Synthesis and superoxide dismutase activity of novel iron complexes

Article abstract of DOI:10.1016/S0022-328X(00)00394-6

We have previously shown that the Fe(II) tetrakis-N,N,N′,N′-(2-pyridylmethyl)ethylenediamine complex (Fe(II)TPEN) has high superoxide dismutase (SOD) activity, using the xanthine-xanthine oxidase-cytochrome c method (cyt. c method) [J. Biol. Chem. 264 (1989) 9243-9249]. X-ray analysis showed that Fe(II)TPEN has two different coordination structures, one in which Fe(II) is coordinated with six nitrogens of TPEN and one in which Fe(II) is coordinated with five nitrogens of TPEN and one oxygen of sulfate anion as the counter anion [Chem. Pharm. Bull. 48 (2000) 223-230]. To investigate the relationship between these two structures and SOD activity, we synthesized novel Fe(II) complexes of TPEN analogues and measured their SOD activity by the cyt. c method. The Fe(II) tetrakis-N,N,N′,N′-(2-pyridylmethyl)trimethylenediamine complex (Fe(II)TPTN) and the Fe(II) tris(2-pyridylmethyi)triazacyclononane complex (Fe(II)TPTCN) had no SOD activity (IC₅₀ = > 100 μM), probably because these two complexes have undistorted steric structure with no easily substituted ligand. On the other hand, other Fe(II) complexes with unsaturated coordination or an easily substituted ligand had high SOD activity (IC₅₀ = 0.4-20 μM). The results indicate that the substitution reaction of a ligand with superoxide or the coordination of superoxide is essential for Fe(II)TPEN analogue complexes to have SOD activity. Moreover, we examined the effect of steric hindrance of the ligands on the SOD activity and the stability of the iron complexes to oxygen.

Full text of DOI:10.1016/S0022-328X(00)00394-6

www.elsevier.nl/locate/jorganchem
Journal of Organometallic Chemistry 611 (2000) 586–592
Synthesis and superoxide dismutase activity of novel iron
complexes
Masakazu Tamura, Yasuteru Urano, Kazuya Kikuchi, Tsunehiko Higuchi,
Masaaki Hirobe, Tetsuo Nagano *
Graduate School of Pharmaceutical Sciences, the Uni6ersity of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Received 8 March 2000; accepted 26 March 2000
Abstract
We have previously shown that the Fe(II) tetrakis-N,N,N%,N%-(2-pyridylmethyl)ethylenediamine complex (Fe(II)TPEN) has high
superoxide dismutase (SOD) activity, using the xanthine–xanthine oxidase–cytochrome c method (cyt. c method) [J. Biol. Chem.
264 (1989) 9243–9249]. X-ray analysis showed that Fe(II)TPEN has two different coordination structures, one in which Fe(II) is
coordinated with six nitrogens of TPEN and one in which Fe(II) is coordinated with five nitrogens of TPEN and one oxygen of
sulfate anion as the counter anion [Chem. Pharm. Bull. 48 (2000) 223–230]. To investigate the relationship between these two
structures and SOD activity, we synthesized novel Fe(II) complexes of TPEN analogues and measured their SOD activity by the
cyt. c method. The Fe(II) tetrakis-N,N,N%,N%-(2-pyridylmethyl)trimethylenediamine complex (Fe(II)TPTN) and the Fe(II) tris(2-
pyridylmethyl)triazacyclononane complex (Fe(II)TPTCN) had no SOD activity (IC50= \100 mM), probably because these two
complexes have undistorted steric structure with no easily substituted ligand. On the other hand, other Fe(II) complexes with
unsaturated coordination or an easily substituted ligand had high SOD activity (IC50=0.4–20 mM). The results indicate that the
substitution reaction of a ligand with superoxide or the coordination of superoxide is essential for Fe(II)TPEN analogue
complexes to have SOD activity. Moreover, we examined the effect of steric hindrance of the ligands on the SOD activity and the
stability of the iron complexes to oxygen. © 2000 Elsevier Science S.A. All rights reserved.
