Bioisosteric replacement of central 1,2,4-oxadiazole ring of high affinity CB2 ligands by regioisomeric 1,3,4-oxadiazole ring

Article abstract of DOI:10.1039/c7md00296c

It has been reported that bioisosteric replacement of an 1,2,4-oxadiazole ring by an 1,3,4-oxadiazole ring leads to higher polarity, reduced metabolic degradation by human liver microsomes and reduced interaction with hERG channels. In a seven to eight st

Full text of DOI:10.1039/c7md00296c

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Bioisosteric replacement of central 1,2,4-oxadiazole ring of high affinity CB2
ligands by regioisomeric 1,3,4-oxadiazole ring
Dominik Heimann,a,§ Corinna Lueg,a,§ Henk de Vries,b Bastian Frehland,a Dirk
Schepmann,a Laura H. Heitmanb and Bernhard Wünscha,c
§ Both authors contributed equally to this work.
a
Institut für Pharmazeutische und Medizinische Chemie der Universität Münster,
Corrensstraße 48, Dꢀ48149 Münster, Germany.
Tel.: +49ꢀ251ꢀ8333311; Fax: +49ꢀ251ꢀ8332144; Eꢀmail: wuensch@uniꢀmuenster.de
b
Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research,
Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
c
CellsꢀinꢀMotion Cluster of Excellence (EXC 1003 – CiM), Westfälische Wilhelmsꢀ
Universität Münster, Germany.
Abstract
It has been reported that bioisosteric replacement of an 1,2,4ꢀoxadiazole ring by an
1,3,4ꢀoxadiazole ring leads to higher polarity, reduced metabolic degradation by
human liver microsomes and reduced interaction with hERG channels. In a seven to
eight step synthesis 1,3,4ꢀoxadiazles 9aꢀc were synthesized as bioisosteric analogs
of highꢀaffinity but rather lipophilic CB2 ligands 1aꢀc containing an 1,2,4ꢀoxadiazole
ring. The 1,3,4ꢀoxadiazole derivatives 9a and 9b show 10ꢀ and 50ꢀfold reduced CB2
affinity compared to the 1,2,4ꢀoxadiazole derivatives 1a and 1b, respectively.

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However, the 1,3,4ꢀoxadiazole 9a has high CB2 affinity (Ki = 25 nM) and high
selectivity over the CB1 receptor.
Key words
CB2 ligands; bioisosterism; 1,2,4ꢀoxadiazoles; 1,3,4ꢀoxadiazoles; carbazolamides;
fluorinated PET tracer;
1.
Introduction
The Gi/o proteinꢀcoupled CB2 receptor belongs to the endogenous cannabinoid
(endocannabinoid) system. After its discovery, it was referred to as the peripheral
cannabinoid receptor since it could initially only be detected in peripheral organs (e.g.
1,2,3,4,5
⁠
⁠
⁠
⁠
⁠
reproductive, cardiovascular, gastrointestinal and respiratory system).
Especially on immune cells (e.g. macrophages, T lymphocytes, B lymphocytes and
6
⁠
natural killer cells) the CB2 receptor is highly expressed. In 2002, the presence of
the CB2 receptor was shown on microglia, i.e. immune cells in the central nervous
7
⁠
system (CNS). Under normal conditions, the CB2 receptor expression in the CNS is
8
⁠
rather low, whereas inflammatory processes let the concentration rise. Antiꢀ
inflammatory effects were observed in numerous in vitro and in vivo models after
9
⁠
activation of central CB2 receptors. Therefore, CB2 agonists are promising
compounds for the treatment of many neurodegenerative, neuroinflammatory and
neuroimmunological diseases.9
Positron emission tomography (PET) is an imaging method that allows the
visualization and timeꢀdependent quantification of tracers, which possess a good

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affinity/selectivity profile towards a specific target (e.g. receptor) and contains a
positronꢀemitting isotope like 18F or 11C. PET tracers can contribute to better
understand biochemical processes like the development and severity of
neuroinflammatory processes.
Figure 1. Lead compound 1a and comparison of the 1,2,4ꢀoxadiazole moiety of 1a
with the 1,3,4ꢀoxadiazole moiety.
In 2013, the fluorineꢀ18 labeled PET tracer [18F]1a for imaging of CB2 receptors has
been reported (Figure 1). Although the 1,2,4ꢀoxadiazole derivative 1a displayed high
CB2 affinity (Ki = 2.3 nM) and high selectivity over the CB1 subtype (Ki > 1 ꢁM),10,11
the high lipophilicity (logD7.4 = 3.8 – 4.2) inhibited its broad application as PET tracer.
In particular, the poor solubility in polar, parenterally administrable solvents (e.g.
physiological saline solution) was recognized as problem.
Very recently, Boström et al. reported the concept of bioisosteric replacement of the
1,2,4ꢀoxadiazole ring by an 1,3,4ꢀoxadiazole ring resulting in reduced lipophilicity,
higher metabolic stability during incubation with human liver microsomes and lower
interactions with the hERG potassium channel.12 Particularly, in case of rather
lipophilic compounds (log D7.4 > 2.0), replacement of the 1,2,4ꢀoxadiazole ring by the

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more polar 1,3,4ꢀoxadiazole ring leads to higher solubility in aqueous systems.
However, the relative orientation of the substituents in 2ꢀ and 5ꢀposition is very
similar in both ring systems.
In order to prove the feasibility of bioisosteric replacement of the 1,2,4ꢀoxadiazole
ring of potent CB2 ligands such as 1, the regioisomeric 1,3,4ꢀoxadiazole derivatives 9
should be synthesized and pharmacologically evaluated (Figure 1).
2.
Synthesis
Scheme 1. Reagents and reaction conditions: (a) 1. SOCl2, DMF, toluene, 95 °C; 2.
N2H4ꢀH2O, CH2Cl2, 40 °C. (b) succinic anhydride, EtOAc, 40 °C, rt. (c) SOCl2, DMF,
MeOH, 0 °C ꢁ rt. (d) SOCl2, DMF, toluene, Na2SO4, 95 °C. (e) CuCN,

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H3CC(O)N(CH3)2, 155 °C. (f) LiOH, THF/H2O, rt. (g) COMU®, NEt3, DMF, 45 °C. (h)
XtalFluorꢀE®, NEt3ꢀ3HF, CH2Cl2, ꢀ78 °C ꢁ rt.
The 1,3,4ꢀoxadiazole derivatives 9aꢀc were synthesized starting from regioisomeric
2,4ꢀdisubstituted benzoic acids 2a and 2b (Scheme 1). After activation with SOCl2,
the benzoyl halides were reacted with N2H4ꢀH2O to afford the benzohydrazides 3a
and 3b. The second acylation of the hydryzine moiety was carried out with succinic
anhydride to give diacylhydrazines 4a and 4b. After esterification of the acids 4a and
4b with methanol, condensation of the diacylhydrazines 5a and 5b was performed
with SOCl2 in the presence of catalytic amounts of DMF providing the 1,3,4ꢀ
oxadiazoles 6a and 6b. The addition of anhydrous Na2SO4 increased the yield from
10 % to 79 % for 6a and 82 % for 6b, respectively. Treatment of the bromo derivative
6a with CuCN under Rosenmund-von-Braun conditions led to the nitrile 6c in 89 %
yield. Hydrolysis of the esters 6aꢀc with LiOH provided the carboxylic acids 7aꢀc,
which were coupled with the carbazolamine 10 and COMU® to yield the amides 8a-c.
Carbazolamine 10 was obtained by hydroxyethylation of carbazole followed by
nitration and catalytic hydrogenation according to literature.13 Deoxofluorination of
alcohols 8a-c with XtalFluoroꢀE® (diethylaminodifluorosulfonium tetrafluoroborate)
gave the aliphatic fluorides 9a-c. The yields of the final reaction step were not
optimized, since we were predominatly interested in very pure samples for biological
testing.

