[go: up one dir, main page]

WO2022129210A2 - Inhibition de protéase virale - Google Patents

Inhibition de protéase virale Download PDF

Info

Publication number
WO2022129210A2
WO2022129210A2 PCT/EP2021/085966 EP2021085966W WO2022129210A2 WO 2022129210 A2 WO2022129210 A2 WO 2022129210A2 EP 2021085966 W EP2021085966 W EP 2021085966W WO 2022129210 A2 WO2022129210 A2 WO 2022129210A2
Authority
WO
WIPO (PCT)
Prior art keywords
alkyl
group
pentyl
butyl
formula
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2021/085966
Other languages
English (en)
Other versions
WO2022129210A3 (fr
Inventor
Grzegorz Maria POPOWICZ
Michael Sattler
Kamyar HADIAN
Oliver Plettenburg
Andre MOURAO
Karl Kenji SCHORPP
Valeria NAPOLITANO
Krzysztof Pyrc
Katarzyna Owczarek
Agnieszka DABROWSKA
Pawel BOTWINA
Aleksandra MILEWSKA
Tony FROEHLICH
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Uniwersytet Jagiellonski
Original Assignee
Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Uniwersytet Jagiellonski
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from LU102310A external-priority patent/LU102310B1/en
Application filed by Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH, Uniwersytet Jagiellonski filed Critical Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Publication of WO2022129210A2 publication Critical patent/WO2022129210A2/fr
Publication of WO2022129210A3 publication Critical patent/WO2022129210A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
    • C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
    • C07D219/08—Nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B15/00—Acridine dyes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a composition, comprising at least one compound according to formula (I) as well as to a composition comprising at least one compound according to formula (I) and/or dimers of compounds according to formula (I), in particular for use in the treatment of diseases caused by betacoronaviruses, leishmaniasis, and trypanosomiasis.
  • Coronaviruses have been considered a potential threat since 2002, when the SARS- CoV virus emerged in southern China to spread through continents and disappear shortly thereafter 1 ,2 rapidly. Ten years later, the second coronavirus - MERS-CoV - gave a final warning to be prepared 3 . Still, the emergence of the SARS-CoV-2 and subsequent pandemic met the public unprepared and paralyzed the modern world in an unprecedented way. At present, 1.4 million fatalities, have already been crossed and the northern hemisphere is facing the long the winter season, which due to the appearance of other comorbidities (low/dry air, coinfections, pollution, dysregulated immune responses) may still increase these numbers drastically 4 ' 6 .
  • the coronaviral genome encodes for several structural and non-structural proteins.
  • the most promising targets are the Spike (S) protein, as the target for neutralizing antibodies and entry inhibitors, the nsp12 polymerase, targeted by several compounds including the remdesivir, nsp14 and nsp16 methyltransferases essential for the capping of viral RNA, and two cysteine proteases M pro and PL pro , responsible for the viral proteome maturation and indispensable for the infection.
  • PLpro is also responsible for type I interferon response attenuation.
  • a potential target is SARS-CoV-2 PL pro .
  • the present invention is related to a composition comprising at least one compound according to formula (I)
  • RT is selected from the group consisting of H, (Ci-C 6 )alkyl, (CH 2 ) 0 (Ci-C 6 )alkyl ,(Ci-C 6 )cycloalkyl, (Ci-C 6 )heterocyclyl, or absent, preferably methyl;; o is 1 to 3, preferably 1 ;
  • R 2 is selected from the group consisting of H, (Ci-C 6 )alkyl,-(CH 2 ) r CONH(CH 2 ) s R6, -(CH- (CH 2 ) p COvinyl, -(CH 2 ) q R 6 , -CO(Ci-C 6 )alkyl preferably H;
  • R 3 is selected from the group consisting of H, (Ci-C 6 )alkyl, -(CH 2 ) r CONH(CH 2 ) s R6, - (CH 2 ) p COvinyl, -(CH 2 ) q R 6 , -CO(Ci-C 6 )alkyl, preferably H; p is 1-3, preferably 1 ; q is 1-3, preferably 1 ; r is 1-3, preferably 1 ; s is 1-3, preferably 1 ;
  • R 4 is selected from the group consisting of H, (Ci-C 6 )alkyl
  • R 5 is selected from the group consisting of H, (Ci-C 6 )alkyl; if R 2 and R 5 are both (Ci-C 6 )alkyl, R 2 and R 5 may be connected to form a 4 to 6 membered ring; if R 3 and R 4 are both (Ci-C 6 )alkyl, R 3 and R 4 may be connected to form a 4 to 6 membered ring; R 6 is selected from the group consisting of
  • X' is an anion or absent ; if X' is absent then RT is absent; for use in the treatment of diseases caused by betacoronaviruses, leishmaniasis, and trypanosomiasis.
  • the invention is further directed to a composition in which at least one compound according to formula (I) and at least one compound according to formula (III) are bonded together via one or two linker systems wherein optionally a) the linker system is (C 2 -Ci 0 )alkyl, preferably (C 2 -Ci 0 )alkenyl, preferably (C 3 -C 8 )alkyl, or (C 3 - 8 )alkenyl; wherein optionally at least one or at least two CH 2 -groups in these alkyl or alkenyl groups are substituted by O, S, S(O)i.
  • the linker system is (C 2 -Ci 0 )alkyl, preferably (C 2 -Ci 0 )alkenyl, preferably (C 3 -C 8 )alkyl, or (C 3 - 8 )alkenyl; wherein optionally at least one or at least two CH 2 -groups in these alkyl or alkeny
  • the invention is further directed to a composition comprising at least one compound according to formula (I)
  • RT is selected from the group consisting of H, (Ci-C 6 )alkyl, (CH 2 ) 0 (Ci-C 6 )alkyl ,(Ci-C 6 )cycloalkyl, (Ci-C 6 )heterocyclalkyl, or absent, preferably methyl; o is 1 to 3, preferably 1 ;
  • R 2 is selected from the group consisting of H, (Ci-C 6 )alkyl,-(CH 2 ) r CONH(CH 2 ) s R6, -(CH- (CH 2 ) p COvinyl, -(CH 2 ) q R 6 , -CO(Ci-C 6 )alkyl preferably H;
  • R 3 is selected from the group consisting of H, (Ci-C 6 )alkyl, -(CH 2 ) r CONH(CH 2 )sR 8 , - (CH 2 ) p COvinyl, -(CH 2 ) q R 6 , -CO(Ci-C 6 )alkyl, preferably H; p is an integer between 1 to 3, preferably 1 ; q is an integer between 1 to 3, preferably 1 ; r is an integer between 1 to 3, preferably 1 ; s is an integer between 1 to 3, preferably 1;
  • R 4 is selected from the group consisting of H, (Ci-C 6 )alkyl
  • R 5 is selected from the group consisting of H, (Ci-C 6 )alkyl; if R 2 and R 5 are both (Ci-C 6 )alkyl, R 2 and R 5 may be connected to form a 4 to 6 membered ring; if R 3 and R 4 are both (Ci-C 6 )alkyl, R 3 and R 4 may be connected to form a 4 to 6 membered ring; ) °>
  • R 6 is selected from the group consisting of ;
  • X' is an anion or absent; if X' is absent then RT is absent; with the provisio that the compound according to formula (I) is not selected from the group
  • the inventive compounds block the replication of the selected betacoronaviruses, including with nanomolar IC 50 , showing high selectivity and good effectiveness (see Figures 8 to 14). Further, it has been shown that the inventive compounds are effective against Trypanosoma and Leishmania parasites.
  • Fig. 1 Inhibiton of PL pro in the presence of ACF.
  • AMC assay using RLRGG-AMC as substrate and PL pro performed in technical triplicates. Vertical Y-axis shows fluorescent signal (rising, as the substrate is proteolytically cleaved). Horizontal X-axis indicate time. An inhibition profile is observable.
  • B As in (A), only with ISG15-AMC instead of RLRGG-AMC
  • C Time course analysis of tri-ubiquitin K48-linked (2 pM) hydrolysis using 100 nM PL pro in the presence of different ACF concentrations.
  • FIG. 2 The inhibition of virus replication by selected compounds.
  • Fig. 3 The crystal structure of SARS-Cov2-PL pro in complex with proflavine.
  • SCoV2- PL pro is represented as a solid surface, whereas proflavine is represented as a stick model.
  • A a zoom-in of the two proflavine molecules in the S3-S5 pockets of the enzyme active site.
  • B a zoom-in on a proflavine molecule between two crystal neighbors of PL pro .
  • Fig. 4 Details of the molecular interactions between the SARS-CoV2-PL pro and proflavines.
  • A, B Both proflavine molecules bind cooperatively in the substrate pocket of the enzyme forming a non-covalent inhibitor. Both polar (gray) and hydrophobic (red) interactions as well as TT-TT stacking (green) are involved. Main residues involved in the interactions are shown as a stick-model.
  • C, D 2D plots of molecular interactions between proflavine-l, proflavine-ll and PL pro .
  • Each proflavine molecule uses different properties for form interaction. The aromatic nitrogen of proflavine-l must be desolvated for binding. This explains the higher affinity of acriflavine than proflavine as it contains N-methylated components.
  • Fig. 5 Both proflavine molecules mimic native substrate interactions.
  • A Comparison of SARS-CoV2-PL pro complexed with proflavine (in gray) and ISG15 host-cell substrate (in cyan; PDB ID:6YVA). The active site is marked by a dashed oval.
  • B Substrate recognition cleft interaction of proflavines and the C-terminal tail of ISG15. Proflavine molecules overlap with the RLRGG motif of the substrate.
  • the two arginines in P3 and P5 turn their lipophilic carbons in the same directions as the aromatic carbons of the proflavine-ll molecule in the S3-S5 pockets.
  • the amide nitrogen H-bond donors of the peptide backbone correspond well with the proflavine-ll donors and the interactions are well preserved.
  • the side chain of the leucin in position P4 points exactly of the proflavine ring in the S4 pocket.
  • Fig. 6 Overlay of 1 H, 15 N TROSY NMR spectra of PL pro with different amount of ACF added. The entire spectrum is shown in the middle, magnification of a few regions above and below. A number of peaks shift with the addition of ACF. The bulk of the spectra remains similar to the reference apo-PL pro . This indicated that the overall fold of the protein is intact and the compound binds in a distinct binding pocket.
  • Fig. 7 The cytotoxicity of ACF in vitro.
  • FIG. 8 The inhibition of virus replication by ACF in A549 ACE2+ (A) and Vero (B) cells.
  • Figure 10 The inhibition of virus replication by ACF in A549 ACE2+ cells.
  • Cells were infected with the SARS-CoV-2 in presence of 500 nM Acriflavine, 10 pM Remdesivir or vessel control for 24 h and 48 h.
  • Cell nuclei are denoted in blue, actin is denoted in red and SARS- CoV-2 N-protein is denoted in green.
  • FIG 11 Antiviral activity of ACF against SARS-CoV-2 in human airway epithelium.
  • A In-house HAE
  • B MucilAirTM.
  • Figure 12 The inhibition of virus replication by ACF in well differentiated HAE cultures.
  • Figure 13 Time of addition study. The inhibition of virus replication in Vero cells by ACF added after post-infection (time denoted on the x-axis).