Keywords: Superoxide; Superoxide dismutase; Iron complex
1. Introduction
as pharmaceuticals. The use of SOD mimics is one
possible approach, and many low-molecular metal com-
plexes, mainly copper and manganese complexes, have
been synthesized to examine their SOD activity in vitro
and in vivo [2–17]. Some complexes have been reported
to have SOD activity in vivo [13,15,18–24]. However,
there are few reports about the structure–activity rela-
tionship (SAR) of SOD mimics. It is important to
investigate SAR in order to develop novel SOD mimics
as pharmaceuticals. We have previously shown that the
Superoxide (O−2 ), a product of cellular respiration,
activates polymorphonuclear leukocytes and endothe-
lial cells, and is involved in the pathology of many
diseases, such as inflammation, ischemia-reperfusion in-
jury and carcinogenesis. Superoxide dismutases (SODs)
catalyze the conversion of superoxide to hydrogen per-
oxide plus dioxygen and protect cells from damage by
O−2 [1]. Therefore, SODs may have potential as phar-
maceuticals, and there have been reports of beneficial
effects of SODs and modified SODs. However, there
are problems intrinsic to their protein nature, such as
cost, stability, membrane permeability and immuno-
genicity, which must be overcome in order to use SODs
Fe(II)
tetrakis-N,N,N%,N%-(2-pyridylmethyl)ethylene-
diamine complex (Fe(II)TPEN) has high SOD activity,
using the xanthine–xanthine oxidase–cytochrome c
(cyt. c) method, and it protected Escherichia coli cells
from paraquat toxicity [25]. We recently revealed that
Fe(II)TPEN has two different structures, depending on
the species of counter anion [26]. In the cases of pen-
tafluorophosphine or perchlorate [27], which have no,
or very low, ability to coordinate, Fe(II) is coordinated
* Corresponding author. Tel.: +81-3-58414850; fax: +81-3-
58414855.
E-mail address: tlong@mol.f.u-tokyo.ac.jp (T. Nagano).
0022-328X/00/$ - see front matter © 2000 Elsevier Science S.A. All rights reserved.
PII: S0022-328X(00)00394-6

M. Tamura et al. / Journal of Organometallic Chemistry 611 (2000) 586–592
587
2. Results and discussion
with six nitrogens of TPEN (Fig. 1(a)). However, in the
case where the sulfate anion is the counter anion, one
pyridine is replaced by the sulfate anion and Fe(II) is
coordinated with five nitrogens of TPEN and one oxy-
gen of the sulfate anion (Fig. 1(b)). This shows that
Fe(II)TPEN has one easily substituted ligand.
A ligand which is readily substituted by an anionic
species may be required for the SOD activity of Fe
complexes. We synthesized some Fe(II) complexes of
TPEN analogues and measured their SOD activity by
the cyt. c method to elucidate the relationship between
the structures of the Fe(II) complexes and the SOD
activity.
2.1. Synthesis of chelators and Fe(II) complexes
We synthesized 11 chelators consisting of pyridine
and alkylamine, as shown in Fig. 2. The synthetic
routes are shown in Scheme 1. TPEN, TPTN and
TPTCN were synthesized by reacting the corresponding
amine and 2-pyridylmethyl chloride hydrochloride at
room temperature in alkaline aqueous solution.
6MeTPEN, BPEN, N,N(6Me)2TPEN, N6MeBPEN
and N%6MeBPEN, were synthesized via the common
intermediate;
N-tert-butoxycarbonylethylenediamine
(2) by Schiff base formation with aldehyde, reduction
of the Schiff base with NaBH4, deprotection and N-
alkylation. N,N%(6Me)2TPEN was prepared by
a
method analogous to that used for 6MeTPEN.
6MeTPTN was synthesized analogously to 6MeTPEN
from N-tert-butoxycarbonyltrimethylenediamine (11).
6MeTPTCN was synthesized via 1-(6-methyl-2-pyridyl-
methyl)triazacyclononane (15), which was prepared by
N-6-methyl-picolylation and deprotection.
Ferrous complexes were prepared by reacting
equimolar FeSO4·7H2O or Fe(ClO4)2·6H2O and the
chelators in MeOH at room temperature for several
minutes, followed by evaporation in vacuo.
Fig. 1. Structures of Fe(II)TPEN(PF6)2 and Fe(II)TPEN(SO4).
