Sharpless asymmetric dihydroxylation as the key step in the enantioselective synthesis of spirocyclic σ1 receptor ligands

Article abstract of DOI:10.1016/j.tetasy.2013.12.009

The enantioselective synthesis of fluorinated spirocyclic σ₁ ligands involved three key steps: (1) the Sharpless asymmetric dihydroxylation of 2-bromostyrene 5 provided enantiomerically pure diols (R)-6 and (S)-6 establishing the stereogenic center; (2) the intramolecular opening of the oxirane ring of (R)-11 and (S)-11, which occurred with excellent regioselectivity and complete inversion of configuration giving access to enantiomerically pure alcohols (S)-7a and (R)-7a; (3) the treatment of alcohols (S)-7b and (R)-7b with DAST, which led to the fluoromethyl derivatives (S)-1 and (R)-1 without racemization. X-ray crystal structure analysis of the tosylate (R)-13 confirmed the absolute configuration of the spirocyclic compounds as well as the enantioselectivity during the Sharpless asymmetric dihydroxylation of 5. The (S)-configured fluoromethyl derivative (S)-1 revealed a high σ₁ affinity (K_i = 1.8 nM), high eudismic ratio (factor 8) and high selectivity over the σ₂ subtype (667-fold).

Full text of DOI:10.1016/j.tetasy.2013.12.009

Tetrahedron: Asymmetry 25 (2014) 268–277
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Sharpless asymmetric dihydroxylation as the key step in
the enantioselective synthesis of spirocyclic r1 receptor ligands
Katharina Holl a, Dirk Schepmann a, Constantin Gabriel Daniliuc b, Bernhard Wünsch a,
⇑
a Institut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms-Universität Münster, Corrensstraße 48, D-48149 Münster, Germany
b Organisch-Chemisches Institut der Westfälischen Wilhelms-Universität Münster, Corrensstraße 40, D-48149 Münster, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 November 2013
Accepted 12 December 2013
The enantioselective synthesis of fluorinated spirocyclic r1 ligands involved three key steps: (1) the
Sharpless asymmetric dihydroxylation of 2-bromostyrene 5 provided enantiomerically pure diols (R)-6
and (S)-6 establishing the stereogenic center; (2) the intramolecular opening of the oxirane ring of (R)-
11 and (S)-11, which occurred with excellent regioselectivity and complete inversion of configuration
giving access to enantiomerically pure alcohols (S)-7a and (R)-7a; (3) the treatment of alcohols (S)-7b
and (R)-7b with DAST, which led to the fluoromethyl derivatives (S)-1 and (R)-1 without racemization.
X-ray crystal structure analysis of the tosylate (R)-13 confirmed the absolute configuration of the spiro-
cyclic compounds as well as the enantioselectivity during the Sharpless asymmetric dihydroxylation of 5.
The (S)-configured fluoromethyl derivative (S)-1 revealed a high r1 affinity (Ki = 1.8 nM), high eudismic
ratio (factor 8) and high selectivity over the r2 subtype (667-fold).
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
pared by nucleophilic substitution of the corresponding enantio-
merically pure tosylates with tetrabutylammonium fluoride. The
The
r
1 receptor is one of two subtypes1 in the class of
r
recep-
resolution of the racemic precursor tosylate was performed by pre-
parative chiral HPLC. The r1 affinity of (R)-2 (Ki = 0.57 nM) is
approximately four times higher than the r1 affinity of (S)-2
(Ki = 2.3 nM).19 These results clearly show that the enantiomers
of the spirocyclic piperidines 2 and 3 behave differently in biolog-
ical systems. Due to these observations, we became interested in
the enantioselective synthesis of enantiomerically pure spirocyclic
tors, which are recognized as a unique receptor class after they had
been ranked among the opioid receptors for more than one
decade.2 The r1 receptor is a 25.3 kDa transmembrane protein3
consisting of 223 amino acids.4 It is found in the central nervous
system5 and in many different peripheral tissues.6 The
r1 receptor
has been associated with numerous neuronal diseases, including
neuropathic pain, schizophrenia, major depression, and neurode-
generative disorders.7 Spirocyclic piperidines represent high-affin-
ity and selective ligands for the r1 receptor,8–11 and, are therefore
regarded to have a high therapeutic potential for the treatment of
these diseases. Moreover, a r1 receptor ligand labeled with a pos-
itron emitter such as [18F] could be used as diagnostic tool in pos-
itron emission tomography (PET).12
r
1 ligands with a 2-benzofuran scaffold (compare compounds 1–4)
on a larger scale (Fig. 1).
(CH2)n-F
1
: n = 1; Ki = 0.74 nM
O
N
A series of homologous (fluoroalkyl) substituted racemic spiro-
cyclic 2-benzofurans 1–4 has been synthesized and evaluated as
2: n = 2; Ki = 0.59 nM
3: n = 3; Ki = 1.4 nM
4: n = 4; Ki = 1.2 nM
[
18F]-labeled PET tracers with promising results.13–17 However,
for in vivo applications, enantiomerically pure drugs and PET trac-
ers are required. The enantiomers of the fluoropropyl derivative 3
have been separated by semi-preparative chiral HPLC. It was
shown that the (S)-configured enantiomer (S)-3 (Ki = 0.59 nM) pos-
sesses a threefold higher
enantiomers of the fluoroethyl derivative fluspidine 2 were pre-
Bn
Figure 1. Homologous fluoroalkyl substituted spirocyclic r1 receptor ligands.
r
1 affinity than (R)-3 (Ki = 1.8 nM).18 The
Herein we report on the synthesis of enantiomerically pure spi-
rocyclic 2-benzofurans (S)-1 and (R)-1 using the Sharpless asym-
metric dihydroxylation of 2-bromostyrene 5 as the key step.
(Fig. 2) The establishment of the spirocyclic ring system (S)-7
⇑
Corresponding author. Tel.: +49 251 8333311; fax: +49 251 8332144.
E-mail address: wuensch@uni-muenster.de (B. Wünsch).
0957-4166/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetasy.2013.12.009

K. Holl et al. / Tetrahedron: Asymmetry 25 (2014) 268–277
269
OH
OH
(DHQ)2PHAL in the asymmetric dihydroxylation step (97% yield).
These yields are slightly higher than those reported for (R)-6 and
(S)-6.23,24 As described in the literature, the enantiomeric purity
of the enantiomeric diols (R)-6 and (S)-6 was determined by chiral
HPLC analysis.25 Both enantiomers showed a high enantiomeric
purity of 98.8% ee for (R)-6 and 97.6% ee for (S)-6.
Sharpless asymmetric
dihydroxylation
bezonfuran ring
formation
Br
Br
6
(R)-
5
In order to obtain the spirocyclic system, the activation of diol
(R)-6 was required. Due to the high reactivity of cyclic sulfates,26
the activation of diol (R)-6 as cyclic sulfate (R)-9 was envisaged.
For this purpose diol (R)-6 was reacted with SOCl2 to give the cyclic
sulfite (R)-8 as a mixture of diastereomers caused by the newly
formed stereogenic center at the S-atom. However, all attempts
to oxidize the cyclic sulfite (R)-8 (e.g., with RuCl3 and various
cooxidants and additives) failed to give the cyclic sulfate (R)-9.
Alternatively diol (R)-6 can be converted into oxirane (R)-11.
Both enantiomers of 11, as well as the racemic mixture, have been
described in the literature.27–29 The reaction of (R)-6 with tosyl
chloride in the presence of dibutyltin oxide (SnBu2O) gave the
monotosylate (R)-10, which was transformed into oxirane (R)-11
in 82% yield upon treatment with NaOCH3.
F
OH
O
N
fluorination
O
N
Bn
R
(S)-1
(S)-7
Figure 2. Synthesis of enantiomerically pure spirocyclic piperidine (S)-1. The key
steps involve the Sharpless asymmetric dihydroxylation, benzofuran ring formation
and fluorination.
requires nucleophilic substitution at the benzylic position, which
has to be controlled carefully. Inversion of configuration should
give access to the (S)-configured spirocyclic 2-benzofuran (S)-7,
which in turn can be transformed into the final fluoromethyl deriv-
ative (S)-1 upon modification of the N-substituent and fluorination.
