Chemical and pharmacological characterization of the TRPML calcium channel blockers ML-SI1 and ML-SI3

Article abstract of DOI:10.1016/j.ejmech.2020.112966

The members of the TRPML subfamily of non-selective cation channels (TRPML1-3) are involved in the regulation of important lysosomal and endosomal functions, and mutations in TRPML1 are associated with the neurodegenerative lysosomal storage disorder muco

Full text of DOI:10.1016/j.ejmech.2020.112966

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Chemical and pharmacological characterization of the TRPML calcium channel
blockers ML-SI1 and ML-SI3
Charlotte Leser, Marco Keller, Susanne Gerndt, Nicole Urban, Cheng-Chang Chen,
Michael Schaefer, Christian Grimm, Franz Bracher
PII:
S0223-5234(20)30938-7
DOI:
https://doi.org/10.1016/j.ejmech.2020.112966
EJMECH 112966
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 August 2020
Revised Date: 6 October 2020
Accepted Date: 21 October 2020
Please cite this article as: C. Leser, M. Keller, S. Gerndt, N. Urban, C.-C. Chen, M. Schaefer, C.
Grimm, F. Bracher, Chemical and pharmacological characterization of the TRPML calcium channel
blockers ML-SI1 and ML-SI3, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
j.ejmech.2020.112966.
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Chemical and pharmacological characterization of the TRPML calcium
channel blockers ML-SI1 and ML-SI3
Charlotte Leser1, Marco Keller1, Susanne Gerndt1, Nicole Urban3, Cheng-Chang Chen1, Michael
Schaefer3, Christian Grimm2, Franz Bracher1*
1Department of Pharmacy – Center for Drug Research, Ludwig-Maximilians University, Munich,
2
Germany; Walter Straub Institute of Pharmacology and Toxicology, Faculty of Medicine, Ludwig-
Maximilians University, Munich, Germany; 3Rudolf-Boehm-Institute for Pharmacology and
Toxicology, University of Leipzig, Leipzig, Germany
Abstract
The members of the TRPML subfamily of non-selective cation channels (TRPML1-3) are involved in
the regulation of important lysosomal and endosomal functions, and mutations in TRPML1 are
associated with the neurodegenerative lysosomal storage disorder mucolipidosis type IV. For in-depth
investigation of functions and (patho)physiological roles of TRPMLs, membrane-permeable chemical
tools are urgently needed. But hitherto only two TRPML inhibitors, ML-SI1 and ML-SI3, have been
published, albeit without clear information about stereochemical details. In this investigation we
developed total syntheses of both inhibitors. ML-SI1 was only obtained as a racemic mixture of
inseparable diastereomers and showed activator-dependent inhibitory activity. The more promising
tool is ML-SI3, hence ML-SI1 was not further investigated. For ML-SI3 we confirmed by
stereoselective synthesis that the trans-isomer is significantly more active than the cis-isomer.
Separation of the enantiomers of trans-ML-SI3 further revealed that the (-)-isomer is a potent inhibitor
of TRPML1 and TRPML2 (IC50 values 1.6 and 2.3 μM) and a weak inhibitor (IC50 12.5 μM) of
TRPML3, whereas the (+)-enantiomer is an inhibitor on TRPML1 (IC50 5.9 μM), but an activator on
TRPML 2 and 3. This renders the pure (-)-trans-ML-SI3 more suitable as a chemical tool for the
investigation of TRPML1 and 2 than the racemate. The analysis of 12 analogues of ML-SI3 gave first
insights into structure-activity relationships in this chemotype, and showed that a broad variety of
modifications in both the N-arylpiperazine and the sulfonamide moiety is tolerated. An aromatic
analogue of ML-SI3 showed an interesting alternative selectivity profile (strong inhibitor of TRPML1
and strong activator of TRPML2).
Key words: TRPML calcium channels; channel blocker; indoline; N-arylpiperazine; stereoselective
synthesis

1. Introduction
Transient receptor potential (TRP) channels are a family of highly diverse cation channels. There are 6
subfamilies in mammals (TRPC, TRPV, TRPM, TRPA, TRPP and TRPML) and many of these
channels are involved in sensory functions [1]. The TRPML subfamily which has three members
(TRPML1-3) is an exception to this mutuality within the TRP family. These channels are mainly
located in the lysosome (TRPML1-3), recycling endosome (TRPML2) and early endosome
(TRPML3). They are involved in the regulation of several lysosomal and endosomal functions such as
lysosomal ion homeostasis, lysosomal and endosomal trafficking and phagocytosis [2]. Mutation with
loss of function of the TRPML1 channel causes the neurodegenerative lysosomal storage disorder
mucolipidosis type IV, which is characterized by psychomotor retardation, corneal clouding, retinal
degeneration and strabismus [3]. Gain of function mutations of TRPML3 cause the varitint-waddler
phenotype in mice, which is characterized by deafness, circling behaviour and coat colour dilution [4],
[5], [6], [7], [8]. There is no identified TRPML3 associated phenotype in humans [9]. For TRPML2 it
is known that it enhances viral entry and trafficking of viruses such as yellow fever virus, dengue virus
and influenza virus type A, which require transport to late endosomes for infection [10].
Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) has been identified as endogenous activator of all
TRPMLs and is a major constituent of the lysosomal membrane. In contrast, phosphatidylinositol 4,5-
bisphosphate (PI(4,5)P2) inhibits TRPML1 and TRPML3 and is located at the plasma membrane [11].
Bisphosphates are not membrane permeable and therefore not suitable tools for pharmacological
investigations in intact cells.
Thus several synthetic activators of the TRPML channels have been generated in recent years, e.g. the
thienylsulfonamide MK6-83 [12], the phthalimidoacetamides SF-51 and ML-SA1 [13], the TRPML2
selective arylisoxazoline-type activator ML2-SA1, and the TRPML3-selective arylisoxazole EVP21
[14], a derivative of the originally identified TRPML3 agonist SN-2 [15]. Besides the many TRPML
agonists, only three synthetic inhibitors (ML-SI1, ML-SI2, ML-SI3) have been identified by Samie et
al. [16] using a calcium imaging-based high-throughput screening. Though only the chemical
structures of ML-SI1 and ML-SI3 were released by Wang et al. in 2015 in the Supporting Information
of their publication [17]. ML-SI1 is a 2,3-disubstituted N-aroylindoline, whereas ML-SI3 is a highly
functionalized 1,2-diaminocyclohexane containing an N-arypiperazine moiety. Both compounds have
two stereocenters, but the authors did not provide any information about the stereochemistry of these
inhibitors (Figure 1).

Figure 1: Structures of the TRPML inhibitors ML-SI1 and ML-SI3, as depicted by Wang et al. [17].
As TRPML inhibitors are valuable tools required for in-depth characterization of the calcium
channels, there is an urgent need to identify the exact structures of the active stereoisomers of ML-SI1
and ML-SI3. Further, synthetic work on structure-activity relationships (SAR) for both inhibitors was
highly attractive, aimed at the possible improvement of both activity and subtype selectivity of these
compounds.
2. Results and discussion
2.1. Chemistry
2.1.1. ML-SI1
A short synthetic approach to ML-SI1 was implemented, as no synthesis of this compound has been
published yet. Following work of Li et al. [18], commercially available indole-3-acetic acid 1 was
converted into the N-acylmorpholine 2 utilizing EDC and DMAP as condensing agents, and
subsequent reduction of the amide group with LiAlH4 gave amine 3 in 50% overall yield. Reduction of
indole 3 to the corresponding indoline 4 was unexpectedly challenging. A couple of protocols for
reduction of related 2,3-disubstituted indoles, in part even claiming to give cis- or trans-configured
products selectively, have been published in the past [19], [20], [21]. The attempted reduction with
H2/Pd to the racemic cis compound failed with 3 as substrate. However, reduction with NaBH3CN [22]
gave indoline 4 in 61% yield. Inspection of the NMR data clearly indicated that this product was
obtained as a 57:43 mixture of diastereomers. This diastereomeric mixture could not be separated by
column chromatography. In a final step, ML-SI1 was obtained as a racemic 55:45 mixture of
diastereomers with a yield of 67% when reacting 4 with 2,3-dichlorobenzoyl chloride following a
procedure of Ohkawa et al. [23] (Scheme 1). Separation of the racemic diastereomers of ML-SI1 was
attempted by column chromatography and preparative HPLC on achiral stationary phases, but failed.

Even analytical separation on achiral HPLC phases failed, but separation on a chiral analytical YMC
Chiral Art Cellulose-SB column (for the chromatogram, see Supplementary Information) clearly
indicated that the compound is a racemic 55:45 mixture of diastereomers. This was important, as the
1H- and 13C NMR spectra of ML-SI1 turned out to be quite complex due to the occurrence of rotamers
at the amide bond.
Scheme 1: Synthesis of ML-SI1 as a racemic mixture of diastereomers.
2.1.2. ML-SI3: Identification of the active stereoisomer
As mentioned above (Figure 1) no information on the relative and absolute stereochemistry of ML-SI3
was available from Wang’s publication [17], nor from earlier or later scientific work describing the
utilization of this inhibitor [16], [24], [25], [26], [27]. This prompted us to develop synthetic
approaches to both cis- and trans-configured ML-SI3 in a first initiative, then to identify the active
form (eutomer) of the active diastereomer, and finally, perform an analysis of structure-activity
relationships by systematic modifications of relevant functional groups in ML-SI3. As screening hit
ML-SI3 arose from a commercial compound library, we speculated that this compound was prepared
via a straightforward synthetic path from commercially available building blocks. Inspection of
published approaches to related N-sulfonyl 1,2-diaminocyclohexane derivatives [28] strongly
indicated that cis-N-phenylsulfonyl cyclohexaneaziridine 7 is a synthetic precursor of ML-SI3, and

imaginable further synthetic transformations should give the hit compound as the racemic trans
isomer. In fact, aziridine 7 (conveniently obtained by direct NBS-mediated sulfonylaziridination of
cyclohexene (5) with chloramine B (6) following a protocol of Thakur et al. [29]) was reacted with 1-
(2-methoxyphenyl)piperazine (8) in hexanes containing DMSO to give, under ring opening of the
aziridine ring and inversion, pure racemic trans-configured product trans-ML-SI3 in 63% yield
(Scheme 2).
Scheme 2: Synthesis of racemic trans-ML-SI3.
For the synthesis of cis-ML-SI3 we attempted to utilize a protocol published by Labrie et al. [30] for
the preparation of closely related products. In this approach,
a trans-configured N-
(arylsulfonylamino)cyclohexanol closely related to 10 was converted into the mesyl ester, and then
converted into an azidocyclohexane, putatively under complete inversion. Reduction of the azido
group followed by N-sulfonylation should open an access to cis-ML-SI3. To our surprise, O-
mesylation of 10 (prepared by reaction of cis-cyclohexene oxide 9 and piperazine 8), followed by
treatment with sodium azide gave a product, which, based on investigation of NMR data (see
Supporting Information) was unambiguously identified as the trans-configured isomer 11. Most likely,
the intermediate trans-configured mesylate underwent, under complete inversion, spontaneous ring
closure by attack at the piperazine nitrogen to a cis-configured spiroaziridinium intermediate, which in
turn then underwent another complete inversion by nucleophilic attack of the azide ion (Scheme 3).
Most likely, the same cascade of reactions took place in Labrie’s work [30], but was not detected. In a
control experiment, we performed reduction of the azido group in 11 to the primary amine (see
compound 19 in Scheme 6), and subsequent N-sulfonylation with benzenesulfonyl chloride in fact
gave pure trans-ML-SI3. Consequently, this approach did not allow synthesis of cis-ML-SI3.