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3.
Receptor affinity
Table 1. CB1 and CB2 receptor affinity of 1,3,4ꢀ and 1,2,4ꢀoxadiazole regioisomers
1aꢀc and 9aꢀc.
Compd
R
X
Y
Ki (hCB2)
displacement
(hCB1)b)
± SEM [nM]a)
1a
Br
F
F
Br
F
2.9 ± 0.41d)
6.7 ± 1.0d)
270 ± 32d)
25 ± 4.1
ꢀ 13 %c)
ꢀ 5 %
1b
1c
CN
Br
F
ꢀ 8 %
9a
F
22 %c)
9b
Br
F
318 ± 55
ꢀ 8 %
9c
CN
219 ± 16
ꢀ 1 %
CP 55,940
WIN 55,212ꢀ2
HU 210
8.44 ± 0.18
8.57 ± 0.16
9.78 ± 0.04
9.26 ± 0.12
8.72 ± 0.24
9.55 ± 0.06
a) The reported Kiꢀvalues are mean values of three independent experiments (n = 3).
b) Due to the low hCB1 affinity, only the radioligand displacement at a test compound
concentration of 1 ꢁM is given as mean value of two independent experiments
(n = 2).
c) Mean value of four experiments (n = 4).
d) The CB2 affinity of lead compounds 1aꢀc has been recorded previously in another
laboratory.11

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The CB2 and CB1 receptor affinities were assessed in competition binding
experiments with fragments of CHOꢀK1 cells expressing the human CB1 or CB2
receptor, respectively. [3H]CPꢀ55,940 served as radioligand in both assays. The nonꢀ
specific binding of the radioligand [3H]CPꢀ55,940 was determined with rimonabant
(SR141716A) and AM630, respectively.
The regioisomeric bromofluorophenyl derivative 1a and 1b containing the 1,2,4ꢀ
oxadiazole ring show high CB2 affinity with Ki values of 2.9 nM and 6.7 nM,
respectively. Introduction of a cyano group as pseudohalogen in 2ꢀposition of the
phenyl ring led to 100ꢀfold decreased CB2 affinity of 1c compared to the bromo
compound 1a.
Replacement of the central 1,2,4ꢀoxadiazle ring of 1 by the regioisomeric 1,3,4ꢀ
oxadiazole ring led to 10ꢀ and 50ꢀfold reduced CB2 affinity of 9a and 9b, respectively.
The nitrile 9c displays almost the same CB2 affinity as the low affinity regioisomer 1c.
Due to the negligible CB1 affinity all compounds show high CB2 : CB1 selectivity,
independent on the structure of the oxadiazole ring and the substitution pattern of the
phenyl ring.
The CB2 affinity of alcohols 8a-c was also determined in the described assay.
However, 8a-c did not compete with the radioligand even at the high concentration of
1 ꢂM. This result is in good agreement with with results obtained for the
regioisomeric 1,2,4ꢀoxadiazoles with hydroxyethyl moiety at the carbazoleꢀNꢀatom.11

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4.
Conclusion
The aim of this study was to investigate, whether the rather lipophilic 1,2,4ꢀ
oxadiazole ring of potent CB2 ligands 1 can be replaced bioisosterically by the more
polar 1,3,4ꢀoxadiazole ring. For this purpose, three pairs of regioisomeric 1,2,4ꢀ and
1,3,4ꢀoxadiazoles 1a-c and 9a-c were prepared and pharmacologically evaluated. In
vitro radioligand binding studies revealed that displacement of the 1,2,4ꢀoxadiazole
ring of the high affinity ligands 1a and 1b by the regioisomeric 1,3,4ꢀoxadiazole ring
in 9a and 9b led to 10ꢀ and 50ꢀfold reduced CB2 affinity, respectively. Nevertheless,
the bromofluoro derivative 9a displays CB2 affinity in the low nanomolar range
(Ki = 25 nM) and high CB2 : CB1 selectivity.
5.
Experimental
Chemistry, General Methods
5.1
Unless otherwise noted, moisture sensitive reactions were conducted under dry
nitrogen. THF was dried with sodium/benzophenone and was freshly distilled before
use. Thin layer chromatography (tlc): Silica gel 60 F254 plates (Merck). Flash
chromatography (fc): Silica gel 60, 40–64 ꢂm (Merck); parentheses include: diameter
of the column, eluent, fraction size, Rf value. Melting point: Melting point apparatus
SMP 3 (Stuart Scientific), uncorrected. MS: MAT GCQ (ThermoꢀFinnigan); IR: IR
1
spectrophotometer 480Plus FTꢀATRꢀIR (Jasco). H NMR (400 MHz), 13C NMR (100
MHz): Unity Mercury Plus 400 spectrometer (Varian); δ in ppm related to
tetramethylsilane; coupling constants are given with 0.5 Hz resolution. HPLC method
for determination of the product purity: Merck Hitachi Equipment; UV detector: Lꢀ
7400; autosampler: Lꢀ7200; pump: Lꢀ7100; degasser: Lꢀ7614; Method: column:
LiChrospher® 60 RPꢀselect B (5 ꢁm), 250ꢀ4 mm cartridge; flow rate: 1.00 mL/min;

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injection volume: 5.0 ꢁL; detection at λ = 210 nm; solvents: A: water with 0.05 % (v/v)
trifluoroacetic acid; B: acetonitrile with 0.05 % (v/v) trifluoroacetic acid: gradient
elution: (A %): 0ꢀ4 min: 90 % , 4ꢀ29 min: gradient from 90 % to 0 %, 29ꢀ31 min: 0 %,
31ꢀ31.5 min: gradient from 0 % to 90 %, 31.5ꢀ40 min: 90 %.
5.2
Synthetic procedures
5.2.1 2-Bromo-4-fluorobenzohydrazide (3a)
Preparation of this compound is described in literature14 following a different
synthesis route.
Under N2, SOCl2 (1.5 mL, 20.7 mmol) was added to a suspension of 2ꢀbromoꢀ4ꢀ
fluorobenzoic acid (2a, 3.0 g, 13.7 mmol) and DMF (0.05 mL) in toluene (25 mL). The
mixture was stirred at 95 °C for 1.5 h. After cooling down to rt, the mixture was
concentrated in vacuo to give 4ꢀbromoꢀ2ꢀfluorobenzoyl chloride. Without further
purification 2ꢀbromoꢀ4ꢀfluorobenzoyl chloride was dissolved in CH2Cl2 (200 mL) and
hydrazine monohydrate (64 % in H2O, 2.8 mL, 35.8 mmol) was added. The reaction
mixture was stirred for 4.5 h at 45 °C. The organic solvent was removed under
reduced pressure and the residue was purified by fc (d = 5.5 cm, l = 10 cm,
cyclohexane/ethyl acetate 10:90, Rf 0.38 (ethyl acetate)). Colorless solid, mp 97 ꢀ
99 °C, yield 2.2 g (68 %). C7H6BrFN2O (233.0 g/mol). Exact mass (APCI):
m/z = calcd. for C7H679BrFN2OH 232.9720 found 232.9699. Purity (HPLC): 83.4 %
1
(tR = 6.29 min). H NMR (DMSOꢀD6): δ (ppm) = 4.49 (s, 2H, NHꢀNH2), 7.31 (td, J =
8.5/2.5 Hz, 1H, 5ꢀH), 7.42 (dd, J = 8.8/6.1 Hz, 1H, 6ꢀH), 7.64 (dd, J = 8.5/2.5 Hz, 1H,
3ꢀH), 9.56 (s, 1H, NHꢀNH2). 13C NMR (DMSOꢀD6): δ (ppm) = 115.3 (d, J = 21.3 Hz,
1C, Cꢀ5), 121.1 (m, 2C, Cꢀ2, Cꢀ3), 131.9 (d, J = 9.1 Hz, 1C, Cꢀ6), 135.1 (d, J = 3.5
Hz, 1C, Cꢀ1), 163.0 (d, J = 251.2 Hz, 1C, Cꢀ4), 166.5 (1C, C=O). IR (neat): ʋ (cmꢀ

