Improvement of σ1 receptor affinity by late-stage C-H-bond arylation of spirocyclic lactones

Article abstract of DOI:10.1016/j.bmc.2013.01.038

The direct C-H-bond arylation of the complex spirocyclic lactones 13, 14, and 18 allows the introduction of diverse aryl moieties in the last step of the synthesis. A selective α-arylation of the thiophene moiety was performed with the catalytic system Pd

Full text of DOI:10.1016/j.bmc.2013.01.038

Bioorganic & Medicinal Chemistry 21 (2013) 1844–1856
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Improvement of
r1 receptor affinity by late-stage C–H-bond arylation
of spirocyclic lactones
Christina Meyer a, Benedikt Neue b, Dirk Schepmann a, Shuichi Yanagisawa c, Junichiro Yamaguchi c,
Ernst-Ulrich Würthwein b, Kenichiro Itami c, , Bernhard Wünsch a,
⇑
a Institut für Pharmazeutische und Medizinische Chemie der Westfälischen, Wilhelms-Universität Münster, Hittorfstraße 58-62, D-48149 Münster, Germany
b Organisch-Chemisches Institut der Westfälischen Wilhelms-Universität Münster, Correnstraße 40, D-48149 Münster, Germany
c Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The direct C–H-bond arylation of the complex spirocyclic lactones 13, 14, and 18 allows the introduction
Received 14 November 2012
Revised 14 January 2013
Accepted 18 January 2013
Available online 31 January 2013
of diverse aryl moieties in the last step of the synthesis. A selective a-arylation of the thiophene moiety
was performed with the catalytic system PdCl2/2,20-bipyridyl/Ag2CO3, whereas the b-position of the thio-
phene ring was addressed by using the alternative catalytic system PdCl2/P[OCH(CF3)2]3/Ag2CO3. Due to
electronic and steric reasons the arylation of the five-membered lactone 18 occurred in both a-positions
providing 40-mono-, 60-mono- and 40,60-diarylated thiophenes 22–26a–c. Compounds with an additional
aryl moiety at the ‘upper left (top)’ position (10-position of 13, 30-position of 14, 40-position of 18) showed
Key words:
r1 Ligands
C–H-bond arylation of complex thiophenes
Spirocyclic lactones
DFT calculations
increased
r
1 affinity compared to the non-arylated parent compounds. A phenyl moiety at the ‘left’ posi-
1 affinity but to a lower extent. A considerable reduction of
tion (20-position in 20a) also increased the
r
r1 affinity was observed after introducing an aryl moiety in 60-position of 18, which might result from
shielding the tertiary amine, which is crucial for interaction with the r1 receptor. The discussion of
the experimental results is supported by high-level quantum chemical DFT-calculations of the NBO-
charges of 13 and 18 and the relative energies of the related arylated products.
Ó 2013 Elsevier Ltd. All rights reserved.
Hydrophobic pocket
1. Introduction
fects after withdrawal of alcohol, cocaine, or methamphetamine
from addicted animals can be reduced by r1 antagonists.7–10 Fur-
The group of
r
receptors has been divided into at least two sub-
thermore, r1 antagonists are valuable tools in cancer research
types, which are termed r1 and r2 receptors. The gene of the hu-
man r1 receptor encodes for a protein of 223 amino acids with a
molecular weight of 25.3 kDa. On the level of amino acid sequence
the r1 receptor is well characterized. The amino and carboxy ter-
mini of the membrane bound r1 receptor protein are linked by
two transmembrane domains and both are located on the intracel-
lular side of the membrane.1–4 Although the r2 receptor is less
characterized than the r1 receptor, the identity of the r2 receptor
and the progesterone receptor membrane component 1 (pgrmc1)
was postulated recently. In 1996 the pgrmc1 was cloned and the
resulting protein consists of 194 amino acids with a molecular
weight of 21.7 kDa.5,6
due to the fact that several human tumor cells produce numerous
copies of r1 (and
r
2) receptors.11,12
Our interest has been focused on the development of novel
compounds with high affinity towards r1 receptors within the
central nervous system. We found that spirocyclic piperidines with
the privileged 2-benzofuran (1) or 2-benzopyran (3) substructure
are potent r1 receptor antagonists binding in the low nanomolar
range (Fig. 1). The methoxy moiety replaced with a carbonyl group
led to the lactones 2 and 4 with 20-fold reduced
r1 receptor affin-
ity.13 Very recently it was shown that an additional aryl moiety at
the ‘upper left (top)’ position of the spirocyclic compounds is well
tolerated by the
r1 receptor protein or even increases the r1 affin-
Ligands which are able to modulate the r1 receptor activity
possess a high potential for the treatment of acute and chronic
neurological disorders (e.g., depression, schizophrenia, (neuro-
pathic) pain, Alzheimer’s disease). Unpleasant and dangerous ef-
ity. It was postulated that the additional aryl moiety of the ligands
might occupy an additional hydrophobic pocket of the
protein.14,15
r1 receptor
Replacement of the benzene substructure of the spirocyclic r1
ligands 1 and 3 with a bioisosteric thiophene moiety (5) led to very
potent
r1 ligands, which even exceed the r1 affinity of the parent
⇑
Corresponding author. Tel.: +49 251 8333311; fax: +49 251 8332144.
E-mail addresses: itami.kenichiro@a.mbox.nagoya-u.ac.jp (K. Itami), wuensch@
uni-muenster.de (B. Wünsch).
benzene derivatives 1 and 3. The
r1 affinity of the spirocyclic thio-
phenes 5 is in the subnanomolar range.15–17 In this project the ef-
fect of an oxo group (6) instead of an acetalic methoxy moiety (5)
Tel./fax: +81 52 788 6098.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.038

C. Meyer et al. / Bioorg. Med. Chem. 21 (2013) 1844–1856
1845
OCH3
O
regioisomeric hydroxyacetals 9 and 10 in 53% and 12% yields,
respectively.15 The formation of the regioisomer 10 is explained
by migration of the lithium atom from the original 4-position to
the thermodynamically more stable 2-position. Addition of this
thiophen-2-yllithium intermediate to 1-benzylpiperidin-4-one
provided the hydroxyacetal 10 (Scheme 1).
The hydroxyacetals 9 and 10 were treated with diluted HCl to
afford the cyclic hemiacetals (lactols) 11 and 12. Since the
oxidation of the lactols 11 and 12 with tetrapropylammonium
perruthenate (TPAP) in presence of an excess of N-methylmorpho-
line-N-oxide (NMMO),18 with oxalyl chloride/DMSO (Swern oxida-
tion),19 or with Dess–Martin-Periodinane20 failed to provide the
lactones 13 and 14, Cr(VI)-salts21–23 were considered as oxidants.
After optimization, the oxidation of lactols 11 and 12 with pyridi-
nium chlorochromate (PCC) provided lactones 13 and 14 in 71%
yield, respectively.
()n
N
()n
N
O
O
Bn
Bn
1
2
(n = 0)
(n = 0)
Ki(σ1) = 1.1 nM
Ki(σ1) = 21 nM
4
(n = 1)
3
(n = 1)
Ki(σ1) = 29 nM
Ki(σ1) = 1.3 nM
3
3
Aryl
3
O
OCH3
O
()n
N
()n
()n
2
2
2
S
S
S
O
O
O
1
1
1
N
Bn
N
Bn
Bn
7
5
6
In addition to the six-membered lactones 13 and 14 the corre-
sponding smaller homologue 18 was envisaged. Both the carbonyl
moiety directly attached to the thiophene ring and the reduced size
of the lactone ring might influence the direct C–H-bond arylation
Figure 1. Potent
r1 ligands with benzofuran and benzopyran structure and design
of novel thiophene annulated lactones with additional aryl substituents.
on the r1 receptor affinity is investigated. Subsequently, a broad
process as well as the
r affinity of the resulting compounds.
selection of aryl moieties with different substituents is introduced
in a- or b-position of the thiophene ring by direct C–H-bond aryla-
A one-pot three step procedure for the synthesis of similar lac-
tones was reported in literature,24 which was followed herein. At
first dibromothiophene 15 was reacted with one equivalent of n-
butyllithium followed by addition of 1-benzylpiperidin-4-one.
The resulting lithium alcoholate 16 was treated with an additional
equivalent of n-butyllithium and the new aryllithium intermediate
tion. This arylation should be performed at the end of the synthesis
to allow the preparation of a large collection of diversely arylated
compounds. A direct C–H-bond arylation of complex spirocyclic
thiophenes with a lactone substructure has not been performed
so far. In particular the compatibility of the lactone moiety with
the basic reaction conditions (Ag2CO3) and the high temperature
(150 °C) should be investigated. The arylation of a constrained
five-membered lactone directly connected to the thiophene ring
(18) is a particular challenge, since the electron density of the thi-
ophene ring is reduced by the carbonyl group. It was found that in
the series of thiophene bioisosteric lactones 6 the r1 affinity was
reduced compared to the r1 affinities of the corresponding meth-
oxy derivatives 5. Therefore it was of particular interest, whether
the introduction of an additional aryl moiety into the lactones 6
is able to compensate the reduced r1 receptor affinities of the
non-arylated lactones 6.
was trapped with CO2. Finally, the formed
c-hydroxyacid 17 was
heated with acetic anhydride to provide the
c-lactone 18 in 44%
yield. During the flash chromatographic purification process small
amounts (3%) of 1-benzyl-30-bromospiro[piperidine-4,40-thie-
no[2,3-c]furan]-60-one were also isolated (Scheme 2).
Pd-catalyzed cross-coupling reactions of metalated arene/het-
eroarene and halogenated arene/heteroarene species are among
the most reliable methods for preparing biaryls and heterobiaryls,
as exemplified by the Suzuki–Miyaura coupling reaction.25 How-
ever, the direct C–H-bond arylation will avoid the lengthy activa-
tion of one arene moiety. After the first pioneering report of Ohta
in 1990,26 a number of catalysts including Pd,27 Rh,28,29 Ir,30 and
Cu31 catalysts were developed promoting the direct
of thiophene derivatives with haloarenes. After our successful Pd-
catalyzed direct -arylation of related spirocyclic thiophenes con-
taining an ether or acetal group,15,32,33 we envisaged the catalytic
system PdCl2/2,20-bipyridyl/Ag2CO3,34,35 for the
-arylation of the
a-arylation
2. Chemistry
a
The synthesis started with the (thiophen-3-yl)acetaldehyde
acetal 8.15 Bromine lithium exchange with n-butyllithium at
ꢀ78 °C and subsequent trapping of the resulting thienyllithium
intermediate with 1-benzylpiperidin-4-one led to the
a
lactones 13, 14 and 18. In particular it was of great interest to
determine whether the basic reaction conditions were tolerated
OCH3
OCH3
OCH3
OCH3
OCH3
OH
S
(a)
O
OCH
3
S
S
OH
Br
8
N
Bn
9
N
Bn
10
N
Bn
(b)
(c)
OH
OH
O
O
S
S
(e)
(d)
O
O
O
O
S
S
N
N
N
N
Bn
Bn
Bn
Bn
11
12
14
13
Scheme 1. Synthesis of the spirocyclic thiophene annulated lactones 13 and 14. Reagents and conditions: (a) n-BuLi, THF, ꢀ78 °C, 15 min, then 1-benzylpiperidin-4-one,
ꢀ78 °C, 3 h, 9 (53%) and 10 (12%); (b) THF, 1 M HCl, rt, 16 h, 76%; (c) THF, 1 M HCl, rt, 16 h, 30%; (d) PCC, CH2Cl2, rt, 16 h, 71%; (e) PCC, CH2Cl2, rt, 16 h, 71%.

