Synthesis and evaluation of guanidine-containing schiff base copper(II), zinc(II), and iron(III) chelates as trypsin inhibitors

Article abstract of DOI:10.1248/cpb.51.625

3-Formyl-4-hydroxyphenylguanidine hydrochloride and its Schiff base copper(II), zinc(II), and iron(III) chelates were synthesized and their inhibitory activity against bovine β-trypsin were determined. Syntheses of Schiff base metal chelates were carried

Full text of DOI:10.1248/cpb.51.625

June 2003
Chem. Pharm. Bull. 51(6) 625—629 (2003)
625
Synthesis and Evaluation of Guanidine-Containing Schiff Base
Copper(II), Zinc(II), and Iron(III) Chelates as Trypsin Inhibitors
*
Eiko TOYOTA, Haruo SEKIZAKI, Kunihiko ITOH, and Kazutaka TANIZAWA
Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido; Ishikari-Tobetsu, Hokkaido 061–0293,
Japan. Received January 9, 2003; accepted March 14, 2003
3-Formyl-4-hydroxyphenylguanidine hydrochloride and its Schiff base copper(II), zinc(II), and iron(III)
chelates were synthesized and their inhibitory activity against bovine b-trypsin were determined. Syntheses of
Schiff base metal chelates were carried out from 3-formyl-4-hydroxyphenylguanidine, various L-amino acids, and
divalent metal acetate. Their structures were established on the basis of spectroscopic evidence and elemental
analysis. The inhibitory activity of these chelates against bovine b-trypsin was determined. The guanidine-con-
taining copper(II) and zinc(II) chelates behaved as potent competitive inhibitors of trypsin. However, similar in-
hibitory activity was not observed for guanidine-containing iron(III) chelates. The inhibition constants (Ki val-
ues, ca. 10؊5 M) of guanidine-containing Schiff base copper(II) and zinc(II) chelates were slightly lower than those
(ca. 10؊6 M) of the corresponding amidine-containing Schiff base chelates with regard to bovine trypsin.
Key words guanidinium group; Schiff base; metal chelate; trypsin; inhibition constant
Studies on trypsin-specific compounds are useful for the gen atom of the Schiff base forms a hydrogen bond with
design of clinically useful compounds, since a variety of Ser195. This result suggests new avenues for the design of
physiologically important enzymes (e.g., enzymes of the even more potent inhibitors based on combining a metal
blood-clotting cascade, kallikrein and urokinase) have chelate motif with a cationic S1 recognition element. For ex-
trypsin-like specificity. Several benzamidine and phenyl- ample, guanidine-containing Schiff base metal chelates may
guanidine derivatives have been reported to be potent in- show higher affinity for trypsin than amidine-containing
hibitors of trypsin-like enzymes.1—12) X-ray crystallographic chelates, because the distance between the positive charge of
studies of trypsin complex prepared with benzamidine deriv- the guanidinium group and the metal ion is approximately
atives indicated that inhibition arises primarily from the in- 1.3 Å longer than that of the amidine-containing Schiff base
teraction between the cationic amidinium group of the in- metal chelate. In the meta-substituted guanidine-based in-
hibitor and the anionic carboxyl group of Asp189 in the S1 hibitors therefore, the increased chain length of the inhibitor
site of trypsin.13—16) The guanidinium group of guanidine- may allow tighter coordination between the metal ion and the
based inhibitors had also electrostatically favorable interac- imidazole nitrogen of His57 of trypsin than amidine-based
tion with the carboxyl group of Asp189.17—19)
inhibitors. Furthermore, the guanidinium group has three ni-
It is well known that metal ions serve to enhance the struc- trogen atoms, which may form hydrogen bonds with S1 site
tural stability of proteins or take part in the catalytic residues of trypsin or solvent molecule, and then the interac-
processes of enzymes.20) Transition metals such as tion between the inhibitor and active site residue of trypsin is
copper(II), zinc(II), and nickel(II) also inhibit trypsin activity expected to be stronger than that of the amidine-based in-
by direct binding to the imidazole nitrogen of histidine hibitor. To confirm the above assumption, guanidine-contain-
residues.16,21,22) Thus we have been interested in the Schiff ing Schiff base copper(II) and iron(III) chelates were synthe-
base metal chelates carrying the amidinium group as trypsin sized. In addition, amidine- and guanidine-containing Schiff
inhibitors. We have previously reported that the Schiff base base zinc(II) chelates were synthesized, since zinc(II) ion is
copper(II) and iron(III) chelates prepared from various a- known to be one of the important elements found in metallo-
amino acids, metal ions, and 4-formyl-3-hydroxybenzami- proteins or metalloenzymes.