Scheme 3: Attempted synthesis of racemic cis-ML-SI3.
Consequently, a novel approach to cis-ML-SI3 had to be worked out. Mono-phenylsulfonylation of
commercially available cis-1,2-diaminocyclohexane (12) gave racemic cis-configured sulfonamide 13.
The piperazine moiety was subsequently constructed by reaction of the primary amino group of 13
with N,N-bis(2-chloroethyl)-2-methoxyaniline (available from 2-methoxyaniline via bis-
hydroxyethylation with oxirane, affording bis(2-hydroxyethyl)-2-aniline 14 and subsequent
chlorination with thionyl chloride [31], [32]) to give pure racemic cis-ML-SI3 (Scheme 4).
Ph-SO2Cl,
NEt3
NH2
NH
NH2
NH2
O S
O
12
13
H3CO
N
OCH3
OH
OH
1) thionyl chloride
N
13
2) , K2CO3
N
NH
O S
14
O
ML-SI3
cis-
(racemic)
Scheme 4: Synthesis of racemic cis-ML-SI3.

Based on the evidence that trans-ML-SI3 is superior to its cis isomer in blocking TRPML channels
(see Chapter 2.2.), we further intended to identify the active enantiomer (eutomer) of trans-ML-SI3.
Separation of the enantiomers of the racemic inhibitor was attempted via chromatographic separation
by chiral HPLC. The separation could be achieved on a YMC Chiral Art Cellulose-SB column (250 ×
10.0 mm, 5 µm) column, and both enantiomers were obtained in pure form (ee values: (-)-trans-ML-
SI3 99 %, (+)-trans-ML-SI3 96 %).
2.1.3. ML-SI3: Chemistry aimed at analysis of structure-activity relationships
Systematic investigation of SAR of ML-SI3 included variation of both the sulfonyl and the N-
arylpiperazine residue. For replacing the phenylsulfonyl group of ML-SI3 by toluenesulfonyl and
thiophene-2-sulfonyl groups the same procedure as described for the synthesis of racemic trans-ML-
SI3 (Scheme 2) was followed in general. The required cis-configured N-tosylsulfonylaziridine 15 was
obtained directly from cyclohexene and chloramine T hydrate and catalytic NBS, as shown in Scheme
2 for its phenylsulfonyl analogue 7. The N-thiophene-2-sulfonyl analogue 16 [33] was prepared from
cis-cyclohexene oxide (9) in three steps (with two inversions of configuration) involving nucleophilic
ring opening with the thiophene-2-sulfonamide under phase-transfer catalysis, O-tosylation and
subsequent K2CO3–mediated aziridine formation [34]. Nucleophilic ring opening of these N-
sulfonylaziridines with N-arylpiperazine 8 gave the desired ML-SI3 analogues 17 and 18 (Scheme 5).
Scheme 5: Modification of the N-sulfonyl residues of ML-SI3.
The role of the sulfonamide group in ML-SI3 was analysed by synthesis of the carboxamide analogue
20. This compound was obtained from the trans-configured azido intermediate 11 that was obtained
inadvertently in our attempts to synthesize cis-ML-SI3 (see Scheme 3). Catalytic hydrogenation of the
azido group gave the primary amine 19, which was directly converted into the benzamide 20 by
treatment with benzoyl chloride/pyridine (Scheme 6).

Scheme 6: Synthesis of the benzamide analogue 20 of ML-SI3.
For investigation of the role of the 2-methoxyphenyl residue on the piperazine ring of ML-SI3, we
prepared, strictly following the chemistry described in Scheme 2 for the synthesis of racemic trans-
ML-SI3, using N-phenylsulfonylaziridine 7 and appropriate N-substituted piperazines, analogues
bearing modified aromatic residues (phenyl compound 21, 3-(trifluoromethyl)phenyl compound 22),
homologous compounds in which the N-aryl piperazine is replaced by an N-arylalkyl piperazine (4-
methylbenzyl compound 23, phenethyl compound 24), and a truncated analogue 25 in which the 2-
methoxyphenyl residue is replaced by a methyl group. Further, the N-arylpiperazine residue was
replaced by 4-arylpiperidines (products 26, 27) utilizing appropriate piperidine building blocks in
combination with N-phenylsulfonylaziridine 7. Replacement of the N-arylpiperazine residue by the
rigid 1,2,3,4-tetrahydroisoquinoline gave compound 28 (Scheme 6).
R
secondary
O
amines
NH
O S
N S
O
O
7
21-28
R =
N
N
CF3
N
N
N
N
21
22
23
H3CO
CH3
N
N
N
N
N
24
25
26
N
N
27
28

Scheme 7: Modification of the (2-methoxyphenyl)piperazinyl residue of trans-ML-SI3.
Finally, we prepared an analogue 31 in which the 1,2-disubstituted cyclohexane moiety of ML-SI3 is
replaced by a 1,2-disubstituted benzene ring. Related compounds were described very recently as
TRPML1 activators in a patent [35]. Buchwald-Hartwig amination of 2-bromonitrobenzene (29) with
1-(2-methoxyphenyl)piperazine
(8) under
Pd(OAc)2/BINAP
catalysis
[36]
gave
(nitrophenyl)piperazine 30, which yielded the desired phenylendiamine product 31 upon catalytic
hydrogenation of the nitro group and subsequent N-sulfonylation with benzenesulfonyl chloride
(Scheme 8).
Scheme 8: Synthesis of the 1,2-phenylenediamine-based analogue 31 of trans-ML-SI3.
2.2. Biological testing
2.2.1. Pre-screening of the activity of ML-SI1 and ML-SI3
The synthesized racemic mixture of ML-SI1, and a commercially available sample of ML-SI3, which
was identified as (±)-trans-ML-SI3, were analyzed in Fura-2 and Fluo-4 calcium imaging
experiments. Fura-2 based single cell calcium imaging experiments confirmed that the synthesized
racemic ML-SI1 (mixture of diastereomers) has an inhibitory effect on hTRPML1 (Figure 2 A, C).
Statistical analysis of the three isoforms of the ion channel showed a strong inhibitory effect on
hTRPML1, weaker effect on hTRPML2 and (at the test concentration of 10 µM) no effect on
hTRPML3, all after activation with ML-SA1 (Figure 2 A). While inhibition after activation with ML-
SA1 showed a robust signal, it was not possible to significantly block MK6-83 induced activation
(Figure 2 B). This indicates an activator dependent inhibition. Comparing the inhibitory effect of ML-
SI1 and commercially available ML-SI3, ML-SI3 is able to block hTRPML1 and 2 around 50%, but
not hTRPML3 (Figure 2 E). Comparing different activators, ML-SI3 is able to block both ML-SA1
and MK6-83 induced activation in the same manner (Figure 2 F, G). Concentration-effect experiments
revealed that ML-SI3 (IC50: 2.6 µM) is more potent than ML-SI1 (IC50: 15 µM) for inhibition of

TRPML1. Furthermore, ML-SI3 can inhibit TRPML3 as well at higher concentrations (IC50: 17 µM),
while ML-SI1 has no effect (Figure 2 D, H). Thus, ML-SI3 seemed the more promising antagonist for
further investigations. As, in addition, ML-SI1 was obtained as an inseparable mixture of
diastereomers with unpredictable individual activities, this inhibitor was not further investigated, and
no further derivatives of ML-SI1 were generated.
Figure 2: Pre-screening results for the synthesized ML-SI1 and commercially available ML-SI3. (A)
Statistical analysis of the inhibitory effect of ML-SI1 (10 µM) on TRPMLs in Fura-2 Ca2+ imaging
experiments (normalized activation) after activation with ML-SA1 (10 µM). HEK293 cells stably
expressing hTRPML2-YFP or hTRPML3-YFP, and transiently transfected hTRPML1ΔNC-YFP cells
were used [1]. (B) Statistical analysis as in (A), using ML-SA1 (10 µM) or MK6-83 (10 µM) for
activation of hTRPML1ΔNC-YFP transiently transfected HEK293 cells. (C) Representative Ca2+ signals
recorded from hTRPML1ΔNC-YFP transiently transfected HEK293 cells. Cells were sequentially
stimulated with ML-SA1 (10 µM, n = 5 transfected and 2 NT cells) or MK6-83 (10 µM, n = 11
transfected and 3 NT cells) and ML-SI1 (10 µM). Highlighted lines represent the mean response from
a population of cells. Shaded traces represent responses of single cells. (D) Concentration-effect
measurements on HEK293 cells stably expressing hTRPML1 and hTRPML3. Cells were treated with
ML-SI1, followed by activation with ML-SA1 (5 µM). (E) Statistical analysis of the inhibitory effect
of ML-SI3 as in (A) using ML-SA1 (10 µM) for activation and ML-SI3 (10 µM) for inhibition. (F)
Statistical analysis as in (B) but inhibition with ML-SI3 (10 µM). (G) Representative Ca2+ signals as in
(C), but performed on a Leica DMi8 live cell microscope and traces are normalized to basal. Cells
were treated with ML-SA1 (10 µM, n = 5 transfected and 8 NT cells) or MK6-83 (10 µM, n = 4

transfected and 5 NT cells) and ML-SI3 (10 µM). (H) Concentration-effect measurements as in (D)
but using ML-SI3 as inhibitor.
2.2.2 Single cell calcium imaging
All compounds of the ML-SI3 series were further tested by single cell Ca2+-imaging using Fura-2-
loaded HEK293 cells stably expressing hTRPML1ΔNC-YFP, hTRPML2-YFP and hTPPML3-YFP
(Figure 3). Activators were tested by adding the compound solution (10 µM) and recording the signals
of Ca-chelated Fura-2 and free Fura-2 for 200 s. For inhibitors, cells were first stimulated with 10 µM
of ML-SA1 or MK6-83, the compound solution added after 200 s, and the signals recorded for
additional 200 s. All compounds, except N-phenethyl piperazine 24, significantly block TRPML1
(Figure 3D). Piperazine 24 is the only compound from this series that activates TRPML1, but the
effect is not significant at 10 µM (Figure 3A). Only weak inhibitory effects on TRPML1 were detected
for cis-ML-SI3, the N-methyl piperazine 25 and the benzamide 20 analogue of trans-ML-SI3.
Surprisingly, on TRPML2 the activity is mainly the opposite, most of the compounds are activating
this channel (Figure 3B). Interestingly, the racemic mixture of trans-ML-SI3 is activating the
TRPML2 channel as efficiently as the known activator ML-SA1. Responsible for this activation is
exclusively the (+)-trans-ML-SI3, which activates TRPML2 stronger than ML-SA1, same as the
toluenesulfonamide 17. In contrast, (-)-trans-ML-SI3, as well as tetrahydroisoquinoline derivative 28,
does not activate but inhibit TRPML2 (Figure 3E). For TRPML3 the activity largely resembles the
activity observed on TRPML2. Here (+)-trans-ML-SI3, toluenesulfonamide 17, N-(4-methylbenzyl)
piperazine 23 and N-phenethyl piperazine 24 are strong activators, whereas (-)-trans-ML-SI3 and
thiophenesulfonamide 18 are the best inhibitors.

