Hydroxyalkylation with cyclic sulfates: Synthesis of carbazole derived CB2 ligands with increased polarity

Article abstract of DOI:10.1002/ardp.201300255

In order to increase the polarity of the potent CB₂ ligand 1a, the homologous hydroxyalkyl carbazoles 2a-c were prepared and pharmacologically evaluated. An important step in the synthesis is the hydroxyalkylation of carbazole with cyclic sulfa

Full text of DOI:10.1002/ardp.201300255

Arch. Pharm. Chem. Life Sci. 2014, 347, 21–31
21
Full Paper
Hydroxyalkylation with Cyclic Sulfates: Synthesis of Carbazole
Derived CB2 Ligands with Increased Polarity
Corinna Lueg1, Fabian Galla1, Bastian Frehland1, Dirk Schepmann1, Constantin G. Daniliuc2,
Winnie Deuther-Conrad3, Peter Brust3, and Bernhard Wünsch1
1
Institut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms-Universität Münster,
Münster, Germany
2
Organisch-chemisches Institut der Westfälischen Wilhelms-Universität Münster, Münster, Germany
Helmholtz-Zentrum Dresden-Rossendorf, Institut für Radiopharmazeutische Krebsforschung Forschungsstelle
3
Leipzig, Abteilung Neuroradiopharmaka, Leipzig, Germany
In order to increase the polarity of the potent CB2 ligand 1a, the homologous hydroxyalkyl carbazoles
2a–c were prepared and pharmacologically evaluated. An important step in the synthesis is the
hydroxyalkylation of carbazole with cyclic sulfates providing the 2-hydroxyethyl and 3-hydroxypropyl
derivatives 5a and 5b in a one-step reaction. The final propionamides 2a–c were prepared using the
recently reported coupling reagent COMU1. The X-ray crystal structure of 2c displays an almost
coplanar arrangement of the 3-phenyl-1,2,4-oxadiazole biaryl system. The increased polarity of 2a is
associated with an almost 100-fold reduced CB2 affinity. The 3-hydroxypropyl derivative 2b represents
the best compromise between lipophilicity and CB2 affinity (Ki ¼ 33 nM).
Keywords: Carbazole derivatives / CB2 receptor ligands / Cyclic sulfates / Structure affinity relationships / X-ray crystal
structure analysis
Received: July 11, 2013; Revised: September 2, 2013; Accepted: September 5, 2013
DOI 10.1002/ardp.201300255
Introduction
been approved as antiemetic and appetite stimulating drug
for AIDS and tumor patients. The semisynthetic D9-THC
analog nabilon (Cesamet1) is clinically used for the treatment
of chemotherapy induced emesis [3–5].
The endocannabinoid system exerts an important role in the
modulation of many physiological responses, in e.g., the
central nervous system, the immune system, and cardiovas-
cular system [1]. It consists of the endogenous ligands
anandamide (N-arachidonoyl ethanolamine, AEA) and 2-O-
arachidonoylglycerol (2-AG), which mediate their effects
through the G-protein coupled cannabinoid CB1 and CB2
receptors. Furthermore, the enzymes catalyzing the key steps
of the biosynthesis (e.g., N-acyltransferase, phosphodiesterase
D, diacylglycerollipase) and degradation of endocannabinoids
(e.g., fatty acid amide hydrolase, monoacylglycerollipase)
belong to the endocannabinoid system [2]. In the last decade,
several clinical studies were performed with cannabinoid
receptor ligands [3–8]. Dronabinol (D9-THC, Marinol1) has
The clinical use of CB1 receptor antagonists and inverse CB1
agonists like rimonabant is limited due to the development of
tolerance and severe side effects including depression and
elevated suicide potential [9]. However, there is an increasing
interest for the development of selective agonists for CB2
receptors, which are found in the immune system [10, 11],
but also in the central nervous system [12–15]. CB2 receptor
agonists show antihyperalgesic effects in different models
of pain, including acute, inflammatory, and neuropathic
pain [16–18]. In different models of neuroinflammation,
CB2 receptor agonists led to anti-inflammatory effects by e.g.,
reduced activation of microglia and reduced expression of
pro-inflammatory cytokines [16, 19–23]. Neuroinflammation
is associated with the progression of numerous chronic
neurodegenerative diseases including Alzheimer’s disease
[20], multiple sclerosis [23], and Huntington’s disease [24].
Considering the role of CB2 receptors in the regulation
of neuronal proliferation and survival [25–27] and the
Correspondence: Dr. Bernhard Wünsch, Institut für Pharmazeutische
und Medizinische Chemie der Westfälischen Wilhelms-Universität Mün-
ster, Corrensstraße, Münster, Germany.
E-mail: wuensch@uni-muenster.de
Fax: þ49 251 8332144
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22
C. Lueg et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 21–31
hydroxybutyl compound 2c was still below the logD value of
the parent ethyl derivative 1a.
CH3
N
Br
O
Results and discussion
F
N
N
H
Synthesis
O
N
The synthesis of the hydroxyalkyl substituted carbazole
derivatives 2a and 2b started with deprotonation of carb-
azole (3) with n-BuLi, reaction of the carbazolyl anion with
ethylene sulfate or trimethylene sulfate and subsequent
hydrolysis with diluted H2SO4 to afford the hydroxyethyl and
hydroxypropyl derivatives 5a and 5b, in 68 and 70% yield,
respectively (Scheme 1). In the literature the synthesis of 5a
and 5b has been reported by alkylation of carbazole (3) with
halogenated ethanol or propanol deriviatives [36–38]. The
crystalline cyclic ethylene sulfate represents a non-toxic but
reactive alternative for haloethanols and gaseous and very
toxic oxirane. Like ethylene sulfate the homologous tri-
methylene sulfate also represents a non-toxic, non-volatile but
reactive solid. However, there are only few examples using
these cyclic sulfates for the introduction of a 2-hydroxy-
ethyl [39–41] or 3-hydroxypropyl moiety [42–44]. Moreover,
hydroxyalkylation of N-nucleophiles with cyclic sulfates
has not been reported so far. The hydroxybutyl derivative
5c was obtained by reaction of the carbazolyl anion with
1,4-dibromobutane and subsequent treatment with NaOH.
Nitration of the hydroxyalkylcarbazoles 5a–c was per-
formed with concentrated nitric acid at 5–10°C to give the
mononitro derivatives 6a–c in 54–67% yields. Reduction of
the nitrocarbazoles 6a–c with H2 in the presence of Pd/C
provided the primary aromatic amines 7a–c, which were
isolated as HCl salts.
The propionic acid 9, which was required for the
preparation of the envisaged CB2 ligands 2, was prepared
according to literature [34]. Addition of hydroxylamine to
the benzonitrile 8 provided an intermediate amide oxime,
which was reacted with succinic anhydride to afford the
oxadiazolylpropionic acid 9 (Scheme 2). The final coupling of
the carbazol-3-amines 7 with the propionic acid 9 was induced
by the recently developed coupling reagent COMU1 [45–47],
which led to the amides 2a–c.
Recrystallization of the 4-hydroxybutyl derivative 2c with
cyclohexane/ethyl acetate led to crystals suitable for X-ray
crystal structure analysis. In Fig. 2 the structure of 2c is shown.
In the solid state the 4-hydroxybutyl and the propionamide
substructures are almost linearly arranged. The dihedral
angle defined by the 2-bromo-4-fluorophenyl and the 1,2,4-
oxadiazolyl planes is 9.7° and can be compared with the
corresponding angle of the cyanophenyl derivative (11.2°)
obtained from X-ray crystal structure experiments [35]. The
almost coplanar orientation of the biaryl substructure is
possible because of the missing substituents in the o-position
1a
OH
n
N
Br
O
F
N
N
H
O
N
2a
: n = 1
2b
: n = 2
2c
: n = 3
Figure 1. Designed CB2 ligands 2 with increased polarity due to an
additional hydroxy moiety in the carbazole N-alkyl side chain.
upregulation of CB2 receptors in neuroinflammatory and
neurodegenerative diseases [28–30], selective CB2 receptor
agonists represent an innovative therapeutic approach for the
treatment of neurodegenerative disorders avoiding psycho-
tropic side effects [31–33].
