Novel metal chelating molecules with anticancer activity. Striking effect of the imidazole substitution of the histidine-pyridine-histidine system

Article abstract of DOI:10.1016/j.bmc.2015.07.044

Previously we have reported a metal chelating histidine-pyridine-histidine system possessing a trityl group on the histidine imidazole, namely HPH-2Trt, which induces apoptosis in human pancreatic adenocarcinoma AsPC-1 cells. Herein the influence of the imidazole substitution of HPH-2Trt was examined. Five related compounds, HPH-1Trt, HPH-2Bzl, HPH-1Bzl, HPH-2Me, and HPH-1Me were newly synthesized and screened for their activity against AsPC-1 and brain tumor cells U87 and U251. HPH-1Trt and HPH-2Trt were highly active among the tested HPH compounds. In vitro DNA cleavage assay showed both HPH-1Trt and HPH-2Trt completely disintegrate pUC19 DNA. The introduction of trityl group decisively potentiated the activity.

Full text of DOI:10.1016/j.bmc.2015.07.044

Accepted Manuscript
Novel metal chelating molecules with anticancer activity. Striking effect of the
imidazole substitution of the histidine-pyridine-histidine system
Taha F.S. Ali, Kana Iwamaru, Halil Ibrahim Ciftci, Ryoko Koga, Masahiro
Matsumoto, Yasunori Oba, Hiromasa Kurosaki, Mikako Fujita, Yoshinari
Okamoto, Kazuo Umezawa, Mitsuyoshi Nakao, Takuichiro Hide, Keishi
Makino, Jun-ichi Kuratsu, Mohamed Abdel-Aziz, Gamal El-Din A.A. Abuo-
Rahma, Eman A.M. Beshr, Masami Otsuka
PII:
S0968-0896(15)00628-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.07.044
BMC 12473
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 May 2015
21 July 2015
24 July 2015
Please cite this article as: Ali, T.F.S., Iwamaru, K., Ciftci, H.I., Koga, R., Matsumoto, M., Oba, Y., Kurosaki, H.,
Fujita, M., Okamoto, Y., Umezawa, K., Nakao, M., Hide, T., Makino, K., Kuratsu, J-i., Abdel-Aziz, M., Abuo-
Rahma, G.E-D., Beshr, E.A.M., Otsuka, M., Novel metal chelating molecules with anticancer activity. Striking
effect of the imidazole substitution of the histidine-pyridine-histidine system, Bioorganic & Medicinal Chemistry
(2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.07.044
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Novel metal chelating molecules with anticancer activity.
Striking effect of the imidazole substitution of the histidine-pyridine-histidine system.
Taha F. S. Alia, Kana Iwamarua, Halil Ibrahim Ciftcia, Ryoko Kogaa, Masahiro Matsumoto,a Yasunori Oba,a
Hiromasa Kurosakib, Mikako Fujitac, Yoshinari Okamotoa, Kazuo Umezawae, Mitsuyoshi Nakaod, Takuichiro
Hidef, Keishi Makinof, Jun-ichi Kuratsuf, Mohamed Abdel-Azizg, Gamal El-Din A. A. Abuo-Rahmag, Eman A. M.
Beshrg,h, Masami Otsukaa*
a
Department of Bioorganic Medicinal Chemistry, Faculty of Life Sciences, Kumamoto University, 5-1
Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
b College of Pharmacy, Kinjo Gakuin University, 2-1723 Omori, Moriyama-ku, Nagoya, Aichi 463-8521, Japan
c Research Institute for Drug Discovery, School of Pharmacy, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku,
Kumamoto 862-0973, Japan
d Department of Molecular Target Medicine Screening, School of Medicine, Aichi Medical University, Nagakute,
Aichi 480-1195, Japan
e
Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University,
Kumamoto 860-0811, Japan
f
Department of Neurosurgery, Faculty of Medicine, Kumamoto University, 1-1-1 Honjo Chuo-ku,
Kumamoto 860-8556, Japan
g Department of Medicinal Chemistry, Faculty of Pharmacy, Minia Uniiversity, 61519 Minia, Egypt
h Department of pharmaceutical chemistry, College of Pharmacy, Umm Al-Qura University, Makkah 21955, Saudi
Arabia
* Corresponding author. E-mail address: motsuka@gpo.kumamoto-u.ac.jp
Keywords
Histidine-pyridine-histidine, AsPC-1, U87, U251, DNA cleavage

Abstract
Previously we have reported a metal chelating histidine-pyridine-histidine system possessing a trityl group
on the histidine imidazole, namely HPH-2Trt, which induces apoptosis in human pancreatic adenocarcinoma
AsPC-1 cells. Herein the influence of the imidazole substitution of HPH-2Trt was examined. Five related
compounds, HPH-1Trt, HPH-2Bzl, HPH-1Bzl, HPH-2Me, and HPH-1Me were newly synthesized and screened
for their activity against AsPC-1 and brain tumor cells U87 and U251. HPH-1Trt and HPH-2Trt were highly
active among the tested HPH compounds. In vitro DNA cleavage assay showed both HPH-1Trt and HPH-2Trt
completely disintegrate pUC19 DNA. The introduction of trityl group decisively potentiated the activity.

