The synthesis, distribution, and anti-hepatic cancer activity of YSL

Article abstract of DOI:10.1016/j.bmc.2004.06.030

(I) HCl/CH₃OH; (II) DCC/HOBt/NMM with corresponding carboxyl component; (III) HCl/CH₃CO₂C₂H₅ (6 mol/L); (IV) NaOH/H₂O/CH₃OH; (V) ³H₂ and 10% Pd/C. YSL was prepared stepwise from C terminal to N terminal with the side chain un-protective amino acids, Boc-Leu-OMe, Boc-Ser-OH, and Boc-Tyr-OH, as the starting materials in 39.5% total yield (31.2 g/per batch). With the side chain un-protective Boc-(3,5-dibromo)-Tyr-OH and HClA·Ser-Leu-OMe as the starting materials (3,5-³H-Tyr)-Ser-Leu-OH was obtained in 29% yield. The determination of radioactive quantity in the urine and feces indicated that even after the administration for 130 h only 8.4% (5.35% in urine and 3.05% in feces) of total radioactive quantity from the metabolite of [3,5-³H-Tyr]-Ser-Leu-OH were monitored. The distribution study revealed the relative accumulation level of the individual tissue was arranged in the sequence of spleen > liver > kidney > lung > heart > muscle > brain. Selecting hepatic cancer as the target YSL significantly increased the survival time of H22 tumor cells implanted mice.

Full text of DOI:10.1016/j.bmc.2004.06.030

Bioorganic & Medicinal Chemistry 12(2004) 4989–4994
The synthesis, distribution, and anti-hepatic cancer activity of YSL
Wenfeng Ding,b Jiali Zhang,c Zhi Yao,d Rong Lu,d Dezhu Wu,e Ginfu Li,e Zilong Shen,c
Yingji Sun,c Gang Lin,f Chao Wang,b Ming Zhaob and Shiqi Penga,*
aCollege of Pharmaceutical Sciences, Capital University of Medical Sciences, Beijing 100054, PRChina
bCollege of Pharmaceutical Sciences, Peking University, Beijing 100083, PRChina
cChina Pharmaceutical University, Nanjing 210009, PRChina
dTianjin Medical University, Tianjin 300070, PRChina
eDepartment of Isotopes, China Institute of Atomic Energy, Beijing 102413, PRChina
fKangzhe Pharmacuetical Ltd Co., Shenzhen 518029, PRChina
Received 23 April 2004; revised 15 June 2004; accepted 18 June 2004
Available online 27 July 2004
Abstract—YSL was prepared stepwise from C terminal to N terminal with the side chain un-protective amino acids, Boc-Leu-OMe,
Boc-Ser-OH, and Boc-Tyr-OH, as the starting materials in 39.5% total yield (31.2g/per batch). With the side chain un-protective
Boc-(3,5-dibromo)-Tyr-OH and HClÆSer-Leu-OMe as the starting materials (3,5-3H-Tyr)-Ser-Leu-OH was obtained in 29% yield.
The determination of radioactive quantity in the urine and feces indicated that even after the administration for 130h only 8.4%
(5.35% in urine and 3.05% in feces) of total radioactive quantity from the metabolite of [3,5-3H-Tyr]-Ser-Leu-OH were monitored.
The distribution study revealed the relative accumulation level of the individual tissue was arranged in the sequence of
spleen>liver>kidney>lung>heart>muscle>brain. Selecting hepatic cancer as the target YSL significantly increased the survival
time of H22 tumor cells implanted mice.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
lished and the therapeutic target has to be defined. As
a practical synthetic route of YSL the starting materials
should be minimal protective and the preparation scale
should be large. In order to define the therapeutic target
of YSL the corresponding in vivo distribution should be
identified. In the in vivo distribution research of peptides
or proteins both ELISA methods7–13 and isotope labe-
ling14,15 may be employed to strengthen the monitor sen-
sitivity. Since the molecular weight of YSL is too low to
obtain the corresponding antibody, the isotope labeling
is obviously the best choice. Though the phenyl group in
Tyr residue of YSL is a convenient function group to be
labeled with isotope 125I, the introduction of 125I at
3 and 5 positions of the phenyl will un-proportionally
increase the molecular weight of YSL from 381 to 581.
In this case the other means such as 3H and 14C labeling
have to be considered. Based on the economical reason
It is well known that animal tissues are the important
source of bioactive peptides.1–5 In the pepsin promotion
degradation of porcine spleen a random peptide library
was obtained, among which one was assigned to Tyr-
Ser-Leu-OH (YSL), which was also obtained 20years
ago from mammalian leucine aminopeptidase promo-
tion degradation of a-BCP (Ala-Pro-Arg-Leu-Arg-Phe-
Tyr-Ser-Leu), a neurotransmitter released by the bag
cells in abdominal ganglion of the marine snail Aplysia6
and no bioactivity was mentioned. In the preliminary
screening YSL exhibited immuno-modulating effects,
such as enhancing concanavalin (ConA) induced prolif-
eration of mouse spleen lymphocytes, phagocytosis of
mouse peritoneal macrophages, and the activity of NK
cells. In our opinion to develop the preliminary screen-
ing results a practical synthetic route has to be estab-
3
the oriental H-labeling YSL is considered for the in
vivo pharmacokinetics process. In the present paper
the normal protective strategy and minimal protective
strategy of the synthesis of YSL were described, the in
vivo distribution was examined by the use of [3,5-3H-
Tyr]-Ser-Leu-OH as the probe, and the effects of YSL
on a tumor model were described.
