Semi-automatic synthesis, antiproliferative activity and DNA-binding properties of new netropsin and bis-netropsin analogues

Article abstract of DOI:10.3390/molecules190811300

A general route for the semi-automatic synthesis of some new potential minor groove binders was established. Six four-numbered sub-libraries of new netropsin and bis-netropsin analogues have been synthesized using a Syncore Reactor. The structures of the

Full text of DOI:10.3390/molecules190811300

Molecules 2014, 19, 11300-11315; doi:10.3390/molecules190811300
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Semi-Automatic Synthesis, Antiproliferative Activity and
DNA-Binding Properties of New Netropsin and
bis-Netropsin Analogues
Jakub Szerszenowicz 1 and Danuta Drozdowska 2,*,†
1
Saint Brunon Farmacy, Bohaterów Westerplatte 4 Str., 11-500 Giżycko, Poland;
E-Mail: jakubszerszenowicz@o2.pl
2
Department of Organic Chemistry, Medical University, Mickiewicza 2A Str.,
15-222 Białystok, Poland
†
In 2006 last name was changed from Bartulewicz.
* Author to whom correspondence should be addressed; E-Mail: danuta.drozdowska@umb.edu.pl;
Tel.: +48-85 748-56-84; Fax: +48-85-748-54-16.
Received: 17 April 2014; in revised form: 15 July 2014 / Accepted: 16 July 2014 /
Published: 31 July 2014
Abstract: A general route for the semi-automatic synthesis of some new potential minor
groove binders was established. Six four-numbered sub-libraries of new netropsin and
bis-netropsin analogues have been synthesized using a Syncore Reactor. The structures of
the all new substances prepared in this investigation were fully characterized by NMR
(1H, 13C), HPLC and LC-MS. The antiproliferative activity of the obtained compounds was
tested on MCF-7 breast cancer cells. The ethidium displacement assay using pBR322
confirmed the DNA-binding properties of the new analogues of netropsin and bis-netropsin.
Keywords: combinatorial chemistry; solid phase synthesis; netropsin analogue;
non-intercalative DNA binding agent; DNA minor groove binder
1. Introduction
The polyamide antibiotics netropsin and distamycin (Figure 1), synthesized by Streptomyces are the
most studied representatives of a series natural products possessing antitumor and antiviral properties [1].
These compounds are also well-known for their strong affinity for DNA. They bind within the minor

Molecules 2014, 19
11301
groove of B-DNA at sites consisting of four or five consecutive AT base pairs [2]. Using netropsin and
distamycin as paradigms, numerous minor groove binders have been designed and synthesized [3,4].
Analogues of these natural antibiotics, as well as other minor groove binders, have found substantial
applications in anti-cancer therapy. Compounds with antibacterial, antifungal, antiviral, and
antiparasitic activity have also been identified. Moreover, their importance in anti-infective therapy
also has been significant and its importance is growing [5].
Figure 1. Structures of netropsin, distamycin and carbocyclic analogues of netropsin.
HN
NH
2
HN
NH
2
H N
2
HN
O
N
R
HN
O
N
HN
O
CH
3
CH
3
HN
O
HN
O
NH
O
R
N
HN
CH
5A R=H
5B R=OCH
3
N
HN
H
CH
3
HN
N
3
N
O
O
HN
H N
O
N
H
netropsin
2
CH
distamycin
carbocyclic analogues of netropsin
3
Distamycin and its analogues have been investigated and described very well [3–6], but
analogues of netropsin have been synthesized less frequently. In the course of our investigation of
minor-groove-binding drugs some netropsin analogues were synthesized and tested for DNA-binding
and anticancer properties (Figure 1) [7]. It has been previously shown that those compounds inhibited
proliferation of the standard MCF-7 mammalian tumor cell line with IC50 values of 24.43 (5A) and
40.73 µM (5B) and act as reversible minor-groove binders with selectivity for AT regions [8].
Dibenzene netropsin analogues 5A and 5B had not inhibited DNA topoisomerases, but they
demonstrated higher DNA binding affinity in comparison to tribenzene distamycin analogues [9]. A
molecular mechanics and molecular dynamics approach was used to examine the structure of the
complexes formed between the d(CGCGAATTCGCG)2 duplex and carbocyclic analogues 5A and 5B.
It could be expected that these compounds would be effectively isohelical with the DNA minor
groove. From the analysis of our model it appears that van der Waals and electrostatic interactions are
more important in stabilizing the complexes than specific hydrogen bond formation. These results
were confirmed by the determination of the association constants of ligands with different
polynucleotides. Compounds 5A and 5B bind to AT sequences with high sequence-selectivity.
Moreover, the high apparent binding constants for T4 coliphage DNA gave evidence of their minor-
groove selectivity [9]. The compounds of this type, containing terminal free amine groups, could be
used as vectors for delivery of the DNA interacting agents. That was illustrated by the syntheses of
carbocyclic analogues of netropsin with a chlorambucil moiety [10]. The syntheses of carbocyclic
analogues with structures related to bis-netropsin were also described [11].
The syntheses were planned based on a previously developed new solid phase synthesis of
distamycin analogues [12]. This simple and general procedure makes it possible to simultaneously
carry out syntheses of many new compounds, permitting automation of the process. In the present

Molecules 2014, 19
11302
paper results of our studies of DNA ligands as potential anticancer drugs and combinatorial synthesis
of new 24-membered netropsin and bis-netropsin analogues library using a Syncore Reactor are
reported. In the same study the antiproliferative activity of compounds 1–24 on MCF-7 breast cancer
cells has also been investigated. An ethidium bromide assay was used to show that these compounds
bind to plasmid pBR322.
2. Results and Discussion
2.1. Preparation of Netropsin Analogues
For the netropsin derivative preparation procedure aromatic amino-nitro compounds A–D, active
ester 1 and selected acid chlorides 2–6, were used as substrates to obtain four six-membered
sub- libraries as shown in Figure 2. Compounds having the structure II were obtained according the
reported procedure [12] from p-nitrophenyl carbonate Wang resin I, as shown in Scheme 1. After
grafting of nitroamines to the resin, reduction of the nitro group of structure II was carried out using
tin (II) chloride dihydrate in DMF.
Figure 2. Structures of substrates and the synthesis schedule.
Cl
N
O
Cl
O
N
Cl
Cl
O
4
Cl
O
Cl
2
Cl
O
Br
Br
5
O
R'O
3
Cl
Cl
1
6
O
O
O2N
A
N
NH2
NH2
NH2
A1
A2
A3
B3
C3
D3
A4
B4
A5
B5
A6
O2N
N
B
N
B1
B2
C2
D2
B6
O2N
C
N
C4
D4
C1
D1
C5
D5
C6
D6
S
NH2
D
O2N
Acylation of four resin-bound amines III, using 3-nitrobenzoyl chloride in the presence of DMAP
in methylene chloride at room temperature produced the resin-bound nitro compounds with structure
IV. Repeated reduction of the nitro groups led to compounds V. The next steps were reactions leading
to obtaining products with structures VI, the final resin-bound analogues of netropsin with different
moieties R1–6.

