WO2015018797A2 - Antiviral compounds - Google Patents

Antiviral compounds Download PDF

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WO2015018797A2
WO2015018797A2 PCT/EP2014/066747 EP2014066747W WO2015018797A2 WO 2015018797 A2 WO2015018797 A2 WO 2015018797A2 EP 2014066747 W EP2014066747 W EP 2014066747W WO 2015018797 A2 WO2015018797 A2 WO 2015018797A2
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compound
alkyl
screening
alkinyl
alkenyl
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PCT/EP2014/066747
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French (fr)
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WO2015018797A3 (en
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Tilmann BÜRCKSTÜMMER
Sejla SALIC
Wolfgang Fischl
Stefan Kubicek
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Haplogen Gmbh
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    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • 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
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • 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
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/498—Pyrazines or piperazines ortho- and peri-condensed with carbocyclic ring systems, e.g. quinoxaline, phenazine
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/527—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim spiro-condensed
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • 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
    • 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 provides compounds for the treatment of viral infections. BACKGROUND
  • Picornaviruses represent a major health burden, yet antiviral treatments are not available.
  • PLA2G16 is a poorly characterized phospholipase A2 whose natural phospholipid substrate remains unknown. Earlier studies suggested a role for PLA2G16 as a suppressor of HRAS signaling (hence its alternative name: HRAS-like suppressor 3; [1 ]), but these data remain poorly documented. More recent studies show that PLA2G16-deficient mice are resistant to high fat diet-induced obesity [2], implying a functional role of PLA2G16 in lipid metabolism.
  • PLA2G16 is a host factor that is shared by many - if not all - Picornaviruses.
  • PLA2G16 was recombinantly expressed in E. coli, purified by affinity chromatography and used for a biochemical assay in which phospholipase A2 activity was read out.
  • the present invention provides compounds for the treatment of viral infections.
  • the invention relates to compounds of general formula I,
  • X is N or C
  • Y is N or C
  • each R-i is independently from one another selected from the group consisting of halogen, -OH, -CN, -NO 2 , -OCH 3 , -CH 2 NH 2 , -NR x R y , -C 1 -3 alkyl, -C 2 - 3 alkenyl, -C 1 -3 alkyl -C 2-3 alkinyl, -NHCH 2 CH 2 OCH 3 , -NHCH 2 CH 2 OH, -SCH 3 , or is selected from a group consisting of halogen, -OH, -CN, -NO 2 , -OCH 3 , -CH 2 NH 2 , -NR x R y , -C 1 -3 alkyl, -C 2 - 3 alkenyl, -C 1 -3 alkyl -C 2-3 alkinyl, -NHCH 2 CH 2 OCH 3 , -NHCH 2 CH 2 OH, -SCH 3 , or is selected from
  • each R x and R y is independently from one another hydrogen or C -3 alkyl, -C 2 . 3 alkenyl, and -C 2 . 3 alkinyl;
  • Ri represents S or NH
  • R 3 represnts N-R y , wherein R y is hydrogen, C -3 alkyl, -C 2-3 alkenyl, -C 2 - 3 alkinyl, or -Ci -4 alkyl-COOH, -Ci- 3 alkyl-0-CH 3 , or optionally with halogen substituted phenyl; and R 4 represents halogen, -N0 2 , Ci -3 alkyl, -C 2 -3alkenyl, -C 2-3 alkinyl, or -OCH 3 , and
  • R 5 represents -OH or -OCH 3 and R 6 is absent
  • R 6 represents an optionally substituted phenyl, wherein the substituents are selected from the group consisting of halogen, -NO 2 , -CF 3 , Ci -3 alkyl, -C 2-3 alkenyl, -C 2-3 alkinyl,
  • R 6 represents naphthyl, Cs-ecycloalkyl, morpholinyl or an optionally substituted pyridinyl, wherein the substituents are selected from the group consisting of halogen, -NO 2 , -CF 3 , C -3 alkyl, -C 2-3 alkenyl, and -C 2-3 alkinyl; or
  • each R-i represents independently from one another halogen, OH, Ci -3 alkyl,
  • each R 2 represents independently from one another halogen, OH,CN, C -3 alkyl, or -COOH;
  • n 0, 1 or 2;
  • Ri represents hydrogen or is selected from the group consisting of
  • Ri represents phenyl, optionally substituted with halogen, -N0 2 , -CF 3 , -NH 2 ,
  • R 2 represents cyclopentenyl or cyclohexenyl
  • a further aspect of the invention is a compound as described above, wherein said compound is a compound of general formula I, preferably a compound selected from the group consisting of D1562, D1716, D1709 and D1708.
  • a further aspect of the invention is a compound as described above, wherein said compound is a compound of general formula II, preferably a compound selected from the group consisting of D1528, D1380, D1532, D1531 , D1529, D1482, D1483, D1477, and D1514.
  • a further aspect of the invention is a compound as described above, wherein said compound is a compound of general formula III, preferably a compound selected from the group consisting of D1547, D1636 and D1664.
  • a further aspect of the invention is a compound as described above, wherein said compound is a compound of general formula IV, preferably D1549.
  • a further aspect of the invention is a compound as described above, wherein said compound is a compound of general formula V, preferably a compound selected from the group consisting ofD1551 , D1580, D1591 and D1583.
  • a further aspect of the invention is a compound as described above for use in the treatment of Picorna virus infections.
  • a further aspect of the invention is the use as described above, wherein the
  • Picornavirus is a Poliovirus, a coxsackievirus, a Rhinovirus or an
  • a further aspect of the invention is a compound as described above for use as
  • a further aspect of the invention is a pharmaceutical preparation, containing as active substance one or more compounds of general formula I, II, III, IV, or V as describe above, or the pharmacologically effective salts thereof, optionally in combination with conventional excipients and/or carriers.
  • Figure 1 A sensitive assay for Poliovirus infection
  • FIG. 1 A sensitive assay for Poliovirus infection
  • Figure 5 A convenient assay for Coxsackievirus B3 (CVB3) using eGFP-CVB3
  • Figure 6 D1562 inhibits Coxsackievirus B3
  • Compound D1380 was identified as an inhibitor with an IC 5 o of approximately 7 ⁇ .
  • the active moiety is presumably the rhodanine (2-thioxothiazolidin-4-one) part.
  • the compound When changed to a Thiobarbituric acid, the compound completely loses activity Scheme 1 ).
  • Compound D1547 inhibits PLA2G16 with an IC 50 of approximately 5 ⁇ .
  • This compound consists of a chalcone (benzalacetophenone) backbone, a class with several important bioactive members.
  • Compound D1549 is a 1 -(1 -(2-(7-ethyl-1 H-indol-3-yl)-2-oxoethyl)piperidin-4-yl)- 1 H-benzo[d]imidazol-2(7aH)-one, a relative large compound with a MW > 400. Some analogs have been tested for this scaffold (Scheme 5).
  • D1552 is a spirooxindole, and 2 related spirooxindoles retain activity (Scheme Commercially available analogs of D1552 are depicted in Scheme 9.
  • Compound D1562 is a dihydroquinolinyl-quinoxaline.
