Design and synthesis of an anthranyl bridged optically active dinuclear iron(II)-ligand and evaluation of DNA-cleaving activity

Article abstract of DOI:10.1016/j.bmcl.2021.127782

It is necessary to design a ligand that is compatible with the target molecule to optimally use the DNA-cleaving ability of metal complexes. In this study, we synthesized an optically active dinuclear ligand, (1R,1′R,2R,2′R)-N¹,N^1′-(anthracene-1,8-diylbis(methylene))bis(N²,N²-bis(pyridin-2-ylmethyl)cyclohexane-1,2-diamine) (R-ABDC, 4a) and its enantiomer (S-ABDC, 4b). We then prepared their Fe(II) complexes by mixing the ligand with FeSO₄·7H₂O in situ and investigated DNA-cleaving activities using plasmid DNA in the presence of excess sodium ascorbate at atmospheric conditions. The Fe(II) complexes efficiently cleaved DNA and selectively recognized two consecutive A and/or T sequences.

Full text of DOI:10.1016/j.bmcl.2021.127782

Bioorg. Med. Chem. Lett. 35 (2021) 127782
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of an anthranyl bridged optically active dinuclear iron
(II)-ligand and evaluation of DNA-cleaving activity
Yoshimi Ichimarua,f, Koichi Katoa,f, Yoshinori Okunob *, Yoshihiro Yamaguchic,
,
,
,*
Wanchun Jina, Mikako Fujitad, Masami Otsukad e, Masanori Imaia, Hiromasa Kurosakia
a College of Pharmacy, Kinjo Gakuin University, 2-1723 Omori, Moriyamaku, Nagoya, Aichi 463-8521, Japan
b Department of Medicinal Chemistry, Yokohama University of Pharmacy, 601 Matano-cho Totsuka-ku, Yokohama 245-0066 Japan
c Environmental Safety Center, Kumamoto University, 39-1 Kurokami 2-Chome, Chuo-ku, Kumamoto 860-8555, Japan
d Medicinal and Biological Chemistry Science Farm Joint Research Laboratory, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto
862-0973, Japan
e Department of Drug Discovery, Science Farm Ltd., 1-7-30 Kuhonji, Chuo-ku, Kumamoto 862-0976, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
It is necessary to design a ligand that is compatible with the target molecule to optimally use the DNA-cleaving
ability of metal complexes. In this study, we synthesized an optically active dinuclear ligand, (1R,1′R,2R,2′R)-N1,
Iron complex
Dinuclear ligand
DNA-cleaving activity
Anthracene
′
N1 -(anthracene-1,8-diylbis(methylene))bis(N2,N2-bis(pyridin-2-ylmethyl)cyclohexane-1,2-diamine) (R-ABDC,
4a) and its enantiomer (S-ABDC, 4b). We then prepared their Fe(II) complexes by mixing the ligand with FeS-
O4⋅7H2O in situ and investigated DNA-cleaving activities using plasmid DNA in the presence of excess sodium
ascorbate at atmospheric conditions. The Fe(II) complexes efficiently cleaved DNA and selectively recognized
two consecutive A and/or T sequences.
AT-rich
Introduction
In this study, we designed and synthesized the optically active
1′
dinuclear ligand, (1R,1′R,2R,2′R)-N1,N -(anthracene-1,8-diylbis(meth-
DNA cleavage by metal complexes has attracted attention as an
important medical bioinorganic chemical reaction. Since the phospho-
diester structure of DNA repels nucleophiles, it is protected against hy-
drolysis by nucleophiles. The DNA cleavage by metal complex occurs
through oxidative cleavage at the 4′-H of deoxyribose.1 One class of
these reagents includes the transition metal ion(s) in a molecule, such as
Fe(II)–EDTA,2 Cu(II)–(OP)2,3 and Ce(IV)–EDTA.4 However, since these
metal complexes do not have selectivity for DNA, they are rarely used for
pharmacotherapy.