The Sharpless asymmetric dihydroxylation is a reliable method
for transforming olefins into enantiomerically pure diols, which
can serve as valuable building blocks for biologically active com-
pounds. Usually very mild reaction conditions are used, side prod-
ucts are not formed and almost all classes of olefins react to give
diols with high enantioselectivities. Phthalazine derivatives of the
‘pseudoenantiomeric’ cinchona alkaloids dihydroquinine (DHQ)
and dihydroquinidine (DHQD), premixed in the commercially
In the next step, oxirane (R)-11 was treated with n-BuLi at
ꢀ78 °C to replace the Br atom with a Li atom. The reaction of this
OH
O
(a)
(b)
O
N
O
Br
N
Boc
Boc
12
(R)-11
(S)-7a
available mixtures, AD-mix-a and AD-mix-b, serve as chiral induc-
tors during the dihydroxylation step, respectively.20,21 The config-
uration obtained in the presence of the respective ligand can be
predicted reliably by the so-called ‘mnemonic device’.22
OH
F
(c)
O
N
O
N
2. Results and discussion
2.1. Synthesis
Bn
Bn
(S)-7b
1
(S)-
According to the literature, the Sharpless asymmetric dihydr-
oxylation of 2-bromostyrene 5 was performed with AD-mix-b con-
taining (DHQD)2PHAL in a 1:1 mixture of tert-butanol and water at
0 °C to afford diol (R)-6 in 92% yield (Scheme 1). The enantiomeric
Scheme 2. Synthesis of fluoromethyl substituted spirocyclic 2-benzofurans.
Reagents and conditions: (a) n-BuLi, THF, then 12 dissolved in THF, 60%; (b) (i) TFA,
CH2Cl2, (ii) PhCH@O, NaBH(OAc)3, 59%; (c) DAST, CH2Cl2, 18%. The (R)-configured
enantiomers were prepared in the same manner starting with (S)-11: (R)-7a: 45%;
(R)-7b: 60%; (R)-1: 23%.
diol (S)-6 was obtained in the same manner using AD-mix-
a
with
O
O
O
O
O
O
S
O
S
Br
Br
(b)
OH
OH
9
(R)-
8
(R)-
(a)
Br
Br
OTos
OH
(c)
6
(R)-
5
O
(d)
Br
Br
(R)-10
11
(R)-
Scheme 1. Sharpless asymmetric dihydroxylation of 2-bromostyrene 5 and synthesis of the reactive ring systems. Reagents and conditions: (a) AD-mix-b, tert-butanol/H2O
1:1, 92%; (b) SOCl2, CH2Cl2, 80%; (c) p-TosCl, NEt3, Bu2SnO, THF, 95%; (d) Na, CH3OH, 82 %. The (S)-configured enantiomers were synthesized in the same manner using
AD-mix-a in the first step: (S)-6: 97%; (S)-10: 81%; (S)-11: 77%.

270
K. Holl et al. / Tetrahedron: Asymmetry 25 (2014) 268–277
aryllithium intermediate with piperidone 12 led to a tertiary alco-
holate, which intramolecularly opened the oxirane ring in a clean
SN2 reaction to form the inverted primary alcohol (S)-7a after
aqueous work-up (Scheme 2). This transformation led exclusively
to 2-benzofuran (S)-7a (yield 45%), although theoretically the oxi-
rane ring opening could also result in a regioisomeric 2-benzopyra-
ne ring system.30,31 The enantiomer (R)-7a was prepared in the
same manner by starting from oxirane (S)-11.
The enantiomeric purity of the primary alcohols (S)-7a and
(R)-7a was determined by chiral HPLC/Daicel Ciralpak IB column),
which showed a high enantiopurity for (S)-7a (96.0% ee) and (R)-7a
(95.0% ee) (Fig. 3). This result indicates a clean SN2 mechanism of
the benzofuran ring closure with concomitant inversion of
configuration.
work-up and purification (e.g., strong tailing of the basic tertiary
amine during chromatography). In order to gain access to (S)-1,
the Boc-group of (S)-7a had to be replaced with a benzyl group.
Thus, the Boc-group of (S)-7a was cleaved off with trifluoroacetic
acid and the benzyl residue was introduced by reductive alkylation
of the resulting secondary amine with benzaldehyde and
NaBH(OAc)3. The N-benzyl substituted piperidine (S)-7b was
isolated in 60% yield. Treatment of alcohol (S)-7b with diethyl-
amino sulfurtrifluoride (DAST) provided the fluoromethyl substi-
tuted spirocyclic compound (S)-1 (Scheme 2).
After preparation of the enantiomer (R)-1 in the same man-
ner by starting from oxirane (S)-11, the enantiomeric purity of
the fluoromethyl derivatives (S)-1 and (R)-1 was analyzed by
chiral HPLC using a Daicel Chiralpak IB column. Compounds
(S)-1 (97.6% ee) and (R)-1 (93.0% ee) showed almost the same
enantiopurity as the starting diols (R)-6 and (S)-6 as well as
the alcohols (S)-7a and (R)-7a (Fig. 4). During the conversion
For the synthesis of the spirocyclic ring system (S)-7a, the Boc-
protected piperidine 12 was used, since the corresponding prod-
ucts formed with 1-benzylpiperidin-4-one caused problems during
A
B
Figure 3. HPLC analysis of enantiomeric alcohols (S)-7a (Fig. 3A) and (R)-7a (Fig. 3B): Daicel Chiralpak IB, isohexane: isopropanol 97: 3, 1.0 mL/min, k = 210 nm). (S)-7a: 96.0%
ee; (R)-7a: 95.0% ee.

K. Holl et al. / Tetrahedron: Asymmetry 25 (2014) 268–277
271
A
B
Figure 4. HPLC analysis of enantiomeric fluoromethyl derivatives (S)-1 (Fig. 4A) and (R)-1 (Fig. 4B): Daicel Chiralpak IB, isohexane: isopropanol 99: 1, 1.0 mL/min,
k = 210 nm). (S)-1: 97.6% ee; (R)-1: 93.0% ee.
of the diols (R)-6 and (S)-6 via alcohols (S)-7a and (R)-7a into
the fluoromethyl derivatives (S)-1 and (R)-1, no racemization
occurred.
OH
OTos
O
N
O
N
(a)
According to the mnemonic device, the Sharpless asymmetric
dihydroxylation of bromostyrene 5 with AD-mix-a should give
the (S)-configured diol (S)-6, which upon inversion of configuration
should lead to the (R)-configured alcohol (R)-7a. In order to
confirm the configuration, alcohol (R)-7a, which could not be
crystallized, was converted into tosylate (R)-13. (Scheme 3)
Recrystallization of tosylate (R)-13 from CH2Cl2 led to crystals
suitable for X-ray crystal structure analysis. (Fig. 5) According to
the X-ray crystal structure, the absolute configuration was (R),
which confirmed the predicted configuration.
Boc
Boc
7a
13
(R)-
(R)-
Scheme 3. Tosylation of alcohol (R)-7a. Reagents and conditions: (a) p-TosCl, NEt3,
4-DMAP, CH2Cl2, 70%. (S)-13, was obtained in the same manner by starting from (S)-
7a: yield 81%.
2.2. Receptor affinity
assay was carried out with membrane homogenates from guinea
pig brains and [3H]-(+)-pentazocine as the
r1-selective radioli-
The r1 and r2 receptor affinities were determined in competi-
tion experiments with radioligands (Table 1). The r1 receptor
gand. Membrane homogenates from the rat liver and the unselec-
tive radioligand [3H]ditolylguanidine ([3H]DTG) were used for the

272
K. Holl et al. / Tetrahedron: Asymmetry 25 (2014) 268–277
3. Conclusion
Herein the first enantioselective synthesis of spirocyclic r1
receptor ligands is reported. The Sharpless asymmetric dihydroxyla-
tion of the terminal alkene 2-bromostyrene 5 was the key step in the
preparation of enantiomerically pure spirocyclic ligands with a 2-
benzofuran scaffold. The ring opening of the oxirane of (R)-11
occurred with high regio- and stereoselectivity (inversion of
configuration) providing spirocyclic 2-benzofurans with high enan-
tiopurity. The very high
r1 affinity of the fluoromethyl derivative
(S)-1 is promising with respect to the development of innovative
CNS drugs. Moreover, the enantiomerically pure alcohols (S)-7 and
(R)-7 represent valuable building blocks that allow the versatile
modification of the substituent at the 3-position of the benzofuran
ring. Enantiomerically pure [18F]-labeled PET tracers are available
by nucleophilic substitution of the tosylates (S)-13 and (R)-13 with
[
18F]fluoride.
