Aromatic Linkers Unleash the Antiproliferative Potential of 3-Chloropiperidines Against Pancreatic Cancer Cells

Article abstract of DOI:10.1002/cmdc.202000457

In this study, we describe the synthesis and biological evaluation of a set of bis-3-chloropiperidines (B?CePs) containing rigid aromatic linker structures. A modification of the synthetic strategy also enabled the synthesis of a pilot tris-3-chloropiperidine (Tri-CeP) bearing three reactive meta-chloropiperidine moieties on the aromatic scaffold. A structure–reactivity relationship analysis of B?CePs suggests that the arrangement of the reactive units affects the DNA alkylating activity, while also revealing correlations between the electron density of the aromatic system and the reactivity with biologically relevant nucleophiles, both on isolated DNA and in cancer cells. Interestingly, all aromatic 3-chloropiperidines exhibited a marked cytotoxicity and tropism for 2D and 3D cultures of pancreatic cancer cells. Therefore, the new aromatic 3-chloropiperidines appear to be promising contenders for further development of mustard-based anticancer agents aimed at pancreatic cancers.

Full text of DOI:10.1002/cmdc.202000457

Accepted Article
Title: Aromatic Linkers Unleash the Antiproliferative Potential of 3-
Chloropiperidines Against Pancreatic Cancer Cells
Authors: Barbara Gatto, Tim Helbing, Caterina Carraro, Alexander
Francke, Alice Sosic, Michele De Franco, Valentina Gandin,
and Richard Göttlich
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: ChemMedChem 10.1002/cmdc.202000457
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0
1/2020

ChemMedChem
10.1002/cmdc.202000457
FULL PAPER
Aromatic Linkers Unleash the Antiproliferative Potential of 3-
Chloropiperidines Against Pancreatic Cancer Cells
[
a]+
[b]+
[a]
[b]
[b]
Tim Helbing
and Caterina Carraro, Alexander Francke, Alice Sosic, Michele De Franco,
[
b]
[a]
[b]
Valentina Gandin, Richard Göttlich* and Barbara Gatto*
[
a]
T. Helbing, A. Francke, Prof. Dr. R. Göttlich
Institute of Organic Chemistry
Justus Liebig University Giessen
Heinrich-Buff-Ring 17, 35392 Giessen (Germany)
E-Mail: richard.göttlich@org.chemie.uni-giessen.de
C. Carraro, Dr. A. Sosic, M. De Franco, Prof. Dr. V. Gandin, Prof. Dr. B. Gatto
Department of Pharmaceutical and Pharmacological Sciences
University of Padova
[
b]
Via Francesco Marzolo 5, 35131 Padova (Italy)
E-Mail: barbara.gatto@unipd.it
+
These authors contributed equally.
Supporting information for this article is given via a link at the end of the document.
Abstract: In the present study we describe the synthesis and
development of bis-3-chloropiperidines (B-CePs) as a new class
[
4]
biological evaluation of a set of bis-3-chloropiperidines (B-CePs)
containing rigid aromatic linker structures. A modification of the
synthetic strategy also enabled the synthesis of a pilot tris-3-
chloropiperidine (Tri-CeP) bearing three reactive chloropiperidine
moieties in meta on the aromatic scaffold. The structure-reactivity
relationship analysis of B-CePs suggests that the arrangement of the
reactive units affects the DNA alkylating activity, while also revealing
correlations between the electron density of the aromatic system and
the reactivity with biologically relevant nucleophiles, both on isolated
DNA and in cancer cells. Interestingly, all aromatic 3-
chloropiperidines exhibited a marked cytotoxicity and tropism for 2D
and 3D cultures of pancreatic cancer cells. Therefore, the new
aromatic 3-chloropiperidines appear to be promising contenders for
further development of mustard based anticancer agents aimed at
pancreatic cancers.
of revisited mustard-based alkylating agents (Figure 1b).
O
a.
b.
O
OH
Cl
Cl
L
OH
NH2
Cl
Cl
N
N
N
N
Chlorambucil
Melphalan
Cl
Cl
L: Aliphatic
N
Aromatic
Cl
Cl
Mechloretamine
c.
HCl
HCl
N
Cl
Cl
N
N
N
N
N
Cl
Cl
Cl
Cl
1
2
5
3
HCl
HCl
HCl
N
O
HCl
N
O
O
HCl
HCl
N
N
Cl
Cl
Cl
N
N
Cl Cl
N
Cl
4
7
6
Introduction
N
Cl
Cl
Cl
Cl
N
N
Recent efforts in the search for targeted anticancer agents led to
the discovery of promising candidates interfering with different
carcinogenic pathways. Nevertheless, persistent drawbacks
undermine the development of these drugs, such as the rapid
emergence of resistance mechanisms and the onerous
N
N
N
Cl
Cl
N
9
Cl
8
[
1]
Figure 1. (a) Chemical structure of chlorambucil, melphalan and
mechloretamine. (b) General structure of B-CePs. Different connecting linkers
manufacturing costs especially for biologics. Consequently,
old” chemotherapeutics still represent a valid choice in first-line
“
(
L) are schematized with different colors. Green line: aliphatic linkers; blue
[
2]
treatments. Among the most diffused alternatives, nitrogen
mustards (NMs, such as those depicted in Figure 1a) represent
one of the first class of alkylating anticancer agents which act by
directly damaging DNA thus preferentially impairing replication
line: aromatic linkers.
chloropiperidines.
(c) Chemical structures of the analyzed 3-
In precedent works, we thoroughly investigated the mechanism
of DNA alkylation by B-CePs. Thanks to their marked
electrophilicity, these compounds efficiently react with
nucleobases, forming mono- and bi-functional adducts
[
2]
and transcription in fast-dividing tumor cells. Unfortunately, the
therapeutic applicability of these drugs is limited by their scarce
selectivity, which is frequently associated to tough side effects
[5]
preferentially with the N7 of guanines. These base lesions
[
2-3]
for patients.
Thus, efforts are required to identify new NM
[5]
rearrange into apurinic sites leading to DNA cleavage, with in
candidates with ameliorated efficacy and safety profiles. Seeking
to improve the therapeutic value of these economically
sustainable antitumor agents, our studies focused on the
vitro potencies modulated by the chemistry of the linker
[4a, 4b]
connecting the two 3-chloropiperidine reactive centers.
Along with potent derivatives characterized by aliphatic linkers,
our previous studies examined the activity
1
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ChemMedChem
10.1002/cmdc.202000457
FULL PAPER
a)
b)
Linker
O
N
H
N
H
H N
Linker
NH2
+
2
1
1
1
1
1
3: 1,2-xylene
4: 1,3-xylene
5: 1,4-xylene
10
23: 1,2-xylene
24: 1,3-xylene
25: 1,4-xylene*
6: 1,4-trans-cyclohexane
7: (CH2)5
26: 5-OMe-1,3-xylene
27: 5-COOMe-1,3-xylene
2
8: 2,6-dimethylpyridine
29: 1,4-trans-cyclohexane*
0: (CH ) *
X
X
+
NH2
Linker
3
2
5
2
2
2
0: 5-OMe-1,3-xylene; X=OMs
1: 5-COOMe-1,3-xylene; X=Br
2: 2,6-dimethylpyridine; X=OMs
12
c)
Cl
Linker
Cl
N
N
d)
Linker
N
N
Cl
Cl
1
: 1,2-xylene (80%)
: 1,3-xylene (69%)
: 1,4-xylene*
31: 1,2-xylene (66% 2 steps)
32: 1,3-xylene (80% 2 steps)
33: 1,4-xylene*
2
3
4
5
6
7
9
: 5-OMe-1,3-xylene (75%)
34: 5-OMe-1,3-xylene (44% 2 steps)
35: 5-COOMe-1,3-xylene (33% 2 steps)
36: 2,6-dimethylpyridine (17% 2 steps)
37: 1,4-trans-cyclohexane*
38: (CH2)5*
: 5-COOMe-1,3-xylene (86%)
: 2,6-dimethylpyridine (76%)
: 1,4-trans-cyclohexane*
: (CH ) *
2
5
Scheme 1. Synthesis of bis-3-chloropiperidines 1-6. Reactants and conditions: a) NaBH(OAc)
0-22 h; c) NCS, dry DCM, 0°C to rt, 2.5-3 h; d) TBAI (cat.), dry CHCl
of compounds 3, 7 and 9 as well as their corresponding precursors has been described elsewhere.
3
, AcOH, dry DCM, 0°C to rt, 16-18 h; b) NaH, dry THF, 0°C to rt,
3
, 60°C (oil bath temperature), 2-2.5 h (inseparable diastereomeric mixture). *The synthesis
[4b]
2
toward DNA of one B-CeP bearing
a
para-xylene linker
influence of a heteroatom in the aromatic bridging linker; finally,
iv) to probe the value of tris-3-chloropiperidines (Tri-CePs), we
synthesized compound 8, bearing three reactive moieties in
meta on the aromatic scaffold, to explore the correlation
between activity and number of reactive centers. For
comparative purpose, we included in our analyses the previously
synthesized aliphatic B-CePs 7, possessing a rigid aliphatic
[
4b]
(
compound 3, Figure 1c).
In vitro, the aromatic rigid linker reduced the DNA cleavage
potency relative to the aliphatic analogues, an attribute worth to
be further explored in the characterization of this class of
[
4b]
compounds. Similarly, the classical nitrogen mustards bearing
aromatic moieties (e.g. chlorambucil and melphalan, Figure 1a)
possess a milder electrophilicity, which makes them less toxic
drugs compared to aliphatic analogues (e.g. mechlorethamine,
[
4b]
cyclohexane linker, and 9, bearing a flexible pentyl linker.
