Appended Aromatic Moieties in Flexible Bis-3-chloropiperidines Confer Tropism against Pancreatic Cancer Cells

Article abstract of DOI:10.1002/cmdc.202000814

Nitrogen mustards (NMs) are an old but still largely diffused class of anticancer drugs. However, spreading mechanisms of resistance undermine their efficacy and therapeutic applicability. To expand their antitumour value, we developed bis-3-chloropiperidines (B-CePs), a new class of mustard-based alkylating agent, and we recently reported the striking selectivity for BxPC-3 pancreatic tumour cells of B-CePs bearing aromatic moieties embedded in the linker. In this study, we demonstrate that such tropism is shared by bis-3-chloropiperidines bearing appended aromatic groups in flexible linkers, whereas esters substituted by aliphatic groups or by efficient DNA-interacting groups are potent but nonselective cytotoxic agents. Besides, we describe how the critical balance between water stability and DNA reactivity can affect the properties of bis-3-chloropiperidines. Together, these findings support the exploitation of B-CePs as potential antitumour clinical candidates.

Full text of DOI:10.1002/cmdc.202000814

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Appended Aromatic Moieties in Flexible Bis-3-
chloropiperidines Confer Tropism against Pancreatic
Cancer Cells
Caterina Carraro,[a] Tim Helbing,[b] Alexander Francke,[b] Ivonne Zuravka,[b] Alice Sosic,[a]
Michele De Franco,[a] Valentina Gandin,[a] Barbara Gatto,*[a] and D. Richard Göttlich*[b]
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Nitrogen mustards (NMs) are an old but still largely diffused
class of anticancer drugs. However, spreading mechanisms of
resistance undermine their efficacy and therapeutic applicabil-
ity. To expand their antitumour value, we developed bis-3-
chloropiperidines (B-CePs), a new class of mustard-based
alkylating agent, and we recently reported the striking
selectivity for BxPC-3 pancreatic tumour cells of B-CePs bearing
aromatic moieties embedded in the linker. In this study, we
demonstrate that such tropism is shared by bis-3-chloropiper-
idines bearing appended aromatic groups in flexible linkers,
whereas esters substituted by aliphatic groups or by efficient
DNA-interacting groups are potent but nonselective cytotoxic
agents. Besides, we describe how the critical balance between
water stability and DNA reactivity can affect the properties of
bis-3-chloropiperidines. Together, these findings support the
exploitation of B-CePs as potential antitumour clinical candi-
dates.
Introduction
Simplicity in synthesis coupled to the possibility of ample
decoration of the molecular structure led us to build a new
library of 3-chloropiperidines as nucleic acids alkylating
agents.[1] Our project has been inspired by antitumour drugs
with renowned therapeutic value such as chlorambucil and
melphalan, old and inexpensive agents still widely used in clinic
(Figure 1a).[2] Nevertheless, widespread resistance phenomena
and unwanted side effects undermined the efficacy of the
oldest mustard-based chemotherapies and new leads are
urgently needed.[3] In this direction, we demonstrated that
bifunctional 3-chloropiperidines (B-CePs) do react with guanine
nucleobases as intended, thus inducing damage on isolated as
well as genomic DNA.[1d,f] Analysing our library on model DNA,
we proved that flexible aliphatic linkers connecting the two
reactive centres of B-CePs (Figure 1b) confer high alkylating
potencies in vitro, whereas restrained flexibility of the linker
decreases the reactivity of the analogues.[1a] Nonetheless,
compounds with lower reactivity showed to be quite cytotoxic
in tumour cells, making them the best suited candidates for
Figure 1. Chemical structures of a) chlorambucil and melphalan and of the
analysed bis-3-chloropiperidines bearing b) linear alkyl and c) Lys ester/
amide linkers.
further development.[1f] Strikingly, B-CePs bearing linkers with
embedded aromatic groups disclosed an unexpected tropism
against a pancreatic cancer cell line (BxPC-3) poorly sensitive to
chlorambucil.[1f] Accordingly, derivative 3 bearing a linear
pentane linker (Figure 1b) lacked the preferential activity
exerted by aromatic analogues against pancreatic cancer
cells.[1f]
Following this line of evidence, we investigate here whether
aromatic moieties appended to the flexible aliphatic side chain
of 3 could reproduce the cytotoxicity and pancreatic tumour
tropism evidenced for rigid aromatic B-CePs.[1f] To do that, we
expanded our library of compounds bearing lysine linkers
[a] 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
Via Francesco Marzolo 5, 35131 Padova (Italy)
E-mail: barbara.gatto@unipd.it
[b] T. Helbing, A. Francke, Dr. I. Zuravka, Prof. D. R. Göttlich
Institute of Organic Chemistry, Justus Liebig University Giessen
Heinrich-Buff-Ring 17, 35392 Giessen (Germany)
E-mail: richard.goettlich@org.chemie.uni-giessen.de
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202000814
© 2020 The Authors. ChemMedChem published by Wiley-VCH GmbH. This is
an open access article under the terms of the Creative Commons Attribution
License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
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variously derivatized on the carboxyl group,[1b,c] and completed
the evaluation of their cytotoxicity and reactivity toward the
biologically relevant nucleophiles.
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3
In detail, the l-Lys ester B-CePs set analysed here included
(Figure 1c): i) compound 5 bearing a methyl ester function and
the aromatic derivatives 7, 9, 10 with increasing distance
between the phenyl ring and the carbonyl centre;[1b] ii) the
naphthyl-substituted compounds 11 and its para-methoxylated
analogue 12;[1b] iii) the anthraquinone (AQ) conjugate 13, whose
ability of binding DNA noncovalently has been already
highlighted.[1c] Along with the previously described compounds,
the new derivative 6, the unnatural d-Lys methyl ester
enantiomer of 5, was synthesised and tested to assess the
possible influence of stereogenicity on the activity of Lys ester
derivatives. In addition, compound 8 bearing a l-Lys amide
linker was synthesised and compared to its ester analogue 7.
Besides, to assess the effect of length in linear alkyl linkers on
the cytotoxicity of derivatives we also tested compounds 1, 2
and 4 (Figure 1b).[1a]
Results confirmed that appended aromatic moieties in the
lysine linker confer to B-CePs preferential activity against the
BxPC-3 pancreatic cell line observed for analogues bearing
embedded aromatic groups.[1f] The test compounds showed
valuable indexes of cytotoxicity in 2D and 3D BxPC-3 cell
cultures, linked to different consumption kinetics in aqueous
buffer, long-term reactivity with DNA and efficient cellular
uptake mediated by transporters. From these premises, this
study completes the investigation on the reactivity of lysine
esters while answering the question of the chemical determi-
nants for the tropism of B-CePs against BxPC-3 cells, further
directing the development of this class of alkylators as
anticancer agents.
