Di(dispirodiazatripiperazine)-triazine derivatives and their use for treatment or prevention of viral infections

Abstract

The present invention provides di(dispirodiazatripiperazine)-triazine compounds and / or pharmaceutical compositions, such compounds and / or compositions for use in methods of treating or preventing diseases, in particular, viral diseases or infections, and methods for manufacturing such compounds and compositions.

Description

[0001] Charite - Universitatsmedizin Berlin

[0002] Korperschaft des bffentlichen Rechts 1 274 305 u22 / n41

[0003] DI(DISPIRODIAZATRIPIPERAZINE)-TRIAZINE DERIVATIVES AND THEIR USE FOR TREATMENT OR PREVENTION OF VIRAL INFECTIONS

[0004] FIELD OF THE INVENTION

[0005] The present invention generally relates to the field of antiviral compounds for the treatment of viral infections.

[0006] BACKGROUND ART

[0007] The biology of viruses unavoidably results in appearance of new pathologic strains. It is impossible to predict the timing of their appearance, genomic variability and antigenic properties. Thus, epidemics and pandemics of new viral infections will always start in the absence of specific immune prophylactics and treatment. This defines the importance of preliminary discovery and development of new antiviral therapies for treatment and prevention, with biological properties of viruses taken into account. The immunity after a viral infection does not usually last long and often does not protect from reinfection or reactivation; this underlines the need in development of drugs with wide antiviral spectrum which would protect the host from viral infection.

[0008] Among the biological aperiodic polymers (nucleic acids, polypeptides, carbohydrates), the carbohydrate aperiodic polymers (glycans, oligosaccharides) have the highest informational capacity due to their structural properties. This ensures high specificity of ligand-receptor interactions of oligosaccharide conjugates. It is suggested that the glycan structure is coded in the eukaryotic genome indirectly. Oligosaccharides synthesis takes place in the Golgi apparatus cisterns with the participation of secondary protein matrices which form functional heterogenic glycosyltransferase associations. Naturally, the special structure of such matrix protein molecules and thus their affinity to glycan synthesis enzymes, may quickly and significantly change under the influence of pH dynamics and redox potential in the Golgi apparatus environment. It should be taken into account that all participants of the human cells’ interaction with viruses (glycoproteins, glycolipids) are copiously decorated by unique glycans which are recognized by viral particles as specific receptors.

[0009] The issue of the participation of glycans in viral adhesion and replication is critical, as this notion may be applied in design and development of a wide spectrum drug against viruses. Such drugs may become a reserve for the current available drugs as well as treatment of new viral infections appearing in the future. Several viruses causing pathology in humans, including types 1 and 2 herpes viruses, human papilloma virus, cytomegalovirus, HIV viruses, respiratory syncytial virus, as well as coronaviruses use a common heparan sulfate-dependent mechanism of adhesion to a wall of a host cell. For this reason, inhibitors of viral / host cell interaction may play an important role in the treatment of multiple viral infections.

[0010] In the prior art, mainly two dispirotripiperazine derivatives are described as potential antiviral agents: an N, A / ’-bisheteryl derivative of dispirotripiperazine (DSTP): A / , / V-bis(1-oxido[1 ,2,5]oxadiazolo[3,4-d]pyrimidin- 7-yl)-3,12-diaza-6,9-diazonia(5,2,5,2)dispirohexadecane dichloride (DSTP-27), and a di(dispiro- tripiperazine) pyrimidine derivative: 3,3’-(2-methyl-5-nitropyrimidine-4,6-diyl)3, 12-bis-6,9-diaza- diazoniadispiro[5.2.5.2]hexadecane tetrachloride dihydrochloride (PDSTP), or derivatives of these. Yet, these compounds have complicated syntheses and issues with stability, e.g., since compounds based on pyrimidine derivatives can be easily degraded, e.g., under slight acid conditions. In addition, many dispiro compounds described in the prior art have a problem with their toxicity (e.g., the most studied derivative DSTP-27 releases nitric oxide).

[0011] Further, many prior art compounds have deficiencies with respect to the potency of their antiviral activity, their toxicity, half-life in vivo, potency, selectivity and / or spectrum of viral infections or diseases that may be treated or prevented.

[0012] In view of the above, the present invention aims at the object of identifying and providing advantageous compounds and / or pharmaceutical compositions useful in the prevention or treatment of viral diseases or infections, preferably overcoming one or more of the drawbacks in the prior art and / or having further advantages.

[0013] SUMMARY OF THE INVENTION

[0014] As a solution, the present invention provides di(dispirodiazatripiperazine)-triazine compounds and / or pharmaceutical compositions according to the appended claims and as described herein below, such compounds and / or or compositions for use in methods of treating or preventing diseases, in particular, but not limited to, viral diseases or infections, and methods for manufacturing such compounds and compositions.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 shows a synthetic pathway for preparing 2-chloro-4,6-di(3,12-diaza-6,9-diazoniadispiro[5.2.5.2] hexadecane-1 -yl)-1 ,3, 5-triazine tetrachloride (Compound 1).

[0017] FIG. 2 shows the carbon atom numbering of 2-chloro-4,6-di(3,12-diaza-6,9-diazoniadispiro[5.2.5.2] hexadecane-1 -yl)-1 ,3, 5-triazine as used in Example 2.

[0018] FIG. 3 shows cytotoxicity of the compounds in various cell lines. Various concentrations of cmpd (1) (grey square) and PDSTP (black triangle) were added to the cells. At seven days treatment, XTT cell proliferation assay was performed. Values are represented as percentage of untreated control cells. Data are mean values from four independent experiments. Error bars represent the standard deviations.

[0019] FIG. 4 shows dose-response-curves of Compound 1 (11826233) and PDSTP against (A) GCV-res. HCMV, (B) HCMV-pp150GFP, (C) HSV-1 on HELF, (D) HSV-1 on Vero E67, (C) OMNV, (F) PRV Ka, (G) PrV AgC, (H) PrV AgG and (I) Zika virus. Cells were preincubated for 30 min. at 37 °C with cmpd (1) and PDSTP and subsequently infected with the selected virus (MOI = 0.001). At 7 d p.i. (HCMV), 3 d p.i. (Zika virus, OMNV) or 2 d p.i. (HSV-1 ; PrV) plaques were normalized to the infected control without pretreatment. Experiments were performed in triplicates and implemented with four independent experiments (N = 4). The data points indicate the avg. ± SD. A summary of the results from these experiments is included in FIG. 6.

[0020] FIG. 5 shows determination of the antiviral activity (EC50) with luciferase assay (A) and dose-response curves of cmpd (1), PDSTP and Didanosin against HX HIV-Luci (B, C), and dose-response inhibition assay results of cmpd (1), PDSTP and another compound (11826234) against HIV-1 HXBc2 on TZM-bl. Didanosin is a nucleoside reverse transcriptase inhibitor (NRTI) used as control. Cells were preincubated for 30 min at 37 °C with cmpd (1), PDSTP or Didanosin and subsequently infected with HX HIV-Luci (MOI =0.01). (A) After 4 d p.i EC50 was analyzed by Luciferase assay. (B) For dose-response curves 4 d p.i. plaques were normalized to the infected control without pretreatment. (C) Results of the percent inhibition are shown. Experiments were performed in triplicates and implemented with three independent experiments (N = 3). The data points indicate the avg. ± SD. (D) Results of shows determination of the antiviral activity (EC50) of compound (1) (11826233), PDSTP and compound 11826234 against HIV-1 HXBc2 on TZM-bl.

[0021] FIG. 6 shows a summary of antiviral effect results for compound 1 relative to PDSTP and 11826234.

[0022] FIG. 7 shows time-of-Addition-Assays (A) Diagram of the experimental settings. (B, C) HELF Fi301 cells treated with EC90 of cmpd (1) or PDSTP or 10 pg / ml Heparin before, during, before and during and after infection (B, GCV-res HCMV; C, HSV-1 ; MOI 0.001) or left untreated were subjected to plaque reduction assays. Shown are mean values and SD of three independent experiments. Statistical significance was calculated using One-Way ANOVA with Dunnett's multiple-comparison test: (****) p < 0.0001 ; (***) p < 0.0002; (**) p < 0.0021 ; (*) p < 0.0332; (ns) < 0.1234.

[0023] FIG. 8 shows time-of-Addition-Assays (A) Diagram of the experimental settings. (B, C) HELF Fi301 cells treated with EC90 of cmpd (1) or compound 11826234 or PDSTP before, during, before and during and after infection (B, GCV-res HCMV; C, HSV-1 ; MOI 0.001) or left untreated were subjected to plaque reduction assays; experimental conditions and data presentation substantially correspond those described in FIG. 7.

[0024] FIG. 9 shows effects of the small molecules on attachment and penetration analysis. Attachment analysis (A-B): Prechilled HELF Fi301 cells were treated with (A) 7.62 pM cmpd (1), 7.08 pM 11826234 and 2.36 pM PDSTP, or (B) 3.88 pM cmpd (1), 13.08 pM 11826234 and 2.44 pM PDSTP, 10 pg / ml Heparin or left untreated (w / o) for 30 min at 4 °C. After removing the inhibitors cells were infected with prechilled (A) GCV- res HCMV (MOI 0.1) or (B) HSV-1 (MOI 0.001) for 2 h at 4 °C. Three washing steps aspirated unattached virus, cells were overlaid with methylcellulose and incubated six days (A) or two days (B). Penetration analysis (C-D): Prechilled HELF Fi301 cells were infected either with GCV-res. HCMV (MOI 0.1) or with HSV-1 (MOI 0.005) for 2 h at 4 °C. Thereafter the cells were treated with EC90 of cmpd (1), 11826234, PDSTP or 10 pg / ml heparin or left untreated (w / o). Penetration was allowed for 10 min at 37 °C and stopped by low pH treatment (pH 3.0). Cells were overlaid with methylcellulose and subjected to plaque reduction assays after six days (C) or 2 days (D). Given are mean values and SD from three independent experiments. Statistical significance was calculated using One-Way ANOVA with Dunnett's multiplecomparison test: (****) p < 0.0001 ; (***) p < 0.0002; (**) p < 0.0021 ; (*) p < 0.0332; (ns) < 0.12345.

[0025] FIG. 10 shows GFP auto-fluorescence, staining of actin filaments and staining of the nuclei with DAPI examined by fluorescence microscopy at indicated time points (48h - 120h) to assess the stability of the compounds. The untreated virus showed plaques starting after 72 h p.i. (B). In contrast, no infected cells were observed upon treatment with the compounds 11826233 (compound 1) and PDSTP (A-D).

[0026] FIG. 11 Dynamics of body weight of animals infected with HSV-1.

[0027] FIG. 12. Dynamics of survival of mice infected with HSV-1. FIG 13. Virus titer in mouse brain tissues taken 5 days after HSV-1 infection

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] All words and terms used herein shall have the same meaning commonly given to them by the person skilled in the art, unless the context indicates a different meaning. All terms used in the singular shall include the plural of that term and vice versa

[0030] Compounds of the di(dispirodiazatripiperazine)-triazine structure discovered during target-driven search of new drugs and disclosed herein specifically block heparan sulfate proteoglycans on the cell membranes of host cells leading to prevention of viral entry. This is a unique mechanism of affecting the viral invasion into a host cell and is based on the blocking of viral adsorption to a target cell due to specific inhibition of heparan sulfate proteoglycans.

[0031] This process may be described as blocking of viral attachment to a host cell; a drug based on the di(dispirodiazatripiperazine)-triazine of the present disclosure interacts with proteoglycans of host cells, which ensures a broad spectrum and generalizability of its mode of action.

[0032] Without wishing to be bound by theory, it is contemplated that the mechanism of action of dispirotripiperazine compounds of the present invention is related to their ability to specifically bind heparan sulfate proteoglycans which leads to dramatic reduction in viral entry and in viral replication. It has been demonstrated that attachment of investigational substances is being antagonized by heparin. The target of diazoniadispiro[5.2.5.2.]hexadecanes is represented by two sulfate groups located in adjacent saccharide residues; for example, for GlcA2S-GlcNS6S, GlcA2S-GlcNS3S, ldoA2S-GlcNAc6S, ldoA2S- GlcNH23SS6S, ldoA2S-GlcNS6S, and ldoA2SGIcNS3S, good electrostatic interaction was demonstrated between the negatively charged sulfate group and positively charged atoms of nitrogen in di(dispirotripiperazine)-derivatives. It has been also demonstrated that similar interaction is possible with a carbonyl group of AUA-GlcNSIdoUA2S-GlcNAc-UA2S-GlcNS-ldoUA2S-GlcNH23S octasaccharide, which represents an area of heparan sulfate essential for penetration of several viruses. Thus, di(dispirodiazatripiperazine)-triazines of the present disclosure are contemplated as blocking key functional groups of heparan sulfate proteoglycans, preventing viral replication and ensuring high antiviral activity. Currently, there are no approved drugs based on this mechanism of action.

[0033] The dispirotripiperazine compounds of the present invention are also contemplated as generally having a moderate immunosuppressive effect, which can be useful for blocking cytokine storm, e.g., caused by coronaviral infection.

[0034] The di(dispirodiazatripiperazine)-triazines of the present disclosure are easily soluble in water, and this property makes them very suitable for use in diverse pharmaceutical forms, e.g., for topical, transmucosal, inhalation or systemic applications.

