Novel Iron Sequestering Agents: Synthesis and Iron-Chelating Properties of Hexadentate Ligands Composed of 1-Hydroxy-2(1H)-pyrimidinone, ω-Amino Carboxylic Acid, and Tris(2-aminoethyl)amine

Article abstract of DOI:10.1021/jo961901m

Novel heterocyclic hexadentate ligands (3HOPY_n: n = 5-7), in which three units of 1-hydroxy-2(1H)-pyrimidinone are linked to tris(2-aminoethyl)amine through an amide bond by an alkyl chain, were synthesized, and their iron-chelating properties were investigated. The stability of their iron complexes (25 to 27 in log K) was significantly larger than that of pyrazinone-containing ligands by virtue of higher pK_a values. On kinetic evaluation of iron removal from human transferrin, 3HOPY₅ showed remarkable efficiency over five times as much as commercially available desferrioxamine B. The conformational analysis of the corresponding Ga(III) complex of Fe(opy₅) by ¹H NMR and by MM and MD calculations are also discussed.

Full text of DOI:10.1021/jo961901m

3618
J . Org. Chem. 1997, 62, 3618-3624
Novel Ir on Sequ ester in g Agen ts: Syn th esis a n d Ir on -Ch ela tin g
P r op er ties of Hexa d en ta te Liga n d s Com p osed of
1-Hyd r oxy-2(1H)-p yr im id in on e, ω-Am in o Ca r boxylic Acid , a n d
Tr is(2-a m in oeth yl)a m in e
J unko Ohkanda,† J un Kamitani,† Takeshi Tokumitsu,† Yoko Hida,† Takeo Konakahara,‡ and
Akira Katoh*,†
Department of Industrial Chemistry, Faculty of Engineering, Seikei University,
Musashino, Tokyo 180, J apan, and Department of Industrial Chemistry,
Faculty of Science and Technology, Science University of Tokyo, Noda, Chiba 278, J apan
Received October 8, 1996X
Novel heterocyclic hexadentate ligands (3HOPYn: n ) 5-7), in which three units of 1-hydroxy-
2(1H)-pyrimidinone are linked to tris(2-aminoethyl)amine through an amide bond by an alkyl chain,
were synthesized, and their iron-chelating properties were investigated. The stability of their iron
complexes (25 to 27 in log K) was significantly larger than that of pyrazinone-containing ligands
by virtue of higher pKa values. On kinetic evaluation of iron removal from human transferrin,
3HOPY5 showed remarkable efficiency over five times as much as commercially available
desferrioxamine B. The conformational analysis of the corresponding Ga(III) complex of Fe(opy5)
1
by H NMR and by MM and MD calculations are also discussed.
In tr od u ction
droxy-2(1H)-pyrimidinone (HOPY) and 5,6-dimethyl-1-
hydroxy-2(1H)-pyrazinone (HOPR) and found them to be
new iron-sequestering agents as bidentate ligands.7 As
Iron deficiency and overload are serious health prob-
lems. Considerable effort has been invested in the
development of new chemotherapeutics for managing
thalassemia.1 Desferrioxamine B (DFO; Desferal), a
microbial siderophore bearing three hydroxamate groups
as iron binding sites, is still the only approved drug for
treatment of patients suffering from acute iron intoxica-
tion or from chronic iron overload as a result of recurrent
blood transfusions. However, DFO has short plasma
half-life2 and cannot be orally administered.3 Further-
more, it possesses a number of side effects such as
septicemia.4 Therefore, the search for new nontoxic and
orally active agents that would be expected to take the
place of DFO has been one of the important issues.
Recently, much attention has been focused on the ap-
plication of nitrogen-containing heterocycles to iron
chelators as therapeutic agents for the iron overload
expected, both diazines showed high water solubility and
acidity (pKa ) 6.1 for HOPY, 4.7 for HOPR). In order to
develop more efficient chelators in low metal concentra-
tion, two types of hexadentate ligands composed of three
HOPRs in a molecule have also been investigated.8 It
was noted that the hexadentate ligands showed remark-
ably high kinetic efficiency of iron removal from human
transferrin, whereas the stability of iron complexes was
disease.5 As for 3-hydroxy-4(1H)-pyridinones,5d,5f-k
a
number of clinical studies are presently ongoing,6 al-
though their clinical safety has not been established.
(5) (a) Sun, Y.; Martell, A. E.; Reibenspies, J .; Welch, M. J .
Tetrahedron 1991, 47, 357. (b) Motekaitis, R. J .; Sun, Y.; Martell, A.
E. Inorg. Chim. Acta 1992, 198, 421. (c) Sun, Y.; Martell, A. E.; Welch,
M. J . Tetrahedron 1991, 47, 8863. (d) Scarrow, R. C.; Riely, P. E.; Abu-
Dari, K.; White, D. L.; Raymond, K. N. Inorg. Chem. 1985, 24, 954. (e)
Scarrow, R. C.; White, D. L.; Raymond, K. N. J . Am. Chem. Soc. 1985,
107, 6540. (f) Hider, R. C.; Kontoghiorghes, G. J .; Silver, J . UK Patent
GB-2118176, 1982. (g) Hider, R. C.; Singh, S.; Porter, J . B.; Huehns,
E. R. Ann. N. Y. Acad. Sci. 1990, 612, 327. (h) Motekaitis, R. J .; Martell,
A. E. Inorg. Chim. Acta 1991, 183, 71. (i) Clarke, E. T.; Martell, A. E.
Inorg. Chim. Acta 1992, 191, 57. (j) Sheppard, L. N.; Kontoghioghes,
G. J . Inorg. Chim. Acta 1991, 188, 177. (k) Faller, B.; Nick, H. J . Am.
Chem. Soc. 1994, 116, 3860. (l) Streater, M.; Taylor, P. D.; Hider, R.
C.; Porter, J . J . Med. Chem. 1990, 33, 1749. (m) Scarrow, R. C.;
Raymond, K. N. Inorg. Chem. 1988, 27, 4140. (n) Clarke, E. T.; Martell,
A. E. Inorg. Chim. Acta 1992, 196, 185.
(6) (a) Olivieri, N. F.; Templeton, D. M.; Koren, G.; Chung, D.;
Hermann, C.; Freedman, M. H.; McClelland, R. A. Ann. N.Y. Acad.
