Selective TMPRSS2 inhibitors and medical use thereof Description The invention relates to novel inhibitors of TMPRSS2 and their use for the prophylaxis and treatment of cancer, influenza virus infection or coronavirus infection, in particular, SARS-CoV2 infection. Background of the invention To enter into airway epithelial cells, influenza, parainfluenza and coronaviruses rely on host cell proteases for activation of the viral protein involved in membrane fusion. Transmembrane protease serine 2 (TMPRSS2) was recently proven to be crucial for hemagglutinin cleavage of some human influenza viruses (W-J Shin et al., Expert Opinion on Drug Discovery, 2017, 12(11), 1139-1152). Since the catalytic sites of the diverse serine proteases linked to influenza, parainfluenza and coronavirus activation are structurally similar, active site inhibitors of these airway proteases could have broad therapeutic applicability against multiple respiratory viruses. A role is now well established for transmembrane protease serine 2 (TMPRSS2), a member of the TTSP family, which are integral membrane proteins with an extracellular C-terminal serine protease domain and an N-terminal cytoplasmic domain. One unique benefit of blocking TMPRSS2 and related airway proteases is that, besides influenza virus, several other respiratory viruses could be targeted. TMPRSS2 was shown to activate the fusion proteins of some paramyxoviruses (i.e. metapneumovirus, trypsin-dependent parainfluenza subtypes and Sendai virus). TMPRSS2 is supported by recent proof, from mouse or clinical studies, that this protease is essential for replication of influenza viruses such as H1N1 and H7N9 viruses (Kouji Sakai et al., Journal of Virology, 2014, 88(10), 5608-5616). In December 2019, Wuhan, in Hubei province, China, became the center of an outbreak of a new coronavirus. This new coronavirus, i.e. SARS-CoV2 causing COVID- 19 and previous experiences with SARS and MERS-CoV, highlight the need for therapeutics for human coronavirus infections that can improve clinical outcomes, reduce risk of disease progression, speed recovery, and reduce the requirements for intensive supportive care and prolonged hospitalisation. Likewise, for some human coronaviruses including the deadly SARS, COVID-19 (SARS-Cov-2) and MERS variants, TMPRSS2 cleavage activates the viral spike (S)- protein at the cell surface enabling cathepsin-independent host cell entry. SARS and COVID-19 uses the SARS-CoV receptor ACE2 for attachement and the serine protease TMPRSS2 for S protein priming and fusion events with the cell membrane. Thus, SARS, MERS and COVID-19 can be inhibited by TMPRSS2 inhibitors. Recently it was shown that camostat could be used effectively in protecting mice against a lethal infection by SARS coronavirus (Hoffmann et al., Cell, 2020, 181, 1-10). Besides this selectivity issue, the safety of serine protease inhibitors largely depends on whether or not the binding is reversible. The classical inhibitors show covalent binding to the catalytic serine, which is in most cases (pseudo-) irreversible, for example with camostat. Such irreversible protease inhibitors may pose a safety issue, as such non-selective serine protease inhibitors also produce strong inhibition of off- tagets such as the related type II transmembrane serine proteases (TTSPs i) ncluding, thrombin, plasmin and factor Xa. Since an antiviral medication for these viruses, in particular coronaviruses is currently lacking, it is neceesary to develop the antiviral drugs. The objective of the present invention is to provide novel and preferably selective inhibitors of TMPRSS2 and methods for the synthesis of said inhibitors as well as their use for the prophylaxis and treatment of viral infections, especially corona virus infections, and in particular COVID-19 (SARS-CoV-2) infections. Said objective is solved by the technical teachings of the independent claims. Further advantageous embodiments, aspects and details of the invention are evident from the dependent claims, the description, the figures and the examples. Surprisingly, it has been found that tetrapeptidomimetics having a benzothiazole warhead as disclosed herein inhibit selectively TMPRSS2 over thrombin and coagulation factor Xa and also inhibit effectively the replication of coronavirus, in particular SARS-CoV2. Thus, the present invention relates to a compound of the general formula (I): wherein

R represent CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ , CH(CH ₃ ) ₂ , O C(CH ₃ ) ₃ , or –cyclo-C ₃ H ₅ ; R5 represents –H, −COOH, or −CONH2; R6 , R7 , and R8 represent independently of each other –H, –CH3, –CH2CH3, –CH ₂ CH ₂ CH ₃ , –CH(CH ₃ ) ₂ , –C(CH ₃ ) ₃ , or –cyclo-C ₃ H ₅ ; or a diastereomer, an enantiomer, a mixture of diastereomers, a mixture of enantiomers, a racemate, a solvate, a hydrate, a prodrug or a pharmaceutically acceptable salt thereof. Preferably, W represent –CHO, –CN, –B(OH) ₂ , The pharmaceutically acceptable salts of the comp ay be formed with organic or inorganic acids or base s. Examples of suitable acids for such acid addition salt formation are hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, malonic acid, salicylic acid, p- aminosalicylic acid, malic acid, fumaric acid, succinic acid, ascorbic acid, maleic acid, sulfonic acid, phosphonic acid, perchloric acid, nitric acid, formic acid, propionic acid, gluconic acid, lactic acid, tartaric acid, hydroxymaleic acid, pyruvic acid, phenylacetic acid, benzoic acid, p-aminobenzoic acid, p-hydroxybenzoic acid, methanesulfonic acid, ethanesulfonic acid, nitrous acid, hydroxyethanesulfonic acid, ethylenesulfonic acid, p- toluenesulfonic acid, naphthylsulfonic acid, sulfanilic acid, camphorsulfonic acid, china acid, mandelic acid, o-methylmandelic acid, hydrogen-benzenesulfonic acid, picric acid, adipic acid, d-o-tolyltartaric acid, tartronic acid, (o, m, p)-toluic acid, naphthylamine sulfonic acid, trifluoroacetic acid, and other mineral or carboxylic acids well known to those skilled in the art. The salts are prepared by contacting the free base form with a sufficient amount of the desired acid to produce a salt in the conventional manner. In the case the inventive compounds bear acidic groups, salts could also be formed with inorganic or organic bases. Examples for suitable inorganic or organic bases are, for example, NaOH, KOH, NH ₄ OH, tetraalkylammonium hydroxide, lysine or arginine and the like. Salts may be prepared in a conventional manner using methods well known in the art, for example by treatment of a solution of the compound of the general formula (I) with a solution of an acid, selected out of the group mentioned above. Preferred are the following compounds of formula (I-1): wherein E* represents W, R4 , R*, A1 , A2 , and A have the same meanings as defined in the formula (I). Preferred are compounds of the general formula (II-1): wherein W represents –CN, –B(OH) ₂ , X represents –H, –CO–OR6 , or –CO–NR7R8; E* represents –R4 , or –COR4; A3 represents R4 represent –CH ₃ , –CH ₂ CH ₃ , –CH ₂ CH ₂ CH ₃ , –CH(CH ₃ ) ₂ , –O-C(CH ₃ ) ₃ , or –cyclo-C ₃ H ₅ ; R5 represents –H, −COOH, or −CONH ₂ ; R6 , R7 , and R8 represents independently of each other –H, –CH ₃ , –CH ₂ CH ₃ , –CH ₂ CH ₂ CH ₃ , –CH(CH ₃ ) ₂ , –C(CH ₃ ) ₃ , or –cyclo-C ₃ H ₅ ; or a diastereomer, an enantiomer, a mixture of diastereomers, a mixture of enantiomers, a racemate, a solvate, a hydrate, a prodrug or a pharmaceutically acceptable salt thereof. More preferred are compound of the general formula (II-1): wherein

or a diastereomer, an enantiomer, a mixture of diastereomers, a mixture of enantiomers, a racemate, a solvate, a hydrate, a prodrug or a pharmaceutically acceptable salt thereof. Also preferred are compounds of the formula (II-2a) or (II-2b): wherein W represents –CN, –B(OH) ₂ , X represents –H, –CO–OR ⁶ , or –CO–NR ⁷ R ⁸ ; E represents –H, –R ⁴ , –COR4 , –A ³ –R ⁴ , or –A ³ –COR ⁴ ; and preferably –H, –R ⁴ , or –COR ⁴ ; A ¹ represents

R4 represent –CH ₃ , –CH ₂ CH ₃ , –CH ₂ CH ₂ CH ₃ , –CH(CH ₃ ) ₂ , –O-C(CH ₃ ) ₃ , or –cyclo-C ₃ H ₅ ; R5 represents –H, −COOH, or −CONH ₂ ; R6 , R7 , and R8 represents independently –H, –CH ₃ , –CH ₂ CH ₃ , –CH ₂ CH ₂ CH ₃ , –CH(CH ₃ ) ₂ , –C(CH ₃ ) ₃ , or –cyclo-C ₃ H ₅ ; or a diastereomer, an enantiomer, a mixture of diastereomers, a mixture of enantiomers, a racemate, a solvate, a hydrate, a prodrug or a pharmaceutically acceptable salt thereof. More preferred, the compound has the formula (II-2a) or (II-2b): wherein E represents –H, –R ⁴ , –COR ⁴ , –A ³ –R4 , or –A ³ –COR ⁴ ; and preferably –H, –R ⁴ , or –COR ⁴ ; R ⁴ and W have the same meanings as defined in the formula (I), or a diastereomer, an enantiomer, a mixture of diastereomers, a mixture of enantiomers, a racemate, a solvate, a hydrate, a prodrug or a pharmaceutically acceptable salt thereof. Also preferred are the compounds of the formulae (III-1) – (III-5):

wherein RN1 represents –H, or R1 and RN1 f ^{orm –C} 2 ^H 4 ^{– , –C} 3 ^H 6 ^{–, –C} 4 ^H 8 ^– , ( ^{i e} ) ^; RN2 represents –H, or R2 and RN2 form –C ₂ H ₄ –, –C ₃ H ₆ –, –C ₄ H ₈ –,

R represents –CH ₃ , –CH ₂ CH ₃ , –CH ₂ CH ₂ CH ₃ , –CH(CH ₃ ) ₂ , –O-C(CH ₃ ) ₃ , or –cyclo-C ₃ H ₅ ; R5 represents –H, −CO ₂ H, or −CONH ₂ ; or a diastereomer, an enantiomer, a mixture of diastereomers, a mixture of enantiomers, a racemate, a solvate, a hydrate, a prodrug or a pharmaceutically acceptable salt thereof. Moreover, in all general formulae disclosed herein it is preferred if A1 is a cyclic amino acid residue such as

residue A or the cyclus-containing amino acid residue A as disclosed directly above is connected to an amino acid residue A2 selected from the group consisting of:

preferably from the group consisting of: Moreover, it is preferred if A2 or A3 represent and more preferably Still more preferred is if A2 represent

The term “prodrug” describes compounds according to the present invention, wherein the compounds comprises at least one carboxylate group which is modified with a rest that is generally known by a person skilled in the art in that way that the carboxylate group of the compound is released under physiological conditions and/or at least one modified hydroxyl group which is modified with a rest that is generally known by a person skilled in the art in that way that the hydroxyl group of the inventive compond is released under physiological conditions, The prodrug includes also amidoxime group of arginine and benzamidine of the compound as follows: mpounds bound to TMPRSS2 reversibly and inhibit TMPRSS2 effectively. The electrophilic warheads can react with highly nucleophilic serine residue in the active site of the TMPRSS2. Therefore, it was found that potential unspecific reactions with off-targets such as the related TTSPs including thrombin, plasmin and factor Xa are reduced. It would be expected that the inventive compounds as reversible TMPRSS2 inhibitor may be less toxic than the non selective transmembrane serine protease inhibitors. Furthermore, the compounds of the present invention also inhibit the replication of coronavirus effectively and thus are useful for the prophylaxis and treatment of virus infection, preferred, wherein virus is influenza virus, parainfluenza virus or coronavirus, in particular SARS-CoV2 infection. Surprisingly, it has been found that tetrapeptidomimetics having a warhead such as benzothiazole, thiazole, aldehyde, nitrile and boric acid as disclosed herein inhibit selectively TMPRSS2 over thrombin, and coagu-lation factor Xa and also inhibit effectively virus infections, preferred influenza virus infection, parainfluenza virus infection, coronavirus infection, in particular COVID-19. More preferred are the compounds of the formulae (IV-1a) – (IV-1e):

wherein R2 – R5 and RN2 have the same meansings as defined in the formula (III-1). More preferred are the compounds of the formulae (IV-2a) – (IV-2f): wherein R2 – R4 and RN2 have the same meansings as defined in the formula (III-2). More preferred are the compounds of the formulae (IV-3a) – (IV-3f): HN NH More preferred are the compounds of the formulae (IV-4a) – (IV-4f): HN NH ₂ NH

w herein R R and R have the same meansings as defined in the formula (III 4). More preferred are the compounds of the formulae (IV-5a) – (IV-5f):

wherein R2 – R5 and RN2 have the same meansings as defined in the formula (III-5). Still more preferred is the compound having any one of the formulae (V-1a) – (V-1b): wherein R1 , R3 , R Still more preferred is the compound having any one of the formulae (V-2a) – (V-2b): w GAR-P04249WO12 PCT Application (without Figures).doc Still more preferred is the compound having any one of the formulae (V-3a) – (V-3b): wherein R1 , R3 , and R4 have the same meanings as defined in the formula (III-3). Still more preferred is the compound having any one of the formulae (V-4a) – (V-4b): ee , , a d ae t e sa e ea gs as de ed t e o ua ( ) Still more preferred is the compound having any one of the formulae (V-1a) – (V-1b): Still more preferred is the compound having any one of the formulae (VI-1a) – (VI-1f):

wherein R and R have the same meanings as defined in formula (III-1). Still more preferred is the compound having any one of the formulae (VI-2a) – (VI-2g):

w Still more preferred is the compound having any one of the formulae (VI-3a) – (VI-3g):

wh Still more preferred is the compound having any one of the formulae (VI-4a) – (VI-4g):

