociation was initiated by the addition of 10 µM JWH-018 after different periods of
3
incubation time. The kinetic equilibrium dissociation constant K for [ H]JWH-018 in rat brain
d
membrane homogenate was calculated as K = k /k , where k is the dissociation rate constant, k
d
d
a
d
a
3
a d
is the association rate constant calculated as k = (kobs-k )/[[ H]JWH-018], kobs is the observed
pseudo-first order rate constant. Saturation binding experiments were performed by measuring
3
the specific binding of [ H]JWH-018 (0.5−35 nM) to rat brain membranes to determine the
equilibrium dissociation constant (K ) and the maximal number of binding sites (Bmax). The
d
specific binding was measured in the presence of 10 µM JWH-018.
Competition binding experiments were carried out by incubating brain membranes with opioid
or cannabinoid receptor specific tritiated radioligands in the presence of increasing
–
11
–5
concentrations (10 –10 M) of various competing unlabeled ligands. MOR competition
3
experiments were performed at 25 °C for 60 min with 2 nM [ H]DAMGO (K = 0.5 nM), DOR
d
3
5,6
competition experiments were performed at 35 °C for 45 min with 3 nM [ H]Ile -deltorphin-2
(
[
K
d
= 2.0 nM) and KOR competition experiments were performed at 25°C for 30 min with 1 nM
3
H]HS-665 (K = 0.64 nM) in 50 mM Tris-HCl binding buffer (pH 7.4) using rat brain (MOR,
d
DOR) or guinea pig brain membrane homogenate (KOR). Non-specific binding was determined
in the presence of 10 µM naloxone (MOR, DOR) or HS-665 (KOR). CB receptor binding
experiments were performed at 30°C for 60 min on rat brain membrane homogenates with 0.6
3
3
d d
nM [ H]JWH-018 (K = 6.5 nM) or with 1.5 nM [ H]WIN-55,212-2 (K = 10.1 nM). Non-specific
binding was determined in the presence of 10 µM JWH-018 or WIN-55,212-2. The competition
experiments were terminated by diluting the suspensions with ice-cold wash buffer (50 mM Tris-
20

ACCEPTED MANUSCRIPT
2
HCl, 2.5 mM EGTA, 5 mM MgCl , 0.5% fatty acid free BSA, pH 7.4 for cannabinoid binding,
or 50 mM Tris-HCl, pH 7.4 for opioid binding) followed by rapid washing and rapid filtration
through Whatman GF/B or GF/C (MOR, KOR) glass fiber filters (Whatman Ltd, Maidstone,
England) presoaked with 0.1% polyethyleneimine (only for CB receptor binding). Filtration was
performed with a 24-well Brandel Cell Harvester (Gaithersburg, MD, USA). Filters were air-
dried and immersed into Ultima Gold MV scintillation cocktail and then radioactivity was
measured with a TRI-CARB 2100TR liquid scintillation analyser (Packard).
3
5
Ligand stimulated [ S]GTPγS binding assay. Rat brain membranes (30 µg protein/tube)
3
5
–10
–5
were incubated with 0.05 nM [ S]GTPγS (PerkinElmer) and 10 –10 M unlabeled ligands in
the presence of 30 µM GDP, 100 mM NaCl, 3 mM MgCl and 1 mM EGTA in 50 mM Tris-HCl
2
35
buffer (pH 7.4) for 60 min at 30 °C. Basal [ S]GTPγS binding was measured in the absence of
ligands and set as 100%. Nonspecific binding was determined by the addition of 10 µM
unlabeled GTPγS and subtracted from total binding. Incubation, filtration and radioactivity
measurement of the samples were carried out as described above.
Cell culture and permeability assay. Primary rat brain endothelial cells, pericytes and
astroglia cells were isolated and cultured according to the method described in our previous
45,46
studies.