Introduction
Bleomycin is an antitumor antibiotic consisting of a metal-binding/oxygen-activating site and a
DNA-binding site, inducing oxidative cleavage of DNA strand.1) Bleomycin forms an iron complex by the
-aminoalanine-pyrimidine--hydroxyhistidine region (Figure 1A). Previously we have designed
-aminoalanine-pyridine--hydroxyhistidine ligands, namely PYML compounds, based on the direct analogy to
the bleomycin metal core (Figure 1B).1) In particular, the Fe(II) complex of PYML-6 activates molecular oxygen
as efficiently as bleomycin does,2) and PYML-8 was found to be superior to bleomycin in oxygen activation.3)
However, although PYML-6-Fe(II) complex showed efficient oxygen activation, it was virtually inactive in the
DNA cleaving reaction.4) This must be due to the lack of the DNA-binding moiety in PYML-6, and, in fact,
introduction of the DNA binding site into the PYML-6 skeleton resulted in the acquisition of the DNA cleaving
capability.1)
For the cellular DNA cleavage, bleomycin must be delivered inside the nucleus to contact DNA. Bleomycin
is capable of this presumably owing to the complicated total structure including the
L-gulose-3-O-carbamoyl-D-mannose disaccharide for the nuclear transport and the bithiazole moiety for the DNA
binding.
Figure 1. Structure of bleomycin (A) and synthetic analogues PYML-6, PYML-8 (B).
For the further investigation of such DNA-binding/oxygen-activation mode of action, we designed a novel
ligands of symmetrized coordination environment histidine-pyridine-histidine ligand, namely HPH compounds

(Figure 2).5) Iron complex of HPH showed efficient oxygen activation that was plausibly optimized by introducing
an electron donating dimethylamino substituent into the pyridine ring, as demonstrated by the ESR spin trapping
experiment.5) Iron complex of unsymmetrical HPH-M showed oxygen-activating property as demonstrated by
ESR spin trapping experiment and pUC19 DNA cleaving activity, producing form II at 10 M and form III at 50
M.6)
Introduction of triphenylmethyl (trityl, Trt) group into the histidine imidazole profoundly altered the activity.
HPH-2Trt induced the cell death of human pancreatic adenocarcinoma AsPC-1 cells in 24 h, causing apoptotic
morphological change and internucleosomal degradation of DNA.7) It was remarkable since AsPC-1 cells are
apoptotic resistant; Bleomycin A2, adriamycin, and vinblastine did not induce apoptosis in AsPC-1 cells even at
8)
high concentrations.7,
HPH-2Trt-induced cell death was not inhibited by antioxidants such as reduced
glutathione and N-acetylcysteine.7) Induction of apoptosis by HPH-2Trt was inhibited by zinc and copper ions.8)
Thus, the imidazole substitution seemed to exert a profound effect on the biological function of HPH. In this
study, we examined the effect of the imidazole substituent by synthesizing novel HPH compounds, i. e., HPH-1Trt,
HPH-1Bzl, HPH-2Bzl, HPH-1Me, HPH-2Me, besides HPH-2Trt.
Figure 2. Structure of Histidine-Pyridine-Histidine compounds.
These compounds were synthesized as shown in the Scheme 1. Whereas pyridinedicarbaldehyde 5 has
previously been prepared starting with chelidamic acid,3,5) we now prepared it by repeating the formylation of
4-dimethylaminopyridine 1. Thus, 1 was treated with t-BuLi followed by DMF to afford monoaldehyde 29) that
was, without further purification, converted to the corresponding acetal 3 in 56% overall yield. The second
formylation of acetal 3 under the same condition gave acetal-aldehyde 4 in 58% yield. Condensation of aldehyde
4 with histidine methyl ester 6 followed by NaBH(OAc)3 reduction furnished secondary amine-acetal 7 (35.8%
yield) which was transformed to aldehyde 8 by acid treatment (66.7% yield). Aldehyde 8 was treated with
imidazole-substituted histidine methyl ester 910),1011), or 1112) to give the corresponding imine which upon
NaBH(OAc)3 reduction gave imidazole-monosubstituted products, HPH-1Trt, HPH-1Bzl, and HPH-1Me in 51%,
11.6%, and 7.7% yields, respectively.
Imidazole-disubstituted compounds were synthesized as follows. Acid treatment of aldehyde-acetal 4 gave
dialdehyde 55) in 98% yield. Treatment of 5 with imidazole-substituted histidine methyl ester 9, 10, or 11 followed
by NaBH(OAc)3 reduction gave imidazole-disubstituted products, HPH-2Trt13), HPH-2Bzl, and HPH-2Me in 22%,
30.7%, and 10.2% yields, respectively. Treatment of HPH-2Trt with acetic acid gave HPH5) in 31.8% yield.

Scheme 1. Synthesis of imidazole-substituted HPHs. Reagents and conditions. a. BF3·OEt2, THF, 0˚C, 1h; t-BuLi,
DMF, -78˚C, 4h; -78˚C to rt, overnight. b. CH(OC2H5)3, p-TsOH, C2H5OH, reflux, 48h. c. BF3.OEt2, THF, 0˚C,