Aside from proposing novel accessible strategies to synthesize
the described set of aromatic derivatives, the present work aims
at extending their biological evaluation beyond the reactivity on
model nucleic acids, investigating the effects of all compounds
against different cancer cell types to gain evidence of biological
activity and tropism toward different tumors. The remarkable
cytotoxicity in the pancreatic tumor-derived cells prompted us to
further explore the valuable biological activity of aromatic B-
CePs. We therefore assessed the involvement of transporters in
the uptake of B-CePs. Furthermore, we inspected their
molecular mechanism of action by checking the presence in
cells of genomic DNA lesions upon treatment. Finally, we
demonstrated in 3D models of pancreatic cancer how the
modulation of reactivity of the aromatic compounds is crucial for
unleashing their antitumor potential.
[
6]
Figure 1a).
From these premises, the present work aims to further
investigate the chemical space and anticancer value of B-CePs
by synthesizing and evaluating a series of derivatives bearing
different linkers intended at modulating the compounds
electrophilicity. The new set is called here for simplicity
“
aromatic B-CePs” as their characteristic feature is the presence
of aromatic linkers connecting the two reactive moieties (Figure
b). The chemical structure of test derivatives is depicted in
1
Figure 1c. In detail, the set includes: i) derivatives with ortho-,
meta- and para- xylene linker arrangements (compounds 1-3) to
reveal the effects of orientation and distance between reactive
centers on the activity of B-CePs; ii) two exploratory derivatives
containing electron-withdrawing functional groups, respectively
bearing
a methoxy group and a methyl ester function
(
compounds 4 and 5) attached in meta position to minimize
steric effects, in order to probe the possible influence of
electronic substitutions on the aromatic system; moreover, iii) we
incorporated a pyridine linker in compound 6 to study the
2
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ChemMedChem
10.1002/cmdc.202000457
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Results and Discussion
The brominated precursor 41 was synthesized by a two-step
procedure from the readily available trimethyl 1,3,5-
benzenetricarboxylate 39 (see Supporting Information for
details), which was then reacted with an excess of the
unsaturated amine 12 to obtain the corresponding secondary
triamine 42. Afterwards, the known synthetic strategy for bis-3-
chloropiperdines, involving the N-chlorination and cyclization,
was adopted and applied to the trifunctional compound,
providing the desired tris-3-chloropiperidine 8 in good yield.
Synthesis of bis- and tris-3-chloropiperidines
The synthetic strategy to obtain the bis-3-chloropiperidines 1-7
[
4]
and 9 remained similar to our previously reported procedures,
but was adapted for the substituted B-CePs 4-6 to further extend
the range of suitable precursors (Scheme 1). The synthesis of
the final products 3, 7 and 9 has already been detailed in our
[
4b]
previous work.
Starting with the diamines 13-17 and the
functionalized aromatic building blocks 20-22 the desired
product was always the corresponding unsaturated diamine 23-
Aromatic and aliphatic B-CePs exhibit different reactivity
3
0, which was prepared by two different strategies. For the
The mechanism of DNA alkylation by B-CePs involves an
intramolecular nucleophilic displacement of the chloride in 3-
position, leading to the formation of the reactive aziridinium ion,
which is readily attacked by nucleophiles such as water or
synthesis of the bis-3-chloropiperidines with varying substitution
pattern 1-3, the preparation started with the corresponding
aromatic diamines 13-15 which were converted to their
secondary analogues 23-25 by a double reductive amination
[
5]
nucleobases. To support our hypothesis that the reactivity of
B-CePs with nucleophiles is influenced by the nature of linker,
we determined the kinetics of B-CePs consumption in aqueous
solution. We employed electrospray ionization mass
spectrometry (ESI-MS) to monitor the formation of compounds
hydrolysis products and intermediates over time. For the
aromatic compounds, we analyzed 1 and 2 to investigate the
influence of ortho- and meta- substitution and compared them
with 7 with 9 to explore the effects of aliphatic linkers on the
dynamics of B-CePs reactivity. Aqueous solutions of mentioned
compounds were incubated at 37 °C and aliquots of the reaction
mixtures were analyzed after different incubation times (0 min,
20 min, 40 min, 60 min, 180 min and overnight) by ESI-MS.
Graphs showing the relative distribution of detected species (U =
using
2,2-dimethylpent-4-enal
10
and
sodium
[
7]
triacetoxyborohydride. This strategy is well known and was
also applied in the previous synthesis of the B-CePs 7 and 9,
[
4b]
starting from their corresponding diamines 16 and 17.
In
contrast, the secondary diamines 26-28 were obtained by
nucleophilic substitution of a suitable leaving group attached to
the substituted aromatic linkers. For this reaction the aldehyde
1
0 was converted to the corresponding primary amine 12, which
was deprotonated using sodium hydride and then reacted with
the brominated or mesylated aromatic precursors 20-22.
Afterwards, the secondary diamines 23-30 were treated with N-
chlorosuccinimide (NCS) to obtain the unsaturated bis-N-
chloroamines 31-38. These products were then converted to the
desired bis-3-chlorpiperidines 1-7 and 9 by iodine catalyzed
+
+
unreacted compound, N
aziridinium ion, OH
=
aziridinium ion, 2N
= double
[
8]
+
cyclization using tetrabutylammoniumiodide.
=
monohydroxylated, N /OH
=
Detailed synthetic procedures for the preparation of the
precursors 10-13 and 18-22 from readily available starting
materials can be found in the Supporting Information.
monohydroxylated/aziridinium ion, 2OH = dihydroxylated) over
time are reported in Figure S1 (Supporting Information). The
kinetics of the reaction of aliphatic versus aromatic compounds
was very different. Derivatives 7 and 9 were rapidly consumed in
water, with the unreacted species coexisting with mono and
dihydroxylated species formed at high rates already from 20
minutes of incubation. At 3 h both compounds were almost
totally reacted. The reactivity of aromatic compounds was slower
compared to the aliphatic analogues, as expected. Besides, we
O
O
R
a)
b)
O
O
R
+
noted the slower kinetic of 1 compared to 2. The N species of 2
O
O
R
formed immediately and turned out to be rather stable, with the
+
ratio between unreacted 2 and its N ion almost preserved
39
41: R=Br (62% 2 steps)
during the first hour of incubation, suggesting the presence of a
pre-equilibrium; the dihydroxylated species formed from 60 min
up to 3 h. Conversely, compound 1 started reacting with water
only after 20 min of incubation leading to the formation of
c)
4
2: R=
Cl
N
H
+
hydroxylated species. Interestingly, the 1 N intermediate was
N
never detected in time, attesting the different reactivity profile of
the ortho compared to the meta analogue. Based on these
findings, we proceeded with the evaluation of reactivity with
biological macromolecules.
N
d)
e)
Cl
N
Meta arrangement increases aromatic B-CePs reactivity
with plasmid DNA
Cl
8
(51% 3 steps)
In line with our precedent studies on 3-chloropiperidines, the
new derivatives were initially evaluated for their ability to react
Scheme 2. Synthesis of tris-3-chloropiperidine 8. Reactants and conditions: a)
LAH, dry THF, 0°C to rt to reflux, 18 h; b) PBr , dry Et O, 0°C to rt, 24 h; c)
,2-dimethylpent-4-en-1-amine 12, dry DCM, 0°C to rt, 68 h; d) NCS, dry DCM,
°C to rt, 2.5 h; e) TBAI (cat.), dry CHCl , reflux, 4 h.
[4, 9]
with nucleic acids and induce DNA strand breaks.
The
3
2
2
0
electrophoretic cleavage assay allows to visualize the
3
3
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ChemMedChem
10.1002/cmdc.202000457
FULL PAPER
compound-mediated conversion of the supercoiled plasmid
substrate (SC) into its retarded nicked and linearized forms (OC
substituent reduces the reactivity exhibited by the parent
compound. Although there is no direct conjugation to the
aromatic system in the case of the substituted B-CePs, the
electronic nature of the aromatic system clearly influences the
reactivity of the compounds, which decreases in presence of
these electron withdrawing groups. Since there is no direct
conjugation between the piperidine-nitrogen and the aromatic
system, the impact of substituents is limited to inductive effects.
Such substituent effects are described by linear free energy
=
open circular, L = linearized). In detail, the pBR322 plasmid
was incubated with increasing concentrations of test compounds
for 3 h at 37 °C and reaction products were run on agarose gel.
The bar chart in Figure 2 reports the EC50 values of test
compounds in ascending order. For the sake of clarity, final
EC50s of the test derivatives are detailed in Table S1.
With the exception of compounds 1 and 8, test derivatives
exhibited EC50 values below 5 µM, thus demonstrating to
efficiently cleave DNA in vitro. In line with the previous
[
10]
relationships derived from the Hammett equation.
Though
initially developed for substituent effects on the ionization of
benzoic acid derivatives, they are also applicable to different
organic reactions and have already been applied to aromatic
[
4b]
observations for 3 with a different substrate,
the new results
confirm that aromatic linkers (blue bars in Figure 2) decrease the
DNA cleavage potency of B-CePs: only the aliphatic compounds
[
11]
nitrogen mustards.
The correlation observed for the
9
and 7 (green bars in Figure 2) possess potencies below 1 µM.
substituted B-CePs compared to compound 2 is not perfect, but
similar to the reactivity of substituted benzylamines as
[
12]
1
00
nucleophiles. This relationship seems to be comparable to our
experiments, since the first step in the reaction of B-CePs is
always the formation of an aziridiunium ion, provided by a
nucleophilic attack of the benzylic piperidine-nitrogen.
Accordingly, the introduction of a nitrogen heteroatom, reducing
the electron density of the aromatic linker in 6, also decreased
the compounds reactivity when compared to analogue 2.
Unintuitively, also the third additional reactive moiety of the Tri-
CeP 8 curtailed its reactivity compared to the bifunctional
analogue 2.
