The synthesis and immunosuppressive activities of steroid-urotoxin linkers

Article abstract of DOI:10.1016/j.bmc.2004.06.012

The urotoxins (Glu-Asp-Gly-OH, His-Gly-Glu-OH, His-Gly-Lys-OH, and His-Gly-Lys-NHNH₂) were introduced into the convenient sites of hydrocortisone and prednisolone via the amidation or condensation reactions to form the corresponding linkers 7a-d, 8a-d, 9a,b, and 10a,b in acceptable yields. The bioassays such as prolongation of heterotopic transplanted cardiac tissue survival in vivo, inhibitory effects on phagocytosis of mouse peritoneal macrophages and concanavalin (ConA) or lipopolysaccharide (LPS) induced proliferation of mouse spleen lymphocytes in vitro show that at the comparable concentrations the immunosuppressive activities of the steroid-urotoxin linkers 7a-d, 8a-d, 9a,b, and 10a,b were higher than that of hydrocortisone, prednisolone, and the urotoxins alone, as well as significantly higher than that of the mixture of hydrocortisone and urotoxins or prednisolone and urotoxins. The so-called 'permissive action' may be responsible for the enhancement of the mentioned bioactivities of the steroid-urotoxin linkers 7a-d, 8a-d, 9a,b, and 10a,b.

Full text of DOI:10.1016/j.bmc.2004.06.012

Bioorganic & Medicinal Chemistry 12 (2004) 4403–4421
The synthesis and immunosuppressive activities of steroid–urotoxin
linkers
Chao Wang,b Ming Zhao,b Xuecai Qiub and Shiqi Penga,*
aCollege of Pharmaceutical Sciences, Capital University of Medical Sciences, Beijing 100054, China
bCollege of Pharmaceutical Sciences, Peking University, Beijing 100083, China
Received 13 April 2004; revised 1 June 2004; accepted 5 June 2004
Available online 2 July 2004
Abstract—The urotoxins (Glu-Asp-Gly-OH, His-Gly-Glu-OH, His-Gly-Lys-OH, and His-Gly-Lys-NHNH2) were introduced into
the convenient sites of hydrocortisone and prednisolone via the amidation or condensation reactions to form the corresponding
linkers 7a–d, 8a–d, 9a,b, and 10a,b in acceptable yields. The bioassays such as prolongation of heterotopic transplanted cardiac
tissue survival in vivo, inhibitory effects on phagocytosis of mouse peritoneal macrophages and concanavalin (ConA) or lipo-
polysaccharide (LPS) induced proliferation of mouse spleen lymphocytes in vitro show that at the comparable concentrations the
immunosuppressive activities of the steroid–urotoxin linkers 7a–d, 8a–d, 9a,b, and 10a,b were higher than that of hydrocortisone,
prednisolone, and the urotoxins alone, as well as significantly higher than that of the mixture of hydrocortisone and urotoxins or
prednisolone and urotoxins. The so-called ‘permissive action’ may be responsible for the enhancement of the mentioned bioactivities
of the steroid–urotoxin linkers 7a–d, 8a–d, 9a,b, and 10a,b.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
The importance of the immunosuppressive lead com-
pounds, the immunosuppressive activity of hydrocorti-
sone, prednisolone, and urotoxins isolated from uremic
fluid, the available synthetic route of hydrocortisone–
urotoxin,6;7 and the preliminary immunosuppressive
activity of four hydrocortisone–urotoxins,8 drives us to
prepare a series of hydrocortisone–urotoxins and pred-
nisolone–urotoxins, to evaluate their in vitro and in vivo
immunosuppressive activities, and to analyze the related
chemical, structural, and biological knowledge.
In the 1950s it was reported that when a peptide was
mixed with a steroid the bioactivity of the peptide was
significantly enhanced by the steroid through increasing
the peptide’s receptor numbers. Since then this kind of
phenomenon was known as ‘permissive action’.1;2
Afterwards it was observed that glucocorticoids exhib-
ited rapid in vivo effects on the integrity of rat lym-
phocyte genomic deoxyribonucleic acid.3 This means
that on the cell membrane both steroid and peptide
hormones have their receptors. All of those observations
encouraged us to synthesize the linkers consisting of
steroid and kyotophin or GHRPs, which exhibited
analgesic or prevented osteoporosis effects, to simulate
the ‘permissive action’ via the chemical combination of
steroids and peptides.4;5 The linkers of steroids and
peptides indeed exhibited interesting biological phe-
nomenon and provided a number of possible modifica-
tions either for peptides or for steroids, which obviously
provides the useful ways to find the lead compounds.
2. Results
2.1. Solution method and stepwise synthesis giving
urotoxins in 78–89% total yields
According to Scheme 1 the protective intermediate Boc-
Glu(OBzl)-Asp(OBzl)-Gly-OEt was prepared via the
solution method and stepwise synthesis (from C-termi-
nal to N-terminal) with Gly-OEt as the starting material
in 76% total yield. After removal of ethyl ester group the
resulted Boc-Glu(OBzl)-Asp(OBzl)-Gly-OH was depro-
tected using the standard procedure in the presence of
TFMSA/TFA to provide Glu-Asp-Gly-OH (1) in 89%
yield. With the substantially similar stepwise synthesis
(from C to N terminal in 78–81% total yield) depicted in
Keywords: Hydrocortisone–urotoxin; Prednisolone–urotoxin; Synthe-
sis; Immunosuppressive activity.
* Corresponding author. Tel.: +86-10-8280-2482; fax: +86-10-8280-
2311; e-mail: sqpeng@mail.bjmu.edu.cn
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.06.012

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C. Wang et al. / Bioorg. Med. Chem. 12 (2004) 4403–4421
Glu-Asp-Gly-OH
Boc-Glu(Bzl)-Asp(Bzl)-Gly-OBzl
Gly-OBzl
1
Scheme 1. Preparation of Glu-Asp-Gly–OH. (1) General procedure for coupling of C-terminal and N-terminal components: The solution of
1.5 mmol of the N-terminal component, 1.5 mmol of HOBt and 1.9 mmol of DCC in 15 mL of anhydrous THF was stirred at 0 °C for 24 h. After the
addition of 1.5 mmol of C-terminal component and 1.9 mmol of N-methylmorpholine the solution was stirred at 0 °C for another 24 h. (2). General
procedure for removal of Boc of the C-terminal component: 1.5 mmol of Boc protected compound were treated with 8 mL of hydrogen chloride in
ethyl acetate (4 mol/L) at room temperature for 3 h. (3) General procedure for removal of all the semi-permanent groups: The solution of 1.0 mmol of
protected peptide were treated with 6 mL of dimethyl sulfide, 6 mL of phenyl methyl ether, and 10 mL of CF3COOH–CF3SO3H (4:1) at 0 °C for 2 h.
A
H-AA1-OBzl
Boc-His(Tos)-Gly-AA1-OBzl
B
Boc-His(Tos)-Gly-Lys(Z)-NH-NH2
B
His-Gly-Lys-NH-NH2
His-Gly-AA-OH
3
2a,b
Scheme 2. Preparation of His-Gly-Glu-OH, His-Gly-Lys-OH, and His-Gly-Lys-NHNH2. The general procedure for coupling of C-terminal and N-
terminal components, general procedure for removal of Boc of the C-terminal component, and general procedure for removal of all the semi-
permanent groups were the same as those described in Scheme 1; (A) 1 N NaOH/MeOH; NH2NH2/MeOH; (B) TFMSA/TFA. Wherein AA1 ¼
Glu(OBzl) and Lys(Z); in 2a, AA ¼ Glu; in 2b AA ¼ Lys.
Scheme 2 His-Gly-Glu-OH (2a), His-Gly-Lys-OH (2b),
and His-Gly-Lys-NHNH2 (3) were obtained.
sulted in the corresponding hydrocortisone-21-O-b-car-
bonylpropionic acid p-nitrophenol ester (6a), and
prednisolone-21-O-b-carbonylpropionic acid p-nitro-
phenol ester (6b) in 95% and 90% yield, respectively. The
amidation of 6a,b with urotoxin 1 provided the corre-
2.2. Introducing urotoxin into 21-O of the steroid via
carbonylpropionyl in 75-80% yield
sponding
hydrocortisone-21-O-b-carbonylpropionyl-
Glu-Asp-Gly-OH (7a) and prednisolone-21-O-b-car-
bonylpropionyl-Glu-Asp-Gly-OH (7b) in 50% and 52%
yield, respectively. The amidation of 6a,b with urotoxin
2a provided the corresponding hydrocortisone-21-O-b-
carbonylpropionyl-His-Gly-Glu-OH (7c) and predniso-
lone-21-O-b-carbonylpropionyl-His-Gly-Glu-OH (7d)
in 48% and 46% yield, respectively.
As indicated in Scheme 3 in the presence of pyridine
hydrocortisone and prednisolone were treated with
succinic anhydride to give hydrocortisone-21-O-b-car-
bonylpropionic acid (5a) and prednisolone-21-O-b-car-
bonylpropionic acid (5b) in 98% and 92% yield,
respectively. The reaction of p-nitrophenol and 5a,b re-
The amidation of 6a and 2b gave the mono-steroid
substituted peptide hydrocortisone-21-O-b-carbonyl-
propionyl-His-Gly-Lys-OH (8a in 45% yield) and the
bis-steroid substituted peptide hydrocortisone-His-Gly-
Lys(hydrocortisone)-OH (8b 21% yield) simultaneously.
The amidation of 6b and 2b gave also the mono-steroid
substituted peptide prednisolone-21-O-b-carbonylpro-
pionyl-His-Gly-Lys-OH (8c in 42% yield) and the
bis-steroid substituted peptide prednisolone-His-Gly-
Lys(prednisolone)-OH (8d in 23% yield) simultaneously
(Scheme 4).
CH2
CH2
OH
OR1
OH
O
O
C
C
R
R
OH
HO
HO
R
R
A
CH2
OR2
OH
CH2
O
B
OR3
O
4a,b
5a,b
O
C
O
C
R
R
HO
OH
HO
R
R
In the presence of dicyclohexylcabodiimide (DCC) and
N-hydroxysuccinimide (HOSu) 5a,b were converted into
hydrocortisone-21-O-b-carbonylpropionic acid HOSu
ester (6c) and prednisolone-21-O-b-carbonylpropionic
acid HOSu ester (6d), which were treated with urotoxin
1 and 2a in situ to provide the corresponding hydro-
cortisone-21-O-b-carbonylpropionyl-Glu-Asp-Gly-OH
(7a), prednisolone-21-O-b-carbonylpropionyl-Glu-Asp-
Gly-OH (7b), hydrocortisone-21-O-b-carbonylpropio-
nyl-His-Gly-Glu-OH (7c), and prednisolone-21-O-b-
carbonylpropionyl-His-Gly-Glu-OH (7d) in 79%, 75%,
80%, and 77% yield, respectively. The amidation of
hydrocortisone-21-O-b-carbonylpropionic acid HOSu
ester (6c) and 2b gave the mono-steroid substituted
C
O
O
6a-d
7a-d
Scheme 3. Preparation of hydrocortisone–urotoxin tripeptide and
prednisolone–urotoxin tripeptide. The general procedure for coupling
of C-terminal and N-terminal components was the same as that de-
scribed in Scheme 1; (A) succinic anhydride/pyridine; (B) DCC/HOSu/
HONp/THF; (C) Glu-Asp-Gly-OH (1) or His-Gly-Glu-OH (2a) in
DMF/H2O; in 4–6a,c, and 7a,c both of R ¼ H and the steroid moi-
ety ¼ hydrocortisone; in 4–6b,d, and 7b,d both of R together constitute
an olefinic bond and the steroid moiety ¼ prednisolone; in 5a,b
R1 ¼ COCH2CH2COOH; in 6a,b R2 ¼ COCH2CH2COO(C6H4-p-
NO2); in 6c,d R2 ¼ COCH2CH2COOSu; in 7a,c R3 ¼ COCH2CH2CO-
Glu-Asp-Gly-OH; in 7b,d R3 ¼ COCH2CH2CO-His-Gly-Glu-OH.

C. Wang et al. / Bioorg. Med. Chem. 12 (2004) 4403–4421
4405
CH2
OR1
OH
CH2
OCO(CH)2COHis-Gly-Lys-OH
OH
O
O
C
C
R
R
HO
HO
R
R
A
O
O
6a,b
8a,c
+
CH2
OCO(CH)2COHis-Gly-Lys-OH
CO(CH2)2COOCH2
O
C
R
O
C
OH
HO
R
OH
HO
R
R
O
O
8b,d
Scheme 4. Preparation of hydrocortisone–His-Gly-Lys-OH, prednisolone–His-Gly-Lys-OH, hydrocortisone–His-Gly-Lys(hydrocortisone)-OH, and
prednisolone–His-Gly-Lys(prednisolone)-OH. The general procedure for coupling of C-terminal and N-terminal components was the same as that
described in Scheme 1; (A) His-Gly-Lys-OH (2b) in DMF/H2O. In 8a,b both of R ¼ H and the steroid moiety ¼ hydrocortisone; in 8c,d both of R
together constitute an olefinic bond and the steroid moiety ¼ prednisolone.
peptide hydrocortisone-21-O-b-carbonylpropionyl-His-
Gly-Lys-OH (8a in 50% yield) and bis-steroid substi-
tuted peptide hydrocortisone-21-O-b-carbonylpropio-
nyl-His-Gly-Lys(hydrocortisone)-OH (8b in 20% yield)
simultaneously. The amidation of prednisolone-21-O-b-
carbonylpropionic acid HOSu ester (6d) and 2b gave
also the mono-steroid substituted peptide prednisolone-
21-O-b-carbonylpropionyl-His-Gly-Lys-OH (8c in 52%
yield) and bis-steroid substituted peptide prednisolone-
His-Gly-Lys(prednisolone)-OH (8d in 21% yield)
simultaneously. The amidation of hydrocortisone-21-O-
b-carbonylpropionic acid HOSu ester (6c) and prednis-
olone-21-O-b-carbonylpropionic acid HOSu ester (6d)
with mono-steroid substituted peptide hydrocortisone-
21-O-b-carbonylpropionyl-His-Gly-Lys-OH (8a) and
prednisolone-21-O-b-carbonylpropionyl-His-Gly-Lys-
OH (8b) gave the corresponding mixed bis-steroid
substituted peptide hydrocortisone-His-Gly-Lys(pred-
2.3. Introducing urotoxin into 3-position of the steroid via
hydrazine in 70–80% yield
In the presence of glacial acetic acid with methanol as
the reaction solvent the selective condensation of the
hydrazine group in Boc-His(Tos)-Gly-Lys(Z)-NHNH2
and His-Gly-Lys-NHNH2 (3) with the 3-carbonyl group
in hydrocortisone and prednisolone provided the 3-
[Boc-His(Tos)-Gly-Lys(Z)-NHN]-substituted hydrocor-
tisone (11a) and 3-[Boc-His(Tos)-Gly-Lys(Z)-NHN]-
substituted prednisolone (11b), 3-(His-Gly-Lys-NHN)-
substituted hydrocortisone (10a), and 3-(His-Gly-Lys-
NHN)-substituted prednisolone (10b) in 80%, 77%,
75%, and 70% yield, respectively. When inorganic acid,
for instance HCl or H2SO4, was used instead of glacial
acetic acid as the catalyst the yield of 10a,b, and 11a,b
was lowed to 15–40%. This dependence may mean that
the protonation of hydrazine group in 3 or Boc-His-
(Tos)-Gly-Lys(Z)-NHNH2 has a negative effect on the
condensation reaction. Using the standard deprotected
procedure in the presence of trifluoromethylsulfonic
nisolone)-OH
Lys(hydrocortisone)-OH (9b) in 78% and 74% yield,
(9a)
and
prednisolone-His-Gly-
respectively (Scheme 5).
CH
OCO(CH) COHis-Gly-Lys-OH
2
2
CH
OCO(CH) COHis-Gly-Lys-OH
2
2
O
CO(CH ) COOCH
C
2 2
2
O
C
R
R
OH
O
C
HO
OH
HO
R1
R
OH
HO
R
6c,d
R1
O
O
O
9a,b
Scheme 5. Preparation of hydrocortisone–His-Gly-Lys(prednisolone)-OH, and prednisolone–His-Gly-Lys(hydrocortisone)-OH. The general pro-
cedure for coupling of C-terminal and N-terminal components was the same as that described in Scheme 1; In 9a both of R ¼ H and the steroid
moiety ¼ hydrocortisone, and both of R1 together constituted an olefinic bond and the steroid moiety ¼ prednisolone; in 9b both of R together
constituted an olefinic bond and the steroid moiety ¼ prednisolone, and both of R ¼ H and the steroid moiety ¼ hydrocortisone.

4406
C. Wang et al. / Bioorg. Med. Chem. 12 (2004) 4403–4421
acid/trifluoroacetic acid (TFMSA/TFA) 11a,b may be
converted into 10a,b in moderate yields (Scheme 6).
microplate with the controls and the synthetic steroid–
urotoxin using the standard procedure.11–13 After 24 h of
incubation and the peritoneal macrophages being acti-
vated to the residue in the cultured well a solution of
neutral red-stained zymosan was added and the neutral
red-stained zymosan phagocytosis of mouse peritoneal
macrophages was reflected by the OD value. The OD
value of the incubation solution was read at 570 nm with
Biorad. The inhibitory effect of the synthetic steroid–
urotoxins on the phagocytosis of mouse peritoneal
macrophages was then represented by the tested OD
value. The data are listed in Tables 5 and 6, and the
statistical analysis of the data are carried out by use of
ANOVA test, p < 0:05 is considered significant. The
results indicated that in the presence of the synthetic
steroid–urotoxins the phagocytosis of mouse peritoneal
macrophages were significantly inhibited (comparing to
hydrocortisone, prednisolone, urotoxin, or the mixture
of hydrocortisone or prednisolone and urotoxin,
P < 0:05 or P < 0:001).
2.4. The steroid–urotoxin significantly inhibiting ConA
and LPS induced lymphocyte proliferation
The spleen cells of BALB/C mice were collected
according to the common method and incubated in a 96-
well microplate with the controls and the synthetic ste-
roid–urotoxin using the standard procedure.9;10 The
inhibitory effect of the synthetic steroid–urotoxins on
ConA or LPS induced proliferation of BALB/C mouse
spleen lymphocytes was evaluated. Based on the
tested optical density (OD) value and the equation,
‘Inhibitory rate ¼ ðODdrug ꢀ ODConA or LPSÞ=ðODConA or
ODLPS ꢀ ODNSÞ’ [wherein ODdrug represents the OD
value derived from the lymphocytes treated with pep-
tide, steroid, or steroid–peptide; ODConA or ODLPS rep-
resents the OD value derived from the lymphocytes
treated with concanavalin A (ConA) or lipopolysac-
charide (LPS); ODNS represents the OD value derived
from the lymphocytes treated with normal saline (NS)],
the inhibitory rate of the synthetic steroid–urotoxin was
calculated. The data are listed in Tables 1–4 and the
statistical analysis of the data is carried out by use of
ANOVA test, p < 0:05 is considered significant. The
results indicated that in the presence of the synthetic
steroid–urotoxins the ConA and LPS induced prolifer-
ations of BALB/C mouse spleen lymphocytes were sig-
nificantly inhibited (comparing to hydrocortisone,
prednisolone, urotoxin, or the mixture of hydrocorti-
sone or prednisolone and urotoxin, P < 0:05, P < 0:01,
or P < 0:001).
2.6. The steroid–urotoxin significantly lengthening the
survival time of the split heart
The split hearts (3 ꢁ 3 mm in size) were obtained from
C57bl/6 mice (ranging in age from near full term fetuses
up to 48 h old) according to the general method and
transplanted into the base of the ‘pocket’ near the
distal edge of the ear of BALB/C mice (male, ranged in
age from 12–14 weeks at the time of transplanta-
tion).14;15 One day after transplantation the mice were
given an injection (ip) of 0.1 mL of DMSO/H2O (1:4, v/
v), of corresponding dosage of hydrocortisone in
0.1 mL of DMSO/H2O (1:4, v/v), and of corresponding
dosage of hydrocortisone–urotoxin in 0.1 mL of
DMSO/H2O (1:4, v/v) per day until the electrocardio-
grams of the grafts disappeared. Six days after trans-
plantation the animals were anesthetized with sodium
pentobarbital at a dosage of 75 lg/g body weight and
the electrocardiograms of the grafts were recorded
2.5. The steroid–urotoxin significantly inhibiting phago-
cytosis of mouse peritoneal macrophages
The peritoneal macrophages were harvested according
to the general method and incubated in a 96-well
CH
OH
2
CH
OH
2
O
C
O
C
R
OH
HO
R
OH
HO
R
R
+
,
H
BocHis(Yos)-Gly-Lys(Z)-NHNH2
BocHis(Yos)-Gly-Lys(Z)-NHN
CH OH
O
11a,b
4a,c
2
O
C
H+
His-Gly-Lys-NHNH2
R1
TFMSA
TFA
OH
HO
R1
His-Gly-Lys-NHN
10a,b
Scheme 6. Preparation of His-Gly-Lys-NHN@hydrocortisone and His-Gly-Lys-NH-N@prednisolone. In 10a and 11a both of R ¼ H and the steroid
moiety ¼ hydrocortisone; in 10b and 11b both of R together constituteed an olefinic bond and the steroid moiety ¼ prednisolone.

