Stereoselective synthesis of conformationally restricted KOR agonists based on the 2,5-diazabicyclo[2.2.2]octane scaffold

Article abstract of DOI:10.1039/c7ob01530e

It has been postulated that the KOR affinity depends on the dihedral angle of the ethylenediamine pharmacophore. Herein, 2,5-diazabicyclooctanes bearing a pyrrolidino moiety in the 7-position were envisaged to study KOR agonists with a conformationally rigid ethylenediamine pharmacophore and thus a defined N(pyrrolidine)-C7-C1-N2 dihedral angle. The first approach with an intramolecular addition at the chiral sulfinylimines 9 failed to give bicyclic products. The key step in the second approach was a Dieckmann analogous cyclization providing mixed methyl silyl ketals 11a-e as key intermediates. The highest KOR affinity was found for the 2,5-dibenzyl substituted derivatives (S,R,S)-16a (K_i = 31 nM) and (R,S,R)-16a (K_i = 74 nM) with the pyrrolidine ring oriented towards N-5. The high KOR affinity of (S,R,S)-16a is unexpected, since the KOR pharmacophoric ethylenediamine system adopts a dihedral angle of about 160°, which is quite different from the angle of the energetically most favored conformer of the flexible and potent KOR agonist 2. (S,R,S)-16a represents a KOR agonist with moderate selectivity over MOR (8-fold) and DOR (5-fold), but high selectivity over both σ receptor subtypes. In the [³⁵S]GTPγS assay (S,R,S)-16a reacted as a full KOR agonist with an EC₅₀ value of 240 nM.

Full text of DOI:10.1039/c7ob01530e

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
rsc.li/obc

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Stereoselective synthesis of conformationally restricted KOR agonists based on
DOI: 10.1039/C7OB01530E
the 2,5-diazabicyclo[2.2.2]octane scaffold
a
a,b
c
d
Christian Wittig, Dirk Schepmann,
Michael Soeberdt, Constantin G. Daniliuc,
Bernhard Wünscha,b*
a
Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-
Universität Münster, Corrensstraße 48, D-48149 Münster, Germany
*
Corresponding author: Tel.: +49-251-8333311; Fax: +49-251-8332144;
E-mail: wuensch@uni-muenster.de
b
Cells-in-Motion Cluster of Excellence (EXC 1003 – CiM), University Münster, Germany
c
Dr. August Wolff GmbH & Co. KG Arzneimittel, Sudbrackstr. 56, D-33611 Bielefeld,
Germany
d
Organisch-chemisches Institut der Westfälischen Wilhelms-Universität Münster,
Corrensstraße 40, D-48149-Münster, Germany
Abstract
It has been postulated that the KOR affinity depends on the dihedral angle of the
ethylenediamine pharmacophore. Herein, 2,5-diazabicyclooctanes bearing a pyrrolidino
moiety in 7-position were envisaged to study KOR agonists with a conformationally rigid
ethylenediamine pharmacophore and thus a defined N(pyrrolidine)-C7-C1-N2 dihedral
angle. The first approach with an intramolecular addition at the chiral sulfinylimines 9
failed to give bicyclic products. The key step in the second approach was a Dieckmann
analogous cyclization providing mixed methyl silyl ketals 11a-e as key intermediates.
The highest KOR affinity was found for the 2,5-dibenzyl substituted derivatives (S,R,S)-

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DOI: 10.1039/C7OB01530E
1
6a (K
i
= 31 nM) and (R,S,R)-16a (K = 74 nM) with the pyrrolidine ring oriented towards
i
N-5. The high KOR affinity of (S,R,S)-16a is unexpected, since the KOR
pharmacophoric ethylenediamine system adopts a dihedral angle of about 160°, which
is quite different from the angle of the energetically most favored conformer of the
flexible and potent KOR agonist 2. (S,R,S)-16a represents a KOR agonist with moderate
selectivity over MOR (8-fold) and DOR (5-fold), but high selectivity over both  receptor
3
5
subtypes. In the [ S]GTPS assay (S,R,S)-16a reacted as full KOR agonist with an EC50
value of 240 nM.
Keywords: KOR agonists; 2,5-diazabicyclo[2.2.2]octanes; chiral pool synthesis;
conformational restriction; ethano bridge; structure affinity relationships; receptor
3
5
selectivity; dihedral angle; [ S]GTPS assay
1. Introduction
Behavioral studies with chronic spinal dogs in 1976 resulted in differentiation of the
opioid receptor into three subtypes, which were termed µ-opioid receptor (MOR, agonist
morphine), -opioid receptor (agonist SKF-10,047) and -opioid receptor (KOR, agonist
1
ketocyclazocine). Later, the  receptor was removed from the class of opioid receptors
and the -opioid receptor (DOR, vas deferens) and the opioid receptor-like 1 (ORL1)
2
receptor (NOR) were added to the class of opioid receptors. In the beginning of the
1990ies, the four opioid receptors were cloned. The homology of the four opioid
3
receptors is higher than 60 % on the level of amino acid sequences. The opioid
receptors belong to the  subfamily of class A (rhodopsin-like) G-protein coupled
4
4
5
6
receptors (GPCRs) and are coupled to G-proteins. The KOR, MOR, DOR, and NOR
i
7
receptors were crystallized with various ligands leading to interesting information about
the three dimensional structure of the receptors and their binding sites.

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DOI: 10.1039/C7OB01530E
The molecular structure of the T4-lysozyme construct of the human KOR in complex
with the antagonist JDTic ((R)-7-hydroxy-N-{(S)-2-[(3R,4R)-4-(3-hydroxyphenyl)-3,4-
dimethylpiperidin-1-yl]-1-isopropylethyl}-1,2,3,4-tetrahydroisoquinoline-3-carboxamide)
was determined as homodimer with a resolution of 2.9 Å. It allows a precise insight into
the binding pocket occupied by the KOR antagonist JDTic. The salt bridge between two
protonated amino moieties of JDTic and the carboxy moiety of Asp138 represents the
key interaction stabilizing binding of the ligand to the binding pocket. Whereas Asp138
8
is conserved in all opioid receptors (“message” structure), lipophilic interactions of the
ligand with lipophilic side chains of the amino acids Val108, Val118, Ile294 and Tyr312
exclusively found in the KOR are responsible for the subtype selectivity (“address”
features).4,8
The strong analgesia mediated by activation of KOR, MOR and DOR opioid receptors
is accompanied by specific receptor subtype side effects. Activation of MOR can cause
respiratory depression, physical dependence and reduced gastrointestinal motility.
Although these very problematic side effects are not observed after activation of KOR,
undesirable centrally mediated side effects such as dysphoria, sedation and diuresis are
mediated by KOR agonists.9
The known KOR agonists belong to four compound classes: peptides derived from the
endogenous KOR agonist dynorphin A (1-17),10 morphinoids with ketocyclazocine,
nalbuphine and buprenorphine as typical representatives,11 the natural product
1
2
salvinorin A isolated from the leaves of Salvia divinorum and ethylenediamines with
1
3
U-50,488 as the first synthetic KOR agonist. (Figure 1) With exception of salvinorin A

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and its derivatives, KOR agonists contain a basic amino group which forms the crucial
DOI: 10.1039/C7OB01530E
ionic interaction with Asp138.4
Figure 1: Prominent KOR agonists.
In case of ethylenediamine KOR agonists the pyrrolidine ring at one end of the ethylene
moiety is featuring the basic functional group. Usually the second N-atom of the central
ethylenediamine unit is bearing the (3,4-dichloropheyl)acetyl moiety representing the
second pharmacophoric substructure of this type of KOR agonists. In addition to U-
1
4
5
0,488, the piperazine derivative GR-89,696 (1) follows this principle. The piperazine
1
with the axially oriented pyrrolidinomethyl moiety in 2-position shows very high KOR
affinity (K
= 0.45 nM).15 Introduction of an additional methyl group into the axially
i