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1) = 3321 (w, NH2), 3159 (m, ꢀNHꢀ), 1654 (s, C=O).
5.2.2 4-Bromo-2-fluorobenzohydrazide (3b)
Preparation of this compound is described in literature14,15 following a different
synthesis route.
Under N2, SOCl2 (1.5 mL, 20.7 mmol) was added to a suspension of 4ꢀbromoꢀ2ꢀ
fluorobenzoic acid (2b, 3.0 g, 13.7 mmol) and DMF (0.05 mL) in toluene (25 mL). The
mixture was stirred at 95 °C for 1.5 h. After cooling down to rt, the mixture was
concentrated in vacuo to give 4ꢀbromoꢀ2ꢀfluorobenzoyl chloride. Without further
purification 2ꢀbromoꢀ4ꢀfluorobenzoyl chloride was solved in CH2Cl2 (200 mL) and
hydrazine monohydrate (64.0 % in H2O, 2.8 mL, 35.8 mmol) was added. The reaction
mixture was stirred for 4.5 h at 45 °C. The organic solvent was removed under
reduced pressure and the residue was purified by fc (d = 4.0 cm, l = 10 cm,
cyclohexane/ethyl acetate 10:90, Rf 0.38 (ethyl acetate)). Colorless solid, mp 98 ꢀ
101 °C, yield 2.1 g (66 %). C7H6BrFN2O (233.0 g/mol). Exact mass (APCI):
m/z = calcd. for C7H679BrFN2OH 232.9720 found 232.9729. Purity (HPLC): 98.7 %
(tR = 8.11 min). 1H NMR (DMSOꢀD6): δ (ppm) = 4.56 (s, 2H, NHꢀNH2) 7.46 ꢀ 7.53 (m,
2H, 3ꢀH, 5ꢀH), 7.47 (d, J = 10.4 Hz, 1H, 6ꢀH), 9.60 (s, 1H, NHꢀNH2). 13C NMR
(DMSOꢀD6): δ (ppm) = 120.2 (d, J = 25.8 Hz, 1C, Cꢀ1), 123.2 (d, J = 15.3 Hz, 1C, Cꢀ
3), 124.4 (d, J = 9.1 Hz, 1C, Cꢀ6), 128.3 (d, J = 3.6 Hz, 1C, Cꢀ5), 132.1 (d, J = 3.9 Hz,
1C, Cꢀ4), 159.6 (d, J = 254.0 Hz, 1C, Cꢀ2), 163.1 (1C, C=O). IR (neat): ʋ (cmꢀ1) =
3321 (w, NH2), 3130 (m, ꢀNHꢀ), 1658 (s, C=O).
5.2.3 4-[2-(2-Bromo-4-fluorobenzoyl)hydrazine-1-yl]-4-oxobutanoic acid (4a)
3a (1.0 g, 4.26 mmol) and succinic anhydride (1.1 g, 10.7 mmol) were suspended in

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ethyl acetate (350 mL) and the mixture was stirred for 3.5 h at rt. The reation mixture
was diluted with diethyl ether (350 mL) and stirred overnight. The resulting precipitate
was filtered off and washed with petroleum ether. The crude product was used
without further purification (Rf 0.37 (ethyl acetate/formic acid 1:0.01)). Colorless solid,
mp 185 ꢀ 186 °C, yield 1.1 g (78 %). C11H10BrFN2O4 (333.1 g/mol). Exact mass
1
(APCI): m/z = calcd. for C11H1079BrFN2O4H 332.9881 found 332.9896. H NMR
(DMSOꢀD6): δ (ppm) = 2.44 (t, J = 5.3 Hz, 2H, CH2CH2CO2H), 2.47 (t, J = 5.2 Hz, 2H,
CH2CH2 CO2H), 7.36 (td, J = 8.5/2.4 Hz, 1H, 5ꢀH), 7.50 (dd, J = 8.5/6.1 Hz, 1H, 6ꢀH),
7.67 (dd, J = 8.7/2.4 Hz, 1H, 3ꢀH), 10.08 (s, 1H, ArꢀC(=O)ꢀNH), 10.27 (s, 1H, ꢀCH2ꢀ
C(=O)ꢀNH).
5.2.4 4-[2-(4-Bromo-2-fluorobenzoyl)hydrazine-1-yl]-4-oxobutanoic acid (4b)
3b (1.0 g, 4.26 mmol) and succinic anhydride (1.1 g, 10.7 mmol) were suspended in
ethyl acetate (350 mL) and the mixture was stirred for 3 h at rt. The reation mixture
was diluted with diethyl ether (350 mL) and stirred overnight. The resulting precipitate
was filtered and washed with petroleum ether. The crude product was used without
further purification (Rf 0.40 (ethyl acetate/formic acid 1:0.01)). Colorless solid, mp
190 ꢀ 194 °C, yield 1.2 g (84 %). C11H10BrFN2O4 (333.1 g/mol). Exact mass (APCI):
m/z = calcd. for C11H1079BrFN2O4H 332.9881 found 332.9878. 1H NMR (DMSOꢀD6): δ
(ppm) = 2.44 (t, J = 6.0 Hz, 2H, CH2CH2CO2H), 2.47 (t, J = 5.5 Hz, 2H,
CH2CH2CO2H), 7.52 ꢀ 7.55 (m, 2H, 3ꢀH, 5ꢀH), 8.07 (d, J = 10.4 Hz, 1H, 6ꢀH), 10.06
(s, 1H, ArꢀC(=O)ꢀNH), 10.27 (s, 1H, ꢀCH2ꢀC(=O)ꢀNH).

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5.2.5 Methyl 4-[2-(2-bromo-4-fluorobenzoyl)hydrazine-1-yl]-4-oxobutanoate
(5a)
Under N2, SOCl2 (0.33 mL, 4.5 mmol) was added to a suspension of 4a (1.0 g,
3.0 mmol) and DMF (0.05 mL) in CH3OH (50 mL) at 0 °C. The reaction mixture was
stirred at rt for 1 h. The mixture was concentrated under reduced pressure. The
residue was diluted with ethyl acetate and washed with brine. The organic solvent
was removed under reduced pressure and the residue was purified by fc (d = 4 cm,
l = 10 cm, cyclohexane/ethyl acetate 10:90, Rf 0.50 (ethyl acetate)). Colorless solid,
mp 146 ꢀ 48 °C, yield 900 mg (86 %). C12H12BrFN2O4 (347.1 g/mol). Exact mass
1
(APCI): m/z = calcd. for C12H1279BrFN2O4H 347.0037 found 347.0033. H NMR
(DMSOꢀD6): δ (ppm) = 2.46 (t, J = 7.0 Hz, 2H, CH2CH2CO2CH3), 2.56 (t, J = 7.0 Hz,
2H, CH2CH2CO2CH3), 3.59 (s, 3H, CH2CH2CO2CH3), 7.36 (td, J = 8.5, 2.5 Hz, 1H, 5ꢀ
H), 7.49 (dd, J = 8.6/6.1 Hz, 1H, 6ꢀH), 7.68 (dd, J = 8.8/2.5 Hz, 1H, 3ꢀH), 10.11 (s,
1H, ArꢀC(=O)ꢀNH), 10.28 (s, 1H, ꢀCH2ꢀC(=O)ꢀNH). IR (neat): ʋ (cmꢀ1) = 3321 (w,
NH2), 3130 (m, ꢀNHꢀ),1720 (m, (C=O)ꢀNH), 1654 (m, C=Oester).
5.2.6 Methyl 4-[2-(4-bromo-2-fluorobenzoyl)hydrazine-1-yl]-4-oxobutanoate
(5b)
Under N2, SOCl2 (0.2 mL, 3.1 mmol) was added to a suspension of 4b (687 mg,
2.1 mmol) and DMF (0.05 mL) in CH3OH (25 mL) at 0 °C. The reaction mixture was
stirred at rt for 40 min. The resulting reaction mixture was stirred at rt for 1 h. The
mixture was concentrated under reduced pressure. The residue was diluted with
ethyl acetate and washed with brine. The organic solvent was removed under
reduced pressure and the residue was purified by fc (d = 3.5 cm, l = 10 cm,
cyclohexane/ethyl acetate 10:90, Rf 0.50 (ethyl acetate)). Colorless solid, mp 148 ꢀ

