CN117642410A

CN117642410A - Ester derivatives of N4-hydroxycytidine and uses thereof

Info

Publication number: CN117642410A
Application number: CN202280040667.7A
Authority: CN
Inventors: 赵祖春; 许凯
Original assignee: Suzhou Chunhai Biopharmaceutical Co ltd
Current assignee: Suzhou Chunhai Biopharmaceutical Co ltd
Priority date: 2021-06-18
Filing date: 2022-06-17
Publication date: 2024-03-01
Also published as: WO2022262845A1

Abstract

The present disclosure relates to N ⁴ Ester derivatives of hydroxycytidine (NHC), pharmaceutical compositions comprising the same, processes for the preparation thereof, and N ⁴ Use and method of ester derivatives of hydroxycytidine for the treatment of viral infections.

Description

Ester derivatives of N4-hydroxycytidine and uses thereof

Cross Reference to Related Applications

The present application claims priority from international application PCT/CN2021/101005 filed on 18/6/2021; the contents of which are incorporated herein by reference in their entirety.

Technical Field

The present disclosure relates to N ⁴ Ester derivatives of hydroxycytidine (NHC), to pharmaceutical compositions comprising same, and N ⁴ Use of an ester derivative of hydroxycytidine for the treatment of a viral infection. These compounds may be administered orally to provide N ⁴ -hydroxycytidine.

Background

Currently, the virus SARS-CoV-2, which causes COVID-19, has been worldwide infected with more than 1 million people and causes hundreds of thousands of people to die without signs of slowing. World economy and human activity have been subject to very large negative impacts. Despite the recent introduction of vaccines, patients infected with oral medications remain highly desirable and may be supplemented with the use of vaccines. N (N) ⁴ Hydroxycytidine (NHC) is a ribonucleoside analogue with broad-spectrum antiviral activity against a number of different RNA viruses, including influenza, ebola, coV and Venezuelan Equine Encephalitis Virus (VEEV), and most importantly human SARS-CoV-2 virus. Although the exact molecular mechanism of action of NHC is still uncertain, it has been proposed that the occurrence of false disasters due to NHC involvement in viral replication is the basis for NHC antiviral activity [ "Characterization of orally efficacious influenza drug with high resistance barrier in ferrets and human airway epithelia", sci tranl med.2019, month 10, 23;11 (515) eaax5866]This results from the tautomeric nature of NHCs:

NHCs can exist in two tautomeric forms, i.e., hydroxylamine in the molecule can be in both oxime and hydroxylamine forms. The oxime form of NHC mimics uridine, matches adenosine upon viral replication (lower left structure), while the other tautomer hydroxylamine mimics cytidine, matches guanosine (lower right structure). Such a mismatch may result in a viral false disaster.

N ⁴ A prodrug of hydroxycytidine (NHC), mo Nuola Wer/EIDD 2801/MK4486, is being tested in clinical trials for the treatment of SARS-CoV-2, a virus responsible for COVID-19. Phase I clinical trial data reported indicate that the molecule is safe and well tolerated [1 ] ]. An ongoing phase III clinical trial for treating patients with early SARS-CoV-2 infection with a planned dose of 800mg twice daily [2 ]]. Both large doses and BID administration require sustained effective concentrations of NHC in humans, thereby inducing a viral false disaster. Thus, there remains a need for more and potentially better prodrugs (i.e., smaller pills, less frequent administration and higher efficacy) for the treatment of viral infections, particularly for the emergency treatment of current worldwide human disasters.

Disclosure of Invention

The present inventors have found a series of N ⁴ Ester derivatives of hydroxycytidine (NHC), which can deliver NHC in the animal's bloodstream with improved bioavailability and prolonged exposure time compared to the parent molecule NHC.

The present disclosure relates to certain ester prodrugs of NHCs, combinations, pharmaceutical compositions, uses, and methods related thereto.

The present disclosure provides compounds of formula (I):

or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, wherein

R ¹ Is rac=o;

R ² is H or RaC=O, R ³ Is H or rac=o; or R is ² And R is ³ In combination with oxygen to which they are attachedTo a 5-membered heterocycloalkyl substituted by C1-6 alkyl-O;

ra is methyl substituted with Ra1, ra2 and Ra 3;

ra1 is selected from H, C _1-6 Alkyl, C _1-6 alkyl-O-C _1-6 Alkyl, C _1-6 -haloalkyl, C _3-6 Cycloalkyl, C _3-6 Halogenated cycloalkyl, 3-6 membered heterocycloalkyl, 3-6 membered halogenated heterocycloalkyl, and C _1-6 alkyl-O- (CH) ₂ ) _n -、C _1-6 alkyl-O-C _1-6 alkyl-O- (CH) ₂ ) _n -、C _1-6 haloalkyl-O- (CH) ₂ ) _n -,C _3-6 cycloalkyl-O- (CH) ₂ ) _n -、C _3-6 Halogenated cycloalkyl-O- (CH) ₂ ) _n -, 3-6 membered heterocycloalkyl-O- (CH) ₂ ) _n -and 3-6 membered haloheterocycloalkyl-O- (CH) ₂ ) _n -；

Ra3 is selected from H, C _1-6 Alkyl and C _1-6 alkyl-O- (CH) ₂ ) _n -；

Or Ra2 and Ra3 together with the carbon to which they are attached form C _3-6 Cycloalkyl or 5-6 membered halogenoalkyl containing 1 ring heteroatom selected from O; and

n is 0 or 1;

provided that when Ra3 is H or C _1-6 In the case of alkyl groups, ra1 is not H, C _1-6 Alkyl, C _1-6 alkoxy-C _1-6 Alkyl, C _1-6 -haloalkyl or C _3-6 Any one of cycloalkyl groups.

The above-mentioned compounds and the compounds disclosed hereinafter (including the compounds of formula (I) and specific compounds, particularly the compounds of the examples) or tautomers, stereoisomers, enantiomers, diastereomers, racemates, geometric isomers, hydrates or solvates thereof or pharmaceutically acceptable salts thereof, are collectively referred to as "the compounds of the present invention" or "the compounds of the present disclosure".

The present disclosure also provides compounds of the invention for use as a medicament.

The present disclosure also provides compounds of the invention for use in the treatment or prevention of RNA viral infection.

The present disclosure also provides pharmaceutical compositions comprising a compound of the present invention, and optionally comprising a pharmaceutically acceptable excipient.

The present disclosure also provides a kit for treating or preventing an RNA viral infection comprising the pharmaceutical composition of the present disclosure and instructions for use.

The present disclosure also provides the use of a compound of the invention in the manufacture of a medicament for the treatment or prophylaxis of an RNA viral infection.

The present disclosure also provides the use of a compound of the invention for the treatment or prevention of RNA viral infection.

The present disclosure also provides a method of treating or preventing an RNA viral infection in a subject comprising administering to a subject in need thereof an effective amount of a compound of the present invention.

The present disclosure also provides for increasing N ⁴ A method of bioavailability of hydroxycytidine for the treatment or prophylaxis of RNA viral infection comprising administering to a subject in need thereof an effective amount of a compound of the invention.

The present disclosure also provides pharmaceutical combinations comprising a compound of the present invention and at least one additional therapeutic agent.

The present disclosure also provides methods of preparing the compounds of the present invention, as well as intermediates for preparing the compounds of the present invention.

Additional advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.

Drawings

The drawings described herein are for illustration purposes only. The drawings are not intended to limit the scope of the present disclosure.

FIG. 1 shows mean+ -SD plasma concentration-time data (N=3/time point) after oral doses of EX-2C, mo Nuola Wer and NHC, respectively, in male CD1 mice for EX-2C, mo Nuola Wer and NHC, examples

Detailed Description

Definition of the definition

As used herein, words, phrases and symbols are generally intended to have the meanings set forth below, unless the context of their use indicates otherwise.

As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The compounds of the present invention may be identified by their chemical structure and/or chemical name. When chemical structures and chemical names conflict with each other, the chemical structure plays a decisive role in the identity of the compound.

Herein the symbolsOr->It is meant that the relevant structures are tautomers that exist in equilibrium and are readily converted from one isomeric form to another. The compounds of the present invention may exist in oxime forms as well as other forms. Thus, the chemical structures described herein include all possible tautomeric forms of the compounds shown, particularly the tautomers of the oxime forms and other forms of tautomers. Regardless of which tautomer is shown, and regardless of the nature of the equilibrium between the tautomers, those skilled in the art understand that the compounds of the invention include oxime forms and other forms.

"bioavailability" refers to the rate and amount of a drug that reaches the systemic circulation of an individual after administration of the drug or a prodrug thereof, and can be determined by evaluating, for example, the plasma or blood concentration versus time profile of the drug. Parameters that may be used to characterize a plasma or blood concentration versus time curve include the area under the curve (AUC), the time to maximum concentration (Tmax), which is the maximum concentration of a drug in the plasma or blood of a subject after administration of a dose or form of the drug to the subject, and the maximum drug concentration (Cmax), which is the time to maximum concentration of the drug in the plasma or blood of the subject after administration of a dose or form of the drug to the subject.

Prodrugs are derivatized forms of a drug that are converted or metabolized in vivo after administration to the active form of the parent drug. Prodrugs are used to modify one or more aspects of the pharmacokinetics of the drug to enhance the therapeutic effect of the parent drug. For example, prodrugs are often used to increase the oral bioavailability of drugs. For therapeutic effects, drugs with poor oral bioavailability may require frequent dosing, high dose administration, or may require administration by a route other than oral, such as intravenous administration. Examples of prodrugs that can be used to improve bioavailability include esters, optionally substituted esters, branched esters, optionally substituted branched esters.

