Diastereoselective synthesis of conformationally restricted KOR agonists

Article abstract of DOI:10.1039/d1ob00398d

In order to analyze the bioactive conformation of flexible KOR agonists the ethylenediamine KOR pharmacophore was conformationally constrained by incorporation into a bicyclic system. For this purpose, 2-azabicyclo[3.2.1.]octan-7-amines were designed, synthesized and pharmacologically evaluated. The primary amine 14 as first key intermediate was prepared in a six-step synthesis starting with methyl cyclopent-3-enecarboxylate 9. Whereas phenylacetamides failed to provide bicyclic compounds, the intramolecular nucleophilic substitution of the sulfonamide 25 was initiated by deprotonation with NaH affording the bicyclic compound 26 in 72% yield. The three-step introduction of the pharmacophoric pyrrolidine ring started with nucleophilic substitution of exo-configured tosylate 26 with NaN3, which unexpectedly occurred under retention of configuration leading to exo-configured azide 31. The final KOR agonists 35 and 36 with exo-configured amino moieties were obtained by removal of the N-tosyl moiety of 33 and introduction of the second pharmacophoric element by acylation with dihalophenylacetyl chlorides. The KOR affinity of the pyrrolidine 35a is in the high nanomolar range (Ki = 862 nM). The low KOR affinity is explained by a non-appropriate dihedral angle of 137°/141° of the N(pyrrolidine)-C-C-N(acyl) system. As observed for stereoisomers of potent KOR agonists, phenylacetamide 35a and more importantly sulfonamides 33a and 33b show moderate affinity at σ1 receptors (Ki = 109-208 nM). This journal is

Full text of DOI:10.1039/d1ob00398d

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Biomolecular Chemistry
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PAPER
Diastereoselective synthesis of conformationally
restricted KOR agonists†
Cite this: Org. Biomol. Chem., 2021,
19, 4082
Denise Ilari,a,b Sarah Maskri,a,c Dirk Schepmann,a,c Jens Köhler,a
Constantin G. Daniliuc, d Oliver Kocha,c and Bernhard Wünsch
*
a,b,c
In order to analyze the bioactive conformation of flexible KOR agonists the ethylenediamine KOR pharma-
cophore was conformationally constrained by incorporation into a bicyclic system. For this purpose,
2-azabicyclo[3.2.1.]octan-7-amines were designed, synthesized and pharmacologically evaluated. The
primary amine 14 as first key intermediate was prepared in a six-step synthesis starting with methyl cyclo-
pent-3-enecarboxylate 9. Whereas phenylacetamides failed to provide bicyclic compounds, the intra-
molecular nucleophilic substitution of the sulfonamide 25 was initiated by deprotonation with NaH
affording the bicyclic compound 26 in 72% yield. The three-step introduction of the pharmacophoric pyr-
rolidine ring started with nucleophilic substitution of exo-configured tosylate 26 with NaN3, which unex-
pectedly occurred under retention of configuration leading to exo-configured azide 31. The final KOR
agonists 35 and 36 with exo-configured amino moieties were obtained by removal of the N-tosyl moiety
of 33 and introduction of the second pharmacophoric element by acylation with dihalophenylacetyl
chlorides. The KOR affinity of the pyrrolidine 35a is in the high nanomolar range (Ki = 862 nM). The low
KOR affinity is explained by a non-appropriate dihedral angle of 137°/141° of the N(pyrrolidine)–C–C–N
(acyl) system. As observed for stereoisomers of potent KOR agonists, phenylacetamide 35a and more
importantly sulfonamides 33a and 33b show moderate affinity at σ1 receptors (Ki = 109–208 nM).
Received 1st March 2021,
Accepted 31st March 2021
DOI: 10.1039/d1ob00398d
rsc.li/obc
tors.1 Further investigations led to the current classification of
opioid receptors in four subtypes, that share more than 60%
1. Introduction
Cultivated already 3000 years B.C. by Sumerians, Papaver som- sequence homology: the µ-opioid receptor (MOR), the κ-opioid
niferum was for millennia praised for the analgesic properties receptor (KOR), the δ-opioid receptor (DOR) and the nociceptin
of its dried latex, called opium. Only in 1806 morphine, the opioid receptor (NOR). MOR, KOR and NOR are named after
most active component of opium, was isolated by the pharma- their first discovered ligands, which are morphine, ketocycla-
cist Friedrich Sertürner and named after Morpheus, the Greek zocine and nociceptin, respectively. DOR derives its name
god of sleep and dreams. In the late 1960s it became clear that from the mouse vas deferens tissue where the receptor was
the pharmacological effects evoked by opioids are caused by characterized first.2–5 They belong to class A (rhodopsin-like)
activation of certain receptors, which were called opioid recep- of the Gi/Go protein-coupled receptors (GPCR) superfamily. A
GPCR consists of seven transmembrane helical domains,
which are connected by three extracellular and three intracellu-
aInstitut für Pharmazeutische und Medizinische Chemie der Universität Münster,
lar loops. Whereas the N-terminal domain is located extracellu-
Corrensstraße 48, D-48149 Münster, Germany. E-mail: wuensch@uni-muenster.de;
larly, the C-terminal domain is found intracellularly.6,7 The
Fax: +49-251-8332144; Tel: +49-251-8333311
bGraduate School of Chemistry, Westfälische Wilhelms-Universität Münster,
Wilhelm-Klemm-Straße 10, D-48149 Münster, Germany
extensive use of morphine and other opioids binding to MOR
soon pointed out the limitations of a long-term therapy. Not
only constipation and nausea, but also more serious effects,
such as respiratory depression, tolerance and physical depen-
dence characterize their pharmacological profile.8 Targeting
KOR seemed to be a promising strategy to develop safer opioid
analgesics.9
The localization of KOR in several peripheral cells, tissues
and organs and in the central nervous system explains its role
in numerous pathways. The presence of KOR in the skin made
it a suitable target for the treatment of itching skin diseases.10
cGRK 2515, Chemical biology of ion channels (Chembion),
Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany
dOrganisch-Chemisches Institut, Westfälische Wilhelms-Universität Münster,
Corrensstraße 40, D-48149 Münster, Germany
†Electronic supplementary information (ESI) available: The purity data of all test
compounds including the corresponding methods, NOE difference spectrum of
10 and NOESY spectra of 35b and 36, crystallographic details of 28, synthesis of
acid chlorides, details of the receptor binding studies, 1H and 13C NMR spectra
of all synthesized compounds and HPLC chromatograms of all test compounds.
CCDC 2063296. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/d1ob00398d
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The localization in immune cells, combined with the fact that
there is an upregulation of opioid receptors during inflam-
mation, provided a new strategy to treat chronic inflammatory
diseases, e.g. rheumatoid arthritis and multiple sclerosis.11
KOR plays a central role in analgesia, since it is widely spread
in the dorsal horn of the spinal cord, where the nociceptive
information is received by primary afferents, and in many
areas of the brain, such as periaqueductal gray and thalamus,
the centers for the elaboration of the ascending nociceptive
information and subsequent descending response.12 The
involvement of KOR in emotions, stress, self-consciousness
and addiction is explained by its location in the limbic system
and cortex.13 While KOR activation leads to severe side effects
such as dysphoria, hallucination, dissociation, sedation and
strong diuresis,14 great efforts have been made to develop KOR
antagonists for migraine prevention15 and for the treatment of
depression, addiction and anxiety disorders.16–18 In order to
pursue the development of safe KOR agonists for the treatment
of pain, two aspects have to be considered. (1) KOR agonists,
which cannot penetrate the blood–brain barrier, remaining
Fig. 1 Prototypical KOR agonists belonging to the class of
ethylenediamines.
peripherally restricted, are devoid of the centrally mediated between the amide and Gln115 and hydrophobic interactions
side effects mentioned above. (2) The second aspect is related between the phenyl ring and the same amino acids that inter-
to the biased agonism. Each ligand stabilizes a specific confor- act with the iodophenyl moiety of MP1104.31 It is expected that
mation of a receptor, which will favor the binding to G pro- GR-89 696 (5), one of the most potent KOR agonists with a Ki
teins, β-arrestins or other signaling molecules, affecting the value of 0.45 nM,32–34 enters into the same binding pocket as
corresponding pharmacological effect.19 It was demonstrated U-50 488 (1), since both molecules contain the same pharma-
that activation of p38 MAPK signaling due to the binding of cophoric key substructure: a pyrrolidine ring connected via an
dynorphin to KOR, is responsible for dysphoria.20
ethylene linker to a (3,4-dichlorophenyl)acetamide moiety.
Due to the adjacent acetamide (allylic strain), the pyrrolidi-
KOR agonists are structurally divided in four classes: pep-
tides derived from the endogenous agonist dynorphin A,21 nylmethyl moiety adopts the axial position at the piperazine
morphinoids including morphinans and benzomorphans, e.g. ring of GR-89 696 (5) and analogs.34,35 However, this pyrrolidi-
ketocyclazocine,22,23 terpenoids such as salvinorin A, a disso- nylmethyl moiety can freely rotate along the marked C–C-bond
ciative and hallucinogenic natural product present in Salvia and the basic pyrrolidine ring, responsible for the crucial ionic
divinorum,24 and ethylenediamines, represented by the proto- interaction with Asp138 of KOR, can adopt several orien-
type U-50 488 (1), the first synthetic KOR agonist with high tations.35 Therefore, a conformational restriction of this substi-
selectivity.25,26 The relative simple pharmacophore of the ethy- tuent leading to a defined dihedral angle between the N-atoms
lenediamine class inspired the synthesis of several derivatives of the pyrrolidine and piperazine rings was designed to
(Fig. 1). ICI-199 441 (2) is 275-fold more potent and more selec- analyze the bioactive conformation of GR-89 696 (5). (Fig. 2)
tive than 1, but its analgesic activity is, unfortunately, centrally Synthesis and pharmacological evaluation of bridged pipera-
mediated and followed by sedative effects.27 Spiradoline (3) zines of type 6 led to KOR affinity of >1 µM and 73 nM for 6
also shows disturbing side effects such as dysphoria, strong with an exo- and endo-configured pyrrolidino moiety, respect-
diuresis and sedation at doses, which are lower than needed ively. The more potent endo-derivative showed a dihedral angle
for the analgesic activity. Moreover, 3 showed antitussive and of 58° (N(pyrrolidine)–C–C–N(amide))36 (Fig. 2).
neuroprotective properties in an animal model of
The question arose, whether the additional basic amino
ischemia.28,29 The only peripherally acting asimadoline (4), moiety in 1-position of 6 (bridgehead N-atom) corresponding
conversely, does not elicit the cited side effects and showed to the carbamate moiety in 5 or an unfavorable dihedral angle
pain relief in a clinical trial for irritable bowel syndrome.30
are responsible for the reduced KOR affinity of 6 compared to
Crystallization of human KOR in complex with epoxymor- 5. Therefore, the bicyclic analog 8 of 6 was designed, where
phinan MP1104, a potent KOR agonist could be achieved with the bridgehead N-atom of 6 was replaced by a C-atom. Bicyclic
the nanobody technology and allowed to study the 3D struc- analogs with a bridgehead C-atom (e.g. 8) are of high interest,
ture of the binding pocket.31 In agreement with previous since the corresponding monocyclic piperidine derivative 7 is
investigations, a key feature is the anchoring in the binding also a potent KOR agonist (Ki = 0.24 nM).37 Herein, we provide
pocket through an ionic interaction (salt bridge) of a basic the diastereoselective synthesis and pharmacological evalu-
amino moiety to Asp138 in TM3.32 A docking study of the ation of conformationally constrained KOR agonists of type 8
prototypical KOR agonist U-50 488 (1) using the active-state with a bridgehead C-atom (Fig. 2). Additionally, the smaller
KOR structure showed two additional interactions: an H-bond bridge containing only two C-atoms results in a modified di-
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Scheme 1 Synthesis of primary amine 14. Reagents and reaction con-
ditions: (a) 1. KMnO4, CH3COCH3, H2O, −18 °C, 1.5 h; 2. 2,2-dimethoxy-
propane, p-toluenesulfonic acid, rt, 16 h, 47%. (b) LiBH4, THF, rt, 16 h,
93%. (c) CH3SO2Cl, DIPEA, CH2Cl2, rt, 2 h, 94%. (d) NaCN, DMSO, 110 °C,
2 h, 99%. (e) LiAlH4, THF, 0 °C → rt, 16 h, 94%.
Fig. 2 Development of new conformationally constrained KOR agonists
of type 8 based on the ethylenediamine pharmacophore. dh = dihedral
angle N(pyrrolidine)–C–C–N(amide).
hedral angle of the KOR pharmacophoric ethylenediamine
system of 8.
In literature only a few syntheses of 2-azabicyclo[3.2.1]
octanes are reported.38,39 Ring expansion of norbornadiene
using tosyl azide provided the azabicyclic framework, which
was further transformed into a polyhydroxylated system.39
A
bicyclic system containing an additional O-atom within the
ring system was obtained by an aza-Prins reaction of N-tosyl
homoallylamine and O,O-dibenzylglyceraldehyde.40 Both
routes do not allow the establishment of the required substi-
tution pattern. Therefore, we developed a novel synthetic route
to prepare 2-azabicyclo[3.21.]octan-7-amines with various sub-
stituents at both N-atoms.
Fig. 3 Nuclear Overhauser effect (NOE) between the indicated protons
confirms the trans-orientation of the ester group relative to the cis-
annulated 1,3-dioxolane ring of 10.
other acetalic CH3 group (δ = 1.20 ppm) with the two methine
protons (δ = 4.61 ppm). The NOE difference spectrum and the
NOESY spectrum are displayed in Fig. S1 (ESI†).
2. Synthesis
In order to obtain the desired bridged piperidines 8 with the
The following modifications were performed to elongate
desired substitution pattern, two synthetic routes with six the ester moiety by one C-atom to obtain 14 with an amino-
common steps at the beginning were investigated (Scheme 1). ethyl side chain. Thus, reduction of the ester 10 with LiBH4
Starting with methyl cyclopent-3-ene-1-carboxylate 9, a cis-dihy- provided the primary alcohol 11, which reacted with mesyl
droxylation with KMnO4 led to a diol, which was not isolated. chloride to yield the mesylate 12. Nucleophilic substitution
However, the color shift of the reaction mixture from violet to with NaCN led to the nitrile 13 introducing an additional
dark brown confirmed the transformation. The cis-configured C-atom into the side chain. The last common step was the
diol was directly protected with 2,2-dimethoxypropane to reduction of the nitrile 13 with LiAlH4 to obtain the primary
afford acetonide 10 with an overall yield of 47%.