1h; t-BuLi, DMF, -78˚C, 4h; -78˚C to rt, overnight. d. His-OMe6, NaBH(OAc)3, dichloroethane, rt, 24h. e. 1N
HCl, reflux, 30min. f. 9, 10, or 11, NaBH(OAc)3, dichloroethane, rt, 24h. g. 6N HCl, C2H5OH, reflux, 4h. h. 9, 10,
or 11, NaBH(OAc)3, dichloroethane, rt, 48h. i. 90% aq AcOH, 60˚C, 1.5hr.
The DNA cleaving capability of the Fe(II) complexes of HPH compounds was studied by the relaxation of
the supercoiled pUC19 DNA to linear DNA (Figure 3). Control experiment using FeSO4, H2O2, and ascorbic acid
omitting the HPH compound showed the formation of nicked form II DNA. Experiments in the presence of HPH,
HPH-1Me, HPH-2Me, HPH-1Bzl, and HPH-2Bzl resulted in the formation of increased amount of form II DNA.
In contrast, trityl substituted HPHs produced smeared DNA. In particular, HPH-2Trt completely disintegrated
pUC19 DNA.
Figure 3. Cleavage of supercoiled pUC19 DNA analyzed by an agarose-gel electrophoresis. pUC19 DNA
(2µg) was treated with HPH compounds (30µM) in water and Tris /boric acid buffer (10mM, pH 8.5) in the
presence and absence of FeSO4·7H2O (30µM), H2O2 (30µM), and ascorbic acid (30µM) as an activator. The
reaction mixture was incubated at 37 ºC for 1.5 h.
In order to examine the oxygen activating property of HPH compounds, ESR spin trapping experiment
using -phenyl-N-tert-butylnitrone (PBN) was carried out (see Supplementary Material). The observed species
exhibited ESR aN and aH hyperfine splitting characteristic to the PBN spin adduct. The magnitude of the signal
obtained by the HPH-1Trt-Fe(II) and HPH-2Trt-Fe(II) complexes was remarkably smaller than that obtained by
the control Fe(II), contrary to the case of imidazole-unsubstituted HPH and HPH-M (Figure S1, S2, S4). ESR
signals of the spin adduct obtained by HPH-2Trt-Fe(III)-H2O2, HPH-1Trt-Fe(III)-H2O2, HPH-2Bzl-Fe(III)-H2O2,
and HPH-2Me-Fe(III)-H2O2, were uniformly smaller than that of the control Fe(III)-H2O2 (Figure S9, S10, S11,
S12, S13). These results indicate that the imidazole substituents suppressed the radical generating property
whereas the pUC19 DNA was completely cleaved by HPH-2Trt. We suppose that the trityl substitution on the
imidazole of HPH might induce DNA affinity.
Previously we prepared Zn complex of HPH and HPH-2Trt and measured high resolution mass spectra.13)
The exact mass obtained was that of 1:1 Zn-ligand complex, Zn-HPH and Zn-HPH-2Trt.13) These results
suggested that HPH and HPH-2 Trt also form 1:1 iron complex. We consider that the two bulky trityl groups serve
as a steric factor that accommodates oxygen, stabilizing the iron-bound oxygen complex, as demonstrated by our

first-generation bleomycin model14) and Collman’s “picket fence” porphyrin.15)
There could be two kind of active oxygen species anticipated, i.e., dispersed oxygen radical (such as
hydroxyl radical) and iron-bound oxygen (such as oxo-ferryl).16) It is most probable that HPH-2Trt generates
oxo-ferryl species predominantly7, 17) concomitant with a small amount of dispersed oxygen. In the spin trapping
experiment, the yield of the spin adduct was small, suggesting the amount of the dispersed oxygen radical
produced was small. In the DNA cleavage experiment, the predominant oxo-ferryl species abstract hydrogen from
the exposed C4 or C5 of the DNA deoxyribose, resulting in the exhaustive DNA cleavage. We also tried the
epoxidation of stilbene using HPH-2Trt-Fe(II)-H2O2, resulting in low yield of the stilbene oxide (data not shown).
Probably stilbene can hardly approach the iron-oxo center because of the steric repulsion between the benzene
rings of stilbene and trityl group.
Anticancer effect of seven synthetic HPH compounds (10 M) were compared (Figure 4). In general,
HPH-2Trt was uniformly effective not only against pancreatic adenocarcinoma AsPC-1 but also brain tumour U87
and U251 cells. HPH-1Trt was less effective and the other HPHs were not effective. This tendency in anticancer
activity is in consistent with the DNA cleavage potency.

