Semisynthetic glycopeptide antibiotics with antiviral activity

Abstract

The present invention relates to compounds for use in the treatment of a disease or condition caused by Zika virus, Dengue virus, West Nile virus, Chikungunya virus, O`nyong`nyong virus, Sindbis virus or SARS-CoV-2 virus.

Description

[0001] Novel semisynthetic glycopeptide antibiotics with antiviral activity

[0002] FIELD OF THE INVENTION

[0003] In particular, the present invention relates to glycopeptide compounds, originally used as antibiotics, for use in the treatment of a disease or condition caused by a broad range of viruses.

[0004] In a preferred embodiment the compounds of the invention are for use in the treatment of a disease or condition caused by Flaviviruses, in particular the Zika virus, Dengue virus and West Nile virus.

[0005] In a preferred embodiment the compounds of the invention are for use in the treatment of a disease or condition caused by Alphaviruses, in particular Chikungunya virus, O'nyong'nyong virus and Sindbis virus.

[0006] In a preferred embodiment the compounds of the invention are for use in the treatment of a disease or condition caused by Zika virus, Dengue virus, West Nile virus, Chikungunya virus, O'nyong'nyong virus or Sindbis virus.

[0007] The invention also relates to pharmaceutical compositions as well as methods for treatment in this indication. The invention also relates to in vitro antiviral uses as well.

[0008] BACKGROUND ART

[0009] Besides developing vaccines to prevent serious viral diseases, antiviral drug development should also be a high priority to save lives. Testing potentially antiviral compounds is essential for the development of new medicines and is particularly relevant for viruses such as Zika virus (ZIKV), Chikungunya virus (CHIKV) and O'nyong'nyong virus (ONNV), to mention a few, for which no specific drugs are available.

[0010] Glycopeptide antibiotics (GPAs, Table 1) are used to treat Gram-positive bacterial infections and have also been shown to have antiviral activity against different viruses including Ebola virus (EBOV), MERS-CoV, and SARS-CoV (Acharya et al., 2022; Bereczki et al., 2021; Wang et al., 2016; Zhou et al., 2016). In addition, it was amply demonstrated that synthetically modified lipophilic derivatives of GPAs, obtained by deglycosylating glycopeptides and attaching hydrophobic groups to the resulting aglycones or pseudoaglycones, show a much more pronounced and much broader antiviral effect than natural antibiotics. Semisynthetic hydrophobic derivatives of the antibiotics vancomycin, teicoplanin, and ristocetin have been shown to inhibit influenza types A and B (IAV, IBV), both human immunodeficiency virus 1 and 2 (HIV-1, HIV-2), hepatitis C (HCV), DENV, YFV, tick-borne encephalitis virus (TBEV), WNV, ZIKV, herpes simplex virus types 1 and 2 (HSV-1, HSV-2), respiratory syncytial virus (RSV), and various coronaviruses including human coronavirus (hCoV) -229E, SARS-CoV, SARS-CoV-2, and MERS (Balzarini et al., 2003; Bereczki et al., 2021; Burghgraeve et al., 2012; N et al., 2016; Sziics et al., 2020). In addition to their ability to inhibit many different viruses, glycopeptide antibiotic derivatives act with different mechanisms, even within the same virus family. Among the inhibitory pathways described, the most common is that they act in the early stages of infection (Bereczki et al., 2021; Burghgraeve et al., 2012; N et al., 2016). The teicoplanin analogue LCTA-949 has been reported to exert its inhibitory effect on the E protein of DENV in the early stages of infection. The E protein of flaviviruses is a promising target for inhibiting virus entry because it is essential for both the virus particle binding to host cell receptors and for the fusion of the viral membrane with the target cell membrane (Burghgraeve et al., 2012). In the case of HCV, which also belongs to the flavivirus family, derivatives of teicoplanin aglycone were observed to inhibit the replication mechanism (Obeid et al., 2011).Togaviridae and Flaviviridae are similar RNA virus families that are characterized by a single mRNA that results in a polyprotein that is posttranslationally modified. Both families comprise enveloped, spherical viruses with positive-sense, single- stranded RNA genomes, which use similar entry (endocytosis) and cytoplasmic replication strategies. These viruses are arboviruses, i.e. arthropod-borne viruses, which means that they replicate arthropod (e.g. mosquito or tick) hosts, however, infect vertebrates as well. Thus, these are typically medically important viruses, which often cause severe diseases like encephalitis, transmitted via mosquito bites (Schmaljohn, A L et al. 1996) (Snyder, J.E. et al. 2014).

[0011] Zika virus (ZIKV), a member of the Flaviviridae family, belongs to the genus Flavivirus, along with other significant pathogens such as Yellow fever virus (YFV), West Nile virus (WNV), and Dengue virus (DENV). These viruses are notorious for causing serious public health concern globally (Kuno et al., 1998). ZIKV has been identified by WHO as a research priority in the context of emergencies and ISIDORE has listed it as a “Priority Preparedness Pathogen’’ (WHO, ISIDORE). ZIKV has an approximately 11 kb positive sense RNA genome. This RNA, when translated in the cytoplasm, forms a large polyprotein. This polyprotein is cleaved into three structural proteins: the capsid (C), pre-membrane / membrane (prM / M), and the envelope (E), along with seven non-structural proteins, namely NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 (Shan et al., 2020). The virus primarily attaches to host cells via the E protein and enters through clathrin-mediated endocytosis (Agrelli et al., 2019). The main vectors of ZIKV are mosquitoes, notably species from the Aedes genus, such as Aedes aegypti and Aedes albopictus (Marchette et al., 1969). ZIKV was first isolated from the serum of a Rhesus monkey in Uganda in 1947 (Dick et al., 1952). The first major mass outbreak was in 2007 on Yap Island Federated States of Micronesia (Duffy et al., 2009). From 2015 to 2016, the indigenous spread of ZIKV extended from Brazil to over 20 Latin American countries and reached North America (Campos et al., 2015; Enfissi et al., 2016; Musso & Gubler, 2016). In human infections, 80% are asymptomatic or present only mild symptoms, including fever, rash, muscle pain, and conjunctivitis (Hayes, 2009). On the other hand ZIKV infection has been associated with significant neurological complications, including Guillain-Barre syndrome (GBS) and microcephaly in fetuses and newborns (Oehler et al., 2014). Since 2013, 31 countries have reported cases of microcephaly and central nervous system malformations linked with ZIKV infection. There are several vaccines under development, based on different generations of technologies, but none is close to being commercialized (Wang et al., 2022). Currently, there is no specific treatment available against ZIKV.

[0012] Chikungunya virus (CHIKV) and O'nyong'nyong virus (ONNV) belonging to the genus Alphavirus in the Togaviridae family are another emerging arthropod-borne viruses, the main vectors of CHIKV, like ZIKV, are A. aegypti and A. albopictus, while ONNV's primary vectors are anopheline mosquitoes, Anopheles funestus and Anopheles gambiae (Powers et al., 2000; Weaver et al., 2018). CHIKV causes acute infection with high fever lasting 3-5 days followed by severe polyarthralgia (Weaver & Lecuit, 2015). The main complications reported are chronic joint pain, severe organ dysfunction and encephalitis in the elderly, and severe neonatal infection. Since 2023, an adult-only chikungunya vaccine has been available for prevention, but there is no specific antiviral drug against CHIKV. ONNV is known to be responsible for sporadic outbreaks in Africa; viral infection, similar to chikungunya fever, is associated with severe arthralgia (Tong Jia Ming et al., 2024). No effective vaccine or drugs are available against ONNV.

[0013] Danubia Ref.: P142354BRIEF DESCRIPTION OF THE INVENTION

[0014] The invention relates to a compound of general formula (I) for use in the treatment of a disease or condition caused by an enveloped, positive strand RNA virus selected from the group consisting of the Togaviridae and Flaviviridae family, said virus being internalized via endosome mediated endocytoses and by membrane fusion triggered by acidic endosomal pH,

[0015] wherein preferably the virus is of the Kitrinoviricota phylum,

[0016]

[0017] wherein

[0018] R1is OH or a substituted or unsubstituted monosaccharide group, preferably a monosaccharide amine, optionally acylated, preferably an N-acetyl monosaccharide amine, in particular of formula a)

[0019]

[0020] R2 is H, Cl -4 alkyl, preferably methyl or ethyl, wherein the R2OOC- may be R or S configuration,

[0021] in particular, R2 is carboxyl or a carboxylate ester having the formula

[0022]

[0023] R3 is H or OH or Cl -4 alkoxy, preferably H or OH,

[0024] X is l,4-diyl-lH-l,2,3-triazole or -(NH)-(CO)-, preferably X is -(NH)-(CO)-,

[0025] alternatively, X is l,4-diyl-lH-l,2,3-triazole, in particular when the compound is for use in the treatment of a disease or condition caused by a flavivirus,

[0026] L is -CH2-O-(CH2-CH2-O)n-CH2-CH2-CH2- or -CH2-(CH2)m-CH2-,

[0027] wherein n is 0 or 1 and m is 0 or 1 ,

[0028] preferably L is -CH2-(CH2)m-CH2- and m is 0 or 1, preferably 0,

[0029] with the provision that if X is l,4-diyl-lH-l,2,3-triazole, then n can not be 1,

[0030] and

[0031] Y is -(CF2)k-CF3

[0032] Danubia Ref.: P142354wherein

[0033] k is 3 to 7, preferably 4 to 7, more preferably 5 to 7, or

[0034] k is 3 to 7, preferably 5 to 7 or 4 to 6, in particular 5 to 6, in particular if the compound is for use in flaviviruses, or pharmaceutically acceptable salts thereof.

[0035] Said viruses have positive-sense, single-stranded RNA genomes.

[0036] Preferably said viruses are arboviruses, i.e. arthropod-borne viruses.

[0037] Preferably, the compound of the invention inhibits acidic pH-induced virion-endosome fusion, thus protecting the capsid protein from protease, preferably trypsin digestion, e.g. in the lyposome.

[0038] Preferably, the compound of the invention inhibits the virus entry process into the cell.

[0039] In a preferred embodiment the subject is in an initial stage of virus infection.

[0040] In a preferred embodiment the virus is a member of the Flaviviridae family, preferably the Flavivirus genus, preferably a Zika virus, a Dengue virus or a West Nile virus, in particular a Zika virus or a Dengue virus, highly preferably a Zika virus.

[0041] Preferably, the compound of the invention is for use in the treatment of a disease or condition caused by a virus of the Flavivirus genus, preferably a Zika virus, a Dengue virus or a West Nile virus, in particular a Zika virus or a Dengue virus, highly preferably a Zika virus.

[0042] In another preferred embodiment the virus is a member of the genus Alphavirus in the Togaviridae family in a subject, preferably a Chikungunya virus, a O'nyong'nyong virus or a Sindbis virus, particularly preferably a Chikungunya virus. Preferably, the compound of the invention is for use in the treatment of a disease or condition caused by an alphavirus, preferably a Chikungunya virus, a O'nyong'nyong virus or a Sindbis virus, particularly preferably a Chikungunya virus. Preferably, the antiviral effect of the compound is shown in an inhibition assay

[0043] In a preferred embodiment the inhibition assay is a CPE inhibition assay.

[0044] The inhibition assay is preferably carried out on a cell line from the species of the subject.

[0045] Preferably, the antiviral effect of the compound is shown by virus membrane fusion inhibition assay, preferably a capsid protection assay, e.g. by a measuring fusion of virions with liposomes.

[0046] In a preferred embodiment the compound of the invention is tested as inhibitory in one or more of these assays. Preferably, the compounds are selected from compounds (1), (2), (3), (4), (5), (6), (7) and (8), in particular (2), (3), (4), (5), (6), (7) and (8), more preferably (3), (4), (6), (7) and (8) (e.g. in case of alphaviruses), or highly preferably compounds (7) and (8).

[0047] In a preferred embodiment, the compound is for use in the treatment of a disease or condition caused by a member of the Togaviridae family, preferably of the Alphavirus genus, in subject. Preferably the subject is an animal, preferably a bird or a mammal, in particular a human, infected by an Alphavirus. Preferably, the antiviral effect of the compound is shown preferably in an inhibition assay, more preferably in a capsid protection or membrane fusion or in a CPE inhibition assay, highly preferably in an inhibition or a CPE inhibition assay in an animal, preferably a bird or a mammal, highly preferably a human cell line.

[0048] In a preferred embodiment the subject is in an initial stage of Alphavirus infection.

[0049] In a more or particularly preferred embodiment the Flavivirus is a Zika virus, a Dengue virus or a West Nile virus, in particular a Zika virus or a Dengue virus, highly preferably a Zika virus,

[0050] Danubia Ref.: P142354In a preferred embodiment, the invention relates to a compound of formula (LI) for use in the treatment of a disease or condition caused by a member of the genus Alphavirus in the Togaviridae family, preferably a Chikungunya virus, a O'nyong'nyong virus or a Sindbis virus, particularly preferably a Chikungunya virus,

[0051]

[0052] wherein

[0053] R1is OH or a substituted or unsubstituted monosaccharide group, preferably a monosaccharide amine, optionally acylated, preferably an N-acetyl monosaccharide amine,

[0054] X is l,4-diyl-lH-l,2,3-triazole or -(NH)-(CO)-,

[0055] L is -CH2-O-(CH2-CH2-O)n-CH2-CH2-CH2- or -CH2-(CH2)m-CH2-,

[0056] Y is -(CF2)k-CF3

[0057] wherein

[0058] n is 0 or 1 ,

[0059] m is 0 or 1, and

[0060] k is 3 to 7, preferably 3 to 6 or 3 to 5 or 3 to 4, or 5 to 6, in particular 3 to 5,

[0061] with the provision that if X is l,4-diyl-lH-l,2,3-triazole and n is 1, then k is less than 5,

[0062] or pharmaceutically acceptable salts thereof, wherein the antiviral effect of the compound is shown preferably in an inhibition assay, more preferably in a CPE inhibition assay, highly preferably in an inhibition or a CPE inhibition assay. In a preferred embodiment,

[0063] X is l,4-diyl-lH-l,2,3-triazole,

[0064] L is -CH2-O-(CH2-CH2-O)n-CH2-CH2-CH2-,

[0065] Y is -(CF2)k-CF3

[0066] wherein

[0067] n is 0 or 1 ,

[0068] k is 3 to 5, in particular 3 or 4 or 5.

[0069] In a further preferred embodiment,

[0070] X is -(NH)-(CO)-,,

[0071] L is -CH2-(CH2)m-CH2-,

[0072] Y is -(CF2)k-CF3

[0073] wherein

[0074] Danubia Ref.: P142354m is 0 or 1, preferably 0,

[0075] k is 3 to 7, in particular 4 to 7, or 5 to 7 or 5 to 6.

[0076] Preferably the monosaccharide group in N-acetyl glucosamine and the compound is a compound of Formula 1.2.

[0077]

[0078] wherein preferably

[0079] X is l,4-diyl-lH-l,2,3-triazole or -(NH)-(CO)-, preferably -(NH)-(CO)-,

[0080] L is -CH2-O-(CH2-CH2-O)n-CH2-CH2-CH2- or -CH2-(CH2)m-CH2-,

[0081] Y is -(CF2)k-CF3

[0082] wherein

[0083] n is 0 or 1,

[0084] m is 0, and

[0085] wherein k is 3 to 7, preferably 3 to 6 or 3 to 5 or 3 to 4, or 5 to 6, in particular 3 to 5,

[0086] with the provision that if X is l,4-diyl-lH-l,2,3-triazole and n is 1, then k is less than 5,

[0087] more preferably

[0088] X is -(NH)-(CO)-,

[0089] L is -CH2-CH2-,

[0090] wherein k is 4 to 7, preferably 5 to 6.

[0091] Preferably, the invention relates to a compound for use according to previous paragraph, wherein the compound is selected from the group consisting of

[0092]

[0093] Compound 3, Danubia Ref.: P142354

[0094]

[0095] or pharmaceutically acceptable salts thereof, or

[0096] a variant thereof having general formula 1.1 wherein R1is OH or a substituted or unsubstituted monosaccharide group, preferably a monosaccharide amine, optionally acylated, preferably an N-acetyl monosaccharide amine.

[0097] In a particular embodiment the compound is selected from the group consisting of compounds 6 and 7, or compounds 4 and 7, highly preferably the compound is compound 7.

[0098] In a preferred embodiment, the compound is for use in the treatment of a disease or condition caused by a member of the Flavivirus genus, in subject. Preferably the subject is an animal, preferably a bird or a mammal, in particular a human, infected by a Flavivirus. Preferably, the antiviral effect of the compound is shown preferably in an inhibition assay, more preferably in a capsid protection or membrane fusion or in a CPE inhibition assay, highly preferably in an inhibition or a CPE inhibition assay in an animal, preferably a bird or a mammal, highly preferably a human cell line.

[0099] Danubia Ref.: P142354In a preferred embodiment the subject is in an initial stage of Flavivirus infection.

[0100] In a more or particularly preferred embodiment the Flavivirus is a Zika virus, a Dengue virus or a West Nile virus, in particular a Zika virus or a Dengue virus, highly preferably a Zika virus,

[0101] Preferably the monosaccharide group in N-acetyl glucosamine and the compound is a compound of Formula 1.1.

