WO2020255016A1 - Combination of hepatitis b virus (hbv) vaccines and dihydropyrimidine derivatives as capsid assembly modulators - Google Patents

Combination of hepatitis b virus (hbv) vaccines and dihydropyrimidine derivatives as capsid assembly modulators Download PDF

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WO2020255016A1
WO2020255016A1 PCT/IB2020/055707 IB2020055707W WO2020255016A1 WO 2020255016 A1 WO2020255016 A1 WO 2020255016A1 IB 2020055707 W IB2020055707 W IB 2020055707W WO 2020255016 A1 WO2020255016 A1 WO 2020255016A1
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hbv
seq
antigen
sequence
nucleic acid
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PCT/IB2020/055707
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French (fr)
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Helen Horton
Jan Martin Berke
Frederik PAUWELS
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Janssen Sciences Ireland Unlimited Company
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This application contains a sequence listing, which is submitted electronically via EFS- Web as an ASCII formatted sequence listing with a file name“065814_40WO1 Sequence
  • Hepatitis B virus is a small 3.2-kb hepatotropic DNA virus that encodes four open reading frames and seven proteins. Approximately 240 million people have chronic
  • hepatitis B infection chronic HBV
  • chronic HBV hepatitis B infection
  • Persistent HBV infection leads to T-cell exhaustion in circulating and intrahepatic HBV-specific CD4+ and CD8+ T-cells through chronic stimulation of HBV-specific T-cell
  • T-cell polyfunctionality is decreased (i.e., decreased levels of IL-2, tumor necrosis factor (TNF)-a, IFN-g, and lack of proliferation).
  • cccDNA covalently closed circular DNA
  • Current therapies targeting the HBV polymerase suppress viremia, but offer limited effect on cccDNA that resides in the nucleus and related production of circulating antigen.
  • HBV surface antigens HBsAg
  • pegylated interferon (pegIFN)-a has proven better in comparison to nucleoside or nucleotide therapy in terms of sustained off-treatment response with a finite treatment course.
  • IFN-a is reported to exert epigenetic suppression of cccDNA in cell culture and humanized mice, which leads to reduction of virion productivity and transcripts (Belloni et al. J. Clin. Invest. (2012) 122(2), 529-537).
  • this therapy is still fraught with side-effects and overall responses are rather low, in part because IFN-a has only poor modulatory influences on HBV-specific T- cells. In particular, cure rates are low ( ⁇ 10%) and toxicity is high.
  • HBV antivirals namely the HBV polymerase inhibitors entecavir and tenofovir
  • HBV polymerase inhibitors entecavir and tenofovir are effective as monotherapy in inducing viral suppression with a high genetic barrier to emergence of drug resistant mutants and consecutive prevention of liver disease progression.
  • cure of chronic hepatitis B defined by HBsAg loss or seroconversion, is rarely achieved with such HBV polymerase inhibitors. Therefore, these antivirals in theory need to be administered indefinitely to prevent reoccurrence of liver disease, similar to antiretroviral therapy for human immunodeficiency virus (HIV).
  • hepatitis B virus particularly chronic HBV
  • the invention satisfies this need by providing therapeutic combinations or compositions and methods for inducing an immune response against hepatitis B viruses (HBV) infection.
  • the immunogenic compositions/combinations and methods of the invention can be used to provide therapeutic immunity to a subject, such as a subject having chronic HBV infection.
  • the application relates to therapeutic combinations or compositions comprising one or more HBV antigens, or one or more polynucleotides encoding the HBV antigens, and a capsid assembly modulator (CAM), for use in treating an HBV infection in a subject in need thereof.
  • CAM capsid assembly modulator
  • the therapeutic combination comprises:
  • a truncated HBV core antigen consisting of an amino acid sequence that is at least 95%, such as at least 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 2,
  • a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide sequence encoding the HBV polymerase antigen
  • R 1 is selected from the group consisting of phenyl, thiophenyl, pyridyl, and pyridonyl, optionally substituted with one or more substituents selected from the group consisting of C 1- 4 alkyl, halogen, and CN;
  • R 2 is C1-4alkyl
  • R 3 is selected from the group consisting of thiazolyl, pyridyl, and oxazolyl, optionally substituted with one or more substituents selected from fluorine, and C1-6alkyl;
  • n is an integer of 0 or 1;
  • R 4 and R 5 are independently selected from the group consisting of H and -COOH; (i.e., the bond between X and Y) is a single bond or a double bond;
  • X is C-SR 9 or C-OR 9
  • Y is N atom
  • R 8 is CF 3 ;
  • R 9 is selected from the group consisting of H, -C 1-6 alkyl, -C 1-6 alkyl-R 10 , -C 1-6 alkoxy- C 1-6 alkyl-R 10 and -(CH 2 ) p -Q-R 10 ;
  • p is an integer of 0, 1, 2, or 3;
  • Q is selected from the group consisting of aryl, heteroaryl, and a 3- to 7- membered saturated ring, optionally containing a heteroatom, the heteroatom being an oxygen or a nitrogen, the nitrogen being substituted with H, -C 1-6 alkyl, -C 1-6 alkoxy-C 1-6 alkyl and -C 1- 6alkylcarbonyl;
  • the truncated HBV core antigen consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, and the HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 7.
  • the therapeutic combination comprises at least one of the first non-naturally occurring nucleic acid molecule comprising the first polynucleotide sequence encoding the truncated HBV core antigen, and the second non-naturally occurring nucleic acid molecule comprising the second polynucleotide sequence encoding the HBV polymerase antigen.
  • the first non-naturally occurring nucleic acid molecule further comprises a polynucleotide sequence encoding a signal sequence operably linked to the N-terminus of the truncated HBV core antigen
  • the second non-naturally occurring nucleic acid molecule further comprises a polynucleotide sequence encoding a signal sequence operably linked to the N-terminus of the HBV polymerase antigen
  • the signal sequence independently comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15, more preferably, the signal sequence is encoded by the polynucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 14, respectively.
  • the first polynucleotide sequence comprises the
  • polynucleotide sequence having at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3.
  • the second polynucleotide sequence comprises a
  • polynucleotide sequence having at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6.
  • a therapeutic combination comprises:
  • polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 95%, such as at least 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 2;
  • a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide sequence encoding an HBV polymerase antigen having an amino acid sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 7, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity; and
  • R 1 is phenyl substituted with one or more substituents selected from halogens and C 1- 6 alkyl;
  • R 2 is methyl or ethyl
  • R 3 is thiazolyl
  • n is an integer of 0 or 1;
  • R 4 and R 5 are H
  • Y is NR 9 ;
  • Z is CH 2 ;
  • R 9 is C 1-6 alkyl-CO 2 H or (CH 2 ) p -Q-R 10 ;
  • the therapeutic combination comprises a) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding an truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; b) a second non-naturally occurring nucleic acid molecule comprising a second
  • the therapeutic combination comprises a first non-naturally occurring nucleic acid molecule comprising a polynucleotide sequence having at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3, and a second non-naturally occurring nucleic acid molecule comprising the polynucleotide sequence having at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6.
  • the therapeutic combination comprises a) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3; b) a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide sequence of SEQ ID NO: 5 or 6; and c) compound 1A:
  • each of the first and the second non-naturally occurring nucleic acid molecules is an RNA molecule, preferably an mRNA or a self-replicating RNA molecule.
  • each of the first and the second non-naturally occurring nucleic acid molecules is independently formulated with a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • the application also relates to a therapeutic combination or kit of the application for use in inducing an immune response against hepatitis B virus (HBV); and use of a therapeutic combination, composition or kit of the application in the manufacture of a medicament for inducing an immune response against hepatitis B virus (HBV).
  • the use can further comprise a combination with another immunogenic or therapeutic agent, preferably another HBV antigen or another HBV therapy.
  • the subject has chronic HBV infection.
  • the application further relates to a therapeutic combination or kit of the application for use in treating an HBV-induced disease in a subject in need thereof; and use of therapeutic combination or kit of the application in the manufacture of a medicament for treating an HBV- induced disease in a subject in need thereof.
  • the use can further comprise a combination with another therapeutic agent, preferably another anti-HBV antigen.
  • the subject has chronic HBV infection, and the HBV-induced disease is selected from the group consisting of advanced fibrosis, cirrhosis, and hepatocellular carcinoma (HCC).
  • the application also relates to a method of inducing an immune response against an HBV or a method of treating an HBV infection or an HBV-induced disease, comprising administering to a subject in need thereof a therapeutic combination according to
  • FIG.1A and FIG.1B show schematic representations of DNA plasmids according to embodiments of the application;
  • FIG.1A shows a DNA plasmid encoding an HBV core antigen according to an embodiment of the application;
  • FIG.1B shows a DNA plasmid encoding an HBV polymerase (pol) antigen according to an embodiment of the application;
  • the HBV core and pol antigens are expressed under control of a CMV promoter with an N- terminal cystatin S signal peptide that is cleaved from the expressed antigen upon secretion from the cell;
  • transcriptional regulatory elements of the plasmid include an enhancer sequence located between the CMV promoter and the polynucleotide sequence encoding the HBV antigen and a bGH polyadenylation sequence located downstream of the polynucleotide sequence encoding the HBV antigen;
  • a second expression cassette is included in the plasmid in reverse orientation including a kanamycin resistance gene under
  • FIG.2A and FIG.2B show the schematic representations of the expression cassettes in adenoviral vectors according to embodiments of the application
  • FIG.2A shows the expression cassette for a truncated HBV core antigen, which contains a CMV promoter, an intron (a fragment derived from the human ApoAI gene - GenBank accession X01038 base pairs 295– 523, harboring the ApoAI second intron), a human immunoglobulin secretion signal, followed by a coding sequence for a truncated HBV core antigen and a SV40 polyadenylation signal
  • FIG.2B shows the expression cassette for a fusion protein of a truncated HBV core antigen operably linked to an HBV polymerase antigen, which is otherwise identical to the expression cassette for the truncated HBV core antigen except the HBV antigen.
  • FIG.3 shows ELISPOT responses of Balb/c mice immunized with different DNA plasmids expressing HBV core antigen or HBV pol antigen, as described in Example 3;
  • the conjunctive term“and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by“and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term“and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term“and/or.”
  • any numerical value such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term“about.”
  • a numerical value typically includes ⁇ 10% of the recited value.
  • a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL.
  • a concentration range of 1 mg/mL to 10 mg/mL includes 0.9 mg/mL to 11 mg/mL.
  • the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.
  • phrases“percent (%) sequence identity” or“% identity” or“% identical to” when used with reference to an amino acid sequence describe the number of matches (“hits”) of identical amino acids of two or more aligned amino acid sequences as compared to the number of amino acid residues making up the overall length of the amino acid sequences.
  • the percentage of amino acid residues that are the same e.g. 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identity over the full-length of the amino acid sequences
  • sequences which are compared to determine sequence identity may thus differ by substitution(s), addition(s) or deletion(s) of amino acids.
  • Suitable programs for aligning protein sequences are known to the skilled person.
  • the percentage sequence identity of protein sequences can, for example, be determined with programs such as CLUSTALW, Clustal Omega, FASTA or BLAST, e.g. using the NCBI BLAST algorithm (Altschul SF, et al (1997), Nucleic Acids Res. 25:3389-3402).
  • the terms and phrases“in combination,”“in combination with,”“co delivery,” and“administered together with” in the context of the administration of two or more therapies or components to a subject refers to simultaneous administration or subsequent administration of two or more therapies or components, such as two vectors, e.g., DNA plasmids, peptides, or a therapeutic combination and an adjuvant.“Simultaneous administration” can be administration of the two or more therapies or components at least within the same day.
  • first therapy or component e.g. first DNA plasmid encoding an HBV antigen
  • first DNA plasmid encoding an HBV antigen a second therapy or component (e.g., second DNA plasmid encoding an HBV antigen), and a third therapy or component (e.g., a CAM) are administered in separate compositions, such as two or three separate compositions.
  • a second therapy or component e.g., second DNA plasmid encoding an HBV antigen
  • a third therapy or component e.g., a CAM
  • a“non-naturally occurring” nucleic acid or polypeptide refers to a nucleic acid or polypeptide that does not occur in nature.
  • A“non-naturally occurring” nucleic acid or polypeptide can be synthesized, treated, fabricated, and/or otherwise manipulated in a laboratory and/or manufacturing setting.
  • a non-naturally occurring nucleic acid or polypeptide can comprise a naturally-occurring nucleic acid or polypeptide that is treated, processed, or manipulated to exhibit properties that were not present in the naturally-occurring nucleic acid or polypeptide, prior to treatment.
  • a“non-naturally occurring” nucleic acid or polypeptide can be a nucleic acid or polypeptide isolated or separated from the natural source in which it was discovered, and it lacks covalent bonds to sequences with which it was associated in the natural source.
  • a “non-naturally occurring” nucleic acid or polypeptide can be made recombinantly or via other methods, such as chemical synthesis.
  • “subject” means any animal, preferably a mammal, most preferably a human, to whom will be or has been treated by a method according to an embodiment of the application.
  • the term“mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans, etc., more preferably a human.
  • operably linked refers to a linkage or a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence operably linked to a nucleic acid sequence of interest is capable of directing the transcription of the nucleic acid sequence of interest, or a signal sequence operably linked to an amino acid sequence of interest is capable of secreting or translocating the amino acid sequence of interest over a membrane.
  • HBV vectors of the application may contain particular components, including, but not limited to, certain promoter sequences, enhancer or regulatory sequences, signal peptides, coding sequence of an HBV antigen, polyadenylation signal sequences, etc. arranged in a particular order, those having ordinary skill in the art will appreciate that the concepts disclosed herein may equally apply to other components arranged in other orders that can be used in HBV vectors of the application.
  • the application contemplates use of any of the applicable components in any combination having any sequence that can be used in HBV vectors of the application, whether or not a particular combination is expressly described.
  • the invention generally relates to a therapeutic combination comprising one or more HBV antigens and at least one CAM.
  • HBV Hepatitis B Virus
  • hepatitis B virus or“HBV” refers to a virus of the hepadnaviridae family.
  • HBV is a small (e.g., 3.2 kb) hepatotropic DNA virus that encodes four open reading frames and seven proteins.
  • the seven proteins encoded by HBV include small (S), medium (M), and large (L) surface antigen (HBsAg) or envelope (Env) proteins, pre-Core protein, core protein, viral polymerase (Pol), and HBx protein.
  • HBV expresses three surface antigens, or envelope proteins, L, M, and S, with S being the smallest and L being the largest.
  • the extra domains in the M and L proteins are named Pre-S2 and Pre-S1, respectively.
  • Core protein is the subunit of the viral nucleocapsid. Pol is needed for synthesis of viral DNA (reverse transcriptase, RNaseH, and primer), which takes place in nucleocapsids localized to the cytoplasm of infected hepatocytes.
  • PreCore is the core protein with an N-terminal signal peptide and is proteolytically processed at its N and C termini before secretion from infected cells, as the so-called hepatitis B e-antigen (HBeAg). HBx protein is required for efficient transcription of covalently closed circular DNA (cccDNA). HBx is not a viral structural protein. All viral proteins of HBV have their own mRNA except for core and polymerase, which share an mRNA. With the exception of the protein pre-Core, none of the HBV viral proteins are subject to post-translational proteolytic processing.
  • the HBV virion contains a viral envelope, nucleocapsid, and single copy of the partially double-stranded DNA genome.
  • the nucleocapsid comprises 120 dimers of core protein and is covered by a capsid membrane embedded with the S, M, and L viral envelope or surface antigen proteins.
  • the virus is uncoated and the capsid- containing relaxed circular DNA (rcDNA) with covalently bound viral polymerase migrates to the nucleus.
  • rcDNA relaxed circular DNA
  • phosphorylation of the core protein induces structural changes, exposing a nuclear localization signal enabling interaction of the capsid with so- called importins.
  • rccDNA covalently closed circular DNA genome from which overlapping transcripts encode for HBeAg, HBsAg, Core protein, viral polymerase and HBx protein.
  • Core protein, viral polymerase, and pre- genomic RNA associate in the cytoplasm and self-assemble into immature pgRNA- containing capsid particles, which further convert into mature rcDNA-capsids and function as a common intermediate that is either enveloped and secreted as infectious virus particles or transported back to the nucleus to replenish and maintain a stable cccDNA pool.
  • HBV is divided into four serotypes (adr, adw, ayr, ayw) based on antigenic epitopes present on the envelope proteins, and into eight genotypes (A, B, C, D, E, F, G, and H) based on the sequence of the viral genome.
  • the HBV genotypes are distributed over different geographic regions. For example, the most prevalent genotypes in Asia are genotypes B and C. Genotype D is dominant in Africa, the Middle East, and India, whereas genotype A is widespread in Northern Europe, sub-Saharan Africa, and West Africa.
  • the terms“HBV antigen,”“antigenic polypeptide of HBV,”“HBV antigenic polypeptide,”“HBV antigenic protein,”“HBV immunogenic polypeptide,” and “HBV immunogen” all refer to a polypeptide capable of inducing an immune response, e.g., a humoral and/or cellular mediated response, against an HBV in a subject.
  • the HBV antigen can be a polypeptide of HBV, a fragment or epitope thereof, or a combination of multiple HBV polypeptides, portions or derivatives thereof.
  • an HBV antigen is capable of raising in a host a protective immune response, e.g., inducing an immune response against a viral disease or infection, and/or producing an immunity (i.e., vaccinates) in a subject against a viral disease or infection, that protects the subject against the viral disease or infection.
  • an HBV antigen can comprise a polypeptide or immunogenic fragment(s) thereof from any HBV protein, such as HBeAg, pre-core protein, HBsAg (S, M, or L proteins), core protein, viral polymerase, or HBx protein derived from any HBV genotype, e.g., genotype A, B, C, D, E, F, G, and/or H, or combination thereof.
  • each of the terms“HBV core antigen,”“HBc” and“core antigen” refers to an HBV antigen capable of inducing an immune response, e.g., a humoral and/or cellular mediated response, against an HBV core protein in a subject.
  • Each of the terms “core,”“core polypeptide,” and“core protein” refers to the HBV viral core protein.
  • Full- length core antigen is typically 183 amino acids in length and includes an assembly domain (amino acids 1 to 149) and a nucleic acid binding domain (amino acids 150 to 183). The 34- residue nucleic acid binding domain is required for pre-genomic RNA encapsidation. This domain also functions as a nuclear import signal.
  • HBV core protein is dimeric in solution, with the dimers self-assembling into icosahedral capsids. Each dimer of core protein has four a-helix bundles flanked by an a-helix domain on either side. Truncated HBV core proteins lacking the nucleic acid binding domain are also capable of forming capsids.
  • an HBV antigen is a truncated HBV core antigen.
  • a“truncated HBV core antigen” refers to an HBV antigen that does not contain the entire length of an HBV core protein, but is capable of inducing an immune response against the HBV core protein in a subject.
  • an HBV core antigen can be modified to delete one or more amino acids of the highly positively charged (arginine rich) C-terminal nucleic acid binding domain of the core antigen, which typically contains seventeen arginine (R) residues.
  • a truncated HBV core antigen comprises a deletion in the C-terminal nucleic acid binding domain, such as a deletion of 1 to 34 amino acid residues of the C-terminal nucleic acid binding domain, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acid residues, preferably a deletion of all 34 amino acid residues.
  • a truncated HBV core antigen comprises a deletion in the C-terminal nucleic acid binding domain, preferably a deletion of all 34 amino acid residues.
  • An HBV core antigen of the application can be a consensus sequence derived from multiple HBV genotypes (e.g., genotypes A, B, C, D, E, F, G, and H).
  • Consensus sequence means an artificial sequence of amino acids based on an alignment of amino acid sequences of homologous proteins, e.g., as determined by an alignment (e.g., using Clustal Omega) of amino acid sequences of homologous proteins. It can be the calculated order of most frequent amino acid residues, found at each position in a sequence alignment, based upon sequences of HBV antigens (e.g., core, pol, etc.) from at least 100 natural HBV isolates.
  • a consensus sequence can be non-naturally occurring and different from the native viral sequences. Consensus sequences can be designed by aligning multiple HBV antigen sequences from different sources using a multiple sequence alignment tool, and at variable alignment positions, selecting the most frequent amino acid.
  • a consensus sequence of an HBV antigen is derived from HBV genotypes B, C, and D.
  • the term“consensus antigen” is used to refer to an antigen having a consensus sequence.
  • An exemplary truncated HBV core antigen according to the application lacks the nucleic acid binding function, and is capable of inducing an immune response in a mammal against at least two HBV genotypes.
  • a truncated HBV core antigen is capable of inducing a T cell response in a mammal against at least HBV genotypes B, C and D. More preferably, a truncated HBV core antigen is capable of inducing a CD8 T cell response in a human subject against at least HBV genotypes A, B, C and D.
  • an HBV core antigen of the application is a consensus antigen, preferably a consensus antigen derived from HBV genotypes B, C, and D, more preferably a truncated consensus antigen derived from HBV genotypes B, C, and D.
  • An exemplary truncated HBV core consensus antigen according to the application consists of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%,
  • SEQ ID NO: 2 and SEQ ID NO: 4 are core consensus antigens derived from HBV genotypes B, C, and D. SEQ ID NO: 2 and SEQ ID NO: 4 each contain a 34-amino acid C-terminal deletion of the highly positively charged (arginine rich) nucleic acid binding domain of the native core antigen.
  • an HBV core antigen is a truncated HBV antigen consisting of the amino acid sequence of SEQ ID NO: 2.
  • an HBV core antigen is a truncated HBV antigen consisting of the amino acid sequence of SEQ ID NO: 4.
  • an HBV core antigen further contains a signal sequence operably linked to the N-terminus of a mature HBV core antigen sequence, such as the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
  • the signal sequence has the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15.
  • the term“HBV polymerase antigen,”“HBV Pol antigen” or“HBV pol antigen” refers to an HBV antigen capable of inducing an immune response, e.g., a humoral and/or cellular mediated response, against an HBV polymerase in a subject.
  • Each of the terms“polymerase,”“polymerase polypeptide,”“Pol” and“pol” refers to the HBV viral DNA polymerase.
  • the HBV viral DNA polymerase has four domains, including, from the N terminus to the C terminus, a terminal protein (TP) domain, which acts as a primer for minus- strand DNA synthesis; a spacer that is nonessential for the polymerase functions; a reverse transcriptase (RT) domain for transcription; and a RNase H domain.
  • TP terminal protein
  • RT reverse transcriptase
  • an HBV antigen comprises an HBV Pol antigen, or any immunogenic fragment or combination thereof.
  • An HBV Pol antigen can contain further modifications to improve immunogenicity of the antigen, such as by introducing mutations into the active sites of the polymerase and/or RNase domains to decrease or substantially eliminate certain enzymatic activities.
  • an HBV Pol antigen of the application does not have reverse transcriptase activity and RNase H activity, and is capable of inducing an immune response in a mammal against at least two HBV genotypes.
  • an HBV Pol antigen is capable of inducing a T cell response in a mammal against at least HBV genotypes B, C and D. More preferably, an HBV Pol antigen is capable of inducing a CD8 T cell response in a human subject against at least HBV genotypes A, B, C and D.
  • an HBV Pol antigen is an inactivated Pol antigen.
  • an inactivated HBV Pol antigen comprises one or more amino acid mutations in the active site of the polymerase domain.
  • an inactivated HBV Pol antigen comprises one or more amino acid mutations in the active site of the RNaseH domain.
  • an inactivated HBV pol antigen comprises one or more amino acid mutations in the active site of both the polymerase domain and the RNaseH domain.
  • the“YXDD” motif in the polymerase domain of an HBV pol antigen that can be required for nucleotide/metal ion binding can be mutated, e.g., by replacing one or more of the aspartate residues (D) with asparagine residues (N), eliminating or reducing metal coordination function, thereby decreasing or substantially eliminating reverse transcriptase function.
  • coordination can be mutated, e.g., by replacing one or more aspartate residues (D) with asparagine residues (N) and/or replacing the glutamate residue (E) with glutamine (Q), thereby decreasing or substantially eliminating RNaseH function.
  • D aspartate residues
  • N asparagine residues
  • E glutamate residue
  • Q glutamine
  • an HBV pol antigen is modified by (1) mutating the aspartate residues (D) to asparagine residues (N) in the“YXDD” motif of the polymerase domain; and (2) mutating the first aspartate residue (D) to an asparagine residue (N) and the first glutamate residue (E) to a glutamine residue (N) in the“DEDD” motif of the RNaseH domain, thereby decreasing or substantially eliminating both the reverse transcriptase and RNaseH functions of the pol antigen.
  • an HBV pol antigen is a consensus antigen, preferably a consensus antigen derived from HBV genotypes B, C, and D, more preferably an inactivated consensus antigen derived from HBV genotypes B, C, and D.
  • An exemplary HBV pol consensus antigen according to the application comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 7, preferably at least 98% identical to SEQ ID NO: 7, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 7.
  • SEQ ID NO: 7 is a pol consensus antigen derived from HBV genotypes B, C, and D comprising four mutations located in the active sites of the polymerase and RNaseH domains.
  • the four mutations include mutation of the aspartic acid residues (D) to asparagine residues (N) in the“YXDD” motif of the polymerase domain; and mutation of the first aspartate residue (D) to an asparagine residue (N) and mutation of the glutamate residue (E) to a glutamine residue (Q) in the“DEDD” motif of the RNaseH domain.
  • an HBV pol antigen comprises the amino acid sequence of SEQ ID NO: 7.
  • an HBV pol antigen consists of the amino acid sequence of SEQ ID NO: 7.
  • an HBV pol antigen further contains a signal sequence operably linked to the N-terminus of a mature HBV pol antigen sequence, such as the amino acid sequence of SEQ ID NO: 7.
  • the signal sequence has the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15.
  • fusion protein or“fusion” refers to a single polypeptide chain having at least two polypeptide domains that are not normally present in a single, natural polypeptide.
  • an HBV antigen comprises a fusion protein comprising a truncated HBV core antigen operably linked to an HBV Pol antigen, or an HBV Pol antigen operably linked to a truncated HBV core antigen, preferably via a linker.
  • Exemplary linkers are polyglycines, particularly (Gly)5, (Gly)8; poly(Gly-Ala), and polyalanines.
  • One exemplary suitable linker as shown in the Examples below is (AlaGly)n, wherein n is an integer of 2 to 5.
  • a fusion protein of the application is capable of inducing an immune response in a mammal against HBV core and HBV Pol of at least two HBV genotypes.
  • a fusion protein is capable of inducing a T cell response in a mammal against at least HBV genotypes B, C and D. More preferably, the fusion protein is capable of inducing a CD8 T cell response in a human subject against at least HBV genotypes A, B, C and D.
  • a fusion protein comprises a truncated HBV core antigen having an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4, a linker, and an HBV Pol antigen having an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, identical to SEQ ID NO: 2 or
  • a fusion protein further comprises a signal sequence operably linked to the N-terminus of the fusion protein.
  • the signal sequence has the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15.
  • a fusion protein comprises the amino acid sequence of SEQ ID NO: 17.
  • HBV vaccines that can be used for the present invention are described in U.S. Patent Application No: 16/223,251, filed December 18, 2018, the contents of the application, more preferably the examples of the application, are hereby incorporated by reference in their entireties.
  • the application provides a non-naturally occurring nucleic acid molecule encoding an HBV antigen useful for an invention according to embodiments of the application, and vectors comprising the non-naturally occurring nucleic acid.
  • a first or second non-naturally occurring nucleic acid molecule can comprise any polynucleotide sequence encoding an HBV antigen useful for the application, which can be made using methods known in the art in view of the present disclosure.
  • a first or second polynucleotide encodes at least one of a truncated HBV core antigen and an HBV polymerase antigen of the application.
  • a polynucleotide can be in the form of RNA or in the form of DNA obtained by recombinant techniques (e.g., cloning) or produced synthetically (e.g., chemical synthesis).
  • the DNA can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequence.
  • the DNA can, for example, comprise genomic DNA, cDNA, or combinations thereof.
  • the polynucleotide can also be a DNA/RNA hybrid.
  • the polynucleotides and vectors of the application can be used for recombinant protein production, expression of the protein in host cell, or the production of viral particles.
  • a polynucleotide is DNA.
  • a first non-naturally occurring nucleic acid molecule comprises a first polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 2, preferably 98%, 99% or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4.
  • a first non- naturally occurring nucleic acid molecule comprises a first polynucleotide sequence encoding a truncated HBV core antigen consisting the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
  • polynucleotide sequences of the application encoding a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 include, but are not limited to, a polynucleotide sequence at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 3, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3, preferably 98%, 99% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3.
  • Exemplary non-naturally occurring nucleic acid molecules encoding a truncated HBV core antigen have the polynucleotide sequence of SEQ
  • a second non-naturally occurring nucleic acid molecule comprises a second polynucleotide sequence encoding an HBV polymerase antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 7, preferably 100% identical to SEQ ID NO: 7.
  • a second non-naturally occurring nucleic acid molecule comprises a second polynucleotide sequence encoding an HBV polymerase antigen consisting of the amino acid sequence of SEQ ID NO: 7.
  • polynucleotide sequences of the application encoding an HBV Pol antigen comprising the amino acid sequence of at least 90% identical to SEQ ID NO: 7 include, but are not limited to, a polynucleotide sequence at least 90% identical to SEQ ID NO: 5 or SEQ ID NO: 6, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 5 or SEQ ID NO: 6, preferably 98%, 99% or 100% identical to SEQ ID NO: 5 or SEQ ID NO: 6.
  • Exemplary non-naturally occurring nucleic acid molecules encoding an HBV pol antigen have the polynucleotide sequence of SEQ ID NOs: 5 or 6.
  • a non-naturally occurring nucleic acid molecule encodes an HBV antigen fusion protein comprising a truncated HBV core antigen operably linked to an HBV Pol antigen, or an HBV Pol antigen operably linked to a truncated HBV core antigen.
  • a non-naturally occurring nucleic acid molecule of the application encodes a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4, more preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO:4; a linker; and an HBV polymerase antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, such as at least 90%, 91%, 92%, 93%, 94%
  • a non-naturally occurring nucleic acid molecule encodes a fusion protein comprising a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, a linker comprising (AlaGly)n, wherein n is an integer of 2 to 5; and an HBV Pol antigen comprising the amino acid sequence of SEQ ID NO: 7.
  • a non- naturally occurring nucleic acid molecule encodes an HBV antigen fusion protein comprising the amino acid sequence of SEQ ID NO: 16.
  • a non- naturally occurring nucleic acid molecule encoding an HBV antigen fusion protein comprises SEQ ID NO: 1 or SEQ ID NO: 3, operably linked to SEQ ID NO: 11, which is further operably linked to SEQ ID NO: 5 or SEQ ID NO: 6.
  • a non-naturally occurring nucleic acid molecule encoding an HBV fusion further comprises a coding sequence for a signal sequence that is operably linked to the N-terminus of the HBV fusion sequence, such as the amino acid sequence of SEQ ID NO: 16.
  • the signal sequence has the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15.
  • the coding sequence for a signal sequence comprises the polynucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 14.
  • the encoded fusion protein with the signal sequence comprises the amino acid sequence of SEQ ID NO: 17.
  • Vectors of the application can contain a variety of regulatory sequences.
  • regulatory sequence refers to any sequence that allows, contributes or modulates the functional regulation of the nucleic acid molecule, including replication, duplication, transcription, splicing, translation, stability and/or transport of the nucleic acid or one of its derivative (i.e. mRNA) into the host cell or organism.
  • this term encompasses promoters, enhancers and other expression control elements (e.g., polyadenylation signals and elements that affect mRNA stability).
  • a vector is a non-viral vector.
  • non-viral vectors include, but are not limited to, DNA plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, etc.
  • non-viral vectors include, but are not limited to, RNA replicon, mRNA replicon, modified mRNA replicon or self-amplifying mRNA, closed linear deoxyribonucleic acid, e.g. a linear covalently closed DNA such as linear covalently closed double stranded DNA molecule.
  • a non-viral vector is a DNA plasmid.
  • A“DNA plasmid”, which is used interchangeably with“DNA plasmid vector,”“plasmid DNA” or“plasmid DNA vector,” refers to a double-stranded and generally circular DNA sequence that is capable of autonomous replication in a suitable host cell.
  • DNA plasmids used for expression of an encoded polynucleotide typically comprise an origin of replication, a multiple cloning site, and a selectable marker, which for example, can be an antibiotic resistance gene.
  • Thermo Fisher Scientific which can be used for production and/or expression in Saccharomyces cerevisiae strains of yeast
  • MAXBAC® complete baculovirus expression system (Thermo Fisher Scientific), which can be used for production and/or expression in insect cells
  • pcDNATM or pcDNA3TM (Life Technologies, Thermo Fisher Scientific), which can be used for high level constitutive protein expression in mammalian cells
  • pVAX or pVAX-1 Life Technologies, Thermo Fisher Scientific
  • the backbone of any commercially available DNA plasmid can be modified to optimize protein expression in the host cell, such as to reverse the orientation of certain elements (e.g., origin of replication and/or antibiotic resistance cassette), replace a promoter endogenous to the plasmid (e.g., the promoter in the antibiotic resistance cassette), and/or replace the polynucleotide sequence encoding transcribed proteins (e.g., the coding sequence of the antibiotic resistance gene), by using routine techniques and readily available starting materials. (See e.g., Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989)).
  • a vector of the application can also be a viral vector.
  • viral vectors are genetically engineered viruses carrying modified viral DNA or RNA that has been rendered non-infectious, but still contains viral promoters and transgenes, thus allowing for translation of the transgene through a viral promoter. Because viral vectors are frequently lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection. Examples of viral vectors that can be used include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, pox virus vectors, enteric virus vectors, Venezuelan Equine Encephalitis virus vectors, Semliki Forest Virus vectors, Tobacco Mosaic Virus vectors, lentiviral vectors, etc.
  • viral vectors examples include, but are not limited to, arenavirus viral vectors, replication-deficient arenavirus viral vectors or replication-competent arenavirus viral vectors, bi-segmented or tri-segmented arenavirus, infectious arenavirus viral vectors, nucleic acids which comprise an arenavirus genomic segment wherein one open reading frame of the genomic segment is deleted or functionally inactivated (and replaced by a nucleic acid encoding an HBV antigen as described herein), arenavirus such as lymphocytic choriomeningitidis virus (LCMV), e.g., clone 13 strain or MP strain, and arenavirus such as Junin virus e.g., Candid #1 strain.
  • the vector can also be a non- viral vector.
  • a viral vector is an adenovirus vector, e.g., a recombinant adenovirus vector.
  • a recombinant adenovirus vector can for instance be derived from a human adenovirus (HAdV, or AdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV) or rhesus adenovirus (rhAd).
  • an adenovirus vector is a recombinant human adenovirus vector, for instance a recombinant human adenovirus serotype 26, or any one of recombinant human adenovirus serotype 5, 4, 35, 7, 48, etc.
  • an adenovirus vector is a rhAd vector, e.g. rhAd51, rhAd52 or rhAd53.
  • a recombinant viral vector useful for the application can be prepared using methods known in the art in view of the present disclosure. For example, in view of the degeneracy of the genetic code, several nucleic acid sequences can be designed that encode the same polypeptide.
  • a polynucleotide encoding an HBV antigen of the application can optionally be codon-optimized to ensure proper expression in the host cell (e.g., bacterial or mammalian cells). Codon-optimization is a technology widely applied in the art, and methods for obtaining codon-optimized polynucleotides will be well known to those skilled in the art in view of the present disclosure.
  • a vector of the application e.g., a DNA plasmid or a viral vector (particularly an adenoviral vector), can comprise any regulatory elements to establish conventional function(s) of the vector, including but not limited to replication and expression of the HBV antigen(s) encoded by the polynucleotide sequence of the vector. Regulatory elements include, but are not limited to, a promoter, an enhancer, a polyadenylation signal, translation stop codon, a ribosome binding element, a transcription terminator, selection markers, origin of replication, etc.
  • a vector can comprise one or more expression cassettes.
  • An“expression cassette” is part of a vector that directs the cellular machinery to make RNA and protein.
  • An expression cassette typically comprises three components: a promoter sequence, an open reading frame, and a 3’-untranslated region (UTR) optionally comprising a polyadenylation signal.
  • An open reading frame is a reading frame that contains a coding sequence of a protein of interest (e.g., HBV antigen) from a start codon to a stop codon.
  • Regulatory elements of the expression cassette can be operably linked to a polynucleotide sequence encoding an HBV antigen of interest.
  • the term“operably linked” is to be taken in its broadest reasonable context, and refers to a linkage of polynucleotide elements in a functional relationship.
  • a polynucleotide is“operably linked” when it is placed into a functional relationship with another polynucleotide.
  • a promoter is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Any components suitable for use in an expression cassette described herein can be used in any combination and in any order to prepare vectors of the application.
  • a vector can comprise a promoter sequence, preferably within an expression cassette, to control expression of an HBV antigen of interest.
  • promoter is used in its conventional sense, and refers to a nucleotide sequence that initiates the transcription of an operably linked nucleotide sequence.
  • a promoter is located on the same strand near the nucleotide sequence it transcribes. Promoters can be a constitutive, inducible, or repressible. Promoters can be naturally occurring or synthetic.
  • a promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals.
  • a promoter can be a homologous promoter (i.e., derived from the same genetic source as the vector) or a heterologous promoter (i.e., derived from a different vector or genetic source).
  • the promoter can be endogenous to the plasmid (homologous) or derived from other sources (heterologous).
  • the promoter is located upstream of the polynucleotide encoding an HBV antigen within an expression cassette.
  • promoters examples include, but are not limited to, a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter (CMV-IE), Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HAV human immunodeficiency virus
  • HSV human immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • LTR long terminal repeat
  • AMV avian leukosis virus
  • CMV cytomegalovirus
  • a promoter can also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metalothionein.
  • a promoter can also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic.
  • a promoter is a strong eukaryotic promoter, preferably a cytomegalovirus immediate early (CMV-IE) promoter.
  • CMV-IE cytomegalovirus immediate early
  • a vector can comprise additional polynucleotide sequences that stabilize the expressed transcript, enhance nuclear export of the RNA transcript, and/or improve transcriptional-translational coupling.
  • sequences include polyadenylation signals and enhancer sequences.
  • a polyadenylation signal is typically located downstream of the coding sequence for a protein of interest (e.g., an HBV antigen) within an expression cassette of the vector.
  • Enhancer sequences are regulatory DNA sequences that, when bound by transcription factors, enhance the transcription of an associated gene.
  • An enhancer sequence is preferably located upstream of the polynucleotide sequence encoding an HBV antigen, but downstream of a promoter seque ce within an expression cassette of the vector.
  • Any polyadenylation signal known to those skilled in the art in view of the present disclosure can be used.
  • the polyadenylation signal can be a SV40
  • a polyadenylation signal is a bovine growth hormone (bGH) polyadenylation signal or a SV40 polyadenylation signal.
  • bGH bovine growth hormone
  • hGH human growth hormone
  • a polyadenylation signal is a bovine growth hormone (bGH) polyadenylation signal or a SV40 polyadenylation signal.
  • a nucleotide sequence of an exemplary bGH polyadenylation signal is shown in SEQ ID NO: 20.
  • a nucleotide sequence of an exemplary SV40 polyadenylation signal is shown in SEQ ID NO: 13.
  • an enhancer sequence can be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer, such as one from CMV, HA, RSV, or EBV.
  • a viral enhancer such as one from CMV, HA, RSV, or EBV.
  • WPRE Woodchuck HBV Post-transcriptional regulatory element
  • ApoAI intron/exon sequence derived from human apolipoprotein A1 precursor
  • HTLV-1) long terminal repeat (LTR) untranslated R-U5 domain of the human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR), a splicing enhancer, a synthetic rabbit b-globin intron, or any combination thereof.
  • an enhancer sequence is a composite sequence of three consecutive elements of the untranslated R-U5 domain of HTLV-1 LTR, rabbit b-globin intron, and a splicing enhancer, which is referred to herein as“a triple enhancer sequence.”
  • a nucleotide sequence of an exemplary triple enhancer sequence is shown in SEQ ID NO: 10.
  • Another exemplary enhancer sequence is an ApoAI gene fragment shown in SEQ ID NO: 12.
  • a vector can comprise a polynucleotide sequence encoding a signal peptide sequence.
  • the polynucleotide sequence encoding the signal peptide sequence is located upstream of the polynucleotide sequence encoding an HBV antigen.
  • Signal peptides typically direct localization of a protein, facilitate secretion of the protein from the cell in which it is produced, and/or improve antigen expression and cross-presentation to antigen- presenting cells.
  • a signal peptide can be present at the N-terminus of an HBV antigen when expressed from the vector, but is cleaved off by signal peptidase, e.g., upon secretion from the cell.
  • a signal peptide can be a cystatin S signal peptide; an immunoglobulin (Ig) secretion signal, such as the Ig heavy chain gamma signal peptide SPIgG or the Ig heavy chain epsilon signal peptide SPIgE.
  • Ig immunoglobulin
  • a signal peptide sequence is a cystatin S signal peptide.
  • Exemplary nucleic acid and amino acid sequences of a cystatin S signal peptide are shown in SEQ ID NOs: 8 and 9, respectively.
  • Exemplary nucleic acid and amino acid sequences of an immunoglobulin secretion signal are shown in SEQ ID NOs: 14 and 15, respectively.
  • a vector such as a DNA plasmid
  • Bacterial origins of replication and antibiotic resistance cassettes can be located in a vector in the same orientation as the expression cassette encoding an HBV antigen, or in the opposite (reverse) orientation.
  • An origin of replication (ORI) is a sequence at which replication is initiated, enabling a plasmid to reproduce and survive within cells. Examples of ORIs suitable for use in the application include, but are not limited to ColE1, pMB1, pUC, pSC101, R6K, and 15A, preferably pUC.
  • An exemplary nucleotide sequence of a pUC ORI is shown in SEQ ID NO: 21.
  • Expression cassettes for selection and maintenance in bacterial cells typically include a promoter sequence operably linked to an antibiotic resistance gene.
  • the promoter sequence operably linked to an antibiotic resistance gene differs from the promoter sequence operably linked to a polynucleotide sequence encoding a protein of interest, e.g., HBV antigen.
  • the antibiotic resistance gene can be codon optimized, and the sequence composition of the antibiotic resistance gene is normally adjusted to bacterial, e.g., E. coli, codon usage.
  • Any antibiotic resistance gene known to those skilled in the art in view of the present disclosure can be used, including, but not limited to, kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr), and tetracycline resistance gene (Tetr), as well as genes conferring resistance to chloramphenicol, bleomycin, spectinomycin, carbenicillin, etc.
  • Kanr kanamycin resistance gene
  • Amr ampicillin resistance gene
  • Tetr tetracycline resistance gene
  • an antibiotic resistance gene in the antibiotic expression cassette of a vector is a kanamycin resistance gene (Kanr).
  • the sequence of Kanr gene is shown in SEQ ID NO: 22.
  • the Kanr gene is codon optimized.
  • An exemplary nucleic acid sequence of a codon optimized Kanr gene is shown in SEQ ID NO: 23.
  • the Kanr can be operably linked to its native promoter, or the Kanr gene can be linked to a heterologous promoter.
  • the Kanr gene is operably linked to the ampicillin resistance gene (Ampr) promoter, known as the bla promoter.
  • An exemplary nucleotide sequence of a bla promoter is shown in SEQ ID NO: 24.
  • Such vector further comprises an antibiotic resistance expression cassette including a
  • polynucleotide encoding an antibiotic resistance gene preferably a Kanr gene, more preferably a codon optimized Kanr gene of at least 90% identical to SEQ ID NO: 23, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 23, preferably 100% identical to SEQ ID NO: 23, operably linked to an Ampr (bla) promoter of SEQ ID NO: 24, upstream of and operably linked to the polynucleotide encoding the antibiotic resistance gene; and an origin of replication, preferably a pUC ori of SEQ ID NO: 21.
  • the antibiotic resistance cassette and the origin of replication are present in the plasmid in the reverse orientation relative to the HBV antigen expression cassette.
  • a vector is a viral vector, preferably an adenoviral vector, more preferably an Ad26 or Ad35 vector, comprising an expression cassette including a polynucleotide encoding at least one of an HBV antigen selected from the group consisting of an HBV pol antigen comprising an amino acid sequence at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96, 97%, preferably at least 98%, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%, identical to SEQ ID NO: 7, and a truncated HBV core antigen consisting of the amino acid sequence at least 95%, such as 95%, 96, 97%, preferably at least 98%, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%
  • a vector such as a plasmid DNA vector or a viral vector (preferably an adenoviral vector, more preferably an Ad26 or Ad35 vector), encodes an HBV Pol antigen having the amino acid sequence of SEQ ID NO: 7.
  • the vector comprises a coding sequence for the HBV Pol antigen that is at least 90% identical to the polynucleotide sequence of SEQ ID NO: 5 or 6, such as 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 5 or 6, preferably 100% identical to SEQ ID NO: 5 or 6.
  • a vector such as a plasmid DNA vector or a viral vector (preferably an adenoviral vector, more preferably an Ad26 or Ad35 vector), encodes a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
  • the vector comprises a coding sequence for the truncated HBV core antigen that is at least 90% identical to the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, such as 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3, preferably 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3.
  • the vector comprises a coding sequence for the fusion, which contains a coding sequence for the truncated HBV core antigen at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 3, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3, preferably 98%, 99% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3, more preferably SEQ ID NO: 1 or SEQ ID NO: 3, operably linked to a coding sequence for the HBV Pol antigen at least 90% identical to SEQ ID NO: 5 or SEQ ID NO: 6, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95
  • the coding sequence for the truncated HBV core antigen is operably linked to the coding sequence for the HBV Pol antigen via a coding sequence for a linker at least 90% identical to SEQ ID NO: 11, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 11, preferably 98%, 99% or 100% identical to SEQ ID NO: 11.
  • polynucleotides and expression vectors encoding the HBV antigens of the application can be made by any method known in the art in view of the present disclosure.
  • a polynucleotide encoding an HBV antigen can be introduced or“cloned” into an expression vector using standard molecular biology techniques, e.g., polymerase chain reaction (PCR), etc., which are well known to those skilled in the art.
  • PCR polymerase chain reaction
  • the application also provides cells, preferably isolated cells, comprising any of the polynucleotides and vectors described herein.
  • the cells can, for instance, be used for recombinant protein production, or for the production of viral particles.
  • Embodiments of the application thus also relate to a method of making an HBV antigen of the application.
  • the method comprises transfecting a host cell with an expression vector comprising a polynucleotide encoding an HBV antigen of the application operably linked to a promoter, growing the transfected cell under conditions suitable for expression of the HBV antigen, and optionally purifying or isolating the HBV antigen expressed in the cell.
  • the HBV antigen can be isolated or collected from the cell by any method known in the art including affinity chromatography, size exclusion chromatography, etc. Techniques used for recombinant protein expression will be well known to one of ordinary skill in the art in view of the present disclosure.
  • the expressed HBV antigens can also be studied without purifying or isolating the expressed protein, e.g., by analyzing the supernatant of cells transfected with an expression vector encoding the HBV antigen and grown under conditions suitable for expression of the HBV antigen.
  • non-naturally occurring or recombinant polypeptides comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 7.
  • isolated nucleic acid molecules encoding these sequences, vectors comprising these sequences operably linked to a promoter, and compositions comprising the polypeptide, polynucleotide, or vector are also contemplated by the application.
  • a recombinant polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2, such as 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 2.
  • a non-naturally occurring or recombinant polypeptide consists of SEQ ID NO: 2.
  • a non-naturally occurring or recombinant polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4, such as 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 4.
  • a non-naturally occurring or recombinant polypeptide comprises SEQ ID NO: 4.
  • a non-naturally occurring or recombinant polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7, such as 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 7.
  • a non-naturally occurring or recombinant polypeptide consists of SEQ ID NO: 7.
  • antibodies or antigen binding fragments thereof that specifically bind to a non-naturally occurring polypeptide of the application.
  • an antibody specific to a non-naturally HBV antigen of the application does not bind specifically to another HBV antigen.
  • an antibody of the application that binds specifically to an HBV Pol antigen having the amino acid sequence of SEQ ID NO: 7 will not bind specifically to an HBV Pol antigen not having the amino acid sequence of SEQ ID NO: 7.
  • the term“antibody” includes polyclonal, monoclonal, chimeric, humanized, Fv, Fab and F(ab ⁇ )2; bifunctional hybrid (e.g., Lanzavecchia et al., Eur. J. Immunol.17:105, 1987), single-chain (Huston et al., Proc. Natl. Acad. Sci. USA 85:5879, 1988; Bird et al., Science 242:423, 1988); and antibodies with altered constant regions (e.g., U.S. Pat. No.5,624,821).
  • an antibody that“specifically binds to” an antigen refers to an antibody that“specifically binds to” an antigen
  • KD refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods in the art in view of the present disclosure.
  • heteroaryldihydropyrimidine (HAP) derivatives relate to heteroaryldihydropyrimidine (HAP) derivatives.
  • Background art on heteroaryldihydropyrimidines for use in the treatment of HBV includes WO
  • heteroaryldihydropyrimidine (HAP) derivatives can function as capsid assembly modulators (CAMs).
  • CAMs capsid assembly modulators
  • the compounds provided herein have potent antiviral activity, exhibit favorable metabolic properties, tissue distribution, safety and pharmaceutical profiles, and are suitable for use in humans.
  • Disclosed compounds may modulate (e.g., accelerate, delay, inhibit, disrupt or reduce) normal viral capsid assembly or disassembly, bind capsid or alter metabolism of cellular polyproteins and precursors. The modulation may occur when the capsid protein is mature, or during viral infectivity.
  • Disclosed compounds can be used in methods of modulating the activity or properties of HBV cccDNA, or the generation or release of HBV RNA particles from within an infected cell.
  • the compounds described herein are suitable for monotherapy and are effective against natural or native HBV strains and against HBV strains resistant to currently known drugs. In addition, the compounds described herein are suitable for use in combination therapy.
  • a capsid assembly modulator is a compound of Formula (I):
  • R 1 is selected from the group consisting of phenyl, thiophenyl, pyridyl, and pyridonyl, optionally substituted with one or more substituents selected from the group consisting of C 1- 4 alkyl, halogen, and CN;
  • R2 is C 1-4 alkyl
  • R3 is selected from the group consisting of thiazolyl, pyridyl, and oxazolyl, optionally substituted with one or more substituents selected from fluorine, and C 1-6 alkyl;
  • n is an integer of 0 or 1;
  • X is C-SR 9 or C-OR 9
  • Y is N atom
  • R 7 is CN
  • R 8 is CF 3 ;
  • R 9 is selected from the group consisting of H, -C 1-6 alkyl, -C 1-6 alkyl-R 10 , -C 1-6 alkoxy- C 1-6 alkyl-R 10 and -(CH 2 ) p -Q-R 10 ;
  • p is an integer of 0, 1, 2, or 3;
  • Q is selected from the group consisting of aryl, heteroaryl, and a 3- to 7- membered saturated ring, optionally containing a heteroatom, the heteroatom being an oxygen or a nitrogen, the nitrogen being substituted with H, -C 1-6 alkyl, -C 1-6 alkoxy-C 1-6 alkyl and -C 1- 6 alkylcarbonyl;
  • capsid assembly modulator refers to a compound that disrupts or accelerates or inhibits or hinders or delays or reduces or modifies normal capsid assembly (e.g., during maturation) or normal capsid disassembly (e.g., during infectivity) or perturbs capsid stability, thereby inducing aberrant capsid morphology and function.
  • a capsid assembly modulator accelerates capsid assembly or disassembly, thereby inducing aberrant capsid morphology.
  • a capsid assembly modulator interacts (e.g.
  • a capsid assembly modulator causes a perturbation in structure or function of CA (e.g., ability of CA to assemble, disassemble, bind to a substrate, fold into a suitable conformation, or the like), which attenuates viral infectivity or is lethal to the virus.
  • the term“pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the coumpound, and is relatively non-toxic, i.e., the material may be adminitered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • the term“pharmaceutically acceptable salt” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • the pharmaceutically acceptable salts of the present invention include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.
  • such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.
  • alkyl by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e., C 1 -C 3 alkyl means an alkyl having one to three carbon atoms, C 1 - C 4 alkyl means an alkyl having one to four carbon) and includes straight and branched chains. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl.
  • Embodiments of alkyl generally include, but are not limited to, C 1 -C 10 alkyl, such as C 1 -C 6 alkyl, such as C 1 -C 4 alkyl.
  • alkenyl by itself or as part of another substituent means, unless otherwise stated, a linear or branched chain of hydrocarbons comprising at least one carbon to carbon double bond, having the number of carbon atoms designated (i.e., C 2 -C 4 alkenyl or C 2-4 alkenyl means an alkenyl having two to four to eight carbon atoms.
  • C 4 -C 8 alkenyl or C 4-8 alkenyl means an alkenyl having four carbon atoms.
  • Embodiments of alkenyl generally include, but are not limited to, C 2 -C 6 alkenyl, such as C 2 -C 4 alkenyl, such as C 2 -C 3 alkenyl.
  • halo or“halogen” alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine, more preferably, fluorine or chlorine.
