IE55292B1 - Enzyme inhibitor - Google Patents

Description

This invention relates to an enzyme inhibitor, and in particular to an inhibitor for an enzyme capable of clotting milk.

The enzyme inhibitor described and claimed herein is useful, for example, in the preparation of a conjugate molecule for use in an enzyme inhibitor labelled immunoassay, such as is described in Patent No 5 5 2 9 1 from which the present Application is divided.

According to the present invention we provide an inhibitor for an enzyme capable of clotting milk wherein the inhibitor is selected from the following group of compounds; P-ser-Z-ala-Q, P-val-Z-val-Q or P-asp-Z-ala-Q wherein .

OH 0 Z is statine or Z is a diradical of the general, formula wherein, Rl is -CH2-NH·; i it H (S) OH 0 C-CH2-, -C-CH2- -S-< 0 i b 0 ll CHy-or -NH-C- (a retroinverso-peptide analogue) and P-2 is -CH2CH2CH2CH3, -CH2CH2SCH3 or -H, and wherein P is selected from A-, his-Α-, pro-his-A·; his-pro-his-Α-, pro-his-pro-his-A-; 3 his-pro-his-pro-his-A? and arg-his-pro-his-pro-his-A-; wherein A is a leucine or valine radical, and wherein Q is selected from -B, -B-pro, -B-pro-pro, -B-pro-pro-lys, -B-pro-pro-lys-lys, and -B-pro-pro-lys-lys-asn, wherein B is an isoleucine or leucine radical.

The inhibitor may be a synthetic polypeptide.

OH Preferably is -(¾-NH- or -&-CH2- H(S) Preferably the inhibitors of the invention are synthetic polypeptides of the general formulae; leu-asp-Z-ala-ile-pro-pro-lys-lys, his-leu-asp-Z-ala-ile-pro-pro-lys-lys, leu-ser-Z-ala-ile-pro-pro-lys-lys or his-leu-ser-Z-ala-ile-pro-pro-lys-lys, wherein Z is as previously defined. Preferably Z is statine.

The following inhibitors of the invention are thus especially preferred his-leu-ser-sta-ala-ile-pro-pro-lys-lys leu-ser-sta-ala-ile-pro-pro-lys-lys The C-terminal and/or the N-terminal amino acid of any of the inhibitors may be substituted. For example the C-terminal amino acid may be esterified and/or the N-terminal amino acid may be acylated.

Some embodiments of the invention are now described by way of Examples.

Two synthetic inhibitors were prepared.

(Unless otherwise stated reagents were obtained from Sigma Chemical Co. Ltd.).

The inhibitors include the amino acid statine (4(S)-amino, 3(S)-hydroxy, 6 methylheptanoic acid) which was prepared in a protected form by the 4 synthesis described in the scheme given below, in the scheme, Z is a benzyloxycarbonyl protecting group, DTBAL represents diisobutylaluminium hydride, THF represents tetrahydrofuran THP represents 5 tetrahydropyranyl and FMOC represents 9-fluorenylmethoxycarbonyl.

ZNHCHC02H GH2N2 ZNHCHC02Me (I) Et20 (II) (a) 0 ZNHCHC-R (III) (II)_-70°DIBAL Toluene (b) OH (III)_-65°LiCH2C02Et ZNHCHCHCH2C02Et THF . (IV) (O (IV) Q/h' OTHP I znhchchch2co 2e t (d) (V) (V) NaOH dioxan (e) OTHP . I .ZNHCHCHCH2C02H (VI) H2/Pd/C (f) OTHP .H2NCHCHCH2C02H (VII) (VI) ΟΤΗΡ (VII) FMOC-C1_ FMOCNHCHCHCH2CO2H (g) (VIII) The reactions described in above scheme were carried out as follows: (a) To an ice-cooled solution of Z-S-leucine I (29g, O.llmol) in ether (200ml) was added a solution of diazomethane in ether ( 6g, O.llmol). After the evolution of nitrogen had ceased, the mixture was concentrated in vacuo to an oil (compound II). Compound II was used in the next reaction without purification. (b) To a vigorously stirred solution of compound II (14.49g, 48.9mmol) in dry toluene (210ml), was added a 1M solution of diisobutylaluminium hydride (DIBAL) (124ml, 124mmol) in hexane at -70°C under nitrogen. After 6 mins, methanol (12ml) was added followed immediately by saturated Rochelle salt solution (500ml). The reaction' mixture was allowed to warm up to room temperature and was then extracted with ether (3x300ml). The organic layers were sequentially washed with saturated sodium chloride solution, dried over sodium sulphate and concentrated to an oil. The yield of compound III was 7.9g. The compound was stored at -20°C and used as soon as possible without further purification. (c) To diisopropylamine (19.2ml, 137mmol) in dry tetrahydrofuran (THF) (45ml) in a dry ice/chloroform bath under nitrogen was added 1.6M n-butyllithium in hexane (85.62ml, 137mmol) by syringe. After one hour, the bath was replaced with a dry ice/ethanol bath and dry ethyl acetate (13.4ml, 13.7mmol) was added slowly whilst keeping the temperature at about -70°C. After the addition, the reaction mixture was 6 left to stir for 15 minutes at which time compound III (22.8g, 92mmol) in dry THF (90ml) was added whilst keeping the temperature below -65°C. The reaction mixture was stirred for 5 minutes at which time 1M hydrochloric acid (300ml) was added and the reaction mixture was allowed to warm up to room temperature and was extracted with ethyl acetate (3x200ml). The organic layers were sequentially washed with saturated sodium chloride solution (1 litre), dried over sodium sulphate and concentrated to an oil of crude material. The yield containing compound IV was 31g. The material was purified twice by silica gel chromatography.

