Development, synthesis and structure–activity-relationships of inhibitors of the macrophage infectivity potentiator (Mip) proteins of Legionella pneumophila and Burkholderia pseudomallei

Article abstract of DOI:10.1016/j.bmc.2016.08.025

The bacteria Burkholderia pseudomallei and Legionella pneumophila cause severe diseases like melioidosis and Legionnaire's disease with high mortality rates despite antibiotic treatment. Due to increasing antibiotic resistances against these and other Gram-negative bacteria, alternative therapeutical strategies are in urgent demand. As a virulence factor, the macrophage infectivity potentiator (Mip) protein constitutes an attractive target. The Mip proteins of B. pseudomallei and L. pneumophila exhibit peptidyl-prolyl cis/trans isomerase (PPIase) activity and belong to the PPIase superfamily. In previous studies, the pipecolic acid moiety proved to be a valuable scaffold for inhibiting this PPIase activity. Thus, a library of pipecolic acid derivatives was established guided by structural information and computational analyses of the binding site and possible binding modes. Stability and toxicity considerations were taken into account in iterative extensions of the library. Synthesis and evaluation of the compounds in PPIase assays resulted in highly active inhibitors. The activities can be interpreted in terms of a common binding mode obtained by docking calculations.

Full text of DOI:10.1016/j.bmc.2016.08.025

Accepted Manuscript
Development, synthesis and structure-activity-relationships of inhibitors of the
macrophage infectivity potentiator (Mip) proteins of Legionella pneumophila
and Burkholderia pseudomallei
Florian Seufert, Maximilian Kuhn, Michael Hein, Matthias Weiwad, Mirella
Vivoli, Isobel H. Norville, Mitali Sarkar-Tyson, Laura E. Marshall, Kristian
Schweimer, Heike Bruhn, Paul Rösch, Nicholas J. Harmer, Christoph A.
Sotriffer, Ulrike Holzgrabe
PII:
S0968-0896(16)30632-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.08.025
BMC 13208
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 June 2016
11 August 2016
18 August 2016
Please cite this article as: Seufert, F., Kuhn, M., Hein, M., Weiwad, M., Vivoli, M., Norville, I.H., Sarkar-Tyson,
M., Marshall, L.E., Schweimer, K., Bruhn, H., Rösch, P., Harmer, N.J., Sotriffer, C.A., Holzgrabe, U., Development,
synthesis and structure-activity-relationships of inhibitors of the macrophage infectivity potentiator (Mip) proteins
of Legionella pneumophila and Burkholderia pseudomallei, Bioorganic & Medicinal Chemistry (2016), doi: http://
dx.doi.org/10.1016/j.bmc.2016.08.025
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Development, synthesis and structure-activity-relationships of
inhibitors of the macrophage infectivity potentiator (Mip) proteins of
Legionella pneumophila and Burkholderia pseudomallei
Florian Seuferta, Maximilian Kuhna, Michael Heina, Matthias Weiwadb, Mirella Vivolic,
Isobel H. Norvilled, Mitali Sarkar-Tysond,e, Laura E. Marshalld, Kristian Schweimerf, Heike
Bruhng, Paul Röschf, Nicholas J. Harmerc, Christoph A. Sotriffera, and Ulrike Holzgrabea
a Institute for Pharmacy and Food Chemistry, University of Würzburg, 97074 Würzburg, Germany
b Research Center for Enzymology of Proteinfolding, Max-Planck-Institute Halle, 06120 Halle, Germany
c School of Biosciences, University of Exeter, Exeter, United Kingdom
d Defence Science and Technology Laboratory, Biomedical Sciences, Porton Down, Salisbury, Wiltshire, SP4 0JQ, United
Kingdom
e Marshall Center for Infectious Disease Research and Training, School of Pathology and Laboratory Medicine, University of
Western Australia, Perth, Australia
f Department of Biopolymers, University of Bayreuth,Universitätsstrasse 30, 95447 Bayreuth, Germany
g Institute for Molecular Infection Biology, University of Würzburg, 97080 Würzburg, Germany
Page 1 of 42

Abstract
The bacteria Burkholderia pseudomallei and Legionella pneumophila cause severe diseases like
melioidosis and Legionnaire’s disease with high mortality rates despite antibiotic treatment. Due to
increasing antibiotic resistances against these and other Gram-negative bacteria, alternative
therapeutical strategies are in urgent demand. As a virulence factor, the macrophage infectivity
potentiator (Mip) protein constitutes an attractive target. The Mip proteins of B. pseudomallei and L.
pneumophila exhibit peptidyl-prolyl cis/trans isomerase (PPIase) activity and belong to the PPIase
superfamily. In previous studies, the pipecolic acid moiety proved to be a valuable scaffold for
inhibiting this PPIase activity. Thus, a library of pipecolic acid derivatives was established guided by
structural information and computational analyses of the binding site and possible binding modes.
Stability and toxicity considerations were taken into account in iterative extensions of the library.
Synthesis and evaluation of the compounds in PPIase assays resulted in highly active inhibitors. The
activities can be interpreted in terms of a common binding mode obtained by docking calculations.
Keywords:
Burkholderia pseudomallei
Legionella pneumophila
macrophage infectivity potentiator protein
synthesis
docking analysis
structure-activity-relationships
Abbrevations: Mip, macrophage infectivity potentiator; PPIase, peptidyl-prolyl cis/trans isomerase;
LpMip, L. pneumophila Mip; BpMip, B. pseudomallei Mip; SAR, structure-activity relationship; HSQC,
Heteronuclear Single Quantum Coherence; NMM, N-methylmorpholine; DIPEA, N, N-
diisopropylethylamine; EDC·HCl, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride;
DMAP dimethylaminopyridine; HOBt, 1-hydroxybenzotriazol; HEPES, 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid
Page 2 of 42

1. Introduction
The diseases melioidosis and Legionnaire’s disease are triggered by the pathogens Burkholderia
pseudomallei and Legionella pneumophila, respectively. B. pseudomallei is endemic throughout the
tropics, and is predicted to cause 89,000 deaths globally per annum.1 Infection can occur by
inoculation of skin lesions or less frequently by inhalation.2 In addition, B. pseudomallei is listed as a
Tier 1 biological agent by the U.S. Centers for Disease Control and Prevention.3
L. pneumophila infections also generally occur following inhalation of contaminated aerosols.4 The
main sources of infection are sanitary facilities such as showers and whirlpools, but also air
conditioning systems and cooling towers.5 While Legionnaire’s disease manifests in an acute
pneumonia, melioidosis symptoms are hard to distinguish from those of tuberculosis. Symptoms
range from localized cutaneous abscesses to multi-organ involvement and septic shocks.6 Despite
protracted treatment with antibiotics, a high mortality rate is reported.7-9
Due to an increasing resistance to antibiotics, the treatment of infections by Gram-negative
bacteria is becoming a difficult issue.10-12 Therefore, it is important to search for new antibacterial
strategies. One particular opportunity is to target virulence factors: these are not the targets for
current antibiotics, commensal organisms should be spared, and the selective pressure for resistance
should be lower.13, 14 An important virulence factor for both B. pseudomallei and L. pneumophila is
the macrophage infectivity potentiator (Mip) protein. The Mip protein from both species shows
peptidyl-prolyl cis/trans isomerase (PPIase) activity.15, 16 Inhibition of the PPIase may prevent the
infection. The already intensively studied L. pneumophila Mip (LpMip) has been shown to play an
important role in invasion and dissemination of the bacteria.17, 18 LpMip is responsible for the
penetration of the lung tissue. A guinea pig infection model with wild-type and an LpMip mutant
revealed a contribution of Mip to bacterial prevalence within the lung. Furthermore, it was shown
that LpMip interacts with collagen IV, the predominant collagen in the human lung.19
Norville et al. discovered two Mip paralogues (BpMip and BPSL0918) in the genome of
B. pseudomallei. Since BPSL0918 did not show any PPIase activity, but knock-out had serious effects
Page 3 of 42

on intracellular survival, it is speculated that BPSL0918 acts as a chaperone that is important for
intracellular survival of the pathogen.20 However, BpMip exhibits PPIase activity and is important in
the infection process. Norville et al.16 demonstrated that without BpMip there is reduced
intracellular survival of B. pseudomallei in macrophage cells. In addition, the BpMip mutant strain
was significantly attenuated in a BALB/c mouse model. Structural comparison between BpMip and
LpMip showed a high homology in the PPIase domain.21, 22 Mip proteins have been associated with
virulence in a range of microbial pathogens, including Trypanosoma cruzi,23 Chlamydia trachomatis,24
and Neisseria gonorrhoeae.25
L. pneumophila and B. pseudomallei Mips belong to the FK506 binding proteins (FKBPs) family.
FKBPs form stable complexes with the immunosuppressive natural products FK506 and rapamycin.
The human FKBP12 complex with these drugs then inhibits the calcineurin and mTOR complex,
respectively.26-29 Both drugs show low nanomolar inhibition of Mips from a range of species. Due to
the immunosuppressive effects, these compounds are not suitable for treatment of Legionnaire’s
disease and melioidosis. However, they demonstrate that Mips are druggable, and so these proteins
are attractive targets for novel inhibitors.
To this end, we previously designed pipecolic acid derivatives to inhibit LpMip using structure-
based design, synthesis, and biological screening. NMR-HSQC experiments confirmed that the
developed compounds bind to the same hydrophobic binding pocket as rapamycin.30 Some of these
pipecolic acid esters were similarly efficient towards the BpMip. In addition, it was demonstrated
that the compounds were not immunosuppressive and that they were able to reduce the cytotoxic
effects of B. pseudomallei in macrophage cells.31
The previously described compounds proved that novel, non-immunosuppressive compounds
targeting Mips can be designed. However, the identified compounds did not have suitable properties
for therapeutic use. In this work, a library of pipecolic acid derivatives was established. Using hot-
spot analyses and exploratory docking experiments against both LpMip and BpMip, possible
substituents were suggested. Stability and toxicity considerations were taken into account in iterative
Page 4 of 42

