DE102011108346A1 - Use of inhibitors of TMPRSS2 as a drug - Google Patents

Abstract

The invention relates to the use of inhibitors according to the general formula I for the treatment of viral infections, especially for the treatment of influenza infections, for the treatment of inflammatory respiratory diseases or for the inhibition of the serine protease TMPRSS2, where X is a CH or N, R1 is a substituted cyclic sulfonyl radical, R2 is a unsubstituted or substituted amidino group and R3 is a secondary amine group coupled as an amide.

Description

The present invention describes the use of inhibitors of serine protease TMPRSS2 for the preparation of medicaments useful in the treatment of diseases involving this protease. Thus inhibitors of TMPRSS2 are suitable for the treatment of virus-related diseases, such as influenza infections. The inhibitors can also be used for the therapy of inflammatory respiratory diseases.

Description and state of the art

The present invention relates to the use of Na-sulfonylated secondary amides of substituted phenylalanines or azaphenylalanines as inhibitors of the serine protease TMPRSS2 for the preparation of medicaments which can be used for the therapy and / or prophylaxis of viral diseases. Likewise, a use of these compounds for the therapy and / or prophylaxis of inflammatory respiratory diseases is possible.

The gene for the serine protease TMPRSS2 was first identified in 1997 ( Paolini-Giacobino, Genomics 44, 309-320 (1997) ). TMPRSS2 has been reported to be involved in viral diseases such as influenza or metapneumovirus infections ( Böttcher et al., J. Virology 80, 9896-9898 (2006) . Böttcher et al. Vaccine 27, 6324-6329 (2009) ; Böttcher et al. J Virol. 84, 5605-5614 (2010) ; Chaipan et al., J. Virology 83, 3200-3211 (2009) . Shirogane et al., J. Virology 82, 8942-8946 (2008) ), but also for infections with the SARS coronavirus (severe acute respiratory syndrome coronavirus, Matsuyama et al., J. Virol. 82 (2010) 12658-12664 ).

The first substrate-analogous synthetic inhibitors of TMPRSS2 have recently been reported to inhibit TMPRSS2 with K _i > 10 nM ( WO 2010/149459 ).

However, these compounds have low selectivity and also inhibit other trypsin-like serine proteases. The invention is therefore based on the object to provide for therapeutic applications, suitable alternative drugs that inhibit the serine protease TMPRSS2 with high activity and therefore are suitable for the treatment of diseases in which this protease is involved.

By screening known inhibitors of trypsin-like serine proteases, which have already been described, for example, as inhibitors of the proteases matriptase, urokinase (uPA) and thrombin (for urokinase: eg. Stürzebecher et al., Bioorg. Med. Chem. Lett. 9, 3147-3152 (1999) ; for matriptase: z. B. Steinmetzer et al., J. Med. Chem. 49, 4116-4126, (2006) ; Steinmetzer et al. Bioorg. Med. Chem. Lett. 19, 67-73, (2009) ; Schweinitz et al. Bioorg. Med. Chem. Lett. 19, 1960-1965, (2009) , for thrombin: Stürzebecher et al. J. Med. Chem. 40, 3091-3099, (1997) ), we have surprisingly found compounds which are effective in inhibiting TMPRSS2 and therefore useful for the treatment of diseases mediated or potentiated by the protease TMPRSS2.

wobei

• • X CH oder N ist, und
• • R₁ ein einfach oder mehrfach substituierter oder unsubstituierter zyklischer Aryl- oder Heteroarylrest ist, der ein bis drei Ringe enthalten kann, die entweder aneinander gebunden oder miteinander fusioniert sind, und
• • R₂ eine Amidinogruppe der folgenden Struktur ist,
  
  in der R₄ bevorzugt H ist, aber auch -OH, -OCH₃ und -O-CO(CH₂)_nCH₃ mit n gleich eine ganze Zahl von 0 bis 5 sein kann, und
• • R₃ eine sekundäre Amingruppe ist, die über eine Amidbindung an das zentrale Phenylalanin- oder Azaphenylalanin-Derivat gebunden ist und mehrfach substituiert oder unsubstituiert sein kann.

As suitable inhibitors of TMPRSS2 and thus for the treatment of viral diseases or respiratory diseases associated with the TMPRSS2, proved to be Na-sulfonylated secondary amides of substituted phenylalanines or Azaphenylalanine the general formula (I),

in which

• • X is CH or N, and
• R _{1 is} a mono- or polysubstituted or unsubstituted cyclic aryl or heteroaryl radical which may contain one to three rings which are either bonded together or fused together, and
• R _{2 is} an amidino group of the following structure,
  
  in which R _{4 is} preferably H, but also -OH, -OCH ₃ and -O-CO (CH ₂ ) _n CH ₃ where n may be an integer from 0 to 5, and
• • R _{3 is} a secondary amine group attached via an amide linkage to the central phenylalanine or azaphenylalanine derivative and may be poly-substituted or unsubstituted.

