Synthesis and analgesic effects of kyotorphin-steroid linkers

Article abstract of DOI:10.1016/S0039-128X(01)00112-X

Kyotorphin (KTP, H-Tyr-Arg-OH) was covalently bonded with hydrocortisone or estrone to form the corresponding hydrocortisone-21-O-yl-succinyl-Tyr-ArgOH or estrone-3-O-yl-acyl-Tyr-Arg-OH. Their analgesic activities were investigated using the tail flick test. The potency of the two linkers were significantly higher than that of KTP and the mixture of KTP and hydrocortisone or estrone in the CNS and/or the periphery administration. Copyright

Full text of DOI:10.1016/S0039-128X(01)00112-X

Steroids 66 (2001) 811–815
Synthesis and analgesic effects of kyotorphin—steroid linkers
Chao Wang, Ming Zhao, Jian Yang, Shiqi Peng*
College of Pharmaceutical Sciences, Peking University, Beijing 100083, P.R. China
Received 5 May 2000; received in revised form 24 January 2001; accepted 31 January 2001
Abstract
Kyotorphin (KTP, H-Tyr-Arg-OH) was covalently bonded with hydrocortisone or estrone to form the corresponding hydrocortisone-
21-O-yl-succinyl-Tyr-ArgOH or estrone-3-O-yl-acyl-Tyr-Arg-OH. Their analgesic activities were investigated using the tail flick test. The
potency of the two linkers were significantly higher than that of KTP and the mixture of KTP and hydrocortisone or estrone in the CNS
and/or the periphery administration. © 2001 Elsevier Science Inc. All rights reserved.
Keywords: Hydrocortisone; Estrone; Kyotorphin; Linker; Analgesia
1. Introduction
In the present paper kyotorphin (KTP, Tyr-ArgOH iso-
lated from bovine brain with a morphine-like analgesic
effect [5]) was coupled with hydrocortisone, and estrone.
The analgesic activities of the corresponding linkers were
investigated using the tail flick test.
As endogenous bioactive substances peptides and ste-
roids play important roles in the normal physiology or
disease processes of mammalian systems. The interactions
between the peptides and steroids were observed. For ex-
ample daily administration of 25 mg of estradiol benzoate to
adult female rats for 9 days led to an approximately three
fold increase in number of pituitary TRH binding sites [1].
Estrogen treatment in vivo leads to an increased sensitivity
of the TSH contents [2]. The phenomenon that the effects of
the peptides were enhanced by the steroids through increas-
ing their receptor numbers was named ‘permissive action’
[3]. Based on this concept in our previous paper the linkers
consisted of hydrocortisone and urotoxin tripeptide, UTP-A,
UTP-B or UTP-C [4], through covalent binding were inves-
tigated. It was found that the bioactivities, such as the
prolongation of heterotopic transplanted cardiac tissue sur-
vival of mouse, the inhibitory effects on phagocytosis of
mouse peritoneal macrophage, and the influence on Con A
induced proliferation of spleen lymphocytes of mouse, were
improved. The results suggested that the linkers of the
steroids and peptides may simulate the ‘permissive action’
mentioned above and this kind of conjugation of them may
provide a special modification for steroids and oligopep-
tides.
2. Experimental
2.1. Chemical synthesis
Estrone, hydrocortisone, and protected L-amino acids
were purchased from Sigma Chemical Co. 1HNMR spectra
were recorded on a VXR-300 instrument with tetramethyl-
silane as the internal standard. IR spectra were recorded
with a Perkin-Elmer 983 instrument, and mass spectra were
recorded with a ZAB-MS (70 eV) spectrometer. Elemental
analyses were carried out on a PE-2400 apparatus. Chro-
matography was performed with Qingdao silica gel H.
2.1.1. Tyrosyl-arginine (1):
Using the same procedure as that in the literature the title
compound was obtained [6].