1846
C. Meyer et al. / Bioorg. Med. Chem. 21 (2013) 1844–1856
Table 2
Br
Br
Yields after
a
- and b-arylation of spirocyclic lactone 14 with various iodoarenes
Br
n-BuLi
O
n-BuLi
CO2
S
S
OLi
3'
5'
O
2'
15
N
Bn
O
S
N
Bn
1'
16
N
Bn
14
O
CO2H
OH
4
6
S
(b)
aryl
(a)
O
Ac2O
NaOAc
44%
S
O
N
Bn
N
Bn
3'
O
O
2'
aryl
7'
O
S
S
17
18
N
Bn
21a,b
N
Bn
20a
Scheme 2. Synthesis of spirocyclic thiophene annulated lactone 18. Reagents and
conditions: n-BuLi, Et2O, ꢀ78 °C, 10 min, 1-benzylpiperidin-4-one, ꢀ78 °C, 30 min;
then n-BuLi, ꢀ78 °C, 10 min, CO2, <ꢀ70 °C, 1 h: then Ac2O, NaOAc, toluene, 110 °C,
16 h, 44%.
(a) PdCl2/2,2’-bipyridyl, Ag2CO3, Aryl-I, m-xylene, 150 °C, 12 h.
(b) PdCl2, P[OCH(CF3)2]3, Ag2CO3, Aryl-I, m-xylene, 150 °C, 12 h.
by the lactone moieties. The direct C–H-bond arylation of the lac-
tones 13, 14 and 18 will allow the step-economical introduction of
diverse aryl substituents as last step of the synthesis.
At first the thieno[3,4-c]pyranone 13 was reacted with iodoben-
zene, PdCl2, 2,20-bipyridyl and Ag2CO3 in boiling m-xylene (150 °C)
to afford the biaryl 19a in 57% yield (Table 1). The regioisomeric
arylation product with the phenyl moiety in 30-position was not
observed. Despite the presence of basic Ag2CO3 the lactone moiety
of 13 was stable during this transformation. Therefore substituted
iodoarenes were also applied for the direct C–H-bond arylation of
lactone 13. Table 1 shows that electron rich (19b), electron defi-
cient (19c) and sterically demanding (19d) aryl moieties could be
Compd
Aryl
Catalyst
Yielda (%)
Yieldb (%)
20a
21a
21b
C6H5
C6H5
p-MeOC6H4
PdCl2/bipy
PdCl2/P[OCH(CF3)2]3
PdCl2/P[OCH(CF3)2]3
47
31
54
33
10c
8c
a
b
c
Yields after gel permeation chromatography.
Yields after preparative thin layer chromatography.
Yields after flash chromatography.
successfully introduced into the
yields were somewhat reduced.
a-position of 13 although the
some unique catalysts have been reported to induce a selective ary-
lation in b-position of the thiophene ring.35–37 However, most of
these catalysts were used exclusively for the arylation of simple
The same reaction conditions were applied for the direct C–H-
bond arylation of the regioisomeric lactone 14 with a thieno[2,3-
c]pyran scaffold. Although the arylation of 14 with iodobenzene
yielded the phenylated lactone 20, a second purification step (pre-
parative thin layer chromatography) had to be performed to achieve
the required purity for pharmacological tests. This additional purifi-
cation step slightly reduced the final yield to 33% (Table 2).
thiophenes without additional functional groups. Herein the cata-
36,37
lytic system PdCl2/P[OCH(CF3)2]3/Ag2CO3
was investigated for
the b-arylation of the key spirocyclic thieno[2,3-c]pyranone 14.
Thus the reaction of lactone 14 with iodobenzene in the pres-
ence of PdCl2/P[OCH(CF3)2]3/Ag2CO3 in boiling m-xylene occurred
regioselectively in 30-position to produce the 30-phenylated thieno-
pyranone 21a. In contrast to the reaction of the lactone 14 with the
donor substituted 1-iodo-4-methoxybenzene providing a moder-
ate yield of the arylation product 21b, the corresponding electron
deficient 4-iodobenzonitrile and the sterically demanding 1-iodo-
4-phenylbenzene did not yield the corresponding arylation prod-
ucts. A second purification procedure (flash chromatography)
was performed to obtain the b-arylated products 21a and 21b in
the requested purity for biological experiments. This purification
step reduced the yields of both arylation products 21 considerably.
Typically, the catalytic system PdCl2/2,20-bipyridyl/Ag2CO3 leads
to a regioselective a-arylation of the thiophene ring. Additionally,
Table 1
Yields after
a
-arylation of the spirocyclic lactone 13 with various iodoarenes
aryl
7'
1'
O
6'
1'
O
(a)
S
2'
S
O
O
After the successful a- and b-arylation of the spirocyclic thieno-
3'
3'
pyranones 13 and 14, the arylation of the corresponding thienof-
uranone 18 was envisaged. It should be investigated whether the
smaller, more constrained five-membered lactone is also stable un-
der the arylation conditions. Moreover, the influence of the car-
bonyl moiety directly attached to the thiophene ring (see Fig. 2)
was evaluated.
N
Bn
N
Bn
13
19a-d
(a) PdCl2/2,2’-bipyridyl, Ag2CO3, Aryl-I, m-xylene, 150 °C, 12 h.
At first the thienofuranone 18 was reacted with iodobenzene
and the catalytic system PdCl2/2,20-bipyridyl/Ag2CO3 in boiling
m-xylene as described for the six-membered lactones 13 and 14.
This transformation led to three arylation products, which were
Compd
Aryl
Yielda (%)
19a
19b
19c
19d
C6H5
57
20
18
25
p-MeOC6H4
p-NC-C6H4
p-C6H5-C6H4
separated by chromatography (Table 3). Unexpectedly, both
a-
positions of the thiophene moiety were attacked by the catalyst
to produce the 40- and 60-monophenylated products 22a and 22b
as well as the diphenylated product 22c.
a
Yields after gel permeation chromatography.

C. Meyer et al. / Bioorg. Med. Chem. 21 (2013) 1844–1856
1847
O
0.250
-0.361
0.486
-0.417
0.239
3. DFT calculations and mechanistic considerations
H
O
0.236
H
-0.404
0.464
O
O
S
S
In order to determine the structural and electronic properties of
compounds 13 and 18 and their arylated derivatives 19 and 22–26,
quantum chemical DFT-calculations at various levels of theory
were performed. The input files were generated using PCModel.38
Structures 13 and 18 were completely optimized at the B3LYP/6-
31G(d) and B3LYP/6-311+G(d,p)39,40 levels of theory employing
the GAUSSIAN09 series of programs.41 Then, SCS-MP2-single point
energies were evaluated.42 Since electronic interaction between
the adjacent aromatic moieties seemed to be important, addition-
ally B97-D/def2-TZVP43,44 optimizations were carried out. The ‘nat-
ural’ charges as calculated from a Natural Bond Orbital analysis45
(‘NBO-charges’) obtained for 13 and 18 at the B97-D level are given
in Figure 2.
-0.407
0.234
H
H
129.9°
N
N
138.5°
13
18
Figure 2. NBO-charges (B97-D/def2-TZVP) at C-, H-, and S-atoms of the thiophene
subunit and the thiophene-piperidine angle in thienopyranone 13 and thienofura-
none 18.
Table 3
Products and yields after a-arylation of spirocyclic lactone 18 with various iodoarenes
At first, the precursor molecules 13 and 18 were inspected with
respect to their NBO-charges. For both exo- and endo-carbon atoms
of thienopyranone 13 (positions 10 and 30) NBO-calculations pro-
vided very similar negative charges, both equally suitable for an at-
tack of an electrophilic arylation reagent like the PdCl2/bipy/
Ag2CO3/Aryl-I system employed in the experiments. In contrast
in thienofuranone 18, due to the adjacent carbonyl moiety, the
NBO-charge at the exo-carbon atom 40 (ꢀ0.36 e) is substantially
smaller compared to that at the endo-position 60 (ꢀ0.42 e) indicat-
ing significantly reduced nucleophilicity at 40-center.
aryl
aryl
4'
4
S
'
O
O
O
4'
S
6'
O
S
S
O 2' (a)
1'
6'
O
O
O
6'
+
+
aryl
aryl
N
Bn
N
Bn
N
Bn
N
Bn
18
22b-26b
22a-26a
22c-26c
(a) PdCl2/2,2’-bipyridyl, Ag2CO3, Aryl-I, m-xylene, 150 °C, 12h.
The calculated structural properties for 13 indicate that the
accessibility at the endo-carbon atom 30 is limited as seen from
the angle between the thiophene and the spirocyclic connected
piperidine ring (129.9°). For 18, the corresponding angle amounts
to 138.5°, thus allowing a less hindered approach of spacious re-
agents in this position (Figure 2).
Compd
Aryl
Position
Yielda (%)
50 (m. r.)
Yield (%)
22a
22b
22c
23a
23b
23c
24a
24b
24c
25a
25b
25c
26a
26b
26c
C6H5
C6H5
C6H5
C-40
C-60
25b
5b
C-40/C-60
C-40
27
25 (m. r.)
21c
16b
8b
In order to evaluate the energetically preferred site for arylation
at the thiophene moieties of 13 and 18 the four possible aryl-
substituted species 19a, 22a, 22b and the experimentally not ob-
served 30-phenyl-substituted lactone 19x were calculated at the
same levels of theory; additionally B2PLYP-D/def2-TZVP-single
point energies46–48 were evaluated (Table 4). As Table 4 indicates,
the exo-aryl-coupling products are generally energetically favored,
although the preference is quite dependent on the computational
method used. This is well in line with the reduced sterical strain
in the exo-position. As the dispersion corrected methods B97-D
and B2PLYP-D are expected to give the more reliable results, a
slightly higher thermodynamic preference for the exo-position is
anticipated from these calculations for the five-membered ring
systems 22–26 compared to the six-membered ring systems
19a–d.
p-MeOC6H4
p-MeOC6H4
p-MeOC6H4
p-NCC6H4
p-NCC6H4
p-NCC6H4
1-Naphthyl
1-Naphthyl
1-Naphthyl
p-C6H5-C6H4
p-C6H5-C6H4
p-C6H5-C6H4
C-60
C-40/C-60
C-40
14
35 (m. r.)
9c
23b
6b
C-60
C-40/C-60
C-40
25
61 (m. r.)
17c
31b
4b
C-60
C-40/C-60
C-40
38
40 (m. r.)
11c
16b
9b
C-60
C-40/C-60
28
20c
(m. r.) Mixture of regioisomers.
a
Yields after gel permeation chromatography.
Yields after preparative thin layer chromatography.
Yields after flash chromatography.
b
c
Thus, the isolation of compounds 19a–d, which result exclu-
sively from attack at the exo-(10)-position of thienopyranone 13,
is well in line with the calculated energetic preference and the re-
stricted accessibility of position 30. However, the formation of the
experimentally obtained mixtures 22a–c, 23a–c, 24a–c, 25a–c,
and 26a–c from thienofuranone 18 indicates that now arylation
at 60-position becomes kinetically competitive due to less steric
hindrance and to the more favorable electronic situation at 60-po-
sition, overcoming the thermodynamic disadvantages.
Very recently, an experimental and theoretical study of the Pd-
catalyzed direct C–H-bond arylation of 2-substituted thiophenes
was published indicating kinetic reasons for the observed C-4/C-
5-selectivity in this carbopalladation pathway.49
The scope of this transformation was investigated by employing
electron rich (1-iodo-4-methoxybenzene), electron deficient
(4-iodobenzonitrile) and sterically demanding (1-iodo-4-phenyl-
benzene, 1-iodonaphthalene) aryl iodides. Table 3 shows that the
arylation conditions were well tolerated by the five-membered
lactone 18. The arylation of 18 with iodobenzene derivatives
always led to both monoarylated 22a,b–26a,b and the diarylated
products 22c–26c. Even sterically demanding aryl iodides like 1-
iodo-4-phenylbenzene and 1-iodonaphthalene led to the 60-arylat-
ed products.
Generally the yields of the arylated products were rather high.
However, due to the formation of product mixtures, careful separa-
tions had to be performed to obtain pure products for the biologi-
cal assays. Generally all compounds were purified by gel
permeation chromatography (gpc) to separate the diarylated com-
pounds 22c–26c. The remaining mixtures of regioisomeric mono-
arylated products were purified either by preparative thin layer
chromatography or flash chromatography.
4. Receptor affinity
The
lactones were recorded in competition experiments with radioli-
gands. In the 1 assay membrane preparations of guinea pig brains
r1 and r2 receptor affinities of the five- and six-membered
r

1848
C. Meyer et al. / Bioorg. Med. Chem. 21 (2013) 1844–1856
Table 4
Relative energies [kcal/mol] for compounds 19a/19x (compound 13 with additional phenyl moiety in 30-position) and 22a/22b obtained by quantum chemical calculations at
various levels of theory (stated in the table)
Compd
B3LYP/6-
31G(d)(ZPE)
B3LYP/6-
311 + G(d,p)(ZPE)
SCS-MP2/6-311 + G(d,p)//B3LYP/6-
311 + G(d,p)(ZPE)
B97-D/def-TZVP
(ZPE)
B2PLYP-D/def2-TZVP//B97-D/
def2-TZVP
19a Pyranone, 10-phenyl 0.00
0.00
5.85
0.00
5.61
0.00
1.70
0.00
0.47
0.00
2.92
0.00
3.74
0.00
3.46
0.00
4.32
(exo)
19x Pyranone, 30-phenyl 6.58
(endo)
22a Furanone 40-phenyl
(exo)
22b Furanone 60-phenyl
(endo)
0.00
5.93
containing
r
1 receptors were incubated with the radioligand [3H]-
pounds 1–4 and important reference compounds (haloperidol, di-
o-tolylguanidine, (+)-pentazocine, progesterone) are given for
(+)-pentazocine and increasing concentrations of the test com-
pound. A large excess of non-tritiated (+)-pentazocine was used
to determine the non-specific binding of the radioligand.50–53 Since
comparison. The
r1 affinities of the thienopyranone 13 and the thi-
enofuranone 18 are in the same range as the r1 affinities of the
benzofuranone 2 and benzopyranone 4. However the regioisomeric
thienopyranone 14 is about 10-fold less active than the other lac-
tones. Obviously the position of the S-atom in the thiophene sys-
tem has a strong impact on the r1 receptor affinity.
r
1 receptors of different species (human, guinea pig, rat) are more
than 92% identical and more than 95% similar at the level of amino
acid sequence,54 the resulting Ki-values can be considered as repre-
sentative for many species. The source for r2 receptors in the r2
assay were rat liver homogenates and [3H]-di-o-tolylguanidine
was employed as radioligand. Due to the interaction of di-o-tolyl-
guanidine with both r1 and r2 receptors a large amount of non-
radiolabeled (+)-pentazocine was added to selectively mask r1
receptors. A large excess of non-tritiated di-o-tolylguanidine was
used to determine the non-specific binding.50–53
Introduction of a phenyl moiety in 10-position of the lactone 13 led
to the very potent r1 ligand 19a with a Ki-value of 2.5 nM. Ligands
with a p-methoxy (19b) and p-cyano (19c) moiety at the new phenyl
group show
the phenylated lactone 19a. The substituted aryl moieties in 10-posi-
tion of 19b and19cstillfavortheinteraction with the 1 receptorpro-
r1 affinities between the unsubstituted lactone 13 and
r
In Table 5 the
r
1 and
r
2 receptor affinities of the spirocyclic lac-
tein although to a lower extend than the non-substituted phenyl
tones are summarized. Furthermore, the affinities of the lead com-
moiety of 19a. The size of the biphenylyl substituent in 10-position
of 19d (Ki = 108 nM) leads to a decreased
r1 receptor affinity.
A phenyl moiety in 20-position of the regioisomeric thienopyra-
none 14 led to a 10-fold increased r1 affinity (20a: Ki = 23 nM)
compared to the r1 affinity of the parent compound 14
(Ki = 255 nM). However, introduction of the phenyl moiety in b-po-
sition of the thiophene ring produced the even more potent lactone
21a (Ki = 5.3 nM). The regioisomeric lactones 19a and 21a bearing
the phenyl moiety in comparable positions show very similar r1
receptor affinities. Obviously a phenyl moiety in the ‘upper left’ po-
sition (10-position in 19a, 30-position in 21a) favors the interaction
Table 5
r
1 and r2 Receptor affinities of the synthesized spirocyclic thiophenes and reference
compounds
Compd
Aryl
Ki SEM (nM) (n = 3)
Selectivity
r1
r2
r1/r2
1a
—
—
—
—
H
H
H
C6H5
p-MeOC6H4
p-CNC6H4
p-Biphenyl
C6H5
C6H5
p-MeOC6H4
C6H5
1.1 0.22
21 3.5
1.3 0.18
29 0.9.0
40 13
255
16 6.8
2.5 0.91
11 3.0
11 3.4
108 46
23 9.9
5.3 0.88
43 13
11 3.2
483
1280 137
1460 108
3500 352
1157 148
732
>1000
>1000
720
>1000
70
>1000
40
18
>4
>60
288
79
>91
>9
>43
57
>23
21
2a
3a
4a
of ligands with the
r1 receptor binding site. As shown for the series
13
14
18
19 a methoxy group within the additional phenyl moiety reduces
the r1 affinity (compare 19b and 21b).
19a
19b
19c
19d
20a
21a
21b
22a
22b
22c
23a
23b
23c
24a
24b
24c
25a
25b
25c
26a
26b
26c
Haloperidol
The five-membered lactone 18 has the highest r1 affinity
(Ki = 16 nM) of the non-arylated lactones. Nevertheless the r1
receptor affinity was further increased by introduction of an aryl
moiety in the ‘upper left (top)’ position (40-position). The phenyl
(22a, Ki = 11 nM), the donor substituted p-methoxyphenyl (23a,
Ki = 9.0 nM), the acceptor substituted p-cyanophenyl (24a,
Ki = 6.3 nM)) and the sterically demanding 1-naphthyl (25a,
Ki = 5.3 nM) substituted derivatives show very similar r1 receptor
affinities in the low nanomolar range. As already observed within
the other series, the p-biphenylyl substituted derivative 26a
872
>1000
>1000
>1000
300
>1000
236
>1000
935
584
C6H5
C6H5
>2
11
65
87 52
9.0 3.2
ꢀ/ꢀ
p-MeOC6H4
p-MeOC6H4
p-MeOC6H4
p-CNC6H4
p-CNC6H4
p-CNC6H4
1-Naphthyl
1-Naphthyl
1-Naphthyl
p-Biphenyl
p-Biphenyl
p-Biphenyl
ꢀ/ꢀ
ꢀ/ꢀ
158
>1000
295
6
(Ki = 777 nM) exhibits a very low
r1 affinity indicating that the bi-
6.3 2.1
190
47
2
—
103
>2
>1
>1
>2
—
phenylyl substituent is too large to fit into the binding pocket of
305
the r1 receptor protein.
>1000
5.3 2.3
639
784
777
402
>1000
6.3 1.6
89 29
5.7 2.2
661 115
>1000
546
Introduction of the phenyl moiety in the second a
-position (60-
>1000
>1000
>1000
>1000
>1000
78 2.3
58 18
—
position) of the thiophene ring led to 22b (Ki = 483 nM) with al-
most negligible r1 receptor affinity. A similar reduction of r1
receptor affinity was also observed for the spirocyclic lactones
23b–26b with substituted or sterically demanding aryl moieties
in 60-position. It is assumed that the phenyl moiety in 60-position
shields the basic N-atom and thus inhibits the formation of an ionic
interaction with the r1 receptor protein, which is crucial for high
receptor affinity.
12
0.7
—
Di-o-tolylguanidine
(+)-Pentazocine
Progesterone
—
a
Ki Values are reported in Ref. 13.