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Scheme 1. Synthesis of new bis-3-chloropiperidine derivatives 6 and 8. a)
Boc2O, 1 M NaOH, H2O/dioxane (1:1), RT, 12 h; b) ClCO2Et, NEt3, benzyl
°
amine, dioxane, RT, 12 h; c) TFA, CH2Cl2, 0 C to RT, 3 h; d) 2,2-dimeth-
oxypropane, conc. HCl, MeOH, reflux to RT, 15 h; e) 2,2-dimethylpent-4-
enal,[5] NaBH(OAc)3, AcOH, dry CH2Cl2, 0 C to RT, 12 h; f) NCS, dry CH2Cl2, 0 C
°
°
°
to RT, 2.5–3.5 h; g) cat. TBAI, dry CHCl3, 60 C (oil bath temperature), 2 h,
(inseparable diastereomeric mixture).
After another double reductive amination with 2,2-dimeth-
ylpent-4-enal the unsaturated diamine 18 was obtained. Both
secondary diamines 18 and 19 where then reacted with N-
chlorosuccinimide and the resulting bis-N-chloroamines con-
verted to their corresponding bis-3-chloropiperidines 6 and 8
by iodine catalysed cyclization.
Appended aromatic groups in B-CePs confer tropism against
BxPC-3 pancreatic cancer cells
To further characterize the antiproliferative properties of B-CePs
and to gain new chemical insights on the mentioned tropism
for pancreatic cancer cells, we investigated the cytotoxicity of
compounds in Figure 1b and 1c against HCT-15 (colorectal
adenocarcinoma), 2008 (ovarian carcinoma) and BxPC-3 (pan-
creatic adenocarcinoma) tumour cell lines. Table 1 reports the
MTT assay IC50 values with associated standard deviations
obtained after 72 h of incubation of cells with test
compounds.[6] Values for compound 3 and the reference nitro-
gen mustard chlorambucil were published in our previous
works.[1e,f]
Noteworthy, all test B-CePs were more active than chlor-
ambucil against HCT-15 and BxPC-3 cells, and most of them
also against 2008 cells, confirming the promising potential for
this class of compounds to improve the therapeutic profile of
NMs. Clearly, no tropism toward pancreatic cancer cells was
observed for B-CePs with linear alkyl linkers (1–4), in line with
our previous observation.[1f] While MTT IC50 values of 1–4 were
similar and attested around 10 μm in 2008 and BxPC-3 cells, the
longer alkyl chains of 3 and 4 improved the cytotoxic effect in
HCT-15 cells.
Results and Discussion
The synthesis of bis-3-chloropiperidines 1–4 as well as 5, 7, 9–
12 and 13 has already been described elsewhere.[1a–c] The new
B-CeP derivatives 6 and 8 were synthesised using a similar
strategy (Scheme 1). To apply our well-established procedure of
N-chlorination and iodine catalysed cyclization[4] the unsatu-
rated diamines 18 and 19 had to be synthesised. For the amide
analogue of compound 7 both free amino groups of l-Lysine
where protected using di-tert-butyl dicarbonate (Boc2O), in
order
to achieve a clean amide coupling with benzyl amine. This
coupling reaction was accomplished by activation of the free
carboxyl group with ethyl chloroformate and provided the Boc-
protected benzylamide derivate 16 in very good yield. After
deprotection with trifluoroacetic acid the resulting free diamine
was converted to the desired unsaturated diamine 19 by
double reductive amination with 2,2-dimethylpent-4-enal.[5]
In contrast, the synthesis of compound 6, the enantiomer of
B-CeP 5, was carried out without any protecting groups. The
starting material, in this case d-Lysine, was directly converted
into its corresponding methyl ester hydrochloride salt by
treatment with 2,2-dimethyoxypropane and hydrochloric acid.
The introduction of the methyl ester group in 5 and 6
increased the cytotoxicity in comparison with their alkyl
analogue 3, regardless of the stereochemistry of the Lys
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In light of the promising activity of 9 and 10 on monolayer
cultures of BxPC-3 cells, the two compounds were tested
against spheroids of the same cancer cell line. Three-dimen-
sional cultures better simulate the complexity of solid tumours,
both in terms of cellular heterogeneity among concentric
spheroid layers and approximation of the microenvironmental
cell-extracellular matrix (ECM) interactions.[7] Cell viability in
spheroids exposed to compounds 9 and 10 for 72 h was
assessed by the acidic phosphatase (APH) assay and IC50s with
associated standard deviations are reported in Table 2.[8]
Compounds 9 and 10 demonstrated to be active also
against BxPC-3 spheroids, an encouraging result considering
the inactivity of chlorambucil up to the maximum tested
concentration of 200 μm. As observed in monolayer cultures, 9
was more active than 10, suggesting that the interplay between
compounds reactivity and lipophilicity are important factors to
be considered in spheroid models.
Table 1. MTT assay IC50 values with associated SDs of test bis-3-
chloropiperidines against HCT-15, 2008 and BxPC-3 cells after 72 h of
treatment. IC50 values were calculated from two independent experiments
by a four-parameter logistic model. Values highlighted in red: IC50 �1.0 μm;
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in yellow: 1.1�IC50 �6.0 μm; in green: 6.1�IC50 �15.0 μm; in blue: IC50
15.1 μm.
�
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Aromatic moieties reduce B-CePs hydroxylation
The highly electrophilic nature of B-CePs accounts for their
ability to react with nucleophiles, such as water molecules and
nucleobases in DNA.[1d,9] In aqueous buffers, 3-chloropiperidines
spontaneously rearrange into the aziridinium ion (N+), which is
readily attacked by water giving rise to mono- (OH) and
dihydroxylated (2 OH) products, irreversibly.[1b,d] Since the side-
reaction with water abates the fraction of compound still able
to alkylate the biological target, the rate of hydroxylation of 3-
chloropiperidine rings is informative both of B-CePs reactivity
and of their consumption kinetics. We therefore investigated by
ESI-MS the hydroxylation of B-CePs 5–13. ESI-MS experiments
with water were performed in BPE pH 7.4, the buffer employed
to assay compounds reactivity with isolated DNA. Freshly-made
solutions of compounds in buffer were analysed immediately or
[a] MTT IC50 from our previous work[1f] [b] Chlorambucil MTT IC50 from our
previous work[1e,f]
bridging linker. The high reactivity of the unsubstituted linear
alkyl B-CeP 3, previously observed in plasmid DNA cleavage
assays,[1b] turned out to be detrimental in cells, possibly
accelerating the premature reaction with competing nucleo-
philes in the cellular milieu. Consistently, given their reported
low potency toward isolated DNA,[1b] B-CePs Lys ester sub-
stituted with aromatic moieties (7–12) showed to be quite
effective against tested tumour cells. The substitution with
appended aromatic groups hallmark the higher toxicity against
the pancreatic tumour cell line, in line with the preferential
activity against BxPC-3 cells reported for B-CePs with embedded
aromatic linkers.[1f] Noticeably, the AQ conjugate 13 was rather
cytotoxic but not cell-line-selective compared to 7–12, a feature
most likely related to its peculiar reactivity empowered by
efficient DNA intercalating properties.[1c] In the ester series 7, 9
and 10, the shorter side chain of 7 appeared to be less suited in
cells, despite the earlier observation that 7 and 9 show similar
reactivity with plasmid DNA.[1b] Indeed, the aromatic com-
pounds 9 and 10 achieved nanomolar IC50s against pancreatic
cancer cells (BxPC-3 IC50 9: 0.6�0.5 μm; BxPC-3 IC50 10: 0.9�
0.2 μm). Concerning the newly synthesised derivatives, we
observed a similar profile between the Lys ester 7 and its amide
analogue 8.