(A)
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Figure 4. Anticancer effect of HPH, HPH-1Me, HPH-2Me, HPH-1Bzl, HPH-2Bzl, HPH-1Trt, and HPH-2Trt
(each 10 M) against AsPC-1 (A), U87 (B), and U251 (C) cells.
Conclusion
As we reported previously, HPH-2Trt induces apoptosis in the human pancreatic adenocarcinoma AsPC-1
cell which is apoptotic resistant.7,8) Herein we prepared a series of HPH compounds possessing various imidazole
substituents, namely HPH-1Trt, HPH-2Bzl, HPH-1Bzl, HPH-2Me, and HPH-1Me, and examined the influence of
the imidazole substitution. HPH-1Trt and HPH-2Trt were highly active against AsPC-1, U87, and U251 cells
while the other HPH compounds were virtually inactive. In vitro DNA cleavage assay showed both HPH-1Trt and
HPH-2Trt strongly disintegrate pUC19 DNA albeit the imidazole substitution did not enhance the apparent
oxygen radical formation. Since the cleavage of the trityl group requires acidic condition and trityl-His could not
be a substrate of enzymes for natural amino acid metabolism, HPH-1Trt and HPH-2Trt are thought to be stable
under physiological condition. The potency in the DNA cleavage and antitumor activity of the trityl-substituted
HPHs was decisively different from that of methyl- and benzyl-substituted HPHs in all-or-none manner.
Experimental
Material and Method
Chemicals were purchased from Aldrich, Fluka, Kanto Chemical, Nacalai Tesque, Tokyo Chemical Industry and
Wako. Thin layer chromatography (TLC) was performed on precoated plates (Merck TLC sheets silica 60 F254
and Fuji silysia chemical Ltd. TLC sheets Chromatorex NH silica). Chromatography was carried out on Silica Gel
60 N (40–100 mesh) and NH silica gel Chromatorex (NH, 100-200mesh) (FUJI SILYSIA CHEMICAL LTD.).
Melting points were determined on a Yanagimoto Melting Point Apparatus and were uncorrected. NMR spectra
(JEOL JNM-AL300MHz) were referenced to TMS. Infra-red spectra were recorded on a JASCO FT/IR-410 or
JEOL JIR-6500W / PerkinElmer Frontier. The samples were prepared as KBr discs or Film or using ATR method.
Mass spectra (EI) and high-resolution mass spectra (HRMS) were recorded by a JEOL JMSDX303HF. HRMS
were recorded by using positive fast atom bombardment (FAB) with 3-nitrobenzyl alcohol (NBA) as the matrix.
4-(Dimethylamino)picolinaldehyde (2).
Under argon atmosphere dissolve 1 (3 g, 24.57 mmol) in dist. THF (160 ml). BF3·OEt2 (4.6 ml, 36.84 mmol) was
added and the mixture was stirred at 0 ˚C for 1h. t-BuLi (26 ml, 49.12 mmol) was added drop wise at -78 ˚C.
DMF (5.0 ml, 98.24 mmol) was added and the mixture was stirred at -78 ˚C for 4h, then from -78 ˚C to rt
overnight. A solution of H2O-saturated aqueous NaHCO3 (1:1, 100ml) was added and excess THF was evaporated
under vacuum then extracted with CH2Cl2 (50 ml X 5) then brine (50 ml). The organic layer was dried over
MgSO4 and concentrated in vacuo. The residue was used in the next step without further purification. A pure
sample for the characterization of 2 was obtained by flash chromatography (EtOAc) to afford 29) as viscous
yellow oil. 1H NMR (300 MHz, CDCl3) δ 9.95 (s, 1H), 8.34 (d, J = 5.8 Hz, 1H), 7.14 (d, J = 2.8 Hz, 1H), 6.62 (dd,
J = 5.9, 2.8 Hz, 1H), 3.03 (s, 6H). FAB-MS (m/z): 151 [M+H]+.
2-(Diethoxymethyl)-N,N-dimethylpyridin-4-amine (3).
To a solution of 2 (crude) in dist. EtOH (140 ml) was added triethyl orthoformate (7.5 ml, 44.18 mmol) and

p-toulenesulfonic acid monohydrate (6.72 g, 35.34 mmol). The mixture was refluxed for 48h under argon
atmosphere, then concentrated in vacuo. A solution of H2O-saturated aqueous NaHCO3 (1:1, 60 ml) was added
and extracted with CH2Cl2 (50 ml X 5) then brine (50 ml). The organic layer was dried over MgSO4 and
concentrated in vacuo. The residue was chromatographed on silica gel (eluted with CH2Cl2 : MeOH = 30:1 to 5 :
1) to afford 3 (3.09 g, 56% 2 steps) as brown oil. 1H NMR (300 MHz, CDCl3) δ 8.18 (d, J = 6.0 Hz, 1H), 6.80 (d,
J = 2.7 Hz, 1H), 6.42 (dd, J = 6.0, 2.7 Hz, 1H), 5.33 (s, 1H), 3.72 (dq, J = 9.5, 7.1 Hz, 2H), 3.59 (dq, J = 9.5, 7.0
Hz, 2H), 2.99 (s, 6H), 1.24 (t, J = 7.1 Hz, 6H).13C NMR (75 MHz, CDCl3) δ 158.52, 155.35, 149.23, 106.57,
103.71, 103.37, 62.61, 39.24, 15.26. HRMS (FAB). Calcd for C12H21N2O2: 224.1525. Found: 224.1608.
6-(Diethoxymethyl)-4-(dimethylamino)picolinaldehyde (4).
Under argon atmosphere dissolve 3 (356 mg, 1.59 mmol) in dist. THF (16 ml). BF3·OEt2 (0.3 ml, 2.4 mmol) was
added and the mixture was stirred at 0 ˚C for 1h. t-BuLi (2.5 ml, 4.72 mmol) was added dropwise at -78 ˚C. DMF
(0.33 ml, 6.48 mmol) was added and the mixture was stirred at -78 ˚C for 4h, then from -78 ˚C to rt overnight. A
solution of H2O-saturated aqueous NaHCO3 (1:1, 60 ml) was added and excess THF was evaporated under
vacuum then extracted with CH2Cl2 (30 ml X 5) then brine (30 ml). The organic layer was dried over MgSO4 and
concentrated in vacuo. The residue was chromatographed on silica gel (eluted with n-hexane:EtOAc = 1:1) to
1
afford 4 (231 mg, 58%) as a pale yellow solid. H NMR (300 MHz, CDCl3) δ 9.99 (s, 1H), 7.12 (d, J = 2.6 Hz,
1H), 6.97 (d, J = 2.6 Hz, 1H), 5.41 (s, 1H), 3.80 – 3.69 (m, 2H), 3.66 – 3.55 (m, 2H), 3.06 (s, 6H), 1.25 (t, J = 7.1
Hz, 6H). 13C NMR (75 MHz, CDCl3) δ 15.2, 39.4, 62.6, 103.1, 104.2, 106.8, 152.6, 155.6, 159.2, 194.7; FAB-MS
(m/z): 253 [M+H]+.
4-(Dimethylamino)pyridine-2,6-dicarbaldehyde (5).
To a solution of 4 (230 mg, 0.91 mmol) in EtOH (1 ml) was added 6N HCl (20 ml). The mixture was refluxed for
4h under an argon atmosphere, then concentrated in vacuo. A solution of H2O-saturated aqueous NaHCO3 (1:1, 60
ml) was added and extracted with EtOAc (50 ml X 3) then brine (50 ml). The organic layer was dried over
1
Na2SO4 and concentrated in vacuo to afford 55) (159 mg, 98%) as a yellow solid. H NMR (300 MHz, CDCl3) δ
10.06 (s, 2H), 7.31 (s, 2H), 3.13 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 194.06, 155.72, 146.91, 107.50, 39.62. IR
(KBr) 3080, 2926, 1670, 1226, 1068, 970, 876, 852, 693 cm-1; EI-MS (m/z):178 [M]+.
Methyl
(S)-2-({[6-(diethoxymethyl)-4-(dimethylamino)pyridin-2-yl]methyl}amino)-3-(1H-imidazol-4-yl)propanoate
(7).
The HCl salt of His-OMe 6 was extracted using saturated Na2CO3 aqueous solution and CHCl3 to give neutral
His-OMe (175 mg, 1.03 mmol), which was subsequently dissolved in 3 ml ClCH2CH2Cl followed by the addition
of 4 (260 mg, 1.03 mmol). NaBH(OAc)3 (250 mg, 1.18 mmol) was added and the mixture was stirred at room
temperature for 24 h under argon atmosphere. A solution of saturated aqueous NaHCO3 (5 ml) was added and the
organic layer was separated. The aqueous layer was extracted with CH2Cl2 (10 ml X 3). The combined organic
layers were dried over Na2SO4 and concentrated in vacuo. The residue was chromatographed on NH silica gel
1
(eluted with CH2Cl2 : MeOH = 30:1) to afford 7 (145 mg, 35.8%) as a yellow oil. H NMR (300 MHz, CDCl3) δ
7.65 (s, 1H), 6.79 (d, J = 0.7 Hz, 1H), 6.73 (d, J = 2.5 Hz, 1H), 6.37 (d, J = 2.1 Hz, 1H), 5.41 (s, 1H), 4.00 (d, J =