[0102]

[0103] wherein

[0104] Ri is OH or a substituted or unsubstituted monosaccharide group, preferably a monosaccharide amine, optionally acylated, preferably an N-acetyl monosaccharide amine,

[0105] X is l,4-diyl-lH-l,2,3-triazole or -(NH)-(CO)-,

[0106] L is -CH2-O-(CH2-CH2-O)n-CH2-CH2-CH2- or -CH2-(CH2)m-CH2-,

[0107] Y is -(CF2)k-CF3

[0108] wherein

[0109] n is 0 or 1 ,

[0110] m is 0 or 1, and

[0111] k is 3 to 7, preferably 5 to 7 or 4 to 6, in particular 5, 6 or 7,

[0112] with the provision that if X is l,4-diyl-lH-l,2,3-triazole, then n can not be 1,

[0113] or pharmaceutically acceptable salts thereof.

[0114] Preferably the monosaccharide group in N-acetyl glucosamine and the compound is a compound of Formula 1.1.

[0115]

[0116] wherein

[0117] Danubia Ref.: P142354R1is a monosaccharide amine, optionally acylated, preferably an N-acetyl monosaccharide amine,

[0118] X is -(NH)-(CO)-,

[0119] L is - -CH2-(CH2)m-CH2-,

[0120] wherein

[0121] m is 0 or 1, and

[0122] Y is -(CF2)k-CF3

[0123] k is 3 to 7, preferably 4 to 7, more preferably 5 to 7,

[0124] or pharmaceutically acceptable salts thereof,

[0125] or pharmaceutically acceptable salts thereof.

[0126] Alternatively, preferably the monosaccharide group in N-acetyl glucosamine and the compound is a compound of Formula 1.2.

[0127]

[0128] wherein preferably

[0129] X is l,4-diyl-lH-l,2,3-triazole or -(NH)-(CO)-, preferably -(NH)-(CO)-,

[0130] L is -CH2-O-(CH2-CH2-O)n-CH2-CH2-CH2- or -CH2-(CH2)m-CH2-,

[0131] Y is -(CF2)k-CF3

[0132] wherein

[0133] n and m is 0,

[0134] m is 0 or 1, and

[0135] wherein k is 4 to 7,

[0136] more preferably

[0137] X is -(NH)-(CO)-,

[0138] L is -CH2-CH2-,

[0139] wherein k is 4 to 7, preferably 5 to 6.

[0140] wherein preferably

[0141] X is -(NH)-(CO)-,

[0142] L is -CH2-(CH2)m-CH2-,

[0143] Y is -(CF2)k-CF3

[0144] Danubia Ref.: P142354wherein

[0145] m is 0 or 1, preferably 0, and

[0146] wherein k is 4 to 7,

[0147] more preferably

[0148] L is -CH2-CH2-,

[0149] Y is -(CF2)k-CF3

[0150] wherein k is 5 to 6.

[0151] Preferably, the invention relates to a compound for use according to previous paragraph, wherein the compound is selected from the group consisting of

[0152] "

[0153]

[0154] "

[0155] Danubia Ref.: P142354

[0156] "" >

[0157] >

[0158]

[0159] or pharmaceutically acceptable salts thereof, or

[0160] a variant thereof having general formula 1.1 wherein R1 is OH or a substituted or unsubstituted monosaccharide group, preferably a monosaccharide amine, optionally acylated, preferably an N-acetyl monosaccharide amine.

[0161] In an embodiment the compound is selected from

[0162] In a particular embodiment the compound is selected from the group consisting of compounds 6 and 7,

[0163] or compounds 4 and 7, highly preferably the compound is compound 7.

[0164] In a further preferred embodiment the compound is a compound of formula (7).

[0165] Alternatively, in a preferred embodiment, the invention relates to a compound of formula (II), for any antiviral use as defined herein

[0166] Danubia Ref.: P142354

[0167] <

[0168] "

[0169]

[0170] wherein

[0171] X is -(NH)-(CO)-,

[0172] L is -CH2-CH2-,

[0173] Y is -(CF2)k-CF3

[0174] wherein k is 3 to 7, preferably 5 to 7,

[0175] or pharmaceutically acceptable salts thereof.

[0176] Highly preferably, The invention preferably relates to a compound having the formula

[0177]

[0178] "" Compound 8 or pharmaceutically acceptable salts thereof.

[0179] Preferably the compound relates to a method of treatment of a disease or condition caused by an enveloped, positive strand RNA virus selected from the group consisting of the Togaviridae and Flaviviridae family, said virus being internalized via endosome mediated endocytoses and by membrane fusion triggered by acidic endosomal pH, wherein preferably the virus is of the Kitrinoviricota phylum,

[0180] said method comprising the administration of the compound for use of the invention in an effective amount to a subject in need thereof, preferably to a subject infected by said virus.

[0181] Preferably, the virus is a member of the Flavivirus genus, preferably a Zika virus, a Dengue virus or a West Nile virus, wherein the antiviral effect of the compound is shown preferably in an inhibition assay, more preferably in a CPE inhibition assay, highly preferably in an inhibition or a CPE inhibition assay in a human cell line.

[0182] Danubia Ref.: P142354In an embodiment the invention relates to a pharmaceutical composition for use in the treatment of a subject infected by a Flavivirus, said composition comprising a compound as defined in any one of the embodiments defined hereinabove for use in the treatment of a disease or condition as defined therein

[0183] and a pharmaceutically acceptable excipient.

[0184] In a further embodiment the invention relates to a method for treatment of a viral disease or condition caused by a Flavivirus, preferably a Zika virus, Dengue virus or West Nile virus, wherein said compound is administered to a subject in need thereof in an effective amount.

[0185] In a particular embodiment the subject is a vertebrate, preferably a mammal infected by said virus. Highly preferably said subject is human.

[0186] Preferably, the virus is a member of the Togaviridae family, preferably an Alphavirus in the Togaviridae family, preferably a Chikungunya virus, a O'nyong'nyong virus or a Sindbis virus, particularly preferably a Chikungunya virus, wherein the antiviral effect of the compound is shown preferably in an inhibition assay, more preferably in a CPE inhibition assay, highly preferably in an inhibition or a CPE inhibition assay in a human cell line.

[0187] In an embodiment the invention relates to a pharmaceutical composition for use in the treatment of a subject infected by an Alphavirus in the Togaviridae family, said composition comprising a compound as defined in any one of the embodiments defined hereinabove for use in the treatment of a disease or condition as defined therein and a pharmaceutically acceptable excipient.

[0188] In a further embodiment the invention relates to a method for treatment of a viral disease or condition caused by an Alphavirus in the Togaviridae family, preferably preferably a Chikungunya virus, a O'nyong'nyong virus or a Sindbis virus, particularly preferably a Chikungunya virus, wherein said compound is administered to a subject in need thereof in an effective amount.

[0189] In a particular embodiment the subject is a vertebrate, preferably a mammal infected by said virus. Highly preferably said subject is human.

[0190] Preferably, in any of these indications and pharmaceutical compositions, the compound is a compound of formula (I-l).

[0191]

[0192] and, highly preferably, the compound is selected from the following compounds.

[0193] Danubia Ref.: P142354

[0194]

[0195] Danubia Ref.: P142354HO,

[0196] "

[0197]

[0198] Alternatively the compounds are selected from the group consisting of: Preferably, the compounds are selected from the group consisting of: (7) and (8)

[0199]

[0200] Danubia Ref.: P142354

[0201]

[0202] In a embodiment the invention relates to a pharmaceutical composition for use in the treatment of a subject infected by a Flavivirus, said composition comprising a compound as defined in any one of the embodiments defined hereinabove for use in the treatment of a disease or condition as defined therein

[0203] and a pharmaceutically acceptable excipient.

[0204] In a further embodiment the invention relates to a method for treatment of a viral disease or condition caused by a Flavivirus, preferably a Zika virus, Dengue virus or West Nile virus, wherein said compound is administered to a subject in need thereof in an effective amount.

[0205] In a particular embodiment the subject is a vertebrate, preferably a mammal infected by said virus. Highly preferably said subject is human.

[0206] In a further embodiment the invention relates to a compound of general formula (I),

[0207]

[0208] wherein

[0209] R1is OH or a substituted or unsubstituted monosaccharide group, preferably a monosaccharide amine, optionally acylated, preferably an N-acetyl monosaccharide amine, in particular of formula a)

[0210]

[0211] Danubia Ref.: P142354R2is H, CM alkyl, preferably methyl or ethyl, wherein the R2OOC- may be R or S configuration, in particular, R2is carboxyl or a carboxylate ester having the formula

[0212]

[0213] orH3COOC R3is H or OH or Cl-4 alkoxy, preferably H or OH,

[0214] X is -(NH)-(CO)-,

[0215] L is -CH2-O-(CH2-CH2-O)„-CH2-CH2-CH2- or -CH2-(CH2)m-CH2-,

[0216] wherein n is 0 or 1 and m is 0 or 1 ,

[0217] preferably L is -CH2-(CH2)m-CH2- and m is 0 or 1, preferably 0,and

[0218] Y is -(CF2)k-CF3

[0219] k is 3 to 7, preferably 4 to 7, more preferably 5 to 7,

[0220] or pharmaceutically acceptable salts thereof.

[0221] Preferably the monosaccharide group in N-acetyl glucosamine and the compound is a compound of Formula 1.1.

[0222]

[0223] wherein

[0224] R1is a monosaccharide amine, optionally acylated, preferably an N-acetyl monosaccharide amine, X is -(NH)-(CO)-,

[0225] L is - -CH2-(CH2)m-CH2-,

[0226] wherein

[0227] m is 0 or 1 , and

[0228] Y is -(CF2)k-CF3

[0229] k is 3 to 7, preferably 4 to 7, more preferably 5 to 7,

[0230] or pharmaceutically acceptable salts thereof,

[0231] or pharmaceutically acceptable salts thereof.

[0232] Alternatively, preferably the monosaccharide group in N-acetyl glucosamine and the compound is a compound of Formula 1.2.

[0233] Danubia Ref.: P142354

[0234]

[0235] wherein preferably

[0236] X is -(NH)-(CO)-,

[0237] L is -CH2-(CH2)m-CH2-,

[0238] Y is -(CF2)k-CF3

[0239] wherein

[0240] m is 0 or 1, preferably 0, and

[0241] wherein k is 4 to 7,

[0242] more preferably

[0243] L is -CH2-CH2-,

[0244] Y is (CF2)k-CF3

[0245] wherein k is 5 to 6.

[0246] In a further preferred embodiment the compound is a compound of formula (7)

[0247]

[0248] or pharmaceutically acceptable salts thereof.

[0249] Alternatively, in a preferred embodiment, the invention relates to a compound of formula (II),

[0250] Danubia Ref.: P142354OH

[0251]

[0252] wherein

[0253] X is -(NH)-(CO)-,

[0254] L is -CH2-CH2-,

[0255] Y is -(CF2)k-CF3

[0256] wherein k is 3 to 7, preferably 5 to 7,

[0257] or pharmaceutically acceptable salts thereof.

[0258] The invention preferably relates to a compound having the formula

[0259]

[0260] or pharmaceutically acceptable salts thereof.

[0261] Preferably, the compounds are selected from the group consisting of:

[0262]

[0263] Danubia Ref.: P142354and

[0264]

[0265] The invention also relates to a compound of general formula (1.1), preferably formula (II) for use in the treatment of a disease or condition caused by Zika virus, Dengue virus, West Nile virus, Chikungunya virus, O'nyong'nyong virus or a Sindbis virus or SARS-CoV-2 virus

[0266] preferably by Zika virus, Dengue virus, Chikungunya virus, O'nyong'nyong virus or a SARS-CoV-2 virus,

[0267]

[0268] wherein

[0269] R1is OH or a substituted or unsubstituted monosaccharide group, preferably a monosaccharide amine, optionally acylated, preferably an N-acetyl monosaccharide amine, preferably OH,

[0270] X is -(NH)-(CO)-,

[0271] L is -CH2-CH2-,

[0272] Y is -(CF2)k-CF3

[0273] wherein k is 3 to 7,

[0274] or pharmaceutically acceptable salts thereof.

[0275] In a preferred embodiment, the compound is a compound of formula (II)

[0276] Danubia Ref.: P142354OH

[0277] "

[0278]

[0279] wherein

[0280] X is -(NH)-(CO)-,

[0281] L is -CH2-CH2-,

[0282] Y is -(CF2)k-CF3

[0283] wherein k is 3 to 7,

[0284] or pharmaceutically acceptable salts thereof.

[0285] The invention also relates to a compound having the formula

[0286]

[0287] or pharmaceutically acceptable salts thereof.

[0288] The invention also relates to a compound of general formula (1.1), preferably (1.2),

[0289] Danubia Ref.: P142354

[0290]

[0291] wherein

[0292] X is -(NH)-(CO)-,

[0293] L is -CH2-CH2-,

[0294] Y is -(CF2)k-CF3

[0295] wherein k is 3 to 7,

[0296] or pharmaceutically acceptable salts thereof.

[0297] Preferably, the invention relates to a compound having formula (1.2)

[0298]

[0299] wherein

[0300] X is -(NH)-(CO)-,

[0301] L is -CH2-CH2-,

[0302] Y is -(CF2)k-CF3

[0303] wherein k is 3 to 7, preferably 4 to 6, highly preferably 5,

[0304] or pharmaceutically acceptable salts thereof.

[0305] Highly preferably the compound is

[0306] Danubia Ref.: P142354

[0307]

[0308] In particular the compound in this embodiment, highly preferably compound 7 is for use in the treatment of a disease or condition caused by a virus as defined above,

[0309] e.g. an enveloped, positive strand RNA virus selected from the group consisting of the Togaviridae and Flaviviridae family, said virus being internalized via endosome mediated endocytoses and by membrane fusion triggered by acidic endosomal pH,

[0310] wherein preferably the virus is of the Kitrinoviricota phylum.

[0311] Preferably, the virus is selected from the group consisting of a Zika virus, Dengue virus, West Nile virus, Chikungunya virus, O'nyong'nyong virus or SARS-CoV-2 virus.

[0312] In an embodiment the invention relates to a pharmaceutical composition comprising a compound as defined in any one of the embodiments defined hereinabove for use in the treatment of a disease or condition as defined in respect of said embodiment as defined by the scope of compounds in said embodiment,

[0313] an a pharmaceutically acceptable excipient.

[0314] In a particular embodiment the subject is a vertebrate, preferably a mammal infected by said virus. Highly preferably said subject is human.

[0315] DEFINITIONS

[0316] A “glycopeptide” refers to a compound that consists of a peptide linked to one or more sugar moiety. Glycopeptides are primarily known for their effectiveness against Gram-positive bacteria, including strains that are resistant to other antibiotics. They act by inhibiting cell wall synthesis in bacteria, thereby preventing their growth and replication. Examples of naturally occurring glycopeptides include vancomycin, teicoplanin and ristocetin.

[0317] An “aglycone” refers to a non-sugar component of a glycoside, which is a compound formed from a sugar and a nonsugar moiety. In connection with the glycosides, the aglycone is a part that remains after the glycosidic bond between the sugar and the aglycone moiety is hydrolyzed. Aglycons can be varios of organic molecules, which often contribute to the biological properties of the glycoside.

[0318] A “perfluoroalkyl group” refers to a functional group that consists of a chain of carbon atoms fully saturated with fluorine atoms. This group is characterized by the formula CnF2n+i, where n is the number of carbon atoms. Exemples of perfluoroalkyl group include, without limitation, perfluoroethyl, perfluorobutyl, perfluorohexyl and perfluorooctyl. Danubia Ref.: P142354A “subject” as used herein is an individual of an animal species, preferably a vertebrate, more preferably a mammalian or avian species, in particular a mammalian species, highly preferably the individual is a primate, a hominid or a human. A “patient” is a subject who is or intended to be under medical or veterinarian observation, supervision, diagnosis or treatment.

[0319] A “treatment” refers to any process, action, application, therapy, or the like, wherein the subject or patient is under aid, in particular medical or veterinarian aid with the object of improving the subject’s or patient’s condition, either directly or indirectly. Improving the subject’s condition may include improving an aesthetic condition (cosmetic treatment) and / or may include, in particular, restoring or maintaining normal function of an organ or tissue, preferably at least partly restoring or maintaining health (medical or veterinarian treatment). Treatment typically refers to the administration of an effective amount of a compound or composition described herein. Treatment may relate to or include medical or veterinarian treatment and cosmetic treatment, in particular medical or veterinarian treatment.

[0320] A “composition” is understood herein as a non-naturally occurring composition of matter which comprises at least one biologically active substance as defined herein in an effective amount. Compositions may also comprise further biologically active substances or a mixture of biologically active substances. Furthermore, the compositions may comprise biologically acceptable carriers, formulation agents, excipients etc. which are well known in the art.

[0321] A “pharmaceutical composition” of the invention is a composition of matter which comprises an active agent, in a particular the antiviral composition as defined herein, e.g. in the brief description of the invention as an active agent and at least one further substance. Preferably the antiviral composition is present in an effective amount. Compositions may also comprise further biologically active substances useful e.g. in a combination therapy. Furthermore, the compositions may comprise biologically acceptable carriers, formulation agents, excipients etc. which may be known in the art. The term “effective amount” qualifies the amount of a compound required to exert the effect of the active agent in a composition. A “therapeutically effective amount” is sufficient to reduce or relieve or prevent (or prevent worsening of) one or more of the symptoms or characteristic parameters of a condition, e.g. a disorder or disease, preferably due to viral infection, or which normalizes physiological responses.

