Patent Application
20250032527
The present disclosure relates to the medical use of citicoline analogs with a N4-hydroxycytidine moiety. These analogs can be used as antiviral drugs for treatment of e.g. Covid-19, human rhinovirus (HRV), influenza and respiratory syncytial virus (RSV).
Citicoline, also known as cytidine diphosphate-choline (CDP-choline) or cytidine 5′-diphosphocholine is an intermediate in the generation of phosphatidylcholine from choline, a common biochemical process in cell membranes. Citicoline is naturally occurring in the cells of human and animal tissue, in particular the organs. Citicoline is commercially available as dietary supplement. When taken as a dietary supplement, citicoline is hydrolyzed into choline and cytidine in the intestine. Once these fragments (i.e. choline, cytidine) cross the blood-brain barrier it is reformed into citicoline by the rate-limiting enzyme in phosphatidylcholine synthesis, CTP-phosphocholine cytidylyltransferase. Citicoline is water-soluble, with more than 90% oral bioavailability and has a very low toxicity profile in animals and humans. Citicoline as the active ingredient is, for example, described as a preventive/remedy for drug-induced neuropathy in WO2004/006940. These advantageous overall properties make citicoline an excellent starting point for drug development. However, no antiviral activity has been mentioned for citicoline. By replacing a hydrogen with a hydroxy group in the 4-position of the cytosine moiety, the resulting compound showed surprisingly antiviral activity against SARS-COV-2, which can be even more improved in a synergistic behavior, when combined with an DHODH inhibitor.
Cytidine can be chemically hydroxylated at the nitrogen in 4-position of the cytosine to form β-D-N4-hydroxycytidine (NHC), also known as EIDD-1931. The isobutyric acid in the 5′-position of the ribose is an prodrug of EIDD-1931, named molnupiravir or EIDD-2801. Both compounds are antiviral drugs exerting their antiviral action through introduction of copying errors during viral RNA replication (Nat. Struct. Mol. Biol. 2021; 28:740). Molnupiravir is currently developed for the treatment of influenza and approved for the treatment of Covid-19. When administered orally, molnupiravir is hydrolyzed to NHC, which is subsequently metabolized to β-D-N4-hydroxycytidine 5′-triphosphate (EIDD-2061). During replication, the virus's RNA polymerase enzyme incorporates EIDD-2061 into newly made RNA instead of using real cytidine.
The disadvantage of molnupiravir is that the prodrug must first be hydrolysed to β-D-NA-hydroxycytidine, which however is not active in the human body but requires intracellular activation by host cell kinases. These enzymes convert the nucleoside analog via the mono-and diphosphate to the ultimately active nucleoside analog triphosphate. The conversion to the monophosphate is the rate-limiting step in this process.
In the present invention, we improve bioactivation by using the diphosphate-choline prodrug (Formula (I)), in which the β-D-N4-hydroxycytidine 5′-diphosphate is formed after cleavage of choline, thus bypassing the rate-limiting step. In addition, we show surprising effects for the nucleoside analog of Formula (I) compared to molnupiravir and NHC, e.g. improved permeability despite low solubility (Example 3) and high concentrations of NHC-DP and NHC-TP in plasma over a long period of time (Example 4). In addition, we describe a straightforward one-step synthesis of a compound according to Formula (I) from readily available and cheap cytidine diphosphate-choline (CDP-choline or citicoline), which is sold as dietary supplement in pharmaceutical grade quality (Example 1 and Example 2).
The compound of the present invention according to Formula (I) is the N4-hydroxy analog of citicoline an be found in SciFinder with CAS-number 13186-92-0 and 13186-58-8 and no other salts are described except the depicted zwitterion.
The only linked reference towards Formula (I) is a publication from Abdelrahman et al. (J. Sep. Sci. 2020; 43:2981), which describes the formation of this compound upon oxidative degradation of citicoline (using 3% hydrogen peroxide) and its use as reference standard in the analytical development and validation of stability-indicating chromatographic methods for simultaneous determination of citicoline and piracetam.
WO2019/113462 (Emory University) describes the preparation of N4-hydroxycytidine and derivatives and antiviral uses related thereto. The application describes many different ester prodrugs of N4-hydroxycytidine including molnupiravir.
Additional patent applications from Emory University WO2017/156380 and WO2016/106050 also describe the reparation of N4-hydroxycytidine prodrugs and antiviral uses related thereto. The 5′-O-position of the ribose moiety can also be substituted with a broad range of residues R1, e.g. monophosphate, diphosphate, triphosphate which can be optionally substituted with one or more residues R20. This R20 mentions a lot of groups including alkyl.
The free acid of β-D-N4-hydroxycytidine 5′-triphosphate (NHC-TP, CAS number 34973-27-8) and β-D-N4-hydroxycytidine 5′-diphosphate (NHC-DP, CAS number 39023-73-9) are commercially available, e.g. from MedChemExpress.
Several former patent applications, e.g. WO2002/100415 (Roche) or WO2016/100569 (Alios) mention the diphosphate moiety, which however can't be substituted to yield an ester.
The present disclosure relates to the medical use of citicoline analogs having a N4-hydroxycytidine moiety. These analogs can be used as antiviral drugs for treatment of e.g. Covid-19, human rhinovirus (HRV), influenza and respiratory syncytial virus (RSV).
Subject matter of the present invention is described by the following embodiments:
1. A compound according to Formula (I):
or a tautomer, solvate or pharmaceutically acceptable salt thereof for use as a medicament.
