WO2016096174A1 - Fluorinated cyclic dinucleotides for cytokine induction - Google Patents

Fluorinated cyclic dinucleotides for cytokine induction Download PDF

Info

Publication number
WO2016096174A1
WO2016096174A1 PCT/EP2015/070635 EP2015070635W WO2016096174A1 WO 2016096174 A1 WO2016096174 A1 WO 2016096174A1 EP 2015070635 W EP2015070635 W EP 2015070635W WO 2016096174 A1 WO2016096174 A1 WO 2016096174A1
Authority
WO
WIPO (PCT)
Prior art keywords
formula
cyclic
sting
cdns
cells
Prior art date
Application number
PCT/EP2015/070635
Other languages
French (fr)
Other versions
WO2016096174A8 (en
Inventor
Fabienne Vernejoul
Gérard TIRABY
Thierry Lioux
Original Assignee
Invivogen
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to EP15762583.1A priority Critical patent/EP3233882B1/en
Priority to CN201580057976.5A priority patent/CN107148424B/en
Priority to ES15762583T priority patent/ES2764178T3/en
Priority to DK15762583.1T priority patent/DK3233882T3/en
Priority to PL15762583T priority patent/PL3233882T3/en
Priority to US15/034,335 priority patent/US10011630B2/en
Application filed by Invivogen filed Critical Invivogen
Priority to EP19174902.7A priority patent/EP3546473A1/en
Publication of WO2016096174A1 publication Critical patent/WO2016096174A1/en
Publication of WO2016096174A8 publication Critical patent/WO2016096174A8/en
Priority to US16/006,465 priority patent/US10562929B2/en
Priority to US16/781,757 priority patent/US11053272B2/en
Priority to US17/365,066 priority patent/US11667664B2/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • 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
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical

Definitions

  • the present invention is in the field of immunotherapy. It concerns cyclic dinucleotides (CDNs) of Formulas I, II and III. In particular, it concerns fluorinated deoxyribo-CDNs and pharmaceutically acceptable salts and prodrugs thereof, which are able to induce production of Type I interferons in human and animal cells.
  • CDNs cyclic dinucleotides
  • fluorinated deoxyribo-CDNs and pharmaceutically acceptable salts and prodrugs thereof, which are able to induce production of Type I interferons in human and animal cells.
  • STING interferon genes
  • Immunotherapy is a rapidly expanding area of medical treatment in which a patient's immune system is deliberately activated, suppressed or otherwise modulated for therapeutic benefit.
  • Immunotherapy agents include cells, antigens (e.g. fragments of bacteria or viruses), antibodies, nucleic acids, peptides, proteins, naturally occurring ligands and synthetic molecules.
  • Cytokines are small glycoprotein messengers known chiefly for their role in orchestrating immune response through complex signaling networks, although they also perform non-immune functions. They have been extensively explored as immunotherapy agents.
  • direct administration of cytokines as immunotherapy is limited by numerous factors, including the very short half-life of cytokines in blood, which must be compensated for with frequent dosing and high doses.
  • One highly promising immunotherapy approach is cytokine induction, whereby the patient is treated with an immunomodulatory agent that triggers the production of one or more therapeutically beneficial cytokines in their body as needed.
  • STING interferon genes
  • E /S E /S, MITA, MPYS, or TM173
  • TMEM173 transmembrane receptor protein
  • IFN-a and IFN- ⁇ via the IRF3 (interferon regulatory factor 3) pathway; and to production of pro-inflammatory cytokines (IL-1a , IL-1 ⁇ , IL-2, IL-6, TNF-a, etc.), via the oncogenic transcription factor NF- ⁇ (nuclear factor kappa-light-chain-enhancer of activated B cells) pathway.
  • STING activates STAT6 (signal transducer and activator of transcription 6) to induce (Th2-type), increase (IL-12) or decrease (IL-10) production of various cytokines, including the chemokines CCL2, CCL20, and CCL26 (Chen et al., 2011 )
  • Human STING is currently known to be activated three ways: via binding of exogenous (3 , 3) cyclic dinucleotides (c- diGMP, c-diAMP and c-GA P) that are released by invading bacteria or archaea (see (Gomelsky, 2011) and references therein); via binding of (2',3')cyclic guanosine monophosphate-adenosine monophosphate ((2',3')c- GAMP), a recently discovered endogenous cyclic dinucleotide that is produced by the enzyme cyclic GMP-AMP synthase (cGAS; also known as C6orf150 or MB21D1) in the presence of exogenous double-stranded DNA (e.g.
  • STING cyclic dinucleotide STING agonists
  • the known cyclic dinucleotide STING agonists are an excellent class of compounds on which to base analogs that might exhibit interesting biological activities or desirable drug-like properties.
  • the present invention comprises novel cyclic dinucleotides for therapeutic use in human and animal health.
  • CDN STING agonists Some examples of cyclic dinucleotide (CDN) STING agonists are described in US/2014/0329889 and WO/2014/189805. However, the authors only chemically synthesized and biologically tested a very small number of the compounds that would be theoretically possible from the extremely general chemical structure drawings that they provide. They do not disclose any detailed structure-activity relationships or describe any class effects of particular structural classes of CDNs. Consequently, the authors provide scant rationale to corroborate the choice of one structural class of CDNs over another in terms of actual STING activity or other properties (e.g. drug-like properties) for a desired STING-related application.
  • CDN cyclic dinucleotide
  • WO/2015/077354 explored STING agonists, including CDNs, all of which are (2',3')-CDNs in which both nucleotides contain a ribose sugar moiety and the two nucleotides are connected by phosphorothioate diester linkages.
  • the present invention is a.
  • the field of STING agonists is ripe for invention.
  • the present invention concerns fluorinated deoxyribo-CDN STING agonists that exhibit unigue, unobvious and previously unreported class effects relative to their corresponding non-fluorinated analogs.
  • CDNs cyclic dinucleotides
  • Xi is H, OH, or F
  • X 2 is H, OH, or F
  • Z is OH, ORi, SH or SRi, wherein:
  • Ri is Na or NH 4 , or
  • Ri is an enzyme-labile group which provides OH or SH in vivo such as pivaloyloxymethyl
  • Bi and B 2 are bases chosen from:
  • cyclic dinucleotide (abbreviated as "CDN") represent a class of cyclic molecules with two phosphodiester linkages, or two phosphorothioate diester linkages, or one phosphodiester linkage and one phosphorothioate diester linkage, between two nucleosides.
  • nucleoside refers to a glycosylamine comprising a nitrogenous base and a five-carbon sugar, wherein the nitrogenous base is bound to the five-carbon sugar via a beta-glycosidic linkage.
  • nucleotide refers to any nucleoside linked to a phosphate group at position 5', 3' or 2' of the sugar moiety.
  • “Pharmaceutically acceptable salts” include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among numerous other acids well known in the pharmaceutical art.
  • pharmaceutically acceptable prodrug herein refers to a compound that is metabolized, for example hydrolyzed or oxidized, in the host (i.e. the human or animal subject that receives the compound) to form the compound of the present invention.
  • prodrugs include compounds that have biologically labile protecting groups on functional moieties of the active compound.
  • Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated or dephosphorylated to produce the active compound.
  • prodrug relates to an inactive or active derivative of a compound represented by the structural Formula (I), (II) or (III) as defined herein above or any one of their specific embodiments, which undergoes spontaneous or enzymatic transformation within the body of an animal, e.g. a mammal such as a human being, in order to release the pharmacologically active form of the compound.
  • a mammal such as a human being
  • prodrugs of the compounds represented by the structural Formula (I), (II) or (III) including any one of the above-described specific embodiments thereof may be formed as described in detail in (Hecker & Erion, 2008).
  • Phosphate prodrug can take the form of an ester, in particular acyloxyalkyl esters (e.g. pivaloyloxymethyl ester (POM)) or S-acylthioethyl (SATE) esters, a carbonate, a carbamate or an amide, such as amino acid prodrugs.
  • acyloxyalkyl esters e.g. pivaloyloxymethyl ester (POM)
  • SATE S-acylthioethyl
  • enzyme-labile protecting group denotes a group designed to enable passive d iffusion of a compound across cellular or parasitic membranes via charge-masking, such that, once inside the cell or parasite interior, the compound undergoes an enzymatic transformation (or deprotection) that affords an OH or SH group.
  • enzyme-labile protecting groups include for instance acyloxyalkyl groups such as pivaloyloxymethyl (POM) or S-acylthioethyl (SATE) or amino acid groups.
  • cyclic dinucleotides of Formula (I), (II) or (III) is considered to also include the pharmaceutically acceptable salts or a pharmaceutically acceptable prodrugs of said cyclic dinucleotides of Formula (I), (II) or (III).
  • a particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Bi or B 2 is Guanine.
  • a particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Bi or B 2 is Adenine.
  • a particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Bi or B 2 are Hypoxanthine.
  • a particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Bi and B 2 are independently chosen from Guanine or Adenine.
  • a particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Bi or B 2 are independently chosen from Guanine or Hypoxanthine.
  • a particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Bi and B 2 are independently chosen from Adenine or Hypoxanthine.
  • a particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Z is OH.
  • a particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Z is SH.
  • a particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Z is OH or SH.
  • a particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Z is SRi, wherein:
  • Ri is Na or NH 4 , or
  • Ri is an enzyme-labile group which provides OH or SH in vivo such as pivaloyloxymethyl.
