WO2021175437A1 - A new class of flavivirus protease inhibitors - Google Patents

A new class of flavivirus protease inhibitors Download PDF

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Publication number
WO2021175437A1
WO2021175437A1 PCT/EP2020/056001 EP2020056001W WO2021175437A1 WO 2021175437 A1 WO2021175437 A1 WO 2021175437A1 EP 2020056001 W EP2020056001 W EP 2020056001W WO 2021175437 A1 WO2021175437 A1 WO 2021175437A1
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group
formula
compound
substituted
ester
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PCT/EP2020/056001
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French (fr)
Inventor
Christian Klein
Nikos KÜHL
Mila LEUTHOLD
Mira BEHNAM
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Universität Heidelberg
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Priority to PCT/EP2020/056001 priority Critical patent/WO2021175437A1/en
Priority to PCT/EP2021/054515 priority patent/WO2021175670A1/en
Publication of WO2021175437A1 publication Critical patent/WO2021175437A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/22—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
    • C—CHEMISTRY; METALLURGY
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/24—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a ring other than a six-membered aromatic ring of the carbon skeleton
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    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/28—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a non-condensed six-membered aromatic ring of the carbon skeleton
    • C07C237/42—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a non-condensed six-membered aromatic ring of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
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    • C07C243/24—Hydrazines having nitrogen atoms of hydrazine groups acylated by carboxylic acids
    • C07C243/26—Hydrazines having nitrogen atoms of hydrazine groups acylated by carboxylic acids with acylating carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C243/34—Hydrazines having nitrogen atoms of hydrazine groups acylated by carboxylic acids with acylating carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of a carbon skeleton further substituted by nitrogen atoms
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    • C07C243/24—Hydrazines having nitrogen atoms of hydrazine groups acylated by carboxylic acids
    • C07C243/38—Hydrazines having nitrogen atoms of hydrazine groups acylated by carboxylic acids with acylating carboxyl groups bound to carbon atoms of six-membered aromatic rings
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    • C07—ORGANIC CHEMISTRY
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    • C07C255/49—Carboxylic acid nitriles having cyano groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C255/54—Carboxylic acid nitriles having cyano groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing cyano groups and etherified hydroxy groups bound to the carbon skeleton
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    • C07C255/58—Carboxylic acid nitriles having cyano groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing cyano groups and singly-bound nitrogen atoms, not being further bound to other hetero atoms, bound to the carbon skeleton
    • C07C255/60—Carboxylic acid nitriles having cyano groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing cyano groups and singly-bound nitrogen atoms, not being further bound to other hetero atoms, bound to the carbon skeleton at least one of the singly-bound nitrogen atoms being acylated
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    • C07C257/00—Compounds containing carboxyl groups, the doubly-bound oxygen atom of a carboxyl group being replaced by a doubly-bound nitrogen atom, this nitrogen atom not being further bound to an oxygen atom, e.g. imino-ethers, amidines
    • C07C257/10—Compounds containing carboxyl groups, the doubly-bound oxygen atom of a carboxyl group being replaced by a doubly-bound nitrogen atom, this nitrogen atom not being further bound to an oxygen atom, e.g. imino-ethers, amidines with replacement of the other oxygen atom of the carboxyl group by nitrogen atoms, e.g. amidines
    • C07C257/18—Compounds containing carboxyl groups, the doubly-bound oxygen atom of a carboxyl group being replaced by a doubly-bound nitrogen atom, this nitrogen atom not being further bound to an oxygen atom, e.g. imino-ethers, amidines with replacement of the other oxygen atom of the carboxyl group by nitrogen atoms, e.g. amidines having carbon atoms of amidino groups bound to carbon atoms of six-membered aromatic rings
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    • C07C259/00—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups
    • C07C259/04—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids
    • C07C259/10—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids having carbon atoms of hydroxamic groups bound to carbon atoms of six-membered aromatic rings
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    • C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C279/12—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by nitrogen atoms not being part of nitro or nitroso groups
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    • C07C279/18—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to carbon atoms of six-membered aromatic rings
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    • C07C311/30—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/37—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
    • C07C311/38—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton
    • C07C311/39—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton having the nitrogen atom of at least one of the sulfonamide groups bound to hydrogen atoms or to an acyclic carbon atom
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/56—Nitrogen atoms
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    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/60—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/72—Nitrogen atoms
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    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/54—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings condensed with carbocyclic rings or ring systems
    • C07D231/56—Benzopyrazoles; Hydrogenated benzopyrazoles
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    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/26—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
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    • C07D277/68—Benzothiazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
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    • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
    • C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
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    • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
    • C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
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    • C07C2601/14—The ring being saturated

Definitions

  • the present invention relates to compounds that inhibit the activity of flavivirus.
  • Dengue virus West Nile virus and Zika virus are attracting more and more attention, which are members of the same genus flavivirus.
  • Dengue and West Nile virus are rapidly spreading global pathogens for which no specific therapeutic treatments are available.
  • a recent analysis estimates 390 million infections per year of which about one third are clinically recognized.
  • Dengue virus (DENV) infections can result in serious and life- threatening diseases like dengue fever, hemorrhagic fever and dengue shock syndrome.
  • a vaccine is approved in several countries, the vaccination of seronegative humans is not recommended.
  • WNV West Nile Virus
  • the technical problem underlying the present invention is the provision of a class of molecules for inhibition of viruses of the flavivirus genus.
  • the present invention provides non-peptidic (4-phenoxy)-phenylglycine and (4-benzyloxy)-phenylglycinederivatives as inhibitor of the NS2B-NS3 serine protease which are useful for inhibition of the protease.
  • NS2B-NS3 is a promising target in drug discovery against flaviviruses, since it is well conserved within this genus.
  • the enzyme cleaves the viral polyprotein into separate functional proteins and is therefore essential for virus replication.
  • the inhibitors can be used for the treatment of several flavivirus related diseases.
  • Compounds according to the present invention have a nanomolar activity in a cellular DENV-2 protease reporter assay, stable stereochemistry under physiological and assay conditions and most of the compounds are non-toxic up to 50 mM and have low off-target activity. These compounds contain only one basic residue and have a much smaller molecular mass than most flaviviral protease inhibitors. Thus, the compounds reach low micromolar to upper nanomolar inhibitory potency in cell-based assays as well as in a DENV-2 virus titer reduction assay, are selective against other serine proteases, e.g. Trypsin and Thrombin, and do not show relevant cytotoxicity.
  • serine proteases e.g. Trypsin and Thrombin
  • the compounds of the present invention are based on a phenylglycine, (4-phenoxy)- phenylglycine or a (4-benzyloxy)-phenylglycine element, and the amino acid is coupled to lipophilic alkyl chains or ring systems via an amide bond or sulfonamide bond, and not to other peptidic elements as described before.
  • the compounds of the present invention are much smaller than previous published DENV protease inhibitors and contain just one or no basic residue.
  • the most active compounds contain a guanidine, a benzamide or a phenylboronic acid group.
  • the compounds of the present invention do not have basic structural elements and therefore have a much more drug-like structure than most other inhibitors. Due to the small size and reduced amount of hydrogen-donating groups, most of the compounds presented here correspond to the Lipinski rule of five. This rule gives an indication of good pharmacokinetic properties of a drug and their oral bioavailability.
  • L 1 is -C(0)NH(CH 2 )mR 1 , -C(0)piperidyl, -NHC(0)(CH 2 )mR 1 or -NH(CH 2 ) m R 1 , wherein m is 0, 1 , 2, 3, 4, 5 or 6;
  • L 2 is -C(0)NH-, -S0 2 NH- or -NH- if L 1 is -C(0)NH(CH 2 ) m R 1 or -C(0)piperidyl; or L 2 is -NHC(O)- if L 1 is -NHC(0)(CH 2 )mR 1 or -NH(CH 2 ) m R 1 ;
  • I is 0 or 1 ;
  • R 2 is independently for each occurrence -CFa, -OCH3, -CN, a halogen atom or a linear or branched alkyl group having 1 to 4 carbon atoms;
  • R 3 is an alkyl group, a substituted or unsubstituted naphthyl group, a substituted or unsubstituted 2-phenylvinyl group, a substituted or unsubstituted 2-phenylethyl group, a heteroaryl group, a substituted or unsubstituted phenyl group or a substituted or unsubstituted benzyl group.
  • Single bonds connecting one residue, moiety, structure, substituent or group of the compound of formula (I) to another are indicated by in the text of the present application or in chemical formula by a dashed line being divided by a perpendicular wavy line or in case a stereocenter is defined by this bond a hashed wedged bond or wedged bond ending at the middle of a wavy line.
  • substituent -Chb- is a methylene bridge
  • -C(O)- is a carbonyl group
  • -NH- is a secondary amine group
  • residue L 1 is either a -C(0)NH(CH2)mR 1 group, a -C(0)piperidyl group, an -NHC(0)(CH2)mR 1 group or an -NH(CH2)mR 1 group, preferably a -C(0)NH(CH2)mR 1 group or a -C(0)piperidyl group and more preferably a -C(0)NH(CH2)mR 1 group.
  • the groups of residue L 1 are bond to the benzylic methine group, which forms the chiral center of the molecule and is bound to the central unsubstituted phenyl group of the compound of formula (I).
  • residue L 2 in the above formula (I) completes the (4-phenoxy)-phenylglycine and (4-benzyloxy)-phenylglycine structure and connects the substituent R 3 to the benzylic methine group, which forms the chiral center of the molecule and is bound to the central unsubstituted phenyl group of the compound of formula (I).
  • Residue L 2 is a -C(0)NH- group, a -SO2NH- group or an - NH- group, preferably a -C(0)NH- group, if residue L 1 is a -C(0)NH(CH2)mR 1 group or a -C(0)piperidyl group.
  • residue L 1 is an -NHC(0)(CH2)mR 1 group or an -NH(CH2)mR 1 group
  • the residue L 2 is an -NHC(O)- group.
  • the -C(0)NH- group and the -SO2NH- group are an amide group and a sulfonamide group, respectively, wherein in both groups the left represents a binding side to substituent R 3 and the right represents a binding side to the benzylic methine group, which forms the chiral center of the molecule and is bound to the central unsubstituted phenyl group of the compound of formula (I).
  • (4-phenoxy)-phenylglycine and (4-benzyloxy)- phenylglycine structures of the compounds of formula (I) can be selected from the structures of the following formulas (la), (lb), (lc), (Id), (le), (If), (Ig) or(lh), preferably (la), (lb), (lc), (Id) or (Ig), more preferably (la) or (Ig) and most preferably (la).
  • the piperidyl group of residue L 1 of formula (I) refers to a substituent derived from piperidine, of which one or more hydrogen atoms may be replaced by a substituent.
  • the substitution pattern and number of substituents are not particularly limited but may preferably be one substituent in 4-position of the piperidyl group of residue L 1 in formula (I).
  • Substituents may preferably be selected independently of each other from 1-pyrrolidinyl, a carboxamidyl group, an N-phenyl carboxamidyl group, trifluoromethyl (-CF3), -CM (cyano), a linear or branched alkyl group having preferably 1 to 4 carbon atoms, an alkyloxy group having preferably 1 to 4 carbon atoms and a halogen atom, most preferably 1-pyrrolidinyl, a carboxamidyl group or an N-phenyl carboxamidyl group.
  • a halogen atom is a group 17 element of the periodic table, preferably fluorine or chlorine.
  • piperidyl group in residue L 1 in formula (I) is preferably selected from the group consisting of:
  • the index “m” in the above formula (I) is selected from 0, 1 , 2, 3, 4, 5 or 6, preferably 0, 1 or 2 more preferably 0 or 1 and most preferably 1.
  • Substituent R 1 in the above formula (I) is preferably a basic or neutral moiety, a chemical residue containing one basic moiety or a non-basic replacement thereof.
  • a basic moiety is an atom or an organic or inorganic substituent which releases hydroxide ions in an aqueous solution or accepts protons from any proton donor.
  • Substituents R 4 and R 5 of -NR 4 R 5 of R 1 in the above formula (I) are preferably selected independently of each other from -H (hydrogen atom) or -Me (methyl). Examples of R 1 are amino, methylamino and dimethylamino.
  • N-containing heteroaryl group refers to any monocyclic or bicyclic aromatic hydrocarbon group in which at least one carbon atom is replaced by a nitrogen atom und optionally one or more carbon atom(s) is/are replaced by a heteroatom such as e.g. nitrogen, oxygen, sulphur. In case two or more carbon atoms are replaced by a heteroatom, the heteroatoms are the same or different.
  • N- containing heteroaryl groups of the present invention are indazole, indole, indoline, benzimidazole, benzoindazole, imidazole, pyrazole, pyrazol, pyridin, pyrimidin, chinolin, isochinolin, furan, thiophen, oxazol, isoxazol, thiazol or thiazin, preferably indazole, indole, indoline, benzimidazole, benzoindazole imidazole or pyrazole, more preferably indazole or benzoindazole.
  • the position of attachment at the N- containing heteroaryl group to -(CH2)m-, or to the nitrogen in case m is 0 of the general formula (I) is not particularly limited.
  • the N-containing heteroaryl group of substituent R 1 is (poly)substituted, i.e. one or more hydrogen atom(s) of the N-containing heteroaryl group are replaced by a substituent selected independently of each other.
  • substituents are preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, amino (-NH2), halogen, cyano (-CN), hydroxyl (-OH), nitro (-NO2), a linear or branched alkoxy group having preferably 1 to 4 carbon atoms, and derivatives thereof, trihalogenmethoxy or trihalogenmethyl.
  • the position and number of the substituent(s) in the heteroaryl group is not particularly limited.
  • substituent R 1 is a group of formula (II).
  • a group of formula (II) of substituent R 1 in the above formula (I) is a substituted pyridinyl derivative (X is nitrogen) or a substituted phenyl derivative (X is CH).
  • CH is a methine bridge.
  • One hydrogen atom of the pyridinyl derivative and phenyl derivative of the group of formula (II) of substituent R 1 is replaced by substituent R 6 .
  • the position of substituent R 6 in the group of formula (II) is not particularly limited, preferably in meta (lib) or para (Ha) position and more preferably in para position (Ha).
  • -OH is a hydroxyl group
  • -CH 2 OH is a hydroxymethyl group
  • -OMe is a methoxy group
  • -NHC(0)NH 2 is a ureido group
  • -C(0)0H is a carboxyl group
  • -C(0)NH 2 is a carboxamidyl group
  • -CH 2 C(0)NH 2 is a carboxamidylmethyl group
  • -C(0)NHNH 2 is a hydrazinecarbonyl group
  • -C(0)NHOH is a hydroxycarbamoyl group
  • -B(OH) 2 is a borono group.
  • substituents -NR 7 R 8 , -CH 2 NR 7 R 8 of substituent R 6 in formula (II) refer to amino derivates and methylamino derivatives, respectively.
  • substituents R 7 and R 8 are preferably selected each independently of each other from -H an -Me.
  • Preferred embodiments of substituents -NR 7 R 8 and -CH 2 NR 7 R 8 of R 6 are amino, methylamino or dimethylamino, and aminomethyl, methylaminomethyl or dimethylaminomethyl.
  • one or more hydrogen atom(s) of the pyridinyl derivative or phenyl derivative of the group of formula (II) are replaced by one or more substituent selected independently of each other.
  • substituents are preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, halogen, cyano (-CN), hydroxyl (-OH), linear or branched alkoxy group having preferably 1 to 4 carbon atoms, and derivatives thereof, trihalogenmethoxy or trihalogenmethyl, more preferably methyl and chlorine.
  • the position and the number of these substituent(s) in the pyridinyl derivative and phenyl derivative of the group of formula (II) are not particularly limited.
  • R 1 is a group of formula (III).
  • a group of formula (III) of substituent R 1 in the above formula (I) is a substituted pyridinyl derivative.
  • substituent R 6 In the pyridinyl derivative of the group of formula (III) of substituent R 1 one hydrogen atom is replaced by substituent R 6 .
  • the position of substituent R 6 in the group of formula (III) is not particularly limited and can be selected from the group consisting of ortho position (Ilia), meta position (Iflb) and para position (lllc), preferably in ortho position (Ilia) to the nitrogen atom of the pyridinyl group.
  • -OH is a hydroxyl group
  • -CH 2 OH is a hydroxymethyl group
  • -OMe is a methoxy group
  • -NHC(0)NH 2 is a ureido group
  • C(0)OH is a carboxyl group
  • -C(0)NH 2 is a carboxamidyl group
  • -CH 2 C(0)NH 2 is a carboxamidylmethyl group
  • -C(0)NHNH 2 is a hydrazinecarbonyl group
  • -C(0)NHOH is a hydroxycarbamoyl group
  • -B(OH) 2 is a borono group.
  • substituents -NR 7 R 8 , -CH 2 NR 7 R 8 of substituent R 6 in formula (III) refer to amino derivates and methylamino derivatives, respectively.
  • substituents R 7 and R 8 are preferably selected each independently of each other from -H an -Me.
  • Preferred embodiments of substituents -NR 7 R 8 and -CH 2 NR 7 R 8 of R 6 are amino, methylamino or dimethylamino, and aminomethyl, methylaminomethyl or dimethylaminomethyl.
  • one or more hydrogen atom(s) of the pyridinyl derivative of the group of formula (III) are replaced by one or more substituent selected independently of each other.
  • substituents are preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, halogen, cyano (-CN), hydroxyl (-OH), a linear or branched alkoxy group having preferably 1 to 4 carbon atoms, and derivatives thereof, trihalogenmethoxy or trihalogenmethyl, more preferably methyl and chlorine.
  • the position and the number of these substituent(s) in the pyridinyl derivative and phenyl derivative of the group of formula (III) are not particularly limited.
  • combinations of -(CH2)mR 1 in formula (I) are preferably selected from the group consisting of:
  • the phenoxy groups of the (4-phenoxy)-phenylglycine (index I is 0) moiety or the benzyloxy substituent of the (4-benzyloxy)-phenylglycine moiety (index I is 1) in formula (I) are (poly)substituted by replacement of one or more hydrogen atoms by one or more substituents R 2 selected independently of each other.
  • Index “n” defines the number of substituents R 2 being 0, 1 , 2, 3, 4 or 5, preferably 0, 1 or 2.
  • the index ⁇ ” in the above formula (I) is selected from 0 or 1, preferably 1.
  • Substitution by one or more substituents R 2 is not limited to a certain substitution pattern.
  • Substitution patterns are preferably ortho, para, meta, ortho-ortho, ortho- meta, ortho-para, meta-para and meta-meta with reference to the -0(CH2)I- group in formula (I) or any combination thereof.
  • Substituents R 2 can preferably be selected independently of each other from trifluoromethyl (-CF3), -CN (cyano), a linear or branched alkyl group having preferably 1 to 4 carbon atoms, like tert-butyl, a linear or branched alkyloxy group having preferably 1 to 4 carbon atoms, like methoxy (-OMe), and an halogen atom, preferably trifluoromethyl and a halogen atom.
  • a halogen atom is a group 17 element of the periodic table, preferably chlorine.
  • Substitution patterns of the benzyloxy residue of the (4-benzyloxy)-phenylglycine moiety are preferably selected from the group consisting of:
  • Configuration of the stereogenic center of the 4-phenoxyphenylglycone derivative or (4-benzyloxy)-phenylglycinederivative does not have to be defined, but may be in D-configuration or L-configuration, preferably in L-configuration.
  • Substituent R 3 in formula (I) can be selected from an alkyl group, a heteroaryl group, a substituted or unsubstituted naphthyl group, a substituted or unsubstituted 2- phenylvinyl group, a substituted or unsubstituted 2-phenylethyl group, a substituted or unsubstituted phenyl group, or a substituted or unsubstituted benzyl group, preferably from an alkyl group, a substituted or unsubstituted phenyl group or a substituted or unsubstituted benzyl group, more preferably from a substituted or unsubstituted phenyl group or a substituted or unsubstituted benzyl group and most preferably a substituted or unsubstituted phenyl group.
  • the alkyl group of R 3 in formula (I) refers to a hydrocarbon group which is linear, cyclic or branched, preferably linear or cyclic and more preferably linear.
  • the chain length is not particularly limited but may be preferably 1 to 15 carbon atoms, more preferably 1 to 9 carbon atoms and most preferably 4 to 9 carbon atoms.
  • the linear, cyclic or branched alkyl group is preferably (poly)substituted, i.e. one or more hydrogen atom(s) of the alkyl group is replaced by one or more substituents selected independently of each other.