Molecules 2014, 19
11303
Scheme 1. General procedure of netropsin analogues syntheses.
NO2
NH2
O
O
NO2
b
a
A-D
A-D
HN
HN
O
O
O
I
O
O
II
III
NH
NH
c
NO2
b
A-D
HN
NH2
O
A-D
HN
O
O
IV
V
O
O
O
R1-6
NH
R1-
NH
e
d
6
NH
A-D
NH
HN
O
A-D
H2N
O
O
VI
O
VII
Reagents and conditions: (a) Aromatic nitroamine A-D, 12 h, DCM, rt; (b) 1 M SnCl2, 4 h DMF, rt;
(c) 3-nitrobenzoyl chloride, 4-DMAP, DCM, 24 h, rt; (d) 1–6, DCM, 2 h 0 °C and rt, overnight;
(e) TFA/DCM (95:5), 2 h, rt.
To combine fragments A-D with R1, the “active ester” of 4-dimethylaminobutyric acid was
prepared, as presented earlier [12]. Compounds with R2 were prepared through acylation reactions
with 4-chlorobutyryl chloride. To join fragments R3 and R4 the chlorides of bromoacrylic acid and
chlorambucil were synthesized, respectively. To synthesise compounds containing linkers R5 and R6;
adipoyl and pimeloyl dichlorides were used. Cleavage by 95% trifluoroacetic acid in dichloromethane
gave the desired compounds having the structure VII in satisfactory yield. The structures, analytical
and spectrometric data are presented in Table 1.
Table 1. Analytical and spectral data of the synthesized compounds.
R1-6
NH
NH
A-D
H2N
O
Library Substrate Fragment Purity
No.
Rt
Formula (Exact Mass)
[M+H]+
No.
A-D
A
R1-R6
[%]
96
98
98
94
87
89
91
89
1
2
3
4
5
6
7
8
1
1
1
1
2
2
2
2
21.98
20.37
20.34
20.35
19.03
19.04
19.01
19.10
C18H23N5O2 (341.4)
C17H22N6O2 (342.4)
C16H21N5O2S (347.4)
C19H24N4O2 (340.4)
C16H17ClN4O2 (332.8)
C15H16ClN5O2 (333.8)
C14H15ClN4O2S (338.8)
C17H18ClN3O2 (331.8)
340.4
341.40
346.4
339.4
331.8
332.8
337.8
330.8
B
1
C
D
A
2
B
C
D

Molecules 2014, 19
11304
Table 1. Cont.
Library Substrate Fragment Purity
No.
Rt
Formula (Exact Mass)
[M+H]+
No.
A-D
A
B
R1-R6
[%]
98
97
95
96
97
93
96
97
99
98
99
97
99
99
98
99
9
3
3
3
3
4
4
4
4
5
5
5
5
6
6
6
6
10.27
10.74
10.95
10.91
15.89
15.98
15.06
15.34
18.80
18.56
18.38
18.42
19.98
19.37
19.34
19.35
C15H13BrN4O2 (361.2)
C14H12BrN5O2 (362.2)
C13H11BrN4O2S (367.2)
C16H14BrN3O2 (360.2)
C26H29Cl2N5O2 (514.4)
C25H28Cl2N6O2 (515.4)
C24H27Cl2N5O2S (520.5)
C27H30Cl2N4O2 (513.4)
C30H30N8O4 (566.6)
C28H28N10O4 (568.6)
C26H26N8O4S (578.7)
C32H32N6O4 (564.6)
C31H32N8O4 (580.6)
C29H30N10O4 (582.6)
C27H28N8O4S2 (592.7)
C33H34N6O4 (578.7)
360.2
365.2
366.2
359.2
513.4
514.4
519.5
512.4
565.6
567.6
577.7
563.6
579.6
581.6
591.7
577.7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
3
C
D
A
B
4
5
6
C
D
A
B
C
D
A
B
C
D
2.2. Antiproliferative Activity of Compounds 1–24
The antiproliferative effects of compounds 1–24 in the standard cell line of human breast cancer
MCF-7 are presented as IC50 values in Table 2.
Table 2. Antiproliferative activity of compounds 1–24 against MCF-7 breast cancer cells.
a
IC50
1
1A
2
3
3A
4
5
6
2A
4A
5A
6A
A
74.71 µM
1B
82.35 µM
2B
75.511 µM
3B
62.73 µM
4B
234.89 µM
5B
97.82 µM
6B
B
C
D
106.42 µM
1C
75.34 µM
2C
85.08 µM
3C
86.28 µM
4C
153.63 µM
5C
96.48 µM
6C
78.35 µM
1D
69.99 µM
2D
71.74 µM
3D
69.59 µM
4D
160.73 µM
5D
70.01 µM
6D
82.31 µM
73.28 µM
70.36 µM
85.27 µM
140.74 µM
93.72 µM
a The results represent the mean (±S.D.) of three independent experiments done in duplicates.
All the tested compounds showed concentration-dependent activity, but none of the compounds
were more active than netropsin with IC50 = 5.40 µM [11]. The concentration of the compounds that
inhibited 50% of colony formation was within the range from 62.73 µM to 234.89 µM. Figure 3 shows
the graph illustrating the compounds 1–24 in order of decreasing IC50 values, that is, according to their
increasing antiproliferative activity.

Molecules 2014, 19
11305
Figure 3. The IC50 values of compounds 1–24.
The activity of the compounds
240
220
200
180
160
140
120
100
80
60
5A 5C 5B 5D 1B 6A 6B 6D 4B 4D 3B 2A 1D 1C 3A 2B 1A 2D 3C 3D 6C 2C 4C 4A
In order to define structure-activity relationships values of IC50 in particular groups of compounds:
groups with different fragments A–D (Figure 4A) and libraries 1–6 (Figure 4B) were compared.
Figure 4. The IC50 values of libraries A–D (A) and 1–6 (B).
Antiproliferating activity of libraries A-D
Antiproliferating activity of libraries 1-6
B
A
230
180
130
80
230
180
130
80
30
30
A
B
C
D
1
2
3
4
5
6
The above data prove that the most active of the compounds is 4A with IC50 = 62.73 µM and
containing a chlorambucil fragment. The least active compounds are in group 5 with (CH2)4 linkers.
Higher activity of bis-netropsin analogues from library 6, containing linker a (CH2)5 in their structures,
in comparison with compounds from library 1, was not observed. Doubling the length of the molecules
also did not result in any increase of their antiproliferative activity. The compounds from libraries 2, 3
and 4 are built with netropsin-like and alkylating fragments. Only a slight increase of activity of these
compounds in comparison to netropsin analogues from library 1 was observed.
2.3. DNA-Binding Effects
The ethidium bromide assay showed that the investigated compounds can bind to plasmid DNA
(Table 3).