ylene))bis(N2,N2-bis(pyridin-2-ylmethyl)cyclohexane-1,2-diamine) (R-
ABDC, 4a) and its enantiomer (S-ABDC, 4b) (Fig. 1). There are two
reasons for synthesizing the above mentioned ligands: first, optically
active molecules are expected to selectively recognize DNA,10 and sec-
ond, anthracene is well known for its intercalation with DNA.11,12
Anthracene ring with large and branched substituents is disadvantaged
in intercalation at GC sites because of the steric clash with the amino
group of guanine in the minor groove.13,14 Anthracenyl derivatives with
substituents at 9- and/or 10-positions selectively intercalate into the AT-
rich region of DNA. However, the 10-phenyl group, which is orthogonal
to the anthracene plane, prevent intercalation.15 There was a concern
that the bulky Fe(II) complex might interfere with the DNA intercalation
of anthracene scaffold. Moreover, anticancer drug actinomycin D, which
has two very bulky substituents at 1- and 9-phenoxizine ring, can
intercalate into DNA.16 Therefore, we designed 4ab that hold the Fe(II)
at the 1- and 8-positions of anthracene. 4ab has an anthracene moiety as
a bridge that connects two optically active mononuclear ligand struc-
tures, (1R,2R)-N1,N1-bis(pyridin-2-ylmethyl)cyclohexane-1,2-diamine
Bleomycin (BLM) is an anticancer agent that chelates an Fe(II) and
then initiates molecular oxygen uptake.5 The incorporated O2 is acti-
vated and cleaves DNA, which results in anticancer activity.5 BLM is
currently used to treat Hodgkin lymphoma6 and testicular cancer.7 It has
DNA recognition regions in the molecule, e.g., the bithiazole region
interacting with guanine; it differs from metal-complex reagents capable
of cleaving DNA.8,9 The design of artificial ligands compatible with the
target molecule is necessary to optimally use the DNA-cleaving ability of
metal complexes.
* Corresponding authors.
E-mail addresses: y.okuno@hamayaku.ac.jp (Y. Okuno), h-kurosaki@kinjo-u.ac.jp (H. Kurosaki).
f
These authors contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2021.127782
Received 8 October 2020; Received in revised form 23 December 2020; Accepted 2 January 2021
Available online 8 January 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

Y. Ichimaru et al.
Bioorganic & Medicinal Chemistry Letters 35 (2021) 127782
and dried, and the residue was reduced with NaBH4 in methanol at 45 ◦C
for 12 h to obtain the desired product 4a in 58% yield, followed by silica
gel column chromatography.18 The enantiomer, 4b, was also synthe-
sized using the same scheme.19
To investigate the DNA-cleaving activity of the synthesized ligands,
the Fe(II)–complexes of 4ab were prepared by mixing half-equimolar
ligand with FeSO4⋅7H2O in situ in the presence of excess sodium ascor-
bate.20 The pUC19 plasmid DNA-cleaving activity was evaluated using
the conversion ratio of the supercoiled form (Form I) into the relaxed
circular form (Form II) and the linear form (Form III) in a 50-mM Tris-
17
◦
borate buffer (pH 8.1) at 37 C. Changes into each form upon DNA
cleavage were detected by fluorescence intensity of ethidium bromide
using a densitometer after electrophoresis. For comparison, the DNA-
cleaving activities of 3ab were also investigated.
First, the concentration dependence of DNA cleavage was investi-
gated. The results are shown in Figs. 2A–E. In the absence of the ligand
(Fig. 2A), the Form I signal gradually decreased to 20% but did not
completely disappear when the Fe(II) concentration was increased from
5 μM to 50 μM. Form II increased in an Fe(II) concentration-dependent
manner, whereas Form III was not observed. When the equimolar
amount of the mononuclear ligand (3a or 3b) and Fe(II) was present, a
decrease in Form I was observed at low Fe(II) concentration (0.1–5 μM)
and completely disappeared at 10
was formed at the Fe(II) concentration of 5
3ab, the DNA-cleaving activity of the dinuclear ligands 4ab was
μ
M Fe(II) (Figs. 2B and C). Form III
μ
M or higher. Compared to
enhanced (Figs. 2D and E). At low Fe(II) concentration (0.1–1 μM), there
was no difference in the DNA-cleaving activity between mononuclear
and binuclear ligands. However, the amount of both Forms I and II
decreased in the presence of 10 μM of Fe(II) with a dinuclear ligand (lane
5 in Figs. 2D and E), and none of DNA strand forms were observed in the
presence of 30–50 M of Fe(II) with a dinuclear ligand. We speculated
μ
that the fragmentation of DNA strand occurred during the incubation.