The trans-configuration of the ester and the O-heterocycle compounds with a (s)-configured pseudochiral center.
was confirmed by nuclear Overhauser effect (NOE) experi-
According to the first synthetic route, the pharmacophoric
amine 14 in 94% yield. Bicyclic compounds 10–14 are achiral
ments (Fig. 3). Irradiation with the resonance frequency of the 3,4-dichlorophenylacetyl group was introduced very early.
CH proton adjacent to the ester moiety (δ = 2.82–2.89 ppm) led Thus, the primary amine 14 was acylated with 2-(3,4-dichloro-
to an increased signal of one of the acetalic CH3 groups (δ = phenyl)acetyl chloride in the presence of ethyldiisopropyl-
1.33 ppm) in the NOE difference spectrum indicating the amine (DIPEA) yielding the amide 15 in 60% yield (Scheme 2).
proximity of these substituents. A NOESY spectrum confirmed The subsequent two steps were performed to introduce a good
this observation and additionally showed the proximity of the leaving group suitable for intramolecular nucleophilic substi-
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Scheme 2 Synthesis of monotosylate 17 and ditosylate 18 and attempts to establish the 2-azabicyclo[3.2.1]octane framework. Reagents and reac-
tion conditions: (a) 2-(3,4-dichlorophenyl)acetyl chloride, DIPEA, CH2Cl2, 0 °C → rt, 16 h, 60%. (b) CH3CO2H, CH3OH, 80 °C, 7 h, 55%. (c)
p-Toluenesulfonyl chloride (TsCl), DIPEA, 4-DMAP, CH2Cl2, rt, 16 h, 52% (17), 26% (18). (d) NaH, THF, rt, 2 d, 0% (19). (e) Dess–Martin periodinane
(DMP), CH2Cl2, rt, 2 h, 82%. (f) NaH, THF, rt, 3 h, 78% (22). Ts = p-tolylsulfonyl (H3CC6H4SO2–). Compounds 15, 16 and 18 are not chiral, but possess
a pseudochiral center with (s)-configuration. Compounds 17 and 19–22 are racemic chiral compounds. For purpose of clarity only one enantiomer
of the racemic mixtures is depicted.
tution by the amide moiety. At first, the acetonide 15 was
Although the early introduction of the KOR pharmacopho-
hydrolyzed with acetic acid in methanol affording the diol 16. ric dichlorophenylacetyl moiety appeared straightforward, as it
Subsequent tosylation of diol 16 with p-toluenesulfonyl chlor- can remain in the final KOR agonists, it was concluded that
ide (TsCl) in the presence of DIPEA and 4-(dimethylamino)pyr- the dichlorophenylacetyl moiety with the rather acidic CH2-
idine (DMAP) led to monotosylate 17 and ditosylate 18. Since moiety between the CvO-group and the phenyl ring is not
tosyl chloride cannot differentiate between the two OH-moi- appropriate to establish bicyclic compounds. Therefore, the
eties of the symmetric diol 16, a racemic mixture of the mono- primary amine 14 was protected and activated by transform-
tosylate 17 was obtained.
ation into the sulfonamide 23 (Scheme 3). The acetonide of 23
The intramolecular nucleophilic substitution of one tosy- was hydrolyzed with concentrated HCl to give the diol 24,
loxy moiety of the pseudochiral ditosylate 18 by the amide was which was reacted with an excess of p-toluenesulfonyl chloride
attempted with NaH in THF (Scheme 2). However, even after 2 to provide the ditosylate 25 in 50% yield. In contrast to the
days, a conversion of the ditosylate 18 could not be observed. tosylation of diol 16, the tosylation of the diol 24 with a sulfon-
This transformation was repeated using various bases and amide in the side chain did not stop at the monotosylated
reaction conditions, but all attempts failed to provide the product. Treatment of the sulfonamide 25 with NaH led to
bicyclic system 19.
deprotonation and the anion reacted in an intramolecular SN2
Therefore, the racemic monotosylate 17, which was isolated reaction to form the bicyclic compound 26, which was
in even higher yields than ditosylate 18, was oxidized with obtained in 72% yield. The trans-orientation of the tosylami-
Dess–Martin periodinane to obtain the ketone 20 in 78% yield noethyl moiety relative to the tosyloxy groups in 25 ((s)-con-
(Scheme 2). Establishment of the bicyclic system by intra- figuration) is a crucial requirement for the successful trans-
molecular nucleophilic substitution of the α-tosyloxyketone 20 formation. Whereas the trisubstituted cyclopentane derivatives
with NaH failed to give 21. Instead, the ketone 22 was isolated 23–25 are achiral compounds with a (s)-configured pseudo-
in 78% yield. The structure of ketone 22 was unequivocally chiral center, the cyclization of 25 to 26 breaks the symmetry
shown by the presence of the additional 4-CH2 moiety in the leading to a racemic mixture.
1H and 13C NMR spectra, 2D-NMR spectra (gHSQC NMR
In the product 26 the remaining OTs moiety was already
spectra) and by MS analysis. The unexpected formation of the activated for the nucleophilic substitution with pyrrolidine. At
ketone 22 was explained by nucleophilic attack of hydride first 26 was reacted with pyrrolidine in the presence of K2CO3
from NaH at the C-atom adjacent to the CvO-moiety leading in DMSO at 90 °C (Scheme 3). The main product of this trans-
to substitution of the tosyloxy moiety.
formation was the carbamate 28, which was isolated in 42%
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Scheme 3 Synthesis of bridged piperidines. Reagents and reaction conditions: (a) p-toluenesulfonyl chloride, pyridine, CH2Cl2, rt, 16 h, 71%. (b) HCl
conc., CH3OH, rt, 16 h, 64%. (c) p-Toluenesulfonyl chloride, DIPEA, 4-DMAP, CH2Cl2, 0 °C → rt, 16 h, 50%. (d) NaH, THF, 0 °C → rt, 3 d, 72%. (e)
Pyrrolidine, K2CO3, DMSO, 90 °C, 7 h, 12% (27), 42% (28), 26% (30). (f) Pyrrolidine, K2CO3, CH3CN, 80 °C, 9 h, 26% (29), 11% (30). Ts = p-tolylsulfonyl
(H3CC6H4SO2–). Pseudochiral compounds 23–25 are (s)-configured. For purpose of clarity only one enantiomer of the racemic mixtures 26–29 is
depicted.
yield. The 1H and 13C NMR spectra of 28 are very similar to the
spectra of the expected substitution product with pyrrolidine.
However, the relative exo-configuration of the substituent in
7-position of carbamate 28, which is the same configuration as
The vapor diffusion method led to colorless crystals of 28 in the tosylate 26, was unexpected. This exo-configuration
suitable for X-ray crystal structure analysis, which confirmed might originate from a neighbor group participation of the sul-
unequivocally the structure of the carbamate 28 (Fig. 4). fonamide leading to a double inversion. Alternatively, a SN1
Carbamate 28 crystallized as a racemic mixture in the centro- reaction, where the intermediate carbenium ion was attacked
ˉ
symmetric space group P1 of the triclinic crystal system. from the less hindered side could result in retention of con-
Moreover, the X-ray crystal structure shows the bicyclic struc- figuration. The formation of the carbamate was explained by
ture of the product 28, proving that the intramolecular nucleo- the reaction of pyrrolidine with CO2 released from K2CO3 and
philic substitution of 25 to produce 26 had been successful. subsequent nucleophilic attack of the formed carbamate anion
The dihedral angle (O1–C7–C1–N1) between the carbamate at the carbenium ion.
unit and the N-tosylamino group is 169.0(1)°.
In addition to the carbamate 28, the ketone 27 (12%) and
the 1-tosylpyrrolidine (30, 26%) were isolated. The ketone 27
could be formed by a Kornblum-like oxidation. After nucleo-
philic replacement of tosylate by the O-atom of dimethyl sulf-
oxide, deprotonation and elimination of dimethyl sulfide will
lead to the ketone 27. Alternatively, deprotonation in
α-position of the tosyloxy moiety of 26 and subsequent elimin-
ation of 4-methylphenylsulfinate could result in the ketone 27.
Attack of pyrrolidine at the S-atom of the sulfonate 26 could
afford 1-tosylpyrrolidine (30).
In order to avoid the oxidation of the tosylate 26, the
nucleophilic substitution with pyrrolidine was repeated in
acetonitrile instead of DMSO. After 9 h, the alcohol 29 and
1-tosylpyrrolidine (30) originating from the nucleophilic attack
of pyrrolidine at the sulfonyl moiety were isolated (Scheme 3).
The desired bicyclic compound containing the pyrrolidinyl
moiety could not be detected or isolated.
The results from the experiments with pyrrolidine led to
the conclusion that a stronger nucleophile was required.
Therefore, the tosylate 26 was reacted with NaN3 in DMSO to
afford the azide 31 in 74% yield. Although the reaction con-
ditions were optimal for a SN2 reaction, the formation of azide
31 occurred with retention of configuration. LiAlH4 reduction
Fig. 4 X-ray crystal structure of carbamate 28 proving the exo-
configuration of the substituent in 7-position. Thermal ellipsoids are set
at 30% probability. Only one enantiomer of the racemic mixture is
shown. CCDC number: 2063296.†
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Scheme 4 Synthesis of KOR agonists 35 and 36. Reagents and reaction conditions: (a) NaN3, DMSO, 100 °C, 2 h, 74%. (b) LiAlH4, THF, 0 °C, 2 h,
97%. (c) 1,4-Diiodobutane, DIPEA, CH3CN, 80 °C, 16 h, 73% (33a). (d) Formalin 37%, NaBH3CN, CH3CO2H, CH3OH, rt, 4 h, 63% (33b). (e) Mg turnings,
CH3OH, reflux, ultrasonic bath, 3 h. (f) NaOH, 2-(3,4-dichlorophenyl)acetyl chloride, THF, H2O, 0 °C → rt, 22% (35a), 17% (35b). (g) NaOH, 2-(3,4-
difluorophenyl)acetyl chloride, THF, H2O, 0 °C → rt, 48%. Ts = p-tolylsulfonyl (H3CC6H4SO2–). All compounds in Scheme 4 are racemic mixtures of
chiral compounds. For purpose of clarity only one enantiomer of the racemic mixtures is depicted.
of the azide 31 led to the primary amine 32, which was alkyl- receptor. The non-specific binding was determined using a
ated with 1,4-diiodobutane to provide the pyrrolidine 33a. large excess of non tritium-labeled U-69 593.34 The KOR
Additionally, the dimethylamine 33b was obtained by reductive affinity values of the bicyclic compounds and some reference
alkylation of the primary amine 32 with formalin and compounds are summarized in Table 1.42–45
NaBH3CN (Scheme 4).
The negligible KOR affinity of sulfonamides 33a and 33b
indicates that replacement of the 3,4-dichlorophenylacetyl
moiety by a tosyl residue is not tolerated by KOR. Nevertheless,
the phenylacetamides 35a and 35b show weak KOR affinity (Ki
= 862 nM and 2400 nM), respectively. The fact that they consti-
tute racemic mixtures and not pure enantiomers cannot
explain the rather low KOR affinity. Since KOR affinity depends
considerably on the stereochemistry, the relative orientation of
the two pharmacophoric elements to each other is of high rele-
vance. It is assumed that the exo-configuration of 35a, 35b and
36 is not favorable for high KOR affinity. Replacement of the
pyrrolidino moiety (35a) by a dimethylamino group (35b) led
to a reduction of KOR affinity. F-substituents (36) instead of Cl-
substituents (35a) reduced the KOR affinity, considerably. Even
at a test compound concentration of 10 µM the difluoro deriva-
tive 36 led only to 11% inhibition of the radioligand binding.
After the establishment of the pyrrolidine ring as first KOR
pharmacophoric structural element, the final introduction of
the second KOR pharmacophoric structural element was per-
formed in a consecutive two-step procedure (Scheme 4). The
tosyl moiety of the sulfonamides 33a and 33b was cleaved off
reductively with Mg0 in methanol and the resulting secondary
amines 34a and 34b were directly acylated with 3,4-dichloro-
phenylacetyl chloride or 3,4-difluorophenylacetyl chloride to
provide the amides 35a, 35b and 36, respectively. The acylation
of the secondary amines 34a and 34b was performed under
Schotten–Baumann conditions.41
The relative configuration of the final dimethylamine 35b
and the pyrrolidine 36 was investigated by NOESY experi-
ments. NOE effects between 7-H and 3-Haxial indicated the
unexpected exo-configuration of the dimethylamino moiety of
35b and the pyrrolidino moiety of 36 (Fig. S2–S4, ESI†). Since
the relative exo-configuration was established during the intro-
duction of the azide (31), either a neighbor group participation
of the sulfonyl moiety (oxazolidine intermediate) leading to
double inversion or a SN1 reaction with favored attack via the
less hindered side were postulated to explain the formation of
the azide 31. The same observations were made during the for-
mation of the carbamate 28.
3.2. Affinity towards MOR, DOR and σ receptors
In order to determine the selectivity of the synthesized com-
pounds, the affinity towards opioid receptors MOR and DOR
was investigated in receptor binding studies.42–45 Up to a test
compound concentration of 10 µM the test compounds 33, 35
and 36 could not compete with the radioligands for MOR and
DOR. At least the low-KOR-affinity compounds 35a and 35b
demonstrate a slight preference for KOR over MOR and DOR.
In addition to the affinity at opioid receptors MOR and
DOR, the interaction with σ receptors was considered, as the σ
receptor was originally classified as third opioid receptor.2
Moreover, modification of the stereochemistry of potent and
3. KOR affinity and selectivity
3.1. KOR affinity
Competitive receptor binding assays were performed in order selective KOR agonists (e.g. U-50 488) resulted in high σ
to establish the KOR affinity of the synthesized bridged piper- affinity.46
idines. Tritium-labeled [3H]U-69 593 was used as radioligand
In receptor binding studies,47–49 the synthesized bicyclic
and membrane preparations of Guinea pig brains provided the compounds displayed considerable affinity toward both σ
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Table 1 Affinities of bicyclic KOR agonists and reference compounds towards KOR and related receptors
Ki SEM [nM]/% inhibition at 1 µMa,b
KOR
MOR
DOR
σ1
σ2
Compd.
NR2
X
[3H]U-69 593
[3H]DAMGO
[3H]DPDPE
(+)-[3H]pentazocine
[3H]DTG
33a
33b
35a
35b
N(CH2CH2)2
N(CH3)2
N(CH2CH2)2
N(CH3)2
—
—
Cl
Cl
F
0%
0%
862
2400
4%
0.34 0.07
7.3 0.40
—
—
—
—
0%
0%
0%
0%
5%
—
2.3 1.1
5.2 1.6
—
0%
0%
0%
0%
0%
—
103
—
1.2 0.5
—
109
157
208
559
867
—
—
—
1.8 µM
10%
167
402
1300
—
—
—
—
—
36
N(CH2CH2)2
U-50 488
Naloxone
Morphine
SNC80
(+)-Pentazocine
Haloperidol
—
5.4 0.5
6.6 0.9
—
—
—
78 2.3
a A value in % reflects the inhibition of the radioligand binding at a test compound concentration of 1 μM. Ki values without SEM values rep-
resent the mean of two experiments (n = 2) and Ki values with SEM values represent the mean of three experiments (n = 3). b Guinea pig brain
membrane preparations were used in the KOR, MOR and σ1 assay. In the DOR assay rat brain and in the σ2 assay rat liver membrane preparations
were used.42–45
receptor subtypes. In particular, 35a and 35b show higher
affinity towards both σ receptor subtypes compared to KOR.