  • the compound is small, synthetically accessible and very potent (-3-5 ⁇ ). It is poor in heteroatoms and both ring systems are unsubstituted.
  • D171 1 showed an IC 5 o of ⁇ 5 ⁇ .
  • CID 5308002 is a 100 nM activator of 5'UTR Stem- Loop Driven Alpha-Synuclein mRNA Translation in H4 Neuroglioblastoma Cells
  • a cDNA for PLA2G16 was custom-synthesized by Genscript (Piscataway, USA) and inserted into a pET-based bacterial expression vector [Moffatt, B.A. and Studier, F.W. (1986) J. Mol. Biol. 189, 1 13-130; Rosenberg, A.H., Lade, B.N., Chui, D., Lin, S., Dunn, J.J., and Studier, F.W. (1987) Gene 56, 125-135], enabling the expression of N-terminally Hexa-His-tagged PLA2G16.
  • Genscript Proliferative bacterial expression vector
  • coli BL21 (DE3) (Agilent) were transformed with pET-His-PLA2G16 according to the manufacturer's instructions and plated on Carbenicillin-containing agar plates (Carbenicillin 100 ⁇ 9/ ⁇ ).
  • Carbenicillin 100 ⁇ 9/ ⁇ A single clone was inoculated in 100 ml LB medium supplemented with 100 ⁇ g/ml Carbenicillin and the inoculated culture was grown at 37° C over night in a rotary shaker.
  • the bacterial culture was diluted 1 :40 in LB medium supplemented with 100 ⁇ g/ml Carbenicillin (50 ml overnight culture in 2 L total culture volume). Bacteria were grown at 30 °C until the OD(600) reaches 0.5.
  • Gene expression was induced by addition of 1 mM isopropyl- -D-thiogalactopyranosid (Fermentas) for 1 h at 20 °C. Bacteria were harvested by centrifugation (5 min; 8,000 x g; 4 °C) and frozen at -80 °C.
  • Bacteria were resuspended in Buffer A (50 mM Tris/HCI pH 7.5, 500 mM NaCI, 5% glycerol, 5 mM ⁇ -Mercaptoethanol and 1 mM PMSF) and lysed by addition of 1 mg/ml lysozyme (Sigma Aldrich) and 100 ⁇ g/ml DNasel (Roche). The sample was incubated at 37 °C for 15 min to allow lysis to occur. Then, the lysate was cleared by centrifugation and incubated with 0.5 ml Ni-NTA agarose suspension (Qiagen) for 15 min. The Ni-NTA agarose was washed with 10 ml Buffer A. Bound protein was eluted by applying a manual gradient of Buffer A supplemented with increasing
  • Red/Green BODIPY® PC-A2 (1 mM) was mixed with equal volumes of DOPC and DOPG (1 :1 :1 ) and vortexed for 5s.
  • the mixture (Red/Green BODIPY® PC- A2/DOPC/DOPG) was diluted in activity assay buffer (dilution 1 :50) using a small- orifice pipette tip under constant vortexing. In the following, this preparation will be referred to as the "substrate solution”.
  • the substrate solution is mixed with different dilutions of the active enzyme. Fluorescence (excitation 480 nm/ emission 530 nm) is recorded over time (between 5-60 min.). The relative increase over time is referred to as the enzyme activity.
  • HeLa cells (ATCC number CCL-2) were seeded in 6-well dishes (5x10 5 cells/well) and incubated at 37 °C over night. The next day, cells were pretreated with the indicated small molecules at 25 ⁇ for 30 min. Next, cells were infected with Coxsackievirus B1 (CVB1 ) at an MOI of 5 TCID 50 /cell for 1 h. Then, cells were washed once with medium to remove input virus and medium containing small molecules was replenished. Cells were washed once with PBS and harvested 6 h post infection.
  • CVB1 Coxsackievirus B1
  • the total cellular RNA was extracted using the SV Total RNA Isolation Kit (Promega) and subjected to coupled reverse transcription and quantitative PCR using the SensiMixTM SYBR No-ROX OneStep Kit (Bioline). The following sets of oligonucleotides were used for qRT-PCR:
  • RNA samples were seeded in 6-well dishes (5x10 5 cells/well) and infected with Picornaviruses (Poliovirus typel (Mahoney strain) or Coxsackievirus B1 ) for 1 h. Then, cells were washed with PBS to remove input virus and the medium (DMEM, 10% FCS) was replenished. Cells and/or supernatants were collected 6-16 h post infection. Viral RNA was isolated using suitable kits (SV Total RNA Isolation Kit (Promega) for cellular RNA; QIAamp Viral RNA Mini Kit (Qiagen) for viral RNA in the supernatant). Viral RNAs were quantified in a coupled reverse transcription/qPCR reaction using the SensiMixTM SYBR No-ROX OneStep Kit (Bioline) (Fig. 1 A).
  • Poliovirus oligonucleotides Poliovirus oligonucleotides:
  • Hela cells (ATCC CCL-2) were seeded in 6-well dishes (5x10 5 cells/well) and treated with 25 ⁇ of the indicated small molecule inhibitors (D1547, D1549, D1551 , D1562) for 30 min. Cells were then infected with Poliovirus type 1 (Mahoney strain; MOI of 1 TCID 50 /cell) for 1 h. Then, cells were washed with PBS to remove input virus and the medium (DMEM, 10% FCS) containing small molecule inhibitors was replenished. Supernatants were collected 8 h post infection.
  • Poliovirus type 1 Poliovirus type 1
  • DMEM 10% FCS
  • Viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen) and quantified in a coupled reverse transcription/qPCR reaction using the SensiMixTM SYBR No-ROX OneStep Kit (Bioline) using the following oligonucleotides as described above (Fig. 2).
  • HeLa cells (ATCC CCL-2) were seeded in 96-well plates (20,000 cells/well) and treated with the indicated small molecule inhibitors (D1547, D1549, D1551 , D1562) at the indicated concentrations for 48 h. Cell viability was assessed using the Cell TiterGlo Luminescent Cell Viability Assay (Promega) according to manufacturer's instructions (Fig. 3).
  • Coxsackievirus B1 oligonucleotides
  • A549 cells (ATCC CCL-185) were treated with the indicated small molecules at 25 ⁇ concentration and infected with Coxsackievirus B1 (MOI 1 ) for 1 h. Cells were washed with PBS and harvested 16 h post infection. Total RNA was extracted using the SV Total RNA Isolation Kit (Promega) and quantified in a coupled reverse transcription/qPCR reaction using the SensiMixTM SYBR No-ROX OneStep Kit (Bioline) using the oligonucleotides as described above (HG2494 and HG2495) (Fig. 7B). Quenching Assay
  • a 1 0 mM fluorescein (3261 5, FLUKA) stock solution in DMSO was prepared. 1 ⁇ of 1 0 mM fluorescein stock solution was diluted in 50 ml of the reaction buffer (50 mM Tris/HCI, 1 00 mM NaCI pH 8.9). 50 ⁇ were pipetted into each well and 5 ⁇ of compound added to each well.
  • the reaction buffer 50 mM Tris/HCI, 1 00 mM NaCI pH 8.9

Abstract

The present invention provides compounds for the treatment of viral infections.

