Synthesis and DNA-binding properties of 1,10-phenanthroline analogues as intercalating-crosslinkers

Article abstract of DOI:10.1002/jhet.5570450654

(Chemical Equation Presented) We synthesized a series of bis(bromomethyl)4 ,10-phenanthrolines as novel anticancer lead compounds and examined their DNA-binding properties. 5,6-Bis(bromomethyl)-1,10-phenanthroline showed DNA intercalating activity and DNA crosslinking activity, furthermore it is stable in aqueous solution.

Full text of DOI:10.1002/jhet.5570450654

Nov-Dec 2008
1889
Synthesis and DNA-binding Properties of 1,10-Phenanthroline
Analogues as Intercalating-Crosslinkers
Toshinori Higashi, Keiko Inami and Masataka Mochizuki*
Division of Organic and Bioorganic Chemistry, Keio University Faculty of Pharmacy
Shibakoen 1-5-30, Minato-ku, Tokyo 105-8512, Japan
Higashi-ts@pha.keio.ac.jp
Received April 22, 2008
DNA crosslinking moiety
CH3
CH2Br
N
N
H3C
BrH2C
N
N
DNA intercalating moiety
We synthesized a series of bis(bromomethyl)-1,10-phenanthrolines as novel anticancer lead compounds
and examined their DNA-binding properties. 5,6-Bis(bromomethyl)-1,10-phenanthroline showed DNA
intercalating activity and DNA crosslinking activity, furthermore it is stable in aqueous solution.
J. Heterocyclic Chem., 45, 1889 (2008).
INTRODUCTION
In the current study, we synthesized
a
novel
crosslinking agent based on the 1,10-phenanthroline
skeleton, and examined both its DNA intercalating and
interstrand crosslinking activity and stability in aqueous
solution.
Cisplatin is a crosslinking agent with high anticancer
activity. However, it causes very serious side effects such
as renal toxicity and nausea. We suspect that some of
these side effects are caused by platinum, a component of
cisplatin, and that a crosslinking agent lacking heavy
metals would therefore be useful as a drug in cancer
chemotherapy.
We have reported the synthesis of DNA crosslinking
agents containing the acridine skeleton [1]. These
compounds possess DNA intercalating and interstrand
crosslinking activities, and inhibit cell proliferation in a
human T cell leukemia cell line. However, these
compounds were hydrolyzed rapidly in aqueous solution.
Therefore, the purpose of the present study was to
synthesize crosslinking compounds which are stable in
aqueous solution.
RESULTS AND DISCUSSION
5,6-Bis(bromomethyl)-1,10-phenanthroline
2
was
synthesized via a radical reaction of 5,6-dimethyl-1,10-
phenanthroline 1 with NBS [20-22] (Scheme 1).
Scheme 1
Br
CH3
H3C
Br
NBS, AIBN / CCl4
The metal-complexing characteristics of 1,10-phenan-
throline have been well-characterized. Some 1,10-phenan-
throline-cuprous complexes oxidatively cleave DNA [2-
6], and possess anticancer activity towards a human
cervical epidermoid carcinoma cell line [7]. Ruthenium
[8], lanthanum [9,10], osmium [11] and vanadium [12]
complexes demonstrate similar effects. 5,6-Dimethyl-
1,10-phenanthroline metal complexes are even more
effective, especially when complexed with cuprous or
ruthenium ions [13-16]. Furthermore, phenanthroline
derivatives, which are crescent-shaped planar aromatic
compounds, have been shown to interact specifically with
a quadruplex and to inhibit telomerase activity [17-19].
Clearly, phenanthroline derivatives possess very
interesting biological activities.
N
N
N
N
1
2
1,10-Phenanthroline-2,9-dicarboxaldehyde
5
was
synthesized via the oxidation of 2,9-dimethyl-1,10-
phenanthroline 3 with selenium dioxide [23]. Compound
5 on hydrolysis afforded 2,9-bis(hydroxymethyl)-1,10-
phenanthroline 7. 2,9-Bis(bromomethyl)-1,10-phenan-
throline 9 was synthesized by a substitution reaction of
compound 7. 4,7-Substitution products 6, 8, 10 were
synthesized by the same routes as compounds 5, 7, 9
(Scheme 2).