  • a particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Bi and B 2 are independently chosen from Adenine or Hypoxanthine and Xi and X 2 are identical.
  • Xi and X 2 are OH or a fluorine atom.
  • Z is OH or SH.
  • a particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Bi and B 2 are independently chosen from Guanine or Hypoxanthine and Xi and X 2 are identical.
  • Xi and X 2 are OH or a fluorine atom.
  • Z is OH or SH.
  • a particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Bi and B 2 are independently chosen from Adenine or Guanine and Xi and X 2 are identical.
  • Xi and X 2 are OH or a fluorine atom.
  • Z is OH or SH.
  • a particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Xi and X 2 are not a fluorine atom.
  • a particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when at least one among Xi and X 2 is a fluorine atom. In one particular embodiment, Xi and X 2 are both a fluorine atom.
  • a particular class of cyclic dinucleotides of Formula (I), (II) or (III) is the following cyclic dinucleotides (each of which is provided with a five-character code of the format "CL###”):
  • a particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when one or both nucleosides are modified at the 2' position.
  • This class comprises the following compounds:
  • CDNs c-[2'FdAMP(S)-2'FdlMP(S)](POM) 2
  • STING agonists we identified a structurally unprecedented subset of these CDNs that showed surprising biological activity and that have never previously been reported as STING agonists.
  • CDNs form the basis for the present invention and are distinct from previously reported CDN STING agonists (see, for example: US/2014/0329889 and WO/2014/189805).
  • the present invention provides CDNs that are defined by all of the following structural criteria: firstly, unlike in naturally occurring CDNs, in which the sugar moiety of each nucleotide is a ribose, in the CDNs of the present invention the sugar moiety of each nucleotide is a 2'-deoxyribose; secondly, in the CDNs of the present invention, either the 2' position of the sugar moiety in both nucleotides must be substituted with a fluorine atom, or the 2' position of the sugar moiety of one nucleotide must be substituted with a fluorine atom while the 2' position of the sugar moiety of the other nucleotide must be substituted with a hydrogen atom; thirdly, in the CDNs of the present invention, the base in each nucleotide is preferably chosen from guanine, adenine or hypoxanthine, with the proviso that the two nucleotides of the CDN cannot contain the same
  • the present invention provides CDNs of Formula (I) according to Claim 1 , i.e. compounds of Formula (I):
  • At least one among Xi and X 2 is a fluorine atom
  • Z is OH, ORi, SH or SRi, wherein:
  • Ri is Na or NH 4 , or
  • Ri is an enzyme-labile group which provides OH or SH in vivo such as pivaloyloxymethyl
  • Bi is a different base than B 2, or a pharmaceutically acceptable salt thereof.
  • Bi is a different base than B 2 " includes Bi is Guanine and B 2 is Adenine or Bi is Guanine and B 2 is Hypoxanthine or Bi is Adenine and B 2 is Guanine or Bi is Adenine and B 2 is Hypoxanthine or Bi is Hypoxanthine and B 2 is Guanine or Bi is Hypoxanthine and B 2 is Adenine.
  • a particular class of cyclic dinucleotides of Formula (I) as defined above is when Bi and B 2 are different and both Xi and X 2 are F.
  • a particular class of cyclic dinucleotides of Formula (I) as defined above is when Bi and B 2 are different, both Xi and X 2 are F and Z is OH.
  • a particular class of cyclic dinucleotides of Formula (I) as defined above is when Bi and B 2 are different, both Xi and X 2 are F and Z is ORi as defined above.
  • a particular class of cyclic dinucleotides of Formula (I) as defined above is when Bi and B 2 are different, both Xi and X 2 are F and Z is SH as defined above.
  • a particular class of cyclic dinucleotides of Formula (I) as defined above is when Bi and B 2 are different, both Xi and X 2 are F and Z is SRi as defined above.
  • a particular class of cyclic dinucleotides of Formula (I) as defined above is when Bi and B 2 are different, Xi and X 2 are different and Z is OH.
  • a particular class of cyclic dinucleotides of Formula (I) as defined above is when Bi and B 2 are different, Xi and X 2 are different and Z is ORi as defined above.
  • a particular class of cyclic dinucleotides of Formula (I) as defined above is when Bi and B 2 are different, Xi and X 2 are different and Z is SH as defined above.
  • a particular class of cyclic dinucleotides of Formula (I) as defined above is when Bi and B 2 are different, Xi and X 2 are different and Z is SRi as defined above.
  • the cyclic dinucleotides of the present invention induce Type I interferons and/or pro-inflammatory cytokines in vitro in human cells, an imal cells and human blood.
  • the cytokine-induction activity of these cyclic dinucleotides requires the presence of STING, as confirmed by in vitro experiments in human or animal cells.
  • the cyclic dinucleotides of the invention are agonists of the receptor STING.
  • agonist refers to any substance that activates a biologic receptor in vitro or in vivo to provoke a physiologic response.
  • STING is an abbreviation of "stimulator of interferon genes", which is also known as “endoplasmic reticulum interferon stimulator (ERIS)", “mediator of IRF3 activation (MITA)”, “MPYS” or “transmembrane protein 173 (TM173)”.
  • ERIS endoplasmic reticulum interferon stimulator
  • MIMA immediate mobile reticulum interferon activation
  • MPYS or “transmembrane protein 173 (TM173).
  • STING is a transmembrane receptor protein that in humans is encoded by the gene TMEM173.
  • Activation of STING by cyclic dinucleotides (CDN) leads to activation of the IRF3 and NF- ⁇ pathways and consequently, to induction of Type I interferons and of pro-inflammatory cytokines, respectively.
  • CDN cyclic dinucleotides
  • STING activates STAT6 (signal transducer and activator of transcription 6) to induce (Th2-type), increase (IL-12) or decrease (IL-10) production of various cytokines, including the chemokines CCL2, CCL20, and CCL26 (Chen et al., 201 1 ).
  • STAT6 signal transducer and activator of transcription 6
  • IL-12 increase
  • IL-10 decrease
  • cytokines including the chemokines CCL2, CCL20, and CCL26 (Chen et al., 201 1 .
  • STING agonist herein refers to a substance that activates the receptor STING in vitro or in vivo. According to the invention, a compound is deemed to be a STING agonist if:
  • Type I interferons in vitro in human or animal cells that do not contain active STING.
  • a typical test to ascertain whether a ligand is a STING agonist is to incubate the ligand in a wild-type human or animal cell line and in the corresponding cell line in which the STING coding gene has been genetically inactivated by small or long base deletions (e.g. a homozygous STING knockout cell line).
  • An agonist of STING will induce Type I interferons in the wild-type cells but will not induce Type I interferons in the cells in which the STING coding gene has been inactivated.
  • the cyclic dinucleotides of the invention induce Type I interferons in vitro in human or animal cells that contain active STING. However, they do not induce Type I interferons in vitro in human or animal cells that do not contain active STING.
  • the present invention is concerned with fluorinated deoxyribo-cyclic dinucleotides (CDNs). Specifically, it is concerned with (3',3')-2'(mono- or di-fluorinated)-2'-deoxyribo-(CDNs).
  • CDNs fluorinated deoxyribo-cyclic dinucleotides
  • the cyclic dinucleotides of Formula (I), (II) or (III) wherein at least one among Xi and X 2 is a fluorine atom, in particular Xi and X 2 are both a fluorine atom, and Z is OH induce more Type I interferon and NF- ⁇ activity than do their non-fluorinated counterparts in human and murine cell lines.
  • Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among numerous other acids well known in the pharmaceutical art.
  • pharmaceutically acceptable prodrugs refers to a compound that is metabolized, for example hydrolyzed or oxidized, in the host (i.e. the human or animal subject that receives the compound) to form the compound of the present invention. Typical examples of prodrugs include compounds that have biologically labile protecting groups on functional moieties of the active compound.
  • Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated or dephosphorylated to produce the active compound.
  • the CDN prodrugs described herein can be administered to additionally increase the activity, bioavailability or stability, or otherwise alter the properties of the CDN monophosphate.
  • CDN prodrug ligands are known.
  • alkylation, acylation or other lipophilic modifications on the phosphate moiety, or the use of other analog of the nucleoside will increase the stability of the nucleotide.
  • substituent groups that can replace one or more hydrogens on the phosphate moiety are alkyl, aryl, steroids, carbohydrates, including, but not limited to, sugars, 1 ,2-diacylglycerol and alcohols. Many are described in (Jones, 1995).
  • Another object of the present invention is the cyclic dinucleotides of Formula (I), (II) or (III) for use in a therapeutic treatment in humans or animals.
  • the compounds of the present invention may be used for therapeutic or diagnostic applications in human or animal health.
  • the following examples serve to illustrate different possible applications of the present invention; however, these examples are not intended to limit the scope of the invention.
  • therapeutic agent refers to one or more substances that are administered to a human or animal in order to achieve some kind of therapeutic effect in that human or animal, including to prevent, cure, or mitigate the effects of, infection or disease, and/or to otherwise improve the health of that human or animal.
  • the term "monotherapy” refers to the use of a single substance and/or strategy to treat a human or animal in any clin ical or medical context, as opposed to the use of multiple substances and/or strategies to treat a human or animal in the same clinical or medical context, regardless of whether the multiple substances and/or strategies are used sequentially in any order or concurrently.
  • chemotherapeutic agent refers to one or more chemical substances that are administered to a human or animal in order to kill tumors, or slow or stop the growth of tumors, and/or slow or stop the division of cancerous cells and/or prevent or slow metastasis. Chemotherapeutic agents are often administered to treat cancer, but are also indicated for other diseases.