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oriented substituent led to the pyrrolidinoethyl derivative 2 with even higher KOR affinity
DOI: 10.1039/C7OB01530E
1
5
(
K = 0.31 nM). (Figure 2)
i
Figure 2: Design of conformationally restricted KOR agonists 4.
Due to free rotation around the axially oriented C-C-bond, the pyrrolidine ring of
piperazines
dichlorophenyl)acetamido moiety. Calculations of the energy depending on the
N(pyrrolidine)-C-C-N(acyl) dihedral angle resulted in an energy minimum at around
1
and
2
can adopt several orientations relative to the
(
1
6
7
0°. In order to learn more about the bioactive conformation of the rather flexible KOR
agonists 1 and 2, we decided to synthesize and pharmacologically evaluate KOR
agonists with reduced conformational flexibility around the axial C-C-bond by connecting
the methyl moiety of 2 with the 5-position of the piperazine ring (2,5-bridged
piperazines). Recently, we have reported on the synthesis of piperazines 3 with a three-
carbon bridge across the piperazine ring. The (1R,4R,5S)-configured stereoisomer
1
(
R,R,S)-3a (R = CO
2
CH
3
) reveals high KOR affinity with a K value of 1.0 nM. The three-
i

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carbon bridge of (R,R,S)-3 can adopt two energetically favored conformations resulting
DOI: 10.1039/C7OB01530E
in dihedral angles of 45° and 97° of the ethylenediamine pharmacophore.17
2. Results and discussion
2.1. Conformational analysis
Herein, we report the synthesis and pharmacological evaluation of piperazines 4 with a
two-carbon bridge instead of the three-carbon bridge of 3. Theoretical calculations led
to N(pyrrolidine)-C-C-NR dihedral angles of 69° and 159° for the diastereomers with (R)-
and (S)-configuration at 7-position, respectively. (Figure 3) In particular the dihedral
angle of (1S,4R,7R)-4a is between the dihedral angles calculated for the conformers of
(
R,R,S)-317 and is close to the dihedral angle of the energetically most favored
conformer of 2 with (S,S)-configuration at both centers of chirality.16
Figure 3: Calculated dihedral angles of the KOR pharmacophoric substructure
N(pyrrolidine)-C-C-N(CH
3
) of diastereomeric model compounds. (1S,4R,7R)-4a: 69°;
(
1S,4R,7S)-4a: 159°. Right: Definition of the dihedral angle.
2
.2. Synthesis plan

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Two strategies were pursued for the synthesis of the appropriately substituted bicyclic
DOI: 10.1039/C7OB01530E
compounds 4. (Figure 4) In a chiral-pool synthesis (R)-aspartate should be converted
into piperazinediones 6 with an acetate side chain. After transformation of the ester 6
into the sulfinylimine 9, intramolecular addition of an enolate at the activated imino
moiety should give diazabicyclo[2.2.2]octanes 10. The last steps comprise
establishment of the pyrrolidine moiety, reduction of the lactam groups and modification
of the substituents at the N-atoms.
Figure 4: Plans for the synthesis of conformationally constrained KOR agonists 4
depicted with (R)-aspartate as starting material.
According to an alternative plan, the ester 6 will be used in a Dieckmann cyclization
resulting in the tricarbonyl compound 12. The keto group in 7-position of 12 should be
converted into the pyrrolidine ring and the lactam groups of the piperazinedione system
should be reduced to end-up with 4.
2
.3. Synthesis

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In Scheme 1 the synthesis of piperazinediones (S)-6a-e with an acetate side chain is
DOI: 10.1039/C7OB01530E
shown starting with the proteinogenic amino acid (S)-aspartate. The chloroacetamides
(
S)-5a and (S)-5e were obtained in four reaction steps comprising esterification, imine
reduction and acylation.18,19 Various primary amines reacted with the
formation, NaBH
4
chloroacetamides (S)-5a and (S)-5e in a domino reaction starting with a nucleophilic
substitution followed by intramolecular aminolysis to provide the piperazinediones (S)-
1
8
6
a-e in 61-83 % yield. (Scheme 1)
Scheme 1: Synthesis of (dioxopiperazinyl)acetates 6 with diverse substituents at the N-
atoms. Reagents and reaction conditions: (a) 4 steps: 1. CH
3
OH, TMS-Cl; 2. PhCH=O
, NEt , CH CN, reflux, 61
2
or 4-CH
3
O(C
6
H
4
)CH=O; 3. NaBH
4
; 4. ClCH
2
COCl. (b) R -NH
2
3
3
1
8
–
83 %. (S)-6c and (S)-6d have already been reported in ref. The enantiomer (R)-6a
was prepared in the same manner starting from (R)-aspartate.
The sulfinylimines 9 represent the key intermediates in the first strategy. Ester (S)-6e
was converted into the N,O-dimethylhydroxamic acid (S)-7 upon treatment with N,O-
dimethylhydroxylamine and Al(CH
3
)
3
. Reduction of (S)-7 with LiAlH
4
led to the aldehyde
(
S)-8, which was condensed with Ellman’s chiral sulfinamide ((R)-2-methylpropane-2-
sulfinamide)20,21 and Ti(OEt)
to give the sulfinylimine (S,R)-9. (Scheme 2).
4

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DOI: 10.1039/C7OB01530E
Scheme 2: Synthesis of sulfinylimines 9 and attempts to obtain bicyclic systems 10.
Reagents and reaction conditions: (a) HN(CH
. (b) LiAlH
Ti(OEt)
3
)OCH
3
.HCl, Al(CH
3
)
3
, CH
2
Cl
2
, rt, 4 h, 97
t
t
%
4
, THF, -78 °C, 16 h, 49 %. (c) (R)- BuS(=O)NH
2
or (S)- BuS(=O)NH
2
,
4
, THF, rt, 3.5 h, 43 % ((S-R)-9); 64 % ((S-S)-9).
Several reaction conditions were investigated to induce the intramolecular addition at
t
the sulfinylimine. Various bases (e.g. LiHMDS, NaHMDS, KO Bu, 1-6 equivalents) at
various temperatures (-78 °C to 25 °C) as well as various additives (e.g. BF
3
,
Ti(OEt) ,ClSiMe ) were tried. However, the bicyclic compound 10 was not formed. In
4
3
order to exclude that (S,R)-9 represents the mismatched configured diastereomer for
this conversion, the diastereomer (S,S)-9 was prepared by condensation of the
aldehyde (S)-8, with (S)-configured Ellman’s chiral sulfinamide ((S)-2-methylpropane-2-
sulfinamide). However, as observed for (S,R)-9 the intramolecular nucleophilic addition
at the sulfinylimine (S,S)-9 to form 2,5-diazabicyclo[2.2.2]octanes 10 could not be
observed. (Scheme 2)

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DOI: 10.1039/C7OB01530E
Scheme 3: Synthesis of 2,5-diazabicyclo[2.2.2]octane-3,6,7-triones 12 by Dieckmann
analogous cyclization of 6. Reagents and reaction conditions: (a) NaHMDS, THF, -78
°
C, 45 min, then TMS-Cl, -78 °C, 1 h, rt, 2 h, yields are given in Scheme. (b) pTosOH,
THF, H O, rt, 16 h, 70 % ((S,S)-12a), 95 % ((S,S)-12b). (c) HCl, THF, rt, 16 h, 74 %
(S,S)-12c). The enantiomers (R,R,S)-11a and (R,R)-12a were prepared in the same
2
(
manner starting from (R)-configured piperazinedione (R)-6a.
According to the second strategy, a Dieckmann cyclization of the piperazinediones (S)-
6
should give the bicyclic compounds (S,S)-12. (Scheme 3) However, reaction of the
piperazinediones (S)-6 with various bases did not lead to bicyclic tricarbonyl compounds
S,S)-12, since the cyclization product could not be stabilized by deprotonation.
(
Therefore, the piperazinediones (S)-6 were treated with NaHMDS at -78 °C and the
primary cyclization products, the anion of hemiketals, was trapped with
chlorotrimethylsilane to yield the mixed methyl silyl ketals (S,S,R)-11. The yields of this
transformation were strongly dependent on the substitution pattern of the
piperazinediones (S)-6. As reported previously, (S,S,R)-11d with a methyl moiety at N-
1
9
2
was obtained in 34 % yield. The dibenzyl derivative (S,S,R)-11a was isolated in
slightly higher yield (39 %) after careful optimization, but a cyclohexylmethyl moiety at