1890
T. Higashi, K. Inami and M. Mochizuki
Vol 45
Scheme 2
Lane
1
2
3
4
5
6
7
Compound 1
CH3
CHO
SeO2 / dioxane
N
N
H3C
OHC
N
N
Compound 3
3
4
: 2,9-substitution
: 4,7-substitution
5: 2,9-substitution
6: 4,7-substitution
Compound 4
CH2OH
Lane 1, open circular plasmid DNA; lane 2, supercoiled plasmid DNA;
lanes 3-7, plasmid DNA treated with 1 mM, 500 μM, 250μM, 100 μM
or 50 μM of the indicated compound. Reactions were performed for 30
minutes.
NaBH4 / EtOH
N
HOH2C
N
7
: 2,9-substitution
: 4,7-substitution
8
Figure 2 DNA intercalating activity of methyl compounds.
Next, we examined DNA interstrand crosslinking
activity [25]. Compound 2 at a concentration of 10ꢀM
generated a band of double stranded DNA, while
compounds 9 and 10 had no effect at the tested
concentrations (Fig. 3). These results suggest that
compound 2 has high crosslinking activity due to its high
DNA affinity, driven by intercalation. Furthermore, the
position of the substitution affects activity.
47% HBr - H2O
CH2Br
N
BrH2C
N
9
: 2,9-substitution
10: 4,7-substitution
We examined the DNA intercalating activity of these
Lane
1
2
3
4
5
6
7
8
9
10 11
12
compounds using an unwinding assay [24]. Compound 2
caused
a partial transition from open circular to
supercoiled plasmid DNA, and demonstrated higher DNA
intercalating activity than did compounds 9 or 10 (Fig. 1).
Comparison of methyl compounds 1, 3 and 4 showed that
compound 1 has the highest activity (Fig. 2). Maheswari
et al. previously demonstrated that 5,6-dimethyl-1,10-
phenanthroline has high affinity for DNA surfaces due to
hydrophobic interactions [13-15]. Our results are in
accordance with their report.
Lane 1, nondenatured (double-stranded) DNA; lane 2, denatured (single-
stranded) DNA; lanes 3-5, plasmid DNA treated with 10, 1 or 0.1 μM
compound 9; lanes 6-8, plasmid DNA treated with 10, 1 or 0.1 μM
compound 10; lanes 9-11, plasmid DNA treated with 10, 1 or 0.1 μM
compound 2; lanes 12, plasmid DNA treated with 0.1 μM cisplatin.
Figure 3. DNA interstrand crosslinking activity.
We previously synthesized 4,5-bis(bromomethyl)-
acridine, which has high DNA crosslinking activity and
inhibits proliferation of a human T cell leukemia cell line
[1]. However, this acridine compound hydrolyzed rapidly
in aqueous solution. We examined the stability of
compound 2 in aqueous solution (Fig. 4) and showed that
it is not hydrolyzed. Hydrolysis is caused by a SN1
reaction. The intermediate carbocation is more stable in
the acridine compound than in 1,10-phenanthroline
compounds because two heteroatoms in the 1,10-phenan-
throline skeleton strongly withdraw electrons. Therefore,
compound 2 is more stable in aqueous solution than the
acridine compound.
Lane
1
2
3
4
5
6
7
Compound 2
Compound 9
Compound 10
In conclusion, we synthesized a novel intercalating-
crosslinker with
a
1,10-phenanthroline skeleton.
Compound 2 has DNA intercalating and crosslinking
activity, and is stable in aqueous solution. These
characteristics make compound 2 a suitable leading
compound as an anticancer agent. We are investigating
the anticancer activity of compound 2 in a human T cell
leukemia cell line.
Lane 1, open circular plasmid DNA; lane 2, supercoiled plasmid DNA;
lanes 3-7, plasmid DNA treated with 1 mM, 500 μM, 250 μM, 100 μM
or 50 μM of the indicated compound. Reactions were performed for 30
minutes.
Figure 1. DNA intercalating activity of crosslinking compounds.

Nov-Dec 2008
Synthesis and DNA-binding Properties of 1,10-Phenanthroline Analogues
1891
2,9-Bis(hydroxymethyl)-1,10-phenanthroline (7).
A
solution of 5 (118 mg, 0.5 mmol) and sodium borohydride (38
mg, 1.0 mmol) in ethanol (10 mL) was heated under reflux for
half an hour. The mixture was extracted with ethyl acetate, and
the organic phase was dried over Na2SO4. The solvent was
evaporated under reduced pressure to give compound 7 as
yellow needles, m.p: 184 ºC dec., yield: 78.2 mg (65.2%).