Keywords: YSL; [3,5-3H-Tyr]-Ser-Leu-OH; Distribution; Anti-hepatic
cancer.
*
Corresponding author. Tel.: +86-10-8280-2482; fax: +86-10-8280-
2311; e-mail: sqpeng@mail.bjmu.edu.cn
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.06.030

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W. Ding et al. / Bioorg. Med. Chem. 12 (2004) 4989–4994
2. Results and discussion
chemistry) were compared and no difference was found
between them.
2.1. The minimal protective strategy is suitable for
preparation of YSL in large scale
2.2. In vivo [3,5-3H-Tyr]-Ser-Leu-OH was degraded
into Tyr and Ser-Leu-OH
In general YSL may be prepared either by normal pro-
tective strategy or by minimal protective strategy.
According to Scheme 1 YSL was prepared stepwise
from C terminal to N terminal with the protective amino
acids, Boc-L-Leu-OBzl, Boc-Ser(Bzl)-OH, and Boc-
Tyr(Dcb)-OH, as the starting materials. The total yield
of YSL was 350mg (44.5%). According to Scheme 2
YSL was prepared stepwise from C terminal to N termi-
nal with the side chain un-protective amino acids, Boc-
L-Leu-OMe, Boc-Ser-OH, and Boc-Tyr-OH, as the
starting materials. The total yield of YSL was 31.2g
(39.5%). As a common strategy for peptide synthesis
when the sequence permits some side chain of amino
acid such as the hydroxyl group of Thr and Ser may
not be protected and its relatively weak nucleophilicity
may be used to realize the selective acylation of amino
group and thus to realize the minimal protective synthe-
sis. The results of the present paper suggested that with
the minimal protective strategy though the yield of YSL
was decreased by 5.4% the scale was enlarged by about
1000 times. In the preparation of (3,5-3H-Tyr)-Ser-Leu-
OH the side chain un-protective Boc-Ser-OH, Boc-L-
Leu-Ome, and Boc-(3,5-dibromo)-Tyr-OH were also
used as the starting materials. The total yield of
(3,5-3H-Tyr)-Ser-Leu-OH was 29% (Scheme 2).
With [3,5-3H-Tyr]-Ser-Leu-OH as tracer the in vivo dis-
position of YSL was investigated. After administration
via intravenous bolus in rat and Rhesus macaque their
plasma radioactivity was determined. Two minutes later
there was no more YSL in the tested plasma. The result
demonstrated that in vivo [3,5-3H-Tyr]-Ser-Leu-OH was
degraded within 2min. The urine and feces of YSL re-
ceived mice were collected. No YSL was found from
the mouse urine and feces collected within 1–6h. The
data supported that in vivo YSL was degraded quickly.
The urine and feces collected after the administration for
130h still gave 8.4% (5.35% in urine and 3.05% in feces)
radioactivity of the total metabolite of [3,5-3H-Tyr]-Ser-
Leu-OH. The data suggested that the excretion of the
metabolites of YSL is rather slow. At 37ꢁC [3,5-3H-
Tyr]-Ser-Leu-OH was incubated in rat plasma for
2min two metabolites were monitored by HPLC, one
gave the same retention time as that of Tyr and another
gave the same retention time as that of Ser-Leu-OH.
Accordingly it could be deduced that in vivo YSL was
degraded into Tyr and Ser-Leu-OH.
2.3. [3,5-3H-Tyr]-Ser-Leu-OH distributed to the tissues
after metabolism
Removing Boc group the protective dipeptide or tripep-
tide was mixed with the solution of hydrogen chloride in
ethyl acetate, the product was immediately separated as
precipitates, and the reaction took less than 1h. Perhaps
the quick precipitation made the product to be out off
the reaction system and thus the acylation of its side
chain was avoided. Preventing racemization the sponifi-
cation of the tripeptidyl ester was carried out at ꢀ4ꢁC
strictly. The specific optical rotation of YSL from solu-
tion method and that of YSL from solid method (Fmoc
After intravenous administration of [3,5-3H-Tyr]-Ser-
Leu-OH solution (9.0·108 dpm/mL, 0.35mg/mL) via
caudal vein the heart, liver, spleen, lung, kidney, brain,
muscle, and genitals of the mice were separated. The tis-
sue dpm value was determined by a scintillation counter
and it was noticed that [3,5-3H-Tyr]-Ser-Leu-OH widely
distributed in the mouse body. The relative accumula-
tion level of the individual tissue was arranged in the
sequence of spleen>liver>kidney>lung>heart>mus-
cle>brain (corresponding to 3.48 1.13, 1.89 0.12,
I
Boc-Ser(Bzl)OH
.
Boc-Ser(Bzl)-Leu-OBzl
II
Boc-Leu-OH
IV
HCl Leu-OBzl
III
II
Boc-Tyr(Dcb)-OH
.
