Synthesis and DNA cleavage activity of bis-3-chloropiperidines as alkylating agents

Article abstract of DOI:10.1002/cmdc.201400034

Nitrogen mustards are an important class of bifunctional alkylating agents routinely used in chemotherapy. They react with DNA as electrophiles through the formation of highly reactive aziridinium ion intermediates. The antibiotic 593A, with potential antitumor activity, can be considered a naturally occurring piperidine mustard containing a unique 3-chloropiperidine ring. However, the total synthesis of this antibiotic proved to be rather challenging. With the aim of designing simplified analogues of this natural product, we developed an efficient bidirectional synthetic route to bis-3-chloropiperidines joined by flexible, conformationally restricted, or rigid diamine linkers. The key step involves an iodide-catalyzed double cyclization of unsaturated bis-N-chloroamines to simultaneously generate both piperidine rings. Herein we describe the synthesis and subsequent evaluation of a series of novel nitrogen-bridged bis-3-chloropiperidines, enabling the study of the impact of the linker structure on DNA alkylation properties. Our studies reveal that the synthesized compounds possess DNA alkylating abilities and induce strand cleavage, with a strong preference for guanine residues.

Full text of DOI:10.1002/cmdc.201400034

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DOI: 10.1002/cmdc.201400034
Synthesis and DNA Cleavage Activity of Bis-3-
chloropiperidines as Alkylating Agents
[a, b]
[a]
[b]
[c]
[c]
Ivonne Zuravka,
Rolf Roesmann, Alice Sosic, Wolfgang Wende, Alfred Pingoud,
[b]
[a]
Barbara Gatto,* and Richard Gçttlich*
Nitrogen mustards are an important class of bifunctional alky-
lating agents routinely used in chemotherapy. They react with
DNA as electrophiles through the formation of highly reactive
aziridinium ion intermediates. The antibiotic 593A, with poten-
tial antitumor activity, can be considered a naturally occurring
piperidine mustard containing a unique 3-chloropiperidine
ring. However, the total synthesis of this antibiotic proved to
be rather challenging. With the aim of designing simplified an-
alogues of this natural product, we developed an efficient bi-
directional synthetic route to bis-3-chloropiperidines joined by
flexible, conformationally restricted, or rigid diamine linkers.
The key step involves an iodide-catalyzed double cyclization of
unsaturated bis-N-chloroamines to simultaneously generate
both piperidine rings. Herein we describe the synthesis and
subsequent evaluation of a series of novel nitrogen-bridged
bis-3-chloropiperidines, enabling the study of the impact of
the linker structure on DNA alkylation properties. Our studies
reveal that the synthesized compounds possess DNA alkylating
abilities and induce strand cleavage, with a strong preference
for guanine residues.
Introduction
Electrophiles readily attack nucleophilic centers of intracellular
biomolecules like DNA and proteins. Their reactivity with DNA
involves the formation of covalent adducts through base alky-
lation that cause DNA damage, leading to inhibition of DNA
[
1]
replication and, eventually, to cell death. This cytotoxic mode
of action has been exploited in cancer treatment. In fact, DNA
alkylating agents are the oldest class of anticancer drugs and
[
2]
are still widely used in chemotherapy. The most potent and
effective drugs, such as the nitrogen mustards chlorambucil
(
Figure 1), cyclophosphamide, and melphalan, react as bifunc-
[
3]
tional alkylating agents. Compared with monoalkylators, they
are capable of forming covalent bonds at two nucleophilic
sites within DNA to induce intra- and interstrand cross-links,
Figure 1. Structures of antibiotic 593A, the nitrogen mustard chlorambucil,
and the synthesized bis-3-chloropiperidines as simplified analogues of 593A.
[4]
which are considered responsible for the cytotoxic effects.
The common mechanism of alkylation by nitrogen mustards is
based on the formation of an electrophilic aziridinium ion,
Over the last decades, several bifunctional nitrogen mustard
analogues and alkylating agents have been synthesized and in-
[
5]
[7]
which is a highly reactive alkylating species. In general, nitro-
gen mustards interact in a relatively nonspecific way with
DNA. In addition, most of them have been found to preferen-
vestigated for their biological properties. In this context, pi-
peridine- and pyrrolidine-based mustards have shown poten-
tial as alkylating antitumor agents via bicyclic aziridinium ion
[
6]
[8]
tially alkylate the N7 position of guanine.
formation. Due to the ring system, the alkylating moiety is
sterically restricted, which in turn may contribute to a more
controlled reactivity and enhance DNA interactions in vivo. In-
troducing conformational rigidity by means of ring constraints
is a popular strategy in medicinal chemistry to improve affinity
and selectivity of drug candidates toward defined biological
[
a] I. Zuravka, Dr. R. Roesmann, Prof. Dr. R. Gçttlich
Institute of Organic Chemistry, Justus Liebig University Giessen
Heinrich-Buff-Ring 58, 35392 Giessen (Germany)
E-mail: richard.goettlich@uni-giessen.de
[b] I. Zuravka, Dr. A. Sosic, Prof. Dr. B. Gatto
[9]
Department of Pharmaceutical and Pharmacological Sciences
University of Padova, Via Francesco Marzolo 5, 35131 Padova (Italy)
E-mail: barbara.gatto@unipd.it
targets.
A structurally related natural product to the synthetic nitro-
gen mustards containing a restricted ring system is the antibi-
otic 593A (Figure 1), isolated from Streptomyces griseoluteus in
[
c] Dr. W. Wende, Prof. Dr. A. Pingoud
Institute for Biochemistry, Justus Liebig University Giessen
Heinrich-Buff-Ring 58, 35392 Giessen (Germany)
[
10]
1
970. This compound is a symmetrical piperazinedione com-
[11]
posed of two unique 3-chloropiperidine rings. Due to its an-
tineoplastic and antibiotic properties, coupled with its remark-
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201400034.
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able structure, 593A has attracted considerable interest in syn-
Previously, our research group established a mild, catalytic,
[
12]
thetic organic and medicinal chemistry. In 1979, Fukuyama
general procedure for the cyclization of unsaturated N-chloroa-
[13]
[15]
et al. successfully achieved a challenging first total synthesis.
mines to 3-chloropiperidines. This synthetic strategy could
Studies of the biological effects of this compound suggest that
the antibiotic acts as an alkylating agent but with a different
mode of action, as 593A was shown to be active in tumors re-
be successfully extended to the synthesis of bis-3-chloropiperi-
dines. Herein we report a new method for a facile bidirectional
synthesis of novel bis-3-chloropiperidines, followed by evalua-
tion of their alkylating activity as well as their DNA sequence
selectivity.