  • a 3-7 membered saturated ring may optionally contain a heteroatom, said heteroatom being an oxygen, or a nitrogen substituted with H, C 1-6 alkyl, or C 1-6 alkoxy-C 1- 6 alkyl.
  • aryl means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two, or three rings), wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene.
  • aryl groups include phenyl, anthracyl, and naphthyl. Preferred examples are phenyl (e.g., C 6 -aryl) and biphenyl (e.g., C 12 -aryl).
  • aryl groups have from six to sixteen carbon atoms.
  • aryl groups have from six to twelve carbon atoms (e.g., C6-C12-aryl).
  • aryl groups have six carbon atoms (e.g., C6-aryl).
  • heteroaryl or“heteroaromatic” refers to a heterocycle having aromatic character.
  • Heteroaryl substituents may be defined by the number of carbon atoms, e.g., C 1 -C 9 -heteroaryl indicates the number of carbon atoms contained in the heteroaryl group without including the number of heteroatoms.
  • a C 1 -C 9 - heteroaryl will include an additional one to four heteroatoms.
  • a polycyclic heteroaryl may include one or more rings that are partially saturated.
  • heteroaryls include pyridyl, pyrazinyl, pyrimidinyl (including, e.g., 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (including, e.g., 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (including, e.g., 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.
  • Non-limiting examples of polycyclic heterocycles and heteroaryls include indolyl (including, e.g., 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (including, e.g., 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (including, e.g., 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (including, e.g., 3-, 4-, 5-, 6- and 7-benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl
  • An embodiment relates to a compound of Formula I as defined herein, wherein R1 is phenyl substituted with one or more substituents selected from halogens and C 1-6 alkyl.
  • An embodiment relates to a compound of Formula I as defined herein, wherein R2 is methyl or ethyl.
  • An embodiment relates to a compound of Formula I as defined herein, wherein R3 is thiazolyl.
  • An embodiment relates to a compound of Formula I as defined herein, wherein R4 and R5 are H.
  • An embodiment relates to a compound of Formula I as defined herein, wherein Z is CH 2 .
  • An embodiment relates to a compound of Formula I as defined herein, wherein R9 is C 1-6 alkyl-CO 2 H or (CH 2 ) p - -R 10 .
  • An embodiment relates to a compound of Formula I as defined herein, wherein Q is phenyl, or wherein Q is a C3-6cycloalkyl, or wherein Q is a 3- to 6- saturated membered ring containing an oxygen.
  • the compound of Formula (I) is compound 1A:
  • the disclosed compounds may possess one or more stereocenters, and each stereocenter may exist independently in either the R or S configuration.
  • the stereochemical configuration at indicated centers has been assigned as“R*”, “S*” when the absolute stereochemistry is undetermined although the compound itself has been isolated as a single stereoisomer and is enantiomerically/diastereomerically pure.
  • compounds described herein are present in optically active or racemic forms. It is to be understood that the compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein.
  • Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase.
  • a mixture of one or more isomer is utilized as the disclosed compound described herein.
  • compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis or separation of a mixture of enantiomers or diastereomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.
  • the disclosed compounds may exist as tautomers. All tautomers are included within the scope of the compounds presented herein.
  • Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes suitable for inclusion in the compounds described herein include and are not limited to 2H, 3H, 11C, 13C, 14C, 36Cl, 18F, 123I, 125I, 13N, 15N, 15O, 17O, 18O, 32P, and 35S.
  • isotopically-labeled compounds are useful in drug or substrate tissue distribution studies.
  • substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements).
  • substitution with positron emitting isotopes is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.
  • Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non- labeled reagent otherwise employed.
  • the application also relates to intermediate compounds for preparation of compounds of Formula (I), such as
  • compositions Compositions, Therapeutic Combinations, and Vaccines
  • the application also relates to compositions, therapeutic combinations, more particularly kits, and vaccines comprising one or more HBV antigens, polynucleotides, and/or vectors encoding one or more HBV antigens according to the application.
  • HBV antigens any of the HBV antigens, polynucleotides (including RNA and DNA), and/or vectors of the application described herein can be used in the compositions, therapeutic combinations or kits, and vaccines of the application.
  • a composition comprises an isolated or non- naturally occurring nucleic acid molecule (DNA or RNA) comprising polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, or an HBV polymerase antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, a vector comprising the isolated or non-naturally occurring nucleic acid molecule, and/or an isolated or non-naturally occurring polypeptide encoded by the isolated or non-naturally occurring nucleic acid molecule.
  • DNA or RNA DNA comprising polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, or an HBV polymerase antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, a vector comprising the isolated or non-naturally
  • a composition comprises an isolated or non- naturally occurring nucleic acid molecule (DNA or RNA) comprising a polynucleotide sequence encoding an HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, preferably 100% identical to SEQ ID NO: 7.
  • a composition comprises an isolated or non- naturally occurring nucleic acid molecule (DNA or RNA) encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4.
  • a composition comprises an isolated or non- naturally occurring nucleic acid molecule (DNA or RNA) comprising a polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4; and an isolated or non-naturally occurring nucleic acid molecule (DNA or RNA) comprising a polynucleotide sequence encoding an HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, preferably 100% identical to SEQ ID NO: 7.
  • DNA or RNA isolated or non-naturally occurring nucleic acid molecule
  • the coding sequences for the truncated HBV core antigen and the HBV Pol antigen can be present in the same isolated or non-naturally occurring nucleic acid molecule (DNA or RNA), or in two different isolated or non-naturally occurring nucleic acid molecules (DNA or RNA).
  • a composition comprises a vector, preferably a DNA plasmid or a viral vector (such as an adenoviral vector) comprising a polynucleotide encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4.
  • a vector preferably a DNA plasmid or a viral vector (such as an adenoviral vector) comprising a polynucleotide encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4.
  • a composition comprises a vector, preferably a DNA plasmid or a viral vector (such as an adenoviral vector), comprising a polynucleotide encoding an HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, preferably 100% identical to SEQ ID NO: 7.
  • a composition comprises a vector, preferably a DNA plasmid or a viral vector (such as an adenoviral vector), comprising a polynucleotide encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4; and a vector, preferably a DNA plasmid or a viral vector (such as an adenoviral vector), comprising a polynucleotide encoding an HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, preferably 100% identical to SEQ ID NO: 7.
  • the vector comprising the coding sequence for the truncated HBV core antigen and the vector comprising the coding sequence for the HBV Pol antigen can be the same vector, or two different vectors.
  • the fusion protein further comprises a linker that operably links the truncated HBV core antigen to the HBV Pol antigen, or vice versa.
  • the linker has the amino acid sequence of (AlaGly)n, wherein n is an integer of 2 to 5.
  • a composition comprises an isolated or non- naturally occurring truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4.
  • a composition comprises an isolated or non- naturally occurring HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, preferably 100% identical to SEQ ID NO: 7.
  • a composition comprises an isolated or non- naturally occurring truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4; and an isolated or non-naturally occurring HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, preferably 100% identical to SEQ ID NO: 7.
  • a composition comprises an isolated or non- naturally occurring fusion protein comprising a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 14, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4, operably linked to an HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, preferably 100% identical to SEQ ID NO: 7, or vice versa.
  • the fusion protein further comprises a linker that operably links the truncated HBV core antigen to the HBV Pol antigen, or vice versa.
  • the linker has the amino acid sequence of (AlaGly)n, wherein n is an integer of 2 to 5.
  • the application also relates to a therapeutic combination or a kit comprising polynucleotides expressing a truncated HBV core antigen and an HBV pol antigen according to embodiments of the application.
  • Any polynucleotides and/or vectors encoding HBV core and pol antigens of the application described herein can be used in the therapeutic combinations or kits of the application.
  • a therapeutic combination or kit for use in treating an HBV infection in a subject in need thereof comprises: i) at least one of:
  • a truncated HBV core antigen consisting of an amino acid sequence that is at least 95% identical to SEQ ID NO: 2, and
  • a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding the truncated HBV core antigen
  • HBV polymerase antigen having an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity, and
  • a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide sequence encoding the HBV polymerase antigen
  • R 1 is selected from the group consisting of phenyl, thiophenyl, pyridyl, and pyridonyl, optionally substituted with one or more substituents selected from the group consisting of C 1- 4alkyl, halogen, and CN;
  • R 2 is C 1-4 alkyl
  • R 3 is selected from the group consisting of thiazolyl, pyridyl, and oxazolyl, optionally substituted with one or more substituents selected from fluorine, and C 1-6 alkyl;
  • n is an integer of 0 or 1;
  • R 4 and R 5 are independently selected from the group consisting of H and -COOH; the bond between X and Y) is a single bond or a double bond;
  • X is C-SR 9 or C-OR 9
  • Y is N atom
  • R 7 is CN
  • R 8 is CF 3 ;
  • R9 is selected from the group consisting of H, -C 1-6 alkyl, -C 1-6 alkyl-R10, -C 1-6 alkoxy- C1-6alkyl-R10 and -(CH2)p-Q-R10;
  • p is an integer of 0, 1, 2, or 3;
  • a therapeutic combination or kit comprises: i) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 95% identical to SEQ ID NO: 2; ii) a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide sequence encoding an HBV polymerase antigen having an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity; and iii) compound 1A:
  • the HBV antigens encoded by the first and second polynucleotides can be expressed from the same vector, such that an HBV core-pol fusion antigen is produced.
  • the core and pol antigens can be joined or fused together by a short linker.
  • the HBV antigens encoded by the first and second polynucleotides can be expressed independently from a single vector using a using a ribosomal slippage site (also known as cis-hydrolase site) between the core and pol antigen coding sequences. This strategy results in a bicistronic expression vector in which individual core and pol antigens are produced from a single mRNA transcript.
  • the core and pol antigens produced from such a bicistronic expression vector can have additional N or C-terminal residues, depending upon the ordering of the coding sequences on the mRNA transcript.
  • ribosomal slippage sites that can be used for this purpose include, but are not limited to, the FA2 slippage site from foot-and-mouth disease virus (FMDV).
  • FMDV foot-and-mouth disease virus
  • Another possibility is that the HBV antigens encoded by the first and second polynucleotides can be expressed independently from two separate vectors, one encoding the HBV core antigen and one encoding the HBV pol antigen.
  • the first and second polynucleotides are present in separate vectors, e.g., DNA plasmids or viral vectors.
  • the separate vectors are present in the same composition.
  • a therapeutic combination or kit comprises a first polynucleotide present in a first vector, a second polynucleotide present in a second vector.
  • the first and second vectors can be the same or different.
  • the vectors are DNA plasmids.
  • the first vector is a first DNA plasmid
  • the second vector is a second DNA plasmid.
  • Each of the first and second DNA plasmids comprises an origin of replication, preferably pUC ORI of SEQ ID NO: 21, and an antibiotic resistance cassette, preferably comprising a codon optimized Kanr gene having a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 23, preferably under control of a bla promoter, for instance the bla promoter shown in SEQ ID NO: 24.
  • the first vector is a viral vector and the second vector is a viral vector.
  • each of the viral vectors is an adenoviral vector, more preferably an Ad26 or Ad35 vector, comprising an expression cassette including the polynucleotide encoding an HBV pol antigen or an truncated HBV core antigen of the application; an upstream sequence operably linked to the polynucleotide encoding the HBV antigen comprising, from 5’ end to 3’ end, a promoter sequence, preferably a CMV promoter sequence of SEQ ID NO: 19, an enhancer sequence, preferably an ApoAI gene fragment sequence of SEQ ID NO: 12, and a polynucleotide sequence encoding a signal peptide sequence, preferably an immunoglobulin secretion signal having the amino acid sequence of SEQ ID NO: 15; and a downstream sequence operably linked to the polynucleotide encoding the HBV antigen comprising a polyadenylation
  • the first and second polynucleotides are present in a single vector, e.g., DNA plasmid or viral vector.
  • the single vector is an adenoviral vector, more preferably an Ad26 vector, comprising an expression cassette including a polynucleotide encoding an HBV pol antigen and a truncated HBV core antigen of the application, preferably encoding an HBV pol antigen and a truncated HBV core antigen of the application as a fusion protein; an upstream sequence operably linked to the polynucleotide encoding the HBV pol and truncated core antigens comprising, from 5’ end to 3’ end, a promoter sequence, preferably a CMV promoter sequence of SEQ ID NO: 19, an enhancer sequence, preferably an ApoAI gene fragment sequence of SEQ ID NO: 12, and a polynucleotide sequence encoding a signal peptide sequence, preferably an immuno
  • a therapeutic combination of the application comprises a first vector, such as a DNA plasmid or viral vector, and a second vector, such as a DNA plasmid or viral vector
  • the amount of each of the first and second vectors is not particularly limited.
  • the first DNA plasmid and the second DNA plasmid can be present in a ratio of 10:1 to 1:10, by weight, such as 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10, by weight.
  • the first and second DNA plasmids are present in a ratio of 1:1, by weight.
  • the therapeutic combination of the application can further comprise a third vector encoding a third active agent useful for treating an HBV infection.
  • compositions and therapeutic combinations of the application can comprise additional polynucleotides or vectors encoding additional HBV antigens and/or additional HBV antigens or immunogenic fragments thereof, such as an HBsAg, an HBV L protein or HBV envelope protein, or a polynucleotide sequence encoding thereof.
  • additional HBV antigens and/or additional HBV antigens or immunogenic fragments thereof such as an HBsAg, an HBV L protein or HBV envelope protein, or a polynucleotide sequence encoding thereof.
  • the compositions and therapeutic combinations of the application do not comprise certain antigens.
  • composition or therapeutic combination or kit of the application does not comprise a HBsAg or a polynucleotide sequence encoding the HBsAg.
  • composition or therapeutic combination of the application does not comprise an HBV envelope protein or a polynucleotide sequence encoding the HBV envelope protein.
  • compositions and therapeutic combinations of the application can also comprise a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier is non-toxic and should not interfere with the efficacy of the active ingredient.
  • Pharmaceutically acceptable carriers can include one or more excipients such as binders, disintegrants, swelling agents, suspending agents, emulsifying agents, wetting agents, lubricants, flavorants, sweeteners, preservatives, dyes, solubilizers and coatings.
  • Pharmaceutically acceptable carriers can include vehicles, such as lipid nanoparticles (LNPs).
  • suitable carriers and additives include water, glycols, oils, alcohols, preservatives, coloring agents and the like.
  • suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like.
  • the aqueous solution/suspension can comprise water, glycols, oils, emollients, stabilizers, wetting agents, preservatives, aromatics, flavors, and the like as suitable carriers and additives.
  • compositions and therapeutic combinations of the application can be formulated in any matter suitable for administration to a subject to facilitate administration and improve efficacy, including, but not limited to, oral (enteral) administration and parenteral injections.
  • the parenteral injections include intravenous injection or infusion, subcutaneous injection, intradermal injection, and intramuscular injection.
  • Compositions of the application can also be formulated for other routes of administration including transmucosal, ocular, rectal, long acting implantation, sublingual administration, under the tongue, from oral mucosa bypassing the portal circulation, inhalation, or intranasal.
  • compositions and therapeutic combinations of the application are formulated for parental injection, preferably subcutaneous, intradermal injection, or intramuscular injection, more preferably intramuscular injection.
  • the plasmid DNA can be present in a concentration of, e.g., 0.5 mg/mL to 5 mg/mL, such as 0.5 mg/mL 1, mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, or 5 mg/mL, preferably at 1 mg/mL.
  • Compositions and therapeutic combinations of the application can be formulated as a vaccine (also referred to as an“immunogenic composition”) according to methods well known in the art. Such compositions can include adjuvants to enhance immune responses.
  • the optimal ratios of each component in the formulation can be determined by techniques well known to those skilled in the art in view of the present disclosure.
  • a composition or therapeutic combination is a DNA vaccine.
  • DNA vaccines typically comprise bacterial plasmids containing a polynucleotide encoding an antigen of interest under control of a strong eukaryotic promoter. Once the plasmids are delivered to the cell cytoplasm of the host, the encoded antigen is produced and processed endogenously. The resulting antigen typically induces both humoral and cell-medicated immune responses.
  • DNA vaccines are advantageous at least because they offer improved safety, are temperature stable, can be easily adapted to express antigenic variants, and are simple to produce. Any of the DNA plasmids of the application can be used to prepare such a DNA vaccine.
  • RNA vaccines typically comprise at least one single- stranded RNA molecule encoding an antigen of interest, e.g., a fusion protein or HBV antigen according to the application. Once the RNA is delivered to the cell cytoplasm of the host, the encoded antigen is produced and processed endogenously, inducing both humoral and cell- mediated immune responses, similar to a DNA vaccine.
  • the RNA sequence can be codon optimized to improve translation efficiency.
  • RNA molecule can be modified by any method known in the art in view of the present disclosure to enhance stability and/or translation, such by adding a polyA tail, e.g., of at least 30 adenosine residues; and/or capping the 5-end with a modified ribonucleotide, e.g., 7-methylguanosine cap, which can be incorporated during RNA synthesis or enzymatically engineered after RNA transcription.
  • An RNA vaccine can also be self-replicating RNA vaccine developed from an alphavirus expression vector.
  • Self-replicating RNA vaccines comprise a replicase RNA molecule derived from a virus belonging to the alphavirus family with a subgenomic promoter that controls replication of the fusion protein or HBV antigen RNA followed by an artificial poly A tail located downstream of the replicase.
  • a further adjuvant can be included in a composition or therapeutic combination of the application, or co-administered with a composition or therapeutic combination of the application.
  • another adjuvant is optional, and can further enhance immune responses when the composition is used for vaccination purposes.
  • Other adjuvants suitable for co-administration or inclusion in compositions in accordance with the application should preferably be ones that are potentially safe, well tolerated and effective in humans.
  • An adjuvant can be a small molecule or antibody including, but not limited to, immune checkpoint inhibitors (e.g., anti-PD1, anti-TIM-3, etc.), toll-like receptor agonists (e.g., TLR 7 agonists and/or TLR8 agonists), RIG-1 agonists, IL-15 superagonists (Altor Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, and IL-7-hyFc.
  • adjuvants can e.g., be chosen from among the following anti-HBV agents: HBV DNA polymerase inhibitors;
  • Immunomodulators include Toll-like receptor 7 modulators; Toll-like receptor 8 modulators; Toll-like receptor 3 modulators; Interferon alpha receptor ligands; Hyaluronidase inhibitors; Modulators of IL-10; HBsAg inhibitors; Toll like receptor 9 modulators; Cyclophilin inhibitors; HBV Prophylactic vaccines; HBV Therapeutic vaccines; HBV viral entry inhibitors; Antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; short interfering RNAs (siRNA), more particularly anti-HBV siRNA; Endonuclease modulators; Inhibitors of ribonucleotide reductase; Hepatitis B virus E antigen inhibitors; HBV antibodies targeting the surface antigens of the hepatitis B virus; HBV antibodies; CCR2 chemokine antagonists; Thymosin agonists; Cytokines, such as IL12; Caps
  • the further adjuvant can e.g., be selected from interferon (for example, interferon-alpha-2a is pegylated interferon-alpha-2a (PEGASYS)), nucleoside or nucleotide or non-nucleos(t)ide polymerase inhibitors, immunomodulatory agents (e.g., IL-12, IL-18, IFN-alpha, -beta, and -gamma and TNF-alpha among others), TLR agonists, siRNAs and antisense oligonucleotides.
  • interferon for example, interferon-alpha-2a is pegylated interferon-alpha-2a (PEGASYS)
  • PEGASYS pegylated interferon-alpha-2a
  • immunomodulatory agents e.g., IL-12, IL-18, IFN-alpha, -beta, and -gamma and TNF-alpha among others
  • TLR agonists e.
  • each of the first and second non-naturally occurring nucleic acid molecules is independently formulated with a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • the application also provides methods of making compositions and therapeutic combinations of the application.
  • a method of producing a composition or therapeutic combination comprises mixing an isolated polynucleotide encoding an HBV antigen, vector, and/or polypeptide of the application with one or more pharmaceutically acceptable carriers.
  • One of ordinary skill in the art will be familiar with conventional techniques used to prepare such compositions.
  • the application also provides methods of inducing an immune response against hepatitis B virus (HBV) in a subject in need thereof, comprising administering to the subject an immunogenically effective amount of a composition or immunogenic composition of the application.
  • HBV hepatitis B virus
  • the term“infection” refers to the invasion of a host by a disease causing agent.
  • a disease causing agent is considered to be“infectious” when it is capable of invading a host, and replicating or propagating within the host.
  • infectious agents include viruses, e.g., HBV and certain species of adenovirus, prions, bacteria, fungi, protozoa and the like.
  • HBV infection specifically refers to invasion of a host organism, such as cells and tissues of the host organism, by HBV.
  • the phrase“inducing an immune response” when used with reference to the methods described herein encompasses causing a desired immune response or effect in a subject in need thereof against an infection, e.g., an HBV infection.“Inducing an immune response” also encompasses providing a therapeutic immunity for treating against a pathogenic agent, e.g., HBV.
  • a pathogenic agent e.g., HBV.
  • the term“therapeutic immunity” or“therapeutic immune response” means that the vaccinated subject is able to control an infection with the pathogenic agent against which the vaccination was done, for instance immunity against HBV infection conferred by vaccination with HBV vaccine.
  • “inducing an immune response” means producing an immunity in a subject in need thereof, e.g., to provide a therapeutic effect against a disease, such as HBV infection.
  • “inducing an immune response” refers to causing or improving cellular immunity, e.g., T cell response, against HBV infection.
  • “inducing an immune response” refers to causing or improving a humoral immune response against HBV infection.
  • “inducing an immune response” refers to causing or improving a cellular and a humoral immune response against HBV infection.
  • the term“protective immunity” or“protective immune response” means that the vaccinated subject is able to control an infection with the pathogenic agent against which the vaccination was done. Usually, the subject having developed a“protective immune response” develops only mild to moderate clinical symptoms or no symptoms at all. Usually, a subject having a“protective immune response” or“protective immunity” against a certain agent will not die as a result of the infection with said agent.
  • compositions and therapeutic combinations of the application will have a therapeutic aim to generate an immune response against HBV after HBV infection or development of symptoms characteristic of HBV infection, e.g., for therapeutic vaccination.
  • an immunogenically effective amount or“immunologically effective amount” means an amount of a composition, polynucleotide, vector, or antigen sufficient to induce a desired immune effect or immune response in a subject in need thereof.
  • An immunogenically effective amount can be an amount sufficient to induce an immune response in a subject in need thereof.
  • An immunogenically effective amount can be an amount sufficient to produce immunity in a subject in need thereof, e.g., provide a therapeutic effect against a disease such as HBV infection.
  • An immunogenically effective amount can vary depending upon a variety of factors, such as the physical condition of the subject, age, weight, health, etc.; the particular application, e.g., providing protective immunity or therapeutic immunity; and the particular disease, e.g., viral infection, for which immunity is desired.
  • An immunogenically effective amount can readily be determined by one of ordinary skill in the art in view of the present disclosure.
  • an immunogenically effective amount refers to the amount of a composition or therapeutic combination which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of an HBV infection or a symptom associated therewith; (ii) reduce the duration of an HBV infection or symptom associated therewith; (iii) prevent the progression of an HBV infection or symptom associated therewith; (iv) cause regression of an HBV infection or symptom associated therewith; (v) prevent the development or onset of an HBV infection, or symptom associated therewith; (vi) prevent the recurrence of an HBV infection or symptom associated therewith; (vii) reduce hospitalization of a subject having an HBV infection; (viii) reduce hospitalization length of a subject having an HBV infection; (ix) increase the survival of a subject with an HBV infection; (x) eliminate an HBV infection in a subject; (xi) inhibit or reduce HBV replication in a subject; and/or (xii
  • An immunogenically effective amount can also be an amount sufficient to reduce HBsAg levels consistent with evolution to clinical seroconversion; achieve sustained HBsAg clearance associated with reduction of infected hepatocytes by a subject’s immune system; induce HBV-antigen specific activated T-cell populations; and/or achieve persistent loss of HBsAg within 12 months.
  • a target index include lower HBsAg below a threshold of 500 copies of HBsAg international units (IU) and/or higher CD8 counts.
  • an immunogenically effective amount when used with reference to a DNA plasmid can range from about 0.1 mg/mL to 10 mg/mL of DNA plasmid total, such as 0.1 mg/mL, 0.25 mg/mL, 0.5 mg/mL.0.75 mg/mL 1 mg/mL, 1.5 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, or 10 mg/mL.
  • an immunogenically effective amount of DNA plasmid is less than 8 mg/mL, more preferably less than 6 mg/mL, even more preferably 3-4 mg/mL.
  • An immunogenically effective amount can be from one vector or plasmid, or from multiple vectors or plasmids.
  • an immunogenically effective amount when used with reference to a peptide can range from about 10 ⁇ g to 1 mg per administration, such as 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 9000, or 1000 ⁇ g per administration.
  • An immunogenically effective amount can be administered in a single composition, or in multiple compositions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 compositions (e.g., tablets, capsules or injectables, or any composition adapted to intradermal delivery, e.g., to intradermal delivery using an intradermal delivery patch), wherein the administration of the multiple capsules or injections collectively provides a subject with an immunogenically effective amount.
  • an immunogenically effective amount can be 3-4 mg/mL, with 1.5-2 mg/mL of each plasmid.
  • prime-boost regimen It is also possible to administer an immunogenically effective amount to a subject, and subsequently administer another dose of an immunogenically effective amount to the same subject, in a so-called prime-boost regimen.
  • This general concept of a prime-boost regimen is well known to the skilled person in the vaccine field. Further booster administrations can optionally be added to the regimen, as needed.
  • the dose of a compound of Formula (I) is from about 1 mg to about 2,500 mg.
  • a dose of a disclosed compound used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg.
  • a dose of a second compound is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
  • a therapeutic combination comprising two DNA plasmids, e.g., a first DNA plasmid encoding an HBV core antigen and second DNA plasmid encoding an HBV pol antigen, can be administered to a subject by mixing both plasmids and delivering the mixture to a single anatomic site.
  • two separate immunizations each delivering a single expression plasmid can be performed.
  • the first DNA plasmid and the second DNA plasmid can be administered in a ratio of 10:1 to 1:10, by weight, such as 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10, by weight.
  • the first and second DNA plasmids are administered in a ratio of 1:1, by weight.
  • a subject to be treated according to the methods of the application is an HBV-infected subject, particular a subject having chronic HBV infection.
  • Acute HBV infection is characterized by an efficient activation of the innate immune system complemented with a subsequent broad adaptive response (e.g., HBV-specific T-cells, neutralizing antibodies), which usually results in successful suppression of replication or removal of infected hepatocytes.
  • HBV-specific T-cells, neutralizing antibodies e.g., HBV-specific T-cells, neutralizing antibodies
  • HBV envelope proteins are produced in abundance and can be released in sub-viral particles in 1,000-fold excess to infectious virus.
  • the phases of chronic HBV infection include: (i) the immune- tolerant phase characterized by high viral load and normal or minimally elevated liver enzymes; (ii) the immune activation HBeAg-positive phase in which lower or declining levels of viral replication with significantly elevated liver enzymes are observed; (iii) the inactive HBsAg carrier phase, which is a low replicative state with low viral loads and normal liver enzyme levels in the serum that may follow HBeAg seroconversion; and (iv) the HBeAg- negative phase in which viral replication occurs periodically (reactivation) with concomitant fluctuations in liver enzyme levels, mutations in the pre-core and/or basal core promoter are common, such that HBeAg is not produced by the infected cell.
  • chronic HBV infection refers to a subject having the detectable presence of HBV for more than 6 months.
  • a subject having a chronic HBV infection can be in any phase of chronic HBV infection.
  • Chronic HBV infection is understood in accordance with its ordinary meaning in the field.
  • Chronic HBV infection can for example be characterized by the persistence of HBsAg for 6 months or more after acute HBV infection.
  • a chronic HBV infection referred to herein follows the definition published by the Centers for Disease Control and Prevention (CDC), according to which a chronic HBV infection can be characterized by laboratory criteria such as: (i) negative for IgM antibodies to hepatitis B core antigen (IgM anti-HBc) and positive for hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), or nucleic acid test for hepatitis B virus DNA, or (ii) positive for HBsAg or nucleic acid test for HBV DNA, or positive for HBeAg two times at least 6 months apart.
  • IgM anti-HBc hepatitis B core antigen
  • HBsAg hepatitis B surface antigen
  • HBeAg hepatitis B e antigen
  • nucleic acid test for hepatitis B virus DNA or
  • positive for HBeAg two times at least 6 months apart.
  • an immunogenically effective amount refers to the amount of a composition or therapeutic combination of the application which is sufficient to treat chronic HBV infection.
  • a subject having chronic HBV infection is undergoing nucleoside analog (NUC) treatment, and is NUC-suppressed.
  • NUC- suppressed refers to a subject having an undetectable viral level of HBV and stable alanine aminotransferase (ALT) levels for at least six months.
  • nucleoside/nucleotide analog treatment include HBV polymerase inhibitors, such as entacavir and tenofovir.
  • a subject having chronic HBV infection does not have advanced hepatic fibrosis or cirrhosis.
  • Such subject would typically have a METAVIR score of less than 3 for fibrosis and a fibroscan result of less than 9 kPa.
  • the METAVIR score is a scoring system that is commonly used to assess the extent of inflammation and fibrosis by histopathological evaluation in a liver biopsy of patients with hepatitis B.
  • the scoring system assigns two standardized numbers: one reflecting the degree of inflammation and one reflecting the degree of fibrosis.
  • an immunogenically effective amount is an amount sufficient to achieve persistent loss of HBsAg within 12 months and significant decrease in clinical disease (e.g., cirrhosis, hepatocellular carcinoma, etc.).
  • Methods according to embodiments of the application further comprises administering to the subject in need thereof another immunogenic agent (such as another HBV antigen or other antigen) or another anti-HBV agent (such as a nucleoside analog or other anti-HBV agent) in combination with a composition of the application.
  • another immunogenic agent such as another HBV antigen or other antigen
  • another anti-HBV agent such as a nucleoside analog or other anti-HBV agent
  • another anti-HBV agent or immunogenic agent can be a small molecule or antibody including, but not limited to, immune checkpoint inhibitors (e.g., anti-PD1, anti-TIM-3, etc.), toll-like receptor agonists (e.g., TLR7 agonists and/oror TLR8 agonists), RIG-1 agonists, IL-15 superagonists (Altor Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, IL12 genetic adjuvant, IL-7-hyFc; CAR-T which bind HBV env (S-CAR cells); capsid assembly modulators; cccDNA inhibitors, HBV polymerase inhibitors (e.g., entecavir and tenofovir).
  • immune checkpoint inhibitors e.g., anti-PD1, anti-TIM-3, etc.
  • toll-like receptor agonists e.g., TLR7 agonist
  • the one or other anti-HBV active agents can be, for example, a small molecule, an antibody or antigen binding fragment thereof, a polypeptide, protein, or nucleic acid.
  • the one or other anti-HBV agents can e.g., be chosen from among HBV DNA polymerase inhibitors; Immunomodulators; Toll-like receptor 7 modulators; Toll-like receptor 8 modulators; Toll-like receptor 3 modulators; Interferon alpha receptor ligands;
  • Hyaluronidase inhibitors Modulators of IL-10; HBsAg inhibitors; Toll like receptor 9 modulators; Cyclophilin inhibitors; HBV Prophylactic vaccines; HBV Therapeutic vaccines; HBV viral entry inhibitors; Antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; short interfering RNAs (siRNA), more particularly anti-HBV siRNA; Endonuclease modulators; Inhibitors of ribonucleotide reductase; Hepatitis B virus E antigen inhibitors; HBV antibodies targeting the surface antigens of the hepatitis B virus; HBV antibodies; CCR2 chemokine antagonists; Thymosin agonists; Cytokines, such as IL12; Capsid Assembly Modulators, Nucleoprotein inhibitors (HBV core or capsid protein inhibitors); Nucleic Acid Polymers (NAPs); Stimulators of retinoi
  • compositions and therapeutic combinations of the application can be administered to a subject by any method known in the art in view of the present disclosure, including, but not limited to, parenteral administration (e.g., intramuscular, subcutaneous, intravenous, or intradermal injection), oral administration, transdermal administration, and nasal administration.
  • parenteral administration e.g., intramuscular, subcutaneous, intravenous, or intradermal injection
  • oral administration e.g., oral administration
  • transdermal administration e.g., transdermal administration
  • nasal administration e.g., by intramuscular injection or intradermal injection
  • compositions and therapeutic combinations are administered parenterally (e.g., by intramuscular injection or intradermal injection) or transdermally.
  • administration can be by injection through the skin, e.g., intramuscular or intradermal injection, preferably intramuscular injection.
  • Intramuscular injection can be combined with electroporation, i.e., application of an electric field to facilitate delivery of the DNA plasmids to cells.
  • electroporation refers to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane.
  • in vivo electroporation electrical fields of appropriate magnitude and duration are applied to cells, inducing a transient state of enhanced cell membrane permeability, thus enabling the cellular uptake of molecules unable to cross cell membranes on their own. Creation of such pores by electroporation facilitates passage of biomolecules, such as plasmids, oligonucleotides, siRNAs, drugs, etc., from one side of a cellular membrane to the other.
  • In vivo electroporation for the delivery of DNA vaccines has been shown to significantly increase plasmid uptake by host cells, while also leading to mild-to-moderate inflammation at the injection site.
  • transfection efficiency and immune response are significantly improved (e.g., up to 1,000 fold and 100 fold respectively) with intradermal or intramuscular electroporation, in comparison to conventional injection.
  • electroporation is combined with intramuscular injection.
  • Administration of a composition, therapeutic combination or vaccine of the application via electroporation can be accomplished using electroporation devices that can be configured to deliver to a desired tissue of a mammal a pulse of energy effective to cause reversible pores to form in cell membranes.
  • the electroporation device can include an electroporation component and an electrode assembly or handle assembly.
  • the electroporation component can include one or more of the following components of electroporation devices: controller, current waveform generator, impedance tester, waveform logger, input element, status reporting element, communication port, memory component, power source, and power switch.
  • Electroporation can be accomplished using an in vivo electroporation device.
  • Examples of electroporation devices and electroporation methods that can facilitate delivery of compositions and therapeutic combinations of the application, particularly those comprising DNA plasmids, include CELLECTRA® (Inovio Pharmaceuticals, Blue Bell, PA), Elgen electroporator (Inovio Pharmaceuticals, Inc.) Tri-GridTM delivery system (Ichor Medical Systems, Inc., San Diego, CA 92121) and those described in U.S. Patent No. 7,664,545, U.S. Patent No.8,209,006, U.S. Patent No.9,452,285, U.S. Patent No.5,273,525, U.S. Patent No.6,110,161, U.S. Patent No.6,261,281, U.S. Patent No.6,958,060, and U.S.
  • in vivo electroporation devices are described in International Patent Application entitled“Method and Apparatus for the Delivery of Hepatitis B Virus (HBV) Vaccines,” filed on the same day as this application with the Attorney Docket Number 688097-405WO, the contents of which are hereby incorporated by reference in their entireties.
  • a pulsed electric field for instance as described in, e.g., U.S. Patent No.6,697,669, which is herein incorporated by reference in its entirety.
  • the method of administration is transdermal.
  • Transdermal administration can be combined with epidermal skin abrasion to facilitate delivery of the DNA plasmids to cells.
  • a dermatological patch can be used for epidermal skin abrasion. Upon removal of the dermatological patch, the composition or therapeutic combination can be deposited on the abraised skin.
  • Methods of delivery are not limited to the above described embodiments, and any means for intracellular delivery can be used.
  • Other methods of intracellular delivery contemplated by the methods of the application include, but are not limited to, liposome encapsulation, lipid nanoparticles (LNPs), etc.
  • a method of inducing an immune response against HBV further comprises administering an adjuvant.
  • adjuvant and “immune stimulant” are used interchangeably herein, and are defined as one or more substances that cause stimulation of the immune system.
  • an adjuvant is used to enhance an immune response to HBV antigens and antigenic HBV polypeptides of the application.
  • an adjuvant can be present in a therapeutic combination or composition of the application, or administered in a separate composition.
  • An adjuvant can be, e.g., a small molecule or an antibody.
  • adjuvants suitable for use in the application include, but are not limited to, immune checkpoint inhibitors (e.g., anti-PD1, anti-TIM-3, etc.), toll-like receptor agonists (e.g., TLR7 and/or TLR8 agonists), RIG-1 agonists, IL-15 superagonists (Altor Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, IL12 genetic adjuvant, and IL-7-hyFc.
  • immune checkpoint inhibitors e.g., anti-PD1, anti-TIM-3, etc.
  • toll-like receptor agonists e.g., TLR7 and/or TLR8 agonists
  • RIG-1 agonists e
  • adjuvants can e.g., be chosen from among the following anti-HBV agents: HBV DNA polymerase inhibitors; Immunomodulators; Toll-like receptor 7 modulators; Toll-like receptor 8 modulators; Toll-like receptor 3 modulators; Interferon alpha receptor ligands; Hyaluronidase inhibitors; Modulators of IL-10; HBsAg inhibitors; Toll like receptor 9 modulators; Cyclophilin inhibitors; HBV Prophylactic vaccines; HBV Therapeutic vaccines; HBV viral entry inhibitors; Antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; short interfering RNAs (siRNA), more particularly anti-HBV siRNA; Endonuclease modulators; Inhibitors of ribonucleotide reductase; Hepatitis B virus E antigen inhibitors; HBV antibodies targeting the surface antigens of the hepatitis B virus; HBV
  • compositions and therapeutic combinations of the application can also be administered in combination with at least one other anti-HBV agent.
  • anti-HBV agents suitable for use with the application include, but are not limited to small molecules, antibodies, and/or CAR-T therapies which bind HBV env (S-CAR cells), capsid assembly modulators, TLR agonists (e.g., TLR7 and/or TLR8 agonists), cccDNA inhibitors, HBV polymerase inhibitors (e.g., entecavir and tenofovir), and/or immune checkpoint inhibitors, etc.
  • the at least one anti-HBV agent can e.g., be chosen from among HBV DNA polymerase inhibitors; Immunomodulators; Toll-like receptor 7 modulators; Toll-like receptor 8 modulators; Toll-like receptor 3 modulators; Interferon alpha receptor ligands; Hyaluronidase inhibitors; Modulators of IL-10; HBsAg inhibitors; Toll like receptor 9 modulators; Cyclophilin inhibitors; HBV Prophylactic vaccines; HBV Therapeutic vaccines; HBV viral entry inhibitors; Antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; short interfering RNAs (siRNA), more particularly anti-HBV siRNA; Endonuclease modulators; Inhibitors of ribonucleotide reductase; Hepatitis B virus E antigen inhibitors; HBV antibodies targeting the surface antigens of the hepatitis B virus; HBV antibodies; CCR
  • Embodiments of the application also contemplate administering an immunogenically effective amount of a composition or therapeutic combination to a subject, and subsequently administering another dose of an immunogenically effective amount of a composition or therapeutic combination to the same subject, in a so-called prime-boost regimen
  • a composition or therapeutic combination of the application is a primer vaccine used for priming an immune response.
  • a composition or therapeutic combination of the application is a booster vaccine used for boosting an immune response.
  • the priming and boosting vaccines of the application can be used in the methods of the application described herein. This general concept of a prime-boost regimen is well known to the skilled person in the vaccine field. Any of the compositions and therapeutic combinations of the application described herein can be used as priming and/or boosting vaccines for priming and/or boosting an immune response against HBV.
  • a composition or therapeutic combination of the application can be administered for priming immunization.
  • the composition or therapeutic combination can be re-administered for boosting immunization. Further booster administrations of the composition or vaccine combination can optionally be added to the regimen, as needed.
  • An adjuvant can be present in a composition of the application used for boosting immunization, present in a separate composition to be administered together with the composition or therapeutic combination of the application for the boosting immunization, or administered on its own as the boosting immunization.
  • the adjuvant is preferably used for boosting immunization.
  • An illustrative and non-limiting example of a prime-boost regimen includes administering a single dose of an immunogenically effective amount of a composition or therapeutic combination of the application to a subject to prime the immune response; and subsequently administering another dose of an immunogenically effective amount of a composition or therapeutic combination of the application to boost the immune response, wherein the boosting immunization is first administered about two to six weeks, preferably four weeks after the priming immunization is initially administered.
  • kits comprising a therapeutic combination of the application.
  • a kit can comprise the first polynucleotide, the second polynucleotide, and the at least one capsid assembly modulator (CAM) in one or more separate compositions, or a kit can comprise the first polynucleotide, the second polynucleotide, and the at least one capsid assembly modulator (CAM) in a single composition.
  • a kit can further comprise one or more adjuvants or immune stimulants, and/or other anti-HBV agents.
  • the ability to induce or stimulate an anti-HBV immune response upon administration in an animal or human organism can be evaluated either in vitro or in vivo using a variety of assays which are standard in the art.
  • assays which are standard in the art.
  • Measurement of cellular immunity can be performed by measurement of cytokine profiles secreted by activated effector cells including those derived from CD4+ and CD8+ T- cells (e.g.
  • IL-10 or IFN gamma-producing cells by ELISPOT
  • T cell proliferation assays by a classical [3H] thymidine uptake or flow cytometry-based assays
  • assaying for antigen-specific T lymphocytes in a sensitized subject e.g. peptide-specific lysis in a cytotoxicity assay, etc.
  • the ability to stimulate a cellular and/or a humoral response can be determined by antibody binding and/or competition in binding (see for example Harlow, 1989, Antibodies, Cold Spring Harbor Press).
  • titers of antibodies produced in response to administration of a composition providing an immunogen can be measured by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the immune responses can also be measured by neutralizing antibody assay, where a neutralization of a virus is defined as the loss of infectivity through reaction/inhibition/neutralization of the virus with specific antibody.
  • the immune response can further be measured by Antibody-Dependent Cellular Phagocytosis (ADCP) Assay.
  • ADCP Antibody-Dependent Cellular Phagocytosis
  • a truncated HBV core antigen consisting of an amino acid sequence that is at least 95%, such as at least 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 2,
  • a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding the truncated HBV core antigen
  • an HBV polymerase antigen having an amino acid sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 7, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity, and
  • R 1 is selected from the group consisting of phenyl, thiophenyl, pyridyl, and pyridonyl, optionally substituted with one or more substituents selected from the group consisting of C 1- 4 alkyl, halogen, and CN;
  • R 3 is selected from the group consisting of thiazolyl, pyridyl, and oxazolyl, optionally substituted with one or more substituents selected from fluorine, and C 1-6 alkyl;
  • n is an integer of 0 or 1;
  • X is C-SR9 or C-OR9, and Y is N atom;
  • R 8 is CF3
  • R is selected from the group consisting of H, -C 1-6 alkyl, -C 1-6 alkyl-R , -C 1-6 alkoxy- C 1-6 alkyl-R10 and -(CH 2 ) p -Q-R10;
  • p is an integer of 0, 1, 2, or 3;
  • Q is selected from the group consisting of aryl, heteroaryl, and a 3- to 7- membered saturated ring, optionally containing a heteroatom, the heteroatom being an oxygen or a nitrogen, the nitrogen being substituted with H, -C 1-6 alkyl, -C 1-6 alkoxy-C 1-6 alkyl and -C 1- 6 alkylcarbonyl;
  • Embodiment 2 is the therapeutic combination of embodiment 1, comprising at least one of the HBV polymerase antigen and the truncated HBV core antigen.
  • Embodiment 3 is the therapeutic combination of embodiment 2, comprising the HBV polymerase antigen and the truncated HBV core antigen.
  • Embodiment 4 is the therapeutic combination of embodiment 1, comprising at least one of the first non-naturally occurring nucleic acid molecule comprising the first polynucleotide sequence encoding the truncated HBV core antigen, and the second non- naturally occurring nucleic acid molecule comprising the second polynucleotide sequence encoding the HBV polymerase antigen.
  • Embodiment 5 is a therapeutic combination for use in treating a hepatitis B virus (HBV) infection in a subject in need thereof, comprising
  • polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 95% identical to SEQ ID NO: 2; and ii) a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide sequence encoding an HBV polymerase antigen having an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity; and
  • 1R is phenyl substituted with one or more substituents selected from halogens and C 1- 6 alkyl;
  • R 2 is methyl or ethyl
  • R 3 is thiazolyl
  • n is an integer of 0 or 1;
  • R 4 and R 5 are H
  • Y is NR9
  • R 9 is C 1-6 alkyl-CO 1
  • p is an integer of 0, 1, 2, or 3;
  • Q is phenyl, a C 3-6 cycloalkyl, or a 3- to 6- saturated membered ring containing an oxygen
  • Embodiment 6 is the therapeutic combination of embodiment 4 or 5, wherein the first non-naturally occurring nucleic acid molecule further comprises a polynucleotide sequence encoding a signal sequence operably linked to the N-terminus of the truncated HBV core antigen.
  • Embodiment 6b is the therapeutic combination of embodiment 6 or 6a, wherein the signal sequence independently comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15.
  • Embodiment 6c is the therapeutic combination of embodiment 6 or 6a, wherein the signal sequence is independently encoded by the polynucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 14.
  • Embodiment 7 is the therapeutic combination of any one of embodiments 1-6c, wherein the HBV polymerase antigen comprises an amino acid sequence that is at least 98%, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, identical to SEQ ID NO: 7.
  • Embodiment 7a is the therapeutic combination of embodiment 7, wherein the HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 7.
  • Embodiment 7c is the therapeutic combination of embodiment 7b, wherein the truncated HBV antigen consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
  • Embodiment 8 is the therapeutic combination of any one of embodiments 1-7c, wherein each of the first and second non-naturally occurring nucleic acid molecules is a DNA molecule.
  • Embodiment 8a is the therapeutic combination of embodiment 8, wherein the DNA molecule is present on a DNA vector.
  • Embodiment 8b is the therapeutic combination of embodiment 8a, wherein the DNA vector is selected from the group consisting of DNA plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, and closed linear deoxyribonucleic acid.
  • Embodiment 8c is the therapeutic combination of embodiment 8, wherein the DNA molecule is present on a viral vector.
  • Embodiment 8d is the therapeutic combination of embodiment 8c, wherein the viral vector is selected from the group consisting of bacteriophages, animal viruses, and plant viruses.
  • Embodiment 8e is the therapeutic combination of any one of embodiments 1-7c, wherein each of the first and second non-naturally occurring nucleic acid molecules is an RNA molecule.
  • Embodiment 8f is the therapeutic combination of embodiment 8e, wherein the RNA molecule is an RNA replicon, preferably a self-replicating RNA replicon, an mRNA replicon, a modified mRNA replicon, or self-amplifying mRNA.
  • the RNA molecule is an RNA replicon, preferably a self-replicating RNA replicon, an mRNA replicon, a modified mRNA replicon, or self-amplifying mRNA.
  • Embodiment 11 is the therapeutic combination of any one of embodiments 4-10, wherein the first polynucleotide sequence comprises a polynucleotide sequence having at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3.
  • Embodiment 11a is the therapeutic combination of embodiment 11, wherein the first polynucleotide sequence comprises a polynucleotide sequence having at least 98%, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3.
  • Embodiment 12 is the therapeutic combination of embodiment 11a, wherein the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
  • Embodiment 13 is the therapeutic combination of any one of embodiments 4 to 12, wherein the second polynucleotide sequence comprises a polynucleotide sequence having at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6.
  • Embodiment 13a is the therapeutic combination of embodiment 13, wherein the second polynucleotide sequence comprises a polynucleotide sequence having at least 98%, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6.
  • Embodiment 14 is the therapeutic combination of embodiment 13a, wherein the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6.
  • Embodiment 15a is the therapeutic combination of any one of embodiments 1 to 14, wherein the compound of Formula (I) is compound 1A:
  • Embodiment 16 is a kit comprising the therapeutic combination of any one of embodiments 1 to 15a, and instructions for using the therapeutic combination in treating a hepatitis B virus (HBV) infection in a subject in need thereof.
  • HBV hepatitis B virus
  • Embodiment 17a is the method of embodiment 17, wherein the treatment induces an immune response against a hepatitis B virus in a subject in need thereof, preferably the subject has chronic HBV infection.
  • Embodiment 17b is the method of embodiment 17 or 17a, wherein the subject has chronic HBV infection.
  • Embodiment 17c is the method of any one of embodiments 17 to 17b, wherein the subject is in need of a treatment of an HBV-induced disease selected from the group consisting of advanced fibrosis, cirrhosis and hepatocellular carcinoma (HCC).
  • HBV-induced disease selected from the group consisting of advanced fibrosis, cirrhosis and hepatocellular carcinoma (HCC).
  • Embodiment 18 is the method of any one of embodiments 17-17c, wherein the therapeutic combination is administered by injection through the skin, e.g., intramuscular or intradermal injection, preferably intramuscular injection.
  • Embodiment 19 is the method of embodiment 18, wherein the therapeutic combination comprises at least one of the first and second non-naturally occurring nucleic acid molecules.
  • Embodiment 19a is the method of embodiment 19, wherein the therapeutic combination comprises the first and second non-naturally occurring nucleic acid molecules.
  • Embodiment 20 is the method of embodiment 19 or 19a, wherein the non-naturally occurring nucleic acid molecules are administered to the subject by intramuscular injection in combination with electroporation.
  • Embodiment 21 is the method of embodiment 19 or 19a, wherein the non-naturally occurring nucleic acid molecules are administered to the subject by a lipid composition, preferably by a lipid nanoparticle.
  • FIG.1A and 1B A schematic representation of the pDK-pol and pDK-core vectors is shown in Fig.1A and 1B, respectively.
  • An HBV core or pol antigen optimized expression cassette containing a CMV promoter (SEQ ID NO: 18), a splicing enhancer (triple composite sequence) (SEQ ID NO: 10), Cystatin S precursor signal peptide SPCS (NP_0018901.1) (SEQ ID NO: 9), and pol (SEQ ID NO: 5) or core (SEQ ID NO: 2) gene was introduced into a pDK plasmid backbone, using standard molecular biology techniques.
  • the plasmids were tested in vitro for core and pol antigen expression by Western blot analysis using core and pol specific antibodies, and were shown to provide consistent expression profile for cellular and secreted core and pol antigens (data not shown).
  • adenovirus vector has been designed as a fusion protein expressed from a single open reading frame. Additional configurations for the expression of the two proteins, e.g. using two separate expression cassettes, or using a 2A-like sequence to separate the two sequences, can also be envisaged.
  • the expression cassettes are comprised of the CMV promoter (SEQ ID NO: 19), an intron (SEQ ID NO:12) (a fragment derived from the human ApoAI gene - GenBank accession X01038 base pairs 295– 523, harboring the ApoAI second intron), followed by the optimized coding sequence– either core alone or the core and polymerase fusion protein preceded by a human immunoglobulin secretion signal coding sequence (SEQ ID NO: 14), and followed by the SV40 polyadenylation signal (SEQ ID NO: 13).
  • VVMP junction sequence
  • the plasmids tested included a pDK-Pol plasmid and pDK-Core plasmid, as shown in FIGS.1A and 1B, respectively, and as described herein in Example 1.
  • the pDK- Pol plasmid encoded a polymerase antigen having the amino acid sequence of SEQ ID NO: 7, and the pDK-Core plasmid encoding a Core antigen having the amino acid sequence of SEQ ID NO: 2.
  • T-cell responses induced by each plasmid individually were tested.
  • the DNA plasmid (pDNA) vaccine was intramuscularly delivered via electroporation to Balb/c mice using a commercially available TriGridTM delivery system-intramuscular (TDS-IM) adapted for application in the mouse model in cranialis tibialis.
  • TDS-IM TriGridTM delivery system-intramuscular
  • HBV Hepatitis B Virus
  • the TDS-IM array of a TDS-IM v1.0 device having an electrode array with a 2.5 mm spacing between the electrodes and an electrode diameter of 0.030 inch was inserted percutaneously into the selected muscle, with a conductive length of 3.2 mm and an effective penetration depth of 3.2 mm, and with the major axis of the diamond configuration of the electrodes oriented in parallel with the muscle fibers.
  • the injection was initiated to distribute DNA (e.g., 0.020 ml) in the muscle.
  • mice Six mice were administered plasmid DNA encoding the HBV core antigen (pDK-core; Group 1), six mice were administered plasmid DNA encoding the HBV pol antigen (pDK-pol; Group 2), and two mice received empty vector as the negative control. Animals received two DNA immunizations two weeks apart and splenocytes were collected one week after the last immunization.
  • Compound I-1 can be prepared by the condensation of aldehyde II, acetoacetate III and amidine IV in the presence of a base such as NaOAc.
  • Compound I-2 was prepared from compound I-1 using brominating reagent such as N-Bromosuccinimide. Coupling of compound I-2 and compound V in the presence of a base such as triethylamine affords compound I.
  • H6-A 400 mg, 90 % purity from 1H NMR, 40 % yield, 100 % stereopure
  • H6-B 400 mg, 95 % purity from 1H NMR, 42 % yield, 99.9 % stereopure
  • hydroimidazo[1,5-a]pyrazine-7(1H)-carboxylate S1-5B (810 mg, 1.89 mmol) in methanol (15 mL) and water (5 mL) was added sodium hydroxide (263 mg, 6.58 mmol) under nitrogen atmosphere at 0 oC. After stirred at room temperature for 6 hours, the mixture was added sodium hydroxide (130 mg, 3.25 mmol) and stirred at 60 oC for 4 hours. Then the reaction was diluted with water (10 mL), removed methanol under vacuo and extracted with ethyl acetate (20 mL) twice.
