Novel multi target-directed ligands targeting 5-HT4 receptors with in cellulo antioxidant properties as promising leads in Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2019.111596

Facing the complexity of Alzheimer's disease (AD), it is now currently admitted that a therapeutic pleiotropic intervention is needed to alter its progression. Among the major hallmarks of the disease, the amyloid pathology and the oxidative stress are closely related. We propose in this study to develop original Multi-Target Directed Ligands (MTDL) able to impact at the same time Aβ protein accumulation and toxicity of Reactive Oxygen Species (ROS) in neuronal cells. Such MTDL were obtained by linking on a central piperidine two scaffolds of interest: a typical aminochlorobenzophenone present in numerous 5-HT₄R agonists, and diverse antioxidant chemotypes. Interestingly, the most active compound 9g possesses a Ki of 12.7 nM towards 5-HT₄R and an antioxidant activity in vitro and in cellulo.

Full text of DOI:10.1016/j.ejmech.2019.111596

Journal Pre-proof
Novel multi target-directed ligands targeting 5-HT receptors with in cellulo
4
antioxidant properties as promising leads in Alzheimer's disease
Caroline Lanthier, Hugo Payan, Irene Liparulo, Bérénice Hatat, Cédric Lecoutey,
Marc Since, Audrey Davis, Christian Bergamini, Sylvie Claeysen, Patrick Dallemagne,
Maria-Laura Bolognesi, Christophe Rochais
PII:
S0223-5234(19)30730-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.111596
EJMECH 111596
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 May 2019
Revised Date: 19 July 2019
Accepted Date: 5 August 2019
Please cite this article as: C. Lanthier, H. Payan, I. Liparulo, Béé. Hatat, Cé. Lecoutey, M. Since, A.
Davis, C. Bergamini, S. Claeysen, P. Dallemagne, M.-L. Bolognesi, C. Rochais, Novel multi target-
directed ligands targeting 5-HT receptors with in cellulo antioxidant properties as promising leads in
4
Alzheimer's disease, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.111596.
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© 2019 Published by Elsevier Masson SAS.

Novel multi target-directed ligands targeting 5-HT4
receptors with in cellulo antioxidant properties as
promising leads in Alzheimer’s disease
Caroline Lanthier,1 Hugo Payan,2 Irene Liparulo,3 Bérénice Hatat,1, 2Cédric Lecoutey,1 Marc
Since, 1 Audrey Davis,1 Christian Bergamini,3 Sylvie Claeysen,2 Patrick Dallemagne,1Maria-
Laura Bolognesi,3 Christophe Rochais1*
1
Centre d’Etudes et de Recherche sur le Médicament de Normandie, Normandie Univ,
UNICAEN, CERMN, 14000 Caen, France
2 IGF, Univ. Montpellier, CNRS, INSERM, Montpellier, France
3 Department of Pharmacy and Biotechnology, Alma Mater Studiorum − University of Bologna,
I-40126 Bologna, Italy
KEYWORDS
Alzheimer’s Disease; 5-HT4 receptor; antioxidant; phenol; MTDL.
ABSTRACT

Facing the complexity of Alzheimer’s disease (AD), it is now currently admitted that a
therapeutic pleiotropic intervention is needed to alter its progression. Among the major hallmarks
of the disease, the amyloid pathology and the oxidative stress are closely related. We propose in
this study to develop original Multi-Target Directed Ligands (MTDL) able to impact at the same
time Aβ protein accumulation and toxicity of Reactive Oxygen Species (ROS) in neuronal cells.
Such MTDL were obtained by linking on a central piperidine two scaffolds of interest: a typical
aminochlorobenzophenone present in numerous 5-HT4R agonists, and diverse antioxidant
chemotypes. Interestingly, the most active compound 9g possesses a Ki of 12.7 nM towards
5-HT4R and an antioxidant activity in vitro and in cellulo.

Introduction
Alzheimer's disease (AD) is the most common form of dementia and affects more than 50 million
people worldwide.1 More than a century after its discovery, AD is now characterized by synaptic
dysfunction and synapse loss correlated with cognitive decline.2 These dysfunction are supposed
to be due to the presence of two peptide deposits in and around neuronal cells. The accumulation
and aggregation of Aβ peptide is at the origin of the amyloid pathology whereas the
neurofibrillary tangles are mainly composed of phosphorylated tau protein.3 These lesions are
generally associated with mitochondria dysfunction, an increase inflammatory response and the
presence of oxidative stress.4 The latter was further particularly linked to the presence of amyloid
aggregates which can produce Reactive Oxygen Species (ROS) in the presence of metal ions and
which ultimately leads to neuronal cell death.5 The control of ROS production is therefore of
particular interest in the prevention or the treatment of AD.
Despite these progresses, the discovery and development of treatments with disease-modifying
effect on AD remain one of the greatest challenges in drug discovery.6 After numerous failures in
clinical trials with compounds targeting mainly amyloid7 or tau pathologies,8 it is now currently
admitted that a pleiotropic approach is needed for this multifactorial neurodegenerative disease.9
In order to impact several biological targets at the same time, single pleiotropic compounds,
known as Multi-Target Directed Ligands (MTDLs),10,11,12 have been developed during the last
decade. Among them, many examples include compounds able to act on oxidative stress and on
another target.13,14,15 On the other hand, many examples have been described in order to limit
amyloid pathology with numerous examples of MTDL acting on β-secretase inhibition,16 or
inhibiting Aβ aggregation.14 In addition to those classical targets, the serotonergic system appears

of particular interest, notably via the modulation of 5-HT4 and 5-HT6 receptors.17,18 The
activation of 5-HT4 receptors (5-HT4R) with partial agonists, such as RS67333, is of particular
interest as it can promote the non-amyloidogenic cleavage of Amyloid Protein Precursor (APP)
and subsequently decrease the amyloid load in AD transgenic mice models.19,20 In this context,
we have in the past modulated the structure of RS67333 in order to introduce another target of
choice for the treatment of AD such as acetylcholinesterase (AChE) inhibitory activity21,22 or 5-
HT6R antagonism.23 This two studies demonstrated that the replacement of the butyl chain of
RS67333 by diverse alky moieties has limited impact on both the affinity and the
pharmacological profile towards 5-HT4R. A similar strategy has been recently followed in order
to generate 5-HT6R antagonists bearing antioxidant activities.24
In this context, we would like to assess the possibility to introduce, on the central piperidine ring
of the targeted structures (Figure 1), various chemical moieties known to exhibit antioxidant
activities. We postulate that such MTDL would be of particular interest combining, for the first
time in a unique compound, antioxidant properties as well as 5-HT4R agonism. Several
antioxidant scaffolds were chosen for this proof of concept: polyphenol,25 hydroxycinnamic
acid26 and lipoic acid.27,28 Following this strategy, the two pharmacophores, will be connected
through a permanent linker in order to optimize the central distribution of the antioxidant moiety.
Indeed, the presence of the piperidine as a linker appears essential for the 5-HT4R activity and the
generated compounds should possess physicochemical properties for CNS application.