"metabolic intermediate" refers to a compound that is formed in vivo by the metabolism of the parent compound and that further reacts in vivo to release the active agent. The compounds of formula (I) are protected ester prodrugs which, after in vivo metabolism, provide the corresponding metabolic intermediates, such as N4-hydroxycytidine (NHC). It is desirable that the reaction product or its metabolite is not toxic.

"individual" refers to a mammal, such as a human.

By "pharmaceutically acceptable" is meant approved or approvable by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

By "pharmaceutically acceptable salt" is meant a salt of a compound that has the desired pharmacological activity of the parent compound. Such salts include acid addition salts formed from one or more protonatable functional groups in the mineral acid and parent compound, such as hydroxylamine. Examples of the inorganic acid include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts may be formed with organic acids such as acetic acid, propionic acid, caproic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1, 2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, t-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like. "pharmaceutically acceptable salts" also include base addition salts formed from compounds of the invention which carry an acidic moiety with a pharmaceutically acceptable cation, such as sodium, potassium, calcium, aluminum, lithium and ammonium.

As used herein, "pharmaceutical combination" refers to a product that is a mixture or combination of more than one therapeutic agent, and includes both fixed and non-fixed combinations of therapeutic agents. The term "fixed combination" refers to a therapeutic agent (e.g., a compound of the invention) and the at least one additional therapeutic agent, both administered to a subject simultaneously in the form of a single entity or dose. The term "non-fixed combination" refers to a therapeutic agent (e.g., a compound of the invention) and the at least one additional therapeutic agent, both administered simultaneously, together or sequentially as separate entities to a subject without specific time constraints, wherein such administration provides therapeutically effective levels of the active agent in the subject.

"preventing" refers to reducing the risk of acquiring a disease or disorder, such as a viral infection, (at least one clinical symptom of the disease does not occur even in individuals who may be exposed to the disease or have a predisposition to the disease but have not yet experienced or displayed symptoms of the disease). In some embodiments, "preventing" refers to reducing symptoms of a disease by taking the compound in a prophylactic manner. Therapeutic application for the prevention of diseases or disorders is referred to as prophylaxis. The compounds provided by the present disclosure may provide excellent preventive effects due to having antiviral activity.

"treating" a disease or disorder, such as a viral infection, refers to preventing or ameliorating a disease or at least one clinical symptom of a disease or disorder, reducing the risk of acquiring at least one clinical symptom of a disease or disorder, reducing the development of at least one clinical symptom of a disease or disorder, or reducing the risk of developing at least one clinical symptom of a disease or disorder. "treating" also refers to inhibiting a disease, which may be physical (e.g., stabilizing a recognizable symptom), physiological (e.g., stabilizing a physical parameter), or both, and inhibiting at least one physical parameter or manifestation, which may or may not be recognizable by an individual. "treating" also refers to delaying the onset of a disease (e.g., viral infection), or at least one or more symptoms thereof, in an individual who may be exposed to or predisposed to a disease or disorder, even though the subject has not experienced or displayed symptoms of the disease.

The term "effective amount" as used herein refers to an amount of a compound of the invention that is effective to "treat" or "prevent" a viral infection in an individual as defined above. An effective amount may cause any observable or measurable change in an individual as described in the definition of "treatment" or "prevention" above. The "effective amount" may vary depending on, for example, the compound, the disease and/or disease symptoms, the severity of the disease and/or disease or disorder symptoms, the age, weight and/or health of the individual to be treated, and the discretion of the prescribing physician. In any given case, the appropriate amount may be determined by one of ordinary skill in the art, or may be determined by routine experimentation.

As used herein, "alkyl" refers to a straight or branched saturated hydrocarbon moiety, such as (C) containing from 1 to 6 carbon atoms _1-6 ) Preferably 1 to 4 carbon atoms (C _1-4 ) Or 1 to 3 carbon atoms (C) _1-3 ) Those of (3). For example, "C _1-6 Alkyl "refers to an alkyl group having 1-6 (including 1, 2, 3, 4, 5, or 6) carbon atoms. Representative straight chain alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl; and branched alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.

The term "cycloalkyl" as used herein means a compound having 3 to 6 ring carbon atoms (C _3-6 ) For example 5 to 6 ring carbon atoms (C _5-6 ) Is a saturated cyclic hydrocarbon moiety of (2). Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. The term "halo-cycloalkyl" herein refers to a cycloalkyl group as defined above wherein one or more, for example 1, 2 or 3, hydrogen atoms are replaced by halogen atoms.

The term "heterocyclyl" as used herein refers to a saturated ring having 3-6 ring atoms (3-6 members), 4-6 ring atoms (4-6 members) or 5-6 ring atoms (5-6 members) wherein one or more, for example 1, 2 or 3, preferably 1 or 2, of the ring atoms are heteroatoms independently selected from N, O and S, preferably O, and the remaining ring atoms are carbon. Examples of heterocyclyl groups include, but are not limited to, morpholinyl, pyrrolidonyl, pyrrolidinyl, piperidinyl, oxiranyl, glycinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothienyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, 1, 3-dioxolane moieties, and the like. Preferably, the heterocyclyl is tetrahydrofuranyl or tetrahydropyranyl. The term "haloheterocycloalkyl" refers to a heterocycloalkyl group as defined above wherein one or more, for example 1, 2 or 3, hydrogen atoms are replaced by halogen atoms.

The terms "halogen" and "halo" refer to fluorine and chlorine.

The term "halo-alkyl" as defined herein refers to an alkyl group as defined herein wherein one or more, for example 1, 2, 3, 4 or 5 hydrogen atoms are replaced by halogen atoms.

The term "substituted" means that at least one hydrogen atom in a molecule is replaced by a substituent. When substituted, one or more groups are "substituents". The molecule may be multiply substituted.

The term "optionally" as used herein means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

All numerical ranges herein should be understood to disclose each and every value within the range, as well as each and every subset of values within the range, whether or not they are specifically disclosed. For example, when referring to any numerical range, it should be taken to refer to each and every value within that numerical range, e.g., each and every integer within that numerical range. The present disclosure includes all numbers falling within these ranges, all smaller ranges, and either the upper or lower limits of the ranges.

Technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains if not specifically defined.

The present disclosureDescription of the embodiments

Embodiment 1. A compound of formula (I):

R ¹ Is rac=o;

R ² is H or RaC=O, R ³ Is H or rac=o; or R is ² And R is ³ Together with the oxygen to which they are attached, form a 5 membered heterocycloalkyl substituted with C1-6 alkyl-O;

ra is methyl substituted with Ra1, ra2 and Ra 3;

Ra3 is selected from H, C _1-6 Alkyl and C _1-6 alkyl-O- (CH) ₂ ) _n -；

n is 0 or 1;

Embodiment 2. The compound according to embodiment 1 or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, wherein

R ¹ Is rac=o and,

R ² and R is ³ Is H, or rac=o in each of R2 and R3; or R2 and R3 together with the oxygen to which they are attached form a C-group _1-6 alkyl-O substituted 5 membered heterocycloalkyl;

ra is methyl substituted with Ra1, ra2 and Ra 3;

ra1 is selected from H, C _1-6 Alkyl, C _1-6 alkyl-O-C _1-6 Alkyl, C _1-6 -haloalkyl, C _3-6 Cycloalkyl, 3-6 membered halogenoalkyl, C _1-6 alkyl-O- (CH) ₂ ) _n -、C _1-6 alkyl-O-C _1-6 alkyl-O- (CH) ₂ ) _n -、C _1-6 haloalkyl-O- (CH) ₂ ) _n -、C _3-6 cycloalkyl-O- (CH) ₂ ) _n -and 3-6 membered heterocycloalkyl-O- (CH) ₂ ) _n -,

Ra2 is C _1-6 Alkyl or C _1-6 alkyl-O- (CH) ₂ ) _n -, and

ra3 is selected from H, C _1-6 Alkyl and C _1-6 alkyl-O- (CH) ₂ ) _n -；

n is 0 or 1;

Embodiment 3. A compound according to any one of the preceding embodiments, or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, provided that

When Ra3 is H or C _1-6 In the case of an alkyl group, the alkyl group,ra1 is not H, C _1-6 Alkyl, C _1-6 alkoxy-C _1-6 Alkyl, C _1-6- Haloalkyl or C _3-6 Any one of cycloalkyl groups; and

When Ra2 and Ra3 together with the carbon to which they are attached form C _3-6 In the case of cycloalkyl, ra1 is not H, C _1-6 Alkyl, C _1-6 alkoxy-C _1-6 Alkyl, C _1-6 -haloalkyl, C _3-6 Cycloalkyl radicals _3-6 Any one of the membered heterocycloalkyl groups.

Embodiment 4. A compound according to any one of the preceding embodiments, or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, wherein

R ¹ Is rac=o and,

R ² and R is ³ Is H, or rac=o in each of R2 and R3;

wherein rac=o is selected from:

raa is selected from C _1-6 Alkyl, C _1-6 Haloalkyl, C _1-6 -alkyl-O-C _1-6 Alkyl-, C _3-6 Cycloalkyl and 3-6 membered heterocycloalkyl; preferably C _1-6 An alkyl group.

Embodiment 5. A compound according to embodiment 4, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, wherein rac=o is selected from:

embodiment 6. A compound according to embodiment 4, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, wherein rac=o is selected from:

embodiment 7. A compound according to embodiment 4, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, wherein rac=o is selected from:

Embodiment 8. A compound according to embodiment 4, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, wherein rac=o is selected from:

embodiment 1. Embodiment 9. A compound according to any one of the preceding embodiments, or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, wherein Raa is selected from C _1-4 Alkyl, C _1-4 Haloalkyl, C _1-4 -alkyl-O-C _1-4 Alkyl-, C _3-5 Cycloalkyl and 4-6 membered heterocycloalkyl.