  • Such substituents are preferably halogen, aryl, heteroaryl, hydroxyl, alkoxy, aryloxy, carboxylic acids and derivatives thereof, acyl, amino, N-acetamido, hydroxy, alkylamino, cyano, trihalogenmethoxy, trihalogenmethyl or oxo and more preferably N-acetamido, hydroxy and oxo.
  • the substitution pattern and the number of substituents are not particularly limited.
  • Preferred alkyl groups of R 3 in formula (I) are methyl, butyl, pentyl, hexyl, heptyl, octly, nonyl, cyclohexylmethyl, cyclobutyl, L-1 -(N-acetoamido)pent-l -yl, L-1-(N- acetoamido)but-1 -yl, butanoyl and 3-methylbutanoyl or 1-(5-(2,5-dimethylphenoxy)- 2-methylpentan-2-yl) and more preferably butyl, pentyl, hexyl, heptyl, octly, nonyl, cyclohexylmethyl, 3-methylbutanoyl 1 -(5-(2,5-dimethylphenoxy)-2-methylpentan-2- yl) and most preferably butyl, pentyl, hexyl, heptyl, oct
  • two or more alkyl groups having preferably 1 to 4 carbon atoms or linear or branched alkoxy groups having preferably 1 to 4 carbon atoms of the substituted phenyl group of substituent R 3 or the substituted benzyl group of substituent R 3 might be bonded to each other to form one or more rings.
  • heteroaryl group of R 3 in formula (I) is thiopen-2-yl
  • one or more hydrogen atoms of the substituted naphthyl group is/are preferably replaced by additional substituents selected independently of each other.
  • the substitution pattern and number of additional substituents are not particularly limited.
  • Such additional substituents are preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, halogen, cyano (-CN), hydroxyl (-OH), a linear or branched alkoxy group having preferably 1 to 4 carbon atoms, amino, dialkylamino, trihalogenmethoxy or trihalogenmethyl.
  • one or more hydrogen atoms of the substituted 2- phenylvinyl group is/are preferably replaced by additional substituents selected independently of each other.
  • the substitution pattern and number of additional substituents are not particularly limited.
  • Such additional substituents are preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, halogen, cyano (-CN), hydroxyl (-OH), linear or branched alkoxy group having preferably 1 to 4 carbon atoms, amino, dialkylamino, trihalogenmethoxy or trihalogenmethyl.
  • one or more hydrogen atoms of the substituted 2- phenylethyl group is/are preferably replaced by additional substituents selected independently of each other.
  • the substitution pattern and number of additional substituents are not particularly limited.
  • Such additional substituents are preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, halogen, cyano (-CN), hydroxyl (-OH), linear or branched alkoxy group having preferably 1 to 4 carbon atoms, amino, dialkylamino, trihalogenmethoxy or trihalogenmethyl.
  • substituted phenyl group of R 3 in formula (I) may be a group of formula (IVa), (IVb) or (IVc), more preferably formula (IVa) or (IVb) and most preferably (IVa).
  • the phenyl groups of formulas (IV) contain at least one substituent selected from R 9 , R 10 and R 11 or combinations thereof. Additionally, one or more hydrogen atoms of the phenyl group of formulas (IV) are preferably further replaced by additional substituents selected independently of each other. The substitution pattern and number of additional substituents are not particularly limited. Such additional substituents are preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, halogen, cyano (-CN), hydroxyl (-OH), linear or branched alkoxy group having preferably 1 to 4 carbon atoms, amino, dialkylamino, trihalogenmethoxy or trihalogenmethyl.
  • substituents R 9 , R 10 and R 11 can be selected from a linear or branched alkyl group having preferably 1 to 4 carbon atoms, a linear or branched alkoxy group having preferably 1 to 6 carbon atoms, aminomethyl group, morpholino, 4-morpholincarbonyl, a substituted or unsubstituted furan-2-yl group, a substituted or unsubstituted pyridiny-5-yl, a substituted or unsubstituted phenyl group, a substituted or unsubstituted benzoyl group, a substituted or unsubstituted phenoxy group or a substituted or unsubstituted benzyloxy group or combinations thereof.
  • substitution pattern and the number of substituents of the substituted furan-2-yl group, the substituted pyridiny-5-yl, the substituted phenyl group, the substituted benzoyl group, the substituted phenoxy group and the substituted benzyloxy group are not particularly limited.
  • such substituents of the substituted furan-2-yl group, the substituted pyridiny-5-yl, the substituted phenyl group, the substituted benzoyl group, the substituted phenoxy group and the substituted benzyloxy group of substituents R 9 , R 10 and R 11 is preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, halogen, cyano (-CN), hydroxyl (-OH), a linear or branched alkoxy group having preferably 1 to 4 carbon atoms, amino, dialkylamino, trihalogenmethoxy or trihalogenmethyl, preferably -OMe and a chlorine atom.
  • Two or more alkyl groups having preferably 1 to 4 carbon atoms or linear or branched alkoxy groups having preferably 1 to 4 carbon atoms of the substituted phenyl group, the substituted benzoyl group, the substituted phenoxy group or the substituted benzyloxy group of substituents R 9 , R 10 and R 11 might be bonded to each other to form one or more rings.
  • Substituent R 9 is preferably a linear or branched alkoxy group having preferably 1 to 6 carbon atoms, a substituted or unsubstituted phenyl group, a substituted or unsubstituted benzoyl group, a substituted or unsubstituted phenoxy group or a substituted or unsubstituted benzyloxy group, more preferably a substituted or unsubstituted phenyl group, a substituted or unsubstituted benzoyl group, a substituted or unsubstituted phenoxy group or a substituted or unsubstituted benzyloxy group and more preferably a substituted or unsubstituted benzoyl group or a substituted or unsubstituted phenoxy group.
  • R 10 is preferably 4-morpholinecarbonyl, a substituted or unsubstituted phenyl group, substituted or unsubstituted phenoxy group or a substituted or unsubstituted benzyloxy group, more preferably a substituted or unsubstituted phenyl group, substituted or unsubstituted phenoxy group or a substituted or unsubstituted benzyloxy group and most preferably a substituted or unsubstituted phenoxy group.
  • R 11 is preferably a substituted or unsubstituted phenyl group, more preferably an unsubstituted phenyl group.
  • the substituted benzyl group of R 3 in formula (I) is a substituted benzyl group of formula (V)
  • Substituent R 12 in formula (V) is preferably a substituted or unsubstituted phenyl.
  • R 12 is a substituted phenyl 1
  • 2, 3, 4 or 5 hydrogen atoms of the phenyl group of R 12 are replaced by 1 , 2, 3, 4 or 5 substituents selected independently of each other.
  • the substitution pattern and number of substituents is not particularly limited.
  • Such substituents are preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, halogen, cyano (-CN), hydroxyl (-OH), a linear or branched alkoxy group having preferably 1 to 4 carbon atoms, amino, dialkylamino, trihalogenmethoxy or trihalogenmethyl.
  • one or more hydrogen atoms of the benzyl group of formula (V) are further replaced by substituents selected independently of each other.
  • substituents are preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, halogen, cyano (-CN), hydroxyl (-OH), a linear or branched alkoxy group having preferably 1 to 4 carbon atoms, amino, dialkylamino, trihalogenmethoxy or trihalogenmethyl.
  • substituted phenyl groups and substituted benzyl groups in formulas (IV) and (V) of R 3 are preferably selected from the group consisting of: Preferred embodiments of the compound of formula (I), according to the present
  • the protease inhibitor according to the present invention is a compound according to formula (I) as described above or a pharmaceutically acceptable salt or ester thereof.
  • the protease inhibitor of the present invention is a pharmaceutically acceptable salt of the compound of formula (I)
  • the salt can be formed with inorganic or organic acids or bases.
  • Preferred embodiments of pharmaceutically acceptable salts comprise, without limitation, non-toxic inorganic or organic salts such as acetate derived from acetic acid, aconitate derived from aconitic acid, ascorbate derived from ascorbic acid, benzoate derived from benzoic acid, cinnamate derived from cinnamic acid, citrate derived from citric acid, embonate derived from embonic acid, enantate derived from heptanoic acid, ethansulfonat derived from ethansulfonic acid ethylenediaminetetraacetate derived from ethylenediaminetetraacetic acid, formiate derived from formic acid, fumarate derived from fumaric acid, gluconat derived from gluconic acid, glutamate derived from glutamic acid, glycolate derived from glycolic acid, chloride derived from hydrochloric acid, bromide derived from hydrobromic acid, lactate derived from lactic acid,
  • salts which are not considered as pharmaceutically acceptable, like oxalate derived from oxalic acid, can be appropriate as intermediates for the production of the protease inhibitor of formula (I) or a pharmaceutically acceptable salt thereof or physiologically functional derivative or a stereoisomer thereof.
  • the ester is a boronic ester and can be formed by the borono group as substituent R 6 and alcohols, preferably diols or polyalcohols, or hydroxycarboxylic acids, preferably alpha-hydroxycarboxylic acids or beta-hydroxycarboxylic acids.
  • the pharmaceutically acceptable ester comprises, without limitation, boronic esters formed by the borono group as substituent R 6 and non-toxic alcohols such as ethylene glycol, propylene glycol, glycerol, butane-2, 3-diol, pinacol, pinanediol, tris(hydroxymethyl)aminomethane, sorbitol, mannitol, other sugar alcohols, sugars such as glucose, mannose, fructose, ribose, arabinose, lyxose, sorbose, xylose and other sugars.
  • non-toxic alcohols such as ethylene glycol, propylene glycol, glycerol, butane-2, 3-diol, pinacol, pinanediol, tris(hydroxymethyl)aminomethane, sorbitol, mannitol, other sugar alcohols, sugars such as glucose, mannose, fructose, ribose, arabinose,
  • the pharmaceutically acceptable ester comprises, without limitation, boronic esters formed by the boro no group as substituent R 6 and hydroxycarboxylic acids, preferably alpha-hydroxycarboxylic acids, such as glycolic acid, malic acid, hexahydromandelic acid, citric acid, isocitric acid, 2-hyd roxyisobutyric acid, mandelic acid, lactic acid, 2-hydroxy-3,3- dimethylbutyric acid, 2-hyd roxy-3-methylbutyric acid, 2-hyd roxyisocaproic acid, tartaric acid, ascorbic acid, gluconic acid, tatronic acid, benzilic acid and others, or beta-hydroxycarboxylic acids, such as malic acid, citric acid, isocitric acid, 3- hydroxybutyric acid, beta-hydroxyisovaleric acid, tartaric acid, ascorbic acid, gluconic acid, mevalonic acid, glucoheptonic acid, maltonic
  • the present invention relates to the compounds of the present invention for use in medicine.
  • the medicine is used as an inhibitor of the flaviviral NS2B-NS3 serine protease.
  • the present invention relates to the use of the compounds of the present invention as a medicament.
  • the present invention relates to the compounds of the present invention for use in the treatment and/or prevention of a condition or disease that is preferably associated with a flavi virus infection.
  • the present invention relates to the compounds of the present invention for use in treating a flavivirus infection.
  • a flavivirus is a virus preferably selected from the group consisting of West Nile virus, Dengue virus, tick- borne encephalitis virus, yellow fever virus and Zika virus, preferably West Nile virus, Zika virus, Dengue virus, Hepacivirus C, Omsk hemorrhagic fever virus, Usuto virus, St.-Louis-Enzephalitis virus, Powassan virus, Louping-lll virus Kyasanur forest disease virus, Gadgets Gully virus, Kedougou virus and Spondweni virus.
  • the invention provides a method of treatment and/or prevention a disease or condition associated with a flavivirus infection. The method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt or ester thereof, thereby treating or preventing the disease or condition.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising one or more of the compounds of the present invention for use in medicine, treatment or prevention of a condition of disease or infection as defined above.
  • the compound or a pharmaceutically acceptable salt or ester thereof according to the present invention is administered to animals, preferably to mammals preferably selected from the group consisting of cat, dog, horse, cattle, pig and humans, and most preferably to humans.
  • the pharmaceutical composition can be administered as therapeutics per se, as mixtures with one another or in the form of pharmaceutical preparations which allow enteral or parenteral use, and which as active constituent contains an effective dose of at least one compound of the present invention as defined above or a pharmaceutically acceptable salt or ester thereof, in addition to customary pharmaceutically innocuous excipients and additives.
  • the compound of the present invention as defined above can also be administered in form of its salts or esters, which are obtainable by reacting the respective compounds with physiologically acceptable acids or alcohols or hydroxycarboxylic acids as defined above.
  • the compound of the present invention as defined above for use in therapy may be administered in the form of the raw chemical compound, it is preferred to introduce the active ingredient, optionally in the form of a physiologically acceptable salt or ester in a pharmaceutical composition together with one or more adjuvants, excipients, carriers, buffers, diluents, and/or other customary pharmaceutical auxiliaries.
  • a physiologically acceptable salt or ester in a pharmaceutical composition together with one or more adjuvants, excipients, carriers, buffers, diluents, and/or other customary pharmaceutical auxiliaries.
  • Such salts or esters of the protease inhibitor as defined above may be anhydrous or solvated.
  • the invention provides medicaments comprising the compound of the present invention as defined above, or a pharmaceutically acceptable salt or ester or physiologically functional derivative thereof, preferably together with one or more pharmaceutically acceptable carriers thereof, and, more preferably other therapeutic and/or prophylactic ingredients.
  • the carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not harmful to the recipient thereof.
  • the compound according to the present invention is administered oral, rectal, bronchial, nasal, topical, buccal, sub-lingual, transdermal, vaginal or parenteral (including cutaneous, subcutaneous, intramuscular, intraperitoneal, intravenous, intraarterial, intracerebral, intraocular injection or infusion), or inhalation or insufflation, including powders and liquid aerosol administration, or by sustained release systems.
  • sustained release systems include semipermeable matrices of solid hydrophobic polymers containing the compound of the invention, which matrices may be in form of shaped articles, e.g. films or microcapsules.
  • the compound of the present invention as defined above, together with a conventional adjuvant, carrier, or diluent, may thus be placed into the form of medicament and unit dosages thereof.
  • Such forms include solids, and in particular tablets, filled capsules, powder and pellet forms, and liquids, in particular aqueous or non-aqueous solutions, suspensions, emulsions, elixirs, and capsules filled with the same, all for oral use, suppositories for rectal administration, and sterile injectable solutions for parenteral use.
  • Such medicament and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms preferably contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.
  • the compound of the present invention as defined above can be administered in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, as the active component, either the compound of the present invention or a pharmaceutically acceptable salt or ester as defined above.
  • pharmaceutically acceptable carriers can be either solid or liquid.
  • Solid form preparations include powders, tablets, pills, capsules, cachets, lozenges, suppositories, and dispersible granules.
  • a solid carrier can be one or more substances which may also act as diluents, flavouring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
  • the carrier is a finely divided solid which is in a mixture with the finely divided active component.
  • the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.
  • Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
  • the term “preparation” is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it.
  • cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.
  • a low melting wax such as a mixture of fatty acid glyceride or cocoa butter
  • the active component is dispersed homogeneously therein, as by stirring.
  • the molten homogenous mixture is then poured into convenient sized moulds, allowed to cool, and thereby to solidify.
  • Liquid preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions.
  • parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.
  • the compound of the present invention as defined above may thus be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative.
  • the compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.
  • Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents, as desired.
  • Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well known suspending agents.
  • solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration.
  • liquid forms include solutions, suspensions, and emulsions.
  • These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
  • the medicament may be applied topically or systemically or via a combination of the two routes.
  • the medicament is applied topically. This reduces possible side effects and limits the necessary treatment to those areas affected.
  • the medicament is prepared in form of an ointment, a gel, a plaster, an emulsion, a lotion, a foam, a cream of a mixed phase or amphiphilic emulsion system (oil/water-water/oil mixed phase), a liposome, a transfersome, a paste or a powder.
  • an ointment a gel, a plaster, an emulsion, a lotion, a foam, a cream of a mixed phase or amphiphilic emulsion system (oil/water-water/oil mixed phase), a liposome, a transfersome, a paste or a powder.
  • Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
  • Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
  • compositions suitable for topical administration in the mouth include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerine or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray.
  • the compositions may be provided in single or multi-dose form. In the latter case of a dropper or pipette, this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray, this may be achieved for example by means of a metering atomising spray pump.
  • Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the active ingredient is provided in a pressurised pack with a suitable propellant such as a chlorofluorocarbon (CFG) for example dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
  • a suitable propellant such as a chlorofluorocarbon (CFG) for example dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
  • the aerosol may conveniently also contain a surfactant such as lecithin.
  • the dose of drug may be controlled by provision of a metered valve.
  • the active ingredients may be provided in the form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP).
  • a powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP).
  • PVP polyvinylpyrrolidone
  • the powder carrier will form a gel in the nasal cavity.
  • the powder composition may be presented in unit dose form, for example in capsules or cartridges of e.g. gelatin, or blister packs from which the powder may be administered by means of an inhaler.
  • the compound In compositions intended for administration to the respiratory tract, including intranasal compositions, the compound will generally have a small particle size for example of the order of 5 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization.
  • compositions adapted to give sustained release of the active ingredient may be employed.
  • the pharmaceutical preparations are preferably in unit dosage forms.
  • the preparation is subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packaged tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. Tablets or capsules for oral administration and liquids for intravenous administration and continuous infusion are preferred compositions.
  • compositions can also contain two or more compounds of the present invention as defined above or their pharmacologically acceptable salts or esters and also other therapeutically active substances.
  • the compound of the present invention as defined above can be used in the form of one compound alone or in combination with other active compounds - for example with medicaments already known for the treatment of the aforementioned diseases, whereby in the latter case a favorable additive, amplifying effect is noticed.
  • Suitable amounts to be administered to humans range from 5 to 1 ,250 mg per day.
  • pharmaceutically inert inorganic or organic excipients can be used.
  • pills tablets, coated tablets and hard gelatin capsules, for example, lactose, corn starch or derivatives thereof, talc, stearic acid or its salts, etc.
  • Excipients for soft gelatin capsules and suppositories are, for example, fats, waxes, semi-solid and liquid polyols, natural or hardened oils etc.
  • Suitable excipients for the production of solutions and syrups are, for example, water, sucrose, invert sugar, glucose, polyols etc.
  • Suitable excipients for the production of injection solutions are, for example, water, alcohols, glycerol, polyols or vegetable oils.
  • the dose can vary within wide limits and is to be suited to the individual conditions in each individual case.
  • the appropriate dosage will vary depending on the mode of administration, the particular condition to be treated and the effect desired.
  • the present invention relates to methods of treating or of preventing any of the conditions and/or diseases as defined above, comprising a step of administering one or more of the compounds or a pharmaceutical composition as defined above, to a subject in need thereof, preferably a mammalian subject and more preferably a human subject.
  • the present invention relates to a pharmaceutical composition as defined above, which is a prophylactic agent against a flavivirus infection or an agent for treating a flavivirus infection.
  • the present invention relates to a compound of the present invention for use of inhibition of infectivity of a flavivirus in vitro. Furthermore, the present invention relates to the use of the compounds of the present invention for inhibition of the NS2B-NS3 serine protease of a flavivirus in vitro.
  • the present invention relates to the compounds of the present invention for use in medicine.
  • these compounds could serve as reference inhibitors in both biochemical and cellular DENV-2 protease assay systems.
  • Viral replication assays are usually performed under variable conditions by different laboratories, impeding the comparison of results, antiviral activity, and assay conditions.
  • reference compounds with good synthetic accessibility provide an opportunity to calibrate results from various laboratories and assay set-ups against a common standard.
  • Figure 1 shows the inhibitory activity of selected compounds at a concentration of 12.5 mM in the DENV-2 virus titer reduction assay.
  • Figure 2 shows the inhibitory activity of selected compounds at a concentration of 12.5 mM in the DENV-2 virus titer reduction assay.
  • Figure 3 shows the inhibitory activity of selected compounds at a concentration of 1 and 12.5 mM in the DENV-2 virus titer reduction assay.
  • Figure 4 shows concentration dependent DENV-2 virus titer reduction and ECso values of compounds NK-189, L-02 and NK-288.
  • Figure 6 shows the concentration-dependent quenching of the fluorescence of the tryptophan residues in the substrate binding region after addition of compounds NK- 119 (A), NK-406 (B) and NK-305 (C) to DENV-2 protease. The fluorescence is partially restored when aprotinin is added.