  • FIG. 14 ACF inhibits betacoronaviruses. Replication of (A) MERS-CoV, (B) HCoV- OC43, HCoV-NL63 (C) and (D) FIPV in vitro in the presence or absence of inhibitors, as assessed with RT-qPCR on cell culture supernatants. Single round of infection was recorded (24 h). All experiments were performed at least in 2 biological repetitions, each in triplicate. The results are presented as average values with standard deviations (error bars). An asterisk (p ⁇ 0.05) indicates values that are significantly different from the non-treated control. ACF: acriflavin, rem: remdesivir.
  • Figure 15 Proflavine molecule at the interface between SARS-CoV2-PL pro asymmetric units.
  • A Most probably due to a crystal packing molecule of proflavine was found on top of the catalytic triad (C111, H272, D286). Important residues are highlighted as stick model. Two water molecules (red spheres) mediate a hydrogen bond with D286. Hydrogen bonds are represented as yellow dashed lines.
  • B 2D plot of the molecular interactions between proflavine and the residues of SARS-CoV2-PL pro .
  • Figure 16 Comparison of SARS-CoV2-PL pro in the proflavine-bound and apo-state. Bound PL pro is colored in gray; whereas the unbound PL pro (PDB ID: 7D47) is colored in yellow.
  • the BL2 loop is involved in an induced fit rearrangement upon the binding mostly due to the movement of the Tyr268.
  • the side-chain of Tyr268 participates in a TT-TT stacking with proflavine molecules.
  • Figure 17 Electron density map showing the fractional presence of additional aromatic proflavine-like molecules TT-TT stacked one on top of the other between two copies of SARS- CoV2-PL pro present in the crystal lattice.
  • 2F O -F C electron density map is contoured at 2CE.
  • the electron density of the identified proflavine molecules is colored in blue; whereas additional electron density is colored in green.
  • the densities are most likely caused by weak and transiently-bound proflavines. We did not model them in the crystal structure as their electron density was much weaker than active site-bound molecules.
  • FIG. 18 SARS-CoV-2 M pro activity is not significantly inhibited by ACF.
  • the digestion of fluorogenic substrate was recorded in the absence and presence of ACF (A). Only small decrease of M pro activity is observed at physiologically irrelevant ACF concentration of 100 pM (B). The vertical shift in signal levels is caused by ACF absorbance.
  • Figure 21 Composition of commercial acriflavine (cACF) sold by Sigma Aldrich (A8251): 56% proflavine (PF), 17% acriflavine (ACF) and about 26% of their side methylated derivatives.
  • cACF commercial acriflavine
  • PF proflavine
  • ACF acriflavine
  • Figure 22 Map of pETM-5a.
  • alkyl refers to a monoradical of a saturated straight or branched hydrocarbon.
  • the alkyl group comprises from 1 to 10 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 9 carbon atoms, more preferably 1 to 5 carbon atoms, such as 1 to 4 or 1 to 2 carbon atoms.
  • Exemplary alkyl groups include methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, n- pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2-dimethyl-propyl, iso-amyl, n-hexyl, iso-hexyl, sechexyl, 2-ethyl-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, and the like.
  • alkylene refers to a diradical of a saturated straight or branched hydrocarbon.
  • the alkylene comprises from 1 to 10 carbon atoms, i.e., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms.
  • Exemplary alkylene groups include methylene, ethylene (i.e., 1 ,1-ethylene, 1,2-ethylene), propylene (i.e., 1,1-propylene, 1,2-propylene (-CH(CH 3 )CH 2 -), 2,2-propylene (-C(CH 3 ) 2 -), and
  • the butylene isomers e.g., 1,1-butylene, 1 ,2-butylene, 2,2-butylene, 1 ,3- butylene, 2,3-butylene (cis or trans or a mixture thereof), 1 ,4-butylene, 1 ,1-iso-butylene, 1 ,2-iso- butylene, and 1,3-iso-butylene
  • the pentylene isomers e.g., 1,1-pentylene, 1 ,2-pentylene, 1,3- pentylene, 1,4-pentylene, 1,5-pentylene, 1,1-iso-pentylene, 1,1 -sec-pentyl, 1,1-neo-pentyl
  • the hexylenisomers e.g., 1,1-hexylene, 1 ,2-hexylene, 1,3-hexylene, 1,4-hexylene, 1,5-hexylene, 1 ,
  • alkenylene refers to a diradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond.
  • the maximal number of carbon-carbon double bonds in the alkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenylene group by 2 and, if the number of carbon atoms in the alkenylene group is uneven, rounding the result of the division down to the next integer.
  • the maximum number of carbon-carbon double bonds is 4.
  • the alkenylene group has 1 to 4, i.e., 1, 2, 3, or 4, carbon-carbon double bonds.
  • the alkenylene group comprises from 2 to 10 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms.
  • the alkenylene group comprises from 2 to 10 carbon atoms and 1 , 2, 3, 4, or 5 carbon-carbon double bonds, more preferably it comprises 2 to 8 carbon atoms and 1 , 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1 , 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds.
  • the carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration.
  • exemplary alkenylene groups include ethen-1 ,2-diyl, vinyliden, 1-propen-1 ,2-diyl, 1-propen-1 ,3-diyl, 1-propen-2,3-diyl, allyliden, 1-buten-1 ,2-diyl, 1-buten-1 ,3-diyl, 1-buten-1 ,4-diyl, 1-buten-2,3-diyl, 1-buten-2,4-diyl, 1-buten-3,4-diyl, 2-buten-1 ,2-diyl, 2-buten-1 ,3-diyl, 2-buten-1 ,4-diyl, 2-buten-2,3-diyl, 2-buten-diyl, 2-buten-
  • cycloalkyl represents cyclic non-aromatic versions of “alkyl” with preferably 3 to 6 carbon atoms, such as 3 to 6 carbon atoms, i.e., 3, 4, 5, or 6, carbon atoms, more preferably 5 to 6 carbon atoms, even more preferably 6 carbon atoms.
  • exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.
  • alkenyl refers to a monoradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond.
  • the maximal number of carbon-carbon double bonds in the alkenyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenyl group by 2 and, if the number of carbon atoms in the alkenyl group is uneven, rounding the result of the division down to the next integer.
  • the maximum number of carbon-carbon double bonds is 4.
  • the alkenyl group has 1 to 4, i.e., 1, 2, 3, or 4, carbon-carbon double bonds.
  • the alkenyl group comprises from 2 to 10 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms.
  • the alkenyl group comprises from 2 to 10 carbon atoms and 1 , 2, 3, 4, or 5 carbon-carbon double bonds, more preferably it comprises 2 to 8 carbon atoms and 1 , 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1 , 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds.
  • the carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration.
  • Exemplary alkenyl groups include vinyl, 1-propenyl, 2-propenyl (i.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5- heptenyl, 6-heptenyl, 1 -octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-non
  • alkenylene refers to a diradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond.
  • the maximal number of carbon-carbon double bonds in the alkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenylene group by 2 and, if the number of carbon atoms in the alkenylene group is uneven, rounding the result of the division down to the next integer.
  • the maximum number of carbon-carbon double bonds is 4.
  • the alkenylene group has 1 to 4, i.e., 1, 2, 3, or 4, carbon-carbon double bonds.
  • the alkenylene group comprises from 2 to 10 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms.
  • the alkenylene group comprises from 2 to 10 carbon atoms and 1 , 2, 3, 4, or 5 carbon-carbon double bonds, more preferably it comprises 2 to 8 carbon atoms and 1 , 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1 , 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds.
  • the carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration.
  • exemplary alkenylene groups include ethen-1 ,2-diyl, vinyliden, 1-propen-1 ,2-diyl, 1-propen-1 ,3-diyl, 1-propen-2,3-diyl, allyliden, 1-buten-1 ,2-diyl, 1-buten-1 ,3-diyl, 1-buten-1 ,4-diyl, 1-buten-2,3-diyl, 1-buten-2,4-diyl, 1-buten-3,4-diyl, 2-buten-1 ,2-diyl, 2-buten-1 ,3-diyl, 2-buten-1 ,4-diyl, 2-buten-2,3-diyl, 2-buten- 2,4-diyl, 2-buten-3,4-diyl, and the like. If an alkenylene group is
  • aryl refers to a monoradical of an aromatic cyclic hydrocarbon.
  • the aryl group contains 6 to 10 (e.g., 6 to 10, such as 5, 6, or 10) carbon atoms which can be arranged in one ring (e.g., phenyl) or two or more condensed rings (e.g., naphthyl).
  • exemplary aryl groups include, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl.
  • aryl refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl.
  • -(Ci-C 6 )alkyl(C6-Ci 0 )aryl means that an aryl group comprising an alkyl substituent is attached to the overall molecule via that alkyl substituent.
  • heteroaryl means an aryl group as defined above in which one or more carbon atoms in the aryl group are replaced by heteroatoms of O, S, or N.
  • heteroaryl refers to a five or six-membered aromatic monocyclic ring wherein 1, 2, or 3 carbon atoms are replaced by the same or different heteroatoms of O, N, or S.
  • it means an aromatic bicyclic or tricyclic ring system wherein 1, 2, 3, 4, or 5 carbon atoms are replaced with the same or different heteroatoms of O, N, or S.
  • heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl (1,2,5- and 1 ,2,3-), pyrrolyl, imidazolyl, pyrazolyl, triazolyl (1 ,2,3- and 1,2,4-), tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl (1,2,3- and 1 ,2,5-), pyridyl, pyrimidinyl, pyrazinyl, triazinyl (1,2,3-, 1,2,4-, and 1,3,5-), benzofuranyl (1- and 2-), indolyl, isoindolyl, benzothienyl (1- and 2-), 1 H
  • Exemplary 5- or 6-memered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl (1 ,2,5- and 1 ,2,3-), pyrrolyl, imidazolyl, pyrazolyl, triazolyl (1 ,2,3- and 1,2,4-), thiazolyl, isothiazolyl, thiadiazolyl (1,2,3- and 1,2,5-), pyridyl, pyrimidinyl, pyrazinyl, triazinyl (1 ,2,3-, 1,2,4-, and 1 ,3,5-), and pyridazinyl.
  • -(Ci-C 6 )alkyl(C5-Ci 0 )heteroaryl means that a heteroaryl group comprising an alkylsubstituent group is attached to the overall molecule via that alkyl substituent.
  • heterocyclyl means a cycloalkyl group as defined above in which from 1 , 2, 3, or 4 carbon atoms in the cycloalkyl group are replaced by heteroatoms of O, S, or N.
  • the maximum number of O atoms is 1
  • the maximum number of S atoms is 1
  • the maximum total number of O and S atoms is 2.
  • heterocyclyl is also meant to encompass partially or completely hydrogenated forms (such as dihydro, tetrahydro or perhydro forms) of the above-mentioned heteroaryl groups.
  • heterocyclyl groups include morpholino, isochromanyl, chromanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, indolinyl, isoindolinyl, di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and tetrahydrooxazolyl, di- and tetrahydroisoxazolyl, di- and tetrahydrooxadiazolyl (1 ,2,5- and 1 ,2,3-), dihydropyrrolyl, dihydroimidazolyl, dihydropyrazolyl, di- and tetrahydrotriazolyl (1 ,2,3- and 1 ,2,4-), di- and tetrahydrothiazolyl, di- and tetrahydrothiazolyl, di- and tetrahydrothiadiazol
