3-Hydroxy-4(1H)-pyridinone-containing linear and cyclic hexapeptides and their iron(III) complexes - Synthesis, property, and the growth-promotion activity-

Article abstract of DOI:10.3987/COM-01-9338

Linear and cyclic hexapeptides composed of ε-(3-hydroxy-1,4-di-hydro-2-methyl-4-oxo-1-pyridyl)-L-norleucine and glycine or β-alanine residue have been successfully synthesized. These hexapeptides formed 1:1 iron(III) complexes at neutral pH region. Glycin

Full text of DOI:10.3987/COM-01-9338

HETEROCYCLES, Vol. 55, No. 11, 2001, pp. 2171 - 2187, Received, 6th September, 2001
3-HYDROXY-4(1H)-PYRIDINONE-CONTAINING LINEAR AND CYCLIC
HEXAPEPTIDES AND THEIR IRON(III) COMPLEXES－ SYNTHESIS,
PROPERTY, AND THE GROWTH-PROMOTION ACTIVITY－
Akira Katoh,*, a Yasushi Hikita,a Manabu Harata,a Junko Ohkanda,a
Taro Tsubomura,a Akon Higuchi,a Ryota Saito,a and Kazuo Haradab
a
Department of Applied Chemistry, Faculty of Engineering, Seikei University,
Musashino, Tokyo 180-8633, Japan
b Department of Life Science, Tokyo Gakugei University, Koganei, Tokyo 184-
8501, Japan
Abstract ̶ Linear and cyclic hexapeptides composed of ε-(3-hydroxy-1,4-di-
hydro-2-methyl-4-oxo-1-pyridyl)-L-norleucine and glycine or β-alanine residue
have been successfully synthesized. These hexapeptides formed 1:1 iron(III)
complexes at neutral pH region.
Glycine linear (8a)- and β-alanine cyclic hexa-
peptide (10)-iron(III) complexes predominantly existed in Δ-configuration, while
β-alanine linear hexapeptide (8b)-iron(III) complex existed in Λ-one. The relative
stability constants of 8a, 8b, and 10-iron(III) complexes were estimated to be
31.6, 33.4, and 31.7, respectively. From iron(III) removal from human transferrin,
linear hexapeptides (8a,b) were found to efficiently remove iron(III) compared with
desferrioxamine B. The growth-promotion activity was also discussed.
Iron is the fourth abundant element in the crust of the earth. Unfortunately, iron exists in extremely
insoluble Fe(OH)3 (Ksp=10-38) at physiological pH under the aerobic environment. In order to solubilize
and uptake such low concentration of ferric iron, microorganisms excrete siderophores, low-molecular-
weight iron-sequestering compounds. They solubilize ferric ion as the form of stable octahedral complexes
and subsequently transport it into cell through the receptor on the surface of membrane. More than 200
siderophores have been isolated and characterized. They are primarily classified into two general structural
classes, hydroxamates and catecholates. Three sets of these bidentate ligands scavenge iron(III) effectively
and selectively by embedding it into an octahedral binding cavity.1-4
On the other hand, the iron overload disease arises from a long term blood transfusionfor treatment of a
certain anemia, particularly β-thalassemia. An excess iron deposit in liver and pancreas causes the damage
of these organs or other serious diseases. Siderophores have paid much attention for treatment of the iron
overload disease. Enterobactin, for example, has been shown to have a high stability constant toward
iron(III) and to remove iron(III) from transferrin, but low acidity and difficulty in dissociation of two
protons on the catechol ring at neutral pH limited the effectiveness of iron-sequestering.5 Desferrioxamine

B (DFB, a commercial name: Desferal) has been shown to be an effective chelating agent for removing of
an excess iron in body, and thus clinically used as a only choice of chemotherapeutic agent for a long time.
Unfortunately it is orally inactive,6 has a short half-life in blood plasma,7 and possesses a number of side
effects such as septicemia.8 Many efforts, therefore, have been devoted to design and synthesize novel
artificial siderophores in place of DFB. Recently, hydroxymonoazine-type ligands have received much
attention because of an effective iron(III) removal, an oral activity, and no apparent toxicity.9 Hexadentate
ligands containing hydroxymonoazines such as 1,2-HOPO,10 3,2-HOPO,11 and 3,4-HOPO,11b, 12 which are
expected to form stable iron(III) complexes by the chelate effect, have been intensively reported. However,
no paper on the synthesis of chiral hexadentate ligands containing hydroxymonoazine-type heterocycles has
been reported, to the best of our knowledge. In addition, N-substituted 2-alkyl-3-hydroxy-4(1H)-
pyridinones were the first compounds among hydroxymonoazine-type heterocycles to enter clinical trials,
and proved their potency as new chemotherapeutic agents for the iron overload disease.9, 13
H
OH
O
N
O
O
N
N
OH
H
OH
3-Hydroxy-2(1H)-
pyridinone (3,2-HOPO)
3-Hydroxy-4(1H)-
pyridinone (3,4-HOPO)
1-Hydroxy-2(1H)-
pyridinone (1,2-HOPO)
A resonance form of monoanion of 3,4-HOPO is isoelectronic to catecholate dianion as shown below. It is,
therefore, quite interesting to examine whether the present hexandentate ligands act as siderophores. A
number of synthetic analogues have been prepared and the growth-promotion activity of them has been
tested using siderophore auxotroph strains.12 Two types of strains, Aureobacterium flavescence (ATCC
25091) and Escherichia coli RW193 (ATCC 33475), are generally used for testing the growth-promotion
activity.14
_
_
_
O
O
O
O
O
O
O
+
-H
+
N
N
_
N
_
H
OH
H
H
In the past decade, we have intensively investigated design of functional molecules based on hydroxyazine-
type heterocycles and application of the heterocycles to chemotherapeutic agents.15,16 We describe here
synthesis and iron(III) complex-forming property of chiral linear and cyclic hexadentate ligands composed
of three sets of ε-(3-hydroxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucine and glycine or β-alanine
in a molecule, including the growth-promotion activity.
RESULTS AND DISCUSSION
Synthesis of Hexapeptide Ligands: The synthetic procedure for linear and cyclic hexapeptide
ligands is depicted in Scheme 1. 3-Benzyloxy-2-methyl-4-pyrone (1) was allowed to react with L-lysine to
give ε-(3-benzyloxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucine (2). The N-tert-butoxy-carbonyl