  • NATA AZ-Acetyl-L-tryptophanamide.
  • C-terminal moieties or caps and N-terminal moieties or caps refer to the residues attached to the carboxylic acid and amino group, respectively, of the (4-phenoxy)-phenylglycine or (4-benzyloxy)-phenylglycine structure of compounds according to formula (I).
  • the present assay - the creation of compound libraries by building block assembly and derivatization - provides a fast track to diverse compounds, which are not easily accessible otherwise, considering for example the use of the racemization-prone phenylglycine in synthetic procedures. While this approach is inspired from amino acids assembly for peptides, it can be effectively applied on other chemical building blocks for lead optimization.
  • exemplary compounds were analyzed, and enantiomeric purity was above 95% in all cases. Additionally, compound NK-305 was tested for its racemization tendency in assay buffers used in this study. Racemization was negligible after incubation for 96 hours at 37°C in phosphate-buffered saline (pH 7.4), biochemical DENV assay buffer (pH 9.0) and DENV2proHel_a media (pH 7.0). A highly basic sodium hydroxide solution (pH 13) and an incubation of 96 hours was required for full racemization of the tested compound.
  • NK-254 and NK- 269 can form similar hydrogen bonds with the protease and were designed as non- basic guanidine replacements.
  • the amidine-containing compound NK- 191 had weak activity.
  • Meta-substituted derivatives had lower potency compared to their para-substituted derivatives (e.g. NK-204, NK-217).
  • a methylene group between the amide nitrogen and the phenyl group was needed for optimal activity (see compounds NK-209 and NK-189).
  • Most compounds have similar activities in the biochemical and the cellular protease assay but compounds NK-201, NK-129 and NK-209 could not be measured due to cytotoxicity at the screening concentration.
  • Table 1 Inhibitory activity of compounds with different -G(0)NH(CH2)mR 1 groups and a -C(0)piperidinyl group of residue L 1 against isolated DENV and WNV proteases, the off-targets thrombin and trypsin, in the cellular DENV protease reporter assay (DENV2proHel_a) and cytotoxicity in HeLa cells.
  • NK-254, NK- 269 and NK-270 are able to form similar H-bonds compared to a guanidine moiety, but just minor inhibition was detected.
  • Cycloalkyl derivatives NK-223 and NK-169 had low activity, except piperidine compound NK-155 with an ECso of around 13 pM.
  • the phenylboronic acid derivatives showed one digit micromolar activities in the cellular DENV2proHel_a assay (table 2). Surprisingly the phenylboronic acid derivatives are promising replacements for the strongly basic guanidine motif at this position.
  • 3-Aminophenylboronic acid and 4-aminomethylphenylboronic acid have high pKa values of 8.9 and 8.3 and are therefore considered as slightly acidic.
  • the electron-withdrawing halogen substituent containing compounds in L- and D-configuration had a 2- to 5-fold higher inhibitory potency in biochemical assays (NK-285, NK-360, NK-344, NK-287).
  • substitutions at the benzyloxy residue of the (4-benzyloxy)-phenylglycine moiety were not well tolerated in the DENV2proHel_a assay.
  • Other substituents like 3- methoxy, 4-cyano or 4-terf-butyl decreased the activity of the compounds in all assays.
  • NK-316 showed a big deviation between the different protease assays:
  • the analog with a 2-(biphenyl-4-yl)acetic acid cap had the highest in vitro activity but reached only an ECso of 20 mM in the DENV2proHeLa system.
  • Some of the caps resulted in cytotoxicity at 50 mM (NK-333, NK-288, NK-307, NK-346, NK-347), but at the DENV2proHel_a screening concentration (12.5 pM), no cytotoxicity was observable.
  • Table 4 The analog with a 2-(biphenyl-4-yl)acetic acid cap had the highest in vitro activity but reached only an ECso of 20 mM in the DENV2proHeLa system.
  • Some of the caps resulted in cytotoxicity at 50 mM (NK-333, NK-288, NK-307, NK-346, NK-347), but at the DENV2proHel_a screening
  • NK-411 contains a (4-phenoxy)-phenylglycine moiety which decreased the activity compared to the (4-benzyloxy)-phenylglycine derivatives.
  • the phenylpiperidine containing compound NK-431 showed lower inhibition, which could be explained with the basic nature of the cap.
  • Table 8 Inhibitory activity of compounds with different R 3 C(0)NH- groups of residue L 2 and different (4-benzyloxy)-phenyiglycine and (4-phenoxy)-phenylglycine moieties against isolated DENV and WNV proteases, the off-targets thrombin and trypsin, in the cellular DENV protease reporter assay (DENV2proHeLa) and cytotoxicity in HeLa cells.
  • the passive membrane permeability of compound NK-189, NK-344, NK-316, NK- 323, NK-365 and a set of drug references was evaluated using the precoated tri layer parallel artificial membrane permeability assay (PAMPA).
  • the permeability of the reference compounds i.e. Caffeine, Carbamazepine and Phenytoin, was in accordance with literature values, but compounds NK-344, NK-316, NK-323 and NK-365 appeared to be non-permeable (see table 11).
  • NK-365 667 not permeable at detectable range
  • Further evaluation of the activity of selected compounds against viral replication in cell culture was performed in a virus titer reduction assay using DENV-2 infected human carcinoma cells (Huh-7). The inhibitors did not show relevant cytotoxicity in Huh-7 cells except compound NK-220. Under its treatment around 60% of the Huh- 7 cells survived.
  • the tested compounds reduced the virus titer with ECso values in the low micromolar or high nanomolar range (see Figures 1 to 5).
  • the pyridin-2-yl guanidine derivative NK-361 and the 4-benzyloxybenzoic acid derivatives NK-307 and NK-347 showed moderate antiviral activity, treatment with, for example, compounds NK-189, NK-200, NK-359, NIK-288 and NK-357 at a concentration of 10 mM reduced viral titers to less than 25% of the control.
  • Antiviral dose-response-curves were obtained for the following compounds that showed the most promising activity profiles in the DENV2proHel_a assay: NK-189, ML-02, and NK-288, NK-375, NK-479, NK-428, NK-433, NK-469, NK-429 and NK-478 (see Figures 4 and 5).
  • compound NK-428 had the highest antiviral activity with an ECso of 0.24 mM and an EC90 of 4.83 mM.
  • COMU [(1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino- carbenium-hexafluorophosphate]
  • HATU 0-(7-Azabenzotriazol-1-yl)-A/,A ,iV,A/'-tetramethyluroniumhexafluoro- phosphate
  • Procedure A Sodium bicarbonate (2 equiv) and the amine were dissolved in water. If the amine was not soluble, MeCN was added dropwise until all solids were dissolved. The solution was cooled with an ice-water bath and a solution of Fmoc- OSu (1.2 equiv) in MeCN was added drop wise. After 20 minutes this mixture was allowed to warm to room temperature and was stirred overnight. The solvent was then removed in vacuo and the resulting suspension was extracted 3 times with ethyl acetate. The organic layer was back extracted twice with saturated sodium bicarbonate solution. The combined aqueous layers were acidified to a pH of 1-3 with a 1 N HCI solution.
  • the resulting suspension was extracted 3 times with ethyl acetate and the combined organic layers were washed with 1 N HCI and water. The organic layer was dried over anhydrous MgSQi and the solvent was removed in vacuo. The protected amine was purified by flash chromatography if necessary.
  • Procedure B Sodium bicarbonate (2 equiv) and the amine were dissolved in HhO/MeCN (1 :1 v/v). The solution was cooled with an ice-water bath and a solution of Fmoc-OSu (1.0 equiv) in MeCN was added drop wise. After 20 minutes this mixture was allowed to warm to room temperature and was stirred overnight. The solvent was then removed in vacuo and the resulting suspension was acidified to a pH of 1-3 with a 1 N HCI solution. This mixture was extracted 3 times with ethyl acetate or DCM and the combined organic layer was washed 2 times with 1 N HCI and water. The organic layer was dried with anhydrous MgSCM and the solvent was removed in vacuo. The protected amine was purified by flash chromatography if necessary.
  • the organic layer was dried over anhydrous MgSC>4, filtered and concentrated in vacuo.
  • the methylester of the carboxylic acid was cleaved by solving the compound in a solution of LiOH (2-3 equiv) in THF/H2O (2:1 , 10 ml_). The solution was stirred for 3 h. The mixture was adjusted to pH 2 with concentrated HCI and a precipitate was formed. The precipitate was filtered, washed with several portions of H2O and dried under reduced pressure. The crude mixture was purified by flash chromatography if necessary or used for subsequent synthetic steps without further purification.
  • Procedure B The respective amine (1.0 equiv), /V,/V'-di-Boc-1H-pyrazole-1- carboxamidine (1.5 equiv), DMAP (0.3 equiv), and DIPEA (1.0 equiv) were dissolved or suspended in MeOH (5-20 mL). The mixture was allowed to stir for 2 or 3 days at room temperature. The solvent was removed in vacuo and the resulting residue was dissolved in ethyl acetate or DCM. The organic phase was washed with 0.1 N HCI, 1 N NaOH and water. The combined organic phases were dried over anhydrous MgSCM and concentrated under reduced pressure.
  • HATU pentan-1 -amine, TMP, DCM/DMF, 0°C -> rt
  • TFA DCM
  • HATU respective acid, TMP, DCM/DMF, 0°C -> rt
  • e piperidine, DCM.
  • the Rink amide resin (loading capacity 0.68 mmol/g) was pre-swollen in DCM for at least 20 min and then washed 3 * with DMF.
  • a piperidine solution (10-20% in DMF) was added 2 c for 10 and 5 min.
  • the resin was washed with 3 c DMF, 3 c DCM, and again 3 c DMF.
  • COMU/TMP were used for coupling steps.
  • the coupling solution contained the M -Fmoc-protected amino acid or N-terminal carboxylic acid capping group (3.0 equiv), COMU (3.0 equiv), and TMP (3.9 equiv) in DMF (-1.0 mt_ per 100 mg of resin). The solution was added to the resin, and the reaction was shaken for 90-180 min. Afterwards, the resin was washed as described before. Fmoc deprotection and coupling steps were iteratively repeated until the desired sequence was obtained.
  • the cleavage solution was dispensed into cold diethyl ether (35 mL per 100 mg of resin), and the resulting precipitate was centrifuged (4000 g, 10 min), washed with diethyl ether, and dried under reduced pressure. If the peptides were soluble in diethyl ether, the ether phase was washed 2 times with a small amount of water and all solvents were removed in vacuo. The resulting residue was co-evaporated with toluene several times (scheme 6).
  • NK-375 (a) piperidine, DMF; (b) COMU, TMP, DMF; (c) Fmoc-4-benzyloxy-L-Phg, COMU, IMP, DMF; (d) 4-phenoxybenzoic acid, COMU, TMP, DMF; (e) TFA, H 2 0, TIPS (95:4:1 v/v).
  • the 2-chlorotrityl chloride resin (loading capacity 1.6 mmol/g) was pre-swollen in DCM for at least 20 min and then washed 3 c with DMF.
  • the first amino acid or respective building block (1.2-3.0 equiv) was added to the resin in the syringe and 1 mL of DMF and TMP (3 equiv) were added. If a hydroxyl functional group was coupled to the solid support in the loading step, 1 mL of pyridine (per 100 mg resin) was added instead of the DMF/TMP mixture. The resin was shaken overnight and capped before the Fmoc deprotection step.
  • the resin was washed with 3 x DMF, 3 x DCM, and again 3 c DMF and a capping solution (DCM/MeOH/TMP 80:15:5 (v/v), 1 mL per 100 mg resin) was added 2 c for 20 min.
  • the resin was washed with 3 c DMF, 3 c DCM, and again 3 c DMF.
  • a piperidine solution (10-20% in DMF) was added 2 c for 10 min.
  • the resin was washed with 3 c DMF, 3 c DCM, and again 3 c DMF. COMU/TMP were used for coupling steps.
  • the coupling solution contained the L/a-Fmoc-protected amino acid or N-terminal carboxylic acid capping group (3.0 equiv), COMU (3.0 equiv), and TMP (3.9 equiv) in DMF ( ⁇ 1.0 mL per 100 mg of resin).
  • the solution was added to the resin, and the reaction was shaken for 90-180 min. Afterwards, the resin was washed as described before. Fmoc deprotection and coupling steps were iteratively repeated until the desired sequence was obtained.
  • the resin loaded with the finished peptide or peptide hybrid was washed 5 c with diethyl ether and dried under reduced pressure.
  • the peptide synthesis with 1-diol resin was performed with Rink amid resin.
  • the first building block (1.3 equiv) was added to the dry resin in the syringe and 1 mL ofTHF and TMP (1.3-2.0 equiv) were added.
  • the resin was shaken overnight and washed with 6 c DMF and 3 c DCM.
  • a piperidine solution (10-20% in DMF) was added 2 c for 10 and 5 min. Following each deprotection or coupling step, the resin was washed with 3 c DMF, 3 x DCM, and again 3 c DMF. COMU/TMP were used for coupling steps.
  • the coupling solution contained the x-Fmoc-protected amino acid or N- terminal carboxylic acid capping group (3.0 equiv), COMU (3.0 equiv), and TMP (3.9, equiv) in DMF ( ⁇ 1.0 mL per 100 mg of resin).
  • the solution was added to the resin, and the reaction was shaken for 90-180 min. Afterwards, the resin was washed as described before. Fmoc deprotection and coupling steps were iteratively repeated until the desired sequence was obtained.
  • the resin loaded with the finished peptide or peptide hybrid was washed 5 c with diethyl ether and dried under reduced pressure.
  • the final product was cleaved off the resin 2-3 times with TFA/H2O/THF solution (10:20:80 (v/v), 1-2 mL per 100 mg resin), and the mixture was shaken for at least 2 hours.
  • the cleavage solution was dispensed into a round bottom flask, washed 2 times with H2O, dried under reduced pressure and co-evaporated with toluene several times (scheme 8).
  • Method B eluent A, water (0.1% TFA); eluent B, acetonitrile (0.1% TFA); flow rate, 8 mL/min; and gradient, 10% B (2.5 min), 100% B (23.5 min), 100% B (26 min), 10% B (26.1 min), and 10% B (30 min). Detection was performed at 214, 254, and 280 nm. After purification, methanol was evaporated, and the peptides were freeze-dried in water and stored at -20 °C. Enantiomeric purity of inhibitors
  • the enantiomeric purity HPLC analysis was carried out on a Jasco HPLC system equipped with a ReproSil Chiral-NR column (8 pm, 150 * 4.6 mm) and a Jasco LJV- 2070 Plus Intelligent UV/VIS Detector.
  • the following method was used: eluent A: water (0.1% TFA); eluent B: methanol (0.1% TFA); injection volume: 20 pL; flow rate: 1 mL/min; isocratic elution with 30% A and 70% B for 25 or 35 minutes. Chromatograms recorded at 254 nm were used for assessment.
  • tryptophan quenching assays with compounds NK-305, NK-119 and NK-406 and competition with aprotinin indicated binding of the active site of the DENV-2 protease. All compounds inhibited the protease in a cellular environment at the one digit micromolar range. The addition of the compounds to DENV-2 protease results in concentration-dependent quenching of the fluorescence of tryptophan residues of the protease (see Figure 6).
  • Compound NK-305 had an ICso of approximately 6 pM against DENV and WNV in in vitro assays. The results of the fluorescence quenching experiments indicate a binding of the inhibitor in the active site of the enzyme.
  • NK-305 As shown in figure 6, the addition of compound NK-305 to DENV-2 protease results in concentration-dependent quenching of the fluorescence of tryptophan residues of the protease.
  • Aprotinin is a known competitive inhibitor for DENV protease. Aprotinin displaces compound NK-305 from the DENV-2 protease, providing further evidence for specific binding at the active site (see figure ⁇ ).
  • Compound NK-305 can be used as quencher for the protease autofluorescence, particularly in displacement assays for compounds that lack moieties capable of interacting with tryptophan fluorescence.
  • the inhibitor offers an advantage through its high potency in both biochemical and cellular assays.
  • the enzymatic activity was monitored for 15 min and determined as a slope of relative fluorescence units per second (RFU/s) for each concentration. Percentage inhibition was calculated relative to a positive control (without the inhibitor), as a mean of the triplicates and respective standard deviation.
  • Dilutions were made starting from 10 mM inhibitor stock solutions in DMSO and final concentrations were measured in triplicates
  • the inhibitors were preincubated for 15 min with the DENV protease (100 nM) or WNV protease (150 nM) in the assay buffer (50 mM Tris-HCI pH 9.0, ethylene glycol (10% v/v), and 0.0016% Brij 58).
  • the enzymatic reaction was then initiated by the addition of the FRET substrate (final concentration 50 mM) to obtain a final assay volume of 100 mI_ per well.
  • the enzymatic activity was monitored for 15 min and determined as a slope of relative fluorescence units per second (RFU/s) for each concentration.
  • the mean and the standard deviation of the triplicates plotted against the corresponding concentration were used to determine the ICso-values on Prism 6.01 (Graphpad Software, Inc.) using non-linear dose-response curves with variable slopes.
  • ICso values of the inhibitor at different substrate concentrations 50, 100, 150, and 200 mM were measured using the previous parameters.
  • K the corrected data considering the inner filter effect was used.
  • Cheng- Prusoffs method was used to calculate the Ki. Linear regressions were performed using Prism 6.01 (Graphpad Software, Inc.).
  • DENV assay flow rate: 1.0 mL/min; eluent A: water (0.1% TFA); eluent B: acetonitrile (0.1% TFA); and gradient: 10% B (0 min), 10% B (1 min), 95% B (9.5 min), 95% B (9.6 min), 10% B (12.6 min), and 10% B (15 min).
  • Different conditions were used for the WNV assay: flow rate: 1.2 mL/min; eluent A: water (0.1 % TFA); eluent B: acetonitrile (0.1% TFA); and gradient: 10% B (0 min), 20% B (1 min), 95% B (5 min), 95% B (6 min), 10% B (6.1 min), and 10% B (8 min). Percentage inhibition was calculated relative to a positive control (without the inhibitor). All experiments were performed in triplicate and the final value was obtained as the average.
  • Thrombin was purchased from Sigma-Aldrich (Germany). Continuous fluorimetric assay was done in black 96 well V-bottom plates (Greiner Bio-One, Germany), using a BMG Labtech Fluostar OPTIMA microtiter fluorescence plate reader. Excitation wavelength of 355 nm and an emission wavelength of 460 nm were used. The inhibitors (final concentration 25 mM, from 10 mM stock solutions in DMSO) were preincubated with thrombin (10 nM) in the assay buffer (50 mM Tris-HCI pH 7.5, 150 mM NaCI, 0.05% Tween 20) for 15 min.
  • Enzymatic cleavage was initiated by the addition of the Boc-Val-Pro-Arg-AMC substrate (Bachem, Germany) to yield a final concentration of 50 pM.
  • the activity of thrombin was monitored for 15 min and determined as a slope of relative fluorescence units per second (RFU/s). All experiments were performed in triplicates and percentage inhibition was calculated as the mean and respective standard deviation of the values. Values were obtained in relation to a positive control.
  • Trypsin was purchased from Sigma-Aldrich (Germany). The inhibition of trypsin was determined as described before. Continuous fluorimetric assay was done in black 96 well V-bottom plates (Greiner Bio-One, Germany), using a BMG Labtech Fluostar OPTIMA microtiter fluorescence plate reader. Excitation wavelength of 355 nm and an emission wavelength of 460 nm were used. The inhibitors (final concentration 50 pM, from 10 mM stock solutions in DMSO) were preincubated with trypsin (1 nM) in the assay buffer (50 mM Tris-HCI pH 7.5, 150 mM NaCI, 0.05% Tween 20) for 15 min.
  • the assay buffer 50 mM Tris-HCI pH 7.5, 150 mM NaCI, 0.05% Tween 20
  • Enzymatic cleavage was initiated by the addition of the Boc-Val-Pro-Arg-AMC substrate (Bachem, Germany) to yield a final concentration of the Boc-Val-Pro-Arg- AMC substrate of 50 mM.
  • the activity of thrombin was monitored for 15 min and determined as a slope of relative fluorescence units per second (RFU/s). All experiments were performed in triplicates and percentage inhibition was calculated as the mean and respective standard deviation of the values. Values were obtained in relation to a positive control (without inhibitor).
