OLIGOPEPTIDIC INHIBITORS OF ELASTASES AND METHODS OF PREPARATION THEREOF

Abstract: The invention relates to oligopeptidic inhibitors of elastases, with potent inhibition of leukocyte elastase. The inhibitors are isobutylamides of tetra- to pentapeptides which have substituted lysine incorporated in the N-terminal portion of the molecule. This dibasic aminoacid is alternatively substituted at both alpha - and epsilon -aminogroups with higher saturated acids or with omega -carboxylalkanoylic residues. In one type of these compounds the epsilon -aminogroup is acylated with an acyl-alanine, where an acyl means a higher saturated acid. The compounds of this invention are the active components of medicinal preparations used mainly in dental practice.

Description: OLIGOPEPTIDIC INHIBITORS OF ELASTASES AND METHODS OF PREPARATION THEREOF Technical Field The invention refers to oligopeptidic inhibitors of elastases, especially leukocyte elastase (LE). Elastolytic enzymes, for instance LE or pancreatic elastase (PE) are the members of the group of serine proteinases which take part in the degradation of the natural substrate elastin; elastin creates the substantial part of protein pool of organism. In the healthy organism this destructive activity is inhibited by endogenic enzymes, for instance α_- proteinase inhibitor (αi-PI). In the case of insufficiency of αi-PI of various causes (infection, nutrition, working environment, genetic factors) the autodigestion of organs essential to life by LE and PE is observed, which is the basis of origin of serious diseases as for instance acute pancreatitis, pulmonary emphysema, arthritis or gingivitis. Background Art One of the possibilities how to reach homeostasis - the balance between elastase and the corresponding endogenic inhibitor (in the case of a pathogenic condition) is the substitution therapy which means the application of the exogenic natural or synthetic inhibitor of type αi-PI. This is carried out by application of either natural (Antilysin R , Trasylol R , Contrykal R ) or synthetic cti-PI like Nafamstat mesylate, FUT (Tori Co, Tokyo, Japan). Recently, the inventors have suggested a synthetic potent inhibitor for the therapy of acute pancreatitis Glt-Ala 3 -NHEt, Inpankin NUFB 16834 (Gut 33, 701-706, 1992). The serious defect of the isolated (natural) inhibitors is not only their antigenicity (α . -PI shows the species specificity), but also the possible contamination by bovine spongioformic encephalopathy (BSE). On the contrary, the synthetic, low molecular inhibitors are lacking these unwanted side effects, are more efficient and more stable. Their chemical structure is strictly defined and characterised in the chemical and physical way. Disclosure oflnvention The invention relates to oligopeptidic inhibitors of elastases of general structure I. wherein X means A or B, wherein A is different from B Y means A or B or A-Ala, wherein A is different from B and if Y is A-Ala then X means B, A means a saturated acid of 8 to 16 carbon atoms, B means the 3-carboxypropionyl or 4-carboxybutyroyl residue. According to a further aspect of the present invention there is provided a method for