  • Exemplary 5- or 6-memered heterocyclyl groups include morpholino, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and tetrahydrooxazolyl, di- and tetrahydroisoxazolyl, di- and tetrahydrooxadiazolyl (1 ,2,5- and 1 ,2,3-), dihydropyrrolyl, dihydroimidazolyl, dihydropyrazolyl, di- and tetrahydrotriazolyl (1 ,2,3- and 1 ,2,4-), di- and tetrahydrothiazolyl, di- and tetrahydroisothiazolyl, di- and tetrahydrothiadiazolyl (1 ,2,3- and 1 ,2,5-), di-
  • halogen means fluoro, chloro, bromo, or iodo; preferably chloro, or fluoro, more preferably fluoro.
  • complex of a compound refers to a compound of higher order which is generated by association of the compound with other one or more other molecules.
  • exemplary complexes of a compound include, but are not limited to, solvates, clusters, and chelates of said compound.
  • solvate refers to an addition complex of a dissolved material in a solvent (such as an organic solvent (e.g., an aliphatic alcohol (such as methanol, ethanol, n-propanol, isopropanol), acetone, acetonitrile, ether, and the like), water or a mixture of two or more of these liquids), wherein the addition complex exists in the form of a crystal or mixed crystal.
  • a solvent such as an organic solvent (e.g., an aliphatic alcohol (such as methanol, ethanol, n-propanol, isopropanol), acetone, acetonitrile, ether, and the like), water or a mixture of two or more of these liquids)
  • a solvent such as an organic solvent (e.g., an aliphatic alcohol (such as methanol, ethanol, n-propanol, isopropanol), acetone, acetonitrile, ether, and the like
  • isotopically labeled compounds one or more atoms are replaced by a corresponding atom having the same number of protons but differing in the number of neutrons.
  • a hydrogen atom may be replaced by a deuterium atom.
  • Exemplary isotopes which can be used in the compounds of the present invention include deuterium, 11 C, 13 C, 14 C, 15 N, 18 F, 32 S, 36 CI, and 125 l.
  • RT is selected from the group consisting of H, (Ci-C 6 )alkyl, (CH 2 ) 0 (Ci-C 6 )alkyl ,(C C 6 )cycloalkyl, (Ci-C 6 )heterocyclyl, or absent, preferably selected from the group consisting of methyl, ethyl, propyl, cyclopropyl, butyl, cyclobutyl, pentyl, cyclopentyl, iso-propyl, sec-propyl, iso-butyl, sec-butyl, tert-butyl, 2-pentyl, 3-pentyl, 2-methylbutyl, 3-methylbutyl (/so-pentyl Oder /so-amyl), 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2-dimethylpropyl
  • R 2 is selected from the group consisting of H, (Ci-C 6 )alkyl,-(CH 2 ) r CONH(CH 2 ) s R6, -(CH- (CH 2 ) p COvinyl, -(CH 2 ) q R 6 , -CO(Ci-C 6 )alkyl; preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl, iso-butyl, sec-butyl, tert-butyl, 2-pentyl, 3-pentyl, 2-methylbutyl, 3-methylbutyl (/so-pentyl Oder /so-amyl), 3-methylbut-2-yl, 2-methylbut-
  • R 3 is selected from the group consisting of H, (Ci-C 6 )alkyl, -(CH 2 ) r CONH(CH 2 ) s R6, - (CH 2 ) p COvinyl, -(CH 2 ) q R 6 , -CO(Ci-C 6 )alkyl, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl, iso-butyl, sec-butyl, tert-butyl, 2-pentyl,
  • p is an integer between 1 to 3, preferably 1 ;
  • q is an integer between 1 to 3, preferably 1 ;
  • r is an integer between 1 to 3, preferably 1 ;
  • s is an integer between 1 to 3, preferably 1 ;
  • R 4 is selected from the group consisting of H, (Ci-C 6 )alkyl
  • R 5 is selected from the group consisting of H, (Ci-C 6 )alkyl; [0059] if R 2 and R 5 are both (Ci-C 6 )alkyl, R 2 and R 5 may be connected to form a 4 to 6 membered ring;
  • R 3 and R 4 are both (Ci-C 6 )alkyl, R 3 and R 4 may be connected to form a 4 to 6 membered ring;
  • R 6 is selected from the group consisting of
  • X' is an anion or absent
  • the compound according to formula (I) is not selected from the
  • X' is preferably selected from the group consisting of napsylate, tetrafluoroborate, formate , trifluoroacetate, glycollylarsanilate, nitrate, benzoate, hexylresorcinate, oleate, bitartrate, hydroxynaphthoate, pantothenate, bicarbonate, hydrabamine, pamoate, camsylate, isethionate, polygalacturonate, propionate, salicylate, lactobionate, stearate, decanoate, edetate, maleate, succinate, estolate, mandelate, teoclate, gluceptate, acetate, glutamate, muscate, aspartate, glycolate, benzenesulfonate, hexanoate, octanoate, sulfonate, chloride, iodide, bromide, phosphate, phosphonate
  • composition in another embodiment, there are at least two compounds according to formula (I) present.
  • Ri is (Ci-C 6 )alkyl, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl, iso-butyl, sec-butyl, tert-butyl, 2-pentyl, 3-pentyl, 2-methylbutyl, 3- methylbutyl (/so-pentyl Oder /so-amyl), 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2-dimethylpropyl, hexyl, 2-hexyl, 3-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl, more preferably methyl; R 2 and R 3 are H; and in
  • the second compound (lb) RT is (Ci-C 6 )alkyl, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl, iso-butyl, sec-butyl, tertbutyl, 2-pentyl, 3-pentyl, 2-methylbutyl, 3-methylbutyl (/so-pentyl Oder /so-amyl), 3-methylbut-2- yl, 2-methylbut-2-yl, 2,2-dimethylpropyl, hexyl, 2-hexyl, 3-hexyl, 2-methylpentyl, 3-methylpentyl,
  • R 2 or R 3 in the second compound is (Ci-C 6 )alkyl, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl, iso-butyl, sec-butyl, tert-butyl, 2-pentyl, 3-pentyl, 2- methylbutyl, 3-methylbutyl (/so-pentyl Oder /so-amyl), 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2- dimethylpropyl, hexyl, 2-hexyl, 3-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, and
  • composition may comprise at least one dimer of the compound according to the formula (I) of claim 1, according to formula (II):
  • R 3 in formula (II) is a linker selected from the group consisting of -(CH 2 ) t -, -(CH 2 ) t Q(CH 2 ) u -, - CO(CH 2 ) t -, -CO(CH 2 ) t CO-; t is an integer between 1 to 4; u is an integer between 1 to 4;
  • (I) and (I)’ are based on formula (I) in claim 1 and may be identical or different, preferably identical.
  • composition may further comprise at least one compound according to formula (III)
  • R 4 is H, halogen, (Ci-C 6 )alkyl, (C 3 -C 6 )cycloalkyl, or -O(Ci-C 6 )alkyl; preferably H.
  • R 5 is H, halogen, (Ci-C 6 )alkyl, (C 3 -C 6 )cycloalkyl, or -O(Ci-C 6 )alkyl; preferably H.
  • R 6 is H, halogen, (Ci-C 6 )alkyl, (C3-C 6 )cycloalkyl, or -O(Ci-C 6 )alkyl; preferably H
  • R 7 is H, (Ci-C 6 ) alkyl, preferably methyl
  • Z is H, halogen, (Ci-C 6 )alkyl, (C3-C 6 )cycloalkyl, -O(Ci-C 6 )alkyl, -(Ci-C 6 )alkyl(C6-Cio)aryl, - (Ci-C 6 )alkyl(C5-Cio)heteroaryl; preferably H.
  • Y is -NH 2 , -NHR 8 , halogen, (Ci-C 6 )alkyl, (C3-C 6 )cycloalkyl; preferably -NH 2
  • R 8 is H, (Ci-C 6 ) alkyl, preferably methyl
  • R 4 is (Ci-C 6 )alkyl, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl, iso-butyl, sec-butyl, tert-butyl, 2-pentyl, 3-pentyl, 2-methylbutyl, 3-methylbutyl (/so-pentyl Oder /so-amyl), 3-methylbut-2-yl, 2-methylbut-
  • R 5 is (Ci-C 6 )alkyl, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl, iso-butyl, sec-butyl, tert-butyl, 2-pentyl,
  • R 6 is (Ci-C 6 )alkyl, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl, iso-butyl, sec-butyl, tert-butyl, 2-pentyl,
  • R 7 is (Ci-C 6 )alkyl, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl, iso-butyl, sec-butyl, tert-butyl, 2-pentyl,
  • R 8 is (Ci-C 6 )alkyl, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl, iso-butyl, sec-butyl, tert-butyl, 2-pentyl, 3-pentyl, 2-methylbutyl, 3-methylbutyl (/so-pentyl Oder /so-amyl), 3-methylbut-2-yl, 2-methylbut-
  • Y is (Ci-C 6 )alkyl, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl, iso-butyl, sec-butyl, tert-butyl, 2-pentyl,
  • Z is (Ci-C 6 )alkyl, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl, iso-butyl, sec-butyl, tert-butyl, 2-pentyl,
  • the molar ratio of the at least one compound according to formula (I) may be 5 to 100, preferably 10 to 40, more preferably 15 to 30 mol-% based on the overall molar ratio of parent compounds (I) and (III) of the composition and compound (III) may be 0 to 95, preferably 60 to 90, more preferably 70 to 85 mol%.-% based on the overall molar ratio of parent compounds (I) and (III) in the composition.
  • At least two compounds according to formula (III) are present; preferably in the first compound (Illa) R 4 , R 5 , R 6 , R 7 , and Z are H; Y is -NH 2 ; in the second compound (lllb) R 4 , R 5 , R 6 , and Z are H; Y is -NH 2 , and R 7 is (Ci-C 6 )alkyl, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl, iso-butyl, sec-butyl, tert-butyl, 2-pentyl, 3-pentyl, 2-methylbutyl, 3-methylbutyl (/so-pentyl Oder /so-amyl), 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2-dimethylpropyl, hexyl, 2-
  • At least two compounds according to formula (I) and at least two compounds according to formula (III) are present, preferably (la), (lb), (Ila) and (lllb) as defined above present.
  • the molar ratio based on the overall molar ratio of compounds (la), (lb), (Illa) and (lllb) in the composition is for
  • Compound (I) and/or (III) may be a solvate, hydrate, salt, complex, or isotopically enriched form, preferably a salt.
  • Compound (I) may be a salt, wherein the salt comprises an anion selected preferably from the group consisting of tetrafluoroborate, formate , trifluoroacetate napsylate, glycollylarsanilate, nitrate, benzoate, hexylresorcinate, oleate, bitartrate, hydroxynaphthoate, pantothenate, bicarbonate, hydrabamine, pamoate, camsylate, isethionate, polygalacturonate, propionate, salicylate, lactobionate, stearate, decanoate, edetate, maleate, succinate, estolate, mandelate, teoclate, gluceptate, acetate, glutamate, muscate, aspartate, glycolate, benzenesulfonate, hexanoate, octanoate, sulfonate, chloride, iod
  • Compound (III) may be a salt, preferably the salt comprises an anion selected preferably from the group consisting of tetrafluoroborate, formate , trifluoroacetate, napsylate, glycollylarsanilate, nitrate, benzoate, hexylresorcinate, oleate, bitartrate, hydroxynaphthoate, pantothenate, bicarbonate, hydrabamine, pamoate, camsylate, isethionate, polygalacturonate, propionate, salicylate, lactobionate, stearate, decanoate, edetate, maleate, succinate, estolate, mandelate, teoclate, gluceptate, acetate, glutamate, muscate, aspartate, glycolate, benzenesulfonate, hexanoate, octanoate, sulfonate, chloride, io
  • a sulfonate used in the present invention may be a sulfonate according to formula (IV) wherein R g is selected from the group consisting of phenyl, 4-nitrophenyl, 4-methylphenyl, 4- trifluoromethyphenyl, trifluoromethyl, and (Ci-C 5 )alkyl.
  • R g is (Ci-C 5 )alkyl and/or
  • (Ci-C 5 )alkyl is selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, isopropyl, sec-propyl, iso-butyl, sec-butyl, tert-butyl, 2-pentyl, 3-pentyl, 2-methylbutyl, 3- methylbutyl (/so-pentyl Oder /so-amyl), 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2-dimethylpropyl, preferably methyl.