4. Experimental
4.1. General
THF was distilled from sodium/benzophenone prior to use. Flash
chromatography (fc): Silica gel 60, 40–63 lm (Macherey-Nagel);
parentheses include: diameter of the column, length of the column,
eluent, fraction size. Melting point: Melting point apparatus SMP 3
(Stuart Scientific), uncorrected. Thin layer chromatography (TLC):
Silica gel 60 F254 plates on aluminum sheets (Merck). Optical rota-
tion: Polarimeter 341 (Perkin Elmer), wavelength: 589 nm, path
length 1 dm, temp. +20 °C; unit [deg mL dmꢀ1 gꢀ1] is omitted; con-
centration [mg/mL] and solvent are given in brackets. Atmospheric
pressure chemical ionization (APCI), MicroTOF QII mass spectrome-
ter (Bruker Daltronics), software: DataAnalysis. Deviations of the
found exact masses from the calculated exact masses: 5 mDa or less.
Electrospray ionization (ESI): LCQ Finnigan MAT mass spectrometer
(Thermo Finnigan), software: Xcalibur 1.3 (Thermo Scientific),
mass-to-charge ratios [m/z] and relative intensity of the signals (%)
are given. 1H NMR (400.3 MHz), 13C NMR (100.7 MHz): Unity Mer-
cury Plus 400 NMR spectrometer (Varian) or AV400 NMR spectrom-
eter (Bruker); d in ppm related to tetramethylsilane. IR: FT/IR
Prestige 21 IR spectrometer (Shidmadzu), ATR technique. X-ray
crystallography: Nonius KappaCCD diffractometer.
Figure 5. X-ray crystal structure analysis of tosylate (R)-13. Thermals ellipsoids are
shown with 30% probability.
Table 1
The r1 and r2 receptor affinities of enantiomerically pure spirocyclic 2-benzofurans
X
O
N
Bn
Entry
Compound
X
Ki SEM (nM)
Selectivity
r1
r2
r1/r2
1
2
3
4
5
6
(R)-1
(S)-1
(R)-7b
(S)-7b
(+)-Pentazocine
Haloperidol
F
F
OH
OH
14 6.2
1.8 1.2
34 4.3
21 7.0
5.7 2.2
6.3 1.6
10%*
1200
6%*
>70
667
>25
>45
—
2%*
4.2. HPLC methods
—
78 2.3
12
HPLC method 1 for the determination of product purity: Merck
Hitachi equipment: pump: L-7100; degasser: L-7614; autosam-
pler: L-7200; UV-Detector: L-7400; interface: D-7000; data trans-
*
Inhibition of the radioligand binding at a concentration of 1 lM.
fer:
LiChrospherÒ 60 RP-select B (5
tridge; Guard Column: LiChrospherÒ 60 RP-select B (5
D-line;
data
acquisition:
m), LiCroCARTÒ 250–4 mm car-
m), LiCro-
HSM-Software;
column:
r2 receptor binding assay. In the r2 assay r1 receptors were
l
masked with non-labeled (+)-pentazocine.9,10
l
Table 1 shows the high
r1 affinity of the enantiomerically pure
CARTÒ 4–4 mm cartridge (No.: 1.50963.0001) using manu-CARTÒ
NT cartridge holder; solvents: A: water with 0.05% (v/v) trifluoro-
acetic 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%; flow rate: 1.0 mL/min; injection volume:
ligands 1 and 7b. The (S)-configured enantiomers (S)-1 and (S)-7b
represent the eutomers, respectively. However, the eudismic ratio
of the fluoromethyl compound (S)-1 (factor 8) is higher than the
eudismic ratio of alcohol (S)-7b (factor 1.5). The higher eudismic
ratio of the fluoromethyl derivative (S)-1 can be explained by the
higher receptor affinity.
The fluoro substituent in the side chain is beneficial for the high
r1 receptor affinity. This tendency has also been observed for the
racemic fluoroalkyl compounds rac-1–4 and their corresponding
alcohols.12–16
5.0
lL; detection wavelength: 210 nm.
HPLC method 2 for the determination of enantiomeric ratios:
equipment: pump: L-6200A; Injection: manual, Rheodyne 7725i;
Diode Array Detector: L-7455; data transfer: D-line; data acquisi-
tion: HSM-Software (all except for Rheodyne 7725i: LaChrom,
Merck Hitachi); wavelength: 210 nm; stop time: 30.0 min; paren-
theses include: column, solvent, flow rate, injection volume.
In addition to the high r1 receptor affinity and the high eudis-
mic ratio of 8, the fluoromethyl derivative (S)-1 displays more than
660-fold selectivity against the r2 subtype.

K. Holl et al. / Tetrahedron: Asymmetry 25 (2014) 268–277
273
LC–HRMS: HPLC system, coupled MicroTOF QII mass spectrom-
eter (Bruker Daltronics); software: Hystar and DataAnalysis 4.0
(Bruker), HPLC equipment: Ultimate 3000 RS Dionex system (Dio-
nex Softron): solvent rack: SRD 3600; pump: DGP-3600RS; auto-
sampler: WPS-3000RS; column oven: TCC-3000RS; diode array
detector: DAD-3000 RS; software: Chromeleon, column: Kinetex™,
NMR (CDCl3): d (ppm) = 66.4 (1C, HOCHCH2OH), 73.7 (1C, HOCHCH2-
OH), 122.1 (1C, C-22-bromophenyl), 127.8 (1C, C-52-bromophenyl),
128.0 (1C, C-62-bromophenyl), 129.5 (1C, C-42-bromophenyl), 132.8 (1C,
~
C-32-bromophenyl), 139.5 (1C, C-12-bromophenyl). IR (neat):
v
[cmꢀ1] =
3267 (m, b,
mO–H), 2940, 2882, (w, mC–H), 748 (s, C1,2-disubst. arom.),
679 (m, C–Br).
m
2.6 lm, C18, 100 Å; 50 mm/2.1 mm, guard column: Security Guard
Standard C18 Cartridge, 4 mm/2 mm, temperature: 30 °C, solvents:
(A) acetonitrile/NH4HCOO (10 mM) = 10:90 + 0.1% (v/v) HCOOH,
(B) acetonitrile/NH4HCOO (10 mM) = 90:10 + 0.1% (v/v) HCOOH,
gradient elution: (A%): 0–5 min: gradient from 100% to 0%, flow
rate: 0.4 mL/min, 5–6.5 min: 0%, flow rate: 0.4 mL/min, 6.5–
7 min: gradient from 0% to 100%, flow rate: 0.4 mL/min, 10 min:
4.3.4. (4R)-4-(2-Bromophenyl)-1,3,2-dioxathiolane 2-oxide
(R)-8
Compound (R)-6 (441 mg, 2.0 mmol) was dissolved in CH2Cl2
(10 mL). Next, SOCl2 (300 lL, 4.1 mmol) was added dropwise and
the mixture was refluxed for 2 h, after which water was added. After
separation of the layers, the aqueous layer was extracted with CH2-
Cl2 (3ꢁ). The combined organic layers were washed with a saturated
sodium bicarbonate solution (2ꢁ) and brine (1ꢁ), dried (Na2SO4), fil-
tered, and the solvent was removed in vacuo. The crude product was
purified by flash column chromatography (3 cm, 15 cm, cyclohex-
ane/ethyl acetate = 9:1, 20 mL). Yellow liquid, yield 426 mg (80%).
C8H7BrO3S (263.1). The ratio of the diastereomers (major:minor)
according to the 1H NMR spectra was 2.5:1. Rf = 0.67 (major),
Rf = 0.46 (minor) (cyclohexane/ethyl acetate = 9:1). Specific
100%, flow rate: 0.6 mL/min, injection volume: 0.5–1 lL, sample
temperature: 5 °C, detection wavelength: 200–350 nm.