  • Compound 3A was prepared from H1-1A and Intermediate S1-B using same condition as for Compound 3B.
  • Compound 3A purified by Prep-HPLC (Column: gilson Xbrige C18 (5 ⁇ m 19 * 150 mm), Mobile phase A: water (+ 0.1 % ammonium bicarbonate), Mobile phase B: acetonitrile, UV: 214 nm, Flow rate: 15 mL/min, Gradient: 10 - 70 % (%B)) to give the title compound (30 mg, 99.6 % purity, 31 % yield) as yellow solids.
  • LC-MS (ESI): R T 3.262 min, mass calcd.
  • Compound 4A was prepared from Intermediate S1-B and intermediate H3-1A using same condition as for Compound 4B and purified by Prep-HPLC (Column: gilson Xbrige C18 (5 ⁇ m 19 * 150 mm), Mobile phase A: water (+ 0.1 % ammonium bicarbonate), Mobile phase B: acetonitrile, UV: 214 nm, Flow rate: 15 mL/min, Gradient: 05 - 95 % (%B)).
  • LC-MS (ESI): R T 3.658 min, mass calcd. for C 27 H 30 ClFN 6 O 4 S 2 620.1, m/z found 621.1.
  • GLS4 (WO 2008154817, example 5; Bioorganic & Medicinal Chemistry, 2017, 25, 1042- 1056, compound 8n) was selected as reference 1; another compound (WO2015132276, example 76) was selected as reference 2. Chemical structure of both reference compounds was shown below.
  • HepG2.2.15 (the HepG2.2.15 cell line can be produced by transfection of the HepG2 cell line as described in Sells, Chen, and Acs 1987 (Proc. Natl. Acad. Sci. USA 84: 1005-1009), and the HepG2 cell line is available from ATCC® under number HB-8065TM).
  • Micro Amp Optical 96-well reaction plate (APPLIED BIOSYSTEMS-4306737)
  • HepG2.2.15 cells were plated into 96-well plate in 2% FBS culture medium at the density of 40,000 cells/well and 5,000cells/well for HBV inhibitory activity and cytotoxicity determination, respectively. After incubation at 37 °C, 5% CO2 overnight, cells were treated with medium containing compounds for 6 days with medium and compounds refreshed after 3 days of treatment. Each compound was tested in a 1:3 serial dilutions at 8 different concentrations in triplicate. The highest concentration of the compounds was 10uM or 1uM for anti-HBV activity assay and 100uM for cytotoxicity determination.
  • CCK-8 assay Cell viability was determined by CCK-8 assay. After 6 days of compounds treatment, 20 ⁇ l CCK-8 reagents were added to each well of cytotoxicity assay plates. Cell plates were incubated at 37°C, 5% CO2 for 2.5 h. The absorbance at 450nm wavelength and the absorbance at 630nm wavelength as reference was measured.
  • the change of HBV DNA level induced by the compounds was assessed by quantitative real- time polymerase chain reaction (qPCR). Briefly, the HBV DNA in the culture medium was extracted using QIAamp 96 DNA Blood Kit according to the manual and then quantified by real-time PCR assay using the primers and probe in the table 2 below.
  • qPCR quantitative real- time polymerase chain reaction
  • EC50 and CC50 values are calculated by the GRAPHPAD PRISM software. If the CV% of DMSO controls is below 15% and the reference compounds shows expected activity or cytotoxicity, the data of this batch of experiment is considered qualified.
  • cryopreserved human hepatocytes cells were thawed in 37°C water bath and diluted with pre-warmed incubation medium to a working cell density of 1 ⁇ 10 ⁇ 6 viable cells/mL.
  • the 198 ⁇ L pre-warmed hepatocyte suspensions were spiked with 2 ⁇ L of 100 ⁇ M compound or reference compound(Verapamil) at a final concentration of 1.0 ⁇ M in a 96- well plate.
  • the plate was incubated at 37°C, 900 rpm. All incubations will be performed in singlet.
  • in vitro half°Clife (in vitro t1/2) is determined from the slope value k:
  • V incubation volume (0.2 mL);
  • N number of hepatocytes per well (0.2 ⁇ 10 ⁇ 6 cells).
  • Control compound verapamil will be included in the assay. Any value of the compound that is not within the specified limits will be rejected and the experiment would be repeated. Result
  • Hepatocyte s metabolic stability test has become the "gold standard" for evaluating hepatic metabolism and toxicity of drugs and other xenobiotics in vitro.
  • compounds 1A, 3B, and 4B showed improved metabolic stability in human hepatocyte cells when comparing with reference 1 and reference 2.
  • EXAMPLE 3 In Vitro Assessment of Cytochrome P450 (Cyp450) Induction in Cryopreserved Human Hepatocytes
  • cryopreserved human hepatocytes were thawed in 37°C water bath and diluted by plating medium to a seeding density of 0.55 ⁇ 10 ⁇ 6 cells/mL.
  • mRNA was prepared and measured using the Cells-to-Ct kit. Add DNase to Lysis solution.
  • sample lysate was added to 35 ⁇ L of Reverse Transcription Master Mix (containing 2 ⁇ RT Buffer, 20 ⁇ RT Enzyme Mix and Nuclease-free Water) for a final 50 ⁇ L reaction volume.
  • PCR cocktails were prepared for CYP3A4; containing the CYP specific probe set and that of ACTB as the endogenous control gene.
  • a typical PCR cocktail contained TaqMan Universal Master Mix (2 ⁇ ), Taqman Gene Expression Assay probe (20 ⁇ , CYP, FAM labeled), Taqman Gene Expression Assay probe (20 ⁇ , ACTB, VIC labeled) and RNase-free water.
  • Percent cell viability (%) (I (sample) -I (background) )/ (I (vehicle) -I (background) ) ⁇ 100
  • RNA qua tificatio For mRNA level determination, the mRNA content in each well is expressed as 2Ct(ACTB)- Ct(CYP).
  • % of positive control [(fold induction of test article)/ (fold induction of positive control)]*100
  • cytochrome P450 cytochrome P450
  • CYP450 cytochrome P450
  • CYP3A4 is by far the most abundant isoform and is responsible for the majority of CYP450-related metabolism of all marketed drugs.
  • the CYP induction activity of compound 1A is far less than two-folds against vehicle control and far less than 20% against the positive control on CYP3A4 isoform.
  • Compound 1A demonstrated no CYP induction effect when comparing with compound reference 2, thus devoid of CYP induction liability.
  • EXAMPLE 4 A Pharmacokinetic and Tissue Distribution Study of compound via Intravenous and Oral administration in male C57BL/6 mice.
  • mice Male C57BL/6 mice with a weight range of 20-25g (Hua Fu Kang, China) were used. Animals were fasted overnight and free access to food 4 hours after dosing.
  • Test compound (correction factor: 1.00) was dissolved in a 20 % hydroxypropyl-b- cyclodextrin (HP-b-CD) at a final concentration of 1 mg/ml for the intravenous (IV) formulation and at final concentrations of 0.5 mg/ml for the oral (PO) formulation.
  • the intravenous formulation was dosed at 2 ml/kg to obtain a dose of 2 mg/kg.
  • the oral Blood samples were taken at 7 and 20 min, 1, 2, 4, 8 and 24 h after intravenous dose administration. Blood and liver samples were taken at 30 min, 1, 2, 4, 8, 12 and 24 h after oral dose administration.
  • Plasma samples were adjusted to pH 3-4 by phosphoric acid and stored at -75 ⁇ 15°C prior to analysis. The whole process was completed within 1 hour.
  • Plasma and liver samples were analyzed using LC-MS/MS methods.
  • the lower limit of quantification (LLOQ) for plasma was 1.0 ng/ml and for liver was 2.5ng/g.
  • LLOQ lower limit of quantification
  • a non- compartmental analysis using the "Linear up log down" rule was used for all data.
  • a limited pharmacokinetic analysis was performed using PhoenixTM Professional (Version 6.1).
  • Results See table 10 below for plasma PK results, and table 11 for PO liver PK results.
  • Test compound (correction factor: 1.00) was dissolved in a 20 % hydroxypropyl-b- cyclodextrin (HP-b-CD) at a final concentration of 1 mg/ml for the intravenous (IV) formulation and at final concentrations of 0.5 mg/ml for the oral (PO) formulation.
  • HP-b-CD hydroxypropyl-b- cyclodextrin
  • the intravenous formulation was dosed at 2 ml/kg to obtain a dose of 2 mg/kg.
  • the oral formulations were dosed at 10 ml/kg to obtain final doses of 5 mg/kg.
  • Blood samples were taken at 5, 15 and 30 min, 1, 2, 4, 8 and 24 h after intravenous dose administration. Blood samples were taken at 15 and 30 min, 1, 2, 4, 8, 12 and 24 h after oral dose administration.
  • NaF Sodium Fluoride
  • KoX Potassium Oxalate
  • Plasma samples were analyzed using LC-MS/MS methods.
  • the lower limit of quantification (LLOQ) for plasma was 1.0 ng/ml.
  • Rat in-vivo PK studies are critical to ensure drug candidates have appropriate PK properties that can be evaluated in preclinical pharmacology and safety studies.
  • Compounds 1A, 3B, and 4B showed a far slower clearance, over two-folds higher dose- normalized AUC (AUC inf/Dose ) and an increased (or equal) bioavailability (F(%)) when comparing with reference 2 compound.

Abstract

Therapeutic combinations of hepatitis B virus (HBV) vaccines and capsid assembly modulators (CAMs), such as dihydropyrimidine derivatives are described. Methods of inducing an immune response against HBV or treating an HBV-induced disease, particularly in individuals having chronic HBV infection, using the disclosed therapeutic combinations are also described.

Description

COMBINATION OF HEPATITIS B VIRUS (HBV) VACCINES AND DIHYDROPYRIMIDINE DERIVATIVES AS CAPSID ASSEMBLY MODULATORS
CROSS REFERENCE TO RELATED APPLICATION
This application claims priority to U.S. Provisional Application No.62/862,794 filed on June 18, 2019, the disclosure of which is incorporated herein by reference in its entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
This application contains a sequence listing, which is submitted electronically via EFS- Web as an ASCII formatted sequence listing with a file name“065814_40WO1 Sequence
Listing” and a creation date of June 5, 2020 and having a size of 46 kb. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety. BACKGROUND OF THE INVENTION
Hepatitis B virus (HBV) is a small 3.2-kb hepatotropic DNA virus that encodes four open reading frames and seven proteins. Approximately 240 million people have chronic
hepatitis B infection (chronic HBV), characterized by persistent virus and subvirus particles in the blood for more than 6 months (Cohen et al. J. Viral Hepat. (2011) 18(6), 377-83).
Persistent HBV infection leads to T-cell exhaustion in circulating and intrahepatic HBV- specific CD4+ and CD8+ T-cells through chronic stimulation of HBV-specific T-cell
receptors with viral peptides and circulating antigens. As a result, T-cell polyfunctionality is decreased (i.e., decreased levels of IL-2, tumor necrosis factor (TNF)-a, IFN-g, and lack of proliferation).
A safe and effective prophylactic vaccine against HBV infection has been available since the 1980s and is the mainstay of hepatitis B prevention (World Health Organization,
Hepatitis B: Fact sheet No.204 [Internet] 2015 March.). The World Health Organization
recommends vaccination of all infants, and, in countries where there is low or intermediate hepatitis B endemicity, vaccination of all children and adolescents (<18 years of age), and of people of certain at risk population categories. Due to vaccination, worldwide infection rates have dropped dramatically. However, prophylactic vaccines do not cure established HBV
infection. Chronic HBV is currently treated with IFN-a and nucleoside or nucleotide analogs, but there is no ultimate cure due to the persistence in infected hepatocytes of an intracellular viral replication intermediate called covalently closed circular DNA (cccDNA), which plays a fundamental role as a template for viral RNAs, and thus new virions. It is thought that induced virus-specific T-cell and B-cell responses can effectively eliminate cccDNA-carrying hepatocytes. Current therapies targeting the HBV polymerase suppress viremia, but offer limited effect on cccDNA that resides in the nucleus and related production of circulating antigen. The most rigorous form of a cure may be elimination of HBV cccDNA from the organism, which has neither been observed as a naturally occurring outcome nor as a result of any therapeutic intervention. However, loss of HBV surface antigens (HBsAg) is a clinically credible equivalent of a cure, since disease relapse can occur only in cases of severe immunosuppression, which can then be prevented by prophylactic treatment. Thus, at least from a clinical standpoint, loss of HBsAg is associated with the most stringent form of immune reconstitution against HBV.
For example, immune modulation with pegylated interferon (pegIFN)-a has proven better in comparison to nucleoside or nucleotide therapy in terms of sustained off-treatment response with a finite treatment course. Besides a direct antiviral effect, IFN-a is reported to exert epigenetic suppression of cccDNA in cell culture and humanized mice, which leads to reduction of virion productivity and transcripts (Belloni et al. J. Clin. Invest. (2012) 122(2), 529-537). However, this therapy is still fraught with side-effects and overall responses are rather low, in part because IFN-a has only poor modulatory influences on HBV-specific T- cells. In particular, cure rates are low (< 10%) and toxicity is high. Likewise, direct acting HBV antivirals, namely the HBV polymerase inhibitors entecavir and tenofovir, are effective as monotherapy in inducing viral suppression with a high genetic barrier to emergence of drug resistant mutants and consecutive prevention of liver disease progression. However, cure of chronic hepatitis B, defined by HBsAg loss or seroconversion, is rarely achieved with such HBV polymerase inhibitors. Therefore, these antivirals in theory need to be administered indefinitely to prevent reoccurrence of liver disease, similar to antiretroviral therapy for human immunodeficiency virus (HIV).
Therapeutic vaccination has the potential to eliminate HBV from chronically infected patients (Michel et al. J. Hepatol. (2011) 54(6), 1286-1296). Many strategies have been explored, but to date therapeutic vaccination has not proven successful. BRIEF SUMMARY OF THE INVENTION
Accordingly, there is an unmet medical need in the treatment of hepatitis B virus (HBV), particularly chronic HBV, for a finite well-tolerated treatment with a higher cure rate. The invention satisfies this need by providing therapeutic combinations or compositions and methods for inducing an immune response against hepatitis B viruses (HBV) infection. The immunogenic compositions/combinations and methods of the invention can be used to provide therapeutic immunity to a subject, such as a subject having chronic HBV infection.
In a general aspect, the application relates to therapeutic combinations or compositions comprising one or more HBV antigens, or one or more polynucleotides encoding the HBV antigens, and a capsid assembly modulator (CAM), for use in treating an HBV infection in a subject in need thereof.
In one embodiment, the therapeutic combination comprises:
i) at least one of:
a) a truncated HBV core antigen consisting of an amino acid sequence that is at least 95%, such as at least 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 2,
b) a first non-naturally occurring nucleic acid molecule comprising a first
polynucleotide sequence encoding the truncated HBV core antigen;
c) an HBV polymerase antigen having an amino acid sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 7, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity, and
d) a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide sequence encoding the HBV polymerase antigen; and
ii) a compound of Formula (I):
Figure imgf000005_0001
or a deuterated isomer, a stereoisomer or tautomeric form thereof, or a pharmaceutically acceptable salt thereof, wherein:
R1 is selected from the group consisting of phenyl, thiophenyl, pyridyl, and pyridonyl, optionally substituted with one or more substituents selected from the group consisting of C1- 4alkyl, halogen, and CN;
R2 is C1-4alkyl;
R3 is selected from the group consisting of thiazolyl, pyridyl, and oxazolyl, optionally substituted with one or more substituents selected from fluorine, and C1-6alkyl;
n is an integer of 0 or 1;
R4 and R5 are independently selected from the group consisting of H and -COOH;
Figure imgf000006_0001
(i.e., the bond between X and Y) is a single bond or a double bond;
when X and Y are linked by a single bond, X is selected from the group consisting of C(=S), C(=NR6), C(=CHR7) and CHR8, and Y is NR9;
when X and Y are linked by a double bond, X is C-SR9 or C-OR9, and Y is N atom; Z is selected from the group consisting of CH2 and C(=O);
R6 is selected from the group consisting of CN, C(=O)CH3, and SO2CH3;
R7 is CN;
R8 is CF3;
R9 is selected from the group consisting of H, -C1-6alkyl, -C1-6alkyl-R10, -C1-6alkoxy- C1-6alkyl-R10 and -(CH2)p-Q-R10;
p is an integer of 0, 1, 2, or 3;
Q is selected from the group consisting of aryl, heteroaryl, and a 3- to 7- membered saturated ring, optionally containing a heteroatom, the heteroatom being an oxygen or a nitrogen, the nitrogen being substituted with H, -C1-6alkyl, -C1-6alkoxy-C1-6alkyl and -C1- 6alkylcarbonyl;
R10 is selected from -COOH, -C(=O)NHS(=O)2-C1-6alkyl, tetrazolyl and carboxylic acid bioisosteres.
In one embodiment, the truncated HBV core antigen consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, and the HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 7.
In one embodiment, the therapeutic combination comprises at least one of the HBV polymerase antigen and the truncated HBV core antigen. In certain embodiments, the therapeutic combination comprises the HBV polymerase antigen and the truncated HBV core antigen.
In one embodiment, the therapeutic combination comprises at least one of the first non-naturally occurring nucleic acid molecule comprising the first polynucleotide sequence encoding the truncated HBV core antigen, and the second non-naturally occurring nucleic acid molecule comprising the second polynucleotide sequence encoding the HBV polymerase antigen. In certain embodiments, the first non-naturally occurring nucleic acid molecule further comprises a polynucleotide sequence encoding a signal sequence operably linked to the N-terminus of the truncated HBV core antigen, and the second non-naturally occurring nucleic acid molecule further comprises a polynucleotide sequence encoding a signal sequence operably linked to the N-terminus of the HBV polymerase antigen, preferably, the signal sequence independently comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15, more preferably, the signal sequence is encoded by the polynucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 14, respectively.
In certain embodiments, the first polynucleotide sequence comprises the
polynucleotide sequence having at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3.
In certain embodiments, the second polynucleotide sequence comprises a
polynucleotide sequence having at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6.
In certain embodiments, compounds useful for the invention, as well as related information such as its structure, production, biological activities, therapeutic applications, etc., are described in International Patent Application PCT/CN2018/122258 filed on
December 20, 2018 and U.S. Patent Application US 62/791,576 filed on January 11, 2019, the content of which are herein incorporated by references in their entireties.
In an embodiment, a therapeutic combination comprises:
a) a first non-naturally occurring nucleic acid molecule comprising a first
polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 95%, such as at least 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 2;
b) a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide sequence encoding an HBV polymerase antigen having an amino acid sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 7, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity; and
c) a compound of Formula (I):
Figure imgf000008_0001
or a deuterated isomer, stereoisomer or tautomeric form thereof, or a pharmaceutically acceptable salt thereof, wherein:
R1 is phenyl substituted with one or more substituents selected from halogens and C1- 6alkyl;
R2 is methyl or ethyl;
R3 is thiazolyl;
n is an integer of 0 or 1;
R4 and R5 are H;
Figure imgf000008_0002
(i.e., the bond between X and Y) is a single bond;
X is C(=S);
Y is NR9;
Z is CH2;
R9 is C1-6alkyl-CO2H or (CH2)p-Q-R10;
p is an integer of 0, 1, 2, or 3;
Q is phenyl, a C3-6cycloalkyl, or a 3- to 6- saturated membered ring containing an oxygen; and
R10 is selected from -COOH, -C(=O)NHS(=O)2-C1-6alkyl, tetrazolyl and carboxylic acid bioisosteres, wherein the carboxylic acid bioisosteres are -S(=O)2(OH), -P(=O)(OH)2, - C(=O)NHOH, -C(=O)NHCN, 1,2,4-oxadiazol-5(4H)-one, or 3-hydroxy-4-methylcyclobut-3- ene-1,2-dione, which refer to the following structures:
Figure imgf000009_0001
. Preferably, the therapeutic combination comprises a) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding an truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; b) a second non-naturally occurring nucleic acid molecule comprising a second
polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 7, and (c) a compound selected from the group consisting of the compounds exemplified in this application, or a deuterated isomer, a stereoisomer or tautomeric form thereof, or a pharmaceutically acceptable salt thereof.
Preferably, the therapeutic combination comprises a first non-naturally occurring nucleic acid molecule comprising a polynucleotide sequence having at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3, and a second non-naturally occurring nucleic acid molecule comprising the polynucleotide sequence having at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6.
More preferably, the therapeutic combination comprises a) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3; b) a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide sequence of SEQ ID NO: 5 or 6; and c) compound 1A:
Figure imgf000009_0002
or a deuterated isomer, a stereoisomer or tautomeric form thereof, or a pharmaceutically acceptable salt thereof In an embodiment, each of the first and the second non-naturally occurring nucleic acid molecules is a DNA molecule, preferably the DNA molecule is present on a plasmid or a viral vector.
In another embodiment, each of the first and the second non-naturally occurring nucleic acid molecules is an RNA molecule, preferably an mRNA or a self-replicating RNA molecule.
In some embodiments, each of the first and the second non-naturally occurring nucleic acid molecules is independently formulated with a lipid nanoparticle (LNP).
In another general aspect, the application relates to a kit comprising a therapeutic combination of the application.
The application also relates to a therapeutic combination or kit of the application for use in inducing an immune response against hepatitis B virus (HBV); and use of a therapeutic combination, composition or kit of the application in the manufacture of a medicament for inducing an immune response against hepatitis B virus (HBV). The use can further comprise a combination with another immunogenic or therapeutic agent, preferably another HBV antigen or another HBV therapy. Preferably, the subject has chronic HBV infection.
The application further relates to a therapeutic combination or kit of the application for use in treating an HBV-induced disease in a subject in need thereof; and use of therapeutic combination or kit of the application in the manufacture of a medicament for treating an HBV- induced disease in a subject in need thereof. The use can further comprise a combination with another therapeutic agent, preferably another anti-HBV antigen. Preferably, the subject has chronic HBV infection, and the HBV-induced disease is selected from the group consisting of advanced fibrosis, cirrhosis, and hepatocellular carcinoma (HCC).
The application also relates to a method of inducing an immune response against an HBV or a method of treating an HBV infection or an HBV-induced disease, comprising administering to a subject in need thereof a therapeutic combination according to
embodiments of the application.
Other aspects, features and advantages of the invention will be apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS The foregoing summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the application is not limited to the precise embodiments shown in the drawings.
FIG.1A and FIG.1B show schematic representations of DNA plasmids according to embodiments of the application; FIG.1A shows a DNA plasmid encoding an HBV core antigen according to an embodiment of the application; FIG.1B shows a DNA plasmid encoding an HBV polymerase (pol) antigen according to an embodiment of the application; the HBV core and pol antigens are expressed under control of a CMV promoter with an N- terminal cystatin S signal peptide that is cleaved from the expressed antigen upon secretion from the cell; transcriptional regulatory elements of the plasmid include an enhancer sequence located between the CMV promoter and the polynucleotide sequence encoding the HBV antigen and a bGH polyadenylation sequence located downstream of the polynucleotide sequence encoding the HBV antigen; a second expression cassette is included in the plasmid in reverse orientation including a kanamycin resistance gene under control of an Ampr (bla) promoter; an origin of replication (pUC) is also included in reverse orientation.
FIG.2A and FIG.2B. show the schematic representations of the expression cassettes in adenoviral vectors according to embodiments of the application; FIG.2A shows the expression cassette for a truncated HBV core antigen, which contains a CMV promoter, an intron (a fragment derived from the human ApoAI gene - GenBank accession X01038 base pairs 295– 523, harboring the ApoAI second intron), a human immunoglobulin secretion signal, followed by a coding sequence for a truncated HBV core antigen and a SV40 polyadenylation signal; FIG.2B shows the expression cassette for a fusion protein of a truncated HBV core antigen operably linked to an HBV polymerase antigen, which is otherwise identical to the expression cassette for the truncated HBV core antigen except the HBV antigen.
FIG.3 shows ELISPOT responses of Balb/c mice immunized with different DNA plasmids expressing HBV core antigen or HBV pol antigen, as described in Example 3;
peptide pools used to stimulate splenocytes isolated from the various vaccinated animal groups are indicated in gray scale; the number of responsive T-cells are indicated on the y-axis expressed as spot forming cells (SFC) per 106 splenocytes; DETAILED DESCRIPTION OF THE INVENTION Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification. All patents, published patent applications and publications cited herein are incorporated by reference as if set forth fully herein.
It must be noted that as used herein and in the appended claims, the singular forms “a,”“an,” and“the” include plural reference unless the context clearly dictates otherwise.
Unless otherwise indicated, the term“at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word“comprise”, and variations such as“comprises” and“comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. When used herein the term“comprising” can be substituted with the term“containing” or“including” or sometimes when used herein with the term“having”.
When used herein“consisting of’ excludes any element, step, or ingredient not specified in the claim element. When used herein,“consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the aforementioned terms of“comprising”,“containing”,“including”, and “having”, whenever used herein in the context of an aspect or embodiment of the application can be replaced with the term“consisting of’ or“consisting essentially of’ to vary scopes of the disclosure.
As used herein, the conjunctive term“and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by“and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term“and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term“and/or.”
Unless otherwise stated, any numerical value, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term“about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, a concentration range of 1 mg/mL to 10 mg/mL includes 0.9 mg/mL to 11 mg/mL. As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.
The phrases“percent (%) sequence identity” or“% identity” or“% identical to” when used with reference to an amino acid sequence describe the number of matches (“hits”) of identical amino acids of two or more aligned amino acid sequences as compared to the number of amino acid residues making up the overall length of the amino acid sequences. In other terms, using an alignment, for two or more sequences the percentage of amino acid residues that are the same (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identity over the full-length of the amino acid sequences) may be determined, when the sequences are compared and aligned for maximum correspondence as measured using a sequence comparison algorithm as known in the art, or when manually aligned and visually inspected. The sequences which are compared to determine sequence identity may thus differ by substitution(s), addition(s) or deletion(s) of amino acids. Suitable programs for aligning protein sequences are known to the skilled person. The percentage sequence identity of protein sequences can, for example, be determined with programs such as CLUSTALW, Clustal Omega, FASTA or BLAST, e.g. using the NCBI BLAST algorithm (Altschul SF, et al (1997), Nucleic Acids Res. 25:3389-3402).
As used herein, the terms and phrases“in combination,”“in combination with,”“co delivery,” and“administered together with” in the context of the administration of two or more therapies or components to a subject refers to simultaneous administration or subsequent administration of two or more therapies or components, such as two vectors, e.g., DNA plasmids, peptides, or a therapeutic combination and an adjuvant.“Simultaneous administration” can be administration of the two or more therapies or components at least within the same day. When two components are“administered together with” or “administered in combination with,” they can be administered in separate compositions sequentially within a short time period, such as 24, 20, 16, 12, 8 or 4 hours, or within 1 hour, or they can be administered in a single composition at the same time.“Subsequent administration” can be administration of the two or more therapies or components in the same day or on separate days. The use of the term“in combination with” does not restrict the order in which therapies or components are administered to a subject. For example, a first therapy or component (e.g. first DNA plasmid encoding an HBV antigen) can be
administered prior to (e.g., 5 minutes to one hour before), concomitantly with or
simultaneously with, or subsequent to (e.g., 5 minutes to one hour after) the administration of a second therapy or component (e.g., second DNA plasmid encoding an HBV antigen), and/or a third therapy or component (e.g., a capsid assembly modulator (CAM)). In some embodiments, a first therapy or component (e.g. first DNA plasmid encoding an HBV antigen), a second therapy or component (e.g., second DNA plasmid encoding an HBV antigen), and a third therapy or component (e.g., a CAM) are administered in the same composition. In other embodiments, a first therapy or component (e.g. first DNA plasmid encoding an HBV antigen), a second therapy or component (e.g., second DNA plasmid encoding an HBV antigen), and a third therapy or component (e.g., a CAM) are administered in separate compositions, such as two or three separate compositions.
As used herein, a“non-naturally occurring” nucleic acid or polypeptide, refers to a nucleic acid or polypeptide that does not occur in nature. A“non-naturally occurring” nucleic acid or polypeptide can be synthesized, treated, fabricated, and/or otherwise manipulated in a laboratory and/or manufacturing setting. In some cases, a non-naturally occurring nucleic acid or polypeptide can comprise a naturally-occurring nucleic acid or polypeptide that is treated, processed, or manipulated to exhibit properties that were not present in the naturally-occurring nucleic acid or polypeptide, prior to treatment. As used herein, a“non-naturally occurring” nucleic acid or polypeptide can be a nucleic acid or polypeptide isolated or separated from the natural source in which it was discovered, and it lacks covalent bonds to sequences with which it was associated in the natural source. A “non-naturally occurring” nucleic acid or polypeptide can be made recombinantly or via other methods, such as chemical synthesis.
As used herein,“subject” means any animal, preferably a mammal, most preferably a human, to whom will be or has been treated by a method according to an embodiment of the application. The term“mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans, etc., more preferably a human.
As used herein, the term“operably linked” refers to a linkage or a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a regulatory sequence operably linked to a nucleic acid sequence of interest is capable of directing the transcription of the nucleic acid sequence of interest, or a signal sequence operably linked to an amino acid sequence of interest is capable of secreting or translocating the amino acid sequence of interest over a membrane.
In an attempt to help the reader of the application, the description has been separated in various paragraphs or sections, or is directed to various embodiments of the application. These separations should not be considered as disconnecting the substance of a paragraph or section or embodiments from the substance of another paragraph or section or embodiments. To the contrary, one skilled in the art will understand that the description has broad application and encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated. The discussion of any embodiment is meant only to be exemplary and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples. For example, while embodiments of HBV vectors of the application (e.g., plasmid DNA or viral vectors) described herein may contain particular components, including, but not limited to, certain promoter sequences, enhancer or regulatory sequences, signal peptides, coding sequence of an HBV antigen, polyadenylation signal sequences, etc. arranged in a particular order, those having ordinary skill in the art will appreciate that the concepts disclosed herein may equally apply to other components arranged in other orders that can be used in HBV vectors of the application. The application contemplates use of any of the applicable components in any combination having any sequence that can be used in HBV vectors of the application, whether or not a particular combination is expressly described. The invention generally relates to a therapeutic combination comprising one or more HBV antigens and at least one CAM. Hepatitis B Virus (HBV)
As used herein“hepatitis B virus” or“HBV” refers to a virus of the hepadnaviridae family. HBV is a small (e.g., 3.2 kb) hepatotropic DNA virus that encodes four open reading frames and seven proteins. The seven proteins encoded by HBV include small (S), medium (M), and large (L) surface antigen (HBsAg) or envelope (Env) proteins, pre-Core protein, core protein, viral polymerase (Pol), and HBx protein. HBV expresses three surface antigens, or envelope proteins, L, M, and S, with S being the smallest and L being the largest. The extra domains in the M and L proteins are named Pre-S2 and Pre-S1, respectively. Core protein is the subunit of the viral nucleocapsid. Pol is needed for synthesis of viral DNA (reverse transcriptase, RNaseH, and primer), which takes place in nucleocapsids localized to the cytoplasm of infected hepatocytes. PreCore is the core protein with an N-terminal signal peptide and is proteolytically processed at its N and C termini before secretion from infected cells, as the so-called hepatitis B e-antigen (HBeAg). HBx protein is required for efficient transcription of covalently closed circular DNA (cccDNA). HBx is not a viral structural protein. All viral proteins of HBV have their own mRNA except for core and polymerase, which share an mRNA. With the exception of the protein pre-Core, none of the HBV viral proteins are subject to post-translational proteolytic processing.
The HBV virion contains a viral envelope, nucleocapsid, and single copy of the partially double-stranded DNA genome. The nucleocapsid comprises 120 dimers of core protein and is covered by a capsid membrane embedded with the S, M, and L viral envelope or surface antigen proteins. After entry into the cell, the virus is uncoated and the capsid- containing relaxed circular DNA (rcDNA) with covalently bound viral polymerase migrates to the nucleus. During that process, phosphorylation of the core protein induces structural changes, exposing a nuclear localization signal enabling interaction of the capsid with so- called importins. These importins mediate binding of the core protein to nuclear pore complexes upon which the capsid disassembles and polymerase/rcDNA complex is released into the nucleus. Within the nucleus the rcDNA becomes deproteinized (removal of polymerase) and is converted by host DNA repair machinery to a covalently closed circular DNA (cccDNA) genome from which overlapping transcripts encode for HBeAg, HBsAg, Core protein, viral polymerase and HBx protein. Core protein, viral polymerase, and pre- genomic RNA (pgRNA) associate in the cytoplasm and self-assemble into immature pgRNA- containing capsid particles, which further convert into mature rcDNA-capsids and function as a common intermediate that is either enveloped and secreted as infectious virus particles or transported back to the nucleus to replenish and maintain a stable cccDNA pool.
To date, HBV is divided into four serotypes (adr, adw, ayr, ayw) based on antigenic epitopes present on the envelope proteins, and into eight genotypes (A, B, C, D, E, F, G, and H) based on the sequence of the viral genome. The HBV genotypes are distributed over different geographic regions. For example, the most prevalent genotypes in Asia are genotypes B and C. Genotype D is dominant in Africa, the Middle East, and India, whereas genotype A is widespread in Northern Europe, sub-Saharan Africa, and West Africa.
HBV Antigens
As used herein, the terms“HBV antigen,”“antigenic polypeptide of HBV,”“HBV antigenic polypeptide,”“HBV antigenic protein,”“HBV immunogenic polypeptide,” and “HBV immunogen” all refer to a polypeptide capable of inducing an immune response, e.g., a humoral and/or cellular mediated response, against an HBV in a subject. The HBV antigen can be a polypeptide of HBV, a fragment or epitope thereof, or a combination of multiple HBV polypeptides, portions or derivatives thereof. An HBV antigen is capable of raising in a host a protective immune response, e.g., inducing an immune response against a viral disease or infection, and/or producing an immunity (i.e., vaccinates) in a subject against a viral disease or infection, that protects the subject against the viral disease or infection. For example, an HBV antigen can comprise a polypeptide or immunogenic fragment(s) thereof from any HBV protein, such as HBeAg, pre-core protein, HBsAg (S, M, or L proteins), core protein, viral polymerase, or HBx protein derived from any HBV genotype, e.g., genotype A, B, C, D, E, F, G, and/or H, or combination thereof.
(1) HBV Core Antigen
As used herein, each of the terms“HBV core antigen,”“HBc” and“core antigen” refers to an HBV antigen capable of inducing an immune response, e.g., a humoral and/or cellular mediated response, against an HBV core protein in a subject. Each of the terms “core,”“core polypeptide,” and“core protein” refers to the HBV viral core protein. Full- length core antigen is typically 183 amino acids in length and includes an assembly domain (amino acids 1 to 149) and a nucleic acid binding domain (amino acids 150 to 183). The 34- residue nucleic acid binding domain is required for pre-genomic RNA encapsidation. This domain also functions as a nuclear import signal. It comprises 17 arginine residues and is highly basic, consistent with its function. HBV core protein is dimeric in solution, with the dimers self-assembling into icosahedral capsids. Each dimer of core protein has four a-helix bundles flanked by an a-helix domain on either side. Truncated HBV core proteins lacking the nucleic acid binding domain are also capable of forming capsids.
In an embodiment of the application, an HBV antigen is a truncated HBV core antigen. As used herein, a“truncated HBV core antigen,” refers to an HBV antigen that does not contain the entire length of an HBV core protein, but is capable of inducing an immune response against the HBV core protein in a subject. For example, an HBV core antigen can be modified to delete one or more amino acids of the highly positively charged (arginine rich) C-terminal nucleic acid binding domain of the core antigen, which typically contains seventeen arginine (R) residues. A truncated HBV core antigen of the application is preferably a C-terminally truncated HBV core protein which does not comprise the HBV core nuclear import signal and/or a truncated HBV core protein from which the C-terminal HBV core nuclear import signal has been deleted. In an embodiment, a truncated HBV core antigen comprises a deletion in the C-terminal nucleic acid binding domain, such as a deletion of 1 to 34 amino acid residues of the C-terminal nucleic acid binding domain, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acid residues, preferably a deletion of all 34 amino acid residues. In a preferred embodiment, a truncated HBV core antigen comprises a deletion in the C-terminal nucleic acid binding domain, preferably a deletion of all 34 amino acid residues.
An HBV core antigen of the application can be a consensus sequence derived from multiple HBV genotypes (e.g., genotypes A, B, C, D, E, F, G, and H). As used herein, “consensus sequence” means an artificial sequence of amino acids based on an alignment of amino acid sequences of homologous proteins, e.g., as determined by an alignment (e.g., using Clustal Omega) of amino acid sequences of homologous proteins. It can be the calculated order of most frequent amino acid residues, found at each position in a sequence alignment, based upon sequences of HBV antigens (e.g., core, pol, etc.) from at least 100 natural HBV isolates. A consensus sequence can be non-naturally occurring and different from the native viral sequences. Consensus sequences can be designed by aligning multiple HBV antigen sequences from different sources using a multiple sequence alignment tool, and at variable alignment positions, selecting the most frequent amino acid. Preferably, a consensus sequence of an HBV antigen is derived from HBV genotypes B, C, and D. The term“consensus antigen” is used to refer to an antigen having a consensus sequence. An exemplary truncated HBV core antigen according to the application lacks the nucleic acid binding function, and is capable of inducing an immune response in a mammal against at least two HBV genotypes. Preferably a truncated HBV core antigen is capable of inducing a T cell response in a mammal against at least HBV genotypes B, C and D. More preferably, a truncated HBV core antigen is capable of inducing a CD8 T cell response in a human subject against at least HBV genotypes A, B, C and D.
Preferably, an HBV core antigen of the application is a consensus antigen, preferably a consensus antigen derived from HBV genotypes B, C, and D, more preferably a truncated consensus antigen derived from HBV genotypes B, C, and D. An exemplary truncated HBV core consensus antigen according to the application consists of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%,
99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4. SEQ ID NO: 2 and SEQ ID NO: 4 are core consensus antigens derived from HBV genotypes B, C, and D. SEQ ID NO: 2 and SEQ ID NO: 4 each contain a 34-amino acid C-terminal deletion of the highly positively charged (arginine rich) nucleic acid binding domain of the native core antigen.
In one embodiment of the application, an HBV core antigen is a truncated HBV antigen consisting of the amino acid sequence of SEQ ID NO: 2. In another embodiment, an HBV core antigen is a truncated HBV antigen consisting of the amino acid sequence of SEQ ID NO: 4. In another embodiment, an HBV core antigen further contains a signal sequence operably linked to the N-terminus of a mature HBV core antigen sequence, such as the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Preferably, the signal sequence has the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15.
(2) HBV Polymerase Antigen
As used herein, the term“HBV polymerase antigen,”“HBV Pol antigen” or“HBV pol antigen” refers to an HBV antigen capable of inducing an immune response, e.g., a humoral and/or cellular mediated response, against an HBV polymerase in a subject. Each of the terms“polymerase,”“polymerase polypeptide,”“Pol” and“pol” refers to the HBV viral DNA polymerase. The HBV viral DNA polymerase has four domains, including, from the N terminus to the C terminus, a terminal protein (TP) domain, which acts as a primer for minus- strand DNA synthesis; a spacer that is nonessential for the polymerase functions; a reverse transcriptase (RT) domain for transcription; and a RNase H domain. In an embodiment of the application, an HBV antigen comprises an HBV Pol antigen, or any immunogenic fragment or combination thereof. An HBV Pol antigen can contain further modifications to improve immunogenicity of the antigen, such as by introducing mutations into the active sites of the polymerase and/or RNase domains to decrease or substantially eliminate certain enzymatic activities.
Preferably, an HBV Pol antigen of the application does not have reverse transcriptase activity and RNase H activity, and is capable of inducing an immune response in a mammal against at least two HBV genotypes. Preferably, an HBV Pol antigen is capable of inducing a T cell response in a mammal against at least HBV genotypes B, C and D. More preferably, an HBV Pol antigen is capable of inducing a CD8 T cell response in a human subject against at least HBV genotypes A, B, C and D.
Thus, in some embodiments, an HBV Pol antigen is an inactivated Pol antigen. In an embodiment, an inactivated HBV Pol antigen comprises one or more amino acid mutations in the active site of the polymerase domain. In another embodiment, an inactivated HBV Pol antigen comprises one or more amino acid mutations in the active site of the RNaseH domain. In a preferred embodiment, an inactivated HBV pol antigen comprises one or more amino acid mutations in the active site of both the polymerase domain and the RNaseH domain. For example, the“YXDD” motif in the polymerase domain of an HBV pol antigen that can be required for nucleotide/metal ion binding can be mutated, e.g., by replacing one or more of the aspartate residues (D) with asparagine residues (N), eliminating or reducing metal coordination function, thereby decreasing or substantially eliminating reverse transcriptase function. Alternatively, or in addition to mutation of the“YXDD” motif, the “DEDD” motif in the RNaseH domain of an HBV pol antigen required for Mg2+
coordination can be mutated, e.g., by replacing one or more aspartate residues (D) with asparagine residues (N) and/or replacing the glutamate residue (E) with glutamine (Q), thereby decreasing or substantially eliminating RNaseH function. In a particular
embodiment, an HBV pol antigen is modified by (1) mutating the aspartate residues (D) to asparagine residues (N) in the“YXDD” motif of the polymerase domain; and (2) mutating the first aspartate residue (D) to an asparagine residue (N) and the first glutamate residue (E) to a glutamine residue (N) in the“DEDD” motif of the RNaseH domain, thereby decreasing or substantially eliminating both the reverse transcriptase and RNaseH functions of the pol antigen. In a preferred embodiment of the application, an HBV pol antigen is a consensus antigen, preferably a consensus antigen derived from HBV genotypes B, C, and D, more preferably an inactivated consensus antigen derived from HBV genotypes B, C, and D. An exemplary HBV pol consensus antigen according to the application comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 7, preferably at least 98% identical to SEQ ID NO: 7, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 7. SEQ ID NO: 7 is a pol consensus antigen derived from HBV genotypes B, C, and D comprising four mutations located in the active sites of the polymerase and RNaseH domains. In particular, the four mutations include mutation of the aspartic acid residues (D) to asparagine residues (N) in the“YXDD” motif of the polymerase domain; and mutation of the first aspartate residue (D) to an asparagine residue (N) and mutation of the glutamate residue (E) to a glutamine residue (Q) in the“DEDD” motif of the RNaseH domain.
In a particular embodiment of the application, an HBV pol antigen comprises the amino acid sequence of SEQ ID NO: 7. In other embodiments of the application, an HBV pol antigen consists of the amino acid sequence of SEQ ID NO: 7. In a further embodiment, an HBV pol antigen further contains a signal sequence operably linked to the N-terminus of a mature HBV pol antigen sequence, such as the amino acid sequence of SEQ ID NO: 7. Preferably, the signal sequence has the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15.
(3) Fusion of HBV Core Antigen and HBV Polymerase Antigen
As used herein the term“fusion protein” or“fusion” refers to a single polypeptide chain having at least two polypeptide domains that are not normally present in a single, natural polypeptide.
In an embodiment of the application, an HBV antigen comprises a fusion protein comprising a truncated HBV core antigen operably linked to an HBV Pol antigen, or an HBV Pol antigen operably linked to a truncated HBV core antigen, preferably via a linker.
For example, in a fusion protein containing a first polypeptide and a second heterologous polypeptide, a linker serves primarily as a spacer between the first and second polypeptides. In an embodiment, a linker is made up of amino acids linked together by peptide bonds, preferably from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. In an embodiment, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. Preferably, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Exemplary linkers are polyglycines, particularly (Gly)5, (Gly)8; poly(Gly-Ala), and polyalanines. One exemplary suitable linker as shown in the Examples below is (AlaGly)n, wherein n is an integer of 2 to 5.
Preferably, a fusion protein of the application is capable of inducing an immune response in a mammal against HBV core and HBV Pol of at least two HBV genotypes. Preferably, a fusion protein is capable of inducing a T cell response in a mammal against at least HBV genotypes B, C and D. More preferably, the fusion protein is capable of inducing a CD8 T cell response in a human subject against at least HBV genotypes A, B, C and D.
In an embodiment of the application, a fusion protein comprises a truncated HBV core antigen having an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4, a linker, and an HBV Pol antigen having an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, identical to SEQ ID NO: 7.
In a preferred embodiment of the application, a fusion protein comprises a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, a linker comprising (AlaGly)n, wherein n is an integer of 2 to 5, and an HBV Pol antigen having the amino acid sequence of SEQ ID NO: 7. More preferably, a fusion protein according to an embodiment of the application comprises the amino acid sequence of SEQ ID NO: 16.
In one embodiment of the application, a fusion protein further comprises a signal sequence operably linked to the N-terminus of the fusion protein. Preferably, the signal sequence has the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15. In one embodiment, a fusion protein comprises the amino acid sequence of SEQ ID NO: 17.
Additional disclosure on HBV vaccines that can be used for the present invention are described in U.S. Patent Application No: 16/223,251, filed December 18, 2018, the contents of the application, more preferably the examples of the application, are hereby incorporated by reference in their entireties. Polynucleotides and Vectors
In another general aspect, the application provides a non-naturally occurring nucleic acid molecule encoding an HBV antigen useful for an invention according to embodiments of the application, and vectors comprising the non-naturally occurring nucleic acid. A first or second non-naturally occurring nucleic acid molecule can comprise any polynucleotide sequence encoding an HBV antigen useful for the application, which can be made using methods known in the art in view of the present disclosure. Preferably, a first or second polynucleotide encodes at least one of a truncated HBV core antigen and an HBV polymerase antigen of the application. A polynucleotide can be in the form of RNA or in the form of DNA obtained by recombinant techniques (e.g., cloning) or produced synthetically (e.g., chemical synthesis). The DNA can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequence. The DNA can, for example, comprise genomic DNA, cDNA, or combinations thereof. The polynucleotide can also be a DNA/RNA hybrid. The polynucleotides and vectors of the application can be used for recombinant protein production, expression of the protein in host cell, or the production of viral particles. Preferably, a polynucleotide is DNA.
In an embodiment of the application, a first non-naturally occurring nucleic acid molecule comprises a first polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 2, preferably 98%, 99% or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4. In a particular embodiment of the application, a first non- naturally occurring nucleic acid molecule comprises a first polynucleotide sequence encoding a truncated HBV core antigen consisting the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
Examples of polynucleotide sequences of the application encoding a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 include, but are not limited to, a polynucleotide sequence at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 3, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3, preferably 98%, 99% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3. Exemplary non-naturally occurring nucleic acid molecules encoding a truncated HBV core antigen have the polynucleotide sequence of SEQ ID NOs: 1 or 3.
In another embodiment, a first non-naturally occurring nucleic acid molecule further comprises a coding sequence for a signal sequence that is operably linked to the N-terminus of the HBV core antigen sequence. Preferably, the signal sequence has the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15. More preferably, the coding sequence for a signal sequence comprises the polynucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 14.
In an embodiment of the application, a second non-naturally occurring nucleic acid molecule comprises a second polynucleotide sequence encoding an HBV polymerase antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 7, preferably 100% identical to SEQ ID NO: 7. In a particular embodiment of the application, a second non-naturally occurring nucleic acid molecule comprises a second polynucleotide sequence encoding an HBV polymerase antigen consisting of the amino acid sequence of SEQ ID NO: 7.
Examples of polynucleotide sequences of the application encoding an HBV Pol antigen comprising the amino acid sequence of at least 90% identical to SEQ ID NO: 7 include, but are not limited to, a polynucleotide sequence at least 90% identical to SEQ ID NO: 5 or SEQ ID NO: 6, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 5 or SEQ ID NO: 6, preferably 98%, 99% or 100% identical to SEQ ID NO: 5 or SEQ ID NO: 6. Exemplary non-naturally occurring nucleic acid molecules encoding an HBV pol antigen have the polynucleotide sequence of SEQ ID NOs: 5 or 6.
In another embodiment, a second non-naturally occurring nucleic acid molecule further comprises a coding sequence for a signal sequence that is operably linked to the N- terminus of the HBV pol antigen sequence, such as the amino acid sequence of SEQ ID NO: 7. Preferably, the signal sequence has the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15. More preferably, the coding sequence for a signal sequence comprises the polynucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 14.
In another embodiment of the application, a non-naturally occurring nucleic acid molecule encodes an HBV antigen fusion protein comprising a truncated HBV core antigen operably linked to an HBV Pol antigen, or an HBV Pol antigen operably linked to a truncated HBV core antigen. In a particular embodiment, a non-naturally occurring nucleic acid molecule of the application encodes a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4, more preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO:4; a linker; and an HBV polymerase antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 7, preferably 98%, 99% or 100% identical to SEQ ID NO: 7. In a particular embodiment of the application, a non-naturally occurring nucleic acid molecule encodes a fusion protein comprising a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, a linker comprising (AlaGly)n, wherein n is an integer of 2 to 5; and an HBV Pol antigen comprising the amino acid sequence of SEQ ID NO: 7. In a particular embodiment of the application, a non- naturally occurring nucleic acid molecule encodes an HBV antigen fusion protein comprising the amino acid sequence of SEQ ID NO: 16.
Examples of polynucleotide sequences of the application encoding an HBV antigen fusion protein include, but are not limited to, a polynucleotide sequence at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 3, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3, preferably 98%, 99% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3, operably linked to a linker coding sequence at least 90% identical to SEQ ID NO: 11, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 11, preferably 98%, 99% or 100% identical to SEQ ID NO: 11, which is further operably linked a polynucleotide sequence at least 90% identical to SEQ ID NO: 5 or SEQ ID NO: 6, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 5 or SEQ ID NO: 6, preferably 98%, 99% or 100% identical to SEQ ID NO: 5 or SEQ ID NO: 6. In particular embodiments of the application, a non- naturally occurring nucleic acid molecule encoding an HBV antigen fusion protein comprises SEQ ID NO: 1 or SEQ ID NO: 3, operably linked to SEQ ID NO: 11, which is further operably linked to SEQ ID NO: 5 or SEQ ID NO: 6.
In another embodiment, a non-naturally occurring nucleic acid molecule encoding an HBV fusion further comprises a coding sequence for a signal sequence that is operably linked to the N-terminus of the HBV fusion sequence, such as the amino acid sequence of SEQ ID NO: 16. Preferably, the signal sequence has the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15. More preferably, the coding sequence for a signal sequence comprises the polynucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 14. In one embodiment, the encoded fusion protein with the signal sequence comprises the amino acid sequence of SEQ ID NO: 17.
The application also relates to a vector comprising the first and/or second non- naturally occurring nucleic acid molecules. As used herein, a“vector” is a nucleic acid molecule used to carry genetic material into another cell, where it can be replicated and/or expressed. Any vector known to those skilled in the art in view of the present disclosure can be used. Examples of vectors include, but are not limited to, plasmids, viral vectors (bacteriophage, animal viruses, and plant viruses), cosmids, and artificial chromosomes (e.g., YACs). Preferably, a vector is a DNA plasmid. A vector can be a DNA vector or an RNA vector. One of ordinary skill in the art can construct a vector of the application through standard recombinant techniques in view of the present disclosure.
A vector of the application can be an expression vector. As used herein, the term “expression vector” refers to any type of genetic construct comprising a nucleic acid coding for an RNA capable of being transcribed. Expression vectors include, but are not limited to, vectors for recombinant protein expression, such as a DNA plasmid or a viral vector, and vectors for delivery of nucleic acid into a subject for expression in a tissue of the subject, such as a DNA plasmid or a viral vector. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
Vectors of the application can contain a variety of regulatory sequences. As used herein, the term“regulatory sequence” refers to any sequence that allows, contributes or modulates the functional regulation of the nucleic acid molecule, including replication, duplication, transcription, splicing, translation, stability and/or transport of the nucleic acid or one of its derivative (i.e. mRNA) into the host cell or organism. In the context of the disclosure, this term encompasses promoters, enhancers and other expression control elements (e.g., polyadenylation signals and elements that affect mRNA stability).