extensions of the library. Synthesis and evaluation of the compounds in LpMip and BpMip inhibition
assays of the best candidates resulted in highly active inhibitors.
2. Results and Discussions
2.1. Library development
Figure 1: Lead structure from the study of Juli et al.30 highlighting the pipecolic acid scaffold and the two regions addressed
for modification and optimization.
Inhibitor CJ168 (see Figure 1) of LpMip30 and BpMip31 was used as starting point to develop a library
of pipecolic acid derivatives for exploring structure-activity relationships (SARs) and, possibly,
obtaining compounds with improved activity, higher solubility, and lower toxicity. While the central
pipecolic acid scaffold should be maintained as essential recognition unit for the MIP binding pocket,
two regions were available for modification (see Figure 1): the phenyl ring of the benzylsulfonamide
group (region A) and the ester part with the alkyl chain and the terminal trimethoxyphenyl group
(region B).
Hot-spot analyses were carried out using GRID32 (version 22c) to determine preferential interaction
sites for various probe atoms in the binding pockets of LpMip and BpMip and identify yet
unaddressed areas. As the binding mode of CJ168 is available from a crystal structure with BpMip,31
which compares very well with the postulated binding mode obtained by docking for LpMip,30
modifications in regions A and B can be suggested on the basis of the determined hot spots. The
binding pockets of BpMip and LpMip are highly homologous (see Figure 2). Marked differences occur
Page 5 of 42

only in the subpocket formed by the Ala94-Gly95-Gly96-Val97-Ile98 loop in BpMip and the Val114-
Gly115-Gly116-Pro117-Ile118 loop in LpMip, respectively (see Figure 2).
Figure 2: Comparison of the binding pockets of BpMip (green, chain C of PDB 4GGQ31) and LpMip (orange, model 4 of PDB
2VCD22). The surface and the ligand (CJ168, grey sticks) are shown for BpMip. The insert at the right highlights the
differences in the loop sequence and conformation.
In BpMip, clear hot spots for hydrophobic substituents in para-position of the phenyl ring of CJ168
were observed (see Figure 3). Furthermore, a suitably placed hydrogen-bond donor function could be
able to address the carbonyl groups of Phe43 and Ala94. In LpMip, the corresponding polar hot spots
were even more pronounced, suggesting donor functions to address the carbonyl groups of Phe65
and Val114 and an acceptor function for an interaction with the Gly116 NH. In contrast, the
hydrophobic hot spots in this region were less well pronounced in LpMip, suggesting at best a small
hydrophobic substituent in meta-position of the phenyl ring.
Modifications of the trimethoxyphenyl ring of region B were planned with the goal of improving
solubility and, possibly, enhancing surface complementarity. In fact, from the BpMip crystal
structures and the LpMip docking modes, the trimethoxyphenyl group appears to bind rather non-
specifically. Although hot spots for chloro probes could be found around this ring, the preference for
(phenolic) OH-probes was much more pronounced. Given the surface exposure of this interaction
area, it is, however, unclear how much an additional hydrogen bond could really contribute to the
Page 6 of 42

affinity. It would already be sufficient if a gain in solubility could be obtained by modifications in this
region without losing much potency.
Figure 3: Characteristic hot spots of BpMip in the binding areas of region A (left) and region B (right) of lead compound
CJ168 (shown in orange in PDB structure 4G503). The following probes were used (with color and contouring level in
kcal/mol specified in parenthesis): methyl (yellow, -4.0), fluoro (cyan, -3.0), chloro (green, -5.0), phenolic OH (blue, -7.0),
carbonyl-O (red, 5.0).
Taking these considerations and synthetic accessibility issues into account, a large variety of
derivatives was considered for preparation, which ultimately led to the compounds summarized in
Table 1 and discussed in more detail below. All suggested molecules were subjected to exploratory
docking calculations (results not shown) to avoid the preparation of unsuitable, non-fitting
compounds. A more detailed docking analysis was carried out for the ultimately prepared
compounds, as further described below.
2.2. Chemistry
Synthesis of racemic compounds. The synthesis of the pipecolic acid derivatives was carried out
following the methods of Juli et al.30 Briefly, synthesis was started with conversion of piperidine-2-
carboxylic acid with 3 equiv. of thionyl chloride in ethanol to prepare ethyl piperidine-2-carboxylate
(see Figure 4). Subsequently, the sulfonamides were obtained by reaction of the ethyl piperidine-2-
carboxylate and 3 equiv. N-methylmorpholine (NMM) and N, N-diisopropylethylamine (DIPEA),
respectively, and equimolar amounts of the corresponding carbonyl or sulfonyl chloride. The ester
Page 7 of 42

was hydrolyzed using lithium hydroxide to give the free pipecolic acids 4a, e-g. The esters 5a-g were
synthesized using 1 equiv. of the free acids, 1 equiv. of the corresponding alcohol, 1.5 equiv. of 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC·HCl) and catalytic amounts of
dimethylaminopyridine (DMAP).
Reagents and conditions: (a) SOCl2, EtOH, reflux, 5 h; (b) corresponding carbonyl or sulfonyl chloride,
NMM or DIPEA, CH2Cl2, 0 °C, 6 - 24 h; (c) LiOH, MeOH, 0 C, 1 h (d) corresponding alcohol, EDC·HCl, DMAP,
CH2Cl2, 0 °C, 24 h, (e) H2, Pd/C, RT, 24 h; (f) Me2CO, Na2SO4, H2, cat. Pd/C, RT, 24 h. (g) corresponding
alcohol, EDC·HCl, DMAP, CH2Cl2, 0 °C, 24 h; (h) TFA, CH2Cl2; (i) corresponding carbonyl or sulfonyl
chloride, NMM or DIPEA, CH2Cl2, 0 °C, 6 - 24 h; (j) phenylglyoxalic acid, EDC·HCl, HOBt, CH2Cl2, 0 °C, 24 h,
(k) corresponding alcohol or amine, EDC·HCl, DMAP or HOBt, CH2Cl2, 0 °C, 24 h.
Figure 4: Synthesis scheme of inhibitors.
Synthesis of compounds 5j and 5k. To reduce the nitro-group to an amino-group, 5f or 5g was
dissolved in EtOAc and hydrogenated at RT and 10 bar hydrogen with catalytic amounts of Pd/C to
obtain 5h, 5i. Using a modified procedure from Fache et al.33 1 equiv. of 5h or 5i, 1.1 equiv. of
Me2CO, 4 equiv. of Na2SO4 and catalytic amounts of Pd/C were hydrogenated at RT and 10 bar
hydrogen to obtain compounds 5j, 5k.
Synthesis of pure S-enantiomers. To synthesize the S-enantiomeric piperidine-2-carboxylate
derivatives, an esterification of (S)-1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid (1 equiv.) was
Page 8 of 42

performed, using the corresponding alcohol (1.0 equiv.), EDC·HCl (1.5 equiv.) and DMAP (0.2 equiv.).
Afterwards, the protection group was cleaved with trifluoroacetic acid in CH2Cl2. Subsequently, the
sulfonamides were obtained by reaction of the ethyl piperidine-2-carboxylate and 3 equiv. NMM or
DIPEA and equimolar amounts of the corresponding sulfonyl chloride.
For the synthesis of S-5l an amidation of S-8a (1 equiv.) with phenylglyoxalic acid (1.0 equiv.) was
performed, using EDC·HCl (1.3 equiv.) and 1-hydroxybenzotriazol (HOBt) (0.5 equiv.). S-5v and S-5w
were obtained in a one pot synthesis: the corresponding alcohol was generated in situ and the ester
established according to the procedures above. For compound S-5y, a benzylic protection group was
used to prevent side reactions with the phenolic moiety. To synthesize compound S-5z in the first
step of the synthesis an amidation was carried out according to the given procedure of S-5l.
Synthesis of corresponding alcohols. To obtain the alcohol derivatives (10q-u), an esterification or
amidation with the corresponding benzoic acid (1 equiv.) was carried out, using the corresponding
alcohol or amine (1.0 equiv.), EDC·HCl (1.5 equiv.) and DMAP or HOBt (0.2 equiv.). The alcohols
(10q-u) were used in the S-enantiomeric synthesis to yield compounds 5q-u. For the derivatives 5v-y
a one pot synthesis combining alcohol synthesis and the first step of the S-enantiomeric synthesis
was established.
Page 9 of 42

Table 1: Substitution pattern of piperidine-2-carboxylic acid derivatives.
racemates
R1
R2
5a
5b
-SO2-CH2-C6H5
-SO2-CH2-C6H5
Trim
Pyr
5c
5d
5e
5f
5g
5h
5i
5j
-SO2-CH2-C6H5
-SO2-CH2-C6H5
-C2H4-OH
-CH(CH3)2
Pyr
-CO-CH2-O-C6H4-p-Cl
-SO2-CH2-C6H4-p-NO2
-SO2-CH2-C6H4-m-NO2
-SO2-CH2-C6H4-p-NH2
-SO2-CH2-C6H4-m-NH2
-SO2-CH2-C6H4-m-NH-CH(CH3)2
-SO2-CH2-C6H4-p-NH-CH(CH3)2
Pyr
Pyr
Pyr
Pyr
Pyr
Pyr
5k
R- or S-enantiomers
S-5a
R-5a
S-5b
S-5l
S-5m*
S-5n
S-5o
S-5p
S-5q
S-5r
-SO2-CH2-C6H5
-SO2-CH2-C6H5
-SO2-CH2-C6H5
-CO-CO-C6H4
Trim
Trim
Pyr
Pyr
Pyr
Pyr
Pyr
Pyr
-SO2-CH2-C6H4-p-Cl
-SO2-CH2-C6H4-m-Cl
-SO2-CH2-C6H4-p-F
-SO2-CH2-C6H4-m-NO2
-SO2-CH2-C6H5
-SO2-CH2-C6H5
-SO2-CH2-C6H5
-SO2-CH2-C6H5
-SO2-CH2-C6H5
-SO2-CH2-C6H5
-SO2-CH2-C6H5
-SO2-CH2-C6H5
-SO2-CH2-C6H5
-SO2-CH2-C6H5
-C2H4-OCO-3-C5H4N
-C2H4-NHCO-3-C5H4N
-C2H4-O-3-C5H4N
S-5s
S-5t
-C2H4-OCO-C6H3-3, 5-(O(CH2)2OCH3)2
-C2H4-NHCO-C6H3-3, 5-(O(CH2)2OCH3)2
-CH(S-CH3)CH2-NHCO-3-C5H4N
-CH(R-CH3)CH2-NHCO-3-C5H4N
-CH(S-CH3)CH2-NHCO-3-C6H2-3,4,5-
-C2H4-NHCO-C6H3-3-OCH3, 4-OH
S-5u
S-5v
S-5w
S-5x
S-5y
S-5z
#
NH-CH(CH3)2
*: Synthesized according to the procedure for racemic compounds. #: Amide instead of ester.
Structure-activity-relationships. Table 2 shows the Mip inhibitory and cytotoxicity data. In previous
studies compound CJ168 (resynthesized here as 5a/S-5a) showed an IC50 value (LpMip) of 9 µM30 for
the racemic compound. Evaluation of the enantiomers of 5a against LpMip indicate an
enantioselective effect (IC50 of S-5a = 6 µM30 versus IC50 of R-5a = 12.8 µM) of the S-enantiomer
which Holt et al.34 had observed for piperidine-2-carboxylates as FKBP inhibitors. Previous docking
studies had already revealed that the R-enantiomer can be placed in the LpMIP pocket only in a
somewhat distorted orientation, leading to a less favorable hydrophobic score.30 With its higher
potency the S-enantiomer served as a starting structure to develop further inhibitors.
Page 10 of 42