[
14]
sistant to cyclophosphamide. However, in the recent past,
no further research has been undertaken in this area. Thus, to
gain a more detailed understanding of this interesting mecha-
nism of action, we synthesized a set of structurally simplified
analogues of antibiotic 593A. Bis-3-chloropiperidines 5a–f
To study the impact of the linker structure on DNA alkyla-
tion, we prepared bis-3-chloropiperidine derivatives with flexi-
ble hydrocarbon linkers, as well as with a more conformation-
ally constrained cyclohexyl and a rigid aromatic linker. The re-
sults demonstrate that the synthesized compounds react with
DNA very efficiently and that alkylation takes place primarily at
guanine residues, in line with what has been pub-
(Figure 1 and Table 1) were selected as proof-of-principle com-
pounds and further characterized with respect to their alkylat-
ing activity toward DNA.
[6]
lished for conventional nitrogen mustards.
Table 1. Bidirectional synthesis of nitrogen-linked bis-3-chloropiperidines 5a–f by re-
ductive amination, followed by chlorination and subsequent iodide-catalyzed cycliza-
tion.
Results and Discussion
Chemistry
Structurally, the antibiotic 593A is a dimer consisting
of a piperazinedione linker connecting two 3-chloro-
piperidines. With the aim of obtaining simplified ana-
logues, our original synthetic plan focused on the
linkage of two 3-chloropiperidine monomers via an
alkyl spacer to the 6-position of each piperidine ring.
However, this strategy did not lead to satisfactory re-
sults, due to poor yields, and was abandoned after
several attempts. One major problem with this syn-
thetic approach can be attributed to the high reac-
tivity and consequently low stability of the 3-chloro-
piperidine ring.
Yield of
Diamine [%]
Yield of
bis-N-chloroamine [%]
Yield of bis-3-chloropiperidine [%]
3
3
3
3
a 95
b 91
c 92
d 95
4a 94
4b 87
4c 77
4d 80
88
88
78
77
Thus, it appeared ideal to prepare both heterocy-
clic rings at the end of the synthesis. We also rea-
soned that the preparation of nitrogen-linked bis-3-
chloropiperidines might provide a more convenient
strategy, as the introduction of protecting groups
would be avoided. To take advantage of the symme-
try of the desired bis-3-chloropiperidines, we pro-
posed a bidirectional route. Accordingly, our synthet-
ic plan involved the simultaneous formation of the
piperidine rings following a well-established proce-
dure in our laboratory for iodide-catalyzed cyclization
[15]
of unsaturated N-chloroamines.
A facile and efficient bidirectional route was de-
signed to enable the synthesis of a series of nitro-
gen-linked bis-3-chloropiperidines from easily acces-
sible starting materials, as outlined in Table 1. We se-
lected a variety of bridging diamine linkers to exam-
ine the effect of linker length and flexibility on the
DNA alkylating properties of the derivatives. To study
these features, we first constructed a set of com-
pounds with three to six carbon atoms between the
two piperidine rings. Furthermore, as the antibiotic
[
c]
3
e 37
4e 78
4 f 91
63
88
3
f 88
[
a] See Experimental Section for the preparation of 2. [b] Oil bath temperature. [c] For
diamine linker 1e, isolation of the imine and its subsequent reduction was found to
be a more effective method: Na SO , RT, 24 h, then NaBH , 2-propanol/MeOH, RT,
2 h, 82% over two steps. NCS=N-chlorosuccinimide, TBAI=tetrabutylammonium
iodide.
2
4
4
1
593A is connected by a rather rigid piperazinedione
system, we targeted the synthesis of bis-3-chloropi-
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peridines with a conformationally restricted cyclohexyl as well
as a rigid aromatic spacer unit, linking the alkylating moieties
through the 1,4-positions, similarly to the natural product.
The precursors could be readily prepared by double reduc-
tive amination of 2,2-dimethylpent-4-enal 2 with the appropri-
ate commercially available diamines 1a–f using sodium triace-
ing of supercoiled plasmid at a concentration of 5 mm. Com-
pound 5c, with a flexible five-carbon linker chain (Figure 2a),
displayed total conversion of the supercoiled form to the open
circular form under these conditions, whereas compound 5 f
(Figure 2b), with an aromatic linker, had no appreciable effect.
An increase in the concentration of alkylating agents led to
DNA fragmentation, resulting in smears or diffused bands in
the agarose gel. In clear contrast, DNA cleavage by the control
substance CA (Figure 2b) could not be detected at the highest
concentration (50 mm). Similar results were observed with dif-
ferent plasmids (not shown). When time course studies were
conducted, complete fragmentation of plasmid DNA was ob-
served at the highest concentration after 15 h of incubation,
while under the same experimental conditions, partial plasmid
linearization was detected for CA (Supporting Information, Fig-
ure S1).
[
16]
toxyborohydride. Aldehyde 2 was obtained from isobutyral-
dehyde and allylic alcohol according to the reported proce-
[
17]
dure. However, a single-stage reductive amination reaction
was not as suitable for trans-1,4-cyclohexanediamine 1e, af-
fording the product in rather low yields. The unsaturated cy-
clohexyl diamine was therefore prepared by imine formation in
a prior step and subsequent reduction in a separate step with
sodium borohydride. Chlorination of 3a–f with N-chlorosucci-
nimide (NCS) yielded the unsaturated bis-N-chloroamines 4a–f,
which were cyclized using a catalytic amount of tetrabutylam-
monium iodide (TBAI) to give the desired bis-3-chloropiperi-
dines 5a–f as an inseparable mixture of stereoisomers.
By comparing the DNA cleavage activities of the investigat-
ed bis-3-chloropiperidines shown in Figure 2, it appears that
the linker length had no apparent influence on alkylating prop-
erties, as a similar level of DNA nicking could be observed
within the group of compounds 5a–d, which vary in linker
length (Figure 2a). However, it should be noted that 5a was
slightly less active than the corresponding derivatives with an
increased linker length (compare 5a with 5b–d). Likewise, the
introduction of a more conformationally restricted cyclohexane
linker did not alter the alkylating efficiency. In contrast, the in-
corporation of a rigid aromatic linker resulted in a decrease of
activity (compare compounds 5e and 5 f, Figure 2b). This dif-
ferent reactivity might be attributed to the fixed geometry of
the planar aromatic linker and potential stacking interactions.