The σ1 receptor affinity of the sulfonamides 33a (Ki = 109 nM)
and 33b (Ki = 157 nM), which do not interact with opioid
receptors, should be emphasized. However, even the Ki values
of the most potent σ receptor ligands 33a (σ1) and 35a (σ2) are
greater than 100 nM.
Fig. 5 Calculated dihedral angles of exo-configured pyrrolidine 35a
and its corresponding endo-configured diastereomer endo-35a.
4. Discussion
In order to obtain conformationally constrained KOR agonists
with the ethylenediamine pharmacophore, bicyclic analogs of
The dihedral angles were calculated using MOE50 and
KOR agonist 7 were designed and prepared in a multi-step syn-
thesis. For the establishment of the bicyclic system by intra-
using the in MOE implemented MMFF94× force field. Based
molecular nucleophilic substitution, the sulfonamide 25
turned out to be the key intermediate, which provided the
hedral angles were calculated, since the orientation of the pyr-
2-azabicyclo[3.2.1]octane 26 in 72% yield. However, this syn-
thetic route required two additional reaction steps, since the
energy-minimized compounds. The minimization was done
on the orientation of the pyrrolidine ring, slightly different di-
rolidine ring slightly influences the energy minimized struc-
ture. The dihedral angles for endo-35a are 44°/54°, and for exo-
tosyl moiety had to be replaced by the second KOR pharmaco-
phoric element, the (3,4-dihalophenyl)acetyl moiety at the end
of the synthesis.
Unexpectedly, the nucleophilic substitution of tosylate 26
with NaN3 occurred with retention of configuration leading to
the exo-configured azide 31 and, subsequently, to exo-config-
ured amines 32–36. This exo-configuration determines the di-
hedral angle of the ethylenediamine pharmacophore
(N(pyrrolidine)–C–C–N(acetyl)), which has a great impact on
the pharmacological activity (Fig. 5).
35a 137°/141° (Fig. 5). In previous studies with the bridged
piperazines 6, the more active KOR agonist endo-6 (Ki = 73 nM)
displayed a smaller dihedral angle (58°, X-ray) than the less
active exo-configured diastereomer exo-6 (Ki > 1 µM, dihedral
angle 180°).36 This observation fits well to the results obtained
for bridged piperidines 35 in this report. As exo-6, the exo-con-
figured diastereomers exo-35 with a large dihedral angle of
137°/141° along the C–C-bond of the central N(pyrrolidine)–C–
C–N(acetyl) moiety show KOR affinity in the high nanomolar
to low micromolar range. The exact size of the dihedral angles
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of 6 and 35 differs slightly due to the different length of the with 0.5 Hz resolution; assignment of 1H and 13C NMR signals
second bridge (3 C-atoms versus 2 C-atoms). The dihedral was supported by 2-D NMR techniques where necessary. IR:
angle O–C–C–N(tosyl) (169.0(1)°) derived from the X-ray crystal FT/IR IR Affinity®-1 spectrometer (Shimadzu, Düsseldorf,
structure of carbamate exo-28 (see Fig. 4) is in the same range Germany) using ATR technique. The X-ray structure was
as the calculated dihedral angle of 35a (137°141°). According measured by the Bruker D8 Venture Photon 100 CMOS diffract-
to these considerations, it is expected that the endo-configured ometer (details see ESI†). The CCDC number of the X-ray
diastereomer endo-35 with a calculated dihedral angle of 44°/ crystal structure of 28 is 2063296.†
54° should exhibit higher KOR affinity than exo-35 presented
herein.
6.2. Synthetic procedures
The rather high σ receptor affinities of 35a and 35b also cor-
6.2.1. Methyl (5S)-2,2-dimethyl-4,5,6,6a-tetrahydro-3aH-
relate well with literature reports. It has been shown that chan- cyclopenta[d][1,3]dioxole-5-carboxylate (10). A solution of
ging the trans-configuration of U-50 488 into cis-configuration methyl cyclopent-3-enecarboxylate 9 (5.0 g, 40 mmol) in
reduces the KOR affinity and increases the σ affinity.46 acetone (125 mL) and H2O (35 mL) was cooled down to
Moreover, the enantiomers of benzomorphan-type KOR ago- −18 °C. KMnO4 (6.9 g, 44 mmol, 1.1 eq.) was slowly added to
nists (e.g. ethylketocyclazocine) do not exhibit KOR affinity, but the solution. After stirring at −18 °C for 1.5 h, the precipitate
high σ affinity (e.g. (+)-pentazocine).51 As discussed above, a was filtered off. 2,2-Dimethoxypropane (390 mL, 3.2 mol) and
higher KOR affinity is expected for the stereoisomers endo-35. p-toluenesulfonic acid (2.0 g, 12 mmol) were added to the
Thus the diastereomers exo-35 with lower KOR affinity should mixture. After stirring at room temperature for 16 h, a satu-
possess considerable σ receptor affinity.
rated NaHCO3 solution (20 mL) was added, and the organic
solvent was removed in vacuo. The remaining aqueous layer
was extracted with CH2Cl2 (2 × 20 mL) and the combined
CH2Cl2 layers were washed with H2O and brine (each 20 mL).
The combined organic layers were dried (Na2SO4), filtered and
5. Conclusion
The design of conformational restriction of the ethylenedia- the solvent was removed in vacuo. The residue was purified by
mine KOR pharmacophore led to 2-azabicyclo[3.2.1]octan-7- flash column chromatography (Ø = 4 cm, h = 15 cm, V =
amines 35 and 36. An unexpected retention of configuration 30 mL, cHex/EtOAc = 2 : 1, Rf = 0.35). Colorless solid, mp
during nucleophilic substitution with azide provided exo-con- 36 °C, yield 3.7 g (47%). C10H16O4 (200.2 g mol−1). 1H NMR
figured products. A dihedral angle of 137°/141° along the C–C- (600 MHz, DMSO-d6): δ (ppm) = 1.20 (s, 3H, CH3CCH3), 1.33
bond of the central N(pyrrolidine)–C–C–N(acetyl) unit resulted (s, 3H, CH3CCH3), 1.59–1.66 (m, 2H, CH2CHCH2), 1.90–1.95
in high-nanomolar to micromolar KOR affinity. It is postulated (m, 2H, CH2CHCH2), 2.82–2.89 (m, 1H, CH2CHCH2), 3.60 (s,
that endo-configured diastereomers with a dihedral angle of 3H, C(vO)OCH3), 4.61 (dt, J = 5.5/2.9 Hz, 2H, OCHCHO). 13C
44°/54° should fit better into the KOR binding pocket.
NMR (151 MHz, DMSO-d6): δ (ppm) = 23.7 (1C, CCH3), 26.0
(1C, CCH3), 36.3 (2C, CH2CHCH2), 40.1 (1C, CH2CHCH2), 51.5
(1C, C(vO)OCH3), 79.3 (2C, OCHCHO), 108.1 (1C, C(CH3)2),
174.4 (1C, C(vO)OCH3). IR (neat): ν (cm−1) = 2944 (C–Haliph),
1730 (CvOester), 1193 (O–CH3 ester), 1038 (O–Cketal). Exact mass
(APCI): m/z = 201.1107 (calcd 201.1121 for C10H17O4 [M + H]+).
6. Experimental
6.1. Chemistry general methods
Moisture and oxygen sensitive reactions were carried out Purity (qNMR): 98.9%.
under nitrogen, dried with molecular sieves (3 or 4 Å, 8 to
6.2.2. [(5S)-2,2-Dimethyl-4,5,6,6a-tetrahydro-3aH-cyclo-
12 mesh, Acros Organics), in dry glassware (Schlenk flasks or penta[d][1,3]dioxol-5-yl]methanol (11). Under N2 atmosphere,
Schlenk tubes, sealed with rubber septa). All solvents were of ester 10 (2.4 g, 12 mmol) was dissolved in dry THF (50 mL)
analytical grade quality. Flash chromatography (FC): silica gel and LiBH4 (1.6 g, 73 mmol, 6 eq.) was added. After stirring the
60, 40–63 µm (Machery Nagel); parentheses include: diameter mixture at room temperature for 16 h, the solvent was removed
of the column (Ø), length of the stationary phase (h), fraction in vacuo. H2O (40 mL) was added and the mixture was
size (V) and eluent. Melting point: Melting point system MP50 extracted with CH2Cl2 (1 × 40 mL). Then, 1 M HCl was added
(Mettler Toledo, Gießen, Germany), open capillary, uncor- to the aqueous layer to reach a pH of 4 and the mixture was
rected. MS: MicroTOFQII mass spectrometer (Bruker extracted again with CH2Cl2 (3 × 40 mL). The combined
Daltonics, Bremen, Germany); deviations of the found exact organic layers were dried (Na2SO4), filtered and the solvent was
masses from the calculated exact masses were 5 ppm or less; removed in vacuo. The residue was purified by flash column
the data were analyzed with DataAnalysis® (Bruker Daltonics). chromatography (Ø = 4 cm, h = 15 cm, V = 30 mL, cHex/EtOAc
NMR: NMR spectra were recorded in deuterated solvents on = 1 : 2, Rf = 0.28). Colorless oil, yield 1.9 g (93%). C9H16O3
Agilent DD2 400 MHz and 600 MHz NMR spectrometers (172.2 g mol−1). 1H NMR (400 MHz, DMSO-d6): δ (ppm) =
(Agilent, Santa Clara CA, USA); chemical shifts (δ) are reported 1.13–1.23 (m, 2H, CH2CHCH2), 1.19 (s, 3H, CH3CCH3), 1.32 (s,
in parts per million (ppm) against the reference substance 3H, CH3CCH3), 1.71 (dd, J = 14.0/5.8 Hz, 2H, CH2CHCH2),
tetramethylsilane and calculated using the solvent residual 2.11–2.24 (m, 1H, CH2CHCH2), 3.35 (t, J = 5.6 Hz, 2H, CH2OH),
peak of the undeuterated solvent; coupling constants are given 4.42 (t, J = 5.3 Hz, 1H, OH), 4.53–4.57 (m, 2H, OCHCHO).
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13C NMR (101 MHz, DMSO-d6): δ (ppm) = 23.7 (1C, CCH3), sphere, a solution of LiAlH4 (0.57 g, 15 mmol, 2 eq.) in dry
26.1 (1C, CCH3), 36.0 (2C, CH2CHCH2), 38.8 (1C, CH2CHCH2), THF (5 mL) was slowly added to a solution of nitrile 13 (1.4 g,
63.4 (1C, CH2OH), 79.9 (2C, OCHCHO), 107.9 (1C, C(CH3)2). IR 7.7 mmol) in dry THF (65 mL) at 0 °C. The mixture was stirred
(neat): ν (cm−1) = 3405 (O–H), 2920 (C–Haliph), 1023 (O–Cketal). at room temperature for 16 h. At 0 °C the excess of LiAlH4 was
Exact mass (APCI): m/z = 173.1136 (calcd 173.1172 for C9H17O3 destroyed by addition of 5 pieces of ice and the mixture was
[M + H]+). Purity (qNMR): 96%.
stirred for 10 min at 0 °C and, then, heated up to reflux for
6.2.3. [(5S)-2,2-Dimethyl-4,5,6,6a-tetrahydro-3aH-cyclo- 30 min. The white precipitate was filtered off and the solvent
penta[d][1,3]dioxol-5-yl]methyl methanesulfonate (12). Under was removed in vacuo. Pale yellow oil, yield 1.3 g (94%). Rf =
N2 atmosphere, primary alcohol 11 (1.9 g, 11 mmol) was dis- 0.24 (CH2Cl2/CH3OH/NH3 25% = 88 : 9 : 3). C10H19NO2 (185.3 g
solved in dry CH2Cl2 (50 mL) and ethyldiisopropylamine mol−1). 1H NMR (600 MHz, CDCl3): δ (ppm) = 1.11–1.19 (m,
(5.0 g, 39 mmol) and mesyl chloride (3.8 g, 33 mmol, 3 eq.) 2H, CH2CHCH2), 1.27 (s, 3H, CH3CCH3), 1.43 (s, 3H,
were added. The mixture was stirred at room temperature for CH3CCH3), 1.80–1.85 (m, 2H, CH2CH2NH2), 1.97 (dd, J = 14.1/
2 h. The excess of mesyl chloride was destroyed by addition of 5.7 Hz, 2H, CH2CHCH2), 2.19 (m, 1H, CH2CHCH2), 2.96–3.02
0.1 M NaOH (20 mL). The aqueous layer was extracted with (m, 2H, CH2CH2NH2), 4.61–4.63 (m, 2H, OCHCHO). Signals for
CH2Cl2 (2 × 20 mL). The combined organic layers were washed the NH2 protons are not seen in the spectrum. 13C NMR
with 1 M HCl (30 mL), dried (Na2SO4), filtered and the solvent (151 MHz, CDCl3): δ (ppm) = 23.8 (1C, CCH3), 26.2 (1C, CCH3),
was removed in vacuo. The residue was purified by flash 32.1 (1C, CH2CH2NH2), 33.7 (1C, CH2CHCH2), 39.4 (1C,
column chromatography (Ø = 4 cm, h = 15 cm, V = 20 mL, CH2CH2NH2), 39.5 (2C, CH2CHCH2), 80.3 (2C, OCHCHO),
cHex/EtOAc = 2 : 1, Rf = 0.44 (cHex/EtOAc = 1 : 2)). Yellow solid, 109.1 (1C, C(CH3)2). IR (neat): ν (cm−1) = 3324 (N–H), 2917 (C–
mp 84 °C, yield 2.6 g (94%). C10H18O5S (250.3 g mol−1). 1H
NMR (400 MHz, DMSO-d6): δ (ppm) = 1.20 (s, 3H, CH3CCH3), (calcd 186.1489 for C10H20NO2 [M + H]+).
Haliph), 1042 (O–Cketal). Exact mass (APCI): m/z = 186.1500
1.23–1.37 (m, 2H, CH2CHCH2), 1.33 (s, 3H, CH3CCH3), 1.79
6.2.6. 2-(3,4-Dichlorophenyl)-N-{2-[(5S)-2,2-dimethyl-4,5,6,6a-
(dd, J = 13.1/5.7 Hz, 2H, CH2CHCH2), 2.34–2.45 (m, 1H, tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-yl]ethyl}acetamide (15).