First Chromatography Conditions 31g of the crude material was applied onto 500g of Merck 9385 silica gel (Trade Mark). (2 litres chloroform, followed by 2 litres chloroform/ethyl acetate (95:5)). This yielded 26g of the unseparated two isomers of compound (IV) (3R, 4S and 3S, 4S) having an Rf of 2.0 (201 ethyl acetate in toluene).

Second Chromatography Conditions 12g of partially purified compound IV was applied onto 200g of Merck 9385 silica gel. (1 litre toluene/ethyl acetate (95:5) followed by 1 litre toluene/ethyl acetate (9:1) followed by 1 litre ethyl acetate (85:15) followed by 2 litres toluene/ethyl acetate (4:1)). Sixty 50ml fractions were collected. 2.4g of the required isomer of compound IV (3R,4S) with an Rf of 0.23 was separated from l.lg of the unrequired isomer (3R, 4S) having an Rf of 0.17' (Elution with-201 ethyl acetate in toluene). (d) To compound IV (2.9g, 8.6 mmoles) in dry dioxan (30ml) was added, with stirring, dihydropyran (4ml, 51.6 mmoles) followed by about 2mg of p-toluenesulphonic acid monohydrate. After two hours stirring in the dark H.P.L.C. showed that the reaction was complete. The HPLC was conducted using an ultrasphere reverse phase HPLC column (30cm x 4mm). Flow 1ml min"l; Solvent A: water; Solvent B: methyl cyanide; :260nm. (0min-60% A, 40% B; 15 min 20% A, 80% B; 20 min-20% A; 80% B). The reaction was complete when the peak due to compound IV, with a retention time of 11 minutes, disappeared. The reaction mixture was well shaken with saturated aqueous sodium hydrogencarbonate (300ml) and extracted with dichloromethane (3 x 200 ml). The organic layer was washed with saturated aqueous sodium hydrogencarbonate (1 x 300ml), and water (lx 200ml) and was concentrated to an oil.

The oil was placed in a high vacuum (about 10“1 Torr) overnight to remove excess dihydropyran. The yield of compound V (with dioxan still present) was 3.8g. The compound was used without further purification, (e) To compound (V) (about 2.7g, about 6 mmoles) in dioxan (35ml) was added with stirring 1M aq. sodium hydroxide (35ml). A clear solution formed after 30 minutes and HPLC showed reaction to be complete after 2 hours. The conditions and protocol of HPLC were as in (d) above 20 ul of the reaction mixture was mixed with 2 ul of Aristar glacial acetic acid and 10 ul of the mixture thus formed was injected onto the column. The reaction was complete when the peak due to compound V, with a retention time of 17 minutes, disappeared. Water (50ml) was added and the aqueous solution was washed with ether (3x50ml), ethyl acetate (2x50ml) and its pH was adjusted with concentrated hydrochloric ,acid to pH 6.00. The resultant oily solution was immediately extracted with ethyl acetate (4x75ml). Immediate extraction is necessary because of the lability of the 8 tetrahydropyran (THP) group. The organic layer was washed with water (1x50ml), dried over sodium sulphate and evaporated to an oil.