[0322] In one aspect, a therapeutically effective amount of a compound of the invention, or a pharmaceutical composition, is an amount which restores a measurable physiological parameter to substantially the same value (preferably to within 30%, more preferably to within 20%, and still more preferably to within 10% of the value) of the parameter in an individual without the condition or pathology in question.

[0323] The singular forms “a”, “an” and “the”, or at least “a”, “an”, include plural reference unless the context clearly dictates otherwise.

[0324] The term “comprising” or “including” given components or elements or species or moieties or method steps is understood herein as having a non-exhaustive meaning and as containing said elements (e.g. features or species or moieties) and optionally further elements as well, i.e. comprising does not exclude the presence of further components or elements or species or moieties or method steps. The terms comprising and including are interchangeable herein.

[0325] The expression “consisting essentially of’ or “comprising substantially” is to be understood as consisting of mandatory components or elements or species or moieties or method steps listed in a list e.g. in a claim whereas allowing to contain additionally other components or elements or species or moieties or method steps which do not materially affect the essential characteristics of the use, method, composition or other subject matter. It is to be understood that “comprises” or

[0326] Danubia Ref.: P142354“comprising” or “including” can be replaced herein by “consisting essentially of’ or “comprising substantially” if so, required without addition of new matter.

[0327] The term comprising can be limited to consisting essentially of or consisting of without addition of new matter. BRIEF DESCRIPTION OF THE FIGURES

[0328] Figure 1 - GPA derivatives broad range of virucidal effect. A: compound 7 strongly inhibits all four viruses in Vero E6 cell line. We measured cell viability and fitted a nonlinear regression curve to our data. B: The inhibitory effect of compound 7 against ZIKV (MOI: 1) on A549 cell line, where EC50 was calculated by non-linear regression analysis. C:

[0329] Antiviral activity of compound 7 was determined using an immunofluorescence assay. The nuclei were stained with Hoechst (blue), and Zika replication was marked with a dsRNA antibody (green). The images were taken with Nikon ECLIPSE Ti-U Serieslipse fluorescence microscope. Each experiment was repeated independently at least three times (± SEM). Scale bar: 40 pm.

[0330] Figure 2 - Early-stage antiviral effects against ZIKV. A: Timeline-of-time of addition assay. Vero E6 cells were infected for 1 hour at MOI of 1. Compound 7 (50 pM) was added at different times. After 24 hours post- infection, we stopped the treatment and examined the viral RNA copy number. B: Result of the time-of-addition assay. The viral RNA expression was measured by qRT-PCR. The significance was calculated by Student's t-test (*p<0.01 ) Bar graphs represent the mean. C: Result of binding and entry assay showed no significant difference. D: Timeline of Binding and Entry assays. Vero E6 cells were infected for 1 hour, at 4 degrees Celsius at MOI of 1. Each experiment was repeated independently at least three times (n=3, ± SEM).

[0331] Figure 3 - A: Result of virion inactivation assay. We performed three times independent plaque assays. The results, of plaque assays were normalized to the virus stock titer. The treated stock has significant differences compared to the non-treated stock. The significance was calculated by Student's t-test *p<0.01. B: Result of destabilization assay. The treated virus stock has no significant differences compared to DMEM treated stock. Compound 7 has no direct virucidal effect. Each experiment was repeated independently at least three times (n=3, ± SEM).

[0332] Figure 4 - Reorganization of the unbound, dimeric form into a membrane-bound, trimeric form is a key step in the membrane fusion mechanism of flaviviruses, illustrated with the respective structures of the Zika (5LBV) and Dengue (1OK8) E proteins, with the full dimer and trimer structures shown in the insets. The binding sites detected by FTMap are shown on the surface of the Zika structure. (See Figure 7 for more detail.)

[0333] Figure 5 - Modeled binding poses of compound 7 in the different binding sites (a: Sitel, b: Site2) with surface (left) and cartoon (right) representations, colored according to the different domains (Domain I: red, domain II: yellow, Domain III: blue). Non-covalent interactions are shown with dotted lines (yellow for hydrogen bonds, purple for salt bridges). Figure 6 - We labelled ZIKV NS1 protein (green) after 4 hour at MOI of 1. Hoechst stain was used to visualize the cells nuclei (red). The images were taken by Zeiss LSM 710 Confocal Microscope. Scale bar: 20 pm.

[0334] Figure 7 - Binding sites identified with FTMap (A, B, C: Sitel, Site2 and Site3 respectively), with surface representation on the left and cartoon representation on the right, colored according to the different domains (Domain I: red, domain II: yellow, Domain III: blue). Hotspots are represented with sticks for the centroids of probe clusters identified by FTMap. D: The structure of ZIKV E protein in the dimeric conformation (red, PDB ID: 5JHM), aligned with the structure of DENV E protein dimer (blue, PDB ID: 1OKE), complexed with n-octyl-P-D-glucoside (B-OG, green sticks).

[0335] Danubia Ref.: P142354The open conformation of the hairpin loop observed in the Dengue structure allows for the binding of a small molecule in this site.

[0336] Figure 8 - A: Binding sites on one chain from the trimeric structure of the Dengue E protein (1OK8). The protein chain is shown in surface representation (red for domain I, yellow for domain II and blue for domain III), and the FTMap consensus clusters are represented with sticks, with a different color for each cluster. The residues corresponding to Sitel on the ZIKV E protein in its dimeric conformation are shown in cyan: clearly, Sitel disappears due to the structural reorganization during trimer formation. (The fusion loop is colored orange.) B: By comparison, Sitel is present in the dimer form of the DENV E-protein (PDB ID: IOAN), at the same location as in the ZIKV E protein.

[0337] Figure 9 - Root mean squared distance (RMSD) values over time for the protein Ca and ligand heavy atoms through the five simulation replicas for Sitel (left) and Site2 (right). For Sitel, both the protein and the ligand reach equilibrium in all simulations. (The anomaly at simulation 1 is due to the ligand traversing through the border of the periodic boundary box.) The relatively large ligand RMSD values (6-8 A) are in line with its size and the flexibility of certain parts, particularly the perfluorinated tail and the glycan unit. For Site2, the ligand is observed to dissociate from the protein in three out of five simulations, as evidenced by RMSD values over 15 A. (The high peak at simulation 1 and the multiple narrow peaks at the end of simulation 4 are, once again, caused by the ligand traversing through the periodic boundary box borders.)

[0338] Figure 10 - The Vero E6 cells were infected (MOI 1) and treated whit the compound at MCC. We incubated them 5 days. In case of compounds 2, 3, 4 and 6 we didn’t observed any CPE. The pictures were taken by CytoSMART™ Omni.

[0339] Figure 11 - Under conditions that did not inhibit fusion (untreated sample or DMSO control), protein C was not detectable by Western blot analysis or appeared only as an extremely weak signal. In contrast, in the presence of the compound under investigation, a strong, concentration-dependent capsid protein signal was observed (indicated by red arrows). This suggests that the compound effectively inhibited acid pH-induced virion-endosome fusion, thereby protecting the capsid protein from trypsin digestion (Figure 11 A). The E protein showed similar intensity in all samples, confirming that the differences observed were not due to differences in virus quantity but to inhibition of the fusion process (Figure 11B).

[0340] DETAILED DESCRIPTION OF THE INVENTION

[0341] Togaviridae and Flaviviridae are similar RNA virus families that are characterized by a single mRNA that results in a polyprotein that is posttranslationally modified. Both families comprise enveloped, spherical viruses with positive-sense, single- stranded RNA genomes. Togaviruses and Flaviviruses (like Dengue, Zika) both use receptor-mediated endocytosis for cell entry. Once internalized via endosome, the viruses rely on the acidic pH in the endosomes to trigger fusion, and use glycoproteins that undergo conformational changes, exposing fusion loops / peptides to insert into the endosomal membrane, ultimately merging viral and cellular membranes to release the RNA genome into the cytoplasm.

[0342] Both virus types utilize the endosome's acidity to activate their surface proteins.

[0343] While both families use different glycoproteins (Alphavirus: E1 / E2, Flavivirus: E protein) they similarly shall undergo conformational changes for acidic pH triggered membrane fusion in the endosomes. Thereby the viral and cellular membranes are merged to release the RNA genome into the cytoplasm.

[0344] Danubia Ref.: P1423541

[0345] These viruses are typically arboviruses, which means that they replicate arthropod (e.g. mosquito or tick) and are typically medically important, often causing severe diseases like encephalitis, transmitted via arthropod bites.

[0346] Their genomic RNA is capped and serves as mRNA for nonstructural proteins (e.g., RNA-dependent RNA polymerase) or even for all proteins (in case of flaviviruses). Complementary (antisense) RNA, made from genomic RNA, serves as a template for progeny genomic RNA. A subgenomic mRNA representing the 3' one-third of the genome encodes the structural proteins. In case of alphaviruses the genomic RNA is polyadenylated, in case of flaviviruses it is not poly adenylated.

[0347] Numerous viral diseases do not have specific antiviral treatments to date. Therefore, continuous efforts to find compounds that inhibit viral replication are inevitable. The broad antiviral effects of glycopeptide antibiotics (GPAs) and their derivatives have already been described previously. In the present description we disclosed the results of our studies on the in vitro viral inhibitory activity of newly synthesized GPAs derivatives against Zika virus (ZIKV), Chikungunya virus (CHKV), O'nyong'nyong virus (ONNV) and Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Cell viability assay was used to determine the EC50 value and cytotoxicity of the active compounds. Seven of the compounds were able to inhibit ZIKV and two compounds could inhibit all of the four tested viruses. One of the teicoplanin pseudoaglycone derivatives, compound 7 was the most effective and exerting an inhibitory effect against all four viruses in the low micromolar range, prompting further investigation into its mechanism of action against ZIKV. Time of addition assay was used to determine the early stage of the compound's activity. Furthermore, by entry and binding assay we determined that the compound does not inhibit either binding or entry. Our assays demonstrated that the compound acts by binding directly to the virion, in a way of attaching to the envelope protein of the virus, thus preventing fusion of the viral membrane and endosome. These results were supported by in silico molecular modelling. The studied GPA derivatives are promising candidates to become broad- spectrum antivirals after further preclinical and clinical testing. In the present description we disclose the antiviral evaluation of hydrophobic derivatives of the aglycones of the antibiotics vancomycin, teicoplanin, and ristocetin against ZIKV, CHIKV, and ONNV. In order to further map the breadth of the activity spectrum of the compounds, we also tested their effects against the pandemic coronavirus, SARS-CoV-2. Perfluorobutyl, -hexyl and -octyl groups were used as hydrophobic units, as it was previously shown that perfluoroalkyl derivatives of teicoplanin displayed good inhibitory effect against IAV, HS V and hCoV viruses at nontoxic concentrations (Bereczki et al., 2020).

[0348] To elicit the mode of action of the GPA derivatives in case of ZIKV, we selected for further testing the observed one of the strongest inhibitory activity compound against ZIKV and could block the other three viruses. We performed a timedependent entry assay, binding assays, as well as computational modeling, to determine whether the compounds inhibit the entry of the virus or hinder a later step of the viral replication cycle.

[0349] A series of hydrophobic glycopeptide antibiotic aglycone derivatives were prepared by attaching perfluoroalkyl groups of different sizes to the A'-terminal amino acid unit of the heptapeptide skeletons of the antibiotics (Figure 2). The series of compounds includes seven teicoplanin pseudoaglycone derivatives (compounds 1-7), one ristocetin aglycone derivative (compound 8) and two vancomycin aglycone derivatives (SI and S2 in Table 2); the latter two are not shown here, as they were completely inactive in antiviral tests. We chose perfluoroalkyl groups as hydrophobic units because they do not have a membrane perturbing effect (Park et al. 2007) and therefore are not expected to make the antibiotic derivatives cytotoxic (Bereczki et al. 2020). The perfluoroalkyl group was either connected directly to the A'-terminal amino group through an

[0350] Danubia Ref.: P142354amide bond (compounds 6-8), or after converting the A'-terminal amino group into an azide, the conjugation was performed by an azide-alkyne click reaction; in the latter case, the fluorinated substituent is connected to the peptide backbone through a linker region consisting of a triazole ring (compound 4) and ethylene glycol (compounds 1-3) or tetraethylene glycol (compound 5). This series of compounds allows us to study the impact of the peptide backbone, the size of the perfluoro group and the linker region on the antiviral activity.

[0351] The synthesis of compounds 1, 3-6 was previously reported (Bereczki et al., 2020, Bereczki et al., 2022), while compounds 2, 7 and 8 are new compounds, designed and produced for the studies disclosed in Examples.

[0352] The structures of studied glycopeptide antibiotic derivatives are listed in Table 1 and Table 2.

[0353] Table 1: The structures of the studied derivatives of naturally occurring glycopeptide antibiotics.

[0354]

[0355] Danubia Ref.: P142354

[0356] <

[0357] >

[0358] >

[0359] >

[0360]

[0361] Vancomycin, ristocetin and teicoplanin are naturally occurring, Actinobacteria-denved glycopeptide antibiotics containing a heptapeptide skeleton. Teicoplanin and vancomycin are used clinically to treat serious Gram-positive infections. Ristocetin, in spite of its good antibacterial activity against Gram-positive strains, was withdrawn from the market in the mid-1960s as it led to thrombocytopenia in some patients due to its platelet-aggregating effect (Blaskovich et al., 2018).

[0362] Danubia Ref.: P142354Table 2: Structures of perfluoroalkyl derivatives of teicoplanin pseudoaglycone (1-7) and ristocetin aglycone (8)

[0363]

[0364] Danubia Ref.: P142354

[0365]

[0366] Danubia Ref.: P142354

[0367] <

[0368]

[0369] The Zika virus continues to infect tens of thousands of people annually as of today (Zika, 2023). Despite decades of familiarity with the virus, vaccine developments against it are currently only in Phase 1 or 2 clinical trials (Peng et al., 2024). Numerous compounds have proven effective in vitro against the virus, but currently, no drugs are available for treating the infection (Baz & Boivin, 2019; Fong & Chu, 2022). This confirms the importance of identifying new potential drug candidates and understanding their mechanism of action. The broader the antiviral activity of a compound, the more valuable it becomes for therapeutic development. Thus, these compounds have been tested against a range of viruses, including CHIKV, ONNV, and SARS-CoV-2, to assess their potential for broad-spectrum antiviral efficacy. Thus, these compounds have been tested against a range of viruses, including CHIKV, ONNV, and SARS-CoV-2, to assess their potential for broad-spectrum antiviral efficacy. We tested for alphaviruses because compounds of this type have not been tested against them before. GPAs and their derivatives have been shown to be effective against many viruses such as DENV, EBOV, HCV, etc. (Burghgraeve et al., 2012; Obeid et al., 2011; Wang et al., 2016). For many compounds, they have been shown to inhibit entry, but the mechanism of action may differ depending on the virus and the type of side chain of the compound (Burghgraeve et al., 2012; Obeid et al., 2011; Zhou et al., 2016). The MTT cell viability assay was used to determine the EC50 values of eight compounds against ZIKV, CHIKV, ONNV, and SARS-CoV-2. Seven of these Danubia Ref.: P142354compounds shown to be effective against ZIKV. The complete inactivity of compounds 5, SI and S2 showed that the long amphiphilic tetraethylene glycol linker was detrimental to the antiviral effect. In line with our previous results (Bereczki et al., 2015), we found that although the peptide backbones of the glycopeptide antibiotics are very similar, there is a significant difference in the antiviral activity and selectivity of their derivatives, which is clearly shown by the different activity profiles of the identically substituted pair of compounds, teicoplanin (compound 6) and ristocetin (compound 8).

[0370] Several compounds have broad-range antiviral effects such as compound 7 which proved effective against ONNV, CHIKV, and SARS-CoV-2 at low micromolar concentrations. We made a further investigation with this compound to understand the underlying mechanism. Firstly, time of addition assay was performed, this assay is widely used to test antiviral activity. We observed compound 7 inhibit only in the early stages of infection. It was likely that they directly affect the virion, as no inhibition was observed either in the pre-treatment or in the post-treatment. Further confirmation came from the binding and entry assay, demonstrating that they do not inhibit binding or entry. The direct interaction between the compound and the virion was then investigated. First, we determined by inactivation assay that it acts directly on the virus in a cell-free environment. The destabilization assay showed us that this cell-free inactivation does not occur through the destruction of the virion.

[0371] Using bioinformatics analysis, we were able to explore further mechanisms. After FTMap scanning of the viral E protein, we identified regions of potentially high affinity for compound 7. We found three sites where the compound is able to bind, all of them take part to inhibit fusion between the lipid membrane of the endosome and the viral envelope. All the experiments that preceded analysis and the microscopic experiments also support this model. We have labeled the NS1 protein, which is only present in cells when translation occurs (Payne, 2017). We have shown that inhibition occurs before translation, preceded by encapsidation. These results further confirm the validity of the bioinformatic model. The compounds plausibly contribute to the development of new antiviral drugs. These compounds not only have inhibitory effects against not only ZIKV but also various alphaviruses. It is plausible that the compounds inhibit virus entry by inhibiting membrane fusion.