Figure 3: Statistical analysis of calcium imaging results. (A, B, C) Activation of TRPMLs in Fura-2
single cell calcium imaging experiments. Mean values of at least three independent experiments are
shown. (D, E, F) Inhibition of TRPMLs, after activation with ML-SA1 (10 µM, TRPML1 and 2) or
MK6-83 (10 µM, TRPML3). DMSO was used as negative control. Activation of ML-SA1 or MK6-83
is normalized to 1. All statistical analyses of Ca2+ imaging experiments are mean values of n=3 or n=4

independent experiments. Test compounds 17-28 were applied in racemic form. *** indicates p <
0.001, ** indicates p < 0.01, * indicates p < 0.05, ns = not significant, one-way ANOVA test followed
by Tukey’s post-hoc test, compared to DMSO.
2.2.3 FLIPR screening
A Fluo-4 calcium-imaging based FLIPR-method was used for determination of concentration-effect
relationships of the ML-SI3-type compounds. First, HEK293 cells stably expressing hTRPML1ΔNC
-
YFP, hTRPML2-YFP or hTPPML3-YFP were treated with the test compound solutions in final
concentrations from 0.098 µM to 100 µM for 10 minutes, followed by activation with the known
activator ML-SA1 (5 µM) for another 10 min. Concentration-effect curves are shown in Figure 4, the
calculated EC50 and IC50 values are presented in Table 1. These data fully confirm the Fura-2 calcium
imaging results shown in Chapter 2.2.2.
Figure 4: ML-SI3 and analogues in FLIPR experiments. Shown are concentration-effect relationships
for Ca2+ increases (Fluo-4) in response to different concentrations of activating ML-SI3 analogs on
HEK293 cells stably expressing hTRPML1-YFP (A), hTRPML2-YFP (B) or hTRPML3-YFP (C).
After 10 minutes, cells were activated with 5 µM ML-SA1 to determine half maximal inhibition
concentration of non-activating compounds on hTRPML1-YFP (D), hTRPML2-YFP (E) or

hTRPML3-YFP (F). Data are calculated from 3 independent experiments, each, and represented as
means ±SEM. EC50 and IC50 values are presented in Table 1.
for
for
Compound
EC50 [µM]
TRPML1 TRPML2
activation
TRPML3
IC50 [µM]
TRPML1 TRPML2
inhibition
TRPML3
-
3.3±1.5
9.4±2.7
-
-
3.1±1.5
18.5±1.9
1.6±0.7
5.9±1.7
5.6±0.5
2.3±1.2
-
28.5±15.9
trans-ML-SI3
-
29.0±4.1
-
-
cis-ML-SI3
-
-
2.3±1.1
12.5±7.6
(-)-trans-ML-SI3
-
2.7±1.4
4.0±0.4
6.3±3
-
10.8±0.5
-
-
-
-
(+)-trans-ML-SI3
-
8.5±0.9
-
17
18
20
21
22
23
24
25
26
27
28
31
-
-
15.9±8.9
-
-
65.1±11.1 26.8±8.7
>100
-
3.6±1.8
9.1±4.2
4.4±1.1
3.6±0.3
-
9.5±3.9
14.6±2.2
5.8±0.9
5.4±1.1
-
14.9±10.5
4.4±2.9
5.7±0.6
-
-
-
-
-
-
-
-
-
-
9.4±0.6
-
-
-
-
-
-
53.3±15.6 40.2±7.9
>100
3.9±1.6
3.7±0.8
-
-
4.8±2.0
8.9±1.5
9.5±2.0
1.8±0.9
-
56.2±16.2
20.7±4.4
9.5±1.4
-
-
-
5.5±2.5
-
-
1.1±0.5
21.7±7.7
Table 1: Activation and inhibition of the three subtypes of TRPML by the ML-SI3-type compounds.
Mean values ± SD of at least three independent experiments are shown. - : no effect.
2.2.4. Analysis of structure-activity relationships of ML-SI3 and its analogues
As evident from Table 1, racemic trans-ML-SI3 is blocking TRPML1 stronger (factor 6) than its
racemic cis isomer. The active (±)-trans-isomer has some selectivity for blocking TRPML1 (factor 9)
over TRPML3, but activates TRPML2. The racemic cis isomer is an activator on both TRPML2 and
TRPML3. Separation of the more potent racemic trans-ML-SI3 into the pure enantiomers led to the
evidence that the levorotatory form (-)-trans-ML-SI3 is the eutomer and the only compound from this
series that is an inhibitor on all three TRPMLs, being virtually equipotent on TRPML1 and 2 and less
potent on TRPML3 (factor of 5-7). The activities of the ML-SI3 isomers on hTRPML1 and hTRPML2
are summarized in Figure 5.

Figure 5: Summary of the activity of the stereoisomers of ML-SI3. (A) Concentration-response curves
for activation TRPML2. (B) Concentration-response curves for inhibition of TRPML1.
Systematic variation of structural motifs in racemic trans-ML-SI3 led to identification of the following
structure activity relationships: (a) most of the (racemic) analogues of trans-ML-SI3 (except 20 and
25) show an activity profile similar to the parent compound; (b) variation of the aromatic moiety of the
sulfonamide (17, 18) is well tolerated, whereas replacing the benzenesulfonamide by a benzamide (20)
leads to dramatic loss of activity; (c) modifications of the substitution pattern of the phenyl ring
located at N-4 of the piperazine (21, 22) do not change activity significantly, but introduction of an
aliphatic spacer (methylene in 23, ethylene in 24) between N-4 and the aromatic ring changes the
activity from inhibition to activation, for compound 24 on all three subtypes, for compound 23 on
TRPML2 and TRPML3; (d) replacement of N-4 of the piperazine by a carbon atom (giving piperidine
analogues 26, 27) leads only to a slight loss of activity on TRPML1, whereas rigidization
(tetrahydroisoquinoline analogue 28) is more detrimental, including a change on TRPML2 from
activation to inhibition; (e) replacement of the N-aryl residue on the piperazine by a methyl group (25 )
leads to a dramatic loss of activity.
In conclusion, the structure-activity relationship seen for the racemic analogues of ML-SI3 is rather
flat. Additional work on optimization of the identified eutomer (-)-trans-ML-SI3 is only promising
when working with pure enantiomers of analogues. This approach is presently hampered by the
lacking access to significant amounts of pure enantiomers. Nevertheless, some of the analogues
showing in their racemic form comparable activity profiles (potency and subtype-selectivity) as
racemic trans-ML-SI3 might provide pure enantiomers with a similar gain in selectivity as seen here

for trans-ML-SI3. Among them are the thiophenesulfonyl analogue 18 and the 4-arylpiperidine
analogues 26 and 27.
Noteworthy, replacement of the cyclohexane moiety of ML-SI3 by a benzene ring gave interesting
results. The resulting achiral phenylenediamine derivative 31 retained inhibitory activity on TRPML1,
equipotent to (-)-trans-ML-SI3. This was not unexpected, since the overall geometries of trans-
configured 1,2-disubstituted cyclohexanes like ML-SI3 (in which both substituents are preferentially
orientated in equatorial position) and of 1,2-disubstituted benzenes (where both residues are coplanar)
are rather similar. However, the activation effect on TRPML2 could not be eliminated for aromatic
analogue 31 as it could for (-)-trans-ML-SI3. No activation of TRPML3 was observed for compound
31 and only weak inhibition. This interesting finding is in contrast to a report on closely related
compounds, which were claimed (without details about activity) as TRPML1 activators very recently
in a patent [35].
3. Conclusions
For in-depth investigation of functions and (patho)physiological roles of TRPMLs versatile chemical
tools are urgently needed. Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) has been identified as
endogenous activator of all TRPMLs, whereas phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)
inhibits TRPML1 and TRPML3. But these physiological compounds are not suitable as chemical tools
for cellular assays due to their high polarity and lack of membrane permeability. In the past few years
several low-molecular, membrane-permeable activators of TRPMLs have been developed by our and
other groups [12], [13], [14], but well characterized lipophilic inhibitors are still missing. Recently,
three synthetic inhibitors (ML-SI1, ML-SI2, ML-SI3) have been reported by Samie et al. [16], but
only the chemical structures of ML-SI1 and ML-SI3 were released by Wang et al. in 2015 [17]. Both
the 2,3-disubstituted N-aroylindoline ML-SI1 and the highly functionalized 1,2-diaminocyclohexane
ML-SI3 contain two stereocenters, but no information about the stereochemistry of these inhibitors
was published (Figure 1). This prompted us to develop syntheses of these inhibitors for elucidation of
the exact structures of the active stereoisomers and for analysis of structure-activity relationships
(SAR).
ML-SI1 was obtained as an inseparable racemic mixture of cis-/trans-isomers (55:45) in a short
synthetic sequence, and its inhibitory activity on hTRPML1 (and a weak effect on TRPML2) after
activation with ML-SA1, but not with the alternative activator MK6-83, was confirmed by calcium
imaging experiments. As concentration-effect experiments revealed that ML-SI1 is inferior to the
second inhibitor of interest, ML-SI3, and the mixture of isomers could not be separated on a
preparative scale for the characterization of the single stereoisomers, ML-SI1 was not further
investigated.

For ML-SI3 we developed stereoselective syntheses of both racemic cis- and trans-isomers, and
identified trans-ML-SI3 as the active TRPML inhibitor. Further, this compound was separated into its
enantiomers by semi-preparative chiral HPLC, and 12 analogues of trans-ML-SI3 were prepared (in
racemic form) on different synthetic routes for an analysis of structure-activity relationships. With few
exceptions (N-methyl derivative 25, benzamide 20) these analogues showed a comparable activity
profile as racemic trans-ML-SI3. However, besides the expected inhibitory activity on subtype
TRPML1, racemic trans-ML-SI3 and most of the analogues showed significant activating effects on
TRPML2 and 3. Finally, detailed characterization of the separated enantiomers of trans-ML-SI3
revealed that (-)-trans-ML-SI3 is an inhibitor on all three subtypes, namely a potent inhibitor of both
TRPML1 und TRPML2 (IC50 values of 1.6 and 2.3 μM) and a weak inhibitor (IC50 12.5 μM) of
TRPML3. In contrast, the (+)-enantiomer is an inhibitor on TRPML1, but an activator on TRPML 2
and 3. This renders the pure (-)-trans-ML-SI3 more suitable as a chemical tool for the investigation of
TRPML1 and 2 than the racemate. Future work in this field will require efficient enantioselective
syntheses of ML-SI3-related compounds, and this will be subject of upcoming investigations.
Further, we identified the achiral aromatic ML-SI3 analogue 31, which has an uncommon selectivity
profile (strong inhibitor of TRPML1 and strong activator of TRPML2).
4. Experimental section
4.1 Chemistry
All chemicals used were of analytical grade and were obtained from abcr (Karlsruhe, Germany),
Fischer Scientific (Schwerte, Germany), Sigma-Aldrich (now Merck, Darmstadt, Germany), TCI
(Eschborn, Germany) or Th. Geyer (Renningen, Germany). HPLC grade and dry solvents were
purchased from VWR (Darmstadt, Germany) or Sigma-Aldrich, all other solvents were purified by
distillation. All reactions were monitored by thin-layer chromatography (TLC) using pre-coated plastic
sheets POLYGRAM® SIL G/UV254 from Macherey-Nagel and detected by irradiation with UV light
(254 nm). Flash column chromatography (FCC) was performed on Merck silica gel Si 60 (0.015 –
0.040 mm).
NMR spectra (1H, 13C, DEPT, H-H-COSY, HSQC/HMQC, HMBC) were recorded at 23 °C on an
Avance III 400 MHz Bruker BioSpin or Avance III 500 MHz Bruker BioSpin instrument. Chemical
shifts δ are stated in parts per million (ppm) and are calibrated using residual protic solvent as an
internal reference for proton (CDCl3: δ = 7.26 ppm, CD2Cl2) and for carbon the central carbon
resonance of the solvent (CDCl3: δ = 77.16 ppm, CD2Cl2). Multiplicity is defined as s = singlet, d =
doublet, t = triplet, q = quartet, p = pentet, m = multiplet. NMR spectra were analysed with NMR
software MestReNova, version 12.0.1-20560 (Mestrelab Research S.L.). High resolution mass spectra
were performed by the LMU Mass Spectrometry Service applying a Thermo Finnigan LTQ FT Ultra