C. Meyer et al. / Bioorg. Med. Chem. 21 (2013) 1844–1856
1849
The lactones 24c–26c with two aryl moieties in both
a
-posi-
from 90%,to 0%, 29–31 min: 0%, 31–31.5 min: 0% to 90%, 31.5–
40 min: 90%. HPLC method B: Agilent TechnologiesÒ; UV and mass
detection: Agilent 1200 Series Variable Wavelength Detector
G13143/G1314C (SL) and Agilent G1978AQ/B Multimode source
(mass detector); autosampler: Agilent 1200 Series High Perfor-
mance autosampler and Micro Well Plate autosampler; quaternary
pump for gradient analysis, degasser: Agilent 1200 Series Micro
Vacuum Degasser; column: ZOBRAX Eclipse Plus C18 150–2 mm,
tions (40- and 60-position) are even less potent than the 60-arylated
compounds 24b–26b. The slightly increased r1 affinity of 22c
leads to the assumption that the additional phenyl group in 40-po-
sition is able to partially compensate the detrimental effects of the
60-phenyl moiety.
The r2 receptor affinities of the arylated lactones are consider-
ably lower than their r1 receptor affinities, that is all compounds
show high selectivity for the
ticular the very potent 1 ligands 19a, 19b, 19c, 21a, 23a, 24a, and
1 receptor selectivities (r1 2 >47).
r
1 receptor over the
r
2 subtype. In par-
3.5 lm; flow rate: 0.40 mL/min; injection volume: 1 lL; detection:
r
r
k = 254 nm, cut off time: 13 min; solvents: A: H2O dest. + 5 mM
NH4HCO2; B: acetonitrile; gradient elution: (A%): 0 min: 80%, 0–
5 min: gradient form 80% to 0%, 5–13 min: 0%, 13–18 min: gradient
from 0% to 80%. The purity of all test compounds was greater than
95%, which was determined by HPLC method A or B.
25a display excellent
/r
5. Conclusion
The six-membered lactones 13 and 14 were selectively arylated
a
- and b-position using the catalytic systems PdCl2/2,20-bipyri-
dyl/Ag2CO3 and PdCl2/P[OCH(CF3)2]3/Ag2CO3, respectively. Unex-
6.1.2. General procedure A for the
thiophenes with iodoarenes
a-arylation of spirocyclic
in
A 20 mL glass vessel was equipped with a magnetic stirring bar
and closed by a J. YoungÒ O-ring tap. The flask was flame-dried un-
der vacuo and filled with Ar after cooling to rt. Under a permanent
stream of Ar the catalyst PdCl2/bipy (10 mol %) and Ag2CO3
(1 equiv) were filled into the vessel and suspended in dry m-xylene
(0.4 mL). This mixture was stirred at 60 °C for 30 min. Finally, a
solution of iodoarene (1.1 equiv) and a solution of the spirocyclic
lactone (1 equiv) in dry m-xylene (0.6 mL in total) were added
dropwise. The vessel was sealed with the O-ring tap and heated
up at 150 °C for 12 h in an 8-well reaction block. After cooling
the vessel to rtk the mixture was filtered through a short silica
pad using EtOAc as the eluent. The filtrate was concentrated in va-
cuo and the crude product was purified by gel permeation chroma-
tography (CHCl3) followed by preparative thin layer
chromatography or fc (short column) to yield the corresponding
arylthiophene in high purity.
pectedly, the direct C–H-bond arylation of the five-membered
lactone 18 afforded both
a-arylated products, thereby giving ac-
cess to the 60-arylated thiophenes 22b,c–26b,c for the first time.
A considerably increased r1 receptor affinity was found for com-
pounds bearing the aryl moiety in the ‘upper left’ position of the
thiophene system (e.g., compounds 19a, 21a, 24a, 25a). Whereas
a phenyl moiety in 20-position (‘left’ position, compound 20a) leads
to moderate r1 affinity, the r1 receptor does not tolerate an aryl
moiety at 60-position (e.g., compounds 22b, 25b). The double ary-
lated compounds 24c–26c show even lower r1 affinities than the
60-arylated compounds 24b–26b.
6. Experimental
6.1. Chemistry
6.1.1. General
6.1.3. General procedure B for the b-arylation of spirocyclic
thiophenes with iodoarenes
Unless otherwise noted, moisture and oxygen sensitive reac-
tions were conducted in dry glassware (Schlenk flask sealed with
a rubber septum) under N2 (dried using phosphorous pentoxide
(GranusicÒ A, Baker)). THF and Et2O were dried using sodium/ben-
zophenone and were freshly distilled before use. Thin layer chro-
matography (tlc): Silica gel 60 F254 plates (Merck). Flash
A 20 mL glass vessel was equipped with a magnetic stirring bar
and closed by a J. YoungÒ O-ring tap. The flask was flame-dried un-
der vacuo and filled with Ar after cooling to rt. Under a permanent
stream of Ar PdCl2 (10 mol %) and Ag2CO3 (1 equiv) were filled into
the vessel. The phosphite ligand P[OCH(CF3)2]3 (20 mol %) and sub-
sequently dry m-xylene (0.5 mL) were added and this mixture was
stirred at 60 °C for 30 min to form the active catalyst. Finally, a
solution of the iodoarene (1.1 equiv) and a solution of the spirocy-
clic lactone (1 equiv) in dry m-xylene (0.5 mL in total) were added.
The vessel was sealed with the O-ring tap and heated up at 150 °C
for 12 h in an 8-well reaction block. After cooling the vessel to rt,
the mixture was filtered through a short silica pad (EtOAc). The fil-
trate was concentrated in vacuo and the crude product was puri-
fied by gel permeation chromatography (CHCl3) followed by
preparative thin layer chromatography or fc (short column) to
yield the corresponding arylthiophene in high purity.
chromatography (fc): Silica gel 60, 40–64 lm (Merck); parentheses
include: diameter of the column [cm], length of the stationary
phase [cm], eluent, fraction size [mL] and retention factor Rf. Gel
permeation chromatography (gpc): LC-9204 instrument (JAI) with
JAIGEL-1H/JAIGEL-2H columns, eluent CHCl3. Preparative thin
layer chromatography (prep. tlc): Wako-gelÒ B5-F silica coated
plates (0.75 mm). IR: IR spectrophotometer 480Plus FT-ATR-IR
(Jasco). 1H NMR (400, 300, 600 MHz) and 13C NMR (100 MHz) spec-
tra were recorded on a Unity Mercury Plus 400 (400 MHz) NMR
spectrometer (Varian), JNM-ECA-400 (400 MHz) spectrometer
(JEOL), Brucker AV 300 (300 MHz), and Varian Unity Plus 600
(600 MHz) operating at 23 °C; chemical shifts d are reported in
parts per million (ppm) against the reference compound tetra-
methylsilane and calculated using the chemical shift of the signal
of the residual non-deuterated solvent. HRMS (ESI): Finnigan
MAT 4200s, Brucker Daltonics Micro Tof and Waters Micromass
Quatro LCZ, peaks are given in m/z (% of basis peak). EI, electron im-
pact, MAT GCQ (Thermo-Finnigan); HRMS: JMS-T100TD instru-
ment (DART). HPLC method A: Merck Hitachi Equipment; UV
detector: L-7400; autosampler:L-7200; pump: L-7100; degasser:
6.1.4. 1-Benzyl-4-[4-(2,2-dimethoxyethyl)thiophen-3-
yl]piperidin-4-ol (9) and 1-benzyl-4-[3-(2,2-dimeth-
oxyethyl)thiophen-2-yl]piperidin-4-ol (10)15
Under N2 and at ꢀ78 °C n-butyllithium in n-hexane (1.26 M,
2.74 mL, 3.44 mmol) was added slowly to a stirred solution of
the bromothiophene 8 (0.67 g, 2.65 mmol) in dry THF (15 mL).
The mixture was stirred for 15 min. Then 1-benzylpiperidin-4-
one (0.57 mL, 3.2 mmol) was added slowly into the solution. After
3 h stirring at ꢀ78 °C, the flask was warmed up to rt and water was
added. The aqueous layer was separated and extracted twice with
CH2Cl2. The combined organic layers were dried using K2CO3, fil-
tered and the solvent was removed in vacuo. The regioisomeric
L-7614; column: LiChrospherÒ 60 RP-select B (5
250–4 mm cartridge; flow rate: 1.000 mL/min; injection volume:
5.0 L; detection at k = 210 nm; solvents: A: water with 0.05% (v/
l
m); LiChroCARTÒ
l
v) trifluoroacetic acid; B: acetonitrile with 0.05% (v/v) trifluoroace-
tic acid: gradient elution: (A%): 0–4 min: 90% 4–29 min: gradient