°
after 1 h, 5 h and overnight (O.N.) incubation at 37 C by ESI-MS
using the protocol reported in previous studies..[1a–c,e,f] Graphs in
Figure 2 show the relative percentage of B-CePs reaction
species, whose chemical structures are schematized in the
figure legend. Graph-associated relative percentages are re-
ported in Table S1 in the Supporting Information.
As attested by results in Figure 2, the hydroxylation of all
Lys derivatives generally slowed down in buffer at physiological
pH compared to the aliphatic control 3 bearing an unsubsti-
tuted pentane linker. In agreement with their comparable
cellular activity, the l-Lys derivative 5 and its unnatural d-Lys
Table 2. APH assay IC50 values with associated SDs against 3D BxPC-3
cultures after 72 h of treatment. IC50 values of compounds 9 and 10 were
calculated from two independent experiments by a four-parameter logistic
model.
Compound
APH IC50 values [μm]
9
10
Chl
71.8�1.9
105.6�0.9
inactive
Chl: chlorambucil, inactive at the maximum tested concentration (200 μm).
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°
Figure 2. Hydroxylation of B-CePs. Time-followed formation of B-CePs reaction species at 37 C in BPE buffer pH 7.4 as detected by ESI-MS. Graphs report the
relative percentages of reaction species (whose symbols and structures are schematized in the legend) resulting from the incubation test B-CePs in BPE buffer
°
pH 7.4 at 37 C for 0, 1, 5 h and overnight (O.N.) detected by ESI-MS.
enantiomer 6 showed a very similar hydroxylation pattern in
the buffered solution at physiological pH.
B-CeP 5, while the presence of appended aromatic groups
strongly curtailed the reactivity of compounds with DNA. This
was particularly evident when increasing the length of the alkyl
spacer between the ester function and the aromatic ring in 7, 9
and 10.[1b] To complete the in vitro characterization of synthes-
ised B-CePs and to explore the suitability of appended linkers to
modulate B-CePs properties, we investigated the new deriva-
tives 6 and 8 at the DNA cleavage assay. To appreciate an
immediate comparison with their published analogues under
the same experimental conditions,[1b] the new compound 6
bearing the d-Lys linker was tested along with its enantiomer 5,
while the new amide derivative 8 was tested along with the
ester analogue 7. The supercoiled pBR322 plasmid was
incubated with increasing concentrations (0.5, 5, 50 μm) of test
The amide 8 exhibited different reactivity profiles than the
ester 7: 8 underwent a faster hydrolysis compared to its ester
analogue 7, showing to be almost totally dihydroxylated after
5 h of incubation. Most likely the hydroxylation of 8 is
accelerated by hydrogen bonding interactions of the amide NH,
which is not possible for the ester analogue 7. Indeed, all
aromatic ester derivatives demonstrated to be mostly unreacted
even after a prolonged overnight incubation in buffer. The
slower consumption kinetics of aromatic ester derivatives 7, 9,
10, 11 and 12 is consistent with their reported limited reactivity
with purified DNA,[1b] which might enable them to “preserve”
their reactivity in the cell cytoplasm context, resulting in the
considerable cytotoxicity shown above. Interestingly, 9 pre-
served its reactivity far longer than 7: the fraction of dihydroxy-
lated 92OH is much lower compared to 72OH after O.N. incubation.
This feature may account for the higher cytotoxicity of 9
compared to 7.
°
compounds for 3 h at 37 C in BPE buffer pH 7.4, as reported
previously.[1a–c,e, The ability of B-CePs to cut DNA leads to a
f]
relaxation of the plasmid to its open circular form, visualized on
1% agarose as a distinct upper band with lower electrophoretic
mobility compared to the supercoiled parent. Results after 3 h
of incubation are shown in Figure 3a (5 vs. 6) and b (7 vs. 8).
The d configuration of Lys linker of 6 seems not to affect its
remarkable DNA cleavage activity compared to natural l-Lys
analogue 5, a behaviour consistent with the similar reactivity in
buffer and the observed cellular effects. On the other hand, the
amide bond in the aromatic Lys linker of 8 substantially
increased its DNA cleavage potency compare to the less potent
ester analogue 7, presumably again due to possible hydrogen
bonding interactions. In both cases, results are in line with the
reactivity observed in BPE buffer at the ESI-MS. In Figure 3b, we
observe slower DNA migration at 50 μM compound, possibility
hinting to noncovalent binding by 7 and 8 to the nucleic acid.
Notably, the AQ conjugate 13, which was demonstrated to
promote a considerably higher plasmid cleavage compared to
the other aromatic Lys ester B-CePs,[1c] showed here a reduced
hydrolysis pattern. This result further endorses the hypothesis
that the improved DNA cleavage activity of 13 might depend
on the optimized targeting of DNA mediated by the intercalat-
ing AQ moiety rather than simply a higher electrophilicity.[1c]
Appended linkers modulate B-CePs reactivity with DNA
In previous studies, we demonstrated the ability of B-CePs to
cleave isolated DNA with potencies dependent on the chemical
structure of the linker.[1a–c,f] Specifically, the insertion of a methyl
ester function in 3 only slightly affected the DNA alkylation of
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compounds: if slowly consumed by competing nucleophiles, B-
CePs preserve the ability to react with their intended nuclear
target.