2.1.2. Hydrocortisone-21-O-succinyl-p-nitrophenol ester
(4):
The solution of 500.0 mg (1.08 mmol) of hydrocorti-
sone-21-O-hemisuccinate (3), which was prepared by use of
the same procedures as that in the literature [7], 165.0 mg
(1.19 mmol) of p-nitrophenol, 228.0 mg (1.10 mmol) of
* Corresponding author. Fax: ϩ86-10-62092311.
E-mail address: sqpeng@mail.bjmu.edu.cn or psqbmu@263.net.cn (S.
Peng).
0039-128X/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(01)00112-X

812
C. Wang et al. / Steroids 66 (2001) 811–815
DCC, and 20 ml of anhydrous tetrahydrofuran (THF) was
stirred at 0°C for 1 h and then at room temperature for an
additional 24 h. After the precipitate of dicyclohexylurea
(DCU) was removed by filtration, the filtrate was evapo-
rated to dryness under reduced pressure. The residue was
triturated with ethyl ether to obtain 600.0 mg (95%) of the
title compound, as colorless solid, m.p. 100 ϳ 102°C,
[␣]D ϭ ϩ108° (c ϭ 1.0, CHCl3). FAB-MS (m/e) 584 [M ϩ
H]ϩ.
compound were obtained, as colorless solid, m.p. 214 ϳ
215°C ( Lit. 214 ϳ 215°C), [␣]D ϭ ϩ159° (c ϭ 0.45, THF),
FAB-MS (m/e) 329 [M ϩ H]ϩ.
2.1.6. Estrone-3-O-acyl-(2,6-dichlorobenzyl-tyrosyl)-N -
␻
nitro-arginine-benzyl ester (9)
The solution of 100.0 mg (0.305 mmol) of 8, 220.0 mg
(0.305 mmol) of H-Tyr(2.6-dich-lorobzl)-Arg(NO2)-OBzl,
70.0 mg (0.335 mmol) of DCC and 10 ml of THF was
stirred at 0°C for 1 h and then at room temperature for an
additional 24 h. After removal of DCU, the filtrate was
evaporated to dryness under reduced pressure. The residue
was sequentially triturated with ethyl ether and ethyl acetate
and then purified by column chromatography (CHCl3:
CH3OH, 10:1) to give 270.0 mg (93%) of the title com-
pound, as colorless solid, m.p. 101 ϳ 103°C, [␣]D ϭ ϩ51°
(c ϭ 0.7, CHCl3), FAB-MS (m/e) 941 [M ϩ H]ϩ.
2.1.3. Hydrocortison-21-O-succinyl-tyrosyl-arginine (5):
The solution of 175.0 mg (0.30 mmol) of 4, 101.0 mg
(0.30 mmol) of 1, 5.0 mg of 1-hydroxybenzotriazole
(HOBt) and 5 ml of N,NЈ-dimethylformamide (DMF) was
adjusted to pH 9 with N-methyl morphiline and stirred at
room temperature for 48 h. When TLC (CHCl3:CH3OH
10:1) indicated that the materials had disappeared com-
pletely, the solvent was removed by evaporation under
reduced pressure. The residue was sequentially triturated
with ethyl ether and ethyl acetate and then purified by
2.1.7. Esrone-3-O-acyl-tyrosyl-arginine (10)
To the solution of 300.0 mg (0.32 mmol) of 9 and 10 ml
of methanol 30.0 mg of 5% palladium charcoal were added.
The mixture was stirred under an atmosphere of hydrogen at
room temperature until no more gas was absorbed. The
catalyst was removed by filtration, and the filtrate was
concentrated in vacuum to dryness. After crystallization of
the residue from acetone-water 180 mg (85%) of the title
compound was obtained, m.p. 137–139°C, [␣]D ϭ ϩ50°
(c ϭ 0.6, CH3OH), FAB-MS (m/e) 648 [M ϩ H]ϩ,
1H NMR ((CD3)2CO) ␦/ppm: 0.81 (s, 3H, 18-CH3); 1.40
(m, 2H, 15-H); 2.83 (t, J ϭ 4.8 Hz, 2H, 16-H); 6.60 ϳ 7.20
(m, 7H, Ar-H); 7.25 ϳ 7.50 (m, 3H, N-H). Anal. Calcd for
C35H45O7N5: C 64.49, H 7.00, N 10.81. Found: C 64.28, H
6.81, N 10.54.
