Exploitation of an additional hydrophobic pocket of σ1 receptors: Late-stage diverse modifications of spirocyclic thiophenes by C-H bond functionalization

Article abstract of DOI:10.1039/c1ob06149f

The hypothesis that the σ₁ receptor will tolerate an additional aryl moiety in position 1 of the spirocyclic system was based on spirocyclic pyrazole derivatives, pharmacophore models of σ₁ receptor ligands and DFT calculations. The strategy of introducing the aryl residue at the final step of the synthesis allowed the preparation of a large set of diverse ligands for the exploitation of the hydrophobic pocket of the σ₁ receptor protein. The catalyst system PdCl ₂/2,2′-bipyridyl/Ag₂CO₃ is able to introduce various aryl groups onto the α-positions of spirocyclic thiophene derivatives 5 and 6 to afford the target aryl-appended spirocyclic thiophenes 3 and 4. Although the σ₁ affinity of the 1-phenyl substituted spirocyclic thiophenes 3a and 4a is slightly reduced compared with the σ₁ affinity of the non-arylated compounds 5 and 6, both compounds represent very potent σ₁ receptor ligands (3a: K _i = 4.5 nM; 4a: K_i = 1.0 nM). This result indicates that an aryl moiety in position 1 is well tolerated by the σ₁ receptor protein. The substitution pattern of the additional phenyl moiety has only weak effects on the σ₁ affinity. Even ligands 3f and 4h with extended naphthyl residue show high σ₁ affinity. However, decrease of σ₁ affinity by extension of the π-system to a biphenylyl substituent (4j: K_i = 30 nM) indicates that the biphenylyl residue is too large for the primary hydrophobic binding pocket of the σ₁ receptor. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c1ob06149f

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PAPER
Exploitation of an additional hydrophobic pocket of r1 receptors: Late-stage
diverse modifications of spirocyclic thiophenes by C–H bond functionalization
Christina Meyer,a Benedikt Neue,b Dirk Schepmann,a Shuichi Yanagisawa,c Junichiro Yamaguchi,c
Ernst-Ulrich Wu¨rthwein,b Kenichiro Itami*c and Bernhard Wu¨nsch*a
Received 12th July 2011, Accepted 30th August 2011
DOI: 10.1039/c1ob06149f
The hypothesis that the s1 receptor will tolerate an additional aryl moiety in position 1 of the
spirocyclic system was based on spirocyclic pyrazole derivatives, pharmacophore models of s1 receptor
ligands and DFT calculations. The strategy of introducing the aryl residue at the final step of the
synthesis allowed the preparation of a large set of diverse ligands for the exploitation of the
hydrophobic pocket of the s1 receptor protein. The catalyst system PdCl2/2,2¢-bipyridyl/Ag2CO3 is
able to introduce various aryl groups onto the a-positions of spirocyclic thiophene derivatives 5 and 6
to afford the target aryl-appended spirocyclic thiophenes 3 and 4. Although the s1 affinity of the
1-phenyl substituted spirocyclic thiophenes 3a and 4a is slightly reduced compared with the s1 affinity
of the non-arylated compounds 5 and 6, both compounds represent very potent s1 receptor ligands (3a:
Ki = 4.5 nM; 4a: Ki = 1.0 nM). This result indicates that an aryl moiety in position 1 is well tolerated by
the s1 receptor protein. The substitution pattern of the additional phenyl moiety has only weak effects
on the s1 affinity. Even ligands 3f and 4h with extended naphthyl residue show high s1 affinity. However,
decrease of s1 affinity by extension of the p-system to a biphenylyl substituent (4j: Ki = 30 nM) indicates
that the biphenylyl residue is too large for the primary hydrophobic binding pocket of the s1 receptor.
stood so far and the pharmacological effects cannot be correlated
to a distinct biochemical mechanism. Nevertheless it has been
1. Introduction
s
Receptors are well established as a non-opioid, non-
shown that s1 receptors are involved in the modulation of various
neurotransmitter systems, including the glutamatergic, dopamin-
ergic and cholinergic neurotransmitter systems.10 Additionally,
a variety of ion channels (e.g. K+-, Na+- and Ca2+ channels) is
regulated by s1 receptors.11–14
We aim to develop novel compounds with high s1 receptor
affinity and high selectivity over the s2 subtype as well as some
other relevant receptors in the central nervous system. Recently
we have shown that the substituent in position 1 of spirocyclic
pyrazoles 1 has a considerable influence on the s1 affinity. The
spirocyclic pyrazole 1b bearing a phenyl moiety in position 1 (Ki =
1.5 nM) is almost 15-fold more active than the corresponding
methyl derivative 1a (Ki = 21 nM). (Fig. 1) We assume that
the phenyl substituent of 1b is able to occupy an additional
hydrophobic pocket of the s1 receptor protein.15
Bioisosteric replacement of the pyrazole ring by a thiophene
ring led to the very potent s1 ligand 2 (Ki = 0.32 nM).16 The
combination of the high affinity thiophene substructure with an
additional aryl substituent as shown for the pyrazole 1b should
lead to a new compound class of s1 ligands with promising activity
and selectivity profile. For this purpose regioisomeric thiophene
derivatives 3 and 4 with various aryl substituents in position 1 were
designed. Aryl substituents with different electronic and steric
properties should allow exploring the character and the size of the
complementary hydrophobic binding pocket of the s1 receptor.
phencyclidine and haloperidol sensitive receptor family with an
unique binding profile and a characteristic distribution in the
central nervous system as well as in endocrine, immune and some
peripheral tissues like kidney, liver, lung and heart. At least two s
receptor subtypes have been identified thus far, and are termed s1
and s2 receptor.1–3
Modulation of s1 receptor activity offers some potential for
the treatment of acute and chronic neurological disorders, like
schizophrenia, depression, Alzheimer’s and Parkinson’s disease,
(neuropathic) pain as well as alcohol and cocaine abuse.4–7
Furthermore, some human tumor cell lines are able to express
a large number of s1 and/or s2 receptors. Consequently s1 (and
s2) ligands may be used for diagnosis and therapy of cancer.8,9
Despite enormous efforts of research, the signal transduction
pathway after activation of s1 receptors is not completely under-
aInstitut fu¨r Pharmazeutische und Medizinische Chemie der Westfa¨lischen
Wilhelms-Universita¨t Mu¨nster, Hittorfstraße 58-62, D-48149, Mu¨nster,
Germany. E-mail: wuensch@uni-muenster.de; Fax: +49-251-8332144;
Tel: +49-251-8333311
bOrganisch-chemisches Institut der Westfa¨lischen Wilhelms-Universita¨t
Mu¨nster, Correnstraße 40, D-48149, Mu¨nster, Germany
cDepartment of Chemistry, Graduate School of Science, Nagoya Uni-
versity, Chikusa-ku, Nagoya, 464-8602, Japan. E-mail: itami.kenichiro@
a.mbox.nagoya-u.ac.jp; Fax: +81-52-788-6098; Tel: +81-52-788-6098
8016 | Org. Biomol. Chem., 2011, 9, 8016–8029
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the determination of the relative energies the SCS-MP2-method
of Grimme26 using the 6-311+G(d,p) basis set was applied (SCS-
MP2/6-311+G(d,p)//B3LYP/6-31G(d) using the GAUSSIAN
0927 package of programs. The relative energies discussed in the
following section include DFT zero point correction.
Among the various conformations and configurations studied,
isomer 4a(I) with the thiophene moiety in the axial position and
the benzyl group in the equatorial orientation of the piperidine
ring in a chair conformation was found to be the lowest-energy
conformer. Isomer 4a(II) bearing both the benzyl group and the
thiophene moiety in equatorial orientation is calculated to be
slightly higher in energy by 0.88 kcal mol-1, whereas the isomers
4a(III) and 4a(IV) with the benzyl group in axial orientation
at N1 are significantly higher in energy by 2.55 and 2.60 kcal
mol-1, respectively. 4a(III) bears the thiophene substructure (C4) in
equatorial orientation, whereas 4a(IV) has the thiophene unit (C4)
in axial orientation of the piperidine ring. As expected, isomers
featuring twist-piperidine conformations are considerably higher
in energy.
Fig. 1 Design of thiophene-based s1 receptor ligands with additional aryl
substituent in position 1.
As described before, Langer et al. generated a 3D computer
model based on the most potent s1 ligands known at the time.
The Langer model was used as a platform to design the structure
of novel s1 ligands. The hydrophobic features of the model are
represented by the thiophene system, the phenyl residue and the
N-benzyl moiety of 4a. The central basic amine is consistent with
the positive ionizable feature in the model. (Fig. 2)
In order to apply the model proposed by Langer for comparison
of 4a with literature data, the distances between the piperidine
nitrogen atom to the center of the phenyl ring of the benzyl moiety,
to the center of the thiophene ring and to the center of the phenyl
ring adjacent to the thiophene were determined.
2. Pharmacophore models of r1 ligands and DFT
calculations of preferred conformations
Various pharmacophore models for s1 receptor ligands have been
reported in literature.17,18 The common features of these models
are a basic amino group and at least two hydrophobic regions
in defined distances to the basic amino group. According to the
˚
model of Glennon the distances are defined as 2.5–3.9 A and 6–
10 A, respectively.19,20 The 3D computer-based s1 pharmacophore
˚
model of Langer et al. postulates four hydrophobic groups and
a positive ionizable feature in definite spatial arrangements and
distances (Fig. 2, top).21 The postulated distances between the
basic amine and three hydrophobic groups are calculated as
The distance 1 (N-Benzyl) is slightly larger in case of equatorial
orientation of the benzyl group (3.765–3.766 A) than in the case of
˚
˚
axial orientation (3.711–3.759 A). The distances 2 and 3 are also
greater in the case of equatorial orientation of the thiophene ring
˚
˚
˚
˚
4.1 A, 6.3 A and 9.8 A and are in good accordance with the
distances in the Glennon model. In the model of Zampieri et al.
five pharmacophoric features are described: one basic amine, two
hydrophobic aromatic groups, one hydrophobic group and an
additional H-bond acceptor.22 The corresponding distances of
(distance 2/N-thiophene: 5.522–5.592 A, distance 3/N-Phenyl:
˚
9.450–9.502 A) than in case of axial orientation (distance 2/N-
˚
˚
thiophene: 4.946–4.947 A, distance 3/N-Phenyl: 9.083–9.093 A).
As an example, the energetically most favorable conformer 4a(I)
with axial orientation of the thiophene moiety and equatorial
orientation of the N-benzyl group is mapped onto the model. The
calculated distances indicate that 4a(I) is in good accordance with
the pharmacophore model of Langer since distance 1 (N-Benzyl:
˚
˚
˚
3.58 A as well as 7.01 A and 8.50 A correlate nicely with the
distances postulated in the Glennon and Langer models.
In order to define the corresponding distances within the
designed spirocyclic thiophene derivative 4a, a conformational
analysis using molecular dynamics simulation was performed
(PCMODEL, option “dynam” (leapfrog Verlet algorithm)).23 The
geometry optimization led to two main conformations, one with
the thienyl moiety in the axial orientation with respect to the
central piperidine ring, and the second one with the thienyl ring
in the equatorial position. The calculated energies of both con-
formations are almost identical. Consequently both conformers
were taken into account to calculate the intramolecular distances.
Furthermore, conformers with an axially arranged benzyl moiety
at the basic piperidine nitrogen atom were also found to be within
an acceptable energy range and therefore were also considered.
To gain further insights into the structure and energy differences
of various conformations of compound 4a, theoreticalcalculations
were performed on several conformers of 4a. (Fig. 2) For geometry
optimization the DFT method B3LYP/6-31G(d)24,25 was used. For
˚
˚
3.765 A), distance 2 (N-Thiophene: 4.946 A) and distance 3 (N-
Phenyl: 9.083 A) agree well with the postulated distances in the
˚
model.
3. Chemistry
In order to synthesize spirocyclic thiophenes 3 and 4 with a large
variety of aryl substituents in position 1, the building blocks 5 and
6 should be prepared and arylated regioselectively in a-position
(Fig. 3). The direct introduction of an aryl moiety in the last step
of synthesis would allow the generation of a diverse set of aryl
substituted spirocyclic s1 receptor ligands.
The synthesis of novel spirocyclic thiophene derivatives 3 and
4 started with commercially available 3,4-dibromothiophene (7).
