Synthesis and analgesic effects of kyotorphin—steroid linkers

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 hydrocortisone21-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 As endogenous bioactive substances peptides and steroids play important roles in the normal physiology or disease processes of mammalian systems. The interactions between the peptides and steroids were observed. For example 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 increasing 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 investigated. It was found that the bioactivities, such as the prolongation of heterotopic transplanted cardiac tissue survival 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 oligopeptides. * Corresponding author. Fax: ⫹86-10-62092311. E-mail address: [email protected] or [email protected] (S. Peng).

In the present paper kyotorphin (KTP, Tyr-ArgOH isolated 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.

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 tetramethylsilane 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. Chromatography 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 hydrocortisone-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

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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 evaporated 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 ⬃ 102°C, [␣]D ⫽ ⫹108° (c ⫽ 1.0, CHCl3). FAB-MS (m/e) 584 [M ⫹ H]⫹. 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,N⬘-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 completely, the solvent was removed by evaporation under reduced pressure. The residue was sequentially triturated with ethyl ether and ethyl acetate and then purified by column chromatography (CHCl3:CH3OH:H2O 1.00:1.00:0.15) to give 120.0 mg (51%) of the title compound, as colorless solid, m.p. 207 ⬃ 209°C, [␣]D ⫽ ⫹67.8° (c ⫽ 0.3, DMF), FAB-MS (m/e) 782 [M ⫹ H]⫹, 1 HNMR (DMSO-d6) ␦/ppm: 0.76 (s, 3H, 18-CH3); 1.37 (s, 3H, 19-CH3); 6.65 (d, J ⫽ 8.1 Hz, 2H, Ar-H); 7.06 (d, J ⫽ 8.1 Hz, 2H, Ar-H); 9.24 (s, 1H, Arg-OH). IR (KBr), 3600 – 2200 cm⫺1 (br. OH); 1650 cm⫺1 (C ⫽ 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 ⬃ 100°C (Lit. 98 ⬃ 100°C), [␣]D ⫽ ⫹138° (c ⫽ 0.5, THF), FAB-MS (m/e) 357 [M ⫹ H]⫹.

compound were obtained, as colorless solid, m.p. 214 ⬃ 215°C ( Lit. 214 ⬃ 215°C), [␣]D ⫽ ⫹159° (c ⫽ 0.45, THF), FAB-MS (m/e) 329 [M ⫹ H]⫹. 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 compound, as colorless solid, m.p. 101 ⬃ 103°C, [␣]D ⫽ ⫹51° (c ⫽ 0.7, CHCl3), FAB-MS (m/e) 941 [M ⫹ H]⫹. 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 ⫽ ⫹50° (c ⫽ 0.6, CH3OH), FAB-MS (m/e) 648 [M ⫹ H]⫹, 1 H NMR ((CD3)2CO) ␦/ppm: 0.81 (s, 3H, 18-CH3); 1.40 (m, 2H, 15-H); 2.83 (t, J ⫽ 4.8 Hz, 2H, 16-H); 6.60 ⬃ 7.20 (m, 7H, Ar-H); 7.25 ⬃ 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.

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 ⫾ 50 g were used. Administration was carried out by intracerebroventricular (i.c.v.) injec-

Table 1 The analgesic effects of the compounds within 1 h after icv injection (X ⫾ SE; n ⫽ 10) Comp.