  • chemotherapy refers to medical treatment of a human or animal with one or more chemotherapeutic agents (see definition above).
  • chemoimmunotherapy refers to the combined use, whether sequentially in any order or concurrently, of chemotherapy substances and/or strategies, and immunotherapy substances and/or strategies. Chemoimmunotherapy is often employed to treat cancer, but can also be employed to treat other diseases.
  • immune system refers to the ensemble, or to any one or more components, of the molecules, substances (e.g. bodily fluids), anatomic structures (e.g. cells, tissue and organs) and physiologic processes involved in preventing infection in the body, in protecting the body during infection or during disease, and/or in helping the body to recuperate after infection or disease.
  • substances e.g. bodily fluids
  • anatomic structures e.g. cells, tissue and organs
  • physiologic processes involved in preventing infection in the body, in protecting the body during infection or during disease, and/or in helping the body to recuperate after infection or disease.
  • immunode agent refers to any endogenous or exogenous substance that can interact with any one or more components of the immune system.
  • the term “immune agent” includes antibodies, antigens, vaccines and their constituent components, nucleic acids, synthetic drugs, natural or synthetic organic compounds, cytokines, natural or modified cells, synthetic analogs thereof, and/or fragments thereof.
  • immunotherapy refers to any medical treatment in which one or more components of a human's or animal's immune system is deliberately modulated in order to directly or indirectly achieve some therapeutic benefit, including systemic and/or local effects, and preventative and/or curative effects.
  • Immunotherapy can involve administering one or more immune agents (see definition above), either alone or in any combination, to a human or animal subject by any route (e.g. orally, intravenously, dermally, by injection, by inhalation, etc.), whether systemically, locally or both.
  • Immunotherapy can involve provoking, increasing, decreasing, halting, preventing, blocking or otherwise modulating the production of cytokines, and/or activating or deactivating cytokines or immune cells, and/or modulating the levels of immune cells, and/or delivering one or more therapeutic or diagnostic substances to a particular location in the body or to a particular type of cell or tissue, and/or destroying particular cells or tissue.
  • Immunotherapy can be used to achieve local effects, systemic effects or a combination of both.
  • the term “immunosuppressed” describes the state of any human or animal subject whose immune system is functionally diminished, deactivated or otherwise compromised, or in whom one or more immune components is functionally diminished, deactivated or otherwise compromised. “Immunosuppression” can be the cause, consequence or byproduct of disease, infection, exhaustion, malnutrition, medical treatment or some other physiologic or clinical state.
  • immunomodulating substance “immunomodulatory substance”, “immunomodulatory agent” and “immunomodulator”, used here synonymously, refer to any substance that, upon administration to a human or animal, directly influences the functioning of the immune system of that human or animal. Examples of common immunomodulators include, but are not limited to, antigens, antibodies and small-molecule drugs.
  • vaccine refers to a biological preparation administered to a human or animal in order to elicit or enhance a specific immune system response and/or protection against one or more antigens in that human or animal.
  • vaccination refers to treatment of a human or animal with a vaccine or to the act of administering a vaccine to a human or animal.
  • adjuvant refers to a secondary therapeutic substance that is administered together (either sequentially in any order, or concurrently) with a primary therapeutic substance to achieve some kind of complimentary, synergic or otherwise beneficial effect that could not be achieved through use of the primary therapeutic substance alone.
  • An adjuvant can be used together with a vaccine, chemotherapy, or some other therapeutic substance.
  • Adjuvants can enhance the efficacy of the primary therapeutic substance, reduce the toxicity or side effects of the primary therapeutic substance, or provide some kind of protection to the subject that receives the primary therapeutic substance, such as, but not limited to, improved functioning of the immune system.
  • the cyclic dinucleotide of Formula (I), (II) or (III) can be administered as immunotherapy to a human or an animal to induce in vivo production of one or more cytokines that are therapeutically beneficial to that human or animal.
  • This type of immunotherapy could be used alone or in combination with other treatment strategies, whether sequentially in any order, or concurrently. It could be used to prevent, cure, and/or mitigate the effects of, infection or disease in that human or animal, and/or to modulate the immune system of that human or animal to achieve some other therapeutic benefit.
  • the cyclic dinucleotides of the present invention can be used for cytokine induction immunotherapy of immunosuppressed individuals.
  • a cyclic dinucleotide of Formula (I) (II) or (III) would be administered to an immunosuppressed human or animal subject to induce in vivo production of one or more cytokines that directly or indirectly enhance the immune system of that human or animal.
  • Subjects that might benefit from such treatment include those suffering from autoimmune disorders, immune system deficiencies or defects, microbial or viral infections, infectious diseases, or cancer.
  • the present invention thus discloses a method for inducing cytokine in immunosuppressed individuals, said method comprising administering to a patient in need thereof a cyclic dinucleotide of Formula (I), (II) or (III) or a pharmaceutically acceptable salt or prodrug thereof.
  • the cyclic dinucleotides of the present invention can be used for cytokine induction immunotherapy in combination with chemotherapy
  • a cyclic dinucleotide of Formula (I) (II) or (III) would be administered together with one or more chemotherapeutic agents, sequentially in any order or concomitantly, to a cancer patient to stop the growth of, shrink and/or destroy tumors in that patient.
  • the chemoimmunotherapy resulting from the combination of cytokine induction, provided by the compound(s) of the present invention, and cytotoxicity, provided by the chemotherapeutic agent(s) might be less toxic to the patient, cause fewer side effects in the patient and/or exhibit greater anti-tumor efficacy than would the chemotherapeutic agent(s) when used as monotherapy.
  • the present invention thus discloses a method for treating cancer, said method comprising administering to a patient in need thereof:
  • chemotherapeutic agent a chemotherapeutic agent
  • a cyclic dinucleotide of Formula (I), (II) or (III) or a pharmaceutically acceptable salt or prodrug thereof a chemotherapeutic agent
  • the cyclic dinucleotides of the present invention can be used for cytokine induction immunotherapy as vaccine adjuvant therapy.
  • a cyclic dinucleotide of Formula (I) (II) or (III) would be administered to a human or animal subject that has received, is receiving or will receive a vaccination.
  • the benefits provided by the present invention might include enhanced efficacy of the vaccination against the target antigen, reduced toxicity of the vaccination, reduced adverse side effects of the vaccination, or enhanced immune protection of the human or animal subject.
  • Another object of the present invention is the cyclic dinucleotides of Formula (I), (II) or (III) for use in the treatment of a bacterial infection, a viral infection or a cancer.
  • cancer refers to the physiological condition in subjects that is characterized by unregulated or dysregulated cell growth or death.
  • the term “cancer” includes solid tumors and blood-born tumors, whether malignant or benign.
  • the cancer is from the following group: bladder cancer, breast cancer, cholangiocellular cancer, leukemia, lung cancer, lymphoma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer and urothelial cancer.
  • the cancer is a solid pancreatic tumor.
  • the present invention thus discloses a method for treating a bacterial infection, a viral infection or a cancer, said method comprising administering to a patient in need thereof a cyclic dinucleotide of Formula (I), (III) or (III) or a pharmaceutically acceptable salt or prodrug thereof.
  • Another object of the present invention is the cyclic dinucleotides of Formula (I), (II) or (III) for use in the treatment of a pathology that may be alleviated by the induction of an immune response via the STING pathway.
  • Another object of the present invention is a kit-of-parts comprising a cyclic dinucleotide of Formula (I), (II) or (III) and a chemotherapeutic agent for use in the treatment of solid pancreatic tumors.
  • kit-of-parts refers to a combined preparation wherein the active ingredients are physically separated for use in a combined therapy by simultaneous administration or sequential administration to the patient.
  • the chemotherapeutic agent and the cyclic dinucleotide or a pharmaceutically acceptable salt or prodrug thereof are administered to the patient in a separate form, either simultaneously, separately or sequentially in any order, for the treatment of cancer.
  • said chemotherapeutic agent is gemcitabine.
  • Natural nucleosides 2'-deoxy-Adenosine (dA), 2'-deoxy-Guanosine (dG), or 2'-deoxy-lnosine (dl) can be used for the synthesis of CDNs of the Formulas (I) and (II).
  • the 3'-deoxy-Adenine, 3'-deoxy-Guanosine, or 3'-deoxy-lnosine for CDNs of the Formula (II) and (III) refers to a nucleoside unit having a sugar moiety, for example a ribosyl moiety or a xylosyl moiety that is modified at the 3' position such that the hydroxyl group (3'-OH) is replaced by an hydrogen group.
  • any STING agonist destined for therapeutic use must be able to penetrate into cells. Furthermore, greater cellular uptake of a compound translates to higher bioavailability, which is a desirable property for clinical use.
  • the fluorinated compounds were designed to explore the possibility that greater cellular uptake conferred by one or two fluorine atoms would lead to greater Type I interferon induction activity than that of the reference compound, c- AIMP, which does not contain any fluorine atoms.
  • the subject CDNs are more active as STING agonists than are their corresponding non-fluorinated analogs.
  • the CDNs of the present invention exhibit greater STING-dependent cytokine induction activity in human cells, animal cells and human blood than do their corresponding non-fluo nated analogs
  • these fluorinated deoxyribo-CDNs consistently showed superior resistance to cleavage by snake-venom phosphodiesterase (SVPD) or nuclease P1 (NP1 ), compared to their corresponding non-fluo nated ribo-CDNs.