2.2. SOD acti6ity of Fe(II) complexes
SOD activity of these ferrous complexes was deter-
mined by the xanthine–xanthine oxidase–cytochrome c
method (Table 1) [28]. The IC50 value is defined as the
concentration of the complex that is required to inhibit
the reduction rate of cytochrome c by 50%, and exerts
SOD activity equivalent to 1 unit of native SOD. SOD
activity of Fe(II)(6MeTPEN) and Fe(II)BPEN was
equal to that of Fe(II)TPEN. The spectral data, espe-
cially molar extinction coefficients, show that
Fe(II)(6MeTPEN) (m=1670, umax=363 nm) and
Fe(II)BPEN (m=1760, umax=396 nm) are both five-co-
ordinated Fe(II) complexes in a high spin state. Al-
though Fe(II)TPEN (m=10 000, umax=416 nm) is a
six-coordinated complex in a low spin state, one ligand
can be easily replaced by O−2 , due to the distorted
structure. Thus, Fe(II)TPEN is considered to have
structural characteristics resembling those of a five-co-
ordinated complex.
The SOD activities of Fe(II)(N,N(6Me)2TPEN),
Fe(II)(N,N%(6Me)2TPEN), Fe(II)(N6MeBPEN) and
Fe(II)(N%6MeBPEN) were lower by one order of mag-
nitude than that of Fe(II)TPEN. These complexes are
five-coordinated, based on the spectral data (m=1200,
1550, 1540 and 1630, respectively). However, the coor-
dination structures of these four complexes are different
from that of five-coordinated Fe(II)BPEN. That is, the
former four complexes are coordinated by four pyridi-
Fig. 2. Structures of chelators.

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M. Tamura et al. / Journal of Organometallic Chemistry 611 (2000) 586–592
Scheme 1.
nes and one 6-methylpyridine, while Fe(II)BPEN is
coordinated by five pyridines. In the case of
Fe(II)(6MeTPEN), 6-methylpyridine is probably not
coordinated to the Fe(II) ion, because the coordination
ability of 6-methylpyridine is lower than that of pyri-
dine. On the other hand, Fe(II)(N,N(6Me)2TPEN),
Fe(II)(N,N%(6Me)2TPEN), Fe(II)(N6MeBPEN) and
Fe(II)(N%6MeBPEN) are coordinated with one 6-
methylpyridine. Thus, the 6-methylpyridine ligand
should hinder the approach of O−2 to the sixth coordi-
nation site of the complexes, which may be the reason
why the SOD activities of the four complexes are lower
by one order of magnitude than those of Fe(II)-
(6MeTPEN) and Fe(II)BPEN. In other words, the
steric hindrance of the methyl group decreases the
reaction rate at the step involving O−2 .
Interestingly, Fe(II)TPTN and Fe(II)TPTCN showed
no SOD activity, while Fe(II)(6MeTPTN) and
Fe(II)(6MeTPTCN) had moderate SOD activity, as
shown in Table 1. Fe(II)TPTN and Fe(II)TPTCN are
undistorted, stable six-coordinated low-spin complexes
[29], of which the m values are 11 000 and 12 000,
respectively. O−2 cannot undergo a substitution reaction
with Fe(II)TPTN and Fe(II)TPTCN. Fe(II)(6Me-
TPTN) and Fe(II)(6MeTPTCN), which are the deriva-
tives of Fe(II)TPTN and Fe(II)TPTCN with 6-
methylpyridine as a ligand, should have unsaturated
coordination or distorted structure, which leads to SOD
activity. This characteristic structure allows O−2 to at-
tack the complex, leading to one-electron oxidation of
Fe(II) to Fe(III), followed by a disproportionation re-
action of O−2 .
Table 1
SOD activity of Fe(II) complexes a
Fe(II) complex
IC50 (mM)
Fe(II)TPEN
0.5
0.4
3.0
3.0
0.4
Fe(II)(6MeTPEN)
Fe(II)(N,N(6Me)2TPEN)
Fe(II)(N,N%(6Me)2TPEN)
Fe(II)BPEN
Fe(II)(N6MeBPEN)
Fe(II)(N%6MeBPEN)
Fe(II)TPTN
Fe(II)(6MeTPTN)
Fe(II)TPTCN
3.0
3.5
\100
2.0
\100
20.0
Fe(II)(6MeTPTCN)
a IC50 value is the concentration of Fe(II) complex required to
inhibit by 50% the reduction rate of cytochrome c by superoxide.