the preparation of compounds of general structure I wherein Y is B, characterised in contacting lysine having protected amino groups with a compound of formula Ala-Ala-Pro- H-iBu, deprotecting the ε-amino group of the product, reacting the resulting product with a compound of formula A-X 1 , wherein A has the above meaning and X 1 is a halogen, deprotecting the α-amino group and contacting the resulting compound of formula Lys(A)-Ala-Ala-Pro-NH-iBu with succinic or glutaric anhydride. The invention also includes a method for the preparation of compounds of general structure I wherein Y means A, characterised in contacting a compound of formula A-Lys(W), wherein W is a protective group and A has the above meaning, with a compound of formula Lys(W), wherein W is a protective group and A has the above meaning, with a compound of formula Ala-Ala-Pro-NH-iBu, deprotecting the product and contacting the resulting compound of formula A-Lys-Ala-Ala- Pro-NH-iBu with succinic or glutaric anhydride. The invention also includes the method for the preparation of compounds of general structure I wherein Y is A-Ala, characterised in contacting a compound of formula A-Ala with a compound of formula W-Lys-Ala-Ala-Pro-NH-iBu, wherein W is a protective group and A has the above meaning, deprotecting the product and contacting the resulting compound of formula Lys(A-Ala)-Ala-Ala-Pro-NH-iBu with succinic or glutaric anhydride. Compounds of general structure I are efficient competitive inhibitors of serine proteinases, especially of leukocyte elastase (LE) and pancreatic elastase (PE). These enzymes are the cause of destructive damage of cell and venous walls and therefore are the cause of serious diseases. Besides the mostly observed pulmonary emphysema, acute pancreatitis and various types of arthritis, the above mentioned enzymes cause hemorrhagic periodontitis. This is the reason why one of the attractive applications of the compounds described is the use of these as the ingredients of various dental preparations, for instance dentifrices or mouth washes. An especially interesting one would be the application of these efficient inhibitors as ingredients of chewing gums; the ingredients in these medicinal preparations ensure the constant level of the active inhibitor in oral cavity also during the daily working activity, when, from the working and time reasons the standard hygienic care of the dental cavity is not possible. Further potential use of LE inhibitors is in blocking of the activation of pro-enzymes, for instance cathepsine B (Biochim.Biophys. Acta 1226, 117-125, 1994) or for inhibition of matrix metalloproteinase 3 (MMP-3) -so called stromelysin from the corresponding zymogen (FEBS Lett. 299, 353-356, 1989). MMP-3 is known as the key enzyme in pathogenesis of various malign diseases and also in sclerosis multiplex. The guarantee of the lack of the unwanted or toxic side effect of the inhibitors described is the fact that these are synthesised only from physiologic, so nontoxic compounds, which means aminoacids (alanine, proline, lysine), higher fatty acids and succinic or glutaric acids. Compounds of general structure I can be used in the