O
N
O
N
OBn
Me
OBn
Me
O
OBn
Me
i
ii, iii
(CH2)4
(CH2)4
O
1
CO2Na
X-HN
H2N
CONH-(CH2)n-COY
H
2
H
3a,b (X=Boc, Y=OEt)
a n=1
b n=2
iv
v
4a,b (X=Boc, Y=OH)
3a,b
5a,b (X=TFA·H, Y=OEt)
4a,b + 5a,b
vi
O
O
OBn
OBn
vii
N
Me
N
Me
(CH2)4
(CH2)4
Boc-[-HN
CONH-(CH2)n-CO-]2-OEt
CONH-(CH2)n-CO-]3-OEt
Boc-[-HN
H
H
6a,b
7a,b
O
O
N
OR
Me
OH
Me
iv, ix, x
viii
N
7a,b
(for 7b)
(CH2)4
(CH2)4
[-HN
xi
CONH-(CH2)2-CO-]3-
H
TFA·H-[-HN
CONH-(CH2)n-CO-]3-OEt
H
8a,b
9 (R=OBn)
10 (R=OH)
Scheme 1 Reagents and conditions: i) L-lysine, NaOH, H2O-EtOH, rt for 4 days; ii) Boc2O,
dioxane, 0 °C for 8 h, and then rt for 2 days; iii) HCl·H2N-(CH2)n-CO2Et, N-methylmorpholine,
WSC·HCl-HOBt, CH2Cl2, rt for 3 days; iv) 1M NaOH, MeOH, rt for 2 h; v) TFA, CH2Cl2, ice-bath
for 1 h; vi) N-methylmorpholine, WSC·HCl-HOBt, CH2Cl2, rt for 3 days; vii) 4a,b, TFA, CH2Cl2,
ice-bath for 2 h, and then N-methylmorpholine, CDI, DMF, rt for 3 days; viii) H2/10% Pd-C, MeOH,
rt for 4 h, and then TFA, CH2Cl2, ice-bath for 2 h; ix) WSC·HCl-HOSu, DMF, rt for 3 days; x) TFA,
CH2Cl2, ice-bath for 2 h, and then pyridine, 75 °C for 5 days (high dilution); xi) H2/10%Pd-C,
MeOH, rt for 3 days.

(Boc) protection of compound (2), and subsequent coupling with glycine or β-alanine by using 1-[3-
.
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (WSC HCl) and 1-hydroxybenzotriazole
(HOBt) gave the dipeptides (3a,b) as the repeating unit of hexapeptides. Dipeptides (3a,b) were
converted to C-terminal free dipeptides (4a,b) by treatment with 1M NaOH. On the other hand,
deprotection of the Boc group of compounds (3a,b) with TFA gave N-terminal free dipeptides (5a,b).
.
Two components (4a,b) and (5a,b) were coupled by means of WSC HCl-HOBt method to afford the
tetrapeptides (6a,b). The Boc group of tetrapeptides (6a,b) were removed by treatment with TFA to give
the corresponding TFA salts, and then the salts were coupled with dipeptides (4a,b) by using 1,1’-
carbonyldiimidazole (CDI) to give the hexapeptides (7a,b). Debenzylation by the catalytic hydrogenation,
followed by deprotection of the Boc group with TFA gave the desired linear hexapeptide ligands (8a,b).
Compounds (7a,b) were treated with 1M NaOH to give the C-terminal free hexapeptides, and then they
were converted into the corresponding O-succinimide esters. After removal of Boc groups of the O-
succinimide esters and subsequent cyclization of the resulting TFA salts were carried out under high dilution
conditions. Any attempts to synthesize cyclic hexapeptide containing the glycine residue were
unsuccessful. It may be attributable to 1) the steric hindrance of the bulky side chain including 4(1H)-
pyridinone ring, 2) the rigidity of the peptide backbone originated from the glycine residue, and 3) the
lability of the methylene protons of the glycine residue by virtue of the activated ester. On the other hand,
cyclization of the hexapeptide containing the β-alanine residue smoothly proceeded to afford the cyclic
hexapeptide (9). Compound (9) was subjected to the catalytic hydrogenation on 10% Pd-C to give the
desired cyclic hexapeptide ligand (10).
Iron(III) Complex Formation: UV-VIS spectra of a 1:1 molar mixture of a hexapeptide and iron(III)
in aqueous solution under various pH conditions were measured, and spectra of 8b-iron(III) complex are
shown in Figure 1. The absorption maxima due to the ligand-to-metal charge transfer were observed at
460-520 nm. With an increase of pH in acidic region, the absorption maximum was shifted to shorter
wavelength with an increase of absorbance, suggesting the transformation of 3-hydroxy-4(1H)-pyridinone :
iron(III) (1:1) complex into a 1:2 and then into a 1:3 complex.
O
O
O
O
O
O
Fe
Fe
Fe
O
HO
O
HO
O
O
N
N
O
N
OH
N
N
O
N
O
O
O
Me
Me
Me
Me
Me
Me
Me
Me
Me
N
N
N
1:1 complex
1:2 complex
1:3 complex
The absorption maximum and extinction coefficient of the complex at pH 7.47 were 460 nm and 4300 M-1
cm-1 as shown in Table 1. These values are comparable to the reported value17 of a 3,4-HOPO-iron(III)
(3:1) complex, indicating that synthetic linear and cyclic hexapeptides formed stable intramolecular 1:1
iron(III) complexes [in other word, iron(III) : 3-hydroxy-4(1H)-pyridinone (1:3) complexes] in the wide

pH range. The mole-ratio plots were also supported 1:1 stoichiometry. (not shown here)
In the case of 8b-iron(III) complex, the absorption spectra showed an isosbestic point at 515 nm in the pH
Table 1 UV-VIS spectral date of iron(III) complexes
Ligand
8a
λmax/nm
460
ε /M- 1 cm- 1
4500
8b
461
4300
10
460
4300
3,4-HOPO*
446
4320
* Ref. 17
range of 2.36-2.93 (Figure 2), reflecting the following equilibrium between Fe(L) and Fe(HL)+ (eq 1);
Fe(L) (1)
H+ Fe(HL)+
+
⇄
where Fe(L) and Fe(HL)+ are tris(1,4-dihydro-4-oxo-3-pyridinolato)iron(III) complex and its mono-
protonated one, respectively. Equation 1 is also expressed by eq 2, KF e(HL) being the protonation
equilibrium constant of the reaction.
KF e(HL) = [Fe(HL)+]/[Fe(L)][H+]
The spectral data was further analyzed using the Schwarzenbach equation (eq 3), given as follows;
Aobs = (A0 – Aobs)/[H+]KF e(HL)
εFe(HL)ctotal (3)
(2)
+
where Aobs is the observed absorbance at each pH value, A0 is the absorbance at pH 7.47, both at 460 nm,
εF e(HL) is the molar absorption coefficient at 460 nm for the monoprotonated species, and ctotal is the total
concentration of the ligand. A straight line was obtained when Aobs was plotted against (A0-Aobs)/[H+], as
shown in Figure 3. KF e(HL) and εF e(HL) values were calculated using the slope and intercept, respectively.
The value of 2800 for εF e(HL) indicates that average increments of 1400 are assignable to the first and second
3-hydroxy-γ-pirinonato ligands; hence, the almost same contribution of 1500 is estimated for the third ligan
upon 1:3 complex. The KF e(HL) value, whose logarithmic value corresponds half protonation pH value
(pH1/ 2), was calculated to be 1.2x102, being much smaller than the reported peptide-based
tris(hydroxamato) iron(III) complexes.18 These data implied that –norleucyl-β-Ala- sequence supplies the
sufficient spacing to form a stable 1:3 complex.
The Relative Stability of Iron(III) Complexes: The relative stability constant (KF e(L)) of hexapeptide
ligands with iron(III) is defined by the following equation 4.
Fe3+ + L3 ⇄ Fe(L)
KF e(L) = [Fe(L)]/[Fe3+][L3 ]
(4)
The competitive reaction between EDTA and these ligands was carried out in order to obtain the stability
constants of iron(III) complexes. Three pKa values of ligands are necessary for calculation. However,
due to the experimental limitation, pKa value (9.8) of N-substituted 3,4-HOPO19 was used for calculation.
The relative stability constants were obtained from the acid dissociation constants20 of EDTA, the stability
constant21 of EDTA-iron(III) complex (log K = 25.1), and the equilibrium constant, and the results are
summarized in Table 2, together with the data19 for DFB. The stability constants were found to be over 30
in logK. These relatively high values are attributable to the pKa value. The relative stability constant of