For the purposes of the application, the compounds of the general formula (I) are to be regarded as prodrugs if the radical R ₄ on the group R ₂ is -OH, -OCH ₃ or -O-CO (CH ₂ ) _n CH ₃ where n is equal to one integer from 0 to 5, is. Their conversion into an unsubstituted amidino group with R ₄ equal to H is described in detail in the literature and leads in the organism to actually effective inhibitor ( Ettmayer et al., J. Med. Chem. 47, 2393-2404 (2004) ).

wobei

• – X und R₂ wie zuvor definiert sind, und
• – Y ein CH oder N ist, falls R₇ direkt an Y gebunden ist, oder Y ein CH₂ oder NH ist, falls R₇ nicht direkt an Y gebunden ist, und
• – m gleich 0, 1 oder 2 ist, und
• – R₅
• – ein Aryl- oder Heteroarylrest ist, der auch teilweise hydriert sein kann, und der mit bis zu zwei Resten unabhängig voneinander substituiert sein kann, die ausgewählt werden aus H, Cl, Br, unverzweigtes oder verzweigtes Alkyl mit 1 bis 4 Kohlenstoffatomen, OH, Alkoxy mit 1 bis 3 Kohlenstoffatomen, NH₂, Amidin, Guanidin, CF₃, CN oder
• – ein Amid der Struktur R₆-CO-NH- ist, wobei R₆ ein unverzweigter oder verzweigter Alkylrest mit 1-4 Kohlenstoffatomen ist, der substituiert sein kann mit NH₂, CH₃NH, (CH₃)₂N, CH₃)₃N⁺, Cl, Br, OH, Alkoxy mit 1 bis 3 Kohlenstoffatomen, CF₃, CN, Amidin, Guanidin oder
• – ein verzweigter oder unverzweigter Alkylrest mit 3–7 Kohlenstoffatomen ist, wobei der Alkylrest substituiert sein kann mit NH₂, CH₃NH, (CH₃)₂N, Cl, Br, OH, Alkoxy mit 1 bis 2 Kohlenstoffatomen, CN, Amidin oder Guanidin, und
• – R₇
• – NH₂, oder ein verzweigter oder unverzweigter NH-Alkyl- oder ein Alkylrest mit 1–6 Kohlenstoffatomen ist, wobei der Alkylrest substituiert sein kann mit NH₂, CO-NH₂, CH₃NH, (CH₃)₂N, Cl, Br, OH, Alkoxy mit 1 bis 2 Kohlenstoffatomen, CN, Amidin, Guanidin oder NH-CO-NH-R₈, und R₈ ein H, Alkyl mit 1 bis 4 Kohlenstoffatomen oder Aryl, Aralkyl, Heteroaryl ist, oder
• – ein -(CH₂)_p-CO-R₉ ist, mit p gleich 0, 1, 2 oder 3 und wobei R₉ OH, O-Alkyl mit 1–7 Kohlenstoffatomen, NH₂ oder NH-Alkyl mit 1 bis 4 Kohlenstoffatomen ist, oder
• – ein -(CH₂)_p-NHCONH-R₁₀ ist, wobei R₁₀ H oder ein Alkyl, Cycloalkyl, Aralkyl, Aryl oder Heteroarylrest ist, der unsubstituiert oder substituiert sein kann, wobei der gegebenenfalls vorhandene Substituent Cl, Br, unverzweigtes oder verzweigtes Alkyl mit 1 bis 4 Kohlenstoffatomen, OH, Alkoxy mit 1 bis 3 Kohlenstoffatomen, NH₂, Amidin, CF₃, oder CN sein kann,
• – im Falle von X = N auch ein verzweigter oder unverzweigter aliphatischer oder aromatischer oder heteroaromatischer Acylrest mit 1–10 Kohlenstoffatomen ist, und der mit bis zu zwei Resten unabhängig voneinander substituiert sein kann, die ausgewählt werden aus H, Cl, Br, unverzweigtes oder verzweigtes Alkyl mit 1 bis 4 Kohlenstoffatomen, OH, Alkoxy mit 1 bis 3 Kohlenstoffatomen, NH₂, Amidin, Guanidin, CF₃ oder CN ist.

Very suitable for use as inhibitors of TMPRSS2, and thus for the treatment of diseases related to the activity of TMPRSS2, have been found to be compounds characterized by the inhibitors having a structure according to formula (II)