form of free acids or (for the reason of higher solubility) in the form of soluble salts created with alkali metals, mostly with sodium. The synthesis of oligopeptidic inhibitors of elastase according to this invention including all the intermediates is described in schemes 1, 2 and 3. The final products and intermediates are characterized by their melting points (m.p.) or by their optical rotations, in some cases by thin layer chromatography (TLC). The values are mentioned in tables I- VI. The key compound for synthesis of all oligopeptidic inhibitors is isobutylamide of alanyl- alanyl-proline (Collection Czechoslov. Chem. Commun. 52, 3034-3041, 1987); acylation of aminoacids by higher fatty residues is described in patent document No CS 280 726. The synthesis of intermediates and of final products is performed in solution and is clear from the examples of preparation. The evaluation (determination of inhibitory constants Ki) by human leukocyte elastase was performed using known procedure (Biol. Chem. Hoppe- Seyler 366, 333-343, 1985), the results of determinations of some compounds are described in table VII. Following abbreviations and symbols were used: Ala = alanine Pro = proline Lys = lysine Sue = succinyl Git = glutaryl Kpl = caproyl Lau = lauroyl Myr = myristoyl Pal = palmitoyl Z = benzyloxycarbonyl Boc = tert.butyloxycarbonyl iBu = isobutyl DCCI N,N' icyclohexylcarbodiimide OHSuc = N-hydroxysuccinimide EtO-CO-Cl = ethyl chloroformate Suc-O = succinic anhydride The standard preparation: the rough reaction product was solubilized in AcOEt and extracted with 1% citric acid, followed by extraction with H 2 O, then with 5% NaHCO 3 and again with H 2 O The product was dried over anhydrous Na 2 SO and evaporated. All the evaporation was done under reduced pressure on a rotary vacuum evaporator. Thin layer chromatography (TLC - silica-gel G) was performed in the system Sf n-butanol-acetic acid-H 2 O (4:1:1). Optical rotation was determined using the Perkin-Elmer 141 polarimeter. Melting points were determined on the Kofϊler block and have not been corrected. Scheme 1 Boc-Lys(Z) + Ala-Ala-Pro-NH-iBu Boc-Lys(Z)-Ala-Ala-Pro-NH-iBu 4-H 2 /Pd Boc-Lys-Ala-Ala-Pro-NH-iBu slPal-Cl Boc-Lys(Pal)-AJa-Ala-Pro-NH-iBu !TFA/anex L 150 Lys(Pal)-Ala-Ala-Pro-NH-iBu i — 1 4- Suc-O Suc-Lys(Pal)-Ala-Ala-Pro-NH-iBu I Scheme 2 A-Lys(Z)-Ala-Ala-Pro-NH-iBu I H 2 /Pd IΙ(a-d) A-Lys-Ala-Ala-Pro-NH-iBu I B - O IΙI(a-d) A-Lys(B)-Ala-Ala-Pro-NH-iBu IV(a-h) Scheme 3 Boc-Lys-Ala-Ala-Pro-NH-iBu I Boc-Lys(Y)-Ala-Ala-Pro-NH-iBu V(a-d) - 1) HCl/AcOH 2) anex (Zerolit) Lys(Y)-Ala-Ala-Pro-NH-iBu VΙ(a-d) I Suc-O Suc-Lys(Y)-Ala-Ala-Pro-NH-iBu VΙI(a-d) Table I II (a-d) M.p. °C a Kpl-Lys(Z)-Ala-Ala-Pro-NH-iBu 205-208 b Lau-Lys(Z)-Ala-Ala-Pro-NH-iBu 188-190 c Myr-Lys(Z)-Ala-Ala-Pro-NH-iBu 178-181 d Pal-Lys(Z)-Ala-Ala-Pro-NH-iBu 169-172 Table 1 II (a-d) M.p. °C a Kpl-Lys(Z)-Ala-Ala-Pro-NH-iBu 205-208 b Lau-Lys(Z)-Ala-Ala-Pro-NH-iBu 188-190 c Myr-Lys(Z)-Ala-Ala-Pro-NH-iBu 178-181 d Pal-Lys(Z)-Ala-Ala-Pro-NH-iBu 169-172 Table II III (a-d) M.p. °C a Kpl-Lys-Ala-Ala-Pro-NH-iBu 177-181 b Lau-Lys-Ala-Ala-Pro-NH-iBu 183-185 c Myr-Lys-Ala-Ala-Pro-NH-iBu 173-176 d Pal-Lys-Ala-Ala-Pro-NH-iBu 