Figure 1. Chemical structure of RS67333 A and targeted structure of the MTDL B.
The influence of these substitutions on 5-HT4R affinity and the antioxidant activity of the
resulting compounds could thus be assessed in vitro and in cellulo.

Results
Chemistry
According to our previous chemical studies, the precursor 7 appears as a central scaffold in the
synthesis of RS67333.22,23 Compound 7 was synthesized in a three-step sequence through
formation of a β-keto ester 2 by reaction between acid 1, activated in situ with CDI, and
potassium 3-ethoxy-3-oxopropanoate.29,30 Subsequent alkylation with a previously synthesized
piperidine chain 6, and followed by reaction with KOH in an EtOH/H2O mixture provided a
saponification-decarboxylation reaction to afford 7.22 From this precursor 7, different moieties
were linked, such as catechol, vanillin or isovanillin widely known for their antioxidant
properties.31,32 The first compounds 9a, 9c, 9d, and 9f were obtained by alkylation with the
corresponding bromobenzyl derivatives at room temperature. A final debenzylation step was
performed with HBr 33% in solution in acetic acid at 0˚C to obtain free alcohols derivatives 9b,
9e, and 9g respectively.
The second goal was to link ferulic and lipoic acid (LA) described as powerful antioxidants in the
literature.33,34,35 In order to link LA and ferulic acid while maintaining the basicity of the nitrogen
of the piperidine, an ethylamine linker was added. According to litterature,23 an ethylamine linker
has no influence on the 5-HT4R affinity and allowed the availability of a free terminal amine for
peptidic coupling. Once the intermediate 8 was obtained, several coupling reactions were
performed with EDCI, HOBt as activators, to synthesize coupled products 10a and 10b. Due to
the sensitivity of LA to air, light, and heat, reactions were performed in dry, cold and dark
conditions.
Scheme 1. Synthesis of the final derivatives 9-10

aReagents, conditions: (a) CDI, THF; (b) KO2CCH2CO2Et, MgCl2, THF, 40°C; (c) LiAlH4, Et2O, reflux; (d) (Boc)2O, DCM; (e) PPh3, I2,
Imidazole, THF; (f) K2CO3, DMF; (g) KOH, EtOH/H2O; (h) TFA, DCM; (i) tert‐butyl N‐(2‐bromoethyl)carbamate, K2CO3, ACN ; (j)
Bromobenzylalcohol, K2CO3, DCM ; (k) EDCI, HOBt, Et3N, 0˚C to rt, in the dark ; (l) HBr 33% in AcOH, DCM, 0˚C..
In vitro evaluation
As previously mentioned, the main goal of our study was to discover a hit that will have
sufficient antioxidant properties, as well as a good affinity toward 5-HT4R. All the synthesized
compounds, were tested on 5-HT4R according to the Grossman et al method.36 The affinity
toward the receptor was evaluated using a displacement assay of the tritiated ligand [3H]-
GR113808, a specific and highly potent 5-HT4R antagonist used to label specific binding sites in
human brain.
Table 1. Binding to h5-HT4R and in vitro antioxidant activities of final derivatives

h5-HT4R
DPPH
ORAC
% inhibition % inhibition
EC50 at
(µMcompo
und / µM
Ki
(nM)
IC50
(nM)
R1
R2
cLogP*
R3
Cmpd
at
at
120 min
(µM)
)
Trolox®
10-8 M
3%
10-6 M
89%
O-Bn
OH
O-Bn
OH
-
-
6.78
3.48
-
-
>250
< 0.2
9a
9b
5.9 ±
0.7
7.5±
0.7
19.6 ±
0.8
3.40 ±
0.02
76%
100%
O-CH2-O
-
-
3.84
5.55
22%
0%
100%
97%
-
-
-
-
>250
>250
< 0.2
< 0.2
9c
O-CH3
O-CH3
O-Bn
OH
O-Bn
OH
9d
180.3 ±
25.5
3.88 ±
0.55
-
-
-
3.92
5.62
3.94
7%
7%
95%
88%
-
-
-
-
9e
9f
O-CH3
O-CH3
>250
< 0.2
12.7 14.5 ± 85.5 ±
± 2.1
3.41 ±
0.59
64%
100%
9g
5.8
2.0
Lipoic
acid
0.76 ±
0.11
-
-
-
-
-
-
-
-
-
-
4.98
26%
39%
0%
-
99%
100%
65%
-
-
-
>250
10a
10b
Ferulic
acid
101
± 8.0
101 ±
3.6
45.5 ±
2.0
1.47 ±
0.25
3.65
246± 323 ±
31
-
-
-
-
-
-
-
-
Serotonin
Trolox
25
22.9 ±
0.9
-
-
1
9.3 ±
1.5
6.50 ±
0.73
-
-
-
-
Quercetin
* calculated logP with ALOGPS 2.1.

Those compounds were also developed for their antioxidant properties and preliminary
antioxidant in vitro test was also performed. Our attention was focused on two tests, DPPH, and
ORAC.37 The ORACFl assay was conducted in an aqueous medium according to well-described
procedure. It evaluates the antioxidant activity via a Hydrogen Atom Transfert (HAT)
mechanism. Calculating the AUC of the fluorescence decrease for 1.5 hours, quantitative and
kinetic aspects are both taken into account. Here, phenol gifted compounds showed a moderate
antioxidant activity compared with a TEAC ranged between Trolox® (6-Hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic Acid), the reference, and quercetin (Table 1), a natural
polyphenol antioxidant. The DPPH assay was performed in an organic solvent and enabled the
evaluation of antioxidant activity following a Single-Electron-Transfer (SET) mechanism.
According to recent literature, our initial procedure was completed here by slight modifications to
ensure a complete evaluation of our compounds. First calculation of the specific absorbance of
DPPH subtracting the absorbance of others components in the solution was realized. The
maximum effect was normalized using a high dose of Trolox® (375 µM) to get the absolute
EC50.38 The kinetic analysis to reach the plateau of % DPPH RSA at infinite time was determined
and
EC50 were calculated using linear or non-linear regression (R²> 0.98) from the dose-effect
curves.
In these conditions, Trolox® and quercetine reached their maximal antioxidant activity very
quickly (immediately and few minutes respectively) compared to other active compounds (T1/2
about 30 min). Their dose-effect curve was a linear (R²>0.99). In our phenol series, when DPPH
EC50 was calculable, EC50 values obtained after 120 min were very spread from 19.6 to >250
µM, varying as a terminal chemical pattern.