Molecules 2014, 19
11306
Table 3. DNA-binding effect of compounds 1–24.
1
2
3
4
5
6
A
B
C
D
1A 85.63% 2A 98.85% 3A 98.80% 4A 70.67% 5A 87.27% 6A 84.67%
1B 85.45% 2B 98.95% 3B 98.90% 4B 74.67% 5B 76.36% 6B 69.36%
1C 77.73% 2C 79.33% 3C 97.33% 4C 85.33% 5C 90.67% 6C 78.18%
1D 83.18% 2D 86.67% 3D 88.00% 4D 64.67% 5D 71.81% 6D 69.33%
Activity of netropsin in this assay is 74.01% [11]. Some of our new compounds were characterized
by a higher binding strength to pBR322 plasmid. This can be seen on the graph presented in Figure 5.
Figure 5. NA-binding activity of compounds 1–24.
DNA-binding activity
100
95
90
85
80
75
2B 3B 2A 3A 3C 5C 3D 5A 2D 1A 1B 4C 6A 1D 2C 6C 1C 5B 4B 5D 4A 6B 6D 4D
70
65
DNA-binding values in particular groups of compounds are shown in the Figure 6: groups with
different fragments A–D (Figure 6A) and libraries 1–6 (Figure 6B).
Figure 6. DNA-binding activity of libraries A–D (A) and 1–6 (B).
Antiproliferating activity of libraries 1-6
Antiproliferating activity of libraries A-D
230
180
130
80
230
180
130
80
30
30
A
B
C
D
1
2
3
4
5
6
(A)
(B)
Compounds 4D, 6D, 6B, 4A and 4B bind stronger or similarly to netropsin. Three of them represent
library 4, amongst which compound 4A is the most active against MCF-7 breast cancer cells. These
data suggest that the chlorambucil fragment has been the most beneficial one in our work. It can be
assumed that presence of this alkylating fragment causes additional interactions with DNA. No clear
relationship between the fragments A–D and DNA binding activity was established. To confirm the

Molecules 2014, 19
11307
binding of the investigated compounds into the minor groove requires additional research, e.g. NMR
or crystallography.
3. Experimental Section
3.1 General Information
All reagents were purchased from Lancaster (Frankfurt am Main, Germany), Fluka (Sigma-Aldrich
sp. z o.o., Poznań, Poland), Merck (Darmstadt, Germany), Alfa Aesar (Karlsruhe, Germany) or Iris
Biotech GmBH (Marktredwitz, Germany) and used without further purification. 4-(4,6-Dimethoxy-
1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate was prepared in Institute of Organic
Chemistry Technical University in Łódź (Poland). Dichloromethane (DCM) and dimethylformamide
1
13
(DMF) were stored under 4 Å molecular sieves. H-NMR and C-NMR spectra were recorded on a
Bruker AC 400F spectrometer (Bruker corp., Fällanden, Switzerland) using TMS as internal standard;
chemical shifts δ are reported in ppm. The spectra were recorded at room temperature. LC-MS spectra
were recorded on Bruker Daltonics Esquire 6000 instrument with electrospray ionization (ESI). A
Shimadzu LC-10A system (Shimadzu corp., Kyoto, Japan) was used for analytical HPLC
(Phenomenex C18, Jupiter 90A, 4 micron, 250 × 10 mm; Phenomenex C18, Jupiter 300A, 5 micron,
250 × 4 mm; solvents: A, 0.1% aqueous TFA; B, 0.1% TFA in acetonitrile, gradient 0% B to 60% B in
A in 30 min, flow rate 1 mL/min, monitored at 220 nm). Ethidium bromide was purchased from Carl
Roth GmbH (Karlsruhe, Germany). Stock cultures of MCF-7 were purchased from the American Type
Culture Collection (Rockville, MD, USA). Dulbecco’s modified Eagle’s medium, fetal bovine serum
(FBS), netropsin, streptomycin and penicillin were products of Sigma. Plasmid pBR322 was purchased
from Fermentas Life Science (Vilnius, Lithuania).
3.2. General Procedure
The solid-phase syntheses of the new compounds are shown in Scheme 1. 4-Nitrophenyl Wang
resin 1 (0.5 g; 0.41 mmol; 0.81 mmol/g) was suspended and swelled for 10 min in dry DCM (10 mL),
then the resin was treated with nitroamines (A, B, C, D; 1.64 mmol; 4 eq) dissolved in DCM (20 mL)
and pyridine (177.22 μL; 2.2 mmol). [A (0.226 g); B (0.225 g); C (0.235 g); D (0.226 g)] Then the
mixture was stirred for 20 h. The products II were washed five times with DCM (20 mL) and three
times with DMF (20 mL). Then reduction of nitro groups of II with the dihydrate of tin (II) chloride in
DMF (1 M; 20 mL) during 4 h at room temperature were carried out. The resins III were filtered off
and then washed five times with DMF (20 mL) and three times with DCM (20 mL). Then the aliquots
of 3-nitrobenzoyl chloride (0.304 g; 1.64 mmol; 4 eq) with DMAP (0.0025 g; 0.0205 mmol) were
dissolved in DCM (20 mL) and added to resin III. The stirring was continued at room temperature
overnight. After that, the resin-bound nitro compounds IV were washing five times with DCM (20 mL)
and three times with DMF (20 mL). The treatment of thes compounds IV with 1 M SnCl2 allowed to
obtain the intermediates V. To prepare the resin-bond compounds VI there were used the substrates
prepared as described below. The resins VII were washed with DCM (5 × 20 mL), DMF (5 × 20 mL)
and DCM (5 × 20 mL), dried and then treated with TFA/DCM (95:5). Subsequent evaporation of the
solvent and the several portions of DCM yielded the products as glaze solids.