Boc-Tyr(Dcb)-Ser(Bzl)-Leu-OBzl
Tyr-Ser-Leu-OH
Ser(Bzl)-Leu-OBzl
HCl
III
Scheme 1. Synthetic route of YSL with normal protective strategy. Reagents: (I) CsCO3, C6H5CH2Br; (II) HCl/CH3CO2C2H5 (6mol/L); (III) DCC/
HOBt/NMM with corresponding carboxyl component; (IV) HF.
IV
III
Boc-Tyr-Ser-Leu-OH
Boc-Tyr-Ser-Leu-OMe
Tyr-Ser-Leu-OH
II
Boc-Tyr-OH
Boc-Ser-OH
II
I
III
.
.
Leu-OMe
HCl
Boc-Ser-Leu-OMe
HCl Ser-Leu-OMe
Leu-OH
II
Boc-(3,5-dibromo)-Tyr-OH
IV
Boc-(3,5-dibromo-Tyr)-Ser-Leu-OMe
Boc-(3,5-dibromo-Tyr)-Ser-Leu-OH
III
V
(3,5-3H-Tyr)-Ser-Leu-OH
(3,5-dibromo-Tyr)-Ser-Leu-OH
Scheme 2. Synthetic route of YSL with minimal protective strategy. Reagents: (I) HCl/CH3OH; (II) DCC/HOBt/NMM with corresponding carboxyl
component; (III) HCl/CH3CO2C2H5 (6mol/L); (IV) NaOH/H2O/CH3OH; (V) 3H2 and 10% Pd/C.

W. Ding et al. / Bioorg. Med. Chem. 12 (2004) 4989–4994
4991
Table 2. The influence of YSL on BEL-7402human hepatic cancer in
nude mice
1.60 0.26, 1.41 0.24, 1.01 0.21, 0.64 0.19, and
0.62 0.18dpm, respectively).
Compound
NS
Dosage (lg/kg)
Tumor weight (g)
2.0258 0.9147
Since in plasma YSL was quickly degraded into Tyr and
Ser-Leu-OH it is critical to define the contribution of
[3,5-3H-Tyr]-Ser-Leu-OH and 3,5-3H-Tyr-OH to the
monitored tissue radioactivity. Accordingly the distribu-
tion of 3,5-3H-Tyr-OH was also observed. After intrave-
nous administration of 3,5-3H-Tyr-OH solution
(9.0·108 dpm/mL, 0.35mg/mL) via caudal vein the
heart, liver, spleen, lung, kidney, brain, muscle, and gen-
itals of the mice were separated. The tissue dpm value
was determined by scintillation count and it was
noticed that 3,5-3H-Tyr-OH also distributed widespread
in the mouse body. The relative accumulation level of
the individual tissue was arranged in another sequence
of lung>kidney>spleen>liver>heart>muscle>brain
(corresponding to 1.49 0.16, 1.33 0.06, 1.24 0.02,
0.77 0.15, 0.68 0.03, 0.60 0.01, 0.51 0.04). The
obviously different distribution of 3,5-3H-Tyr-OH and
[3,5-3H-Tyr]-Ser-Leu-OH suggested that after the
administration of [3,5-3H-Tyr]-Ser-Leu-OH the moni-
tored tissue radioactivity reflected rather the level of it-
self than that of its metabolite 3,5-3H-Tyr-OH. Thus
the metabolism of YSL possibly follows its distribution.
Based on the high affinity of [3,5-3H-Tyr]-Ser-Leu-OH
with the mouse liver in the present study hepatic cancer
was selected as its target.
0.5mL
Cyclophosphamide
YSL
20
5
0.2611 0.1484a
0.8345 0.4957
0.6327 0.4970a
0.5627 0.3230a
80
160
n=10.
a Compare to NS, p<0.05.
2.5. YSL significantly decreased the weight of BEL-7402
human hepatic tumor in nude mice
To the small tear at the subcutaneous part of the
abdominal cavity of the nude mice a 2–4mm3 BEL-
7402tumor mass was implanted. The animals were
given an injection (ip) with YSL per day for therapeutic
group, with sterile saline for control group, and with
cyclophosphamide for positive control. The experiments
showed that 80lg/kg/d of YSL significantly decreased
the weight of BEL-7402human hepatic tumor in nude
mice18,19 (Table 2). Because of its low dosage (80lg/
kg) and low metabolism stability though a lot of efforts
were made no YSL can be detected from the BEL-7402
human hepatic tumor cells in nude mice.
2.4. YSL significantly prolong the survival time of H22
tumor cells implanted mice
2.6. Experimental
All chemicals were commercially available. The palla-
dium on activated carbon (10%) was prepared by our-
selves. Ultraviolet spectra were recorded on the
UV-210 spectrometer using water as solvent. Radio-
chemical purity was determined in a thin layer radio-
scanner Model RTLS-A. Tritium was counted using a
Packard Liquid Scintillation counter, Model FG-
353G. Elemental analysis was determined with Elemen-
tary Analyzer Model 1106. MS spectra were recorded on
HP-5988 GC–MS spectrometer.