(Scheme 1) Halogen-metal exchange with n-BuLi and subsequent
trapping of the resulting thienyllithium intermediate with DMF
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Fig. 2 Top: Pharmacophore model according to Langer et al.21 Bottom: Calculated distances of pharmacophoric elements and relative energies of four
energetically favored conformers of spirocyclic thiophene 4a (SCS-MP2/6-311+G(d,p)//B3LYP/6-31G(d), energies in [kcal mol-1]). 4a(I): thiophene
moiety in axial and benzyl group in equatorial orientation; 4a(II): both thiophene moiety and benzyl group in equatorial orientation; 4a(III): thiophene
moiety in equatorial and benzyl group in axial orientation; 4a(IV): both thiophene moiety and benzyl group in axial orientation. * B3LYP/6-31G(d)+ZPE
[kcal mol-1]. ** SCS-MP2/6-311G(d,p)//B3LYP/6-31G(d)+ZPE [kcal mol-1].
regioisomeric hydroxy acetals 11 and 12. The formation of
undesired regioisomer 11 could be explained by intermolecular
deprotonation of the thienyllithium intermediate to generate
an anion at the thermodynamically more sTable 2-position.
The ratio of regioisomers was considerably influenced by the
reaction temperature. At -78 ◦C the ratio of 11/12 was 20 : 80,
whereas increasing the reaction temperature to -50 ◦C led to
a decreased ratio of 40 : 60. Finally at -100 ◦C the ratio of
11/12 remained at 20 : 80, but the experimental procedure was
Fig. 3 Synthesis strategy for thiophene-based s1 ligands with additional
aryl moiety in position 1.
less convenient. After isolation by flash chromatography, hydroxy
acetal 12 was cyclized with p-toluenesulfonic acid in methanol to
provide the spirocyclic piperidine 6 in 43% yield over two reaction
steps.
Cyclic hemiacetal 13 was obtained by cyclization of hydroxy
acetal 12 with diluted HCl. (Scheme 2) Treatment of 13 with
methanesulfonyl chloride and triethylamine in CH2Cl2 afforded
the enol ether 14 in 51% yield. Subsequently, 14 was reduced with
H2 in the presence of Pd/C to provide the spirocyclic thienopyran
5 in 42% yield.
afforded thiophenecarbaldehyde 8 in 84% yield.28 Homologa-
+
tion of the resulting aldehyde 8 with Ph3PCH2OCH3 Cl- and
KOtBu16,29 led to a mixture of (E)- and (Z)-configured enol ethers
9, which was then converted into the dimethyl acetal 10 upon
reaction with methanol.
A second halogen-metal exchange with n-BuLi at -78 ◦C in
THF followed by addition to 1-benzylpiperidin-4-one afforded
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Table 1 Yields after a-arylation of the non-acetalic spirocyclic thiophene
5 with various iodoarenes
Compd.
Aryl
Yield (gpc)a
Yield (ptlc)b
3a
3b
3c
3d
3e
3f
C6H5
54%
65%
49%
48%
59%
67%
35%
32%
34%
11%
49%
35%
p-MeOC6H4
p-MeC6H4
p-NO2C6H4
p-CNC6H4
1-naphthyl
Scheme 1 Synthesis of the spirocyclic thiophene derivative 6. Reagents
and conditions: (a) n-BuLi, Et2O, -78 ◦C, 15 min, then DMF, -78 ◦C,
3 h, 84%. (b) Ph3PCH2OCH3 Cl-, KOtBu, Et2O, -20 ◦C, 2 h, 90%. (c)
+
a gpc: yields after gel permeation chromatography; b ptlc: yields after
preparative thin layer chromatography.
CH3OH, p-TosOH, 65 ◦C, 18 h, 90%. (d) n-BuLi, THF, -78 ◦C, 15 min, then
1-benzylpiperidin-4-one, -78 ◦C, 3 h, 53% (12), 12% (11). (e) p-TosOH,
CH3OH, rt, 24 h, 51%.
selective arylation have also been established recently.38–40 In view
of broad substrate scope, high a-regioselectivity, and operational
simplicity, we decided to apply one of our catalytic systems,
PdCl2/2,2¢-bipyridyl/Ag2CO3,38,41 to the arylation of spirocyclic
thiophenes 5 and 6. In particular electron-rich and electron-poor
aryl moieties were taken into account, which is of great interest
both for synthetic and pharmacological features.
At first the spirocyclic system 5 without an alkoxy sub-
stituent at the pyran moiety was reacted with iodobenzene,
PdCl2, 2,2¢-bipyridyl and Ag2CO3 in boiling m-xylene (150 ◦C)
to provide the phenylated product 3a in 54% yield (Table 1).
Similar yields were obtained when electron-rich iodoarenes (e.g.
1-iodo-4-methoxybenzene, → 3b), electron-deficient iodoarenes
(e.g. p-iodobenzonitrile, → 3e), and extended p-systems (e.g. 1-
iodonaphthalene, → 3f) were used. These results clearly indicate
that the applied reaction conditions are compatible with the
tertiary amine of the piperidine ring and the ether moiety of the
pyran system.
The a-arylation of spirocyclic thiophene 6 was expected to
be more problematic, due to the additional methoxy substituent
in position 6 of the pyran ring (acetal). However, treatment
of 6 with iodobenzene, PdCl2, 2,2¢-bipyridyl and Ag2CO3 in
m-xylene led to the arylated product 4a in even higher yields
(67%) (Table 2). As demonstrated for the unsubstituted spirocyclic
system 5, the acetalic compound 6 reacted with electron-rich
and electron-deficient iodoarenes as well as with extended p-
systems to produce the arylated compounds 4a–4j in 27–79%
yields. These transformations represent the first examples of
the a-arylation of highly functionalized thiophenes using the
PdCl2/2,2¢-bipyridyl/Ag2CO3 method. The tolerance of the acid-
labile acetal substructure of 6 is of particular importance.
All compounds were purified by gel permeation chromatogra-
phy (gpc) to isolate the arylated products. In order to use pure com-
pounds in the receptor binding studies, these arylated products
were further purified by preparative thin layer chromatography
(ptlc) before testing.
Scheme 2 Synthesis of spirocyclic thiophene derivatives without acetalic
substructure. Reagents and conditions: (a) HCl, H2O, THF, rt, 18 h, 76%.
(b) MeSO2Cl, NEt3, CH2Cl2, rt, 3 h, 51%. (c) H2, balloon, Pd/C, CH3OH,
rt, 2 h, 42%.
With the requisite core spirocyclic thiophene structures (5 and
6) in hand, we questioned whether we should conduct a lengthy
but reliable cross-coupling technology or search for a direct C–H
bond functionalization methodology for the introduction of aryl
groups onto the thiophene core of 5 and 6. The Pd-catalyzed
cross-coupling reactions of metalated arene/heteroarene and
halogenated arene/heteroarene species as exemplified by Suzuki–
Miyaura coupling are undoubtedly among the most reliable
methods for making biaryls and heterobiaryls,30 the C–H bond
arylation of arenes and heteroarenes holds significant potential for
streamlining overall synthetic routes.31 Indeed, the quest for such
methods has been the driving force behind enormous efforts in the
synthetic community including our group, culminating in a wealth
of useful catalytic systems for reactions assembling arenes through
C–H bond functionalization. Thus, we envisaged that direct C–H
bond arylation of thiophenes 5 and 6 would allow us to access
the target spirocyclic thiophenes 3 and 4 in a step-economical and
late-stage-diversifying manner.
The palladium-catalyzed direct C–H bond arylation of thio-
phenes with haloarenes was first discovered by the group of Ohta
in 1990.32 After this pioneering work, a number of research groups
reported various catalysts (Pd,33 Rh,34,35 Ir,36 Cu37) promoting the
C–H bond arylation of thiophenes with haloarenes. Although
these arylation reactions typically occur regioselectively at the a-
position of thiophene ring, several unique catalysts promoting b-
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compound compete with the potent and selective radioligand [3H]-
(+)-pentazocine for s1 receptors in a membrane preparation of
guinea pig brains. Non-specific binding was determined in the
presence of a large excess of non-tritiated (+)-pentazocine.42–45
Membrane preparations of guinea pig brains represent the stan-
dard source for s1 receptors in the s1 assay. The resulting Ki-
values are rather reliable, since s1 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.46 In the s2
assay homogenates of rat liver served as the source for s2 receptors
and [3H]-di-o-tolylguanidine was employed as radioligand. Due
to the low s receptor selectivity of di-o-tolylguanidine, an excess
amount of non-radiolabeled (+)-pentazocine was added to mask
s1 receptors. A large excess of non-tritiated di-o-tolylguanidine
was used for the determination of non-specific binding.42–45
In Table 3 the s1 and s2 receptor affinities of the synthesized
compounds are summarized. The s1 receptor affinities of the non-
arylated regioisomeric thiophenes 5 (Ki = 0.35 nM) and 6 (Ki =
0.22 nM) are in the same range as the s1 receptor affinity of the
lead compound 2 (Ki = 0.32 nM). Although introduction of a
phenyl moiety in position 1 led to a slightly reduced s1 affinity,
the resulting compounds 3a (Ki = 4.5 nM) and 4a (Ki = 1.0 nM)
still represent very potent s1 ligands. Obviously, the s1 receptor
tolerates a lipophilic phenyl moiety in position 1 of the thiophene
system. In contrast to the pyrazole series of compounds 1, the s1
affinity is not increased by the additional phenyl moiety. A reason
might be that the s1 affinity of the parent compounds 5 and 6 is
already very high.
Table 2 Yields after a-arylation of the acetalic spirocyclic thiophene 6
with various iodoarenes
Compd.
Aryl
Yield (gpc)a
yield (ptlc)b
4a
4b
4c
4d
4e
4f
C6H5
67%
52%
42%
80%
48%
47%
38%
45%
60%
40%
19%
46%
18%
38%
p-MeOC6H4
p-MeC6H4
p-NO2C6H4
p-AcC6H4
p-CNC6H4
p-CF3C6H4
1-naphthyl
3-pyridyl
50%
38%
72%
27%
39%
4g
4h
4i
4j
p-biphenyl
a gpc: yields after gel permeation chromatography; b ptlc: yields after
preparative thin layer chromatography.
4. Receptor Affinity
The s1 and s2 receptor affinities of the spirocyclic thienopyran
derivatives 3 and 4 as well as the corresponding synthetic precur-
sors 5,6,13 and 14 were determined in competition experiments
with radioligands. In the s1 assay different amounts of the test
In both series of compounds introduction of an electron rich
or electron poor substituent into the phenyl moiety has only
little influence on the s1 affinity. In the series of compounds
Table 3 s1 and s2 receptor affinities of the synthesized spirocyclic thiophenes and reference compounds
Ki SEM [nM] (n = 3)
s1
Selectivity
Compd.
X
Aryl
s2
s1/s2
1a
OCH3
OCH3
OCH3
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
CH3
C6H5
H
C6H5
21 2.3
> 1 mM
> 1 mM
> 1 mM
> 1 mM
926
1.6 mM
> 1 mM
> 1 mM
51
> 47
> 660
> 3125
> 222
617
1b
1.5 0.08
0.32 0.10
4.5 2.9
1.5 0.54
3.6 0.40
1.7 0.79
3.4 0.90
4.0 1.9
1.0 0.40
2.2 0.13
2.0 0.81
1.0 0.16
1.6 0.86
0.25 0.14
5.7 2.3
5.0 0.50
2.2 0.42
30 18
2
3a
3b
p-MeOC6H4
p-MeC6H4
p-NO2C6H4
p-CNC6H4
1-naphthyl
C6H5
p-MeOC6H4
p-MeC6H4
p-NO2C6H4
p-AcC6H4
p-CNC6H4
p-CF3C6H4
1-naphthyl
3-pyridyl
p-biphenyl
H
3c
444
3d
> 588
> 294
13
> 1000
341
> 500
> 1000
> 625
3692
> 175
420
> 450
> 33
657
3e
3f
4a
>1 mM
751
4b
4c
> 1 mM
> 1 mM
> 1 mM
923
>1 mM
2.1 mM
> 1 mM
> 1 mM
230
806
266
84.6 25.4
78 2.0
42 15
4d
4e
4f
4g
4h
4i
4j
5
6
13
14
0.35 0.06
0.22 0.06
3.2 0.41
1.9 0.66
3.9 1.5
OCH3
OH
H
H
H
3664
83
45
20
0.7
HC3 C4H
haloperidol
di-o-tolylguanidine
61
8
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3, the methoxyphenyl derivative 3b is slightly more potent than
the unsubstituted phenyl derivative 3a, whereas in the methoxy
substituted series 4 an opposite tendency is observed. In both
series, extension of the p-system to a 1-naphthyl system (3f,4h)
does not reduce s1 affinity significantly. However, extension of
the phenyl substituent to a 4-phenylphenyl (biphenylyl) residue
led to 4j with the lowest s1 affinity (Ki = 30 nM) within this
compound class. The p-cyanophenyl substituted derivative 4f (Ki =
0.25 nM) shows the same s1 affinity as the non-arylated parent
compound 6 and represents the most potent s1 ligand of this series.