Sci. 1990, 612, 369. (b) Tondury, P.; Kontoghiorghes, G. J .; Ridolfi-
Luthy, A.; Hirt, A.; Hoffbrand, A. V.; Lottenbach, A. M.; Sonderegger,
T.; Wagner, H. P. Br. J . Haematol. 1990, 76, 550. (c) Al-Refaie, F. N.;
Wonke, B.; Hoffbrand, A. V.; Wickens, D. G.; Nortey, P.; Kon-
toghiorghes, G. J . Blood 1992, 80, 593.
On the other hand, N-hydroxy amide-containing diaz-
ines can be regarded as cyclic hydroxamic acids. On
chelating iron under physiological conditions, they would
be expected to have several advantages: higher water
solubility and acidity than those of monoazine, arising
from their -electron deficient ring system. Previously,
we have reported the synthesis of 4,6-dimethyl-1-hy-
* Author to whom correspondence should be addressed.
† Seikei University.
‡ Science University of Tokyo.
X Abstract published in Advance ACS Abstracts, May 1, 1997.
(1) For a recent review on iron chelators for clinical use, see: The
Development of Iron Chelators for Clinical Use; Bergeron, R. J .,
Brittenham, G. M., Eds.; CRC Press: Boca Raton, 1992.
(2) Summers, M. R.; J acobs, A.; Tudway, D.; Perera, P.; Ricketts,
C. Br. J . Haematol. 1979, 42, 547.
(3) (a) Callender, S. T.; Weatherall, D. J . Lancet 1980, Sept. 27, 689.
(b) J acobs, A.; Ting, W. C. Lancet 1980, Oct. 11, 794.(c) Kattamis, C.;
Fitsialos, J .; Sinopoulou, C. Lancet 1981, J an. 3, 51.
(4) Miller, M. J .; Malouin F. In The Development of Iron Chelators
for Clinical Use; Bergeron, R. J ., Brittenham, G. M., Eds.; CRC Press:
Boca Raton, 1992; p 277.
(7) Ohkanda, J .; Tokumitsu, T.; Mitsuhashi, K.; Katoh, A. Bull.
Chem. Soc. J pn. 1993, 66, 841.
(8) (a) Ohkanda, J .; Katoh, A. J . Org. Chem. 1995, 60, 1583. (b)
Ohkanda, J .; Katoh, A. Tetrahedron 1995, 51, 12995. (c) Ohkanda, J .;
Katoh, A. Chem. Lett. 1996, 423.
S0022-3263(96)01901-9 CCC: $14.00 © 1997 American Chemical Society

Novel Iron Sequestering Agents
J . Org. Chem., Vol. 62, No. 11, 1997 3619
Sch em e 1
O-succinimide esters 6, followed by coupling with tris-
(2-aminoethyl)amine under mild conditions gave tripodal
compounds 7a -c as shown in Scheme 2. Finally, de-
benzylation of 7a -c by the catalytic hydrogenation in
MeOH under reflux afforded the desired hexadentate
ligands, 3HOPYn (n ) 5-7).
In order to clarify whether intramolecular hydrogen
bonds exist in the free ligands or not, the temperature
dependence of 1H NMR chemical shifts was examined for
the amide and amino protons of 3HOPY5. 1H NMR
spectra of 3HOPY5 in DMSO-d6 at various temperatures
from 23 to 90 °C exhibited one set of signals, indicating
that 3HOPY5 possesses the pseudo-C3-symmetrical struc-
ture at a range of the temperatures. The plots of
chemical shifts of these amide and amino protons versus
the temperatures gave straight lines with the tempera-
ture coefficients -4.6 × 10-3 and -5.1 × 10-3 ppm/deg,
respectively. These large values indicate that no par-
ticular hydrogen bond exists in DMSO-d6 solution.10
Ir on Com p lex F or m a tion . Iron complexation ability
of 3HOPYn in aqueous solution was demonstrated by
means of spectrophotometric analysis. As for 3HOPY5
shown in Figure 1, UV-vis spectra of a 1:1 molar mixture
of ligand with ferric ion under various pH conditions
showed the characteristic LMCT (ligand to metal charge
transfer) band of a ferric complex around at 465 nm (ꢀ
very low due to their high acidity. The results prompted
us to note the other bidentate ligand HOPY, which has
higher pKa value than that of HOPR. The pyrimidine
bases consist of uracil, thymine, and cytosine which are
regarded as 4-hydroxy-, 4-hydroxy-5-methyl-, and 4-amino-
2(1H)-pyrimidinone, respectively, and thus 2(1H)-pyri-
midinones seem to be attractive compounds from the
viewpoint of biological interest. However, no multiden-
tate ligand bearing 2(1H)-pyrimidinone has been re-
ported, to the best of our knowledge. Thus, we designed
novel hexadentate ligand 3HOPYn in which three units
of 1-hydroxy-2(1H)-pyrimidinone were linked to tris(2-
aminoethyl)amine (TREN) through an amide bond by an
alkyl chain. In this paper, we report the synthesis and
thermodynamic and kinetic results on iron-chelating of
3HOPYn. The effect of the methylene chain length (n )
5-7) on the stability constant and the conformation of
the iron complex is also discussed.
4550 at pH 5.6). The observed
and ꢀ values, which
max
were comparable to those of Fe(3-opr(Me)) (ꢀ 3000 at
450 nm),8a indicated the formation of an intramolec-
ular 1:1 iron complex. The 1:1 stoichiometry was con-
firmed by the mole ratio plot. No apparent change in
and absorbance was observed over a wide pH
max
range, especially under acidic to neutral conditions, in
contrast to the corresponding bidentate ligand, HOPY.
This observation suggests that hexadentate ligand,
3HOPY5, can form a stable 1:1 complex by virtue of its
structural effect which is entropically superior to biden-
tate one.
