Chiral multinuclear macrocyclic polyamine complexes: Synthesis, characterization and their interaction with plasmid DNA

Article abstract of DOI:10.1016/j.bmc.2006.10.057

A series of multinuclear macrocyclic polyamine metal (Zn²⁺, Cu²⁺, Co²⁺) complexes containing chiral dipeptide linkage were synthesized and used as artificial nuclease enzyme model. The interaction between the complexes and

Full text of DOI:10.1016/j.bmc.2006.10.057

Bioorganic & Medicinal Chemistry 15 (2007) 696–701
Chiral multinuclear macrocyclic polyamine complexes: Synthesis,
characterization and their interaction with plasmid DNA
Yu-Guo Fang,a Ji Zhang,a Shan-Yong Chen,a Ning Jiang,b Hong-Hui Lin,b,*
Yu Zhanga and Xiao-Qi Yua,c,
*
aDepartment of Chemistry, Key Laboratory of Green Chemistry and Technology (Ministry of Education),
Sichuan University, 29 Wangjiang Road, Chengdu, Sichuan 610064, PR China
bCollege of Life Science, Sichuan University, Chengdu 610064, PR China
cState Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University,
Chengdu, Sichuan 610041, PR China
Received 5 August 2006; revised 25 October 2006; accepted 28 October 2006
Available online 1 November 2006
Abstract—A series of multinuclear macrocyclic polyamine metal (Zn2+, Cu2+, Co2+) complexes containing chiral dipeptide linkage
were synthesized and used as artificial nuclease enzyme model. The interaction between the complexes and plasmid DNA (pUC19)
was studied, and the results revealed that these complexes could act as powerful catalysts for the cleavage of plasmid DNA under
physiological conditions.
ꢀ 2006 Published by Elsevier Ltd.
1. Introduction
mononuclear Zn(II) complexes, which bind to peptide
chains tethered to a rhodium complex, could act as
intercalator and promote DNA cleavage. Scrimin and
coworkers synthesized dinuclear Zn2+ complexes and
used the complexes as artificial nucleases, they proved
that these complexes were more effective in the cleav-
age of RNA and DNA under physiological conditions
than mononuclear complex, which revealed that a high
degree of synergistic effect existed between the two met-
al ion centers.15–17 Suh and coworkers reported that
Cu(II) or Co(III) complexes of polyamines could be
used as catalytic drugs to promote the selective cleav-
age of protein.18,19 The heteroatoms on amino acid
or peptide side chains and backbone could enhance
the ability of ligands to coordinate with metal ions.20
Many dinuclear metal complexes were studied as
enzyme models,2,21–23 however, to the best of our
knowledge, the reports of studies of multinuclear metal
complexes with chiral peptide linkage and their uses in
the cleavage of DNA remain rare. Due to the folded
structure of the peptide, the two catalytic centers might
have an appropriate distance and angle in the catalytic
process. In this paper, we synthesized a series of multi-
nuclear metal complexes with chiral dipeptide spacers
and used them as artificial nuclease to study their inter-
action with plasmid DNA (pUC19).
With the high abilities in recognizing specific DNA se-
quence and catalyzing the hydrolysis of phosphate dies-
ter bonds, chemical nucleases have rapidly become an
invaluable research tool in the fields of biology, bioor-
ganic chemistry, therapy, and molecular biology.1,2
Many complexes have been designed and studied as
artificial enzyme models. In the commonly used
ligands, 1,4,7,10-tetraazacyclododecane (cyclen) has a
special structure and can coordinate with most metal
cations (transition metal ions and lanthanide ions3–6).
Therefore, metal–cyclen complexes were widely used
in DNA recognition,7,8 cleavage,9 and enzyme
mimics.10–13
Many nucleases carry one or more metal centers. In
most cases, synergistic effects exist between the metal
centers.14 It has been reported that Zn(II) is a key met-
al ion for the development of artificial metallonucleas-
es. Barton and coworkers proved that a series of
Keywords: 1,4,7,10-Tetraazacyclododecane (cyclen); Macrocyclic poly-
amine; Multinuclear complexes; DNA cleavage; Chemical nuclease.