Description

Antiviral Compounds
The present invention provides compounds for the treatment of viral infections. BACKGROUND
Picornaviruses represent a major health burden, yet antiviral treatments are not available. We used gene-trap mutagenized human haploid cells for a screen to identify picornaviral host dependency factors. Knockout of such genes confers resistance to Picornavirus infection. Those host factors represent attractive drug targets as the virus is heavily dependent on them and drug resistant viral variants are less likely to occur.
A screen for gene knockouts that confer resistance to Poliovirus infection yielded the Poliovirus receptor (PVR) and the phospholipase PLA2G16 as major host factors in human cells. PLA2G16 is a poorly characterized phospholipase A2 whose natural phospholipid substrate remains unknown. Earlier studies suggested a role for PLA2G16 as a suppressor of HRAS signaling (hence its alternative name: HRAS-like suppressor 3; [1 ]), but these data remain poorly documented. More recent studies show that PLA2G16-deficient mice are resistant to high fat diet-induced obesity [2], implying a functional role of PLA2G16 in lipid metabolism.
Human cells lacking PLA2G16 are largely resistant to Poliovirus infection.
Interestingly, these cells also loose susceptibility to all other Picornaviruses tested so far (Coxsackieviruses, Rhinoviruses, Encephalomyocarditis virus), but not to Non- Picornaviruses such as Vesicular Stomatitis Virus, Influenza Virus, Adenovirus or Herpes Simplex Virus. This indicates that PLA2G16 is a host factor that is shared by many - if not all - Picornaviruses. Reconstitution of PLA2G16-deficient cells with PLA2G16 wild-type, but not with a catalytically inactive mutant, restores susceptibility to Picornavirus infection, indicating that the enzymatic activity of PLA2G16 is required for Picornavirus infection. These findings were summarized in patent application WO201 1/160043.
Based on these observations, a drug discovery program was initiated to find small molecule inhibitors of PLA2G16. PLA2G16 was recombinantly expressed in E. coli, purified by affinity chromatography and used for a biochemical assay in which phospholipase A2 activity was read out. The screen of 24,000 unrelated and highly diverse compounds yielded several primary hits, of which six were selected for further optimization. DETAILED DESCRIPTION OF THE INVENTION
The present invention provides compounds for the treatment of viral infections. In one aspects the invention relates to compounds of general formula I,
Com ounds of general formula I,
Figure imgf000003_0001
wherein
X is N or C, and
Y is N or C, and
each R-i is independently from one another selected from the group consisting of halogen, -OH, -CN, -NO2, -OCH3, -CH2NH2, -NRxRy, -C1 -3alkyl, -C2-3alkenyl, -C1 -3alkyl -C2-3alkinyl, -NHCH2CH2OCH3, -NHCH2CH2OH, -SCH3, or is selected from a group
Figure imgf000003_0002
each R2 is independently from one another selected from the group consisting of H, -NH2, -OCH3, -OCH2CH3, -NO2, -NRxRy, -NHC(=O)CH3, -Ci-3alkyl, -C2.3alkenyl, and -C2.3alkinyl; and
each Rx and Ry is independently from one another hydrogen or C -3alkyl, -C2.3alkenyl, and -C2.3alkinyl; and
Rz is hydrogen, halogen, C1 -3alkyl, -C2.3alkenyl, -C2.3alkinyl, or -NHC(=O)CH3; n is 0, 1 or 2, or
compounds of general formula II
Figure imgf000004_0001
wherein
Ri represents S or NH, and
R2 represents =S or NRX, wherein Rx is Cs-ecycloalkyl, and
R3 represnts N-Ry, wherein Ry is hydrogen, C -3alkyl, -C2-3alkenyl, -C2-3alkinyl, or -Ci-4alkyl-COOH, -Ci-3alkyl-0-CH3, or optionally with halogen substituted phenyl; and R4 represents halogen, -N02, Ci-3alkyl, -C2-3alkenyl, -C2-3alkinyl, or -OCH3, and
R5 represents S, O, 0-CH2, 0-(CH2)2, 0-CH2-C(=0); or
R5 represents -OH or -OCH3 and R6 is absent; and
R6 represents an optionally substituted phenyl, wherein the substituents are selected from the group consisting of halogen, -NO2, -CF3, Ci-3alkyl, -C2-3alkenyl, -C2-3alkinyl,
Figure imgf000004_0002
or R6 represents naphthyl, Cs-ecycloalkyl, morpholinyl or an optionally substituted pyridinyl, wherein the substituents are selected from the group consisting of halogen, -NO2, -CF3, C -3alkyl, -C2-3alkenyl, and -C2-3alkinyl; or
compounds of general formula III
Figure imgf000004_0003
wherein
each R-i represents independently from one another halogen, OH, Ci-3alkyl,
-C2-3alkenyl, -C2-3alkinyl, or
Figure imgf000004_0004
and
each R2 represents independently from one another halogen, OH,CN, C -3alkyl, or -COOH; and
n is 0, 1 or 2; or
compounds of general formula IV
Figure imgf000005_0001
wherein
Ri represents hydrogen or is selected from the group consisting of
Figure imgf000005_0002
H and H ; or
l formula V
Figure imgf000005_0003
(V) wherein
Ri represents phenyl, optionally substituted with halogen, -N02, -CF3, -NH2,
-N(CH3)2, Ci-3alkyl, -C2-3alkenyl, or -C2-3alkinyl, or thiophenyl, optionally substituted with halogen; and
R2 represents cyclopentenyl or cyclohexenyl; and
R3 represents hydrogen, -CHO, =N-NH2, =N-0-CH3, cyclopentenyl, or is selected from the roup consisting of
Figure imgf000005_0004
Figure imgf000006_0001
for use in the treatment of viral infections.
A further aspect of the invention is a compound as described above, wherein said compound is a compound of general formula I, preferably a compound selected from the group consisting of D1562, D1716, D1709 and D1708.
A further aspect of the invention is a compound as described above, wherein said compound is a compound of general formula II, preferably a compound selected from the group consisting of D1528, D1380, D1532, D1531 , D1529, D1482, D1483, D1477, and D1514.
A further aspect of the invention is a compound as described above, wherein said compound is a compound of general formula III, preferably a compound selected from the group consisting of D1547, D1636 and D1664.
A further aspect of the invention is a compound as described above, wherein said compound is a compound of general formula IV, preferably D1549.
A further aspect of the invention is a compound as described above, wherein said compound is a compound of general formula V, preferably a compound selected from the group consisting ofD1551 , D1580, D1591 and D1583.
A further aspect of the invention is a compound as described above for use in the treatment of Picorna virus infections.
A further aspect of the invention is the use as described above, wherein the
Picornavirus is a Poliovirus, a coxsackievirus, a Rhinovirus or an
Encephalomyocarditis virus.
A further aspect of the invention is a compound as described above for use as
PLA2G16 inhibitor.
A further aspect of the invention is a pharmaceutical preparation, containing as active substance one or more compounds of general formula I, II, III, IV, or V as describe above, or the pharmacologically effective salts thereof, optionally in combination with conventional excipients and/or carriers.