CH2CHCH2), 3.16 (s, 3H, OSO2CH3), 4.18 (d, J = 6.3 Hz, 2H, Under N2 atmosphere, ethyldiisopropylamine (0.70 g,
CH2OSO2CH3), 4.57–4.62 (m, 2H, OCHCHO). 13C NMR 5.4 mmol, 2.0 eq.) was added dropwise to a solution of
(101 MHz, DMSO-d6): δ (ppm) = 23.7 (1C, CCH3), 26.0 (1C, primary amine 14 (0.50 g, 2.7 mmol) in dry CH2Cl2 (40 mL) at
CCH3), 35.5 (2C, CH2CHCH2), 35.7 (1C, CH2CHCH2), 36.4 (1C,
0 °C. A solution of 2-(3,4-dichlorophenyl)acetyl chloride
OSO2CH3), 72.3 (1C, CH2OSO2CH3), 79.5 (2C, OCHCHO), 108.1 (0.94 g, 4.2 mmol, 1.6 eq.) in dry CH2Cl2 (10 mL) was slowly
(1C, C(CH3)2). IR (neat): ν (cm−1) = 2921 (C–Haliph), 1347 added to the mixture at 0 °C. After stirring at room tempera-
(OvSvO), 1171 (O–S), 1041 (O–Cketal). Exact mass (APCI): m/z ture for 16 h, 1 M NaOH (13 mL) was added. The organic
= 251.0928 (calcd 251.0948 for C10H19O5S [M + H]+). Purity phase was separated and the aqueous layer was extracted with
(qNMR): 97.1%.
CH2Cl2 (3 × 10 mL). The combined organic layers were dried
6.2.4. 2-[(5S)-2,2-Dimethyl-4,5,6,6a-tetrahydro-3aH-cyclo- (Na2SO4), filtered and the solvent was removed in vacuo. The
penta[d][1,3]dioxol-5-yl]acetonitrile (13). Under N2 atmo- residue was purified by flash column chromatography (Ø =
sphere, a solution of mesylate 12 (2.4 g, 9.7 mmol) in anh. 4 cm, h = 15 cm, V = 20 mL, cHex/EtOAc = 2 : 1, Rf = 0.5
DMSO (4 mL) was added to a solution of NaCN (1.2 g, (CH2Cl2/CH3OH = 9 : 1). Yellow oil, yield 0.61 g (60%).
24 mmol, 2.5 eq.) in anh. DMSO (76 mL) at 85 °C. The mixture C18H23Cl2NO3 (372.3 g mol−1).
was heated up to 110 °C for 2 h. H2O was added (10 mL) and
1H NMR (600 MHz, DMSO-d6): δ (ppm) = 1.00–1.07 (m, 2H,
the mixture was extracted with Et2O (4 × 40 mL). The combined CH2CHCH2), 1.18 (s, 3H, CH3CCH3), 1.29 (s, 3H, CH3CCH3),
organic layers were dried (Na2SO4), filtered and the solvent was 1.41 (q, J = 7.2 Hz, 2H, CH2CH2NH), 1.78 (dd, J = 13.9/5.7 Hz,
removed in vacuo. The residue was purified by flash column 2H, CH2CHCH2), 1.96–2.05 (m, 1H, CH2CHCH2), 3.01–3.05 (m,
chromatography (Ø = 4 cm, h = 15 cm, V = 30 mL, cHex/EtOAc 2H, CH2NH), 3.41 (s, 2H, CH2C(vO)), 4.51–4.54 (m, 2H,
= 1 : 2, Rf = 0.47). Colorless solid, mp 112 °C, yield 1.7 g (99%). OCHCHO), 7.23 (dd, J = 8.3/2.1 Hz, 1H, 6-CHarom), 7.50 (d, J =
C10H15NO2 (181.2 g mol−1). 1H NMR (600 MHz, DMSO-d6): δ 2.1 Hz, 1H, 2-CHarom), 7.54 (d, J = 8.3 Hz, 1H, 5-CHarom), 8.07
(ppm) = 1.20 (s, 3H, CH3CCH3), 1.22–1.29 (m, 2H, CH2CHCH2), (t, J = 5.5 Hz, 1H, NH). 13C NMR (151 MHz, DMSO-d6): δ (ppm)
1.33 (s, 3H, CH3CCH3), 1.83 (dd, J = 13.9/5.7 Hz, 2H, = 23.7 (1C, CCH3), 26.0 (1C, CCH3), 33.3 (1C, CH2CHCH2), 33.4
CH2CHCH2), 2.24–2.32 (m, 1H, CH2CHCH2), 2.59 (d, J = 6.5 Hz, (1C, CH2CH2NH), 38.0 (1C, CH2NH), 39.0 (2C, CH2CHCH2),
2H, CH2CN), 4.57–4.61 (m, 2H, OCHCHO). 13C NMR (151 MHz, 41.1 (1C, CH2CO), 79.8 (2C, OCHCHO), 107.9 (1C, C(CH3)2),
DMSO-d6): δ (ppm) = 19.5 (1C, CH2CN), 23.6 (1C, CCH3), 26.0 129.0 (1C, C-4arom), 129.4 (1C, C-6arom), 130.3 (1C, C-5arom),
(1C, CCH3), 32.3 (1C, CH2CHCH2), 38.1 (2C, CH2CHCH2), 79.6 130.6 (1 C, C-3arom), 131.0 (1C, C-2arom), 137.6 (1C, C-1arom),
(2C, OCHCHO), 108.1 (1C, C(CH3)2), 119.7 (1C, CN). IR (neat): ν 168.9 (1C, C(vO)). IR (neat): ν (cm−1) = 3298 (N–H), 2924 (C–
(cm−1) = 2978 (C–Haliph), 2242 (CN), 1033 (O–Cketal). Exact
Haliph), 1041 (O–Cketal). Exact mass (APCI): m/z = 372.1096
mass (APCI): m/z = 182.1179 (calcd 182.1176 for C10H16NO2 [M (calcd 372.1128 for C18H2435Cl2NO3 [M + H]+). Purity (HPLC):
+ H]+). Purity (qNMR): 98.5%.
71% (tR = 19.8 min).
6.2.5. 2-[(5S)-2,2-Dimethyl-4,5,6,6a-tetrahydro-3aH-cyclo-
6.2.7. 2-(3,4-Dichlorophenyl)-N-{2-[(1S)-3,4-dihydroxycyclo-
penta[d][1,3]dioxol-5-yl]ethan-1-amine (14). Under N2 atmo- pentyl]ethyl}acetamide (16). Acetic acid (10 mL, 0.18 mol) was
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added to a solution of 15 (0.61 g, 1.6 mmol) in CH3OH (7 mL) C-1arom), 145.1 (2C, C-4tosyl), 169.8 (1C, NC(vO)). IR (neat):
and the mixture was stirred for 7 h at 80 °C. 1 M NaOH was ν (cm−1) = 3256 (N–H), 2928 (C–H aliph), 1339 (OvSvO), 814
added to neutralize the mixture (pH 7) and, then, it was (1,4-disubst. arom). Exact mass (APCI): m/z = 640.0955 (calcd
extracted with EtOAc (2 × 30 mL). The combined organic layers 640.0992 for C29H3235Cl2NO7S2 [M + H]+). Purity (HPLC):
were dried (Na2SO4), filtered and the solvent was removed in 96.4% (tR = 23.5 min).
vacuo. The residue was purified by flash column chromato-
Compound 18 (Rf = 0.47 (CH2Cl2/CH3OH = 9 : 1). Yellow oil,
graphy (Ø = 2 cm, h = 15 cm, V = 10 mL, CH2Cl2/CH3OH = 9 : 1, yield 0.23 g (52%)). C22H25Cl2NO5S (486.4 g mol−1). 1H NMR
Rf = 0.25 (CH2Cl2/CH3OH = 9 : 1). Colorless solid, mp 127 °C, (600 MHz, DMSO-d6): δ [ppm] = 1.37–1.51 (m, 4H, CH2CHCH2
yield 0.30 g (55%). C15H19Cl2NO3 (332.2 g mol−1). 1H NMR (2H), CH2CH2NH (2H)), 1.91 (ddd, J = 14.1/8.6/3.9 Hz, 1H,
(400 MHz, DMSO-d6): δ (ppm) = 1.27 (dt, J = 12.8/6.3 Hz, 2H, CH2CHOH), 2.04 (ddd, J = 14.6/9.2/5.5 Hz, 1H, CH2CHOSO2),
CH2CHCH2), 1.35 (q, J = 7.5 Hz, 2H, CH2CH2NH), 1.68 (ddd, J = 2.24–2.32 (m, 1H, CH2CHCH2), 2.46 (s, 3H, CH3), 3.14–3.18 (m,
13.0/8.9/4.1 Hz, 2H, CH2CHCH2), 2.11 (hept, J = 7.8 Hz, 1H, 2H, CH2NH), 3.47 (s, 2H, CH2C(vO)), 4.18–4.22 (m, 1H,
CH2CHCH2), 3.03–2.96 (m, 2H, CH2NH), 3.41 (s, 2H, CH2C CHOH), 4.73 (ddd, J = 7.2/5.5/3.8 Hz, 1H, CHOSO2), 5.35–5.40
(vO)), 3.81–3.87 (m, 2H, OCHCHO), 4.25 (d, J = 4.4 Hz, 2H, (m, 1H, NH), 7.10 (dd, J = 8.2/2.2 Hz, 1H, 6-CHarom), 7.34–7.38
OHCHCHOH), 7.22 (dd, J = 8.3/2.1 Hz, 1H, 6-CHarom), 7.50 (d, J (m, 3H, 2-CHarom, 3-Htosyl, 5-Htosyl), 7.41 (d, J = 8.2 Hz, 1H,
= 2.0 Hz, 1H, 2-CHarom), 7.55 (d, J = 8.3 Hz, 1H, 5-CHarom), 8.02 5-CHarom), 7.80 (d, J = 8.4 Hz, 2H, 2-Htosyl, 6-Htosyl). The signal
(t, J = 5.5 Hz, 1H, NH). 13C NMR (101 MHz, DMSO-d6): δ (ppm) for the OH proton is not seen in the spectrum. 13C NMR
= 31.5 (1C, CH2CHCH2), 36.5 (1C, CH2CH2NH), 37.6 (2C, (151 MHz, DMSO-d6): δ [ppm] = 21.9 (1C, CH3), 31.7 (1C,
CH2CHCH2), 37.8 (1C, CH2NH), 41.1 (1C, CH2C(vO)), 72.7 CH2CHCH2), 34.9 (1C, CH2CHOSO2), 36.5 (1C, CH2CH2NH),
(2C, OCHCHO), 129.0 (1C, C-4arom), 129.4 (1C, C-6arom), 130.2 37.3 (1C, CH2CHOH), 38.7 (1C, CH2NH), 42.8 (1C, CH2C(vO)),
(1C, C-5arom), 130.6 (1C, C-3arom), 131.0 (1C, C-2arom), 137.7 72.9 (1C, CHOH), 83.7 (1C, CHOSO2), 128.0 (2C, C-2tosyl
,
(1C, C-1arom), 168.9 (1C, C(vO)). IR (neat): ν (cm−1) = 3371 (O– C-6tosyl), 128.9 (1C, C-6arom), 130.2 (2C, C-3tosyl, C-5tosyl), 131.0
H, N–H), 3298 (N–CvO), 2936 (C–Haliph). Exact mass (APCI): (1C, C-5arom), 131.4 (1C, C-2arom), 131.8 (1C, C-4arom), 133.1
m/z = 332.0791 (calcd 332.0815 for C15H2035Cl2NO3 [M + H]+). (1C, C-3arom), 133.5 (1C, C-1tosyl), 135.1 (1C, C-1arom), 145.3 (1C,
Purity (HPLC): 94.9% (tR = 15.6 min).
6.2.8. (4S)-4-{2-[2-(3,4-Dichlorophenyl)acetamido]ethyl}- 20.3 min).