1HNMR (CDCl3, 500MHz): ꢀ 5.10 (s, 4H, -CH2-), 7.60 (d, 2H,
phenyl protons), 7.79 (s, 2H, phenyl protons), 8.24 (d, 2H,
phenyl protons). EI MS: m/z: 240, 239, 210, 209, 193.
10
9
8
7
4,7-Bis(hydroxymethyl)-1,10-phenanthroline (8).
A
0
50
100
150
200
300
250
solution of 6 (118 mg, 0.5 mmol) and sodium borohydride (38
mg, 1.0 mmol) in ethanol (10 mL) was heated under reflux for
half an hour. The mixture was extracted with ethyl acetate, and
the organic phase was dried over Na2SO4. The solvent was
evaporated under reduced pressure to give compound 8 as
yellow needles, m.p: 220 ºC dec., yield: 31.2 mg (26.0%).
1HNMR (CD3OD, 500MHz): ꢀ 5.23 (s, 4H, -CH2-). EI MS: m/z:
240, 239, 210, 209, 193. HRMS: 240.0917 (calcd. for
C14H12O2N2: 240.0900) Anal. Calcd. for C14H12O2N2 (240.26).
0.2 H2O: C, 68.95; H, 5.12; Found: C, 68.86; H, 5.12.
Reaction time (min)
Solid circle, compound 2; open circle, 4,5-bis(bromomethyl)acridine.
Figure 4. Stability in aqueous solution.
EXPERIMENTAL
1H NMR spectra were obtained on a Jeol JNM-A500 spec-
trometer using tetramethylsilane as an internal reference. Mass
spectra were determined using a Shimadzu GCMS-QP5050A
spectrometer. Compounds 1, 3 and 4 were purchased from
Tokyo Kasei, Inc. (Tokyo, Japan). Silica gel 60 and aluminum
oxide 90 were purchased from Merck & Co. (Rahway, NJ,
USA). pBR322 plasmid DNA and topoisomerase I were
purchased from Takara-Bio, Inc. (Tokyo, Japan). L-column 2
was purchased from Chemicals Evaluation and Research
Institute, Japan (Tokyo, Japan).
2,9-Bis(bromomethyl)-1,10-phenanthroline (9). A solution
of 7 (24 mg, 0.1 mmol) and hydrobromic acid (15 mL, 47%)
was heated under reflux for an hour, then cooled on ice and
treated with solid sodium carbonate at pH 10. The mixture was
extracted with dichloromethane and the organic phase was dried
over Na2SO4. The solvent was evaporated under reduced
pressure, and the residue was purified by silica gel column
chromatography in 5:1 dichloromethane / ethyl acetate to obtain
9 as yellow needles, m.p: 105 ºC dec., yield: 26.0 mg (71.0%).
1HNMR (CDCl3, 500MHz): ꢀ 4.97 (s, 4H, -CH2-), 7.82 (s, 2H,
phenyl protons), 7.92 (d, 2H, phenyl protons), 8.29 (d, 2H,
phenyl protons). EI MS: m/z: 368, 366, 364, 287, 285, 206.
4,7-Bis(bromomethyl)-1,10-phenanthroline (10). A solution
of 8 (12 mg, 0.05 mmol) and hydrobromic acid (10 mL, 47%)
was heated under reflux for an hour and then cooled on ice and
treated with solid sodium carbonate at pH 10. The mixture was
extracted with dichloromethane, and the organic phase was dried
over Na2SO4. The solvent was evaporated under reduced
pressure, and the residue was purified by aluminum oxide
column chromatography in dichloromethane to obtain 10 as a
yellow powder, m.p: 135 ºC dec., yield: 7.0 mg (38.3%).
1HNMR (CDCl3, 500MHz): ꢀ 4.94 (s, 4H, -CH2-), 7.66 (d, 2H,
phenyl protons), 8.27 (s, 2H, phenyl protons), 9.18 (d, 2H,
phenyl protons). EI MS: m/z: 368, 366, 364, 287, 285, 206.
HRMS: 363.9197 (calcd. for C14H10N2Br2: 363.9211) Anal.
Calcd. for C14H10N2Br2 (366.04): C, 45.93; H, 2.75; Found: C,
46.09; H, 3.01.
Unwinding assay. Negatively supercoiled pBR322 DNA
(0.25 μg/reaction) was first relaxed at 37 ºC by incubation with
5 units of calf thymus topoisomeraseꢀ at pH 8.0 for 30 minutes.