SHORT DESCRIPTION OF THE FIGURES
Figure 1 : A sensitive assay for Poliovirus infection
Figure 2: A sensitive assay for Poliovirus infection
Figure 3: Selected compounds inhibiting Poliovirus Figure 4: Cell viability assay for selected compounds
Figure 5: A convenient assay for Coxsackievirus B3 (CVB3) using eGFP-CVB3 Figure 6: D1562 inhibits Coxsackievirus B3
Figure 7: Activity of selected compounds on HeLa cells
EXAMPLES
High Throughput Screening
So far, 23,640 Compounds have been screened at the PLACEBO Platform for inhibition of PLA2G16. Compounds originate from chemical diversity libraries (1 ,000 compounds Otava, 20,000 compounds Enamine); focused libraries for kinases (287 compounds) chromatin modifiers (891 compounds); the NIH clinical collection (723 compounds); chemist-contributed compounds and compounds selected from literature and virtual approaches.
Typically compounds were tested in a single replicate at a concentration of 25 μΜ in a commercial PLA2 activity assay (EnzChek Phospholipase A2 assay kit, Invitrogen). Hit compounds that inhibited PLA2G16 at this concentration were first confirmed in the original assay. Confirmed hits were then tested for false positives in a quenching assay followed by an 8-point dose response against PLA2G16 and bee venom phospholipase as control.
For compounds that appeared selective, hit expansion was done by obtaining commercially available analogs. This resulted in structure-activity relationships for several of the series, described below.
D1380/D1528 series
Structure activity relationship
Compound D1380 was identified as an inhibitor with an IC5o of approximately 7 μΜ. The active moiety is presumably the rhodanine (2-thioxothiazolidin-4-one) part. When changed to a Thiobarbituric acid, the compound completely loses activity Scheme 1 ).
Figure imgf000007_0001
D1478 inactive Scheme 1 : Rhodanine as active group in D1380/D1528.
A series of 17 analogs resulted in clear SAR (Scheme 2): Different substitutions on the 2 phenyl-rings are permissible, whereas the rhodanine thiooxo group appears essential. Interestingly, substitution of the rhodanine nitrogen with an acidic group (but not hydrophobic group) is possible. Compound D1528 has an IC5o of approximately 4 μΜ.
Figure imgf000009_0001
Scheme 2: D1380/D1528 analogs tested. The active moiety of D1380 and D1528 is presumably the rhodanine (2- thioxothiazolidin-4-one) part.
Figure imgf000010_0001
Scheme 3: Commercially available analogs of D1380/D1528 D1547 series
Compound D1547 inhibits PLA2G16 with an IC50 of approximately 5 μΜ. This compound consists of a chalcone (benzalacetophenone) backbone, a class with several important bioactive members.
There is some SAR with a limited set of analogs (Scheme 4).
Figure imgf000011_0001
IC50 : 5 μΜ IC50 : 20 μΜ Inactive
Figure imgf000011_0002
Com ound
Figure imgf000011_0003
Scheme 4: SAR of D1547 analogs D1549 series
Compound D1549 is a 1 -(1 -(2-(7-ethyl-1 H-indol-3-yl)-2-oxoethyl)piperidin-4-yl)- 1 H-benzo[d]imidazol-2(7aH)-one, a relative large compound with a MW > 400. Some analogs have been tested for this scaffold (Scheme 5).
Compound IC50
Figure imgf000012_0001
Scheme 5: SAR of D1549 analogs
Additionally following commercially available compounds (Scheme 6) are tested.
Figure imgf000013_0001
Scheme 6: Commercially available D1549 analogs
Compound D1551 and analogs
Compound D1551 , (E)-4-(3-(4-bromobenzylidene)-[1 ,1 '-bi(cyclopentane)]-1 ,1 '- dien-2-yl)morpholine, is a highly conjugated system. The SAR series (Scheme 7) have been tested.
D1551 D1580/D1611 D1572/1605
Figure imgf000014_0001
Compound R-| R2 R3 Compound R^ R2 R3
Figure imgf000014_0002
Scheme 7: SAR of D1551 analogs
D1552 series
D1552 is a spirooxindole, and 2 related spirooxindoles retain activity (Scheme Commercially available analogs of D1552 are depicted in Scheme 9.
Figure imgf000015_0001
D1552 543745-35-3 897091
Scheme 9: Commercially available D1552 analogs
Compound D1562 and analogs
Compound D1562 is a dihydroquinolinyl-quinoxaline. The compound is small, synthetically accessible and very potent (-3-5 μΜ). It is poor in heteroatoms and both ring systems are unsubstituted. D171 1 showed an IC5o of < 5 μΜ.
Figure imgf000015_0002
So far, more than 100 compounds related to D1562 have been tested (overview Scheme 1 1 ). Direct substitutions of the ring systems have only been tested on the 3- position of the quinoxaline and the 3- and 5-positions of the dihydroquinoline (Scheme 12). Changing the backbone from a quinoxaline to a quinazoline gives active compounds (Scheme 12). One of the few actives identified from other backbones is D1668 which is more potent than the original hit (Scheme 10).
Figure imgf000016_0001
Scheme 10: Overview of SAR data for D1562 analogs
Figure imgf000017_0001
IC50 : 3 μΜ IC50 : 30 μΜ inactive
Figure imgf000017_0002
2-(3,4-dihydroisoquinolin-2(1/-/)-yl)quinoxaline
2-quinoxaline dihydroisoquinoline 2-quinoxaline dihydroisoquinoline e
Figure imgf000017_0003
Scheme 1 1 : D1562 analogs tested
Figure imgf000018_0001
Figure imgf000018_0002
2-(3,4-dihydroisoquinolin-2(1 /-/)-yl)quinazoline
2-quinazoline substituent dihydroisoquinoline substituent
Figure imgf000018_0003
- wNH2
D1705/ H
N none
D1706
X>H
D1707 4- none
H
Figure imgf000018_0004
Scheme 1 2: Quinazoline derivatives of D1562
In total 547 dihydroquinolinyl-quinoxalines (analogs of D1562) have been published, however, most (502) are derivatives having a fused ring system.
Figure imgf000018_0005
HP002P
Figure imgf000019_0001
Scheme 13: 43 compounds containing a dihydroisoquinoliny-quinoxalin structure currently in the literature.
For D1668, 335 derivatives are in the literature, of which 258 are commercially available.
One related compound (CID 5308002) is a 100 nM activator of 5'UTR Stem- Loop Driven Alpha-Synuclein mRNA Translation in H4 Neuroglioblastoma Cells
Interestingly, 2 related compounds (CID 1932237=D1614, CID
2096996=D1620) have been described as hits in screens for human phospholipase A2
(http://pubchem.ncbi.nlm.nih.qov/assav/assay.cqi?aid=588400) with IC5oS in the 10 μΜ range. These c PLA2G16
Figure imgf000020_0001
Albano uil CID 5308002
Figure imgf000020_0002
CID 1932237 CID 2096996
Scheme 14: Biologically active analogs of D1562/D1668
Commercial analogs are depicted in Scheme 15.