Fourier Transform Ion Cyclotron Resonance device at 250 °C for ESI. IR spectra were recorded on a
Perkin Elmer FT-IR Paragon 1000 instrument as neat materials. Absorption bands were reported in
wave number (cm-1) with ATR PRO450-S. Melting points were determined by the open tube capillary
method on a Büchi melting point B-540 apparatus and are uncorrected. HPLC purities were
determined using an HP Agilent 1100 HPLC with a diode array detector and an Agilent Zorbax
Eclipse plus C18 column (150 × 4.6 mm; 5 µm) with methanol/water in different proportions adjusted
to pH = 9 with NaOH or neutral as mobile phase. Determination of ee values was performed using a
Daicel Chiralcel-OD column (250 × 4.6 mm, 10 µm). Semi-preparative HPLC was performed on a
YMC Chiral Art Cellulose-SB column (250 × 10.0 mm, 5 µm) using a VWR LaPrep P110 system
with a UV Detector P311. Values for specific rotation [α] were measured at 23 °C at a wavelength of
λ = 589 nm (Na-D-line) using a Perkin Elmer 241 Polarimeter instrument. All samples were dissolved
in chloroform (layer thickness l = 10 cm), the concentration is stated in g/100 mL.
4.1.1 Synthesis
General procedure A: Synthesis of aziridines from cyclohexene
According to Thakur et al. [29] the appropriate chloramine hydrate was dried at 60 °C for 24 h and
suspended in acetonitrile (~2 M). Cyclohexene (1.0 eq) and NBS (0.2 eq) were added to the
suspension and the resulting mixture stirred at rt for 22 h. The solvent was removed in vacuo and the
residue taken up in ethyl acetate. The organic layer was washed with water and brine, dried over
Na2SO4 and the solvent removed in vacuo. The residue was purified by FCC using petrol ether/ethyl
acetate (7:3) as an eluent to afford the desired product.
General procedure B: Synthesis of trans-1,2-diaminocyclohexanes from aziridines
The aziridine was suspended in dry hexanes (~0.3 M), then DMSO (5 eq) and the appropriate
secondary amine were added. The resulting mixture was stirred at rt for 24 h. After completion (TLC
control) ethyl acetate was added and the organic layer washed three times with water and dried over
Na2SO4. The solvent was removed in vacuo and the crude product recrystallized from ethanol to afford
the desired product.
4-(2-(5-Methoxy-2-methylindolin-3-yl)ethyl)morpholine (EY19)
4-(2-(5-Methoxy-2-methyl-1H-indol-3-yl)ethyl)morpholine (100 mg, 0.364 mmol) was dissolved in
2.5 mL trifluoroacetic acid and cooled to 0°C. Then, sodium borohydride (133 mg, 2.00 mmol) was
added portionwise. The reaction mixture was stirred for 2 hours, then quenched with 2 N NaOH (10
mL) and extracted with ethyl acetate (3 x 70 mL). The combined organic layers were washed with
water (100 mL) and brine (100 mL), dried by filtration and the solvent was removed in vacuo.
Purification by FCC using methylene chloride/methanol (9:1) as an eluent gave EY19 (62 mg) as a

1
brown oil (yield: 61 %). H NMR (400 MHz, CD2Cl2): δ (ppm) = 6.69 (m, 1H, 4-H), 6.59 – 6.46 (m,
2H, 6-H, 7-H), 3.91 (p, J = 7.6, 6.5 Hz, 0.6H, 2-H), 3.71 (d, J = 0.9 Hz, 3H, O-CH3), 3.67 (m, 4H, 3’’-
H, 5’’-H), 3.58 (p, J = 6.3 Hz, 0.4H, 2-H), 3.12 (q, J = 7.5 Hz, 0.6H, 3-H), 2.83 (q, J = 6.6 Hz, 0.4H,
3-H), 2.49 – 2.33 (m, 6H, 2’-H, 2’’-H, 6’’-H), 1.92 – 1.70 (m, 2H, 1’-H), 1.23 (d, J = 6.2 Hz, 1.2H,
13
CH3), 1.12 (d, J = 6.5 Hz, 1.8H, CH3); C NMR (101 MHz, CD2Cl2): δ (ppm) = 153.7 (C-5), 153.7
(C-5), 145.0 (C-7a), 144.8 (C-7a), 134.6 (C-3a), 134.5 (C-3a), 112.5 (C-6), 112.2 (C-4), 112.0 (C-6),
111.7 (C-4), 109.9 (C-7), 109.9 (C-7), 67.4 (C-3’’, C-5’’), 61.7 (C-2), 59.3 (C-2), 57.3 (C-1’), 56.8 (C-
1’), 56.2 (O-CH3), 56.2 (O-CH3), 54.3 (C-2’’, C-6’’), 48.6 (C-3), 43.7 (C-3), 31.3 (C-1’), 25.1 (C-1’),
22.4 (CH3), 16.3 (CH3). IR (ATR): ν̃
[cm-1] = 2956, 1489, 1270, 1230, 1116, 1034, 915; HR-MS (EI):
calcd. for C16H24N2O2: 276.1838, found 276.1832; purity (HPLC): >96 %.
(2,3-Dichlorophenyl)(5-methoxy-2-methyl-3-(2-morpholinoethyl)indolin-1-yl)methanone (ML-
SI1) (racemic mixture of diastereomers)
EY19 (38 mg, 0.14 mmol) was dissolved in 5 mL dichloromethane and cooled to 0°C. Triethylamine
(20 µL, 0.14 mmol) and 2,3-dichlorobenzoyl chloride (35 mg, 0.17 mmol) were added portionwise.
The reaction mixture was stirred at room for 12 hours, then poured into brine (30 mL) and extracted
with dichloromethane (3 x, 20 mL). The combined organic layers were dried by filtration and the
solvent was removed in vacuo. Purification by FCC using methylene chloride/methanol (9:1) as an
eluent gave the product (41 mg) as a yellow solid (yield: 67 %). ML-SI1 was obtained as a 55:45
1
13
mixture of cis/trans isomers. In the H- and C NMR spectra we saw a set of signals, caused by the
1
diastereomers and their rotamers. mp: 68-69 °C. H NMR (500 MHz, CD2Cl2): δ (ppm) = 8.21-8.02
(m, 0.58H), 7.64-7.54 (m, 1H), 7,51-7.21 (m, 2H), 7,16-7.09 (m, 0.16H), 6.87-6.74 (m, 1.7H), 6.42-
6.32 (m, 0.4H), 5.71-5.58 (m, 0.4H), 5.15-5.07 (m, 0.23H), 4.75-4.68 (m, 0.12 H), 4.12-4.05 (m,
0.28H), 3.86-3.49 (m, 8H), 2.09-2.79 (m, 0.51H), 2.61-2.30 (m, 8H), 2.21-2.08 (m, 0.77H), 1.88-1.59
(m, 2.52H), 1.36 (dd, J1 = 16.1 Hz, J2 = 6.5 Hz, 0.49H), 1.21 (dd, J1 = 15.1 Hz, J2 = 6.5 Hz, 0.79H),
1.08 (d, J = 6.4 Hz, 0.97H), 0.95 (d, J = 6.4 Hz, 0.83H); 13C NMR (126 MHz, CD2Cl2): δ (ppm) =
164.4, 164,0 163.7, 163.5, 163.2, 163.2, 162.7, 157.9, 157.6, 157.3, 157.2, 157.0, 156.9, 139.3,
139.2, 139.2, 139.0, 138.9, 138.9, 138.7, 138.6, 138.4, 137.7, 137.6, 137.3, 134.8, 134.5, 134.3, 134.3,
134.2, 134.0, 133.9, 133.7, 133.4, 133.3, 133.0, 133.0, 131.8, 131.7, 131.7, 131.4, 130.2, 130.1, 129.9,
129.1, 129.0, 128.9, 128.9, 128.8, 128.8, 128.7, 128.7, 128.6, 128.4, 127.8, 127.6, 127.2, 127.0, 126.3,
126.0, 126.0, 119.1, 119.0, 115.3, 115.1, 115.1, 114.8, 112.9, 112.8, 112.8, 112.3, 112.3, 112.0, 111.9,
111.9, 111.7, 110.9, 110.8, 110.4, 67.3, 67.3, 67.3, 64.1, 63.0, 62.4, 62.2, 62.0, 61.2, 60.5, 60.1, 57.6,
57.5, 57.5, 56.8, 56.5, 56.3, 56.2, 56.0, 56.0, 55.9, 55.9, 55.9, 55.8, 54.3, 54.3, 54.2, 54.2, 47.7, 46.1,
45.8, 43.5, 42.9, 42.4, 41.9, 41.5, 33.1, 33.0, 32.9, 32.6, 30.1, 24.3, 24.3, 21.9, 21.3, 20.0, 19.6, 15.2,
14.8, 13.4, 12.8; IR (ATR): ν̃
[cm-1] = 1636, 1485, 1398, 1277, 1115, 1029, 795, 686, 630, 617, 607,
596, 581, 570; HR-MS (ESI): calcd. for C23H26Cl2N2O3: 449.1399 [M+H]+, found 449.1389; purity
(HPLC): >96 %.

7-(Phenylsulfonyl)-7-azabicyclo[4.1.0]heptane (7)
Following general procedure A, chloramine B hydrate (6.9 g, 30 mmol), cyclohexene (3.0 mL,
30 mmol) and NBS (1.1 g, 6.0 mmol) were reacted in 150 mL acetonitrile. Purification by FCC gave 7
as a colourless oil (yield: 45%). 1H NMR (400 MHz, CDCl3): δ (ppm) = 7.98 – 7.92 (m, 2H, 2‘H, 6‘-
H), 7.65 – 7.59 (m, 1H, 4‘-H), 7.57 – 7.51 (m, 2H, 3‘-H, 5‘-H), 3.01 (p, J = 1.6 Hz, 2H, 1-H, 6-H),
1.79 (tt, J = 6.3, 1.3 Hz, 4H, 2-H, 5-H), 1.46 – 1.35 (m, 2H, 3-H, 4-H), 1.28 – 1.17 (m, 2H, 3-H, 4-H);
13C NMR (101 MHz, CDCl3): δ (ppm) = 139.1 (C-1‘), 133.3 (C-4‘), 129.1 (C-3‘,C-5‘), 127.7 (C-2‘, C-
6‘), 40.1 (C-1, C-6), 22.9 (C-2, C-5), 19.5 (C-3, C-4); IR (ATR): ν̃
[cm-1] = 2938, 2862, 1446, 1318,
1153, 1091; HR-MS (ESI): calcd. for C12H16NO2S+: 238.0896 [M+H]+, found 238.0897; purity
(HPLC): >96 %.
trans-N-(2-(4-(2-Methoxyphenyl)piperazin-1-yl)cyclohexyl)benzenesulfonamide (trans-ML-SI3)
Following general procedure B, 7-(phenylsulfonyl)-7-azabicyclo[4.1.0]heptane (7, 122 mg,
0.514 mmol) and 1-(2-methoxyphenyl)piperazine (8, 119 mg, 0.617 mmol) were reacted to give trans-
ML-SI3 as a white solid (yield: 63 %). mp: 150-151 °C. 1H NMR (500 MHz, CDCl3): δ (ppm) = 7.94
– 7.89 (m, 2H, 2-H, 6-H), 7.60 – 7.56 (m, 1H, 4-H), 7.53 (ddt, J = 8.4, 6.6, 1.5 Hz, 2H, 3-H, 5-H), 7.02
(td, J = 7.6, 1.7 Hz, 1H, 4‘‘‘-H), 6.95 (td, J = 7.5, 1.5 Hz, 1H, 5‘‘‘-H), 6.89 (dd, J = 7.8, 1.7 Hz, 1H,
6‘‘‘-H), 6.85 (dd, J = 8.0, 1.4 Hz, 1H, 3‘‘‘-H), 6.11 (s, 1H, NH), 3.83 (s, 3H, OCH3), 3.12 – 2.10 (m,
13
11H), 1.91 – 1.64 (m, 3H, CH2 cyclohexane), 1.35 – 1.04 (m, 4H, CH2 cyclohexane). C NMR (126
MHz, CDCl3): δ (ppm) = 152.3 (C-2‘‘‘), 141.2 (C-1‘‘‘), 139.9 (C-1), 132.7 (C-4), 129.1 (C-3, C-5),
127.4 (C-2, C-6), 123.3 (C-4‘‘‘), 121.2 (C-5‘‘‘), 118.4 (C-6‘‘‘), 111.3 (C-3‘‘‘), 66.8 (C-2‘), 55.5
(OCH3), 53.5 (C-1‘), 51.2 (CH2 piperazine), 33.0 (CH2 cyclohexane), 25.5 (CH2 cyclohexane), 24.3
(CH2 cyclohexane), 23.2 (CH2 cyclohexane). IR (ATR): ν̃
[cm-1] = 3171, 2932, 2809, 1497, 1447,
1339, 1307, 1021, 757, 725, 690. HR-MS (ESI): calcd. for C23H32N3O3S+: 430.2159 [M+H]+; found:
430.2159. purity (HPLC): >96 %.
trans-2-(4-(2-Methoxyphenyl)piperazin-1-yl)cyclohexan-1-ol (10)
Cyclohexene oxide (0.43 mL, 4.2 mmol) and 1-(2-methoxyphenyl)piperazine (8, 0.41 g, 2.1 mmol)
were suspended in water (10 mL), heated to reflux for 1 h. After cooling to rt the water was decanted
off and the precipitate recrystallized from ethanol to afford 10 as an off-white solid (yield: 85 %). mp:
1
109-110 °C. H NMR (400 MHz, CDCl3): δ (ppm) = 7.04 – 6.83 (m, 4H; Ar-H), 4.04 (s, 1H, OH),
3.86 (s, 3H, OCH3), 3.41 (td, J = 9.8, 4.5 Hz, 1H, 2-H), 3.20 – 2.83 (m, 6H, CH2 piperazine), 2.62 (dt,
J = 10.2, 4.7 Hz, 2H, CH2 piperazine), 2.32 – 2.22 (m, 1H, 1-H), 2.18 – 2.10 (m, 1H, CH2
cyclohexane), 1.92 – 1.68 (m, 3H, CH2 cyclohexane), 1.32 – 1.17 (m, 4H, CH2 cyclohexane). 13C NMR
(101 MHz, CDCl3): δ (ppm) = 152.2 (C-2‘‘), 141.3 (C-1‘‘), 123.0 (C-4‘‘), 121.0 (C-5‘‘), 118.3 (C-
6‘‘), 111.1 (C-3‘‘), 70.3 (C-1), 68.6 (C-2), 55.3 (OCH3), 51.3 (CH2 piperazine), 48.6 (CH2 piperazine),
33.3 (CH2 cyclohexane), 25.6 (CH2 cyclohexane), 24.1 (CH2 cyclohexane), 22.5 (CH2 cyclohexane). IR

