Bis-3-chloropiperidines containing bridging lysine linkers: Influence of side chain structure on DNA alkylating activity

Article abstract of DOI:10.1016/j.bmc.2015.01.050

A series of bis-3-chloropiperidines containing lysine linkers was synthesised as DNA alkylating model compounds by using a bidirectional synthetic strategy. These novel piperidine mustard based agents have been evaluated for their alkylating properties towards nucleic acids and were shown to alkylate and cleave DNA with strong preference for guanine residues. Our studies reveal that the introduction of aromatic groups in the side chain of the lysine linker has an impact on DNA alkylating activity. Analysis by ESI mass spectrometry enabled the verification of the reactive aziridinium ion formation. Overall, the results confirm our recently proposed reaction mechanism of bis-3-chloropiperidines.

Full text of DOI:10.1016/j.bmc.2015.01.050

Bioorganic & Medicinal Chemistry 23 (2015) 1241–1250
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Bis-3-chloropiperidines containing bridging lysine linkers: Influence
of side chain structure on DNA alkylating activity
Ivonne Zuravka , Rolf Roesmann , Alice Sosic , Richard Göttlich , Barbara Gatto b,
a,b
a
b
a,
⇑
⇑
a
Institute of Organic Chemistry, Justus Liebig University Giessen, Heinrich-Buff-Ring 58, 35392 Giessen, Germany
Dipartimento di Scienze del Farmaco, Università di Padova, via Marzolo 5, 35131 Padova, Italy
b
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of bis-3-chloropiperidines containing lysine linkers was synthesised as DNA alkylating model
compounds by using a bidirectional synthetic strategy. These novel piperidine mustard based agents
have been evaluated for their alkylating properties towards nucleic acids and were shown to alkylate
and cleave DNA with strong preference for guanine residues. Our studies reveal that the introduction
of aromatic groups in the side chain of the lysine linker has an impact on DNA alkylating activity. Analysis
by ESI mass spectrometry enabled the verification of the reactive aziridinium ion formation. Overall, the
results confirm our recently proposed reaction mechanism of bis-3-chloropiperidines.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 20 November 2014
Revised 27 January 2015
Accepted 28 January 2015
Available online 7 February 2015
Keywords:
Bis-3-chloropiperidines
Alkylating agents
Nitrogen mustards
Aziridinium ion
DNA cleavage
1
. Introduction
In the search for more effective alkylating agents with less sys-
temic toxicity, various mustard analogues have been investigated.7
Alkylating agents which interact directly with DNA to form
covalent bonds have an important therapeutic role in anticancer
1
–3
treatment.
Nitrogen mustards were the first effective antineo-
4
(a)
(b)
plastic drugs and are still commonly used in chemotherapy. Their
mechanism of action is based on the formation of a highly reactive
electrophilic aziridinium ion.5 This reactive species can be readily
attacked by multiple nucleophilic sites in DNA. Previous studies
Cl
O
CH
N
3
OH
,6
Cl
Cl
N
established that most alkylating agents react particularly with
Mechlorethamine
Chlorambucil
the N7 position of guanine.7–9 In the first place base alkylation pre-
Cl
vents DNA replication and induces DNA fragmentation by hydro-
lytic reactions, which ultimately leads to cell death. However, the
linker
emergence of resistance to this class of drugs is also a substantial
Cl
Cl
2 3
B1: linker = -(CH ) -
N
N
challenge in cancer therapy.1
0,11
As a result there are ongoing
B2: linker = -(CH
2 4
) -
B3: linker = -(CH ) -
research efforts to develop more effective antitumour drugs.
An important strategy in drug design is based on gaining a dee-
per insight into the underlying mechanism of action. In this regard
mechanistic understanding can be derived from chemical ana-
logues of the original drug which can serve as model compounds.
As an example, the well-known chemotherapeutic drug chloram-
2 5
2 6
B4: linker = -(CH ) -
(
c)
O
N
OR
1: R = -CH3
2: R = -CH C H
5
2
6
Cl
Cl
bucil was developed as a more stable analogue of mechloretha-
2 2 6 5
3: R = -(CH ) C H
N
mine, the prototype of the nitrogen mustards (Fig. 1a).12
4:
5
6
R = -(CH
: R = -1-naphthyl
: R = -1-naphthyl-4-OCH
2 3 6 5
) C H
3
⇑
Corresponding authors. Tel.: +49 641 993 4340; fax: +49 641 993 4349 (R.G.);
tel.: +39 049 827 5717; fax: +39 049 827 5366 (B.G.).
E-mail addresses: richard.goettlich@uni-giessen.de (R. Göttlich), barbara.gat-
to@unipd.it (B. Gatto).
Figure 1. (a) Chemical structures of the nitrogen mustards mechlorethamine and
chlorambucil. (b) Bis-3-chloropiperidines B1–B4 investigated in a previous study.14
(c) Series of newly synthesised compounds 1–6 with bridging lysine linkers.
http://dx.doi.org/10.1016/j.bmc.2015.01.050
0
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

1
242
I. Zuravka et al. / Bioorg. Med. Chem. 23 (2015) 1241–1250
In particular the aromatic nitrogen mustards have taken a signifi-
cant role in cancer therapy.13 Still, further identification of the
molecular interactions between DNA and agents is needed to bet-
ter understand their mechanisms of action.
As discussed in the previous paragraph, it is apparent that struc-
tural modifications of leading compounds are a fundamental con-
cept in order to increase biological activity and potency. In a
recent proof-of-principle study we demonstrated that nitrogen-
linked bis-3-chloropiperidines (B1–B4, Fig. 1b), which can be con-
sidered as piperidine-based analogues of nitrogen mustards, are
more reactive towards DNA than chlorambucil.14
preparation of the target compounds 1–6 started with the
amino-protection of readily available -lysine 7 by using di-tert-
butyl dicarbonate (Boc O) to afford the di-Boc- -lysine 8. In order
L
2
L
to introduce aromatic groups to the carboxylic acid side chain of
lysine we planned the synthesis of a series of lysine ester deriva-
tives. Thus, coupling of 8 with the appropriate aromatic alcohols
9–13 in the presence of hydroxybenzotriazole (HOBt) and dicyclo-
hexyl carbodiimide (DCC) furnished the corresponding lysine
esters 14–18. Subsequently, the BOC protecting groups were
cleaved with trifluoroacetic acid (TFA) to yield the free diamines
19–23 as their TFA salt.
These new alkylating agents induce highly efficient cleavage in
double-stranded DNA with dominating preference for guanine
sites. In addition, investigations of linker structure on DNA alkyl-
ation activity revealed that the flexibility of the linker is important
for alkylation efficiency, whereas the direct introduction of an aro-
matic group as linker leads to a decrease in activity.14 Due to our
experimental observations it appeared promising to attempt the
synthesis of novel bis-3-chloropiperidines containing a flexible lin-
ker which offers the possibility of placing the aromatic moiety in
the side chain (1–6, Fig. 1c). The attachment of an aromatic unit
to a side chain of the linker as in the case of chlorambucil instead
of its placement in the linker structure itself may enable a control
of the biochemical reactivity of the agents and enhance as well as
modulate DNA affinity.
Following the three-step procedure previously reported, the
desired bis-3-chloropiperidines 2–6 were readily prepared from
the precursors 19–23. The bidirectional method involves the
1
4
In our previous study it has been shown that the bis-3-chloro-
piperidine B3 with a flexible pentyl hydrocarbon linker exhibited
highly efficient DNA alkylating activity (see also gel electrophoresis
data below, Fig. 2a).1 The amino acid
4
L-lysine 7 is a suitable linker
structure for the preparation of a new set of bis-3-chloropiperi-
dines. The carboxylic acid functionality of lysine provides a posi-
tion at which the side chain can be modified. For instance,
chemical derivatisation can be easily achieved through esterifica-
tion of the carboxyl group with various alcohols. Consequently,
lysine represents a flexible linker which allows an experimental
comparison between the bis-3-chloropiperidines of the present
study 1–6 with the previous series of compounds B1–B4 (in partic-
ular B3).
To examine the influence of aromatic motifs on the bridging
lysine esters we analysed DNA alkylating activity of the set of com-
pounds 1–6. The lysine derivatives were chosen as it is relatively
easy to prepare different esters and thereby study the impact of
different groups on the reactivity towards DNA. Thus, we first
planned the synthesis of bis-3-chloropiperidines 2, 3 and 4 by
introducing a simple phenyl group tethered through flexible
hydrocarbon spacers to the lysine linker. The lysine methyl ester
1
was selected for comparison purposes in the biochemical assays.
Design of compounds 5 and 6 was based on the knowledge that
naphthalene chromophores, present in the antitumour antibiotics
neocarzinostatin and azinomycin A and B, contribute significantly
to reinforce the affinity for DNA.1
5–17
Additionally, the methoxy
group in the 4-position of the naphthalene moiety 6 can participate
in hydrogen bonding interactions.
In the present paper we report the synthesis and the evaluation
of DNA alkylating properties of these novel lysine-linked bis-3-
chloropiperidines as prototype model compounds to better under-
stand the molecular mechanism of action of bis-3-chloropiperidine
derivatives.
Figure 2. DNA cleavage activity of bis-3-chloropiperidines containing different
flexible linker structures (a) increasing carbon chain length B1–B4 (b) methyl and
phenyl lysine esters 1–4 (c) naphthoate derivatives 5 and 6. The supercoiled form of
plasmid DNA pBR322 (3.5 nM) was incubated with the compounds at 37 °C for 3 h
2
2
. Results and discussion
.1. Synthesis of bis-3-chloropiperidines
The synthesis of bis-3-chloropiperidines B1–B4 has been
in BPE buffer, pH 7.4, at various concentrations (0.5, 5, 50
lM). Chlorambucil (CA,
50 l
M) was used as a control. Cleavage of DNA was analysed by agarose (1%) gel
electrophoresis in 1ꢀ TAE (Tris–acetate–EDTA). C = supercoiled DNA control,
1
4
reported elsewhere. The synthetic route (Scheme 1) for the
L = linear DNA control, OC = open circular (nicked) DNA control.