dine or 3-formyl-4-hydroxybenzamidine are potent inhibitors
This paper deals with both the syntheses of Schiff base
of bovine b-trypsin.23,24) To elucidate further the structure– metal chelates and the study their inhibitory activity against
activity relationships in this novel series of inhibitors, we have bovine b-trypsin with the final goal of discovering clinically
studied the crystal structures of trypsin complex with ami- useful chelate compounds.
dine-containing Schiff base copper(II) or iron(III) chelate.25)
It became clear that the difference in the positioning of the Results and Discussion
amidinium group on the benzene ring has dramatic effects on
Syntheses and Characterization 3-Formyl-4-hydroxy-
the binding modes. In the Schiff base copper(II) chelates de- phenylguanidine hydrochloride (6), a key compound in this
rived from 4-formyl-3-hydroxybenzamidine (para position), study, was prepared from 2-hydroxy-5-nitrobenzaldehyde (1)
the amino acid moiety as a part of the Schiff base is situated as shown in Chart 1. The formyl group in compound 1 was
near the active site of trypsin, whereas the copper(II) ion is protected with ethylene glycol in the presence of p-toluene-
positioned in the region away from the active site residues of sulfonic acid in benzene to give rise to the acetal (2). Amine
trypsin. In contrast, in the Schiff base copper(II) chelates de- compound 3 was obtained by catalytic hydrogenation of 2 in
rived from 3-formyl-4-hydroxybenzamidine (meta position), dioxane–EtOH. Compound 3 was reacted with the amidina-
the copper(II) ion is directly coordinated to the imidazole ni- tion reagent 1-[N,NЈ-bis(Z)amidino]pyrazole, which was pre-
trogen of His57 (bond distance, 3.1 Å), and the phenolic oxy- pared according to the reported procedure26,27) in THF to give
* To whom correspondence should be addressed. e-mail: tanizawa@hoku-iryo-u.ac.jp
© 2003 Pharmaceutical Society of Japan

626
Vol. 51, No. 6
4. Deprotection of the bis(Z) compound 4 was carried out by iron(II) acetate to a solution of a stoichiometric amount of 6
catalytic hydrogenation over 10% Pd–C in EtOH–Et2O to and L-amino acids. The amidine-containing Schiff base
give 5, and then treated with 2 M HCl to give 6. Since com- zinc(II) chelates (10a—i) were prepared from 3-formyl-4-
pound 6 undergoes rapid decomposition when converted to hydroxybenzamidine hydrochloride,23) L-amino acids, and
the free base, the compound must be kept acidic during pu- zinc(II) acetate.
rification and storage. The structure of 6 was assigned on the
Structural formulae of the metal chelates (7—10) are
basis of IR and NMR spectra and elemental analysis. The IR shown in Fig. 1, and the physical and spectral data are sum-
spectrum of 6 is shown by broad strong bands in the region marized in Table 2. The structure of copper(II) and iron(III)
of 3150—3400 cmϪ1 due to typical amine absorptions (NH chelates (7, 9) is readily assigned on the basis of their ele-
stretching) and the strong band at 1657 cmϪ1 is assigned to mental analysis and spectral properties. In our previous
CϭN streching.28) The physical and spectral data of com- studies of amidine-containing copper(II) (11) and iron(III)
pounds 2—6 are listed in Table 1.