Pain threshold variation at corresponding dose (␮mol/kg) 0.2

1 5 10 2 6 1⫹2 1⫹6 Control a

0.4

12.8 ⫾ 1.6 * 28.1 ⫾ 2.2a***,b*** 33.7 ⫾ 4.1a***,b*** 0.8 ⫾ 2.3 5.1 ⫾ 2.8 26.1 ⫾ 3.0a***,b** 22.3 ⫾ 3.3a***,b* a

0.8

31.4 ⫾ 2.5 ** 41.7 ⫾ 2.1a***,b** 46.3 ⫾ 2.9a***,b** ⫺10.3 ⫾ 10.5 1.7 ⫾ 3.4 36.8 ⫾ 5.3a*** 47.3 ⫾ 4.0a***,b** 0.2 ⫾ 4.0

Compared to control; b compared to 1; * P ⬍ 0.05; ** P ⬍ 0.01; *** P ⬍ 0.001.

a

70.8 ⫾ 2.5a*** 84.2 ⫾ 2.8a***,b* 88.1 ⫾ 3.1a***,b*** 10.7 ⫾ 1.9 6.1 ⫾ 2.0 78.8 ⫾ 2.5a***,b* 67.9 ⫾ 2.8a***

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Table 2 The analgesic effects of the examined compounds within 3 h after ip injection (X ⫾ SE; n ⫽ 10) Comp.

Pain threshold variation at corresponding dose (mmol/kg)

1 5 10 2 6 1⫹2 1⫹6 Control

0.16

0.32

0.64

1.10 ⫾ 4.94 13.15 ⫾ 5.69c*** 52.82 ⫾ 9.74a***,b***,d*** 2.57 ⫾ 3.11 3.35 ⫾ 4.85 1.64 ⫾ 4.91 0.96 ⫾ 4.56

1.01 ⫾ 3.71 20.07 ⫾ 7.99a*,b**,c*** 62.31 ⫾ 11.01a***,b***,d*** 1.54 ⫾ 4.43 5.84 ⫾ 3.94 0.58 ⫾ 5.46 2.41 ⫾ 3.89 ⫺0.68 ⫾ 5.06

10.02 ⫾ 5.09 44.94 ⫾ 10.64a**,b**,c*** 80.48 ⫾ 12.58a***,b***,d*** 5.18 ⫾ 2.97 8.34 ⫾ 4.87 9.81 ⫾ 6.94 0.19 ⫾ 7.38

a Compared with control; b compared with 1; c compared with 1 ⫹ 2; d compared with 1 ⫹ 6. * P ⬍ 0.05; ** P ⬍ 0.01; *** P ⬍ 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

Pain threshold variation ⫽

sured 3 times and these readings were averaged and constituted 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 expressed with the pain threshold variation. The pain threshold variation was calculated according to the following formula:

After administration pain threshold value ⫺ Basic pain threshold value ⫻ 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. 3.1.1. Administration in periphery Male ICR mice weighing 25 ⫾ 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 evaluated by TFL. Each animal was placed in turn in a cylindrical 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 ⬃ 6 s by a transformer and a control reaction time was established before the animals receiving the tested compound. Each animal was mea-

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.

Fig. 1. The time course of analgesia within 1 h after icv injection at a dose of 0.2 ␮mol/kg.

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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 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 hydrocortisone (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 hydrocortisone or estrone. On the other hand the analgesic effects of the hydrocortisone-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

Scheme 1. Amidation of hydrocortisone-21-O-ylsuccinic acid p-nitrophenol ester with Tyr-ArgOH directly to prepare hydrocortisone-21-O-ylsuccinyl-Tyr-Arg, wherein (a) Succinic anhydride, Pyridine; (b) p-Nitrophenol, DCC, THF; (c). H-Tyr-Arg-OH, N-methyl morpholine, DMF.

Scheme 2. Catalytic hydrogenation of the protected linker to give estrone3-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.

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 exhibited good analgesia (Fig. 2), suggesting that even though with i.p. injection the ‘permissive action’ of KTP and hydrocortisone 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 internal 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 hydrocortisone and succinic anhydride gave hydrocortisone-21O-hemisuccinate (3) in high yield. With DCC as the coupling agent 3 was converted into the corresponding p-nitrophenol ester (4) smoothly. In the presence of Nmethyl morpholine KTP was treated with 4 hydrocortisone21-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-dichlorobzl)-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).

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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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