Sta bility of Ir on Com p lex. The stability constant
of the complex of the hexadentate ligand with iron is
defined by the following equilibrium:
Resu lts a n d Discu ssion
Fe3+ + L3- y
\
Kz FeL
K )
[FeL]
Syn th esis. 1-(Benzyloxy)uracil (2) was prepared from
the reaction of N-(benzyloxy)amine and sodium cyanate
and subsequent cyclization of N-(benzyloxy)urea (1) with
3,3-dimethoxypropionic acid methyl ester in the presence
of sodium ethoxide as shown in Scheme 1. Treatment of
2 with 1,2,4-triazole in the presence of phosphorus
oxychloride9 and Et3N in dry MeCN gave 1-(benzyloxy)-
4-(1,2,4-triazol-1-yl)-2(1H)-pyrimidinone (3) in 61% yield.
In order to introduce a spacer group to the heterocyclic
ring system, 3 was reacted with -amino carboxylic acid
methyl ester (n ) 5-7) to give 4a -c. Two types of
signals at 3.19 and 3.43 ppm (1:5 integrated intensity)
assignable to CH2N protons were observed in the 1H
NMR of 4a in CDCl3, suggesting that 4a existed in a
tautomeric equilibrium between 4-imino and 4-amino
forms. The major peak at 3.43 ppm appeared as a
quartet, indicating that 4a predominantly exists in the
4-amino form. Two NH proton signals at 6.14 and 6.75
were D2O-exchangeable, with changing of the quartet of
CH2N into a triplet. In DMSO-d6 solution, 4a exclusively
existed in the 4-amino form. The hydrolysis of 4 with 1
M NaOH gave 5a -c. Conversion to the corresponding
[Fe3+][L3-
]
In order to estimate the stability constants of com-
plexes of 3HOPYn with iron, the competitive reactions
between EDTA and these ligands were carried out.
Although three pKa values of 3HOPYn are necessary for
the calculation, measurement of these pKa values was
impossible due to experimental limitation. Consequently,
1-hydroxy-4-(butylamino)-2(1H)-pyrimidinone (8) was pre-
pared as a model compound, and its pKa value (7.5) was
used for the calculation. This relatively higher pKa than
that of HOPY (6.1) may be attributable to the inductive
effect of the electron-donating amino group at the C-4
position of the pyrimidinone ring. The competitive
reaction of 3HOPY5 and 3HOPY6 with EDTA were car-
ried out in water, while the measurement of stability of
Fe(3opy7) in water was precluded due to poor water
solubility of 3HOPY7. Thus, the reaction was also carried
out in 40% aqueous MeOH in the same fashion of those
in water. The results are summarized in Table 1. As
expected, the stability constants (25 to 27 in log K) were
(9) Harden, M. R.; J ennings, L. J .; Parkin, A. J . Chem. Soc., Perkin
Trans. 1 1990, 2175.
(10) Llinas, M. Structure and Bonding; Springer-Verlag: New York,
1974; Vol. 17, p 142.

3620 J . Org. Chem., Vol. 62, No. 11, 1997
Ohkanda et al.
Sch em e 2
Ta ble 1. Estim a ted Sta bility Con sta n ts of Ir on (III)
a
Com p lexes of 3HOP Yn
log K
ligand
3HOPYn
in H2O
25.7
in 40% aqueous MeOH
n ) 5
25.1
27.0
27.1
n ) 6
26.9
n ) 7
3-HOPR(Me)b
3HOPRnCMec
HOPYd
22.5
20.6-21.7
22.1
a
Initial concentrations of Fe(3opyn) and EDTA, 0.30 mM; in
acetate buffer (pH 4.0) at 20 °C, ) 0.04 with KNO3 solution.
b
d
Reference 8a. c n ) 2-6; reference 8b. log 3; reference 7.
F igu r e 1. Spectral change of Fe(3opy5) in aqueous solution
under various pH conditions.
considerably greater than those of pyrazinone-containing
hexadentate ligands, 3-HOPR(Me) (Figure 2),8a by at
least 3 orders of magnitude. Interestingly, the longer
spacer was slightly advantageous to the stable chelation
with iron. This observation disagreed with the consid-
eration that we have previously proposed. As for 3HOP-
RnCMe (Figure 2), the stability of iron complex increased
with decrease of spacer length, suggesting that the
shorter spacer was suitable for stabilization entropically
of the complex by virtue of the chelating effect.8b The
different propensity of the effect of spacer length on the
stability constant could be explained in terms of the
difference of the spacer position in each heterocyclic ring
system. As shown in Figure 2a, the pyrazinone deriva-
tives were linked to the spacer group at the meta position
F igu r e 2.
toward the NOH moiety, and they could form an octa-
hedral 3:1 iron complex with hydroxamate moieties in
the molecule. In contrast, the pyrimidinone derivatives
had the spacer group at the para position toward the
NOH (Figure 2b), so that the shorter spacer should
prevent complexation. Therefore, in the case of 3HOPYn,
the longer spacer would be favorable to form an octahe-
dral complex.
Con for m a tion of Ga lliu m Com p lex. The examina-
tion using molecular model showed that the conformation

Novel Iron Sequestering Agents
J . Org. Chem., Vol. 62, No. 11, 1997 3621
F igu r e 3. 1H NMR spectra of (a) Ga(3opy5) complex and (b) free ligand (3HOPY5) in CD3OD.
of Fe(3opyn) should possess the C3 symmetry. In order
to understand the conformation in a solution, a Ga(III)
complex was prepared from 3HOPY5 and Ga(NO3)3, and
1
the H NMR spectrum of the complex was measured in
CD3OD at room temperature. The spectrum is shown
in Figure 3 together with that of free ligand, 3HOPY5.
Interestingly, two doublet peaks at 6.0-6.2 assignable
to the 5-H of the pyrimidinone ring were observed, while
the other protons showed only simple sets of signals. In
general, two isomers are possible for a Ga(III) complex,
cis and trans forms. On the basis of the molecular model
examination, it is evident that simultaneous existence
of both cis and trans forms is possible by virtue of rather
long spacers in the molecule. Thus, the existence of cis
and trans isomers would reflect the separation of the 5-H
doublet.
F igu r e 4. Optimized structures of Fe(3opy5) by MM calcula-
tion (a view from the top of the complex: all hydrogens were
excluded for clarity).