We assume that the s1 receptor protein accepts both electron-rich
and electron-poor phenyl moieties in position 1 of the thiophene
system. Moreover, the size of the aromatic system in position 1
may be expanded to a naphthyl residue without loss of s1 affinity,
but not to a biphenylyl residue.
The similar s1 affinities of non-arylated and arylated spirocyclic
thiophenes are surprising. We assume that there is a hydrophobic
pocket within the s1 receptor protein which is not addressed
by the small non-arylated derivatives. However, an additional
aryl moiety can open the hydrophobic pocket and allow further
lipophilic interactions. These additional interactions of the aryl
moiety do only modulate the ligand - s1 receptor interac-
tions, which are mainly determined by the residual spirocyclic
framework.
DFT-calculations demonstrated that the spirocyclic thiophene
4a fits well onto the s1 pharmacophore model described by
Langer.
6. Experimental
6.1. Chemistry
6.1.1. General. Unless otherwise noted, moisture and oxy-
gen sensitive reactions were conducted in dry glassware (Schlenk
flask sealed with a rubber septum) under N2 (dried with phospho-
R
rous pentoxide (Granusicꢀ A, Baker)). THF and Et2O were dried
with sodium/benzophenone and were freshly distilled before use.
Methanol was dried with magnesium and iodine and was used
freshly distilled. DMF was dried with CaH2, filtered, distilled and
˚
stored over molecular sieves 3 A. The concentration of n-BuLi
was determined by titration with 1,3-diphenyl-2-propanone p-
toluenesulfonylhydrazone in THF under N2-atmosphere.47 Thin
layer chromatography (tlc): Silica gel 60 F254 plates (Merck).
Flash chromatography (fc): Silica gel 60, 40–64 mm (Merck);
parentheses include: diameter of the column [cm], length of
the stationary phase [cm], eluent, fraction size [mL] and re-
tention factor Rf. Gel permeation chromatography (gpc): LC-
9204 instrument (JAI) with JAIGEL-1H/JAIGEL-2H columns,
eluent CHCl3; preparative thin layer chromatography (prep. tlc):
The s2 receptor affinities of all investigated compounds are con-
siderably lower than their s1 affinities indicating high selectivity
against the s2 subtype. As a general rule, the methoxy group in
position 6 (compound series 4) leads to reduced s2 affinity and thus
increased s1 : s2 selectivity compared with compounds without a
substituent in position 6 (compare compounds 4f (s1 : s2 = 3692)
with 3e (s1 : s2 = > 294) and 6 (s1 : s2 = 3664) with 5 (s1 : s2 =
657) and 14 (s1 : s2 = 45)). Introduction of an aryl moiety with an
increased p-system in position 1 (1-naphthyl, biphenylyl residue)
results in reduced s1 : s2 selectivity. The naphthyl substituted
compound 3f without a substituent in position 6 shows the lowest
s1 : s2 selectivity (13) of this series of compounds. A polar OH-
moiety in position 6 (compound 13) also favors the interaction
with s2 receptors.
R
Wako-gelꢀ B5-F silica coated plates (0.75 mm) prepared in the
Itami laboratory; IR: IR spectrophotometer 480Plus FT-ATR-
IR (Jasco). H NMR (400 MHz, 300 MHz, 600 MHz) and 13C
NMR (100 MHz) spectra 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
1
◦
Varian Unity Plus 600 (600 MHz) operating at 23 C. Chemical
shifts d are reported in parts per million (ppm) against the
reference compound tetramethylsilane and calculated using the
chemical shift of the signal of the residual non-deuterated solvent.
Data are reported as follows: chemical shift, multiplicity (s =
singlet, d = doublet, dd = doublet of doublets, t = triplet, q =
quartet, m = multiplet), coupling constant (Hz) and integration.
MS: HRMS (ESI): Finnigan MAT 4200 s, Brucker Daltonics
Micro Tof and Waters Micromass Quatro LCZ, peaks are given in
m/z (% of basis peak). EI, electron impact, MAT GCQ (Thermo-
Finnigan); HRMS: JMS-T100TD instrument (DART); HPLC:
Merck Hitachi Equipment; UV detector: L-7400; autosampler:L-
5. Conclusion
In summary, the regioselective a-arylation of the thiophene
derivatives 5 and 6 allows the late-stage introduction of diverse
aromatic substituents into the spirocyclic s1 ligands without
previous activation of the C–H bond. The phenylated thiophenes
3a and 4a show a slightly reduced s1 receptor affinity compared
to the extraordinarily potent, non-arylated spirocyclic thiophenes
5 and 6. Nevertheless, both phenylated compounds 3a and 4a
interact in the low nanomolar range with s1 receptors (3a: Ki =
4.5 nM, 4a: Ki = 1.0 nM). The substitution pattern of the aryl
residue does not considerably influence the s1 affinity. Whereas
the larger naphthyl residue was also well accepted by the s1
receptor protein, the biphenylyl substituted compound 4j showed
reduced s1 affinity. With exception of naphthyl derivative 3f all
compounds have very high selectivity against the s2 subtype. The
most promising compounds of this series are the non-arylated
compound 6 (Ki = 0.22 nM) and the p-cyanophenyl substituted
compound 4f (Ki = 0.25 nM), possessing subnanomolar s1 affini-
ties. Furthermore, molecular modelling investigations including
R
7200; pump: L-7100; degasser: L-7614; column: LiChrospherꢀ
60 RP-select B (5 mm); LiChroCARTꢀ 250-4 mm cartridge; flow
R
rate: 1.000 mL min-1; injection volume: 5.0 mL; detection at l =
210 nm; solvents: A: water with 0.05% (v/v) trifluoroacetic acid; B:
acetonitrile with 0.05% (v/v) trifluoroacetic acid: gradient elution:
(A%): 0–4 min: 90%, 4–29 min: gradient from 90%,to 0%, 29–31
min: 0%, 31–31.5 min: 0% to 90%, 31.5–40 min: 90%. The purity
of test compounds was greater than 95%, which was determined
by the given HPLC method.
6.1.2. 4-Bromothiophene-3-carbaldehyde (8)28. Under N2
3,4-dibromothiophene (7, 22.2 g, 92 mmol) was dissolved in dry
Et2O (35 mL) and cooled down to -78 ◦C. After stirring the
◦
mixture at -78 C for 10 min a solution of n-butyllithium in n-
hexane (1.6 M, 57.2 mL, 92 mmol) was added dropwise and the
mixture was stirred for another 15 min. The resulting aryllithium
intermediate was subsequently trapped with freshly distilled DMF
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(7.08 mL, 92 mmol) and the mixture was stirred for 3 h at -78 ◦C.
The solution was warmed up to rt, water (40 mL) was added,
the organic layer was separated, the aqueous layer was extracted
twice with Et2O, the combined organic layers were dried (K2CO3),
concentrated in vacuo and the residue was purified by high vacuum
6.1.5. 1-Benzyl-4-[3-(2,2-dimethoxyethyl)thiophen-2-yl]piperi-
din-4-ol (11) and 1-Benzyl-4-[4-(2,2-dimethoxyethyl)thiophen-3-
yl]piperidin-4-ol (12). Under N2 and at -78 ◦C n-BuLi in n-
hexane (1.26 M, 2.74 mL, 3.44 mmol) was added slowly to a
stirred solution of bromoacetal 10 (0.67 g, 2.65 mmol) in dry
THF (15 mL). The mixture was stirred for 15 min at -78 ◦C.
Then 1-benzylpiperidin-4-one (0.57 mL, 3.2 mmol) was added
◦
distillation. Pale yellow oil, bp 41 C (9 ¥ 10-3 mbar), yield 14.8
g (84%). C5H3BrOS (191.0 g mol-1). MS (EI): m/z = 189 [79BrM+
◦
- H], 191 [81BrM+ - H]. IR (neat): v (cm-1) = 3103 (C–Haryl), 2848
slowly. After stirring at -78 C for 3 h, the mixture was warmed
1
(C–H), 1683 (C O). H NMR (CDCl3): d (ppm) = 7.37 (d, J =
to rt and water was added. The aqueous layer was separated and
extracted twice with CH2Cl2. The combined organic layers were
dried (K2CO3), filtered and the solvent was removed in vacuo.
The regioisomeric alcohols were separated by fc (5 cm, 15 cm,
cyclohexane : EtOAc = 8 : 2, 20 mL).
11 (Rf 0.35, cyclohexane : EtOAc = 3 : 7): Pale yellow oil, yield
0.11 g (12%). C20H27NO3S (361.5 g mol-1). MS (EI): m/z = 361
[M+], 328 [M+ - HOCH3, - H], 298 [M+ - OCH3 - HOCH3],
91 [+CH2Ph]. IR (neat): v (cm-1) = 3430 (O–H), 3026 (C–Haryl),
2934 (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,
3-H-thioph), 7.09 (d, J = 5.1 Hz, 1H, 2-H-thioph), 7.29–7.37 (m,
5H, Ph-H).
3.4 Hz, 1H, 5-H-thioph), 8.16 (d, J = 3.5 Hz, 1H, 2-H-thioph),
9.95 (s, 1H, CH O).
6.1.3. (E)- and (Z)-3-Bromo-4-(2-methoxyvinyl)thiophene (9).
Under N2 dried MeOCH2PPh3 Cl- (42.6 g, 120 mmol) was
+
suspended in dry Et2O (200 mL) and the mixture was cooled
down to -20 ◦C. KOtBu (13.9 g, 120 mmol) was dissolved in dry
THF (77 mL) to give a 1.6 M solution. This solution was added
dropwise to the stirred suspension◦of the Wittig reagent so that the
temperature was kept below -10 C and the mixture was stirred
for 30 min after complete addition of KOtBu. Next the aldehyde
8 (14.8 g, 80 mmol) was dissolved in dry Et2O (3–5 mL) and
the solution was added slowly to the suspension. The mixture was
stirred for 2 h at -20 ◦C and overnight at rt. After addition of water
the aqueous layer was extracted twice with Et2O. The combined
organic layers were dried (K2CO3), concentrated in vacuo and the
side product Ph3PO was removed by multiple recrystallization. The
remaining◦oil was purified by high vacuum distillation. Yellow oil,
bp 58–62 C (1.2 ¥ 10-2 mbar), yield 15.8 g (90%). C7H7BrOS
(219.1 g mol-1). MS (EI): m/z = 218 [79BrM+], 220 [91BrM+], 175
[79BrMH+ - CH(OCH3)], 177 [81BrMH+ - CH(OCH3)]. IR (neat):
v (cm-1) = 3110 (C–Haryl), 2831 (OCH3), 1639 (C C), 1216 (C–
12 (Rf 0.22, cyclohexane : EtOAc = 3 : 7): Pale yellow oil, yield
0.50 g (53%). C20H27NO3S (361.5 g mol-1). MS (EI): m/z = 361
[M+], 330 [M+ - OCH3], 298 [M+ - OCH3 - HOCH3], 91 [+CH2Ph].
IR (neat): v (cm-1) = 3442 (O–H), 3027 (C–Haryl), 2926 (C–H), 1119
1
(C–O), 698 (C–H). H 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), 7.07 (m, 2H, 2-H-thioph,
5-H-thioph), 7.26–7.37 (m, 5H, Ph-H).
1
O), 1098 (C–O). H NMR (CDCl3): d (ppm) = 3.69 (s, 3·0.6H,
OCH3, (E)-isomer), 3.80 (s, 3·0.4H, OCH3, (Z)-isomer), 5.44 (d,
J = 6.8 Hz, 0.4H, thiophCH CH, (Z)-isomer), 5.75 (d, J =
13.0 Hz, 0.6H, thiophCH CH, (E)-isomer), 6.24 (d, J = 6.8
Hz, 0.4H, thiophCH CH, (Z)-isomer), 6.97 (d, J = 12.9 Hz,
0.6H, thiophCH CH, (E)-isomer), 7.01 (d, J = 3.4 Hz, 0.6H,
5-H-thioph, (E)-isomer), 7.18 (d, J = 3.5 Hz, 0.4H, 5-H-thioph,
(Z)-isomer) 7.23 (d, J = 3.4 Hz, 0.6H, 2-H-thioph, (E)-isomer),
7.71 (d, J = 3.5 Hz, 0.4H, 2-H-thioph, (Z)-isomer). The ratio of
(E)-9 and (Z)-9 is 6 : 4.