1850
C. Meyer et al. / Bioorg. Med. Chem. 21 (2013) 1844–1856
alcohols 9 and 10 were separated by fc (5 cm, h = 15 cm, cyclohex-
ane/EtOAc = 4:1, 20 mL).
(C–H). 1H NMR (CDCl3): d (ppm) = 1.89–2.15 (m, 4H, N(CH2CH2)2),
2.42–2.63 (m, 2H, N(CH2CH2)2), 2.68 (dd, 15.5/7.4 Hz, 1H, thi-
ophCH2CH), 2.72–2.82 (m, 2H, N(CH2CH2)2), 2.96 (dd, J = 15.5/
3.2 Hz, 1H, thiophCH2CH), 3.59 (sbroad, 2H, NCH2Ph), 5.33 (dd,
J = 7.4/3.2 Hz, 1H, thiophCH2CH), 6.75 (d, J = 5.0 Hz, 1H, 30-H-thi-
oph), 7.17 (d, J = 5 Hz, 1H, 20-H-Thioph), 7.27–7.40 (m, 5H, Ph-H). A
signal for the OH-proton is not visible. 13C NMR (CDCl3): d
(ppm) = 34.0 (1C, thiophCH2CH), 37.7 (1C, N(CH2CH2)2), 40.9 (1C,
N(CH2CH2)2), 49.2 (1C, N(CH2CH2)2), 49.3 (1C, N(CH2CH2)2), 63.3
(1C, NCH2Ph), 74.6 (1C, thiophCspiro), 90.4 (1C, thiophCH2CH),
123.8 (1C, C-20-thioph), 126.8 (1C, C-30-thioph), 127.3 (1C, Ph-CH),
128.4 (2C, Ph-CH), 129.5 (2C, Ph-CH), 131.0 (1C, Cquart), 141.4 (1C,
Cquart). One signal for a quaternary carbon atom is not visible.
Compound 10 (Rf = 0.35, cyclohexane/EtOAc = 3:7): Pale yellow
oil, yield 0.11 g (12%). C20H27NO3S (361.5 g/mol). MS (EI): m/
z = 361 [M+], 328 [M+ –OCH3,–2H], 298 [M+ –(OCH3)2, –H], 91
[+CH2Ph]. IR (neat): v (cmꢀ1) = 3430 (O–H), 3026 (C–Haryl), 2934,
2812 (C–H), 1118 (C–O), 697 (C–H). 1H NMR (CDCl3):
d
(ppm) = 1.90–1.97 (m, 2H, N(CH2CH2)2), 2.07–2.16 (m, 2H,
N(CH2CH2)2), 2.49–2.56 (m, 2H, N(CH2CH2)2), 2.71–2.80 (m, 2H,
N(CH2CH2)2), 3.22 (d, J = 5.5 Hz, 2H, thiophCH2CH), 3.35 (s, 6H,
CH(OCH3)2), 3.57 (s, 2H, NCH2Ph), 3.62 (s, 1H, OH), 4.48 (t,
J = 5.5 Hz, 1H, thiophCH2CH), 6.81 (d, J = 5.1 Hz, 1H, 4-H-thioph),
7.09 (d, J = 5.1 Hz, 1H, 5-H-thioph), 7.29–7.37 (m, 5H, Ph-H).
Compound 9 (Rf = 0.22, cyclohexane/EtOAc = 3:7): Pale yellow
oil, yield 0.50 g (53%). C20H27NO3S (361.5 g/mol). MS (EI): m/
z = 361 [M+], 330 [M+ –OCH3], 298 [M+ –(OCH3)2, –H], 91 [+CH2Ph].
IR (neat): v (cmꢀ1) = 3442 (O–H), 3027 (C–Haryl), 2926, 2814 (C–H),
1119 (C–O), 698 (C–H). 1H NMR (CDCl3): d (ppm) = 1.88–1.95 (m,
2H, N(CH2CH2)2), 2.06 (td, J = 12.9/4.3 Hz, 2H, N(CH2CH2)2), 2.51
(td, J = 11.9/2.5 Hz, 2H, N(CH2CH2)2), 2.70–2.78 (m, 2H,
N(CH2CH2)2), 3.24 (d, J = 5.6 Hz, 2H, thiophCH2CH), 3.35 (s, 6H,
CH(OCH3)2), 3.51 (s, 1H, OH), 3.57 (s, 2H, NCH2Ph), 4.52 (t,
J = 5.6 Hz, 1H, thiophCH2CH), 7.06 (d, J = 3.2 Hz, 1H, 5-H-thioph),
7.08 (d, J = 3.2 Hz, 1H, 2-H-thioph), 7.26–7.37 (m, 5H, Ph-H).
6.1.7. 1-Benzylspiro[piperidine-4,40-thieno[3,4-c]pyran]-60(70H)-
one (13)
Under N2 PCC (274 mg, 1.27 mmol) was suspended in dry
CH2Cl2 (5 mL) and stirred for 2 min. Then lactol 11 (309 mg,
0.98 mmol) was suspended in CH2Cl2 (10 mL) and added to the
PCC suspension rapidly. This mixture was stirred at rt for 16 h.
Then saturated NaHCO3 solution (30 mL) was added and the aque-
ous layer was extracted twice with Et2O. The combined organic
layers were filtered, dried using K2CO3 and filtered again. The sol-
vent was removed in vacuo and the residue was purified by fc
(3 cm, h = 15 cm, cyclohexane/EtOAc = 7.5:2.5, NEt3 2%, 20 mL,
Rf = 0.24 cm). Pale yellow oil, yield 216 mg (71%). C18H19NO2S
(313.4 g/mol). Purity (HPLC, method A): 99.3%, tR = 14.4 min MS
(ESI): m/z = 314 [MH+]. IR (neat): v (cmꢀ1) = 3100 (C–Haryl), 2924,
6.1.5. 1-Benzyl-60,70-dihydrospiro[piperidine-4,40-thieno[3,4-
c]pyran]-60-ol (11)
The hydroxyacetal 9 (1.06 g, 2.94 mmol) was dissolved in THF
(5 mL) and HCl (1 M, 30 mL, 30 mmol) was added. The mixture
was stirred at rt for 16 h. Subsequently 2 M NaOH was added drop-
wise until pH 8, the aqueous layer was separated and extracted
twice with CH2Cl2. The combined organic layers were dried using
K2CO3, filtered and the solvent was removed in vacuo. The remain-
ing paste was purified by fc (6 cm, h = 15 cm, cyclohexane/
EtOAc = 3:2, 20 mL, Rf = 0.18). Colorless solid, mp 146 °C, yield
703.1 mg (76%). C18H21NO2S (315.4 g/mol). Purity (HPLC, method
A): 97.3%, tR = 14.0 min MS (EI): m/z = 315 [M+], 224 [M+ –CH2Ph],
91 [+CH2Ph]. IR (neat): v (cmꢀ1) = 3079 (O–H), 2937 (C–H), 2836
(C–H), 1051 (C–O), 698 (C–H). 1H NMR (CDCl3): d (ppm) = 1.91–
2.20 (m, 4H, N(CH2CH2)2), 2.44–2.61 (m, 2H, N(CH2CH2)2), 2.68–
2.85 (m, 3H, thiophCH2CH, N(CH2CH2)2), 3.05 (dd, J = 15.3/3.0 Hz,
1H, thiophCH2CH), 3.59 (sbroad, 2H, NCH2Ph), 5.30 (dd, J = 7.4/
3.0 Hz, 1H, thiophCH2CH), 6.94 (d, J = 3.0 Hz, 30-H-thioph), 6.99
(d, J = 2.9 Hz, 1H, 10-H-Thioph), 7.28–7.41 (m, 5H, Ph-H). A signal
for the OH-proton is not visible. 13C NMR (CDCl3): d (ppm) = 34.3
(1C, thiophCH2CH), 37.3 (1C, N(CH2CH2)2), 39.9 (1C, N(CH2CH2)2),
49.3 (1C, N(CH2CH2)2), 49.5 (1C, N(CH2CH2)2), 63.4 (1C, NCH2Ph),
74.6 (1C, thiophCspiro), 90.5 (1C, thiophCH2CH), 118.7 (1C, C-10-thi-
oph), 120.3 (1C, C-30-thioph), 127.4 (Ph-CH), 128.5 (Ph-CH), 129.6
(Ph-CH), 133.2 (1C, Cquart), 142.9 (1C, Cquart). One signal for a qua-
ternary carbon atom is not visible.
2816 (C–H), 1735 (C@O), 698 (C–H). 1H NMR (CDCl3):
d
(ppm) = 2.03 (dd, J = 14.7/2.3 Hz, 2H, N(CH2CH2)2), 2.15 (td,
J = 12.4/4.4 Hz, 2H, N(CH2CH2)2), 2.59 (td, J = 12.0/2.4 Hz, 2H,
N(CH2CH2)2), 2.80 (d, J = 11.6 Hz, 2H, N(CH2CH2)2), 3.59 (s, 2H,
NCH2Ph), 3.76 (s, 2H, thiophCH2), 7.04 (d, J = 2.8 Hz, 1H, 30-H-thi-
oph), 7.12 (d, J = 2.9 Hz, 1H, 10-H-thioph), 7.27–7.37 (m, 5H, Ph-
H). 13C NMR (CDCl3): d (ppm) = 32.8 (1C, thiophCH2C@O), 37.2
(2C, N(CH2CH2)2), 48.7 (2C, N(CH2CH2)2), 63.1 (1C, NCH2Ph), 81.4
(1C, thiophCspiro), 119.2 (1C, C-10-thioph), 120.8 (1C, C-30-thioph),
127.5 (1C, Ph-C), 128.5 (2C, Ph-C), 129.5 (2C, Ph-C), 130.7 (1C,
Cquart), 146.0 (1C, Cquart), 169.3 (1C, C@O). One signal for a quater-
nary carbon atom is not visible.
6.1.8. 1-Benzylspiro[piperidine-4,70-thieno[2,3-c]pyran]-50(40H)-
one (14)
Under N2 PCC (152 mg, 0.7 mmol) was suspended in dry CH2Cl2
(5 mL) and stirred for 2 min. Then lactol 12 (170 mg, 0.54 mmol)
was suspended in CH2Cl2 (5 mL) and added to the PCC suspension
rapidly. This mixture was stirred at rt for 16 h. Then saturated
NaHCO3 solution (20 mL) was added and the aqueous layer was ex-
tracted twice with Et2O. The combined organic layers were filtered,
dried using K2CO3 and filtered again. The solvent was removed in
vacuo and the residue was purified by fc (3 cm, h = 15 cm, cyclo-
hexane/EtOAc = 4:1, NEt3 2%, 15 mL, Rf = 0.27 cm). Pale yellow oil,
yield 120 mg (71%). C18H19NO2S (313.4 g/mol). Purity (HPLC, meth-
od A): 98.2%, tR = 14.4 min. Exact mass (APCI): m/z = calcd for
6.1.6. 1-Benzyl-40,50-dihydrospiro[piperidine-4,70-thieno[2,3-
c]pyran]-50-ol (12)
The alcohol 10 (1.99 g, 5.5 mmol) was dissolved in THF (10 mL)
and HCl (1 M, 55 mL, 55 mmol) was added. The mixture was stirred
at rt for 16 h. Subsequently, 2 M NaOH was added dropwise until pH
8, the aqueous layer was separated and extracted twice with CH2Cl2.
The combined organic layers were dried using K2CO3, filtered and
the solvent was removed in vacuo. The remaining solid was purified
by flash chromatography (Ø = 5.5 cm, h = 15 cm, cyclohexane/
EtOAc = 4:1 (NEt3 2%), 30 mL, Rf = 0.26) followed by recrystalliza-
tion. Colorless solid, mp 168 °C, yield 515 mg (30%). C18H21NO2S
(315.4 g/mol). Purity (HPLC, method A): 99.2%, tR = 14.3 min MS
(EI): m/z = 315 [M+], 224 [M+ –CH2Ph], 91 [+CH2Ph]. IR (neat): v
(cmꢀ1) = 3104 (O–H), 2936 (C–H), 2840 (C–H), 1055 (C–O), 695
C v
18H19NO2S [MH+] 314.1209, found 314.1217. IR (neat):
(cmꢀ1) = 3099 (C–Haryl), 2918, 2815 (C–H), 1737 (C@O), 697 (C–
H). 1H NMR (CDCl3): d (ppm) = 2.05 (dd, J = 14.6/2.3 Hz, 2H,
N(CH2CH2)2), 2.14 (td, J = 12.0/4.4 Hz, 2H, N(CH2CH2)2), 2.61 (td,
J = 11.8/2.6 Hz, 2H, N(CH2CH2)2), 2.79 (d, J = 11.6 Hz, 2H,
N(CH2CH2)2), 3.59 (s, 2H, NCH2Ph), 3.70 (s, 2H, thiophCH2), 6.81
(d, J = 5.0 Hz, 1H, 30-H-thioph), 7.27–7.38 (m, 6H, 20-H-thioph, Ph-
H). 13C NMR (CDCl3): d (ppm) = 31.9 (1C, thiophCH2C@O), 39.2
(2C, N(CH2CH2)2), 48.7 (2C, N(CH2CH2)2), 63.2 (1C, NCH2Ph), 81.8
(1C, thiophCspiro), 125.3 (1C, CH-20-thioph), 125.9 (1C, C-30-thioph),
127.3 (Ph-CH), 128.5 (Ph-CH), 129.3 (Ph-CH), 129.7 (1C, Cquart),
138.2 (1C, Cquart), 138.4 (1C, Cquart), 169.1 (1C, C@O).