in which

• X and R _{2 are} as previously defined, and
• Y is CH or N if R _{7 is} directly attached to Y, or Y is CH ₂ or NH if R _{7 is} not directly attached to Y, and
• - m is 0, 1 or 2, and
• - R ₅
• An aryl or heteroaryl radical which may also be partially hydrogenated and which may be substituted independently of one another by up to two radicals selected from H, Cl, Br, unbranched or branched alkyl having 1 to 4 carbon atoms, OH, Alkoxy of 1 to 3 carbon atoms, NH ₂ , amidine, guanidine, CF ₃ , CN or
• An amide of structure R ₆ -CO-NH-, wherein R _{6 is} an unbranched or branched alkyl radical of 1-4 carbon atoms which may be substituted with NH ₂ , CH ₃ NH, (CH ₃ ) ₂ N, CH ₃ ) ₃ N ⁺ , Cl, Br, OH, alkoxy of 1 to 3 carbon atoms, CF ₃ , CN, amidine, guanidine or
• - is a branched or unbranched alkyl radical having 3-7 carbon atoms, wherein the alkyl radical may be substituted with NH ₂ , CH ₃ NH, (CH ₃ ) ₂ N, Cl, Br, OH, alkoxy having 1 to 2 carbon atoms, CN, amidine or guanidine, and
• - R ₇
• - NH ₂ , or a branched or unbranched NH-alkyl or alkyl radical having 1-6 carbon atoms, wherein the alkyl radical may be substituted with NH ₂ , CO-NH ₂ , CH ₃ NH, (CH ₃ ) ₂ N, Cl , Br, OH, alkoxy 1 to 2 carbon atoms, CN, amidine, guanidine or NH-CO-NH-R ₈ , and R _{8 is} H, alkyl of 1 to 4 carbon atoms or aryl, aralkyl, heteroaryl, or
• - a - (CH ₂ ) _p -CO-R ₉ , where p is 0, 1, 2 or 3 and where R _{9 is} OH, O-alkyl of 1-7 carbon atoms, NH ₂ or NH-alkyl of 1 to 4 Carbon atoms is, or
• - a - (CH ₂ ) _p -NHCONH-R ₁₀ , wherein R _{10 is} H or an alkyl, cycloalkyl, aralkyl, aryl or heteroaryl radical which may be unsubstituted or substituted, wherein the optionally present substituent Cl, Br, unbranched or branched alkyl of 1 to 4 carbon atoms, OH, alkoxy of 1 to 3 carbon atoms, NH ₂ , amidine, CF ₃ , or CN may be,
• In the case of X = N, also a branched or unbranched aliphatic or aromatic or heteroaromatic acyl radical having 1-10 carbon atoms, and which may be substituted independently of one another by up to two radicals selected from H, Cl, Br, unbranched or branched alkyl of 1 to 4 carbon atoms, OH, alkoxy of 1 to 3 carbon atoms, NH ₂ , amidine, guanidine, CF ₃ or CN.

worin

• – X wie zuvor definiert ist, bevorzugt aber CH ist, und
• – R₇ definiert ist wie zuvor, bevorzugt aber ein -(CH₂)_n-NH₂ oder -(CH₂)_n-Guanidin mit n = eine ganze Zahl von 4 bis 5 ist, oder ein -(CH₂)₃-CO-NHCH₃, und
• – R₁₀ und R₁₁ unabhängig voneinander ausgewählt werden aus H, Cl, Br, unverzweigtes oder verzweigtes Alkyl mit 1 bis 4 Kohlenstoffatomen, OH, Alkoxy mit 1 bis 3 Kohlenstoffatomen, NH₂, H₂N-CH₂, Amidin, Guanidin, CF₃ oder CN, wobei sie bevorzugt Cl sind.

Especially suitable for use as inhibitors of TMPRSS2 and thus for the treatment of diseases related to the activity of TMPRSS2 have been compounds characterized by the inhibitors having a structure according to formula (III)

wherein

• X is as defined above, but preferably CH is, and
• R ₇ is as defined above, but is preferably a - (CH ₂ ) _n -NH ₂ or - (CH ₂ ) _n guanidine with n = an integer from 4 to 5, or a - (CH ₂ ) ₃ - CO-NHCH ₃ , and
• R ₁₀ and R _{11 are} independently selected from H, Cl, Br, unbranched or branched alkyl having 1 to 4 carbon atoms, OH, alkoxy having 1 to 3 carbon atoms, NH ₂ , H ₂ N-CH ₂ , amidine, guanidine, CF ₃ or CN, preferably being Cl.

The compounds of the invention according to formulas (I) to (III) and their salts of inorganic and organic acids are inhibitors of the serine protease TMPRSS2 and can in the form of their pharmaceutically acceptable salts for the preparation of medicaments for patients for the therapy and / or prophylaxis of viral diseases, for example Influenza infections as well as for the treatment of inflammatory respiratory diseases or diseases caused by the SARS-CoV. The term "patient" refers equally to humans and vertebrates. Thus, the drugs can be used in human and veterinary medicine.

Pharmaceutically acceptable salts of the compounds according to the invention for use as inhibitors of the serine protease TMPRSS2 according to formulas (I) to (III) are the corresponding salts of organic and inorganic acids which, according to reliable medical assessment, do not trigger excessive toxicity, irritations or allergic reactions on the patient.