Recently Cheng et al. [34] reported carbazole based CB2
agonists of type 1 bearing different substituents at the phenyl
ring in 3-position of the 1,2,4-oxadiazole ring. Compounds
of type 1 reveal high CB2 agonistic activity and selectivity
against the CB1 subtype (Fig. 1). According to the method of
Cheng et al., the 2-bromo-4-fluorophenyl derivative 1a was
prepared and
a Ki-value (CB2 affinity) of 4.3 nM was
determined [35]. In order to improve the bioavailability of
the potent but very lipophilic CB2 agonist 1a, we planned to
enhance the polarity of 1a. For this purpose the ethyl side
chain in 9-position of the carbazole moiety should be replaced
by a hydroxyalkyl substituent (2). A particular aim of this
project was to study whether a rather polar hydroxy group
within the side chain of the lipophilic carbazole region is
tolerated by the CB2 receptor.
At first the logD-values (pH 7.4) of the CB2 agonist 1a and the
envisaged hydroxyalkyl analogs 2a–c were calculated with
ChemDraw Ultra 12. Table 1 shows that introduction of a
hydroxy group into the ethyl side chain (2a) decreased the
logD value by almost one order of magnitude from 5.99 (1a) to
5.13 (2a). Elongation of the side chain to a 3-hydroxpropyl (2b)
and furthermore a 4-hydroxybutyl (2c) group led to increased
lipophilicity, but even the logD value of the most lipophilic
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Arch. Pharm. Chem. Life Sci. 2014, 347, 21–31
CB2 Ligands by Hydroxyalkylation with Cyclic Sulfates
23
Scheme 1. Reagents and conditions: (a) n-BuLi, THF, ꢀ78°C, 30 min, then ethylene sulfate or trimethylene sulfate, ꢀ78°C, 1 h, then rt, 24 h,
then H2SO4, reflux, 24 h, 68% (5a), 70% (5b). (b) n-BuLi, THF, ꢀ78°C, 30 min, then Br(CH2)4Br, 0°C, 1 h, then rt, 24 h, 62%. (c) NaOH, Bu4NI,
diglyme, reflux, 45 min, 90% (5c). (d) HNO3 conc., CH2Cl2, 5–10°C, 54–67%. (e) H2 (balloon), Pd/C, THF, rt, 24 h, 90–96% of HCl salt.
of the 1,2,4-oxadiazole ring, although a large bromo atom is
present in o-position of the phenyl moiety. The distance
between the oxadiazole N-atom and the bromo atom (N3-Br1)
is 3.005 Å, indicating a weak interaction between these atoms.
This interaction could contribute to the planar orientation of
the two aromatic rings.
of a second molecule. The distance between the proton
and the O-atom is 2.840 Å and the corresponding angle
–
N–H···O( C) is 164°. Furthermore the amide is not coplanar
–
with the tricyclic system. The torsion angle is around 50°,
which might be due to the crystal packing effect.
The amide group of 2c displays a classical intermolecular
Receptor affinity
hydrogen bond between the N–H moiety and the O C moiety
Competition experiments with the radioligand [3H]CP-55,940
were performed to determine the CB2 receptor affinities of
the carbazole derivatives 2a–c. In the CB2 assay the test
compounds competed with the radioligand [3H]CP-55,940 to
interact with a rather low amount of CB2 receptors of a
membrane preparation generated from CHO cells stably
transfected with the gene for the human CB2 receptor [48, 49].
The CB2 affinities of the carbazole derivatives are summarized
in Table 1.
–
–
The data in Table 1 show that introduction of a polar
hydroxy moiety into the ethyl side chain of 1a (Ki ¼ 4.3 nM)
leads to a considerable reduction of the CB2 affinity. The
2-hydroxyethyl derivative 2a is almost 100-fold less active than
the ethyl derivative 1a. The lower CB2 affinity of 2a might be
due to the higher polarity: the additional hydroxy moiety
in the ethyl side chain may inhibit lipophilic interactions of
the carbazole part of the molecule with lipophilic parts of the
binding pocket of the CB2 receptor.
Scheme 2. Reagents and conditions: (a) 1. NH2OH·HCl, Na2CO3,
H2O, CH3OH, 86°C, workup; 2. succinic anhydride, DMF, 148°C,
30 min, 45%. (b) COMU1, NEt3, DMF, <10°C, 24 h, 30–80%.
Elongation of the N-substituent to a 3-hydroxypropyl side
chain (2b) leads to increased lipophilicity and CB2 affinity
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Arch. Pharm. Chem. Life Sci. 2014, 347, 21–31
Table 1. CB2 receptor affinities of carbazole derivatives 1a and 2a–c and reference compounds.
Compd.
R1
n
CB2 affinity Ki ꢁ SEM (nM) (n ¼ 3)
logDa) (pH 7.4)
1a
2a
2b
2c
H
1
1
2
3
4.3 ꢁ 3.035
5.99
5.13
5.24
5.69
OH
OH
OH
367b)
33 ꢁ 11
96 ꢁ 29
27 ꢁ 6
CP 55,940
WIN 55,212
HU 210
70 ꢁ 27
18 ꢁ 5
a)
Calculated (ChemDraw) logD values at a pH of 7.4.
Due to low affinity, the CB2 affinity was only recorded once.
b)
Figure 2. A view of the molecular structure of the 4-hydroxybutyl derivative 2c: selected bond length (Å) and angles (°): N1-C1 1.368(8);
N1-C12 1.387(6); N1-C24 1.431(8); N2-C9 1.427(8); N2-C13 1.323(8); O1-C13 1.223(7); N3-O2 1.424(6); N3-C17 1.307(8); N4-C17 1.386
(7); C19-Br1 1.891(8); C21-F1 1.332(9); C1-N1-C24-C25 -84.7(8); N4-C17-C18-C19 169.9(6): N3-C17-C18-C19 -10.3(10); C13-N2-C9-C10
49.5(5); C13-N2-C9-C8 ꢀ133.0(8)°. N3-Br1 3.005(5); C17-N3-Br1 91.3; C19-Br1-N3 73.1. Intermolecular H-bond: N2-H02…O1 2.840(7) Å,
164(5).
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Arch. Pharm. Chem. Life Sci. 2014, 347, 21–31
CB2 Ligands by Hydroxyalkylation with Cyclic Sulfates
25
compared with 2a. Although the lipophilicity is further
increased by the 4-hydroxybutyl side chain of 2c, the CB2
affinity is reduced (Ki ¼ 96 nM). The 3-hydroxypropyl deriva-
tive 2b represents the best compromise between lipophilicity
and CB2 affinity.
NaOH, brine was added and the mixture was extracted with
CH2Cl2. The organic layers were dried (Na2SO4), concentrated
under reduced pressure and the residue was purified by fc.
General Procedure B for the nitration of 9-(hydroxyalkyl)-
carbazoles 5
The respective 9-(hydroxyalkyl)carbazole
5 (1.0 equiv.) was
Conclusions
dissolved in CH2Cl2 (2 mL per 100 mg) and cooled down to
5–10°C. Concentrated nitric acid (1.5 equiv.) was added dropwise
under vigorous stirring. Stirring was continued at <10°C until
the starting material was transformed completely. H2O (0.25 mL
per 1 mL CH2Cl2) was added, the reaction mixture was
neutralized with NaHCO3, brine was added and the mixture
was extracted with CHCl3. The organic layer was dried (Na2SO4),
concentrated under reduced pressure, the residue was purified
by fc.
Introduction of a hydroxy moiety into the alkyl side chain
attached to the carbazole N-atom leads to CB2 ligands with
moderate affinity compared with the CB2 agonist 1a bearing
an ethyl substituent at the carbazole ring system. However the
hydroxy group is tolerated by the CB2 receptor, since the CB2
affinity of the hydroxypropyl derivative 2b is still very high.
The novel developed strategy of the one-pot introduction
of hydroxyalkyl substituents via cyclic sulfates allows the
fast access of these type of ligands and, additionally,
the further modification of the properties of the carbazole
N-substituent.
General Procedure C for the reduction of 3-nitro-9-
(hydroxyalkyl)carbazoles 6
The respective 3-nitro-9-(hydroxyalkyl)carbazole 6 (1.0 equiv.) was
dissolved in THF (2 mL per 100 mg) and Pd/C (10 wt.% loading)
was added. The reaction mixture was stirred for 24 h at rt under
H2 (balloon). The catalyst was removed by filtration over Celite1
and the filtrate was concentrated under reduced pressure.
The residue was dissolved in Et2O. Under N2, HCl · Et2O (2 M,
2.0 equiv.) was added dropwise under vigorous stirring to
produce the respective HCl salt. The precipitate was filtered,
washed with cold MeOH, dried (Na2SO4) and used without
further purification.