Molecules 2014, 19
3.2.1. Library 1
11308
To introduce the 4-(dimethylamino)butanamido- fragment, the “superactive ester” 1 was prepared as
described earlier [12]. 4-Dimethylaminobutyric acid (0.137 g; 0.82 mmol) and NMM (902 μL;
0.82 mmol) were added to a vigorously stirred solution of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-
methylmorpholinium tetrafluoroborate (0.269 g; 0.82 mmol) in DMF (20 mL) and cooled to 0 °C. The
stirring was continued for an additional 2 h in 0 °C, after which time these mixtures were added to the
resins V and stirred together for an additional 2h at 0 °C and overnight at room temperature.
N-(6-Aminopyridin-3-yl)-3-(4-(dimethylamino)butanamido)benzamide (A1). Yield: 0.050 g (36%).
1H-NMR (DMSO-d6) δ = 10.10 (s,1H, NH), 7.22 (t,1H, NH), 6.80–7.70 (m, 7H, Ar-H), 3.95 (s, 2H,
NH2), 2.99 (t, 2H, CH2), 2.72 (s, 6H, CH3), 2.32 (t, 2H, CH2); 1.85–1.88 (m, 2H, CH2). 13C-NMR
(DMSO-d6) δ = 173.45 (CONH), 166.93 (CONH), 159.90 (C), 157.89 (C), 149.90 (CH), 139.72(C),
134.58 (CH), 131.85 (C), 129.61 (CH), 123.36 (CH), 119.71 (CH), 117.02 (CH), 115.64 (CH), 59.87
(CH2), 42.05 (CH3), 30.46 (CH2), 19.70 (CH2).
N-(2-Aminopyrimidin-5-yl)-3-(4-(dimethylamino)butanamido)benzamide (B1). Yield: 0.045 g (32%).
1H-NMR (DMSO-d6) δ = 9.48 (s,1H, NH), 7.27 (t,1H, NH), 6.85–7.35 (m, 6H, Ar-H), 3.30–4.10 (bs,
13
2H, NH2), 2.99 (t, 2H, CH2), 2.77 (s, 6H, CH3), 2.31 (t, 2H, CH2); 1.78–1.88 (m, 2H, CH2). C-NMR
(DMSO-d6) δ = 173.38 (CONH), 167.48 (CONH), 159.20 (C), 157. 96 (C), 139.72(C), 134.55 (CH),
134.33 (CH), 131.52 (C), 129.10 (CH), 119.71 (CH), 116.98 (CH), 115.63 (CH), 59.85 (CH2), 42.23
(CH3), 30.35 (CH2), 19.36 (CH2).
N-(2-Aminothiazol-5-yl)-3-(4-(dimethylamino)butanamido)benzamide (C1). Yield: 0.040 g (28%).
1H-NMR (DMSO-d6) δ = 9.48 (s,1H, NH), 7.95 (t,1H, NH), 6.83–7.37 (m, 5H, Ar-H), 3.94 (s, 2H,
NH2), 2.99 (t, 2H, CH2), 2.77 (s, 6H, CH3), 2.28 (t, 2H, CH2); 1.80–1.86 (m, 2H, CH2). 13C-NMR
(DMSO-d6) δ = 173.44 (CONH), 167.50 (CONH), 162.30 (C), 157.85 (C), 134.58 (C), 131.53 (C),
129.16 (CH), 120.04 (CH), 119.29 (CH), 116.49 (CH), 115.63 (CH), 59.87 (CH2), 42.22 (CH3), 30.36
(CH2), 19.37 (CH2).
N-(3-Aminophenyl)-3-(4-(dimethylamino)butanamido)benzamide (D1). Yield:
0.049 g (35%).
1H-NMR (DMSO-d6) δ = 9.48 (s,1H, NH), 7.22 (t,1H, NH), 6.84–7.95 (m, 8H, Ar-H), 4.54 (s, 2H,
NH2), 2.99 (t, 2H, CH2), 2.73 (s, 6H, CH3), 2.31 (t, 2H, CH2); 1.85–1.88 (m, 2H, CH2). 13C-NMR
(DMSO-d6) δ = 173.45 (CONH), 166.93 (CONH), 159.90 (C), 157.89 (C), 149.90 (CH), 139.72(C),
134.58 (CH), 131.85 (C), 129.61 (CH), 123.36 (CH), 119.71 (CH), 117.02 (CH), 115.64 (CH), 59.87
(CH2), 42.05 (CH3), 30.46 (CH2), 19.70 (CH2).
3.2.2. Library 2
4-Chlorobutanamido derivatives were obtained by direct acylation of resins V by 4-chlorobutanoyl
chloride (2, 2 mmol; 0.282 g; 0.224 mL). The above acid chloride was dissolved in DCM (20 mL),
chilled to 0 °C and every portion was added to chilled resins V with DMAP (0.0025 g; 0.0205 mmol).
These mixtures were stirred for 2 h in 0 °C and overnight at room temperature.