[0372] The development of broad- spectrum antiviral drugs is essential for the treatment of current and future expected epidemics.

[0373] Particularly preferred compounds are selected from the group consisting of compounds (2), (3), (4), (5), (6), (7) and (8), compounds (2), (3), (4), (6), (7) and (8), preferably compounds (3), (4), (6), (7) and (8), preferably compounds (4), (6), (7) and (8), highly preferably compounds (7) and (8).

[0374] Compound 7 was also tested in human cell line (A549), and we found similar EC50 as in Vero E6 cell line. In summary, we have found a mechanism of action different from that of any glycopeptide antibiotic derivative previously studied. In the case of ZIKV, our compound binds directly to the viral E protein, independent of the cellular environment and can inhibit viral fusion and uncoating. Despite the ability of the compound 7 to act directly on the virus in a cell-free environment, other GPAs derivatives with similar lipophilic side chains have been found to act inside cells. From these we conclude that, despite the large size of our molecule, it may be able to enter the cell (N et al., 2016; Vanderlinden et al., 2012).

[0375] Dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV) belong to the flavivirus genus. All three viruses are transmitted by mosquitoes (DENV and ZIKV by Aedes sp. mosquitoes). Flaviviruses are enveloped viruses. The E proteins are the outermost proteins in the structure of flaviviruses and are capable of attaching to the lipid envelope of the

[0376] Danubia Ref.: P142354cell. This protein is responsible for the entry of the virus into the cell. DENV and ZIKV E proteins share significant structural similarities and play similar roles in host cell entry and interaction with the immune system.

[0377] During our studies of the mechanism of action, we came to the conclusion that a compound having inhibitory effect against studied viruses is able to bind to the E protein of the virus, and subsequently this will prevent membrane fusion in the endosome.

[0378] In the case of WNV, its E protein differs to a greater extent compared to ZIKV, but it can be assumed that the E protein, in the regions where compounds effective against ZIKV bind, does not differ to such an extent that the compound has no effect.

[0379] EXAMPLES EXAMPLE 1 - Materials and Methods

[0380] General information

[0381] Teicoplanin was purchased from Beijing Mesochem Technology Co., Ltd (Yizhuang, Beijing), vancomycin was purchased from BLD Pharmatech GmbH (Reinbek, Germany), allyl alcohol, perfluorohexyl iodide and perfluorooctyl iodide were purchased from Merck Life Science Kft., an affiliate of Merck KGaA (Darmstadt, Germany). The synthesis of teicoplanin pseudoaglycone, teicoplanin pseudoaglycone azide, ristocetin aglycone, perfluroalkyl teicoplanin derivatives compounds 1, 3-6, perfluoroalkyl vancomycin derivatives SI and S2 and compounds 9, 14 and 19 were published previously (Bereczki et al., 2020, 2022; Naesens et al., 2009; Pinter et al., 2009; Wanner et al., 2003). Synthesis of teicoplanin derivatives compounds 2 and 7, and ristocetin derivative compound 8 are shown in Schemes 1-3. TLC was performed on Kieselgel 60 F254 (Merck) with detection either by immersing into ammonium molybdate- sulfuric acid solution followed by heating or by using Pauly’s reagent for detection. Flash column chromatography was performed using Silica gel 60 (Merck 0.040-0.063 mm). The photoinitiated reactions were carried out in a borosilicate vessel by irradiation with a Hg-lamp giving maximum emission at 365 nm.

[0382] The 'H NMR (400, 500 and 700 MHz),13C NMR (100, 125 and 176 MHz) and19F (659 MHz, CPMG) spectra were recorded using Bruker’s DRX-400, Avance-II 500 and NEG-700 spectrometers at 298 K or 310 K in CDCL or DMSO-de solvent. Assignments were aided with 2D NMR spectra (’H-’H COSY, ROESY, ’H-^C HSQC, HSQC-TOCSY, HMBC, and!H-19F HSQC) using manufacturer’s Topspin program and pulse programs. Chemical shifts are referenced to Me4Si (0.00 ppm for II) or to solvent residual signals. NMR spectra and signal assignments of compounds 2, 7 and 8 are as shown in the Supporting information. MALDLTOF MS measurements were carried out with a Bruker Autoflex Speed mass spectrometer equipped with a time-of-flight (TOF) mass analyzer. In all cases 19 kV (ion source voltage 1) and 16.65 kV (ion source voltage 2) were used. For reflectron mode, 21 kV and 9.55 kV were applied as reflector voltage 1 and 2, respectively. A solid phase laser (355 nm, >100 pJ / pulse) operating at 500 Hz was applied to produce laser desorption.

[0383] 2,5-Dihydroxybenzoic acid (DHB) was used as matrix and FsCCOONa as cationising agent in DMF. HRMS measurements were carried out on a maXis II UHR ESLQTOF MS instrument (Bruker), in positive ionization mode. The following parameters were applied for the electrospray ion source: capillary voltage: 3.5 kV; end plate offset: 500 V; nebulizer pressure: 0.8 bar; dry gas temperature: 200 °C and dry gas flow rate: 4.5 l / min. Constant background correction was applied for each spectrum, the background was recorded before each sample by injecting the blank sample matrix (solvent). Na-formate calibrant was injected after each sample, which enabled internal calibration during data evaluation.

[0384] Danubia Ref.: P142354Mass spectra were recorded by otofControl version 4.1 (build: 3.5, Bruker) and processed by Compass DataAnalysis version 4.4 (build: 200.55.2969).

[0385] Virus and cell lines

[0386] Vero E6 cell line, derived from the kidney epithelial cells of the African green monkey Cercopithecus aethiops) (ATCC® CRL-1586™) and A549, human lung adenocarcinoma epithelial cell line (ATCC, CCL-185) were used in our in vitro antiviral screens. The cells were maintained at 37 °C under 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, Lonza, Switzerland), supplemented with 10% fetal bovine serum (FBS, Biosera, France) and 1% penicillin / streptomycin antibiotics (PS, Lonza, Switzerland). Antiviral tests were utilized ZIKV (MR766, Uganda), SARS-CoV-2 (B.1.5 (G) isolate, Hungarian), CHIKV (Guatemala), ONNV (UVE / ONNV / UNK / SN / Dakar 234) viruses. All virus stock were propagated on Vero E6 cells and stored at -80 C°. Upon infection cell culture media were replaced with media containing 2% FBS and 1% PS.

[0387] Cytotoxicity assay

[0388] To measure the cytotoxicity effect of compounds, the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colorimetric cell viability assay (MTT Cell Proliferation Assay, Roche, Germany) was performed. To determine the minimal cytotoxicity concentration, MCC (compound concentration producing minimal changes in cell morphology) we used microscopic observation by inverted Nikon ECLIPSE Ti-U Serieslipse fluorescence microscope (Nikon, Japan).

[0389] Plaque assay

[0390] To determine the infectious viral titer of the virus stock plaque assay was used. Cells were grown in a 24-well plate at a density of 3xl05for one day (37 °C, 5% CO2) before the infection. We prepared a tenfold serial dilution of the virus stock solutions. After one hour of infection, the wells were covered with carboxymethyl cellulose to achieve a final concentration of 1%, and the plates were incubated for five days. Following fixation with paraformaldehyde, the cells were stained with 0.25% crystal violet and plaques were counted. The infectious viral titer was then calculated with the following formula: pfu / ml=avg. plaque number*(l / dilution)*(1000 / infection volume (pl)).

[0391] CPE inhibition assay

[0392] In the context of ZIKV, IxlO4Vero E6 cells per well were seeded in 96- well tissue culture plate and incubated (37 °C, 5% CO2) overnight. For SARS-CoV-2, CHIKV, ONNV 3.5xl04Vero E6 cells were seeded the day before the infection. To measure the inhibitory effect of compounds, following a microscopic observation, MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colorimetric cell viability assay (MTT Cell Proliferation Assay, Roche, Germany) was employed. The cells were treated with the compounds diluted at indicated concentrations (from 0.3 p M to 60 pM) for the entire duration of the experiment. The control group was treated with an equal concentration of DMSO. After two hours of ZIKV infection at a multiplicity of infection (MOI) of 1, the supernatant was discarded, and the cells were incubated for five days with the compounds. SARS-CoV-2 and CHIKV infections were carried out with MOI of 0.1 for half an hour, while ONNV for 1 hour also with MOI of 0.1 and incubated for 3 days. Following the appropriate incubation time and microscopic examination, our measurements were carried out using 50 pl MTT working solution per 100 pl sample. The absorbance was measured at 560 nm with Crocodile 5inl mini Workstation (Berthold, Germany). Three biological replicates of each concentration were used. Three biological replicates of the positive and negative controls were used.

[0393] Danubia Ref.: P142354Time of drug addition assay

[0394] Vero E6 cells were seeded on 96-well plate 3.5xl04well and incubated (37 °C, 5% CO2) overnight. The cells were infected for 1 hour with MOI of 1. Cells were treated with 50 pM of compound 7. Cells were treated at three different times: 1 hour before infection, concurrently with the infection, and 1 hour post-infection. After the infection, the supernatant was discarded and the cells were incubated for 24 hours whit the media, then RNA was isolated from the supernatant and quantified it using qRT-PCR (see below).

[0395] Entry and Binding assay

[0396] Vero E6 cells were seeded on 96 well plate (3,5xl04) well and incubated (37 °C, 5% CO2) overnight. 1 hour before the infection we put the cells at 4 °C. During entry assay cells were infected for 1 hour at 4 °C MOI of 1, then washed them twice with PBS. Afterward, they were treated with 50 M of compound 7, lasted for 1 hour (37 °C, 5% CO2). While during binding assay the cells were infected as before for 1 hour at 4 °C and same time treated them with 50 pM of compound 7. Following both of the treatments, the cells were washed once with PBS, then RNA was isolated from the cells. Viral RNA was quantified using qRT-PCR (see below).

[0397] RNA isolation and quantitative reverse transcription PCR (qRT-PCR)

[0398] For viral RNA isolation Total RNA Miniprep Kit (New England Biolabs, USA) was used, following on the manufacturer’s instruction. After RNA elution, samples were stored at -80 °C until use. For the determination of viral RNA quantity, we used the Luna® Universal Probe One-Step RT-qPCR Kit (New England Biolabs, USA). The primers used were ZIKV NS1 F: AAA AGG AAA CGA GAG ATG TGG CA, ZIKV NS1 R: CAT TCT CCT CTA GGA TAG CAT, and the probe: FAM5’-CCC GCA GAT-3’ / ZEN / 5’-TGG CAG CAG-3’. The reaction was performed as follows: 1 cycle at 55 °C for 10 minutes, 1 cycle at 95 °C for 1 minute, followed by 40 cycles at 95 °C for 10 seconds and 60 °C for 30 seconds, using the CFX Opus Dx Real-Time PCR Systems (Bio-Rad, USA). To determine the copy number, we prepared a standard curve. This involved initially purifying viral cDNA (DNA Gel Extraction Kit, New England Biolabs, USA) using gel electrophoresis, followed by determining the DNA concentration with the aid of a Qubit dsDNA BR Assay kit (ThermoFisher, Invitrogen, USA) based on the manufacturer’s protocol. Then we made a series of ten-fold dilutions, which were included alongside each PCR reaction.

[0399] Inactivation assay

[0400] Anti-ZIKV effects of the compound 7 in interaction with the virus itself was studied, in a cell-free environment. The virus stock (apx. 5000000 PFU / ml) was incubated in solutions of 50 pM compound 7 for 2 hours at 37 °C. Then it was diluted 100-fold to eliminate the compounds from the sample and plaque assays were performed as previously described.

[0401] Virion destabilization assay

[0402] Undiluted 50 pl virus stock solution was incubated in the presence or absence 50 pM of compound 7 for 1 hour. After treatment, 80 pg / ml RNase A (Sigma-Aldrich, USA) was added. One hour later Proteinase K (New England Biolabs, USA) was added at 1 mg / ml for Ih incubation to inactivate the RNase. Then we isolated the viral RNA and performed qRT-PCR (see above). As a positive control we treated our stock solution with 20% ethanol. The optimisation is based on the following work (Lu et al., 2021).

[0403] Immunofluorescence assay (IF A)

[0404] Cells were fixed with ice-cold methanol for 30 minutes. 1% BSA (ThermoFisher) in phosphate-buffered saline (PBS) solution was used for blocking. Subsequently, the cells were stained using a primary dsRNA antibody (CliniScience, Cat#

[0405] Danubia Ref.: P14235410020200, 1:1000) or Zika virus NS1 Monoclonal Antibody (Invitrogen, Cat# MA5-24585, 1:100) for 1 hour at 37 °C and secondary Goat Anti-Mouse IgG H&L Alexa Fluor® 488 (Abeam, Cat# abl50113, 1:1000) for 30 minutes at 37 °C. Finally, the cells were stained with Hoechst 33342 Solution (Invitrogen, Cat# H1399) at room temperature for 10 minutes. Between each step, the cells were washed five times with PBS. Images of the cells were captured using an inverted Nikon ECLIPSE Ti-U Series Eclipse fluorescence microscope (Nikon, Japan) and Zeiss LSM 710 Confocal Microscope (Plan Apochromat lOx, 20x, and 63x objectives (NA: 0.45, 0.8, 1.4, Carl Zeiss Inc., Jena, Germany)) with normalized laser power and filter settings with open pinhole in low magnification or by making 0.5 pm thin optical sections.

[0406] Statistical analysis

[0407] For the statistical analysis, we used version 9.5 of GraphPad Prism (GraphPad Software, Boston, MA, USA). To determine the ECso in the CPE inhibition assay, we employed non-linear regression analysis. qRT-PCR results were analysed using a Student t-test. qPCR data represent the mean and standard error of the mean (±SEM) and p < 0.05 was marked as statistically significant.

[0408] Molecular modelling

[0409] Briefly, protein structures were prepared with the Protein Preparation Wizard of the Schrodinger suite with default settings (Sastry et al., 2013), and prospective binding sites were mapped with the FTMap webserver (Brenke et al., 2009; Ngan et al., 2012). The structure of compound 7 was prepared with Schrodinger Ligprep (Johnston et al., 2023), conformationally sampled with Macromodel and docked to the respective binding sites by either Glide SP (Friesner et al., 2004; Halgren et al., 2004), or the Induced Fit Docking protocol of the Schrodinger suite (Sherman et al., 2006), and the binding poses were rescored with the Prime MM / GBSA protocol (Li et al., 2011).

[0410] Protein structure preparation

[0411] The structures were refined with the Protein Preparation Wizard of the Schrodinger suite with default settings (Sastry et al., 2013). Here, missing heavy atoms and hydrogen atoms were added, missing residues and loops were modelled, and the structure was relaxed in the OPLS4 force field (Lu et al., 2021). All nonprotein entries and waters were removed from the structures. There was a longer loop missing from the deposited 5JHM structure on both chains (147-161), which was further refined using the Prime loop refinement application (Jacobson et al., 2004).

[0412] Ligand preparation

[0413] For the docking studies, we generated the model of compound 7 with Schrodinger Ligprep, here the whole 3D atomic model of the molecule was built with the most probable protomer assigned with Epik at physiological pH (Johnston et al., 2023). Next we ran a conformational sampling search with the Macromodel mixed torsional and low-mode sampling protocol on default settings, in the OPLS4 forcefield (Mohamadi et al., 1990; Sastry et al., 2013). Here, the molecule’s conformational space is explored by changing a conformation by varying Monte Carlo or low-mode steps. The lowest energy conformations are retained and used for further conformer generation until convergence. Conformers with less than 0.5 root mean square deviation (RMSD) between identical atoms in the macrocyclic core after alignment were filtered out. Every docking run was started with the resulting conformers as input. (The molecule’s hydrophobic tail for the induced fit docking (IFD) calculation was copied into an individual model from the structure generated by Ligprep.) Hotspot mapping with FTmap

[0414] FTmap is a computational solvent mapping algorithm to locate energetically robust binding regions (hotspots) on protein surfaces, by placing multiple small organic probe molecules with different chemical properties on a dense grid

[0415] Danubia Ref.: P142354around a protein (Brenke et al., 2009; Ngan et al., 2012). It then finds probe positions and orientations with low binding energies, first based on an empirical energy function, then by default it minimizes the 2000 lowest-energy positions for every probe based on the CHARMM potential with a continuum electrostatic term. It then clusters the positions of every probe type based on distance, and then clusters all of the probe clusters based on distance, resulting in so-called consensus clusters, which pinpoint the location of binding hotspots. The stronger the hotspot, the more consensus clusters it contains. One binding site can be composed of multiple nearby hotspots. In this study, we used FTmap in PPI mode.

[0416] Ligand docking with Glide

[0417] For ligand docking, Glide uses a series of hierarchical filtering steps to search for possible binding conformations of a given ligand (pose), with more and more accurate scoring of the selected poses (Friesner et al., 2004; Halgren et al., 2004). The properties of the receptor are represented on a pre-generated grid, by different sets of fields. By default, ligand flexibility is handled by a systematic rotamer generation before docking. The top-scored poses are submitted to a multi-step energy minimization process. Finally, the best poses are scored with the Emodel scoring function and the best-scored pose is re-scored with the Glide scoring function and retained.