Experimental
Chemistry, general
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 mm (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 (Thermo-Finnigan); IR: IR spectropho-
tometer 480Plus FT-ATR-IR (Jasco). 1H NMR (400 MHz), 13C NMR
(100 MHz): Unity Mercury Plus 400 spectrometer (Varian); d 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: L-7400;
autosampler: L-7200; pump: L-7100; degasser: L-7614; Method:
column: LiChrospher1 60 RP-select B (5 mm), 250–4 mm cartridge;
flow rate: 1.00 mL minꢀ1; injection volume: 5.0 mL; detection at
l ¼ 210 nm; solvents: A: water with 0.05% v/v trifluoroacetic acid;
B: acetonitrile with 0.05% v/v trifluoroacetic acid: gradient
elution: (A%): 0–4 min: 90%, 4–29 min: gradient from 90 to 0%,
29–31 min: 0%, 31–31.5 min: gradient from 0 to 90%, 31.5–40 min:
90%.
General Procedure D for the synthesis of 3-(3-phenyl)-
1,2,4-oxadiazol-5-yl)propanoic acid 9 [34]
NH2OH · HCl (1.0 equiv.) was dissolved in H2O (0.4 mL per mmol),
Na2CO3 (0.5 equiv.) was added and the mixture was stirred for
25 min at rt. A solution of benzonitrile 8 (1.0 equiv.) in CH3OH
(1.8 mL per 1 mmol) was added under vigorous stirring and the
mixture was stirred at 86°C until the nitrile 8 was converted.
Then, CH3OH was removed under reduced pressure, the residue
was diluted with brine and the mixture was extracted with
CH2Cl2. The organic layer was dried (Na2SO4) and concentrated
in vacuo. Succinic anhydride (0.9 equiv.) and DMF (0.5 mL) were
added to the residue and the reaction mixture was stirred
for 30 min at 148°C. The solvent was removed in vacuo and the
residue was purified by fc (cyclohexane/ethyl acetate/formic acid
50:50:0.01). The final product was purified by recrystallization
(CH2Cl2/H2O).
General Procedure E for the COMU1 coupling of 1,2,4-
General procedures
General Procedure A for the hydroxyalkylation of
carbazole 3
Under N2 9H-carbazole (3, 1.0 equiv.) was dissolved in THF (1 mL
per 100 mg) and cooled down to ꢀ78°C. n-BuLi (ca. 1.6 M in
n-hexane, 1.2 equiv.) was added dropwise and the mixture was
stirred for 30 min at ꢀ78°C. Then a solution of the respective
cyclic sulfate (1.1 equiv.) in THF (15 mL) was added and the
mixture was stirred for 1 h at ꢀ78°C and 24 h at rt. H2O (5 mL) was
added, the mixture was acidified with H2SO4 conc. and heated to
reflux for 24 h. Afterwards, the solution was neutralized with 5 M
oxadiazole carboxylic acid 9 with 3-aminocarbazoles 7
COMU1 (1.2–1.5 equiv.) was added to a mixture of 3-(1,2,4-
oxadiazolyl)propanooic acid 9 (1.0 equiv.) and triethylamine (2.0–
3.0 equiv.) in DMF and the mixture was stirred for 30 min at rt.
The reaction mixture was cooled down to 0°C and a solution of
the respective 3-aminocarbazole hydrochloride 7 (1.0 equiv.) in
DMF was added dropwise. This mixture was stirred for 24 h at
<10°C. Then H2O and brine were added, the mixture was
extracted with CHCl3, the combined organic layers were dried
(Na2SO4), concentrated in vacuo and the residue was purified by fc
and finally by recrystallization with CH2Cl2.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 21–31
Experimental procedures
9-(4-Bromobutyl)-9H-carbazole (4)
2H, NCH2CH2CH2OH), 4.25 (t, J ¼ 6.6 Hz, 2H, NCH2CH2CH2OH),
7.04 (m, 4H, 2-H, 3-H, 6-H, 7-H), 7.27 (d, 2H, 1-H, 8-H), 7.91
(d, J ¼ 7.7 Hz, 2H, 4-H, 5-H). 13C NMR (CDCl3): d (ppm) ¼ 31.5
(1C, NCH2CH2CH2OH), 39.3 (1C, NCH2CH2CH2OH), 59.7 (1C,
NCH2CH2CH2OH), 108.8 (2C, C-1, C-8), 119.0 (2C, C-3, C-6), 120.5
(2C, C-4, C-5), 123.0 (2C, C-4a, C-4b), 125.8 (2C, C-2, C-7), 140.6
(2C, C-8a, C-9a). IR (neat): ~n (cmꢀ1) ¼ 3344 (m, O–H), 3047, 3020
(w, C–H, arom), 2943 (m, C–H, aliph).
Under N2 9H-carbazole (6.0 g, 35.9 mmol) was dissolved in THF
(60 mL) and cooled down to ꢀ78°C. n-BuLi (1.6 M in n-hexane,
29.2 mL, 46.7 mmol) was added dropwise and the mixture was
stirred for 30 min at ꢀ78°C. This mixture was added via a cannula
to a solution of 1,4-dibromobutane (16.5 mL, 140 mmol) in THF
(15 mL) at 0°C and the mixture was stirred for 1 h at 0°C and for
24 h at rt. Then H2O (5 mL) was added, the solution was
neutralized with 5 M NaOH, brine was added and the mixture
was extracted with CH2Cl2. The organic layers were dried
(Na2SO4), concentrated in vacuo and the residue was purified by
fc [d ¼ 8 cm, l ¼ 14 cm, cyclohexane, Rf 0.43 (cyclohexane/ethyl
acetate 90:10)]. Colorless needles, mp 62–64°C, yield 7.3 g (62%).
4-(9H-Carbazol-9-yl)butan-1-ol (5c)
Solid NaOH (79.4 mg, 1.99 mmol) was added to a solution of
bromide
4 (120 mg, 0.397 mmol) and tetrabutylammonium
iodide (44 mg, 0.12 mmol) in diglyme (5 mL) and the mixture
was heated to reflux for 45 min. After neutralization with 5 M HCl
and saturated solution of NaHCO3, brine was added to the
reaction mixture. The mixture was extracted with CHCl3, the
organic layers were dried (Na2SO4), concentrated under reduced
pressure and the residue was purified by fc [d ¼ 2 cm, l ¼ 14 cm,
cyclohexane/ethyl acetate 75:25, Rf 0.25 (cyclohexane/ethyl
acetate 60:40)]. Colorless solid, mp 68–69°C, yield 85.4 mg
(90%). C16H17NO (239.3 g/mol). Exact mass (APCI): m/z ¼ calcd.
for C16H17NOH 240.1388; Found: 240.1383. Purity (HPLC): 99.2%
C
16H16BrN (302.2 g molꢀ1). Exact mass (APCI): m/z ¼ calcd. for
C16H1679BrNH 302.0539; Found: 302.0521. Purity (HPLC): 95.61%
(tR ¼ 19.58 min). 1H NMR (CDCl3): d (ppm) ¼ 1.88–1.97 (m, 2H,
NCH2CH2CH2CH2Br), 2.03–2.12 (m, 2H, NCH2CH2CH2CH2Br), 3.39
(t, J ¼ 6.5 Hz, 2H, NCH2CH2CH2CH2Br), 4.37 (t, J ¼ 6.9 Hz, 2H,
NCH2CH2CH2CH2Br), 7.24 (t, J ¼ 6.3 Hz, 2H, 3-H, 6-H), 7.41
(t, J ¼ 8.2 Hz, 2H, 2-H, 7-H), 7.46–7.49 (m, J ¼ 7.6 Hz, 2H, 1-H,
8-H), 8.11 (d, J ¼ 7.8 Hz, 2H, 4-H, 5-H). 13C NMR (CDCl3): d (ppm) ¼ 30.4
(1C, NCH2CH2CH2CH2Br), 31.1 (1C, NCH2CH2CH2CH2Br), 33.3
(1C, NCH2CH2CH2CH2Br), 42.3 (1C, NCH2CH2CH2CH2Br), 108.8
(2C, C-1,C-8), 120.5 (2C, C-4,C-5), 118.7 (2C, C-3, C-6), 123.0 (2C,
C-4a, C-4b), 125.9 (2C, C-2, C-7), 140.4 (2C, C-8a, C-9a). IR (neat):
~n (cmꢀ1) ¼ 3051 (m, C–H, arom), 2958, 2927 (m, C–H, aliph), 651,
617 (s, CH2–Br).