2. The compound of Formula (I) or a tautomer, solvate or pharmaceutically acceptable salt thereof for use as a medicament according to embodiment 1, in the prevention and/or treatment of a disease caused by viral infection in a mammalian subject, wherein said viral infection is caused by an RNA virus, such as, for example HIV, HCV, Ebola, rotavirus, Zika virus, polio virus, rhinovirus, hepatitis A virus, measles virus, mumps virus, RSV, rabies, Lassa virus, hantavirus, or influenza, in particular a single stranded RNA virus, such as HCoV-229E, HCoV-NL63, or betacoronaviruses, such as HCoV-OC43, SARS-COV-1, HCoV-HKU1, MERS-CoV or SARS-COV-2.
3. The compound of Formula (I) or a tautomer, solvate or pharmaceutically acceptable salt thereof for use as a medicament according to embodiment 1 or 2, in the prevention and/or treatment of a disease caused by viral infection with coronaviruses in a human.
4. The compound of Formula (I) or a tautomer, solvate or pharmaceutically acceptable salt thereof for use as a medicament according to any of embodiments 1 to 3, in the prevention and/or treatment of a disease, wherein said disease is selected from AIDS, hepatitis, ebola, polio, diarrhea, measles, mumps, rabies, Lassa fever, viral flu, respiratory disease, acute respiratory disease, sepsis, acute respiratory distress syndrome, long COVID, and adverse immune reactions, in particular moderate to severe cases of said diseases, and wherein said disease preferably is selected from diseases causes by SARS-COV-2, in particular COVID-19, such as acute respiratory disease, sepsis, acute respiratory distress syndrome, long COVID and adverse immune reactions, such as a cytokine storm.
5. The compound of Formula (I) or a tautomer, solvate or pharmaceutically acceptable salt thereof for use as a medicament according to any of embodiments 1 to 4, in the prevention and/or treatment of a disease, wherein said prevention and/or treatment is in combination with a standard antiviral therapy (SAT).
6. The compound of Formula (I) or a tautomer, solvate or pharmaceutically acceptable salt thereof for use as a medicament of a compound according to any of embodiments 1 to 4, in the prevention and/or treatment of a disease, wherein said prevention and/or treatment is in combination with an DHODH inhibitor, and optionally with a standard antiviral therapy (SAT).
7. The compound of Formula (I) or a tautomer, solvate or pharmaceutically acceptable salt thereof for use as a medicament according to embodiment 6, wherein in said combination the DHODH inhibitor consists of Formula (II):
is selected from a 5-membered heteroaryl, cyclopentenyl and heterocyclopentenyl, having one or more hydrogen atoms optionally replaced by deuterium, said A is unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of halogen, CN, NO2, oxo, OH, C1-4-alkyl, O-C1-4-alkyl, fluoro-C1-4-alkyl and O-fluoro-C1-4-alkyl, CO2H and SO3H, having one or more hydrogen atoms in alkyl optionally replaced by deuterium;
8. The compound of Formula (I) or a tautomer, solvate or pharmaceutically acceptable salt thereof for use as a medicament according to embodiment 7, wherein in said combination the DHODH inhibitor is selected from
9. A pharmaceutical composition comprising the compound of Formula (I) or a tautomer, solvate or pharmaceutically acceptable salt thereof for use as a medicament according to any of embodiments 1 to 8, such as a tablet, capsule, granule, powder, sachet, reconstitutable powder, dry powder inhaler and/or chewable.
10. A pharmaceutical composition according to embodiment 9, wherein said pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients.
11. A compound according to Formula (I)
which is obtainable by reacting citicoline or salts thereof with hydroxylamine or salts thereof.
12. A compound according to Formula (I):
or a tautomer, solvate or pharmaceutically acceptable salt thereof for use in the treatment of a disease selected from AIDS, hepatitis, ebola, polio, diarrhea, measles, mumps, rabies, Lassa fever, viral flu, respiratory disease, acute respiratory disease, sepsis, acute respiratory distress syndrome, long COVID, and adverse immune reactions, in particular moderate to severe cases of said diseases, and wherein said disease preferably is selected from diseases causes by SARS-COV-2, in particular COVID-19, such as acute respiratory disease, sepsis, acute respiratory distress syndrome, long COVID and adverse immune reactions, such as a cytokine storm.
13. Use of a compound according to Formula (I):
or a tautomer, solvate or pharmaceutically acceptable salt thereof as a medicament.
14. The use of a compound according to Formula (I) or a tautomer, solvate or pharmaceutically acceptable salt thereof as a medicament according to embodiment 13, in the prevention and/or treatment of a disease caused by viral infection in a mammalian subject, wherein said viral infection is caused by an RNA virus, such as, for example HIV, HCV, Ebola, rotavirus, Zika virus, polio virus, rhinovirus, hepatitis A virus, measles virus, mumps virus, RSV, rabies, Lassa virus, hantavirus, or influenza, in particular a single stranded RNA virus, such as HCoV-229E, HCoV-NL63, or betacoronaviruses, such as HCoV-OC43, SARS-COV-1, HCoV-HKU1, MERS-CoV or SARS-COV-2.
15. The use of a compound according to Formula (I) or a tautomer, solvate or pharmaceutically acceptable salt thereof as a medicament according to embodiment 13 or 14, in the prevention and/or treatment of a disease caused by viral infection with coronaviruses in a human.