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N-2 led to considerably reduced yields ((S,S,R)-11c, 15 % yield). The piperazinediones
DOI: 10.1039/C7OB01530E
(
S)-6b and (S)-6e with one p-methoxybenzyl moiety were prepared in order to have
orthogonal protective groups at the N-atoms. Despite intensive optimization, the mixed
methyl silyl ketals (S,S,R)-11b and (S,S,R)-11e were obtained in only 12 % and 0.3 %
yield, respectively. It was concluded that the p-methoxybenzyl moiety in particular at N-
5
is detrimental for this cyclization reaction. Hydrolysis of the mixed methyl silyl ketals
(
S,S,R)-11a-c provided the ketones (S,S)-12a-c on 70-95 % yields.
Scheme 4: Synthesis of 7-pyrrolidino substituted 2,5-diazabicyclo[2.2.2]octanes
containing KOR-pharmacophoric structural elements. Reagents and reaction
conditions: (a) NaBH
4
, CH
3
OH, 5-10 °C, 30 min, 84 %. (b) CH
3
SO
2
Cl, CH
2
Cl
2
, NEt
3
,
DMAP, rt, 16 h, 78 %. (c) Pyrrolidine, NaBH(OAc)
3
, THF, AcOH, rt, 3 d or 16 h, fc

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separation, 56 % (S,S,R)-15a and 6 % (S,S,S)-15a; 79 % (S,S,R)-15b and 8 % (S,S,S)-
DOI: 10.1039/C7OB01530E
1
5b. (d) LiAlH
f) 2-(3,4-dichlorophenyl)acetyl chloride, CH
S,R,S)-16a, (S,R,R)-16a and (S,R,S)-18 were prepared in the same manner starting
4
, THF, reflux, 16 h, 30-67 %. (e) H
2
, balloon, Pd/C, THF, rt, 16 h, 74 %.
(
(
2
Cl , rt, 16 h, 45 %. The enantiomers
2
from (R,R)-configured trione (R,R)-12a.
NaBH reduction of the ketone (S,S)-12a with two benzyl moieties led to a mixture of
4
diastereomeric alcohols (S,S,R)-13a and (S,S,S)-13a (ratio 80:20), which was reacted
with methanesulfonyl chloride. (Scheme 4) After chromatographic purification, only the
major diastereomer (S,S,R)-14a was isolated in 78 % yield. However, all attempts failed
to convert the mesylate (S,S,R)-14a into pyrrolidine derivatives 15a.
Next, the direct reductive amination of the ketone (S,S)-12a with pyrrolidine and
NaBH(OAc) 22
of an acid. The yields and the ratio of diastereomers obtained with the additives HOAc
(S,S,R)-15a: 56 %; (S,S,S)-15a: 6 %) and Ti(OiPr) ((S,S,R)-15a: 55 %; (S,S,S)-15a: 9
) were very similar. Therefore, HOAc, which could be handled and removed easily,
was investigated. This transformation was successful only after addition
3
(
4
%
was used preferably for the synthesis of the pyrrolidines (S,S,R)-15a and (S,S,S)-15a in
larger amounts, for the synthesis of the enantiomeric pyrrolidines (R,R,S)-15a and
(
R,R,R)-15a and the p-methoxybenzyl substituted analogs (S,S,R)-15b and (S,S,S)-
5b. (Scheme 4)
1
Reduction of the dilactams (S,S,R)-15a,b and (S,S,S)-15a with LiAlH led to the
4
diazabicyclooctanes (R,S,R)-16a,b and (R,S,S)-16a in 30-67 % yields. Hydrogenolytic
removal of the benzyl moieties of the major diastereomer (R,S,R)-16a resulted in the
unsubstituted bicyclic compound (R,S,R)-17, which was acylated with

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DOI: 10.1039/C7OB01530E
(
dichlorophenyl)acetyl chloride to afford the bis[(dichlorophenyl)acetamide] (R,S,R)-18.
(Scheme 4)
The enantiomers (S,R,S)-16a, (S,R,R)-16a, and (S,R,S)-18 were prepared in the same
manner starting from the enantiomeric amino acid (R)-aspartate.
2.4. Structure determination
Figure 5: Molecular structure of (R,R,S)-15a, the main diastereomer after reductive
amination of ketone (R,R)-12a with pyrrolidine and NaBH(OAc) . Thermal ellipsoids are
3
shown at 30 % probability. P43 chiral space group with Flack parameter 0.01(5).
Dihedral angles: N31-C7-C1-N2: 179,5(1)°; H7-C7-C1-N2: 59.0°.
The configuration of the major diastereomer obtained by reductive amination of the
ketone (R,R)-12a was assigned unequivocally by recording an X-ray crystal structure,
which is shown in Figure 5. The crystal structure displays the pyrrolidine ring in anti-
periplanar orientation to N-2 of the bicyclic framework. This orientation leads to (S)-
configuration of the newly formed center of chirality in 7-position. (S)-configuration in 7-

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position originates from Re-face attack of the reducing agent at the intermediate iminium
DOI: 10.1039/C7OB01530E
ion. This result fits nicely to the hypothesis that the large benzyl moiety at 5-position
inhibits the attack of the nucleophile along the Bürgi-Dunitz trajectory of 107° from the
1
7
SI-face. The dihedral angle of the ethylenediamine pharmacophore in the solid state
is 179.5°.
Figure 6: Molecular structure of racemic 18. Thermal ellipsoids are shown at 30 %
probability. The crystal contains both enantiomers in the ratio 1:1. Centrosymmetric
space group P2/c. Dihedral angles: N31-C7-C1-N2: 178.4(3)°; H7-C7-C1-N2: - 61.0°.
1
Analysis of the structure of diamides (S,R,S)-18 and (R,S,R)-18 by H NMR
spectroscopy was difficult due to broad signals caused by rotational isomerism along
the N-C(=O) bonds. Therefore, an X-ray crystal structure was recorded. Due to partial
racemization of diamide (S,R,S)-18 during the synthesis, a racemic mixture (S,R,S)-
18/(R,S,R)-18 was crystallized; the elemental unit contained the enantiomers in a 1:1
ratio. The molecular structure of 18 shows two acyl moieties at the ring N-atoms
confirming the constitution of the final product. (Figure 6) Moreover, the orientation of
the pyrrolidine ring towards N-5 of the bicyclic framework was proved by the crystal
structure. The amide moieties together with the adjacent substituents form an almost

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planar structure. According to the molecular structure, the ethylenediamine
DOI: 10.1039/C7OB01530E
pharmacophoric system adopts a dihedral angle of 178.4°.
2.5. Pharmacological evaluation
KOR affinity
Competitive radioligand receptor binding studies were used to determine the KOR
3
affinity of 2,5-diazabicyclocotanes 15-18. [ H]U-69,593 served as radioligand and
guinea pig brain membrane preparations as receptor material. A large excess of U-
69,593 (10 µM) was used to determine the non-specific binding of the radioligand in the
assay.16,17 Table 1 summarizes the KOR affinity recorded for the test compounds 15-18
and the reference compounds 2, U-69,593, naloxone and morphine.
Table 1: Affinities of 2,5-diazabicyco[2.2.2]octanes with pyrrolidine moiety towards KOR
and related receptors.
K
i
± SEM (nM)a,b
DORc)
Compd.
R1
R2
KORb)
[3
H]U-
MORc)

1

2
[3H]-(+)-
3
3
3
6
9,593
0 %
0 %
0 %
4 %
5 %
0 %
[ H]DAMGO [ H]DPDPE pentazocine [ H]DTG
(
S,S,R)-15a
S,S,S)-15a
R,R,S)-15a
R,R,R)-15a
S,S,R)-15b
S,S,S)-15b
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
24 %
0 %
8 %
4 %
0 %
0 %
0 %
0 %
0 %
0 %
0 %
0 %
0 %
5 %
4 %
3 %
(
(
Bn
0 %
0 %
(
Bn
0 %
16 %
12 %
3 %
(
PMB
PMB
38 %
0 %
(