chelates (12),23,24) formation of the Schiff base metal chelate
Syntheses of the guanidine-containing Schiff base cop- was ascertained by the appearance of a marked absorption
per(II) (7a—i) and zinc(II) chelates (8a—i) were achieved by at 360—380 nm for copper(II) chelates and at 430—475 nm
mixing equimolar amounts of 6, L-amino acids, and for iron(III) chelates, which can be attributed to a p®p*
copper(II) or zinc(II) acetate, respectively. The correspond- transition originating mainly from the azomethine chro-
ing iron(III) chelates (9a—i) were obtained by adding mophore. The absorption spectra of 7 and 9 are consistent
with those of 11 and 12, respectively. In the IR spectra,
the copper(II) and iron(III) chelates (7b, 9b) showed charac-
teristic bands at 3100—3450 cmϪ1, ca. 1670 cmϪ1, ca.
1640 cmϪ1, ca. 1620 cmϪ1, and ca. 1300 cmϪ1. The broad
band in the region of 3100—3450 cmϪ1 is due to n(H2O)
and n(NH). The band at ca. 1670 cmϪ1 is assigned to be
n(CϭN) of the guanidinium group. The bands at ca.
1640 cmϪ1 and ca. 1620 cmϪ1 are assigned to n(CϭN) and
n(COOϪ), respectively, indicating coordination through
imine nitrogen and carboxyl oxygen to the metal ion.29,30) The
phenolic C–O vibration band in chelates appeared at ca.
1300 cmϪ1. This is evidence of the bonding of the metal ion
with phenolic oxygen, since the phenolic n(C–O) in the free
Schiff base is usually found near 1280 cmϪ1.31) In addition,
elemental analysis of chelates indicated the ratio of metal to
Schiff base ligand is 1 : 1 for copper(II) chelate and 1 : 2 for
iron(III) chelate. From these observations, the coordination
of copper(II) chelates was revealed to be a square planar
geometry, and copper(II) ion coordinates to carboxyl oxygen,
imine nitrogen, phenolic oxygen of Schiff base ligand, and a
Chart 1
Table 1. Physical and Spectral Data of Compounds 2—6
1H-NMR
Analysis (%) Calcd (Found)
Formula
Comp.
No.
mp (dec.)
(°C)
IR (KBr)
n (cmϪ1
)
d (ppm), J (Hz)
Solventa)
1)
C
H
N
Cl
2
3
72—73
3248
2894
1594
4.1—4.2 (4H, m), 6.00 (1H, s),
6.96 (1H, d, 8.8),
8.15 (1H, dd, 2.9, 8.8),
8.21 (1H, d, 2.9), 8.59 (1H, s)
3.85 (2H, m), 3.98 (2H, m),
4.45 (2H, s), 5.84 (1H, s),
6.42 (1H, dd, 2.5, 8.8),
C9H9NO5
C9H11NO3
51.19
(51.18
4.31
4.37
6.63
6.62
—
— )
116—118
3329
3290
3200
2904
2)
1)
59.66
(59.71
6.12
6.20
7.73
7.73
—
— )
6.52 (1H, d, 8.8),
6.62 (1H, d, 2.5), 8.52 (1H, s)
4.0—4.1 (4H, m), 5.13 (2H, s),
5.22 (2H, s), 5.91 (1H, s),
6.83 (1H, d, 8.8),
4
79—81
2900—3400
1728
1717
C26H25N3O7
63.53
(63.68
5.13
5.27
8.56
8.62
—
— )
1644
7.2—7.4 (12H, m), 7.74 (1H, s),
10.07 (1H, s), 11.90 (1H, s)
3.8—4.0 (4H, m), 5.90 (1H, s),
6.64 (2H, s), 6.79 (1H, s)
7.13 (1H, d, 8.8),
5
6
(164—165)
200—202
2900—3400
1670
3150—3400
1657
2)
2)
C10H13N3O3
53.80
(53.40
44.56
5.87
6.07
4.67
4.83
18.83
18.62
19.51 16.44
19.77 16.28)
—
— )
C8H10ClN3O2
7.37 (1H, dd, 2.4, 8.8),
(44.67
1629
7.44 (5H, s), 9.80 (1H, s),
10.27 (1H, s), 11.08 (1H, s)
a) Solvents: 1) CDCl3, 2) DMSO-d6.