There was no difference in the DNA-cleaving activity between enan-
tiomers in both mononuclear and dinuclear ligands. These results indi-
cated that the Fe(II) complexes of dinuclear ligands 4ab have higher
DNA-cleaving activity than those of mononuclear ligands 3ab. The
two cleaving sites connected by an anthracene bridge in 4ab would
orient to each strand of the supercoiled plasmid pUC19 DNA. In addi-
tion, the Fe(II) complexes of 4ab may easily gain accessibility to DNA
owing to the Fe(II) ion valence.
Fig. 1. Chemical structures of the novel ligands and Fe(II) complexes.
(R-DC, 3a) or its enantiomer (S-DC, 3b), respectively. The DNA-cleaving
activity of 4ab in the presence of FeSO4⋅7H2O and excess sodium
ascorbate under atmospheric conditions were investigated17 and
compared to those of 3ab to elucidate the efficacy of the anthracene
bridge.
Second, the reaction time dependence of the DNA-cleaving activity
was investigated. 3a (R-DC) and 4a (R-ABDC) were used for the time
dependence analysis because there was no difference in the DNA-
cleaving activities between the Fe(II) complexes of enantiomers. Fe(II)
The synthetic route for 4a is shown in Scheme 1. Briefly, the treat-
ment of benzyl (1R,2R)-2-aminocyclohexylcarbamate (1a) with two
equimolar amounts of 2-(chloromethyl) pyridine in the presence of
K2CO3 at 80 ◦C for 12 h led to the isolation of pyridylmethyl derivate 2a
in 60% yield, followed by silica gel column chromatography. The
deprotection of the carbobenzoxy group of 2a with 30% HBr-AcOH
yielded the mononuclear intermediate 3a. 3a was condensed with 1,8-
diformylanthracene in acetonitrile at 80 ◦C in the presence of a molec-
ular sieve (4A 1/16). The resulting crude Schiff base was concentrated
concentration was maintained at 30 μM, and the corresponding amounts
of ligands, 3a or 4a, were added. The results are shown in Fig. 3. When
the mononuclear ligand 3a coexisted with the Fe(II), the Form II signal
increased in response to the incubation time-dependent decrease in the
Form I signal. The Form III signal was not observed upon incubation for
0–15 min. In contrast, when the dinuclear ligand 4a coexisted with the
Fe(II), Form I almost disappeared and converted to Forms II and III
Scheme 1. Synthetic scheme of R-ABDC (4a). (a) Carbobenzoxy chloride (Cbz-Cl), aqueous EtOH, rt, 82%; (b) 2-(Chloromethyl)pyridine, K2CO3, CH3CN, 80 ◦C, 12
h, 60%; (c) 30% HBr-AcOH, 2 h, rt, 93%; (d) 1,8-Diformylanthracene, molecular sieve, CH3CN, 80 ◦C, 12 h, without further purification; (e) NaBH4, MeOH, 45 ◦C, 12
h, 58%.
2

Y. Ichimaru et al.
Bioorganic & Medicinal Chemistry Letters 35 (2021) 127782
Fig. 2. Concentration dependence of the DNA-cleaving activity. A–E) The pUC19 plasmid DNA strand (0.025 μM) was treated with A) absence of ligand, B) R-DC
(3a), C) S-DC (3b), D) R-ABDC (4a), and E) S-ABDC (4b) in the presence of FeSO4⋅7H2O and excess sodium ascorbate for 60 min at 37 ◦C.
enantiomers.