In some embodiments of the application, a vector is a non-viral vector. Examples of non-viral vectors include, but are not limited to, DNA plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, etc. Examples of non-viral vectors include, but are not limited to, RNA replicon, mRNA replicon, modified mRNA replicon or self-amplifying mRNA, closed linear deoxyribonucleic acid, e.g. a linear covalently closed DNA such as linear covalently closed double stranded DNA molecule. Preferably, a non-viral vector is a DNA plasmid. A“DNA plasmid”, which is used interchangeably with“DNA plasmid vector,”“plasmid DNA” or“plasmid DNA vector,” refers to a double-stranded and generally circular DNA sequence that is capable of autonomous replication in a suitable host cell. DNA plasmids used for expression of an encoded polynucleotide typically comprise an origin of replication, a multiple cloning site, and a selectable marker, which for example, can be an antibiotic resistance gene. Examples of DNA plasmids suitable that can be used include, but are not limited to, commercially available expression vectors for use in well-known expression systems (including both prokaryotic and eukaryotic systems), such as pSE420 (Invitrogen, San Diego, Calif.), which can be used for production and/or expression of protein in Escherichia coli; pYES2
(Invitrogen, Thermo Fisher Scientific), which can be used for production and/or expression in Saccharomyces cerevisiae strains of yeast; MAXBAC® complete baculovirus expression system (Thermo Fisher Scientific), which can be used for production and/or expression in insect cells; pcDNATM or pcDNA3TM (Life Technologies, Thermo Fisher Scientific), which can be used for high level constitutive protein expression in mammalian cells; and pVAX or pVAX-1 (Life Technologies, Thermo Fisher Scientific), which can be used for high-level transient expression of a protein of interest in most mammalian cells. The backbone of any commercially available DNA plasmid can be modified to optimize protein expression in the host cell, such as to reverse the orientation of certain elements (e.g., origin of replication and/or antibiotic resistance cassette), replace a promoter endogenous to the plasmid (e.g., the promoter in the antibiotic resistance cassette), and/or replace the polynucleotide sequence encoding transcribed proteins (e.g., the coding sequence of the antibiotic resistance gene), by using routine techniques and readily available starting materials. (See e.g., Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989)).
Preferably, a DNA plasmid is an expression vector suitable for protein expression in mammalian host cells. Expression vectors suitable for protein expression in mammalian host cells include, but are not limited to, pcDNATM, pcDNA3TM, pVAX, pVAX-1, ADVAX, NTC8454, etc. Preferably, an expression vector is based on pVAX-1, which can be further modified to optimize protein expression in mammalian cells. pVAX-1 is commonly used plasmid in DNA vaccines, and contains a strong human intermediate early cytomegalovirus (CMV-IE) promoter followed by the bovine growth hormone (bGH)-derived polyadenylation sequence (pA). pVAX-1 further contains a pUC origin of replication and kanamycin resistance gene driven by a small prokaryotic promoter that allows for bacterial plasmid propagation.
A vector of the application can also be a viral vector. In general, viral vectors are genetically engineered viruses carrying modified viral DNA or RNA that has been rendered non-infectious, but still contains viral promoters and transgenes, thus allowing for translation of the transgene through a viral promoter. Because viral vectors are frequently lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection. Examples of viral vectors that can be used include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, pox virus vectors, enteric virus vectors, Venezuelan Equine Encephalitis virus vectors, Semliki Forest Virus vectors, Tobacco Mosaic Virus vectors, lentiviral vectors, etc. Examples of viral vectors that can be used include, but are not limited to, arenavirus viral vectors, replication-deficient arenavirus viral vectors or replication-competent arenavirus viral vectors, bi-segmented or tri-segmented arenavirus, infectious arenavirus viral vectors, nucleic acids which comprise an arenavirus genomic segment wherein one open reading frame of the genomic segment is deleted or functionally inactivated (and replaced by a nucleic acid encoding an HBV antigen as described herein), arenavirus such as lymphocytic choriomeningitidis virus (LCMV), e.g., clone 13 strain or MP strain, and arenavirus such as Junin virus e.g., Candid #1 strain. The vector can also be a non- viral vector.
Preferably, a viral vector is an adenovirus vector, e.g., a recombinant adenovirus vector. A recombinant adenovirus vector can for instance be derived from a human adenovirus (HAdV, or AdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV) or rhesus adenovirus (rhAd). Preferably, an adenovirus vector is a recombinant human adenovirus vector, for instance a recombinant human adenovirus serotype 26, or any one of recombinant human adenovirus serotype 5, 4, 35, 7, 48, etc. In other embodiments, an adenovirus vector is a rhAd vector, e.g. rhAd51, rhAd52 or rhAd53. A recombinant viral vector useful for the application can be prepared using methods known in the art in view of the present disclosure. For example, in view of the degeneracy of the genetic code, several nucleic acid sequences can be designed that encode the same polypeptide. A polynucleotide encoding an HBV antigen of the application can optionally be codon-optimized to ensure proper expression in the host cell (e.g., bacterial or mammalian cells). Codon-optimization is a technology widely applied in the art, and methods for obtaining codon-optimized polynucleotides will be well known to those skilled in the art in view of the present disclosure.
A vector of the application, e.g., a DNA plasmid or a viral vector (particularly an adenoviral vector), can comprise any regulatory elements to establish conventional function(s) of the vector, including but not limited to replication and expression of the HBV antigen(s) encoded by the polynucleotide sequence of the vector. Regulatory elements include, but are not limited to, a promoter, an enhancer, a polyadenylation signal, translation stop codon, a ribosome binding element, a transcription terminator, selection markers, origin of replication, etc. A vector can comprise one or more expression cassettes. An“expression cassette” is part of a vector that directs the cellular machinery to make RNA and protein. An expression cassette typically comprises three components: a promoter sequence, an open reading frame, and a 3’-untranslated region (UTR) optionally comprising a polyadenylation signal. An open reading frame (ORF) is a reading frame that contains a coding sequence of a protein of interest (e.g., HBV antigen) from a start codon to a stop codon. Regulatory elements of the expression cassette can be operably linked to a polynucleotide sequence encoding an HBV antigen of interest. As used herein, the term“operably linked” is to be taken in its broadest reasonable context, and refers to a linkage of polynucleotide elements in a functional relationship. A polynucleotide is“operably linked” when it is placed into a functional relationship with another polynucleotide. For instance, a promoter is operably linked to a coding sequence if it affects the transcription of the coding sequence. Any components suitable for use in an expression cassette described herein can be used in any combination and in any order to prepare vectors of the application.
A vector can comprise a promoter sequence, preferably within an expression cassette, to control expression of an HBV antigen of interest. The term "promoter" is used in its conventional sense, and refers to a nucleotide sequence that initiates the transcription of an operably linked nucleotide sequence. A promoter is located on the same strand near the nucleotide sequence it transcribes. Promoters can be a constitutive, inducible, or repressible. Promoters can be naturally occurring or synthetic. A promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter can be a homologous promoter (i.e., derived from the same genetic source as the vector) or a heterologous promoter (i.e., derived from a different vector or genetic source). For example, if the vector to be employed is a DNA plasmid, the promoter can be endogenous to the plasmid (homologous) or derived from other sources (heterologous). Preferably, the promoter is located upstream of the polynucleotide encoding an HBV antigen within an expression cassette.
Examples of promoters that can be used include, but are not limited to, a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter (CMV-IE), Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. A promoter can also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metalothionein. A promoter can also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic.
Preferably, a promoter is a strong eukaryotic promoter, preferably a cytomegalovirus immediate early (CMV-IE) promoter. A nucleotide sequence of an exemplary CMV-IE promoter is shown in SEQ ID NO: 18 or SEQ ID NO: 19.
A vector can comprise additional polynucleotide sequences that stabilize the expressed transcript, enhance nuclear export of the RNA transcript, and/or improve transcriptional-translational coupling. Examples of such sequences include polyadenylation signals and enhancer sequences. A polyadenylation signal is typically located downstream of the coding sequence for a protein of interest (e.g., an HBV antigen) within an expression cassette of the vector. Enhancer sequences are regulatory DNA sequences that, when bound by transcription factors, enhance the transcription of an associated gene. An enhancer sequence is preferably located upstream of the polynucleotide sequence encoding an HBV antigen, but downstream of a promoter seque ce within an expression cassette of the vector. Any polyadenylation signal known to those skilled in the art in view of the present disclosure can be used. For example, the polyadenylation signal can be a SV40
polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human b- globin polyadenylation signal. Preferably, a polyadenylation signal is a bovine growth hormone (bGH) polyadenylation signal or a SV40 polyadenylation signal. A nucleotide sequence of an exemplary bGH polyadenylation signal is shown in SEQ ID NO: 20. A nucleotide sequence of an exemplary SV40 polyadenylation signal is shown in SEQ ID NO: 13.
Any enhancer sequence known to those skilled in the art in view of the present disclosure can be used. For example, an enhancer sequence can be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer, such as one from CMV, HA, RSV, or EBV. Examples of particular enhancers include, but are not limited to, Woodchuck HBV Post-transcriptional regulatory element (WPRE), intron/exon sequence derived from human apolipoprotein A1 precursor (ApoAI), untranslated R-U5 domain of the human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR), a splicing enhancer, a synthetic rabbit b-globin intron, or any combination thereof. Preferably, an enhancer sequence is a composite sequence of three consecutive elements of the untranslated R-U5 domain of HTLV-1 LTR, rabbit b-globin intron, and a splicing enhancer, which is referred to herein as“a triple enhancer sequence.” A nucleotide sequence of an exemplary triple enhancer sequence is shown in SEQ ID NO: 10. Another exemplary enhancer sequence is an ApoAI gene fragment shown in SEQ ID NO: 12.
A vector can comprise a polynucleotide sequence encoding a signal peptide sequence. Preferably, the polynucleotide sequence encoding the signal peptide sequence is located upstream of the polynucleotide sequence encoding an HBV antigen. Signal peptides typically direct localization of a protein, facilitate secretion of the protein from the cell in which it is produced, and/or improve antigen expression and cross-presentation to antigen- presenting cells. A signal peptide can be present at the N-terminus of an HBV antigen when expressed from the vector, but is cleaved off by signal peptidase, e.g., upon secretion from the cell. An expressed protein in which a signal peptide has been cleaved is often referred to as the“mature protein.” Any signal peptide known in the art in view of the present disclosure can be used. For example, a signal peptide can be a cystatin S signal peptide; an immunoglobulin (Ig) secretion signal, such as the Ig heavy chain gamma signal peptide SPIgG or the Ig heavy chain epsilon signal peptide SPIgE.
Preferably, a signal peptide sequence is a cystatin S signal peptide. Exemplary nucleic acid and amino acid sequences of a cystatin S signal peptide are shown in SEQ ID NOs: 8 and 9, respectively. Exemplary nucleic acid and amino acid sequences of an immunoglobulin secretion signal are shown in SEQ ID NOs: 14 and 15, respectively.
A vector, such as a DNA plasmid, can also include a bacterial origin of replication and an antibiotic resistance expression cassette for selection and maintenance of the plasmid in bacterial cells, e.g., E. coli. Bacterial origins of replication and antibiotic resistance cassettes can be located in a vector in the same orientation as the expression cassette encoding an HBV antigen, or in the opposite (reverse) orientation. An origin of replication (ORI) is a sequence at which replication is initiated, enabling a plasmid to reproduce and survive within cells. Examples of ORIs suitable for use in the application include, but are not limited to ColE1, pMB1, pUC, pSC101, R6K, and 15A, preferably pUC. An exemplary nucleotide sequence of a pUC ORI is shown in SEQ ID NO: 21.
Expression cassettes for selection and maintenance in bacterial cells typically include a promoter sequence operably linked to an antibiotic resistance gene. Preferably, the promoter sequence operably linked to an antibiotic resistance gene differs from the promoter sequence operably linked to a polynucleotide sequence encoding a protein of interest, e.g., HBV antigen. The antibiotic resistance gene can be codon optimized, and the sequence composition of the antibiotic resistance gene is normally adjusted to bacterial, e.g., E. coli, codon usage. Any antibiotic resistance gene known to those skilled in the art in view of the present disclosure can be used, including, but not limited to, kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr), and tetracycline resistance gene (Tetr), as well as genes conferring resistance to chloramphenicol, bleomycin, spectinomycin, carbenicillin, etc.
Preferably, an antibiotic resistance gene in the antibiotic expression cassette of a vector is a kanamycin resistance gene (Kanr). The sequence of Kanr gene is shown in SEQ ID NO: 22. Preferably, the Kanr gene is codon optimized. An exemplary nucleic acid sequence of a codon optimized Kanr gene is shown in SEQ ID NO: 23. The Kanr can be operably linked to its native promoter, or the Kanr gene can be linked to a heterologous promoter. In a particular embodiment, the Kanr gene is operably linked to the ampicillin resistance gene (Ampr) promoter, known as the bla promoter. An exemplary nucleotide sequence of a bla promoter is shown in SEQ ID NO: 24. In a particular embodiment of the application, a vector is a DNA plasmid comprising an expression cassette including a polynucleotide encoding at least one of an HBV antigen selected from the group consisting of an HBV pol antigen comprising an amino acid sequence at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96, 97%, preferably at least 98%, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%, identical to SEQ ID NO: 7, and a truncated HBV core antigen consisting of the amino acid sequence at least 95%, such as 95%, 96, 97%, preferably at least 98%, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%, identical of SEQ ID NO: 2 or SEQ ID NO: 4; an upstream sequence operably linked to the polynucleotide encoding the HBV antigen comprising, from 5’ end to 3’ end, a promoter sequence, preferably a CMV promoter sequence of SEQ ID NO: 18, an enhancer sequence, preferably a triple enhancer sequence of SEQ ID NO: 10, and a polynucleotide sequence encoding a signal peptide sequence, preferably a cystatin S signal peptide having the amino acid sequence of SEQ ID NO: 9; and a downstream sequence operably linked to the polynucleotide encoding the HBV antigen comprising a
polyadenylation signal, preferably a bGH polyadenylation signal of SEQ ID NO: 20. Such vector further comprises an antibiotic resistance expression cassette including a
polynucleotide encoding an antibiotic resistance gene, preferably a Kanr gene, more preferably a codon optimized Kanr gene of at least 90% identical to SEQ ID NO: 23, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 23, preferably 100% identical to SEQ ID NO: 23, operably linked to an Ampr (bla) promoter of SEQ ID NO: 24, upstream of and operably linked to the polynucleotide encoding the antibiotic resistance gene; and an origin of replication, preferably a pUC ori of SEQ ID NO: 21. Preferably, the antibiotic resistance cassette and the origin of replication are present in the plasmid in the reverse orientation relative to the HBV antigen expression cassette.
In another particular embodiment of the application, a vector is a viral vector, preferably an adenoviral vector, more preferably an Ad26 or Ad35 vector, comprising an expression cassette including a polynucleotide encoding at least one of an HBV antigen selected from the group consisting of an HBV pol antigen comprising an amino acid sequence at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96, 97%, preferably at least 98%, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%, identical to SEQ ID NO: 7, and a truncated HBV core antigen consisting of the amino acid sequence at least 95%, such as 95%, 96, 97%, preferably at least 98%, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%, identical of SEQ ID NO: 2 or SEQ ID NO: 4; an upstream sequence operably linked to the polynucleotide encoding the HBV antigen comprising, from 5’ end to 3’ end, a promoter sequence, preferably a CMV promoter sequence of SEQ ID NO: 19, an enhancer sequence, preferably an ApoAI gene fragment sequence of SEQ ID NO: 12, and a polynucleotide sequence encoding a signal peptide sequence, preferably an immunoglobulin secretion signal having the amino acid sequence of SEQ ID NO: 15; and a downstream sequence operably linked to the polynucleotide encoding the HBV antigen comprising a polyadenylation signal, preferably a SV40 polyadenylation signal of SEQ ID NO: 13.
In an embodiment of the application, a vector, such as a plasmid DNA vector or a viral vector (preferably an adenoviral vector, more preferably an Ad26 or Ad35 vector), encodes an HBV Pol antigen having the amino acid sequence of SEQ ID NO: 7. Preferably, the vector comprises a coding sequence for the HBV Pol antigen that is at least 90% identical to the polynucleotide sequence of SEQ ID NO: 5 or 6, such as 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 5 or 6, preferably 100% identical to SEQ ID NO: 5 or 6.
In an embodiment of the application, a vector, such as a plasmid DNA vector or a viral vector (preferably an adenoviral vector, more preferably an Ad26 or Ad35 vector), encodes a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Preferably, the vector comprises a coding sequence for the truncated HBV core antigen that is at least 90% identical to the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, such as 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3, preferably 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3.
In yet another embodiment of the application, a vector, such as a plasmid DNA vector or a viral vector (preferably an adenoviral vector, more preferably an Ad26 or Ad35 vector), encodes a fusion protein comprising an HBV Pol antigen having the amino acid sequence of SEQ ID NO: 7 and a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Preferably, the vector comprises a coding sequence for the fusion, which contains a coding sequence for the truncated HBV core antigen at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 3, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3, preferably 98%, 99% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3, more preferably SEQ ID NO: 1 or SEQ ID NO: 3, operably linked to a coding sequence for the HBV Pol antigen at least 90% identical to SEQ ID NO: 5 or SEQ ID NO: 6, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 5 or SEQ ID NO: 6, preferably 98%, 99% or 100% identical to SEQ ID NO: 5 or SEQ ID NO: 6, more preferably SEQ ID NO: 5 or SEQ ID NO: 6. Preferably, the coding sequence for the truncated HBV core antigen is operably linked to the coding sequence for the HBV Pol antigen via a coding sequence for a linker at least 90% identical to SEQ ID NO: 11, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 11, preferably 98%, 99% or 100% identical to SEQ ID NO: 11. In particular embodiments of the application, a vector comprises a coding sequence for the fusion having SEQ ID NO: 1 or SEQ ID NO: 3 operably linked to SEQ ID NO: 11, which is further operably linked to SEQ ID NO: 5 or SEQ ID NO: 6.
The polynucleotides and expression vectors encoding the HBV antigens of the application can be made by any method known in the art in view of the present disclosure. For example, a polynucleotide encoding an HBV antigen can be introduced or“cloned” into an expression vector using standard molecular biology techniques, e.g., polymerase chain reaction (PCR), etc., which are well known to those skilled in the art.
Cells, Polypeptides and Antibodies
The application also provides cells, preferably isolated cells, comprising any of the polynucleotides and vectors described herein. The cells can, for instance, be used for recombinant protein production, or for the production of viral particles.
Embodiments of the application thus also relate to a method of making an HBV antigen of the application. The method comprises transfecting a host cell with an expression vector comprising a polynucleotide encoding an HBV antigen of the application operably linked to a promoter, growing the transfected cell under conditions suitable for expression of the HBV antigen, and optionally purifying or isolating the HBV antigen expressed in the cell. The HBV antigen can be isolated or collected from the cell by any method known in the art including affinity chromatography, size exclusion chromatography, etc. Techniques used for recombinant protein expression will be well known to one of ordinary skill in the art in view of the present disclosure. The expressed HBV antigens can also be studied without purifying or isolating the expressed protein, e.g., by analyzing the supernatant of cells transfected with an expression vector encoding the HBV antigen and grown under conditions suitable for expression of the HBV antigen.
Thus, also provided are non-naturally occurring or recombinant polypeptides comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 7. As described above and below, isolated nucleic acid molecules encoding these sequences, vectors comprising these sequences operably linked to a promoter, and compositions comprising the polypeptide, polynucleotide, or vector are also contemplated by the application.
In an embodiment of the application, a recombinant polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2, such as 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 2. Preferably, a non-naturally occurring or recombinant polypeptide consists of SEQ ID NO: 2.
In another embodiment of the application, a non-naturally occurring or recombinant polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4, such as 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 4. Preferably, a non-naturally occurring or recombinant polypeptide comprises SEQ ID NO: 4.
In another embodiment of the application, a non-naturally occurring or recombinant polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7, such as 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 7. Preferably, a non-naturally occurring or recombinant polypeptide consists of SEQ ID NO: 7. Also provided are antibodies or antigen binding fragments thereof that specifically bind to a non-naturally occurring polypeptide of the application. In an embodiment of the application, an antibody specific to a non-naturally HBV antigen of the application does not bind specifically to another HBV antigen. For example, an antibody of the application that binds specifically to an HBV Pol antigen having the amino acid sequence of SEQ ID NO: 7 will not bind specifically to an HBV Pol antigen not having the amino acid sequence of SEQ ID NO: 7.
As used herein, the term“antibody” includes polyclonal, monoclonal, chimeric, humanized, Fv, Fab and F(ab¢)2; bifunctional hybrid (e.g., Lanzavecchia et al., Eur. J. Immunol.17:105, 1987), single-chain (Huston et al., Proc. Natl. Acad. Sci. USA 85:5879, 1988; Bird et al., Science 242:423, 1988); and antibodies with altered constant regions (e.g., U.S. Pat. No.5,624,821).
As used herein, an antibody that“specifically binds to” an antigen refers to an
-7
antibody that binds to the antigen with a KD of 1×10 M or less. Preferably, an antibody that “specifically binds to” an antigen binds to the antigen with a KD of 1×10-8 M or less, more preferably 5×10-9 M or less, 1×10-9 M or less, 5×10-10M or less, or 1×10-10 M or less. The term“KD” refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods in the art in view of the present disclosure. For example, the KD of an antibody can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a Biacore® system, or by using bio-layer interferometry technology, such as a Octet RED96 system.
The smaller the value of the KD of an antibody, the higher affinity that the antibody binds to a target antigen.
Capsid Assembly Modulators (CAMs)
The application relates to heteroaryldihydropyrimidine (HAP) derivatives. Background art on heteroaryldihydropyrimidines for use in the treatment of HBV includes WO
2015/13226, WO2013/102655 and WO99/54326, all of which are herein incorporated by reference in their entireties.
According to this application, heteroaryldihydropyrimidine (HAP) derivatives can function as capsid assembly modulators (CAMs). The compounds provided herein are believed to modulate or disrupt HBV assembly and other HBV core protein functions necessary for HBV replication or the generation of infectious particles and/or may disrupt HBV capsid assembly leading to empty capsids with greatly reduced infectivity or replication capacity.
The compounds provided herein have potent antiviral activity, exhibit favorable metabolic properties, tissue distribution, safety and pharmaceutical profiles, and are suitable for use in humans. Disclosed compounds may modulate (e.g., accelerate, delay, inhibit, disrupt or reduce) normal viral capsid assembly or disassembly, bind capsid or alter metabolism of cellular polyproteins and precursors. The modulation may occur when the capsid protein is mature, or during viral infectivity. Disclosed compounds can be used in methods of modulating the activity or properties of HBV cccDNA, or the generation or release of HBV RNA particles from within an infected cell.
The compounds described herein are suitable for monotherapy and are effective against natural or native HBV strains and against HBV strains resistant to currently known drugs. In addition, the compounds described herein are suitable for use in combination therapy.
According to embodiments of the application, a capsid assembly modulator (CAM) is a compound of Formula (I):
Figure imgf000038_0001
or a deuterated isomer, a stereoisomer or tautomeric form thereof, or a pharmaceutically acceptable salt thereof, wherein:
R1 is selected from the group consisting of phenyl, thiophenyl, pyridyl, and pyridonyl, optionally substituted with one or more substituents selected from the group consisting of C1- 4alkyl, halogen, and CN;
R2 is C1-4alkyl;
R3 is selected from the group consisting of thiazolyl, pyridyl, and oxazolyl, optionally substituted with one or more substituents selected from fluorine, and C1-6alkyl;
n is an integer of 0 or 1; R4 and R5 are independently selected from the group consisting of H and -COOH; (i.e., the bond between X and Y) is a single bond or a double bond; when X and Y are linked by a single bond, X is selected from the group consisting of C(=S), C(=NR6), C(=CHR7) and CHR8, and Y is NR9;
when X and Y are linked by a double bond, X is C-SR9 or C-OR9, and Y is N atom; Z is selected from the group consisting of CH2 and C(=O);
R6 is selected from the group consisting of CN, C(=O)CH3, and SO2CH3;
R7 is CN;
R8 is CF3;
R9 is selected from the group consisting of H, -C1-6alkyl, -C1-6alkyl-R10, -C1-6alkoxy- C1-6alkyl-R10 and -(CH2)p-Q-R10;
p is an integer of 0, 1, 2, or 3;
Q is selected from the group consisting of aryl, heteroaryl, and a 3- to 7- membered saturated ring, optionally containing a heteroatom, the heteroatom being an oxygen or a nitrogen, the nitrogen being substituted with H, -C1-6alkyl, -C1-6alkoxy-C1-6alkyl and -C1- 6alkylcarbonyl;
R10 is selected from -COOH, -C(=O)NHS(=O)2-C1-6alkyl, tetrazolyl and carboxylic acid bioisosteres.
As used herein, the term“capsid assembly modulator” refers to a compound that disrupts or accelerates or inhibits or hinders or delays or reduces or modifies normal capsid assembly (e.g., during maturation) or normal capsid disassembly (e.g., during infectivity) or perturbs capsid stability, thereby inducing aberrant capsid morphology and function. In an embodiment, a capsid assembly modulator accelerates capsid assembly or disassembly, thereby inducing aberrant capsid morphology. In another embodiment, a capsid assembly modulator interacts (e.g. binds at an active site, binds at an allosteric site, modifies or hinders folding and the like) with the major capsid assembly protein (CA), thereby disrupting capsid assembly or disassembly. In yet another embodiment, a capsid assembly modulator causes a perturbation in structure or function of CA (e.g., ability of CA to assemble, disassemble, bind to a substrate, fold into a suitable conformation, or the like), which attenuates viral infectivity or is lethal to the virus.
As used herein, the term“pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the coumpound, and is relatively non-toxic, i.e., the material may be adminitered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
As used herein, the term“pharmaceutically acceptable salt” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present invention include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.
As used herein, the term“alkyl,” by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e., C1-C3alkyl means an alkyl having one to three carbon atoms, C1- C4alkyl means an alkyl having one to four carbon) and includes straight and branched chains. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl. Embodiments of alkyl generally include, but are not limited to, C1-C10 alkyl, such as C1-C6 alkyl, such as C1-C4 alkyl.
As used herein, the term“alkenyl,” by itself or as part of another substituent means, unless otherwise stated, a linear or branched chain of hydrocarbons comprising at least one carbon to carbon double bond, having the number of carbon atoms designated (i.e., C2-C4 alkenyl or C2-4alkenyl means an alkenyl having two to four to eight carbon atoms. C4-C8 alkenyl or C4-8alkenyl means an alkenyl having four carbon atoms. Embodiments of alkenyl generally include, but are not limited to, C2-C6 alkenyl, such as C2-C4 alkenyl, such as C2-C3 alkenyl. As used herein, the term“halo” or“halogen” alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine, more preferably, fluorine or chlorine.
As used herein, the term“3-7 membered saturated ring” refers to a mono cyclic non- aromatic saturated radical, wherein each of the atoms forming the ring (i.e., skeletal atoms) is a carbon atom, unless such ring contains one or more heteroatoms if so further defined. 3-7 Membered saturated rings include groups having 3 to 7 ring atoms. Monocyclic 3-7 membered saturated rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl.
As used herein, a 3-7 membered saturated ring may optionally contain a heteroatom, said heteroatom being an oxygen, or a nitrogen substituted with H, C1-6alkyl, or C1-6alkoxy-C1- 6alkyl.
As used herein, the term“aromatic” refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, i.e., having (4n + 2) delocalized p (pi) electrons, where n is an integer.
As used herein, the term“aryl,” employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two, or three rings), wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples of aryl groups include phenyl, anthracyl, and naphthyl. Preferred examples are phenyl (e.g., C6-aryl) and biphenyl (e.g., C12-aryl). In some embodiments, aryl groups have from six to sixteen carbon atoms. In some embodiments, aryl groups have from six to twelve carbon atoms (e.g., C6-C12-aryl). In some embodiments, aryl groups have six carbon atoms (e.g., C6-aryl).
As used herein, the term“heteroaryl” or“heteroaromatic” refers to a heterocycle having aromatic character. Heteroaryl substituents may be defined by the number of carbon atoms, e.g., C1-C9-heteroaryl indicates the number of carbon atoms contained in the heteroaryl group without including the number of heteroatoms. For example, a C1-C9- heteroaryl will include an additional one to four heteroatoms. A polycyclic heteroaryl may include one or more rings that are partially saturated. Non-limiting examples of heteroaryls include pyridyl, pyrazinyl, pyrimidinyl (including, e.g., 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (including, e.g., 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (including, e.g., 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.
Non-limiting examples of polycyclic heterocycles and heteroaryls include indolyl (including, e.g., 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (including, e.g., 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (including, e.g., 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (including, e.g., 3-, 4-, 5-, 6- and 7-benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (including, e.g., 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (including, e.g., 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl (including, e.g., 2-benzimidazolyl), benzotriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.
As used herein, the term“substituted” means that an atom or group of atoms has replaced hydrogen as the substituent attached to another group.
As used herein, the terminology“selected from…” (e.g.,“R4 is selected from A, B and C”) is understood to be equivalent to the terminology“selected from the group consisting of…” (e.g.,“R4 is selected from the group consisting of A, B and C”).
An embodiment relates to a compound of Formula I as defined herein wherein the carboxylic acid bioisosteres are -S(=O)2(OH), -P(=O)(OH)2, -C(=O)NHOH, -C(=O)NHCN, 1,2,4-oxadiazol-5(4H)-one, or 3-hydroxy-4-methylcyclobut-3-ene-1,2-dione. This refers to the following structures:
Figure imgf000042_0001
. An embodiment relates to a compound of Formula I as defined herein, wherein R1 is phenyl substituted with one or more substituents selected from halogens and C1-6alkyl.
An embodiment relates to a compound of Formula I as defined herein, wherein R2 is methyl or ethyl.
An embodiment relates to a compound of Formula I as defined herein, wherein R3 is thiazolyl.
An embodiment relates to a compound of Formula I as defined herein, wherein R4 and R5 are H.
An embodiment relates to a compound of Formula I as defined herein, wherein X is C(=S). An embodiment relates to a compound of Formula I as defined herein, wherein Z is CH2.
An embodiment relates to a compound of Formula I as defined herein, wherein R9 is C1-6alkyl-CO2H or (CH2)p- -R10 .
An embodiment relates to a compound of Formula I as defined herein, wherein Q is phenyl, or wherein Q is a C3-6cycloalkyl, or wherein Q is a 3- to 6- saturated membered ring containing an oxygen.
An embodiment relates to a compound selected from the group consisting of compound satisfying the following formulae:
Figure imgf000043_0001
Figure imgf000044_0001
42
Figure imgf000045_0001
43
Figure imgf000046_0001
An embodiment relates to a compound selected from the group consisting of compound satisfying the following formulae:
Figure imgf000047_0001

Figure imgf000048_0001

Figure imgf000049_0001

Figure imgf000050_0002
In a preferred embodiment, the compound of Formula (I) is compound 1A:
Figure imgf000050_0001
The disclosed compounds may possess one or more stereocenters, and each stereocenter may exist independently in either the R or S configuration. For some compounds, the stereochemical configuration at indicated centers has been assigned as“R*”, “S*” when the absolute stereochemistry is undetermined although the compound itself has been isolated as a single stereoisomer and is enantiomerically/diastereomerically pure. In an embodiment, compounds described herein are present in optically active or racemic forms. It is to be understood that the compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein.
Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. In an embodiment, a mixture of one or more isomer is utilized as the disclosed compound described herein. In another embodiment, compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis or separation of a mixture of enantiomers or diastereomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.
When the absolute R or S stereochemistry of a compound cannot be determined, it can be identified by the retention time after chromatography under particular chromatographic conditions as determined by chromatography column, eluent etc.
In an embodiment, the disclosed compounds may exist as tautomers. All tautomers are included within the scope of the compounds presented herein.
Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include and are not limited to 2H, 3H, 11C, 13C, 14C, 36Cl, 18F, 123I, 125I, 13N, 15N, 15O, 17O, 18O, 32P, and 35S. In an embodiment, isotopically-labeled compounds are useful in drug or substrate tissue distribution studies. In another embodiment, substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements).
In yet another embodiment, substitution with positron emitting isotopes, such as 11C, 18F, 15O and 13N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non- labeled reagent otherwise employed.
In an embodiment, the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.
The compounds described herein, and other related compounds having different substituents are synthesized using techniques and materials described herein and techniques known to a person skilled in the art. General methods for the preparation of compound as described herein are modified by the use of appropriate reagents and conditions, for the introduction of the various moieties found in the formula as provided herein.
Compounds of Formula (I) are synthesized using any suitable procedures starting from compounds that are available from commercial sources or are prepared using procedures described herein.
The application also relates to intermediate compounds for preparation of compounds of Formula (I), such as
Figure imgf000052_0001
Figure imgf000053_0001
51
Figure imgf000054_0001
Compositions, Therapeutic Combinations, and Vaccines
The application also relates to compositions, therapeutic combinations, more particularly kits, and vaccines comprising one or more HBV antigens, polynucleotides, and/or vectors encoding one or more HBV antigens according to the application. Any of the HBV antigens, polynucleotides (including RNA and DNA), and/or vectors of the application described herein can be used in the compositions, therapeutic combinations or kits, and vaccines of the application.
In an embodiment of the application, a composition comprises an isolated or non- naturally occurring nucleic acid molecule (DNA or RNA) comprising polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, or an HBV polymerase antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, a vector comprising the isolated or non-naturally occurring nucleic acid molecule, and/or an isolated or non-naturally occurring polypeptide encoded by the isolated or non-naturally occurring nucleic acid molecule.
In an embodiment of the application, a composition comprises an isolated or non- naturally occurring nucleic acid molecule (DNA or RNA) comprising a polynucleotide sequence encoding an HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, preferably 100% identical to SEQ ID NO: 7.
In an embodiment of the application, a composition comprises an isolated or non- naturally occurring nucleic acid molecule (DNA or RNA) encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4.
In an embodiment of the application, a composition comprises an isolated or non- naturally occurring nucleic acid molecule (DNA or RNA) comprising a polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4; and an isolated or non-naturally occurring nucleic acid molecule (DNA or RNA) comprising a polynucleotide sequence encoding an HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, preferably 100% identical to SEQ ID NO: 7. The coding sequences for the truncated HBV core antigen and the HBV Pol antigen can be present in the same isolated or non-naturally occurring nucleic acid molecule (DNA or RNA), or in two different isolated or non-naturally occurring nucleic acid molecules (DNA or RNA).
In an embodiment of the application, a composition comprises a vector, preferably a DNA plasmid or a viral vector (such as an adenoviral vector) comprising a polynucleotide encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4.
In an embodiment of the application, a composition comprises a vector, preferably a DNA plasmid or a viral vector (such as an adenoviral vector), comprising a polynucleotide encoding an HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, preferably 100% identical to SEQ ID NO: 7.
In an embodiment of the application, a composition comprises a vector, preferably a DNA plasmid or a viral vector (such as an adenoviral vector), comprising a polynucleotide encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4; and a vector, preferably a DNA plasmid or a viral vector (such as an adenoviral vector), comprising a polynucleotide encoding an HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, preferably 100% identical to SEQ ID NO: 7. The vector comprising the coding sequence for the truncated HBV core antigen and the vector comprising the coding sequence for the HBV Pol antigen can be the same vector, or two different vectors.
In an embodiment of the application, a composition comprises a vector, preferably a DNA plasmid or a viral vector (such as an adenoviral vector), comprising a polynucleotide encoding a fusion protein comprising a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4, operably linked to an HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, preferably 100% identical to SEQ ID NO: 7, or vice versa. Preferably, the fusion protein further comprises a linker that operably links the truncated HBV core antigen to the HBV Pol antigen, or vice versa. Preferably, the linker has the amino acid sequence of (AlaGly)n, wherein n is an integer of 2 to 5.
In an embodiment of the application, a composition comprises an isolated or non- naturally occurring truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4.
In an embodiment of the application, a composition comprises an isolated or non- naturally occurring HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, preferably 100% identical to SEQ ID NO: 7.
In an embodiment of the application, a composition comprises an isolated or non- naturally occurring truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4; and an isolated or non-naturally occurring HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, preferably 100% identical to SEQ ID NO: 7.
In an embodiment of the application, a composition comprises an isolated or non- naturally occurring fusion protein comprising a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 14, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4, operably linked to an HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, preferably 100% identical to SEQ ID NO: 7, or vice versa. Preferably, the fusion protein further comprises a linker that operably links the truncated HBV core antigen to the HBV Pol antigen, or vice versa. Preferably, the linker has the amino acid sequence of (AlaGly)n, wherein n is an integer of 2 to 5.
The application also relates to a therapeutic combination or a kit comprising polynucleotides expressing a truncated HBV core antigen and an HBV pol antigen according to embodiments of the application. Any polynucleotides and/or vectors encoding HBV core and pol antigens of the application described herein can be used in the therapeutic combinations or kits of the application.
According to embodiments of the application, a therapeutic combination or kit for use in treating an HBV infection in a subject in need thereof, comprises: i) at least one of:
a) a truncated HBV core antigen consisting of an amino acid sequence that is at least 95% identical to SEQ ID NO: 2, and
b) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding the truncated HBV core antigen
c) an HBV polymerase antigen having an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity, and
d) a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide sequence encoding the HBV polymerase antigen; and
ii) a compound of Formula (I):
Figure imgf000057_0001
or a deuterated isomer, a stereoisomer or tautomeric form thereof, or a pharmaceutically acceptable salt thereof, wherein:
R1 is selected from the group consisting of phenyl, thiophenyl, pyridyl, and pyridonyl, optionally substituted with one or more substituents selected from the group consisting of C1- 4alkyl, halogen, and CN;
R2 is C1-4alkyl;
R3 is selected from the group consisting of thiazolyl, pyridyl, and oxazolyl, optionally substituted with one or more substituents selected from fluorine, and C1-6alkyl;
n is an integer of 0 or 1;
R4 and R5 are independently selected from the group consisting of H and -COOH;
Figure imgf000057_0002
the bond between X and Y) is a single bond or a double bond;
when X and Y are linked by a single bond, X is selected from the group consisting of C(=S), C(=NR6), C(=CHR7) and CHR8, and Y is NR9;
when X and Y are linked by a double bond, X is C-SR9 or C-OR9, and Y is N atom; Z is selected from the group consisting of CH2 and C(=O);
R6 is selected from the group consisting of CN, C(=O)CH3, and SO2CH3;
R7 is CN;
R8 is CF3;
R9 is selected from the group consisting of H, -C1-6alkyl, -C1-6alkyl-R10, -C1-6alkoxy- C1-6alkyl-R10 and -(CH2)p-Q-R10;
p is an integer of 0, 1, 2, or 3;
Q is selected from the group consisting of aryl, heteroaryl, and a 3- to 7- membered saturated ring, optionally containing a heteroatom, the heteroatom being an oxygen or a nitrogen, the nitrogen being substituted with H, -C1-6alkyl, -C1-6alkoxy-C1-6alkyl and -C1- 6alkylcarbonyl;
R10 is selected from -COOH, -C(=O)NHS(=O)2-C1-6alkyl, tetrazolyl and carboxylic acid bioisosteres. In a particular embodiment of the application, a therapeutic combination or kit comprises: i) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 95% identical to SEQ ID NO: 2; ii) a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide sequence encoding an HBV polymerase antigen having an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity; and iii) compound 1A:
Figure imgf000058_0001
or a deuterated isomer, a stereoisomer or tautomeric form thereof, or a pharmaceutically acceptable salt thereof. According to embodiments of the application, the polynucleotides in a vaccine combination or kit can be linked or separate, such that the HBV antigens expressed from such polynucleotides are fused together or produced as separate proteins, whether expressed from the same or different polynucleotides. In an embodiment, the first and second polynucleotides are present in separate vectors, e.g., DNA plasmids or viral vectors, used in combination either in the same or separate compositions, such that the expressed proteins are also separate proteins, but used in combination. In another embodiment, the HBV antigens encoded by the first and second polynucleotides can be expressed from the same vector, such that an HBV core-pol fusion antigen is produced. Optionally, the core and pol antigens can be joined or fused together by a short linker. Alternatively, the HBV antigens encoded by the first and second polynucleotides can be expressed independently from a single vector using a using a ribosomal slippage site (also known as cis-hydrolase site) between the core and pol antigen coding sequences. This strategy results in a bicistronic expression vector in which individual core and pol antigens are produced from a single mRNA transcript. The core and pol antigens produced from such a bicistronic expression vector can have additional N or C-terminal residues, depending upon the ordering of the coding sequences on the mRNA transcript. Examples of ribosomal slippage sites that can be used for this purpose include, but are not limited to, the FA2 slippage site from foot-and-mouth disease virus (FMDV). Another possibility is that the HBV antigens encoded by the first and second polynucleotides can be expressed independently from two separate vectors, one encoding the HBV core antigen and one encoding the HBV pol antigen.
In a preferred embodiment, the first and second polynucleotides are present in separate vectors, e.g., DNA plasmids or viral vectors. Preferably, the separate vectors are present in the same composition.
According to preferred embodiments of the application, a therapeutic combination or kit comprises a first polynucleotide present in a first vector, a second polynucleotide present in a second vector. The first and second vectors can be the same or different. Preferably the vectors are DNA plasmids.
In a particular embodiment of the application, the first vector is a first DNA plasmid, the second vector is a second DNA plasmid. Each of the first and second DNA plasmids comprises an origin of replication, preferably pUC ORI of SEQ ID NO: 21, and an antibiotic resistance cassette, preferably comprising a codon optimized Kanr gene having a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 23, preferably under control of a bla promoter, for instance the bla promoter shown in SEQ ID NO: 24. Each of the first and second DNA plasmids independently further comprises at least one of a promoter sequence, enhancer sequence, and a polynucleotide sequence encoding a signal peptide sequence operably linked to the first polynucleotide sequence or the second polynucleotide sequence. Preferably, each of the first and second DNA plasmids comprises an upstream sequence operably linked to the first polynucleotide or the second polynucleotide, wherein the upstream sequence comprises, from 5’ end to 3’ end, a promoter sequence of SEQ ID NO: 18 or 19, an enhancer sequence, and a polynucleotide sequence encoding a signal peptide sequence having the amino acid sequence of SEQ ID NO: 9 or 15. Each of the first and second DNA plasmids can also comprise a polyadenylation signal located downstream of the coding sequence of the HBV antigen, such as the bGH polyadenylation signal of SEQ ID NO: 20.
In one particular embodiment of the application, the first vector is a viral vector and the second vector is a viral vector. Preferably, each of the viral vectors is an adenoviral vector, more preferably an Ad26 or Ad35 vector, comprising an expression cassette including the polynucleotide encoding an HBV pol antigen or an truncated HBV core antigen of the application; an upstream sequence operably linked to the polynucleotide encoding the HBV antigen comprising, from 5’ end to 3’ end, a promoter sequence, preferably a CMV promoter sequence of SEQ ID NO: 19, an enhancer sequence, preferably an ApoAI gene fragment sequence of SEQ ID NO: 12, and a polynucleotide sequence encoding a signal peptide sequence, preferably an immunoglobulin secretion signal having the amino acid sequence of SEQ ID NO: 15; and a downstream sequence operably linked to the polynucleotide encoding the HBV antigen comprising a polyadenylation signal, preferably a SV40 polyadenylation signal of SEQ ID NO: 13.
In another preferred embodiment, the first and second polynucleotides are present in a single vector, e.g., DNA plasmid or viral vector. Preferably, the single vector is an adenoviral vector, more preferably an Ad26 vector, comprising an expression cassette including a polynucleotide encoding an HBV pol antigen and a truncated HBV core antigen of the application, preferably encoding an HBV pol antigen and a truncated HBV core antigen of the application as a fusion protein; an upstream sequence operably linked to the polynucleotide encoding the HBV pol and truncated core antigens comprising, from 5’ end to 3’ end, a promoter sequence, preferably a CMV promoter sequence of SEQ ID NO: 19, an enhancer sequence, preferably an ApoAI gene fragment sequence of SEQ ID NO: 12, and a polynucleotide sequence encoding a signal peptide sequence, preferably an immunoglobulin secretion signal having the amino acid sequence of SEQ ID NO: 15; and a downstream sequence operably linked to the polynucleotide encoding the HBV antigen comprising a polyadenylation signal, preferably a SV40 polyadenylation signal of SEQ ID NO: 13.
When a therapeutic combination of the application comprises a first vector, such as a DNA plasmid or viral vector, and a second vector, such as a DNA plasmid or viral vector, the amount of each of the first and second vectors is not particularly limited. For example, the first DNA plasmid and the second DNA plasmid can be present in a ratio of 10:1 to 1:10, by weight, such as 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10, by weight. Preferably, the first and second DNA plasmids are present in a ratio of 1:1, by weight. The therapeutic combination of the application can further comprise a third vector encoding a third active agent useful for treating an HBV infection.
Compositions and therapeutic combinations of the application can comprise additional polynucleotides or vectors encoding additional HBV antigens and/or additional HBV antigens or immunogenic fragments thereof, such as an HBsAg, an HBV L protein or HBV envelope protein, or a polynucleotide sequence encoding thereof. However, in particular embodiments, the compositions and therapeutic combinations of the application do not comprise certain antigens.
In a particular embodiment, a composition or therapeutic combination or kit of the application does not comprise a HBsAg or a polynucleotide sequence encoding the HBsAg.
In another particular embodiment, a composition or therapeutic combination or kit of the application does not comprise an HBV L protein or a polynucleotide sequence encoding the HBV L protein.
In yet another particular embodiment of the application, a composition or therapeutic combination of the application does not comprise an HBV envelope protein or a polynucleotide sequence encoding the HBV envelope protein.
Compositions and therapeutic combinations of the application can also comprise a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is non-toxic and should not interfere with the efficacy of the active ingredient. Pharmaceutically acceptable carriers can include one or more excipients such as binders, disintegrants, swelling agents, suspending agents, emulsifying agents, wetting agents, lubricants, flavorants, sweeteners, preservatives, dyes, solubilizers and coatings. Pharmaceutically acceptable carriers can include vehicles, such as lipid nanoparticles (LNPs). The precise nature of the carrier or other material can depend on the route of administration, e.g., intramuscular, intradermal, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., gut), intranasal or intraperitoneal routes. For liquid injectable preparations, for example, suspensions and solutions, suitable carriers and additives include water, glycols, oils, alcohols, preservatives, coloring agents and the like. For solid oral preparations, for example, powders, capsules, caplets, gelcaps and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. For nasal sprays/inhalant mixtures, the aqueous solution/suspension can comprise water, glycols, oils, emollients, stabilizers, wetting agents, preservatives, aromatics, flavors, and the like as suitable carriers and additives.
Compositions and therapeutic combinations of the application can be formulated in any matter suitable for administration to a subject to facilitate administration and improve efficacy, including, but not limited to, oral (enteral) administration and parenteral injections. The parenteral injections include intravenous injection or infusion, subcutaneous injection, intradermal injection, and intramuscular injection. Compositions of the application can also be formulated for other routes of administration including transmucosal, ocular, rectal, long acting implantation, sublingual administration, under the tongue, from oral mucosa bypassing the portal circulation, inhalation, or intranasal.
In a preferred embodiment of the application, compositions and therapeutic combinations of the application are formulated for parental injection, preferably subcutaneous, intradermal injection, or intramuscular injection, more preferably intramuscular injection.
According to embodiments of the application, compositions and therapeutic combinations for administration will typically comprise a buffered solution in a pharmaceutically acceptable carrier, e.g., an aqueous carrier such as buffered saline and the like, e.g., phosphate buffered saline (PBS). The compositions and therapeutic combinations can also contain pharmaceutically acceptable substances as required to approximate physiological conditions such as pH adjusting and buffering agents. For example, a composition or therapeutic combination of the application comprising plasmid DNA can contain phosphate buffered saline (PBS) as the pharmaceutically acceptable carrier. The plasmid DNA can be present in a concentration of, e.g., 0.5 mg/mL to 5 mg/mL, such as 0.5 mg/mL 1, mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, or 5 mg/mL, preferably at 1 mg/mL. Compositions and therapeutic combinations of the application can be formulated as a vaccine (also referred to as an“immunogenic composition”) according to methods well known in the art. Such compositions can include adjuvants to enhance immune responses. The optimal ratios of each component in the formulation can be determined by techniques well known to those skilled in the art in view of the present disclosure.
In a particular embodiment of the application, a composition or therapeutic combination is a DNA vaccine. DNA vaccines typically comprise bacterial plasmids containing a polynucleotide encoding an antigen of interest under control of a strong eukaryotic promoter. Once the plasmids are delivered to the cell cytoplasm of the host, the encoded antigen is produced and processed endogenously. The resulting antigen typically induces both humoral and cell-medicated immune responses. DNA vaccines are advantageous at least because they offer improved safety, are temperature stable, can be easily adapted to express antigenic variants, and are simple to produce. Any of the DNA plasmids of the application can be used to prepare such a DNA vaccine.
In other particular embodiments of the application, a composition or therapeutic combination is an RNA vaccine. RNA vaccines typically comprise at least one single- stranded RNA molecule encoding an antigen of interest, e.g., a fusion protein or HBV antigen according to the application. Once the RNA is delivered to the cell cytoplasm of the host, the encoded antigen is produced and processed endogenously, inducing both humoral and cell- mediated immune responses, similar to a DNA vaccine. The RNA sequence can be codon optimized to improve translation efficiency. The RNA molecule can be modified by any method known in the art in view of the present disclosure to enhance stability and/or translation, such by adding a polyA tail, e.g., of at least 30 adenosine residues; and/or capping the 5-end with a modified ribonucleotide, e.g., 7-methylguanosine cap, which can be incorporated during RNA synthesis or enzymatically engineered after RNA transcription. An RNA vaccine can also be self-replicating RNA vaccine developed from an alphavirus expression vector. Self-replicating RNA vaccines comprise a replicase RNA molecule derived from a virus belonging to the alphavirus family with a subgenomic promoter that controls replication of the fusion protein or HBV antigen RNA followed by an artificial poly A tail located downstream of the replicase.
In certain embodiments, a further adjuvant can be included in a composition or therapeutic combination of the application, or co-administered with a composition or therapeutic combination of the application. Use of another adjuvant is optional, and can further enhance immune responses when the composition is used for vaccination purposes. Other adjuvants suitable for co-administration or inclusion in compositions in accordance with the application should preferably be ones that are potentially safe, well tolerated and effective in humans. An adjuvant can be a small molecule or antibody including, but not limited to, immune checkpoint inhibitors (e.g., anti-PD1, anti-TIM-3, etc.), toll-like receptor agonists (e.g., TLR7 agonists and/or TLR8 agonists), RIG-1 agonists, IL-15 superagonists (Altor Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, and IL-7-hyFc. For example, adjuvants can e.g., be chosen from among the following anti-HBV agents: HBV DNA polymerase inhibitors;
Immunomodulators; Toll-like receptor 7 modulators; Toll-like receptor 8 modulators; Toll- like receptor 3 modulators; Interferon alpha receptor ligands; Hyaluronidase inhibitors; Modulators of IL-10; HBsAg inhibitors; Toll like receptor 9 modulators; Cyclophilin inhibitors; HBV Prophylactic vaccines; HBV Therapeutic vaccines; HBV viral entry inhibitors; Antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; short interfering RNAs (siRNA), more particularly anti-HBV siRNA; Endonuclease modulators; Inhibitors of ribonucleotide reductase; Hepatitis B virus E antigen inhibitors; HBV antibodies targeting the surface antigens of the hepatitis B virus; HBV antibodies; CCR2 chemokine antagonists; Thymosin agonists; Cytokines, such as IL12; Capsid Assembly Modulators, Nucleoprotein inhibitors (HBV core or capsid protein inhibitors); Nucleic Acid Polymers (NAPs); Stimulators of retinoic acid-inducible gene 1; Stimulators of NOD2; Recombinant thymosin alpha-1; Hepatitis B virus replication inhibitors; PI3K inhibitors; cccDNA inhibitors; immune checkpoint inhibitors, such as PD-L1 inhibitors, PD-1 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, Lag3 inhibitors, CTLA-4 inhibitors; Agonists of co-stimulatory receptors that are expressed on immune cells (more particularly T cells), such as CD27 and CD28; BTK inhibitors; Other drugs for treating HBV; IDO inhibitors; Arginase inhibitors; and KDM5 inhibitors.
In certain embodiments, the further adjuvant can e.g., be selected from interferon (for example, interferon-alpha-2a is pegylated interferon-alpha-2a (PEGASYS)), nucleoside or nucleotide or non-nucleos(t)ide polymerase inhibitors, immunomodulatory agents (e.g., IL-12, IL-18, IFN-alpha, -beta, and -gamma and TNF-alpha among others), TLR agonists, siRNAs and antisense oligonucleotides.
In certain embodiments, each of the first and second non-naturally occurring nucleic acid molecules is independently formulated with a lipid nanoparticle (LNP). The application also provides methods of making compositions and therapeutic combinations of the application. A method of producing a composition or therapeutic combination comprises mixing an isolated polynucleotide encoding an HBV antigen, vector, and/or polypeptide of the application with one or more pharmaceutically acceptable carriers. One of ordinary skill in the art will be familiar with conventional techniques used to prepare such compositions.
Methods of Inducing an Immune Response or Treating an HBV Infection
The application also provides methods of inducing an immune response against hepatitis B virus (HBV) in a subject in need thereof, comprising administering to the subject an immunogenically effective amount of a composition or immunogenic composition of the application. Any of the compositions and therapeutic combinations of the application described herein can be used in the methods of the application.
As used herein, the term“infection” refers to the invasion of a host by a disease causing agent. A disease causing agent is considered to be“infectious” when it is capable of invading a host, and replicating or propagating within the host. Examples of infectious agents include viruses, e.g., HBV and certain species of adenovirus, prions, bacteria, fungi, protozoa and the like.“HBV infection” specifically refers to invasion of a host organism, such as cells and tissues of the host organism, by HBV.