I. Zuravka et al. / Bioorg. Med. Chem. 23 (2015) 1241–1250
1243
O
OH
a)
O
OH
b)
O
OR
c)
O
OR
R'-O H
H2N
NH2
BocHN
NHBoc
BocHN
NHBoc
H2N
NH2
·
2 TFA
7
8
9
: R' = -CH2 C6H5
14: R = -CH C H
2
6
5
2 6 5
19: R = -CH C H
d)
10: R' = -(CH ) C H
15: R = -(CH2)2C6 H5
16: R = -(CH2)3C6 H5
17: R = -1-naphthyl
20: R = -(CH2)2 C6H5
21:
2
2
6
5
1
1: R' = -(CH2)3C6H5
R = -(CH2)3 C6H5
1
1
2: R' = -1-naphthyl
22: R = -1-naphthyl
3: R' = -1 naphthyl 4-OCH
18: R = -1-naphthyl 4-OCH3
3
23: R = -1-naphthyl-4-OCH3
O
OCH3
H2N
NH2
·
2 HCl
O
e)
e)
35
24
O
N
OR
O
N
OR
3
O
OR
Cl
Cl
g)
N
f)
N
H
N
N
3
3
H
Cl
Cl
3
3
3
3
3
3
7: R = -CH3
36: R = -CH3
1-6
0: R = -CH2C6H5
25: R = -CH2C6H5
1: R = -(CH2)2 C6H5
2: R = -(CH2)3 C6H5
3: R = -1-naphthyl
26: R = -(CH2)2 C6H5
27: R = -(CH2)3 C6H5
28: R = -1-naphthyl
4: R = -1-naphthyl-4-OCH
29: R = -1-naphthyl-4-OCH
3
3
Scheme 1. Synthesis of bis-3-chloropiperidines 1–6 (see Fig. 1c for the residues R). Reactants and conditions: (a) Boc
aromatic alcohols (9–13, respectively) DCC, HOBt, Et N, dry CH Cl
dimethoxypropane, MeOH, concd HCl, reflux to rt, 15 h, 88%; (e) 2,2-dimethylpent-4-enal (24), NaBH(OAc)
1% (27), 90% (28) 84% (29); (f) NCS, dry CH Cl
temperature), 2 h, 54% (1), 64% (2), 68% (3), 72% (4), 56% (5)50% (6) (inseparable diastereomeric mixture).
2
O, 1 N NaOH, H
Cl
, 0 °C to rt, 12 h, 89% (36), 65% (25), 89% (26),
, 60 °C (oil bath
2
O/dioxan (1:1), rt, 12 h, 92%; (b)
3
2
2
, rt, 1–3 d, 88% (14), 89% (15), 72% (16), 84% (17), 86% (18); (c) TFA, CH
2
2
, 0 °C to rt, 12 h, quant.; (d) 2,2-
18
3
2 2
, AcOH, dry CH Cl
8
2
2
, 0 °C to rt, 2.5 h, 29% (37), 29% (30), 30% (31), 28% (32), 21% (33), 36% (34); (g) TBAI (cat.), dry CHCl
3
double reductive amination of 2,2-dimethylpent-4-enal18 24 with
the appropriate lysine ester 19–23 using sodium triacetoxyborohy-
dride, followed by chlorination of diamines 25–29 with N-chloro-
succinimide (NCS). The resulting unsaturated bis-N-chloroamines
resulted in DNA fragmentation giving rise to smeary diffuse bands
on the agarose gel, as expected. Within the series, varying the lin-
ker chain length did not alter the reactivity, since compounds B1–
B4 exert similar alkylating activity (Fig. 2a), with B2 and B3 being
slightly more active. These data are consistent with our findings in
recently published DNA cleavage studies employing a different
3
2
0–34 were eventually converted into the cyclisation products
–6 in the presence of a catalytic amount of tetrabutylammonium
1
4
iodide (TBAI). The desired bis-3-chloropiperidines were obtained
as an inseparable mixture of diastereomers.
plasmid (pAT153).
Figure 2b and c show the agarose gel electrophoresis pattern
for the novel lysine-bridged bis-3-chloropiperidines 1–6. Com-
pounds 1–3 generated single-strand nicks in supercoiled DNA,
whereas 4 showed no significant effect (Fig. 2b). Compound 1,
lacking the aromatic group in the side chain of the lysine linker,
proved to be the most active agent in this series of bis-3-chloro-
piperidines (compare 1 with 2–6 in Fig. 2b and c) and displayed
almost complete conversion of the supercoiled plasmid to the
For the synthesis of compound 1 a different approach has been
chosen. The
L-lysine methyl ester 35 was prepared according to a
published method by treatment of
L
-lysine 7 with 2,2-dimethoxy-
propane and concentrated hydrochloric acid.19 The bis-3-chloropi-
peridine derivative 1 was then generated via the precursor
compounds 36 and 37 using the same three-step procedure as
described before (Scheme 1).
open circular form at the relatively low concentration of 5 lM
2
.2. Bis-3-chloropiperidines exhibit DNA nicking activity
and 3 h (Fig. 2b). However, it seems that the incorporation of
the ester functionality in the lysine linker reduces the alkylation
potency, for compound 1 displayed less activity than the corre-
We previously demonstrated that compounds B1–B4 induced
1
4
highly efficient nicking of supercoiled plasmid pAT153. In our
present study we wanted to explore if the incorporation of an aro-
matic group into the side chain of a bridging lysine linker as well as
the side chain spacer length would affect the reactivity of the
agents with DNA. Thus, the DNA alkylating activities of the syn-
thesised bis-3-chloropiperidines 1–6 were analysed by electropho-
retic assays in agarose gels using the supercoiled plasmid pBR322.
For comparison also the control compounds B1–B4 were tested in
sponding derivative B3 (compare B3 in Fig. 2a with 1 in
Fig. 2b). Moreover, a correlation between the spacer length and
alkylating properties could be ascertained within the group of
bis-3-chloropiperidines 2–4, where the alkyl chain joining the
lysine linker and the aromatic moiety was varied from one to
three carbons. As can be seen in Figure 2b, increasing the spacer
length resulted in a loss of DNA nicking activity (compare 2–4),
which may be related to entropic effects. Yet there was no appar-
ent conversion of the pBR322 plasmid into an open circular form
by the naphthoate derivatives 5 and 6 (Fig. 2c), indicating that
the compounds exert no DNA nicking activity under these condi-
these conditions. The nitrogen mustard chlorambucil (CA, 50 lM)
was used as a control. In our experiment, the supercoiled form of
pBR322 was incubated with gradually increasing concentrations
(
0.5, 5, 50
lM) of alkylating agents at 37 °C for 3 h in bisphos-
tions. However, at a concentration of 50 lM the occurrence of
phate–EDTA (BPE) buffer at pH 7.4.
DNA precipitation can be observed (visible ethidium bromide-
stained bands in the gel wells, Fig. 2c). This might be attributed
to possible binding of the agents 5 and 6 to DNA, thereby increas-
ing its molecular weight and consequently leading to precipita-
tion in the wells.
As seen in Figure 2, that depicts the results after gel electropho-
resis, bis-3-chloropiperidines B1–B4 displayed significant nicking
of the supercoiled plasmid at the lowest tested concentration of
0.5 lM (Fig. 2a). Increasing concentrations of agents B1–B4

1
244
I. Zuravka et al. / Bioorg. Med. Chem. 23 (2015) 1241–1250
suggest that the size of the ester substituent has a perceptible
effect on alkylation activity and that naphthoate analogues are able
to interact with DNA.
2
.3. Bis-3-chloropiperidines react with guanines in DNA
We recently demonstrated by polyacrylamide gel electrophore-
sis (PAGE) that bis-3-chloropiperidines preferentially induce DNA
1
4
cleavage through reactions with guanines. In order to clarify
whether the alkylating patterns of this new class of agents are con-
sistent, we carried out the same experiment used in our previous
evaluation of bis-3-chloropiperidines, exploring their effects on a
2
2-mer oligonucleotide duplex containing a G-rich sequence.14
Subsequent sequence specificity analysis by high-resolution PAGE
revealed that the alkylating potency of the set of test compounds
correlates with the DNA nicking activity (compare Figs. 2 and S1 ).
0
In fact, the results for bis-3-chloropiperidines 1–6 toward the 5 -
FAM-labelled double-stranded oligonucleotide demonstrated that
the lysine methyl ester 1 displayed significantly higher alkylation
activity than its aromatic counterparts (compare 1 with 2–6 in
Fig. S1 ).
The shifted gel bands with lower mobility than the control band
(
C, Fig. S1 ) are comparable to those obtained from our previous
study and suggest the formation of DNA adducts (DNA adducts,
1
4
Fig. S1 ). In addition, there were no marked differences in selec-
14
tivity of DNA alkylation compared to our previous analysis. Our
current results confirm that bis-3-chloropiperidines induce DNA
strand cleavage primarily at guanine sites (compare Fig. S1 ).
Figure 3. DNA cleavage activity of bis-3-chloropiperidines containing different
lysine linker structures (a) methyl and phenyl lysine esters 1–4 (b) naphthoate
derivatives 5 and 6. The supercoiled form of plasmid DNA pBR322 (3.5 nM) was
incubated with the compounds at ambient temperature for 24 h in BPE buffer, pH
2
.4. ESI-MS analysis of reactive aziridinium ion formation
The proposed mechanism of DNA alkylation by bis-3-chloropi-
7
.4, at various concentrations (0.5, 5, 50
l
M). Chlorambucil (CA, 50
l
M) was used as
peridines involves an intramolecular nucleophilic displacement
of chloride by nitrogen to afford a reactive bicyclic aziridinium
ion. Once formed, this electrophilic intermediate can be rapidly
attacked by nucleophiles. To prove this hypothesis and to inves-
tigate the chemical behaviour of the lysine-linked bis-3-chloropi-
a control. Cleavage of DNA was analysed by agarose (1%) gel electrophoresis in 1ꢀ
TAE (Tris–acetate–EDTA). C = supercoiled DNA control, L = linear DNA control,
OC = open circular (nicked) DNA control.