8b-iron(III) complex increased two orders of magnitude over that of 8a-iron(III) one, indicating that the
change in the peptide chain length remarkably affected the stability of the complex. Considering from the
moleculer model, only four isomers are possible for 8a-iron(III) complex, while all cis and trans isomers
are possible for 8b-iron(III) complex. Further, the relative stability constant of 10-iron(III) complex was
not so high as expected. It may be attributed that cyclic structure is so rigid that iron(III) ion can not be
tightly held in the form of 3:1 complex.
Table 2 Stability constants of iron(III) complexes
Ligand
8a
log K
31.6
33.4
31.7
30.5
8b
10
DFB*
* Ref. 19
Configuration of Iron(III) Complexes: The absolute configuration of iron(III) complexes in
aqueous solutions was examined by means of CD spectroscopy. The CD spectra of iron(III) complexes
were shown in Figure 4. The iron(III) complex of linear hexapeptide (8a) showed a negative band at 488
nm and a positive band at 349 nm and, indicating that 8a-iron(III) complex predominantly exists in a Δ-
configuration in an aqueous solution. A cyclic hexapeptide 10-iron(III) complex also gave the similar CD
pattern. On the contrary, 8b-iron(III) complex showed the opposite CD pattern (a possitive band at 485
nm and a negative band at 366 nm), indicating the preference of a Λ-configuration.
2
1
0
-1
-2
400
500
600
Wavelength / nm
Fugure 4 CD spectra of iron(III) complexes in aqueous
solution at pH 7.4; ( ); 8a-iron(III), ( ); 8b-iron(III);
(
); 10-iron(III)

Previously, Shanzer and co-workers demonstrated factors for determining and stabilizing the configuration
of iron(III) complex by using synthetic ferrichrome analogues in terms of 1) the chirality of amino acid
residues in a molecule and 2) a strong intramolecular hydrogen bond.22 Further, we showed that the
difference in the side-chain bulkiness of the amino acid residue of ligands causes change in absolute
configuration in the case of tripodal ligands.23 It is noteworthy that the difference in the amino acid residue
(Gly vs. β-Ala) in the peptide chain causes change in absolute configuration, although the reason has
remained obscure.
Iron(III) Removal from Transferrin: Diferric transferrin was prepared from commercially available
human apotransferrin according to the literatures.24 An addition of hexapeptides to a solution of diferric
transferrin (0.002 mM) result in an increase of absorbance at 460 nm characteristic of hexapeptide-iron(III)
complexes. The concentration of ligands was 7-fold excess over diferric transferrin. Plots of log[(A∞-
Aobs)/(A∞-A0)] as a function of time gave a good linear relationship as shown in Figure 5, indicating that the
reaction of iron removal from transferrin by 8a proceeded in the pseudo-first-order kinetics. The kinetic
results were summarized in Table 3, together with the data for DFB. It is noteworthy that these ligands
efficiently removed iron from transferrin even at less concentration of the ligand after the reaction was
performed for 30 min. Further, there was remarkable difference between 8a and 8b. As mentioned above,
8a- and 8b-iron(III) complexes had Δ- and Λ-configuration, respectively. We have demonstrated that the
rates of iron(III) removal from transferrin by L- or D-alanine residue-containing tripodal ligands were
affected by the absolute configuration (Δ or Λ) of iron(III) complexes.23 The present data was consistent
with the reported phenomenon. Unfortunately, kinetic data for cyclic hexapeptide (10) could not be
obtained because of low water solubility under the employed conditions.
Table 3 Iron(III) removal from transferrin at pH 7.4
Ligand
[ligand]/[Fe2.00Tf]
kobs
(x10- 3 min- 1)
1.72
% Fe removed
(at a point 30 min)
8 a
8b
7
7
7
14
5
3.08
DFB*
* Ref. 24a
100
0.66
The Growth-Promotion Activity: As mentioned above, Aureobacterium flavescence (ATCC 25091) and
Escherichia coli RW193 (ATCC 33475) were commonly used for assay of the growth-promotion activity.
A. flavescence was shown to require hydroxamate-type siderophores and has a ferrichrome receptor,
although it does not synthesize any detectable siderophore and does not response to catecholate-type
siderophores.
E. coli RW193 showed activity against both hydroxamate- and catecholate-type
siderophores. These strains, therefore, are useful for diagnosis whether a synthetic compound acts as an
artificial siderophore. The assay was performed according to the standared procedure.14,22,25,26 The
activity was evaluated by measuring the diameter of the halo of exhibition of growth, and the results are
presented in Table 4. Natural siderophore DFB exhibited 55 mm halo of growth, but 8b-iron(III)

complex showed no response, indicating that synthetic hexapeptide (8b) is not an auxotrophe for A.
flavescence. On the other hand, although less than DFB, E. coli RW193 showed significant growth-
promotion activity against 8b-iron(III) complex. This result may be attributable that microorganism
recognizes 3-hydroxy-4(1H)-pyridinone as catecholate-type ligand. The assay was performed only using
8b-iron(III) complex, because other complexes were insoluble in water under the employed conditions.
Table 4 The growth-promotion activity
Ligand
Diameter (mm)
A. flavescence
E. coli
12
8b
<6
<6
55
DW
DFB
<6
18
*20 µL iron(III) complex solutions (1 mM) were absorbed onto a
6 mm paper discs. DFB-iron(III) solution was used as a reference,
and distilled water (DW) as a blank. The diameter of the halo of
growth was measured.
EXPERIMENTAL
Melting points were recorded on a Mel-Temp apparatus in open capillaries and are uncorrected. IR spectra
1
were recorded on JASCO FT/IR-230 infrared spectrophotometer.
H-NMR spectra were recorded on a
JEOL GX-270 and JNM-LA400D spectrometers using Me Si or sodium 3-trimethylsilyl-1-propane-
4
sulfonate sodium salt as an internal standard. Optical rotations were measured on a JASCO DIP-370
digital polarimeter. UV-VIS spectra were recorded on a JASCO Ubest V-550 spectrophotometer. CD
spectra were measured with a JASCO J-500 spectropolarimeter. Column chromatography was carried out
with Merck Kieselgel 60 (230-400 mesh). Combustion analysis was performed on a Perkin Elmer 2400 II
CHNS Analyzer. 3-Benzyloxy-2-methyl-4-pyrone (1) was prepared according to literature methods.13, 27
ε-(3-Benzyloxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucine sodium salt (2): To
a solution of NaOH (0.64 g, 16 mmol) in H2O : EtOH (2:1) mixture (100 mL) was added compound (1)
.
(1.4 g, 6.5 mmol) and H-L-Lys-OH HCl (1.1 g, 6.1 mmol), and then the reaction mixture was stirred at rt for
4 days. The reaction mixture was washed with AcOEt (45 mL x 2), and then the aqueous layer was
evaporated under reduced pressure to give the sodium salt, which was used for the next reaction without
1
further purification: H-NMR (δ, D2O, 270 MHz): 1.33 (2H, m), 1.57-1.86 (4H, m), 1.93 (3H, s), 3.54
(1H, t, J=7.0 Hz), 3.59 (2H, t, J=7.8 Hz), 5.02 (2H, s), 6.56 (1H, d, J=8.7 Hz), 7.40 (5H, m), and 7.72
(1H, d, J=8.7 Hz).
General procedure for compounds (3a,b);
A typical example: ε-(3-Benzyloxy-1,4-
ethyl ester
dihydro-2-methyl-4-oxo-1-pyridyl)-N-tert-butoxycarbonyl-L-norleucylglycine
(3a): To a solution of compound (2) (9.32 mmol) in H2O (30 mL) was added Boc2O (2.43 g, 11.0 mmol)
in dioxane (20 mL) on an ice-bath. The reaction mixture was stirred at 0 °C for 8 h and then at rt for
another 2 days. H2O (50 mL) was added to the mixture and the solution was washed with CHCl3 (100
mL x 2). The aqueous solution was adjusted to pH 4 with 5% citric acid, and then extracted with CHCl3