(ATR): ν
̃
[cm-1] = 3471, 2931, 2838, 2817, 1593, 1499, 1448, 1240, 1121, 1080, 1017, 949, 751, 741.
HR-MS (ESI): calcd. for C17H27N2O2 : 291.2067 [M+H]+; found: 291.2066. Purity (HPLC): >96 %.
+
trans-1-(2-Azidocyclohexyl)-4-(2-methoxyphenyl)piperazine (11)
To a solution of 10 (0.35 g, 1.2 mmol) and triethylamine (0.34 mL, 2.4 mmol) in methylene chloride
(12 mL) was added mesyl chloride (0.19 mL, 2.4 mmol) dropwise at 0 °C. After warming up to rt, the
solution was stirred for 12 h. After addition of 1N NaOH (15 mL) the phases were separated and the
aqueous layer extracted twice with methylene chloride (20 mL). The combined organic phases were
dried over Na2SO4 and the solvent removed in vacuo. The residue was dissolved in dimethyl
formamide (10 mL) and sodium azide (0.39 g, 6.0 mmol) added. The resulting mixture was heated to
reflux for 24 h. After cooling to rt 1N NaOH (50 mL) was added and the aqueous layer extracted with
methylene chloride (3 x 50 mL). The combined organic layers were washed with water (100 mL) and
brine (100 mL), dried over Na2SO4 and the solvent removed in vacuo. Purification by FCC using
petrol ether /ethyl acetate/triethylamine (7:3:0.1) as an eluent gave 11 as a white solid (yield: 55 %).
mp: 77-88 °C. 1H NMR (500 MHz, CDCl3): δ (ppm) = 7.02 – 6.89 (m, 3H, 4‘‘-H, 5‘‘-H, 6‘‘-H), 6.85
(dd, J = 8.0, 1.4 Hz, 1H, 3‘‘-H), 3.86 (s, 3H, OCH3), 3.35 (td, J = 10.6, 4.4 Hz, 1H, 2‘-C), 3.10 (s, 4H;
CH2 piperazine), 2.93 (dt, J = 10.1, 4.7 Hz, 2H, CH2 piperazine), 2.76 – 2.70 (m, 2H, CH2 piperazine),
2.46 (ddd, J = 11.7, 10.3, 3.7 Hz, 1H, 1‘-H), 2.07 – 1.93 (m, 2H, CH2 cyclohexane), 1.84 – 1.66 (m,
2H, CH2 cyclohexane), 1.32 – 1.14 (m, 4H, CH2 cyclohexene). 13C NMR (126 MHz, CDCl3): δ (ppm)
= 152.4 (C-1‘‘), 141.7 (C-1‘‘), 122.9 (C-4‘‘), 121.1 (C-5‘‘), 118.5 (C-6‘‘), 111.1 (C-3‘‘), 68.5 (C-1),
60.8 (C-2), 55.4 (OCH3), 51.0 8 (CH2 piperazine), 48.8 (CH2 piperazine), 32.6 (CH2 cyclohexane),
25.2 (CH2 cyclohexane), 24.9 (CH2 cyclohexane), 24.0 (CH2 cyclohexane). IR (ATR): ν̃
[cm-1] = 2924,
2823, 2091, 1593, 1497, 1446, 1265, 1236, 1143, 1113, 1026, 740. HR-MS (ESI): calcd. for
C17H26N5O+: 316.2132 [M+H]+; found: 316.2130. Purity (HPLC): >96 %.
cis-N-(2-Aminocyclohexyl)benzenesulfonamide (13)
To a solution of cis-1,2-cyclohexanediamine (0.59 g, 5.0 mmol) and triethylamine (2.8 mL, 20 mmol)
in methylene chloride (30 mL) was added benzenesulfonyl chloride (0.64 mL, 5.0 mmol) at 0 °C and
the solution stirred for 10 min. The solution was concentrated in vacuo and the residue taken up in
ethyl acetate. The organic layer was washed with sat. aq. NaHCO3 solution (20 mL), subsequently
HCl conc. aq. (approx.. 1 mL) added and extracted twice with water (20 mL). The combined aqueous
layers were brought to pH 9 with a 50 %NaOH in water solution and extracted three times with ethyl
acetate (30 mL). The combined organic layers were dried over Na2SO4 and the solvent removed in
1
vacuo, to afford 13 as a light brown solid (yield: 81%). mp: 75-76 °C H NMR (400 MHz, CDCl3): δ
(ppm) = 7.93 – 7.85 (m, 2H, 2-H, 6-H), 7.59 – 7.53 (m, 1H, 4-H), 7.53 – 7.47 (m, 2H, 3-H, 5-H), 5.29
(bs, 1H, SO2NH), 3.16 (dt, J = 7.7, 3.8 Hz, 1H, CH cyclohexane), 2.82 (dt, J = 7.3, 3.7 Hz, 1H, CH
cyclohexane), 1.66 – 0.99 (m, 10H, CH2 cyclohexane, NH2). 13C NMR (101 MHz, CDCl3): δ (ppm) =
141.1 (C-1), 132.5 (C-4), 129.1 (C-3, C-5), 127.2 (C-2, C-6), 54.6 (CH cyclohexane), 50.1 (CH

cyclohexane), 31.6 (CH2), 28.6 (CH2), 22.1 (CH2), 21.3 (CH2). IR (ATR): ν̃
[cm-1] = 3375, 2931, 1445,
1307, 1151, 1091, 930, 758. HR-MS (ESI): calcd. for C12H19N2O2S+ : 255,1162 [M+H]+; found:
255.1161. Purity (NMR): >96%.
N,N-Bis(2-hydroxyethyl)-2-methoxyaniline (14)
2-Methoxyaniline (0.37 g, 3.0 mmol) and ethylene oxide (4.6 mL of an approx. 3.3 M solution in
THF, ~15 mmol) in 1N aqueous acetic acid (10 mL) were stirred for 40 h, then the solution poured
into brine (15 mL) and extracted three times with ethyl acetate (30 mL). The combined organic layers
were dried over Na2SO4 and the solvent removed in vacuo. The residue was purified by FCC using
ethyl acetate as an eluent to afford 14 as a colourless oil (yield: 86%). 1H NMR (400 MHz, CDCl3): δ
(ppm) = 7.21 (dd, J = 7.8, 1.7 Hz, 1H, 6-H), 7.16 (ddd, J = 8.2, 7.5, 1.7 Hz, 1H, 4-H), 6.98 (td, J = 7.6,
1.4 Hz, 1H, 3-H), 6.93 (dd, J = 8.2, 1.4 Hz, 1H, 5-H), 3.88 (s, 3H, OCH3), 3.52 – 3.48 (m, 4H, 2
CH2O), 3.23 – 3.07 (m, 6H, 2 CH2N, OH). 13C NMR (101 MHz, CDCl3): δ (ppm) = 155.8 (C-2), 138.2
(C-1), 126.4 (C-4), 125.4 (C-6), 121.9 (C-3), 111.7 (C-5), 59.9 (NCH2), 57.8 (OCH2), 55.8 (OCH3). IR
+
(ATR): ν
̃
[cm-1] = 3359, 2926, 1593,1497, 1236, 1024, 746. HR-MS (ESI): calcd. for C11H18NO3 :
212,1281 [M+H]+; found: 212.1280. Purity (HPLC): >96%.
cis-N-(2-(4-(2-Methoxyphenyl)piperazin-1-yl)cyclohexyl)benzenesulfonamide (cis-ML-SI3)
14 (450 mg, 2.13 mmol) was dissolved in methylene chloride (5 mL) and a solution of thionyl chloride
(0.326 mL, 4.47 mmol) in methylene chloride (4.5 mL) added dropwise. The solution was heated for
1 h at 40 °C, then the solvent and excess of thionyl chloride were removed in vacuo. The residue was
dissolved in ethanol (5 mL), K2CO3 and a solution of compound 13 (1.08 g, 4.26 mmol) in ethanol
(10 mL) added. The reaction mixture was heated to reflux for 72 h and then the solvent removed in
vacuo. The residue was taken up in methylene chloride, the inorganic salts filtered off and the solvent
removed in vacuo. The residue was purified by FCC using petrol ether /ethyl acetate/triethylamine
(6:4:0.1) as an eluent, followed by recrystallisation from ethanol to afford cis-ML-SI3 as a pale yellow
solid (yield: 42 %). mp:129-130 C. 1H NMR (500 MHz, CDCl3): δ (ppm) = 7.95 – 7.86 (m, 2H, 2-H,
6-H), 7.60 – 7.56 (m, 1H, 4-H), 7.52 (ddt, J = 8.4, 6.5, 1.5 Hz, 2H, 3-H, 5-H), 7.01 (td, J = 7.7, 1.7 Hz,
1H, 4’’’-H), 6.94 (td, J = 7.6, 1.5 Hz, 1H, 5’’’-H), 6.86 (ddd, J = 8.5, 7.9, 1.6 Hz, 2H, 6’’’-H, 3’’’-H ),
5.45 (s, 1H, NH), 3.83 (s, 3H, OCH3), 3.34 (q, J = 3.4 Hz, 1H, 1’-H), 2.83 (s, 4H, CH2 piperazine),
2.60 – 2.34 (m, 3H, CH2 piperazine, 6’-H), 2.23 (dt, J = 10.5, 4.8 Hz, 2H, CH2 piperazine), 2.10 (dt, J
= 12.0, 3.9 Hz, 1H, 2’-H), 1.84 – 1.75 (m, 2H, CH2 cyclohexane), 1.64 – 1.55 (m, 1H, CH2
cyclohexane), 1.39 – 1.15 (m, 4H, CH2 cyclohexane). 13C NMR (126 MHz, CDCl3): δ (ppm) = 152.4
(C-2’’’), 141.1 (C-1’’’), 139.6 (C-1), 132.7 (C-4), 129.1 (C-3, C-5), 127.5 (C-2, C-6), 123.3 (C-4’’’),
121.2 (C-5’’’), 118.2 (C-6’’’), 111.3 (C-3’’’), 63.6 (C-2’), 55.5 (OCH3), 51.0 (CH2 piperazine), 49.5
(CH2 piperazine), 49.1 (C-1’), 28.7 (C-6’), 25.0 (CH2 cyclohexane), 24.7 (CH2 cyclohexane), 18.9
(CH2 cyclohexane). IR (ATR): ν
found: 430.2152. Purity (HPLC): >96 %.
̃
[cm-1] =. HR-MS (ESI): calcd. for C23H32N3O3S+: 430.2159 [M+H]+;