Embodiment 10. A compound according to any one of the preceding embodiments, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, wherein Raa is selected from methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, 2-methoxyethyl, fluoro-substituted ethyl, fluoro-substituted propyl, cyclopropyl, cyclobutyl, cyclopentyl, glycidylyl, tetrahydro-2-furyl, tetrahydro-3-furyl, or tetrahydro-2H-pyranyl; methyl, ethyl, propyl, isopropyl, glycidylyl and tetrahydro-2H-pyran-4-yl are preferred.

Embodiment 11. A compound according to any one of the preceding embodiments, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, wherein Raa is selected from methyl, ethyl, propyl, isopropyl, n-butyl, and sec-butyl.

Embodiment 12. The compound according to embodiments 1-3, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, wherein

Ra1 and Ra3 are independently selected from H, C _1-6 Alkyl, C _1-6 alkyl-O-and C _1-6 alkyl-O-CH ₂ -；

Ra2 is selected from C _1-6 Alkyl, C _1-6 alkyl-O-and C _1-6 alkyl-O-CH ₂ -; or (b)

Ra2 and Ra3 together with the carbon to which they are attached form C _3-6 Cycloalkyl, or a 5-6 membered halogenated heterocyclyl containing one ring heteroatom selected from O;

provided that when Ra3 is H or C _1-6 Ra1 is not H or C when alkyl _1-6 An alkyl group.

Embodiment 13. A compound according to any one of the preceding embodiments, or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, wherein

Ra1 is selected from C _1-6 Alkyl, C _1-6 alkyl-O-and C _1-6 Alkyl O-CH2-.

Embodiment 14. A compound according to any one of the preceding embodiments, or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, wherein

Ra3 is selected from C _1-6 Alkyl, C _1-6 alkyl-O-and C _1-6 Alkyl O-CH2-.

Embodiment 15. The compound according to embodiments 1-3, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, wherein

Ra1 is C _1-6 alkyl-O-or C _1-6 alkyl-O-CH ₂ -,

Ra2 is selected from C _1-6 Alkyl, C _1-6 alkyl-O-and C _1-6 Alkyl O-CH2-, and

ra3 is selected from H、C _1-6 Alkyl, C _1-6 alkyl-O-and C _1-6 alkyl-O-CH ₂ -。

Embodiment 16. The compound according to embodiments 1-3, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, wherein

Ra1 is C _1-6 alkyl-O-,

ra2 is C _1-6 Alkyl group, and

ra3 is H or C _1-6 alkyl.

Embodiment 17. The compound according to embodiments 1-3, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, wherein

Ra1 is C _1-6 alkyl-O-CH ₂ -, and

ra2 and Ra3 are each C _1-6 An alkyl group.

Embodiment 18. A compound according to any one of the preceding embodiments, or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, wherein

Ra1 is C _1-6 alkyl-O-or C _1-6 alkyl-O-CH ₂ -, and

ra2 and Ra3 are each C _1-6 alkyl-O-CH ₂ -。

Embodiment 19. A compound according to any one of the preceding embodiments, or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, wherein

Ra1 is C _1-6 alkyl-O-CH ₂ -, and

one of Ra2 and Ra3 is C _1-6 Alkyl and the other is-C _1-6 alkyl-O-CH 2.

Embodiment 20. A compound according to any one of the preceding embodiments, or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, wherein

Ra1 is C _1-6 alkyl-O-, and

ra2 and Ra3 are independently C _1-3 Alkyl, preferably Ra2 and Ra3 are the same.

Embodiment 21. A compound according to any one of the preceding embodiments, or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, wherein

Ra1 is C _1-6 alkyl-O-or C _1-6 alkyl-O-CH ₂ -, and

ra2 and Ra3 together with the carbon to which they are attached form C _3-6 Cycloalkyl groups.

Embodiment 22. A compound according to any one of the preceding embodiments, or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, wherein

Ra1 is selected from H, C _1-6 Alkyl, C _1-6 Alkoxy and C1-6 alkyl-O-CH 2-; and

ra2 and Ra3 together with the carbon to which they are attached form a 5-6 membered haloheterocycloalkyl comprising 1 ring heteroatom selected from O;

preferably, ra1 is selected from C _1-6 Alkyl, C _1-6 alkyl-O-and C _1-6 Alkyl O-CH2-.

Embodiment 23. A compound according to any one of the preceding embodiments, or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, wherein R ¹ Is rac=o, and each of R2 and R3 is H.

Embodiment 24. A compound according to any one of the preceding embodiments, or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, wherein R ¹ 、R ² And R is ³ Is rac=o.

Embodiment 25. A compound according to any one of the preceding embodiments, or a tautomer, stereoisomer, racemate or pharmaceutically acceptable salt thereof, wherein Ra1 is C _1-6 alkyl-O-.

Embodiment 26. A compound according to any one of the preceding embodiments, or a tautomer, stereoisomer, racemate or a pharmaceutically acceptable salt thereof, wherein rac=o is selected from:

/>

embodiment 27. A compound according to embodiments 1-4 and 12, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, wherein said compound is selected from the group consisting of:

/>

embodiment 28. A pharmaceutical composition comprising a compound of any one of embodiments 1-27, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, and optionally comprising a pharmaceutically acceptable excipient.

Embodiment 29. Use of a compound of any one of embodiments 1-27, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prevention of an RNA viral infection.

Embodiment 30. The use according to embodiment 29, wherein the RNA virus is a coronavirus, such as a human coronavirus, SARS coronavirus or MERS coronavirus, an alpha virus, such as eastern equine encephalitis virus, western equine encephalitis virus, venezuelan equine encephalitis virus, chikungunya virus or ross river virus or Barmah Forest virus, a filoviridae virus, such as ebola virus, an orthomyxoviridae virus (such as influenza virus, influenza a virus or influenza b virus), a paramyxoviridae virus, such as Respiratory Syncytial Virus (RSV), a flavivirus, such as zika virus or berva mulberry virus; preferred are SARS-CoV-2/New coronapneumovirus, alpha variant SARS-CoV-2/New coronapneumovirus, beta variant SARS-CoV-2/New coronapneumovirus, gamma variant SARS-CoV-2/New coronapneumovirus, delta variant SARS-family V-2/New coronapneumovirus or any other variant SARS-coronavirus 2/New coronapneumovirus.

Embodiment 31. A method of treating or preventing an RNA viral infection in a subject, comprising administering to a subject in need thereof an effective amount of a compound of any one of embodiments 1-27, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof.

Embodiment 32. The use according to embodiment 31, wherein the RNA virus is a coronavirus, such as a human coronavirus, SARS coronavirus or MERS coronavirus, an alpha virus, such as eastern equine encephalitis virus, western equine encephalitis virus, venezuelan equine encephalitis virus, chikungunya virus or ross river virus, a filoviridae virus, such as ebola virus, an orthomyxoviridae virus, such as influenza virus, influenza a virus or influenza b virus, a paramyxoviridae virus, such as Respiratory Syncytial Virus (RSV), a flaviviridae, such as zhai card virus; preferably SARS-CoV-2/COVID-19 virus, alpha variant SARS-CoV-2/COVID-19 virus, beta variant SARS-CoV-2/COVID-19 virus, gamma variant SARS-CoV-2/COVID-19 virus, delta variant SARS-CoV-2/COVID-19 virus or any other variant SARS-CoV-2/COVID-19 virus.

Embodiment 33. A compound of any one of embodiments 1-27 or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, for use as a medicament.

Embodiment 34. A compound of any one of embodiments 1-27, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of an RNA viral infection.

Embodiment 35. The use according to embodiment 34, wherein the RNA virus is a coronavirus, such as a human coronavirus, SARS coronavirus or MERS coronavirus, an alpha virus, such as eastern equine encephalitis virus, western equine encephalitis virus, venezuelan equine encephalitis virus, chikungunya virus or ross river virus, a filoviridae virus, such as ebola virus, an orthomyxoviridae virus, such as influenza virus, influenza a virus or influenza b virus, a paramyxoviridae virus, such as Respiratory Syncytial Virus (RSV), a flaviviridae, such as zhai card virus; preferably SARS-CoV-2/COVID-19 virus, alpha variant SARS-CoV-2/COVID-19 virus, beta variant SARS-CoV-2/COVID-19 virus, gamma variant SARS-CoV-2/COVID-19 virus, delta variant SARS-CoV-2/COVID-19 virus or any other variant SARS-CoV-2/COVID-19 virus.

Embodiment 36. For increasing N for the treatment or prevention of RNA viral infection ⁴ A method of bioavailability of hydroxycytidine comprising administering to a subject in need thereof an effective amount of a compound of any one of embodiments 1-27, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof.

Embodiment 37. A pharmaceutical combination comprising a compound of any one of embodiments 1-27, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, and at least one additional therapeutic agent.

Embodiment 38. The pharmaceutical combination according to embodiment 37, wherein the additional therapeutic agent is selected from the group consisting of:

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application method

According to the present disclosure, the RNA virus is a coronavirus, such as a human coronavirus, SARS coronavirus or MERS coronavirus, an alpha virus, such as eastern equine encephalitis virus, western equine encephalitis virus, venezuelan equine encephalitis virus, chikungunya virus or ross river virus, a filoviridae virus, such as ebola virus, an orthomyxoviridae virus, such as influenza virus, influenza a virus (including H1N1, H3N2, H7N9 or H5N1 subtypes), influenza virus B or influenza virus C, a paramyxoviridae virus, such as Respiratory Syncytial Virus (RSV), a flaviviridae, such as zika virus, rotavirus, such as rotavirus a, rotavirus B, rotavirus C, rotavirus D, rotavirus E; preferably SARS-CoV-2/COVID-19 virus, alpha variant SARS-CoV-2/COVID-19 virus, beta variant SARS-CoV-2/COVID-19 virus, gamma variant SARS-CoV-2/COVID-19 virus, delta variant SARS-CoV-2/COVID-19 virus or any other variant SARS-CoV-2/COVID-19 virus.