(200 mL x 3). The organic layer was washed with saturated NaCl solution (200 mL), and then dried over
anhydrous Na2SO4. Removal of the solvent by evaporation under reduced pressure and subsequent
recrystallization from EtOH gave the product, ε-(3-benzyloxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-N-
20
tert-butoxycarbonyl-L-norleucine (1.67 g, 37%): mp 172-174 °C; [α]D -2.9° (c=1 in MeOH); IR (KBr):
1
1705, 1624, 753, and 704 cm-1; H-NMR (δ, CDCl3, 270 MHz): 1.30-1.39 (2H, m), 1.44 (9H, s), 1.61-
1.72 (2H, m), 1.80-1.92 (2H, m), 2.13 (3H, s), 3.48 (1H, m), 3.80-3.91 (2H, m), 4.20 (1H, br s), 5.14
(2H, s), 5.4 (1H, d, J=8.1 Hz), 6.6 (1H, d, J=8.1 Hz), and 7.30-7.42 ppm (5H, m). Anal. Calcd for
C23H32N2O6: C, 63.87; H, 7.45; N, 6.47. Found: C, 64.07; H, 7.24; N, 6.23.
.
To a solution of HCl H-Gly-OEt (252 mg, 1.80 mmol) and N-methylmorpholine (192 mg, 1.90 mmol) in
dry CH2Cl2 (1.5 mL) was added the above Boc-norleucine (801 mg, 1.8 mmol) and HOBt (257 mg, 1.90
.
mmol) at -10 °C. A solution of WSC HCl (346 mg, 1.80 mmol) in dry CH2Cl2 (3 mL) was added to the
reaction mixture at -10 °C, and then the reaction mixture was stirred for 3 days at rt. After removal of the
solvent under reduced pressure, the residue was dissolved in CHCl3 (150 mL). The organic layer was
successively washed with 5% citric acid (80 mL x 3), 5% NaHCO3 (80 mL x 3), water (80 mL x 3),
saturated NaCl solution (100 mL), and then dried over anhydrous Na2SO4. After removal of the solvent
under reduced pressure, the crude product was purified by column chromatography on silica gel with
20
CHCl3 : MeOH (10:1) mixture as an eluent to afford the pure product (3a) (701 mg, 75%): [α]D -3.3°
1
(c=1 in MeOH); IR(KBr): 1738, 1718, 1679, 1628, 754, and 703 cm-1; H-NMR (δ, CDCl3, 400 MHz):
1.26 (3H, t, J=7.6 Hz), 1.35-1.40 (2H, m), 1.43 (9H, s), 1.58-1.72 (2H, m), 1.86-1.98 (2H, m), 2.07
s), 3.72 (2H, t, J=7.9 Hz), 3.96 and 4.02 (2H, ABq, J=19 Hz), 4.17 (2H, q, J=7.6 Hz), 4.19 (1H,
5.18 (2H, s), 5.26-5.33 (1H, m), 6.39 (1H, d, J=7.3 Hz), 7.15-7.17 (1H, m), 7.19 (1H, d, J=7.3
(3H,
m),
Hz),
and 7.29-7.42 ppm (5H, m). Anal. Calcd for C28H39N3O7·2H2O: C, 58.52; H, 7.27; N, 7.31.
Found: C,
58.68; H, 7.18; N, 7.27.
N-tert-Butoxycarbonyl-ε-(3-benzyloxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleu-
22
cyl-β-alanine ethyl ester (3b): 85%; mp 101-103 °C; [α]D -6.5° (c=0.5 in MeOH); IR(KBr): 1735,
1
1707, 1661, 1626, 754, and 704 cm-1; H-NMR (δ, CDCl3, 400 MHz): 1.26 (3H, t, J=7.2 Hz), 1.35 (2H,
m), 1.44 (9H, s), 1.62 (2H, m), 1.84 (2H, m), 2.08 (3H, s), 2.52 (2H, t, J=6 Hz), 3.53 (2H, t, J=6 Hz), 3.70
(2H, t, J=7 Hz), 4.07 (1H, m), 4.14 (2H, q, J=7.2 Hz), 5.20 (2H, s), 5.22 (1H, br s), 6.38 (1H, d, J=7.6 Hz),
6.90 (1H, br s), 7.17 (1H, d, J=7.6 Hz), and 7.33 ppm (5H, m). Anal. Calcd for
C29H41N3O7: C, 64.09;
H, 7.55; N, 7.73. Found: C, 63.84; H, 7.87; N, 8.07.
General procedure for compounds (4a,b); A typical example: N-tert-Butoxycarbonyl-ε- (3-
benzyloxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucylglycine (4a): To a solution of
compound (3a) (260 mg, 0.49 mmol) in MeOH (5 mL) was added 1M NaOH (0.75 mL, 0.75 mmol), and
then the reaction mixture was stirred at rt for 2 h. The solution was adjusted to pH 8 with 5% citric acid,
and then the solvent was removed under reduced pressure. The solution of the residue in H2O (5 mL) was
adjusted pH 4 with 6M HCl on an ice-bath, and then extracted with CHCl3 (15 mL x 4). The organic layer
was washed with saturated NaCl solution (20 mL), and then dried over anhydrous Na2SO4. Removal of