These findings indicate that the flexibility of the linker compo-
nent is an important structural feature of bis-3-chloropiperi-
dines with regard to DNA alkylation efficiency.
Bis-3-chloropiperidines induce nicking of supercoiled plas-
mid DNA
The activity of the synthetic bis-3-chloropiperidines toward
DNA was tested analyzing the conversion of supercoiled DNA
in its different topological forms. DNA strand cleavage can be
ascertained from the different electrophoretic migration of su-
percoiled, open circular and linear forms of the plasmid DNA
in agarose gels. The anticancer drug chlorambucil (CA), which
serves as a representative of the bifunctional nitrogen mus-
tards, was used as a control. In our experiment, the supercoiled
form of pAT153 was incubated with increasing concentrations
(0.5, 5, 50 mm) of alkylating agents at 378C for 3 h in bisphos-
phate-EDTA (BPE) buffer at pH 7.4. The results shown in
Figure 2 demonstrate that the tested compounds efficiently
nick the plasmid, a feature connected to DNA alkylation, fol-
lowed by destabilization of the nucleobases, resulting in strand
Mechanism of DNA alkylation by 3-chloropiperidines
[
1a]
cleavage. Bis-3-chloropiperidines 5a–e induced efficient nick-
3-Chloropiperidines 5 react with DNA via a highly electrophilic
bicyclic aziridinium intermediate 6, formed from intramolecular
nucleophilic displacement of chloride by nitrogen as indicated
in Scheme 1. Detailed mechanistic studies have confirmed the
[18]
existence of the aziridinium ion. This reactive species can be
readily attacked by nucleophilic centers of the DNA bases to
give five-membered 7 and six-membered 8 ring adducts
(
Scheme 1). The favored alkylation product depends on the
Figure 2. DNA cleavage activity of bis-3-chloropiperidine derivatives with
a) flexible carbon chain linkers and b) restricted linkers. The supercoiled form
of plasmid DNA pAT153 (120 ng) was incubated with the compounds at
Scheme 1. Proposed alkylation mechanism of 3-chloropiperidines 5. The re-
action proceeds via a bicyclic aziridinium ion intermediate 6, which is readily
attacked by nucleophiles (Nuc). The resulting product can be a pyrrolidine 7
or a piperidine 8 adduct, depending on the steric influence of both the R
group and the nucleophile.
378C for 3 h in BPE buffer, pH 7.4, at various concentrations (0.5, 5, 50 mm).
Chlorambucil (CA) was used as a control. Cleavage of DNA was analyzed by
agarose (1%) gel electrophoresis in 1ꢁ TBE. C=supercoiled DNA control,
L=linear DNA control, OC=open circular (nicked) DNA control.
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bulkiness of the R-group attached to the nitrogen, as well as
[
8a,18]
on the nature of the attacking nucleophile.
Steric hin-
drance almost certainly diverts the alkylation reaction to the
sterically less hindered side of the aziridinium ion 6, affording
the pyrrolidine adduct 7.
In addition, the N-substituent (R) influences the basicity of
the nitrogen atom, which in turn has a severe impact on the
formation of the respective aziridinium ions. Accordingly, elec-
tron-donating groups should lead to an increased formation
rate and stabilization of the reactive species, as the R-group
will push electron density into the aziridinium moiety. On the
other hand, electron-withdrawing substituents should have the
opposite effect by destabilizing the positive charge of the aziri-
dinium ion. As a result, the nitrogen center in aromatic nitro-
gen mustards like CA is less basic than in the aliphatic ana-
logues. Thus, the latter alkylating agents are much more readi-
[
19]
ly activated.
Consequently, the formation of DNA alkylation products de-
pends mainly on the reactivity and the nature of the alkylating
agent. However, due to a general instability of these covalent
DNA adducts in solution, the structural characterization is diffi-
cult. Previous studies established that guanine is the most re-
active site for DNA alkylation by many cytotoxic alkylating
[
1a,6]
agents.
One effect of guanine alkylation is an increase in
the electrophilicity of neighboring positions, which can lead to
hydrolytic DNA cleavage reactions. Alternatively, alkylation can
destabilize the nucleobases, followed by deglycosylation and
[
1a,20]
subsequent DNA strand scission.
DNA alkylation by 3-chloropiperidines
Figure 3. Representative denaturing polyacrylamide (20%) gel in 1ꢁ TBE,
showing time- and concentration-dependent cleavage of a 22-mer double-
stranded oligonucleotide caused via guanine alkylation by bis-3-chloropiper-
idine 5a. The 5’-FAM-labeled scrambled duplex oligonucleotide (4 mm), GGA
TGT GAG TGT GAG TGT GAG G, was treated with compound 5a at 378C in
BPE buffer, pH 7.4, at 5 and 50 mm for incubation times as indicated. Chlor-
ambucil (CA) was used as a control. Arrows indicate the position of fast-mi-
grating fragment bands. C=untreated duplex oligonucleotide control.
To obtain experimental evidence of DNA alkylation by the syn-
thesized bis-3-chloropiperidines, we examined their effects on
a 22-mer oligonucleotide duplex containing a G-rich sequence.
After incubation, the reaction products were resolved by poly-
acrylamide gel electrophoresis (PAGE), enabling identification
of the specific alkylated bases. Alkylation reactions were carried
out in BPE buffer, pH 7.4, at 378C for a range of incubation
times from 1 h to 24 h at two distinct concentrations (5 and
5
0 mm). Sequence specificities were analyzed by high-resolu-
tion PAGE. Figure 3 shows the results for bis-3-chloropiperidine
a, which was selected as a representative example for this
matic mustard CA, which is in agreement with our experimen-
tal observations.
5
Interestingly, in addition to DNA fragmentation, the appear-
ance of diffuse gel bands with lower mobility than the control
band corresponding to the untreated duplex oligonucleotide
(C, Figure 3) were observed at the highest dose of compounds.