7-(4-Methylbenzenesulfonyl)-7-azabicyclo[4.1.0]heptane (15)
Following general procedure A, chloramine T hydrate (1.2 g, 4.8 mmol), cyclohexene (0.5 mL,
4.8 mmol) and NBS (0.17 g, 0.96 mmol) were reacted in 25 mL acetonitrile. Purification by FCC gave
1
15 as a white solid (yield: 34%). mp: 56-57 °C (lit.: 55-57 °C; [29]). H NMR (400 MHz, CDCl3): δ
(ppm) = 7.86 – 7.77 (m, 2H, 2-H, 6-H), 7.35 – 7.29 (m, 2H, 3-H, 5-H), 2.97 (p, J = 1.5 Hz, 2H, 1‘-H,
2‘-H), 2.44 (s, 3H; CH3), 1.81 – 1.74 (m, 4H; 2‘-H, 5‘-H), 1.45 – 1.34 (m, 2H, 3‘-H or 4‘-H), 1.27 –
13
1.16 (m, 2H, 3‘-H or 4‘-H). C NMR (101 MHz, CDCl3): δ (ppm) = 144.1 (C-4), 136.0 (C-1), 129.7
(C-3, C-5), 127.8 (C-2, C-6), 39.9 (C-1‘, C-2‘), 22.9 (C-3‘, C-6‘), 21.8 (CH3), 19.6 (C-4‘, C-5‘); IR
(ATR): ν̃
[cm-1] = 2946, 2864, 1315, 1288, 1155, 1087, 961, 921,849, 721, 664; HR-MS (ESI): calcd.
for C13H18NO2S+: 252.1053 [M+H]+, found 252.1053; purity (HPLC): >96 %.
trans-N-(2-Hydroxycyclohexyl)thiophene-2-sulfonamide
Cyclohexene oxide (0.30 mL, 3.0 mmol), thiophene-2-sulfonamide (0.59 mg, 3.6 mmol),
benzyltriethylammonium chloride (68 mg, 0.30 mmol) and K2CO3 (50 mg, 0.30 mmol) were
suspended in dioxane (2 mL). The reaction mixture was heated to 90 °C and stirred for 16 h. After
cooling to rt the mixture was diluted with methylene chloride, the solids filtered off and the solvent
removed in vacuo. The residue was purified by FCC using diethyl ether/ petrol ether as an eluent to
1
give the target compound as a white solid (yield: 100 %). mp: 110-111 °C. H NMR (500 MHz,
CDCl3): δ (ppm) = 7.66 (dd, J = 3.7, 1.3 Hz, 1H, 3-H), 7.61 (dd, J = 5.0, 1.3 Hz, 1H, 5-H), 7.10 (dd, J
= 5.0, 3.7 Hz, 1H, 4-H), 4.79 (d, J = 6.8 Hz, 1H, NH), 3.38 – 3.29 (m, 1H, 2‘-H), 3.00 – 2.93 (m, 1H,
1‘-H), 2.35 (d, J = 3.5 Hz, 1H, OH), 2.07 – 2.00 (m, 1H, CH2 cyclohexane), 1.90 – 1.81 (m, 1H, CH2
cyclohexane), 1.73 – 1.60 (m, 2H, CH2 cyclohexane), 1.30 – 1.13 (m, 4H, CH2 cyclohexane).13C NMR
(126 MHz,CDCl3): δ (ppm) = 141.3 (C-2), 132.7 (C-3), 132.3 (C-5), 127.6 (C-4), 73.4 (C-2‘), 60.3 (C-
1‘), 33.7 (C-3‘), 32.0 (C-6‘), 24.8 (C-4‘ or C-5‘), 24.0 (C-4‘ or C-5‘). IR (ATR): ν̃
[cm-1] = 3487,
3141, 2942, 2858, 1449, 1402, 1311, 1150, 1138, 1062, 727, 672. HR-MS (ESI): calcd. for
-
C10H14NO3S2 : 260.0421 [M-H]-, found: 260.0419. purity (HPLC): >96 %.
7-(Thiophen-2-ylsulfonyl)-7-azabicyclo[4.1.0]heptane (16)
p-Toluenesulfonyl chloride (2.9 g, 15 mmol) was suspended in methylene chloride (15 mL) and
pyridine (1.2 mL, 15 mmol) added dropwise at 0 °C. The resulting solution was stirred for 20 min at
0 °C, then a solution of trans-N-(2-hydroxycyclohexyl)thiophene-2-sulfonamide in methylene chloride
(10 mL) added dropwise. After stirring at 0 °C for 20 min, the solution was heated to 40 °C and stirred
at this temperature for 72 h. After cooling to rt, the mixture was washed with 2 N HCl and brine, dried
over Na2SO4 and the solvent removed in vacuo. The residue was purified by FCC and directly used for
the next step. The crude tosylate was dissolved in acetonitrile (20 mL), K2CO3 (0.70 g, 1.7 mmol) was
added, and the mixture heated to reflux for 4 h. Acetonitrile was removed in vacuo, the residue taken
up in methylene chloride, the organic layer washed with water and brine, dried over Na2SO4 and the

solvent removed in vacuo. Purification by FCC using petrol ether/ethyl acetate as an eluent gave 16 as
1
a white solid (yield: 59 %). mp: 67-68 °C (lit. 71-71.5°C) [33]. H NMR (400 MHz, CDCl3-d): δ
(ppm) = 7.70 (dd, J = 3.7, 1.4 Hz, 1H, 3-H), 7.66 (dd, J = 5.0, 1.4 Hz, 1H, 5-H), 7.12 (dd, J = 5.0, 3.8
Hz, 1H, 4-H), 3.02 (tt, J = 1.9, 1.1 Hz, 2H, 1‘-H, 2‘-H ), 1.90 – 1.78 (m, 4H; 3‘-H, 6‘-H), 1.49 – 1.39
(m, 2H, 4‘-H or 5‘-H), 1.29 – 1.19 (m, 2H, 4‘-H or 5‘-H). 13C NMR (101 MHz, CDCl3-d): δ (ppm) =
139.1 (C-2‘), 133.2 (C-3), 133.0 (C-5), 127.4 (C-4), 40.8 (C-1‘, C-2‘), 22.9 (C-3‘, C-6‘), 19.5 (C-4‘,
C-5‘). IR (ATR): ν
̃
[cm-1] = 3121, 2949, 1401, 1312, 1143, 1016, 964, 923, 849, 737, 714. HR-MS
+
(ESI): calcd. for C10H14NO2S2 : 244.0460 [M+H]+; found: 244.0460. Purity (NMR): >96 %.
trans-N-(2-(4-(2-Methoxyphenyl)piperazin-1-yl)cyclohexyl)-4-methylbenzenesulfonamide (17)
Following general procedure B, 7-(4-methylbenzenesulfonyl)-7-azabicyclo[4.1.0]heptane (15, 139 mg,
0.551 mmol) and 1-(2-methoxyphenyl)piperazine (8, 127 mg, 0.662 mmol) were reacted to give 17
(yield: 63 %). mp: 132-133 °C. 1H NMR (400 MHz, CDCl3): δ (ppm) = 7.81 – 7.75 (m, 2H, 3-H, H-5),
7.34 – 7.29 (m, 2H; 2-H, 6-H), 7.02 (ddd, J = 7.9, 7.3, 1.8 Hz, 1H, H-4‘‘‘), 6.95 (td, J = 7.6, 1.5 Hz,
1H, H-5‘‘‘), 6.90 (dd, J = 7.8, 1.8 Hz, 1H, H-3‘‘‘), 6.86 (dd, J = 8.0, 1.5 Hz, 1H, H-6‘‘‘), 6.08 (s, 1H,
NH), 3.84 (s, 3H, OCH3), 3.06 – 2.64 (m, 5H), 2.52 – 2.16 (m, 9H), 1.91 – 1.64 (m, 3H, CH2
cyclohexane), 1.35 – 1.03 (m, 4H, CH2 cyclohexane). 13C NMR (101 MHz, CDCl3): δ (ppm) = 152.3
(C-2‘‘‘), 143.4 (C-4), 141.2 (C-1‘‘‘), 136.9 (C-1), 129.7 (C-2, C-6), 127.4 (C-3, C-5), 123.3 (C-4‘‘‘),
121.2 (C-5‘‘‘), 118.4 (C-3‘‘‘), 111.2 (C-6‘‘‘), 66.8 (C-2‘), 55.5 (OCH3), 53.5 (C-1‘), 51.2 (CH2
piperazine), 33.0 (CH2 cyclohexane), 25.5 (CH2 cyclohexane), 24.3 (CH2 cyclohexane), 23.2 (CH2
cyclohexane), 21.7 (CH3). IR (ATR): ν̃
[cm-1] = 3183, 2933, 2809, 1498, 1448, 1339, 1239, 1167,
1022, 753, 704. HR-MS (ESI): calcd. for C24H34N3O3S+: 444.2315 [M+H]+; found: 444.2316. purity
(HPLC): >96 %.
trans-N-(2-(4-(2-Methoxyphenyl)piperazin-1-yl)cyclohexyl)thiophene-2-sulfonamide (18)
Following general procedure B, 7-(thiophen-2-ylsulfonyl)-7-azabicyclo[4.1.0]heptane (16, 125 mg,
0.514 mmol) and 1-(2-methoxyphenyl)piperazine (8, 116 mg, 0.603 mmol) were reacted to give 18 as
1
an off-white solid (yield: 78 %). mp: 149-150 °C. H NMR (400 MHz, CDCl3): δ (ppm) = 7.62 (dd, J
= 3.7, 1.3 Hz, 1H, 3-H), 7.58 (dd, J = 5.0, 1.3 Hz, 1H, 5-H), 7.11 (dd, J = 5.0, 3.7 Hz, 1H, 4-H), 7.02
(ddd, J = 8.0, 7.2, 1.9 Hz, 1H,4‘‘‘-H), 6.94 (td, J = 7.5, 1.4 Hz, 1H, 5‘‘‘-H), 6.89 (dd, J = 7.8, 1.9 Hz,
1H, 6‘‘‘-H), 6.85 (dd, J = 8.0, 1.5 Hz, 1H, 3‘‘‘-H), 6.21 (s, 1H; NH), 3.84 (s, 3H; OCH3), 3.06 – 2.21
(m, 11H), 1.96 – 1.66 (m, 3H, CH2 cyclohexane), 1.39 – 1.11 (m, 4H, CH2 cyclohexane). 13C NMR
(101 MHz, CDCl3): δ (ppm) = 152.3 (C-2‘‘‘), 141.2 (C-1‘‘‘), 140.9 (C-1), 132.0 (C-3), 131.7 (C-5),
127.6 (C-4), 123.3 (C-4‘‘‘), 121.2 (C-5‘‘‘), 118.4 (C-6‘‘‘), 111.2 (C-3‘‘‘), 66.7 (C-2‘), 55.5 (OCH3),
54.0 (C-1‘), 51.2 (CH2 piperazine), 33.0 (C-6‘), 25.5 (CH2 cyclohexane), 24.4 (CH2 cyclohexane), 23.2
(CH2 cyclohexane). IR (ATR): ν
̃
[cm-1] = 3159, 2936, 2873, 2831, 1344, 1239, 1227, 1158, 1141,
+
1032, 1016, 729, 706. HR-MS (ESI): calcd. for C21H30N3O3S2 : 436.1723 [M+H]+; found: 436.1723.
purity (HPLC) >96 %.