13.9 Hz, 1H), 3.77 (d, J= 13.9 Hz, 1H), 3.73 (s, 3H), 3.71 – 3.56 (m, 5H), 3.10 (dd, J = 15.3, 3.8 Hz, 1H), 3.01 (s,
6H), 2.93 (dd, J = 15.3, 7.9 Hz, 1H), 1.23 (t, J = 7.1 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ 174.49, 158.85,
158.29, 155.89, 135.95, 130.00, 121.60, 104.94, 102.81, 102.67, 62.17, 61.99, 60.65, 52.71, 52.16, 39.37, 15.28,
28.70. IR (Film) 3570, 2977, 2886, 1733, 1608, 1549, 1509, 1441, 1391, 1173, 1110, 1060, 734 cm-1; MS (FAB)
m/z 406 (M+H)+; HRMS (FAB). Calcd for C20H32N5O4: 406.2454. Found: 406.2484.
Methyl
(8).
(S)-2-({[4-(dimethylamino)-6-formylpyridin-2-yl]methyl}amino)-3-(1H-imidazol-4-yl)propanoate
A solution of 7 (477 mg, 1.18 mmol) in aqueous 1N HCl (20 ml) was heated to reflux for 30 min. After cooling to
room temperature, the mixture was adjusted to pH 8 with solid NaHCO3 and was extracted with CH2Cl2 (20 ml X
3). The organic layer was dried over Na2SO4 and concentrated in vacuo to afford 8 (260 mg, 66.7%) as yellow
1
solid. H NMR (300 MHz, CDCl3) δ 10.01 (s, 1H), 7.58 (s, 1H), 7.08 (d, J = 2.6 Hz, 1H), 6.84 (s, 1H), 6.60 (s,
1H), 4.06 (d, J = 13.9 Hz, 1H), 3.82 (d, J = 14.2 Hz, 1H), 3.74 (s, 3H), 3.64 (dd, J = 8.7, 4.0 Hz, 1H), 3.15 – 3.08
(m, 1H), 3.07 (s, 6H), 2.93 (dd, J = 15.3, 7.8 Hz, 1H). IR (Film) 3416, 3102, 2952, 1734, 1608, 1543, 1510, 1439,
1397, 1214, 1102, 1027, 983, 937 cm-1. MS (FAB) m/z 332 (M+H)+.
Methyl
(S)-2-({[6-({[(S)-3-(1H-imidazol-4-yl)-1-methoxy-1-oxopropan-2-yl]amino}methyl)-4-(dimethylamino)pyridi
n-2-yl]methyl}amino)-3-(1-trityl-1H-imidazol-4-yl)propanoate (HPH-1Trt).
To a solution of 910) (452 mg, 1.1 mmol) in 10 ml ClCH2CH2Cl was added 8 (365 mg, 1.1 mmol) and
NaBH(OAc)3 (266 mg, 2.2 mmol). The mixture was stirred at room temperature for 48 h under argon atmosphere.
A solution of saturated aqueous NaHCO3 (10 ml) was added and the organic layer was separated. The aqueous
layer was extracted with CH2Cl2 (20 ml X 3). The combined organic layers were dried over Na2SO4 and
concentrated in vacuo. The residue was chromatographed on NH silica gel (eluted with EtOAc : MeOH = 10:1) to
afford HPH-1Trt (413mg, 52%) as a low melting white solid. 1H NMR (300 MHz, CD3OD) δ 7.55 (d, J = 1.1Hz,
1H), 7.35 (d, J = 1.5 Hz, 1H), 7.34 – 7.08 (m, 15H), 6.82 (s, 1H), 6.67 (d, J = 1.4 Hz, 1H), 6.53 (d, J = 2.4 Hz,
1H), 6.52 (d, J = 2.4 Hz, 1H), 3.76 (dd, J = 14.2, 4.4 Hz, 2H),3.72 (s, 3H), 3.56 (s, 3H), 3.34 – 3.29 (m, 4H), 2.95
(s, 6H), 3.01 – 2.88 (m, 4H). 13C NMR (75 MHz, CD3OD) δ 175.93, 175.86, 159.49, 157.62, 143.77, 139.57,
137.94, 136.37, 131.00, 129.48, 129.39, 121.38, 104.87, 104.77, 76.85, 62.29, 62.04, 53.74, 53.66, 52.32, 52.29,
39.38, 32.69. IR (KBr) 3201, 1736, 1604, 1550, 1442, 1392, 1169, 995, 829, 756, 702 cm-1. MS (FAB) m/z 727.3
(M+H)+ ; HRMS (FAB). Calcd for C42H47N8O4: 727.3720. Found: 727.3721.
Methyl
(S)-2-({[6-({[(S)-3-(1H-imidazol-4-yl)-1-methoxy-1-oxopropan-2-yl]amino}methyl)-4-(dimethylamino)pyridi
n-2-yl]methyl}amino)-3-(1-benzyl-1H-imidazol-4-yl)propanoate (HPH-1Bzl).
To a solution of 1011) (156 mg, 0.6 mmol) in 20 ml ClCH2CH2Cl was added 8 (199 mg, 0.6 mmol) and
NaBH(OAc)3 (255 mg, 1.2 mmol). The mixture was stirred at room temperature for 48 h under argon atmosphere.
A solution of saturated aqueous NaHCO3 (10 ml) was added and the organic layer was separated. The aqueous
layer was extracted with CH2Cl2 (20 ml X 3). The combined organic layers were dried over Na2SO4 and
concentrated in vacuo. The residue was chromatographed on NH silica gel (eluted with EtOAc : MeOH = 10:1) to