Preferably, in accordance with the present disclosure, the RNA virus is a human coronavirus, SARS coronavirus, MERS coronavirus, eastern equine encephalitis virus, western equine encephalitis virus, venezuelan equine encephalitis virus, chikungunya virus, ross river virus, orthomyxoviridae virus, paramyxoviridae virus, RSV virus, influenza a virus, influenza b virus, filoviridae virus, or ebola virus.

More preferably, according to the present disclosure, the RNA virus is a human coronavirus, a SARS-CoV-2/COVID-19 virus, an alpha variant SARS-CoV-2/COVID-19 virus, a beta variant SARS-CoV-2/COVID-19 virus, a gamma variant SARS-CoV-2/COVID-19 virus, a delta variant SARS-CoV-2/COVID-19 virus or any other variant SARS-CoV-2/COVID-19 virus.

According to the present disclosure, an individual is at risk of, exhibits symptoms of, or is diagnosed with: SARS-CoV-2/COVID-19 virus, influenza A virus including subtype H1N1, H3N2, H7N9 or H5N1, influenza B virus, influenza C virus, rotavirus A, rotavirus B, rotavirus C, rotavirus D, rotavirus E, human coronavirus, SARS coronavirus, MERS coronavirus, human adenovirus type (HAdV-1 to 55), human Papilloma Virus (HPV) type 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59, parvovirus B19, infectious soft wart virus, JC virus (JCV), BK virus, mexico cell polyoma virus, coxsackie A virus, norovirus, rubella virus, lymphocytic choriomeningitis virus (V), dengue virus, zika virus, chikungunya virus, eastern Equine Encephalitis Virus (EEEV), western Equine Encephalitis Virus (WEEV), venezuelan Equine Encephalitis Virus (VEEV), ross river virus, baer Ma Senlin virus, yellow fever virus, measles virus, mumps virus, respiratory syncytial virus, rinderpest virus, california encephalitis virus, hantavirus, rabies virus, ebola virus, marburg virus, herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), varicella Zoster Virus (VZV), epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, roselight rash virus or Kaposi sarcoma-associated herpes virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, and hepatitis B virus, hepatitis E Virus or Human Immunodeficiency Virus (HIV), human T-lymphotropic Virus type I (HTLV-1), frandspleen focus forming Virus (SFFV) or xenotropic MuLV-related Virus (XMLV). In some embodiments, the individual is at risk of, shows symptoms of, or is diagnosed with a zika virus infection.

According to the invention, an individual is diagnosed with a SARS-CoV-2/COVID-19 viral infection, including an alpha variant SARS-CoV-2/COVID-19 virus, a beta variant SARS-CoV-2/COVID-19 virus, a gamma variant SARS-CoV-2/COVID-19 virus, a delta variant SARS-CoV-2/COVID-19 virus, or any variant SARS-CoV-2/COVID-19 virus infection, which may be treated with a compound of formula (I) or a medicament comprising a compound of formula (I).

According to the invention, an individual is diagnosed with influenza A virus, including subtypes H1N1, H3N2, H7N9, H5N1 (low-pathway) and H5N1 (high-pathway), influenza B virus, influenza C virus, rotavirus A, rotavirus B, rotavirus C, rotavirus D, rotavirus E, SARS coronavirus, MERS-CoV, human adenovirus type (HAdV-1 to 55), human Papilloma Virus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59, parvovirus B19, infectious soft wart virus, JCV (JCV), BK virus, mekel cell polyoma virus, coxsackie A virus, norovirus, rubella virus, lymphocytic choriomeningitis virus (V), yellow fever virus, measles virus, mumps virus, respiratory syncytial virus, parainfluenza viruses 1 and 3, rinderpest virus, chikungunya virus, eastern Equine Encephalitis Virus (EEEV), venezuelan Equine Encephalitis Virus (VEEV), western Equine Encephalitis Virus (WEEV), california encephalitis virus, japanese encephalitis virus, rift Valley Fever Virus (RVFV), hantavirus, dengue virus serotypes 1, 2, 3 and 4, zika virus, west nile virus, tacrolimus Bei Bingdu, hooning virus, rabies virus, ebola virus, marburg virus, adenovirus, herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), varicella Zoster Virus (VZV), epstein Barr Virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, rose virus or Kaposi's sarcoma-associated herpes virus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E or Human Immunodeficiency Virus (HIV). In certain embodiments, the individual is diagnosed with a zika virus infection.

According to the invention, the individual is diagnosed with gastroenteritis, acute respiratory disease, severe acute respiratory syndrome, post viral infection fatigue syndrome, viral hemorrhagic fever, acquired immunodeficiency syndrome or hepatitis.

Pharmaceutical composition and administration

The compounds of the present invention (e.g., any of the compounds in the examples herein) may be formulated into pharmaceutical compositions, alone or in combination with one or more additional therapeutic agents. The pharmaceutical composition comprises: (a) an effective amount of a compound of the present invention; (b) Pharmaceutically acceptable excipients (e.g., one or more pharmaceutically acceptable carriers); and optionally (c) at least one additional therapeutic agent.

Pharmaceutically acceptable excipients refer to excipients that are compatible with the active ingredient in the composition (in certain embodiments, the active ingredient may be stabilized) and that are not deleterious to the subject to be treated. Suitable pharmaceutically acceptable excipients are disclosed in standard references in the art (e.g., remington's Pharmaceutical Sciences, remington: the Science and Practice of pharmacy.) and include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, fragrances, flavoring agents, diluents and other known additives to provide a perfect presentation of a drug (i.e., a compound of the present invention or pharmaceutical composition thereof) or to aid in the preparation of a pharmaceutical product (i.e., a drug).

The compounds of the invention may be administered in a variety of known ways, for example orally, parenterally, inhaled or by pulmonary, i.e., pulmonary, nasal, sublingual, lingual, buccal, rectal, dermal, transdermal, conjunctival, otic routes, or as implants or stents. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion.

Oral or parenteral administration is preferred, in particular oral administration.

The compounds of the present invention may be administered in any convenient formulation, such as tablets, powders, capsules, pills, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, aqueous buffers, such as saline or phosphate buffers, and the like. Such compositions may contain conventional components in pharmaceutical formulations, such as diluents, carriers, pH modifying agents, sweeteners, fillers and additional active agents.

Generally, it has been found that in the case of parenteral administration, the amount administered is about 0.001 to 20mg/kg body weight, preferably about 0.01 to 10mg/kg body weight, to achieve an effective effect. In the case of oral administration, the dosage is about 0.01 to 100mg/kg body weight, preferably about 0.01 to 20mg/kg body weight, and most preferably 0.1 to 15mg/kg body weight.

Combination therapy

The compounds described herein may be co-administered with at least one additional therapeutic agent.

Additional therapeutic agents include, but are not limited to, analgesics, anti-inflammatory agents, antipyretics, antidepressants, antiepileptics, antihistamines, antimigraine agents, antimuscarinics, anxiolytics, sedatives, hypnotics, antipsychotics, bronchodilators, anti-asthmatics, cardiovascular agents, corticosteroids, dopaminergic agents, electrolytes, gastrointestinal agents, muscle relaxants, nutritional agents, vitamins, parasympathetic agents, stimulants, anorexics, anti-somnolence agents, and antiviral agents. In particular embodiments, the antiviral agent is a non-CNS-targeted antiviral compound. As used herein, "co-administration" means that the compound may be administered in the same dosage form or in a different dosage form than one or more other active agents. The additional therapeutic agent may be formulated for immediate release, controlled release, or a combination thereof.

The compounds and pharmaceutical compositions of the present invention may be administered in combination with at least one additional therapeutic agent, such as an antiviral agent, such as abacavir, acyclovir, adefovir, amantadine, amprenavir, an Puli, arbidol, atazanavir, atripla, balapiravir, BCX4430, boscalid, cidofovir, bispidotive, dacarbazine, darunavir, dasabavir, desquamation, didanosine, behenone, edestin, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, fomivirgine, fosamprenavir, foscarnet, sodium phosphonate, ganciclovir, GS-5734, ibatabine, isoprinosine, iodoside, imiquimod, indinavir, inosine, type III interferon, type II interferon, type I interferon, lamivudine, dipivudine, lopinamide Weiluo maraviroc, moroxydine, mevaltiazem, nelfinavir, nevirapine, nexavir, NITD008, obetavir, oseltamivir, paliprevir, polyethylene glycol interferon alpha-2 a, penciclovir, peramivir, praecoratide, podophyllotoxin, raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, cimiprevir, sofosbuvir, stavudine, tebiprevir, tebipine, tenofovir disoproxil, tenofovir, exalide, telavaavir, triamcinolone, triadamine, valacyclovir, ganciclovir, valganciclovir, valicarb, vidone, vidarabine, wei Lami, zalcitabine, zanamivir, mo Nuola Wei Huoji docodine and combinations thereof.

The compounds and pharmaceutical compositions of the invention disclosed herein may be administered in combination with any of the compounds disclosed in WO2012119559 for the treatment of SARS-CoV-2/covd-19 infection.

The compounds and pharmaceutical compositions of the invention disclosed herein may be administered in combination with any of the compounds disclosed in WO2012119559 for the prevention of SARS-CoV-2/covd-19 infection.

The compounds and pharmaceutical compositions of the invention disclosed herein may be administered in combination with prochloraz for the treatment of SARS-CoV-2/COVID-19 infection.

The compounds and pharmaceutical compositions of the invention disclosed herein may be administered in combination with prochloraz for the prevention of SARS-CoV-2/COVID-19 infection.

The compounds and pharmaceutical compositions of the invention disclosed herein may be administered in combination with compound-X for the treatment of SARS-CoV-2/COVID-19 infection.