By replacing the trimethoxyphenyl group of 5a with a pyridinyl group (5b) the activity was
maintained but the toxicity in macrophages (J774.1) was reduced significantly (IC50 value => 100 µM).
Furthermore, this substitution reduced the molecular weight and the logP, and dramatically
increased the solubility of the compound. Accordingly, the pyridinyl group was preferred over the
trimethoxyphenyl group for all subsequent derivatives exploring region A.
Table 2: PPIase activity, cytotoxicity and selectivity index of piperidine-2-carboxylic acid derivatives.
BpMip
LpMip
Cytotoxicity-
cytotoxicity,
J774.1
Cytotoxicity-
affinity (CA)
comp.
IC50 [µM]
Ki [µM]
IC50 [µM]
affinity (CA)
IC50, [µM]
44.5
48.4
5a30
S-5a30
R-5a
5b
S-5b
5c
0.8 ± 0.1
0.12 ± 0.09
1.9 ± 0.3
0.69 ± 0.3
0.26 ±0.08
1.6 ± 0.3
4.7 ± 1.2
6.3 ± 4
0.3 ± 0.1
n. d.
3.5 ± 1.7
n. d.
1.25 ± 0.3
0.16 ± 0.02
1.2 ± 0.3
0.57 ± 0.16
0.15 ± 0.03
2.8 ± 0.4
1.8 ± 0.6
17 ± 4
0.17 ± 0.03
n. d.
4.2 ± 0.7
35.6
303
55.8
> 175
> 667
> 35.7
> 55.6
3.0
334
-
> 23.8
-
13.3
9.8
16.0
259
241
> 1031
105
> 1099
> 345
> 400
> 170
> 102
> 385
> 124
> 102-
> 1075
> 10.9
9.0 ± 0.7
6.0 ± 0.7
12.8 ± 6.2
10.7 ± 1.8
5.8 ± 0.5
94 ± 26
79.2 ± 3.6
89.3 ± 5.7
3.9 ± 0.3
8.7 ± 0.7
21.4 ± 3.9
> 100
41.5 ± 9.7
44.5 ± 16
142 ± 17.5
2.4 ± 0.1
7.5 ± 1.0
5.7 ± 0.8
7.9 ± 1.2
2.6 ± 0.6
11.4 ± 1.7
7.7 ± 2.5
8.6 ± 1.3
5.6 ± 0.7
3.5 ± 0.5
11.5 ± 2.2
2.2 ± 0.1
3.2 ± 0.4
> 100
4.9
8.1
5.2
> 9.4
> 17.2
> 1.1
> 1.3
0.6
14.5
5.4
> 4.7
< 0.6
1.4
1.0
0.6
14.0
5.8
> 17.5
6.1
> 38.5
> 8.8
> 13
> 9.3
> 18.0
> 28.6
> 8.7
> 46
> 31.3
1
66.9
> 100
> 100
> 100
> 100
50.9
5d
5e
5f
56.7
5g
46.7
5h
5i
> 100
62.3
n. d.
4.3 ± 0.5
4.5 ± 1.7
5.5 ± 1.1
5j
57.4
3.8 ± 1.5
5k
44.3
-
S-5l
S-5m
S-5n
S-5o
S-5p
S-5q
S-5r
S-5s
S-5t
S-5u
S-5v
S-5w
S-5x
S-5y
S-5z
88.1
33.6
43.3
> 100
48.2
> 100
> 100
> 100
> 80*
> 100
> 100
> 100
> 100
> 100
> 100
3.2 ± 1.2
0.21 ± 0.08
0.23 ± 0.08
0.072 ± 0.03
0.48 ± 0.17
0.22 ± 0.08
0.42 ± 0.18
2.2 ± 0.4
0.42 ± 0.05
0.58 ± 0.08
0.32 ± 0.12
0.99 ± 0.4
0.58 + 0.16
0.16 ± 0.08
6.2 ± 0.7
0.13 ± 0.02
0.18 ± 0.01
0.097 ± 0.02
0.46 ± 0.06
0.091 ± 0.03
0.29 ± 0.06
0.25 ± 0.06
0.47 ± 0.07
0.98 ± 0.29
0.26 ± 0.04
0.81 ± 0.13
0.74 + 0.25
0.093 ± 0.01
9.2 ± 1.2
* Precipitation at given concentration. n.d.: not determined
From comparison of PPIase assay data of LpMip and BpMip it could be concluded that para-
substituted benzyl sulfonamides showed slightly better inhibitory effects than meta-substituted
counterparts (cf. S-5m and S-5n, 5f and 5g, 5h and 5i). Additionally, several compounds with para-
substituents exhibited better IC50 values than their unsubstituted counterparts. In fact, for LpMip the
compound S-5m showed the best inhibition compared to the unsubstituted substance S-5b. For
BpMip the compound with the smallest halogen S-5o (fluorine) has the best IC50 value (0.072 µM).
Page 11 of 42

Moreover, larger substituents like isopropyl in para- or meta-position significantly reduced activity
(5j, 5k). To evaluate the necessity of the benzyl sulfonamide for activity, the amide (5e) and the
diketone (S-5l) were synthesized. Both of them, as well as other amide and diketone derivatives not
reported here, showed only weak inhibition of LpMip and BpMip. This indicated that the benzyl
sulfonamide is the moiety of choice for inhibiting these two Mips.
The influence of the moiety at the ester of the piperidine-2-carboxylate (region B) was also
investigated. Compounds with a second ester or an amide group connected to the aryl ring, but also
with ethers in the alkyl chain were synthesized and the aromatic substituent at the phenyl group was
exchanged. Compounds carrying small isopropyl and 2-hydroxyethyl moieties showed significantly
lower anti-PPIase-activity. This indicated that, in spite of the rather exposed position of the
piperidine-2-carboxylate function30 (region B in Figure 1; cf. also Figure 2), it makes a significant
contribution to the overall binding affinity.
Nearly all diester compounds showed a higher activity against LpMip than the references 5b/S-5b or
5a/S-5a. For BpMip, S-5q is the compound exhibiting the best anti-PPIase-activity. However, these
results have to be discussed critically because the diester compounds showed limited stability in
buffer media (see below). Since a substantial inhibitory activity is observed, it is likely that the
hydrolysis product is still active. By exchanging the benzoic ester with an amide (S-5q ↔ S-5r, S-5t
↔ S-5u) the chemical stability could be enhanced significantly. Furthermore, compound S-5y, being
most similar to rapamycin, showed the best Ki value for BpMip which was 0.093 µM. To further
improve the chemical stability, a methyl group was introduced next to the ester moiety of the
piperidine-2-carboxylate. An S-configuration of the stereo center showed a better anti-PPIase-activity
than the R-configured one (S-5v ↔ S-5w). S-5s, a representative of the ether derivatives, revealed a
good activity for BpMip ranging between the diester and the amide derivative. With other variations
(e.g., substituting the piperidine-2-carboxylate for an amide) no increase in activity was detected (S-
5z ↔ 5d). To conclude, substitution of region B is worthwhile to improve in vitro activity.
Page 12 of 42

NMR-HSQC analysis. To vertify the binding of the newly synthesized compounds to LpMip NMR-
HSQC experiments were carried out according to.30 Therefore, the chemical shift changes induced by
the Mip inhibitors were determined. Figure 5 displays the patterns of chemical shift changes of
compounds S-5b and S-5q, representatively. In analogy to Juli et al.30 the titration of 15N labeled
MIP77-213 with the substances S-5b and S-5q resulted in significant chemical shift changes of the
amide resonances which mirror the affinity to the binding pocket. Since not only the protons being
shifted are the same but also the extent of shift is similar, a strong binding to the Mip domain and
the Mip cavity is very likely. Moreover, the affected residues are located in the binding pocket for
rapamycin. Akin results were obtained for all new inhibitors which were subjected to the NMR
measurement. Due to solubility problems a determination of valid dissociation constant was not
possible.
0.3
S-5b
0.2
0.1
0
0
20
40
60
80
100
120
sequence
0.3
0.2
0.1
0
S-5q
0
20
40
60
80
100
120
sequence
Figure 5: NMR-HSQC experiments with S-5b and S-5q.
Docking analysis. To investigate the binding modes of the inhibitors and to analyze the binding
model as well as the scoring approach in light of the experimental activity data, docking calculations
Page 13 of 42

were carried out for all compounds reported in Table 1 as described in the experimental section
(4.4). The crystal structure of the lead compound CJ168 (= S-5a, corresponding to ligand CJ168)
complexed with BpMip (PDB structure 4G5031) served as reference for the entire analysis and
provided the possibility to evaluate the docking protocol by redocking ligand CJ168 back to the
uncomplexed 4G50 structure. As shown in Figure 6, the experimental binding mode is reproduced
very well, indicating a reliable docking protocol. A more detailed discussion of this finding and further
redocking results are provided in the Supplementary Material (Table S1).
Figure 6: Redocking of CJ168 to BpMip (crystal pose of PDB structure 4G50 chain A shown in lime, docked pose in yellow).
Docking to BpMip: For all compounds, docking poses showing the same binding mode as the lead
structure CJ168 were obtained with good scores (the scores for the top pose of each compound are
reported in Table S2 of the Supplementary Material). A quantitative correlation between the scores
and the experimental affinities cannot be expected, given the narrow affinity range of less than 2.3
pKi units for BpMIP (1.8 pIC50 units for LpMIP). Nevertheless, correlations within particular subsets
can be observed and pairwise comparisons reflect structure-activity relationship (SAR) trends. These
are discussed in detail in the Supplementary Material.
Docking to LpMip: For virtually all compounds, the scores obtained for the LpMip poses are less
favorable than the scores for the corresponding BpMip poses. This may be related to the fact that
only a complex structure with rapamycin is available for docking to LpMip, whereas for BpMip the
Page 14 of 42