The suspension of 4·107 H22 viable tumor cells and
0.2mL sterile saline were implanted intraperitoneally
into female Kun-Ming mice (18–22g, 6weeks old). Each
animal was given an injection (ip) with YSL per day for
therapeutic group, with sterile saline for control group,
and with cyclophosphamide for positive control. The
survival time was recorded in days following tumor
implantation. The antitumor activity of YSL was repre-
sented by the mean survival time.16,17 The data are
listed in Table 1 and the statistical analysis of the data
were carried out by the use of ANOVA test, p<0.05
is considered significant. The experiments showed
that 10lg/kg/d of YSL significantly prolonged the
survival time of murine H22 tumor cells implantation
mice.
2.7. 3,5-Dibromotyrosine
The solution of 3.0mL of bromine (3.12g, 0.056mol) in
15mL of acetic acid was dropped into the stirred suspen-
sion of 5.0g (0.028mol) of L-tyrosine and a clear red
solution was obtained. The reaction mixture was stirred
at 50–60ꢁC for 2h and then allowed to cool. The formed
precipitates of 3,5-dibromotyrosine hydrobromide were
collected by filtration and washed with ether to give
7.0g of the title compound. The filtrate was evaporated
and the residue was treated with ether to provide an-
other 3.8g of the title compound. The total yield of
crude title compound was 92%. The crude product was
dissolved in 30mL of water and the solution was ad-
justed to pH6 with ammoniasol union (25%). The
formed crystals were collected by filtration and washed
with ether to provide 6.5g (69.4%) of pure title com-
pound. IR (KBr): 3359, 3352–3221, 3011, 1760, 1686,
Table 1. The influence of YSL on H22 tumor in mice
Compound
NS
Dosage (lg/kg)
Survival time (day)
18.53 1.37
0.5mL
Cyclophosphamide
YSL
20
10
20
40
80
21.94 4.54b
25.53 14.14a
25.82 14.29a
30.47 17.89b
35.06 20.90b,c
n=17.
a Compare to NS, p<0.05.
b Compare to NS, p<0.01.
c Compare to cyclophosphamide, p<0.05.
1
1600, 1595, 1492, 1460, 1380, 760, 702cmꢀ1. H NMR
(DMSO-d6): d=11.25 (s, 1H), 11.02 (s, 1H), 9.50 (d,
J=6.42Hz, 2H), 7.08 (s, 2H), 4.02 (m, J=5.68Hz,

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W. Ding et al. / Bioorg. Med. Chem. 12 (2004) 4989–4994
1H), 3.12(d, J=5.75Hz, 2H). FAB-MS (m/e), 340
(4mol/L) was stirred at 0ꢁC for 2h. The colorless precip-
itates were collected by filtration and washed with ethyl
acetate and ethyl ether repeatedly to give 28.5g (99%) of
the title compound, as a colorless powder. Mp 128–
129ꢁC. IR (KBr): 3434, 3423, 3357, 1735, 1630, 1394,
20
[M+H]+, ½aŠ +1.3 (c 5, 4% HCl). Anal. Calcd for
D
C9H9O3NBr2: C, 31.89; H, 2.68; N, 4.13. Found: C,
31.81; H, 2.72; N, 4.06.
2.8. Boc-3,5-dibromotyrosine
1386cmꢀ1
.
1H NMR (DMSO-d6): d=9.42(d,
J=
6.87Hz, 2H), 8.01 (d, J=6.75Hz, 1H), 4.45 (m,
J=5.75Hz, 1H), 4.05 (m, J=5.75Hz, 1H), 3.60 (s,
3H), 3.06 (d, J=5.47Hz, 2H), 2.18 (s, 1H), 1.85 (t,
The solution of 2.1g (0.00478mol) of L-3,5-dibromotyr-
osine in 20mL of NaOH/H2O solution (1mol/L) was
stirred at 0ꢁC for 0.5h, to which the solution of 1.78g
(0.00817mol) of Boc2O in 2mL of THF were added.
The reaction mixture was stirred at room temperature
for another 24h. After evaporation in vacuum the resi-
due was extracted with ether to remove the tert-butyl
carboxylic acid and the aqueous phase was acidified
with a dilute solution of KHSO4 to pH2–3. The sepa-
rated aqueous phase was extracted with ethyl acetate
for three times. The organic layer was combined, washed
with water twice, dried over anhydrous sodium sulfate,
filtered, and evaporated in vacuum to give 2.4g
(85.2%) of the title compound as a colorless powder.
Mp 105–108ꢁC. 1H NMR (DMSO-d6): d=11.23 (s,
1H), 11.05 (s, 1H), 8.25 (d, J=6.45Hz, 1H), 7.09 (s,
2H), 4.02 (m, J=5.64Hz, 1H), 3.12(d, J=5.66Hz,
J=5.64Hz, 2H), 1.81 (m, 1H), 1.04 (d, J=5.55Hz,
20
6H). FAB-MS (m/e): 233 [M+H]+, ½aŠ ꢀ18 (c 2,
D
MeOH). Anal. Calcd for C10H21ClN2O4: C, 44.69; H,
7.88; N, 10.42. Found: C, 44.54; H, 7.64; N, 10.21.
