The usefulness of cyclic diamidines with different core-substituents as antitumor agents

Article abstract of DOI:10.1016/j.bioorg.2008.05.002

A series of related polycationic compounds has been screened for potential antitumor activity by the NCI's in vitro testing (one dose primary anticancer assay and the NCI-60 full panel screening). The GI₅₀ values of triazines 3 and 4 are on average 1.9 μM and 2.4 μM, respectively. Furan 8 deserves mention too (1.9 μM). The biological test results showed that carbazole 10 possessed cytotoxic activity in the nanomolar range, much better than the other compounds tested, only against several cancer cell lines: CCRF-CEM, HL-60(TB), MOLT-4, NCI-H522, COLO 205, SF-268, but the average GI₅₀ value was higher (15 μM). The activity appears closely dependent on the core-shape and length of the bisimidazoline molecules (important for both high cytotoxicity and DNA binding). The mechanism of DNA minor-groove binding of diamidines 1-12, based on the anticancer parameters, is highly probable.

Full text of DOI:10.1016/j.bioorg.2008.05.002

Bioorganic Chemistry 36 (2008) 183–189
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
The usefulness of cyclic diamidines with different core-substituents
as antitumor agents
*
Jarosław Spychała
´
Department of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 February 2008
Available online 20 June 2008
A series of related polycationic compounds has been screened for potential antitumor activity by the
NCI’s in vitro testing (one dose primary anticancer assay and the NCI-60 full panel screening). The GI50
values of triazines 3 and 4 are on average 1.9
lM and 2.4 lM, respectively. Furan 8 deserves mention
too (1.9 M). The biological test results showed that carbazole 10 possessed cytotoxic activity in the
nanomolar range, much better than the other compounds tested, only against several cancer cell lines:
CCRF-CEM, HL-60(TB), MOLT-4, NCI-H522, COLO 205, SF-268, but the average GI50 value was higher
(15 lM). The activity appears closely dependent on the core-shape and length of the bisimidazoline mol-
ecules (important for both high cytotoxicity and DNA binding). The mechanism of DNA minor-groove
binding of diamidines 1–12, based on the anticancer parameters, is highly probable.
Ó 2008 Elsevier Inc. All rights reserved.
l
Keywords:
Antitumor
Carbazoles
Fluorenes
Furans
Imidazolines
Piperidines
Pyrenes
Tetrahydropyrimidines
Triazines
Xylenes
1. Introduction
cationic groups lie between phosphate groups on each strand of
the duplex [24,25]. The complex results in the inhibition of the
Anticancer drugs have been classified as antitumor antibiotics,
antimetabolites, antitubulin agents, topoisomerase inhibitors, and
alkylating agents [1–5]. They often exhibit multiple mechanisms
of action in tumor cells. During the past few decades, simple and
cyclic amidines and their derivatives have been widely tested as
antitumor agents in vitro and in vivo treatment [6–23]. Other
exemplary compounds, containing the amidine N@C–N unit (e.g.,
pyrimidines, purines, triazines, guanidines, ureas), are usually con-
sidered separately.
microbial topoisomerase II enzyme. Other findings of a molecular
modeling study are in agreement with the crystal structure de-
scribed [26].
Additional bifurcated hydrogen bonding interactions between
the indole NH hydrogen and the thymine O2-carbonyl groups over-
stabilize the B-form helix and increase the
DTm values of the DAPI-
DNA complex [27]. The complexation can occur in vivo and it
causes inhibition of DNA synthesis at therapeutic levels in humans,
in treatment of far advanced cancer cases.
From a general structure-activity standpoint, the above groups of
amidinesdemonstratethatthestructuralvariationscanresultinsig-
nificant changes in specificity and potency with regard to anticancer
activity. The active bisamidines increase the denaturation tempera-
ture of the calf thymus DNA (stimulate topoisomerase II—mediated
DNA cleavage). A general strategy for the synthesis of drugs having a
specific activity has been elaborated [20,24].
Hydrogen bond formation is involved in many biological pro-
cesses. A model has been described for the biological activity
against Giardia lamblia in which only one nitrogen atom of both
amidine groups is in hydrogen bonding distance to the thymine
O2 atom at AT rich sites of the minor-groove of DNA and the two
The amidinium moiety is known to contribute to the stabiliza-
tion of DNA recognition element through electrostatic and hydro-
gen bonding interactions [28–32]. Therefore, hydrogen bonds are
frequently used as recognition elements due to their directionality.