DMSO was used as a co-solvent.

M. Tamura et al. / Journal of Organometallic Chemistry 611 (2000) 586–592
589
Fe(II)TPEN, Fe(II)TPTN and Fe(II)TPTCN in H2O.
The spectra of Fe(II)TPEN were changed and the
absorbance was decreased by the addition of hydroxide
anion. However, the addition of hydroxide anion had
no effect on the spectra of Fe(II)TPTN and
Fe(II)TPTCN. This indicates that Fe(II)TPTN and
Fe(II)TPTCN do not have any easily replaced ligands
and therefore should have no SOD activity.
To confirm this interpretation, Fe(II)(6MeTPTN)
and Fe(II)(6MeTPTCN) were also examined in the
presence of hydroxide anion, because these two com-
plexes are expected to be easily substituted owing to the
steric hindrance of the methyl functional group intro-
duced at the 6-position of one pyridine ligand. As
expected, the spectra of Fe(II)(6MeTPTN) and
Fe(II)(6MeTPTCN), which have SOD activity, were
changed like that of Fe(II)TPEN by the addition of
hydroxide anion (Fig. 4). Large amounts of hydroxide
anion were required to obtain a spectral change in the
case of Fe(II)(6MeTPTCN) compared with Fe(II)-
(6MeTPTN), which is consistent with the SOD activi-
ties of Fe(II)(6MeTPTN) (IC50=2.0 mM) and
Fe(II)(6MeTPTCN) (IC50=20.0 mM). These results
support the idea that it is essential for complexes to
have an easily replaced ligand(s) in order to show SOD
activity.
Fig. 3. Spectral changes of Fe(II)TPEN (a), Fe(II)TPTN (b) and
Fe(II)TPTCN (c) by hydroxide anion. The spectra were measured in
H2O. The concentration of Fe(II) complex was 0.1 mM. (a) Line 1,
no additive; line 2, 1.0 mM NaOH; line 3, 2.5 mM NaOH; line 4, 5.0
mM NaOH; line 5, 10 mM NaOH; line 6, 20 mM NaOH; line 7, 50
mM NaOH; line 8, 75 mM NaOH; line 9, 100 mM NaOH. (b) line 1,
no additive; line 2, 1.0 mM NaOH; line 3, 2.0 mM NaOH; line 4, 3.0
mM NaOH; line 5, 4.0 mM NaOH; line 6 5.0 mM NaOH. (c) line 1,
no additive; line 2, 1.0 mM NaOH; line 3, 2.0 mM NaOH; line 4, 3.0
mM NaOH; line 5, 4.0 mM NaOH; line 6 5.0 mM NaOH.
2.3. Spectral changes of Fe(II) complexes induced by
hydroxide anion
Fe(II)TPEN has two different structures and one
pyridine of Fe(II)TPEN can be replaced by another
anion which has coordination ability. Fe(II)TPEN-
(ClO4)2 and Fe(II)TPEN(SO4) give the same spectra in
an aqueous solvent, while they show quite different
spectra in MeOH (Fe(II)TPEN(ClO4); m=10 000,
umax=416 nm, (Fe(II)TPEN(SO4); m=3640, umax=403
nm). These observations suggest that the replacement
of one pyridine of Fe(II) complexes by an anion such as
sulfate or hydroxide anion changes the spectra. We
measured the spectral change induced by hydroxide
anion in H2O. Fig. 3 shows the visible spectra of
Fig. 4. Spectral changes of Fe(II)(6MeTPTN) (a) and
Fe(II)(6MeTPTCN) (b) by hydroxide anion. The spectra were mea-
sured in H2O. The concentration of Fe(II) complex was 0.1 mM. (a)
line 1, no additive; line 2, 0.1 mM NaOH; line 3, 0.2 mM NaOH; line
4, 0.3 mM NaOH. (b) line 1, no additive; line 2, 5.0 mM NaOH; line
3, 25 mM NaOH; line 4, 50 mM NaOH; line 5, 100 mM NaOH; line
6, 150 mM NaOH; line 7, 200 mM NaOH.