Still more preferred is the compound having any one of the formulae (VI-5a) – (VI-5g):

w Still more preferred are compounds of any one of the formulae (VII-1a) – (VII-1i):

Still more preferred are compounds of any one of the formulae (VII-2a) – (VII-2i):

Still more preferred are compounds of any one of the formulae (VII5a) (VII5i):

Still more preferred are compounds of any one of the formulae (VIII-1a) (VIII-1i): GA R4 represents –CH ₃ or –O-tert-Bu. Still more preferred are compounds of any one of the formulae (VIII-3a) – (VIII-3i): wherein R2 represents R4 represents –CH ₃ o r –O-tert-Bu. Still more preferred are compounds of any one of the formulae (VIII-4a) – (VIII-4i):

Still more preferred are compounds of any one of the formulae (VIII-5a) – (VIII-5i):

Still more preferred are compounds of any one of the formulae (VIII-6a) – (VIII-6i):

It is well-known for a person skilled in the art that the proline moiety of the compounds of any of the above-mentioned formulae (IV-1c), (IV-2c), (IV-3c), (IV-4c), (VI-1c), (VI-2c), (VI-3c), (VI-4c), (VI-5c), (VII-1g), (VII-2g), (VII-3g), (VII-4g), (VII-5g), (VII-6g), (VIII-1g), (VIII-2g), (VIII-3g), (VIII-4g), (VIII-5g) and (VIII-6g) can be equivalently replaced with any of the following groups:

Most preferred are the compounds selected from the group consisting of:

GAR-P04249WO12 PCT Application (without Figures).doc or a diastereomer, an enantiomer, a mixture of diastereomers, a mixture of enantiomers, a racemate, a solvate, a hydrate, a prodrug or a pharmaceutically acceptable salt thereof. A further aspect of the present invention relates to the production of compounds of the general formula (III-1). As shown in Scheme 1, a method for producing the compound of the present invention comprises the steps: Step A: providing a benzothiazole precursor 1* and a tripeptide precursor 2*; , , p g g p , RN1 , RN2 , R4 and R5 have the same meanings as defined herein, Step B: performing a coupling reaction with the benzothiazole precursor 1* with the tripeptide precursor 2* to obtain an intermediate compound 3*; Step C: removing protecting groups PG ₀ , PG ₁ , PG ₂ and PG ₃ of the intermediate compound to produce the compound of the formular (III-1) Optionally, pur ifying and isolsating step may be performed after the Step C as follows: Step D: purifiying and isolating the compound of the formula (III-1) obtained by the Step C. Herein the tripeptide precursor is obtained by solid phase peptide synthesis. All protecting groups PG ₀ , PG ₁ , PG ₂ and PG ₃ are selectively or simultaneously removed. The term “protecting groups” as used herein refers to commonly used protection groups in organic synthesis, preferably for amino and carboxyl groups. Amino protecting gorups may be selected from the group consisting of or comprising: acetyl, benzoyl, benzyloxycarbonyl (Cbz), tert-butylcarbonyl, tert-butyloxycarbonyl (Boc), trimethylbenzenesulfonyl(Mtr) and fluorenylmethylenoxy group (Fmoc). Carboxyl protecting groups may be selected from the group consisting of or comprising: methoxy, ethoxy, isobutoxy, tert-butoxy, benzyloxy; preferably, tert-butoxy group. PG ₀ preferably is suitable amino protecting group. In coupling reaction in Step B, carboxyl acid is activated and promote the coupling reaction with amino group of intermediate compound. Preferably, carboxyl acid is activated in situ and it is well-known in peptide chemistry. Any of the following coupling reagent can be used to introduce activating group AG1: BOP, PyBOP, AOP, PyAOP, TBTU, EEDQ, Polyphosphoric Acid (PPA), DPPA, HATU, HOBt, HOAt, DCC, EDCI, BOP-Cl, TFFH, Brop, PyBrop, and CIP.

Medical Use The particular suitability of the inventive compounds of the general formula (I) is connected to the sterical and electronical properties which result from the molecule structure. The electrophilic benzothiazole warhead group is an essential unit of the reversible TMPRSS2 inhibitors, and, especially in combination with the certain peptidomimetic backbone, the arginine backbone for the P1 pocket and the certain specfic amino acids at the P2 postion which result in potent TMPRSS2 Selectivity is obtained by implementing said components at selected positions within the backbone. Thus, the inventive compound inhibit selectively TMPRSS2 over thrombin, and coagulation factor Xa and also inhibit effectively the replication of influenza virus, or coronavirus, in particular SARS-CoV2. It is known from the literature on proteases that certain warheads form covalent but reversible complexes with the active site cysteine or serin. This is particularly relevant to provide affinity to the target while forming a thiohemiacetal or hemiacetal respectively. In the biological example B-1 and B-2, it is proven that the inventive compounds as reversible serine protease inhibitor effectively inhibit the activity of TTSP family, especially TMPRSS2 (see Table 1). Furthermore, the biological example B-3 demonstrate that the inventive compounds effectively inhibit the infection of CaCo-2 cells casued by the LV(Luc)-CoV2 which is based on an infection-deficient lentiviral backbone that bears SARS-CoV2 spike protein (see Table 2). All of the inventive compound as TMPRSS2 inhibitors effectively LV(Luc)-CoV2-S infection in a dose-dependent manner. In particular Compound 2p2i shows similar activity to camostat mesylate. Furthermore, by microscopy: no apparent cytotoxicity of the inventive compound was observed. Most of all, the biological example B-4 demonstrate that the inventive compounds effectively inhibit SARS-CoV2 infection (see Table 3). Some human coronaviruses including the deadly SARS, COVID-19 (SARS-Cov-2) and MERS variants, TMPRSS2 cleavage activates the viral spike (S)-protein at the cell surface enabling cathepsin-independent host cell entry. SARS and SARS-Cov2 uses the receptor ACE2 for attachement and the serine protease TMPRSS2 for S protein priming. Thus, the SARS and COVID-19 (SARS-CoV-2) can be inhibited by TMPRSS2 inhibitors. Therefore, another aspect of the present invention relates to compounds according to the general formula (I) as medicament as well as their use in medicine. Especially preferred is the use as selective inhibitors of TMPRSS2 over thrombin, and coagulation factor Xa and also inhibit effectively the replication of virus such as influenza virus or coronavirus. The compounds according to general formula (I) (I)