23
the solvent under reduced pressure afforded the dipeptide (4a) (223 mg, 94%): mp 124-127 °C; [α]D -3.2°
1
(c=0.5 in MeOH); IR(KBr): 1706, 1659, 1614 , 754 , and 704 cm-1; H-NMR (δ, CDCl3, 400 MHz): 1.31
(2H, m), 1.39 (9H, s), 1.63 (2H, m), 1.76 (2H, m), 2.10 (3H, s), 3.81 (2H, m), 3.99 (2H, m), 4.24
(1H, m), 5.11 (2H, s), 5.79 (1H, m), 6.68 (1H, d, J=7.8 Hz), 7.34 (5H, m), 7.29-7.38 (1H, m), and
7.55 ppm (1H, d, J=7.8 Hz). Anal. Calcd for C26H35N3O7·1.5H2O: C, 59.08; H, 6.96; N, 7.95.
Found: C, 59.21, H, 6.98; N, 7.88.
N-tert-Butoxycarbonyl-ε-(3-benzyloxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucyl-β-
22
alanine (4b): 98%; mp 63-65 °C; [α]D -12.1° (c=0.5 in MeOH); IR(KBr): 1706, 1659, 1625,
1
754, and 704 cm-1; H-NMR (δ, CDCl3, 400 MHz): 1.26 (2H, m), 1.45 (9H, s), 1.70 (4H, m), 2.08 (3H,
s), 2.53 (2H, m), 3.33 (1H, m), 3.49 (1H, m), 3.7 (2H, m), 4.11 (1H, m), 5.10 (2H, s), 5.51 (1H, br s),
6.59 (1H, d, J=7 Hz), 6.91 (1H, br s), 7.31 (5H, m), and 7.44 ppm (1H, d, J=7 Hz). Anal. Calcd for
C27H37N3O7·H2O: C, 60.77; H, 7.37; N, 7.87. Found: C, 60.94, H, 7.23; N, 7.78.
General procedure for compounds (5a,b); A typical example: ε-(3-Benzyloxy-1,4-di-
hydro-2-methyl-4-oxo-1-pyridyl)-L-norleucine ethyl ester TFA salt (5a):
To a solution of
compound (3a) (350 mg, 0.66 mmol) in CH2Cl2 (2.5 mL) was added TFA (3.39 g, 29.7 mmol), and then
the reaction mixture was stirred on an ice-bath for 1 h. After removal of the solvent under reduced
pressure, dry EtOH (20 mL) was added to the residue, and then the solvent was removed under reduced
pressure to afford the TFA salt (5a). This salt was used for the next reaction without further purification.
Compound (3b) was similarly converted into the corresponding TFA salt (5b).
General preparation procedure for compounds (6a,b); A typical example: N-tert-Butoxy-
carbonyl-ε-(3-benzyloxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucylglycyl-ε-(3-
benzyloxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucylglycine ethyl ester (6a): To
a solution of compound (5a) (789 mg, 104 mmol) and N-methylmorpholine (304 mg, 3.01 mmol) in dry
CH2Cl2 (1.5 mL) was added compound (4a) (515 mg, 1.03 mmol) and HOBt (154 mg, 1.14 mmol) at -10
°C. A solution of WSC·HCl (219 mg, 1.14 mmol) in dry CH2Cl2 (1.5 mL) was added to the mixture at -10
°C, and then the reaction mixture was stirred for 3 days at rt. After removal of the solvent under reduced
pressure, the residue was dissolved in CHCl3 (250 mL). The organic layer was successively washed with
5% citric acid (100 mL x 3), 5% NaHCO3 (100 mL x 3), saturated NaCl solution (150 mL), and then dried
over anhydrous Na2SO4. After removal of the solvent under reduced pressure, the crude product was
purified by column chromatography on silica gel with CHCl3:MeOH (6:1) mixture as an eluent to afford the
tetrapeptide (6a) (729 mg, 75%): mp 98-101 °C; [α]D27 -8.6° (c=0.5 in MeOH); IR(KBr): 1671, 1664, 1651,
-1
1
1626, 754, and 703 cm ; H-NMR (δ, CDCl3, 400 MHz): 1.22 (3H, t, J=7 Hz), 1.24- 1.39 (4H, m), 1.40
(9H, s), 1.53-1.98 (8H, m), 2.06 (6H, s), 3.71 (4H, m), 3.80-3.98 (4H, m), 4.12
(1H, m), 4.49 (1H, q, J=8 Hz,), 5.12 (4H, s), 5.93 (1H, d, J=8 Hz), 6.37 (2H, d,
7.37 (2H, d, J=7 Hz), 7.49 (1H, br s), 8.03 (1H, br s), and 8.28 ppm (1H, br
C49H64N6O11·H2O: C, 60.29; H, 6.97; N, 8.61. Found: C, 60.23; H, 6.79; N, 8.21.
(2H, q, J=7 Hz),4.19
J=7 Hz), 7.35 (10H, m),
s). Anal. Calcd for

N-tert-Butoxycarbonyl-ε-(3-benzyloxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-nor-
leucyl-β-alanyl-ε-(3-benzyloxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucyl-β-
alanine
ethyl ester (6b): 78%; mp 64-66 °C; [α]D22 -3.2° (c=0.5 in MeOH); IR(KBr): 1710, 1694, 1654, 1626,
-1
754, and 705 cm ; H-NMR (δ, CDCl3, 400 MHz): 1.23 (3H, t, J=7 Hz), 1.37 (4H, m), 1.43 (9H, s),
1
1.65 (8H, m), 2.17 (6H, s), 2.41 (2H, m), 2.51 (2H, t, J=6 Hz), 3.39 (4H, m), 3.95 (4H,
3.99 (1H, m), 4.10 (2H, q, J=7 Hz), 4.21 (1H, m), 5.08 (4H, s), 6.51 (2H, d, J=8 Hz),
t, J=7.2 Hz),
7.33 (10H, m),
and 7.71 (2H, d, J=8 Hz). Anal. Calcd for C51H68N6O11·2H2O: C, 62.29; H, 7.43; N, 8.60. Found: C,
62.67; H, 7.10; N, 8.83.
General procedure for compounds (7a,b); N-tert-Butoxycarbonyl-ε-(3-benzyloxy-1,4-di-
hydro-2-methyl-4-oxo-1-pyridyl)-L-norleucylglycyl-ε-(3-benzyloxy-1,4-dihydro-2-meth-
yl-
4-oxo-1-pyridyl)-L-norleucylglycyl-ε-(3-benzyloxy-1,4-dihydro-2-methyl-4-oxo-1-
norleucylglycine ethyl ester (7a): To a solution of compound (6a) (129 mg, 0.14
pyridyl)-L-
mmol) in dry
CH2Cl2 (2 mL) was added TFA (684 mg, 6.0 mmol), and then the reaction mixture was stirred for 2 h on
an ice-bath. After removal of the solvent under reduced pressure, dry EtOH (10 mL) was added to the
residue, and then the solvent was removed under reduced pressure to afford the TFA salt of N-terminal free
tetrapeptide,N-tert-butoxycarbonyl-ε-(3-benzyloxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-
norleucylglycyl-ε-(3-benzyloxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucylglycine eth- yl ester
TFA salt. This salt was used for the next reaction without further purification.
To a solution of the above TFA salt of N-terminal free tetrapeptide (0.175 mmol) and N-methylmorpholine
(110 mg, 1.08 mmol) in dry DMF (2.5 mL) was added compound (4a) (87.9 mg, 0.175 mmol) and CDI
(56.7 mg, 0.35 mmol) at 0 °C, and then the reaction mixture was stirred for 3 days at rt. After removal of
the solvent under reduced pressure, the residue was dissolved in CHCl3 (100 mL). The organic layer was
successively washed with 5% citric acid (50 mL x 3), 5% NaHCO3 (50 mL x 3), water (50 mL x 3),
saturated NaCl solution (80 mL), and then dried over anhydrous Na2SO4. After removal of the solvent
under reduced pressure, the crude product was purified by gel chromatography on Sephadex LH-20 with
22
MeOH as an eluent to afford the hexapeptide (7a) (136mg, 60%): mp 109-111 °C; [α]D -12.1° (c=0.5 in
-1
1
MeOH); IR(KBr): 1738, 1690, 1679, 1663, 1654, 1642, 1626, 753, and 704 cm ; H-NMR (δ, CD3OD,
400 MHz): 1.24 (3H, t, J=8 Hz), 1.31 (6H, m), 1.38 (9H, s), 1.57-1.82 (12H, m), 2.06 (9H, s), 3.70
(6H, m), 3.74 (2H, m), 3.96 (4H, m), 4.11 (2H, q, J=8 Hz), 4.23 (2H, m), 4.46 (1H, m), 5.10 (6H, s),
6.02 (1H, br s), 6.81 (3H, m), 7.32 (15 H, m), 7.34 (3H, m), 7.51 (1H, br s),7.94 (1H, br s), 8.06 (1H,
br s), 8.21 (1H, br s), and 8.52 ppm (1H, br s). Anal. Calcd for C70H89N9O15·2H2O: C, 63.09; H, 7.03; N,
9.46. Found: C, 62.84; H, 7.33; N, 9.21.
N-tert-Butoxycarbonyl-ε-(3-benzyloxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleu-
cyl-β-alanyl-ε-(3-benzyloxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucyl-β-alan-
yl-ε-(3-benzyloxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucyl-β-alanineethyl
22
ester (7b): 76%; mp 88-90 °C; [α] D +4.4° (c=0.5 in MeOH); IR(KBr): 1720, 1702, 1655, 1626, 754,
-1
1
and 704 cm ; H-NMR (δ, CD3OD, 400 MHz): 1.22 (3H, t, J=8 Hz), 1.36 (6H, m), 1.41 (9H, s), 1.65
(12H, m), 2.15 (9H, s), 2.40 (4H,m), 2.50 (2H, m), 3.39 (6H, m), 3.93 (6H, t, J=7 Hz), 4.03 (1H, m),