complex with lead compound CJ168 provides a protein conformation which is already better adapted
to the class of ligands investigated here. Besides that, it is also more difficult to correctly capture the
relative trends in LpMip activity since the range of the IC50 values spans less than two orders of
magnitude (2.2 – 142 µM). Nevertheless, the relatively low (i.e., less favorable) scores obtained for
the smaller ligands (5c, 5d, S-5z) and the “non-sulfonamide” compounds (5e, S-5l) correctly point to
the weaker activity of these substances. Further details are available as Supplementary Material.
Stability of compounds. For the different assays, analytical methods have been developed to assess
the stability of the synthesized substances. To study the stability of the compounds in the PPIase
assay, different dilutions were prepared, and the substances were incubated at 25 °C and 40 °C for
24 h in methanol with 10 mM HEPES buffer (pH = 7.8). Samples were analysed by HPLC after 0 and
24 h. Whilst the parent compounds S-5a and S-5b (99 %) were highly stable in buffer for 24 h, the
more active derivatives S-5q (40 %) and S-5t (85 %) showed a considerable decomposition in buffer.
All other examined samples 5c, 5d, S-5o, S-5s, S-5u, S-5v and S-5z showed stabilities higher than
95 %. Of note, all unstable compounds carry two ester functions in the molecule (see Figure 7).
Figure 7: basic structure of the unstable compounds.
In addition, the stability of the inhibitors in the infection assay was evaluated. Compounds were
incubated with murine macrophages (J774.1) at 37 °C in RPMI medium for 0, 1, and 24 h. HPLC
analysis mirrored the results of the buffer stability study. As expected, the diesters showed the
lowest stability (S-5q and S-5t). The other tested inhibitors (5c, S-5r, S-5s, and S-5v) were stable in the
applied assay systems (see Figure 8). It is remarkable that compound 5c displayed a good stability,
indicating that the ester at the piperidine ring is stable under these experimental conditions. Similar
incubation experiments were performed for S-5r in human plasma. A decrease of the S-5r
Page 15 of 42

concentration was observed after 1, 4 and 24 h, being 5, 20 and 50 percent respectively. These data
demonstrate that most of the inhibitors, except the diester compounds, show a sufficient stability in
buffer, cell media, and plasma.
Stability of SF compounds in macrophages
98
97
95
95
100
80
60
40
20
0
88
86
82
82
81
77
59
50
5c
SF197
S-5q
SF110
S-5t
SF272
S-5s
SF276
S-5r
SF235
S-5v
SF299
time of incubation 1 h
time of incubation 24 h
Figure 8: Stability of inhibitors in cell media.
3. Conclusions
Mips are challenging targets which is mirrored in the flat SARs obtained and which can be explained
by binding pockets which do not provide a lot of interaction partners. Nevertheless, the established
compound library revealed Mip inhibitors with promising anti-PPIase activity in the nanomolar range
of concentration and low cytotoxicity. A structure-activity relationship analysis showed that a para-
substitution of the benzyl moiety of the sulfonamide is slightly preferred to a meta-substitution.
Large substituents like isopropyl at the benzyl moiety of the sulfonamide in meta- or para-position
resulted in a poor inhibition of the PPIase activity. Diester-compounds showed good activities;
however, stability tests demonstrated that they were unstable in buffer media and therefore are not
considered suitable for further investigations. Applying a replacement of the ester with an amide, the
chemical stability could be clearly improved while the inhibitory activity remains almost the same.
Moreover, the compound S-5x showed the best cytotoxicity-affinity (CA) value for LpMip
(IC50 = 2.2 µM, CA ≥ 46), whilst S-5o Ki = 0.0937 µM, CA ≥> 1099)
CA ≥> 1075) had the best CA value for BpMip.
4. Experimental Section.
and S-5y (Ki = 0.093 µM,
Page 16 of 42

4.1. General information
All chemicals were purchased from Sigma-Aldrich Chemicals (Schnelldorf, Germany), Acros Organics
(Geel, Belgium), Bachem (Bubendorf, Switzerland), and VWR International (Darmstadt, Germany),
1
and were used without further purification. H and 13C NMR spectra were recorded on an AV 400
nuclear magnetic resonance spectrometer (1H 400.132 MHz, 13C 100.613 MHz) (Bruker BioSpin MRI
GmbH, Ettlingen, Germany). As internal standard, the signals of the deuterated solvent were used
(chloroform-d: 1H 7.26 ppm, 13C 77.16 ppm; DMSO-d6: 1H 2.50 ppm, 13C 39.52 ppm). The
abbreviations (s) singlet, (d) doublet, (t) triplet, (q) quartet, (dd) doublet of doublet, (ddd) doublet of
doublet of doublet, (br) broad signal, (m) multiplet were applied during spectra interpretation.
Coupling constants are given in Hertz. Thin layer chromatography (TLC) was carried out on SIL G-25
silica gel plates (Macherey-Nagel, Düren, Germany) using petrol ether and ethyl acetate or ethyl
acetate and methanol as eluent. For purification, the Puriflash-430-system and Puriflash-Silica-STD
columns (10 - 50 µm, 12 and 25 g) were used (Interchim, Montluçon, France). IR spectra were
recorded on a Jasco FT-IR-6100 FT-IR spectrometer (Jasco, Gross-Umstadt, Germany). Melting points
were determined with the MPD350:BM 3.5 melting point apparatus (Sanyo, Gallenkamp BV,
Netherland) and are reported uncorrected. ESI-MS was conducted in positive mode; for LC/MS, the
following conditions were used: column: Agilent Zorbax SB-CN (50 x 4.6 mm, 3.5 µm particle size
(Agilent Technologies, Böblingen, Germany); mobile phase: A) Acetonitrile + 0.1 % acetic acid, B) H2O
+ 0.1 % acetic acid; gradient: 95 % A (0 - 5 min), 95 % A  10 % A (5 - 10 min), 10 % A (10 - 15 min),
flow: 0.4 ml/min. MS detection mode: ESI, nebulizer pressure: 40 psi, drying gas flow: 5 L/min, drying
gas temperature: 350 °C. Purity of compounds was determined by HPLC using an Agilent HPLC
system (1100 series, Agilent Technologies, Böblingen, Germany) with UV detection at λ = 254 nm. An
Inertsil ODS-2 C18 column (150 × 4.6 mm, 5 µm particle size; MZ-Analysetechnik, Mainz, Germany)
was used as the stationary phase. The mobile phase was a mixrure of A) a 10 mM KH2PO4 buffer
solution (pH = 7.4) and B) methanol. A gradient elution (method I; 0-15 min: 30  90 % (B), 15-18
Page 17 of 42

min: 90 % (B), 18-20 min: 90  30 % (B), 20-25 min: 30 % (B), as well as an isocratic elution (method
II) with 30 % of A) and 70 % of (B) was applied. All final products had a purifty ≥ 95 %.
4.2. Synthesis procedures
4.2.1. Synthesis of Ethyl piperidine-2-carboxylate (2). Piperidine-2-carboxylic acid (1 equiv.) was
dissolved in 40 mL of anhydrous ethanol. Thionyl chloride (3 equiv.) was added dropwise and
refluxed. After the reaction was completed (TLC control), the solvent was removed in vacuo. The
residue was suspended in 20 mL of aqueous NaHCO3 solution and washed with 4 x 30 mL CHCl3. The
organic layers were dried over anhydrous Na2SO4, and the solvent was removed in vacuo to yield
compound 2.
Spectroscopic data was acquired following the methods of Juli et al.30
4.2.2. General Procedure for the synthesis of Compounds 3a, e-g. Compound 2 (1 equiv.) was
dissolved in 40 mL of anhydrous CH2Cl2, and NMM or DIPEA (3 equiv.) was added at 0 °C followed by
the corresponding carbonyl or sulfonyl chloride (1 equiv.). The mixture was stirred until completion
of the reaction (TLC control) and subsequently washed with 4 x 30 mL 2 M HCl and H2O. The organic
layer was dried over anhydrous Na2SO4, and the solvent was removed in vacuo. If necessary, a
subsequent purification by flash-chromatography was performed to obtain compounds 3a, e-g.
The
preparation
of
1-(Benzylsulfonyl)piperidine-2-carboxylate
(3a)
and
(S)-1-
(Benzylsulfonyl)piperidine-2-carboxylate (S-3a) has already been described.30
4.2.2.1. Ethyl (R)-1-(benzylsulfonyl)piperidine-2-carboxylate (R-3a) was obtained as a yellow oil
1
(0.34 g, 57 %). H-NMR (400 MHz, CDCl3) δ 7.43-7.50 (m, 2H), 7.31-7.42 (m, 3H), 4.57 (d, 1H, J = 3.9
Hz), 4.26-4.19 (m, 4H), 3.42-3.49 (m, 1H), 3.16 (ddd, 1H, J = 12.7, 12.7, 3.1 Hz), 2.12-2.22 (m, 1H),
1.53-1.74 (m, 3H), 1.37-1.52 (m, 1H), 1.20-1.27 (m, 4H); 13C-NMR (100 MHz, CDCl3) δ 171.4, 131.0
Page 18 of 42