C. Meyer et al. / Bioorg. Med. Chem. 21 (2013) 1844–1856
1851
6.1.9. 1-Benzylspiro[piperidine-4,10-thieno[3,4-c]furan]-30-one
6.1.10. 1-Benzyl-10-phenylspiro[piperidine-4,40-thieno[3,4-
(18) and 1-benzyl-30-bromospiro[piperidine-4,40-thieno[2,3-c]
furan]-60-one
c]pyran]-60(70H)-one (19a)
According to General Procedure A the spirocyclic thiophene 13
(32.2 mg, 0.103 mmol) was reacted with iodobenzene (12.6 L,
In a three-necked flask 3,4-dibromothiophene (15, 654 mg,
2.7 mmol) was dissolved in freshly distilled Et2O (8 mL) and cooled
down under N2 to ꢀ78 °C. After 15 min stirring at ꢀ78 °C, n-butyl-
lithium in n-hexane (1.35 M, 2 mL, 2.7 mmol) was added and the
mixture was stirred for 10 min. 1-Benzylpiperidin-4-one (511 mg,
2.7 mmol), dissolved in Et2O (3 mL), was added dropwise into the
solution and the solution was stirred for 30 min (?16). Next the
reaction mixture was diluted with Et2O (15 mL). For the second
halogen-metal-exchange-reaction a second equivalent of n-butyl-
lithium in n-hexane (1.35 M, 2 mL, 2.7 mmol) was added slowly
and the mixture was stirred for 10 min at ꢀ78 °C. Finally CO2 (gen-
erated from dry ice and dried by H2SO4) was bubbled through the
mixture for 1 h while the temperature was kept below ꢀ70 °C. For
the workup the flask was warmed up to rt and the solvent was re-
moved in vacuo almost completely. The remaining crude product
was poured into water (20 mL), basified with 2 M NaOH and ex-
tracted twice with Et2O. Next the aqueous layer was acidified with
2 M HCl and washed several times with Et2O. The aqueous layer
was concentrated in vacuo and the intermediate 17 was directly
used for cyclization without purification. The crude product was
dissolved in a mixture of Ac2O (3 mL) and NaOAc (532 mg,
6.5 mmol) in toluene (20 mL) and the mixture was heated to reflux
for 16 h. For the workup the reaction mixture was cooled down to
rt, poured into saturated K2CO3 solution and the mixture was stir-
red for 1 h. When the gas formation was finished, the toluene layer
was separated and the aqueous layer was extracted twice with
CH2Cl2. Finally the combined organic layers were dried using
K2CO3, filtered and the solvent was removed in vacuo. The remain-
ing solid was purified by fc (4.5 cm, h = 15 cm, cyclohexane/
EtOAc = 7:3 (NHEt2 2%), 30 mL). In addition to the desired lactone
18, traces of the side product 1-benzyl-30-bromospiro[piperidine-
l
0.11 mmol), Ag2CO3 (29.7 mg, 0.11 mmol) and PdCl2/bipy
(3.4 mg, 0.01 mmol) in m-xylene (1.2 mL). The crude product was
purified by CHCl3-gpc (yield 22.6 mg, 57%) and fc (1.5 cm,
h = 5 cm, hexane/EtOAc = 4:1, Rf = 0.20). Colorless solid, mp 124–
125 °C, yield 13.4 mg (34%). C24H23NO2S (389.5 g/mol). Purity
(HPLC, method B): 98.4%, tR = 7.09 min. Exact MS (APCI): m/
z = calcd for C24H24NO2S [MH+] 390.1522, found 390.1528. 1H
NMR (CDCl3): d (ppm) = 2.09 (dd, J = 14.8/2.2 Hz, 2H, N(CH2CH2)2),
2.19 (td, J = 13.7/4.6 Hz, 2H, N(CH2CH2)2), 2.61 (td, J = 11.9/2.6 Hz,
2H, N(CH2CH2)2), 2.82 (dd, J = 9.0/2.4 Hz, 2H, N(CH2CH2)2), 3.60
(s, 2H, NCH2Ph), 3.83 (s, 2H, thiophCH2), 7.10 (s, 1H, 30-H-thioph),
7.30–7.47 (m, 10H, Ph-H). 13C NMR (CDCl3): d (ppm) = 32.7 (1C,
thiophCH2C@O), 37.1 (2C, N(CH2CH2)2), 48.7 (2C, N(CH2CH2)2),
63.2 (1C, NCH2Ph), 81.2 (1C, thiophCspiro), 117.7 (1C, CH-30-thioph),
126.4 (1C, Cquart), 127.4 (Ph-CH), 128.4 (Ph-CH), 128.6 (Ph-CH),
128.8 (Ph-CH), 129.2 (Ph-CH), 129.5 (Ph-CH), 133.1 (1C, Cquart),
138.1 (1C, Cquart), 141.3 (1C, Cquart), 169.5 (1C, C@O). One signal
for a quaternary carbon atom is not visible.
6.1.11. 1-Benzyl-10-(4-methoxyphenyl)spiro-[piperidine-4,40-
thieno[3,4-c]pyran]-60(70H)-one (19b)
According to General Procedure A the spirocyclic thiophene 13
(33.2 mg, 0.106 mmol) was reacted with p-iodoanisole (30.9 mg,
0.13 mmol), Ag2CO3 (34.2 mg, 0.12 mmol) and PdCl2/bipy
(3.3 mg, 0.01 mmol) in m-xylene (1.2 mL). The crude product was
purified by CHCl3-gpc (yield 8.8 mg, 20%) and fc (1.5 cm,
h = 5 cm, hexane/EtOAc = 1:1, 3 mL, Rf = 0.48). Colorless solid, mp
159–160 °C, yield 4.6 mg (10%). C25H25NO3S (419.5 g/mol). Purity
(HPLC, method B): 97%, tR = 7.07 min. Exact MS (APCI): m/z = calcd
for C25H26NO3S [MH+] 420.1628, found 420.1665. 1H NMR (CDCl3):
d (ppm) = 2.08 (dd, J = 14.4/2.2 Hz, 2H, N(CH2CH2)2), 2.18 (td,
J = 12.5/4.2 Hz, 2H, N(CH2CH2)2), 2.60 (td, J = 11.9/2.5 Hz, 2H,
N(CH2CH2)2), 2.82 (d, J = 11.4 Hz, 2H, N(CH2CH2)2), 3.60 (s, 2H,
NCH2Ph), 3.79 (s, 2H, thiophCH2), 3.85 (s, 3H, OCH3), 6.94–6.97
(m, 2H, o-OCH3-Ph-H), 7.04 (s, 1H, 30-H-thioph), 7.28–7.38 (m,
7H, Ph-H). 13C NMR (CDCl3): d (ppm) = 32.7 (1C, thiophCH2CO),
37.1 (2C, N(CH2CH2)2), 48.7 (2C, N(CH2CH2)2), 55.5 (1C, OCH3),
63.2 (1C, NCH2Ph), 81.2 (1C, Cspiro), 114.6 (Ph-CH), 116.9 (1C, CH-
30-thioph), 125.5 (1C, Cquart), 125.6 (1C, Cquart), 127.4 (Ph-CH),
128.6 (Ph-CH), 129.5 (Ph-CH), 130.0 (Ph-CH), 138.6 (1C, Cquart),
139.2 (1C, Cquart), 141.2 (1C, Cquart), 159.9 (1C, Ph-Cquart-OCH3),
169.7 (1C, C@O).
4,40-thieno[2,3-c]furan]-60-one
(Rf = 0.60,
cyclohexane/
EtOAc = 3:7) were separated.
Compound 18 (Rf = 0.42, cyclohexane/EtOAc = 3:7), colorless so-
lid, mp 95 °C, yield 353 mg (44%, calculated from 15). C17H17NO2S
(299.1 g/mol). Purity (HPLC, method A): 98.7%, tR = 13.7 min. Exact
MS (APCI): m/z = calcd for C17H17NO2S [MH+] 300.1053, found
300.1081. IR (neat): v (cmꢀ1) = 3102 (C–Haryl), 2922 (C–H), 2811
(C–H), 1758 (C@O), 698 (C–H). 1H NMR (CDCl3): d (ppm) = 1.96
(d, J = 13.5 Hz, 2H, N(CH2CH2)2), 2.08 (td, J = 10.0/3.9 Hz, 2H,
N(CH2CH2)2), 2.61 (t, J = 9.8 Hz, 2H, N(CH2CH2)2), 2.75 (d,
J = 11.7 Hz, 2H, N(CH2CH2)2), 3.61 (s, 2H, NCH2Ph), 7.07 (d,
J = 2.3 Hz, 1H, 60-H-thioph), 7.27–7.39 (m, 5H, Ph-H). 7.88 (d,
J = 2.4 Hz, 1H, 40-H-thioph). 13C NMR (CDCl3): d (ppm) = 36.8 (2C,
N(CH2CH2)2), 49.7 (2C, N(CH2CH2)2), 63.1 (1C, NCH2Ph), 83.7 (1C,
Cspiro), 115.5 (1C, C-60-thioph), 126.6 (1C, C-40-thioph), 127.4 (1C,
Ph-C), 128.5 (2C, Ph-C), 129.4 (2C, Ph-C), 133.3 (1C, Cquart), 138.0
(1C, Cquart)(Ph-C), 154.7 (1C, Cquart), 162.8 (1C, C@O).
6.1.12. 4-(1-Benzyl-60-oxo-60,70-dihydrospiro-[piperidine-4,40-
thieno[3,4-c]pyran]-10-yl) benzonitrile (19c)
According to General Procedure A the spirocyclic thiophene 13
(33.2 mg, 0.106 mmol) was reacted with p-iodobenzonitrile
(30.5 mg, 0.13 mmol), Ag2CO3 (33.1 mg, 0.12 mmol) and PdCl2/
bipy (3.5 mg, 0.01 mmol) in m-xylene (1.2 mL). The crude product
was purified by CHCl3-gpc (yield 7.8 mg, 18%) and fc (1.5 cm,
h = 10 cm, hexane/EtOAc = 7:3, 3 mL, Rf = 0.08). Colorless solid,
mp 183–184 °C, yield 5.1 mg (12%). C25H22N2O2S (414.5 g/mol).
Purity (HPLC, method B): 96.2%, tR = 6.66 min. Exact MS (APCI):
m/z = calcd for C25H23N2O2S [MH+] 415.1475, found 415.1469. 1H
NMR (CDCl3): d (ppm) = 2.08 (d, J = 12.3 Hz, 2H, N(CH2CH2)2),
2.18 (td, J = 12.5/4.5 Hz, 2H, N(CH2CH2)2), 2.61 (td, J = 11.9/2.3 Hz,
2H, N(CH2CH2)2), 2.83 (d, J = 11.3 Hz, 2H, N(CH2CH2)2), 3.60 (s,
2H, NCH2Ph), 3.82 (s, 2H, thiophCH2), 7.21 (s, 1H, 30-H-thioph),
7.27–7.38 (m, 5H, Ph-H), 7.50 (d, J = 8.2 Hz, 2H, m-NC-Ph-H), 7.73
(d, J = 8.2 Hz, 2H, o-NC-Ph-H). 13C NMR (CDCl3): d (ppm) = 32.8
(1C, thiophCH2), 37.1 (2C, N(CH2CH2)2), 48.6 (2C, N(CH2CH2)2),
1-Benzyl-30-bromospiro[piperidine-4,40-thieno[2,3-c]furan]-60-
one (Rf = 0.60, cyclohexane/EtOAc = 3:7): Colorless solid, mp
105 °C, yield 28.4 mg (3%, referring to 15).
C17H16BrNO2S
(378.2 g/mol). Purity (HPLC, method A): 97.5%, tR = 15.7 min. Ex-
act MS (APCI): m/z = calcd for C17H16BrNO2S [MH+] 378.0158,
found 378.0163. IR (neat):
(C@O), 1051 (C-Br), 697 (C–H). 1H NMR (CDCl3):
v
(cmꢀ1) = 3112 (C–Haryl), 1763
d
(ppm) = 1.68 (d, J = 10.6 Hz, 2H, N(CH2CH2)2), 2.44–2.58 (m, 4H,
N(CH2CH2)2), 2.91 (d, J = 7.3 Hz, 2H, N(CH2CH2)2), 3.61 (s, 2H,
NCH2Ph), 7.27–7.40 (m, 5H, Ph-H), 7.69 (s, 1H, 20-H-thioph).
13C NMR (CDCl3): d (ppm) = 33.8 (2C, N(CH2CH2)2), 49.3 (2C,
N(CH2CH2)2), 63.1 (1C, NCH2Ph), 85.1 (1C, Cspiro), 103.6 (1C, Br-
Cquart), 127.3 (Ph-CH), 128.4 (Ph-CH), 129.2 (Ph-CH), 131.2 (1C,
Cquart), 136.9 (1C, CH-20-thioph), 138.3 (1C, Cquart), 140.4 (1C,
Cquart), 163.7 (1C, C@O).
63.2 (1C, NCH2Ph), 81.1 (1C, thiophCspiro), 111.9 (1C, Ph-Cquart
-

