New thrombin inhibiting compounds.

Technical Field

The present invention is related to new thrombin inhibiting N^α-arylsulfonyl-p-guandinophenylalanine amides of the general formula: <chemistry id="chem0001" num="0001"><img file="EP0097630A2_D0001.tif" /></chemistry>wherein Ar is o-, m- or p-tolyl, naphthyl-1, naphthyl-2 or 5-dimethylamino-l-naphthyl and R₁ and R₂ is an alkyl group having 1-5 carbon atoms <chemistry id="chem0002" num="0002"><img file="EP0097630A2_D0002.tif" /></chemistry>is a ringsystem <chemistry id="chem0003" num="0003"><img file="EP0097630A2_D0003.tif" /></chemistry>wherein n=2 or 3 and X is a single bond, CH₂, CH-CH₃, CH-C₂H₅, CH-C₃H₅, 0,NH or N-CH₃.

Background Art

Thrombin plays an important role in the coagulation process, where it as last enzyme, in the coagulation cascade, transfers fibrinogen to polymerizable fibrin. This is done by splitting of Arg-bonds. Thrombin has just close to its active center a "specificity pocket" with great affinity for the positively charged guanidino group in Arg. This knowledge has been used for construction of synthetic substrates and inhibitors of thrombin. Since a long time it is known that substituted Arg esters can be split by thrombin, e.g. Bz-Arg-OEt (BAEE) and Tos-Arg-OMe (TAME). The last-named ester acts, by being a competitive substrate, also as inhibitor to the reaction of thrombin with fibrinogen. The plasma coagulation time is prolonged e.g. by addition of TAME. A better thrombin inhibitor can be obtained by making the Arg-bond not cleavable. Okamoto^(1,2) has described thrombin inhibitors, where the carboxylic group of Arg is bound to sec amines: <chemistry id="chem0004" num="0004"><img file="EP0097630A2_D0004.tif" /></chemistry>

Markwardt^(3,4) has modified the structure II by inserting the synthetic amino acid p-amidinophenylalanine (Aph), an analog to Arg, instead of Arg. The compounds (III) thereby obtained seem to be about as good thrombin inhibitors as the corresponding compounds (II) made by Okamoto. <chemistry id="chem0005" num="0005"><img file="EP0097630A2_D0005.tif" /></chemistry>

Another arginine anolog, p-guanidinophenylalanine (Gph), was first synthezised by Elliot⁽⁵⁾. Klausner(6) syntezised the derivative Tos-Gph-OMe, which appeared to be a good substrate for trypsin, but on the other hand a bad substrate for thrombin but with ability to inhibit the amidase- and esterase activity of thrombin. Tsunematso⁽⁷⁾ has synthezised Bz-Gph-OEt too and has found that it as substrate for trypsin is comparable with Tos-Gph-OMe and Tos-Arg-OMe (TAME).

Description of the invention

Now we have found that the new compounds (I) according to the invention have very good thrombininhibiting properties which to advantage can be compared with the corresponding Arg- as well as Aph-derivatives (II and III). Table 1 compares Gph- and Arg-derivatives with respect to the inhibition constant (Ki) for thrombin and prolongation of coagulation time initiated by thrombin (thrombin time). Especially the thrombin times show the advantages of the Gph- derivates. Table 2 gives Ki (thrombin) for the corresponding Gph- and Aph-derivatives. Also in this comparison the Gph-derivatives show a distinct advantage.

The new prepared compounds according to the invention have a very specific thrombin inhibiting effect. E.g. factor Xa is inhibited up to 1000 times less than thrombin. The compounds are therefor very useful as selective inhibitors for thrombin when determining factor Xa in a medium (e.g. blood), where thrombin may be present and disturb the determination of factor Xa. Besides this diagnostic use of the new compounds as thrombin inhibitors they are also useful for a direct specific determination of thrombin in blood.

The new aryl-sulfonyl-L-p-guanidinophenylalanine amides and the pharmaceutically acceptable acid addition salts thereof according to the invention can in their capacity as good thrombin inhibitors also be used as anticoagulants in therapy and prophylax of thrombosis.

In the synthesis of the new thrombin inhibitors, protecting groups and coupling methods traditionally well-known in chemistry can be used. The C-terminal amide residues are also coupled using methods of synthesis which are well-known in the organic chemistry. Purifying of intermediates and end products is made by precipation, crystallization or gel filtration chromatography.