These pharmaceutically acceptable salts of organic and inorganic acids are particularly suitable for the preparation of medicaments due to their higher water solubility compared to the starting or basic compounds. These salts must have a pharmaceutically acceptable anion. Suitable pharmaceutically acceptable acid addition salts of the compounds of the invention are, for. As salts of inorganic acids such as hydrochloric acid, hydrobromic acid, phosphorus, metaphosphorus, nitric, sulfonic and Sulfuric acid or salts of organic acids such. Acetic, benzenesulfonic, benzoic, citric, ethanesulfonic, fumaric, gluconic, glycolic, isethionic, lactic, lactobionic, maleic, methanesulfonic, succinic, p-toluenesulfonic, tartaric, trifluoroacetic acids. The term "physiologically functional derivative" used below denotes any physiologically acceptable derivative of a compound of the formula (I) is (III), z. An ester which, when administered to a mammal, such as. Human being, is able to form (directly or indirectly) a compound of formula I or an active metabolite thereof and is suitable for use as inhibitors of the serine protease TMPRSS2. The compounds according to general formula I can furthermore be used in various forms, e.g. B. as amorphous and crystalline polymorphic forms.

Hereinafter, all references to the use of compounds of the formula (I) to (III) as described above and salts, solvates and physiologically functional derivatives as described herein relate.

The use of active ingredients according to the present invention which are both therapeutically effective and pharmaceutically acceptable can be administered to the patient as part of a pharmaceutically acceptable composition. Administration may be peroral, parenteral, intravenous, intramuscular, subcutaneous, intracisternal, intravaginal, intraperitoneal, intravascular, intrathecal, intratracheal, intravesical, topical, topical (powder, ointment or drops) or in spray form (aerosol). The intravenous, subcutaneous, intraperitoneal or intrathecal administration can be carried out continuously by means of a pump or dosing unit. Dosage forms for topical administration of the compounds of the invention include ointments, powders, suppositories, sprays and inhalants. The active component is mixed under sterile conditions with a physiologically acceptable carrier and possible preservatives, buffers, diluents and propellants as needed. The active component may be compounds according to formula (I) to (III), their pharmaceutically acceptable salts or mixtures of compounds of formula (I) to (III) and their pharmaceutically acceptable salts.

The invention will be described below with reference to the specified embodiments.

embodiments

Methods for analysis and purification of the compounds

Analytical HPLC

For analytical reversed-phase HPLC was a HPLC system LC-10A Shimadzu, consisting of the subsystems CTO-10A column oven, LC-10ATvp pumps (2 ×), DGU-14A degasser, SIL-10Axl autoinjector, SCL-10Avp System controller, SPD-M10Avp photodiode array detector and a column 250 / 4,6 Nucleodur 100-5 C18 ec Macherey-Nagel, Germany, using the associated software Shimadzu CLASS-VP, Version 7.2.1, used. The detection was carried out at 220 nm. The eluant used was water with 0.1% TFA (A) and acetonitrile with 0.1% TFA (B), the analysis being carried out at a flow rate of 1 ml / min with a linear gradient (1%). B / min) and 10% B as start condition.

Preparative HPLC

All final inhibitors were purified by preparative RP-HPLC and are available as TFA salts. To this was added a Varian HPLC system consisting of Varian PrepStar Model 218 preparative pumps (2x), Varian ProStar Model 320 UV-Vis detector, Varian fraction collector Model 701, and a VP 250/32 Nucleodur 100-5 column C8 ec Macherey-Nagel (dunes, Germany), using the associated Star Software V. 6.0. The detection was carried out at 220 nm. Eluent also used water with 0.1% TFA (A) and acetonitrile with 0.1% TFA (B) at a flow rate of 20 mL / min and a suitable gradient.

Mass spectrometry

The spectra were recorded with an Applied Biosystems device (QTrap 2000).

used abbreviations

• Boc

  tert-butyloxycarbonyl

  Cbz

  benzyloxycarbonyl

  DIPEA

  diisopropylethylamine

  DCM

  dichloromethane

  DMF

  N, N-dimethylformamide

  HPLC

  High performance liquid chromatography

  i. V.

  in a vacuum

  LM

  solvent

  MS

  mass spectroscopy

  NMM

  N-methylmorpholine

  PyBOP

  Benzotriazol-1-yl-N-oxy-tris (pyrrolidino) phosphonium hexafluorophosphate

  RT

  room temperature

  TFA

  trifluoroacetic

All of the amino acids, coupling reagents and other reagents used in the synthesis were purchased from Peptech, Senn, Aldrich, Fluka or Acros.

example 1

Synthesized compounds

The basic structures of the compounds or inhibitors used for the experiments are already known and were therefore synthesized by the methods known from the literature, finally purified by preparative HPLC and are present as TFA salts ( Stürzebecher et al., Bioorg. Med. Chem. Lett. 9, 3147-3152 (1999) ; S teinmetzer et al., J. Med. Chem. 49, 4116-4126, (2006) ; Steinmetzer et al. Bioorg. Med. Chem. Lett. 19, 67-73, (2009) ; S chweinitz et al. Bioorg. Med. Chem. Lett. 19, 1960-1965, (2009) ; Stürzebecher et al. J. Med. Chem. 40, 3091-3099, (1997) ). Compounds with a 3-Amidinoazaphenylalanin as a central building block are also synthesized by literature methods (Zega et al., Bioorg Med Med Chem 14 (2004) 1563-1567).