C. Wang et al. / Bioorg. Med. Chem. 12 (2004) 4403–4421
4407
Table 1. Effect of hydrocortisone–urotoxin on ConA induced spleen lymphocyte proliferation (XꢂSD%)
Compound
10ꢀ8 mol/L
10ꢀ7 mol/L
10ꢀ6 mol/L
10ꢀ5 mol/L
10ꢀ4 mol/L
4a
6.3 ꢂ 5.1
14.7 ꢂ 4.3
5.0 ꢂ 5.3
36.0 ꢂ 5.2
21.7 ꢂ 4.1
22.4 ꢂ 5.3
34.5 ꢂ 3.5
20.6 ꢂ 4.2
30.0 ꢂ 6.2
28.4 ꢂ 6.1
32.9 ꢂ 5.3
24.5 ꢂ 5.6
49.2 ꢂ 3.5c;d
51.2 ꢂ 3.1c;f
31.4 ꢂ 5.1
49.0 ꢂ 5.0b;g
41.6 ꢂ 6.2a;i
40.0 ꢂ 5.4a;i
56.4 ꢂ 4.2
32.8 ꢂ 4.9
35.9 ꢂ 6.3
37.4 ꢂ 5.6
33.7 ꢂ 5.4
46.4 ꢂ 7.2
40.9 ꢂ 5.8
42.1 ꢂ 6.9
38.7 ꢂ 6.2
67.9 ꢂ 6.0b;d
77.9 ꢂ 5.7c;f
68.6 ꢂ 5.0b;g
67.1 ꢂ 4.9b;g
73.0 ꢂ 6.9b;i
71.8 ꢂ 7.1b;i
70.6 ꢂ 4.6
55.1 ꢂ 6.1
55.7 ꢂ 4.2
62.4 ꢂ 4.1
54.9 ꢂ 5.0
60.6 ꢂ 6.6
60.1 ꢂ 7.3
65.5 ꢂ 7.1
62.9 ꢂ 7.0
78.5 ꢂ 6.0a;d
77.7 ꢂ 5.5a;f
79.8 ꢂ 5.6a;g
78.9 ꢂ 4.9a;g
79.7 ꢂ 5.8a;i
78.3 ꢂ 4.4a;i
72.9 ꢂ 5.5
64.0 ꢂ 6.8
65.2 ꢂ 7.2
64.3 ꢂ 8.2
63.8 ꢂ 6.9
64.9 ꢂ 4.5
67.0 ꢂ 6.9
66.4 ꢂ 7.9
65.6 ꢂ 7.3
88.8 ꢂ 5.4c;d
77.0 ꢂ 5.7e
89.2 ꢂ 6.1c;g
90.1 ꢂ 6.0c;g
81.3 ꢂ 6.5h
79.2 ꢂ 5.9h
1
2a
2b
3
8.8 ꢂ 4.2
4.9 ꢂ 4.3
4aþ1
4aþ2a
4aþ2b
4aþ3
7a
16.9 ꢂ 4.8
7.3 ꢂ 6.1
10.8 ꢂ 5.4
5.8 ꢂ 5.3
36.2 ꢂ 4.0c;d
37.8 ꢂ 3.6c;f
29.3 ꢂ 5.9c;g
29.9 ꢂ 5.5c;g
31.7 ꢂ 4.8c;i
30.7 ꢂ 4.6c;i
7b
8a
8b
9a
10a
n ¼ 6.
a Compare to 4a, P < 0:05.
b Compare to 4a, P < 0:01.
c Compare to 4a, P < 0:001.
d Compare to 1 and 4aþ1, P < 0:001.
e Compare to 2a and 4aþ2a, P < 0:01.
f Compare to 2a and 4aþ2a, P < 0:001.
g Compare to 2b and 4aþ2b, P < 0:001.
h Compare to 3 and 4aþ3, P < 0:01.
i Compare to 3 and 4aþ3, P < 0:001.
Table 2. Effect of prednisolone–urotoxin on ConA induced spleen lymphocyte proliferation (XꢂSD%)
Compound
1/4 ꢁ 10ꢀ8 mol/L
1/4 ꢁ 10ꢀ7 mol/L/4
1/4 ꢁ 10ꢀ6 mol/L
1/4 ꢁ 10ꢀ5 mol/L
1/4 ꢁ 10ꢀ4 mol/L
4b
6.0 ꢂ 4.9
14.7 ꢂ 4.3
5.0 ꢂ 5.3
34.1 ꢂ 6.1
21.7 ꢂ 4.1
22.45.3
52.3 ꢂ 5.2
32.8 ꢂ 4.9
35.9 ꢂ 6.3
37.4 ꢂ 5.6
33.7 ꢂ 5.4
44.9 ꢂ 6.6
39.6 ꢂ 5.4
41.7 ꢂ 5.6
39.0 ꢂ 5.7
68.2 ꢂ 5.9b;d
78.4 ꢂ 6.0c;f
69.1 ꢂ 5.3b;g
66.9 ꢂ 4.7b;g
72.7 ꢂ 6.6b;i
72.2 ꢂ 6.8b;i
68.9 ꢂ 5.7
55.1 ꢂ 6.1
55.7 ꢂ 4.2
62.4 ꢂ 4.1
54.9 ꢂ 5.0
59.4 ꢂ 5.3
59.8 ꢂ 6.8
64.7 ꢂ 6.9
63.2 ꢂ 6.9
77.9 ꢂ 6.2a;d
78.3 ꢂ 5.7a;f
80.0 ꢂ 5.7a;g
78.1 ꢂ 4.8a;g
80.3 ꢂ 6.1a;i
77.8 ꢂ 4.6a;i
75.0 ꢂ 6.4
64.0 ꢂ 6.8
65.2 ꢂ 7.2
64.3 ꢂ 8.2
63.8 ꢂ 6.9
63.8 ꢂ 4.3
66.8 ꢂ 6.4
65.0 ꢂ 6.7
66.1 ꢂ 6.9
89.1 ꢂ 5.6c;d
76.8 ꢂ 5.5e
88.8 ꢂ 5.7c;g
89.0 ꢂ 5.8c;g
80.9 ꢂ 6.6h
78.9 ꢂ 6.0h
1
2a
2b
3
8.8 ꢂ 4.2
34.5 ꢂ 3.5
20.6 ꢂ 4.2
28.9 ꢂ 5.8
26.9 ꢂ 5.8
33.2 ꢂ 5.6
23.8 ꢂ 5.3
48.7 ꢂ 3.6c;d
50.9 ꢂ 3.4c;f
30.8 ꢂ 4.8
48.8 ꢂ 4.7b;g
42.0 ꢂ 6.3a;i
40.9 ꢂ 5.6a;i
4.9 ꢂ 4.3
4bþ1
4bþ2a
4bþ2b
4bþ3
7c
15.2 ꢂ 4.4
7.1 ꢂ 5.9
11.0 ꢂ 5.6
6.1 ꢂ 5.5
35.9 ꢂ 3.8c;d
38.2 ꢂ 3.8c;f
30.1 ꢂ 6.2c;g
29.5 ꢂ 5.4c;g
32.2 ꢂ 5.1c;i
31.2 ꢂ 4.8c;i
7d
8c
8d
9b
10b
n ¼ 6.
a Compare to 4b, P < 0:05.
b Compare to 4b, P < 0:01.
c Compare to 4b, P < 0:001.
d Compare to 1 and 4bþ1, P < 0:001.
e Compare to 2a and 4bþ2a, P < 0:01.
f Compare to 2a and 4bþ2a, P < 0:001.
g Compare to 2b and 4bþ2b, P < 0:001.
h Compare to 3 and 4bþ3, P < 0:01.
i Compare to 3 and 4bþ3, P < 0:001.
using the physiography. The interval between the tissue
transplantation and the disappearance of pulsatile
activity was as defined the survival time of the graft.
The data are listed in Tables 7 and 8, and the statistical
analysis of the data are carried out by use of ANOVA
test, p < 0:05 is considered significant. The results
indicated that after the administration of hydrocorti-
sone–urotoxin and prednisolone-urotoxin the survival
time of the grafts was significantly lengthened (com-
paring to hydrocortisone, prednisolone, urotoxin, or
the mixture of hydrocortisone or prednisolone and
urotoxin, P < 0:01, or P < 0:001).

4408
C. Wang et al. / Bioorg. Med. Chem. 12 (2004) 4403–4421
Table 3. Effect of hydrocortisone–urotoxin on LPS induced spleen lymphocyte proliferation (XꢂSD%)
Compound
10ꢀ8 mol/L
10ꢀ7 mol/L
10ꢀ6 mol/L
10ꢀ5 mol/L
10ꢀ4 mol/L
4a
10.3 ꢂ 6.4
4.7 ꢂ 4.3
35.7 ꢂ 6.6
21.7 ꢂ 4.1
ꢀ5.0 ꢂ 8.1
ꢀ5.4 ꢂ 3.0
19.3 ꢂ 4.0
27.1 ꢂ 8.2
10.1 ꢂ 7.8
6.8 ꢂ 5.4
51.6 ꢂ 6.2
32.8 ꢂ 4.9
60.9 ꢂ 5.3
45.1 ꢂ 6.1
66.8 ꢂ 5.6
54.0 ꢂ 6.8
1
2a
ꢀ7.5 ꢂ 7.0
ꢀ2.5 ꢂ 4.2
5.9 ꢂ 4.3
ꢀ15.9 ꢂ 16.6
15.6 ꢂ 2.4
ꢀ12.1 ꢂ 13.4
23.3 ꢂ 7.0
ꢀ15.8 ꢂ 17.5
33.4 ꢂ 9.1
2b
3
33.2 ꢂ 5.3
44.8 ꢂ 5.1
53.7 ꢂ 6.8
4aþ1
4aþ2a
4aþ2b
4aþ3
7a
7.4 ꢂ 5.5
39.9 ꢂ 8.8
54.3 ꢂ 6.9
60.2 ꢂ 8.6
6.9 ꢂ 5.5
5.9 ꢂ 6.9
7.1 ꢂ 6.0
5.8 ꢂ 7.5
6.5 ꢂ 4.8
25.0 ꢂ 9.8
43.3 ꢂ 17.9
54.0 ꢂ 9.9
43.4 ꢂ 19.6
62.2 ꢂ 9.9
8.8 ꢂ 7.5
26.8 ꢂ 8.2
49.2 ꢂ 3.5b;d
43.5 ꢂ 6.7e
47.4 ꢂ 2.4b;f
44.0 ꢂ 5.0a;f
46.0 ꢂ 4.6a;g
44.7 ꢂ 3.7a;g
43.2 ꢂ 15.1
65.9 ꢂ 6.0b;d
64.2 ꢂ 5.9b;e
58.5 ꢂ 3.8a;f
65.1 ꢂ 4.9b;f
66.3 ꢂ 4.9b;g
64.9 ꢂ 5.3b;g
36.2 ꢂ 4.0c;d
33.9 ꢂ 10.7c;e
17.5 ꢂ 5.1a;f
29.9 ꢂ 5.5c;f
32.7 ꢂ 9.2c;g
31.3 ꢂ 9.5c;g
72.5 ꢂ 6.0b;d
70.9 ꢂ 6.1a;e
83.2 ꢂ 12.5b;f
72.9 ꢂ 5.9b;f
74.8 ꢂ 5.6b;g
73.2 ꢂ 5.1b;g
78.8 ꢂ 5.4b;d
64.5 ꢂ 2.6e
77.3 ꢂ 6.6a;f
80.1 ꢂ 6.0b;f
81.4 ꢂ 5.7b;g
79.2 ꢂ 6.5b;g
7b
8a
8b
9a
10a
n ¼ 6.
a Compare to hydrocortisone, P < 0:05.
b Compare to hydrocortisone, P < 0:01.
c Compare to hydrocortisone, P < 0:001.
d Compare to 1 and hydrocortisoneþ1, P < 0:001.
e Compare to 2a and hydrocortisoneþ2a, P < 0:001.
f Compare to 2b and hydrocortisoneþ2b, P < 0:001.
g Compare to 3 and hydrocortisoneþ3, P < 0:001; minus quantity means enhanced action.
Table 4. Effect of prednisolone–urotoxin on LPS induced spleen lymphocyte proliferation (X ꢂ SD%)
Compound
1/4 ꢁ 10ꢀ8 mol/L
1/4 ꢁ 10ꢀ7 mol/L
1/4 ꢁ 10ꢀ6 mol/L
1/4 ꢁ 10ꢀ5 mol/L
1/4 ꢁ 10ꢀ4 mol/L
4b
11.1 ꢂ 6.2
36.2 ꢂ 6.4
22.2 ꢂ 4.4
ꢀ5.8 ꢂ 7.4
ꢀ6.2 ꢂ 3.6
18.7 ꢂ 4.3
27.8 ꢂ 7.6
10.6 ꢂ 7.6
6.9 ꢂ 5.0
52.1 ꢂ 6.6
61.4 ꢂ 5.6
44.8 ꢂ 5.8
ꢀ12.8 ꢂ 13.1
22.7 ꢂ 6.8
45.2 ꢂ 5.3
53.9 ꢂ 6.6
7.7 ꢂ 5.9
66.1 ꢂ 5.4
1
2a
5.1 ꢂ 4.5
33.3 ꢂ 5.1
53.7 ꢂ 6.3
ꢀ7.2 ꢂ 6.7
ꢀ4.2 ꢂ 3.9
6.3 ꢂ 4.5
ꢀ16.5 ꢂ 16.0
14.9 ꢂ 2.6
)16.4 ꢂ 16.9
32.9 ꢂ 8.7
2b
3
34.5 ꢂ 5.4
54.5 ꢂ 6.6
4bþ1
4bþ2a
4bþ2b
4bþ3
7c
7.2 ꢂ 5.2
40.2 ꢂ 8.5
59.9 ꢂ 7.9
7.0 ꢂ 5.4
6.1 ꢂ 6.0
6.7 ꢂ 7.1
6.9 ꢂ 4.6
24.8 ꢂ 7.6
42.8 ꢂ 16.8
53.6 ꢂ 9.3
73.0 ꢂ 5.8b;d
42.6 ꢂ 18.1
63.0 ꢂ 9.0
9.2 ꢂ 7.2
27.1 ꢂ 7.9
48.8 ꢂ 3.3b;d
44.4 ꢂ 6.5e
46.9 ꢂ 2.5b;f
44.6 ꢂ 5.2a;f
45.8 ꢂ 4.4a;g
45.5 ꢂ 3.6a;g
42.8 ꢂ 14.6
65.1 ꢂ 5.6b;d
63.9 ꢂ 6.0b;e
59.3 ꢂ 3.9a;f
64.7 ꢂ 4.6b;f
67.0 ꢂ 4.6b;g
64.4 ꢂ 5.0b;g
35.7 ꢂ 4.2c;d
34.4 ꢂ 9.5c;e
18.1 ꢂ 5.3a;f
30.3 ꢂ 5.4c;f
33.2 ꢂ 8.8c;g
32.2 ꢂ 9.3c;g
78.0 ꢂ 5.2b;d
63.8 ꢂ 3.1e
78.0 ꢂ 6.4a;f
79.9 ꢂ 5.9b;f
81.0 ꢂ 5.6b;g
78.8 ꢂ 6.2b;g
;e
7d
8c
71.4 ꢂ 5.8a
82.7 ꢂ 11.7b;f
73.4 ꢂ 5.7b;f
75.4 ꢂ 5.5b;g
72.6 ꢂ 5.0b;g
8d
9b
10b
n ¼ 6.
a Compare to 4b, P < 0:05.
b Compare to 4b, P < 0:01.
c Compare to 4b, P < 0:001.
d Compare to 1 and 4bþ1, P < 0:001.
e Compare to 2a and 4bþ2a, P < 0:001.
f Compare to 2b and 4bþ2b, P < 0:001.
g Compare to 3 and 4bþ3, P < 0:001; minus quantity means enhanced action.
3. Discussion
intermediates were converted into urotoxins in satisfac-
tory yield. The amidation of hydrocortisone-21-O-b-
carbonylpropionic acid p-nitrophenol ester and pred-
nisolone-21-O-b-carbonylpropionic acid p-nitrophenol
ester with urotoxins provided the desirable linkers in
moderate yield. If hydrocortisone-21-O-b-carbonyl-
propionic acid HOSu ester and prednisolone-21-O-b-
carbonylpropionic acid HOSu ester were used instead of
hydrocortisone-21-O-b-carbonylpropionic acid p-nitro-
3.1. The amidation of urotoxins preferring the HOSu
ester to the p-nitrophenol ester
Using the solution method via stepwise synthesis (from
C-terminal to N-terminal) the protective intermediates of
urotoxins were smoothly obtained in relatively high
yield. In the solution of TFMSA/TFA the protective