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5
6
7
Appended aromatic moieties in B-CePs promote noncovalent
DNA interactions
8
9
The retarded open circular plasmid form visualized in Figure 3b
suggested possible noncovalent interactions between aromatic
B-CePs and DNA, as already proved for the naphthyl-substituted
derivatives 11 and 12 and, more evidently, for the AQ conjugate
13.[1b,c] To verify noncovalent DNA-interacting properties of
appended mono-cyclic aromatic B-CePs, we incubated the lysine
esters 7, 9 and 10 with a double-strand G-rich oligonucleotide
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for 24 h at 37 C, and then observed results on a denaturing
Figure 3. DNA cleavage activity of bis-3-chloropiperidines a) 5 versus 6 and
b) 7 versus 8. The supercoiled pBR322 plasmid was incubated with
polyacrylamide gel (Figure 5). For comparative purposes, we
analysed also the recently published derivative bearing an
embedded aromatic linker (P, whose chemical structure is
reported in Figure 5),[1f] and the aliphatic ester 5.
°
increasing concentrations of test compounds at 37 C for 3 h in BPE buffer.
Lower band: supercoiled plasmid form, upper bands: open circular plasmid
species. C: supercoiled pBR322 plasmid control.
Only B-CePs bearing aromatic moieties demonstrated to
interact noncovalently with the oligonucleotide, as attested by
the appearance of a weak but distinctive uppermost band with
retarded gel migration. This band, not detected upon incuba-
tion with 5, was more pronounced in the case of the
compounds bearing appended phenyl-methyl and phenyl-ethyl
substituent (7 and 9), while minor for the B-CePs P bearing an
embedded aromatic linker, suggesting that only sufficiently
spaced and unhindered aromatic rings can establish stacking
interactions with DNA in B-CePs series. The higher reactivity of
5 with DNA is re-attested here by the concentration dependent
decrease of the intact oligo intensity and concurrent increased
intensity of cleaved oligo bands.
Water-stable B-CePs retain long-term DNA cleavage
properties
Having demonstrated that aromatic ester and amide B-CePs do
cleave DNA and in light of the new findings on their reduced
hydroxylation in buffered conditions, we checked if reactivity
with DNA is retained even after a prolonged incubation in
aqueous buffer. To do this, we compared the amide 8 with the
esters 7 and 9: as shown in Figure 2, they have quite different
hydroxylation kinetics in buffer, with 8 being faster consumed
in BPE than 7 and 9.
The results of the plasmid cleavage assay by this set of
compounds after a prolonged DNA-free preincubation in buffer
°
at 37 C is reported in Figure 4.
As expected, the DNA cleavage was clearly reduced by the
overnight preincubation in aqueous buffer, as the sustained
hydrolysis abates the fraction of compound still able to react
with the plasmid. However, 7 and 9 preserved higher long-term
DNA cleavage ability, as lower was their water consumption
detected by ESI-MS relative to 8 (Figure 2). The balance
between hydroxylation rate and reactivity with DNA is one of
the key elements to determine the biological value of these
Figure 5. Denaturing polyacrylamide gel showing concentration-dependent
cleavage of a 22-mer double-stranded oligonucleotide caused by bis-3-
chloropiperidine alkylation of guanines. The 5’-FAM-labelled scrambled
duplex oligonucleotide (4 μm), GGA TGT GAG TGT GAG TGT GAG G, was
Figure 4. DNA cleavage activity of bis-3-chloropiperidines 8–9 after over-
°
night preincubation of compounds at 37 C in BPE buffer (pH 7.4).
Preincubated compounds were added at increasing concentrations to the
°
treated with indicated compounds at 37 C in BPE buffer pH 7.4 at 5 and
°
pBR322 plasmid, and the mixture was incubated at 37 C for 3 h. C:
supercoiled pBR322 plasmid control.
50 μm for 24 h. Products were run on denaturing polyacrylamide gel 20% in
TBE buffer. C: untreated duplex oligonucleotide control.
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Transporters mediate entry of Lys ester B-CePs in BxPC-3 cells
transporter-mediated compound distribution across inner layers
is critical.
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7
8
9
B-CePs were recently demonstrated to exploit mechanisms of
transporter-mediated uptake in BxPC-3 cells.[1f] In order to
confirm such mode of entry for the new set of Lys linker Conclusion
derivatives, a modified MTT assay was performed on the highly
active compounds 7, 9 and 10.[1f,10] As reported in previous
B-CePs are a class of DNA alkylating agents developed to
works, cells were seeded in two microplates and incubated in
overcome the limitations of available chemotherapeutics.[1a–d,f]
To this extent, we recently reported the promising anticancer
properties of a set of B-CePs with embedded aromatic linkers
and their notable tropism against BxPC-3 pancreatic tumour
cells.[1f] As a step forward, in this study we analysed Lys esters
along with an amide analogue to further characterize their
mechanism of action and establish the tropism against BxPC-3
cells.
[1f]
°
°
parallel at 37 C or 4 C for 5 h. Afterwards, fresh medium was
added to rinsed wells, microplates were further incubated at
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37 C and cell viability was assessed at 72 h. Results are reported
in Figure 6.
Given the almost identical values of formazan absorbance
°
between untreated controls in plates incubated at 37 and 4 C,
we excluded that differences in viability might depend on the
°
sole initial temperature gap. At 37 C (red line), the intracellular
By virtue of their electrophilic nature, B-CePs react with
nucleophiles, such as water and DNA nucleobases:[1a,d] the
balance between nucleic acids alkylation and hydroxylation of
3-chloropiperidine rings contributes to delineate the pharmaco-
logical activity of these compounds. The cellular evaluation of
B-CePs demonstrated their generally improved potency com-
pared to the therapeutic NM chlorambucil against the tested
colorectal, ovarian and pancreatic cancer cell lines. In detail, we
observed that the Lys methyl ester linker increased the
cytotoxicity in all cell lines compared to the linear alkyl
analogue, whose sustained hydroxylation leads to premature
consumption in aqueous environment. In this sense, the limited
reactivity with water implies a much slower consumption of
compounds in buffered conditions and the retention of long-
term reactivity with DNA.
Notably, we point out in this study the remarkable activity
in pancreatic cancer cells by all B-CePs substituted with
appended aromatic linkers: the aromatic lysine esters 9 and 10
resulted the most active among the test derivatives, exhibiting
nanomolar IC50 values against the pancreatic cancer cell line. It
is significant that these two derivatives were demonstrated to
be active against the 3D model of BxPC-3 spheroids, possibly
thanks to efficient cell accumulation. Aromatic moieties ap-
pended to the flexible linkers can therefore reproduce the
pancreatic tumour tropism and cytotoxicity evidenced for rigid
embedded aromatic B-CePs,[1f] suggesting the existence of
collateral mechanisms of toxicity along with DNA alkylation and
damage. In fact, the efficiency of DNA repair machineries, drug
permeability and efflux mechanisms as well as the metabolic
status of cells are known to determine distinct sensitivities to
treatments in different cancer types, even in the case of drugs
exerting non-targeted mechanism of action, such as NMs.[11]
Transcriptome profiling to identify cancer vulnerabilities is
gaining increasing relevance and applicability in drug
discovery.[12] Studies on the dynamics of the transcriptional and
chromatin states in cells exposed to these cytotoxic compounds
are ongoing to mine the complex basis of the tropism of
aromatic B-CePs for the pancreatic cancer cell line.