For example, the particularly effective inhibitor 2 is synthesized according to the following scheme (Scheme 1).

Scheme 1: Synthesis of inhibitor 2: (a) Dioxane / 1N NaOH, stir at 0 ° C for 1 h and at RT overnight; (b) PyBOP, 2.0 eq. DIPEA in DMF, 15 min at 0 ° C and 3 h at RT; (c) 2 eq. Baric acid, 1 mol% Pd (OAc) ₂ and 2 mol% S-Phos in toluene with 3 eq. 2 M Cs ₂ CO ₃ solution, 4 h at reflux; (d) i: 2.5 eq. Hydroxylamine × HCl and 2.5 eq. DIPEA, reflux in ethanol, 4 h, stir at RT overnight, followed by concentration of the solvent i. V., ii: 3 eq. Ac ₂ O in acetic acid, 30 min RT, concentration of the solvent; iii: zinc dust in 90% acetic acid, stirring at 30 ° C overnight; iv: 90% TFA, RT, 1 h, concentration of the solvent and purification of the product by preparative RP-HPLC and lyophilization.

The compounds having C-terminal urea structure (e.g., 14, 15, 22-24, 27-31) in the C-terminal secondary amide residue R _{3 of} the general structure (i) are prepared by reacting with Intermediate A, the synthesis thereof previously described (see Steinmetzer et al., J. Med. Chem. 49, 4116-4126, (2006) , Intermediate 10 in Scheme 2 of this publication) cleaves off the Boc protective group with TFA or 1N HCl in glacial acetic acid, reacting the liberated amine with variously substituted isocyanates and finally building the amidino group from the nitrile by known methods. Scheme 2 exemplifies the synthesis of C-terminal ethyl urea inhibitor 15. For the synthesis of inhibitor 14, on the other hand, intermediate B (Scheme 2) was reacted with N-succinimidyl-N-methylcarbamate, while in the case of inhibitor 27, intermediate B was treated with N-methyl-N-nitrosourea. The final structure of the amidino group was carried out analogously to the procedure described in Scheme 1.

Scheme 2: Synthesis of inhibitor 15: (a) 1N HCl in acetic acid, 1 h, RT; (b) 1.5 eq. Ethyl isocyanate and 2 eq. TEA in DCM overnight at RT; (c) i: 2.5 eq. Hydroxylamine × HCl and 2.5 eq. DIPEA, reflux in ethanol, 4 h, stir at RT overnight, followed by concentration of the solvent i. V., ii: 3 eq. Ac ₂ O in acetic acid, 30 min RT, concentration of the solvent; iii: H ₂ at 10% by weight of 10% Pd / C, stirring at RT overnight in 90% acetic acid; iv: Concentration of the solvent and purification of the product by preparative RP-HPLC and lyophilization.

The structures and analytical data of the synthesized inhibitors are summarized in Table 1.

Table 1: Overview and analytical characterization of the synthesized inhibitors (n.b. = not determined). All compounds are present as TFA salts.

Example 2

Recombinant production of the protease domain of TMPRSS2

Cloning of the protease domain of TMPRSS2:
The serine protease domain was amplified from the expression plasmid pCAGGS-TMPRSS2, wherein
5'-GGATATCATATGAAACATCACCATCACCATCACATCGTGGGCGGTGAGAG-3 'and 5'-GGATATGAATTCTTAGCCGTTTGCCTTCATTTG-3' were used as sense and antisense primers. The primers were chosen such that at the 5 'end of the protease domain, an Nde1 site and at the 3' end an EcoR1 site. The ~ 750 base pair amplification product was purified, digested with the restriction enzymes Nde1 and EcoR1 and cloned into a pET24-b vector (Novagen, Merck Biosciences, Bad Soden, Germany) for protein expression in Escherichia coli.

The catalytic domain of TMPRSS2 is expressed in the form of inclusion bodies, as described below, then denatured, purified, refolded and activated.

Expression, Purification, Refolding and Activation of the TMPRSS2 Catalytic Domain:
BL21 (DE3) codon plus cells containing the expression vector were incubated in LB medium, 30 μg / ml kanamycin and 35 μg / ml chloramphenicol at 37 and 220 rpm. Expression was induced by addition of 1 mM IPTG at a cell density of OD ₆₀₀ = 1.2 and incubation continued for an additional three hours.

The cells were harvested, resuspended in buffer A (50 mM Tris HCl, pH 7.5, 0.9% NaCl), disrupted by ultrasonication, and the DNA digested by means of benzonase (25 U / g cell pellet, Novagen). The inclusion bodies were washed and denatured in buffer B (8 M urea, 100 mM NaH ₂ PO ₄ , pH 8.0, 10 mM Tris, 5 mM DTT, 5 ml per 1 ml pellet). The insoluble components were separated by centrifugation from the denatured protein.