5
2
0
0
1
0
5
.0
.5
.0
2
0
7
9
2
3
6
4
5
8
1
Compound
a.
Figure 2. DNA cleavage EC50 values of analyzed B-CePs. The supercoiled
pBR322 plasmid was incubated with increasing concentrations of test
compounds at 37 °C for 3 h in BPE buffer. EC50 values were calculated by
comparing the intensities of the supercoiled species band to the negative
control as a function of compound concentration. Average EC50s and standard
deviations result from two independent experiments. Green bars: aliphatic
B-CePs; blue bars: aromatic B-CePs; blue checkered bar: Tri-CeP.
C
1
2
3
0.5ꢀꢀꢀꢀ 5ꢀꢀꢀꢀꢀ50 0.5ꢀꢀꢀꢀ 5ꢀꢀꢀꢀꢀ50 0.5ꢀꢀꢀꢀꢀ5ꢀꢀꢀꢀ50ꢀꢀꢀ[µM]ꢀꢀꢀꢀ
OC
SC
A comparison between the isomers 1, 2 and 3, respectively
bearing ortho-, meta- and para-xylene linkers, allowed to
evaluate the influence of different substitution patterns on the
reactivity of aromatic B-CePs. Resulting EC50s demonstrated
that, while the meta- and para- substitutions conferred a similar
potency to compounds 2 and 3, the activity of the ortho
positional isomer 1 toward DNA decreased consistently with the
reduced reactivity with water. It that conditions, after the first
hydroxylation of 1, intramolecular hydrogen bonding interactions
between the newly formed hydroxyl group and the nitrogen
atoms of both chloropiperidine rings might reduce the reactivity
of the second 3-chloropiperidine moiety. Such interactions
cannot be established when increasing the distance between the
two reactive groups as in the case of the meta isomer 2. In
addition, the restrained rotability and steric hindrance of 1 may
prevent an efficient attack to guanine residues in DNA. Given
the limited cleavage of 1 observed at 3 h, the reactivity of 1, 2
and 3 was indeed investigated at the longer incubation time of
b.
C
1
2
3
0.5ꢀꢀꢀꢀꢀ5ꢀꢀꢀꢀ 50
0.5ꢀꢀꢀꢀ 5ꢀꢀꢀꢀꢀ50 0.5ꢀꢀꢀꢀꢀ5ꢀꢀꢀꢀ50ꢀꢀꢀ[µ
M]ꢀꢀꢀꢀ
OC
L
SC
Figure 3. DNA cleavage assay for compounds 1, 2 and 3. DNA cleavage
assay upon incubation with pBR322 after 3 h (a) and 18 h (b) at 37 °C at
increasing compound concentrations (0.5, 5, 50 µM). SC: supercoiled plasmid;
L: linearized plasmid; OC: open circular plasmid; C: supercoiled pBR322
plasmid control.
1
8 h. Resulting gel images are shown in Figure 3a (3 h) and 3b
(
18 h). Though much less reactive than its analogues,
compound 1 demonstrated to extensively fragment the plasmid
at the longer incubation time of 18 h, as clearly attested by the
appearance of an intense smear. This suggests that the reaction
with nucleic acids still occurs, although it needs time to achieve
DNA cleavage compared to the meta and para isomers.
Compounds 4 and 5, tested to investigate the influence of
substitution of the xylene linker, show higher EC50 values than 2,
indicating that the insertion of the ether group and the ester
Aromatic linkers address B-CePs toxicity against pancreatic
cancer cells
After analyzing the reactivity of compounds with water and with
purified DNA, the biological value of compounds was assessed
by the MTT assay on a panel of human cancer cell lines, namely
colorectal adenocarcinoma HCT-15, pancreatic adenocarcinoma
BxPC-3 and ovarian carcinoma 2008 cells. Table 1 reports the
4
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FULL PAPER
IC50s observed after 72 h of treatment with tested B-CePs along
with previously determined IC50s of the reference nitrogen
Results concerning the pancreatic cell line are quite divergent:
very interestingly, all and only the aromatic derivatives showed
nanomolar IC50s against pancreatic tumor cells, showing to be
on average 200-times more toxic for BxPC-3 than chlorambucil,
a difference not so evident considering HCT-15 and 2008 cells.
To highlight the unexpected tropism toward the pancreatic
cancer cell line, the preferential activity index (P.A.I.) expressed
as ratio between the IC50 of each compound against 2008 or
HCT-15 relative to BxPC-3 cells is reported in Figure 4.
[
9]
mustard chlorambucil (Chl).
For its poor solubility and
compatibly with the use of maximum 0.5% DMSO in cell culture,
compound 5 was not tested over 25 µM.
Table 1. MTT assay IC50s with the associated standard deviations on HCT-15,
BxPC-3 and 2008 cells of test B-CePs on BxPC-3 cells after treatment for 72 h.
IC50 values were calculated by a four-parameter logistic model.
Compound
MTT IC50 values / µM
6
4
2
0
0
0
HCT-15
2008
BxPC-3
1
2
3
4
5
6
7
8
9
23.8±7.2
3.3±1.1
3.0±0.9
9.5±2.7
>25
16.1±5.6
2.8±1.1
2.3±0.3
4.0±1.9
>25
0.4±0.1
0.3±0.2
0.4±0.1
0.5±0.2
0.4±0.1
0.4±0.1
5.5±1.1
0.3±0.2
9.4±1.6
75.3±5.1
HCT-15/BxPC-3
008/BxPC-3
2
1
0
1
2
3
4
6
7
8
9
Chl
Compound
Figure 4. Preferential activity indexes (P.A.I.) of 3-chloropiperidines. P.A.I are
expressed as ratio between the compound MTT IC50 against HCT-15 and
BxPC-3 cells (solid bars), as well as 2008 and BxPC-3 cells (dashed bars).
Chl: Chlorambucil. Green bars: aliphatic B-CePs; blue bars: aromatic B-CePs;
grey bar: Chl.
14.2±2.2
3.2±2.5
9.3±2.9
6.8±2.1
49.7±3.3
10.7±2.7
2.6±0.8
14.3±5.5
11.3±2.2
12.5±2.1
Notably, we observe a marked selectivity for the pancreatic cell
line (Figure 4), with the least DNA reactive compounds 1 and 8
among the most selective for BxPC-3 cells. On the other hand,
the aliphatic compounds 7 and 9 (green bars in Figure 4)
showed no different behavior throughout the screened panel,
resulting in no selectivity and much higher IC s for pancreatic
[
a]
Chl
[
a]
.[9]
Reference IC50s reported in our previous work
5
0
Chl: Chlorambucil
cancer cells.
Given the encouraging cytotoxicity observed against the
pancreatic cancer cell line, we proceeded to better characterize
the biological profile of the tested compounds.
Of note, all derivatives demonstrated a valuable cytotoxicity
against the three human tumor cell lines, in most cases
[
9]
improved compared to the reference drug chlorambucil.
Interestingly, results suggest a separate analysis for HCT-15
and 2008 compared to BxPC-3 tumor cells: in fact, B-CePs
behaved similarly against colorectal and ovarian cancer cells,
exhibiting IC50 values between 1 and 20 µM, whereas the new
set was one or two orders of magnitude more potent against the
pancreatic cancer cells. Considering the former two cell lines,
B-CePs exploit transporter-mediated uptake
A modified MTT assay was performed on selected derivatives to
investigate their mode of entry in BxPC-3 cells to disclose
[13]
possible mechanisms of transporter-mediated uptake.
We
analyzed the aromatic B-CePs 1, 2, 4, 6 together with compound
as representative of the aliphatic subset. The BxPC-3 selective
aromatic derivatives exhibit
a positive correlation between
9
cytotoxicity and reactivity. The poorly reactive isomer 1 bearing
the ortho-xylene linker turned out to be less cytotoxic than its
meta- and para-analogues 2 and 3. The ether and ester
substituents led to a decrease both in the reactivity and
cytotoxicity of 4 and 5 compared to compound 2, following the
ranking 2>4>5. Moreover, compound 2 resulted more cytotoxic
and more reactive than its pyridine analogue 6. Finally, the
trifunctional compound 8 was also examined. Given the almost
equivalent reactivity and cytotoxicity profile between 2 and 3, the
latter compound was not included in this analysis. Besides,
being a non-optimal candidate for its poor solubility, compound 5
was not further investigated. Cells were seeded in two
microplates and incubated for 5 h in parallel at 37 °C or 4 °C.
Given the short incubation time, the adopted range of
concentrations was increased compared to the IC50s obtained at
bifunctional agent
2 was also more cytotoxic than the
trifunctional derivative 8, always in line with cleavage results.
However, when considering the aliphatic derivatives 7 and 9, it is
evident that the overall reactivity and cytotoxicity rankings do not
match: unsurprisingly, this suggests that factors other than
reactivity finally contribute to determine the cellular activity of B-
CePs. When considering the aromatic series, we can conclude
that for the colorectal and ovarian cancer models our results
corroborate the mechanism of action of this class of alkylators
and confirm the feasibility of modulating the activity of B-CePs
by acting on the chemistry of the linker.
7
2 h. At the end of the incubation time the wells were rinsed with
PBS, fresh medium was added and both microplates were
incubated at 37 °C in the absence of compound. Finally, cell
viability was assessed at 72 h and resulting curves are shown in
Figure 5.
5
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.202000457
FULL PAPER
THP
1
031
TH
A
P
2
F
0
2
31
TH
Aromatic B-CePs directly damage cellular DNA
1
00
100
100
100
1
00
37 °C
9
0
0
90
90
4 °C
9
8
7
6
5
0
0
0
0
0
9
In support of the activity observed on isolated DNA and as proof
of principle of their mechanism of action, the alkaline Single-Cell
Gel Electrophoresis (SCGE) assay was performed to evaluate
8
0
0
80
80
8
7
0
0
70
70
7
AF2
THP036
6
0
0
60
60
6
1
00
100
90
[14]
3
7 °C
4 °C
3
7 °C
A
A
5
50
0
FJ
0
FJ
37 °C
A
3
A
2
2
4
4
potential genomic DNA lesions induced by B-CePs in cells.