accumulation of compounds takes advantage of both passive
diffusion and transporter-mediated uptake. Conversely, since
transporters have been shown to be inhibited at low temper-
°
atures, the cytotoxicity observed at 4 C (blue line) depends
almost exclusively on the passive diffusion of compounds.[10]
Thus, the elative contribution of transporters to the intracellular
accumulation of B-CePs is described by the difference in
viability resulting from the 5 h parallel incubation of microplates
°
°
at 37 C or 4 C. On these premises, graphs in Figure 6 confirm
that also appended aromatic Lys ester derivatives exploit
transporters to re-enter BxPC-3 cells. The contribution of
transporters to B-CePs
intracellular accumulation is comparable between 7 and 9,
while the role of transporters in the uptake of 10 is less
relevant. In fact, most likely for the higher lipophilicity conferred
by its longer benzyl side chain, 10 takes advantage of an
enhanced passive diffusion, a slower and less efficient process.
This feature could partly explain the higher cytotoxicity of 9
compared to 10 especially against spheroids, where an efficient
Figure 6. BxPC-3 cell viability upon exposure to increasing concentrations of
°
selected compounds 7, 9 and 10 for 5 h at 37 or 4 C, followed by a gentle
rinse of wells with PBS and addition of fresh RPMI medium. The MTT assay
was performed after 72 h. Average viability percentages with associated SDs
from three experimental replicates are reported.
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Acid phosphatase (APH) assay: The APH assay was performed to
Experimental Section
1
2
3
4
5
6
7
8
9
assess cell viability of 3D BxPC-3 cultures treated with test B-CePs
following previously reported protocols.[1f,8] Cells were seeded (1.5×
103 cells/well) in phenol red-free RPMI medium (Euroclone)
containing antibiotics and l-glutamine 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 incubated with defined concentration of compound freshly
dissolved in DMSO to a final solvent concentration of 0.5%. After
72 h of treatment, spheroids were incubated with 100 μL of the
Materials and methods
MS studies: B-CePs stability and hydrolysis rate upon incubation at
°
37 C in BPE (NaH2PO4 0.2 mm, Na2HPO4 0.6 mm, Na2EDTA 0.1 mm)
pH 7.4 was followed in time by ESI-MS. A fresh solution of test
compounds at the concentration of 80 μm was prepared in BPE
°
buffer from 1 mm DMSO stocks and incubated at 37 C. Samples
were taken after 0 h, 1 h, 5 h and overnight incubation, diluted
1:10 with methanol and analysed by ESI-MS. Measurements were
performed in positive ion mode using a Xevo G2-XS Qtof instru-
ment (Waters) and calculations were performed using the MassLynx
software (Waters). The relative abundances of detected reaction
species at different experimental conditions were reported as
percentages.[1f,13]
°
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
microplate reader). Compounds IC50s were extrapolated applying a
four-parameter logistic (4-PL) model and average values with
associated SDs of two independent experiments (each one with
three replicates) were reported.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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29
30
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37
38
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40
41
42
43
44
45
46
47
48
49
50
51
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57
Cleavage assay: The DNA cleavage properties of test B-CePs were
assessed on the supercoiled plasmid pBR322 (Inspiralis Ltd). The
cleavage assay was performed following previously reported
materials and protocols.[1b,d, e] B-CePs dilutions were freshly prepared
from a 10 mm DMSO stock in Milli-Q water.
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 chromatography
silica gel 60 (Merck) was used. 1H and 13C NMR spectra were
recorded at Bruker Avance II 200 spectrometer (1H at 200 MHz; 13C
at 50 MHz) and Bruker Avance II 400 spectrometer (1H at 400 MHz;
13C at 100 MHz) in deuterated solvents. Chemical shifts were
determined by reference to the residual solvent signals. High-
resolution ESI mass spectra (HRMS) were recorded in methanol
using a ESImicroTOF spectrometer (Bruker Daltonics) in positive ion
mode. The synthesis and analytical data of the bis-3-chloropiper-
idines 1–5, 7, 9–12[1b] and 13[1c] have been described elsewhere.
Sequencing gel analysis: The sequencing gel analysis was
performed according to previously reported materials and
protocols.[1a,c,d] The 5’-FAM-labelled scrambled oligonucleotide (5’-
FAM-GGA TGT GAG TGT GAG TGT GAG G-3’, Eurogentec) was slowly
annealed with its complementary co-scrambled oligonucleotide (5’-
CCT CAC ACT CAC ACT CAC ATC C-3’, Eurogentec): equimolar
amounts of both sequences were mixed in BPE buffer, denatured at
°
95 C for 5 min and left to cool to room temperature to obtain the
final duplex. The 5’-FAM-labelled duplex oligonucleotide (4 μm) was
incubated with 50 μm of test derivatives in BPE buffer for 24 h at
°
37 C. Samples were dried in a vacuum centrifuge (UNIVAPO 100H,
UniEquip), resuspended in denaturing gel loading buffer (10 mm
Tris-HCl, 80% formamide, 0.025% bromophenol blue), and loaded
on a 20% denaturing polyacrylamide gel (7 m urea) in TBE buffer.
The fluorescence of the oligonucleotide bands was detected by
scanning using Storm Scanner Control (STORM 840, Molecular
Dynamics).
1
Copies of the H and 13C NMR spectra as well as the HRMS spectra
of the new B-CeP derivatives 6 and 8 are included in the supporting
information.
Cell cultures: Colon (HCT-15) and pancreatic (BxPC-3) carcinoma
cell lines were purchased from ATCC (American Type Culture
Collection) while the human ovarian 2008 cancer cell line was
provided by G. Marverti (Department of Biomedical Science,
University of Modena and Reggio Emilia, Modena, Italy). Cell lines
Synthetic procedures
d-Lysine methylester dihydrochloride (14): To a suspension of d-
lysine hydrochloride (5.00 g, 22.82 mmol) in methanol (55 mL) was
added 2,2-dimethoxypropane (35 ml) and conc. HCl (9 mL). The
mixture was heated to reflux for 3 h and was then stirred at room
temperature for 12 h. Afterwards the solvent was removed under
reduced pressure. The residue was dissolved in a small amount of
°
were maintained in logarithmic phase at 37 C under 5% CO2 using
RPMI-1640 medium (Euroclone) containing 10% foetal calf serum
(FCS, Euroclone), antibiotics (50 units/mL penicillin and 50 μg/mL
streptomycin), and 2 mm l-glutamine.