The phrase“inducing an immune response” when used with reference to the methods described herein encompasses causing a desired immune response or effect in a subject in need thereof against an infection, e.g., an HBV infection.“Inducing an immune response” also encompasses providing a therapeutic immunity for treating against a pathogenic agent, e.g., HBV. As used herein, the term“therapeutic immunity” or“therapeutic immune response” means that the vaccinated subject is able to control an infection with the pathogenic agent against which the vaccination was done, for instance immunity against HBV infection conferred by vaccination with HBV vaccine. In an embodiment,“inducing an immune response” means producing an immunity in a subject in need thereof, e.g., to provide a therapeutic effect against a disease, such as HBV infection. In certain embodiments, “inducing an immune response” refers to causing or improving cellular immunity, e.g., T cell response, against HBV infection. In certain embodiments,“inducing an immune response” refers to causing or improving a humoral immune response against HBV infection. In certain embodiments,“inducing an immune response” refers to causing or improving a cellular and a humoral immune response against HBV infection. As used herein, the term“protective immunity” or“protective immune response” means that the vaccinated subject is able to control an infection with the pathogenic agent against which the vaccination was done. Usually, the subject having developed a“protective immune response” develops only mild to moderate clinical symptoms or no symptoms at all. Usually, a subject having a“protective immune response” or“protective immunity” against a certain agent will not die as a result of the infection with said agent.
Typically, the administration of compositions and therapeutic combinations of the application will have a therapeutic aim to generate an immune response against HBV after HBV infection or development of symptoms characteristic of HBV infection, e.g., for therapeutic vaccination.
As used herein,“an immunogenically effective amount” or“immunologically effective amount” means an amount of a composition, polynucleotide, vector, or antigen sufficient to induce a desired immune effect or immune response in a subject in need thereof. An immunogenically effective amount can be an amount sufficient to induce an immune response in a subject in need thereof. An immunogenically effective amount can be an amount sufficient to produce immunity in a subject in need thereof, e.g., provide a therapeutic effect against a disease such as HBV infection. An immunogenically effective amount can vary depending upon a variety of factors, such as the physical condition of the subject, age, weight, health, etc.; the particular application, e.g., providing protective immunity or therapeutic immunity; and the particular disease, e.g., viral infection, for which immunity is desired. An immunogenically effective amount can readily be determined by one of ordinary skill in the art in view of the present disclosure.
In particular embodiments of the application, an immunogenically effective amount refers to the amount of a composition or therapeutic combination which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of an HBV infection or a symptom associated therewith; (ii) reduce the duration of an HBV infection or symptom associated therewith; (iii) prevent the progression of an HBV infection or symptom associated therewith; (iv) cause regression of an HBV infection or symptom associated therewith; (v) prevent the development or onset of an HBV infection, or symptom associated therewith; (vi) prevent the recurrence of an HBV infection or symptom associated therewith; (vii) reduce hospitalization of a subject having an HBV infection; (viii) reduce hospitalization length of a subject having an HBV infection; (ix) increase the survival of a subject with an HBV infection; (x) eliminate an HBV infection in a subject; (xi) inhibit or reduce HBV replication in a subject; and/or (xii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy.
An immunogenically effective amount can also be an amount sufficient to reduce HBsAg levels consistent with evolution to clinical seroconversion; achieve sustained HBsAg clearance associated with reduction of infected hepatocytes by a subject’s immune system; induce HBV-antigen specific activated T-cell populations; and/or achieve persistent loss of HBsAg within 12 months. Examples of a target index include lower HBsAg below a threshold of 500 copies of HBsAg international units (IU) and/or higher CD8 counts.
As general guidance, an immunogenically effective amount when used with reference to a DNA plasmid can range from about 0.1 mg/mL to 10 mg/mL of DNA plasmid total, such as 0.1 mg/mL, 0.25 mg/mL, 0.5 mg/mL.0.75 mg/mL 1 mg/mL, 1.5 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, or 10 mg/mL.
Preferably, an immunogenically effective amount of DNA plasmid is less than 8 mg/mL, more preferably less than 6 mg/mL, even more preferably 3-4 mg/mL. An immunogenically effective amount can be from one vector or plasmid, or from multiple vectors or plasmids. As further general guidance, an immunogenically effective amount when used with reference to a peptide can range from about 10 µg to 1 mg per administration, such as 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 9000, or 1000 µg per administration. An immunogenically effective amount can be administered in a single composition, or in multiple compositions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 compositions (e.g., tablets, capsules or injectables, or any composition adapted to intradermal delivery, e.g., to intradermal delivery using an intradermal delivery patch), wherein the administration of the multiple capsules or injections collectively provides a subject with an immunogenically effective amount. For example, when two DNA plasmids are used, an immunogenically effective amount can be 3-4 mg/mL, with 1.5-2 mg/mL of each plasmid. It is also possible to administer an immunogenically effective amount to a subject, and subsequently administer another dose of an immunogenically effective amount to the same subject, in a so-called prime-boost regimen. This general concept of a prime-boost regimen is well known to the skilled person in the vaccine field. Further booster administrations can optionally be added to the regimen, as needed.
As yet further general guidance, the dose of a compound of Formula (I) is from about 1 mg to about 2,500 mg. In some embodiments, a dose of a disclosed compound used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound (i.e., another drug for HBV treatment) as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
A therapeutic combination comprising two DNA plasmids, e.g., a first DNA plasmid encoding an HBV core antigen and second DNA plasmid encoding an HBV pol antigen, can be administered to a subject by mixing both plasmids and delivering the mixture to a single anatomic site. Alternatively, two separate immunizations each delivering a single expression plasmid can be performed. In such embodiments, whether both plasmids are administered in a single immunization as a mixture of in two separate immunizations, the first DNA plasmid and the second DNA plasmid can be administered in a ratio of 10:1 to 1:10, by weight, such as 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10, by weight. Preferably, the first and second DNA plasmids are administered in a ratio of 1:1, by weight.
Preferably, a subject to be treated according to the methods of the application is an HBV-infected subject, particular a subject having chronic HBV infection. Acute HBV infection is characterized by an efficient activation of the innate immune system complemented with a subsequent broad adaptive response (e.g., HBV-specific T-cells, neutralizing antibodies), which usually results in successful suppression of replication or removal of infected hepatocytes. In contrast, such responses are impaired or diminished due to high viral and antigen load, e.g., HBV envelope proteins are produced in abundance and can be released in sub-viral particles in 1,000-fold excess to infectious virus.
Chronic HBV infection is described in phases characterized by viral load, liver enzyme levels (necroinflammatory activity), HBeAg, or HBsAg load or presence of antibodies to these antigens. cccDNA levels stay relatively constant at approximately 10 to 50 copies per cell, even though viremia can vary considerably. The persistence of the cccDNA species leads to chronicity. More specifically, the phases of chronic HBV infection include: (i) the immune- tolerant phase characterized by high viral load and normal or minimally elevated liver enzymes; (ii) the immune activation HBeAg-positive phase in which lower or declining levels of viral replication with significantly elevated liver enzymes are observed; (iii) the inactive HBsAg carrier phase, which is a low replicative state with low viral loads and normal liver enzyme levels in the serum that may follow HBeAg seroconversion; and (iv) the HBeAg- negative phase in which viral replication occurs periodically (reactivation) with concomitant fluctuations in liver enzyme levels, mutations in the pre-core and/or basal core promoter are common, such that HBeAg is not produced by the infected cell.
As used herein,“chronic HBV infection” refers to a subject having the detectable presence of HBV for more than 6 months. A subject having a chronic HBV infection can be in any phase of chronic HBV infection. Chronic HBV infection is understood in accordance with its ordinary meaning in the field. Chronic HBV infection can for example be characterized by the persistence of HBsAg for 6 months or more after acute HBV infection. For example, a chronic HBV infection referred to herein follows the definition published by the Centers for Disease Control and Prevention (CDC), according to which a chronic HBV infection can be characterized by laboratory criteria such as: (i) negative for IgM antibodies to hepatitis B core antigen (IgM anti-HBc) and positive for hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), or nucleic acid test for hepatitis B virus DNA, or (ii) positive for HBsAg or nucleic acid test for HBV DNA, or positive for HBeAg two times at least 6 months apart.
Preferably, an immunogenically effective amount refers to the amount of a composition or therapeutic combination of the application which is sufficient to treat chronic HBV infection.
In some embodiments, a subject having chronic HBV infection is undergoing nucleoside analog (NUC) treatment, and is NUC-suppressed. As used herein,“NUC- suppressed” refers to a subject having an undetectable viral level of HBV and stable alanine aminotransferase (ALT) levels for at least six months. Examples of nucleoside/nucleotide analog treatment include HBV polymerase inhibitors, such as entacavir and tenofovir. Preferably, a subject having chronic HBV infection does not have advanced hepatic fibrosis or cirrhosis. Such subject would typically have a METAVIR score of less than 3 for fibrosis and a fibroscan result of less than 9 kPa. The METAVIR score is a scoring system that is commonly used to assess the extent of inflammation and fibrosis by histopathological evaluation in a liver biopsy of patients with hepatitis B. The scoring system assigns two standardized numbers: one reflecting the degree of inflammation and one reflecting the degree of fibrosis.
It is believed that elimination or reduction of chronic HBV may allow early disease interception of severe liver disease, including virus-induced cirrhosis and hepatocellular carcinoma. Thus, the methods of the application can also be used as therapy to treat HBV- induced diseases. Examples of HBV-induced diseases include, but are not limited to cirrhosis, cancer (e.g., hepatocellular carcinoma), and fibrosis, particularly advanced fibrosis characterized by a METAVIR score of 3 or higher for fibrosis. In such embodiments, an immunogenically effective amount is an amount sufficient to achieve persistent loss of HBsAg within 12 months and significant decrease in clinical disease (e.g., cirrhosis, hepatocellular carcinoma, etc.).
Methods according to embodiments of the application further comprises administering to the subject in need thereof another immunogenic agent (such as another HBV antigen or other antigen) or another anti-HBV agent (such as a nucleoside analog or other anti-HBV agent) in combination with a composition of the application. For example, another anti-HBV agent or immunogenic agent can be a small molecule or antibody including, but not limited to, immune checkpoint inhibitors (e.g., anti-PD1, anti-TIM-3, etc.), toll-like receptor agonists (e.g., TLR7 agonists and/oror TLR8 agonists), RIG-1 agonists, IL-15 superagonists (Altor Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, IL12 genetic adjuvant, IL-7-hyFc; CAR-T which bind HBV env (S-CAR cells); capsid assembly modulators; cccDNA inhibitors, HBV polymerase inhibitors (e.g., entecavir and tenofovir). The one or other anti-HBV active agents can be, for example, a small molecule, an antibody or antigen binding fragment thereof, a polypeptide, protein, or nucleic acid. The one or other anti-HBV agents can e.g., be chosen from among HBV DNA polymerase inhibitors; Immunomodulators; Toll-like receptor 7 modulators; Toll-like receptor 8 modulators; Toll-like receptor 3 modulators; Interferon alpha receptor ligands;
Hyaluronidase inhibitors; Modulators of IL-10; HBsAg inhibitors; Toll like receptor 9 modulators; Cyclophilin inhibitors; HBV Prophylactic vaccines; HBV Therapeutic vaccines; HBV viral entry inhibitors; Antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; short interfering RNAs (siRNA), more particularly anti-HBV siRNA; Endonuclease modulators; Inhibitors of ribonucleotide reductase; Hepatitis B virus E antigen inhibitors; HBV antibodies targeting the surface antigens of the hepatitis B virus; HBV antibodies; CCR2 chemokine antagonists; Thymosin agonists; Cytokines, such as IL12; Capsid Assembly Modulators, Nucleoprotein inhibitors (HBV core or capsid protein inhibitors); Nucleic Acid Polymers (NAPs); Stimulators of retinoic acid-inducible gene 1; Stimulators of NOD2; Recombinant thymosin alpha-1; Hepatitis B virus replication inhibitors; PI3K inhibitors; cccDNA inhibitors; immune checkpoint inhibitors, such as PD-L1 inhibitors, PD-1 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, Lag3 inhibitors, and CTLA-4 inhibitors; Agonists of co-stimulatory receptors that are expressed on immune cells (more particularly T cells), such as CD27, CD28; BTK inhibitors; Other drugs for treating HBV; IDO inhibitors; Arginase inhibitors; and KDM5 inhibitors.
Methods of Delivery
Compositions and therapeutic combinations of the application can be administered to a subject by any method known in the art in view of the present disclosure, including, but not limited to, parenteral administration (e.g., intramuscular, subcutaneous, intravenous, or intradermal injection), oral administration, transdermal administration, and nasal administration. Preferably, compositions and therapeutic combinations are administered parenterally (e.g., by intramuscular injection or intradermal injection) or transdermally.
In some embodiments of the application in which a composition or therapeutic combination comprises one or more DNA plasmids, administration can be by injection through the skin, e.g., intramuscular or intradermal injection, preferably intramuscular injection. Intramuscular injection can be combined with electroporation, i.e., application of an electric field to facilitate delivery of the DNA plasmids to cells. As used herein, the term “electroporation” refers to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane. During in vivo electroporation, electrical fields of appropriate magnitude and duration are applied to cells, inducing a transient state of enhanced cell membrane permeability, thus enabling the cellular uptake of molecules unable to cross cell membranes on their own. Creation of such pores by electroporation facilitates passage of biomolecules, such as plasmids, oligonucleotides, siRNAs, drugs, etc., from one side of a cellular membrane to the other. In vivo electroporation for the delivery of DNA vaccines has been shown to significantly increase plasmid uptake by host cells, while also leading to mild-to-moderate inflammation at the injection site. As a result, transfection efficiency and immune response are significantly improved (e.g., up to 1,000 fold and 100 fold respectively) with intradermal or intramuscular electroporation, in comparison to conventional injection. In a typical embodiment, electroporation is combined with intramuscular injection. However, it is also possible to combine electroporation with other forms of parenteral administration, e.g., intradermal injection, subcutaneous injection, etc.
Administration of a composition, therapeutic combination or vaccine of the application via electroporation can be accomplished using electroporation devices that can be configured to deliver to a desired tissue of a mammal a pulse of energy effective to cause reversible pores to form in cell membranes. The electroporation device can include an electroporation component and an electrode assembly or handle assembly. The electroporation component can include one or more of the following components of electroporation devices: controller, current waveform generator, impedance tester, waveform logger, input element, status reporting element, communication port, memory component, power source, and power switch. Electroporation can be accomplished using an in vivo electroporation device. Examples of electroporation devices and electroporation methods that can facilitate delivery of compositions and therapeutic combinations of the application, particularly those comprising DNA plasmids, include CELLECTRA® (Inovio Pharmaceuticals, Blue Bell, PA), Elgen electroporator (Inovio Pharmaceuticals, Inc.) Tri-GridTM delivery system (Ichor Medical Systems, Inc., San Diego, CA 92121) and those described in U.S. Patent No. 7,664,545, U.S. Patent No.8,209,006, U.S. Patent No.9,452,285, U.S. Patent No.5,273,525, U.S. Patent No.6,110,161, U.S. Patent No.6,261,281, U.S. Patent No.6,958,060, and U.S. Patent No.6,939,862, U.S. Patent No.7,328,064, U.S. Patent No.6,041,252, U.S. Patent No. 5,873,849, U.S. Patent No.6,278,895, U.S. Patent No.6,319,901, U.S. Patent No.6,912,417, U.S. Patent No.8,187,249, U.S. Patent No.9,364,664, U.S. Patent No.9,802,035, U.S. Patent No.6,117,660, and International Patent Application Publication WO2017172838, all of which are herein incorporated by reference in their entireties. Other examples of in vivo electroporation devices are described in International Patent Application entitled“Method and Apparatus for the Delivery of Hepatitis B Virus (HBV) Vaccines,” filed on the same day as this application with the Attorney Docket Number 688097-405WO, the contents of which are hereby incorporated by reference in their entireties. Also contemplated by the application for delivery of the compositions and therapeutic combinations of the application are use of a pulsed electric field, for instance as described in, e.g., U.S. Patent No.6,697,669, which is herein incorporated by reference in its entirety.
In other embodiments of the application in which a composition or therapeutic combination comprises one or more DNA plasmids, the method of administration is transdermal. Transdermal administration can be combined with epidermal skin abrasion to facilitate delivery of the DNA plasmids to cells. For example, a dermatological patch can be used for epidermal skin abrasion. Upon removal of the dermatological patch, the composition or therapeutic combination can be deposited on the abraised skin.
Methods of delivery are not limited to the above described embodiments, and any means for intracellular delivery can be used. Other methods of intracellular delivery contemplated by the methods of the application include, but are not limited to, liposome encapsulation, lipid nanoparticles (LNPs), etc.
Adjuvants
In some embodiments of the application, a method of inducing an immune response against HBV further comprises administering an adjuvant. The terms“adjuvant” and "immune stimulant" are used interchangeably herein, and are defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance an immune response to HBV antigens and antigenic HBV polypeptides of the application.
According to embodiments of the application, an adjuvant can be present in a therapeutic combination or composition of the application, or administered in a separate composition. An adjuvant can be, e.g., a small molecule or an antibody. Examples of adjuvants suitable for use in the application include, but are not limited to, immune checkpoint inhibitors (e.g., anti-PD1, anti-TIM-3, etc.), toll-like receptor agonists (e.g., TLR7 and/or TLR8 agonists), RIG-1 agonists, IL-15 superagonists (Altor Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, IL12 genetic adjuvant, and IL-7-hyFc. Examples of adjuvants can e.g., be chosen from among the following anti-HBV agents: HBV DNA polymerase inhibitors; Immunomodulators; Toll-like receptor 7 modulators; Toll-like receptor 8 modulators; Toll-like receptor 3 modulators; Interferon alpha receptor ligands; Hyaluronidase inhibitors; Modulators of IL-10; HBsAg inhibitors; Toll like receptor 9 modulators; Cyclophilin inhibitors; HBV Prophylactic vaccines; HBV Therapeutic vaccines; HBV viral entry inhibitors; Antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; short interfering RNAs (siRNA), more particularly anti-HBV siRNA; Endonuclease modulators; Inhibitors of ribonucleotide reductase; Hepatitis B virus E antigen inhibitors; HBV antibodies targeting the surface antigens of the hepatitis B virus; HBV antibodies; CCR2 chemokine antagonists; Thymosin agonists; Cytokines, such as IL12; Capsid Assembly Modulators, Nucleoprotein inhibitors (HBV core or capsid protein inhibitors); Nucleic Acid Polymers (NAPs); Stimulators of retinoic acid-inducible gene 1; Stimulators of NOD2; Recombinant thymosin alpha-1; Hepatitis B virus replication inhibitors; PI3K inhibitors; cccDNA inhibitors; immune checkpoint inhibitors, such as PD-L1 inhibitors, PD-1 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, Lag3 inhibitors, and CTLA-4 inhibitors; Agonists of co- stimulatory receptors that are expressed on immune cells (more particularly T cells), such as CD27, CD28; BTK inhibitors; Other drugs for treating HBV; IDO inhibitors; Arginase inhibitors; and KDM5 inhibitors.
Compositions and therapeutic combinations of the application can also be administered in combination with at least one other anti-HBV agent. Examples of anti-HBV agents suitable for use with the application include, but are not limited to small molecules, antibodies, and/or CAR-T therapies which bind HBV env (S-CAR cells), capsid assembly modulators, TLR agonists (e.g., TLR7 and/or TLR8 agonists), cccDNA inhibitors, HBV polymerase inhibitors (e.g., entecavir and tenofovir), and/or immune checkpoint inhibitors, etc.
The at least one anti-HBV agent can e.g., be chosen from among HBV DNA polymerase inhibitors; Immunomodulators; Toll-like receptor 7 modulators; Toll-like receptor 8 modulators; Toll-like receptor 3 modulators; Interferon alpha receptor ligands; Hyaluronidase inhibitors; Modulators of IL-10; HBsAg inhibitors; Toll like receptor 9 modulators; Cyclophilin inhibitors; HBV Prophylactic vaccines; HBV Therapeutic vaccines; HBV viral entry inhibitors; Antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; short interfering RNAs (siRNA), more particularly anti-HBV siRNA; Endonuclease modulators; Inhibitors of ribonucleotide reductase; Hepatitis B virus E antigen inhibitors; HBV antibodies targeting the surface antigens of the hepatitis B virus; HBV antibodies; CCR2 chemokine antagonists; Thymosin agonists; Cytokines, such as IL12; Capsid Assembly Modulators, Nucleoprotein inhibitors (HBV core or capsid protein inhibitors); Nucleic Acid Polymers (NAPs); Stimulators of retinoic acid-inducible gene 1; Stimulators of NOD2; Recombinant thymosin alpha-1; Hepatitis B virus replication inhibitors; PI3K inhibitors; cccDNA inhibitors; immune checkpoint inhibitors, such as PD-L1 inhibitors, PD-1 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, Lag3 inhibitors, and CTLA-4 inhibitors; Agonists of co-stimulatory receptors that are expressed on immune cells (more particularly T cells), such as CD27, CD28; BTK inhibitors; Other drugs for treating HBV; IDO inhibitors; Arginase inhibitors; and KDM5 inhibitors. Such anti-HBV agents can be administered with the compositions and therapeutic combinations of the application simultaneously or sequentially.
Methods of Prime/Boost Immunization
Embodiments of the application also contemplate administering an immunogenically effective amount of a composition or therapeutic combination to a subject, and subsequently administering another dose of an immunogenically effective amount of a composition or therapeutic combination to the same subject, in a so-called prime-boost regimen Thus, in an embodiment, a composition or therapeutic combination of the application is a primer vaccine used for priming an immune response. In another embodiment, a composition or therapeutic combination of the application is a booster vaccine used for boosting an immune response. The priming and boosting vaccines of the application can be used in the methods of the application described herein. This general concept of a prime-boost regimen is well known to the skilled person in the vaccine field. Any of the compositions and therapeutic combinations of the application described herein can be used as priming and/or boosting vaccines for priming and/or boosting an immune response against HBV.
In some embodiments of the application, a composition or therapeutic combination of the application can be administered for priming immunization. The composition or therapeutic combination can be re-administered for boosting immunization. Further booster administrations of the composition or vaccine combination can optionally be added to the regimen, as needed. An adjuvant can be present in a composition of the application used for boosting immunization, present in a separate composition to be administered together with the composition or therapeutic combination of the application for the boosting immunization, or administered on its own as the boosting immunization. In those embodiments in which an adjuvant is included in the regimen, the adjuvant is preferably used for boosting immunization.
An illustrative and non-limiting example of a prime-boost regimen includes administering a single dose of an immunogenically effective amount of a composition or therapeutic combination of the application to a subject to prime the immune response; and subsequently administering another dose of an immunogenically effective amount of a composition or therapeutic combination of the application to boost the immune response, wherein the boosting immunization is first administered about two to six weeks, preferably four weeks after the priming immunization is initially administered. Optionally, about 10 to 14 weeks, preferably 12 weeks, after the priming immunization is initially administered, a further boosting immunization of the composition or therapeutic combination, or other adjuvant, is administered.
Kits
Also provided herein is a kit comprising a therapeutic combination of the application. A kit can comprise the first polynucleotide, the second polynucleotide, and the at least one capsid assembly modulator (CAM) in one or more separate compositions, or a kit can comprise the first polynucleotide, the second polynucleotide, and the at least one capsid assembly modulator (CAM) in a single composition. A kit can further comprise one or more adjuvants or immune stimulants, and/or other anti-HBV agents.
The ability to induce or stimulate an anti-HBV immune response upon administration in an animal or human organism can be evaluated either in vitro or in vivo using a variety of assays which are standard in the art. For a general description of techniques available to evaluate the onset and activation of an immune response, see for example Coligan et al. (1992 and 1994, Current Protocols in Immunology; ed. J Wiley & Sons Inc, National Institute of Health). Measurement of cellular immunity can be performed by measurement of cytokine profiles secreted by activated effector cells including those derived from CD4+ and CD8+ T- cells (e.g. quantification of IL-10 or IFN gamma-producing cells by ELISPOT), by determination of the activation status of immune effector cells (e.g. T cell proliferation assays by a classical [3H] thymidine uptake or flow cytometry-based assays), by assaying for antigen-specific T lymphocytes in a sensitized subject (e.g. peptide-specific lysis in a cytotoxicity assay, etc.).
The ability to stimulate a cellular and/or a humoral response can be determined by antibody binding and/or competition in binding (see for example Harlow, 1989, Antibodies, Cold Spring Harbor Press). For example, titers of antibodies produced in response to administration of a composition providing an immunogen can be measured by enzyme-linked immunosorbent assay (ELISA). The immune responses can also be measured by neutralizing antibody assay, where a neutralization of a virus is defined as the loss of infectivity through reaction/inhibition/neutralization of the virus with specific antibody. The immune response can further be measured by Antibody-Dependent Cellular Phagocytosis (ADCP) Assay. EMBODIMENTS
The invention provides also the following non-limiting embodiments. Embodiment 1 is a therapeutic combination for use in treating a hepatitis B virus (HBV) infection in a subject in need thereof, comprising:
i) at least one of:
a) a truncated HBV core antigen consisting of an amino acid sequence that is at least 95%, such as at least 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 2,
b) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding the truncated HBV core antigen
c) an HBV polymerase antigen having an amino acid sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 7, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity, and
d) a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide sequence encoding the HBV polymerase antigen; and ii) a compound of Formula (I):
Figure imgf000077_0001
or a deuterated isomer, a stereoisomer or tautomeric form thereof, or a pharmaceutically acceptable salt thereof, wherein:
R1 is selected from the group consisting of phenyl, thiophenyl, pyridyl, and pyridonyl, optionally substituted with one or more substituents selected from the group consisting of C1- 4alkyl, halogen, and CN;
R2 is C1-4alkyl;
R3 is selected from the group consisting of thiazolyl, pyridyl, and oxazolyl, optionally substituted with one or more substituents selected from fluorine, and C1-6alkyl;
n is an integer of 0 or 1;
R4 and R5 are independently selected from the group consisting of H and -COOH; (i.e., the bond between X and Y) is a single bond or a double bond;
when X and Y are linked by a single bond, X is selected from the group consisting of C(=S), C(=NR6), C(=CHR7) and CHR8, and Y is NR9;
when X and Y are linked by a double bond, X is C-SR9 or C-OR9, and Y is N atom; Z is selected from the group consisting of CH2 and C(=O);
R6 is selected from the group consisting of CN, C(=O)CH3, and SO2CH3;
R7 is CN;
R8 is CF3;
9 10
R is selected from the group consisting of H, -C1-6alkyl, -C1-6alkyl-R , -C1-6alkoxy- C1-6alkyl-R10 and -(CH2)p-Q-R10;
p is an integer of 0, 1, 2, or 3;
Q is selected from the group consisting of aryl, heteroaryl, and a 3- to 7- membered saturated ring, optionally containing a heteroatom, the heteroatom being an oxygen or a nitrogen, the nitrogen being substituted with H, -C1-6alkyl, -C1-6alkoxy-C1-6alkyl and -C1- 6alkylcarbonyl;
R10 is selected from -COOH, -C(=O)NHS(=O)2-C1-6alkyl, tetrazolyl and carboxylic acid bioisosteres.
Embodiment 2 is the therapeutic combination of embodiment 1, comprising at least one of the HBV polymerase antigen and the truncated HBV core antigen.
Embodiment 3 is the therapeutic combination of embodiment 2, comprising the HBV polymerase antigen and the truncated HBV core antigen.
Embodiment 4 is the therapeutic combination of embodiment 1, comprising at least one of the first non-naturally occurring nucleic acid molecule comprising the first polynucleotide sequence encoding the truncated HBV core antigen, and the second non- naturally occurring nucleic acid molecule comprising the second polynucleotide sequence encoding the HBV polymerase antigen.
Embodiment 5 is a therapeutic combination for use in treating a hepatitis B virus (HBV) infection in a subject in need thereof, comprising
i) a first non-naturally occurring nucleic acid molecule comprising a first
polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 95% identical to SEQ ID NO: 2; and ii) a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide sequence encoding an HBV polymerase antigen having an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity; and
iii) a compound of Formula (I):
Figure imgf000079_0001
or a deuterated isomer, stereoisomer or tautomeric form thereof, or a pharmaceutically acceptable salt thereof, wherein:
1R is phenyl substituted with one or more substituents selected from halogens and C1- 6alkyl;
R2 is methyl or ethyl;
R3 is thiazolyl;
n is an integer of 0 or 1;
R4 and R5 are H;
Figure imgf000079_0002
( , the bond between X and Y) is a single bond;
X is C(=S);
Y is NR9;
Z is CH2;
R9 is C1-6alkyl-CO 1
2H or (CH2)p-Q-R10;
p is an integer of 0, 1, 2, or 3;
Q is phenyl, a C3-6cycloalkyl, or a 3- to 6- saturated membered ring containing an oxygen; and
R10 is selected from -COOH, -C(=O)NHS(=O)2-C1-6alkyl, tetrazolyl and carboxylic acid bioisosteres, wherein the carboxylic acid bioisosteres are -S(=O)2(OH), -P(=O)(OH)2, - C(=O)NHOH, -C(=O)NHCN, 1,2,4-oxadiazol-5(4H)-one, or 3-hydroxy-4-methylcyclobut-3- ene-1,2-dione, which refer to the following structures:
Figure imgf000080_0001
. Embodiment 6 is the therapeutic combination of embodiment 4 or 5, wherein the first non-naturally occurring nucleic acid molecule further comprises a polynucleotide sequence encoding a signal sequence operably linked to the N-terminus of the truncated HBV core antigen.
Embodiment 6a is the therapeutic combination of any one of embodiments 4 to 6, wherein the second non-naturally occurring nucleic acid molecule further comprises a polynucleotide sequence encoding a signal sequence operably linked to the N-terminus of the HBV polymerase antigen.
Embodiment 6b is the therapeutic combination of embodiment 6 or 6a, wherein the signal sequence independently comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15.
Embodiment 6c is the therapeutic combination of embodiment 6 or 6a, wherein the signal sequence is independently encoded by the polynucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 14.
Embodiment 7 is the therapeutic combination of any one of embodiments 1-6c, wherein the HBV polymerase antigen comprises an amino acid sequence that is at least 98%, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, identical to SEQ ID NO: 7.
Embodiment 7a is the therapeutic combination of embodiment 7, wherein the HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 7.
Embodiment 7b is the therapeutic combination of any one of embodiments 1 to 7a, wherein the truncated HBV core antigen consists of the amino acid sequence that is at least 98%, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, identical to SEQ ID NO: 2.
Embodiment 7c is the therapeutic combination of embodiment 7b, wherein the truncated HBV antigen consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
Embodiment 8 is the therapeutic combination of any one of embodiments 1-7c, wherein each of the first and second non-naturally occurring nucleic acid molecules is a DNA molecule. Embodiment 8a is the therapeutic combination of embodiment 8, wherein the DNA molecule is present on a DNA vector.
Embodiment 8b is the therapeutic combination of embodiment 8a, wherein the DNA vector is selected from the group consisting of DNA plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, and closed linear deoxyribonucleic acid.
Embodiment 8c is the therapeutic combination of embodiment 8, wherein the DNA molecule is present on a viral vector.
Embodiment 8d is the therapeutic combination of embodiment 8c, wherein the viral vector is selected from the group consisting of bacteriophages, animal viruses, and plant viruses.
Embodiment 8e is the therapeutic combination of any one of embodiments 1-7c, wherein each of the first and second non-naturally occurring nucleic acid molecules is an RNA molecule.
Embodiment 8f is the therapeutic combination of embodiment 8e, wherein the RNA molecule is an RNA replicon, preferably a self-replicating RNA replicon, an mRNA replicon, a modified mRNA replicon, or self-amplifying mRNA.
Embodiment 8g is the therapeutic combination of any one of embodiments 1 to 8f, wherein each of the first and second non-naturally occurring nucleic acid molecules is independently formulated with a lipid composition, preferably a lipid nanoparticle (LNP).
Embodiment 9 is the therapeutic combination of any one of embodiments 4-8g, comprising the first non-naturally occurring nucleic acid molecule and the second non- naturally occurring nucleic acid molecule in the same non-naturally occurring nucleic acid molecule.
Embodiment 10 is the therapeutic combination of any one of embodiments 4-8g, comprising the first non-naturally occurring nucleic acid molecule and the second non- naturally occurring nucleic acid molecule in two different non-naturally occurring nucleic acid molecules.
Embodiment 11 is the therapeutic combination of any one of embodiments 4-10, wherein the first polynucleotide sequence comprises a polynucleotide sequence having at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3.
Embodiment 11a is the therapeutic combination of embodiment 11, wherein the first polynucleotide sequence comprises a polynucleotide sequence having at least 98%, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3.
Embodiment 12 is the therapeutic combination of embodiment 11a, wherein the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
Embodiment 13 is the therapeutic combination of any one of embodiments 4 to 12, wherein the second polynucleotide sequence comprises a polynucleotide sequence having at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6.
Embodiment 13a is the therapeutic combination of embodiment 13, wherein the second polynucleotide sequence comprises a polynucleotide sequence having at least 98%, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6.
Embodiment 14 is the therapeutic combination of embodiment 13a, wherein the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6.
Embodiment 15 is the therapeutic combination of any one of embodiments 1 to 14, wherein the compound of Formula (I) is selected from the group consisting of the exemplified compounds described herein or in International Patent Application
PCT/CN2018/122258 filed on December 20, 2018 and U.S. Patent Application US 62/791,576 filed on January 11, 2019, the content of which are herein incorporated by references in their entireties, or a deuterated isomer, stereoisomer or tautomeric form thereof, or a pharmaceutically acceptable salt thereof.
Embodiment 15a is the therapeutic combination of any one of embodiments 1 to 14, wherein the compound of Formula (I) is compound 1A:
Figure imgf000083_0001
, p aceutically acceptable salt thereof. Embodiment 16 is a kit comprising the therapeutic combination of any one of embodiments 1 to 15a, and instructions for using the therapeutic combination in treating a hepatitis B virus (HBV) infection in a subject in need thereof.
Embodiment 17 is a method of treating a hepatitis B virus (HBV) infection in a subject in need thereof, comprising administering to the subject the therapeutic combination of any one of embodiments 1 to 15a.
Embodiment 17a is the method of embodiment 17, wherein the treatment induces an immune response against a hepatitis B virus in a subject in need thereof, preferably the subject has chronic HBV infection.
Embodiment 17b is the method of embodiment 17 or 17a, wherein the subject has chronic HBV infection.
Embodiment 17c is the method of any one of embodiments 17 to 17b, wherein the subject is in need of a treatment of an HBV-induced disease selected from the group consisting of advanced fibrosis, cirrhosis and hepatocellular carcinoma (HCC).
Embodiment 18 is the method of any one of embodiments 17-17c, wherein the therapeutic combination is administered by injection through the skin, e.g., intramuscular or intradermal injection, preferably intramuscular injection.
Embodiment 19 is the method of embodiment 18, wherein the therapeutic combination comprises at least one of the first and second non-naturally occurring nucleic acid molecules.
Embodiment 19a is the method of embodiment 19, wherein the therapeutic combination comprises the first and second non-naturally occurring nucleic acid molecules. Embodiment 20 is the method of embodiment 19 or 19a, wherein the non-naturally occurring nucleic acid molecules are administered to the subject by intramuscular injection in combination with electroporation.
Embodiment 21 is the method of embodiment 19 or 19a, wherein the non-naturally occurring nucleic acid molecules are administered to the subject by a lipid composition, preferably by a lipid nanoparticle. EXAMPLES
It will be appreciated by those skilled in the art that changes could be made to the embodiments described herein without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the present description.
Example 1. HBV core plasmid & HBV pol plasmid
A schematic representation of the pDK-pol and pDK-core vectors is shown in Fig.1A and 1B, respectively. An HBV core or pol antigen optimized expression cassette containing a CMV promoter (SEQ ID NO: 18), a splicing enhancer (triple composite sequence) (SEQ ID NO: 10), Cystatin S precursor signal peptide SPCS (NP_0018901.1) (SEQ ID NO: 9), and pol (SEQ ID NO: 5) or core (SEQ ID NO: 2) gene was introduced into a pDK plasmid backbone, using standard molecular biology techniques.
The plasmids were tested in vitro for core and pol antigen expression by Western blot analysis using core and pol specific antibodies, and were shown to provide consistent expression profile for cellular and secreted core and pol antigens (data not shown).
Example 2. Generation of Adenoviral Vectors Expressing a Fusion of Truncated HBV Core Antigen with HBV Pol Antigen
The creation of an adenovirus vector has been designed as a fusion protein expressed from a single open reading frame. Additional configurations for the expression of the two proteins, e.g. using two separate expression cassettes, or using a 2A-like sequence to separate the two sequences, can also be envisaged.
Design of expression cassettes for adenoviral vectors
The expression cassettes (diagrammed in FIG.2A and FIG.2B) are comprised of the CMV promoter (SEQ ID NO: 19), an intron (SEQ ID NO:12) (a fragment derived from the human ApoAI gene - GenBank accession X01038 base pairs 295– 523, harboring the ApoAI second intron), followed by the optimized coding sequence– either core alone or the core and polymerase fusion protein preceded by a human immunoglobulin secretion signal coding sequence (SEQ ID NO: 14), and followed by the SV40 polyadenylation signal (SEQ ID NO: 13).
A secretion signal was included because of past experience showing improvement in the manufacturability of some adenoviral vectors harboring secreted transgenes, without influencing the elicited T-cell response (mouse experiments).
The last two residues of the Core protein (VV) and the first two residues of the Polymerase protein (MP) if fused results in a junction sequence (VVMP) that is present on the human dopamine receptor protein (D3 isoform), along with flanking homologies.
The interjection of an AGAG linker between the core and the polymerase sequences eliminates this homology and returned no further hits in a Blast of the human proteome. Example 3. In Vivo Immunogenicity Study of DNA Vaccine in Mice
An immunotherapeutic DNA vaccine containing DNA plasmids encoding an HBV core antigen or HBV polymerase antigen was tested in mice. The purpose of the study was designed to detect T-cell responses induced by the vaccine after intramuscular delivery via electroporation into BALB/c mice. Initial immunogenicity studies focused on determining the cellular immune responses that would be elicited by the introduced HBV antigens.
In particular, the plasmids tested included a pDK-Pol plasmid and pDK-Core plasmid, as shown in FIGS.1A and 1B, respectively, and as described herein in Example 1. The pDK- Pol plasmid encoded a polymerase antigen having the amino acid sequence of SEQ ID NO: 7, and the pDK-Core plasmid encoding a Core antigen having the amino acid sequence of SEQ ID NO: 2. First, T-cell responses induced by each plasmid individually were tested. The DNA plasmid (pDNA) vaccine was intramuscularly delivered via electroporation to Balb/c mice using a commercially available TriGridTM delivery system-intramuscular (TDS-IM) adapted for application in the mouse model in cranialis tibialis. See International Patent Application Publication WO2017172838, and U.S. Patent Application No.62/607,430, entitled“Method and Apparatus for the Delivery of Hepatitis B Virus (HBV) Vaccines,” filed on December 19, 2017 for additional description on methods and devices for intramuscular delivery of DNA to mice by electroporation, the disclosures of which are hereby incorporated by reference in their entireties. In particular, the TDS-IM array of a TDS-IM v1.0 device having an electrode array with a 2.5 mm spacing between the electrodes and an electrode diameter of 0.030 inch was inserted percutaneously into the selected muscle, with a conductive length of 3.2 mm and an effective penetration depth of 3.2 mm, and with the major axis of the diamond configuration of the electrodes oriented in parallel with the muscle fibers. Following electrode insertion, the injection was initiated to distribute DNA (e.g., 0.020 ml) in the muscle. Following completion of the IM injection, a 250 V/cm electrical field (applied voltage of 59.4 -65.6 V, applied current limits of less than 4 A, 0.16 A/sec) was locally applied for a total duration of about 400 ms at a 10% duty cycle (i.e., voltage is actively applied for a total of about 40 ms of the about 400 ms duration) with 6 total pulses. Once the electroporation procedure was completed, the TriGridTM array was removed and the animals were recovered. High-dose (20 µg) administration to BALB/c mice was performed as summarized in Table 1. Six mice were administered plasmid DNA encoding the HBV core antigen (pDK-core; Group 1), six mice were administered plasmid DNA encoding the HBV pol antigen (pDK-pol; Group 2), and two mice received empty vector as the negative control. Animals received two DNA immunizations two weeks apart and splenocytes were collected one week after the last immunization.
Table 1: Mouse immunization experimental design of the pilot study.
Figure imgf000086_0001
Antigen-specific responses were analyzed and quantified by IFN-g enzyme-linked immunospot (ELISPOT). In this assay, isolated splenocytes of immunized animals were incubated overnight with peptide pools covering the Core protein, the Pol protein, or the small peptide leader and junction sequence (2µg/ml of each peptide). These pools consisted of 15 mer peptides that overlap by 11 residues matching the Genotypes BCD consensus sequence of the Core and Pol vaccine vectors. The large 94 kDan HBV Pol protein was split in the middle into two peptide pools. Antigen-specific T cells were stimulated with the homologous peptide pools and IFN-g-positive T cells were assessed using the ELISPOT assay. IFN-g release by a single antigen-specific T cell was visualized by appropriate antibodies and subsequent chromogenic detection as a colored spot on the microplate referred to as spot-forming cell (SFC).
Substantial T-cell responses against HBV Core were achieved in mice immunized with the DNA vaccine plasmid pDK-Core (Group 1) reaching 1,000 SFCs per 106 cells (FIG. 3). Pol T-cell responses towards the Pol 1 peptide pool were strong (~1,000 SFCs per 106 cells). The weak Pol-2-directed anti-Pol cellular responses were likely due to the limited MHC diversity in mice, a phenomenon called T-cell immunodominance defined as unequal recognition of different epitopes from one antigen. A confirmatory study was performed confirming the results obtained in this study (data not shown).
The above results demonstrate that vaccination with a DNA plasmid vaccine encoding HBV antigens induces cellular immune responses against the administered HBV antigens in mice. Similar results were also obtained with non-human primates (data not shown).
Preparative Examples
Exemplary compounds useful in methods of the invention will now be described by reference to the illustrative synthetic schemes for their general preparation below and the specific examples to follow.
General Scheme
Figure imgf000088_0001
The preparation of compound I is shown in the above general scheme.
Compound I-1 can be prepared by the condensation of aldehyde II, acetoacetate III and amidine IV in the presence of a base such as NaOAc. Compound I-2 was prepared from compound I-1 using brominating reagent such as N-Bromosuccinimide. Coupling of compound I-2 and compound V in the presence of a base such as triethylamine affords compound I. Preparation of ethyl 4-(2-chloro-3-fluorophenyl)-6-methyl-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate (H1)
Figure imgf000088_0002
To a solution of 2-chloro-3-fluorobenzaldehyde (8.8 g, 55.7 mmol), ethyl 3-oxobutanoate (7.24 g, 55.7 mmol) in isopropanol (40 mL) was added piperidine (473 mg, 5.57 mmol) and AcOH (334 mg, 5.57 mmol). After stirred at room temperature for 4 hours, the mixture was added thiazole-2-carboximidamide (6.4 g, 39 mmol) and triethylamine (5.62 g, 55.7 mmol) at room temperature over 15 minutes. The reaction mixture was stirred at 75 oC for 12 hours. It was cooled to room temperature, extracted with ethyl acetate, washed with brine, dried over Na2SO4 and purified by silica gel column chromatography (petroleum ether : ethyl acetate = 20 : 1) to give the title compound H1 (5.45 g, 95 % purity from 1H NMR, 26 % yield) as yellow solids. LC-MS (ESI): RT = 1.74 min, mass calcd. for C17H15ClFN3O2S 379.1, m/z found 380.1 [M+H]+ .1H NMR (400 MHz, CDCl3) d 7.84 - 7.80 (m, 1.7H), 7.50 (d, J = 3.6 Hz, 0.3H), 7.47 (s, 0.3H), 7.44 (d, J = 3.2 Hz, 0.7H), 7.23 - 7.14 (m, 2H), 7.09 - 7.01 (m, 1H), 6.27 (s, 0.7H), 6.14 (d, J = 2.4 Hz, 0.3H), 4.13 - 3.98 (m, 2H), 2.57 (s, 0.7H), 2.52 (s, 2.3H), 1.13 - 1.10 (m, 3H). Chiral separation of ethyl 4-(2-chloro-3-fluorophenyl)-6-methyl-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate (H1) The racemic mixture ethyl 4-(2-chloro-3-fluorophenyl)-6-methyl-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate H1 (5.45 g, 13.7 mmol) was separated by chiral separation (separation condition: column: Chiralpak IC 5 µm 20 * 250 mm; Mobile Phase: Hex : EtOH : DEA = 95 : 5 : 0.3 at 28 mL/ min, Temp: 30 °C, Wavelength: 254 nm) to give H1-A (2.5 g, 90 % purity from 1HNMR, 46 % yield, 100 % ee) and H1-B (2.48 g, 90 % purity from 1HNMR, 46 % yield, 92.1 % ee) as yellow solids.
H1-A: LC-MS (ESI): RT = 3.886 min, mass calcd. for C17H15ClFN3O2S 379.06, m/z found 380.1 [M+H]+ . Chiral analysis (Column: Chiralpak IA 5 µm 4.6 * 250 mm; Mobile Phase: Hex : EtOH : DEA = 90 : 10 : 0.2 at 1.0 mL/ min; Temp: 30 oC; Wavelength: 254 nm, RT = 7.438 min).1H NMR (400 MHz, CDCl3) d 7.84 - 7.80 (m, 1.7H), 7.51 - 7.44 (m, 1.3H), 7.22 - 7.14 (m, 2H), 7.09 - 7.01 (m, 1H), 6.27 (s, 0.7H), 6.14 (s, 0.3H), 4.05 - 4.00 (m, 2H), 2.57 (s, 0.7H), 2.52 (s, 2.3H), 1.13 - 1.10 (m, 3H).
H1-B: LC-MS (ESI): RT = 3.887 min, mass calcd. for C17H15ClFN3O2S 379.06, m/z found
+
380.1 [M+H] . Chiral analysis (Column: Chiralpak IA 5 µm 4.6 * 250 mm; Mobile Phase: Hex : EtOH : DEA = 90 : 10 : 0.2 at 1.0 mL/ min; Temp: 30 oC; Wavelength: 254 nm, RT = 6.903 min).1H NMR (400 MHz, CDCl3) d 7.84 - 7.80 (m, 1.7H), 7.51 - 7.43 (m, 1.3H), 7.22 - 7.14 (m, 2H), 7.09 - 7.01 (m, 1H), 6.27 (s, 0.7H), 6.14 (s, 0.3H), 4.10 - 3.98 (m, 2H), 2.57 (s, 0.7H), 2.51 (s, 2.3H), 1.13 - 1.10 (m, 3H). Preparation of ethyl 6-(bromomethyl)-4-(2-chloro-3-fluorophenyl)-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate (H1-1A) (single enantiomer)
Figure imgf000090_0001
To a solution of ethyl 4-(2-chloro-3-fluorophenyl)-6-methyl-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate H1-A (300 mg, 90 % purity, 0.711 mmol) in carbon tetrachloride (5 mL) was added N-bromosuccinimide (120 mg, 0.674 mmol). After stirred at 60 oC for 1 hour, the reaction mixture was concentrated to give a residue, which was purified by gel column chromatography (petroleum ether : ethyl acetate = 20 : 1 to 10 : 1) to give the title compound (H1-1A) (240 mg, 90 % purity from HNMR, 66 % yield) as yellow solids. LC-MS (ESI): RT = 1.852 min, mass calcd. for C17H14BrClFN3O2S 456.9, m/z found 457.9 [M+H]+ .1H NMR (400 MHz, CDCl3) d 8.26 (s, 0.3H), 7.84 (d, J = 2.8 Hz, 1H), 7.53 - 7.46 (m, 1.7H), 7.24 - 7.14 (m, 2H), 7.09 - 7.01 (m, 1H), 6.26 (s, 0.3H), 6.17 (s, 0.7H), 4.92 (d, J = 8.0 Hz, 1H), 4.76 (d, J = 11.2 Hz, 0.3H), 4.60 (d, J = 8.0 Hz, 0.7H), 4.12 (q, J = 7.2 Hz, 2H), 1.14 (t, J = 11.2 Hz, 3H). Using the same procedure, the following intermediates were prepared.
Figure imgf000090_0002
Figure imgf000091_0001
89
Figure imgf000092_0001
90 Intermediate H2 : Ethyl 4-(3-fluoro-2-methylphenyl)-6-methyl-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate was prepared using same condition as for H1.
1H NMR (400 MHz, DMSO-d6) d 9.86 (s, 0.8H), 9.52 (d, J = 2.8 Hz, 0.2H), 8.00 - 7.98 (m, 0.4H), 7.96 (d, J = 3.2 Hz, 0.8H), 7.88 (d, J = 2.8 Hz, 0.8H), 7.20 - 7.15 (m, 1.2H), 7.06 - 6.99 (m, 1.8H), 5.83 (s, 0.8H), 5.73 (d, J = 3.2 Hz, 0.2H), 3.99 - 3.93 (m, 2H), 2.48 (s, 2.4H), 2.45 (s, 1.2H), 2.44 (s, 1.2H), 2.41 (s, 0.3H), 2.40 (s, 0.3H), 2.37 (s. 0.6H), 1.08 - 1.02 (m, 3H).
Intermediate H2 was separated by chiral Prep-HPLC (separation condition: Column: Chiralpak OJ-H 5 µm 20 * 250 mm; Mobile Phase: Hex : EtOH : DEA = 90 : 10 : 0.3 at 15 mL/min; Temp: 30 °C; Wavelength: 214 nm) to afford H2-A and H2-B as yellow solids. Intermediate H2-A: Chiral analysis (Column: Chiralpak OJ-H 5 µm 4.6 * 250 mm; Mobile Phase: Hex : EtOH : DEA = 85 : 15 : 0.2 at 1.0 mL/min; Temp: 30 °C; Wavelength: 230 nm, RT = 7.251 min). H2-A was certificated to absolute S stereochemistry by the following chemical resolution which is consistent with reported data (J. Med. Chem., 2017, 60 (8), pp 3352–3371). Optical rotation: [a] 20
D - 24º (c 0.10, MeOH).
Intermediate H2-B: Chiral analysis (Column: Chiralpak OJ-H 5 µm 4.6 * 250 mm; Mobile Phase: Hex : EtOH : DEA = 85 : 15 : 0.2 at 1.0 mL/min; Temp: 30 °C; Wavelength: 230 nm, RT = 9.072 min). Optical rotation: [a] 20
D + 35º (c 0.10, MeOH). Intermediate H2-1A
(R)-Ethyl 6-(bromomethyl)-4-(3-fluoro-2-methylphenyl)-2-(thiazol-2-yl)-1,4-dihy dropyrimidine-5-carboxylate was prepared from H2-A using same condition as for H1-1A. LC-MS (ESI): RT = 1.84 min, mass calcd. for C18H17BrFN3O2S 437.0, m/z found 440.0 [M+H]+ . 1H NMR (400 MHz, CDCl3) d 8.22 (s, 0.5H), 7.82 (d, J = 3.2 Hz, 1H), 7.53 (s, 0.4H), 7.44 (s, 0.6H), 7.25 - 7.08 (m, 2.5H), 6.96 - 6.92 (s, 1H), 5.99 (s, 0.6H), 5.93 (s, 0.4H), 4.92 - 4.77 (m, 1.6H), 4.67 - 4.65 (m, 0.4H), 4.13 - 4.07 (m, 2H), 2.53 (s, 1.7H), 2.41 (s, 1.3H), 1.14 (t, J = 7.2 Hz, 3H). Optical rotation: [a] 20
D + 0.093º (c 0.10, MeOH). Intermediate H3 : Methyl 4-(2-chloro-4-fluorophenyl)-6-methyl-2-(thiazol-2-yl)-1,4- dihydropyrimid ine-5-carboxylate (racemic) was prepared using same condition as for H1. LC-MS (ESI): RT = 1.70 min, mass calcd. for C16H13ClFN3O2S 365.04, m/z found 366.1 [M+H]+ .1H NMR (400 MHz, CDCl3) d 7.84 - 7.83 (m, 0.9H), 7.81 - 7.80 (m, 0.8H), 7.55 - 7.50 (m, 0.6H), 7.44 - 7.43 (m, 0,7H), 7.33 - 7.26 (m, 1H), 7.13 - 7.11 (m, 1H), 6.95 - 6.88 (m, 1H), 6.18 (s, 0.7H), 6.05 (s, 0.3H), 3.63 (s, 0.8H), 3.60 (s, 2.2H), 2.57 (s, 0.8H), 2.51 (s, 2.2H).
Racemic H3 (20 g, 95 % purity, 51.9 mmol) was separated by chiral Prep-HPLC (Column: Chiralpak IG 5 µm 30 * 250 mm; Mobile Phase: CO2 : MeOH = 70 : 30 at 55 g/min; Col. Temp: 40 °C; Wavelength: 230 nm, Back pressure: 100 bar) to afford the title compounds H3-A (9.46 g, 95 % purity from NMR, 47 % yield, 100 % ee) and H3-B (9.5 g, 95 % purity from NMR, 48 % yield, 98.0 % ee) as yellow solids.