6.1.6. 1-Benzyl-6¢-methoxy-6¢,7¢-dihydrospiro[piperidine-4,4¢-
thieno[3,4-c]pyran] (6). Hydroxy acetal 12 (421 mg, 1.16 mmol)
was dissolved in dry MeOH (25 mL) and p-toluenesulfonic acid
(251 mg, 1.45 mmol) was added. The solution was stirred for
24 h at rt. Then 2 M NaOH was added until pH 8 and the
solution was diluted with water (20 mL). The aqueous layer was
separated and extracted twice with CH2Cl2. The combined organic
layers were dried (K2CO3), filtered and the solvent was removed
in vacuo. The remaining oil was purified by fc (2.5 cm, 15 cm,
cyclohexane : EtOAc = 3 : 2, 20 mL, Rf 0.22). Pale yellow oil,
yield 194 mg (51%). C19H23NO2S (329.5 g mol-1). Purity (HPLC):
6.1.4. 2-(4-Bromothiophen-3-yl)acetaldehyde dimethyl acetal
(10). Enol ether 9 (360 mg, 1.62 mmol) was dissolved in dry
MeOH (30 mL) and catalytic amounts of p-toluenesulfonic acid
were added. The mixture was stirred overnight under reflux.
After cooling down to rt CH2Cl2 (100 mL) was added and the
solution was alkalized with 2 M NaOH. The aqueous layer
was extracted twice with CH2Cl2. The combined organic layers
were dried (K2CO3), filtered and purified by fc (3 cm, 15 cm,
cyclohexane : EtOAc = 19 : 1, 20 mL, Rf 0.22). Colorless oil, yield
369 mg (90%). C8H11BrO2S (251.1 g mol-1). MS (EI): m/z =
98.5%, tr = 16.8 min. MS (EI): m/z = 329 [M+], 314 [M+
-
CH3], 298 [M+ - OCH3], 238 [M+ - CH2Ph], 91 [+CH2Ph]. IR
(neat): v (cm-1) = 3026 (C–Haryl), 2921, 2809 (C–H), 1067 (C–
1
O), 698 (C–H). H NMR (CDCl3): d (ppm) = 1.82–1.98 (m, 2H,
219 [79BrM+ - OCH3], 221 [81BrM+ - OCH3], 175 [79BrM+
-
N(CH2CH2)2), 2.02–2.16 (m, 2H, N(CH2CH2)2), 2.44 (td, J =
12.8/2.4 Hz, 1H, N(CH2CH2)2), 2.55 (td, J = 12.0/2.6 Hz, 1H,
N(CH2CH2)2), 2.72–2.82 (m, 3H, thiophCH2CH, N(CH2CH2)2),
2.96 (dd, J = 15.3/3.1 Hz, 1H, thiophCH2CH), 3.53 (s, 3H,
OCH3), 3.57 (d, J = 13.0 Hz, 1H, NCH2Ph), 3.61 (d, J = 13.6
Hz, 1H, NCH2Ph), 4.84 (dd, J = 7.4/3.1 Hz, 1H, thiophCH2CH),
6.91 (d, J = 3.0 Hz, 1H, 1-H-thioph), 6.96 (d, J = 3.0 Hz, 1H,
CH(OCH3)2], 177 [81BrM+ - CH(OCH3)2], 140 [M+ - Br, - OCH3].
IR (neat): v (cm-1) = 3107 (C–Haryl), 2830 (C–O), 1059 (Caryl–Br). 1H
NMR (CDCl3): d (ppm) = 2.93 (d, J = 5.6 Hz, 2H, thiophCH2CH),
3.36 (s, 6H, CH(OCH3)2), 4.61 (t, J = 5.6 Hz, 1H, thiophCH2CH),
7.16 (d, J = 3.4 Hz, 1H, 2-H-thioph), 7.24 (d, J = 3.4 Hz, 1H,
5-H-thioph).
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3-H-thioph), 7.27–7.39 (m, 5H, Ph-H). 13C NMR (CDCl3): d
(ppm) = 32.8 (1C, thiophCH2CH), 37.7 (1C, N(CH2CH2)2),
40.0 (1C, N(CH2CH2)2), 49.4 (1C, N(CH2CH2)2), 49.4 (1C,
N(CH2CH2)2), 56.6 (1C, OCH3), 63.5 (1C, NCH2Ph), 74.3 (1C,
thiophCspiro), 97.4 (1C, thiophCH2CH), 118.5 (1C, C-1¢-thioph),
120.0 (1C, C-3¢-thioph), 127.3 (Ph-CH), 128.5 (Ph-CH), 129.6
(Ph-CH), 133.5 (1C, Cquart), 138.5 (1C, Cquart), 143.4 (1C, Cquart).
(2C, Ph-C), 129.5 (2C, Ph-C), 131.2 (1C, Cquart), 138.2 (1C, Cquart),
139.9 (1C, Cquart), 142.4 (1C, thiophCH CH). The solvent signals
probably overlap with the quaternary Cspiro signal.
6.1.9. 1-Benzyl-6¢,7¢-dihydrospiro[piperidine-4,4¢-thieno[3,4-c]-
pyran] (5). The enol ether 14 (91.7 mg, 0.31 mmol) was dissolved
in dry MeOH (10 mL) and 10% Pd/C (50 mg) was added.
The suspension was stirred under H2 atmosphere (balloon) for
2 h. Afterwards the catalyst was filtered off and the remaining
residue was washed with 2 M HCl and with water. The filtrate
was alkalized with 2 M NaOH and extracted twice with CH2Cl2.
The combined organic layers were dried over K2CO3, the solvent
was removed in vacuo and the crude product was purified by fc
(2.5 cm, 15 cm, cylcohexane : EtOAc = 7 : 3, 15 mL, Rf 0.25).
Colorless oil, yield 38.4 mg (42%). C18H21NOS (299.4 g mol-1).
Purity (HPLC): 97.9%, tr = 17.5 min. MS (EI): m/z = 299 [M+],
208 [M+ - CH2Ph], 91 [+CH2Ph]. IR (neat): v (cm-1) = 3028 (C–
6.1.7. 1-Benzyl-6¢,7¢-dihydrospiro[piperidine-4,4¢-thieno[3,4-
c]pyran]-6¢-ol (13). Hydroxy acetal 12 (1.06 g, 2.94 mmol) was
dissolved in THF (5 mL) and hydrochloric acid (1 M, 30 mL,
30 mmol) was added. The mixture was stirred at rt overnight.
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 (K2CO3), filtered and the
solvent was removed in vacuo. The remaining resin was purified
by fc (6 cm, 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-1). Purity (HPLC): 97.3%, tr = 14.0 min. MS (EI):
m/z = 315 [M+], 224 [M+ - CH2Ph], 91 [+CH2Ph]. IR (neat):
v (cm-1) = 3079 (O–H), 2937 (C–H), 2836 (C–H), 1051 (C–O),
1
Haryl), 2922 (C–H), 1080 (C–O), 697 (C–H). H NMR (CDCl3):
d (ppm) = 1.88 (dd, J = 14.9/2.4 Hz, 2H, N(CH2CH2)2), 1.96
(td, J = 14.5/4.2 Hz, 2H, N(CH2CH2)2), 2.39 (td, J = 12.1/2.9
Hz, 2H, N(CH2CH2)2), 2.66 (t, 5.2 Hz, 2H, thiophCH2CH2), 2.72
(d, J = 11.6 Hz, 2H, N(CH2CH2)2), 3.56 (s, 2H, NCH2Ph), 3.72
(t, J = 5.6 Hz, 2H, thiophCH2CH2), 6.79 (d, 3.0 Hz, 1H, 3¢-H-
thioph), 6.87 (d, 3.0 Hz, 1H, 1¢-H-thioph), 7.27–7.38 (m, 5H, Ph-
H). 13C NMR (CDCl3): d (ppm) = 27.1 (1C, thiophCH2CH2),
37.3 (2C, N(CH2CH2)2), 49.1 (2C, N(CH2CH2)2), 59.2 (1C,
thiophCH2CH2), 63.1 (1C, NCH2Ph), 73.6 (1C, thiophCspiro),
118.9 (1C, CH-1¢-thioph), 119.4 (1C, CH-3¢-thioph), 127.5 (1C,
Ph-CH), 128.5 (2C, Ph-CH), 129.8 (2C, Ph-CH), 134.8 (1C, Cquart),
137.2 (1C, Cquart), 143.7 (1C, Cquart).
1
1
698 (C–H). H NMR (CDCl3): d (ppm) = H 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, 3¢-H-thioph), 6.99 (d, J = 2.9 Hz, 1H, 1¢-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-1¢-
thioph), 120.3 (1C, C-3¢-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 quaternary carbon atom is not visible.
6.1.10. General procedure A for the a-arylation of spirocyclic
thiophene derivatives 5 and 6. A 20 mL glass vessel was equipped
R
with a magnetic stirring bar and closed by a J. Youngꢀ O-ring tap.
The flask was flame-dried under vacuo and filled with Ar after
cooling to rt. Under a permanent stream of Ar the Pd-catalyst
(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 the iodoarene (1.1 equiv.)
and a solution of the spirocyclic thiophene 5 or 6 (1 equiv.) in
dry m-xylene (0.6 mL in total) were added dropwi◦se. The vessel
was sealed with the O-ring tap and heated at 150 C for 12 h in
a 8-well reaction block. After cooling the vessel to rt the mixture
was filtered through a short silica pad (EtOAc). The filtrate was
concentrated in vacuo and the crude product was purified by gel
permeation chromatography (CHCl3) followed by preparative thin
layer chromatography to yield the corresponding arylthiophene in
high purity.
6.1.8. 1-Benzylspiro[piperidine-4,4¢-thieno[3,4-c]pyran] (14).
Under N2 the hemiacetal 13 (284 mg, 0.9 mmol) was dissolved in
dry CH2Cl2 (10 mL) and cooled down to 0 ◦C. After 10 min NEt3
(0.32 mL, 2.28 mmol) and MeSO2Cl (0.12 mL, 1.52 mmol) were
added dropwise. The solution was stirred for 2 h at rt and finally
heated to reflux for 1 h. After cooling down to rt the mixture was
alkalized with saturated NaHCO3 solution. The aqueous layer
was separated and extracted twice with CH2Cl2. The combined
organic layers were dried (K2CO3), filtered and the solvent was
removed in vacuo. The residue was purified by fc (3 cm, 15 cm,
cyclohexane : EtOAc = 7 : 3, 20 mL, Rf 0.34). Colorless solid, mp
◦
85 C, yield 137 mg (51%). C18H19NOS (297.4 g mol-1). Purity
(HPLC): 95.1%, tr = 17.6 min. MS (EI): m/z = 297 [M+], 206
[M+ - CH2Ph], 91 [+CH2Ph]. R (neat): v (cm-1) = 3089 (C–H),
2923 (C–H), 1615 (C C), 1041 (C–O). 1H NMR (CDCl3): d
(ppm) = 1.93 (td, J = 13.4/4.6 Hz, 2H, N(CH2CH2)2), 2.16 (dd,
J = 14.0/2.4 Hz, 2H, N(CH2CH2)2), 2.42 (td, J = 12.0/2.4 Hz,
2H, N(CH2CH2)2), 2.70–2.77 (m, 2H, N(CH2CH2)2), 3.57 (s, 2H,
NCH2Ph), 5.80 (d, J = 5.9 Hz, 1H, 7-H), 6.44 (d, J = 5.9 Hz, 1H,
6-H), 6.77 (d, J = 2.7 Hz, 1H, 1-H-thioph), 6.91 (d, J = 2.1 Hz,
1H, 3-H-thioph), 7.29–7.38 (m, 5H, Ph-H). 13C NMR (CDCl3):
d (ppm) = 35.7 (2C, N(CH2CH2)2), 49.1 (2C, N(CH2CH2)2),
63.4 (1C, NCH2Ph), 101.0 (1C, thiophCH CH), 115.5 (1C,
C-3¢-thioph), 117.9 (1C, C-1¢-thioph), 127.3 (1C, Ph-C), 128.4
6.1.11. 1-Benzyl-1¢-phenyl-6¢,7¢-dihydrospiro[piperidine-4,4¢-
thieno[3,4-c]pyran] (3a). According to General procedure A
spirocyclic thiophene 5 (27.7 mg, 0.093 mmol) was reacted with
iodobenzene (11.3 mL, 0.10 mmol), Ag2CO3 (28 mg, 0.10 mmol)
and PdCl2/2,2¢-bipyridyl (3.6 mg, 0.01 mmol) in m-xylene (1.2
mL). The crude product was purified by CHCl3-gpc and prep.
tlc (h = 15 cm, hexane : EtOAc = 9 : 1, Rf 0.42). Colorless solid,
yield 18.8 mg (54%) after gpc; yield 12.0 mg (35%) after prep. tlc.
