WO2022232518A1 - Compositions de calxinine et méthode de traitement d'infections virales - Google Patents

Compositions de calxinine et méthode de traitement d'infections virales Download PDF

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WO2022232518A1
WO2022232518A1 PCT/US2022/026943 US2022026943W WO2022232518A1 WO 2022232518 A1 WO2022232518 A1 WO 2022232518A1 US 2022026943 W US2022026943 W US 2022026943W WO 2022232518 A1 WO2022232518 A1 WO 2022232518A1
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compound
infection
calxinin
viral infection
pharmaceutical composition
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PCT/US2022/026943
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English (en)
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Steven BRADFUTE
Ravi Venkata Durvasula
Prakasha Kempaiah
Brijesh RATHI
Poonam FNU
Yash GUPTA
Agam Prasad SINGH
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Unm Rainforest Innovations
Hansraj College (University Of Delhi)
Loyola University Of Chicago
National Institute Of Immunology
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Publication of WO2022232518A1 publication Critical patent/WO2022232518A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • 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
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/125—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/13—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • 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

  • composition formulated for treating a viral infection.
  • the composition includes 3-amino-4-phenyl-1-(4-(4- (trifluoromethyl)benzyl)piperazin-1-yl)butan-2-ol formulated for treating a viral infection.
  • the composition includes the (2S, 3S) isomer of 3-amino-4- phenyl-1-(4-(4-(trifluoromethyl)benzyl)piperazin-1-yl)butan-2-ol.
  • the viral infection includes infection by SARS-CoV-2, influenza, rhinovirus, or enterovirus.
  • the viral infection includes infection by a flavivirus.
  • the flavivirus is Zika virus (ZIKV).
  • this disclosure describes a method of treating a subject having, or at risk of having, a viral infection.
  • the method includes administering to the subject any embodiment of the pharmaceutical composition summarized above in an amount effective to ameliorate at least one symptom or clinical sign of the viral infection.
  • the viral infection includes infection by SARS-CoV-2, influenza, rhinovirus, or enterovirus.
  • the viral infection includes infection by a flavivirus.
  • the flavivirus is Zika virus (ZIKV).
  • treating the subject results in fewer side effects than treatment with ribavirin, sofosbuvir, or 6-methylmercaptopurine riboside.
  • this disclosure describes a method of synthesizing a disubstituted piperazine compound.
  • the method includes providing a substituted piperazine compound, opening the piperazine ring and thereby generating an activated substituted piperazine compound that includes a reactive amine, and reacting the activated substituted piperazine compound with a hydroxyethylamine-based epoxide compound, thereby producing the disubstituted piperazine compound.
  • opening the piperazine ring involves subjecting the substituted piperazine compound to microwave irradiation.
  • the hydroxyethylamine-based epoxide compound includes a protective group on the reactive amine.
  • the protective group includes a tert-butyloxycarbonyl (BOC) protecting group.
  • the method further includes removing the protective group.
  • the substituted piperazine compound includes a (trifluoromethyl)benzyl group.
  • the method further includes recovering the disubstituted piperazine compound using a chiral column.
  • FIG.2 Schematic roadmap of the drug repurposing a HEA/piperazine/cyclobutanone- based synthetic library screening against the eight SARS-CoV-2 protein targets. The strategy can be expanded to cover assembly, invasion, and host factors playing important role in COVID-19 infection.
  • FIG.3. Protein-Ligand Contacts.
  • FIG.4. Piperazine sub-structure strongly binds to triple aspartate core of COVID-19 ribonucleotide binding region (RdRp).
  • FIG.5. Preliminary anti-SARS-CoV-2 screening. Vero cell rescue assay infected with SARS-CoV-2 after being treated with the indicated compounds.
  • FIG.6 Fragment-based toxicity predictions of calxinin enantiomers by Osiris property explorer (Nicolotti, Orazio, et al. “REACH and in silico methods: an attractive opportunity for medicinal chemists.” Drug Discovery Today 19.11 (2014): 1757-1768). Lighter sphere below “Toxicity Risks” indicates no risk.
  • A The compound has no substructure with known mutagenic, tumorigenic, or irritant properties.