Thus the compounds according to the invention can be prepared by a) reaction between a compound of the formula

<chemistry id="chem0006" num="0006"><img file="EP0097630A2_D0006.tif" /></chemistry>

wherein R₁ and R₂ are as defined previously, and a compound of the formula <chemistry id="chem0007" num="0007"><img file="EP0097630A2_D0007.tif" /></chemistry>wherein Y is a reactive group, e.g. halogen as Cl which together with H₂N can form a sulfonamide group and where Ar is as defined previously.

b) by removal of the NO₂-group e.g. by hydrogenation in a compound of the formula <chemistry id="chem0008" num="0008"><img file="EP0097630A2_D0008.tif" /></chemistry>wherein R₁, R₂ and Ar are as defined previosly.

The starting material according to a) above can be obtained by removal of the protecting group Z from a compound of the formula <chemistry id="chem0009" num="0009"><img file="EP0097630A2_D0009.tif" /></chemistry>by reaction with e.g. trifluoroacetic acid, wherein R₁ and R₂ are as defined previously and Z is a removable group as -COOR°, wherein R° is a hydrocarbon residue e.g. alkyl.

The starting'material according to b) above can be obtained by e.g. a reaction between a compound of the formula <chemistry id="chem0010" num="0010"><img file="EP0097630A2_D0010.tif" /></chemistry>and a compound of formula <chemistry id="chem0011" num="0011"><img file="EP0097630A2_D0011.tif" /></chemistry>wherein Z, R₁ and R₂ are as defined previously, to formation of a compound of the formula <chemistry id="chem0012" num="0012"><img file="EP0097630A2_D0012.tif" /></chemistry>which protecting group Z thereafter is removed to formation of a compound <chemistry id="chem0013" num="0013"><img file="EP0097630A2_D0013.tif" /></chemistry>which thereafter by reaction with a compound of the formula <chemistry id="chem0014" num="0014"><img file="EP0097630A2_D0014.tif" /></chemistry>wherein R₁, R₂₉ Y and Z are as defined previously, is transferred to the starting material according to b).

The new compounds according to the invention show a asymmetric center and appear therefore in two stereoisomeric forms as well as in form of racemate. The two optical antipodes can be separated with help of known methods and the invention includes the racemate as well as these antipodes separately as free base as well as salt. Specially preferred is the L-form.

Many different organic and inorganic acids can be employed to form acid addition salts of the new N^a-arylsulfonyl-p-guanidinophenylalanine amides of this invention. The product of the reactions described above can be isolated in free form or as acid addition salts. In addition, the product can be obtained as pharmaceutically acceptable acid addition salts by reacting one of the free bases with an acid, such as hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, acetic, citric, maleic, succinic, lactic, tartaric, gluconic, benzoic, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic acid or the like. Similarly, a treatment of the acid addition salts by reaction of a base gives as result a reformation of the free amide or ester.

The invention is described from the following not limiting examples.

Abbreviations

<ul id="ul0001" list-style="none"><li>Arg = L-Arginine</li><li>Phe = L-Phenylalanine</li><li>Aph = p-Amidino-Phe</li><li>Gph = p-Guanidino-L-Phe</li><li>Dahsyl = 5-Dimethylamino-l-naphthalenesulfonyl</li><li>Bz = Benzoyl</li><li>Et = Ethyl</li><li>Me = Methyl</li><li>BOC = t-Butyloxycarbonyl</li><li>Ac = Acetyl</li><li>DCCI = Dicyclohexylcarbodiimide</li><li>DCU = Dicyclohexylurea</li><li>DMF = Dimethylformamide</li><li>HOBT = N-Hydroxybenzotriazole</li><li>TFA = Trifluoroacetic acid</li><li>TLC = Thin Layer Chromatography</li></ul>

Used methods for Thin Layer Chromatography

At TLC-analysis pre-fabricated glass plates, with silica gel F₂₅₄ (Merck) as an absorption medium, are used. The solvent systems used are (volume ratios): A: n-Butanol : AcOH : H₂0 (3:2:1) Pa₆: Chloroform : MeOH : AcOH : H20 (34:4:9:2)

Following chromatography, the plate is inspected in UV light (254 nm) and then developed with chlorine/o-tuluidin reagent according to normal procedure. The given R_f values are the result of single experiments.