[0418] Here, we used Glide in the SP precision, with default settings, in peptide docking mode, which retains more conformations during the docking process. For grid generation at Sitel and Site2, we set the center of the grid to be the center of the atomic coordinates of the consensus cluster representations at the given site. For Site3, we set the center of the grid to be the center of the atomic coordinates of the consensus cluster representation and the atomic coordinates of the B-OG ligand in the aligned structure of the 1OKE complex to the given structure with the ‘align’ function of Pymol.

[0419] Induced Fit Docking (IFD)

[0420] To generate an appropriate binding pose in Site3, we selected the A chain of 5JHM as a starting structure, because it contains the highest-ranking consensus cluster near the cryptic site. We first opened the hairpin loop (274-286) by rebuilding it as a homology model, using the open hairpin conformation in PDB entry 1OKE (269-281) as a template. Next, we docked the fluorinated tail of Compound 7 into the open hydrophobic pocket with the Induced Fit Docking protocol of the Schrodinger suite (Sherman et al., 2006) (https: / / sciwheel.com / work / citation?ids=1608895&pre=&suf=&sa=0&dbf=0), with the grid center set to be the center of the atomic coordinates of the B-OG ligand in the aligned structure of the 1OKE complex to the open protein with Pymol align. Next, the 274-286 loop was refined using Prime with the docked ligand in place. Then, every sidechain from 5.0 A of the docked ligand were refined. Finally, the ligand was redocked to the refined protein structure with Glide, using the same settings as before. The poses were ranked according to the IFD score, which is the Glide score + 5% of the Prime energy of the protein. From the resulting complexes, we selected the one with the best IFD score and used its protein structure for docking calculations for the whole ligand.

[0421] Prime MM / GBSA calculations

[0422] Prime MM / GBSA implements a rescoring protocol for ligand-protein complexes for a more accurate ranking of ligands. The MM / GBSA dGbind score is calculated using the following expression:

[0423] -GbindCOmplex(minimized) ^ligand(minimized^ ^protein(mimmized^ Here, the respective terms mean the Prime energies calculated for the minimized complex, ligand and protein. The energies are extended with a solvation term, calculated with the VSGB implicit solvation model (Li et al., 2011). Residues

[0424] Danubia Ref.: P142354within a 5.0 A distance from the ligand in the complex were treated fully flexible, and the forcefield was set to OPLS4 (Lu et al., 2021).

[0425] Molecular Dynamics simulations

[0426] Molecular dynamics (MD) simulations were performed using the Desmond simulation suite to evaluate the stability of the determined binding poses, using the minimized protein-ligand complex acquired from the MM / GBSA calculations. Production runs of 100 ns were conducted on five replica systems with randomly sampled initial velocities for each pose, with the OPLS4 force field at 300K and atmospheric pressure. The simulations were set up using the default simulation protocol in Desmond, as follows (Bowers et al., 2006). The systems were set up with an orthorombic simulation box with 10 A padding, solvated with TIP4P explicit waters (Jorgensen et al., 1983) and neutralized by adding 22 Na+ ions. The salt concentration was set to 0.15 M by adding further Na+ and Cl- ions. The recording interval was set to 100 ps resulting in 1000 recorded frames per simulation. The simulations were conducted according to the default Desmond settings using a 2 fs timestep RESPA integrator, Nose-Hoover thermostat (Hoover, 1985) and Martyna-Tobias-Klein barostat (Martyna et al., 1994). Coulombic interactions were neglected between charges farther than 9.0 A. The systems were subjected to the default Desmond relaxation protocol before the actual simulation. For each binding site, five parallel NPT simulations with random initial velocities were carried out for 100 ns with the OPLS4 force field (Lu et al., 2021) at 300K and atmospheric pressure. The simulations were assessed by plotting the root mean squared distance (RMSD) values of the protein Ca, as well as the ligand heavy (non-hydrogen) atoms over time, after superimposing the protein to the starting frame.

[0427] Capsid protection assay to monitor fusion of virions with liposomes

[0428] To study virion-endosome fusion, we prepared liposomes that mimic the composition of late endosomal membranes. The liposomes were prepared using l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), soybean-derived phosphatidylinositol (PI), bis(monoacylglycero)phosphate (BMP, S,R isomer), and cholesterol in a molar ratio of 4:1:1:2:4. The lipid mixture was suspended in TAN buffer (20 mM triethanolamine, 100 mM NaCl, pH 8.0) (Pi-Chun Li et al., 2019).

[0429] The liposomes were prepared by five consecutive freeze-thaw cycles, and then the mixture was extruded through a membrane with a pore size of 0.2 pm to ensure uniform size distribution. For liposomes containing trypsin, 3.8 mg of trypsin was added to 0.38 mL of lipid suspension (total 2.8 mg of lipid) immediately after the fifth freeze-thaw cycle, prior to extrusion. Unincorporated trypsin was separated using a qEV size-exclusion chromatography-based system that allows automated fraction collection (Izon Science Ltd.).

[0430] The virus samples were incubated with the tested compounds at concentrations of 0, 10, and 50 pM for 45 minutes at 37 °C in TAN buffer (pH 8.0). Subsequently, 20 pL of liposome containing trypsin was added to each sample, and the mixture was incubated for another 5 minutes. To trigger the fusion process, 2.5 pL of 1 M sodium acetate (pH 5.0) was added to the samples, lowering the pH to 5.3 to allow fusion to occur, and the mixture was incubated for 5 minutes. The pH was then restored by adding 2.5 pL of 2 M triethanolamine (pH 8.1), and the samples were incubated at 37 °C for an additional 20 minutes to allow trypsin digestion.

[0431] The reaction was stopped by adding Laemmli sample buffer (supplemented with 2-mercaptoethanol), and the samples were boiled at 95°C for 20 minutes. The proteins were separated by SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane using a semi-dry transfer device. Anti-ZIKV capsid antibody (1:600) was used to detect the Zika

[0432] Danubia Ref.: P142354virus capsid (C) protein, while anti-ZIKV envelope antibody (1:1200) was used to detect the envelope (E) protein. The membranes were developed with enhanced chemiluminescence (ECL) detection reagents (Bio-Rad) and the signals were recorded using an Azure 200 chemiluminescence imaging system.

[0433] EXAMPLE 2 - Synthesis of the compound 2

[0434] Commercially available perfluorohexyl iodide was conjugated to olefin compound 9 by a light- induced radical addition reaction (Beniazza et al., 2016), and the resulting compound 10 was deiodinated by catalytic hydrogenation to give compound 11 (Scheme 1). Next, fluorinated alcohol compound 11 was converted to propargyl ether compound 12 by alkylation with propargyl bromide. Finally, a copper(I) -catalyzed 1,3-dipolar cycloaddition reaction of compound 12 with teicoplanin pseudoaglycone azide (Pinter et al. 2009) provided the perfluorohexyl teicoplanin derivative compound 2 having a triazolyl ethyleneglycol linker region.

[0435]

[0436] Scheme 1

[0437] Scheme 1 shows the synthesis of perfluorohexyl derivative compound 2 from teicoplanin pseudoaglycone azide by Cu(I)-catalyzed azide-alkyne click reaction.

[0438] Compound 10: Allyloxyethanol 9 (1.02 ml, 10 mmol) and n-perfluorohexyl iodide (2.6 ml, 12 mmol, 1.2 equiv.) were dissolved in methanol (15 ml) and benzophenone (18 mg, 0.1 mmol, 0.01 equiv.) was added. Argon gas was bubbled Danubia Ref.: P142354through the solution for 10 minutes and the reaction mixture was irradiated with UV light for 20 minutes. The solvent was evaporated and the product was purified by flash column chromatography (hexane / acetone 8:2) to yield compound 10 (4.0 g, 73%) as a colorless syrup. Rf = 0.56 (hexane / acetone 7:3); 'HNMR (400 MHz, CDCh): d (ppm) 4.40 (p, 1H, J = 6.4 Hz, CI77), 3.83-3.59 (m, 6H, 3CH2), 3.12-2.94 (m, 1H, CH2), 2.84-2.65 (m, 1H, CH2), 2.23 (s, 1H, OH);13C NMR (100 MHz, CDCh): <5 (ppm) from 120.4 to 108.1 highly splitted signs (6C, CF2and CF3), 75.8, 72.4, 61.8 (3C, CH2), 37.9 (t, 1C, CH2), 15.0 (1C, CHI); HRMS: calculated for CnHi0Fi3IO2Na+570.9410 [M+Na]+; found: 570.9401 m / z.

[0439] Compound 11: Compound 10 (4 g, 0.73 mol) was dissolved in methanol (25 ml) and Argon gas was bubbled through the solution for 10 minutes. Palladium on activated charcoal (10%, 800 mg, 2.5 equiv.) and NaHCCh (1.5 g, 1.82 mmol, 2.5 equiv.) were added. The reaction mixture was stirred overnight under H2athmosphere, then filtered through Celite, and the solvent was evaporated. The residue was dissolved in dichloromethane (100 ml) and the solution was washed with distilled water (2x20 ml), dried over anhydrous Na2SC>4, filtered and the solvent was evaporated in vacuum. The product was purified by flash column chromatography (hexane / acetone 8:2) to yield 2.3 g (77%) compound 11 as a colorless syrup. Rf= 0.5 (hexane / acetone 7:3); 'H NMR (400 MHz, CDCh) <5 (ppm) 3.78-3.71 (m, 2H, CH2), 3.60-3.52 (m, 4H, 2CH ).

[0440] 2.31-2.11 (m, 3H, CH2, OH), 1.96-1.86 (m, 2H, CH,)13C NMR (100 MHz, CDCh) <5 (ppm) from 121.2 to 108.9 highly splitted signs (6C, CF2and CF3), 72.2, 69.8, 61.9 (3C, CH2), 28.1 (t, 1C, CH2), 20.9 (1C, CH2); HRMS: calculated for CiiHnFi3O2Na+445.0444 [M+Na]+; found: 445.0433 m / z.

[0441] Compound 12: Compound 11 (1 g, 2.36 mmol) was dissolved in anhydrous THF and cooled in an ice-water bath. Then argon gas was bubbled through the solution for 10 minutes, then NaH (226 mg, 5.796 mmol, 60% dispersion in mineral oil) was added and the mixture was stirred for 30 minutes under argon atmosphere. After 30 minutes stirring, 1.2 mmol propargyl bromide (420 pl, 2.832 mmol, 1.2 equiv., 80% solution in toluene) was added and the reaction mixture was stirred overnight at room temperature. Then methanol (2 ml) and water (10 ml) were added to the mixture and it was stirred for 15-15 minutes. The solvent was evaporated, the residue was dissolved in dichloromethane (200 ml) and the solution was washed with distilled water (2x20 ml). The organic phase was dried over anhydrous Na2SC>4, filtered and evaporated in vacuum. The product was purified by flash column chromatography (hexane / ethyl acetate 95:5) to yield compound 12 (838 mg, 77%) as a colorless syrup. Rf = 0.55 (hexane / ethyl acetate 9:1); 'H NMR (500 MHz, CDCI3) 8 (ppm) 4.21 (d, 2H, J= 2.4 Hz, CH2propargyl), 3.73-3.68 (m, 2H, CH2), 3.65-3.60 (m, 2H, CH2), 3.56 (t, 2H, 7= 6.1 Hz, CH2), 2.43 (t, 1H, 7 = 2.4 Hz, CH propargyl), 2.27-2.13 (m, 2H, CH2), 1.94-1.85 (m, 2H, CH2);13C NMR (100 MHz, CDCh) <5 (ppm) from 120.7 to 108.8 highly split signals (6C, CF2and CF3),79.6 (1C, Cqpropargyl), 74.6 (1C, CH propargyl), 70.2, 69.9, 69.2 (3C, CH2), 58.5 (1C, CH2propargyl) 28.1 (t, 1C, CH2), 20.9 (1C, CH2); HRMS: calculated for Ci4Hi3Fi3O2Na+483.0600 [M+Na]+; found: 483.0595 m / z.

[0442] Compound 2: Teicoplanin pseudoaglycone (TC) azide (143 mg, 0.1 mmol) and compound 12 (55 mg, 0.12 mmol, 1.2 equiv.) were dissolved in anhydrous dimethylformamide (5 ml). Triethylamine (2 equiv., 28 pl, 0.2 mmol) and Cu(I)-iodide (4 mg, 0.02 mmol) were added, and the reaction mixture was stirred under argon athmosphere for 2 days. Then Na2S (5 mg, 0.064 mmol) dissolved in 1 ml of water was added. After 10 minutes stirring the solvent was evaporated in vacuum (butanol was added to prevent foaming) and the residue was purified by flash column chromatography (acetonitrile / water 95:5— >93:7) to yield compound 2 (44 mg, 23%) as a yellowish solid foam. Rf = 0.33 (acetonitrile / water 9:1); HRMS: Calculated for CsoHesChFiaNioChsNa^: 1931.3316 [M-H+2Na]+; found: 1931.3324 m / z. The structure of compound 2 with numbering for peak assignment:

[0443] Danubia Ref.: P142354

[0444]

[0445] Compound 2

[0446] Table 1: 'H-^C HSQC NMR assignment of compound 2.

[0447]

[0448] Danubia Ref.: P142354

[0449]

[0450] HSQC (125 MHz DMSO-d6)

[0451] EXAMPLE 3 - Synthesis of the compound 7

[0452] For the synthesis of the perfluorohexyl teicoplanin derivative 7, the fluorous carboxylic acid 15 was prepared from allyl alcohol and perfluorohexyl iodide in three steps including photoinitiated addition, catalytic hydrogenolysis and oxidation with the TEMPO-BAIB reagent combination. (Scheme 2). Compound 15 was converted to the active ester 16 with A-hydroxysuccinimide in the presence of EDC. Finally, by acylating the A-terminal amino group of the teicoplanin pseudoaglycone with the active ester 16, compound 7 was obtained, in which the perfluorohexyl group is directly connected to the glycopeptide skeleton.

[0453] Danubia Ref.: P142354

[0454] > < "

[0455] "

[0456]

[0457] Scheme 2

[0458] Scheme 2 shows the synthesis of perfluorohexyl derivative compound 7 from teicoplanin pseudoaglycone via amide formation. TEMPO: tetramethylpiperidine 1-oxyl, BAIB: bis(acetoxy)iodobenzene, NHS: A-hydroxy succinimide, EDC: l-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0459] Compound 15: Compound 14 (293 mg, 0.775 mmol) was dissolved in dichloromethane (5 ml) and distilled water (2.5 ml) was added. TEMPO (2,2,6,6-tetramethylpiperidine 1-oxyl, 20 mg, 0.139 mmol, 0.18 equiv.) and BAIB ((diacetoxyiodo)benzene, 749 mg, 2.325 mmol, 3 equiv.) were added and the reaction mixture was stirred for 5 h. Then it was neutralized with 10% aqueous solution of Na2.S2Os (3 ml), diluted with dichloromethane (100 ml) shaked, then separated. The aqueous phase was extracted with dichloromethane (2x100 ml), the combined organic phase was dried over Na2SC>4, filtered and the solvent was evaporated. The product was purified by flash column chromatography (hexane / acetone 8:2) to yield compound 15 (259 mg, 85%) as a white powder. A’r = 0.4 (hexane / acetone 7:3);13C NMR (100 MHz, methanol-d4): 3 (ppm) 175.5 (1C, C=O), 119.7, from 120.0 to 109.8 highly split signals (6C, CF2and CF3), 27.5 (t, 1C, CH2), 26.0 (1C, CH2).

[0460] Compound 16: Compound 15 (200 mg, 0.51 mmol) was dissolved in the mixture of anhydrous dichloromethane (5 ml) and anhydrous THF (5 ml) and under argon atmosphere N-hydroxy succinimide (65 mg, 0.561 mmol, 1.1 equiv.) was

[0461] Danubia Ref.: P142354added and the mixture was cooled to 0 °C. After 10 min, EDC (l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 108 mg, 0.561 mmol, 1.1 equiv.) was added and the reaction mixture was stirred overnight. Then the solvent was evaporated and the product was purified by flash column chromatography (hexane / acetone 8:2) to yield compound 16 (224 mg, 90%) as a white powder.

[0462] 0.32 (hexane / acetone 7:3); ’HNMR (400 MHz, CDCh): <5 (ppm) 3.02-2.94 (m, 2H, CH2), 2.86 (s, 4H, succinimide CH2), 2.71-2.50 (m, 2H, CH2);13C NMR (100 MHz, CDCh): <5 (ppm) 168.9 (2C, succinimide C=O), 166.9 (1C, C=O), from 120.0 to 111.1 highly split signals (6C, CF2and CF3), 26.4 (t, 1C, CH2), 25.7 (2C, succinimide CH2), 23.0 (1C, CH2).

[0463] Compound 7: Teicoplanin pseudoaglycone (TC) (136 mg, 0.1 mmol) was dissolved in anhydrous dimethylformamide (3 ml) and triethylamine (21 pl, 0.15 mmol, 1.5 equiv.) was added. Under argon atmosphere compound 16 (62 mg, 0.13 mmol, 1.3 equiv.) was added, and the reaction mixture was stirred overnight. Then the solvent was evaporated and the product was purified by flash column chromatography to yield compound 7 (119 mg, 69%) as a dirty white solid. A’r = 0.2 (toluene / methanol 1:1 containing 1.0 v / v% acetic acid); MALDI-TOF-MS: calculated for C75H61O2F13N8O24 1797.286 [M+Na]+; found 1797.271. The structure of compound 7 with numbering for peak assignment NM:

[0464] " " "

[0465]

[0466] Compound 7

[0467] Table 2:19F NMR assignment of compound 7.