16. The use of a compound according to Formula (I) or a tautomer, solvate or pharmaceutically acceptable salt thereof as a medicament according to any of embodiments 13 to 15, in the prevention and/or treatment of a disease, wherein said disease is selected from AIDS, hepatitis, ebola, polio, diarrhea, measles, mumps, rabies, Lassa fever, viral flu, respiratory disease, acute respiratory disease, sepsis, acute respiratory distress syndrome, long COVID, and adverse immune reactions, in particular moderate to severe cases of said diseases, and wherein said disease preferably is selected from diseases causes by SARS-COV-2, in particular COVID-19, such as acute respiratory disease, sepsis, acute respiratory distress syndrome, long COVID and adverse immune reactions, such as a cytokine storm.
17. The use of a compound according to Formula (I) or a tautomer, solvate or pharmaceutically acceptable salt thereof as a medicament according to any of embodiments 13 to 16, in the prevention and/or treatment of a disease, wherein said prevention and/or treatment is in combination with a standard antiviral therapy (SAT).
18. The use of a compound according to Formula (I) or a tautomer, solvate or pharmaceutically acceptable salt thereof as a medicament according to any of embodiments 13 to 16, in the prevention and/or treatment of a disease, wherein said prevention and/or treatment is in combination with an DHODH inhibitor, and optionally with a standard antiviral therapy (SAT).
19. The use of a compound according to Formula (I) or a tautomer, solvate or pharmaceutically acceptable salt thereof as a medicament according to embodiment 18, wherein in said combination the DHODH inhibitor consists of Formula (II):
or an enantiomer, diastereomer, tautomer, prodrug, solvate, or pharmaceutically acceptable salt thereof, wherein
R1 and R2 are independently selected from H and D;
is selected from a 5-membered heteroaryl, cyclopentenyl and heterocyclopentenyl, having one or more hydrogen atoms optionally replaced by deuterium,
20. The use of a compound according to Formula (I) or a tautomer, solvate or pharmaceutically acceptable salt thereof as a medicament according to embodiment 19, wherein in said combination the DHODH inhibitor is selected from
21. The use of a compound according to Formula (I) or a tautomer, solvate or pharmaceutically acceptable salt thereof as a medicament according to embodiment 13 to 20, wherein said compound(s) is/are provided as a suitable pharmaceutical composition, such as a tablet, capsule, granule, powder, sachet, reconstitutable powder, dry powder inhaler and/or chewable.
22. The use of a compound according to Formula (I) or a tautomer, solvate or pharmaceutically acceptable salt thereof as a medicament according to embodiment 21, wherein said pharmaceutical composition further include one or more pharmaceutically acceptable carriers or excipients.
23. The use of a compound according to Formula (I):
24. A method of treating or prevention a disease caused by viral infection in a mammalian subject, wherein said viral infection is caused by an RNA virus, such as, for example HIV, HCV, Ebola, rotavirus, Zika virus, polio virus, rhinovirus, hepatitis A virus, measles virus, mumps virus, RSV, rabies, Lassa virus, hantavirus, or influenza, in particular a single stranded RNA virus, such as HCoV-229E, HCoV-NL63, or betacoronaviruses, such as HCoV-OC43, SARS-CoV-1, HCoV-HKU1, MERS-COV or SARS-COV-2 comprising administering to a patient in need of such treatment or prevention a therapeutically or prophylactically effective amount of a compound according to Formula (I):
25. The method according to embodiment 24, wherein said disease caused by viral infection with coronaviruses in a human.
26. The method according to embodiment 24 or 25, wherein said disease is selected from AIDS, hepatitis, ebola, polio, diarrhea, measles, mumps, rabies, Lassa fever, viral flu, respiratory disease, acute respiratory disease, sepsis, acute respiratory distress syndrome, long COVID, and adverse immune reactions, in particular moderate to severe cases of said diseases, and wherein said disease preferably is selected from diseases causes by SARS-CoV-2, in particular COVID-19, such as acute respiratory disease, sepsis, acute respiratory distress syndrome, long COVID and adverse immune reactions, such as a cytokine storm.
27. The method according to any of embodiments 24 to 26, wherein said prevention and/or treatment is in combination with a standard antiviral therapy (SAT).
28. The method according to any of embodiments 24 to 26, wherein said prevention and/or treatment is in combination with an DHODH inhibitor, and optionally with a standard antiviral therapy (SAT).
29. The method according to embodiment 28, wherein in said combination the DHODH inhibitor consists of Formula (II):
is selected from a 5-membered heteroaryl, cyclopentenyl and heterocyclopentenyl, having one or more hydrogen atoms optionally replaced by deuterium,
30. The method according to embodiment 29, wherein in said combination the DHODH inhibitor is selected from
31. A method of treatment of disease selected from AIDS, hepatitis, ebola, polio, diarrhea, measles, mumps, rabies, Lassa fever, viral flu, respiratory disease, acute respiratory disease, sepsis, acute respiratory distress syndrome, long COVID, and adverse immune reactions, in particular moderate to severe cases of said diseases, and wherein said disease preferably is selected from diseases causes by SARS-COV-2, in particular COVID-19, such as acute respiratory disease, sepsis, acute respiratory distress syndrome, long COVID and adverse immune reactions, such as a cytokine storm comprising administering to a patient in need of such treatment or prevention a therapeutically or prophylactically effective amount of a compound according to Formula (I):
The invention further relates to the use of a compound according to Formula (I):
An DHODH inhibitor for combination with a compound according to Formula (I) is selected from vidofludimus, vidofludimus calcium (IMU-838), teriflunomide, leflunomide, emvododstat (PTC299), brequinar, farudostat (ASLAN003), PP-001, RP7214, orludodstat (BAY2402234), JNJ-74856665, AG-636, MEDS433, or a compound according to Formula (II), which have been described in provisional application EP21167690. Preferably, the DHODH inhibitor is a compound according to Formula (II). More preferably, the DHODH inhibitor is selected from
or a solvate
Examples of other therapeutic agents used in a standard antiviral therapy (SAT) that may be combined with the compound of Formula (I), either administered separately, co-administered or in the same pharmaceutical composition include, but are not limited to a:
Any formula or structure given herein, is also intended to represent isotopically labelled atoms. Examples of isotopes that can be incorporated into compounds of the disclosure include further isotopes of hydrogen (i.e. deuterium or tritium), as well as isotopes of carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as, but not limited to 11C, 13C, 14C, 15N, 18F, 31P, 32P, 35S, 36Cl and 125I. The disclosure further comprises various isotopically labelled compounds into which radioactive isotopes such as 3H, 13C and 14C are incorporated. Such isotopically labelled compounds may be useful in metabolic studies, reaction kinetic studies, detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays or radioactive treatment of patients.