  • SVPD snake-venom phosphodiesterase
  • NP1 nuclease P1
  • the 2'-deoxy-2'-fluoro of Adenosine, Guanosine or Inosine for CDNs of Formula (I) and (II) refers to a nucleoside with a modification at the 2' position, such that, the hydroxyl group (2'-OH) is replaced by a fluoro group (2'-F).
  • the 2'-fluoronucleoside derivatives for the synthesis of CDNs of Formula (I) and (II) may be prepared by any of the methods known in the art (see, for example: (Herdewijna, 1989; Thomas, 1994)and(Ross, 1997)).
  • the 3'-deoxy-3'-fluoro of Adenosine, Guanosine or Inosine for CDN of Formula (II) and (III) refers to a nucleoside with a modification at the 3' position, such that the hydroxyl group (3'-OH) is replaced by a fluoro group (3'-F).
  • One or both of the internucleotide linkages that are present in the cyclic dinucleotides can be phosphorothioate internucleotide linkages.
  • the phosphorous atom in a phosphodiester linkage of CDNs can be described as being "pro-chiral.” Once a non- bonding oxygen atom of the phosphodiester linkage is replaced or modified, a chiral sugar-phosphate linkage is generated. The resulting intersugar linkage is either an Sp intersugar linkage or an Rp intersugar linkage. Replacement of a non-bonding oxygen atom in the natural phosphodiester linkage, with a sulfur atom to obtain a phosphorothioate linkage generates a chiral center and consequently, affords Sp and Rp diastereomers. Molecules wherein substantially all of the phosphorous atoms in the sugar backbone are either Sp or Rp are referred to herein as "chirally pure".
  • Cyclic dinucleotides are enzymatically degraded by nucleases and/or phosphodiesterases (see, for example: (Li et al., 2014) (Diner et al., 2013) (Danilchanka & Mekalanos, 2013) (Shanahan, Gaffney, Jones, & Strobel, 2013) (Simm, Morr, Kader, Nimtz, & Romling, 2004)) and therefore, when used as therapeutic agents, these compounds can suffer from diminished half-life.
  • the compounds CL655 and CL656 were chosen to enable maximal half-life, and possibly higher activity, in vivo, as they contain phosphorothioate (also known as "P(S)" or "thiophosphate”) internucleotide linkages.
  • P(S) also known as "P(S)” or "thiophosphate”
  • the use of such linkages is a known strategy to circumvent enzymatic hydrolysis (see, for example: US 2014/0205653 A1 ).
  • An example of a phosphorothioate compound that is more resistant to enzymatic hydrolysis than its phosphodiester analog is (2',3')c-G s A s MP or (2',3') c-GAMP(S) (InvivoGen catalog code: tlrl-scga; Li, 2014).
  • the phosphorothioate linkage introduces an additional chiral center on the phosphorus atom, which yields a diastereoisomer pair ([Rp] and [Sp]) at each phosphorothioate linkage.
  • CL655 and CL656 were obtained and tested as racemic mixtures.
  • protecting group refers to a chemical functional group that is attached to an oxygen, nitrogen or phosphorus atom to prevent further reaction of that atom, or for other purposes.
  • a wide variety of protecting groups for oxygen and for nitrogen are known to those skilled in the art of organic synthesis, and are described, for example, in (Wuts, Greene, & Greene, 2014).
  • cyclic dinucleotides or prodrugs thereof of the Formulas (I), (II) or (III) are prepared by first preparing the corresponding nucleoside, then preparing the 5'-hydroxy group, the functional group (OH) at the 2' position or 3' position, and then, if necessary, protecting the exocyclic amine of the purine base. Then, the appropriately protected nucleosides are converted to the corresponding 3'-phosphoramidite, 2'-phosphoramidite, 3'-H-phosphonate or 2'-H- phosphonate, which constitute the starting material for the preparation of the cyclic dinucleotides described herein.
  • Scheme 1 is a non-limiting example of the synthesis of active compounds of the present invention, and in particular, a synthetic approach to CDNs of Formula (I), where Bi, B 2 , Xi , X ⁇ , and Z are defined above.
  • Str is a trityl derivative such as dimethoxytrityl (DMTr)
  • p is a protecting group
  • R ⁇ is an alkyl group such as an isopropyl group
  • R3 is a protecting group such as cyanoethyl
  • R 4 is a protecting group such as allyl
  • X 3 is O or S.
  • Scheme 2 is a non-limiting example of the synthesis of active compounds of the present invention, and in particular, an alternate synthetic approach to CDNs of Formula (I), where Bi, B 2 , Xi, X 2 , and Z are defined above.
  • Str is a trityl derivative such as dimethoxytrityl (DMTr)
  • p is a protecting group
  • R 2 is an alkyl group such as an isopropyl group
  • R 4 is a protecting group such as cyanoethyl
  • R 5 is a protecting group such as allyl
  • X 3 is O or S.
  • CDNs of Formulas (I) and (II) are prepared by first preparing appropriately protected phosphoramidite of Formula (l A ) or H-phosphonate of Formula (l F ):
  • Scheme 3 is a non-limiting example of the synthesis of phosphoramidites of Formula (l A ) and of H-phosphonates of Formula (l F ), where B 2 , Yp, Xp, R 7 , Re, and Str are defined above.
  • Phosphoramidites of the Formula ( ) and H-phosphonates of the Formula (IF) can be synthesized by first preparing the appropriately protected nucleosides of the Formula (IV), which can be accomplished by one of ordinary skill in the art. Firstly, the base, which has an exocyclic amine, is protected, and then the 3'- and 5'-hydroxyl groups are simultaneously or selectively protected.
  • Scheme 4 is a non-limiting example of the synthesis of appropriately protected nucleoside of Formula (IV), where B 2 , X 2 , p, and Str are defined above.
  • R 5 and R 6 are protecting groups.
  • Nucleosides of the Formula (IV A ) can be prepared by methods outlined in: (Chu, 2002; Rajagopalan, 2003; Schinazi, 2004; Vorbriiggen, 2001 ).
  • FIGURE 1 STING signaling in the cell. Activation of STING by cyclic dinucleotides (CDN) leads to activation of the IRF3 and NF- ⁇ pathways and consequently, to induction of Type I interferons and of pro-inflammatory cytokines, respectively.
  • CDN cyclic dinucleotides
  • FIGURE 2 In vitro Type I interferon induction activity in THP1 -DualTM cell cultures: non-fluorinated vs. fluorinated cyclic dinucleotides.
  • FIGURE 3 In vitro NF- ⁇ pathway induction in THP1 -DualTM cell cultures: non-fluorinated vs. fluorinated cyclic dinucleotides.
  • FIGURE 4 In vitro Type I interferon induction activity in wild-type vs. STING knockout B16 cells. It shows relative ISG54 activity (as an indirect measurement of Type I interferon induction) of non-fluorinated vs. fluorinated cyclic dinucleotides incubated in cultures of wild-type (left-side of graph) or STING-knockout (right-side of graph) B16 cells for 24 h. WT: wild-type; SKO: STING knockout (homozygous).
  • FIGURE 5 In vitro NF- ⁇ pathway induction in THP1 -DualTM cell cultures: non-fluorinated vs. fluorinated cyclic dinucleotides.
  • FIGURE 4 In vitro Type I interferon induction activity in
  • Type I interferon induction activity in wild-type vs. STING-knockout RAW cells shows relative ISG54 activity (as an indirect measurement of Type I interferon induction) of non-fluorinated vs. fluorinated cyclic dinucleotides incubated in cultures of wild-type (left-side of graph) or STING-knockout (right-side of graph) RAW cells for 24 h.
  • WT wild-type
  • SKO STING knockout (homozygous).
  • FIGURE 6 Type I interferon induction activity of cyclic dinucleotides in mice. Measurement of Type I interferon induction in sera from mice at 4 h post-treatment.
  • FIGURE 7 IL-6 induction activity of cyclic dinucleotides in mice. Measurement of IL-6 induction in sera from mice at 4 h post-treatment.
  • FIGURE 8 In vivo elimination of cyclic dinucleotides in mice. Temporal change in plasma concentration of cyclic dinucleotides in treated mice.
  • FIGURE 9 Cytokine induction activity of CDNs in human whole blood.
  • FIGURES 10 A-D Resistance of distinct CDNs to cleavage by the enzymes SVPD and NP1 over time, as monitored by HPLC. Compare CL614 and CL656 to c-AIMP, for resistance to SVPD (A) or NP1 (B). Compare CL603 and CL656 to c-GAMP, for resistance to SVPD (C) or NP1 (D).
  • FIGURE 11 In vitro activity of distinct CDNs in THP-1 DualTM cells before and after incubation with the enzymes SVPD and NP1.
  • the invention will be illustrated by the non-limiting following examples.
  • Anhydrous solvents and reagents suitable for nucleoside and nucleotide synthesis were purchased and were handled under dry argon or nitrogen using anhydrous technique.
  • Amidite coupling reactions and cyclizations were performed in anhydrous acetonitrile or pyrid ine under dry argon or nitrogen.
  • the starting materials for all reactions in dry pyridine were dried by concentration (three times) from pyrid ine.
  • Preparative silica-gel flash chromatography was performed using Fluka 60 A high-purity grade or Merck Grade 9385 silica using gradients of methanol in dichloromethane.
  • Analytical LC/ES MS was performed on an Agilent 1290 Infinity UHPLC system coupled to a diode array detector (DAD) Agilent 1260 Infinity and an Agilent 6130 Quadrupole mass spectrometer equipped with an electrospray ionization source (ESI) and controlled by Chemstation software.