Organic & Biomolecular Chemistry
Page 16 of 66
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(
R,S,R)-16a
Bn
Bn
Bn
Bn
Bn
Bn
Bn
74 ± 14
0 %
>1 µM
25 %
241
27 %
23 ± 9
270
260
-
318
>1 µM
146
75
D
5
OI: 10.1039/C
>
7
1
OB
µ
01530E
M
(
(
R,S,S)-16a
S,R,S)-16a
13 %
>1 µM
Bn
31 ± 9
0 %
882
>1 µM
(
S,R,R)-16a
R,S,R)-16b
R,S,R)-18
Bn
>1 µM
30 ± 13
>1 µM
10 %
-
>1 µM
>1 µM
(
PMB
155
343
>1 µM
c
c
c
(
DCPA DCPA
92 ± 26
253
0 %
3 %
c
(
S,R,S)-18
DCPA DCPA
5 %
0 %
2
0.31 ± 0.04
0.88 ± 0.10
-
-
-
-
-
-
U-69,593
naloxone
-
-
-
6.9 ± 0.50 2.1 ± 0.50 2.4 ± 0.50
-
morphine
35 ± 6.0
3.9 ± 2.1
2.0 ± 0.30
-
(
+)-pentazocine
-
-
-
-
-
-
5.4 ± 0.5
haloperidol
6.6 ± 0.90 78 2.3
a)
In case of high affinity (K < 100 nM) the experiments were repeated twice (n = 3)
i
and SEM values are given in Table 1.
b)
Values in % reflect the inhibition of the radioligand binding at a test compound
concentration of 1 μM.
c)
DCPA = 2-(3,4-dichlorophenyl)acetyl
The data in Table 1 indicate very low KOR affinity for compounds 15 with two carbonyl
moieties within the bicyclic framework. After reduction, pyrrolidine derivatives 16 display
promising KOR affinity, which is strongly dependent on the configuration: Only
compounds (S,R,S)-16a and (R,S,R)-16a with the pyrrolidine ring oriented towards N-5
show high KOR affinity. (S,R,S)-16a represents the eutomer with a K value of 31 nM.
i
Taking into account that partial racemization had occurred, the eudismic ratio is
expected to be higher than 2. The dihedral angle of the ethylenediamine pharmacophore
of (S,R,S)-16a is in the range 159-180° (compare Figures 3, 5, and 7). This result is
unexpected, as this arrangement is quite different from the energetically favored
conformation (70°) of the flexible KOR agonist 2. A possible explanation is a revers

Page 17 of 66
Organic & Biomolecular Chemistry
17
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orientation of (S,R,S)-16a in the KOR binding pocket with N-5 together with the
DOI: 10.1039/C7OB01530E
pyrrolidine ring representing the KOR-pharmacophoric structural elements. Although the
dihedral angle of the diastereomer (S,R,R)-16a (69°, see Figure 3) is close to the
energetically favored conformation (70°), its KOR affinity is negligible.
Replacement of the benzyl groups of the KOR agonists (S,R,S)-16a and (R,S,R)-16a by
(dichlorophenyl)acetyl moieties resulted in reduced KOR affinity. Although (S,R,S)-18
contains both pharmacophoric elements, an 8-fold reduction of KOR affinity was
detected. It is postulated that the large second (dichlorophenyl)acetyl moiety at N-5
might inhibit the interaction with KOR.
Receptor selectivity
The MOR and DOR affinity was recorded in receptor binding studies using the
3
3
23
radioligands [ H]DAMGO and [ H]DPDPE, respectively. The bislactams 15 as well as
the dibenzyl derivatives (R,S,S)-16a and (S,R,R)-16a without KOR affinity did not
display any MOR and DOR affinity. However, the KOR agonists (S,R,S)-16a and
(
R,S,R)-16a showed moderate MOR and DOR affinity leading for the most potent KOR
agonist (S,R,S)-16a to KOR/MOR and KOR/DOR selectivity factors of 8-fold and 5-fold
respectively.
Introduction of a methoxy moiety into the N-2 benzyl group of (R,S,R)-16a led to (R,S,R)-
16b with lightly reduced KOR affinity, but considerably increased MOR and DOR affinity.
Altogether, (R,S,R)-16b represents a ligand with 5- to 6-fold preference for MOR and
DOR compared with KOR. An increased MOR affinity was also observed for the
(dichlorophenyl)acetyl derivatives (R,S,R)-18 and (S,R,S)-18 leading to unselective
ligands.

Organic & Biomolecular Chemistry
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DOI: 10.1039/C7OB01530E
1
Originally,  receptors were regarded as opioid receptor subtype. Moreover, the ligand
binding profile of opioid receptors, in particular KORs and  receptors is often very
similar.24-27 Therefore, determination of 
and  receptor affinity was included into this
1
2
3
3
study. [ H](+)-pentazocine and [ H]di-o-tolylguanidine were employed as radioligands in
the receptor binding studies.28-30 As described for MOR and DOR affinity the bislactams
1
5 and the dibenzyl derivatives (R,S,S)-16a and (S,R,R)-16a without KOR affinity did
not interact with both  receptor subtypes. Weak  receptor affinity was detected for
R,S,R)-16a and (S,R,S)-16a resulting in 10- and 25-fold KOR/ receptor selectivity,
respectively. The (dichlorophenyl)acetamides (R,S,R)-18 and (S,R,S)-18 did not show
any  or  receptor affinity.
1
(
1
1
2
Intrinsic activity
The intrinsic activity of the most promising KOR ligands (S,R,S)-16a and (R,S,R)-16a
3
5
35
was investigated with the [ S]GTPS ([ S]-guanosine-5´-3-O-(thio)triphosphate)
binding assay.31,32
U-69,593
U-69,593
(
R,S,R)-16a
(
S,R,S)-16a
3
5
Figure 7: KOR agonistic activity of (S,R,S)-16a and of (R,S,R)-16a in the [ S]GTPS
3
5
assay. Left: Comparison of [ S]GTPγS binding after activation of KORs by U-69,593
red curve) and by (S,R,S)-16a (blue curve, EC50 = 240 nM); Right: Comparison of
(

Page 19 of 66
Organic & Biomolecular Chemistry
19
[35
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DOI: 10.1039/C7OB01530E
S]GTPγS binding after activation of KORs by U-69,593 (red curve) and by (R,S,R)-
16a (blue curve, EC50 = 540 nM).
In this assay both KOR ligands (S,R,S)-16a and (R,S,R)-16a behaved as full agonists
with the same intrinsic activity as the reference compound U-69,593. (S,R,S)-16a, the
ligand with higher KOR affinity, led to an EC50 value of 240 nM, whereas the lower KOR
affinity enantiomer (R,S,R)-16a provided an EC50 value of 540 nM. (Figure 7) Although
these data show a nice correlation between KOR affinity and activation of KORs, the
EC50 values are considerably higher than the EC50 value of U-69-593 (EC50 = 2.5.nM).
However, it is demonstrated that ethylenediamine ligands without the
dichlorophenylacetyl KOR pharmacophoric group are able to activate KORs.
Conclusion
The synthesis of ethano bridged piperazine derivatives is rather difficult, due to high
conformational constraints. Trapping of intermediate hemiketal anions resulted in very
low to moderate yields of mixed methyl silyl ketals 11. Although both pharmacophoric
elements (pyrrolidine ring, (dichlorophenyl)acetamide) are present in (R,S,R)-18 and
(
S,R,S)-18, only moderate to low KOR affinity was found. It is postulated that either an
unfavorable dihedral angle of 178° (Figure 6) or the large substituent at N-5 is
responsible for the low KOR affinity. The most promising KOR agonists are the 2,5-
dibenzyl substituted derivatives (R,S,R)-16a and (S,R,S)-16a with K values of 74 nM
i
and 31 nM, respectively. The nanomolar KOR affinity of (S,R,S)-16a was unexpected,
since the (dichlorophenyl)acetyl moiety is missing and the dihedral angle of the
ethylenediamine KOR pharmacophore (159-180°) differs considerably from the
expected value. (S,R,S)-16a requires higher concentrations (EC50 = 240 nM) than U-
69,593 (EC50 = 2.5 nM) to reach the same full KOR agonistic effects as U-69,593, which
is in accordance to the 30-fold lower KOR affinity of (S,R,S)-16a.