column
1.00:1.00:0.15) to give 120.0 mg (51%) of the title com-
pound, as colorless solid, m.p. 207 ϳ 209°C, [␣]D
chromatography
(CHCl3:CH3OH:H2O
ϭ
ϩ67.8° (c ϭ 0.3, DMF), FAB-MS (m/e) 782 [M ϩ H]ϩ,
1HNMR (DMSO-d6) ␦/ppm: 0.76 (s, 3H, 18-CH3); 1.37 (s,
3H, 19-CH3); 6.65 (d, J ϭ 8.1 Hz, 2H, Ar-H); 7.06 (d, J ϭ
8.1 Hz, 2H, Ar-H); 9.24 (s, 1H, Arg-OH). IR (KBr), 3600–
2200 cmϪ1 (br. OH); 1650 cmϪ1 (C ϭ O). Anal. Calcd for
C40H55O11N5 : C 60.44, H 7.09, N 8.96. Found: C 60.21, H
6.84, N 8.59.
2.1.4. Ethyl estrone-3-O-ylacetate (7)
Using the same procedure as that in the literature [8]
from 200.0 mg (0.74 mmol) of estrone and 370.0 mg (2.20
mmol) of ethyl bromoacetate 239 mg (91%) of the title
compound were obtained, as colorless solid, m.p. 98 ϳ
100°C (Lit. 98 ϳ 100°C), [␣]D ϭ ϩ138° (c ϭ 0.5, THF),
FAB-MS (m/e) 357 [M ϩ H]ϩ.
3. Bioassay
3.1. Administration in the central nervous system
2.1.5. Estrone-3-O-ylacetic acid (8)
Using the same procedure as that in the literature [8]
from 200.0 mg (0.62 mmol) of 7 199.0 mg (98%) of the title
Male rats weighing 250 Ϯ 50 g were used. Administra-
tion was carried out by intracerebroventricular (i.c.v.) injec-
Table 1
The analgesic effects of the compounds within 1 h after icv injection (X Ϯ SE; n ϭ 10)
Comp.
Pain threshold variation at corresponding dose (␮mol/kg)
0.2
0.4
31.4 Ϯ 2.5a**
0.8
1
5
10
2
12.8 Ϯ 1.6a*
70.8 Ϯ 2.5a***
84.2 Ϯ 2.8a***,b*
88.1 Ϯ 3.1a***,b***
10.7 Ϯ 1.9
28.1 Ϯ 2.2a***,b***
33.7 Ϯ 4.1a***,b***
0.8 Ϯ 2.3
41.7 Ϯ 2.1a***,b**
46.3 Ϯ 2.9a***,b**
Ϫ10.3 Ϯ 10.5
1.7 Ϯ 3.4
6
5.1 Ϯ 2.8
6.1 Ϯ 2.0
1 ϩ 2
1 ϩ 6
Control
26.1 Ϯ 3.0a***,b**
22.3 Ϯ 3.3a***,b*
36.8 Ϯ 5.3a***
47.3 Ϯ 4.0a***,b**
0.2 Ϯ 4.0
78.8 Ϯ 2.5a***,b*
67.9 Ϯ 2.8a***
a Compared to control; b compared to 1; * P Ͻ 0.05; ** P Ͻ 0.01; *** P Ͻ 0.001.

C. Wang et al. / Steroids 66 (2001) 811–815
813
Table 2
The analgesic effects of the examined compounds within 3 h after ip injection (X Ϯ SE; n ϭ 10)
Comp.