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149 °C, yield 613 mg (88 %). C12H12BrFN2O4 (347.1 g/mol). Exact mass (APCI):
1
m/z = calcd. for C12H1279BrFN2O4H 347.0037 found 347.0039. H NMR (CDCl3): δ
(ppm) = 2.89 (t, J = 7.0 Hz, 2H, CH2CH2CO2CH3), 3.12 (t, J = 7.0 Hz, 2H,
CH2CH2CO2CH3), 3.72 (s, 3H, CH2CH2CO2CH3), 7.37 (dd, J = 8.5/2.5 Hz, 1H, ꢀCH2ꢀ
C(=O)ꢀNH), 7.45 (dd, J = 8.4/1.8 Hz, 1H, ArꢀC(=O)ꢀNH), 7.98 (t, J = 8.3 Hz, 1H, 6ꢀH),
8.80 (d, J = 5.9 Hz, 1H, 3ꢀH), 9.15 ꢀ 9.25 (m, 1H, 5ꢀH). IR (neat): ʋ (cmꢀ1) = 3321 (w, ꢀ
NH2), 3130 (m, ꢀNHꢀ),1720 (m, (C=O)ꢀNH), 1654 (m, C=Oester).
5.2.7 Methyl 3-[5-(2-bromo-4-fluorophenyl)-1,3,4-oxadiazol-2-yl]propanoate (6a)
Under N2, a mixture of 5a (200 mg, 0.6 mmol), DMF (0.05 mL) and toluene (15 mL)
was heated to 75 °C. Na2SO4 (ca. 0.5 – 1.0 g) and SOCl2 (0.04 mL, 0.5 mmol) were
added and the mixture was heated to 95 °C for 2.5 h. The mixture was concentrated
in vacuo. The residue was dissolved in CH2Cl2 and the solution was washed with
brine. The organic solvent was removed under reduced pressure and the residue
was purified by fc (d = 4 cm, l = 10 cm, cyclohexane/ethyl acetate 10:90, Rf 0.75
(ethyl acetate)). Colorless solid, mp 75 ꢀ 77 °C, yield 150 mg (79 %). C12H10BrFN2O3
(329.0 g/mol). Exact mass (APCI): m/z = calcd. for C12H1079BrFN2O3H 328.9932
1
found 328.9922. Purity (HPLC): 96.9 % (tR = 17.57 min). H NMR (DMSOꢀD6): δ
(ppm) = 2.89 (t, J = 7.0 Hz, 2H, CH2CH2CO2CH3), 3.12 (t, J = 7.0 Hz, 2H,
CH2CH2CO2CH3), 3.63 (s, 3H, CH2CH2CO2CH3), 7.51 (td, J = 8.5/2.5 Hz, 1H, 5ꢀH),
7.89 (dd, J = 8.6/6.1 Hz, 1H, 6ꢀH), 7.96 (dd, J = 8.8/2.5 Hz, 1H, 3ꢀH). 13C NMR
(DMSOꢀD6): δ (ppm) = 20.4 (1C, CH2CH2CO2CH3), 29.5 (1C, CH2CH2CO2CH3), 51.7
(1C, CH2CH2CO2CH3), 115.8 (d, J = 21.9 Hz, 1C, Cꢀ5), 121.7 (d, J = 3.6 Hz, 1C, Cꢀ
1), 121.7 (d, J = 25.3 Hz, 1C, Cꢀ3), 121.9 (d, J = 10.3 Hz, 1C, Cꢀ2), 133.5 (d, J = 9.6
Hz, 1C, Cꢀ6), 162.1 (1C, Cꢀ5oxadiazole), 163.2 (d, J = 249.3 Hz, 1C, Cꢀ4), 166.3 (1C, Cꢀ

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2oxadiazole), 171.8 (1C, CO2CH3). IR (neat): ʋ (cmꢀ1) = 3074 (m, CꢀH, arom), 2958 (m,
CꢀH, aliph), 1735 (s, C=O).
5.2.8 Methyl 3-[5-(4-bromo-2-fluorophenyl)-1,3,4-oxadiazol-2-yl]propanoate
(6b)
Under N2, a mixture of 5b (200 mg, 0.6 mmol), DMF (0.05 mL) and toluene (15 mL)
was heated to 75 °C. Na2SO4 (ca. 0.5 – 1.0 g) and SOCl2 (0.04 mL, 0.5 mmol) were
added and the mixture was heated to 95 °C for 2.5 h. The mixture was concentrated
in vacuo. The residue was dissolved in CH2Cl2 and the solution was washed with
brine. The organic solvent was removed under reduced pressure and the residue
was purified by fc (d = 5 cm, l = 10 cm, cyclohexane/ethyl acetate 10:90, Rf 0.75
(ethyl acetate)). Colorless solid, mp 76 ꢀ 77 °C, yield 156 mg (82 %). C12H10BrFN2O3
(329.0 g/mol). Exact mass (APCI): m/z = calcd. for C12H1079BrFN2O3H 328.9932
1
found 328.9945. Purity (HPLC): 98.9 % (tR = 15.17 min). H NMR (DMSOꢀD6): δ
(ppm) 2.89 (t, J = 7.0 Hz, 2H, CH2CH2CO2CH3), 3.12 (t, J = 7.0 Hz, 2H,
CH2CH2CO2CH3), 3.63 (s, 3H, CH2CH2CO2CH3), 7.51 (td, J = 8.5/2.5 Hz, 1H, 6ꢀH),
7.89 (dd, J = 8.6/6.1 Hz, 1H, 3ꢀH), 7.96 (dd, J = 8.8/6.0 Hz, 1H, 5ꢀH). 13C NMR
(DMSOꢀD6): δ (ppm) = 20.4 (1C, CH2CH2CO2CH3), 29.5 (1C, CH2CH2CO2CH3), 51.7
(1C, CH2CH2CO2CH3), 115.82 (d, J = 21.9 Hz, 1C, Cꢀ3), 121.4 – 122.1 (m, 3C, Cꢀ1,
Cꢀ4, Cꢀ5), 133.5 (d, J = 9.7 Hz, 1C, Cꢀ6), 162.5 (d, J = 12.3 Hz, 1C, Cꢀ5oxadiazole),
163.2 (d, J = 249.3 Hz, 1C, Cꢀ2), 166.3 (1C, Cꢀ2oxadiazole), 171.7 (1C, CO2CH3). IR
(neat): ʋ (cmꢀ1) = 3074 (m, CꢀH, arom), 2958 (m, CꢀH, aliph), 1735 (s, C=O).
5.2.9 Methyl 3-[5-(2-cyano-4-fluorophenyl)-1,3,4-oxadiazol-2-yl]propanoate (6c)
6a (200 mg, 0.6 mmol) and CuCN (271 mg, 3.1 mmol) were suspended in N,Nꢀ

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dimethylacetamide (5 mL) under N2. The mixture was stirred at 155 °C for 12.5 h
before cooling to room temperature. Saturated NH4Cl (2 mL) and then ethyl acetate
(8 mL) were added. The precipitated CuCN was filtered off and the organic layer was
collected and washed once with brine. The organic layer was dried (Na2SO4),
evaporated under reduced pressure and the residue was purified by fc (d = 2.5 cm,
l = 10 cm, cyclohexane/ethyl acetate 75:25, Rf 0.68 (ethyl acetate)). Pale yellow oil,
yield 147 mg (89 %). C13H10FN3O3 (275.2 g/mol). Exact mass (APCI): m/z = calcd. for
C13H10FN3O3H 276.0779 found 276.0808. Purity (HPLC): 96.7 % (tR = 15.15 min). 1H
NMR (CDCl3): δ (ppm) = 2.96 (t, J = 7.3 Hz, 2H, CH2CH2CO2CH3), 3.31 (t, J = 7.3
Hz, 2H, CH2CH2CO2CH3), 3.63 (s, 3H, CH2CH2CO2CH3), 7.47 (td, J = 8.9/7.6/2.6 Hz,
1H, 5ꢀH), 7.55 (dd, J = 7.8/2.6 Hz, 1H, 3ꢀH), 8.26 (dd, J = 8.9/5.2 Hz, 1H, 6ꢀH). 13C
NMR (DMSOꢀD6): δ (ppm) = 21.6 (1C, CH2CH2CO2CH3), 30.7 (1C, CH2CH2CO2CH3),
51.8 (1C, CH2CH2CO2CH3), 111.9 (d, J = 10.3 Hz, 1C, Cꢀ2), 116.1 (1C, CN), 121.6
(d, J = 21.9 Hz, 1C, Cꢀ5), 122.5 (d, J = 26.1 Hz, 1C, Cꢀ3), 125.0 (d, J = 3.6 Hz, 1C,
Cꢀ1), 132.6 (d, J = 9.4 Hz, 1C, Cꢀ6), 162.9 (d, J = 252.1 Hz, 1C, Cꢀ4), 165.0 (1C, Cꢀ
5oxadiazole), 171.8 (1C, Cꢀ2oxadiazole), 179.9 (1C, CO2CH3). IR (neat): ʋ (cmꢀ1) = 2233 (w,
CN), 1735 (s, C=O).
5.2.10 3-[5-(2-Bromo-4-fluorophenyl)-1,3,4-oxadiazol-2-yl]propanoic acid (7a)
6a (550 mg, 1.7 mmol) and LiOH (350 mg, 8.4 mmol) were dissolved in THF (60 mL)
and H2O (20 mL) and the mixture was stirred for 20 min at rt. Afterwards, the mixture
was neutralized with 1 M H2SO4, diluted with ethyl acetate and washed twice with
brine. The organic layer was dried (Na2SO4) and evaporated under reduced pressure.
The residue was washed with CH2Cl2 and purified by fc (ethyl acetate/formic acid
1:0.01, Rf 0.78). Colorless solid, mp 152 ꢀ 154 °C, yield 500 mg (96 %).