The compounds of the present invention are partly subject to tautomerism. For example, if a heteroaromatic group containing a nitrogen atom in the ring is substituted with a hydroxy or amino group on the carbon atom adjacent to the nitrogen atom, the following tautomerism can appear:
The term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Thus, the compounds of the present disclosure which contain acidic groups can be present on these groups and can be used according to the disclosure, for example, as alkali metal salts, alkaline earth metal salts or ammonium salts. More precise examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines such as, for example, ethylamine, ethanolamine, triethanolamine or amino acids. The respective salts can be obtained by customary methods which are known to the person skilled in the art like, for example, by contacting these with an organic or inorganic base in a solvent or dispersant, or by cation exchange with other salts. The present disclosure also includes all salts of the compounds of the present disclosure which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts.
Further the compounds of the present disclosure may be present in the form of solvates, such as those which include as solvate water, or pharmaceutically acceptable solvates, such as alcohols, in particular ethanol. A stoichiometric or non-stoichiometric amount of solvent is bound by non-covalent intermolecular forces. When the solvent is water, the “solvate” is a “hydrate”. It is understood, that a “pharmaceutically acceptable salts” can in addition optionally contain a “solvate”.
The term “effective amount” is meant to include the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of a disorder, disease, or condition being treated. The term “effective amount” also refers to the amount of a compound that is sufficient to elicit the biological or medical response of a cell, tissue, system, animal, or human, which is being sought by a researcher, veterinarian, medical doctor, or clinician.
As used herein, the term “subject” refers to any member of the animal kingdom including humans. In some embodiments, “subject” refers to humans, at any stage of development. In some embodiments, “subject” refers to a human patient. In some embodiments, “subject” refers to non-human animals. In some embodiments, the non-human animal is a mammal (e.g. a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate or a pig). In some embodiments, subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish or worms. In some embodiments, a subject may be a transgenic animal, genetically engineered animal or a clone.
As used herein, the term “treating” or “treatment” includes, but is not limited to, methods and manipulations to produce beneficial changes in a recipient's health status. The changes can be either subjective or objective and can relate to features such as symptoms or signs of the viral infection being treated. Preventing the deterioration of a recipient's status is also included by the term. Treating, as used herein, also includes administering the compound of Formula (I) alone or in combination with an additional therapeutic agent to a subject having a viral infection or is at risk of becoming a viral infection.
As used herein, the term “administering” includes activities associated with providing a patient an amount of a compound described herein, e.g. a compound of Formula (I). Administering includes providing unit dosages of compositions set forth herein to a patient in need thereof. Administering includes providing effective amounts of compounds, e.g. a compound of Formula (I), for a specified period of time, e.g. for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more days, or in a specified sequence, e.g. administration a compound of Formula (I) followed by the administration of one or more antiviral drugs, or vice versa.
As used herein, the term “co-administering” includes sequential or simultaneous administration of two or more structurally different compounds. For example, two or more structurally different pharmaceutically active compounds can be co-administered by administering a pharmaceutical composition adapted for oral administration that contains two or more structurally different active pharmaceutically active compounds. As another example, two or more structurally different compounds can be co-administered by administering one compound and then administering the other compound. In some instances, the co-administered compounds are administered by the same route. In other instances, the co-administered compounds are administered via different routes. For example, one compound can be administered orally, and the other compound can be administered, e.g. sequentially or simultaneously, via intravenous or intraperitoneal injection.
The compound of Formula (I) or optionally the additional therapeutic agents can be administered in admixture with suitable pharmaceutical diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral administration. The compound of Formula (I) or optionally the additional therapeutic agents can be administered alone but is generally mixed with a pharmaceutically acceptable carrier and co-administered in the form of a tablet or capsule, liposome or as an agglomerated powder. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents flow-inducing agents and melting agents.
Compositions may further comprise one or more pharmaceutically acceptable additional ingredient(s) such as alum, stabilizers, antimicrobial agents, buffers, coloring agents, flavoring agents, adjuvants, and the like.
Compositions may be in the form of tablets or lozenges formulated in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients including, but not limited to, binding agents, fillers, lubricants, disintegrants and wetting agents. Binding agents include, but are not limited to, syrup, acacia, gelatin, sorbitol, tragacanth, mucilage of starch and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, maize starch, calcium phosphate, and sorbitol. Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica. Disintegrants include, but are not limited to, potato starch and sodium starch glycollate. Wetting agents include, but are not limited to, sodium lauryl sulfate). Tablets may be coated according to methods well known in the art.