°
methanol and TBME (250 mL) was added at 0 C to induce
crystallization. The crude product was then recrystallized from
TBME/methanol to obtain the title compound (14) as a white solid
(5.05 g, 21.68 mmol, 95%). 1H NMR (400.1 MHz, D2O): δ=4.20 (t, J=
6.5 Hz, 1H), 3.87 (s, 3H), 3.04 (t, J=7.7 Hz, 2H), 2.12–1.91 (m, 2H),
1.75 (p, J=7.7 Hz, 2H), 1.62–1.42 (m, 2H) ppm; 13C NMR (100.6 MHz,
D2O): δ=170.6, 53.6, 52.6, 39.0, 29.2, 26.2, 21.5 ppm; HRMS(ESI): m/z
calcd for C7H17N2O2: 161.1285, found 161.1297 [M+H+].
MTT assay: The MTT assay was performed as in previously reported
6]
materials and protocols.[1e,f, 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. Compounds MTT IC50s were
extrapolated applying a four-parameter logistic (4-PL) model and
average values with associated SDs of two independent experi-
ments (each one with three replicates) were reported. We applied
modifications to the classical MTT assay to investigate the uptake of
B-CePs. BxPC-3 cells were incubated with test compounds for 5 h,
(2S)-2,6-Bis(tert-butoxycarbonylamino)lysine (15): To a solution of
l-lysine monohydrate (5.09 g, 31.0 mmol) in water/dioxane (1:1,
100 mL) were added di-tert-butyl dicarbonate (16.9 g, 78.0 mmol)
and 1 M NaOH(aq) (35 mL). The reaction mixture was stirred at room
temperature for 12 h and then concentrated in vacuo until
approximately 50 mL remained. The pH was adjusted to 1–2 by
careful addition of an aqueous KHSO4 solution (150 g/L). The
suspension was extracted three times with ethyl acetate. The
combined organic layers were dried over MgSO4 and the solvent
was removed under reduced pressure to afford the title compound
°
either at 37 or 4 C in parallel microplates. Then, wells were rinsed
with PBS, fresh RPMI was added and microplates were incubated at
°
37 C in the absence of compound. After 72 h from seeding, cell
viability was assessed and average values with associated SDs from
three experimental replicates were reported.[1f,10]
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1
(15; 9.89 g, 28.5 mmol, 92%) as a colourless oil. H NMR ([D6]DMSO,
200.1 MHz): δ=12.40 (s, 1H, OH), 6.98 (d, J=7.9 Hz, 1H), 6.76 (t, J=
5.4 Hz, 1H), 3.75–3.86 (m, 1H), 2.50–2.83 (m, 2H), 1.37 (s, 18H), 1.14–
1.68 (m, 6H) ppm; 13C NMR ([D6]DMSO, 50.3 MHz): δ=173, 155.1,
77.4, 76.8, 52.9, 29.9, 28.6, 27.7, 22.4 ppm (one signal, probably
around 40 ppm, is hidden by the solvent signal). HRMS(ESI): m/z
calcd for C16H30N2O6Na+: 369.1996, found 369.1998 [M+Na+].
11.4 Hz, 1H), 2.34 (s, 2H), 2.08 (d, J=11.4 Hz, 1H), 2.04–1.92 (m, 4H),
1.68–1.13 (m, 8H), 0.94- 0.79 (m, 12H) ppm; 13C NMR (100.6 MHz,
CDCl3): δ=176.6, 135.7, 135.6, 116.9, 116.9, 62.6, 60.4, 58.4, 51.6,
50.8, 44.9, 44.5, 34.6, 34.4, 33.6, 29.8, 25.7, 25.5, 25.4, 23.8 ppm;
HRMS(ESI): m/z calcd for C21H41N2O2+: 353.3163, found 353.3176 [M
+H+].
1
2
3
4
5
6
7
8
9
(2S)-N-Benzyl-2,6-bis(2,2-dimethylpent-4-enylamino)hexanamide
(19): Was prepared according to general procedure A from (2S)-N-
benzyl-2,6-diaminohexanamide trifluoroacetic acid salt (17; 1.15 g,
2.48 mmol) and 2,2-dimethyl-4-pentenal (0.67 g, 5.95 mmol) yield-
ing the title compound (19) as a pale yellow oil (0.84 g, 1.96 mmol,
(2S)-N-Benzyl-2,6-bis(tert-butoxycarbonylamino)hexanamide (16):
To a solution of (2S)-2,6-bis(tert-butoxycarbonylamino)lysine (15;
1.00 g, 2.89 mmol) in 1,4-dioxane (20 mL) triethylamine (0.35 g,
3.47 mmol) as well as ethyl chloroformate (0.38 g, 3.47 mmol) were
added. The solution was stirred at room temperature for 20 min.
Afterwards benzylamine (0.37 g, 3.47 mmol) was added dropwise,
and the mixture was stirred at room temperature for 12 h. The
solvent was removed under reduced pressure and the residue
dissolved in ethyl acetate (30 mL). The solution was washed with
distilled water, followed by 1 M NaOH(aq), 1 M HCl(aq) and distilled
water. The organic phase was separated, dried over MgSO4 and the
solvent was removed under reduced pressure. The crude product
was purified by flash column chromatography (pentane/ethyl
acetate 1:1) to obtain the title compound (16) as a colourless oil
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
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47
48
49
50
51
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1
79%). H NMR (400.1 MHz, CDCl3): δ=7.57 (t, J=6.0 Hz, 1H), 7.36–
7.29 (m, 2H), 7.29–7.21 (m, 3H), 5.94–5.62 (m, 2H), 5.08–4.90 (m, 4H),
4.47 (dd, J=14.7, 6.3 Hz, 1H), 4.38 (dd, J=14.7, 5.6 Hz, 1H), 3.01 (dd,
J=7.9, 4.9 Hz, 1H), 2.66 (t, J=7.5 Hz, 2H), 2.41 (s, 2H), 2.37 (d, J=
11.4 Hz, 1H), 2.18 (d, J=11.4 Hz, 1H), 2.07–2.00 (m, 2H), 1.97–1.88
(m, 2H), 1.84–1.71 (m, 1H), 1.65–1.49 (m, 3H), 1.47–1.33 (m, 2H), 0.93
(s, 6H), 0.81 (s, 6H) ppm; 13C NMR (100.6 MHz, CDCl3): δ=174.5,
138.7, 135.5, 135.2, 128.8, 127.9, 127.5, 117.4, 117.2, 63.7, 59.8, 59.5,
50.4, 44.8, 44.8, 43.1, 34.3, 34.2, 33.8, 28.9, 25.6, 25.4, 23.9 ppm;
HRMS(ESI): m/z calcd for C27H46N3O2+: 428.3635, found 428.3621 [M
+H+].