To induce BBB characteristics the isolated cells were co-cultured with the help of
1
2-well tissue culture inserts (Transwell, polycarbonate membrane, 3 µm pore size, Corning
Costar, USA). After two days of co-culture brain endothelial cells became confluent and 550 nM
hydrocortisone (Sigma) was added to the culture medium and one day before the experiment
cells were treated with CPT-cAMP (250 mM, Sigma) and RO 201724 (17.5 mM; Sigma) for 24
4
7
h to tighten junctions and elevate transendothelial resistance. Permeability tests on the co-
cultured BBB model were performed when transendothelial electrical resistance values
2
expressed to the surface area of the inserts reached 123.8 ± 12.9 Ωcm , n= 16. The resistance of
cell-free inserts was subtracted from the measured data. During the permeability assay the
culture medium was changed with the same as used in the growth period, but it also contained
3
3
1
0% serum. Compounds [ H]11 and [ H]19 were applied in the upper compartment in a final
concentration of 0.25 and 0.75 µM. Compound permeability was measured from the AB (from
blood to brain) direction. After 15, 30 and 60 min samples were collected both from the upper
3
3
and lower compartments and the transport of [ H]11 and [ H]19 was determined by measuring
the radioactivity using a TRI-CARB 2100TR liquid scintillation analyser (Packard). Flux of the
21

ACCEPTED MANUSCRIPT
compounds across coated, cell-free inserts was also measured. Endothelial permeability
3
3
coefficients (P ) were calculated from clearance values of [ H]11 and [ H]19 as described
e
4
7
previously.
Hot plate test. Thermal nociception in the hot plate test was assessed with a commercially
available apparatus consisting of a metal plate 25 × 25 cm (Ugo Basile, Italy) heated to a
constant temperature of 55.0 ± 0.1 °C, on which a plastic cylinder (20 cm diameter, 18 cm high)
was placed. The time of latency (s) was recorded from the moment the animal was placed in the
cylinder on the hot plate until it licked its paws or jumped; the cut-off time was 60 s. The
baseline was calculated as the mean of three readings recorded before testing at intervals of 15
min. The time course of latency was then determined at 15, 30, 45, 60, 90 and 120 min after
compound treatment. Data were analyzed as time-course curves of the percentage of maximum
effect (%MPE= (post drug latency – baseline latency) / (cut-off time – baseline latency) × 100).
CD-1 male mice (Harlan, Italy) weighing 25 g were used for the hot plate test. The research
protocol was approved by the Service for Biotechnology and Animal Welfare of the Istituto
Superiore di Sanità and authorized by the Italian Ministry of Health, according to Legislative
Decree 26/14, which implemented the EU Directive 2010/63/EU on the protection of laboratory
animals in Italy. Bivalent compounds 11 and 19 were injected intravenously (i.v.) at the dose of
1
0 mg/kg in a volume of 10 mL/kg. The control animals were injected i.v. with the vehicle of the
compounds (physiological saline containing 5% DMSO, 10 mL/kg).
Nociceptive test in rats at spinal level. The procedures involved in the animal surgery and
testing were approved by the Institutional Animal Care Committee of the University of Szeged,
Faculty of Medicine. Surgical procedures including intrathecal (i.t.) catheterization and the
monosodium iodoacetate (MIA)-induced inflammation are described in Supporting Information.
Mechanical allodynia (von Frey test) was determined using a dynamic plantar aesthesiometer
(Ugobasile, Comerio, Italy). Prior to baseline testing, each rat was habituated to a testing box
with a wiremesh grid floor for 20 min. Straight metal filament was used for the measurements
that exerts an increasing upward force at a constant rate (6.25 g/s) with a maximum cut-off force
of 50 g. The filament was placed under the plantar surface of the hind paw. Measurement was
stopped when the paw was withdrawn, and the results were expressed as paw withdrawal
thresholds in grams. The pain thresholds were registered before the i.t. drug injections (baseline
at 0 min) and then in every 15 min for 90 min. The parent drugs (oxycodone (20 µg), JWH-018
22

ACCEPTED MANUSCRIPT
2
(20 µg), Tyr-D-Ala-Gly-Phe-NH (7 µg)) and the bivalent compounds 11 (20 µg) and 19 (20 µg)
were injected over 120 s in a volume of 10 µL, followed by 8 µL flush of physiological saline
within 60 s. The control animals were injected with the vehicle of the compounds (physiological
saline containing 5% DMSO). The i.t. drug effect was analysed on the MIA-injected hind paws,
since none of the treatments influenced the pain threshold at the contralateral side.