MM Ca lcu la tion . In order to confirm the hypothesis
described above, MM calculation of the iron complex was
carried out. The conformation of cis and trans isomers
of Fe(3opy5) was optimized by MM2 and MD calcula-
trans form was slightly distorted (Figure 4). The heats
of formation were -96.34 and -92.23 kcal/mol for cis and
trans isomers, respectively. Energy gap of 4 kcal/mol
between the isomers indicated that the cis isomer is
superior to trans one at least in vacuum system. How-
ever, when other factors that determine the stability of
the complex in solution are considered, such as the
interaction with the solvent, the difference in heat of
formation is regarded to be not enough to allow the
formation of the cis isomer predominantly in solution.
tions.11 The X-ray crystallographic data of Fe(1,2-opo)3
5m
were used for the Cartesian coordinate of each atom
around iron. The optimized structure of the cis form
completely possessed C3 symmetry, while that of the
(11) The MM2 and MD calculations were performed on Macintosh
Quadra 950 with coprocessor CXP40M using MM2 in CAChe (Ver. 3.6)
argumented MM2 force field (CAChe Scientific) and MD in CAChe
(Ver. 3.6).
(12) Carrano, C. J .; Raymond, K. N. J . Am. Chem. Soc. 1979, 101,
5401.

3622 J . Org. Chem., Vol. 62, No. 11, 1997
Ohkanda et al.
Elmer Series II 2400 CHNS Analyzer. FAB mass spectra were
taken on a J EOL DX303 with a DA 5000 data system by using
a
Xe beam at 6 keV and a nitrobenzyl alcohol matrix.
N-(Benzyloxy)urea (1) was prepared by the same procedure
described in the literature.13
Distilled deionized water was used at all times. For all
experiments, observed pH was measured as -log [H+] with a
calibrated Horiba combination electrode (Horiba-6378) using
a Horiba F-12 meter.
1-(Ben zyloxy)u r a cil (2). N-(Benzyloxy)urea (9.97 g, 0.06
mol) was added to a solution of sodium (1.5 g, 0.07 mol) in
absolute EtOH (90 mL) at 30 °C under a nitrogen atmosphere.
After addition of 3,3-dimethoxypropionic acid methyl ester
(9.78 g, 0.07 mol), the reaction mixture was stirred for 3 h at
20 °C, then refluxed for another 17 h, and finally cooled to 10
°C and maintained at the temperature for 4 h. The precipi-
tated sodium salt was collected by filtration and dissolved in
H2O (120 mL). The pH of the aqueous solution was adjusted
to 4 with AcOH. The resulting precipitate was collected by
filtration and then recrystallized from an EtOH-H2O (1:1)
mixture to give the product 2; 1.52 g (67%): mp 180-183 °C
(lit.14 mp 185 °C); IR (KBr) 3234, 1633, 738, 699 cm-1; 1H NMR
(CDCl3) 5.18 (s, 2H), 5.40 (d, J ) 9 Hz, 1H), 6.98 (d, J ) 9
Hz, 1H), 7.41 (s, 5H).
F igu r e 5. Plots of log[(A∞ - Abs)]/[(A∞ - A0)] vs time on iron
removal of 3HOPYn from TfFe2.0
.
Ta ble 2. Ir on Rem ova l fr om Tr a n sfer r in a t p H 7.4
ligand
3HOPYn, n )
kobs
% Fe
a
[L]/[TfFe2.0
]
(×10-3 min-1
)
removedb
1-(Be n zyloxy)-4-(1,2,4-t r ia zol-1-yl)-2(1H )-p yr im id i-
n on e (3). A solution of 1,2,4-triazole (6.22 g, 90 mmol) in dry
MeCN (25 mL) was treated with POCl3 (2.5 mL, 27 mmol) and
Et3N (12.5 mL, 90 mmol) at 0 °C. A solution of 2 (1.96 g, 9
mmol) in dry MeCN (100 mL) was added to the mixture at 0
°C. The pH of the solution was adjusted to around 9 by
addition of Et3N (ca. 5 mL), and the reaction mixture was
stirred for 24 h at room temperature. Et3N (12.5 mL) and H2O
(5 mL) were successively added to the reaction mixture in order
to quench the reaction, and MeOH was added to the emulsion
until the solution became clear. After removal of the solvents,
the residue was washed with cold water (2 × 40 mL) and
recrystallized from EtOH to give the product 3; 1.47 g (61%):
5
6
60
60
4.5
4.0
27
24
7
60
100
1.7
0.66
11
DFO
5 (5)c
a
[TfFe2.0]0 ) 0.02 mM, TfFe2.0 was prepared from human serum
b
apotransferrin (Sigma). At a point 30 min after the reaction was
initiated. c Reference 12.
Therefore, the two isomers might be in the equilibrium
1
in the system, as observed in the H NMR spectrum.
Ir on Rem ova l fr om Tr a n sfer r in . There is great
interest to the potential application of 3HOPYn to a
chemotherapeutic agent for the iron overload. High
kinetic efficiency of iron removal from transferrin is one
of the quite important requisites of a nontoxic drug.
Therefore, kinetic evaluation of the iron removal ability
of each ligand from human transferrin was carried out
in physiological conditions. As shown in Figure 5, there
mp 206-209 °C; IR (KBr) 1680, 740, 695 cm-1 1H NMR
;
(CDCl3) 5.36 (s, 2H), 6.75 (d, J ) 8 Hz, 1H), 7.40 (s, 5H),
7.55 (d, J ) 8 Hz, 1H), 8.07 (s, 1H), 9.22 (s, 1H). Anal. Calcd
for C13H11N5O2: C, 57.99; H, 4.12; N, 26.01. Found: C, 57.75;
H, 4.36; N, 25.77.