1852
C. Meyer et al. / Bioorg. Med. Chem. 21 (2013) 1844–1856
C„N), 118.6 (1C, C„N), 119.6 (1C, CH-30-thioph), 127.4 (Ph-CH),
128.1 (1C, Cquart), 128.6 (Ph-CH), 129.1 (Ph-CH), 129.4 (Ph-CH),
133.0 (Ph-CH), 137.1 (1C, Cquart), 137.6 (1C, Cquart), 138.4 (1C,
Cquart), 141.9 (1C, Cquart), 168.9 (1C, C@O).
3.8 Hz, 2H, N(CH2CH2)2), 2.63 (td, J = 11.6/2.7 Hz, 2H, N(CH2CH2)2),
2.82 (d, J = 11.6 Hz, 2H, N(CH2CH2)2), 3.61 (s, 2H, NCH2Ph), 3.70 (s,
2H, thiophCH2), 7.23 (s, 1H, 20-H-thioph), 7.27–7.48 (m, 10H, Ph-H).
13C NMR (CDCl3): d (ppm) = 32.0 (1C, thiophCH2C@O), 38.8 (2C,
N(CH2CH2)2), 48.7 (2C, N(CH2CH2)2), 63.2 (1C, NCH2Ph), 81.4 (1C,
thiophCspiro), 121.9 (CH-20-thioph), 127.4 (Ph-CH), 128.0 (Ph-CH),
128.3 (Ph-CH), 128.5 (Ph-CH), 129.0 (Ph-CH), 129.2 (1C, Cquart),
129.4 (Ph-CH), 135.2 (1C, Cquart), 138.5 (1C, Cquart), 139.5 (1C,
Cquart), 141.1 (1C, Cquart), 169.3 (1C, C@O).
6.1.13. 1-Benzyl-10-(1,10-biphenyl-4-yl)-spiro-[piperidine-4,40-
thieno[3,4-c]pyran]-60 (70H)-one (19d)
According to General Procedure A the spirocyclic thiophene 13
(30.5 mg, 0.097 mmol) was reacted with 4-iodo-1,10-biphenyl
(33.9 mg, 0.12 mmol), Ag2CO3 (32.3 mg, 0.12 mmol) and PdCl2/
bipy (3.3 mg, 0.01 mmol) in m-xylene (1.2 mL). The crude product
was purified by CHCl3-gpc (yield 11.3 mg, 25%) and prep. tlc
(h = 15 cm, hexane/EtOAc = 1:1, Rf = 0.20). Colorless solid, mp
200–201 °C, yield 5.6 mg (12%). C30H27NO2S (465.6 g/mol). Purity
(HPLC, method B): 95.5%, tR = 8.15 min. Exact MS (APCI): m/
z = calcd for C30H28NO2S [MH+] 466.1835, found 466.1792. 1H
NMR (CDCl3): d (ppm) = 2.10 (dd, J = 14.3/1.8 Hz, 2H, N(CH2CH2)2),
2.20 (td, J = 14.4/4.6 Hz, 2H, N(CH2CH2)2), 2.62 (td, J = 11.7/2.3 Hz,
2H, N(CH2CH2)2), 2.83 (d, J = 11.3 Hz, 2H, N(CH2CH2)2), 3.61 (s,
2H, NCH2Ph), 3.88 (s, 2H, thiophCH2), 7.12 (s, 1H, 30-H-thioph),
7.28–7.41 (m, 6H, Ph-H), 7.44–7.50 (m, 4H, Ph-H), 7.60–7.70 (m,
4H, Ph-H). 13C NMR (CDCl3): d (ppm) = 32.8 (1C, thiophCH2CO),
37.1 (2C, N(CH2CH2)2), 48.7 (2C, N(CH2CH2)2), 63.2 (1C, NCH2Ph),
81.2 (1C, thiophCspiro), 117.8 (1C, CH-30-thioph), 126.2 (1C, Cquart),
126.4 (1C, Cquart), 127.3 (Ph-CH), 127.4 (Ph-CH), 127.9 (Ph-CH),
127.9 (Ph-CH), 128.6 (Ph-CH), 129.1 (Ph-CH), 129.2 (Ph-CH),
129.5 (Ph-CH), 138.6 (1C, Cquart), 140.5 (1C, Cquart), 141.3 (1C,
Cquart), 141.4 (1C, Cquart), 169.5 (1C, C@O). One signal for a quater-
nary carbon atom is not visible.
6.1.16. 1-Benzyl-30-(4-methoxyphenyl)spiro[piperidine-4,70-
thieno-[2,3-c]pyran]-50 (40H)-one (21b)
According to General Procedure B the spirocyclic thiophene 14
(29.1 mg, 0.093 mmol) was reacted with p-iodoanisole (30.8 mg,
0.13 mmol), Ag2CO3 (31.1 mg, 0.11 mmol), PdCl2 (1.8 mg,
0.01 mmol) and P[OCH(CF3)2]3 (6.3 lL, 0.02 mmol) in m-xylene
(1.2 mL). The crude product was purified by CHCl3-gpc (yield
21.2 mg, 54%) and fc (1.5 cm, h = 5 cm, hexane/EtOAc = 7:3, 3 mL,
Rf = 0.13). Colorless solid, mp 155–156 °C, yield 3.1 mg (8%).
C25H25NO3S (419.5 g/mol). Purity (HPLC, method B): 99.5%,
tR = 7.18 min. Exact MS (APCI): m/z = calcd for C25H26NO3S [MH+]
420.1628, found 420.1671. 1H NMR (CDCl3): d (ppm) = 2.10 (d,
J = 12.4 Hz, 2H, N(CH2CH2)2), 2.18 (td, J = 12.4/4.0 Hz, 2H,
N(CH2CH2)2), 2.63 (td, J = 11.5/2.9 Hz, 2H, N(CH2CH2)2), 2.82 (d,
J = 11.7 Hz, 2H, N(CH2CH2)2), 3.60 (s, 2H, NCH2Ph), 3.68 (s, 2H, thi-
ophCH2), 3.84 (s, 3H, OCH3), 6.95 (d, J = 8.8 Hz, 2H, m-H3CO-Ph-H),
7.15 (s, 1H, 20-H-thioph), 7.22–7.25 (m, 2H, o-H3CO-Ph-H), 7.29–
7.39 (m, 5H, Ph-H). 13C NMR (CDCl3): d (ppm) = 32.0 (1C, thi-
ophCH2C@O), 38.8 (2C, N(CH2CH2)2), 48.7 (2C, N(CH2CH2)2), 55.5
(1C, OCH3), 63.2 (1C, NCH2Ph), 81.5 (1C, thiophCspiro), 114.4 (Ph-
CH), 121.1 (CH-20-thioph), 127.4 (Ph-CH), 127.7 (1C, Cquart), 128.4
(1C, Cquart), 128.5 (Ph-CH), 129.4 (Ph-CH), 129.5 (Ph-CH), 138.5
(1C, Cquart), 139.3 (1C, Cquart), 140.8 (1C, Cquart), 159.5 (1C, Cquart),
169.4 (1C, C@O).
6.1.14. 1-Benzyl-20-phenylspiro[piperidine-4,70-thieno[2,3-
c]pyran]-50 (40H)-one (20a)
According to General Procedure A the spirocyclic thiophene 14
(30.8 mg, 0.098 mmol) was reacted with iodobenzene (12.1 lL,
0.11 mmol), Ag2CO3 (30.2 mg, 0.11 mmol) and PdCl2/bipy
(3.4 mg, 0.01 mmol) in m-xylene (1.2 mL). The crude product was
purified by CHCl3-gpc (yield 17.8 mg, 47%) and fc (1.5 cm,
h = 5 cm, hexane/EtOAc = 4:1, 3 mL, Rf = 0.20). Colorless solid, mp
140–141 °C, yield 12.5 mg (33%). C24H23NO2S (389.5 g/mol). Purity
(HPLC, method B): 97.3%, tR = 7.42 min. Exact MS (APCI): m/
z = calcd for C24H24NO2S [MH+] 390.1522, found 390.1531. 1H
NMR (CDCl3): d (ppm) = 2.08 (dd, J = 14.5/2.3 Hz, 2H, N(CH2CH2)2),
2.17 (td, J = 14.2/4.4 Hz, 2H, N(CH2CH2)2), 2.62 (td, J = 11.7/2.8 Hz,
2H, N(CH2CH2)2), 2.81 (d, J = 11.5 Hz, 2H, N(CH2CH2)2), 3.60 (s,
2H, NCH2Ph), 3.70 (s, 2H, thiophCH2), 7.00 (s, 1H, 30-H-thioph),
6.1.17. 1-Benzyl-40-phenylspiro[piperidine-4,10-thieno[3,4-
c]furan]-30-one (22a) and 1-benzyl-60-phenylspiro[piperidine-
4,10-thieno[3,4-c]furan]-30-one (22b) and 1-benzyl-40,60-
diphenylspiro[piperidine-4,10-thieno[3,4-c]furan]-30-one (22c)
According to General Procedure A the spirocyclic thiophene 18
(31.9 mg, 0.107 mmol) was reacted with iodobenzene (13.1 lL,
0.12 mmol), Ag2CO3 (32.5 mg, 0.12 mmol) and PdCl2/bipy
(4.0 mg, 0.01 mmmol) in m-xylene (1.2 mL). The residue was puri-
fied by CHCl3-gpc to yield 22c in the first fraction (yield 13.2 mg,
27%) and 22a and 22b in the second fraction of the gpc (yield
19.9 mg, 50%, mixture of regioisomers). 22a and 22b were sepa-
rated by prep. tlc (h = 15 cm, hexane/EtOAc = 7:3, Rf (22a) = 0.22,
Rf (22b) = 0.48).
Compound 22a colorless solid, mp 136–137 °C, yield 10.1 mg
(25%). C23H21NO2S (375.5 g/mol). Purity (HPLC, method B): 97%,
tR = 7.37 min. Exact MS (APCI): m/z = calcd for C23H22NO2S [MH+]
376.1366, found 376.1352. 1H NMR (CDCl3): d (ppm) = 1.99 (d,
J = 13.4 Hz, 2H, N(CH2CH2)2), 2.12 (td, J = 12.0/3.8 Hz, 2H,
N(CH2CH2)2), 2.64 (td, J = 12.7/2.6 Hz, 2H, N(CH2CH2)2), 2.80 (d,
J = 11.8 Hz, 2H, N(CH2CH2)2), 3.63 (s, 2H, NCH2Ph), 6.93 (s, 1H, 60-
H-thioph), 7.27–7.49 (m, 8H, Ph-H), 8.02–8.08 (m, 2H, Ph-H). 13C
7.27–7.43 (m, 8H, Ph-H), 7.49–7.58 (m, 2H, Ph-H). 13C NMR13
NMR (CDCl3): (ppm) = 31.7 (1C, thiophCH2C@O), 39.0 (2C,
C
d
N(CH2CH2)2), 48.7 (2C, N(CH2CH2)2), 63.2 (1C, NCH2Ph), 81.8 (1C,
thiophCspiro), 121.7 (1C, CH-30-thioph), 126.0 (Ph-CH), 127.4 (Ph-
CH), 128.3 (Ph-CH), 128.6 (Ph-CH), 129.3 (Ph-CH), 129.4 (Ph-CH),
130.5 (1C, Cquart), 133.8 (1C, Cquart), 137.5 (1C, Cquart), 138.6 (1C,
Cquart), 144.7 (1C, Cquart), 169.1 (1C, C@O).
6.1.15. 1-Benzyl-30-phenylspiro[piperidine-4,70-thieno[2,3-
c]pyran]-50 (40H)-one (21a)
According to General Procedure B the spirocyclic thiophene 14
(30.6 mg, 0.098 mmol) was reacted with iodobenzene (12
0.11 mmol), Ag2CO3 (29.2 mg, 0.11 mmol), PdCl2 (1.7 mg,
0.01 mmol) and P[OCH(CF3)2]3 (6.3 L, 0.02 mmol) in m-xylene
(1.2 mL). The crude product was purified by CHCl3-gpc (yield
11.8 mg, 31%) and prep. tlc (h = 15 cm, hexane/EtOAc = 7:3,
Rf = 0.06, 4 runs). Colorless solid, mp 114–115 °C, yield 3.7 mg
l
L,
NMR (CDCl3): d (ppm) = 36.9 (2C, N(CH2CH2)2), 49.6 (2C,
N(CH2CH2)2), 63.1 (1C, NCH2Ph), 82.1 (1C, thiophCspiro), 112.4 (1C,
CH-60-thioph), 126.5 (1C, Cquart), 127.5 (Ph-CH), 128.2 (Ph-CH),
128.6 (Ph-CH), 129.2 (Ph-CH), 129.5 (Ph-CH), 129.8 (Ph-CH),
131.4 (1C, Cquart), 138.1 (1C, Cquart), 148.4 (1C, Cquart), 156.7 (1C,
Cquart), 163.1 (1C, C@O).
l
(9.7%).
96.3%, tR = 7.25 min. Exact MS (APCI): m/z = calcd for C24H24NO2S
[MH+] 390.1522, found 390.1545. 1H NMR (CDCl3):
(ppm) = 2.11 (d, J = 12.8 Hz, 2H, N(CH2CH2)2), 2.19 (td, J = 14.4/
C
24H23NO2S (389.5 g/mol). Purity (HPLC, method B):
Compound 22b colorless solid, mp 122–123 °C, yield 2.2 mg
(5%). C23H21NO2S (375.5 g/mol). Purity (HPLC, method B): 95.2%,
tR = 7.20 min. Exact MS (APCI): m/z = calcd for C23H22NO2S [MH+]
376.1366, found 376.1393. 1H NMR (CDCl3): d (ppm) = 1.84 (d,
d