A3 represents R represent CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, O-C(CH3)3, or –cyclo-C ₃ H ₅ ; R5 represents –H, −COOH, or −CONH2; R6 , R7 , and R8 represent independently of each other –H, –CH3, –CH2CH3, –CH ₂ CH ₂ CH ₃ , –CH(CH ₃ ) ₂ , –C(CH ₃ ) ₃ , or –cyclo-C ₃ H ₅ ; or a diastereomer, an enantiomer, a solvate, a hydrate, a prodrug or a pharmaceutically acceptable salt thereof; are especially suitable for use in the treatment or prophylaxis of virus infections, preferred, wherein the virus is influenza virus, parainfluenza virus or coronavirus. Influenza virus is preferably influenza A virus or influenza B virus, more preferably influenza A. Influenza A virus includes H1N1, H3N2, and H7N9. In a preferred embodiment, the inventive compound is used for the treatment or prophylaxis of of virus infection, preferred, wherein virus is coronavirus, preferably, coronavirus is coronavirus 229E, SARS, MERS, or SARS-CoV2, more preferably, SARS-CoV2 causing COVID-19. Preferred, SARS-CoV2 also includes mutants containing any of mutations K417N, E484K, E484Q, L452R, N501Y, D614G, preferred any of mutations S13I, L18F, P26S, I33T, Q52R, ΔH69/ΔV70, T77A, V82A, D89A, T93M, D118H, D138Y, G142D, Y144/145Del, W152C, E154K, R190S, D215G, L242-244del, R246I, K259R, P323L, K417N, K417T, M429I, L452R, E484K, E484Q, N501Y, A570D, D614G, H655Y, Q677H, P681H, P681R, A701V, T716I, T749I, F888L, S982A, T1027I, Q1071H, H1101D, D1118H, V1176F, D1183Y, and L4205V. More preferred, SARS-CoV2 mutants includes, but not limited to B.1.1.7 (emerged in the UK) and B.1.351 (emerged in South Africa), B.1.1.248 (P.1, emerged in Brazil), B.1.429 (emerged in Califonia, USA), B.1.525 (emerged in Angola), and B.1.617 (emerged in India). Preferred, the compound of any of the formulae (I), (I-1), (II-1), (II-2a), (II-2b), (III-1) – (III-5), (IV-1a) – (IV-1e), (IV-2a) – (IV-2f), (IV-3a) – (IV-3f), (IV-4a) – (IV-4f), (IV-5a) – (IV-5f), (V-1a) – (V-1b), (V-2a) – (V-2b), (V-3a) – (V-3b), (V-4a) – (V-4b), (V-5a) – (V- 5b), (VI-1a) – (VI-1f), (VI-2a) – (VI-2g), (VI-3a) – (VI-3g), (VI-4a) – (VI-4g), (VI-5a) – (VI- 5g), (VII-1a) – (VII-1i), (VII-2a) – (VII-2i), (VII-3a) – (VII-3i), (VII-4a) – (VII-4i), (VII-5a) – (VII-5i), (VII-6a) – (VII-6i), (VIII-1a) – (VIII-1i), (VIII-2a) – (VIII-2i), (VIII-3a) – (VIII-3i), (VIII-4a) – (VIII-4i), (VIII-5a) – (VIII-5i), (VIII-6a) – (VIII-6i) is used for the treatment or prophylaxis of coronavirus infection, wherein the coronavirus is coronavirus 229E, SARS, MERS, or SARS-CoV2, more preferably, SARS-CoV2 causing COVID-19. More preferred, the compounds 1, 2, 3, 4, 5, 6, 7, ST1, ST1a, ST1a-S, ST1a-R, ST2, ST3, ST3b, ST3c, ST3d, ST3e, ST4, ST5, ST6, ST7, ST8, ST9, ST10, ST11, ST12, ST13, ST14, ST15, and ST16 are used for the treatment or prophylaxis of of coronavirus infection, wherein the coronavirus is coronavirus 229E, SARS, MERS, or SARS-CoV2, more preferably, SARS-CoV2 causing COVID-19. In a preferred embodiment, the inventive compound is used for the treatment and prophylaxis of the above-mentioned virus infection, preferrably, coronavirus in combination with at least one active agent selected from antiviral agent, anti-HIV agent, anti-malarial agent, antibaterial agent, and monoclonal antibody. Examples for antiviral drugs are: remdesivir, favipiravir, arbidol (umnifenovir), arbidol salts (hydrochloride), Oseltamivir(Tamiflu), DASS181, galidesivir, Virazole® (ribavirin for inhalation solution), Camostat mesylate, Selinexor, emtricitabine/tenofovir. Examples for anti-HIV agents are: l (opinavir/ ritonavir (Kaletra), Darunavir, Cobicistat, ASC09F. For example, Kaletra lopinavir / ritonavir) is expected to interact with SARS- CoV-2 proteases. The therapeutic effect of ritonavir and lopinavir could be mainly due to its inhibitory effect on coronavirus endopeptidase C30. Examples for antiparasite and anti-malarial agents are: chloroquine, chloroquine salts (phosphate) hydroxychloroquine, hydroxychloroquine salts (sulfate), atovaquone, nitazoxanide and artemisinin. Examples for antibacterial agents are: azithromycin (Zithromax), cararimycin, clindamycin, and telemedicine. Preferably, the inventive compound is used in combination with at least one active agent, wherein the at least one active agent is selected from remdesivir, favipiravir (avigan), osteltamivir (tamiflu) lopinavir/ritonavir (Kaletra), darunavir, cobicistat, atovaquone, azithromycin, cararimycin, and clindamycin. Furthermore, the inventive compound can be used further in combination with at least one further active agent as the follows to to obtain a synergistic effect or reduce the other symptoms caused by the virus, in particular coronavirus. Examples for such further active agents are: ACE inhibitor, angiotensin receptor blocker, janus knase inhibitor, anti-interleukin durgs, and anti-fibrosis drugs. The monoclonal antibodies include: monoclonal antibody specific for spike protein of COVID-19 such as bamlanivimab, etesevimab, casirivimab, imdevimab, sotrovimab, regdanvimab, PD-1 blocking antibody and monoclonal antibody against CD14 – IC14 such as , pembrolizumab, nivolumab. For the treatment against the cytokine storm, ruxolitinib, Lenzilumab TJ003234 (anti- GM-CSF mAb), BMS-986253, leronlimab, Canakinumab can be used. Further active agents which can be combined with the inventive compounds are: ACE inhibitor, Angiotensin receptor blocker, Angiotensin-converting enzyme inhibitors (ACE-I) and Angiotensin II Receoptor Blockers (ARB) – losartan, losartan potassium, recombinant human angiotensin-converting Enzyme 2 (rhACE2) APN01; Anti-inflammatory drugs such as: indomethacin, Piclidenoson, prednisone; TLR agonist - PUL-042. For treatment of acute respiratory distress syndrome or failure caused by COVID-19 the following active agents can be administered together with a compound of the present application: Chlorpromazine, sirolimus, dexamethasone, Bromhexine, Nitazoxanide, Levamisole, methylprednisolone (glucocorticoid therapy), Tacrolimus, colchicine, captopril, and/or Anti-interleukin (IL-6) drugs such as: bevacizumab, clazakizumab, sarilumab, siltuximab, tocilizumab, Gimsilumab, eculizumab, Anakinra, trimethoprim / sulfamethoxazole, and/or immune modulating drugs such as: fingolimod or methotrexate. For the treatment of the cytokine storm the following active agents could be used together with a compound of the present application: ruxolitinib, Lenzilumab TJ003234 (anti-GM-CSF mAb), BMS-986253, leronlimab, Canakinumab. Still further active agents could be administered in combination with at least one compound of the present application. Examples for such further active agents are: Pyridostigmine bromide (for the treatment of myasthenia gravis) , Kerecis (as oral or nasal spray), chlorhexidine (as oral or nasal spray), Hydrocortisone (for the treatment of sever hypoxia), Vazegepant, Tetrandrine, Tranexamic acid, Isotretinoin. Still further Kinase inhibitors as active agents could be administered in combination with at least one compound of the present application. Kinase inhibitors: baricitinib, imatinib, Tofacitinib (JAK inhibitor), Acalabrutinib (BTK inhibitor). Baricitinib, Janus kinase inhibitor, showing high affinity for AAK1. Disruption of AAK1 (one of the known regulators of viral endocytosis) could block passage of SARS-CoV-2 to cells and also the intracellular assembly of virus particles. Furthermore, it has the capacity to bind cyclin G-associated kinase, another regulator of endocytosis. Systemic inflammatory response and cytokine production can be limited by inhibiting the JAK-STAT3 pathway. Antiviral properties of Imatinib have been shown in early stages of infection against SARS-CoV and MERS-CoV, phylogenetically related to SARS-CoV2. In addition, it has been linked to reduced inflammation and improved endothelial barrier and pulmonary edema. Still further active agents could be administered in combination with at least one compound of the present application. Anti-thrombosis agents and anti-fibrosis agents such as: nintedanib, defibrotide (pneumonia); Anticoagulation agents such as: Enoxaparin, heparine, angiotensin-(1,7); PD-1 blocking antibodies such as: monoclonal antibody against CD14 – IC14, Interferon beta-1A, 1B, peginterferon lambda-1a; Colchicine (for reducing myocardinal injury); Valsartan (Diovan), simvastatin. As supplements vitamin C, vitamin D (cholecalciferol), folic acid, zinc gluconate, 25-OH cholecalciferol, Melatonin, N-acetylcysteine could be present in the pharmaceutical composition. The pharmaceutical compositions according to the present invention comprise at least one compound according to the present invention. Preferably, the pharmaceutical compositions according to the present invention comprise at least one compound according to the present invention as an active ingredient together with at least one pharmaceutically acceptable (i.e. non-toxic) carrier, excipient and/or diluent. Optionally, the pharmaceutical composition according to the present invention further comprises at least one active agent selected from antiviral agent, anti-HIV agent, anti- malarial agent, antibacterial agent, and monoclonal antibody as mentioned above. The pharmaceutical composition according to the present invention is useful for the treatment or prophylaxis of virus infection, preferred, wherein virus is influenza virus, parainfluenza virus or coronavirus. Influenza virus is preferably influenza A virus or influenza B virus, more preferably influenza A. Influenza A virus includes H1N1, H3N2, and H7N9. Preferred, the pharmaceutical composition according to the present invention is used for the treatment or prophylaxis of of virus infection, preferred, wherein virus is coronavirus, preferrably, coronavirus is coronavirus 229E, SARS, MERS, or COVID-19, more preferrably, COVID-19. The pharmaceutical compositions of the present invention can be prepared in a conventional solid or liquid carrier or diluent and a conventional pharmaceutically made adjuvant at suitable dosage level in a known way. The preferred preparations are adapted for inhalation or oral application or intraveuous or subcutaneous injections. These administration forms include, for example, pills, tablets, film tablets, coated tablets, capsules, powders and deposits, solutions, microparticles or other inhalative forms using an inhalator or spray or any other device for applying the compound into the airways and preferably deep into the lungs. The present invention also includes pharmaceutical preparations for parenteral application, including dermal, intradermal, intragastral, intracutaneous, intravasal, intravenous, intramuscular, intraperitoneal, intranasal, intravaginal, intrabuccal, percutan, rectal, subcutaneous, sublingual, topical, or transdermal application, which preparations in addition to typical vehicles and/or diluents contain at least one compound according to the present invention and/or a pharmaceutical acceptable salt thereof as active ingredient. The pharmaceutical compositions according to the present invention containing at least one compound according to the present invention and/or a pharmaceutical acceptable salt thereof as active ingredient will typically be administered together with suitable carrier materials selected with respect to the intended form of administration, i.e. for oral administration in the form of tablets, capsules (either solid filled, semi-solid filled or liquid filled), powders for constitution, gels, elixirs, dispersable granules, syrups, suspensions, and the like, and consistent with conventional pharmaceutical practices. For example, for oral administration in the form of tablets or capsules, the active drug component may be combined with any oral non-toxic pharmaceutically acceptable carrier, preferably with an inert carrier like lactose, starch, sucrose, cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, talc, mannitol, ethyl alcohol (liquid filled capsules) and the like. Moreover, suitable binders, lubricants, disintegrating agents and coloring agents may also be incorporated into the tablet or capsule. Powders and tablets may contain about 5 to about 95-weight % of the derivatives according to the general formula (I) or analogues compound thereof or the respective pharmaceutically active salt as active ingredient. The preferred formulation of the inhalation application for the treatment of respiratory diseases such as COVID-19 are often formulated as dry powders and are delivered using a dry-powder inhaler (DPI). The medicaments are micronized such as to have a respirable aerodynamic diameter which is typically in the region of 0.5 to 10 μm. Such micronized particles tend to be cohesive and have poor flow properties. To increase flowability and dosing accuracy the fine drug particles of respirable size are typically mixed with coarser excipient particles to form an ordered mixture, wherein fine drug particles are attached to the coarser excipient particles. Thus, the inventive compound as the active ingredient may be in micronized form, i.e. having particle size lower than about 10 μm, for example in the range from about 0.5 to about 10 μm, particularly in the range from about 1 to about 6 μm, such as to be able to deposit target areas in the lungs. Conventional methods, such as milling, can be used to provide the active ingredient in micronized form. The amount of the active ingredient in the dry powder inhalation composition can vary depending e.g. on the active ingredient and the type of dry powder inhaler used. Generally, the amount of the active ingredient in the dry powder inhalation composition is within the range of 0.02 to 30 %, typically from 0.05 to 10 , more typically from 0.1 to 5 %, per weight of the composition. The excipient used in the dry powder inhalation composition is a mono- or disaccharide, particularly lactose or mannitol, for example alpha lactose monohydrate. In general, the particle size of the excipient is preferably such that it can be entrained in the air stream but not enter deeply into the lung. However, a small proportion of particles with respirable size (< 10 μm) can be present in the excipient as such fine particles of the excipient may help in attaining higher fine particle dose (FPD) values. The volume median diameter (VMD) of the excipient, such as lactose, to be used in the composition is suitably in the range of, for example, from about 30 to about 150 μm. The excipient of desired VMD can be obtained from commercial sources or can be prepared using methods known in the art such as by blending together excipient powders of known particle size or by sieving. Suitable binders include starch, gelatin, natural sugars, corn sweeteners, natural and synthetic gums such as acacia, sodium alginate, carboxymethylcellulose, polyethylene glycol and waxes. Among suitable lubricants there may be mentioned boric acid, sodium benzoate, sodium acetate, sodium chloride, and the like. Suitable disintegrants include starch, methylcellulose, guar gum, and the like. Sweetening and flavoring agents as well as preservatives may also be included, where appropriate. The disintegrants, diluents, lubricants, binders etc. are discussed in more detail below. Moreover, the pharmaceutical compositions of the present invention may be formulated in sustained release form to provide the rate controlled release of any one or more of the components or active ingredients to optimise the therapeutic effect(s), e.g. anti- cancer activity or activity against cancer metastases and the like or antiviral activity or activity against the spreading of the virus. Suitable dosage forms for sustained release include tablets or inhalative forms having layers of varying disintegration rates or controlled release, polymeric matrices impregnated with the active components and shaped in tablet form or capsules containing such impregnated or encapsulated porous polymeric matrices. Liquid form preparations include solutions, suspensions, and emulsions. As an example, there may be mentioned water or water/propylene glycol solutions for parenteral injections or addition of sweeteners and opacifiers for oral solutions, suspensions, and emulsions. Liquid form preparations may also include solutions for intranasal administration. Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be present in combination with a pharmaceutically acceptable carrier such as an inert, compressed gas, e.g. nitrogen. The term capsule as recited herein refers to a specific container or enclosure made e.g. of methylcellulose, polyvinyl alcohols, or denatured gelatins or starch for holding or containing compositions comprising the active ingredient(s). Capsules with hard shells are typically made of blended of relatively high gel strength gelatins from bones or pork skin. The capsule itself may contain small amounts of dyes, opaquing agents, plasticisers and/or preservatives. Under tablet a compressed or moulded solid dosage form is understood which comprises the active ingredients with suitable diluents. The tablet may be prepared by compression of mixtures or granulations obtained by wet granulation, dry granulation, or by compaction well known to a person of ordinary skill in the art. Oral gels refer to the active ingredients dispersed or solubilised in a hydrophilic semi- solid matrix. Powders for constitution refers to powder blends containing the active ingredients and suitable diluents which can be suspended e.g. in water or in juice. Suitable diluents are substances that usually make up the major portion of the composition or dosage form. Suitable diluents include sugars such as lactose, sucrose, mannitol, and sorbitol, starches derived from wheat, corn, rice, and potato, and celluloses such as microcrystalline cellulose. The amount of diluent in the composition can range from about 5 to about 95 % by weight of the total composition, preferably from about 25 to about 75 weight %, and more preferably from about 30 to about 60 weight %. The term disintegrants refers to materials added to the composition to support break apart (disintegrate) and release the pharmaceutically active ingredients of a medicament. Suitable disintegrants include starches, “cold water soluble” modified starches such as sodium carboxymethyl starch, natural and synthetic gums such as locust bean, karaya, guar, tragacanth and agar, cellulose derivatives such as methylcellulose and sodium carboxymethylcellulose, microcrystalline celluloses, and cross-linked microcrystalline celluloses such as sodium croscaramellose, alginates such as alginic acid and sodium alginate, clays such as bentonites, and effervescent mixtures. The amount of disintegrant in the composition may range from about 2 to about 20 weight % of the composition, more preferably from about 5 to ca.10 weight %. Binders are substances which bind or “glue” together powder particles and make them cohesive by forming granules, thus serving as the “adhesive” in the formulation. Binders add cohesive strength already available in the diluent or bulking agent. Suitable binders include sugars such as sucrose, starches derived from wheat, corn, rice and potato, natural gums such as acacia, gelatin and tragacanth, derivatives of seaweed such as alginic acid, sodium alginate and ammonium calcium alginate, cellulose materials such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethylcellulose, polyvinylpyrrolidone, and inorganic compounds such as magnesium aluminum silicate. The amount of binder in the composition may range from about 2 to about 20 weight % of the composition, preferably from about 3 to about 10 weight %, and more preferably from about 3 to about 6 weight %. Lubricants refer to a class of substances which are added to the dosage form to enable the tablet granules etc. after being compressed to release from the mould by reducing friction or wear. Suitable lubricants include metallic stearates such as magnesium stearate, calcium stearate, or potassium stearate, stearic acid, high melting point waxes, and other water soluble lubricants such as sodium chloride, sodium benzoate, sodium acetate, sodium oleate, polyethylene glycols and D,L-leucine. Lubricants are usually added at the very last step before compression, since they must be present at the surface of the granules. The amount of lubricant in the composition may range from about 0.2 to about 5 weight % of the composition, preferably from about 0.5 to about 2 weight %, and more preferably from about 0.3 to about 1.5 weight % of the composition. Glidents are materials that prevent caking of the components of the pharmaceutical composition and improve the flow characteristics of granulate so that flow is smooth and uniform. Suitable glidents include silicon dioxide and talc. The amount of glident in the composition may range from about 0.1 to about 5 weight % of the final composition, preferably from about 0.5 to about 2 weight %. Coloring agents are excipients that provide coloration to the composition or the dosage form. Such excipients can include food grade dyes adsorbed onto a suitable adsorbent such as clay or aluminum oxide. The amount of the coloring agent may vary from about 0.1 to about 5 weight % of the composition, preferably from about 0.1 to about 1 wgt %. Description of Figures Figure 1 shows 4 subfamilies of transmembrane serine proteases (17 proteases in total). This Figure was taken from the Atlas of genetics and cytogenetics in oncology and haematology in 2013; Huret JL, Ahmad M, Arsaban M, Bernheim A, Cigna J, Desangles F, Guignard JC, Jacquemot-Perbal MC, Labarussias M, Leberre V, Malo A, Morel-Pair C, Mossafa H, Potier JC, Texier G, Viguié F, Yau Chun Wan-Senon S, Zasadzinski A, Dessen P; Nucleic Acids Res. 2013 Jan;41(Database issue):D920-4; PMID:23161685. Figure 2: A) a crstal structure of hepsin; B) TMPRSS2 model structure. TMPRSS2 belongs to Hepsin/TMPRSS subfamily and thus crystal strucutre of hepsin can provides good templates for TMPRSS2 modelling. Figure 3 shows IC ₅₀ vaules of the compounds 1 to 6 and reference compound against thrombin. Figure 4 shows IC ₅₀ vaules of the compounds 1 to 6 and reference compound against factor Xa. Figure 5 shows the general synthetic method of the tetrapeptidomimetic compounds. Figure 6. Predicted binding mode of a) reference binder ace-D-Arg-Pro-Arg-aldehyde. in complex with TMPRSS2 homology model (white carbon atoms and surface). light gray dashed lines indicate hydrogen bonds, the gray dashed line the distance between catalytic serine nucleophile oxygen and carbon of the P1|P1’ amide bond. For clear view, only residues forming polar contacts with the ligand and His14/57 of the catalytic triad are labeled and depicted as lines.; b) camostat mesylate (CM, Ref. 5) to matriptase as surrogate for TMPRSS2 (white carbon atoms and surface). For clear view only residues forming polar interactions (dashed lines), and the catalytic residues Ser-195 and His-57 are depicted as lines. Distance between nucleophilic serine oxygen and electrophilic carbon atom of the serine trap in angstrom is illustrated by a dashed blue line.. Figure 7. Correlation of the pK _i values from compounds ST1, ST1a, ST5, ST6, ST8, ST10, ST14 and ST16, CM (Ref. 5) and FOY-251(Ref. 8) against TMPRSS2 and matriptase. A linear regression fit (R ² = 0.96) was plotted through the data points excluding ST1 and FOY-251 (Ref.8). Figure 8. Peptidomimetic inhibitors block protease activity of Caco-2 cells. Peptidomimetic compounds ST1a, ST6, ST8, ST16 as well as camostat mesylate (CM, Ref.5) and FOY-251 (Ref.8) were added to Caco-2 cells. After 30 min, the fluorogenic reference substrate Boc-Gln-Ala-Arg-AMC was added, and the reaction rate of substrate degradation was assessed by recording the fluorescence intensity within 2 h. Shown are the means ± SEM of triplicate measurements. Calculated IC ₅₀ values for each compound are presented in Table 10. Figure 9. Peptidomimetic inhibitors reduce SARS-CoV-2 spike driven entry. Peptidomimetic inhibitors and the small molecule camostat mesylate (CM) were added to Caco-2 cells. After 1 h, cells were transduced with lentiviral SARS-CoV-2 pseudoparticles carrying the spike protein of SARS-CoV-2 wildtype (a), the B1.1.7 (b) or B.1.351 (c) variant of concern. Transduction rates were assessed 2 days post transduction by measuring luciferase activity in cell lysates. Shown are the means ± SEM of two independent experiments, each performed in triplicates. Calculated IC ₅₀ values for each compound are presented in Table 11. Figure 10. Peptidomimetic inhibitors reduce SARS-CoV-2 infection. Peptidomimetic inhibitors and the small molecule camostat mesylate (CM) were added to Caco-2 cells. After 1 h, cells were infected with SARS-CoV-2 WT (a), SARS-CoV-2 bearing the spike D614G mutation (b), or the variants of concern B.1.1.7 (c) and B.1.351 (d). Infection rates were determined 2 days post transduction by in cell ELISA for the viral N protein. Shown are the means ± SEM of three independent experiments, each performed in triplicates. Calculated IC ₅₀ values for each compound are listed in Table 12. Figure11. Inhibition of reference substrate degradation by matriptase with compound ST1a (left panel) and ST16 (right panel) at c = 1665 nM after incubation in blood serum at various time points (a). Positive control = inhibitor without incubation, negative control = no inhibitor. Residual inhibitory activity K _i for compound ST1a and compound ST16 against matriptase after 0 min and 10 days incubation in 25% serum or 25% plasma in RPMI medium or pure RPMI medium (b). Figure 12. a) Re-docking result for reference ligand-matriptase complex (Protein data bank PDB-ID: 6N4T). Docking pose and, crystallographic binding mode, FlexX-score = 67.2 kJ/mol, RMSD = 1.8 Å. b) ROC curve for binder vs non-binder discrimination of molecular docking using a TMPRSS2 homology model (56 binder and 314 non-binder successfully docked, ROC AUC = 0.98). c) ROC curve for binder vs non-binder discrimination of molecular docking using matriptase-1 surrogate model (PDB-ID: 6N4T, 56 binder and 314 non-binder successfully docked, ROC AUC = 0.96). FPR: false positive rate, TPR: true positive rate. Figure 13. Michaelis-Menten constant (K _M ) of the fluorogenic reference substrate Boc- Gln-Ala-Arg-AMC for TMPRSS2. The data points were plotted using the Michaelis- Menten equation in GraphPad Prism prism version 8.4.2 (San Diego, California). Shown are the means ± SD of triplicate measurements. Figure 14. Peptidomimetic inhibitors block activity of purified TMPRSS2 (a), matriptase (b), thrombin (c) and factor Xa (d). Peptidomimetic inhibitors, camostat mesylate (CM) and FOY-251 were added to isolated enzymes. After 30 min, the fluorogenic reference substrate Boc-Gln-Ala-Arg-AMC was added to matriptase and TMPRSS2 and the chromogenic substrates, D-Phe-Homopro-Arg-pNA or Bz-Ile-Glu-Gly-Arg-pNA, were added to thrombin or factor Xa, respectively. The velocity of substrate degradation was assessed by recording the fluorescence intensity at 460 nm or the absorbance at 405 nm within 2 h. Shown are the means ± SEM of triplicate measurements. Figure 15. Influence of serine trap on biological activity of compound ST16 against matriptase, thrombin and factor Xa. Peptidomimetic inhibitors with a ketobenzothiazole (ST16), ketothiazole (ST16b) or alcohol (Ref. 10) serine trap residue were added to isolated enzymes. After 30 minutes, the fluorogenic reference substrate Boc-Gln-Ala- Arg-AMC was added to matriptase and TMPRSS2 and the chromogenic substrates, D- Phe-Homopro-Arg-pNA or Bz-Ile-Glu-Gly-Arg-pNA, were added to thrombin or factor Xa, respectively. The velocity of substrate degradation was assessed by recording the fluorescence intensity at 460 nm or the absorbance at 405 nm within 2 h. Shown are the means ± SD of triplicate measurements. Figure 16. Biological activity of virtual screening hits against matriptase, thrombin and factor Xa. After 30 minutes, the fluorogenic reference substrate Boc-Gln-Ala-Arg-AMC was added to matriptase and TMPRSS2 and the chromogenic substrates, D-Phe- Homopro-Arg-pNA or Bz-Ile-Glu-Gly-Arg-pNA, were added to thrombin or factor Xa, respectively. The velocity of substrate degradation was assessed by recording the fluorescence intensity at 460 nm or the absorbance at 405 nm within 2 h. Shown are the means ± SD of triplicate measurements. Figure 17. The transmembrane serine protease TMPRSS2 is expressed on the surface of Caco-2 cells. The Figure shows the detection of the transmembrane serine protease TMPRSS2 on human Caco-2 cells, which was performed by first incubating the cells with different amounts of a TMPRSS2 antibody (rabbit anti-human) followed by the detection of bound TMPRSS2 antibodies on the cell surface via an anti-rabbit FITC- labeled secondary antibody. Only secAB (secondary antibody) FITC and only TMPRSS2 were used as negative control groups. a) shows the percentage of FITC- positive Caco-2 cells, where the bars represent the percentage of gated cell measurements generated by flow cytometry (n = 3, ± standard deviation). b) shows an overlay of the normalized FITC signal histograms for each group (one out of three histograms is exemplary shown for each group). Figure 18. Impact of DMSO on SARS-CoV-2 spike mediated entry and infection. DMSO was added to Caco2 cells at concentrations corresponding to the maximum DMSO concentrations in transduction/infection experiments. After 1 h cells were either transduced with lentiviral SARS-CoV-2 pseudoparticles or infected with SARS-CoV-2 wildtype. Transduction/infection rates were assessed 2 days later by measuring luciferase activity in cell lysates or by ELISA, respectively. Shown are the mean ± SEM of two experiments, performed in triplicates. Figure 19. Cytotoxicity of TMPRSS2 inhibitors. TMRPSS2 inhibitors and the small molecule camostat mesylate (CM) were added to Caco2 cells. Cell viability was assessed 2 days post addition by measuring ATP content in cell lysates. Due to low stock concentration compounds ST1, ST10 and ST14 were tested at a maximum concentration of 20,000 nM. As solvent control, DMSO concentrations corresponding to the maximum DMSO concentration of cells were assessed. Shown is the mean ± SD of one experiment, performed in triplicates.