The strategy approach for the development of new drugs for the
treatment of diseases, which are induced by RNA viruses, is based
on the design of agents which bind to specific stem or loop units in
the viral RNA genome [33,34].
The binding affinities and specificities observed suggest that the
incorporation of a variety of moieties will lead to substances that
interact with RNA targets. This approach greatly expands the util-
ity of the amidines and related polycationic compounds for the
construction of drugs in general. Hydrogen bonding is an attractive
approach to the biological activity [35]. If there is the right size of a
drug, the hydrogen bonds become stronger.
*
Fax: +48 618658008.
E-mail address: jjspychala@wp.pl
0045-2068/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2008.05.002

184
J. Spychała / Bioorganic Chemistry 36 (2008) 183–189
It has been shown that various bisamidines can be effective for
design strong DNA binders by means of various anticancer
parameters.
opportunistic infections and as antifungal, antileishmanial, anti-
plasmodial, antiprotozoal, and antitrypanosomal agents [36–43].
The structures, which possess adequate proportions of interactions
among DNA [44] (e.g., Berenil, DAPI, Furamidine DB75, Hoechst
33258, Pentamidine), are mainly related to several antibiotics
[45,46] (Amidinomycin, Anthelvencin A and B, Congocidin, Dista-
mycin A, Kikumycin A and B, Noformycin) and their derivatives
[32,47,48].
The radius of curvature of several defined groups in the mimic
molecule shows that the shape of the latter affects the strength
of nucleic acid binding. Therefore, many classes of drugs have sub-
stantial curvature, high DNA affinity, binding to the minor-groove,
and interference with DNA-associated enzymes. Very often, the
cytotoxicity remains at the micromolar level and confirms the re-
sults provided by the Tm studies [20].
This paper highlights usefulness in medicinal chemistry of the
direct reaction of formation of cyclic amidines and secondary
amides from nitriles. The practical value of this complex reaction
has been demonstrated by preparation of some bisimidazolines
[49] and tetrahydropyrimidines [50]. The reaction is an alternative
method to the existing methodologies based on sulfur containing
reagents [9,51–57].
2. Materials and methods
2.1. Materials
Melting points were uncorrected and determined on a Boetius
melting point apparatus. NMR spectra were recorded on a Varian
Gemini (300 MHz) spectrometer with D2O solutions using TMS as
the internal standard. High-resolution mass spectra were obtained
using an AMD 402 or 602 mass spectrometer in the EI or FAB mode,
respectively. Thin layer chromatography was performed with
Merck silica gel 60 F254 plates (0.25 mm thickness). The reagents
were purchased from Aldrich. Compounds 1–11 are the author
samples in the literature cited [9,24,42,49]. All final compounds
were dried in an oven at 120 °C for several hours and then stored
in a vacuum desiccator over P2O5.
2.2. Representative procedures for the preparation of amidines 12 and
13
Polycationic compounds containing an a,
a0 -xylene linker have
been found to exhibit various inhibitory effects [34,63]. Monocy-
clam and bicyclam have proven to be effective against HIV-1 and
HIV-2 and have been tested in clinical trials [64–66]. They interfere
with nucleic acid binding via non-covalent interactions. All xylene
and toluene derivatives described in this paper were prepared fol-
lowing the synthetic way outlined in Fig. 2.
The syntheses of the cyclic amidines were previously disclosed
by the author [49]. To illustrate the methodology, the synthetic
scheme and experimental procedures for the representative com-
pounds 12 and 13 are included. As starting compounds, the appro-
priate nitriles and diaminoalkanes, saturated with hydrogen
sulfide, have been used for their preparation.