Organic & Biomolecular Chemistry
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DOI: 10.1039/C7OB01530E
5
. Experimental Chemistry
.1. General Methods
5
Unless otherwise noted, reactions were conducted under dry nitrogen in absolute
solvents. THF was dried with sodium/benzophenone and was freshly distilled before
use.
5
.2. Instruments
Thin layer chromatography (tlc): Silica gel 60 F254 plates (Merck). Flash chromatography
fc): Silica gel 60, 40–64 µm (Merck); parentheses include: diameter of the column,
eluent, fraction size, R value. Melting point: Melting point apparatus SMP 3 (Stuart
(
f
Scientific), uncorrected. MS: MAT GCQ (Thermo-Finnigan); IR: IR spectrophotometer
1
4
80Plus FT-ATR-IR (Jasco). H NMR (400 MHz): Unity Mercury Plus 400 spectrometer
(
Varian);  in ppm related to tetramethylsilane; coupling constants are given with 0.5 Hz
resolution. Optical rotation: Polarimeter 341 (Perkin Elmer); 1.0 dm tube; concentration
c in g/100 mL; T = 20 °C; wavelength 589 nm (D-line of Na light); the unit of the specific
rotation ([] T
deg mL dm g ) is omitted for clarity.
.
.
-1. -1
D
5.3. HPLC method for the determination of the purity
Merck Hitachi Equipment; UV detector: L-7400; autosampler:L-7200; pump: L-7100;
®
®
degasser: L-7614; column: LiChrospher 60 RP-select B (5 µm); LiCroCART 250-4
mm cartridge; flow rate: 1.00 mL/min; injection volume: 5.0 µL; detection at λ = 210 nm;
solvents: A: water with 0.05% (v/v) trifluoroacetic acid; B: acetonitrile with 0.05% (v/v)
trifluoroacetic acid: gradient elution: 0.0 min: 90.0 % of A, 10.0 % of B; 4.0 min: 90.0 %
of A, 10.0 % of B; 29.0 min: 0.0 % of A, 100.0 % of B; 31.0 min: 0.0 % of A, 100.0 % of

Page 21 of 66
Organic & Biomolecular Chemistry
21
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B; 31.5 min: 90.0 % of A, 10.0 % of B; 40.0 min: 90.0 % of A, 10.0 % of B. According to
DOI: 10.1039/C7OB01530E
this HPLC method the purity of all test compounds was greater than 95 %.
5.4. X-ray crystallography
Data sets for compounds (R,R,S)-15a, and 18 were collected with a D8 Venture Dual
Source 100 CMOS diffractometer. Programs used: data collection: APEX2 V2014.5-0
3
3
33
(
Bruker AXS Inc., 2014); cell refinement: SAINT V8.34A (Bruker AXS Inc., 2013);
data reduction: SAINT V8.34A (Bruker AXS Inc., 2013);33 absorption correction,
SADABS V2014/2 (Bruker AXS Inc., 2014);33 structure solution SHELXT-2014
(
Sheldrick, 2014);34 structure refinement SHELXL-2014 (Sheldrick, 2014)34 and
3
5
graphics, XP (Bruker AXS Inc., 2014). R-values are given for observed reflections, and
2
wR values are given for all reflections. Thermals ellipsoids are shown with 30 %
probability.
5
.6. Synthetic Procedures
Dimethyl (S)-2-[N-(2-chloroacetyl)-N-(4-methoxybenzyl)amino]butanedioate ((S)-
e)
Dimethyl (S)-aspartate HCl (6.9 g, 32.6 mmol) was dissolved in CH
Triethylamine (4.5 mL, 32.6 mmol) and freshly distilled p-methoxybenzaldehyde (4.0
mL, 32.6 mmol) were added successively. Afterwards, Na SO was added and the
mixture was stirred for 16 h at room temperature. Then, the suspension was filtered and
the filtrate was concentrated in vacuo. The resulting residue was suspended in Et O and
the mixture was filtered again. Removing of the solvent of the filtrate in vacuo gave the
imine (8.4 g, 30.1 mmol) as a colorless oil. The imine was dissolved in CH OH (50 mL).
NaBH (1.9 g, 51.2 mmol) was added over 45 min under ice cooling. Afterwards, the
mixture was stirred at room temperature for further 5 min, before the solvent was
5
.
2
Cl (75 mL).
2
2
4
2
3
4

Organic & Biomolecular Chemistry
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DOI: 10.1039/C7OB01530E
removed in vacuo. For work-up, H
layer was extracted with Et O (6 x 30 mL). The combined organic layers were dried
CO ), filtered and the solvent was removed under reduced pressure to give the
secondary amine (8.1 g, 28.8 mmol) as a colorless solid, which was dissolved in CH Cl
75 mL). Under N -atmosphere and ice cooling, chloroacetyl chloride (3.7 mL, 46.1
2
O (60 mL) was added to the residue and the aqueous
2
(
K
2
3
2
2
(
2
mmol) and triethylamine (3.2 mL, 23.0 mmol) were added slowly and the mixture was
then stirred for 2.5 h at room temperature. Afterwards the solvent was removed in vacuo
and diethyl ether was added to the resulting residue. This suspension was filtered and
the filtrate was concentrated in vacuo. The orange oily residue was purified by fc (Ø
6
cm, h = 19 cm, v = 65 mL, cyclohexane : EtOAc = 2:1, R
f
= 0.25). Orange oil, yield 6.3
= -83.7 (c = 0.41; CH OH). HPLC:
): δ (ppm) = 2.69 (dd, J = 17.1 /
2
0
g (55 % over 3 steps). C16
H20ClNO
6
(357.8). []
D
3
1
Purity 90.9 %, t
R
= 17.5 min. H NMR (600 MHz, CDCl
3
HR
6
.6 Hz, 0.85H, CHCH
2
CO
2
CH
3
), 2.86 (dd, J = 17.3 / 4.6 Hz, 0.15H,
CHCH
2
CO
2
CH
3
NR), 3.08 (dd, J = 17.3 / 4.4 Hz, 0.15H, CHCH
2
CO
2
CH
NR), 3.28 (dd, J =
3
HR
NR), 3.59 (s, 3 x 0.15H,
1
7.1, 6.6 Hz, 0.85H, CHCH
2
CO
NR), 3.65 (s, 3 x 0.85H, OCH
NR), 3.81 (s, 3 x 0.85H, OCH
2
CH
3
), 3.58 (s, 3 x 0.15H, OCH
3
OCH
3
3
3
HR), 3.67 (s, 3 x 0.85H, OCH
3
HR), 3.77 (s, 3 x 0.15H,
HR
OCH
3
HR), 4.07 (d, J = 12.7 Hz, 0.85H, O=CCH
Cl ), 4.14
2
HR
NR
(
d, J = 12.6 Hz, 0.85H, O=CCH
2
Cl ), 4.23 (d, J = 15.5 Hz, 0.15H, NCH
2
Ar ) ,4.42 (s,
NR
HR
broad, 2 x 0.15H, O=CCH
2
Cl ), 4.45 (t, J = 6.6 Hz, 0.85H, CHCH
2
CO
2
CH
3
), 4.60 (d,
ArHR ), 4.66 (d, J = 16.2 Hz, 0.85H, NCH
Ar ), 4.77 (d, J =
HR
J = 16.2 Hz, 0.85H, NCH
2
2
1
8
2
8
=
4
5.4 Hz, 0.15H, NCH
2
ArNR ), 5.00 (t, J = 7.5 Hz, 0.15H, CHCH
2
CO
2
CH
NR), 6.80 (d, J =
3
NR
HR
.4 Hz, 2 x 0.15H, Ar-H ), 6.90 (d, J = 8.6 Hz, 2 x 0.85H, Ar-H ), 7.12 (d, J = 8.3 Hz,
NR
HR
x 0.15H, Ar-H ), 7.28 (d, J = 8.6 Hz, 2 x 0.85H, Ar-H ). The ratio of rotamers is
1
3
5:15. HR = major rotamer, NR = minor rotamer. C NMR (101 MHz, CDCl
3
): δ (ppm)
HR), 34.6 (1C, CHCH
CO
CH
NR), 41.5 (1C, O=CCH
Cl ),
HR
34.4 (1C, CHCH
2
CO
2
CH
3
2
2
3
2
NR
NR
HR), 52.3 (1C, OCH NR),
2.0 (1C, O=CCH
2
Cl ), 47.0 (1C, NCH
2
Ar ), 52.1 (1C, OCH
3
3

