New triple-helix DNA stabilizing agents

Article abstract of DOI:10.1016/j.bmcl.2004.12.019

Several substituted quinolin-4-amines and heteroaromatic analogs were synthesized and evaluated for interaction with triplex polydA·2polydT and duplex polydA·polydT by using UV-thermal melting experiments. Excellent triple-helix DNA ligands with high affinity toward T·A·T triplets and triple/duplex selectivity were designed through a rational approach.

Full text of DOI:10.1016/j.bmcl.2004.12.019

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1097–1100
New triple-helix DNA stabilizing agents
Lucjan Strekowski,* Maryam Hojjat, Ewa Wolinska,ꢀ Alesia N. Parker,
Ekaterina Paliakov, TadeuszGorecki, Farial A. Tanious and W. David Wilson
*
Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA
Received 27 October 2004; revised 2 December 2004; accepted 8 December 2004
Available online 29 December 2004
Abstract—Several substituted quinolin-4-amines and heteroaromatic analogs were synthesized and evaluated for interaction with
triplex polydAÆ2polydT and duplex polydAÆpolydT by using UV-thermal melting experiments. Excellent triple-helix DNA ligands
with high affinity toward TÆAÆT triplets and triple/duplex selectivity were designed through a rational approach.
Ó 2004 Elsevier Ltd. All rights reserved.
Intermolecular triple helices are formed by the sequence-
specific hydrogen bonding of a single stranded DNA in
the major groove of duplex DNA. This interaction is
quite weak under physiological conditions. The stabil-
ization of triple-helical DNA is currently of immense
interest in various biotechnology applications.1 A suc-
cessful strategy for increasing the interaction strength
is to use triplex-specific ligands that bind strongly to tri-
plex but only weakly to duplex DNA. Examples of selec-
tive triplex intercalators are quinolines 12 and 23 that
have been synthesized and tested by us previously. The
terminal dimethylamino group and the quinoline N1
atom in compounds 1 and 2 are protonated under phys-
iological conditions and, due to the cationic nature,
these compounds do not intercalate with the cationic
C+ÆGC triplets.1–3 As a result, these compounds prefer
triplex over duplex and show absolute selectivity for
TÆAT triplets in the presence of C+ÆGC triplets. An ideal
TÆAT intercalator would not only bind strongly with the
triplex but also would show no significant interaction
with the duplex. Unfortunately, this is not the case with
1 and 2 and a number of their analogs that show signif-
icant binding with duplex DNA.1–5 Additional triplex
stabilizing agents that suffer from substantial affinity to-
ward duplex have been developed by other groups.
These are cationic derivatives of benzopyridoindoles,
benzopyridiquinoxalines, dibenzophenanthrolines, cora-
lyne, and anthraquinone, as briefly reviewed.6
NMe2
6
7
HN
R
N
8
5
4
2
9
N
3
10
1
1
NMe2
N Me
HN
2: 2-phenanthryl, R =
3: 2-phenanthryl, R =
N
NMe2
4: 3-phenanthryl, R = HN
5: 9-phenanthryl, R =
NMe2
HN
Based on the observations described above, a series of
new heteroaromatic amines were designed and synthe-
sized. The compounds were designed to test the impor-
tance of stacking surface/planar aromatic system,
rotational freedom, and charge that can complement
the base triplet structure. The position of the cationic
groups, which fit into the triplex grooves, was varied
to find the optimum interactions for triplex-specific
binding. In our search for a rapid and accurate method
to evaluate triplex affinity as well as specificity of triplex
over duplex binding, we have selected thermal denatur-
ation studies of polydTÆdAÆdT. This DNA exists as a tri-
plex at low temperature but as the temperature is
increased, the third strand dissociates. At higher temper-
atures the melting temperature of the duplex can be
determined in the same experiment. The interaction of
a compound can thus be evaluated with both triplex
and duplex in a well-controlled experiment by determin-
ing its effects on both the triplex and duplex melting
temperatures. The method is rapid, accurate, and
*
Corresponding authors. Tel.: +1 404 651 0999; fax: +1 404 651
1416; e-mail addresses: Lucjan@gsu.edu; chewdw@langate.gsu.edu
ꢀ On leave of absence from the University of Podlasie, Poland.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.12.019