In several papers, the formation of a range of cyclic amidines
from thioamides or imidates has been described [58,59]. Both thio-
amides and nitriles were important synthetic intermediates for the
preparation of simple amidines [60–62]. This paper describes the
practical results obtained from anticancer screening of the prod-
ucts of the cited reactions. The presence of cationic groups and
other core structural features make these compounds very suitable
probes for nucleic acid binding modes. Therefore, it is desirable to
Table 1
Growth percentages of 1–13 (one dose primary anticancer assay)
Compound
NSC Number
NCI-H460 (Lung)
MCF7 (Breast)
SF-268 (CNS)
2.2.1. ( )-N,N0 -Bis[4-(4,5-dihydro-4-methyl-1H-imidazol-2-yl)
1
2
3
4
5
6
7
8
9
10
11
12
13
NSC 710604
NSC 710605
NSC 710607
NSC 710608
NSC 711986
NSC 711987
NSC 711989
NSC 714616
NSC 714620
NSC 715653
NSC 715654
NSC 717052
NSC 717046
12
23
ꢀ90
ꢀ90
20
10
17
ꢀ79
ꢀ91
14
8
30
ꢀ85
ꢀ96
31
benzyl]-4,40 -propylenebispiperidine tetrahydrochloride (12)
Starting from 4,40 -propylenebispiperidine and
a-bromo-p-tol-
unitrile, N,N0 -bis(4-cyanobenzyl)-4,40 -propylenebispiperidine (mp
128 °C) was obtained by refluxing the reaction mixture in metha-
nol in the presence of potassium carbonate [49]. The reaction of
the bisnitrile with the ( )-1,2-diaminopropane-H2S reagent in eth-
anol was carried out under literature conditions [49] and the title
compound was obtained (90%): mp > 30 °C; 1H NMR d 1.24–1.48
(m, 14H), 1.50 (d, 6H, J = 6.3 Hz, CH3), 1.63 (m, 2H), 2.19 (m, 4H),
3.08 (m, 3H), 3.55 (m, 3H), 3.76 (dd, 2H, J = 11.3, 8.0 Hz, imidazo-
line), 4.28 (t, 2H, J = 11.3 Hz, imidazoline), 4.45 (s, 4H, NCH2),
20
7
36
ꢀ74
ꢀ74
ꢀ74
49
ꢀ66
8
14
29
57
ꢀ80
ꢀ48
9
3
ꢀ45
ꢀ49
72
ꢀ33
16
100
98
102
Table 2
Overview of the results of the in vitro antitumor screening for compounds 1–12 scheduled automatically for evaluation against the full panel of about 60 human tumor cell lines
Compound
No. of the cell lines investigated
Number of the cell lines giving log GI50, log TGI, and log LC50 < ꢀ4
ꢀlog GI50
ꢀlog TGI
ꢀlog LC50
No.
Range
No.
Range
No.
Range
1
2
3
4
5
6
7
8
9
58
58
58
58
58
58
58
58
58
57
57
59
43
45
58
58
38
48
57
58
43
50
52
41
4.06–4.75
4.04–4.70
4.78–6.44
4.63–6.07
4.09–4.88
4.00–5.24
4.12–4.86
4.55–5.92
4.01–5.65
4.11–8.00
4.09–5.76
4.07–4.83
15
11
58
58
3
12
49
54
2
4.01–4.41
4.00–4.30
4.45–5.78
4.22–5.66
4.17–4.23
4.01–4.41
4.02–4.53
4.10–5.60
4.02–4.87
4.20–8.00
4.02–4.72
4.04–4.54
2
0
49
52
0
4.06–4.07
<4.00
4.05–5.31
4.06–5.31
<4.00
4.01–4.05
4.01–4.26
4.24–5.30
<4.00
4.04–7.25
4.01–4.21
4.01–4.25
2
23
49
0
3
12
7
10
11
12
5
35
20

J. Spychała / Bioorganic Chemistry 36 (2008) 183–189
185
4.55–4.72 (m, 2H, imidazoline), 7.78 (d, 4H, J = 8.5 Hz), 7.95 (d, 4H,
8.3 Hz); 13C NMR d 22.7, 25.4, 32.0, 35.5, 37.7, 54.3, 55.9, 56.5, 62.6,
126.8, 131.7, 135.0, 138.1, 167.6; LRMS (EI): 554 (24), 382 (25), 381
(86), 174 (100),
propane (61%): mp > 300 °C; 1H NMR d 2.53 (br quintet, 8H,
J = 5.1 Hz, CH2), 4.00 (br t, 16H, J = 5.2 Hz, NCH2), 8.75 (s, 2H,
C2H, C7H), 8.82 (s, 4H, CH); 13C NMR d 20.6, 42.6, 126.7, 128.6,
129.8, 129.9, 133.6, 162.3; HRMS (FAB, NBA): MH+, found
531.2961. C32H35N8 requires 531.2985.
158 (15); HRMS (EI): M+, found 554.4067. C35H50N6 requires
554.4097.
2.3. Determination of growth percentages in the 3-cell line, one dose
primary anticancer assay
2.2.2. 1,3,6,8-Tetrakis(1,4,5,6-tetrahydro-2-pyrimidinyl)pyrene
tetrahydrochloride (13)
This compound was prepared by analogy to the 2-imidazoline
homologues [49] from 1,3,6,8-tetracyanopyrene and 1,3-diamino-
As a primary screening, compounds 1–13 were submitted to the
National Cancer Institute (NCI) cell line screen for evaluation of
Fig. 1. Compounds 1–11, active against most tumor cell lines, may serve as useful lead compounds for the search of more powerful anticancer agents. The presence of
opposite cationic groups in the molecule is essential for cytotoxicity.