It is known that anthracene derivatives are AT-rich sequences spe-
cific DNA intercalator.22,23 Cationic amine substituted anthracene de-
rivatives have more than two-fold higher affinity to the minor groove of
double-stranded tetramers d(ATAT)2 than d(GCGC)2.22 Moreover,
selectivity is also exhibited in anthracene scaffold derivatives such as
aza-anthracenedione.23 In this study, we designed optically active li-
gands containing an anthracene bridge hoping to achieve sequence-
specific DNA recognition. As intended, the AT-rich specific DNA cleav-
age of Fe(II) complexes of dinuclear ligands was derived from the
anthracene bridge moiety of the ligand. These results suggested that the
anthracene moiety contributes to increase the DNA affinity of dinuclear
ligand more than optically active moiety. However, the sequence spec-
ificity of 4ab can be improved further. Oligopyrrole carboxamide
compounds such as distamycin analogs can specifically recognize AT-
rich sequence.24 These compounds, which are also called the minor
groove binders, have a concave-shaped framework that fits into the
convex-shaped DNA minor groove comprising the amino terminus at the
adenine. The Fe(II) complex of penta-N-methylpyrrolecarboxamide-
EDTA cleaves 3–5 base pairs contiguous to a 6-base-pair sequence 5′-
TTTTTA-3′.25 Compared with the minor groove binders, the Fe(II)–
complexes of 4ab roughly recognizes the sequence containing AT and
cleave it.
Fig. 3. Incubation time dependence of the DNA-cleaving activity. The super-
coiled pUC19 plasmid DNA strand (0.025 M) was treated with FeSO4⋅7H2O
(30 M), A) R-DC (30 M) (3a), B) R-ABDC (15 M) (4a), and sodium ascorbate
(4 mM) at 37 ◦C for the indicated period of time.
μ
μ
μ
μ
within 0–1 min of incubation. Forms II and III were gradually frag-
mented and unobservable within 15 min. Thus, the DNA-cleaving rate of
the Fe(II) complex of 3a was lower than that of 4a.
Kamitori and co-worker have reported the co-crystal structure of
actinomycin D, which prevent the transcription of DNA, with d
(GAAGCTTC)2.16 In the co-crystal structure, the phenoxazine-3-one ring
in actinomycin D intercalates into the middle sequence 5′-GC-3′ from the
Finally, to determine the sequence selectivity of the Fe(II) complexes
of 3ab and 4ab, a 254-base pair pBluescript II SK+ DNA fragment was
prepared using PCR.21 Owing to the differences in the DNA-cleaving
activities of mononuclear and dinuclear ligands, different concentra-
minor groove of DNA (Fig. 5A). The two cyclic depsipeptide rings (α- and
β-rings) lie on both sides of the minor groove and cover the four base
pairs. ABDC resembles actinomycin D in some structural features.
Therefore, it is presumed that the double-stranded DNA binding mode of
ABDC is analogous to that of actinomycin D. This configuration is ex-
pected to be appropriate to the cleavage for both chains of double-strand
DNA because the Fe(II) are located near difference strands (Fig. 5B).
There was no significant difference in DNA-cleaving ability between
the enantiomers. In Fe(II) uncoordinated state, the conformation of the
cyclohexane ring is flexible to a certain degree by ring flipping. Upon the
tions of Fe(II) were used, i.e., 30
μM of Fe(II) with 30 μM of 3ab and 10
μ
M of Fe(II) with 5 μM of 4ab. Cleavage sites are shown in Fig. 4. The Fe
(II) complex of 3ab cleaved DNA with a subtle sequence for 5′-CT-3′
(Fig. 4A). In contrast, the Fe(II) complex of 4ab may recognize AT-rich
sequences, which results in DNA cleavage (Fig. 4B). In particular, two
consecutive bases, 5′-AATTCCGGAA-3′ and 5′-TTAATT-3′, seem to be
specifically recognized by the Fe(II) complex of 4ab. There was no
critical difference in DNA-cleaving sequence specificity between
3

Y. Ichimaru et al.
Bioorganic & Medicinal Chemistry Letters 35 (2021) 127782
Fig. 4. Sequence-specificity of DNA cleaving by Fe(II)-synthesized ligand complexes. A) The pBluescript II SK+ DNA strand (0.025
μM) was treated with FeSO4⋅7H2O
(30
μ
M), R-DC (30
μ
M) (3a) or S-DC (30
μ
M) (3b), and sodium ascorbate (4 mM) for 60 min at 37 ◦C. B) The pBluescript II SK+ DNA strand (0.025
μM) was treated
with FeSO4⋅7H2O (10
μ
M), R-ABDC (5 μM) (4a) or S-ABDC (5 μ
M) (4b), and sodium ascorbate (4 mM) for 30 min at 37 ◦C.