[0468]

[0469] 19F NMR (658 MHz DMSO-d6)

[0470] Danubia Ref.: P142354Table 3: 'H-^C HSQC NMR assignment of compound 7

[0471]

[0472] Danubia Ref.: P142354

[0473]

[0474] HSQC (125 MHz DMSO-d6)

[0475] 13C (125 MHz DMSO-d6)

[0476] EXAMPLE 4 - Synthesis of the compound 8

[0477] Ristocetin aglycone derivative compound 8 containing a perfluorooctyl substituent was prepared in the same way as the synthesis of compound 7, but using perfluorooctyl iodide as the fluorous reactant (Scheme 3). Active ester 19, prepared from perfluorooctyl iodide and allyl alcohol in four steps, was conjugated to ristocetin aglycone (Naesens et al., 2009) by amide coupling to provide compound 8 bearing the perfluorous group directly connected to the glycopeptide skeleton. This compound is the ristocetin counterpart of teicoplanin derivative compound 6 containing exactly the same hydrophobic group.

[0478] 1. C8F17I

[0479] benzophenone

[0480]

[0481] Danubia Ref.: P142354Scheme 3 shows the synthesis of compound 8. (TEMPO: tetramethylpiperidine 1-oxyl, BAIB: bis(acetoxy)iodobenzene, EDC: l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, THF: tetrahydrofuran.) Compound 8: Ristocetin aglycone (100 mg, 0.085 mmol) was dissolved in anhydrous dimethylformamide (3 ml) and triethylamine (18 pl, 0.13 mmol, 1.5 equiv.) was added. Under argon atmosphere compound 19 (65 mg, 0.11 mmol, 1.3 equiv.) was added, and the reaction mixture was stirred overnight. Then the solvent was evaporated and the product was purified by flash column chromatography to yield compound 8 (60 mg, 43%) as a dirty white solid. A’r = 0.69 (toluene / methanol 1:1 containing 1.0 v / v% acetic acid); MALDI-TOF-MS: calculated for C71H54F17N7O20 1670.304 [M+Na]+; found 1670.408. The structure of compound 8 with numbering for peak assignment:

[0482] <

[0483]

[0484] Compound 8

[0485] Table 4:19F NMR assignment of compound 8

[0486]

[0487] 19F NMR (658 MHz DMSO-d6)

[0488] Table 5: ’H-^C HSQC and ’H-’H HMBC NMR assignment of compound 8

[0489]

[0490] Danubia Ref.: P142354

[0491]

[0492] Danubia Ref.: P142354

[0493]

[0494] HSQC (125 MHz DMSO-d6)

[0495] 13C (125 MHz DMSO-d6)

[0496] EXAMPLE 5 - Study on antiviral effects and cytotoxicity

[0497] In this example we disclose the results of studies on the antiviral effects and cytotoxicity of teicoplanin, vancomycin, ristocetin and their deglycosylated hydrophobic derivatives (overall 16 glycopeptide antibiotic derivatives). The studied compounds showed a broad range of inhibitory effects. Vero E6 cells were infected with ZIKV (MOI: 1), SARS-CoV-2 (MOI: 0.1), CHIKV (MOI: 0.1), and ONNV (MOI: 0.1). Because of the differences in virus infection dynamics, we used different MOI in ZIKV. Among the natural glycopeptide antibiotics and their aglycones, only the native teicoplanin complex showed some antiviral activity, and the hydrophobic vancomycin derivatives SI and S2 were also completely inactive (see Table 2). At the same time, we observed that most of the semi-synthetic, hydrophobic teicoplanin derivatives (compounds 1-7) did not exhibit any cytotoxic effects at concentrations below 50 pM, but their antiviral effects were noticeable. The hydrophobic ristocetin derivative compound 8 showed an inhibitory effect against all tested viruses but was also slightly cytotoxic. It's important to note that we only tested the virus-inhibitory effects of the compounds at concentrations where no cytotoxic effects were observed. During the experiments, we concurrently examined the GPAs on both infected and uninfected cells, where in two cases, inhibitory effects against all four viruses were observed (Table 6). The compounds were tested against different viruses in a 7-element concentration series. We measured cell viability by MTT assay and calculated EC50 (half maximal effective concentration) and CC50 (50% cytotoxic concentration) from the OD (optical density) values. SI (selectivity index) values are calculated and only those above 10 are considered suitable for further testing (Indrayanto et al., 2021). In the case of ZIKV, the assay was also performed on A549 cell line with compound 7. The EC 50 value (5.832 pM) was very similar to the result obtained on Vero E6 (5.18 pM) (Fig. 1). No cytopathic effects (CPE) were observed for compounds 2, 3, 4, 6 and 7, after 5 days of ZIKV infection, even at the highest concentrations, where compounds caused no cytotoxicity (Figure 10).

[0498] Danubia Ref.: P142354Table 6: Inhibitory effect of GPA derivatives against various viruses based on the CPE inhibition assay on Vero E6 cell line.

[0499] ZIKV SARS-COV-2 CHIKV ONNV >

[0500] >

[0501] >

[0502] >

[0503] >

[0504]

[0505] 6 >60 174.60 6.14 28.44 4.70 37.15 61.07 2.86 n.a.

[0506] 7 ( >60 203.30 5.18 39.25 10.29 19.76 6.49 31.33 5.37 37.86 8 >20 62.63 42.3 1.48 9.88 6.34 14.04 4.46 39.50 1.59

[0507] Abbreviations: MCC - compound concentration producing minimal changes in cell morphology, as estimated by microscopy; EC50 - half maximal effective concentration; n.a. - not active; SI - selectivity index; CC50 50% - cytotoxic concentration.

[0508] Table 7 below shows additional data about inhibitory effect of GPA derivatives against Sindbis virus.

[0509] Table 7: inhibitory effect of GPA derivatives against Sindbis virus

[0510]

[0511] Abbreviations: EC50 - half maximal effective concentration; n.a. - not active.

[0512] Based on the results it can be seen that the studied GPAs show antiviral activities amongst different emerging viruses.

[0513] EXAMPLE 6 - Time of-addition assay

[0514] After establishing the potent anti-ZIKV activity of compound 7, we performed a time of-addition assay to identify at which stage of the viral replication cycle the compound possesses inhibitory activity. We divided the samples into five groups based on the timing of treatment: no-treatment (ZIKV), pre-treatment for 1 hour (pre), co-treatment (co), posttreatment for 1 hour (post), and full treatment (full) (Fig. 2). After 24 hours post-infection (pi), we isolated the viral RNA from supernatant and quantified it using qRT-PCR. The virus was added at MOI of 1 and for the treatment compound 7 was used at 50 p M. The results indicated significant differences in viral RNA levels compared to no-treatment group only in the case of co-treatment, leading us to conclude that the compound probably exerts its effects during the early stages of infection (Fig. 4B).

[0515] The results show that the studied GPA derivative compound 7 reduces the viral RNA level during the early stages of ZIKV infection.

[0516] Danubia Ref.: P142354EXAMPLE 7 - Binding and entry assays

[0517] For a more precise understanding of the mechanism of action of compound 7, we performed binding and entry assays. In the binding assay, infection and treatment were conducted simultaneously for 1 hour at 4 °C. This low temperature was chosen because it restricts the virion entry and beneficial for evaluating the impact of virus binding (Lu et al., 2021). For the entry assay, cells were infected for 1 h at 4 °C, washed twice with PBS, treated with compound 7 and incubated for 1 h at 37°C (Fig 2D). Vero E6 cells were infected at MOI of 1. For both assays, after the incubation, cells were washed with PBS before RNA extraction, and the results were analysed using qRT-PCR. We observed no significant difference between the treated and control groups in both assays (Fig. 24C). This led us to conclude that the GPA derivatives directly restrict the virion. To prove this, we performed an inactivation assay. For this, we pre-treated the virus stock with 50 pM of 37 °C for 2 hours. After that, we diluted the virus-drug mixtures 100-fold to eliminate our compound from the system. Then we determined the plaque-forming units (PFU) of the control and treated stocks by using a plaque assay. The results showed that compound 7 reduced the PFU by an order of magnitude (Fig. 3). This indicates that this compound directly reduces the stability of the virion. To test the virion inactivation effect, a destabilization assay was performed. The virus stock (apx. 5000000 PFU / ml) was treated with 50 pM compound 7 for 1 hour at 37 °C, then RNase A was added and incubated for 1 hour at 37 °C. Protein K was added at the end for RNase A digestion for Ih at 37 °C, followed by RNA extraction and measurement of viral RNA by qRT-PCR. As a positive control, 20% ethanol was used, while DMEM was used as a negative control in place of the compound. No significant difference was obtained in the results of our study compared to the negative control, the viral RNA copy number was not decreased (Fig. 3). We can conclude that the compound does not directly degrade the virion.

[0518] The result shows that the virucidal effect of the studied GPA derivative compound 7 occurs through the inactivation of the ZIKV virions, but not destabilising them.

[0519] EXAMPLE 8 - Mechanistic investigation of the effect of compound 7

[0520] The ZIKV envelope protein (E protein) contains three domains (Dai et al., 2016): a central B-barrel domain (domain I), a long finger- like domain (domain II) and an immunoglobulin-like domain (domain III) at the C-terminus (Fig. 4, there is an additional stem region and transmembrane anchor region of the protein at the C-terminus, which is not shown). Infected cells internalize ZIKV particles by envelope protein-mediated endocytosis (Lindenbach & Rice, 2003). Before uptake, the envelope protein of the virus sits on the viral surface in the form of antiparallel dimers, with their fusion loop buried. The dimers dissociate on the lower pH of the endosome, which exposes the fusion loop region of the monomers. The fusion loop can integrate into the endosomal membrane, which causes the formation of E protein trimers (Barba-Spaeth et al., 2016). Then, by proposedly irreversible conformational changes, the trimers fuse the endosomal and viral membranes, releasing the genetic content of the virus inside the cytosol. This is illustrated by the E protein trimer structure of the DENV, whose domain III closes itself onto domain I, with its C-terminus pushed towards the fusion loop (Fig. 4). Also, there is a slight rotation of domain II towards domain I.

[0521] To investigate the mechanistic details of the effect of compound 7 upon the function of the envelope protein, we utilized state-of-the-art binding site detection and ligand docking tools, as well as the X-ray crystal structures of the Zika virus E protein dimer from the Protein Data Bank (PDB IDs 5JHM (Dai et al., 2016) and 5LBV (Barba-Spaeth et al., 2016)).

[0522] Danubia Ref.: P142354First we mapped possible binding sites on the surface of every deposited chain in the two complexes using the FTmap protocol (Brenke et al., 2009; Ngan et al., 2012). FTMap is based on the conformational sampling of small, organic probe molecules on the protein’s surface, in order to identify energetically favourable regions, so-called hotspots, that are particularly suitable for small-molecule or macromolecular ligand binding. Here, FTMap identified three binding sites (Fig. 4): Sitel is found at the interface between domains I and III, Site2 is found only on the chains of the 5LBV structure at the region connecting domains I and III near the glycan loop (which is not present in structure 5JHM), while Site3 appears as a weaker, shallow binding pocket on domain II, near its interface with domain I, near a possible cryptic, hydrophobic pocket, hidden by a P-hairpin loop (residues 274-286 in 5JHM and 5LBV) (Modis et al., 2004).

[0523] While we did not find any prior studies describing Sitel, it apparently disappears during the dimer-trimer reorganization upon membrane fusion (Figure 9). To corroborate this, we mapped the surface of the trimeric DENV E protein structure (PDB ID:1OK8) with FTMap, where indeed, this site was not found (Figure 9). Therefore, we propose that ligand binding at this location could stabilize the dimeric form and thereby inhibit dimer-trimer conversion, and consequently, membrane fusion.

[0524] Site2 was identified by Sharma and co-workers by computational means, and described as the likely binding site of a natural compound, and a further inhibitor discovered by virtual screening, found to inhibit ZIKV infection in vitro (Carneiro et al., 2016; Sharma et al., 2017, 2020). The proposed mechanism of action is that ligand binding reduces the flexibility of the linker that connects domains I and III (residues 298-305), which in turn is needed for domain rearrangement and membrane fusion (Zheng et al., 2011).

[0525] The cryptic pocket next to Site3 was detected experimentally on the Dengue virus E protein by co-crystalizing it with the detergent n-octyl- -D-glucoside ( -OG) (Modis et al., 2004). Several Dengue membrane fusion inhibitors were found to bind to this site and it was proposed as a general binding site for anti-flaviviral drugs, due to several conserved residues found here (de Wispelaere et al., 2018). Here, we hypothesized that the poly-fluorinated tail region of compound 7 could bind to this cryptic site similarly to P-OG, while the multicyclic core could fit the shallower binding site identified by FTMap.

[0526] To assess the binding mode of compound 7, we used Schrodinger Glide (and for Site3, Induced Fit Docking, see EXAMPLE 1 - Materials and Methods) to dock the molecule to the three binding sites found. We ranked the resulted docking poses for each chain based on their Glide scores, and selected the best 20 ligand-protein complexes, which were finally evaluated by MM / GBSA dGbind scores and visual inspection. The results can be seen in Table 8.

[0527] Table 8: MM / GBSA dGbind (kcal / mol) scores at different sites for compound 7

[0528]

[0529] The different binding modes predicted for compound 7 are shown in Fig. 5. We were not able to produce a viable binding pose for Site3, so we excluded this binding site from further analysis. From the remaining two binding pockets, Sitel produced a better MM / GBSA score (see Table 8) and showed a tighter complementarity with the pocket. Here, several contacts are detected with residues from domains I and III, including a salt bridge with the sidechain of K340. At Site2, the calculated dGbind score is almost half of that at Sitel, and focusing on the linker loop between domains I and III

[0530] Danubia Ref.: P142354(needed for trimer formation), the modeled pose most notably shows a hydrogen bond interaction with K301. However, due to the highly flexible nature of its H-bonding partner (amide linker connecting the fluorinated tail of compound 7 to its core unit), we cannot safely assume that this binding mode would be strong enough to exert any functional effect by interacting with this loop. This was further corroborated by equilibrium MD simulations of the predicted binding modes of compound 7, showing overall stability for the binding mode in Sitel, and multiple dissociation events in Site2 (Figure 11). Therefore, we propose Sitel as the main binding site for compound 7, with the inhibition of the E protein dimer-trimer conversion as the basis for its detected in vitro antiviral activity.

[0531] EXAMPLE 9

[0532] Our aim was to support the bioinformatic modelling with experiments. We previously demonstrated that the amount of viral RNA in the cell does not change after 2 hours of treatment with compound 7 so the same amount of virus is in the cells as in untreated. This suggests that the treated virus may be able to enter the endosome. Based on this observation, and accepting the hypothesis that the compound inhibits membrane fusion through the inhibition of E protein domain rearrangement, the virus should be trapped in the endosomes, without entering the cell via membrane fusion. To corroborate this hypothesis experimentally, we examined the position of the NS1 protein by confocal microscopy. In positive sense single stranded RNA viruses, translation must go through before transcription. If the virus doesn't leave the endosome, translation won't occur, and the NS1 protein can't be labeled. Unfortunately, we did not have any additional markers that could directly prove the virus was trapped in the endosome. The NS1 protein is crucial for the virus to replicate (Sirohi & Kuhn, 2017). We infected Vero E6 cells for 4 hours (MOI: 1) and treated them with 50 pM compound 7. Our images clearly show that the intensity of the NS1 protein signal was much lower in the treated case than the not treated (Fig. 6). Based on these results, it can be concluded that the compound prevent translation.

[0533] EXAMPLE 10 - Capsid protection assay to monitor fusion of virions with liposomes

[0534] To study virion-endosome fusion and the inhibiting effect of the compounds of the invention thereon, using compound 7 as an example, a capsid protection assay to monitor fusion of virions with liposomes was used, as described in the Materials and Methods.

[0535] Briefly, we prepared liposomes that mimic the composition of late endosomal membranes. The lipid mixture was suspended in TAN buffer.

[0536] The liposomes of uniform size distribution were prepared by extruding through a membrane with a pore size of 0.2 pm, and trypsin was added immediately prior to extrusion, after the freeze-thaw cycles.

[0537] The virus samples were incubated with the tested compounds also in TAN buffer, then liposome containing trypsin was added to each sample, and the mixture was incubated. A virus fusion is induced by low pH in the endosomes, in this model experiment the pH was lowered to pH 5.3 to allow fusion to occur, and the mixture was incubated for 5 minutes. The pH was then restored by adding triethanolamine (pH 8.1), and the samples were incubated at 37 °C to allow trypsin digestion. The reaction was stopped and the Zika virus envelope (E) and capsid (C) proteins were separated and quantified using Western blot analysis.

[0538] The theoretical basis of the experiment is that successful virion-endosome fusion results in the release of the nucleocapsid, making the capsid protein accessible to trypsin inside the liposomes, which breaks it down. Accordingly,

[0539] Danubia Ref.: P142354under conditions that do not inhibit fusion (untreated or DMSO control), the C protein is not detectable on Western blot, or is detectable only as an extremely weak signal. In contrast, in samples where the fusion was inhibited, e.g. when the compound of the invention (here: compound 7) was present and active, a strong concentration-dependent capsid protein signal was observed.