  • the LC system was equipped with an Aquity CSH C18 50x2.1 m m 1 .7 ⁇ column using gradients of 10 mM ammonioum formate and acetonitrile at 300 ⁇ /min flow.
  • the UV detection wavelength was 254 nm.
  • the mass spectrometer was operated in positive and negative ESI modes
  • Preparative HPLC was performed on a Waters preparative 150Q HPLC system monitoring at 254 nm on a SunFire Prep C18 5 ⁇ OBD 30 x 150mm column using gradients of 10 mM ammonium formate and acetonitrile at a flow rate 60 mL/min.
  • the 1 H NMR spectra were acquired on either a Bruker 300 MHz (Fourrier 300) at room temperature and reported in ppm downfield.
  • Molecular sieves (MS) 3A were employed after drying the commercially supplied product at 250 °C for 12 h under vacuum.
  • the commercial nucleoside phosphoramidites were supplied from Chemgenes.
  • EXAMPLE 1 Synthesis of the compounds of the invention
  • Example 1.A General protocol for preparation of phosphotriester: The appropriately protected phosphoramidite or commercially available phosphoramidite was coevaporated three times with dry ACN, and the resulting solid was dissolved in a solution of Activator42 ® (0.1 mol/L, 2 equiv.) in the presence of molecular sieves 3A. To the solution was added allyl alcohol (2 equiv.) and the resulting mixture was stirred for 30 min.
  • Activator42 ® 0.1 mol/L, 2 equiv.
  • the appropriately protected phosphoramidite or commercial phosphoramidite was dissolved in a solution of ACN. To the solution were added water (2 equiv.) and pyridium TFA (1.2 equiv.), and the mixture was stirred for 15 min. Then, the solvents were removed in vacuo. The residue was treated with a solution of DCA/DCM (3%) in the presence of water (10 equiv.) for 15 min. The reaction was quenched with addition of MeOH and pyridine. The solvents were removed in vacuo and the residue was purified by silica-gel column chromatography, using DCM/MeOH as eluent. The structure of the compound was confirmed by LC-ES/MS analysis with ions at [M-H] " and/or [M+H] + .
  • Example 1.D Alternative protocol for the dinucleotide synthesis: To a solution of compound of Example 1.B in a solution of Activator42 ® (0.1 mol/L, 2 equiv.) in the presence of molecular sieves 3A was added in one portion an appropriately protected phosphoramidite or commercial phosphoramidite. The mixture was stirred for 30 min.
  • Activator42 ® 0.1 mol/L, 2 equiv.
  • Example 1.G Protocol for cyclization of the dinucleotide:
  • Example 1.E or 1.F was coevaporated three times with dry pyrid ine and then, dry ACN.
  • the residue was suspended in THF, and to the resulting heterogeneous mixture were successively added N- methylimidazole (10 equiv.) and 2,4,6-triisopropylbenzenesulfonyl chloride (10 equiv.).
  • the resulting mixture was stirred at 25 °C for 3 h to 36 h.
  • the solvent was removed in vacuo and the residue was triturated with EtOAc.
  • the resulting colorless precipitate was collected by filtration and washed with chilled EtOAc. This precipitate was used in the next step without any further purification.
  • the structure of the compound was confirmed by LC-ES/MS analysis with ions at [M-H] " and/or [M+H] + .
  • Example 1.H Alternative protocol for cyclization of the dinucleotide:
  • Example 1 .E or 1.F The solid obtained from Example 1 .E or 1.F was coevaporated three times with dry pyridine. The residue was suspended in dry pyridine, and to the resulting solution was added 1 -(Mesitylene-2-sulfonyl)-3-nitro-1 ,2,4-triazole (MSNT) (5 equiv.). The resulting mixture was stirred at 25 °C for 3 h to 18 h. Then, the solvent was removed in vacuo, and the resulting product used in the next step without any further purification. The structure of the compound was confirmed by LC-ES/MS analysis with ions at [M-H] " and/or [M+H] + .
  • MSNT 1 -(Mesitylene-2-sulfonyl)-3-nitro-1 ,2,4-triazole
  • Example 1.1 Alternative protocol for cyclization of the dinucleotide:
  • Example 1 The solid obtained in Example 1 .D or appropriately protected compound was coevaporated three times with dry pyridine. The residue was suspended in dry pyridine, and to the resulting solution was added 5,5-dimethyl-2-oxo-2- chloro-1 ,3,2-dioxaphosphinane (DMOCP) (3 equiv.). The resulting mixture was stirred at 25 °C for 3 h to 18 h.
  • DMOCP 5,5-dimethyl-2-oxo-2- chloro-1 ,3,2-dioxaphosphinane
  • Example 1.J Protocol for the deprotection and purification of cyclic dinucleotides:
  • Example 1.K Protocol for the deprotection and purification of cyclic dinucleotides:
  • Intermediate 2 was prepared from intermediate 1 and the commercially available phosphoramidite of Inosine using a sim ilar procedure to that described in Example 1.D to provide 8.04 g (68% yield) of intermediate 2.
  • the intermediate 8 (4.93 g, 6.41 mmol) was dissolved in a solution of ZnBr 2 (0.5 M) in DCIW/PrOH (1/1 ) (40 mL, 19.3 mmol). The solution was stirred for 40 min at rt. The reaction was neutralized with 1 N NaHC0 3 solution. The different layers were separated and the organic layer was washed with water and brine, dried over MgSC filtered, and then concentrated in vacuo. The residue was purified by silica-gel column chromatography, using DCM/MeOH as eluent to give 2.68 g (90% yield) of intermediate 9.
  • Intermediate 21 was prepared from intermediate 1 and the commercially available phosphoramidite of Inosine using a sim ilar procedure to that described in Example 1 .D to provide 1.05 g (65% yield) of intermediate 21.
  • Intermediate 27 was prepared from intermediate 23 and commercially available phosphoramidite of 2'-deoxy-2'- fluorolnosine using a similar procedure to that described in Example 1.C to provide 3.16 g (62% yield) of intermediate 27.
  • Intermediate 31 was prepared from intermediate 30 and commercial phosphoramidite of 2'-deoxy-2'-fluorolnosine using a similar procedure to that described in Example 1.C to provide 2.53 g (55% yield) of intermediate 31.
  • Intermediate 33 was prepared from intermediate 32 using a similar procedure to that described in Example 1.G to provide 1.40 g (99% yield) of intermediate 33.
  • Intermediate 35 was prepared from intermediate 34 and commercially available phosphoramidite of 2'-deoxy-2'- fluorolnosine using a similar procedure to that described in Example 1.C to provide 447 mg (35% yield) of intermediate 35.
  • Rt 6.20 min
  • m/z 964 [M+Hf
  • Rt 6.20 min
  • m/z 964 [M+Hf
  • Rt 6.20 min
  • m/z 964 [M+Hf
  • Intermediate 45 was prepared from the commercially available phosphoramidite of 2'-deoxy-2'-fluoroGuanosine using a similar procedure to that described in Example 1.A to provide 3.75 g (60% yield) of intermediate 45.
  • Intermediate 47 [3'-0-(CE)phosphotriester-N 2 (iB)-2'-deoxy- 2'fluoroGuanosine]-(3',5')-[3'-0- (CE)phosphodiester-N 2 (iB)-2'-deoxy-2 - fluoroGuanosine]
  • Intermediate 54 [3'-0-(CE)phosphorothioate-triester-N e (Bz)2'- deoxy-2'fluoroAdenosine]-(3',5')-[3'-0- (CE)phosphorothioate-diester-N 2 (iB)-2'-deoxy- 2'-fluoroGuanosine]
  • Example 1.1 was prepared from intermediate 3 using a similar procedure to that described in Example 1.J to provide 2.4 g (60% yield) of example 1.1.
  • Example 1.2 was prepared from intermediate 13 using a similar procedure to that described in Example 1 J to provide 22.5 mg (21% yield) of example 1.2.
  • Example 1.3 was prepared from intermediate 17 using a similar procedure to that described in Example 1.J to provide 17.5 mg (22% yield) of example 1.3.
  • Example 1.4 was prepared from intermediate 20 using a similar procedure to that described in Example 1 J to provide 50 mg (19% yield) of example 1.4.
  • Example 1.5 was prepared from intermediate 22 using a similar procedure to that described in Example 1 J to provide 7mg (20% yield) of example 1.5.
  • Example 1.6 was prepared from intermediate 26 using a similar procedure to that described in Example 1.K to provide 90 mg (31 % yield) of Example 1 .6.
  • Example 1.7 was prepared from intermediate 29 using a similar procedure to that described in Example 1.K to provide 40 mg (21 % yield) of Example 1 .7.
  • Example 1.8 was prepared from intermediate 33 using a similar procedure to that described in Example 1.K to provide 104 mg (10% yield) of Example 1.8.
  • Example 1.9 was prepared from intermediate 37 using a similar procedure to that described in Example 1.K to provide 10 mg (18% yield) of Example 1 .9.
  • Example 1.10 was prepared from intermediate 40 using a similar procedure to that described in Example 1.J to provide 50 mg (19% yield) of Example 1 .10.
  • Example 1.1 1 was prepared from intermediate 44 using a similar procedure to that described in Example 1.K to provide 157 mg (35% yield) of Example 1.1 1.
  • Example 1.12 was prepared from intermediate 48 using a similar procedure to that described in Example 1.K to provide 15 mg (5% yield) of Example 1 .12.