1
(tR ¼ 19.33 min). H NMR (DMSO-D6): d (ppm) ¼ 1.39–1.47 (m, 2H,
NCH2CH2CH2CH2OH), 1.75–1.84 (m, 2H, NCH2CH2CH2CH2OH),
3.38 (m, 2H, NCH2CH2CH2CH2OH), 4.41 (t, J ¼ 5.2 Hz, 2H,
NCH2CH2CH2CH2OH), 7.19 (t, J ¼ 6.3 Hz, 2H, 3-H, 6-H), 7.45
(t, J ¼ 7.5 Hz, 2H, 2-H, 7-H), 7.60 (d, J ¼ 8.2 Hz, 2H, 1-H, 8-H), 8.14
(d, J ¼ 7.8 Hz, 2H, 4-H, 5-H). 13C NMR (DMSO-D6): d (ppm) ¼ 25.4
(s, 1C, NCH2CH2CH2CH2OH), 30.1 (s, 1C, NCH2CH2CH2CH2OH), 42.3
(s, 1C, NCH2CH2CH2CH2OH), 60.6 (s, 1C, NCH2CH2CH2CH2OH),
109.4 (s, 2C, C-1, C-8), 118.8 (s, 2C, C-3, C-6), 120.4 (s, 2C, C-4, C-5),
122.1 (s, 2C, C-4a, C-5a), 125.8 (s, 2C, C-2, C-7), 140.1 (s, 2C, C-8a,
C-9a). IR (neat): ~n (cmꢀ1) ¼ 3244 (m, O–H), 3051 (m, C–H, arom),
2935 (m, C–H, aliph).
2-(9H-Carbazol-9-yl)ethan-1-ol (5a)
Following the General Procedure A, n-BuLi (1.2 M in n-hexane,
32.4 mL, 38.9 mmol, 1.2 equiv.) was added dropwise to 9H-carb-
azole (5.43 g, 32.5 mmol, 1.0 equiv.) in THF (55 mL) and, after
30 min,
a solution of ethylene sulfate (4.51 g, 36.3 mmol,
1.1 equiv.) in THF (15 mL) was added. The crude product was
purified by fc (d ¼ 8 cm, l ¼ 12 cm, cyclohexane/ethyl acetate
65:35, Rf 0.42). Pale yellow solid, mp 79–81°C, yield 4.66 g (68%).
C14H13NO (211.3 g molꢀ1). Exact mass (APCI): m/z ¼ calcd. for
C14H13NOH 212.1069; Found: 212.1060. Purity (HPLC): 98.1%
2-(3-Nitro-9H-carbazol-9-yl)ethan-1-ol (6a)
According to the General Procedure B a solution of 5a
(5.11 g, 24.2 mmol) in CH2Cl2 (100 mL) was treated with
HNO3 conc. (1.7 mL, 36.3 mmol). The product was purified
by fc (d ¼ 8 cm, l ¼ 12 cm, cyclohexane/ethyl acetate 50:50,
Rf 0.27). Pale yellow solid, mp 230°C, yield 3.34 g (54%).
C14H12N2O3 (256.3 g molꢀ1). Exact mass (APCI): m/z ¼ calcd. for
C14H12N2O3Na 279.0740; Found: 279.0740. Purity (HPLC): 97.8%
(tR ¼ 18.87 min). 1H NMR (CDCl3): d (ppm) ¼ 4.13 (t, J ¼ 5.3 Hz,
2H, NCH2CH2OH), 4.55 (t, J ¼ 5,3 Hz, 2H, NCH2CH2OH), 7.38
(t, J ¼ 8.0 Hz, 1H, 6-H), 7.51–7.60 (m, 3H, 1-H, 7-H, 8-H), 8.13
(d, J ¼ 7.5 Hz, 1H, 5-H), 8.39 (dd, J ¼ 8.9/2.0 Hz, 1H, 2-H), 9.03
(d, J ¼ 2.2 Hz, 1H, 4-H). 13C NMR (DMSO-D6): d (ppm) ¼ 39.4
(1C, NCH2CH2OH), 59.6 (1C, NCH2CH2OH), 110.2 (1C, C-8), 110.9
(1C, C-1), 117.3 (2C, C-4, C-6), 120.7 (1C, C-5), 121.3 (1C, C-2), 122.0
(1C, C-4b), 122.3 (1C, C-7), 127.4 (1C, C-4a), 139.9 (1C, C-3), 141.8
(1C, C-8a), 144.1 (1C, C-9a). IR (neat): ~n (cmꢀ1) ¼ 3414 (s, O–H), 3051
(m, C–H, arom), 1492, 1323 (s, NO2).
1
(tR ¼ 19.28 min). H NMR (DMSO-D6): d (ppm) ¼ 3.86 (quart, J ¼ 5.9
Hz, 2H, NCH2CH2OH), 4.47 (t, J ¼ 5.5 Hz, 2H, NCH2CH2OH), 4.99
(t, J ¼ 5.0Hz, 1H, NCH2CH2OH), 7.24 (t, J ¼ 7.4Hz, 2H, 3-H, 6-H), 7.48
(t, J ¼ 7.6 Hz, 2H, 2-H, 7-H), 8.18 (d, J ¼ 7.8Hz, 2H, 4-H, 5-H). 13C NMR
(CDCl3): d (ppm) ¼ 45.3 (1C, NCH2CH2OH), 59.6 (1C, NCH2CH2OH),
109.6 (2C, C-1, C-8), 118.6 (2C, C-3, C-6), 120.2 (2C, C-4, C-5),
122.2 (2C, C-4a, C-4b), 125.0 (2C, C-2, C-7), 140.5 (2C,C-8a, C-9a). IR
(neat): n (cmꢀ1) ¼ 3356 (m, O–H), 3045, 3019 (w, C–H, arom), 2943
~
(w, C–H, aliph).
3-(9H-Carbazol-9-yl)propan-1-ol (5b)
Following the General Procedure A, n-BuLi (1.3 M in n-hexane,
29.9 mL, 38.9 mmol, 1.2 equiv.) was added dropwise to 9H-
carbazole (5.43 g, 32.5 mmol, 1.0 equiv.) in THF (55 mL) and, after
30 min a solution of trimethylene sulfate (5.01 g, 36.3 mmol,
1.1 equiv.) in THF (15 mL) was added. The crude product was
purified by fc (d ¼ 8 cm, l ¼ 12 cm, cyclohexane/ethyl acetate
65:35, Rf 0.45). Colorless solid, mp 102–105°C, yield 5.1 g (70%).
C15H15NO (225.3 g molꢀ1). Exact mass (APCI): m/z ¼ calcd.
for C15H15NOH 226.1226; Found: 226.1129. Purity (HPLC):
3-(3-Nitro-9H-carbazol-9-yl)propan-1-ol (6b)
According to the General Procedure B a solution of 5b (5.0 g,
25.0 mmol) in CH2Cl2 (100 mL) was treated with HNO3 conc.
(1.75 mL, 37.5 mmol). The product was purified by fc (d ¼ 5 cm,
l ¼ 11 cm, cyclohexane/ethyl acetate 50:50, Rf 0.31). Yellow solid,
mp 188–190°C, yield 4.25 g (62%). C15H14N2O3 (270.3 g molꢀ1).
97.8% (tR ¼ 19.35 min). 1H NMR (CDCl3):
d
(ppm) ¼ 1.90
(dt, J ¼ 12.4/6.4/6.0 Hz, 2H, NCH2CH2CH2OH), 3.39 (t, J ¼ 5.9 Hz,
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2014, 347, 21–31
CB2 Ligands by Hydroxyalkylation with Cyclic Sulfates
27
Exact mass (APCI): m/z ¼ calcd. for C15H14N2O3H 271.1077; Found:
3-(3-Amino-9H-carbazol-9-yl)propan-1-ol hydrochloride
(7b·HCl)
1
279.1066. Purity (HPLC): 95.6% (tR ¼ 19.58 min). H NMR (CDCl3):
d (ppm) ¼ 1.60 (s, 1H, NCH2CH2CH2OH), 2.14 (quint, J ¼ 6.3 Hz, 2H,
NCH2CH2CH2OH), 3.64 (m, 2H, NCH2CH2CH2OH), 4.54 (t, J ¼ 6.7
Hz, 2H, NCH2CH2CH2OH), 7.35 (t, J ¼ 7.8 Hz, 1H, 6-H), 7.49–
7.60 (m, 3H, 1-H, 7-H, 8-H), 8.15 (d, J ¼ 8.1 Hz, 1H, 5-H),
8.37 (dd, J ¼ 9.1/2.3 Hz, 1H, 2-H), 9.00 (d, J ¼ 2.2 Hz, 1H, 4-H).