154-158 Table III IV (a-h) M.p. °C a Kpl-Lys(Suc)-Ala-Ala-Pro-NH-iBu 141-144 b Lau-Lys(Suc)-Ala-Ala-Pro-NH-iBu 143-145 c Myr-Lys(Suc)-Ala-Ala-Pro-NH-iBu 148-151 d Pal-Lys(Suc)-Ala-Ala-Pro-NH-iBu 142-144 e Kpl-Lys(Glt)-Ala-Ala-Pro-NH-iBu 126-128 f Lau-Lys(Glt)-Ala-Ala-Pro-NH-iBu 152-155 g Myr-Lys(Glt)-Ala-Ala-Pro-NH-iBu 147-150 h Pal-Lys(Glt)-Ala-Ala-Pro-NH-iBu 124-126 Table IV V (a-d) M.p. °C a Boc-Lys(Kpl-Ala)-Ala-Ala-Pro-NH-iBu 179-182 b Boc-Lys(Lau-Ala)-Ala-Ala-Pro-NH-iBu 181-184 c Boc-Lys(Myr-Ala)-Ala-Ala-Pro-NH-iBu 133-135 d Boc-Lys(Pal-Ala)-Ala-Ala-Pro-NH-iBu 146-150 Table V VI (a-d) M.p. °C a Lys(Kpl-Ala)-Ala-Ala-Pro-NH-iBu 185-188 b Lys(Lau-Ala)-Ala-Ala-Pro-NH-iBu 189-191 c Lys(Myr-Ala)-Ala-Ala-Pro-NH-iBu 184-187 d Lys(Pal-Ala)-Ala-Ala-Pro-NH-iBu 158-161 Table VI VII (a-d) M.p. °C a Suc-Lys(Kpl-Ala)-Ala-Ala-Pro-NH-iBu 182-186 b Suc-Lys(Lau-Ala)-Ala-Ala-Pro-NH-iBu 188-189 c Suc-Lys(Myr-Ala)-Ala-Ala-Pro-NH-iBu 181-184 _d Suc-Lys(Pal-Ala)-Ala-Ala-Pro-NH-iBu 171-174 Table VII Inhibitory constants (Ki) of human leukocyte elastase Ilia Kpl-Lys-Ala-Ala-Pro-NH-iBu 2,27.10 '5 M IVa Kpl-Lys(Suc)-Ala-Ala-Pro-NH-iBu 4,50. lO^ M IVb Lau-Lys(Suc)-Ala-Ala-Pro-NH-iBu 5,32. lO^ M IVc Myr-Lys(Suc)-Ala-Ala-Pro-NH-iBu 6,82.10- 6 M IVe Kpl-Lys(Glt)-Ala-Ala-Pro-NH-iBu 9,96. lO^ M IVf Lau-Lys(Glt)-Ala-Ala-Pro-NH-iBu 7,35.10 "6 M IVh Pal-Lys(Glt)-Ala-Ala-Pro-NH-iBu 1,46.10 '5 M VIb Lys(Lau-Ala)-Ala-Ala-Pro-NH-iBu 5,38.10 "7 M Vllb Suc-Lys(Lau-Ala)-Ala-Ala-Pro-NH-iBu 6,92.10- 7 M VId Lys(Pal-Ala)-Ala-Ala-Pro-NH-iBu 2,47.10- 5 M Vlld Suc-Lys(Pal-Ala)-Ala-Ala-Pro-NH-iBu 1,19.10- 5 M Examples Example 1 Boc-Lys(Z)-Ala-Ala-Pro-NH-iBu To the solution of Boc-Lys(Z) (20 mmol) and N-Etp (2,8 ml) in DMFA (50 ml) cooled to -10 °C EtOCO-Cl (2 ml) was added; after 5 min stirring (-10 °C) to the reaction mixture the solution of Ala- Ala-Pro-NH-iBu (6,3 g, 20 mmol) in 20 ml DMFA was added. The mixture was stirred for 30 min (0 °C) and then the stirring continued for 2 h (room temperature). After this, the reaction mixture was evaporated, the residue solubilized in the mixture of AcOEt and H 2 O and standard procedure I followed. The residue was crystallised from 2- propanol and light petroleum. 5,7 g of the product (m.p. 168-170 °C) were obtained. Boc-Lys-Ala-Ala-Pro-NH-iBu To the solution of Boc-Lys(Z)-Ala-Ala-Pro-NH-iBu (3,4 g; 5 mmol) in methanol (300 ml) the suspension 5% Pd/C (0,3 g) in toluene (10 ml) was added and the reaction mixture was hydrogenated in autoclave 20 min, under the pressure 2 MPa. After removing of the catalyst by filtration, the methanol solution was evaporated. 2,8 g of product (m.p. 150-153 °C) was obtained. Boc-Lys(Pal)-Ala-Ala-Pro-NH-iBu To the solution of Boc-Lys-Ala-Ala-Pro-NH-iBu (1,1 g, 2 mmol) in DMFA (4 ml) and 1 M NaOH (3,5 ml) Pal-Cl (1 ml) during 30 min was added (room temperature); after further 30 min of stirring the reaction mixture was acidified (10% AcOH, pH 5) and the crude precipitate formed was separated by filtration. 