In cellulo evaluation
The pharmacological profile of the selected compounds was assayed towards h5-HT4R
transiently expressed in COS-7 cells (Figure 2). Three compounds behaved as partial agonists of
the receptor with EC50 of 2.8 ± 1.3 nM, 1.4 ± 0.8 nM and 4.5 ± 2.1 nM for 9b, 9g and 10b,
respectively (Figure 4). Compound 10a also behaved as a partial agonist of the 5-HT4R, but its
irregular profile suggested that it may engage other targets in the COS-7 and no clear
pharmacological profile was established. Compound 10b presented an Emax of 47 ± 8% that is
similar to the one of RS67333, the 5-HT4R agonist of reference. Compounds 9b and 9g showed
greater efficacy than the reference to induce cAMP production with Emax of 72 ± 10% and 57 ±
3%, respectively.
150
5-HT
RS67333
100
9b
9g
10b
50
0
-50
-11 -10 -9 -8 -7 -6 -5 -4
0
log [Drugs] (M)
Figure 2. Determination of the 5-HT4R pharmacological profiles for compounds 9b, 9g and 10b,
by quantification of cAMP accumulation. The agonism activity of the compounds was
determinated with serotonin (5-HT) and the partial agonist RS67333 as references. Data
presented are the mean of three independent experiments performed in duplicate.

The first in vitro antioxidant assays allowed us to select 9b, 9g, 10a-b, to complete their
antioxidant evaluation in a cellular context. To define the suitable concentration, the cytotoxicity
of 9b, 9g, 10a-b was determined by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl
tetrazolium bromide) assay39 in a neuroblastoma cell line, SH-SY5Y. As seen on Figure 3,
treatment of the cells with compounds 9b, 9g, 10a (0.5 to 10 µM) did not show cytotoxicity. A
little decrease of 12% of viability compared to control cells was seen with compound 10b. While
at a dose of 50 µM all compounds seem to show a small cytotoxicity with a decreasing of 37%,
40%, 48%, 68% for 9b, 9g, 10a, 10b respectively. Therefore, a single dose of 10 µM was chosen
to perform the in cellulo antioxidant assay.
Figure 3 Figure 3 Cell viability determined by MTT assay. Cells were grown into 96-well plates
and treated with 9b, 9g, 10a and 10b for 24 hours at the concentration of 0.5, 1, 10 and 50 µM or
vehicle (DMSO). Data are presented as percentage of viable cells in comparison with vehicle
treated controls. Error bars indicate ± SEM.

****
****
****
***
Figure 4 Reactive oxygen species determination in live SH-SY5Y cells. Cells were grown in 96
wells plates and incubated for 24 hours with compounds 9b 9g 10a and 10b, Trolox or vehicle
,
,
(CTRL). Oxidative stress was induced by treating the cells with 100µM TBH for 30 minutes and
detected by staining the cells with the fluorogenic probe H2DCF-DA. Data are presented as
Arbitrary Fluorescence Units normalized on protein content measured by Lowry method. Error
bars indicate ± SEM. *** P < 0.001, **** P < 0.0001
The antioxidant activity of selected compounds was evaluated on live SH-SY5Y cells using the
fluorogenic probe H2DFCDA. The cell-permeant H2DCFDA passively diffuses into cells and is
retained in the intracellular level after cleavage by intracellular esterases. Upon oxidation by

ROS, the nonfluorescent H2DCF is converted to the highly fluorescent 2',7'-dichlorofluorescein
(DCF) as described by Wang in 1999.40 The level of oxidative stress is directly correlated with
the intensity of DCF fluorescence.
To evaluate the antioxidant activity, cells were incubated for 24 hours with the selected
compounds, Trolox or vehicle. Then cells were incubated with 10µM H2DCFDA probe for 30
minutes. After this time the cells were carefully washed with PBS and the oxidative stress was
induced by 30 minutes treatment with tert-butyl hydroxyperoxide (t-BuOOH or TBH). The
fluorescence intensity was normalized on protein content (Figure 4).
Discussion
We have demonstrated in our previous study22 that the introduction of a non-susbtituted benzyl
group instead of the butyl group present in RS67333 has little impact on affinity towards the 5-
HT4R and on Acetylcholinesterase inhibition. Based on these results a first series of hydroxyl or
methoxy substituted benzylated derivatives was generated. In a previous study, we have
demonstrated that the replacement of the butyl substituent of the piperidine of RS67333 by
benzyl or methylenecyclohexyl moiety leads to potent AChE inhibitors with good
physicochemical properties and in vivo promnesiant activities.22 In order to verify the impact of
these modulations on drugability parameters, cLogP were determined. If the protected benzylated
compounds 9a, 9d and 9f appear particularly lipophilic, all final compounds are compatible with
optimal CNS parameters.41 If benzyl piperidine scaffolds are well known pharmacophores for
cholinesterase interaction,42 we have verified that the RS67333 analogs were selective towards
butyrylcholinesterase.22 Docking studies in AChE binding site revealed that the orientation of
those ligands was similar to Donepezil and reproduced its interactions. In particular, the benzyl

moiety is located at the bottom of the aromatic gorge of AChE and engaged in a hydrophobic
interaction with Trp86. We postulate that the introduction of polar or bulky substituents on the
benzyl group of our final compounds will be detrimental for their activities. In order to verify this
hypothesis, all final compounds 9a-g and 10a-b were evaluated as inhibitors of human AChE. As
expected, these modulations were particularly detrimental for the affinity of the ligands towards
AChE (See Table S1 in Supporting Informations). Concerning the impact of these modulations on
5-HT4R activity, as shown in Table 1, all compounds exhibited good inhibition rates at 10-6 M.
However, the benzyl protecting group present on the scaffold of compounds 9a
, 9d and 9f has
detrimental impact on affinity towards 5-HT4R receptors probably due to its steric hindrance. On
the contrary, smaller substituents lead to enhanced activity. In this assay only 9b
, 9g, and 10b
exhibited more than 35% of inhibition rate at 10-8 M allowing us to calculate further data such as
IC50 and K
isovanillin analog 9g. On the contrary, 9b and 9g are really promising with K
nM. According to our hypothesis, compounds 9b 9g and 10a behave as partial agonist for the 5-
i. Surprisingly the vanillin derivative 9e showed a decreased potency compared to the
i
values under 15
,
HT4R, the first two being able to induce cAMP production with a greater efficiency than the
reference RS67333 (Figure 2).
All compounds were then engaged in two complementary in vitro antioxidant biological assays.
In the DPPH assay, a SET mechanism test, hydroxylated compounds 9b, 9e, 9g and 10b were the
only one to exert antioxidant activity, the catechol 9b being the most potent. In this assay, the
lipoic acid derivative 10a did show limited antioxidant activity. In the ORAC evaluation, the
antioxidant activities of all compounds were compared to Trolox®, a widely used antioxidant
reference compound. The same compounds 9b, 9e and 9g appeared to be 3-fold more potent than
Trolox®, whereas ferulic derivative 10g demonstrated similar activity than the reference. Both