Molecules 2014, 19
11309
1
N-(6-Aminopyridin-3-yl)-3-(4-chlorobutanamido)benzamide (A2). Yield: 0.037 g (27%). H-NMR
(DMSO-d6) δ = 10.08 (s,1H, NH), 7.40 (t,1H, NH), 6.88–8.22 (m, 7H, Ar-H), 3.90–4.40 (bs, 2H,
NH2), 3.70 (t, 2H, CH2), 2.36 (t, 2H, CH2); 1.88–1.98 (m, 2H, CH2). 13C-NMR (DMSO-d6) δ = 170.43
(CONH), 167.13 (CONH), 162.29 (CH), 148.79 (C), 139.37 (C), 131.30 (C), 131.23 (CH), 131.23 (C),
128.90 (CH), 123.13 (CH), 117.93 (CH), 116.59 (CH), 114.41 (CH), 45.01 (CH2), 33.35 (CH2),
27.83 (CH2).
1
N-(2-Aminopyrimidin-5-yl)-3-(4-chlorobutanamido)benzamide (B2). Yield: 0.047 g (34%). H-NMR
(DMSO-d6) δ = 10.09 (s,1H, NH), 7.21 (t,1H, NH), 6.82–8.35 (m, 6H, Ar-H), 3.30–4.10 (bs, 2H, NH2),
3.75 (t, 2H, CH2), 2.35 (t, 2H, CH2); 1.88–1.98 (m, 2H, CH2). 13C-NMR (DMSO-d6) δ = 171.38
(CONH), 167.79 (CONH), 159.88 (CH), 157. 96 (C), 139.72(C), 136.64 (CH), 132.24 (C), 129.38 (C),
129.18 (CH), 119.81 (CH), 117.12 (CH), 114.81 (CH), 59.85 (CH2), 42.23 (CH3), 33.37 (CH2),
27.86 (CH2).
1
N-(2-Aminothiazol-5-yl)-3-(4-chlorobutanamido)benzamide (C2). Yield: 0.032 g (23%). H-NMR
(DMSO-d6) δ = 10.09 (s,1H, NH), 7.41 (t,1H, NH), 6.87–8.81 (m, 5H, Ar-H), 3.20–3.70 (bs, 2H,
NH2), 3.80 (t, 2H, CH2), 2.35 (t, 2H, CH2); 1.85–1.96 (m, 2H, CH2). 13C-NMR (DMSO-d6) δ = 171.44
(CONH), 167.66 (CONH), 167.13 (C), 139.37 (C), 134.58 (C), 131.53 (C), 129.16 (CH), 120.04 (CH),
119.29 (CH), 115.34 (CH), 115.32(CH), 44.95 (CH2), 33.34 (CH2), 27.83 (CH2).
N-(3-Aminophenyl)-3-(4-chlorobutanamido)benzamide (D2). Yield: 0.042 g (31%). 1H-NMR (DMSO-d6)
δ = 10.06 (s,1H, NH), 7.42 (t,1H, NH), 6.76–8.31 (m, 8H, Ar-H), 4.34 (s, 2H, NH2), 3.75 (t, 2H, CH2),
2.36 (t, 2H, CH2); 1.81–1.85 (m, 2H, CH2). 13C-NMR (DMSO-d6) δ = 174.35 (CONH), 167.78
(CONH), 167.12 (C), 149.90 (C), 139.72(C), 134.58 (CH), 131.31 (C), 129.61 (CH), 128.88 (CH),
123.36 (CH), 119.71 (CH), 118.09 (CH), 117.02 (CH), 115.64 (CH), 44.93 (CH2), 33.34 (CH2),
27.82 (CH2).
3.2.3. Library 3
Bromoacrylic acid chloride (3) was used to carry out the acylation reaction of resins V.
2-Bromoacrylic acid (2 mmol; 0.302 g) was dissolved in dry THF (20 mL), then oxalyl chloride (4 mmol;
0.508 g; 0.343 mL) was added, and warming to mild reflux under a drying tube for 2 h. The excess
oxalyl chloride and solvent were removed under reduced pressure and the residue co-evaporated with
dry DCM (5 mL, twice). The above acid chloride was dissolved in DCM (20 mL), chilled to 0 °C and
such portions were added to chilled resins V with DMAP (0.0025 g; 0.0205 mmol). The mixtures were
stirred for 2 h in 0 °C and overnight at room temperature.
1
(E)-N-(6-Aminopyridin-3-yl)-3-(3-bromoacrylamido)benzamide (A3). Yield: 0.044 g (30%). H-NMR
(DMSO-d6) δ = 10.35 (s,1H, NH), 9.65 (s,1H, NH), 6.78–8.31 (m, 7H, Ar-H), 7.80 (d, 1H, CH), 7.65
(d, 1H, CH), 3.25–3.65 (bs, 2H, NH2). 13C-NMR (DMSO-d6) δ = 167.71 (CONH), 162.26 (CH),
160.74 (CONH), 148.79 (C), 139.37(C), 131.30 (C), 131.23 (C), 129.10 (CH), 128.86 (CH), 124.36
(CH), 123.20 (CH), 118.34 (CH), 117.93 (CH), 116.59 (CH), 114.41 (CH).

Molecules 2014, 19
11310
(E)-N-(2-Aminopyrimidin-5-yl)-3-(3-bromoacrylamido)benzamide (B3). Yield: 0.045 g (30%).
1H-NMR (DMSO-d6) δ = 10.36 (s,1H, NH), 9.66 (s,1H, NH), 6.62–8.31 (m, 6H, Ar-H), 7.80 (d, 1H,
13
CH), 7.65 (d, 1H, CH), 3.20–3.45 (bs, 2H, NH2). C-NMR (DMSO-d6) δ = 167.78 (CONH), 162.26
(CH), 160.74 (CONH), 148.56 (C), 139.72(C), 136.64 (CH), 132.24 (C), 131.30 (C), 129.38 (CH),
128.81 (CH), 119.81 (CH), 118.05 (CH), 116.74 (CH), 114.81 (CH).
1
(E)-N-(2-Aminothiazol-5-yl)-3-(3-bromoacrylamido)benzamide (C3). Yield: 0.042 g (28%). H-NMR
(DMSO-d6) δ = 10.87 (s,1H, NH), 9.66 (t,1H, NH), 6.80–8.31 (m, 5H, Ar-H), 7.80 (d, 1H, CH), 7.64
(d, 1H, CH), 3.30–3.70 (bs, 2H, NH2). 13C-NMR (DMSO-d6) δ = 167.75 (CONH), 162.28 (CH),
160.76 (CONH), 148.11 (C), 139.37 (C), 134.58 (C), 131.34 (C), 128.87 (CH), 120.04 (CH), 119.29
(CH), 118.33 (CH), 117.09 (CH), 114.79(CH).
1
(E)-N-(3-Aminophenyl)-3-(3-bromoacrylamido)benzamide (D3). Yield: 0.041 g (28%). H-NMR
(DMSO-d6) δ = 10.10 (s,1H, NH), 9.12 (s,1H, NH), 6.76–8.31 (m, 8H, Ar-H), 7.80 (d, 1H, CH), 7.64
(d, 1H, CH), 3.30–3.50 (bs, 2H, NH2). 13C-NMR (DMSO-d6) δ = 167.80 (CONH), 162.30 (CH),
160.77 (CONH), 149.80 (C), 132.72 (C), 132.58 (CH), 131.32 (2C), 129.61 (CH), 128.87 (CH),
123.36 (CH), 119.71 (CH), 118.18 (CH), 116.90 (CH), 115.604 (CH), 114.64 (CH).
3.2.4. Library 4
Chlorambucil chloride (4) was used to carry out the acylation reaction of resins V. Chlorambucil
(2 mmol; 0.608 g) was dissolved in dry THF (20 mL), then oxalyl chloride (4 mmol; 0.508 g; 0.343 mL)
was added, and warming to mild reflux under a drying tube for 2 h. The excess oxalyl chloride and
solvent were removed under reduced pressure and the residue co-evaporated with dry DCM (5 mL,
twice). The above acid chloride was dissolved in DCM (20 mL), chilled to 0 °C and such portions
were added to chilled resins V with DMAP (0.0025 g; 0.0205 mmol). The mixture were stirred for 2 h in
0 °C and overnight at room temperature.
N-(6-Aminopyridin-3-yl)-3-(4-(4-(bis(2-chloroethyl)amino)phenyl)butanamido)benzamide (A4). Yield:
0.060 g (28%). 1H-NMR (DMSO-d6) δ = 10.08 (s,1H, NH), 7.40 (t,1H, NH), 6.65–8.42 (m, 11H, Ar-H),
3.70–4.30 (bs, 2H, NH2), 3.69 (t, 8H, CH2), 2.47 (t, 2H, CH2), 2.17 (t, 2H, CH2), 1.65–1.88 (m, 2H,
13
CH2). C-NMR (DMSO-d6) δ = 174.31 (CONH), 167.17 (CONH), 161.89 (CH), 157.10 (C), 144.46
(C), 139.52 (C), 137.91 (C), 131.35 (C), 131.23 (CH), 131.20 (C), 129.54 (CH), 129.28 (CH), 129.00
(CH), 128.84 (CH), 125.32 (C), 124.53 (C), 123.13 (CH), 123.09 (CH), 121.08 (CH), 119.81 (CH),
111.97 (CH), 52.24 (2CH2), 41.17 (2CH2), 33.60 (CH2), 33.08 (CH2), 26.57 (CH2).
N-(2-Aminopyrimidin-5-yl)-3-(4-(4-(bis(2-chloroethyl)amino)phenyl)butanamido)benzamide (B4). Yield:
0.056 g (26%). 1H-NMR (DMSO-d6) δ = 10.09 (s,1H, NH), 7.30 (t,1H, NH), 6.65–8.43 (m, 10H, Ar-H),
3.30–4.10 (bs, 2H, NH2), 3.70 (t, 8H, CH2), 2.47 (t, 2H, CH2), 2.19 (t, 2H, CH2), 1.64–1.80 (m, 2H, CH2).
13C-NMR (DMSO-d6) δ = 174.26 (CONH), 167.14 (CONH), 161.87 (CH), 157. 08 (C), 144.44 (C),
139.72 (C), 137.89 (C), 132.24 (C), 131.18 (C), 129.52 (CH), 129.26 (CH), 128.82 (CH), 125.29 (CH),
124.51 (CH), 123.11 (CH), 121.07 (CH), 119.79 (CH), 111.95 (CH), 51.99 (2CH2), 40.88 (2CH2),
33.58 (CH2), 33.06 (CH2), 26.54 (CH2).