Example 1 N

α

-Dansyl-p-guanidino-Phe-piperidide hydrochloride la BOC-p-(nitroguanidino)-Phe-piperidide

1.3 g of BOC-p-(nitroguanidino)-Phe-OH (Moore S. et al. J. Chem. Soc. Perkin I 1977, 2025-30) in 15 ml of DMF is cooled to -15°C. 0.5 ml of Et₃N and then 0.5 ml of isobutylchloroformiate is added with stirring. After 15 min. 0.4 ml of piperidine is added and the solution is stirred at continued cooling for 1 hour and then further 1 hour at room temperature. The reaction mixture is evaporated in vacuum to an oil which is triturated with water and dried. The residue is dissolved in methanol and chromatographed on a Sephadex^R LH20 column in methanol with methanol as medium for eluation. The fraction containing the compound la is evaporated to dryness in vacuum, the product is obtained as an amorphous powder. .Yield: 0.95 g (62%) of compound la TLC: Rf = 0.79 (Pa₆)

lb N^α-Dansyl-p-(nitroguanidino)Phe-piperidide

1,5 g of Ia is suspended in a 25% solution of TFA in dichloromethane and is stirred at room temperature for 30 min. Dichloromethane is evaporated. The residue is cooled on an ice-bath and ca 1000 ml dry ether is added under vigorous stirring. The precipitate formed is filtered and washed with dry ether and dried in vacuum over NaOH. The obtained TFA salt of H-p-(nitroguanidino)-Phe-piperidide is dissolved in 20 ml tetrahydrofurane-water (9:1) and neutralized at a low temperature (about -10°C) of 0.55 ml of ET₃N. 1.2 g of dansylchloride and further 0.6 ml of Et₃N is added. The solution is stirred for 2 hours at a low temperature and then at room temperature over night. The precipitated product is filtered and washed carefully with water and thereafter with ether. Yield: 1.8 g (92%) of compound lb TLC: Rf = 0.84 (Pa₆)

1. N^α-Dansyl-p-guanidino-Phe-piperidide hydrochloride 200 mg of Ib is suspended in 10 ml of methanol and 0,07 ml of conc. HC1 and 20 mg palladium on carbon (10%) are added. The solution is hydrogenated at room temperature and at athmospheric pressure for 48 h. The catalyst is filtered off and the reaction mixture is evaporated .The residue is dissolved in 5 ml of methanol and chromotographed on Sephadex LH20 in methanol with methanol as medium for eluation. The fraction containing compound 1 is evaporated. The residue is dissolved in 5 ml ethanol:water (1:1) and is ion exchanged on QAE Sephadex^R A25 in chloride form in ethanol:water (1:1) with the same medium for eluation. The fraction containing the pure compound 1 is evaporated and the residue is lyophilized from water. Yield: 150 mg (71%) of compound 1 TLC: R_f = 0,43 (A) shows one spot.

Data of analysis vide table 3.

Example 2 Na-(Naphthalene-2-sulfonyl)-p-guanidino-Phe-4-methyl-piperidide hydrochloride

2a BOC-p-guanidino-Phe-4-methyl-eieeridine hydrochloride 1,1 g of 4-methylpiperidide in 20 ml of DMF is acidified with 1 ml of conc. HC1 and is then evaporated to dryness in vacuum. The residue together with 3,2 g of BOC-p-guanidino-Phe-OH [Klausner Y.S. et al. Biochem. J. 169, 157-67 (1978)] and 1,35 g of HOBT are dissolved in 20 ml DMF. After cooling in an ice bath 2,5 g of DCCI is added. The reaction mixture is stirred for 2 hours at a low temperature and then at room temperature over night. The prepared DCU is filtered off and the reaction mixture is evaporated in vacuum to an oil. The oil is dissolved in 80 ml of n-butanol. The butanol solution is washed in sequence with 10% NaCl in water, 0,5 M NaHC0₃ in 5% NaCl in water and 10% NaCl in water. The butanol phase is dried over Na₂S0₄ and evaporated in vacuum. Chromatography on Sephadex^R LH20 in methanol and on QAE Sephadex^R A50 in chloride form in ethanol-water (1:1) gives after lyophilizing from water a pure compound 2a. Yield: 3,0 g (68%) of compound 2a TLC: R_f = 0,64 (Pa₆)