  • Cytotoxicity of the above described compounds was tested by determination of the Cell’s viability in Huh-7 or Hela cells in presence of compound dilutions, which was determined using CellTiter-Blue® (Promega) in accordance to the manufacturer’s instructions. Plates were prepared in parallel to the cell based DENV reporter or virus titer reduction assay with analogous treatment. Each concentration was assayed in triplicates. Cells were incubated with the compounds for 24 hours (HeLa cells) or 48 hours (Huh-7 cells). Afterwards the medium was removed and resazurin was added to the cells and the cells were incubated for 1 hour.
  • CCso Conversion of the dye resazurin to resorufin by metabolically active cells results in the generation of a fluorescent product.
  • CCso a serial dilution of the compound was prepared and by plotting the relative signal compared to the untreated control in a non-linear dose-response curve, a compound concentration at which 50% of the cells are viable (CCso) can be determined.
  • Huh-7 cells were seeded into 96-well plates at a density of 1 * 10 4 cells in 50 pi per well and incubated overnight. The next day, cells were infected with DENV serotype 2 for 2 h at a MOI of 1 , before medium change and compound addition. Infected cells were then incubated for 48 h in the presence of compound in triplicate wells. In order to determine the ECso, a range of serial diluted compound concentrations starting at 50 mM was used. After 48 h incubation, supernatants were harvested and triplicates pooled. These virus containing supernatants were used to determine the virus titer (reduction) by plaque assay.
  • VeroE6 cells were seeded into 24-well plates at a density of 2.5 c 10 s cells/well and the next day infected for 1 h with 10-fold serial dilutions of virus supernatant ranging from 10 1 to 10 ⁇ 6 . After medium exchange and addition of the plaque medium VeroE6 cells were incubated for 7 days. Cells were subsequently fixed with formaldehyde and plaques visualized by crystal violet stain. At a suitable dilution, plaques were counted, the virus titer calculated and plotted against the respective compound concentration. Plaques are readily visible where the cells have been lysed by viral infection. The titer of a virus stock can be calculated in plaque-forming units per milliliter.
  • the plaques are counted in an appropriate dilution (e.g. 10 5 ).
  • the resulting virus titer is calculated from the dilution factor and the number of counted plaques.
  • Virus titer reduction is determined by comparing the virus titer in compound-treated wells with the virus titer of untreated wells (in the same dilution series, e.g. 10 5 ). ECso values were derived from fitting data to a dose-response curve (variable-slope, nonlinear regression model) using Prism 6.01 (Graphpad Software, Inc.).
  • Huh-7 and Vero E6 cells were maintained in DMEM supplemented with 100 U/mL penicillin, 100 pg/mL of streptomycin, and 10% heat-inactivated FCS. During infection of Huh-7 cells, DMEM was supplemented with 10 mM HEPES.
  • DENV protease (0.2 mM) was titrated with different concentrations (0, 1.25, 2.5, 5, 10, 20, 30, 40, 50 mM) of the inhibitor in assay buffer (50 mM Tris-HCI pH 9.0 for compounds NK-119 and NK- 406; 50 mM Tris-HCI pH 9.0, ethylene glycol (10% v/v), and 0.0016% Brij® 58 for compound NK-305) and the samples were incubated for 1 h at room temperature.
  • the protease was incubated with the inhibitor (50 pM) and aprotinin (10 pM) under the same conditions.

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Abstract

The present invention relates to compounds that inhibit the activity of flavivirus.

Description

A NEW CLASS OF FLAVIVIRUS PROTEASE INHIBITORS
The present invention relates to compounds that inhibit the activity of flavivirus.
The global incidence of the mosquito-borne viral disease has grown rapidly in past years. Among these viral diseases Dengue virus, West Nile virus and Zika virus are attracting more and more attention, which are members of the same genus flavivirus. In particular, Dengue and West Nile virus are rapidly spreading global pathogens for which no specific therapeutic treatments are available. A recent analysis estimates 390 million infections per year of which about one third are clinically recognized. Dengue virus (DENV) infections can result in serious and life- threatening diseases like dengue fever, hemorrhagic fever and dengue shock syndrome. Despite an increasing impact, there are no specific therapeutic treatments available. Although a vaccine is approved in several countries, the vaccination of seronegative humans is not recommended. Similar to DENV, the mosquito-borne West Nile Virus (WNV) is a pathogen of global occurrence. Although most infections with WNV are asymptomatic, many patients develop flulike symptoms or more severe neuroinvasive diseases like encephalitis. Unfortunately, neither an effective antiviral therapy nor a vaccine is available. Hence, there is an unmet medical need for antiviral agents against infections with DENV and other flaviviruses like West Nile and Zika virus.
In view of the above, the technical problem underlying the present invention is the provision of a class of molecules for inhibition of viruses of the flavivirus genus.
The solution to the above technical problem is achieved by the embodiments characterized by the claims.
In particular, the present invention provides non-peptidic (4-phenoxy)-phenylglycine and (4-benzyloxy)-phenylglycinederivatives as inhibitor of the NS2B-NS3 serine protease which are useful for inhibition of the protease. NS2B-NS3 is a promising target in drug discovery against flaviviruses, since it is well conserved within this genus. The enzyme cleaves the viral polyprotein into separate functional proteins and is therefore essential for virus replication. Thus, the inhibitors can be used for the treatment of several flavivirus related diseases.
Most of the inhibitors developed so far contain highly charged recognition elements, frequently with a di- or polybasic motif, since the flaviviral proteases are serine proteases with a preference for substrates with dibasic motifs. The combination of basic peptide sequences with an electrophilic group such as an aldehyde, capable to form covalent interactions with the catalytic serine, results in high-affinity inhibitors. The efficacy of such inhibitors in cellular assay systems is often limited by pharmacokinetic liabilities due to the high molecular weight and pronounced polarity of the highly basic side chains and the ECso values from cell-based assays are often significantly high (micromolar range).
Compounds according to the present invention have a nanomolar activity in a cellular DENV-2 protease reporter assay, stable stereochemistry under physiological and assay conditions and most of the compounds are non-toxic up to 50 mM and have low off-target activity. These compounds contain only one basic residue and have a much smaller molecular mass than most flaviviral protease inhibitors. Thus, the compounds reach low micromolar to upper nanomolar inhibitory potency in cell-based assays as well as in a DENV-2 virus titer reduction assay, are selective against other serine proteases, e.g. Trypsin and Thrombin, and do not show relevant cytotoxicity.
The compounds of the present invention are based on a phenylglycine, (4-phenoxy)- phenylglycine or a (4-benzyloxy)-phenylglycine element, and the amino acid is coupled to lipophilic alkyl chains or ring systems via an amide bond or sulfonamide bond, and not to other peptidic elements as described before. The compounds of the present invention are much smaller than previous published DENV protease inhibitors and contain just one or no basic residue. The most active compounds contain a guanidine, a benzamide or a phenylboronic acid group. The compounds of the present invention do not have basic structural elements and therefore have a much more drug-like structure than most other inhibitors. Due to the small size and reduced amount of hydrogen-donating groups, most of the compounds presented here correspond to the Lipinski rule of five. This rule gives an indication of good pharmacokinetic properties of a drug and their oral bioavailability.
In the present invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt or ester thereof:
Figure imgf000004_0001
wherein
L1 is -C(0)NH(CH2)mR1 , -C(0)piperidyl, -NHC(0)(CH2)mR1 or -NH(CH2)mR1, wherein m is 0, 1 , 2, 3, 4, 5 or 6;
R1 is -NR4R5, -(HN)(H2N)C=NH, an N-containing heteroaryl group or a group of formula (II) or formula (III)
Figure imgf000004_0002
wherein X is nitrogen or CH,
L2 is -C(0)NH-, -S02NH- or -NH- if L1 is -C(0)NH(CH2)mR1 or -C(0)piperidyl; or L2 is -NHC(O)- if L1 is -NHC(0)(CH2)mR1 or -NH(CH2)mR1;
I is 0 or 1 ;
R6 is -NR7R8, -CH2NR7R8, -(HN)(H2N)C=NH, -CH2(HN)(H2N)C=NH, -OH, -CH2OH, -OMe, -NHC(0)NH2, -C(0)NHNH2, -C(0)NH0H, -C(0)OH -C(0)NH2, -CH2C(0)NH2 or -B(OH)2, wherein R7 and R8 are each independently -H or -Me, wherein R4 and R5 are each independently -H or -Me; n is 0, 1, 2, 3, 4 or 5;
R2 is independently for each occurrence -CFa, -OCH3, -CN, a halogen atom or a linear or branched alkyl group having 1 to 4 carbon atoms;
R3 is an alkyl group, a substituted or unsubstituted naphthyl group, a substituted or unsubstituted 2-phenylvinyl group, a substituted or unsubstituted 2-phenylethyl group, a heteroaryl group, a substituted or unsubstituted phenyl group or a substituted or unsubstituted benzyl group.
Single bonds connecting one residue, moiety, structure, substituent or group of the compound of formula (I) to another are indicated by in the text of the present application or in chemical formula by a dashed line being divided by a perpendicular wavy line or in case a stereocenter is defined by this bond a hashed wedged bond or wedged bond ending at the middle of a wavy line.
Substituents R1, R2, R3 and indices I, m and n of the above formula (I) can be selected independently of each other.
In the present invention the substituent -Chb- is a methylene bridge, -C(O)- is a carbonyl group and -NH- is a secondary amine group.
According to the present invention residue L1 is either a -C(0)NH(CH2)mR1 group, a -C(0)piperidyl group, an -NHC(0)(CH2)mR1 group or an -NH(CH2)mR1 group, preferably a -C(0)NH(CH2)mR1 group or a -C(0)piperidyl group and more preferably a -C(0)NH(CH2)mR1 group. The groups of residue L1 are bond to the benzylic methine group, which forms the chiral center of the molecule and is bound to the central unsubstituted phenyl group of the compound of formula (I).
According to the present invention residue L2 in the above formula (I) completes the (4-phenoxy)-phenylglycine and (4-benzyloxy)-phenylglycine structure and connects the substituent R3 to the benzylic methine group, which forms the chiral center of the molecule and is bound to the central unsubstituted phenyl group of the compound of formula (I). Residue L2 is a -C(0)NH- group, a -SO2NH- group or an - NH- group, preferably a -C(0)NH- group, if residue L1 is a -C(0)NH(CH2)mR1 group or a -C(0)piperidyl group. In case residue L1 is an -NHC(0)(CH2)mR1 group or an -NH(CH2)mR1 group the residue L2 is an -NHC(O)- group. In residue L2 the -C(0)NH- group and the -SO2NH- group are an amide group and a sulfonamide group, respectively, wherein in both groups the left represents a binding side to substituent R3 and the right represents a binding side to the benzylic methine group, which forms the chiral center of the molecule and is bound to the central unsubstituted phenyl group of the compound of formula (I).
In a preferred embodiment of the (4-phenoxy)-phenylglycine and (4-benzyloxy)- phenylglycine structures of the compounds of formula (I) can be selected from the structures of the following formulas (la), (lb), (lc), (Id), (le), (If), (Ig) or(lh), preferably (la), (lb), (lc), (Id) or (Ig), more preferably (la) or (Ig) and most preferably (la).
Figure imgf000006_0001
The piperidyl group of residue L1 of formula (I) refers to a substituent derived from piperidine, of which one or more hydrogen atoms may be replaced by a substituent. The substitution pattern and number of substituents are not particularly limited but may preferably be one substituent in 4-position of the piperidyl group of residue L1 in formula (I). Substituents may preferably be selected independently of each other from 1-pyrrolidinyl, a carboxamidyl group, an N-phenyl carboxamidyl group, trifluoromethyl (-CF3), -CM (cyano), a linear or branched alkyl group having preferably 1 to 4 carbon atoms, an alkyloxy group having preferably 1 to 4 carbon atoms and a halogen atom, most preferably 1-pyrrolidinyl, a carboxamidyl group or an N-phenyl carboxamidyl group. In this context a halogen atom is a group 17 element of the periodic table, preferably fluorine or chlorine.
According the present invention the piperidyl group in residue L1 in formula (I) is preferably selected from the group consisting of:
Figure imgf000007_0001
The index “m” in the above formula (I) is selected from 0, 1 , 2, 3, 4, 5 or 6, preferably 0, 1 or 2 more preferably 0 or 1 and most preferably 1.
Substituent R1 in the above formula (I) is preferably a basic or neutral moiety, a chemical residue containing one basic moiety or a non-basic replacement thereof. According to the present invention a basic moiety is an atom or an organic or inorganic substituent which releases hydroxide ions in an aqueous solution or accepts protons from any proton donor. Substituent R1 in the above formula (I) is -NR4R5, -(HN)(H2N)C=NH, an N-containing heteroaryl group, a group of formula (II) or a group of formula (III), wherein R1 is preferably an N-containing heteroaryl group, a group of formula (II) or a group of formula (III), more preferably a group of formula (II) or a group of formula (III) and most preferably a group of formula (II).
Substituents R4 and R5 of -NR4R5 of R1 in the above formula (I) are preferably selected independently of each other from -H (hydrogen atom) or -Me (methyl). Examples of R1 are amino, methylamino and dimethylamino. Substituent -(HN)(H2N)C=NH of R1 in the above formula (I) is a guanidinyl group in which one or more hydrogens may be preferably substituted independently of each other by -Me. The substitution pattern and number of substituents are not particularly limited.
In the present application “N-containing heteroaryl group” refers to any monocyclic or bicyclic aromatic hydrocarbon group in which at least one carbon atom is replaced by a nitrogen atom und optionally one or more carbon atom(s) is/are replaced by a heteroatom such as e.g. nitrogen, oxygen, sulphur. In case two or more carbon atoms are replaced by a heteroatom, the heteroatoms are the same or different. N- containing heteroaryl groups of the present invention are indazole, indole, indoline, benzimidazole, benzoindazole, imidazole, pyrazole, pyrazol, pyridin, pyrimidin, chinolin, isochinolin, furan, thiophen, oxazol, isoxazol, thiazol or thiazin, preferably indazole, indole, indoline, benzimidazole, benzoindazole imidazole or pyrazole, more preferably indazole or benzoindazole. The position of attachment at the N- containing heteroaryl group to -(CH2)m-, or to the nitrogen in case m is 0 of the general formula (I) is not particularly limited.
In a preferred embodiment of the present invention, the N-containing heteroaryl group of substituent R1 is (poly)substituted, i.e. one or more hydrogen atom(s) of the N-containing heteroaryl group are replaced by a substituent selected independently of each other. Such substituents are preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, amino (-NH2), halogen, cyano (-CN), hydroxyl (-OH), nitro (-NO2), a linear or branched alkoxy group having preferably 1 to 4 carbon atoms, and derivatives thereof, trihalogenmethoxy or trihalogenmethyl. The position and number of the substituent(s) in the heteroaryl group is not particularly limited.
In a preferred embodiment of the present invention substituent R1 is a group of formula (II). A group of formula (II) of substituent R1 in the above formula (I) is a substituted pyridinyl derivative (X is nitrogen) or a substituted phenyl derivative (X is CH). In this context, CH is a methine bridge. One hydrogen atom of the pyridinyl derivative and phenyl derivative of the group of formula (II) of substituent R1 is replaced by substituent R6. The position of substituent R6 in the group of formula (II) is not particularly limited, preferably in meta (lib) or para (Ha) position and more preferably in para position (Ha).
Figure imgf000009_0001
In a preferred embodiment of the present invention substituent R6 of the group of formula (II) of substituent R1 in formula (I) can be selected from -NR7R8, -CH2NR7R8, -(HN)(H2N)C=NH, -CH2(HN)(H2N)C=NH, -OH, -CH2OH, -OMe, -NHC(0)NH2, -C(0)NHNH2, -C(0)NH0H, -C(0)0H, -C(0)NH2,
-CH2C(0)NH2 or -B(OH)2. Substituent R6 in the group of formula (II) is preferably selected from -(HN)(H2N)C=NH, -CH2(HN)(H2N)C=NH, -C(0)NH2, -CH2C(0)NH2 or -B(OH)2 and more preferably selected from-(HN)(H2N)C=NH, -C(0)NH2 or -B(OH)2.
In this context -OH is a hydroxyl group, -CH2OH is a hydroxymethyl group, -OMe is a methoxy group, -NHC(0)NH2 is a ureido group, -C(0)0H is a carboxyl group, -C(0)NH2 is a carboxamidyl group, -CH2C(0)NH2 is a carboxamidylmethyl group, -C(0)NHNH2 is a hydrazinecarbonyl group, -C(0)NHOH is a hydroxycarbamoyl group and -B(OH)2 is a borono group.
In a preferred embodiment, substituents -NR7R8, -CH2NR7R8 of substituent R6 in formula (II) refer to amino derivates and methylamino derivatives, respectively. In -NR7R8 and -CH2NR7R8 of R6 substituents R7 and R8 are preferably selected each independently of each other from -H an -Me. Preferred embodiments of substituents -NR7R8 and -CH2NR7R8 of R6 are amino, methylamino or dimethylamino, and aminomethyl, methylaminomethyl or dimethylaminomethyl. In a preferred embodiment, substituent -(HN)(H2N)C=NH of substituent R6 in formula (II) is a guanidinyl group as defined above.
In a preferred embodiment of the present invention, substituent -CH2(HN)(H2N)C=NH of substituent R6 in formula (II) is a guanidinylmethyl group which bears an additional methylene bridge to the guanidinyl group as defined above.
In a preferred embodiment, one or more hydrogen atom(s) of the pyridinyl derivative or phenyl derivative of the group of formula (II) are replaced by one or more substituent selected independently of each other. Such substituents are preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, halogen, cyano (-CN), hydroxyl (-OH), linear or branched alkoxy group having preferably 1 to 4 carbon atoms, and derivatives thereof, trihalogenmethoxy or trihalogenmethyl, more preferably methyl and chlorine. The position and the number of these substituent(s) in the pyridinyl derivative and phenyl derivative of the group of formula (II) are not particularly limited.
In a preferred embodiment of the present invention R1 is a group of formula (III). A group of formula (III) of substituent R1 in the above formula (I) is a substituted pyridinyl derivative.
Figure imgf000010_0001
In the pyridinyl derivative of the group of formula (III) of substituent R1 one hydrogen atom is replaced by substituent R6. The position of substituent R6 in the group of formula (III) is not particularly limited and can be selected from the group consisting of ortho position (Ilia), meta position (Iflb) and para position (lllc), preferably in ortho position (Ilia) to the nitrogen atom of the pyridinyl group.
Figure imgf000010_0002
In a preferred embodiment of the present invention substituent R6 of the group of formula (II) of substituent R1 in formula (I) can be selected from -NR7R8, -CH2NR7R8, -(HN)(H2N)C=NH, -CH2(HN)(H2N)C=NH, -OH, -CH2OH, -OMe, -NHC(0)NH2I -C(0)NHNH2, -C(0)NHOH, -C(0)OH, -C(0)NH2I
-CH2C(0)NH2 or -B(OH)2. Substituent R6 in the group of formula (II) is preferably selected from -(HN)(H2N)C=NH, -CH2(HN)(H2N)C=NH, -C(0)NH2, -CH2C(0)NH2 or -B(OH)2 and more preferably selected from -(HN)(H2N)C=NH, -C(0)NH2 or -B(OH)2.
In this context -OH is a hydroxyl group, -CH2OH is a hydroxymethyl group, -OMe is a methoxy group, -NHC(0)NH2 is a ureido group, C(0)OH is a carboxyl group, -C(0)NH2 is a carboxamidyl group, -CH2C(0)NH2 is a carboxamidylmethyl group, -C(0)NHNH2 is a hydrazinecarbonyl group, -C(0)NHOH is a hydroxycarbamoyl group and -B(OH)2 is a borono group.
In a preferred embodiment, substituents -NR7R8, -CH2NR7R8 of substituent R6 in formula (III) refer to amino derivates and methylamino derivatives, respectively. In -NR7R8 and -CH2NR7R8 of R6 substituents R7 and R8 are preferably selected each independently of each other from -H an -Me. Preferred embodiments of substituents -NR7R8 and -CH2NR7R8 of R6 are amino, methylamino or dimethylamino, and aminomethyl, methylaminomethyl or dimethylaminomethyl.
In a preferred embodiment, substituent -(HN)(H2N)C=NH of substituent R6 in formula (III) is a guanidinyl group as defined above.
In a preferred embodiment of the present invention, substituent -CH2(HN)(H2N)C=NH of substituent R6 in formula (III) is a guanidinylmethyl group which bears an additional methylene bridge to the guanidinyl group as defined above.