The yield of compound VI was 1.92g. The compound was used without further purification. (f) To compound (VI) (1.920g, 4.7 mmoles) in methanol (25ml) was added 5%Pd/C catalyst (0.50g). Hydrogen was passed through the mixture, with stirring for one hour. The reaction mixture was filtered through celite and washed with 10% water in methanol (50ml). The collected filtrate was concentrated to a solid. TLC ethanol/hexane, 2:1 v/v) developed with a ninhydrin spray gave a product spot at Rf 0.13.

The yield of compound VII was l.llg. The compound was used without further purification. (g) To a stirred solution of compound VII (Q.526g, 2mmol) in 10% (v/v) aq. sodium carbonate (5.4ml) and dioxan (2ml), was added 9-fluorenylmethoxycarbonyl chloride FM0C-C1 (0,544g, 2.1mmol) in dioxan (5ml) over a period of 20 minutes. During this addition more 10% (w/v) aq. sodium carbonate (13ml) was added over the same period in order to maintain a clear solution. After 60 minutes stirring, water (20ml) was added and the mixture was concentrated to about half its original volume. More water (200ml) was added before the mixture was washed with ether (4x200ml). The aqueous layer was adjusted carefully with hydrochloric'acid to pH6.5 and then immediately extracted with ethyl acetate (4x150ml). The organic layer was washed with water (100ml), dried over sodium sulphate and concentrated to a solid (0.740g).

The product compound VIII was purified by reverse phase chromatography, under the following conditions: 2.5cm x 50cm column packed with Lichroprep RP18 30¾ methanol/water - 100% methanol over 2 litres. 100% methanol 1 litre 20ml fractions were collected.

Fractions 101-118, which contained product, were pooled, concentrated to half its original volume, added to water (500ml) and extracted with dichloromethane (3 x 500ml). The organic layer was washed with water (100ml), dried over sodium sulphate and concentrated to a solid (0.450g).

Nmr (CDCI3): T 9.0 (6H,d, (013)2(¾); 8.6 - 8.0 (9H,M,THP); 7.4 (2H,d, CH2 - C02H); 6.5 (2H, d, CH20C(0)); 2.7 - 2.0 (8H,M (C6H4)2CH2; 1.1 (1H, 6brs, C02H) .

The protected statine produced by the above steps (a)-(g) was used in the preparation of a polyamide supported, partially protected decapeptide IX essentially by the method described by Atherton et al (Proc. 17th European Peptide symp., eds. Blaha, K. and Malon, P. pp 241-246 (1982)). (0cBu » tertiary butoxy, TFA - trifluoroacetic acid (T) « polyamide support).

A polyamide supported, partially protected nonapeptide X was also prepared, using the same method.

H-leu-ser- (X) 0cBu Z Z (Z « benzyloxycarbonyl).

Compounds X and IX are inhibitors of the activity of chymosin. 1C Enzyme Inhibition data was obtained using synthetic peptide substrates containing a chromophoric nitrophenylalanine residue in the position of the scissile bond so that hydroysis can be followed spectrophotometrically. (The chromogenic peptide substrates were supplied by Dr.B.M.Dunn, Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, U.S.A.)· Hydrolysis was followed at 300 nm and the substrate can be used at any pH value (usually between 2-6) at which the enzyme is active. Kinetic parameters for the hydrolysis of a chromogenic peptide substrate by different types of mammalian aspartic proteinases have been reported J. Kay, M. J. Valler and B.M.Dunn, Naturally-occurring inhibitors of Aspartic Proteinases in Proteinase Inhibitors; N.Katunuma, H. Umezawa and H. Holzer Eds., 1983 Springer-Verlag, Berlin (in press). The compound Ac-his-leu-ser-stat-ala-ile-pro-pro-lys-lys was shown to exhibit a Kj. of 0.006 (yjM) at pH 3.1 with Endothia parasiticaprotease.

BIBLIOGRAPHY I. Evolution in the Structure and Function of Carboxyl Proteases. Jordan Tang Mol. Cell. Blochem., 1979, 26(2) 93-109. 2. The inactivation of Pepsin by Diazoacetyl-norleucine Methyl Ester, T.G.

Rajagopalan, W.H.Stein and S. Moore J. Biol. Chem 1966, 241, 4295-4297. 3. Acid Proteases, Structure, Function and Biology, Edited by Jordan Tang, Plenum Press, New York and London, 1977. 4. Proteinases and their inhibitors, Structure, Function and Applied Aspects. Proc. Intern. Symp. 11 Portoroz, Yugoslavia, 1980. Published 1981, Perganon Press.

. Effect of Pepstatin on Acid Proteases T. Aoyagi, 5. Kunimoto, H. Morishina, T. Takeuchi and H.