afford HPH-1Bzl (40 mg, 11.6%) as a yellow oil. 1H NMR (300 MHz, CDCl3) δ 7.51 (d, J= 0.9 Hz, 1H), 7.44 (d,
J= 1.3 Hz, 1H), 7.37 – 7.29 (m, 3H), 7.15 – 7.09 (m, 2H), 6.82 (s, 1H), 6.67 (d, J= 1.1 Hz, 1H), 6.44 (d, J = 1.8
Hz, 1H), 6.28 (d, J = 2.0 Hz, 1H), 5.02 (s, 2H), 3.92 (d, J = 13.7 Hz, 1H), 3.87 (d, J = 11.9 Hz, 1H), 3.74 – 3.60
(m, 4H), 3.72 (s, 3H), 3.61 (s, 3H), 3.08 (dd, J= 15.0 Hz, 4.0 Hz, 1H), 3.03 – 2.88 (m, 3H), 2.96 (s, 6H). 13C NMR
(75 MHz, CDCl3) δ 175.17, 174.62, 159.13, 158.64, 155.70, 138.78, 137.12, 136.36, 135.38, 129.12, 128.39,
127.42, 117.09, 103.90, 103.69, 61.26, 60.54, 53.75, 53.07, 52.03, 51.72, 50.82, 39.24, 32.22. IR (ATR) 3079,
2931, 1731, 1602, 1548 cm-1. MS (FAB) m/z 575.5 (M+H)+ ; HRMS (FAB). Calcd for C30H39N8O4: 575.3094.
Found: 575.3065.
Methyl
(S)-2-({[6-({[(S)-3-(1H-imidazol-4-yl)-1-methoxy-1-oxopropan-2-yl]amino}methyl)-4-(dimethylamino)pyridi
n-2-yl]methyl}amino)-3-(1-methyl-1H-imidazol-4-yl)propanoate (HPH-1Me).
To a solution of 1112) (85 mg, 0.46 mmol) in 15 ml DCE was added 8 (152 mg, 0.46 mmol) and NaBH(OAc)3
(195 mg, 0.92 mmol). The mixture was stirred at room temperature for 48 h under argon atmosphere. A solution of
saturated aqueous NaHCO3 (10 ml) was added and the organic layer was separated. The aqueous layer was
extracted with CH2Cl2 (15 ml X 3). The combined organic layers were dried over Na2SO4 and concentrated in
vacuo. The residue was chromatographed on NH silica gel (eluted with EtOAc : MeOH = 5:1) to afford
HPH-1Me (17.7 mg, 7.7%) as a pale yellow oil. 1H NMR (300 MHz, CD3OD) δ 7.56 (d, J = 1.1 Hz, 1H), 7.47 (d,
J = 0.8 Hz, 1H), 6.85 (d, J = 1.2 Hz, 1H), 6.84 (s, 1H), 6.54 (d, J = 2.4 Hz, 1H), 6.52 (d, J = 2.5 Hz, 1H), 3.76 (dd,
J = 14.3, 4.2 Hz, 2H), 3.65 (s, 3H), 3.64 (s, 3H), 3.64 (s, 3H), 3.61 – 3.56 (m, 4H), 3.00 (s, 6H), 2.98-2.81 (m, 4H).
13C NMR (75 MHz, CD3OD) δ 176.01, 175.95, 159.37, 159.34, 157.64, 139.00, 138.58, 136.39, 119.86, 104.90,
62.01, 53.58, 52.28, 52.25, 39.34, 33.58, 32.59. IR (ATR) 3212, 2950, 1731, 1606 cm-1. MS (FAB) m/z 499.4
(M+H)+ ; HRMS (FAB). Calcd for C24H35N8O4: 499.2781. Found: 499.2798.
Dimethyl
(2S,2’S)-2,2'-({[4-(dimethylamino)pyridine-2,6-diyl]bis(methylene)}bis(azanediyl))bis[3-(1-trityl-1H-imidaz
ol-4-yl)propanoate] (HPH-2Trt).
To a solution of 910) (1.032 g, 2.51 mmol) in 20 ml ClCH2CH2Cl was added 5 (224 mg, 1.26 mmol) and
NaBH(OAc)3 (1.1 g, 5.02 mmol). The mixture was stirred at room temperature for 48 h under argon atmosphere.
A solution of saturated aqueous NaHCO3 (20 ml) was added and the organic layer was separated. The aqueous
layer was extracted with CH2Cl2 (25 ml X 3). The combined organic layers were dried over Na2SO4 and
concentrated in vacuo. The residue was chromatographed on NH silica gel (eluted with CH2Cl2 : MeOH = 100:1)
to afford HPH-2Trt13) (269 mg, 22%) as a yellow solid. 1H NMR (300 MHz, CD3OD) δ 7.36 (s, 2H), 7.33 – 7.09
(m, 30H), 6.69 (s, 2H), 6.55 (s, 2H), 3.74 (d, J = 14.2 Hz, 2H), 3.55 (s, 6H), 3.65 – 3.50 (m, 4H), 2.93 (s, 6H),
2.99 – 2.87 (m, 4H). 13C NMR (75 MHz, CD3OD) δ 175.81, 159.67, 157.57, 143.77, 139.55, 137.96, 131.00,
129.47, 129.39, 121.38, 104.73, 76.85, 62.27, 53.87, 52.30, 39.41, 32.72. MS (FAB) m/z 727.3 (M+H)+ ; HRMS
(FAB). Calcd for C61H61N8O4: 969.4816. Found: 969.4824.
Dimethyl
(2S,2’S)-2,2'-({[4-(dimethylamino)pyridine-2,6-diyl]bis(methylene)}bis(azanediyl))bis[3-(1-benzyl-1H-imida