1098
L. Strekowski et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1097–1100
requires very little sample. A large data base of DTm val-
ues, determined by this procedure, is available for com-
parison with new compound results. The correlation
between DTm values and fundamental equilibrium mea-
surements has been shown to validate the screening
method.1–7
OLi
F
CF3
N
H2N
N
N
11
12
1. Chemistry
NMe2
H2N
Our previous work has shown that quinoline-4-amines
substituted at position 2 with an aryl and at N4 with
an aminoalkyl group, such as 1 and 2, are good triplex
intercalators. As part of this work, additional phenanth-
ryl-substituted quinolines 3–5 were synthesized by using
the general procedure reported for 17 and 2.3
NMe2
HN
N
N
13
Compounds 8–10 (Scheme 1) and 13 (Scheme 2) are
substituted phenanthrolines in which the heteroaromatic
subunit is larger than the quinoline in triplex intercala-
tors synthesized by us previously. A simple approach
to the synthesis of 8–10 is by construction of a 4,6-phe-
nanthroline ring system by the reaction of 4-trifluoro-
methylquinolin-4-amine (6) with a lithium enolate deri-
ved from an aryl methyl ketone followed by nucleophilic
displacement of fluoride from the resultant 3-aryl-1-flu-
oro-4,6-phenanthroline 7 by treatment with an amine.8
A 4,7-phenanthroline derivative 13 was prepared in a
similar way from 5-trifluoromethylquinolin-6-amine
(11). In all these cases crude fluorophenanthrolines 7
and 12 were allowed to react with an appropriate amine,
and the final products 8–10, 13 were purified by chromato-
graphy (silica gel eluting with hexanes/EtOH/Et3N,
18:1:1) and subsequent crystallization from hexanes/
Et2O.
Scheme 2.
synthesized for comparison. Compound 19 was synthe-
sized starting with Schiff base 14 derived from 2-trifluo-
romethylaniline and a-tetralone.9 The intermediate
products 17 and 18 were obtained by using well-devel-
oped general methodologies.10,11 A final Mannich reac-
tion of 18 furnished the desired product 19. A similar
strategy was used to prepare compounds 22 and 23
starting with appropriate Schiff bases 15 and 16 derived
from 2-trifluoromethylaniline and methoxy-substituted
a-tetralones. A Mannich reaction of the intermediate
products 20 and 21 yielded the respective products 22
and 23. The unfused analog 24 was synthesized in a
similar way starting with Schiff base obtained by the
reaction
methoxyacetophenone.
of
2-trifluoromethylaniline
and
4-
Compounds 19, 22, 23 contain a nonplanar 5,6-dihydro-
benz[c]acridine ring system (Scheme 3). In addition, they
are at least tricationic under physiological conditions for
an increased electrostatic interaction with a highly
charged polyanionic triplex DNA. A polyamine-substi-
tuted unfused analog 24 (structure in Scheme 3) was also
Finally, a simple acridine 25 was prepared as shown in
Eq. 1. Since acridines can intercalate efficiently with du-
plex DNA,12 it was thought that the presence of a bulky
bromine atom in the molecule 25 would inhibit this
interaction.
NMe2
Cl
HN
NMe2
Br
Br
H2N
ð1Þ
CF3
NH2
N
N
25
N
6
All products 19, 22–25 were isolated by chromatography
as mentioned above, then transformed into hydrobro-
mide salts,13 and the salts were crystallized from 95%
EtOH. All final compounds described in this report gave
satisfactory results of elemental analysis, and their struc-
OLi
Ar
R
1
HN
tures were fully consistent with MS, H NMR, and 13C
F
R
NH2
NMR data. The compositions and mpꢁs (°C) of the ana-
lytically pure samples, as used in DNA binding studies,
are given as follows: 3Æ2HBrÆ3H2O, 135–137; 4Æ2HBrÆ3-
H2O, 120–122; 5Æ2HBrÆ3H2O, 110–111; 8, 142–143; 9,
169–170; 10, 139–140; 13, 160–161; 19Æ5HBrÆ3H2O,
241–243; 22Æ4HBrÆ2H2O, 247–248; 23Æ6HBrÆH2O, 222–
224; 24Æ6HBrÆ3H2O, 244–245 (dec); 25Æ2HBrÆ1/2H2O,
250–252.
N
N
N
Ar
N
Ar
7
8: Ar = phenyl, R = CH2CH2NMe2
9: Ar = 2-naphthyl, R = CH2CH2NMe2
10: Ar = 2-naphthyl, R = CH2C(Me)2CH2NMe2
Scheme 1.