2.11. Boc-Tyr-Ser-Leu-OMe
At 0ꢁC to the stirring solution of 27.45g (0.097mol) of
Boc-L-Tyr-OH, 13.85g (0.102mol) of HOBt, 26.23g
(0.097mol) of HClÆSer-Leu-OMe, 15.6mL (0.121mmol)
of N-methylmorpholine, and 125mL of anhydride THF
21.13g (0.102mol) of DCC were added. The reaction
mixture was adjusted to pH8–9 with N-methylmorpho-
line. After stirring at 0ꢁC for 3h the reaction mixture
was stirred at room temperature for 12h. The precipi-
tated N,N0-dicyclohexylurea was removed by filtration
and the filtrate was evaporated in vacuum. The residue
was dissolved in ethyl acetate and the solution was
washed successively with 5% sodium bicarbonate, 5% cit-
ric acid, and saturated sodium chloride and the organic
phase was separated and dried over anhydrous sodium
sulfate. After filtration and evaporation under reduced
pressure 47.5g (99%) of the title compound were ob-
tained as a colorless powder. IR (KBr): 3434, 3350,
3346, 3012, 1737, 1627, 1600, 1591, 1489, 1452, 1396,
2H), 1.45 (s, 9H). FAB-MS (m/e): 450 [M+H]+,
20
D
½aŠ +13 (c 2, MeOH). Anal. Calcd for C14H17Br2NO5:
C, 38.29; H, 3.90; N, 3.19. Found: C, 38.40; H, 3.79; N,
3.30.
2.9. Boc-Ser-Leu-OMe
The solution of 20.02g (0.121mmol) of L-leucine methyl
ester hydrochloride, 17.2g (0.127mmol) of HOBt,
24.87g (0.121mol) of Boc-Ser-OH, and 15.6mL
(0.121mmol) of N-methylmorpholine were dissolved in
100mL of anhydride tetrahydrofurane. At 0ꢁC to the
stirring solution 26.24g (0.127mol) of DCC were added.
The reaction mixture was adjusted to pH8–9 with N-
methylmorpholine. The reaction mixture was stirred at
0ꢁC for 3h and then at room temperature for 12h.
The precipitated N,N0-dicyclohexylurea was removed
by filtration and the filtrate was evaporated in vacuum.
The residue was dissolved in ethyl acetate and the solu-
tion was washed successively with 5% sodium bicarbo-
nate, 5% citric acid, and saturated sodium chloride
and the organic phase was separated and dried over
anhydrous sodium sulfate. After filtration and evapora-
tion under reduced pressure 39.3g (99%) of the title
compound were obtained as a syrup. IR (KBr):
1
1385, 710cmꢀ1. Mp 147–148ꢁC. H NMR (DMSO-d6):
d=11.00 (s, 1H), 8.18 (d, J=6.83Hz, 1H), 8.16 (d,
J=6.75Hz, 1H), 8.10 (d, J=6.85Hz, 1H), 7.31
(d, J=7.26Hz, 2H), 7.18 (d, J=7.26Hz, 2H), 4.90 (d,
J=5.69Hz, 1H), 4.84 (m, J=5.61Hz, 1H), 4.65 (m,
J=5.84Hz, 1H), 4.04 (d, J=5.66Hz, 2H), 3.62 (s, 3H),
3.13 (d, J=5.47Hz, 2H), 2.02 (s, 1H), 1.85 (t,
J=5.65Hz, 2H), 1.81 (m, 1H), 1.42 (s, 9H), 1.05 (d,
20
D
J=5.57Hz, 6H). ½aŠ ꢀ22 (c 2, MeOH), FAB-MS (m/
e): 496 [M+H]+. Anal. Calcd for C24H37N3O8: C, 58.17;
H, 7.53; N, 8.48. Found: C, 58.11; H, 7.38; N, 8.31.
2.12. Boc-Tyr-Ser-Leu-OH
The solution of 49.5g (0.10mol) of Boc-Tyr-Ser-Leu-
OMe, 500mL of methanol, and 300mL of aqueous
NaOH (2mol/L) was stirred at 0 ꢁC for 5h. The reaction
mixture was adjusted to pH6 with KHSO4 (2mol/L) and
evaporated in vacuum. The residue was again adjusted to
pH1 with KHSO4 (2mol/L). The formed precipitates
were collected by filtration to offer 38.0g (96%) of the
title compound as a colorless powder. Mp 103–105ꢁC.
IR (KBr): 3432, 3353, 3344–2109, 1735, 1622, 1605,
3434, 3350, 1730, 1635, 1396, 1385cmꢀ1 1H NMR
.
(DMSO-d6): d=8.21 (d, J=6.85Hz, 1H), 8.15 (d,
J=6.79Hz, 1H), 4.90 (m, J=5.88Hz, 1H), 4.66 (m,
J=5.86Hz, 1H), 3.60 (s, 3H), 3.08 (d, J=5.45Hz, 2H),
2.02 (s, 1H), 1.78 (t, J=5.63Hz, 2H), 1.82 (m, 1H),
1.42(s, 9H), 1.03 (d, J=5.55Hz, 6H). FAB-MS (m/e):
20
333 [M+H]+, ½aŠ ꢀ36 (c 2, MeOH). Anal. Calcd for
D
C15H28N2O6: C, 54.20; H, 8.49; N, 8.43. Found: C,
54.33; H, 8.29; N, 8.61.
1591, 1492, 1450, 1394, 1387, 710cmꢀ1 1H NMR
.