zol-4-yl)propanoate] (HPH-2Bzl).
To a solution of 1011) (458 mg, 1.77 mmol) in 10 ml ClCH2CH2Cl was added 5 (157 mg, 0.88 mmol) and
NaBH(OAc)3 (750 mg, 3.54 mmol). The mixture was stirred at room temperature for 24 h under argon
atmosphere. A solution of saturated aqueous NaHCO3 (10 ml) was added and the organic layer was separated. The
aqueous layer was extracted with CH2Cl2 (15 ml X 3). The combined organic layers were dried over Na2SO4 and
concentrated in vacuo. The residue was chromatographed on NH silica gel (eluted with CH2Cl2 : MeOH = 100:1)
1
to afford HPH-2Bzl (0.18 g, 30.7%) as a brown oil. H NMR (300 MHz, CDCl3) δ 7.34 (s, 2H), 7.29 – 7.19 (m,
6H), 7.10 – 6.98 (m, 4H), 6.62 (s, 2H), 6.37 (s, 2H), 4.94 (s, 4H), 3.76 (d, J = 14.1 Hz, 2H), 3.59 (dd, J = 14.4, 7.8
Hz, 4H), 3.52 (s, 6H), 2.90 (d, J= 6.6 Hz, 4H), 2.86 (s, 6H), 2.60 (bs, 2H). 13C NMR (75 MHz, CDCl3) δ 175.12,
158.97, 155.65, 138.95, 136.98, 136.47, 128.98, 128.20, 127.27, 116.94, 103.22, 61.12, 53.58, 51.53, 50.65, 39.16,
32.23. IR (ATR) 2947, 1730, 1599, 1498, 1392, 1163, 984, 820, 727, 692 cm-1. MS (FAB) m/z 665.4 (M+H)+ ;
HRMS (FAB). Calcd for C37H45N8O4: 665.3564. Found: 665.3569.
Dimethyl
(2S,2’S)-2,2'-({[4-(dimethylamino)pyridine-2,6-diyl]bis(methylene)}bis(azanediyl))bis[3-(1-methyl-1H-imida
zol-4-yl)propanoate] (HPH-2Me).
To a solution of 1112) (331 mg, 1.81 mmol) in 20 ml ClCH2CH2Cl was added 5 (187 mg, 1.05 mmol) and
NaBH(OAc)3 (750 mg, 3.54 mmol). The mixture was stirred at room temperature for 72 h under argon
atmosphere. A solution of saturated aqueous NaHCO3 (15 ml) was added and the organic layer was separated. The
aqueous layer was extracted with CH2Cl2 (25 ml X 3). The combined organic layers were dried over Na2SO4 and
concentrated in vacuo. The residue was chromatographed on NH silica gel (eluted with CH2Cl2 : MeOH = 100:0
to 30:1) to afford HPH-2Me (55 mg, 10.2%) as a brown oil. 1H NMR (300 MHz, CD3OD) δ 7.49 (s, 2H), 6.86 (s,
2H), 6.65 (s, 2H), 3.86 (dd, J = 15.2, 2.6 Hz, 2H), 3.72 (dd, J = 15.2, 2.6 Hz, 2H),3.65 (s, 12H), 3.60 (t, J= 6.7 Hz,
2H), 3.08 (s, 6H), 2.94 (dd, J = 14.4, 6.7 Hz, 2H), 2.86 (dd, J = 14.4, 6.7 Hz, 2H). 13C NMR (75 MHz, CD3OD) δ
175.97, 158.54, 156.66, 156.57, 139.06, 138.60, 119.89, 104.96, 62.10, 52.33, 51.46, 39.75, 33.60, 32.47. IR
(ATR) 2918, 1728, 1601, 1547 cm-1. MS (FAB) m/z 513.5 (M+H)+ ; HRMS (FAB). Calcd for C25H37N8O4:
513.2938. Found: 513.2928.
Dimethyl
(2S,2’S)-2,2'-({[4-(dimethylamino)pyridine-2,6-diyl]bis(methylene)}bis(azanediyl))bis[3-(1H-imidazol-4-yl)p
ropanoate] (HPH).
A solution of HPH-2Trt (130 mg, 0.13 mmol) in 90% aqueous AcOH (10 ml) was heated to 60 ˚C for 1.5 h. After
cooling to room temperature, the mixture was adjusted to pH 8 with saturated aqueous Na2CO3 and was extracted
with CH2Cl2 (15 ml X 3). The organic layer was dried over Na2SO4 and concentrated in vacuo. The residue was
chromatographed on NH silica gel (eluted with CH2Cl2 : MeOH = 20:1) to afford HPH5) (20 mg, 31.8%) as a
brown oil. 1H NMR (300 MHz, CDCl3) δ 7.46 (s, 2H), 6.75 (s, 2H), 6.31 (d, J = 3.0 Hz, 2H), 3.88 (d, J = 13.7 Hz,
2H), 3.85-3.54 (m, 10H), 3.13-2.79 (m, 10H). 13C NMR (75 MHz, CDCl3) δ 174.78, 158.71, 158.68, 155.73,
135.34, 131.51, 120.45, 104.03, 60.84, 53.35, 52.07, 39.26, 29.66. HRMS (FAB). Calcd for C23H33N8O4:
485.2625. Found: 485.2636.