Compositions may also be liquid formulations including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs. The compositions may also be formulated as a dry product for constitution with water or other suitable vehicle before use, such liquid preparations may contain additives including, but not limited to, suspending agents, emulsifying agents, nonaqueous vehicles and preservatives. Suspending agent include, but are not limited to, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel, and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Nonaqueous vehicles include, but are not limited to, edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol. Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid.
As used herein the viral infection, especially acute viral infection, is selected from coronavirus infections, SARS-COV-2 (COVID-19), SARS, flu/influenza (and avian influenza), HIV/Aids, chickenpox (Varicella), cytomegalovirus, Dengue Fever, German measles (Rubella), hand-foot-mouth disease, hantavirus infections, all forms of hepatitis, Lassa fever, Marburg virus infections, measles, meningitis, MERS-COV, mumps, norovirus infections, herpes simplex virus infections, smallpox, monkeypox, rotavirus infections, Ebola virus, poliovirus infections, rhinovirus infections, parainfluenzavirus infections, RSV infections, HCMV infections and bannavirus infections. Preferred is SARS-COV-2 (COVID-19), flu/influenza, rhinovirus and respiratory syncytial virus (RSV) infections. More preferred is SARS-COV-2 (COVID-19), flu/influenza and rhinovirus infections, most preferred is SARS-COV-2 (COVID-19). It is understood, that also mutated forms of the virus (e.g. of SARS-COV-2) are covered.
A virus, especially SARS-COV-2, is constantly mutating, which many increase virulence and transmission rates. Drug-resistant variants of viruses may emerge after prolonged treatment with an antiviral agent. Drug resistance may occur by mutation of a gene that encodes for an enzyme used in viral replication. The efficacy of a drug against an RNA virus infection in certain cases can be prolonged, augmented or restored by administering the compound in combination or alternation with another and perhaps even two or three other, antiviral compounds that induce a different mutation or act through a different pathway, from that of the principal drug. A variant of a known virus can refer to a virus carrying one or more nucleotide mutations in the viral genome as compared to the known virus, for instance at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 60, 100, 200, 300 or even more nucleotide mutations. Mutations can refer to nucleotide deletion, insertion, or substitution. In some cases, a variant can have at most 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2% or 1% of the viral genome different than the genome of a known virus.
In particular embodiments of the present invention, a compound according to Formula (I) is for use as a medicament and/or for use in therapy. In a more particular embodiment of the present invention, the therapy is to prevent and/or treat a diseases caused by viral infection in a mammalian subject. In an even more particular embodiment of the present invention, the disease is caused by an RNA virus, such as, for example HIV, HCV, Ebola, rotavirus, Zika virus, polio virus, rhinovirus, hepatitis A virus, measles virus, mumps virus, RSV, rabies, Lassa virus, hantavirus, or influenza, in particular a single stranded RNA virus, such as HCoV-229E, HCoV-NL63, or betacoronaviruses, such as HCoV-OC43, SARS-COV-1, HCoV-HKU1, MERS-CoV or SARS-COV-2. In an even more particular embodiment of the present invention, the disease is caused by SARS-COV-2, rhinovirus, RSV and influenza virus. In a most particular embodiment of the present invention, the disease is caused by SARS-COV-2.
In other particular embodiments of the present invention, a compound according to Formula (I) is for use as a medicament and/or for use in therapy. In a more particular embodiment of the present invention, the therapy is to prevent and/or treat a diseases caused by viral infection in a mammalian subject. In an even more particular embodiment of the present invention, the disease is selected from AIDS, hepatitis, ebola, polio, diarrhea, measles, mumps, rabies, Lassa fever, viral flu, respiratory disease, acute respiratory disease, sepsis, acute respiratory distress syndrome, long COVID, and adverse immune reactions, in particular moderate to severe cases of said diseases, and wherein said disease preferably is selected from diseases causes by SARS-COV-2, in particular COVID-19, such as acute respiratory disease, sepsis, acute respiratory distress syndrome, long COVID and adverse immune reactions, such as a cytokine storm. In an even more particular embodiment of the present invention, the disease is COVID-19, viral flu, respiratory disease and acute respiratory disease. In a most particular embodiment of the present invention, the disease is COVID-19.
In other particular embodiments of the present invention, a compound according to Formula (I) is for use as a medicament in combination with a standard antiviral therapy (SAT). In a more particular embodiment of the present invention, this combination is with an DHODH inhibitor, and optionally with a standard antiviral therapy (SAT). In a more particular embodiment of the present invention, the DHODH inhibitor is selected from vidofludimus, vidofludimus calcium (IMU-838), teriflunomide, leflunomide, emvododstat (PTC299), brequinar, farudostat (ASLAN003), PP-001, RP7214, orludodstat (BAY2402234), JNJ-74856665, AG-636, MEDS433. In an equally more particular embodiment of the present invention, the DHODH inhibitor consists of Formula (II).
In other particular embodiments of the present invention, a compound according to Formula (1) is prepared by reacting citicoline or salts thereof with hydroxylamine or salts thereof. In a more particular embodiment of the present invention, a compound according to Formula (I) is prepared by reacting citicoline or salts thereof with hydroxylamine free base, hydroxylamine sulfate or hydroxylamine hydrochloride. In an even more particular embodiment of the present invention, a compound according to Formula (I) is prepared by reacting citicoline or salts thereof with 1 to 6 equivalents of hydroxylamine sulfate or hydroxylamine hydrochloride.