Paw withdrawal thresholds on the inflamed side were transformed to % maximum possible
effect (%MPE) by the following formula: %MPE= [(observed threshold – baseline threshold) /
(
50 – baseline threshold)] × 100. It was calculated for the early (15–45 min) and late phase (60–
0 min) after drug administration. Therefore, 100% MPE means perfect relief of allodynia
equivalent to a cut-off value of 50 g for all measurements), while 0% MPE means that the
9
(
observed threshold is equivalent to the baseline value.
Data analysis. The direct saturation isotherms were determined to obtain the equilibrium
dissociation constant (K ) and the receptor density (Bmax). In competition binding studies, the
d
i
inhibitory constants (K ) were calculated from the inflexion points of the displacement curves
using nonlinear least-square curve fitting option and the Cheng-Prusoff equation as K = EC /(1
i
50
3
5
+
d
[ligand]/K ). In [ S]GTPγS binding studies, data were expressed as the percentage stimulation
3
5
of the specific [ S]GTPγS binding over the basal activity. Each experiment was performed in
triplicate and analyzed with the sigmoid dose-response curve fitting option to obtain potency
(ED50) and efficacy (Emax). Statistical comparison of Emax and EC50 values were performed by
one-way ANOVA followed by the Bonferroni’s multiple comparison test (*** P< 0.001; ** P<
0
.01); Emax values of 11 and 19 in the presence of 10 µM naloxone were compared to the basal
#
activity by unpaired Student’s t-test, P< 0.05).
The time-course data sets of the hot plate test and the von Frey tests were examined by two-
way ANOVA. The significance of differences between the experimental and control groups was
calculated by using the Fisher LSD test for post hoc comparison (P< 0.05 was considered
significant). All data and curves were analyzed by the GraphPad Prism 5.0 Software, San Diego,
CA, USA.
Results and Discussion
The bivalent compounds were prepared in a convergent way. The MOR and CB agonists were
conjugated via short spacers of different length (4 – 15 atoms) and polarity (Scheme 1).
23

ACCEPTED MANUSCRIPT
Oxycodone and JWH-018 were modified at the 6-oxo and at the N-pentyl groups, respectively, to
obtain the key intermediates. In the case of the peptidic compounds the C-terminal carboxyl
function of the peptide acids was used for the conjugation. Condensation of oxycodone with 2-
(aminooxy)acetic acid in EtOH resulted in the linker conjugated O-carboxymethyl ketoxime 1.
4
8,49
Due to the α-effect the ketoximes are stable at physiological pH,
therefore the bivalent
ligands are probably stable against hydrolysis. Then the carboxymethyl group of 1 was activated
as an O-benztriazolyl ester that was used for the N-acylation of the mono- protected diamine
spacers N-Boc-ethylenediamine, N-Boc-1,6-diaminohexane and N-Boc-4,7,10-trioxa-1,13-
tridecanediamine. The final acidolytic removal of the Boc protecting group resulted in the
amines 5–7.
JWH-018 was functionalized by introducing a terminal carboxyl group to the N-pentyl
substituent of the indole ring (Scheme 1). This modification does not affect the aromatic groups
a
Scheme 1. Preparation of oxycodone – JWH-018 bivalent compounds
a
Reagents and conditions: a) EtOH, pyridine, 80 °C, 75 min, 93%; b) HOBt, DIC, DIEA, DMF,
5
0 °C, 16 h, 81% (2), 77% (3), 66% (4); c) TFA/DCM (1:1), rt, 30 min, 95% (5), 96% (6), 95%
7); d) 6-bromohexanoic acid, TEA, ACN, 80 °C, 16 h, 77% (8); e) 1-naphthoyl chloride,
Et AlCl, DCM, 0 °C, 16 h, 42% (9); f) HOBt, DIC, DIEA, DMF, 50 °C, 16 h, 79% (10), 71%
11), 61% (12).