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M. Tamura et al. / Journal of Organometallic Chemistry 611 (2000) 586–592
3. Experimental
3.1. Materials
Catalase, cytochrome c (type III), and xanthine
sodium salt were purchased from Sigma. Xanthine ox-
idase was purchased from Boehringer Mannheim
GmbH. The other chemicals were all reagent-grade
products from Aldrich, Tokyo Kasei Kogyo Ltd, and
Wako Ltd.
3.2. Assay of SOD acti6ity
SOD activity was measured according to the method
of Ref. [28], except for omitting EDTA and adding
catalase. The reaction mixture was prepared with 50
mM potassium phosphate buffer (pH 7.4) containing 10
mM cytochrome c, 50 mM xanthine and catalase (1200 U
ml−1). The reaction was started by adding xanthine
oxidase at 25°C, and reduction of cytochrome c was
monitored at 550 nm. The amount of xanthine oxidase
added was adjusted so as to give a rate of A550nm=0.025
min−1 in the absence of a SOD mimic. SOD activity
(IC50) was determined as the concentration which in-
hibits by 50% the reduction rate of cytochrome c.
Fig. 5. Spectral change of Fe(II)BPEN (a) and Fe(II)(N%6MeBPEN)
(b) in the presence of O2. The spectra were measured in 50 mM
potassium phosphate buffer (pH 7.4). The concentration of Fe(II)
complex was 1.0 mM. (a) The spectra were measured after 0, 0.5, 1,
1.5, 2, 3, 4, 5, 6 and 7 h. (b) The spectra were measured after 0, 1, 2,
3, 4, 5, 6, 7, 8 and 9 h.
2.4. Stability of Fe(II) complexes to oxygen
3.3. Synthesis
In order to investigate the steric effect of a methyl
group, the stability of Fe(II)BPEN and Fe(N%6Me-
BPEN) to oxygen was examined. Fig. 5 shows that
Fe(II)BPEN was auto-oxidized, while Fe(N%6MeBPEN)
was stable, which indicates that the methyl functional
group increased the stability of the complex to oxygen.
The methyl group of 6-methylpyridine can apparently
block the approach of oxygen to the Fe(II) complex.
Fe(II)TPEN was stable in the presence of oxygen for
more than 1 month, and Fe(II)TPTN and Fe(II)-
TPTCN were also non-reactive with oxygen. These
three complexes are considered not to react with oxygen
because of their saturated coordination structure.
In summary, we synthesized 11 chelators and mea-
sured the SOD activity of their Fe(II) complexes in
order to clarify the relationship between the two struc-
tures of Fe(II)TPEN and SOD activity. It is important
for an Fe(II) complex to have an easily replaced ligand
in order to exhibit SOD activity. Moreover, coordina-
tion of 6-methylpyridine to Fe(II) makes the complex
stable to oxygen, owing to steric hindrance.
3.3.1. N,N,N%,N%-Tetrakis(2-pyridylmethyl)-
ethylenediamine (TPEN)
TPEN was synthesized according to Ref. [30]. 2-
Pyridylmethyl chloride hydrochloride (31.0 g, 189
mmol) was dissolved in 10 ml H2O. To this solution,
NaOH solution (5 N 100 ml), ethylenediamine (2.86 g,
47 mmol) and cetyltrimethylammonium chloride (323
mg) were added. The reaction mixture was stirred at
room temperature (r.t.) under Ar for 24 h and the crude
product was extracted with CH2Cl2. The CH2Cl2 phase
was dried over anhydrous K2CO3 and evaporated. The
residue was chromatographed on alumina and recrystal-
lized from benzene to afford TPEN (45.3%). M.p.
1
115–117°C. H-NMR (400 MHz, CDCl3/TMS) 2.76 (s,
4H), 3.78 (s, 8H), 7.11 (dd, 4H), 7.45 (d, 4H), 7.57 (ddd,
4H), 8.48 (d, 4H); MS 424 [M+]; Anal. Calc. for
C26H28N6: C, 73.56; H, 6.65; N, 19.80. Found: C, 73.55;
H, 6.70; N, 19.60%.