June 2003
627
Fig. 1. Chemical Formulae of the Schiff Base Metal Chelates
Table 2. Physical and Spectral Data of Metal Chelates 7b—10b
Absorption
spectra (H2O)
nm (e)
Analysis (%) Calcd (Found)
mp (dec.)
IR (KBr)
n (cmϪ1
Compound
(°C)
Formula
)
C
H
N
Cl
7b
8b
9b
[Cu(gua-sal-ala)(H2O)]HCl
[Zn(gua-sal-ala)(H2O)]0.5H2O·HCl
K[Fe(gua-sal-ala)2]3H2O
(224—226) 3100—3450 359 (4750)
C11H15ClCuN4O4
C11H16ClN4O4.5Zn
C22H30FeKN8O9
C11H15ClN3O4.5Zn
36.22
(36.07
4.09
4.13
15.30
15.30
9.95
9.68)
1676, 1636
1617, 1298
(279—281) 3100—3450 354 (4710)
1670, 1637
1610, 1292
(190—191) 3100—3400 476 (3470)
35.21
(35.03
4.19
4.28
14.57
14.86
8.98
9.40)
41.17
(40.93
4.71
4.68
17.15
17.36
—
— )
1670, 1629
1300
(254—256) 3100—3400 341 (5360)
1670, 1640
10b [Zn(ami-sal-ala)(H2O)]0.5H2O·HCl
36.49
(36.69
4.18
4.31
11.61
11.61
9.79
9.38)
1608, 1300
Abbreviations: (gua-sal-ala), m-guanidinosalicylidene-L-alaninato; (ami-sal-ala), m-amidinosalicylidene-L-alaninato.
H2O molecule as shown in Fig. 1.23,32,33) On the other hand,
Trypsin The inhibitory activity of the chelates (7—10)
against bovine b-trypsin was determined according to the re-
ported procedure,23,24) and the Ki values are listed in Table 3.
The Ki values (ca. 10Ϫ5 M) of the guanidine-containing Schiff
base copper(II) (7a—i) and zinc(II) chelates (8a—i) indicate
strong inhibition. On the other hand, remarkable inhibitory
activity was not observed for iron(III) chelates except for 9a
and 9b.
From the result of crystal structures of the amidine-based
Schiff base inhibitor-trypsin complexes,25) we anticipated that
the increase in chain length between the guanidinium nitro-
gens, which form an ion pair with carboxyl oxygens of
Asp189 in trypsin, and metal ion in the inhibitors must result
in tighter interaction with the catalytic site and S1 site of
trypsin. Especially, the bond distance of metal-His57 must be
shorter than that of the amidine-based inhibitor, that is, the
inhibitory activity of guanidine-based inhibitors must be
higher than corresponding amidine-based inhibitors. Unex-
pectedly, the inhibition against bovine b-trypsin of guani-
dine-containing copper(II) (7a—i) and zinc(II) (8a—i)
the CϭN stretching band of the iron(III) chelate (9b) is
shifted to a higher-frequency region (1629 cmϪ1) than that
(1636 cmϪ1) of 7b, and the n(COOϪ) band of 9b is broad-
ened by overlapping with n(CϭN) stretching. This is typical
behavior of the bis(salicylideneamino acidato)iron(III)
chelates.24,30) Thus the iron(III) ion was concluded to have
octahedral coordination geometry with two Schiff base lig-
ands, as shown in Fig. 1.24,30,34,35)
Schiff base zinc(II) chelates derived from salicylaldehyde
and amino acid are considered to have a four-coordination
structure on the basis of their spectral properties, though X-
ray diffraction data have not been available. The IR and ab-
sorption spectra of zinc(II) chelates (8b, 10b) were very sim-
ilar to those of [(salicylideneamino acidato)(H2O)]zinc(II)
chelates.36—38) Furthermore, elemental analysis of 8b and
10b indicated the ratio of metal to Schiff base ligand is 1 : 1.
These results indicate that zinc(II) ion is tetrahedrally coordi-
nated by a Schiff base ligand and a H2O molecule.