cyclopentane-1,2-diyl bis(4-methylbenzenesulfonate) (18) and 6.2.9. (1RS,4RS)-4-{2-[2-(3,4-Dichlorophenyl)acetamido]-
(1RS,2SR,4SR)-4-{2-[2-(3,4-dichlorophenyl)acetamido]ethyl}-2- ethyl}-2-oxocyclopentyl 4-methylbenzenesulfonate (20). Dess–
hydroxycyclopentyl 4-methylbenzenesulfonate (17). Martin periodinane (0.32 g, 0.75 mmol, 1.7 eq.) was added to a
C-4tosyl), 169.8 (1C, C(vO)). Purity (HPLC): 61.6% (tR =
Ethyldiisopropylamine (0.13 g, 1.0 mmol, 1.1 eq.), p-toluene- solution of 17 (0.21 g, 0.44 mmol) in CH2Cl2 (5 mL) and the
sulfonyl chloride (0.22 g, 1.2 mmol, 1.3 eq.) and 4-(dimethyl- mixture was stirred for 2 h at room temperature. A saturated
amino)pyridine (0.066 g, 0.54 mmol, 0.60 eq.) were added to a NaHCO3 solution (5 mL) and two spatulas of Na2S2O3 were
solution of diol 16 (0.30 g, 0.90 mmol) in dry CH2Cl2 (50 mL). added. After stirring the mixture for 15 min, the aqueous layer
The mixture was stirred for 16 h at room temperature. A satu- was extracted with CH2Cl2 (3 × 5 mL). The combined organic
rated NaHCO3 solution (30 mL) was added and the aqueous layers were dried (Na2SO4), filtered and the solvent was
layer was extracted with CH2Cl2 (2 × 30 mL). The combined removed in vacuo. The residue was purified by flash column
organic layers were dried (Na2SO4), filtered and the solvent was chromatography (Ø = 2 cm, h = 15 cm, V = 10 mL, CH2Cl2/
removed in vacuo. The residue was purified by flash column CH3OH = 99 : 1, Rf = 0.48 (CH2Cl2/CH3OH = 9 : 1)). Yellow oil,
chromatography (Ø = 3 cm, h = 15 cm, V = 10 mL, cHex/EtOAc = yield 0.17 g (82%). C22H23Cl2NO5S (484.4 g mol−1). 1H NMR
3 : 1). At first bistosylated 17, then monotosylated 18 was eluted. (400 MHz, CDCl3): δ (ppm) = 1.54–1.56 (m, 2H, CH2CH2NH),
Compound 17 (Rf = 0.55 (CH2Cl2/CH3OH = 9 : 1). Colorless 1.88–1.98 (m, 2H, CH2CHCH2), 2.25–2.34 (m, 1H,
solid, mp 82 °C, yield 0.15 g (26%)). C29H31Cl2NO7S2 (640.6 g CH2CHOSO2), 2.34–2.41 (m, 1H, CH2CHCH2), 2.45 (s, 3H,
mol−1). 1H NMR (600 MHz, CDCl3): δ [ppm] = 1.41–1.49 (m, CH3), 2.46–2.53 (m, 1H, CHCH2C(vO)), 3.21–3.33 (m, 2H,
4H, CH2CH2NH (2H), CH2CHCH2 (2H)), 2.13 (ddd, J = 14.0/9.0/ CH2NH), 3.49 (s, 2H, PhCH2C(vO)), 4.67–4.73 (m, 1H,
4.1 Hz, 2H, CH2CHCH2), 2.26–2.36 (m, 1H, CH2CHCH2), 2.45 CHOSO2), 5.50–5.58 (m, 1H, NH), 7.11 (dd, J = 8.2/2.1 Hz, 1H,
(s, 6H, CH3), 3.09–3.15 (m, 2H, CH2NH), 3.46 (s, 2H, CH2C 6-CHarom), 7.33–7.38 (m, 3H, 2-CHarom (1H), 3-Htosyl (1H),
(vO)), 4.68–4.73 (m, 2H, OCHCHO), 5.38–5.44 (m, 1H, NH), 5-Htosyl (1H)), 7.42 (d, J = 8.2 Hz, 1H, 5-CHarom), 7.80 (d, J = 8.3
7.10 (dd, J = 8.2/1.3 Hz, 1H, 6-CHarom), 7.31 (d, J = 8.0 Hz, 4H, Hz, 2H, 2-Htosyl (1H), 6-Htosyl (1H)). 13C NMR (101 MHz,
3-Htosyl, 5-Htosyl), 7.34–7.36 (m, 1H, 2-CHarom), 7.42 (d, J = 8.2 CDCl3): δ (ppm) = 21.9 (1C, CH3), 30.3 (1C, CH2CHCH2), 35.6
Hz, 1H, 5-CHarom), 7.70 (d, J = 8.2 Hz, 4H, 2-Htosyl, 6-Htosyl). 13
C
(1C, CH2CH2NH), 35.9 (1C, CH2CHOSO2), 38.3 (1C, CH2NH),
NMR (151 MHz, CDCl3): δ [ppm] = 21.9 (2C, CH3), 31.3 (1C, 42.0 (1C, CHCH2C(vO)), 42.7 (1C, PhCH2C(vO)), 78.7 (1C,
CH2CHCH2), 34.9 (2C, CH2CHCH2), 36.3 (1C, CH2CH2NH), CHOSO2), 128.2 (2C, C-2tosyl, C-6tosyl), 128.9 (1C, C-6arom), 130.0
38.6 (1C, CH2NH), 42.7 (1C, CH2C(vO)), 80.3 (2C, OCHCHO), (2C, C-3tosyl, C-5tosyl), 130.1 (1C, C-4arom), 131.0 (1C, C-5arom),
128.0 (4C, C-2tosyl, C-6tosyl), 128.9 (1C, C-6arom), 130.0 (4C, 131.4 (1C, C-2arom), 131.8 (1C, C-3arom), 133.4 (1C, C-1tosyl),
C-3tosyl, C-5tosyl), 130.0 (1C, C-5arom), 131.4 (1C, C-2arom), 131.8 135.0 (1C, C-1arom), 145.4 (1C, C-4tosyl), 170.0 (1C, NC(vO)),
(1C, C-4arom), 133.0 (1C, C-3arom), 133.5 (2C, C-1tosyl), 135.0 (1C, 208.8 (1C, CHC(vO)).
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6.2.10. (RS)-2-(3,4-Dichlorophenyl)-N-[2-(3-oxocyclopentyl)
Organic & Biomolecular Chemistry
340.1577 for C17H26NO4S [M + H]+). Purity (HPLC): 83.4% (tR =
ethyl]acetamide (22). NaH 60% dispersion (8.0 mg, 19.1 min).
0.33 mmol, 3.3 eq.) dissolved in dry THF (3 mL) was added to
6.2.12. N-{2-[(1S)-3,4-Dihydroxycyclopentyl]ethyl}-4-methyl-
a solution of 20 (50 mg, 0.10 mmol) in dry THF (3 mL) at room benzenesulfonamide (24). Conc. HCl (8 mL) was added to a
temperature. The mixture was stirred for 3 h at room tempera- solution of acetonide 23 (0.22 g, 0.63 mmol) in CH3OH
ture. 1 M HCl was added to reach pH 7 and the mixture was (20 mL) and the mixture was stirred at room temperature for
extracted with EtOAc (3 × 10 mL). The combined organic layers 16 h. 5 M NaOH (20 mL) was added to neutralize the solution
were dried (Na2SO4), filtered and the solvent was removed in (pH 7) and the mixture was extracted with CH2Cl2 (2 × 20 mL).
vacuo. The residue was purified by flash column chromato- The combined organic layers were dried (Na2SO4), filtered and
graphy (Ø = 2 cm, h = 15 cm, V = 5 mL, CH2Cl2/CH3OH = 9 : 1, the solvent was removed in vacuo. The residue was purified by
Rf = 0.55 (CH2Cl2/CH3OH = 9 : 1)). Yellowish oil, yield 25 mg flash column chromatography (Ø = 2 cm, h = 15 cm, V =
(78%). C15H17Cl2NO2 (314.2 g mol−1). 1H NMR (600 MHz, 10 mL, cHex/EtOAc = 1 : 9, Rf = 0.36 (CH2Cl2/CH3OH = 90 : 10)).
CDCl3): δ (ppm) = 1.49–1.55 (m, 1H, CH2CH2C(vO)), 1.58–1.66 Colorless oil, yield 0.12 g (64%). C14H21NO4S (299.4 g mol−1).
(m, 2H, CH2CH2NH), 1.77–1.83 (m, 1H, CHCH2C(vO)), 1H NMR (600 MHz, DMSO-d6): δ (ppm) = 1.15–1.21 (m, 2H,
2.09–2.21 (m, 3H, CHCH2C(vO) (1H), CH2CH2C(vO) (1H), CH2CHCH2), 1.31 (q, J = 7.5 Hz, 2H, CH2CH2NH), 1.59–1.65
CH2CH2C(vO) (1H)), 2.26–2.33 (m, 1H, CH2CH2C(vO)), (m, 2H, CH2CHCH2), 2.05–2.13 (m, 1H, CH2CHCH2), 2.38 (s,
2.36–2.42 (m, 1H, CHCH2C(vO)), 3.26–3.34 (m, 2H, CH2NH), 3H, CH3), 2.61–2.66 (m, 2H, CH2CH2NH), 3.78–3.82 (m, 2H,
3.50 (s, 2H, PhCH2C(vO)), 5.41–5.49 (m, 1H, NH), 7.12 (dd, J = OCHCHO), 7.38 (d, J = 7.9 Hz, 2H, 3-Htosyl, 5-Htosyl), 7.65 (d, J =
8.2/2.1 Hz, 1H, 6-CHarom), 7.37 (d, J = 2.1 Hz, 1H, 2-CHarom), 8.3 Hz, 2H, 2-Htosyl, 6-Htosyl). Signals for the OH and NH
7.42 (d, J = 8.2 Hz, 1H, 5-CHarom). 13C NMR (151 MHz, CDCl3): protons are not seen in the spectrum. 13C NMR (151 MHz,
δ (ppm) = 29.5 (1C, CH2CH2C(vO)), 35.0 (1C, CHCH2C(vO)), DMSO-d6): δ (ppm) = 21.0 (1C, ArCH3), 31.1 (1C, CH2CHCH2),
35.7 (1C, CH2CH2NH), 38.5 (1C, CH2CH2C(vO)), 38.6 (1C, 36.5 (1C, CH2CH2NH), 37.4 (2C, CH2CHCH2), 41.5 (1C,
CH2NH), 42.8 (1C, PhCH2C(vO)), 45.0 (1C, CHCH2C(vO)), CH2CH2NH), 72.6 (2C, OCHCHO), 126.5 (2C, C-2tosyl, C-6tosyl),
128.9 (1C, C-6arom), 131.0 (1C, C-5arom), 131.4 (1C, C-2arom), 129.6 (2C, C-3tosyl, C-5tosyl), 137.6 (1C, C-1tosyl), 142.5 (1C,
131.8 (1C, C-4arom), 133.1 (1C, C-3arom), 135.1 (1C, C-1arom), C-4tosyl). IR (neat): ν (cm−1) = 3518 (O–H, N–H), 2927 (C–H
169.8 (1C, NHC(vO)), 218.8 (1C, CHCH2C(vO)). IR (neat): ν aliph), 1315 (OvSvO), 814 (1,4-disubst. arom). Exact mass
(cm−1) = 3287 (N–H), 2924 (C–Haliph), 1643 (CvO).
6.2.11. N-{2-[(5S)-2,2-Dimethyl-4,5,6,6a-tetrahydro-3aH- H]+). Purity (HPLC): 83% (tR = 13.9 min).
(APCI): m/z = 300.1264 (calcd 300.1264 for C14H22NO4S [M +
cyclopenta[d][1,3]dioxol-5-yl]ethyl}-4-methylbenzenesulfonamide
6.2.13. (4S)-4-{2-[(4-Methylphenyl)sulfonamido]ethyl}cyclo-
(23). Under N2 atmosphere, primary amine 14 (1.6 g, pentane-1,2-diyl bis(4-methylbenzenesulfonate) (25). Under N2
8.6 mmol) was dissolved in dry CH2Cl2 (80 mL). At 0 °C, pyri- atmosphere, ethyldiisopropylamine (0.42 g, 3.3 mmol, 2.1 eq.),
dine (1.1 g, 14 mmol, 1.6 eq.) and p-toluenesulfonyl chloride p-toluenesulfonyl chloride (0.65 g, 3.4 mmol, 2.1 eq.) and 4-(di-
(3.2 g, 17 mmol, 2 eq.) were added to the mixture. After stirring methylamino)pyridine (0.24 g, 2.0 mmol, 1.3 eq.) were added
for 16 h at room temperature, a saturated NaHCO3 solution to a solution of diol 24 (0.49 g, 1.6 mmol) in dry CH2Cl2
(80 mL) was added and the aqueous layer was extracted with (120 mL) at 0 °C. The reaction was stirred for 16 h at room
CH2Cl2 (2 × 80 mL). The combined organic layers were dried temperature. A saturated NaHCO3 solution (100 mL) was
(Na2SO4), filtered and the solvent was removed in vacuo. The added, the organic layer was separated and the aqueous layer
residue was purified by flash column chromatography (Ø = was extracted with CH2Cl2 (3 × 120 mL). The combined organic
6 cm, h = 15 cm, V = 30 mL, cHex/EtOAc = 1 : 1, Rf = 0.39 layers were dried (Na2SO4), filtered and the solvent was
(CH2Cl2/CH3OH = 90 : 10)). Yellow solid, mp 72 °C, yield 2.0 g removed in vacuo. The residue was purified by flash column
(71%). C17H25NO4S (339.5 g mol−1). 1H NMR (600 MHz, chromatography (Ø = 4 cm, h = 15 cm, V = 30 mL, cHex/EtOAc
DMSO-d6): δ (ppm) = 0.96–1.03 (m, 2H, CH2CHCH2), 1.17 (s, = 2 : 1, Rf = 0.44 (cHex/EtOAc = 1 : 2)). Colorless solid, mp
3H, CH3CCH3), 1.30 (s, 3H, CH3CCH3), 1.37 (q, J = 7.4 Hz, 2H, 119 °C, yield 0.49 g (50%). C28H33NO8S3 (607.8 g mol−1). 1H
CH2CH2NH), 1.72 (dd, J = 13.9/5.7 Hz, 2H, CH2CHCH2), NMR (600 MHz, DMSO-d6): δ [ppm] = 1.30 (q, J = 7.2 Hz, 2H,
1.93–2.03 (m, 1H, CH2CHCH2), 2.38 (s, 3H, CH3), 2.65–2.70 (m, CH2CH2NH), 1.35–1.43 (m, 2H, CH2CHCH2), 1.74 (ddd, J =
2H, CH2CH2NH), 4.49–4.52 (m, 2H, OCHCHO), 7.39 (d, J = 7.9 13.6/9.3/4.2 Hz, 2H, CH2CHCH2), 2.11–2.20 (m, 1H,
Hz, 2H, 3-Htosyl, 5-Htosyl), 7.66 (d, J = 8.4 Hz, 2H, 2-Htosyl
,
CH2CHCH2), 2.38 (s, 3H, CH3N-tosyl), 2.42 (s, 6H, CH3, 2×
6-Htosyl). A signal for the proton of the NH proton is not seen O-tosyl,) 2.57 (m, 2H, CH2CH2NH), 4.74–4.79 (m, 2H,
in the spectrum. 13C NMR (151 MHz, DMSO-d6): δ (ppm) = OCHCHO), 7.37 (d, J = 7.8 Hz, 2H, 3-HN-tosyl, 5-HN-tosyl), 7.44 (d,
21.0 (1C, ArCH3), 23.7 (1C, CCH3), 26.0 (1C, CCH3), 33.0 (1C, J = 8.0 Hz, 4H, 2× 3-HO-tosyl, 2× 5-HO-tosyl), 7.63 (d, J = 8.2 Hz,
CH2CHCH2), 33.4 (1C, CH2CH2NH), 38.8 (2C, CH2CHCH2), 2H, 2-HN-tosyl, 6-HN-tosyl), 7.67 (d, J = 8.3 Hz, 4H, 2× 2-HO-tosyl
41.8 (1C, CH2CH2NH), 79.7 (2C, OCHCHO), 107.8 (1C, 2× 6-HO-tosyl). A signal for the NH proton is not seen in the
C(CH3)2), 126.5 (2C, C-2tosyl, C-6tosyl), 129.6 (2C, C-3tosyl
spectrum. 13C NMR (151 MHz, DMSO-d6): δ [ppm] = 20.9 (1C,
,
,
C-5tosyl), 142.5 (1C, C-1tosyl), 145.8 (1C, C-4tosyl). IR (neat): ν CH3N-tosyl), 21.1 (2C, CH3, 2× O-tosyl), 30.4 (1C, CH2CHCH2),
(cm−1) = 3264 (N–H), 1330 (OvSvO), 1157 (O–Cketal), 814 (1,4- 33.9 (2C, CH2CHCH2), 35.2 (1C, CH2CH2NHTs), 41.0 (1C,
disubst. arom). Exact mass (APCI): m/z = 340.1591 (calcd CH2CH2NH), 80.7 (2C, OCHCHO), 126.4 (2C, C-2N-tosyl
,
4092 | Org. Biomol. Chem., 2021, 19, 4082–4099
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Organic & Biomolecular Chemistry
C-6N-tosyl), 127.4 (4C, 2× C-2O-tosyl
Paper
,
C-6O-tosyl), 129.6 (2C, 1.68–1.74 (m, 1H, 4-CH2), 1.74–1.79 (m, 1H, 8-CH2), 1.86 (br. s,
C-3N-tosyl, C-5N-tosyl), 130.1 (4C, 2× C-3O-tosyl, C-5O-tosyl), 133.0 4H, N(CH2CH2)2), 1.91 (ddd, J = 14.8/7.1/2.3 Hz, 1H, 6-CH2),
(2C, 2× C-1O-tosyl), 137.6 (1C, C-1N-tosyl), 142.5 (1C, C-4N-tosyl), 2.42 (s, 3H, CH3), 2.43–2.47 (m, 1H, 5-CH), 2.56 (td, J = 12.4/4.6
144.9 (2C, 2× C-4O-tosyl). IR (neat): ν (cm−1) = 3267 (N–H), 2947 Hz, 1H, 3-CH2ax), 3.32 (br. s, 4H, N(CH2CH2)2), 3.64 (dd, J =
(C–Haliph), 1350 (OvSvO), 810 (1,4-disubst. arom). Exact mass 12.4/6.4 Hz, 1H, 3-CH2eq), 4.35 (d, J = 4.4 Hz, 1H, 7-CH), 4.46
(APCI): m/z = 608.1490 (calcd 608.1441 for C28H34NO8S3 [M + (dt, J = 6.8/2.2 Hz, 1H, 1-CH), 7.30 (d, J = 8.0 Hz, 2H, 3-Htosyl
,
H]+). Purity (HPLC): 77.1% (tR = 23.0 min). 5-Htosyl), 7.74 (d, J = 8.2 Hz, 2H, 2-Htosyl, 6-Htosyl). 13C NMR
6.2.14. [(1RS,5SR,7RS)-2-Tosyl-2-azabicyclo[3.2.1]octan-7- (151 MHz, CDCl3): δ [ppm] = 21.7 (1C, CH3), 25.2 (2C,
yl]-4-methylbenzenesulfonate (26). Under N2 atmosphere, NaH N(CH2CH2)2), 29.7 (1C, C-4), 33.6 (1C, C-5), 36.6 (1C, C-8), 37.9
60% dispersion (0.58 g, 15 mmol) was added to a solution of (1C, C-6), 40.4 (1C, C-3), 46.1 (2C, N(CH2CH2)2), 60.5 (1C, C-7),
25 (0.17 g, 0.28 mmol) in dry THF (50 mL) at 0 °C and the 76.0 (1C, C-1), 127.6 (2C, C-2tosyl, C-6tosyl), 129.9 (2C, C-3tosyl
,
mixture was stirred for 3 d at room temperature. 1 M HCl was C-5tosyl), 136.1 (1C, C-1tosyl), 143.6 (1C, C-4tosyl), 153.9 (1C,
added to neutralize the mixture (pH 7) and the organic solvent C(vO)). IR (neat): ν (cm−1) = 2932 (C–Haliph), 1715 (CvO), 342
was removed in vacuo. The residual aqueous solution was (OvSvO), 814 (1,4-disubst. arom). Exact mass (APCI): m/z =
extracted with CH2Cl2 (3 × 20 mL) and the combined CH2Cl2 379.1702 (calcd 279.1686 for C19H27N2O4S [M + H]+). Purity
layers were washed with H2O (1 × 20 mL). The combined (HPLC): 95.2% (tR = 21.2 min).