Examples Following abbreviations used in the examples have the following meaning. BOC tert-butyloxycarbonyl DIEA diisopropylethyl amide EDCI ethylcarbodiimide hydrochloride HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyri dinium 3-oxid hexafluorophosphate Mtr trimethylbenzenesulfonyl Pbf 2,2,4,6,7-pentamethyIdlhydrobenzofuran-5-sulfonyl PyBOP benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate Chemical Examples The following examples are intended to illustrate the invention with selected compounds without limiting the protecting scope of the present intellectual property right on these concrete examples. It is clear for a person skilled in the art that analogous compounds and compounds produced according to analogous synthetic ways fall under the protecting scope of the present intellectual property right. Example 1. Synthesis of arginine modified serine trap with mtr protected guanidino group (serine trap precursor) 1.1Synthesis of arginine weinreb amide* Boc-NH- roxyl-amine hydrochloride (460 mg, 4.72 mmol, 2.0 equiv), and 1-hydroxybenzotriazole [HOBt] (350 mg, 2.59 mmol, 1.1 equiv) were dissolved in 25 mL THF at room temperature. N, N- diisopropylethylamine [DIEA] (1.22 mL, 7.00 mmol, 3 equiv) was added via syringe and the solution was stirred until it was homogeneous. 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride [EDCI] (470 mg, 2.45 mmol, 1.05 equiv) was added in one portion and the reaction was allowed to stir at room temperature for 4.5 h. THF may be first removed in vacuo, then the reaction mixture is diluted with ethyl acetate (150 mL), washed with 5% aqueous acetic acid (1 × 75 mL), sat. aqueous NaHCO ₃ (1 × 75 mL), water (1 × 75 mL), and brine (1 × 75 mL). The organic layer was dried over MgSO4, filtered and concentrated in vacuo to afford the title Weinreb amide. Mass = 858 mg, yield: 69%, purity > 95% Scientific name: (S)-tert-butyl-1-(methoxy(methyl)amino)-5-(3-(4-methoxy-2,5 dimethylphenylsulfonyl)guanidino)-1-oxopentan-2-ylcarbamate. 1H NMR (300 MHz, CD3OD): d=1.43 (s, 9 H) 1.45–1.67 (m, 4 H) 1.87 (s, 1 H) 2.13 (s, 3 H) 2.61 (s, 3 H) 2.67 (s, 3 H) 3.17 (m, 3 H) 3.74 (s, 3 H) 3.90 (s, 3 H) 6.67 ppm (s, 1 H); MS(ESI): found: [M+H]+ = 530.2, [M+H+DIEA]+ = 659.4, [2M+H]+ = 1059.4 1.2 Synthesis of Boc-Arg(Mtr) ketobenzothiazole Weinreb-Nahm ketone synthesis* At -78,8°C, n on of benzothiazol e (0.8 g, 1.52 mmol) in THF (50 mL) over 15 min. After the mixture was stirred for an additional 30 min, the solution of Boc-Arg(Mtr) Weinreb amide (0.8 g, 1.52 mmol) in THF (15 mL) was added slowly over 50 min. The mixture was stirred at - 78,8°C for 3h. The reaction was quenched with aqueous NH ₄ Cl and the aqueous layer was extracted with EtOAc. The organic phase was collected, dried with Na ₂ SO ₄ , and then concentrated. The resulting residue was purified by silica gel chromatography with CHCl ₂ /MeOH combination as eluent to give the title compound. 1H NMR (300 MHz, CD3OD): d=1.43 (s, 9 H) 1.53–1.78 (m, 4 H) 2.05 (s, 3 H) 2.55 (s, 3 H) 2.61 (s, 3 H) 3.17–3.29 (m, 2 H) 3.82 (s, 3 H) 5.19–5.43 (m, 1 H) 6.57 (s, 1 H) 7.53–7.71 (m, 2 H) 8.06–8.28 ppm (m, 2 H). MS(ESI): found: [M+H]+ , 603.8, [2M+H]+ = 1206.5 1.3 Synthesis of HCl·H-Arg(Mtr) ketobenzothiazole To 100 mg of Boc-Arg(Mtr) ketobenzothiazole, a solution of 5 ml (1.5 M HCl/Dioxane) was added. The mixture was stirred at room temperature for 18h. The solvent was removed the resulting residue was dried in vacuo. 1H NMR (300 MHz, CD3OD): d 1.60 1.86 (m, 2 H) 2.00 2.20 (m, 4 H) 2.24 2.36 (m, 1 H) 2.53 (s, 3 H) 2.61 (s, 3 H) 3.84 (s, 3 H) 5.14–5.40 (m, 1 H) 6.65 (s, 1 H) 7.59–7.78 (m, 2 H) 8.10–8.34 ppm (m, 2 H). MS(ESI): found: [M +H]+ = 502.6, [2M+H]+ = 1006.5 1.4 Purification and lyophilization of serine trap precursor All compounds were purified with semi-preparative HPLC. The following gradient was applied: 95%H ₂ O/5%ACN to 5%H ₂ O/95%ACN in 30 minutes. Column used: Zobrax Eclipse XDB C-189.4x250 mm 5μm, company: Agilent Technologies. Detector: UV Vis detector model S-3702, company: Soma. For the detection of the compounds a wavelength of 220 nm was used. After purification with HPLC, the fractions were collected and freeze dried overnight. The purified and lyophilized compounds were stored in the freezer at -20°C. The mass of the purified compounds was determined with MS-ESI. Model used: expression-L compact mass spectrometer, company Advion. Peptides were dissolved to a concentration of c = 0.01 mg/ml in MeOH + 0.1% formic acid. Injection was done by a syringe pump with a flow rate of 10 µl/min. Data analysis ^ MS ESI data were analyzed with the program Advion Mass/Data express ^ HPLC data were analyzed with Graph Pad Prism 7 Example 2. Synthesis of tripeptides with protected side chains, acetylated N- terminus and free carboxyl group (tripeptide precursors) 2.1 Chlorination of tritylhydroxid resin (Trt-OH)* A 20 ml glass vial was rinsed out with dry DCM.5 g of Trt-OH (1.33 mmol/g, mesh 100- 200, company: Iris Biotech) was weighed and put into glas vial. The resin was suspended in a mixture of 50% DCM and 50% toluene, just enough to double the resin volume and 2 ml acetyl chloride (30 eq) was added. The glass vial was sealed and agitated for 24 h. The next day, the resin was dried and thoroughly washed with DCM (4 x 5 ml). Resin was stored in freezer. 2.2 Addition of first amino acid to Trt-Cl 10 ml of dry DCM were added to 200 mg Trt-Cl (subst.1.33 mmol/g) and shaken for 10 min. 10 eq. (2.66 mmol) of amino acid was dissolved and 20 eq. (5.32 mmol) of activator base was added. The coupling reaction was shaken for 4 h at room temperature. After the reaction, the resin was washed with DMF (2 x 5 ml) and DCM (2 x 5 ml). The resin was swollen in 10 ml DMF for 1h prior to use for solid phase peptide synthesis. 2.3 Solid phase peptide synthesis Fmoc protected amino acids: All fmoc protected amino acids were purchased from Carl Roth / Novabiochem / Merck and dissolved in DMF to a concentration of c = 0.2 M. Deprotection solution for fmoc group: 20% Piperidine in DMF (peptide synthesis grade) Coupling reagents: Activator PyBOP (Company: Merck) was dissolved in DMF to a concentration of 1 M and the activator base diisopropylethyl amide (DIEA) was dissolved in DMF to a concentration of 0.5M. Peptide synthesizer: Microwave assisted peptide synthesizer from the company CEM. Model: Liberty. The standard procedure for solid phase peptide synthesis was used with slight modifications to couple two additional amino acids to the loaded resin. Please see the attached synthesis protocols for the respective experiment. Conditions for Fmoc deprotection: Conditions for coupling reaction for all amino acids except Fmoc-His(Trt)-OH, Fmoc- Cys(Trt)-OH and Fmoc-Arg(Pbf)-OH: temperature power (microwave) time Conditions for coupling reaction Fmoc-His(Trt)-OH and Fmoc-Cys(Trt)-OH: step temperature power (microwave) time Conditions for coupling reaction Fmoc-Arg(Pbf)-OH: step temperature power (microwave) time 2.4 Ac etyl capping of the tripeptides* Acetyl capping solution: 0.5 M Ac ₂ O/DMF and 1 M iPr2NEt/DMF 200 mg of tripeptide resin was suspended in 20 ml acetyl capping solution. The mixture was shaken at RT for 1 h. The resin was filtered and washed with DMF (3 x 5 ml) followed by DCM (3 x 5 ml) (ChemMedChem, 2016, 11(6), 685) 2.5 Cleavage of tripeptide from resin** Cleavage solution: 20% Hexafluoroisopropanol (HFIP) in CH ₂ Cl ₂ ) 20 ml of cleavage solution was freshly prepared (6 ml HFIP / 24 ml DCM) and added to 200 mg resin. Suspension was put on orbital shaker for 3h and then filtered. The solvent was taken off with rotovap. 2.6 Purification and lyophilization of tripeptide precursors All tripeptide precursors were purified with semi-preparative HPLC. The following gradient was applied: 95%H ₂ O/5%ACN to 5%H ₂ O/95%ACN in 30 minutes. Column used: Zobrax Eclipse XDB C-18 9.4x250 mm 5μm, company: Agilent Technologies. Detector: UV Vis detector model S-3702, company: Soma. For the detection of the peptides a wavelength of 220 nm was used. After purification with HPLC, the fractions were collected and freeze dried overnight. The purified and lyophilized tripeptide precursors were stored in the freezer at -20°C. The mass of the purified compounds was determined with MS-ESI. Model used: expression-L compact mass spectrometer, company Advion. Peptides were dissolved to a concentration of c = 0.01 mg/ml in MeOH + 0.1% formic acid. Injection was done by a syringe pump with a flow rate of 10 µl/min. Data analysis ● MS ESI data were analyzed with the program Advion Mass/Data express ● HPLC data were analyzed with Graph Pad Prism 7 Table A. Synthesized tripeptide precursors with highest calculated score values of P- sid library P4 P3 P2 score of related MS data a natural substrate of hepsin, b reference sequence of known substrate, c negative control. d Spike protein recognition sequence of TMPRSS2 * purity >99%, lyophilized Table B. MS data of the compounds of the invention compound sequence structure MS data 1 A RQLR / 366 c Example 3. Synthesis of tripeptides with protected side chains, acetylated N- terminus and free carboxyl group (tripeptide precursors) 3.1 Synthesis of Boc-Arg(Mtr) ketothiazole (S4) tert-Butyl-(5-(3-((4-methoxy-2,3,6-trimethylphenyl)sulfonyl) guanidino)-1-oxo-1-(thiazol- 2-yl)pentan-2-yl)carbamate To a solution of 2-bromothiazo l (0.211 g, 1.29 mmol, 3.3 equiv.) in dry THF (10 mL) n- BuLi (2.5 M, 0.52 mL, 1.29 mmol, 3.3 equiv.) was added dropwise under inert atmosphere at –78 °C. The reaction mixture stirred for 1.5 h at –78 °C, followed by dropwise addition of compound S1 (0.205 g, 0.39 mmol, 1 equiv.) at the same temperature. The resulting solution was stirred 2 h at –78 °C, after which sat. aqueous NH ₄ Cl (10 mL) was added. The organic phase was separated and the aqueous phase was extracted three times with EtOAc. The combined organic extracts were washed with brine (30 mL), dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified on a silica column eluting with EtOAC/cyclohexane (4:1 v/v), to afford the compound S4 (0.12 g, 0.22 mmol, yield: 56%) as a white foam Purity (LC, 254 nm) > 95%.1H NMR (300 MHz, CDCl ₃ ): δ = 8.04 (d, 1 H), 7.72 (d, 1 H), 6.53 (s, 1 H), 5.64 (d, 1 H), 5.41 (s, 1 H), 3.83 (s, 3 H), 3.26 (m, 2 H), 2.67 (s, 3 H), 2.59 (s, 3 H), 2.12 (s, 3 H), 1.76 – 1.57 (m, 4 H), 1.41 (s, 9 H). ppm. MS (ESI): m/z: calcd for C H N O S [M+H]+ 554.2, + 2 _{4 35 5 6 2} found [M+H] 554.2. 3.2 Synthesis of H ₂ N-Arg(Mtr) ketothiazole (S5) N-(N-(4-amino-5-oxo-5-(thiazol-2-yl)pentyl)carbamimidoyl)-4- methoxy-2,3,6- trimethylbenzenesulfonamide