C. Wang et al. / Bioorg. Med. Chem. 12 (2004) 4403–4421
4409
Table 5. The phagocytosis of mouse peritoneal macrophages after treatment of hydrocortisone–urotoxin (X ꢂ SD)
Compound
10ꢀ9 mol/L
10ꢀ8 mol/L
10ꢀ7 mol/L
10ꢀ6 mol/L
10ꢀ5 mol/L
NS
4a
0.78 ꢂ 0.04
0.66 ꢂ 0.03a
0.80 ꢂ 0.03
0.74 ꢂ 0.02
0.81 ꢂ 0.03
0.76 ꢂ 0.03
0.78 ꢂ 0.04
0.73 ꢂ 0.03
0.80 ꢂ 0.04
0.75 ꢂ 0.04
0.58 ꢂ 0.03c;f ;g
0.55 ꢂ 0.03c;f ;h
0.68 ꢂ 0.04a;i
0.64 ꢂ 0.04c;i
0.60 ꢂ 0.04c;j
0.65 ꢂ 0.03c;j
0.56 ꢂ 0.04c
0.67 ꢂ 0.02a
0.65 ꢂ 0.05
0.53 ꢂ 0.05c
0.63 ꢂ 0.03a
0.63 ꢂ 0.03a
0.62 ꢂ 0.05a
0.64 ꢂ 0.03a
0.62 ꢂ 0.04a
0.64 ꢂ 0.03a
0.63 ꢂ 0.03a
0.62 ꢂ 0.04a
0.44 ꢂ 0.02c;f ;g
0.41 ꢂ 0.02c;f ;h
0.45 ꢂ 0.03c;f ;i
0.43 ꢂ 0.03c;f ;i
0.42 ꢂ 0.04c;f ;j
0.44 ꢂ 0.03c;f ;j
0.50 ꢂ 0.06c
0.63 ꢂ 0.04a
0.64 ꢂ 0.04a
0.55 ꢂ 0.04c
0.64 ꢂ 0.04a
0.62 ꢂ 0.05a
0.63 ꢂ 0.05a
0.54 ꢂ 0.03c
0.63 ꢂ 0.04a
0.48 ꢂ 0.03c;g
0.45 ꢂ 0.05c;d;h
0.40 ꢂ 0.05c;f ;i
0.40 ꢂ 0.03c;f ;i
0.42 ꢂ 0.04c;d;j
0.44 ꢂ 0.05c;d;j
0.46 ꢂ 0.03c
0.60 ꢂ 0.03b
0.59 ꢂ 0.03c
0.54 ꢂ 0.03c
0.61 ꢂ 0.03b
0.61 ꢂ 0.04b
0.58 ꢂ 0.03c
0.55 ꢂ 0.02c
0.60 ꢂ 0.03b
0.43 ꢂ 0.03c;d;c
0.38 ꢂ 0.04c;f ;h
0.41 ꢂ 0.03c;f ;i
0.40 ꢂ 0.02c;f ;i
0.40 ꢂ 0.03c;e;j
0.41 ꢂ 0.04c;e;j
1
2a
2b
3
0.66 ꢂ 0.02a
0.66 ꢂ 0.04
4aþ1
4aþ2a
4aþ2b
4aþ3
7a
0.66 ꢂ 0.03a
0.63 ꢂ 0.06
0.65 ꢂ 0.02a
0.65 ꢂ 0.04
0.53 ꢂ 0.04c;g
0.52 ꢂ 0.04c;d;h
0.52 ꢂ 0.02a;e;i
0.51 ꢂ 0.02c;f ;i
0.51 ꢂ 0.03c;j
0.53 ꢂ 0.04c;j
7b
8a
8b
9a
10a
n ¼ 12; NS ¼ vehicle.
a Compared to NS, P < 0:05.
b Compared to NS, P < 0:01.
c Compared to NS, P < 0:001.
d Compared to 4a, P < 0:05.
e Compared to 4a, P < 0:01.
f Compared to 4a, P < 0:001.
g Compared to 1 and 4aþ1, P < 0:001.
h Compared to 2a and 4aþ2a, P < 0:001.
i Compared to 2b and 4aþ2b, P < 0:001.
j Compared to 3 and 4aþ3, P < 0:001.
Table 6. The phagocytosis of mouse peritoneal macrophages after treatment of prednisolone-urotoxin (X ꢂ SD)
Compound
1/4 ꢁ 10ꢀ9 mol/L
1/4 ꢁ 10ꢀ8 mol/L
1/4 ꢁ 10ꢀ7 mol/L
1/4 ꢁ 10ꢀ6 mol/L
1/4 ꢁ 10ꢀ5 mol/L
NS
4b
0.78 ꢂ 0.04
0.64 ꢂ 0.03a
0.80 ꢂ 0.03
0.74 ꢂ 0.02
0.81 ꢂ 0.03
0.76 ꢂ 0.03
0.80 ꢂ 0.05
0.74 ꢂ 0.04
0.79 ꢂ 0.05
0.77 ꢂ 0.04
0.57 ꢂ 0.03c;f ;g
0.56 ꢂ 0.03c;f ;h
0.66 ꢂ 0.04a;i
0.65 ꢂ 0.04c;i
0.62 ꢂ 0.03c;j
0.63 ꢂ 0.03c;j
0.58 ꢂ 0.05c
0.67 ꢂ 0.02a
0.65 ꢂ 0.05
0.56 ꢂ 0.04c
0.63 ꢂ 0.03a
0.63 ꢂ 0.03a
0.62 ꢂ 0.05a
0.64 ꢂ 0.03a
0.66 ꢂ 0.05a
0.66 ꢂ 0.04a
0.65 ꢂ 0.03a
0.64 ꢂ 0.04a
0.45 ꢂ 0.02c;f ;g
0.42 ꢂ 0.02c;f ;h
0.44 ꢂ 0.03c;f ;i
0.44 ꢂ 0.03c;f ;i
0.43 ꢂ 0.03c;f ;j
0.43 ꢂ 0.03c;f ;j
0.52 ꢂ 0.05c
0.63 ꢂ 0.04a
0.64 ꢂ 0.04a
0.55 ꢂ 0.04c
0.64 ꢂ 0.04a
0.64 ꢂ 0.05a
0.65 ꢂ 0.05a
0.55 ꢂ 0.03c
0.64 ꢂ 0.04a
0.46 ꢂ 0.03c;g
0.44 ꢂ 0.04c;d;h
0.40 ꢂ 0.04c;f ;i
0.41 ꢂ 0.03c;f ;i
0.43 ꢂ 0.04c;d;j
0.43 ꢂ 0.05c;d;j
0.48 ꢂ 0.04c
0.60 ꢂ 0.03b
0.59 ꢂ 0.03c
0.54 ꢂ 0.03c
0.61 ꢂ 0.03b
0.63 ꢂ 0.04b
0.57 ꢂ 0.04c
0.57 ꢂ 0.03c
0.62 ꢂ 0.03b
0.44 ꢂ 0.03c;d;g
0.37 ꢂ 0.04c;f ;h
0.40 ꢂ 0.03c;f ;i
0.41 ꢂ 0.02c;f ;i
0.41 ꢂ 0.03c;e;j
0.42 ꢂ 0.03c;e;j
1
2a
2b
3
0.66 ꢂ 0.02a
0.66 ꢂ 0.04
4bþ1
4bþ2a
4bþ2b
4bþ3
7c
0.68 ꢂ 0.04a
0.62 ꢂ 0.05
0.67 ꢂ 0.03a
0.66 ꢂ 0.04
0.52 ꢂ 0.03c;g
0.51 ꢂ 0.04c;d;h
0.52 ꢂ 0.02c;e;i
0.50 ꢂ 0.02c;f ;i
0.50 ꢂ 0.03c;j
0.51 ꢂ 0.03c;j
7d
8c
8d
9b
10b
n ¼ 12; NS ¼ vehicle.
a Compared to NS, P < 0:05.
b Compared to NS, P < 0:01.
c Compared to NS, P < 0:001.
d Compared to 4b, P < 0:05.
e Compared to 4b, P < 0:01.
f Compared to 4b, P < 0:001.
g Compared to 1 and 4bþ1, P < 0:001.
h Compared to 2a and 4bþ2a, P < 0:001.
i Compared to 2b and 4bþ2b, P < 0:001.
j Compared to 3 and 4bþ3, P < 0:001.
phenol ester and prednisolone-21-O-b-carbonylprop-
ionic acid p-nitrophenol ester the yield of the amidation
was increased by about 30%. These results indicated that
for the amidation of urotoxins the HOSu ester is pref-
erable to the p-nitrophenol ester.
3.2. The amidation of His-Gly-Lys-OH preferring the a-
amino group of His to the x-amino group of Lys
In principle the a-amino group at His residue and the x-
amino group at Lys residue of His-Gly-Lys-OH may be

4410
C. Wang et al. / Bioorg. Med. Chem. 12 (2004) 4403–4421
Table 7. Survival time of the transplantation grafts after treatment of hydrocortisone–urotoxin (X ꢂ SD, h)
Compound
0.15 (mmol/20 g/d)
0.75 (mmol/20 g/d)
1.50 (mmol/20 g/d)
DMSO/H2O (1:4)
4a
210.9 ꢂ 7.2
235.2 ꢂ 7.7b
220.9 ꢂ 5.7a
222.0 ꢂ 7.8a
225.2 ꢂ 7.3b
221.2 ꢂ 6.7a
225.9 ꢂ 6.7b
226.0 ꢂ 8.8b
227.2 ꢂ 7.5b
223.2 ꢂ 7.1a
245.9 ꢂ 6.7b;c;e
246.1 ꢂ 6.1b;c;f
250.1 ꢂ 6.5b;d;g
247.7 ꢂ 5.7b;d;g
250.5 ꢂ 6.7b;d;h
246.7 ꢂ 6.4b;d;h
250.6 ꢂ 6.1b
280.1 ꢂ 7.2b
1
2a
231.6 ꢂ 6.4b
250.6 ꢂ 7.3b
234.8 ꢂ 7.3b
247.2 ꢂ 6.4b
2b
3
232.6 ꢂ 6.4b
252.1 ꢂ 5.9b
230.6 ꢂ 7.0b
250.1 ꢂ 6.2b
4aþ1
4aþ2a
4aþ2b
4aþ3
7a
232.6 ꢂ 6.6b
252.6 ꢂ 7.5b
235.2 ꢂ 7.1b
250.1 ꢂ 6.5b
234.6 ꢂ 6.6b
254.1 ꢂ 6.4b
232.0 ꢂ 6.9b
252.2 ꢂ 6.0b
270.8 ꢂ 7.4b;d;e
269.6 ꢂ 6.1b;d;f
268.0 ꢂ 5.8b;d;g
269.9 ꢂ 5.5b;d;g
273.2 ꢂ 5.9b;d;h
270.5 ꢂ 6.3b;d;h
299.4 ꢂ 5.2b;d;e
301.4 ꢂ 7.0b;d;f
302.7 ꢂ 5.4b;d;g
303.4 ꢂ 6.0b;d;g
305.1 ꢂ 6.3b;d;h
301.5 ꢂ 5.9b;d;h
7b
8a
8b
9a
10a
n ¼ 12; DMSO/H2O (1:4) ¼ vehicle.
a Compare to vehicle, P < 0:01.
b Compare to vehicle, P < 0:001.
c Compare to 4a, P < 0:01.
d Compare to 4a, P < 0:001.
e Compare to 1 and 4aþ1, P < 0:001.
f Compare to 2a and 4aþ2a, P < 0:001.
g Compare to 2b and 4aþ2b, P < 0:001.
h Compare to 3 and 4aþ3, P < 0:001.
Table 8. Survival time of the transplantation grafts after treatment of prednisolone–urotoxin (XꢂSD, h)
Compound
0.04 (mmol/20 g/d)
0.19 (mmol/20 g/d)
0.38 (mmol/20 g/d)
DMSO/H2O (1:4)
4b
210.9 ꢂ 7.2
232.4 ꢂ 7.4b
220.9 ꢂ 5.7a
222.0 ꢂ 7.8a
225.2 ꢂ 7.3b
221.2 ꢂ 6.7a
223.6 ꢂ 6.5b
225.6 ꢂ 8.6b
226.7 ꢂ 7.5b
222.7 ꢂ 6.8a
246.3 ꢂ 6.6b;c;e
246.7 ꢂ 6.1b;c;f
251.3 ꢂ 6.4b;d;g
248.4 ꢂ 5.8b;d;g
251.3 ꢂ 6.6b;d;h
247.2 ꢂ 6.3b;d;h
248.9 ꢂ 5.9b
278.8 ꢂ 7.0b
1
2a
231.6 ꢂ 6.4b
250.6 ꢂ 7.3b
234.8 ꢂ 7.3b
247.2 ꢂ 6.4b
2b
3
232.6 ꢂ 6.4b
252.1 ꢂ 5.9b
230.6 ꢂ 7.0b
250.1 ꢂ 6.2b
4bþ1
4bþ2a
4bþ2b
4bþ3
7c
231.8 ꢂ 6.4b
251.6 ꢂ 7.2b
234.8 ꢂ 6.9b
249.6 ꢂ 6.3b
233.9 ꢂ 6.4b
253.5 ꢂ 6.2b
231.4 ꢂ 6.7b
251.7 ꢂ 5.9b
271.7 ꢂ 7.5b;d;e
268.9 ꢂ 6.0b;d;f
267.9 ꢂ 5.7b;d;g
269.2 ꢂ 5.4b;d;g
274.0 ꢂ 6.0b;d;h
271.2 ꢂ 6.1b;d;h
298.9 ꢂ 5.1b;d;e
302.0 ꢂ 7.1b;d;f
303.4 ꢂ 5.5b;d;g
304.3 ꢂ 6.1b;d;g
304.8 ꢂ 6.1b;d;h
302.2 ꢂ 5.8b;d;h
7d
8c
8d
9b
10b
n ¼ 12; DMSO/H2O (1:4) ¼ vehicle.
a Compare to vehicle, P < 0:01.
b Compare to vehicle, P < 0:001.
c Compare to 4b, P < 0:01.
d Compare to 4b, P < 0:001.
e Compare to 1 and 4bþ1, P < 0:001.
f Compare to 2a and 4bþ2a, P < 0:001.
g Compare to 2b and 4bþ2b, P < 0:001.
h Compare to 3 and 4bþ3, P < 0:001.
acylated either individually or simultaneously by the
HOSu or p-nitrophenol ester of hydrocortisone- and
prednisolone-21-O-b-carbonylpropionic acid and may
result in the mono-steroid substituted peptides hydro-
cortisone-21-O-b-carbonylpropionyl-His-Gly-Lys-OH,
prednisolone-21-O-b-carbonylpropionyl-His-Gly-Lys-
OH, and bis-steroid substituted peptides hydrocorti-
sone-His-Gly-Lys(hydrocortisone)-OH and predniso-
lon-His-Gly-Lys(prednisolone)-OH simultaneously. The
ratio of the mono-steroid substituted peptide to bis-
steroid substituted peptide depended on the ratio of 6 to
1 used in the amidation. When the ratio of raw material
6 to 1 was 1:1 the ratio of the mono-steroid substituted
peptide to bis-steroid substituted peptide was about 2:1,
when the ratio of raw material 6 to 1 was 1.5:1 the ratio
of the mono-steroid substituted peptide to bis-steroid

C. Wang et al. / Bioorg. Med. Chem. 12 (2004) 4403–4421
4411
substituted peptide was 1.5:1, when the ratio of raw
material 6 to 1 was 2:1 the ratio of the mono-steroid
substituted peptide to bis-steroid substituted peptide
was 1:1. In all cases no mono-steroid substituted peptide
with hydrocortisone- and prednisolone-21-O-b-carbon-
ylpropionyl at the x-amino group of the Lys residue was
formed. The rather weak nucleophilicity of x-amino
group of the Lys residue may be responsible for the lack
of the mono-steroid substituted peptide with hydrocor-
tisone- and prednisolone-21-O-b-carbonylpropionyl at
the x-amino group of the Lys residue. When the ami-
dations of urotoxins were carried out by use of hydro-
cortisone-21-O-b-carbonylpropionic acid HOSu ester as
the amidation agent the yields of the linkers may be
obviously increased.
toxin, or the mixture of hydrocortisone or prednisolone
and urotoxins. The rodent heterotopic ear–heart trans-
plant assay in vivo suggested that the immunosuppres-
sive effects, which was demonstrated by the survival
times of grafts, of 7a–d, 8a–d, 9a,b, and 10a,b were again
significantly higher than that of hydrocortisone, pred-
nisolone, urotoxin, or the mixture of hydrocortisone or
prednisolone and urotoxins. In conclusion, comparing
to hydrocortisone, prednisolone, urotoxin, or the mix-
ture of hydrocortisone or prednisolone and urotoxins,
hydrocortisone–urotoxins, and prednisolone–urotoxins
exhibited better immunosuppressive activities in vitro
and in vivo, namely the immunosuppressive activities of
both steroids and urotoxins were enhanced by the
chemical combination of the steroids and urotoxins.
3.3. The condensation of the hydrazine and the 3-carbonyl
group depending on the catalyst
3.5. The possible chemical combination of steroids and
peptides commonly enhancing bioactivity
The yield of the condensation of the hydrazine group in
His-Gly-Lys-NHNH2 and Boc-His-(Tos)-Gly-Lys(Z)-
NHNH2 with the 3-carbonyl group in hydrocortisone
and prednisolone was significantly dependent on the
catalyst. With glacial acetic acid as the catalyst the yields
of 3-(His-Gly-Lys-NHN)-substituted hydrocortisone, 3-
(His-Gly-Lys-NHN)-substituted prednisolone, 3-[Boc-
His(Tos)-Gly-Lys(Z)-NHN]-substituted hydrocortisone,
In our previous studies it was found that the analgesic
effects of hydrocortisone–KTP and estrone–KTP were
significantly higher than that of KTP, hydrocortisone or
estrone, and the mixture of KTP and hydrocortisone or
estrone;4 the anti-osteoporosis effects of estrogen–
GHRPs were significantly higher than that of GHRPs,
estrogen, and the mixture of estrogen and GHRPs.5
According to the data from the in vitro and in vivo assays
it is clear that the immunosuppressive activities of 7a–c,
8a–c, 10a, and 10b are at the same level and again sig-
nificantly higher than that of hydrocortisone, predniso-
lone, and urotoxins. These observations indicated that
not only the combination of steroid and peptide may
result in an enhancement of the activity but also the
substitution position of peptides at steroids gave no effect
on the enhancing activity. The data from the in vitro and
in vivo assays clearly point that the immunosuppressive
activities of 9a and 9b are also at the same level as that of
7a–c, 8a–c, 10a, and 10b and again significantly higher
than that of hydrocortisone, prednisolone, and urotox-
ins, meaning the increase of steroid gave no effect on the
enhancing activity neither. Thus all the results suggested
that the ‘permissive action’ should be generally simulated
by the possibly chemical combination of steroids and
peptides. Considering the immunosuppressive activities
of 7a–c, 8a–c, 9a,b, 10a, and 10b are significantly higher
than that of the mixture of hydrocortisone or predniso-
lone and urotoxins, and the chemical stability of C–N
bond in the 3-position of 10a and 10b, it is likely that the
synthetic steroid–urotoxin in its entirety should be
responsible to its immunosuppressive action. Thus the
chemical combinations of steroids and peptides via
simple ester bond are able to provide the useful way for
the design of steroid and peptide related lead compounds
enhancing the desirable bioactivity.
and
3-[Boc-His-(Tos)-Gly-Lys(Z)-NHN]-substituted
prednisolone were within 70–80%. When inorganic acid,
for instance HCl or H2SO4, was used instead of glacial
acetic acid as the catalyst their yields were lowed to 15–
40%. This dependence perhaps means that the proton-
ation of hydrazine group in His-Gly-Lys-NHNH2 and
Boc-His(Tos)-Gly-Lys(Z)-NHNH2 has a negative effect
on the condensation reaction.
3.4. Permissive action enhancing the immunosuppressive
effect of both steroids and urotoxins
Both ConA or LPS induced proliferation of mouse
spleen lymphocytes in vitro were used for evaluating the
immunosuppressive effects of the synthetic steroid–uro-
toxins. The data in Tables 1 and 2 show that at the
concentrations of 10ꢀ8–10ꢀ4 mol/L the inhibition rates
of 7a–d, 8a–d, 9a,b, and 10a,b for ConA or LPS induced
proliferation of mouse spleen lymphocytes in vitro are
significantly higher than that of hydrocortisone, pred-
nisolone, urotoxins, or the mixture of hydrocortisone or
prednisolone and urotoxins. On the other hand 2a (at
the concentrations of 10ꢀ8–10ꢀ4 mol/L) and 2b (at the
concentrations of 10ꢀ8–10ꢀ7 mol/L) exhibited (the minus
inhibition rate) an enhanced action rather for LPS than
for ConA induced proliferation of mouse spleen lym-
phocytes. This may mean that the immunosuppressive
effects of 2a,b on LPS and ConA induced proliferation
of mouse spleen lymphocytes via distinct pathway. The
phagocytosis of mouse peritoneal macrophages assay in
4. Experimental
vitro indicated that at the concentration of 10ꢀ6
–
4.1. Synthesis of peptides and steroid–urotoxins
10ꢀ2 mol/L the inhibition effects of 7a–d, 8a–d, 9a,b, and
10a,b on the macrophage activity were also significantly
higher than that of hydrocortisone, prednisolone, uro-
4.1.1. General. The protected amino acids were of
L
-configuration. The purity of the intermediates and the