  • the bioavailability (cLogP), solubility and topological polar surface area (TPSA) are all in desired range. Drug score and drug likeness which depends on Lipinski’s rule of five is also in a good range.
  • FIG.7 Calxinin anti-COVID-19 activity. At 10 ⁇ M, there is 88% reduction in infection and at 50 ⁇ M there is a complete reduction in infection.
  • FIG.8 In vitro effects of calxinin on hemostatic parameters.
  • Calxinin did not induce an anticoagulant effect following supplementation to whole blood at concentrations up to 20 ⁇ g/ml.
  • A Prothrombin time.
  • B Activated partial thromboplastin time.
  • FIG.9. In vitro effects of calxinin on hemostatic parameters. Calxinin did not induce an anticoagulant effect following supplementation to whole blood at concentrations up to 20 ⁇ g/ml.
  • A Thrombin time.
  • Calxinin did not affect clot formation as assessed by thrombelastography (20 ⁇ g/ml).
  • FIG.10 Calxinin did not produce any effect on the agonist-induced aggregation of human platelets (20 ⁇ g/ml).
  • FIG.11. Schematic summarizing toxicity study protocol.
  • FIG.12. No adverse and notable behavioral changes were seen after seven days of calxinin treatment. Mice were sacrificed after 14 days for further evaluations. There was no indication of acute toxicity in the mice since body weights remained constant. No behavior changes were observed. Motor and feeding activities were normal. Pathological examination of the major organs of the mice in the calxinin treated groups did not indicate an increase or decrease of Kuepfer cells in the liver (FIG.13), loss of structural integrity in the kidney (FIG. 14) or lining of stomach (FIG.15). Overall, there was no indication of toxicity by calxinin at 1200 mg/kg (300 mg/kg administered for four consecutive days. FIG.13. Histopathology of liver of mice treated as in FIG.11.
  • FIG.14 Histopathology of kidney of mice treated as in FIG.11.
  • A Histopathology of kidney from vehicle-treated mice.
  • B Histopathology of kidney from calxinin-treated mice.
  • FIG.15 Histopathology of stomach of mice treated as in FIG.11.
  • A Histopathology of stomach from vehicle-treated mice.
  • B Histopathology of stomach from calxinin-treated mice.
  • FIG.16 Calxinin activity against enterovirus (CVB3), rhinovirus (HRV2), and flavivirus (ZIKV).
  • FIG.17 In silico docking analysis.50 nanosecond (ns) molecular dynamics (MD) simulations showed that Influenza A, HCV-2a, and Enterovirus all have strong predicted interaction and energetically favorable dynamics.
  • FIG.18 Ligand-protein and protein-ligand contacts between Influenza A 100 ns MD simulation of calxinin with RdRp.
  • FIG.20 Protein-ligand and ligand-protein contacts between HCV_2a 100 ns MD simulation of calxinin with RdRp.
  • FIG.21 Structure of analogs having potency against ZIKV infection.
  • FIG.22 The structure of Compound VI and protease inhibitors of HIV highlighting pharmacophores.
  • HEA-based analogs Compound VI and Compound VII have similar toxicity profile in epithelial (Vero) cells.
  • Cell viability was determined by the MTT method. Briefly, 96- well microplates were seeded with Vero cells (1 ⁇ 10 4 cells/well) and were then treated with various concentrations of either Compound VI or Compound VII. After 72 hours of incubation at 37°C, the culture medium was removed and was replenished with 50 ⁇ L of MTT working solution (1 mg/mL) and was further incubated for four hours. The optical density was determined by spectrophotometry at 570 nm. Values are the mean ⁇ standard deviation of three independent experiments.
  • A Compound VI.
  • B Compound VII.
  • FIG.24 Compound VII.
  • HEA-based Compound VI inhibits infectious ZIKV production in Vero cells in comparison to the 6-Methylmercaptopurine riboside (6MMPr).
  • 6MMPr 6-Methylmercaptopurine riboside
  • A Infectious progeny titer as determined by the Median Tissue Culture Infective Dose (TCID 50 ) method.
  • B Reduction in virus pfu upon treatment with Compound VI. Vero cells were plated in 24-well tissue culture plates and were infected with ZIKV at a multiplicity of infection (MOI) of 0.1 for two hours.