13C NMR (DMSO-D6): d (ppm) ¼ 31.6 (1C, NCH2CH2CH2OH), 39.3
(1C, NCH2CH2CH2OH), 57.7 (1C, NCH2CH2CH2OH), 109.5 (1C, C-8),
110.3 (1C, C-1), 117.3 (1C, C-6), 120.6 (1C, C-4), 121.3 (1C, C-5), 121.4
(1C, C-2)121.8 (1C, C-4b), 122.2 (1C, C-7), 127.4 (1C, C-4a),
139.9 (1C, C-3), 141.6 (1C, C-8a), 143.3 (1C, C-9a). IR (neat):
Following the General Procedure C, 6b (1.3 g, 4.8 mmol, 1.0 equiv.)
was dissolved in THF (26 mL), Pd/C (260 mg) was added and the
mixture was stirred under H2 atmosphere (balloon) for 24 h.
The amine was precipitated with HCl · Et2O [2 mol Lꢀ1, 4.8 mL,
9.6 mmol, 2.0 equiv., Rf 0.37 (cyclohexane/ethyl acetate 15:85)].
Colorless solid, mp 228–230°C, yield 1.21 g (91%). C14H15ClN2O
(276.8 g molꢀ1). Exact mass (APCI): m/z ¼ calcd. for C15H17N2O
241.1335; Found 241.1324. Purity (HPLC): 97.8% (tR ¼ 12.64 min).
1H NMR (7b · HCl, DMSO-D6): d (ppm) ¼ 1.91 (quint, J ¼ 6.5 Hz, 2H,
NCH2CH2CH2OH), 3.78 (t, J ¼ 5.5 Hz, 2H, NCH2CH2CH2OH), 4.47
(t, J ¼ 5.5Hz, 2H, NCH2CH2CH2OH), 7.24 (t, J ¼ 7.4 Hz, 1H, 6-H), 7.45
(dd, J ¼ 8.7, 2.1 Hz, 1H, 2-H), 7.50 (t, J ¼ 7,7 Hz, 1H, 7-H), 7.66
(d, J ¼ 8.3 Hz, 1H, 1-H), 7.73 (d, J ¼ 8.7Hz, 1H, 8-H), 8.13 (d, J ¼ 2.1 Hz,
1H, 4-H), 8.19 (d, J ¼ 7.7Hz, 1H, 5-H), 10.4 (s, 2H, –NH2). 13C NMR
(7b · HCl, DMSO-D6): d (ppm) ¼ 31.7 (s, 1C, NCH2CH2CH2OH), 45.5
(s, 1C, NCH2CH2CH2OH), 57.9 (s, 1C, NCH2CH2CH2OH), 109.7 (s, 1C,
C-4), 110.2 (s, 1C, C-8), 114.8 (s, 1C, C-1), 119.2 (s, 1C, C-2), 120.6 (s, 1C,
C-6), 121.3 (s, 1C, C-5), 122.2 (s, 1C, C-4b), 122.8 (s, 1C, C-7), 126.6
(s, 2C, C-4a, C-9a), 139.1 (s, 1C, C-8a), 140.7 (s, 1C, C-3). IR (neat):
~n (cmꢀ1) ¼ 3263 (s, –NH2), 2889 (m, C–H, aliph).
n (cmꢀ1) ¼ 3344 (s, O–H), 3041 (m, C–H, arom), 2935 (m, C–H,
~
aliph), 1495, 1323 (s, NO2).
4-(3-Nitro-9H-carbazol-9-yl)butan-1-ol (6c)
According to the General Procedure B a solution of 5c (2.5 g,
10.45 mmol) in CH2Cl2 (50 mL) was treated with HNO3 conc.
(0.73 mL, 15.68 mmol). The product was purified by fc (d ¼ 6 cm,
l ¼ 12 cm, cyclohexane/ethyl acetate 50:50, Rf 0.28). Yellow solid,
mp 87–89°C, yield 1.99g (67%). C16H16N2O3 (270.3 g molꢀ1). Exact
mass (APCI): m/z ¼ calcd. for C16H16N2O3H 285.1234; Found:
279.1238. Purity (HPLC): 99.4% (tR ¼ 19.50 min). 1H NMR (DMSO-
D6): d (ppm) ¼ 1.43 (m, 2H, NCH2CH2CH2CH2OH), 1.83 (m, 2H,
NCH2CH2CH2CH2OH), 3.38 (m, 2H, NCH2CH2CH2CH2OH), 4.43
(t, J ¼ 5.1 Hz, 1H, NCH2CH2CH2CH2OH), 4.52 (t, J ¼ 7.1 Hz, 2H,
NCH2CH2CH2CH2OH), 7.34 (t, J ¼ 7.5 Hz, 1H, 6-H), 7.59 (t, J ¼ 7.7 Hz,
1H, 7-H), 7.75 (d, J ¼ 8.3 Hz, 1H, 8-H), 7.82 (d, J ¼ 9.1 Hz, 1H, 5-H), 8.35
(dd, J ¼ 9.1/2.3 Hz 1H, 2-H), 8.42 (d, J ¼ 7.9 Hz, 1H, 1-H), 9.19
(d, J ¼ 2.2 Hz, 1H, 4-H). 13C NMR (DMSO-D6): d (ppm) ¼ 25.4
(1C, NCH2CH2CH2CH2OH), 29.9 (1C, NCH2CH2CH2CH2OH), 42.9
(1C, NCH2CH2CH2CH2OH), 60.5 (1C, NCH2CH2CH2CH2OH), 109.7
(1C, C-8), 110.6 (1C, C-1), 117.5 (1C, C-6), 120.7 (1C, C-4), 121.4 (1C,
C-5), 121.6 (1C, C-2), 122.0 (1C, C-4b), 122.3 (1C, C-7), 127.6 (1C, C-4a),
140,0 (1C, C-3), 141.5 (1C, C-8a), 143.4 (1C, C-9a). IR (neat):
4-(3-Amino-9H-carbazol-9-yl)butan-1-ol hydrochloride
(7c·HCl)
Following the General Procedure C, 6c (500 mg, 1.71 mmol,
1.0 equiv.) was dissolved in THF (10 mL), Pd/C (150 mg) was added
and the mixture was stirred under H2 atmosphere (balloon) for
24 h. The amine was precipitated with HCl · Et2O [2 mol Lꢀ1
,
1.7 mL, 3.4 mmol, 2.0 equiv., Rf 0.37 (cyclohexane/ethyl acetate
15:85)]. Colorless solid, mp 182°C, yield 448.3 mg (90%).
C
16H19ClN2O (290.8 g molꢀ1). Exact mass (APCI): m/z ¼ calcd. for
C16H19N2O 255.1492; Found: 241.1475. Purity (HPLC): 97.6%
(tR ¼ 13.16 min). 1H NMR (7c · HCl, DMSO-D6): d (ppm) ¼ 1.41 (quint,
J ¼ 6.5 Hz, 2H, NCH2CH2CH2CH2OH), 1.80 (quint, J ¼ 6.5Hz, 2H,
NCH2CH2CH2CH2OH), 3.39 (t, J ¼ 5.5Hz, 2H, NCH2CH2CH2CH2OH),
4.44 (t, J ¼ 5.5Hz, 2H, NCH2CH2CH2CH2OH), 7.24 (t, J ¼ 7.4 Hz, 1H,
6-H), 7.46 (dd, J ¼ 8.7/2.1 Hz, 1H, 2-H), 7.52 (t, J ¼ 7,7 Hz, 1H, 7-H),
7.66 (d, J ¼ 8.3 Hz, 1H, 1-H), 7.74 (d, J ¼ 8.7 Hz, 1H, 8-H), 8.12
(d, J ¼ 2.1 Hz, 1H, 4-H), 8.20 (d, J ¼ 7.7 Hz, 1H, 5-H), 10.4 (s, 2H,
n (cmꢀ1) ¼ 3244 (s, O–H), 3043 (m, C–H, arom), 2935 (m, C–H, aliph),
~
1495, 1323 (s, NO2).