5
0
0
0
5
9
8
7
6
5
0
0
0
0
0
0
4 °C
3
6
9
1
2
1
5
4 °C
3
6
6
0
3
6
9
12
15
5
0
3
6
9
1
2
1
5
1
1
d
1
0
o
o
0
0
n
n
0
c
The highly cytotoxic derivative 2 was chosen as representative
of the aromatic B-CePs set. BxPC-3 cells were exposed for 6 h
to 5 µM of compound 2: this short incubation time allows
observing only direct compound-mediated DNA damage (DNA
strand breaks, alkaline-labile sites), while excluding apoptotic
fragmentation. Lesioned genomic DNA subjected to alkaline
treatment and electrophoresis leads to the appearance of a
characteristic comet tail upon staining with SYBR Green I. The
same experiment was performed in presence of 0.5% DMSO
and of the reference drug chlorambucil (Chl) at the concentration
of 100 µM, representing the negative and positive controls
respectively. Figure 6 shows the results of the SCGE assay:
Figure 6a reports the relative percentage of comets, i.e. the
number of cells forming a comet relative to the total number of
cells detected in two randomly captured fields from two
independent experiments per each condition, while Figure 6b
reports representative images of the comets in each condition.
Compound 2 harshly damages cellular DNA: in fact, upon 6 h of
incubation with 5 µM of compound 2, at least one third of treated
cells gave rise to a fragmented comet. This phenomenon was
much reduced in case of the positive control chlorambucil: even
at the much higher concentration of 100 µM, only 15% of cells
nuclei got damaged by the drug, and the damage was less
severe, as attested by the representative images reported in
Figure 6b. Overall, these cellular evidences endorse the
mechanism of action of this class of DNA alkylators and justify
the significant difference in cytotoxicity observed between our
new set of derivatives and chlorambucil.
Compound c
Compound co
C
o
o
m
m
p
p
o
o
n
d
d c
c
o
o
n
en
n
t
o
io
n
M
µ
]
M
80
70
C
C
C
C
o
o
o
o
m
m
m
m
p
p
p
p
o
o
o
o
u
u
u
u
n
nd
n
n
d
d
c
c
c
c
o
o
n
n
c
c
e
e
e
e
n
n
n
n
tr
tr
a
r at i
r at i
a
t
o
o
io
io
n
n
n
[µ
[µ
M
M
µM
µ
]
]
M
C
un
u
nc
e
c
t rt r
a
a
ti
n
[µ
/
c
t
t
n
-
/
AF2
THP036
TH
9
9
0
P
0
036
T
T
H
H
P
P
4
0
0
54
5
4
A
A
THP
F
F
J
J
A
6
A24
054
24
RO
RO
1
00
0
100
60 100
100
37 °C
4 °C
37 °C
° C° C
4 °C
° C° C
37 °C
° C° C
4 °C
° C° C
1
1
0
0
0
1
1
0
0
0
0
10
9 09 0
1
0
0
37
3
7
8
8
0
0
3
3
7
7
9
0
0
0
90
50 90
90
9
9
9
9
0
0
4
4
4
4
0
3
6
9
12
15
0
3
6
9
12
15
8
0
80
80
C
8
8
8
0
o
0
0
mpound concentration [µM]
C
8
o
0
mpound concentration [µM]
70
70
80
80
37 °C
4 °C
7
0
0
7
7
0
7
7
0
0
70
70
7
0
70
AFJA24
ROE 321
60
60
6
0
0
60
6
6
0
6
6
0
0
60
60
6
0
60
1
00 AFJA24
100
5
0
37 °C
50 37 °C
37 °C
5
5
0
0
5
5
0
0
50
50
50
50
5
90 50
0
3
6
9
12
15 90 50
0
3
6
9
12
15
15
0
0
4 °C
3
3
6
6
9
9
1
1
2
2
1
1
5
5
0
0
4 °C
3
3
6
6
9
9
12
12
1
2
1
5
0
4 °C
5
5
10
10
0
3
6
9
12
15
0
3
6
9
15
0
Compound concentration [µM]
Compound conc
0
entration
0
3
[
3
µM]
6
6
9
9
C
Compound concentration [µM] 80
C
C
o
o
o
m
m
m
p
p
p
o
o
ou
u
u
n
n
n
d
d
d
c
c
o
o
o
n
n
n
c
c
c
e
e
e
n
n
n
t
t
t
r
r
r
a
a
a
t
t
i
t
i
o
i
o
o
n
n
n
[
[
µ
µ
/
M
M
µ
]
]
Compound concentration / µM
Compound concentration [µM]
Compound concentration [µM]
Compound concentration [µM]
37 °C
Compound co
Compound c
8
7
6
5
0
0
0
0
Compound concentra
Compound concentra
4
°C
9
F
ROE 321
AFJ
8
A24
70
60
50
ROE 321
1
00
100
90
100
37 °C
37 °C
4 °C
9
0
90
4 °C
0
3
6
9
12
15
0
5
10
15
20
25
30
8
0
80
C
8
o
0
mpound concentration [µM]
Compound concentration [µM]
7
0
70
7
0
6
0
60
50
6
0
5
0
0
5
5
0
3
6
9
12
12
25
15
30
0
5
10
15
20
25
30
5
0
5
10
15
20
6
9
15
Compound concentration [µM]
Compound concentration [µM]
Compound concentration / µM
CC oo mm pp oo uu nn d cc oo nn cce en nt rt ar at i toi on n[ µ /M µ] M
und concentration [µM]
Figure 5. Cell viability curve for BxPC-3 cells upon treatment at 37 °c vs 4 °C
with test compounds (1, 2, 4, 6, 8, 9). BxPC-3 cell viability upon exposure to
increasing concentrations of selected compounds (from 3 µM to 12 µM of rigid
derivatives 1, 2, 4, 6, 8 and from 10 µM to 50 µM of 9) for 5 h at 37 °C (red
curve) or 4 °C (blue curve), followed by a gentle rinse of wells with PBS and
addition of fresh RPMI medium. The MTT assay was performed at 72 h.
The differential viability resulting from the incubation of twin
microplates at 37 °C or 4 °C allows to estimate the relative
contribution of transporters to the net intracellular accumulation
of the compounds. The formazan absorbance of untreated
controls was almost identical between plates incubated at 37 °C
and 4 °C, suggesting that the sole initial temperature gap was
not responsible for differences in viability, measured as
metabolic state. At 37 °C (red line), both passive diffusion and
transporter-mediated uptake could contribute to the net
accumulation of cytotoxic agents in cells. On the other hand, the
cytotoxicity observed at 4 °C (blue line) is almost exclusively a
result of the passive diffusion of compounds across the cell
membrane, since transporters are inhibited at low temperatures.
A substantial drop in cell viability was observed upon treatment
with all B-CePs derivatives at 37 °C compared to 4 °C (Figure 5),
suggesting a key role for membrane transporters in determining
intracellular accumulation of tested B-CePs. Nevertheless, the
a.
4
3
2
1
0
0
0
0
0
**
*
2
Chl
MSO
D
b.
DMSO
+ꢀChl (100ꢀµM)
+ꢀ2 (5ꢀµM)
residual cytotoxicity observed at
4 °C demonstrates that
compounds are also capable of passively diffusing through the
cell membrane.
Interestingly,
a comparable uptake profile was highlighted
between 2 and 9, respectively aromatic and aliphatic analogues.
This evidence confutes the hypothesis that the tropism toward
BxPC-3 cells observed for aromatic but not aliphatic derivatives
could depend on an enhanced cell penetration of the former.
Results suggest that the pyridine linker enhances the
intracellular accumulation of 6 by fostering its passive diffusion.
Interestingly, the third reactive moiety of 8 apparently enhances
its passive diffusion while clearly decreasing its transporter-
mediated uptake compared to other bifunctional analogues.
Figure 6. Alkaline Single-Cell Gel Electrophoresis (SCGE) assay. Alkaline
SCGE assay was performed on BxPC-3 cells upon incubation with DMSO
0.5%, chlorambucil (Chl) and compound
2 for 6 h at the indicated
concentrations. (a) The bar chart reports the relative percentage of comets (n°
cells forming a comet/total n° of cells) detected in two randomly captured fields
from two independent experiments per condition. (b) Representative images
(40x) of treated samples (including negative and positive controls) with comets
are also reported. Paired t-test: * p<0.05 ** p<0.001
6
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ChemMedChem
10.1002/cmdc.202000457
FULL PAPER
3
-chloropiperidines exhibit remarkable cytotoxicity against
function to hamper potential self-stacking interactions between
aromatic molecules, thus increasing the efficiency of spheroid
penetration.
pancreatic cancer spheroids
In light of the promising cytotoxicity observed against BxPC-3
2
D monolayer cultures, the activity of the aromatic B-CePs was
Conclusions
further evaluated on 3D BxPC-3 spheroids, a more sophisticated
model for the in vitro screening of therapeutics that better
[
15]
approximates the milieu and hypoxic core of solid tumors. The
Acidic Phosphatase assay (APH) was performed to assess
viability of BxPC-3 spheroids grown in 96-well round-bottom
microplates for two days and then incubated with selected
This study highlighted the anticancer potential of a new set of 3-
chloropiperidines designed to improve the efficacy of mustard-
based chemotherapeutics obtained through
a convenient
synthetic strategy. Different linker substituents and substitution
patterns demonstrated to modulate the reactivity of B-CePs
toward isolated DNA. Most interestingly, meta and para xylene
isomers demonstrated to provide a similar and efficient potency
of alkylation on isolated DNA, while the ortho analogue and B-
CePs incorporating electron withdrawing substituents on the
aromatic linker exhibited a delayed reactivity. Noteworthy, we
demonstrated for the first time the potent antiproliferative effect
of B-CePs on a panel of tumor cell lines. In the case of aromatic
B-CePs, a clear correlation between reactivity and cytotoxicity
was observed against ovarian 2008 and colorectal HCT-15
cancer cells. Unexpectedly, aromatic but not aliphatic linkers
confer to B-CePs nanomolar toxicities against pancreatic BxPC-
[
16]
compounds for 72 h.
are reported in table 2.