Next, the DNA was treated with the test compound dissolved in
DMSO at pH 8.0 for 30 minutes at 37 ºC. The reaction was
terminated by the addition of sodium dodecyl sulfate (0.5% final
concentration) followed by dilution and extraction to remove the
drugs. The DNA samples were separated by 1% agarose gel
electrophoresis, and the DNA bands were visualized by staining
with ethidium bromide.
Crosslinking assay Linearized pBluescript® DNA (460 ng)
was treated with each test compound dissolved in DMSO at 37
ºC for 6 hours. After the reaction, the DNA was precipitated
with ethanol (95%) and cooled for 24 hours before being
5,6-Bis(bromomethyl)-1,10-phenanthroline (2). A mixture
of 1 (104 mg, 0.5 mmol), N-bromosuccinimide (270 mg, 1.5
mmol) and 2,2'-azobis(isobutyronitrile) (32 mg, 0.2 mmol) in
tetrachloromethane (30 mL) was refluxed under nitrogen for 15
minutes, then the mixture was allowed to cool to room
temperature. The solvent was evaporated, and the residue was
purified by aluminum oxide column chromatography in
dichloromethane to obtain 2 as yellow needles, m.p: 170 ºC dec.,
1
yield: 67.5 mg (36.9%). HNMR (CDCl3, 500MHz): ꢀ 5.08 (s,
4H, -CH2-), 7.77 (dd, 2H, phenyl protons), 8.57 (dd, 2H, phenyl
protons), 9.25 (dd, 2H, phenyl protons). EI MS: m/z: 368, 366,
364, 287, 285, 206.
1,10-Phenanthroline-2,9-dicarboxaldehyde (5). A mixture
of 3 (545 mg, 2.5 mmol) and selenium dioxide (1.11 g, 10
mmol) in dioxane containing 4% H2O (50 mL) was heated under
reflux for an hour and then filtered through celite while hot. A
solid separated in the cold filtrate and was recrystallized from
dioxane containing 4% H2O to give compound 5 as white
needles, m.p: 237ºC dec., yield: 395.9 mg (67.1%). 1HNMR
(CDCl3, 500MHz): ꢀ 8.05 (s, 2H, phenyl protons), 8.39 (d, 2H,
phenyl protons), 8.52 (d, 2H, phenyl protons), 10.57 (s, 2H, -
CHO). EI MS: m/z: 236, 208, 180.
1,10-phenanthroline-4,7-dicarboxaldehyde (6). A mixture
of 4 (104 mg, 0.5 mmol) and selenium dioxide (224 mg, 2.0
mmol) in dioxane containing 4% H2O (10 mL) was heated under
reflux for 30 minutes and then filtered through celite while hot.
A solid separated in the cold filtrate and recrystallized from
dioxane containing 4% H2O to give compound 6 as yellow
1
needles, m.p: 160 ºC dec., yield: 212.5 mg (90.0%). HNMR
(CDCl3, 500MHz): ꢀ 8.10 (d, 2H, phenyl protons), 9.23 (s, 2H,
phenyl protons), 9.54 (d, 2H, phenyl protons), 10.65 (s, 2H, -
CHO). EI MS: m/z: 236, 208, 179.

1892
T. Higashi, K. Inami and M. Mochizuki
Vol 45
[8] Karki, S. S.; Thota, S.; Darj, S. Y.; Balzarini, J.; Clercq, E. D.
Bioorg. Med. Chem. 2007, 15, 6632.
[9] Heffeter, P.; Jakupec, M. A.; Körner, W.; Wild, S.;
Keyserlingk, N. G.; Elbling, L.; Zorbas, H.; Korynevska, A.;
Knasmüller, S.; Sutterlüty, H.; Micksche, M.; Keppler, B. K.; Berger,
W. Biochem. Pharmacol. 2006, 71, 426.
[10] Heffeter, P.; Jakupec, M. A.; Körner, W.; Chiba, P.; Pirker,
C.; Dornetshuber, R.; Elbling, L,; Sutterlüty, H.; Micksche, M.; Keppler,
B. K.; Berger, W. Biochem. Pharmacol. 2007, 73, 1873.
[11] Peacock, A. F. A.; Habtemariam, A.; Moggach, S. A.;
Prescimone, A.; Parsons, S.; Sadler, P. J. Inorg. Chem. 2007, 46, 4049.
[12] Sam, M.; Hwang, J. H.; Chanfreau, G.; Abu-Omar, M. M.