  • MOI multiplicity of infection
  • C Reduction in virus pfu upon treatment with 6MMPr. Vero cells were plated in 24-well tissue culture plates and were infected with ZIKV at a multiplicity of infection (MOI) of 0.1 for two hours.
  • FIG.25 Representative hematoxylin and eosin (H&E)-stained images of major organs - liver, kidney, and stomach. (A) H&E staining of mice treated with control.
  • FIG.26 Schematic representation of 2D interaction maps against protease enzyme of ZIKV infection.
  • A Compound VI (identified potent analog).
  • FIG.27 Root mean square deviations (RMSD) difference between the proteins of ZIKV infections and bound ligand VI during 100 ns MD simulation Graphs show the RMSD value of ligand (darker line) from the protein backbone (lighter line).
  • A Compound VI-protease complex. The Compound VI-protease complex quickly stabilized to a low energy state (within 25 ns) and was highly stable throughout the simulation.
  • B Compound VI-helicase complex.
  • compositions and methods for treating viral infection includes administering to a subject having, or at risk of having, a viral infection a composition that includes calxinin, a synthetic hydroxyethylamine-based compound.
  • Calxinin targets the RNA-directed RNA polymerase (RdRp) of SARS-CoV-2 (NSP11).
  • RdRp RNA-directed RNA polymerase
  • SARS-CoV-2 SARS-CoV-2
  • Calxinin also reduces viral titers in human liver cells infected with Enterovirus (e.g., Coxsackievirus (CVB3)), Rhinovirus (HRV2), and Flavivirus (ZIKV).
  • Enterovirus e.g., Coxsackievirus (CVB3)
  • HRV2 Rhinovirus
  • ZIKV Flavivirus
  • Calxinin also binds to the ribonucleotide binding region of the RdRp of Influenza A, Enterovirus (EV-71), and hepatitis C virus (HCV_2a).
  • Calxinin may be formulated with a pharmaceutically acceptable carrier.
  • carrier includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial, and/or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, and the like. The use of such media and/or agents for pharmaceutical active substances is well known in the art.
  • compositions can be pharmaceutically acceptable.
  • pharmaceutically acceptable refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with calxinin without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. Treating a condition can be prophylactic or, alternatively, can be initiated after the subject exhibits one or more symptoms or clinical signs of infection.
  • the term “at risk” refers to a subject that may or may not actually possess the described risk.
  • a subject “at risk” of infectious condition is a subject present in an area where other individuals have been identified as having the infectious condition and/or is likely to be exposed to the infectious agent even if the subject has not yet manifested any detectable indication of infection and regardless of whether the subject may harbor a subclinical infection.
  • Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.
  • a pharmaceutical composition that includes calxinin can be administered before, during, or after the subject first exhibits a symptom or clinical sign of infection.
  • a pharmaceutical composition that includes calxinin can be administered before, during, or after the subject first comes in contact with the infectious agent. Treatment initiated before the subject first exhibits a symptom or clinical sign associated with infection may result in decreasing the likelihood that the subject experiences clinical evidence of infection compared to a subject to which the composition is not administered, decreasing the severity of symptoms and/or clinical signs of infection, and/or completely resolving the infection.
  • Treatment initiated after the subject first exhibits a symptom or clinical sign associated with infection may result in decreasing the severity of symptoms and/or clinical signs of infection compared to a subject to which the composition is not administered, and/or completely resolving the infection.
  • the method includes administering an effective amount of a pharmaceutical composition containing calxinin to a subject having, or at risk of having, a particular infection.
  • an “effective amount” is an amount effective to reduce, limit progression, ameliorate, or resolve, to any extent, a symptom or clinical sign related to the infection.
  • Calxinin may be formulated into a pharmaceutical composition adapted to a preferred route of administration.
  • a composition can be administered via known routes including, for example, oral, parenteral (e.g., intradermal, transcutaneous, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transcutaneous, rectally, etc.).
  • a pharmaceutical composition can be administered to a mucosal surface, such as by administration to, for example, the nasal or respiratory mucosa (e.g., by spray or aerosol).
  • a composition also can be administered via a sustained or delayed release.