The compounds and pharmaceutical compositions of the invention disclosed herein may be administered in combination with compound-X for the prevention of SARS-CoV-2/COVID-19 infection.

The compounds and pharmaceutical compositions of the invention disclosed herein may be administered in combination with PF-07321332 for the treatment of SARS-CoV-2/COVID-19 infection.

The compounds and pharmaceutical compositions of the invention disclosed herein may be administered in combination with PF-07321332 for preventing SARS-CoV-2/COVID-19 infection.

Accordingly, the present disclosure also provides a pharmaceutical combination comprising a compound of the present invention and at least one additional therapeutic agent. Examples of additional therapeutic agents include, but are not limited to, those mentioned above, preferably pramipexole, compound-X and PF-07321332.

Unless otherwise indicated, percentages in the following tests and examples are by weight; the portion is a weight portion. The solvent ratio, dilution ratio and concentration data of the liquid/liquid solution are in each case based on volume.

Each embodiment and technical means described in the present disclosure and features in each embodiment and technical means should be understood as being capable of being combined with each other in any way, and those technical means obtained by such combination are included in the scope of the present disclosure as if each technical means obtained by such combination were specifically and individually listed unless the context clearly shows otherwise.

All patents, patent applications, publications, and other references cited or referred to herein are incorporated by reference in their entirety to the extent permitted by law. The discussion of these references is merely to summarize the assertions made therein. No admission is made that any such patent, patent application, publication or reference, or any portion thereof, is relevant material or prior art. The claims and their equivalents are expressly set forth in such patents, patent applications, publications and other references to the accuracy and pertinence of any claim as a matter of related material or prior art.

Examples

The following examples are presented to illustrate compositions, methods, and results according to the disclosed subject matter. These examples are not intended to include all aspects of the subject matter disclosed herein, but rather are intended to illustrate representative methods, compositions, and results. It will be apparent to those skilled in the art that these embodiments are not intended to exclude equivalents and variations of the invention.

Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless otherwise indicated, the parts are weight parts. There are many variations and combinations of reaction conditions, e.g., component concentrations, temperatures, pressures, and other reaction ranges and conditions, that can be used to optimize the purity and yield of the product obtained from the process. Such process conditions can be optimized by only reasonable routine experimentation.

Unless otherwise indicated, all reagents and starting materials used in the present invention are commercially available or prepared according to the prior art.

¹ H NMR spectra at Bruker 4Measurement on a 00MHz instrument, chemical shift relative to the corresponding solvent peak: CDCl3 (δ7.27), DMSO-D6 (δ2.50), CD3OD (δ3.31), D2O (δ4.79). The following abbreviations are used to describe the coupling: s=singlet, d=doublet, t=triplet, q=quartet, quin=quintet, m=multiplet, br=broad. ¹³ The C NMR spectrum was measured on a Bruker instrument at 100MHz and the chemical shift was measured relative to the corresponding solvent peak: CDCl3 (δ77.0), DMSOd6 (δ39.5), CD3OD (δ49.0).

Abbreviations and acronyms:

aq. aqueous solution

calc calculated value

br s broad unimodal (in NMR)

DCI direct chemical ionization (in MS)

dec decomposition Point

DMF dimethylformamide

DMSO dimethyl sulfoxide

DSC differential scanning calorimetry

eq. Equivalent weight

ESI electrospray ionization (in MS).

Et ethyl group

fnd. Found values

h hours

HPLC high pressure high performance liquid chromatography

HRMS high resolution mass spectrometry

Conc. concentrated

LC-MS liquid chromatography-coupled mass spectrometry

LiHMDS hexamethyldisilyl lithium amide

Me methyl group

Min minutes

MS mass spectrometry

NMR Spectroscopy

Pd2 dba3 tris (dibenzylideneacetone) dipalladium

Ph phenyl

PLM polarized light microscope

RT room temperature

Rt retention time (in HPLC)

TGA thermogravimetric analysis

THF tetrahydrofuran

UV spectroscopy

v/v volume to volume ratio (solution)

Preparation of starting materials and intermediates

Preparation 1: synthesis of alkoxy substituted propionic acids and anhydrides

The synthesis method of (R) -2-methoxypropionic acid (I-3) and anhydride (I-4) comprises the following steps:

(S) -2-chloropropionic acid (80.0 g,738mmol,1 eq, 98%) was added to a two-necked round bottom flask under nitrogen. 25wt% sodium methoxide (506 mL,2.212mol,3 eq.) was slowly added. The reaction was heated to 60 ℃ for 16 hours and the conversion was monitored until starting material remained <2%. When sufficient conversion is reached, the reaction vessel is cooled to room temperature and the pH is adjusted with 4M hydrochloric acid in dioxane (200 mL, 99%) such that the pH is just from>12 to 7, indicating that excess sodium methoxide was neutralized without protonating the sodium carboxylate salt. The reaction mixture was filtered to remove salts and the filter cake was washed twice with 5mL methanol. The filtrate was concentrated, redissolved in water, acidified with 6M HCl to pH = -2, and extracted with EtOAc. The organic layer was dried over sodium sulfate and concentrated to give compound (I-3) (73 g, 95%) as a liquid, which was pure enough to be used without purification. ¹ H NMR(CD ₃ OD) delta 3.67 (q, 1H), 3.33 (s, 3H), and 1.33 (d, 3H).

In a 2 liter four-necked glass reactor equipped with a thermometer and a stirrer, 500g of methylene chloride, 104.1g (1.0 mol) of (R) -2-methoxypropionic acid (3) and 57.3g (0.5 mol) of methanesulfonyl chloride were charged under a nitrogen atmosphere. The mixture was cooled to 5 ℃.

Then, 101.3g (1.0 mol, 1 equivalent to the acid formed from methanesulfonyl chloride) of triethylamine was added dropwise over 2 hours, and the temperature of the reaction mixture was controlled at 30℃or lower. After the completion of the dropwise addition, stirring was carried out for 1 hour, and the same temperature was maintained. The reaction mixture was analyzed by Gas Chromatograph (GC) and the result showed >95% conversion of (R) -2-methoxypropionic acid (3).

After completion of the reaction, 200g of water was added to the reaction mixture to wash the reaction mixture. The reaction mixture was further washed twice with 200g of water each time, followed by distillation to remove methylene chloride. 85.6g of (R) -2-methoxypropionic anhydride (I-4) are obtained as a yellow liquid, which is used for the acylation step without further purification.

The following 2-alkoxy-substituted propionic anhydride was prepared in the same manner as described in preparation 1.

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Preparation 2: synthesis of alkoxy substituted isobutyric acids and anhydrides

The synthesis method of 2-ethoxyisobutyric acid/2-ethoxy-2-methylpropanoic acid (I-21) and anhydride (I-22) comprises the following steps:

2-ethoxyisobutyric acid is described in reference (Ragan, john A.; ide, nathan D.; cai, weiling; cawley, james J.; colon-Cruz, roberto; kumar, rajesh; peng, zhihui; vanderplas, brian C.; organic process research and development,2010, volume 14, #6, pages 1402-1406)]) Preparation: in a 500mL three-necked round bottom flask, 2-bromo-2-methylpropanoic acid (I-20) (40 g,239.5 mmol) was dissolved in ethanol (320 mL) and cooled to 0 to 5℃followed by dropwise addition of DIPEA (87.4 mL,502.9 mmol) at 0 to 5℃and stirring of the reaction mixture at 0℃for 30 min. The reaction mixture was warmed to room temperature for 16 hours. After 16 hours, the reaction mixture was cooled to room temperature and the ethanol was removed in vacuo leaving a thick white slurry. Diethyl ether and water were added to the slurry and cooled to 0 ℃. Acidification of the mixture with 10% HC1 (50 mL) The organic layer was separated and washed with brine. 10% NaHSO was added to the organic phase ₃ Aqueous solution, and the mixture was stirred at room temperature for 6 hours. The biphasic mixture was acidified with 10% HC1 (50 mL) to a pH of 1.0±0.5. The organic phase was washed with brine (100 mL), dried over sodium sulfate, filtered and concentrated to give 30g of 2-ethoxy-2-methylpropanoic acid (I-21). The product 2-ethoxy-2-methylpropanoic acid (I-21) was used in the next step without further purification.

In a 2 liter four-necked glass reactor equipped with a thermometer and a stirrer, 300g of methylene chloride, 66.1g (0.5 mol) of 2-ethoxy-2-methylpropanoic acid (I-21) and 28.65g (0.25 mol) of methanesulfonyl chloride were charged under a nitrogen atmosphere. The mixture was cooled to 5 ℃.

Then, 50.65g (0.5 mol, 1 equivalent to the acid formed from methanesulfonyl chloride) of triethylamine was added dropwise over 2 hours, and the temperature of the reaction mixture was controlled at 30℃or lower. After the completion of the dropwise addition, the mixture was stirred for 1 hour, and the same temperature was maintained. Analysis of the reaction mixture by Gas Chromatography (GC) showed >95% conversion of 2-ethoxy-2-methylpropanoic acid (I-21).

After completion of the reaction, 100g of water was added to the reaction mixture to wash the reaction mixture. The reaction mixture was further washed twice with 100g of water each time, followed by distillation to remove methylene chloride. 51g of 2-ethoxy-2-methylpropanoic anhydride (I-22) are obtained as a yellow liquid which is used in the acylation step without further purification.

In the same manner as in preparation 1, the following 2-alkoxy-substituted 2-methylpropanoic acid/2-alkoxy-substituted isobutyric anhydride was prepared:

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preparation 3: synthesis of 4-alkoxytetrahydro-2H-pyran-4-carboxylic acid and anhydride

The synthesis method of 4-methoxytetrahydro-2H-pyran-4-carboxylic acid (I-36) and anhydride (I-37) comprises the following steps:

commercially available methyl tetrahydro-2H-pyran-4-carboxylate is brominated according to the method described in Organic Letters,2020, volume 22, #10, pages 3922-3925. The ester is then hydrolyzed to the corresponding alpha-bromoacid (I-35). The α -bromoacid (I-35) is then converted to the corresponding acid (I-36) and anhydride (I-37) according to the method of preparation 2.