186
J. Spychała / Bioorganic Chemistry 36 (2008) 183–189
their anticancer activity [67]. From the data analysis [68] it follows
that approximately 95% of the actives from the 60 cell line screen
can be identified using only three cell lines. Thus a 48 h continuous
drug exposure protocol and a protein-binding dye sulforhodamine
B (SRB) were used to estimate cell growth. In a preliminary test at a
was obtained. Overall, the results show that the presence of amidi-
nium groups is not the unique requirement for the compounds to
induce activity. Inactive tetraamidine 13 led to consider the shape
of molecules responsible for the activity exerted by the
compounds.
single concentration (100
l
M) against three human cell lines (NCI-
Most cell lines were sensitive to the agents. It appeared, as
indicated in Table 2, that weak structural modifications were
responsible for the activity variation. The range between the
least sensitive and most sensitive cell lines was about 1–2 log
units for 3, 4, 6, 8, 9, 10, and 11. The range was smaller, less
than 1 log unit, for the other compounds. Thus, the growth
inhibition results allow classification of the compounds accord-
ing to their activity profiles.
Averaged values, designated as mean graph midpoints
(MG_MID), are calculated for each of the three parameters men-
tioned by averaging the log parameters of all cell lines. All antican-
cer agents possessed three different average potencies at the
parameters pGI50, pTGI, and pLC50, respectively: 1 (4.33, 4.06,
4.00), 2 (4.34, 4.03, 4.00), 3 (5.73, 5.17, 4.54), 4 (5.62, 5.13, 4.59),
5 (4.30, 4.01, 4.00), 6 (4.41, 4.04, 4.00), 7 (4.66, 4.29, 4.05), 8
(5.72, 5.31, 4.73), 9 (4.28, 4.02, 4.00), 10 (4.83, 4.15, 4.11), 11
(4.77, 4.21, 4.02), 12 (4.37, 4.09, 4.01).
Among all compounds, the data for selected members 1, 2, 3, 4,
8, and 10 are summarized in Table 3. The assays of 3 and 4 were
repeated once; other compounds were assayed once. At the pGI50
endpoint isomers 3 and 4 were of approximately equal overall po-
tency; the ranges of the ꢀlog molar concentrations for all cell lines
were from 4.78 to 6.44 for 3 (average 5.73) and 4.63 to 6.07 for 4
(average 5.62), compared to the average potency of 8, which was
5.72, ranging from 4.55 to 5.92.
H460 lung cancer, MCF7 breast cancer, and SF-268 glioma), a com-
pound is considered active when it reduces the growth of any of
the cell lines to 32% or less.
By these criteria, 12 compounds reported were active (six com-
pounds: 3, 4, 7, 8, 10, and 11 with negative numbers indicate cell
kills) and passed on for evaluation in the full panel of 57–59 human
tumor cell lines. Pyrene 13, which bears four tetrahydropyrimidine
groups on the pyrene residue, did not inhibit growth of the tumor
cells. The results of 1–13 are summarized in Table 1.
2.4. Methodology of the NCI-60 in vitro cancer screening
The panel is organized into nine subpanels representing diverse
histologies: leukemia, melanoma, and cancers of lung, colon, kid-
ney, ovary, breast, prostate, and central nervous system [68]. The
test compounds 1–12 were dissolved in DMSO and evaluated using
five concentrations at 10-fold dilutions, the highest being 10ꢀ4
M
and the others being 10ꢀ5, 10ꢀ6, 10ꢀ7, and 10ꢀ8 M. Table 2 reports
the results obtained with this test expressed as the ꢀlog of the mo-
lar concentration that inhibited the cell growth by 50% (pGI50), that
caused total cytostasis (pTGI, total growth inhibition), or that killed
half of the cells (pLC50) when compared with values of untreated
control cells. For the calculation of the MG_MID values, insensitive
cell lines are included with the highest test concentration.
For the most active compounds, there were considerable differ-
ences between log GI50 and log LC50 values (MG_MID parameters).
At the pTGI endpoint the average log molar potencies were very
close to the pLC50 in most cases. The ranges at the pTGI between
the least sensitive and the most sensitive cell lines were tighter
than the ranges seen at the pGI50, though the overall patterns
showed less difference among the cell lines of the same tumor, ex-
cept for 10 (data shown in Tables 2–3).