4.3. Synthetic procedures
4.3.1. (R)-1-(2-Bromophenyl)ethane-1,2-diol (R)-623,24
AD-mix-b (38.3 g) was added to a mixture of tert-butyl alcohol
(137 mL) and water (137 mL). The mixture was cooled to 0 °C, after
which 2-bromostyrene 5 (5.0 g, 27.3 mmol) was added and the
reaction mixture was stirred at 0 °C for 19 h. Next, sodium sulfite
(41.0 g) was added and the mixture was allowed to warm to room
temperature and stirred for 20 min. Ethyl acetate was then added
to the reaction mixture, and after separation of the layers, the
aqueous layer was extracted with ethyl acetate (3ꢁ). The com-
bined organic layers were dried (Na2SO4), filtered, and the solvent
was removed in vacuo. The crude product was purified by flash col-
umn chromatography (8 cm, 15 cm, cyclohexane/ethyl ace-
tate = 2:1, 100 mL). Colorless solid, melting point: 49 °C, yield
5.43 g (92%). C8H9BrO2 (217.1). Rf = 0.25 (cyclohexane/ethyl ace-
rotation: ½a 2D0
¼ ꢀ7:5 (c 4.6, CH2Cl2). Purity (HPLC method 1):
ꢂ
tR = 19.2 min (22%), tR = 19.4 min (77%), purity 99.4%. HRMS:
m/z = 198.9750 (calcd 198.9753for C8H879BrO [M-SO2+H]+),
200.9730 (calcd 200.9733 for C8H881BrO [M-SO2+H]+). 1H NMR
(CDCl3): d (ppm) = 4.24 (dd, J = 8.6/5.6 Hz, 1H, CHCH2Ominor), 4.34
(dd, J = 10.0/9.0 Hz, 1H, CHCH2Omajor), 5.02 (dd, J = 9.0/6.7 Hz, 1H,
CHCH2Omajor), 5.15 (dd, J = 8.6/6.7 Hz, 1H, CHCH2Ominor), 5.84
(dd, J = 10.0/6.7 Hz, 1H, CHCH2Omajor), 6.25 (t, J = 6.1 Hz, 1H,
CHCH2Ominor), 7.21–7.28 (m, 2H, Harom/major (1), Harom/minor (1)),
7.35–7.46 (m, 3H, Harom/major (1), Harom/minor (2)), 7.53–7.62 (m, 2H,
Harom/major (1), Harom/minor (1)), 7.73 (dd, J = 7.8/1.3 Hz, 1H,
tate = 2:1). Specific rotation: ½a D20
ꢂ
¼ ꢀ63:2 (c 8.0 CH2Cl2). Purity
Harom/major). The ratio of the diastereomers (major:minor) according
(HPLC method 1): tR = 11.8 min, purity 99.8%. Enantiomeric ratio
to the 1H NMR spectra is 2.5:1. 13C NMR (CDCl3): d (ppm) = 70.8 (1C,
CHCH2Omajor), 72.9 (1C, CHCH2Ominor), 80.3 (1C, CHCH2Ominor), 84.2
(1C, CHCH2Omajor), 121.4 (1C, C-22-bromophenyl/minor), 121.8 (1C,
C-22-bromophenyl/major), 127.1 (1C, Carom/minor), 128.2 (1C, Carom/minor),
128.6 (1C, Carom/major), 128.7 (1C, Carom/major), 130.6 (1C, Carom/minor),
130.6 (1C, Carom/major), 132.8 (1C, Carom/major), 133.2 (1C, Carom/minor),
135.1 (1C, C-12-bromophenyl/major), 135.7 (1C, C-12-bromophenyl/minor). IR
~
(HPLC method 2, Daicel Chiralcel OD, 10 lm, 250 mm/4.6 mm, iso-
hexane/isopropanol = 9:1, 0.8 mL/min, 7.5 lL): tR = 11.1 min,
(R):(S) = 99.4:0.6.
4.3.2. (S)-1-(2-Bromophenyl)ethane-1,2-diol (S)-623,24
AD-mix- (9.3 g) was added to a mixture of tert-butyl alcohol
a
(33 mL) and water (33 mL). The mixture was then cooled to 0 °C,
after which 2-bromostyrene 5 (1.22 g, 6.7 mmol) was added and
the reaction mixture was stirred at 0 °C for 21.5 h. Next, sodium
sulfite (9.5 g) was added and the mixture was allowed to warm
to room temperature and stirred for 40 min. Ethyl acetate was then
added to the reaction mixture, and after separation of the layers,
the aqueous layer was extracted with ethyl acetate (3ꢁ). The com-
bined organic layers were dried (Na2SO4), filtered, and the solvent
was removed in vacuo. The crude product was purified by flash col-
umn chromatography (3.5 cm, 15 cm, cyclohexane/ethyl ace-
tate = 2:1, 30 mL). Colorless solid, melting point 50 °C, yield
1.40 g (97%). C8H9BrO2 (217.1). Rf = 0.25 (cyclohexane/ethyl ace-
(neat):
(s, C1,2-disubst. arom.), 652 (s,
v
[cmꢀ1] = 3067 (w, mC–H arom.), 2955 (w, mC–H aliph.), 748
mC–Br).
4.3.5. [(R)-2-(2-Bromophenyl)-2-hydroxyethyl] 4-methylbenze-
nesulfonate (R)-10
Compound (R)-6 (300 mg, 1.4 mmol) was dissolved in THF
(10 mL). Triethylamine (250 lL, 1.7 mmol), dibutyltin oxide
(21 mg, 0.08 mmol), and 4-toluenesulfonyl chloride (427 mg,
2.2 mmol) were then added and the mixture was stirred overnight
at ambient temperature. Next, water was added to the reaction
mixture and after separation of the layers, the aqueous layer was
extracted with CH2Cl2 (3ꢁ). The combined organic layers were
dried (Na2SO4), filtered, and the solvent was removed in vacuo.
The crude product was purified by flash column chromatography
(3 cm, 15 cm, cyclohexane/ethyl acetate = 5:1, 20 mL). Colorless
oil, yield 483 mg (95%). C15H15BrO4S (371.2). Rf = 0.27 (cyclohex-
tate = 2:1). Specific rotation: ½a D20
¼ þ61:5 (c 6.2, CH2Cl2). Purity
ꢂ
(HPLC method 1): tR = 11.1 min, purity 99.9%. Enantiomeric ratio
(HPLC method 2, Daicel Chiralcel OD, 10 lm, 250 mm/4.6 mm, iso-
hexane/isopropanol = 9:1, 0.8 mL/min, 7.5 lL): tR = 14.0 min,
(R):(S) = 1.2:98.8.
ane/ethyl acetate = 5:1). Specific rotation: ½a D20
¼ ꢀ53:6 (c 28.1,
ꢂ
CH2Cl2). Purity (HPLC method 1): tR = 19.9 min, purity 96.9%.
4.3.3. Spectroscopic data for (R)-6 and (S)-6
LC–HRMS: m/z = 238.9681 (calcd 238.9678 for C8H979BrNaO2
[M+Na]+), 240.9661 (calcd 240.9658 for C8H981BrNaO2 [M+Na]+).
1H NMR (CDCl3): d (ppm) = 2.44 (s br, 1H, OH), 3.03 (s br, 1H, OH),
3.52–3.59 (m, 1H, HOCHCH2OH), 3.86–3.94 (m, 1H, HOCHCH2OH),
5.16–5.21 (m, 1H, HOCHCH2OH), 7.13–7.18 (m, 1H, 4-H2-bromophenyl),
7.32–7.37 (m, 1H, 5-H2-bromophenyl), 7.52 (dd, J = 7.9/1.2 Hz, 1H,
3-H2-bromophenyl), 7.58 (dd, J = 7.7/1.7 Hz, 1H, 6-H2-bromophenyl). 13C
4.3.6. [(S)-2-(2-Bromophenyl)-2-hydroxyethyl] 4-methylbenze-
nesulfonate (S)-10
Compound (S)-6 (2.71 g, 12.5 mmol) was dissolved in THF
(50 mL). Triethylamine (3.5 mL, 25.3 mmol), dibutyltin oxide
(155 mg, 0.62 mmol), and p-toluenesulfonyl chloride (3.58 g,
18.8 mmol) were then added and the mixture was stirred 3.5 h at
ambient temperature. Next, water and CH2Cl2 were added to the

274
K. Holl et al. / Tetrahedron: Asymmetry 25 (2014) 268–277
reaction mixture and after separation of the layers, the aqueous
phase was extracted with CH2Cl2 (3ꢁ). The combined organic layers
were dried (Na2SO4), filtered, and the solvent was removed in vacuo.