organic layers were dried (Na2SO4), filtered and the solvent was
Ketone 27 (Rf = 0.48 (cHex/EtOAc = 1 : 2). Colorless solid,
removed in vacuo. The residue was purified by flash column yield 0.004 g (12%)). C14H17NO3S (279.4 g mol−1). 1H NMR
chromatography (Ø = 4 cm, h = 15 cm, V = 20 mL, cHex/EtOAc (600 MHz, DMSO-d6): δ [ppm] = 1.57–1.62 (m, 1H, 4-CH2), 1.69
= 5 : 1, Rf = 0.58 (cHex/EtOAc = 1 : 2)). Colorless oil, yield (dd, J = 12.4/3.8 Hz, 1H, 8-CH2), 1.79–1.86 (m, 1H, 4-CH2),
0.087
g (72%). C21H25NO5S2 (435.6 g
mol−1). 1H NMR 1.88–1.93 (m, 2H, 8-CH2 (1H), 6-CH2 (1H)), 2.22 (dd, J = 18.9/
(600 MHz, CDCl3): δ [ppm] = 1.30–1.35 (m, 1H, 4-CH2), 6.7 Hz, 1H, 6-CH2), 2.40 (s, 3H, CH3), 2.55–2.59 (m, 1H, 5-CH),
1.50–1.54 (m, 1H, 8-CH2), 1.63–1.71 (m, 1H, 4-CH2), 1.72–1.77 2.66 (td, J = 12.7/4.6 Hz, 1H, 3-CH2ax), 3.61 (dd, J = 12.9/6.6 Hz,
(m, 1H, 8-CH2), 1.78–1.81 (m, 2H, 6-CH2), 2.43–2.44 (m, 1H, 1H, 3-CH2eq), 3.97 (d, J = 4.7 Hz, 1H, 1-CH), 7.40 (d, J = 7.9 Hz,
5-CH), 2.45 (s, 3H, CH3N-tosyl), 2.46 (s, 3H, CH3O-tosyl), 2.51 (td, J 2H, 3-Htosyl, 5-Htosyl), 7.65 (d, J = 8.3 Hz, 2H, 2-Htosyl, 6-Htosyl).
= 12.3/4.7 Hz, 1H, 3-CH2ax), 3.63 (dd, J = 12.5/6.4 Hz, 1H, 13C NMR (151 MHz, DMSO-d6): δ [ppm] = 21.0 (1C, CH3), 28.7
3-CH2eq), 4.38 (d, J = 4.4 Hz, 1H, 1-CH), 4.49–4.53 (m, 1H, (1C, C-4), 29.9 (1C, C-5), 35.5 (1C, C-8), 40.2 (1C, C-3), 40.6 (1C,
7-CH), 7.31 (d, J = 7.9 Hz, 2H, 3-HN-tosyl, 5-HN-tosyl), 7.35 (d, J = C-6), 58.9 (1C, C-1), 127.5 (2C, C-2tosyl, C-6tosyl), 129.5 (2C,
7.9 Hz, 2H, 3-HO-tosyl, 5-HO-tosyl), 7.68 (d, J = 8.3 Hz, 2H, C-3tosyl, C-5tosyl), 135.3 (1C, C-1tosyl), 143.3 (1C, C-4tosyl), 213.7
2-HN-tosyl, 6-HN-tosyl), 7.74 (d, J = 8.3 Hz, 2H, 2-HO-tosyl
,
(1C, C(vO)). Exact mass (APCI): m/z = 280.1014 (calcd
6-HO-tosyl). 13C NMR (151 MHz, CDCl3): δ [ppm] = 21.7 (1C, 280.1002 for C14H18NO3S [M + H]+). Purity (HPLC): 98% (tR
CH3N-tosyl), 21.8 (1C, CH3O-tosyl), 29.3 (1C, C-4), 33.4 (1C, C-5), 18.5 min).
=
35.8 (1C, C-8), 37.2 (1C, C-6), 40.1 (1C, C-3), 60.2 (1C, C-1), 82.5
6.2.16. (1RS,5SR,7RS)-2-Tosyl-2-azabicyclo[3.2.1]octan-7-ol
(1C, C-7), 127.7 (2C, C-2N-tosyl, C-6N-tosyl), 127.9 (2C, C-2O-tosyl
C-6O-tosyl), 130.0 (2C, C-3O-tosyl, C-5O-tosyl), 130.1 (2C, C-3N-tosyl
,
,
(29). Under N2 atmosphere, pyrrolidine (9.0 mg, 0.13 mmol,
3.2 eq.) and K2CO3 (11 mg, 0.082 mmol, 2.0 eq.) were added to
C-5N-tosyl), 134.2 (1C, C-1O-tosyl), 135.8 (1C, C-1N-tosyl), 143.0 (1C, a solution of 26 (18 mg, 0.041 mmol) in dry CH3CN (10 mL).
C-4N-tosyl), 145.0 (1C, C-4O-tosyl). IR (neat): ν (cm−1) = 2932 (C–H The mixture was stirred for 10 h at 80 °C. 1 M HCl was added
aliph), 1342 (OvSvO), 814 (1,4-disubst. arom). Exact mass to neutralize the mixture (pH 7) and the organic solvent was
(APCI): m/z = 436.1263 (calcd 436.1247 for C21H26NO5S2 [M + removed in vacuo. The residual aqueous layer was extracted
H]+). Purity (HPLC): 98.4% (tR = 22.8 min).
with CH2Cl2 (2 × 10 mL). The combined organic layers were
6.2.15. [(1RS,5SR,7RS)-2-Tosyl-2-azabicyclo[3.2.1]octan-7-yl] dried (Na2SO4), filtered and the solvent was removed in vacuo.
pyrrolidine-1-carboxylate (28) and (1RS,5SR)-2-tosyl-2-azabicy- The residue was purified by flash column chromatography (Ø
clo[3.2.1]octan-7-one (27). Under N2 atmosphere, pyrrolidine = 2 cm, h = 15 cm, V = 10 mL, cHex/EtOAc = 1 : 1, Rf = 0.52
(0.042 g, 0.60 mmol, 5.0 eq.) and K2CO3 (0.018 g, 0.13 mmol, (cHex/EtOAc = 1 : 2)). Yellow solid, mp 101 °C, yield 3.0 mg
1.1 eq.) were added to a solution of 26 (0.052 g, 0.12 mmol) in (26%). C14H19NO3S (281.4 g mol−1). 1H NMR (600 MHz,
anh. DMSO (15 mL). The mixture was stirred for 8 h at 100 °C. DMSO-d6): δ [ppm] = 1.22–1.29 (m, 2H, 4-CH2 (1H), 8-CH2
0.1 M HCl was added to neutralize the mixture (pH 7) and it (1H)), 1.30–1.36 (m, 1H, 6-CH2), 1.45–1.52 (m, 1H, 4-CH2),
was extracted with Et2O (3 × 20 mL). The combined organic 1.63–1.71 (m, 2H, 8-CH2 (1H), 6-CH2 (1H)), 2.28–2.32 (m, 1H,
layers were dried (Na2SO4), filtered and the solvent was 5-CH), 2.41 (s, 3H, CH3), 2.46 (td, J = 12.4/4.6 Hz, 1H, 3-CH2ax),
removed in vacuo. The residue was purified by flash column 3.36–3.40 (m, 1H, 7-CH), 3.48 (dd, J = 12.4/6.4 Hz, 1H,
chromatography (Ø = 2 cm, h = 15 cm, V = 10 mL, cHex/EtOAc 3-CH2eq), 3.91 (d, J = 4.4 Hz, 1H, 1-CH), 4.71 (d, J = 3.5 Hz, 1H,
= 4 : 1). At first, ketone 27, then carbamate 28 was eluted.
OH), 7.42 (d, J = 8.2 Hz, 2H, 3-Htosyl, 5-Htosyl), 7.69 (d, J = 8.3
Carbamate 28 (Rf = 0.39 (cHex/EtOAc = 1 : 2). Colorless Hz, 2H, 2-Htosyl, 6-Htosyl). 13C NMR (151 MHz, DMSO-d6): δ
solid, mp 139 °C, yield 0.019 g (42%)). C19H26N2O4S (378.5 g [ppm] = 21.0 (1C, CH3), 29.0 (1C, C-4), 32.9 (1C, C-5), 34.7 (1C,
mol−1). 1H NMR (600 MHz, CDCl3): δ [ppm] = 1.32–1.38 (m, C-8), 38.5 (1C, C-6), 40.1 (1C, C-3), 62.2 (1C, C-1), 71.4 (1C,
1H, 4-CH2), 1.57–1.63 (m, 2H, 8-CH2 (1H), 6-CH2, (1H)), C-7), 127.1 (2C, C-2tosyl, C-6tosyl), 129.8 (2C, C-3tosyl, C-5tosyl),
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136.0 (1C, C-1tosyl), 143.1 (1C, C-4tosyl). Exact mass (APCI): m/z 818 (1,4-disubst. arom). Exact mass (APCI): m/z = 281.1336
= 282.1141 (calcd 282.1158 for C14H20NO3S [M + H]+). Purity (calcd 281.1318 for C14H21N2O2S [M + H]+). Purity (HPLC):
(HPLC): 70.3% (tR = 17.0 min).
59.7% (tR = 13.8 min).