Intermediate H3-A : LC-MS (ESI): RT = 1.69 min, mass calcd. for C16H13ClFN3O2S 365.0, m/z found 366.0. Chiral analysis (Column: Chiralpak IA 5 µm 4.6 * 250 mm; Mobile Phase: Hex: EtOH = 80 : 20 at 1.0 mL/ min; Temp: 30 °C; Wavelength: 254 nm, RT = 5.593 min).1H NMR (400 MHz, CDCl3) d 7.84 - 7.83 (m, 1H), 7.80 (d, J = 2.8 Hz, 0.7H), 7.52 - 7.50 (m, 0.5H), 7.44 (d, J = 2.8 Hz, 0.7H), 7.34 - 7.30 (m, 1H), 7.15 - 7.11 (m, 1H), 6.96 - 6.88 (m, 1H), 6.19 (s, 0.7H), 6.06 (d, J = 2.4 Hz, 0.3H), 3.63 (s, 0.8H), 3.60 (s, 2.2H), 2.57 (s, 0.8H), 2.51 (s, 2.2H).
Intermediate H3-B: LC-MS (ESI): RT = 1.68 min, mass calcd. for C16H13ClFN3O2S 365.0, m/z found 366.0. Chiral HPLC (Column: Chiralpak IA 5 µm 4.6 * 250 mm; Mobile Phase: Hex: EtOH = 80 : 20 at 1.0 mL/ min; Temp: 30 °C; Wavelength: 254 nm, RT = 6.827 min). 1H NMR (400 MHz, CDCl3) 7.85 - 7.82 (m, 1H), 7.80 (d, J = 3.2 Hz, 0.7H), 7.54 - 7.50 (m, 0.5H), 7.43 (d, J = 3.2 Hz, 0.7H), 7.34 - 7.30 (m, 1H), 7.14 - 7.11 (m, 1H), 6.96 - 6.88 (m, 1H), 6.18 (s, 0.7H), 6.06 (d, J = 2.4 Hz, 0.3H), 3.62 (s, 0.8H), 3.60 (s, 2.2H), 2.57 (s, 0.8H), 2.50 (s, 2.2H). Intermediate H3-1A: methyl 6-(bromomethyl)-4-(2-chloro-4-fluorophenyl)-2-(thiazol-2- yl)-1,4-dihydropyrimidine-5-carboxylate was prepared from H3-A using same condition as for H1-1A.
LC-MS (ESI): RT = 1.802 min, mass calcd. for C16H12BrClFN3O2S 442.9, m/z found 443.9 [M+H]+ . 1H NMR (400 MHz, CDCl3) d 8.29 (br s, 0.3H), 7.84 (d, J = 3.2 Hz, 1H), 7.59 - 7.53 (m, 1.4H), 7.47 (br s, 0.3H), 7.41 - 7.31 (m, 1H), 7.14 (d, J = 8.4 Hz, 1H), 6.99 - 6.90 (m, 1H), 6.18 (s, 0.3H), 6.09 (d, J = 2.0 Hz, 0.7H), 4.93 (d, J = 8.4 Hz, 1H), 4.74 (d, J = 11.2 Hz, 0.3H), 4.58 (d, J = 8.4 Hz, 0.7H), 3.67 (s, 2.1H), 3.65 (s, 0.9H). Intermediate H4: Methyl 4-(3-fluoro-2-methylphenyl)-6-methyl-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate (racemic) was prepared using same condition as for H1. 1 NMR (400 MHz, CDCl3) d 7.93 (d, J = 3.2 Hz, 0.1H), 7.80 - 7.77 (m, 1.8H), 7.52 - 7.50 (m, 0.1H), 7.41 (d, J = 3.2 Hz, 0.9H), 7.20 (br s, 0.1H), 7.16 - 7.00 (m, 2H), 6.94 - 6.87 (m, 1H), 6.00 (s, 0.9H), 5.90 (s, 0.1H), 3.60 (s, 3H), 2.55 - 2.49 (m, 5.8H), 2.40 (br s, 0.2H). A racemic mixture of methyl 4-(3-fluoro-2-methylphenyl)-6-methyl-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate H4 (1.30 g, 95 % purity, 3.58 mmol) was separated by chiral Prep-HPLC (separation condition: Column: Chiralpak AS-H 5 µm 30 * 250 mm; Mobile Phase: Hex : EtOH = 75 : 25 at 15 mL/min; Temp: 30 oC; Wavelength: 214 nm) to afford the title compounds (H4-A)(610 mg, 95 % purity from H NMR, 44 % yield, 100 % stereopure) and (H4-B) (520 mg, 95 % purity from 1H NMR, 40 % yield, 97.7 % stereopure) as yellow oil.
Intermediate H4-A: Chiral analysis (Column: Chiralpak AS 5 µm 4.6 * 250 mm; Mobile Phase: Hex : EtOH = 80 : 20 at 1 mL/min; Temp: 30 oC; Wavelength: 254 nm, RT = 5.247 min). 1 NMR (400 MHz, CDCl3) d 7.93 (d, J = 2.8 Hz, 0.1H), 7.80 (br s, 0.9H), 7.78 (d, J = 2.8 Hz, 1H), 7.52 - 7.50 (m, 0.1H), 7.41 (d, J = 3.2 Hz, 0.9H), 7.10 - 7.02 (m, 2H), 6.92 - 6.87 (m, 1H), 6.00 (s, 0.9H), 5.91 (s, 0.1H), 3.61 (s, 3H), 2.55 (s, 3H), 2.53 (s, 3H).
Intermediate H4-B: Chiral analysis (Column: Chiralpak AS 5 µm 4.6 * 250 mm; Mobile Phase: Hex : EtOH = 80 : 20 at 1 mL/min; Temp: 30 oC; Wavelength: 254 nm, RT = 9.049 min).1H NMR (400 MHz, CDCl3) d 7.78 (d, J = 3.2 Hz, 2H), 7.42 (d, J = 2.4 Hz, 1H), 7.10 - 7.05 (m, 2H), 6.92 - 6.89 (m, 1H), 5.99 (s, 1H), 3.61 (s, 3H), 2.54 (s, 3H), 2.53 (m, 3H). Intermediate H4-1B:
Methyl 6-(bromomethyl)-4-(3-fluoro-2-methylphenyl)-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate was prepared from H4-B using same condition as for H1-1A.1H NMR (400 MHz, CDCl3) d 8.23 (s, 1H), 7.82 (d, J = 3.2 Hz, 1H), 7.53 - 7.44 (m, 1H), 7.12 - 7.07 (m, 2H), 6.93 (s, 1H), 5.98 - 5.94 (m, 1H), 4.89 - 4.66 (m, 2H), 3.65 (s, 3H), 2.53 - 2.41 (m, 3H). Intermediate H5: Methyl 4-(2-chloro-3,4-difluorophenyl)-6-methyl-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate, 1H NMR (400 MHz, CD3OD) d 8.08 (d, J = 2.8 Hz, 0.1H), 7.98 (d, J = 2.8 Hz, 0.1H), 7.93 (d, J = 2.8 Hz, 0.9H), 7.72 (d, J = 2.8 Hz, 0.9H), 7.26 - 7.18 (m, 2H), 6.13 (s, 0.9H), 6.09 (s, 0.1H), 3.61 (s, 3H), 2.53 (s, 3H). Racemic H5 (1.10 g, 2.90 mmol) was separated by chiral Prep-HPLC (separation condition: Column: Chiralpak IC 5 µm 20 * 250 mm; Mobile Phase: Hex : EtOH = 90 : 10 at 18 mL/min; Temp: 30 °C; Wavelength: 214 nm) to afford the title compounds H5-A (450 mg, 41 % yield, 100 % stereopure) and H5-B (450 m g, 41 % yield, 99.8 % stereopure) as yellow solids.
Intermediate H5-A: Chiral analysis (Column: Chiralpak IC 5 µm 4.6 * 250 mm; Mobile Phase: Hex : EtOH = 90 : 10 at 1.0 mL/min; Temp: 30 °C; Wavelength: 254 nm, RT = 6.457 min).
Intermediate H5-B: Chiral analysis (Column: Chiralpak IC 5 µm 4.6 * 250 mm; Mobile Phase: Hex : EtOH = 90 : 10 at 1.0 mL/min; Temp: 30 °C; Wavelength: 254 nm, RT = 7.641 min). Intermediate H5-1A: Methyl 6-(bromomethyl)-4-(2-chloro-3,4-difluorophenyl)-2- (thiazol-2-yl)-1,4-dihydropyrimidine-5-carboxylate was prepared from H5-A using same condition as for H1-1A, 1H NMR (400 MHz, CD3OD) d 7.92 (d, J = 3.2 Hz, 1H), 7.80 (d, J = 3.2 Hz, 0.5H), 7.70 (d, J = 3.2 Hz, 0.5H), 7.32 - 7.17 (m, 2H), 6.11 (s, 0.5H), 6.09 (s, 0.5H), 4.91 (d, J = 10.0 Hz, 0.5H), 4.81 (d, J = 10.0 Hz, 1H), 4.57 (d, J = 8.4 Hz, 0.5H), 3.64 (s, 1.5H), 3.62 (s, 1.5H). Intermediate H6: Methyl 4-(3,4-difluoro-2-methylphenyl)-6-methyl-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate
LC-MS (ESI): RT = 1.58 min, mass calcd. for C17H15F2N3O2S 363.3, m/z found 364.0 [M+H]+ .1H NMR (400 MHz, CDCl3) d 7.80 - 7.78 (m, 2H), 7.42 (d, J = 3.2 Hz, 1H), 7.00 - 6.85 (m, 2H), 5.93 (s, 1H), 3.61 (s, 3H), 2.58 (s, 1.5H), 2.57 (s, 1.5H), 2.53 (s, 1.5H), 2.51 (s, 1.5H).
Racemic H6 (1.00 g, 90 % purity, 2.48 mmol) was separated by chiral Prep-HPLC (separation condition: Column: Chiralpak IH 5 µm 30 * 250 mm; Mobile Phase: Hex : EtOH = 90 : 10 at 18 mL/min; Temp: 30 oC; Wavelength: 214 nm) to afford the desired products H6-A (400 mg, 90 % purity from 1H NMR, 40 % yield, 100 % stereopure) and H6-B (400 mg, 95 % purity from 1H NMR, 42 % yield, 99.9 % stereopure) as yellow solids.
Intermediate H6-A: Chiral analysis (Column: Chiralpak IH 5 µm 4.6 * 150 mm; Mobile Phase: Hex : EtOH = 90 : 10 at 1 mL/min; Temp: 30 oC; Wavelength: 230 nm, RT = 4.809 min).1H NMR (400 MHz, CDCl3) d 7.84 (br s, 1H), 7.78 (d, J = 3.2 Hz, 1H), 7.42 (d, J = 3.2 Hz, 1H), 6.96 - 6.86 (m, 2H), 5.93 (s, 1H), 3.61 (s, 3H), 2.57 (d, J = 1.6 Hz, 3H), 2.52 (s,
3H).
Intermediate H6-B: Chiral analysis (Column: Chiralpak IH 5 µm 4.6 * 150 mm; Mobile
Phase: Hex : EtOH = 90 : 10 at 1 mL/min; Temp: 30 oC; Wavelength: 230 nm, RT = 7.018
min).1H NMR (400 MHz, CDCl3) d 7.82 (br s, 1H), 7.79 (d, J = 3.2 Hz, 1H), 7.42 (d, J = 3.2
Hz, 1H), 6.97 - 6.88 (m, 2H), 5.93 (s, 1H), 3.61 (s, 3H), 2.58 (d, J = 2.0 Hz, 3H), 2.52 (s,
3H). Intermediate H6-1B: Methyl 6-(bromomethyl)-4-(3,4-difluoro-2-methylphenyl)-2- (thiazol-2-yl)-1,4-dihydropyrimidine-5-carboxylate was prepared from H6-B using same
condition as for H1-1A, 1H NMR (400 MHz, CDCl3) d 8.24 (s, 1H), 7.83 (d, J = 3.6 Hz, 1H),
7.54 - 7.45 (m, 1H), 7.00 - 6.93 (m, 2H), 5.91 (s, 1H), 4.94 - 4.80 (s, 21H), 3.66 (s, 3H), 2.56
- 2.45 (m, 3H). Intermediate H7: ethyl 4-(2-bromo-4-fluorophenyl)-6-methyl-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate, LC-MS (ESI): RT = 3.63 min, mass calcd. for
C17H15BrFN3O2S 423.0, m/z found 423.9 [M+H]+ .1H NMR (400 MHz, DMSO-d6) d 9.95 (s,
1H), 7.97 (d, J = 2.8 Hz, 1H), 7.90 (d, J = 3.2 Hz, 1H), 7.57 - 7.54 (m, 1H), 7.37 - 7.33 (m,
1H), 7.26 - 7.23 (m, 1H), 5.96 (s, 0.9H), 5.89 (s, 0.1H), 3.93 (q, J = 7.2 Hz, 2H), 2.47 (s,
2.7H), 2.39 (s, 0.3H), 1.03 (t, J = 7.2 Hz, 3H). Racemic H7 (55.7 g, 127 mmol) was separated by chiral Prep-HPLC (separation condition:
Column: OZ-H 5 µm 30 * 250 nm; Mobile Phase: CO2 : MeOH (0.1 % NH .
3H2O) = 70 : 30 at
60 mL/min; Temp: 38 oC; Wavelength: 254 nm) to give the title compounds H7-A (30.0 g,
100 % purity, 99.2 % ee, 56 % yield) as yellow solids and H7-B (27.0 g, 100 % purity, 99.5
% ee, 50 % yield) as light brown oil.
Intermediate H7-A: LC-MS (ESI): RT = 1.66 min, mass calcd. for C17H15BrFN3O2S 423.0,
m/z found 424.0 [M+H]+ . Chiral analysis (Column: Chiralpak IE 5 µm 4.6 * 250 mm; Mobile
Phase: Hex : EtOH = 90 : 10 at 1 mL/min; Temp: 30 oC; Wavelength: 230 nm, RT = 8.259
min).1H NMR (400 MHz, CDCl3) d 7.83 - 7.80 (m, 1.7H), 7.51 - 7.43 (m, 1.3H), 7.35 - 7.30
(m, 2H), 6.99 - 6.94 (m, 1H), 6.17 (s, 0.7H), 6.05 (s, 0.3H), 4.08 - 4.01 (m, 2H), 2.57 (s,
0.8H), 2.52 (s, 2.2H), 1.13 (t, J = 7.2 Hz, 3H). Optical rotation: [a] D 25 - 36° (c 0.30, MeOH): Intermediate H7-B: LC-MS (ESI): RT = 1.65 min, mass calcd. for C17H15BrFN3O2S 423.0, +
m/z found 424.0 [M+H] . Chiral analysis (Column: Chiralpak IE 5 µm 4.6 * 250 mm; Mobile Phase: Hex : EtOH = 90 : 10 at 1 mL/min; Temp: 30 oC; Wavelength: 230 nm, RT = 10.485 min).1H NMR (400 MHz, CDCl3) 7.85 - 7.79 (m, 1.7H), 7.57 - 7.43 (m, 1.3H), 7.35 - 7.30 (m, 2H), 6.99 - 6.94 (m, 1H), 6.17 (s, 0.7H), 6.05 (s, 0.3H), 4.11 - 4.02 (m, 2H), 2.57 (s, 0.8H), 2.51 (s, 2.2H),1.13 (t, J = 7.2 Hz, 3H). Intermediate H7-1A: Ethyl 4-(2-bromo-4-fluorophenyl)-6-(bromomethyl)-2-(thiazol-2- yl)-1,4-dihydropyrimidine-5-carboxylate was prepared from H7-A using same condition as for H1-1A, 1H NMR (400 MHz, CDCl3) d 8.23 (br s, 0.3H), 7.87 - 7.82 (m, 1H), 7.54 - 7.53 (m, 1H), 7.51 (br s, 0.7H), 7.45 - 7.39 (m, 1H), 7.34 - 7.31 (m, 1H), 7.05 - 6.98 (m, 1H), 6.18 (s, 0.3H), 6.08 (d, J = 2.4 Hz, 0.7H), 5.00 - 4.92 (m, 1H), 4.75 (d, J = 10.8 Hz, 0.3H), 4.59 (d, J = 8.4 Hz, 0.7H), 4.14 - 4.09 (m, 2H), 1.16 (t, J = 6.8 Hz, 3H). Intermediate H8: Ethyl 4-(2-chloro-3,4-difluorophenyl)-6-methyl-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate, 1H NMR (400 MHz, CDCl3)d7.83 - 7.81 (m, 1.8H), 7.52 - 7.44 (m, 1.2H), 7.13 - 7.10 (m, 1H), 7.08 - 7.00 (m, 1H), 6.20 (s, 0.8H), 6.08 (s, 0.2H), 4.11 - 4.00 (m, 2H), 2.57 (s, 0.5H), 2.51 (s, 2.5H), 1.13 (t, J = 7.2 Hz, 3H). Racemic H8 (1.00 g, 2.51 mmol) was separated by chiral Prep-HPLC (Column: Chiralpak IC 5 µm 20 * 250 mm; Mobile Phase: Hex : EtOH = 90 : 10 at 18 mL/min; Temp: 30 °C; Wavelength: 214 nm) to give the desired compound H8-A (353 mg, 35 % yield, 98.1 % stereopure) and H8-B (321 mg, 32 % yield, 99.8 % stereopure) as yellow solids.
Intermediate H8-A: Chiral analysis (Column: Chiralpak IC 5 µm 4.6 * 250 mm; Mobile Phase: Hex : EtOH = 90 : 10 at 1.0 mL/min; Temp: 30 °C; Wavelength: 254 nm, RT = 5.901 min).
Intermediate H8-B: Chiral analysis (Column: Chiralpak IC 5 µm 4.6 * 250 mm; Mobile Phase: Hex : EtOH = 90 : 10 at 1.0 mL/min; Temp: 30 °C; Wavelength: 254 nm, RT = 6.914 min). Intermediate H8-1: Ethyl 6-(bromomethyl)-4-(2-chloro-3,4-difluorophenyl)-2-(thiazol-2- yl)-1,4-dihydropyrimidine-5-carboxylate was prepared from H8 using same condition as for H1-1A, 1H NMR (400 MHz, CDCl3) d 8.25 (s, 0.3H), 7.85 (d, J = 2.8 Hz, 1H), 7.54 - 7.44 (m, 1.5H), 7.20 - 7.04 (m, 2.2H), 6.19 - 6.11 (m, 1H), 4.98 - 4.95 (m, 1H), 4.74 - 4.72 (m, 0.4H), 4.58 - 4.56 (m, 0.6H), 4.13 - 4.11 (m, 2H), 1.19 - 1.15 (m, 3H). Intermediate H8-1A: Ethyl 6-(bromomethyl)-4-(2-chloro-3,4-difluorophenyl)-2-(thiazol- 2-yl)-1,4-dihydropyrimidine-5-carboxylate was prepared from H8-A using same condition as for H1-1A, 1H NMR (400 MHz, CDCl3) d 8.25 (s, 0.3H), 7.85 (d, J = 3.2 Hz, 1H), 7.54 (d, J = 3.2 Hz, 0.6H), 7.47 -7.45 (m, 0.9H), 7.22 - 7.00 (m, 2.2H), 6.19 (s, 0.4H), 6.11 (d, J = 2.4 Hz, 0.6H), 4.97 (d, J = 11.2 Hz, 0.4H), 4.94 (d, J = 8.8 Hz, 0.6H), 4.73 (d, J = 11.2 Hz, 0.4H), 4.56 (d, J = 8.4 Hz, 0.6H), 4.16 - 4.04 (m, 2H), 1.19 - 1.13 (m, 3H). Intermediate H9: Ethyl 4-(3,4-difluoro-2-methylphenyl)-6-methyl-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate, LC-MS (ESI): RT = 1.78 min, mass calcd. for C18H17F2N3O2S 377.4, m/z found 378.1 [M+H]+ . 1H NMR (400 MHz, CDCl3) d 7.81- 7.76 (m, 2H), 7.42 (d, J = 3.2 Hz, 1H), 6.98 - 6.86 (m, 2H), 5.94 (s, 1H), 4.11 - 4.00 (m, 2H), 2.58 (s, 1.5H), 2.57 (s, 1.5H), 2.52 (s, 3H), 1.14 (t, J = 7.2 Hz, 3H). Racemic H9 (1.20 g, 90 % purity, 2.86 mmol) was separated by chiral Prep-HPLC (separation condition: Column: Chiralpak IC 5 µm 30 * 250 mm; Mobile Phase: Hex : IPA = 95 : 5 at 18 mL / min; Temp: 30 oC; Wavelength: 214 nm) to afford the desired compounds H9-A (580 mg, 90 % purity, 48 % yield, 97.8 % ee) as yellow solids and H9-B (500 mg, 90 % purity, 42 % yield, 99.4 % ee) as yellow solids.
Intermediate H9-A: Chiral analysis (Column: Chiralpak IC 5 µm 4.6 * 250 mm; Mobile Phase: Hex : IPA = 95 : 5 at 1 mL/min; Temp: 30 oC; Wavelength: 230 nm, RT = 7.550 min). 1H NMR (400 MHz, CDCl3) d 7.79 - 7.77 (m, 2H), 7.42 (d, J = 3.6 Hz, 1H), 7.00 - 6.88 (m, 2H), 5.94 (s, 1H), 4.08 - 4.01 (m, 2H), 2.58 (s, 2.5H), 2.55 (s, 0.5H), 2.52 (s, 3H), 1.14 (t, J = 7.2 Hz, 3H).
Intermediate H9-B: Chiral analysis (Column: Chiralpak IC 5 µm 4.6 * 250 mm; Mobile Phase: Hex : IPA = 95 : 5 at 1 mL/min; Temp: 30 oC; Wavelength: 230 nm, RT = 8.495 min). 1H NMR (400 MHz, CDCl3) d 7.79 - 7.75 (m, 2H), 7.42 (d, J = 2.8 Hz, 1H), 6.98 - 6.86 (m, 2H), 5.94 (s, 1H), 4.08 - 4.00 (m, 2H), 2.58 (d, J = 2.0 Hz, 3H), 2.52 (s, 3H), 1.14 (t, J = 7.2 Hz, 3H). Intermediate H9-1A: Ethyl 6-(bromomethyl)-4-(3,4-difluoro-2-methylphenyl)-2- (thiazol-2-yl)-1,4-dihydropyrimidine-5-carboxylate was prepared from H9-A using same condition as for H1-1A, LC-MS (ESI): RT = 1.85 min, mass calcd. for C18H16BrF2N3O2S 455.0, m/z found 456.0 [M+H]+ .1H NMR (400 MHz, CDCl3) d 7.83 (d, J = 2.8 Hz, 1H), 7.54 (d, J = 2.8 Hz, 0.4H), 7.44 (d, J = 2.8 Hz, 0.6H), 7.21 - 7.06 (m, 1H), 7.02 - 6.89 (m, 2H), 5.93 (s, 0.6H), 5.87 (d, J = 2.0 Hz, 0.4H), 4.93 (d, J = 11.6 Hz, 0.6H), 4.81 - 4.78 (m, 1H), 4.61 (d, J = 8.4 Hz, 0.4H), 4.11 - 4.06 (m, 2H), 2.56 (d, J = 2.0 Hz, 2H), 2.45 (d, J = 2.0 Hz, 1H), 1.19 - 1.13 (m, 3H). Intermediate H9-1B: Ethyl 6-(bromomethyl)-4-(3,4-difluoro-2-methylphenyl)-2-(thiazol- 2-yl)-1,4-dihydropyrimidine-5-carboxylate was prepared from H9-B using same condition as for H1-1A, LC-MS (ESI): RT = 1.85 min, mass calcd. for C18H16BrF2N3O2S 455.0, m/z found 456.0 [M+H]+ .1H NMR (400 MHz, CDCl3) d 7.83 (d, J = 3.2 Hz, 1H), 7.54 - 7.44 (m, 1H), 7.20 - 7.10 (m, 1H), 7.00 - 6.89 (m, 2H), 5.92 - 5.88 (m, 1H), 4.91 - 4.63 (m, 2H), 4.11 - 4.08 (m, 2H), 2.56 (s, 2H), 2.45 (s, 1H), 1.17 - 1.14 (m, 3H). Intermediate H10: Methyl 4-(2-bromo-4-fluorophenyl)-6-methyl-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate, 1H NMR (400 MHz, CDCl3) d 7.89 - 7.75 (m, 1.7H), 7.62 - 7.55 (m, 0.3H), 7.49 - 7.40 (m, 1H), 7.33 - 7.29 (m, 2H), 7.00 - 6.94 (m, 1H), 6.15 (s, 0.7H), 6.03 (s, 0.3H), 3.61 (s, 3H), 2.52 (s, 3H).
Racemic H10 (1.80 g, 90 % purity, 3.95 mmol) was separated by chiral Prep-HPLC (Column: Chiralpak IG 5 µm 20 mm * 250 mm; Mobile Phase: CO2 : MeOH = 75 : 25 at 50 g/min; Col. Temp: 40 oC; Wavelength: 230 nm, Back pressure: 100 bar) to afford the title compounds H10-A (850 mg, 90 % purity from 1H NMR, 47 % yield, 99.6 % ee) and H10-B (850 mg, 90 % purity from 1H NMR, 47 % yield, 99.4 % ee) as yellow solids.
Intermediate H10-A: LC-MS (ESI): RT = 1.717 min, mass calcd. for C16H13BrFN3O2S 409.0, m/z found 410.0 [M+H]+ . Chiral analysis (Column: Chiralpak IG 5 µm 4.6 * 250 mm; Mobile Phase: CO2 : MeOH = 75 : 25 at 3 g/min; Temp: 40 oC; Wavelength: 230 nm; Back pressure: 100 bar, RT = 3.92 min).1H NMR (400 MHz, CDCl3) d 7.87 - 7.84 (m, 1H), 7.80 (d, J = 3.2 Hz, 0.7H), 7.57 (br s, 0.3H), 7.51 (d, J = 3.2 Hz, 0.3H), 7.44 (d, J = 3.2 Hz, 0.7H), 7.34 - 7.29 (m, 2H), 7.01 - 6.93 (m, 1H), 6.16 (s, 0.7H), 6.02 (d, J = 2.4 Hz, 0.3H), 3.62 (s, 1H), 3.60 (s, 2H), 2.57 (s, 1H), 2.51 (s, 2H). Intermediate H10-B: LC-MS (ESI): RT = 1.713 min, mass calcd. for C16H13BrFN3O2S +
409.0, m/z found 410.0 [M+H] . Chiral analysis (Column: Chiralpak IG 5µm 4.6 * 250 mm; Mobile Phase: CO2 : MeOH = 75 : 25 at 3 g/min; Temp: 40 oC; Wavelength: 230 nm; Back pressure: 100 bar, RT = 4.92 min). 1H NMR (400 MHz, CDCl3) d 7.88 - 7.83 (m, 1H), 7.80 (d, J = 3.2 Hz, 0.7H), 7.58 (br s, 0.3H), 7.50 (d, J = 3.2 Hz, 0.3H), 7.44 (d, J = 3.2 Hz, 0.7H), 7.34 - 7.29 (m, 2H), 7.01 - 6.93 (m, 1H), 6.16 (s, 0.7H), 6.02 (d, J = 2.0 Hz, 0.3H), 3.62 (s, 1H), 3.60 (s, 2H), 2.57 (s, 1H), 2.51 (s, 2H). Intermediate H10-1A: Methyl 4-(2-bromo-4-fluorophenyl)-6-(bromomethyl)-2-(thiazol- 2-yl)-1,4-dihydropyrimidine-5-carboxylate was prepared from H10-A using same condition as for H1-1A, 1H NMR (400 MHz, CDCl3) d 7.85 (d, J = 3.2 Hz, 1H), 7.52 (d, J = 2.8 Hz, 1H), 7.40 - 7.36 (m, 1H), 7.34 - 7.32 (m, 1H), 7.04 - 6.99 (m, 1H), 6.09 (s, 1H), 4.95 (d, J = 9.2 Hz, 1H), 4.63 (d, J = 8.4 Hz, 1H), 3.67 (s, 3H). Intermediate H10-1B: Methyl 4-(2-bromo-4-fluorophenyl)-6-(bromomethyl)-2-(thiazol- 2-yl)-1,4-dihydropyrimidine-5-carboxylate was prepared from H10-B using same condition as for H1-1A, 1H NMR (400 MHz, CDCl3) d 7.85 (d, J = 3.2 Hz, 1H), 7.60 (br s, 1H), 7.56 - 7.47 (m, 1H), 7.40 - 7.37 (m, 1H), 7.34 - 7.31 (m, 1H), 7.03 - 6.99 (m, 1H), 6.08 (s, 1H), 4.94 (d, J = 9.2 Hz, 1H), 4.64 (br s, 1H), 3.67 (s, 3H). Compound 1A: 3-(7-(((S)-5-(ethoxycarbonyl)-6-(3-fluoro-2-methylphenyl)-2-(thiazol-2- yl)-3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)-2,2-dimethylpropanoic acid (single enantiomer)
Figure imgf000101_0001
Preparation of 2,2-dimethyl-3-(3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)-yl)propanoic
Figure imgf000102_0001
Intermediate S1-1: 1-benzyl 4-(tert-butyl) (S)-2-(hydroxymethyl)piperazine-1,4- dicarboxylate
To the solution of (S)-tert-butyl 3-(hydroxymethyl)piperazine-1-carboxylate (10.0 g, 46.2 mmol) and saturated sodium bicarbonate aqueous solution (64 mL) in tetrahydrofuran (106 mL) was added dropwise benzyl chloroformate (9.16 g, 53.7 mmol) at 0 oC under nitrogen atmosphere. After stirred at room temperature overnight, the mixture was concentrated under reduced pressure to remove tetrahydrofuran, added water (50 mL) and extracted with ethyl acetate (50 mL) for three times. The combined organic layers were washed with brine (100 mL), dried over Na2SO4(s) and filtered. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether : ethyl acetate = 4 : 1 to 1 : 1) to give the title compound S1-1 (14.8 g, 82 % yield) as colorless oil. LC-MS (ESI): RT = 2.056 min, mass calcd. for C18H26N2O5350.2, m/z found 373.1[M+Na] + .1H NMR (400 MHz, CDCl3) d 7.43 - 7.30 (m, 5H), 5.17 (d, J = 12.4 Hz, 1H), 5.12 (d, J = 12.4 Hz, 1H), 4.31 - 4.12 (m, 2H), 4.07 - 3.84 (m, 2H), 3.73 - 3.50 (m, 2H), 3.15 - 2.79 (m, 3H), 1.47 (s, 9H). Intermediate S1-2: 1-Benzyl 4-tert-butyl 2-formylpiperazine-1,4-dicarboxylate (mixture of 2 enantiomers)
To a solution of anhydrous dimethyl sulfoxide (38.5 g, 493 mmol) in anhydrous dichloromethane (300 mL) was added dropwise oxalyl dichloride (57.8 g, 455 mmol) at -78 oC. After stirred at -78 oC under nitrogen atmosphere for 1.5 hours, a solution of (S)-1-benzyl 4-tert-butyl 2-(hydroxymethyl)piperazine-1,4-dicarboxylate S1-1 (28.8 g, 90 % purity, 73.9 mmol) in anhydrous dichloromethane (50 mL) was added dropwise. The mixture was stirred o
at -78 C for 1.5 hours and triethylamine (60.9 g, 602 mmol) was then added. After stirred at room temperature for 0.5 hour, the reaction mixture was diluted with ice water (100 mL) and neutralized with 1 M hydrochloride aqueous solution to pH 6 ~ 7, extracted with dichloromethane (150 mL) for three times. The combined organic phases were washed with saturated sodium bicarbonate (100 mL) and brine (100 mL) for three times, dried over Na2SO4(s), filtered and evaporated to give the title compound S1-2 (28.8 g, 89 % yield) as light yellow oil. LC-MS (ESI): RT = 1.68 min, mass calcd. for C18H24N2O5348.2, m/z found
+ 1
293.1[M+H-56] . H NMR (400 MHz, CDCl3) d 9.60 (d, J = 7.2 Hz, 1H), 7.37 - 7.29 (m, 5H), 5.18 (s, 1H), 5.14 (s, 1H), 4.91 - 4.51 (m, 2H), 4.07 - 3.82 (m, 2H), 3.29 - 3.07 (m, 2H), 3.00 - 2.79 (m, 1H), 1.44 (s, 9H). Intermediate S1-3: 1-Benzyl 4-tert-butyl 2-(((3-ethoxy-2,2-dimethyl-3- oxopropyl)amino)methyl)piperazine-1,4-dicarboxylate (mixture of 2 enantiomers) To a solution of ethyl 3-amino-2,2-dimethylpropanoate hydrochloride (17.7 g, 97.4 mmol) in methanol (200 mL) was added triethylamine (9.86 g, 97.4 mmol) at room temperature. After stirred at room temperature under nitrogen atmosphere for 0.5 hour, a solution of 1-benzyl 4- tert-butyl 2-formylpiperazine-1,4-dicarboxylate S1-2 (29.5 g, 80 % purity, 67.7 mmol) in methanol (100 mL) was added and stirred at room temperature for 1 hour. Then sodium cyanoborohydride (9.84 g, 157 mmol) was added at 0 oC and the mixture was stirred at room temperature for 2 hours, quenched with ice water (100 mL), removed methanol under vacuo and extracted with ethyl acetate (100 mL) for three times. The combined organic layers were dried over Na2SO4(s) and filtered. The filtrate was concentrated and purified by silica gel chromatography (petreleum ether : ethyl acetate = 8 : 1 to 2 : 1) to give the title compound S1-3 (29.6 g, 82 % yield) as light yellow oil. LC-MS (ESI): RT = 2.533 min, mass calcd. for C25H39N3O6477.3, m/z found 478.3 [M+H]+ .1H NMR (400 MHz, CDCl3) d 7.39 - 7.31 (m, 5H), 7.27 (s, 1H), 5.83 - 5.77 (m, 0.7H), 5.67 - 5.62 (m, 0.3H), 5.16 (s, 2H), 4.30 (t, J = 6.4 Hz, 2H), 3.68 (s, 3H), 2.89 (t, J = 7.2 Hz, 0.5H), 2.86 (t, J = 6.4 Hz, 1.5H), 2.71 - 2.62 (m, 1H), 2.46 - 2.42 (m, 2H), 2.40 - 2.33 (m, 2H), 2.31 (s, 1H), 2.30 (s, 2H), 2.17 - 2.12 (m, 1H), 1.92 - 1.79 (m, 1H). Intermediate S1-4: tert-Butyl 3-(((3-ethoxy-2,2-dimethyl-3- oxopropyl)amino)methyl)piperazine-1-carboxylate (mixture of 2 enantiomers) To a solution of 1-benzyl 4-tert-butyl 2-(((3-ethoxy-2,2-dimethyl-3-oxopropyl)amino) methyl)piperazine-1,4-dicarboxylate S1-3 (17.6 g, 33.2 mmol) in ethanol (300 mL) was added 20 % wt. palladium hydroxide on carbon (8.0 g, 11.4 mmol) and then the mixture was stirred at 60 oC under 60 psi hydrogen atmosphere overnight. Another 20 % palladium hydroxide on carbon (500 mg, 0.712 mmol) was added and stirring continued at 60 oC under 60 psi hydrogen atmosphere overnight. Then the reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give the title compound S1-4 (11.7 g, 82 % yield) as colorless oil. LC-MS (ESI): RT = 1.362 min, mass calcd. for C17H33N3O4343.2, m/z found 44.11 [M+H] . N HMR1 (400 MHz, CDCl3) d 4.12 (q, J = 7.2 Hz, 2H), 4.02 - 3.80 (m, 2H), 2.99 - 2.96 (m, 1H), 2.94 - 2.81 (m, 1H), 2.74 - 2.63 (m, 4H), 2.60 - 2.47 (m, 3H), 1.46 (s, 9H), 1.25 (t, J = 7.2 Hz, 3H), 1.19 (s, 3H), 1.17 (s, 3H). Intermediate S1-5: tert-Butyl 2-(3-ethoxy-2,2-dimethyl-3-oxopropyl)-3- thioxohexahydroimidazo[1,5-a]pyrazine-7(1H)-carboxylate
To a solution of tert-butyl 3-(((3-ethoxy-2,2-dimethyl-3-oxopropyl)amino)methyl)pip erazine-1-carboxylate S1-4 (3.70 g, 8.62 mmol) and triethylamine (2.72 g, 26.9 mmol) in dichloromethane (25 mL) was added a solution of thiophosgene (1.48 g, 12.9 mmol) in dichloromethane (5 mL) at 0 oC under nitrogen atmosphere. After stirred at room temperature overnight, the mixture was diluted with ice water (20 mL) and extracted with dichloromethane (15 mL) for three times. The combined organic layers were washed with brine (30 mL), dried over Na2SO4(s) and filtered. The filtrate was concentrated and purified by C18 column (acetonitrile : water = 5 % to 100 %) to give the title compound S1-5 (2.1 g, 57 % yield) as white solids. LC-MS (ESI): RT = 2.380 min, mass calcd. for C18H31N3O4S 385.2, m/z found 386.2 [M+H]+ .1H NMR (400 MHz, CDCl3) 4.49 - 4.45 (m, 1H), 4.16 (q, J = 7.2 Hz, 2H), 4.11 - 4.10 (m, 1H), 4.08 - 4.00 (m, 1H), 3.94 (d, J = 14.4 Hz, 1H), 3.87 (d, J = 14.0 Hz, 1H), 3.78 - 3.69 (m, 1H), 3.60 (t, J = 9.6 Hz, 1H), 3.11 - 3.07 (m, 1H), 3.03 - 2.99 (m, 1H), 2.92 - 2.78 (m, 1H), 2.67 - 2.51 (m, 1H), 1.46 (s, 9H), 1.28 (t, J = 7.2 Hz, 3H), 1.25 (s, 3H), 1.24 (s, 3H). A racemic mixture of tert-butyl 2-(3-ethoxy-2,2-dimethyl-3-oxopropyl)-3-thioxohexa hydroimidazo[1,5-a]pyrazine-7(1H)-carboxylate S1-5 (7.3 g, 90 % purity, 17.0 mmol) was separated by chiral Prep-HPLC (separation condition: Column: Chiralpak IF 5 µm 20 * 250 mm; Mobile Phase: Hex : EtOH : DEA = 80 : 20 : 0.3 at 15 mL/ min; Temp: 30 °C; Wavelength: 230 nm) to afford the title compound S1-5A (4.38 g) as white solids and S1-5B (1.89 g) as white solids.