test confirm that the in vitro activity is clearly linked to the introduction of a phenol group as all
benzylated compounds are inactive in this assay.
Based on the results we have chosen to test the in cellulo antioxidant activity of these compounds
9b, 9g, 10a and 10b. Compound 9e was not selected because of its lower affinity towards 5-
HT4R. As already demonstrated for prucalopride, another 5-HT4R agonist, we have chosen to
evaluate this antioxidant and neuroprotective activities in SH-SY5Y neuroblastoma cell line.43
Lipoic acid antioxidant activity is dependent of mitochondrial activity44 and therefore cannot be
highlighted by an in vitro assay. For that reason, 10a was also selected despite no sign of in vitro
antioxidant effect.
A non-toxic dose of 10µM was chosen for the in cellulo antioxidant activity assay, after a general
MTT viability assay. Oxidative stress was generated using TBH and the ability of the compounds
to limit ROS induction was compared with the well-known antioxidant compound Trolox®.45
Trolox® was used at 100 µM and, as expected, exerts a significant reduction of ROS production.
Used at a concentration of 10µM in this assay, compounds 9b
of 10µM significantly decreased ROS production by at least 50% compared to the control group.
However, no activity was obtained in this assay with the lipoic acid substituted compound 10a
, 9g and 10b used at a concentration
.
These results are in accordance with the pharmacological profile of the compounds on 5-HT4R,
towards which no clear profile was established for compound 10a. The three other coupounds
demonstrated clear agonism toward the receptor with the catechol substituted compound 9b and
the isovanillin substituted compound 9g being interesting candidates to further in vivo evaluation.
Conclusion

This study demonstrates for the time the possibility to generate a series of potent 5-HT4R ligands
with promising antioxidant activity. Among them compound 9g appears as the best compromise
with a Ki of 12.7 nM toward 5-HT4R and an antioxidant activity in cellulo. If the balance of
activity between the in cellulo or in vitro antioxidant assay (micro molar range) and the in vitro
assay for 5-HT4R affinity (nanomolar range) might be improved, these series of compound show
great promises in more challenging models. In the experiment, the amount of oxidative stress
needs to be high enough to be visible with DCF-DA. In the brain, the level of oxidative stress
caused by AD is not properly quantified and is lower than the one induced by t-BuOOH. On the
base of the obtained data, it seems plausible that, with a suitable pharmacokinetic and a proper
Blood Brain Barrier crossing according to its adequate LogP, 9g could have an antioxidant effect
at a nanomolar concentration and will be tested in a near future in preclinical animal models of
AD.

Experimental section
Chemistry. All commercially available compounds were used without further purification.
Melting points were determined on a Stuart melting point apparatus (SMP50). Analytical thin-
layerchromatography (TLC) was performed on silica gel 60 F254 on aluminium plates (Merck)
and visualized with UV light (254 nm). Flash chromatography was conducted on a VWR SPOT
II Essential instrument with silica gel 60 (40-63µm). Column size and flow rate followed the
manufacturer’s recommendations. NMR spectra were recorded at 295K, at 400 or 500 MHz
1
(Bruker Avance III 400/500 MHz) for H NMR and at 100 or 126 MHz for 13C NMR in
chloroform-d, methanol-d4 or DMSO-d6 with chemical shifts (δ) given in parts per million (ppm)
relative to TMS as internal standard and recorded. The following abbreviations are used to
describe peak splitting patterns when appropriate: br = broad, s = singlet, d = doublet, t = triplet,
q = quartet, m = multiplet, dd = doublet of doublet, dt = doublet of triplet. Coupling constants
J
are reported in hertz units (Hz). Infrared spectra (IR) were obtained on a PERKIN-ELMER FT-IR
spectrometer and are reported in terms of frequency of absorption (cm-1) using KBr discs. High-
resolution mass spectra (HRMS) were obtained by electrospray (ESI, sampling cone 50V,
capillary 0.3kV) on a Xevo G2-XS QTof WATERS mass spectrometer. LC-MS (ESI) analyses
were realized with Waters Alliance 2695 as separating module using the following gradients: A
(95%)/B (5%) to A (5%)/B (95%) in 4.00min. This ratio was hold during 1.50 min before return
to initial conditions in 0.50 min. Initial conditions were then maintained for 2.00 min (A = H2O,
B = CH3CN; each containing HCOOH : 0.1 %; column XBridge BEH C18 2.5 µm/3.0 x 50 mm;
flow rate 0.8 mL/min). MS were obtained on a SQ detector by positive ESI. Mass spectrum data
are reported as m/z.
General procedure for deprotection/alkylation of the piperidine moiety A

To a stirred solution of tert-butyl piperidine-1-carboxylate derivative (1.0 eq.) in DCM (20
mL/mmol) was added TFA (2 mL/mmol). The resulting mixture was stirred at room temperature
for 1h. Removal of the solvent under vacuum afforded the crude product, which was directly
engaged in the next step. The residue obtained (1.0 eq.) was dissolved in DCM or ACN (10
mL/mmol) and bromo-benzyl-alcohol derivatives (1.1-1.5 eq.) and K2CO3 (10.0 eq.) were added.
The resulting mixture was stirred at RT or 70°C depending on the product for 2-72h, then
concentrated in vacuo. The crude was directly purified by chromatography on silica gel column
and concentrated under reduced pressure to afford alkylated compound (isolated yields).
The general procedure to obtain unprotected cathecol B
Appropriate benzylated product (1 eq) was stirred in bromhydric acid 33 % diluted in acetic acid
(10 mL.mmol-1, 0.10M) and DCM (50 mL.mmol-1, 0.02M) at 0°C for 1 to 4h. The mixture was
evaporated in vacuo at rt. Then the crude was directly purified on silica gel column
(DCM/MeOH; from 100:0 to 80:20).
Ethyl 3
̶
̶
To a solution of 4-amino-5-chloro-2-methoxybenzoic acid (5.00 g, 24.8 mmol, 1.0 eq.) in THF
(200 mL, 0.12 M) was added CDI (4.42 g, 27.3 mmol, 1.1 eq.) portionwise. The mixture was
stirred at rt for 4h. Then, ethyl potassium malonate (5.06 g, 29.7 mmol, 1.2 eq.) and MgCl2 (2.83
g, 29.7 mmol, 1.2 eq.) were added portionwisely. The resulting mixture was stirred 16h at 40°C.
Solvent were evaporated under vacuum. The residue was diluted with water and then extracted
with DCM three times. Organics layers were combined and washed with NaHCO3 sat. solution,
then brine, and dried over MgSO4. Solvent was evaporated under vaccum to afford a crude