Figure imgf000021_0001
2-quinoxaline dihydroisoquinoline
862197-63-5 3-CI none
1212328-42-1 none 3-CONH2
4-OH, 5,8-di-OMe
Figure imgf000021_0002
Figure imgf000021_0003
2-quinoline dihydroisoquinoline 2-quinoline dihydroisoquinoline
1240875-85-7 3- CN none
1154583-56-8
603970-52-1 3-CN, 6,7-diOme none
938120-70-8 3-CH2NH2 none 1031567-58-4 6-
Figure imgf000021_0004
di-OMe NH2
Figure imgf000021_0005
418774-75-1 4-Me, 7-CI none
418782-81-7 4-Me, 7-OMe none
418782-52-2 4-Me, 8-OMe 6,7-di-OMe
418772-90-4 4-Me, 5, 7-di-OMe none
418785-89-4 4-Me, 5,8-di-OMe none
418780-99-1 4-Me, 5, 8-di-OMe 6,7-di-OMe
1332613-14-5 4-Me, 6,7-di-OMe 6,7-di-OEt
418782-29-3 4-Me, 5,6,7-triOMe none
Scheme 15: Commercial D1562/D1668 analogs Purification and activity assay for PLA2G16
A cDNA for PLA2G16 (NM_007069.3) was custom-synthesized by Genscript (Piscataway, USA) and inserted into a pET-based bacterial expression vector [Moffatt, B.A. and Studier, F.W. (1986) J. Mol. Biol. 189, 1 13-130; Rosenberg, A.H., Lade, B.N., Chui, D., Lin, S., Dunn, J.J., and Studier, F.W. (1987) Gene 56, 125-135], enabling the expression of N-terminally Hexa-His-tagged PLA2G16. E. coli BL21 (DE3) (Agilent) were transformed with pET-His-PLA2G16 according to the manufacturer's instructions and plated on Carbenicillin-containing agar plates (Carbenicillin 100 μ9/ιηΙ). A single clone was inoculated in 100 ml LB medium supplemented with 100 μg/ml Carbenicillin and the inoculated culture was grown at 37° C over night in a rotary shaker. On the next day, the bacterial culture was diluted 1 :40 in LB medium supplemented with 100 μg/ml Carbenicillin (50 ml overnight culture in 2 L total culture volume). Bacteria were grown at 30 °C until the OD(600) reaches 0.5. Gene expression was induced by addition of 1 mM isopropyl- -D-thiogalactopyranosid (Fermentas) for 1 h at 20 °C. Bacteria were harvested by centrifugation (5 min; 8,000 x g; 4 °C) and frozen at -80 °C.
Bacteria were resuspended in Buffer A (50 mM Tris/HCI pH 7.5, 500 mM NaCI, 5% glycerol, 5 mM β-Mercaptoethanol and 1 mM PMSF) and lysed by addition of 1 mg/ml lysozyme (Sigma Aldrich) and 100 μg/ml DNasel (Roche). The sample was incubated at 37 °C for 15 min to allow lysis to occur. Then, the lysate was cleared by centrifugation and incubated with 0.5 ml Ni-NTA agarose suspension (Qiagen) for 15 min. The Ni-NTA agarose was washed with 10 ml Buffer A. Bound protein was eluted by applying a manual gradient of Buffer A supplemented with increasing
concentrations of imidazole (Sigma Aldrich; 25/50/75/100/250 mM). Purified fractions were analyzed by Coomassie staining to assess the purity. Activity of the protein was assessed as follows:
Stock solutions:
1 mM Red/Green BODIPY® PC-A2 (Invitrogen) in DMSO
10 mM Dioleoylphosphatidylcholine (DOPC; Sigma Aldrich) in Ethanol
10 mM Dioleoylphosphatidylglycerol (DOPG; Sigma Aldrich) in Ethanol
5x Activity Assay Buffer (250 mM Tris/HCI pH 8.9, 500 mM NaCI)
Red/Green BODIPY® PC-A2 (1 mM) was mixed with equal volumes of DOPC and DOPG (1 :1 :1 ) and vortexed for 5s. The mixture (Red/Green BODIPY® PC- A2/DOPC/DOPG) was diluted in activity assay buffer (dilution 1 :50) using a small- orifice pipette tip under constant vortexing. In the following, this preparation will be referred to as the "substrate solution".
For the kinetic measurement, the substrate solution is mixed with different dilutions of the active enzyme. Fluorescence (excitation 480 nm/ emission 530 nm) is recorded over time (between 5-60 min.). The relative increase over time is referred to as the enzyme activity.
In order to evaluate their inhibitory potential, different small molecule inhibitors were dissolved at 10 mM concentration in DMSO. To assess their inhibitory potential, they were added to the enzyme (PT or BV) in different concentrations (20 μΜ, 10 μΜ, 5 μΜ, 2.5 μΜ, 1 .3 μΜ, 0.6 μΜ, 0.3 μΜ) before addition of the substrate solution. The small molecule inhibitor concentration at which inhibition is half-maximal is referred to
Infection model for Coxsackievirus B1
HeLa cells (ATCC number CCL-2) were seeded in 6-well dishes (5x105 cells/well) and incubated at 37 °C over night. The next day, cells were pretreated with the indicated small molecules at 25 μΜ for 30 min. Next, cells were infected with Coxsackievirus B1 (CVB1 ) at an MOI of 5 TCID50/cell for 1 h. Then, cells were washed once with medium to remove input virus and medium containing small molecules was replenished. Cells were washed once with PBS and harvested 6 h post infection. The total cellular RNA was extracted using the SV Total RNA Isolation Kit (Promega) and subjected to coupled reverse transcription and quantitative PCR using the SensiMix™ SYBR No-ROX OneStep Kit (Bioline). The following sets of oligonucleotides were used for qRT-PCR:
CVB1
Forward: 5'-TCCTCCGGCCCCTGAATG-3' (HG2494)
Reverse: 5'-GAAACACGGACACCCAAAGTA-3' ( HG2495)
GAPDH
Forward: 5'-GAAGGTGAAGGTCGGAGT-3' (HG2538)
Reverse: 5'-GAAGATGGTGATGGGATTTC-3' (HG2539)
A sensitive assay for Picornavirus replication
(A) Cells (HeLa, ATCC CCL-2; A549, ATCC CCL-185) were seeded in 6-well dishes (5x105 cells/well) and infected with Picornaviruses (Poliovirus typel (Mahoney strain) or Coxsackievirus B1 ) for 1 h. Then, cells were washed with PBS to remove input virus and the medium (DMEM, 10% FCS) was replenished. Cells and/or supernatants were collected 6-16 h post infection. Viral RNA was isolated using suitable kits (SV Total RNA Isolation Kit (Promega) for cellular RNA; QIAamp Viral RNA Mini Kit (Qiagen) for viral RNA in the supernatant). Viral RNAs were quantified in a coupled reverse transcription/qPCR reaction using the SensiMix™ SYBR No-ROX OneStep Kit (Bioline) (Fig. 1 A).