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C11H8BrFN2O3 (315.0 g/mol). Exact mass (APCI): m/z = calcd. for C11H879BrFN2O3H
1
314.9775 found 314.9773. Purity (HPLC): 90.5 % (tR = 18.41 min). H NMR (DMSOꢀ
D6): δ (ppm) = 3.00 (t, J = 7.2 Hz, 2H, CH2CH2CO2H), 3.28 (t, J = 7.1 Hz, 2H,
CH2CH2CO2H), 7.18 (td, J = 8.8/2.5 Hz, 1H, 5ꢀH), 7.49 (dd, J = 8.2/2.5 Hz, 1H, 3ꢀH),
7.85 (dd, J = 8.8/5.9 Hz, 1H, 6ꢀH). 13C NMR (DMSOꢀD6): δ (ppm) = 21.4 (1C,
CH2CH2CO2H), 29.8 (1C, CH2CH2CO2CH3), 115.8 (d, J = 21.9 Hz, 1C, Cꢀ5), 121.6 (d,
J = 24.7 Hz, 1C, Cꢀ3), 121.7 (d, J = 3.6 Hz, 1C, Cꢀ1), 122.0 (d, J = 10.4 Hz, 1C, Cꢀ2),
133.5 (d, J = 9.6 Hz, 1C, Cꢀ6), 162.1 (1C, Cꢀ5oxadiazole), 163.2 (d, J = 254.7 Hz, 1C, Cꢀ
4), 166.6 (1C, Cꢀ2oxadiazole), 171.8 (1C, CO2H). IR (neat): ʋ (cmꢀ1) = 3170ꢀ2350 (m,
COOH), 1705 (s, C=O).
5.2.11 3-[5-(4-Bromo-2-fluorophenyl)-1,3,4-oxadiazol-2-yl]propanoic acid (7b)
6b (450 mg, 1.4 mmol) and LiOH (290 mg, 6.9 mmol) were dissolved in THF (60 mL)
and H2O (20 mL) and the mixture was stirred for 20 min at rt. Afterwards, the mixture
was neutralized with 1 M H2SO4, diluted with ethyl acetate and washed twice with
brine. The organic layer was dried (Na2SO4) and evaporated under reduced pressure.
The residue was washed with CH2Cl2 and purified by fc (ethyl acetate/formic acid
1:0.01, Rf 0.80). Colorless solid, mp 152 ꢀ 155 °C, yield 409 mg (93 %).
C11H8BrFN2O3 (315.0 g/mol). Exact mass (APCI): m/z = calcd. for C11H879BrFN2O3H
1
314.9775 found 314.9760. Purity (HPLC): 98.6 % (tR = 15.19 min). H NMR (DMSOꢀ
D6): δ (ppm) = 3.02 (t, J = 7.2 Hz, 2H, CH2CH2CO2H), 3.29 (t, J = 7.1 Hz, 2H,
CH2CH2CO2H), 7.32 – 7.47 (m, 2H, 3ꢀH, 5ꢀH), 7.93 (t, J = 7.9 Hz, 1H, 6ꢀH). 13C NMR
(DMSOꢀD6): δ (ppm) = 21.9 (1C, CH2CH2CO2H), 30.9 (1C, CH2CH2CO2CH3), 120.6
(d, J = 24.3 Hz, 2C, Cꢀ1, Cꢀ3), 128.5 (d, J = 3.8 Hz, 1C, Cꢀ5), 131.8 (d, J = 11.4 Hz,
2C, Cꢀ4, Cꢀ6), 156.0 (d, J = 10.6 Hz, 1C, Cꢀ5oxadiazole), 160.5 (d, J = 260.6 Hz, 1C, Cꢀ

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2), 175.4 (1C, Cꢀ2oxadiazole), 178.1 (1C, CO2H). IR (neat): ʋ (cmꢀ1) = 3170ꢀ2310 (m,
COOH), 1705 (s, C=O).
5.2.12 3-[5-(2-Cyano-4-fluorophenyl)-1,3,4-oxadiazol-2-yl]propanoic acid (7c)
6c (550 mg, 2.0 mmol) and LiOH (420 mg, 10.0 mmol) were dissolved in THF
(60 mL) and H2O (20 mL) and the mixture was stirred for 15 min at rt. Afterwards, the
reaction was neutralized with 1 M H2SO4, diluted with CH2Cl2 and washed twice with
brine. The organic layer was dried (Na2SO4) and evaporated under reduced pressure.
The residue was washed with CH2Cl2 and purified by fc (ethyl acetate/formic acid
1:0.01, Rf 0.66). Colorless solid, mp 139 ꢀ 140 °C, yield 450 mg (86 %). C12H8FN3O3
(261.2 g/mol). Exact mass (APCI): m/z = calcd. for C12H8FN3O3H 262.0622 found
1
262.0635. Purity (HPLC): 86.8 % (tR = 17.41 min). H NMR (CDCl3): δ (ppm) = 3.01
(t, J = 7.1 Hz, 2H, CH2CH2CO2H), 3.30 (t, J = 7.1 Hz, 2H, CH2CH2CO2H), 7.43 (ddd, J
= 8.7/7.7/2.7 Hz, 1H, 5ꢀH), 7.54 (dd, J = 8.2/2.7 Hz, 1H, 3ꢀH), 8.16 (dd, J = 8.7/6.0
Hz, 1H, 6ꢀH). 13C NMR (DMSOꢀD6): δ (ppm) = 20.5 (1C, CH2CH2 CO2H), 29.7 (1C,
CH2CH2 CO2H),, 111.35 (d, J = 10.4 Hz, 1C, Cꢀ2), 115.9 (1C, CN), 121.7 (d, J = 22.1
Hz, 1C, Cꢀ5), 122.3 (d, J = 3.5 Hz, 1C, Cꢀ1), 122.6 (d, J = 26.2 Hz, 1C, Cꢀ3), 131.7
(d, J = 9.4 Hz, 1C, Cꢀ6), 161.0 (1C, Cꢀ5oxadiazole), 162.9 (d, J = 252.8 Hz, 1C, Cꢀ4),
165.0 16.9 (1C, Cꢀ2oxadiazole), 172.8 (1C, CO2H). IR (neat): ʋ (cmꢀ1) = 2229 (w, CN),
1728 (s, C=O).
5.2.13 3-[5-(2-Bromo-4-fluorophenyl)-1,3,4-oxadiazol-2-yl]-N-[9-(2-hydroxyethyl)-
9H-carbazol-3-yl]propanamide (8a)
COMU® (489 mg, 1.1 mmol) was added to a mixture of carboxylic acid 7a (400 mg,
1.0 mmol) and triethylamine (0.35 mL, 2.5 mmol) in DMF (15 mL), and the mixture

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was stirred for 30 min at rt. The reaction mixture was cooled down to 0 °C and a
solution of carbazole hydrochloride 10ꢀHCl (215 mg, 0.9 mmol) in DMF was added
dropwise. This mixture was stirred for 24 h at 45 °C. Then H2O and brine were added
and the resulting precipitate was filtered off and dissolved in ethyl acetate. Brine was
added and the aqueous layer was extracted with ethyl acetate until the product was
extracted completely. The combined organic layers were washed with brine, dried
(Na2SO4) and concentrated under reduced pressure. The residue was purified by fc
(d = 8 cm, l = 7 cm, cyclohexane/ethyl acetate 75:25, Rf 0.62 (ethyl acetate)). The
product was recrystallized from CH2Cl2. Colorless solid, mp 213ꢀ215 °C, yield 200 mg
(38 %). C25H20BrFN4O3 (523.4 g/mol). Exact mass (ESI): m/z = calcd. for
C25H2079BrFN4O3H
523.0776
found
523.0768.
Purity
(HPLC):
98.1 %
(tR = 19.09 min). 1H NMR (DMSOꢀD6): δ (ppm) = 2.95 (t, J = 7.0 Hz, 2H,
CH2CH2CONH), 3.29 (t, J = 7.1 Hz, 2H, CH2CH2CONH), 3.70 – 7.34 (m, 2H,
NCH2CH2OH), 4.40 (t, J = 5.0 Hz, 2H, NCH2CH2OH), 4.87 (t, J = 5.2 Hz, 1H,
NCH2CH2OH), 7.16 (t, J = 7.3 Hz, 1H, 6ꢀHcarb), 7.42 (t, J = 7.5 Hz, 1H, 7ꢀHcarb), 7.48
(td, J = 8.9/1.8 Hz, 1H, 5ꢀHphenyl), 7.49 – 7.55 (m, 2H, 1ꢀHcarb, 2ꢀHcarb), 7.57 (d, J = 8.2
Hz, 1H, 8ꢀHcarb), 7.87 (dd, J = 8.6/2.1 Hz, 1H, 3ꢀHphenyl), 7.97 (dd, J = 8.5/6.1 Hz, 1H,
6ꢀHphenyl), 8.03 (d, J = 7.8 Hz, 1H, 5ꢀHcarb), 8.41 (s, 1H, 4ꢀHcarb), 10.13 (s, 1H, CONH).
13C NMR (DMSOꢀD6): δ (ppm) = 21.4 (1C, CH2CH2CONH), 32.7 (1C,
CH2CH2CONH), 46.0 (1C, NCH2CH2OH), 60.2 (1C, NCH2CH2OH), 110.2 (1C, Cꢀ
8carb), 110.3 (1C, Cꢀ1carb), 111.5 (1C, Cꢀ4carb), 116.5 (d, J = 21.8 Hz, 1C, Cꢀ5phenyl),
119.2 (1C, Cꢀ2carb), 119.3 (1C, Cꢀ6carb), 120.6 (1C, Cꢀ5carb), 122.2 (1C, Cꢀ4acarb),
122.4 (3C, Cꢀ1phenyl, Cꢀ2phenyl, Cꢀ3phenyl), 122.6 (1C, Cꢀ4bcarb), 126.3 (1C, Cꢀ3carb),
131.7 (1C, Cꢀ7carb), 134.2 (d, J = 9.6 Hz, 1C, Cꢀ6phenyl), 137.7 (1C, Cꢀ9acarb), 141.5
(1C, Cꢀ8acarb), 162.76 (1C, Cꢀ2oxadiazole), 163.8 (d, J = 253.4 Hz, 1C, Cꢀ4phenyl), 167.6