(2C), 129.4, 128.5 (2C), 128.4, 61.4, 58.8, 56.0, 43.4, 27.8, 25.1, 20.3, 14.2; IR (ATR, [cm-1]): 3026,
2949, 2864, 1708, 1377, 1325, 1180, 1125, 797, 696.
4.2.2.2. 1-(2-(4-Chlorophenoxy)acetyl)piperidine-2-carboxylat (3e) was obtained as a yellow oil
1
1
(0.54 g, 87 %). The H-NMR showed the presence of a 77 to 23 mixture of rotamers. Rotamer a: H-
NMR (400 MHz, CDCl3) δ 7.20-7.25 (m, 2H), 6.85-6.91 (m, 2H), 5.28 (d, 1H, J = 5.4 Hz), 4.65-4.80 (m,
3H), 4.10-4.25 (m, 2H),3.77-3.85 (m, 1H), 3.31 (ddd, 1H, J = 13.0, 13.0, 2.9 Hz), 2.23-2.34 (m, 1H),
1.56-1.77 (m, 3H), 1.27-1.55 (m, 5H); 13C-NMR (100 MHz, CDCl3) δ 170.8, 167.6, 156.7, 129.4 (2C),
126.5, 116.0 (2C), 67.4, 61.3, 52.3, 43.0, 26.5, 25.2, 20.9, 14.2; rotamer b (only different signals to a
are shown): 1H-NMR (400 MHz, CDCl3) δ 4.45-4.52 (m, 1H); 13C-NMR (100 MHz, CDCl3) δ 170.6, 167.5,
156.5, 129.5 (2C), 126.6, 115.9 (2C), 68.2, 61.7, 55.8, 40.0, 27.3, 24.5, 20.8; IR (ATR, [cm-1]): 3055,
2941, 2861, 1732, 1660, 1491, 1367, 1227, 1161, 1018, 823.
4.2.2.3. Ethyl 1-((4-nitrobenzyl)sulfonyl)piperidine-2-carboxylate (3f) was obtained as a yellow oil
1
(0.2 g, 88 %). H-NMR (400 MHz, CDCl3) δ 8.20-8.26 (m, 2H), 7.66-7.72 (m, 2H), 4.70 (d, 1H, J = 4.4
Hz), 4.41 (AB-spin system, 1H, JAB = 13.6 Hz), 4.39 (AB-spin system, 1H, JAB = 13.6 Hz), 4.20-4.33 (m,
2H), 3.45-3.55 (m, 1H), 3.10 (ddd, 1H, J = 12.6, 12.6, 2.9 Hz), 2.21-2.29 (m, 1H), 1.69-1.81 (m, 2H),
1.43-1.65 (m, 2H), 1.33 (t, 3H, J = 7.1 Hz), 1.21-1.28 (m, 1H); 13C-NMR (100 MHz, CDCl3) δ 171.4,
148.0, 136.7, 132.1 (2C), 123.7 (2C), 61.7, 58.0, 56.2, 43.5, 28.0, 25.1, 20.4, 14.2; IR (ATR, [cm-1]):
3080, 2938, 2857, 1732 (m) 1520, 1344, 1320, 1180, 1128, 857.
4.2.2.4. Ethyl 1-((3-nitrobenzyl)sulfonyl)piperidine-2-carboxylate (3g) was obtained as a yellow oil
1
(0.7 g, 93 %). H-NMR (400 MHz, CDCl3) δ 8.37 (t, 1H, J = 2.0 Hz), 8.22 (ddd, 1H, J = 8.0, 2.0, 1.0 Hz),
7.81-7.86 (m, 1H), 7.56 (t, 1H, J = 8.0 Hz), 4.68 (d, 1H, J = 4.5 Hz), 4.41 (AB-spin system, 1H, JAB = 13.6
Hz), 4.38 (AB-spin system, 1H, JAB = 13.6 Hz), 4.20-4.35 (m, 2H), 3.46-3.57 (m, 1H), 3.13 (ddd, 1H,
J = 12.6, 12.6, 3.1 Hz), 2.19-2.31 (m, 1H), 1.69-1.80 (m, 2H), 1.42-1.66 (m, 2H), 1.33 (t, 3H, J = 7.1 Hz),
Page 19 of 42

1.23-1.30 (m, 1H); 13C-NMR (100 MHz, CDCl3) δ 171.4, 148.3, 137.1, 131.6, 129.5, 126.0, 123.4, 61.7,
57.7, 56.2, 43.5, 27.9, 25.2, 20.4, 14.2; IR (ATR, [cm-1]): 3089, 2942, 2862, 1732, 1527, 1351, 1336,
1208, 1181, 1148, 1129, 811.
4.2.3. General Procedure for the Synthesis of Compounds 4a, e-g. Compounds 4a, e-g were
synthesized using a procedure described by Holt et al.34 Therefore, compound 3a, e-g (1 equiv.) was
dissolved in 30 mL of methanol and cooled to 0 °C. To this mixture a solution of lithium hydroxide (10
mL, 1 M) was added dropwise and stirred for 1 h. After the reaction was completed (TLC control),
10% HCl was added to adopt a pH value of 1. The free carboxylic acid was extracted with 4 x 30 mL
CH2Cl2. The combined organic layers were washed with 4 x 30 mL H2O and brine and then dried over
anhydrous Na2SO4. The solvent was removed in vacuo to give compounds 4a, e-g.
The preperation of 1-(Benzylsulfonyl)piperidine-2-carboxylic acid (4a) and (S)-1-
(Benzylsulfonyl)piperidine-2-carboxylic acid (S-4a) has already been described.30
4.2.3.1. (R)-1-(Benzylsulfonyl)piperidine-2-carboxylic acid (R-4a) was obtained as a yellow oil (0.31 g,
1
99 %). H-NMR (400 MHz, CDCl3) δ 7.41-7.47 (m, 2H), 7.33-7.41 (m, 3H), 4.58 (d, 1H, J = 5.0 Hz), 4.27
(s, 2H), 3.42-3.52 (m, 1H), 3.15 (ddd, 1H, J = 12.7, 12.7, 3.1 Hz), 2.12-2.23 (m, 1H), 1.55-1.78 (m, 3H),
1.37-1.50 (m, 1H), 1.26-1.37 (m, 1H); 13C-NMR (100 MHz, CDCl3) δ 176.1, 130.9 (2C), 129.1, 128.6
(3C), 58.9, 55.6, 43.5, 27.5, 24.9, 20.3; IR (ATR, [cm-1]): 3027, 2950, 2864, 2560, 1708, 1444, 1326,
1228, 1180, 1125, 696.
4.2.3.2. 1-(2-(4-Chlorophenoxy)acetyl)piperidine-2-carboxylic acid (4e) was obtained as a yellow oil
1
1
(0.21 g, 92 %). The H-NMR showed the presence of a 77 to 23 mixture of rotamers. Rotamer a: H-
NMR (400 MHz, CDCl3) δ 7.19-7.25 (m, 2H), 6.84-6.89 (m, 2H), 5.33 (d, 1H, J = 5.0 Hz), 4.62-4.86 (m,
2H), 3.78-3.86 (m, 1H), 3.30 (ddd, 1H, J = 12.8, 12.8, 2.7 Hz), 2.26-2.35 (m, 1H), 1.60-1.81 (m, 3H),
1.34-1.55 (m, 2H); 13C-NMR (100 MHz, CDCl3) δ 175.9, 168.3, 156.5, 129.4 (2C), 126.6, 116.1 (2C),
Page 20 of 42

1
67.2, 52.3, 43.1, 26.3, 25.0, 20.8; Rotamer b (only different signals to a are shown): H-NMR (400
MHz, CDCl3) δ 4.82-4.86 (m, 3H); 13C-NMR (100 MHz, CDCl3) δ 175.4, 167.9, 156.4, 129.5 (2C), 126.7,
115.9 (2C), 68.1, 55.6, 40.1, 27.1, 24.4; IR (ATR, [cm-1]): 3027, 2933, 2863, 1707, 1490, 1422, 1227,
1160, 1016, 823.
4.2.3.3. 1-((4-Nitrobenzyl)sulfonyl)piperidine-2-carboxylic acid (4f) was obtained as a yellow oil
1
(0.07 g, 95 %). H-NMR (400 MHz, CDCl3) δ 8.20-8.26 (m, 2H), 7.61-7.67 (m, 2H), 4.71 (d, 1H, J = 4.6
Hz), 4.38 (s, 2H), 3.47-3.58 (m, 1H), 3.13 (ddd, 1H, J = 12.7, 12.7, 3.0 Hz), 2.21-2.32 (m, 1H), 1.61-1.82
(m, 1H), 1.42-1.56 (m, 1H), 1.27-1.40 (m, 1H); 13C-NMR (100 MHz, CDCl3) δ 175.7, 148.0, 136.7, 131.9
(2C), 123.7 (2C), 58.1, 55.8, 43.6, 27.8, 25.0, 20.4; IR (ATR, [cm-1]): 3076, 2945, 2880, 1716, 1598,
1519, 1343, 1318, 1125, 1109, 841.
4.2.3.4. 1-((3-Nitrobenzyl)sulfonyl)piperidine-2-carboxylic acid (4g) was obtained as a yellow oil
(0.22 g, 96 %). 1H-NMR (400 MHz, CDCl3) δ 8.32 (t, 1H, J = 2.0 Hz), 8.20 (ddd, 1H, J = 8.1, 2.0, 1.0 Hz),
7.78-7.82 (m, 1H), 7.54 (t, 1H, J = 8.1 Hz), 4.77 (d, 1H, J = 4.9 Hz), 4.36 (s, 2H), 3.44-3.52 (m, 1H), 3.09
(ddd, 1H, J = 12.7, 12.7, 3.0 Hz), 2.21-2.29 (m, 1H), 1.57-1.81 (m, 3H), 1.17-1.57 (m, 2H); 13C-NMR
(100 MHz, CDCl3) δ 175.9, 148.2, 137.1, 131.3, 129.6, 126.0, 123.5, 57.9, 55.8, 43.7, 27.8, 25.1, 20.4;
IR (ATR, [cm-1]): 3088, 2943, 2862, 1715, 1504, 1334, 1258, 1182, 1129, 960, 840.
4.2.4. General Procedure for the Synthesis of Compounds 5a-g. For synthesis of compounds 5a-g a
procedure from Dong et al.35 was used. Compound 4a, e-g (1 equiv.), the corresponding alcohol (1.0
equiv.), EDC·HCl (1.5 equiv.), and DMAP (0.2 equiv.), were dissolved in 20 mL of anhydrous CH2Cl2.
This mixture was stirred overnight. After the reaction was completed (TLC control) a subsequent
purification by flash-chromatography was performed to obtain compound 5a-g.
Page 21 of 42