C24H25NOS (375.5 g mol-1). Exact MS (HRMS): m/z = calcd. for
C24H25NOS [M+] 375.1657, found 375.1643. 1H NMR (CDCl3): d
(ppm) = 1.95–2.06 (m, 4H, N(CH2CH2)2), 2.42 (td, J = 10.8/4.9
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Hz, 2H, N(CH2CH2)2), 2.74 (d, J = 11.5 Hz, 2H, N(CH2CH2)2),
2.86 (t, J = 5.5 Hz, 2H, thiophCH2CH2), 3.58 (s, 2H, NCH2Ph),
3.82 (t, J = 5.5 Hz, 2H, thiophCH2CH2), 6.97 (s, 1H, 3¢-H-
thioph), 7.27–7.50 (m, 10H, Ph-H). 13C NMR (CDCl3): d (ppm) =
27.8 (1C, thiophCH2CH2), 37.9 (2C, N(CH2CH2)2), 49.3 (2C,
N(CH2CH2)2), 59.3 (1C, thiophCH2CH2), 63.6 (1C, NCH2Ph),
73.9 (1C, thiophCspiro), 118.0 (1C, CH-3¢-thioph), 127.3 (Ph-CH),
127.5 (Ph-CH), 128.5 (Ph-CH), 128.6 (Ph-CH), 128.9 (Ph-CH),
129.6 (Ph-CH), 131.1 (1C, Cquart), 134.6 (1C, Cquart), 137.5 (1C,
with p-iodonitrobenzene (28.7 mg, 0.12 mmol), Ag2CO3 (28.1 mg,
0.10 mmol) and PdCl2/2,2¢-bipyridyl (4.0 mg, 0.012 mmol) in
m-xylene (1.2 mL). The crude product was purified by CHCl3-
gpc and prep. tlc (h = 15 cm, hexane : EtOAc = 9 : 1, NHEt2
2%, Rf 0.60). Pale yellow solid, yield 18.7 mg (48%) after gpc;
yield 4.4 mg (11%) after prep. tlc. C24H24N2O3S (420.5 g mol-1).
Exact MS (HRMS): m/z = calcd. for C24H24N2O3S [M+] 420.1508,
found 420.1523. 1H NMR (CDCl3): d (ppm) = 1.95–2.06 (m,
4H, N(CH2CH2)2), 2.42 (td, J = 10.8/5.6 Hz, 2H, N(CH2CH2)2),
2.75 (d, J = 10.8 Hz, 2H, N(CH2CH2)2), 2.88 (t, J = 5.3 Hz,
2H, thiophCH2CH2), 3.58 (s, 2H, NCH2Ph), 3.84 (t, J = 5.4
Hz, 2H, thiophCH2CH2), 7.11 (s, 1H, 3¢-H-thioph), 7.27–7.40
(m, 5H, Ph-H), 7.62 (d, J = 7.9 Hz, 2H, m-NO2-Ph-H), 8.26 (d,
J = 7.8 Hz, 2H, o-NO2-Ph-H). 13C NMR (CDCl3): d (ppm) =
28.1 (1C, thiophCH2CH2), 37.8 (2C, N(CH2CH2)2), 49.2 (2C,
N(CH2CH2)2), 59.0 (1C, thiophCH2CH2), 63.5 (1C, NCH2Ph),
73.9 (1C, thiophCspiro), 120.5 (1C, CH-3¢-thioph), 124.3 (Ph-CH),
127.3 (Ph-CH), 128.5 (Ph-CH), 128.7 (Ph-CH), 129.6 (Ph-CH),
133.6 (1C, Cquart), 135.1 (1C, Cquart), 138.6 (1C, Cquart), 141.3 (1C,
C
quart), 138.6 (1C, Cquart), 145.5 (1C, Cquart).
6.1.12. 1-Benzyl-1¢-(4-methoxyphenyl)-6¢,7¢-dihydrospiro[pipe-
ridine-4,4¢-thieno[3,4-c]pyran] (3b). According to General proce-
dure A spirocyclic thiophene 5 (28.4 mg, 0.095 mmol) was reacted
with p-iodoanisole (26.7 mg, 0.11 mmol), Ag2CO3 (30.2 mg, 0.11
mmol) and PdCl2/2,2¢-bipyridyl (4.1 mg, 0.012 mmol) in m-xylene
(1.2 mL). The crude product was purified by CHCl3-gpc and prep.
tlc (h = 15 cm, hexane : EtOAc = 9 : 1, Rf 0.22). Colorless solid, yield
25.0 mg (65%) after gpc; 12.3 mg (32%) after prep. tlc. C25H27NO2S
(405.6 g mol-1). Exact MS (HRMS): m/z = calcd. for C25H27NO2S
C
quart), 146.4 (1C, Cquart), 146.7 (1C, Cquart).
1
[M+] 405.1762, found 405.1779. H NMR (CDCl3): d (ppm) =
6.1.15. 4-(1-Benzyl-6¢,7¢-dihydrospiro[piperidine-4,4¢-thieno-
1.96–2.04 (m, 4H, N(CH2CH2)2), 2.42 (td, J = 11.1/4.4 Hz, 2H,
N(CH2CH2)2), 2.74 (d, J = 11.5 Hz, 2H, N(CH2CH2)2), 2.81 (t,
J = 5.5 Hz, 2H, thiophCH2CH2), 3.58 (s, 2H, NCH2Ph), 3.81 (t,
J = 5.5 Hz, 2H, thiophCH2CH2), 3.83 (s, 3H, OCH3), 6.91 (s,
1H, 3¢-H-thioph), 6.93 (d, J = 8.9 Hz, 2H, o-H3CO-Ph-H), 7.27–
7.41 (m, 7H, Ph-H, H3CO-Ph-H). 13C NMR (CDCl3): d (ppm) =
27.7 (1C, thiophCH2CH2), 37.9 (2C, N(CH2CH2)2), 49.3 (2C,
N(CH2CH2)2), 55.4 (1C, Ph-OCH3), 59.3 (1C, thiophCH2CH2),
63.6 (1C, NCH2Ph), 73.9 (1C, thiophCspiro), 114.3 (2C, o-H3CO-
Ph-CH), 117.1 (1C, CH-3¢-thioph), 127.1 (1C, Cquart), 127.3 (Ph-
CH), 128.5 (Ph-CH), 129.6 (Ph-CH), 129.8 (Ph-CH), 130.3 (1C,
[3,4-c]pyran]-1¢-yl) benzonitrile (3e). According to General pro-
cedure A spirocyclic thiophene 5 (23.9 mg, 0.080 mmol) was
reacted with p-iodobenzonitrile (21.8 mg, 0.09 mmol), Ag2CO3
(23.8 mg, 0.09 mmol) and PdCl2/2,2¢-bipyridyl (3.3 mg, 0.01
mmol) in m-xylene (1.0 mL). The crude product was purified
by CHCl3-gpc and prep. tlc (h = 15 cm, hexane : EtOAc = 9 : 1,
NHEt2 2%, Rf 0.36). Colorless solid, yield 19 mg (59%) after gpc;
yield 15.7 mg (49%) after prep. tlc. C25H24N2OS (400.5 g mol-1).
Exact MS (HRMS): m/z = calcd. for C25H24N2OS [M+] 400.1609,
found 400.1606. 1H NMR (CDCl3): d (ppm) = 1.93–2.04 (m,
4H, N(CH2CH2)2), 2.42 (td, J = 11.0/4.5 Hz, 2H, N(CH2CH2)2),
2.74 (d, J = 11.5 Hz, 2H, N(CH2CH2)2), 2.86 (t, J = 5.5 Hz,
2H, thiophCH2CH2), 3.58 (s, 2H, NCH2Ph), 3.83 (t, J = 5.5
Hz, 2H, thiophCH2CH2), 7.08 (s, 1H, 3¢-H-thioph), 7.27–7.41
(m, 5H, Ph-H), 7.56 (d, J = 8.2 Hz, 2H, m-NC-Ph-H), 7.68 (d,
J = 8.1 Hz, 2H, o-NC-Ph-H). 13C NMR (CDCl3): d (ppm) =
28.0 (1C, thiophCH2CH2), 37.9 (2C, N(CH2CH2)2), 49.2 (2C,
N(CH2CH2)2), 59.0 (1C, thiophCH2CH2), 63.6 (1C, NCH2Ph),
73.9 (1C, thiophCspiro), 110.7 (1C, Ph-Cquart-C N), 119.0 (1C,
C
quart), 137.3 (1C, Cquart), 145.4 (1C, Cquart), 159.2 (1C, Cquart). One
signal for a quaternary carbon atom is not visible.
6.1.13. 1-Benzyl-1¢-(4-methylphenyl)-6¢,7¢-dihydrospiro[piperi-
dine-4,4¢-thieno[3,4-c]pyran] (3c). According to General proce-
dure A spirocyclic thiophene 5 (22.9 mg, 0.077 mmol) was reacted
with p-iodotoluene (23.0 mg, 0.11 mmol), Ag2CO3 (23.5 mg, 0.09
mmol) and PdCl2/2,2¢-bipyridyl (3.1 mg, 0.01 mmol) in m-xylene
(1.0 mL). The crude product was purified by CHCl3-gpc and
prep. tlc (h = 15 cm, hexane : EtOAc = 12 : 1, NHEt2 2%, Rf 0.56).
Colorless solid, yield 14.6 mg (49%) after gpc; yield 10.0 mg (34%)
C
N), 120.0 (1C, CH-3¢-thioph), 127.3 (Ph-CH), 128.5 (Ph-CH),
128.8 (Ph-CH), 129.6 (Ph-CH), 132.7 (Ph-CH), 133.1 (1C, Cquart),
135.4 (1C, Cquart), 138.7 (1C, Cquart), 139.3 (1C, Cquart), 146.2 (1C,
1
after prep. tlc. C25H27NOS (389.6 g mol-1). H NMR (CDCl3): d
(ppm) = 1.96–2.05 (m, 4H, N(CH2CH2)2), 2.37 (s, 3H, H3C-Ph),
2.42 (td, J = 11.5/4.4 Hz, 2H, N(CH2CH2)2), 2.74 (d, J = 11.5 Hz,
2H, N(CH2CH2)2), 2.84 (t, J = 5.5 Hz, 2H, thiophCH2CH2), 3.58
(s, 2H, NCH2Ph), 3.81 (t, J = 5.5 Hz, 2H, thiophCH2CH2), 6.94 (s,
1H, 3¢-H-thioph), 7.20 (d, J = 7.9 Hz, 2H, o-H3C-Ph-H), 7.27–7.39
(m, 7H, Ph-H, H3C-Ph-H). 13C NMR (CDCl3): d (ppm) = 21.2
(1C, Ph-CH3), 27.8 (1C, thiophCH2CH2), 37.9 (2C, N(CH2CH2)2),
49.4 (2C, N(CH2CH2)2), 59.3 (1C, thiophCH2CH2), 63.6 (1C,
NCH2Ph), 74.0 (1C, thiophCspiro), 117.5 (1C, CH-3¢-thioph), 127.2
(Ph-CH), 128.5 (Ph-CH), 128.5 (Ph-CH), 129.6 (Ph-CH), 130.7
(1C, Cquart), 131.7 (1C, Cquart), 137.3 (1C, Cquart), 137.6 (1C, Cquart),
138.8 (1C, Cquart), 145.5 (1C, Cquart).
C
quart).
6.1.16. 1-Benzyl-1¢-(naphthalen-1-yl)-6¢,7¢-dihydrospiro[pipe-
ridine-4,4¢-thieno [3,4-c]pyran] (3f). According to General proce-
dure A spirocyclic thiophene 5 (34.7 mg, 0.11 mmol) was reacted
with 1-iodonaphthalene (19 mL, 0.13 mmol), Ag2CO3 (34.8 mg,
0.13 mmol) and PdCl2/2,2¢-bipyridyl (4.0 mg, 0.01 mmol) in m-
xylene (1.2 mL). The crude product was purified by CHCl3-gpc and
prep. tlc (h = 15 cm, hexane : EtOAc = 11 : 1, NHEt2 2%, Rf 0.40).