  • calxinin may be provided in any suitable form including but not limited to a solution, a suspension, an emulsion, a spray, an aerosol, or any form of mixture.
  • the composition may be delivered in formulation with any pharmaceutically acceptable excipient, carrier, or vehicle.
  • the formulation may be delivered in a conventional topical dosage form such as, for example, a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion, and the like.
  • the formulation may further include one or more additives including such as, for example, an adjuvant, a skin penetration enhancer, a colorant, a fragrance, a flavoring, a moisturizer, a thickener, and the like.
  • a formulation may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy.
  • Methods of preparing a composition with a pharmaceutically acceptable carrier include the step of bringing the calxinin into association with a carrier that constitutes one or more accessory ingredients.
  • a formulation may be prepared by uniformly and/or intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations.
  • the amount of calxinin administered can vary depending on various factors including, but not limited to, the weight, physical condition, and/or age of the subject, and/or the route of administration.
  • the absolute weight of calxinin included in a given unit dosage form can vary widely, and depends upon factors such as the species, age, weight, and physical condition of the subject, and/or the method of administration. Accordingly, it is not practical to set forth generally the amount that constitutes an amount of calxinin effective for all possible applications. Those of ordinary skill in the art, however, can readily determine the appropriate amount with due consideration of such factors.
  • calxinin may be administered at the same dose and frequency for which the drug has received regulatory approval for another indication.
  • One can alter the dosages and/or frequency as needed to achieve a desired level of antiviral activity.
  • the method can include administering sufficient calxinin to provide a dose of, for example, from about 100 ng/kg to about 1000 mg/kg to the subject, although in some embodiments the methods may be performed by administering calxinin in a dose outside this range.
  • the method includes administering sufficient calxinin to provide a dose of from about 100 mg/kg to about 1000 mg/kg to the subject, for example, a dose of from about 300 mg/kg to about 1000 mg/kg.
  • the methods may be performed by administering an effective dose.
  • an effective dose may be defined as the amount required to reduce viral titer as measured in plaque-forming units by at least 99%.
  • a single dose may be administered all at once, continuously for a prescribed period of time, or in multiple discrete administrations.
  • the amount of each administration may be the same or different.
  • a dose of 1 mg per day may be administered as a single administration of 1 mg, continuously over 24 hours, as two or more equal administrations (e.g., two 0.5 mg administrations), or as two or more unequal administrations (e.g., a first administration of 0.75 mg followed by a second administration of 0.25 mg).
  • the interval between administrations may be the same or different.
  • the active agent may be administered, for example, from a single dose to multiple doses per week, although in some embodiments the method can involve a course of treatment that includes administering doses of the active agent at a frequency outside this range.
  • the amount of each dose may be the same or different.
  • a course of treatment can include a loading dose initial dose, followed by a maintenance dose that is lower than the loading dose.
  • the interval between doses may be the same or be different.
  • calxinin may be administered from about once per month to about five times per week.
  • this disclosure describes the synthesis of hydroxyethylamine (HEA) analogs and their use in treating, either therapeutically or prophylactically, viral infections.
  • the HEA pharmacophore has been used to develop novel therapeutic candidates against various infectious diseases, some of which are depicted in FIG.22.
  • To prepare the compounds ring-opening reactions were performed under microwave conditions (FIG.1).
  • the microwave irradiation provided fast, convenient, small to large scale (1- 2 g) synthesis, and easy work-up procedures compared to reported conventional syntheses of HEA compounds.
  • the reaction conditions include the use of ethanol as a greener, economic solvent and a reaction time of 30 minutes (FIG.1).
  • Amine mediated ring-opening of epoxide follows a regioselective attack on the less hindered carbon through SN2 reaction mechanism that inverts the stereochemistry at the attacked carbon. Similar ring opening trends were realized while ring-opening of epoxides in the presence of 1-(4-(trifluoromethyl)benzyl)piperazine (I) under microwave irradiations.
  • Compound VI was synthesized starting from (2R,3S)-N-BOC-3- amino-1,2-epoxy-4-phenylbutane (II), while VII was synthesized starting from (2S,3S)-N-BOC- 3-amino-1,2-epoxy-4-phenylbutane.