S1-5A: LC-MS (ESI): RT = 1.74 min, mass calcd. for C18H31N3O4S 385.2, m/z found 386.3 [M+H]+ . Chiral analysis (Column: Chiralpak IF 5 µm 4.6 * 250 mm; Mobile Phase: Hex : EtOH : DEA = 80 : 20 : 0.2 at 1 mL / min; Temp: 30 °C; Wavelength: 254 nm, RT = 9.710 min).1H NMR (400 MHz, CDCl3) d 4.48 - 4.46 (m, 1H), 4.28 - 4.18 (m, 1H), 4.16 (q, J = 7.2 Hz, 2H), 4.11 - 4.00 (m, 1H), 3.94 (d, J = 14.0 Hz, 1H), 3.87 (d, J = 14.40 Hz, 1H), 3.78 - 3.679 (m, 1H), 3.610 (t, J = 9.6 Hz, 1H), 3.11 - 3.07 (m, 1H), 3.03 - 2.97 (m, 1H), 2.92 - 2.75 (m, 1H), 2.69 - 2.51(m, 1H), 1.47 (s, 9H), 1.28 (t, J = 7.2 Hz, 3H), 1.25 (s, 3H), 1.24 (s, 3H). S1-5B: LC-MS (ESI): RT = 1.74 min, mass calcd. for C18H31N3O4S 385.2, m/z found 386.3 [M+H]+ . Chiral analysis: (Column: Chiralpak IF 5 µm 4.6 * 250 mm; Mobile Phase: Hex : EtOH : DEA = 80 : 20 : 0.2 at 1 mL / min; Temp: 30 °C; Wavelength: 254 nm, RT = 7.397 min).1H NMR (400 MHz, CDCl3) d 4.49 - 4.46 (m, 1H), 4.33 - 4.18 (m, 1H), 4.16 (q, J = 7.2 Hz, 2H), 4.11 - 3.99 (m, 1H), 3.94 (d, J = 14.4 Hz, 1H), 3.87 (d, J = 14.0 Hz, 1H), 3.79 - 3.69 (m, 1H), 3.60 (t, J = 9.6 Hz, 1H), 3.11 - 3.07 (m, 1H), 3.03 - 2.97 (m, 1H), 2.92 - 2.75 (m, 1H), 2.68 - 2.50 (m, 1H), 1.47 (s, 9H), 1.28 (t, J = 7.2 Hz, 3H), 1.25 (s, 3H), 1.24 (s, 3H). Intermediate S1-6A: 3-(7-(tert-butoxycarbonyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin- 2(3H)-yl)-2,2-dimethylpropanoic acid To a solution of tert-butyl 2-(3-ethoxy-2,2-dimethyl-3-oxopropyl)-3-thioxohexahy droimidazo[1,5-a]pyrazine-7(1H)-carboxylate S1-5A (4.38 g, 10.2 mmol) in methanol (30 mL) and water (10 mL) was added sodium hydroxide (1.43 g, 35.8 mmol) under nitrogen atmosphere at 0 oC. After stirred at room temperature for 6 hours, the mixture was added sodium hydroxide (700 mg, 17.5 mmol) and stirred at 60 oC for 4 hours. Then the reaction was diluted with water (20 mL), removed methanol under vacuo and extracted with ethyl acetate (20 mL) twice. The combined aqueous phase were acidified with saturated citric acid aqueous solution to pH 3 ~ 4, extracted with ethyl acetate (20 mL) for three times. The combined organic layers were washed with brine (30 mL), dried over Na2SO4(s) and filtered. The filtrate was concentrated to give the title compound S1-6A (3.6 g, 90 % purity from 1H NMR, 89 % yield) as white solids. LC-MS (ESI): RT = 1.612 min, mass calcd. For C16H27N3O4S 357.2, m/z found 358.2 [M+H]+ .1H NMR (400 MHz, DMSO-d6) d 12.47 (br s, 1H), 4.25 - 4.21 (m, 1H), 4.06 - 4.02 (m, 1H), 3.95 - 3.92 (m, 1H), 3.81 (d, J = 14.0 Hz, 1H), 3.79 - 3.74 (m, 1H), 3.73 (d, J = 13.6 Hz, 1H), 3.65 (t, J = 9.6 Hz, 1H), 3.18 - 3.13 (m, 1H), 2.99 - 2.92 (m, 1H), 2.80 - 2.54 (m, 2H), 1.41 (s, 9H), 1.12 (s, 3H), 1.11 (s, 3H). Intermediate S1-6B: 3-(7-(tert-Butoxycarbonyl)-3-thioxohexahydroimidazo[1,5- a]pyrazin-2(3H)-yl)-2,2-dimethylpropanoic acid
To a solution of tert-butyl 2-(3-ethoxy-2,2-dimethyl-3-oxopropyl)-3-thioxohexa
hydroimidazo[1,5-a]pyrazine-7(1H)-carboxylate S1-5B (810 mg, 1.89 mmol) in methanol (15 mL) and water (5 mL) was added sodium hydroxide (263 mg, 6.58 mmol) under nitrogen atmosphere at 0 oC. After stirred at room temperature for 6 hours, the mixture was added sodium hydroxide (130 mg, 3.25 mmol) and stirred at 60 oC for 4 hours. Then the reaction was diluted with water (10 mL), removed methanol under vacuo and extracted with ethyl acetate (20 mL) twice. The combined aqueous phase were acidified with saturated citric acid aqueous solution to pH 3 ~ 4, extracted with ethyl acetate (20 mL) for three times. The combined organic layers were washed with brine (20 mL), dried over Na2SO4(s) and filtered. The filtrate was concentrated to give the title compound S1-6B (650 mg, 87 % yield) as white solids. LC-MS (ESI): RT = 1.654 min, mass calcd. For C16H27N3O4S 357.2, m/z found 358.2 [M+H]+ .1H NMR (400 MHz, DMSO-d6) d 12.46 (br s, 1H), 4.25 - 4.21 (m, 1H), 4.10 - 4.00 (m, 1H), 3.95 - 3.92 (m, 1H), 3.81 (d, J = 13.6 Hz, 1H), 3.79 - 3.74(m, 1H), 3.73 (d, J = 14.0 Hz, 1H), 3.65 (t, J = 10.0 Hz, 1H), 3.18 - 3.14(m, 1H), 2.99 - 2.92 (m, 1H), 2.80 - 2.55 (m, 2H), 1.41 (s, 9H), 1.12 (s, 3H), 1.11 (s, 3H). Intermediate S1-A: 2,2-Dimethyl-3-(3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)propanoic acid hydrochloride
3-(7-(tert-butoxycarbonyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)-yl)-2,2- dimethylpropanoic acid S1-6A (3.6 g, 9.06 mmol) was added into 3 M hydrochloride in ethyl acetate (50 mL, 150 mmol). The reaction was stirred at room temperature under nitrogen atmosphere for 5 hours, the completed reaction was concentrated under reduced pressure to give the title compound (2.9 g, 98 % yield) as white solids. LC-MS (ESI): RT = 0.513 min, mass calcd. for C11H20ClN3O2S 293.1, m/z found 258.1 [M+H-HCl]+ . 1H NMR (400 MHz, DMSO-d6) d 12.41 (br s, 1H), 9.62 (br s, 2H), 4.39 - 4.35 (m, 1H), 4.23 - 4.13 (m, 1H), 3.82 (d, J = 13.6 Hz, 1H), 3.74 - 3.69 (m, 2H), 3.54 - 3.39 (m, 2H), 3.33 - 3.24 (m, 2H), 2.88 - 2.73 (m, 2H), 1.40 (s, 3H), 1.12 (s, 3H). Intermediate S1-B: 2,2-Dimethyl-3-(3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)propanoic acid hydrochloride
(R)-3-(7-(tert-butoxycarbonyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)-yl)-2,2- dimethylpropanoic acid S1-6B (650 mg, 1.64 mmol) was added into 3 M hydrochloride in ethyl acetate (20 mL, 60 mmol). The reaction was stirred at room temperature under nitrogen atmosphere for 4 hours, the completed reaction was concentrated under reduced pressure to give the title compound Intermediate S1-B (530 mg, 90 % purity from HNMR, 99 % yield) as white solids. LC-MS (ESI): RT = 0.82 min, mass calcd. for C11H20ClN3O2S 293.1, m/z found 258.1 [M+H-HCl] . H NMR (400 MHz, DMSO-d6) d 12.52 (br s, 1H), 9.41 (br s, 2H), 4.40 - 4.36 (m, 1H), 4.21 - 4.10 (m, 1H), 3.83 (d, J = 14.0 Hz, 1H), 3.74 - 3.69 (m, 2H), 3.39 - 3.35 (m, 2H), 3.28 - 3.24 (m, 2H), 2.91 - 2.77 (m, 2H), 1.41 (s, 3H), 1.12 (s, 3H). Preparation of Compounds Compound 1A: 3-(7-(((S)-5-(ethoxycarbonyl)-6-(3-fluoro-2-methylphenyl)-2-(thiazol-2-yl)- 3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)-yl)-2,2- dimethylpropanoic acid
Figure imgf000107_0001
To the solution of 2,2-dimethyl-3-(3-thioxohexahydroimidazo[1,5-a]pyrazin-2
(3H)-yl)propanoic acid hydrochloride Intermediate S1-A (1.87 g, 5.73 mmol) in tetrahydrofuran (160 mL) was added triethylamine (3.4 mL, 24.5 mmol). The mixture was stirred at room temperature for 10 minutes before (S)-ethyl 6-(bromomethyl)-4-(3-fluoro-2- methylphenyl)-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-carboxylate (H2-1A) (2.5 g, 5.14 mmol) was added. After stirred at 40 oC under nitrogen atmosphere for 2.5 hours and then stirred at room temperature overnight, the mixture was filtered and the filtrate was concentrated and purified by C18 column (acetonitrile : water (+ 0.05 % hydrochloride) = 45 % ~ 50 %) to give the desired compound (1.69 g, 48 % yield) as light yellow solids. LC-MS (ESI): RT = 8.325 min, mass calcd. for C29H35FN6O4S2 614.8, m/z found 615.2 [M+H]+ . Chiral analysis: (Column: Chiralpak IE 5 µm 4.6 * 250 mm; Mobile Phase: Hex : IPA : TFA = 50 : 50 : 0.2 at 1 mL / min; Temp: 30 °C; Wavelength: 254 nm, RT = 11.063 min).1H NMR (400 MHz, DMSO-d6) d 12.45 (s, 1H), 9.58 (s, 0.9H), 9.53 (d, J = 3.2 Hz, 0.1H), 8.01 - 7.92 (m, 2H), 7.21 - 7.16 (m, 1H), 7.06 - 7.01 (m, 2H), 5.88 (s, 0.9H), 5.77 (d, J = 3.2 Hz, 0.1H), 4.35 (d, J = 11.6 Hz, 0.9H), 4.22 (d, J = 14 Hz, 0.1H), 4.02 - 3.88 (m, 5H), 3.81 - 3.73 (m, 2H), 3.66 - 3.61 (m, 1H), 3.18 - 3.12 (m, 2H), 3.06 - 3.03 (m, 0.1H), 2.95 - 2.89 (m, 1.9H), 2.45 (d, J = 1.6 Hz, 2.8H), 2.39 (d, J = 1.6 Hz, 0.2H), 2.27 (dt, J = 11.6, 3.2 Hz, 1H), 2.07 (t, J = 10.8 Hz, 1H), 1.13 - 1.04 (m, 9H). Compound 1B: 3-(7-(((S)-5-(ethoxycarbonyl)-6-(3-fluoro-2-methylphenyl)-2-(thiazol-2- yl)-3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)-2,2-dimethylpropanoic acid (single enantiomer)
Figure imgf000108_0001
Compound 1B was prepared using H2-1A and S1-B under condition for compound 1A, purified by Prep-HPLC (Column: Xbridge C18 (5 µm 19 * 150 mm), Mobile Phase A: water (0.1 % ammonium bicarbonate), Mobile Phase B: acetonitrile, UV: 214 nm, Flow rate: 15 mL / min, Gradient: 30 - 75 % (%B)), LC-MS (ESI): RT = 3.915 min, mass calcd. for C29H35FN6O4S2 614.2, m/z found 615.2 [M+H]+ . Chiral analysis: (Column: Chiralpak IE 5 µm 4.6 * 250 mm; Mobile Phase: Hex : EtOH : TFA = 70 : 30 : 0.2 at 1 mL / min; Temp: 30 °C; Wavelength: 254 nm, RT = 19.029 min).1H NMR (400 MHz, DMSO-d6) d 12.22 (br s, 1H), 9.62 (s, 1H), 8.01 - 7.99 (m, 1H), 7.94 (d, J = 2.8 Hz, 1H), 7.21 - 7.15 (m, 1H), 7.07 - 7.02 (m, 2H), 5.89 (s, 0.9H), 5.76 (s, 0.1H), 4.30 - 4.27 (m, 1H), 4.04 - 3.89 (m, 5H), 3.82 - 3.74 (m, 2H), 3.72 - 3.67 (m, 1H), 3.22 - 3.17 (m, 1H), 3.14 - 3.04 (m, 2H), 2.78 - 2.75 (m, 1H), 2.45 (s, 3H), 2.22 - 2.12 (m, 2H), 1.14 (s, 3H), 1.13 (s, 3H), 1.05 (t, J = 7.2 Hz, 3H). Compound 2: 3-(3-(cyanoimino)-7-(((S)-5-(ethoxycarbonyl)-6-(3-fluoro-2- methylphenyl)-2-(thiazol-2-yl)-3,6-dihydropyrimidin-4- yl)methyl)hexahydroimidazo[1,5-a]pyrazin-2(3H)-yl)-2,2-dimethylpropanoic acid (miture of 2 diastereomers)
Figure imgf000109_0001
Preparation of intermediate S2: 3-(3-(cyanoimino)hexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)-2,2-dimethylpropanoic acid
Figure imgf000109_0002
Intermediate S2-1: tert-Butyl 3-(cyanoimino)-2-(3-ethoxy-2,2-dimethyl-3- oxopropyl)hexahydroimidazo[1,5-a]pyrazine-7(1H)-carboxylate
To a solution of tert-butyl 3-(((3-ethoxy-2,2-dimethyl-3-oxopropyl)amino)methyl)piperazine- 1-carboxylate S1-4 (3.00 g, 6.99 mmol) in 1,4-dioxane (30 mL) was added dimethyl cyanocarbonimidodithioate (1.30 g, 8.89 mmol). After heated to reflux and stirred overnight, the reaction mixture was cooled down to room temperature and diluted with water (150 mL). The mixture was extracted with ethyl acetate (50 mL) twice. The combined organic layers were washed with brine (50 mL), dried over Na2SO4(s), filtered and concentrated to afford a crude product (4.00 g, 83 % yield) as yellow oil. LC-MS (ESI): RT = 1.62 min, mass calcd. for C19H31N5O4393.2, m/z found 394.2 [M+H]+ . Intermediate S2-2: 3-(7-(tert-Butoxycarbonyl)-3-(cyanoimino)hexahydroimidazo-[1,5- a]pyrazin-2(3H)-yl)-2,2-dimethylpropanoic acid
To a solution of tert-butyl 3-(cyanoimino)-2-(3-ethoxy-2,2-dimethyl-3- oxopropyl)hexahydroimidazo[1,5-a]pyrazine-7(1H)-carboxylate S2-1 (4.68 g, 6.66 mmol) in methanol (50 mL) was added a solution of sodium hydroxide (1.10 g, 27.5 mmol) in water (20 mL) at 0 oC. After stirred at 40 oC overnight, method was removed and the remained aqueous phase was extracted with ethyl acetate (50 mL). The aqueous lasyer was separated and acidified by 2 M hydrochloride solution to pH ~3, then extracted with ethyl acetate (50 mL) twice. The combined organic layers were dried over Na2SO4(s), filtered and concentrated to give the crude product (2.60 g, 86 % purity, 91 % yield) as white solids. LC-MS (ESI): RT = 1.46 min, mass calcd. for C17H27N5O4365.2, m/z found 366.2 [M+H]+ . Intermediate S2-3: tert-Butyl 3-(cyanoimino)-2-(3-ethoxy-2,2-dimethyl-3- oxopropyl)hexahydroimidazo[1,5-a]pyrazine-7(1H)-carboxylate
To a mixture of 3-(7-(tert-butoxycarbonyl)-3-(cyanoimino)hexahydroimidazo[1,5-a]pyrazin- 2(3H)-yl)-2,2-dimethylpropanoic acid S2-2 (2.60 g, 6.12 mmol) and potassium carbonate (1.30 g, 9.41 mmol) in N,N-dimethylformamide (30 mL) at 0 oC was added iodoethane (1.00 g, 6.41 mmol) by dropwise. After stirred at room temperature for 3 hours, the mixture was diluted with water (150 mL), extrated with ethyl acetate (150 mL) twice. The combined extracts were washed with brine (150 ml) twice, dried over Na2SO4(s), filtered and concentrated to give the crude product, which was purifed by C18 (acetonitrile : water = 5 % to 45 %) to give the title compound (2.40 g, 89 % yield) as white solids. LC-MS (ESI): RT = 1.60 min, mass calcd. for C19H31N5O4 393.2, m/z found 394.3 [M+H]+ .1H NMR (400 MHz, CDCl3) d 4.64 (d, J = 11.6 Hz, 1H), 4.31 - 3.96 (m, 4H), 3.65 - 3.52 (m, 4H), 3.11 - 3.03 (m, 2H), 2.89 - 2.56 (m, 2H), 1.47 (s, 9H), 1.28 (t, J = 7.2 Hz, 3H), 1.22 (s, 6H). Intermediate S2-4: 3-(7-(tert-Butoxycarbonyl)-3-(cyanoimino)hexahydroimidazo-[1,5- a]pyrazin-2(3H)-yl)-2,2-dimethylpropanoic acid
To a solution of tert-butyl 3-(cyanoimino)-2-(3-ethoxy-2,2-dimethyl-3- oxopropyl)hexahydroimidazo[1,5-a]pyrazine-7(1H)-carboxylate S2-3 (500 mg, 1.14 mmol) in methanol (20 mL) at 0 oC was added a solution of sodium hydroxide (180 mg, 4.50 mmol) in water (10 mL). After stirred at 40 oC overnight, methanol was removed and the remained aqueous phase was extracted with ethyl acetate (30 mL). The aqeous phase was separated and acidified by 2 M hydrochloride aqueous solution to pH ~ 3, extracted with ethyl acetate (50 mL) twice. The combined extracts were dried over Na2SO4(s), filtered and concentrated to give the crude product (400 mg, 92 % yield) as white solids. LC-MS (ESI): RT = 1.21 min, mass calcd. for C17H27N5O4365.2, m/z found 364.2 [M-H]+ . Intermediate S2: 3-(3-(Cyanoimino)hexahydroimidazo[1,5-a]pyrazin-2(3H)-yl)-2,2- dimethylpropanoic acid hydrochloride
A mixture of 3-(7-(tert-butoxycarbonyl)-3-(cyanoimino)hexahydroimidazo[1,5-a]pyrazin- 2(3H)-yl)-2,2-dimethylpropanoic acid S2-4 (150 mg, 0.398 mmol) in 3 M hydrochloride in ethyl acetate (6 mL, 18.0 mmol) was stirred at room temperature for 3 hours. Then the mixture was concentrated to give the desired product (120 mg, 99 % yield) as white solids. The crude product was used for next step directly. LC-MS (ESI): RT = 0.87 min, mass calcd. for C12H20ClN5O2301.1, m/z found 266.2 [M+H-HCl]+ . Compound 2: 3-(3-(Cyanoimino)-7-((5-(ethoxycarbonyl)-6-(3-fluoro-2-methylphenyl)-2- (thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)-hexahydroimidazo[1,5-a]pyrazin- 2(3H)-yl)-2,2-dimethylpropanoic acid (miture of 2 diastereomers)
Figure imgf000111_0001
To a mixture of 3-(3-(cyanoimino)hexahydroimidazo[1,5-a]pyrazin-2(3H)-yl)-2,2- dimethylpropanoic acid hydrochloride S2 (120 mg, 0.398 mmol) in dichloromethane (10 mL) was added triethanolamine (300 mg, 2.01 mmol). After stirred for 0.5 hour at room temperature, (S)-ethyl 6-(bromomethyl)-4-(3-fluoro-2-methylphenyl)-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate (H2-1A) (150 mg, 0.308 mmol) was added. After stirred at room temperature overnight, the reaction mixture was diluted by dichloromethane (50 mL), washed by brine (50 mL) twice, dried over Na2SO4(s), filtered and concentrated to give a residue, which was purified was by C18 column (acetonitrile : water = 5 % to 45 %) to give the title compound (48 mg, 97.4 % purity, 18 % yield) as yellow solids. LC-MS (ESI): RT = 3.677 min, mass calcd. for C30H35FN8O4S 622.3, m/z found 623.3 [M+H]+ .1H NMR (400 MHz, DMSO-d6) d 9.60 - 9.52 (m, 1H), 8.01 - 8.00 (m, 1H), 7.93 - 7.92 (m, 1H), 7.21 - 7.15 (m, 1H), 7.06 - 7.01 (m, 2H), 5.89 - 5.88 (m, 1H), 4.47 - 4.36 (m, 1H), 4.04 - 3.91 (m, 4H), 3.85 - 3.76 (m, 1H), 3.64 - 3.43 (m, 3H), 3.22 - 2.91 (m, 4H), 2.45 (s, 3H), 2.39 - 2.13 (m, 2H), 1.13 - 1.04 (m, 9H). Compound 3A: 3-(7-((6-(2-chloro-3-fluorophenyl)-5-(ethoxycarbonyl)-2-(thiazol-2-yl)- 3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)-yl)- 2,2-dimethylpropanoic acid (single enantiomer)
Figure imgf000112_0001
Compound 3A was prepared from H1-1A and Intermediate S1-B using same condition as for Compound 3B. Compound 3A: purified by Prep-HPLC (Column: gilson Xbrige C18 (5 µm 19 * 150 mm), Mobile phase A: water (+ 0.1 % ammonium bicarbonate), Mobile phase B: acetonitrile, UV: 214 nm, Flow rate: 15 mL/min, Gradient: 10 - 70 % (%B)) to give the title compound (30 mg, 99.6 % purity, 31 % yield) as yellow solids. LC-MS (ESI): RT = 3.262 min, mass calcd. for C28H32ClFN6O4S2634.2, m/z found 635.2.1H NMR (400 MHz, DMSO-d6) d 9.67 (s, 1H), 8.03 (d, J = 3.2 Hz, 1H), 7.95 (d, J = 3.2 Hz, 1H), 7.38 - 7.25 (m, 3H), 6.11 (s, 0.97H), 6.00 (s, 0.03H), 4.31 - 4.28 (m, 1H), 4.02 - 3.89 (m, 5H), 3.82 - 3.74 (m, 2H), 3.72 - 3.67 (m, 1H), 3.22 - 3.18 (m, 1H), 3.15 - 3.04 (m, 2H), 2.81 - 2.78 (m, 1H), 2.21 - 2.14 (m, 2H), 1.14 (s, 6H), 1.03 (t, J = 7.2 Hz, 3H). Compound 3B: 3-(7-((6-(2-chloro-3-fluorophenyl)-5-(ethoxycarbonyl)-2-(thiazol-2-yl)- 3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)-yl)- 2,2-dimethylpropanoic acid (single enantiomer)
Figure imgf000113_0001
To a solution of 2,2-dimethyl-3-(3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)propanoic acid hydrochloride Intermediate S1-A (100 mg, 0.31 mmol) in dichloromethane (3 mL) was added triethanolamine (230 mg, 1.54 mmol). After stirred at 40 oC for 30 minutes, a solution of ethyl 6-(bromomethyl)-4-(2-chloro-3-fluorophenyl)-2- (thiazol-2-yl)-1,4-dihydropyrimidine-5-carboxylate (H1-1A) (157 mg, 90 % purity, 0.279 mmol) in dichloromethane (2 mL) was added dropwise. After stirred at 40 oC for 16 hours, the reaction mixture was concentrated to give a residue, which was purified by Prep-HPLC (Column: Waters Xbridge C18 (5 µm 19 *150 mm), Mobile Phase A: Water (0.1 % ammonium bicarbonate), Mobile Phase B: acetonitrile, UV: 214 nm, Flow rate: 15 mL/min, Gradient: 20 - 60 % (%B)) give the title compound 3B (34.8 mg. 17.8% yield) as yellow solids. LC-MS (ESI): RT = 3.542 min, mass calcd. for C28H32ClFN6O4S2 634.2, m/z found 635.2 [M+H]+ . 1H NMR (400 MHz, DMSO-d6) d 9.67 (br s, 1H), 8.02 (d, J = 3.2 Hz, 1H), 7.94 (d, J = 3.2 Hz, 1H), 7.39 - 7.29 (m, 2H), 7.29 - 7.24 (m, 1H), 6.10 (s, 1H), 4.35 (d, J = 11.6 Hz, 1H), 4.00 - 3.87 (m, 5H), 3.78 (d, J = 14.0 Hz, 1H), 3.74 (d, J = 14.0 Hz, 1H), 3.64 (t, J = 9.6 Hz, 1H), 3.19 - 3.12 (m, 2H), 2.95 - 2.92 (m, 2H), 2.32 - 2.21 (m, 1H), 2.08 (t, J = 10.8 Hz, 1H), 1.12 (s, 6H), 1.05 (t, J = 7.2 Hz, 3H). Compound 4A: 3-(7-((6-(2-chloro-4-fluorophenyl)-5-(methoxycarbonyl)-2-(thiazol-2-yl)- 3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)-yl)- 2,2-dimethylpropanoic acid (single enantiomer)
Figure imgf000114_0001
Compound 4A was prepared from Intermediate S1-B and intermediate H3-1A using same condition as for Compound 4B and purified by Prep-HPLC (Column: gilson Xbrige C18 (5 µm 19 * 150 mm), Mobile phase A: water (+ 0.1 % ammonium bicarbonate), Mobile phase B: acetonitrile, UV: 214 nm, Flow rate: 15 mL/min, Gradient: 05 - 95 % (%B)). LC-MS (ESI): RT = 3.658 min, mass calcd. for C27H30ClFN6O4S2 620.1, m/z found 621.1. 1H NMR (400 MHz, DMSO-d6) d 9.71 (br s, 0.9H), 8.03 (d, J = 3.2 Hz, 1H), 8.01 (s, 0.1H), 7.95 (d, J = 3.2 Hz, 1H), 7.45 - 7.39 (m, 2H), 7.17 (td, J = 8.4, 2.4 Hz, 1H), 6.05 (s, 0.97H), 5.93 (s, 0.03H), 4.30 - 4.27 (m, 1H), 4.02 - 3.89 (m, 3H), 3.81 - 3.67 (m, 3H), 3.52 (s, 3H), 3.22 - 3.18 (m, 1H), 3.15 - 3.04 (m, 2H), 2.79 - 2.76 (m, 1H), 2.22 - 2.14 (m, 2H), 1.13 (s, 6H). Compound 4B: 3-(7-((6-(2-chloro-4-fluorophenyl)-5-(methoxycarbonyl)-2-(thiazol-2-yl)- 3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)-yl)- 2,2-dimethylpropanoic acid (single enantiomer)
Figure imgf000115_0001
To a solution of 2,2-dimethyl-3-(3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)propanoic acid hydrochloride Intermediate S1-A (80 mg, 0.250 mmol) in dichloromethane (3 mL) was added triethanolamine (184 mg, 1.23 mmol) at room temperature and the resulting mixture was stirred at 40 oC for 30 minutes. Then a solution of (R)-methyl 6-(bromomethyl)-4-(2-chloro-4-fluorophenyl)-2-(thiazol-2-yl)- 1,4-dihydropyrimidine-5-carboxylate (H3-1A) (122 mg, 0.250 mmol) in dichloromethane (2 mL) was added dropwise. After stirred at 40 oC for 16 hours, the reaction mixture was concentrated to give a residue, which was purified by Prep-HPLC (Column: Waters Xbridge C18 (5 µm 19 *150 mm), Mobile Phase A: water (0.1 % ammonium bicarbonate), Mobile Phase B: acetonitrile, UV: 214 nm, Flow rate: 15 mL/min, Gradient: 20 - 55 % (%B)) give the title compound (6.1 mg, 99.3 % purity, 4 % yield) as yellow solids. LC-MS (ESI): RT = 3.754 min, mass calcd. for C27H30ClFN6O4S2620.1, m/z found 621.2 [M+H]+ .1H NMR (400 MHz, CD3OD) d7.84 (d, J = 2.8 Hz, 1H), 7.64 (d, J = 3.6 Hz, 1H), 7.31 (dd, J = 8.8, 6.0 Hz, 1H), 7.12 (dd, J = 8.8, 2.8 Hz, 1H), 6.97 - 6.92 (m, 1H), 6.05 (s, 1H), 4.43 - 4.39 (m, 1H), 4.01 - 3.93 (m, 2H), 3.84 - 3.73 (m, 3H), 3.60 - 3.56 (m, 1H), 3.49 (s, 3H), 3.18 - 3.11 (m, 2H), 2.87 - 2.77 (m, 2H), 2.35 - 2.30 (m, 1H), 2.10 - 2.04 (m, 1H), 1.13 (s, 3H), 1.12 (s, 3H). Compound 5: 1-((7-((6-(2-Chloro-3-fluorophenyl)-5-(ethoxycarbonyl)-2-(thiazol-2-yl)- 3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)methyl)cyclopropanecarboxylic acid
Figure imgf000116_0001
Intermediate S3-1: 1-Benzyl 4-tert-butyl 2-((((1- (ethoxycarbonyl)cyclopropyl)methyl)amino)methyl) piperazine-1,4-dicarboxylate
To a solution of ethyl 1-(aminomethyl)cyclopropanecarboxylate hydrochloride (2.04 g, 11.4 mmol) in ethanol (50 mL) was added triethylamine (1.15 g, 11.4 mmol) at room temperature. After stirred at room temperature under nitrogen atmosphere for 0.5 hour, a solution of 1- benzyl 4-tert-butyl 2-formylpiperazine-1,4-dicarboxylate S1-2 (3.10 g, 7.56 mmol) in ethanol (10 mL) was added and stirred at room temperature for 1.5 hours. Then sodium cyanoborohydride (1.12 g, 17.8 mmol) was added at 0 oC. After stirred at room temperature for 2 hours, the mixture was quenched with ice water (15 mL), then removed ethanol under vacuo. The residue was diluted with water (40 mL) and extracted with ethyl acetate (20 mL) for three times. The combined organic layers were dried over Na2SO4(s) and filtered. The filtrate was concentrated to give a residue, which was purified by C18 column (acetonitrile : water = 65 % to 95 %) to give the title compound (2.00 g, 50 % yield) as yellow oil. LC-MS (ESI): RT = 1767 min mass calcd for C25H37N3O6 4753 m/z found 4763 [M+H]+ 1H NMR (400 MHz, CDCl3) d 7.36 - 7.32 (m, 5H), 5.14 (s, 1H), 4.27 - 3.93 (m, 6H), 3.05 - 2.66 (m, 7H), 1.71 (br s, 1H), 1.46 (s, 9H), 1.23 - 1.19 (m, 5H), 0.81 - 0.68 (m, 2H). Intermediate S3-2: tert-Butyl 3-((((1-(ethoxycarbonyl)cyclopropyl)-methyl)amino)- methyl)piperazine-1-carboxylate
To a solution of 1-benzyl 4-tert-butyl 2-((((1-(ethoxycarbonyl)cyclopropyl)- methyl)amino)methyl)piperazine-1,4-dicarboxylate (S3-1) (1.80 g, 3.41 mmol) in ethanol (80 mL) was added 20 % palladium hydroxide on carbon (2.00 g, 2.85 mmol) under nitrogen atmosphere. After stirred at 50 oC under hydrogen atmosphere (50 psi) overnight, the mixture was cooled to room temperature. Then the catalyst was filtered, and the filtrate was concentrated to give the desired compound (1.10 g, 85 % yield) as yellow oil. LC-MS (ESI): RT = 1.374 min, mass calcd. for C17H31N3O4341.2, m/z found 342.2 [M+H]+ .1H NMR (300 MHz, CDCl3) d 4.13 (q, J = 7.2 Hz, 2H), 3.93 - 3.90 (m, 1H), 3.00 - 2.43 (m, 8H), 2.25 (br s, 2H), 1.46 (s, 9H), 1.26 - 1.21 (m, 4.6H), 0.81 - 0.77 (m, 1.4H). Intermediate S3-3: tert-Butyl 2-((1-(ethoxycarbonyl)cyclopropyl)methyl)-3- thioxohexahydroimidazo[1,5-a]pyrazine-7(1H)-carboxylate
To a solution of tert-butyl 3-((((1-(ethoxycarbonyl)cyclopropyl)methyl)amino)- methyl)piperazine-1-carboxylate (S3-2) (1.10 g, 2.90 mmol) and triethylamine (900 mg, 8.89 mmol) in dichloromethane (25 mL) was added a solution of thiophosgene (550 mg, 4.78 mmol) in dichloromethane (5 mL) at 0 oC under nitrogen atmosphere. After stirred at room temperature overnight, the mixture was diluted with ice water (40 mL) and extracted with dichloromethane (10 mL) for three times. The combined organic layers were washed with brine (20 mL), dried over Na2SO4(s) and filtered. The filtrate was concentrated to give a residue, which was purified by silica gel column chromatography (petroleum ether : ethyl acetate = 8 : 1 to 2 : 1) to give the crude compound, which was further purified by C18 column (acetonitrile : water = 45 % to 95 %) to give the title compound (650 mg, 53 % yield) as yellow solids. LC-MS (ESI): RT = 1.701 min, mass calcd. for C18H29N3O4S 383.2, m/z found 384.2 [M+H]+ . 1H NMR (400 MHz, CDCl3) d 4.44 (d, J = 11.6 Hz, 1H), 4.15 - 4.10 (m, 4H), 3.97 (s, 2H), 3.87 - 3.82 (m, 1H), 3.78 - 3.71 (m, 1H), 3.30 - 3.26 (m, 1H), 3.04 - 2.98 (m, 1H), 2.86 - 2.81 (m, 1H), 2.65 - 2.58 (m, 1H), 1.47 (s, 9H), 1.31 (s, 2H), 1.26 - 1.19 (m, 5H). Intermediate S3-3A and S3-3B:
A racemic mixture of tert-butyl 2-((1-(ethoxycarbonyl)cyclopropyl)methyl)-3- thioxohexahydroimidazo[1,5-a]pyrazine-7(1H)-carboxylate S3-3 (400 mg, 0.939 mmol) was separated by chiral Prep-HPLC (separation conditon: Column: Chiralpak ID 5 µm 20 * 250 mm; Mobile Phase: Hex : EtOH : DEA = 85 : 15 : 0.3 at 18 mL/min; Temp: 35 °C; Wavelength: 214 nm) to give the title compounds S3-3A (90 mg, 90 % purity from 1H NMR, 23 % yield, 100 % stereopure) and S3-3B (204 mg, 90 % purity from 1H NMR, 51 % yield, 99.2 % stereopure).
Intermediate S3-3A: LC-MS (ESI): RT = 1.71 min, mass calcd. for C18H29N3O4S 383.2, m/z found 384.1 [M+H]+ . Chiral analysis (Column: Chiralpak IE 5 um 4.6 * 250 mm; Mobile Phase: Hex : EtOH : DEA = 85 : 15 : 0.2 at 1 mL/min; Temp: 30 oC; Wavelength: 254 nm, RT = 15.778 min).1H NMR (400 MHz, CDCl3) d 4.46 - 4.43 (m, 1H), 4.26 - 4.03 (m, 4H), 3.97 (s, 2H), 3.87 - 3.82 (m, 1H), 3.80 - 3.68 (m, 1H), 3.31 - 3.26 (m, 1H), 3.05 - 2.98 (m, 1H), 2.89 - 2.78 (m, 1H), 2.69 - 2.54 (m, 1H), 1.47 (s, 9H), 1.32 - 1.19 (m, 7H).
Intermediate S3-3B: LC-MS (ESI): RT = 1.71 min, mass calcd. for C18H29N3O4S 383.2, m/z found 384.1 [M+H]+ . Chiral analysis (Column: Chiralpak IE 5 um 4.6 * 250 mm; Mobile Phase: Hex : EtOH : DEA = 85 : 15 : 0.2 at 1 mL/min; Temp: 30 oC; Wavelength: 254 nm, RT = 18.449 min).1H NMR (400 MHz, CDCl3) d 4.46 - 4.43 (m, 1H), 4.27 - 4.02 (m, 4H), 3.97 (s, 2H), 3.85 - 3.82 (m, 1H), 3.78 - 3.68 (m, 1H), 3.31 - 3.26 (m, 1H), 3.05 - 2.98 (m, 1H), 2.92 - 2.77 (m, 1H), 2.70 - 2.55 (m, 1H), 1.47 (s, 9H), 1.32 - 1.19 (m, 7H). Intermediate S3-4: 1-((7-(tert-Butoxycarbonyl)-3-thioxohexahydroimidazo[1,5- a]pyrazin-2(3H)-yl)methyl)cyclopropanecarboxylic acid
To a solution of tert-butyl 2-((1-(ethoxycarbonyl)cyclopropyl)methyl)-3- thioxohexahydroimidazo[1,5-a]pyrazine-7(1H)-carboxylate (S3-3) (100 mg, 0.235 mmol) in tetrahydrofuran (1 mL), methanol (2 mL) and water (1 mL) was added lithium hydroxide monohydrate (40 mg, 0.953 mmol) under nitrogen atmosphere. After stirred at room temperature overnight, the reaction was concentrated at 35 oC to give a residue, which was purified by C18 column (acetonitrile : water = 30 % to 90 %) to give the desired compound (88 mg) as light yellow solids. LC-MS (ESI): RT = 1.24 min, mass calcd. for C16H25N3O4S 355.2, m/z found 356.2 [M+H]+ . Intermediate S3-4A was prepared from S3-3A using same condition as for S3-4. LC-MS (ESI): RT = 1.21 min, mass calcd. for C16H25N3O4S 355.2, m/z found 356.1 [M+H]+ . Intermediate S3-4B was prepared from S3-3B using same condition as for S3-4. LC-MS (ESI): RT = 1.24 min, mass calcd. for C16H25N3O4S 355.2, m/z found 356.1 [M+H]+ . Intermediate S3: 1-((7-(tert-Butoxycarbonyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin- 2(3H)-yl)methyl)cyclopropanecarboxylic acid hydrochloride To a solution of 1-((7-(tert-butoxycarbonyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)methyl)cyclopropanecarboxylic acid (S3-4) (88 mg, 0.235 mmol) in dichloromethane (3 mL) was added 4 M hydrochloride in ethyl acetate (2 mL, 8 mmol) under nitrogen atmosphere. After stirred at room temperature for 1 hour, the reaction mixture was concentrated to give the title compound (63 mg, 78 % yield) as white solids. 1H NMR (400 MHz, CD3OD) d 4.67 - 4.63 (m, 0.5H), 4.62 - 4.60 (m, 0.5H), 4.21 - 4.12 (m, 1H), 3.99 - 3.88 (m, 3H), 3.59 - 3.34 (m, 4H), 3.06 - 2.85 (m, 2H), 1.31 - 1.26 (m, 2H), 1.18 - 1.13 (m, 2H). Intermediate S3-A was prepared from S3-4A using same condition as for intermediate S3.1H NMR (400 MHz, DMSO-d6) d 12.61 - 12.13 (m, 1H), 10.14 - 9.27 (m, 2H), 4.37 - 4.33 (m, 1H), 4.25 - 4.11 (m, 1H), 3.85 - 3.78 (m, 2.4H), 3.73 - 3.65 (m, 0.6H), 3.40 - 3.29 (m, 4H), 2.87 - 2.69 (m, 2H), 1.18 - 1.10 (m, 2H), 1.09 - 1.02 (m, 2H). Intermediate S3-B was prepared from S3-4B using same condition as for intermediate S3.1H NMR (400 MHz, DMSO-d6) d 12.77 - 12.05 (m, 1H), 10.16 - 9.64 (m, 2H), 4.39 - 4.32 (m, 1H), 4.26 - 4.15 (m, 1H), 3.85 - 3.77 (m, 2.4H), 3.72 - 3.65 (m, 0.6H), 3.47 - 3.29 (m, 4H), 2.85 - 2.70 (m, 2H), 1.16 - 1.14 (m, 2H), 1.07 - 1.01 (m, 2H). Compound 5: 1-((7-((6-(2-Chloro-3-fluorophenyl)-5-(ethoxycarbonyl)-2-(thiazol-2-yl)- 3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)methyl)cyclopropanecarboxylic acid
Figure imgf000120_0001
To a solution of ethyl 6-(bromomethyl)-4-(2-chloro-3-fluorophenyl)-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate (H1-1A) (110 mg, 0.216 mmol) in tetrahydrofuran (3 ml) was added 1-((3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)methyl)cyclopropanecarboxylic acid hydrochloride (S3) (63 mg, 0.194 mmol) and triethylamine (110 mg, 1.09 mmol) under nitrogen atmosphere. After stirred at 40 oC under nitrogen atmosphere for 2.5 hours and then stirred at room temperature overnight, the reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (10 mL) twice. The combined organic layers were washed with brine (10 mL), dried over Na2SO4(s), filtered and concentrated to give a residue, which was purified by C18 column (acetonitrile : water = 40 % to 70 %) to give the title compound (24.2 mg, 17 % yield) as yellow solids. LC- MS (ESI): RT = 3.723 min, mass calcd. for C28H30ClFN6O4S2632.1, m/z found 633.2
[M+H]+ .1H NMR (400 MHz, CD3OD) d 7.85 (d, J = 3.2 Hz, 1H), 7.64 (d, J = 3.2 Hz, 1H), 7.22 - 7.14 (m, 2H), 7.06 - 7.02 (m, 1H), 6.13 (s, 0.4H), 6.12 (s, 0.6H), 4.41 - 4.37 (m, 0.6H), 4.34 - 4.30 (m, 0.4H), 4.05 - 3.90 (m, 4H), 3.85 - 3.70 (m, 4H), 3.34 - 3.25 (m, 1.2H), 3.18 - 3.13 (m, 0.8H), 2.99 - 2.94 (m, 0.4H), 2.87 - 2.81 (m, 1H), 2.79 - 2.66 (m, 0.6H), 2.36 - 2.30 (m, 0.5H), 2.24 - 2.05 (m, 1.5H), 1.21 - 1.15 (m, 2H), 1.07 - 0.99 (m, 5H). Compound 6: 3-((S)-2-(((S)-5-(ethoxycarbonyl)-6-(3-fluoro-2-methylphenyl)-2-(thiazol- 2-yl)-3,6-dihydropyrimidin-4-yl)methyl)-6-thioxohexahydro-2H-pyrazino[1,2- c]pyrimidin-7(6H)-yl)-2,2-dimethylpropanoic acid
Figure imgf000121_0002
Preparation of intermediate S4:
(S)-2,2-Dimethyl-3-(6-thioxotetrahydro-1H-pyrazino[1,2-c]pyrimidin-7(2H,6H,8H)- yl)propanoic acid hydrochloride
Figure imgf000121_0001
Intermediate S4-1: (S)-2-(Piperazin-2-yl)ethanol
To a solution of (S)-2-(4-benzylpiperazin-2-yl)ethanol (1.50 g, 6.82 mmol, cas#477220-33-0) in methanol (30 mL) was added 10 % palladium on charcoal wt. (500 mg). The reaction mixture was stirred at room temperature under hydrogen atmosphere (50 psi) overnight. Then it was filtered and concentrated to give the title compound (900 mg, 92 % yield) as white colorless oil. LC-MS (ESI): RT = 0.31 min, mass calcd. for C6H14N2O 130.1, m/z found 131.0 [M+H]+ .1H NMR (400 MHz, CDCl3) d 3.84 - 3.74 (m, 1H), 3.69 - 3.66 (m, 1H), 2.99 - 2.91 (m, 2.3H), 2.84 - 2.61 (m, 4.1H), 2.55 - 2.49 (m, 0.6H), 1.70 - 1.67 (m, 1H), 1.60 - 1.56 (m, 1H). Intermediate S4-2: (S)-tert-Butyl 3-(2-hydroxyethyl)piperazine-1-carboxylate
To a solution of (S)-2-(Piperazin-2-yl)ethanol dihydrochloride S4-1 (750 mg, 3.33 mmol) in methanol (15 mL) was added triethylamine (660 mg, 6.53 mmol) and di-tert-butyl dicarbonate (654 mg, 3.00 mmol) at -10 oC. Then the mixture was warmed to 0 oC and stirred overnight. The mixture was evaporated to give a residue, which was diluted with dichloromethane (20 mL) and washed with brine (20 mL), dried over Na2SO4(s), filtered and concentrated to give the title compound (800 mg, 83 % yield) as yellow oil. LC-MS (ESI): RT = 1.19 min, mass calcd. for C11H22N2O3 230.2, m/z found 231.1 [M+H]+ . 1H NMR (400 MHz, CD3OD) d 3.86 - 3.83 (m, 2H), 3.80 - 3.77 (m, 2H), 3.58 - 3.55 (m, 2H), 2.84 - 2.81 (m, 1H), 2.76 - 2.73 (m, 1H), 2.65 - 2.54 (m, 3H), 1.52 - 1.47 (m, 2H), 1.36 (s, 9H). Intermediate S4-3: (S)-1-Benzyl 4-tert-butyl 2-(2-hydroxyethyl)piperazine-1,4- dicarboxylate
To a solution of (S)-tert-butyl 3-(2-hydroxyethyl)piperazine-1-carboxylate S4-2 (800 mg, 2.78 mmol) and sodium bicarbonate (2.60 g, 13.9 mmol) in tetrahydrofuran (10 mL) and water (5 mL) was added benzyl chloroformate (709 mg, 4.17 mmol) at 0 oC. After stirred at room temperature overnight, the mixture was diluted with water (50 mL) and extracted with ethyl acetate (30 mL) for three times. The combined organic layers were washed with brine (50 mL), dried over Na2SO4(s), filtered and concentrated to give a residue, which was purified by silica gel column chromatography (petroleum ether : ethyl acetate = 2 : 1) to give the title compound (760 mg, 75 % yield) as colorless oil. LC-MS (ESI): RT = 1.58 min, mass calcd. for C19H28N2O5 364.2, m/z found 365.2 [M+H]+ .1H NMR (400 MHz, CDCl3) d 7.39 - 7.32 (m, 5H), 5.16 (s, 2H), 4.36 (s, 1H), 3.95 - 3.93 (m, 2H), 3.65 - 3.59 (m, 1H), 3.37 - 2.83 (m, 5H), 1.87 - 1.81 (m, 2H), 1.48 (s, 9H). Intermediate S4-4: (S)-1-Benzyl 4-tert-butyl 2-(2-oxoethyl)piperazine-1,4-dicarboxylate To a solution of oxalyl chloride (619 mg, 4.88 mmol) in dichloromethane (15 mL) was added a solution of dimethyl sulfoxide (533 mg, 6.83 mmol) in dichloromethane (50 mL) at - 78 °C. After stirred at - 78 oC for 1 hour, a solution of (S)-1-benzyl 4-tert-butyl 2-(2- hydroxyethyl)piperazine-1,4-dicarboxylate S4-3 (750 mg, 1.95 mmol) was added at - 78 oC. After stirring at - 78 oC for 3 hours, triethylamine (1.50 g, 14.6 mmol) was added dropwise to quench the reaction. The reaction mixture was allowed to warm to room temperature and extracted with dichloromethane (30 mL) for three times. The combined organic layers were dried over anhydrous Na2SO4(s), filtered and concentrated to give the title compound (750 mg, 95 % yield) as light yellow oil. LC-MS (ESI): RT = 1.59 min, mass calcd. for C19H26N2O5 362.2, m/z found 363.2 [M+H]+ .1H NMR (400 MHz, CDCl3) d 9.74 (s, 1H), 7.39 - 7.30 (m, 5H), 5.14 (s, 2H), 4.74 - 4.71 (m, 1H), 4.11 - 3.97 (m, 3H), 3.05 - 2.84 (m, 2H), 2.83 - 2.74 (m, 2H), 2.61 - 2.57 (m, 1H), 1.46 (s, 9H). Intermediate S4-5: (S)-1-Benzyl 4-tert-butyl 2-(2-((3-ethoxy-2,2-dimethyl-3- oxopropyl)amino)-ethyl)piperazine-1,4-dicarboxylate
To a solution of ethyl 3-amino-2,2-dimethylpropanoate hydrochloride (378 mg, 2.08 mmol) in ethanol (5 mL) was added triethylamine (263 mg, 2.60 mmol) at room temperature. After stirred at room temperature under nitrogen atmosphere for 0.5 hour, a solution of (S)-1- benzyl 4-tert-butyl 2-(2-oxoethyl)piperazine-1,4-dicarboxylate S4-4 (750 mg, 1.86 mmol) in ethanol (5 mL) was added. The mixture was stirred at room temperature for 1 hours, then sodium cyanoborohydride (269 mg, 4.28 mmol) was added at 0 oC. After stirred at room temperature for 2 hours, the mixture was quenched with ice water (5 mL), concentrated under vacuo. The residue was diluted with water (15 mL) and extracted with ethyl acetate (20 mL) for three times. The combined organic layers were dried over Na2SO4(s), filtered and concentrated to give a residue, which was purified by silica gel column chromatography (dichloromethane : methanol = 30 : 1) to give the title compound (600 mg, 66 % yield) as colorless oil. LC-MS (ESI): RT = 1.89 min, mass calcd. for C26H41N3O6 491.3, m/z found 492.3 [M+H]+ .1H NMR (400 MHz, CDCl3) d 7.39 - 7.29 (m, 5H), 5.14 (s, 2H), 4.27 - 4.13 (m, 1H), 4.12 - 4.09 (q, J = 7.2 Hz, 2H), 4.08 - 3.94 (m, 2H), 3.06 - 3.00 (m, 2H), 2.95 - 2.79 (m, 2H), 2.62 - 2.57 (m, 4H), 1.75 - 1.69 (m, 2H), 1.45 (s, 9H), 1.23 (t, J = 7.2 Hz, 3H), 1.15 (s, 6H). Intermediate S4-6: (S)-tert-Butyl 3-(2-((3-ethoxy-2,2-dimethyl-3- oxopropyl)amino)ethyl)piperazine-1-carboxylate To a solution of (S)-1-benzyl 4-tert-butyl 2-(2-((3-ethoxy-2,2-dimethyl-3- oxopropyl)amino)ethyl)piperazine-1,4-dicarboxylate S4-5 (600 mg, 1.16 mmol) in ethanol (10 mL) was added 20 % palladium hydroxide on carbon (300 mg) under nitrogen atmosphere. After stirred at 50 oC under hydrogen atmosphere (60 psi) overnight, the mixture was cooled to room temperature. Then the catalyst was filtered, and the filtrate was concentrated to give the title compound (430 mg, 93 % yield) as yellow oil. LC-MS (ESI): RT = 1.66 min, mass calcd. for C18H35N3O4 357.3, m/z found 358.4 [M+H]+ . 1H NMR (400 MHz, CDCl3) d 4.12 (q, J = 7.2 Hz, 2H), 3.92 (br s, 2H), 2.96 - 2.94 (m, 1H), 2.85 - 2.78 (m, 2H), 2.75 - 2.61 (m, 6H), 2.28 (br s, 2H), 1.57 - 1.51 (m, 2H), 1.46 (s, 9H), 1.25 (t, J = 7.2 Hz, 3H), 1.19 (s, 6H). Intermediate S4-7: (S)-tert-Butyl 7-(3-ethoxy-2,2-dimethyl-3-oxopropyl)-6- thioxohexahydro-1H-pyrazino[1,2-c]pyrimidine-2(6H)-carboxylate
To a solution of (S)-tert-butyl 3-(2-((3-ethoxy-2,2-dimethyl-3- oxopropyl)amino)ethyl)piperazine-1-carboxylate S4-6 (330 mg, 90 % purity, 0.83 mmol) and triethylamine (268 mg, 2.66 mmol) in dichloromethane (25 mL) was added a solution of thiophosgene (153 mg, 1.33 mmol) in dichloromethane (10 mL) at 0 oC under nitrogen atmosphere. After stirred at room temperature overnight, the mixture was diluted with ice water (10 mL) and extracted with dichloromethane (20 mL) for three times. The combined organic layers were washed with brine (20 mL), dried over Na2SO4(s) and filtered. The filtrate was concentrated to give a residue, which was purified by silica gel column chromatography (petroleum ether : ethyl acetate = 4 : 1) to give the title compound (135 mg, 41 % yield) as yellow oil. LC-MS (ESI): RT = 1.73 min, mass calcd. for C19H33N3O4S 399.2, m/z found 400.3 [M+H]+ .1H NMR (400 MHz, CDCl3) d 5.42 - 5.39 (m, 1H),4.37 - 4.29 (m, 2H), 4.14 (q, J = 6.8 Hz, 2H), 3.97 - 3.93 (m, 2H), 3.46 - 3.38 (m, 1H), 3.28 - 3.25 (m, 2H), 3.07 - 2.99 (m, 2H), 2.63 - 2.60 (m, 1H), 2.14 - 2.09 (m, 1H), 1.75 - 1.66 (m, 1H), 1.47 (s, 9H), 1.29 - 1.26 (m, 9H). Intermediate S4-8: (S)-3-(2-(tert-Butoxycarbonyl)-6-thioxotetrahydro-1H-pyrazino[1,2- c]pyrimidin-7(2H,6H,8H)-yl)-2,2-dimethylpropanoic acid
To a solution of (S)-tert-butyl 7-(3-ethoxy-2,2-dimethyl-3-oxopropyl)-6-thioxohexahydro- 1H-pyrazino[1,2-c]pyrimidine-2(6H)-carboxylate S4-7 (170 mg, 0.405 mmol) in methanol (3 mL) and water (1 mL) was added sodium hydroxide (51 mg, 1.28 mmol) under nitrogen atmosphere. After stirred at 40 oC overnight, the reaction was concentrated to give a residue, which was diluted with water (5 mL) and acidified with 1 N hydrochloride aqueous solution to pH ~ 3. The aqueous phase was extracted with ethyl acetate (20 mL) for three times. The combined organic layers were dried over Na2SO4(s), filtered and concentrated to give the desired compound (130 mg, 78 % yield) as yellow solids. LC-MS (ESI): RT = 1.17 min, mass calcd. for C17H29N3O4S 371.2, m/z found 370.3 [M-H]- .1H NMR (400 MHz, CDCl3) d 5.40 - 5.37 (m, 1H), 4.40 - 3.96 (m, 2H), 4.02 - 3.96 (m, 2H), 3.45 - 3.40 (m, 1H), 3.37 - 3.34 (m, 2H), 3.07 - 3.02 (m, 2H), 2.60 (br s, 1H), 2.18 - 2.11 (m, 1H), 1.76 - 1.72 (m, 1H), 1.47 (s, 9H), 1.31 (m, 6H). Intermediate S4: (S)-2,2-Dimethyl-3-(6-thioxotetrahydro-1H-pyrazino[1,2-c]pyrimidin- 7(2H,6H,8H)-yl)propanoic acid hydrochloride
To a solution of (S)-3-(2-(tert-butoxycarbonyl)-6-thioxotetrahydro-1H-pyrazino[1,2- c]pyrimidin-7(2H,6H,8H)-yl)-2,2-dimethylpropanoic acid S4-8 (130 mg, 0.315 mmol) in 1,4- dioxane (2 mL) was added 4 M hydrochloride in 1,4-dioxane (2 mL) under nitrogen atmosphere. After stirred at room temperature under nitrogen atmosphere for 2 hour, the reaction mixture was concentrated to give the title compound (102 mg, 95 % yield) as yellow solids. 1H NMR (400 MHz, CD3OD) d 5.56 - 5.52 (m, 1H), 4.26 (d, J = 14.0 Hz, 1H), 4.16 (d, J = 14.0 Hz, 1H), 3.78 - 3.72 (m, 1H), 3.40 - 3.36 (m, 1H), 3.34 - 3.27 (m, 3H), 3.17 - 3.13 (m, 1H), 3.06 - 2.99 (m, 1H), 2.82 - 2.76 (t, J = 12.4 Hz, 1H), 2.20 - 2.14 (m, 1H), 1.74 - 1.65 (m, 1H), 1.16 (s, 3H), 1.15 (s, 3H).