The APH IC50 values for test derivatives
Table 2. BxPC-3 3D cultures cytotoxicity. APH assay IC50 values with the
associated standard deviations of selected compounds on BxPC-3 cancer cell
3
D cultures after treatment for 72 h. IC50 values were calculated by a four-
parameter logistic model.
Compound
APH IC50 values / µM
BxPC-3
1
2
40.1±7.6
122.4±5.7
133.1±4.4
46.2±0.6
3
cells, resulting into valuable indexes of preferential activity for
3
this cancer cell line. As proof of principle of their mechanism of
4
action, the representative aromatic derivative 2 demonstrated to
damage cellular DNA to
a higher extent compared to
6
112.7±2.4
68.2±4.6
chlorambucil even at a much lower concentration. The tropism
toward pancreatic cancer cells is exceptionally relevant
considering the aggressiveness of this tumor type and recalls
what observed with monofunctional 3-chloropiperidines (M-
8
Chl
Inactive
Chl: Chlorambucil, totally inactive at the maximum tested concentration (200
[9]
CePs) against the same cell line.
µM).
In this study we also investigated the new trifunctional 3-
chloropiperidine 8, which showed a valuable activity against the
advanced model of pancreatic 3D spheroid cultures, most likely
by virtue of its lower reactivity, a feature shared with the low
reactive B-CeP 1. This attests that restrained reactivity and good
uptake are determinants for the effectiveness of 3-
chloropiperidines in this model, a precious knowledge for the
potential development of such compounds against solid tumors.
To conclude, the empowered anticancer features of aromatic B-
CePs compared to aliphatic analogues provide a versatile
starting point for the advanced synthesis of such compounds.
This exclusivity seems not to rely on differences in the
intracellular accumulation of aromatic compared to aliphatic
compounds, since both showed a very similar uptake profile.
Nevertheless, studies are ongoing to reveal which distinctive
mechanisms prior or following DNA damage are responsible for
the unexpected activity of aromatic B-CePs against BxPC-3
pancreatic cancer cells. Preferential targeting by nitrogen
mustards has recently been reported for chlorambucil against
BRCA 1/2-deficient tumors, thus demonstrating that even agents
commonly known as aspecific could exhibit cancer type
Aromatic B-CePs confirmed their cytotoxicity also against BxPC-
3
spheroids. Passing from 2D to 3D cultures, we can appreciate
more pronounced inter-compound differences: in spheroids,
compound 1 bearing the ortho-xylene linker was the most
cytotoxic, followed by 4, bearing the meta-methoxy xylene linker,
and by the new Tri-CeP agent 8. Compounds 2, 3 and 6
exhibited IC50 values over 100 µM, a relevant result considering
that chlorambucil was completely inactive up to the maximum
tested concentration of 200 µM. To understand this result, we
should reason about the requisites necessary for compounds to
be effective against spheroids. Being three-dimensional, these
systems are constituted by stratified cell layers and we can
speculate that B-CePs must efficiently penetrate them while
keeping their reactivity intact for a sufficient time. In this sense, a
lower or delayed reactivity and a good uptake profile turn out to
be crucial for the biological activity of these new set of alkylators
in 3D models. Compound
1 was the least reactive with
nucleophiles but could demonstrate effective time-dependent
DNA damaging activity (Figure 3b). Compound 8 showed also a
restrained reactivity at the DNA cleavage assay but it
demonstrated to almost exclusively enter cells by passive
diffusion, a slow and less efficient process. Noteworthy, 1 and 8
were highlighted to be among the most selective against the
pancreatic cancer cell line (Figure 4), thus showing to be
promising leads for upgraded studies as anticancer agents for
pancreatic tumors. Although more reactive than 1 and 8, the
methyl-ether compound 4 showed a valuable activity against
spheroids: this result might depend on the ability of the ether
[
17]
tropisms. In this sense, we plan to employ an omic approach
to elucidate the molecular determinants of sensitivity for
aromatic 3-chloropiperidines against BxPC-3 cells. Perturbations
of the transcriptional profile and chromatin accessibility state will
be evaluated upon cell stimulation with selected derivatives. This
signature-driven strategy may open new perspectives in the
refinement and exploration of the mechanism of action of these
new chemical entities, facilitating the mindful repositioning of B-
CePs as potential therapeutics.
7
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.202000457
FULL PAPER
spotted with 50 µL of cells-LMPA mixture and allowed to set at
Experimental Section
4
°C for 30 min. Slides were immersed in lysis buffer (Trevigen)
for 45 min, then incubated for 20 min in an alkaline
electrophoresis solution (1 mM EDTA, 300 mM NaOH) followed
by alkaline electrophoresis (1 V/cm) at 4 °C for 30 min. Slides
Materials and Methods
MS studies. The formation of B-CePs reactive species upon
were then washed twice in deionized H
for 5 min and air-dried. DNA was stained with SYBR Green I for
min at °C. Slides were examined at 5x and 40x
2
O, fixed in 70% ethanol
incubation at 37 °C in water was followed in time by ESI-MS as
[
4a]
reported precedently. A 80 µM solution of test compounds was
prepared in MilliQ water from 1 mM DMSO stocks and incubated
at 37 °C for 3 h. Small samples were taken after 0 min, 20 min,
1
0
4
magnification in a Zeiss LSM 800 confocal microscope using the
Zeiss ZEN 2.3 software system. The relative % of comets (n°
cells forming a comet/ total n° of cells) detected in two randomly
captured fields (including at least 250 cells/field) from two
independent experiments per condition was analyzed.
4
0 min, 60 min and 180 min, diluted 1:10 with methanol, and
analysed by ESI-MS. Measurements were performed in positive
ion mode using a Xevo G2-XS Qtof instrument (Waters).
Cleavage assay. The ability of compounds to cleave the
supercoiled plasmid pBR322 (Inspiralis Ltd) was investigated at
the electrophoretic cleavage assay. The assay was performed
Acid Phosphatase (APH) assay. The APH assay was
employed to assess cell viability of spheroids treated with test
[
16]
[
4a, 4b]
compounds following previously reported protocols.
BxPC-3
following previously reported materials and protocols.
B-
3
cells were seeded (1.5 x 10 cells/well) in phenol red-free RPMI
medium (Euroclone) containing antibiotics and L-glutamine (as
previously specified) as well as 10% FCS and 20% methyl
cellulose in round-bottom non-tissue culture treated 96 well-
plates (Greiner Bio-one). After 72 h from seeding, spheroids
were treated at the defined concentration of compound freshly
dissolved in DMSO to a final solvent concentration of 0.5%,
which had no detectable effect on cell killing. Upon 72 h of
incubation, spheroids were incubated with 100 µL of the assay
buffer for 3 h at 37°C (0.1 M sodium acetate, 0.1% Triton-X-100,
supplemented with ImmunoPure p-nitrophenyl phosphate by
Sigma). Subsequently, 10 µL of 1 M NaOH were added before
measuring the absorbance of each well at 405 nm (BioRad 680
CePs dilutions were freshly prepared from a 10 mM DMSO stock
in Milli-Q water, resulting in a 0.5% DMSO concentration in the
final reaction volume. Diversely, a 5 mM DMSO stock was
prepared in the case of the poorly soluble compound 5, resulting
in a final 1% DMSO concentration in the assay volume. EC50
values were calculated considering the intensity of the
supercoiled
plasmid
band
at
increasing
compound
concentrations expressed as percentage compared to the
untreated control. Experiments were performed in duplicate to
calculate average values and standard deviations.
Cell cultures. Colon (HCT-15) and pancreatic (BxPC-3)
carcinoma cell lines were purchased from ATCC (American
Type Culture Collection). The human ovarian 2008 cancer cell
line was kindly provided by G. Marverti (Department of
Biomedical Science, University of Modena and Reggio Emilia,
Modena, Italy). Cell lines were maintained in logarithmic phase
at 37 °C in a 5% carbon dioxide atmosphere using RPMI-1640
medium (Euroclone) containing 10% fetal calf serum (FCS,
Euroclone), antibiotics (50 units/mL penicillin and 50 µg/mL
streptomycin), and 2 mM L-glutamine.
microplate reader). Compounds IC50
s
were extrapolated
applying a four-parameter logistic (4-PL) model and average
values of two experimental replicates were reported.
Chemistry
All solvents were purified by distillation prior to use and in case
of anhydrous solvents dried and stored under nitrogen
atmosphere. Commercially available reagents were used as
supplied if not stated different. Synthesis using anhydrous
solvents were carried out under Schlenk conditions. For
purification by flash column chromatography silica gel 60
MTT assay. The 72 h MTT assay was performed following
[
9, 18]
previously reported materials and protocols.
Test
compounds were dissolved in DMSO and added at defined
concentrations to the cell growth medium to a final solvent
concentration of 0.5%, which had no detectable effect on cell
killing. A modified MTT assay was employed to investigate the
uptake of test compounds. BxPC-3 cells were incubated either in
the absence or presence of the compound for 5 h at 37 °C or
1
13
(
Merck) was used. H and C NMR spectra were recorded at
1
13
Bruker Avance II 200 spectrometer ( H at 200 MHz; C at
1
5
0 MHz) and Bruker Avance II 400 spectrometer ( H at 400
1
3
MHz; C at 100 MHz) in deuterated solvents. Chemical shifts
were determined by reference to the residual solvent signals.