  • (2S,3S)-3-amino-4-phenyl-1-(4-(4-(trifluoromethyl)benzyl)piperazin-1- yl)butan-2-ol is variably referred to as “Compound VI” and “calxinin.”
  • Compound VI 2S,3S-3-amino-4-phenyl-1-(4-(4-(trifluoromethyl)benzyl)piperazin-1- yl)butan-2-ol
  • calxinin 2S,3S)-3-amino-4-phenyl-1-(4-(4-(trifluoromethyl)benzyl)piperazin-1- yl)butan-2-ol
  • the molecular docking analysis revealed that Compound VI has shown H-bond interaction with amino acid residues, Asp83 and Asp1075 of protease protein (FIG.26). Further, a total of three molecular dynamics (MD) simulation were performed for a time period of 100 ns each to authenticate the docking results. All the ligand-protein complexes were analyzed for interactions throughout the simulation. The results indicate a stable protein-ligand complex between Compound VI and ZIKV protease protein, as interaction between Compound VI and protease in the complex remained highly stable throughout the simulation (FIG.27A).
  • MD molecular dynamics
  • the method includes obtaining a piperazine compound, opening the piperazine ring to generate a reactive amine, and reacting the opened ring with a hydroxyethylamine-based epoxide.
  • the ring is opened using microwave irradiation.
  • the hydroxyethylamine-based epoxide may contain a reactive amine.
  • the hydroxyethylamine-based epoxide may also be substituted with a protective group.
  • the protective group can be a tert-butyloxycarbonyl protecting group. The protective group may be removed after synthesis of the disubstituted piperazine compound to produce a reactive amine.
  • the protective group may be removed by reacting the compound with trifluoroacetic acid (TFA).
  • TFA trifluoroacetic acid
  • the piperazine compound may be substituted with a number of moieties.
  • the substituted groups may be a (trifluoromethyl)benzyl group, an aryl halide, or another halogenated organic group.
  • the compound may be further recovered using chemical separation techniques.
  • the compound may be recovered from a chiral column. Cytotoxicity and Toxicology
  • calxinin may be administered to treat infection by the Zika virus (ZIKV).
  • ZIKV belongs to the Flaviviridae family and, in humans, causes Zika fever.
  • Zika fever is a vector-borne disease primarily transmitted by a bite of daytime-active Aedes mosquitoes, mainly Aedes aegypti.
  • Evidence of ZIKV infection of varying intensities have been reported in more than 80 countries.
  • preventive measures remain the only options for the human population to avoid the infection in case of emergency.
  • Drug repurposing of several FDA-approved therapeutics as temporary treatments for ZIKV infections is still in clinical development.
  • ribavirin a broad-spectrum antiviral agent that targets RNA-dependent RNA polymerase (RdRp), inhibits ZIKV replication in cell-based assays.
  • Ribavirin exhibited an IC50 value of 31.6 ⁇ M in Vero cells with a selectivity index (SI) of 4.97 (FIG.21).
  • SI selectivity index
  • ribavirin and other antivirals agents e.g., favipiravir and interferon
  • Sofosbuvir an antiviral drug that also targets RdRp, displays activity against ZIKV infection without any cytotoxic effects.
  • Sofosbuvir displayed an IC 50 value of 3.9 ⁇ M from plaque reduction assay performed on ZIKV infected Huh7 cells.
  • sofosbuvir failed to inhibit viral infection in Vero and A549 cell lines (FIG.21).
  • 6-methylmercaptopurine riboside (6MMPr), a thiopurine nucleoside analog, provided an IC 50 value against ZIKV infection of 20.3 ⁇ M in SH- SY5Y cells and 24.5 ⁇ M in Vero cells (FIG.21).6MMPr CC 50 values, a measure of toxicity, were 291 ⁇ M in Vero cells (FIG.21) and 460.3 ⁇ M in SH-SY5Y cells. However, the potency of 6MMPr was inadequate to use as a prophylactic measure towards the ZIKV infection. Overall, there is no effective drug or vaccines for therapeutic or prophylactic treatment of ZIKV infection.
  • 6MMPr 6-methylmercaptopurine riboside
  • This disclosure describes the synthesis of hydroxyethylamine (HEA) analogs and their use in treating, either therapeutically or prophylactically, viral infections.