C. Meyer et al. / Bioorg. Med. Chem. 21 (2013) 1844–1856
1853
J = 12.9 Hz, 2H, N(CH2CH2)2), 2.17 (t, J = 14.2 Hz, 2H, N(CH2CH2)2),
2.49 (t, J = 9.2 Hz, 2H, N(CH2CH2)2), 2.78 (d, J = 9.0 Hz, 2H,
N(CH2CH2)2), 3.53 (s, 2H, NCH2Ph), 7.27–7.34 (m, 4H, Ph-H),
7.39–7.56 (m, 6H, Ph-H), 7.84 (s, 1H, 40-H-thioph). Due to the low
amount of 22b recording of a 13C NMR-spectrum was not possible.
Compound 22c was purified by fc (1.5 cm, h = 5 cm, hexane/
EtOAc = 9:1, 3 mL, Rf = 0.12). Colorless solid, mp 182–183 °C, yield
10.2 mg (21%). C29H25NO2S (451.6 g/mol). Purity (HPLC, method
B): 96.6%, tR = 8.62 min. Exact MS (APCI): m/z = calcd for
(ppm) = 1.85 (d, J = 12.4 Hz, 2H, N(CH2CH2)2), 2.14 (td, J = 13.3/
4.3 Hz, 2H, N(CH2CH2)2), 2.50 (td, J = 12.3/1.9 Hz, 2H, N(CH2CH2)2),
2.77 (d, J = 10.6 Hz, 2H, N(CH2CH2)2), 3.54 (s, 2H, NCH2Ph), 3.85 (s,
3H, OCH3), 3.88 (s, 3H, OCH3), 6.97 (m, 4H, m- H3CO-Ph-H), 7.27–
7.34 (m, 5H, Ph-H), 7.41(d, J = 8.7 Hz, 2H, o-H3CO-Ph-H), 8.01 (d,
J = 8.8 Hz, 2H, o-H3CO-Ph-H). 13C NMR (CDCl3): d (ppm) = 36.4
(2C, N(CH2CH2)2), 49.4 (2C, N(CH2CH2)2), 55.5 (1C, OCH3), 55.5
(1C, OCH3), 63.1 (1C, NCH2Ph), 82.8 (1C, thiophCspiro), 114.5 (Ph-
CH), 123.8 (1C, Cquart), 124.3 (1C, Cquart), 127.3 (Ph-CH), 128.5
(Ph-CH), 129.4 (Ph-CH), 129.9 (Ph-CH), 131.2 (Ph-CH), 138.6 (1C,
Cquart), 140.3 (1C, Cquart), 150.5 (1C, Cquart), 160.4 (1C, Cquart),
160.9 (1C, Cquart), 163.4 (1C, C@O). Two signals for quaternary car-
bon atoms are not visible.
C
29H26NO2S [MH+] 452.1679, found 452.1687. 1H NMR (CDCl3): d
(ppm) = 1.87 (d, J = 12.0 Hz, 2H, N(CH2CH2)2), 2.17 (td, J = 13.3/
4.7 Hz, 2H, N(CH2CH2)2), 2.52 (td, J = 12.3/2.2 Hz, 2H, N(CH2CH2)2),
2.78 (dd, J = 11.1/4.2 Hz, 2H, N(CH2CH2)2), 3.54 (s, 2H, NCH2Ph),
7.27–7.56 (m, 13H, Ph-H), 8.03–8.08 (m, 2H, Ph-H). 13C NMR
(CDCl3): d (ppm) = 36.4 (2C, N(CH2CH2)2), 49.3 (2C, N(CH2CH2)2),
63.0 (1C, NCH2Ph), 83.0 (1C, thiophCspiro), 127.3 (Ph-CH), 127.3
(1C, Cquart), 128.4 (Ph-CH), 128.5 (Ph-CH), 129.1 (Ph-CH), 129.2
(Ph-CH), 129.2 (Ph-CH), 129.3 (Ph-CH), 129.7 (Ph-CH), 129.9 (Ph-
CH), 131.3 (1C, Cquart), 131.6 (1C, Cquart), 132.7 (1C, Cquart), 138.6
(1C, Cquart), 146.9 (1C, Cquart), 151.0 (1C, Cquart), 163.0 (1C, C@O).
6.1.19. 4-(1-Benzyl-30-oxospiro[piperidine-4,10-thieno[3,4-c]
furan]-40-yl)-benzonitrile (24a) and 4-(1-benzyl-30-oxospiro
[piperidine-4,10-thieno[3,4-c] furan]-60-yl)benzonitrile (24b)
and 4,40-(1-benzyl-30-oxospiro[piperidine-4,10-thieno[3,4-c]
furan]-40,60diyl)dibenzonitrile (24c)
According to General Procedure A the spirocyclic thiophene 18
(27.8 mg, 0.093 mmol) was reacted with p-iodobenzonitrile
(30.1 mg, 0.13 mmol), Ag2CO3 (29.9 mg, 0.11 mmol) and PdCl2/
bipy (4.2 mg, 0.01 mmol) in m-xylene (1.2 mL). The residue was
purified by CHCl3-gpc to yield 24c in the first fraction (11.8 mg,
25%) and 24a and 24b in the second fraction of the gpc (yield
13.0 mg, 35%, mixture of regioisomers). 24a and 24b were sepa-
rated by prep. tlc (h = 15 cm, hexane/EtOAc = 7:3, NHEt2 2%, Rf
(24a) = 0.18, Rf (24b) = 0.08, 3 runs).
6.1.18. 1-Benzyl-40-(4-methoxyphenyl)spiro[piperidine-4,10-
thieno[3,4-c]furan]-30-one (23a) and 1-benzyl-60-(4-methoxy
phenyl)spiro[piperidine-4,10-thieno[3,4-c]furan]-30-one (23b)
and 1-benzyl-40,60-bis(4-methoxyphenyl)spiro[piperidine-4,
10-thieno[3,4-c]furan]-30-one (23c)
According to General Procedure A the spirocyclic thiophene 18
(30.7 mg, 0.103 mmol) was reacted with p-iodoanisole (26.5 mg,
0.11 mmol), Ag2CO3 (30.5 mg, 0.11 mmol) and PdCl2/bipy
(4.7 mg, 0.01 mmol) in m-xylene (1.2 mL). The residue was purified
by CHCl3-gpc to yield 23c in the first fraction (yield 7.4 mg, 14%)
and 23a and 23b in the second fraction of the gpc (yield 10.5 mg,
25%, mixture of regioisomers). 23a and 23b were separated by
prep. tlc (h = 15 cm, hexane/EtOAc = 7:3, Rf (23a) = 0.18, Rf
(23b) = 0.34, 3 runs).
Compound 23a colorless solid, mp 158–159 °C, yield 6.7 mg
(16%). C24H23NO3S (405.5 g/mol). Purity (HPLC, method B): 99.1%,
tR = 7.38 min. Exact MS (APCI): m/z = calcd for C24H24NO3S [MH+]
406.1471, found 406.1592. 1H NMR (CDCl3): d (ppm) = 1.98 (d,
J = 13.5 Hz, 2H, N(CH2CH2)2), 2.08 (td, J = 10.8/4.1 Hz, 2H,
N(CH2CH2)2), 2.62 (m, 2H, N(CH2CH2)2), 2.78 (d, J = 11.7 Hz, 2H,
N(CH2CH2)2), 3.61 (s, 2H, NCH2Ph), 3.85 (s, 3H, OCH3), 6.83 (s, 1H,
60-H-thioph), 6.95 (d, J = 8.9 Hz, 2H, m-OCH3-Ph-H), 7.27–7.38 (m,
5H, Ph-H), 8.03 (d, J = 8.9 Hz, 2H, o-OCH3-Ph-H). 13C NMR (CDCl3):
d (ppm) = 36.9 (2C, N(CH2CH2)2), 49.6 (2C, N(CH2CH2)2), 55.5 (1C,
OCH3), 63.2 (1C, NCH2Ph), 82.1 (1C, thiophCspiro), 111.0 (1C, CH-
60-thioph), 114.5 (Ph-CH), 124.3 (1C, Cquart), 125.3 (1C, Cquart),
127.4 (Ph-CH), 128.5 (Ph-CH), 129.4 (Ph-CH), 129.7 (Ph-CH),
138.3 (1C, Cquart), 148.7 (1C, Cquart), 156.5 (1C, Cquart), 160.9 (1C,
Cquart), 163.4 (1C, C@O).
Compound 23b colorless solid, yield 3.5 mg (8%). C24H23NO3S
(405.5 g/mol). Purity (HPLC, method B): 87.6%, tR = 7.16 min. Exact
MS (APCI): m/z = calcd for C24H24NO3S [MH+] 406.1471, found
406.1503. 1H NMR (CDCl3): d (ppm) = 1.82 (dd, J = 14.0/1.6 Hz,
2H, N(CH2CH2)2), 2.15 (td, J = 13.2/4.5 Hz, 2H, N(CH2CH2)2), 2.48
(td, J = 12.3/2.4 Hz, 2H, N(CH2CH2)2), 2.77 (d, J = 10.5 Hz, 2H,
N(CH2CH2)2), 3.54 (s, 2H, NCH2Ph), 3.88 (s, 3H, OCH3), 6.98 (d,
J = 8.6 Hz, 2H, m-H3CO-Ph-H), 7.27–7.33 (m, 5H, Ph-H), 7.39 (d,
J = 8.6 Hz, 2H, o-H3CO-Ph-H), 7.79 (s, 1H, 40-H-thioph). Due to the
low amount of 23b recording of a 13C NMR -spectrum was not
possible.
Compound 24a colorless solid, mp 1659–160 °C, yield 8.4 mg
(23%).
96.2%, tR = 7.02 min. Exact MS (APCI): m/z = calcd for C24H21N2O2S
[MH+] 401.1318, found 401.1358. 1H NMR (CDCl3):
C24H20N2O2S (400.5 g/mol). Purity (HPLC, method B):
d
(ppm) = 1.98 (d, J = 13.6 Hz, 2H, N(CH2CH2)2), 2.10 (td, J = 11.4/
3.6 Hz, 2H, N(CH2CH2)2), 2.61 (td, J = 11.5/2.3 Hz, 2H, N(CH2CH2)2),
2.79 (d, J = 11.9 Hz, 2H, N(CH2CH2)2), 3.61 (s, 2H, NCH2Ph), 7.07 (s,
1H, 60-H-thioph), 7.27–7.39 (m, 5H, Ph-H), 7.72 (d, J = 8.3 Hz, 2H,
m-CN-Ph-H), 8.21 (d, J = 8.3 Hz, 2H, o-CN-Ph-H). 13C NMR (CDCl3):
d (ppm) = 36.9 (2C, N(CH2CH2)2), 49.6 (2C, N(CH2CH2)2), 63.2 (1C,
NCH2Ph), 82.8 (1C, thiophCspiro), 112.8 (1C, Ph-Cquart-C„N), 114.4
(1C, CH-60-thioph), 118.7 (1C, C„N), 127.5 (Ph-CH), 128.5 (Ph-
CH), 128.6 (Ph-CH), 129.4 (Ph-CH), 132.9 (Ph-CH), 135.5 (1C, Cquart),
138.3 (1C, Cquart), 145.0 (1C, Cquart), 157.4 (1C, Cquart), 162.8 (1C,
C@O). One signal for a quaternary carbon atom is not visible.
Compound 24b colorless solid, mp 163–164 °C, yield 2.3 mg
(6%). C24H20N2O2S (400.5 g/mol). Purity (HPLC, method B): 96.2%,
tR = 6.67 min. Exact MS (APCI): m/z = calcd for C24H21N2O2S [MH+]
401.1318, found 401.1350. 1H NMR (CDCl3): d (ppm) = 1.86 (d,
J = 12.8 Hz, 2H, N(CH2CH2)2), 2.12 (td, J = 13.3/4.6 Hz, 2H,
N(CH2CH2)2), 2.49 (td, J = 12.4/1.8 Hz, 2H, N(CH2CH2)2), 2.79 (d,
J = 9.2 Hz, 2H, N(CH2CH2)2), 3.54 (s, 2H, NCH2Ph), 7.27–7.35 (m,
5H, Ph-H), 7.62 (d, J = 8.3 Hz, 2H, m-CN-Ph-H), 7.77 (d, J = 8.3 Hz,
2H, o-CN-Ph-H), 7.93 (s, 1H, 40-H-thioph). 13C NMR (CDCl3): d
(ppm) = 36.6 (2C, N(CH2CH2)2), 49.3 (2C, N(CH2CH2)2), 63.1 (1C,
NCH2Ph), 84.4 (1C, thiophCspiro), 113.0 (1C, Ph-Cquart-C„N), 118.3
(1C, C„N), 126.9 (1C, CH-40-thioph), 127.4 (Ph-CH), 128.5 (Ph-
CH), 129.3 (Ph-CH), 130.1 (Ph-CH), 133.0 (Ph-CH), 133.6 (1C, Cquart),
134.9 (1C, Cquart), 136.4 (1C, Cquart), 138.3 (1C, Cquart), 150.5 (1C,
Cquart), 162.3 (1C, C@O).
Compound 24c was purified by fc (1.5 cm, h = 5 cm, hexane/
EtOAc = 3:2, 3 mL, Rf = 0.49). Colorless solid, mp 222–123 °C,
7.9 mg (17%). C31H23N3O2S (501.6 g/mol). Purity (HPLC, method
B): 98.9%, tR = 7.47 min. Exact MS (APCI): m/z = calcd for
Compound 23c was purified by fc (1.5 cm, h = 5 cm, hexane/
EtOAc = 3:2, 3 mL, Rf = 0.58). Pale yellow solid, mp 213–214 °C,
yield 4.5 mg (9%). C31H29NO4S (511.6 g/mol). Purity (HPLC, method
B): 98.9%, tR = 8.44 min. Exact MS (APCI): m/z = calcd for
C
31H24N3O2S [MH+] 502.1584, found 502.1638. 1H NMR (CDCl3):
d (ppm) = 1.89 (d, J = 12.5 Hz, 2H, N(CH2CH2)2), 2.11 (td, J = 13.3/
4.6 Hz, 2H, N(CH2CH2)2), 2.50 (td, J = 11.8/2.3 Hz, 2H, N(CH2CH2)2),
2.80 (dd, J = 10.7/3.3 Hz, 2H, N(CH2CH2)2), 3.55 (s, 2H, NCH2Ph),
C
31H30NO4S [MH+] 512.1890, found 512.1935. 1H NMR (CDCl3): d