4412
C. Wang et al. / Bioorg. Med. Chem. 12 (2004) 4403–4421
products was confirmed by TLC (Merck silica gel plates of
type 60 F254, 0.25 mm layer thickness) and HPLC (waters,
C18 column 3.9 ꢁ 150 mm). Melting points were measured
on a XT5 hot stage microscope (Beijing keyi electro-optic
factory), and are uncorrected. Infrared spectra were re-
corded with a Perkin–Elmer 983 instrument. FAB-MS
was determined by a VG-ZAB-MS and a HPES-5989 ꢁ
instrument. 1H NMR spectra were determined by a
Varian INOVA-500 MHz spectrometer. Optical rota-
tions were determined at 20 °C on a SchmidtþHaensch
Polartronic D instrument. The amino acid analysis was
performed by a Hitachi 835-50 instrument.
(OBzl)-Gly-OBzl 880 mg (87%) of the title compound
were obtained as a colorless powder. Mp 78–80 °C, ½aꢃ
20
D
þ4.0 (c 1, CHCl3), FAB-MS (m=z) 690 [MþH]þ. IR
(KBr) 3351, 3346, 3328, 3088, 3072, 3031, 1741, 1688,
1
1605, 1504, 1459, 1448, 1396, 1366, 740, 691 cmꢀ1. H
NMR (DMSO-d6): d ¼ 8:312 (d, J ¼ 6:31 Hz, 1H),
8.215 (d, J ¼ 6:52 Hz, 1H), 8.069 (t, J ¼ 6:55 Hz, 1H),
7.346 (t, J ¼ 7:90 Hz, 1H), 7.340 (t, J ¼ 7:92 Hz, 1H),
7.337 (t, J ¼ 7:88 Hz, 1H), 7.304 (t, J ¼ 7:90 Hz, 2H),
7.295 (t, J ¼ 7:92 Hz, 2H), 7.254 (t, J ¼ 7:88 Hz, 2H),
7.245 (d, J ¼ 7:87 Hz, 2H), 7.191 (d, J ¼ 7:89 Hz, 2H),
7.174 (d, J ¼ 7:86 Hz, 2H), 5.162 (s, 2H), 5.142 (s, 2H),
5.135 (s, 2H), 4.921 (dt, J ¼ 6:31 Hz, J ¼ 6:66 Hz, 1H),
4.715 (d, J ¼ 6:55 Hz, 2H), 4.705 (d, J ¼ 6:60 Hz, 1H),
2.904 (t, J ¼ 6:60 Hz, 2H), 2.721 (d, J ¼ 6:52 Hz, 2H),
2.230 (m, J ¼ 6:60 Hz, 2H), 1.455 (s, 9H). Amino acid
analysis: calcd, Glu/Asp/Gly ¼ 1.0:1.0:1.0. Found, Glu/
Asp/Gly ¼ 0.98:0.99:1.00. Anal. Calcd for C37H43N3O10:
C, 64.43; H, 6.28; N, 6.09. Found: C, 64.22; H, 6.15; N,
6.14.
4.2. General procedure for coupling C-terminal and N-
terminal components
To a solution of 1.5 mmol of the N-terminal component
in 15 mL of anhydrous THF at 0 °C 1.5 mmol of HOBt
and 1.9 mmol of DCC were added. The reaction mixture
was stirred at 0 °C for 24 h and the precipitates of DCU
were removed by filtration. The filtrate was evaporated
under reduced pressure and the residue was triturated
with petroleum ether to provide the corresponding ac-
tive ester. To the solution of the active ester in 30 mL of
anhydrous THF 1.5 mmol of C-terminal component and
1.9 mmol of N-methylmorpholine were added. The
reaction mixture was stirred at room temperature for
24 h. After evaporation the residue was dissolved in
50 mL of ethyl acetate. The solution was washed suc-
cessively with 5% sodium bicarbonate, 5% citric acid,
and saturated sodium chloride and the organic phase
was dried over anhydrous sodium sulfate. After filtra-
tion and evaporation under reduced pressure the residue
was purified by chromatography (CHCl3/CH3OH, 30:1)
to provide the protective intermediates.
4.5. Boc-Gly-Glu(OBzl)-OBzl
Using the general procedure for coupling C-terminal
and N-terminal components from 261 mg (1.5 mmol) of
Boc-Gly-OH and 489 mg (1.5 mmol) of Glu(OBzl)-OBzl
617 mg (85%) of the title compound were obtained as a
20
D
colorless oil, ½aꢃ þ8.0 (c 1, CHCl3), FAB-MS (m=z):
485 [MþH]þ. IR (KBr) 3351, 3346, 3091, 3070, 3027,
1745, 1690, 1601, 1502, 1462, 1450, 1394, 1365, 741,
.
690 cmꢀ1
1H NMR (DMSO-d6): d ¼ 8:202 (t,
J ¼ 6:32 Hz, 1H), 8.101 (d, J ¼ 6:55 Hz, 1H), 7.325 (t,
J ¼ 7:78 Hz, 1H), 7.315 (t, J ¼ 7:81 Hz, 1H), 7.313 (t,
J ¼ 7:58 Hz, 2H), 7.306 (t, J ¼ 7:60 Hz, 2H), 7.301 (d,
J ¼ 7:52 Hz, 2H), 7.234 (d, J ¼ 7:54 Hz, 2H), 5.152 (s,
2H), 5.148 (s, 2H), 4.930 (dt, J ¼ 6:55 Hz, J ¼ 6:78 Hz,
1H), 4.906 (d, J ¼ 6:32 Hz, 2H), 2.901 (t, J ¼ 6:54 Hz,
2H), 2.202 (m, J ¼ 6:54 Hz, 2H), 1.454 (s, 9H). Amino
acid analysis: calcd, Gly/Glu ¼ 1.0:1.0. Found, Gly/
Glu ¼ 1.00:0.98. Anal. Calcd for C26H32N2O7: C, 64.45;
H, 6.66; N, 5.78. Found: C, 64.60; H, 6.80; N, 5.58.
4.3. Boc-Asp(OBzl)-Gly-OBzl
Using the general procedure for coupling C-terminal
and N-terminal components from 450 mg (1.5 mmol) of
Boc-Asp(OBzl)-OH and 248 mg (1.5 mmol) of Gly-OBzl
556 mg (80%) of the title compound were obtained as a
20
D
colorless powder. Mp 93–95 °C, ½aꢃ þ16.0 (c 1, CHCl3),
FAB-MS (m=z) 471 [MþH]þ. IR (KBr) 3344, 3336,
4.6. Boc-His(Tos)-Gly-Glu(OBzl)-OBzl
3090, 3075, 3029, 1735, 1691, 1600, 1500, 1460, 1450,
1395, 1365, 742, 693 cmꢀ1
.
1H NMR (DMSO-d6):
Using the general procedure for coupling C-terminal
and N-terminal components from 588 mg (1.5 mmol) of
Boc-His(Tos)-OH and 630 mg (1.5 mmol) of HClÆGly-
Glu(OBzl)-OBzl 907 mg (78%) of the title compound
d ¼ 8:013 (d, J ¼ 6:39 Hz, 1H), 8.005 (t, J ¼ 6:54 Hz,
1H), 7.335 (t, J ¼ 7:92 Hz, 1H), 7.322 (t, J ¼ 7:96 Hz,
1H), 7.310 (t, J ¼ 7:92 Hz, 2H), 7.305 (t, J ¼ 7:96 Hz,
2H), 7.257 (d, J ¼ 7:84 Hz, 2H), 7.220 (d, J ¼ 7:80 Hz,
2H), 5.181 (s, 2H), 5.172 (s, 2H), 4.904 (dt, J ¼ 4:56 Hz,
J ¼ 6:39 Hz, 1H), 3.921 (d, J ¼ 6:54 Hz, 2H), 2.901 (d,
J ¼ 5:96 Hz, 2H), 1.456 (s, 9H). Amino acid analysis:
calcd, Asp/Gly ¼ 1.0:1.0. Found, Asp/Gly ¼ 1.00:0.99.
Anal. Calcd for C25H30N2O7: C, 63.82; H, 6.43; N, 5.95.
Found: C, 63.76; H, 6.50; N, 6.08.
were obtained as a colorless powder. Mp 98–100 °C,
20
½aꢃ þ2.5 (c 1, CHCl3), FAB-MS (m=z) 776 [MþH]þ. IR
D
(KBr) 3355, 3344, 3338, 3093, 3072, 3031, 1740, 1691,
1600, 1504, 1460, 1453, 1395, 1384, 1367, 740, 692 cmꢀ1
.
1H NMR (DMSO-d6) d ¼ 8:406 (d, J ¼ 6:58 Hz, 1H),
8.221 (t, J ¼ 6:52 Hz, 1H), 8.032 (d, J ¼ 6:60 Hz, 1H),
7.776 (t, J ¼ 7:83 Hz, 1H), 7.423 (t, J ¼ 7:82 Hz, 1H),
7.341 (s, 1H), 7.29 (d, J ¼ 7:78 Hz, 2H), 7.320 (t,
J ¼ 7:79 Hz, 2H), 7.247 (t, J ¼ 7:76 Hz, 2H), 7.244 (d,
J ¼ 7:67 Hz, 2H), 7.244 (d, J ¼ 7:78 Hz, 2H), 7.244 (d,
J ¼ 7:76 Hz, 2H), 6.905 (s, 1H), 5.160 (s, 2H), 5.146 (s,
2H), 4.944 (dt, J ¼ 6:60 Hz, J ¼ 6:54 Hz, 1H), 4.909 (dt,
J ¼ 6:58 Hz, J ¼ 6:52 Hz, 1H), 4.524 (d, J ¼ 6:52 Hz,
4.4. Boc-Glu(OBzl)-Asp(OBzl)-Gly-OBzl
Using the general procedure for coupling C-terminal
and N-terminal components from 507 mg (1.5 mmol) of
Boc-Glu(OBzl)-OH and 599 mg (1.5 mmol) of HClÆAsp

C. Wang et al. / Bioorg. Med. Chem. 12 (2004) 4403–4421
4413
2H), 3.130 (d, J ¼ 6:52 Hz, 2H), 2.656 (t, J ¼ 6:54 Hz,
2H), 2.371 (s, 3H), 1.812 (m, J ¼ 6:54 Hz, 2H), 1.457 (s,
9H). Amino acid analysis: calcd, His/Gly/Glu ¼ 1.0:1.0.
Found, His/Gly/Glu ¼ 0.97:1.00:0.98. Anal. Calcd for
C39H45N5O10S: C, 60.37; H, 5.85; N, 9.03. Found: C,
60.19; H, 5.68; N, 9.22.
hydrate (50%) were mixed and stirred at room temper-
ature for 60 h. After evaporation the residue was dis-
solved in 30 mL of ethyl acetate. The solution was
washed successively with 5% sodium bicarbonate, 5%
citric acid, and saturated sodium chloride and the or-
ganic phase was dried over anhydrous sodium sulfate.
After filtration and evaporation under reduced pressure
the residue was triturated with ether repeatedly to pro-
4.7. Boc-Gly-Lys(Z)-OBzl
vide 579 mg (78%) of the title compound as a colorless
20
D
oil, ½aꢃ ꢀ7.8, FAB-MS (m=z): 743 [MþH]þ. IR (KBr)
Using the general procedure for coupling C-terminal and
N-terminal components from 261 mg (1.5 mmol) of Boc-
Gly-OH and 555 mg (1.5 mmol) of Lys(Z)-OBzl 696 mg
3431, 3410, 3365, 3342, 3334, 3090, 3072, 3031, 1750,
1690, 1609, 1507, 1450, 1438, 1395, 1380, 1365, 740,
692 cmꢀ1
.
1H NMR (DMSO-d6) d ¼ 8:422 (d,
(88%) of the title compound were obtained as a colorless
J ¼ 6:58 Hz, 1H), 8.223 (t, J ¼ 6:61 Hz, 1H), 8.158 (d,
J ¼ 6:68 Hz, 1H), 8.112 (s, 1H), 8.012 (t, J ¼ 6:56 Hz,
1H), 7.780 (d, J ¼ 7:84 Hz, 2H), 7.431 (t, J ¼ 7:82 Hz,
1H), 7.391 (t, J ¼ 7:75 Hz, 2H), 7.315 (s, 1H), 7.308 (d,
J ¼ 7:75 Hz, 2H), 7.228 (d, J ¼ 7:82 Hz, 2H), 6.760 (s,
1H), 5.112 (s, 2H), 4.935 (dt, J ¼ 6:58 Hz, J ¼ 6:78 Hz,
1H), 4.722 (d, J ¼ 6:61 Hz, 2H), 4.526 (dt, J ¼ 6:68 Hz,
J ¼ 5:50 Hz, 1H), 3.985 (d, J ¼ 6:78 Hz, 2H), 3.084 (dt,
J ¼ 6:56 Hz, J ¼ 5:40 Hz, 2H), 3.062 (m, J ¼ 5:50 Hz,
2H), 2.369 (s, 3H), 1.884 (m, J ¼ 5:56 Hz, 2H), 1.545 (m,
J ¼ 5:40 Hz, 2H), 1.326 (m, J ¼ 5:50 Hz, 2H), 1.459 (s,
20
D
oil, ½aꢃ ꢀ2.6 (c 1, CHCl3), FAB-MS (m=z): 528
[MþH]þ. IR (KBr) 3351, 3340, 3088, 3070, 3030, 1745,
1690, 1605, 1500, 1463, 1450, 1397, 1364, 744, 696 cmꢀ1
.
1H NMR (DMSO-d6) d ¼ 8:301 (t, J ¼ 6:88 Hz, 1H),
8.175 (d, J ¼ 6:79 Hz, 1H), 8.100 (t, J ¼ 6:82 Hz, 1H),
7.366 (t, J ¼ 7:82 Hz, 1H), 7.336 (t, J ¼ 7:80 Hz, 1H),
7.340 (t, J ¼ 7:77 Hz, 2H), 7.318 (t, J ¼ 7:78 Hz, 2H),
7.315 (d, J ¼ 7:77 Hz, 2H), 7.312 (d, J ¼ 7:77 Hz, 2H),
5.139 (s, 2H), 5.056 (s, 2H), 4.935 (dt, J ¼ 6:88 Hz,
J ¼ 6:60 Hz, 1H), 4.515 (d, J ¼ 6:79 Hz, 2H), 2.965 (dt,
J ¼ 6:82 Hz, J ¼ 5:02 Hz, 2H), 1.674 (m, J ¼ 5:02 Hz,
2H), 1.540 (m, J ¼ 4:96 Hz, 2H), 1.521 (m, J ¼ 4:98 Hz,
2H), 1.460 (s, 9H). Amino acid analysis: calcd, Gly/
Lys ¼ 1.0:1.0. Found, His/Gly/Lys ¼ 1.00:0.98. Anal.
Calcd for C28H37N3O7: C, 63.74; H, 7.07; N, 7.96, O,
21.23. Found: C, 63.52; H, 7.18; N, 7.96, O, 21.07.
9H).
Amino
acid
analysis:
calcd,
His/Gly/
Lys ¼ 1.0:1.0:1.0. Found, His/Gly/Lys ¼ 0.98:1.00:0.98.
Anal. Calcd for C34H46N8O9S: C, 54.97; H, 6.24; N,
15.08. Found: C, 54.79; H, 6.40; N, 15.22.
4.10. General procedure for removal of Boc of the
C-terminal component
4.8. Boc-His(Tos)-Gly-Lys(Z)-OBzl
The solution of 1.5 mmol of Boc protected compound in
8 mL of hydrogen chloride in ethyl acetate (4 mol/L) was
stirred at room temperature for 3 h. The reaction mix-
ture was evaporated to remove the solvent. The residue
was dissolved in 20 mL of ethyl acetate and the solution
was evaporated to dry. The resulted solid was used for
coupling reaction directly.
Using the general procedure for coupling C-terminal
and N-terminal components from 612 mg (1.5 mmol) of
Boc-His(Tos)-OH and 695 mg (1.5 mmol) of HClÆGly-
Lys(Z)-OBzl 1067 mg (87%) of the title compound were
20
D
obtained as a colorless powder. Mp 70–72 °C, ½aꢃ ꢀ6.3
(c 1, CHCl3), FAB-MS (m=z) 819 [MþH]þ. IR (KBr)
3363, 3347, 3329, 3092, 3068, 3029, 1751, 1693, 1600,
1
1512, 1458, 1451, 1395, 1381, 1365, 748, 690 cmꢀ1. H
4.11. HClꢄAsp(OBzl)-Gly-OBzl
NMR (DMSO-d6) d ¼ 8:412 (d, J ¼ 6:59 Hz, 1H), 8.241
(t, J ¼ 6:54 Hz, 1H), 8.169 (d, J ¼ 6:60 Hz, 1H), 8.112
(t, J ¼ 6:55 Hz, 1H), 7.662 (d, J ¼ 7:80 Hz, 2H), 7.422
(t, J ¼ 7:88 Hz, 1H), 7.360 (t, J ¼ 7:85 Hz, 1H), 7.352 (s,
1H), 7.338 (d, J ¼ 7:80 Hz, 2H), 7.328 (t, J ¼ 7:86 Hz,
2H), 7.310 (t, J ¼ 7:84 Hz, 2H), 7.234 (d, J ¼ 7:90 Hz,
2H), 7.230 (d, J ¼ 7:89 Hz, 2H), 6.776 (s, 1H), 5.126 (s,
2H), 5.107 (s, 2H), 4.942 (dt, J¼ 6:78 Hz, J ¼ 6:59 Hz,
1H), 4.752 (dt, J ¼ 6:60 Hz, J ¼ 5:90 Hz, 1H), 4.520 (d,
J ¼ 6:54 Hz, 2H), 3.162 (dt, J ¼ 6:55 Hz, J ¼ 5:40 Hz,
2H), 2.873 (m, J ¼ 5:90 Hz, 2H), 2.373 (s, 3H), 2.015 (d,
J ¼ 6:78 Hz, 2H), 1.943 (m, J ¼ 5:41 Hz, 2H), 1.524 (m,
J ¼ 5:52 Hz, 2H), 1.462 (s, 9H). Amino acid analysis:
calcd, His/Gly/Lys ¼ 1.0:1.0:1.0. Found, His/Gly/Lys ¼
0.99:1.00:0.98. Anal. Calcd for C41H50N6O10S: C, 60.13;
H, 6.15; N, 10.26. Found: C, 60.25; H, 6.03; N, 10.11.
Using the general procedure for removal of Boc of the
C-terminal component from 693 mg (1.5 mmol) of Boc-
Asp(OBzl)-Gly-OBzl 543 mg (91%) of the title com-
pound were obtained as a colorless powder. Mp 72–
20
D
75 °C, ½aꢃ þ4.6 (c 1, CHCl3), FAB-MS (m=z) 363
[MþH]þ.
4.12. HClꢄGly-Glu(OBzl)-OBzl
Using the general procedure for removal of Boc of the C-
terminal component from 725 mg (1.5 mmol) of Boc-Gly-
Glu(OBzl)-OBzl 566 mg (90%) of the title compound
were obtained as a colorless powder. Mp 105–107 °C,
20
½aꢃ þ4.0 (c 1, CH3OH), FAB-MS (m=z) 384 [MþH]þ.
D
4.9. Boc-His(Tos)-Gly-Lys(Z)-NHNH2
4.13. HClꢄGly-Lys(Z)-OBzl
The solution of 818 mg (1.0 mmol) of Boc-His(Tos)-Gly-
Lys(Z)-OBzl in 8 mL of methanol and 3 mL of hydrazine
Using the general procedure for removal of Boc of the
C-terminal component from 791 mg (1.5 mmol) of