Compound S4 (0.256 g, 0.46 mmol) was stirred in DCM (3 mL) at 0 °C and TFA (1 mL) was added. The reaction mixture stirred for 1 h at ambient temperature, then isopropyl alcohol (0.5 mL) was added. The solution was concentrated in vacuo and triturated with diethyl ether. The supernatant was decanted and the residue was purified by RP- HPLC. The obtained compound S5 was used for the following peptide couplings. MS(ESI): MS (ESI): m/z: calcd for C + + 20H27N5O4S2 [M+H] 453.2, found [M+H] 454.1. Retention time 0.08 min. Purity (254 nm) > 95%. 3.3 Synthesis of Boc-Arg(Mtr) alcohol (S6) (S)-tert-Butyl-(1-hydroxy-5-(3-((4-methoxy-2,3,6-trimethylph enyl)sulfonyl)guanidino) pentan-2-yl)carbamate To a solution of Boc-Arg(Mtr)-OH v.) in dry THF (5 mL) were added NMM (0.063 g, 0.62 mmo l, 1 equiv.) and EtOCOCl (0.067 g, 0.62 mmol, 1 equiv.) at – 15 °C under argon. The reaction mixture stirred for 1 h at – 15 °C, then transferred dropwise via canula into a stirred solution of NaBH ₄ (0.047 g, 1.24 mmol, 2 equiv.) in water (15 mL). The resulting solution was stirred 5 min at 0 °C and then diluted with water (15 mL). The aqueous phase was extracted twice with EtOAc (10 mL). The combined organic extracts were dried over Na ₂ SO ₄ and concentrated in vacuo to obtain the compound S6 (0.24 g, 0.5 mmol, yield: 81%) as a colourless oil. Purity (LC, 254 nm) 98%. 1H NMR (300 MHz, CDCl ₃ ) δ = 6.52 (s, 1 H), 6.33 (s, 2 H) 5.15 (d, 1 H), 3.82 (s, 3 H), 3.55 (s, 2 H), 3.21 (s, 1 H), 2.69–2.66 (m, 5 H), 2.59 (s, 3 H), 2.12 (s, 3 H), 1.55 (s, 4 H), 1.40 (s, 9 H) ppm. LC-MS: m/z: calcd for C + + 21H36N4O6S [M+H] 473.2, found [M+H] 473.2. 3.4 Synthesis of H ₂ N-Arg(Mtr)-OH (S7) ((S)-N-(N-(4-amino-5-hydroxypentyl)carbamimidoyl)-4-methoxy- 2,3,6- trimethylbenzenesulfonamide) Compound S6 (0.22 g, 0.47 mmol) as st ed C (3 mL) at 0 °C and TFA (1 mL) was added. The reaction mixture stirred for 2 h at ambient temperature, then isopropyl alcohol (0.5 mL) was added. The solution was concentrated in vacuo and triturated with diethyl ether. The supernatant was decanted and the residue was purified by RP- HPLC. The received compound S7 was used for the following peptide couplings. Preparation of inhibitors. Respective serine traps (1.5 equiv.) were coupled with dipeptides (1.0 equiv.) bearing standard protection groups using PyBOP (1.5 equiv.) and DIEA (3 equiv.) in DMF. After the reaction was agitated for 4 h at room temperature, 3 mL of a deprotection solution was added (93% TFA, 3.5%TIPS, 3.5% H ₂ O) and further agitated for 8 h at room temperature. After concentrating, the crude inhibitor was precipitated in 50 mL cold diethylether and afterwards purified using RP- HPLC. Purification, lyophilization and analysis of peptide- and warhead precursors and inhibitors. All precursor compounds were purified with a semi-preparative RP-HPLC. The following gradient was applied: 95% H ₂ O / 5% ACN to 5% H ₂ O / 95% ACN in 30 min. Trifluoracetic acid was dissolved in the water to a concentration of 0.1%. Column used: Zorbax Eclipse XDB C-18 9.4x250 mm 5 μm, company: Agilent Technologies. Detector: UV Vis detector model S-3702, company: Soma. For the detection a wavelength of 220 nm was used. After chromatographic purification, the fractions were collected and freeze dried overnight. The purified and lyophilized precursor compounds were stored in the freezer at -20 °C. The mass of the purified compounds was determined with MS-ESI. Model used: expression-L compact mass spectrometer, company Advion. Peptides were dissolved to a concentration of c = 0.01 mg/mL in MeOH + 0.1% formic acid. Injection was done by a syringe pump with a flow rate of 10 µL/min. Table B2. MS data of the compounds of the invention