  • the invention is further directed to a composition in which at least one compound according to formula (I) and at least one compound according to formula (III) are bonded together via one or two linker systems.
  • the linker system may be -(C 2 -Ci 0 )alkylene or (C 2 -Ci 0 )alkenylene, preferably (C 3 - C 8 )alkylene, or (C 3-8 )alkenylene; wherein optionally at least one or at least two CH 2 -groups in these alkyl or alkenyl groups are substituted by O, S, S(O)i.
  • the acridine compounds (III) of the invention can be produced by applying suitable known method of synthesizing acridine derivatives, e.g. as described in Prager, R. H., Williams, C. M., Science of Synthesis (2005) 15, 987; Gensicka-Kowalewska M., Cholewinski G, Dzierzbicka, K. RSC Adv. (2017), 7, 15776 Matejova, M.; Janovec, L; Imrich, J. ARKIVOC 2015 (v), 134 and references cited therein.
  • anilineacetophenones coupled to aryl bromides e.g. via Ullman coupling and subsequently performing a cyclization under appropriate conditions e.g. acid catalysis (Ullmann, F; Torre, A. L., Ber. Dtsch. Chem. Ges., (1904) 37, 2922, Mayer, F; Freund, W., Ber. Dtsch. Chem. Ges., (1922) 55, 2049) or via 2-anilinobenzaldehyde derivatives, e.g. obtained by the McFadyen-Stevens reaction, followed by conversion to acridine derivatives (Graboyes, H.; Anderson, E. L.; Levinson, S.
  • acid catalysis Ullmann, F; Torre, A. L., Ber. Dtsch. Chem. Ges., (1904) 37, 2922, Mayer, F; Freund, W., Ber. Dtsch. Chem. Ges., (1922) 55, 2049
  • acridin- 9(10H)-ones are useful precursors of acridine derivatives, which can be obtained by various methods (e.g. described in Prager, R.H.; Williams, C.M. Science of Synthesis (2005), 1029).
  • Acridine derivatives modified in position 9 can for example be obtained by reacting the corresponding acridine-9(10H)-one derivatives, e.g. with phosphorous oxychloride or thionyl chloride to give 9-CI derivatives (Anuradha, S., Poonam, PChem. Biol. Drug Des. (2017), 90, 926; Nakajima, M., Nagasawa, S., Matsumoto, K., Kuribara, T., Muranaka, A., Uchiyama, M., Nemoto, T, Angew. Chem. Int. Ed.
  • Fused derivatives containing two acridine rings can be synthesized by adaption of methods described in the literature, e.g. starting from diphenyl anilines and reacting these with dicarboxylic acids, using the Bernthsen condensation (Eldho, N. V.; Saminathan, M.; Ramaiah, D., Synth. Commun., (1999) 29, 4007).
  • linker systems with two activated functionalities e.g. malonyl chloride, succinic anhydride, glutaric acid, succinyl chloride, 1,3-dibromopropane, 1,4-dibromobutane, glutaroyl dischloride, 1,3-diiodopropane, 1 ,4-diiodobutane, glutaraldehyde, 4-chlorobutanolyl chloride or 5-chloropentanoyl chloride, e.g. Frohlich, T.; Reiter, C.; Saeed, M. E.
  • two activated functionalities e.g. malonyl chloride, succinic anhydride, glutaric acid, succinyl chloride, 1,3-dibromopropane, 1,4-dibromobutane, glutaroyl dischloride, 1,3-diiodopropane, 1 ,4-dii
  • the compounds of the present invention may be for use in the treatment of diseases caused by betacoronaviruses, leishmaniasis, trypanosomiasis.
  • the treatment is caused by human and veterinary coronaviruses that belong to subgenera hibecovirus, nobecovirus, embecovirus, merbecovirus and sarbecovirus, preferably coronaviruses.
  • the treatment is caused by human coronavirus HKLI1 (HCoV- HKLI1), human coronavirus OC43 (HCoV-OC43), Middle East respiratory syndrome-related coronavirus (MERS-CoV).
  • HCV-HKLI1 human coronavirus HKLI1
  • HoV-OC43 human coronavirus OC43
  • MERS-CoV Middle East respiratory syndrome-related coronavirus
  • the treatment is caused by severe acute respiratory syndrome-related coronaviruses, preferably SARS-CoV, more preferably SARS-CoV-2.
  • the treatment is caused by a virus that evolve or mutate from the species described in the three paragraphs above.
  • the disease to be treated is the severe acute respiratory syndrome, preferably SARS-CoV or SARS-CoV-2, more preferably SARS-CoV-2.
  • the disease to be treated is the Middle East respiratory syndrome (MERS-CoV).
  • MERS-CoV Middle East respiratory syndrome
  • the disease to be treated is pneumonia.
  • the invention is directed to a composition comprising at least the composition as descibed above and at least one pharmaceutically acceptable carrier, thus to a pharmaceutical composition.
  • “Pharmaceutical composition” refers to one or more active ingredients, and one or more inert ingredients that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound or composition of the present invention and a pharmaceutically acceptable carrier.
  • Carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered orally.
  • Saline and aqueous dextrose are preferred carriers when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are preferably employed as liquid carriers for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
  • the amount of active ingredient in particular, the amount of the compound of the present invention, optionally together with other therapeutically active agents, if present in the pharmaceutical formulations/compositions
  • the amount of active ingredient may range from about 0.01% to about 99%, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30%, wherein the reminder is preferably composed of the one or more pharmaceutically acceptable excipients.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start with doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a composition of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect.
  • Such an effective dose will generally depend upon the factors described above.
  • Administration may carried out oral, by inhalation, intravenous, intramuscular, intraperitoneal, or subcutaneous, preferably administered proximal to the site of the target.
  • the effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more subdoses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation/composition.
  • the amount of active ingredient, e.g., a compound of the invention, in a unit dosage form and/or when administered to an indiviual or used in therapy, may range from about 0.1 mg to about 1000mg (for example, from about 1mg to about 500mg, such as from about 10mg to about 200mg) per unit, administration or therapy.
  • a suitable amount of such active ingredient may be calculated using the mass or body surface area of the individual, including amounts of between about 1mg/Kg and 10mg/Kg (such as between about 2mg/Kg and 5mg/Kg), or between about 1mg/m 2 and about 400mg/m 2 (such as between about 3mg/m 2 and about 350mg/m 2 or between about 10mg/m 2 and about 200mg/m 2 ).
  • the invention is directed to a composition for use as described above, formulated as an inhalative drug.
  • composition for use as described above is formulated as an oral drug.
  • Scheme 1 Synthetic route for the preparation of different 3,6-diaminoacridin-10-ium derivatives alkylated at the N10 position.
  • Scheme 2 Synthetic routes for the preparation of 3,6-diaminoacridin-10-ium derivatives that are dual alkylated at the N6 position.
  • Air and water sensitive reactions were performed in flame-dried glassware under an argon atmosphere.
  • Solvents used for column chromatography, extractions and recrystallization were purchased in technical grade and were distilled prior to use.
  • Solvents used for reversed phase chromatography and HPLC-MS analyses were purchased from Thermofisher Scientific in HPLC-quality. Reagents and dry solvents were purchased from Sigma Aldrich, ABCR, Alfa Aesar, Thermofisher Scientific, TCI, Carl Roth and Merck and were used without further purification.
  • Analytical thin layer chromatography was performed on silica coated plates (silica gel 60 F254) purchased from VWR. Compounds were detected by ultraviolet (UV) irradiation at 254 or 366 nm. Manual flash column chromatography was performed using silica gel 60 (particle size: 0.040-0.063 mm) available from VWR. Automated preparative chromatography was performed on a Grace Reveleris Prep purification system using linear gradient elution and Buchi Reveleris Silica 40 pm cartridges for normal-phase and Buchi Reveleris C1840 pm cartridges for reverse-phase separations.
  • AVHD400 or AVHD500 spectrometer operating at either 400 MHz or 500 MHz.
  • HPLC-UV/MS analyses were performed on a Dionex UltiMate 3000 HPLC system coupled with a Thermo ScientificTM ISQTM EC Single Quadrupole Mass Spectrometer, using the following methods: Thermo ScientificTM AccucoreTM RP-MS LC-column (2.1 x 50 mm, 2.6 pm); gradient method A): 5 to 95% of acetonitrile + 0.1% formic acid v/v in water + 0.1% formic acid v/v over 5 min period; flow rate: 0.6 mL/min; gradient method B): 5 to 10% of acetonitrile + 0.1% formic acid v/v in water + 0.1% formic acid v/v over 9.5 min period; 10 to 20% of acetonitrile + 0.1% formic acid v/v in water + 0.1% formic acid v/v over 4.0 min period; 20 to 95% of acetonitrile + 0.1% formic acid v/v in water + 0.1% for
  • Neutral proflavine (nPF) was obtained from commercially available proflavine hemisulfate monohydrate, which was purchased from Sigma Aldrich. The procedure is as follows: Proflavine hemisulfate monohydrate (2.00 g, 7.24 mmol) was dissolved in water (100 mL) and then aqueous ammonia (10%) was added until the pH reached a value of 8. The formed orange precipitate was filtered off, washed with water (3 x 20 mL) and dried in vacuo to yield neutral proflavine (nPF) (1.26 g, 6.02 mmol, 83%).
  • the resulting reaction mixture was allowed to reach room temperature and stirred for 15 h. After this time period, the reaction was quenched by pouring the mixture into an aqueous solution of NaHCO 3 (40%, 300 mL), which was then stored at 5 °C overnight. The following day, the obtained precipitate was filtered, washed with water (3 x 50 mL) and dried. The crude product was recrystallized from ethanol in order to yield pure pivalate amide protected proflavine TF 139 as a beige solid (1.52 g, 4.03 mmol, 48%).
  • TF 156 3,6-Diamino-10-ethylacridin-10-ium chloride (TF 156).
  • Pivalate protected diaminoacridine derivative TF 153 400 mg, 0.811 mmol, 1.0 eq
  • ethanol 10.0 mL
  • hydrochloric acid 6.0 M in H 2 O, 3.38 mL, 20.28 mmol, 25.0 eq
  • the resulting reaction mixture was heated until reflux and stirred overnight. The following day, the solvent was removed under reduced pressure and the crude material was taken up in a mixture of EtOH and dichloromethane and stored for 1 day at 8 °C.
  • the obtained deep red precipitate was filtered off, washed with Et 2 O (2 x 10.0 mL) and dried in vacuum.
  • the desired target compound TF 163 (106 mg, 0.338 mmol, 74%) exhibited a purity greater than 95% and therefore no further purification was necessary.