The crude product was purified by flash column chromatography
(6 cm, 16 cm, cyclohexane/ethyl acetate = 5:1, 65 mL). Colorless
oil, yield 3.74 g (81%). C15H15BrO4S (371.2). Rf = 0.27 (cyclohexane/
sphere. A solution of n-butyllithium in hexanes (1.6 m, 10 mL,
16.0 mmol) was added dropwise and the mixture was stirred for
15 min. Next, tert-butyl 4-oxopiperidine-1-carboxylate 12 (2.7 g,
13.6 mmol) was added. The mixture was stirred at ꢀ78 °C for
1.5 h, then warmed to ambient temperature and stirred for 1.5 h.
The reaction was stopped by the addition of water and worked
up as described for (R)-7a. The crude product was purified by flash
column chromatography (8 cm, 18 cm, n-hexane/ethyl ace-
tate = 7:3 ? ethyl acetate, 100 mL). Colorless oil, yield 124 mg
ethyl acetate = 5:1). Specific rotation: ½a D20
¼ þ54:1 (c 27.1, CH2Cl2).
ꢂ
Purity (HPLC method 1): tR = 19.7 min, purity 97.3%.
4.3.7. Spectroscopic data for (R)-10 and (S)-10
(60%). C18H25NO4 (319.4). Rf = 0.21 (cyclohexane/ethyl ace-
MS (ESI): m/z (%) = 393 (M(79Br)+Na+, 100), 395 (M(81Br) + Na+,
94). 1H NMR (CDCl3): d (ppm) = 2.45 (s, 3H, CH3), 2.74 (s br, 1H,
OH), 3.96 (dd, J = 10.6/8.4 Hz, 1H, HOCHCH2O), 4.27 (dd, J = 10.6/
2.6 Hz, 1H, HOCHCH2O), 5.31 (dd, J = 8.4/2.6 Hz, 1H, HOCHCH2O),
7.16 (td, J = 7.8/1.7 Hz, 1H, 4-H2-bromophenyl), 7.31–7.35 (m, 3H,
tate = 7:3). Specific rotation: ½a D20
¼ þ4:2 (c 12.4, CH2Cl2). Purity
ꢂ
(HPLC method 1): tR = 18.4 min, purity 97.0%. Enantiomeric ratio
(HPLC method 2, Daicel Chiralpak IB, 5
lm, 250 mm/4.6 mm,
isohexane/isoproanol = 97:3, 1.0 mL/min, 10
l
L): tR = 20.8 min,
(R):(S) = 2.0:98.0.
5-H2-bromophenyl
3-H2-bromophenyl), 7.57 (dd, J = 7.8/1.7 Hz, 1H, 6-H2-bromophenyl),
7.76–7.81 (m, 2H, 2-Htosyl
6-Htosyl). 13C NMR (CDCl3):
, 3-Htosyl, 5-Htosyl), 7.48 (dd, J = 7.8/1.1 Hz, 1H,
4.3.12. tert-Butyl(R)-3-(hydroxymethyl)-3H-spiro[[2]benzofur-
an-1,40-piperidine]-10-carboxylate (R)-7a
,
d
(ppm) = 21.8 (1C,CH3), 71.1 (1C, HOCHCH2O), 72.9 (1C, HOCHCH2O),
121.9 (1C, C-22-bromophenyl), 128.0 (1C, C-52-bromophenyl), 128.1 (2C,
Compound (S)-11 (173 mg, 0.87 mmol) was dissolved in THF
(7 mL). The solution was then cooled to ꢀ78 °C under N2 atmo-
C-2tosyl
,
C-6tosyl), 128.3 (1C, C-62-bromophenyl), 130.0 (1C,
sphere. A solution of n-butyllithium in hexanes (1.4 m, 870 lL,
C-42-bromophenyl), 130.1 (2C, C-3tosyl, C-5tosyl), 132.7 (1C, C-1tosyl),
1.2 mmol) was added dropwise and the mixture was stirred for
20 min. Next, tert-butyl 4-oxopiperidine-1-carboxylate 12
(208 mg, 1.0 mmol) was added. The mixture was stirred at
ꢀ78 °C for 1.5 h, and then warmed to ambient temperature. The
reaction was stopped by the addition of water. After separation
of the layers, the aqueous layer was extracted with CH2Cl2 (3ꢁ).
The combined organic layers were dried (Na2SO4), filtered, and
the solvent was removed in vacuo. The crude product was purified
by flash column chromatography (2.25 cm, 15 cm, n-hexane/ethyl
acetate = 7:3, 10 mL). Colorless oil, yield 124 mg (45%).
132.8 (1C, C-32-bromophenyl), 137.4 (1C, C-4tosyl), 145.2 (1C,
C-12-bromophenyl). IR (neat):
2986, 2901, (w,
C1,2-disubst. arom.), 660 (m,
v
[cmꢀ1] = 3518 (w, b,
m
O–H),
~
m
C–H), 1344 (m), 1173 (s,
C–Br).
mO@S@O), 756 (s,
m
4.3.8. (R)-2-(2-Bromophenyl)oxirane (R)-11
Compound (R)-10 (4.7 g, 12.7 mmol) was dissolved in methanol
(135 mL). Next, sodium (291 mg, 12.7 mmol) was added. The mix-
ture was stirred at ambient temperature for 1.5 h. The solvent was
then removed in vacuo and the crude product was purified by flash
column chromatography (3 cm, 16 cm, petrolether/ethyl ace-
tate = 50:1, 20 mL). Colorless liquid, yield 2.1 g (82%). C8H7BrO
C
18H25NO4 (319.4). Rf = 0.21 (cyclohexane/ethyl acetate = 7:3).
Specific rotation: ½a D20
¼ ꢀ4:3 (c 14.1, CH2Cl2). Purity (HPLC meth-
ꢂ
od 1): tR = 18.3 min, purity 99.0%. Enantiomeric ratio (HPLC meth-
(199.0). Rf = 0.18 (cyclohexane). Specific rotation: ½a D20
ꢂ
¼ ꢀ49:9 (c
od 2, Daicel Chiralpak IB, 5
l
m, 250 mm/4.6 mm, isohexane/
63.6, CH2Cl2). Purity: HPLC method 1: tR = 18.4 min, purity 99.4%.
isoproanol = 97:3,
1.0 mL/min, 10 L): tR = 18.1 min,
l
(R):(S) = 97.5:2.5.
4.3.9. (S)-2-(2-Bromophenyl)oxirane (S)-11
Compound (S)-10 (9.0 g, 24.3 mmol) was dissolved in methanol
(250 mL). Next, sodium (558 mg, 24.3 mmol) was added. The mix-
ture was stirred overnight at ambient temperature. The solvent
was then removed in vacuo. The crude product was purified by
flash column chromatography (8 cm, 16 cm, petroleum ether/ethyl
acetate = 50:1, 100 mL). Colorless liquid, yield 3.7 g (77%). C8H7BrO
4.3.13. Spectroscopic data for (S)-7a and (R)-7a
HRMS: m/z = 310.1851 (calcd 320.1856 for C18H26NO4 [M+H]+).
1H NMR (CDCl3): d (ppm) = 1.49 (s, 9H, CO2C(CH3)3), 1.64–1.72 (m,
2H, N(CH2CH2)2), 1.77 (td, J = 13.1/4.7 Hz, 1H, N(CH2CH2)2), 1.96
(td, J = 13.1/4.7 Hz, 1H, N(CH2CH2)2), 3.12–3.28 (m, 2H,
N(CH2CH2)2), 3.76 (dd, J = 11.6/5.7 Hz, 1H, CH2OH), 3.94 (dd,
J = 11.6/3.3 Hz, 1H, CH2OH), 4.00–4.19 (m, 2H, N(CH2CH2)2), 5.27–
5.32 (m, 1H, ArCHO), 7.07–7.12 (m, 1H, Harom.), 7.17–7.23 (m, 1H,
(199.0). Rf = 0.18 (cyclohexane). Specific rotation: ½a D20
¼ þ50:4 (c
ꢂ
29.4, CH2Cl2). Purity: HPLC method 1: tR = 18.1 min, purity 99.2%.