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
We are grateful to Ms. Yukiko Ogata (Kumamoto University) for her
great contribution to the experimental work. This work was supported
by JSPS KAKENHI Grant Number JP145720341. This work was also
supported in part by the Kinjo Gakuin University-Parent Teacher Asso-
ciation Research Grant. The authors would like to thank Enago (www.
enago.jp) for the English language review.
Conflicts of interests
None declared.
References
Fig. 5. The binding mode of DNA intercalators to double-stranded DNA. A)
Crystal structure of the DNA–actinomycin D complex (PDB ID: 173D); B) Pro-
posed structure of DNA–Fe(II)-R-ABDC complex. These structures were visual-
ized by PyMol software (http://pymol.sourceforgen.net/). DNA strands are
represented in ribbon, and actinomycin D and R-ABDC are shown in stick.
1
2
3
4
5
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9
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coordination of Fe(II) with the ligand, the steric configuration of Fe(II)
ion may be fixed regardless of the absolute configuration of the
cyclohexane-1,2-diamine moiety. Consequently, each Fe(II) position is
considered to be the same against DNA strand. However, the cleavage
activity for the 5′-GTTAATTG-3′ sequence of S-ABDC was less than that
of R-ABDC. The elucidation of the mechanism for this difference is
helpful for more specific DNA-cleavage reagents. In addition, from our
preliminary study, we know that typical radical scavengers, such as
glycerol26 or SOD,27 do not decrease the DNA-cleaving activity (data not
shown). Therefore, we expected that diffusible radicals would not be
involved in DNA cleaving by Fe(II) complexes. Further studies are
needed to elucidate the effect of optical activity on DNA-cleaving ability
and the active species involved in DNA cleaving.
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17 Ida S, Iwamaru K, Fujita M, et al. Bioorg Chem. 2015;62:8.
In summary, we designed and synthesized novel optically active li-
gands, 4ab. We also investigated the DNA-cleaving ability of the Fe(II)
complexes of 4ab. Compared to the mononuclear Fe(II) complexes of
3ab, the dinuclear Fe(II) complexes of 4ab enhanced the DNA-cleaving
activity. Furthermore, the Fe(II) complex of 4ab recognized AT-rich
sequences without DNA recognition regions, such as a bithiazole moi-
ety in BLM. These results suggested the possibility of applying small-
molecule chemotherapy agents and artificial restriction enzymes via
oxidative cleavage. Future research will focus on elucidating the specific
ligand–DNA interaction and the active species for DNA cleaving.
18. 1H NMR (CDCl3, 500 MHz, ppm from TMS): δ 1.18-1.39 (8H, m), 1.74 (2H, d, J =
12.20 Hz), 1.84 (2H, d, J = 12.20 Hz), 2.08 (2H, d, J = 12.20 Hz), 2.35 (2H, d, J =
12.82 Hz), 2.56 (2H, ddd, J = 3.05 and 10.68 Hz), 2.70 (2H, ddd, J = 3.66 and
10.07 Hz), 3.68 and 3.78 (8H, d, J = 14.64 and 14.65 Hz), 3.97 and 4.52 (4H, d, J =
13.43 and 14.03), 6.84 (4H, t, J = 4.88 and 6.71 Hz), 7.02 (4H, d, J = 7.32 Hz), 7.09
(4H, ddd, J = 1.83 and 7.63 Hz), 7.40 (2H, t, J = 6.72 Hz), 7.49 (2H, d, J = 6.11 Hz),
7.88 (2H, d, J = 8.54 Hz), 8.27 (4H, d, J = 4.27 Hz), 8.36 (H, s), 8.97 (H, s). 13
C
NMR (CDCl3, 125 MHz, ppm from TMS): δ 23.40, 24.78, 25.72, 32.08, 48.63, 56.27,
58.70, 64.21, 119.02, 121.50, 122.52, 124.28, 124.88, 127.31, 127.39, 130.11,
131.80, 135.93, 137.13, 148.71, 160.03. FAB-MS [M+]: m/z 795. [
(% in MeOH).