  • Example 1.13 was prepared from intermediate 51 using a similar procedure to that described in Example 1.K to provide 320 mg (31 % yield) of Example 1.13.
  • Example 1.14 was prepared from intermediate 55 using a similar procedure to that described in Example 1.K to provide 15 mg (28% yield) of Example 1 .14.
  • Example 1.14 To a solution of Example 1.14 (7 mg, 9.85 ⁇ ) in water (200 ⁇ _) was added dropwise a solution of intermediate 56 (19 mg, 79.0 ⁇ ) in acetone (500 ⁇ _). The mixture was stirred overnight in the dark. Then the mixture was neutralized with a saturated solution of Na 2 S 2 0 3 (15 ⁇ _) and subsequently diluted with water (10 mL). The aqueous layer was extracted three times with EtOAc (3 x 10mL). The organic layers were pooled, dried over MgS0 4 , filtered and concentrated in vacuo to provide 4 mg (45% Yield) of Example 1 .15.
  • Example 1.9 To a solution of Example 1.9 (5 mg, 7.19 ⁇ ) in water (200 ⁇ ) was added dropwise a solution of intermediate 56 (17 mg, 70 ⁇ ) in acetone (500 ⁇ ). The mixture was stirred overnight in the dark. Then the mixture was neutralized with a saturated solution of Na 2 S 2 0 3 (15 ⁇ ) and subsequently diluted with water (10 mL). The aqueous layer was extracted three times with EtOAc (3 x 10mL). The organic layers were pooled, dried over MgS0 4 , filtered and concentrated in vacuo to provide 5 mg (78% Yield) of Example 1.16.
  • EXAMPLE 2 Biological assays We have determined that several cyclic d inucleotides of the present invention induce the production of multiple cytokines in human or animal cells. Specifically, these cyclic dinucleotides induce the production of Type I interferons and/or pro-inflammatory cytokines. The in vitro cytokine-induction activity of a representative set of these cyclic dinucleotides is reported here to require the presence of the eukaryotic cellular receptor stimulator of interferon genes (STING).
  • STING eukaryotic cellular receptor stimulator of interferon genes
  • cytokine-induction activities of non-fluorinated vs. fluorinated cyclic dinucleotides disclosed in this invention have been demonstrated by using different reporter cell lines. The cell lines and experiments are explained below.
  • THP1 -DualTM (InvivoGen catalog code: thpd-nfis): These cells were derived from the human monocytic cell line THP- 1 by stable integration of two inducible reporter constructs. They enable simultaneous study of the two main signaling pathways for STING: the NF- ⁇ pathway, by monitoring the activity of secreted embryonic alkaline phosphatase (SEAP); and the IRF pathway, by assessing the activity of a secreted luciferase (Lucia).
  • SEAP secreted embryonic alkaline phosphatase
  • IRF pathway by assessing the activity of a secreted luciferase (Lucia).
  • Both reporter proteins are readily measurable in the cell culture supernatant when using QUANTI-BlueTM (InvivoGen catalog code: rep-qb1 ), a SEAP detection reagent that turns purple/blue in the presence of SEAP (quantified by measuring the optical density from 620 nm to 655 nm), and QUANTI-LucTM (InvivoGen; catalog code: rep-qld ), a luminometric enzyme assay that measures luciferase expression to report on ISG54 expression (as an indicator of IFN- ⁇ / ⁇ production).
  • Lucia ISG cell lines Each of the following two cell lines expresses a secreted luciferase (Lucia) reporter gene under control of an IRF-inducible promoter.
  • This composite promoter comprises five IFN-stimulated response elements (ISREs) fused to a mthel promoter of the human ISG54 gene, which is unresponsive to activators of the NF- ⁇ or AP-1 pathways.
  • ISREs IFN-stimulated response elements
  • these cells enable monitoring of the IRF pathway based on luciferase (Lucia) activity.
  • monitoring of the IRF pathway is used to measure the STING agonist activity of the subject cyclic dinucleotides.
  • RAW-LuciaTM ISG InvivoGen catalog code: rawl-isg
  • RAW-LuciaTM ISG-KO-STING InvivoGen catalog code: rawl-kostg: These cells were generated from the RAW-LuciaTM ISG54 cell line (see above), through stable homozygous knockout of the STING gene.
  • B16 BlueTM ISG54 cell lines Each of the following two cell lines expresses a SEAP reporter gene under a promoter: the I-ISG54 reporter, which comprises the IFN-inducible ISG54 promoter enhanced by a multimeric ISRE. Stimulation of these cells with interferons, or inducers of type I interferons or of the NF- ⁇ pathway, triggers activation of the I- ISG54 promoter (and consequently, production of SEAP) or of the IFN- ⁇ minimal promoter (and consequently, production of TNF-a).
  • the I-ISG54 reporter which comprises the IFN-inducible ISG54 promoter enhanced by a multimeric ISRE. Stimulation of these cells with interferons, or inducers of type I interferons or of the NF- ⁇ pathway, triggers activation of the I- ISG54 promoter (and consequently, production of SEAP) or of the IFN- ⁇ minimal promoter (and consequently, production of TNF-a).
  • the levels of SEAP in the supernatant can be easily determined using QUANTI- BlueTM (InvivoGen catalog code: rep-qb1 ), a reagent that turns purple/blue in the presence of SEAP, by measuring the optical density from 620 nm to 655 nm. 1 .
  • B16-BlueTM ISG InvivoGen catalog code: bb-ifnabg: These cells are derived from the murine B16 F1 melanoma cell line. Production of Type I interferons in these cells is measured using QUANTI-BlueTM.
  • B16-BlueTM ISG-KO-STING InvivoGen catalog code: bb-kostg: These cells were generated from the B16- BlueTM ISG cell line (see above), through stable homozygous knockout of the STING gene. Production of Type I interferons in these cells is measured using QUANTI-BlueTM.
  • HEK-BlueTM cell lines The following three cell lines also were used for biological evaluation of the CDNs.
  • HEK-Blue IFN-a/p-KO-STING These cells are derived from HEK-Blue IFN- ⁇ / ⁇ cells (InvivoGen catalog code: hkb-ifnab), in which the STING gene has been inactivated.
  • HEK-BlueTM IFN- ⁇ / ⁇ cells enable detection of bioactive human type I IFNs through monitoring of activation of the ISGF3 pathway. These cells were generated by stable transfection of HEK293 cells with the human STAT2 and IRF9 genes to obtain a fully active type I IFN signaling pathway. The other genes of the pathway (IFNAR1 , IFNAR2, JAK1 , TyK2 and STAT1 ) are naturally expressed in sufficient amounts.
  • the cells were further transfected with a SEAP reporter gene under control of an IFN-a/p-inducible ISG54 promoter. Stimulation of HEK-BlueTM IFN- ⁇ / ⁇ cells with human IFN-a or IFN- ⁇ activates the JAK/STAT/ISGF3 pathway and subsequently induces production of SEAP. Production of Type I interferons in these cells is measured using QUANTI-BlueTM.
  • HEK-BlueTM IL-1 R (InvivoGen catalog code: hkb-il1 r): HEK-BlueTM IL-1 R cells were designed to detect bioactive human and murine IL- ⁇ through monitoring of activation of the NF- ⁇ and AP-1 pathways.
  • HEK-BlueTM IL-1 R cells can detect IL-1a and IL-1 ⁇ , as these cytokines bind to the same receptor, IL-1 R. These cells derive from HEK-BlueTM IL-1 ⁇ cells (InvivoGen catalog code: hkb-il1 b), in which the TNF-a response is blocked. Therefore, HEK-BlueTM IL-1 R cells respond specifically to IL-1 . These cells endogenously express the human IL-1 receptor and were stably transfected with the murine IL-1 receptor, rendering them very sensitive to both human and murine IL-1 ⁇ .
  • HEK-BlueTM IL-1 R cells express a SEAP reporter gene under control of an IFN- ⁇ minimal promoter fused to five NF- ⁇ and five AP-1 binding sites. Binding of IL-1 ⁇ to IL- 1 R on the surface of HEK-BlueTM IL-1 R cells triggers a signaling cascade that leads to the activation of NF- KB and subsequent production of SEAP. Production of IL-1 ⁇ in these cells is measured using QUANTI- BlueTM.
  • HEK-Blue TNF-a (InvivoGen catalog code: hkb-tnfdmyd): HEK-BlueTM TNF-a cells enable detection of bioactive human and murine TNF-a through monitoring of activation of the NF- ⁇ pathway. These cells were generated by stable transfection of HEK293 cells with a SEAP reporter gene under control of an IFN- ⁇ minimal promoter fused to five NF- ⁇ and five AP-1 binding sites. They were further rendered unresponsive to IL-1 ⁇ by knocking out the MyD88 gene. Stimulation of HEK-BlueTM TNF-a cells with TNF-a triggers activation of the NF-KB-inducible promoter and production of SEAP. Production of TNF-a in these cells is measured using QUANTI-BlueTM. Quantification of IL-6 in experiments
  • lnterleukin-6 was quantified using an enzyme-linked immunoassay (ELISA) according to the manufacturer's instructions (R&D Systems).
  • ELISA enzyme-linked immunoassay
  • Example 2.1 Measuring cytokine induction in treated cell cultures
  • the cyclic dinucleotides disclosed in the present invention do not induce cytokine production in vitro in the supernatant of cells that lack the receptor STING.