Colorless solid, yield 33.4 mg (67%) after gpc; yield 17.4 mg (35%)
after prep. tlc. C28H27NOS (425.6 g mol-1). Exact MS (HRMS):
m/z = calcd. for C28H27NOS [M+] 425.1813, found 425.1805. H
1
NMR (CDCl3): d (ppm) = 2.01–2.14 (m, 4H, N(CH2CH2)2), 2.41–
2.50 (m, 4H, N(CH2CH2)2, thiophCH2CH2), 2.78 (d, J = 11.4
Hz, 2H, N(CH2CH2)2, 3.60 (s, 2H, NCH2Ph), 3.79 (t, J = 5.6 Hz,
2H, thiophCH2CH2), 7.09 (s, 1H, 3¢-H-thioph), 7.27–7.31 (m, 1H,
6.1.14. 1-Benzyl-1¢-(4-nitrophenyl)-6¢,7¢-dihydrospiro[piperi-
dine-4,4¢-thieno[3,4-c]pyran] (3d). According to General proce-
dure A spirocyclic thiophene 5 (27.8 mg, 0.093 mmol) was reacted
8024 | Org. Biomol. Chem., 2011, 9, 8016–8029
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Ar-H), 7.32–7.42 (m, 4H, Ar-H), 7.43–7.54 (m, 4H, Ar-H),
7.76 (d, J = 8.2 Hz, 1H, Ar-H), 7.82–7.93 (m, 2H, Ar-H).
13C NMR (CDCl3): d (ppm) = 26.8 (1C, thiophCH2CH2),
37.9 (2C, N(CH2CH2)2), 49.3 (2C, N(CH2CH2)2), 59.1 (1C,
thiophCH2CH2), 63.6 (1C, NCH2Ph), 73.9 (1C, thiophCspiro),
118.4 (1C, CH-3¢-thioph), 125.4 (1C, Ar-CH), 126.2 (1C, Ar-CH),
126.3 (1C, Ar-CH), 126.6 (1C, Ar-CH), 127.3 (1C, Ar-CH), 128.5
(1C, Ar-CH), 128.6 (1C, Ar-CH), 128.9 (1C, Ar-CH), 129.3 (1C,
Ar-CH), 129.6 (1C, Ar-CH), 131.8 (1C, Cquart), 132.5 (1C, Cquart),
133.2 (1C, Cquart), 133.9 (1C, Cquart), 134.9 (1C, Cquart), 138.7 (1C,
N(CH2CH2)2), 40.1 (1C, N(CH2CH2)2), 49.4 (2C, N(CH2CH2)2),
55.5 (1C, Ph-OCH3), 56.6 (1C, OCH3), 63.5 (1C, NCH2Ph),
74.2 (1C, thiophCspiro), 97.6 (1C, thiophCH2CH), 114.4 (Ph-CH),
116.7 (1C, CH-3¢-thioph), 126.8 (Ph-CH), 127.3 (1C, Cquart), 128.5
(Ph-CH), 128.6 (1C, Cquart), 129.4 (1C, Cquart), 129.6 (Ph-CH),
129.9 (Ph-CH), 138.0 (1C, Cquart), 144.6 (1C, Cquart), 159.4 (1C,
Ph-Cquart-OCH3).
6.1.19. 1-Benzyl-6¢-methoxy-1¢-(4-methylphenyl)-6¢,7¢-dihy-
drospiro[piperidine-4,4¢-thieno[3,4-c]pyran] (4c). According to
General procedure A spirocyclic thiophene 6 (28.3 mg, 0.086
mmol) was reacted with p-iodotoluene (22.5 mg, 0.10 mmol),
Ag2CO3 (25.5 mg, 0.09 mmol) and PdCl2/2,2¢-bipyridyl (3.7 mg,
0.011 mmol) in m-xylene (1.2 mL). The crude product was purified
by CHCl3-gpc and prep. tlc (h = 15 cm, hexane : EtOAc = 4 : 1,
NEt3 2%, Rf 0.62). Colorless solid, yield 15.2 mg (42%) after gpc;
13.7 mg (38%) after prep. tlc. C26H29NO2S (419.6 g mol-1). Exact
MS (HRMS): m/z = calcd. for C26H29NO2S [M+] 419.1919, found
419.1912. 1H NMR (CDCl3): d (ppm) = 1.90 (td, J = 13.3/3.9 Hz,
1H, N(CH2CH2)2), 1.99 (dd, J = 13.7/2.7 Hz, 1H, N(CH2CH2)2),
2.04–2.15 (m, 2H, N(CH2CH2)2), 2.37 (s, 3H, H3C-Ph), 2.46 (td,
J = 12.7/2.3 Hz, 1H, N(CH2CH2)2), 2.57 (td, J = 12.1/2.7 Hz,
1H, N(CH2CH2)2), 2.74–2.83 (m, 2H, N(CH2CH2)2), 2.86 (dd,
J = 15.5/7.5 Hz, 1H, thiophCH2CH), 3.00 (dd, J = 15.5/3.1 Hz,
1H, thiophCH2CH), 3.52 (s, 3H, OCH3), 3.58 (d, J = 13.0 Hz,
1H, NCH2Ph), 3.62 (d, J = 13.1 Hz, 1H, NCH2Ph), 4.79 (dd, J =
7.4/3.1 Hz, 1H, thiophCH2CH), 6.93 (s, 1H, 3¢-H-thioph), 7.19
(d, J = 7.9 Hz, 2H, o-H3C-Ph-H), 7.27–7.40 (m, 7H, Ph-H, H3C-
Ph-H). 13C NMR (CDCl3): d (ppm) = 21.2 (1C, Ph-CH3), 33.3 (1C,
thiophCH2CH), 37.6 (1C, N(CH2CH2)2), 40.1 (1C, N(CH2CH2)2),
49.4 (2C, N(CH2CH2)2), 56.6 (1C, OCH3), 63.6 (1C, NCH2Ph),
74.2 (1C, thiophCspiro), 97.5 (1C, thiophCH2CH), 117.0 (1C, CH-
3¢-thioph), 127.3 (Ph-CH), 128.5 (Ph-CH), 128.5 (Ph-CH), 128.9
(1C, Cquart), 129.6 (Ph-CH), 129.6 (Ph-CH), 131.3 (1C, Cquart),
137.5 (1C, Cquart), 138.2 (1C, Cquart), 138.6 (1C, Cquart), 144.6
(1C, Cquart).
C
quart), 144.5 (1C, Cquart).
6.1.17. 1-Benzyl-6¢-methoxy-1¢-phenyl-6¢,7¢-dihydrospiro[pipe-
ridine-4,4¢-thieno[3,4-c]pyran] (4a). According to General proce-
dure A spirocyclic thiophene 6 (87.8 mg, 0.27 mmol) was reacted
with iodobenzene (35.2 mg, 0.17 mmol), Ag2CO3 (49.3 mg, 0.18
mmol) and PdCl2/2,2¢-bipyridyl (5.5 mg, 0.016 mmol) in m-xylene
(1.5 mL). The crude product was purified by CHCl3-gpc and prep.
tlc (h = 15 cm, hexane : EtOAc = 3 : 2, Rf 0.4). Pale yellow resin,
yield 46.3 mg (67%) after gpc; yield 33.2 mg (48%) after prep.tlc.
C25H27NO2S (405.6 g mol-1). Exact MS (HRMS): m/z = calcd. for
1
C25H27NO2S [M+] 405.1762, found 405.1782. H NMR (CDCl3):
d (ppm) = 1.91 (td, J = 13.6/4.6 Hz, 1H, N(CH2CH2)2), 1.96–
2.06 (m, 1H, N(CH2CH2)2), 2.06–2.19 (m, 2H, N(CH2CH2)2),
2.47 (td, J = 11.8/2.3 Hz, 1H, N(CH2CH2)2), 2.58 (td, J =
11.9/2.7 Hz, 1H, N(CH2CH2)2), 2.80 (dd, J = 13.5/11.4 Hz, 2H,
N(CH2CH2)2), 2.88 (dd, J = 15.5/7.4 Hz, 1H, thiophCH2CH), 3.02
(dd, J = 15.5/3.0 Hz, 1H, thiophCH2CH), 3.52 (s, 3H, OCH3),
3.58 (d, J = 13.0 Hz, 1H, NCH2Ph), 3.62 (d, J = 13.0 Hz, 1H,
NCH2Ph), 4.80 (dd, J = 7.4/3.1 Hz, 1H, thiophCH2CH), 6.96 (s,
1H, 3¢-H-thioph), 7.27–7.46 (m, 10H, Ph-H). 13C NMR (CDCl3):
d (ppm) = 33.3 (1C, thiophCH2CH), 37.5 (1C, N(CH2CH2)2),
39.9 (1C, N(CH2CH2)2), 49.4 (2C, N(CH2CH2)2), 56.7 (1C,
OCH3), 63.4 (1C, NCH2Ph), 74.0 (1C, thiophCspiro), 97.5 (1C,
thiophCH2CH), 117.5 (1C, CH-3¢-thioph), 127.5 (Ph-CH), 128.1
(Ph-CH), 128.2 (Ph-CH), 128.5 (Ph-CH), 128.5 (Ph-CH), 128.8
(Ph-CH), 129.1 (Ph-Cquart), 129.6 (Ph-CH), 131.6 (Ph-CH), 134.0
(1C, Cquart), 138.1 (1C, Cquart)(Ph-C), 138.4 (1C, Cquart)(Ph-C), 144.2
(1C, Cquart).
6.1.20. 1-Benzyl-6¢-methoxy-1¢-(4-nitrophenyl)-6¢,7¢-dihydro-
spiro-[piperidine-4,4¢-thieno [3,4-c]pyran] (4d). According to
General procedure A spirocyclic thiophene 6 (30.5 mg, 0.093
mmol) was reacted with p-iodo-nitrobenzene (26.1 mg, 0.10
mmol), Ag2CO3 (26.0 mg, 0.09 mmol) and PdCl2/2,2¢-bipyridyl
(3.5 mg, 0.01 mmol) in m-xylene (1.2 mL). The crude product was
purified by CHCl3-gpc and prep. tlc (h = 15 cm, hexane : EtOAc =
3 : 2, NEt3 2%, Rf 0.33). Pale yellow solid, yield 33.3 mg (80%)
after gpc; 18.8 mg (45%) after prep. tlc. C25H26N2O4S (450.5 g
mol-1). Exact MS (HRMS): m/z = calcd. for C25H26N2O4S [M+]
450.1613, found 450.1595. 1H NMR (CDCl3): d (ppm) = 1.90 (td,
J = 13.3/4.0 Hz, 1H, N(CH2CH2)2), 1.99 (dd, J = 13.9/2.4 Hz,
1H, N(CH2CH2)2), 2.03–2.15 (m, 2H, N(CH2CH2)2), 2.46 (td,
J = 12.8/2.5 Hz, 1H, N(CH2CH2)2), 2.57 (td, J = 11.9/2.2 Hz, 1H,
N(CH2CH2)2), 2.80 (dd, J = 14.5/12.6 Hz, 2H, N(CH2CH2)2), 2.92
(dd, J = 15.7/6.8 Hz, 1H, thiophCH2CH), 3.03 (dd, J = 15.5/3.1
Hz, 1H, thiophCH2CH), 3.52 (s, 3H, OCH3), 3.58 (d, J = 13.1 Hz,
1H, NCH2Ph), 3.62 (d, J = 13.1 Hz, 1H, NCH2Ph), 4.84 (dd, J =
6.7/3.1 Hz, 1H, thiophCH2CH), 7.11 (s, 1H, 3¢-H-thioph), 7.28–
7.40 (m, 5H, Ph-H), 7.59 (d, J = 8.3 Hz, 2H, m-NO2-Ph-H), 8.25
(d, J = 8.1 Hz, 2H, o-NO2-Ph-H). 13C NMR (CDCl3): d (ppm) =
33.5 (1C, thiophCH2CH), 37.8 (1C,N(CH2CH2)2), 40.0 (1C,
N(CH2CH2)2), 49.3 (2C, N(CH2CH2)2), 56.7 (1C, OCH3), 63.5
6.1.18. 1-Benzyl-6¢-methoxy-1¢-(4-methoxyphenyl)-6¢,7¢-dihy-
drospiro[piperidine-4,4¢-thieno[3,4-c]pyran] (4b). According to
General procedure A spirocyclic thiophene 6 (20.0 mg, 0.061
mmol) was reacted with p-iodoanisole (15.9 mg, 0.07 mmol),
Ag2CO3 (16.9 mg, 0.06 mmol) and PdCl2/2,2¢-bipyridyl (2.2 mg,
0.007 mmol) in m-xylene (1.0 mL). The crude product was purified
by CHCl3-gpc and prep. tlc (h = 15 cm, hexane : EtOAc = 3 : 2,
NEt3 2%, Rf 0.36). Colorless solid, yield 13.8 mg (52%) after
gpc; 12.5 mg (47%) after prep. tlc. C26H29NO3S (435.6 g mol-1).