3. Results and discussion
A current trend in chemical modifications has appending the
examples that have already been tested. Efforts were directed to-
wards extending the approach to the synthesis of other biologically
interesting antibiotic analogues. Molecules used in the present as-
say and their numbering are given in Figs. 1–2. Since it has been
well known that amidines in general exhibit biological and phar-
macological activities, it was interesting to investigate the antican-
cer properties of some polycationic molecules and to compare their
activities with the relevant literature data.
The average TGI values for triazines 3 and 4 were 6.8
M, respectively. The hexahydrobenzimidazolyl to tetrahydro-
pyrimidinyl change in 8 (4.9 M) to 9 (95.5 M) causes such a large
increase in the TGI. The Tm data from biophysical studies with
DNA and 8 ( Tm 24.5 °C) or 9 ( Tm > 28 °C) [24] show that these
lM and
7.4
l
l
l
D
D
D
Consequently, the parent structures are altered by varying the
core-substituents (shape of the molecules). On the basis of the
growth inhibition parameters, a structure-activity relationship
compounds exhibit dissimilar binding affinities. It is reflected in
their different dose response curves (Fig. 3). These results support
Fig. 2. Representative syntheses of 12 and 13. Reagents: (A) K2CO3, MeOH; (B) H2NCH2CH(CH3)NH2-H2S, EtOH; (C) Br2, CCl4; (D) Cu2(CN)2, quinoline; (E) H2N(CH2)3NH2-H2S.

J. Spychała / Bioorganic Chemistry 36 (2008) 183–189
187
Table 3
Inhibition of in vitro cancer lines by active compounds 1, 2, 3, 4, 8, and 10 (ꢀlog GI50 > 4.00 for active compounds)
Panel Cell lines (cytotoxicity: ꢀlog GI50 > 4.00)
2,7-Bis(4,5-dihydro-4-methyl-1H-imidazol-2-yl)fluorene dihydrochloride (1)
Leukemia
CCRF-CEM (<4.00), K-562 (4.46), MOLT-4 (4.42), RPMI-8226 (4.44), SR (4.37)
Non-small cell lung cancer
A549/ATCC (<4.00), EKVX (<4.00), HOP-62 (4.39), HOP-92 (<4.00), NCI-H226 (4.25), NCI-H23 (4.43), NCI-H322M (4.63), NCI-H460 (4.58), NCI-
H522 (4.65)
Colon cancer
CNS cancer
COLO 205 (4.27), HCC-2998 (4.74), HCT-116 (4.50), HCT-15 (<4.00), HT29 (4.21), KM12 (4.69), SW-620 (4.25)
SF-268 (4.58), SF-295 (4.14), SNB-19 (4.44), SNB-75 (4.27), U251 (4.71)
Melanoma
LOX IMVI (4.33), MALME-3M (4.72), M14 (4.40), SK-MEL-2 (4.63), SK-MEL-28 (4.06), SK-MEL-5 (4.14), UACC-257 (4.49), UACC-62 (4.54)
IGROV1 (<4.00), OVCAR-3 (4.68), OVCAR-4 (4.72), OVCAR-5 (<4.00), OVCAR-8 (4.43), SK-OV-3 (4.42)
786-0 (4.26), A498 (<4.00), ACHN (<4.00), CAKI-1 (<4.00), RXF 393 (<4.00), SN12C (4.32), TK-10 (<4.00), UO-31 (<4.00)
PC-3 (4.36), DU-145 (4.18)
MCF7 (4.75), NCI/ADR-RES (<4.00), MDA-MB-231/ATCC (4.47), HS 578T (4.42), MDA-MB-435 (4.59), MDA-N (4.46), BT-549 (<4.00), T-47D
(4.45)
Ovarin cancer
Renal cancer
Prostate cancer
Breast cancer
2,7-Bis(4,5-dihydro-4,4-dimethyl-1H-imidazol-2-yl)fluorene dihydrochloride (2)