1
(1.16 g, 2.66 mmol, 92%). H NMR (400.1 MHz, CDCl3): δ=7.36–7.19
(m, 5H), 6.61 (s, 1H), 5.17 (s, 1H), 4.60 (s, 1H), 4.47–4.36 (m, 2H), 4.07
(s, 1H), 3.08 (t, J=6.7 Hz, 2H), 1.93–1.79 (m, 1H), 1.72–1.57 (m, 1H),
1.53–1.45 (m, 2H), 1.41 (overlap 2 s, 18H), 1.39–1.30 (m, 2H) ppm;
13C NMR (100.6 MHz, CDCl3): δ=172.2, 156.3, 156.0, 138.2, 128.8,
127.8, 127.6, 80.3, 79.4, 54.7, 43.6, 40.1, 32.0, 29.9, 28.6, 28.6, 28.4,
22.8 ppm; HRMS(ESI): m/z calcd for C23H37N3O5Na+: 458.2625, found
458.2623 [M+Na+].
General procedure B: Synthesis of bis-N-chloroamines (20 and
21): Under nitrogen, the crude unsaturated diamine was dissolved
in anhydrous dichloromethane (10 mL/mmol of diamine), and
freshly recrystallized N-chlorosuccinimide (2.2 equiv) was added
°
°
portion wise at 0 C. The mixture was stirred at 0 C for 30 min and
for an additional 2–3 h at room temperature. The solvent was
removed under reduced pressure and the crude product was
purified by flash column chromatography.
(2S)-N-Benzyl-2,6-diaminohexanamide trifluoroacetic acid salt
(17): To a solution of (2S)-N-benzyl-2,6-bis(tert-butoxycarbonylami-
no)hexanamide (16; 1.00 g, 2.30 mmol) in CH2Cl2 (30 mL) trifluoro-
(2R)-Methyl-2,6-bis(N-chloro-N-(2,2-dimethylpent-4-enyl)amino)-
hexanoate (20): Was prepared according to general procedure B
from (2R)-methyl-2,6-bis(2,2-dimethylpent-4-enylamino)hexanoate
(18; 2.73 g, 7.74 mmol) and purified by flash column chromatog-
raphy (pentane/TBME 9:1) yielding the title compound (20) as a
pale yellow oil (1.84 g, 4.37 mmol, 57%). 1H NMR (400.1 MHz,
CDCl3): δ=5.87–5.73 (m, 2H), 5.08–4.96 (m, 4H), 3.75 (s, 3H), 3.49 (t,
J=7.2 Hz, 1H), 3.13 (d, J=15.2 Hz, 1H), 2.97-2.81 (m, 5H, H-6), 2.05
(d, J=7.5, 1.3 Hz, 4H), 1.89–1.78 (m, 2H), 1.72–1.62 (m, 2H), 1.61–
1.50 (m, 1H), 1.45–1.34 (m, 1H), 0.98–0.89 (m, 12H) ppm; 13C NMR
(100.6 MHz, CDCl3): δ=171.5, 135.4, 135.3, 117.5, 117.4, 75.0, 73.4,
72.4, 66.6, 51.8, 45.0, 44.7, 36.0, 35.7, 30.5, 27.9, 25.9, 25.7, 25.5,
23.5 ppm; HRMS(ESI): m/z calcd for C21H39Cl2N2O2+: 421.2389, found
421.2380 [M+H+].
°
acetic acid (10 mL) was added dropwise at 0 C. The mixture was
stirred at room temperature for 3 h. The solvent was removed
under reduced pressure, the residue was redissolved in CH2Cl2, and
the solvent was removed under reduced pressure once again to
obtain the title compound (17) as a pale yellow oil (0.97 g,
2.09 mmol, 91%). 1H NMR (400.1 MHz, [D6]DMSO): δ=8.95 (t, J=
5.9 Hz, 1H), 8.19 (d, J=5.3 Hz, 3H), 7.78 (s, 3H), 7.44–7.12 (m, 5H),
4.44–4.38 (m, 2H), 3.78 (q, J=6.0 Hz, 1H), 2.82–2.61 (m, 2H), 1.82–
1.65 (m, 2H), 1.57–1.47 (m, 2H), 1.38–1.25 (m, 2H). ppm; 13C NMR
(100.6 MHz, [D6]DMSO): δ=168.4, 138.5, 128.4, 127.5, 127.2, 52.1,
42.4, 38.5, 30.5, 26.54, 21.25. ppm; HRMS(ESI): m/z calcd for
C13H22N3O+: 236.17595, found 236.1766 [M+H+].
General procedure A: Synthesis of unsaturated diamines (18 and
(2S)-N’-Benzyl-2,6-bis(N-chloro-N-(2,2-dimethylpent-4-enyl)
amino)hexanamide (21): Was prepared according to general
procedure B from (2S)-N-Benzyl-2,6-bis(2,2-dimethylpent-4-enylami-
no)hexanamide (19; 1.20 g, 2.81 mmol) and purified by flash
column chromatography (pentane/TBME 4:1) yielding the title
compound (21) as a pale yellow oil (0.41 g, 0.82 mmol, 29%). 1H
NMR (400.1 MHz, CDCl3): δ=7.42–7.27 (m, 5H), 6.88 (t, J=5.9 Hz,
1H), 5.91–5.68 (m, 2H), 5.10–4.90 (m, 4H), 4.60–4.39 (m, 2H), 3.41
(dd, J=7.3, 5.4 Hz, 1H), 3.03–2.86 (m, 4H), 2.84 (s, 2H), 2.04 (ddt, J=
15.7, 7.5, 1.3 Hz, 5H), 1.88 (ddt, J=13.7, 9.8, 5.8 Hz, 1H), 1.76–1.64
(m, 2H), 1.63–1.45 (m, 2H), 0.99–0.88 (m, 12H) ppm; 13C NMR
(100.6 MHz, CDCl3): δ=170.6, 138.3, 135.4, 134.9, 128.8, 127.8,
127.6, 117.7, 117.5, 75.4, 75.0, 72.0, 66.5, 45.0, 45.0, 43.5, 35.9, 35.7,
29.0, 28.2, 25.9, 25.8, 24.7 ppm; HRMS(ESI): m/z C27H44Cl2N3O+:
496.2856, found 496.2850 [M+H+].
19): Under
a
nitrogen atmosphere 2,2-dimethylpent-4-enal[5]
(2.4 equiv) as well as the corresponding diamine as its HCl/TFA salt
were dissolved in anhydrous CH2Cl2 (10 mL/mmol of diamine) and
sodium triacetoxyborohydride (3 equiv) was added portion wise at
°
0 C, followed by acetic acid (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 over MgSO4. The solvent was removed
under reduced pressure and the crude product was obtained,
which was used in the next step without further purification.