[0035] The dispiro derivatives of the present disclosure are accessible via a synthesis that is easy to scale up, and are based on readily available, cheap triazines. The derivatives also have a high level of stability, beneficial half-life, bioavailability, bioactivity, solubility, and improved antiviral and biological properties. The present compounds having a “6-6-6 system” are advantageous over related systems, e.g., compounds having homopiperazine ring(s), such as a 7-6-7 dispiro system, in terms of physico-chemical properties, biological properties and / or synthesis. Exemplary compounds having a 7-6-7 dispiro system include, e.g., 3-{4- chloro-6-(3,7,10,13-tetraaza-7,10-dispiro[6.2.6.2]octadecanediium-3-yl)-1 ,3,5-triazin-2-yl}-3,7,10,13-tetraaza- 7,10-dispiro[6.2.6.2]octadecanediium tetrabromide and 3-{4-chloro-6-(3,7,10,13-tetraaza-7,10-dispiro [6.2.6.2]octadecanediium-3-yl)-1 ,3,5-triazin-2-yl}-3,7,10,13-tetraaza-7,10-dispiro[6.2.6.2]octadecanediium tetrachloride dihydrochloride. Compounds having homopiperazine ring(s) are much more complicated to synthesize compared to the present compounds. Synthetically, homopiperazine rings present many steric problems for the closure of the central piperazine ring, and the yield of such reactions is as low as 20%, which is not comparable with the >60% yield of the 6-6-6 system as in the present invention. Further, 7-membered homopiperazine cycles place significant stress on the central six-membered ring, leading to its instability and Hoffmann opening in aqueous solutions (which can be observed by NMR after 1 hour in water neutral solution). In contrast, the present 6-6-6 system is very stable and aqueous solutions are stable for at least one year at acidic or neutral pH without observation of Hoffmann cleavage. Overall, this shows that the present compounds, which have a 6-6-6 system attached to a triazine core, are much easier to synthesize, more stable and more suitable as pharmaceutical APIs due to their advantageous stability.

[0036] The derivatives of the disclosure can be used as medication for the treatment and / or prevention of different viral diseases or infections, in particular those caused by one or more enveloped and / or HSPG-dependent virus(es), such as cytomegalovirus, herpes simplex virus type 1 , Kaposi sarcoma associated virus (HHV- 8), as well as other viruses using heparan sulfate proteoglycans for attachment to a host cell. Particularly well-suited viral diseases or infections may include, but are not limited to, those caused by SARS-CoV2, Nipah virus, hepatitis B virus (HBV), Human immunodeficiency virus (HIV), papillomavirus (in particular, human: HPV), human orthopneumovirus (respiratory syncytial virus, RSV), Pseudorabies virus (PrV), Herpes simplex virus 1 (HSV-1), Zika virus (ZIKV), Dengue virus, O’nyong-nyong virus (ONV), Chikungunya virus, Adenovirus Type 5, Influenza A virus, Influenza B virus, and Mpox (MPXV).

[0037] The compounds and compositions of the present invention are advantageously applicable as broadspectrum antiviral agents, exhibiting good inhibition (EC50 values) against a multitude of viruses.

[0038] Beneficial effects in viral clearance are achieved through inhibition of viral attachment and / or viral entry mediated by the compound of the invention.

[0039] A skilled person could not have derived from common knowledge or the prior art that the inventive compounds would provide such broad-spectrum antiviral activity while at same time being effective at low dosing of the active compounds and compositions, such as in nM range. The compounds maintain efficacy over a broad range whilst having low or even non-detectable side effects. As is evident from the experimental support provided herein, even low doses of the active compounds, for example, between 0.2 and 10% of the maximum non-toxic dose in humans or animals, may be effectively employed. Viral clearance increased when administered in the inventive composition. Even when administered in low doses, the desired effect of an inhibition of viral entry remained.

[0040] The compounds and compositions of the present invention generally exhibit a low cytotoxicity, as confirmed by their CC50 (50% cytotoxic concentration) which is many fold (e.g., > 63-fold, preferably > 100-fold, more preferably 100 to 360-fold) higher than the compound concentration used for virus inhibition / treatment / prevention; and a low in vivo toxicity, as confirmed by the LD50.

[0041] The compounds of the present disclosure generally exhibit a selectivity index (SI) of > 10, i.e., they are biologically active and sufficiently non-toxic. In many cases, their SI is > 20, > 60, > 80, > 100, > 150, > 170 or even > 360.

[0042] Furthermore, the compounds and compositions of the present invention exhibit advantageous or improved antiviral activity, in particular: against viruses belonging to Orthoherpesviridae, Togaviridae, Flavivirdae and / or Orthoretrovirinae, such as HSV-1 , ONNV, PrV, ZIKV, and / or HIV-1 , especially when compared to related or prior art compounds. The compounds and compositions of the present invention exhibit advantageous or improved selectivity, in particular: against viruses belonging to Orthoherpesviridae, Togaviridae, and / or Flavivirdae, HCMV, HSV-1 , ONNV and ZIKV, especially when compared to related or prior art compounds. The compounds and compositions of the present invention exhibit advantageous or improved selectivity, in particular: before, before and during or during infection, and in particular with viruses belonging to Orthoherpesviridae, e.g., HCMV.

[0043] Furthermore, the compounds and compositions of the present invention exhibit advantageous or improved inhibition of virus cell penetration and / or attachment, at significant amount (e.g., compared to the untreated control, or even improved as compared to prior art compounds).

[0044] The present invention provides in particular the following embodiments.

[0045] Compounds

[0046] The present invention provides a compound which is a pharmaceutically acceptable salt of the following structure of Formula I: or a hydrate, solvate and / or acid addition salt thereof, wherein:

[0047] X is selected from halogen, alkoxy and haloalkoxy;

[0048] Y1and Y2are each independently a substituent, preferably selected from alkyl;

[0049] Z1, Z2, Z3and Z4are each independently selected from H and alkyl; n1and n2are each independently 0 or an integer of > 1 , preferably selected from 1 , 2, 3 and 4, n3and n4are each independently 0 or 1 ; if Z3is present, the nitrogen atom to which Z3is bound is positively charged; and if Z4is present, the nitrogen atom to which Z4is bound is positively charged. In the above notation, Y1and Y2may be attached to any of the piperazine rings. If Z3is present, the nitrogen atom to which Z1and Z3are bound is positively charged, absent, the nitrogen atom

[0050] In one embodiment, the compound is a pharmaceutically acceptable salt of Formula II: or a hydrate, solvate and / or acid addition salt thereof. In the above notation, Y1and Y2may be attached to any carbon atom of the respective terminal piperazine ring (i.e., the respective piperazine ring most distant from the triazine core).

[0051] In one embodiment, in any of the structures disclosed herein, X is selected from F, Br, Cl, OMe, OCF3 and OEt.

[0052] In one embodiment, in any of the structures disclosed herein, Y1and Y2are each independently selected from Ci alkyl, C2 alkyl, C3 alkyl, C4 alkyl and C5 alkyl.

[0053] In one embodiment, in any of the structures disclosed herein, Z1, Z2, Z3and Z4are each independently selected from H and Ci alkyl, C2 alkyl, C3 alkyl, C4 alkyl and C5 alkyl.

[0054] In one embodiment, in any of the structures disclosed herein, n1and n2are each 0. In another embodiment, n3and n4are each 0. In another embodiment, n1, n2, n3and n4are each 0.

[0055] In one embodiment, in any of the structures disclosed herein, Z1and Z2are independently selected from H and Ci alkyl, C2 alkyl, C3 alkyl, C4 alkyl and C5 alkyl, and Z3and Z4are absent.

[0056] Exemplary structures include those according to Formula Ila, lib or He:

[0057] Ila lib

[0058] Preferred structures include those listed in the following table: Preferred concrete compounds are listed in the following table:

[0059] Most preferably, the compound has a structure of Formula III (also referred to herein as “compound 1 ”, “cmpd (1)” or “11826233”): or a hydrate, solvate and / or acid addition salt thereof.

[0060] Pharmaceutically acceptable salts; acid addition salts

[0061] Since the structure of Formula I is cationic, the compounds of the present invention are provided as pharmaceutically acceptable salts, i.e., with suitable counterions. “Pharmaceutically acceptable” refers to molecular entities, compositions, salts or corresponding acids which retain the biological effectiveness and properties of the compound, and which are not biologically undesirable, such as do not produce a severe allergic or similar untoward reaction when administered to a subject. Suitable counterions may be derived from inorganic or organic acids. Preferable counterions include halogenide anions, more preferably chloride or bromide, most preferably chloride. Further suitable counterions may include non-nucleophilic pharmaceutically acceptable anions (e.g., mesylate, esylate, isethionate, tosylate, napsylate, benzenesulfonate).

[0062] Preferably, the pharmaceutically acceptable salt anion corresponds to group X, e.g., a chloride when X = Cl, and a bromide when X = Br. In such cases, there is a reduced likelihood of reactive changes during storage (e.g., exchange of a chlorine atom on the triazine moiety changes to bromine in the solid state, as observed with some prior art compounds).

[0063] In cases where n3and n4are each 0 (i.e., Z3and Z4are each absent), the structure of Formula I is a tetracation; accordingly, four monovalent pharmaceutically acceptable counterions are required. Preferably, all counterions are the same, e.g., tetrachloride.

[0064] Tertiary nitrogen atoms, if not quaternized (i.e., if present as free bases), may form acid addition salts with pharmaceutically acceptable acids, i.e., acids corresponding to suitable anions as above. The acid addition salt is preferably selected from a hydrochloride and a hydrobromide, more preferably dihydrochloride. Preferably, the anion in the acid of the acid addition salt is the same as counterion in the pharmaceutically acceptable salt, e.g., tetrachloride dihydrochloride.

[0065] Hydrates and solvates

[0066] The compounds of the present invention may be present in an anhydrous state or as hydrates and / or solvates. Often crystallizations may produce a hydrate or solvate of the compound of the invention. As used herein, the term “solvate” refers to an aggregate that comprises one or more molecules of a compound of the invention with one or more molecules of solvent. In some embodiments, the solvent is water, in which case the solvate is a hydrate. Alternatively, in other embodiments, the solvent is an organic solvent. In some embodiments, a combination of water and organic co-solvent molecules may be comprised in the solvate. In some aspects, the compound of the invention is a true solvate, while in other cases, the compound of the invention merely retains adventitious water or is a mixture of water plus some adventitious solvent. The compounds of the present invention may exist as a hydrate, e.g., comprising up to 10 mol of water per one mol of the compound, including a monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, pentahydrate, hexahydrate, heptahydrate, octahydrate, nonahydrate, decahydrate, and the like, as well as the corresponding (co-)solvated forms.

[0067] Pharmaceutical compositions and dosage forms

[0068] The present invention further provides a pharmaceutical composition comprising a compound as described herein (that is, the pharmaceutically acceptable salt of any of the structures disclosed herein, or a hydrate, solvate and / or acid addition salt thereof) and a pharmaceutically excipient.

[0069] The term “pharmaceutical composition” refers to a combination of the compound as described herein with a pharmaceutically acceptable carrier. As used herein, "carrier" or “carrier substance” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions.

[0070] Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable excipients (carrier(s), diluent(s), auxiliaries) which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Any pharmaceutically acceptable techniques, carriers, and excipients are used as suitable to formulate the pharmaceutical compositions, e.g., as described herein: Remington: The Science and Practice of Pharmacy, 23rdEd. (Adejare, A. (Editor): Academic Press, 2020); Rowe, R.C., Sheskey, P.J. and Quinn, M.E.: Handbook of Pharmaceutical Excipients, 6th Ed., (Pharmaceutical Press, 2009), each of which is incorporated herein by reference in its entirety.

[0071] In some embodiments, the pharmaceutical composition is a formulation for one or more of oral, parenteral, topical, inhalative, rectal, vaginal, sublingual, ear, ophthalmic, nasal, intrathecal and epidural administration.

[0072] In one embodiment, the pharmaceutical composition is a formulation for parenteral or systemic administration, preferably an injection formulation, more preferably an intravenous injection or intramuscular injection formulation. The formulation can be a ready-to-use formulation or a (solid) drug product for reconstitution with a diluent. One or more compounds may be formulated in a solution (e.g., a ready-to-use formulation). In specific embodiments, the aqueous solution is selected from, by way of example only, a solution in water (e.g., water for injection), a physiologically compatible buffer, such as Hank’s solution, Ringer’s solution, a physiological saline solution or buffer, or in another solvent, e.g., DMSO, or combinations of any of the foregoing. In other embodiments, the composition is provided as a drug product (e.g., a solid or lyophilized product, e.g., in a prepackaged vial) and intended for reconstitution with a diluent, e.g., immediately prior to use. In such cases, the diluent may be selected, e.g., amongst the water for injection and any of the foregoing solutions and solvents. The diluent may be optionally provided in a separate container, e.g., as a part of a kit together with the drug product.