(
2
(
24

ACCEPTED MANUSCRIPT
5
0
of JWH-018 that are responsible for aromatic interactions with the CB receptors. Furthermore,
the introduction of heteroatoms to the alkyl group may be tolerated by CB receptors as in the
1
5
1,52
case of the morpholino group of WIN-55,212-2.
The carboxyl derivative of JWH-018 (9)
5
0
was prepared in a way analogous to that reported by Huffman et al. The N-alkylation of indole
was achieved with 6-bromohexanoic acid, then 8 was selectively acylated at position 3 with 1-
naphthoyl chloride in the presence of Et
2
AlCl. Finally, 9 was activated as an O-benztriazolyl
ester and it was used for the N-acylation of the amines 5–7 resulting in the bivalent compounds
1
0–12.
The peptidic compounds 18–21 were prepared also in a convergent way (Scheme 2). Glycine,
-aminopropanoic acid or 4-aminobutanoic acid were used as spacers between the opioid and
3
cannabinoid pharmacophores. Indole was regioselectively acylated with 1-naphthoyl chloride
and the resulting 3-(α-naphthoyl)-indole (13) was N-alkylated with N-Boc-5-bromopentane-1-
amine (14). Acidolytic deprotection of the carbamate 15 resulted in the JWH-018 derivative 16
with a
a
Scheme 2. Preparation of peptide – JWH-018 bivalent compounds
a
2
Reagents and conditions: a) 1-Naphthoyl chloride, Et AlCl, DCM, 0 °C, 16 h, 70%; b) MsCl,
TEA, DCM, –10 °C, 5 h; c) LiBr, THF, reflux, 16 h, 72% (14); d) NaH, DMF, 80 °C, 18 h, 85%;
e) TFA/DCM (1:1), rt, 30 min, 97%; f) Ac O, TEA, DCM, rt, 16 h, 91% (17), or Boc stepwise
2
peptide synthesis: EDC, HOBt.H O, NMM, DMF, DCM, and deprotection with TFA/DCM (1:1),
2
rt, 30 min; overall yields 21% (18), 14% (19), 25% (20), 12% (21).
25

ACCEPTED MANUSCRIPT
2
terminal amine in the N-pentyl group. The N-acetylation of 16 with Ac O resulted in the control
compound 17. The elongation of 16 with the opioid peptide or with a spacer amino acid followed
by the opioid peptide were achieved in stepwise Boc/tBu solution phase peptide synthesis using
EDC and HOBt as coupling agents.
3
Preparation and validation of [ H]JWH-018. The in vitro characterization of the bivalent
compounds in radioligand displacement studies required appropriate opioid and cannabinoid
radioligands. The most commonly used CB radioligands in heterologous competition binding
3
3
3
3
experiments are [ H]CP-55,940, [ H]HU-243, [ H]WIN-55,212-2, [ H]SR-141716A
3
3
53
(
rimonabant), [ H]SR-144528 and [ H]Sch225336. The structural diversity of the CB receptor
5
4
55
ligands and the presence of allosteric site on the CB receptors prompted us to prepare a novel
radioligand relevant for the investigation of the CB receptor binding affinities of the JWH-018
containing bivalent compounds. JWH-018 was labeled with tritium as outlined in Scheme 3 and
the resulting radioligand was validated in vitro. N-Alkylation of 5-bromoindole with 1-
iodopentane was achieved in the presence of triethylamine followed by acylation with 1-
naphthoyl chloride that resulted in the brominated precursor 25. Then 25 was dehalogenated with
tritium gas
a
Scheme 3. Tritium labeling of JWH-018
a
Reagent and conditions: a) (22) 1-iodopentane, TEA, ACN, 80 °C, 16 h, 75%, (23) 1-
iodopentane, NaOH, DMF, rt, 4 h, 66%; b) Et AlCl, 1-naphthoyl chloride, DCM, 0 °C, 16 h,
2
3
7
2% (24), 82% (25); c) H2(g), Pd/C, EtOAc, TEA, rt, 4 h.
3
under heterogeneous catalytic conditions and [ H]JWH-018 (26) was obtained with a specific
activity of 1.48 TBq/mmol. In a similar way, JWH-018 (24) was also prepared for the
radioligand binding experiments.