Biological Examples Example B-1. Inhibitory activity of the inventive compounds against thrombin Protease Thrombin inhibiton assay – Calculation of IC ₅₀ values Materials Protease: Recomb. Human Thrombin, Bought 30.01.2020; Art. 1473-SE-010; Company: R&D systems Reference substrate: D-Phe-Homopro-Arg-pNA; Company: Bachem; Art no.: 4008145.0050 Plates for plate reader: Greiner 96 well plates, PS, F-Bottom (Chimney well), µclear, black, cellstar. Cat. No.: 655090/655096/655097 Tubes: Eppendorf, Protein LoBind Tube 1.5ml/2ml. Cat. No.: 022431081 Platereader: Device: Infinite M1000 Application: Tecan i-control Firmware: V_2.09_04/2011_S3LCE (May 22011/09.25.56) Preparation of solutions 1. Preparation of Thrombin solution: ^ Reconstitute at 100 µg/ml (1428.57 nM) in sterile 50 mM HEPES and 640 mM NaCl ^ Diluted to a stock solution c = 0.04 µg/µl with TNC buffer 2. Preparation of reference substrate solution: ^ Dilute and aliquote 2.1 mg of reference substrate to a concentration of c = 2000 µM in TNC. Dilute and aliquote further to a concentration of c = 200 µM in TNC buffer. Endconcentration in well: c = 100 µM. 3. Preparation of inhibitor solutions: ^ Inhibitor was diluted with pure DMSO to a concentration of 10 mM ^ This 10 mM stock solution was further diluted: concentration of inhibitor concentration of inhibitor (stock solution) in well* *Total v olume per well = 100 µl, so the dilution factor of the stock solutions is 100x 2. Preparation of TNC buffer: ^ 25 mM Tris, 150 mM NaCl, 5 mM CaCl ₂ , 0,01% Triton X-100, pH = 8 (stored at 8°C) Platereader setup ^ Total volume per well: 100µl ^ Temperature: 25°C ^ Kinetic interval: every 2 minutes ^ Absorption: 405 nm ^ Number of flashes: 25 ^ Shaking before measurememt: 3s ^ Shaking amplitude: 5 mm Experimental setup ^ Per well: add 50 µl of Thrombin/TNC solution (c = 0.04 µg/µl), add 1 µl of inhibitor. ^ Incubate for 30 minutes at room temperature ^ After incubation, add 1 µl of c = 200 µM reference substrate Endconcentration of substances per well: c = 100 µM of reference substrate, c = 0.002 µg of Factor Xa solution, 1% DMSO Data analysis ^ Graph pad prism 7 was used to analyze the data. The velocity (change of absorbance over time) was plotted against the concentration. A non linear regression was performed with the following equation: [inhibitor] vs. response – Variable slope (four parameters) Example B-2. Inhibitory activity of the inventive compounds against Factor Xa Protease Factor Xa inhibiton assay – Calculation of IC ₅₀ values Materials Protease: Recomb. Human Coag. Factor Xa protein, CF: Bought 09.12.2020; Art. 1063-SE-010; Company: Bio-Techne GmbH Reference substrate: Bz-Ile-Glu-Gly-Arg-pNA; Company: Bachem; Art no.: 4003874.00051000025708 Plates for plate reader: Greiner 96 well plates, PS, F-Bottom (Chimney well), µclear, black, cellstar. Cat. No.: 655090/655096/655097 Tubes: Eppendorf, Protein LoBind Tube 1.5ml/2ml. Cat. No.: 022431081 Platereader: Device: Infinite M1000 Application: Tecan i-control Firmware: V_2.09_04/2011_S3LCE (May 22011/09.25.56) Preparation of solutions 1. Preparation of Factor Xa solution: ^ Reconstitute at 100 µg/ml in sterile 25 mM MES, 150 mM NaCl, 5 mM CaCl2, pH6 ^ Diluted to a stock solution of 1000 nM with TNC buffer ^ This stock solution was further diluted with TNC buffer to a concentration of 35 nM and aliquoted. Stored at -20°C. Endconcentration in well: c = 0.35 nM 2. Preparation of reference substrate solution: ^ Delivered: 5mg. Dilute and aliquote reference substrate to a concentration of 10 mM in pure DMSO. Endconcentration in well: c = 200 µM (= K _M for Factor Xa) 3. Preparation of inhibitor solutions: ^ Inhibitor was diluted with pure DMSO to a concentration of 10 mM ^ This 10 mM stock solution was further diluted: concentration of inhibitor concentration of inhibitor (stock solution) in well* *Total volume per well = 100 µl, so the dilution factor of the stock solutions is 100x 2. Preparation of TNC buffer: ^ 25 mM Tris, 150 mM NaCl, 5 mM CaCl ₂ , 0,01% Triton X-100, pH = 8 (stored at 8°C) Platereader setup ^ Total volume per well: 100µl ^ Temperature: 25°C ^ Kinetic interval: every 2 minutes ^ Absorption: 405 nm ^ Number of flashes: 25 ^ Shaking before measurememt: 3s ^ Shaking amplitude: 5 mm Experimental setup ^ Per well: add 97 µl TNC buffer , add 1 µl of c = 35 nM solution of Factor Xa, add 1 µl of inhibitor ^ Incubate for 30 minutes at room temperature ^ After incubation, add 1 µl of c = 10 mM reference substrate Endconcentration of substances per well: c = 200 µM of reference substrate, c = 0.35 nM of Factor Xa solution, 2% DMSO Data analysis Graph pad prism 7 was used to analyze the data. The velocity (change of absorbance over time) was plotted against the concentration. A non linear regression was performed with the following equation: [inhibitor] vs. response – Variable slope (four parameters) Table 1. IC50 values of inventive inhibitors with respect to TMPRSS2, thrombin and Factor Xa arhead. with TMPRSS2, thrombin and factor Xa. IC ₅₀ values were calculated from nonlinear regressions with the following equation: [Inhibitor] vs. Response – Variable slope (four parameters) usig the program Prism 7 * NS : natural substrate (P-site from hepatocyte growth factor) ** RS : reference substrate (P-site from preferred substrate for hepsin) *** SA : SARS CoV-2 S Protein-TMPRSS2 recognition sequence **** FOY251 : active form of Camostate mesylate Example B-3. Inhibitory activity against a pseudotyped particle LV(Luc)-CoV2 in CaCo-2 cells Generation of lentiviral SARS-CoV-2 pseudoparticles All cells were cultured in a humidified incubator at 37 °C, 5 % CO ₂ . To generate lentiviral pseudoparticles harboring the SARS-CoV-2 Spike (termed LV(Luc)-CoV-2-S), 900 000 HEK293T (human embryonal kidney) cells were seeded in 2 ml of Dulbecco´s Modified Eagle Medium (DMEM, Gibco) supplemented with 10 % FCS (Gibco), 2 mM glutamine, 100 U/ml Penicillin and 100 µg/ml Streptomycin (Pan Biotech) in 6-well plates. The next day, medium was refreshed and cells were transfected using Polyethyleneimine (PEI) transfection reagent. A total of 1 µg DNA comprising 2 % pCG1-SARS-2-S (encoding SARS-CoV-2 Spike of isolate Wuhan-HU-1, NCBI Reference Sequence: YP_009724390.1) (Hoffmann M, et. al., Cell, 2020, 181:271-280.e8), pSEW-luc2 (a crippled lentiviral vector expressing luciferase (Abel T, et. al., Blood, 2013, 122:2030– 2038; Demaison C, et. al., Hum Gene Ther., 2002, 13:803–813; Münch RC, et. al., Mol Ther, 2011, 19:686–693.) and pCMVdR8.91 (a Gag-Pol expression plasmid) (Romain Zufferey, et. al., Nat. Biotechnol., 1997, 3:3-8) at a 1:1 ratio was mixed in 1.5 ml serum reduced medium (Opti-MEM, Gibco). PEI was added to the DNA at a PEI:DNA ratio of 3:1. The mixture was briefly vortexed, incubated for 20 min at RT and added dropwise to the cells. At 8 h post transfection, cells were washed with PBS and 2 ml of DMEM supplemented with 2.5 % FCS, 2 mM glutamine, 100 U/ml Penicillin and 100 µg/ml Streptomycin were added. At 48 h post transfection, pseudoparticle containing supernatants were collected and clarified by centrifugation at 1500 rpm for 5 min. For transduction experiments in presence of TMPRSS2 inhibitors, 10 000 Caco2 (human colon) cells were seeded in 100 µl of DMEM supplemented with 10 % FCS (Gibco), 2 mM glutamine, 100 U/ml Penicillin, 100 µg/ml Streptomycin, 1x non-essential amino acids (NEAA) and 1 mM sodium-pyruvate (ThermoFischer) in 96-well flat-bottom plates. The next day medium was removed and replaced by 60 µl fresh medium in the presence of 20 µl TMPRSS2 inhibitors, EK1 peptide inhibitor control (Xia S, et. al., 2019, A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike. Sci Adv 5:eaav4580.) or equivalent volumes of DMSO solvent control. After 2 h of incubation, cells were transduced with 20 µl of fresh, infectivity normalized LV(Luc)-CoV-2-S. At 48 h post transduction, transduction rates were assessed by measuring luciferase activity of cell lysates using a commercially available kit (Promega). Briefly, cells were washed with PBS and incubated with 40 µl cell culture lysis reagent for 10 min at RT. 30 µl of lysates were transferred to opaque 96-well plates and mixed with 50 µl of Luciferase assay substrate. Luminescence was recorded immediately for 0.1 s/well in an Orion II Microplate luminometer (Berthold). Luciferase activities in absence of inhibitors were set to 100 % and IC ₅₀ s were determined by linear regression using GraphPad Prism version 8.4.2. LV(Luc)-CoV2 is based on an infection-deficient lentiviral backbone that bears SARS- CoV2 spike protein, furthermore, it harbors a luciferase reporter gene as readout of infection . Protocol: -day 0: seed 10000 Caco2 cells per well in 100 µl Caco medium in a 96 F-well plate -day 1: remove medium from cells and add 60 µl of Caco medium -dilute inhibitors (stock 10 mM) to 100 µM and titrate in 1:5 steps -add 20 µl inhibitors to cells in duplicates and incubate for 2 h at 37 °C -add 20 µl of LV(Luc)-CoV2 pseudoparticle -at 4 h post infection: measure luciferase activity Caco Medium: ml DMEM The inventive compounds effectively inhibit the infection of CaCo-2 cells casued by the LV(Luc)-CoV2 which is based on an infection-deficient lentiviral backbone that bears SARS-CoV2 spike protein (see Table 2). All of the inventive compound as TMPRSS2 inhibitors effectively LV(Luc)-CoV2-S infection in a dose-dependent manner. In particular Compound 2p2i shows similar activity to camostatmesylate. Furthermore, by microscopy: no apparent cytotoxicity of the inventive compound was observed. Table 2. IC ₅₀ values of the inventive compounds against a pseudotyped particle LV(Luc)-CoV2 in CaCo-2 cells Ref.3: Leupeptin – naturally occruing inhibitor of Ser, Cys and Thr protease Ref.4: Benzamidine – inhibitor of trypsin, trypsin-like enzyme and Ser protease Ref.6: PPack: irreversible thrombin inhibitor Ref.9: EK1 peptide: EK1 protein binds to SARS-COV2 spike protein Example B-4. Inhibition of SARS-CoV2 WT infection Protocol: • Seed 30000 cells/well in 96 well plate • Serially dilute TMPRSS2 inhibitors and controls in 1:5 steps from 500 to 0.0064 µM • Add 20 µl of TMPRSS2 inhibitors to 60 µl cells • Incubate for 2 h at 37 °C • Infect with 20 µl of SARS-CoV2 at an MOI of 0.005 • Incubate for 48 h at 37 °C • Check cells in microscope & measure ELISA Virus isolate used: BetaCoV/France/IDF0372/2020 Readout: In-cell ELISA • Immunodetection of viral antigens in cells • 48 h post infection • Principle: • primary CoV2-S Antibody detects Spike protein • Secondary antibody (labelled) detects primary antibody • Readout by measuring signal of secondary antibody Table 3. IC ₅₀ values of the inventive compounds against SARS-COV2 WT infection IC ₅₀ • TMPRSS2 inhibitors are active against SARS-CoV2 WT • IC ₅₀ s are higher in all instances, this could be caused by: • High infection, leading to cell death and therefore lower signal in wells with little/ no inhibitor ^ normalization to wells without inhibitor makes it look like infection is enhanced with higher conc of inhibitor before inhibition can be seen • The higher number of cells seeded (3x more than in Pseudoparticle test) • IC ₅₀ of comopund 4(P5i) appears quite high, however last datapoint of the compound 4 at 10 µM has tremendous SD Example C Molecular modelling. For TMPRSS2, a homology model was built using the Swiss- Model web server (Waterhouse, A. et al., Nucleic Acids Res 2018, 46 (W1), W296- W303). The template structure was selected based on the serine protease hepsin in complex with N-acetyl-6-ammonio-L-norleucyl-L-glutaminyl-N-[(1S)-4-w-1- (chloroacetyl)butyl]-L-leucinamide (PDB-ID: 1Z8G) (Herter, S. et al., Biochem J 2005, 390 (Pt 1), 125-36.)with a sequence identity to TMPRSS2 of 42.49% and a 1.5 Å resolution. For subsequent docking studies, both the homology model and the crystal structure of matriptase in complex with N-(3-phenylpropanoyl)-3-(1,3-thiazol-4-yl)-L- alanyl-N-[(1S,2S)-1-(1,3-benzothiazol-2-yl)-5-carbamimidamid o-1-hydroxypentan-2-yl]- L-valinamide (PDB-ID: 6N4T) (Beliveau, F. et al., Cell Chem Biol 2019, 26 (11), 1559- 1572 e9) as a surrogate model were used. The focused serine-protease inhibitor library was derived from the ZINC15 database (Sterling, T. et. al., J Chem Inf Model 2015, 55 (11), 2324-37). Commercially available compounds with reported affinity for factor Xa, hepsin, kallikrein, plasminogen, thrombin, matriptase-1, matriptase-2, trypsin and urokinase were pooled and after duplicate removal a total of 315 trypsin-like serine protease inhibitors were obtained. Sequences of tripeptides for docking studies on TMPRSS2 homology/surrogate were generated using CycloPs and included proteinogenic and non-proteinogenic amino acids (aa). The generated SMILES were modified to carry a N-terminal acetyl cap (ace) and a C-terminal aldehyde warhead. Prior to docking, all molecules were protonated and energetically minimized using MOE2019. Hereby, the MMFF94x forcefield was used for small molecules and AMBER14:EHT for peptidic molecules. For molecular docking with LeadIT-2.3.2, the binding site was defined to include all residues within 6 Å around the reference ligand of the hepsin homology model (PDB-ID (1Z8G)) (Maier, J. A. et al., J Chem Theory Comput 2015, 11 (8), 3696-71). For matriptase, all residues within 6.5 Å around the crystallographic reference ligand (PDB-ID: 6N4T) and water molecules forming at least three interactions with the target and ligand were included. Structures were protonated with the Protoss module within LeadIT-2.3.2. All dockings were performed using standard settings and the enthalpy-entropy hybrid approach. The docking strategy was validated for matriptase surrogate model by redocking of the ligand (PDB-ID: 6N4T), and for TMPRSS2 homology model and matriptase surrogate model by docking of the substrate ace-D-Arg-Pro/Gly-Arg-nme and by a binder vs non-binder discrimination using 56 published