[0073] In one embodiment, the pharmaceutical composition is an inhalation formulation, preferably selected from aerosol, inhaler, vaporizer and nebulizer formulation. Various forms suitable for administration by inhalation include, but are not limited to, aerosols, mists or powders. Pharmaceutical compositions of any compound of the invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In specific embodiments, the dosage unit of a pressurized aerosol is determined by providing a valve to deliver a metered amount. In certain embodiments, capsules and cartridges of, such as, by way of example only, gelatin for use in an inhaler or insufflator are formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0074] In one embodiment, the pharmaceutical composition is a spray, preferably selected from nasal spray and skin spray. A spray dosage unit may be provided with a (ready-to-use) aqueous solution of one or more compounds of the invention, optionally along with further excipients, or with a drug substance for reconstitution with an (aqueous) diluent, which may optionally be provided in a separate container, e.g., as a part of a kit. In one embodiment, the pharmaceutical composition is a topical or external formulation, preferably selected from solution, emulsion, gel, drops, paste, cream, ointment, liniment, balm, lotion, foam, patch, powder and dust. Such pharmaceutical compositions optionally contain one or more solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives. In some embodiments, one or more compound(s) of the invention is / are formulated for transmucosal administration, e.g., intranasal or buccal administration. In specific embodiments, transmucosal formulations include penetrants that are appropriate to the barrier to be permeated.

[0075] In some embodiments, a patient may receive therapy for the treatment and / or management of the viral infection before, during or afterthe administration of the therapeutically effective regimen of the compound of the invention, or a pharmaceutically acceptable salt thereof. Non-limiting examples of such a therapy include pain management, anti-inflammatory drugs, antibody therapy, immunotherapy, targeted therapy (i.e. therapy directed toward a specific target or pathway, e.g. virus replication, etc.), and any combination thereof. In some embodiments, the patient has not previously received a therapy for the treatment and / or management of virus infection.

[0076] Viruses

[0077] Enveloped virus refers to a virus possessing a lipid bilayer membrane derived from host cellular membranes, typically acquired during the budding process from the host cell. The viral envelope is embedded with viral glycoproteins, which serve as mediators for binding, fusion, and entry into host cells. The envelope confers sensitivity to chemical and environmental agents, in particular detergents and solvents that disrupt lipid membranes. Enveloped viruses may carry DNA or RNA genomes and exhibit diverse mechanisms for cellular entry, but all are unified by the presence of the lipid envelope surrounding their capsid. A non-limiting list of human pathogenic and / or animal pathogenic enveloped viruses is listed in the following table.

[0078] Non-limiting list of examples for human pathogenic and / or animal pathogenic enveloped viruses

[0079] HSPG-dependent viruses refer to viruses whose entry into host cells is facilitated or mediated by a specific interaction between viral surface proteins and cell-surface heparan sulfate proteoglycans (HSPG). This interaction may serve as the primary or an auxiliary attachment mechanism, increasing viral concentration on the cell surface and promoting subsequent internalization steps. HSPG-dependency can range from obligatory cellular entry mechanism to enhancing infectivity or tropism. A non-limiting list of human pathogenic and / or animal pathogenic HSPG-dependent viruses is listed in the following table.

[0080] Non-limiting list of examples for human pathogenic and / or animal pathogenic HSPG-dependent viruses. Heparan sulfate proteoglycans (HSPGs) play a pivotal role in the entry of HSPG-dependent viruses by acting as primary attachment factors on the surface of almost all mammalian cells. The highly sulfated and negatively charged heparan sulfate chains enable viruses to bind electrostatically, thus concentrating viral particles at the cell surface and increasing the likelihood of interaction with specific entry receptors. In certain viruses, such as herpes simplex virus and dengue virus, HSPG binding is essential for efficient cell infection, either as the initial contact or sometimes as the direct entry receptor. By facilitating viral attachment and sometimes entry, HSPGs are a key determinant in tissue tropism and pathogenesis for a variety of human and animal viruses. HSPGs are cell- and tissue-specific. Both the protein structure and the heparan sulphate chains of HSPGs depend on the cell and tissue type, which determines their function and interactions.

[0081] The advantages for a compound targeting HSPGs comprise broad-spectrum potential, early viral lifecycle blockade, reduced resistance risk, host cell protection.

[0082] Many viruses exploit HSPGs for cell entry, drugs or biologies that block these interactions could inhibit multiple virus families simultaneously. Targeting the initial attachment step prevents infection from gaining a foothold, potentially reducing viral replication and spread at its earliest stage. As HSPGs are host cell factors rather than viral components, viral escape mutations are less likely, making resistance development slower. Interventions targeting HSPG-virus interactions may shield the host cell without directly affecting normal cellular functions, leading to fewer side effects.

[0083] Viruses can be enveloped and HSPG-dependent viruses characterized by the presence of a lipid envelope as described above, wherein the entry process into host cells additionally requires or is enhanced by specific binding of viral envelope proteins to cell-surface heparan sulfate proteoglycans. Such viruses leverage HSPG molecules for initial attachment followed by envelope-mediated fusion with the host membrane. A non-limiting list of human pathogenic and / or animal pathogenic viruses being enveloped and HSPG-dependent is listed in the following table.

[0084] Non-limiting list of examples for human pathogenic and / or animal pathogenic viruses being enveloped and HSPG-dependent viruses

[0085] Virus attachment refers to the specific or non-specific binding interaction between viral surface components and molecules present on the host cell membrane. This is the initial and requisite step for viral entry, enabling the virus to dock and position itself for subsequent penetration.

[0086] Attachment of enveloped viruses is mediated by viral envelope glycoproteins that recognize and bind to specific protein, carbohydrate, or lipid receptors on the host cell surface, establishing close proximity of viral and cellular membranes.

[0087] Attachment of HSPG-dependent viruses relies primarily on interactions between viral surface proteins and heparan sulfate proteoglycans (HSPG), negatively charged polysaccharides ubiquitously present on host cell surfaces, which serve as initial attachment factors that facilitate viral concentration and affinity for secondary entry receptors. Enveloped and HSPG-dependent viruses attach via envelope glycoproteins binding to cell surface HSPG, often as a preliminary docking step preceding engagement of additional viral receptors needed for entry.

[0088] Viral entry refers to the multi-step process by which a virus gains access to the interior of a host cell to initiate infection. This process begins with binding of the virus to cell surface molecules, followed by breaching the cellular membrane barrier to deliver the viral genome into the host cell cytoplasm or nucleus. Viral entry may proceed via direct fusion with the plasma membrane, endocytosis, or other host-mediated uptake mechanisms, depending on the virus type.

[0089] Entry of enveloped viruses involves fusion of the viral lipid envelope with the host cell membrane, either at the cell surface or within endosomal compartments, mediated by specialized viral glycoproteins (fusogens) that undergo conformational changes to catalyze membrane merging. This fusion releases the viral nucleocapsid into the cytoplasm.

[0090] Entry of HSPG-dependent viruses is facilitated by initial attachment to heparan sulfate proteoglycans (HSPG) on the host cell surface, which serve as attachment factors or co-receptors concentrating the virus on the cell surface. Subsequent internalization and entry steps may involve receptor-mediated endocytosis or other mechanisms independent of membrane fusion.

[0091] Enveloped and HSPG-dependent viruses utilize envelope glycoproteins that specifically bind HSPG on host cells to initiate attachment, followed by membrane fusion-mediated entry. The HSPG-binding concentrates virus particles on the cell surface, enhancing subsequent fusion and viral genome release.

[0092] Penetration, as used herein, refers to the process by which a virus crosses the host cell membrane barrier to deposit its nucleic acid or nucleocapsid inside the host cell cytoplasm or nucleus, thereby initiating infection. Penetration of enveloped viruses is accomplished by fusion of the viral envelope with the host cell membrane, a process catalyzed by viral fusion proteins that mediate merging of lipid bilayers, forming a fusion pore that allows translocation of the capsid into the cytoplasm.

[0093] Penetration of HSPG-dependent viruses often occurs via endocytic uptake pathways triggered after attachment to HSPG, followed by membrane disruption or conformational changes in viral proteins that enable release of the viral genome from endosomal compartments into the cytoplasm.

[0094] Enveloped and HSPG-dependent viruses penetration entails binding of envelope glycoproteins to HSPG molecules, triggering fusion-mediated translocation of the viral core through the host membrane, often coupled to receptor-mediated endocytosis and pH-dependent activation of fusion proteins for genome release.

[0095] Plaque reduction refers to a laboratory assay method used to measure the decrease in infectious virus particles based on counting plaques formed in a cell culture. A plaque is a clear zone caused by virus- induced cell lysis in an infected cell monolayer. In plaque reduction assays, the virus is exposed to a treatment (e.g., antiviral antibodies or disinfectants), and the decrease in the number of plaques compared to untreated control reflects the reduction in infectious virus titer. This assay directly measures infectious virions capable of causing cytopathic effects. A high plaque reduction in vitro assay indicates the potential effectiveness of a treatment or step in reducing infectious virus and is a component of demonstrating viral clearance.

[0096] Viral clearance refers to the overall reduction or removal of virus particles from a process or system, such as during pharmaceutical manufacturing or purification. Viral clearance studies measure how effectively viruses are inactivated or removed by specific process steps, considering all infectious and non-infectious virus particles. The reduction is typically reported as a log reduction value (LRV), calculated from virus titers before and after a removal / inactivation step.

[0097] The compound is highly effective in plaque reduction thus indicating an effective inhibition in inhibiting viral entry, viral attachment, and viral clearance in vivo. In some embodiments, the compound exhibit a plaque reduction of at least 50%, 60%, 70%, 80%, 90%, 95% or 100%. In some embodiments, the plaque reduction of the compound is an indicator for viral entry, viral attachment, and viral clearance in vivo. Medical uses

[0098] The present invention also provides the compounds and / or pharmaceutical compositions described herein for use a medicament, i.e., for treating or preventing diseases of the human or animal body. All embodiments of medical uses described herein equally disclose methods of treating the respective disease or condition in a human or animal subject in need thereof, or of preventing a human or animal subject from contracting the respective disease or condition, the methods involving a therapeutically or prophylactically effective amount of the compound or composition to the subject. All embodiments of medical uses described herein equally disclose use of the compounds and / or pharmaceutical compositions described herein in the manufacture of a medicament for treating the respective disease or condition in a human or animal subject. In some embodiments, the respective disease or condition is caused by a virus or related to a virus entering a host. The compounds and / or pharmaceutical compositions are provided for use in the treatment and / or prevention of a viral infection in a subject and / or a medical condition associated with a viral infection. In embodiments, the compounds of the present invention can be administered prophylactically or therapeutically, preferably in an amount that is effective against the mentioned disorders, to a warm-blooded animal, for example a human, requiring such treatment, the compounds preferably being used in the form of pharmaceutical compositions. As used herein, the term "subject" refers to a mammal, such as humans, but can also be another animal, such as a companion animal (e.g., a dog, cat orthe like), a livestock animal (e.g., a cow, sheep, pig, horse or the like) or a laboratory animal (e.g., a monkey, rat, mouse, rabbit, guinea pig or the like).

[0099] As used herein, terms “medical use”, “treatment”, “therapy”, “prevention “ and “prophylaxis” and the like encompass methods performed on a human or animal subject, i.e., possible uses in both human medicine and veterinary medicine are encompassed. The subject is preferably a human, a livestock animal, e.g., pig, or a companion animal, e.g., dog or cat, more preferably a human. The term "patient" refers to a "subject" suffering from or suspected of suffering from a viral disease and / or a viral infection. In one embodiment, the subject is a patient.

[0100] The phrase "therapeutically effective" is intended to include, within the scope of sound medical judgment, excessive toxicity, irritation, allergic reactions, and / or other problems or complications, but commensurate with a reasonable benefit / risk ratio. As used herein to refer to the compounds, compositions, combinations and / or dosage forms suitable for use in contact with a subject that produces a result that in and of itself helps to treat, to prevent and / or cure a disease or condition.

[0101] A "therapeutically relevant amount", “therapeutically relevant dosage” or "therapeutically effective amount" of an agent ortherapeutic means, such as a chemical compound i.e. compound cmpd (1) orthe analogues thereof, is an amount sufficient to produce the desired effect, e.g., inhibition of viral entry into a host cell. It refers for a compound, i.e. compound cmpd (1) or the analogues thereof, compositions, combinations and I or dosage forms suitable for use in contact with a subject that produces a result that in and of itself helps to prevent, to treat and / or cure a medical condition, or for example show other beneficial effect against a medical condition, prevention of onset of a disease, condition or symptom in a host, inhibition of a symptom of a condition, prevention of progression of the symptom, amelioration of a symptom of a condition, or induction of regression of the disease and / or symptom. The phrases "therapeutically relevant amount", “therapeutically relevant dosage” or "therapeutically effective amount" can be used interchangeably and are intended to include, in some embodiments and within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0102] In the present invention, "therapy" includes arbitrary treatments of diseases or conditions in mammals, in particular, humans, for example, the following treatments (a) to (c): (a) Prevention of onset of a disease, condition or symptom in a patient; (b) Inhibition of a symptom of a condition, that is, prevention of progression of the symptom; (c) Amelioration of a symptom of a condition, that is, induction of regression of the disease or symptom.

[0103] In particular, the treatment described herein relates to viral clearance either by blockade of viral cell entry and / or viral attachment. Therefore, the surrogate parameter viral load will be assessed on a daily basis. The prophylactic therapy as described herein is intended to encompass prevention or reduction of risk of infection with a virus, preferably a virus disclosed herein, due to a blockade of viral cell entry and / or viral attachment after treatment with the compounds described herein. The treatment of the present invention may be employed or administered as deemed suitable by a skilled practitioner. For example, in some embodiments, a time interval between treatments comprises a period of several hours (2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) several days (2, 3, 4, 5, 6, or 7), several weeks (1 , 2, 3, 4, 5, 6, 7, or 8), or months (2, 3, 4, 5, or 6), preferably 1 day. In some embodiments, the time interval from one treatment to the next subsequent treatment can be the same, nearly the same or can change.