Exact MS (HRMS): m/z = calcd. for C26H29NO3S [M+] 435.1868,
found 435.1877. 1H NMR (CDCl3): d (ppm) = 1.90 (td, J =
13.5/3.0 Hz, 1H, N(CH2CH2)2), 1.96–2.15 (m, 3H, N(CH2CH2)2),
2.46 (td, J = 12.1/1.6 Hz, 1H, N(CH2CH2)2), 2.57 (td, J =
11.5/1.3 Hz, 1H, N(CH2CH2)2), 2.74–2.89 (m, 3H, N(CH2CH2)2,
thiophCH2CH), 2.98 (dd, J = 15.4/3.0 Hz, 1H, thiophCH2CH),
3.52 (s, 3H, OCH3), 3.58 (d, J = 12.5 Hz, 1H, NCH2Ph), 3.60 (d,
J = 13.4 Hz, 1H, NCH2Ph), 3.83 (s, 3H, OCH3), 4.79 (dd, J =
7.4/3.1 Hz, 1H, thiophCH2CH), 6.90 (s, 1H, 3¢-H-thioph), 6.92
(d, J = 8.7 Hz, 2H, o-H3CO-Ph-H), 7.30–7.40 (m, 7H, Ph-H). 13
NMR (CDCl3): d (ppm) = 33.2 (1C, thiophCH2CH), 37.6 (1C,
C
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(1C, NCH2Ph), 74.1 (1C, thiophCspiro), 97.2 (1C, thiophCH2CH),
120.0 (1C, CH-3¢-thioph), 124.3 (Ph-CH), 127.4 (Ph-CH), 128.5
(Ph-CH), 128.9 (Ph-CH), 129.6 (Ph-CH), 130.5 (1C, Cquart), 131.6
(1C, Cquart), 135.7 (1C, Cquart), 140.9 (1C, Cquart), 145.3 (1C, Cquart),
146.83 (1C, Cquart).
(Ph-CH), 128.9 (Ph-CH), 129.5 (Ph-CH), 131.2 (1C, Cquart), 132.7
(Ph-CH), 136.0 (1C, Cquart), 138.5 (1C, Cquart), 138.9 (1C, Cquart),
145.3 (1C, Cquart).
6.1.23. 1-Benzyl-6¢-methoxy-1¢-[4-(trifluoromethyl)phenyl]-6¢,
7¢-dihydrospiro[piperidine-4,4¢-thieno[3,4-c]pyran] (4g). Accord-
ing to General procedure A spirocyclic thiophene 6 (20.1 mg, 0.061
mmol) was reacted with p-iodo-trifluoromethylbenzene (10 mL,
0.07 mmol), Ag2CO3 (18.1 mg, 0.07 mmol) and PdCl2/2,2¢-
bipyridyl (2.2 mg, 0.007 mmol) in m-xylene (1.0 mL). The crude
product was purified by CHCl3-gpc and prep. tlc (h = 15 cm,
hexane : EtOAc = 3 : 2, Rf 0.43). Colorless solid, yield 10.9 mg
(38%) after gpc; 5.4 mg (19%) after prep. tlc. C26H26F3NO2S (473.6
g mol-1). Exact MS (HRMS): m/z = calcd. for C26H26F3NO2S
6.1.21. 1-[4-(1-Benzyl-6¢-methoxy-6¢,7¢-dihydrospiro[piperi-
dine-4,4¢-thieno[3,4-c]pyran]-1¢-yl)phenyl]ethanone (4e). Accord-
ing to General procedure A spirocyclic thiophene 6 (21.4 mg,
0.065 mmol) was reacted with p-iodo-acetophenone (17.9 mg, 0.07
mmol), Ag2CO3 (17.0 mg, 0.06 mmol) and PdCl2/2,2¢-bipyridyl
(2.1 mg, 0.006 mmol) in m-xylene (1.0 mL). The crude product
was purified by prep. tlc (h = 15 cm, hexane : EtOAc = 3 : 2,
NEt3 2%, Rf 0.52). Colorless solid, yield 17.4 mg (60%) after
prep. tlc. C27H29NO3S (447.6 g mol-1). Exact MS (HRMS): m/z =
calcd. for C27H29NO3S [M+] 447.1868, found 447.1890. 1H NMR
(CDCl3): d (ppm) = 1.91 (td, J = 13.3/4.1 Hz, 1H, N(CH2CH2)2),
1.99 (dd, J = 13.8/2.6 Hz, 1H, N(CH2CH2)2), 2.05–2.16 (m, 2H,
N(CH2CH2)2), 2.47 (td, J = 12.4/2.7 Hz, 1H, N(CH2CH2)2), 2.53–
2.59 (m, 1H, N(CH2CH2)2), 2.61 (s, 3H, CH3C O), 2.74–2.86 (m,
2H, N(CH2CH2)2), 2.92 (dd, J = 15.6/7.1 Hz, 1H, thiophCH2CH),
3.03 (dd, J = 15.6/3.1 Hz, 1H, thiophCH2CH), 3.52 (s, 3H, OCH3),
3.58 (d, J = 13.1 Hz, 1H, NCH2Ph), 3.62 (d, J = 13.1 Hz, 1H,
NCH2Ph), 4.82 (dd, J = 7.1/3.1 Hz, 1H, thiophCH2CH), 7.04 (s,
1H, 3¢-H-thioph), 7.27–7.40 (m, 5H, Ph-H), 7.53 (d, J = 8.4 Hz,
2H, m-CH3C O-Ph-H), 7.98 (d, J = 8.3 Hz, 2H, o-CH3C O-Ph-
H). 13C NMR (CDCl3): d (ppm) = 26.6 (1C, CH3C O), 33.5 (1C,
thiophCH2CH), 37.8 (1C, N(CH2CH2)2), 40.1 (1C, N(CH2CH2)2),
49.4 (2C, N(CH2CH2)2), 56.6 (1C, OCH3), 63.5 (1C, NCH2Ph),
74.2 (1C, thiophCspiro), 97.4 (1C, thiophCH2CH), 118.9 (1C, CH-
3¢-thioph), 127.4 (Ph-CH), 128.5 (Ph-CH), 129.0 (Ph-CH), 129.6
(Ph-CH), 130.7 (1C, Cquart), 135.9 (1C, Cquart), 136.9 (1C, Cquart),
138.5 (1C, Cquart), 139.0 (1C, Cquart), 145.1 (1C, Cquart), 197.8 (1C,
1
[M+] 473.1636, found 473.1648. H NMR (CDCl3): d (ppm) =
1.90 (td, J = 13.4/4.2 Hz, 1H, N(CH2CH2)2), 1.96–2.16 (m, 3H,
N(CH2CH2)2), 2.46 (td, J = 12.6/2.4 Hz, 1H, N(CH2CH2)2), 2.57
(td, J = 12.2/2.7 Hz, 1H, N(CH2CH2)2), 2.80 (dd, J = 13.6/11.7 Hz,
2H, N(CH2CH2)2), 2.89 (dd, J = 15.5/7.1 Hz, 1H, thiophCH2CH),
3.00 (dd, J = 15.5/3.1 Hz, 1H, thiophCH2CH), 3.52 (s, 3H, OCH3),
3.58 (d, J = 13.3 Hz, 1H, NCH2Ph), 3.62 (d, J = 12.9 Hz, 1H,
NCH2Ph), 4.82 (dd, J = 7.0/3.2 Hz, 1H, thiophCH2CH), 7.03
(s, 1H, 3¢-H-thioph), 7.27–7.42 (m, 5H, Ph-H), 7.54 (d, J = 8.1
Hz, 2H, m-F3C-Ph-H), 7.64 (d, J = 8.3 Hz, 2H, o-F3C-Ph-H). 13
C
NMR (CDCl3): d (ppm) = 33.3 (1C, thiophCH2CH), 37.8 (1C,
N(CH2CH2)2), 40.1 (1C, N(CH2CH2)2), 49.4 (2C, N(CH2CH2)2),
56.6 (1C, OCH3), 63.6 (1C, NCH2Ph), 74.2 (1C, thiophCspiro),
97.3 (1C, thiophCH2CH), 118.8 (1C, CH-3¢-thioph), 125.9 (Ph-
C-CF3), 126.3 (1C, Cquart), 127.4 (Ph-CH), 128.5 (Ph-CH), 128.7
(Ph-CH), 129.2 (1C, Cquart), 129.6 (Ph-CH), 130.5 (1C, Cquart), 136.4
(1C, Cquart), 137.8 (1C, Cquart), 145.0 (1C, Cquart).
6.1.24. 1-Benzyl-6¢-methoxy-1¢-(naphthalen-1-yl)-6¢,7¢-dihy-
drospiro[piperidine-4,4¢-thieno[3,4-c]pyran] (4h). According to
General procedure A spirocyclic thiophene 6 (35.2 mg, 0.11
mmol) was reacted with 1-iodonaphthalene (17.2 mL, 0.12 mmol),
Ag2CO3 (26.9 mg, 0.10 mmol) and PdCl2/2,2¢-bipyridyl (3.3
mg, 0.01 mmol) in m-xylene (1.2 mL). The crude product was
purified by CHCl3-gpc and prep. tlc (h = 15 cm, hexane : EtOAc =
9 : 1, NHEt2 2%, Rf 0.28). Colorless solid, yield 35.1 mg (72%)
after gpc; 22.3 mg (46%) after prep. tlc. C29H29NO2S (455.6 g
mol-1). Exact MS (HRMS): m/z = calcd. for C29H29NO2S [M+]
C
quart, CH3C O).
6.1.22. 4-(1-Benzyl-6¢-methoxy-6¢,7¢-dihydrospiro[piperidine-
4,4¢-thieno[3,4-c]pyran]-1¢-yl)benzonitrile (4f). According to
General procedure A spirocyclic thiophene 6 (33.5 mg, 0.102
mmol) was reacted with p-iodobenzonitrile (24.8 mg, 0.11 mmol),
Ag2CO3 (26.1 mg, 0.09 mmol) and PdCl2/2,2¢-bipyridyl (3.1 mg,
0.009 mmol) in m-xylene (1.2 mL). The crude product was purified
by CHCl3-gpc and prep. tlc (h = 15 cm, hexane : EtOAc = 9 : 1,
NEt3 2%, Rf 0.22). Colorless solid, yield 21.7 mg (50%) after gpc;
yield 17.4 mg (40%) after prep. tlc. C26H26N2O2S (430.6 g mol-1).