  • the vector map can be found at HMGU-PEPF website.
  • the plasmids were transformed into E. coli BL21 (DE3), and the transformed cells were cultured at 37 °C in terrific broth (TB) media containing 100 mg/L kanamycin. After the GD600 reached 2, the culture was cooled to 18 °C and supplemented with 0.25 mM IPTG.
  • the vector petM5a yielded more soluble protein and it was used for all protein expression.
  • preculture was gown in M9 minimal media followed by inoculation (OD 6 oo 0.05) into 1 L of D2O M9 minimal media supplemented with 15N-Ammoniun chloride.
  • GD600 reached 0.8
  • the culture was cooled to 18°C and supplemented with 0.25mM IPTG.
  • the cells were harvested through centrifugation, and the pellets were resuspended in lysis buffer (20 mM Tris-HCI, pH 8.5, 350 mM NaCI, 10% glycerol, 10mM imidazole, 5mM betamercaptoethanol) and sonicated at 4 °C.
  • the insoluble material was removed through centrifugation at 24,000 rpm.
  • the fusion protein was first purified by Ni-NTA affinity chromatography, the supernatant was applied to nickel resin and washed with 10 times the column volume with lysis buffer followed by a wash step of lysis buffer supplemented with 20mM imidazol.
  • the protein was eluted with 3 times column volume by a buffer containing high imidazole concentration, 20 mM Tris-HCI, pH 8.5, 350 mM NaCI, 5% glycerol, 350mM imidazole, 5mM beta-mercaptoethanol.
  • TEV protease 1mg was added and the solution was dialyzed overnight at 4°C against a buffer containing low imidazole concentration, 20 mM Tris- HCI, pH 8.5, 150 mM NaCI, 5% glycerol, 10mM imidazole, 1mM Beta mercaptoethanol. The next day, the protein was applied to a nickel column and the flow though was collected followed by concentration using a top centrifuge concentrator with a 30kDa cut off up to a volume of 2mL.
  • the protein was applied to a size exclusion chromatography column, High load S75 (GE- Helathcare, chigaco, USA), pre-equilibrated with the final buffer, 20mM Tris pH 8.0, 40mM NaCI and 2mM DTT. The purity of the protein was accessed by SDS page gel.
  • TEV and uncut M pro were removed by a second NiNTA purification step.
  • the protein was further purified by size exclusion chromatography (Superdex s75, GE Healthcare) in 50mM Tris pH 7.4, 150mM NaCI, 2mM P-mercaptoethanol.
  • the assay was designed to measure p
  • the assay buffer contained 50 mM Tris (pH 8.0), 0.01 % (w/v) BSA and 10 mM DTT.
  • RLRGG-AMC was used as fluorogenic substrate for PLPro 40 pl of PLPro protein (end concentration 60 nM) was incubated with 10 pl RLRGG-AMC substrate (end concentration 400 nM).
  • the assay final volume 50 pl was incubated for 30 min.
  • the release of AMC (Ex. 360 nm I Em. 487 nm) was measured on an Envision plate reader (Perkin Elmer, Waltham, MA).
  • the assay was designed to measure PL PRO protease activity under screening conditions in white 384-well Optiplates.
  • the assay buffer contained 50 mM Tris (pH 8.0), 0.01 % (w/v) BSA and 10 mM DTT.
  • RLRGG-AMC was used as fluorogenic substrate for PLPro 40 pl of PLPro protein (end concentration 60 nM) was incubated with 10 pl RLRGG-AMC substrate (end concentration 400 nM).
  • the assay final volume 50 pl was incubated for 30 min.
  • the release of AMC (Ex. 360 nm I Em. 487 nm) was measured on an Envision plate reader (Perkin Elmer, Waltham, MA).
  • RLRGG-AMC or ISG15-AMC was used as substrate for PL PRO and the release of AMC fluorescence was measured (Ex. /Em. 360/487 nm) on an Envision plate reader.
  • 40 pl of a 75 nM PLpro solution in assay buffer 50 mM Tris (pH 8,0), 0.01% (w/v) BSA and 10 mM DTT was pipeted into 384 well plates and different concentration of ACF (50 pM - 0 pM, final concentration) was added. The mixture was incubated for 1 hour at RT.
  • reaction was initiated by adding 10 pl of 2 pM RLRGG-AMC (400 nM final) or 10 pl of 0.5 pM ISG15-AMC (100 nM, final), respectively.
  • Initial velocities of AMC release were normalized to the DMSO control.
  • IC 5 o value was calculated using GraphPad Prism. The experiment was repeated three times.
  • RLRGG-AMC or ISG15-AMC was used as substrate for PL PRO and the release of AMC fluorescence was measured (Ex./Em. 360/487 nm) on an Envision plate reader.
  • 40 pl of a 75 nM PLpro solution in assay buffer 50 mM Tris (pH 8,0), 0.01% (w/v) BSA and 10 mM DTT was pipetted into 384 well plates and different concentration of ACF (50 pM - 0 pM, final concentration) was added. The mixture was incubated for 1 hour at RT.
  • reaction was initiated by adding 10 pl of 2 pM RLRGG-AMC (400 nM final) or 10 pl of 0.5 pM ISG15-AMC (100 nM, final), respectively.
  • Initial velocities of AMC release were normalized to the DMSO control.
  • IC 50 value was calculated using GraphPad Prism. The experiment was repeated three times.
  • Vero (Cercopithecus aethiops’ kidney epithelial; ATCC CCL-81), HRT-18 (ATCC CRL-11663) cells, derivative of HRT-18 (ileocecal colorectal adenocarcinoma; ATCC CCL-244), CRFK (Felis catus, kidney cortex; ATCC® CCL-94) were cultured in Dulbecco’s MEM (Thermo Fisher Scientific, Tru) supplemented with 5% fetal bovine serum (heat-inactivated; Thermo Fisher Scientific, Poland) and antibiotics: penicillin (100 U/ml), streptomycin (100 pg/ml), and ciprofloxacin (5 pg/ml).
  • A549 cells with ACE2 overexpression (A549 ACE2+ ) 31 were cultured in the same manner with supplementation with G418 (5 mg/ml; BioShop, Canada).
  • LLC-MK2 cells (ATCC CCL-7; Macaca mulatta kidney epithelial cells) were maintained in minimal essential medium (MEM; two parts Hanks' MEM and one part Earle's MEM [Life Technologies, Tru]) 5% fetal bovine serum (heat-inactivated; Thermo Fisher Scientific, Tru), penicillin (100 ll/rnl), streptomycin (100 ll/rnl ), and ciprofloxacin (5 pg/ml).
  • MEM minimal essential medium
  • fetal bovine serum heat-inactivated
  • penicillin 100 ll/rnl
  • streptomycin 100 ll/rnl
  • ciprofloxacin 5 pg/ml
  • HSF Primary human skin fibroblasts
  • MEM Thermo Fisher Scientific, Tru
  • fetal bovine serum heat-inactivated; Thermo Fisher Scientific, Poland
  • 1% nonessential amino acids Life Technologies
  • antibiotics penicillin (100 ll/rnl), streptomycin (100 pg/ml), and ciprofloxacin (5 pg/ml).
  • HAE Human airway epithelial
  • MucilAirTM- Bronchial (Epithelix Sari, Switzerland) HAE cultures were also used for the ex vivo analysis. MucilAirTM cultures were maintained as suggested by the provider in MucilAirTM culture medium.
  • SARS-CoV-2 strain used in the study was isolated in house and is designated PL_P07 [GISAID Clade G, Pangolin lineage B.1] (accession numbers for the GISAID database: hCoV-19/Poland/PL_P07/2020).
  • Reference SARS-CoV-2 strain 026V-03883 was kindly granted by Christian Drosten, Charite - Universitatstechnik Berlin, Germany by the European Virus Archive - Global (EVAg); https://www.european-virus-archive.com/).
  • All SARS-CoV-2 stocks were generated by infecting monolayers of Vero cells. The cells were incubated at 37 °C under 5% CO 2 . The virus-containing liquid was collected at day 2 post-infection (p.i.), aliquoted and stored at -80°C. Control samples from mock-infected cells was prepared in the same manner.
  • MERS-CoV stock (isolate England 1 , 1409231v, National Collection of Pathogenic Viruses, Public Health England, United Kingdom) was generated by infecting monolayers of Vero cells. The cells were incubated at 37°C under 5% CO 2 . The virus-containing liquid was collected at day 3 p.i., aliquoted and stored at -80°C. Control samples from mock- infected cells were prepared in the same manner.
  • FIPV stock strain 79-1146 was generated by infecting CRFK cells in 90% confluency. The cells were incubated at 37 °C under 5% CO 2 . The virus-containing liquid was collected at day 3 p.i., aliquoted and stored at -80°C. Control samples from mock-infected cells were prepared in the same manner.
  • the HCoV-NL63 stock (isolate Amsterdam 1) was generated by infecting monolayers of LLC-MK2 cells. The cells were incubated at 32 °C under 5% CO 2 and then lysed by two freeze-thaw cycles at 6 days p.i. The virus-containing liquid was aliquoted and stored at -80°C. A control LLC-MK2 cell lysate from mock-infected cells was prepared in the same manner.
  • the HCoV-OC43 stock (ATCC: VR-1558) was generated by infecting monolayers of HRT-18 cells. The cells were incubated at 32 °C under 5% CO 2 and then lysed by two freezethaw cycles at 5 days post-infection (p.i.). The virus-containing liquid was aliquoted and stored at -80°C. A control HRT-18G cell lysate from mock-infected cells was prepared in the same manner.
  • Virus yields were assessed by titration on fully confluent cells in 96-well plates, according to the method of Reed and Muench. Plates were incubated at 32°C or 37°C for times indicated above, and the cytopathic effect (CPE) was scored by observation under an inverted microscope.
  • CPE cytopathic effect
  • Cell viability was evaluated using the XTT Cell Viability Assay kit (Biological Industries, Cromwell, CT, USA) according to the manufacturer’s protocol. Vero, A549 ACE2+ , CRFK, HRT-18, LLC-MK2 and HSF cells were cultured on 96-well plates. Cells were incubated with ACF for 24 h at 37°C in an atmosphere containing 5% CO 2 . After incubation, the medium was discarded and 100 pL of fresh medium was added to each well.
  • Vero, A549 ACE2+ , CRFK, HRT-18, LLC-MK2 and HSF cells were cultured on 96-well plates. Cells were incubated with ACF for 24 h at 37°C in an atmosphere containing 5% CO 2 . After incubation, the medium was discarded and 100 pL of fresh medium was added to each well.
  • Vero cells were seeded in culture medium on 96-well plates (TPP, Trasadingen, Switzerland) at 2 days before infection. Subconfluent cells were infected with SARS-CoV-2 viruses at 1600 50% tissue culture infectious dose (TCID 50 )/mL. Infection was performed in the presence of 100 nM, 1 pM and 10 pM concentration of compounds listed in Table 2. After 2 h of incubation at 37°C, cells were rinsed twice with PBS and fresh medium without compounds was added. The infection was carried out for 48 h and the cytopathic effect (CPE) was assessed. Culture supernatants were collected from wells where CPE reduction was observed.
  • CPE cytopathic effect
  • SARS-CoV-2 virus at 5000 TCID 50 /ml in the presence of ACF, remdesivir or PBS.
  • Two concentrations of ACF (400 nM and 500 nM) and the controls were added to the apical side of the inserts followed by the addition of the virus diluted in PBS. Infection time was of 2 hours at 37°C. After the infection, the apical side of the HAE were washed three times with PBS and each compound was re-applied and incubated again for 30 minutes at 37°C. After the last incubation with the ACF, the samples (50pL) were collected and the HAE were left in air-liquid interphase. Every 24 hours the HAE were incubated for 30 minutes with the ACF dilutions or controls, and the samples were collected. After collecting last samples cells were fixed with 3.7% paraformaldehyde and stained as described below.