Inorg. Chem. 2004, 43, 8447.
centrifuged for 20 minutes. The supernatant was removed, the
samples were washed with ethanol (70%) and spun for 20
minutes. The supernatant was removed and the DNA was
lyophilized to dryness. The DNA was then dissolved in
separation buffer (30% DMSO and 1 mM ethylenediamine-
N,N,N',N'-tetraacetic acid), heated at 95 ºC for 5 minutes, and
immediately placed in an ice bath. The DNA samples were
separated by 1% agarose gel electrophoresis, and the bands were
visualized by staining with ethidium bromide.
Stability of intercalating-crosslinker The stability of
compound 2 toward nucleophilic reagents was examined by
measuring the decomposition rate in aqueous solution. Test
compound (final concentration of 100 μM) was added to an
aqueous solution (70:30 mixture of 0.01 M sodium phosphate
buffer [pH 7.4] and acetonitrile), and 6 μL of the solution was
injected onto an HPLC L-column 2 (5 μm) at regular intervals.
Compounds were eluted with a 2:1 mixture of methanol and
water.
[13] Maheswari, P. U.; Palaniandavar, M. Inorg. Chim. Acta 2004,
357, 901.
[14] Maheswari, P. U.; Rajendiran, V.; Palaniandavar, M.;
Thomas, R.; Kulkarni, G. U. Inorg. Chim. Acta 2006, 359, 4601.
[15] Maheswari, P. U.; Rajendiran, V.; Palaniandavar, M.;
Parthasarathi, R.; Subramanian, V. J. Inorg. Biochem. 2006, 100, 3.
[16] Selvakumar, B.; Rajendiran, V.; Maheswari, P. U.; Stoeckli-
Evans, H.; Palaniandavar, M. J. Inorg. Biochem. 2006, 100, 316.
[17] Mergny, J.; Lacroix, L.; Teulade-Fichou, M.; Hounsou, C.;
Guittat, L.; Hoarau, M.; Arimondo, P. B.; Vigneron, J.; Lehn, J.; Riou,
J.; Garestier, T.; Hélène, C. Proc. Natl. Acad. Sci. USA 2001, 98, 3062.
[18] Kaiser, M.; Cian, A. D.; Sainlos, M.; Renner, C.; Mergny, J.;
Teulade-Fichou, M. Org. Biomol, Chem. 2006, 4, 1049.
[19] Kaiser, M.; Sainlos, M.; Lehn, J.; Bombard, S.; Teulade-
Fichou, M. ChemBioChem 2006, 7, 321.
[20] Beer, R. H.; Jimenez, J.; Drago, R. S. J. Org. Chem. 1993,
58, 1746.
[21] Mukkala, V.; Sund, C.; Kwiatkowski, M. Helv. Chim. Acta.
1992, 75, 1621.
[22] Hartl, F.; Vernier, S.; Belser, P. Collect. Czech. Chem.
Commun. 2005, 70, 1891.
[23] Chandler, C. J.; Deady, L. W.; Reiss, J. A. J. Heterocycl.
Chem. 1981, 18, 599.
REFERENCES
[1] Higashi, T.; Sakamoto, M.; Mochizuki, M. Heterocycles
2008, 75, 1943.
[2] Kuwabara, M.; Yoon, C.; Goyne, T.; Thederahn, T.; Sigman,
D. S. Biochemistry 1986, 25, 7401.
[3] Sigman, D. S.; Graham, D. R.; Aurora, V. D.; Stern, A. M. J.
Biol. Chem. 1979, 254, 12269.
[4] Goyne, T. E.; Sigman, D. S. J. Am. Chem. Soc. 1987, 109,
2846.
[5] Chen, T.; Greenberg, M. M. J. Am. Chem. Soc. 1998, 120,
3815.
[6] Oyoshi, T.; Sugiyama, H. J. Am. Chem. Soc. 2000, 122,
6313.
[7] Rajendiran, V.; Karthik, R.; Palaniandavar, M.; Stoeckli-
Evans, H.; Periasamy, V. S.; Akbarsha, M. A.; Srinag, B. S.;
Krishnamurthy, H. Inorg. Chem. 2007, 46, 8208.
[24] Vance, J. R.; Bastow, K. F. Biochem. Pharmacol. 1999, 58,
703.
[25] Hertley, J. A.; Berardini, M. D.; Souhami, R. L. Anal.
Biochem. 1991, 193, 131.