(B) HeLa cells (ATCC CCL-2) were infected with Poliovirus type 1 (Mahoney strain) for 1 h. Supernatants were collected at different time points post infection. Viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen) and quantified in a coupled reverse transcription/qPCR reaction using the SensiMix™ SYBR No-ROX OneStep Kit (Bioline) using the following oligonucleotides (Fig. 1 B):
Poliovirus oligonucleotides:
Forward (HG1404): 5'-CCACTGGCTTCAGTGTTT-3'
Reverse (HG1405): 5'-AGGTCAGATGCTTGAAAGC-3'
An assay for small molecule inhibitors of PLA2G 6 inhibiting Poliovirus infection
Hela cells (ATCC CCL-2) were seeded in 6-well dishes (5x105 cells/well) and treated with 25 μΜ of the indicated small molecule inhibitors (D1547, D1549, D1551 , D1562) for 30 min. Cells were then infected with Poliovirus type 1 (Mahoney strain; MOI of 1 TCID50/cell) for 1 h. Then, cells were washed with PBS to remove input virus and the medium (DMEM, 10% FCS) containing small molecule inhibitors was replenished. Supernatants were collected 8 h post infection. Viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen) and quantified in a coupled reverse transcription/qPCR reaction using the SensiMix™ SYBR No-ROX OneStep Kit (Bioline) using the following oligonucleotides as described above (Fig. 2).
Determining the cytotoxicity of small molecule inhibitors of PLA2G 6
HeLa cells (ATCC CCL-2) were seeded in 96-well plates (20,000 cells/well) and treated with the indicated small molecule inhibitors (D1547, D1549, D1551 , D1562) at the indicated concentrations for 48 h. Cell viability was assessed using the Cell TiterGlo Luminescent Cell Viability Assay (Promega) according to manufacturer's instructions (Fig. 3).
Structure-activity relationship for D1562
(A) Purified PLA2G16 or Bee Venom-PLA2 (BV-PLA2) were incubated with the indicated small molecules at 20 μΜ concentration and enzymatic activity was measured as described under Materials & Methods (Fig. 4A). (B) HeLa cells were treated with the indicated small molecules (Fig. 4B) and infected with Poliovirus as described above.
Structure-activity relationship for D1562
(A) Schematic representation (Wang et al., Antiviral Res. 2012 Feb;93(2):270- 9): HeLa cells were infected with recombinant Coxsackievirus B3 expressing eGFP for 16 h. Cells were analyzed for GFP expression by fluorescence microscopy (Fig. 5A).
(B) HeLa cells were treated with the indicated small molecules for 30 min and subsequently infected with recombinant recombinant Coxsackievirus B3 expressing eGFP for 16 h. GFP fluorescence, indicative of a productive viral infection, was visualized by microscopy (Fig. 5B). All images were taken with identical settings to properly reflect differences in viral infection.
Structure-activity relationship for D1562
(A) Structures of active and inactive members of the D1562 series, as determined by PLA2G16 enzymatic activity assays (Fig. 6A).
(B) HeLa cells were treated with the indicated small molecules at 25 μΜ concentration and infected with Coxsackievirus B1 (MOI 10 TCID50/cell) for 1 h. Cells were washed with PBS and harvested 8 h post infection. Total RNA was extracted using the SV Total RNA Isolation Kit (Promega) and quantified in a coupled reverse transcription/qPCR reaction using the SensiMix™ SYBR No-ROX OneStep Kit (Bioline) using the following oligonucleotides (Fig. 6B):
Coxsackievirus B1 oligonucleotides:
Forward: 5'-TCCTCCGGCCCCTGAATG-3' (HG2494)
Reverse: 5'-GAAACACGGACACCCAAAGTA-3' (HG2495)
Structure-activity relationship for D1528
(A) Structures of active and inactive members of the D1528 series, as determined by PLA2G16 enzymatic activity assays (Fig. 7A).
(B) A549 cells (ATCC CCL-185) were treated with the indicated small molecules at 25 μΜ concentration and infected with Coxsackievirus B1 (MOI 1 ) for 1 h. Cells were washed with PBS and harvested 16 h post infection. Total RNA was extracted using the SV Total RNA Isolation Kit (Promega) and quantified in a coupled reverse transcription/qPCR reaction using the SensiMix™ SYBR No-ROX OneStep Kit (Bioline) using the oligonucleotides as described above (HG2494 and HG2495) (Fig. 7B). Quenching Assay
A 1 0 mM fluorescein (3261 5, FLUKA) stock solution in DMSO was prepared. 1 μΙ of 1 0 mM fluorescein stock solution was diluted in 50 ml of the reaction buffer (50 mM Tris/HCI, 1 00 mM NaCI pH 8.9). 50 μΙ were pipetted into each well and 5 μΙ of compound added to each well.
Measurement settings: - Endpoint
- Excitation : 485 nm/ Emission : 530 nm
Commercial Analogs of D1 528:
CASNr Vendor CatNr
1055978-16-9 AKos Building Blocks Product List AKOS000415539
Aurora Screening Library K00.914.100
ChemBridge Screening Library 5715013
ChemDiv Screening Collection 8008-1864
Interchim Screening Library 5715013
Ryan Scientific High Throughput Screening
Compound Library A1683/0071864
TimTec Compound Collection ST079219
Vitas-M Laboratory Screening Collection STK075526
1055977-25-7 AKos Screening Library AKOS005105968
Aurora Screening Library K02.535.035
Interchim Screening Library JS-0858
Ryan Scientific High Throughput Screening
Compound Library JS-0858
TimTec Compound Collection ST019140
939238-45-6 AKos Screening Library AKOS002608961
Aurora Screening Library K03.375.685
883053-88-1 Aurora Screening Library K02.249.1 19
Interchim Screening Library XAX00168
Maybridge Screening Collection XAX00168
Ryan Scientific High Throughput Screening
Compound Library XAX00168
882073-18-9 AKos Screening Library AKOS005108502
Aurora Screening Library K02.039.060
DiscoveryCPR from Aldrich R126578
Interchim Screening Library JS-2192
Ryan Scientific High Throughput Screening
Compound Library JS-2192
330203-03-7 AKos Building Blocks Product List AKOS000409001
Aurora Screening Library K04.818.739
ChemDiv Screening Collection 2531 -0026
Interchim Screening Library STK031034
Princeton Express Stock OSSK_334048
Ryan Scientific High Throughput Screening
Compound Library STK031034
Vitas-M Laboratory Screening Collection STK031034 292172-84-0 AKos Building Blocks Product List AKOS000409184
Aurora Screening Library K04.817.177
ChemDiv Screening Collection 3229-1640
Interchim Screening Library AK-968/36945014
Ryan Scientific High Throughput Screening
Compound Library AK-968/36945014
Specs Compounds For Screening AK-968/36945014
286860-13-7 AKos Screening Library AKOS005108529
Aurora Screening Library K02.039.061