4.09 (2H, q, J=8 Hz), 4.19 (2H, m), 5.05 (6H, s), 6.44 (3H, d, J=7 Hz), 7.32 (15 H, m), and 7.66 ppm (3H, d,
J=7 Hz). Anal. Calcd for C73H95N9O15·3H2O: C, 62.95; H, 7.31; N, 9.05. Found: C, 62.79; H, 7.03; N, 8.85.
ε-3-Hydroxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucylglycyl-ε-(3-hydroxy-
1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucylglycyl-ε-(3-hydroxy-1,4-dihydro-2-
methyl-4-oxo-1-pyridyl)-L-norleucylglycine ethyl ester (8a): A suspension of 10% Pd-C (13
mg) in MeOH (5 mL) was prehydrogenated with H2 for 0.5 h. To the suspension was added a solution of
compound (7a) (127 mg, 0.10 mmol) in MeOH (2 mL). After hydrogenation under H2 atmospheric
pressure for 4 h, the catalyst was removed by filtration. The filtrate was evaporated, and then the residue
was purified by gel chromatography on Sephadex LH-20 with MeOH as an eluent to afford the
corresponding debenzylated product, N-tert-butoxycarbonyl-ε-(3-hydroxy-1,4-dihydro-2-methyl-4-oxo-1-
pyridyl)-L- norleucylglycyl –ε-(3-hydroxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucylglycyl-ε-(3-
hydroxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucylglycine ethyl ester:(27.3 mg, 67%): mp 125-
-1
22
1
127 °C (decomp): [α]D -12.9° (c=0.1 in MeOH); IR(KBr): 1738, 1658, and 1634 cm ; H-NMR (δ,
CD3OD, 400 MHz): 1.24 (3H, t, J=7 Hz), 1.42 (6H, m), 1.42 (9H, s), 1.75 (12H, m), 2.45 (9H, s), 3.86
(6H, m), 4.04 (6H, m), 4.14 (2H, q, J=7 Hz), 4.15 (1H, m), 4.26 (1H, m), 4.39 (1H, m), 6.39 (3H, d,
J=8 Hz), and 7.62 ppm (3H, d, J=8 Hz). Anal. Calcd for C49H71N9O15·2H2O: C, 55.41; H, 7.12; N, 11.87
. Found: C, 55.44; H, 7.20; N, 11.79.
To a solution of the above debenzylation product (5.9 mg, 6 x 10-3 mmol) in dry CH2Cl2 (1 mL) was added
TFA (23 mg, 0.2 mmol), and then the reaction mixture was stirred for 2 h on an ice-bath. After removal
of the solvent under reduced pressure, dry EtOH (10 mL) was added to the residue, and then the solvent
was removed by evaporation under reduced pressure to afford the desired linear hexapeptide (8a) (100%)
22
1
-22.6° (c=0.1 in MeOH); H-NMR (δ, CD3OD, 400 MHz): 1.23 (3H, t, J=7 Hz), 1.45
as an oil: [α]D
(6H, m), 1.86 (12H, m), 2.57 (9H, s), 4.05 (6H, m), 4.16 (2H, q, J=7 Hz), 4.19 (1H, m), 4.27 (1H, m),
4.33 (7H, m), 7.06 (3H, d, J=8 Hz), and 8.00 ppm (3H, d, J=8 Hz). This TFA salt was used for spectral
measurement without further purification.
ε-(3-Hydroxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucyl-β-alanyl-ε-(3-hydro-
xy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucyl-β-alanyl-ε-(3-hydroxy-1,4-di-
hydro-2-methyl-4-oxo-1-pyridyl)-L-norleucyl-β-alanine ethyl ester (8b):
A removal of the
benzyl protecting group of compound (7b) was carried out under the same manner as compoud (7a) to give
the corresponding debenzylation product, N-tert-butoxycarbonyl-ε-(3-hydroxy-1,4-dihydro-2-methyl-4-
oxo-1-pyridyl)-L-norleucyl-β-alanyl-ε-(3-hydroxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucyl-β-
alanyl-ε-(3-hydroxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucyl-β-alanine ethyl ester: 72%: mp
119-121 °C; [α]D22 -14.3° (c=0.1 in MeOH); IR(KBr): 1718, 1700, 1685, 1655, 1648, 1637 and 1630 cm- ;
1H-NMR (δ, DMSO-d6, 400 MHz): 1.17 (3H, t, J=8 Hz), 1.24 (6H, m), 1.36 (9H, s), 1.61 (12H, m), 2.29
(9H, s), 2.31 (4H, m), 2.43 (2H, t, J=7 Hz), 3.28 (6H, m), 3.91 (7H, m), 4.06 (2H, q, J=8 Hz),
4.17 (2H, m), 6.18 (3H, d, J=8 Hz), 6.79 (1H, br s), 7.59 (3H, d, J=8 Hz), 7.84 (1H, br s), 7.96 (1H,