3.3.2. N,N,N%,N%-Tetrakis(2-pyridylmethyl)-
trimethylenediamine (TPTN)
We are now searching for more active complexes than
Fe(II)TPEN by introducing an electron-donating group
such as a dimethylamino, methoxy or methyl group at
the 4-position of pyridine of TPEN. In addition, further
studies on biological applications of these complexes are
in progress using biological assay systems such as
paraquat toxicity in E. coli.
TPTN was prepared analogously to TPEN from
trimethylenediamine (59.6%). M.p. 84.5–86.0°C (cyclo-
hexane). H-NMR (400 MHz, CDCl3/TMS) 1.81 (m,
2H), 2.55 (t, 4H, J=7.6 Hz), 3.75 (s, 8H), 7.11 (ddd,
4H), 7.41 (d, 4H), 7.58 (ddd, 4H), 8.48 (dd, 4H); MS 438
[M+]; Anal. Calc. for C27H30N6: C, 73.94; H, 6.89; N,
19.16. Found: C, 74.04; H, 7.00; N, 19.01%.
1

M. Tamura et al. / Journal of Organometallic Chemistry 611 (2000) 586–592
591
3.3.6. N-(6-Methyl-2-pyridylmethyl)-N,N%,N%-tris-
(2-pyridylmethyl)ethylenediamine (6MeTPEN)
3.3.3. 1,4,7-Tris(2-pyridylmethyl)trimethylenediamine
(TPCTN)
N-(6-Methyl-2-pyridylmethyl)ethylenediamine (399
mg, 1.56 mmol) and 2-pyridylmethyl chloride hydro-
chloride (768 mg, 4.68 mmol) were added to 5 N NaOH
aqueous solution 10 ml and the mixture was stirred at
r.t. for 4 days. H2O (10 ml) was added and the crude
product was extracted with CH2Cl2. The CH2Cl2 phase
was dried over anhydrous K2CO3 and evaporated. The
residue was chromatographed on alumina and recrys-
tallization from n-hexane afforded 6MeTPEN (43.9%).
TPTCN was prepared from 1,4,7-triazacyclononane
(quant). 1H-NMR (400 MHz, CDCl3/TMS) 2.90 (s,
12H), 3.84 (s, 6H), 7.15 (dd, 3H), 7.51 (d, 3H), 7.65
(ddd, 3H), 8.51 (d, 3H); MS 402 [M+].
3.3.4. N-tert-Butoxycarbonylethylenediamine (2)
To EtOH 20 ml containing ethylenediamine 1 (16 ml,
239 mmol) and triethylamine (1.03 g, 10.2 mmol), di-
tert-butyl dicarbonate (2.22 g, 10.2 mmol) was added
dropwise at 0°C. After having been stirred at r.t. for 1
h, the reaction mixture was concentrated. The residue
was dissolved in CH2Cl2 and the product was extracted
with 1 N AcOH aqueous solution. The aqueous phase
was basified with NaOH aqueous solution and the
product was again extracted with CH2Cl2. The CH2Cl2
phase was dried over anhydrous K2CO3 and evaporated
1
M.p. 86.0–87.0°C. H-NMR (400 MHz, CDCl3/TMS)
2.49 (s, 3H), 2.76 (s, 4H), 3.75 (s, 2H), 3.77 (s, 6H), 6.97
(d, 1H), 7.11 (ddd, 3H), 7.28 (d, 1H), 7.44–7.48 (m,
4H), 7.56 (ddd, 3H), 8.48–8.49 (m, 3H); MS 438 [M+];
Anal. Calc. for C27H30N6: C, 73.94; H, 6.89; N, 19.16.
Found: C, 74.12; H, 6.94; N, 19.09%.
3.3.7. N-Benzyl-N,N%,N%-tris(2-pyridylmethyl)-
ethylenediamine (BPEN)
1
to afford 2. H-NMR (CDCl3/TMS) 1.22 (s, 2H), 1.42
BPEN was prepared analogously to 6MeTPEN from
1
(s, 9H), 2.50–2.85 (m, 2H), 2.95–3.20 (m, 2H), 5.60
(brs, 1H).