L. Strekowski et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1097–1100
1099
HO
CF3
N
Cl
1) t-BuOK
2) pTsOH
3) POCl3/PCl5
R1
NH
HN
N Me
1) p-anisidine
2) BBr3
N
CH2O
N
R2
17
18
Me
N
14: R1= R2 = H
N
15: R1= H, R2 = OMe
16: R1 = OMe, R2 = H
Me
HO
N
N
NH
NHLi
1)
H2N
NMe2
2) BBr3
HN
N
NMe2
N
HN
N
19
HN
N Me
22
N
OH
N
NMe2
HN
CH2O
R1
N
Me
N
Me
R2
20: R1 = H, R2 = OH
21: R1 = OH, R2 = H
N
N
N
NMe2
N
HN
OH
Me
24
N
N
N
N
Me
N
OH
Me
(prepared in a similar way from a Schiff base
derived from 2-trifluoromethylaniline
and 4-methoxyacetophenone)
23
N
Me
Scheme 3.
Table 1. Tm increases for interaction of compounds 1–5, 8–10, 13, 19,
22–25 with triplex polydAÆ2polydT and duplex polydAÆpolydT
2. DNA binding studies
Interaction of ligands with duplex polyAÆpolydT and tri-
plex polydAÆ2polydT were evaluated by UV-thermal
experiments under conditions identical with those re-
ported previously for quinolines 1 and 2, for direct com-
parison.2,3 Briefly, compound stabilization of DNA
samples was compared by the increase in Tm (DTm = Tm
of the complex ꢀ Tm of the free nucleic acid) they pro-
duce in PIPES 20 buffer with 0.2 M NaCl (pH = 7.0)
at saturating amounts of the compound (a ratio of
0.2 mol of compound to nucleic acid base duplets or
triplets). DTm values are reproducible to 0.5 °C. The
Tm of the triplex is 41 °C and of the duplex is 74 °C
under these conditions. The results are shown in Table 1.
Although the correlation between compound binding
affinity and the increase in DNA Tm is not completely
linear, the agreement is quite good within any series of
compounds. For example, the high selectivity of quino-
line 1 toward triplex DNA in the presence of duplex
DNA, as derived from competition dialysis experiments,
was confirmed by using Tm measurements. Moreover,
the relative binding affinities of 1 with duplex and triplex
DNA1 parallel the corresponding Tm values given in
Table 1.
No
DTm (°C)
Triplex
Duplex
1a
2b
3
35.6
35.3
18.6
28.1
15.4
27.5
23.1
8.2
5.5
5.2
0.3
13.6
0.5
0.0
0.0
0.0
0.0
0.0
0.3
0.7
0.7
0.0
4
5
8
9
10
13
19
22
23
24
25
24.0
17.8
10.7
1.6
3.6
16.6
a Taken from Ref. 2.
b Taken from Ref. 3.
pounds showing a substantial stabilization of duplex
DNA. The selectivity is slightly improved for the 2-
and 9-phenanthryl analogs 3, 5 of 2 albeit at the expense
of a decreased triplex affinity relative to that for 2. Sur-
prisingly, the 2-(3-phenanthryl)quinoline 4 stabilizes du-
plex DNA strongly. On the other hand, the
phenanthroline derivatives 8, 9, and 13 show strong
As can be seen from Table 1, a 2-(2-naphthyl)quinoline
1 and a 2-(2-phenanthryl)quinoline 2 exhibit virtually
identical triplex/duplex selectivities with both com-