1
4
peridines described,
a 80 lM solution of the most active
We repeated the experiment for compounds 1–6 at lower tem-
peratures and increasing the incubation time to 24 h to verify DNA
cleavage and to enhance possible stacking interactions between
DNA bases and naphthyl groups. The obtained electrophoretic pat-
terns, shown in Figure 3, were similar to those seen after 3 h of
incubation at 37 °C for 1–4 (compare Fig. 2b with Fig. 3a), further
validating our results. Gradually increasing the concentrations of
the naphthoate derivatives 5 and 6 resulted again in precipitation
of DNA, confirming our suggestion that 5 and 6 interact with DNA.
This phenomenon became significantly obvious with the meth-
compound (1) in distilled water was prepared and incubated for
a time consistent with the reaction with DNA. Small samples were
taken at different incubation times (0 min, 15 min, 30 min, 45 min,
60 min and 120 min, at 37 °C) and analysed by ESI-MS (Figs. 4 and
S2–S6 ).
The results of ESI-MS analysis demonstrate the fast formation of
the reactive species: while the major peak in the ESI mass spec-
trum of Figure 4 is assigned to the hydroxyl substituted derivative
(1OH), we observe the appearance of the electrophilic aziridinium
+
ion (1N OH), detected already after 15 min of incubation. A minor
oxy-substituted naphthoate 6 at a concentration of 50
l
M (Fig. 2c
peak assigned to the dihydroxyl substituted compound (12OH) is
also apparent at 15 min (Fig. 4). Further hydrolysis leads to a sub-
sequent increase in intensity of the dihydroxyl substituted product
peak (12OH) coexisting with the reactive aziridinium species
and Fig. 3b), implying that the incorporation of a methoxy group
can possibly improve DNA interactions. Compounds 5 and 6
seemed to be somewhat more active under the chosen conditions
+
(
compare Fig. 2c with Fig. 3b). These findings might be explained
(1N OH, compare Figs. S2–S6 corresponding to longer incubation
by the prolonged contact of the agents with DNA due to longer
incubation time. Therefore, the slightly higher activity of 5 and 6
may be related to an improved positioning of the alkylating species
with respect to the DNA. This supports the idea that combining
alkylating agents with DNA-affinity moieties is a promising possi-
bility to design DNA-recognizing hybrid molecules with nucleic
acids alkylation capability.
In summary, these findings revealed that aromatic groups at the
linker side chain are compatible with DNA alkylating activity,
although its positioning influences the reactivity with DNA. Consis-
tent with our previous data, the antitumour drug chlorambucil
time). These observations, and the rapid formation of the reactive
species followed by the nucleophilic attack of water, are in good
correlation with our hypothesis and with studies on DNA reactivity
1
4
shown here and in previous paper.
Consequently, the present investigations are further supporting
our proposed mechanism of reaction. While more work is needed
to determine the reactivity in complex biological conditions
reflecting in vivo environment, the current studies serve as an
experimental model system to demonstrate the efficacy of bis-3-
chloropiperidines as DNA alkylating agents.
(
CA) had no detectable activity on plasmid DNA under the chosen
experimental conditions (Figs. 2 and 3). In addition, our results
See Supplementary data S1–S6.

I. Zuravka et al. / Bioorg. Med. Chem. 23 (2015) 1241–1250
1245
Figure 4. ESI-MS analysis of a sample taken after an incubation time of 15 min at 37 °C in a hydrolysis experiment of lysine methyl ester 1. The ESI mass spectrum displays
+
+
+
the hydroxyl substituted derivative (1OH m/z 403 [M+H] and m/z 425 [M+Na] ) and the disubstituted hydrolysis product (12OH m/z 385 [M+H] ) as well as the reactive
þ
aziridinium species (1 OH m/z 367 [M] ).
N
+
3
. Conclusions
A series of new lysine-bridged bis-3-chloropiperidines were
studies might represent valuable starting points for further devel-
opment and optimisation of novel alkylating derivatives based on
the bis-3-chloropiperidine scaffold.
prepared as model compounds in order to explore the influence
of aromatic moieties on DNA alkylating activity. Accordingly, a
phenyl group was attached by esterification to the lysine side chain
via hydrocarbon spacers of different chain length (compounds 2–
4
4
. Experimental section
.1. General remarks
4). In addition, two derivatives (5 and 6) containing a naphthoate
unit were synthesised to analyse whether the incorporation of this
known DNA-binding chromophore might increase reactivity
toward DNA. Thus, we evaluated DNA alkylation activity by a
DNA cleavage assay with a supercoiled plasmid and sequencing
gel analysis with a 22-mer duplex oligonucleotide. Results with
nucleic acids are consistent with ESI-MS analysis, confirming the
fast formation of the reactive species in solution. Our studies
revealed a clear correlation between linker structure and extent
of DNA alkylation.
As a proof-of-principle, the results demonstrated that the intro-
duction of aromatic groups to the linker side chain curtail the reac-
tivity of bis-3-chloropiperidines in comparison to their related
counterparts containing symmetrical non-aromatic linkers. Fur-
thermore, it was shown that an increase in the length of the alkyl
spacer chain attached to the linker also lowers the activity (com-
pounds 2–4). Within the current series, the most active compound
was the derivative 1 suggesting that bis-3-chloropiperidines with-
out an aromatic group seem to be significantly more potent alkyl-
ating agents. These findings are consistent with recently published
data and can be particularly advantageous in terms of modulating
the reactivity of bis-3-chloropiperidines towards DNA.14
Commercially available reagents were used as supplied. All sol-
vents were purified by distillation and dried, if necessary, by stan-
dard methods. Reactions requiring the use of anhydrous solvents
were carried out in heat-gun-dried glassware under a nitrogen
atmosphere (Schlenk technique). Products were purified by flash
chromatography on silica gel 60 (Merck). Melting points were
measured using Digital Melting Point Analyzer KSP1N apparatus
1
13
(
Krüss Optronic) and are uncorrected. H and C NMR spectra
1
were recorded on a Bruker Avance II 200 spectrometer ( H at
1
3
1
2
00 MHz; C at 50 MHz), Bruker Avance II 400 spectrometer ( H
1
3
at 400 MHz; C at 100 MHz) and Bruker Avance III 600 spectrom-
1
13
eter ( H at 600 MHz; C at 150 MHz) in the deuterated solvent sta-
ted using TMS as internal standard. Chemical shifts (d is expressed
in part per million) were determined by reference to the residual
solvent resonances. High-resolution ESI mass spectrometry data
were obtained with ESImicrOTOF (Bruker Daltonics) mass spec-
trometer. The samples were dissolved in methanol and analysed
in positive ion mode. All elemental analysis (CHN) were performed
on a Carlo Erba Modell 1106 instrument.
It is noteworthy that although the naphthoate analogues 5 and
4.2. Synthetic procedures
6
proved to be less potent DNA alkylating agents our experiments
indicate that these compounds interact with DNA. This suggests
that the presence of a DNA-affinity moiety in the bis-3-chloropi-
peridine molecule can contribute to DNA recognition and might
direct the location of the alkylating unit near to suitable DNA
bases.
Moreover, the present results also confirm those from our pre-
vious study showing that the examined compounds induce cleav-
age of double-stranded DNA, primarily through reactions toward
guanine residues through a DNA nicking mechanism.14 Currently,
additional biochemical investigations and measurements of cyto-
toxicity are in progress to provide further insight into the mecha-
nism of action. Utilizing the knowledge obtained from these
4.2.1. 2,2-Dimethylpent-4-enal (24)
Freshly distilled isobutyraldehyde (108 g, 1.5 mol) and allyl
alcohol (58.0 g, 1.0 mol) were added to a solution of p-toluenesul-
fonic acid (0.25 g) in p-cymene (200 g). The mixture was heated to
reflux for 32 h under a Dean–Stark trap until no more water was
separated and a sump temperature of about 140 °C was reached.
After vacuum distillation (76 °C at 200 mbar) through a 50 cm Vig-
reux column the aldehyde (81.4 g, 0.73 mol, 73%) was obtained as a
1
colourless liquid. H NMR (CDCl
3
, 200 MHz): d = 9.47 (s, 1H), 5.60–
5.70 (m, 1H), 5.01–5.05 (m, 2H), 2.21 (d, J = 7.3 Hz, 2H), 1.05 (s, 6H)
1
3
ppm; C NMR (CDCl
3
, 50 MHz): d = 205.9, 133.1, 118.4, 45.7, 41.4,
18
21.1 ppm. These data are consistent with published data.

1
246
I. Zuravka et al. / Bioorg. Med. Chem. 23 (2015) 1241–1250
4
.2.2.
,2-Dimethoxypropane (70 mL) and concentrated hydrochloric
acid (18 mL) were added to a suspension of -lysine monohydro-
L
-Lysine methyl ester dihydrochloride (35)
38 2 6
pale yellow oil; (Found: C, 63.85; H, 8.66; N, 6.18. C24H N O
1
2
requires C, 63.98; H, 8.50; N, 6.22%);
3
H NMR (CDCl ,
L
400.1 MHz): d = 7.27–7.31 (m, 2H), 7.19–7.24 (m, 3H), 4.20–4.40
(m, 3H), 3.01– 3.11 (m, 2H), 2.94 (t, J = 7.0 Hz, 2H), 1.51–1.75 (m,
chloride (10.0 g, 54.7 mmol) in methanol (110 mL). The reaction
mixture was heated under reflux for 3 h and stirred 12 h at room
temperature. The solvent was removed under reduced pressure
and the resulting oil was dissolved in a small amount of methanol.
Addition of ice-cold tert-butyl methyl ether (450 mL) resulted in
crystallisation of the desired product. The compound was recrys-
tallised from methanol/tert-butyl methyl ether (11.2 g, 48.0 mmol,
1
3
3
2H), 1.43 (s, 18H), 1.06–1.38 (m, 4H) ppm; C NMR (CDCl ,
100.6 MHz): d = 172.5, 155.8, 155.2, 137.3, 128.8, 128.7, 126.5,
79.6, 78.9, 65.4, 63.5, 39.9, 34.8, 32.1, 29.3, 28.2, 22.2 ppm; HRMS
+
(ESI): m/z calcd for C24
[M+Na] .