The introduction of the N4-hydroxy moiety into a cytidine derivative has been described e.g. by Paymode et al. (Org. Process Res. Dev. 2021; 25:1822) using hydroxylamine sulfate in water at elevated temperatures or by Purohit et al. (J. Med. Chem. 2012; 55:9988) or Vasudevan et al. (Chem. Commun. 2020; 56:13363) using hydroxylammonium acetate in water at 37° C. to 40° C. at pH 5.5 to 6.0.
Citicoline sodium (CAS: 33818-15-4) is commercially available, e.g. from MedChemExpress (Art.-Nr.: HY-B0739A) and has the following analytical data: 1H-NMR (400 MHZ, D2O) 0 7.84 (d, J=7.6 Hz, 1H), 6.01 (d, J=7.6 Hz, 1H), 5.89 (d, J=4.0 Hz, 1H), 4.30-4.06 (m, 7H), 3.59-3.57 (m, 2H), 3.12 (s, 9H). ESI-MS m/z calcd for [C14H27N4O11P2+] [M] +: 489.1; found: 489.0.
2-(((((((2R,3S,4R,5R)-3,4-Dihydroxy-5-(4-(hydroxyamino)-2-oxopyrimidin-1 (2H)-yl) tetrahydrofuran-2-yl) methoxy) (hydroxy) phosphoryl) oxy) oxidophosphoryl) oxy)-N,N,N-trimethylethan-1-aminium mono sodium salt (1)
To a suspension of citicoline sodium (200 mg, 392 umol) in EtOH (5 mL) was added 5N aqueous hydroxylamine hydrochloride until the solution was adjusted to pH=5 to 6. The mixture was stirred for 8 days at 37° C. with adding 5N aqueous hydroxylamine hydrochloride intermittently to maintain the pH value. LCMS analysis indicated that most of the starting material was consumed. The solvent was removed under reduced pressure and the residue was purified by chromatography using diethylaminoethyl (DEAE) Sephadex A-25 (CAS 12609-80-2) (100 mM triethylammonium bicarbonate/H2O gradient from 0/1 to 3/7, 20 mL/min, UV detection 254 nm). Lyophilization of the fractions gave a triethylammonium salt of the desired product. The prepared triethylammonium salt was dissolved in water and eluted through the ion-exchange column (Amberlite IR120 sodium form). Fractions containing the product were combined and lyophilized to give compound 1 (60.4 mg, 24%) as an off-white solid. 1H-NMR (400 MHZ, D2O)δ7.08 (d, J=8.4 Hz, 1H), 5.87-5.84 (m, 1H), 5.73 (d, J=8.4 Hz, 1H), 4.30-4.21 (m, 4H), 4.14-4.13 (m, 1H), 4.08-4.03 (m, 2H), 3.59-3.57 (m, 2H), 3.12 (s, 9H). ESI-MS m/z calcd for [C14H27N4012P2+] [M] +: 505.1; found: 505.0. Elemental analysis:
Found: C: 26.19% H: 5.99% N: 8.68%
Calc.: C: 26.14% H: 5.95% N: 8.71% for C14H25N4NaO12P2·6.5H2O
2-(((((((2R,3S,4R,5R)-3,4-Dihydroxy-5-(4-(hydroxyamino)-2-oxopyrimidin-1 (2H)-yl) tetrahydrofuran-2-yl) methoxy) (hydroxy) phosphoryl) oxy) oxidophosphoryl) oxy)-N,N,N-trimethylethan-1-aminium triethylammonium salt (2)
To a solution of citicoline (10.0 g) in H2O (100 mL) was added hydroxylamine hydrochloride (2.8 g, 2 eq.) at 38° C. The mixture was stirred at 38° C. for 5 days. Another batch of hydroxylamine hydrochloride (2.6 g, 50% in water, 2 eq.) was added and the reaction mixture was stirred 38° C. for another 2 days. The third batch of hydroxylamine hydrochloride (2.6 g, 50% in water, 2 eq.) was added and the reaction mixture was stirred 38° C. for another 2 days. The mixture was concentrated under reduced pressure and washed with MeOH (30 mL) to give a crude product. The resulting crude was dissolved in H2O and directly purified by prep-HPLC (column: Phenomenex Luna C18 250 mm×100 mm×10 um; mobile phase: [water (20 mM triethylammonium bicarbonate)-ACN]; B %: 0%-5%, 20 min) to get pure compound 2 (1.0 g, 7.3%, as 1.65 TEA salt) as a white solid. HPLC: 214 nm: 98.52% purity; 254 nm: 99.87% purity; 260 nm: >99.9% purity.
The aqueous solubility (in PBS, PH 7.4-cat ref: 435; in simulated intestinal fluid-cat ref: 2062 and in simulated gastric fluid-cat ref: 2061), partition coefficient (logD, n-octanol/PBS, PH 7.4; cat ref: 417) and permeability (Caco-2, pH 6.5/7.4; cat ref: 3318 and 3320) of Example 1 and comparative example (see Scheme 1) were measured at Eurofins according their standard procedures and furnished following results:
Conclusion: Although Example 1 is similar lipophilicity compared to molnupiravir and NHC according the logD, the aqueous solubility in PBS, simulated intestinal fluid and simulated gastric fluid is much worser (i.e. practically insoluble). The solubility of NHC and molnupiravir is above the measurement limit of the assay, and indeed, for molnupiravir a high solubility of 121 mM (39.7 mg/mL) in water has been mentioned in the assessment report for use of molnupiravir for the treatment of COVID-19 published by the Committee for Medicinal Products for Human Use (CHMP):
www.ema.europa.eu/en/documents/referral/lagevrio-also-known-molnupiravir-mk-4482-covid-19-article-53-procedure-assessment-report_en.pdf
For NHC a high solubility of 58 mM (15 mg/mL) in water has been mentioned upon slight warming: www.cellsignal.com/products/activators-inhibitors/beta-d-n4-hydroxycytidine/81178 Nevertheless, the permeability of zwitterionic Example 1 is improved by about two orders of magnitude compared to molnupiravir, NHC and NHC-DP, allowing a beneficial biopharmaceutics classification of Class Il (low solubility, high permeability).