  • One identified compound (VI; FIG.1) exhibited an IC 50 value of 340 nM that was lower than the positive control, 6MMPr, with minimal cytotoxicity (CC50).
  • the activity of both the isomers (VI and VII, FIG.1) was tested individually against ZIKV in culture, and the results were compared to inculcate the stereospecific activity of two stereoisomers. Further, acute and subacute cytotoxicity studies were performed using a mouse model.
  • Vero cells were infected at a multiplicity of infection (MOI) of 0.1 viruses/cell then treated with the CC20 of each compound. After 72 hours, the supernatant was collected and titrated by Median Tissue Culture Infectious Dose (TCID 50 ).
  • TCID 50 Median Tissue Culture Infectious Dose
  • Table 2 The results are depicted in Table 2.
  • Compound VI S,S isomer
  • 6MMpr displayed 100% inhibition at a concentration of 60.5 ⁇ M concentration.
  • another compound VII demonstrated an 81.0% reduction of virus titer at 4.49 ⁇ M concentration.
  • mice For acute toxicity, single doses of 100 mg/Kg/week, 300 mg/Kg/week, and 1000 mg/Kg/week of Compound VI were given orally to the experimental mice. None of the doses had any toxic effects on the mice as evidence by the weight of the mice remained constant, and feeding activity and motor activity were also unaltered. In a parallel experiment, subacute toxicity was evaluated by orally administering a dose of 300 mg/Kg for four consecutive days. The mice exhibited normal feeding and motor activities and weight maintenance. Further, a serum biochemistry study was performed for the mice administered with the highest dose (1200 mg/kg) to monitor potential toxic effects on liver and kidney function of the experimental mice in comparison to the control set of mice. No biochemical differences were observed between the control and treated sets of mice.
  • the characterization of the stereochemical isomers included spectroscopic studies such as NMR, SOR, mass-spectrometry HRMS, and, finally, the formation of single stereoisomer in the particular reaction was confirmed with extensive HPLC studies on chiral columns.
  • the inhibitory activity assay against ZIKV infection demonstrated that out of the two stereochemical isomers, Compound VI (S,S) inhibits ZIKV infection more efficiently than Compound VII (R,S), highlighting the importance of stereochemistry in biological activities of the compounds.
  • Compound VI displayed 72-fold better inhibitory potency compared to positive control (6MMPr) with a SI of 22.41.
  • the term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended—i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). In the preceding description, particular embodiments may be described in isolation for clarity.
  • a virtual compound library was assembled by combining 150 compounds from an in-house HEA/piperazine/cyclobutanone-based library were pooled with the world approved compound library.
  • molecular dynamics (MD) simulations docking the compound library to identified essential viral enzymes were performed.
  • An example of a molecule (Compound VI) found to strongly bind the core of COVID-19 ribonucleotide binding region (RdRp) is shown in FIG.3 and FIG.4.
  • MD simulations were carried out for the RdRp of several other viruses of interest, including Influenza A, enterovirus, and Hepatitis C (FIG.17-20).
  • Fragment based toxicity was predicted of top scoring compounds by Osiris property explorer (Nicolotti, Orazio, et al.2014). Several enantiomers of identified compounds were predicted to have acceptable bioavailability (cLogP), solubility, and TPSA (FIG.6). Top scored drug compounds were selected from in-silico computer-aided drug design (CADD) analysis and assayed for antiviral activity measured by cell viability assay (MTT assay) (FIG.5). Briefly, 10 ⁇ M of selected drug compounds were added to Vero cells one hour before cells were infected with SARS-CoV-2 and incubated for 48 hours. After 48 hours, XTT was added and O.D.
  • CCD computer-aided drug design
  • calxinin a synthetic HEA-based compound was top performer (close to 60%; depicted within the bar) and bisindolylmaleimide (close to 45%; depicted within the bar), an approved drug is second best performer (FIG.5).
  • FOG.5 bisindolylmaleimide
  • Testing of calxinin demonstrated potent anti-SARS-CoV-2 activity in a viral pfu assay.
  • the IC50 was calculated to be 0.293 ⁇ M (FIG.7).