Gen er a l P r oced u r e for th e Cou p lin g of 3 w ith Meth yl
ω-Am in o Ca r boxyla te Hyd r och lor id e 4a -c. A Typ ica l
Exa m p le: 4-[(5-(Meth oxyca r bon yl)p en tyl)a m in o]-1-(ben -
zyloxy)-2(1H )-p yr im id in on e (4a ). Methyl 6-aminohex-
anoate hydrochloride (2.12 g, 11.7 mmol) and Et3N (1.18 g,
11.7 mmol) were added to a solution of 3 (2.7 g, 10 mmol) in
dry THF (60 mL). The reaction mixture was refluxed for 44
h and then concentrated. H2O (30 mL) was added to the
residue, and the aqueous solution was extracted with CHCl3
(5 × 50 mL). The combined organic layers were dried over
anhydrous MgSO4. After evaporation of the solvent, the
resulting residual solid was recrystallized from EtOH to give
the product 4a ; 2.41 g (70%): mp 144-146 °C; IR (KBr) 3020,
are good linear relationships on the plots of log[(A∞
-
Abs)/(A∞ - A0)] as a function of time, indicating that the
iron removal from transferrin by 3HOPYn proceeded in
the pseudo-first-order kinetics. From the slope of the
straight line, kobs was obtained. The kinetic results are
summarized in Table 2 together with data of DFO. It is
noteworthy that 3HOPYn, especially 3HOPY5, efficiently
removed iron from transferrin over five times as much
as commercially available DFO did even at a lower
concentration of the ligand than that of DFO. This
difference is apparently observed in the percentages of
iron removal after the reactions were performed for 30
min (27% for 3HOPY5; 5% for DFO).
1
1670, 1630, 750, 700 cm-1; H NMR (CDCl3) 1.30-1.45 (m,
2H), 1.55-1.70 (m, 4H), 2.32 (t, J ) 8 Hz, 2H), 3.19 (br s, 1/5H),
3.43 (q, J ) 8 Hz, 4/5H), 3.65 (s, 3H), 5.20 (s, 2H), 5.41 (br s,
1/5H), 5.56 (d, J ) 8 Hz, 4/5H), 6.14 (br s, 4/5H), 6.75 (br s,
1/5H), 6.90 (d, J ) 8 Hz, 4/5H), 7.07 (d, J ) 8 Hz, 1/5H), 7.38
(s, 5H); (DMSO-d6) 1.25-1.38 (m, 2H), 1.40-1.60 (m, 4H),
2.3 (t, J ) 8 Hz, 2H), 3.18 (q, J ) 8 Hz, 2H), 3.58 (s, 3H), 5.05
(s, 2H), 5.45 (d, J ) 8 Hz, 1H),7.35-7.46 (m, 5H), 7.55 (d, J )
8 Hz, 1H), and 7.67 (br s, 1H). Anal. Calcd for C18H23N3O4:
C, 62.59; H, 6.71; N, 12.17. Found: C, 62.61; H, 6.74; N, 12.22.
Exp er im en ta l Section
Gen er a l. Melting points were recorded on a Mel-Temp
apparatus in open capillaries and are uncorrected. IR spectra
were recorded on a J ASCO IR-700 infrared spectrometer. UV
spectra were recorded on a J ASCO Ubest V-550 UV/vis
Spectrophotometer. 1H NMR were recorded on J EOL GX-270
NMR spectrometer in CDCl3, DMSO-d6, D2O, and CD3OD.
Chemical shifts were reported in ppm ( ) downfield from
internal tetramethylsilane (TMS) or 2,2-dimethyl-2-silapen-
tane-5-sulfonate (DSS). Thin layer chromatography (TLC)
analyses were performed on silica gel 60F-254 with a 0.2 mm
layer thickness. Column chromatography was carried out with
Merck kieselgel 60 (230-400 mesh). Combustion analyses
were performed on a Yanaco MT-3 CHN CORDER and Perkin-
4-[(6-(Met h oxyca r b on yl)h exyl)a m in o]-1-(b en zyloxy)-
2(1H)-p yr im id in on e (4b); 50%: mp 110-111 °C; IR (KBr)
3252, 1737, 1645, 702 cm-1; 1H NMR (CDCl3) 1.25-1.35 (m,
4H), 1.52-1.64 (m, 4H), 2.33 (t, 2H), 3.15 (br s, 2/5H), 3.40
(m, 8/5H), 3.65 (s, 3H), 5.20 (s, 2H), 5.55 (d, J ) 8 Hz, 1H),
6.41 (br s, 1H), 6.68 (br s, 1/5H), 6.89 (d, J ) 8 Hz, 4/5H), 7.10
(13) Behrend, R.; Leuchs, K. J ustus Liebigs Ann. Chem. 1890, 257,
203.
(14) Klo¨tzer, W. Monatsh. Chem. 1964, 95, 1729.

Novel Iron Sequestering Agents
J . Org. Chem., Vol. 62, No. 11, 1997 3623
(d, J ) 8 Hz, 1/5H), 7.37 (s, 5H). Anal. Calcd for C19H25N3O4:
C, 63.49; H, 7.01; N, 11.69. Found: C, 63.46; H, 7.21; N, 11.66.
4-[(7-(Meth oxyca r bon yl)h ep tyl)a m in o]-1-(ben zyloxy)-
2(1H)-p yr im id in on e (4c). The crude product was purified
by column chromatography on silica gel (eluent: CHCl3-
acetone-EtOH ) 100:10:2) to afford the pure product 4c;
60%: mp 132-135 °C; IR (KBr) 3250, 1737, 1671, 1642, 749,
40 with MeOH to give the pure product 7a ; 296 mg (52%): IR
(KBr) 3500, 2940, 2860, 1760, 1630, 750, and 700 cm-1
;
1H
NMR (CDCl3) 1.18-1.32 (m, 6H), 1.45-1.65 (m, 12H), 2.15-
2.25 (m, 6H), 2.55-2.70 (m, 6H), 3.25-3.40 (m, 12H), 5.15 (s,
6H), 5.65 (d, J ) 8 Hz, 3H), 6.90 (d, J ) 8 Hz, 3H), 7.25 (s,
15H), 7.72 (br s, 3H). Anal. Calcd for C57H75N13O9‚3H2O: C,
60.04; H, 7.16; N, 15.97. Found: C, 60.22; H, 6.98; N, 15.71.