4414
C. Wang et al. / Bioorg. Med. Chem. 12 (2004) 4403–4421
Boc-Gly-Lys(Z)-OBzl 577 mg (90%) of the title com-
pound were obtained as a colorless powder. Mp 116–
His(Tos)-Gly-Lys(Z)-OBzl 286 mg (84%) of the title
compound were obtained as a colorless powder. Mp
20
D
20
D
119 °C, ½aꢃ ꢀ10.0 (c 1, CH3OH), FAB-MS (m=z) 428
226 °C (dec.), ½aꢃ ꢀ16.0 (c 0.5, H2O), FAB-MS 341
[MþH]þ.
[MþH]þ. IR (KBr): 3350, 3344, 3339, 3331, 3021, 2489,
1
1648, 1460 cmꢀ1. H NMR (DMSO-d6): d ¼ 12:755 (s,
1H), 10.350 (s, 1H), 8.666 (d, J ¼ 6:80 Hz, 2H), 8.617
(2H), 8.502 (t, J ¼ 6:57 Hz, 1H), 7.895 (d, J ¼ 6:68 Hz,
1H), 7.645 (s, 1H), 6.833 (s, 1H), 4.652 (dt, J ¼ 6:68 Hz,
J ¼ 4:55 Hz, 1H), 4.474 (d, J ¼ 6:57 Hz, 2H), 4.015 (dt,
J ¼ 6:80 Hz, J ¼ 4:86 Hz, 1H), 3.103 (d, J ¼ 4:86 Hz,
2H), 2.795 (t, J ¼ 4:59 Hz, 2H), 1.804 (m, J ¼ 4:78 Hz,
2H), 1.534 (m, J ¼ 4:66 Hz, 2H), 1.267 (m, J ¼ 4:58 Hz,
4.14. General procedure for removal of all the semi-
permanent groups
The protected peptide (1.0 mmol) was mixed with 6 mL
of dimethyl sulfide, 6 mL of phenyl methyl ether, and
10 mL of CF3COOH–CF3SO3H (4:1). The mixture was
stirred at 0 °C for 2 h. After removal of CF3COOH–
CF3SO3H the residue was triturated with ether and the
residue was purified on Sephadex G10 and HPLC to
provide the title compound as a colorless powder, among
which 5b–11b, 7d, 8d, 9a, and 11a are new compounds.
2H).
Amino
acid
analysis:
calcd,
His/Gly/
Lys ¼ 1.0:1.0:1.0. Found, His/Gly/Lys ¼ 0.98:1.00:0.98.
Anal. Calcd for C14H24N6O4: C, 49.40; H, 7.11;
N, 24.69, O, 18.80. Found: C, 49.40; H, 7.11; N, 24.69.
4.18. His-Gly-Lys-NHNH2 (3)
4.15. Glu-Asp-Gly-OH (1)
Using the general procedure for removal of all the semi-
permanent groups from742 mg (1.0 mmol) of Boc-His-
(Tos)-Gly-Lys(Z)-NHNH2 287 mg (81%) of the title
Using the general procedure for removal of all the semi-
permanent groups from 673 mg (1.0 mmol) of Boc-
Glu(OBzl)-Asp(OBzl)-Gly-OBzl 284 mg (89%) of the
compound were obtained as a colorless powder. Mp 96–
title compound were obtained as a colorless powder. Mp
20
D
(KBr) 3348, 3340, 3335, 3330, 3316, 2489, 1652, 1500,
20
D
98 °C ½aꢃ ꢀ8.0 (c 1.0, H2O). FAB-MS 355 [MþH]þ. IR
143–145 °C (dec.), ½aꢃ þ20.0 (c 0.5, H2O), FAB-MS 320
[MþH]þ. IR (KBr) 3356, 3345, 3336, 3332, 3019, 2598,
1
1460, 1410 cmꢀ1. H NMR (DMSO-d6): d ¼ 12:801 (s,
1
2568, 2492, 1755, 1649, 1460 cmꢀ1. H NMR (DMSO-
1H), 8.553 (t, J ¼ 6:60 Hz, 1H), 8.475 (s, 2H), 8.422 (s,
2H), 7.995 (d, J ¼ 6:56 Hz, 1H), 7.943 (s, 1H), 7.494 (s,
1H), 6.446 (s, 1H), 4.776 (dt, J ¼ 6:76 Hz, J ¼ 5:96 Hz,
1H), 4.352 (dt, J ¼ 6:56 Hz, J ¼ 4:76 Hz, 1H), 4.293 (d,
J ¼ 6:60 Hz, 2H), 3.012 (d, J ¼ 4:86 Hz, 2H), 2.795 (t,
J ¼ 4:50 Hz, 2H), 2.245 (m, J ¼ 4:76 Hz, 2H), 1.804 (m,
J ¼ 4:64 Hz, 2H), 1.534 (m, J ¼ 4:52 Hz, 2H), 1.267 (m,
J ¼ 4:64 Hz, 2H). Amino acid analysis: calcd, His/Gly/
Lys ¼ 1.0:1.0:1.0. Found, His/Gly/Lys ¼ 0.98:1.00:0.99.
Anal. Calcd for C14H26N8O3: C, 47.45; H, 7.39; N,
31.62. Found: C, 47.30; H, 7.17; N, 31.44.
d6): d ¼ 10:621 (s, 1H), 10.605 (s, 1H), 10.401 (s, 1H),
8.668 (d, J ¼ 6:87 Hz, 2H), 8.044 (d, J ¼ 6:68 Hz, 1H),
7.986 (t, J ¼ 6:49 Hz, 1H), 4.246 (m, J ¼ 6:55 Hz, 1H),
4.035 (dt, J ¼ 6:68 Hz, J ¼ 5:21 Hz, 1H), 4.012 (d,
J ¼ 6:49 Hz, 2H), 2.670 (d, J ¼ 5:21 Hz, 2H), 2.354 (t,
J ¼ 4:96 Hz, 2H), 2.165 (m, J ¼ 4:75 Hz, 2H). Amino
acid analysis: calcd, Glu/Asp/Gly ¼ 1.0:1.0:1.0. Found,
Glu/Asp/Gly ¼ 0.98:0.98:1.00.
Anal.
Calcd
for
C11H17N3O8: C, 41.38; H, 5.37; N, 13.16. Found: C,
41.50; H, 5.19; N, 13.33.
4.16. His-Gly-Glu-OH (2a)
4.19. Hydrocortisone-21-O-b-carbonylpropionic acid (5a)
Using the general procedure for removal of all the semi-
permanent groups from 775 mg (1.0 mmol) of Boc-
His(Tos)-Gly-Glu(OBzl)-OBzl 266 mg (78%) of the title
The solution of 4.0 g (11.0 mmol) of hydrocortisone,
5.0 g (50.2 mmol) of succinic anhydride and 25 mL of
pyridine was stirred at room temperature for 24 h. The
reaction mixture was poured into the mixture of 100 g of
ice, 30 mL of concentrated hydrochloric acid and
100 mL of water. The formed precipitates were collected
compound were obtained as a colorless powder. Mp
20
D
220 °C (dec.) ½aꢃ þ8.0 (c 0.5, H2O). FAB-MS 342
[MþH]þ. IR (KBr): 3352, 3341, 3335, 3325, 3010, 2605,
2489, 1652, 1463 cmꢀ1
.
1H NMR (DMSO-d6):
d ¼ 12:787 (s, 1H), 10.445 (s, 1H), 10.356 (s, 1H), 8.654
(d, J ¼ 6:78 Hz, 2H), 8.230 (t, J ¼ 6:59 Hz, 1H), 8.206 (d,
J ¼ 6:70 Hz, 1H), 7.554 (s, 1H), 6.785 (s, 1H), 4.460 (m,
J ¼ 5:22 Hz, 1H), 4.369 (t, J ¼ 6:59 Hz, 2H), 3.846 (dt,
J ¼ 6:70 Hz, J ¼ 4:80 Hz, 1H), 3.204 (t, J ¼ 4:89 Hz,
2H), 2.678 (t, J ¼ 4:85 Hz, m, J ¼ 4:90 Hz, 2H), 1.976
(m, J ¼ 4:54 Hz, 2H). Amino acid analysis: calcd,
and dried to provide 4.90 g (98%) of the title compound
20
D
as a colorless powder. Mp 165–166 °C ½aꢃ 152.3 (c 1.0,
CHCl3). FAB-MS 463 [MþH]þ. IR (KBr) 3230, 3210,
1
3016, 1742, 1708, 1671, 1460 cmꢀ1. H NMR (DMSO-
d6): d ¼ 10:188 (s, 1H), 5.628 (s, 1H), 4.804 (s, 2H),
3.954 (dt, J ¼ 12:88 Hz, J ¼ 2:33 Hz, 2H), 3.628 (dt,
J ¼ 12:88 Hz, J ¼ 2:33 Hz, 2H), 3.156 (m, J ¼ 7:99 Hz,
1H), 2.602 (m, J ¼ 8:12 Hz, 2H), 2.454 (m, J ¼ 7:98 Hz,
2H), 2.384 (m, J ¼ 8:60 Hz, 2H), 2.212 (m, J ¼ 8:06 Hz,
1H), 2.123 (m, J ¼ 7:96 Hz, 2H), 2.112 (s, 1H), 2.101 (s,
1H), 1.943 (m, J ¼ 8:12 Hz, 1H), 1.851 (m, J ¼ 7:95 Hz,
2H), 1.741 (m, J ¼ 8:04 Hz, 1H), 1.708 (m, J ¼ 7:03 Hz,
2H), 1.236 (s, 3H), 1.152 (s, 3H), 0.966 (m, J ¼ 8:00 Hz,
2H). Anal. Calcd for C25H34O8: C, 64.92; H, 7.41; O,
27.67. Found: C, 64.79; H, 7.22.
His/Gly/Glu ¼ 1.0:1.0:1.0.
Found,
His/Gly/Glu ¼
0.98:1.00:0.99. Anal. Calcd for C13H19N5O6: C, 45.75; H,
5.61; N, 20.52. Found: C, 45.75; H, 5.61; N, 20.52.
4.17. His-Gly-Lys-OH (2b)
Using the general procedure for removal of all the semi-
permanent groups from 818 mg (1.0 mmol) of Boc-

C. Wang et al. / Bioorg. Med. Chem. 12 (2004) 4403–4421
4415
4.20. Hydrocortisone-21-O-b-carbonylpropionic acid
p-nitrophenolic ester (6a)
1H), 4.845 (s, 2H), 4.275 (m, J ¼ 7:55 Hz, 1H), 3.985 (dt,
J ¼ 11:98 Hz, J ¼ 3:42 Hz, 2H), 3.622 (dt, J ¼ 11:98 Hz,
J ¼ 3:42 Hz, 2H), 3.367 (s, 1H), 3.335 (m, J ¼ 8:20 Hz,
2H), 3.320 (s, 1H), 2.263 (m, J ¼ 7:94 Hz, 2H), 2.245 (m,
J ¼ 8:10 Hz, 1H), 1.922 (m, J ¼ 8:12 Hz, 1H), 1.902 (m,
J ¼ 7:90 Hz, 2H), 1.782 (m, J ¼ 8:00 Hz, 1H), 1.701 (m,
J ¼ 7:00 Hz, 2H), 1.030 (m, J ¼ 7:45 Hz, 2H), 1.246 (s,
3H), 0.978 (2H), 0.695 (s, 3H). Anal. Calcd for
C31H37NO10: C, 63.80; H, 6.39; N, 2.40. Found: C,
63.67; H, 6.18; N, 2.31.
The solution of 500 mg (1.1 mmol) of hydrocortisone-
21-O-carbonyl-b-propionic acid and 165 mg (1.2 mmol)
of p-nitrophenol in 5 mL of anhydrous THF was stirred
at 0 °C and 240 mg (1.2 mmol) of DCC were added. The
reaction mixture was stirred at 0 °C for 2 h and then at
room temperature for 24 h. The reaction mixture was
filtrated and the filtrate was evaporated to dryness. The
obtained residue was triturated with ether repeatedly to
give 600 mg (95%) of the title compound as a colorless
powder. Mp 100–102 °C ½aꢃ20 108.2 (c 1.0, CHCl3). FAB-
4.23. Hydrocortisone-21-O-b-carbonylpropionyl-Glu-
Asp-Gly-OH (7a)
D
MS 584 [MþH]þ. IR (KBr) 3230, 3210, 3030, 3010,
1712, 1708, 1641, 1601, 1580, 1520, 1500, 1490, 1460
.
1350 cmꢀ1
1H NMR (DMSO-d6): d ¼ 8:092 (d,
(A) Under stirring the solution of 200 mg (0.63 mmol) of
Glu-Asp-Gly-OH in 1 mL of water, 1 mL of THF and
0.5 mL of DMF was adjusted to pH 9 with N-meth-
ylmorpholine. To the solution 408 mg (0.7 mmol) of
hydrocortisone-21-O-b-carbonylpropionic acid p-nitro-
phenolic ester were added. The reaction mixture was
stirred at room temperature for 40 h and TLC (CHCl3–
CH3OH–H2O ¼ 1:1:0.15) indicated the complete disap-
pearance of Glu-Asp-Gly-OH. After evaporation the
residue was washed with ether and ethyl acetate to
provide the crude product. The crude product was then
purified by silica gel chromatography (CHCl3–CH3OH–
J ¼ 7:88 Hz, 2H), 8.084 (d, J ¼ 7:88 Hz, 2H), 5.655 (s,
1H), 4.845 (s, 2H), 4.321 (m, J ¼ 6:88 Hz, 1H), 3.985 (dt,
J ¼ 12:78 Hz, J ¼ 3:62 Hz, 2H), 3.622 (dt, J ¼ 12:78 Hz,
J ¼ 3:62 Hz, 2H), 3.323 (s, 1H), 3.144 (m, J ¼ 7:00 Hz,
1H), 2.441 (m, J ¼ 7:69 Hz, 2H), 2.403 (m, J ¼ 7:96 Hz,
2H), 2.224 (s, 1H), 2.145 (s, 2H), 1.863 (m, J ¼ 7:24 Hz,
2H), 1.757 (m, J ¼ 7:09 Hz,, 1H), 1.711 (m, J ¼ 7:81 Hz,
2H), 1.246 (s, 3H), 0.978 (m, J ¼ 6:98 Hz, 2H), 0.655 (s,
3H).
4.21. Prednisolone-21-O-b-carbonylpropionic acid (5b)
H2O ¼ 1:1:0.15) to provide 240 mg (50%) of the title
20
D
compound. Mp 218–220 °C, ½aꢃ þ88 (c 0.3, DMF),
Using the same procedure as that used for preparation
of hydrocortisone-21-O-b-carbonylpropionic acid with
4.0 g (11.0 mmol) of prednisolone instead of hydrocor-
FAB-MS (m=z) 764 [MþH]þ, IR (KBr) 3342, 3320,
3315, 3006, 1740, 1702, 1669, 1655, 1460 cmꢀ1. 1H NMR
(DMSO-d6): d ¼ 10:601 (s, 1H), 10.600 (s, 1H), 10.421
(s, 1H), 8.144 (d, J ¼ 6:81 Hz, 1H), 8.084 (d,
J ¼ 6:70 Hz, 1H), 7.986 (t, J ¼ 6:50 Hz, 1H), 5.507 (s,
1H), 4.986 (s, 2H), 4.265 (dt, J ¼ 6:81 Hz, J ¼ 4:88 Hz,
1H), 4.010 (dt, J ¼ 6:70 Hz, J ¼ 5:00 Hz, 1H), 4.109 (d,
J ¼ 6:50 Hz, 2H), 3.898 (dt, J ¼ 12:44 Hz, J ¼ 3:42 Hz,
2H), 3.564 (dt, J ¼ 12:44 Hz, J ¼ 3:42 Hz, 2H), 3.215
(m, J ¼ 7:93 Hz, 1H), 2.654 (d, J ¼ 5:15 Hz, 2H), 2.616
(m, J ¼ 8:03 Hz, 2H), 2.450 (m, J ¼ 7:888 Hz, 2H),
2.347 (m, J ¼ 8:442 Hz, 2H), 2.312 (s, 1H), 2.208 (s, 1H),
2.204 (m, J ¼ 8:04 Hz, 1H), 2.140 (m, J ¼ 7:94 Hz, 2H),
2.134 (m, J ¼ 4:88 Hz, 2H), 2.059 (t, J ¼ 4:94 Hz, 2H),
1.932 (m, J ¼ 8:12 Hz, 1H), 1.825 (m, J ¼ 7:87 Hz, 2H),
1.727 (m, J ¼ 8:01 Hz, 1H), 1.716 (m, J ¼ 7:00 Hz, 2H),
1.256 (s, 3H), 0.955 (m, J ¼ 7:95 Hz, 2H), 0.680 (s, 3H).
Anal. Calcd for C36H49N3O15: C, 56.61; H, 6.47; N, 5.50;
O, 31.42. Found: C, 56.45; H, 6.28; N, 5.67.
tisone 4.66 g (92%) of the title compound were obtained
20
D
as a colorless powder. Mp 174–176 °C ½aꢃ 131.7 (c 1.0,
CHCl3). FAB-MS 461 [MþH]þ. IR (KBr) 3239, 3209,
1
3016, 1740, 1700, 1642, 1461 cmꢀ1. H NMR (DMSO-
d6): d ¼ 10:202 (s, 1H), 7.244 (d, J ¼ 7:66 Hz, 1H), 6.142
(d, J ¼ 7:66 Hz, 1H), 6.065 (s, 1H), 4.812 (s, 2H), 4.321
(m, J ¼ 7:68 Hz, 1H), 3.948 (dt, J ¼ 12:01 Hz,
J ¼ 3:56 Hz, 2H), 3.615 (dt, J ¼ 12:01 Hz, J ¼ 3:56 Hz,
2H), 3.346 (s, 1H), 3.340 (s, 1H), 3.335 (m, J ¼ 8:20 Hz,
2H), 2.345 (m, J ¼ 7:94 Hz, 2H), 2.136 (m, J ¼ 7:90 Hz,
1H), 1.910 (m, J ¼ 7:84 Hz, 2H), 1.901 (m, J ¼ 7:88 Hz,
1H), 1.801 (m, J ¼ 8:10 Hz, 1H), 1.730 (m, J ¼ 8:01 Hz,
2H), 1.312 (s, 3H), 1.105 (m, J ¼ 7:98 Hz, 2H), 0.781 (s,
3H).
4.22. Prednisolone-21-O-b-carbonylpropionic acid p-
nitrophenolic ester (6b)
(B) Under stirring and at 0 °C to the solution of 291 mg
(0.63 mmol) of hydrocortisone-21-O-b-carbonylprop-
ionic acid and 81 mg (0.7 mmol) of N-hydroxysuccini-
mide in 10 mL of anhydride THF 140 mg (0.7 mmol) of
DCC were added. The reaction mixture was stirred at
0 °C for 2 h and then at room temperature for 24 h. The
reaction mixture was filtrated and to the filtrate 200 mg
(0.63 mmol) of Glu-Asp-Gly-OH were added. The
reaction mixture was stirred at room temperature for
40 h and TLC (CHCl3–CH3OH–H2O ¼ 1:1:0.15) indi-
cated the complete disappearance of Glu-Asp-Gly-OH.
After evaporation the residue was washed with ether
and ethyl acetate to provide the crude product. The crude
product was then purified by silica gel chromatography
Using the same procedure as that used for preparation
of hydrocortisone-21-O-b-carbonylpropionic acid p-ni-
trophenolic ester with 500 mg (1.1 mmol) of predniso-
lone-21-O-b-carbonylpropionic
acid
instead
of
hydrocortisone-21-O-b-carbonylpropionic acid 575 mg
(90%) of the title compound were obtained as a colorless
20
D
powder. Mp 114–116 °C, ½aꢃ þ95.8 (c 1.0, CHCl3).
FAB-MS 582 [MþH]þ. IR (KBr) 3232, 3215, 3025,
3009, 1710, 1705, 1638, 1604, 1578, 1517, 1500, 1486,
.
1460 1352 cmꢀ1
1H NMR (DMSO-d6): d ¼ 8:092 (d,
J ¼ 7:62 Hz, 2H), 8.084 (d, J ¼ 7:62 Hz, 2H), 7.233 (d,
J ¼ 7:64 Hz, 1H), 6.130 (d, J ¼ 7:64 Hz, 1H), 6.040 (s,