(2R)-Methyl-2,6-bis(2,2-dimethylpent-4-enylamino)hexanoate
(18): Was prepared according to general procedure A from d-lysine
methylester dihydrochloride (14; 2.13 g, 9.14 mmol) and 2,2-
dimethyl-4-pentenal (2.46 g, 21.93 mmol) yielding the title com-
General procedure C: Synthesis of bis-3-chloropiperidines (6 and
8): 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
1
pound (18) as a pale yellow oil (2.73 g, 7.74 mmol, 85%). H NMR
(400.1 MHz, CDCl3): δ=5.89–5.66 (m, 2H), 5.06–4.90 (m, 4H), 3.70 (s,
3H), 3.12 (t, J=6.8 Hz, 1H), 2.58 (t, J=7.0 Hz, 2H), 2.39 (d, J=
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°
added. The mixture was heated to 60 C (oil bath temperature) for
2 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 diastereomers.
Keywords: anticancer agents
mustards · pancreatic cancer · spheroids
· DNA alkylation · nitrogen
1
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5
6
7
8
(2R)-Methyl-2,6-bis(5-chloro-3,3-dimethylpiperidin-1-yl)hexa-
noate (6): Was prepared according to general procedure C from
(2R)-methyl-2,6-bis(N-chloro-N-(2,2-dimethylpent-4-enyl)amino)hex-
anoate (20; 1.84 g, 4.37 mmol) and purified by flash column
chromatography (pentane/TBME 9:1) yielding the title compound
9
(6) as
a
pale yellow oil (0.88 g, 2.09 mmol, 48%). 1H NMR
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
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39
40
41
42
43
44
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47
48
49
50
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54
55
56
57
(400.1 MHz, CDCl3) mixture of isomers: δ=4.12–3.91 (m, 2H), 3.68
(2x s, total 3H), 3.22–3.08 (m, 3H), 2.51–2.17 (m, 6H), 1.97–1.89 (m,
3H), 1.73–1.60 (m, 3H), 1.50–1.37 (m, 3H, H-8), 1.37–1.25 (m, 3H),
1.01 (2x s, total 6H), 0.90 (2x s, total 6H) ppm; 13C NMR (100.6 MHz,
CDCl3, selected signals) major isomer: δ=172.8, 66.9, 64.9, 64.2,
62.5, 61.7, 57.7, 55.2, 54.6, 54.3, 51.2, 48.8, 33.8, 33.4, 29.6, 29.2, 26.6,
25.4, 24.0 ppm; HRMS(ESI): m/z C21H39Cl2N2O2+: 421.2392, found
421.2388 [M+H+].
(2S)-N-Benzyl-2,6-bis(5-chloro-3,3-dimethylpiperidin-1-yl)hexana-
mide (8): Was prepared according to general procedure C from
(2S)-N’-benzyl-2,6-bis(N-chloro-N-(2,2-dimethylpent-4-enyl)amino)-
hexanamide (21; 0.70 g, 1.41 mmol) and purified by flash column
chromatography (pentane/TBME 1:1) yielding the title compound
(8) as
a
pale yellow oil (0.47 g, 0.95 mmol, 67%). 1H NMR
(400.1 MHz, CDCl3) ~1:1 isomeric mixture of 8a and 8b: δ=7.35–
7.25 (m, 10H: 5H 8a and 5H 8b), 7.08 (s, 1H 8a), 6.93 (s, 1H 8b), 4.51–
4.35 (m, 4H: 2H 8a and 2H 8b), 4.11–3.88 (m, 4H: 2H 8a and 2H 8b),
3.16-3.03 (m, 4H: 2H 8a and 2H 8b), 3.01 (dd, J=7.6, 5.0 Hz, 1H 5H
8a), 2.88 (t, J=6.4 Hz, 1H 8b), 2.46–2.21 (m, overlapping signals 9H:
4H 8a and 4H 8b and 1H 8a), 2.15–2.08 (m, 1H 8b), 2.07–1.97 (m, 2H:
1H 8a and 1H 8b), 1.95–1.85 (m, 6H: 3H 8a and 3H 8b), 1.77–1.59 (m,
6H: 3H 8a and 3H 8b), 1.51–1.28 (m, 12H: 6H 8a and 6H 8b), 1.02 (s,
6H 8a), 0.91 (s, 6H 8b), 0.87 (d, J=10.0 Hz, 6H 8a), 0.82 (s, 6H 8b)
ppm; 13C NMR (100.6 MHz, CDCl3, selected signals) major isomer:
δ=172.7, 138.6, 128.8, 128.1, 127.7, 69.2, 64.8, 62.5, 62.4, 57.7, 57.5,
54.5, 53.9, 48.5, 48.0, 43.5, 33.4, 33.2, 29.5, 28.5, 27.2, 25.4, 24.4 ppm;
HRMS(ESI): m/z C27H44Cl2N3O+: 496.2856, found 496.2856 [M+H+].
Acknowledgements
A.S. received funding from the European Union’ s Horizon2020
Research and Innovation programme under the Marie Skłodow-
ska-Curie grant agreement no. 751931. Open access funding
enabled and organized by Projekt DEAL.
[13] a) N. Hagan, D. Fabris, Biochemistry 2003, 42, 10736–10745; b) A. Sosic, I.
Saccone, C. Carraro, T. Kenderdine, E. Gamba, G. Caliendo, A. Corvino, P.
Di Vaio, F. Fiorino, E. Magli, E. Perissutti, V. Santagada, B. Severino, V.
Spada, D. Fabris, F. Frecentese, B. Gatto, Bioconjugate Chem. 2018, 29,
2195–2207.
Conflict of Interest
Manuscript received: October 16, 2020
Accepted manuscript online: November 17, 2020
Version of record online: ■■■, ■■■■
The authors declare no conflict of interest.
ChemMedChem 2020, 15, 1–10
www.chemmedchem.org
9
© 2020 The Authors. ChemMedChem published by Wiley-VCH GmbH
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FULL PAPERS
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Balancing act: Following our devel-
opment of bis-3-chloropiperidines (B-
CePs), a new class of sustainable
mustard-based alkylating agents, we
demonstrate that B-CePs bearing
appended aromatic groups in linkers
show tropism toward pancreatic
tumour models. In addition, we
describe how the critical balance
between DNA reactivity and water
stability can affect the properties of
this class of compounds.
C. Carraro, T. Helbing, A. Francke, Dr. I.
Zuravka, Dr. A. Sosic, M. De Franco,
Prof. Dr. V. Gandin, Prof. Dr. B. Gatto*,
Prof. D. R. Göttlich*
1 – 10
Appended Aromatic Moieties in
Flexible Bis-3-chloropiperidines
Confer Tropism against Pancreatic
Cancer Cells
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