Exact MS (HRMS): m/z = calcd. for C26H26N2O2S [M+] 430.1715,
found 430.1693. 1H NMR (CDCl3): d (ppm) = 1.84–2.02 (m, 2H,
N(CH2CH2)2), 2.03–2.18 (m, 2H, N(CH2CH2)2), 2.46 (td, J =
12.6/2.7 Hz, 1H, N(CH2CH2)2), 2.57 (td, J = 11.9/2.6 Hz, 1H,
N(CH2CH2)2), 2.80 (dd, J = 14.0/11.4 Hz, 2H, N(CH2CH2)2),
2.89 (dd, J = 15.5/6.9 Hz, 1H, thiophCH2CH), 3.00 (dd, J =
15.5/3.2 Hz, 1H, thiophCH2CH), 3.52 (s, 3H, OCH3), 3.58 (d,
J = 11.7 Hz, 1H, NCH2Ph), 3.62 (d, J = 13.1 Hz, 1H, NCH2Ph),
4.82 (dd, J = 6.9/3.2 Hz, 1H, thiophCH2CH), 7.07 (s, 1H,
3¢-H-thioph), 7.27–7.40 (m, 5H, Ph-H), 7.53 (d, J = 8.6 Hz,
2H, m-NC-Ph-H), 7.67 (d, J = 8.5 Hz, 2H, o-NC-Ph-H). 13C
NMR (CDCl3): d (ppm) = 33.4 (1C, thiophCH2CH), 37.8 (1C,
N(CH2CH2)2), 40.1 (1C, N(CH2CH2)2), 49.3 (2C, N(CH2CH2)2),
56.6 (1C, OCH3), 63.5 (1C, NCH2Ph), 74.1 (1C, thiophCspiro),
97.2 (1C, thiophCH2CH), 110.9 (1C, Ph-Cquart-C N), 119.0
(1C, C N), 119.5 (1C, CH-3¢-thioph), 127.3 (Ph-CH), 128.5
1
455.1919, found 455.1938. H NMR (CDCl3): d (ppm) = 1.98
(td, J = 13.3/4.2 Hz, 1H, N(CH2CH2)2), 2.09 (dd, J = 13.7/2.5
Hz, 1H, N(CH2CH2)2), 2.12–2.28 (m, 2H, N(CH2CH2)2), 2.49
(td, J = 13.6/3.0 Hz, 1H, N(CH2CH2)2), 2.56 (d, J = 5.7 Hz, 2H,
thiophCH2CH), 2.57–2.65 (m, 1H, N(CH2CH2)2), 2.83 (dd, J =
14.0/11.9 Hz, 2H, N(CH2CH2)2), 3.48 (s, 3H, OCH3), 3.60 (d,
J = 13.7 Hz, 1H, NCH2Ph), 3.64 (d, J = 13.8 Hz, 1H, NCH2Ph),
4.81 (t, J = 5.3 Hz, 1H, thiophCH2CH), 7.09 (s, 1H, 3¢-H-thioph),
7.27–7.53 (m, 9H, Ar-H), 7.77 (d, J = 8.2 Hz, 1H, Ar-H), 7.88
(td, J = 7.1/2.2 Hz, 2H, Ar-H). 13C NMR (CDCl3): d (ppm) =
32.5 (1C, thiophCH2CH), 37.8 (1C, N(CH2CH2)2), 40.1 (1C,
N(CH2CH2)2), 49.4 (1C, N(CH2CH2)2), 49.5 (1C, N(CH2CH2)2),
56.5 (1C, OCH3), 63.6 (1C, NCH2Ph), 74.3 (1C, thiophCspiro),
97.5 (1C, thiophCH2CH), 117.9 (1C, CH-3¢-thioph), 125.3 (Ar-
CH), 126.2 (Ar-CH), 126.7 (Ar-CH), 127.3 (Ar-CH), 128.4 (1C,
C
quart), 128.5 (Ar-CH), 128.6 (Ar-CH), 129.0 (Ar-CH), 129.2 (Ar-
CH), 129.6 (Ar-CH), 130.1 (1C, Cquart), 131.4 (1C, Cquart), 131.8
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(Ar-CH), 132.5 (1C, Cquart), 133.9 (Ar-CH), 135.5 (1C, Cquart), 138.7
(1C, Cquart), 143.8 (1C, Cquart).
138.7 (1C, Cquart), 140.4 (1C, Cquart), 140.8 (1C, Cquart), 144.9 (1C,
C
quart).†
6.2. Receptor binding studies
6.1.25. 1-Benzyl-6¢-methoxy-1¢-(pyridin-3-yl)-6¢,7¢-dihydro-
spiro[piperidine-4,4¢-thieno [3,4-c]pyran] (4i). According to Gen-
eral procedure A spirocyclic thiophene 6 (34 mg, 0.103 mmol) was
reacted with 3-iodopyridine (20.4 mg, 0.10 mmol), Ag2CO3 (26.8
mg, 0.10 mmol) and PdCl2/2,2¢-bipyridyl (3.4 mg, 0.01 mmol) in
m-xylene (1.2 mL). The crude product was purified by CHCl3-gpc
and prep. tlc (h = 15 cm, hexane : EtOAc = 7 : 3, NEt3 2%, Rf 0.44).
Colorless solid, yield 11.4 mg (27%) after gpc; 7.4 mg (18%) after
prep. tlc. C24H26N2O2S (406.5 g mol-1). Exact MS (HRMS): m/z =
calcd. for C24H26N2O2S [M+] 406.1715, found 406.1703. 1H NMR
(CDCl3): d (ppm) = 1.83–2.17 (m, 4H, N(CH2CH2)2), 2.47 (td, J =
12.1/2.4 Hz, 1H, N(CH2CH2)2), 2.57 (td, J = 11.9/2.6 Hz, 1H,
N(CH2CH2)2), 2.73–2.94 (m, 3H, N(CH2CH2)2), thiophCH2CH),
2.99 (dd, J = 15.4/3.2 Hz, 1H, thiophCH2CH), 3.51 (s, 3H, OCH3),
3.57 (d, J = 14.0 Hz, 1H, NCH2Ph), 3.62 (d, J = 13.5 Hz, 1H,
NCH2Ph), 4.83 (dd, J = 6.8/3.3 Hz, 1H, thiophCH2CH), 7.05
(s, 1H, 3¢-H-thioph), 7.28–7.42 (m, 6H, Ph-H, 5-H-Pyr), 7.73 (d,
J = 7.9 Hz, 1H, 4-H-Pyr), 8.54 (d, J = 4.8 Hz, 1H, 6-H-Pyr),
8.70 (s, 1H, 2-H-Pyr). 13C NMR (CDCl3): d (ppm) = 33.0 (1C,
thiophCH2CH), 37.9 (1C, N(CH2CH2)2), 40.1 (1C, N(CH2CH2)2),
49.4 (2C, N(CH2CH2)2), 56.6 (1C, OCH3), 63.5 (1C, NCH2Ph),
74.2 (1C, thiophCspiro), 97.4 (1C, thiophCH2CH), 118.8 (1C, CH-
3¢-thioph), 123.6 (1C, CH-5¢-Pyr), 127.4 (Ph-CH), 128.5 (Ph-CH),
129.6 (Ph-CH), 130.4 (1C, Cquart), 130.6 (1C, Cquart), 134.1 (1C,
6.2.1. Materials and general procedures. Guinea pig brains
and rat livers were commercially available (Harlan-Winkelmann,
Germany). Homogenizer: Elvehjem Potter (B. Braun Biotech
International). Centrifuge: High-speed cooling centrifuge model
Sorvall RC-5C plus (Thermo Finnigan). Filter: Printed Filtermat
Type A (Perkin Elmer), presoaked in 0.5% aqueous polyethylen-
imine for 2 h at rt before use. The filtration was carried out
with a MicroBeta FilterMate-96 Harvester (Perkin Elmer). The
scintillation analysis was performed using Meltilex (Type A) solid
scintillator (Perkin Elmer). The solid scintillator was melted on the
filtermat at a temperature of 95 ◦C for 5 min. After solidification
of the scintillator at rt, the scintillation was measured using
a MicroBeta Trilux scintillation analyzer (Perkin Elmer). The
overall counting efficiency was 20%.
6.2.2 Membrane preparation for the r1 assay42–45
.
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 1200 x g for 10 min at 4 ◦C.
The supernatant was separated and centrifuged at 23500 x g
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
23500 x g (20 min, 4 ◦C). This procedure was repeated twice.
The final pellet was resuspended in 5–6 volumes of buffer, the
protein concentration was determined according to the method
of Bradford48 using bovine serum albumin as standard, and
subsequently the preparation was frozen (-80 ◦C) in 1.5 mL
portions containing about 1.5 mg protein/mL.
C
quart), 135.7 (1C, CH-4¢-Pyr), 144. 9 (1C, Cquart), 148.7 (1C, CH-
2¢-Pyr), 149.4 (1C, CH-6¢-Pyr). One signal for a quaternary carbon
atom is not visible.
6.1.26. 1-Benzyl-1¢-(1,1¢-biphenyl-4¢-yl)-6¢-methoxy-6¢,7¢-di-
hydrospiro[piperidine-4,4¢-thieno[3,4-c]pyran] (4j). According to
General procedure A spirocyclic thiophene 6 (31.0 mg, 0.094
mmol) was reacted with 4-iodo-1,1¢-biphenyl (32.4 mg, 0.12
mmol), Ag2CO3 (33.0 mg, 0.12 mmol) and PdCl2/2,2¢-bipyridyl
(3.2 mg, 0.01 mmol) in m-xylene (1.2 mL). The crude product
was purified by CHCl3-gpc and flash chromatography (Ø = 1.5
cm, h = 5 cm, hexane : EtOAc = 4 : 1, 3 mL, Rf 0.22). Pale yellow
6.2.3. Performance of the r1 assay42–45
. The test was per-
formed with the radioligand [3H]-(+)-pentazocine (42.5 Ci/mmol;
Perkin Elmer). The thawed membrane preparation (about 75 mg
of the protein) was incubated with various concentrations of test
compounds, 2 nM [3H]-(+)-pentazocine, and buffer (50 mM TRIS,
pH 7.4) in a total volume of 200 mL for 180 min at 37 ◦C.
The incubation was terminated by rapid filtration through the
presoaked filtermats by using the cell harvester. After washing
each well five times with 300 mL of water, the filtermats were
dried at 95 ◦C. Subsequently, the solid scintillator was put on the
filtermat and melted at 95 ◦C. After 5 min, the solid scintillator was
allowed to solidify at rt. The bound radioactivity trapped on the
filters was counted in the scintillation analyzer. The non-specific
binding was determined with 10 mM unlabeled (+)-pentazocine.
The Kd-value of the radioligand [3H]-(+)-pentazocine is 2.9 nM.49
◦
solid, mp 161.2 C, yield 17.7 mg (39%) after gpc; yield 17.4 mg
(38%) after flash chromatography. C31H31NO2S (481.6 g mol-1).
Purity (HPLC†): 96.7%, tR = 10.11 min. Exact MS (APCI):
m/z = calcd. for C31H32NO2S [MH+] 482.2148, found 482.2188.
1H NMR (CDCl3): d (ppm) = 1.91 (td, J = 12.6/4.9 Hz, 1H,
N(CH2CH2)2), 2.01 (dd, J = 13.7/2.7 Hz, 1H, N(CH2CH2)2),
2.07–2.19 (m, 2H, N(CH2CH2)2), 2.47 (td, J = 12.6/2.8 Hz, 1H,
N(CH2CH2)2), 2.58 (td, J = 12.0/2.6 Hz, 1H, N(CH2CH2)2),
2.80 (dd, J = 13.4/11.5 Hz, 2H, N(CH2CH2)2), 2.93 (dd, J =
15.5/7.4 Hz, 1H, thiophCH2CH), 3.07 (dd, J = 15.4/3.1 Hz, 1H,
thiophCH2CH), 3.53 (s, 3H, OCH3), 3.59 (d, J = 12.9 Hz, 1H,
NCH2Ph), 3.62 (d, J = 13.0 Hz, 1H, NCH2Ph), 4.82 (dd, J =
7.4/3.1 Hz, 1H, thiophCH2CH), 6.98 (s, 1H, 3¢-H-thioph), 7.27–
7.65 (m, 14H, Ph-H). 13C NMR (CDCl3): d (ppm) = 33.4 (1C,
thiophCH2CH), 37.7 (1C, N(CH2CH2)2), 40.2 (1C, N(CH2CH2)2),
49.5 (2C, N(CH2CH2)2), 56.7 (1C, OCH3), 63.6 (1C, NCH2Ph),
74.3 (1C, thiophCspiro), 97.5 (1C, thiophCH2CH), 117.5 (1C, CH-
3¢-thioph), 127.2 (Ph-CH), 127.3 (Ph-CH), 127.6 (Ph-CH), 127.7
(Ph-CH), 128.5 (Ph-CH), 129.0 (Ph-CH), 129.1 (Ph-CH), 129.5
(1C, Cquart), 129.5 (Ph-CH), 133.2 (1C, Cquart), 137.8 (1C, Cquart),
6.2.4. Membrane preparation for the r2 assay42–45
.
Two rat
livers were cut into small pieces and homogenized with a potter
(500–800 rpm, 10 up-and-down strokes) in 6 volumes of cold 0.32
M sucrose. The suspension was centrifuged at 1200 x g for 10
min at 4 ◦C. The supernatant was separated and centrifuged at
† HPLC Agilent TechnologiesꢀR , quaternary pump; UV detector: Agilent
1200 Series; autosampler: Agilent 1200 Series High Performance autosam-
pler; column: ZORBAX Eclipse Plus C18 2.1 ¥ 150 mm, 3.5 mm; flow rate:
0.40 mL min-1; detection at l = 254 nm; cut off time: 13 min; solvents: A:
H2O with 5 mM NH4HCO2; B: CH3CN; gradient elution (A%): 0–5 min:
gradient from 80% to 0%; 5–13 min: 0%; 13–18 min: 0% to 80%.
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31000 x g for 20 min at 4 ◦C. The pellet was resuspended in
buffer (50 mM TRIS, pH 8.0) and incubated at rt for 30 min.
After the incubation, the suspension was centrifuged again at
31000 x g for 20 min at 4 ◦C. The final pellet was resuspended
in buffer, the protein concentration was determined according to
the method of Bradford48 using bovine serum albumin as standard,
and subsequently the preparation was frozen (-80 ◦C) in 1.5 mL
portions containing about 2 mg protein/mL.
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◦
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Acknowledgements
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 the IRTG and
this project by the Deutsche Forschungsgemeinschaft is gratefully
acknowledged.
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