[0104] According to the present invention, an "inhibitor" in the context of "inhibitor of viral replication" is considered to be any agent, substance, compound, molecule, or other means that results in slowing, repressing, blocking, or otherwise interfering with or negatively affecting the viral entry, viral attachment to the cell surface, viral binding, viral penetration and / or viral replication. The terms “agent”, “substance”, “compound”, “molecule” can be used interchangeably. The terms “analogue” and “derivatives” can be used interchangeably. For example, the chemical compound, i.e. compound cmpd (1) and the analogues thereof, can affect attachment step of entry to a host cell. The inhibitors as described herein may also be termed “agents”. Preferred inhibitors are those described herein.

[0105] As used herein, the term "viral infection" preferably describes a disease state or condition in which a virus invades healthy cells. Viruses use the cell's reproductive machinery to multiply or replicate and finally dissolve the cell, leading to cell death, the release of viral particles and infection of other cells by the newly produced progeny viruses. The term “viral load” refers to a quantitative measure of viral genomes per invaded cell. The determination of viral genomic material may be employed in such a method. A latent infection by certain viruses is also a possible consequence of a viral infection. The term “viral growth” relates to the infection and replication of a virus and the production of viral particles during and after the infection of a host cell.

[0106] The terms "host cell" and “target cell”, as used herein, refer to a single cell or cell culture that may be or has been a recipient of at least one of the agents described herein, individually or in combination. Host cells include progeny of a single host cell, which progeny may not necessarily be completely identical (in morphology or overall DNA complement) to the original parent cell due to natural, random or intentional mutations and / or changes. In one embodiment, the host cell also refers to a cell into which an infectious agent (e.g. a virus) has invaded or is capable of invading. In some embodiments, a host cell can be a human host cell or a non-human host cell. In some embodiments, a host cell is a cell from a subject.

[0107] Therapeutic and / or prophylactic indications

[0108] The compounds or compositions of the present invention are useful in methods of treating or preventing a viral infection or disease.

[0109] The viral infection or disease to be treated or prevented may be caused by various viruses. It is contemplated that the compounds or compositions of the present invention are particularly well-suited for viral infections or diseases caused by at least one enveloped virus(es) or virus(es) dependent on or using heparan sulfate proteoglycan(s) for attachment to host cells (HSPG-dependent virus(es)).

[0110] A non-exhaustive list of HSPG-dependent viruses (as described, e.g., in Cagno, V.; Tseligka, E.D.; Jones, S.T.; Tapparel, C. Viruses 2019, 11 , 596, incorporated herein by reference) includes Herpes simplex virus, Cytomegalovirus, Human herpes virus-8 (Kaposi sarcoma herpes virus), Foot and mouth disease virus, John Cunningham polyomavirus, Respiratory syncytial virus, Dengue virus, Pseudorabies virus, Human papillomavirus, Venezuelan equine encephalitis virus, Enterovirus 71 , Parainfluenza virus 3, Echovirus 5, Merkel cell polyomavirus, Hepatitis C virus, Sindbis virus, Human metapneumovirus, Echovirus 6, Hepatitis B virus / hepatitis Delta virus, Adeno-associated virus 2, Semliki forest virus, Zika virus, North American eastern equine encephalitis virus, Vaccinia virus, Human immunodeficiency virus, Rhinovirus C15, Adenovirus 5, Adenovirus 2, Filoviruses, Rhinovirus 8, Coronavirus NL63, Norovirus genogroup II, Akabane virus, Rhinovirus 89, Schmallenberg virus, Rift valley fever virus, Coxsackie virus B3, Rabies virus, Rhinovirus 54, Yellow fever virus, Swine vesicular disease virus, Enterovirus 71 , Japanese encephalitis virus, Theiler murine encephalomyelitis virus, Coxsackie virus A9, West Nile virus, Human parechovirus 1 , Hendra and Nipah viruses, Tick-borne encephalitis virus, Porcine reproductive and respiratory syndrome virus, Human T cell leukemia virus type 1 , Coronavirus group 1 , Porcine circovirus 2, Hepatitis E virus, Coronavirus OC43, Chikungunya virus, and Murray Valley encephalitis virus. The MPox virus is also dependent heparan sulfate (Montanuy I, Alejo A, Alcami A. FASEB J. 2011 ;25(6): 1960-1971 , incorporated herein by reference). Coronaviruses, such as SARS-CoV2, are also known to use HPSG for cell entry.

[0111] A non-exhaustive list of further examples of enveloped and / or a HSPG-dependent viruses particularly suitable for being addressed with compounds or compositions of the present invention include cytomegalovirus, herpes simplex virus type 1 , Kaposi sarcoma associated virus (HHV-8), SARS-CoV2, Nipah virus, hepatitis B virus (HBV), Human immunodeficiency virus (HIV), papillomavirus (in particular, human: HPV), human orthopneumovirus (respiratory syncytial virus, RSV), Pseudorabies virus (PrV), Herpes simplex virus 1 (HSV-1), Zika virus (ZIKV), Dengue virus, O’nyong-nyong virus (ONV), Chikungunya virus, Adenovirus Type 5, Influenza A virus, Influenza B virus, and Mpox (MPXV).

[0112] In one embodiment, the viral infection or disease to be treated or prevented is caused by at least one virus(es) belonging to one or more of the taxonomic groups Alphaherpesvirinae, Betaherpesvirinae, Bornaviridae, Gammaherpesvirinae, Flavivirdae, Togaviridae, Orthoretrovirinae Adenoviridae, Orthoherpesviridae, Orthomyxoviridae, Orthoparamyxovirinae, Papillomaviridae, Picomaviridae, Pneumoviridae, Poxviridae, and Coronaviridae.

[0113] Preferably, the viral infection or disease to be treated or prevented is caused by at least one virus(es) belonging to one or more ofthe taxonomic groups Varicellovirus, Simplexvirus, Cytomegalovirus, Flavivirus, Alphavirus, Lentivirus, Mastadenovirus, Rhadinovirus, Henipavirus, Hepatovirus, Alphainfluenzavirus, Betainfluenzavirus, Orthohepadnavirus, Alphapapillomavirus, Orthopneumovirus, Orthopoxvirus, Orthobornavirus and Betacoronavirus.

[0114] More preferably, the viral infection or disease to be treated or prevented is caused by at least one virus(es) selected from Pseudorabies virus (PrV), suid alphaherpesvirus 1 , animal alpha-herpesvirus(es), Equid alphaherpesvirus(es), Bovine alphaherpesvirus(es), Varizella Zoster Virus, Herpes simplex virus 1 (HSV- 1), human alphaherpesvirus 1 , HSV-1 KOS, Human cytomegalovirus (HCMV), human betaherpesvirus 5, GCV-resistant HCMV, Zika virus (ZIKV), Orthoflavivirus zikaense, ZIKV H / PF / 2013, Dengue virus, O’nyong-nyong virus (ONV), Chikungunya virus, Human immunodeficiency virus 1 (HIV-1), CXCR4-tropic HIV-1 virus, Adenovirus Type 5, human adenovirus 5, Kaposi sarcoma associated virus, Human gammaherpesvirus 8, HHV-8, Nipah virus, Henipavirus nipahense, Influenza A virus, Influenza B virus, Hepatitis B virus (HBV), Hepatitis A virus (HAV), Human papillomavirus (HPV), Human orthopneumovirus, Respiratory syncytial virus (RSV), Mpox (MPXV), Borna Disease Virus 1 (BoDV-1), and SARS-CoV2.

[0115] In the context of viral infection or disease to be treated or prevented being infection(s) caused by Equid alphaherpesvirus(es), it is particularly preferred that the viral infection is caused by Equid alphaherpesvirus 1 , also called Equine herpesvirus 1 (EHV-1). In one embodiment, the equid alphaherpesvirus 1 may be of either the D752 or the N752 strain.

[0116] In the context of treating or preventing viral infections and / or diseases herein, it is important to highlight that such infections and / or diseases may be caused by one or more viruses described herein, and optionally further viruses. The infection and / or disease can be active with and without symptoms, latent or quiescent. For instance, for organ-transplanted patients or immunosuppressed patients (e.g., during anticancer therapy), reactivation of not only HCMV but also of other viruses (e.g., EBV, HSV) might be an issue, and these different viruses may be simultaneously suppressed by the compounds of the present invention. A further advantage may be present, e.g., for HIV positive patients because the present compounds have not only direct anti-HIV activity but also may protect these patients from other pathogenic viruses.

[0117] Routes of administration In the context of the therapeutic and / or prophylactic uses described herein, the compound or composition is administered via various routes.

[0118] Suitable routes of administration include one or more of parenteral route, systemic route, injection, inhalation, topical route, external route and mucosal route, preferably by intravenous or intramuscular injection or by application to the skin or a mucous membrane, more preferably to the nasal mucosa.

[0119] Methods of manufacture

[0120] An exemplary synthetic route leading to compound 1 is depicted in FIG. 1 and described in more detail in Example 1 . A person skilled in the art will appreciate that certain steps, reagents and / or conditions may be suitably varied, e.g., when preparing further compounds according to the present invention having a structure different from that of compound 1 .

[0121] A general method of manufacturing a compound according to the present invention may comprise one or more, preferably all of the following steps:

[0122] (Y1'2)n

[0123] PhOCH2-LG

[0124] PhOCN ' NCHO

[0125] (Y1 / ,2)n- (Y1pn- acid, preferably HHal,

[0126] A more preferably HCI

[0127] PhOCN ' NCHO PhOCN^ ' / NCHO in polar protic solvent, preferably alcohol,

[0128] (b) more preferably EtOH more preferably EtOH

[0129] In each of the above, LG is a suitable leaving group, e.g., selected from halogen (Hal), OSOsalkyl, OSO2alkyl, OSO2aryl, OSO2alkylaryl and OTf, preferably Cl or Br, more preferably Cl. Y1 / 2denotes Y1or Y2, and n1 / 2denotes n1or n2, as the case may be. A person skilled in the art will appreciate that the method of manufacturing may be further modified as appropriate, e.g., to allow for alternative substitution by one or more (further) Y1or Y2at a different position in the compounds of the invention.

[0130] It will also be appreciated by those skilled in the art that in the processes for preparing the compounds described herein the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include, but are not limited to, hydroxy, amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (for example, t- butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino include t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include C(O)R” (where R” is alkyl, aryl or arylalkyl), p- methoxybenzyl, trityl, and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups are optionally added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one skilled in the art would appreciate, the protecting group may also be a polymer resin such as a Wang resin, Rink resin or a 2-chlorotrityl-chloride resin.

[0131] Definitions

[0132] “Alkyl” refers to a straight, branched or cyclic hydrocarbon chain residue consisting solely of carbon and hydrogen atoms, having preferably from 1 to 12 carbon atoms (C1-C12 alkyl), more preferably 1 to 8 carbon atoms (Ci-Ca alkyl), most preferably 1 to 5 carbon atoms (C1-C5 alkyl), and which is attached to the rest of the molecule by a single bond; e.g., methyl, ethyl, n-propyl, 1-methylethyl ( / so-propyl), n-butyl, n-pentyl, 1 ,1 -dimethylethyl (f-butyl), cyclopropyl, cyclopropylmethyl, 2-cyclopropyleth-1-yl, cyclobutyl, cyclobutylmethyl, 2-cyclobutyleth-1-yl, cyclopentyl, and the like. Unless stated otherwise, an alkyl group is optionally substituted.

[0133] “Alkoxy” refers to a residue of the formula -ORawhere Rais an alkyl residue as defined above, preferably containing one to twelve carbon atoms. Unless stated otherwise, an alkoxy group is optionally substituted.

[0134] Halo” or “halogen” refers to a halogen atom, preferably F, Cl, Br or I.

[0135] “Haloalkoxy” refers to a residue of the formula -ORawhere Rais an alkyl residue as defined above, preferably containing one to twelve carbon atoms, wherein one or more, preferably all H atoms of the alkyl are substituted by halogen atom(s), preferably F, Cl, Br or I; e.g., that is substituted by one or more halo radicals, as defined above, e.g., trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2 trifluoroethyl, 1 ,2 difluoroethyl, 3 bromo 2 fluoropropyl, 1 ,2 dibromoethyl, and the like. Unless stated otherwise specifically in the specification, a haloalkyl group is optionally substituted. Unless stated otherwise, haloalkoxy group is optionally substituted.