  • Example 2.2 Measuring cytokine induction in CDN-treated wild-type or STING knockout cells Cytokine reporter cell lines used: B16-Blue ISG and RAW-Lucia ISG
  • the cyclic dinucleotides disclosed in the present invention induce cytokines in vivo in mice.
  • Example 2.3 Measuring cytokine induction in CDN-treated mice
  • Cytokines evaluated IFN- ⁇ / ⁇ (using RAW ISG54 reporter cells) and IL-6 (by ELISA).
  • mice Twenty-one mice (Swiss; female; mean age: 8 weeks) were divided into seven groups of three: one group served as control (saline) and the other six groups were each treated with a cyclic dinucleotide (either c-AIMP, CL604, CL606, CL609, CL61 1 or CL614).
  • a cyclic dinucleotide either c-AIMP, CL604, CL606, CL609, CL61 1 or CL614
  • blood samples for basal cytokine levels were collected from all mice and stored at -20 °C until analysis.
  • the mice On Day 1 , the mice were treated with either 200 ⁇ of physiologic serum (containing 0.9% NaCI) or 200 ⁇ of a solution of a cyclic dinucleotide (dose: 10 mg/kg) in physiologic serum (containing 0.9% NaCI), by intravenous (i.v.) injection.
  • Blood samples were collected from the mice at 4 h post-injection, and then stored at
  • mice We have measured the in vivo elim ination of representative cyclic dinucleotides of the present invention in mice.
  • Cyclic dinucleotides tested CL603, CL609, CL614, CL626 and CL656 Reference compounds: c-AIMP (manufactured by InvivoGen).
  • mice Thirty m ice (C57BL/6) were divided into six groups of five. Within each group, each mouse was treated with a different cyclic dinucleotide (50 mg/kg; i.v. bolus). Each group of mice was sacrificed at a different time point post-treatment: either 2 min, 5 min, 15 min, 30 min or 1 h. Just before sacrifice, a sample of blood (500 ⁇ ) was collected. The blood samples were collected in heparin tubes, and then centrifuged. The supernatant (plasma) was stored at - 20 °C until analysis. Before analysis by high performance liquid chromatography-mass spectrometry (HLPC/MS), the plasma samples were processed as follows:
  • Plasma each sample was treated with methanol at a ratio of 1 :4, shaken and filtered (0.22 ⁇ ).
  • One ⁇ _ of the processed sample was injected onto the HPLC/MS column.
  • the following HPLC gradient was used (A: 10 mM ammonium formate; B: acetanitrile; total time: 6 min): 100% A for 1 min; followed by 100% A to 100% B in 4 min; followed by 100% B for 1 min.
  • Each cyclic dinucleotide eluted at a different time and was detected by measuring the absorbance at 254 nm.
  • Type I IFN induction HEK-BlueTM IFN-a/p-KO-STING
  • IL-1 induction HEK- Blue IL-1 R
  • TNF-a induction HEK-Blue TNF-a
  • Each blood sample was diluted (1 :1 ) in RPMI medium and aliguoted into 96-well plates (180- ⁇ . wells) containing each CDN at six different concentrations (30 ⁇ g mL, 10 ⁇ g mL, 3 ⁇ g mL, 1 ⁇ g mL, 0.3 ⁇ g mL and 0.1 ⁇ g mL).
  • the plates were incubated at 37 °C in a C0 2 incubator for 18 to 20 hours. The next day the supernatants were collected, transferred into the corresponding wells of round-bottom 96-well plates, and stored at -80 °C.
  • a new 96-well plate was prepared for each of the three reporter cell lines tested, as follows: 10 ⁇ of supernatant from the previous plate (containing the incubated CDNs and plasma) were added to the corresponding well in the new reporter cell plate. Then, a 180- ⁇ . aliquot of cells of the desired reporter cell line, previously harvested in medium containing heat-inactivated serum and counted, was added to each well (approximately 50,000 cells/well), and the plate was incubated for approximately 20 hours. The desired cytokine induction activity was determined using the QUANTI-BlueTM Assay, as previously described. Briefly, 20 ⁇ L of supernatant from the previously incubated plate was transferred to the corresponding well of a new 96-well plate in which 180 ⁇ L of QUANTI-BlueTM reagent had previously been added.
  • Figure 9 illustrates three cytokine induction activities (Type I IFN, IL-1 and TNF-a) for representative fluorinated CDNs of the present invention and for related reference CDNs.
  • the values shown are total averages for all 20 donors over the full concentration range tested (30 ⁇ g mL, 10 ⁇ g mL, 3 ⁇ g mL, 1 ⁇ g mL, 0.3 ⁇ g mL, 0.1 ⁇ g mL and 0.03 ⁇ g mL).
  • the activity of the CDNs is expressed in terms of "active concentration” (defined here as the CDN giving a SEAP intensity value of at least 0.5 at the indicated concentration), from the lowest of the seven tested concentrations (0.03 ⁇ g mL) up to the highest tested concentration (30 ⁇ g mL).
  • active concentration defined here as the CDN giving a SEAP intensity value of at least 0.5 at the indicated concentration
  • the Figure reveals two principal findings: firstly, that with in the tested concentration range, each one of the CDNs tested induces each one of the cytokines assessed; and secondly, that for each cytokine induction activity, each fluorinated CDN is more active than is its corresponding non-fluorinated analog (compare CL603 with c-GAMP; CL614 with c-AIMP; and CL656 with c-AIMP[s]).
  • Example 2.6 Comparison of fluorinated deoxyribo-CDNs and non-fluorinated ribo-CDNs for their resistance to enzymatic cleavage by SVPD or NP1 , as monitored by UHPLC-MS
  • Enzymes used snake-venom phosphodiesterase (SVPD) and nuclease P1 (NP1 )
  • Each CDN (7 ⁇ g) was separately incubated with a solution (21 ⁇ ) of either enzyme (either 160 ⁇ g SVPD in PBS buffer containing 0.6 mM MgCI 2 ; or 2.5 mU NP1 in 30 mM acetate buffer containing 2 mM ZnCI 2 [pH 5.3]), or with water (as control) in a water bath at 37 °C. Aliquots of the reaction mixture were collected at various time points from 0 to 120 hours, heated at 100 °C for 2 min, and then frozen at 0 °C.
  • enzyme either 160 ⁇ g SVPD in PBS buffer containing 0.6 mM MgCI 2 ; or 2.5 mU NP1 in 30 mM acetate buffer containing 2 mM ZnCI 2 [pH 5.3]
  • water as control
  • the fluorinated deoxyribo-CDNs of the present invention surprisingly exhibit unique, unobvious and previously unreported class effects that could be exploited for therapeutic, diagnostic and research applications that involve manipulation of STING activity.
  • the fluorinated CDNs are more active, as we have ascertained in vitro by measuring cytokine induction in different cell lines and in whole blood. Moreover, they are eliminated more slowly in vivo, as we have determined in mice. Lastly, they exh ibit superior resistance to enzymatic cleavage.
  • the innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING.
  • Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Mol Cell, 51(2), 226-235.

Abstract

The invention concerns a cyclic dinucleotide compound of Formula (I) wherein X1 is H or F; X2 is H or F; at least one among X1 and X2 is a fluorine atom; Z is OH, OR1, SH or SR1, wherein: R1 is Na or NH4, or R1 is an enzyme-labile group which provides OH or SH in vivo such as pivaloyloxymethyl; B1 and B2 are bases chosen from Adenine, Hypoxanthine or Guanine, and B1 is a different base than B2 and a pharmaceutically acceptable salt thereof. The invention also concerns pharmaceutical compositions comprising said cyclic dinucleotide, as well as their use in the treatment of a bacterial infection, a viral infection or a cancer.

Description

FLUORINATED CYCLIC DINUCLEOTIDES FOR CYTOKINE INDUCTION FIELD OF THE INVENTION
The present invention is in the field of immunotherapy. It concerns cyclic dinucleotides (CDNs) of Formulas I, II and III. In particular, it concerns fluorinated deoxyribo-CDNs and pharmaceutically acceptable salts and prodrugs thereof, which are able to induce production of Type I interferons in human and animal cells. The cytokine-induction activity of these cyclic dinucleotides requires the presence of the eukaryotic cellular receptor stimulator of interferon genes (STING), as demonstrated in vitro.
BACKGROUND OF THE INVENTION
Cytokine induction immunotherapy
Immunotherapy is a rapidly expanding area of medical treatment in which a patient's immune system is deliberately activated, suppressed or otherwise modulated for therapeutic benefit. Immunotherapy agents include cells, antigens (e.g. fragments of bacteria or viruses), antibodies, nucleic acids, peptides, proteins, naturally occurring ligands and synthetic molecules. Cytokines are small glycoprotein messengers known chiefly for their role in orchestrating immune response through complex signaling networks, although they also perform non-immune functions. They have been extensively explored as immunotherapy agents. However, direct administration of cytokines as immunotherapy is limited by numerous factors, including the very short half-life of cytokines in blood, which must be compensated for with frequent dosing and high doses. One highly promising immunotherapy approach is cytokine induction, whereby the patient is treated with an immunomodulatory agent that triggers the production of one or more therapeutically beneficial cytokines in their body as needed.