3-(3,4,5-Trimethoxyphenyl)propyl 1-(benzylsulfonyl)piperidine-2-carboxylate (5a) and (S)-3-(3,4,5-
Trimethoxyphenyl)propyl 1-(benzylsulfonyl)piperidine-2-carboxylate (S-5a) have already been
described.30
4.2.4.1. (R)-3-(3,4,5-Trimethoxyphenyl)propyl 1-(benzylsulfonyl)piperidine-2-carboxylate (R-5a)
1
was obtained as a yellow oil (0.45 g, 81 %). H-NMR (400 MHz, CDCl3) δ 7.41-7.48 (m, 2H), 7.32-7.40
(m, 3H), 6.41 (s, 2H), 4.53 (d, 1H, J = 4.7 Hz), 4.27 (s, 2H), 4.16-4.24 (m, 2H), 3.84 (s, 6H), 3.82 (s, 3H),
3.40-3.49 (m, 1H), 3.17 (ddd, 1H, J = 12.8, 12.8, 3.0 Hz), 2.65 (t, 2H, J = 7.6 Hz), 2.11-2.18 (m, 1H),
1.94-2.03 (m, 2H), 1.54-1.73 (m, 3H), 1.37-1.49 (m, 1H), 1.17-1.30 (m, 1H); 13C-NMR (100 MHz, CDCl3)
δ 171.5, 153.2 (2C), 136.7, 131.0 (2C), 129.3, 128.5 (3C), 105.4 (2C), 64.6, 60.8, 58.9, 56.1 (3C), 43.5,
32.4, 30.3, 27.8, 25.0, 20.4; IR (ATR, [cm-1]): 3064, 2940, 2859, 1731, 1589, 1455, 1335, 1237, 1177,
1122, 698 (m); HPLC purity: 98 % (method I); ESI-MS: m/z 492.4 [M+H]+.
4.2.4.2. 3-(Pyridin-3-yl)propyl 1-(benzylsulfonyl)piperidine-2-carboxylate (5b) was obtained as a
1
colourless oil (0.66 g, 93 %). H-NMR (400 MHz, CDCl3) δ 8.45-8.48 (m, 2H), 7.51-7.56 (m, 1H),
7.43-7.48 (m, 2H), 7.33-7.40 (m, 3H), 7.24 (ddd, 1H, J = 7.8, 4.9, 0.7 Hz), 4.50 (d, 1H, J = 4.4 Hz),
4.13-4.31 (m, 4H), 3.39-3.50 (m, 1H), 3.16 (ddd, 1H, J = 12.8, 12.8, 3.0 Hz), 2.73 (t, 2H, J = 7.8 Hz),
2.09-2.16 (m, 1H), 1.96-2.03 (m, 2H), 1.55-1.72 (m, 3H), 1.35-1.49 (m, 1H), 1.15-1.23 (m, 1H); 13C-
NMR (100 MHz, CDCl3) δ 171.4, 149.7, 147.5, 136.3, 136.1, 130.9 (2C), 129.3, 128.5 (3C), 123.5, 64.3,
58.9, 56.1, 43.5, 29.9, 29.3, 27.7, 24.9, 20.4; IR (ATR, [cm-1]): 3032, 2945, 2860, 1732, 1575, 1335,
1147, 1127, 1109, 738, 698 (m); HPLC purity: > 99 % (method I); ESI-MS: m/z 403.4 [M+H]+.
4.2.4.3. 2-Hydroxyethyl 1-(benzylsulfonyl)piperidine-2-carboxylate (5c) was obtained as a colourless
1
oil (0.41 g, 71 %). H-NMR (400 MHz, CDCl3) δ 7.34-7.45 (m, 5H), 4.34-4.43 (m, 1H), 4.26 (s, 2H),
4.18-4.28 (m, 2H), 3.83 (q, 2H, J = 4.8 Hz), 3.34-3.43 (m, 1H), 3.13 (ddd, 1H, J = 12.8, 12.7, 2.9 Hz),
2.46 (t, 1H, J = 5.6 Hz), 2.11-2.20 (m, 1H), 1.34-1.72 (m, 4H), 1.18-1.29 (m, 1H); 13C-NMR (100 MHz,
Page 22 of 42

CDCl3) δ 171.4, 130.9 (2C), 129.1, 128.7, 128.6 (2C), 66.9, 60.8, 58.8, 56.3, 43.8, 27.4, 24.8, 20.2; IR
(ATR, [cm-1]): 3491 (br), 3065, 3034, 2944, 2861, 1733, 1455, 1334, 1322, 1198, 1177, 1147, 1125,
1059, 739, 697 (s); HPLC purity: > 99 % (method I); ESI-MS: m/z 328.3 [M+H]+.
4.2.4.4. Isopropyl 1-(benzylsulfonyl)piperidine-2-carboxylate (5d) was obtained as a white powder
1
(0.09 g, 39 %); mp 64-68 °C. H-NMR (400 MHz, CDCl3) δ 7.44-7.49 (m, 2H), 7.32-7.39 (m, 3H), 5.10
(sept, 1H, J = 6.3 Hz), 4.54 (d, 1H, J = 4.5 Hz), 4.28 (s, 2H), 3.42-3.49 (m, 1H), 3.16 (ddd, 1H, J = 12.7,
12.7, 3.1 Hz), 2.12-2.21 (m, 1H), 1.54-1.72 (m, 1H), 1.38-1.51 (m, 1H), 1.16-1.31 (m, 7H); 13C-NMR
(100 MHz, CDCl3) δ 170.9, 131.0 (2C), 129.4, 128.5 (2C), 128.4, 69.1, 58.7, 56.1, 43.4, 27.8, 25.1, 21.8
(2C), 20.3; IR (ATR, [cm-1]): 3041, 2934, 2853, 1721, 1498, 1336, 1210, 1183, 1142, 1126, 735, 697
(m); HPLC purity: 96 % (method II); ESI-MS: m/z 326.2 [M+H]+.
4.2.4.5. 3-(Pyridin-3-yl)propyl 1-(2-(4-chlorophenoxy)acetyl)piperidine-2-carboxylate (5e)
was
1
obtained as a yellow oil (0.26 g, 88 %). The H-NMR showed the presence of a 77 to 23 mixture of
rotamers. Rotamer a: 1H-NMR (400 MHz, CDCl3) δ 8.42-8.48 (m, 2H), 7.44-7.51 (m, 1H), 7.19-7.24 (m,
3H), 6.84-6.90 (m, 2H), 5.30 (d, 1H, J = 5.1 Hz), 4.65-4.82 (m, 2H), 4.07-4.19 (m, 2H), 3.79-3.87 (m,
1H), 3.31 (ddd, 1H, J = 13.0, 13.1, 3.0 Hz), 2.61-2.76 (m, 2H), 2.21-2.32 (m, 1H), 1.87-1.98 (m, 2H),
1.60-1.79 (m, 3H), 1.27-1.53 (m, 2H); 13C-NMR (100 MHz, CDCl3) δ 170.8, 167.7, 156.6, 149.7, 147.6,
136.4, 135.9, 129.4, 126.5, 123.4, 116.0, 67.4, 64.2, 52.4, 43.1, 29.8, 29.2, 26.5, 25.2, 20.9. Rotamer b
1
(only different signals to a are shown): H-NMR (400 MHz, CDCl3) δ 4.65-4.82 (m, 3H), 4.47-4.53 (m,
1H); 13C-NMR (100 MHz, CDCl3) δ 170.6, 167.5, 156.5, 129.5 (2C), 115.9 (2C), 68.4, 64.6, 55.9, 40.1,
29.3, 27.3, 24.5, 20.8; IR (ATR, [cm-1]): 3031, 2942, 2860, 1732, 1661, 1594, 1491, 1422, 1227, 1161,
1016, 824 (m); HPLC purity: 96 % (method II); ESI-MS: m/z 417.4 [M+H]+.
4.2.4.6. 3-(Pyridin-3-yl)propyl 1-((4-nitrobenzyl)sulfonyl)piperidine-2-carboxylate (5f) was obtained
1
as a yellow oil (0.22 g, 52 %). H-NMR (400 MHz, CDCl3) δ 8.45-8.49 (m, 2H), 8.20-8.25 (m, 2H),
Page 23 of 42

7.65-7.70 (m, 2H), 7.49-7.54 (m, 1H), 7.24 (ddd, 1H, J = 7.8, 4.8, 0.7 Hz), 4.66 (d, 1H, J = 4.7 Hz), 4.39
(AB-spin system, 1H, JAB = 13.6 Hz), 4.37 (AB-spin system, 1H, JAB = 13.6 Hz), 4.15-4.28 (m, 2H),
3.45-3.52 (m, 1H), 3.11 (ddd, 1H J = 12.7, 12.7, 3.0 Hz), 2.73 (t, 2H, J = 7.7 Hz), 2.16-2.27 (m, 1H),
1.98-2.08 (m, 2H), 1.58-1.79 (m, 3H), 1.41-1.55 (m, 1H), 1.15-1.29 (m, 1H); 13C-NMR (100 MHz, CDCl3)
δ 171.4, 149.9, 148.0, 147.8, 136.6, 136.0, 135.8, 132.0 (2C), 123.6 (2C), 123.5, 64.6, 58.0, 56.2, 43.6,
29.8, 29.3, 28.0, 25.0, 20.4; IR (ATR, [cm-1]): 3031, 2943, 2858, 1732, 1606, 1519, 1446, 1344, 1197,
1177, 1128, 812 (m); HPLC purity: > 99 % (method II); ESI-MS: m/z 448.4 [M+H]+.
4.2.4.7. 3-(Pyridin-3-yl)propyl 1-((3-nitrobenzyl)sulfonyl)piperidine-2-carboxylate (5g) was obtained
1
as a yellow oil (0.82 g, 65 %). H-NMR (400 MHz, CDCl3) δ 8.45-8.51 (m, 2H), 8.36 (t, 1H, J = 1.8 Hz),
8.22 (ddd, 1H, J = 8.1, 1.8, 0.9 Hz), 7.80-7.87 (m, 1H), 7.53-7.60 (m, 1H), 7.23-7.30 (m, 1H), 4.63 (d,
1H, J = 3.4 Hz), 4.39 (AB-spin system, 1H, JAB = 14.0 Hz), 4.37 (AB-spin system, 1H, JAB = 14.0 Hz),
4.17-4.30 (m, 2H), 3.46-3.54 (m, 1H), 3.13 (ddd, 1H, J = 12.7, 12.7, 3.0 Hz), 2.74 (t, 2H, J = 7.7 Hz),
2.17-2.28 (m, 1H), 1.99-2.07 (m, 2H), 1.59-1.79 (m, 3H), 1.42-1.57 (m, 1H), 1.16-1.30 (m, 1H); 13C-
NMR (100 MHz, CDCl3) δ 171.3, 149.5, 148.3, 147.4, 137.1, 136.3, 136.2, 131.5, 129.6, 126.0, 123.6,
123.5, 64.6, 57.8, 56.2, 43.6, 29.9, 29.3, 27.9, 25.1, 20.4; IR (ATR, [cm-1]): 3087, 3030, 2944, 2860,
1732, 1526, 1351, 1336, 1301, 1148, 1128, 811 (m); HPLC purity: 99 % (method I); ESI-MS: m/z 448.4
[M+H]+.
4.2.4.8. 3-(Pyridin-3-yl)propyl 1-((4-aminobenzyl)sulfonyl)piperidine-2-carboxylate
(5h)
was
1
obtained as a orange oil (0.05 g, 38 %). H-NMR (400 MHz, CDCl3) δ 8.42-8.48 (m, 2H), 7.49-7.53 (m,
1H), 7.18-7.24 (m, 3H), 6.62-6.67 (m, 2H), 4.47 (d, 1H, J = 4.9 Hz), 4.08-4.25 (m, 4H), 3.67 (s, 2H),
3.39-3.46 (m, 1H), 3.16 (ddd, 1H, J = 12.8, 12.8, 3.0 Hz), 2.72 (t, 2H, J = 7.8 Hz), 2.06-2.16 (m, 1H),
1.93-2.16 (m, 2H), 1.54-1.72 (m, 3H), 1.13-1.49 (m, 1H); 13C-NMR (100 MHz, CDCl3) δ 171.5, 149.8,
147.6, 146.8, 136.2, 135.9, 131.9 (2C), 123.4, 118.6, 114.9 (2C), 64.2, 58.0, 56.0, 43.5, 29.9, 29.2,
Page 24 of 42