DiscoveryCPR from Aldrich R126586
Interchim Screening Library JS-2193
Maybridge Screening Collection XAX00138
Ryan Scientific High Throughput Screening
Compound Library JS-2193
204322-27-0 Aurora Screening Library K02.039.059
DiscoveryCPR from Aldrich R 126543
Interchim Screening Library XAX00152
Maybridge Screening Collection XAX00152
Ryan Scientific High Throughput Screening
Compound Library XAX00152
168548-99-0 AKos Building Blocks Product List AKOS001710203
Aurora Screening Library K01 .105.392
ChemDiv Screening Collection 8006-4639
Chemical Block Stock Library A1 178/0054639
Interchim Screening Library A1 178/0054639
Ryan Scientific High Throughput Screening
Compound Library A1 178/0054639
TimTec Compound Collection ST026957
Vitas-M Laboratory Screening Collection STK754177
81 154-09-8 AKos Building Blocks Product List AKOS000307455
Aurora Building Blocks A00.784.751
ChemBridge Building Block Library 3018324
ChemDiv Stock Building Blocks BB55-4495
DiscoveryCPR from Aldrich R61 1522
Innovapharm Stock Screening Compounds STT-00128392
Ryan Scientific Intermediate and Building
Block Compounds B018324
Vitas-M Laboratory Stock Building Blocks BBL017195
34709-44-9 DiscoveryCPR from Aldrich S21513
2Daybiochem Product List DAYB24650
Commercial Analogs of D1 549
CASNr Vendor CatNr
924438-53-9 Ambinter Stock Screening Collection Amb486170
Aurora Screening Library K02.685.192 Enamine Advanced HTS Collection Z126942084 Ryan Scientific High Throughput Screening T5629556 Compound Library
924193-52-2 Ambinter Stock Screening Collection Amb489901
Aurora Screening Library K02.675.573
Ryan Scientific High Throughput Screening
Compound Library T56461 15
1 158067-18-5 Ambinter Stock Screening Collection Amb9663682
Aurora Building Blocks A01 .270.248
Ryan Scientific Intermediate and Building Block BBV- Compounds 27271900
1252191 -94-8 Ambinter Stock Screening Collection Amb15719501
Aurora Screening Library K08.240.468 919949-21 -6 Ambinter Stock Screening Collection Amb491213
Aurora Screening Library K02.708.257
Enamine HTS Collection Z130193342
Ryan Scientific High Throughput Screening
Compound Library T5651 197
Commercial Analogs of D1 552
CASNr Vendor CatNr
543745-35-3 Ambinter Stock Screening Collection Amb9678130
Aurora Screening Library K05.927.812 Bionet Screening and Fragments Library NA-0701
Ryan Scientific Fragment Compound Library NA-0701
897091 -78-0 Aurora Screening Library K00.794.199
Commercial Analogs of D1 562
CASNr Vendor CatNr
862197-63-5 Ambinter Stock Screening Collection Amb3914249
1212328-42-1 Ambinter Stock Screening Collection Amb10663629
Aurora Screening Library K08.247.132
Enamine HTS Collection Z728964882
1 197625-78-7 Ambinter Stock Screening Collection Amb10628537
Aurora Screening Library K07.102.854
Enamine HTS Collection Z246381930
131527-22-5 AKos Building Blocks Product List AKOS001643008
Ambinter Stock Screening Collection Amb610730
Aurora Screening Library K00.425.465
ChemDiv Screening Collection 4282-0018
Interbioscreen Compound Library STOCK2S-01 105
Vitas-M Laboratory Screening Collection STK835195
1240875-85-7 Ambinter Stock Screening Collection Amb1 1202581
Aurora Screening Library K08.403.864
Enamine HTS Collection Z954440076
1332599-62-8 APAC Pharmaceutical Product List 602950
1 1 15897-47-6 AKos Building Blocks Product List AKOS002140124
Ambinter Stock Screening Collection Amb16454727 Aurora Screening Library K06.108.633
ChemDiv Screening Collection L759-0053
1082550-22-8 Ambinter Stock Screening Collection Amb17072797
Aurora Building Blocks A00.619.820
1018516-32-9 Ambinter Stock Screening Collection Amb17050528
Aurora Building Blocks A00.588.500
1018263-65-4 Ambinter Stock Screening Collection Amb17050505
Aurora Building Blocks A00.588.477
938120-70-8 Ambinter Stock Screening Collection Amb5759928
Aurora Building Blocks A00.672.072
Otava Building Blocks 7020618017
92461 1 -46-1 Ambinter Stock Screening Collection Amb7703333
Aurora Screening Library K02.181 .810
924591 -07-1 Ambinter Stock Screening Collection Amb7702443
Aurora Screening Library K02.178.838
924561 -07-9 Ambinter Stock Screening Collection Amb7702822
Aurora Screening Library K02.175.051
864430-96-6 Ambinter Stock Screening Collection Amb10818019
603970-52-1 AKos Screening Library AKOS000757138
Ambinter Stock Screening Collection Amb9098292
Aurora Screening Library K01 .741 .566
418791 -78-3 Ambinter Stock Screening Collection Amb10818257
418785-73-6 Ambinter Stock Screening Collection Amb10818102
Aurora Screening Library K1 1 .142.621
ChemBridge Screening Library 5564707
Interchim Screening Library 5564707
418782-81 -7 Ambinter Stock Screening Collection Amb10817963
418782-52-2 Ambinter Stock Screening Collection Amb10817953
418774-75-1 Ambinter Stock Screening Collection Amb1 1033935
Aurora Screening Library K1 1 .142.469
ChemBridge Screening Library 5537763
Interchim Screening Library 5537763
418772-90-4 Ambinter Stock Screening Collection Amb10817609
Aurora Screening Library K1 1 .142.439
ChemBridge Screening Library 5533370
Interchim Screening Library 5533370
353466-23-6 AKos Screening Library AKOS005440244
Ambinter Stock Screening Collection Amb3360442
Aurora Screening Library K01 .910.373
Interchim Screening Library STK332693
TimTec Compound Collection ST45059302
Vitas-M Laboratory Screening Collection STK332693
332181 -58-5 APAC Pharmaceutical Product List 602951
300573-66-4 Ambinter Stock Screening Collection Amb341706
Aurora Screening Library K00.1 19.877
Enamine HTS Collection Z31 194745 1332613-14-5 APAC Pharmaceutical Product List 653234
1225834-49-0 Ambinter Stock Screening Collection Amb17322225
Aurora Building Blocks A02.336.01 1
1 179062-75-9 Aurora Building Blocks A01 .722.744
Ryan Scientific Intermediate and Building Block
Compounds BBV-32300070
ZereneX Molecular Building Blocks ZXW041049
1 172278-23-7 Ambinter Stock Screening Collection Amb9980689
1 171562-83-6 Ambinter Stock Screening Collection Amb9980726
1 170944-57-6 Ambinter Stock Screening Collection Amb9978085
1 170939-17-9 Ambinter Stock Screening Collection Amb9978084
1 154583-56-8 Ambinter Stock Screening Collection Amb9310132
Aurora Building Blocks A00.916.729
Ryan Scientific Intermediate and Building Block
Compounds BBV-22164965
1031567-58-4 AKos Building Blocks Product List AKOS004944799
Aurora Screening Library K04.933.249
ChemDiv Screening Collection F052-0086