702 cm-1 1H NMR (CDCl3) 1.25-1.35 (m, 6H), 1.53-1.65
;
Tr is(2-(6-(1-(1-(ben zyloxy)-2-oxo-1,2-d ih yd r op yr im id i-
(m, 4H), 2.32 (t, 2H), 3.16 (br s, 2/5H), 3.41 (m, 8/5H), 3.66 (s,
3H), 5.19 (s, 2H), 5.40 (d, J ) 8 Hz, 1/5H), 5.56 (d, J ) 8 Hz,
4/5H), 6.41 (br s, 4/5H), 6.68 (br s, 1/5H), 6.88 (d, J ) 8 Hz,
4/5H), 7.10 (d, J ) 8 Hz, 1/5H), 7.37 (s, 5H). Anal. Calcd for
C20H27N3O4: C, 64.32; H, 7.29; N, 11.25. Found: C, 64.26; H,
7.30; N, 11.02.
4-yl)a m in o)h ep ta n a m id o)eth yl)a m in e (7b); 86%: IR (KBr)
1
3284, 1636, 753, 702 cm-1; H NMR (CDCl3) 1.18-1.32 (m,
12H), 1.48-1.65 (m, 12H), 2.22 (m, 6H), 2.58-2.68 (m, 6H),
3.20-3.35 (m, 12H), 5.12 (s, 6H), 5.71 (d, J ) 8 Hz, 3H), 6.92
(d, J ) 8 Hz, 3H), 7.36 (s, 15H), 7.51 (br s, 3H), 7.71 (br s,
3H). Anal. Calcd for C60H81N13O9‚3H2O: C, 60.94; H, 7.42
N, 15.40. Found: C, 61.14; H, 7.61; N, 15.46.
Gen er a l P r oced u r e for P r ep a r a tion of 5a -c. A Typ i-
ca l Exa m p le: 4-[(5-Ca r boxyp en tyl)a m in o]-1-(ben zyloxy)-
2(1H)-p yr im id in on e (5a ). To a solution of compound 4a
(2.41 g, 6.98 mmol) in MeOH (100 mL) was added 1M NaOH
(7.7 mL, 7.7 mmol), and the reaction mixture was stirred for
1 h at room temperature. Additional 1 M NaOH (15.4 mL,
15.4 mmol) was added to the mixture, and the reaction mixture
was stirred for another 14 h at room temperature. After
evaporation of the solvent, the residue was dissolved in H2O
(40 mL). The pH of the aqueous solution was adjusted to 3
with 5% HCl. The precipitated crude solid was recrystallized
from EtOH to give the product 5a ; 2.16 g (93%): mp 192-193
Tr is(2-(6-(1-(1-(ben zyloxy)-2-oxo-1,2-d ih yd r op yr im id i-
4-yl)a m in o)octa n a m id o)eth yl)a m in e (7c); 71%: IR (KBr)
1
3284, 1636, 753, 703 cm-1; H NMR (CDCl3) 1.20-1.32 (m,
18H), 1.45-1.65 (m, 12H), 2.22 (m, 6H), 2.48-2.55 (m, 6H),
3.20-3.40 (m, 12H), 5.12 (s, 6H), 5.72 (d, J ) 8 Hz, 3H), 6.93
(d, J ) 8 Hz, 3H), 7.35 (s, 15H), 7.64 (br s, 3H), 7.71 (br s,
3H). Anal. Calcd for C63H87N13O9‚0.5H2O: C, 64.16; H, 7.52
N, 15.44. Found: C, 63.99; H, 7.50; N, 15.58.
1-Hyd r oxy-4-(N-bu tyla m in o)-2(1H)-p yr im id in on e (8).
A solution of 3 (0.63 g, 2.34 mmol) and butylamine (0.21 g,
2.87 mmol) in dry THF (20 mL) was refluxed for 4 h. The
solvent was evaporated, and H2O (10 mL) was added to the
residue. The aqueous solution was extracted with CHCl3 (4
× 50 mL), and then the combined organic layers were dried
over anhydrous Na2SO4. After removal of the solvent, the
crude product was recrystallized from EtOH to give 1-(ben-
zyloxy)-4-(N-butylamino)-2(1H)-pyrimidinone; 75%: mp 160-
162 °C; IR (KBr) 3246, 1644, 747, 699 cm-1; 1H NMR (CDCl3)
0.94 (t, J ) 8 Hz, 3H), 1.3-1.41 (m, 2H), 1.50-1.60 (m, 2H),
3.10-3.50 (m, 2H), 5.22 (s, 3H, Bn and 5-H), 6.90 (d, J ) 8
Hz, 1H), 7.40 (s, 5H). Anal. Calcd for C15H19N3O2: C, 65.91;
H, 7.01; N, 15.37. Found: C, 65.80; H, 7.18; N, 15.46.
A suspension of 10% Pd-C (25 mg) in distilled MeOH (10
mL) was prehydrogenated with H2 for 0.5 h. A solution of
1-(benzyloxy)-4-(N-butylamino)-2(1H)-pyrimidinone (252 mg,
0.92 mmol) in distilled MeOH (20 mL) was added to the
suspension. After hydrogenation with H2 under reflux for 2
h, the catalyst was removed by filtration. The filtrate was
evaporated to give the product 8; 100%: mp 120-123 °C.
IR (KBr) 3300-2700, 3284, and 1636 cm-1; 1H NMR (DMSO-
°C; IR (KBr) 3200-2800, 1710, 1642, 752, 701 cm-1 1H
;
NMR (DMSO-d6) 1.18-1.39 (m, 2H), 1.43-1.61 (m, 4H), 2.19
(t, 2H), 3.17-3.30 (m, 2H), 5.17 (s, 2H), 5.51 (d, J ) 7 Hz,
1H), 7.32-7.47 (m, 6H), 7.67 (br s, 1H). Anal. Calcd for
C17H21N3O4‚0.3H2O: C, 60.80; H, 6.58; N, 12.45. Found: C,
60.63; H, 6.46; N, 12.45.
4-[(6-Ca r boxyh exyl)a m in o]-1-(ben zyloxy)-2(1H)-p yr i-
m id in on e (5b); 69%: mp 134-137 °C; IR (KBr) 3200-2700,
1
1716, 1673, 739, 700 cm-1; H NMR (DMSO-d6) 1.28-1.48
(m, 8H), 2.20 (t, 2H), 5.08 (s, 2H), 5.63 (d, J ) 8 Hz, 1H), 7.40
(s, 5H), 7.68 (d, J ) 8 Hz, 1H). Anal. Calcd for C18H23N3O4:
C, 56.68; H,7.13; N, 11.02. Found: C, 56.73; H, 6.77; N, 11.19.