Compound 6: 3-((S)-2-(((S)-5-(Ethoxycarbonyl)-6-(3-fluoro-2-methylphenyl)-2-(thiazol- 2-yl)-3,6-dihydropyrimidin-4-yl)methyl)-6-thioxotetrahydro-1H-pyrazino[1,2- c]pyrimidin-7(2H,6H,8H)-yl)-2,2-dimethylpropanoic acid
Figure imgf000126_0001
To a solution of (S)-ethyl 6-(bromomethyl)-4-(3-fluoro-2-methylphenyl)-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate (H2-1A) (106 mg, 0.205 mmol) in tetrahydrofuran (3 ml) was added (S)-2,2-dimethyl-3-(6-thioxotetrahydro-1H-pyrazino[1,2-c]pyrimidin- 7(2H,6H,8H)-yl)propanoic acid hydrochloride S4 (70 mg, 0.205 mmol) and triethylamine (80 mg, 0.792 mmol) under nitrogen atmosphere. After stirred at 40 oC under nitrogen atmosphere for 2 hours, the reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (10 mL) twice. The combined organic layers were washed with brine (10 mL), dried over Na2SO4(s), filtered and concentrated to give a residue, which was purified by pre-HPLC (Column: Waters Xbrige C18 (5 µm 19 * 150 mm), Mobile phase A: water (0.1 % ammonium bicarbonate), Mobile phase B: acetonitrile, UV: 214 nm, Flow rate: 15 mL/min, Gradient: 20 - 50 % (%B)) to give the title compound (30 mg, 98.5 % purity, 23 % yield, 99.6 % stereopure) as yellow solids. LC-MS (ESI): RT = 3.764 min, mass calcd. for
C30H37FN6O4S2628.2, m/z found 629.3 [M+H]+ . Chiral HPLC (Column: Chiralpak IE, 5 µm 4.6 * 250 mm; Mobile Phase: Hex : EtOH : TFA = 60 : 40 : 0.2 at 1 mL/min; Temp: 30 °C; Wavelength: 254 nm; RT = 8.668 min).1H NMR (400 MHz, CDCl3) d 9.54 (s, 1H), 7.81 (d, J = 3.2 Hz, 1H), 7.41 (d, J = 3.2 Hz, 1H), 7.08 - 7.02 (m, 1H), 6.99 - 6.97 (m, 1H), 6.90 (t, J = 8.4 Hz, 1H), 6.02 (s, 1H), 5.50 - 5.47 (m, 1H), 4.38 - 4.35 (m, 2H), 4.09 - 4.02 (m, 3H), 3.89 (d, J = 16.8 Hz, 1H), 3.72 - 3.65 (m, 1H), 3.41 - 3.38 (m, 2H), 3.26 - 3.21 (m, 1H), 2.91 - 2.88 (m, 1H), 2.80 - 2.77 (m, 1H), 2.55 (s, 3H), 2.41 (t, J = 9.2 Hz, 1H), 2.29 (t, J = 10.8 Hz, 1H), 2.18 - 2.13 (m, 1H), 1.82 - 1.78 (m, 1H), 1.33 (s, 6H), 1.12 (t, J = 7.2 Hz, 3H). Compound 7: 3-(3-(Cyanomethylene)-7-(((S)-5-(ethoxycarbonyl)-6-(3-fluoro-2- methylphenyl)-2-(thiazol-2-yl)-3,6-dihydropyrimidin-4- yl)methyl)hexahydroimidazo[1,5-a]pyrazin-2(3H)-yl)-2,2-dimethylpropanoic acid (mixtur of 2 diastereomers)
Figure imgf000127_0001
Preparation of intermediate S5:
Figure imgf000127_0002
Intermediate S5-1: Ethyl 2-cyanoacetimidate hydrochloride
To a solution of malononitrile (3.00 g, 45.4 mmol) and ethanol (2.09 g, 45.4 mmol) in diethyl ether (15 mL) was added 6 M hydrochloride in diethyl ether (10 mL, 60 mmol) at 0 oC. After stirred at 0 oC for 0.5 hour, the mixture was warmed up to room temperature and stirred at room temperature overnight. It was filtered and the cake was washed with cooled diethyl ether (20 mL) twice, then suspended in diethyl ether (20 mL), and filtered again, then dried to give the title compound (6.13 g, 70 % purity from 1H NMR, 64 % yield) as white solids which was used in the next step without further purification.1H NMR (400 MHz, DMSO-d6) d 4.20 - 4.14 (m, 2H), 4.03 (s, 2H), 1.24 - 1.20 (m, 3H). Intermediate S5-2: tert-Butyl 3-(cyanomethylene)-2-(3-ethoxy-2,2-dimethyl-3- To a solution of ethyl 2-cyanoacetimidate hydrochloride S5-1 (710 mg, 3.345 mmol) and triethylamine (450 mg, 4.447 mmol) in acetonitrile (20 mL) was added tert-butyl 3-(((2,2- dimethyl-3-oxo-3-propoxypropyl)amino)methyl)piperazine-1-carboxylate S1-4 (500 mg, 1.17 mmol). After stirred at 50 oC overnight, the mixture was concentrated to give a residue, which was diluted with ethyl acetate (15 mL), washed with brine (100 mL), dried over Na2SO4 (s) and filtered. The filtrate was concentrated in vacuo to give a residue, which was purified by by silica gel column chromatography (petroleum ether : ethyl acetate = 2 : 1) to give the title compound (244 mg, 48 % yield) as yellow oil. LC-MS (ESI): RT = 1.52 min, mass calcd. for C20H32N4O4 392.2, m/z found 396.3 [M+H]+ . 1H NMR (400 MHz, CDCl3) 4.57 (d, J = 12.0 Hz, 0.6H), 4.29 - 4.22 (m, 0.4H), 4.18 - 4.00 (m, 3.4H), 3.90 - 3.87 (m, 0.6H), 3.46 - 3.34 (m, 2.4H), 3.21 - 2.81 (m, 5.6H), 2.71 - 2.58 (m, 1H), 1.47 (s, 9H), 1.31 - 1.26 (m, 6H), 1.21 (s, 3H). Intermediate S5-3: Ethyl 3-(3-(cyanomethylene)hexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)-2,2-dimethylpropanoate hydrochloride
To a solution of tert-butyl 3-(cyanomethylene)-2-(3-ethoxy-2,2-dimethyl-3-oxoprop yl)hexahydroimidazo[1,5-a]pyrazine-7(1H)-carboxylate S5-2 (123 mg, 0.282 mmol) in dichloromethane (1 mL) was added 6 M hydrochloride in diethyl ether (3 mL, 18 mmol) at 0 oC. After stirred at room temperature for 2 hours, the reaction mixture was concentrated to give the title compound (96 mg, 99 % yield) as yellow solids which was used in the next step without further purification. LC-MS (ESI): RT = 0.95 min, mass calcd. for C15H25ClN4O2 328.2, m/z found 293.4 [M-HCl+H]+ . Intermediate S5: (4S)-Ethyl 6-((3-(cyanomethylene)-2-(3-ethoxy-2,2-dimethyl-3- oxopropyl)hexahydroimidazo[1,5-a]pyrazin-7(1H)-yl)methyl)-4-(3-fluoro-2- methylphenyl)-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-carboxylate
To a solution of ethyl 3-(3-(cyanomethylene)hexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)-2,2-dimethylpropanoate hydrochloride S5-3 (96 mg, 0.280 mmol) in N,N- dimethylformamide (1 mL) was added (S)-ethyl 6-(bromomethyl)-4-(3-fluoro-2- methylphenyl)-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-carboxylate (H2-1A) (100 mg, 0.205 mmol), N-ethyl-N-isopropylpropan-2-amine (225 mg, 1.74 mmol) at room temperature. After stirred at room temperature for 3 hours, the mixture poured into water (20 mL), extracted with ethyl acetate (20 mL) twice. The combined organic layers were washed with water (10 mL), brine (10 mL), dried over Na2SO4(s) and filtered. The filtrate was concentrated to give a residue, which was purified by silica gel column chromatography (petroleum ether : acetone = 10 : 1 to 5 : 1) to give the title compound (71 mg, 48 % yield) as yellow solids. LC- MS (ESI): RT = 1.84 min, mass calcd. for C33H40FN7O4S 649.3, m/z found 650.2 [M+H]+ .1H NMR (400 MHz, CDCl3) 9.48 - 9.43 (m, 1H), 7.82 (d, J = 2.8 Hz, 1H), 7.43 (d, J = 3.2 Hz, 1H), 7.12 - 7.06 (m, 1H), 6.99 - 6.97 (m, 1H), 6.93 - 6.88 (m, 1H), 6.05 - 6.01 (m, 1H), 4.72 - 4.60 (m, 0.6H), 4.18 - 3.87 (m, 6.4H), 3.76 - 3.58 (m, 1H), 3.46 - 3.30 (m, 2H), 3.21 - 3.02 (m, 2.6H), 2.97 - 2.70 (m, 3H), 2.59 - 2.50 (m, 3.4H), 2.48 - 2.31 (m, 1H), 2.22 - 2.15 (m, 1H), 1.33 - 1.21 (m, 9H), 1.12 (t, J = 7.2 Hz, 3H). Compound 7: 3-(3-(Cyanomethylene)-7-(((S)-5-(ethoxycarbonyl)-6-(3-fluoro-2- methylphenyl)-2-(thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)- hexahydroimidazo[1,5-a]pyrazin-2(3H)-yl)-2,2-dimethylpropanoic acid (mixtur of 2 diastereomers)
Figure imgf000129_0001
To a solution of (4S)-ethyl 6-((3-(cyanomethylene)-2-(3-ethoxy-2,2-dimethyl-3-oxopr opyl)hexahydroimidazo[1,5-a]pyrazin-7(1H)-yl)methyl)-4-(3-fluoro-2-methylphenyl)-2- (thiazol-2-yl)-1,4-dihydropyrimidine-5-carboxylate S5 (71 mg, 0.079 mmol) in ethanol (0.9 mL) and water (0.3 mL) was added lithium hydroxide monohydrate (19 mg, 0.453 mmol). After stirred at room temperature overnight, the mixture was concentrated and diluted with waster (15 mL), adjusted with 0.1 M hydrochloric aqueous solution to pH 5 ~ 6, extracted with ethyl acetate (20 mL) twice. The combined organic layers were concentrated to give a residue, which was purified by Prep-HPLC (Column: waters Xbrige C18 (5 µm 19 * 150 mm), Mobile Phase A: water (0.1 % ammonium hydroxide), Mobile Phase B: acetonitrile, (4.9 mg, 93.6 % purity, 8 % yield) as yellow solids. LC-MS (ESI): RT = 3.554 min, mass calcd. for C31H36FN7O4S 621.3, m/z found 621.9 [M+H]+ . 1H NMR (400 MHz, DMSO-d6) 12.26 (br s, 1H), 9.59 - 9.51 (m, 1H), 8.04 - 7.92 (m, 2H), 7.22 - 7.15 (m, 1H), 7.06 - 7.01 (m, 2H), 5.88 (s, 1H), 4.47 (d, J = 12.8 Hz, 0.6H), 4.04 - 3.91 (m, 4.4H), 3.75 (s, 0.6H), 3.62 - 3.50 (m, 1.4H), 3.43 - 3.37 (m, 2H), 3.24 - 3.17 (m, 2H), 2.99 - 2.90 (m, 3H), 2.45 (s, 3H), 2.39 - 2.33 (m, 1H), 2.14 - 2.04 (m, 1H), 1.18 - 1.04 (m, 9H). Compound 8: (S)-3-(3-(Acetylimino)-7-((5-(ethoxycarbonyl)-6-(3-fluoro-2- methylphenyl)-2-(thiazol-2-yl)-3,6-dihydropyrimidin-4- yl)methyl)hexahydroimidazo[1,5-a]pyrazin-2(3H)-yl)-2,2-dimethylpropanoic acid (mixtur of 2 diastereomers)
Figure imgf000130_0001
Preparation of intermediate S6
Figure imgf000130_0002
Intermediate S6-1: tert-Butyl 2-(3-ethoxy-2,2-dimethyl-3-oxopropyl)-3- iminohexahydroimidazo[1,5-a]pyrazine-7(1H)-carboxylate hydrobromide
To a mixture of tert-butyl 3-(((3-ethoxy-2,2-dimethyl-3-oxopropyl)amino)methyl)pip erazine-1-carboxylate S1-4 (1.6 g, 4.19 mmol) in dichloromethane (2 mL) at room temperature was added the solution of cyanic bromide (666 mg, 6.29 mmol) in dichloromethane (2 mL) dropwise. After stirred at room temperature overnight, the mixture concentrated under reduced pressure to give the title compound (1.61 g, 77 % yield) as white soilds. LC-MS (ESI): RT = 1.732 min, mass calcd. for C18H32N4O4 368.2, m/z found 369.2 [M+H]+ .1H NMR (300 MHz, DMSO-d6) d 8.37 (s, 2H), 4.14 (q, J = 6.9 Hz, 3H), 4.00 - 3.90 (m, 2H), 3.87 - 3.71 (m, 1H), 3.64 (t, J = 9.6 Hz, 1H), 3.56 - 3.45 (m, 2H), 3.20 - 3.05 (m, 2H), 2.95 - 2.67 (m, 1H), 1.44 (s, 9H), 1.25 (t, J = 6.9 Hz, 3H), 1.20 (s, 6H). Intermediate S6-2: tert-Butyl 3-(acetylimino)-2-(3-ethoxy-2,2-dimethyl-3- oxopropyl)hexahydroimidazo[1,5-a]pyrazine-7(1H)-carboxylate
To a solution of tert-butyl 2-(3-ethoxy-2,2-dimethyl-3-oxopropyl)-3- iminohexahydroimidazo[1,5-a]pyrazine-7(1H)-carboxylate hydrobromide S6-1 (245 mg, 0.436 mmol) in dichloromethane (10 mL) was added triethylamine (140 mg, 1.38 mmol) and acetyl chloride (35 mg, 0.446 mmol) at room temperature. After stirred at room temperature for 1 hour, the mixture was poured into water (30 mL) and extracted with dichloromethane (30 mL) twice. The combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4(s), filtered and concentrated in vacuo to give the title compound (190 mg, 96 % yield) as brown oil. LC-MS (ESI): RT = 1.460 min, mass calcd. for C20H34N4O5410.3, m/z found 411.2 [M+H]+ .1H NMR (400 MHz, CDCl3) d 4.42 - 4.34 (m, 1H), 4.18 - 4.13 (m, 4H), 3.99 - 3.92 (m, 2H), 3.65 - 3.55 (m, 2H), 3.31 - 3.19 (m, 2H), 2.99 - 2.75 (m, 2H), 2.35 (s, 3H), 1.46 (s, 9H), 1.30 - 1.24 (m, 9H). Intermediate S6-3: tert-Butyl 3-(acetylimino)-2-(3-ethoxy-2,2-dimethyl-3- oxopropyl)hexahydroimidazo[1,5-a]pyrazine-7(1H)-carboxylate
A mixture of tert-butyl 3-(acetylimino)-2-(3-ethoxy-2,2-dimethyl-3- oxopropyl)hexahydroimidazo[1,5-a]pyrazine-7(1H)-carboxylate S6-2 (190 mg, 0.417 mmol) in tetrahydrofuran (3 mL) and methanol (3 mL) was added a solution of lithium hydroxide monohydrate (40 mg, 0.953 mmol) in water (1 mL). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 1 hour. Then the reaction mixture was acidified to pH = 5 with 0.5 M hydrochloride aqueous solution. The mixture was extracted with ethyl acetate (15 mL) for three times and the combined organic layers were concentrated in vacuo to give the title compound (120 mg, 56 % yield) as yellow oil. LC-MS (ESI): RT = 1.068 min, mass calcd. for C18H30N4O5382.2, m/z found 383.2 [M+H]+ . Intermediate S6: 3-(3-(Acetylimino)hexahydroimidazo[1,5-a]pyrazin-2(3H)-yl)-2,2- dimethylpropanoic acid hydrochloride
To a solution of tert-butyl 3-(acetylimino)-2-(3-ethoxy-2,2-dimethyl-3- oxopropyl)hexahydroimidazo[1,5-a]pyrazine-7(1H)-carboxylate S6-3 (120 mg, 0.235 mmol) in 3 M hydrochloride in 1,4-dioxane (7 mL, 21 mmol) was stirred at room temperature for 30 minutes. The mixture was concentrated in vacuo to give the title compound (80 mg, 90 % yield) as brown solids. LC-MS (ESI): RT = 0.226 min, mass calcd. for C13H22N4O3282.2, m/z found 283.2 [M+H]+ . Compound 8: (S)-3-(3-(Acetylimino)-7-((5-(ethoxycarbonyl)-6-(3-fluoro-2- methylphenyl)-2-(thiazol-2-yl)-3,6-dihydropyrimidin-4- yl)methyl)hexahydroimidazo[1,5-a]pyrazin-2(3H)-yl)-2,2-dimethylpropanoic acid (mixtur of 2 diastereomers)
Figure imgf000132_0001
To a solution of 3-(3-(acetylimino)hexahydroimidazo[1,5-a]pyrazin-2(3H)-yl)-2,2- dimethylpropanoic acid hydrochloride S6 (60 mg, 0.159 mmol) in N,N-dimethylformamide (3 mL) was added N,N-diisopropylethylamine (150 mg, 1.16 mmol) and (S)-ethyl 6- (bromomethyl)-4-(3-fluoro-2-methylphenyl)-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5- carboxylate (H2-1A) (65 mg, 0.141 mmol) at room temperature. After stirred at room temperature overnight, the mixture was concentrated and purified by C18 column (acetonitrile : water = 5 % to 95 %) to afford the title compound (6.7 mg, 95.1 % purity, 7 % yield) as yellow solids. LC-MS (ESI): RT = 3.522 min, mass calcd. for C31H38FN7O5S 639.3, m/z found 640.3 [M+H]+ . 1H NMR (400 MHz, CD3OD) d 7.82 - 7.81 (m, 1H), 7.62 (d, J = 3.2 Hz, 1H), 7.04 - 6.98 (m, 2H), 6.85 - 6.80 (m, 1H), 5.87 (s, 0.3H), 5.86 (s, 0.7H), 4.15 - 4.03 (m, 2H), 3.95 (q, J = 7.2 Hz, 2H) , 3.87 - 3, 71 (m, 2H), 3.55 - 3,42 (m, 2H), 3.36 - 3.28 (m, 3H), 3.10 - 3.03 (m, 0.5H), 2.95 - 2.88 (m, 1.5H), 2.45 - 2.36 (m, 4H), 2.26 - 2.20 (m, 1H), 1.99 (s, 2H), 1.97 (s, 1H), 2.11 - 2.11 (m, 2H), 1.09 (s, 4H), 1.02 (t, J = 7.2 Hz, 3H). Compound 9A: 1-((7-(((S)-5-(Ethoxycarbonyl)-6-(3-fluoro-2-methylphenyl)-2-(thiazol-2- yl)-3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)methyl)cyclopropanecarboxylic acid (single enantiomer)
Figure imgf000133_0001
To a solution of (S)-ethyl 6-(bromomethyl)-4-(3-fluoro-2-methylphenyl)-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate (H2-1A) (90 mg, 0.185 mmol) in tetrahydrofuran (4 ml) was added 1-((3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)methyl)cyclopropanecarboxylic acid hydrochloride S3-A (56 mg, 0.173 mmol) and triethylamine (133 mg, 0.891 mmol) under nitrogen atmosphere. After stirred at 40 oC under nitrogen atmosphere for 2.5 hours and then stirred at room temperature overnight, the reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (10 mL) twice. The combined organic layers were washed with brine (10 mL), dried over Na2SO4(s), filtered and concentrated to give a residue, which was purified by C18 column (acetonitrile : water = 40 % to 70 %) to give the title compound (19 mg, 16 % yield) as yellow solids. LC- MS (ESI): RT = 3.924 min, mass calcd. for C29H33FN6O4S2 612.2, m/z found 613.2 [M+H] . 1H NMR (400 MHz, CD3OD) d 7.81 (d, J = 3.2 Hz, 1H), 7.61 (d, J = 3.2 Hz, 1H), 7.05 - 6.98 (m, 2H), 6.84 - 6.80 (m, 1H), 5.87 (s, 1H), 4.32 - 4.29 (m, 1H), 4.03 - 3.92 (m, 4H), 3.89 - 3.79 (m, 4H), 3.37 - 3.31 (m, 1H), 3.16 - 3.13 (m, 1H), 2.96 - 2.93 (m, 1H), 2.67 - 2.64 (m, 1H), 2.41 (s, 3H), 2.22 - 2.13 (m, 2H), 1.08 - 1.07 (m, 2H), 1.02 (t, J = 6.8 Hz, 3H), 0.87 - 0.86 (m, 2H). Compound 9B: 1-((7-(((S)-5-(Ethoxycarbonyl)-6-(3-fluoro-2-methylphenyl)-2-(thiazol-2- yl)-3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)methyl)cyclopropanecarboxylic acid (single enantiomer)
Figure imgf000134_0001
This compound was prepared from intermediate H2-1A and S3-B under same condition as for 9A. LC-MS (ESI): RT = 3.890 min, mass calcd. for C29H33FN6O4S2 612.2, m/z found 613.2 [M+H]+ .1H NMR (400 MHz, CD3OD) d 7.94 (d, J = 3.2 Hz, 1H), 7.73 (d, J = 3.2 Hz, 1H), 7.18 - 7.10 (m, 2H), 6.96 - 6.92 (m, 1H), 5.99 (s, 1H), 4.52 - 4.49 (m, 1H), 4.15 - 4.03 (m, 4H), 3.97 - 3.89 (m, 3H), 3.88 - 3.81 (m, 1H), 3.41 - 3.36 (m, 1H), 3.32 - 3.27 (m, 1H), 3.01 - 2.88 (m, 2H), 2.52 (s, 3H), 2.48 - 2.41 (m, 1H), 2.19 - 2.13 (m, 1H), 1.22 - 1.19 (m, 2H), 1.13 (t, J = 7.2 Hz, 3H), 1.03 - 0.94 (m, 2H). Compound 10B: 3-(-7-((6-(3-Fluoro-2-methylphenyl)-5-(methoxycarbonyl)-2-(thiazol-2- yl)-3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)-2,2-dimethylpropanoic acid (single enantiomer)
Figure imgf000134_0002
To a solution of methyl 6-(bromomethyl)-4-(3-fluoro-2-methylphenyl)-2-(thiazol-2-yl)-1,4- dihydropyrimidine -5-carboxylate (H4-1B) (100 mg, 90% purity, 0.212 mmol) in dichloromethane (6 mL) was added 2,2-dimethyl-3-(3-thioxohexahydroimidazo[1,5- a]pyrazin-2(3H)-yl)propanoic acid hydrochloride salt S1-A (69 mg, 90 % purity, 0.211 mmol) and triethanolamine (348 mg, 2.33 mmol) at room temperture. After stirred at room temperture overnight, the mixture was diluted with ethyl acetate (30 mL) and washed with brine (30 mL), dried over Na2SO4(s) and filtered. The filtrate was concentrated under reduced presure to give a residue, which was purified by Prep-HPLC (Column: Xtimate C18 (10 µm 50 * 250 mm); Mobile phase A: water (0.1 % ammonium bicarbonate), Mobile phase B: acetonitrile; UV: 254 nm, Flow rate: 15 mL/min, Gradient: 20 - 60 % (%B)) to afford the desired product (42 mg, 98.7 % purity, 33 % yield) as yellow solids. LC-MS (ESI): RT = 3.762 min, mass calcd. for C28H33FN6O4S2 600.7, m/z found 601.2 [M+H]+ 1H NMR (400 MHz, CD3OD) d 7.95 (d, J = 3.2 Hz, 1H), 7.74 (d, J = 3.2 Hz, 1H), 7.18 - 7.08 (m, 2H), 6.96 - 6.92 (m, 1H), 5.98 (s, 1H), 4.54 - 4.51 (m, 1H), 4.13 - 4.04 (m, 2H), 3.96 - 3.84 (m, 3H), 3.70 (t, J = 10.4 Hz, 1H), 3.62 (s, 3H), 3.30 - 3.25 (m, 2H), 2.98 - 2.96 (m, 1H), 2.90 - 2.87 (m, 1H), 2.53 (d, J = 2.0 Hz, 3H), 2.49 - 2.42 (m, 1H), 2.18 (t, J = 10.8 Hz, 1H), 1.22 (s, 3H), 1.21 (s, 3H). Compound 11A: 3-(7-((6-(2-Chloro-3,4-difluorophenyl)-5-(methoxycarbonyl)-2-(thiazol- 2-yl)-3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin- 2(3H)-yl)-2,2-dimethylpropanoic acid (single enantiomer)
Figure imgf000135_0001
To a solution of 2,2-dimethyl-3-(3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)propanoic acid hydrochloride S1-A (50 mg, 0.153 mmol) in tetrahydrofuran (5 mL) was added triethylamine (60 mg, 0.593 mmol). After stirred for 5 minutes, methyl 6- (bromomethyl)-4-(2-chloro-3,4-difluorophenyl)-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5- carboxylate (H5-1A) (82 mg, 0.161 mmol) was added. The mixture was stirred at 40 °C for 2.5 hours, then acidified to pH = 3 with 1 M hydrochloride aqueous solution and extracted with ethyl acetate (10 mL) for three times. The combined organic layers were washed with brine (10 mL), dried over Na2SO4(s), filtered and concentrated to give a residue, which was purified by Prep-HPLC (Column: Gilson Xbrige C18 (5 µm 19 * 150 mm), Mobile phase A: water (+ 0.1 % ammonium bicarbonate), Mobile phase B: acetonitrile, UV: 214 nm, Flow rate: 15 mL/min, Gradient: 20 - 60 % (%B)) to give the title compound (32.0 mg, 28 % yield) as yellow solids. LC-MS (ESI): RT = 3.512 min, mass calcd. for C27H29ClF2N6O4S2 638.1, m/z found 639.1[M+H]+ .1H NMR (400 MHz, CD3OD) d 7.94 (d, J = 3.2 Hz, 1H), 7.74 (d, J = 3.2 Hz, 1H), 7.23 - 7.20 (m, 2H), 6.15 (s, 1H), 4.52 - 4.49 (m, 1H), 4.11 - 3.83 (m, 5H), 3.68 (t, J = 9.6 Hz, 1H), 3.59 (s, 3H), 3.30 - 3.21 (m, 2H), 2.96 - 2.87 (m, 2H), 2.43 (td, J = 11.6, 3.2 Hz, 1H), 2.17 (t, J = 11.2 Hz, 1H), 1.22 (s, 3H), 1.21 (s, 3H). Compound 12B: 3-(7-((6-(3,4-Difluoro-2-methylphenyl)-5-(methoxycarbonyl)-2- (thiazol-2-yl)-3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5- a]pyrazin-2(3H)-yl)-2,2-dimethylpropanoic acid (single enantiomer)
Figure imgf000136_0001
To a solution of methyl 6-(bromomethyl)-4-(3,4-difluoro-2-methylphenyl)-2-(thiazol-2-yl)- 1,4-dihydropyrimidine-5-carboxylate (H6-1B) (100 mg, 90 % purity, 0.203 mmol) in dichloromethane (6 mL) was added 2,2-dimethyl-3-(3-thioxohexahydroimidazo[1,5- a]pyrazin-2(3H)-yl)propanoic acid hydrochloride Intermediate S1-A (66 mg, 90 % purity, 0.203 mmol) and triethanolamine (334 mg, 2.24 mmol) at room temperture. After stirred at room temperture overnight, the mixture was dissolved in ethyl acetate (30 mL) and washed with brine (30 mL), dried over Na2SO4(s) and filtered. The filtrate was concentrated under reduced presure to give a residue which was purified by Prep-HPLC (Column: Xtimate C18 (10 µm 50 * 250 mm); Mobile phase A: water (0.1 % ammonium bicarbonate), Mobile phase B: acetonitrile; UV: 254 nm, Flow rate: 15 mL/min, Gradient: 30 - 80 % (%B)) to afford the desired product (18 mg, 14 % yield) as yellow solids. LC-MS (ESI): RT = 3.474 min, mass calcd. for C28H32F2N6O4S2 618.7, m/z found 619.2 [M+H]+ . 1H NMR (400 MHz, CD3OD) d 7.95 (d, J = 3.2 Hz, 1H), 7.74 (d, J = 3.2 Hz, 1H), 7.05 - 7.02 (m, 2H), 5.93 (s, 1H), 4.54 - 4.51 (m, 1H), 4.14 - 4.03 (m, 2H), 3.96 (s, 0.6H), 3.91 (s, 0.4H), 3.88 - 3.83 (m, 3H), 3.70 (t, J = 10.0 Hz, 1H), 3.62 (s, 3H), 3.29 - 3.25 (m, 2H), 2.98 - 2.95 (m, 1H), 2.88 - 2.86 (m, 1H), 2.57 (d, J = 2.4 Hz, 3H), 2.49 - 2.42 (m, 1H), 2.18 (t, J = 10.8 Hz, 1H), 1.21 (s, 3H), 1.19 (s, 3H). Compound 13A: 3-(7-((6-(2-Bromo-4-fluorophenyl)-5-(ethoxycarbonyl)-2-(thiazol-2-yl)- 3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)-yl)- 2,2-dimethylpropanoic acid (single enantiomer)
Figure imgf000137_0001
To a solution of 2,2-dimethyl-3-(3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)propanoic acid hydrochloride Intermediate S1-A (100 mg, 0.306 mmol) in tetrahydrofuran (10 mL) were added triethylamine (149 mg, 1.48 mmol) and ethyl 4-(2- bromo-4-fluorophenyl)-6-(bromomethyl)-2-(thiazol-2-yl)-1,4-dihydro
pyrimidine-5-carboxylate (H7-1A) (200 mg, 0.358 mmol) at room temperature. After heated at room temperature overnight under nitrogen atmosphere, the reaction mixture was quenched with water (20 mL) slowly and extracted with ethyl acetate (20 mL) for three times. The separated organic layer was washed with brine (20 mL), dried over Na2SO4(s), filtered and concentrated under reduced pressure to give a residue, which was purified by Prep-HPLC (Column: waters Xbrige C18 (5 µm 19 * 150 mm), Mobile Phase A: water (0.1 % ammonium bicarbonate), Mobile Phase B: acetonitrile, UV: 214 nm, Flow rate: 15 mL/min, Gradient: 20 - 60 % (%B)) to afford desired product (70 mg, 29 % yield) as yellow solids. LC-MS (ESI): RT = 3.865 min, mass calcd. For C28H32BrFN6O4S2 678.1, m/z found 679.1 [M+H]+ .1H NMR (400 MHz, DMSO-d6) d 9.63 (br s, 1H), 8.03 (d, J = 2.8 Hz, 1H), 7.95 (d, J = 2.8 Hz, 1H), 7.59 - 7.56 (m, 1H), 7.42 - 7.39 (m, 1H), 7.26 - 7.22 (m, 1H), 6.03 (s, 1H), 4.36 (d, J = 14.4 Hz, 1H), 4.00 - 3.93 (m, 5H), 3.77 (d, J = 2.8 Hz, 2H), 3.64 (t, J = 10.0 Hz, 1H), 3.18 - 3.13 (m, 2H), 2.96 - 2.91 (m, 2H), 2.29 - 2.24 (m, 1.6H), 2.10 - 2.05 (m, 1.4H), 1.13 (s, 6H), 1.05 (t, J = 6.8 Hz, 3H). Compound 14: 3-(7-((6-(2-Chloro-3,4-difluorophenyl)-5-(ethoxycarbonyl)-2-(thiazol-2- yl)-3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)-2,2-dimethylpropanoic acid (mixtur of 2 diastereomers)
Figure imgf000138_0001
To a solution of ethyl 6-(bromomethyl)-4-(2-chloro-3,4-difluorophenyl)-2-(thiazol-2-yl)-1,4- dihydropyrimidine-5-carboxylate H8-1 (95 mg, 0.199 mmol) in dichloromethane (10 mL) was added 2,2-dimethyl-3-(3-thioxohexahydroimidazo [1,5-a]pyrazin-2(3H)-yl)propanoic acid hydrochloride S1-A (58 mg, 0.197 mmol), triethanolamine (90 mg, 0.604 mmol) at room temperature. After stirred at 40 oC overnight, the mixture was concentrated under reduced pressure to give a residue, which was purified by C18 column (acetonitrile : water = 20 % to 40 %) to give the desired compound (34.6 mg, 98.8 % purity, 26 % yield) as yellow solids. LC-MS (ESI): RT = 3.747 min, mass calcd. for C28H31ClF2N6O4S2 652.2, m/z found 653.2 [M+H]+ .1H NMR (400 MHz, CD3OD) d 7.94 (d, J = 2.8 Hz, 1H), 7.74 (d, J = 3.2 Hz, 1H), 7.24 - 7.20 (m, 2H), 6.17 (s, 0.5H), 6.16 (s, 0.5H), 4.52 - 4.42 (m, 1H), 4.13 - 4.00 (m, 4H), 3.94 - 3.86 (m, 3H), 3.77 - 3.66 (m, 1H), 3.30 - 3.22 (m, 2H), 3.05 - 2.78 (m, 2H), 2.45 - 2.40 (m, 0.5H), 2.32 - 2.27 (m, 1H), 2.19 - 2.14 (m, 0.5H), 1.23 - 1.21 (m, 6H), 1.12 (t, J = 7.2 Hz, 3H). Compound 14A: 3-(7-((6-(2-Chloro-3,4-difluorophenyl)-5-(ethoxycarbonyl)-2-(thiazol-2- yl)-3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)-2,2-dimethylpropanoic acid (single enantiomer)
Figure imgf000139_0001
This compound ws prepared from H8-1A and S1-A under same condition as for compound 14 and purified by C18 column (acetonitrile : water = 20 % to 40 %) to give the desired compound (19.9 mg, 97.1 % purity, 17 % yield) as yellow solids. LC-MS (ESI): RT = 3.481 min, mass calcd. for C28H31ClF2N6O4S2 652.2, m/z found 653.2 [M+H]+ . 1H NMR (400 MHz, CD3OD) d 7.94 (d, J = 3.2 Hz, 1H), 7.74 (d, J = 2.8 Hz, 1H), 7.24 - 7.21 (m, 2H), 6.16 (s, 1H), 4.52 - 4.48 (m, 1H), 4.11 - 4.01 (m, 4H), 3.94 - 3.80 (m, 3H), 3.71 - 3.66 (m, 1H), 3.30 - 3.22 (m, 2H), 2.96 - 2.87 (m, 2H), 2.45 - 2.39 (m, 1H), 2.19 - 2.14 (m, 1H), 1.21 (s, 6H), 1.12 (t, J = 7.2 Hz, 3H). Compound 15A: 3-(7-((6-(3,4-Difluoro-2-methylphenyl)-5-(ethoxycarbonyl)-2-(thiazol- 2-yl)-3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin- 2(3H)-yl)-2,2-dimethylpropanoic acid (single enantiomer)
Figure imgf000140_0001
To a solution of ethyl 6-(bromomethyl)-4-(3,4-difluoro-2-methylphenyl)-2-(thia
zol-2-yl)-1,4-dihydropyrimidine-5-carboxylate H9-1A (100 mg, 0.197 mmol) in tetrahydrofuran (4 mL) was added 2,2-dimethyl-3-(3-thioxohexahydroimidazo[1,5-a]pyrazin- 2(3H)-yl)propanoic acid hydrochloride S1-A (74 mg, 0.227 mmol) and triethylamine (0.14 mL, 0.97 mmol) at 40 oC for 2 hours. Then stirred at room temperture overnight, the mixture was dissolved in ethyl acetate (10 mL) and washed with brine (10 mL), dried over Na2SO4(s) and filtered. The filtrate was concentrated under reduced presure to give a residue, which was purified by Prep-HPLC (separation condition: Column: Xtimate C18, 10 µm 50 mm * 250 mm; Mobile Phase: acetonitrile : water (0.1 % ammonium bicarbonate) = 30 % - 80 % at 15 mL/min; Temp: 35 oC; Wavelength: 254 nm) to afford the desired product (31 mg, 97.9 % purity, 24 % yield) as yellow solids. LC-MS (ESI): RT = 3.710 min, mass calcd. for C29H34F2N6O4S2 632.7, m/z found 633.7 [M+H]+ . 1H NMR (400 MHz, CD3OD) d 7.95 (d, J = 2.8 Hz, 1H), 7.74 (d, J = 3.2 Hz, 1H), 7.10 - 7.00 (m, 2H), 5.94 (s, 1H), 4.53 (d, J = 14.8 Hz, 1H), 4.14 - 4.02 (m, 4H), 3.97 - 3.87 (m, 3H), 3.70 (t, J = 10.0 Hz, 1H), 3.30 - 3.23 (m, 2H), 2.98 (d, J = 11.2 Hz, 1H), 2.89 (d, J = 6.8 Hz, 1H), 2.58 (s, 1.5H), 2.57 (s, 1.5H), 2.45 (td, J = 11.2, 3.6 Hz, 1H), 2.18 (t, J = 10.0 Hz, 1H), 1.24 (s, 3H), 1.23 (s, 3H), 1.15 (t, J = 6.8 Hz, 3H). Compound 15B: 3-(7-((6-(3,4-Difluoro-2-methylphenyl)-5-(ethoxycarbonyl)-2-(thiazol- 2-yl)-3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin- 2(3H)-yl)-2,2-dimethylpropanoic acid (single enantiomer)
Figure imgf000141_0001
This compound was prepared from H9-1B and S1-A using same condition as for compound 15A and purified by Prep-HPLC (separation condition: Column: Xtimate C18, 10 µm 50 mm * 250 mm; Mobile Phase: acetonitrile : water (0.1 % ammonium bicarbonate) = 30 % - 80 % at 15 mL/min; Temp: 35 oC; Wavelength: 254 nm) to afford the desired product (30 mg, 98.2 % purity, 24 % yield) as yellow solids. LC-MS (ESI): RT = 3.539 min, mass calcd. for C29H34F2N6O4S2632.7, m/z found 633.7 [M+H]+ .1H NMR (400 MHz, CD3OD) d 7.93 (d, J = 3.2 Hz, 1H), 7.73 (d, J = 2.8 Hz, 1H), 7.07 - 6.97 (m, 2H), 5.92 (s, 1H), 4.53 (d, J = 14.4 Hz, 1H), 4.14 - 4.03 (m, 4H), 3.93 - 3.88 (m, 3H), 3.75 (t, J = 9.6 Hz, 1H), 3.29 - 3.25 (m, 2H), 3.04 (d, J = 10.0 Hz, 1H), 2.78 (d, J = 11.2 Hz, 1H), 2.55 (s, 3H), 2.34 - 2.24 (m, 2H), 1.24 (s, 3H), 1.23 (s, 3H), 1.13 (t, J = 7.2 Hz, 3H). Compound 16A: 3-(7-((6-(2-Bromo-4-fluorophenyl)-5-(methoxycarbonyl)-2-(thiazol-2- yl)-3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)-2,2-dimethylpropanoic acid (single enantiomer)
Figure imgf000142_0001
To a solution of 2,2-dimethyl-3-(3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)propanoic acid hydrochloride S1-A (126 mg, 0.386 mmol) in tetrahydrofuran (5 mL) were added triethylamine (195 mg, 1.93 mmol) and methyl 4-(2-bromo-4-fluorophenyl)-6- (bromomethyl)-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-carboxylate H10-1A (210 mg, 0.386 mmol) at room temperature. After stirred at room temperature overnight under nitrogen atmosphere, the reaction mixture was quenched with water (20 mL) slowly and extracted with ethyl acetate (20 mL) for three times. The separated organic layer was washed with brine (20 mL), dried over Na2SO4(s), filtered and concentrated under reduced pressure to give a residue, which was purified by C18 column (acetonitrile : water (0.1 % ammonium bicarbonate) = 05 % to 95 %) to give the title compound (33 mg, 99.7 % purity, 13 % yield) as yellow solids. LC-MS (ESI): RT = 3.095 min, mass calcd. for C27H30BrFN6O4S2 664.1, m/z found 665.1 [M+H]+ . 1H NMR (400 MHz, CD3OD) d 7.98 - 7.90 (m, 1H), 7.78 - 7.70 (m, 1H), 7.46 - 7.36 (m, 2H), 7.14 - 7.04 (m, 1H), 6.14 (s, 1H), 4.53 - 4.48 (m, 1H), 4.11 - 4.02 (m, 2H), 3.94 - 3.85 (m, 3H), 3.68 (t, J = 9.6 Hz, 1H), 3.59 (s, 3H), 3.24 - 3.15 (m, 2H), 2.98 - 2.86 (m, 2H), 2.48 - 2.41 (m, 1H), 2.23 - 2.15 (m, 1H), 1.23 (s, 6H). Compound 16B: 3-(7-((6-(2-Bromo-4-fluorophenyl)-5-(methoxycarbonyl)-2-(thiazol-2- yl)-3,6-dihydropyrimidin-4-yl)methyl)-3-thioxohexahydroimidazo[1,5-a]pyrazin-2(3H)- yl)-2,2-dimethylpropanoic acid (single enantiomer)
Figure imgf000143_0001
This compound was prepared from H10-1B and S1-A under same condition as for compound 16A and purified by Prep-HPLC (Column: sunfire C18 (5 µm 19 * 150 mm), Mobile Phase A: water (0.1 % trifluoroacetic acid), Mobile Phase B: acetonitrile, UV: 214 nm, Flow rate: 15 mL/min, Gradient: 35 - 45 % (%B)) to afford the product, which was further purified by C18 column (acetonitrile : water (0.1 % ammonium bicarbonate) = 05 % to 95 %) to give the title compound (47 mg, 99.4 % purity, 18 % yield) as yellow solids. LC- MS (ESI): RT = 3.096 min, mass calcd. for C27H30BrFN6O4S2 664.1, m/z found 665.1 [M+H]+ .1H NMR (400 MHz, CD3OD) d 7.94 (d, J = 3.6 Hz, 1H), 7.73 (d, J = 3.2 Hz, 1H), 7.42 - 7.38 (m, 2H), 7.10 - 7.06 (m, 1H), 6.14 (s, 1H), 4.46 - 4.42 (m, 1H), 4.12 - 4.03 (m, 2H), 3.92 - 3.85 (m, 3H), 3.75 (t, J = 10.0 Hz, 1H), 3.59 (s, 3H), 3.28 - 3.25 (m, 2H), 3.06 - 3.03 (m, 1H), 2.81 - 2.74 (m, 1H), 2.33 - 2.26 (m, 2H), 1.23 (s, 3H), 1.22 (s, 3H). GLS4 (WO 2008154817, example 5; Bioorganic & Medicinal Chemistry, 2017, 25, 1042- 1056, compound 8n) was selected as reference 1; another compound (WO2015132276, example 76) was selected as reference 2. Chemical structure of both reference compounds was shown below.
Figure imgf000144_0001
Bioassay Example
EXAMPLE 1: anti-viral assay in HepG2.2.15 cells
Materials and Equipments
1) Cell line
HepG2.2.15 (the HepG2.2.15 cell line can be produced by transfection of the HepG2 cell line as described in Sells, Chen, and Acs 1987 (Proc. Natl. Acad. Sci. USA 84: 1005-1009), and the HepG2 cell line is available from ATCC® under number HB-8065™).
2) Reagents
DMEM/F12 (INVITROGEN-11330032)
FBS (GIBCO-10099-141)
Dimethyl sulfoxide(DMSO) (SIGMA-D2650)
Penicillin-streptomycin solution (HYCLONE-SV30010)
NEAA (INVITROGEN-1114050)
L-Glutamine (INVITROGEN-25030081)
Geneticin Selective Antibiotic (G418, 500mg/ml) (INVITROGEN-10131027)
Trypsinase digestion solution (INVITROGEN-25300062)
CCK8 (BIOLOTE-35004)
QIAamp 96 DNA Blood Kit (12) (QIAGEN-51162)
FastStart Universal Probe Mast Mix (ROCHE-04914058001)
3) Consumables
96-well cell culture plate (COSTAR- 3599)
Micro Amp Optical 96-well reaction plate (APPLIED BIOSYSTEMS-4306737)
Micro Amp Optical 384-well reaction plate (APPLIED BIOSYSTEMS) 4) Equipment
Plate reader (MOLECULAR DEVICES, SPECTRAMAX M2e)
Centrifuge (BECKMAN, ALLEGRA-X15R)
Real Time PCR system (APPLIED BIOSYSTEMS, QUANTSTUDIO 6)
Real Time PCR system (APPLIED BIOSYSTEMS, 7900HT)
Methods
1) Anti-HBV activity and cytotoxicity determination
HepG2.2.15 cells were plated into 96-well plate in 2% FBS culture medium at the density of 40,000 cells/well and 5,000cells/well for HBV inhibitory activity and cytotoxicity determination, respectively. After incubation at 37 °C, 5% CO2 overnight, cells were treated with medium containing compounds for 6 days with medium and compounds refreshed after 3 days of treatment. Each compound was tested in a 1:3 serial dilutions at 8 different concentrations in triplicate. The highest concentration of the compounds was 10uM or 1uM for anti-HBV activity assay and 100uM for cytotoxicity determination.
Cell viability was determined by CCK-8 assay. After 6 days of compounds treatment, 20 µl CCK-8 reagents were added to each well of cytotoxicity assay plates. Cell plates were incubated at 37℃, 5% CO2 for 2.5 h. The absorbance at 450nm wavelength and the absorbance at 630nm wavelength as reference was measured.
The change of HBV DNA level induced by the compounds was assessed by quantitative real- time polymerase chain reaction (qPCR). Briefly, the HBV DNA in the culture medium was extracted using QIAamp 96 DNA Blood Kit according to the manual and then quantified by real-time PCR assay using the primers and probe in the table 2 below.
Table 2
Figure imgf000145_0001
2) DATA analysis
EC50 and CC50 values are calculated by the GRAPHPAD PRISM software. If the CV% of DMSO controls is below 15% and the reference compounds shows expected activity or cytotoxicity, the data of this batch of experiment is considered qualified.
RESULTS: See Table 3 below.
Table 3
Figure imgf000146_0001
As the potency data shown in table 3, all these compounds demonstrated highly potent in vitro activities against HBV HepG2.2.15 cell. EXAMPLE 2: Metabolic stability of test compound in Human Hepatocyte cell
Materials and reagents: see table 4 below.
Table 4
Figure imgf000146_0002
Figure imgf000147_0001
Study Design
1. The cryopreserved human hepatocytes cells were thawed in 37°C water bath and diluted with pre-warmed incubation medium to a working cell density of 1 × 10^6 viable cells/mL.
2. The 198 µL pre-warmed hepatocyte suspensions were spiked with 2µL of 100 µM compound or reference compound(Verapamil) at a final concentration of 1.0 µM in a 96- well plate. The plate was incubated at 37°C, 900 rpm. All incubations will be performed in singlet.
3. 25 µL aliquots of well contents were collected at time points of 0, 15, 30, 60, 90 and 120 minutes. The reactions were stopped by the addition of 6-fold volumes of cold acetonitrile with internal standards.
4. After centrifugation for 25 minutes at 3,220 g. Aliquot of 100 µL of the supernatant was mixed with 100 µL of ultra-pure H2O and then used for LC-MS/MS analysis.
Data Analysis
All calculations were carried out using Microsoft Excel. Peak areas were determined from extracted ion chromatograms. Determine the in vitro half-life (t1/2) of parent compound by regression analysis of the percent parent disappearance vs. time curve.
The in vitro half℃life (in vitro t1/2) is determined from the slope value k:
in vitro t1/2 = 0.693 / k
Conversion of the in vitro t1/2 (in min) into the in vitro intrinsic clearance (in vitro CLint, in µL/min/10^6 cells) is done using the following equation:
in vitro CLint = kV/N
V = incubation volume (0.2 mL);
N = number of hepatocytes per well (0.2 × 10^6 cells).
Conversion of the in vitro t1/2 (in min) into the scale-up intrinsic clearance (CLint(liver), in mL/min/kg) was done using the following equation:
CLint(liver) = kV/N × scaling factor
Table 5. Scaling factors for in vivo intrinsic clearance prediction are listed below: Hepatocyte Liver blood Liver Weight Scaling
Species Concentration flow (Q,
(g liver/kg body weight) Factor
(106cells/g liver) mL/min/kg) Human 25.7 99 2544.3 20.7
Control compound verapamil will be included in the assay. Any value of the compound that is not within the specified limits will be rejected and the experiment would be repeated. Result
Table 6: Results Summary of Metabolic Stability of Compounds in Human Hepatocytes
Figure imgf000148_0001
Hepatocyte s metabolic stability test has become the "gold standard" for evaluating hepatic metabolism and toxicity of drugs and other xenobiotics in vitro. As the human hepatocyte stability data shown in table 6, compounds 1A, 3B, and 4B showed improved metabolic stability in human hepatocyte cells when comparing with reference 1 and reference 2. EXAMPLE 3: In Vitro Assessment of Cytochrome P450 (Cyp450) Induction in Cryopreserved Human Hepatocytes
Materials: See table 7 below.
Table 7.
Figure imgf000148_0002
Figure imgf000149_0001
Equipment:
Infinite 200 PRO microplate reader, Tecan
7500 QPCR system, Applied Biosystems.
Study design
Preparation and plating of Human Hepatocytes
1. The cryopreserved human hepatocytes were thawed in 37°C water bath and diluted by plating medium to a seeding density of 0.55 × 10^6 cells/mL.
2. Transfer 100 µL to each well of collagen I coated 96-well plate. Place plate(s) in incubator and incubate at 37°C for 4-6 hours.
3. After incubation, observe cell morphology, agitate plate(s) to loosen debris, and replace medium. Place plate in incubator and incubate for 18 hours.
Incubation with test compound(s)
1. Prepare dilute test compound and positive control inducers with 37°C prepared incubation medium to respective working concentrations (Table 8). Final concentration of DMSO in the treatment group will be 0.1%. Prepare 25 mM chlorpromazine in DMSO and dilute 1000-fold with incubation medium as a cytotoxicity control.
Table 8: Test compound and positive control inducer concentrations
Figure imgf000149_0002
2. Remove the Hepatocyte plate from the incubator. Observe cell morphology. Replace the medium in the appropriate wells with 125 µL of the toxicity controls, DMSO controls, inducers, or test article solutions, each in triplicate.
3. After 24 hours and 48 hours, remove the Hepatocyte plate from the incubator and observe cell morphology. Renew the medium with test articles that freshly diluted from DMSO stocks. Return plate to the incubator.
3. Cell viability assessment
After 72 hours of treatment, warm the incubation medium to 37°C. Remove the induction plate(s) from the incubator. Observe cell morphology. Cell viability was assessed by CellTiter-Fluor™ Cell Viability Assay kit.
4. mRNA preparation and RT-PCR
1. mRNA was prepared and measured using the Cells-to-Ct kit. Add DNase to Lysis solution.
2. 15µL of sample lysate was added to 35µL of Reverse Transcription Master Mix (containing 2× RT Buffer, 20× RT Enzyme Mix and Nuclease-free Water) for a final 50µL reaction volume.
3. Separate PCR cocktails were prepared for CYP3A4; containing the CYP specific probe set and that of ACTB as the endogenous control gene. A typical PCR cocktail contained TaqMan Universal Master Mix (2×), Taqman Gene Expression Assay probe (20×, CYP, FAM labeled), Taqman Gene Expression Assay probe (20×, ACTB, VIC labeled) and RNase-free water.
4. 4 µL cDNA samples or RT mix without cell lysate (negative control) were added to PCR cocktail to make the final volume of 20µL. Templates for standard curve are prepared from a 3-fold serial dilution of the cDNA sample mixture of respective Rifampicin induced samples at highest concentration.
5. Reactions were analyzed on an Applied Biosystems Real Time PCR system (AB 7500).
Each PCR was performed in triplicate.
Data Analysis
All calculations are carried out using Microsoft Excel.
1) Cell viability
Percent cell viability (%) = (I(sample)-I(background))/ (I(vehicle)-I(background)) × 100
Where“I” means fluorescence intensity.
2) RNA qua tificatio For mRNA level determination, the mRNA content in each well is expressed as 2Ct(ACTB)- Ct(CYP).
Fold of induction = mRNA(induced) / mRNA(vehicle)
3) The percent adjusted positive control is determined by:
% of positive control = [(fold induction of test article)/ (fold induction of positive control)]*100
Result
Table 9. Induction potential of CYP3A4 by test compound based on mRNA level determination
Figure imgf000151_0001
Induction of cytochrome P450 (CYP450) enzymes is associated with an increased prevalence of clinical drug-drug interactions and may result in therapeutic failure. CYP3A4 is by far the most abundant isoform and is responsible for the majority of CYP450-related metabolism of all marketed drugs. The CYP induction activity of compound 1A is far less than two-folds against vehicle control and far less than 20% against the positive control on CYP3A4 isoform. Compound 1A demonstrated no CYP induction effect when comparing with compound reference 2, thus devoid of CYP induction liability.
EXAMPLE 4: A Pharmacokinetic and Tissue Distribution Study of compound via Intravenous and Oral administration in male C57BL/6 mice.
Materials and methods
Male C57BL/6 mice with a weight range of 20-25g (Hua Fu Kang, China) were used. Animals were fasted overnight and free access to food 4 hours after dosing.
Test compound (correction factor: 1.00) was dissolved in a 20 % hydroxypropyl-b- cyclodextrin (HP-b-CD) at a final concentration of 1 mg/ml for the intravenous (IV) formulation and at final concentrations of 0.5 mg/ml for the oral (PO) formulation. The intravenous formulation was dosed at 2 ml/kg to obtain a dose of 2 mg/kg. The oral Blood samples were taken at 7 and 20 min, 1, 2, 4, 8 and 24 h after intravenous dose administration. Blood and liver samples were taken at 30 min, 1, 2, 4, 8, 12 and 24 h after oral dose administration.
Approximately 0.020 mL blood will be collected into BD blood collection tubes containing K3-EDTA at each time point. Samples were placed immediately on melting ice and plasma was obtained following centrifugation at 4 °C for 5 minutes at approximately 4000 x g. Plasma samples were adjusted to pH 3-4 by phosphoric acid and stored at -75±15°C prior to analysis. The whole process was completed within 1 hour.
Liver samples were collected at adopted time point, and the vial containing the tissues sample was snap-frozen in liquid nitrogen right away and kept at -75±15°C prior to analysis. All liver samples were weighed and homogenized with phosphoric acid solution (pH to 3-4) by liver weight (g) to phosphoric acid solution volume (mL) ratio 1:4 before analysis.
Plasma and liver samples were analyzed using LC-MS/MS methods. The lower limit of quantification (LLOQ) for plasma was 1.0 ng/ml and for liver was 2.5ng/g.A non- compartmental analysis using the "Linear up log down" rule was used for all data. A limited pharmacokinetic analysis was performed using PhoenixTM Professional (Version 6.1).
Results: See table 10 below for plasma PK results, and table 11 for PO liver PK results.
Table 10. Summary of IV/PO Pharmacokinetic results in mice plasma.
Figure imgf000153_0001
Table 11. Summary of PO liver Pharmacokinetic results in mice.
Figure imgf000153_0002
Mouse in-vivo PK studies are critical to ensure drug candidates have appropriate PK properties that can be evaluated in preclinical pharmacology and safety studies. When comparing with compounds reference 1 and reference 2, compounds 1A and 3B showed a far slower clearance, over 3-folds higher dose-normalized AUC and increased bioavailability in plasma, and far increased dose-normalized Cmax and dose-normalized AUCinf in liver. EXAMPLE 5: A Pharmacokinetic Study of test compound after Intravenous and Oral Administration in Male SD Rats
Materials and methods
Male SD rats with a weight range of 250-300 g (Si Bei Fu Laboratory Animal Technology Co. Ltd, China) were used. Animals were fasted overnight and free access to food 4 hours after dosing.
Test compound (correction factor: 1.00) was dissolved in a 20 % hydroxypropyl-b- cyclodextrin (HP-b-CD) at a final concentration of 1 mg/ml for the intravenous (IV) formulation and at final concentrations of 0.5 mg/ml for the oral (PO) formulation.
The intravenous formulation was dosed at 2 ml/kg to obtain a dose of 2 mg/kg. The oral formulations were dosed at 10 ml/kg to obtain final doses of 5 mg/kg.
Blood samples were taken at 5, 15 and 30 min, 1, 2, 4, 8 and 24 h after intravenous dose administration. Blood samples were taken at 15 and 30 min, 1, 2, 4, 8, 12 and 24 h after oral dose administration.
Approximately 0.20 mL blood will be collected into BD blood collection tubes containing Sodium Fluoride (NaF), Potassium Oxalate(KoX) and K3-EDTA at each time point. Samples were placed immediately on melting ice and plasma was obtained following centrifugation at 4 °C for 5 minutes at approximately 4000 x g. Plasma samples were adjusted to pH 3-4 by phosphoric acid and stored at -75±15°C prior to analysis. The whole process was completed within 1 hour.
Plasma samples were analyzed using LC-MS/MS methods. The lower limit of quantification (LLOQ) for plasma was 1.0 ng/ml.
A non-compartmental analysis using the "Linear up log down" rule was used for all data. A limited pharmacokinetic analysis was performed using PhoenixTM Professional (Version 6.1). Results: See table 12 below for plasma PK results. Table 12. Summary of IV/PO Pharmacokinetic results in rat plasma.
Figure imgf000155_0001
Rat in-vivo PK studies are critical to ensure drug candidates have appropriate PK properties that can be evaluated in preclinical pharmacology and safety studies.
Compounds 1A, 3B, and 4B showed a far slower clearance, over two-folds higher dose- normalized AUC (AUCinf/Dose) and an increased (or equal) bioavailability (F(%)) when comparing with reference 2 compound.
It is understood that the examples and embodiments described herein are for illustrative purposes only, and that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the invention as defined by the appended claims.

Claims

CLAIMS It is claimed:
1. A therapeutic combination for use in treating a hepatitis B virus (HBV) infection in a subject in need thereof, comprising:
i) at least one of:
a) a truncated HBV core antigen consisting of an amino acid sequence that is at least 95% identical to SEQ ID NO: 2,
b) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding the truncated HBV core antigen.
c) an HBV polymerase antigen having an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity, and d) a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide sequence encoding the HBV polymerase antigen; and
ii) a compound of Formula (I):
Figure imgf000157_0001
or a deuterated isomer, a stereoisomer or tautomeric form thereof, or a pharmaceutically acceptable salt thereof, wherein:
R1 is selected from the group consisting of phenyl, thiophenyl, pyridyl, and pyridonyl, optionally substituted with one or more substituents selected from the group consisting of C1-4alkyl, halogen, and CN;
R2 is C1-4alkyl; R3 is selected from the group consisting of thiazolyl, pyridyl, and oxazolyl, optionally substituted with one or more substituents selected from fluorine, and C1-6alkyl; n is an integer of 0 or 1;
R4 and R5 are independently selected from the group consisting of H and -COOH;
(i.e., the bond between X and Y) is a single bond or a double bond;
when X and Y are linked by a single bond, X is selected from the group consisting of C(=S), C(=NR6), C(=CHR7) and CHR8, and Y is NR9;
when X and Y are linked by a double bond, X is C-SR9 or C-OR9, and Y is N atom;
Z is selected from the group consisting of CH2 and C(=O);
R6 is selected from the group consisting of CN, C(=O)CH3, and SO2CH3;
R7 is CN;
R8 is CF3;
R9 is selected from the group consisting of H, -C1-6alkyl, -C1-6alkyl-R10, -C1- 6alkoxy-C1-6alkyl-R10 and -(CH2)p-Q-R10;
p is an integer of 0, 1, 2, or 3;
Q is selected from the group consisting of aryl, heteroaryl, and a 3- to 7- membered saturated ring, optionally containing a heteroatom, the heteroatom being an oxygen or a nitrogen, the nitrogen being substituted with H, -C1-6alkyl, -C1-6alkoxy-C1-6alkyl and -C1- 6alkylcarbonyl; and
R10 is selected from -COOH, -C(=O)NHS(=O)2-C1-6alkyl, tetrazolyl and
carboxylic acid bioisosteres.
2. The therapeutic combination of claim 1, comprising at least one of the HBV
polymerase antigen and the truncated HBV core antigen.
3. The therapeutic combination of claim 2, comprising the HBV polymerase antigen and the truncated HBV core antigen.
4. The therapeutic combination of claim 1, comprising at least one of the first non- naturally occurring nucleic acid molecule comprising the first polynucleotide sequence encoding the truncated HBV core antigen and the second non-naturally occurring nucleic acid molecule comprising the second polynucleotide sequence encoding the HBV polymerase antigen
5. A therapeutic combination for use in treating a hepatitis B virus (HBV) infection in a subject in need thereof, comprising
i) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 95% identical to SEQ ID NO: 2; and
ii) a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide sequence encoding an HBV polymerase antigen having an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity; and
iii) a compound of Formula (I):
i
Figure imgf000159_0001
or a deuterated isomer, stereoisomer or tautomeric form thereof, or a
pharmaceutically acceptable salt thereof, wherein:
R1 is phenyl substituted with one or more substituents selected from halogens and C1-6alkyl;
R2 is methyl or ethyl;
R3 is thiazolyl;
n is an integer of 0 or 1;
R4 and R5 are H;
Figure imgf000159_0002
(i.e., the bond between X and Y) is a single bond;
X is C(=S);
Y i NR9 Z is CH2;
R9 is C1-6alkyl-CO2H or (CH2)p-Q-R10;
p is an integer of 0, 1, 2, or 3;
Q is phenyl, a C3-6cycloalkyl, or a 3- to 6- saturated membered ring containing an oxygen; and
R10 is selected from -COOH, -C(=O)NHS(=O)2-C1-6alkyl, tetrazolyl and carboxylic acid bioisosteres, wherein the carboxylic acid bioisosteres are -S(=O)2(OH), - P(=O)(OH)2, -C(=O)NHOH, -C(=O)NHCN, 1,2,4-oxadiazol-5(4H)-one, or 3-hydroxy-4- methylcyclobut-3-ene-1,2-dione, which refer to the following structures:
Figure imgf000160_0001
.
6. The therapeutic combination of claim 4 or 5, wherein the first non-naturally
occurring nucleic acid molecule further comprises a polynucleotide sequence encoding a signal sequence operably linked to the N-terminus of the truncated HBV core antigen, and the second non-naturally occurring nucleic acid molecule further comprises a polynucleotide sequence encoding a signal sequence operably linked to the N-terminus of the HBV polymerase antigen, preferably, the signal sequence independently comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15, preferably the signal sequence is independently encoded by the polynucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 14.
7. The therapeutic combination of any one of claims 1-6, wherein
a) the truncated HBV core antigen consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; and
b) the HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 7.
8. The therapeutic combination of any one of claims 1-7, wherein each of the first, and second non-naturally occurring nucleic acid molecules is a DNA molecule, preferably the DNA molecule is present on a plasmid or a viral vector.
9. The therapeutic combination of any one of claims 4 to 8, comprising the first non- naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule in the same non-naturally nucleic acid molecule.
10. The therapeutic combination of any one of claims 4 to 8, comprising the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule in two different non-naturally occurring nucleic acid molecules.
11. The therapeutic combination of any one of claims 4 to 10, wherein the first
polynucleotide sequence comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3.
12. The therapeutic combination of claim 11, wherein the first polynucleotide
sequence comprises the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
13. The therapeutic combination of any one of claims 4 to 12, wherein the second polynucleotide sequence comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6.
14. The therapeutic combination of claim 13, wherein the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6.
15. The therapeutic combination of any one of claims 1-14, wherein the compound is selected from the group consisting of:
Figure imgf000161_0001
Figure imgf000162_0001
Figure imgf000163_0001
Figure imgf000164_0001
Figure imgf000165_0001
Figure imgf000166_0001
Figure imgf000167_0001
Figure imgf000168_0001
or a deuterated isomer, stereoisomer or tautomeric form thereof, or a
pharmaceutically acceptable salt thereof.
16. A kit comprising the therapeutic combination of any one of claims 1-15, and instructions for using the therapeutic combination in treating a hepatitis B virus (HBV) infection in a subject in need thereof.
17. The therapeutic combination of any one of claims 1 to 15 for use in treating a hepatitis B virus (HBV) infection in a subject in need thereof.
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