₂. N^α-(Naphthalene-2-sulfonyl)-p-guanidino-Phe-4-methylieridide hydrochloride 1,3 g of compound 2a is deprotected with 30 ml of 25% TFA in dichloromethane according to lb. The TFA salt of H-p-guanidino-Phe-4-methyl-piperidide is dissolved in 25 ml of DMF and after cooling 0,42 mg of Et₃N is added to give a weak basic reaction. First 3,3 g of naphthalene-2-sulfonylchloride and then 0,45 ml of Et₃N are added to the solution. The reaction mixture is stirred for 1 hour at a low temperature and for 1 hour at room temperature. After cooling Et₃N hydrochloride is filtered off and the DMF-solution is evaporated in vacuum. Chromatography and lyophilizing according to ex. 1 gives a pure compound 2. Yield: 1,24 g (79%) of compound 2 TLC: R_f = 0,51 (A) shows one spot Data of analysis vide table 3.

Determination of thrombin inhibition

Data and test results of other synthesized aryl-p-guanidinophenylalanine amides are compared in table 3, where compound V is prepared according_.to ex.1 and the other compounds are prepared according to ex. 2.

Determination of Ki

The inbibition of reaction of the enzymes (human thrombin; Sigma Chemical Co., St. Louis, USA and human factor Xa; KabiVitrum AB, Stockholm) with the substrates (S-2238 resp. S-2222, Kabi Diagnostica, Stockholm) was determined at three different substrate concentrations from 0,3 to 2 Km. Six different inhibitor concentrations, which give inhibition from 40% to 90%, were incubated with the enzyme for 30 seconds at 37°C. The buffer composition, ionic strength, pH and enzyme concentration were the same as those recommended in the booklets from Kabi Diagnostica. The initial reaction velocity (ΔA/min) was measured on a recorder at 405 nm. Ki values were then graphically determined from Dixon and Lineweaver-Burk diagrams.

Thrombin time

A volume of 200 µl citrate-plasma was heated for 1 minute at 37°C. The coagulation was started by adding 10 µl fresh thrombin solution (about 5 NIH U/ml)and the coagulation time was registrated by a fibrino- meter. At the inhibition experiments the plasma was incubated for 30 seconds with 10 µl inhibitor solution at different concentrations before the thrombin solution was added. <tables id="tabl0001" num="0001"><img file="EP0097630A2_D0015.tif" /></tables>

Thrombin inhibition by Arg- and Gph-derivatives

Ki of thrombin in mol/1 "Thrombin time" designates the concentration (mol/1) of the inhibitor, which doubles the thrombin time of dog plasma.

The Arg derivates have been synthesized according to ref. 1 and 2. Tos-Gph-OMe has been synthesized according to ref. 6. <tables id="tabl0002" num="0002"><img file="EP0097630A2_D0016.tif" /></tables>

Thrombin inhibition constants of Aph- and Gph-derivate

Ki of thrombin is given in mol/l Ki-values of Aph-derivatives are taken from ref. 3 They refer to the racemates of the compounds and are determined by bovine thrombin and DL-benzoylarginine-p-nitroanilide. <tables id="tabl0003" num="0003"><img file="EP0097630A2_D0017.tif" /></tables><tables id="tabl0004" num="0004"><img file="EP0097630A2_D0018.tif" /></tables>

REFERENCES

<ul id="ul0002" list-style="none"><li>1. Okamoto, S., et al SE patent appl. 760891-5</li><li>2. Okamoto, S., et al. J.Med.Chem. 23, 830-836 (1980)</li><li>3.Sturzenbecher, J., et al. Pharmazie 36, 639-641 (1981)</li><li>4. Hauptmann, J.,Thromb. Haem. 43, 118-123 (1980)</li><li>5. Elliot, D.F., och Harrington, C., J.Chem.Soc. 1949, 1374-1478</li><li>6. Klausner, Y.S., et al. Biochem.J. 169, 157-167 (1978)</li><li>7. Tsunematsu, H., et al. J.Biochem. 88, 1773-1783 (1980)</li><li>8. Moore, S., et al. J.Chem.Soc.Perkin I, 1977, 2025-2030.</li></ul>