Leukemia
CCRF-CEM (4.32), K-562 (4.61), MOLT-4 (4.50), RPMI-8226 (4.60), SR (4.49)
Non-small cell lung cancer
A549/ATCC (<4.00), EKVX (4.22), HOP-62 (4.47), HOP-92 (4.04), NCI-H226 (4.31), NCI-H23 (4.45), NCI-H322M (4.60), NCI-H460 (4.51), NCI-
H522 (4.52)
Colon cancer
CNS cancer
COLO 205 (4.55), HCC-2998 (4.58), HCT-116 (4.48), HCT-15 (<4.00), HT29 (4.49), KM12 (4.55), SW-620 (4.46)
SF-268 (4.46), SF-295 (<4.00), SNB-19 (4.45), SNB-75 (4.04), U251 (4.66)
Melanoma
LOX IMVI (4.43), MALME-3M (4.61), M14 (4.35), SK-MEL-2 (4.70), SK-MEL-28 (4.41), SK-MEL-5 (4.22), UACC-257 (4.51), UACC-62 (4.60)
IGROV1 (<4.00), OVCAR-3 (4.45), OVCAR-4 (4.48), OVCAR-5 (<4.00), OVCAR-8 (4.43), SK-OV-3 (4.35)
786-0 (4.30), A498 (4.11), ACHN (<4.00), CAKI-1 (<4.00), RXF 393 (<4.00), SN12C (4.44), TK-10 (<4.00), UO-31 (<4.00)
PC-3 (4.40), DU-145 (<4.00)
MCF7 (4.63), NCI/ADR-RES (<4.00), MDA-MB-231/ATCC (4.41), HS 578T (4.19), MDA-MB-435 (4.48), MDA-N (4.40), BT-549 (<4.00), T-47D
(4.21)
Ovarin cancer
Renal cancer
Prostate cancer
Breast cancer
4,6-Bis [3-(4,5-dihydro-4-methyl-1H-imidazol-2-yl)phenyl]-2-dimethylamino-1,3,5-triazine tetrahydrochloride (3)
Leukemia
CCRF-CEM (6.33), HL-60(TB) (5.69), K-562 (6.21), MOLT-4 (6.44), RPMI-8226 (5.55)
Non-small cell lung cancer
Colon cancer
CNS cancer
A549/ATCC (5.53), EKVX (5.56), HOP-62 (5.92), HOP-92 (5.63), NCI-H23 (5.60), NCI-H322M (5.98), NCI-H460 (5.69), NCI-H522 (5.97)
COLO 205 (5.90), HCC-2998 (5.94), HCT-116 (6.26), HCT-15 (5.01), HT29 (5.88), KM12 (5.81), SW-620 (6.12)
SF-268 (5.56), SF-295 (5.36), SF-539 (6.34), SNB-19 (6.07), SNB-75 (5.76), U251 (6.15)
Melanoma
Ovarin cancer
Renal cancer
Prostate cancer
Breast cancer
LOX IMVI (5.96), MALME-3M (5.29), M14 (5.45), SK-MEL-2 (5.66), SK-MEL-28 (5.44), SK-MEL-5 (5.69), UACC-257 (5.65), UACC-62 (5.73)
IGROV1 (5.53), OVCAR-3 (5.67), OVCAR-4 (5.91), OVCAR-5 (5.55), OVCAR-8 (5.69), SK-OV-3 (5.69)
786-0 (5.67), A498 (5.63), ACHN (5.47), CAKI-1 (4.78), RXF 393 (5.57), SN12C (5.91), TK-10 (5.63), UO-31 (5.48)
PC-3 (6.13), DU-145 (5.66)
MCF7 (6.06), NCI/ADR-RES (4.78), MDA-MB-231/ATCC (5.86), HS 578T (5.55), MDA-MB-435 (5.85), MDA-N (5.82), BT-549 (5.43), T-47D (5.67)
4,6-Bis[4-(4,5-dihydro-4-methyl-1H-imidazol-2-yl)phenyl]-2-dimethylamino-1,3,5-triazine dihydrochloride (4)
Leukemia
CCRF-CEM (6.06), HL-60(TB) (5.58), K-562 (5.79), MOLT-4 (6.02), RPMI-8226 (5.37)
Non-small cell lung cancer
Colon cancer
CNS cancer
A549/ATCC (5.47), EKVX (5.46), HOP-62 (5.82), HOP-92 (5.70), NCI-H23 (5.54), NCI-H322M (5.79), NCI-H460 (5.45), NCI-H522 (5.82)
COLO 205 (5.93), HCC-2998 (5.70), HCT-116 (5.81), HCT-15 (4.77), HT29 (5.64), KM12 (5.75), SW-620 (5.82)
SF-268 (5.64), SF-295 (5.42), SF-539 (6.07), SNB-19 (5.72), SNB75 (5.78), U251 (5.94)
Melanoma
Ovarin cancer
Renal cancer
Prostate cancer
Breast cancer