*
Corresponding authors. Fax: +86 28 85415886; e-mail: xqyu@
tfol.com
0968-0896/$ - see front matter ꢀ 2006 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2006.10.057

Y.-G. Fang et al. / Bioorg. Med. Chem. 15 (2007) 696–701
697
2. Results and discussion
Then we studied the complexes 8b-M. Unlike 8a-M, 8b-
Cu instead of 8b-Co was the best catalyst for DNA
cleavage. A solution containing plasmid DNA was incu-
bated in a 0.5 mL tube with catalyst 8b-Zn, 8b-Cu and
8b-Co (0.14 mM) in a Na2HPO4/NaH2PO4 buffer
(100 mM, pH 7.0) under 37 ꢁC for 48 h. The amount
of nicked DNA (Form II) was 37.37%, 60.57%, and
31.31%, respectively. So, our subsequent efforts focused
on the catalytic ability of complex 8b-Cu under physio-
logical conditions. Figures 4 and 5 show the effects of
reaction time and concentration of catalyst in 8b-Cu-
catalyzed cleavage reaction. The results showed that
complex 8b-Cu was an excellent chemical nuclease and
the amount of nicked DNA (Form II) was more than
the reaction catalyzed by 8a-Co. On the other hand, sim-
ilar to 8a-Co, the catalytic ability of 8b-Cu was increased
in association with the raise of reaction time or concen-
tration of complex. However, when we increased the two
factors to a special level, the linear DNA (Form III)
appeared.
2.1. Synthesis
The synthetic route of these multinuclear ligands is
shown in Scheme 1. The Z-(benzylacetate) group was
introduced to be an effective protecting group for the
carboxyl acid on side chain. Compounds 4 were ob-
tained by the reaction between 2 and Z-protected
amines 3 in the presence of (DCC/HOBt). The desired
products were purified by silica gel column chromatog-
raphy to avoid the loss from washing by citric acid
and aqueous NaHCO3 solution. Deprotected products
5 were obtained by catalytic method under H2 atmo-
sphere with Pd/C. Subsequent preparation was pro-
cessed through the traditional way of peptide synthesis
(DCC/HOBt). Complexes 7a and 7b were obtained
in around 65–74% yields. The Boc protective groups
were removed by adding the solution of trifluoroacetic
acid (TFA) dropwise in dichloromethane. Multinuclear
metal complexes were obtained by adding the excessive
salt [Zn(ClO4)2, Co(NO3)2 or Cu(NO3)2] solution under
refluxing conditions for 2 h. All new compounds
and metal complexes were confirmed by analytic
methods.
By comparing the results of reactions catalyzed by the
complexes derived from 8a and 8b, we can conclude that
the peptide linkage did play an important role in the
DNA cleavage process. The structure of the bridge spac-
er may influence the character of the complexes. When
using serine to build the spacer, the hydroxyl group
might change the coordinative property, and, as a result,
the metal complexes derived from 8b showed different
catalytic ability from those derived from 8a. In the cat-
alytic cleavage process, Cu2+ was chosen to be the most
suitable metal ion for 8b, while Co2+ was most suitable
for 8a. The results of parallel experiments are shown in
Figure 6. The amine group might also have the ability to
promote the performance of metal complex in the cleav-
age of DNA under physiological conditions. Mean-
while, due to the special folded structure of the
peptide, the two catalytic centers might have an appro-
priate distance and angle in the catalytic process, so a
synergistic effect might exist in the system.
2.2. Interaction between Zn(II), Cu(II), Co(II) complexes
and plasmid pUC19 DNA
The interaction of dinuclear metal complexes with
pUC19 was studied by monitoring the conversion of cir-
cular supercoiled DNA (Form I) to nicked (Form II)
and linear (Form III) DNA. The amount of strand scis-
sion was assessed by agarose gel electrophoresis.
Frist, we choose the complexes 8a-M (M = Zn, Cu, or
Co) as the catalyst to optimize the conditions of DNA
cleavage. Different metal complexes displayed different
catalytic effect, the results are shown in Figure 1. Among
the three complexes, 8a-Co is the best catalyst for DNA
cleavage. When we increase the concentration of com-
plexes from 0.14 to 0.28 mM, only 8a-Co showed obvi-
ous increase of catalytic effect, the conversion of Form I
increased from 40% to 51%. On the contrary, the cata-
lytic ability of 8a-Zn and 8a-Cu was slightly decreased
with the raise of concentration. Our subsequent efforts
focused on the reactivity of complex 8a-Co under phys-
iological conditions.