The shifted bands may result from formation of DNA adducts
(DNA adducts, Figure 3). These lower mobility adducts were
transformed in fragmented oligonucleotides over time, as evi-
denced by the smearing of the DNA band concurrent with the
occurrence of fast migrating bands corresponding to shorter
DNA fragments (arrows, Figure 3) cleaved at guanines. Al-
though a certain degree of precipitation may have occurred,
the band diffusion and the smear in the lanes moving to lower
molecular weight with time was consistent with fragmentation
of DNA, which was observed in all our experimental condi-
tions. At the same G position in the gel, relatively weak frag-
ment bands were observed for CA.
study, toward the 5’-FAM-labelled double-stranded oligonu-
cleotide. The tested compound, 5a, showed alkylation fol-
lowed by cleavage at guanines. The effect is dependent on
time and concentration, with the oligonucleotide being de-
graded after 24 h alkylation at a concentration of 50 mm. In
comparison, the control substance, CA, displayed no apprecia-
ble effect on the oligonucleotide at this concentration or even
at a higher concentration of 100 mm. This finding supports the
above-mentioned assumption that formation of the aziridinium
ion in the case of the bis-3-chloropiperidines with aliphatic N-
substituents is more favorable than the activation of CA. The
nitrogen lone pair in CA is delocalized into the aromatic ring
[19]
and is therefore less available for electrophilic attack.
Ac-
cordingly, it can be expected that the bis-3-chloropiperidines
in this study possess higher alkylating activities than the aro-
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1
The alkylation patterns from the sequencing gel analysis re-
vealed that bis-3-chloropiperidines preferentially induce DNA
cleavage through reactions with guanines, sustaining our pro-
posed mechanism of reaction. In general, the sequence selec-
tivity is similar to that observed previously for chemotherapeu-
73%) was obtained as a colorless liquid. H NMR (200 MHz, CDCl
):
3
d=9.47 (s, 1H; CH=O), 5.70 (m, 1H), 5.05 (m, 2H), 2.21 (d, J=
.3 Hz, 2H), 1.05 ppm (s, 6H); C NMR (50 MHz, CDCl ): d=205.9,
33.1, 118.4, 45.7, 41.4, 21.1 ppm. These data are consistent with
13
7
3
1
[16,18]
published data.
[
6]
tic nitrogen mustards. It should be pointed out here that the
alkylating potency of the set of compounds examined follow
the same pattern observed in the supercoiled DNA nicking
assay (data not shown).
General procedure for the synthesis of diamines 3a–f: Sodium
triacetoxyborohydride (2.5 equiv) was added portionwise to a solu-
tion of the unsaturated aldehyde 2 (2 equiv) and the appropriate
À1
diamine in anhydrous CH Cl (7 mLmmol of diamine) at 08C, fol-
2
2
lowed by addition of acetic acid (1–2 equiv). The reaction mixture
was stirred at room temperature under argon atmosphere for 12 h
and was then quenched with 20% NaOH solution. The phases
were separated, and the aqueous layer was extracted three times
with 20 mL CH Cl . The combined organic phases were first
Conclusions
With the aim of designing simplified analogues of the antineo-
plastic antibiotic 593A, we successfully synthesized a series of
bis-3-chloropiperidines by a three-step route using a bidirec-
tional strategy. Relationships between the linker structure of
compounds and DNA alkylation activity were determined by
a DNA cleavage assay with a supercoiled plasmid and sequenc-
ing gel analysis with a 22-mer duplex oligonucleotide. The re-
sults demonstrate that examined bis-3-chloropiperidines 5a–
e alkylate DNA with high efficiency, involving the induction of
strand cleavage primarily at guanine sites. In addition, our
studies reveal that linker length does not affect the alkylating
properties, whereas a change in flexibility by insertion of an ar-
omatic rigid linker structure causes a decrease in reactivity
2
2
washed with brine, then with water, and were dried over Na SO .
2 4
The solvent was removed under reduced pressure to afford the
corresponding product, which was used in the next step without
further purification.
1
3
N ,N -Bis(2,2-dimethylpent-4-enyl)propane-1,3-diamine
(3a):
Compound 3a was obtained from 2 and 1,3-diaminopropane as
1
a colorless liquid (2.22 g, 8.32 mmol, 95%): H NMR (400 MHz,
CDCl ): d=5.80 (ddt, J=15.8 Hz, J=11.5 Hz, J=7.5 Hz, 2H), 4.99
3
(
1
m, 4H), 2.64 (t, J=6.8 Hz, 4H), 2.33 (s, 4H), 1.99 (d, J=7.5 Hz, 4H),
.65 (quin, J=6.8 Hz, 2H), 0.87 ppm (s, 12H); C NMR (100 MHz,
13
CDCl ): d=135.6, 116.7, 60.7, 49.7, 44.8, 34.2, 30.2, 25.5 ppm; HRMS
3
(
EI): m/z calcd for C H N : 266.2722; found: 266.2714.
17 34 2
1
4
(
5 f). Significantly, all of the tested bis-3-chloropiperidines were
N ,N -Bis(2,2-dimethylpent-4-enyl)butane-1,4-diamine (3b): Com-
more reactive toward DNA than the anticancer drug CA. Fur-
ther investigations into the reactivity of bis-3-chloropiperidines
with DNA are underway to provide a better understanding of
the molecular mechanism of action, useful for directing the
synthesis of new molecules with therapeutic effect.
pound 3b was obtained from 2 and 1,4-diaminobutane as a color-
1
less liquid (5.84 g, 20.8 mmol, 91%): H NMR (400 MHz, CDCl ): d=
3
5
.76 (ddt, J=16.1 Hz, J=11.9 Hz, J=7.5 Hz, 2H), 4.95 (m, 4H), 2.54
(
t, J=6.3 Hz, 4H), 2.30 (s, 4H), 1.95 (d, J=7.5 Hz, 4H), 1.44 (m, 4H),
13
0
5
2
.83 ppm (s, 12H); C NMR (100 MHz, CDCl ): d=135.6, 116.7, 60.6,
3
1.2, 44.2, 34.7, 27.7, 25.6 ppm; HRMS (EI): m/z calcd for C H N :
1
8
36
2
80.2878; found: 280.2847.