product which was purified on silica gel column (Cyclohexane/EtOAc from 100:0 to 60:40). The
compound was obtained as a light yellow powder (3.76 g, 60% yield).1H NMR (CDCl3, 400
MHz)
δ
7.92 (s, 1H), 6.23 (s, 1H), 4.55 (s, 2H), 4.17 (q,
= 7.1 Hz, 3H).13C NMR (CDCl3, 100 MHz)
117.3, 111.7, 97.0, 60.8, 55.4, 50.5, 14.2 MS m/z [M+H]+ 272.51 IR (neat, cm-1)
J = 7.1 Hz, 2H), 3.87 (s, 2H), 3.82 (s,
3H), 1.23 (t,
J
δ 189.6, 168.6, 159.9, 148.7, 132.5,
ν
3463, 3363,
3223, 2983, 1725, 1648, 1622, 1573, 1479, 1457 1326, 1220, 837, 654 Mp 117°C. Spectral and
analytical data matched with literature.21
(piperidin-4-yl)methanol (4)
To a solution of ethyl isonipecotate (2 mL, 13.0 mmol, 1.0 eq.) in Et2O (150 mL) was added
portion-wise LiAlH4 (1.48 g, 39.0 mmol, 3.0 eq.) at 0°C and the reaction mixture was refluxed
for 7h. The mixture was then cooled to 0°C and the reaction was then slowly quenched with 1.5
mL of water, 1.5 mL of 15% NaOH solution followed by 4.5 mL of water to afford a granular
inorganic precipitate. The white solid was filtered and washed several times with EtOAc.
Evaporation of the filtrate under reduced pressure provided 1.38 g (92% yield) of compound (
1)
as a colorless oil. 1H NMR (CDCl3, 400 MHz)
δ
3.45 (d, J = 5.9 Hz, 2H), 3.08 (m, 2H), 2.59 (dt,
J
= 12.0 Hz,
J
= 3.0 Hz, 2H), 1.96 (br s, 1H), 1.71 (m, 2H), 1.60-1.55 (m, 1H), 1.17-1.07 (m,
66.9, 45.9 (2C), 38.8, 29.7 (2C); IR (neat, cm-1)
ν 3369 ,
2H); 13C NMR (CDCl3, 100 MHz)
δ
2923, 2855, 1636, 1531, 1425, 1266, 1036. Spectral and analytical data matched with literature.46
Tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate (5)
To a solution of (
1) (1.14 g, 9.96 mmol, 1.0 eq.) in DCM (40 mL) was added di-tert-
butyldicarbonate (2.39 g, 10.9 mmol, 1.1 eq.). The solution was stirred for 15h at room

temperature. The mixture was then diluted with water and the aqueous layer was extracted
several times with DCM. The organic layer was dried over MgSO4 and concentrated in vacuo.
The crude product was purified by chromatography on silica gel column (cyclohexane/EtOAc,
gradient 100:0 to 70:30) to give 1.34 g (62% yield) of compound (2) as white solid.
1H NMR (CDCl3, 400 MHz)
1H, OH), 1.69 (m, 2H), 1.63 (m, 1H), 1.44 (s, 9H), 1.12 (m, 2H); 13C NMR (CDCl3, 100 MHz)
155.0, 79.5, 67.6, 43.9 (2C), 38.9, 28.7 (2C), 28.6 (3C); MS m/z [M+H]+ 216.47; [M-Boc+H]+
-butyl+H]+ 160.47; IR (neat, cm-1)
δ 4.10 (m, 2H), 3.48 (d, J = 6.1 Hz, 2H) 2.68 (m, 2H), 1.80 (br s,
δ
116.47; [M-
t
ν 3470, 2926, 2859, 1672, 1422, 1168, 1045 ,
572; Mp 85°C.
Tert-butyl 4-(iodomethyl)piperidine-1-carboxylate (6)
To a solution of (2) (2.25 g, 10.5 mmol, 1.0 eq.), imidazole (858 mg, 12.6 mmol, 1.2 eq.) and
triphenylphosphine (3.30 g, 12.6 mmol, 1.2 eq.) in dry THF (30 mL) was added drop-wise a
solution of iodine (3.20 g, 12.6 mmol, 1.2 eq.) in dry THF (30 mL) at 0°C. The reaction mixture
was stirred at room temperature for 14h. The mixture was quenched with a saturated Na2S2O3
solution. The aqueous layer was extracted with EtOAc. The organic layer was washed with water,
dried over MgSO4, and concentrated in vacuo. The crude was several times diluted with cold
Et2O, and white crystals were filtered. After concentration of filtrate in vacuo,3.13 g of the
compound (3) was obtained as a colorless oil (92% yield).
1H NMR (CDCl3, 400 MHz)
δ
4.11 (m, 2H), 3.10 (d,
J
= 5.8 Hz, 2H), 2.68 (m, 2H), 1.83 (m,
13
2H), 1.64-1.61 (m, 1H), 1.46 (s, 9H), 1.19-1.09 (m, 2H); C NMR (CDCl3, 100 MHz)
δ
154.6,
79.5, 43.4 (2C), 38.6, 32.5 (2C), 28.4 (3C), 13.5; IR (neat, cm-1)
1243, 1157, 965. Spectral and analytical data matched with literature.47
ν 2975, 2932, 2850, 1692, 1422 ,

Tert-butyl 4-[3-(4-amino-5-chloro-2-methoxyphenyl)-3-oxopropyl]piperidine-1-carboxylate
(7)
To a solution of ethyl 3-(4-amino-5-chloro-2-methoxyphenyl)-3-oxopropanoate (
4) (3.14 g, 11.6
mmol, 1.0 eq.) in DMF (30 mL) and tert-butyl 4-(iodomethyl)piperidine-1-carboxylate (
3
) (4.14
g, 12.7 mmol, 1.1 eq.) was added K2CO3 (3.19 g, 23.10 mmol, 2 eq.). The mixture was stirred
72h at rt. The reaction mixture was diluted with water, and product was extracted with EtOAc.
Combined organic layers were washed with water and brine and then dried over MgSO4.
Evaporation of the solvent gave a crude mixture which was directly dissolved in EtOH (500 mL),
H2O (100 mL) and KOH (3.23 g, 57.5 mmol, 5.5 eq.) were added. The reaction mixture was
refluxed for 3h, EtOH was evaporated. The mixture was diluted with H2O and extracted with
EtOAc. The organic layer was washed with H2O and brine. Evaporation of the solvent provided a
crude product, which was purified by column on silica gel, eluting on DCM/EtOAc from 100:0 to
60:40 to give the product
1H NMR (CDCl3, 400 MHz)
(s, 3H), 2.91 (dd, = 8.3, 6.9 Hz, 2H), 2.67 (t,
5 as a colorless solid (3.11 g, 67% isolated yield).
δ
7.79 (s, 1H), 6.26 (s, 1H), 4.46 (s, 2H), 4.18 – 3.98 (m, 2H), 3.85
J
J
= 12.4 Hz, 2H), 1.72 – 1.64 (m, 2H), 1.63 – 1.58
= 12.4, 4.3 Hz, 2H).13C NMR (CDCl3,
(m, 2H), 1.58 – 1.54 (m, 1H), 1.45 (s, 9H), 1.10 (qd,
J
100.4 MHz)
32.1, 31.1, 28.5.MS m/z [M+H]+ 397.60 ; [M-Boc+H]+ 297.51; [M-
cm-1)
967, 738; Mp 136°C. Spectral and analytical data matched with literature.21
δ 198.7, 159.5, 154.9, 147.7, 132.2, 118.9, 111.3, 97.5, 79.2, 55.7, 44.0, 40.6, 35.8,
t-butyl+H]+ 341.55; IR (neat,
ν
3470, 3353, 2975, 2930, 2851, 1673, 1624, 1588, 1421, 1366, 1311, 1250, 1216, 1173,