Molecules 2014, 19
11311
N-(2-Aminothiazol-5-yl)-3-(4-(4-(bis(2-chloroethyl)amino)phenyl)butanamido)benzamide (C4). Yield:
1
0.061 g (29%). H-NMR (DMSO-d6) δ = 10.08 (s,1H, NH), 7.40 (t,1H, NH), 6.65–8.83 (m, 9H, Ar-H),
3.20–3.80 (bs, 2H, NH2), 3.65 (t, 2H, CH2), 2.45 (t, 2H, CH2), 2.18 (t, 2H, CH2), 1.65–1.80 (m, 2H, CH2).
13C-NMR (DMSO-d6) δ = 171.44 (CONH), 171.26 (CONH), 167.16 (C), 162.30 (C), 144.45 (C),
139.37 (C), 134.58 (C), 131.53 (C), 129.69 (CH), 129.16 (CH), 128.93 (CH), 128.84 (CH), 120.04
(CH), 119.29 (CH), 111.94 (2CH), 111.66 (CH), 52.23 (2CH2), 40.94 (2CH2), 33.33 (CH2), 33.07 (CH2),
26.56 (CH2).
N-(3-Aminophenyl)-3-(4-(4-(bis(2-chloroethyl)amino)phenyl)butanamido)benzamide (D4). Yield:
0.059 g (28%). 1H-NMR (DMSO-d6) δ = 10.06 (s,1H, NH), 7.42 (t,1H, NH), 6.64–8.24 (m, 12H, Ar-H),
4.20–4.90 (bs, 2H, NH2), 3.69 (t, 2H, CH2), 2.35 (t, 2H, CH2); 1.64–1.85 (m, 2H, CH2). 13C-NMR
(DMSO-d6) δ = 174.32 (CONH), 171.32 (CONH), 162.30 (CH), 158.42 (C), 157.74 (C), 145.21 (C),
144.46 (C), 139.72 (C), 131.20 (C), 129.77 (CH), 129.61 (CH), 129.11 (CH), 128.86 (CH), 123.72
(CH), 123.12 (CH), 119.79 (CH), 119.16 (CH), 113.30 (CH), 111.93 (CH), 111.54 (CH), 52.22
(2CH2), 39.52 (2CH2), 33.34 (CH2), 33.07 (CH2), 26.57 (CH2).
3.2.5. Library 5
Bis-netropsin derivatives in library 5 were prepared by direct acylation of resins V by adipoyl
chloride (5, 2 mmol; 0.366 g; 0.293 mL). The above acid chloride was dissolved in DCM (20 mL),
chilled to 0 °C and this solution was added to chilled resins V with DMAP (0.0025 g; 0.0205 mmol).
The mixtures were stirred for 2 h in 0 °C and overnight at room temperature.
1
N1,N6-bis(3-((6-Aminopyridin-3-yl)carbamoyl)phenyl)adipamide (A5). Yield: 0.042 g (36%). H-NMR
(DMSO-d6) δ = 10.08 (s, 2H, NH), 7.62 (t, 2H, NH), 6.71–8.06 (m, 14H, Ar-H), 5.20–5.50 (bs, 4H,
NH2), 2.37 (t, 4H, CH2); 1.66 (t, 4H, CH2). 13C-NMR (DMSO-d6) δ = 174.32 (2CONH), 171.29
(2CONH), 164.99 (2C), 153.67 (2C), 139.38(2C), 135.93 (2CH), 130.69 (2C), 128.58 (2CH), 122.18
(2CH), 121.72 (CH), 120.84 (2CH), 118.49 (2CH), 115.15 (2CH), 114.95 (CH), 36.25 (2CH2),
24.83 (2CH2).
N1,N6-bis(3-((2-Aminopyrimidin-5-yl)carbamoyl)phenyl)adipamide (B5). Yield: 0.047 g (40%).
1H-NMR (DMSO-d6) δ = 10.09 (s, 2H, NH), 7,60 (t, 2H, NH), 6.71–8.15 (m, 12H, Ar-H), 4.10–5.00
(bs, 4H, NH2), 2.37 (t, 4H, CH2); 1.66 (t, 4H, CH2). 13C-NMR (DMSO-d6) δ = 174.32 (2CONH),
171.29 (2CONH), 164.99 (2C), 153.676 (2C), 139.37 (2C), 135.93 (CH), 130.68 (2C), 128.59 (2CH),
122.17 (2CH), 121.73 (2CH), 118.49 (2CH), 115.14 (2CH), 36.26 (2CH2), 24.83 (2CH2).
1
N1,N6-bis(3-((2-Aminothiazol-4-yl)carbamoyl)phenyl)adipamide (C5). Yield: 0.034 g (29%). H-NMR
(DMSO-d6) δ = 10.08 (s, 2H, NH), 7.60 (t, 2H, NH), 6.81–8.22 (m, 10H, Ar-H), 3.30–3.90 (bs, 4H,
NH2), 2.36 (t, 4H, CH2); 1.51 (t, 4H, CH2). 13C-NMR (DMSO-d6) δ = 174.30 (2CONH), 171.30
(2CONH), 167.16 (2C), 162.30 (2C), 139.47 (2C), 131.53 (2C), 128.88 (2CH), 123.11 (2CH), 119.79
(2CH), 115.82 (2CH), 115.63 (2CH), 36.23 (2CH2), 24.75 (2CH2).
1
N1,N6-bis(3-((4-Aminophenyl)carbamoyl)phenyl)adipamide (D5). Yield: 0.048 g (41%). H-NMR
(DMSO-d6) δ = 10.08 (s, 2H, NH), 7.60 (t, 2H, NH), 6.71–8.05 (m, 16H, Ar-H), 3.90–4.50 (bs, 4H,