The following 4-alkoxytetrahydro-2H-pyran-4-carboxylic acid and anhydride were prepared in a similar manner.

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Preparation 4: synthesis of 4-alkyltetrahydro-2H-pyran-4-carboxylic acid and anhydride

The synthesis method of 4-methyltetrahydro-2H-pyran-4-carboxylic acid (I-46) and anhydride (I-47):

commercially available methyl tetrahydro-2H-pyran-4-carboxylate (I-33) is methylated in the same way as described in example 64.1A in US 9434690. The methyl ester was then hydrolyzed with aqueous NaOH and acidified with HCl to give 4-methyltetrahydro-2H-pyran-4-carboxylic acid (I-46) as an off-white solid.

4-methyltetrahydro-2H-pyran-4-carboxylic acid anhydride (I-47) was prepared as a pale yellow oil according to the procedure of preparation 2.

The following 4-alkyltetrahydro-2H-pyran-4-carboxylic acid anhydrides were prepared in a similar manner.

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Preparation 5: synthesis of 2-ethyl-2-alkoxy-butyric acid and anhydride

The synthesis method of 2-ethyl-2-methoxy-butyric acid (I-62) and anhydride (I-63) comprises the following steps:

2-ethyl-2-bromo-butyric acid (I-61) is commercially available or may be according to Doran; shonle was prepared as described in Journal of Organic Chemistry,1938, volume 3, page 195.

2-ethyl-2-bromo-butyric acid (I-61) was first converted to ethyl-2-methoxy-butyric acid (I-62) and then to ethyl-2-methoxy-butyric anhydride (I-63) as a pale yellow oil, as described in preparation 2.

The following acids and anhydrides were prepared in a similar manner.

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Preparation 6: synthesis of 2-methyl-2-alkoxy-butyric acid and anhydride

A method for synthesizing 2-methyl-2-methoxybutyric acid (I-72) and anhydride (I-73). Commercially available (R, S) -2-hydroxy-2-methylbutanoic acid (I-70) is resolved into enantiomerically pure R and S isomers (I-71) and then esterified to methyl ester (I-72) according to the procedure described in preparation 74 of US 2008114005.

Alternatively, commercially available 2-bromo-2-methylbutanoic acid is converted to (R, S) -2-methoxy-2-methylbutanoic acid (I-78) according to the procedure disclosed in preparation 2, and (I-78) is resolved into enantiomers (I-80) and (I-77) according to the procedure described in preparation 74 of U.S. Pat. No. 5, 2008114005. The chiral acid (I-75) was then converted to the anhydride (I-76) as described in preparation 2 as an oil.

The following acids and anhydrides were prepared in a similar manner.

Preparation 7: synthesis of 2-alkyltetrahydrofuran-2-carboxylic acid and anhydride.

The synthesis method of 2-methyltetrahydrofuran-2-formic acid (I-112), (I-114) and anhydride (I-113), (I-115): according to Pohl; enantiomerically pure 2-methyltetrahydrofuran-2-carboxylic acids (I-112) and (I-114) were prepared by the method described in Wollweber, european Journal of Medicinal Chemistry,1976, vol.11, pp.163, 168, 169. The acid was then converted to the corresponding anhydride (I-113) and (I-115) in a similar manner as described in preparation 2.

The following 2-alkyltetrahydrofuran-2-carboxylic acid and anhydride were prepared in a similar manner:

preparation 8: synthesis of 2-methyl-2-alkoxymethylpropanoic acid and anhydride.

The synthesis method of 2-methyl-2-methoxymethyl propionic acid (I-130) and anhydride (I-131) comprises the following steps:

commercially available methyl 2-methyl-2-hydroxymethylpropionate (I-128) was first methylated and the ester was then hydrolyzed using the methods described in examples 55 and 56 of WO2009/77608, 2009 to give 2-methyl-2-methoxymethylpropanoic acid (I-130).

2-methyl-2-methoxymethylpropanoic acid (I-130) was then converted to anhydride (I-131) according to the method of preparation 2, which was obtained as an oil.

The following 2-methyl-2-alkoxymethylpropanoic anhydride was prepared in a similar manner.

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Preparation 9: synthesis of 1-alkyl-2, 2-dialkoxy-isobutyric acid and anhydride

The synthesis method of 1-methyl-2, 2-dimethoxy-isobutyric acid (I-142) and anhydride (I-143) comprises the following steps:

1-methyl-2, 2-dimethoxy-isobutyric acid (I-142) was prepared according to the method described in reference example 14 of US 2004248941.

Alternatively, 1-methyl-2, 2-dimethoxy-isobutyric acid (I-142) was prepared from commercially available 2, 2-bis (hydroxymethyl) propionic acid according to reference example 14 of EP 1437352.

1-methyl-2, 2-dimethoxy-isobutyric acid (I-142) was then converted to anhydride (I-143) according to the method of preparation 2, which was obtained as an oil.

The following 1-alkyl-2, 2-dialkoxy-isobutyric acid and anhydride were prepared in a similar manner.

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Preparation 10: synthesis of 1- (alkoxymethyl) cyclopropane-1-carboxylic acid and anhydride.

The synthesis method of 1- (methoxymethyl) cyclopropane-1-carboxylic acid (I-225) and anhydride (I-226) comprises the following steps:

methyl 1- (hydroxymethyl) cyclopropane-1-carboxylate (I-223) was prepared according to the method described in reference example 22-1 of US 9546155. The hydroxy groups were then alkylated with methyl iodide using a similar procedure as described by Shen, peng-Xiang et al, journal of the American Chemical Society,2018, vol.140, #21, pages 6545-6549. The ester was then hydrolyzed to give 1- (methoxymethyl) cyclopropane-1-carboxylic acid (I-225).

1- (methoxymethyl) cyclopropane-1-carboxylic acid (I-225) was then converted to anhydride (I-226) according to the procedure of preparation 2, yielding (I-226) as an oil.

The following 1- (alkoxymethyl) cyclopropane-1-carboxylic acid and anhydride were prepared in a similar manner.

Preparation 11: synthesis of 1- (alkoxymethyl) cyclobutane-1-carboxylic acid and anhydride.

The synthesis method of 1- (methoxymethyl) cyclobutane-1-carboxylic acid (I-238) and anhydride (I-239):

methyl 1- (hydroxymethyl) cyclobutane-1-carboxylate (I-236) was prepared according to the method described in reference example 22-4 of US 9546155. The hydroxy groups were then alkylated with methyl iodide and then subjected to ester hydrolysis in a similar manner to that described in US10040791 with reference to example K-19 to give 1- (methoxymethyl) cyclobutane-1-carboxylic acid (I-238).

1- (methoxymethyl) cyclobutane-1-carboxylic acid (I-238) was then converted to anhydride (I-239) according to the method of preparation 2, which was obtained as an oil.

The following 1- (alkoxymethyl) cyclobutane-1-carboxylic acid and anhydride were prepared in a similar manner.

Preparation 12: synthesis of 1, 2-trialkoxy-isobutyric acid and anhydride

The synthesis method of the 1-methoxy-2, 2-diethoxy-isobutyric acid (223) and the anhydride (224) comprises the following steps:

ethyl 1-hydroxy-2, 2-diethoxy-isobutyrate (I-249) was prepared according to the method described by Bernardon, C.et al, comptes Rendus des Seances de l' Academie des Sciences, series C. Sciences Chimiques,1968, volume 266, pages 1502-1505. The hydroxyl groups were then alkylated with methyl iodide in a similar manner as described in US10040791 with reference to example K-19. The ester is then hydrolyzed to give 1-methoxy-2, 2-diethoxy-isobutyric acid (I-251).

1-methoxy-2, 2-diethoxy-isobutyric acid (I-251) was then converted to the anhydride (I-252) according to the method of preparation 2, giving it as an oil.

The following 1-alkoxy-2, 2-dialkoxy-isobutyric acid and anhydride were prepared in a similar manner.

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Preparation 13: synthesis of 1-alkoxycyclobutanecarboxylic acids and anhydrides

The synthesis method of 1-methoxy cyclopropanecarboxylic acid (I-291) and anhydride (I-292) comprises the following steps:

commercially available methyl 2-methoxyacetate (I-289) was alkylated with dibromoethane in the same manner as described in example 26A of US10464914 to give methyl 1-methoxycyclopropanecarboxylate which was then hydrolyzed under basic conditions to give the corresponding acid (I-291). The acid (I-291) was then converted to the corresponding anhydride (I-292) according to the procedure of preparation 2, giving it as an oil.

In the same manner as described for the preparation above, the following 1-methoxycyclopropanecarboxylic acid and anhydride and 1-alkoxycyclobutanecarboxylic acid and anhydride can be prepared:

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preparation of 14:2',3' -O-isopropylidenediamine (I-318)

To a solution of uridine (97.6 g,400 mmol) and a catalytic amount of p-toluenesulfonic acid (TsOH. H2O,7.60g,40.0 mmol) in acetone (2000 mL) was added 2, 2-dimethoxypropane (89.2 g, 433 mmol) and the suspension was slowly heated to gentle reflux for 48 hours, then the solution was cooled to room temperature, treated with sodium bicarbonate (60.72 g, 72 mmol) and stirred at room temperature for 0.5 hours, the solid was filtered and the crude solution was concentrated under reduced pressure to give a pale yellow residue, the residue was dissolved in 1000mL EtOAc, washed with water x2, sodium bicarbonate x2, water, brine x2 and dried over sodium sulfate, and the colorless solution was concentrated under reduced pressure to give 97 g (85%). 1H-NMR (MeOD, 400 MHz) delta (ppm): 7.82 (1H, d), 5.86 (1H, d), 5.67 (1H, d), 4.19 (1H, t), 3.54 m, 3.34H (3.54H), 3.34H (3H, 54H).