[0136] “Substituted” or “substituent” used refers to substitution, i.e., replacement of at least one hydrogen atom, by replaced by a bond to a non-hydrogen atoms, preferably, but not limited to: a halogen atom, preferably F, Cl, Br, or I; an oxygen atom in groups, preferably hydroxyl groups, alkoxy groups, or ester groups; a sulfur atom in groups, preferably thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, or sulfoxide groups; a nitrogen atom in groups, preferably amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, or enamines; a silicon atom in groups, preferably trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, or triarylsilyl groups; and other heteroatoms in various other groups. “Substituted” may also mean that one or more hydrogen atoms are replaced by a higher-order bond (e.g., a double- or triple-bond) to a heteroatom such as oxygen, preferably in oxo, carbonyl, carboxyl, and ester groups; and nitrogen, preferably in imines, oximes, hydrazones, and nitriles. Preferably, “substituted” or “substituent” refers to substitution with one or more groups selected from aminyl, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, heteroalkyl, alkoxy, alkylaminyl, dialkylaminyl, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, / V-heterocyclyl, heterocyclylalkyl, heteroaryl, / V-heteroaryl and / or heteroarylalkyl group. In each of the preceding groups, preferably “alkyl” is a C1-5 alkyl; heteroalkyl is a C1-5 alkyl further containing one or more heteroatoms selected from halogen, N, O and S as substituents and / or in between C atoms of the alkyl chain; heterocyclyl is a 5- or6-membered heterocyclic group with one or more heteroatoms selected from N, O and S; aryl is a C6-10 monocyclic or bicyclic aryl; heteroaryl is a 5- or 6-membered heteroaromatic group with one or more heteroatoms selected from N, O and S. In addition, each of the foregoing substituents, if chemically possible, may also be optionally substituted with one or more of the foregoing substituents.

[0137] It is understood that in the present description, combinations of substituents and / or variables of the depicted formulae are permissible only if such contributions result in stable compounds.

[0138] As used herein, the meaning of the term "comprising" encompasses three alternatives, namely, "comprising", "consisting of and "consisting essentially of.

[0139] As used herein, the terms “improved”, in the context of an effect being achieved by a certain subject-matter or feature, preferably refers to an enhancement, increase or advantage (quantitative or qualitative) achieved as compared to a situation where the subject-matter or feature in question is absent and / or as compared to the prior art.

[0140] EXAMPLES

[0141] Example 1 : Synthesis of 2-chloro-4,6-di(3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecan-1-yl)- 1 ,3,5-triazine tetrachloride

[0142] To a solution of 15.2 g (0.133 M) 1 -formylpiperazine in 267 ml of chloroform, 12.26 g (0.146 M) of sodium bicarbonate are added. The mixture is cooled down to 10°C with water, prior to dripping a solution of 17 ml (0.146 M) of benzoylchloride in 27 ml of chloroform to the mixture for 0.5 hours. The mixture is then being mixed for 16 hours (nighttime included) at room temperature. The resulting mass is being washed twice, using 150 ml water each time; the organic layer is being dried above sodium sulphate for 0.5 hours, mixing continuously. After evaporating chloroform, 200 ml of hexane are added, the solution is mixed at 4°C, the hexane is decanted. This step of preparation is repeated. After the addition of the 3rd hexane portion, the mixture is being mixed for 0.5 hours. Resulting precipitation is filtrated, washed with 20 ml of hexane and 20 ml of ether, and left to air-dry. The process results in obtaining 22.4 g (77%) of 4-benzoylpiperazine-1- carbaldehyde with melting point 87°C.

[0143] 10.9 g (0.05 M) of 4-benzoylpiperazine-1-carbaldehyde are being dissolved in 60 ml of mixture of MeOH and concentrated HCI (HCI:MeOH ration 1 :11) and mixed for 24 hours at room temperature. The resulting precipitate is being filtered and washed by 2x5 ml of methanol and 2x10 ml of acetone, then dried in a drying oven for 4 hours at 90°C. This yields 7.55 g (63%) of hydrochloride 1 -benzoylpiperazine with melting point 315°C.

[0144] Hydrochloride benzoylpiperazine 18 g (0.0795 M) is added to a solution of 5.34 g (0.0954 M) KOH in 55 ml of ethanol and mixed for 0.5 hours at 20-22°C. After that, 13.3 ml (0.199 M) of ethilenchlorhydrine are added, and a solution of 11.6 g (0.207 M) KOH in 98 ml of ethanol is being dripped for 1 hour; the temperature of the mass cannot exceed 20°C. Twenty hours later, the resulting KOI is being filtered and washed by 25 ml absolute ethanol. The filtrate is cooled down to 10°C, after which 58 ml of 12% HCI / EtOH (control pH 2-3) are slowly added, mixed for 1 hour at 5°C, and left for a night in the fridge. The precipitate is being filtered, washed with 2x10 ml of absolute ethanol, and dried on air (48 hours) or in a drying oven (4-5 hours at 50-55°C). This process yields 15.6 g (72%) of hydrochloride 1-benzoyl-4-(p- oxyethyl)piperazine with melting point 215°C.

[0145] To a suspension of 13.5 g (0.05 M) hydrochloride 1-benzoyl-4-(p-oxyethyl)piperazine in 96 ml of chloroform, 8 ml of SOCh is being dripped for 0.5 hours, with continuous mixing; at the same time, the temperature of reacting mass is being increased up to 45°C by means of an oil bath. The mixture stays at this temperature for 0.5 hours, is then heated to 55°C and left for 0.5 hours, and is then heated to 70°C and mixed for 3 hours. After that the reacting mass is being cooled down to 20°C and left in a fridge for 16 hours. The precipitate is filtered, washed with 2x30 ml of chloroform, then dried for 3 hours at 40-45°C in a drying oven. This process yields 11.7 g (81 %) of hydrochloride 1-benzoyl-4-(p-chloroethyl)piperazine with melting point 230°C.

[0146] To a suspension of 7.35 g (0.0254 M) hydrochloride 1-benzoyl-4-(p-chloroethyl)piperazine in 15 ml ethanol, a solution of 1 .12 g (0.028 M) NaOH in 19 ml of 96% ethanol is added and mixed for 1 .5 hours at 20-25 °C. Thereafter, NaCI is filtered and washed by 2x5 ml of absolute ethanol. The resulting filtrate is boiled with continuous mixing for 1 hour, and then evaporated on a rotary evaporator at 80 °C, until dry. The rests are heated to 120°C for 16 hours. After cooling down, 15 ml of distilled water are added and mixed while boiling up to complete dissolution. To this solution, 0.7 g of activated charcoal are added and boiled for 10 minutes. The charcoal is filtered and washed by 2x5 ml of hot water. The mother solution is cooled down and left in the fridge. The precipitate is filtered and washed with water (2x5 ml) and ethanol (2x5 ml), then dried for 2 hours at 100°C. This process yields 3.1 g (45%) of dichloride / V, / V”-dibenzoyl- / V( / V”-dispirotripiperazinium in dihydrate form, with melting point >360°C (with decomposition). A mixture of 3.1 g (0.0057 M) dichloride A / ,A / ”-dibenzoyl-A / ’,A / ”-dispirotripiperazinium in dihydrate form and 20 ml 10% hydrochloric acid is obtained by means of mixing 7 ml of concentrated hydrochloric acid and 13 ml distilled water; the mixture is boiled for 4 hours. The reacting mixture is cooled down to 10-15 °C using an ice bath; the precipitating benzoic acid is filtered and washed with water. The filtrate is evaporated on a rotary evaporator until dry. Solid residue is mixed with 10 ml of methanol, the precipitate is filtered and washed with 5 ml of methanol, then dried for 2 hours at 100 °C. This yields 1.9 g (82%) of dichloride N’,N”- dispirotripiperazinium dihydrochloride dihydrate with melting point >330°C (with decomposition).

[0147] To a solution of 1 .9 g (0.0047 M) dichloride / V’, / V”-dispirotripiperazinium dihydrochloride dihydrate in 3.2 ml water, 0.26 g (0.0108 M) of LiOH is being added in small portions, with continuous mixing at 20 °C (pH 9). Then 0.17 g of activated charcoal are added, mixed for 0.5 hours, after which the charcoal is filtered and washed with 2x1 ml water. The mother solution is diluted with 30 ml of methanol and left for 16 hours at 5 °C in the fridge. The resulting precipitate is filtered and washed by 5 ml methanol on the filter, then dried for 2 hours at 100°C. This yields 1.1 g (79%) 3,12-diaza-6,9-diazonidiaspiro[5.2.5.2]hexadecane dichloride with melting point 350 °C (with decomposition).

[0148] To a solution of 2,4,6-trichlorotriazine (0.12 M) in 940 ml of ethanol, a solution of 3,12-diaza-6,9- diazonidiaspiro[5.2.5.2]hexadecane dichloride (0.24 M) in 220 ml of water is added with intensive mixing. The suspension is heated for 4 hours at 70 °C. Afterthat, the mixture is cooled down to room temperature, the precipitate is filtered and washed with ethanol, then dried for 18 hours at 110 °C and left on air for 4x24 hours. The yield of 2-chloro-4,6-di(3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecan-1-yl)-1 ,3,5-triazine tetrachloride is 93%. Melting point 216-220°C, with decomposition.

[0149] Example 2: Spectroscopic analysis of 2-chloro-4,6-di(3,12-diaza-6,9- diazoniadispiro[5.2.5.2]hexadecan-1 -y I )-1 ,3,5-triazine tetrachloride

[0150] After successful synthesis of 2-chloro-4,6-di(3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecan-1-yl)-1 ,3,5- triazine tetrachloride, analytical data was collected using standard protocols.

[0151] MS (m / z): 564.2127 (M+-4CI ) C27H50CI5N11.1H NMR (200 MHz, D2O) 6 3.80 (br s, 8H, H2C(11 , 13, 11 ’, 13’)), 3.98 (br s, 8H, H2C(2, 4, 2’, 4’)), 4.20 - 4.52 (m, 32H, H2C(1 , 5, 7, 8, 10, 14, 15, 16, T, 5’, 7’, 8’, 10’, 14’, 15’, 16’,)).13C NMR (50 MHz, D2O) 6 37.98 (C(11 , 13, 11 ’, 13’)), 38.57 (C(2, 4, 2’, 4’)), 52.92 (C(8, 15, 8’, 15’)), 53.62 (C(7, 16, 7’, 16’)), 57.30 (br s, C(10, 14, 10’, 14’)), 60.15 (br s, C(1 , 5, T, 5’)), 166.94 (C(2”, 4”)), 172.04 (C(6”)). Analysis of microelements (%). Estimated: C27H50CI5N11: C, 45.93; H, 7.14; N, 21.82. Measured: C, 45.86; H, 7.16; N, 21.84. Karl Fischer titration. Measured: 0.02 %.

[0152] In the following, 2-chloro-4,6-di(3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecan-1-yl)-1 ,3,5-triazine tetrachloride (also referred to as “compound 1 ”, “cmpd (1)” or 11826233), compound 11826234 and / or PDSTP are tested and compared.

[0153] 11826234 PDSTP

[0154] Example 3: Cytotoxicity

[0155] The cytotoxicity of Compound 1 was investigated in human cell lines derived from different organs. Cells (1 x 104) were seeded in 96-well plates. Subconfluent cells (70%) were incubated with 25, 50, 100, 200, 350, 500, 750, 900 pM of cmpd (1) in a final volume of 200 pl for seven days at 37 °C. Cytotoxicity (50% cytotoxic concentration, CC50) profiling of the compounds was determined by the use of Cell Proliferation Kit II (XTT, Roche) as recommended by the manufacturer. Briefly, after the incubation time, 50 pl of the labelling solution (XTT labeling reagent and electron coupling reagent, 50:1) was added to the cells, followed by incubation of 4 h at 37 °C. The absorbance of 492 nm with a reference wavelength of 650 nm was measured using a microplate reader. The mean 50% cytotoxic concentration (CC50) was in lung cells (human lung epithelial cells A549 / human fetal lung fibroblast HELF Fi301) 237.85 / >200 pM with 11826233, and 192.59 / 89.75 pM with PDSTP. In liver cells (HUH7), the values for CC50 were 273.74 pM with 11826233, and 278.43pM with PDSTP. In blood vessel cells (HUVEC), the CC50 values were 330.24 pM with 11826233 and 450.88 pM with PDSTP. The mean CC50 in kidney cells (HEK 293T / PK15 / VERO E6Z VERO E76) was 257.34 pM, 436.94 pM, 332.75pM and 277.72 pM, respectively, with 11826233, and 434.86 pM, 687.77 pM, 340.92 pM and 461.55 pM, respectively, with PDSTP.

[0156] These results demonstrated that the triazine derivative was not cytotoxic below > 230 pM in different cells.

[0157] The results are shown in FIG. 3 and the table below.

[0158] Example 4: Antiviral activity

[0159] To characterize the antiviral activity of cmpd (1) and PDSTP, analyses of viral plaque formation were performed. HELF FI301 , PK15, Vero E6 or Vero E64 (5 x 104cells per well) were treated in triplicate with various concentrations (1.0, 2.0, 3.0, 4.0, 5.0, 10.0, 15.0 and 20.0 pM) of cmpd (1) or PDSTP for 30 min or left untreated. The culture media containing compounds was discarded and the cells were washed three times with PBS prior to infection with GCV-res. HCMV (FIG. 4A), HCMV TB40 / E-pp150GFP (FIG. 4B), HSV-1 KOS (FIG. 4C-D), O'nyong-nyong virus (OMNV; Fig. 3E), PrV Ka (FIG. 4F), PrV AgC, lacking the attachment protein of PrV (FIG. 4G), PrV AgG (FIG. 4H), ZIKA H / PF / 2013 (FIG. 4I) or HX HIV-Luci (FIG. 5). After 1 h p.i. the inoculum was discarded, the cells were washed three times with PBS and overlaid with 2 ml 0,5 % (w / v) methylcellulose (Methocel MC; Fluka) containing DMEM with 7.5 % FBS. After incubation for seven (HCMV), three days (Zika virus, OMNV) or two days (HSV-1 , PrV) p.i., the cells were fixed with an ethanol-acetone composition (95:5) prior to staining with crystal violet (2% w / v) for 15 min at room temperature. Plaques were counted by using a microscope and compound effects were calculated by comparing compound-treated cells versus untreated cells. Dose-response curves for various cell types together with EC50 values are shown in FIG. 4 and 5; the results are summarized in FIG. 6.