Molecules 2014, 19
11312
NH2), 2.37 (t, 4H, CH2); 1.65 (t, 4H, CH2). 13C-NMR (DMSO-d6) δ = 174.33 (2CONH), 171.29
(2CONH), 164.98 (2C), 153.66 (2C), 139.38 (2C), 135.93 (2CH), 130.68 (2C), 128.59 (2CH), 122.17
(2CH), 121.73 (2CH), 120.83 (2CH), 120.75 (2CH), 118.49 (2CH), 114.95 (2CH), 36.26 (2CH2),
24.82 (2CH2).
3.2.6. Library 6
Derivatives in library 6 were obtained using pimeloyl chloride (6, 2 mmol; 0.394 g; 0.327 mL), by
direct acylation of resin V. The above acid chloride was dissolved in DCM (20 mL), chilled to 0 °C
and then added to a chilled resin V with DMAP (0.0025 g; 0.0205 mmol). The mixtures were stirred
for 2 h in 0 °C and overnight at room temperature.
N1,N7-bis(3-((6-Aminopyridin-3-yl)carbamoyl)phenyl)heptanediamide (A6). Yield: 0.045 g (38%). 1H-
NMR (DMSO-d6) δ = 10.08 (s, 2H, NH), 7.95 (t, 2H, NH), 6.97–8.23 (m, 14H, Ar-H), 3.10–3.80 (bs,
4H, NH2), 2.33 (t, 4H, CH2); 1.48–1.56 (m, 4H, CH2), 1.15–1.45 (m, 2H, CH2). 13C-NMR (DMSO-d6)
δ = 174.41 (2CONH), 171.46 (2CONH), 167.20 (2C), 156.96 (2C), 139.53 (2C), 139.13 (2CH), 131.24
(2C), 128.88 (2CH), 123.75 (2CH), 123.10 (4CH), 119.79 (2CH), 106.95 (2CH), 36.26 (CH2), 33.56
(CH2), 28.12 (CH2), 24.83 (CH2), 24.23 (CH2).
N1,N7-bis(3-((2-Aminopyrimidin-5-yl)carbamoyl)phenyl)heptanediamide (B6). Yield: 0.056 g (47%).
1H-NMR (DMSO-d6) δ = 10.07 (s, 2H, NH), 7,60 (t, 2H, NH), 6.95–8.23 (m, 12H, Ar-H), 3.10–3.70
(bs, 4H, NH2), 2.34 (t, 4H, CH2); 1.38–1.66 (m, 4H, CH2), 1.15–1.40 (m, 2H, CH2). 13C-NMR
(DMSO-d6) δ = 174.40 (2CONH), 171.46 (2CONH), 167.19 (2C), 156.97 (2C), 139.37 (2C), 139.14
(2CH), 131.25 (2C), 128.88 (2CH), 123.75 (2CH), 123.11 (2CH), 119.80 (2CH), 106.96 (2CH), 36.26
(CH2), 33.56 (CH2), 28.11 (CH2), 24.80 (CH2), 24.23 (CH2).
N1,N7-bis(3-((2-Aminothiazol-4-yl)carbamoyl)phenyl)heptanediamide (C6). Yield: 0.052 g (43%).
1H-NMR (DMSO-d6) δ = 10.10 (s, 2H, NH), 7.60 (t, 2H, NH), 6.95–8.23 (m, 10H, Ar-H), 3.10–3.20
(bs, 4H, NH2), 2.33 (t, 4H, CH2); 1.40–1.70 (m, 4H, CH2), 1.15–1.40 (m, 2H, CH2). 13C-NMR
(DMSO-d6) δ = 174.44 (2CONH), 171.52 (2CONH), 167.24 (2C), 157.01 (2C), 139.59 (2C), 139.17
(2CH), 131.59 (2C), 128.88 (2CH), 123.14 (2CH), 119.85 (2CH), 115.68 (2CH), 106.99 (2CH), 36.30
(CH2), 33.61 (CH2), 28.16 (CH2), 24.85 (CH2), 24.28 (CH2).
N1,N7-bis(3-((4-Aminophenyl)carbamoyl)phenyl)heptanediamide (D6). Yield: 0.044 g (37%). 1H-NMR
(DMSO-d6) δ = 10.11 (s, 2H, NH), 7.40 (t, 2H, NH), 6.55–8.23 (m, 16H, Ar-H), 3.00–3.50 (bs, 4H,
NH2), 2.34 (t, 4H, CH2); 1.40–1.70 (m, 4H, CH2), 1.15–1.40 (m, 2H, CH2). 13C-NMR (DMSO-d6)
δ = 174.38 (2CONH), 171.46 (2CONH), 167.18 (2C), 156.94 (2C), 149.73 (2C), 139.53 (2CH), 139.07
(2CH), 131.22 (2C), 128.86 (2CH), 123.72 (2CH), 123.09 (2CH), 119.77 (2CH), 115.63 (2CH),
106.95 (2CH), 36.23 (CH2), 33.55 (CH2), 28.17 (CH2), 24.79 (CH2), 24.22 (CH2).