Page 23 of 66
Organic & Biomolecular Chemistry
23
HR), 53.1 (2C, NCH
ArHR+NR), 55.4 (1C, OCH
HR
), 55.5 (1C, OCH
ViewNARrticle Online
),
5
2.7 (1C, OCH
3
2
3
3
DOI: 10.1039/C7OB01530E
NR), 57.1 (1C, CHCH
CO
CH
3
HR
), 114.0 (1C, Ar-C ), 114.4
NR
5
6.9 (1C, CHCH
2
CO
2
CH
3
2
2
HR
HR
HR
NR
(
2C, Ar-C ), 127.0 (1C, Ar-C
q
), 129.1 (2C, Ar-C ), 129.2 (1C, Ar-C ), 159.7 (1C,
HR
HR
NR
CH
HR),
Ar-C ), 167.0 (1C, O=CCH
2
Cl ), 167.8 (1C, O=CCH
2
Cl ), 169.8 (1C, CO
2
3
169.9 (1C, CO
2
CH
3
NR), 170.6 (1C, CO
2
CH
3
NR), 171.8 (1C, CO
2
CH
3
HR). HR = major
rotamer, NR = minor rotamer. Exact mass (APCI): m/z = 358.1057 (calcd. 358.1052 for
3
5
+
+
-1
C
16
H
21Cl NO
s, C=Oamide), 1512 (m, C=C arom.), 1435 (m, C-Harom.), 1246, 1173 (s, C-Oether),
18 (m, C-Harom. disubst.) 798 (w, C-Cl).
6
[MH] ). IR (neat): ῦ [cm ] = 2951 (w, C-Haliph.), 1732 (s, C=Oester), 1654
(
8
Methyl (S)-2-(1,4-dibenzyl)-3,6-dioxopiperazin-2-yl)acetate ((S)-6a)
Under N , (S)-5a (9.6 g, 29.1 mmol) was dissolved in dry acetonitrile (molecular sieves
Å, 200 mL). Benzylamine (4.8 mL, 43.7 mmol) and triethylamine (2.6 mL, 34.9 mmol)
2
3
were added dropwise. The mixture was heated to 70 °C for 1 h and then stirred at room
temperature for further 16 h. The solvent was removed in vacuo and the residue was
dissolved in EtOAc (150 mL). The organic layer was washed with 0.5 M HCl (2 x 30 mL),
0
.5 M NaOH (30 mL) and brine (30 mL). After drying (Na
concentrated under reduced pressure. The residue was purified by fc (Ø 8cm, h = 12
cm, v = 65 mL, cyclohexane : EtOAc = 3 : 2  2 : 3  0 : 1, R = 0.22 (cyclohexane :
EtOAc = 3:2)). Yellow solid, mp 97-98 °C, yield 8.9 g (83 %). C21
+34.8 (c = 0.75; CH Cl ). Purity by HPLC: 94.2 %, t = 19.5 min.
2
SO ), it was filtered and
4
f
2
0
H
22
N
2
O
4
(366.4). []
D
=
2
2
R
Methyl (R)-2-(1,4-dibenzyl-3,6-dioxopiperazin-2-yl)acetate ((R)-6a)

Organic & Biomolecular Chemistry
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Under N
2
, (R)-5a (18.6 g, 56.7 mmol) was dissolved in dry acetonitrile (molecular sieves
DOI: 10.1039/C7OB01530E
3
Å, 220 mL). Benzylamine (9.3 mL, 85.1 mmol) and triethylamine (9.4 mL, 68.04 mmol)
were added dropwise. The mixture was heated to 70 °C for 1 h and then stirred at room
temperature for further 16 h. The solvent has then been removed in vacuo and the
residue was dissolved in EtOAc (150 mL). The organic layer was washed with 0.5 M
HCl (2 x 30 mL), 0.5 M NaOH (30 mL) and brine (30 mL). After drying (Na
filtered and concentrated under reduced pressure. The residue was purified by fc (Ø
cm, h = 20 cm, v = 65 mL, cyclohexane:EtOAc = 3:2  2:3  0:1, R = 0.22
cyclohexane : EtOAc = 3 : 2)). Pale yellow solid, mp 99-100 °C, yield 18.2 g (88 %).
2
SO
4
), it was
8
f
(
C
21
H
22
N
2
O
4
(366.4). [] 20
D
= -25.9 (c = 1.58; CH
3
OH). Purity by HPLC: 94.0 %, t
R
= 19.1
min.
Spectroscopic data of (S)-6a and (S+R)-6a
1H NMR (400 MHz, CDCl
3
): δ (ppm) = 2.82 (dd, J = 17.1 / 5.0 Hz, 1H, CHCH
2
CO
2
CH ),
3
3
.08 (dd, J = 17.1 / 3.3 Hz, 1H, CHCH
2
CO
N ), 4.15 (d, J = 16.6 Hz, 1H, O=CCH
Ph), 4.51 (d, J = 14.5 Hz, 1H, NCH
Ph), 5.07 (d, J = 15.1 Hz, 1H, NCH Ph), 7.21 – 7.38 (m,
0H, Ar-H). C NMR (101 MHz, CDCl ): δ (ppm) = 35.3 (1C, CHCH CO CH ), 47.3 (1C,
Ph), 49.5 (1C, O=CCH N), 49.8 (1C, NCH Ph), 52.2 (1C, OCH ), 56.2 (1C,
CHCH CO CH ), 128.19 (2C, Ar-C), 128.20 (1C, Ar-C), 128.3 (1C, Ar-C), 128.7 (2C, Ar-
C), 129.0 (2C, Ar-C), 129.1 (2C, Ar-C), 135.1 (1C, C-1(benzyl)), 135.6 (1C, C-1(benzyl)),
2
CH
3
), 3.54 (s, 3H, OCH
N), 4.16 (t, J = 4.34 Hz, 1H,
Ph),
3
), 3.87 (d, J = 17.3
Hz, 1H, O=CCH
2
2
CHCH
2
CO
2
CH
3
), 4.21 (d, J = 15.1 Hz, 1H, NCH
2
2
4
1
.71 (d, J = 14.5 Hz, 1H, NCH
2
2
1
3
3
2
2
3
NCH
2
2
2
3
2
2
3
164.5 (1C, C=O), 165.5 (1C, C=O), 170.2 (1C, CO
2
CH
3
). Exact mass (APCI): m/z =
+
+
-1
367.1660 (calcd. 367.1652 for C21
H
23
N
2
O
4
[MH] ). IR (neat): ῦ [cm ] = 2947 (w, C-
Haliph.), 1732 (s, C=Oester), 1655 (s, C=Oamide), 1204, 1180 (s, C-Oester), 752, 702 (m, C-
Harom. monosubst.).

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DOI: 10.1039/C7OB01530E
Methyl (S)-2-[1-benzyl-4-(4-methoxybenzyl)-3,6-dioxopiperazin-2-yl]acetate
(S)-6b)
Under N
Å, 230 mL). p-Methoxybenzylamine (11.0 mL, 84.3 mmol) and triethylamine (9.4 mL,
7.5 mmol) were added dropwise. The mixture was heated to 70 °C for 1 h and then
(
2
, (S)-5a (18.4 g, 56.2 mmol) was dissolved in dry acetonitrile (molecular sieves
3
6
stirred at room temperature for further 16 h. The solvent was removed in vacuo and the
residue was dissolved in EtOAc (150 mL). The organic layer was washed with 0.5 M
HCl (2 x 30 mL), 0.5 M NaOH (30 mL) and brine (30 mL). After drying (Na
filtered and concentrated under reduced pressure. The residue was purified by fc (Ø
cm, h = 15 cm, v = 65 mL, cyclohexane/EtOAc = 2:1  1:1  1:2, R = 0.16
cyclohexane : EtOAc = 1 : 1)). Colorless solid, mp 99-101 °C, yield 17.5 g (79 %).
2
SO
4
), it was
8
f
(
C
22
H
24
N
2
O
5
(396.4). [] 20
D
= +69.8 (c = 1.2; CH
): δ (ppm) = 2.80 (dd, J = 17.1 / 5.1 Hz, 1H,
), 3.06 (dd, J = 17.1 / 3.3 Hz, 1H, CHCH CO CH ), 3.54 (s, 3H,
), 3.80 (s, 3H, Ar-OCH ), 3.85 (d, J = 17.2 Hz, 1H, O=CCH N), 4.11 (d, J = 17.5
Hz, 1H, O=CCH N), 4.14 (t, J = 4.1 Hz, 1H, CHCH CO CH ), 4.20 (d, J = 15.1 Hz, 1H,
NCH Ph), 4.43 (d, J = 14.4 Hz, 1H, NCH PMB), 4.65 (d, J = 14.4 Hz, 1H,
CH NCH PMB), 5.07 (d, J = 15.1 Hz, 1H, NCH Ph), 6.87 (d, J = 8.4 Hz, 2H, 3-H, 5-H (p-
3
OH). Purity by HPLC: 92.9 %, t = 19.9
R
1
min. H NMR (400 MHz, CDCl
3
CHCH
2
CO
2
CH
3
2
2
3
CO CH
2
3
3
2
2
2
2
3
2
2
2
2
2
methoxybenzyl)), 7.20 (d, J = 8.2 Hz, 2H, 2-H, 6-H (p-methoxybenzyl)), 7.22 – 7.37 (m, 5H, Ar-H).
13C NMR (101 MHz, CDCl
CHNCH Ar), 49.2 (1C, CH NCH
CO CH ), 56.2 (1C, CHCH CO
): δ (ppm) = 35.3 (1C, CHCH
Ar), 49.3 (1C, O=CCH N), 52.1 (1C, OCH
CH
CO
CH
), 47.3 (1C,
3
2
2
3
2
2
2
2
3
), 55.4 (1C,
2
3
2
2
3
), 114.3 (2C, C-3(p-methoxybenzyl), C-5(p-methoxybenzyl)),
27.1 (1C, C-1(p-methoxybenzyl)), 128.18 (2C, C-2, C-6(benzyl)), 128.20 (1C, C-4(benzyl)), 129.1
2C, C-3, C-5(benzyl)), 130.1 (2C, C-2, C-6(p-methoxybenzyl)), 135.6 (1C, C-1(benzyl)), 159.6 (1C,
C-4(p-methoxybenzyl)),164.5 (1C, C=O), 165.4 (1C, C=O), 170.2 (1C, CO
1
(
2
CH ). Exact mass
3