br s), and 8.01 ppm (3H, br s). Anal. Calcd for C49H71N9O15·2H2O: C, 55.41; H, 7.12; N, 11.87.
Found: C, 55.44; H, 7.20; N, 11.79.
A removal of the Boc protecting group of the above debenzylation product with TFA afforded the desired
linear hexapeptide (8b) as an oil: 100%; [α]D22 -4.9° (c=0.1 in MeOH); 1H-NMR (δ, DMSO-d6, 400 MHz):
1.17 (3H, t, J=7 Hz), 1.32 (6H, m), 1.73 (12H, m), 2.33 (4H, m), 2.43 (2H, t, J=8 Hz), 2.51 (9H, s),
3.28 (6H, m), 3.73 (6H, m), 4.04 (2H, q, J=7 Hz), 4.17 (2H, m), 4.28 (6H, m), 7.17 (3H, d, J=8 Hz),
8.06 (4H, m), 8.20 ppm (3H, d, J=8 Hz), and 8.53 ppm (1H br s). This TFA salt was used for spectral
measurement without further purification.
Cyclo[ε-(3-benzyloxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucyl-β-alanyl-ε-(3-
benzyloxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucyl-β-alanyl-ε-(3-benzyloxy-
1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucyl-β-alanyl] (9): To a solution of compound
(7b) (180 mg, 0.135 mmol) in MeOH (3 mL) was added 1M NaOH (0.75 mL, 0.75 mmol), and then the
reaction mixture was stirred at rt for 2 h. The solution was adjusted to pH 8 with 5% citric acid, and then
the solvent was removed under reduced pressure. The solution of the residue in H2O (5 mL) was adjusted
to pH 2 with 6M HCl on an ice-bath, and then extracted with CHCl3 (250 mL) by using liquid-liquid
Soxlet’s extractor. The organic layer was dried over anhydrous Na2SO4. Removal of the solvent under
reduced pressure afforded the corresponding C-terminal free hexapeptide (171 mg 97%): mp 135-136 °C;
23
[α]D -1.7° (c=0.5 in MeOH); IR(KBr): 1708, 1657, 1640, 1625, 753, and 705 cm-1 ; 1H-NMR (δ,
CD3OD, 400 MHz): 1.37 (6H, m), 1.42 (9H, s), 1.73 (12H, m), 2.37 (9H, s), 2.50 (6H, m), 3.43 (6H,
m), 3.99 (1H, m), 4.19 (6H, t, J=7 Hz), 4.49 (2H, m), 5.14 (6H, s), 6.94 (3H, d, J=8 Hz), 7.39 (15H,
m), and 8.12 ppm (3H, d, J=8 Hz). Anal. Calcd for C71H91N9O15·3H2O: C, 62.49; H, 7.16; N, 9.24.
Found: C, 62.49; H, 7.04; N, 9.08.
To a solution of the above C-terminal free hexapeptide (170 mg, 0.128 mmol) in dry DMF (5 mL) was
added HOSu (29.4 mg, 0.256 mmol) at -10 °C. A solution of WSC·HCl (42.2 mg, 0.256 mmol) in dry
CH2Cl2 (2 mL) was added to the reaction mixture at -10 °C, and then the reaction mixture was stirred for 3
days at rt. After removal of the solvent under reduced pressure, the residue was dissolved in CHCl3 (200
mL). The organic layer was successively washed with water (80 mL x 3), saturated NaCl solution (100
mL), and then dried over anhydrous Na2SO4. Removal of the solvent under reduced pressure afforded the
corresponding O-succinimide ester (100%): IR(KBr): 1813, 1782, and 1737 cm-1. This product was used
for the next reaction without further purification.
To a solution of the O-succinimide ester (171 mg, 0.12 mmol) in dry CH2Cl2 (1.5 mL) was added TFA
(684 mg, 6.0 mmol), and then the reaction mixture was stirred for 2 h on an ice-bath. Removal of the
solvent under reduced pressure afforded the corresponding TFA salt. This salt was used for the next
reaction without further purification.
A solution of the TFA salt (220 mg, 0.153 mmol) in dry DMF (5 mL) was added dropwise to pyridine (175
mL), and then the reaction mixture was stirred for 5 days at 75 °C. After removal of the solvent, the crude
product was purified by column chromatography on silica gel with MeOH followed by gel chromatography
on Sephadex LH-20 with MeOH as an eluent to give the cyclic peptide (9) (60 mg, 33%): mp 141-144 °C;

22
1
[α]D +11.7° (c=0.1 in MeOH); IR(KBr): 1655, 1624, 753, and 703 cm-1; H-NMR (δ, CD3OD, 400
MHz): 1.39 (6H, m), 1.67 (12H, m), 2.17 (9H, s), 2.30 (3H, m), 2.55 (3H, m), 3.30 (3H, m), 3.53 (3H,
m), 3.94 (6H, t, J=7 Hz), 4.00 (3H, t, J=6 Hz), 5.06 (6H, s), 6.44 (3H, d, J=8 Hz), 7.34 (15H, m), and
7.67 ppm (3H, t, J=8 Hz). Anal. Calcd for C66H81N9O12·2H2O: C, 64.53; H, 6.97; N, 10.26. Found: C,
64.33; H, 6.78; N, 10.11.
Cyclo[ε-(3-hydroxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucyl-β-alanyl-ε-(3-
hydroxy-1,4-dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucyl-β-alanyl-ε-(3-hydroxy-1,4-
dihydro-2-methyl-4-oxo-1-pyridyl)-L-norleucyl-β-alanyl] (10): A suspension of 10% Pd-C
(15 mg) in MeOH (2 mL) was prehydrogenated with H2 for 0.5 h. To the suspension was added a
solution of compound (9) (90 mg, 0.08 mmol) in MeOH (3 mL). After hydrogenation under H2
atmospheric pressure for 4 h, the catalyst was removed by filtration. The filtrate was evaporated, and the
residue was purified by gel chromatography on Sephadex LH-20 with DMF as an eluent to give the desired
22
cyclic hexapeptide 10 (40 mg, 57%): mp 183-185 °C (decomp); [α]D +12.3° (c=0.1 in MeOH); IR(KBr):
1
1654, and 1637 cm-1; H-NMR (δ, DMSO-d6, 400 MHz): 1.30 (6H, m), 1.60 (12H, m), 2.14 (3H, m),
2.26 (9H, s), 2.40 (3H, m), 3.13 (3H, m), 3.43 (3H, m), 3.88 (6H, t, J=8 Hz), 3.91 (3H, m), 6.10 (3H,
d, J=8 Hz), 7.53 ppm (3H, d, J=8 Hz), 7.71 (3H, m), and 8.06 (3H, m). Anal. Calcd for
C45H63N9O12·4H2O: C, 54.37; H, 7.20; N, 12.68. Found: C, 54.33; H, 7.06; N, 12.45.
General Procedure for Spectral Measurement of 1:1 Mixtures of Hexapeptide Ligands and
Iron(III)23: 3 mg of each hexapeptide ligand and an equimolar amount of Fe(NO3)3 (2.99 x 10-3M) were
mixed and diluted to 10.0 mL with distilled water. The pH was adjusted to an appropriate pH value with
0.01 or 0.1 M NaOH and 0.01 or 0.1 M HNO3 solutions before spectral measurement.
Iron(III) Exchange Reaction23: A buffered solution (2 mL Tris buffer (pH 7.4), and [KNO3] =
0.04 M) containing 1 x 10-4 M of iron(III) complex was mixed with EDTA solution (1 mL Tris buffer (pH
7.4), and [EDTA] = 0.1 M). Iron(III) exchange reaction was monitored by a decrease of absorbance at 460
nm. The relative stability constant of iron(III) complex was calculated by using the stability constant of
Fe(edta), pKa values of EDTA, pKa value of 3,4-HOPO, and the equilibrium point.
Iron(III) Removal from Transferrin23: The solutions of each hexapeptide ligand (1.0 mL, 1 mM) and
TfF e2.00 (2.0 mL, 0.02 mM) in Tris buffer were mixed, and then the absorbance of the solution was
monitored at 460 nm. The pseudo-first-order rate constant (kobs) was calculated from the slope of the plots
of log[(A∞-Abs)/(A∞-A0)] as a function of time.
The Growth-Promotion Activity26: The biological activity of 8b-iron(III) complex was evaluated by
the standard paper-disc method by using Aureobacterium flavescens and E. Coli. On nutrient agar (ATCC
Medium No. 424: A. flavescens, ATCC Medium No. 33475: E. coli) (0.7 % agar) containing the strain
(~108 cells mL-1) laid over nutrient agar plate (1.5 % agar) was placed filter paper discs (6 mm diam.).