Molecules 2014, 19
11313
3.3. Pharmacology
3.3.1. Cell Culture
Human breast cancer MCF-7 cells maintained in Dulbecco’s modified Eagle’s medium
supplemented with 10% FBS, 50 μg/mL streptomycin, 100 U/mL penicillin at 37 °C, atmosphere
containing 5% CO2. Cells were cultivated in Costar flasks and subconfluent cells were detached with
0.05% trypsin and 0.02% EDTA in calcium-free phosphate-buffered saline. The study was carried out
using cells growing as monolayer in six-well plates (Nunc, Sarstedt, Newton, NC, USA) (5 × 105 cells
per well) and preincubated 24 h without phenol red.
3.3.2. Determination of IC50 Values
The compounds were dissolved in DMSO/H2O (10:90) and used at concentrations of 5, 10, 15, 50
and 100 μM. Microscopic observations of cells monolayers were performed with a Nikon optiphot
microscope (Nikon Instruments Inc., Melville, NY, USA). Wright-Giemsa staining was performed
using the Fischer Leuko Stat Kit (Fisher Diagnostics, Orangeburg, NY, USA). After 24 h of drug
treatment MCF-7 cells were mixed with a dye mixture (10 μM acridine orange and 10 μM ethidium
bromide, prepared in phosphate-buffered saline). At the end of each experimental time point, all of the
media was removed and cells were harvested by incubation with 0.05% trypsin and 0.02% EDTA for 1
min and washed with the medium. Then, 250 μL of cells suspension was mixed with 10 μL of the dye
mix and 200 cells per sample were examined by fluorescence microscopy. There were counted
percentage of non-viable (apoptotic and necrotic) cells and the concentrations which inhibited 50% of
colony formation (IC50 values) were counted. The results were submitted to statistical analysis using
the method of the smallest squares.
3.3.3. Ethidium Bromide Assay
Each well of 96-well plate was loaded with Tris buffer containing ethidium bromide (0.1 M Tris,
1 M NaCl, pH 8.0, 0.5 mM EtBr final concentration, 100 μL). To each well was added 15 μg plasmid
pBR322 as water solution (0.05 μg/μL). Then to each well was added netropsin or compound 1–24 (1 μL
of a 1 mM solution in water, 10 μM final concentrations). After the incubation at 25 °C for 30 min.
the. fluorescence of each well was read on a Infinite M200 fluorescence spectrophotometer (TECAN,
Männedorf, Switzerland) (ex. 546 nm, em. 595 nm) in duplicate experiments with two control wells
(no drug = 100% fluorescence, no DNA = 0% fluorescence). Fluorescence readings are reported as %
fluorescence relative to the controls.
4. Conclusions
The synthesis and testing of minor groove binders analogues are the subject of constant active
research. A short and efficient synthesis of 24 different netropsin analogues was developed. The
presented compounds have linear chains like the natural antibiotic netropsin based on readily available
building blocks, such as: pyridine, pyrimidine, thiazole and benzene rings. The replacement of
N-methylpyrrole rings in netropsin by other rings yields lexitropsins with changed affinity to A-T

Molecules 2014, 19
11314
pairs, in comparison to netropsin [11]. The introduction of the terminal 4-(dimethylamino)butanamido-
fragment leads to netropsin-like compounds. Some of the new compounds (libraries 2, 3 and 4) have
different connected alkylating groups. Compounds from libraries 5 and 6 are analogues of
bis-netropsin. This protocol can be no doubt very useful in terms of speed, materials availability and
generality, as well, as may provide compounds with better properties.
The antiproliferative activity of the new compounds was determined and it was confirmed that the
obtained netropsin analogues act against MCF-7 breast cancer cells, though all of them are weaker
than netropsin. The ethidium displacement assay demonstrated the DNA-binding properties of the
newly obtained analogues of netropsin and bis-netropsin. Five among the obtained derivatives showed
stronger binding ability to DNA than the used model compound. Further investigations and testing are
required to confirm whether the newly obtained compounds bind to DNA in the minor groove or in
any other way.
Acknowledgments
This studies were supported by the grant N N405 355537 donated by M.S.H.E.
Author Contributions
JS and DD designed research. JS performed research and analyzed the data: NMR, DNA-binding
activity and IC50. JS and DD wrote the paper. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Cory, M. DNA Minor-Groove Binding Compounds as Antitumor Agents. In Cancer
Chemotherapeutic Agents, 1st ed.; Foye, W.O., Ed.; American Chemical Society: Washington,
DC, USA, 1995, pp. 311–344.
2. Bailly, C.; Chaires, J.B. Sequence-specific DNA minor groove binders. Design and synthesis of
netropsin and distamycin analouges. Bioconjugate Chem. 1998, 9, 513–538.
3. Baraldi, P.G.; Bovero, A.; Fruttarolo, F.; Preti, D.; Tabrizi, M.A.; Pavani, M.G.; Romagnoli, R.
DNA minor groove binders as potential antitumor and antimicrobial agents. Med. Res. Rev. 2004.
24, 475–528.
4. Khan, G.S.; Shah, A.; Rehman, Z.; Barker, D. Chemistry of DNA minor groove binding agents.
J. Photochem. Photobiol. B 2012, 115, 105–118.
5. Barrett, M.P.; Gemmel, C.G.; Suckling, C.J. Minor groove binders as anti-infective agents.
Pharmacol. Ther. 2013, 130, 12–23.
6. Cortesi R.; Esposito, E. Distamycins: Strategies for possible enhancement of activity and
specificity. Mini-Rev. Med. Chem. 2010, 10, 218–231.

Molecules 2014, 19
11315
7. Drozdowska, D. The analogues of DNA minor–groove binders as antineoplastic compounds. In
Breast Cancer—Current and Alternative Therapeutic Modalities, 1st ed.; Gunduz, E., Gunduz, M.,
Eds.; INTECHWEB.ORG: Rijeka, Croatia, 2011; pp. 133–148.
8. Bartulewicz, D.; Markowska, A.; Wołczyński, S.; Dąbrowska, M.; Różański, A. Molecular
modelling, synthesis and antitumour activity of carbocyclic analogues of netropsin and
distamycin—New carriers of alkylating elements. Acta Biochim. Polon. 2000, 47, 23–35.
9. Bartulewicz, D.; Bielawski, K.; Bielawska, A. Carbocyclic Analogues of Netropsin and
Distamycin: DNA-Binding Properties and Inhibition of DNA Topoisomerases. Arch. Pharm.
Pharm. Med. Chem. 2001, 9, 422–426.
10. Bartulewicz, D.; Bielawski, K.; Bielawska, A.; Różański, A. Synthesis, molecular modelling, and
antiproliferative and cytotoxic effects of carbocyclic derivatives of distamycin with chlorambucil
moiety. Eur. J. Med. Chem. 2001, 36, 461–467.
11. Drozdowska, D.; Rusak, M.; Bielawski, T.; Midura-Nowaczek, K. Carbocyclic potential DNA
minor groove binders and their biological evaluation. J. Enz. Inhib. Med. Chem. 2010, 25,
629–634.
12. Drozdowska, D. New solid phase synthesis of distamycin analogues. Molecules 2011, 16, 3066–3076.
Sample Availability: Samples of the compounds 1–24 are available from the authors.
© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).