Organic & Biomolecular Chemistry
Page 26 of 66
26
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(
APCI): m/z = 397.1810 (calcd. 397.1758 for C22
947 (w, C-Haliph.), 1732 (s, C=Oester), 1651 (s, C=Oamide), 1474 (m, C-Harom.), 1246, 1173
s, C-Oester), 818 (m, C-Harom. disubst.), 729, 702 (m, C-Harom. monosubst.).
H
25
N
2
O [MH] ). IR (neat): ῦ [cm ] =
5
DOI: 10.1039/C7OB01530E
2
(
Methyl (S)-2-[1-benzyl-4-(cyclohexylmethyl)-3,6-dioxopiperazin-2-yl]acetate
(S)-6c)18
(
Under N
2
, (S)-5a (5.5 g, 16.8 mmol) was dissolved in dry acetonitrile (molecular sieves
3
2
1
Å, 200 mL). Cyclohexylmethylamine (2.8 mL, 21.8 mmol) and triethylamine (2.8 mL,
0.1 mmol) were added dropwise and the mixture was stirred at room temperature for
6 h. The solvent was removed in vacuo and the residue was dissolved in EtOAc (150
mL). The organic layer was washed with 0.5 M HCl (2 x 30 mL), 0.5M NaOH (30 mL)
and brine (30 mL). After drying (Na SO ), it was filtered and concentrated under reduced
pressure. The residue was purified by fc (Ø 5.5 cm, h = 17 cm, v = 65 mL, cyclohexane
2
4
:
EtOAc = 1 : 1, R
C, yield 4.6 g (73 %). C21
= 20.5 min. H NMR (600 MHz, CDCl
11), 1.10 – 1.23 (m, 3H, NCH 11), 1.62 – 1.74 (m, 6H, NCH
dd, J = 17.1 / 5.1 Hz, 1H, CHCH CH ), 3.00 (dd, J = 17.1 / 3.5 Hz, 1H,
CHCH CO CH ), 3.19 (dd, J = 13.5 / 6.7 Hz, 1H, NCH 11), 3.26 (dd, J = 13.5 / 7.6
Hz, 1H, NCH 11), 3.59 (s, 3H, OCH ), 3.92 (d, J = 17.2 Hz, 1H, O=CCH N), 4.07 –
.10 (m, 1H, CHCHCO CH ), 4.23 (d, J = 15.1 Hz, 1H, NCH Ph), 4.33 (d, J = 17.2 Hz,
H, O=CCH N), 5.05 (d, J = 15.1 Hz, 1H, NCH Ph), 7.20 – 7.34 (m, 5H, Ar-H). C NMR
): δ (ppm) = 25.80 (1C, NCH 11), 25.83 (1C, NCH 11), 26.4 (1C,
11), 30.6 (1C, NCH 11), 30.8 (1C, NCH 11), 35.2 (1C, CHCHCO CH ),
5.5 (1C, NCH 11), 47.4 (1C, NCH Ph), 50.9 (1C, O=CCH N), 52.1 (1C, OCH ), 52.8
1C, NCH 11), 56.3 (1C, CHCHCO CH ), 128.1 (2C, Ar-C), 128.2 (1C, Ar-C), 129.0
f
= 0.40 (cyclohexane : EtOAc = 1 : 1)). Colorless solid, mp 93 – 94
2
0
°
H
28
N
2
O
4
(372.5). []
D
= +57.3 (c = 1.12; CH
3
OH). Purity by
1
HPLC: 97.5 %, t
NCH
R
3
): δ (ppm) = 0.89 – 1.01 (m, 2H,
2
C
6
H
2
C
6
H
2
6
C H11), 2.77
(
2
CO
2
3
2
2
3
2
6
C H
2
C
6
H
3
2
4
1
2
3
2
1
3
2
2
(
151 MHz, CDCl
3
2
C
6
H
2
6
C H
NCH
2
C
6
H
2
C
6
H
2
C
6
H
2
3
3
2
C
6
H
2
2
3
(
2
C
6
H
2
3

Page 27 of 66
Organic & Biomolecular Chemistry
27
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DOI: 10.1039/C7OB01530E
(
2C, Ar-C), 135.7 (1C, Ar-C
q
), 164.7 (1C, C=O), 165.5 (1C, C=O), 170.3 (1C, CO
2
CH ).
3
+
+
Exact mass (APCI): m/z = 373.2159 (calcd. 373.2122 for C21
H
29
N
2
O
4
[MH] ). IR (neat):
-1
ῦ [cm ] = 2920, 2851 (w, C-Haliph.), 1732 (s, C=Oester) 1647 (s, C=Oamide), 1200 (m, C-
,
O
ester), 725, 698 (m, C-Harom. monosubst.).
Methyl (S)-2-(1-benzyl-4-methyl-3,6-dioxopiperazin-2-yl)acetate ((S)-6d)18
Under N , (S)-5a (3.6 g, 11.0 mmol) was dissolved in dry acetonitrile (molecular sieves
Å, 200 mL). A solution of methylamine in THF (2 M, 7.2 mL, 14.4 mmol) and
triethylamine (1.8 mL, 13.2 mmol) were added dropwise and the reaction was stirred for
d at room temperature. The solvent was removed in vacuo and the residue was
dissolved in EtOAc (150 mL). The organic layer was washed with 0.5 M HCl (2 x 30 mL),
.5 M NaOH (30 mL) and brine (30 mL). After drying (Na SO ), it was filtered and
concentrated under reduced pressure. The residue was purified by fc (Ø 4 cm, h = 18
cm, v = 30 mL, EtOAc : acetone = 4 : 1, R = 0.44). Colorless solid, mp 99-100°C, yield
= +92.7 (c = 1.86; CH OH). Purity by HPLC:
= 14.5 min. H NMR (600 MHz, CDCl ): δ (ppm) = 2.80 (dd, J = 17.4 / 4.9 Hz,
H, CHCH CO CH ), 2.97 (s, 3H, NCH ), 3.04 (dd, J = 17.4 / 3.3 Hz, 1H,
CO CH ), 3.60 (s, 3H, OCH ), 3.95 (d, J = 17.1 Hz, 1H, O=CCH N), 4.04 (t, J =
.9 Hz, 1H, CHCH CO CH ), 4.15 (d, J = 15.1 Hz, 1H, NCH Ar), 4.33 (d, J = 17.1 Hz,
H, O=CCH N), 5.12 (d, J = 15.1 Hz, 1H, NCH Ar), 7.20 – 7.35 (m, 5H, Ar-H). C NMR
): δ(ppm) = 33.7 (1C, NCH ), 34.9 (1C, CHCH CO CH ), 47.1 (1C,
Ar), 52.1 (1C, O=CCH N), 52.2 (1C, OCH ), 55.7 (1C, CHCH CO CH ), 128.2 (3C,
Ar-C), 129.1 (2C, Ar-C), 135.5 (1C, Ar-C
2
3
3
0
2
4
f
2
0
2.0 g (61 %). C15
H
18
N
2
O
4
(290.3). []
D
3
1
9
1
7.0 %, t
R
3
2
2
3
3
CHCH
2
2
3
3
2
3
1
2
2
3
2
1
3
2
2
(
151 MHz, CDCl
3
3
2
2
3
NCH
2
2
3
2
2
3
q
), 164.3 (1C, C=O), 165.6 (1C, C=O), 170.4
+
(
1C, CO
2
CH
2
). Exact mass (APCI): m/z = 291.1344 (calcd. 291.1339 for C15
H
19
N
2
O
4
+
-1
[
MH] ). IR (neat): ῦ [cm ] = 2951, 2920 (w, C-Haliph.), 1728 (s, C=Oester), 1643 (s,
C=Oamide), 1196 (m, C-Oester), 725, 698 (m, C-Harom. monosubst.).