DNA Cleavage Assay
The DNA cleavage activities of compounds were studied using supercoiled pUC19 DNA and analyzed by an
agarose-gel electrophoresis. pUC19 DNA (2µg) was treated with compounds in water and Tris/boric acid
buffer (10mM, pH 8.5) in the presence and absence of FeSO4·7H2O (30µM), H2O2 (30µM) and ascorbic acid
(30µM) as an activator. The reaction mixture was incubated at 37 ºC for 1,5h before the addition of EDTA
and loading buffer. Agarose gel electrophoresis of pUC 19 DNA was performed at 100 V for 40mins in 1%
slab gels containing ethidium bromide in Tris/Boronic acid/EDTA buffer. DNA was visualized by
photographing the fluorescence of intercalated ethidium bromide under a UV illuminator.
Cell culture and drug treatment
U251 and U87 human glioblastoma cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM)
(Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10 % fetal calf serum (Equitech-Bio,
Texas, U.S.). AsPC-1 human pancreas adenocarcinoma cells were incubated RPMI 1640 supplemented with
10 % fetal calf serum. All media were supplemented with 89µg/mL streptomycin (Meiji Seika Pharma,
Tokyo, Japan) and cells were incubated at 37 ºC in a humidified atmosphere of 95 % air and 5 % CO2.
Growing cells were plated at 1 x 105 cells/mL into 24-well microtiter tissue culture plates (Iwaki, Japan) and
incubated for 24 h before the addition of the drugs (the optimum cell number for cytotoxicity assays was
determined in preliminary experiments). Stock solutions of compounds were prepared in dimethyl sulfoxide
(DMSO; Wako Pure Chemical Industries, Osaka, Japan) and was added to fresh culture medium. The
concentration of DMSO in the final culture medium was 1 %.
MTT assay for cytotoxicity of compounds
The level of cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(Dojindo, Kumamoto, Japan) was quantified as previously described in the literature with small
modifications.18) The tested compounds were incubated with cells to give a final concentration in the range
10 µM for 24 h. At the end of this period, MTT was added to cells in culture at a final concentration 0.275
mg/mL and incubated for further 4 h at 37 ºC. The medium was removed and the formazan crystals were
solubilized by addition of 100µL DMSO to each well. After solubilized crystals were diluted (1:10) with
DMSO, 100µL was transferred to wells of 96-well microtiter plates and the absorbance at 550 nm (ref 630
nm) was measured using a microtiter plate spectrophotometer (Infinitive M1000 plate reader).
Acknowledgment
This work was supported by a Grant-in-Aid for Exploratory Research (B) (23390028) (to M. O.) and a
Grant-in-Aid for Scientific Research (C) (19659025) (to M. F.) from the Japan Society for the Promotion of
Science. We acknowledge support from the Egyptian Ministry of Higher Education through a Joint
Supervision Channel Program (to T. F. S. A.).
Supplementary data
1H/13C NMR spectra of new compounds
ESR spin trapping experiment using -phenyl-N-tert-butylnitrone (PBN)

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Graphical abstract