Tert-butyl
N-(2-{4-[3-(4-amino-5-chloro-2-methoxyphenyl)-3-oxopropyl]piperidin-1-
yl}ethyl)carbamate (8)
Following procedure
A
with tert-butyl 4-[3-(4-amino-5-chloro-2-methoxyphenyl)-3-
) (2.37 g, 5.98 mmol, 1 eq.) and tert-butyl N-(2-
oxopropyl]piperidine-1-carboxylate (
7
bromoethyl)carbamate (1.61 g, 7.18 mmol, 1.2 eq.) synthesized following the literature, the
reaction was stirred at rt for 16h. After a purification by chromatography on silica gel column
(Cyclohexane/EtOAC, gradient 100:0 to 0:100), the compound (8) was obtained as a light yellow
solid (780 mg, 30% isolated yield).
1H NMR (CDCl3, 400 MHz)
δ 7.71 (s, 1H), 6.20 (s, 1H), 4.98 (br s, 1H, NH), 4.49 (s, 2H, NH2),
3.77 (s, 3H), 3.14 (t,
J = 6.0 Hz, 2H), 2.86 – 2.72 (m, 4H), 2.35 (t, J = 6.0 Hz, 2H), 1.92 – 1.76
(m, 2H), 1.67 – 1.54 (m, 2H), 1.56 – 1.44 (m, 2H), 1.38 (s, 9H), 1.24 – 1.08 (m, 3H).13C NMR
(CDCl3, 100.0 MHz)
δ 199.0, 159.4, 156.0, 147.7, 132.2, 118.9, 111.2, 97.5, 79.1, 57.5, 55.6,
53.8 (2C), 40.9, 37.4, 35.6, 32.2 (2C), 31.3, 28.5 (3C); MS m/z [M+H]+ 441.8; IR (neat, cm-1)
ν
3486, 3376, 3325, 2980, 2925, 2855, 1690, 1625, 1573, 1467, 1367, 1311, 1175, 1216, 1114, 969,
610; Mp 119°C; HRMS/ESI: m/z calcd. for C22H34ClN3O4 [M+H]+ 440.2316, found 440.2324;
1-(4-amino-5-chloro-2-methoxy-phenyl)-3-[1-[(3,4-dibenzyloxyphenyl)methyl]-4-
piperidyl]propan-1-one (9a)
Following procedure
A
with tert-butyl 4-[3-(4-amino-5-chloro-2-methoxyphenyl)-3-
) (200 mg, 0.50 mmol, 1 eq.) and 1,2-bis(benzyloxy)-4-
oxopropyl]piperidine-1-carboxylate (
7
(bromomethyl)benzene (230 mg, 0.60 mmol, 1.2 eq.) synthesized following the literature, the
reaction was stirred at RT for 10h. After a purification by chromatography on silica gel column

(DCM/MeOH, gradient 100:0 to 90:10), the compound (9a) was obtained as a light yellow solid
(141 mg, 47% isolated yield).
1H NMR (CDCl3, 400 MHz) 7.78 (s, 1H), 7.49 – 7.40 (m, 4H), 7.40 – 7.24 (m, 6H), 7.01 (d,
J =
2.0 Hz, 1H), 6.87 (d, = 8.2 Hz, 1H), 6.81 (dd, = 8.2, 2.0 Hz, 1H), 6.25 (s, 1H), 5.18 (s, 2H),
J
J
5.14 (s, 2H), 4.51 (s, 2H), 3.82 (s, 3H), 3.49 (s, 2H), 2.95 – 2.79 (m, 4H), 1.97 (t,
J = 10.5 Hz,
2H), 1.66 (d,
MHz)
J
= 10.5 Hz, 2H), 1.64 – 1.51 (m, 2H), 1.47 – 1.20 (m, 4H). 13C NMR (CDCl3, 100
δ
198.9, 159.54 148.6, 148.4, 147.8, 137.3, 137.3, 132.2, 129.5, 128.5 (2C), 128.4 (2C),
127.8 (2C), 127.7 (2C), 127.5, 127.3, 122.7, 118.7, 116.6, 114.6, 111.2, 97.5, 71.3, 71.2, 62.5,
55.6, 53.3 (2C), 40.7, 35.0, 31.5 (2C), 31.0;.MS m/z [M+H]+ 599.7; IR (neat, cm-1)
ν
3482, 3368,
3220, 2921, 2847,2798, 1618, 1585, 1508, 1453, 1419, 1257, 1214, 1184, 1015, 735, 695; Mp
104.5°C. HRMS/ESI: m/z calcd. for C36H39ClN2O4 [M+H]+ 599.2677, found 599.2673;
1-(4-amino-5-chloro-2-methoxy-phenyl)-3-[1-[(3,4-dihydroxyphenyl)methyl]-4-
piperidyl]propan-1-one (9b)
From general procedure
B
with 1-(4-amino-5-chloro-2-methoxy-phenyl)-3-[1-[(3,4-
dibenzyloxyphenyl)methyl]-4-piperidyl]propan-1-one 9a (125 mg, 0.21 mmol) to afford 71 mg
of
1-(4-amino-5-chloro-2-methoxy-phenyl)-3-[1-[(3,4-dihydroxyphenyl)methyl]-4-
piperidyl]propan-1-one 9b as a pale yellow powder with a yield of 81%.1H NMR (MeOD, 400
MHz)
δ 7.66 (s, 1H), 6.92 (s, 1H), 6.85 – 6.80 (m, 2H), 6.45 (s, 1H), 4.06 (s, 2H), 3.86 (s, 3H),
3.39 (d,
J = 11.8 Hz, 2H), 2.97 – 2.81 (m, 4H), 1.95 (m, 2H), 1.66 – 1.52 (m, 3H), 1.51 – 1.36 (m,
2H). 13C NMR (MeOD, 100 MHz)
δ
198.78, 160.32, 150.43, 146.74, 145.57, 131.46, 122.57,
120.61, 117.59, 116.27, 115.16, 110.26, 96.64, 60.35, 54.75, 51.86 (2C), 39.86, 33.28, 30.16,