The His-tagged protease was purified on metal chelate chromatography (NiNTA Agarose, Qiagen, Hilden, Germany) and the fractions containing TMPRSS2 pooled. to Renaturation was the rapid dilution method in Renaturierungspuffer (50 mM Tris, pH 7.5, 0.5 M L-arginine, 20 mM CaCl ₂ , 1 mM EDTA, 100 mM NaCl, glutathione added fresh) to a final concentration of 50 ug / ml , After three days of incubation at 4 ° C., the protein solution was filtered to a concentration of> 300 μg / ml by tangential filtration (vivaflow 200.10 kDa cut-off, Sartorius, Göttingen, Germany) and ultrafiltration (vivaspin 20, 10 kDa cut-off , Sartorius) and the buffer was changed to activation buffer (50 mM Tris, pH 7.5, 1 M NaCl, 0.05% Brij 58).

Since a free N-terminus is essential for the activity of the serine protease, the TMPRSS2 must be activated by cleavage of the n-terminal peptide sequence MK (His) ₆ . This was achieved by incubating the protease for seven hours with 2.5 U / ml of activated DAPase ^™ (Qiagen) followed by separation of activated non-activated TMPRSS2 and His-tag bound DAPase via chelate chromatography.

The yield of this protocol is about 0.6 mg of active catalytic domain per 2 L cell culture.

For analysis of the purified protein, an SDS-Page was carried out with subsequent Western Blot ( 1 ) using TMPRSS2-specific antibodies.

Example 3

Enzyme kinetic studies to determine TMPRSS2 inhibitory activity

The determination of the TMPRSS2 inhibitory activity was carried out with a fluorescence plate reader Safire ² from Tecan (λ _Ex = 380 nm, λ _Em = 460 nm) and H-dCha-Pro-Arg-AMC × 2 TFA as substrate. The enzyme used was the recombinantly produced protease domain of TMPRSS2.

Tabelle 2: K_i-Werte ausgewählter Inhibitoren für die Hemmung der TMPRSS2. Inhibitor K_i(nM) 1 4,0 2 0,93 3 17,0 4 4,8 5 7,8 6 19,0 7 40,1 8 207,2 9 71,8 10 7,8 11 71,6 12 28,5 13 27,5 14 9,6 15 10,9 16 4,5 17 0,95 18 19,0 19 2,7 20 36,2 21 8,9 22 6,5 23 22,3 24 17,6 25 7,3 26 5,4 27 14,0 28 77,6 29 72,5 30 36,2 31 8,0 32 214 33 17400 34 357 35 14870 36 146 37 24970 38 853 39 16,5 40 28,8 41 56,2 For the determination of the inhibition constants, the measurement buffer (50 mM Tris HCl, pH 8.0, 154 mM NaCl) was combined with substrate (concentrations 200, 100, 50 μM in the batch) with different inhibitor concentrations, which were varied at least over the range of one order of magnitude. After enzyme addition, the steady-state rates were determined by linear regression. Parallel to each inhibitor measurement, K _m and V _max (in the unit ΔRFU / s) were determined by means of a v / S characteristic. The K _i values were calculated by adapting the determined rates as a function of the inhibitor and substrate concentrations to the rate equation for competitively reversibly binding inhibitors:

Table 2: K _i values of selected inhibitors of inhibition of TMPRSS2. inhibitor K _i (nM) 1 4.0 2 0.93 3 17.0 4 4.8 5 7.8 6 19.0 7 40.1 8th 207.2 9 71.8 10 7.8 11 71.6 12 28.5 13 27.5 14 9.6 15 10.9 16 4.5 17 0.95 18 19.0 19 2.7 20 36.2 21 8.9 22 6.5 23 22.3 24 17.6 25 7.3 26 5.4 27 14.0 28 77.6 29 72.5 30 36.2 31 8.0 32 214 33 17400 34 357 35 14870 36 146 37 24970 38 853 39 16.5 40 28.8 41 56.2

Example 4

Inhibition of TMPRSS2-Mediated Virus Propagation in the Presence of Synthetic Serine Protease Inhibitors

MDCK-TMPRSS2 cells with inducible expression of protease TMPRSS2 were used for the experiments. MDCK-TMPRSS2 cells were isolated by stable transfection of MDCK cells (Madin Darby Canine Kidney) with the plasmids pcEFTet-On / NEO and pTRE2pur-TMPRSS2-FLAG ( Böttcher et al. Vaccine 27, 6324-6329 (2009) ; Böttcher et al. J Virol 84, 5605-5614 (2010) ) generated. The expression of TMPRSS2 in these cells can be induced by adding doxycycline (Dox) to the culture medium (Tet-On expression system, Gossen and Bujard, Science 1995).

To analyze the efficacy of synthetic serine protease inhibitors on the inhibition of influenza virus proteolytic activation by TMPRSS2, multicyclic replication and virus spread in MDCK-TMPRSS2 cells in the presence of the inhibitors was examined. MDCK-TMPRSS2 cells were first cultured in 24-well plates for 24 h in the presence and absence of 0.2 μg / ml doxycycline. The cells were subsequently infected with the human influenza isolate A / Memphis / 14/96 (H1N1) and incubated for 24 h in the presence or absence of various inhibitors at 37 ° C and 5% CO ₂ . Subsequently, infected cells were immunohistochemically stained against the viral nucleoprotein. It could a concentration-dependent inhibition of virus proliferation and spread by the synthetic inhibitors used. Exemplary is in 2 inhibition of virus spread in inhibitor 2 treated cells.