  • Virus yield was measured using the RT-qPCR method described below.
  • RNA was isolated according to the manufacturer’s instructions.
  • Viral RNA was quantified using quantitative PCR coupled with reverse transcription (RT-qPCR) (GoTaq Probe 1-Step RT-qPCR System, Promega, Poland) using CFX96 Touch real-time PCR detection system (Bio-Rad, Poland). The reaction was carried out in the presence of the probes and primers indicated in the Table 1. The heating scheme was as follows: 15 min at 45°C and 2 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 58°C or 60°C (specified in Table 1). In order to assess the copy number for the N gene, standards were prepared and serially diluted.
  • Vero cells were seeded on coverslips in 12-well plate (TPP, Trasadingen, Switzerland) at 2 days before experiment. ACF at 1 pM or DMSO at 0,01% concentration were applied on cells. After 1h cells were fixed with 3.7% paraformaldehyde (PFA) for 15 min. Fluorescent images were acquired under EVOS XL Core Imaging System.
  • A549 ACE2+ cells were seeded on coverslips in 12 well plate (TPP, Trasadingen,
  • Subconfluent cells were infected with SARS-CoV— 2 in the presence of ACF or remdesivir. After 2h infection unbound virions were washed off twice with PBS and fresh medium supplemented with compounds was added. The infection was carried out for 24 h whereupon cells were fixed with 3.7% paraformaldehyde (PFA) for 1 h. Fixed cells were permeabilized using 0,5% Tween-20 (10 min, room temperature [RT]) and unspecific binding sites were blocked with 5% bovine serum albumin (BSA) in PBS (4°C, overnight) prior to staining.
  • PFA paraformaldehyde
  • Fluorescent images were acquired under Zeiss LSM 880 confocal microscope.
  • Drug-resistant SARS-CoV-2 was obtained by serial passages of the virus in the presence of increasing concentrations of ACF or remdesivir, starting at a concentration equivalent to their IC 50 .
  • Vero cells were seeded in 12 well plate and infected with SARS-CoV-2 in the presence of the drug or PBS. When CPE was observed, samples were collected, aliquoted, frozen and used to infect cells for the next passage. Infection was repeated with increasing concentrations of the compound (IC 50 dose was doubled every 2 passages). After 5 passages, culture supernatants were collected, RNA was isolated and viral RNA was sequenced (NGS, Illumina). All experiments were carried out in triplicate.
  • Crystals of PL pro -proflavine complex grew at room temperature in 0.05 M Hepes sodium salt pH 7, 0.05M magnesium sulfate and 1.6 M lithium sulfate. Crystals suitable for testing were moved in cryo-protectant solution containing the harvesting solution supplemented with 25% (v/v) glycerol and snap frozen in liquid nitrogen.
  • PL pro -proflavine crystals were measured at Swiss Light Source (SLS, Villigen, Switzerland), beamline PXIII. The best dataset was collected at 2.7 A resolution and it was indexed and integrated using XDS software 34 ; scaled and merged using STARANISO webserver 35 . Crystal belongs to space group P6 5 22. Matthews coefficient analysis suggested the presence of two PL pro -proflavine molecules in the asymmetric unit. Molecular replacement solution was found using Phaser 36,37 and the apo PL pro structure (PDB code: 6W9C) as searching model. Model and restraints for proflavine was prepared using Lidia, the ligand builder in Coot 38 .
  • the initial model was subjected to several iterations of manual and automated refinement cycles using COOT and REFMAC5, respectively 39,40 . Throughout the refinement, 5% of the reflections were used for cross-validation analysis 41 , and the behavior of Rfree was employed to monitor the refinement strategy.
  • the commercial ACF is a mixture of 3,6-diaminoadridin-10-ium (proflavine) and
  • the NMR analysis is based on the fact that the protons of the methyl group at position 10 of acriflavine (ACF) have a very distinct chemical shift of 3.94 ppm (4.04 ppm for side methylated ACF), whereas the protons of the methyl group connected to the amino group at position 3, like it is the case in side methylated PF and ACF, have a much lower chemical shift of 2.87 and 2.98 ppm, respectively.
  • ACF acriflavine
  • the IC 50 of ACF with ISG15-AMC (1.46 pM) was comparable to the IC 50 that was determined with the RLRGG-AMC substrate.
  • AMC assays are fluorescence-based assays that are susceptible to autofluorescence or quenching effects of the compounds, further conducted gelbased de-ubiquiniting assays have been conducted to confirm the results.
  • the protease activity of PL pro and the inhibitory potential of ACF on tri-ubiquitin K48 chains have been tested. First, K48 tri-ubiqiutin has ben incubated with PL pro and samples of different time points were taken to analyze the cleavage of these chains in Western Blot assays.
  • the hit compounds were tested in cell culture and cytotoxicity was verified on Vero cells at three concentrations (100 nM, 1 pM, 10 pM) using the XTT assay. At the same time, the cytopathic effect (CPE) reduction assay was carried out. Amongst 13 compounds, three hampered the development of the cytopathic effect, but the initial RT-qPCR analysis revealed that only ACF inhibited virus replication ( Figure 2).
  • Table 3 CPE reduction assay. Initial screen of 13 proposed compounds in given concentrations. Table shows results of cytopathic effect (CPE) reduction assay obtained by microscopic observations. CPE - cytopathic effect; RED - CPE reduction; TOX - toxicity.
  • CPE cytopathic effect
  • NMR ligand-based binding analysis was carried out to validate PL pro -ligand interaction.
  • the direct interaction of ACF with PL pro was also observed by NMR ligand based assay (peaks of the ACF partially disappear in the presence of the protein) and the results are presented in Figure 3.
  • ACF is not a SARS-CoV-2 M pro inhibitor
  • the second molecule, Kir proflavine- II is TT-TT stacked at 3.5A on top of the other and occupies the S3 and S5 pockets ( Figure 4 C, D).
  • Gly163 and Asp164 form a hydrogen bond with the primary amine group at position 3 (2.9 A) and the imine group of the acridine moiety (2.9 A), respectively.
  • Tyr268 forms a T-shape TT-TT staked interaction (5.1 A) with this proflavine-ll.
  • this provides unique binding model where two proflavines, tightly TT-TT stacked, cooperate in blocking the substrate pocket. It is clear that, both molecules are requested for the inhibition.
  • the electron density analysis shows weaker trace of at least two more proflavines that can be allocated on top of the other two at optimal distance for TT-TT staking forming a continuous, DNA-like stacking from one to another PLpro molecule in the same asymmetric unit. Their electron density is much weaker and does not allow to build these molecules unambiguously ( Figure 17). However, they are not involved in any interaction with PL pro .
  • Shin et al. solved the crystal structure of SARS-CoV2-PL pro in complex with ISG15 (interferon-induced gene 15) bearing the RLRGG recognition motif at the C-terminus (1).
  • the amino group at position 3 of proflavine-l I is located in similar position as amide nitrogen of the glycine P2.
  • Side-methylated proflavine also present in commercial acriflavine preparations (see Figure 21), would, therefore, mimic the P2 amino acid.
  • Vero cells which are broadly used simian model and human A549 ACE2+ cells overexpressing the ACE2 receptor 43 . All experiments were carried out in parallel.
  • the IC 50 value calculated based on the presented data was 64 nM for the Vero cells and 86 nM for A549 ACE2+ cells.
  • Selectivity index (SI) values for Vero and A549 ACE2+ models are 53 and 36, respectively.
  • ACF hampers SARS-CoV-2 replication in the HAE ex vivo model.
  • a lower viral yield was detected in the cultures treated with ACF in comparison with the PBS control.
  • ACF treated HAE showed an even higher inhibition of virus replication than the positive control with 10 pM remdesivir after 144 h of infection.
  • the virus was under the detection limits in the ACF 500 nM treatment.
  • ACF may be used as a generic anticoronaviral drug
  • its activity against other betacoronaviruses (MERS-CoV, HCoV-OC43) and alphacoronaviruses (HCoV- NL63 and feline infectious peritonitis virus (FIPV) was tested.
  • IC 50 21 nM
  • SI 162
  • No effect on any of tested alphacoronaviruses replication in tested concentrations (Figure 14) was observed.
  • rhodesiense STIB 900 in 50 pL was added to each well and the plate incubated at 37 °C under a 5% CO 2 atmosphere for 70 h.
  • 10 pL resazurin solution (resazurin, 12.5 mg in 100 mL double-distilled water) was then added to each well and incubation continued for a further 2-4 h. 45 Then the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wave length of 536 nm and an emission wave length of 588 nm.
  • Rat skeletal myoblasts (L-6 cells) were seeded in 96-well microtitre plates at 2000 cells/well in 100 pL RPMI 1640 medium with 10% FBS and 2 mM L-glutamine. After 24 h the medium was removed and replaced by 100 pL per well containing 5000 trypomastigote forms of T. cruzi Tulahuen strain C2C4 containing the p-galactosidase (Lac Z) gene. 47 After 48 h the medium was removed from the wells and replaced by 100 pL fresh medium with or without a serial drug dilution of eleven 3-fold dilution steps covering a range from 100 to 0.002 pg/mL.
  • Amastigotes of L. donovani strain MHOM/ET/67/L82 are grown in axenic culture at 37 °C in SM medium 48 at pH 5.4 supplemented with 10% heat-inactivated fetal bovine serum under an atmosphere of 5% CO 2 in air.
  • One hundred microlitres of culture medium with 105 amastigotes from axenic culture with or without a serial drug dilution are seeded in 96-well microtitre plates.
  • Serial drug dilutions of eleven 3-fold dilution steps covering a range from 100 to 0.002 pg/mL are prepared. After 70 h of incubation the plates are inspected under an inverted microscope to assure growth of the controls and sterile conditions.
  • Assays were performed in 96-well microtiter plates, each well containing 100 pl of RPMI 1640 medium supplemented with 1 % L-glutamine (200 mM) and 10% fetal bovine serum, and 4000 L-6 cells (a primary cell line derived from rat skeletal myoblasts). 49 Serial drug dilutions of eleven 3-fold dilution steps covering a range from 100 to 0.002 pg/mL were prepared. After 70 h of incubation the plates were inspected under an inverted microscope to assure growth of the controls and sterile conditions. 10 pL of resazurin was then added to each well and the plates incubated for another 2 h.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Molecular Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