trans-N-(2-(4-(2-Methoxyphenyl)piperazin-1-yl)cyclohexyl)benzamide (20)
Azide 11 (0.43 g, 1.5 mmol) was dissolved in ethanol (30 mL), 10 % Pd/C (~50 mg) added, and the
mixture stirred under hydrogen atmosphere for 72 h. The catalyst was removed by filtration over a pad
of celite, and the solvent removed in vacuo. A part of the crude product (0.11 g, 0.39 mmol) was
dissolved in pyridine (3 mL), cooled to 0 °C and benzoyl chloride (90 µL, 0.76 mmol) added. The
solution was stirred and warmed up to rt over 3 h. Satd. aqueous NaHCO3 solution (15 mL) and ethyl
acetate (20 mL) were added and the phases separated. The organic layer was washed with water
(15 mL) and brine (15 mL), dried over Na2SO4 and the solvent removed in vacuo. The crude product
1
was recrystallized from ethanol to afford 20 as a pale yellow solid (yield: 42 %). mp: 144-145 °C. H
NMR (400 MHz, CDCl3) δ (ppm) = 7.83 – 7.78 (m, 2H, 2-H, 6-H), 7.50 – 7.45 (m, 1H, 4-H), 7.45 –
7.39 (m, 2H, 3-H, 5-H), 7.04 (d, J = 4.1 Hz, 1H, NH), 7.01 – 6.95 (m, 1H, 4’’’-H), 6.91 – 6.82 (m, 3H,
6’’’-H, 5’’’-H, 3’’’-H), 3.85 (s, 3H, OCH3), 3.70 (tt, J = 10.8, 4.0 Hz, 1H, 1’-H), 3.14 – 2.86 (m, 6H,
CH2 piperazine), 2.73 (d, J = 12.6 Hz, 1H, 6’-H), 2.62 (dt, J = 10.3, 4.5 Hz, 2H, CH2 piperazine), 2.49
(td, J = 11.0, 3.1 Hz, 1H, 2’-H), 2.02 (d, J = 11.4 Hz, 1H, CH2 cyclohexane), 1.88 (d, J = 11.4 Hz, 1H,
CH2 cyclohexane), 1.74 (d, J = 13.4 Hz, 1H, CH2 cyclohexane), 1.48 – 1.12 (m, 4H, CH2
13
cyclohexane). C NMR (101 MHz, CDCl3) δ (ppm) = 167.9 (OCNH), 152.3 (C-2’’), 141.4 (C-1’’’),
135.5 (C-1), 131.3 (C-4), 128.7 (C-3, C-5), 127.0 (C-2, C-6), 123.1 (C-4’’’), 121.0 (C-5’’’), 118.3 (C-
6’’’), 111.3 (C-3’’’), 67.5 (C-2’), 55.5 (OCH3), 51.6 (CH2 piperazine), 51.3 (C-1’), 48.2 (CH2
piperazine), 32.8 (CH2 cyclohexane), 25.8 (CH2 cyclohexane), 24.8 (CH2 cyclohexane), 23.5 (CH2
cyclohexane). IR (ATR): ν̃
[cm-1] = 3310, 2923, 2818, 1626, 1542, 1503, 1240. HR-MS (ESI): calcd.
+
for C24H32N3O2 : 394,2489 [M+H]+; found: 394.2483. Purity (HPLC): >96%.
trans-N-(2-(4-Phenylpiperazin-1-yl)cyclohexyl)benzenesulfonamide (21)
Following general procedure B, 7-(phenylsulfonyl)-7-azabicyclo[4.1.0]heptane (7, 125 mg,
0.527 mmol) and 1-phenylpiperazine (92 µL, 0.60 mmol) were reacted to give 21 as a white solid
(yield: 77 %). mp: 154-155 °C. 1H NMR (500 MHz, CDCl3): δ (ppm) = 7.96 – 7.91 (m, 2H, 2-H, 6-H),
7.65 – 7.61 (m, 1H, 4-H), 7.58 – 7.53 (m, 2H, 3-H, 5-H), 7.32 – 7.26 (m, 2H, 3‘‘‘-H, 5‘‘‘-H), 6.93 –
6.87 (m, 3H, 2‘‘‘-H, 4‘‘‘-H, 6‘‘‘-H), 6.04 (s, 1H, NH), 3.07 – 2.92 (m, 4H, CH2 piperazine), 2.78 (td, J
= 10.6, 4.1 Hz, 1H, 1‘-H), 2.55 – 2.50 (m, 1H, 6‘-H), 2.42 – 2.26 (m, 5H), 1.90 – 1.69 (m, 3H, CH2
cyclohexane), 1.37 – 1.05 (m, 4H, CH2 cyclohexane). 13C NMR (126 MHz, CDCl3): δ (ppm) = 151.2
(C-1‘‘‘), 139.8 (C-1), 132.6 (C-4), 129.2 (C-3‘‘‘, C-5‘‘‘), 129.1 (C-3, C-5), 127.2 (C-2, C-6), 120.1
(C-4‘‘‘), 116.3 (C-2‘‘‘, C-6‘‘‘), 66.6 (C-2‘), 53.4 (C-1‘), 49.7 (CH2 piperazine), 47.6 (CH2 piperazine),
32.9 (C-6‘), 25.2 (CH2 cyclohexane), 24.2 (CH2 cyclohexane), 22.8 (CH2 cyclohexane). IR (ATR): ν
[cm-1] = 3183, 2935, 2852, 1600, 1504, 1449, 1159, 754, 724, 689. HR-MS (ESI): calcd. for
C22H30N3O2S+: 400.2053 [M+H]+; found: 400.2053. purity (HPLC): >96 %.
̃
trans-N-(2-(4-(3-(Trifluoromethyl)phenyl)piperazin-1-yl)cyclohexyl)benzenesulfonamide (22)

Following general procedure B, 7-(phenylsulfonyl)-7-azabicyclo[4.1.0]heptane (7, 120 mg,
0.506 mmol) and 1-(3-(trifluoromethyl)phenyl)piperazine (140 mg, 0.608 mmol) were reacted to give
22 as a white solid (yield: 77 %). mp: 111-112 °C. 1H NMR (500 MHz, CDCl3): δ (ppm) = 7.93 – 7.89
(m, 2H, 2-H, 6-H), 7.64 – 7.59 (m, 1H, H-4), 7.57 – 7.51 (m, 2H, 3-H, 5-H), 7.37 – 7.32 (m, 1H, 5‘‘-
H), 7.11 – 7.07 (m, 1H, 4‘‘-H), 7.05 – 7.02 (m, 1H, 2‘‘-H), 6.99 (dd, J = 8.3, 2.6 Hz, 1H, 6‘‘-H), 5.95
(s, 1H, NH), 3.08 – 2.92 (m, 4H), 2.80 (td, J = 10.5, 4.1 Hz, 1H; 1‘-H), 2.51 – 2.44 (m, 1H, 6‘-H), 2.41
– 2.25 (m, 5H), 1.87 – 1.65 (m, 3H, CH2 cyclohexane), 1.34 – 1.04 (m, 4H; CH2 cyclohexane). 13C
NMR (126 MHz, CDCl3): δ (ppm) = 151.2 (C-1‘‘), 139.9 (C-1), 132.7 (C-4), 131.5 (q, J = 31.7 Hz, C-
3‘‘) , 129.6 (C-5‘‘), 129.1 (C-3, C-5), 127.2 (C-2, C-6), 124.3 (q, J = 272.4 Hz, CF3), 118.9 (C-6‘‘),
116.2 (q, J = 3.8 Hz, C-4‘‘), 112.5 (q, J = 3.9 Hz, C-2‘‘), 66.58 (C-2‘), 53.4 (C-1‘), 49.2 (CH2
piperazine), 47.4 (CH2 piperazine), 33.0 (CH2 cyclohexane), 25.2 (CH2 cyclohexane), 24.1 (CH2
cyclohexane), 22.9 (CH2 cyclohexane). IR (ATR): ν̃
[cm-1] = 2939, 1446, 1346, 1308, 1156, 1119,
1092, 756, 729, 689. HR-MS (ESI): calcd. for C23H29F3N3O2S+: 468.1927 [M+H]+; found: 468.1928.
purity (HPLC): >96 %.
trans-N-(2-(4-(4-Methylbenzyl)piperazin-1-yl)cyclohexyl)benzenesulfonamide (23)
Following general procedure B, 7-(phenylsulfonyl)-7-azabicyclo[4.1.0]heptane (7, 125 mg,
0.527 mmol) and 1-(4-methylbenzyl)piperazine (117 mg, 0.615 mmol) were reacted to give 23 as a
pale yellow solid (yield: 49 %). mp: 97-98 °C. 1H NMR (400 MHz, CDCl3): δ (ppm) = 7.90 – 7.86 (m,
2H, 2-H, 6-H), 7.58 – 7.53 (m, 1H, 4-H), 7.52 – 7.47 (m, 2H, 3-H, 5-H), 7.18 – 7.11 (m, 4H, Ar-H),
6.06 (s, 1H, NH), 3.42 (s, 2H, 1‘‘‘-H), 2.65 (td, J = 10.5, 4.0 Hz, 1H, 1‘-H), 2.53 – 1.87 (m, 13H), 1.81
– 1.64 (m, 3H; CH2 cyclohexane), 1.35 – 0.90 (m, 4H, CH2 cyclohexane). 13C NMR (101 MHz,
CDCl3): δ (ppm) = 139.8 (C-1), 136.9 (C-4‘‘‘‘), 134.9 (C-1‘‘‘‘), 132.6 (C-4), 129.3 (C-3, C-5), 129.1
(C-Ar), 127.4 (C-2, C-6), 66.6 (C-2‘), 62.9 (C-1‘‘‘), 53.5 (C-1‘, CH2 piperazine), 32.9 (C-6‘), 25.4
(CH2 cyclohexane), 24.3 (CH2 cyclohexane), 23.0 (CH2 cyclohexane), 21.3 (CH3). IR (ATR): ν̃
[cm-1]
= 3204, 2929, 2817, 1448, 1401, 1332, 1317, 1169, 1098, 1010, 758. HR-MS (ESI): calcd. for
C24H34N3O2S+: 428.2366 [M+H]+; found: 428.2366. purity (HPLC): >96 %.
trans-N-(2-(4-Phenethylpiperazin-1-yl)cyclohexyl)benzenesulfonamide (24)
Following general procedure B, 7-(phenylsulfonyl)-7-azabicyclo[4.1.0]heptane (7, 130 mg,
0.548 mmol) and 1-phenethylpiperazine (121 mg, 0.636 mmol) were reacted to give 24 as an off-white
solid (yield: 45 %). mp: 92-93 °C. 1H NMR (500 MHz, CDCl3): δ (ppm) = 7.92 – 7.87 (m, 2H, 2-H, 6-
H), 7.59 – 7.54 (m, 1H; 4-H), 7.52 – 7.48 (m, 2H, 3-H, 5-H), 7.32 – 7.27 (m, 2H, 3‘‘‘‘-H, 5‘‘‘‘-H),
7.23 – 7.18 (m, 3H, 2‘‘‘‘-H, 4‘‘‘‘-H, 6‘‘‘‘-H), 6.04 (s, 1H, NH), 2.83 – 2.09 (m, 15H), 1.84 – 1.64 (m,
3H, CH2 cyclohexane), 1.31 – 1.00 (m, 4H, CH2 cyclohexane). 13C NMR (126 MHz, CDCl3): δ (ppm)
= 140.2 (C-1‘‘‘‘), 139.7 (C-1), 132.5 (C-4), 129.0 (C-3, C-5), 128.7 (C-3‘‘‘‘, C-5‘‘‘‘), 128.5 (C-2‘‘‘‘,
C-6‘‘‘‘), 127.2 (C-2, C-6), 126.1 (C-4‘‘‘‘), 66.4 (C-2‘), 60.4 (C-1‘‘‘), 53.6 (CH2 piperazine), 53.4 (C-
1‘), 33.7 (C-2‘‘‘), 32.8 (C-6‘), 25.3 (CH2 cyclohexane), 24.2 (CH2 cyclohexane), 22.8 (CH2