Pain threshold variation at corresponding dose (mmol/kg)
0.16 0.32
1.10 Ϯ 4.94 1.01 Ϯ 3.71
0.64
1
5
10
2
10.02 Ϯ 5.09
13.15 Ϯ 5.69c***
52.82 Ϯ 9.74a***,b***,d***
2.57 Ϯ 3.11
20.07 Ϯ 7.99a*,b**,c***
62.31 Ϯ 11.01a***,b***,d***
1.54 Ϯ 4.43
44.94 Ϯ 10.64a**,b**,c***
80.48 Ϯ 12.58a***,b***,d***
5.18 Ϯ 2.97
6
3.35 Ϯ 4.85
5.84 Ϯ 3.94
8.34 Ϯ 4.87
1 ϩ 2
1 ϩ 6
Control
1.64 Ϯ 4.91
0.96 Ϯ 4.56
0.58 Ϯ 5.46
2.41 Ϯ 3.89
Ϫ0.68 Ϯ 5.06
9.81 Ϯ 6.94
0.19 Ϯ 7.38
a Compared with control; b compared with 1; c compared with 1 ϩ 2; d compared with 1 ϩ 6.
* P Ͻ 0.05; ** P Ͻ 0.01; *** P Ͻ 0.001.
tion as described in details in earlier reports [9]. Each
evaluated compound was dissolved in the mixture of DMF
and water (1:1). Each animal in the drug-receiving groups
was given a single injection of the analgesic agent at a dose
of 0.2, 0.4, or 0.8 ␮mol/kg in 10 ␮l of the mixture of DMF
and water (1:1). The control group was given an injection of
the mixture of DMF and water (1:1).
The analgesic effects of the examined compounds were
evaluated by the tail-flick latency (TFL) test [10,11]. After
the administration, the pain thresholds were measured at 5
sured 3 times and these readings were averaged and consti-
tuted the basic pain threshold values. After the compounds
were administered, the pain thresholds were re-tested at 10
min intervals. This measurement was carried out for 180
min. In order to prevent the heat induced damage to the
animal tail the heat stimulation should be no longer than 15
s. The analgesia potency of the tested compound was ex-
pressed with the pain threshold variation. The pain thresh-
old variation was calculated according to the following
formula:
After administration pain threshold value Ϫ Basic pain threshold value
Pain threshold variation ϭ
ϫ 100%
Basic pain threshold value
min intervals and total measurement was carried out for 60
min. The details of TFL measurement and pain threshold
variety calculations were similar to those of the following
administration in periphery.
All values of the pain threshold variation for each animal
were averaged and constituted one sample. The statistical
analysis of the data were carried out by using standard
analysis of variance methods. Student’s t-test was used for
the comparison of the difference between the individual
groups. The results are summarized in Tables 1 and 2.
3.1.1. Administration in periphery
Male ICR mice weighing 25 Ϯ 2 g were used. The
experiments were performed at room temperature. Each
animal in the drug-receiving groups was given a single
intraperitoneal (i.p.) injection of analgesic agent at a dose of
0.16, 0.32, or 0.64 mmol/kg in 0.2 ml of the mixture of
DMF and water (1:1). The mice in the control group were
given an injection of the mixture of DMF and water.
The analgesic effects of the tested compounds were eval-
uated by TFL. Each animal was placed in turn in a cylin-
drical cage, with the tail extending from the end of the cage.
A beam of heat, from a 12V 50W bulb was focused on the
tip of the tail of each animal and the time needed for the heat
inducing the tail flick was measured. The lamp was adjusted
to give a normal reaction time of 4 ϳ 6 s by a transformer
and a control reaction time was established before the ani-
mals receiving the tested compound. Each animal was mea-
Fig. 1. The time course of analgesia within 1 h after icv injection at a dose
of 0.2 ␮mol/kg.

814
C. Wang et al. / Steroids 66 (2001) 811–815
Fig. 2. The time course of analgesia within 2 h after ip injection at a dose
of 0.16 mmol/kg.