H
38
N
2
O
6
Na : 473.2628; found: 473.2623
+
1
9
1
8
8%); mp 204 °C (lit., 203–205 °C); H NMR (CD
d = 4.06 (t, J = 6.5 Hz, 1H), 3.82 (s, 3H), 2.94 (t, J = 7.5 Hz, 2H), 1.86–
.01 (m, 2H), 1.71 (dt, J = 15.3 Hz, J = 7.7 Hz, 2H), 1.44–1.61 (m, 2H)
3
OD, 400.1 MHz):
4.2.4.3.
(S)-3-Phenylpropyl
2,6-bis(tert-butoxycarbonyl-
amino)hexanoate (16). Prepared according to the general pro-
cedure from 8 (2.00 g, 5.77 mmol) and 3-phenylpropyl alcohol
(393 mg, 2.89 mmol) yielding 16 (969 mg, 2.09 mmol, 72%) after
43 h of reaction time as a colourless oil; (Found: C, 64.68; H,
2
1
3
ppm; C NMR (CD
3
OD, 100.6 MHz): d = 170.8, 53.7, 53.6, 40.2,
3
0.9, 27.9, 12.1 ppm. These data are consistent with published
1
9
data.
8.67; N, 5.97. C25
H
40
N
2
O
6
requires C, 64.63; H, 8.68; N, 6.03%);
1
3
H NMR (CDCl , 400.1 MHz): d = 7.26–7.31 (m, 2H), 7.16–7.21 (m,
2
6
4
.2.3. N ,N -Bis(tert-butoxycarbonyl)-
To a solution of -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 N NaOH aq (35 mL). The reaction mix-
L-lysine (8)
3H), 4.24–4.29 (m, 1H), 4.14 (t, J = 6.5 Hz, 2H), 3.07–3.14 (m, 2H),
L
2.66–2.73 (m, 2H), 1.76–2.08 (m, 4H), 1.59–1.68 (m, 2H), 1.44 (d,
1
3
J = 3.7 Hz, 18H), 1.32–1.40 (m, 2H) ppm;
3
C NMR (CDCl ,
(
100.6 MHz): d = 172.8, 156.0, 140.9, 128.4, 128.3, 126.0, 79.8,
ture was stirred at room temperature 12 h and then concentrated
in vacuo until approximately 50 mL remained. The pH was
64.5, 62.2, 40.0, 34.2, 32.0, 30.0, 29.5, 28.4, 28.3, 22.4 ppm; HRMS
+
(ESI): m/z calcd for C25
[M+H] .
H
41
N
O
2 6
: 487.2784; found: 487.2736
+
adjusted to 1–2 by careful addition of an aqueous KHSO
150 g/L). The suspension was extracted three times with ethyl
acetate. The combined organic layers were dried over MgSO and
the solvent was removed under reduced pressure to afford the
product (9.89 g, 28.5 mmol, 92%) as an oil. 1H NMR (DMSO-d
00.1 MHz): d = 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,
8H), 1.14–1.68 (m, 6H) ppm; 13C NMR (DMSO-d
, 50.3 MHz):
4
solution
(
4
4.2.4.4.
(S)-Naphthalen-1-yl
2,6-bis((tert-butoxycarbonyl)-
amino)hexanoate (17). Prepared according to the general proce-
dure from 8 (2.00 g, 5.77 mmol) and naphthalen-1-ol (416 mg,
6
,
2
2.89 mmol) yielding 17 (1.91 g, 2.41 mmol, 84%) after 68 h of reac-
1
tion time as a yellow solid; H NMR (CDCl
3
, 400.1 MHz): d = 7.85–
1
6
7.91 (m, 2H), 7.74 (d, J = 7.8 Hz, 1H), 7.50–7.54 (m, 2H), 7.45 (t,
J = 7.8 Hz, 1H), 7.22 (d, J = 7.0 Hz, 1H), 5.24 (br s, 1H), 3.18 (br s,
2H), 2.10–2.17 (m, 1H), 1.91–2.00 (m, 1H), 1.57–1.64 (m, 4H), 1.49
d = 173, 155.1, 77.4, 76.8, 52.9, 29.9, 28.6, 27.7, 22.4 ppm (one sig-
nal, probably around 40 ppm, is hidden by the solvent signal).
13
(
3
s, 9H), 1.45 (m, 9H) ppm; C NMR (CDCl , 100.6 MHz): d = 171.7,
4
.2.4. General synthetic procedure for compounds 14–18
171.1, 156.1, 155.7, 146.4, 134.6, 127.9, 126.6, 126.5, 126.3, 125.3,
2
6
N ,N -Bis(tert-butoxycarbonyl)-
L-lysine (8) (2 equiv) was dis-
121.2, 117.8, 80.2, 79.2, 53.7, 40.0, 32.1, 29.7, 28.4, 28.3, 22.7 ppm;
+
solved in anhydrous dichloromethane (10 mL/mmol of 8). Triethyl-
amine (2 equiv) was added, followed by the appropriate alcohol
HRMS (ESI): m/z calcd for
495.2471 [M+Na] .
C
26
H
36
N
2
NaO
6
:
495.2471; found:
+
(
1 equiv). The mixture was stirred for a couple of minutes under
nitrogen atmosphere and then cooled to 0 °C. Hydroxybenzotria-
zole (HOBt, 2 equiv) and dicyclohexyl carbodiimide (DCC, 2 equiv)
were added simultaneously. The reaction mixture was stirred at
room temperature for the times indicated below. The precipitate
was removed by filtration and discarded. The filtrate was washed
4.2.4.5. (S)-4-Methoxynaphthalen-1-yl 2,6-bis((tert-butoxy-car-
bonyl)amino)hexanoate (18). Prepared according to the general
procedure from 8 (2.00 g, 5.77 mmol) and 4-methoxynaphthalen-
1-ol (503 mg, 2.89 mmol) yielding 18 (1.25 g, 2.49 mmol, 86%)
after 68 h of reaction time as a pale red, gel-like solid; 1H NMR
successively with NaHCO
NaHCO aq (satd) and finally with water. The organic phase was
dried over MgSO and the solvent was removed under reduced
pressure. The resulting crude product was purified by flash chro-
matography (pentane/ethyl acetate 1:1).
3
aq (satd), then NaHSO
4
aq (150 g/L),
3
(CDCl , 400.1 MHz): d = 8.24–8.27 (m, 1H), 7.79 (d, J = 7.9 Hz, 1H),
3
7.47–7.55 (m, 2H), 7.13 (d, J = 8.5 Hz, 1H), 6.75 (d, J = 8.3 Hz, 1H),
5.27 (br s, 1H), 4.64–4.65 (m, 1H), 3.99 (s, 3H), 3.17 (s, 2H), 2.08–
2.16 (m, 1H), 1.70–1.85 (m, 1H), 1.53–1.63 (m, 4H), 1.48 (s, 9H),
4
1
3
1.44 (m, 9H) ppm; C NMR (CDCl
56.1, 155.7, 153.6, 139.7, 127.3, 127.1, 126.2, 125.8, 122.3,
120.0, 117.4, 80.1, 55.7, 53.7, 32.2, 29.7, 28.4, 28.3, 21.0 ppm;
3
, 100.6 MHz): d = 172.0, 171.1,
1
4
.2.4.1. (S)-Benzyl 2,6-bis(tert-butoxycarbonylamino)-hexano-
ate (14). Prepared according to the general procedure from 8
405 mg, 1.17 mmol) and benzyl alcohol (63.2 mg, 0.59 mmol)
yielding 14 (225 mg, 0.52 mmol, 88%) after 21 h of reaction time
+
HRMS (ESI): m/z calcd for
27 38 2 7
C H N NaO : 525.2577; found:
+
(
525.2574 [M+Na] .
1
as a colourless oil. H NMR (CDCl
3
, 400.1 MHz): d = 7.31–7.39 (m,
H), 5.11–5.22 (m, 2H), 4.29–4.35 (m, 1H), 3.03–3.09 (m 2H),
.77–1.89 (m, 2H), 1.59–1.69 (m, 2H), 1.43 (s, 18H), 1.29–1.37
4.2.5. General synthetic procedure for compounds 19–23
(S)-2,6-Bis(tert-butoxycarbonyl)-lysine ester was dissolved in
dichloromethane (5 mL/mmol) and trifluoroacetic acid (1 mL/
mmol) was slowly added at 0 °C. The reaction mixture was stirred
12 h at room temperature. Removal of the solvent under reduced
pressure gave the pure product in quantitative yield.
5
1
(
1
3
m, 2H) ppm; C NMR (CDCl
3
, 100.6 MHz): d = 172.6, 156.0,
1
2
4
35.4, 128.6, 128.4, 128.3, 79.6, 78.8, 66.9, 60.4, 40.1, 32.3, 29.5,
+
8.4, 28.3, 22.4 ppm; HRMS (ESI): m/z calcd for C23
H
36
N
2
O
6
Na :
+
59.2471; found: 459.2466 [M+Na] .
4
.2.5.1. (S)-Benzyl 2,6-diaminohexanoate TFA salt (19). Fol-
4
.2.4.2. (S)-Phenethyl 2,6-bis(tert butoxycarbonylamino)-hexa-
lowing the general procedure, 14 (210 mg, 0.48 mmol) was depro-
noate (15). Prepared according to the general procedure from 8
8.80 g, 25.4 mmol) and 2-phenylethyl alcohol (1.55 g, 12.7 mmol)
yielding 15 (5.10 g, 11.3 mmol, 89%) after 48 h of reaction time as a
tected to provide the corresponding TFA salt 19 in quantitative
1
(
yield. H NMR (DMSO-d
6
, 400.1 MHz): d = 8.47 (s, 2H), 7.79 (s,
2H), 7.35–7.43 (5H), 5.24 (s, 2H), 4.08–4.10 (m, 1H), 2.67–2.74

I. Zuravka et al. / Bioorg. Med. Chem. 23 (2015) 1241–1250
1247
(
m, 2H), 1.73–1.84 (m, 2H), 1.48–1.55 (m, 2H), 1.23–1.44 (m, 2H)
4.2.6.1. (S)-Methyl 2,6-bis(2,2-dimethylpent-4-enylamino)-hex-
anoate (36). Prepared according to the general procedure from
1
3
ppm;
6
C NMR (DMSO-d , 50.3 MHz): d = 168.8, 134.6, 128.0,
1
27.9, 127.8, 66.6, 51.2, 51.2, 28.9, 25.8, 20.7 ppm. HRMS (ESI):
2,2-dimethyl-4-pentenal 24 (5.57 g, 49.6 mmol) and L-lysine
methyl ester dihydrochloride 35 (4.82 g, 20.7 mmol) yielding 36
(6.46 g, 18.3 mmol, 89%) as a pale yellow liquid; (Found: C,
+
+
m/z calcd for C13
one signal, probably around 40 ppm, is hidden by the solvent
signal).