LOX IMVI (5.83), MALME-3M (5.42), M14 (5.46), SK-MEL-2 (5.69), SK-MEL-28 (5.43), SK-MEL-5 (5.50), UACC-257 (5.69), UACC-62 (5.75)
IGROV1 (5.51), OVCAR-3 (5.68), OVCAR-4 (5.79), OVCAR-5 (5.41), OVCAR-8 (5.63), SK-OV-3 (5.71)
786-0 (5.62), A498 (5.60), ACHN (5.48), CAKI-1 (4.79), RXF 393 (5.59), SN12C (5.69), TK-10 (5.57), UO-31 (5.31)
PC-3 (5.74), DU-145 (5.76)
MCF7 (5.75), NCI/ADR-RES (4.63), MDA-MB-231/ATCC (5.84), HS 578T (5.54), MDA-MB-435 (5.81), MDA-N (5.67), BT-549 (5.40), T-47D (5.65)
2,5-Bis[4-(4,5,6,7,8,9-hexahydro-1H-benzimidazol-2-yl)phenyl]furan dihydrochloride (8)
Leukemia
CCRF-CEM (5.75), HL-60(TB) (5.64), K-562 (5.85), MOLT-4 (5.74), RPMI-8226 (5.75)
Non-small cell lung cancer
A549/ATCC (5.77), EKVX (5.77), HOP-62 (5.82), HOP-92 (5.84), NCI-H226 (5.73), NCI-H23 (5.80), NCI-H322M (5.87), NCI-H460 (5.74), NCI-
H522 (5.79)
Colon cancer
CNS cancer
Melanoma
Ovarin cancer
Renal cancer
Prostate cancer
Breast cancer
COLO 205 (5.82), HCC-2998 (5.89), HCT-116 (5.82), HCT-15 (5.28), HT29 (5.86), KM12 (5.84), SW-620 (5.59)
SF-268 (5.81), SF-295 (5.74), SF-539 (5.76), SNB-19 (5.83), SNB-75 (5.77), U251 (5.84)
LOX IMVI (5.81), MALME-3M (5.76), M14 (5.78), SK-MEL-2 (5.74), SK-MEL-28 (5.74), SK-MEL-5 (5.80), UACC-257 (5.76), UACC-62 (5.77)
OVCAR-3 (5.74), OVCAR-4 (5.81), OVCAR-5 (5.68), OVCAR-8 (5.75), SK-OV-3 (5.79)
786-0 (5.83), A498 (5.54), ACHN (5.63), CAKI-1 (5.23), RXF 393 (5.71), SN12C (5.83), TK-10 (5.29), UO-31 (5.53)
PC-3 (5.84), DU-145 (5.80)
MCF7 (5.92), NCI/ADR-RES (4.55), MDA-MB-231/ATCC (5.82), HS 578T (5.81), MDA-MB-435 (5.73), MDA-N (5.78), BT-549 (5.67), T-47D (5.70)
3,6-Bis(4,5-dihydro-1H-imidazol-2-yl)-9-methylcarbazole dihydrochloride (10)
Leukemia
CCRF-CEM (>8.00), HL-60(TB) (>8.00), K-562 (4.58), MOLT-4 (6.20), RPMI-8226 (4.61), SR (4.74)
Non-small cell lung cancer
A549/ATCC (4.34), EKVX (4.77), HOP-62 (4.82), HOP-92 (4.87), NCI-H226 (4.52), NCI-H23 (5.38), NCI-H322M (4.90), NCI-H460 (5.27), NCI-
H522 (7.11)
Colon cancer
CNS cancer
Melanoma
Ovarin cancer
Renal cancer
Prostate cancer
Breast cancer
COLO 205 (6.14), HCT-116 (4.66), HCT-15 (<4.00), HT29 (5.11), KM12 (5.81), SW-620 (4.75)
SF-268 (7.33), SF-295 (4.37), SF-539 (4.53), SNB-19 (4.37), SNB-75 (4.11), U251 (4.83)
LOX IMVI (4.31), MALME-3M (4.74), M14 (4.31), SK-MEL-2 (4.47), SK-MEL-28 (4.62), SK-MEL-5 (4.50), UACC-257 (4.36)
IGROV1 (4.61), OVCAR-3 (4.38), OVCAR-4 (4.87), OVCAR-5 (4.42), OVCAR-8 (4.54), SK-OV-3 (4.36)
786-0 (4.41), A498 (<4.00), ACHN (4.42), CAKI-1 (<4.00), RXF 393 (4.46), SN12C (5.20), TK-10 (4.22), UO-31 (4.25)
PC-3 (4.46), DU-145 (4.58)
NCI/ADR-RES (<4.00), MDA-MB-231/ATCC (4.39), HS 578T (4.97), MDA-MB-435 (4.21), MDA-N (4.28), BT-549 (<4.00), T-47D (5.10)
the putative mechanism that minor-groove binding to DNA (topo-
isomerase inhibition) is a component of the anticancer activity.