High-resolution ESI mass spectra were recorded in methanol
4
°C in parallel microplates. At the end of the incubation, fresh
RPMI was added to wells replacing the medium containing the
compound. Microplates were then incubated both at 37 °C up to
using
a ESImicroTOF spectrometer (Bruker Daltonics) in
7
2 h and the MTT assay was performed. The method was
positive ion mode. All elemental analysis (CHN) were performed
on a Thermo FlashEA – 1112 series instrument. NMR spectra of
all final products, as well as the synthetic procedures of the
precursors 10-13, 18-22 and 40-43 are included in the
Supporting Information. The synthesis of compounds 3, 7 and 9
as well as their corresponding precursors has been described
validated in this modified version using cisplatin as positive
internal control (Figure S2). This drug is known to exploit
[
13, 19]
transporters to enter cells.
Alkaline Single-Cell Gel Electrophoresis (SCGE). The
alkaline SCGE assay allowed to investigate the possible direct
damage of genomic DNA induced by B-CePs on BxPC-3 cells.
Experiments were performed following a previously reported
[
4b]
elsewhere.
[
20]
5
protocol with only minor modifications.
3 x 10 cells were
2
Synthetic procedures
seeded in 25 cm flasks and incubated after 24 h with the
defined concentration of compound or 0.5% DMSO for 6 h.
Subsequently, cells were washed in PBS, harvested, centrifuged
General procedure A: Synthesis of diamines (23-24)
5
Under a nitrogen atmosphere 2,2-dimethylpent-4-enal (10) (2.1-
and resuspended at 1 x 10 in 1% low melting point agarose
2
.4 equiv) as well as the corresponding diamine were dissolved
(
LMPA, Trevigen). Pretreated comet slides (Trevigen) were
8
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.202000457
FULL PAPER
in anhydrous dichloromethane (10 mL/mmol of diamine) and
sodium triacetoxyborohydride (2.6-3 equiv) was added portion
wise at 0 °C, followed by acetic acid (2.2-2.4 equiv). The mixture
was stirred at room temperature for 16-18 h and was then
quenched by the addition of 20% NaOH solution. The phases
were separated and the aqueous layer was extracted three
times with dichloromethane. The combined organic extracts
were washed with brine, followed by distilled water and dried
6.84 (s, 2H), 5.81 – 5.73 (m, 2H), 5.03 – 4.98 (m, 4H), 3.82 –
3.81 (m, 7H), 2.40 (s, 4H), 2.04 – 2.02 (m, 4H), 0.91 (s, 12H)
1
3
ppm; C NMR (101 MHz, CDCl
3
) δ = 160.04, 135.39, 120.61,
117.19, 112.60, 59.35, 55.42, 54.41, 44.72, 34.37, 25.57 ppm;
+
HRMS (ESI): m/z calcd for
23 39 2
C H N O : 359.3057; found:
+
359.3053 [M+H] .
Methyl 3,5-bis-[(2,2-dimethylpent-4-enyl)aminomethyl]-
benzoate (27)
4
over MgSO . The solvent was removed under reduced pressure
and the crude product was obtained, which was used in the next
step without further purification.
Was prepared according to the general procedure B from 3,5-
Bis-(bromomethyl)benzoic acid methyl ester (21) (1.17 g,
3
.63 mmol) and 2,2-dimethylpent-4-en-1-amine (12) (0.99 g,
1
,2-Bis-[(2,2-dimethylpent-4-enyl)aminomethyl]benzene (23)
8.72 mmol) yielding the title compound as a yellow oil (1.38 g,
1
Was prepared according to the general procedure A from 1,2-
bis(aminomethyl)benzene (13) (0.71 g, 5.23 mmol) and 2,2-
dimethylpent-4-enal (10) (1.41 g, 12.55 mmol) yielding the title
3
3.56 mmol). H NMR (400 MHz, CDCl ) δ = 7.89 – 7.85 (m, 2H),
7.58 – 7.53 (m, 1H), 5.83 – 5.73 (m, 2H), 5.03 – 4.97 (m, 4H),
3.91 (s, 3H), 3.83 (s, 4H), 2.35 (s, 4H), 2.02 (d, J = 7.5 Hz, 4H),
1
13
compound as a pale red oil (1.50 g, 4.58 mmol). H NMR (400
3
0.89 (s, 12H) ppm; C NMR (101 MHz, CDCl ) δ = 167.45,
MHz, CDCl
3
) δ = 7.33 – 7.29 (m, 2H), 7.26 – 7.22 (m, 2H), 5.84
141.33, 135.60, 132.53, 130.33, 127.93, 117.01, 59.67, 54.34,
–
2
5.74 (m, 2H), 5.03 – 4.96 (m, 4H), 3.83 (s, 4H), 2.41 (s, 4H),
52.22, 44.76, 34.50, 25.67 ppm; HRMS (ESI): m/z calcd for
1
3
+
+
.01 (d, J = 7.5 Hz, 4H), 0.89 (s, 12H) ppm; C NMR (101 MHz,
) δ = 139.21, 135.65, 129.78, 127.19, 116.96, 60.33,
2.93, 44.86, 34.54, 25.69 ppm; HRMS (ESI): m/z calcd for
24 39 2 2
C H N O : 387.3006; found: 387.3008 [M+H] .
CDCl
3
5
2,6-Bis-[(2,2-dimethylpent-4-enyl)aminomethyl]pyridine (28)
Was prepared according to the general procedure B from 2,6-
Bis-[(methylsulfonyloxy)methyl]pyridine (22) (1.29 g, 4.39 mmol)
and 2,2-dimethylpent-4-en-1-amine (12) (1.19 g, 10.53 mmol)
+
+
C
22
H
37
N
2
: 329.2951; found: 329.2950 [M+H] .
1
,3-Bis-[(2,2-dimethylpent-4-enyl)aminomethyl]benzene (24)
Was prepared according to the general procedure A from 1,3-
bis(aminomethyl)benzene (14) (1.03 g, 7.56 mmol) and 2,2-
dimethylpent-4-enal (10) (1.75 g, 15.60 mmol) yielding the title
yielding the title compound as a yellow oil (1.41 g, 4.28 mmol).
1
3
H NMR (400 MHz, CDCl ) δ = 7.58 (t, J = 7.6 Hz, 1H), 7.18 (d, J
= 7.6 Hz, 2H), 5.85 – 5.74 (m, 2H), 5.03 – 4.98 (m, 4H), 3.88 (s,
1
compound as a colourless oil (2.18 g, 6.64 mmol). H NMR (200
4H), 2.39 (s, 4H), 2.03 (d, J = 7.5 Hz, 4H), 0.91 (s, 12H) ppm;
1
3
MHz, CDCl
3
) δ = 7.42 – 7.28 (m, 4H), 5.90 – 5.65 (m, 2H), 5.11
C NMR (101 MHz, CDCl
3
) δ = 159.67, 136.80, 135.59, 120.33,
–
4
1
3
3
4.93 (m, 4H), 4.13 (s, 4H), 2.93 (s, 4H), 2.08 (d, J = 7.2 Hz,
117.00, 60.16, 56.03, 44.82, 34.50, 25.62 ppm; HRMS (ESI):
1
3
+
+
H), 0.95 (s, 12H) ppm; C NMR (50 MHz, CDCl
3
) δ = 137.79,
36 3
m/z calcd for C21H N : 330.2904; found: 330.2901 [M+H] .
35.33, 130.18, 128.80, 128.39, 117.43, 73.42, 70.49, 45.03,
+
5.75, 25.95 ppm; HRMS (ESI): m/z calcd for C22
H
37
N
2
:
General procedure C: Synthesis of bis-N-chloroamines
(31-36)
+
29.2951; found: 329.2958 [M+H] .
Under a nitrogen atmosphere the crude unsaturated diamine
was dissolved in anhydrous dichloromethane (10 mL/mmol of
diamine) and freshly recrystallized N-chlorosuccinimde
(2.4 equiv) was added portion wise at 0 °C. The mixture was
stirred at 0 °C for 30 min and for an additional 2-2.5 h at room
temperature. The solvent was removed under reduced pressure
and the crude product was purified by flash column
chromatography.
General procedure B: Synthesis of diamines (26-28)
Under a nitrogen atmosphere sodium hydride (2.8 equiv) was
suspended in anhydrous tetrahydrofuran (10 mL/100mg of
sodium hydride) and 2,2-dimethylpent-4-en-1-amine (12)
(
2.4 equiv) was added dropwise at 0 °C. The mixture was stirred
at 0 °C for 30 min and the corresponding dibromide or bis-
mesylate, dissolved in anhydrous tetrahydrofuran (2 mL/mmol of
dibromide/bis-mesylate), was added dropwise at
0
°C.
Afterwards the mixture was stirred for 1 h at 0 °C and for an
additional 20 h at room temperature. The reaction was then
quenched by the addition of 10% NaOH solution. The phases
were separated and the aqueous layer was extracted three
times with ethyl acetate. The combined organic extracts were
washed with distilled water, followed by brine and dried over
1,2-Bis-[N-chloro(2,2-dimethylpent-4-
enyl)aminomethyl]benzene (31)
Was prepared according to the general procedure C from 1,2-
Bis-[(2,2-dimethylpent-4-enyl)aminomethyl]benzene (23) (1.48 g,
4.49 mmol) and purified by flash column chromatography
(pentane/TMBE 20:1), yielding the title compound as a pale
1
MgSO
4
. The solvent was removed under reduced pressure and
yellow oil (1.35 g, 4.10 mmol, 66 % over 2 steps). H NMR (400
the crude product was obtained, which was used in the next step
without further purification.