H
21
N
O
2 2
: 237.1603; found: 237.1590 [M+H]
(
71.16; H, 11.53; N, 7.82. C21
H
40
N
2
O
2
requires C, 71.54; H, 11.44;
1
N, 7.95%); H NMR (CDCl
3
, 400.1 MHz): d = 5.73–5.87 (m, 2H),
4
(
.2.5.2. (S)-Phenethylhexane 2,6-diaminohexanoate TFA salt
20). Following the general procedure, 15 (925 mg, 2.05 mmol)
was deprotected to provide the corresponding TFA salt 20 in quan-
4.97–5.06 (m, 4H), 3.69 (s, 3H), 3.12 (t, J = 6.8 Hz, 1H), 2.52 (t,
J = 6.8 Hz, 2H), 2.34 (d, J = 11.3 Hz, 1H), 2.32 (s, 2H), 2.07 (d,
J = 11.5 Hz, 1H), 1.95–2.02 (m, 4H), 1.29–1.65 (m, 6H), 0.84 and
1
13
titative yield. H NMR (DMSO-d
6
, 400.1 MHz): d = 8.44 (s, 2H), 7.83
0.88 (2ꢀ s, total 12H) ppm;
3
C NMR (CDCl , 100.6 MHz):
(
s, 2H), 7.22–7.34 (m, 5H), 4.32–4.81 (m, 2H), 3.87–4.04 (m, 1H),
d = 176.2, 135.3, 116.6, 116.5, 62.3, 60.2, 58.0, 51.3, 50.5, 44.5,
2
1
1
3
2
.95 (t, J = 6.5 Hz, 2H), 2.66–2.73 (m, 2H), 1.64–1.70 (m, 2H),
44.1, 34.2, 34.0, 33.3, 29.6, 25.3, 25.1, 25.0, 23.6 ppm.
13
.42–1.48 (m, 2H), 1.13–1.36 (m, 2H) ppm; C NMR (DMSO-d
6
,
00.6 MHz): d = 169.0, 137.1, 128.4, 127.9, 126.1, 65.6, 51.2, 37.9,
4.2.6.2. (S)-Benzyl 2,6-bis(2,2-dimethylpent-4-enylamino)-hex-
anoate (25). Prepared according to the general procedure from
19 (850 mg, 1.83 mmol) and 2,2-dimethyl-4-pentenal 24
+
3.6, 29.0, 26.6, 20.7 ppm; HRMS (ESI): m/z calcd for C14
51.1760; found: 251.1715 [M+H] .
H
23
N
2
O
2
:
+
(
493 mg, 4.39 mmol) yielding 25 (510 mg, 1.19 mmol, 65%) as a
1
4
.2.5.3. (S)-3-Phenylpropyl 2,6-diaminohexanoate TFA salt
3
pale yellow oil. H NMR (CDCl , 400.1 MHz): d = 7.30–7.38 (m,
(
21). Following the general procedure, 16 (190 mg, 0.41 mmol)
5H), 5.70–5.81 (m, 2H), 5.12–5.16 (m, 2H), 4.95–5.10 (m, 4H),
3.10–3.13 (m, 1H), 2.86–2.90 (m, 2H), 2.38 (d, J = 11.3 Hz, 1H),
2.15 (d, J = 7.8 Hz, 2H), 2.01–2.03 (m, 1H), 1.93–1.96 (m, 2H),
1.76–1.84 (m, 2H), 1.15–1.69 (m, 6H), 1.07 (s, 6H), 0.82 (2ꢀ s, total
was deprotected to provide the corresponding TFA salt 21 in quan-
titative yield. H NMR (DMSO-d
1
6
, 400.1 MHz): d = 8.44 (s, 2H), 7.78
(
s, 2H), 7.28–7.31 (m, 2H), 7.18–7.22 (m, 3H), 4.16 (t, J = 6.5 Hz,
1
3
2
1
1
1
2
2
H), 3.99–4.05 (m, 1H), 2.73–2.80 (m, 2H), 2.66 (t, J = 7.5 Hz, 2H),
3
6H) ppm; C NMR (CDCl , 100.6 MHz): d = 175.3, 135.8, 134.4,
.89–1.96 (m, 2H), 1.75–1.81 (m, 2H), 1.51–1.58 (m, 2H), 1.30–
133.3, 128.5, 128.3, 118.9, 116.8, 66.3, 62.2, 58.1, 57.3, 49.1, 44.3,
.47 (m, 2H) ppm; 13C NMR (DMSO-d
, 50.3 MHz): d = 169.1,
40.6, 128.0, 127.9, 125.6, 64.6, 59.4, 51.4, 30.8, 29.2, 29.1, 26.0,
43.5, 34.4, 33.5, 32.7, 29.6, 25.2, 25.1, 23.8 ppm; HRMS (ESI): m/z
6
+
+
calcd for C27
H
45
N
2
O
2
: 429.3481; found: 429.3454 [M+H] .
+
0.4 ppm; HRMS (ESI): m/z calcd for C15
H
25
N
2
O
2
: 265.1916; found:
+
65.1902 [M+H] .
4.2.6.3.
(S)-Phenethyl 2,6-bis(2,2-dimethylpent-4-enyl-
amino)hexanoate (26). Prepared according to the general pro-
cedure from 20 (980 mg, 2.05 mmol) and 2,2-dimethyl-4-pentenal
4
.2.5.4. (S)-Naphthalen-1-yl 2,6-diaminohexanoate TFA salt
(
22). Following the general procedure, 16 (190 mg, 0.41 mmol)
24 (551 mg, 4.92 mmol) yielding 26 (810 mg, 1.83 mmol, 89%) as a
1
was deprotected to provide the corresponding TFA salt 21 in quan-
titative yield. H NMR (DMSO-d
(
2
1
1
1
2
2
3
pale yellow oil. H NMR (CDCl , 400.1 MHz): d = 7.28–7.33 (m, 2H),
1
6
, 400.1 MHz): d = 8.44 (s, 2H), 7.78
7.10–7.25 (m, 3H), 5.73–5.88 (m, 2H), 4.97–5.07 (m, 4H), 4.35 (t,
J = 7.0 Hz, 2H), 3.03–3.15 (m, 1H), 2.90–2.97 (m, 2H), 2.72 (s, 1H),
2.07–2.36 (m, 5H), 1.95–2.03 (m, 4H), 1.74–1.83 (m, 2H, 3-H),
s, 2H), 7.28–7.31 (m, 2H), 7.18–7.22 (m, 3H), 4.16 (t, J = 6.5 Hz,
H), 3.99–4.05 (m, 1H), 2.73–2.80 (m, 2H), 2.66 (t, J = 7.5 Hz, 2H),
13
.89–1.96 (m, 2H), 1.75–1.81 (m, 2H), 1.51–1.58 (m, 2H), 1.30–
1.30–1.65 (m, 4H), 0.84 and 0.89 (2ꢀ s, total 12H) ppm; C NMR
(CDCl , 100.6 MHz): d = 175.3, 137.1, 134.9, 134.7, 128.3, 127.9,
126.0, 116.5, 116.2, 71.1, 64.2, 50.1, 44.2, 43.8, 42.8, 34.6, 33.8,
.47 (m, 2H) ppm; 13C NMR (DMSO-d
, 50.3 MHz): d = 169.1,
40.6, 128.0, 127.9, 125.6, 64.6, 59.4, 51.4, 30.8, 29.2, 29.1, 26.0,
6
3
+
+
0.4 ppm; HRMS (ESI): m/z calcd for C15
H
25
N
2
O
2
: 265.1916; found:
33.6, 24.7, 24.6, 23.2 ppm; HRMS (ESI): m/z calcd for C28
443.3638; found: 443.3620 [M+H] .
H
47
N
2
O
2
:
+
+
65.1902 [M+H] .
4
.2.5.5. (S)-4-Methoxynaphthalen-1-yl 2,6-diamino-hexanoate
4.2.6.4. (S)-3-Phenylpropyl 2,6-bis(2,2-dimethylpent-4-enyl-
amino)hexanoate (27). Prepared according to the general
TFA salt (23). Following the general procedure, 18 (968 mg,
1
2
8
2
1
.93 mmol) was deprotected to provide the corresponding TFA salt
procedure from 21 (1.00 g, 2.03 mmol) and 2,2-dimethyl-4-pente-
1
3 in quantitative yield. H NMR (DMSO-d
6
, 400.1 MHz): d = 8.19–
nal 24 (547 mg, 4.87 mmol) yielding 27 (755 mg, 1.65 mmol, 81%)
1
.23 (m, 1H), 7.93–7.96 (br s, 2H), 7.88–7.90 (m, 1H), 7.56–7.65 (m,
H), 7.32 (d, J = 8.4 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H), 4.55–4.60 (m,
H), 4.17–4.22 (m, 4H), 4.00 (s, 1H), 2.84–2.86 (m, 2H), 2.03–2.18
as a yellow-brown oil. H NMR (CDCl
3
, 400.1 MHz): d = 7.26–7.30
(m, 2H), 7.16–7.21 (m, 3H), 5.73–5.81 (m, 2H), 4.96–5.07 (m,
4H), 4.09–4.15 (m, 2H), 3.08 (dd, J = 7.7 Hz, J = 5.5 Hz, 1H), 2.92–
2.96 (m, 2H), 2.66–2.69 (m, 4H), 2.16–2.43 (m, 4H), 1.82–2.09
(m, 8H), 1.32–1.69 (m, 4H), 1.09 (s, 6H), 0.85 (2ꢀ s, total 6H)
13
(
m, 2H), 1.55–1.72 (m, 4H);
6
C NMR (DMSO-d , 50.3 MHz):
d = 168.8, 158.6, 158.3, 153.2, 138.7, 127.2, 126.5, 126.1, 125.3,
1
1
3
21.0, 118.2, 115.2, 103.5, 55.9, 51.9, 33.7, 29.6, 26.6, 21.5 ppm.