3. Experimental
3.1. General information
All reagents were purchased from commercial sources
and used without further purification. Anhydrous aceto-
nitrile (CH3CN), chloroform (CHCl3), dichloromethane
(CH2Cl2), and tetrahydrofuran (THF) were dried and
purified under nitrogen by using standard methods
and were distilled immediately before use. All aqueous
solutions were prepared from deionized or distilled
water. Electrophoresis grade agarose and plasmid
DNA (pUC19) were purchased from Takara Biotech-
nology Company. MS (ESI) mass spectral data were
recorded on a Finnigan LCQDECA mass spectrometer.
HRMS spectral data were recorded on Bruker Daltonics
Figures 2 and 3 show the effects of reaction time and
concentration of complex in the cleavage reaction cata-
lyzed by 8a-Co. The results revealed that dinuclear mac-
rocyclic polyamine complexes as chemical nucleases are
capable to accelerate the plasmid DNA dramatically.
Increasing the reaction time resulted in the increase of
catalytic ability. But when extending the reaction time
to 72 h, the amount of Form II was slightly decreased.
So the best interaction time is 48 h. Figure 3 indicated
that the best concentration of complex 8a-Co is
0.028 mM. Further increase of concentration led to the
decrease of Form II. The reason might be that the part
of DNA was incised to pieces in high concentration of
catalyst.
1
Bio TOF. H NMR spectral data were measured on a
Varian INOVA-400 spectrometer and chemical shifts
in ppm are reported relative to internal Me4Si (CDCl3).
Elemental analyses were performed using a Carlo-Elba
1106 elemental analytical instrument. IR spectra were

698
Y.-G. Fang et al. / Bioorg. Med. Chem. 15 (2007) 696–701
R
*
O
Boc
N
HO
N
H
O
Ph
3a R=CH2CH(CH3)2
3b R=CH2OH
N
H
(Boc)2O
Et3N
O
Boc N
HN
NH
HN
H
N
DCC/HOBt, THF
N
Boc
2
1
Boc
Boc
N
Boc
N
R
*
O
R
*
N
OH
Pd/C
Boc
N
N
Boc
N
N
Boc
N
N
MeOH
N
H
O
Ph
NH2
O
N
N
N
O
O
Boc
Boc
Boc
6
5a R=CH2CH(CH3)2
5b R=CH2OH
4a R=CH2CH(CH3)2
4b R=CH2OH
Boc
N
Boc
N
O
H
N
CF3COOH
CH2Cl2
6, DCC/HOBt
*
N
N Boc
Boc
N
N
CH2Cl2
N
O
N
R
Boc
Boc
7a R=CH2CH(CH3)2
7b R=CH2OH
O
N
H
H
N
H
8a-Co2+
8b-Co2+
N
relative metal salts
*
NH
N
8a-Zn2+
8a-Cu2+
8b-Zn2+
8b-Cu2+
N
HN
.7CF3COOH
H
N
H
O
N
R
8a R=CH2CH(CH3)2
8b R=CH2OH
Scheme 1. Preparation of chiral multinuclear macrocyclic polyamine complexes.
A
Form II
Form I
Form II
Form I
lane1
lane1
2
3
4
5
6
7
2
3
4
5
6
7
8
0.6
0.5
0.4
0.3
0.2
0.1
0
B
0.8
B
0.7
0.6
0.5
0.4
0.3
0.2
0.1
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
8
Figure 1. Cleavage reaction of pUC19 DNA (5 lg/mL) in Tris–HCl
buffer (100 mM, pH 7.4) catalyzed by different metal complexes at
37 ꢁC for 52 h. (A) Diagram of agarose gel electrophoresis. Lane 1,
DNA control; lane 2, 0.14 mM 8a-Zn2+; lane 3, 0.14 mM 8a-Cu2+; lane
4, 0.14 mM 8a-Co2+; lane 5, 0.28 mM 8a-Zn2+; lane 6, 0.28 mM 8a-
Cu2+; lane 7, 0.28 mM 8a-Co2+. (B) Quantitation of % plasmid
relaxation relative to plasmid DNA per lane.
Figure 2. Effect of reaction time on the cleavage of pUC19 DNA (5 lg/
mL) with 8a-Co (0.14 mM) in a Na2HPO4/NaH2PO4 buffer (100 mM,
pH 7.0) at 37 ꢁC. (A) Agarose gel electrophoresis diagrams: lane 1,
DNA control; lanes 2–8, complex 8a-Co, 1, 2, 4, 8, 12, 24, 48, and 72 h.