Example 5

Inhibition of TMPRSS2-Mediated Virus Propagation by Synthetic Serine Protease Inhibitors in Human Airway Epithelial Cells

Calu-3 cells (human respiratory epithelial cells, endogenous expression of TMPRSS2) were used to demonstrate the inhibitory effect of TMPRSS2-mediated viral spread. For this, the cells were cultured in 6-well plates and infected with the human influenza virus isolate A / Memphis / 14/96 (H1N1) in the presence and absence of inhibitor 2 (50 μM) for 72 h and at various times the virus titer in Cell culture supernatant by plaque assay (determination of infectious virus per ml, pfu: plaque forming units) determined. A significant delay in virus replication and a 1000-fold decrease in the virus titer in the presence of inhibitor 2 compared to the control without inhibitor (mock) could be observed. This is in 3 shown.

Figure legends

Fig. 1

• 1. MDCK-Zellen ohne TMPRSS2-Expression
• 2. MDCK-Zellen mit TMPRSS2-Expression
• 3. Active TMPRSS2 exprimiert in E. coli

For analysis of the purified protein, an SDS-Page followed by Western Blot was performed using TMPRSS2 specific antibodies. The western blot is in 1 shown. 1 shows the SDS-Page and the detection of the protease domain of TMPRSS2 by Western Blot.
G
Western blot using specific TMPRSS2 antibody:

• 1. MDCK cells without TMPRSS2 expression
• 2. MDCK cells with TMPRSS2 expression
• 3. Active TMPRSS2 expressed in E. coli

Fig. 2

2 shows the inhibition of multicyclic replication and virus spread in MDCK-TMPRSS2 cells in the presence of inhibitor 2.

Fig. 3

3 shows the inhibition of TMPRSS2-dependent virus spread in Calu-3 cells by inhibitor 2.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• WO 2010/149459 [0004]

Cited non-patent literature

• Paolini-Giacobino, Genomics 44, 309-320 (1997) [0003]
• Böttcher et al., J. Virology 80, 9896-9898 (2006) [0003]
• Böttcher et al. Vaccine 27, 6324-6329 (2009) [0003]
• Böttcher et al. J Virol. 84, 5605-5614 (2010) [0003]
• Chaipan et al., J. Virology 83, 3200-3211 (2009) [0003]
• Shirogane et al., J. Virology 82, 8942-8946 (2008) [0003]
• Matsuyama et al., J. Virol. 82 (2010) 12658-12664 [0003]
• Stürzebecher et al., Bioorg. Med. Chem. Lett. 9, 3147-3152 (1999) [0006]
• Steinmetzer et al., J. Med. Chem. 49, 4116-4126, (2006) [0006]
• Steinmetzer et al. Bioorg. Med. Chem. Lett. 19, 67-73, (2009) [0006]
• Schweinitz et al. Bioorg. Med. Chem. Lett. 19, 1960-1965, (2009) [0006]
• Stürzebecher et al. J. Med. Chem. 40, 3091-3099, (1997) [0006]
• Ettmayer et al., J. Med. Chem. 47, 2393-2404 (2004) [0008]
• Stürzebecher et al., Bioorg. Med. Chem. Lett. 9, 3147-3152 (1999) [0021]
• teinmetzer et al., J. Med. Chem. 49, 4116-4126, (2006) [0021]
• Steinmetzer et al. Bioorg. Med. Chem. Lett. 19, 67-73, (2009) [0021]
• chweinitz et al. Bioorg. Med. Chem. Lett. 19, 1960-1965, (2009) [0021]
• Stürzebecher et al. J. Med. Chem. 40, 3091-3099, (1997) [0021]
• Steinmetzer et al., J. Med. Chem. 49, 4116-4126, (2006) [0024]
• Böttcher et al. Vaccine 27, 6324-6329 (2009) [0038]
• Böttcher et al. J Virol 84, 5605-5614 (2010) [0038]

Claims (10)

Use of compounds according to the general formula (I) and of pharmaceutically suitable salts of these compounds,

where X is CH or N, and R _{1 is} a mono- or polysubstituted or unsubstituted cyclic aryl or heteroaryl radical which may contain one to three rings which are either bonded together or fused together, and R _{2 is} an amidino group of following structure is

in which R _{4 is} preferably H, but also -OH, -OCH ₃ and -O-CO (CH ₂ ) _n CH ₃ where n may be an integer from 0 to 5, and • R _{3 is} a secondary amine group, which is bound via an amide bond to the central phenylalanine or azaphenylalanine derivative and may be multiply substituted or unsubstituted, for inhibiting the serine protease TMPRSS2 and as a medicament for the treatment of influenza infections or for the treatment of inflammatory respiratory diseases and infections with the Sars coronavirus. Use according to claim 1, characterized in that the inhibitors have a structure according to formula (II), wherein