cyclohexane). IR (ATR): ν̃
[cm-1] = 3578, 3168, 2827, 1645, 1454, 1336, 1310, 1163, 1089, 1005, 762,
723. HR-MS (ESI): calcd. for C24H34N3O2S+: 428.2366 [M+H]+; found: 428.2366. purity (HPLC):
>96 %.
trans-N-(2-(4-Methylpiperazin-1-yl)cyclohexyl)benzenesulfonamide (25)
Following general procedure B, 7-(phenylsulfonyl)-7-azabicyclo[4.1.0]heptane (7, 123 mg,
0.518 mmol) and 1-methylpiperazine (69 µL, 0.62 mmol) reacted and the crude product purified by
FCC using methylene chloride/ methanol /triethylamine as an eluent to give 25as a white solid (yield:
57 %). mp: 106-107 °C. 1H NMR (500 MHz, CDCl3): δ (ppm) = 7.92 – 7.88 (m, 2H, 3-H, 5-H), 7.61 –
7.55 (m, 1H, 4-H), 7.52 (ddt, J = 8.4, 6.6, 1.4 Hz, 2H, 2-H, 6-H), 6.00 (s, 1H, NH), 2.68 (td, J = 10.6,
4.1 Hz, 1H, 1‘-H), 2.49 – 2.04 (m, 12H), 1.82 – 1.63 (m, 3H, CH2), 1.30 – 0.99 (m, 5H, CH2). 13C
NMR (126 MHz, CDCl3): δ (ppm) = 139.9 (C-1), 132.7 (C-4), 129.1 (C-2,-6), 127.4 (C-3,-5), 66.6 (C-
1‘), 55.5 (CH2 piperazine), 53.5 (C-2‘), 46.1 (CH3), 32.9 (CH2 cyclohexane), 25.4 (CH2 cyclohexane),
24.3 (CH2 cyclohexane), 22.9 (CH2 cyclohexane). IR (ATR): ν̃
[cm-1] = 2923, 2819, 1448, 1162, 757,
719, 691. HR-MS (ESI): calcd. for C17H28N3O2S+: 338.1897 [M+H]+; found 338.1898. purity (HPLC):
>96 %.
trans-N-(2-(4-(2-Methoxyphenyl)piperidin-1-yl)cyclohexyl)benzenesulfonamide (26)
Following general procedure B, 7-(phenylsulfonyl)-7-azabicyclo[4.1.0]heptane (7, 121 mg,
0.510 mmol) and 4-(2-methoxyphenyl)piperidine (120 mg, 0.609 mmol) were reacted to give 26 as a
1
white solid (yield: 85 %). mp: 131-132 °C. H NMR (400 MHz, CDCl3): δ (ppm) = 7.94 – 7.88 (m,
2H, 2-H, 6-H), 7.57 – 7.47 (m, 3H, 3-H, 4-H, 5-H), 7.23 – 7.15 (m, 2H, 4‘‘‘-H, 6‘‘‘-H), 6.98 (td, J =
7.5, 1.1 Hz, 1H, 5‘‘-H), 6.86 (dd, J = 8.2, 1.1 Hz, 1H; 3‘‘‘-H), 6.21 (s, 1H, NH), 3.81 (s, 3H, OCH3),
2.79 (tt, J = 12.0, 3.7 Hz, 1H,4‘‘-H), 2.72 – 2.57 (m, 3H), 2.55 – 2.46 (m, 1H, 6‘-H), 2.27 – 2.18 (m,
1H, 2‘-H), 1.97 (td, J = 11.5, 2.5 Hz, 1H, CH2 piperidine), 1.88 – 1.63 (m, 6H), 1.51 – 1.44 (m, 1H, 6‘-
H), 1.35 – 0.99 (m, 5H). 13C NMR (101 MHz, CDCl3): δ (ppm) = 156.9 (C-2‘‘‘), 139.8 (C-1), 134.2
(C-1‘‘‘), 132.6 (C-4), 129.1 (C-3, C-5), 127.4 (C-2, C-6), 127.1 (C-4‘‘‘oder C-6‘‘‘), 126.7 (C-4‘‘‘oder
C-6‘‘‘), 120.9 (C-5‘‘‘), 110.5 (C-3‘‘‘), 67.2 (C-2‘), 55.5 (OCH3), 53.7 (C-1‘), 53.1 (CH2 piperidine),
44.9 (CH2 piperidine), 35.5 (C-4‘‘), 33.0 (C-6‘), 32.7 (CH2 piperidine), 32.7 (CH2 piperidine), 25.5
(CH2 cyclohexane), 24.4 (CH2 cyclohexane), 22.9 (CH2 cyclohexane). IR (ATR): ν̃
[cm-1] = 3167,
2935, 2839, 1493, 1336, 1230, 1163, 1092, 1032, 757, 691. HR-MS (ESI): calcd. for C24H33N2O3S+:
429.2206 [M+H]+; found: 429.2205. purity (HPLC): >96 %.
trans-N-(2-(4-Phenylpiperidin-1-yl)cyclohexyl)benzenesulfonamide (27)
Following general procedure B, 7-(phenylsulfonyl)-7-azabicyclo[4.1.0]heptane (7, 120 mg,
0.506 mmol) and 4-phenylpiperidine (99 mg, 0.61 mmol) were reacted to give 27 as white solid (yield:
72 %). mp: 129-130 °C. 1H NMR (500 MHz, CDCl3): δ (ppm) = 7.96 – 7.88 (m, 2H, 2-H, 6-H), 7.58 –
7.48 (m, 3H, 3-H, 4-H, 5-H), 7.37 – 7.29 (m, 2H, 3‘‘‘-H, 5‘‘‘-H ), 7.24 – 7.14 (m, 3H, 2‘‘‘-H, 4‘‘‘-H,

6‘‘‘-H), 6.14 (s, 1H, NH), 2.74 – 2.46 (m, 4H), 2.35 (tt, J = 12.1, 3.6 Hz, 1H, 4‘‘-H), 2.23 (td, J = 11.8,
3.2 Hz, 1H, 2‘-H), 1.98 – 1.48 (m, 9H; CH2), 1.34 – 1.02 (m, 5H, CH2). 13C NMR (126 MHz, CDCl3):
δ (ppm) = 146.1 (C-1‘‘‘), 139.8 (C-1), 132.7 (C-4), 129.1 (C-3, C-5), 128.6 (C-3‘‘‘, C5‘‘‘), 127.3 (C-
2, C-6), 126.9 (C-2‘‘‘, C-6‘‘‘), 126.4 (C-4‘‘‘), 67.2 (C-2‘), 53.7 (C-1‘), 52.9 (CH2 piperidine), 44.7
(CH2 piperidine), 42.9 (C-4‘‘), 34.1 (CH2 piperidine), 34.1 (CH2 piperidine), 33.0 (CH2 cyclohexane),
25.5 (CH2 cyclohexane), 24.4 (CH2 cyclohexane), 22.9 (CH2 cyclohexane). IR (ATR): ν̃
[cm-1] = 2932,
1344, 1313, 1164, 1086, 1068, 905, 759, 733, 691. HR-MS (ESI): calcd. for C23H31N2O2S+: 399.2101
[M+H]+; found: 399.2099. purity (HPLC): >96 %.
trans-N-(2-(1,2,3,4-Tetrahydroisoquinolin-2-yl)cyclohexyl)benzenesulfonamide (28)
Following general procedure B, 7-(phenylsulfonyl)-7-azabicyclo[4.1.0]heptane (7, 122 mg,
0.514 mmol) and 1,2,3,4-tetrahydroisoquinoline (77 µL, 0.62 mmol) were reacted to give 28 as a
1
yellow solid (yield: 24 %). mp: 113-114 °C. H NMR (500 MHz, CDCl3): δ (ppm) = 7.84 – 7.77 (m,
2H, 2-H, 6-H), 7.62 – 7.55 (m, 1H, 4-H), 7.46 (t, J = 7.8 Hz, 2H, 3-H, 5-H), 7.12 (pd, J = 7.3, 1.8 Hz,
2H, 6‘‘-H, 7‘‘-H), 7.08 – 7.05 (m, 1H, 5‘‘-H), 6.80 (d, J = 7.2 Hz, 1H, 8‘‘-H), 6.10 (s, 1H, NH), 3.48 –
3.29 (m, 2H, 1‘‘-H), 2.83 – 2.38 (m, 7H), 1.91 – 1.67 (m, 3H, CH2 cyclohexane), 1.35 – 1.11 (m, 4H,
CH2 cyclohexane). 13C NMR (126 MHz, CDCl3): δ (ppm) = 139.7 (C-1), 134.4 (C-4‘‘a or C-8‘‘a),
134.2 (C-4‘‘a or C-8‘‘a), 132.5 (C-4), 129.0 (C-2, C-5), 128.7 (C-5‘‘), 127.2 (C-2, C-6), 126.6 (C-8‘‘),
126.2 (C-6‘‘ or C-7‘‘), 125.5 (C-6‘‘ or C-7‘‘), 66.6 (C-2‘), 53.6 (C-1‘), 50.3 (C-1‘‘), 32.83 (C-3‘‘),
32.8 (C-6‘), 29.8 (C-4‘‘), 25.29 (CH2 cyclohexane), 24.2 (CH2 cyclohexane), 22.6 (CH2 cyclohexane).
IR (ATR): ν̃
[cm-1] = 2929, 1446, 1340, 1307; 1159; 1091; 903, 741, 724, 689. HR-MS (ESI): calcd.
for C21H27N2O2S+: 371.1788 [M+H]+ found: 371.1788. purity (HPLC): >96 % (210 nm).
1-(2-Methoxyphenyl)-4-(2-nitrophenyl)piperazine (30)
A
oven-dried Schlenk tube containing 2-bromonitrobenzene (0.20 g, 1.0 mmol), 1-(2-
methoxyphenyl)piperazine (0.27 g, 1.4 mmol), Pd(OAc)2 (23 mg, 0.10 mmol), BINAP (93 mg,
0.15 mmol) and Cs2CO3 (0.46 g, 1.4 mmol) was degassed in vacuo and purged with N2, then dry
toluene (12 mL) was added and the resulting suspension stirred for 30 min at rt, then at 80 °C for 16 h.
After completion (TLC control) the mixture was filtered over a pad of celite, and the celite washed
several times with ethyl acetate (~30 mL). The filtrate was washed with water (20 mL) and brine
(20 mL), dried over Na2SO4 and the solvent removed in vacuo. The residue was purified by FCC using
petrol ether /ethyl acetate as an eluent to afford 30 as an orange solid (yield: 94 %). mp: 117-118 °C.
1H NMR (500 MHz, CDCl3): δ (ppm) = 7.78 (dd, J = 8.1, 1.7 Hz, 1H, 3’-H), 7.50 (ddd, J = 8.2, 7.3,
1.6 Hz, 1H, 5’-H), 7.22 (dd, J = 8.3, 1.3 Hz, 1H, 6’-H), 7.08 – 6.98 (m, 3H, 4’-H, 4’’-H, 6’’-H), 6.97 –
6.93 (m, 1H, 5’’-H), 6.89 (dd, J = 8.0, 1.4 Hz, 1H, 3’’-H), 3.89 (s, 3H, OCH3), 3.28 – 3.16 (m, 8H,
CH2). 13C NMR (126 MHz, CDCl3): δ (ppm) = 152.4 (C-2’’), 146.3 (C-1’), 143.8 (C-2’), 141.1 (C-
1’’), 133.7 (C-5’), 126.0 (C-3’), 123.4 (C-4’), 122.0 (C-5’’), 121.2 (C-6’), 118.6 (C-4’’ ,C-6’’), 111.4
(C-3’’), 55.5 (OCH3), 52.2 (C-2, C-6), 50.9 (C-3, C-5). IR (ATR): ν̃
[cm-1] = 3373, 2930, 1480, 1444,