  • chloroquine another known SARS-CoV-2 antiviral compound
  • IC50 1.3 ⁇ M. Therefore, the CADD-based screening revealed at least six compounds from the preliminary screening having encouraging results (FIG.6). There is room for further improving these drug compounds through pharmacophore-based design and synthesis, in addition to exploring more virtual screening.
  • Example 2 Organic synthesis Piperazine I (1 eq., 1.9 mmol), (2R,3S)-N-BOC-3-amino-1,2-epoxy-4-phenylbutane (II) or (2S,3S)-N-BOC-3-amino-1,2-epoxy-4-phenylbutane (III) (1 eq., 1.9 mmol) and were dissolved in ethanol and the contents were refluxed in microwave at 80°C (300 W) for 30 minutes. The progress of the reaction was monitored by thin layer chromatography (TLC). Later, the reaction mixture was cooled to reach room temperature and remaining solvent was evaporated under reduced pressure.
  • TLC thin layer chromatography
  • Example 3 - Toxicity Evaluation in Mice Testing of acute toxicity study Administration of Compound VI (calxinin) or 0.2 ml of vehicle (10% DMSO in PBS) was performed by oral gavage at a dose of 300 mg/kg for four consecutive days as follows. Calxinin was evaluated for its toxicity in BALB/c mice aged six to eight weeks and weighing 20-22 g. Mice were divided in into two groups of three mice (FIG. 11). Before oral administration of a single dose of compound, the mice were fasted for one or two hours.
  • mice in Group 1 were given orally 0.2 ml of vehicle (10% DMSO in PBS) and the mice in Group 3 were given a single dose of 100 mg/kg/week, 300 mg/kg/week, or 1000 mg/kg/week orally. Mice were observed continuously for one hour after the treatment, intermittently for six hours, and thereafter over a period of 24 hours. Several parameters were observed such as weight loses, behavior change, hair erection, reduction in feed and motor activity (FIG.12). Again, mice were randomly divided in two groups. Each group consisted of three mice. Administration of calxinin was performed by oral gavage at a dose of 300 mg/kg for four consecutive days.
  • Example 4 Antiviral Activity Calxinin was assayed in vitro for antiviral activity against enterovirus (coxsackievirus, CVB3), rhinovirus (HRV2), and flavivirus (ZIKV).
  • enterovirus coxsackievirus, CVB3
  • HRV2 rhinovirus
  • ZIKV flavivirus
  • Huh7 human liver cells were treated with Compound I four hours prior to infection with virus.24 hours after infection, viral content of infected cells was measured virus by plaque assay. Calxinin was shown to have inhibitory activity against all viruses tested (FIG.16). No virus was detected (ND) in cells infected with ZIKA and treated with calxinin.
  • Vero cells were seeded in 24-well plates a day prior to infection at the density of 5 ⁇ 10 4 cells/well.
  • the cells were infected with the ZIKV PE243 strain at a multiplicity of infection (MOI) of 0.1 viruses/cell and incubated for two hours (37°C, 5% CO 2 ). After virus internalization, viral inoculum was removed, cells were washed twice with DMEM, and the supernatant was replaced with fresh medium containing 6MMPr, Compound VI, or Compound VII. At 72 hours post-infection (hpi), the cells supernatant was collected and titrated by TCID 50 (FIG.24). Stock solution of the compounds were prepared in Milli-Q H 2 O (MilliporeSigma, St.
  • MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to determine the cytotoxicity of bioactive compounds. Vero cells were used due to the susceptibility and permissiveness of arbovirus infection.96-well cell plates were cultured for 24 hours at 37oC and 5% atm of CO2 and compounds (VI and VII) in DMEM culture medium in concentrations in quadruplicate.
  • the culture medium was removed from the wells and a new medium with MTT (1 mg/mL) was added 50 ⁇ L/well and incubated over a period of 3.5 hours.
  • the medium was replaced by DMSO (100 ⁇ L/well) to solubilize the formazan crystals formed by the viable cells present in the wells.
  • the plates were incubated with shaking for 15 minutes at 35oC and 120 RPM. The wavelength used was 570 nm and statistical analysis was performed using EXCEL (Microsoft Corp., Redmond, WA) and PRISM v.8.0 (GraphPad Software, San Diego, CA).