1
5
Experimental Section
N ,N -Bis(2,2-dimethylpent-4-enyl)pentane-1,5-diamine
(3c):
Compound 3c was obtained from 2 and 1,5-diaminopentane as
a colorless liquid (3.81 g, 12.9 mmol, 92%): H NMR (400 MHz,
Chemistry
1
CDCl ): d=5.75 (m, 2H), 4.93 (m, 4H), 2.52 (t, J=7.1 Hz, 4H), 2.28
Commercially available reagents were used as supplied. All sol-
vents were purified by distillation and dried, if necessary, prior to
use. Reactions requiring the use of anhydrous solvents were car-
ried out in heat gun-dried glassware under an argon atmosphere
3
(s, 4H), 1.94 (d, J=7.5 Hz, 4H), 1.43 (dt, J=14.7 Hz, J=7.4 Hz, 4H),
1
.28 (ddd, J=12.1 Hz, J=7.3, J=2.4 Hz, 2H), 0.82 ppm (s, 12H);
1
3
C NMR (100 MHz, CDCl ): d=135.5, 116.7, 60.3, 50.9, 44.7, 34.1,
3
2
9.9, 25.5, 25.0 ppm; HRMS (EI): m/z calcd for C H N : 294.3035;
19 38 2
(
Schlenk technique). Products were purified by flash chromatogra-
1
13
found: 294.3015.
phy on silica gel 60 (Merck). H and C NMR spectra were recorded
on a Bruker Avance II 200 spectrometer ( H at 200 MHz; C at
5
at 100 MHz), and a Bruker Avance III 600 spectrometer ( H at
6
TMS as an internal standard. Chemical shifts were determined by
reference to the residual solvent resonances. High-resolution EI
mass spectrometry data were obtained with a Finnigan MAT 95
1
13
1
6
1
13
N ,N -Bis(2,2-dimethylpent-4-enyl)hexane-1,6-diamine
Compound 3d was obtained from 2 and 1,6-diaminohexane as
a colorless liquid (8.43 g, 27.3 mmol, 95%): H NMR (400 MHz,
CDCl ): d=5.77 (m, 2H), 4.96 (m, 4H), 2.53 (t, J=7.5 Hz, 4H), 2.30
(3d):
0 MHz), a Bruker Avance II 400 spectrometer ( H at 400 MHz;
C
1
1
1
3
00 MHz; C at 150 MHz) in the stated deuterated solvent using
3
(s, 4H), 1.96 (d, J=7.5 Hz, 4H), 1.42 (m, 4H), 1.27 (m, 4H),
13
0
5
.84 ppm (s, 12H); C NMR (100 MHz, CDCl ): d=135.6, 116.7, 60.4,
3
0.9, 44.8, 34.2, 30.0, 27.3, 25.5 ppm; HRMS (EI): m/z calcd for
(
70 eV); all ESI mass spectra were obtained with a Finnigan
C H N : 308.3191; found: 308.3195.
20
40
2
LCQDuo mass spectrometer. NMR spectra of all synthesized com-
pounds are included in the Supporting Information.
1
4
trans-N ,N -Bis(2,2-dimethylpent-4-enyl)cyclohexane-1,4-diamine
(3e)*: Compound 3e was obtained from 2 and trans-1,4-cyclohexa-
nediamine as a colorless liquid (1.02 g, 3.34 mmol, 37%). *Isolation
of the 3e-imine and its subsequent reduction afforded a signifi-
cantly higher yield of 3e (82% over two steps) than the single-
stage reductive amination procedure. The method for synthesis of
3e-imine and its reduction for preparation of corresponding bis-N-
diamine 3e is below:
2
,2-Dimethylpent-4-enal (2): Freshly distilled isobutyraldehyde
(
108 g, 1.5 mol) and allyl alcohol (58.0 g, 1.0 mol) were added to
a solution of p-toluenesulfonic acid (0.25 g) in p-cymene (200 g).
The mixture was heated at reflux for 32 h under a Dean–Stark trap
until no more water was separated and the sump temperature
reached ~1408C. After vacuum distillation (768C at 200 mbar)
through a 50 cm Vigreux column, aldehyde 2 (81.4 g, 0.73 mol,
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1
4
1
5
1
5
trans-N ,N -Bis(2,2-dimethylpent-4-enylidene)cyclohexane-1,4-di-
amine (3e-imine): trans-1,4-Cyclohexanediamine (1.50 g,
3.1 mmol, 1 equiv) was added portionwise to a solution of 2,2-di-
N ,N -Dichloro-N ,N -bis(2,2-dimethylpent-4-enyl)pentane-1,5-di-
amine (4c): Compound 4c was obtained from 3c according to the
1
general procedure as a colorless oil (1.45 g, 3.99 mmol, 77%):
H NMR (400 MHz, CDCl ): d=5.80 (ddt, J=16.7 Hz, J=10.4 Hz, J=
7.5 Hz, 2H), 5.02 (m, 4H), 2.93 (t, J=7.1 Hz, 4H), 2.84 (s, 4H), 2.06
(d, J=7.5 Hz, 4H), 1.66 (q, J=7.3, 4H), 1.40 (m, 2H), 0.94 ppm (s,
12H); C NMR (100 MHz, CDCl ): d=135.2, 117.3, 74.8, 66.6, 44.8,
1
methyl-4-pentenal 2 (2.94 g, 26.2 mmol, 2 equiv) in 20 mL anhy-
drous CH Cl at 08C, followed by one spatula (~1 g) of Na SO . The
reaction mixture was stirred at room temperature for 24 h. After re-
3
2
2
2
4
13
moving the Na SO by filtration, the solvent was removed under
2
4
3
reduced pressure to afford pure product 3e-imine in 93% yield
35.6, 27.9, 25.7, 23.8 ppm.
1
(
3.69 g, 12.2 mmol) as a colorless liquid: H NMR (400 MHz, CDCl ):
3
1
6
1
6
N ,N -Dichloro-N ,N -bis(2,2-dimethylpent-4-enyl)hexane-1,6-di-
amine (4d): Compound 4d was obtained from 3d according to
d=7.51 (s, 2H), 5.69 (m, 2H), 4.95 (m, 4H), 2.90 (m, 2H), 2.10 (d,
J=7.4 Hz, 4H), 1.57 (m, 8H), 0.98 ppm (s, 12H); C NMR (100 MHz,
13
the general procedure as a colorless oil (1.51 g, 3.99 mmol, 80%):
CDCl ): d=169.3, 134.7, 117.2, 69.1, 44.7, 38.6, 32.6, 24.7 ppm;
3
1
H NMR (400 MHz, CDCl ): d=5.81 (ddt, J=16.7 Hz, J=10.4 Hz, J=
3
HRMS (EI): m/z calcd for C H N : 302.2722; found: 302.2749.