(B) Quantitation of % plasmid relaxation (Form II) relative to plasmid
DNA per lane.

Y.-G. Fang et al. / Bioorg. Med. Chem. 15 (2007) 696–701
699
A
Form II
Form I
lane1
2
3
4
5
6
7
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
FormIII
FormII
B
0.7
0.6
0.5
0.4
0.3
0.2
B
0.1
0
1
2
3
4
5
6
7
1
2
3
4
5
6
7
8
Figure 3. Effect of concentration on the cleavage of pUC19 DNA
(5 lg/mL) with 8a-Co in a Na2HPO4/NaH2PO4 buffer (100 mM, pH
7.0) and at 37 ꢁC for 48 h. (A) Agarose gel electrophoresis diagrams:
lane 1, DNA control; lanes 2–7, 0.014, 0.028, 0.070, 0.14, 0.28, and
1.14 mM. (B) Quantitation of % plasmid relaxation relative to plasmid
DNA per lane.
Figure 5. Effect of concentration on the cleavage of pUC19 DNA
(5 lg/mL) with 8b-Cu in a Na2HPO4/NaH2PO4 buffer (100 mM, pH
7.0) at 37 ꢁC for 72 h. (A) Agarose gel electrophoresis diagrams: lane 1,
DNA control; lanes 2–8, 0.007, 0.014, 0.028, 0.07, 0.14, 0.28, 0.57. (B)
Quantitation of open circular form (Form II) and linear form (Form
III) relative to plasmid DNA per lane.
A
A
Form II
Form III
Form II
Form II
Form III
Form I
Form I
Form I
lane 1
2
3
4
5
6
7
lane 1
2
3
4
5
6
7
8
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
B
FormIII
FormII
B
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
Figure 4. Effect of reaction time on the cleavage of pUC19 DNA (5 lg/
mL) with 8b-Cu (0.14 mM) in a Na2HPO4/NaH2PO4 buffer (100 mM,
pH 7.0) at 37 ꢁC. (A) Agarose gel electrophoresis diagrams: lane 1,
DNA control; lanes 2–8, complex 8a-Cu, 1, 2, 4, 8, 12, 24, 48, and 72.
(B) Quantitation of % plasmid relaxation open circular form (Form II)
and linear form (Form III) relative to plasmid DNA per lane.
Figure 6. Effect of different complexes on the cleavage of pUC19 DNA
(5 lg/mL) in a Na2HPO4/NaH2PO4 buffer (100 mM, pH 7.0) at 37 ꢁC
for 24 h. (A) Agarose gel electrophoresis diagrams: lane 1, DNA
control; lanes 2–7, 0.28 mM Cu(NO3)2, 0.28 mM Co(NO3)2, 0.14 mM
8a-Cu, 0.14 mM 8b-Cu, 0.14 mM 8a-Co, 0.14 mM 8b-Co. (B) Quan-
titation of open circular form (Form I) and linear form (Form II)
relative to plasmid DNA per lane.
recorded on a Shimadzu FTIR-4200 spectrometer as
KBr pellets or thin films on KBr plates. Melting points
were determined using a micro-melting point apparatus
without any corrections. Cyclen and 1,4,7-tris(tert-
butyloxycaronyl)-1,4,7,10-tetraazacyclododecan24,25 were
prepared as previously reported. The compound 6
(4,7,10-tris(tert-butoxycarbonyl)-1,4,7,10-tetraazacyclo-
dodecan-1-yl) acetic acid was synthesized according to
the literature.26
3.2. General procedure for the synthesis of compounds 4
Compounds 4 were synthesized by the following general
method. A THF solution of 3 (4.22 mmol), 3Boc-cyclen
(4.22 mmol), and 1-hydroxybenzotriazole-1-hydrox-
ybenzotriazole (HOBt) (5.06 mmol) was cooled to 0 ꢁC
in an ice bath. N,N0-dicyclohexylcarbodiimide (DCC)
(5.06 mmol) was dissolved in 20 mL THF and added

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Y.-G. Fang et al. / Bioorg. Med. Chem. 15 (2007) 696–701
dropwise to the solutions. The mixture was stirred at
0 ꢁC for 2 h and warmed to room temperature, and stir-
red at room temperature overnight. The suspension was
filtered and the precipitate was washed twice with small
amount of cold THF. The filtrate was purified by silica
gel column chromatography (EtOAC/hexane = 1:2) to
give colorless amorphous solid 4.
at room temperature for 24 h under N2 atmosphere. The
reaction mixture was filtered, the filtrate was dried, and
the solvent was removed under reduced pressure. The
residue was purified by silica gel column chromatogra-
phy (7a: EtOAc/hexane = 2:1; 7b: EtOAc/hexane = 4:1).