[0160] The mean 50% effective concentration (EC50) was 3.15 pM for cmpd (1) and 0.97 pM for PDSTP against GCV-res. HCMV (FIG. 4A), 2.00 pM for cmpd (1) and 0.91 pM for PDSTP against HCMV TB40 / E- pp150GFP (FIG. 4B), 1 .17 to 3.36 pM for cmpd (1) and 0.99 to 1 .73 pM for PDSTP against HSV-1 (FIG. 4C, D), 2.16 for cmpd (1) and 3.10 for PDSTP against OMNV (FIG. 4E), 1 .21 pM for cmpd (1) and 0.66 pM for PDSTP against PrV (FIG. 4F), 1.07pM for cmpd (1) and 0.69 pM for PDSTP against PrV AG (FIG. 4H), against Zika virus the EC50 was 12.27 pM for cmpd (1) and 13.33 pM for standard drug PDSTP (FIG. 4I) and 1 .61 pM for cmpd (1) and 1 ,49pM for PDSTP against HX HIV-Luci (FIG. 5). As expected, experiments with the negative control PrV AgC did not detect an EC50 (FIG. 4G). The selectivity index (SI) is the ratio of the cytotoxicity of the compound against its effective concentration. In general, an SI >10 indicates a selective bioactive compound. The higher the SI ratio of a compound, the higher the efficacy in inhibition of virus replication is.

[0161] In FIG. 4G, a control experiment involving PrV AgC is shown. PrV glycoprotein C is the attachment protein of PrV. The mutant with a deletion of gC is no longer able to infect the host cell. This virus was used as a perfect control for our compound. The compound blocks the glycoprotein C in wild-type PrV.

[0162] Antiviral properties against HIV are shown in FIG. 5.

[0163] The results demonstrate that cmpd (1) is bioactive against all tested viruses with the exception of the negative control lacking the attachment protein gC (FIG. 6). This result demonstrates that cmpd (1) is active against DNA and RNA viruses.

[0164] Example 5: Time of addition

[0165] HELF Fi301 cells (5 x 104cells per well) were seeded in 24-well plates and grown until confluence. The EC90 against GCV-res. HCMV (cmpd (1): 7.62 pM, PDSTP: 2.36 pM), against HSV-1 (cmpd (1): 3.88 pM, PDSTP: 2.44 pM) or 10 pg / ml Heparin were added: (i) 30 min before infection and the compound was removed by three washing steps with PBS before infection (before inf.), (ii) during infection (during inf.), (iii) before and during infection (before + during inf.) or (iv) after infection in the methylcellulose overlay (after inf.) (FIG. 7A). Infection was performed with GCV-res HCMV or HSV-1 strain KOS for 60 min at 37 °C. Then, three washing steps with PBS removed the inoculum containing non-adsorbed virus. Afterwards, 2 ml of methylcellulose (Methocel MC) containing DMEM with 7.5 % FBS was added and plaque assay was performed.

[0166] Addition of the compounds before infection (before inf.) resulted in plaque reduction of > 80% for both viruses, whereas heparin led to a reduction of <20% (FIG. 7B-C). Similar observations were made by addition of compounds 30 min before and 90 min during infection (before +during inf.), and addition during infection (during inf.) resulted as well in plaque reduction of 80-90% (FIG. 7B-C). However, addition after infection (after inf.) had only marginal effects (reduction between 20-40%) (FIG. 7B-C). Corresponding results compared with compound 11826234 are shown in FIG. 8. This analysis indicates that cmpd (1) prevents viral replication at an early event of host-pathogen interactions.

[0167] Example 6: Attachment and penetration

[0168] To determine if the anti-herpesvirus effects of cmpd (1), compound 11826234, and PDSTP are due to inhibition of viral entry, attachment and penetration analyses were performed.

[0169] Prechilled HELF FI301 cells were treated with inhibitors at EC90, 10 pg / ml Heparin or left untreated on ice for 30 min. After removal of the small molecules, cells were infected with prechilled GCV-res. HCMV or HSV-1 (MOI 0.001) for 2 h at 4 °C. Three washing steps removed unattached virus and the cells were incubated under plaque assay conditions. All three inhibitors led to a plaque reduction of about 90% in HCMV infected cells, whereas in HSV-1 infected cells the reduction was approximately 60-65 (FIG. 9A-B). Heparin prevents virus attachment to 50% (FIG. 9A-B).

[0170] Penetration assay was performed with infection of prechilled cells with either GCV-res. HCMV or HSV-1 (KOS) (MOI 0.001 or 0.0003) for 2 h at 4 °C. HCMV and HSV-1 infected cells were treated with EC90 (Table 1) of cmpd (1), 11826234, PDSTP, treated with Heparin or left untreated. Penetration was allowed by a shift to 37 °C and an incubation of 10 min. The reaction was stopped by low pH treatment (pH 3.0). All three inhibitors had only marginal effect on HCMV penetration (FIG. 9C). In contrast, in HSV-1 infected cells penetration is reduced approximately 30-55% (FIG. 9D). While heparin treatment led to a not significant reduction of 15% in HCMV infected cells, the reduction of ~50% is more obvious in HSV- infected cells. These results indicate that cmpd (1) blocks viral infection at the entry step of attachment to the host cell.

[0171] Example 7: Toxicity studies

[0172] Acute toxicity of drug 11826233 (cmpd (1)) was experimentally studied for intragastric administration in white mice. The test drug is white crystals soluble in water. The compound was solubilized in saline to be administrated to animals.

[0173] Studies were performed in accordance with FSA NRCMPQC guidelines (“Guide for experimental (preclinical) study of new pharmaceutical products”, Moscow, 2018). All animal work was conducted under protocols approved by the Federal Research Center Fundamentals of Biotechnology RAS (IACUC protocol #007-2022) according to the institution’s guidelines for animal use, the state industry standards GOST 33215-2014 and GOST 33216-2014 (in harmonization with EU Directive 2010 / 63 / EU on the protection of animals used for scientific purposes).

[0174] Female white mice were provided by Andreevka branch of SI Scientific center for biomedical technologies RAMS. The animals were kept in vivarium according to sanitary regulations and on standard ration with dry pelleted feed (OOO Laboratorkorm, Moscow) in accordance to order No. 1179 of RF Ministry of Healthcare dated 2017-10-10. Animal handling accorded to order No. 775 of USSR Ministry of Healthcare dated 2017- 08-12.

[0175] The purpose of investigation was the evaluation of acute toxicity of compound 1 , depending on dose and observation time after single per oral administration.

[0176] Preparation of soluble drug form

[0177] Solution in saline.

[0178] Experimental animals

[0179] Animals: 40 mice. Strain Balb / C. Sex: female. Age: 7 weeks. Weight: 20-21 g. Mice were marked with picric acid for identification under laboratory conditions.

[0180] Keeping the animals

[0181] Size of cages: 25 x 40 cm. Number of animals in one cage: 8. Food: in granules, ration by RAMN. Drinking water: in bottles. Change of drinking 3 times a week. Litter: in the cage sawdust. Change of litter: 2 or 3 times a week. Light cycle: 10 / 14 (day / night). Humidity of air: natural. Room temperature: 20 ± 2 °C. Cleaning of the cage: 2 / 3 times a week. Cage marks: Label with the cage number, dose and date of introducing into.

[0182] Procedure of the experiment

[0183] Ways of administering compound: 11826233 (cmpd (1)) per os. Number of doses: 4. Dosing: 500-750-1000-1500 mg / kg. Volume introduced into 0.3 ml / 20 g (15 ml / kg).

[0184] Data recorded: behavior, weight changes, rectal temperature, mortality.

[0185] Experimental period: 14 days

[0186] Results of the experiments Example 8: Further assays

[0187] To further confirm the activity and utility of the compounds, analysis against SARS-CoV2, influenza virus A and B by plaque reduction assay; immunofluorescence analysis for determination HCMV spread; and organ-on-a chip analysis to determine the effect concerning preclinical analysis is performed.

[0188] Example 9: Stability of treatment To address the stability of the compounds, HELF Fi301 cells infected with HCMV TB40 / E-pp150GFP (MOI 0.3) were examined by fluorescence microscopy at indicated time points (48h - 120h). The read-out was the GFP auto-fluorescence, staining of actin filaments and staining of the nuclei with DAPI. The untreated virus showed plaques starting after 72 h p.i. (Figure 10B). In contrast, no infected cells were observed upon treatment with the compounds 11826233 (compound 1) and PDSTP (Figure 10A-D). Treatment with heparin served as a control. These results demonstrated that our compound prevents viral spread and implicates that they are stable in cell culture at least for 120 hours. HELF Fi301 cells were grown on glass cover slips (1 x 105). Cells were cooled down to 4°C and treated with 10.71 pM 11826233, 2.63 pM PDSTP, 10 pg / ml Heparin or left untreated for 30 min. After removing the compounds by washing steps with pre-cooled medium cells were infected with pre-cooled HCMV TB40 / E-pp150GFP (MOI 0.25) for 2 h at 4°C. After washing with medium the cells were shifted to 37°C. After 48 h, 72 h, 96 h and 120 h of infection, the cells were fixed with 4 % paraformaldehyde. Detection of actin filament was carried out with anti beta-actin, rabbit mAb (Biomol GmbH, Hamburg, Germany) for 45 min with Cy3-conjugated goat anti rabbit F(ab)2 fragments (Merck, Darmstadt, Germany). HCMV pp150 was detected by the GFP tagged virus. The samples were mounted in Fluoroprep (bioMerieux, Deutschland GmbH, Nurtingen, Germany) with 2.5 % (w / v) 1 ,4-Diazabicyclo[2.2.2]octan Samples were examined under a confocal laser scanning microscope TCS SPE (Leica Camera AG, Wetzlar, Germany) and images were captured with LAS-X program (Leica Camera AG, Wetzlar, Germany).

[0189] Example 10: Comparative stability of 11826095 and 11826233 (compound 1)

[0190] Studied samples:

[0191] 3,3'-(6-chloro-1 ,3,5-triazine-2,4-diyl)bis-3,13-diaza-7,10-diazoniadispiro[6.2.6.2]octadecane tetrabromide (11826095) CAS 3033410-92-0

[0192] 3,3'-(6-chloro-1 ,3,5-triazine-2,4-diyl)bis-3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecane tetrachloride (11826233)

[0193] Aim of the study:

[0194] 1- Investigate the physical and chemical properties of the substances at elevated temperature.

[0195] 2- Comparison stability of 11826095 and 11826233

[0196] 3- Establish an experimental expiration date for the substances

[0197] Conditions for the study:

[0198] Storage temperature: (55 ± 2) °C.

[0199] Type of packaging:

[0200] Double layer polyethylene bag. The package is placed in a bag made of laminated aluminum foil. Each package is wrapped in wrapping paper.

[0201] Tested samples:

[0202] Series: 3,3'-(6-chloro-1 ,3,5-triazine-2,4-diyl)bis-3,13-diaza-7,10-diazoniadispiro[6.2.6.2]octadecane tetrabromide (11826095)

[0203] Series: 3,3'-(6-chloro-1 ,3,5-triazine-2,4-diyl)bis-3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecane tetrachloride (11826233)

[0204] Intervals: Substance samples were analyzed after 6, 12, 18, 24 days after experiment start, which is equivalent to 6, 12, 18, 24 months when stored at 5±3 °C, respectively.

[0205] Conclusion:

[0206] According to the results of the stability study, using the accelerated aging method, we can conclude that: • The substance of 3,3'-(6-chloro-1 ,3,5-triazine-2,4-diyl)bis-3,13-diaza-7,10- diazoniadispiro[6.2.6.2]octadecane tetrabromide (11826095) is unstable under short-term changes in the storage conditions investigated;

[0207] • The substance of 3,3'-(6-chloro-1 .S.S-triazine^A-diyObis-S.^-diaza-S.Q- diazoniadispiroIS^.S^hexadecane tetrachloride (11826233) is stable under short-term changes in the storage conditions investigated and experimental shelf life at a temperature of 20±3 °C is more 2 years.

[0208] The results are summarized in the tables below:

[0209]

[0210]

[0211] Example 11 : Study of the effectiveness of the substance 11826233 on the model of herpes encephalitis in mice

[0212] The experiment assessed the effectiveness of compound 11826233 at doses of 50 and 100 mg / kg (once a day) in a therapeutic and prophylactic regimen and various routes of administration, on the model herpes simplex virus type I - induced encephalitis in mice. The antiviral drug was used as a positive control: Zovirax® (Acyclovir).

[0213] The object of the study is the herpes simplex virus type I.

[0214] Subject of study: compound 11826233 (compound 1).

[0215] The aim of the work was to evaluate the effectiveness of the compound on a model of herpes encephalitis in mice at doses of 50 and 100 mg / kg (once a day).

[0216] The objectives of the study included :

[0217] Modeling of herpes encephalitis in mice.