MHz, CDCl
3
) δ = 7.39 – 7.35 (m, 2H), 7.32 – 7.28 (m, 2H), 5.78
– 5.66 (m, 2H), 5.02 – 4.95 (m, 4H), 4.28 (s, 4H), 2.94 (s, 4H),
1
3
2
.03 (d, J = 7.4 Hz, 4H), 0.92 (s, 12H) ppm; C NMR (101 MHz,
CDCl ) δ = 136.74, 135.22, 130.86, 127.92, 117.41, 73.59,
67.96, 45.19, 35.61, 26.03 ppm; HRMS (ESI): m/z calcd for
5
-Methoxy-1,3-bis-[(2,2-dimethylpent-4-enyl)aminomethyl]-
3
benzene (26)
+
+
Was prepared according to the general procedure B from 5-
22 2 2
C H35Cl N : 397.2172; found: 397.2170 [M+H] .
Methoxy-1,3-bis-[(methylsulfonyloxy)methyl]benzene
(20)
2.50 g, 7.71 mmol) and 2,2-dimethylpent-4-en-1-amine (12)
2.09 g, 18.46 mmol) yielding the title compound as a yellow oil
(
(
(
1,3-Bis-[N-chloro(2,2-dimethylpent-4-
enyl)aminomethyl]benzene (32)
1
3
2.71 g, 7.55 mmol). H NMR (400 MHz, CDCl ) δ = 6.92 (s, 1H),
9
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.202000457
FULL PAPER
Was prepared according to the general procedure C from 1,3-
Bis-[(2,2-dimethylpent-4-enyl)aminomethyl]benzene (24) (1.47 g,
added. The mixture was heated to 60 °C (oil bath temperature)
for 2-2.5 h and the solvent was removed under reduced
pressure. The product was purified by flash column
chromatography. The resulting bis-3-chloropiperidines were
obtained as an inseparable mixture of diastereomeres.
4
.48 mmol) and purified by flash column chromatography
pentane/TMBE 10:1), yielding the title compound as
colourless oil (1.63 g, 4.10 mmol, 80 % over 2 steps). H NMR
400 MHz, CDCl ) δ = 7.38 – 7.30 (m, 4H), 5.83 – 5.71 (m, 2H),
.05 – 4.97 (m, 4H), 4.13 (s, 4H), 2.93 (s, 4H), 2.08 (d, J = 7.6
(
a
1
(
3
5
1,2-Bis-[(3-Chloro-5,5-dimethylpiperidin-1-
yl)methyl]benzene (1)
1
3
3
Hz, 4H), 0.95 (s, 12H) ppm; C NMR (50 MHz, CDCl ) δ =
1
4
C
37.79, 135.33, 130.18, 128.81, 128.40, 117.43, 73.42, 70.49,
Was prepared according to the general procedure D from 1,2-
bis-[N-chloro(2,2-dimethylpent-4-enyl)aminomethyl]benzene (31)
5.02, 35.75, 25.95 ppm; HRMS (ESI): m/z calcd for
+
+
22
H
35Cl
2
N
2
: 397.2172; found: 397.2178 [M+H] .
(1.34 g,
3.36 mmol)
and
purified
by
flash
column
chromatography (pentane/TMBE 20:1), yielding the title
1
5
-Methoxy-1,3-bis-[N-chloro(2,2-dimethylpent-4-
compound as a pale yellow oil (1.07 g, 2.69 mmol, 80%).
H
enyl)aminomethyl]benzene (34)
NMR (400 MHz, CDCl ) δ = 7.32 – 7.26 (m, 2H), 7.24 – 7.19 (m,
3
Was prepared according to the general procedure C from 5-
Methoxy-1,3-bis-[(2,2-dimethylpent-4-enyl)aminomethyl]benzene
2H), 4.09 – 3.98 (m, 2H), 3.74 (d, J = 13.1 Hz, 1H), 3.59 (q, J =
13.2 Hz, 2H), 3.43 (d, J = 13.1 Hz, 1H), 3.13 – 3.03 (m, 2H),
2.42 – 2.32 (m, 2H), 2.00 – 1.89 (m, 4H), 1.83 – 1.75 (m, 2H),
1.36 (t, J = 12.3 Hz, 2H), 1.04 (d, J = 7.4 Hz, 6H), 0.91 (s, 6H)
(
26) (1.54 g, 4.28 mmol) and purified by flash column
chromatography (pentane/TMBE 20:1), yielding the title
1
3
13
compound as a pale yellow oil (827 mg, 1.93 mmol, 44 % over 2
ppm; C NMR (101 MHz, CDCl
CDCl ) δ 137.46, 130.32, 127.08, 77.16, 65.59, 61.74, 60.04,
54.36, 48.49, 33.60, 29.45, 25.52 ppm; HRMS (ESI): m/z calcd
3
) δ = C NMR (101 MHz,
1
steps). H NMR (400 MHz, CDCl
3
) δ = 6.94 – 6.92 (m, 1H), 6.88
3
–
4
7
1
5
C
6.86 (m, 2H), 5.82 – 5.71 (m, 2H), 5.03 – 5.02 (m, 2H), 5.01 –
+
+
.97 (m, 2H), 4.09 (s, 4H), 3.82 (s, 3H), 2.91 (s, 4H), 2.07 (d, J =
for C22
analysis calcd (%) for C22
found: C 66.23; H 8.54; N 6.72.
H
35Cl
2
N
2
:397.2172; found: 397.2175 [M+H] ; elemental
1
3
.4 Hz, 4H), 0.94 (s, 12H) ppm; C NMR (101 MHz, CDCl
3
) δ =
2 2
H34Cl N
: C 66.49; H 8.62; N 7.05;
59.74, 139.19, 135.33, 122.34, 117.41, 114.18, 73.44, 70.50,
5.43, 45.07, 35.75, 25.98 ppm; HRMS (ESI): m/z calcd for
+
+
23
H
36Cl
2
N
2
ONa : 449.2097; found: 449.2100 [M+Na] .
1,3-Bis-[(3-Chloro-5,5-dimethylpiperidin-1-yl)methyl]-
benzene (2)
Methyl 3,5-bis-[N-chloro(2,2-dimethylpent-4-enyl)-
aminomethyl]benzoate (35)
Was prepared according to the general procedure D from 1,3-
bis-[N-chloro(2,2-dimethylpent-4-enyl)aminomethyl]benzene (32)
(950 mg, 2.39 mmol) and purified by flash column
chromatography (pentane/TMBE 10:1), yielding the title
Was prepared according to the general procedure C from Methyl
3
,5-bis-[(2,2-dimethylpent-4-enyl)aminomethyl]benzoate
(27)
1
(
1.37 g, 3.55 mmol) and purified by flash
column
compound as a pale yellow oil (651 mg, 1.64 mmol, 69%). H
chromatography (pentane/TMBE 10:1), yielding the title
3
NMR (400 MHz, CDCl ) δ = 7.27 – 7.15 (m, 4H), 4.16 – 4.06 (m,
compound as a colourless oil (546 mg, 1.20 mmol, 33% over 2
2H), 3.58 – 3.43 (m, 4H), 3.20 – 3.12 (m, 2H), 2.43 – 2.35 (m,
2H), 2.04 – 1.92 (m, 4H), 1.76 (dd, J = 10.9, 6.7 Hz, 2H), 1.35 (t,
J = 12.3 Hz, 2H), 1.06 (d, J = 2.8 Hz, 6H), 0.89 (d, J = 1.5 Hz,
1
steps). H NMR (400 MHz, CDCl
3
) δ = 7.95 (d, J = 1.5 Hz, 2H),
7
2
4
1
5
C
.62 – 7.60 (m, 1H), 5.04 – 5.03 (m, 2H), 5.00 (d, J = 5.4 Hz,
1
3
H), 4.15 (s, 4H), 3.93 (s, 3H), 2.94 (s, 4H), 2.08 (d, J = 7.5 Hz,
6H) ppm; C NMR (101 MHz, CDCl
3
) δ = 138.61, 129.15,
1
3
3
H), 0.95 (s, 12H) ppm; C NMR (101 MHz, CDCl ) δ = 167.00,
128.25, 127.58, 64.69, 62.43, 62.08, 54.36, 48.56, 33.53, 29.43,
+
38.31, 135.24, 134.59, 130.44, 129.90, 117.52, 73.71, 69.99,
25.27 ppm; HRMS (ESI): m/z calcd for C22
H
35Cl
2
N
2
:397.2172;
+
2.35, 45.06, 35.83, 25.95 ppm; HRMS (ESI): m/z calcd for
found: 397.2176 [M+H] .
+
+
24
H
37Cl
2
N
2
O
2
:455.2227; found: 455.2231 [M+H] .