La présente invention concerne une composition comprenant au moins un composé selon la formule (I) ainsi qu'une composition comprenant au moins un composé selon la formule (I) et/ou des dimères de composés selon la formule (I) en particulier pour une utilisation dans le traitement de maladies provoquées par des bétacoronavirus, la leishmaniose et la trypanosomiase.
PCT/EP2021/085966 2020-12-15 2021-12-15 Inhibition de protéase virale Ceased WO2022129210A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP20214108 2020-12-15
EP20214108.1 2020-12-15
LU102310A LU102310B1 (en) 2020-12-17 2020-12-17 Inhibition of virus protease
LULU102310 2020-12-17

Publications (2)

Publication Number Publication Date
WO2022129210A2 true WO2022129210A2 (fr) 2022-06-23
WO2022129210A3 WO2022129210A3 (fr) 2022-07-28

Family

ID=79287793

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2021/085966 Ceased WO2022129210A2 (fr) 2020-12-15 2021-12-15 Inhibition de protéase virale

Country Status (1)

Country Link
WO (1) WO2022129210A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120118126A (zh) * 2025-05-13 2025-06-10 上海健康医学院 咔唑金属配合物及其制备方法和在制备抗新型冠状病毒的药物中的应用

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2711527A1 (fr) * 1993-10-22 1995-05-05 Richard Laboratoire M Utilisation de l'acriflavine comme agent anti HIV.
US7544712B1 (en) * 2003-05-28 2009-06-09 National Health Research Insitutes Treatment of coronavirus infection
US7601498B2 (en) * 2005-03-17 2009-10-13 Biotium, Inc. Methods of using dyes in association with nucleic acid staining or detection and associated technology
US7666886B2 (en) * 2005-07-15 2010-02-23 The Regents Of The University Of California Compounds and methods for the diagnosis and treatment of amyloid associated diseases
US9933417B2 (en) * 2014-04-01 2018-04-03 Howard Hughes Medical Institute Azetidine-substituted fluorescent compounds
KR102608609B1 (ko) * 2016-04-26 2023-12-04 에스에프씨 주식회사 아크리딘계 화합물, 이를 포함하는 생체분자 표지용 염료, 키트 및 조영제 조성물

Non-Patent Citations (68)