[0218] Evaluation of mortality and body weight dynamics in all experimental groups over 14 days.

[0219] Calculation of the average life expectancy of animals (ALS) for each treatment option.

[0220] Evaluation of the virus titer in the brain of animals on the 5th day after infection of animals with the virus.

[0221] The study found that oral administration of compound 11826233 at a dose of 50 mg / kg had no significant effect on body weight dynamics, survival, average lifespan, and virus titer in brain tissue. Oral administration of compound 11826233 at a dose of 100 mg / kg, although not protecting animals from body weight loss and virus proliferation in brain tissue, contributed to stable body weight restoration in animals, statistically significantly increased the average lifespan of mice infected with HSV-1 herpetic encephalitis, and maintained the percentage of protection against death at a statistically significant level up to the 8th day of the experiment. The most effective was intraperitoneal therapy with compound 11826233 at a dose of 50 mg / kg. This therapy statistically significantly protected the animals from death throughout the experiment, significantly increased the average lifespan of the animals, and also reduced the replication of the virus in brain tissue compared to a group of untreated mice with herpes encephalitis.

[0222] 1. MATERIALS AND METHODS

[0223] 1.1 Samples and their preparation

[0224] The reference drug for animal studies, Zovirax® (acyclovir), lyophilisate for the preparation of infusion solution, was purchased through a pharmacy network. To prepare the comparison drug, the powder was dissolved in sterile distilled water. Doses were prepared and indicated based on the content of pure substance in the dosage form. The drugs were weighed to an accuracy of 0.1 mg on an analytical scale. After receiving the results of the study, the remains of the tested samples were disposed of. 1.2. Viruses and cells.

[0225] Cell culture. The study used a continuous culture of green monkey kidney cells Vero obtained from ATTC (American Cell Culture and Virus Collection). The cells were cultured in a growth medium, which was Eagle's MEM medium ( BioLot , Russian Federation) with the addition of 10% heat-inactivated fetal calf serum, 2 mM L - glutamine ( Sigma , USA ) and antibiotics (100 U / ml penicillin and 100 pg / ml strep tomycin). The support medium contained all the above ingredients and 2% FBS. The cells were incubated in a thermostat in an atmosphere of 5% CO2 at +37°C.

[0226] Virus. The experiments used the herpes simplex virus HSV-1 antigen type, strain L2 (number in collection: 2118; strain lineage: isolated from a Herpes labialis patient's bladder in 1954; control date: 2018), obtained from the Federal Collection of Viruses at the Federal State Budgetary Institution "Gamaleya National Research Center for Epidemiology and Microbiology " of the Ministry of Health of Russia.

[0227] Virus titers were estimated by the standard method (according to Reed and Muench ) by the micromethod in 96-well culture plates using cell culture and were expressed as Ig TCID50 / ml. The minimum virus dilution causing 50% damage to the cell monolayer in the absence of cell monolayer degeneration in the control without infection (50% tissue cytopathic dose of virus TCID50) was taken as TCID50 of the virus. The experiment used the virus in the form of a virus-containing suspension with an infectious titer of 6.0 Ig TCID50 I ml. All obtained viruses were stored frozen in aliquots.

[0228] Work with the virus was carried out in laminar flow hoods of the 2nd class of biological protection, using disposable consumables.

[0229] The efficacy of compound 11826233 was studied using an infecting dose of 5 MLD50 HSV-1.

[0230] 1. 3. Animals

[0231] In this study, 6-8 week old sexually mature female BALB I C mice weighing 14-16 g were used. Animals were supplied from the Andreevka branch of the Federal State Budgetary Scientific Institution "National Center for Biomedical Technologies" of the Federal Medical and Biological Agency of Russia (Moscow Region). The supplier has a veterinary certificate of compliance of the activities of the facility supervised by the State Veterinary Supervision Service with veterinary requirements, rules and regulations. When forming experimental groups, each animal was assigned an individual number from 1 to 13 in each group. The animals were distributed into groups by weight. Animals were marked using fucorcin staining. The name of the study, group number and the drug administered were indicated on the card of the cage in which the animals were kept.

[0232] 1.4. Keeping animals

[0233] The animals were kept in accordance with the rules adopted by the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Strasbourg, 1986) and the rules for the design, equipment and maintenance of experimental biological clinics (vivariums) (GOST 33216- 2014 "Guidelines for the care and maintenance of laboratory animals. Rules for the care and maintenance of laboratory rodents and rabbits"). The mice were placed in plastic cages (size LxWxH 35x20x15 cm) with sterilized fine shavings as bedding in accordance with the placement standards. The following conditions were maintained in the animal keeping room: ambient temperature 20-24 °C; automatic change of 12-hour light period (08:00-20:00 - day, 20:00-08:00 - night); relative humidity 45-65%; ventilation without recirculation with air change of 7-12 room volumes per hour. The animals were fed with briquetted feed in accordance with the approved standards. The animals were given distilled water to drink. The animals had unlimited access to water and food.

[0234] The mice were adapted in the institute's vivarium in a separate room for 3 days before the start of introducing the analyzed samples. During this period, the animals' external condition was examined daily and they were clinically examined before the stage of distribution into groups. No animals with deviations detected during the examination were found.

[0235] Euthanasia (painless killing of the animal) was performed by the responsible person in accordance with the requirements adopted at the institute, by dislocation of the cervical vertebrae with preliminary ether anesthesia. Euthanasia was carried out promptly after the end of the experiments, without causing suffering.

[0236] 1.5. Doses, regimens and route of administration of drugs to animals

[0237] Depending on the doses and methods of administration, the experiment included the following groups:

[0238] Group 1 viral control - a group of animals infected with HSV-1 , strain L2 and receiving a placebo in the form of distilled water (viral control);

[0239] Group 2 - a group of animals infected with HSV-1 , strain L2 and treated comparison drug Acyclovir 50 mg / kg / day for 7 days.

[0240] Group 3 - a group of animals infected with HSV-1 , strain L2 and treated with compound 11826233 50 mg / kg once a day for 7 days orally; Group 4 - a group of animals infected with HSV-1 , strain L2 and treated with compound 11826233 100 mg / kg once a day for 7 days orally;

[0241] Group 5 - a group of animals infected with HSV-1 , strain L2 and treated with compound 11826233 50 mg / kg once a day for 7 days intraperitoneally ;

[0242] Oral administration to animals was carried out using a disposable insulin syringe with a feeding tube for animals.

[0243] The drug was given once a day for 7 days.

[0244] The comparison drug (Acyclovir) was given at a dose of 50 mg / kg / day for 7 days. Oral administration to animals was performed intragastrically using a disposable insulin syringe with a probe ( lavage ).

[0245] To prepare solutions for administration, the samples were dissolved in distilled water before the experiment. The doses of the studied samples were calculated in relative weight units - mg / kg of animal body weight per day. The animals were given solutions orally and intraperitoneally in a volume of 200 pl. The animals of the control group were given distilled water orally in a volume of 200 pl. 1.6. DETERMINATION OF DRUG EFFICACY IN A MOUSE HERPES ENCEPHALITIS MODEL

[0246] In preliminary experiments The dose of virus causing 100% mortality in the viral control group was determined to be 10LD50, which was then used in experiments to evaluate the efficacy of the compound.

[0247] Each animal within a group was assigned an individual serial number, mice were distributed randomly into groups, body weight was used as an inclusion criterion so that the individual weight value did not deviate from the average by more than ±10%.

[0248] BALB I c mice , females weighing 14-16 g, with no signs of health deviations, were divided into 10 groups of 13 individuals each, taking into account their body weight, so that individual body weight values did not deviate from the average value by more than 10%.

[0249] Pre-weighed mice were infected intranasally under anesthesia with herpes simplex virus type I , 25 pl of virus-containing fluid in each nostril.

[0250] The activity of the compounds in the mouse influenza pneumonia model was assessed according to the following criteria: animal survival, increase in average life expectancy, weight loss dynamics, virus titer in the lungs on day 5 after infection.

[0251] The mortality rate was determined as the ratio of the number of dead animals to the total number of infected individuals in the group. The average lifespan of animals was determined based on the total number of days of observation of animals (after infection) according to the formula: MSD = f ( d -1) / n , where f is the number of mice that died on day d , surviving mice are also included in f and d in this case is 14, n is the number of mice in the group.

[0252] Each group contained 13 animals (3 animals per virus titer and 10 animals per survival rate), and 1 experiment was conducted.

[0253] 1.7. Animal mass estimation

[0254] Mice were weighed in the morning on the day of infection (day 0) and the day after until the end of the experiment (on days 2, 4, 6, 8, 10, 12, 14 after infection with the virus). Weight loss or gain was calculated separately for each mouse and expressed as a percentage. The animal's weight before infection (i.e., the weight of the animals on the day of infection) was taken as 100%. The average weight change (as a percentage) was determined for all mice in one group. The body weight of the animals whose lungs were taken to determine the virus titer was not used to plot the graphs. 1 .8. Taking mouse brain samples for study and determining viral titer

[0255] Infection, 3 mice in each group were humanely sacrificed and their brains were removed under sterile conditions, homogenized, and resuspended in 1 ml of cold sterile 0.01 M PBS. The suspension was cleared of cell debris by centrifugation at 2000 g for 10 min. 0.1 ml of the supernatant was used to determine the infectious titer of the virus in Vero cell culture. The samples were stored at 4°C for no more than a week before experiments.

[0256] To determine the infectious titer of the virus, Vero cells were plated in 96-well Costar plates at an average density of 30,000-35,000 cells per well and grown in Eagle's minimal medium (MEM) in the presence of 5% fetal calf serum, 10 mM glutamine and antibiotics (penicillin 100 lU / ml and streptomycin 100 pg / ml) until a complete monolayer was formed. Before infection with the virus, the cell culture was washed twice with MEM medium without serum. Ten-fold dilutions of each virus sample from the lungs were prepared (whole up to 109). The resulting dilutions were used to infect monolayers of 4 wells of a 96-well plate. After incubation at 37°C in an atmosphere of 5% CO2 for 72 hours, the cells were washed three times with PBS and fixed with 10% formaldehyde solution at a temperature of 18-23°C for 5 min. After removing the formaldehyde solution, 100 pl of 1 % crystal violet solution were added to each well of the plate and kept at a temperature of 18-23°C for 5 min. After washing with water and drying the plate, 0.1 ml of 96% alcohol was added to the wells, the mixture was incubated with shaking (room temperature for 20 min), and then the optical density was measured at a wavelength of 570 nm. The wells were considered "positive" if the optical density in them was 20% less than the optical density in the cell control. The infectious titer of the virus was determined in 4 replicates for each sample representing the material from one animal, for each sample according to the Reed and Muench method and expressed as Ig TCID50 / 0.1 ml (tissue cytopathic infectious dose 50). Then the average titer value was calculated for five identical samples.

[0257] 1.9. Performance criteria

[0258] The primary criterion of the antiviral effect was considered to be an increase in the survival rate of animals (p<0.05) is considered as a reliable difference from the untreated control and / or an increase in the percentage of survival in the group of treated animals by 30% from the viral control group [1]). A decrease in weight loss indicates a decrease in the severity of the disease, and a decrease in the viral titer in the lungs of infected animals by 1 .5 or more Ig TCID50 Z0.1 ml after the introduction of experimental samples compared to the control group of infected untreated animals were assessed as indicators of the effectiveness of the disease.

[0259] 1.10 Data Analysis

[0260] The Kaplan-Meier log-rank test was used to determine the statistical significance of survival. The nonparametric Kruskal-Wallis test with Bonferroni correction was used to determine the statistical difference in the dynamics of animal weight, significance level p <0.05. Kaplan-Meier curves were used to visualize survival data. Normality distributions data was assessed using test Shapiro- Wilk. All graphs were plotted in Microsoft Excel. Virus titer was calculated using the Ramakrishnan MA formula in Excel. Descriptive statistics parameters included the mean value of the indicator in the group and the standard deviation (SD). 2 RESULTS

[0261] 2.1 Initial clinical examination of laboratory animals

[0262] The mice were brought from the bio-nursery in the first half of the day. Upon receipt, each animal underwent a general clinical examination of the body to identify possible pathologies.

[0263] During the examination of animals, no pathologies were detected, all animals were assigned the status of "clinically healthy". After the clinical examination, the mice were placed in the vivarium of the Institute randomly in groups in plastic cages, in accordance with GOST 33216-2014, to undergo a 3-day quarantine. After quarantine , before the start of experimental work with laboratory mice, the animals underwent a repeated clinical examination, during which the animals were also assigned the status of "clinically healthy". During the quarantine, no deaths or deterioration in the condition of the mice were observed.

[0264] 2.2 Effect of compound 11826233 therapy on body weight dynamics in mice infected with HSV-1.

[0265] Throughout the experiment, visual inspection of the animals and recording of deaths were carried out daily, and changes in body weight were recorded once every two days. Detailed data on the dynamics of body weight of each animal are given in Appendix 1 .