2-(3-Amino-9H-carbazol-9-yl)ethan-1-ol hydrochloride
(7a HCl)
–NH2). 13C NMR (7c · HCl, DMSO-D6):
d
(ppm) ¼ 25.4 (1C,
Following the General Procedure C, 6a (500 mg, 1.95 mmol,
1.0 equiv.) was dissolved in THF (10 mL), Pd/C (100 mg) was
added and the mixture was stirred under H2 atmosphere
(balloon) for 18 h. The amine was precipitated with HCl · Et2O
[2 mol Lꢀ1, 2 mL, 4 mmol, 2.0 equiv., Rf 0.35 (cyclohexane/ethyl
acetate 30:70)]. Colorless solid, mp 230°C, yield 492 mg (96%).
C14H15ClN2O (262.7 g molꢀ1). Exact mass (APCI): m/z ¼ calcd.
NCH2CH2CH2CH2OH), 30.0 (1C, NCH2CH2CH2CH2OH), 42.5 (1C,
NCH2CH2CH2CH2OH), 60.5.9 (1C, NCH2CH2CH2CH2OH), 109.9 (1C,
C-4), 110.4 (1C, C-8), 114.9 (1C, C-1), 119.4 (1C, C-2), 120.7 (1C, C-6),
120.8 (1C, C-5), 121.4 (1C, C-4b), 122.3 (1C, C-7), 122.8 (1C, C-4a),
126.8 (1C, C-9a), 139.2 (1C, C-8a), 140.8 (1C, C-3). IR (neat):
n (cmꢀ1) ¼ 3282 (s, –NH2), 2885 (m, C–H, aliph).
~
for
C14H15N2O 217.1179; Found 217.1179. Purity (HPLC):
99.0% (tR ¼ 11.64 min). 1H NMR (7a · HCl, DMSO-D6):
d (ppm) ¼ 3.78 (t, J ¼ 5.5 Hz, 2H, NCH2CH2OH), 4.47 (t, J ¼ 5.5 Hz,
2H, NCH2CH2OH), 7.24 (t, J ¼ 7.4 Hz, 1H, 6-H), 7.45 (dd, J ¼ 8.7/
2.1 Hz, 1H, 2-H), 7.50 (t, J ¼ 7.7 Hz, 1H, 7-H), 7.66 (d, J ¼ 8.3 Hz,
1H, 1-H), 7.74 (d, J ¼ 8.7 Hz, 1H, 8-H), 8.12 (d, J ¼ 2.1 Hz, 1H, 4-H),
8.18 (d, J ¼ 7.7 Hz, 1H, 5-H), 10.4 (s, 2H, –NH2). 13C NMR
(7a · HCl, DMSO-D6): d (ppm) ¼ 45.5 (1C, NCH2CH2OH), 59.7
(1C, NCH2CH2OH), 110.2 (1C, C-1), 110.8 (1C, C-8), 114.8
(1C, C-4), 119.3 (1C, C-2), 120.6 (1C, C-6), 121.5 (1C, C-5), 122.3
(1C, C-4b), 122.8 (1C, C-7), 126.7 (2C, C-4a, C-9a), 139.7 (1C, C-8a),
141.2 (1C, C-3). IR (neat): ~n (cmꢀ1) ¼ 3344 (s, O–H), 3041 (m, C–H,
arom), 2935 (m, C–H, aliph).
3-[3-(2-Bromo-4-fluorophenyl)-1,2,4-oxadiazol-5-yl]-
propanoic acid (9) [34]
According to General Procedure D, 2-bromo-4-fluorobenzonitrile
(8, 5.0 g, 25.0 mmol) was reacted with NH2OH · HCl (5.21 g,
75.0 mmol) and Na2CO3 (3.98 g, 37.5 mmol) in H2O (15 mL) and
CH3OH (45 mL). The intermediate was purified by fc (d ¼ 8 cm,
l ¼ 12 cm, cyclohexane/ethyl acetate 50:50, Rf 0.27, yield 492 mg,
39%) and subsequently reacted with succinic anhydride (2.5 g,
25.0 mmol) in DMF (0.5 mL). The product was purified by
recrystallization [CH2Cl2/H2O, Rf 0.46 (cyclohexane/ethyl acetate/
formic acid 50:50:0.01)]. Colorless solid, mp 114–116°C, yield 2.9 g
(45%). C11H8BrFN2O3 (315.1 g/mol). Exact mass (APCI): m/z ¼ calcd.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

28
C. Lueg et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 21–31
for C11H879BrFN2O3H 314.9775; Found: 314.9760. Purity (HPLC):
7.52–7.54 (m, 2H, 1-Hcarb, 2-Hcarb), 7.57 (d, J ¼ 8.3 Hz, 1H, 8-Hcarb),
99.9% (tR ¼ 17.15 min). 1H NMR (CDCl3):
d
(ppm) ¼ 3.02 (t,
7.78–7.90 (m, J ¼ 8.6/2.5 Hz, 1H, 3-Hphenyl
, 6-Hphenyl), 8.01
J ¼ 7.1 Hz, 2H, CH2CH2CO2H), 3.29 (t, J ¼ 7.1 Hz, 2H, CH2CH2CO2H),
7.15 (ddd, J ¼ 8.7/7.7/2.6 Hz, 1H, 5-H), 7.47 (dd, J ¼ 8.2/2.6 Hz, 1H, 3-
H), 7.85 (dd, J ¼ 8.7/6.0 Hz, 1H, 6-H). 13C NMR (DMSO-D6):
(d, J ¼ 7.8 Hz, 1H, 5-Hcarb), 8.38 (s, 1H, 4-Hcarb), 10.12 (s, 1H,
CONH). 13C NMR (DMSO-D6): d (ppm) ¼ 21.1 (1C, CH2CH2CONH),
31.8 (1C, NCH2CH2CH2OH), 32.0 (1C, CH2CH2CONH), 58.0 (1C,
NCH2CH2CH2OH), 109.1 (1C, C-8carb), 109.3 (1C, C-1carb), 111.1 (1C,
C-4carb), 115.5 (d, J ¼ 21.8 Hz, 1C, C-5phenyl), 118.5 (1C, C-2carb),
118.9 (1C, C-6carb), 120.1 (1C, C-5carb), 121.4 (d, J ¼ 24.6 Hz, 1C,
C-3phenyl), 121.7 (1C, C-4acarb), 121.9 (1C, C-4bcarb), 122.2
(d, J ¼ 10.1 Hz, 1C, C-2phenyl), 122.3 (d, J ¼ 8.7 Hz, 1C, C-2phenyl),
125.8 (d, J ¼ 10.6 Hz, 1C, C-6phenyl), 126.9 (1C, C-7carb), 129.8 (1C,
C-8carb), 131.1 (d, J ¼ 3.8 Hz, 1C, C-1phenyl), 136.7 (1C, C-9acarb),
141.4 (1C, C-8acarb), 163.7 (d, J ¼ 258.9 Hz, 1C, C-4phenyl), 167.4
(1C, Cꢀ-31oxadiazole), 168.6 (1C, CONH), 179.9 (1C, C-5oxadiazole). IR (neat):
d
(ppm) ¼ 21.6 (1C, CH2CH2CO2H), 29.8 (1C, CH2CH2CO2H),
114.7 (d, J ¼ 21.5 Hz, 1C, C-5), 121.5 (d, J ¼ 24.7 Hz, 1C, C-3),
127.2 (1C, C-2), 129.4 (1C, C-1), 133.2 (d, J ¼ 9.1 Hz, 1C, C-6), 163.2
(d, J ¼ 255.7 Hz, 1C, C-4), 167.0 (1C, C-3oxadiazole), 175.6 (1C, CO2H),
177.6 (1C, C-5oxadiazole). IR (neat): n (cmꢀ1) ¼ 3097–2403 (m, COOH),
~
–
1708 (s, C O).
–
3-[3-(2-Bromo-4-fluorophenyl)-1,2,4-oxadiazol-5-yl]-N-
[9-(2-hydroxyethyl)-9H-carbazol-3-yl]propanamide (2a)
According to the General Procedure E, a solution of propanoic
acid 9 (400 mg, 1.3 mmol), 7a · HCl (222 mg, 0.9 mmol), COMU1
(652 mg, 1.5 mmol) and triethylamine (0.54 mL, 3.9 mmol) in
DMF (15 mL) was stirred for 24 h at <10°C. The residue was
purified by fc [d ¼ 5 cm, l ¼ 15 cm, cyclohexane/ethyl acetate
15:85, Rf 0.51 (ethyl acetate)]. Colorless solid, mp 163–165°C, yield
368 mg (80%). C25H20BrFN4O3 (523.4 g molꢀ1). Exact mass (ESI):
m/z ¼ calcd. for C25H2079BrFN4O3H 523.0776; Found: 523.0783.