X and R _{2 are} as previously defined, and Y is CH or N if R _{7 is} directly attached to Y, or Y is CH ₂ or NH if R _{7 is} not directly attached to Y, and m is 0, 1 or 2, and - R ₅ - is an aryl or heteroaryl radical, which may also be partially hydrogenated, and may be substituted with up to two independently selected radicals selected from H, Cl, Br, unbranched or branched alkyl of 1 to 4 carbon atoms, OH, alkoxy of 1 to 3 carbon atoms, NH ₂ , amidine, guanidine, CF ₃ , ON or - an amide of the structure R ₆ -CO-NH-, where R _{6 is} unbranched or branched Is alkyl radical having 1-4 carbon atoms, which may be substituted by NH ₂ , CH ₃ NH, (CH ₃ ) ₂ N, CH ₃ ) ₃ N ⁺ , Cl, Br, OH, alkoxy having 1 to 3 carbon atoms, CF ₃ , CN, amidine, guanidine or - is a branched or unbranched alkyl radical having 3-7 carbon atoms, wherein the alkyl radical may be substituted with NH ₂ , CH ₃ NH, (CH ₃ ) ₂ N, Cl, Br, OH, alkoxy with 1 to 2 carbon atoms, CN, amidine or guanidine, and - R ₇ - NH ₂ , or a branched or unbranched NH-alkyl or alkyl radical having 1-6 carbon atoms, wherein the alkyl radical may be substituted with NH ₂ , CO-NH ₂ , CH ₃ NH, (CH ₃ ) ₂ N, Cl, Br, OH, alkoxy of 1 to 2 carbon atoms, CN, amidine, guanidine or NH-CO-NH-R ₈ , and R _{8 is} H, alkyl of 1 to 4 carbon or aryl, aralkyl, heteroaryl, or - a - (CH ₂ ) _p -CO-R ₉ , where p is 0, 1, 2 or 3 and where R _{9 is} OH, O-alkyl having 1-7 carbon atoms, NH ₂ or NH-alkyl of 1 to 4 carbon atoms, or - is - (CH ₂ ) _p -NHCONH-R ₁₀ wherein R ₁₀ is H or an alkyl, cycloalkyl, aralkyl, aryl or heteroaryl group which is unsubstituted or substituted where the optional substituent Cl, Br, unbranched or branched alkyl having 1 to 4 carbon atoms, OH, alkoxy having 1 to 3 carbon atoms, NH ₂ , amidine, CF ₃ , or CN, - in the case of X = N also is a branched or unbranched aliphatic or aromatic or heteroaromatic acyl radical having 1-10 carbon atoms, and which may be independently substituted with up to two radicals selected from H, Cl, Br, straight or branched alkyl having 1 to 4 carbon atoms , OH, alkoxy of 1 to 3 carbon atoms, NH ₂ , amidine, Gua nidine, CF ₃ or CN. Use according to claims 1 to 2, characterized in that the inhibitors have a structure according to formula (III)

wherein - X is as defined above, and - R _{7 is} defined as before, but is preferably a - (CH ₂ ) _n -NH ₂ or - (CH ₂ ) _n guanidine with n = an integer from 0 to 5, or a - (CH ₂ ) ₃ -CO-NHCH ₃ , and - R ₁₀ and R _{11 are} independently selected from H, Cl, Br, straight or branched alkyl of 1 to 4 carbon atoms, OH, alkoxy of 1 to 3 carbon atoms , NH ₂ , H ₂ N-CH ₂ , amidine, guanidine, CF ₃ or CN, preferably being Cl. Use according to claims 1 to 3, characterized in that X in the inhibitors is a CH and the resulting central 3-Amidinophenylalanin derivative is preferably in the L-configuration. Use according to claims 1 to 4, characterized in that it is one of the following structures in the compounds of formula I to III

Use of a pharmaceutically acceptable composition comprising compounds according to any one of claims 1 to 5 and / or their pharmaceutically acceptable salts of inorganic and organic acids or bases as the active component and a physiologically acceptable carrier for the manufacture of a medicament for the treatment of viral infections or respiratory inflammatory diseases. Use of a pharmaceutically acceptable composition according to claim 6, characterized in that it is administered perorally, parenterally, intravenously, intramuscularly, subcutaneously, intracisternally, intravaginally, intraperitoneally, intravascularly, intrathecally, intratracheally, topically, locally or as an aerosol. Use of compounds according to any one of claims 1 to 7 and their pharmaceutically acceptable salts of inorganic and organic acids for the manufacture of a medicament for the therapy and / or prophylaxis of viral diseases, especially of influenza infections. Use of compounds according to any one of claims 1 to 7 and their pharmaceutically acceptable salts of inorganic and organic acids for the manufacture of a medicament for the treatment of inflammatory respiratory diseases. Use of compounds according to any one of claims 1 to 7 and their pharmaceutically acceptable salts of inorganic and organic acids for inhibiting the serine protease TMPRSS2.

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