(DMSO-d6): d=11.04 (s, 1H), 11.00 (s, 1H), 8.12(d,
J=6.85Hz, 1H), 8.05 (d, J=6.76Hz, 1H), 7.89 (d, J=
2.10. HClÆSer-Leu-OMe
6.84Hz, 1H), 7.32(d,
J=7.28Hz, 2H), 7.16 (d,
The solution of 41.2g (0.124mol) of Boc-Ser-Leu-OMe
and 4mL of hydrogen chloride in ethyl acetate
J=7.28Hz, 2H), 4.92 (d, J=5.67Hz, 1H), 4.64 (m,
J=5.63Hz, 1H), 4.49 (m, J=5.82Hz, 1H), 4.02 (d,

W. Ding et al. / Bioorg. Med. Chem. 12 (2004) 4989–4994
4993
J=5.68Hz, 2H), 3.03 (d, J=5.52Hz, 2H), 2.04 (s, 1H),
1.81 (t, J=5.66Hz, 2H), 1.79 (m, 1H), 1.44 (s, 9H), 1.02
2.15. Boc-(3,5-dibromo-Tyr)-Ser-Leu-OH
20
D
(d, J=5.52Hz, 6H). FAB-MS (m/e): 481 [M+H]+, ½aŠ
The solution of 2.1g (3.22mmol) of Boc-(3,5-dibromo-
Tyr)-Ser-Leu-OMe, 50mL of methanol, and 20mL of
aqueous NaOH (2.0mol/L) was stirred at 0ꢁC for 5h.
The reaction mixture was adjusted to pH6 with KHSO4
(2mol/L) and evaporated in vacuum. The residue was
again adjusted to pH1 with KHSO4 (2mol/L). The sep-
arated aqueous phase is extracted with ethyl acetate for
three times. The extractions were pooled, washed with
water twice, dried over anhydrous sodium sulfate, fil-
tered, and evaporated in vacuum to give 1.9g (98.5%)
of the title compound as a colorless powder. Mp 102–
105ꢁC. IR (KBr): 3441, 3352, 3347–2245, 3011, 1740,
1628, 1605, 1590, 1486, 1455, 1392, 1384, 1097,
ꢀ7 (c 2, MeOH). Anal. Calcd for C23H35N3O8: C,
57.37; H, 7.33; N, 8.73. Found: C, 57.44; H, 7.55; N, 8.58.
2.13. Tyr-Ser-Leu-OH
The solution of 38.0g (0.08mol) of Boc-Tyr-Ser-Leu-
OH and 400mL of the solution of hydrogen chloride
in ethyl acetate (6mol/L) was stirred at room tempera-
ture for 3h. The reaction mixture was evaporated in vac-
uum and the residue was dissolved in 40mL of water.
The solution was adjusted to pH6 with ammonia spirit,
the formed precipitates were collected by filtration and
recrystallized in ethanol to provide 31.2g (85%) of the
title compound as a colorless powder. The purity was
higher than 98%. IR (KBr): 3405, 3362, 3359, 3352–
3221, 3012, 1762, 1684, 1602, 1591, 1496, 1462, 1381,
710cmꢀ1 1H NMR (DMSO-d6): d=11.02(s, 1H),
.
11.00 (s, 1H), 8.13 (d, J=6.84Hz, 1H), 8.08 (d,
J=6.74Hz, 1H), 7.94 (d, J=6.83Hz, 1H), 7.33 (s, 2H),
4.91 (d, J=5.65Hz, 1H), 4.66 (m, J=5.65Hz, 1H),
4.47 (m, J=5.84Hz, 1H), 4.05 (d, J=5.66Hz, 2H),
3.05 (d, J=5.54Hz, 2H), 2.02 (s, 1H), 1.83 (t,
1
762, 706cmꢀ1. H NMR (DMSO-d6): d=11.02(s, 1H),
10.41 (s, 1H), 9.66 (d, J=6.83Hz, 2H), 8.21 (d,
J=6.74Hz, 1H), 8.01 (d, J=6.82Hz, 1H), 7.30 (d,
J=7.31Hz, 2H), 7.14 (d, J=7.31Hz, 2H), 4.65
(d, J=5.66Hz, 1H), 4.56 (m, J=5.65Hz, 1H), 4.05 (m,
J=5.78Hz, 1H), 4.10 (d, J=5.66Hz, 2H), 3.17 (d,
J=5.56Hz, 2H), 2.06 (s, 1H), 1.83 (t, J=5.64Hz, 2H),
J=5.65Hz, 2H), 1.76 (m, 1H), 1.42 (s, 9H), 1.04 (d,
20
D
J=5.54Hz, 6H). FAB-MS (m/e): 641 [M+H]+, ½aŠ ꢀ1
(c 2, MeOH). Anal. Calcd for C23H33Br2N3O8: C,
43.21; H, 5.20; N, 6.57. Found: C, 43.40; H, 5.32; N,
6.41.
1.76 (m, 1H), 1.05 (d, J=5.52Hz, 6H). FAB-MS (m/e):
20
382[M+H] +, ½aŠ ꢀ6 (c 2 , HO). Anal. Calcd for
2.16. (3,5-Dibromo-Tyr)-Ser-Leu-OH
2
D
C18H27N3O6Æ1/2H2O: C, 55.37; H, 7.14; N, 10.76.