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(1C, Cꢀ5oxadiazole), 169.2 (1C, CONH). IR (neat): ʋ (cmꢀ1) = 3305 (m, NꢀH), 3051 (m,
CꢀH, arom), 2928 (m, CꢀH, aliph), 1685 (s, C=O).
5.2.14 3-[5-(4-Bromo-2-fluorophenyl)-1,3,4-oxadiazol-2-yl]-N-[9-(2-hydroxyethyl)-
9H-carbazol-3-yl]propanamide (8b)
COMU® (290 mg, 1.2 mmol) was added to a mixture of carboxylic acid 7b (300 mg,
0.9 mmol) and triethylamine (0.4 mL, 2.8 mmol) in DMF (15 mL), and the mixture was
stirred for 30 min at rt. The reaction mixture was cooled down to 0 °C and a solution
of carbazole hydrochloride 10ꢀHCl (200 mg, 0.9 mmol) in DMF was added dropwise.
This mixture was stirred for 24 h at 45 °C. Then H2O and brine were added and the
resulting precipitate was filtered off and dissolved in ethyl acetate. Brine was added
and the aqueous layer was extracted with ethyl acetate until the product was
extracted completely. The combined organic layers were washed with brine, dried
(Na2SO4) and concentrated under reduced pressure. The residue was purified by fc
(d = 5 cm, l = 6 cm, cyclohexane/ethyl acetate 50:50, Rf 0.18 (ethyl acetate)). The
product was recrystallized from CH2Cl2. Colorless solid, mp 202 ꢀ 203 °C, yield
220 mg (24 %). C25H20BrFN4O3 (523.4 g/mol). Exact mass (ESI): m/z = calcd. for
C25H2079BrFN4O3H
523.0776
found
523.0763.
Purity
(HPLC):
84.1 %
(tR = 19.07 min). 1H NMR (DMSOꢀD6): δ (ppm) = 2.95 (t, J = 6.9 Hz, 2H,
CH2CH2CONH), 3.28 (t, J = 7.0 Hz, 2H, CH2CH2CONH), 3.71 – 3.79 (m, 2H,
NCH2CH2OH), 4.40 (t, J = 5.6 Hz, 2H, NCH2CH2OH), 4.86 (t, J = 5.6 Hz, 1H,
NCH2CH2OH), 7.15 (t, J = 7.4 Hz, 1H, 6ꢀHcarb), 7.42 (t, J = 7.2 Hz, 1H, 7ꢀHcarb), 7.48 –
7.55 (m, 2H, 1ꢀHcarb, 2ꢀHcarb), 7.57 (d, J = 8.3 Hz, 1H, 8ꢀHcarb), 7.65 (dd, J = 8.4/1.4
Hz, 1H, 5ꢀHphenyl), 7.89 (dd, J = 10.3/1.7 Hz, 1H, 3ꢀHphenyl), 7.95 (t, J = 8.1 Hz, 1H, 6ꢀ
Hphenyl), 8.02 (d, J = 7.8 Hz, 1H, 5ꢀHcarb), 8.40 (s, 1H, 4ꢀHcarb), 10.12 (s, 1H, CONH).

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13C NMR (DMSOꢀD6): δ (ppm) = 21.7 (1C, CH2CH2CONH), 32.0 (1C,
CH2CH2CONH), 45.3 (1C, NCH2CH2OH), 59.6 (1C, NCH2CH2OH), 109.5 (1C, Cꢀ
8carb), 109.7 (1C, Cꢀ1carb), 110.9 (1C, Cꢀ4carb), 111.3 (d, J = 21.7 Hz, 1C, Cꢀ3phenyl),
118.5 (1C, Cꢀ2carb), 118.7 (1C, Cꢀ6carb), 120.0 (1C, Cꢀ5carb), 120.7 (d, J = 24.9 Hz, 1C,
Cꢀ1phenyl), 121.8 (d, J = 10.1 Hz, 1C, Cꢀ4phenyl), 125.6 (2C, Cꢀ4acarb, Cꢀ4bcarb), 125.9
(d, J = 10.7 Hz, 1C, Cꢀ6phenyl), 128.7 (d, J = 3.6 Hz, 1C, Cꢀ5phenyl), 130.1 (1C, Cꢀ3carb),
130.7 (1C, Cꢀ7carb), 137.0 (1C, Cꢀ9acarb), 140.9 (1C, Cꢀ8acarb), 158.9 (d, J = 260.8 Hz,
1C, Cꢀ2phenyl), 160.0 (d, J = 5.5 Hz, 1C, Cꢀ2oxadiazole), 166.9 (1C, Cꢀ5oxadiazole), 168.6
(1C, CONH). IR (neat): ʋ (cmꢀ1) = 3305 (m, NꢀH), 2924 (m, CꢀH, aliph), 1685 (s,
C=O).
5.2.15 3-[5-(2-Cyano-4-fluorophenyl)-1,3,4-oxadiazol-2-yl]-N-[9-(2-hydroxyethyl)-
9H-carbazol-3-yl]propanamide (8c)
COMU® (550 mg, 1.3 mmol) was added to a mixture of carboxylic acid 7c (280 mg,
1.1 mmol) and triethylamine (0.4 mL, 2.8 mmol) in DMF (15 mL), and the mixture was
stirred for 30 min at rt. The reaction mixture was cooled down to 0 °C and a solution
of carbazole hydrochloride 10ꢀHCl (200 mg, 0.9 mmol) in DMF was added dropwise.
This mixture was stirred for 24 h at 45 °C. Then H2O and brine were added and the
resulting precipitate was filtered off and dissolved in ethyl acetate. Brine was added
and the aqueous layer was extracted with ethyl acetate until the product was
extracted completely. The combined organic layers were washed with brine, dried
(Na2SO4) and concentrated under reduced pressure. The residue was purified by fc
(d = 5 cm, l = 7 cm, cyclohexane/ethyl acetate 50:50, Rf 0.28 (ethyl acetate)). The
product was recrystallized from CH2Cl2. Colorless solid, mp 215 °C, yield 121 mg
(24 %). C26H20FN5O3 (469.5 g/mol). Exact mass (APCI): m/z = calcd. for