1100
L. Strekowski et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1097–1100
interaction with triplex DNA with virtually no stabiliza-
tion of duplex DNA. Although groove binding of these
compounds with the duplex cannot be excluded, because
of Tm measurement limitations, almost certainly they
stabilize triplex DNA by intercalation. The diminished
affinity toward the triplex of ligand 10 substituted with
a bulky aminoalkyl group is consistent with intercala-
tion of phenanthridine ligands from the narrow minor
groove of the triplex. Following intercalation of the phe-
nanthridine system of 10 with triplex, the bulky cationic
substituent would interact poorly with the minor
groove, thus lowering the complex stability, as observed.
excellent triplex/duplex selectivity of ligand 25 can be ex-
plained in terms of the lack of intercalation with duplex,
because of the presence of a bulky bromine atom in the
molecule, as already suggested.
References and notes
1. Chaires, J. B.; Ren, J.; Henary, M.; Zegrocka, O.; Bishop,
G. R.; Strekowski, L. J. Am. Chem. Soc. 2003, 125, 7272,
and references cited therein.
2. Wilson, W. D.; Tanious, F. A.; Mizan, S.; Yao, S.;
Kiselyov, A. S.; Zon, G.; Strekowski, L. Biochemistry
1993, 32, 10614.
Preliminary molecular modeling of systems related to 1
and 13 (AM1 molecular orbital, Spartan software) indi-
cate correlated differences in twist and stereoelectronic
effects in the two compounds. Steric clash between the
side chain NH moiety and a proton on the pyrido group
of 13 causes the side chain to twist out of the aromatic
plane. A much smaller twist is observed for the quino-
line system of 1. In addition, the pyrido group and twist
of 13 give it different stereoelectronic and stacking prop-
erties than 1. All of these effects could lead to the ob-
served decrease in triplex Tm for 13. Clearly, a full
understanding of the differences in DNA binding prop-
erties of these compounds will require more detailed
studies that are in progress.
3. Strekowski, L.; Gulevich, Y.; Baranowski, T. C.; Parker,
A. N.; Kiselyov, A. S.; Lin, S.-Y.; Tanious, F. A.; Wilson,
W. D. J. Med. Chem. 1996, 39, 3980.
4. Strekowski, L.; Parker, A. N.; Hojjat, M.; Say, M.;
Zegrocka-Stendel, O.; Patterson, S. E.; Tanious, F. A.;
Wilson, W. D. Acta Pol. Pharm., in press.
5. Strekowski, L.; Say, M.; Zegrocka, O.; Tanious, F. A.;
Wilson, W. D.; Manzel, L.; Macfarlane, D. E. Bioorg.
Med. Chem. 2003, 11, 1079.
6. Keppler, M.; Zegrocka, O.; Strekowski, L.; Fox, K. R.
FEBS Lett. 1999, 447, 223.
7. Wilson, W. D.; Zhao, M.; Patterson, S. E.; Wydra, R. L.;
Janda, L.; Strekowski, L. Med. Chem. Res. 1992, 2,
102.
8. Strekowski, L.; Kiselyov, A. S.; Hojjat, M. J. Org. Chem.
1994, 59, 5886.
Compound 19 is also a good triplex stabilizing agent.
The ring system of this compound is complementary
with the TÆAÆT base triplet and the cationic group must
fit well into a triplex groove. On the other hand, an in-
creased charge concentration by protonation of 22–24
as well as unfavorable steric interactions within the tri-
plex grooves results in an adverse effect on triplex stabil-
ization. These polycations apparently bind weakly and
externally to both duplex and triplex by electrostatic
interaction.14
9. Strekowski, L.; Wydra, R. L.; Harden, D. B.; Honkan, V.
A. Heterocycles 1990, 31, 1565.
10. Strekowski, L.; Zegrocka, O.; C. Windham, C.; Czarny,
A. Org. Process Res. Dev. 1997, 1, 384.
11. Paliakov, E.; Strekowski, L. Tetrahedron Lett. 2004, 45,
4093.
12. Keppler, M. D.; McKeen, C. M.; Zegrocka, O.; Strekow-
ski, L.; Brown, T.; Fox, K. R. Biochim. Biophys. Acta
1999, 1447, 137, and references cited therein.
13. Strekowski, L.; Mokrosz, J. L.; Honkan, V. A.; Czarny,
A.; Cegla, M. T.; Wydra, R. L.; Patterson, S. E.; Schinazi,
R. F. J. Med. Chem. 1991, 34, 1739.
Acridines are known to intercalate with duplex DNA
and triplex DNA with little selectivity.12 In part, the
14. Thomas, T.; Thomas, T. J. Biochemistry 1993, 32, 14068,
and references cited therein.