0,9 g of product (chromatographic homogeneity, R f = 0,71/S-) was obtained. Lys(Pal)-Ala-Ala-Pro-NH-iBu To the solution of Boc-Lys(Z)-Ala-Ala-Pro-NH-iBu (390 mg) in AcOH (0,5 ml) TFA (0,5 ml) was added. After 1 h, when the mixture was standing at the room temperature it was evaporated and dried over P 2 O 5 . Then, the trifluoracetate obtained was solubilized in methanol and deionised using slightly basic anex (L 150). 180 mg of product (base, m.p. 139-142 °C; [α] 20 D -78,3° (c = 0,2; methanol) was obtained. Suc-Lys(Pal)-Ala-Ala-Pro-NH-iBu To the solution of Lys(Pal)-Ala-Ala-Pro-NH-iBu (140 mg, 0,2 mmol) in DMFA (20 ml) succinic anhydride (25 mg) was added; the reaction mixture was kept at the temperature 80 °C for 30 min, then the reaction mixture was evaporated and gel residue was crystallised from the solution of DMFA and AcOEt. 60 mg of product (m.p. 157-160 °C; [α] 20 D -73,5° /c = 0,2; methanol/) was obtained. Example 2 Myr-Lys(Z)-Ala-Ala-Pro-NH-iBu (lie) To the solution of Myr-Lys(Z) (5,05 g, 10 mmol) in DMFA (150 ml), HOSuc (1,15 g) in DMFA (20 ml) was added after cooling to 0 °C DCCI (2,2 g) was added. After 1 h stirring (0 °C) Ala-Ala-Pro-NH-iBu (3,15 g, 10 mmol) in DMFA (25 ml) was added. The reaction mixture was left to stand for 12 h at room temperature, then AcOH (0,2 ml) was added and the reaction suspension was left at 0 °C for 30 min. Then it was filtered, washed by DMFA and evaporated. The gel residue was crystallised from hot DMFA (40 ml). 4,7 g of product (m.p. 178-181 °C) was obtained. The similar procedure was used for the preparation of compounds II (a,b,d). Myr-Lys-Ala-Ala-Pro-NH-iBu (IIIc) To the solution of Myr-Lys(Z)-Ala-Ala-Pro-NH-iBu (4 g; 5 mmol) in methanol (300 ml), the suspension of 5% Pd/C (0,3 g) was added the pressure hydrogenolysis was performed (2 MPa) as described in example 1. 2,9 g of product (m.p. 173-176 °C) was obtained. The similar procedure was used for the preparation of compounds III (a,b,d). Myr-Lys(Suc)-Ala-Ala-Pro-NH-iNH-iBu (IVc) To the solution of Myr-Lys-Ala-Ala-Pro-NH-iBu (1,35 g, 2 mmol) in dioxane (30 ml) succinic anhydride (380 mg) was added and the reaction mixture was heated for 30 min under rellux. After cooling, 1,75 g of product (m.p. 148-151 °C) was obtained. Similar procedure was used for preparation of compounds IV (a,b,d). Pal-Lys(Glt)-Ala-Ala-Pro-NH-iBu (IVh) To the solution of Pal-Lys-Ala-Ala-Pro-NH-iBu (2.05 g, 3 mmol) in dioxane (30 ml) glutaric anhydride (600 mg) was added and the reaction mixture was heated for 30 min under the reflux. After cooling 2,1 g of product was obtained (m.p. 124-126 °C; [α] 2 ° D - 34,6° /c = 0,2; DMFA/). Similar procedure was used for the preparation of compounds IV (e-g). Example 3 Boc-Lys(Pal-Ala)-Ala-Ala-Pro-NH-iBu (Vd) To the solution Pal-Ala (985 mg, 3 mmol) in DMFA (25 ml) OHSuc (350 mg) was added, after cooling to -5 °C DCCI (660 mg) was added, after 30 min of stirring (-5 °C) the solution of Boc-Lys-Ala-Ala-Pro-NH-iBu (1,65 g, 3 mmol) in DMFA (30 ml) was added. The mixture was stirred at room temperature for 3 h. The created precipitate was separated by filtration, washed DMFA and water (50 ml) was added. The precipitate was separated by filtration and crystallised from 2-propanol. 