In a preferred embodiment, one or more hydrogen atom(s) of the pyridinyl derivative of the group of formula (III) are replaced by one or more substituent selected independently of each other. Such substituents are preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, halogen, cyano (-CN), hydroxyl (-OH), a linear or branched alkoxy group having preferably 1 to 4 carbon atoms, and derivatives thereof, trihalogenmethoxy or trihalogenmethyl, more preferably methyl and chlorine. The position and the number of these substituent(s) in the pyridinyl derivative and phenyl derivative of the group of formula (III) are not particularly limited.
According the present invention combinations of -(CH2)mR1 in formula (I) are preferably selected from the group consisting of:
Figure imgf000012_0001
In a preferred embodiment, the phenoxy groups of the (4-phenoxy)-phenylglycine (index I is 0) moiety or the benzyloxy substituent of the (4-benzyloxy)-phenylglycine moiety (index I is 1) in formula (I) are (poly)substituted by replacement of one or more hydrogen atoms by one or more substituents R2 selected independently of each other. Index “n” defines the number of substituents R2 being 0, 1 , 2, 3, 4 or 5, preferably 0, 1 or 2.
The index Ί” in the above formula (I) is selected from 0 or 1, preferably 1.
Substitution by one or more substituents R2 is not limited to a certain substitution pattern. Substitution patterns are preferably ortho, para, meta, ortho-ortho, ortho- meta, ortho-para, meta-para and meta-meta with reference to the -0(CH2)I- group in formula (I) or any combination thereof. Substituents R2 can preferably be selected independently of each other from trifluoromethyl (-CF3), -CN (cyano), a linear or branched alkyl group having preferably 1 to 4 carbon atoms, like tert-butyl, a linear or branched alkyloxy group having preferably 1 to 4 carbon atoms, like methoxy (-OMe), and an halogen atom, preferably trifluoromethyl and a halogen atom. In this context a halogen atom is a group 17 element of the periodic table, preferably chlorine. Substitution patterns of the benzyloxy residue of the (4-benzyloxy)-phenylglycine moiety are preferably selected from the group consisting of:
Figure imgf000013_0001
Configuration of the stereogenic center of the 4-phenoxyphenylglycone derivative or (4-benzyloxy)-phenylglycinederivative does not have to be defined, but may be in D-configuration or L-configuration, preferably in L-configuration.
Substituent R3 in formula (I) can be selected from an alkyl group, a heteroaryl group, a substituted or unsubstituted naphthyl group, a substituted or unsubstituted 2- phenylvinyl group, a substituted or unsubstituted 2-phenylethyl group, a substituted or unsubstituted phenyl group, or a substituted or unsubstituted benzyl group, preferably from an alkyl group, a substituted or unsubstituted phenyl group or a substituted or unsubstituted benzyl group, more preferably from a substituted or unsubstituted phenyl group or a substituted or unsubstituted benzyl group and most preferably a substituted or unsubstituted phenyl group.
The alkyl group of R3 in formula (I) refers to a hydrocarbon group which is linear, cyclic or branched, preferably linear or cyclic and more preferably linear. The chain length is not particularly limited but may be preferably 1 to 15 carbon atoms, more preferably 1 to 9 carbon atoms and most preferably 4 to 9 carbon atoms. The linear, cyclic or branched alkyl group is preferably (poly)substituted, i.e. one or more hydrogen atom(s) of the alkyl group is replaced by one or more substituents selected independently of each other. Such substituents are preferably halogen, aryl, heteroaryl, hydroxyl, alkoxy, aryloxy, carboxylic acids and derivatives thereof, acyl, amino, N-acetamido, hydroxy, alkylamino, cyano, trihalogenmethoxy, trihalogenmethyl or oxo and more preferably N-acetamido, hydroxy and oxo. The substitution pattern and the number of substituents are not particularly limited. Preferred alkyl groups of R3 in formula (I) are methyl, butyl, pentyl, hexyl, heptyl, octly, nonyl, cyclohexylmethyl, cyclobutyl, L-1 -(N-acetoamido)pent-l -yl, L-1-(N- acetoamido)but-1 -yl, butanoyl and 3-methylbutanoyl or 1-(5-(2,5-dimethylphenoxy)- 2-methylpentan-2-yl) and more preferably butyl, pentyl, hexyl, heptyl, octly, nonyl, cyclohexylmethyl, 3-methylbutanoyl 1 -(5-(2,5-dimethylphenoxy)-2-methylpentan-2- yl) and most preferably butyl, pentyl, hexyl, heptyl, octly, nonyl or cyclohexylmethyl
1-(5-(2,5-dimethylphenoxy)-2-methylpentan-2-yl).
In a preferred embodiment two or more alkyl groups having preferably 1 to 4 carbon atoms or linear or branched alkoxy groups having preferably 1 to 4 carbon atoms of the substituted phenyl group of substituent R3 or the substituted benzyl group of substituent R3 might be bonded to each other to form one or more rings.
In a preferred embodiment the heteroaryl group of R3 in formula (I) is thiopen-2-yl,
2-thiophenyl-thiophen-5-yl, benzothiophen-2-yl or a derivative thereof.
In a preferred embodiment one or more hydrogen atoms of the substituted naphthyl group is/are preferably replaced by additional substituents selected independently of each other. The substitution pattern and number of additional substituents are not particularly limited. Such additional substituents are preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, halogen, cyano (-CN), hydroxyl (-OH), a linear or branched alkoxy group having preferably 1 to 4 carbon atoms, amino, dialkylamino, trihalogenmethoxy or trihalogenmethyl.
In a preferred embodiment one or more hydrogen atoms of the substituted 2- phenylvinyl group is/are preferably replaced by additional substituents selected independently of each other. The substitution pattern and number of additional substituents are not particularly limited. Such additional substituents are preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, halogen, cyano (-CN), hydroxyl (-OH), linear or branched alkoxy group having preferably 1 to 4 carbon atoms, amino, dialkylamino, trihalogenmethoxy or trihalogenmethyl.
In a preferred embodiment one or more hydrogen atoms of the substituted 2- phenylethyl group is/are preferably replaced by additional substituents selected independently of each other. The substitution pattern and number of additional substituents are not particularly limited. Such additional substituents are preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, halogen, cyano (-CN), hydroxyl (-OH), linear or branched alkoxy group having preferably 1 to 4 carbon atoms, amino, dialkylamino, trihalogenmethoxy or trihalogenmethyl.
In another preferred embodiment the substituted phenyl group of R3 in formula (I) may be a group of formula (IVa), (IVb) or (IVc), more preferably formula (IVa) or (IVb) and most preferably (IVa).
Figure imgf000015_0001
The phenyl groups of formulas (IV) contain at least one substituent selected from R9, R10 and R11 or combinations thereof. Additionally, one or more hydrogen atoms of the phenyl group of formulas (IV) are preferably further replaced by additional substituents selected independently of each other. The substitution pattern and number of additional substituents are not particularly limited. Such additional substituents are preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, halogen, cyano (-CN), hydroxyl (-OH), linear or branched alkoxy group having preferably 1 to 4 carbon atoms, amino, dialkylamino, trihalogenmethoxy or trihalogenmethyl.
In a preferred embodiment, substituents R9, R10 and R11 can be selected from a linear or branched alkyl group having preferably 1 to 4 carbon atoms, a linear or branched alkoxy group having preferably 1 to 6 carbon atoms, aminomethyl group, morpholino, 4-morpholincarbonyl, a substituted or unsubstituted furan-2-yl group, a substituted or unsubstituted pyridiny-5-yl, a substituted or unsubstituted phenyl group, a substituted or unsubstituted benzoyl group, a substituted or unsubstituted phenoxy group or a substituted or unsubstituted benzyloxy group or combinations thereof.
The substitution pattern and the number of substituents of the substituted furan-2-yl group, the substituted pyridiny-5-yl, the substituted phenyl group, the substituted benzoyl group, the substituted phenoxy group and the substituted benzyloxy group are not particularly limited.
In preferred embodiments, such substituents of the substituted furan-2-yl group, the substituted pyridiny-5-yl, the substituted phenyl group, the substituted benzoyl group, the substituted phenoxy group and the substituted benzyloxy group of substituents R9, R10 and R11 is preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, halogen, cyano (-CN), hydroxyl (-OH), a linear or branched alkoxy group having preferably 1 to 4 carbon atoms, amino, dialkylamino, trihalogenmethoxy or trihalogenmethyl, preferably -OMe and a chlorine atom. Two or more alkyl groups having preferably 1 to 4 carbon atoms or linear or branched alkoxy groups having preferably 1 to 4 carbon atoms of the substituted phenyl group, the substituted benzoyl group, the substituted phenoxy group or the substituted benzyloxy group of substituents R9, R10 and R11 might be bonded to each other to form one or more rings.
Substituent R9 is preferably a linear or branched alkoxy group having preferably 1 to 6 carbon atoms, a substituted or unsubstituted phenyl group, a substituted or unsubstituted benzoyl group, a substituted or unsubstituted phenoxy group or a substituted or unsubstituted benzyloxy group, more preferably a substituted or unsubstituted phenyl group, a substituted or unsubstituted benzoyl group, a substituted or unsubstituted phenoxy group or a substituted or unsubstituted benzyloxy group and more preferably a substituted or unsubstituted benzoyl group or a substituted or unsubstituted phenoxy group.
R10 is preferably 4-morpholinecarbonyl, a substituted or unsubstituted phenyl group, substituted or unsubstituted phenoxy group or a substituted or unsubstituted benzyloxy group, more preferably a substituted or unsubstituted phenyl group, substituted or unsubstituted phenoxy group or a substituted or unsubstituted benzyloxy group and most preferably a substituted or unsubstituted phenoxy group.
R11 is preferably a substituted or unsubstituted phenyl group, more preferably an unsubstituted phenyl group.
In a preferred embodiment of the present invention, the substituted benzyl group of R3 in formula (I) is a substituted benzyl group of formula (V)
Figure imgf000017_0001
Substituent R12 in formula (V) is preferably a substituted or unsubstituted phenyl. In case R12 is a substituted phenyl 1, 2, 3, 4 or 5 hydrogen atoms of the phenyl group of R12 are replaced by 1 , 2, 3, 4 or 5 substituents selected independently of each other. The substitution pattern and number of substituents is not particularly limited. Such substituents are preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, halogen, cyano (-CN), hydroxyl (-OH), a linear or branched alkoxy group having preferably 1 to 4 carbon atoms, amino, dialkylamino, trihalogenmethoxy or trihalogenmethyl.
In a preferred embodiment, additionally one or more hydrogen atoms of the benzyl group of formula (V) are further replaced by substituents selected independently of each other. Such substituents are preferably a linear or branched alkyl group having preferably 1 to 4 carbon atoms, halogen, cyano (-CN), hydroxyl (-OH), a linear or branched alkoxy group having preferably 1 to 4 carbon atoms, amino, dialkylamino, trihalogenmethoxy or trihalogenmethyl.
According to the invention substituted phenyl groups and substituted benzyl groups in formulas (IV) and (V) of R3 are preferably selected from the group consisting of: Preferred embodiments of the compound of formula (I), according to the present
5 invention are:
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
The protease inhibitor according to the present invention is a compound according to formula (I) as described above or a pharmaceutically acceptable salt or ester thereof. In case the protease inhibitor of the present invention is a pharmaceutically acceptable salt of the compound of formula (I), the salt can be formed with inorganic or organic acids or bases. Preferred embodiments of pharmaceutically acceptable salts comprise, without limitation, non-toxic inorganic or organic salts such as acetate derived from acetic acid, aconitate derived from aconitic acid, ascorbate derived from ascorbic acid, benzoate derived from benzoic acid, cinnamate derived from cinnamic acid, citrate derived from citric acid, embonate derived from embonic acid, enantate derived from heptanoic acid, ethansulfonat derived from ethansulfonic acid ethylenediaminetetraacetate derived from ethylenediaminetetraacetic acid, formiate derived from formic acid, fumarate derived from fumaric acid, gluconat derived from gluconic acid, glutamate derived from glutamic acid, glycolate derived from glycolic acid, chloride derived from hydrochloric acid, bromide derived from hydrobromic acid, lactate derived from lactic acid, laurate derived from lauric acid, malate derived from malic acid, maleate derived from maleic acid, malonate derived from malonic acid, mandelate derived from mandelic acid, methanesulfonate derived from methanesulfonic acid, mucate derived from mucic acid, naphthaline-2-sulfonate derived from naphtha!ine-2- sulfonic acid, nitrate derived from nitric acid, oleate derived from oleic acid, palmitate derived from palmitic acid, perchlorate derived from perchloric acid, phosphate derived from phosphoric acid, phthalate derived from phthalic acid, propionate derived from propionic acid, pyruvate derived from pyruvic acid, salicylate derived from salicylic acid, sorbate derived from sorbic acid, stearate derived from stearic acid, succinate derived from succinic acid, sulfanilate derived from sulfaniiic acid, sulphate derived from sulphuric acid, tannate derived from tannic acid, tartrate derived from tartaric acid, toluene-p-sulfonate derived from p-toluenesulfonic acid, valerate derived from valeric acid, polymeric salts derived from polymeric acids, e.g. carboxymethyl cellulose, and others or combinations thereof. Such salts can be readily produced by methods known to a person skilled in the art.
Other salts which are not considered as pharmaceutically acceptable, like oxalate derived from oxalic acid, can be appropriate as intermediates for the production of the protease inhibitor of formula (I) or a pharmaceutically acceptable salt thereof or physiologically functional derivative or a stereoisomer thereof.
In case the protease inhibitor of the present invention is a pharmaceutically acceptable ester of the compound of formula (I), the ester is a boronic ester and can be formed by the borono group as substituent R6 and alcohols, preferably diols or polyalcohols, or hydroxycarboxylic acids, preferably alpha-hydroxycarboxylic acids or beta-hydroxycarboxylic acids. In a preferred embodiment the pharmaceutically acceptable ester comprises, without limitation, boronic esters formed by the borono group as substituent R6 and non-toxic alcohols such as ethylene glycol, propylene glycol, glycerol, butane-2, 3-diol, pinacol, pinanediol, tris(hydroxymethyl)aminomethane, sorbitol, mannitol, other sugar alcohols, sugars such as glucose, mannose, fructose, ribose, arabinose, lyxose, sorbose, xylose and other sugars. In another preferred embodiment the pharmaceutically acceptable ester comprises, without limitation, boronic esters formed by the boro no group as substituent R6 and hydroxycarboxylic acids, preferably alpha-hydroxycarboxylic acids, such as glycolic acid, malic acid, hexahydromandelic acid, citric acid, isocitric acid, 2-hyd roxyisobutyric acid, mandelic acid, lactic acid, 2-hydroxy-3,3- dimethylbutyric acid, 2-hyd roxy-3-methylbutyric acid, 2-hyd roxyisocaproic acid, tartaric acid, ascorbic acid, gluconic acid, tatronic acid, benzilic acid and others, or beta-hydroxycarboxylic acids, such as malic acid, citric acid, isocitric acid, 3- hydroxybutyric acid, beta-hydroxyisovaleric acid, tartaric acid, ascorbic acid, gluconic acid, mevalonic acid, glucoheptonic acid, maltonic acid, lactobionic acid, galactaric acid, embonic acid, 1 -hydroxy-2-naphtoic acid, 3-hydroxy-2-naphtoic acid, salicylic acid and others. Such esters can be readily produced by methods known to a person skilled in the art. Furthermore, complex boro nates can be formed by reaction with /V-methyliminodiacetic acid and its congeners.
Further, the present invention relates to the compounds of the present invention for use in medicine. In a preferred embodiment, the medicine is used as an inhibitor of the flaviviral NS2B-NS3 serine protease.
Furthermore, the present invention relates to the use of the compounds of the present invention as a medicament.
Moreover, the present invention relates to the compounds of the present invention for use in the treatment and/or prevention of a condition or disease that is preferably associated with a flavi virus infection.
In a preferred embodiment, the present invention relates to the compounds of the present invention for use in treating a flavivirus infection. A flavivirus is a virus preferably selected from the group consisting of West Nile virus, Dengue virus, tick- borne encephalitis virus, yellow fever virus and Zika virus, preferably West Nile virus, Zika virus, Dengue virus, Hepacivirus C, Omsk hemorrhagic fever virus, Usuto virus, St.-Louis-Enzephalitis virus, Powassan virus, Louping-lll virus Kyasanur forest disease virus, Gadgets Gully virus, Kedougou virus and Spondweni virus. The invention provides a method of treatment and/or prevention a disease or condition associated with a flavivirus infection. The method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt or ester thereof, thereby treating or preventing the disease or condition.
Moreover, according the invention, said compounds are preferably administered in combination with one or more further compounds. Further, the present invention relates to a pharmaceutical composition comprising one or more of the compounds of the present invention for use in medicine, treatment or prevention of a condition of disease or infection as defined above.
In a preferred embodiment, the compound or a pharmaceutically acceptable salt or ester thereof according to the present invention is administered to animals, preferably to mammals preferably selected from the group consisting of cat, dog, horse, cattle, pig and humans, and most preferably to humans. The pharmaceutical composition can be administered as therapeutics per se, as mixtures with one another or in the form of pharmaceutical preparations which allow enteral or parenteral use, and which as active constituent contains an effective dose of at least one compound of the present invention as defined above or a pharmaceutically acceptable salt or ester thereof, in addition to customary pharmaceutically innocuous excipients and additives. The compound of the present invention as defined above can also be administered in form of its salts or esters, which are obtainable by reacting the respective compounds with physiologically acceptable acids or alcohols or hydroxycarboxylic acids as defined above.
The production of medicaments containing the compound of the present invention as defined above and its application can be performed according to well-known pharmaceutical methods.
While the compound of the present invention as defined above for use in therapy may be administered in the form of the raw chemical compound, it is preferred to introduce the active ingredient, optionally in the form of a physiologically acceptable salt or ester in a pharmaceutical composition together with one or more adjuvants, excipients, carriers, buffers, diluents, and/or other customary pharmaceutical auxiliaries. Such salts or esters of the protease inhibitor as defined above may be anhydrous or solvated.
The invention provides medicaments comprising the compound of the present invention as defined above, or a pharmaceutically acceptable salt or ester or physiologically functional derivative thereof, preferably together with one or more pharmaceutically acceptable carriers thereof, and, more preferably other therapeutic and/or prophylactic ingredients. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not harmful to the recipient thereof.
In a preferred embodiment, the compound according to the present invention is administered oral, rectal, bronchial, nasal, topical, buccal, sub-lingual, transdermal, vaginal or parenteral (including cutaneous, subcutaneous, intramuscular, intraperitoneal, intravenous, intraarterial, intracerebral, intraocular injection or infusion), or inhalation or insufflation, including powders and liquid aerosol administration, or by sustained release systems. Suitable examples of sustained release systems include semipermeable matrices of solid hydrophobic polymers containing the compound of the invention, which matrices may be in form of shaped articles, e.g. films or microcapsules.
The compound of the present invention as defined above, together with a conventional adjuvant, carrier, or diluent, may thus be placed into the form of medicament and unit dosages thereof. Such forms include solids, and in particular tablets, filled capsules, powder and pellet forms, and liquids, in particular aqueous or non-aqueous solutions, suspensions, emulsions, elixirs, and capsules filled with the same, all for oral use, suppositories for rectal administration, and sterile injectable solutions for parenteral use. Such medicament and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms preferably contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.
The compound of the present invention as defined above can be administered in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, as the active component, either the compound of the present invention or a pharmaceutically acceptable salt or ester as defined above.
For preparing a medicament from the compound of the present invention as defined above, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, lozenges, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavouring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term “preparation” is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.
For preparing suppositories, a low melting wax, such as a mixture of fatty acid glyceride or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogenous mixture is then poured into convenient sized moulds, allowed to cool, and thereby to solidify. Liquid preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.
The compound of the present invention as defined above may thus be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.
Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents, as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well known suspending agents.
Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
The medicament may be applied topically or systemically or via a combination of the two routes. In a particular embodiment of the present invention, the medicament is applied topically. This reduces possible side effects and limits the necessary treatment to those areas affected.
Preferably the medicament is prepared in form of an ointment, a gel, a plaster, an emulsion, a lotion, a foam, a cream of a mixed phase or amphiphilic emulsion system (oil/water-water/oil mixed phase), a liposome, a transfersome, a paste or a powder.
Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
Compositions suitable for topical administration in the mouth include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerine or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The compositions may be provided in single or multi-dose form. In the latter case of a dropper or pipette, this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray, this may be achieved for example by means of a metering atomising spray pump.
Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the active ingredient is provided in a pressurised pack with a suitable propellant such as a chlorofluorocarbon (CFG) for example dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by provision of a metered valve.
Alternatively, the active ingredients may be provided in the form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP). Conveniently the powder carrier will form a gel in the nasal cavity. The powder composition may be presented in unit dose form, for example in capsules or cartridges of e.g. gelatin, or blister packs from which the powder may be administered by means of an inhaler.
In compositions intended for administration to the respiratory tract, including intranasal compositions, the compound will generally have a small particle size for example of the order of 5 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization.
When desired, compositions adapted to give sustained release of the active ingredient may be employed.
The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packaged tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. Tablets or capsules for oral administration and liquids for intravenous administration and continuous infusion are preferred compositions.
Pharmaceutical compositions can also contain two or more compounds of the present invention as defined above or their pharmacologically acceptable salts or esters and also other therapeutically active substances. Thus, the compound of the present invention as defined above can be used in the form of one compound alone or in combination with other active compounds - for example with medicaments already known for the treatment of the aforementioned diseases, whereby in the latter case a favorable additive, amplifying effect is noticed. Suitable amounts to be administered to humans range from 5 to 1 ,250 mg per day.
To prepare the pharmaceutical preparations, pharmaceutically inert inorganic or organic excipients can be used. To prepare pills, tablets, coated tablets and hard gelatin capsules, for example, lactose, corn starch or derivatives thereof, talc, stearic acid or its salts, etc. can be used. Excipients for soft gelatin capsules and suppositories are, for example, fats, waxes, semi-solid and liquid polyols, natural or hardened oils etc. Suitable excipients for the production of solutions and syrups are, for example, water, sucrose, invert sugar, glucose, polyols etc. Suitable excipients for the production of injection solutions are, for example, water, alcohols, glycerol, polyols or vegetable oils.
The dose can vary within wide limits and is to be suited to the individual conditions in each individual case. For the above uses the appropriate dosage will vary depending on the mode of administration, the particular condition to be treated and the effect desired.
Additionally, the present invention relates to methods of treating or of preventing any of the conditions and/or diseases as defined above, comprising a step of administering one or more of the compounds or a pharmaceutical composition as defined above, to a subject in need thereof, preferably a mammalian subject and more preferably a human subject.
Further, the present invention relates to a pharmaceutical composition as defined above, which is a prophylactic agent against a flavivirus infection or an agent for treating a flavivirus infection.
Moreover, the present invention relates to a compound of the present invention for use of inhibition of infectivity of a flavivirus in vitro. Furthermore, the present invention relates to the use of the compounds of the present invention for inhibition of the NS2B-NS3 serine protease of a flavivirus in vitro.
Further, the present invention relates to the compounds of the present invention for use in medicine.
Besides the above-mentioned application, these compounds could serve as reference inhibitors in both biochemical and cellular DENV-2 protease assay systems. Viral replication assays are usually performed under variable conditions by different laboratories, impeding the comparison of results, antiviral activity, and assay conditions. Considering this background, reference compounds with good synthetic accessibility provide an opportunity to calibrate results from various laboratories and assay set-ups against a common standard.
Figures
Figure 1 shows the inhibitory activity of selected compounds at a concentration of 12.5 mM in the DENV-2 virus titer reduction assay.
Figure 2 shows the inhibitory activity of selected compounds at a concentration of 12.5 mM in the DENV-2 virus titer reduction assay.
Figure 3 shows the inhibitory activity of selected compounds at a concentration of 1 and 12.5 mM in the DENV-2 virus titer reduction assay.
Figure 4 shows concentration dependent DENV-2 virus titer reduction and ECso values of compounds NK-189, L-02 and NK-288.
Figure imgf000030_0001
compounds NK-375, NK-479, NK-428, NK-433, NK-469, NK-429, NK-478 (A) and
ECso and ECgo values for those compounds (B). Figure 6 shows the concentration-dependent quenching of the fluorescence of the tryptophan residues in the substrate binding region after addition of compounds NK- 119 (A), NK-406 (B) and NK-305 (C) to DENV-2 protease. The fluorescence is partially restored when aprotinin is added. NATA = AZ-Acetyl-L-tryptophanamide.
The present invention will be further illustrated by the following example without being limited thereto.
Examples
Various series of compounds were evaluated in biochemical enzymatic assays against DENV and WNV proteases. Thrombin and trypsin were evaluated as potential off-targets. All compounds with low cytotoxicity (>80% signal compared to non-treated control) at the screening concentration were further evaluated in a DENV2proHel_a assay, preferably as described in Richter, M., Leuthold, M.M., Graf, D.K., Bartenschlager, R., Klein, C.D., Prodrug Activation by a Viral Protease: Evaluating Combretastatin Peptide-Hybrids to Selectively Target Infected Cells, ACS Med. Chem. Lett. 2019, 10, 8, 1115-1121. The SAR exploration was focused on basic C-terminal moieties [-NH-(CH2)m-R1], different substitutions of the benzyl residue and variation of lipophilic N-terminal caps (R3-CO). In this context C-terminal moieties or caps and N-terminal moieties or caps refer to the residues attached to the carboxylic acid and amino group, respectively, of the (4-phenoxy)-phenylglycine or (4-benzyloxy)-phenylglycine structure of compounds according to formula (I). From a medicinal chemistry perspective, the present assay - the creation of compound libraries by building block assembly and derivatization - provides a fast track to diverse compounds, which are not easily accessible otherwise, considering for example the use of the racemization-prone phenylglycine in synthetic procedures. While this approach is inspired from amino acids assembly for peptides, it can be effectively applied on other chemical building blocks for lead optimization.
Exemplary compounds were analyzed, and enantiomeric purity was above 95% in all cases. Additionally, compound NK-305 was tested for its racemization tendency in assay buffers used in this study. Racemization was negligible after incubation for 96 hours at 37°C in phosphate-buffered saline (pH 7.4), biochemical DENV assay buffer (pH 9.0) and DENV2proHel_a media (pH 7.0). A highly basic sodium hydroxide solution (pH 13) and an incubation of 96 hours was required for full racemization of the tested compound.
Variation of -C(0)NHfCH2)mR1 groups and -CfQtoiperidinyi groups of residue L1
Basic residues were primarily evaluated in residue L1 (see Table 1). The lysine and alkylamine analogs NK-187, NK-130, NK-304 and NK-246 showed no or minor inhibition in the biochemical assays, similarto non-basic or weakly basic compounds (e.g., NK-136 NK-158, NK-190, NK-188) The inhibition increased with higher basicity of the residues. Especially phenylalkylamines or phenylguanidines led to potent inhibitors. K\ values of 3.8 mM for NK-129 and 9.8 mM for NK-189 at DENV-2 protease were determined using Cheng-Prusoff plots. Compounds NK-254 and NK- 269 can form similar hydrogen bonds with the protease and were designed as non- basic guanidine replacements. Surprisingly, the amidine-containing compound NK- 191 had weak activity. Meta-substituted derivatives had lower potency compared to their para-substituted derivatives (e.g. NK-204, NK-217). A methylene group between the amide nitrogen and the phenyl group was needed for optimal activity (see compounds NK-209 and NK-189). Most compounds have similar activities in the biochemical and the cellular protease assay but compounds NK-201, NK-129 and NK-209 could not be measured due to cytotoxicity at the screening concentration. Noteworthy is the uppernanomolar DENV2proHel_a ECso of compound NK-189. The A/,A/-dimethylamine derivative NK-195 showed no inhibition in the in vitro assays but a high inhibition in the cell-based assay, which could be due to intracellular demethylation to yield the monomethylamine derivative of compound NK-129. The latter compound was a potent inhibitor in the biochemical assays, but could not be evaluated in cells due to its low CCso of 4.8 mM. Various heterocycies were evaluated: The 1 H-indazole containing compound NK-299 had an ECso of around 7 mM. The pyridine containing compounds NK-158 and NK-356 inhibited 50% of the protease at 12.5 mM in cells whereas the 1 -(pyridin-2- yl)guanidine containing NK-361 had an ICso of 6 mM. Table 1. Inhibitory activity of compounds with different -G(0)NH(CH2)mR1 groups and a -C(0)piperidinyl group of residue L1 against isolated DENV and WNV proteases, the off-targets thrombin and trypsin, in the cellular DENV protease reporter assay (DENV2proHel_a) and cytotoxicity in HeLa cells.
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
According to the present invention less basic or neutral residues in L1 can be applied (table 2). Whereas the (4-chloro) and unsubstituted phenyl derivatives had no relevant activity, the (4-methoxy), (4-hydroxy) and 4-(phenyl)methanol derivatives gave ECso values between 10-20 mM. For para and meta substituted benzamide compounds one digit micromolar inhibition values were observed and are therefore promising non-charged guanidine replacements at this position (NK-124 - NK- 244.2). Urea, hydrazide and hydroxamic acid containing compound NK-254, NK- 269 and NK-270 are able to form similar H-bonds compared to a guanidine moiety, but just minor inhibition was detected. Cycloalkyl derivatives NK-223 and NK-169 had low activity, except piperidine compound NK-155 with an ECso of around 13 pM. The phenylboronic acid derivatives showed one digit micromolar activities in the cellular DENV2proHel_a assay (table 2). Surprisingly the phenylboronic acid derivatives are promising replacements for the strongly basic guanidine motif at this position. 3-Aminophenylboronic acid and 4-aminomethylphenylboronic acid have high pKa values of 8.9 and 8.3 and are therefore considered as slightly acidic.
Table 2. Inhibitory activity of compounds with different -C(0)NH(CH2)mR1 groups and different -C(0)piperidinyl groups of residue L1 against isolated DENV and WNV proteases, the off-targets thrombin and trypsin, in cellular DENV protease reporter assay (DENV3proHELA) and cytotoxicity in HeLa cells.
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
t
Figure imgf000043_0001
Variations in the (4-phenoxy)-phenylqlvcine and (4-benzyloxy)-phenylglvcine moieties by different substituents R2
Different (4-phenoxy)-phenylglycine moieties and different (4-benzyloxy)- phenylglycine moieties having different substituents R2 are listed in table 3. The phenylglycine moiety containing compound NK-334 was inactive in all assays. Further, the unsubstituted L-derivative (NK-284) had a 20-fold lower inhibition compared to the L-isomer (NK-189). This is in strong contrast to previously published inhibitor structures, which showed about 3-fold higher activity for D- derivatives and may be seen as an indication for a fundamentally different binding mode of the present, new chemotype. The electron-withdrawing halogen substituent containing compounds in L- and D-configuration had a 2- to 5-fold higher inhibitory potency in biochemical assays (NK-285, NK-360, NK-344, NK-287). However, substitutions at the benzyloxy residue of the (4-benzyloxy)-phenylglycine moiety were not well tolerated in the DENV2proHel_a assay. Other substituents like 3- methoxy, 4-cyano or 4-terf-butyl decreased the activity of the compounds in all assays.
Table 3. Inhibitory activity of compounds having different (4-phenoxy)-phenylglycine and different (4-benzyloxy)-phenylglycine moieties against isolated DENV and WNV protease, the off-targets thrombin and trypsin, in the cellular DENV protease reporter assay (DENV2proHel_a) and cytotoxicity in HeLa cells.
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Variation of the lipophilic R3CfQ)NH- group of residue L2
Mostly lipophilic alkyl groups and phenyl residues have been evaluated as R3 in the R3C(G)NH- group of residue L2 (table 4). The length of the alkyl chains had a major influence on the activity, with a positive correlation between lipophilicity and activity (NK-366 to NK-377). The minor inhibition by the acetyl analog in the cell-based assay could be increased by extending the chain length. The hexanoic acid cap (NK-189) showed a nearly 6-fold higher activity compared to the valeric acid cap (NK-367). The heptanoic and octanoic acid caps gave comparable ECso values of 0.5 mM and 0.8 mM, respectively. Those compounds are therefore novel promising structures with high activity in cellular environment without relevant cytotoxicity. A benzoic acid cap led to low inhibition whereas cyclohexyloxy-, phenyloxy- and benzyloxy-benzoic acid derivatives showed a 2- or 3-fold higher inhibition in the biochemical assays compared to compound NK-189. For these inhibitors, comparable ECso values were measured in the cellular protease assay. With a 4- chloro substituent (NK-346) a nearly 5-fold higher inhibition in biochemical assays was achieved whereas the 3-methoxy substituent (NK-347) led to comparable inhibition to the unsubstituted derivative (NK-307). Similar values were observed in the cellular protease assay for those derivatives except for NK-346, which were significantly less potent. An up to 9-fold higher activity was observed with the biphenyl-4-carboxylic acid and 2-(biphenyl-4-yl)acetic acid caps in the DENV biochemical assay and an up to 7-fold higher activity in the WNV assay. The incorporation of biphenyl-2-carboxylic acid as a cap led to 2-fold lower inhibition compared to compound NK-189. Most improvements of the biphenyl caps could not be followed up in the DENV2proHeLa assay. Here the long alkyl chain cap containing compounds achieved the highest activity (e.g. NK-189). Especially compound NK-316 showed a big deviation between the different protease assays: The analog with a 2-(biphenyl-4-yl)acetic acid cap had the highest in vitro activity but reached only an ECso of 20 mM in the DENV2proHeLa system. Some of the caps resulted in cytotoxicity at 50 mM (NK-333, NK-288, NK-307, NK-346, NK-347), but at the DENV2proHel_a screening concentration (12.5 pM), no cytotoxicity was observable. Table 4. Inhibitory activity of compounds with variations of R3C(0)NH- groups of residue LAagainst isolated DENV and WNV proteases, the off-targets thrombin and trypsin, in the cellular DENV protease reporter assay (DENV2proHeLa) and cytotoxicity in HeLa cells.
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Selected fragments of the compounds described in Tables 1 to 4 were merged, thus combining the most active residues in the biochemical assays (see table 5). Compound NK-199 showed promising inhibition in biochemical assays. Compounds NK-323 and NK-365 are combinations of the most active fragments in the biochemical assays. The combination of 2,6-dichloro-(4-benzyloxy)-phenylglycine, 2-(biphenyl-4-yl)acetic acid and the phenylguanidine moiety afforded compound NK-365 with upper nanomolar potencies in biochemical assays. Ki values were measured in DENV and WNV biochemical assays and were found to be 0.5 mM and 1.0 mM, respectively. In spite of the promising potency in in vitro assays, compound NK-365 had significantly lower activity (ECso: 39 mM) in the DENV2proHeLa assay.
The same observation was made for compound NK-323. The high molecular masses of these compounds (683 g/mol and 667 g/mol) may result in an inferior pharmacokinetic profile and low membrane permeability. Compounds NK-344 and NK-316, which contain the same structural moieties as compound NK-365, were also significantly less potent in the cell-based assay in comparison to compound NK-189.
Table 5. Inhibitory activity of merged compounds against isolated DENV and WNV proteases, the off-targets thrombin and trypsin, in the cellular DENV protease reporter assay (DENV2proHel_a) and cytotoxicity in HeLa cells.
Figure imgf000054_0001
Different (4-benzyloxy)-phenylglycine derivatives and different R3C(0)NH- groups were merged to further increase activity. 4-Benzamide and 3-benzamide derivatives were evaluated as R1 in the -C(0)NH(CH2)mR1 group of residue L1 (see table 6, 7 and 8). Whereas polar caps in the R3C(0)NH- group of residue L2 like 2-(2- methoxyethoxy)acetic acid or 6-aminohexanoic acid decreased the activity, especially long alkyl chains and ring systems increased it. Residues at the (4- benzyloxy) residue were not well tolerated. Only the 4-(trifluoromethy!)- and the 1 ,3- dichloro-derivatives showed comparable activities to NK-84. L-configured compounds showed higher activity compared to their D-substituted enantiomers. Compound NK-220 caused 50% inhibition of DENV protease activity at 1 mM but also a relevant cytotoxicity (CCso 18 mM). To overcome this, efa-substituted benzamide derivatives in R1 were evaluated (table 6). Compound NK-375 in contrast to NK-220 gave a CCso higher than 100 mM. Most of the evaluated phenoxyphenyl- or biphenyl analog substituents R3 in the R3C(0)NH-group showed high activities with ECso values at about 0.6 - 2 micromolar (except NK-466). The 4-benzoylbenzoic acid analog (NK-469) as well as compound NK-375 revealed high nanomolar inhibition. The 4-(benzo[d][1,3]dioxol-5-yloxy)benzoic acid analog (NK- 433) had significantly higher activity than the 4-(2,3-dihydrobenzo[b][1 ,4]dioxin-6- yloxy)benzoic acid analog (NK-480). NK-411 contains a (4-phenoxy)-phenylglycine moiety which decreased the activity compared to the (4-benzyloxy)-phenylglycine derivatives. The phenylpiperidine containing compound NK-431 showed lower inhibition, which could be explained with the basic nature of the cap.
Table 6. Inhibitory activity of compounds of Formula (I) with different R3C(0)NH-, R3SC>2NH- or R3NH- groups of residue L2 und different (4-benzyloxy)-phenylg lyci ne , phenylglycine and phenylalanine moieties against isolated DENV and WNV proteases, the off- targets thrombin and trypsin, in the cellular DENY protease reporter assay (DENV2proHeLa) and cytotoxicity in HeLa cells.
Figure imgf000056_0002
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Table 7. Inhibitory activity of compounds with different R3C(0)NH- groups of residue L2 und different (4-benzyfoxy)-phenylglycine moieties against isolated DENV and WNV proteases, the off-targets thrombin and trypsin, in the cellular DENV protease reporter assay (DENV2proHeLa) and cytotoxicity in HeLa cells.
Figure imgf000062_0001
Figure imgf000063_0002
Figure imgf000063_0001
Table 8. Inhibitory activity of compounds with different R3C(0)NH- groups of residue L2 and different (4-benzyloxy)-phenyiglycine and (4-phenoxy)-phenylglycine moieties against isolated DENV and WNV proteases, the off-targets thrombin and trypsin, in the cellular DENV protease reporter assay (DENV2proHeLa) and cytotoxicity in HeLa cells.
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0002
Figure imgf000068_0001
Different phenylboronic acids were evaluated as non-basic replacements in substituent R1 of the -C(0)NH(CH2)mR1 group of residue L1 (see table 9). 4- (Aminomethyl)phenylboronic acid derivatives had the highest activities but most derivatives showed high activities at one digit micromolar levels. The 4- (aminomethyl)phenylboronic acid compounds revealed slightly higher activities than the one carbon longer 4-(2-aminoethyl) derivatives. Phenoxyphenyl- and biphenyl analogous groups in substituent R3 in the R3C(0)NH- group of residue L2 further increased the activities of the compounds. Compound NK-388 even had a promising nanomolar ECso of 0.5 mM and this therefore a new promising lead compound against DENV infections. 3,4-dichloro-substituents in the benzyloxy group of the (4- benzyloxy)-phenylglycinewere tolerated. None of the compounds caused cytotoxicity at the screening concentration. Nevertheless the 4-(benzo[c/j[1 ,3]dioxol- 5-yloxy)benzoic acid analogous group as substituent R3 in compound (NK-434) lowered the cytotoxicity even more but also decreased the activity about 3-fold. The ester compound NK-290 showed about 3-fold lower activity compared to the amide compound NK-371 , possibly due to the expected lower stability of the ester motif.
Table 9. Inhibitory activity of compounds with a phenylboronic acid in residue L1 and different R3C(0)NH- groups of residue L2 based on different (4-benzyloxy)-pheny!glycine moieties against isolated DENV and WNV proteases, the off-targets thrombin and trypsin, in the cellular DENV protease reporter assay (DENV2proHeLa) and cytotoxicity in HeLa cells.
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0002
Figure imgf000072_0001
A set of compounds with reversed amide bonds (L1 is -NHC(0)(CH2)mR1 or -NH(CH2)mR1; L2 is R3NHC(0)-) was evaluated (table 10). Activities in the cellular assay up to one digit micromolar range were observed. Different basic moieties as well as benzamide and phenylboronic acids were evaluated as substituent R1 in residue L1. In compound NK-298 one amide bond was exchanged to a secondary amine reaching about 2-fold lower activity than compound NK-371 .
Table 10. Inhibitory activity of compounds with reversed amide bonds with different -NHC(0)(CH2)mR1 and -NHCH2(CH2)R1 groups of residue L1 against isolated DENV and WNV proteases, the off-targets thrombin and trypsin, in the cellular DENV protease reporter assay (DENV2proHel_a) and cytotoxicity in HeLa cells.