* Cited by examiner, † Cited by third party
Title
AHMED, S. A. ET AL., J. IMMUNOL. METHODS
ANURADHA, S., POONAM, PCHEM. BIOL. DRUG DES., vol. 90, 2017, pages 926
BALTZ, T. ET AL., EMBO JOURNAL, vol. 4, 1985, pages 1273 - 1277
BENCHABANE, Y.DI GIORGIO, C.BOYER, G.SABATIER, A. S.ALLEGRO, D.PEYROT, V.DE MEO, M., EUR. J. MED. CHEM., vol. 44, 2009, pages 2459 - 2467
BERNAL, F. A.KAISER, M.WUNSCH, B.SCHMIDT, T. J.: "Structure-Activity Relationships of Cinnamate Ester Analogues as Potent Antiprotozoal Agents", CHEMMEDCHEM, vol. 15, 2020, pages 68 - 78
BERNTHSEN, A., JUSTUS LIEBIGS ANN. CHEM., vol. 224, no. 1, 8 January 1984 (1984-01-08)
BROWNING, C. H.: "Aminoacridine Compounds as Surface Antiseptics", BRITISH MEDICAL, vol. 1, 1943, pages 341 - 343
BROWNING, C. H.: "Proflavine and acriflavine", BRITISH MEDICAL JOURNAL, vol. 2, 1967, pages 111 - 111
BRUNGER, A. T.: "Free R value: a novel statistical quantity for assessing the accuracy of crystal structures", NATURE, vol. 355, 1992, pages 472 - 475
BUCKNER, F. S. ET AL., ANTIMICROB. AGENTS CHEMOTHER, vol. 40, 1996, pages 2592 - 2597
CLINICAL ASSESSMENT OF URINARY ANTISEPTICS METHENAMINE AND METHYLTHIONINIUM IN, 2020, Retrieved from the Internet <URL:https://clinicaltrials.gov/ct2/show/NCT03379389>
CORSELLO, S. M. ET AL.: "The Drug Repurposing Hub: a next-generation drug library and information resource", NAT MED, vol. 23, 2017, pages 405 - 408
CUNNIGHAM, I., J. PROTOZOOL., vol. 24, 1977, pages 325 - 329
DANA, S. ET AL.: "Potent antimalarial activity of acriflavine in vitro and in vivo", ACS CHEM BIOL, vol. 9, 2014, pages 2366 - 2373
DROSTEN, C. ET AL.: "Identification of a novel coronavirus in patients with severe acute respiratory syndrome", N ENGL J MED, vol. 348, 2003, pages 1967 - 1976, XP002288120, DOI: 10.1056/NEJMoa030747
ELDHO, N. V.SAMINATHAN, M.RAMAIAH, D., SYNTH. COMMUN., vol. 29, 1999, pages 4007
EMSLEY, P.: "Tools for ligand validation in Coot", ACTA CRYSTALLOGR D STRUCT BIOL, vol. 73, 2017, pages 203 - 210
EMSLEY, P.COWTAN, K.: "Coot: model-building tools for molecular graphics", ACTA, 2004, pages 2126 - 2132
FROHLICH, T.HAHN, F.BELMUDES, L.LEIDENBERGER, M.FRIEDRICH, O.KAPPES, B.COUTE, YMARSCHALL, M.TSOGOEVA, S. B., CHEM. EUR. J., vol. 24, 2018, pages 8103 - 8113
FROHLICH, T.REITER, C.SAEED, M. E. M.HUTTERER, C.HAHN, F.LEIDENBERGER, M.FRIEDRICH, O.KAPPES, B.MARSCHALL, M.EFFERTH, T., ACS MED. CHEM. LETT., vol. 9, 2017, pages 534 - 539
FUNATSUKI, K. ET AL.: "acrB mutation located at carboxyl-terminal region of gyrase B subunit reduces DNA binding of DNA gyrase", J BIOL CHEM, vol. 272, 1997, pages 13302 - 13308
GENSICKA-KOWALEWSKA MCHOLEWINSKI GDZIERZBICKA, K, RSC ADV, vol. 7, 2017, pages 15776
GRABOYES, H.ANDERSON, E. L.LEVINSON, S. H.RESNICK, T. M., J. HETEROCYCL. CHEM., vol. 12, 1975, pages 1225
HORBY, P. ET AL.: "Effect of Hydroxychloroquine in Hospitalized Patients with Covid-19", N ENGL J MED, vol. 383, 2020, pages 2030 - 2040
HUBER, W. ET AL., ACTA TROPICA, vol. 55, 1993, pages 257 - 261
JAWDOSIUK, M.CZYZEWSKI, J.MAKOSZA, M.: "Chem. Commun.", J. CHEM. SOC., 1973, pages 794
KABSCH, W., ACTA CRYSTALLOGR D BIOL CRYSTALLOGR, vol. 66, 2010, pages 125 - 132
KAWAI, M.YAMAGISHI, J.: "Mechanisms of action of acriflavine: electron microscopic study of cell wall changes induced in Staphylococcus aureus by acriflavine", MICROBIOL IMMUNOL, vol. 53, 2009, pages 481 - 486
KISHIMOTO, M.KONDO,K.AKITA, M.YOSHIZAWA, M., CHEM. COMMUN., vol. 53, 2017, pages 1425
KSIAZEK, T. G. ET AL.: "A novel coronavirus associated with severe acute respiratory syndrome", N ENGL J MED, vol. 348, 2003, pages 1953 - 1966, XP002486657, DOI: 10.1056/NEJMoa030781
LACOBUCCI, G.: "Covid-19: Risk of death more than doubled in people who also had flu, English data show", BMJ, vol. 370, 2020, pages m3720
LEE, K.: "Acriflavine inhibits HIF-1 dimerization, tumor growth, and vascularization", PROC NATL ACAD SCI U S A, vol. 106, 2009, pages 17910 - 17915, XP055450319, DOI: 10.1073/pnas.0909353106
LU, D. Y. ET AL.: "HAART in HIV/AIDS Treatments: Future Trends", INFECT DISORD DRUG TARGETS, vol. 18, 2018, pages 15 - 22
MA, L. ET AL.: "Coinfection of SARS-CoV-2 and Other Respiratory Pathogens", INFECT DRUG RESIST, vol. 13, 2020, pages 3045 - 3053
MAHMOUD, A. B., DANTON, O., KAISER, M., KHALID, S., HAMBURGER, M., MASER, P.: "HPLC-Based Activity Profiling for Antiprotozoal Compounds Cuscuta hyalina. Front. Pharmacol. ", CUSCUTA HYALINA. FRONT. PHARMACOL., vol. 11, 2020, pages 1246
MANCHESTER, T.CAVALCANTI, D. P.ZOGOVICH, M.W, D. E. S.MOTTA, M. C.: "Acriflavine treatment promotes dyskinetoplasty in Trypanosoma cruzi as revealed by ultrastructural analysis", PARASITOLOGY, vol. 140, 2013, pages 1422 - 1431
MATEJOVA, MJANOVEC, LIMRICH, J, ARKIVOC, vol. 134, 2015
MATHE, G: "The non-enumerable described retrovirus integrase inhibitors are not a lure, as evidenced by ten years of clinical experience", BIOMEDICINE & PHARMACOTHERAPY, vol. 54, 2000, pages 3 - 6, Retrieved from the Internet <URL:https://doi.orq/10.1016/S0753-3322(00)88633-3>
MAYER, F.FREUND, W., BER. DTSCH. CHEM. GES., vol. 55, 1922, pages 2049
MCCOY, A. J.: "Phaser crystallographic software", J APPL CRYSTALLOGR, vol. 40, 2007, pages 658 - 674
MCCOY, A. J.: "Solving structures of protein complexes by molecular replacement with Phaser", ACTA CRYSTALLOGR D BIOL CRYSTALLOGR, vol. 63, 2007, pages 32 - 41
MILEWSKA, A. ET AL.: "HTCC as a Polymeric Inhibitor of SARS-CoV-2 and MERS-CoV", J VIROL, 2020
MILEWSKA, A. ET AL.: "Human coronavirus NL63 utilizes heparan sulfate proteoglycans for attachment to target cells", J VIROL, vol. 88, 2014, pages 13221 - 13230
MIRZAEI, R. ET AL.: "Bacterial co-infections with SARS-CoV-2", IUBMB LIFE, 2020
MURSHUDOV, G. N.: "REFMAC5 for the refinement of macromolecular crystal structures", ACTA CRYSTALLOGR D BIOL CRYSTALLOGR, vol. 67, 2011, pages 355 - 367
NAKAJIMA, M.NAGASAWA, S.MATSUMOTO, K.KURIBARA, T.MURANAKA, A.UCHIYAMA, M.NEMOTO, T., ANGEW. CHEM. INT. ED., vol. 59, 2020, pages 6847
NIETLISPACH, D.: "Suppression of anti-TROSY lines in a sensitivity enhanced gradient selection TROSY scheme", J BIOMOL NMR, vol. 31, 2005, pages 161 - 166, XP019249516
ORHAN, I.$ENER, B.KAISER, M.BRUN, R.TASDEMIR, D.: "Inhibitory activity of marine sponge-derived natural products against parasitic protozoa", MAR. DRUGS, vol. 8, 2010, pages 47 - 58
PAGE, B. ET AL., INT. J. ONCOL., vol. 3, 1993, pages 473 - 476
PAN, H. ET AL.: "Repurposed Antiviral Drugs for Covid-19 - Interim WHO Solidarity Trial Results", N ENGL J MED, vol. 382, 2020, pages 1787 - 1799
PEPIN, G.: "Activation of cGAS-dependent antiviral responses by DNA intercalating agents", NUCLEIC ACIDS RES, vol. 45, 2017, pages 198 - 205
PERSINOTI, G. F. ET AL.: "RNA-sequencing analysis of Trichophyton rubrum transcriptome in response to sublethal doses of acriflavine", BMC GENOMICS, vol. 15, 2014, pages S1, XP021200699, DOI: 10.1186/1471-2164-15-S7-S1
PERVUSHIN, K.RIEK, R.WIDER, G.WUTHRICH, K.: "Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution", PROC NATL ACAD SCI U S A, vol. 94, 1997, pages 12366 - 12371, XP002108361, DOI: 10.1073/pnas.94.23.12366
POULALLION, P.GALY, J.-P.VINCENT, E.-J.GALY, A.-M.BARBE, J.ATASSI, G. J., HETEROCYCLIC CHEM, vol. 23, 1986, pages 1141
PRAGER, R. H.WILLIAMS, C. M., SCIENCE OF SYNTHESIS, vol. 15, 2005, pages 1029
RATIA, K.: "Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme", PROC NATL ACAD SCI U S A, vol. 103, 2006, pages 5717 - 5722
RAZ, B. ET AL., ACTA TROPICA, vol. 68, 1997, pages 139 - 147
SAHA, R. P. ET AL.: "Repurposing Drugs, Ongoing Vaccine, and New Therapeutic Development Initiatives Against COVID-19", FRONT PHARMACOL, vol. 11, 2020, pages 1258, XP055873445, DOI: 10.3389/fphar.2020.01258
SHIN, D. ET AL.: "Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity", NATURE, vol. 587, 2020, pages 657 - 662, XP055845080, DOI: 10.1038/s41586-020-2601-5
SIMONOVICH, V. A. ET AL.: "A Randomized Trial of Convalescent Plasma in Covid-19 Severe Pneumonia", N ENGL J MED, 2020
TICKLE, I. J. ET AL.: "STARANISO", 2018, GLOBAL PHASING LTD
TRIPATHI, R. P. ET AL.: "Search of antitubercular activities in tetrahydroacridines: synthesis and biological evaluation", BIOORG MED CHEM LETT, vol. 16, 2006, pages 5144 - 5147, XP025107232, DOI: 10.1016/j.bmcl.2006.07.025
ULLMANN, F.TORRE, A. L., BER. DTSCH. CHEM. GES., vol. 37, 1904, pages 2922
VERMA, M.: "A short translational ramp determines the efficiency of protein synthesis", NATURE COMMUNICATIONS, vol. 10, 2019, pages 5774
WAINWRIGHT, M: "Acridine-a neglected antibacterial chromophore", J ANTIMICROB CHEMOTHER, vol. 47, 2001, pages 1 - 13, XP002715987, DOI: 10.1093/jac/47.1.1
YANG, H.: "Design of wide-spectrum inhibitors targeting coronavirus main proteases", PLOS BIOI, vol. 3, 2005, pages e324
ZAKI, A. M.VAN BOHEEMEN, S.BESTEBROER, T. M.OSTERHAUS, A. D.FOUCHIER, R. A.: "Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia", N ENGL J MED, vol. 367, 2012, pages 1814 - 1820, XP055274765, DOI: 10.1056/NEJMoa1211721
ZHANG, Y.WERLING, U.EDELMANN, W.: "Seamless Ligation Cloning Extract (SLiCE) cloning method", METHODS MOL BIOL, vol. 1116, 2014, pages 235 - 244

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120118126A (zh) * 2025-05-13 2025-06-10 上海健康医学院 咔唑金属配合物及其制备方法和在制备抗新型冠状病毒的药物中的应用

Also Published As

Publication number Publication date
WO2022129210A3 (fr) 2022-07-28

Similar Documents

Publication Publication Date Title
EP3428170B1 (fr) Dérivé de pyridone polycyclique anti-grippal et promédicament de celui-ci
Nimesh et al. Synthesis and biological evaluation of novel bisbenzimidazoles as Escherichia coli topoisomerase IA inhibitors and potential antibacterial agents
AU2014350729B2 (en) N-benzyl tryptanthrin derivative, and preparation method and application thereof
JP2015524457A (ja) ジヒドロキシピリミジン炭酸誘導体及びウイルス性疾患の治療、改善又は予防における使用
WO2014074926A1 (fr) Hydroxyquinolones thérapeutiques
JP2019196390A (ja) 非アポトーシス性の制御された細胞死の阻害剤としてのスピロキノキサリン誘導体
US12358897B2 (en) Compounds and method of treating COVID-19
US10011598B2 (en) Small molecules targeting repeat r(CGG) sequences
WO2022129210A2 (fr) Inhibition de protéase virale
CA2959548C (fr) Agents antiviraux et leurs utilisations
AU2019393784B2 (en) Compounds for the treatment of arenavirus infection
JP7261529B1 (ja) ウイルス増殖阻害作用を有するトリアジン誘導体の製造方法
Wu et al. Synthesis of Pyrazine‐1, 3‐thiazine Hybrid Analogues as Antiviral Agent Against HIV‐1, Influenza A (H1N1), Enterovirus 71 (EV 71), and Coxsackievirus B3 (CVB 3)
JP2016504376A (ja) ナフチリジノン誘導体及びウイルス性疾患の治療、改善又は予防におけるそれらの使用
WO2023048151A1 (fr) Peptide cyclique présentant une activité inhibitrice de la prolifération virale
AU2017348434B2 (en) Peripheral modifications on pocket-redesigned vancomycin analogs synergistically improve antimicrobial potency and durability
WO2017046362A1 (fr) Pyrazolopyrazines et leur utilisation dans le traitement, le soulagement ou la prévention d&#39;une maladie virale
CN115160301A (zh) 一种山荷叶素衍生物、其制备方法及用途
EP3826717A2 (fr) Dérivés d&#39;oxynitidine utiles comme inhibiteurs de la topoisomérase ib (top1) et de la tyrosyl-adn phosphodiestérase 1 (tdp1)
Khachatryan et al. Novel derivatives of thiohydantoin‐containing tetrahydro‐β‐carboline possess activity against influenza virus at late stages of viral cycle without affecting viral neuraminidase
EP3039012A1 (fr) Inhibiteurs d&#39;histone désacétylase à base de dérivés de polyhydroacridine tricyclique et d&#39;analogues possédant des cycles à cinq et sept éléments saturés et fusionnés
US20240366540A1 (en) Mitofusin inhibitors and uses thereof
US20240352013A1 (en) Bicyclic fused pyrazole derivatives for the treatment of respiratory infections including rsv
Aydın et al. Chemical Structure and Synthesis of Baloxavir Marboxil, A Novel Endonuclease Inhibitor For The Treatment Influenza: An Overview
LU102310B1 (en) Inhibition of virus protease

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21839911

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21839911

Country of ref document: EP

Kind code of ref document: A2