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C. Meyer et al. / Bioorg. Med. Chem. 21 (2013) 1844–1856
7.27–7.35 (m, 5H, Ph-H), 7.65 (d, J = 8.4 Hz, 2H, m-CN-Ph-H), 7.75
(d, J = 8.5 Hz, 2H, m-CN-Ph-H), 7.81 (d, J = 8.3 Hz, 2H, o-NC-Ph-H),
8.20 (d, J = 8.5 Hz, 2H, o-NC-Ph-H). 13C NMR (CDCl3): d (ppm)
= 36.6 (2C, N(CH2CH2)2), 49.2 (2C, N(CH2CH2)2), 63.1 (1C, NCH2Ph),
83.4 (1C, thiophCspiro), 113.3 (1C, Ph-Cquart-C„N), 113.5 (1C, Ph-
Cquart-C„N), 118.2 (1C, C„N), 118.5 (1C, C„N), 127.4 (Ph-CH),
128.5 (Ph-CH), 128.8 (Ph-CH), 129.3 (Ph-CH), 129.7 (1C, Cquart),
130.3 (Ph-CH), 132.1 (1C, Cquart), 133.0 (Ph-CH), 133.0 (Ph-CH),
134.8 (1C, Cquart), 135.7 (1C, Cquart), 138.2 (1C, Cquart), 144.9 (1C,
Cquart), 152.9 (1C, Cquart), 162.1 (1C, C@O).
NCH2Ph), 83.0 (1C, thiophCspiro), 125.3 (Ar-CH), 125.3 (Ar-CH),
125.7 (Ar-CH), 125.9 (Ar-CH), 126.6 (Ar-CH), 126.9 (Ar-CH), 127.1
(Ar-CH), 127.2 (Ar-CH), 127.2 (Ar-CH), 127.8 (1C, Cquart), 128.1
(1C, Cquart), 128.4 (Ar-CH), 128.7 (Ar-CH), 128.8 (Ar-CH), 129.1
(1C, Cquart), 129.4 (Ar-CH), 129.7 (Ar-CH), 130.0 (Ar-CH), 130.4
(Ar-CH), 130.5 (Ar-CH), 131.4 (1C, Cquart), 131.7 (1C, Cquart), 133.6
(1C, Cquart), 133.9 (1C, Cquart), 134.0 (1C, Cquart), 138.1 (1C, Cquart),
144.8 (1C, Cquart), 152.1 (1C, Cquart), 162.3 (C@O).
6.1.21. 1-Benzyl-40-(1,10-biphenyl-4-yl)spiro[piperidine-4,10-
thieno-[3,4-c]furan]-30-one (26a) and 1-benzyl-60-(1,10-biphe-
nyl-4-yl)spiro[piperidine-4,10-thieno[3,4-c] furan]-30-one (26b)
and 1-benzyl-40,60-di(1,10-biphenyl-4-yl)spiro[piperidine-4,
10-thieno[3,4-c]furan]-30-one (26c)
According to General Procedure A the spirocyclic thiophene 18
(33.7 mg, 0.11 mmol) was reacted with 4-iodo-1,10-biphenyl
(33.0 mg, 0.12 mmol), Ag2CO3 (33.0 mg, 0.12 mmol) and PdCl2/
bipy (3.5 mg, 0.01 mmmol) in m-xylene (1.2 mL). The residue
was injected into the CHCl3-gpc to yield 26c in the first fraction
(yield 19.2 mg, 28%) and 26a and 26b in the second fraction of
the gpc (yield 20.5 mg, 40%, mixture of regioisomers). Compound
26a and 26b were separated by prep. tlc (h = 15 cm, hexane/
EtOAc = 4:1, Rf (26a) = 0.10, Rf (26b) = 0.46).
Compound 26a colorless solid, mp 187–188 °C, yield 8 mg
(16%). C29H25NO2S (451.6 g/mol). Purity (HPLC, method B): 97.2%,
tR = 8.38 min. Exact MS (APCI): m/z = calcd for C29H26NO2S [MH+]
452.1679, found 452.1736. 1H NMR (CDCl3): d (ppm) = 2.00 (d,
J = 13.5 Hz, 2H, N(CH2CH2)2), 2.09 (td, J = 10.4/3.9 Hz, 2H,
N(CH2CH2)2), 2.63 (td, J = 12.3/2.9 Hz, 2H, N(CH2CH2)2), 2.79 (d,
J = 11.8 Hz, 2H, N(CH2CH2)2), 3.62 (s, 2H, NCH2Ph), 6.93 (s, 1H, 60-
H-thioph), 7.27–7.40 (m, 6H, Ph-H), 7.46 (t, J = 7.5 Hz, 2H, Ph-H),
7.60–7.70 (m, 4H, Ph-H), 8.12–8.17 (m, 2H, Ph-H). 13C NMR
(CDCl3): d (ppm) = 37.0 (2C, N(CH2CH2)2), 49.7 (2C, N(CH2CH2)2),
63.2 (1C, NCH2Ph), 82.3 (1C, thiophCspiro), 112.2 (1C, CH-60-thioph),
126.6 (1C, Cquart), 127.2 (Ph-CH), 127.4 (Ph-CH), 127.8 (Ph-CH),
128.0 (Ph-CH), 128.5 (Ph-CH), 128.6 (Ph-CH), 129.1 (Ph-CH),
129.4 (Ph-CH), 130.4 (1C, Cquart), 138.4 (1C, Cquart), 140.4 (1C,
Cquart), 142.4 (1C, Cquart), 148.0 (1C, Cquart), 156.8 (1C, Cquart),
163.2 (1C, C@O).
6.1.20. 1-Benzyl-40-(naphthalen-1-yl)-spiro[piperidine-4,10-
thieno[3,4-c]furan]-30-one (25a) and 1-benzyl-60-(naphthalen-
1-yl)-30H-spiro[piperidine-4,10-thieno[3,4-c]furan]-30-one (25b)
and 1-benzyl-40,60-di(naphthalen-1-yl)-spiro[piperidine-4,10-
thieno-[3,4-c]furan]-30-one (25c)
According to General Procedure A the spirocyclic thiophene 18
(32.9 mg, 0.11 mmol) was reacted with 1-iodonaphthalene
(17.6 lL, 0.12 mmol), Ag2CO3 (33.7 mg, 0.12 mmol) and PdCl2/bipy
(4.6 mg, 0.01 mmol) in m-xylene (1.2 mL). The residue was purified
by CHCl3-gpc to yield 25c in the first fraction (yield 23.1 mg, 38%)
and 25a and 25b in the second fraction of the gpc (yield 28.4 mg,
61%, mixture of regioisomers). Compound 25a and 25b were sepa-
rated by prep. tlc (h = 15 cm, hexane/EtOAc = 7:3, Rf (25a) = 0.16, Rf
(25b) = 0.40, 3 runs).
Compound 25a colorless solid, mp 165–166 °C, yield 14.7 mg
(31%). C27H23NO2S (425.5 g/mol). Purity (HPLC, method B): 96.6%,
tR = 7.56 min. Exact MS (APCI): m/z = calcd for C27H24NO2S [MH+]
426.1522, found 426.1555. 1H NMR (CDCl3): d (ppm) = 2.07 (m,
2H, N(CH2CH2)2), 2.16 (td, J = 10.6/4.5 Hz, 2H, N(CH2CH2)2), 2.67
(m, 2H, N(CH2CH2)2), 2.81 (d, J = 11.9 Hz, 2H, N(CH2CH2)2), 3.63
(s, 2H, NCH2Ph), 7.13 (s, 1H, 60-H-thioph), 7.27–7.41 (m, 5H, Ar-
H), 7.49–7.57 (m, 3H, Ar-H), 7.65 (dd, J = 7.1/1.2 Hz, 1H, Ar-H),
7.89–7.98 (m, 2H, Ar-H), 8.00–8.06 (m, 1H, Ar-H). 13C NMR (CDCl3):
d (ppm) = 37.0 (2C, N(CH2CH2)2), 49.7 (2C, N(CH2CH2)2), 63.2 (1C,
NCH2Ph), 82.5 (1C, thiophCspiro), 114.5 (1C, CH-60-thioph), 125.3
(Ar-CH), 125.6 (Ar-CH), 126.6 (Ar-CH), 127.1 (Ar-CH), 127.5 (Ar-
CH), 127.9 (1C, Cquart), 128.6 (Ar-CH), 128.7 (Ar-CH), 129.5 (Ar-
CH), 129.6 (Ar-CH), 130.5 (Ar-CH), 131.5 (1C, Cquart), 134.0 (1C,
Cquart), 138.4 (1C, Cquart), 145.2 (1C, Cquart), 155.5 (1C, Cquart),
162.4 (1C, C@O). One signal for a quaternary carbon atom is not
visible.
Compound 25b colorless solid, mp 145–146 °C, yield 1.9 mg
(4%). C27H23NO2S (425.5 g/mol). Purity (HPLC, method B): 95.8%,
tR = 7.75 min. Exact MS (APCI): m/z = calcd for C27H24NO2S [MH+]
426.1522, found 426.1580. 1H NMR (CDCl3): d (ppm) = 1.78 (m,
4H, N(CH2CH2)2), 2.41 (m, 2H, N(CH2CH2)2), 2.62 (d, J = 11.3 Hz,
2H, N(CH2CH2)2), 3.41 (s, 2H, NCH2Ph), 7.11–7.24 (m, 5H, Ar-H),
7.43–7.64 (m, 5H, Ar-H), 7.88–8.04 (m, 2H, Ar-H), 7.98 (s, 1H, 40-
H-thioph). 13C NMR (CDCl3): d (ppm) = 35.9 (2C, N(CH2CH2)2),
49.1 (2C, N(CH2CH2)2), 63.1 (1C, NCH2Ph), 84.3 (1C, thiophCspiro),
125.2 (Ar-CH), 125.7 (Ar-CH), 126.5 (Ar-CH), 126.7 (Ar-CH), 127.2
(Ar-CH), 127.2 (Ar-CH), 128.1 (1C, Cquart), 128.4 (Ar-CH), 128.6
(Ar-CH), 129.4 (Ar-CH), 129.9 (Ar-CH), 130.4 (Ar-CH), 133.0 (1C,
Cquart), 133.3 (1C, Cquart), 133.5 (1C, Cquart), 133.8 (1C, Cquart),
138.0 (1C, Cquart), 151.6 (1C, Cquart), 162.9 (1C, C@O).
Compound 26b pale yellow solid, mp 172–173 °C, yield 4.4 mg
(9%). C29H25NO2S (451.6 g/mol). Purity (HPLC, method B): 97%,
tR = 8.09 min. Exact MS (APCI): m/z = calcd for C29H26NO2S [MH+]
452.1679, found 452.1736. 1H NMR (CDCl3): d (ppm) = 1.87 (d,
J = 12.5 Hz, 2H, N(CH2CH2)2), 2.24 (td, J = 13.3/4.6 Hz, 2H,
N(CH2CH2)2), 2.51 (td, J = 12.4/1.9 Hz, 2H, N(CH2CH2)2), 2.80 (dd,
J = 10.9/3.8 Hz, 2H, N(CH2CH2)2), 3.55 (s, 2H, NCH2Ph), 7.27–7.73
(m, 14H, Ph-H), 7.86 (s, 1H, 40-H-thioph). 13C NMR (CDCl3): d
(ppm) = 36.4 (2C, N(CH2CH2)2), 49.4 (2C, N(CH2CH2)2), 63.1 (1C,
NCH2Ph), 84.6 (1C, thiophCspiro), 125.5 (1C, CH-40-thioph), 127.3
(Ph-CH), 127.3 (Ph-CH), 127.8 (Ph-CH), 128.1 (Ph-CH), 128.5 (Ph-
CH), 129.2 (Ph-CH), 129.4 (Ph-CH), 130.1 (Ph-CH), 130.6 (1C, Cquart),
134.4 (1C, Cquart), 136.0 (1C, Cquart), 138.4 (1C, Cquart), 142.0 (1C,
Cquart), 149.2 (1C, Cquart), 157.5 (1C, Cquart), 162.5 (1C, C@O).
Compound 26c was purified by fc (1.5 cm, h = 5 m, hexane/
EtOAc = 7:3, 3 mL, Rf = 0.54). Pale yellow solid, mp 117–118 °C,
13.3 mg (20%). C41H33NO2S (603.8 g/mol). Purity (HPLC, method
B): 97.7%, tR = 10.82 min. Exact MS (APCI): m/z = calcd for
Compound 25c was purified by fc (1.5 cm, h = 5 cm, hexane/
EtOAc = 3:2, NHEt2 2%, 3 mL, Rf = 0.40). Colorless solid, mp 230 °C,
yield 6.5 mg (11%). C37H29NO2S (551.7 g/mol). Purity (HPLC, meth-
od C): 96%, tR = 9.20 min. Exact MS (APCI): m/z = calcd for
C
41H34NO2S [MH+] 604.2305, found 604.2340. 1H NMR (CDCl3): d
(ppm) = 1.92 (d, J = 12.8 Hz, 2H, N(CH2CH2)2), 2.26 (td, J = 13.3/
4.6 Hz, 2H, N(CH2CH2)2), 2.55 (td, J = 12.4/1.8 Hz, 2H, N(CH2CH2)2),
2.81 (d, J = 8.8 Hz, 2H, N(CH2CH2)2), 3.56 (s, 2H, NCH2Ph), 7.27–7.54
(m, 11H, Ph-H), 7.56–7.74 (m, 10H, Ph-H), 8.16 (m, 2H, Ph-H). 13C
C
37H30NO2S [MH+] 552.1992, found 552.2050. 1H NMR (CDCl3): d
(ppm) = 1.82–1.94 (m, 4H, N(CH2CH2)2), 2.45 (td, J = 11.0/3.7 Hz,
2H, N(CH2CH2)2), 2.65 (d, J = 11.5 Hz, 2H, N(CH2CH2)2), 3.43 (s,
2H, NCH2Ph), 7.13–7.25 (m, 5H, Ar-H), 7.50–7.66 (m, 7H, Ar-H),
7.76–7.84 (m, 2H, Ar-H), 7.91–8.24 (m, 5H, Ar-H). 13C NMR (CDCl3):
d (ppm) = 36.1 (2C, N(CH2CH2)2), 49.1 (2C, N(CH2CH2)2), 63.1 (1C,
NMR (CDCl3):
d (ppm) = 36.4 (2C, N(CH2CH2)2), 49.4 (2C,
N(CH2CH2)2), 63.1 (1C, NCH2Ph), 83.1 (1C, thiophCspiro), 127.3
(Ph-CH), 127.3 (Ph-CH), 127.3 (Ph-CH), 127.5 (1C, Cquart), 127.8
(Ph-CH), 127.8 (Ph-CH), 128.0 (Ph-CH), 128.1 (Ph-CH), 128.5 (Ph-

C. Meyer et al. / Bioorg. Med. Chem. 21 (2013) 1844–1856
1855
CH), 128.8 (Ph-CH), 129.1 (Ph-CH), 129.2 (Ph-CH), 129.4 (Ph-CH),
130.2 (Ph-CH), 130.3 (1C, Cquart), 130.5 (1C, Cquart), 132.4 (1C, Cquart),
138.5 (1C, Cquart), 140.3 (1C, Cquart), 140.5 (1C, Cquart), 142.0 (1C,
Cquart), 142.4 (1C, Cquart), 146.6 (1C, Cquart), 151.1 (1C, Cquart),
163.1 (1C, C@O).
6.2.5. General protocol for the binding assays
The test compound solutions were prepared by dissolving
approximately 10 mol (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-
l
6.2. Receptor binding studies50–53
6.2.1. 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
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, Ger-
many). Chemicals and reagents were purchased from different
commercial sources and of analytical grade.
says were performed by addition of 50
buffer, 50 L test compound solution in various concentrations
(10ꢀ5, 10ꢀ6, 10ꢀ7, 10ꢀ8, 10ꢀ9 and 10ꢀ10 mol/L), 50
L of corre-
sponding radioligand solution and 50 L of the respective receptor
preparation into each well of the multiplate (total volume 200 L).
lL of the respective assay
l
l
l
l
The receptor preparation was always added last. During the incu-
bation, 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 har-
vester. During the filtration each well was washed five times with
300 lL of water. Subsequently, the filtermats were dried at 95 °C.
The solid scintillator was melted on the dried filtermats at a tem-
perature of 95 °C for 5 min. After solidifying of the scintillator at
room temperature, the trapped radioactivity in the filtermats was
measured with the scintillation analyzer. Each position on the fil-
termat 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 pro-
gram GraphPad PrismÒ 3.0 (GraphPad Software, San Diego, CA,
USA) by non-linear regression analysis. Subsequently, the IC50 val-
ues were transformed into Ki-values using the equation of Cheng
and Prusoff.57 The Ki-values are given as mean value SEM from
three independent experiments.
6.2.2. 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 su-
crose. The suspension was centrifuged at 1200ꢁg for 10 min at
4 °C. The supernatant was separated and centrifuged at 23,500ꢁg
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 23,500ꢁg
(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 about 1.5 mg protein/mL.
6.2.6. Protocol of the r1 receptor binding assay
The assay was performed with the radioligand [3H]-(+)-Pentaz-
ocine (22.0 Ci/mmol; Perkin Elmer). The thawed membrane prepa-
ration of guinea pig brain cortex (about 100 lg of protein) was
incubated with various concentrations of test compounds, 2 nM
[3H]-(+)-Pentazocine, and TRIS buffer (50 mM, pH 7.4) at 37 °C.
6.2.3. Preparation of membrane homogenates from 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 1200ꢁg for
10 min at 4 °C. The supernatant was separated and centrifuged at
31,000ꢁg for 20 min at 4 °C. The pellet was resuspended in 5–
6 volumes of buffer (50 mM TRIS, pH 8.0) and incubated at room
temperature for 30 min. After the incubation, the suspension was
centrifuged again at 31,000ꢁg 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 about 2 mg protein/mL.
The non-specific binding was determined with 10 lM unlabeled
(+)-Pentazocine. The Kd-value of (+)-Pentazocine is 2.9 nM.58
6.2.7. Protocol of the r2 receptor binding assay
The assays were performed with the radioligand [3H]DTG (spe-
cific activity 50 Ci/mmol; ARC, St. Louis, MO, USA). The thawed
membrane preparation of rat liver (about 100 lg of protein) was
incubated with various concentrations of the test compound,
3 nM [3H]DTG and buffer containing (+)-pentazocine (500 nM
(+)-pentazocine in 50 mM TRIS, pH 8.0) at room temperature.
The non-specific binding was determined with 10 lM non-labeled
DTG. The Kd value of [3H]DTG is 17.9 nM.59
6.2.4. Protein determination
The protein concentration was determined by the method of
Bradford,55 modified by Stoscheck.56 The Bradford solution was
prepared by dissolving 5 mg of Coomassie Brilliant Blue G 250 in
2.5 mL of EtOH (95%, v/v). 10 mL deionized H2O and 5 mL phospho-
ric acid (85%, m/v) were added to this solution, the mixture was
stirred and filled to a total volume of 50.0 mL with deionized
water. The calibration was carried out using bovine serum albumin
as a standard in 9 concentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0
Acknowledgment
This work was performed within the framework of the Interna-
tional Research Training Group ‘Complex Functional Systems in
Chemistry: Design, Synthesis and Applications’ in collaboration
with the University of Nagoya. Financial support of this project
by the IRTG Münster—Nagoya and the Deutsche Forschungsgeme-
inschaft is gratefully acknowledged.
and 4.0 mg/mL). In a 96-well standard multiplate, 10
l
L of the cal-
L of the membrane receptor preparation
L of the Bradford solution, respectively.
ibration solution or 10
were mixed with 190
l
l
References and notes
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