The present study aimed at the determination of pharmacokinetic parameters of Example 2 and it's metabolites (NHC, NHC-MP, NHC-DP and NHC-TP) in blood after single dose oral administration of 96.5 μmol/kg to female C57BL/6 mice (body weight 20 to 21 g). Blood samples were obtained from the retrobulbar venous plexus into Li-Heparin tubes (Minivette POCT, SARSTEDT). The vehicle for preparation of the dose formulations was PBS. The samples were analysed by high resolution LC-MS at Pharmacelsus GmbH, Germany.
After oral administration of 96.5 μmol/kg Example 2 the test item was not found in all blood samples (below lower limit of quantification of 14.4 nM). The metabolite NHC-DP reached a maximal blood concentration of 923+138 nM 4 h post dose and it's metabolite NHC-TP reached a maximal blood concentration of 611+100 nM 4 h post dose. The elimination half-life and the AUC (0-inf) were not calculable in both metabolites. Furthermore it's metabolite NHC-MP reached a maximal blood concentration of 274+41.4 nM 30 min post dose. The elimination half-life was 7.3 h and the AUC (-inf) was 1624 nM*h. The metabolite NHC reached a maximal blood concentration of 4239+902 nM 30 min post dose. The elimination half-life was 1.7 h and the AUC (0-inf) was 5761 nM*h.
Conclusion:
Step 1:2-Fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) aniline (50a)
To a solution of 4-bromo-2-fluoroaniline (4.00 g, 21.1 mmol) in 1,4-dioxane (30 mL) was added bis (pinacolato) diboron (5.38 g, 21.2 mmol), KOAc (6.23 g, 63.5 mmol) and Pd (dppf) Cl2 (776 mg, 1.1 mmol). Then the mixture was heated at 90° C. for 1 h, cooled to rt, filtered, concentrated and purified by FCC(PE:EA=8:1) to give compound 50a as a white solid.
Step 2:3-Fluoro-3′-(methoxy-d3)-[1,1′-biphenyl]-4-amine (50b)
To a solution of compound 50a (800 mg, 3.37 mmol) in 1,4-dioxane (10 mL) and H2O (1 mL) was added 1-bromo-3-(methoxy-d3) benzene (638 mg, 3.36 mmol), Na2CO3 (1.07 g, 10.1 mmol) and Pd (dppf) Cl2 (124 mg, 0.17 mmol) and then the mixture was heated at 90° C. for 2 h, cooled to rt, filtered, concentrated and purified by FCC (PE:EA=10:1) to give compound 50b as an oil.
Step 3:2-((3-Fluoro-3′-(methoxy-d3)-[1,1′-biphenyl]-4-yl) carbamoyl) cyclopent-1-ene-1-carboxylic acid (50)
A solution of compound 50b (120 mg, 545 μmol) and 1-cyclopentene-1,2-dicarboxylic anhydride (74 mg, 540 μmol) in DCM (2.5 mL) was heated at 40° C. for 4 h. The mixture was filtered and the filter cake washed with MeCN (2×2 mL). The solid was dried in vacuum to afford compound 50 as a pale yellow solid. 1H-NMR (400 MHZ, DMSO-d6) δ13.04 (br s, 1H), 10.58 (s, 1H), 8.07 (t, J=8.4 Hz, 1H), 7.63 (d, J=12.4 Hz, 1H), 7.53 (d, J=8.4 Hz, 1H), 7.37 (t, J=8.0 Hz, 1H), 7.27-7.23 (m, 2H), 6.94 (dd, J=8.0, 2.0 Hz, 1H), 2.80 (br s, 2H), 2.69 (br s, 2H), 1.93-1.85 (m, 2H). LCMS (ESI): m/z 359.0 (M+H)*.
Step 1:2,6-Difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) aniline (51a)
To a solution of 4-bromo-2,6-difluoroaniline (10 g, 48 mmol) in 1,4-dioxane (100 mL) was added bis (pinacolato) diboron (12.8 g, 50.4 mmol), CH3COOK (14.1 g, 144 mmol) and Pd (dppf) Cl2 (1.0 g, 2.40 mmol). The mixture was stirred at 90° C. under N2 for 2 h, cooled to rt, concentrated and purified by FCC (PE:EA=10:1) to give compound 51a as a yellow solid. Step 2:3,5-Difluoro-3′-(methoxy-d3)-[1,1′-biphenyl]-4-amine (51b)
To a solution of compound 51a (4.5 g, 13.3 mmol) in 1,4-dioxane (50 mL) and H2O (5 mL) was added 1-bromo-3-(methoxy-d3) benzene (3.34 g, 13.3 mmol), Na2CO3 (5.61 g, 39.4 mmol) and Pd (dppf) Cl2 (400 mg, 0.67 mmol). The mixture was stirred at 90° C. under N2 for 2 h, cooled to rt, concentrated and purified by FCC (PE:EA=10:1) to give compound 51b as a yellow solid. LCMS (ESI): m/z 239.1 (M+H)*.