  • PBMCs peripheral blood mononuclear cells
  • HEK293 immortalized human kidney cells
  • Huh7.1 and HepG2 immortalized liver cells
  • mice Six-week-old BALB/C mice were housed at 22 ⁇ 2 oC with a 12-hour light/dark cycle and fed standard rodent chow and water ad libitum.
  • mice were observed continuously for one hour after the treatment, intermittently for six hours, and thereafter over a period of 24 hours. Several parameters were observed such as weight loses, behavior change, hair erection, reduction in feed, and motor activity.
  • mice were prepared for the study as described for the acute toxicity study, then randomly divided in two groups. Mice in the control group were given 0.2 ml of vehicle (10% DMSO in PBS) orally for four consecutive days. The experimental mice were given dosage of 300 mg/kg/day for four consecutive days. After monitoring the motor and feeding activity, mice were sacrificed to isolate their liver, kidney, and stomach. Organ samples were analyzed for histopathology studies. Serum was also taken for biochemistry studies.

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Abstract

Une composition pharmaceutique selon la présente invention comprend de la calxinine formulée pour traiter une infection virale. La composition pharmaceutique peut être utilisée pour traiter un sujet ayant une infection virale ou présentant un risque d'avoir une infection virale. Dans certains modes de réalisation, l'infection virale comprend une infection par le SARS-CoV-2. Dans certains modes de réalisation, l'infection virale comprend une infection par le virus Zika.
PCT/US2022/026943 2021-04-29 2022-04-29 Compositions de calxinine et méthode de traitement d'infections virales WO2022232518A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020128270A1 (en) * 1996-12-17 2002-09-12 Fujisawa Pharmaceutical Co., Ltd. Piperazine compounds as inhibitors of MMP or TNF
US20200360381A1 (en) * 2017-05-15 2020-11-19 Dana-Farber Cancer Institute, Inc. Compounds for treating dengue virus infection and other viral infections

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020128270A1 (en) * 1996-12-17 2002-09-12 Fujisawa Pharmaceutical Co., Ltd. Piperazine compounds as inhibitors of MMP or TNF
US20200360381A1 (en) * 2017-05-15 2020-11-19 Dana-Farber Cancer Institute, Inc. Compounds for treating dengue virus infection and other viral infections

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Title
CLAIRE-LISE CIANA, SIEGRIST ROMAIN, AISSAOUI HAMED, MARX LÉO, RACINE SOPHIE, MEYER SOLANGE, BINKERT CHRISTOPH, DE KANTER RUBEN, FI: "Novel in vivo active anti-malarials based on a hydroxy-ethyl-amine scaffold", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, PERGAMON, vol. 23, no. 3, 11 December 2012 (2012-12-11), pages 658 - 662, XP055111645, ISSN: 0960894X, DOI: 10.1016/j.bmcl.2012.11.118 *
KUMAR SUMIT, SHARMA PREM PRAKASH, SHANKAR UMA, KUMAR DHRUV, JOSHI SANJEEV K., PENA LINDOMAR, DURVASULA RAVI, KUMAR AMIT, KEMPAIAH : "Discovery of New Hydroxyethylamine Analogs against 3CL pro Protein Target of SARS-CoV-2: Molecular Docking, Molecular Dynamics Simulation, and Structure–Activity Relationship Studies", JOURNAL OF CHEMICAL INFORMATION AND MODELING, AMERICAN CHEMICAL SOCIETY , WASHINGTON DC, US, vol. 60, no. 12, 28 December 2020 (2020-12-28), US , pages 5754 - 5770, XP055968696, ISSN: 1549-9596, DOI: 10.1021/acs.jcim.0c00326 *
SINGH ANIL K., RATHORE SUMIT, TANG YAN, GOLDFARB NATHAN E., DUNN BEN M., RAJENDRAN VINOTH, GHOSH PRAHLAD C., SINGH NEELU, LATHA N.: "Hydroxyethylamine Based Phthalimides as New Class of Plasmepsin Hits: Design, Synthesis and Antimalarial Evaluation", PLOS ONE, vol. 10, no. 10, 26 October 2015 (2015-10-26), pages e0139347, XP055981482, DOI: 10.1371/journal.pone.0139347 *

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