4-[(7-Ca r boxyh ep tyl)a m in o]-1-(ben zyloxy)-2(1H)-p yr i-
m id in on e (5c); 80%: IR (KBr) 3200-2700, 1739, 1636, 739,
1
703 cm-1; H NMR (DMSO-d6) 1.28-1.46 (m, 10H), 2.19 (t,
2H), 3.13-3.18 (m, 2H), 5.06 (s, 2H), 5.50 (d, J ) 8 Hz, 1H),
7.42 (s, 5H), 7.55 (d, J ) 8 Hz, 1H), 7.68 (br s, 1H), 11.95 (br
s, 1H). Anal. Calcd for C19H25N3O4‚0.5H2O: C, 61.94; H,7.11;
N, 11.41. Found: C, 62.07; H, 7.07; N, 11.15.
Gen er a l P r oced u r e for P r ep a r a tion of O-Su ccin im id e
Ester s 6a -c. A Typ ica l Exa m p le: 4-[(5-Ca r boxyp en tyl)-
a m in o]-1-(ben zyloxy)-2(1H)-p yr im id in on e O-Su ccin im -
id e Ester (6a ). To a solution of compound 5a (700 mg, 2.11
mmol) and HOSu (486 mg, 4.22 mmol) in dry DMF (10 mL)
was added a solution of 1-[3-(dimethylamino)propyl]-3-ethyl-
carbodiimide hydrochloride (WSC‚HCl; 810 mg, 4.22 mmol) in
CH2Cl2 (20 mL) at -10 °C. The reaction mixture was stirred
for 14 h at room temperature, the solvent was evaporated, and
then the resulting solid was dissolved in CHCl3 (150 mL). The
organic layer was washed with H2O (2 × 50 mL) and dried
over anhydrous Na2SO4. After removal of the solvent, the
O-succinimide ester 6a was obtained and used for the next
reaction without further purification; 740 mg (82%): IR
d6)
0.85 (t, J ) 7 Hz, 3H), 1.25-1.51 (m, 4H), 3.20 (q,
J ) 7 Hz, 2H), 5.55 (d, J ) 7 Hz, 1H), 7.51 (br s, 1H) 7.65 (d,
J
) 7 Hz, 1H), and 10.85 (br s, 1H). Anal. Calcd for
.
C8H13N3O2 0.2H2O: C 51.44, H 7.23, N 22.49. Found: C 51.62,
H 7.09, N 22.75.
Gen er a l P r oced u r e for P r ep a r a tion of Hexa d en ta te
Liga n d s (3HOP Yn ). A Typ ica l Exa m p le: Tr is(2-(6-(1-(1-
h yd r oxy-2-oxo-1,2-d ih yd r op yr im id i-4-yl)a m in o)h exa n -
a m id o)eth yl)a m in e (3HOP Y5). A suspension of 10% Pd-C
(35 mg) in MeOH (20 mL) was prehydrogenated with H2 for
0.5 h. To the suspension was added a solution of compound
7a (300 mg, 0.28 mmol) in MeOH (10 mL). After hydrogena-
tion with H2 under atmospheric pressure for 1 h under reflux,
the catalyst was removed by filtration. The filtrate was
concentrated to give the residue, which was purified by gel
chromatography on Sephadex LH-20 with MeOH to afford
the product 3HOPY5; 150 mg (66%): IR (KBr) 3500-3200,
(CDCl3) 1810, 1780, 1720, 1620, 740, 690 cm-1
.
4-[(6-Ca r boxyh exyl)a m in o]-1-(ben zyloxy)-2(1H)-p yr i-
m id in on e O-su ccin im id e ester (6b); 100%: IR (CDCl3)
1810, 1770, 1720 cm-1
.
3282, 1636 cm-1 1H NMR (DMSO-d6) 1.20-1.30 (m, 6H),
:
4-[(7-Ca r boxyh ep tyl)a m in o]-1-(ben zyloxy)-2(1H)-p yr i-
m id in on e O-su ccin im id e ester (6c); 100%: IR (CDCl3)
1.41-1.53 (m, 12H), 2.08 (t, J ) 7 Hz, 6H), 2.40-2.52 (m, 6H),
3.05-3.22 (m, 12H), 5.55 (d, J ) 8 Hz, 3H), 7.55 (br s, 3H),
7.65 (d, J ) 8 Hz, 3H), 7.73 (br s, 3H). Anal. Calcd for
C36H57N13O9‚0.5H2O: C, 52.42; H, 7.09; N, 22.07. Found: C,
52.29; H, 7.37; N, 22.23.
1808, 1772, 1720 cm-1
.
Gen er a l P r oced u r e for P r ep a r a tion of Tr ip od a l Com -
p ou n d s 7a -c. A Typ ica l Exa m p le: Tr is(2-(6-(1-(1-(ben -
zyloxy)-2-oxo-1,2-d ih yd r op yr im id i-4-yl)a m in o)h exa n -
a m id o)eth yl)a m in e (7a ). A solution of tris(2-aminoethyl)-
amine (76 mg, 0.52 mmol) in DMF (5 mL) was added to a
solution of compound 6a (740 mg, 0.52 mmol) in DMF (15 mL),
and then the mixture was stirred for 48 h at 38 °C. After
removal of the solvent, the residue was purified by column
chromatography on silica gel with a CHCl3-MeOH (5:1)
mixture, followed by gel chromatography on Toyopearl HW-
Tr is(2-(6-(1-(1-h yd r oxy-2-oxo-1,2-d ih yd r op yr im id i-4-
yl)a m in o)h ep ta n a m id o) eth yl)a m in e (3HOP Y6); 66%: IR
(KBr) 3500-3200, 3284, 1635 cm-1 1H NMR (DMSO-d6)
;
1.21-1.32 (m, 12H), 1.40-1.55 (m, 12H), 2.10 (t, J ) 7 Hz,
6H), 2.39-2.50 (m, 6H), 3.05-3.25 (m, 12H), 5.55 (d, J ) 8
Hz, 3H), 7.55 (br s, 3H), 7.65 (d, J ) 8 Hz, 3H), 7.70 (br s,
3H), 10.90 (br s, 3H, OH); MS (FAB) m/ z 858 (M+). Anal.