Step 3:2-((3,5-Difluoro-3′-(methoxy-d3)-[1,1′-biphenyl]-4-yl) carbamoyl) cyclopent-1-ene-1-carboxylic acid (51)
To a solution of compound 51b (3.40 g, 14.3 mmol) in DCM (20 mL) were added 1-cyclopentene-1,2-dicarboxylic anhydride (1.90 g, 14.3 mmol) and then the mixture was stirred at rt for 2 h. The mixture was filtered and the filter cake washed with MeCN. The solid was dried in vacuum to afford compound 51 as a white solid. 1H-NMR (500 MHZ, DMSO-d6) δ12.95 (br s, 1H), 10.13 (s, 1H), 7.55 (d, J=8.0 Hz, 2H), 7.39 (t, J=7.8 Hz, 1H), 7.32-2.83 (m, 2H), 6.99 (dd, J=1.8, 8.3 Hz, 1H), 2.81-2.79 (m, 2H), 2.69-2.66 (m, 2H), 1.97-1.89 (m, 2H). LCMS (ESI): m/z 377.3 (M+H)*.
The in vitro inhibition of hDHODH was measured using an N-terminally truncated recombinant hDHODH enzyme as described in J. Med. Chem. 2006; 49:1239. Briefly, the hDHODH concentration was adjusted in a way that an average slope of approximately 0.2 AU/min served as the positive control (e.g. without inhibitor). The standard assay mixture contained 60 UM 2,6-dichloroindophenol, 50 μM decylubiquinone and 100 UM dihydroorotate. The hDHODH enzyme with or without at least six different concentrations of the compounds was added and measurements were performed in 50 mM TrisHCI, 150 mM KCl and 0.1% Triton X-100 at pH 8.0 and at 30° C. The reaction was started by adding dihydroorotate and measuring the absorption at 600 nm for 2 min. For the determination of the IC50 values, each data point was recorded in triplicate. For the determination of the inhibitory constant Ki, the KM values for DHO and decylubichinon were determined. Afterwards, the compounds were diluted in a dilution series depending on their IC50 values in DMSO. The dilution was: 0 ×IC50 , 1/4×IC50 , 1/2×IC50 , 1×IC50 , 2×IC50 , 4×IC50 . In addition, the substrate concentration for DHO and decylubichinon were varied 1/4×KM, 1/2×KM, 1×KM, 2×KM, 4×KM in further dilution series with separate measurement of DHO and decylubiquinone. Each data point was recorded in duplicate.
The Ki values for examples of the present invention were in the range of the non-deuterated matched pair (Example C26 from WO2003/006425):
As shown above, the DHODH inhibition of deuterated analog 50 compared to the non-deuterated matched pair (Example C26 from WO2003/006425) is not affected, however the microsomal stability is improved.
The viral replication (YFP) and cell viability assay has been described in Pathogens 2021; 10:1076 and furnished the following results:
Contrary to citicoline (C1), its analog 1 showed antiviral activity against SARS-COV-2. In addition, the DHODH inhibitors 50 and 51 show similar antiviral activity.
The synergistic potential of compound 1 together with the DHODH inhibitor 50
The method of combinatorial drug assessment by a viral replication inhibition assay has been published in Pathogens 2021; 10:1076. Caco-2 cells were cultivated in 96-well plates at 25000 cells/well, infected with SARS-COV-2 d6-YFP at an MOI of 0.003 and treated with Example 50, Example 1 or a combination of the drugs, starting at the respective 4×EC50 concentrations of the single compounds. Viral replication was determined as 30 h post infection (p.i.) by quantitative fluorescence detection of virus-driven YFP expression in the fixed cells. Inhibitory profiles of viral replication measured through virus-encoded YFP reporter expression are presented in a bar chart of quadruplicate determinations (mean+SD). The combinatorial drug assessment was calculated by using the CompuSyn algorithm as described in Int. J. Mol. Sci. 2021; 22:575.
A representative experiment is shown in
In collaboration with the National Institute of Health (NIH), the antiviral activity of the nucleoside analog Example 1 was tested against human rhinovirus-14 (HRV-14), influenza A and respiratory syncytial virus (RSV) A2. Hela cells were used for HRV-14, MDCK cells for influenza A and MA-104 cells for RSV A2. Cells were treated with a serial log 10 dilution of Example 1 and subsequently infected with human rhinovirus 14 (HRV-14), influenza A (H1N1, California/07/2009) or RSV A2. EC50 concentration was determined by linear regression and CC50 concentration by neutral red. For Example 1, the following measured values were obtained:
Furthermore, in vitro combination assays (AP2B) with DHODH inhibitor Example 50 and nucleoside analog Example 1 were performed to investigate antiviral activity against RSV and HRV-14. For both viruses, there was an improvement in antiviral activity after combining the two compounds.
RSV: MA-104 cells were treated either in monotherapy with a serial dilution of Example 50 or Example 1, or a combination of 25 UM of Example 50 with serial dilution of Example 1 and then infected with RSV A2. Virus concentrations were determined by endpoint titration (CCID50) and yielded the following results:
HRV-14: Hela cells were treated either in monotherapy with a serial dilution of Example 50 or Example 1, or a combination of 25 UM of Example 50 with serial dilution of Example 1 and then infected with HRV-14. Virus concentrations were determined by endpoint titration (CCID50) and yielded the following results:
Conclusion: Combination of a DHODH inhibitor with Example 1 showed a synergistic behavior also on human rhinovirus-14 (HRV-14) and respiratory syncytial virus (RSV).
| Number | Date | Country | Kind |
|---|---|---|---|
| 21205795.4 | Nov 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/080430 | 11/1/2022 | WO |