731012-34-3 Ambinter Stock Screening Collection Amb62162
Aurora Screening Library K00.062.536
418785-89-4 Ambinter Stock Screening Collection Amb10818104
418782-29-3 Ambinter Stock Screening Collection Amb1081791 1
ChemBridge Screening Library 5553336
418780-99-1 Ambinter Stock Screening Collection Amb10817897
ChemBridge Screening Library 5552015
302785-76-8 AKos Screening Library AKOS005708920
Ambinter Stock Screening Collection Amb745576
Aurora Screening Library K02.267.021
Vitas-M Laboratory Screening Collection STL055824
Ryan Scientific High Throughput Screening
Compound Library STOCK4S-85803

Claims

Claims:
1 . Com ounds of general formula I,
Figure imgf000031_0001
wherein
X is N or C, and
Y is N or C, and
each Ri is independently from one another selected from the group consisting of halogen, -OH, -CN, -NO2, -OCH3, -CH2NH2, -NRxRy, -Ci-3alkyl, -C2-3alkenyl, -Ci-3alkyl -C2-3alkinyl, -NHCH2CH2OCH3, -NHCH2CH2OH, -SCH3, or is selected from a group
Figure imgf000031_0002
each R2 is independently from one another selected from the group consisting of H, -NH2, -OCH3, -OCH2CH3, -NO2, -NRxRy, -NHC(=O)CH3, -d-salkyl, -C2.3alkenyl, and -C2.3alkinyl; and
each Rx and Ry is independently from one another hydrogen or C1 -3alkyl, -C2.3alkenyl, and -C2.3alkinyl; and
Rz is hydrogen, halogen, C^alkyl, -C2.3alkenyl, -C2.3alkinyl, or -NHC(=O)CH3;
n is 0, 1 or 2, or
compounds of general formula II
Figure imgf000032_0001
wherein
R-i represents S or NH, and
R2 represents =S or NRX, wherein Rx is Cs-ecycloalkyl, and
R3 represnts N-Ry, wherein Ry is hydrogen, Ci-3alkyl, -C2-3alkenyl, -C2-3alkinyl, or -Ci-4alkyl-COOH, -Ci-3alkyl-0-CH3, or optionally with halogen substituted phenyl; and R4 represents halogen or -N02, Ci-3alkyl, -C2-3alkenyl, -C2-3alkinyl, or -OCH3, and R5 represents S, O, 0-CH2, 0-(CH2)2, 0-CH2-C(=0); or
Rs represents -OH or -OCH3 and R6 is absent; and
R6 represents an optionally substituted phenyl, wherein the substituents are selected from the group consisting of halogen, -NO2, -CF3, Ci-3alkyl, -C2-3alkenyl, -C2-3alkinyl, and -OCH3,
or R6 represents naphthyl, Cs-ecycloalkyl, morpholinyl or an optionally substituted pyridinyl, wherein the substituents are selected from the group consisting of halogen, -NO2, -CF3, Ci-3alkyl, -C2-3alkenyl, and -C2-3alkinyl; or
compounds of general formula III
Figure imgf000032_0002
wherein
each R-i represents independently from one another halogen, OH, C -3alkyl,
-C2-3alkenyl, -C2-3alkinyl, or -CH2C(=O)OCi-3alkyl; and
each R2 represents independently from one another halogen, OH,CN, Ci-3alkyl, or -COOH; and
n is 0, 1 or 2; or
compounds of general formula IV
Figure imgf000033_0001
wherein
Ri represents hydrogen or is selected from the group consisting of
Figure imgf000033_0002
H ; or
l formula V
Figure imgf000033_0003
(V) wherein
Ri represents phenyl, optionally substituted with halogen, -N02, -CF3, -NH2,
-N(CH3)2, Ci-3alkyl, -C2-3alkenyl, or -C2-3alkinyl, or thiophenyl, optionally substituted with halogen; and
R2 represents cyclopentenyl or cyclohexenyl; and
R3 represents hydrogen, -CHO, =N-NH2, =N-0-CH3, cyclopentenyl, or is selected from the roup consisting of
Figure imgf000033_0004
Figure imgf000034_0001
for use in the treatment of viral infections.
2. A compound according to claim 1 , wherein said compound is a compound of general formula I, preferably a compound selected from the group consisting of D1562, D1716, D1709 and D1708.
3. A compound according to claim 1 , wherein said compound is a compound of general formula II, preferably a compound selected from the group consisting of D1528, D1380, D1532, D1531 , D1529, D1482, D1483, D1477, and D1514.
4. A compound according to claim 1 , wherein said compound is a compound of general formula III, preferably a compound selected from the group consisting of D1547, D1636 and D1664.
5. A compound according to claim 1 , wherein said compound is a compound of general formula IV, preferably D1549.
6. A compound according to claim 1 , wherein said compound is a compound of general formula V, preferably a compound selected from the group consisting ofD1551 , D1580, D1591 and D1583.
7. A compound according to any one of claims 1 to 6 for use in the treatment of Picorna virus infections.
8. The use according to claim 7, wherein the Picornavirus is a Poliovirus, a coxsackievirus, a Rhinovirus or an Encephalomyocarditis virus.
9. A compound according to any one of claims 1 to 6 for use as PLA2G16 inhibitor.
10. Pharmaceutical preparation, containing as active substance one or more compounds of general formula I, II, III, IV, or V according to any one of the claims 1 to 6, or the pharmacologically effective salts thereof, optionally in combination with conventional excipients and/or carriers.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019068841A1 (en) * 2017-10-05 2019-04-11 Haplogen Gmbh Antiviral compounds
US11648254B2 (en) 2021-03-02 2023-05-16 Kumquat Biosciences Inc. Substituted pyrido[2,3-d]pyrimidines as inhibitors of Ras pathway signaling
US11912708B2 (en) 2022-04-20 2024-02-27 Kumquat Biosciences Inc. Macrocyclic heterocycles and uses thereof

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GB1383409A (en) * 1972-09-09 1974-02-12 Pfizer Ltd Derivatives of 2-amino- and 4-amino-quinazoline and pharmaceutical compositions containing them
GB8716972D0 (en) * 1987-07-17 1987-08-26 Pfizer Ltd Treatment of cardiac arrhythmias
EP1363899B1 (en) * 2001-01-02 2005-05-11 F.Hoffmann-La Roche Ag Quinazolone derivatives as alpha 1a/b adrenergic receptor antagonists
WO2006019955A2 (en) * 2004-07-14 2006-02-23 President And Fellows Of Harvard College Antiviral methods and compositions
EP2283002A2 (en) * 2008-04-15 2011-02-16 Intermune, Inc. Novel inhibitors of hepatitis c virus replication
CA2805409A1 (en) * 2010-06-18 2011-12-22 Whitehead Institute For Biomedical Research Pla2g16 as a target for antiviral compounds

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019068841A1 (en) * 2017-10-05 2019-04-11 Haplogen Gmbh Antiviral compounds
US11180479B2 (en) 2017-10-05 2021-11-23 Haplogen Gmbh Antiviral compounds
US11648254B2 (en) 2021-03-02 2023-05-16 Kumquat Biosciences Inc. Substituted pyrido[2,3-d]pyrimidines as inhibitors of Ras pathway signaling
US11912708B2 (en) 2022-04-20 2024-02-27 Kumquat Biosciences Inc. Macrocyclic heterocycles and uses thereof

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