3
ppm; C NMR (CDCl , 100.6 MHz): d = 175.4, 140.9, 135.4, 135.2,
1
28.4, 128.3, 126.0, 117.1, 116.9, 63.9, 62.3, 58.2, 49.1, 44.3, 44.2,
4
.2.6. General synthetic procedure for compounds 25–29 and 36
To a solution of the unsaturated aldehyde 24 (2.4 equiv) and the
43.3, 34.4, 33.5, 32.8, 32.1, 25.3, 25.2, 23.7, 23.4 ppm; HRMS
+
(ESI): m/z calcd for C29
[M+H] .
H
49
N
2
O
2
: 457.3794; found: 457.3758
+
appropriate diamine in anhydrous dichloromethane (7 mL/mmol
of diamine) was added sodium triacetoxyborohydride (3 equiv)
portion wise at 0 °C, followed by acetic acid (1.2 equiv). The reac-
tion mixture was stirred at room temperature under an nitrogen
atmosphere for 12 h and was then quenched with 20% NaOH solu-
tion. The phases were separated and the aqueous layer was
extracted three times with 20 mL dichloromethane. The combined
organic phases were first washed with brine, then with water and
4.2.6.5. (S)-Naphthalen-1-yl 2,6-bis((2,2-dimethylpent-4-en-1-
yl)amino)hexanoate (28). Prepared according to the general pro-
cedure from 22 (765 mg, 1.53 mmol) and 2,2-dimethyl-4-pentenal
24 (412 mg, 3.67 mmol) yielding 28 (638 mg, 1.37 mmol, 90%) as
1
a yellow-orange oil. H NMR (CDCl
3
, 400.1 MHz): d = 7.85–7.90 (m,
2H), 7.74–7.76 (m, 1H), 7.45–7.54 (m, 3H), 7.25 (d, J = 7.2 Hz, 1H),
5.77–5.91 (m, 2H), 4.99–5.06 (m, 4H), 3.54–3.58 (m, 1H), 2.64–
2.69 (m, 3H), 2.32–2.38 (m, 3H), 1.97–2.09 (m, 5H), 1.81–1.89 (m,
1H), 1.56–1.73 (m, 4H), 1.32–1.55 (m, 2H), 0.94 (2ꢀ s, total 6H),
4
dried over MgSO . The solvent was removed under reduced pres-
sure to afford the corresponding product, which was used for the
next step without further purification.

1
248
I. Zuravka et al. / Bioorg. Med. Chem. 23 (2015) 1241–1250
0
1
1
2
C
.90 (s, 6H) ppm; 13C NMR (CDCl
3
, 100.6 MHz): d = 174.6, 146.6,
2H), 2.06 (d, J = 7.5 Hz, 2H), 2.04 (d, J = 8.5 Hz, 2H), 1.77–1.85 (m,
35.5, 135.4, 134.7, 128.1, 126.8, 126.4, 126.0, 125.4, 121.1, 118.0,
17.0, 116.8, 62.9, 60.4, 58.5, 50.8, 44.8, 44.5, 34.6, 34.2, 33.7,
9.9, 25.5, 25.4, 25.3, 23.9 ppm; HRMS (ESI): m/z calcd for
2H), 1.60–1.69 (m, 2H), 1.49–1.57 (m, 1H), 1.31–1.41 (m, 1H),
1
3
0.95 (s, 6H), 0.91 (2ꢀ s, total 6H) ppm;
3
C NMR (CDCl ,
100.6 MHz): d = 170.9, 141.0, 135.2, 135.1, 128.5, 128.4, 126.1,
+
+
30
H
45
N
2
O
2
: 465.3481; found: 465.3484 [M+H] .
117.3, 117.2, 74.8, 73.4, 66.4, 64.1, 44.8, 44.6, 35.9, 35.6, 32.3,
3
0.4, 30.3, 27.8, 27.0, 25.7, 25.6, 25.4, 23.3 ppm; HRMS (ESI): m/z
+
+
4
4
.2.6.6. (S)-4-Methoynaphthalen-1-yl 2,6-bis((2,2-dimethyl-pent-
-en-1-yl)amino)hexanoate (29). Prepared according to the gen-
2 2 2
calcd for C28H45Cl N O : 511.2858; found: 511.2828 [M+H] .
eral procedure from 23 (1.00 g, 1.88 mmol) and 2,2-dimethyl-4-
pentenal 24 (508 mg, 4.52 mmol) yielding 29 (785 mg, 1.59 mmol,
4
4
.2.7.4. (S)-3-Phenylpropyl 2,6-bis(N-chloro(2,2-dimethyl-pent-
-en-1-yl)amino)hexanoate (32). Prepared according to the
8
8
4%) as a pale brown oil. 1H NMR (CDCl
3
, 400.1 MHz): d = 8.24–
.28 (m, 1H), 7.74–7.79 (m, 1H), 7.48–7.55 (m, 2H), 7.11 (d,
general procedure from 27 (750 mg, 1.64 mmol) yielding 32
245 mg, 0.45 mmol, 28%) as a clear and colourless viscous liquid.
(
J = 8.5 Hz, 1H), 6.76 (d, J = 8.3 Hz, 1H), 5.76–5.91 (m, 2H), 4.99–
1
H NMR (CDCl
3
, 400.1 MHz): d = 7.32–7.37 (m, 2H), 7.24–7.29 (m,
H), 5.81–5.92 (m, 2H), 5.05–5.11 (m, 4H), 4.20–4.30 (m, 2H),
.54 (t, J = 7.3 Hz, 1H), 2.97–3.03 (m, 3H), 2.90 (s, 2H), 2.79 (t,
5
2
1
.07 (m, 4H), 4.00 (s, 3H), 3.51–3.55 (m, 1H), 2.62–2.67 (m, 2H),
.38 (s, 2H), 2.31–2.35 (m, 1H), 1.93–2.11 (m, 5H), 1.78–1.87 (m,
3
3
1
3
H), 1.56–1.74 (m, 5H), 0.90 and 0.93 (2ꢀ s, total 12H) ppm;
, 100.6 MHz): d = 174.9, 153.4, 139.9, 135.5, 127.5,
26.9, 126.2, 125.7, 122.5, 120.8, 117.6, 116.9, 116.8, 102.8, 71.7,
2.8, 60.3, 58.5, 55.7, 50.7, 44.8, 44.5, 43.4, 34.6, 34.2, 25.5, 25.4,
C
J = 7.5 Hz, 2H), 2.10–2.13 (m, 4H), 2.05–2.10 (m, 2H), 1.88–1.93
m, 2H), 1.70–1.78 (m, 2H), 1.43–1.52 (m, 1H), 1.25 (s, 2H), 0.99
NMR (CDCl
3
(
1
6
2
4
13
and 1.01 (2ꢀ s, total 12H) ppm; C NMR (CDCl
3
, 100.6 MHz):
d = 170.9, 141.0, 135.2, 135.1, 128.5, 128.4, 126.1, 117.3, 117.2,
+
5.3, 23.9, 23.8 ppm; HRMS (ESI): m/z calcd for C31
H
47
N
2
O
3
:
7
2
C
4.8, 73.4, 66.4, 64.1, 44.8, 44.6, 35.9, 35.6, 35.4, 32.3, 30.4, 30.3,
7.8, 27.0, 25.7, 25.6, 25.4, 23.3 ppm; HRMS (ESI): m/z calcd for
+
95.3587; found: 495.3581 [M+H] .
+
+
29
H47Cl
2
N
2
O
2
: 525.3015; found: 525.2954 [M+H] .
4
3
.2.7. General procedure for the synthesis of bis-N-chloroamines
0–34 and 37
4
.2.7.5. (S)-Naphthalen-1-yl 2,6-bis(chloro(2,2-dimethyl-pent-4-
N-Chlorosuccinimide (2.2 equiv) was added to a cooled (0 °C)
en-1-yl)amino)hexanoate (33). Prepared according to the gen-
eral procedure from 28 (620 mg, 1.33 mmol) yielding 33
solution of the corresponding diamine in anhydrous dichlorometh-
ane (10 mL/mmol of diamine). The reaction mixture was stirred
first for half an hour at 0 °C and for additional 2 h at room temper-
ature. After removal of the solvent in vacuo, the product was iso-
lated from the residue by flash chromatography (pentane/TBME
1
(
150 mg, 0.28 mmol, 21%) as a pale yellow oil. H NMR (CDCl
3
,
4
00.1 MHz): d = 7.98–8.00 (m, 1H), 7.87–7.90 (m, 1H), 7.76 (d,
J = 8.3 Hz, 1H), 7.51–7.56 (m, 2H), 7.48 (t, J = 7.8 Hz, 1H), 7.29–
.31 (m, 1H), 5.77–5.91 (m, 2H), 5.00–5.08 (m, 4H), 3.92–4.30
m, 1H), 3.32–3.36 (m, 1H), 3.12–3.16 (m, 1H), 3.01 (t, J = 6.2 Hz,
H), 2.88 (s, 2H), 2.12–2.14 (m, 3H), 2.07–2.09 (m, 2H), 1.74–1.82
m, 3H), 1.58–1.69 (m, 2H), 1.00 (2ꢀ s, total 6H), 0.96 (s, 6H)
7
(
2
(
1
0:1).
4
.2.7.1.