Urnezawa, J. Antiblot., 1971, ^4(10), 687-694. 6. Synthesis of Analogues of the Carboxyl Protease inhibitor Pepstatin. Effect of Structure on Inhibition of Pepsin and Renin. D.H.Rich, E.T.O. Sun and E. Uln. J. Med. Chem. 1980, 23 27-33. 7. Mechanism of Inhibition of Pepsin by Pepstatin.

Effect of Inhibitor structure on dissocition constant and Time-Dependent Inhibition. D.H.Rich and E.T.O. Sun, Biochm. Pharmacol., 1980, 29 2205-2212. 8. New Renin Inhibitors homologous with pepstatin. 15 M. Eid., G. Evin, B. Castro, J. Menard and P.

Corvol., Biochem J 1981, 197 465-471. 9. The active Site of Acid Proteinases.

T.L.Blundell, H.B.Jones, C. Khan, G. Taylor, B.T. Sewell, L.H.Pearl and S.P. Wood. FEBS Proc, 1980, 20 60, 281-288.

. Synthesis of a 3-oxo-4(s)-amino acid analog of pepstatin. A New Inhibitor of Carboxyl (acid) proteases. D.H.Rich, A.S.Boparai and M.S.Bernatowicz, Biochem. Biophys. Res. Commun. 1982, 25 104(3), 1127-1133. 11. Spin-labelled Pepstatin Binding to Pepsin. A study by Electron spin Resonance and Nuclear Magnetic Resonance. P.G.Schmidt, M.S.Bernatowicz and D.H.

Rich Biochemistry, 1982, 21, 1830-1835. . 12. Solid-phase synthesis of a soluble pepstatin derivative suitable for therapeutic use. B.M.Austen, T*F. Ford, D.A.W. Grant and J. Hermon-Taylor. 12 Biosclence Reports, 1982, 2, 427-432. 13. Inhibition of Cathepsin D by synthetic Oligopeptides. T-Y. Lin and H.R.Williams, J.Biol. Chem., 1979, 11875-11883.

Claims (11)

1. ;5

2. 1. An inhibitor for an enzyme capable of clotting milk wherein the inhibitor is selected from the following group of compounds

3. 5 P-ser-Z-ala-Q, P-val-Z-val-Q or P-asp-Z-ala-Q wherein Z is statine or Z is a diradical of the general formula Ro 0 lZ II -NH-CH-Rl-CH-C- CH2 10 wherein Rl is -CHi-NH-, OH -C-CHjg H (S) 0. ii -c-ch21 OH I H

4. 0 I -p-o- i_ 0 0. o c I ii n 2”» -S-CH2> -S-CH2- or -NH-C- (a retroinverso. 0 peptide analogue) and 15 20 fs -CH2CH2CH2CH3, -CH2CH2S CH3 or -H, and wherein P is selected from A-, his-Α-, pro-his-A-his-pro-his-Α-, pro-his-pro-his-A·; his-pro-his-pro-his-Α-, and arg-his-pro-his-pro-his-A·; wherein A is a leucine or valine radical, and wherein Q is selected from -B, -B-pro, -B-pro-pro, -B-pro-pro-lys, -B-pro-pro-lys-lys, and -B-pro-pro-lys-lys-asn, wherein B is an isoleucine or leucine radical.

5. 2. An inhibitor according to claim 1 wherein Ri is OH -CH2-NH- or -C-CH2- h(s)

6. 3. An inhibitor according to claim 1 or 2 selected from one of the following group of compounds 25 leu-asp-Z-ala-ile-pro-pro-lys-lys, his-leu-asp-Z-ala-ile-pro-pro-lys-lys, leu-ser-Z-ala-ile-pro-pro-lys-lys and his-leu-ser-Z-ala-ile-pro-pro-lys-lys, 5 wherein Z is as defined in claim 1.

7. 4. An inhibitor according to claim 3 wherein Z is statine.

8. 5. A compound having the amino acid sequence -his-leu-ser-sta-ala-ile-pro-pro-lys-lys. 10 6. A compound having the amino acid sequence - leu-ser-sta-ala-ile-pro-pro-lys-lys.

9. 7. An inhibitor according to any one of the preceding claims wherein the C-terminal amino acid is esterified and/or the N-terminal amino acid is 15 acylated.

10. 8. An inhibitor according to claim 1, substantially as hereinbefore described. Dated this the21stday of August, 1986. F. R. KELLY & CO., BY: G&'VA/M

11. 27 Clyde Road, AGENTS FOR THE APPLICANTS.