Inhibition Constant (Ki) of Chelates against Bovine b-

628
Vol. 51, No. 6
Table 3. Inhibition Constants of Copper(II), Zinc(II), and Iron(III) Chelates for Trypsin-Catalyzed Hydrolysis of Benzoyl-L-arginine p-Nitroanilide at pH
8.0
Guanidine-containing chelates
Zn(II)
Amidine-containing chelates
Zn(II)
Cu(II)
Fe(III)
Amino acid
No.
Ki (M)
No.
Ki (M)
No.
Ki (M)
No.
Ki (M)
Gly
7a
7b
7c
7d
7e
7f
7g
7h
7i
2.8ϫ10Ϫ5
3.1ϫ10Ϫ5
5.0ϫ10Ϫ5
3.5ϫ10Ϫ5
2.6ϫ10Ϫ5
1.1ϫ10Ϫ5
1.1ϫ10Ϫ5
4.7ϫ10Ϫ5
4.0ϫ10Ϫ5
8a
8b
8c
8d
8e
8f
8g
8h
8i
9.5ϫ10Ϫ5
7.3ϫ10Ϫ5
8.8ϫ10Ϫ5
7.3ϫ10Ϫ5
9.3ϫ10Ϫ5
8.6ϫ10Ϫ5
7.8ϫ10Ϫ5
9.7ϫ10Ϫ5
8.8ϫ10Ϫ5
9a
9b
9c
9d
9e
9f
9g
9h
9i
7.8ϫ10Ϫ5
1.8ϫ10Ϫ4
1.1ϫ10Ϫ3
3.4ϫ10Ϫ3
3.0ϫ10Ϫ3
2.5ϫ10Ϫ3
1.1ϫ10Ϫ3
1.1ϫ10Ϫ3
1.1ϫ10Ϫ3
10a
10b
10c
10d
10e
10f
10g
10h
10i
7.1ϫ10Ϫ6
7.6ϫ10Ϫ6
7.5ϫ10Ϫ6
4.4ϫ10Ϫ6
9.8ϫ10Ϫ6
6.0ϫ10Ϫ6
2.4ϫ10Ϫ5
1.7ϫ10Ϫ5
1.8ϫ10Ϫ5
L-Ala
L-Val
L-Leu
L-Ile
L-Phe
L-Met
L-Ser
L-Thr
chelates was less than that of the corresponding amidine- Further details of structural requirements for trypsin-in-
containing copper(II) (11a—h) and zinc(II) chelates (10a— hibitor complex using the X-ray diffraction method will be
i). This result suggests that the conformation of the active described in a future paper.
site of trypsin is a better fit for the amidine-based inhibitor
In this study, the design of guanidine-based inhibitors with
than the guanidine-based inhibitor. Previous X-ray crystallo- higher inhibitory activity than amidine-based inhibitors
graphic studies indicated that the cationic guanidinium group against bovine b-trypsin was not successful, although this
formed salt bridge interactions with the carboxylate group of approach will hopefully lead to the final goal of discovering
Asp189 in trypsin.17,18) Thus a difference in the pKa value be- clinically useful chelate compounds that inhibit trypsin-like
tween the amidinium group and guanidinium group is the enzymes.
most important reason why the inhibitory activity does not
increase with guanidine-based inhibitors. The reported pKa
Experimental
Materials 2-Hydroxy-5-nitrobenzaldehyde and iron(II) acetate were
values for p-hydroxyphenylguanidinium ion and p-hydroxy-
purchased from Aldrich Chemical Co., Ltd. Bovine b-trypsin (EC 3.4.21.4)
benzamidinium ion are 9.05 and 12.69, respectively.39,40)
was purchased from Worthington Biochemical Corp. (twice crystallized).
In the iron(III) chelates, the Ki values of 9a and 9b derived
from Gly or Ala were comparable to those of the copper(II)
and zinc(II) chelates, although the inhibitory activity of 9c—
i was very weak. At a higher concentration of 9c—i, compet-
itive inhibition with Ki values of ca. 10Ϫ3 M, which is compa-
rable to that for the binding of an aromatic compound to
Instruments Melting points were measured on a Yanagimoto micro
melting point apparatus. H-NMR spectra were recorded on a JNM-FX-400
spectrometer (JEOL). IR and absorption spectra were recorded on a FT/IR
VALOR-III spectrometer (JASCO) and a U-2000 spectrophotometer (Hi-
tachi), respectively.