Example 1:

synthesis of N4-hydroxycytidine (NHC) or 1- (3, 4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -4- (hydroxyamino) pyrimidin-2-one.

Cytidine (20.0 g,82.24mmol,1.0 eq.) and NH ₂ AcOH (23 g,246.7mmol,3.0 eq.) in H ₂ The mixture in O (350 mL) was stirred at 40℃for 48 hours. The reaction was monitored by HPLC and, after completion of the reaction,the water was evaporated under vacuum in a rotary evaporator to give a thick slurry, which was then suspended in 100mL of water and left to crystallize in a refrigerator for 24 hours. The solid thus crystallized was filtered off with cold H ₂ O (about 15.0 mL) and dried under vacuum to give the desired N4-hydroxycytidine (NHC/EX-1) as a white solid (8.48 g,40% yield). 1H NMR (400 MHz, D) ₂ O) δ7.0 (d, 1H), 5.75 (d, 1H), 5.60 (d, 1H), 4.2 (t, 1H), 4.06 (t, 1H), 3.89 (m, 1H), 3.75 (dd, 1H), 3.60 (dd, 1H); purity 98% (assessed by HPLC).

Example 2: preparation of Compound (EX-2A)

To a solution of 2',3' -O-isopropylidene uridine (I-318) (10 g,35.2 mmol) in a mixture of CH2Cl2 (150 mL) and pyridine (60 mL) was added anhydride (I-22) (17.3 g,70.4 mmol) and DMAP (0.5 g,3.6 mmol) at 0deg.C. The resulting mixture was warmed to room temperature and stirred at 20-40 ℃ for 48 hours. After completion (TLC, 10:1CHCl 3-MeOH), the reaction mixture was washed three times with aqueous HCl to remove all pyridine, then with aqueous NaHCO3 and aqueous NaCl. The organic layer was concentrated. The product (I-2-1) (12.6 g) was used in the next step without further purification.

To a 500mL round bottom flask was added the above crude product (20.24 g,50.8 mmol) and MeCN (200 mL). The reaction mixture was stirred until all starting materials were dissolved. Next, 1,2, 4-triazole (25.3 g, 365.8 mmol) was added followed by N, N-diethylamine (56.6 ml, 406.4 mmol). The reaction mixture was stirred at rt until all solids were dissolved. The reaction was then cooled to 0 ℃ using an ice bath. Phosphorus oxychloride (12.2 ml, 76.2 mmol) was slowly added. The resulting slurry was stirred under argon while slowly warming to room temperature. The reaction was then stirred by TLC (EtOAc) until complete. The reaction was then quenched by the addition of 60mL of water. The slurry then turned into a dark solution, which was then concentrated under reduced pressure. The residue was dissolved in DCM and washed with water and brine. The organics were then dried over sodium sulfate, filtered, and concentrated under reduced pressure. The product was purified by silica gel chromatography (500 g column). The product-containing fractions were collected and concentrated under reduced pressure to give (I-2-2).

To a 300mL round bottom flask was added the product from the previous step (5.8 g,14.6 mmol) and isopropanol (80 mL). The reaction mixture was stirred at rt until all solids were dissolved. Next, hydroxylamine (0.67 mL,22 mmol) was added and stirring continued at ambient temperature. When the reaction was complete (HPLC), some of the solvent was removed under high vacuum at ambient temperature. The remaining solvent was removed under reduced pressure at 45 ℃. The resulting residue was dissolved in EtOAc and washed with water and brine. The organics were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give an oil (I-2-3).

To a 100mL round bottom flask was added the product from the previous step (4.0 g,10 mmol) and formic acid (60 mL). The reaction mixture was stirred at room temperature overnight. The progress of the reaction was monitored by TLC. The reaction mixture was concentrated under reduced pressure <35 ℃ to give an oil. Next, 20mL of ethanol was added. The solvent was then removed under reduced pressure. The resulting residue was purified on a silica gel column using a CH2Cl2-MeOH gradient (100:0 to 10:1 v/v) solvent system as eluent to give (EX-2A) as a white solid. 1H NMR (400 MHz, DMSO). Delta.10.0 (s, 1H), 9.6 (s, 1H), 6.9 (d, 1H), 5.72 (d, 1H), 5.52 (d, 1H), 5.40 (d, 1H), 5.25 (d, 1H), 4.20 (d, 2H), 4.05-3.85 (m, 3H), 1.40 (s, 6H), 1.00 (t, 3H); purity 98% (assessed by HPLC).

The following exemplary compounds (I-24), (I-26), (I-28) (I-30) and (I-32) were prepared in analogy to example 2 using the anhydrides.

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Example 3: preparation of Compound (EX-3A)

The compound (EX-3A) was prepared as a white solid 1H NMR (400 MHz, DMSO) delta according to the method of example 2, but using anhydride (I-4)

The following exemplary compounds (I-6), (I-8) and (I-10) were prepared similarly to example 2 by using the acid anhydride.

Example 4: preparation of Compound (EX-4A)

Following the procedure of example 2, but using anhydride (I-307), compound (EX-4A) was prepared as a white solid by 1H NMR (400 MHz, D ₂ O)δ

The following exemplary compounds (I-310), (I-313) and (I-316) were prepared similarly to the above by using an acid anhydride.

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Example 5: preparation of Compound (EX-5A)

Following the procedure of example 2, but using anhydride (I-51), compound (EX-5A) was prepared as a white solid by 1H NMR (400 MHz, D ₂ O)δ

The following exemplary compounds (I-46), (I-55), (I-59), (I-37), (I-39), (I-41) and (I-43) were prepared in analogy to example 2 by using tetrahydro-2H-pyran-4-carboxylic anhydride, anhydride.

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Example 6: preparation of Compound (EX-6A)

Following the procedure of example 2, but using anhydride (I-63), compound (EX-6A) was prepared as a white solid by 1H NMR (400 MHz, D ₂ O)δ

The following exemplary compounds (I-65), (I-67), (I-69), (I-76), (I-90), (I-103) and (I-111) were prepared analogously to example 2 by using the anhydrides.

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Example 7: preparation of Compound (EX-7A)

Following the procedure of example 2, but using anhydride (I-115), compound (EX-7A) was prepared as a white solid by 1H NMR (400 MHz, D ₂ O)δ

The following exemplary compounds (I-119), (I-123) and (I-127) were prepared in analogy to example 2 using (R) -tetrahydro-2-furoic acid anhydride, (S) -tetrahydroxy-2-furoic acid anhydride and anhydride.

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Example 8: preparation of Compound (EX-8A)

Following the procedure of example 2, but using anhydride (I-131), compound (EX-8A) was prepared as a white solid by 1H NMR (400 MHz, D ₂ O)δ

The following exemplary compounds (I-134), (I-137) and (I-140) were prepared analogously to example 2 by using anhydrides.

Example 9: preparation of Compound (EX-9A)

Following the procedure of example 2, but using anhydride (I-226), compound (EX-9A) was prepared as a white solid by 1H NMR (400 MHz, D ₂ O)δ

The following exemplary compounds (I-229), (I-232) and (I-235) were prepared in analogy to example 2 by using the anhydride.

Example 10: preparation of Compound (EX-10A)

Following the procedure of example 2, but using anhydride (I-239), compound (EX-10A) was prepared as a white solid by 1H NMR (400 MHz, D ₂ O)δ

The following exemplary compounds (I-242), (I-245) and (I-248) were prepared similarly to example 2 by using the acid anhydride.

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Example 11: preparation of Compound (EX-11A)

Following the procedure of example 2, but using anhydride (I-265), compound (EX-11A) was prepared as a white solid by 1H NMR (400 MHz, D ₂ O)δ

The following exemplary compounds (I-268), (I-271), (I-274), (I-143), (I-149), (I-152), (I-155), (I-188), (I-191), (I-194), (I-197), (I-252), (I-255), (I-258), (I-261), (I-160), (I-163), (I-166), (I-169), (I-200), (I-203), (I-206), (I-209), (I-278), (I-281), (I-174), (I-177), (I-212), (I-215), (I-285), (I-288), (I-182), (I-185), (I-218) and (I-221) were prepared in analogy to example 2 by using the anhydrides.

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Example 12: preparation of Compound (EX-12A)

Prepared as a white solid according to a similar procedure to that described in example 2 using a commercially available 2-furoyl chloride compound (EX-12A). 1H NMR (400 MHz, DMSO). Delta.10.0 (s, 1H), 9.6 (s, 1H), 8.0 (s, 1H), 7.40 (brs, 1H), 6.9 (d, 1H), 6.70 (brs, 1H), 5.75 (d, 1H), 5.52 (d, 1H), 5.40 (d, 1H), 5.25 (d, 1H), 4.40 (m, 2H), 4.0 (m, 3H).

Example 13: preparation of Compound (EX-13A)

Following a procedure analogous to that described in example 2 using commercially available (2R) -tetrahydrofuran-2-carboxylic acid chloride, the white solid compound (EX-13A) was prepared. 1H NMR (400 MHz, D) ₂ O)δ

Example 14: preparation of Compound (EX-14A)

According to a procedure similar to that described in example 2In the step (1H NMR) (400 MHz, D) of the compound (EX-14A) was prepared as a white solid using commercially available (2S) -tetrahydrofuran-2-carboxylic acid chloride ₂ O)δ

Example 15: preparation of Compound (EX-15A)

Compound (EX-15A) as a white solid was prepared by following a procedure similar to that described in example 2 using anhydride (I-15-2). HNMR (400 MHz, CDCl) ₃ )δ8.7(s,1H),8.3(s,1H),6.9(d,1H),6.1(d,1H),5.7(d,2H),5.05(d,1H),4.9(d,1H),4.40(m,3H),4.1(multiple singlets,6H),3.5(three singlets,9H).