Compound 7a, colorless amorphous solid. Yield: 67%.
IR (KBr, cmꢀ1): 3448, 2974, 2930, 1687, 1465, 1413,
1366,1250, 1165, 781.1H NMR (400 MHz, CDCl3,
TMS) d = 4.88–4.92 (m, 1H, –NH); 3.21–3.53 (m, 30H,
–CH2); 2.84 (s, 4H, –CH2); 1.69 (m, 3H, CH–CH2);
1.44–1.48 (m, 54H, OC(CH3)3); 0.98 (d, J = 6.4 Hz,
3H, CH3); 0.92 (J = 6.4 Hz, 3H, CH3). HRMS-ESI of
C54H99N9O14: 1120.7204 [M+Na]+. Found: 1120.7185
[M+Na]+.
Compound 4a, colorless amorphous solid. Yield: 78%.
IR (KBr, cmꢀ1): 3421, 2974, 2931, 1696, 1647, 1466,
1411, 1366, 1250, 1164, 1043, 966, 864, 775. H NMR
1
(400 MHz, CDCl3, TMS) d = 7.33 (m, 5H, Ph–H);
5.44 (d, J = 8.8 Hz, 1H, N–H); 5.07 (s, 2H, –CH2–Ph);
4.63 (t, J = 8.4 Hz, 1H, CH); 3.49 (m, 16H, –CH2);
1.74–1.76 (m, 2H, –CH2); 1.45–1.48 (m, 27H,
OC(CH3)3); 1.01 (d, J = 6.4 Hz, 3H, –CH3); 0.93 (d,
J = 6.8 Hz, 3H, –CH3). HRMS-ESI of C37H61N5O9:
742.4361 [M+Na]+. Found: 742.4349 [M+Na]+.
Compound 7b, colorless amorphous solid. Yiled: 65%.
IR (KBr, cmꢀ1): 3447, 2975, 2930, 1699, 1465, 1414,
1366, 1249, 1165, 776. 1H NMR (400 MHz, CDCl3,
TMS) d = 4.92 (d, J = 4.8 Hz, 1H, NH); 3.73–3.81 (m,
5H, CH2CH); 3.26–3.54 (m, 26H, CH2); 2.78 (s, 4H,
CH2); 1.81 (s, 2H, CH2OH). ESI-MS of C51H93N9O15:
1094.7 [M+Na]+. Found 1094.7 [M+Na]+.
Compound 4b, colorless amorphous solid. Yield: 72%.
IR (KBr, cmꢀ1): 3437, 2975, 2931, 1687, 1639, 1466,
1413, 1366, 1250, 1164, 1019, 966, 858, 777. H NMR
1
(400 MHz, CDCl3, TMS) d = 7.26–7.37 (m, 5H, Ph–
H); 5.73 (d, J = 7.6 Hz, 1H, N–H); 5.12 (s, 2H, –CH2–
Ph); 4.69–4.74 (m, 1H, –OH); 3.39–3.80 (m, 15H,
–CH2); 2.65 (s, 4H, –CH2); 1.44–1.48 (m, 27H,
OC(CH3)3). HRMS-ESI of C34H55N5O10: 716.3841
[M+Na]+. Found: 716.3868 [M+Na]+.
3.5. General procedure for the synthesis of compounds 8
Trifluoroacetic acid (0.66 mmol) was added dropwise to
a solution of 7 (0.11 mmol) in CH2Cl2 (5 mL) at 0 ꢁC
under N2 atmosphere. After stirring overnight at room
temperature, the reaction mixture was concentrated un-
der reduced pressure below 40 ꢁC to give the crude prod-
uct. And the resulting yellow solid was recrystallized
from anhydrous ether and washed three times with
anhydrous ether (5 mL).
3.3. General procedure for the synthesis of compounds 5
A 50 mL two-necked round-bottomed flask was charged
with the solid of Pd/C (0.14 g) under N2 atmosphere,
then the solution of 4 (1.9 mmol) in MeOH was injected.