1,75 g of product (m.p. 146-150 °C) was obtained. The similar procedure was used for the preparation of compounds N(a-c). Lys(Pal-Ala)-Ala-Ala-Pro-ΝH-iBu (IVd) To the solution of Boc-Lys(Pal-Ala)-Ala-Ala-Pro NH-iBu (1,7 g; 2 mmol) in AcOH (20 ml) the solution of 0,6 M HCl/AcOH (4 ml) was added. After 3 h of standing at room temperature, the created hydrochloride was precipitated by means of ether, separated by filtration and dried in the presence of P 2 O 5 and NaOH. Then, the hydrochloride was deionised by strong basic anex (Zerolit FF/OH-cycle) in methanol. The solution was evaporated and the residue was crystallised from 2-propanol and light petroleum. The base (820 mg) was obtained (m.p. 158-161 °C). The similar procedure was used for the preparation of compounds VΙ(a-c). Suc-Lys(Pal-Λla)-Ala-Ala-Pro-NH-iBu (Vlld) To the solution of Lys(Pal-Ala)-Ala-Ala-Pro-NH-iBu (750 mg; 1 mmol) in DMFA (20 ml) succinic anhydride (160 mg) was added. After 2 h of stirring at the room temperature the product was precipitated by addition of H 2 O (50 ml) and crystallised from 2-propanol and AcOEt. 690 mg of the final product was obtained (m.p. 171-174 °C). The similar procedure was used for the preparation of compounds VΙI(a-c).

Claims: C L A I M S 1. Oligopeptidic inhibitors of elastases of general structure 1 Y-NH (I), χ _ NH CO-Ala-Ala-Pro-NH-iBu wherein X means A or B, wherein A is different from B Y means A or B or A-Ala, wherein A is different from B and if Y is A-Ala then X means B, A means a saturated acid of 8 to 16 carbon atoms, B means the 3-carboxypropionyl or 4-carboxybutyroyl residue. 2. A method for the preparation of compounds of general structure I wherein Y is B, characterised in contacting lysine having protected amino groups with a compound of formula Ala-Ala-Pro-NH-iBu, deprotecting the ε-amino group of the product, reacting the resulting product with a compound of formula A-X 1 , wherein A has the above meaning and X 1 is a halogen, deprotecting the α-amino group and contacting the resulting compound of formula Lys(A)-Ala-Ala-Pro-NH-iBu with succinic or glutaric anhydride. 3. A method for the preparation of compounds of general structure I wherein Y means A, characterised in contacting a compound of formula A-Lys(W), wherein W is a protective group and A has the above meaning, with a compound of formula Ala-Ala-Pro-NH-iBu, deprotecting the product and contacting the resulting compound of formula A-Lys- Ala-Ala-Pro-NH-iBu with succinic or glutaric anhydride. 4. A method for the preparation of compounds of general structure I wherein Y is A-Ala, characterised in contacting a compound of formula A-Ala with a compound of formula W-Lys-Ala-Ala-Pro-NH-iBu, wherein W is a protective group and A has the above meaning, deprotecting the product and contacting the resulting compound of formula Lys(A-Ala)-Ala-Ala-Pro-NH-iBu with succinic or glutaric anhydride.

Inventors: KASAFIREK EVZEN, PIHERA PAVEL, SEDO ALEKSI, STURC ANTONIN, KASAFIREK, EVZEN, PIHERA, PAVEL, SEDO, ALEKSI, STURC, ANTONIN
Applicants: KASAFIREK EVZEN, PIHERA PAVEL, SEDO ALEKSI, STURC ANTONIN, VUFB AS, KASAFIREK, EVZEN, PIHERA, PAVEL, SEDO, ALEKSI, STURC, ANTONIN, VUFB, A.S.
Application year: 1998
Main classification: C07K14
IPC codes: A23G4, A61K38, A61P1, A61P43, C07K1, C07K5, C07K7, C07K14