27.7, 25.0, 20.3; IR (ATR, [cm-1]): 3452, 3372, 3031, 2927, 2856, 1731, 1613, 1517, 1445, 1323,
1298, 1176, 1126, 823 (m); HPLC purity: 97 % (method II); ESI-MS: m/z 418.4 [M+H]+.
4.2.4.9. 3-(Pyridin-3-yl)propyl 1-((3-aminobenzyl)sulfonyl)piperidine-2-carboxylate
(5i)
was
1
obtained as a orange oil (0.16 g, 69 %). H-NMR (400 MHz, CDCl3) δ 8.44-8.49 (m, 2H), 7.52-7.56 (m,
1H), 7.24 (ddd, 1H, J = 7.8, 4.9, 0.8 Hz), 7.10-7.15 (m, 1H), 6.77-6.81 (m, 2H), 6.63-6.67 (m, 1H), 4.51
(d, 1H, J = 4.7 Hz), 4.08-4.25 (m, 4H), 3.44-3.52 (m, 1H), 3.32 (s, 2H), 3.19 (ddd, 1H, J = 12.8, 12.8, 3.0
Hz), 2.73 (t, 2H, J = 7.8 Hz), 2.08-2.15 (m, 1H), 1.95-2.03 (m, 2H), 1.56-1.72 (m, 3H), 1.37-1.50 (m, 1H),
1.16-1.29 (m, 1H); 13C-NMR (100 MHz, CDCl3) δ 171.5, 149.6, 147.4, 146.6, 136.4, 136.2, 130.2, 129.4,
123.5, 121.1, 117.3, 115.2, 64.2, 58.9, 56.1, 43.5, 29.9, 29.2, 27.7, 24.9, 20.4; IR (ATR, [cm-1]): 3447,
3370, 3031, 2944, 2859, 1731, 1625, 1606, 1590, 1462, 1322, 1147, 1126, 965, 767 (m); HPLC purity:
>99 % (method I); ESI-MS: m/z 418.4 [M+H]+.
4.2.4.10. (S)-3-(Pyridin-3-yl)propyl 1-((4-chlorobenzyl)sulfonyl)piperidine-2-carboxylate (S-5m) was
1
obtained as a yellow oil (0.20 g, 86 %). H-NMR (400 MHz, CDCl3) δ 8.46-8.51 (m, 2H), 7.57-7.62 (m,
1H), 7.38-7.43 (m, 2H), 7.32-7.36 (m, 2H), 7.28-7.32 (m, 1H), 4.55 (d, 1H, J = 4.9 Hz), 4.14-4.28 (m,
4H), 3.40-3.50 (m, 1H), 3.14 (ddd, 1H, J = 12.8, 12.8, 3.0 Hz), 2.75 (t, 2H, J = 7.8 Hz), 2.12-2.20 (m, 1H),
1.97-2.06 (m, 2H), 1.57-1.76 (m, 3H), 1.38-1.50 (m, 1H), 1.15-1.25 (m, 1H); 13C-NMR (100 MHz, CDCl3)
δ 171.4, 149.1, 147.0, 136.7, 136.6, 134.7, 132.3 (2C), 128.8 (2C), 127.8, 123.7, 64.3, 58.1, 56.1, 43.6,
29.9, 29.3, 27.9, 25.0, 20.4; IR (ATR, [cm-1]): 3031, 2944, 2859, 1732, 1492, 1335, 1301, 1176, 1128,
826 (m); HPLC purity: 97 % (method I); ESI-MS: m/z 437.4 [M+H]+.
4.2.5. General Procedure for the Synthesis of Compounds 5j and 5k. To reduce the nitro to an amino
group the starting material 5f or 5g was dissolved in 20 mL EtOAc and hydrogenated at RT and 10 bar
hydrogen with catalytic amounts of Pd/C. After the reaction was completed the Pd/C was filtrated
and the solvent was evaporated to yield compound 5h or 5i. Without further purification a modified
Page 25 of 42

procedure from Fache et al.33 was used to synthesize compounds 5j and k. Therefore, 1 equiv. of 5h
or 5i, respectively, 1.1 equiv. of Me2CO, 4 equiv. of Na2SO4 and catalytic amounts of Pd/C were
dissolved in 20 mL CH2Cl2 and hydrogenated at RT and 10 bar hydrogen. After the reaction was
finished (TLC control) Pd/C was filtered off and the crude product was purified by flash-
chromatography to obtain compounds 5j, 5k.
4.2.5.1. 3-(Pyridin-3-yl)propyl 1-((3-(isopropylamino)benzyl)sulfonyl)piperidine-2-carboxylate (5j)
was obtained as a colouless oil (0.01 g, 9 %). 1H-NMR (400 MHz, CDCl3) δ 8.43-8.49 (m, 2H), 7.48-7.55
(m, 1H), 7.22 (ddd, 1H, J = 7.8, 4.8, 0.7 Hz), 7.13 (t, 1H, J = 7.7), 6.67-6.71 (m, 1H), 6.65-6.67 (m, 1H),
6.55 (ddd, 1H, J = 7.7, 2.2, 0.7 Hz), 4.51 (d, 1H, J = 4.7 Hz), 4.11-4.25 (m, 4H), 3.63 (sept, 1H, J = 6.3
Hz), 3.45-3.53 (m, 1H), 3.19 (ddd, 1H, J = 12.8, 12.8, 3.0 Hz), 2.72 (t, 2H, J = 7.8 Hz), 2.07-2.15 (m, 1H),
1.94-2.05 (m, 2H), 1.55-1.72 (m, 3H), 1.33-1.50 (m, 1H), 1.13-1.29 (m, 7H); 13C-NMR (100 MHz, CDCl3)
δ 171.5, 149.9, 147.6, 147.6, 136.3, 135.9, 130.1, 129.3, 123.4, 119.4, 115.5, 113.3, 64.2, 59.2, 56.0,
44.1, 43.4, 30.0, 29.2, 27.7, 24.9, 22.9 (2C), 20.3; IR (ATR, [cm-1]): 3389, 3031, 2961, 2861, 1732,
1605, 1491, 1323, 1175, 1127, 792 (m); HPLC purity: 95 % (method II); ESI-MS: m/z 460.3 [M+H]+.
4.2.5.2. 3-(Pyridin-3-yl)propyl 1-((4-(isopropylamino)benzyl)sulfonyl)piperidine-2-carboxylate (5k)
1
was obtained as a colourless oil (0.04 g, 21 %). H-NMR (400 MHz, CDCl3) δ 8.43-8.47 (m, 2H),
7.49-7.53 (m, 1H), 7.18-7.23 (m, 3H), 6.51-6.56 (m, 2H), 4.47 (d, 1H, J = 4.8 Hz), 4.08-4.24 (m, 4H),
3.61 (sept, 1H, J = 6.4 Hz), 3.39-3.47 (m, 1H), 3.16 (ddd, 1H, J = 12.8, 12.8, 3.0 Hz), 2.71 (t, 2H, J = 7.8
Hz), 2.05-2.14 (m, 1H), 1.93-2.03 (m, 2H), 1.52-1.71 (m, 3H), 1.35-1.49 (m, 1H), 1.13-1.28 (m, 7H); 13C-
NMR (100 MHz, CDCl3) δ 171.5, 149.8, 147.7, 147.6, 136.2, 135.9, 131.8 (2C), 123.4, 116.7, 112.9
(2C), 64.1, 58.5, 56.0, 44.1, 43.4, 29.9, 29.2, 27.7, 24.9, 22.8 (2C), 20.3; IR (ATR, [cm-1]): 3389, 3029,
2927, 2858, 1732, 1613, 1520, 1323, 1174, 1127, 826 (m); HPLC purity: 96 % (method II); ESI-MS: m/z
460.4 [M+H]+.
Page 26 of 42