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C26H20FN5O3H 470.1652 found 470.1623. Purity (HPLC): 93.2 % (tR = 19.76 min). 1H
NMR (DMSOꢀD6): δ (ppm) = 2.97 (t, J = 7.0 Hz, 2H, CH2CH2CONH), 3.29 (t, J = 7.1
Hz, 2H, CH2CH2CONH), 3.76 (q, J = 5.5 Hz, 2H, NCH2CH2OH), 4.40 (t, J = 5.7 Hz,
2H, NCH2CH2OH), 4.85 (t, J = 5.4 Hz, 1H, NCH2CH2OH), 7.15 (t, J = 7.5 Hz, 1H, 6ꢀ
Hcarb), 7.42 (t, J = 7.6 Hz, 1H, 7ꢀHcarb), 7.51 – 7.55 (m, 2H, 1ꢀHcarb, 2ꢀHcarb), 7.57 (d, J
= 8.2 Hz, 1H, 8ꢀHcarb), 7.81 (td, J = 8.5/2.7 Hz, 1H, 5ꢀHphenyl), 8.02 (d, J = 7.7 Hz, 1H,
5ꢀHcarb), 8.16 (dd, J = 8.7/2.7 Hz, 1H, 3ꢀHphenyl), 8.20 (dd, J = 8.9/5.3 Hz, 1H, 6ꢀ
13
Hphenyl), 8.40 (s, 1H, 4ꢀHcarb), 10.12 (s, 1H, CONH). C NMR (DMSOꢀD6): δ (ppm) =
20.8 (1C, CH2CH2CONH), 31.8 (1C, CH2CH2CONH), 45.3 (1C, NCH2CH2OH), 59.5
(1C, NCH2CH2OH), 109.4 (1C, Cꢀ8carb), 109.6 (1C, Cꢀ1carb), 110.9 (1C, Cꢀ4carb), 115.8
(d, J = 3.1 Hz, 1C, CN), 116.2 (d, J = 10.2 Hz, 1C, Cꢀ2phenyl), 118.4 (1C, Cꢀ2carb),
118.7 (1C, Cꢀ6carb), 120.0 (1C, Cꢀ5carb), 121.6 (d, J = 20.4 Hz, 1C, Cꢀ3phenyl), 121.7
(1C, Cꢀ4acarb), 121.9 (1C, Cꢀ4bcarb), 122.3 (d, J = 3.3 Hz, 1C, Cꢀ1phenyl), 122.5 (d, J =
26.7 Hz, 1C, Cꢀ5phenyl), 125.6 (1C, Cꢀ7carb), 131.0 (1C, Cꢀ3carb), 131.7 (d, J = 9.3 Hz,
1C, Cꢀ6phenyl), 137.0 (1C, Cꢀ9acarb), 140.8 (1C, Cꢀ8acarb), 160.9 (1C, Cꢀ2oxadiazole),
162.9 (d, J = 252.6 Hz, 1C, Cꢀ4phenyl), 167.3 (1C, Cꢀ5oxadiazole), 168.5 (1C, CONH). IR
(neat): ʋ (cmꢀ1) = 3464 (w, OꢀH), 3324 (m, NꢀH), 2935 (m, CꢀH, aliph), 2233 (w, CN),
1689 (s, C=O).
5.2.16 General procedure for the fluorination of the alcohols 8a-c with
XtalFluor-E®
Under N2, diethylaminodifluorosulfonium tetrafluoroborate (XtalFluorꢀE®, 1.5 ꢀ 3.0 eq.)
was suspended in CH2Cl2. Triethylamine trihydrofluoride (1.5 ꢀ 3.0 eq.) and a solution
of the respective alcohol 8 (1.0 eq.) in CH2Cl2 were added to the suspension via
cannula at ꢀ 78 °C. The resulting mixture was warmed up to rt during 1 or 3 h. An

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aqueous solution of Na2CO3 (5 % m/m) was added and the reaction mixture was
stirred for 15 min at rt. After addition of brine the mixture was extracted with CH2Cl2
until the product was extracted completely. The organic layer was dried (Na2SO4),
the organic concentrated in vacuo, the product was recrystallized from ethyl acetate.
5.2.17 3-[5-(2-Bromo-4-fluorophenyl)-1,3,4-oxadiazol-2-yl]-N-[9-(2-fluoroethyl)-
9H-carbazol-3-yl]propanamide (9a)
According to the General Procedure, 8a (120 mg, 0.2 mmol) was treated with
XtalFluorꢀE® (78 mg, 0.3 mmol) and triethylamine trihydrofluoride (0.1 mL, 0.6 mmol)
in CH2Cl2 (20 mL) at ꢀ 78 °C. The product was purified by fc (d = 3 cm, l = 10 cm,
cyclohexane/ethyl acetate 25:75, Rf 0.60 (ethyl acetate)). Colorless solid, mp 203 ꢀ
205 °C, yield 60 mg (49 %). C25H19BrF2N4O2 (525.3 g/mol). Exact mass (APCI):
m/z = calcd. for C25H1979BrF2N4O2H 525.0732 found 525.0738. Purity (HPLC): 93.0 %
(tR = 21.86 min). 1H NMR (DMSOꢀD6): δ (ppm) = 2.95 (t, J = 7.0 Hz, 2H,
CH2CH2CONH), 3.29 (t, J = 7.1 Hz, 2H, CH2CH2CONH), 4.70 (dt, J = 14.7/4.3 Hz,
2H, NCH2CH2F), 4.79 (dt, J = 38.5/4.4 Hz, 2H, NCH2CH2F), 7.18 (t, J = 7.1 Hz, 1H,
6ꢀHcarb), 7.44 (t, J = 7.2 Hz, 1H, 7ꢀHcarb), 7.48 (ddd, J = 8.7/8.3/2.7 Hz Hz, 1H, 5ꢀ
Hphenyl), 7.54 (dd, J = 8.0, 1H, 1ꢀHcarb), 7.57 (d, J = 8.8/1.8, 1H, 2ꢀHcarb), 7.60 (d, J =
8.2 Hz, 1H, 8ꢀHcarb), 7.88 (dd, J = 8.6/2.6 Hz, 1H, 3ꢀHphenyl), 7.97 (dd, J = 8.8/6.0 Hz,
1H, 6ꢀHphenyl), 8.05 (d, J = 7.6 Hz, 1H, 5ꢀHcarb), 8.43 (s, 1H, 4ꢀHcarb), 10.15 (s, 1H,
CONH). 13C NMR (DMSOꢀD6): δ (ppm) = 20.7 (1C, CH2CH2CONH), 32.0 (1C,
CH2CH2CONH), 42.9 (d, J = 20.3 Hz, 1C, NCH2CH2F), 82.6 (d, J = 167.4 Hz, 1C,
NCH2CH2F), 109.5 (1C, Cꢀ8carb), 109.6 (1C, Cꢀ1carb), 110.8 (1 C, Cꢀ4carb), 115.8 (d, J
= 21.8 Hz,1C, Cꢀ5phenyl), 119.2 (1C, Cꢀ2carb), 119.3 (1C, Cꢀ6carb), 120.0 (1C, Cꢀ5carb),
121.2 – 121.4 (5C, Cꢀ4acarb, Cꢀ4bcarb, Cꢀ1phenyl, Cꢀ2phenyl, Cꢀ3phenyl), 125.8 (1C, Cꢀ7carb),

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131.4 (1C, Cꢀ3carb), 133.5 (d, J = 9.6 Hz, 1C, Cꢀ6phenyl), 136.7 (1C, Cꢀ9acarb), 140.6
(1C, Cꢀ8acarb), 162.1 (1C, Cꢀ2oxadiazole), 163.2 (d, J = 254.6 Hz, 1C, Cꢀ4phenyl), 166.9
(1C, Cꢀ5oxadiazole), 168.6 (1C, CONH). IR (neat): ʋ (cmꢀ1) = 3267 (m, NꢀH), 2958 (m,
CꢀH, aliph), 1689 (s, C=O).
5.2.18 3-[5-(4-Bromo-2-fluorophenyl)-1,3,4-oxadiazol-2-yl]-N-[9-(2-fluoroethyl)-
9H-carbazol-3-yl]propanamide (9b)
According to the General Procedure, 8b (200 mg, 0.4 mmol) was treated with
XtalFluorꢀE® (130 mg, 0.6 mmol) and triethylamine trihydrofluoride (0.2 mL,
1.2 mmol) in CH2Cl2 (30 mL) at ꢀ 78 °C. The product was purified by fc (d = 3 cm,
l = 12 cm, cyclohexane/ethyl acetate 50:50, Rf 0.60 (ethyl acetate)). Colorless solid,
mp 207 ꢀ 208 °C, yield 110 mg (54 %). C25H19BrF2N4O2 (525.3 g/mol). Exact mass
(APCI): m/z = calcd. for C25H1979BrF2N4O2H 525.0732 found 525.0732. Purity
(HPLC): 95.2 % (tR = 21.83 min). 1H NMR (DMSOꢀD6): δ (ppm) = 2.95 (t, J = 7.1 Hz,
2H, CH2CH2CONH), 3.29 (t, J = 6.9 Hz, 2H, CH2CH2CONH), 4.69 (dt, J = 14.4/4.5
Hz, 2H, NCH2CH2F), 4.79 (dt, J = 34.8/4.5 Hz, 2H, NCH2CH2F), 7.18 (t, J = 7.4 Hz,
1H, 6ꢀHcarb), 7.43 (t, J = 7.7 Hz, 1H, 7ꢀHcarb), 7.49 ꢀ 7.58 (m, 3H, 1ꢀHcarb, 2ꢀHcarb, 5ꢀ
Hphenyl), 7.60 (d, J = 8.3 Hz, 1H, 8ꢀHcarb), 7.64 (dd, J = 8.7/1.7 Hz, 1H, 3ꢀHphenyl), 7.95
(t, J = 8.1 Hz, 1H, 6ꢀHphenyl), 8.04 (d, J = 7.8 Hz, 1H, 5ꢀHcarb), 8.41 (s, 1H, 4ꢀHcarb),
10.15 (s, 1H, CONH). 13C NMR (DMSOꢀD6): δ (ppm) = 21.9 (1C, CH2CH2CONH),
31.9 (1C, CH2CH2CONH), 42.9 (d, J = 19.7 Hz, 1C, NCH2CH2F), 82.6 (d, J = 167.9
Hz, 1C, NCH2CH2F), 109.5 (1C, Cꢀ8carb), 109.6 (1C, Cꢀ1carb), 111.0 (1 C, Cꢀ4carb),
111.8 (d, J = 10.3 Hz,1C, Cꢀ4phenyl), 116.2 (1C, Cꢀ2carb), 118.9 (1C, Cꢀ6carb), 120.1
(1C, Cꢀ5carb), 121.5 (d, J = 21.7 Hz, 1C, Cꢀ1phenyl), 121.9 (1C, Cꢀ4acarb), 122.1 (1C, Cꢀ
4bcarb), 122.5 (d, J = 26.1 Hz, 1C, Cꢀ3phenyl), 125.1 (d, J = 3.5 Hz,1C, Cꢀ5phenyl), 125.8