The mixture was stirred for 4 h under H2 atmosphere at
room temperature. After filtration, the filtrate was
evaporated off and purified by silica gel column
chromatography (CHCl3/MeOH = 9:1).
Compound 8a. Yield: 84.0%. 1H NMR (400 MHz, D2O)
d = 4.72–4.74 (m, 1H, –CH); 3.58–3.63 (m, 2H, –CH2);
3.21–3.34 (2H, –CH2); 3.04–3.12 (m, 4H, –CH2); 1.69–
1.77 (m, 2H, CH2); 1.56–1.59 (m, 1H, –CH); 0.99–1.01
(m, 6H, CH3). HRMS-ESI of C24H51N9O2: 498.4238
[M+H]+. Found: 498.4229 [M+H]+.
Compound 5a, colorless amorphous solid. Yield: 79%.
IR (KBr, cmꢀ1): 3433, 2974, 2931, 1695, 1647, 1467,
1411, 1366, 1249, 1164, 966, 787. H NMR (400 MHz,
1
1
Compound 8b. Yield: 82%. H NMR (400 MHz, D2O)
CDCl3, TMS) d = 3.17–3.75 (m, 17H, –CH, –NCH2);
1.78–1.86 (m, 3H, –CH2–CH); 1.47 (m, 27H,
OC(CH3)3); 0.94–0.97 (m, 6H, –CH3). HRMS-ESI of
C29H55N5O7: 586.4174 [M+H]+. Found: 586.4189
[M+H]+.
d = 4.11–4.15 (m, 1H, –CH); 3.83–3.94 (m, 4H, –CH2);
3.66–3.69 (m, 2H, –CH2); 3.48–3.56 (m, 2H, –CH2);
3.26–3.27 (m, 12H, –CH2); 3.16–3.21 (m, 8 H, –CH2);
3.01 (s, 8H, CH2). HRMS-ESI of C21H45N9O3:
472.3718 [M+H]+. Found: 472.3710 [M+H]+.
Compound 5b, colorless amorphous solid. Yield: 80%.
IR (KBr, cmꢀ1): 3420, 2975, 2930, 1696, 1467, 1412,
1367, 1249, 1162, 1108, 1055, 978, 847, 787. H NMR
3.6. General procedure for the preparation of metal
complexes 8-Zn, 8-Co, and 8-Cu
1
(400 MHz, CDCl3, TMS) d = 3.97 (t, J = 7.2 Hz, 1H,
–CH); 3.79–3.83 (m, 1H, –OH); 3.37–3.71 (m, 20H,
–CH2, –CH2N); 1.45–1.46 (m, 27H, OC(CH3)3).
HRMS-ESI of C26H49N5O8: 560.3654 [M+H]+. Found
560.3650 [M+H]+.
The trifluoroacetic acid salts of ligand 8 (0.9 mmol) were
dissolved in 5 mL of water and adjusted the aqueous
solution to alkaline (pH P 12) with 50% aqueous
NaOH. The solutions were extracted with CH2Cl2
(4 · 10 mL). Then organic layer was dried over anhy-
drous Na2SO4 and the solutions were concentrated
under reduced pressure to give yellow oil. To the ethanol
3.4. General procedure for the synthesis of compounds 7
To a solution of 6 (1.30 mmol) in CH2Cl2 was added
DCC (1.56 mmol), then 5 (1.30 mmol) in CH2Cl2 and
HOBt (1.56 mmol) were added at 0 ꢁC and the mixture
was stirred for 2 h at same temperature, and then stirred
solutions (5 mL) of 8 was added excessive salt
[Zn(ClO4)2, Co(NO3)2 or Cu(NO3)2] solution in
10 mL of ethanol and stirred overnight under room
temperature and then refluxed for 2 h. The solution

Y.-G. Fang et al. / Bioorg. Med. Chem. 15 (2007) 696–701
701
was concentrated under reduced pressure, the solid was
purified by centrifugal device, and washed with ethanol
References and notes
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(2 · 10 mL).
8a-Co2+: Anal. Calcd for C24H51N9O2Æ3Co(NO3)Æ3(OH)
(911.19): C, 31.62; H, 5.97; N, 18.44. Found: C, 31.27;
H, 5.94; N, 18.38. Atomic absorption spectrometry for
Co%: 19.40. Found: 19.78. IR (KBr, cmꢀ1): 3433,
2924, 2854, 1627, 1562, 1472, 1384.