6.2.17. (1RS,5RS,7RS)-7-Azido-2-tosyl-2-azabicyclo[3.2.1]-
6.2.19. (1RS,5RS,7RS)-7-(Pyrrolidin-1-yl)-2-tosyl-2-azabicyclo-
octane (31). Under N2 atmosphere, sodium azide (0.090 g, [3.2.1]octane (33a). Under N2 atmosphere, 1,4-diiodobutane
1.4 mmol, 2.0 eq.) was added to a solution of 26 (0.30 g, (0.17 g, 0.54 mmol, 1.2 eq.) and ethyldiisopropylamine
0.69 mmol) in anh. DMSO (10 mL) and the mixture was stirred (0.086 g, 0.67 mmol, 1.5 eq.) were added to a solution of
for 2 h at 100 °C. After cooling down to 0 °C, 1 M NaOH (5 mL) primary amine 32 (0.13 g, 0.45 mmol) in dry CH3CN (5 mL).
was added and the mixture was extracted with Et2O (3 × The mixture was stirred for 16 h at 80 °C and, then, the solvent
15 mL). The combined organic layers were dried (Na2SO4), fil- was removed in vacuo. 0.1 M NaOH (5 mL) was added and the
tered and the solvent was removed in vacuo. The residue was aqueous layer was extracted with CH2Cl2 (3 × 5 mL). The com-
purified by flash column chromatography (Ø = 2 cm, h = bined organic layers were dried (Na2SO4), filtered and the
15 cm, V = 10 mL, cHex/EtOAc = 2 : 1, Rf = 0.38). Colorless solvent was removed in vacuo. The residue was purified by
solid, mp 79 °C, yield 0.16 g (74%). C14H18N4O2S (306.4 g flash column chromatography (Ø = 2 cm, h = 15 cm, V = 3 mL,
mol−1). 1H NMR (600 MHz, CDCl3): δ [ppm] = 1.37–1.42 (m, CH2Cl2/CH3OH = 9 : 1, Rf = 0.35). Pale orange oil, yield 0.11 g
1H, 4-CH2), 1.55–1.62 (m, 2H, 8-CH2 (1H), 6-CH2 (1H)), (73%). C18H26N2O2S (334.5 g mol−1). 1H NMR (600 MHz,
1.65–1.70 (dtd, J = 11.9/4.8/2.1 Hz, 1H, 8-CH2), 1.71–1.78 (m, DMSO-d6): δ [ppm] = 1.24 (d, J = 11.1 Hz, 1H, 6-CH2),
2H, 4-CH2 (1H), 6-CH2 (1H)), 2.44–2.48 (m, 4H, CH3 (3H), 5-CH 1.30–1.36 (m, 1H, 4-CH2), 1.47–1.54 (m, 1H, 4-CH2), 1.54–1.63
(1H)), 2.52 (td, J = 12.2/4.6 Hz, 1H, 3-CH2ax), 3.09–3.13 (m, 1H, (m, 7H, 8-CH2 (2H), 6-CH2 (1H), N(CH2CH2)2 (4H)), 2.16–2.21
7-CH), 3.72 (dd, J = 12.2/6.4 Hz, 1H, 3-CH2eq), 4.19 (d, J = 4.4 (m, 1H, 7-CH), 2.24–2.32 (m, 5H, N(CH2CH2)2 (4H), 5-CH
Hz, 1H, 1-CH), 7.36 (d, J = 8.2 Hz, 2H, 3-Htosyl, 5-Htosyl), 7.71 (1H)), 2.40 (s, 3H, CH3), 2.77 (td, J = 12.5/4.7 Hz, 1H, 3-CH2ax),
(d, J = 8.2 Hz, 2H, 2-Htosyl, 6-Htosyl). 13C NMR (151 MHz, 3.57 (dd, J = 12.5/6.3 Hz, 1H, 3-CH2eq), 4.14 (d, J = 4.2 Hz, 1H,
CDCl3): δ [ppm] = 21.7 (1C, CH3), 29.6 (1C, C-4), 33.8 (1C, C-5), 1-CH), 7.42 (d, J = 7.9 Hz, 2H, 3-Htosyl, 5-Htosyl), 7.66 (d, J = 8.3
35.9 (1C, C-6), 36.2 (1C, C-8), 40.5 (1C, C-3), 60.9 (1C, C-1), 62.1 Hz, 2H, 2-Htosyl, 6-Htosyl). 13C NMR (151 MHz, DMSO-d6): δ
(1C, C-7), 127.5 (2C, C-2tosyl, C-6tosyl), 130.2 (2C, C-3tosyl
,
[ppm] = 21.0 (1C, CH3), 22.8 (2C, N(CH2CH2)2), 29.2 (1C, C-4),
C-5tosyl), 135.6 (1C, C-1tosyl), 143.9 (1C, C-4tosyl). IR (neat): ν 33.2 (1C, C-5), 34.0 (1C, C-8), 35.7 (1C, C-6), 39.9 (1C, C-3), 51.8
(cm−1) = 2920 (C–Haliph), 2095 (N3), 1330 (OvSvO), 822 (1,4- (2C, N(CH2CH2)2), 58.5 (1C, C-1), 67.1 (1C, C-7), 127.1 (2C,
disubst. arom). Exact mass (APCI): m/z = 307.1239 (calcd C-2tosyl, C-6tosyl), 129.7 (2C, C-3tosyl, C-5tosyl), 136.7 (1C, C-1tosyl),
307.1223 for C14H19N4O2S [M + H]+). Purity (HPLC): 96.6% (tR 143.1 (1C, C-4tosyl). IR (neat): ν (cm−1) = 2924 (C–Haliph), 1327
= 21.8 min).
(OvSvO), 814 (1,4-disubst. arom). Exact mass (APCI): m/z =
6.2.18. (1RS,5RS,7RS)-2-Tosyl-2-azabicyclo[3.2.1]octan-7- 335.1801 (calcd 335.1788 for C18H27N2O2S [M + H]+). Purity
amine (32). Under N2 atmosphere, LiAlH4 (7.0 mg, 0.18 mmol, (HPLC): 95.7% (tR = 15.6 min).
1.2 eq.) suspended in dry THF (1 mL) was added to a solution
6.2.20. 2-(3,4-Dichlorophenyl)-1-[(1RS,5RS,7RS)-7-(pyrrolidin-
of azide 31 (46 mg, 0.15 mmol) in dry THF (9 mL) at 0 °C and 1-yl)-2-azabicyclo[3.2.1]octan-2-yl]ethan-1-one (35a). Under N2
the mixture was stirred for 2 h at 0 °C. 0.1 M NaOH (3 mL) was atmosphere, Mg turnings (22 mg, 0.90 mmol) were added to a
added and the mixture was extracted with CH2Cl2 (2 × 10 mL). solution of 33a (30 mg, 0.090 mmol) in dry CH3OH (3 mL) and
The organic layers were washed with a saturated solution of the mixture was heated to reflux in the ultrasonic bath for 1 h.
potassium sodium tartrate (4 mL). The aqueous layer was After cooling to room temperature, additional Mg turnings
extracted with CH2Cl2 (2 × 10 mL). The combined organic (22 mg, 0.90 mmol) were added and the mixture was heated to
layers were dried (Na2SO4), filtered and the solvent was reflux in the ultrasonic bath for 1 h. Additional Mg turnings
removed in vacuo. Yellow oil, yield 41 mg (97%). Rf = 0.24 (30 mg, 1.2 mmol) were added and the mixture was heated to
(CH2Cl2/CH3OH = 9 : 1). C14H20N2O2S (280.4 g mol−1). 1H NMR reflux in the ultrasonic bath for 1 h. After cooling to room
(400 MHz, DMSO-d6): δ [ppm] = 1.08 (ddd, J = 13.4/6.4/3.6 Hz, temperature, H2O (6 mL) and NaOH (11 mg, 0.27 mmol) were
1H, 6-CH2), 1.21–1.32 (m, 2H, 4-CH2 (1H), 8-CH2 (1H)), added and the mixture was cooled down to 0 °C. A solution of
1.46–1.54 (m, 1H, 4-CH2), 1.58 (ddd, J = 13.5/7.6/2.1 Hz, 1H, 2-(3,4-dichlorophenyl)acetyl chloride (80 mg, 0.36 mmol) in
6-CH2), 1.79 (dtd, J = 11.1/4.5/1.9 Hz, 1H, 8-CH2), 2.22–2.28 (m, THF (2 mL) was added dropwise and the mixture was stirred at
1H, 5-CH), 2.41 (s, 3H, CH3), 2.42–2.47 (m, 2H, 3-CH2ax (1H), room temperature for 16 h. Additional 2-(3,4-dichlorophenyl)
7-CH (1H)), 3.46 (dd, J = 12.2/6.3 Hz, 1H, 3-CH2eq), 3.75 (d, J = acetyl chloride (80 mg, 0.36 mmol) and NaOH (40 mg,
4.3 Hz, 1H, 1-CH), 7.42 (d, J = 7.9 Hz, 2H, 3-Htosyl, 5-Htosyl), 1.0 mmol) were added and the mixture was stirred at room
7.71 (d, J = 8.2 Hz, 2H, 2-Htosyl, 6-Htosyl). Signals for the NH2 temperature for 1 h. Then, the mixture was extracted with
protons are not seen in the spectrum. 13C NMR (101 MHz, EtOAc (3 × 15 mL). The combined organic layers were dried
DMSO-d6): δ [ppm] = 21.0 (1C, CH3), 29.3 (1C, C-4), 33.3 (1C, (Na2SO4), filtered and the solvent was removed in vacuo. The
C-5), 34.6 (1C, C-8), 38.3 (1C, C-6), 40.1 (1C, C-3), 52.5 (1C, residue was purified by flash column chromatography (Ø =
C-7), 63.6 (1C, C-1), 127.1 (2C, C-2tosyl, C-6tosyl), 129.8 (2C, 2 cm, h = 15 cm, V = 3 mL, CH2Cl2/CH3OH = 9 : 1, Rf = 0.50
C-3tosyl, C-5tosyl), 135.8 (1C, C-1tosyl), 143.0 (1C, C-4tosyl). IR (CH2Cl2/CH3OH = 9 : 1)). CH2Cl2 (2 mL) was added to the
(neat): ν (cm−1) = 3374 (N–H), 2946 (C–Haliph), 1326 (OvSvO), residue and the organic layer was washed with 0.1 M NaOH (2
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× 2 mL). The organic layer was dried (Na2SO4), filtered and the 57.5 (1C, C-1), 69.5 (1C, C-5), 127.6 (2C, C-2tosyl, C-6tosyl), 129.7
solvent was removed in vacuo. Pale yellow oil, yield 6.0 mg (2C, C-3tosyl, C-5tosyl), 137.0 (1C, C-1tosyl), 143.4 (1C, C-4tosyl). IR
(22%). C19H24Cl2N2O (367.3 g mol−1). 1H NMR (600 MHz, (neat): ν (cm−1) = 2959 (C–Haliph), 1331 (OvSvO), 1015 (C–N),
CDCl3): δ [ppm] = 1.28–1.31 (m, 1H, 8-CH2), 1.36–1.46 (m, 1H, 799 (1,4-disubst. arom). Exact mass (APCI): m/z = 309.1625
4-CH2), 1.56–1.65 (m, 1H, 4-CH2), 1.72–1.92 (m, 7H, (calcd 309.1631 for C16H25N2O2S [M + H]+). Purity (HPLC):
N(CH2CH2)2 (4H), 6-CH2 (2H), 8-CH2 (1H)), 2.39–2.52 (m, 3H, 95.2% (tR = 14.7 min).
5-CH (1H), N(CH2CH2)2 (2H)), 2.57 (br. s, 0.54H, 7-CH), 2.63
6.2.22. 2-(3,4-Dichlorophenyl)-1-[(1RS,5RS,7RS)-7-(di-
(br. s, 0.46H, 7-CH), 2.66–2.79 (m, 2.46H, 3-CH2ax (0.46H), methylamino)-2-azabicyclo[3.2.1]octan-2-yl]ethan-1-one (35b).
N(CH2CH2)2 (2H)), 3.09 (ddd, J = 13.3/11.6/5.4 Hz, 0.54H, Under N2 atmosphere, Mg turnings (33 mg, 1.4 mmol) were
3-CH2ax), 3.54–3.61 (m, 1.08H, 3-CH2eq (0.54H), C(vO)CH2Ar added to a solution of 33b (42 mg, 0.14 mmol) in dry CH3OH
(0.54H)), 3.64 (d, J = 15.5 Hz, 0.54H, C(vO)CH2Ar), 3.66 (d, J = (3 mL) and the mixture was heated to reflux in the ultrasonic
15.5 Hz, 0.46H, C(vO)CH2Ar), 3.72 (d, J = 15.5 Hz, 0.46H, bath for 1 h. After cooling to room temperature, additional Mg
C(vO)CH2Ar), 4.16 (d, J = 4.2 Hz, 0.46H, 1-CH), 4.40 (dd, J = turnings (33 mg, 1.4 mmol) were added and the mixture was
14.1/6.8 Hz, 0.46H, 3-CH2eq), 5.13 (br. d, J = 4.3 Hz, 0.54H, heated to reflux in the ultrasonic bath for 1 h. Additional Mg
1-CH), 7.07 (dd, J = 8.2/2.1 Hz, 0.54H, 6-CHarom), 7.10 (dd, J = turnings (33 mg, 1.4 mmol) were added and the mixture was
8.2/2.1 Hz, 0.46H, 6-CHarom), 7.32 (br. d, J = 2.1 Hz, 0.54H, heated to reflux in the ultrasonic bath for 1 h. After cooling to
2-CHarom), 7.35 (br. d, J = 2.1 Hz, 0.46H, 2-CHarom), 7.38 (d, J = room temperature, H2O (6 mL) and NaOH (16 mg, 0.4 mmol)
8.2 Hz, 0.54H, 5-CHarom), 7.39 (d, J = 8.2 Hz, 0.46H, 5-CHarom). were added and the mixture was cooled down to 0 °C. A solu-
Ratio of the rotational isomers = 54 : 46. 13C NMR (151 MHz, tion of 2-(3,4-dichlorophenyl)acetyl chloride (0.12 g,
CDCl3): δ [ppm] = 23.4/23.5 (2C, N(CH2CH2)2), 30.0HR/30.6LR 0.54 mmol) in THF (2 mL) was added dropwise and the
(1C, C-4), 34.0LR/34.5HR (1C, C-5), 35.7/35.9 (1C, C-6), 36.8 (1C, mixture was stirred at room temperature for 16 h. Additional 2-
C-8), 37.1LR (1C, C-3), 40.3 (1C, C(vO)CH2Ar), 40.8HR (1C, C-3), (3,4-dichlorophenyl)acetyl chloride (0.16 g, 0.72 mmol) in
53.2/53.5 (2C, N(CH2CH2)2), 54.8HR (1C, C-1), 59.6LR (1C, C-1), CH3OH (1 mL) and NaOH (30 mg, 0.8 mmol) were added and
70.8 (1C, C-7), 128.4HR/128.5LR (1C, C-6arom), 130.6/130.7 (1C, the mixture was stirred at room temperature for 16 h. 0.1 M
C-5arom), 131.0 (1C, C-2arom), 132.7 (1C, C-4arom), 135.4 (1C, NaOH (20 mL) was added and the mixture was extracted with
C-3arom), 135.6 (1C, C-1arom), 167.8 (1C, CH2C(vO)). Signals of EtOAc (3 × 20 mL). The combined organic layers were dried
the major rotational isomer are marked with HR, signals of (Na2SO4), filtered and the solvent was removed in vacuo. The
the minor rotational isomer are marked with LR. IR (neat): ν residue was purified by flash column chromatography (Ø =
(cm−1) = 2928 (C–Haliph), 1632 (CvO), 1030 (C–N), 679 and 799 2 cm, h = 15 cm, V = 3 mL, CH2Cl2/CH3OH = 9 : 1). CH2Cl2
(C–Harom). Exact mass (APCI): m/z = 367.1309 (calcd 367.1338 (2 mL) was added to the residue and the organic layer was
for C19H2535Cl2N2O [M + H]+). Purity (HPLC): 94.0% (tR
=
washed with 0.1 M NaOH (3 × 2 mL). The organic layer was
17.0 min).
dried (Na2SO4), filtered and the solvent was removed in vacuo.