1
Synthesis of 2-(1,4-Dioxa-5-cyclopentyl)-4-nitrophenol (2) A solution
of 2-hydroxy-5-nitrobenzaldehyde (1) (8.3 g, 49.7 mmol), ethylene glycol
trypsin, was found.41) In the model fitting of trypsin with 9b, (15.5 g, 250 mmol), and p-toluenesulfonic acid (0.2 g, 1.1 mmol) in benzene
(400 ml) was refluxed for 4 h using the distilling receiver for water. The solu-
it was simulated that one of the Schiff base ligands would
orient closely to the catalytic triad (His57, Asp102, Ser195)
in such a manner that the guanidinium nitrogens of 9b form
tion was transferred to a separatory funnel and the benzene layer was sepa-
rated, washed with water and saturated NaCl solution, dried over anhydrous
Na2SO4, and evaporated to dryness in vacuo. The residue was purified by re-
an ion pair with carboxyl oxygens of Asp189. Probably, octa-
hedrally coordinated iron(III) chelates derived from amino
acids (which have bulky side chains on the a-carbon) such as
Val, Leu, Ile, and Phe could not be included in the active site
of trypsin due to unfavorable interactions with the active site
residues. This is supported by the results of crystal structures
of bis(m-amidinosalicylidene-L-alaninato)iron(III) (12b) and
bis(p-amidinosalicylidene-L-alaninato)iron(III) (13b) bound
to bovine trypsin.25) The refined structure of the trypsin-12b
complex showed that inhibitor 12b did not bind to the
trypsin, whereas inhibitor 13b was bound. In the case of the
trypsin-12b complex, the iron(III) ion was replaced by mag-
nesium ion which was present in the mother liquor, and one
of the Schiff base ligands was replaced by solvent molecules
to avoid unfavorable interactions with the active site residues.
crystallization from benzene to give 2 (7.8 g, yield 74%) as a colorless crys-
talline powder. Physical properties and spectral data are given in Table 1.
4-Amino-2-(1,4-dioxa-5-cyclopentyl)phenol (3) Palladium-activated
carbon (Pd 10%) (200 mg) was added to a solution of 2 (2.1 g, 9.9 mmol) in
dioxane–EtOH (5 : 1) (300 ml), and the mixture was vigorously stirred for
1 h under an atmosphere of hydrogen at room temperature. The catalyst was
filtered off, and the filtrate was evaporated to dryness in vacuo. The residue
was purified by recrystallization from EtOH–hexane to give 3 (1.6 g, yield
89%) as pale brown needles. Physical properties and spectral data are given
in Table 1.
4-[N؅,N؆-Bis(benzyloxycarbonyl)guanidino]-2-(1,4-dioxa-5-cy-
clopentyl)phenol (4) A solution of 3 (0.9 g, 5.0 mmol) and 1-[N,NЈ-
bis(Z)amidino]pyrazole (1.7 g, 5.0 mmol) in anhydrous THF (1.0 ml) was
stirred for 1 h at room temperature. The reaction mixture was concentrated
in vacuo. The residue was purified by silica gel column chromatography.
The eluate [benzene–ethyl acetate (6 : 1)] was evaporated to dryness in
vacuo and the solid residue was recrystallized from benzene–hexane to give
4 (2.2 g, yield 90%) as pale yellow prisms. Physical properties and spectral
The Ki values of guanidine- or amidine-containing Schiff data are given in Table 1.
2-(1,4-Dioxa-5-cyclopentyl)-4-guanidinophenol (5) A solution of 4
base zinc(II) chelates (8a—i, 10a—i) were somewhat larger
than those of the corresponding copper(II) chelates (7a—i)
and (11a—i),23) respectively (Table 3). This observation im-
plies that the coordination geometry of copper(II) ion is a
better fit for the active site of trypsin than that of zinc(II) ion.