4416
C. Wang et al. / Bioorg. Med. Chem. 12 (2004) 4403–4421
(CHCl3–CH3OH–H2O ¼ 1:1:0.15) to provide 380 mg
20
J ¼ 6:64 Hz, 2H), 3.928 (dt, J ¼ 12:10 Hz, J ¼ 3:55 Hz,
2H), 3.564 (dt, J ¼ 12:10 Hz, J ¼ 3:55 Hz, 2H), 3.248
(m, J ¼ 7:93 Hz, 2H), 3.101 (d, J ¼ 4:78 Hz, 2H), 2.586
(m, J ¼ 8:05 Hz, 2H), 2.451 (m, J ¼ 7:88 Hz, 2H), 2.404
(m, J ¼ 8:44 Hz, 2H), 2.224 (t, J ¼ 4:78 Hz, 2H), 2.200
(m, J ¼ 8:02 Hz, 1H), 2.116 (s, 1H), 2.101 (s, 1H), 2.093
(m, J ¼ 4:78 Hz, 2H), 2.125 (m, J ¼ 7:88 Hz, 2H), 1.934
(m, J ¼ 8:09 Hz, 1H), 1.861 (m, J ¼ 7:94 Hz, 2H), 1.745
(m, J ¼ 8:00 Hz, 1H), 1.710 (m, J ¼ 7:02 Hz, 2H),
1.288 (s, 3H), 1.026 (m, J ¼ 7:85 Hz, 2H), 0.685 (s,
3H). Anal. Calcd for C38H51N5O13: C, 58.08; H, 6.54;
N, 8.91; O, 26.47. Found: C, 58.25; H, 6.33; N,
8.72.
(79%) of the title compound as a colorless powder. Mp
218–220 °C, ½aꢃ þ88 (c 0.3, DMF), FAB-MS (m=z) 764
D
[MþH]þ.
4.24. Prednisolone-21-O-b-carbonylpropionyl-Glu-Asp-
Gly-OH (7b)
(A) Using the same procedure as (A) used for the
preparation of hydrocortisone-21-O-b-carbonylpropio-
nyl-Glu-Asp-Gly-OH with 408 mg (0.7 mmol) of pred-
nisolone-21-O-b-carbonylpropionic acid p-nitrophenolic
ester instead of hydrocortisone-21-O-b-carbonylprop-
ionic acid p-nitrophenolic ester 237 mg (48%) of the title
(B) Using the same procedure as (B) used for the prep-
aration of hydrocortisone-21-O-b-carbonylpropionyl-
Glu-Asp-Gly-OH with 215 mg (0.63 mmol) of His-Gly-
Glu-OH instead of Glu-Asp-Gly-OH 396 mg (80%) of
compound were obtained as a colorless powder. Mp
20
D
178–180 °C, ½aꢃ þ64 (c 0.3, DMF), FAB-MS (m/
e) ¼ 762 [MþH]þ. IR (KBr) 3410, 3339, 3006, 1740,
1700, 1645, 1461 cmꢀ1
.
1H NMR (DMSO-d6):
the title compound were obtained as a colorless powder.
20
D
d ¼ 10:554 (s, 1H), 10.506 (s, 1H), 10.491 (s, 1H), 8.232
(d, J ¼ 6:78 Hz, 1H), 8.101 (d, J ¼ 6:70 Hz, 1H), 7.937
(t, J ¼ 6:51 Hz, 1H), 7.232 (d, J ¼ 7:62 Hz, 1H), 6.138
(d, J ¼ 7:62 Hz, 1H), 6.043 (s, 1H), 4.998 (s, 2H), 4.270
(dt, J ¼ 6:78 Hz, J ¼ 4:69 Hz, 1H), 4.033 (dt,
J ¼ 6:70 Hz, J ¼ 5:10 Hz, 1H), 4.008 (d, J ¼ 6:51 Hz,
2H), 3.908 (dt, J ¼ 12:50 Hz, J ¼ 3:46 Hz, 2H), 3.570
(dt, J ¼ 12:50 Hz, J ¼ 3:46 Hz, 2H), 3.335 (s, 1H), 3.323
(m, J ¼ 8:55 Hz, 2H), 3.315 (s, 1H), 2.670 (d,
J ¼ 5:10 Hz, 2H), 2.338 (m, J ¼ 8:03 Hz, 1H), 2.252 (m,
J ¼ 7:90 Hz, 2H), 2.230 (t, J ¼ 4:97 Hz, 2H), 2.131 (m,
J ¼ 4:85 Hz, 2H), 1.902 (m, J ¼ 8:04 Hz, 1H), 1.834 (m,
J ¼ 7:85 Hz, 2H), 1.775 (m, J ¼ 7:96 Hz, 1H), 1.688 (m,
J ¼ 7:02 Hz, 2H), 1.298 (s, 3H), 1.005 (m, J ¼ 7:95 Hz,
2H), 0.677 (s, 3H). Anal. Calcd for C36H47N3O15: C,
56.76; H, 6.22; N, 5.52; O, 31.50. Found: C, 56.57; H,
6.35; N, 5.66.
Mp 157–158 °C, ½aꢃ þ82 (c 0.3, DMF), FAB-MS (m/e)
¼ 786 [MþH]þ.
4.26. Prednisolone-21-O-b-carbonylpropionyl-His-Gly-
Glu-OH (7d)
(A) Using the same procedure as (A) used for the
preparation of hydrocortisone-21-O-b-carbonylprop-
ionyl-His-Gly-Glu-OH with 408 mg (0.7 mmol) of pred-
nisolone-21-O-b-carbonylpropionic acid p-nitrophenolic
ester instead of hydrocortisone-21-O-b-carbonylprop-
ionic acid p-nitrophenolic ester 227 mg (46%) of the title
compound were obtained as a colorless powder. Mp
20
D
172–174 °C, ½aꢃ þ55.4 (c 0.3, DMF), FAB-MS (m/e)
784 [MþH]þ, IR (KBr) 3433, 3355, 3340, 3008, 1744,
1702, 1656, 1640, 1600, 1589, 1463 cmꢀ1 1H NMR
.
(DMSO-d6): d ¼ 12:323 (s, 1H), 10.114 (s, 1H), 10.088
(s, 1H), 8.234 (d, J ¼ 6:74 Hz, 1H), 8.022 (d,
J ¼ 6:69 Hz, 1H), 7.996 (t, J ¼ 6:65 Hz, 1H), 7.430 (s,
1H), 7.321 (d, J ¼ 7:52 Hz, 1H), 6.881 (s, 1H), 6.140 (d,
J ¼ 7:52 Hz, 1H), 6.088 (s, 1H), 4.981 (s, 2H), 4.557 (dt,
J ¼ 6:74 Hz, J ¼ 4:75 Hz, 1H), 4.325 (m, J ¼ 7:90 Hz,
1H), 4.314 (dt, J ¼ 6:69 Hz, J ¼ 4:82 Hz, 1H), 3.956 (d,
J ¼ 6:65 Hz, 2H), 3.915 (dt, J ¼ 12:55 Hz, J ¼ 3:51 Hz,
2H), 3.570 (dt, J ¼ 12:55 Hz, J ¼ 3:51 Hz, 2H), 3.508 (s,
1H), 3.392 (m, J ¼ 8:44 Hz, 2H), 3.102 (d, J ¼ 4:75 Hz,
2H), 3.318 (s, 1H), 2.305 (m, J ¼ 7:95 Hz, 1H), 2.235 (t,
J ¼ 4:58 Hz, 2H), 2.230 (m, J ¼ 7:86 Hz, 2H), 2.023 (m,
J ¼ 4:82 Hz, 2H), 1.910 (m, J ¼ 7:78 Hz, 2H), 1.878 (m,
J ¼ 8:08 Hz, 1H), 1.741 (m, J ¼ 7:84 Hz, 1H), 1.706 (m,
J ¼ 6:98 Hz, 2H), 1.305 (s, 3H), 1.043 (m, J ¼ 7:69 Hz,
2H), 0.687 (s, 3H). Anal. Calcd for C38H49N5O13: C,
58.23; H, 6.30; N, 8.93. Found: C, 58.05; H, 6.16; N,
8.77.
(B) Using the same procedure as (B) used for the prep-
aration of hydrocortisone-21-O-b-carbonylpropionyl-
Glu-Asp-Gly-OH with 291 mg (0.63 mmol) of prednis-
olone-21-O-b-carbonylpropionic acid instead of hydro-
cortisone-21-O-b-carbonylpropionic acid 370 mg (75%)
of the title compound were obtained as a colorless
20
D
powder. Mp 178–180 °C, ½aꢃ þ64 (c 0.3, DMF), FAB-
MS (m/e) ¼ 784 [MþH]þ.
4.25. Hydrocortisone-21-O-b-carbonylpropionyl-His-Gly-
Glu-OH (7c)
(A) Using the same procedure as (A) used for the
preparation of hydrocortisone-21-O-b-carbonylpropio-
nyl-Glu-Asp-Gly-OH with 215 mg (0.63 mmol) of His-
Gly-Glu-OH instead of Glu-Asp-Gly-OH 257 mg (52%)
of the title compound were obtained as a colorless
20
D
powder. Mp 157–158 °C, ½aꢃ þ82 (c 0.3, DMF), FAB-
(B) Using the same procedure as that in (B) used for the
preparation of hydrocortisone-21-O-b-carbonylprop-
ionyl-Glu-Asp-Gly-OH with 291 mg (0.63 mmol) of
prednisolone-21-O-b-carbonylpropionylpropionic acid
instead of hydrocortisone-21-O-b-carbonylpropionic
MS (m/e) 786 [MþH]þ, IR (KBr) 3422, 3350, 3344,
3008, 1735, 1700, 1671, 1658, 1465 cmꢀ1 1H NMR
.
(DMSO-d6): d ¼ 12:305 (s, 1H), 10.060 (s, 1H), 10.056
(s, 1H), 8.243 (d, J ¼ 6:76 Hz, 1H), 8.015 (d,
J ¼ 6:62 Hz, 1H), 7.988 (t, J ¼ 6:64 Hz, 1H), 5.624 (s,
1H), 4.986 (dt, J ¼ 6:76 Hz, J ¼ 4:78 Hz, 1H), 4.966 (s,
2H), 4.562 (dt, J ¼ 6:62 Hz, J ¼ 4:98 Hz, 1H), 3.996 (d,
acid 380 mg (77%) of the title compound were obtained
20
D
as a colorless powder. Mp 172–174 °C, ½aꢃ þ55.4 (c 0.3,
DMF), FAB-MS (m/e) 784 [MþH]þ.