3624 J . Org. Chem., Vol. 62, No. 11, 1997
Ohkanda et al.
Calcd for C39H63N13O9‚1.8H2O: C, 52.61; H, 7.54; N, 20.45.
3HOPYn), and the pH of the solution at an equilibrium point
at 20 °C. The experiments for all ligands were carried out by
using 40% MeOH in an H2O solution in the same fashion as
described above, except for using MeOH to dilute to 10 mL in
the preparation of the iron complex solution. The reaction
solution after being combined with the same amount of EDTA
stock solution totally contained MeOH in 40%. The apparent
pH value of the solution was used for the calculation without
corrections.
Found: C, 52.73; H, 7.73; N, 20.25.
Tr is(2-(6-(1-(1-h yd r oxy-2-oxo-1,2-d ih yd r op yr im id i-4-
yl)a m in o)octa n a m id o)eth yl)a m in e (3HOP Y7); 61%: IR
(KBr) 3500-3200, 3284, 1636 cm-1 1H NMR (DMSO-d6)
;
1.16-1.30 (m, 18H), 1.40-1.55 (m, 12H), 2.08 (t, J ) 7 Hz,
6H), 2.41-2.51 (m, 6H), 3.05-3.25 (m, 12H), 5.57 (d, J ) 8
Hz, 3H), 7.56 (br s, 3H), 7.65 (d, J ) 8 Hz, 3H), 7.65 (br s,
3H), 10.86 (br s, 3H, OH); MS (FAB) m/ z 900 (M+). Anal.
Calcd for C42H69N13O9‚2H2O: C, 53.89; H, 7.86; N, 19.45.
Found: C, 53.83; H, 7.85; N, 19.39.
Ga lliu m Com p lex F or m a tion . 3HOPY5 (6 mg) and Ga-
(NO3)3 (4 mg) were dissolved in 10% CD3OD/D2O (1:9; 0.5 mL).
The pD was adjusted to 6 with freshly prepared 0.4% NaOD
in D2O.16 The 1H NMR spectrum was measured at room
temperature. Ga(3opy5): 1.35 (m, 6H), 1.60 (m, 12H), 2.25
(m, 6H), 3.40 (m, 6H), 3.55 (m, 6H), 6.00 and 6.12 (d each, J
) 8 Hz each, 3H), 7.87 (d, J ) 8 Hz, 3H).
Mea su r em en t of p Ka Va lu e of 8. Compound 8 (30 mg)
was dissolved in deionized water (30 mL). The pH of the
solution was measured after every 0.1 mL addition of a 0.08
M NaOH solution at room temperature under an Ar atmo-
sphere. The pKa value was calculated from the pH value at
the midpoint of neutralization.
Kin et ics of Ir on R em ova l fr om TfF e by 3HOPYn .
A
2.0
Typ ica l Exa m p le. A commercially available human serum
transferrin (98%, Sigma) was used. Fe2.0Tf was prepared
according to the literature reported in detail by Raymond.17
The stock solution of 3HOPY5 (1.0 mL, 1.52 × 10-3 M) and
TfFe2.0 (1.0 mL, 3.886 × 10-5 M) were prepared in Tris chloride
buffer and combined. The absorbance of the solution was
monitored at 460 nm. The pseudo-first-order rate constant
(kobs) was obtained from the slope of the plots of log[(A∞ - Abs)/
(A∞ - A0)] against time (min).
Gen er a l P r oced u r e for Sp ectr a l Mea su r em en t of 1:1
Mixtu r es of Ir on a n d Hexa d en ta te Liga n d s. A sample
(13-15 mg) of each hexadentate ligand was dissolved in
deionized water (5.0 mL). The sample solution (1.0 mL) was
mixed with an equimolar amount of ferric nitrate solution (3.28
mM) and diluted to 10.0 mL (0.3 mM). The pH of the solution
was adjusted to an appropriate value with 0.1 or 0.01 M NaOH
or 0.1 or 0.01 M HNO3 before spectral measurement.
Ir on Exch a n ge Rea ction . An iron complex solution (0.30
mM) of the hexadentate ligands (3HOPY5 and 3HOPY6) was
prepared by mixing a stock solution of the ligand (3.28 mM)
with an equimolar amount of ferric nitrate solution (3.28 mM)
and 1.0 mL of 0.4 M KNO3 and then diluting the solution to
10.0 mL with acetate buffer (pH 4.0). An EDTA solution was
prepared by dissolving (EDTA)2-‚2Na+‚2H2O in acetate buffer
solution (ionic strength 0.04, pH 4.0) to give a concentration
of 0.30 mM. The competitive reaction was initiated by
combination the iron complex solution (2.0 mL) with the EDTA
solution (2.0 mL). The iron exchange reaction was checked
by monitoring the decrease of absorbance at 450 nm. The
relative stability constants of the iron complexes were calcu-
lated by using the stability constant of Fe(edta) (log K 25.1),15
the pKa of the corresponding bidentate ligand (pKa 7.5 of 8 for
Ack n ow led gm en t. The authors are grateful to Dr.
T. Takahashi and K. Yamada of Lion Corporation for
measuring FAB mass spectra. This work was partially
supported by a Grant-in-Aid for Scientific Research from
the Ministry of Education, Science, Sports, and Culture,
J apan (08740506), Saneyoshi Scholarship Foundation
(0268), and Japan Private School Promotion Foundation.
J O961901M
(15) Martell, A. E., Smith, R. M. In Critical Stability Constants;
Plenum: New York, 1974; Vol. 1.
(16) Hou, Z.; Whisenhunt, J r., D. W.; Xu, J .; Raymond, K. N. J . Am.
Chem. Soc. 1994, 116, 840.
(17) Nguyen, S. A. K.; Craig, A.; Raymond, K. N. J . Am. Chem. Soc.
1993, 115, 6758.