TMPRSS2 inhibitors and 314 decoys generated for four inhibitors present at ZINC using the database of useful decoys enhanced (DUD-E; Figure 12a-c). Results were analyzed by FlexX score and visual pose inspection to select molecules for purchase and synthesis. Figures were made with PyMOL. Enzymes. Recombinant human hepsin protein and factor Xa protein were purchased from Bio-Techne GmbH (Wiesbaden, Germany). Recombinant human thrombin protein and matriptase protein were purchased from R&D Systems (Minneapolis, MN, USA). Determination of inhibitory constant K _i . The activity of the compounds against the recombinant human enzymes was determined in enzyme inhibition assays. Here, a ten- point dilution series for the inhibitors was prepared and incubated for 30 min with the enzyme in TNC buffer (25 mM Tris, 150 mM NaCl, 5 mM CaCl ₂ , 0.01% Triton X-100, pH = 8) prior to adding a fluorogenic reference substrate Boc-Gln-Ala-Arg-AMC for matriptase and TMPRSS2 or a chromogenic substrate D-Phe-Homopro-Arg-pNA, Bz- Ile-Glu-Gly-Arg-pNA for thrombin and factor Xa, respectively. The measurements were performed on a Tecan infinite® M1000 and the fluorescence intensity was measured by exciting the AMC fluorophore at 380 nm wavelength and recording emission at 460 nm wavelength. The absorption of pNA was measured at 405 nm. Fluorescence intensities and absorption were measured every 2 min for 2 h. The end concentrations of the enzymes were 0.2 nM (matriptase) and 0.2 nM (TMPRSS2) in 20 µL total volume and 0.6 nM (thrombin), 0.35 nM (factor Xa) in 100 µL total volume. The end concentration of the reference substrate was 100 µM (matriptase), 100 µM (TMPRSS2), 200 µM (factor Xa) and 100 µM (thrombin). To determine the IC ₅₀ values, the concentration-response data were plotted with the program GraphPad prism version 8.4.2 (San Diego, California) and a nonlinear regression fit with the equation [Inhibitor] vs. normalized response was applied. The inhibitory constant K _i was calculated from the IC ₅₀ values using the Cheng-Prusoff equation (K _i = IC ₅₀ /[S]/K _M ) for competitive reversible inhibitors. The K _M value was determined to be 77 µM for TMPRSS2 (Figure 13). Analysis of cellular TMPRSS2 expression. The human colorectal adenocarcinoma cell line Caco-2 from the Collection of Microorganisms and Cell Cultures (DSMZ, Germany) was maintained in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% FBS, 100 U /mL penicillin, 100 mg/mL streptomycin, and 2 mM glutamine (all Invitrogen, Germany). For the validation of the expression of the transmembrane serine protease TMPRSS2, 100,000 Caco-2 cells were resuspended in 100 µL of Dulbecco's phosphate buffered saline (DPBS, Sigma-Aldrich) and incubated with the TMPRSS2 antibody (rabbit anti-human IgG from ThermoFisher Scientific, PA5- 14264) at final concentrations of 10 µg/mL, 20 µg/mL, 40 µg/mL, and 100 µg/mL for 30 min at 4 °C. After the separation from unbound antibody molecules by centrifugation (200 × g for 3 min) and resuspension of the cells in 100 µL DPBS, 1 µL of a FITC- labeled secondary donkey anti-rabbit IgG (ThermoFisher, A16024) was added and incubated for 30 min at 4 °C. Following a final centrifugation (200 × g for 3 min), the cells were resuspended in 1 mL DPBS and analyzed by flow cytometry. The measurements were performed on an Attune™ NxT cytometer (ThermoFisher) with a 488 nm laser for excitation of bound secondary antibody molecules (FITC) and a 530/30 nm band pass filter for emission detection. Using the Attune™ NxT software (ThermoFisher), Caco-2 cells were selected by the FSC/SSC plot, thereby excluding cell debris. From this dot plot gating of Caco-2 cells, a histogram plot of the BL1-H emission filter signal was generated. The signal of untreated Caco-2 cells (autofluorescence) was gated to one percent, whereby all other samples refer to the percentage of events within this gate. For the data analysis, GraphPad prism version 8.4.2 was applied. Analysis of inhibition of cellular TMPRSS2 activity.10,000 Caco-2 cells in 100 µL EMEM medium supplemented with 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mM glutamine (all Invitrogen, Germany) were seeded per well in a 96-well plate and incubated at 37 °C for four days until full confluency of the cells. The cells were washed two times with PBS and EMEM medium without FBS was added. For the determination of IC ₅₀ values, 1 µL of inhibitor was incubated for 30 min at room temperature prior to adding 2 µL of 10 mM reference substrate (Boc-Gln-Ala-Arg-AMC). The fluorescence intensity was measured as described above. SARS-CoV-2 pseudoparticles. To generate replication deficient lentiviral pseudoparticles carrying the SARS-CoV-2 spike protein (LV(Luc)-CoV-2), 900,000 HEK293T cells were seeded in 2 mL DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mM glutamine. The next day, the medium was refreshed and cells were transfected with a total of 1 µg DNA using polyethyleneimine (PEI). To this end, 2% of SARS-2 spike plasmid (encoding the spike protein of SARS-CoV-2 isolate Wuhan-Hu-1, NCBI reference sequence YP_009724390.1, SARS-CoV-2 variant B.1.1.7 or B.1.351) were mixed with pCMVdR8_91 (encoding HIV structural proteins gag and pol) and pSEW-Luc2 (crippled lentiviral vector encoding the luciferase reporter gene) in a 1:1 ratio in serum-free medium. Plasmid DNA was mixed with PEI at a DNA:PEI ratio of 1:3 (3 μg PEI per 1 μg DNA), incubated for 20 min at room temperature and added to cells dropwise. At 8 h post transfection, the medium was removed, cells were washed with 2 mL of PBS and 2 mL of HEK293T medium with 2.5% FCS were added. At 48 h post transfection, pseudoparticles containing supernatants were harvested and clarified by centrifugation for 5 min at 1500 rpm. SARS-CoV-2 strains and propagation. Viral isolates BetaCoV/Netherlands/01/NL/2020 of the pandemic D614G variant (#010V-03903), BetaCoV/France/IDF0372/2020 (#014V-03890) and virus lineage B.1.1.7 hCoV- 19/Netherlands/NH-RIVM-20432/2020 (#014V-04031) were obtained from the European Virus Archive global. The virus lineage B.1.351 2102-cov-IM-r1-164 was isolated, sequenced and kindly provided by Michael Schindler. All strains were propagated on Vero E6 or Caco-2 cells. To this end, 70-90 % confluent cells in 75 cm² cell culture flasks were inoculated with SARS-CoV-2 isolate (multiplicity of infection (MOI) of 0.03-0.1) in 3.5 mL serum-free medium. Cells were incubated for 2 h at 37 °C, before adding 20 mL medium containing 15 mM HEPES. Cells were incubated at 37 °C and supernatant harvested when a strong cytopathic effect (CPE) was visible. Supernatants were centrifuged for 5 min at 1,000 × g to remove cellular debris, and then aliquoted and stored at -80 °C as virus stocks. Infectious virus titer was determined as plaque forming units (PFU) on Vero E6 cells, which was used to calculate MOI. Pseudovirus inhibition assay. 10,000 Caco-2 cells were seeded in 100 µL DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, 2 mM glutamine, 1× non-essential amino acids and 1mM sodium pyruvate. The next day, medium was replaced by 60 µL of fresh medium and cells were treated with 20 µL of serial dilutions of TMPRSS2 inhibitors or small molecule protease inhibitors for 2 h at 37 °C, followed by transduction with 20 µL of infectivity normalized LV(Luc)-CoV-2 pseudoparticles. Transduction rates were assessed after 48 h by measuring luciferase activity in cell lysates with a commercially available kit (Promega). Briefly, cells were washed with PBS and incubated with 40 µL cell culture lysis reagent for 10 min at RT. 30 µL of lysates were transferred to opaque 96-well plates and mixed with 50 µL of Luciferase assay substrate. Luminescence was recorded immediately for 0.1 s/well in an Orion II Microplate luminometer (Berthold) with simplicity 4.2 software. Luciferase activities in absence of inhibitors were set to 100% and IC ₅₀ were determined by linear regression using GraphPad Prism version 8.4.2. SARS-CoV-2 inhibition assay. 25,000 Caco-2 cells were seeded in 100 µL respective medium. The next day 40 µL of medium were removed and cells were treated with 20 µL of serial dilutions of TMPRSS2 inhibitors or small molecule protease inhibitors for 2 h at 37 °C, followed by infection with 20 µL SARS-CoV-2 of the respective virus strain at a multiplicity of infection (MOI) of 5 × 10-4. Infection rates were assessed at 2 days post infection by in-cell ELISA for SARS-CoV-2 nucleocapsid or spike. Briefly, cells were fixed by adding 180 µL 8% paraformaldehyde (PFA) for 30 min at room temperature and permeabilized by incubation with 100 µL 0.1% Triton-X for 5 min. After washing once with PBS, cells were stained with 1:5,000 diluted anti-spike protein antibody 1A9 (Biozol GTX-GTX632604) or anti-nucleocapsid antibody (Sinobiological 40143-MM05) in antibody buffer (10 % FCS and 0.3% Tween 20 in PBS) for 1 h at 37 °C. After 2 washes with 0.3% Tween 20 in PBS, the secondary HRP- conjugated antibody (Thermo Fisher #A16066) (1:15,000) was incubated for 1 h at 37 °C. Cells were washed 3 times with 0.3% Tween 20 in PBS, TMB peroxidase substrate (Medac #52-00-04) was added for 5 min and the reaction stopped using 0.5 M H ₂ SO ₄ . The optical density (OD) was recorded at 450 nm - 620 nm using the Asys Expert 96 UV microplate reader (Biochrom) with DigiRead 1.26 software. Values were corrected for the background signal derived from uninfected cells and untreated controls were set to 100% infection. Cytotoxicity assay 10,000 Caco-2 cells were seeded in 100 µL respective medium. The next day, medium was replaced by 80 µL of fresh medium and cells were treated with 20 µL of serial dilutions of peptidomimetic TMPRSS2 inhibitors or small molecule protease inhibitors. Cell viability was assessed after 48 h with a commercially available kit (Promega). Briefly, medium was removed and cells were lysed with 100 µL CellTiter- Glo reagent for 10 min at room temperature. 50 µL of lysates were transferred to opaque 96-well plates and luminescence was recorded immediately for 0.1 s/well in an Orion II Microplate luminometer (Berthold) with simplicity 4.2 software. Luciferase activities in absence of inhibitors were set to 100%. Stability of inhibitors in serum, plasma and cell culture medium. The stability of the inhibitors was measured according to a modified procedure from Jensen et al..52 In short, TMPRSS2 inhibitor solutions of 1 mM were prepared by dissolving the peptide in pure dimethyl sulfoxide. 10 µL of the peptide solution were added to 1 mL RPMI medium 1640 supplemented with 25% (v/v) human serum, citrate plasma or only RPMI medium and incubated at 37 °C. At indicated intervals 100 µL samples were taken and mixed with200 µL ethanol for precipitation of proteins. The cloudy solution was cooled at 4 °C for 15 min and centrifuged at 14,800 rpm for 2 min. The supernatant was aspirated and analyzed using analytical HPLC. The residual inhibitory constants K _i after 10 days incubation in biological fluids was measured as described above. Results Structure-based design of TMPRSS2 inhibitors. For the identification of novel peptide based TMPRSS2 inhibitors as a potential treatment of SARS-CoV-2 infection, molecular docking studies were performed. As no crystal structure of TMPRSS2 is available in the protein data bank (PDB) we used matriptase-1 as a surrogate model. (Berman, H. M, et al., The Protein Data Bank. Nucleic Acids Research 2000, 28 (1), 235-242; Beliveau, F. et. al., Cell Chem Biol 2019, 26 (11), 1559-1572 e9) Matripase-1 shares 41% sequence identity with TMPRSS2 and previously described substrate analogue TMPRSS2 inhibitors showed no selectivity over matriptase (Meyer, D. et al., Biochem J 2013, 452 (2), 331-43.). Additionally, a homology model of TMPRSS2 was built using hepsin (43% sequence identity) as a template. The docking procedure of those crystal structures was tested by redocking of reference ligands for matriptase and TMPRSS2 and receiver operator characteristic (ROC) curve analysis which indicated a very strong ability to discriminate between known binders (Figure 12a-c; Table 4). Table 4. FlexX-scores of tripeptidic substrate-analogue ligands for docking receptor validation. Scores are in kJ/mol, ace: N-terminal acetyl-cap, nme: C-terminal N- methylamide cap. aPredicted binding modes is shown in Figure 6a;14. Li d Fl X Fl X t i t A reference binder comprising a N-terminal acetyl-cap and a C-terminal aldehyde serine trap with the sequence ace-D-Arg-Gly/Pro-Arg-aldehyde was designed and docked to the matriptase-based TMPRSS2 surrogate model (Figure 12a) and TMPRSS2 homology model (Figure 14). Our dockings show that both the reference binder and the published TMPRSS2 inhibitor Ref. 5(CM) bind to the reactive center of the surrogate and the homology model (Figure 12b; Figure 14). To optimize the binding affinity of the reference binder, we altered the residues at P1-P3 position using both proteinogenic and non-proteinogenic amino acids and docked the resulting structures to both, the matriptase surrogate and TMPRSS2 homology model. Compounds were then ranked based on their binding score (Table S2-S4), the plausibility of their binding mode to the S1-S3 sub-pockets of the TMPRSS2 active site and proximity of the aldehyde serine trap to the catalytic Ser186/195 (TMPRSS2 homology model/matriptase enumeration), as well as commercial availability of their building blocks. Overall, the D-configuration for P3 residue was favored to improve metabolic stability. The most promising compounds were chosen for solid phase synthesis, whereby the aldehyde serine trap used for in silico modelling was exchanged by a well- established ketobenzothiazole, yielding a library of peptidomimetic inhibitors (Table 5). To identify literature-known inhibitors for TMPRSS2, a focused library consisting of 315 commercially available inhibitors reported for the closely related trypsin-like serine