Found: C, 55.68; H, 7.28; N, 10.76.
The solution of 1.9g (2.97mmol) of Boc-(3,5-dibromo-
Tyr)-Ser-Leu-OH and 20mL of the solution of hydrogen
chloride in ethyl acetate (4mol/L) was stirred at 0ꢁC for
3h. The reaction mixture was filtrated to collect the pre-
cipitates. The precipitates were washed with ethyl ace-
tate and ethyl ether repeatedly to provide 1.5g (88%)
of the title compound as a colorless powder. IR (KBr):
3438, 3354, 3352, 3342–2245, 3010, 1730, 1629, 1608,
2.14. Boc-(3,5-dibromo-Tyr)-Ser-Leu-OMe
At 0ꢁC to the stirring solution of 817mg (3.043mmol) of
HClÆSer-Leu-OMe, 484mg (3.585mmol) of HOBt,
1600mg (3.64mmol) of Boc-3,5-dibromotyrosine,
0.39mL (3.045mol) of N-methylmorpholine and 40mL
of anhydrous tetrahydrofurane, 739mg (3.59mmol) of
DCC was added. The reaction mixture was adjusted to
pH8–9 with N-methylmorpholine. The reaction mixture
was stirred at 0ꢁC for 3h and then at room temperature
for 12h. The precipitated N,N0-dicyclohexylurea was re-
moved by filtration and the filtrate was evaporated in
vacuum. The residue was dissolved in 100mL of ethyl
acetate and washed with saturated solution of NaHCO3,
saturated solution of NaCl, and the solution was washed
successively with 5% sodium bicarbonate, 5% citric acid,
and saturated sodium chloride. The organic phase was
separated and dried over anhydrous sodium sulfate, fil-
tered, and evaporated in vacuum to offer 2.1g (87%) of
the title compound as a colorless powder. Mp 91–93ꢁC.
IR (KBr): 3438, 3354, 3349, 3010, 1730, 1625, 1604,
1590, 1487, 1459, 1380, 1098, 712cmꢀ1 1H NMR
.
(DMSO-d6): d=11.04 (s, 1H), 10.40 (s, 1H), 9.64 (d,
J=6.85Hz, 2H), 8.23 (d, J=6.76Hz, 1H), 8.05 (d,
J=6.84Hz, 1H), 7.32(s, 2H), 4.63 (d, J=5.67Hz, 1H),
4.54 (m, J=5.67Hz, 1H), 4.07 (m, J=5.76Hz, 1H),
4.11 (d, J=5.67Hz, 2H), 3.16 (d, J=5.58Hz, 2H), 2.04
(s, 1H), 1.85 (t, J=5.66Hz, 2H), 1.74 (m, 1H), 1.07 (d,
20
J=5.54Hz, 6H). FAB-MS 540 [M+H]+, ½aŠ +11 (c 2,
D
H2O). Anal. Calcd for C18H25Br2N3O6: C, 40.09; H,
4.67; N, 7.79. Found: C, 40.00; H, 4.81; N, 7.62.
2.17. [3,5-3H-Tyr]-Ser-Leu-OH
In a 20mL reaction flask 5mg (0.01mmol) of H-(3,5-di-
bromoTyr)-Ser-Leu-OH, 12mg of calcium carbonate,
12mg of 10% Pd/C, and 2mL of DMF–water (1:1) were
mixed. The reaction flask was put into liquid nitrogen to
cool and then evacuated to 1·10ꢀ3 mmHg. To the reac-
tion flask tritium was introduced and 650mmHg of vac-
uum was maintained untill the sample thawed. The
reaction mixture was stirred at room temperature for
1h and then cooled with liquid nitrogen. The excess tri-
tium was recovered and backed into the tritium storage
tank. The catalyst Pd/C was removed. To the residue
ethanol was added repeatedly followed by evaporation
to remove the labile tritium and give the crude product,
1592, 1484, 1456, 1394, 1386, 1092, 710cmꢀ1 1H
.
NMR (DMSO-d6): d=11.30 (s, 1H), 8.17 (d,
J=6.85Hz, 1H), 8.14 (d, J=6.76Hz, 1H), 8.11 (d,
J=6.84Hz, 1H), 7.36 (s, 2H), 4.92 (d, J=5.67Hz, 1H),
4.82(m, J=5.63Hz, 1H), 4.67 (m, J=5.86Hz, 1H),
4.02(d, J=5.67Hz, 2H), 3.63 (s, 3H), 3.12 (d,
J=5.46Hz, 2H), 2.01 (s, 1H), 1.86 (t, J=5.67Hz, 2H),
1.82(m, 1H), 1.43 (s, 9H), 1.03 (d,
J=5.56Hz,
20
6H). FAB- MS (m/e): 655 [M+H]+, ½aŠ ꢀ8 (c 2,
D
MeOH). Anal. Calcd for C24H35Br2N3O8: C, 44.12; H,
5.40; N, 6.43. Found: C, 44.30; H, 5.38; N 6.29.