4.2.5.3. (S)-3-(Pyridin-3-yl)propyl 1-(2-oxo-2-phenylacetyl)piperidine-2-carboxylate (S-5l). For the
synthesis of S-5l an amidation with 8a (1 equiv.) and phenylglyoxalic acid (1.0 equiv.) in 20 mL
anhydrous CH2Cl2 was performed at 0 °C using EDC·HCl (1.3 equiv.) and HOBt (0.5 equiv.). The
mixture was stirred until completion (TLC control) and after purification by means of flash-
chromatography the compound was obtained as a yellow oil (0.20 g, 62 %). The 1H-NMR showed the
1
presence of a 77 to 23 mixture of rotamers. Rotamer a: H-NMR (400 MHz, CDCl3) δ 8.40-8.50 (m,
2H), 8.01-8.05 (m, 2H), 7.58-7.67 (m, 1H), 7.43-7.55 (m, 3H), 7.19-7.25 (m, 1H), 5.42 (d, 1H, J = 5.2
Hz), 4.26 (t, 2H, J = 6.4 Hz), 3.48-3.55 (m, 1H), 3.26 (ddd, 1H, J = 13.0, 13.1, 3.0 Hz), 2.75 (t, 2H, J = 7.8
Hz), 2.01-2.10 (m, 2H), 1.31-1.94 (m, 6H); 13C-NMR (100 MHz, CDCl3) δ 191.6, 170.4, 167.4, 149.9,
147.8, 136.1, 135.9, 134.8, 133.2, 129.7 (2C), 129.1 (2C), 123.4, 64.6, 51.6, 44.3, 30.0, 29.3, 26.4,
1
24.9, 21.2. Rotamer b (only different signals to a are shown): H-NMR (400 MHz, CDCl3) δ 7.96-7.99
(m, 2H), 4.61-4.68 (m, 1H), 4.39 (d, 1H, J = 4.9), 3.03 (ddd, 1H, J = 13.3, 13.3, 3.0 Hz), 2.61 (t, 2H,
J = 7.6 Hz); 13C-NMR (100 MHz, CDCl3) δ 191.2, 170.3, 147.7, 135.8, 133.1, 129.9 (2C), 128.9 (2C),
123.3, 64.5, 56.6, 39.3, 29.8, 29.1, 27.3, 24.4, 21.0; IR (ATR, [cm-1]): 3058, 3031, 2943, 2860, 1734,
1678, 1639, 1444, 1228, 1161, 794, 714 (m); HPLC purity: 98 % (method I); ESI-MS: m/z 381.3 [M+H]+.
4.2.6. General Procedure for the Synthesis of Compounds S-5a, b, n-y. According to a procedure of
Choi et al.36 (S)-1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid (1 equiv.) was dissolved in 20 mL
anhydrous CH2Cl2 and at 0 °C the corresponding alcohol (1.0 equiv.), EDC·HCl (1.5 equiv.) and DMAP
(0.2 equiv.) were added. After the reaction was completed (TLC control) the organic layer was
washed twice with water and the solvent was evaporated in vacuo. The boc-protection group was
cleaved with 2 mL trifluoroacetic acid in 20 mL CH2Cl2. After 24 h the reaction was neutralized with
saturated NaHCO3 extracted with 3 x 30 mL CH2Cl2. The combined organic layers were dried over
Na2SO4 and the solvent was evaporated to yield compound 8a, b, n-y which was used in the next step
without further purification. Compound 8a, b, n-y (1 equiv.) was dissolved in 40 mL of anhydrous
CH2Cl2, and NMM or DIPEA (3 equiv.) was added at 0 °C followed by corresponding sulfonyl chloride
Page 27 of 42

(1 equiv.), respectively. The mixture was stirred until completion (TLC) and subsequently the solvent
removed in vacuo. After purification by means of flash-chromatography compounds S-5a, b, n-y were
obtained.
4.2.6.1. (S)-3-(Pyridin-3-yl)propyl 1-(benzylsulfonyl)piperidine-2-carboxylate (S-5b) was obtained as
1
a colourless oil (0.22 g, 80 %). H-NMR (400 MHz, CDCl3) δ 8.45-8.48 (m, 2H), 77.52 (ddd, 1H, J = 7.8,
2.2, 1.7 Hz), 7.43-7.48 (m, 2H), 7.33-7.40 (m, 3H), 7.23 (ddd, 1H, J = 7.8, 4.9, 0.7 Hz), 4.51 (d, 1H,
J = 4.3 Hz), 4.13-4.31 (m, 4H), 3.39-3.50 (m, 1H), 3.16 (ddd, 1H, J = 12.8, 12.8, 3.0 Hz), 2.73 (t, 2H,
J = 7.8 Hz), 2.09-2.16 (m, 1H), 1.96-2.03 (m, 2H), 1.55-1.72 (m, 3H), 1.35-1.49 (m, 1H), 1.14 (ddq, 1H,
J = 12.4, 12.4, 3.2 Hz); 13C-NMR (100 MHz, CDCl3) δ 171.5, 149.7, 147.7, 136.2, 135.9, 131.0 (2C),
129.3, 128.6 (3C), 123.4, 64.3, 58.9, 56.1, 43.5, 30.0, 29.3, 27.8, 25.0, 20.4; IR (ATR, [cm-1]): 3032,
2945, 2859,1732, 1575, 1335, 1147, 1127, 1109, 738, 697 (m); HPLC purity: 97 % (method I); ESI-MS:
m/z 403.4 [M+H]+.
4.2.6.2. (S)-3-(pyridin-3-yl)propyl 1-((3-chlorobenzyl)sulfonyl)piperidine-2-carboxylate (S-5n) was
obtained as a yellow oil (0.85 g, 83 %). 1H-NMR (400 MHz, CDCl3) δ 8.46-8.49 (m, 2H), 7.54 (ddd, 1H,
J = 7.8, 2.2, 1.7 Hz), 7.46 (t, 1H, J = 1.6 Hz), 7.28-7.37 (m, 3H), 7.23-7.27 (m, 1H), 4.55 (d, 1H, J = 4.8
Hz), 4.08-4.29 (m, 4H), 3.43-3.50 (m, 1H), 3.16 (ddd, 1H, J = 12.8, 12.8, 3.0 Hz), 2.73 (t, 2H, J = 7.8 Hz),
2.10-2.24 (m, 1H), 1.95-2.07 (m, 2H), 1.57-1.77 (m, 3H), 1.38-1.52 (m, 1H), 1.15-1.30 (m, 1H); 13C-
NMR (100 MHz, CDCl3) δ 171.4, 149.6, 147.4, 136.3, 136.2, 134.3, 131.2, 131.0, 129.8, 129.2, 128.7,
123.5, 64.4, 58.2, 56.1, 43.6, 29.9, 29.3, 27.8, 25.0, 20.4; IR (ATR, [cm-1]): 3031, 2944, 2860, 1732,
1597, 1423, 1335, 1177, 1148, 1128, 795 (m); HPLC purity: 97 % (method II); ESI-MS: m/z 437.5
[M+H]+.
4.2.6.3. (S)-3-(Pyridin-3-yl)propyl 1-((4-fluorobenzyl)sulfonyl)piperidine-2-carboxylate (S-5o) was
obtained as a yellow oil (0.23 g, 91 %). 1H-NMR (400 MHz, CDCl3) δ 8.44-8.49 (m, 2H), 7.53 (ddd, 1H,
Page 28 of 42

J = 7.8, 2.0, 1.7 Hz), 7.41-7.46 (m, 2H), 7.22-7.26 (m, 1H), 7.02-7.09 (m, 2H), 4.55 (d, 1H, J = 4.8 Hz),
4.08-4.28 (m, 4H), 3.40-3.47 (m, 1H), 3.13 (ddd, 1H, J = 12.8, 12.8, 3.0 Hz), 2.73 (t, 2H, J = 7.8 Hz),
2.12-2.20 (m, 1H), 1.95-2.06 (m, 2H), 1.55-1.75 (m, 3H), 1.36-1.52 (m, 1H), 1.14-1.28 (m, 1H); 13C-
NMR (100 MHz, CDCl3) δ 171.4, 162.9 (d, 1JCF=246.4 Hz), 149.7, 147.5, 136.3, 136.1, 132.7 (2C, d, JCF =
8.3 Hz), 125.2 (d, JCF = 3.3 Hz), 123.5, 115.6 (2C, d, JCF = 21.5 Hz), 64.3, 57.9, 56.1, 43.5, 29.9, 29.3,
27.9, 25.0, 20.4; IR (ATR, [cm-1]): 3031, 2946, 2861, 1732, 1508, 1335, 1176, 1127, 841 (m); HPLC
purity: 98 % (method II); ESI-MS: m/z 421.4 [M+H]+.
4.2.6.4. (S)-3-(Pyridin-3-yl)propyl 1-((3-nitrobenzyl)sulfonyl)piperidine-2-carboxylate (S-5p) was
1
obtained as a yellow oil (0.27 g, 68 %). H-NMR (400 MHz, DMSO-d6) δ 8.42 (d, 1H, J = 1.8 Hz), 8.40
(dd, 1H, J = 4.8, 1.8 Hz), 8.33-8.36 (m, 1H), 8.24 (ddd, 1H, J = 8.1, 2.3, 0.9 Hz), 7.86-7.91 (m, 1H), 7.71
(t, 1H, J = 8.1 Hz), 7.60-7.66 (m, 1H), 7.30 (ddd, 1H, J = 7.8, 4.8, 0.6 Hz), 4.71 (AB-spin system, 1H,
JAB = 13.6 Hz), 4.66 (AB-spin system, 1H, JAB = 13.6 Hz), 4.38 (d, 1H, J = 5.0 Hz), 4.04-4.18 (m, 2H),
3.39-3.47 (m, 1H), 3.14 (ddd, 1H, J = 12.8, 12.7, 2.7 Hz), 2.67 (t, 2H, J = 7.7 Hz), 1.96-2.03 (m, 1H),
1.87-1.96 (m, 2H), 1.50-1.67 (m, 3H), 1.21-1.36 (m, 1H), 1.03-1.20 (m, 1H); 13C-NMR (100 MHz,
DMSO-d6) δ 170.9, 149.5, 147.4, 147.2, 137.5, 136.4, 135.7, 132.3, 129.7, 125.5, 123.3, 123.0, 63.9,
56.2, 55.1, 42.7, 29.4, 28.3, 27.1, 24.2, 19.6; IR (ATR, [cm-1]): 3087, 3031, 2943, 2860, 1732, 1576,
1526, 1351, 1336, 1301, 1148, 1128, 880 (w); HPLC purity: 97 % (method I); ESI-MS: m/z 448.4
[M+H]+.
4.2.6.5. (S)-2-((1-(Benzylsulfonyl)piperidine-2-carbonyl)oxy)ethyl nicotinate (S-5q) was obtained as
a colourless oil (0.17 g, 58 %). 1H-NMR (400 MHz, CDCl3) δ 9.22 (dd, 1H, J = 2.2, 0.8 Hz), 8.78 (dd, 1H,
J = 4.9, 1.7 Hz), 8.35 (td, 1H, J = 8.0, 1.9 Hz), 7.31-7.46 (m, 6H), 4.45-4.63 (m, 5H), 4.24 (s, 2H),
3.39-3.47 (m, 1H), 3.15 (ddd, J = 12.8, 12.8, 3.1 Hz), 2.08-2.18 (m, 1H), 1.51-1.69 (m, 3H), 1.34-1.47
(m, 1H), 1.13-1.25 (m, 1H); 13C-NMR (100 MHz, CDCl3) δ 171.4, 164.6, 152.9, 150.3, 137.9, 130.9 (2C),
129.2, 128.5 (3C), 125.8, 123.7, 3.0, 62.7, 58.8, 56.0, 43.4, 27.8, 24.9, 20.3; IR (ATR, [cm-1]): 3062,
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