(S)-Methyl
2,6-bis(N-chloro-N-(2,2-dimethylpent-4-
enyl)amino)hexanoate (37). Prepared according to the general
1
3
ppm; C NMR (CDCl
3
, 100.6 MHz): d = 169.6, 146.4, 135.2, 135.1,
procedure from 36 (2.50 g, 7.09 mmol) yielding 37 (880 mg,
1
134.7, 128.0, 126.7, 126.6, 126.5, 126.2, 125.3, 121.3, 118.0,
2
.09 mmol, 29%) as a pale yellow oil. H NMR (CDCl
3
, 400.1 MHz):
1
2
5
17.3, 117.2, 74.9, 73.7, 66.4, 44.8, 36.0, 35.6, 30.6, 27.8, 25.7,
5.6, 25.5, 23.5 ppm; HRMS (ESI): m/z calcd for C30
55.2521; found: 555.2517 [M+Na] .
d = 5.75–5.85 (m, 2H), 4.99–5.04 (m, 4H), 3.75 (s, 3H), 3.49 (t,
J = 7.3 Hz, 1H), 3.11–3.17 (m, 1H), 2.88–2.95 (m, 3H), 2.83 (s, 2H),
H42Cl
2
N
2
NaO+
2
:
+
2
1
.04 (d, J = 7.5 Hz, 4H), 1.81–1.87 (m, 2H), 1.63–1.71 (m, 2H),
1
3
.27–1.45 (m, 2H), 0.92, 0,93 and 0.94 (3ꢀ s, total 12H) ppm;
C
4
.2.7.6. (S)-4-Methoxynaphthalen-1-yl 2,6-bis(chloro(2,2-dim-
NMR (CDCl
3
, 100.6 MHz): d = 171.3, 135.2, 135.1, 117.3, 117.2,
ethylpent-4-en-1-yl)amino)hexanoate (34). Prepared accord-
ing to the general procedure from 29 (769 mg, 1.55 mmol)
yielding 34 (320 mg, 0.57 mmol, 36%) as a colourless oil. 1H NMR
7
2
4
4.8, 73.3, 72.2, 66.4, 44.8, 44.5, 35.8, 35.6, 30.3, 27.7, 25.7, 25.5,
+
5.4, 23.3 ppm; HRMS (ESI): m/z calcd for
21.2389; found: 421.2320 [M+H] .
21 2 2 2
C H39Cl N O :
+
(
7
5
CDCl
3
, 400.1 MHz): d = 8.24–8.31 (m, 1H), 7.85–7.92 (m, 1H),
.47–7.59 (m, 2H), 7.17 (d, J = 8.3 Hz, 1H), 6.78 (d, J = 8.5 Hz, 1H),
.75–5.91 (m, 2H), 4.98–5.08 (m, 4H), 4.01 (s, 3H), 3.91 (t,
4
.2.7.2. (S)-Benzyl 2,6-bis(N-chloro(2,2-dimethylpent-4-en-1-
yl)amino)hexanoate (30). Prepared according to the general
procedure from 25 (188 mg, 0.28 mmol) yielding 30 (40 mg,
J = 7.5 Hz, 1H), 3.29–3.35 (m, 1H), 3.09–3.17 (m, 1H), 3.00 (t,
J = 6.5 Hz, 2H), 2.87 (s, 2H), 2.08 (dd, J = 10.2 Hz, 7.0 Hz, 6H),
0
.80 mmol, 29%) as a clear and colourless viscous liquid. 1H NMR
1
6
1
1
3
C
.72–1.82 (m, 2H), 1.59 (br s, 2H), 0.99 (2ꢀ s, total 6H), 0.95 (s,
(
CDCl
3
, 400.1 MHz): d = 7.31–7.39 (m, 5H), 5.73–5.85 (m, 2H),
13
3
H) ppm; C NMR (CDCl , 100.6 MHz): d = 170.0, 153.6, 139.8,
5
.19–5.23 (m, 2H), 5.00–5.05 (m, 3H), 4.97–5.00 (m, 1H), 3.53 (t,
35.2, 135.1, 127.4, 127.1, 126.2, 125.8, 122.4, 121.1, 117.7,
17.5, 117.3, 102.8, 74.9, 73.7, 72.4, 66.4, 55.7, 44.8, 44.7, 36.0,
0.6, 27.0, 25.7, 25.6, 25.5, 23.5 ppm; HRMS (ESI): m/z calcd for
J = 6.8 Hz, 1H), 3.11–3.15 (m, 1H), 2.86–2.92 (m, 3H), 2.83 (s, 1H),
2
1
.04 (t, J = 7.3 Hz, 3H), 1.83–1.89 (m, 2H), 1.62–1.70 (m, 2H),
.50–1.59 (m, 1H), 1.25–1.43 (m, 3H), 0.91 (2ꢀ s, total 12H)
+
+
1
3
31 2 2 3
H45Cl N O : 563.2807; found: 563.2807 [M+H] .
3
ppm; C NMR (CDCl , 100.6 MHz): d = 170.7, 135.6, 135.2, 135.1,
1
3
28.5, 128.3, 117.3, 117.2, 74.8, 73.3, 72.3, 66.5, 66.4, 44.8, 44.7,
6.8, 35.6, 30.4, 27.7, 25.7, 25.5, 25.4, 23.3 ppm; HRMS (ESI): m/z
4.2.8. General procedure for the synthesis of bis-3-
chloropiperidines 1–6
+
+
2 2 2
calcd for C27H43Cl N O : 497.2702; found: 497.2763 [M+H] .
The bis-N-chloroamine was dissolved in anhydrous chloroform
(10 mL/mmol of bis-N-chloroamine) and tetrabutylammonium
iodide (10 mol %) was added to the solution. The resulting mixture
was then heated at 60 °C (oil bath temperature) for 2 h. After
removal of the solvent under reduced pressure, the residue was
purified by flash chromatography (pentane/TBME 10:1). The bis-
3-chloropiperidine was obtained as a mixture of inseparable
diastereomers.
4
1
.2.7.3. (S)-Phenethyl 2,6-bis(N-chloro(2,2-dimethylpent-4-en-
-yl)amino)hexanoate (31). Prepared according to the general
procedure from 26 (810 mg, 1.83 mmol) yielding 31 (281 mg,
.55 mmol, 30%) as a clear and colourless viscous liquid. 1H NMR
CDCl , 400.1 MHz): d = 7.30–7.34 (m, 2H), 7.22–7.27 (m, 3H),
.74–5.87 (m, 2H), 4.99–5.06 (m, 4H), 4.37–4.46 (m, 2H), 3.44 (t,
0
(
5
3
J = 7.0 Hz, 1H), 3.00–3.07 (m, 2H), 2.92 (t, J = 6.5 Hz, 2H), 2.85 (s,

I. Zuravka et al. / Bioorg. Med. Chem. 23 (2015) 1241–1250
1249
4
.2.8.1. (S)-Methyl 2,6-bis(5-chloro-3,3-dimethylpiperidin-1-
signals, 6H) ppm; 13C NMR (CDCl
3
, 50.3 MHz, selected signals)
yl)hexanoate (1). Prepared according to the general procedure
from 37 (750 mg, 1.78 mmol) yielding 1 (406 mg, 0.96 mmol,
major isomer: d = 171.0, 146.3, 134.7, 128.1, 126.7, 126.5, 126.0,
125.3, 121.1, 118.1, 67.0, 64.8, 63.9, 62.3, 57.7, 54.3, 48.6, 48.4,
5
4%) as a pale yellow oil; (Found: C, 59.65; H, 9.09; N, 6.31.
43
33.3, 29.1, 26.5, 25.2, 24.1 ppm; HRMS (ESI): m/z calcd for C30H -
Cl N O : 533.2702; found: 533.2702 [M+H] .
2 2 2
1
+
+
C
21
H38Cl
2
N
2
O
2
requires C, 59.85; H, 9.09; N, 6.65%); H NMR
, 400.1 MHz) mixture of diastereomers: d = 3.92–4.10 (m,
H), 3.68 (s, 3H), 3.08–3.19 (m, 3H), 2.36–2.46 (m, 2H), 2.22–
.27 (m, 2H), 1.87–1.97 (m, 4H), 1.53–1.74 (m, 4H), 1.26–1.49
(
CDCl
3
2
2
4.2.8.6. (2S)-4-Methoxynaphthalen-1-yl 2,6-bis(5-chloro-3,3-
dimethylpiperidin-1-yl)hexanoate (6). Prepared according to
(
6
m, 6H), 0.99 and 1.01 (2ꢀ s, total 6H), 0.90 and 0.91 (2ꢀ s, total
the general procedure from 34 (320 mg, 0.57 mmol) yielding 6
1
3
1
H) ppm; C NMR (CDCl
3
, 100.6 MHz, selected signals) major iso-
(160 mg, 0.28 mmol, 50%) as a pale green oil. H NMR (CDCl
3
,
mer: d = 172.7, 66.8, 64.7, 64.1, 62.3, 61.5, 57.6, 55.0, 54.3, 51.0,
400.1 MHz) mixture of diastereomers: d = 8.23–8.31 (m, 1H),
7.74–7.79 (m, 1H), 7.49–7.56 (m, 2H), 7.11–7.17 (m, 1H), 6.78 (d,
J = 8.3 Hz, 1H), 4.04–4.18 (m, 2H), 4.01 (s, 3H), 3.58–3.65 (m, 1H),
4
8.6, 48.3, 33.2, 29.3, 26.4, 25.2, 24.9, 23.8 ppm; HRMS (ESI): m/z
+
+
2 2 2
calcd for C21H39Cl N O : 421.2389; found: 421.2388 [M+H] .
3
.32–3.41 (m, 1H), 3.16–3.19 (m, 1H), 2.51–2.61 (m, 2H), 2.28–
4
.2.8.2. (2S)-Benzyl 2,6-bis(5-chloro-3,3-dimethylpiperidin-1-
2.43 (m, 3H), 1.89–2.00 (m, 4H), 1.68–1.75 (m, 1H), 1.52–1.58
(m, 4H), 1.23–1.48 (m, 4H), 1.03–1.11 (overlapping signals, 6H),
yl)hexanoate (2). Prepared according to the general procedure
from 30 (125 mg, 0.25 mmol) yielding 2 (80 mg, 0.16 mmol, 64%)
as a pale yellow oil. H NMR (CDCl
1
3
0.92–0.96 (overlapping signals, 6H) ppm;
3
C NMR (CDCl ,
1
3
, 400.1 MHz) mixture of diaste-
50.3 MHz, selected signals) major isomer: d = 171.3, 153.5, 139.7,
reomers: d = 7.32–7.40 (m, 5H), 5.11–5.14 (m, 2H), 3.92–4.05 (m,
127.4, 126.9, 126.2, 125.7, 122.5, 120.8, 117.7, 102.8, 67.0, 64.9,
2
1
H), 3.10–3.12 (m, 1H), 2.24–2.47 (m, 6H), 1.85–1.96 (m, 4H),
.50–1.73 (m, 4H), 1.29–1.48 (m, 6H), 0.97–1.00 (overlapping sig-
62.9, 62.3, 61.3, 55.7, 55.2, 54.3, 48.4, 33.8, 33.3, 29.1, 26.5, 25.2,
+
24.1 ppm; HRMS (ESI): m/z calcd for C31
found: 563.2804 [M+H] .