C. Wang et al. / Bioorg. Med. Chem. 12 (2004) 4403–4421
4417
4.27. Hydrocortisone-21-O-b-carbonylpropionyl-His-Gly-
Lys-OH (8a) and Na-(hydrocortisone-21-O-b-carbonyl-
propionyl)-His-Gly-Ne-(hydrocortisone-21-O-b-carbonyl-
propionyl)-Lys-OH (8b)
3H). Anal. Calcd for C64H88N6O18: C, 62.52; H, 7.21; N,
6.84. Found: C, 62.36; H, 7.35; N, 6.96.
(B) Using the same procedure as that in (B) used for the
preparation of hydrocortisone-21-O-b-carbonylpropion-
yl-Glu-Asp-Gly-OH with 214 mg (0.63 mmol) of His-
(A) Using the same procedure as (A) used for the
preparation of hydrocortisone-21-O-b-carbonylprop-
ionyl-Glu-Asp-Gly-OH with 214 mg (0.63 mmol) of His-
Gly-Lys-OH instead of Glu-Asp-Gly-OH 222 mg (45%)
of 8a and 163 mg (21%) of 8b were obtained as a col-
orless powder.
Gly-Lys-OH instead of Glu-Asp-Gly-OH 247 mg (50%)
20
of 8a (Mp 125–127 °C, ½aꢃ þ70 (c 0.2, DMF), FAB-MS
D
(m=z) 785 [MþH]þ) and 142 mg (20%) of 8b (Mp 111–
113 °C, FAB-MS (m=z) ¼ 1229 [MþH]þ) were obtained
as a colorless powder.
20
Compound 8a: Mp 125–127 °C, ½aꢃ þ70 (c 0.2, DMF),
D
FAB-MS (m=z) 785 [MþH]þ. IR (KBr) 3420, 3415,
4.28. Prednisolone-21-O-b-carbonylpropionyl-His-Gly-
Lys-OH (8c) and Na-(Prednisolone-21-O-b-carbonylprop-
ionyl)-His-Gly- Ne-(prednisolone-21-O-b-carbonylprop-
ionyl)-Lys-OH (8d)
3358, 3350, 3344, 3010, 1742, 1700, 1670, 1658, 1641,
1
1460 cmꢀ1. H NMR (DMSO-d6): d ¼ 12:331 (s, 1H),
12.020 (m, 2H), 10.513 (s, 1H), 8.204 (d, J ¼ 6:79 Hz,
1H), 8.116 (d, J ¼ 6:66 Hz, 1H), 8.011 (t, J ¼ 6:55 Hz,
1H), 7.411 (s, 1H), 6.739 (s, 1H), 6.061 (s, 1H), 4.977 (s,
2H), 4.501 (dt, J ¼ 6:79 Hz, J ¼ 4:82 Hz, 1H), 4.345 (m,
J ¼ 7:91 Hz, 1H), 4.305 (dt, J ¼ 6:66 Hz, J ¼ 4:69 Hz,
1H), 3.992 (d, J ¼ 6:55 Hz, 2H), 3.920 (dt, J ¼ 12:32 Hz,
J ¼ 3:61 Hz, 2H), 3.568 (dt, J ¼ 12:32 Hz, J ¼ 3:61 Hz,
2H), 3.347 (m, J ¼ 8:41 Hz, 2H), 3.336 (s, 1H), 3.320 (s,
1H), 3.168 (d, J ¼ 4:82 Hz, 2H), 2.802 (m, J ¼ 4:33 Hz,
2H), 2.622 (m, J ¼ 8:03 Hz, 2H), 2.423 (m, J ¼ 7:76 Hz,
2H), 2.275 (m, J ¼ 7:88 Hz, 2H), 2.238 (m, J ¼ 8:07 Hz,
1H), 1.936 (m, J ¼ 8:08 Hz, 1H), 1.905 (m, J ¼ 7:85 Hz,
2H), 1.888 (m, J ¼ 4:77 Hz, 2H), 1.782 (m, J ¼ 8:02 Hz,
1H), 1.703 (m, J ¼ 6:97 Hz, 2H), 1.586 (m, J ¼ 4:66 Hz,
2H), 1.322 (s, 3H), 1.275 (m, J ¼ 4:67 Hz, 2H), 1.045 (m,
J ¼ 7:98 Hz, 2H), 0.674 (s, 3H). Anal. Calcd for
C39H56N6O11: C, 59.68; H, 7.19; N, 10.71. Found: C,
59.52; H, 7.30; N, 10.54.
(A) Using the same procedure as (A) used for the
preparation of hydrocortisone-21-O-b-carbonylpropio-
nyl-His-Gly-Lys-OH with 408 mg (0.7 mmol) of pred-
nisolone-21-O-b-carbonylpropionic acid p-nitrophenolic
ester instead of hydrocortisone-21-O-b-carbonylprop-
ionic acid p-nitrophenolic ester 206 mg (42%) of 8c and
114 mg (23%) of 8d were obtained as a colorless powder.
20
Compound 8c: Mp 134–136 °C, ½aꢃ þ49 (c 0.2, DMF),
D
FAB-MS (m=z) 783 [MþH]þ, IR (KBr) 3434, 3421,
3360, 3346, 3340, 3010, 1745, 1700, 1665, 1652, 1640,
.
1600, 1580, 1464 cmꢀ1
1H NMR (DMSO-d6):
d ¼ 12:385 (s, 1H), 12.269 (s, 2H), 10.321 (s, 1H), 8.212
(d, J ¼ 6:85 Hz, 1H), 8.103 (d, J ¼ 6:62 Hz, 1H), 7.992
(t, J ¼ 6:55 Hz, 1H), 7.415 (s, 1H), 7.149 (d,
J ¼ 7:62 Hz, 1H), 6.812 (s, 1H), 6.165 (d, J ¼ 7:62 Hz,
1H), 6.101 (s, 1H), 4.966 (s, 2H), 4.502 (dt, J ¼ 6:85 Hz,
J ¼ 4:82 Hz, 1H), 4.412 (dt, J ¼ 6:62 Hz, J ¼ 4:74 Hz,
1H), 4.356 (m, J ¼ 7:94 Hz, 1H), 4.021 (d, J ¼ 6:55 Hz,
2H), 3.993 (dt, J ¼ 12:88 Hz, J ¼ 2:35 Hz, 2H), 3.651
(dt, J ¼ 12:88 Hz, J ¼ 2:35 Hz, 2H), 3.340 (s, 1H), 3.319
(s, 1H), 3.196 (m, J ¼ 8:44 Hz, 2H), 3.05 (d,
J ¼ 4:82 Hz, 2H), 2.804 (t, J ¼ 4:49 Hz, 2H), 2.338 (m,
J ¼ 7:86 Hz, 2H), 2.285 (m, J ¼ 8:08 Hz, 1H), 1.916 (m,
J ¼ 8:10 Hz, 1H), 1.906 (m, J ¼ 7:88 Hz, 2H), 1.829 (m,
J ¼ 7:88 Hz, 2H), 1.756 (m, J ¼ 7:98 Hz, 1H), 1.732 (m,
J ¼ 4:74 Hz, 2H), 1.702 (m, J ¼ 7:02 Hz, 2H), 1.612 (m,
J ¼ 4:58 Hz, 2H), 1.305 (s, 3H), 1.114 (m, J ¼ 7:66 Hz,
2H), 0.688 (s, 3H). Anal. Calcd for C39H54N6O11: C,
59.83; H, 6.95; N, 10.73; O, 22.48. Found: C, 59.66; H,
7.02; N, 10.54.
Compound 8b: Mp 111–113 °C, FAB-MS (m=z) 1229
[MþH]þ. IR (KBr) 3434, 3428, 3346, 3331, 3324, 3010,
1745, 1702, 1669, 1654, 1643, 1462 cmꢀ1 1H NMR
.
(DMSO-d6) d ¼ 12:331 (s, 1H), 10.045 (s, 1H), 8.463 (d,
J ¼ 6:70 Hz, 1H), 8.126 (t, J ¼ 6:55 Hz, 1H), 8.112 (d,
J ¼ 6:65 Hz, 1H), 8.005 (t, J ¼ 6:44 Hz, 1H), 7.432 (s,
1H), 6.741 (s, 1H), 5.603 (s, 1H), 5.593 (s, 1H), 4.998 (s,
2H), 4.994 (s, 2H), 4.493 (dt, J ¼ 6:70 Hz, J ¼ 4:84 Hz,
1H), 4.127 (dt, J ¼ 6:65 Hz, J ¼ 4:64 Hz, 1H), 3.984 (d,
J ¼ 6:55 Hz, 2H), 3.915 (dt, J ¼ 12:40 Hz, J ¼ 3:58 Hz,
2H), 3.896 (dt, J ¼ 12:43 Hz, J ¼ 3:62 Hz, 2H), 3.612
(dt, J ¼ 12:40 Hz, J ¼ 3:58 Hz, 2H), 3.609 (dt,
J ¼ 12:43 Hz, J ¼ 3:62 Hz, 2H), 3.604 (m, J ¼ 7:88 Hz,
1H), 3.514 (m, J ¼ 7:78 Hz, 1H), 3.442 (m, J ¼ 4:61 Hz,
2H), 2.810 (t, J ¼ 4:62 Hz, 2H), 2.607 (m, J ¼ 8:08 Hz,
2H), 2.600 (m, J ¼ 8:02 Hz, 2H), 2.460 (m, J ¼ 7:81 Hz,
2H), 2.428 (m, J ¼ 7:69 Hz, 2H), 2.407 (m, J ¼ 8:44 Hz,
2H), 2.395 (m, J ¼ 8:54 Hz, 2H), 2.210 (m, J ¼ 8:00 Hz,
1H), 2.207 (m, J ¼ 8:02 Hz, 1H), 2.133 (m, J ¼ 7:88 Hz,
2H), 2.124 (m, J ¼ 7:87 Hz, 2H), 2.115 (s, 1H), 2.111 (s,
1H), 2.101 (s, 1H), 2.008 (s, 1H), 1.961 (m, J ¼ 8:10 Hz,
1H), 1.942 (m, J ¼ 8:09 Hz, 1H), 1.861 (m, J ¼ 7:55 Hz,
2H), 1.840 (m, J ¼ 7:58 Hz, 2H), 1.747 (m, J ¼ 7:12 Hz,
2H), 1.740 (m, J ¼ 7:05 Hz, 2H), 1.762 (m, J ¼ 7:01 Hz,
1H), 1.743 (m, J ¼ 8:01 Hz, 1H), 1.728 (m, J ¼ 4:65 Hz,
2H), 1.702 (m, J ¼ 4:66 Hz, 2H), 1.315 (m, J ¼ 6:62 Hz,
2H), 1.224 (s, 3H), 1.215 (s, 3H), 0.945 (m, J ¼ 7:98 Hz,
2H), 0.921 (m, J ¼ 7:88 Hz, 2H), 0.654 (s, 3H), 0.651 (s,
Compound 8d: Mp 119–121 °C, FAB-MS (m=z) 1225
[MþH]þ. IR (KBr) 3436, 3431, 3366, 3355, 3342, 3010,
1742, 1738, 1706, 1695, 1660, 1648, 1640, 1601, 1591,
1
1464 cmꢀ1. H NMR (DMSO-d6): d ¼ 12:248 (s, 1H),
10.110 (s, 1H), 8.224 (d, J ¼ 6:77 Hz, 1H), 8.137 (d,
J ¼ 6:66 Hz, 1H), 8.121 (t, J ¼ 4:40 Hz, 1H), 7.902 (t,
J ¼ 6:50 Hz, 1H), 7.438 (s, 1H), 7.334 (d, J ¼ 7:54 Hz,
1H), 7.330 (d, J ¼ 7:57 Hz, 1H), 6.786 (s, 1H), 6.140 (d,
J ¼ 7:54 Hz, 1H), 6.139 (d, J ¼ 7:57 Hz, 1H), 6.062 (s,
1H), 6.055 (s, 1H), 4.998 (s, 2H), 4.996 (s, 2H), 4.877 (dt,
J ¼ 6:77 Hz, J ¼ 4:85 Hz, 1H), 4.477 (dt, J ¼ 6:66 Hz,
J ¼ 4:45 Hz, 1H), 4.352 (m J ¼ 7:94 Hz, 1H), 4.350 (m
J ¼ 7:91 Hz, 1H),
2
3.991 (dt, J ¼ 12:85 Hz,

4418
C. Wang et al. / Bioorg. Med. Chem. 12 (2004) 4403–4421
J ¼ 2:42 Hz, 2H), 3.988 (dt, J ¼ 12:80 Hz, J ¼ 2:55 Hz,
2H), 3.929 (d, J ¼ 6:50 Hz, 2H), 3.891 (dt, J ¼ 4:65 Hz,
J ¼ 4:54 Hz, 2H), 3.651 (dt, J ¼ 12:85 Hz, J ¼ 2:42 Hz,
2H), 3.648 (dt, J ¼ 12:80 Hz, J ¼ 2:55 Hz, 2H), 3.401
(m, J ¼ 8:52 Hz, 2H), 3.388 (m, J ¼ 8:33 Hz, 2H), 3.341
(s, 1H), 3.340 (s, 1H), 3.316 (s, 1H), 3.310 (s, 1H), 3.021
(d, J ¼ 4:85 Hz, 2H), 2.311 (m, J ¼ 8:02 Hz, 1H), 2.320
(m, J ¼ 8:01 Hz, 1H), 2.218 (m, J ¼ 7:88 Hz, 2H), 2.216
(m, J ¼ 7:86 Hz, 2H), 1.912 (m, J ¼ 8:00 Hz, 1H), 1.908
(m, J ¼ 8:02 Hz, 1H), 1.914 (m, J ¼ 7:81 Hz, 2H), 1.910
(m, J ¼ 7:83 Hz, 2H), 1.786 (m, J ¼ 4:76 Hz, 2H), 1.755
(m, J ¼ 7:98 Hz, 1H), 1.737 (m, J ¼ 7:96 Hz, 1H), 1.728
(m, J ¼ 7:01 Hz, 2H), 1.715 (m, J ¼ 7:04 Hz, 2H), 1.706
(m, J ¼ 4:66 Hz, 2H), 1.514 (m, J ¼ 4:55 Hz, 2H), 1.314
(s, 3H), 1.310 (s, 3H), 1.033 (m, J ¼ 8:00 Hz, 2H), 1.031
(m, J ¼ 8:02 Hz, 2H), 0.755 (s, 3H), 0.749 (s, 3H). Anal.
Calcd for C64H84N6O18: C, 62.73; H, 6.91; N, 6.86.
Found: C, 62.55; H, 6.79; N, 6.94.
J ¼ 8:00 Hz, 1H), 1.927 (m, J ¼ 8:10 Hz, 1H), 1.903 (m,
J ¼ 7:87 Hz, 2H), 1.859 (m, J ¼ 7:92 Hz, 2H), 1.838 (m,
J ¼ 4:84 Hz, 2H), 1.769 (m, J ¼ 7:96 Hz, 1H), 1.733 (m,
J ¼ 8:01 Hz, 1H), 1.731 (m, J ¼ 7:01 Hz, 2H), 1.698 (m,
J ¼ 7:01 Hz, 2H), 1.510 (m, J ¼ 4:74 Hz, 2H), 1.369 (m,
J ¼ 4:80 Hz, 2H), 1.309 (s, 3H), 1.220 (s, 3H), 1.031 (m,
J ¼ 8:00 Hz, 2H), 0.950 (m, J ¼ 7:98 Hz, 2H), 0.709 (s,
3H), 0.656 (s, 3H). Anal. Calcd for C64H86N6O18: C,
62.63; H, 7.06; N, 6.85. Found: C, 62.47; H, 7.18; N,
6.72.
4.30. Na-(Prednisolone-21-O-b-carbonylpropionyl)-His-
Gly-Ne-(hydrocortisone-21-O-b-carbonylpropionyl)-Lys-
OH (9b)
Using the same procedure as (B) used for the prepara-
tion of hydrocortisone-21-O-b-carbonylpropionyl-Glu-
Asp-Gly-OH from 494 mg (0.63 mmol) of prednisolone-
21-O-b-carbonylpropionyl-His-Gly-Lys-OH and 291 mg
(0.63 mmol) of hydrocortisone-21-O-b-carbonylprop-
ionic acid 570 mg (total yield 74%) of the mixed bis-
steroid substituted peptide prednisolone-His-Gly-Lys-
(hydrocortisone)-OH (9b) were obtained as a colorless
powder. Mp 130–132 °C, FAB-MS (m=z) 1227 [MþH]þ.
IR (KBr) 3434, 3421, 3362, 3349, 3343, 3008, 1745,
1706, 1668, 1650, 1642, 1603, 1583, 1465 cmꢀ1. 1H NMR
(DMSO-d6) d ¼ 12:202 (s, 1H), 10.101 (s, 1H), 8.471 (d,
J ¼ 6:78 Hz, 1H), 8.198 (d, J ¼ 6:70 Hz, 1H), 8.131 (t,
J ¼ 4:60 Hz, 1H), 8.121 (t, J ¼ 6:56 Hz, 1H), 7.413 (s,
1H), 6.751 (s, 1H), 7.365 (d, J ¼ 7:55 Hz, 1H), 6.138 (d,
J ¼ 7:55 Hz, 1H), 6.058 (s, 1H), 5.597 (s, 1H), 4.898 (s,
2H), 4.879 (s, 2H), 4.857 (dt, J ¼ 6:78 Hz, J ¼ 4:84 Hz,
1H), 4.518 (dt, J ¼ 6:70 Hz, J ¼ 4:74 Hz, 1H), 4.322 (m,
J ¼ 7:92 Hz, 1H), 3.981 (dt, J ¼ 12:66 Hz, J ¼ 2:62 Hz,
2H), 3.939 (d, J ¼ 6:56 Hz, 2H), 3.979 (dt, J ¼ 12:62 Hz,
J ¼ 2:60 Hz, 2H), 3.887 (dt, J ¼ 4:65 Hz, J ¼ 4:58 Hz,
2H), 3.615 (dt, J ¼ 12:66 Hz, J ¼ 2:62 Hz, 2H), 3.602
(dt, J ¼ 12:62 Hz, J ¼ 2:60 Hz, 2H), 3.385 (m,
J ¼ 8:52 Hz, 2H), 3.342 (s, 1H), 3.321 (s, 1H), 3.145 (m,
J ¼ 7:88 Hz, 1H), 3.021 (d, J ¼ 4:84 Hz, 2H), 2.611 (m,
J ¼ 8:08 Hz, 2H), 2.430 (m, J ¼ 7:92 Hz, 2H), 2.351 (m,
J ¼ 8:54 Hz, 2H), 2.346 (m, J ¼ 7:90 Hz, 2H), 2.212 (m,
J ¼ 8:00 Hz, 1H), 2.210 (m, J ¼ 8:02 Hz, 1H), 2.138 (s,
1H), 2.135 (m, J ¼ 7:95 Hz, 2H), 2.107 (s, 1H), 1.939 (m,
J ¼ 8:08 Hz, 1H), 1.912 (m, J ¼ 8:05 Hz, 1H), 1.906 (m,
J ¼ 7:88 Hz, 2H), 1.838 (m, J ¼ 7:88 Hz, 2H), 1.832 (m,
J ¼ 4:76 Hz, 2H), 1.777 (m, J ¼ 7:96 Hz, 1H), 1.734 (m,
J ¼ 8:00 Hz, 1H), 1.711 (m, J ¼ 6:98 Hz, 2H), 1.687 (m,
J ¼ 7:01 Hz, 2H), 1.512 (m, J ¼ 4:67 Hz, 2H), 1.309 (s,
3H), 1.280 (m, J ¼ 7:84 Hz, 2H), 1.210 (s, 3H), 0.926 (m,
J ¼ 8:01 Hz, 2H), 0.754 (s, 3H), 0.685 (s, 3H). Anal.
Calcd for C64H86N6O18: C, 62.63; H, 7.06; N, 6.85.
Found: C, 62.79; H, 7.17; N, 6.68.
(B) Using the same procedure as (B) used for the prep-
aration of hydrocortisone-21-O-b-carbonylpropionyl-
His-Gly-Lys-OH with 291 mg (0.63 mmol) of predniso-
lone-21-O-b-carbonylpropionic acid instead of hydro-
cortisone-21-O-b-carbonylpropionic acid 256 mg (52%)
20
of 8c (Mp 134–136 °C, ½aꢃ þ49 (c 0.2, DMF), FAB-MS
D
(m=z) 783 [MþH]þ) and 162 mg (21%) of 8d were ob-
tained as a colorless powder (Mp 119–121 °C, FAB-MS
(m=z) 1225 [MþH]þ).
4.29. Na-(Hydrocortisone-21-O-b-carbonylpropionyl)-
His-Gly-Ne-(prednisolone-21-O-b-carbonylpropionyl)-
Lys-OH (9a)
Using the same procedure as (B) used for the prepara-
tion of hydrocortisone-21-O-b-carbonylpropionyl-Glu-
Asp-Gly-OH from 494 mg (0.63 mmol) of hydrocorti-
sone-21-O-b-carbonylpropionyl-His-Gly-Lys-OH and
291 mg (0.63 mmol) of prednisolone-21-O-b-carbonyl-
propionic acid 600 mg (total yield 78%) of the mixed bis-
steroid substituted peptide hydrocortisone-His-Gly-Lys-
(prednisolone)-OH (9a) were obtained as a colorless
powder. Mp 122–125 °C, FAB-MS (m=z) 1227 [MþH]þ.
IR (KBr) 3436, 3425, 3361, 3354, 3346, 3010, 1746,
1710, 1666, 1650, 1642, 1600, 1585, 1464 cmꢀ1. 1H NMR
(DMSO-d6): d ¼ 12:249 (s, 1H), 10.12 (s, 1H), 8.209 (d,
J ¼ 6:79 Hz, 1H), 8.119 (d, J ¼ 6:65 Hz, 1H), 8.121 (t,
J ¼ 4:52 Hz, 1H), 7.997 (t, J ¼ 6:59 Hz, 1H), 7.459 (s,
1H), 7.307 (d, J ¼ 7:69 Hz, 1H), 6.836 (s, 1H), 6.139 (d,
J ¼ 7:69 Hz, 1H), 6.071 (s, 1H), 5.606 (s, 1H), 4.989 (s,
2H), 4.893 (s, 2H), 4.887 (dt, J ¼ 6:79 Hz, J ¼ 4:84 Hz,
1H), 4.518 (dt, J ¼ 6:65 Hz, J ¼ 4:74 Hz, 1H), 4.352 (m,
J ¼ 7:94 Hz, 1H), 3.987 (d, J ¼ 6:59 Hz, 2H), 3.979 (dt,
J ¼ 12:72 Hz, J ¼ 2:60 Hz, 2H), 3.965 (dt, J ¼ 12:80 Hz,
J ¼ 2:65 Hz, 2H), 3.887 (dt, J ¼ 6:65 Hz, J ¼ 4:74 Hz,
2H), 3.641 (dt, J ¼ 12:72 Hz, J ¼ 2:60 Hz, 2H), 3.637
(dt, J ¼ 12:80 Hz, J ¼ 2:65 Hz, 2H), 3.380 (m,
J ¼ 8:44 Hz, 2H), 3.339 (s, 1H), 3.215 (m, J ¼ 7:88 Hz,
1H), 3.322 (s, 1H), 3.020 (d, J ¼ 4:84 Hz, 2H), 2.611 (m,
J ¼ 8:10 Hz, 2H), 2.465 (m, J ¼ 7:92 Hz, 2H), 2.343 (m,
J ¼ 8:44 Hz, 2H), 2.224 (m, J ¼ 8:04 Hz, 1H), 2.214 (m,
J ¼ 7:90 Hz, 2H), 2.149 (m, J ¼ 8:04 Hz, 1H), 2.128 (m,
J ¼ 7:95 Hz, 2H), 2.123 (s, 1H), 2.104 (s, 1H), 1.957 (m,
4.31. 3-(His-Gly-Lys-NH-N)-Hydrocortisone (10a)
(A) The solution of 120 mg (0.35 mmol) of His-Gly-Lys-
NH-NH2, 134 mg (0.35 mmol) of hydrocortisone in
5 mL of methanol was adjusted to pH 2 with glacial
acetic acid and stirred at room temperature for 48 h and
TLC (CHCl3–CH3OH–H2O ¼ 1:1:0.15) indicated the