α]
= - 105.4˚
D
25
19. 1H NMR (CDCl3, 500 MHz, ppm from TMS): δ 1.20-1.36 (8H, m), .174 (2H, d, J =
9.76 Hz), 1.84 (2H, d, J = 9.76 Hz), 2.08 (2H, d, J = 9.76 Hz), 2.38 (2H, m), 2.61
(2H, m), 2.73 (2H, m), 3.68 and 3.78 (8H, d, J = 14.65 Hz), 4.01 and 4.54 (4H, d, J
= 14.04 and 13.43 Hz), 6.82 (4H, t, J = 6.11 Hz), 7.00 (4H, d, J = 7.32 Hz), 7.08
(4H, t, J = 7.02 Hz), 7.41 (2H, t, J = 7.63 Hz), 7.52 (2H, d, J = 6.72 Hz), 7.89 (2H,
d, J = 8.54 Hz), 8.24 (4H, d, J = 3.67 Hz), 8.36 (H, s), 8.95 (H, s). 13C NMR (CDCl3,
125 MHz, ppm from TMS): δ 23.40, 24.70, 25.59, 31.74, 48.33, 56.19, 58.66, 64.05,
Declaration of Competing Interest
The authors declare that they have no known competing financial
4

Y. Ichimaru et al.
Bioorganic & Medicinal Chemistry Letters 35 (2021) 127782
118.82, 121.47, 122.49, 124.37, 124.85, 127.38, 127.42, 129.98, 131.70, 135.90,
ddCTP, and ddGTP). General protocol for DNA sequencing experiment; Reaction
mixtures containing the pBluescript II SK+ DNA strand (final concentration; 0.025
25
136.21, 148.63, 150.87. FAB-MS [M+]: m/z 795. [
α
]
= + 106.2˚(% in MeOH).
20 General protocol for DNA cleavage experiment; ReactionD mixtures containing 0.52
μM), FeSO4⋅7H2O and ligand in CH3OH (final concentration; 30 μM), and sodium
μ
g of pUC19 DNA in 50 mM Tris-borate buffer (pH 8.1), FeSO4⋅7H2O and ligand in
ascorbate aq (final concentration; 4 mM) were incubated at 37 ◦C for 30 min. After
the reaction, the aliquots of extracted samples were electrophoresed in a Long
Ranger polyacrylamide denaturing gel.
CH3OH, and 24 mM sodium ascorbate aq (final concentration; 4 mM) were incubated
at 37 ◦C for predetermined period. After the reaction, loading buffer was added and
then aliquots were electrophoresed in a 1% agarose gel (containing ethidium
bromide 0.5 mg /mL) at 100 V for 60 min. The fluorescence intensity of ethidium
bromide was detected using a densitometer (AE-6900M, ATTO Co., Ltd. Japan).
21 Preparation of pBluescript II SK+ DNA strand; Forward primer: 5’-
22 Chen KX, Gresh N, Pullman B. Nucleic Acids Res. 1986;14:9103.
23 Antonini I, Santoni G, Lucciarini R, et al. J Med Chem. 2008;51:997.
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26 Shah SV, Walker PD. Am J Physiol. 1988;255:F438.
CACGACGTTGTAAAACGAC-3’ (19 mer); Reverse primer: 5’-
´
´
´ ´
˜
GGATAACAATTTCACACAGG-3’ (20 mer); Sequence markers were prepared by PCR
methods using fluorescently labeled 2’,3’-deoxyribonucleotide (ddATP, ddGTP,
27 Feher J, Lang I, Nekam K, Gergely P, Muzes G. Tokai J Exp Clin Med. 1990;15:129.
5