(2.0 g, 4.1 mmol) in EtOH–Et2O (1 : 1) (250 ml) containing 10% Pd–C
(100 mg) was vigorously stirred for 1 h under an atmosphere of hydrogen at
room temperature. The catalyst was filtered off, and the filtrate was evapo-
rated to dryness in vacuo. The solid residue was recrystallized from
MeOH–Et2O to give 5 (0.8 g, yield 87%) as a pale brown crystalline powder.

June 2003
629
Physical properties and spectral data are given in Table 1.
6) Tidwell R. R., Geratz J. D., Dubovi E. J., J. Med. Chem., 26, 294—298
(1983).
7) Nochi S., Shimomura N., Hattori T., Sato T., Miyake Y., Tanizawa K.,
Chem. Pharm. Bull., 37, 2855—2857 (1989).
8) Matsumoto O., Masuda H., Taga T., Kuroda Y., Machida K., Chem.
Pharm. Bull., 38, 2015—2017 (1990).
3-Formyl-4-hydroxyphenylguanidine Hydrochloride (6) To a solution
of 5 (1.12 g, 5.0 mmol) in EtOH (5 ml) was added 2 M HCl (5 ml), and the
precipitate was collected. Recrystallization from EtOH–Et2O give a pale yel-
low crystalline powder (0.77 g, yield 71%). Physical properties and spectral
data are given in Table 1.
m-Guanidinosalicylidene-L-alaninato(aqua)copper(II) Hydrochloride
(7b) Synthesis was carried out following the procedure reported for m-
9) Senokuchi K., Nakai H., Nakayama Y., Odagaki Y., Sasaki K., Kato
M., Maruyama T., Miyazaki T., Ito H., Kamiyasu K., Kim S., Kawa-
mura M., Hamanaka N., J. Med. Chem., 38, 2521—2523 (1995).
10) Odagaki Y., Nakai H., Senokuchi K., Kawamura M., Hamanaka N.,
Nakamura M., Tomoo K., Ishida T., Biochemistry, 34, 12849—12853
(1995).
amidinosalicylidene-L-alaninato(aqua)copper(II).23) To
a solution of 6
(0.22 g, 1.0 mmol) and L-alanine (0.09 g, 1.0 mmol) in water (2 ml) was
added a solution of copper(II) acetate monohydrate (0.2 g, 1.0 mmol) in
water (2 ml) and the mixture was stirred for 3 h at 50 °C. The reaction mix-
ture was concentrated and subjected to Sephadex LH-20 column chromatog- 11) Stürzebecher J., Prasa D., Wikström P., Vieweg H., J. Enzyme Inhib., 9,
raphy using MeOH. The eluate was evaporated to dryness in vacuo, and the 87—99 (1995).
solid residue was recrystallized from H2O–EtOH to give 7b (0.30 g, yield 12) Stürzebecher J., Prasa D., Hauptmann J., Vieweg H., Wikström P., J.
84%) as a green crystalline powder. Physical properties and spectral data are
given in Table 2.
m-Guanidinosalicylidene-L-alaninato(aqua)zinc(II) Hemihydrate Hy-
Med. Chem., 40, 3091—3099 (1997).
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temperature. The reacted precipitate was collected by filtration. Complex 16) Katz B. A., Clark J. M., Finer-Moore J. S., Jenkins T. E., Johnson C.
was purified by recrystallization from H2O–EtOH and dried under a vacuum.
A white powder was obtained (0.2 g, yield 53%). Physical properties and
spectral data are given in Table 2.