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Chem. Rev. 2001, 216–217, 287.
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1993, 115, 6730.
8a-Zn2+: Anal. Calcd for C24H51N9O2Æ2Zn(ClO4)2Æ2Z-
n(OH)2Æ5H2O (1314.98): C, 21.92; H, 4.98; N, 9.58.
Found: C, 22.10; H, 4.74; N, 9.45. Atomic absorption
spectrometry for Zn%: 19.89. Found: 19.84. IR (KBr,
cmꢀ1): 3450, 2927, 1639, 1460, 1143, 1120, 1088, 627.
8. Shionoya, M.; Ikeda, T.; Kimura, E.; Shiro, M. J. Am.
Chem. Soc. 1994, 116, 3848.
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R. M.; Horrocks, W. D.; Morrow, J. R. Inorg. Chem.
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Yamado, Y. J. Am. Chem. Soc. 1992, 114, 10134.
12. Meyer, M.; Dahaoui-Gindrey, V.; Lecomete, C.; Guilard,
R. Coord. Chem. Rev. 1998, 178–180, 1313.
13. Schrodt, A.; Neubrand, A.; van Eldik, R. Inorg. Chem.
1997, 36, 4579.
14. Saki, N.; Akkaya, E. U. J. Mol. Catal. A: Chem. 2004,
219, 227.
15. Boseggia, E.; Gatos, M.; Lucatello, L.; Mancin, F.; Moro,
S.; Palumbo, M.; Sissi, C.; Tecilla, P.; Tonellato, U.;
Zagotto, G. J. Am. Chem. Soc. 2004, 126, 4543.
16. Sissi, C.; Rossi, P.; Felluga, F.; Formaggio, F.; Palumbo,
M.; Tecilla, P.; Toniolo, C.; Scrimin, P. J. Am. Chem. Soc.
2001, 121, 3169.
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M.; Toniolo, C.; Scrimin, P. J. Am. Chem. Soc. 1999, 121,
6948.
8a-Cu2+: Anal. Calcd for C24H51N9O2Æ2Cu(OH)2Æ2Cu-
(NO3)2 (1067.10): C, 26.99; H, 5.19; N, 17.05. Found:
C, 26.97; H, 4.91; N, 16.12. Atomic absorption spec-
trometry for Cu%: 23.80. Found: 23.66. IR (KBr,
cmꢀ1): 3438, 3231, 2933, 1621, 1384, 1359, 1073.
8b-Co2+: Anal. Calcd for C21H45N9O3Æ3Co(NO3)Æ3(OH)
(885.14): C, 28.48; H, 5.46; N,18.98. Found: C, 29.43; H,
5.32; N, 19.23. Atomic absorption spectrometry for
Co%: 19.97. Found: 19.78. IR (KBr, cmꢀ1): 3430,
2927, 2879, 1620, 1578, 1384.
8b-Zn2+: Anal. Calcd for C21H45N9O3ÆZn(ClO4)2ÆZn-
(ClO4)(OH) C2H5OH (963.11): C, 28.66; H, 5.44; N,
13.08. Found: C, 28.31; H, 5.04; N, 12.77. Atomic
absorption spectrometry for Zn%: 13.57. Found:
13.54. IR (KBr, cmꢀ1): 3436, 2921, 2867, 1639, 1452,
1117, 1108, 619.
8b-Cu2+: Anal. Calcd for C21H45N9O3Æ2Cu(OH)2Æ2-
Cu(NO3)2 (1041.87): C, 24.21; H, 4.74; N, 17.48.
Found: C, 24.57; H, 4.71; N, 16.67. Atomic absorp-
tion spectrometry for Cu%: 24.40. Found: 25.05. IR
(KBr, cmꢀ1): 3432, 3243, 2933, 1633, 1420, 1384,
1347, 1074.
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Acknowledgments
We thank the financial support from the National Nat-
ural Science Foundation of China (Grant Nos.
20372051, 20471038, and 20572075), Program for New
Century Excellent Talents in University, Specialized
Research Fund for the Doctoral Program of Higher
Education, and Scientific Fund of Sichuan Province
for Outstanding Young scientist, and YZ thanks the
sustentation of Student Innovation Found of Sichuan
University.
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Soc. 1997, 119, 3068.
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