H45Cl
2
N
2
O
3
: 563.2807;
1
3
+
nals, 6H), 0.86–0.90 (overlapping signals, 6H) ppm; C NMR
CDCl 50.3 MHz, selected signals) major isomer: d = 171.8,
35.9, 128.5, 128.4, 128.3, 66.8, 65.9, 63.9, 62.8, 61.4, 57.6, 54.4,
4.0, 48.6, 48.5, 33.6, 33.2, 29.6, 26.9, 25.2, 24.9, 23.8 ppm; HRMS
(
1
5
3
,
4.3. Bioassays
+
(
[
ESI): m/z calcd for C27
M+H] .
H43Cl
2
2
N O
2
: 497.2702; found: 497.2698
The water used in all biochemical experiments was prepared
from the Milli-Q Synthesis (Millipore) water purification system.
Chlorambucil and chemicals for preparing buffer solutions were
purchased from Sigma–Aldrich (St. Louis, MO, USA), Agarose D-1
Low EEO was purchased from Eppendorf (Hamburg, Germany),
and acrylamide-bis ready-to-use solution (40%, 19:1) was pur-
chased from Merck (Darmstadt, Germany). Oligonucleotides were
purchased from Eurogentec (Seraing, Liège, Belgium) and stored
at ꢁ20 °C in TE (10 mM Tris–HCl, 1 mM EDTA). The sequence of
+
4
1
.2.8.3. (2S)-Phenethyl 2,6-bis(5-chlor-3,3-dimethyl-piperidin-
-yl)hexanoate (3). Prepared according to the general procedure
from 31 (250 mg, 0.49 mmol) yielding 3 (169 mg, 0.33 mmol, 68%)
as a pale yellow oil. H NMR (CDCl
reomers: d = 7.29–7.33 (m, 2H), 7.21–7.25 (m, 3H), 4.28–4.42 (m,
2
2
1
1
3
, 400.1 MHz) mixture of diaste-
H), 3.88–4.10 (m, 2H), 3.01–3.14 (m, 3H), 2.94–2.98 (m, 2H),
.12–2.39 (m, 6H), 1.59–1.94 (m, 6H), 1.19–1.35 (m, 6H), 0.84–
0
0
the scrambled oligonucleotide used for 5 -FAM labeling was: 5 -
13
0
.01 (overlapping signals, 12H) ppm;
C
NMR (CDCl
3
,
FAM-GGA TGT GAG TGT GAG TGT GAG G-3 ; the complementary
co-scrambled oligonucleotide sequence was: 5 -CCT CAC ACT CAC
ACT CAC ATC C-3 . The 5 -FAM labeled scrambled oligonucleotide
was mixed with equimolar amounts of its complementary co-
0
1
1
4
00.6 MHz, selected signals) major isomer: d = 171.9, 137.3,
28.5, 128.2, 126.3, 66.5, 64.4, 64.1, 63.6, 62.0, 57.3, 54.0, 48.2,
8.1, 34.8, 32.9, 29.6, 26.4, 25.2, 24.9, 23.5 ppm; HRMS (ESI): m/z
0
0
+
+
calcd for C28
H45Cl
2
N
2
O
2
: 511.2858; found: 511.2851 [M+H] .
scrambled oligonucleotide in BPE buffer (2 mM NaH
2
PO
4
ꢂ2H
2
O,
6
mM Na HPO O, 1 mM Na EDTAꢂ2H O, pH 7.4; used at 1:5
2
4
ꢂ12H
2
2
2
4
.2.8.4. (S)-3-Phenylpropyl 2,6-bis(5-chlor-3,3-dimethyl-piperi-
dilution), denatured at 95 °C for 5 min, and then left to cool to room
temperature (slow annealing). This step ensured the formation of
the duplex DNA through annealing of scrambled and co-scrambled
oligonucleotides. Dilutions of bis-3-chloropiperidines and chloram-
bucil were freshly prepared from a DMSO stock solution (8 mM) in
water. Alkylation reactions were carried out in BPE buffer.
din-1-yl)-hexanoate (4). Prepared according to the general pro-
cedure from 32 (220 mg, 0.42 mmol) yielding
4
(158 mg,
0
.30 mmol, 72%) as a pale yellow oil; (found: C, 66.20; H, 8.83; N,
1
5
.19. C29
H46Cl
2
N
2
O
2
requires C, 66.27; H, 8.82; N, 5.33%);
H
NMR (CDCl
3
, 400.1 MHz) mixture of diastereomers: d = 7.27–7.31
(
m, 2H), 7.18–7.22 (m, 3H), 4.12 (t, J = 6.2 Hz, 2H), 3.94–4.08 (m,
2
1
1
5
1
3
H), 3.12–3.21 (m, 3H), 2.68–2.72 (m, 2H), 2.26–2.51 (m, 6H),
.90–2.01 (m, 6H), 1.58–1.70 (m, 8H), 1.39–1.49 (m, 4H), 1.00–
4.3.1. DNA cleavage assay
DNA cleavage assays were performed using pBR322 plasmid.
pBR322 (3.5 nM) was incubated with increasing concentrations
(0.5, 5, 50 mM) of agent for 3 h at 37 °C in BPE buffer. The linear
DNA standard was generated by EcoRI (Promega) digestion of
pBR322, according to the manufacturer’s instructions. In order to
obtain the open circular form of pBR322, 250 ng of supercoiled
.02 (overlapping signals, 6H), 0.90 (s, 6H) ppm; 13C NMR (CDCl
3
,
0.3 MHz, selected signals) major isomer: d = 171.9, 141.0, 128.4,
28.3, 126.0, 66.8, 64.7, 63.5, 61.6, 57.6, 54.1, 48.5, 48.3, 33.2,
2.3, 30.3, 29.4, 26.5, 25.2, 24.0, 23.9 ppm; HRMS (ESI): m/z calcd
+
+
2 2 2
for C29H47Cl N O : 525.3015; found: 525.2999 [M+H] .
plasmid was incubated on ice for 5 min with 0.001 U/
DNase–RNase-free (Promega) in 1ꢀ RQ1 buffer in a total reaction
volume of 10 L. Nicking reaction was stopped adding 5 L EGTA
(ethylene glycol tetraacetic acid) 20 mM (pH 8.0). Gel loading buf-
fer (10 mm Tris–HCl, 50% glycerol, 0.025% bromophenol blue) was
added to all reaction tubes, and the samples were loaded onto a 1%
agarose gel. Electrophoresis was conducted in 1ꢀ TAE (Tris–HCl
40 mM, acetic acid 20 mM, EDTA 1 mM). DNA in the gel system
lL RQ1
4
.2.8.5. (2S)-Naphthalen-1-yl 2,6-bis(5-chloro-3,3-dimethyl-
piperidin-1-yl)hexanoate (5). Prepared according to the general
procedure from 33 (138 mg, 0.26 mmol) yielding (77 mg,
, 400.1 MHz)
l
l
5
1
0
.14 mmol, 56%) as a colourless oil. H NMR (CDCl
3
mixture of diastereomers: d = 7.84–7.92 (m, 2H), 7.74 (d,
J = 7.5 Hz, 1H), 7.51–7.53 (m, 2H), 7.47 (t, J = 8.5 Hz, 1H), 7.24–
7
(
3
(
.26 (m, 1H), 3.99–4.20 (m, 2H), 3.61–3.67 (m, 1H), 3.34–3.42
m, 1H), 3.14–3.23 (m, 1H), 2.49–2.64 (m, 2H), 2.28–2.48 (m,
H), 1.89–2.04 (m, 4H), 1.71–1.77 (m, 1H), 1.58 (br s, 6H), 1.26
s, 2H), 1.03–1.11 (overlapping signals, 6H), 0.88–0.99 (overlapping
was detected by staining with ethidium bromide (0.5 lg/mL) for
30 min with visualization by a Geliance 600 imaging system (Perk-
inElmer, Waltham, MA, USA).

1
250
I. Zuravka et al. / Bioorg. Med. Chem. 23 (2015) 1241–1250
4
.3.2. Sequencing gel analysis
The 5 -FAM-labelled duplex oligonucleotide (2
Supplementary data
0
l
M final concen-
tration) was incubated with each alkylating agent (final concentra-
tions of 5 and 50 M) in BPE buffer (final dilution 1:5) for 24 h at
7 °C. The samples were dried in a vacuum centrifuge (UNIVAPO
00H, UniEquip), resuspended in 5 mL of denaturing gel loading
Supplementary data (sequencing gel analysis and ESI-MS spec-
tra of the hydrolysis reaction of compound 1) associated with this
article can be found, in the online version, at http://dx.doi.org/10.
1016/j.bmc.2015.01.050.
l
3
1
buffer (10 mM Tris–HCl, 80% formamide, 0.025% bromophenol
blue), and loaded on a 20% denaturing polyacrylamide gel (7 M
urea) in 1ꢀ TBE (Tris–HCl 89 mM, borate 89 mM, EDTA 2 mM).
The fluorescence of the oligonucleotide bands were detected by
scanning using Storm Scanner Control (STORM 840, Molecular
Dynamics).
References and notes
1
2
3
.
.
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A 80
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1
2. Silverman, R. B. The Organic Chemistry of Drug Design and Drug Action; Elsevier
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Acknowledgments
1
1
This work was supported financially by MIUR, Progetti di Ric-
erca di Interesse Nazionale (Grant 2010W2KM5L_006) to B.G.
The authors would like to acknowledge the assistance of Dr. Erwin
Röcker with mass spectrometric measurements. I.Z. thanks the
German National Academic Foundation (Studienstiftung des deuts-
chen Volkes) for providing a PhD scholarship, as well as the Inter-
national Giessen Graduate Centre for the Life Sciences (GGL) and
the German Academic Exchange Service (DAAD) for a travel grant.
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1