3
Before its application in radioligand competition assays, [ H]JWH-018 was characterized in
various in vitro receptor binding experiments. Association and dissociation binding experiments
3
were performed to characterize the interaction of [ H]JWH-018 with membrane receptors using
26

ACCEPTED MANUSCRIPT
5
6–59
rat brain membrane homogenate that contains both CB
1
and CB
2
receptors.
Association
3
binding experiments were carried out in the presence of 0.6 nM [ H]JWH-018 at 30 °C and they
3
revealed specific binding of [ H]JWH-018 to rat brain membranes (Figure 1A). At this
temperature the specific binding determined in the presence of 10 µM 24 reached steady-state
after 40 min, and it remained stable up to 90 min, the longest incubation time investigated (not
shown). The specific binding was found to be 65% of the total binding at 0.6 nM radioligand
concentration under equilibrium conditions. Analyzing the association curve provided an
observed pseudo-first
3
Figure 1. Binding of [ H]JWH-018 to rat whole brain membrane homogenates (0.45 mg/mL
3
protein). (A) Association and (B) dissociation time courses of [ H]JWH-018 at 30 °C; (C)
3
saturation isotherm of specific CB receptor binding of [ H]JWH-018 at 30 °C for 60 min
incubation. Data are means ± SEM (n≥ 3).
–
1
order rate constant (k ) of 0.124 ± 0.01 min . In the dissociation experiments, rat brain
obs
3
membranes were incubated with 0.6 nM of [ H]JWH-018 at 30 °C for 60 min and dissociation of
the ligand–receptor complex was initiated by the addition of 10 µM 24 at different incubation
periods (Figure 1B). It was found that 60% of the radioligand dissociated from the membranes.
Dissociation proceeded with a monophasic kinetics and it resulted in a dissociation rate constant
-
1
(
k ) of 0.105 ± 0.01 min . The equilibrium dissociation constant (K ) calculated from the kinetic
d
d
data was 3.4 nM under our experimental conditions. Saturation binding experiments were then
27

ACCEPTED MANUSCRIPT
Cannabinoid ligands
K (nM)
Cannabinoid ligands
AM 251
K (nM)
i
i
JWH-018 (24)
3.4 ± 0.8
69 ± 9.1
27 ± 2.2
>10000
WIN-55,212-2
7.2 ± 2.8
82 ± 4.5
59 ± 3.3
43 ± 5.5
AM 630
9
∆
-THC
Hemopressin (1-7)
Hemopressin (1-9)
RVD-Hemopressin
2
5
2793 ± 41
Rimonabant
>10000
Figure 2. Characterization of JWH-018 binding sites in competition binding experiments in rat
3
or guinea pig ([ H]HS665) whole brain membrane homogenates. (A-C) The specific binding of
3
[
H]JWH-018 in the presence of unlabeled cannabinoid or opioid ligands. (D) The specific
3
3
5,6
binding of the MOR, DOR and KOR specific radioligands [ H]DAMGO, [ H]Ile -deltorphin-2
and [ H]HS-665, respectively, in the presence of JWH-018 (filled symbols) or in the presence of
3
the corresponding unlabeled opioid ligand (open symbols). Data are mean percentage of specific
3
binding ± SEM (n≥ 3). Table shows the calculated inhibitory constants against [ H]JWH-018. K
i
i d d
values were as K = EC50/(1 + [ligand]/K ), where K = 6.5 nM was obtained from the saturation
experiment, data are means ± SEM, n≥ 3.
performed to determine the K and B
values. The radioligand was incubated with rat brain
max
d
membranes at increasing concentrations (0–35 nM) in the absence or presence of 24. The
3
specific binding of [ H]JWH-018 was found to be saturable and of high affinity in the nanomolar
range (Figure 1C). A single-site binding was calculated from the non-linear fitting of the specific
binding data and resulted in an apparent K
Bmax) of 1120 ± 89 fmol/mg protein.
d
value of 6.5 ± 1.22 nM and a high receptor density
(
28