Purity (HPLC): 97.7% (tR ¼ 20.24 min). 1H NMR (DMSO-D6):
d (ppm) ¼ 2.99 (t, J ¼ 7.0 Hz, 2H, CH2CH2CONH), 3.35 (t, J ¼ 7.1 Hz,
2H, CH2CH2CONH), 3.76 (q, J ¼ 5.6 Hz, 2H, NCH2CH2OH), 4.40
(t, J ¼ 5.5 Hz, 2H, NCH2CH2OH), 4.86 (t, J ¼ 5.4 Hz, 1H,
NCH2CH2OH), 7.15 (t, J ¼ 7.4 Hz, 1H, 6-Hcarb), 7.38–7.48 (m, 2H,
–
~
n (cm ) ¼ 3667 (m, O–H), 3290 (m, N–H), 1654 (s, NH–C O).
–
3-[3-(2-Bromo-4-fluorophenyl)-1,2,4-oxadiazol-5-yl]-N-
[9-(4-hydroxybutyl)-9H-carbazol-3-yl]propanamide (2c)
According to the General Procedure E, a solution of propanoic
acid 9 (275 mg, 0.87 mmol), 7c · HCl (270 mg, 1.2 mmol), COMU1
(560 mg, 1.3 mmol) and triethylamine (0.36 mL, 3.6 mmol) in
DMF (15 mL) was stirred for 22.5 h at <10°C. The residue was
purified by fc [d ¼ 5 cm, l ¼ 15 cm, cyclohexane/ethyl acetate
15:85, Rf 0.43 (ethyl acetate)]. Colorless solid, mp 158–161°C, yield
158 mg (33%). C28H26BrFN4O3 (551.4 g molꢀ1). Exact mass (ESI):
m/z ¼ calcd. for C28H2679BrFN4O3H 551.1089; Found: 551.1052.
Purity (HPLC): >99%. 1H NMR (DMSO-D6): d (ppm) ¼ 1.37–1.45
(m, 2H, NCH2CH2CH2CH2OH), 1.73–1.82 (m. 2H, NCH2CH2CH2CH2OH),
2.98 (t, J ¼ 7.0Hz, 2H, CH2CH2CONH), 3.36 (t, J ¼ 7.0 Hz, 2H,
CH2CH2CONH), 4.36 (q, J ¼ 6.7Hz, 2H, NCH2CH2CH2CH2OH),
4.46 (t, J ¼ 6.7 Hz, 1H, NCH2CH2CH2CH2OH), 7.16 (t, J ¼ 7.7 Hz,
1H, 6-Hcarb), 7.40–7.47 (m, 2H, 7-Hcarb, 5-Hphenyl), 7.52–7.54
(m, 2H, 1-Hcarb, 2-Hcarb), 7.56 (d, J ¼ 8.2Hz, 1H, 8-Hcarb), 7.81
(dd, J ¼ 8.4/2.4 Hz, 1H, 3-Hphenyl), 7.87 (dd, J ¼ 8.7/6.0 Hz, 1H,
6-Hphenyl), 8.02 (d, J ¼ 7.6 Hz, 1H, 5-Hcarb), 8.38 (s, 1H, 4-Hcarb),
10.16 (s, 1H, CONH). 13C NMR (DMSO-D6): d (ppm) ¼ 21.81
(1C, CH2CH2CONH), 25.36 (1C, NCH2CH2CH2CH2OH), 29.98
(1C, NCH2CH2CH2CH2OH), 31.97 (1C, CH2CH2CONH), 42.19
(1C, NCH2CH2CH2CH2OH), 60.43 (1C, NCH2CH2CH2CH2OH),
109.2 (1C, C-8carb), 109.4 (1C, C-1carb), 111.0 (1C, C-4carb), 115.5
(d, J ¼ 21.6 Hz, 1C, C-5phenyl), 118.5 (1C, C-2carb), 118.8 (1C, C-6carb),
120.1 (1C, C-5carb), 121.4 (d, J ¼ 25.1 Hz, 1C, C-3phenyl), 121.7 (1C,
C-4acarb), 121.9 (1C, C-4bcarb), 122.2 (d, J ¼ 10.1 Hz, 1C, C-2phenyl),
124.5 (d, J ¼ 3.5 Hz, 1C, C-1phenyl), 125.7 (1C, C-7carb), 131.0 (1C,
C-3carb), 133.6 (d, J ¼ 9.5 Hz, 1C, C-6phenyl), 136.1 (1C, C-9acarb), 140.4
(1C, C-8acarb), 162.9 (d, J ¼ 253.3 Hz, 1C, C-4phenyl), 166.6 (1C,
C-3oxadiazole), 168.5 (1C, CONH), 179.6 (1C, C-5oxadiazole).
7-Hcarb, 5-Hphenyl), 7.49–7.55 (m, 2H, 1-Hcarb, 2-Hcarb), 7.57
(d, J ¼ 8.3 Hz, 1H, 8-Hcarb), 7.83 (dd, J ¼ 8.6/2.6 Hz, 1H, 3-Hphenyl),
7.89 (dd, J ¼ 8.7/6.1 Hz, 1H, 6-Hphenyl), 8.02 (d, J ¼ 7.8 Hz, 1H,
5-Hcarb), 8.39 (s, 1H, 4-Hcarb), 10.13 (s, 1H, CONH). 13C NMR
(DMSO-D6):
d
(ppm) ¼ 21.8 (1C, CH2CH2CONH), 32.0 (1C,
CH2CH2CONH), 45.3 (1C, NCH2CH2OH), 59.6 (1C, NCH2CH2OH),
109.5 (1C, C-8carb), 109.7 (1C, C-1carb), 110.9 (1C, C-4carb), 115.5
(d, J ¼ 21.6 Hz,1C, C-5phenyl), 118.5 (1C, C-2carb), 118.7 (1C, C-6carb),
120.0 (1C, C-5carb), 121.4 (d, J ¼ 24.9 Hz, 1C, C-3phenyl), 121.7 (1C,
C-4acarb), 122.0 (1C, C-4bcarb), 122.2 (d, J ¼ 10.1 Hz, 1C, C-2phenyl),
124.5 (d, J ¼ 3.4 Hz,1C, C-1phenyl), 125.6 (1C, C-7carb), 131.0 (1C,
C-3carb), 133.6 (d, J ¼ 9.3 Hz, 1C, C-6phenyl), 137.0 (1C, C-9acarb),
140.9 (1C, C-8acarb), 162.9 (d, J ¼ 253.4 Hz, 1C, C-4phenyl), 166.4
(1C, C-3oxadiazole), 168.5 (1C, CONH), 179.6 (1C, C-5oxadiazole).
IR (neat): n (cmꢀ1) ¼ 3421 (m, O–H), 3329 (m, N–H), 3078
~
–
–
(m, C-H, arom), 2935 (m, C–H, aliph), 1678 (s, NH–C O).
3-[3-(2-Bromo-4-fluorophenyl)-1,2,4-oxadiazol-5-yl]-N-
[9-(3-hydroxypropyl)-9H-carbazol-3-yl]propanamide (2b)
According to the General Procedure E, a solution of propanoic
acid 9 (453 mg, 1.4 mmol), 7b · HCl (400 mg, 1.4 mmol), COMU1
(740 mg, 1.7 mmol) and triethylamine (0.4 mL, 2.8 mmol) in
DMF (15 mL) was stirred for 24 h at <10°C. The residue was
purified by fc [d ¼ 6 cm, l ¼ 10 cm, cyclohexane/ethyl acetate 5:95,
Rf 0.20 (ethyl acetate)]. Colorless solid, mp 155–157°C, yield
229 mg (30%). C26H22BrFN4O3 (537.4 g molꢀ1). Exact mass (ESI):
m/z ¼ calcd. for C26H2279BrFN4O3H 537.0935; Found: 537.0932.