20
34
2
7
.5 Hz, 2H), 5.03 (m, 4H), 2.91 (t, J=6.9 Hz, 4H), 2.83 (s, 4H), 2.06
1
4
trans-N ,N -Bis(2,2-dimethylpent-4-enyl)cyclohexane-1,4-diamine
3e): Sodium borohydride (462 mg, 1 equiv) was added portion-
(d, J=7.5 Hz, 4H), 1.64 (m, 4H), 1.36 (m, 4H), 0.94 ppm (s, 12H);
13
(
C NMR (100 MHz, CDCl ): d=135.2, 117.2, 74.7, 66.7, 44.8, 35.6,
3
wise to a solution of 3e-imine (3.69 g, 12.2 mmol) in 30 mL 2-prop-
anol and 5 mL MeOH at 08C. The reaction mixture was stirred at
room temperature for 12 h and was then hydrolyzed with 20%
NaOH solution. The phases were separated, and the aqueous layer
was extracted three times with 20 mL diethyl ether. The combined
organic phases were washed with brine and dried over Na SO .
28.0, 26.5, 25.7 ppm.
1
4
1
4
trans-N ,N -Dichloro-N ,N -bis(2,2-dimethylpent-4-enyl)cyclo-
hexane-1,4-diamine (4e): Compound 4e was obtained from 3e
according to the general procedure as a colorless viscous oil
1
(3.13 g, 8.34 mmol, 78%): H NMR (400 MHz, CDCl ): d=5.81 (m,
3
2
4
2
2
H), 5.02 (m, 4H), 2.79 (s, 4H), 2.63 (m, 2H), 2.05 (d, J=7.5 Hz, 4H),
.00 (d, J=6.5 Hz, 4H), 1.53 (m, 4H), 0.93 ppm (s, 12H); C NMR
The solvent was removed under reduced pressure to obtain the
desired compound 3e as a colorless liquid (3.27 g, 10.7 mmol,
1
3
(
100 MHz, CDCl ): d=135.3, 117.2, 71.2, 70.5, 44.7, 35.4, 27.3,
8
8%), which was used without further purification for the next
3
1
25.5 ppm.
step: H NMR (400 MHz, CDCl ): d=5.71 (m, 2H), 4.95 (m, 4H), 2.31
3
(
1
1
s, 4H), 2.27 (m, 2H), 1.94 (d, J=7.5 Hz, 4H), 1.85 (d, J=6.2 Hz, 4H),
N,N’-(1,4-Phenylenebis(methylene))bis(N-chloro-2,2-dimethyl-
pent-4-en-1-amine) (4 f): Compound 4 f was obtained from 3 f ac-
13
.05 (m, 4H), 0.82 ppm (s, 12H); C NMR (100 MHz, CDCl ): d=
3
35.6, 116.6, 57.7, 44.6, 34.1, 32.3, 25.5 ppm; HRMS (EI): m/z calcd
cording to the general procedure as a colorless oil (1.61 g,
for C H N : 306.3035; found: 306.3021.
1
2
0
38
2
4
.06 mmol, 91%): H NMR (400 MHz, CDCl ): d=7.35 (s, 4H), 5.76
3
(
ddt, J=15.3 Hz, J=11.0 Hz, J=7.5 Hz, 2H), 5.00 (m, 4H), 4.11 (s,
N,N’-(1,4-Phenylenebis(methylene))bis(2,2-dimethylpent-4-en-1-
amine) (3 f): Compound 3 f was obtained from 2 and 1,4-bis(ami-
4
H), 2.92 (s, 4H), 2.07 (d, J=7.5 Hz, 4H), 0.94 ppm (s, 12H);
1
3
C NMR (100 MHz, CDCl ): d=137.2, 135.2, 129.1, 117.3, 73.3, 70.2,
nomethyl)benzene according to the general procedure as a color-
3
1
44.8, 35.6, 25.8 ppm.
less liquid (4.18 g, 12.7 mmol, 88%): H NMR (400 MHz, CDCl ): d=
3
7
2
.29 (s, 4H) 5.79 (m, 2H), 4.99 (m, 4H), 3.78 (s, 4H), 2.37 (s, 4H),
.02 (d, J=7.5 Hz, 4H), 0.89 ppm (s, 12H); C NMR (100 MHz,
General procedure for the synthesis of bis-3-chloropiperidines
13
5
a–f: The bis-N-chloroamine was dissolved in anhydrous chloro-
CDCl3): d=139.4, 135.6, 127.9, 116.7, 59.7, 54.5, 44.6, 34.3,
À1
form (10 mLmmol of bis-N-chloroamine), and tetrabutylammoni-
um iodide (10 mol%) was added to the solution. The resulting mix-
ture was then heated at 608C (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 diastereomers.
2
5.5 ppm; HRMS (EI): m/z calcd for C H N : 328.2878; found:
22 36 2
3
28.2888.
General procedure for the synthesis of bis-N-chloroamines 4a–f:
N-Chlorosuccinimide (2.2 equiv) was added to a cooled (08C) solu-
tion of the corresponding bis-N-diamine in anhydrous CH Cl
2
2
À1
(
10 mLmmol of bis-N-diamine). The reaction mixture was stirred
1
,3-Bis(5-chloro-3,3-dimethylpiperidin-1-yl)propane (5a): Com-
first for half an hour at 08C, and then for an additional 2 h at room
temperature. After removal of the solvent in vacuo, the product
was isolated from the residue by flash chromatography (pentane/
TBME, 10:1).
pound 5a was obtained from 4a according to the general proce-
dure as a colorless oil (310 mg, 0.92 mmol, 88%, mixture of diaste-
1
reomers): H NMR (400 MHz, CDCl ): d=4.06 (ttd, J=8.9 Hz, J=
3
4
.4 Hz, J=1.0 Hz, 2H), 3.13 (dd, J=10.4 Hz, J=4.3, 2H), 2.35 (m,
1
3
1
3
N ,N -Dichloro-N ,N -bis(2,2-dimethylpent-4-enyl)propane-1,3-di-
amine (4a): Compound 4a was obtained from 3a according to
6H), 1.93 (m, 4H), 1.71 (d, J=11.0 Hz, 2H), 1.58 (quin, J=7.2 Hz,
2H), 1.32 (t, J=12.3 Hz, 2H), 1.02 (s, 6H), 0.91 ppm (s, 6H);
13
the general procedure as a colorless oil (354 mg, 1.06 mmol, 94%):
C NMR (100 MHz, CDCl ): d=64.8, 62.3, 62.2, 55.7, 55.6, 54.3, 48.4,
3
1
H NMR (400 MHz, CDCl ): d=5.81 (ddt, J=16.7 Hz, J=10.5 Hz, J=
33.3, 29.4, 25.2, 24.3 ppm; HRMS (ESI): m/z calcd for C17
H : 335.2015; found: 335.2015.