The highest sensitivity of 10 described here was found for leu-
MOLT-4, GI50 0.6
H522, GI50 0.08
CNS cancer (SF-268, GI50 0.05
was 15 M. Only three cancer cell lines possessed values repre-
l
M) (Fig. 4), non-small cell lung cancer (NCI-
M), colon cancer (COLO 205, GI50 0.7 M), and
M), but the average GI50 value
l
l
l
kemia (CCRF-CEM, GI50 < 0.01
lM; HL-60 (TB), GI50 < 0.01
lM;
l

188
J. Spychała / Bioorganic Chemistry 36 (2008) 183–189
Fig. 3. Dose–response curves of 8 (top) and 9 (bottom) for all nine subpanels of cell
lines. Horizontal lines are provided at the PG (Percentage Growth) values of +50, 0,
and ꢀ50. The concentrations corresponding to points where the curves cross these
lines are the GI50, TGI, and LC50, respectively.
senting concentrations at which the PG is ꢀ50: CCRF-CEM, 0.2
lM;
HL-60(TB), 0.06 M; NCI-H460, 90 M. None of the three response
l
l
parameters can be obtained by interpolation in several cases (the
PGs in a given row exceed +50).
In the current article, the following has to be noted regarding
the tumor cell growth inhibition data with two carbazoles. The
substitution of the carbazole unit at the position 9 by the cyclo-
hexylmethyl group led to less interesting compound 11 (the lowest
GI50 is 1.7
ues were very close (17
24.0 °C [42]) and 11 ( Tm 16.8 °C [42]) possess the MG_MID
parameters similar to 9. The presence of two amidine groups is
important concerning the activity of this class of derivatives, but
care should be also taken to steric differences between the active
compounds and to the distance of the cationic groups.
l
M for COLO-205), nevertheless their average GI50 val-
l
M for 11). Both carbazoles 10 ( Tm
D
Fig. 4. In vitro activity on leukemia cell lines. Triazine 3 (top), selected for further
studies with the hollow fiber assay, in comparison with furan 8 (middle) and car-
bazole 10 (bottom).
D
4. Conclusion
The activity may be due to a sufficient structural similarity to
the amidine antibiotics (the pyrene series is not comparable in
terms of structure-activity relationships). The average cytotoxic ef-
fects of 3, 4, and 8 against all cell lines investigated were compara-
ble to those of N-alkylated furamidine derivatives [20]. They
remain at the low-micromolar level and confirm the results pro-
In summary, the group of active compounds comprises 12 cyclic
diamidines. Compounds 1–12 exhibited activity against most of
cancer cell lines and, in particular, the in vitro anticancer data in
the low-micromolar range prove usefulness of the cationic system
in the design of active anticancer agents 3, 4, and 8. Triazines 3 and
4 were the most promising agents (point of view of the NCI’s Bio-
logical Evaluation Committee for Cancer Drugs), and carbazole 10
was of special interest because of its high activity in the nanomolar
range against some cell lines: leukemia, non-small cell lung, colon,
and CNS cell lines. Compounds 3, 4, and furan 8 exhibited activities
against all tumor cell lines investigated.
The anticancer screening data presented are well correlated
with mode of binding to DNA. They can be used to explore the
strength of the DNA interactions by comparison of the NCI’s activ-
ity profiles (all dose response curves). The strength of these inter-
actions is likely a sum of both hydrogen bonding capabilities and
vided by the previous
DTm studies [24,34,42,53]. The high values
indicate strong DNA affinities for these molecules.
It appeared that the most active compounds are DNA associated
[69]. The poorer binding to DNA may be due to the presence of the
out-of-shape less active molecules which prevent a suitable fit into
the electronegative minor-groove of the DNA helix. In addition,
some agents show significant affinity for RNA and have weaker
affinity for DNA (nonspecific interaction mode) [34,53]. The discov-
ery of antibiotic analogs with new selectivity patterns sets the
stage for further effort to explore therapeutic potential, especially
for AIDS-defining cancers [70].

J. Spychała / Bioorganic Chemistry 36 (2008) 183–189
189
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