Harom.), 7.28–7.34 (m, 2H, Harom.). A signal for the OH proton is
4.3.10. Spectroscopic data for (R)-11 and (S)-11
not visible in the spectrum. 13C NMR (CDCl3): d (ppm) = 28.6 (3C,
CO2C(CH3)3), 37.1 (1C, N(CH2CH2)2), 38.0 (1C, N(CH2CH2)2), 40.5
(br, 1C, N(CH2CH2)2), 41.0 (br, 1C, N(CH2CH2)2), 66.1 (1C, CH2OH),
79.7 (1C, CO2C(CH3)3), 82.6 (1C, ArCHO), 84.7 (1C, ArCO), 121.1
(1C, Carom.), 121.6 (1C, Carom.), 128.2 (1C, Carom.), 128.4 (1C, Carom.),
138.1 (1C, 3a-Cbenzofuran), 146.2 (1C, 7a-Cbenzofuran), 155.1 (1C,
~
HRMS: m/z = 198.9750 (calcd for 198.9753 C8H879BrO [M+H]+),
200.9731 (calcd 200.9733 for C8H881BrO [M+H]+). 1H NMR (CDCl3):
d (ppm) = 2.65 (dd, J = 5.7/2.6 Hz, 1H, CHCH2O), 3.19 (dd, J = 5.7/
4.1 Hz, 1H, CHCH2O), 4.15 (dd, J = 4.1/2.6 Hz, 1H, CHCH2O), 7.16
(td, J = 7.9/1.9 Hz, 1H, 4-H2-bromophenyl), 7.23 (dd, J = 7.8/1.9 Hz,
1H, 6-H2-bromophenyl), 7.28–7.32 (m, 1H, 5-H2-bromophenyl), 7.58 (dd,
CO2C(CH3)3). IR (neat):
(w, C–H), 1670 (s,
(s, C–O–C, ether), 756 (s, C1,2-disubst. arom.).
v mO–H), 2974, 2920
[cmꢀ1] = 3441 (w, b,
J = 7.9/1.2 Hz, 1H, 3-H2-bromophenyl). 13C NMR (CDCl3):
d
m
mC–O–C), 1238, 1165 (s, mC–O–C, ester), 1042
(ppm) = 50.1 (1C, CHCH2O), 52.4 (1C, CHCH2O), 122.7 (1C,
C-22-bromophenyl), 126.1 (1C, C-62-bromophenyl), 127.8 (1C,
C-52-bromophenyl), 129.3 (1C, C-42-bromophenyl), 132.4 (1C,
[cm-1] =
~
v
m
4.3.14. (S)-(10-Benzyl-3H-spiro[[2]-benzofuran-1,40-piperidin]-
3-yl)methanol (S)-7b
C-32-bromophenyl), 137.3 (1C, C-12-bromophenyl). IR (neat):
3055 (w,
664 (m,
m
C–H arom.), 2990 (w,
m
C–H aliph.), 748 (s, C1,2-disubst. arom.),
Compound (S)-7a (250 mg, 0.78 mmol) was dissolved in CH2Cl2.
Next, trifluoroacetic acid (1 mL) was added and the mixture was
stirred for 1 h at ambient temperature. A 2 M aqueous solution of
sodium hydroxide was then added, and after separation of the lay-
ers, the aqueous layer was extracted with CH2Cl2 (3ꢁ). The com-
bined organic layers were dried (Na2SO4), filtered, and the
solvent was removed in vacuo. The residue was dissolved in CH2Cl2
m
C–Br).
4.3.11. tert-Butyl (S)-3-(hydroxymethyl)-3H-spiro[[2]benzofur-
an-1,40-piperidine]-10-carboxylate (S)-7a
Compound (R)-11 (2.3 g, 11.6 mmol) was dissolved in THF
(75 mL). The solution was then cooled to ꢀ78 °C under N2 atmo-

K. Holl et al. / Tetrahedron: Asymmetry 25 (2014) 268–277
275
(30 mL), after which benzaldehyde (316
l
L, 3.12 mmol) and, after
4.3.18. (R)-10-Benzyl-3-(fluoromethyl)-3H-spiro[[2]benzofuran-
1,40-piperidine] (R)-1
5 min, sodium triacetoxyborohydride (1.3 g, 6.2 mmol) were added
and the mixture was stirred overnight at ambient temperature. The
reaction was stopped by the addition of a 2 M aqueous solution of
NaOH, and after separation of the layers, the aqueous layer was ex-
tracted with CH2Cl2 (3ꢁ). The combined organic layers were dried
(Na2SO4), filtered, and the solvent was removed in vacuo. The
crude product was purified by flash column chromatography
(2.75 cm, 17 cm, cyclohexane/ethyl acetate = 5:5, 20 mL). Pale
yellow oil, yield 145 mg (60%). C20H23NO2 (309.4). Rf = 0.17 (cyclo-
At first, CH2Cl2 (3 mL) was cooled to ꢀ78 °C and diethylamino-
sulfur trifluoride (100 lL, 0.76 mmol) was added. After 45 min, (R)-
7b (55 mg, 0.18 mmol), dissolved in CH2Cl2 (2 mL), was added and
the solution was stirred for 1 h at ꢀ78 °C. Next, the mixture was
allowed to warm to ambient temperature and stirred overnight.
The reaction was then stopped by the addition of a 2 M aqueous
solution of NaOH, and after separation of the layers, the aqueous
layer was extracted with CH2Cl2 (4ꢁ). The combined organic layers
were dried (Na2SO4), filtered, and the solvent was removed in va-
cuo. The crude product was purified by flash column chromatogra-
phy twice (1. 1.25 cm, 16 cm, cyclohexane/ethyl acetate = 9:1,
5 mL; 2. 1 cm, 15 cm, cyclohexane/ethyl acetate = 9:1, 5 mL). Col-
orless oil, yield 13 mg (23%). C20H22FNO (311.4). Rf = 0.52 (cyclo-
hexane/ethyl acetate = 5:5). Specific rotation: ½a D20
¼ þ5:9 (c 29.4,
ꢂ
CH2Cl2). Purity (HPLC method 1): tR = 13.8 min, purity 97.3%.
4.3.15. (R)-(10-Benzyl-3H-spiro[[2]benzofuran-1,40-piperidin]-3-
yl)methanol (R)-7b
Compound (R)-7a (210 mg, 0.66 mmol) was dissolved in CH2Cl2.
Trifluoroacetic acid (1 mL) was then added and the mixture was
stirred for 2.5 h at ambient temperature. The reaction was worked
up as described for (S)-7b. The residue was dissolved in CH2Cl2
hexane/ethyl acetate = 5:5). Specific rotation: ½a D20
¼ þ4:6 (c 4.8,
ꢂ
CH2Cl2). Purity (HPLC method 1): tR = 16.8 min, purity 95.1%. Enan-
tiomeric ratio (HPLC method 2, Daicel Chiralpak IB, 5
4.6 mm, isohexane:isopropanol 99:1, 1.0 mL/min, 10
min, (R):(S) = 96.5:3.5.
l
l
m, 250 mm/
L): tR = 9.9 -
(30 mL), after which benzaldehyde (278 lL, 2.74 mmol) and, after
1 h, sodium triacetoxyborohydride (1.1 g, 5.2 mmol) were added
and the mixture was stirred overnight at ambient temperature.
Next, the reaction was worked up as described for (S)-7b. The
crude product was purified by flash column chromatography
(3 cm, 17 cm, cyclohexane/ethyl acetate = 5:5, 20 mL). Pale yellow
oil, yield 121 mg (59%). C20H23NO2 (309.4). Rf = 0.17 (cyclohexane/
4.3.19. Spectroscopic data for (S)-1 and (R)-1
HRMS: m/z = 310.1773 (312.1758 calcd for C20H23FNO [M+H]+).
1H NMR (CDCl3): d (ppm) = 1.71–1.81 (m, 2H, N(CH2CH2)2), 2.02–
2.29 (m, 2H, N(CH2CH2)2), 2.50–2.69 (m, 2H, N(CH2CH2)2), 2.86–
2.00 (m, 2H, N(CH2CH2)2), 3.69 (s, 2H, NCH2Ph), 4.56 (ddd,
J = 47.3/9.7/4.9 Hz, 1H, CH2F), 4.61 (ddd, J = 47.3/9.7/3.7 Hz, 1H,
CH2F), 5.40 (dt, J = 20.3/4.3 Hz, 1H, ArCHO), 7.17–7.25 (m, 2H,
ethyl acetate = 5:5). Specific rotation: ½a D20
¼ ꢀ5:7 (c 28.2, CH2Cl2).
ꢂ
Purity (HPLC method 1): tR = 13.7 min, purity 95.6%.