H
32
N
2
Cl
2
+
3
+
7
.5 Hz, 2H), 5.03 (m, 4H), 3.02 (t, J=6.7 Hz, 4H), 2.85 (s, 4H), 2.06
(
d, J=7.5 Hz, 4H), 1.98 (quin, J=6.7 Hz, 2H), 0.95 ppm (s, 12H);
1
3
1,4-Bis(5-chloro-3,3-dimethylpiperidin-1-yl)butane (5b): Com-
pound 5b was obtained from 4b according to the general proce-
C NMR (100 MHz, CDCl ): d=135.2, 117.3, 74.8, 63.7, 44.9, 35.6,
3
2
6.7, 25.8 ppm.
dure as a colorless oil (1.51 g, 4.33 mmol, 88%, mixture of diaste-
1
4
1
4
1
N ,N -Dichloro-N ,N -bis(2,2-dimethylpent-4-enyl)butane-1,4-di-
amine (4b): Compound 4b was obtained from 3b according to
reomers): H NMR (400 MHz, CDCl ): d=4.06 (ttd, J=11.8 Hz, J=
3
4.4 Hz, J=1.1 Hz, 2H), 3.13 (dd, J=10.6 Hz, J=4.3, 2H), 2.38 (d, J=
11.0 Hz, 2H), 2.29 (m, 4H), 1.91 (m, 4H), 1.67 (dd, J=11.1 Hz, J=
2.7 Hz, 2H), 1.44 (m, 4H), 1.31 (t, J=12.3 Hz, 2H), 1.02 (s, 6H),
the general procedure as a colorless oil (1.73, 4.94 mmol, 87%):
1
H NMR (400 MHz, CDCl ): d=5.74 (ddt, J=16.7 Hz, J=10.5 Hz, J=
3
13
7
7
.5 Hz, 2H), 4.95 (m, 4H), 2.88 (m, 4H), 2.77 (s, 4H), 2.00 (d, J=
0.90 ppm (s, 6H); C NMR (100 MHz, CDCl ): d=64.8, 64.7, 62.3,
3
13
.5 Hz, 4H), 1.63 (m, 4H), 0.87 ppm (s, 12H); C NMR (100 MHz,
62.2, 57.7, 57.6, 54.3, 48.4, 33.3, 29.4, 25.2, 24.5 ppm; HRMS (ESI):
+
CDCl ): d=135.2, 117.3, 74.8, 66.4, 44.9, 35.6, 25.7, 25.2 ppm.
m/z calcd for C H N Cl +H : 349.2172; found: 349.2172.
3
18 34
2
2
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ChemMedChem 0000, 00, 1 – 9
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1
,5-Bis(5-chloro-3,3-dimethylpiperidin-1-yl)pentane (5c): Com-
ensured the formation of the duplex DNA through annealing of
scrambled and co-scrambled oligonucleotides. Dilutions of bis-3-
chloropiperidines and chlorambucil were freshly prepared from
a DMSO stock solution (8 mm) in water. Alkylation reactions were
carried out in BPE buffer.
pound 5c was obtained from 4c according to the general proce-
dure as a colorless oil (1.24 g, 3.40 mmol, 78%, mixture of diaste-
reomers): H NMR (400 MHz, CDCl ): d=4.07 (tt, J=11.3 Hz, J=
1
3
4
2
.4 Hz, 2H), 3.14 (dd, J=10.6 Hz, J=4.4, 2H), 2.39 (d, J=11.1 Hz,
H), 2.30 (m, 4H), 1.91 (m, 4H), 1.68 (d, J=11.1 Hz, 2H), 1.44 (quin,
Preparation of plasmid DNA: Plasmid pAT153 was transformed
into E. coli strain JM109 (Promega). After fermentation, the plasmid
DNA was isolated using the Promega PureYield Plasmid Midiprep
System according to the instructions of the supplier. The concen-
tration of plasmid was determined using a NanoDrop 1000 spec-
trophotometer (Thermo Scientific).
J=7.1 Hz, 4H), 1.31 (m, 4H), 1.02 (s, 6H), 0.91 ppm (s, 6H);
1
3
C NMR (100 MHz, CDCl ): d=64.7, 62.3, 57.9, 54.4, 48.4, 33.2, 29.4,
3
+
2
6.8, 25.2, 25.1 ppm; HRMS (ESI): m/z calcd for C H N Cl +H :
19
36
2
2
3
63.2328; found: 363.2328.
1
,6-Bis(5-chloro-3,3-dimethylpiperidin-1-yl)hexane (5d): Com-
pound 5d was obtained from 4d according to the general proce-
DNA cleavage assay: DNA cleavage assays were performed using
pAT153 plasmid obtained as described above. pAT153 (120 ng)
was incubated with increasing concentrations (0.5, 5, 50 mm) of
agent for 3 h at 378C in BPE buffer. Plasmid pAT153 nicked by
Nb·Bpu10I (Fermentas) was used as a marker for the open circular
DNA form, while the linearized standard was obtained by HindIII
(NEB) digestion, according to the manufacturer’s instructions. Gel
loading buffer (10 mm Tris-HCl, 50% glycerol, 0.025% bromophe-
nol blue) was added to all reaction tubes, and the samples were
loaded onto a 1% agarose gel. Electrophoresis was conducted in
TBE 1X (Tris-HCl 89 mm, borate 89 mm, EDTA 2 mm). DNA in the
gel system was detected by staining with ethidium bromide
dure as a colorless oil (1.16 g, 3.07 mmol, 77%, mixture of diaste-
1
reomers): H NMR (400 MHz, CDCl ): d=4.06 (ddt, J=11.9 Hz, J=
3
1
0.8 Hz, J=4.4 Hz, 2H), 3.13 (dd, J=10.6 Hz, J=4.4, 2H), 2.39 (d,
J=11.0 Hz, 2H), 2.29 (m, 4H), 1.91 (m, 4H), 1.67 (d, J=11.1 Hz, 2H),
13
1
.43 (m, 4H), 1.30 (m, 6H), 1.02 (s, 6H), 0.90 ppm (s, 6H); C NMR
(
2
100 MHz, CDCl ): d=64.7, 62.4, 57.9, 54.4, 48.4, 33.2, 29.4, 27.2,
6.9, 25.2 ppm; HRMS (ESI): m/z calcd for C H N Cl +H :
20 38 2 2
3
+
3
77.2485; found: 377.2485.