Figure imgf000074_0001
Figure imgf000075_0001
The passive membrane permeability of compound NK-189, NK-344, NK-316, NK- 323, NK-365 and a set of drug references was evaluated using the precoated tri layer parallel artificial membrane permeability assay (PAMPA). The permeability of the reference compounds, i.e. Caffeine, Carbamazepine and Phenytoin, was in accordance with literature values, but compounds NK-344, NK-316, NK-323 and NK-365 appeared to be non-permeable (see table 11). This offers an explanation for the lower activity of these compounds in the DENV2proHel_a system compared to their inhibition in the biochemical DENV assay, although an active transport or endocytotic uptake of these basic compounds is possible, especially in hepatocytes such as the Huh-7 cell line used for the viral replication experiments. Compound NK-189, which had the highest activities in the DENV2proHel_a system, is somewhat permeable, but significantly less than the tested standards caffeine, carbamazepine and phenytoin. Notably, the molecular mass of the cell-active compound NK-189 (502 g/mol) is very close to the Lipinski criteria, whereas compounds NK-344, NK-316, NK-323 and NK-365 violate this rule.
Table 11. Passive membrane permeability of selected compounds determined in the PAM PA assay.
Figure imgf000076_0001
Carbamazepine 236 35.7±5.2 171.6±6.7 8.1 2,3
Figure imgf000076_0003
NK-189 502 7.5±0.7 171.0±11.4 1.6 12.0
Figure imgf000076_0004
NK-316 598 not permeable at detectable range
Figure imgf000076_0005
NK-365 667 not permeable at detectable range
Figure imgf000076_0002
Further evaluation of the activity of selected compounds against viral replication in cell culture was performed in a virus titer reduction assay using DENV-2 infected human carcinoma cells (Huh-7). The inhibitors did not show relevant cytotoxicity in Huh-7 cells except compound NK-220. Under its treatment around 60% of the Huh- 7 cells survived.
In accordance with inhibition in the cellular DENV-2 protease assay (DENV2proHeLa), the tested compounds reduced the virus titer with ECso values in the low micromolar or high nanomolar range (see Figures 1 to 5). Whereas the pyridin-2-yl guanidine derivative NK-361 and the 4-benzyloxybenzoic acid derivatives NK-307 and NK-347 showed moderate antiviral activity, treatment with, for example, compounds NK-189, NK-200, NK-359, NIK-288 and NK-357 at a concentration of 10 mM reduced viral titers to less than 25% of the control. Antiviral dose-response-curves were obtained for the following compounds that showed the most promising activity profiles in the DENV2proHel_a assay: NK-189, ML-02, and NK-288, NK-375, NK-479, NK-428, NK-433, NK-469, NK-429 and NK-478 (see Figures 4 and 5). Of these, compound NK-428 had the highest antiviral activity with an ECso of 0.24 mM and an EC90 of 4.83 mM.
Compound Synthesis
The following abbreviations for reagents and compounds are used:
COMU: [(1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino- carbenium-hexafluorophosphate]
Fmoc-OSu: N-(9H-Fluoren-9-ylmethoxycarbonyloxy)-succinimide
HATU: 0-(7-Azabenzotriazol-1-yl)-A/,A ,iV,A/'-tetramethyluroniumhexafluoro- phosphate,
2-Abz: 2-aminobenzoic acid Nle: norleucine
General procedure for the synthesis of (4-benzyloxy)-phenylqlvcine derivatives
A solution of 4-hydroxyphenylglycine (1 equiv) in 1 M NaOH (1 equiv) and a solution of CuS04-5H20 (0.67 equiv) in water (10 mL) were warmed to 50 °C under stirring. Both solutions were combined, and the reaction mixture was stirred for 30 min at 50 °C. After cooling on an ice-water bath, the blue precipitate of the amino acid Cu- complex separated immediately. The precipitate was isolated, washed with water, and dried. The Cu-complex was dissolved in MeOH (25 mL) and 1 M NaOH (1 equiv). The corresponding benzyl bromide (1.1 equiv) was added, and the mixture was stirred at room temperature overnight. The insoluble Cu-complex of the resulting ether was collected by filtration, washed with MeOH, and then with water to remove excess of the unreacted starting material. Finally, 1 M HCI (2 equiv) was added to release the product from the Cu-complex. The precipitated product was washed with water and dried under reduced pressure. The crude product was used for subsequent synthetic steps without further purification.
Scheme 1 : Synthesis of (4-benzyloxy)-phenylglyeine derivatives
Figure imgf000078_0001
(a) 1 eq NaOH, 1 eq CuSC -S H2O; (b) 1 eq NaOH, respective alkylating agent, MeOH; (c) HCI; (d) Fmoc-OSu, DIPEA, H20/MeCN ; (e) HCI.
General procedure for the synthesis of Ng-Fmoc-protected amino acids A solution of the amino acid or its hydrochloride salt (1 equiv) and DIPEA (2-3 equiv) in 30 mL of ACN/water (1 :1) was stirred at room temperature for 20 min, then Fmoc- OSu (0.9-0.95 equiv) was added. After 30 min, most of the solids were dissolved. When the reaction was completed (1 ,5-2h), the solution was acidified to pH 1-2 using 1 N aqueous HCI, 10 mL of water were added, and the mixture was allowed to stir for an additional hour. Finally, the resulting precipitate was collected by filtration, washed with water, dried under reduced pressure, and used as crude product without further purification for solid phase peptide synthesis. General procedure for the synthesis of (4-phenvioxy)-phenylqlvcine derivatives 4 A molecular sieves were given to a mixture of A/a-Fmoc-(4-hydroxy)-phenylglycine (1 equiv), the respective arylboronic acid (2 equiv) and Cu(OAc)2 (2 equiv). 10 ml_ DCM was added and the resulting colored suspension was treated with 4 equivalents pyridine. After stirring the reaction mixture over night at ambient atmosphere, the suspension was dried under reduced pressure. The resulting residue was suspended in ethyl acetate, filtered and the filtrate was washed 3 times with 1 N HCI (scheme 2). The organic layer was dried over anhydrous MgSCK filtered and concentrated in vacuo. The crude mixture was purified by flash chromatography if necessary or used for subsequent synthetic steps without further purification.
Scheme 2: Synthesis of (4-phenyloxy)phenylglycine derivatives
Figure imgf000079_0001
(a) Fmoc-OSu, DIPEA, FfcO/MeCN; (b) HCI; (c) phenyl boronic acid, Cu(OAc)2, pyridine, DCM.
General procedures for Fmoc-protection of building blocks
Procedure A: Sodium bicarbonate (2 equiv) and the amine were dissolved in water. If the amine was not soluble, MeCN was added dropwise until all solids were dissolved. The solution was cooled with an ice-water bath and a solution of Fmoc- OSu (1.2 equiv) in MeCN was added drop wise. After 20 minutes this mixture was allowed to warm to room temperature and was stirred overnight. The solvent was then removed in vacuo and the resulting suspension was extracted 3 times with ethyl acetate. The organic layer was back extracted twice with saturated sodium bicarbonate solution. The combined aqueous layers were acidified to a pH of 1-3 with a 1 N HCI solution. The resulting suspension was extracted 3 times with ethyl acetate and the combined organic layers were washed with 1 N HCI and water. The organic layer was dried over anhydrous MgSQi and the solvent was removed in vacuo. The protected amine was purified by flash chromatography if necessary.
Procedure B: Sodium bicarbonate (2 equiv) and the amine were dissolved in HhO/MeCN (1 :1 v/v). The solution was cooled with an ice-water bath and a solution of Fmoc-OSu (1.0 equiv) in MeCN was added drop wise. After 20 minutes this mixture was allowed to warm to room temperature and was stirred overnight. The solvent was then removed in vacuo and the resulting suspension was acidified to a pH of 1-3 with a 1 N HCI solution. This mixture was extracted 3 times with ethyl acetate or DCM and the combined organic layer was washed 2 times with 1 N HCI and water. The organic layer was dried with anhydrous MgSCM and the solvent was removed in vacuo. The protected amine was purified by flash chromatography if necessary.
General procedure for hydrogenation of building blocks
To a suspension of a nitrile (5.0 mmol) and Pd/C (10 mg) in ethanol (20 ml_) was added 2 mL of concentrated HCI. This mixture was stirred under H2 at room temperature overnight. The mixture was filtered through celite and concentrated to obtain the corresponding amine.
General procedure for the benzylation of hvdroxybenzoic acids To a suspension of the respective hydroxybenzoic acid (1 equiv) and K2CO3 (5 equiv) in DMF was added the respective benzyl bromide (2.1 equiv). The solution was warmed to 80°C and was stirred for 2-3 days. The reaction mixture was diluted with ethyl acetate, washed with 1 N HCI, saturated NaHCCb and water. The organic layer was dried over anhydrous MgSC and concentrated to give an oil. This was dissolved in a mixture of MeOH and 2 N NaOH (1 :1 ) and was stirred at 50°C for 2 hours. After the pH was adjusted to 2 with concentrated HCI, the resulting precipitate was collected by filtration and dried under reduced pressure.
General procedure for the synthesis of f4-ohenyloxy¾benzoic acid derivatives 4 A molecular sieves were given to a mixture of Methyl 4-hyd roxybenzoate (1 equiv), the respective arylboronic acid (2 equiv) and Cu(OAc)2 (2 equiv). 10-15 mL DCM was added and the resulting colored suspension was treated with 4 equivalents pyridine. After stirring the reaction mixture overnight at ambient atmosphere, the suspension was dried under reduced pressure. The resulting residue was suspended in H2O and ethyl acetate. The suspension was filtered and the filtrate was washed 2 times with 1 N HCI, 0.2 N NaOH and brine. The organic layer was dried over anhydrous MgSC>4, filtered and concentrated in vacuo. The methylester of the carboxylic acid was cleaved by solving the compound in a solution of LiOH (2-3 equiv) in THF/H2O (2:1 , 10 ml_). The solution was stirred for 3 h. The mixture was adjusted to pH 2 with concentrated HCI and a precipitate was formed. The precipitate was filtered, washed with several portions of H2O and dried under reduced pressure. The crude mixture was purified by flash chromatography if necessary or used for subsequent synthetic steps without further purification.
General procedure for peptide coupling of (4-benzyloxy)-phenylglvcine containing compounds in solution
The respective carboxylic acid (1.0 equiv) and HATU (1.2 equiv) were suspended in DCM (2-5 mL). The mixture was cooled to 0°C and the respective amine (1.2 equiv) was added prior to the drop wise addition of TMP (1 .3-3.2 equiv). DMF was added until all solids were dissolved and the mixture was stirred at room temperature for 2 hours. The reaction was quenched with the addition of a solution of TFA in DCM (5%, 5 mL) (scheme 3). All solvents were removed in vacuo and the resulting residue was dissolved in ethyl acetate which was washed 2 times with 1 N HCI, 0.05 N NaOH and water. The combined organic phase was dried over anhydrous MgS04, concentrated under reduced pressure and purified by preparative HPLC. After purification, all organic solvents were evaporated and the peptides were freeze- dried in water. Scheme 3: Exemplary peptide coupling in solution
Figure imgf000082_0001
(a) HATU, respective amine, TMP, DCM/DMF, 0°C -> rt; (b) TEA, DCM. General procedure for guanidinylation of amines
Procedure A: To the respective amine (0.02 mmol) and A/,/V-di-(ferf- butoxycarbonyl)-S-methylisothiourea (0.02 mmol) in THF (5 mL) was added triethylamin (0.06 mmol). The solution was cooled on an ice-water bath and mercuric chloride (0.02 mmol) was added. After 15 min the stirred suspension was allowed to warm to room temperature and stirred until no leftover the amine was observed. The solvent was removed in vacuo and the residue was suspended in ethyl acetate and filtered through celite. The filtrate was washed several times with water and brine, dried over MgSC and concentrated under reduced pressure. The resulting residue was dissolved in TFA/DCM (1:1, 10 mL) and allowed to stir for 3 hours (scheme 4). The mixture was concentrated and further co-evaporated with several portions of toluene. The residue was purified by preparative HPLC and freeze-dried in water.
Procedure B: The respective amine (1.0 equiv), /V,/V'-di-Boc-1H-pyrazole-1- carboxamidine (1.5 equiv), DMAP (0.3 equiv), and DIPEA (1.0 equiv) were dissolved or suspended in MeOH (5-20 mL). The mixture was allowed to stir for 2 or 3 days at room temperature. The solvent was removed in vacuo and the resulting residue was dissolved in ethyl acetate or DCM. The organic phase was washed with 0.1 N HCI, 1 N NaOH and water. The combined organic phases were dried over anhydrous MgSCM and concentrated under reduced pressure. The resulting solid or oil was dissolved in TFA/DCM (1 :1 , 5-10 ml_) and was stirred for 2-3 hours. The mixture was concentrated and further co-evaporated with several portions of toluene. The residue was purified by preparative HPLC and freeze-dried in water.
Scheme 4: Exemplary guanidinylation reactions of amines
Figure imgf000083_0001
NK-189, NK-285, NK-288, NK-307 NK-200
(a) /V,A/'-Di-Boc-S-methylisothiourea, TEA, HgCh, THF, 24 h; (b) TFA, DCM (1 :1); (c) L/,L/' -Di-Boc-1 H-pyrazole-1 -carboxamidine, DMAP, DIPEA, MeOH, 48 h.
Synthesis of compounds with reversed amino acids
Compounds with reversed amino acids were synthesized analogous to the general procedure for peptide coupling of (4-benzyloxy)-phenylglycine containing compounds in solution. For Fmoc deprotection in solution the corresponding starting material (1 .6 mmol) was added to a solution of piperidine in DCM (20%, 15 ml_). When the reaction was completed, all solvents were removed in vacuo and the resulting residue was co-evaporated several times with toluene. Hexane was added to the residue and the resulting suspension was centrifuged. The supernatant was removed and hexane was added a second time to the solid and it was centrifuged again. After removal of the supernatant the resulting oil was dried under reduced pressure and purified (scheme 5).
Scheme 5: Exemplary synthesis of compounds with reversed amide bonds
Figure imgf000084_0001
INK-250 NK-260 0H
(a) HATU, pentan-1 -amine, TMP, DCM/DMF, 0°C -> rt; (b) TFA, DCM; (c) piperidine, DCM; (d) HATU, respective acid, TMP, DCM/DMF, 0°C -> rt; (e) piperidine, DCM.
General procedure for the synthesis of inhibitors and intermediates on solid support
All peptide and compound sequences were assembled by stepwise solid-phase synthesis on Rink amide, 2-chlorotrityl chloride resin or 1-diol resin using the standard Fmoc-strategy. Solid phase synthesis was done manually in plastic syringes equipped with a frit; all steps were performed at room temperature under continuous shaking.
Synthesis of compounds with C-terminal amides (L1 is -CfO)NH(CH2)mR1¾
The Rink amide resin (loading capacity 0.68 mmol/g) was pre-swollen in DCM for at least 20 min and then washed 3 * with DMF. For Fmoc group deprotection, a piperidine solution (10-20% in DMF) was added 2 c for 10 and 5 min. Following each deprotection or coupling step, the resin was washed with 3 c DMF, 3 c DCM, and again 3 c DMF. COMU/TMP were used for coupling steps. In detail, the coupling solution contained the M -Fmoc-protected amino acid or N-terminal carboxylic acid capping group (3.0 equiv), COMU (3.0 equiv), and TMP (3.9 equiv) in DMF (-1.0 mt_ per 100 mg of resin). The solution was added to the resin, and the reaction was shaken for 90-180 min. Afterwards, the resin was washed as described before. Fmoc deprotection and coupling steps were iteratively repeated until the desired sequence was obtained. For the sequences containing 4-(OH)-phenylglycine, a final treatment with piperidine solution (25% in DMF) for 30 min was carried out after coupling of the N-terminal cap to cleave any formed esters at the unprotected hydroxyl group. The resin loaded with the finished peptide or peptide hybrid was washed 5 * with diethyl ether and dried under reduced pressure. The final product was cleaved off the resin with TFA/triisopropylsi!ane/hkO solution (95:1:4, 1-2 mL per 100 mg resin) or TFA/H2O/DCM solution (95:2.5:2.5, 1-2 mL per 100 mg resin), and the mixture was shaken for at least 2 h. The cleavage solution was dispensed into cold diethyl ether (35 mL per 100 mg of resin), and the resulting precipitate was centrifuged (4000 g, 10 min), washed with diethyl ether, and dried under reduced pressure. If the peptides were soluble in diethyl ether, the ether phase was washed 2 times with a small amount of water and all solvents were removed in vacuo. The resulting residue was co-evaporated with toluene several times (scheme 6).
Scheme 6: Exemplary solid phase peptide synthesis on Rink amide resin
Figure imgf000085_0001
NK-375 (a) piperidine, DMF; (b) COMU, TMP, DMF; (c) Fmoc-4-benzyloxy-L-Phg, COMU, IMP, DMF; (d) 4-phenoxybenzoic acid, COMU, TMP, DMF; (e) TFA, H20, TIPS (95:4:1 v/v).
Synthesis of compounds with C-terminal amines, carboxylic acids and alcohols (L1 is -C(0¾NH(CH2)mR1 )
The 2-chlorotrityl chloride resin (loading capacity 1.6 mmol/g) was pre-swollen in DCM for at least 20 min and then washed 3 c with DMF. The first amino acid or respective building block (1.2-3.0 equiv) was added to the resin in the syringe and 1 mL of DMF and TMP (3 equiv) were added. If a hydroxyl functional group was coupled to the solid support in the loading step, 1 mL of pyridine (per 100 mg resin) was added instead of the DMF/TMP mixture. The resin was shaken overnight and capped before the Fmoc deprotection step. Therefore, the resin was washed with 3 x DMF, 3 x DCM, and again 3 c DMF and a capping solution (DCM/MeOH/TMP 80:15:5 (v/v), 1 mL per 100 mg resin) was added 2 c for 20 min. The resin was washed with 3 c DMF, 3 c DCM, and again 3 c DMF. For Fmoc group deprotection, a piperidine solution (10-20% in DMF) was added 2 c for 10 min. Following each deprotection or coupling step, the resin was washed with 3 c DMF, 3 c DCM, and again 3 c DMF. COMU/TMP were used for coupling steps. In detail, the coupling solution contained the L/a-Fmoc-protected amino acid or N-terminal carboxylic acid capping group (3.0 equiv), COMU (3.0 equiv), and TMP (3.9 equiv) in DMF (~1.0 mL per 100 mg of resin). The solution was added to the resin, and the reaction was shaken for 90-180 min. Afterwards, the resin was washed as described before. Fmoc deprotection and coupling steps were iteratively repeated until the desired sequence was obtained. The resin loaded with the finished peptide or peptide hybrid was washed 5 c with diethyl ether and dried under reduced pressure. The final product was cleaved off the resin 3 times with TFA/DCM solution (1 :99, 1-2 mL per 100 mg resin), and the mixture was shaken for at least 30 minutes. The cleavage solution was dispensed into a round bottom flask, washed 2 times with H2O, dried under reduced pressure and co-evaporated with toluene several times (scheme 7). Scheme 7: Exemplary solid phase peptide synthesis on chlorotrityl chloride resin
Figure imgf000087_0001
(a) TMP, DMF, 16h; (b) DCM/MeOH/TMP (80:15:5 v/v); (c) piperidine, DMF; (d) Fmoc-4-benzyloxy-i-Phg, COMU, TMP, DMF; (e) hexanoic acid, COMU, TMP, DMF; (f) TFA, DCM; (g) pyridine, DMF, 48h.