[0540] This suggests that the compound effectively inhibited acid pH-induced virion-endosome fusion, thus protecting the capsid protein from trypsin digestion. The presence of envelope protein showed similar intensity in all samples, confirming that the observed differences were not due to differences in virus quantity but to inhibition of the fusion process.

[0541] The Western blot pattern presented clearly supports that the compound tested effectively blocks Zika virus endosomal fusion, thereby preventing nucleocapsid release. These results suggest that one of the key mechanisms of the compound's antiviral effect is the inhibition of the virus entry process.

[0542] SEQUENCES

[0543] Primer for ZIKV NS1 (artificial sequence):

[0544] Forward primer: AAA AGG AAA CGA GAG ATG TGG CA,

[0545] Reverse primer: CAT TCT CCT CTA GGA TAG CAT,

[0546] probes:

[0547] FAM: 5’-CCC GCA GAT-3’

[0548] ZEN: 5’-TGG CAG CAG-3’.

[0549] REFERENCES

[0550] Acharya, Y., Bhattacharyya, S., Dhanda, G., & Haidar, J. (2022). Emerging Roles of Glycopeptide Antibiotics: Moving beyond Gram-Positive Bacteria. ACS Infectious Diseases, <S(1), 1-28. https: / / doi.org / 10.1021 / acsinfecdis.lc00367 Agrelli, A., de Moura, R. R., Crovella, S., & Brandao, L. A. C. (2019). ZIKA virus entry mechanisms in human cells. Infection, Genetics and Evolution, 69, 22-29. https: / / doi.Org / 10.1016 / j.meegid.2019.01.018

[0551] Balzarini, J., Pannecouque, C., De Clercq, E., Pavlov, A. Y., Printsevskaya, S. S., Miroshnikova, O. V., Reznikova, M. I., & Preobrazhenskaya, M. N. (2003). Antiretroviral Activity of Semisynthetic Derivatives of Glycopeptide Antibiotics. Journal of Medicinal Chemistry, 46(13), 2755-2764. https: / / doi.org / 10.1021 / jm0300882

[0552] Barba-Spaeth, G., Dejnirattisai, W., Rouvinski, A., Vaney, M.-C., Medits, I., Sharma, A., Simon-Loriere, E., Sakuntabhai, A., Cao-Lormeau, V.-M., Haouz, A., England, P., Stiasny, K., Mongkolsapaya, J., Heinz, F. X., Screaton, G. R., & Rey, F. A. (2016). Structural basis of potent Zika-dengue virus antibody cross-neutralization. Nature, 536(7614), 48-53. http s : / / doi . org / 10.1038 / nature 18938

[0553] Beniazza, R., Atkinson, R., Absalon, C., Castet, F., Denisov, S. A., McClenaghan, N. D., Lastecoueres, D., & Vincent, J.-M. (2016). Benzophenone vs. Copper / Benzophenone in Light-Promoted Atom Transfer Radical Additions (ATRAs): Highly Effective lodoperfluoroalkylation of Alkenes / Alkynes and Mechanistic Studies. Advanced Synthesis & Catalysis, 355(18), 2949-2961. https: / / doi.org / 10.1002 / adsc.201600501

[0554] Bereczki, I., Csavas, M., Sziics, Z., Roth, E., Batta, G., Ostorhazi, E., Naesens, L., Borbas, A., & Herczegh, P. (2020). Synthesis of Antiviral Perfluoroalkyl Derivatives of Teicoplanin and Vancomycin. ChemMedChem, 15(11), 1661-1671. https: / / doi.org / 10.1002 / cmdc.202000260

[0555] Danubia Ref.: P142354Bereczki, I., Mandi, A., Roth, E., Borbas, A., Fizil, A., Komaromi, I., Sipos, A., Kurtan, T., Batta, G., Ostorhazi, E., Rozgonyi, F., Vanderlinden, E., Naesens, L., Sztaricskai, F., & Herczegh, P. (2015). A few atoms make the difference: Synthetic, CD, NMR and computational studies on antiviral and antibacterial activities of glycopeptide antibiotic aglycon derivatives. European Journal of Medicinal Chemistry, 94, 73-86. https: / / doi.Org / 10.1016 / j.ejmech.2015.02.028 Bereczki, I., Papp, H., Kuczmog, A., Madai, M., Nagy, V., Agocs, A., Batta, G., Milankovits, M., Ostorhazi, E., Mitrovic, A., Kos, J., Zsigmond, A., Hajdu, I., Lorincz, Z., Bajusz, D., Keserii, G. M., Hodek, J., Weber, J., Jakab, F., Borbas, A. (2021). Natural Apocarotenoids and Their Synthetic Glycopeptide Conjugates Inhibit SARS-CoV-2 Replication. Pharmaceuticals, 14( ), Article 11. https: / / doi.org / 10.3390 / phl4111111

[0556] Bereczki, I., Vimberg, V., Lorincz, E., Papp, H., Nagy, L., Keki, S., Batta, G., Mitrovic, A., Kos, J., Zsigmond, A., Hajdu, I., Lorincz, Z., Bajusz, D., Petri, L., Hodek, J., Jakab, F., Keserii, G. M., Weber, J., Naesens, L., ... Borbas, A. (2022). Semisynthetic teicoplanin derivatives with dual antimicrobial activity against SARS-CoV-2 and multiresistant bacteria. Scientific Reports, 12(f), 16001. https: / / doi.org / 10.1038 / s41598-022-20182-y

[0557] Blaskovich, M. A. T., Hansford, K. A., Butler, M. S., Jia, Z., Mark, A. E., & Cooper, M. A. (2018). Developments in Glycopeptide Antibiotics. ACS Infectious Diseases, 4(5), 715-735. https: / / doi.org / 10.1021 / acsinfecdis.7b00258 Bowers, K. J., Chow, D. E., Xu, H., Dror, R. O., Eastwood, M. P., Gregersen, B. A., Klepeis, J. L., Kolossvary, L, Moraes, M. A., Sacerdoti, F. D., Salmon, J. K., Shan, Y., & Shaw, D. E. (2006). Scalable Algorithms for Molecular Dynamics Simulations on Commodity Clusters. SC ’06: Proceedings of the 2006 ACM / IEEE Conference on Supercomputing, 43-43. https: / / doi.org / 10.1109 / SC.2006.54

[0558] Brenke, R., Kozakov, D., Chuang, G.-Y., Beglov, D., Hall, D., Landon, M. R., Mattos, C., & Vajda, S. (2009). Fragment-based identification of druggable ‘hot spots’ of proteins using Fourier domain correlation techniques. Bioinformatics (Oxford, England), 25(5), 621-627. https: / / doi.org / 10.1093 / bioinformatics / btp036

[0559] Burghgraeve, T. D., Kaptein, S. J. F., Ayala-Nunez, N. V., Mondotte, J. A., Pastorino, B., Printsevskaya, S. S., Lamballerie, X. de, Jacobs, M., Preobrazhenskaya, M., Gamarnik, A. V., Smit, J. M., & Neyts, J. (2012). An Analogue of the Antibiotic Teicoplanin Prevents Flavivirus Entry In Vitro. PLOS ONE, 7(5), e37244. https: / / doi.org / 10.1371 / journal.pone.0037244

[0560] Campos, G. S., Bandeira, A. C., & Sardi, S. I. (2015). Zika Virus Outbreak, Bahia, Brazil. Emerging Infectious Diseases, 27(10), 1885-1886. https: / / doi.org / 10.3201 / eid2110.150847

[0561] Carneiro, B. M., Batista, M. N., Braga, A. C. S., Nogueira, M. L., & Rahal, P. (2016). The green tea molecule EGCG inhibits Zika virus entry. Virology, 496, 215-218. https: / / doi.Org / 10.1016 / j.virol.2016.06.012

[0562] Dai, L., Song, J., Lu, X., Deng, Y.-Q., Musyoki, A. M., Cheng, H., Zhang, Y., Yuan, Y., Song, H., Haywood, J., Xiao, H., Yan, J., Shi, Y., Qin, C.-F., Qi, J., & Gao, G. F. (2016). Structures of the Zika Virus Envelope Protein and Its Complex with a Flavivirus Broadly Protective Antibody. Cell Host & Microbe, 79(5), 696-704. https: / / doi.Org / 10.1016 / j.chom.2016.04.013

[0563] de Wispelaere, M., Lian, W., Potisopon, S., Li, P.-C., Jang, J., Ficarro, S. B., Clark, M. J., Zhu, X., Kaplan, J. B., Pitts, J. D., Wales, T. E., Wang, J., Engen, J. R., Marto, J. A., Gray, N. S., & Yang, P. L. (2018). Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein. Cell Chemical Biology, 25(8), 1006-1016. e8. https: / / doi.Org / 10.1016 / j.chembiol.2018.05.011

[0564] Danubia Ref.: P142354Dick, G. W. A., Kitchen, S. F., & Haddow, A. J. (1952). Zika Virus (I). Isolations and serological specificity. Transactions of The Royal Society of Tropical Medicine and Hygiene, 46(5), 509-520. https: / / doi.org / 10.1016 / 0035-9203(52)90042-4

[0565] Duffy, M. R., Chen, T.-H., Hancock, W. T., Powers, A. M., Kool, J. L., Lanciotti, R. S., Pretrick, M., Marfel, M., Holzbauer, S., Dubray, C., Guillaumot, L., Griggs, A., Bel, M., Lambert, A. J., Laven, J., Kosoy, O., Panella, A., Biggerstaff, B. J., Fischer, M., & Hayes, E. B. (2009). Zika virus outbreak on Yap Island, Federated States of Micronesia. The New England Journal of Medicine, 360(24), 2536-2543. https: / / doi.org / 10.1056 / NEJMoa0805715

[0566] Enfissi, A., Codrington, J., Roosblad, J., Kazanji, M., & Rousset, D. (2016). Zika virus genome from the Americas. The Lancet, 357(10015), 227-228. https: / / doi.org / 10.1016 / S0140-6736(16)00003-9

[0567] Friesner, R. A., Banks, J. L., Murphy, R. B., Halgren, T. A., Klicic, J. J., Mainz, D. T., Repasky, M. P., Knoll, E. H., Shelley, M., Perry, J. K., Shaw, D. E., Francis, P., & Shenkin, P. S. (2004). Glide: A new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. Journal of Medicinal Chemistry, 47(7), 1739-1749. https: / / doi.org / 10.1021 / jm0306430

[0568] Halgren, T. A., Murphy, R. B., Friesner, R. A., Beard, H. S., Frye, L. L., Pollard, W. T., & Banks, J. L. (2004). Glide: A New Approach for Rapid, Accurate Docking and Scoring. 2. Enrichment Factors in Database Screening. Journal of Medicinal Chemistry, 47(7), 1750-1759. https: / / doi.org / 10.1021 / jm030644s

[0569] Hayes, E. B. (2009). Zika Virus Outside Africa. Emerging Infectious Diseases, 15(9), 1347-1350. https: / / doi.org / 10.3201 / eidl509.090442

[0570] Hoover, W. G. (1985). Canonical dynamics: Equilibrium phase- space distributions. Physical Review A, 31(3), 1695-1697. https: / / doi.org / 10.1103 / PhysRevA.31.1695

[0571] Indrayanto, G., Putra, G. S., & Suhud, F. (2021). Validation of in-vitro bioassay methods: Application in herbal drug research. In A. A. Al-Majed (Ed.), Profiles of Drug Substances, Excipients and Related Methodology (Vol. 46, pp. 273-307). Academic Press, https: / / doi.org / 10.1016 / bs.podrm.2020.07.005

[0572] Jacobson, M. P., Pincus, D. L., Rapp, C. S., Day, T. J. F., Honig, B., Shaw, D. E., & Friesner, R. A. (2004). A hierarchical approach to all-atom protein loop prediction. Proteins, 55(2), 351-367. https: / / doi.org / 10.1002 / prot.10613 Johnston, R. C., Yao, K., Kaplan, Z., Chelliah, M., Leswing, K., Seekins, S., Watts, S., Calkins, D., Chief Elk, J., Jerome, S. V., Repasky, M. P., & Shelley, J. C. (2023). Epik: pKa and Protonation State Prediction through Machine Learning. Journal of Chemical Theory and Computation, 19(8), 2380-2388. https: / / doi.org / 10.1021 / acs.jctc.3c00044 Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W., & Klein, M. L. (1983). Comparison of simple potential functions for simulating liquid water. The Journal of Chemical Physics, 79(2), 926-935. https: / / doi.org / 10.1063 / L445869

[0573] Kuno, G., Chang, G.-J. J., Tsuchiya, K. R., Karabatsos, N., & Cropp, C. B. (1998). Phylogeny of the Genus Flavivirus. Journal of Virology, 72(1), 73-83. https: / / doi.org / 10.1128 / jvi.72.L73-83.1998

[0574] Li, J., Abel, R., Zhu, K., Cao, Y., Zhao, S., & Friesner, R. A. (2011). The VSGB 2.0 model: A next generation energy model for high resolution protein structure modeling. Proteins, 79(10), 2794-2812. https: / / doi.org / 10.1002 / prot.23106 Lindenbach, B. D., & Rice, C. M. (2003). Molecular biology of flaviviruses. Advances in Virus Research, 59, 23-61. https: / / doi.org / 10.1016 / s0065-3527(03)59002-9

[0575] Danubia Ref.: P142354Lu, J.-W., Chen, Y.-C., Huang, C.-K., Lin, K.-C., & Ho, Y.-J. (2021). Synergistic in-vitro antiviral effects of combination treatment using anidulafungin and T-1105 against Zika virus infection. Antiviral Research, 195, 105188. https: / / doi.Org / 10.1016 / j.antiviral.2021.105188

[0576] Lu, C., Wu, C., Ghoreishi, D., Chen, W., Wang, L., Damm, W., Ross, G. A., Dahlgren, M. K., Russell, E., Von Bargen, C. D., Abel, R., Friesner, R. A., & Harder, E. D. (2021). OPLS4: Improving Force Field Accuracy on Challenging Regimes of Chemical Space. Journal of Chemical Theory and Computation, 17(1), 4291-4300. https: / / doi.org / 10.1021 / acs.jctc.lc00302

[0577] Marchette, N. J., Garcia, R., & Rudnick, A. (1969). Isolation of Zika virus from Aedes aegypti mosquitoes in Malaysia. The American Journal of Tropical Medicine and Hygiene, 18(3), 411-415. https: / / doi.org / 10.4269 / ajtmh.1969.18.411 Martyna, G. J., Tobias, D. J., & Klein, M. L. (1994). Constant pressure molecular dynamics algorithms. The Journal of Chemical Physics, 101(5), 4177^1189. https: / / doi.org / 10.1063 / L467468

[0578] Modis, Y ., Ogata, S., Clements, D., & Harrison, S. C. (2004). Structure of the dengue virus envelope protein after membrane fusion. Nature, 427(6912), 313-319. https: / / doi.org / 10.1038 / nature02165

[0579] Mohamadi, F., Richards, N. G. J., Guida, W. C., Liskamp, R., Lipton, M., Caufield, C., Chang, G., Hendrickson, T., & Still, W. C. (1990). Macromodel — An integrated software system for modeling organic and bioorganic molecules using molecular mechanics. Journal of Computational Chemistry, 11( ), 440-467. https: / / doi.org / 10.1002 / jcc.540110405 Musso, D., & Gubler, D. J. (2016). Zika Virus. Clinical Microbiology Reviews, 29(3), 487-524. https: / / doi.org / 10.1128 / cmr.00072-15

[0580] Zhou N, Pan T, Zhang J, Li Q, Zhang X, Bai C, Huang F, Peng T, Zhang J, Liu C, Tao L, Zhang H. (2016). Glycopeptide Antibiotics Potently Inhibit Cathepsin L in the Late Endosome / Lysosome and Block the Entry of Ebola Virus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV). The Journal of Biological Chemistry, 291(11). https: / / doi.org / 10.1074 / jbc.M116.716100 Naesens, L., Vanderlinden, E., Roth, E., Jeko, J., Andrei, G., Snoeck, R., Pannecouque, C., Illyes, E., Batta, G., Herczegh, P., & Sztaricskai, F. (2009). Anti-influenza virus activity and structure-activity relationship of aglycoristocetin derivatives with cyclobutenedione carrying hydrophobic chains. Antiviral Research, 82(1), 89-94. https: / / doi.Org / 10.1016 / j.antiviral.2009.01.003

[0581] Ngan, C. H., Bohnuud, T., Mottarella, S. E., Beglov, D., Villar, E. A., Hall, D. R., Kozakov, D., & Vajda, S. (2012). FTMAP: Extended protein mapping with user-selected probe molecules. Nucleic Acids Research, 40(fNe)o Server issue), W271-W275. https: / / doi.org / 10.1093 / nar / gks441

[0582] Obeid, S., Printsevskaya, S. S., Olsufyeva, E. N., Dallmeier, K., Durantel, D., Zoulim, F., Preobrazhenskaya, M. N., Neyts, J., & Paeshuyse, J. (2011). Inhibition of hepatitis C virus replication by semi- synthetic derivatives of glycopeptide antibiotics. Journal of Antimicrobial Chemotherapy, 66(6), 1287-1294. https: / / doi.org / 10.1093 / jac / dkrl04 Oehler, E., Watrin, L., Larre, P., Leparc-Goffart, L, Lastere, S., Valour, F., Baudouin, L., Mallet, H. P., Musso, D., & Ghawche, F. (2014). Zika virus infection complicated by Guillain-Barre syndrome - case report, French Polynesia, December 2013. Eurosurveillance, 19(9), 20720. https: / / doi.Org / 10.2807 / 1560-7917.ES2014.19.9.20720