4. Results and discussion
Scheme 2. Catalytic hydrogenation of the protected linker to give estrone-
3-O-ylacylTyr-Arg wherein (a) Ethyl bromoacetate, Sodium ethoxide,
THF; (b) KOH/EtOH-H2O; and HCl/H2O; (c) H-Tyr(2,6-DichloroBzl)-
Arg(NO2)OBzl, DCC, THF; (d) H2/Pd/C, MeOH.
The data in Table 1 show the analgesic effects of KTP (1)
as determined by thermal irradiation-tail flick method after
intracerebroventricular injection in rats. Even through hy-
drocortisone (2) or estrone (6) alone exhibited no analgesic
effect, the mixture of hydrocortisone or estrone with KTP
did, and the potencies of the mixtures were higher than that
of KTP alone. This evidence suggested that the analgesic
effects of KTP could be enhanced in the presence of hydro-
cortisone or estrone.
On the other hand the analgesic effects of the hydrocor-
tisone-KTP (5) and estrone-KTP (10) were significantly
higher than that of KTP, not only with respect to potency
but also in the duration of analgesia (Fig. 1).
In general, the means of administration may effect the
bio-activity. After i.p. injections of compounds in mice, the
analgesic effects were changed significantly. The data in
Table 2 indicated that i.p. injections of hydrocortisone,
estrone, KTP, and the mixture of KTP with hydrocortisone
or estrone exhibited no analgesic activity at all, implicating
that with i.p. injection there was no ‘permissive action’
between KTP and hydrocortisone or estrone. In this case
KTP may not be transported from the periphery to the
central neural system.
In contrast hydrocortisone-KTP and estrone-KTP exhib-
ited good analgesia (Fig. 2), suggesting that even though
with i.p. injection the ‘permissive action’ of KTP and hy-
drocortisone or estrone can be observed. In the case both
pharmacodynamics and pharmacokinetics were improved
significantly.
There was no loss of body weight in any of male mice
given i.p. injections at daily dose of 1.6 mmol/kg or male
rats given i.c.v. injections at daily dose of 2.0 ␮mol/kg of
hydrocortisone-KTP or estrone-KTP for five days. The in-
ternal organs and other soft tissues had a normal appearance
on dissection. These results mean that the linkers exhibit no
obvious toxicity.
In order to avoid problems that might arise from cleaving
protected groups in strong acid, two synthetic tactics were
used for preparation of the linkers. The reaction of hydro-
cortisone and succinic anhydride gave hydrocortisone-21-
O-hemisuccinate (3) in high yield. With DCC as the cou-
pling agent 3 was converted into the corresponding
p-nitrophenol ester (4) smoothly. In the presence of N-
methyl morpholine KTP was treated with 4 hydrocortisone-
21-O-succinyl-Tyr-Arg (5) was obtained in 51% yield
(Scheme 1). In the presence of ethanolic sodium ethoxide
the reaction of estrone and ethyl bromoacetate afforded
ethyl estrone-3-O-ylacetate (7) in 91% yield. Treating the
solution of 7 in ethanol was treated with sodium hydroxide
estrone-3-O-ylacetic acid (8) was obtained quantitatively. In
the presence of DCC 8 was amidated with Tyr(2,6-dichlo-
robzl)-Arg(NO2)-Obzl and the protected linker 9 in 93%
yield. After catalytic hydrogenation 9 was converted into
estrone-3-O-ylacyl-Tyr-Arg (10) in 93% yield (Scheme 2).
Scheme 1. Amidation of hydrocortisone-21-O-ylsuccinic acid p-nitrophe-
nol ester with Tyr-ArgOH directly to prepare hydrocortisone-21-O-ylsuc-
cinyl-Tyr-Arg, wherein (a) Succinic anhydride, Pyridine; (b) p-Nitrophe-
nol, DCC, THF; (c). H-Tyr-Arg-OH, N-methyl morpholine, DMF.

C. Wang et al. / Steroids 66 (2001) 811–815
815
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Acknowledgments
The author Peng Shiqi wishes to thank the Key Research
Project (G 1998051111) of China for financial support.
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