2. M.p. 84.0–87.0°C (n-hexane). H-NMR (400 MHz,
CDCl3/TMS) 2.70–2.74 (m, 4H), 3.58 (s, 2H), 3.71 (s,
2H), 3,77 (s, 4H), 7.09–7.13 (m, 3H), 7.20–7.30 (m,
5H), 7.43 (d, 2H), 7.46 (d, 1H), 7.54–7.59 (m, 3H),
8.47–8.49 (m, 3H); MS 423 [M+]; Anal. Calc. for
C27H29N5: C, 76.56; H, 6.90, N, 16.53. Found: C, 76.70;
H, 6.80; N, 16.40%.
3.3.5. N-(6-Methyl-2-pyridylmethyl)ethylenediamine (3)
To MeOH solution containing 2 (5.394 g, 33.7 mmol)
,
and 3 A molecular sieves, 6-methylpyridine-2-carbox-
aldehyde (4.083 g, 33.8 mmol) in MeOH was added
dropwise under Ar. The reaction mixture was refluxed
for 1.5 h. After removal of the molecular sieves by
filtration, NaBH4 (3.93 g, 103.4 mmol) was added and
the whole was stirred overnight at r.t. MeOH was
evaporated and 2 N Na2CO3 aqueous solution was
added to the residue. The crude product was extracted
with CH2Cl2. The CH2Cl2 phase was dried over anhy-
drous K2CO3 and evaporated. The residue was chro-
matographed on alumina to afford N-tert-butoxy-
carbonyl-N%-(6-methyl-2-pyridylmethyl)ethylenediamine
3.3.8. N,N-Bis(6-methyl-2-pyridylmethyl)-N%N%-bis(2-
pyridylmethyl)ethylenediamine (N,N(6Me)2TPEN)
N,N(6Me)2TPEN was prepared analogously to
6MeTPEN from N,N-bis(2-pyridylmethyl)ethylene-
1
diamine. M.p. 85.5–87.0°C (n-hexane). H-NMR (400
MHz, CDCl3/TMS) 2.49 (s, 6H), 2.75 (s, 4H), 3.75 (s,
4H), 3.77 (s, 4H), 6.96 (d, 2H, J=7.2 Hz), 7.10 (t, 2H,
J=7.2 Hz), 7.29 (d, 2H, J=7.2 Hz), 7.45 (t, 2H,
J=7.6 Hz), 7.56 (ddd, 2H, J=5.0, 2.0 Hz), 8.48 (d,
2H, J=8.0 Hz); MS 452 [M+].
1
(30.1%). H-NMR (CDCl3/TMS) 1.33 (s, 9H), 2.07 (s,
3.3.9. N,N%-Bis(6-methyl-2-pyridylmethyl)-N%,N%-bis(2-
pyridylmethyl)ethylenediamine (N,N%(6Me)2TPEN)
N,N%(6Me)2TPEN was prepared analogously to
6MeTPEN. M.p. 93.5–94.5°C (n-hexane). 1H-NMR
(400 MHz, CDCl3/TMS) 2.49 (s, 6H), 2.75 (s, 4H), 3.75
(s, 4H), 3.77 (s, 4H), 6.97 (d, 2H), 7.11 (dd, 2H), 7.29
(d, 2H), 7.44–7.47 (m, 4H), 7.56 (ddd, 2H), 8.48 (d,
2H); MS 452 [M+]; Anal. Calc. for C28H32N6: C, 74.31;
H, 7.13; N, 18.57. Found: C, 74.09; H, 7.26; N, 18.37%.
1H), 2.54 (s, 3H), 2.65–2.95 (m, 2H), 3.07–3.35 (m,
2H), 3.85 (s, 2H), 5.20 (brs, 1H), 7.13 (dd, 2H, J=7
Hz, 3 Hz), 7.54 (t, 1H, J=7 Hz).
N - tert - Butoxycarbonyl - N% - (6 - methyl - 2 - pyridyl-
methyl)ethylenediamine (616 mg, 2.32 mmol) in CH2Cl2
was added dropwise to trifluoroacetic acid 20 ml at
0°C. The mixture was stirred at r.t. for 1.5 h, then
concentrated. The residue was taken up in 2 N Na2CO3
aqueous solution and the crude product was extracted
with CH2Cl2. The CH2Cl2 phase was dried over anhy-
drous K2CO3 and evaporated. The residue was used in
the next reaction without further purification. 1H-NMR
(CDCl3/TMS) 1.78 (s, 3H), 2.55 (s, 3H), 2.68–2.88 (m,
4H), 3.85 (s, 2H), 5.20 (brs, 1H), 6.98 (d, 1H, J=7 Hz),
7.08 (d, 1H, J=7 Hz), 7.50 (t, 1H, J=7 Hz).