Synthesis of compounds with C-terminal phenylboronic acids (L1 is -C(Q¾NHfCH2)mR1i
The peptide synthesis with 1-diol resin (loading capacity 0.6 mmol/g) was performed with Rink amid resin. The first building block (1.3 equiv) was added to the dry resin in the syringe and 1 mL ofTHF and TMP (1.3-2.0 equiv) were added. The resin was shaken overnight and washed with 6 c DMF and 3 c DCM. For Fmoc group deprotection, a piperidine solution (10-20% in DMF) was added 2 c for 10 and 5 min. Following each deprotection or coupling step, the resin was washed with 3 c DMF, 3 x DCM, and again 3 c DMF. COMU/TMP were used for coupling steps. In detail, the coupling solution contained the x-Fmoc-protected amino acid or N- terminal carboxylic acid capping group (3.0 equiv), COMU (3.0 equiv), and TMP (3.9, equiv) in DMF (~1.0 mL per 100 mg of resin). The solution was added to the resin, and the reaction was shaken for 90-180 min. Afterwards, the resin was washed as described before. Fmoc deprotection and coupling steps were iteratively repeated until the desired sequence was obtained. The resin loaded with the finished peptide or peptide hybrid was washed 5 c with diethyl ether and dried under reduced pressure. The final product was cleaved off the resin 2-3 times with TFA/H2O/THF solution (10:20:80 (v/v), 1-2 mL per 100 mg resin), and the mixture was shaken for at least 2 hours. The cleavage solution was dispensed into a round bottom flask, washed 2 times with H2O, dried under reduced pressure and co-evaporated with toluene several times (scheme 8).
Scheme 8: Exemplary solid phase peptide synthesis on 1-diol resin
Figure imgf000088_0001
(a) DIPEA, THF, overnight; (b) Fmoc-4-benzyloxy-L-Phg, COMU, TMP, DMF; (c) piperidine, DMF; (d) 4-phenoxybenzoic acid, COMU, TMP, DMF; (e)TFA, H2O, THF.
All crude peptides were purified by preparative HPLC on an AKTA Purifier, GE Healthcare (Germany), with an RP-18 pre and main column (Rephospher, Dr. Maisch GmbH, Germany, C18-DE, 5 pm, 30 mm c 16 mm and 120 mm c 16 mm). The following conditions were used: Method A: eluent A, water (0.1% TFA); eluent B, methanol (0.1% TFA); flow rate, 8 mL/min; and gradient, 10% B (2.5 min), 100% B (23.5 min), 100% B (26 min), 10% B (26.1 min), and 10% B (30 min). Method B: eluent A, water (0.1% TFA); eluent B, acetonitrile (0.1% TFA); flow rate, 8 mL/min; and gradient, 10% B (2.5 min), 100% B (23.5 min), 100% B (26 min), 10% B (26.1 min), and 10% B (30 min). Detection was performed at 214, 254, and 280 nm. After purification, methanol was evaporated, and the peptides were freeze-dried in water and stored at -20 °C. Enantiomeric purity of inhibitors
The enantiomeric purity HPLC analysis was carried out on a Jasco HPLC system equipped with a ReproSil Chiral-NR column (8 pm, 150 * 4.6 mm) and a Jasco LJV- 2070 Plus Intelligent UV/VIS Detector. The following method was used: eluent A: water (0.1% TFA); eluent B: methanol (0.1% TFA); injection volume: 20 pL; flow rate: 1 mL/min; isocratic elution with 30% A and 70% B for 25 or 35 minutes. Chromatograms recorded at 254 nm were used for assessment.
Selectivity and Binding Mode
To determine the selectivity of the inhibitors, biochemical assays with the serine proteases thrombin and trypsin were performed. None of the compounds showed significant inhibition of thrombin at 25 pM, which stands in marked contrast to some of the flaviviral protease inhibitor series before, in particular the peptide boronic acids. Three of the compounds were moderately active against trypsin in a screen (above 50% inhibition at 50 pM). The IC50 of compounds NK-191, NK-284 and NK- 335 against trypsin were determined to be 44.6, 43.5 and 31.1 pM, respectively. All other compounds had low off-target activities.
Cheng-Prusoff plots of several inhibitors against DENV and WNV proteases indicate a competitive inhibition mechanism for this inhibitor series.
Furthermore, tryptophan quenching assays with compounds NK-305, NK-119 and NK-406 and competition with aprotinin indicated binding of the active site of the DENV-2 protease. All compounds inhibited the protease in a cellular environment at the one digit micromolar range. The addition of the compounds to DENV-2 protease results in concentration-dependent quenching of the fluorescence of tryptophan residues of the protease (see Figure 6). Compound NK-305 had an ICso of approximately 6 pM against DENV and WNV in in vitro assays. The results of the fluorescence quenching experiments indicate a binding of the inhibitor in the active site of the enzyme. As shown in figure 6, the addition of compound NK-305 to DENV-2 protease results in concentration-dependent quenching of the fluorescence of tryptophan residues of the protease. Aprotinin is a known competitive inhibitor for DENV protease. Aprotinin displaces compound NK-305 from the DENV-2 protease, providing further evidence for specific binding at the active site (see figure ø). Compound NK-305 can be used as quencher for the protease autofluorescence, particularly in displacement assays for compounds that lack moieties capable of interacting with tryptophan fluorescence. In comparison to commercially available inhibitors for the flaviviral protease (e.g. aprotinin), the inhibitor offers an advantage through its high potency in both biochemical and cellular assays.
DENV and WNV Protease relative inhibition assays
Relative inhibition of proteases at the specific concentration of an inhibitor to an untreated control is given by “% (concentration)" The DENV and WNV protease relative inhibition assays were performed in black 96-well V-bottom plates (Greiner Bio-One, Germany) using a BMG Labtech Fluostar OPTIMA Microtiter fluorescence plate reader at an excitation wavelength of 320 nm and a monitored emission wavelength of 405 nm. Stock solutions of the inhibitors (10 mM in DMSO) were diluted to a final concentration of 50 mM in triplicates, and preincubated for 15 min with the DENV protease (100 nM) or WNV protease (150 nM) in the assay buffer [50 mM Tris-HCI pH 9, ethylene glycol (10% v/v), and 0.0016% Brij 58 (Polyethylene glycol hexadecyl ether). The reaction was then initiated by the addition of the FRET substrate (final concentration of the FRET substrate 50 mM) to obtain a final assay volume of 100 mI_ per well. The enzymatic activity was monitored for 15 min and determined as a slope of relative fluorescence units per second (RFU/s) for each concentration. Percentage inhibition was calculated relative to a positive control (without the inhibitor), as a mean of the triplicates and respective standard deviation.
Measurements of the ICso values for DENV and WNV inhibitors were performed in black 96-well V-bottom plates (Greiner Bio-One, Germany) using a BMG Labtech Fluostar OPTIMA Microtiter fluorescence plate reader at an excitation wavelength of 320 nm and a monitored emission wavelength of 405 nm. Eight inhibitor concentrations covering the range 0-3, 0-6, 0-10, 0-15, 0-30 or 0-50 mM were chosen for analysis. Dilutions were made starting from 10 mM inhibitor stock solutions in DMSO and final concentrations were measured in triplicates The inhibitors were preincubated for 15 min with the DENV protease (100 nM) or WNV protease (150 nM) in the assay buffer (50 mM Tris-HCI pH 9.0, ethylene glycol (10% v/v), and 0.0016% Brij 58). The enzymatic reaction was then initiated by the addition of the FRET substrate (final concentration 50 mM) to obtain a final assay volume of 100 mI_ per well. The enzymatic activity was monitored for 15 min and determined as a slope of relative fluorescence units per second (RFU/s) for each concentration. The mean and the standard deviation of the triplicates plotted against the corresponding concentration were used to determine the ICso-values on Prism 6.01 (Graphpad Software, Inc.) using non-linear dose-response curves with variable slopes.
FRET substrates synthesis
Dengue and WNV protease FRET substrates peptides, with respective sequences 2-Abz-Nle-Lys-Arg-Arg-Ser-(3-N02)-Tyr-NH2 (Km = 105 mM) and 2-Abz-Gly-Lys-
Lys-Arg-Gly-(3-N03)-Tyr-Ala-Lys-NH2 (Km = 36 mM), were synthesized according to standard Fmoc solid phase peptide synthesis procedure. Purity was determined by RP-HPLC using method A.
Determination of the dissociation constant (Ki)
To determine the dissociation constant of the enzyme-inhibitor complex ( ), ICso values of the inhibitor at different substrate concentrations (50, 100, 150, and 200 mM) were measured using the previous parameters. For the calculation of the K, values, the corrected data considering the inner filter effect was used. Cheng- Prusoffs method was used to calculate the Ki. Linear regressions were performed using Prism 6.01 (Graphpad Software, Inc.).
HPLC-based DENV and WNV protease assays
After performing the fluorimetric assay (15 min), the enzymatic reaction was stopped by adding 10 pL of TFA (4%) to each well, and the samples were cooled to 4 °C for 30 min. The HPLC analysis was carried out on a Jasco HPLC system equipped with a RP-18 column Phenomenex Luna Cis(2) (5 pm, 150 c 3 mm) and a FP-2020 plus fluorescence detector (excitation, 320 nm; emission, 405 nm). The following conditions were used for DENV assay: flow rate: 1.0 mL/min; eluent A: water (0.1% TFA); eluent B: acetonitrile (0.1% TFA); and gradient: 10% B (0 min), 10% B (1 min), 95% B (9.5 min), 95% B (9.6 min), 10% B (12.6 min), and 10% B (15 min). Different conditions were used for the WNV assay: flow rate: 1.2 mL/min; eluent A: water (0.1 % TFA); eluent B: acetonitrile (0.1% TFA); and gradient: 10% B (0 min), 20% B (1 min), 95% B (5 min), 95% B (6 min), 10% B (6.1 min), and 10% B (8 min). Percentage inhibition was calculated relative to a positive control (without the inhibitor). All experiments were performed in triplicate and the final value was obtained as the average.
Thrombin assay
Thrombin was purchased from Sigma-Aldrich (Germany). Continuous fluorimetric assay was done in black 96 well V-bottom plates (Greiner Bio-One, Germany), using a BMG Labtech Fluostar OPTIMA microtiter fluorescence plate reader. Excitation wavelength of 355 nm and an emission wavelength of 460 nm were used. The inhibitors (final concentration 25 mM, from 10 mM stock solutions in DMSO) were preincubated with thrombin (10 nM) in the assay buffer (50 mM Tris-HCI pH 7.5, 150 mM NaCI, 0.05% Tween 20) for 15 min. Enzymatic cleavage was initiated by the addition of the Boc-Val-Pro-Arg-AMC substrate (Bachem, Germany) to yield a final concentration of 50 pM. The activity of thrombin was monitored for 15 min and determined as a slope of relative fluorescence units per second (RFU/s). All experiments were performed in triplicates and percentage inhibition was calculated as the mean and respective standard deviation of the values. Values were obtained in relation to a positive control.
Trypsin assay
Trypsin was purchased from Sigma-Aldrich (Germany). The inhibition of trypsin was determined as described before. Continuous fluorimetric assay was done in black 96 well V-bottom plates (Greiner Bio-One, Germany), using a BMG Labtech Fluostar OPTIMA microtiter fluorescence plate reader. Excitation wavelength of 355 nm and an emission wavelength of 460 nm were used. The inhibitors (final concentration 50 pM, from 10 mM stock solutions in DMSO) were preincubated with trypsin (1 nM) in the assay buffer (50 mM Tris-HCI pH 7.5, 150 mM NaCI, 0.05% Tween 20) for 15 min. Enzymatic cleavage was initiated by the addition of the Boc-Val-Pro-Arg-AMC substrate (Bachem, Germany) to yield a final concentration of the Boc-Val-Pro-Arg- AMC substrate of 50 mM. The activity of thrombin was monitored for 15 min and determined as a slope of relative fluorescence units per second (RFU/s). All experiments were performed in triplicates and percentage inhibition was calculated as the mean and respective standard deviation of the values. Values were obtained in relation to a positive control (without inhibitor).
Cytotoxicity
Cytotoxicity of the above described compounds was tested by determination of the Cell’s viability in Huh-7 or Hela cells in presence of compound dilutions, which was determined using CellTiter-Blue® (Promega) in accordance to the manufacturer’s instructions. Plates were prepared in parallel to the cell based DENV reporter or virus titer reduction assay with analogous treatment. Each concentration was assayed in triplicates. Cells were incubated with the compounds for 24 hours (HeLa cells) or 48 hours (Huh-7 cells). Afterwards the medium was removed and resazurin was added to the cells and the cells were incubated for 1 hour. Conversion of the dye resazurin to resorufin by metabolically active cells results in the generation of a fluorescent product. The fluorescence or absorption signal of the resulting reduced dye that was incubated with treated cells was compared with the fluorescence or absorption of untreated cells. For CCso determination of compounds a serial dilution of the compound was prepared and by plotting the relative signal compared to the untreated control in a non-linear dose-response curve, a compound concentration at which 50% of the cells are viable (CCso) can be determined.
Virus titer reduction assays
For virus titer reduction assays Huh-7 cells were seeded into 96-well plates at a density of 1 * 104 cells in 50 pi per well and incubated overnight. The next day, cells were infected with DENV serotype 2 for 2 h at a MOI of 1 , before medium change and compound addition. Infected cells were then incubated for 48 h in the presence of compound in triplicate wells. In order to determine the ECso, a range of serial diluted compound concentrations starting at 50 mM was used. After 48 h incubation, supernatants were harvested and triplicates pooled. These virus containing supernatants were used to determine the virus titer (reduction) by plaque assay. In a plaque assay VeroE6 cells were seeded into 24-well plates at a density of 2.5 c 10s cells/well and the next day infected for 1 h with 10-fold serial dilutions of virus supernatant ranging from 101 to 10~6. After medium exchange and addition of the plaque medium VeroE6 cells were incubated for 7 days. Cells were subsequently fixed with formaldehyde and plaques visualized by crystal violet stain. At a suitable dilution, plaques were counted, the virus titer calculated and plotted against the respective compound concentration. Plaques are readily visible where the cells have been lysed by viral infection. The titer of a virus stock can be calculated in plaque-forming units per milliliter. To determine the virus titer, the plaques are counted in an appropriate dilution (e.g. 105). The resulting virus titer is calculated from the dilution factor and the number of counted plaques. Virus titer reduction is determined by comparing the virus titer in compound-treated wells with the virus titer of untreated wells (in the same dilution series, e.g. 105). ECso values were derived from fitting data to a dose-response curve (variable-slope, nonlinear regression model) using Prism 6.01 (Graphpad Software, Inc.).
Cell culture experiments
For cell culture experiments HeLa, Huh-7 and Vero E6 cells were maintained in DMEM supplemented with 100 U/mL penicillin, 100 pg/mL of streptomycin, and 10% heat-inactivated FCS. During infection of Huh-7 cells, DMEM was supplemented with 10 mM HEPES.
Tryptophan fluorescence quenching
For tryptophan fluorescence quenching assays DENV protease (0.2 mM) was titrated with different concentrations (0, 1.25, 2.5, 5, 10, 20, 30, 40, 50 mM) of the inhibitor in assay buffer (50 mM Tris-HCI pH 9.0 for compounds NK-119 and NK- 406; 50 mM Tris-HCI pH 9.0, ethylene glycol (10% v/v), and 0.0016% Brij® 58 for compound NK-305) and the samples were incubated for 1 h at room temperature. For the fluorescence displacement experiment, the protease was incubated with the inhibitor (50 pM) and aprotinin (10 pM) under the same conditions. For inhibitors that lack absorbance at 340 nm and are not able to quench the emission signal from the active site tryptophan residues, the specific binding was demonstrated indirectly. Those inhibitors (50 pM) displace a less-affine (or lower concentrated) tryptophanquenching ligand, for which specific interaction with the protease active site has been confirmed. In this case the following compound was applied as the less-affine tryptophan-quenching ligand at a concentration of 10 mM:
Figure imgf000095_0001
Fluorescence of the enzymes (0.2 mM DENV) in the presence of aprotinin (10 mM), without the inhibitor, was also determined and used to correct the results of aprotinin displacement; in order to exclude aprotinin intrinsic fluorescence or inner filter effects. Fluorescence emission at 340 nm was monitored on a Tecan Safire II instrument (excitation at 280 nm). All experiments were performed in triplicates and the values were obtained in relative fluorescence units (RFU). Tryptophan quenching was plotted as a curve of the mean and standard deviation of the values against the respective concentrations of the inhibitor in comparison to the fluorescence displacement experiment using Prism 6.01 (Graphpad Software, Inc.).

Claims

Claims
1 . A compound of formula (I) or a pharmaceutically acceptable salt or ester thereof
Figure imgf000096_0001
wherein
L1 is -C(0)NH(CH2)mR1 » -C(0)piperidyl, -NHC(0)(CH2)mR1 or -NH(CH2)mR1 , wherein m is 0, 1 , 2, 3, 4, 5 or 6;
R1 is -NR4R5, -(HN)(H2N)C=NH, an N-containing heteroaryl group ora group of formula (II) or formula (III)
Figure imgf000096_0002
wherein X is nitrogen or CH,
L2 is -C(0)NH-, -SO2NH- or -NH- if L1 is -C(0)NH(CH2)mR1 or -C(0)piperidyf; or L2 is -NHC(O)- if L1 is -NHC(0)(CH2)mR1 or -NH(CH2)mR1;
I is 0 or 1 ;
R6 is -NR7R8, -CH2NR7R8, -(HN)(H2N)C=NH, -CH2(HN)(H2N)C=NH, -OH, -CH2OH, -OMe, -NHC(0)NH2I -C(0)NHNH2I -C(0)NHOH, -C(0)0H, -C(0)NH2 -CH2C(0)NH2 or -B(OH)2, wherein R7 and R8 are each independently -H or -Me wherein R4 and R5 are each independently -H or -Me; n is 0, 1 , 2, 3, 4 or 5;
R2 is independently for each occurrence -CF3, -OCH3, -CN, a halogen atom or a linear or branched alkyl group having 1 to 4 carbon atoms;
R3 is an alkyl group, a substituted or unsubstituted naphtyl group, a substituted or unsubstituted 2-phenylvinyl group, a substituted or unsubstituted 2-phenylethyl group, a heteroaryl group, a substituted or unsubstituted phenyl group or a substituted or unsubstituted benzyl group.
2. The compound of formula (I) or a pharmaceutically acceptable salt or ester thereof according to claim 1 , wherein m is 0 or 1 .
3. The compound of formula (I) or a pharmaceutically acceptable salt or ester thereof according to claim 1 or 2, wherein L1 is -C(0)NH(CH2)mR1 or -C(0)piperidyl.
4. The compound of formula (I) or a pharmaceutically acceptable salt or ester thereof according to anyone of claims 1 to 3, wherein R1 is a group of formula (Ha) or (lib)
Figure imgf000097_0001
wherein X and R6 are defined in claim 1.
5. The compound of formula (!) or a pharmaceutically acceptable salt or ester thereof according to anyone of claims 1 to 4, wherein n is 0, 1 or 2.
6. The compound of formula (I) or a pharmaceutically acceptable salt or ester thereof according to anyone of claims 1 to 5, wherein R2 is -CF3 or a halogen atom.
7. The compound of formula (I) or a pharmaceutically acceptable salt or ester thereof according to anyone of claims 1 to 6, wherein the substituted phenyl group of R3 is a group of formula (IVa), (IVb) and (IVc)
Figure imgf000097_0002
wherein R9 is a linear or branched alkoxy group having 1 to 6 carbon atoms, a substituted or unsubstituted phenyl group or a substituted or unsubstituted benzyloxy group, R10 is a substituted or unsubstituted phenyl group or a substituted or unsubstituted benzyloxy group and R11 is a substituted or unsubstituted phenyl, or the substituted benzyl group of R3 is a group of formula (V) wherein R12 is a substituted or unsubstituted phenyl.
8. Use of the compound of formula (I) or a pharmaceutically acceptable salt or ester thereof according to anyone of claims 1 to 7 for inhibiting the infectivity of a flavivirus in vitro.
9. Use of the compound of formula (I) or a pharmaceutically acceptable salt or ester thereof according to anyone of claims 1 to 7 for inhibiting the NS2B-NS3 serine protease of a flavivirus in vitro.
10. The compound of formula (I) or a pharmaceutically acceptable salt or ester thereof according to anyone of claims 1 to 9 for use in medicine.
11. The compound of formula (I) or a pharmaceutically acceptable salt or ester thereof according to anyone of claims 1 to 9 for use in a method of treating or preventing a flavivirus infection.
12. The compound of formula (I) or a pharmaceutically acceptable salt or ester thereof for use in a method of treating or preventing a flavivirus infection, wherein the flavivirus is selected from the group consisting of Dengue virus, West Nile virus, Zika virus or Tick-borne encephalitis virus.
13. Pharmaceutical composition containing the compound of formula (I) or a pharmaceutically acceptable salt or ester thereof according to anyone of claims 1 to 9.
14. Pharmaceutical composition according to claim 12, which is a prophylactic agent against a flavivirus infection.
15. Pharmaceutical composition according to claim 12, which is an agent for treating a flavivirus infection
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