R., Ross M. J., Luong C., Moore W. R., Stroud R. M., Nature (Lon-
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Potassium Bis(m-guanidinosalicylidene-L-alaninato)iron(III) Trihy-
drate (9b) Synthesis was carried out following the procedure reported for
potassium bis(m-amidinosalicylidene-L-alaninato)iron(III) (12b).24) A mix- 18) Matsumoto O., Taga T., Matsushima M., Higashi T., Machida K.,
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Chem. Pharm. Bull., 38, 2253—2255 (1990).
droxide (0.23 g, 4.1 mmol) in H2O–EtOH (15 ml) was stirred for several 19) Turk D., Sturzebecher J., Bode W., FEBS Lett., 287, 133—138 (1991).
minutes at room temperature. To the reaction mixture, an ethanolic solution 20) Glusker J. P., Adv. Protein Chem., 42, 1—76 (1991).
of iron(II) acetate enneahydrate (0.17 g, 1.0 mmol) was added over about 21) Brinen L. S., Willett W. S., Craik C. S., Fletterick R. J., Biochemistry,
15 min. The solution was stirred for 8 h at room temperature and evaporated
35, 5999—6009 (1996).
to dryness. The residue was subjected to a column chromatography on 22) Katz B. A., Luong C., J. Mol. Biol., 292, 669—684 (1999).
Sephadex LH-20 (MeOH). The eluate was concentrated until precipitation 23) Toyota E., Chinen C., Sekizaki H., Itoh K., Tanizawa K., Chem.
of the product occurred. The precipitate was collected by filtration, washed
with EtOH and Et2O, and dried under a vacuum. A reddish brown powder
was obtained (0.4 g, yield 31%). Physical properties and spectral data are
given in Table 2.
Pharm. Bull., 44, 1104—1106 (1996).
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m-Amidinosalicylidene-L-alaninato(aqua)zinc(II) Hemihydrate Hy-
drochloride (10b) Equimolar amounts of 3-formyl-4-hydroxybenzamidine 26) Bernatowicz M. S., Wu Y., Matsueda G. R., J. Org. Chem., 57, 2497—
hydrochloride, L-alanine, and zinc(II) acetate dihydrate (1.0 mmol) were dis- 2502 (1992).
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temperature. The complex was purified by recrystallization from 3389—3392 (1993).
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(0.2 g, yield 55%). 725 (1957).
Determination of Inhibitory Activity (Ki) of Chelates Enzyme activ- 29) Heinert D., Martell A. E., J. Am. Chem. Soc., 84, 3257—3263 (1962).
ity was determined in 50 mM Tris–HCl buffer containing 20 mM CaCl2 (pH
30) Burrows R. C., Bailar J. C., Jr., J. Am. Chem. Soc., 88, 4150—4155
8) using benzoyl-L-arginine p-nitroanilide (BAPA) as a substrate. Determina-
(1966).
tion of Ki values was carried out as follows: Concentrations of the inhibitor 31) Kovacic J. E., Spectrochim. Acta, Part A, 23, 183—187 (1967).
and trypsin used in the kinetic analysis were 10Ϫ4—10Ϫ5 M and 10Ϫ6 M, re-
32) Nakahara A., Bull. Chem. Soc. Jpn., 32, 1195—1199 (1959).
spectively. Hydrolytic rates in the presence of chelate were determined and 33) Ueki T., Ashida T., Sasada Y., Kakudo M., Acta Crystallogr., 22,
the Ki values were calculated according to the method of Dixon42) using a
870—878 (1967).
linear regression program.
34) Hämäläinen R., Turpeinen U., Acta Chem. Scand., 43, 15—18 (1989).
35) Bowden F. L., Carpenter R. P., Parish R. V., Pollock R. D., Inorg.
Chim. Acta, 23, 35—36 (1977).
Acknowledgments This work was supported in part by the Akiyama
Foundation and the High Technology Research Program from the Ministry 36) O’Connor M. J., Ernst R. E., Schoenborn J. E., Holm R. H., J. Am.
of Education, Culture, Sports, Science, and Technology of Japan.
Chem. Soc., 90, 1744—1752 (1968).
37) Leussing D. L., Bai K. S., Anal. Chem., 40, 575—581 (1968).
38) Casella L., Gullotti M., J. Am. Chem. Soc., 103, 6338—6347 (1981).
39) Koike H., Nippon Kagaku Zasshi, 83, 917—923 (1962).
40) Rogana E., Nelson D. L., Leite L. F. F., Mares-Guia M., J. Chem. Res.
(S), 1985, 286—287.
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