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(1C, Cꢀ7carb), 131.3 (1C, Cꢀ3carb), 132.5 (d, J = 9.4 Hz, 1C, Cꢀ6phenyl), 136.8 (1C, Cꢀ
9acarb), 140.6 (1C, Cꢀ8acarb), 162.8 (d, J = 252.1 Hz, 1C, Cꢀ2phenyl), 165.0 (1C, Cꢀ
2oxadiazole), 168.5 (1C, Cꢀ5oxadiazole), 180.6 (1C, CONH). IR (neat): ʋ (cmꢀ1) = 3267 (m,
NꢀH), 2958 (m, CꢀH, aliph), 1689 (s, C=O).
5.2.19 3-[5-(2-Cyano-4-fluorophenyl)-1,3,4-oxadiazol-2-yl]-N-[9-(2-fluoroethyl)-
9H-carbazol-3-yl]propanamide (9c)
According to the General Procedure, 8c (100 mg, 0.2 mmol) was treated with
XtalFluorꢀE® (73 mg, 0.3 mmol) and triethylamine trihydrofluoride (0.1 mL, 0.6 mmol)
in CH2Cl2 (30 mL) at ꢀ 78 °C. The product was purified by fc (d = 3 cm, l = 12 cm,
cyclohexane/ethyl acetate 50:50, Rf 0.60 (ethyl acetate)). Colorless solid, mp 209 ꢀ
211 °C, yield 25 mg (3 %). C26H19F2N5O2 (471.2 g/mol). Exact mass (APCI): m/z =
calcd. for C26H19F2N5O2H 472.1580 found 472.1596. Purity (HPLC): 95.5 % (tR =
1
20.13 min). H NMR (DMSOꢀD6): δ (ppm) = 3.01 (t, J = 6.9 Hz, 2H, CH2CH2CONH),
3.34 (t, J = 6.9 Hz, 2H, CH2CH2CONH), 4.69 (dt, J = 14.6/4.2 Hz, 2H, NCH2CH2F),
4.78 (dt, J = 38.5/4.1 Hz, 2H, NCH2CH2F), 7.18 (t, J = 7.4 Hz, 1H, 6ꢀHcarb), 7.44 (t, J =
7.6 Hz, 1H, 7ꢀHcarb), 7.54 (dd, J = 8.9/ 2.0 Hz, 1H, 2ꢀHcarb), 7.56 (d, J = 9.1 Hz, 1H, 1ꢀ
Hcarb), 7.60 (d, J = 8.4 Hz, 1H, 8ꢀHcarb), 7.81 (ddd, J = 8.7/8.3/2.7 Hz, 1H, 5ꢀHphenyl),
8.04 (d, J = 7.8 Hz, 1H, 5ꢀHcarb), 8.15 (dd, J = 8.6/2.7 Hz, 1H, 3ꢀHphenyl), 8.20 (dd, J =
8.9/5.3 Hz, 1H, 6ꢀHphenyl), 8.41 (s, 1H, 4ꢀHcarb), 10.15 (s, 1H, CONH). 13C NMR
(DMSOꢀD6): δ (ppm) = 20.8 (1C, CH2CH2CONH), 31.9 (1C, CH2CH2CONH), 45.3 (d,
J = 19.7 Hz, 1C, NCH2CH2F), 82.6 (d, J = 167.8 Hz, 1C, NCH2CH2F), 109.4 (1C, Cꢀ
8carb), 109.6 (1C, Cꢀ1carb), 109.6 (d, J = 7.2 Hz, 1C, Cꢀ2phenyl), 111.0 (1C, Cꢀ4carb),
118.8 (2C, Cꢀ2carb, Cꢀ6carb), 119.6 (d, J = 5.3 Hz, 1C, CN),120.0 (1C, Cꢀ5carb), 121.6
(d, J = 22.0 Hz, 1C, Cꢀ5phenyl), 121.9 (1C, Cꢀ4acarb), 122.3 (1C, Cꢀ4bcarb), 122.5 (d, J =

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3.1 Hz, 1C, Cꢀ1phenyl), 122.5 (d, J = 26.6 Hz, 1C, Cꢀ3phenyl), 125.8 (1C, Cꢀ7carb), 131.7
(1C, Cꢀ3carb), 131.8 (d, J = 10.8 Hz, 1C, Cꢀ6phenyl), 136.8 (1C, Cꢀ9acarb), 140.6 (1C, Cꢀ
8acarb), 160.1 (1C, Cꢀ2oxadiazole), 161.7 (d, J = 252.6 Hz, 1C, Cꢀ4phenyl), 167.3 (1C, Cꢀ
5
oxadiazole), 168.6 (1C, CONH). IR (neat): ʋ (cmꢀ1) = 3348 (m, NꢀH), 2924 (m, CꢀH,
aliph), 2221 (w, CN), 1615 (s, C=O).
5.3
Receptor binding studies to determine CB1 and CB2 receptor
affinity
[3H]CP55940 displacement assays were used for the determination of affinity (Ki)
values of ligands for the cannabinoid CB1 and CB2 receptors. Membrane aliquots
containing 5 ꢂg (CHOK1hCB1_bgal) or 1 ꢂg (CHOK1hCB2_bgal) of membrane
protein in 100 ꢂL assay buffer (50 mM Tris–HCl, 5 mM MgCl2, 0.1 % BSA, pH 7.4)
were incubated at 30 °C for 1 h, in presence of 3.5 nM [3H]CP55940
(CHOK1hCB1_bgal) or 1.5 nM [3H]CP55940 (CHOK1hCB2_bgal). Initially, 1 ꢁM of
competing ligand was used, followed by six concentrations of competing ligand
(between 10ꢀ5.5 M and 10ꢀ10.5 M) when more than 50 % displacement was found at
1 ꢁM. Nonꢀspecific binding was determined in the presence of 10 ꢂM AM630
(CHOK1hCB2_bgal) or 10 ꢂM SR141716A (CHOK1hCB1_bgal). Incubation was
terminated by rapid filtration through GF/C filters (Whatman International, Maidstone,
UK), and followed by extensive washing using a Filtermate 96ꢀwell harvester (Perkin
Elmer, Groningen, The Netherlands). Filterꢀbound radioactivity was determined by
scintillation spectrometry using a 1450 Microbeta Wallac Trilux scintillation counter
(Perkin Elmer).
Data analysis was performed by using the nonlinear regression curve fitting program

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GraphPad Prism 7.0 (GraphPad Software, Inc., San Diego, CA). From displacement
assays, IC50 values were obtained by nonꢀlinear regression analysis of the
displacement curves. The obtained IC50 values were converted into Ki values using
the Cheng Prusoff equation16 to determine the affinity of the ligands using a KD value
of [3H]CP55940 of 0.93 nM at CB2R.
Acknowledgement
Financial support by the Deutsche Forschungsgemeinschaft (DFG, collaborative
research center 656 “Molecular Cardiovascular Imaging”) is gratefully acknowledged.
Conflict of interest
The authors declare no competing interests.
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Graphical Abstract
Highlights
Three pairs of regioisomeric 1,2,4ꢀ and 1,3,4ꢀoxadiazoles were synthesized as
selective CB2 ligands. Although the 1,3,4ꢀoxadiazoles should have better
physicochemical and pharmacokinetic properties, the CB2 affinity was reduced by the
bioisosteric replacement.