C. Wang et al. / Bioorg. Med. Chem. 12 (2004) 4403–4421
4419
20
complete disappearance of His-Gly-Lys-NH-NH2. After
evaporation the residue was washed with ethyl acetate to
provide the crude product. The crude product was
crystallized in DMF–ether (1:5) to provide 180 mg (75%)
colorless powder. Mp 233–235 °C (dec.), ½aꢃ þ145.5 (c
0.3, DMF), FAB-MS (m=z) 697 [MþH]þD. IR (KBr)
3441, 3429, 3359, 3350, 3340, 3000, 1740, 1702, 1662,
1653, 1640, 1605, 1583, 1460 cmꢀ1. H NMR (DMSO-
1
of the title compound as a colorless powder. Mp 218–
d6) d ¼ 12:221 (s, 1H), 12.146 (d, J ¼ 4:70 Hz, 2H),
12.120 (s, 2H), 10.190 (s, 1H), 8.006 (d, J ¼ 6:69 Hz,
1H), 7.983 (t, J ¼ 6:55 Hz, 1H), 7.464 (s, 1H), 7.308 (d,
J ¼ 7:58 Hz, 1H), 6.775 (s, 1H), 6.127 (d, J ¼ 7:58 Hz,
1H), 6.076 (s, 1H), 4.677 (s, 2H), 4.667 (dt, J ¼ 6:69 Hz,
J ¼ 4:58 Hz, 1H), 4.354 (m, J ¼ 7:86 Hz, 1H), 4.049 (t,
J ¼ 4:84 Hz, 1H), 3.979 (d, J ¼ 6:55 Hz, 2H), 3.398 (m,
J ¼ 8:44 Hz, 2H), 3.332 (s, 1H), 3.311 (s, 1H), 3.044 (d,
J ¼ 4:84 Hz, 2H), 2.835 (m, J ¼ 4:58 Hz, 2H), 2.454 (s,
1H), 2.235 (m, J ¼ 8:03 Hz, 1H), 2.218 (m, J ¼ 7:95 Hz,
2H), 1.922 (m, J ¼ 7:99 Hz, 1H), 1.906 (m, J ¼ 7:89 Hz,
2H), 1.779 (m, J ¼ 4:77 Hz, 2H), 1.773 (m, J ¼ 8:01 Hz,
1H), 1.725 (m, J ¼ 7:01 Hz, 2H), 1.559 (m, J ¼ 4:57 Hz,
2H), 1.451 (m, J ¼ 4:64 Hz, 2H), 1.299 (s, 3H), 1.033 (m,
J ¼ 7:87 Hz, 2H), 0.738 (s, 3H). Anal. Calcd for
C35H52N8O7: C, 60.33; H, 7.52; N, 16.08. Found: C,
60.42; H, 7.41; N, 16.22.
20
220 °C (dec.), ½aꢃ þ173 (c 0.3, DMF), FAB-MS (m=z)
D
699 [MþH]þ. IR (KBr) 3442, 3435, 3358, 3350, 3342,
3008, 1740, 1709, 1661, 1648, 1640, 1462 cmꢀ1. 1H NMR
(DMSO-d6): d ¼ 12:312 (S, 1H), 12.234 (S, 2H), 12.107
(t, J ¼ 4:45 Hz, 2H), 10.451 (s, 1H), 8.002 (d,
J ¼ 6:60 Hz, 1H), 7.976 (t, J ¼ 6:65 Hz, 1H), 7.395 (s,
1H), 6.824 (s, 1H), 5.478 (s, 1H), 4.845 (s, 2H), 4.558 (dt,
J ¼ 6:60 Hz, J ¼ 4:75 Hz, 1H), 4.051 (t, J ¼ 4:86 Hz,
1H), 3.993 (d, J ¼ 6:65 Hz, 2H), 3.167 (m, J ¼ 7:88 Hz,
1H), 3.054 (d, J ¼ 4:85 Hz, 2H), 2.878 (m, J ¼ 4:61 Hz,
2H), 2.650 (m, J ¼ 8:08 Hz, 2H), 2.415 (s, 1H), 2.401 (m,
J ¼ 7:87 Hz, 2H), 2.325 (m, J ¼ 8:54 Hz, 2H), 2.238 (m,
J ¼ 8:00 Hz, 1H), 2.126 (m, J ¼ 7:87 Hz, 2H), 2.115 (s,
1H), 2.103 (s, 1H), 1.942 (m, J ¼ 8:08 Hz, 1H), 1.786 (m,
J ¼ 4:76 Hz, 2H), 1.868 (m, J ¼ 7:89 Hz, 2H), 1.742 (m,
J ¼ 8:01 Hz, 1H), 1.720 (m, J ¼ 4:59 Hz, 2H), 1.708 (m,
J ¼ 7:02 Hz, 2H), 1.542 (m, J ¼ 4:61 Hz, 2H), 1.270 (s,
3H), 0.921 (m, J ¼ 7:98 Hz, 2H), 0.665 (s, 3H). Anal.
Calcd for C35H54N8O7: C, 60.15; H, 7.79; N, 16.03.
Found: C, 60.02; H, 7.65; N, 16.21.
(B) Using the same procedure as that in (B) used for the
preparation of 3-(His-Gly-Lys- NH-N)-hydrocortisone
with 134 mg (0.35 mmol) of prednisolone instead of
hydrocortisone 195 mg (80%) of the title compound as a
20
D
(B) The solution of 260 mg (0.35 mmol) of Boc-His-
(Tos)-Gly-Lys(Z)-NHNH2, 134 mg (0.35 mmol) of
hydrocortisone in 15 mL of methanol was adjusted to
pH 2 with glacial acetic acid and stirred at room tem-
perature for 48 h and TLC (CHCl3–CH3OH–
H2O ¼ 1:1:0.15) indicated the complete disappearance of
Boc-His(Tos)-Gly-Lys(Z)-NHNH2. After evaporation
the residue was dissolved in 50 mL of ethyl acetate. The
solution was washed successively with 5% sodium
bicarbonate, 5% citric acid, and saturated sodium
chloride and the organic phase was dried over anhy-
drous sodium sulfate. After filtration and evaporation
under reduced pressure the resulted residue was purified
by silica gel chromatography (CHCl3/CH3OH, 30:1) to
provide the protective intermediates, which was mixed
with 6 mL of dimethyl sulfide, 6 mL of phenyl methyl
ether, and 10 mL of CF3COOH–CF3SO3H (4:1). The
mixture was stirred at 0 °C for 2 h. After removal of
CF3COOH–CF3SO3H the residue was triturated with
ether and the residue was purified on Sephadex G10 and
HPLC to provide 200 mg (total yield 82%) of the title
colorless powder. Mp 233–235 °C (dec.), ½aꢃ þ145.5 (c
0.3, DMF), FAB-MS (m=z) 697 [MþH]þ.
(C) Using the general procedure for removal of all the
semi-permanent groups from 380 mg (0.35 mmol) of 11b
200 mg (82%) of the title compound were obtained as a
20
D
colorless powder. Mp 233–235 °C (dec.), ½aꢃ þ145.5 (c
0.3, DMF), FAB-MS (m=z) 697 [MþH]þ.
4.33. 3-[Boc-His(Tos)-Gly-Lys(Z)-NH-N]-Hydrocorti-
sone (11a)
Using the same procedure as that used for the prepa-
ration of 3-(His-Gly-Lys-NH-N)-hydrocortisone with
260 mg (0.35 mmol) of Boc-His(Tos)-Gly-Lys(Z)-
NHNH2 instead of His-Gly-Lys-NH-NH2 304 mg (80%)
of the title compound were obtained as a colorless
powder. Mp 133–135 °C, ½aꢃ þ125.6 (c 0.3, DMF),
20
D
FAB-MS (m=z) 1087 [MþH]þ. IR (KBr) 3443, 3438,
3354, 3346, 3340, 3002, 1740, 1703, 1662, 1650, 1640,
1609, 1507, 1462, 1450, 1438, 1392, 1378, 1365, 741,
690 cmꢀ1. 3431, 3410, 3365, 3342, 3334, 3090, 3072,
3031, 1750, 1690 cmꢀ1. 1H NMR (DMSO-d6): d ¼ 12:24
(s, 1H), 8.661 (d, J ¼ 6:58 Hz, 1H), 8.112 (d,
J ¼ 6:58 Hz, 1H), 8.100 (t, J ¼ 6:55 Hz, 1H), 7.988 (t,
J ¼ 6:56 Hz, 1H), 7.805 (d, J ¼ 7:84 Hz, 2H), 7.891 (s,
1H), 7.326 (d, J ¼ 7:78 Hz, 2H), 7.240 (t, J ¼ 7:85 Hz,
1H), 7.224 (t, J ¼ 7:81 Hz, 2H), 7.105 (d, J ¼ 7:86 Hz,
2H), 7.015 (s, 1H), 5.480 (s, 1H), 5.255 (s, 2H), 4.564 (s,
2H), 4.501 (dt, J ¼ 6:80 Hz, J ¼ 6:58 Hz, 1H), 4.306 (dt,
J ¼ 6:58 Hz, J ¼ 5:87 Hz, 1H), 3.975 (d, J ¼ 6:56 Hz,
2H), 3.395 (m, J ¼ 6:55 Hz, 2H), 3.176 (m, J ¼ 7:94 Hz,
1H), 3.022 (d, J ¼ 6:80 Hz, 2H), 2.588 (m, J ¼ 8:08 Hz,
2H), 2.401 (m, J ¼ 7:97 Hz, 2H), 2.386 (s, 3H), 2.230 (s,
1H), 2.310 (m, J ¼ 8:55 Hz, 2H), 2.123 (m, J ¼ 7:88 Hz,
2H), 2.115 (s, 1H), 2.102 (s, 1H), 2.206 (m, J ¼ 7:88 Hz,
compound as a colorless powder. Mp 218–220 °C (dec.),
20
½aꢃ þ173 (c 0.3, DMF), FAB-MS (m=z) 699 [MþH]þ.
D
(C) Using the general procedure for removal of all the
semi-permanent groups from380 mg (0.35 mmol) of 11a
195 mg (80%) of the title compound were obtained as a
20
D
colorless powder. Mp 218–220 °C (dec.), ½aꢃ þ173 (c
0.3, DMF), FAB-MS (m=z) 699 [MþH]þ.
4.32. 3-(His-Gly-Lys-NH-N)-Prednisolone (10b)
(A) Using the same procedure as that in (A) used for the
preparation of 3-(His-Gly-Lys- NH-N)-hydrocortisone
with 134 mg (0.35 mmol) of prednisolone instead of
hydrocortisone 171 mg (70%) of the title compound as a

4420
C. Wang et al. / Bioorg. Med. Chem. 12 (2004) 4403–4421
1H), 1.945 (m, J ¼ 8:09 Hz, 1H), 1.848 (m, J ¼ 7:91 Hz,
2H), 1.744 (m, J ¼ 7:99 Hz, 1H), 1.781 (m, J ¼ 5:88 Hz,
2H), 1.704 (m, J ¼ 7:02 Hz, 2H), 1.706 (m, J ¼ 7:01 Hz,
2H),1.530 (m, J ¼ 5:40 Hz, 2H), 1.432 (s, 9H), 1.276 (s,
3H), 0.972 (m, J ¼ 7:89 Hz, 2H), 0.706 (s, 3H). Anal.
Calcd for C55H74N8O13S: C, 60.76; H, 6.86; N, 10.31.
Found: C, 60.88; H, 7.01; N, 10.17.
To each well in a 96-well microplate 100 lL of the cell
suspension, 100 L of RPMI-1640 containing ConA (final
concentration 5 lg/mL) or LPS (final concentration
10 lg/mL) and know concentration solution of tested
compounds were added to make a final cell count of
2 ꢁ 105 cells/well. The plates were incubated at 37 °C in a
humidified incubator containing air/CO2 (95:5). After
44 h of incubation to each well 10 lL of the solution of
MTT (5 mg/mL) were added and the plate was incu-
bated for an additional 4 h. After centrifugation (1000g,
10 min) the precipitates were dried and the residue was
dissolved with 150 lL of DMSO and the solution was
gently shaken. The optical density (OD) of the solution
was read at 570 nm with Biorad and their inhibitory
activities were represented by the tested OD values.
4.34. 3-[Boc-His(Tos)-Gly-Lys(Z)-NH-N]-prednisolone
(11b)
Using the same procedure as that used for the prepa-
ration of 3-(His-Gly-Lys-NH-N)-prednisolone with
260 mg (0.35 mmol) of Boc-His(Tos)-Gly-Lys(Z)-
NHNH2 instead of His-Gly-Lys-NH-NH2 292 mg (77%)
of the title compound were obtained as a colorless
20
D
powder. Mp 145–147 °C ½aꢃ þ120.6 (c 0.3, DMF),
5.2. Phagocytosis of mouse peritoneal macrophages
FAB-MS (m=z) 1085 [MþH]þ. IR (KBr) 3440, 3432,
3356, 3349, 3335, 3002, 1744, 1702, 1660, 1650, 1641,
1601, 1580, 1462, 1395, 1380, 1365, 740, 692 cmꢀ1. H
BALB/C mice were injected intraperitoneally with
1.5 mL of sodium thioglycollate solution (3%). On day 4
the peritoneal macrophages were harvested in Hank’s
balanced salt solution (HBSS). After washing with
RPMI-1640 (supplemented with fetal calf serum and
antibiotics, penicillin and streptomycin) for three times
the cells were suspended in RPMI-1640 and the cell
density was adjusted to 2 ꢁ 106 cells/mL. To each well in
a 96-well microplate 100 lL of the cell suspension were
added and then cultured in the presence of 10 lL of
known concentration solution of the tested compound
in NS at 37 °C with air/CO2 (95:5). After 24 h to the
residue in the cultured well 100 lL of 0.1% solution of
neutral red-stained zymosan were added and incubated
at 37 °C for another 30 min. After washing with HBSS
for three times 100 lL of triton X-100 (10%) were added.
The solution was stood over night and then gently
shaken. The OD of the solution was read at 570 nm with
Biorad and the inhibitory activity was represented by
the tested OD value.
1
NMR (DMSO-d6) d ¼ 12:561 (s, 1H), 8.656 (d,
J ¼ 6:78 Hz, 1H), 8.113 (d, J ¼ 6:70 Hz, 1H), 8.102 (t,
J ¼ 4:61 Hz, 1H), 8.006 (t, J ¼ 6:60 Hz, 1H), 7.852 (s,
1H), 7.758 (d, J ¼ 7:80 Hz, 2H), 7.330 (d, J ¼ 7:72 Hz,
2H), 7.321 (d, J ¼ 7:59 Hz, 1H), 7.242 (t, J ¼ 7:74 Hz,
1H), 7.227 (d, J ¼ 7:70 Hz, 2H), 7.114 (d, J ¼ 7:80 Hz,
2H), 7.030 (s, 1H), 6.140 (d, J ¼ 7:74 Hz, 1H), 6.059 (s,
1H), 5.152 (s, 2H), 4.856 (s, 2H), 4.508 (dt, J ¼ 6:78 Hz,
J ¼ 4:85 Hz, 1H), 4.355 (m, J ¼ 7:94 Hz, 1H), 4.295 (dt,
J ¼ 6:70 Hz, J ¼ 4:77 Hz, 1H), 3.985 (m, J ¼ 4:51 Hz,
2H), 3.982 (d, J ¼ 6:61 Hz, 2H), 3.394 (m, J ¼ 8:55 Hz,
2H), 3.346 (s, 1H), 3.321 (s, 1H), 3.068 (d, J ¼ 4:85 Hz,
2H), 2.388 (s, 3H), 2.320 (s, 1H), 2.245 (m, J ¼ 7:90 Hz,
1H), 2.233 (m, J ¼ 7:96 Hz, 1H), 1.922 (m, J ¼ 8:11 Hz,
1H), 1.906 (m, J ¼ 7:78 Hz, 2H), 1.786 (m, J ¼ 4:77 Hz,
2H), 1.772 (m, J ¼ 7:96 Hz, 1H), 1.704 (m, J ¼ 7:01 Hz,
2H), 1.556 (m, J ¼ 4:56 Hz, 2H), 1.44 (s, 9H), 1.356 (m,
J ¼ 4:66 Hz, 2H), 1.315 (s, 3H), 1.032 (m, J ¼ 7:88 Hz,
2H), 0.702 (s, 3H). Anal. Calcd for C55H72N8O13S: C,
60.87; H, 6.69; N, 10.32. Found: C, 60.67; H, 6.54; N,
10.50.
5.3. Cardiac tissue transplantation
BALB/C mice (male, ranged in age from 12 to 14 weeks
at the time of transplantation, from the Animal Center
of Beijing Medical University) were used as the host
animals, which were housed six animals in a wooden
cage 6 ꢁ 12 ꢁ 6 inches and the normal food and water
were available at all times. Donor tissue was obtained
from C57bl/6 mice ranging in age from near full term
fetuses up to 48 h old. Utilizing one ear of a given host
mouse the ‘pockets’ were on the dorsum of the ear was
made by slitting the skin over the auricular artery at the
base of the ear. The split-heart transplanted was
approximately 3 ꢁ 3 mm in size. All tissue was grafted
promptly following removal from the donor except for a
period of 1–3 min in 0.9% saline solution while the host
site was prepared. The donor tissue was then eased into
the base of the ‘pocket’ near the distal edge of the ear.
Usually within 3–5 days pulsatile activity of the trans-
plant could be observed. One day after transplantation
the mice were randomly divided into 11 groups. The
animals of negative control were given an injection (ip)
5. Bioassays of the peptides and steroid–urotoxins
5.1. Lymphocyte proliferation assay
The spleen of BALB/C mice euthanized by means of
carbon dioxide was collected. The collected spleen was
pushed gently through a stainless steel mesh screen and
the cells were suspended with RPMI-1640 solution in a
sterile petri dish (50 mL). The suspensions of the spleen
cells were then layered on top of a Histopaque solution
(3 mL) in a sterile conical tube. After centrifugation
(1000g, 10 min) the lymphocyte band was taken and
washed with RPMI-1640 via an additional centrifuga-
tion (1000g, 10 min). The final cell pellet was suspended
in RPMI-1640, which contained penicillin (100 IU/mL),
streptomycin (100 mg/mL), heat-inactivated rat serum
(2.5%), and 2-mercaptoethanol (5 ꢁ 10ꢀ5 M). The cells
were counted using a hemocytometer.

C. Wang et al. / Bioorg. Med. Chem. 12 (2004) 4403–4421
4421
of 0.1 mL of DMSO/H2O (1:4, v/v), the animals of po-
References and notes
sitive control were given an injection (ip) of a corre-
sponding dosage of hydrocortisone in 0.1 mL of DMSO/
H2O (1:4, v/v), and the animals of steroid–urotoxin
treating were given an injection (ip) of a corresponding
dosage of hydrocortisone–urotoxin or prednisolone–
urotoxin in 0.1 mL of DMSO/H2O (1:4, v/v) per day
until the electrocardiograms of the grafts disappeared.
Six days after transplantation the animals were anes-
thetized with sodium pentobarbital at a dosage of 75 lg/
g body weight and the electrocardiograms of the grafts
were recorded using the physiography. The interval be-
tween the tissue transplantation and the disappearance
of pulsatile activity was defined as the survival time of a
graft. The data are listed in Tables 7 and 8. The statis-
tical analysis of the data were carried out by the use of
ANOVA test, p < 0:05 is considered significant.
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Acknowledgements
The author, S.P., wishes to thank the pre-explore of key
Basic Research Project (2003CCA04400) of China and
National Natural Scientific Foundation of China
(20232020) for financial support.
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