6.2.21. (1RS,5RS,7RS)-N,N-Dimethyl-2-tosyl-2-azabicyclo- Colorless oil, yield 8.0 mg (17%). Rf = 0.53 (CH2Cl2/CH3OH =
[3.2.1]octan-7-amine (33b). NaBH3CN (16 mg, 0.25 mmol) 9 : 1). C17H22Cl2N2O (341.3 g mol−1). 1H NMR (600 MHz,
was added to a solution of formalin (37%, 0.15 g, 5.0 mmol) in CDCl3): δ [ppm] = 1.27–1.31 (m, 1H, 8-CH2), 1.37–1.47 (m, 1H,
CH3OH (7 mL). The pH value of the mixture was adjusted with 4-CH2), 1.56–1.68 (m, 2H, 4-CH2 (1H), 8-CH2 (1H)), 1.72–1.87
acetic acid to pH 5. A solution of 32 (55 mg, 0.25 mmol) in (m, 2H, 6-CH2), 2.21 (s, 2.52H, N(CH3)2), 2.37 (s, 3.48H,
CH3OH (7 mL) was added and the mixture was stirred at room N(CH3)2), 2.45–2.53 (m, 1.58H, 5-CH (1H), 7-CH (0.58H)), 2.65
temperature for 4 h. A saturated NaHCO3 solution (10 mL) was (br. s, 0.42H, 7-CH), 2.75 (ddd, J = 14.2/12.6/5.4 Hz, 0.42H,
added and the mixture was stirred at room temperature for 3-CH2ax), 3.08 (ddd, J = 13.4/11.7/5.4 Hz, 0.58H, 3-CH2ax),
15 min. After evaporation of the organic solvent, the aqueous 3.54–3.61 (m, 1.16H, 3-CH2eq (0.58H), C(vO)CH2Ar (0.58H)),
phase was extracted with CH2Cl2 (5 × 10 mL). The combined 3.65 (d, J = 15.5 Hz, 0.58H, C(vO)CH2Ar), 3.69 (d, J = 15.5 Hz,
organic layers were dried (Na2SO4), filtered and the solvent was 0.42H, C(vO)CH2Ar), 3.73 (d, J = 15.5 Hz, 0.42H, C(vO)
removed in vacuo. The residue was purified by flash column CH2Ar), 4.19 (d, J = 4.3 Hz, 0.42H, 1-CH), 4.41 (dd, J = 14.2/6.9
chromatography (Ø = 2 cm, h = 15 cm, V = 10 mL, CH2Cl2/ Hz, 0.42H, 3-CH2eq), 5.18 (d, J = 4.3 Hz, 0.58H, 1-CH), 7.07 (dd,
CH3OH = 9 : 1, Rf = 0.54). Colorless oil, yield 38 mg (63%). J = 8.2/2.1 Hz, 0.58H, 6-CHarom), 7.10 (dd, J = 8.3/2.1 Hz, 0.42H,
C16H24N2O2S (308.4 g mol−1). 1H NMR (600 MHz, CDCl3): δ 6-CHarom), 7.32 (d, J = 2.1 Hz, 0.58H, 2-CHarom), 7.35 (d, J = 2.1
[ppm] = 1.35–1.41 (m, 1H, 4-CH2), 1.45 (d, J = 11.9 Hz, 1H, Hz, 0.42H, 2-CHarom), 7.38 (d, J = 8.2 Hz, 0.42H, 5-CHarom),
8-CH2), 1.59–1.77 (m, 4H, 6-CH2 (2H), 4-CH2 (1H), 8-CH2 (1H)), 7.39 (d, J = 8.2 Hz, 0.58H, 5-CHarom). Ratio of the rotational
2.21 (s, 6H, N(CH3)2), 2.34–2.41 (m, 1H, 5-CH), 2.41–2.43 (m, isomers = 58 : 42. 13C NMR (151 MHz, CDCl3): δ [ppm] =
1H, 7-CH), 2.43 (s, 3H, CH3tosyl), 2.83 (td, J = 12.5/4.8 Hz, 1H, 30.0HR/30.5LR (1C, C-4), 34.1/34.4 (1C, C-5), 34.5/35.2 (1C, C-6),
3-CH2ax), 3.65 (dd, J = 12.6/6.6 Hz, 1H, 3-CH2eq), 4.37 (d, J = 4.5 37.1 (2C, C-8, C-3LR), 40.2/40.3 (1C, C(vO)CH2Ar), 40.8 (1C,
Hz, 1H, 1-CH), 7.30 (d, J = 8.2 Hz, 2H, 3-Htosyl, 5-Htosyl), 7.70 C-3HR), 43.8LR/44.0HR (2C, N(CH3)2), 53.2HR/58.1LR (1C, C-1),
(d, J = 8.3 Hz, 2H, 2-Htosyl, 6-Htosyl). 13C NMR (151 MHz, 71.5LR/71.6HR (1C, C-7), 128.4HR/128.6LR (1C, C-6arom), 130.6/
CDCl3): δ [ppm] = 21.7 (1C, CH3), 29.8 (1C, C-4), 34.0 (1C, C-7), 130.7 (1C, C-5arom), 130.9HR/131.1LR (1C, C-2arom), 132.7 (1C,
34.2 (1C, C-6), 36.6 (1C, C-8), 40.3 (1C, C-3), 43.8 (2C, N(CH3)2), C-4arom), 135.3 (1C, C-3arom), 135.6 (1C, C-1arom), 167.7/168.0
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Organic & Biomolecular Chemistry
(1C, C(vO)CH2Ar). Signals of the major rotational isomer are and 791 (C–Harom). Exact mass (APCI): m/z = 335.1929 (calcd
marked with HR, signals of the minor rotational isomer are 335.1929 for C19H25F2N2O [M + H]+). Purity (HPLC): 98.6% (tR
marked with LR. IR (neat): ν (cm−1) = 2932 (C–Haliph), 1636 = 15.0 min).
(CvO), 1030 (C–N), 679 and 729 (C–Harom). Exact mass (APCI):
m/z = 341.1160 (calcd 341.1182 for C17H2335Cl2N2O [M + H]+).
6.3. Receptor binding studies
Purity (HPLC): 98.1% (tR = 16.3 min).
6.3.1. Preparation of membrane homogenates from Guinea
pig brain.43–45 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 centri-
fuged at 1200g for 10 min at 4 °C. The supernatant was separ-
ated and centrifuged at 23 500g for 20 min at 4 °C. The pellet
was resuspended in 5–6 volumes of buffer (50 mM TRIS, pH
7.4) and centrifuged again at 23 500g (20 min, 4 °C). This pro-
cedure 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 per mL.
6.3.2. Determination of KOR affinity (Guinea pig
brain).43–45 The assay was performed with the radioligand [3H]
U-69 593 (55 Ci mmol−1, BIOTREND). The thawed Guinea pig
brain membrane preparation (about 100 μg of the protein) was
incubated with various concentrations of test compounds, 1
nM [3H]U-69 593, and TRIS-MgCl2-buffer (50 mM TRIS, 8 mM
MgCl2, pH 7.4) at 37 °C. The non-specific binding was deter-
mined with 10 μM unlabeled U-69 593. The Kd value of
U-69 593 is 0.69 nM.
6.2.23. 2-(3,4-Difluorophenyl)-1-[(1RS,5RS,7RS)-7-(pyrrolidin-
1-yl)-2-azabicyclo[3.2.1]octan-2-yl]ethan-1-one (36). Under N2
atmosphere, Mg turnings (39 mg, 1.6 mmol) were added to a
solution of 33a (54 mg, 0.16 mmol) in dry CH3OH (4 mL) and
the mixture was heated to reflux in the ultrasonic bath for 1 h.
After cooling to room temperature, additional Mg turnings
(39 mg, 1.6 mmol) were added and the mixture was heated to
reflux in the ultrasonic bath for 1 h. Additional Mg turnings
(39 mg, 1.6 mmol) were added and the mixture was heated to
reflux in the ultrasonic bath for 1 h. After cooling to room
temperature, H2O (3 mL) and NaOH (19 mg, 0.48 mmol) were
added and the mixture was cooled down to 0 °C. A solution of
2-(3,4-difluorophenyl)acetyl chloride (0.12 g, 0.64 mmol) in
THF (2 mL) was added dropwise and the mixture was stirred at
room temperature for 16 h. Additional NaOH (27 mg,
0.68 mmol) were added and the mixture was stirred at room
temperature for 30 min. H2O and brine (10 mL each) were
added and the mixture was extracted with EtOAc (3 × 20 mL).
The combined organic layers were dried (Na2SO4), filtered and
the solvent was removed in vacuo. The residue was purified by
flash column chromatography (Ø = 2 cm, h = 15 cm, V = 4 mL,
CH2Cl2/CH3OH = 9 : 1, Rf = 0.34). Colorless oil, yield 26 mg
(48%). C19H24F2N2O (334.4 g mol−1). 1H NMR (600 MHz,
CDCl3): δ [ppm] = 1.22–1.28 (m, 0.43H, 8-CH2), 1.36–1.47 (m,
1.57H, 4-CH2 (1H), 8-CH2 (0.57H)), 1.54–1.68 (m, 1H, 4-CH2),
1.72–1.89 (m, 5.86H, N(CH2CH2)2 (4H), 6-CH2 (1.43H), 8-CH2
(0.43H)), 1.90–1.97 (m, 0.57H, 6-CH2), 2.00–2.12 (m, 0.57H,
6.3.3. Affinity towards MOR, DOR,36,43–45 σ1 and σ2
recetors.47–49 Materials, preparation of membrane homo-
genates from various tissues, protein determination, and the
general procedure for binding assays are detailed in the ESI.†
Abbreviations
8-CH2), 2.43–2.49 (m, 2.43H, 5-CH (0.43H), N(CH2CH2)2 (2H)), DIPEA Ethyldiisopropylamine
2.49–2.53 (m, 0.57H, 5-CH), 2.62–2.69 (m, 1H, 7-CH), 2.73 DMAP 4-Dimethylaminopyridine
(ddd, J = 14.1/12.6/5.3 Hz, 0.43H, 3-CH2ax), 2.77–2.92 (2H, DMP
N(CH2CH2)2), 3.10 (ddd, J = 13.3/11.1/5.5 Hz, 0.57H, 3-CH2ax), DOR
Dess–Martin periodinane
δ-Opioid receptor
3.54–3.60 (m, 1.14H, 3-CH2eq (0.57H), C(vO)CH2Ar (0.57H)), GPCR G protein-coupled receptor
3.63 (d, J = 15.8 Hz, 0.57H, C(vO)CH2Ar), 3.66 (d, J = 16.0 Hz,
h
length of the stationary phase
0.43H, C(vO)CH2Ar), 3.71 (d, J = 15.4 Hz, 0.43H, C(vO) KOR
κ-Opioid receptor
CH2Ar), 4.18 (d, J = 4.2 Hz, 0.43H, 1-CH), 4.39 (dd, J = 14.2/6.8 MAPK Mitogen-activated protein kinase
Hz, 0.43H, 3-CH2eq), 5.11 (d, J = 4.3 Hz, 0.57H, 1-CH), MOR
6.89–6.96 (m, 1H, 6-Harom), 7.01–7.11 (m, 2H, 2-Harom (1H), NOE
5-Harom (1H)). Ratio of the rotational isomers = 57 : 43. 13C NOR
NMR (151 MHz, CDCl3): δ [ppm] = 23.4/23.6 (2C, N(CH2CH2)2), PBS
µ-Opioid receptor
Nuclear Overhauser effect
Nociceptin opioid receptor
Phosphate buffered saline
29.9LR/30.5HR (1C, C-4), 33.8HR/34.4LR (1C, C-5), 35.0HR (1C, TBAF Tetrabutylammonium fluoride
C-8), 35.5LR/35.7HR (1C, C-6), 36.8LR (1C, C-8), 37.0LR (1C, C-3), TsCl
p-Toluenesulfonyl chloride
40.3 (1C, C(vO)CH2Ar), 40.5HR (1C, C-3), 53.1/53.5 (2C,
N(CH2CH2)2), 54.7HR/59.5LR (1C, C-1), 70.7/70.9 (1C, C-7), 117.4
(dd, J = 17.1/13.2 Hz, 1C, C-5arom), 118.0 (dd, J = 17.6/3.2 Hz,
1C, C-2arom), 125.0 (m, 1C, C-6arom), 132.0/132.3 (m, 1C,
C-1arom), 149.4 (dd, J = 247.3/12.6 Hz, 1C, C-4arom), 150.3 (dd, J
V
Fraction size
Author contributions
= 248.4/12.7 Hz, 1C, C-3arom), 168.0LR/168.7HR (1C, C(vO)). Denise Ilari: Synthesis, analytical data, preparation of the
Signals of the major rotational isomer are marked with HR, manuscript. Sarah Maskri: Conformational analysis, calcu-
signals of the minor rotational isomer are marked with LR. IR lation of dihedral angles. Dr Dirk Schepmann: Biological
(neat): ν (cm−1) = 2932 (C–Haliph), 1632 (CvO), 1115 (C–N), 775 activity. Dr Jens Köhler: NOE difference spectra, NOESY
4096 | Org. Biomol. Chem., 2021, 19, 4082–4099
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Paper
spectra, Q-NMR studies, including interpretation of the 11 J. L. Wilson, V. Nayanar and J. S. Walker, The site of anti-
results. Dr Constantin G. Daniliuc: X-ray crystal structure ana-
lysis. Dr Oliver Koch: Theoretical analysis, supervisor of Sarah
Maskri. Bernhard Wünsch: Idea of the project, supervision
Denise Ilari, writing of the manuscript.
arthritic action of the κ-opioid, U-50 488H, in adjuvant
arthritis: importance of local administration, Br. J.
Pharmacol., 1996, 118, 1754–1760.
12 L. M. Snyder, M. C. Chiang, E. Loeza-Alcocer, Y. Omori,
J. Hachisuka, T. D. Sheahan, J. R. Gale, P. C. Adelman,
E. I. Sypek, S. A. Fulton, R. L. Friedman, M. C. Wright,
M. G. Duque, Y. S. Lee, Z. Hu, H. Huang, X. Cai,
K. A. Meerschaert, V. Nagarajan, T. Hirai, G. Scherrer,
D. H. Kaplan, F. Porreca, B. M. Davis, M. S. Gold,
H. R. Koerber and S. E. Ross, Kappa Opioid Receptor
Distribution and Function in Primary Afferents, Neuron,
2018, 99, 1274–1288.
Conflicts of interest
The authors declare no conflict of interests.
Acknowledgements
13 A. Mansour, C. A. Fox, H. Akil and S. J. Watson, Opioid-
receptor mRNA expression in the rat CNS: anatomical
and functional implications, Trends Neurosci., 1995, 18,
22–29.
14 Y. Wang, J. Sun, Y. Tao, Z. Chi and J. Liu, The role of
kappa-opioid receptor activation in mediating antinocicep-
tion and addiction, Acta Pharmacol. Sin., 2010, 31, 1065–
1070.
This work was supported by the Research Training Group
“Chemical biology of ion channels (Chembion)” funded by the
Deutsche Forschungsgemeinschaft (DFG), which is gratefully
acknowledged.
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