Purity (HPLC): 96.6% (tR ¼ 20.53 min). 1H NMR (DMSO-D6):
d (ppm) ¼ 1.82–1.91 (m, 2H, NCH2CH2CH2OH), 2.97 (t, J ¼ 7.1 Hz,
2H, CH2CH2CONH), 3.12 (t, J ¼ 7.2 Hz, 2H, CH2CH2CONH), 3.33–
3.36 (m, 2H, NCH2CH2CH2OH), 4.40 (t, J ¼ 6.7 Hz, 2H,
NCH2CH2CH2OH), 4.86 (t, J ¼ 4.9 Hz, 1H, NCH2CH2OH), 7.13
(t, J ¼ 7.5 Hz, 1H, 6-Hcarb), 7.38–7.47 (m, 2H, 7-Hcarb, 5-Hphenyl),
X-ray diffraction
X-ray diffraction, general
Datasets were collected with a Nonius KappaCCD diffractom-
eter. Programs used: data collection, COLLECT [50], data
reduction Denzo-SMN [51]; absorption correction, Denzo [52];
structure solution SHELXS-97 [53]; structure refinement
SHELXL-97 [54]; and graphics, XP (BrukerAXS, 2000). Thermals
ellipsoids are shown with 30% probability, R values are given
for observed reflections, and wR2 values are given for all
reflections.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2014, 347, 21–31
CB2 Ligands by Hydroxyalkylation with Cyclic Sulfates
29
Exceptions and special features
(DMEM) containing 10% of standardized FCS (Biochrom AG,
Berlin, Germany) and 250 mg mLꢀ1 Geneticin. The cells were
harvested by scraping off the surface, resuspended in PBS
buffer and pelleted (10 min, 5000 ꢂ g) and the number of cells
was determined using an improved Neubauer’s counting
chamber (VWR, Darmstadt, Germany). Subsequently, the cells
were lysed by sonication (4°C, 6 ꢂ 10 s cycles with breaks of
10 s). The resulting cell fragments were centrifuged with a
high performance cool centrifuge (20,000 ꢂ g, 4°C). The
supernatant was discarded and the pellet resuspended in a
defined volume of TRIS buffer (50 mM TRIS, 12 mM MgCl2,
4 mM EDTA, pH 7.4) yielding cell fragments of approximately
2,000,000 cells mLꢀ1. The suspension of membrane homoge-
nates was sonicated again (4°C, 2 ꢂ 10 s cycles with breaks of
10 s) and stored at ꢀ80°C.
An unidentified disordered solvent molecule was found in the
asymmetrical unit and could not be satisfactorily refined. The
program SQUEEZE [55] was therefore used to remove
mathematically the effect of the solvent. The Flack parameter
was refined to 0.01(2). The CH2-OH group at C26 atom is
disordered over two positions. Several restraints (SADI, SAME,
and SIMU) were used in order to improve refinement stability.
X-ray crystal structure analysis of 2c
Formula C27H24BrFN4O3, M ¼ 551.41, colorless crystal,
0.44 ꢂ 0.08 ꢂ 0.06 mm, a ¼ 4.7209(1), b¼ 14.4710(4), c ¼ 39.7695
(13) Å, V¼ 2716.90(13) Å3, rcalc ¼ 1.348 g cmꢀ3, m ¼ 1.554 mmꢀ1
,
empirical absorption correction (0.548ꢃ T ꢃ 0.912), Z ¼ 4,
orthorhombic, space group P212121 (No. 19), l ¼ 0.71073 Å,
T¼ 223(2) K, v and w scans, 13,647 reflections collected (ꢁh, ꢁk,
ꢁl), [(sinu)/l] ¼ 0.60 Åꢀ1, 3835 independent (Rint ¼ 0.053) and
3414 observed reflections [I> 2s(I)], 350 refined parameters,
R ¼ 0.067, wR2 ¼ 0.169, max. (min.) residual electron density
0.31 (ꢀ0.42) e Åꢀ3, the hydrogen at N2 atom was refined freely;
others were calculated and refined as riding atoms. CCDC
947453.
General protocol for the binding assays
The test compound solutions were prepared by dissolving
approximately 10 mmol (usually 2–4 mg) of test compound in
DMSO so that a 10 mM stock solution was obtained. To obtain
the required test solutions for the assay, the DMSO stock
solution was diluted with the respective assay buffer. The
filtermats were presoaked in 0.5% aqueous polyethylenimine
solution for 2 h at room temperature before use. All binding
experiments were carried out in duplicates in 96-well
multiplates. The concentrations given are the final concen-
trations in the assay. Generally, the assays were performed by
addition of 50 mL of the respective assay buffer, 50 mL test
Experimental receptor binding studies
Materials
The recombinant CHO cells expressing the CB2 receptor were
a generous donation of Prof. Paul Prather (Little Rock,
Arkansas, USA). Cell incubator: Heracell 120 (Thermo Fisher
Scientific, Langenselbold, Germany). Homogenizers: Elvehjem
Potter (Braun Biotech International, Melsungen, Germany)
and Soniprep 150 (MSE, London, UK). Centrifuges: cooling
centrifuge model Rotina 35R (Hettich, Tuttlingen, Germany)
and high-speed cooling centrifuge model Sorvall RC-5C plus
(Thermo Fisher Scientific, Langenselbold, Germany). Multi-
plates: standard 96-well multiplates (Diagonal, Muenster,
Germany). Shaker: self-made device with adjustable tempera-
ture and tumbling speed (scientific workshop of the institute).
Vortexer: Vortex Genie 2 (Thermo Fisher Scientific, Langen-
selbold, Germany). Harvester: MicroBeta FilterMate-96 Har-
vester. Filter: Printed Filtermat Typ A and B. Scintillator:
Meltilex (Typ A or B) solid state scintillator. Scintillation
analyzer: MicroBeta Trilux (all Perkin Elmer LAS, Rodgau-
Jügesheim, Germany). Chemicals and reagents were
purchased from different commercial sources and were of
analytical grade.
compound solution in various concentrations (10ꢀ5, 10ꢀ6
,
10ꢀ7, 10ꢀ8, 10ꢀ9, and 10ꢀ10 mol Lꢀ1), 50 mL of corresponding
radioligand solution and 50 mL of the respective receptor
preparation into each well of the multiplate (total volume
200 mL). The receptor preparation was always added last.
During the incubation, the multiplates were shaken at a
speed of 500–600 rpm at the specified temperature. Unless
otherwise noted, the assays were terminated after 120 min by
rapid filtration using the harvester. During the filtration each
well was washed five times with 300 mL of water. Subsequent-
ly, the filtermats were dried at 95°C. The solid scintillator was
melted on the dried filtermats at a temperature of 95°C for
5 min. After solidifying of the scintillator at room tempera-
ture, the trapped radioactivity in the filtermats was measured
with the scintillation analyzer. Each position on the filtermat
corresponding to one well of the multiplate was measured for
5 min with the [3H]-counting protocol. The overall counting
efficiency was 20%. The IC50 values were calculated with the
program GraphPad Prism1 3.0 (GraphPad Software, San
Diego, CA, USA) by non-linear regression analysis. Subse-
quently, the IC50 values were transformed into Ki values using
the equation of Cheng and Prusoff [56]. The Ki values of most
active compounds are given as mean value ꢁ SEM from three
independent experiments.
Cell culture and preparation of membrane
homogenates from CB2 cells
The cell culture was modified according to ref. [49].
CHO cells stably transfected with the gene for the human
CB2 receptor were grown in Dulbecco Modified Earl’s Medium
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

30
C. Lueg et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 21–31
Protocol of the CB2 receptor binding assay
The assay was modified according to ref. [48]:
[21] E. Nunez, C. Benito, M. R. Pazos, A. Barbachano, O. Fajardo,
S. Gonzalez, R. M. Tolon, J. Romero, Synapse 2004, 53, 208–
213.
The assay was performed with the radioligand [3H]CP-
55,940 (Perkin Elmer). The CB2 receptor containing cell
membrane fragments (fragments of approximately
100000 cells wellꢀ1) were incubated with various concentra-
tions of the test compound, 1 nM [3H]CP-55,940 and binding
buffer (50 TRIS pH 7.4, 12 mM MgCl2, 4 EDTA and 2% BSA) at
37°C. The non-specific binding was determined with 10 mM
non-labeled CP-55,940. The Kd value of [3H]CP-55,940 is
11.8 nM (determined by a saturation experiment).
[22] V. Di Marzo, Nat. Rev. Drug Discovery 2008, 7, 438–455.
[23] A. G. Granja, F. Carrillo-Salinas, A. Pagani, M. Gómez-Cañas,
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We are very grateful to Prof. Paul Prather, Institute of Pharmacology
and Toxicology, University of Arkansas, Little Rock, Arkansas, USA, for
supplying us with CB2 cells. This work was supported by the Deutsche
Forschungsgemeinschaft, which is gratefully acknowledged.
[26] J. Palazuelos, N. Davoust, B. Julien, E. Hatterer, T. Aguado,
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The authors have declared no conflict of interest.
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