5
-Methoxy-1,3-bis-[(3-Chloro-5,5-dimethylpiperidin-1-
2
,6-Bis-[N-chloro(2,2-dimethylpent-4-
yl)methyl]benzene (4)
enyl)aminomethyl]pyridine (36)
Was prepared according to the general procedure D from 5-
Methoxy-1,3-bis-[N-chloro(2,2-dimethylpent-4-
Was prepared according to the general procedure C from 2,6-
Bis-[(2,2-dimethylpent-4-enyl)aminomethyl]pyridine (28) (1.31 g,
enyl)aminomethyl]benzene (34) (1.31 g, 3.06 mmol) and purified
by flash column chromatography (pentane/TMBE 10:1), yielding
3
.98 mmol) and purified by flash column chromatography
(
pentane/TMBE 10:1), yielding the title compound as
a
the title compound as a pale orange oil (981 mg, 2.29 mmol,
1
1
colourless oil (284 mg, 0.71 mmol, 17 % over 2 steps). H NMR
400 MHz, CDCl ) δ = 7.73 (t, J = 7.7 Hz, 1H), 7.47 (d, J = 7.7
Hz, 2H), 5.82 – 5.72 (m, 2H), 5.03 – 4.97 (m, 4H), 4.28 (s, 4H),
75 %). H NMR (400 MHz, CDCl
3
) δ = 6.84 – 6.80 (m, 1H), 6.80
(
3
– 6.75 (m, 2H), 4.17 – 4.06 (m, 2H), 3.80 (s, 3H), 3.56 – 3.37 (m,
4H), 3.20 – 3.11 (m, 2H), 2.43 – 2.34 (m, 2H), 2.05 – 1.91 (m,
4H), 1.78 – 1.70 (m, 2H), 1.35 (t, J = 12.3 Hz, 2H), 1.07 (s, 6H),
1
3
3
.01 (s, 4H), 2.07 (d, J = 7.5 Hz, 4H), 0.94 (s, 12H) ppm;
C
1
3
NMR (101 MHz, CDCl
3
) δ = 156.85, 137.11, 135.25, 122.34,
0.89 (s, 6H) ppm; C NMR (101 MHz, CDCl
140.21, 121.37, 112.77, 77.16, 64.64, 62.33, 55.34, 54.36, 48.51,
33.52, 29.42, 25.27 ppm; HRMS (ESI): m/z calcd for
3
) δ = 159.87,
1
17.51, 73.96, 71.93, 44.97, 35.85, 25.85 ppm; HRMS (ESI):
+
+
2 3
m/z calcd for C21H34Cl N : 398.2125; found: 398.2126 [M+H] .
+
+
C
23
H
37Cl
2
N
2
O : 427.2278; found: 427.2281 [M+H] ; elemental
analysis calcd (%) for C23 O: C 64.63; H 8.49; N 6.55;
found: C 64.96; H 8.29; N 6.62.
General procedure D: Synthesis of bis-3-chloropiperidines
1-6)
2 2
H36Cl N
(
Under a nitrogen atmosphere the appropriate bis-N-chloroamine
was dissolved in anhydrous chloroform (10 mL/mmol of bis-N-
chloroamine) and tetrabutylammonium iodide (10 mol%) was
Methyl 3,5-bis-[(3-Chloro-5,5-dimethylpiperidin-1-yl)methyl]-
benzoate (5)
1
0
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.202000457
FULL PAPER
Was prepared according to the general procedure D from Methyl
CDCl
3
) δ = 7.32 – 7.27 (m, 3H), 5.81 – 5.71 (m, 3H), 5.03 – 4.97
3
,5-bis-[N-chloro(2,2-dimethylpent-4-enyl)aminomethyl]benzoate
(m, 6H), 4.12 (s, 6H), 2.92 (s, 6H), 2.06 (d, J = 7.5 Hz, 6H), 0.94
1
3
(
35) (514 mg, 1.13 mmol) and purified by flash column
(s, 18H) ppm; C NMR (101 MHz, CDCl
3
) δ = 137.84, 135.34,
chromatography (pentane/TMBE 10:1), yielding the title
129.73, 117.43, 77.16, 73.37, 70.39, 45.06, 35.75, 25.96 ppm;
1
+
compound as a pale yellow oil (447 mg, 0.98 mmol, 86%). H
HRMS (ESI): m/z calcd for C30
H
48Cl
3
N
3
Na : 578.2806; found:
+
NMR (400 MHz, CDCl
3
) δ = 7.87 – 7.82 (m, 2H), 7.48 (d, J = 7.8
578.2806 [M+H] .
Hz, 1H), 4.10 (s, 2H), 3.92 (s, 3H), 3.61 – 3.46 (m, 4H), 3.18 –
3
.09 (m, 2H), 2.36 (t, J = 9.9 Hz, 2H), 2.06 – 1.92 (m, 4H), 1.80
1,3,5-Tris-[(3-chloro-5,5-dimethylpiperidin-1-yl)methyl]-
–
1.73 (m, 2H), 1.35 (t, J = 12.3 Hz, 2H), 1.06 (d, J = 3.3 Hz, 6H), benzene (8)
1
3
0
.89 (s, 6H) ppm; C NMR (101 MHz, CDCl
3
) δ = 167.33,
Under
a
nitrogen atmosphere 1,3,5-Tris-[N-chloro-(2,2-
(43) (1.03 g,
1
39.19, 133.65, 130.36, 128.82, 77.16, 64.68, 62.01, 54.18,
dimethylpent-4-enyl)aminomethyl]benzene
5
2.27, 48.45, 33.53, 29.40, 25.25 ppm; HRMS (ESI): m/z calcd
1.85 mmol) was dissolved in anhydrous chloroform (20 mL) and
tetrabutylammonium iodide (81 mg, 14 mol%) was added. The
mixture was heated to reflux for 4 h and the solvent was
removed under reduced pressure. The product was purified by
flash column chromatography (pentane/TBME 10:1) and was
+
+
2 2 2
for C24H37Cl N O : 455.2227; found: 455.2228 [M+H] .
2
,6-Bis-[(3-Chloro-5,5-dimethylpiperidin-1-yl)methyl]-
pyridine (6)
1
Was prepared according to the general procedure D from 2,6-
Bis-[N-chloro(2,2-dimethylpent-4-enyl)aminomethyl]pyridine (36)
obtained as a pale yellow oil (0.70 g, 1.26 mmol, 68%). H NMR
3
(400 MHz, CDCl ) δ = 7.14 – 7.06 (m, 3H), 4.14 – 4.06 (m, 3H),
(
240 mg, 0.60 mmol) and purified by flash column
3.56 – 3.40 (m, 6H), 3.19 – 3.11 (m, 3H), 2.41 – 2.33 (m, 3H),
chromatography (pentane/TMBE 3:1), yielding the title
2.03 – 1.91 (m, 6H), 1.74 (dt, J = 11.0, 5.4 Hz, 3H), 1.34 (t, J =
1
13
compound as a pale yellow oil (183 mg, 0.46 mmol, 76 %). H
12.3 Hz, 3H), 1.05 (t, J = 3.1 Hz, 9H), 0.89 (s, 9H) ppm;
NMR (101 MHz, CDCl
54.39, 48.53, 33.53, 29.44, 25.31 ppm; HRMS (ESI): m/z calcd
C
NMR (400 MHz, CDCl
3
) δ = 7.64 (t, J = 7.7 Hz, 1H), 7.34 (d, J =
.1 Hz, 2H), 4.19 – 4.10 (m, 2H), 3.76 – 3.59 (m, 4H), 3.19 (d, J
8.5 Hz, 2H), 2.40 (d, J = 10.9 Hz, 2H), 2.14 (t, J = 10.6 Hz, 2H), for C30
.98 – 1.86 (m, 4H), 1.37 (t, J = 12.3 Hz, 2H), 1.09 (s, 6H), 0.90 analysis calcd (%) for C30
3
) δ = 138.51, 128.02, 64.66, 62.41, 62.10,
7
=
1
+
+
H
49Cl
3
N
3
: 556.2987; found: 556.2988 [M+H] ; elemental
: C 64.68; H 8.69; N 7.44;
H
3 3
48Cl N
1
3
(
s, 6H) ppm; C NMR (101 MHz, CDCl
3
) δ = 158.30, 137.07,
found: C 64.58; H 8.52; N 7.44.
1
21.00, 64.79, 63.95, 62.18, 54.02, 48.26, 33.53, 29.39, 25.30
+
ppm; HRMS (ESI): m/z calcd for C21
H
34Cl
2
N
3
: 398.2125; found:
+
3
98.2124 [M+H] .
Acknowledgements
1
,3,5-Tris-[(2,2-dimethylpent-4-enyl)aminomethyl]-
AS has received funding from the European Union’ s Horizon
020 Research and Innovation programme under the Marie
Skłodowska-Curie grant agreement No. 751931.
benzene (42)
2
Under a nitrogen atmosphere 1,3,5-tris(bromomethyl)benzene
(
41) (1.00 g, 2.81 mmol) was dissolved in anhydrous
dichloromethane (60 mL) and 2,2-dimethylpent-4-en-1amine
12) (1.90 g, 16.78 mmol) was added dropwise at 0 °C. The
(
mixture was stirred at room temperature for 68 h and was then References
quenched by the addition of distilled water (50 mL). The layers
were separated, the organic phase washed with brine and was
then dried over MgSO . The solvent was removed under
[1]
a) N. Saijo, Cancer Res Treat 2012, 44, 1-10; b) M. Huang,
A. J. Shen, J. Ding, M. Y. Geng, Trends Pharmacol Sci
4
2
014, 35, 41-50.
B. A. Chabner, T. G. Roberts, Jr., Nat Rev Cancer 2005, 5,
5-72.
reduced pressure and the crude product was obtained as a
[
2]
yellow oil, which was used in the next step without further
6
1
purification (1.17 g, 2.59 mmol). H NMR (400 MHz, CDCl
3
) δ =
[3]
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5
9.88, 54.81, 44.78, 34.50, 25.67 ppm; HRMS (ESI): m/z calcd
+
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52 3
for C30H N : 454.4156; found: 454.4154 [M+H] .
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ChemMedChem
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Keywords
Aromatic 3-chloropiperidines • alkylating agents • DNA damage •
structure-activity relationship • pancreatic cancer spheroids.
1
2
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ChemMedChem
10.1002/cmdc.202000457
FULL PAPER
Entry for the Table of Contents
In this study we synthesized and evaluated a new set of aromatic 3-chloropiperidines designed to improve the therapeutic potential of
mustard-based DNA alkylating agents. These small molecules exhibited a marked anti-proliferative effect preferentially against BxPC-3
pancreatic adenocarcinoma cells in 2D and 3D cultures, demonstrating to be promising candidates for the further development of sustainable
chemotherapeutics active against pancreatic tumors.
1
3
This article is protected by copyright. All rights reserved.