4994
W. Ding et al. / Bioorg. Med. Chem. 12 (2004) 4989–4994
which was purified on silica paper (ethyl acetate–pyr-
idine–acetic acid–water=30:20:6:11 v/v, Rf =0.56) and
then washed with water. The chemical quantity was
determined by UV spectrometer and the radioactivity
was counted with liquid scintillation counter. The radio-
chemical purity was determined by paper chromato-
graphy followed by scanning with thin layer
radioscanner, and the radiochemical purity and specific
activity were >98% and 36Ci/mmol, respectively. HPLC
analysis (chromatograph column: waters symmetry C18,
5lm, 3.9mm·150mm; mobile phase: A, 0.1%TFA/
H2O; B, 0.1%TFA/CH3CN; gradient elution: 0–20min
from 95%B to 65%B, 20–30min from 65%B to 95%B;
flow rate: 1.5mL/min; detector: waters 2487 dual k
absorbance detector; detection wavelength: 215nm) con-
firmed that both YSL and its labeled derivative had
same retention time (13min).
On the abdominal cavity of the nude mice a small tear
was made into which a 2–4mm3 BEL-7402tumor mass
was implanted. On the next day of the tumor implanta-
tion the animal of cyclophosphamide treating group was
injected (ip) 20lg/kg of YSL per day for 60days, the
animal of NS treating group was injected (ip) 0.5mL
of NS per day for 60days, the animal of YSL treating
group was injected (ip) 10, 5, 80, or 160lg/kg of YSL
per day for 60days. Then the nude mice were executed
and the tumors were taken out and weighed.
Acknowledgements
The author, Shiqi Peng, wishes to thank National Nat-
ural Scientific Foundation of China (20232020 and
30372433) for financial support.
2.18. Distribution study in mice
References and notes
Fifty mice were divided into five groups (10/group) in
random. The work solution (9.0·108 dpm/mL,
0.35mg/mL) was prepared and administrated (151.6lg/
kg, 2.5·109 dpm/kg) via caudal vein of the mice. The
mice in each group were put to death at 20min, 1, 1.5,
4, and 12h after administration, respectively. The heart,
liver, spleen, lung, kidney, brain, muscle, and genitals of
the mice were separated and washed with water. From
each tissue 100mg of sample were taken and its dpm
value was determined by scintillation. The relatively
accumulation level was calculated according to the fol-
lowing equation.
1. Morioka, K.; Kinoshita, Y.; Tominaga, K.; Hato, F.;
Itoh, T. Cell. Mol. Biol. 1989, 35, 523.
2. Goldstein, A. L.; Low, T. L.; McAdoo, M.; McClure, J.;
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3. Hughes, J.; Smith, T. W.; Kosterlitz, H. W.; Fothergill,
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1991, 266, 22355.
relative accumulation level
dpm in tissue=weight of tissue
7. Tijssen, P. In Practice and Theory of Enzyme Immuno-
assays; Elsevier Science: Amsterdam, 1985; p 298.
8. Briand, J. P.; Muller, S.; Van Regenmortel, M. H. V.
J. Immunol. Meth. 1985, 78, 59.
¼
ꢁ 100%
total dpm given=weight of mouse
9. Sondergard, A. J.; Lauritzen, E.; Lind, K. J. Immunol.
Meth. 1990, 131, 99.
2.19. Anti murine H22 tumor assay
10. McMartin, C. In Handbook of Experimental Pharmacol-
ogy; Academic: Berlin, 1992; p 41.
11. Tang, Z. M.; Liu, X. W.; Cai, B. X. Chin. J. Pharmacol.
Toxicol. 1996, 10, 161.
12. Wills, R. J.; Ferraiolo, B. L. Clin. Pharmacokinet. 1992,
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13. Nicol, P.; Fadil, F. A. E.; Charpin, G. Peptides 1994, 16,
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14. Burchiel, S. W.; Oette, D.; Day, P. W. Int. J. Immuno-
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15. Tang, G. H.; Jiang, G. H.; Zhang, Y. Chin. Pharm. J.
1998, 33, 227.
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Kun-Ming mice (18–22g, 6weeks old) were divided into
six groups (20/group) in random. The suspension of
4·107 H22 viable tumor cells suspended in 0.2mL ster-
ile saline was implanted intraperitoneally into the mice.
One day later the animal of cyclophosphamide treating
group was injected (ip) 20lg/kg of cyclophosphamide
per day for at most 60days. After 5days the animal of
NS treating group was injected (ip) 0.5mL of NS per
day for 60days. After 5days the animal of YSL treating
group was injected (ip) 10, 20, 40, or 80lg/kg of YSL
per day for at most 60days. The survival time of the
mice in the individual group was recorded in days fol-
lowing tumor implantation.
17. Yang, J. B.; Li, X. W.; Dong, W. H.; Kong, T. H.; Song,
H. X.; Zheng, X. Y.; Liu, G. T. Zhongguo Zhong Yao Za
Zhi 2000, 25, 736.
2.20. Inhibition effect of YSL on BEL-7402 human
hepatic tumor in nude mice
18. Wang, Q.; Wu, L. M.; Li, A. Y.; Zhao, Y.; Wang, N. P.
Zhongguo Zhong Yao Za Zhi 2001, 26, 707.
19. Jiang, M. Zhonghua Zhong Liu Za Zhi 1988, 10, 357.
Health BALB/C(nu/nu) nude mice (18–22g, 4–5week
old) were divided into five groups (10/group) in random.