H
arom.), 7.28–7.47 (m, 7H, Harom.). 13C NMR (CDCl3):
d
(ppm) = 37.8 (1C, N(CH2CH2)2), 38.3 (1C, N(CH2CH2)2), 50.0 (1C,
N(CH2CH2)2), 50.1 (1C, N(CH2CH2)2), 63.2 (1C, NCH2Ph), 80.8 (d,
J = 20.1 Hz, 1C, ArCHO), 85.2 (1C, ArCO), 85.5 (d, J = 175.1 Hz, 1C,
CH2F), 121.4 (1C, Carom.), 121.7 (1C, Carom.), 127.7 (1C, Carom.),
128.2 (1C, Carom.), 128.6 (2C, Carom.), 128.7 (1C, Carom.), 129.9 (2C,
4.3.16. Spectroscopic data for (S)-7b and (R)-7b
HRMS: m/z = 310.1825 (calcd 310.1802 for C20H24NO2 [M+H]+).
1H NMR (CDCl3): d (ppm) = 1.70–1.78 (m, 2H, N(CH2CH2)2), 2.00
(td, J = 13.0/4.4 Hz, 1H, N(CH2CH2)2), 2.19 (td, J = 13.0/4.4 Hz, 1H,
N(CH2CH2)2), 2.48–2.57 (m, 2H, N(CH2CH2)2), 2.83–2.91 (m, 2H,
N(CH2CH2)2), 3.62 (s, 2H, NCH2Ph), 3.75 (dd, J = 11.6/56 Hz, 1H,
CH2OH), 3.94 (dd, J = 11.6/3.5 Hz, 1H, CH2OH), 5.29 (dd, J = 5.6/
3.5 Hz, 1H, ArCHO), 7.15–7.20 (m, 2H, Harom.), 7.26–7.45 (m, 7H,
C
arom.), 137.0 (1C, Carom.), 138.5 (1C, Carom.), 146.7 (1C, Carom.). IR
[cmꢀ1] = 2940, 2808 (w,
m
C–H), 1053 (s,
m
C–O–C), 737 (s,
~
(neat):
v
C1,2-disubst. arom.), 698 (s, Cmonosubst. arom.).
4.3.20. tert-Butyl (S)-3-[(tosyloxy)methyl]-3H-spiro[[2]benzo-
furan-1,40-piperidine]-10-carboxylate (S)-13
Compound (S)-7a (210 mg, 0.66 mmol) was dissolved in CH2Cl2
(20 mL), after which 4-dimethylaminopyridine (21 mg, 0.17 mmol),
H
arom.). A signal for the OH proton is not visible in the spectrum.
13C NMR (CDCl3): d (ppm) = 37.2 (1C, N(CH2CH2)2), 38.1 (1C, N(CH2-
CH2)2), 49.8 (1C, N(CH2CH2)2), 50.0 (1C, N(CH2CH2)2), 63.2 (1C,
NCH2Ph), 66.1 (1C, CH2OH), 82.5 (1C, ArCHO), 84.5 (1C, ArCO),
121.3 (1C, Carom.), 121.5 (1C, Carom.), 127.5 (1C, Carom.), 128.1 (1C,
triethylamine (460 lL, 3.3 mmol), and 4-toluenesulfonyl chloride
(251 mg, 1.3 mmol) were added and the mixture was stirred at
ambient temperature for 17.5 h. The reaction was then stopped by
the addition of a 2 M aqueous solution of NaOH and worked up as
described for (R)-13. The crude product was purified by flash col-
umn chromatography (2.25 cm, 16 cm, n-hexane/ethyl ace-
tate = 9:1, 10 mL). Colorless solid, yield 252 mg (81%). C25H31NO6S
(473.6). Rf = 0.28 (cyclohexane/ethyl acetate = 8:2). Melting point:
C
arom.), 128.3 (1C, Carom.), 128.5 (2C, Carom.), 129.8 (2C, Carom.),
137.5 (1C, Carom.), 138.2 (1C, Carom.), 129.8 (1C, Carom.). IR (neat):
~
v
(s,
[cmꢀ1] = 3325 (w, b,
mO–H), 2924, 2816 (w,
mC–H), 1045
mC–O–C), 741 (s, C1,2-disubst. arom.), 698 (s, Cmonosubst. arom.).
4.3.17. (S)-10-Benzyl-3-(fluoromethyl)-3H-spiro[[2]benzofuran-
1,40-piperidine] (S)-1
124 °C. Specific rotation: ½a D20
¼ þ19:2 (c 7.6, CH2Cl2). Purity (HPLC
ꢂ
Diethylaminosulfur trifluoride (64 mg, 0.40 mmol) was dis-
solved in CH2Cl2 (1 mL). The solution was cooled to ꢀ78 °C, after
which (S)-7b (50 mg, 0.16 mmol), dissolved in CH2Cl2 (1 mL), was
added. The solution was stirred for 1 h at ꢀ78 °C. Next, the mixture
was warmed to ambient temperature, stirred overnight, and
worked up as described for (R)-2. The crude product was purified
by flash column chromatography three times (1. 1 cm, 15 cm,
cyclohexane/ethyl acetate = 7:3, 5 mL; 2. 1 cm, 18 cm, cyclohex-
ane/ethyl acetate = 9:1, 5 mL; 3. 1 cm, 18 cm, cyclohexane/ethyl
acetate = 9:1, 5 mL). Colorless oil, yield 9 mg (18%). C20H22FNO
(311.4). Rf = 0.52 (cyclohexane/ethyl acetate = 5:5). Specific rota-
method 1): tR = 22.8 min, purity 98.8%.
4.3.21. tert-Butyl (R)-3-[(tosyloxy)methyl]-3H-spiro[[2]benzo-
furan-1,40-piperidine]-10-carboxylate (R)-13
Compound (R)-7a (4.5 g, 14.1 mmol) was dissolved in CH2Cl2
(400 mL), after which 4-dimethylaminopyridine (434 mg,
3.56 mmol), triethylamine (9 mL, 65.0 mmol), and 4-toluenesulfo-
nyl chloride (5.4 g, 28.4 mmol) were added and the mixture was
stirred overnight at ambient temperature. The reaction was then
stopped by the addition of a 2 M aqueous solution of NaOH and
after separation of the layers, the aqueous layer was extracted with
CH2Cl2 (3ꢁ). The combined organic layers were dried (Na2SO4), fil-
tered, and the solvent was removed in vacuo. The crude product
was purified by flash column chromatography (6 cm, 17 cm,
tion:
½
a 2D0
ꢂ
¼ ꢀ3:9 (c 4.5, CH2Cl2). Purity (HPLC method 1):
tR = 16.7 min, purity 94.6%. Enantiomeric ratio (HPLC method 2,
Daicel Chiralpak IB, 5
99:1, 1.0 mL/min, 10
l
m, 250 mm/4.6 mm, isohexane:isopropanol
lL): tR = 8.0 min, (R):(S) = 1.2:98.8.

276
K. Holl et al. / Tetrahedron: Asymmetry 25 (2014) 268–277
n-hexane/ethyl acetate = 8:2, 65 mL) and crystallized from CH2Cl2.
Colorless solid, yield 4.7 g (70%). C25H31NO6S (473.6). Rf = 0.28
(cyclohexane/ethyl acetate = 8:2). Melting point: 125 °C. Specific
cooling centrifuge model Sorvall RC-5C plus (Thermo Fisher Scien-
tific, Langenselbold, Germany). Multiplates: standard 96-well mul-
tiplates (Diagonal, Muenster, Germany). Shaker: self-made device
with adjustable temperature and tumbling speed (scientific work-
shop of the institute). Vortexer: Vortex Genie 2 (Thermo Fisher Sci-
entific, Langenselbold, Germany). Harvester: MicroBeta FilterMate-
96 Harvester. 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 of analytical grade.
rotation: ½a 2D0
¼ ꢀ19:3 (c 7.2, CH2Cl2). Purity (HPLC method 1):
ꢂ
tR = 22.3 min, purity 99.8%.