29.05 (2C); MS m/z [M+H]+ 419.8; IR (neat, cm-1)
ν 3432, 3366, 3206, 2938, 2725, 1619, 1584,
1453, 1419, 1295, 1261, 1216, 1184.Mp 115.9°C. HRMS/ESI: m/z calcd. for C22H28ClN2O4
[M+H]+ 419.1738, found 419.1737;
1-(4-amino-5-chloro-2-methoxy-phenyl)-3-[1-(1,3-benzodioxol-5-ylmethyl)-4-
piperidyl]propan-1-one (9c)
tert-butyl 4-(3-(4-amino-5-chloro-2-methoxyphenyl)-3-oxopropyl)piperidine-1-carboxylate (200
mg, 0.50 mmol, 1 eq.) was dissolved in 4 mL of DCM, then 1 mL of TFA was added. The
solution was stirred 15 min at RT, then the solvent was reduced under pressure. The resulting
yellow oil was dissolved in DMF (10 mL), then K2CO3 (691.0 mg, 5.00 mmol, 10 eq.) and 5-
(bromomethyl)benzo[d][1,3]dioxole (129.4 mg, 0.60 mmol, 1.2 eq.) were added dropwisely. The
reaction mixture was warmed at 110°C for 3h.
Then water was added and the solution was extracted three times with EtOAc, then dried over
MgSO4, concentrated under vacuum to afford a crude product which was purified on neutral
alumine column with a 100% DCM isocratic gradient. The product was obtained as a yellowish
1
powder (65 mg, 30% isolated yield) H NMR (CDCl3, 400 MHz)
δ 7.77 (s, 1H), 6.84 (s, 1H),
6.74 (d, J = 7.5 Hz, 1H), 6.72 (d, J = 7.5 Hz, 1H), 6.24 (s, 1H), 5.93 (s, 2H), 4.46 (br s, 2H), 3.82
(s, 3H), 3.38 (s, 2H), 2.93 – 2.80 (m, 4H), 1.90 (m, 2H), 1.87 (m, 1H), 1.66 (d, = 9.2 Hz, 2H),
J
1.58 (dd,
J
= 7.2, 6.4 Hz, 2H), 1.31 – 1.15 (m, 2H). 13C NMR (CDCl3, 100 MHz)
δ 199.1, 159.5,
147.7, 147.5, 146.4, 132.5, 132.2, 122.3, 118.9, 111.2, 109.6, 107.8, 100.8, 97.5, 63.3, 55.6, 53.8
(2C), 40.9, 35.6, 32.3 (2C), 31.3; MS m/z [M+H]+ 431.6; IR (neat, cm-1)
ν
3470, 3358, 3216,
3013, 2922, 2873, 2854, 2810, 2775, 1715, 1642, 1619, 1573, 1440, 1421, 1332, 1312, 1261,

1214, 1178, 986, 932, 738, 567; Mp 124°C. HRMS/ESI: m/z calcd. for C23H28ClN2O4 [M+H]+
431.1738, found 431.1747;
1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1-{[4-(benzyloxy)-3-
methoxyphenyl]methyl}piperidin-4-yl)propan-1-one (9d)
Following procedure A with
7 (200 mg, 0.50 mmol, 1 eq.) and 1-(benzyloxy)-4-(bromomethyl)-
2-methoxybenzene (185 mg, 0.60 mmol, 1.2 eq.), the reaction was stirred at RT for 16h. After a
purification by chromatography on silica gel column (DCM/MeOH, gradient 100:0 to 80:20), the
compound (9d) was obtained as a light yellow oil (74 mg, 28% isolated yield).
1H NMR (CDCl3, 400 MHz)
δ 7.78 (s, 1H), 7.49 – 7.28 (m, 5H), 6.96 – 6.88 (m, 1H), 6.81 (d, J =
8.2 Hz, 1H), 6.75 (dd, = 8.2, 1.9 Hz, 1H), 6.24 (s, 1H), 5.13 (s, 2H), 4.46 (s, 2H), 3.89 (s, 3H),
J
3.82 (s, 3H), 3.43 (s, 2H), 2.98 – 2.78 (m, 4H), 1.92 (s, 2H), 1.67 (m, 2H), 1.59 (m, 2H), 1.28 (s,
3H). 13C NMR (CDCl3, 100 MHz)
δ
199.1, 159.5, 149.4, 147.7, 147.2, 137.3, 132.2, 129.5,
128.5(2C), 127.78, 127.23 (2C), 121.4, 118.9, 113.5, 112.9, 111.2, 97.5, 71.1, 63.2, 56.1, 55.6,
53.8 (2C), 40.9, 35.6, 32.2 (2C), 31.3.MS m/z [M+H]+ 523.6; IR (neat, cm-1)
ν
3.449, 2.919,
2.853, 2.788, 1.616, 1.586, 1.463, 1.450, 1.414, 1.261, 1.216, 1.177, 735; HRMS/ESI: m/z calcd.
for C30H36ClN2O4 [M+H]+ 523.2364, found 523.2363;
1-(4-amino-5-chloro-2-methoxyphenyl)-3-{1-[(4-hydroxy-3-
methoxyphenyl)methyl]piperidin-4-yl}propan-1-one (9e)

From general procedure B with 9d (80 mg, 0.16 mmol, 1.0 eq.) to afford 9e as yellow powder
with a yield of 85%. 1H NMR (MeOD, 400 MHz)
δ
7.66 (s, 1H), 7.10 (d, J = 2.0 Hz, 1H), 6.91
(dd, J = 8.1, 2.0 Hz, 1H), 6.85 (d, J = 8.1 Hz, 1H), 6.45 (s, 1H), 4.13 (s, 2H), 3.90 (s, 3H), 3.86 (s,
3H), 3.40 (d,
J = 12.6 Hz, 2H), 3.00 – 2.79 (m, 4H), 1.96 (d, J = 13.4 Hz, 2H), 1.62 (m, 3H), 1.46
(m, 2H). 13C NMR (MeOD, 100 MHz )
δ
198.78, 160.3, 150.4, 148.0, 147.9, 131.5, 124.0, 120.5,
116.3, 115.1, 114.0, 110.3, 96.7, 60.5, 55.1, 54.8, 51.9 (2C), 39.9, 33.3, 30.2, 29.1 (2C); MS m/z
[M+H]+ 432.8; MP: 120.5˚C; IR (neat, cm-1)
ν
3360, 3220, 2932, 2855, 1620, 1586, 1420, 1254,
1216, 1177, 1136, 1084, 735; HRMS/ESI: m/z calcd. for C23H30ClN2O4+ [M+H]+ 433.1894,
found 433.1894;
1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1-{[3-(benzyloxy)-4-
hydroxyphenyl]methyl}piperidin-4-yl)propan-1-one (9f)
Following procedure A with
7 (200 mg, 0.05 mmol, 1 eq.) and 2-(benzyloxy)-4-(bromomethyl)-
1-methoxybenzene (185 mg, 0.60 mmol, 1.2 eq.), the reaction was stirred at rt for 16h. After a
purification by chromatography on silica gel column (DCM/MeOH, gradient 100:0 to 80:20), the
compound (9f) was obtained as a light yellow oil (100 mg, 38% isolated yield). 1H NMR (CDCl3,
400 MHz)
δ 7.78 (s, 1H), 7.44 (d, J = 7.2 Hz, 2H), 7.34 (t, J = 7.4 Hz, 2H), 7.28 (d, J = 7.2 Hz,
1H), 6.94 (s, 1H), 6.83 (s, 2H), 6.25 (s, 1H), 5.17 (s, 2H), 4.46 (s, 2H), 3.87 (s, 3H), 3.84 (s, 3H),
3.45 (s, 2H), 2.87 (m, 4H), 1.96 – 1.84 (m, 2H), 1.65 (d,
1.28 (s, 3H). 13C NMR (CDCl3, 100 MHz)
J = 10.2 Hz, 2H), 1.62 – 1.56 (m, 2H),
δ
199.0, 159.5, 149.0, 147.9, 147.7, 137.1, 132.1,
129.3, 128.5 (2C), 127.8, 127.5 (2C), 122.3, 118.7, 115.5, 111.4, 111.2, 97.5, 70.9, 62.7, 56.0,
55.7, 53.4 (2C), 40.8, 35.2, 31.7 (2C), 31.1; MS m/z [M+H]+ 523.6; IR (neat, cm-1)
ν
3360, 2923,