Each disc was impregnated with aliquots (20 µL) of 1 mM complex solutions in water. The diameter of
halo of growth was measured after incubation at 30 °C for 2 days.
ACKNOWLEDGEMENT
This work was partially supported by the Japan Private School Promotion Fundation.
REFERENCES AND NOTES
1. ‘Iron Transport in Microbes, Plants and Animals’, ed. by G. Winkelman, D. van der Helm, and J. B.
Neilands, VCH, Weinheim, 1987.
2. ‘Bioinorganic Chemistry: Inorganic Elements in the Chemistry of Life’, ed. by W. Kaim and B.
Schwederski, John Wiley & Sons, New York, 1991.
3. ‘Handbook of Microbial Iron Chelators’, ed. by G.Winkelmann, CRC Press, Boca Raton, 1991.
4. ‘Transition Metals in Microbial Metabolism’ ed. by G. Winkelmann and C. J. Carrano, 1997.
5. R. J. Motekaitis and A. E. Martel, Inorg. Chim. Acta, 1991, 183, 71.
6. (a) S. T. Callender and D. J. Weatherall, Lancet, 1980, 689. (b) A. Jacobs and W. C. Ting, Lancet,
1980, 794. (c) C. Kattamis, J. Fitsialos, and C. Sinopoulou, Lancet, 1981, 51.
7. M. R. Summers, A. Jacobs, D. Tudway, P. Perera, and C. Rickett, Br. J. Haematol., 1979, 42, 547.
8. M. J. Miller and F. Malouin, in ‘The Development of Iron Chelators for Clinical Use’, ed. by R. J.
Bergeron and G. M. Brittenham, CRC Press, Boca Raton, 1992, p. 277.
9. R. C. Hider, J. B. Porter, and S. Singh, in ‘The Development of Iron Chelators for Clinical Use’, ed.
by R. J. Bergeron and G. M. Brittenham, CRC Press, Boca Raton, 1992, p. 353.
10. (a) J. Xu, B. Kullgren, P. W. Durbin, and K. N. Raymond, J. Med. Chem., 1995, 38, 2606 (b) H.
Weizman and A. Shanzer, Chem. Commun., 2000, 2013.
11. (a) M. Streater, P. D. Taylor, R. C. Hider, and J. Porter, J. Med. Chem., 1990, 33, 1749. (b) A. E.
Martell, R. J. Motekaitis, Y. Sun, and E. T. Clarke, in ‘The Development of Iron Chelators for Clinical
Use’, ed. by R. J. Bergeron and G. M. Brittenham, CRC Press, Boca Raton, 1992, p. 329. (c) J.
Xu, S. J. Franklin, D. W. Whisenhunt. Jr., and K. N. Raymond, J. Am. Chem. Soc., 1995, 117,
7245. (d) G. Xiao, D. van der Helm, R. C. Hider, and P. S. Dobbin, Inorg. Chem., 1995, 34,
1268. (e) M. Meyer, J. R. Telford, S. M. Cohen, D. J. White, J. Xu, and K. N. Raymond, J. Am.
Chem. Soc., 1997, 119, 10093. (f) A. R. Johnson, B. O’Sullivan, and K. N. Raymond, Inorg. Chem.,
2000, 39, 2652. (g) S. Hajela, M. Botta, S. Giraudo, J. Xu, K. N. Raymond, and S. Aime,
Chem. Soc., 2000, 122, 11238.
J. Am.
12. L. N. Sheppard and G. J. Kontoghioghes, Inorg. Chim. Acta, 1991, 188, 177.
13. B. L. Rai, L. S. Dekhordi, H. Khodr, Y. Jin, Z. Liu, and R. C. Hider, J. Med. Chem., 1998, 41,
3347.
14. K. N. Raymond, G. Muller, and B. F. Matzanke, Top. Curr. Chem., 1984, 123, 49 and references cited
therein.
15. J. Ohkanda and A. Katoh, Rev. Heteroatom Chem., 1998, 18, 87 and references cited therein.

16. (a) J. Ohkanda, H. Shibui, and A. Katoh, Chem. Commun., 1998, 375. (b) A. Katoh, Y. Hida, J.
Kamitani, and J. Ohkanda, J. Chem. Soc., Dalton Trans., 1998, 3859. (c) A. Katoh, K. Taguchi, H.
Okada, M. Harata, Y. Fujisawa, T. Takino, and H. Sakurai, Chem. Lett., 2000, 866.
17. R. C. Scarrow, P. E. Riley, K. Abu-Dari, D. L. White, and K. N. Raymond, Inorg. Chem., 1985, 24,
954.
18. (a) M. Akiyama, A. Katoh, M. Iijima, T. Takagi, K. Natori, and T. Kojima, Bull. Chem. Soc. Jpn.,
1992, 65, 1356. (b) Y. Hara, L. Shen, A. Tsubouchi, and M. Akiyama, Inorg. Chem., 2000, 39, 5074.
19. P. S. Dobbin, R. C. Hider, A. D. Hall, P. D. Taylor, P. Sarpong, J. B. Poter, G. Xiao, and D. van
der Helm, J. Med. Chem., 1993, 36, 2448.
20. G. Anderegg, F. L’Eplattenier, and G. Schwarzenbach, Helv. Chim. Acta, 1963, 46, 1400 and 1409.
21. ‘Critical Stability Constants’, ed. by A. E. Martell and R. M. Smith, Plenum Press, New York, 1974.
22. A. Shanzer, J. Libman, R. Lazar, and Y. Tor, Pure Appl. Chem., 1989, 61, 1529.
23. (a) J. Ohkanda and A. Katoh, J. Org. Chem., 1995, 60, 1583. (b) J. Ohkanda and A. Katoh, Chem.
Lett., 1996, 423.
24. (a) K. N. Raymond and C. J. Carano, J. Am. Chem. Soc., 1979, 101, 5401. (b) K. N. Raymond,
S. A. Nguyen, and A. Crag, J. Am. Chem. Soc., 1993, 115, 6758.
25. J. B. Neiland, Struct. Bonding (Berlin), 1984, 58, 1.
26. A. Katoh and M. Akiyama, J. Chem. Soc., Perkin Trans. 1, 1991, 1839.
27. R. L. N. Harris, Aust J. Chem., 1976, 29, 1329.