Example 16: preparation of Compound (EX-16A)

Using anhydride (I-24) and uridine, compound (EX-16A) was prepared as a white solid according to a procedure similar to that described in example 2. 1H NMR (400 MHz, CDCl) ₃ )δ8.80-9.0(brs,2H),7.05(d,1H),6.15(d,1H),5.72(d,1H),5.52(m,1H),5.35(m,1H),4.45(m,1H),4.35(m,1H),4.25(m,2H),3.28(s,3H),3.27(s,3H),3.20(multiple singlets,9H),1.30-1.50(multiple singlets,18H).

Example 17: preparation of Compound (EX-17A)

Using anhydride (I-24) and uridine, compound (EX-17A) was prepared as a white solid according to a procedure similar to that described in example 2. 1H NMR (400 MHz, CDCl) ₃ )δ8.80(brs,2H),7.05(d,1H),6.2(d,1H),5.72(d,1H),5.4(m,2H),4.4(m,1H),4.3(m,2H),3.5(m,6H),1.30-1.50(multiple singlets,18H),1.2(m,9H).

Example 18: preparation of Compound (EX-18A)

Intermediate (I-18-1) was prepared according to the procedure of reference example 1 of US 4962193. Using isobutyric anhydride and (I-18-1), compound (EX-18A) was prepared as a white solid according to a procedure similar to that described in example 2. NMR (400 MHz, CDCl) ₃ )δ8.7(s,1H),8.2(s,1H),6.6(d,1H),6.0(s,1H),5.7(d,2H),5.0(d,1H),4.8-4.9(d,1H),4.30(m,3H),3.3(s,3H),2.5(m,1H),1.20(s,6H)。

Example 19: preparation of Mo Nuola-Weir

Mo Nuola of Wair was prepared according to the procedure of example 10 of US2020276219 using 2',3' -O-isopropylidene uridine (I-318) and isobutyric anhydride to give a white solid. 1H NMR (400 MHz, D) ₂ O)δ7.08(d,1H),6.09(d,1H),5.67(d,1H),4.33(t,1H),4.06(t,1H),3.89-3.86(m,2H),3.76(dd,1H)；Purity:98％(assessed by HPLC)

EXAMPLE 20 plasma stability

Preparation of the solution:stock solutions (10 mM) of each test compound were prepared in DMSO. Stock solutions of each compound were then diluted to 100 μm with acetonitrile.

Plasma incubation:plasma incubations were performed in 96-well plates at 37 ℃ in duplicate. Plasma was pre-warmed at 37 ℃ for 5 minutes in total volume of 198 μl, then 2 μl of 100 μΜ of test compound was added to the incubation wells containing plasma, mixed with a pipette to obtain a homogeneous suspension, and immediately 20 μl of incubation was transferred as a 0 minute sample to the wells of the "quench" plate, then 200 μl acetonitrile, a metola Zong Zuowei Internal Standard (IS) was added, and mixed with a pipette. At 2, 5, 60 and 90 minutes At clock time, the incubations were mixed with a pipette and 20 μl of the incubate sample series at each time point was transferred to the wells of another "quench" plate, then 200 μl of acetonitrile, a metola Zong Zuowei internal standard, was added and mixed with a pipette.

Sample analysis:the 96-well plate was centrifuged at 6000g for 10 minutes. The supernatant was injected into an LC-MS/MS system for analysis.

EXAMPLE 21 microsomal stability

Microparticle incubation:the incubation mixture was prepared to a total volume of 200 μl and the final component concentrations were as follows: 0.1M PBS (pH 7.4), NADPH (2 mM) and liver microsomes (0.2 mg/mL) and test compound (1. Mu.M) or Munolavir (1. Mu.M) were used as positive controls, wherein NADPH was added after all other components were preincubated at 37℃for 5 minutes. Mix with a pipette to obtain a homogeneous suspension and immediately transfer 20 μl of incubation as a 0 minute sample into the wells of a "quench" plate, then add 200 μl acetonitrile with trimethoprim as IS, mix with a pipette. At 2, 5, 10 and 45 minutes, incubates were mixed with a pipette and 20 μl of incubate samples at each time point were serially transferred to wells of a separate "quench" plate, then 200 μl acetonitrile, metolazone as IS, was added and mixed with a pipette.

Example 22 mouse PK study: NHC, example compound and Mo Nuola wei

A carrier having the following composition was prepared: solution A (PEG 400/Tween 80 (90%/10%): solution B)

(30% solutol/10% dmac) =1:1 (v/v). The example compounds NHC and Mo Nupi Lavir were suspended in the carrier at 0.4mol/10mL, respectively.

CD1 mice, 20-25g, 6-7 week old, male, total n=45, n=9/compound, purchased from Shanghai family planning institute animals, fasted overnight and fed 4 hours after dosing. Mice were given a dose of 0.4ml/kg, 10ml/kg by oral gavage. Animals were manually restrained and approximately 150 μl of blood per time point was collected into pre-cooled EDTA-K2 tubes by retroorbital injection. Blood samples were centrifuged at 4 ℃ (4000 g,5 min) and plasma was obtained within 15min after sample collection. The plasma samples were stored at about-80 ℃ until analysis. Plasma samples were analyzed using LC-MS/MS. The test results are shown in fig. 1 and the following table, where PK parameters were estimated by a non-compartmental model using Winnonlin software.

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Example 23: pharmacokinetic studies in cynomolgus monkeys

Example 24: treatment with NHC, example Compounds in Ferset/mouse model of influenza infection

Example 25: treatment with NHC, example compounds in a feret/mouse model of severe acute respiratory syndrome coronavirus type 2 infection.

Claims

1. The compound of the formula (I),

R ¹ Is rac=o and,

ra is methyl substituted with Ra1, ra2 and Ra 3;

Ra2 is C _1-6 Alkyl or C _1-6 alkyl-O- (CH) ₂ ) _n -, and

ra3 is selected from H, C _1-6 Alkyl and C _1-6 alkyl-O- (CH) ₂ ) _n -；

n is 0 or 1;

2. The compound of claim 1, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, wherein

R ¹ Is rac=o and,

R ² and R is ³ Is H, or rac=o in each of R2 and R3;

wherein rac=o is selected from:

3. The compound of claim 2, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, wherein rac=o is selected from:

4. the compound of claim 2, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, wherein rac=o is selected from:

5. a compound according to any one of the preceding claims, or a tautomer, stereoisomer or racemate thereof, or a pharmaceutically acceptable salt thereof, wherein Raa is selected from methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, 2-methoxyethyl, fluoro-substituted ethyl, fluoro-substituted propyl, cyclopropyl, cyclobutyl, cyclopentyl, epoxypropyl, tetrahydro-2-furyl, tetrahydro-3-furyl or tetrahydro-2H-pyranyl; methyl, ethyl, propyl, isopropyl, glycidylyl and tetrahydro-2H-pyran-4-yl are preferred.

6. A compound according to any one of the preceding claims, or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, wherein Raa is selected from methyl, ethyl, propyl, isopropyl, n-butyl and sec-butyl.

7. A compound according to claims 1-2, or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, wherein

8. A compound according to any one of the preceding claims, or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, wherein

Ra1 is C _1-6 alkyl-O-, and

9. A compound according to any one of the preceding claims, or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, wherein R ¹ Is rac=o, and R ² And R is ³ Each is H.

10. A compound according to any one of the preceding claims, or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, wherein Ra1 is C _1-6 alkyl-O-.

11. A compound according to claims 1-2, or a tautomer, stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

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12. a pharmaceutical composition comprising a compound according to any one of claims 1-11, or a tautomer, stereoisomer or racemate thereof, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient.

13. Use of a compound according to any one of claims 1 to 11, or a tautomer, stereoisomer or racemate thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment or prophylaxis of RNA viral infection.

14. A method of treating or preventing an RNA viral infection in a subject, comprising administering to a subject in need thereof an effective amount of a compound of any one of claims 1-28, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof.

15. A compound according to any one of claims 1 to 11, or a tautomer, stereoisomer or racemate thereof, or a pharmaceutically acceptable salt thereof, for use as a medicament.

16. A compound according to any one of claims 1 to 11, or a tautomer, stereoisomer or racemate thereof, or a pharmaceutically acceptable salt thereof, for use in the treatment or prophylaxis of RNA viral infections.

17. Use according to claim 13, method according to claim 14 or compound for use according to claim 16, wherein the RNA virus is a coronavirus, such as a human coronavirus, SARS coronavirus or MERS coronavirus, an alpha virus, such as eastern equine encephalitis virus, western equine encephalitis virus, venezuelan equine encephalitis virus, chikungunya virus or ross river virus, a filoviridae virus, such as ebola virus, an orthomyxoviridae virus, such as influenza virus, influenza a virus or influenza b virus, a paramyxoviridae virus, such as Respiratory Syncytial Virus (RSV), a flaviviridae, such as zika virus; preferably SARS-CoV-2/COVID-19 virus, alpha variant SARS-CoV-2/COVID-19 virus, beta variant SARS-CoV-2/COVID-19 virus, gamma variant SARS-CoV-2/COVID-19 virus, delta variant SARS-CoV-2/COVID-19 virus or any other variant SARS-CoV-2/COVID-19 virus.

18. A pharmaceutical combination comprising a compound according to any one of claims 1-11, or a tautomer, stereoisomer, or racemate thereof, or a pharmaceutically acceptable salt thereof, and at least one additional therapeutic agent.

19. The pharmaceutical combination according to claim 18, wherein the additional therapeutic agent is selected from the group consisting of:

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