proteases factor Xa, hepsin, kallikrein, plasminogen, thrombin, matriptase-1, matriptase-2, trypsin and urokinase was virtually screened. The most promising virtual screening (VS) hits based on score, binding pose and availability were subsequently purchased and tested (Table 6). Table 5. Assembled peptidomimetic inhibitor library selected for synthesis. alanine; Pip: Pipecolinic acid; kbt: ketobenzothiazole. The bond between the P1 position and warhead is the site of nucleophilic attack by the protease catalytic triad. Table 6. Virtual screening hits selected for testing. aPotential covalent inhibiton, bcontrol compound as low affinity reference. Scores are in kJ/mol. Ref. No. Synonym / FlexX-score FlexX-score D esg ed peptdo etc bto s boc SS a d at ptase act ty We next investigated the impact of the peptidomimetic inhibitors on the activity of closely related matriptase and TMPRSS2 enzymes. To this end, the respective purified proteases were incubated with the compoundsST1, ST1a, ST5, ST6, ST8, ST10, ST14 and ST16, followed by adding a protease specific reporter substrate that allowed monitoring of protease activity over time. Overall, the compounds suppressed TMPRSS2 activity in the low nanomolar range (Ki = 2.5 - 215.9 nM) and inhibit matriptase with comparable activity (Table 7; Figure 14a, b). Compound ST1 which contains the peptide sequence of the reference binder showed an activity of K _i = 86.7 nM, while the compounds ST1a, ST5, ST6, ST8, ST10, and ST16 were most active against isolated TMPRSS2, with inhibitory constants of 2.5 - 57.5 nM. The compounds ST1a (K _i = 3.8 nM) and ST8 (K _i = 2.5 nM) were 2-3-fold more active than the active metabolite of CM, FOY-251 (K _i = 9.7 nM). Compounds ST10 and ST14 were the least active with K _i values of 71.5 and 215.9 nM, respectively. The activity of the inhibitors against TMPRSS2 correlated with their activity against matriptase and revealed a linear correlation (Figure 7). Yet, compound ST1 showed the highest selectivity (~51-fold) for matriptase while Ref. 8 (FOY-251) showed the highest selectivity (~18-fold) for TMPRSS2. For systemic administration of the protease inhibitors, high selectivity over off-target proteases is required to reduce side effects. To investigate potential interference of the peptidomimetic inhibitors with serine proteases involved in coagulation, we assessed their activity against thrombin and factor Xa (Table 7; Figure 14c, d). All compounds excluding compound ST1 display a > 100-fold selectivity against thrombin (Table 7). Table 7. Overview of inhibitory activity (K _i ) of synthesized TMPRSS2 inhibitors ST1a, ST1, ST5, ST6, ST8, ST10, ST14, and ST16 against TMPRSS2, matriptase, thrombin and factor Xa. Selectivity indices represent the quotient of K _i values of matriptase, thrombin, and factor Xa by the K _i value of TMPRSS2. K _i [nM] selectivity indices Compound ST1 shows no selectivity over factor Xa while compounds ST1a, ST5, ST6, ST8, and ST16 reveal 1.6 to 38.9-fold selectivity compared to TMPRSS2. Truncation of the ketobenzothiazole serine trap moiety to a ketothiazole did not improve activity against matriptase, nor thrombin/factor Xa selectivity and further reduction to an alcohol abolished antiprotease activity (Figure 15; Table 8). Considering the inhibitory constants and selectivity over potential off-target coagulation proteases, the compounds ST1a, ST6, ST8, and ST16 were further analyzed for inhibition of cellular TMPRSS2 activity. Table 8. Influence of serine trap on biological activity of compound ST16 against matriptase, thrombin and factor Xa. The ketobenzothiazole (ST16) serine trap moiety was truncated to ketothiazole (ST16b) and further reduced to the alcohol (Ref. 10) for complete abolishment of electrophilicity. N.i = no inhibition within concentration range. Among the selected, high scoring literature known inhibitors, only Leupeptin and PPack showed activity against matriptase with K _i = 1452 nM and K _i = 12.4 nM, respectively (Figure 16; Table 9). The anticoagulant drugs PPack, Dabigatran and Melagatran show inhibitory activities comparable to literature known values. Due to low activity against matriptase and poor selectivity against the coagulation proteases, those inhibitors were not further investigated. Table 9. K _i values of reference inhibitors against matriptase, TMPRSS2, thrombin and factor Xa. N.i = no inhibition within concentration range. K _i [nM] Having dem MPRSS2 activity, we next analyzed inhibition of cell associated enzyme activity. For this we used SARS-CoV-2 permissive Caco-2 cells, which show upregulation of the TMPRSS2 gene and express TMPRSS2 on the cell surface (Figure 17). Cells were incubated with the respective inhibitors and treated with fluorogenic protease substrate. The most potent inhibitors against matriptase and TMPRSS2 also efficiently prevented cell-mediated proteolysis of the fluorogenic substrate with half maximum inhibitory concentrations (IC ₅₀ ) of 12.7 - 234.2 nM, with compounds ST1a (IC ₅₀ = 32 nM) and ST8 (IC ₅₀ = 12.7 nM) being most active (Figure 8; Table 10). Table 10. IC ₅₀ values of peptidomimetic TMPRSS2 inhibitors, camostat mesylate (CM, Ref.5) and FOY-251 (Ref.8) measured on Caco-2 cells. Taken together, our results demonstrate that the synthesized peptidomimetic inhibitors potently reduce the activity of purified matriptase and TMPRSS2 while showing no activity against thrombin. Further, the selected most potent inhibitors display selectivity over factor Xa and reduce cellular protease activity. TMPRSS2-specific peptidomimetic inhibitors block SARS-CoV-2 infection. We next analyzed whether compounds ST1, ST1a, ST5, ST6, ST8, ST10, ST14 and ST16 may inhibit SARS-CoV-2 spike driven viral entry. For this, Caco-2 cells treated with serial dilutions of the compounds (and CM as control) were inoculated with luciferase encoding lentiviral pseudoparticles carrying the wildtype SARS-CoV-2 spike protein. Transduction rates were determined 2 days later by measuring cell-associated luciferase activity and showed a concentration dependent inhibition of viral entry for all analyzed compounds (Figure 9a). Compound ST8 was most efficient with an IC ₅₀ value of 467.2 nM and was even more potent than CM (IC ₅₀ ~ 747.5 nM). Compounds ST1, ST1a, ST5, ST6, and ST16 suppressed spike driven entry with IC ₅₀ values between 1,200 and 2,068 nM while compounds ST10 and ST14 were the least antivirally active with IC ₅₀ values between 5,604-12,085 nM, respectively (Figure 9a; Table 11). Table 11. IC ₅₀ values of peptidomimetic TMPRSS2 inhibitors and camostat mesylate (CM, Ref.5) against pseudotype lentivirus. n.d.:not determined IC ₅₀ [nM] Wildtyp B.1.1. B.1.35 C d As the wildtype virus has largely been replaced by other SARS-CoV-2 clades, and variants of concern (VOC) with increased transmissibility and virulence emerged, we also determined the activity of selected compounds against the B.1.1.7 (emerged in the UK) and B.1.351 (emerged in South Africa) spikes. TMPRSS2 inhibitors ST1a, ST6, ST8 and ST16 suppressed cell entry mediated by both spike variants in a dose- dependent manner with IC ₅₀ values ranging between 260.7-1,597 nM. (Figure 9b, c; Table 11). Collectively these data show that the designed TMPRSS2 inhibitors suppress SARS-CoV-2 spike driven viral transduction. We next analyzed whether the inhibitors may also block authentic SARS-CoV-2 infection. To this end, Caco-2 cells were supplemented with serial dilutions of the compounds ST1, ST1a, ST5, ST6, ST8, ST10, ST14 and ST16 or Ref. 5 (CM) and were then infected with a wildtype SARS-CoV-2 isolate obtained from France. Infection rates were determined 2 days later by quantifying intracellular viral protein expression by ELISA. All compounds including Ref. (CM) suppressed SARS-CoV-2 infection in a concentration dependent manner (Figure 10 a). The IC ₅₀ values were, however, generally higher as compared to the pseudotype experiment (Table 12). Compounds ST1a, ST6, and ST8 were the most potent inhibitors with IC ₅₀ values of 4.6, 5.7 and 4,7 µM, respectively, similar to Ref. 5, CM (3.6 µM). The remaining compounds were less active with IC ₅₀ values > 10 µM. The DMSO solvent control neither affected transduction of cells with SARS-CoV-2 pseudoparticles nor the infection with wildtype virus (Figure 18) and we did not observe cytotoxic effects from the compounds tested that exceed toxicity of the solvent control (Figure 19). Finally, we determined the antiviral activity of compounds ST1a, ST6, ST8, and ST16 against a SARS-CoV-2 isolate harboring the D614G mutation, which increases viral infectivity (Figure 10b), and the VOCs B.1.1.7 (Figure 10c) or B.1.351 (Figure 10d). The four selected compounds as well as CM inhibited all three tested SARS-CoV-2 isolates. Compounds ST1a and ST8 suppressed “D614G” infection with IC ₅₀ values of 17.1 and 11.9 µM, respectively and were even more active than CM (26.7 µM) (Table 12). SARS-CoV-2 variants B.1.1.7 and B.1.351 were most efficiently inhibited by compound ST8 (IC ₅₀ of 6.8 and 6.3 µM, respectively) and CM (IC ₅₀ of 16.4 and 9.3 µM, respectively). Compounds ST1a, ST6 and ST16 showed moderately higher IC ₅₀ as compared to CM and compound ST8, (Table 12), but were still capable of blocking infection entirely. Thus, the designed peptidomimetic TMPRSS2 inhibitors prevent SARS-CoV-2 infection with comparable antiviral activity as CM (Ref. 5), which is currently evaluated in clinical trials as COVID-19 therapeutic. Table 12. IC ₅₀ values of peptidomimetic TMPRSS2 inhibitors and camostat mesylate (CM, Ref. 5) against SARS-CoV-2 wildtype and variants of concerns. n.d.:not determined Discussion We here describe novel, potent and stable peptidomimetic inhibitors of TMPRSS2 that block SARS-CoV-2 infection. Targeting TMPRSS2 is a promising antiviral strategy because the protease is not only essential for SARS-CoV-2 entry, but also primes glycoproteins of various other viruses for subsequent fusion and infection. Since TMPRSS2 is a host and not a viral protein TMPRSS2-targeting therapeutics that block its enzymatic activity should be less likely to induce resistance mutations. To develop TMPRSS2 inhibitors, we designed a reference binder, which was used as a template for the preparation of peptidomimetic libraries, which then were screened in silico against the binding cavity of matriptase as a surrogate model for TMPRSS2 and against a TMPRSS2 homology model. A library of recognition sequences was compiled by incorporation of the identified top scoring amino acids in the template. The recognition sequences were connected with an electrophilic ketobenzothiazole warhead moiety as a reactive functional group to yield a panel of inhibitors. We tested the inhibitors against isolated enzymes and our data identified the four compounds ST1a, ST6, ST8, and ST16 as potential hits with high activity against TMPRSS2 and matriptase, and good off-target selectivity against coagulation proteins thrombin and factor Xa. Ref. 5 (CM) and its rapidly forming active metabolite Ref. 8 (FOY-251) in blood were used for comparison since they have been shown previously to efficiently inhibit TMPRSS2 proteolytic activity and CM is currently evaluated in clinical trials for COVID-19. Our best candidates show inhibitory activities in the same range as Ref. 5 (CM), and the compounds ST1a and ST8 even show a 2-3-fold higher activity against isolated TMPRSS2 than Ref. 8 (FOY-251). The ketobenzothiazole serine trap moiety revealed the highest activity which may be attributed to a preferential fit in the hydrophobic S1' pocket in contrast to the less hydrophobic and smaller ketothiazole. Substitutions on the benzothiazole with amino acids to interact more specifically with the S'-site might be of interest Furthermore, the high activity of the top compounds was confirmed through cleavage of a fluorogenic substrate on Caco-2 epithelial cells, which serve as an intestinal model carrying the TMPRSS2 protease on the surface. Finally, the inhibitors blocked SARS- CoV-2 spike driven viral entry into Caco-2 cells and infection of Caco-2 cells by authentic SARS-CoV-2 wildtype and variants of concern in a concentration dependent manner. Thus, the designed inhibitors likely block TMPRSS2 mediated proteolytic priming of the viral spike protein, thereby preventing subsequent receptor binding and fusion. These data also show that SARS-CoV-2 VOCs are still dependent on TMPRSS2 as essential cofactor for cell entry and demonstrate that VOCs that escape from preexisting immunity are equally sensitive to entry inhibitors, as previously shown for soluble ACE2 or fusion-inhibiting peptide EK1 and EK1C4. Two compounds (ST1a and ST16) were incubated in body fluids for up to 10 days and still showed residual inhibitory activity in the sub nanomolar range which is remarkable considering the literature known stability issues of peptide therapeutics. The rapid epimerization of the compounds in blood serum did not alter the activity significantly, suggesting that structurally simplified inhibitors may be developed. The high stability in body fluids and potent anti-TMPRSS2 and anti-SARS-CoV-2 activity warrant further preclinical development of selected compounds. Of note, the TMPRSS2 inhibitors will not only act against SARS-CoV-2 but potentially also block other TMPRSS2-dependent coronaviruses such as SARS-CoV and MERS-CoV, and likely also future novel emerging coronaviruses, and also TMPRSS2-dependent viruses from other viral families. In sum, TMPRSS2 represents an attractive drug target in COVID-19 and downregulation of its enzymatic activity with active and selective inhibitors should significantly improve health rehabilitation. Here we showed a new direction for the fast development of peptidomimetic inhibitors and our results offer potential candidates with comparable activities to CM whose efficacy may be further elucidated in in vivo studies.