2834, 2788, 1618, 1587, 1463, 1450, 1414, 1261, 1216, 1177, 735; HRMS/ESI: m/z calcd. for
C30H36ClN2O4 [M+H]+ 523.2364, found 523.2365;
1-(4-amino-5-chloro-2-methoxyphenyl)-3-{1-[(3-hydroxy-4-
methoxyphenyl)methyl]piperidin-4-yl}propan-1-one (9g)
From general procedure B with 9f (80 mg, 0.16 mmol, 1 eq.) to afford acetate salt of 9g as a pale
yellow powder with a yield of 70%. 1H NMR (CDCl3, 400 MHz)
δ 7.76 (s, 1H), 6.95 (d, J = 2.0
Hz, 1H), 6.86 – 6.76 (m, 2H), 6.26 (s, 1H), 4.54 (br s, 2H), 3.86 (s, 3H), 3.82 (s, 3H), 3.74 (s,
2H), 3.16 (d,
J
= 11.5 Hz, 2H), 2.87 (t,
J
= 7.4 Hz, 2H), 2.27 (t,
J
= 11.5 Hz, 2H), 2.04 (s, 3H),
13
1.74 –1.72 (m, 2H), 1.62 – 1.60 (m, 2H), 1.54 – 1.43 (m, 2H), 1.37 (m, 1H). C NMR (CDCl3,
100 MHz)
110.7, 97.5, 61.0, 55.9, 55.7, 52.1 (2C), 40.4, 34.2, 30.5, 30.1 (2C), 22.9; MS m/z [M+H]+ 432.8;
MP: 80.8˚C; IR (neat, cm-1)
δ 198.5, 176.9, 159.6, 147.9, 147.1, 145.9, 132.2, 125.78, 122.2, 118.6, 116.8, 111.3,
ν
3360, 3220, 2932, 2855, 1620, 1586, 1420, 1254, 1216, 1177,
1136, 1084, 735; HRMS/ESI: m/z calcd. for C23H31ClN2O4+ [M+H]+ 433.1894, found 433.1894;
N-(2--{4-[3-(4-amino-5-chloro-2-methoxyphenyl)-3-oxopropyl]piperidin-1-yl}ethyl)-5-[(3R)-
1,2-dithiolan-3-yl]pentanamide (10a)
To a stirred solution of
8 (125 mg, 0.28 mmol, 1.1 eq.) in DCM (20 mL/mmol) was added TFA (2
mL/mmol). The resulting mixture was stirred at room temperature for 1h. Removal of the solvent
under vacuum afforded the crude product, which was directly engaged in the next step. To a
solution of LA (53 mg, 0.26 mmol, 1.0 eq.) in DCM (5 mL) were successively added

hydroxybenzotriazole (HOBt) (42 mg, 0.31 mmol, 1.2 eq.) and 1-ethyl-3-[3-
(dimethylamino)propyl]carbodiimide hydrochloride (EDCI) (49 mg, 0.31 mmol, 1.2 eq.), the
mixture was stirred at 0˚C in the dark for 30min, then the free amine was added, and the reaction
was stirred at rt for 16h. Solvent was evaporated under vaccum then the product was purified on
silica gel column with DCM/MeOH/NH3 (97.5:2.5:0.25), 10a was obtained as white powder
1
with a yield of 44%. H NMR (CDCl3, 400 MHz)
δ
7.81 (s, 1H), 6.28 (s, 1H), 6.11 (br s, 1H),
4.49 (s, 2H), 3.87 (s, 3H), 3.59 (dq,
J
= 8.5, 6.5 Hz, 1H), 3.35 (q, = 5.7 Hz, 2H), 3.24 – 3.07 (m,
J
2H), 2.97 – 2.82 (m, 4H), 2.47 (m, 3H), 2.24 – 2.17 (m, 2H), 2.01 – 1.90 (m, 3H), 1.76 – 1.65 (m,
= 8.5, 6.5 Hz, 2H), 1.57 – 1.42 (m, 2H), 1.33 – 1.17 (m, 3H). 13C NMR (CDCl3,
6H), 1.62 (dt,
J
100 MHz)
40.9, 40.3, 38.5, 36.5, 36.1, 35.6, 34.7, 32.3, 31.2 (2C), 28.9, 25.5;.MS m/z [M+H]+ 527.8; MP:
122.5˚C; IR (neat, cm-1)
δ 198.9, 172.7, 159.5, 147.7, 132.2, 118.9, 111.3, 97.6, 56.87, 56.5, 55.7, 53.7 (2C),
ν
3217, 2926, 2844, 1642, 1586, 1462, 1419, 1316, 1254, 1250, 1170;
HRMS/ESI: m/z calcd. for C23H39ClN3O3S2 [M+H]+ 528.2121, found 528.2118;
(2E)-N-(2-{4-[3-(4-amino-5-chloro-2-methoxyphenyl)-3-oxopropyl]piperidin-1-yl}ethyl)-3-
(4-hydroxy-3-methoxyphenyl)prop-2-enamide (10b)
Following the same procedure as 10a, from N-(2-{4-[3-(4-amino-5-chloro-2-methoxyphenyl)-3-
oxopropyl]piperidin-1-yl}ethyl)carbamate (8) (125 mg, 0.28 mmol, 1.1 eq.) and ferulic acid (39
mg, 0.20 mmol , 1.eq.) with EDCI, HOBt, and Et3N. The crude was purified on silica gel column
with DCM/MeOH/NH3 (92.5:7.5:0.75) to afford a bright yellow powder with a yield of 42%. 1H
NMR (CDCl3, 400 MHz)
δ
7.72 (s, 1H), 7.43 (d,
J = 15.5 Hz, 1H), 6.96 (dd, J = 8.2, 2.0 Hz, 1H),
6.90 (d, = 2.0 Hz, 1H), 6.80 (d,
J
J
= 8.2 Hz, 1H), 6.37 (br s, 1H), 6.23 – 6.07 (m, 2H), 4.40 (br s,