[0583] Payne, S. (2017) Introduction to RNA Viruses. Viruses, 97-105. https: / / doi.org / 10.1016 / B978-0-12-803109-4.00010- 6

[0584] Danubia Ref.: P142354Pinter, G., Batta, G., Keki, S., Mandi, A., Komaromi, I., Takacs-Novak, K., Sztaricskai, F., Roth, E., Ostorhazi, E., Rozgonyi, F., Naesens, L., & Herczegh, P. (2009). Diazo Transfer-Click Reaction Route to New, Lipophilic Teicoplanin and Ristocetin Aglycon Derivatives with High Antibacterial and Anti-influenza Virus Activity: An Aggregation and Receptor Binding Study. Journal of Medicinal Chemistry, 52(19), 6053-6061. https: / / doi.org / 10.1021 / jm900950d Powers, A. M., Brault, A. C., Tesh, R. B., & Weaver, S. C. (2000). Re-emergence of Chikungunya and O’nyong-nyong viruses: Evidence for distinct geographical lineages and distant evolutionary relationships. The Journal of General Virology, <S7(Pt 2), 471-479. https: / / doi.org / 10.1099 / 0022-1317-81-2-471

[0585] Sastry, G. M., Adzhigirey, M., Day, T., Annabhimoju, R., & Sherman, W. (2013). Protein and ligand preparation: Parameters, protocols, and influence on virtual screening enrichments. Journal of Computer-Aided Molecular Design, 27(3), 221-234. https: / / doi.org / 10.1007 / sl0822-013-9644-8

[0586] Sastry, G. M., Adzhigirey, M., Day, T., Annabhimoju, R., & Sherman, W. (2013). Protein and ligand preparation: Parameters, protocols, and influence on virtual screening enrichments. Journal of Computer-Aided Molecular Design, 27(3), 221-234. https: / / doi.org / 10.1007 / slQ822-013-9644-8

[0587] Schmaljohn, Alan L. and McClain, David: Alphaviruses (Togaviridae) and Flaviviruses (Flaviviridae) Chapter 54 in Medical Microbiology, 4th edition, Baron, Samuel (Ed.) Galveston (TX): University of Texas Medical Branch at Galveston; 1996. ISBN-10: 0-9631172-1-1

[0588] Shan, C., Xia, H., Haller, S. L., Azar, S. R., Liu, Y., Liu, J., Muruato, A. E., Chen, R., Rossi, S. L., Wakamiya, M., Vasilakis, N., Pei, R., Fontes-Garfias, C. R., Singh, S. K., Xie, X., Weaver, S. C., & Shi, P.-Y. (2020). A Zika virus envelope mutation preceding the 2015 epidemic enhances virulence and fitness for transmission. Proceedings of the National Academy of Sciences, 117(33), 20190-20197. https: / / doi.org / 10.1073 / pnas.2005722117

[0589] Sharma, N., Murali, A., Singh, S. K., & Giri, R. (2017). Epigallocatechin gallate, an active green tea compound inhibits the Zika virus entry into host cells via binding the envelope protein. International Journal of Biological Macromolecules, 104(Pt A), 1046-1054. https: / / doi.Org / 10.1016 / j.ijbiomac.2017.06.105

[0590] Sharma, N., Prosser, O., Kumar, P., Tuplin, A., & Giri, R. (2020). Small molecule inhibitors possibly targeting the rearrangement of Zika virus envelope protein. Antiviral Research, 182, 104876. https: / / doi.Org / 10.1016 / j.antiviral.2020.104876

[0591] Sherman, W., Day, T., Jacobson, M. P., Friesner, R. A., & Farid, R. (2006). Novel procedure for modeling ligand / receptor induced fit effects. Journal of Medicinal Chemistry, 49(2), 534-553. https: / / doi.org / 10.1021 / jm050540c Sirohi, D., & Kuhn, R. J. (2017). Zika Virus Structure, Maturation, and Receptors. The Journal of Infectious Diseases, 276(suppl_10), S935-S944. https: / / doi.org / 10.1093 / infdis / jix515

[0592] Snyder, J.E. Jose, J. Kuhn, R.J. The Togaviridae and the Flaviviridae, Reference Module in Biomedical Sciences, Elsevier, 2014, ISBN 9780128012383, https: / / doi.org / 10.1016 / B978-0-12-801238-3.00085-4.

[0593] Sziics, Z., Naesens, L., Stevaert, A., Ostorhazi, E., Batta, G., Herczegh, P., & Borbas, A. (2020). Reprogramming of the Antibacterial Drug Vancomycin Results in Potent Antiviral Agents Devoid of Antibacterial Activity. Pharmaceuticals, 13(7), Article 7. https: / / doi.org / 10.3390 / phl3070139

[0594] Tong Jia Ming, S., Tan Yi Jun, K., & Carissimo, G. (2024). Pathogenicity and virulence of O’nyong-nyong virus: A less studied Togaviridae with pandemic potential. Virulence, 15(1), 2355201. https: / / doi.org / 10.1080 / 21505594.2024.2355201

[0595] Danubia Ref.: P142354Vanderlinden, E., Vanstreels, E., Boons, E., ter Veer, W., Huckriede, A., Daelemans, D., Van Lommel, A., Roth, E., Sztaricskai, F., Herczegh, P., & Naesens, L. (2012). Intracytoplasmic Trapping of Influenza Virus by a Lipophilic Derivative of Aglycoristocetin. Journal of Virology, 86(17), 9416-9431. https: / / doi.org / 10.1128 / jvi.07032-ll Wang, Y., Cui, R., Li, G., Gao, Q., Yuan, S., Altmeyer, R., & Zou, G. (2016). Teicoplanin inhibits Ebola pseudovirus infection in cell culture. Antiviral Research, 125, 1-7. https: / / doi.Org / 10.1016 / j.antiviral.2015.ll.003

[0596] Wang, Y ., Ling, L., Zhang, Z., & Marin-Lopez, A. (2022). Current Advances in Zika Vaccine Development. Vaccines, 70(11), Article 11. https: / / doi.org / 10.3390 / vaccinesl0111816

[0597] Wanner, J., Tang, D., McComas, C. C., Crowley, B. M., Jiang, W., Moss, J., & Boger, D. L. (2003). A new and improved method for deglycosidation of glycopeptide antibiotics exemplified with vancomycin, ristocetin, and ramoplanin. Bioorganic & Medicinal Chemistry Letters, 13(6), 1169-1173. https: / / doi.org / 10.1016 / S0960-894X(03)00051-9

[0598] Weaver, S. C., Charlier, C., Vasilakis, N., & Lecuit, M. (2018). Zika, Chikungunya, and Other Emerging Vector-Borne Viral Diseases. Annual Review of Medicine, 69, 395-408. https: / / doi.org / 10.1146 / annurev-med-050715-105122 Weaver, S. C., & Lecuit, M. (2015). Chikungunya Virus and the Global Spread of a Mosquito-Borne Disease. New England Journal of Medicine, 372(13), 1231-1239. https: / / doi.org / 10.1056 / NEJMral406035

[0599] Zheng, Y., Sanchez-San Martin, C., Qin, Z., & Kielian, M. (2011). The Domain LDomain III Linker Plays an Important Role in the Fusogenic Conformational Change of the Alphavirus Membrane Fusion Protein’. Journal of Virology, <S5(13), 6334-6342. https: / / doi.org / 10.1128 / JVI.00596-ll

[0600] Zhou, N., Pan, T., Zhang, J., Li, Q., Zhang, X., Bai, C., Huang, F., Peng, T., Zhang, J., Liu, C., Tao, L., & Zhang, H. (2016). Glycopeptide Antibiotics Potently Inhibit Cathepsin L in the Late Endosome / Lysosome and Block the Entry of Ebola Virus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) *. Journal of Biological Chemistry, 291(17), 9218-9232. https: / / doi.org / 10.1074 / jbc.M116.716100

[0601] Danubia Ref.: P142354

Claims

1. CLAIMS1. A compound of general formula (I.) for use in the treatment of a disease or condition caused by an enveloped, positive strand RNA virus selected from the group consisting of the Togaviridae and Flaviviridae family, said virus being internalized via endosome mediated endocytoses and by membrane fusion triggered by acidic endosomal pH, wherein preferably the virus is of the Kitrinoviricota phylum,preferably a member of the Flavivirus genus, preferably Zika virus, Dengue virus or West Nile virus,preferably a member of the Togaviridae family, preferably of the Alphavirus genus, preferably a Chikungunya virus, a O'nyong'nyong virus or a Sindbis virus, particularly preferably a Chikungunya virus,whereinR1is OH or a substituted or unsubstituted monosaccharide group, preferably a monosaccharide amine, optionally acylated, preferably an N-acetyl monosaccharide amine, in particular of formula a)R2is H, Ci-4 alkyl, preferably methyl or ethyl, wherein the R2OOC- may be R or S configuration,in part .i.cu ,lar, p R2i •s car Kboxy 1l or a carboxylate ester having the formu 1la H • *0vO ■'C**' or H3COOC' iR3is H or OH or Cl -4 alkoxy, preferably H or OH,X is l,4-diyl-lH-l,2,3-triazole or -(NH)-(CO)-,L is -CH2-O-(CH2-CH2-O)n-CH2-CH2-CH2- or -CH2-(CH2)m-CH2-,L is -CH2-O-(CH2-CH2-O)n-CH2-CH2-CH2- or -CH2-(CH2)m-CH2-,wherein n is 0 or 1 and m is 0 or 1 ,preferably L is -CH2-(CH2)m-CH2- and m is 0 or 1, preferably 0,andDanubia Ref.: P142354Y is -(CF2)k-CF3whereinn is 0 or 1 ,m is 0 or 1, andk is 3 to 7, preferably 5 to 7 or 4 to 6, in particular 5 to 6, in particular in case of Flaviviriedae, preferably Flaviviruses or k is 3 to 7, preferably 4 to 7, more preferably 5 to 7, in particular in case of Togaviridae, preferably Alphaviruses, with the provision that if X is l,4-diyl-lH-l,2,3-triazole, then n can not be 1, in particular in case of Flaviviriedae, preferably Flaviviruses,wherein preferably X is -(NH)-(CO)- in case of Togaviridae, preferably Alphaviruses,or pharmaceutically acceptable salts thereof.

2. A compound of general formula (1.1) according to claim 1 for use in the treatment of a disease or condition caused by a member of the Flavivirus genus, preferably Zika virus, Dengue virus or West Nile virus.

3. A compound for use of general formula (1.1) according to claim 1 for use in the treatment of a disease or condition caused by a member of the genus Alphavirus in the Togaviridae family, preferably a Chikungunya virus, O'nyong'nyong virus or Sindbis virus.

4. The compound according to any of claims 1 to 3, wherein the compound is a compound of Formula LI.whereinR1is OH or a substituted or unsubstituted monosaccharide group, preferably a monosaccharide amine, optionally acylated, preferably an N-acetyl monosaccharide amine, in particular of formula a)Danubia Ref.: P142354X is l,4-diyl-lH-l,2,3-triazole or -(NH)-(CO)-,L is -CH2-O-(CH2-CH2-O)n-CH2-CH2-CH2- or -CH2-CH2-,Y is -(CF2)k-CF3wherein n is 0 or 1, andk is 3 to 7, preferably 5 to 7 or 4 to 6, in particular 5, 6 or 7,with the provision that if X is l,4-diyl-lH-l,2,3-triazole, then n can not be 1 in particular for use in a condition or disease caused by a member of the Flavivirus genus, andin n is 1, then k is less than 5 in particular for use in a condition or disease caused by a member of the Alphaviridae genus,or pharmaceutically acceptable salts thereof.whereinR1is a monosaccharide amine, optionally acylated, preferably an N-acetyl monosaccharide amine,X is -(NH)-(CO)-,L is - -CH2-(CH2)m-CH2-,whereinm is 0 or 1, andY is -(CF2)k-CF3k is 3 to 7, preferably 4 to 7, more preferably 5 to 7,or pharmaceutically acceptable salts thereof.

5. The compound for use according to any of claims 1 to 4 wherein preferably the monosaccharide group in N-acetyl glucosamine and the compound is a compound of Formula 1.2.wherein preferablyX is -(NH)-(CO)-,L is -CH2-(CH2)m-CH2-,Y is -(CF2)k-CF3whereinDanubia Ref.: P142354m is 0 or 1, preferably 0, andwherein k is 4 to 7,more preferablyL is -CH2-CH2-,Y is -(CF2)k-CF3wherein k is 5 to 6or pharmaceutically acceptable salts thereof.wherein preferablyX is l,4-diyl-lH-l,2,3-triazole or -(NH)-(CO)-, preferably -(NH)-(CO)-,L is -CH2-O-(CH2-CH2-O)n-CH2-CH2-CH2- or -CH2-(CH2)m-CH2-,Y is -(CF2)k-CF3whereinn and m is 0,m is 0 or 1, andwherein k is 4 to 7, in particular in case of Flaviviriedae, preferably Flaviviruses; orn is 0 or 1 ,m is 0, andwherein k is 3 to 7, preferably 3 to 6 or 3 to 5 or 3 to 4, or 5 to 6, in particular 3 to 5, in particular in case of Togaviridae, preferably Alphaviruses,with the provision that if X is l,4-diyl-lH-l,2,3-triazole and n is 1, then k is less than 5, andhighly preferablyX is -(NH)-(CO)-,L is -CH2-CH2-,wherein k is 4 to 7, preferably 5 to 6.

6. The compound for use according to any of claims 1 to 5, wherein the compound is a compound of formula (II)OH"Danubia Ref.: P142354whereinX is -(NH)-(CO)-,L is -CH2-CH2-,Y is -(CF2)k-CF3wherein k is 3 to 7, preferably 5 to 7 in case of Flaviviruses,or pharmaceutically acceptable salts thereof.

7. The compound for use according to any of claims 1 to 6, wherein the compound is selected from the group consisting of>"Danubia Ref.: P142354"Danubia Ref.: P142354<> "or pharmaceutically acceptable salts thereof.

8. The compound for use according to any of the previous claims wherein the compound is selected from the group consisting of Compounds 2, 3, 4, 6, 7 and 8, preferably Compounds 3, 4, 6, 7 and 8, more preferably Compounds 6, 7 and 8.

9. The compound for use according to claim 8, wherein the virus is a virus of the Flavivirus genus, preferably Zika virus, Dengue virus or West Nile virus.

10. The compound for use according to claim 8, wherein the virus is a virus of the Alphaviridae genus, said compound selected from Compounds 3, 4, 6, 7 and 8, for use in an Alphavirus of the Togiviridae family preferably a Chikungunya virus, O'nyong'nyong virus or Sindbis virus..

11. The compound for use according to any of the previous claims wherein the compound is selected from the group consisting of Compounds 7 and 8.

12. The compound according to any of the previous claims wherein the antiviral effect of the compound is shown in an inhibition assay.Danubia Ref.: P14235413. The compound according to any of the previous claims wherein the antiviral effect of the compound is shown in an assay selected from the group consisting ofa CPE inhibition assay, highly preferably a CPE inhibition assay in a human cell line, anda capsid protection assay to monitor membrane fusion of virions, preferably with liposomes.

14. A pharmaceutical composition comprising a compound as defined in any one of the previous claims for use in the treatment of a disease or condition as defined therein,and a pharmaceutically acceptable excipient.

15. The pharmaceutical composition according to claim 14, wherein the subject is an animal, preferably a bird or a mammal, infected by a virus which is an enveloped, positive strand RNA virus selected from the group consisting of the Togaviridae and Flaviviridae family, said virus being internalized via endosome mediated endocytoses and by membrane fusion triggered by acidic endosomal pH,wherein preferably the virus is of the Kitrinoviricota phylum,16. The pharmaceutical composition according to claim 14 or 15, for use in the treatment of a disease or condition caused by a member of the Flavivirus genus, preferably Zika virus, Dengue virus or West Nile virus,17. The pharmaceutical composition according to claim 14 or 15, for use in the treatment of a disease or condition caused by a member of the genus Alphavirus in the Togaviridae family, preferably a Chikungunya virus, O'nyong'nyong virus or Sindbis virus, wherein preferably the monosaccharide group in the compound is N-acetyl glucosamine18. The pharmaceutical composition according to claim 16, wherein the Flavivirus is a Zika virus19. A compound, having the structure of formula (II),whereinX is -(NH)-(CO)-,L is -CH2-CH2-,Danubia Ref.: P142354Y is -(CF2)k-CF3wherein k is 3 to 7, preferably 5 to 7,or pharmaceutically acceptable salts thereof.

20. A compound according to claim 19, wherein said compound is compound (8)Compound 8 or pharmaceutically acceptable salts thereof.

21. A compound as defined in of claims 1 to 13, wherein said compound is compound (7)22. The compound for use according to any of claim 7, wherein the disease is caused by a bacterial infection or viral infection.Danubia Ref.: P142354

Images