[0266] In the viral control group, significant weight loss was observed, which reached 23.8 % after day 6 after infection. Dosages of substance 11826233 50 mg / kg / day and 100 mg / kg / day when administered orally did not have a significant effect on preventing body weight loss in infected animals; at a dose of 100 mg / kg / day orally, animals lost less weight, but this loss was not statistically significant (the difference with the viral control was 3.1 and 4.4%, respectively). Despite this, in animals treated with compound 11826233 at a dosage of 100 mg / kg, stable positive dynamics of body weight restoration was recorded after the 8th day of the experiment. The best indicators for maintaining and further restoring body weight were in the group of therapy with compound 11826233 50 mg / kg with intraperitoneal administration. On day 6 post-infection, when the viral control group recorded the maximum body weight loss (23.8%), therapy with compound 11826233 50 mg / kg i.o. significantly reduced the weight loss of treated animals, differing from the viral control by 6%. However , the reduction in weight loss in animals in this group was close to the group with therapy with the gold standard of herpes therapy - Acyclovir and did not differ significantly from it throughout the experiment (p>0.05) (Fig. 11).

[0267] 2.3 Effect of compound 11826233 therapy on survival and lifespan of mice infected with HSV-1.

[0268] On the 8th day of the experiment, when there was 100% mortality of animals in the viral control group. On this day, by the day of death of the viral control, in the groups with compound 11826233 therapy in all studied dosages and routes of administration, survival was observed, estimated as reliable (30%) compared to the viral control group. On the 8th day, therapy with compound 11826233 orally in a lower dosage of 50 mg I kg protected 30% of animals from death. With an increase in the dosage of the compound to 100 mg / kg, the percentage of survival increased and amounted to 60% protection. The best indicators were with the use of compound 11826233 intraperitoneally 50 mg / kg - in this group, the percentage of animal protection from death was 80%, which was comparable to the protective effect of Acyclovir. During further observation, a decrease in survival was recorded in the study groups by day 14, however, the percentage of survival in the group treated with compound 11826233 50 mg / kg with intraperitoneal administration remained high, significantly different from the viral control group, which indicates the therapeutic efficacy and neuroprotective properties of the studied compound 11826233 at this dose and route of administration in relation to HSV-1 (Fig. 12, and Table below).

[0269] Results of survival registration.

[0270] Calculation of the average lifespan of mice infected with HSV-1 showed that the use of compound 11826233 increases the average lifespan of mice when administered orally at a dose of 50 mg / kg by 1 day, but this increase was not significant. An increase in the dose of 100 mg / kg orally led to an increase in the average lifespan - by 2.6 days (p<0.05), and with intraperitoneal administration at a dose of 50 mg / kg - by 4.2 days, which is a statistically significant difference (p<0.05).

[0271] Results of registration of the following indicators: life expectancy, incubation period, protection index, and change in body weight and virus titer. 2.4 Effect of therapy with the study drug on the virus titer in the brain tissue of animals

[0272] On the 5th day after viral infection, brain tissues of infected animals were taken to determine the amount of virus in it. The difference in the virus titer was considered statistically significant at a difference of 1.5 Ig TCID50 / ml.

[0273] Data on virus replication in the brain of animals correlated with data on survival, weight loss and life expectancy of animals.

[0274] In the virus control group, in which all animals died, the virus titer was maximum and amounted to 5.4±0.2 Ig TCID50 The control drug Acyclovir suppressed virus replication by more than 2 Ig.

[0275] Therapy with compound 11826233 at doses of 50 and 100 mg / kg orally, although it somewhat reduced the amount of virus in the brain of infected animals, did not differ significantly from the viral control group (4.7 and 4.6 vs. 5.4 Ig TCID50 , respectively). At the same time, the use of compound 11826233 orally at a dose of 50 mg / kg contributed to a reliable decrease in the amount of virus in brain tissue by 1.8 Ig TCID50 compared to the viral control. (3.6 Ig vs 5.4 Ig respectively).

[0276] CONCLUSION

[0277] In the course of the research work, the efficacy of the compound was assessed on a model of herpes encephalitis in mice induced by the herpes simplex virus type I. The use of compound 11826233 orally at a dose of 50 mg I kg did not have a significant effect on the dynamics of body weight, survival, average lifespan and virus titer in brain tissues. Oral administration of compound 11826233 at a dosage of 100 mg I kg did not protect animals from body weight loss and virus proliferation in brain tissues, but contributed to a stable restoration of body weight in animals, statistically significantly increased the average lifespan of mice infected with HSV-1 herpes encephalitis, and maintained the percentage of protection from death at a statistically significant level up to the 8th day of the experiment. The most effective was intraperitoneal therapy with compound 11826233 at a dosage of 50 mg / kg. This therapy statistically significantly protected the animals from death throughout the experiment, significantly increased the average lifespan of the animals, and also reduced the replication of the virus in brain tissue compared to a group of untreated mice with herpes encephalitis.

[0278] APPENDIX

[0279] Dynamics of body weight and survival of mice

[0280] Legend: “+” - animal death; “ high ” - animal lungs taken for seeding.

[0281] Group 1 : Viral control Group 2: Acyclovir orally, 50 mg / kg

[0282] Group 3: Compound 11826233 50 mg / kg per os

[0283] Group 4: Compound 11826233 100 mg / kg per os

[0284] Group 5: Compound 11826233 50 mg / kg intraperitoneally Example 12: Further experiments with addition viruses

[0285] To get a further insight on the effects of the compound according to the present invention, analyses with RNA viruses, such as SARS-CoV-2 (different strains), Hepatitis A virus (different strains), Borna disease virus 1 (BoDV-1), Respiratory Syncytial Virus (RSV), and DNA viruses, such as Adenovirus Type 5 or Equine Herpesviruses (EHV-1) are performed, e.g., by techniques including Plaque reduction assays, quantitative PCR and / or determination of TCID50 values.

[0286] Example 13: Organ-on-a-Chip

[0287] 1. Preclinical analysis with Organ-on-a-Chip Modell

[0288] To analyze the permeability of the inhibitor, biochips from Dynamic42 will be used. The vascular model has already been established on Dynamic42 Vasculature-on-Chips. This chip consists of a chamber adjacent to a porous membrane. The pores (1 x 105 per cm2) are randomly distributed and have a diameter of 8 pM. Additionally, there are inflow and outflow channels that mimic blood flow. Primary endothelial cells (HUVEC) have been successfully seeded in this chamber.

[0289] 1.1 Cultivation of cells

[0290] For a vascular model, endothelial cells will be seeded into the chamber and cultured with daily medium changes. After three days, cell-type-specific macrophages can be added.

[0291] Another option is co-cultivation of endothelial cells with fibroblasts. This will be performed in a second step of the analysis.

[0292] 1.2 Effect of the compound on Permeability

[0293] After the biochips have been established, the inhibitor is analyzed for its uptake into the cells and its translocation into the medium. For this purpose, the medium is removed from the respective chamber 48 hours after administration of the inhibitor and analyzed by HPLC. This allows for a quantitative evaluation.

[0294] 1.3 Influence on immune cells

[0295] Furthermore, the supernatants will be used to analyze their effect on immune cells. Forthis purpose, ELISA standard kits will be used to detect the cytokines IL-6, IFN-beta, IL-10, and TNF-a. The analysis will be performed according to the manufacturer's instructions. The data will be generated using a plate reader.

[0296] 1.4 Antiviral activity in biochips

[0297] The biochips cultured with cells should first be pre-incubated with or without the inhibitors for 30 minutes. The inhibitors are then removed, and the pretreated endothelial cells on the chip are infected with TB40 / E- pp150EGFP for 1 hour. After removing the virus suspension, further cultivation should continue for 4 days. Subsequently, both the supernatants in the respective chambers and the cells themselves should be removed. The antiviral effect will be demonstrated using fluorescence microscopy and plaque assay.

Claims

43CLAIMS1 . A compound which is a pharmaceutically acceptable salt of the following structure of Formula I:or a hydrate, solvate and / or acid addition salt thereof, wherein:X is selected from halogen, an alkoxy and a haloalkoxy;Y1and Y2are each independently a substituent, preferably selected from alkyl;Z1, Z2, Z3and Z4are each independently selected from H and an alkyl; n1and n2are each independently 0 or an integer of > 1 , preferably selected from 1 , 2, 3 and 4, n3and n4are each independently 0 or 1 ; if Z3is present, the nitrogen atom to which Z3is bound is positively charged; and if Z4is present, the nitrogen atom to which Z4is bound is positively charged.

2. The compound of claim 1 which is a pharmaceutically acceptable salt of Formula II:or a hydrate, solvate and / or acid addition salt thereof.

3. The compound of claim 1 or 2, wherein:X is selected from F, Br, Cl, OMe, OCF3 and OEt;Y1and Y2are each independently selected from Ci alkyl, C2 alkyl, C3 alkyl, C4 alkyl and C5 alkyl; andZ1, Z2, Z3and Z4are each independently selected from H and Ci alkyl, C2 alkyl, C3 alkyl, C4 alkyl and C5 alkyl.

4. The compound of any one of the preceding claims, wherein: n1, n2, n3and n4are each 0; andZ1and Z2are independently selected from H, Ci alkyl, C2 alkyl, C3 alkyl, C4 alkyl and C5 alkyl.

445. The compound of any one of the preceding claims, wherein:(a) the pharmaceutically acceptable salt is selected from a chloride and a bromide, preferably wherein the pharmaceutically acceptable salt is a chloride when X = Cl, and is a bromide when X = Br, more preferably tetrachloride; and / or(b) the acid addition salt is selected from a hydrochloride and a hydrobromide, preferably wherein the anion in the acid is the same as counter anion of the pharmaceutically acceptable salt, more preferably dihydrochloride.

6. The compound of any one of the preceding claims, wherein the hydrate comprises up to 10 mol of water per one mol of the compound.

7. The compound of any one of the preceding claims having a structure of Formula III:or a hydrate, solvate and / or acid addition salt thereof.

8. Pharmaceutical composition comprising the compound of any one of the preceding claims and a pharmaceutically excipient.

9. The pharmaceutical composition of claim 1 , which is selected from: a formulation for parenteral or systemic administration, preferably selected from an injection formulation, a ready-to-use formulation and a solid drug product for reconstitution with a diluent, more preferably an intravenous injection or intramuscular injection formulation; an inhalation formulation, preferably selected from aerosol, inhaler, vaporizer and nebulizer formulation; a spray, preferably selected from nasal spray and skin spray; a topical or external formulation, preferably selected from solution, emulsion, gel, drops, paste, cream, ointment, liniment, balm, lotion, foam, patch, powder and dust.4510. The compound or composition of any one of the preceding claims for use as a medicament.11 . The compound or composition of any one of claims 1-9 for use in a method of treatment or prevention of a viral infection or disease.

12. The compound or composition of any one of claims 1-9 for use in the method as defined in claim 11 , wherein the viral infection or disease is caused by at least one virus dependent on or using heparan sulfate proteoglycan(s) for attachment to host cells (HSPG-dependent virus(es)).

13. The compound or composition of any one of claims 1-9 for use in the method as defined in claim 11 or 12, wherein the viral infection or disease is caused by at least one enveloped virus(es).

14. The compound or composition of any one of claims 1-9 for use in the method as defined in any of claims 11 to 13, wherein the viral infection or disease is caused by at least one virus(es) belonging to one or more of:(a) Alphaherpesvirinae, Betaherpesvirinae, Gammaherpesvirinae, Flavivirdae, Togaviridae, Orthoretrovirinae Adenoviridae, Orthoherpesviridae, Orthomyxoviridae, Orthoparamyxovirinae, Papillomaviridae, Picornaviridae, Pneumoviridae, Poxviridae, Coronaviridae,(b) Varicellovirus, Simplexvirus, Cytomegalovirus, Flavivirus, Alphavirus, Lentivirus, Mastadenovirus, Rhadinovirus, Henipavirus, Hepatovirus, Alphainfluenzavirus, Betainfluenzavirus, Orthohepadnavirus, Alphapapillomavirus, Orthopneumovirus, Orthopoxvirus, Betacoronavirus,(c) Pseudorabies virus (PrV), suid alphaherpesvirus 1 , animal alpha-herpesvirus(es), Equid alphaherpesvirus(es), Bovine alphaherpesvirus(es), Varizella Zoster Virus, Herpes simplex virus 1 (HSV-1), human alphaherpesvirus 1 , HSV-1 KOS, Human cytomegalovirus (HCMV), human betaherpesvirus 5, GCV-resistant HCMV, Zika virus (ZIKV), Orthoflavivirus zikaense, ZIKV H / PF / 2013, Dengue virus, O’nyong-nyong virus (ONV), Chikungunya virus,Human immunodeficiency virus 1 (HIV-1), CXCR4-tropic HIV-1 virus, Adenovirus Type 5, human adenovirus 5, Kaposi sarcoma associated virus, Human gammaherpesvirus 8, HHV-8, Nipah virus, Henipavirus nipahense, Influenza A virus, Influenza B virus, Hepatitis B virus (HBV), Hepatitis A virus (HAV), Human papillomavirus (HPV), Human orthopneumovirus, Respiratory syncytial virus (RSV), Mpox (MPXV), and SARS-CoV2.

15. The compound or composition of any one of claims 1-9 for use in the method as defined in any of claims 10-14, wherein:(a) the compound or composition is administered via one or more of parenteral route, systemic route, injection, inhalation, topical route, external route and mucosal route, preferably by intravenous or intramuscular injection or by application to the skin or a mucous membrane, more preferably to the nasal mucosa; and / or(b) the method is performed on a human or animal subject, preferably a human, a livestock animal, e.g., pig, or a companion animal, e.g., dog or cat.

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