trans-1,4-Bis(5-chloro-3,3-dimethylpiperidin-1-yl)cyclohexane
5e): Compound 5e was obtained from 4e according to the gen-
(
eral procedure as a colorless solid (176 mg, 0.47 mmol, 63%, mix-
1
À1
ture of diastereomers): H NMR (600 MHz, CDCl ): d=4.01 (ddd, J=
(0.5 mgmL ) for 30 min with visualization by a Geliance 600 imag-
3
1
2
1
4
5.7 Hz, J=11.0 Hz, J=4.4 Hz, 2H), 3.09 (dd, J=10.4 Hz, J=4.1,
H), 2.30 (d, J=11.0 Hz, 2H), 2.26 (m, 2H), 2.20 (t, J=10.5 Hz, 2H),
.97 (d, J=11.0 Hz, 2H), 1.91 (dd, J=12.5 Hz, J=4.0, 2H), 1.80 (m,
H), 1.26 (m, 2H), 1.23 (m, 4H), 0.99 (s, 6H), 0.89 ppm (s, 6H);
ing system (PerkinElmer, Waltham, MA, USA).
Sequencing gel analysis: The 5’-FAM-labelled duplex oligonucleo-
tide (4 mm) was incubated with each alkylating agent (final concen-
trations of 5 and 50 mm) in BPE buffer. The reaction was analyzed
at different incubation times: 1, 2, 4, 7, 15, and 24 h at 378C. The
samples were dried in a vacuum centrifuge (UNIVAPO 100H, UniE-
quip), resuspended in 5 mL of denaturing gel loading buffer
1
3
C NMR (150 MHz, CDCl ): d=63.5, 60.5, 58.8, 55.2, 48.9, 33.3, 29.4,
3
2
7.9, 27.8, 27.2, 27.1, 25.0 ppm; HRMS (ESI): m/z calcd for
+
C H N Cl +H : 375.2328; found: 375.2328.
2
0
36
2
2
(
10 mm Tris-HCl, 80% formamide, 0.025% bromophenol blue), and
loaded on a 20% denaturing polyacrylamide gel (7m urea) in TBE
X. The fluorescence of the oligonucleotide bands were detected
1
,4-Bis((5-chloro-3,3-dimethylpiperidin-1-yl)methyl)benzene (5 f):
Compound 5 f was obtained from 4 f according to the general pro-
cedure as a colorless oil (1.41 g, 3.55 mmol, 88%, mixture of diaste-
reomers): H NMR (400 MHz, CDCl ): d=7.24 (s, 4H), 4.11 (ddd, J=
1
1
by scanning using Storm Scanner Control (STORM 840, Molecular
Dynamics).
3
1
6.0 Hz, J=11.3 Hz, J=4.4 Hz, 2H), 3.49 (q, J=13.4 Hz, 4H), 3.16
(
dd, J=10.0 Hz, J=4.0, 2H), 2.39 (d, J=10.9 Hz, 2H), 1.97 (m, 4H),
1
0
6
.76 (d, J=11.0 Hz, 2H), 1.35 (t, J=12.3 Hz, 2H), 1.06 (s, 6H),
1
3
Acknowledgements
.89 ppm (s, 6H); C NMR (100 MHz, CDCl ): d=137.3, 128.5, 64.6,
3
2.0, 61.9, 54.3, 48.4, 33.4, 29.3, 25.1 ppm; HRMS (ESI): m/z calcd
+
for C H N Cl +H : 397.2172; found: 397.2172.
I.Z. thanks Mert Yanik for helpful discussions and gratefully ac-
knowledges financial support from the German National Aca-
demic Foundation (Studienstiftung des deutschen Volkes) through
a PhD scholarship, as well as the International Giessen Graduate
Centre for the Life Sciences (GGL) and the German Academic Ex-
change Service (DAAD) for a travel grant. B.G. thanks MIUR for fi-
nancial support (grant 2010W2KM5L_006).
2
2
34
2
2
Bioassays
The water used in all biochemical experiments was prepared from
the Milli-Q Synthesis (Millipore) water purification system. Chloram-
bucil 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 acrylam-
ide-bis ready-to-use solution (40%, 19:1) was purchased from
Merck (Darmstadt, Germany). Oligonucleotides were purchased
from Eurogentec (Seraing, Liꢂge, Belgium) and stored at À208C in
TE (10 mm Tris-HCl, 1 mm EDTA). The sequence of the scrambled
oligonucleotide used for 5’-FAM labeling was: 5’-FAM-GGA TGT
GAG TGT GAG TGT GAG G-3’; the complementary co-scrambled oli-
gonucleotide sequence was: 5’-CCT CAC ACT CAC ACT CAC ATC C-
Keywords: antitumor agents · cyclization · DNA cleavage ·
nitrogen heterocycles · structure–activity relationships
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’. The 5’-FAM labeled scrambled oligonucleotide was mixed with
equimolar amounts of its complementary co-scrambled oligonu-
cleotide in BPE buffer (2 mm NaH PO ·2H O, 6 mm Na HPO ·12H O,
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005.
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4
2
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mm Na EDTA·2H O, pH 7.4), denatured at 958C for 5 min, and
2 2
then left to cool to room temperature (slow annealing). This step
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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ÞÞ
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CHEMMEDCHEM
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Published online on && &&, 0000
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 9
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FULL PAPERS
It’s all about Gs! An elegant and simple
bidirectional synthetic approach to bis-
I. Zuravka, R. Roesmann, A. Sosic,
W. Wende, A. Pingoud, B. Gatto,*
R. Gçttlich*
3-chloropiperdines with various linker
structures is reported. These new alky-
lating agents induce cleavage of
double-stranded DNA, primarily through
reactions toward guanine sites via the
formation of electrophilic aziridinium
ion intermediates.
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Synthesis and DNA Cleavage Activity
of Bis-3-chloropiperidines as
Alkylating Agents
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 9
&
9
&
ÞÞ
These are not the final page numbers!