3.3.10. N-Benzyl-N-(6-methyl-2-pyridylmethyl)-N%,N%-
bis(2-pyridylmethyl)ethylenediamine (N6MeBPEN)
N6MeBPEN was prepared analogously to 6Me-
TPEN from N-benzyl-N-(6-methyl-2-pyridylmethyl)-
ethylenediamine. 1H-NMR (400 MHz, CDCl3/TMS)
2.49 (s, 3H), 2.69–2.74 (m, 4H), 3.58 (s, 2H), 3.69 (s,

592
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2H), 3.76 (s, 4H), 6.96 (d, 1H, J=7.2 Hz), 7.09–7.59
(m, 13H), 8.48–8.50 (m, 1H).
[4] E. Kimura, A. Yatsunami, A. Watanabe, R. Machida, T. Koike,
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3.3.11. N-Benzyl-N%-(6-methyl-2-pyridylmethyl)-N%,N%-
bis(2-pyridylmethyl)ethylenediamine (N%6MeBPEN)
N%6MeBPEN was prepared analogously to 6Me-
TPEN from N-benzyl-N%-(6-methyl-2-pyridylmethyl)-
ethylenediamine. M.p. 86.5–88.0°C (n-hexane). 1H-
NMR (400 MHz, CDCl3/TMS) 2.50 (s, 6H), 2.69–2.74
(m, 4H), 3.58 (s, 2H), 3.71 (s, 2H), 3.74 (s, 4H), 3.75 (s,
2H), 3.76 (s, 4H), 6.97 (d, 1H), 7.09–7.12 (m, 2H),
7.19–7.29 (m, 6H), 7.43–7.48 (m, 3H), 7.56 (d, 2H),
8.46–8.49 (m, 2H); MS 437 [M+]; Anal. Calc. for
C28H31N5: C, 76.86; H, 7.14; N, 16.00. Found: C, 76.80;
H, 7.12; N, 15.81%.
3.3.12. N-(6-Methyl-2-pyridylmethyl)-N,N%,N%-
tris(2-pyridylmethyl)trimethylenediamine (6MeTPTN)
6MeTPTN was prepared analogously to 6MeTPEN.
1H-NMR (400 MHz, CDCl3/TMS) 1.80 (m, 2H), 2.50
(s, 3H), 2.55 (t, 4H, J=6.8 Hz), 3.73 (s, 2H), 3.75 (s,
6H), 6.96 (d, 1H, J=7.6 Hz), 7.11 (ddd, 3H, J=6.4,
1.2 Hz), 7.43 (d, 1H, J=7.6 Hz), 7.42 (m, 3H), 7.47 (t,
1H, J=7.6 Hz), 7.58 (ddd, 3H, J=7.6, 1.6 Hz), 8.48
(m, 3H); MS 452 [M+].
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1208.
3.3.13. 1-(6-Methyl-2-pyridylmethyl)-4,7-
bis(2-pyridylmethyl)triazacyclononane (6MeTPTCN)
6MeTPTCN was also prepared analogously from
1-(6-methyl-2-pyridylmethyl)triazacyclononane
(15).
1H-NMR (300 MHz, CDCl3/TMS) 2.52 (s, 3H), 2.85–
2.90 (m, 12H), 3.80 (s, 2H), 3.83 (s, 4H), 7.00 (d, 1H,
J=7.53 Hz), 7.11–7.16 (m, 2H), 7.26 (d, 1H, J=7.68
Hz), 7.50–7.56 (m, 3H), 7.62–7.67 (m, 2H), 8.49–8.52
(m, 2H); MS 416 [M+].
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3.3.14. Formation of ferrous complexes
Ferrous complexes were prepared by reacting
equimolar FeSO4·7H2O or Fe(ClO4)2·6H2O and the
chelators in MeOH at r.t. for several minutes, evaporat-
ing the solvent and drying the product in vacuo.
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