4.3.22. Spectroscopic data for (S)-13 and (R)-13
HRMS: m/z = 474.1969 (calcd 474.1945 for C25H32NO6S
[M+H]+). 1H NMR (CDCl3): d (ppm) = 1.48 (s, 9H, CO2C(CH3)3),
1.51–1.70 (m, 2H, N(CH2CH2)2), 1.70 (td, J = 13.1/4.8 Hz, 1H, N(CH2-
CH2)2), 1.82 (td, J = 13.1/4.8 Hz, 1H, N(CH2CH2)2), 2.44 (s, 3H, CH3),
3.02 (td, J = 12.9/2.9 Hz, 1H, N(CH2CH2)2), 3.12 (td, J = 12.9/2.9 Hz,
1H, N(CH2CH2)2), 3.95–4.07 (m, 2H, N(CH2CH2)2), 4.15 (dd,
J = 10.2/5.3 Hz, 1H, CH2OTos), 4.24 (dd, J = 10.2/4.1 Hz, 1H, CH2-
OTos), 5.37 (t, J = 4.7 Hz 1H, ArCHO), 7.04–7.08 (m, 1H, Harom.),
7.12–7.17 (m, 1H, Harom.), 7.24–7.35 (m, 4H, 3-Htosyl, 5-Htosyl, Harom.
(2H)), 7.71–7.75 (m, 2H, 2-Htosyl, 6-Htosyl). 13C NMR (CDCl3): d
(ppm) = 21.8 (1C, CH3), 28.6 (3C, CO2C(CH3)3), 37.2 (1C, N(CH2-
CH2)2), 37.9 (1C, N(CH2CH2)2), 40.4 (1C, N(CH2CH2)2), 40.6 (1C,
N(CH2CH2)2), 72.2 (1C, CH2OTos), 79.4 (1C, ArCHO), 79.6 (1C, CO2-
C(CH3)3), 85.4 (1C, ArCO), 121.1 (1C, Carom.), 122.0 (1C, Carom.),
128.1 (2C, C-2tosyl, C-6tosyl), 128.3 (1C, Carom.), 128.8 (1C, Carom.),
130.0 (2C, C-3tosyl, C-5tosyl), 133.0 (1C, Carom.), 136.9 (1C, Carom.),
145.1 (1C, Carom.), 145.8 (1C, Carom.), 155.1 (1C, CO2C(CH3)3). IR
~
4.5.2. Preparation of membrane homogenates from guinea pig
brain
Five guinea pig brains were homogenized with the potter (500–
800 rpm, 10 up-and-down strokes) in 6 volumes of cold 0.32 M
sucrose. The suspension was centrifuged at 1200g for 10 min at
4 °C. The supernatant was separated and centrifuged at 23,500g
for 20 min at 4 °C. The pellet was resuspended in 5–6 volumes of
buffer (50 mM TRIS, pH 7.4) and centrifuged again at 23,500g
(20 min, 4 °C). This procedure was repeated twice. The final pellet
was resuspended in 5–6 volumes of buffer and frozen (ꢀ80 °C) in
1.5 mL portions containing approximately 1.5 mg protein/mL.
(neat):
O@S@O), 1234, 1173 (m, mC–O–C, ester), 1069 (m, mC–O–C, ether), 768
(m, C1,2-disubst. arom.).
v
[cmꢀ1] = 2978, 2870 (w,
mC–H), 1686 m,
mC@O), 1362 (m,
4.5.3. Preparation of membrane homogenates from the rat liver
Two rat livers were cut into small pieces and homogenized with
the potter (500–800 rpm, 10 up-and-down strokes) in 6 volumes of
cold 0.32 M sucrose. The suspension was centrifuged at 1200g for
10 min at 4 °C. The supernatant was separated and centrifuged at
31,000g for 20 min at 4 °C. The pellet was resuspended in 5–6 vol-
umes of buffer (50 mM TRIS, pH 8.0) and incubated at room tem-
perature for 30 min. After the incubation, the suspension was
centrifuged again at 31,000g for 20 min at 4 °C. The final pellet
was resuspended in 5–6 volumes of buffer and stored at ꢀ80 °C
in 1.5 mL portions containing approximately 2 mg protein/mL.
m
4.4. X-ray crystal structure analysis of (R)-13
Data sets were collected with a Nonius KappaCCD diffractome-
ter. Programs used: data collection, COLLECT (R. W. W. Hooft, Bru-
ker AXS, 2008, Delft, The Netherlands); data reduction Denzo-SMN
(Otwinowski, Z., Minor, W., Methods Enzymol. 1997, 276, 307–326);
absorption correction, Denzo (Otwinowski, Z., Borek, D., Majewski,
W., Minor, W., Acta Crystallogr. 2003, A59, 228–234); structure
solution SHELXS-97 (Sheldrick, G. M., Acta Crystallogr. 1990, A46,
467–473); structure refinement SHELXL-97 (Sheldrick, G. M., Acta
Crystallogr. 2008, A64, 112–122) and graphics, XP (BrukerAXS,
2000). Thermal ellipsoids are shown with 30% probability, R-values
are given for observed reflections, and wR2 values are given for all
reflections.
4.5.4. Protein determination
The protein concentration was determined by the method of
Bradford,32 and modified by Stoscheck.33 The Bradford solution
was prepared by dissolving 5 mg of Coomassie Brilliant Blue G
250 in 2.5 mL of EtOH (95%, v/v). Next, 10 mL of deionized H2O
and 5 mL of phosphoric acid (85%, m/v) were added to this solu-
tion, after which the mixture was stirred and filled to a total vol-
ume of 50.0 mL with deionized water. The calibration was
carried out using bovine serum albumin as a standard in 9 concen-
trations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, and 4.0 mg /mL). In a 96-
X-ray crystal structure analysis of (R)-13: formula C25H31NO6S,
M = 473.57, colorless crystal, 0.20 ꢁ 0.04 ꢁ 0.02 mm, a = 7.2889(5),
b = 10.5174(3), c = 32.5260(30) Å, V = 2493.5(3) Å3,
qcalc = 1.262
g cmꢀ3
(0.756 6 T 6 0.971), Z = 4, orthorhombic, space group P212121 (No.
19), k = 1.54178 Å, T = 223(2) K, and scans, 15495 reflections
collected ( h, k, l), [(sinh)/k] = 0.59 Åꢀ1
3674 independent
(Rint = 0.072) and 2921 observed reflections [I > 2 (I)], 362 refined
,
l
= 1.481 mmꢀ1
,
empirical absorption correction
well standard multiplate, 10
lL of the calibration solution or 10
lL
of the membrane receptor preparation were mixed with 190
lL of
x
u
the Bradford solution, respectively. After 5 min, the UV absorption
of the protein-dye complex at k = 595 nm was measured with a
platereader (Tecan Genios, Tecan, Crailsheim, Germany).
,
r
parameters, R = 0.047, wR2 = 0.107, max. (min.) residual electron
density 0.15 (ꢀ0.17) e.Åꢀ3, hydrogen atoms were calculated and re-
fined as riding atoms. Flack parameter: ꢀ0.02(3). CCDC 956667 con-
tains the supplementary crystallographic data for this paper. These
data can be obtained free of charge from the Cambridge Crystallo-
graphic Data Centre.
4.5.5. General protocol for the binding assays
The test compound solutions were prepared by dissolving
approximately 10 lmol (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 car-
ried out in duplicates in 96-well multiplates. The concentrations
given are the final concentrations in the assay. Generally, the as-
4.5. Receptor binding studies9,10
4.5.1. Materials
The guinea pig brains and rat liver for the r1 and r2 receptor
binding assays were commercially available (Harlan–Winkelmann,
Borchen, Germany). Homogenizer: Elvehjem Potter (B. Braun Bio-
tech International, Melsungen, Germany). Cooling centrifuge mod-
el Rotina 35R (Hettich, Tuttlingen, Germany) and High-speed
says were performed by the addition of 50
say buffer, 50 L test compound solution in various concentrations
(10ꢀ5, 10ꢀ6, 10ꢀ7, 10ꢀ8, 10ꢀ9 and 10ꢀ10 mol/L), 50
L of the corre-
sponding radioligand solution and 50 L of the respective receptor
lL of the respective as-
l
l
l

K. Holl et al. / Tetrahedron: Asymmetry 25 (2014) 268–277
277
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The receptor preparation was always added last. During the incu-
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was 20%. The IC50-values were calculated with the program Graph-
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incubated with various concentrations of test compounds, 2 nM
[3H]-(+)-Pentazocine, and TRIS buffer (50 mM, pH 7.4) at 37 °C.
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membrane preparation of the rat liver (about 100 lg of protein)
was incubated with various concentrations of the test compound,
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(+)-pentazocine in 50 mM TRIS, pH 8.0) at room temperature.
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Acknowledgement
This work was supported by the Deutsche Forschungsgemeins-
chaft (DFG), which is gratefully acknowledged.
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