Organic & Biomolecular Chemistry
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DOI: 10.1039/C7OB01530E
Methyl (S)-2-[4-benzyl-1-(4-methoxybenzyl)-3,6-dioxopiperazin-2-yl]acetate
(S)-6e)
Under N
Å, 200 mL). Freshly distilled benzylamine (3.4 mL, 30.7 mmol) and triethylamine (4.3
(
2
, (S)-5e (7.9 g, 22 mmol) was dissolved in dry acetonitrile (molecular sieves
3
mL, 30.7 mmol) were added dropwise. The reaction mixture was heated to reflux for 16
h. After cooling to room temperature, the solvent was removed in vacuo and the residue
was dissolved in EtOAc (100 mL). The organic layer was washed with 0.5 M HCl (2 x
3
0 mL), 0.5 M NaOH (1 x 30 mL) and brine (1 x 30 mL). After drying (Na
filtered and concentrated under reduced pressure. The residue was purified by fc (Ø
cm, h = 17 cm, v = 65 mL, cyclohexane/EtOAc = 1:1  1:2, R = 0.44
cyclohexane:EtOAc = 1:2)). Colorless solid, mp 97-98 °C, yield 6.7 g (77 %).
2
SO ), it was
4
6
f
(
C
22
H
24
N
2
O
5
(396.4). [] 20
D
= +59.0 (c = 0.24; CH
): δ (ppm) = 2.83 (dd, J = 17.1 / 5.1 Hz, 1H,
), 3.07 (dd, J = 17.0 / 3.3 Hz, 1H, CHCH CO CH ), 3.54 (s, 3H, CO CH ),
), 3.85 (d, 1H, J = 17.2 Hz, 1H, O=CCH N ), 4.10 (d, 1H, J = 15.0
N), 4.15 (m, 1H, CHCH CO CH ),
Ph), 5.05 (d,
3
OH). Purity by HPLC: 94.3 %, t = 19.5
R
1
min. H NMR (600 MHz, CDCl
3
CHCH
2
CO
2
CH
3
2
2
3
2
3
3.79 (s, 3H, Ar-OCH
3
2
Hz, NCH
2
ArOCH
3
), 4.12 (d, J = 17.9 Hz, 1H, O=CCH
2
2
2
3
4
.48 (d, J = 14.5 Hz, 1H, CHNCH
2
Ph), 4.71 (d, J = 14.6 Hz, 1H, CHNCH
2
1
H, J = 15.0 Hz, NCH ArOCH ), 6.85 (d, J = 8.6 Hz, 2H, 3-H, 5-H (p-methoxybenzyl)), 7.16 (d,
2
3
1
3
J = 8.4 Hz, 2H, 2-H, 6-H (p-methoxybenzyl)), 7.24 – 7.37 (m, 5H, Ar-H). C NMR (151 MHz,
CDCl
O=CCH
CHCH CO
3
): δ (ppm) = 35.2 (1C, CHCH
N), 49.8 (1C, CHNCH Ar), 52.1 (1C, CO
CH
2
CO
2
CH
3
), 46.6 (1C, CH
2
NCH
2
ArOCH
3
), 49.5 (1C,
), 55.8 (1C,
2
2
2
CH ), 55.4 (1C, Ar-OCH
3
3
2
2
3
), 114.5 (2C, C-3(p-methoxybenzyl), C-5(p-methoxybenzyl)), 127.4 (1C, C-1(p-
methoxybenzyl)), 128.2 (1C, C-4(benzyl)), 128.6 (2C, C-2, C-6(benzyl)), 128.9 (2C, C-3, C-5(benzyl)),
129.6 (2C, C-2, C-6(p-methoxybenzyl)), 135.0 (1C, C-1(benzyl)), 159.6 (1C, C-4(p-methoxybenzyl)),
164.4 (1C, C=O), 165.6 (1C, C=O), 170.2 (1C, CO
2
CH ). Exact mass (APCI): m/z =
3

Page 29 of 66
Organic & Biomolecular Chemistry
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397.1780 (calcd. 397.1758 for C22
H
25
N
2
O
5
[MH] ). IR (neat): ῦ [cm ] = 2978 (w, C-
DOI: 10.1039/C7OB01530E
Haliph.), 1736 (s, C=Oester), 1651 (s, C=Oamide), 1450 (m, C-Harom.), 1234 (s, C-Oether), 1180,
1165 (s, C-Oester), 826 (w, C-Harom. disubst.), 745, 710 (w, C-Harom. monosubst.).
(
S)-2-[4-Benzyl-1-(4-methoxybenzyl)-3,6-dioxopiperazin-2-yl]-N-methoxy-N-
methylacetamide ((S)-7)
Under N and ice cooling, Al(CH
added slowly to a solution of H Cl (60 mL).
2
3
)
3
solution (2 M in toluene, 16.0 mL, 32 mmol) was
.HCl (3.2 g, 32.7 mmol) in dry CH
3
CNHOCH
3
2
2
After stirring for 30 min at room temperature, a solution of (S)-6e (4.3 g, 10.9 mmol) in
dry CH Cl (60 mL) was added and the mixture was stirred for 4 h at room temperature.
2
2
An aqueous sodium potassium tartrate solution (10 %, 50 mL) was added and the
resulting suspension was stirred for 1 h. Then the mixture was filtered through Celite®
using CH
2
Cl as eluent. The filtrate was concentrated under reduced pressure and the
2
residue was purified by fc (Ø 6 cm, h = 18 cm, v = 65 mL, cyclohexane:EtOAc = 2:9, R
f
2
0
=
0.20). Colorless solid, mp 89-90 °C, yield 4.5 g (97 %). C23
38.4 (c = 0.17; CH CN). Purity by HPLC: 99.2 %, t
): δ (ppm) = 2.96 (dd, J = 17.3 / 3.8 Hz, 1H, CHCH
), 3.04 (dd, J = 16.7 / 3.9 Hz, 1H, CHCH CON(OCH
.73 (s, 3H, Ar-OCH ), 3.77 (d, J = 17.0 Hz, 1H, O=CCH
CHCH CON(OCH )CH ), 4.17 (d, J = 12.7 Hz, 1H, NCH Ar), 4.20 (d, J = 16.9 Hz, 1H,
O=CCH N), 4.40 (d, J = 14.7 Hz, 1H, NCH Ph), 4.68 (d, J = 14.7 Hz, 1H, NCH Ph), 4.87
d, J = 14.9 Hz, 1H, NCH
H
27
N
3
O
5
(425.5). []
D
=
1
+
3
R
= 19.3 min. H NMR (400 MHz,
CON(OCH )CH ), 3.03 (s, 3H,
)CH ), 3.47 (s, 3H, NOCH ),
N), 4.15 (t, J =3.8 Hz, 1H,
CDCl
3
2
3
3
NCH
3
2
3
3
3
3
3
2
2
3
3
2
2
2
2
(
2
Ar), 6.79 (d, J = 8.6 Hz, 2H, 3-H, 5-H (p-methoxybenzyl)), 7.11 (d, J
1
3
=
8.6 Hz, 2H, 2-H, 6-H (p-methoxybenzyl)), 7.20 – 7.30 (m, 5H, Ar-H). C NMR (101 MHz,
CDCl
NCH
CHCH
3
): δ (ppm) = 32.1 (1C, NCH
Ar), 49.7 (1C, NCH Ph), 49.8 (1C, O=CCH
CON(OCH )CH ), 61.3 (1C, NOCH
3
), 32.9 (1C, CHCH
2
CON(OCH
3
)CH
3
), 46.9 (1C,
), 56.1 (1C,
2
2
2
N), 55.5 (1C, Ar-OCH
3
2
3
3
3
), 114.4 (2C, C-3, C-5(p-methoxybenzyl)), 128.0