STING, cytokines and immune response
A major player in physiological production of cytokines is stimulator of interferon genes (STING; also known as E /S, MITA, MPYS, or TM173), a transmembrane receptor protein that is paramount in innate immunity. Human STING is encoded by the gene TMEM173. Activation of STING leads to production of Type I interferons {e.g. IFN-a and IFN-β), via the IRF3 (interferon regulatory factor 3) pathway; and to production of pro-inflammatory cytokines (IL-1a , IL-1 β , IL-2, IL-6, TNF-a, etc.), via the oncogenic transcription factor NF-κΒ (nuclear factor kappa-light-chain-enhancer of activated B cells) pathway. Moreover, researchers recently reported that in response to viral infection, STING activates STAT6 (signal transducer and activator of transcription 6) to induce (Th2-type), increase (IL-12) or decrease (IL-10) production of various cytokines, including the chemokines CCL2, CCL20, and CCL26 (Chen et al., 2011 )
STING agonists
Human STING is currently known to be activated three ways: via binding of exogenous (3 , 3) cyclic dinucleotides (c- diGMP, c-diAMP and c-GA P) that are released by invading bacteria or archaea (see (Gomelsky, 2011) and references therein); via binding of (2',3')cyclic guanosine monophosphate-adenosine monophosphate ((2',3')c- GAMP), a recently discovered endogenous cyclic dinucleotide that is produced by the enzyme cyclic GMP-AMP synthase (cGAS; also known as C6orf150 or MB21D1) in the presence of exogenous double-stranded DNA (e.g. that released by invading bacteria, viruses or protozoa) or of self-DNA in mammals (see, for example: (Ablasser et al., 2013) and (Zhang et al., 2013)); or via binding of synthetic ligands, such as analogs of the aforementioned naturally- occurring cyclic dinucleotides (see, for example: (Dubensky, Kanne, & Leong, 2013) and (Li et al., 2014)). Modulation of STING in immunotherapy
Inspired by the interplay among STING, cytokines and immune response, as well as by the ever-growing body of knowledge on the clinical implications of STING and its mutations, researchers have very recently begun to explore STING as a therapeutic target for myriad indications. New STING agonists are being pursued as therapeutic agents for human and animal health in areas such as cancer or infectious diseases. The known cyclic dinucleotide STING agonists are an excellent class of compounds on which to base analogs that might exhibit interesting biological activities or desirable drug-like properties. The present invention comprises novel cyclic dinucleotides for therapeutic use in human and animal health. DETAILED DESCRIPTION OF THE INVENTION
CDN STING agonists
Some examples of cyclic dinucleotide (CDN) STING agonists are described in US/2014/0329889 and WO/2014/189805. However, the authors only chemically synthesized and biologically tested a very small number of the compounds that would be theoretically possible from the extremely general chemical structure drawings that they provide. They do not disclose any detailed structure-activity relationships or describe any class effects of particular structural classes of CDNs. Consequently, the authors provide scant rationale to corroborate the choice of one structural class of CDNs over another in terms of actual STING activity or other properties (e.g. drug-like properties) for a desired STING-related application. More recently, WO/2015/077354 (PCT/US2014/066436) explored STING agonists, including CDNs, all of which are (2',3')-CDNs in which both nucleotides contain a ribose sugar moiety and the two nucleotides are connected by phosphorothioate diester linkages.
Knowledge gaps on CDN STING agonists
Very little is known about the biological activity of different structural classes of CDNs. Specifically, there is scant patent precedent on class effects according to CDN structural class, nor is there any patent precedent on how such class effects might be specifically exploited for specific therapeutic, diagnostic or research applications related to STING activity. Such information will be critical for discovery and exploitation of novel CDN STING agonists with desirable properties for therapeutic, diagnostic or research applications based on manipulation of STING activity.
The present invention
Considering the paucity of reported structure-activity relationships for STING agonists, the field of STING agonists is ripe for invention. In this context, the present invention concerns fluorinated deoxyribo-CDN STING agonists that exhibit unigue, unobvious and previously unreported class effects relative to their corresponding non-fluorinated analogs. In our work exploring cyclic dinucleotides (CDNs) as immunomodulatory compounds and potential agonists of STING, we initially sought to synthesize and assay CDNs of the following Formulas (I), (II) and (III):
Figure imgf000005_0001
Formula (I) Formula (II) Formula (III) wherein:
Xi is H, OH, or F;
X2 is H, OH, or F;
Z is OH, ORi, SH or SRi, wherein:
i) Ri is Na or NH4, or
ii) Ri is an enzyme-labile group which provides OH or SH in vivo such as pivaloyloxymethyl; Bi and B2 are bases chosen from:
Figure imgf000005_0002
Adenine, Hypoxanthine, or
Guanine, with the proviso that:
- in Formula (I): Xi and X2 are not OH,
- in Formula (II): when Xi and X2 are OH, Bi is not Adenine and B2 is not Guanine, and
- in Formula (III): when Xi and X2 are OH, Bi is not Adenine, B2 is not Guanine and Z is not OH.
In the present invention, the term "cyclic dinucleotide" (abbreviated as "CDN") represent a class of cyclic molecules with two phosphodiester linkages, or two phosphorothioate diester linkages, or one phosphodiester linkage and one phosphorothioate diester linkage, between two nucleosides. This includes (3',5')-(3',5') nucleotide linkages (abbreviated as (3',3')); (3',5')-(2',5') nucleotide linkages (abbreviated as (3',2')); (2',5')-(3',5') nucleotide linkages (abbreviated as (2',3')); and (2',5')-(2',5') nucleotide linkages (abbreviated as (2',2')).
The term "nucleoside" refers to a glycosylamine comprising a nitrogenous base and a five-carbon sugar, wherein the nitrogenous base is bound to the five-carbon sugar via a beta-glycosidic linkage.
The term "nucleotide" refers to any nucleoside linked to a phosphate group at position 5', 3' or 2' of the sugar moiety. "Pharmaceutically acceptable salts" include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among numerous other acids well known in the pharmaceutical art. The term "pharmaceutically acceptable prodrug" herein refers to a compound that is metabolized, for example hydrolyzed or oxidized, in the host (i.e. the human or animal subject that receives the compound) to form the compound of the present invention. Typical examples of prodrugs include compounds that have biologically labile protecting groups on functional moieties of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated or dephosphorylated to produce the active compound.
The term "prodrug", as used herein, relates to an inactive or active derivative of a compound represented by the structural Formula (I), (II) or (III) as defined herein above or any one of their specific embodiments, which undergoes spontaneous or enzymatic transformation within the body of an animal, e.g. a mammal such as a human being, in order to release the pharmacologically active form of the compound. For a comprehensive review, see (Rautio et al., 2008).
In particular for the purpose of the present invention, prodrugs of the compounds represented by the structural Formula (I), (II) or (III) including any one of the above-described specific embodiments thereof, may be formed as described in detail in (Hecker & Erion, 2008).
Phosphate prodrug can take the form of an ester, in particular acyloxyalkyl esters (e.g. pivaloyloxymethyl ester (POM)) or S-acylthioethyl (SATE) esters, a carbonate, a carbamate or an amide, such as amino acid prodrugs.
The expression "enzyme-labile protecting group" denotes a group designed to enable passive d iffusion of a compound across cellular or parasitic membranes via charge-masking, such that, once inside the cell or parasite interior, the compound undergoes an enzymatic transformation (or deprotection) that affords an OH or SH group. Examples of enzyme-labile protecting groups include for instance acyloxyalkyl groups such as pivaloyloxymethyl (POM) or S-acylthioethyl (SATE) or amino acid groups.
In the present description, it is considered that the expression "cyclic dinucleotides of Formula (I), (II) or (III)" is considered to also include the pharmaceutically acceptable salts or a pharmaceutically acceptable prodrugs of said cyclic dinucleotides of Formula (I), (II) or (III). A particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Bi or B2 is Guanine.
A particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Bi or B2 is Adenine.
A particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Bi or B2 are Hypoxanthine.
A particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Bi and B2 are independently chosen from Guanine or Adenine.
A particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Bi or B2 are independently chosen from Guanine or Hypoxanthine.
A particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Bi and B2 are independently chosen from Adenine or Hypoxanthine.
A particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Z is OH. A particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Z is SH.
A particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Z is OH or SH.
A particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Z is SRi, wherein:
i) Ri is Na or NH4, or
ii) Ri is an enzyme-labile group which provides OH or SH in vivo such as pivaloyloxymethyl.
A particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Bi and B2 are independently chosen from Adenine or Hypoxanthine and Xi and X2 are identical. Preferably, Xi and X2 are OH or a fluorine atom. In one particular embodiment, Z is OH or SH.
A particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Bi and B2 are independently chosen from Guanine or Hypoxanthine and Xi and X2 are identical. Preferably, Xi and X2 are OH or a fluorine atom. In one particular embodiment, Z is OH or SH.
A particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Bi and B2 are independently chosen from Adenine or Guanine and Xi and X2 are identical. Preferably, Xi and X2 are OH or a fluorine atom. In one particular embodiment, Z is OH or SH.
A particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when Xi and X2 are not a fluorine atom.
A particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when at least one among Xi and X2 is a fluorine atom. In one particular embodiment, Xi and X2 are both a fluorine atom.
A particular class of cyclic dinucleotides of Formula (I), (II) or (III) is the following cyclic dinucleotides (each of which is provided with a five-character code of the format "CL###"):
Figure imgf000007_0001
CL606 CL611
(3',2')c-AIMP (2',2')c-AIMP
Figure imgf000008_0001
CL602 CL655
(2',3')c-AIMP c-AIMP(S) or a pharmaceutically acceptable salt or prodrug thereof.
A particular class of cyclic dinucleotides of Formula (I), (II) or (III) is when one or both nucleosides are modified at the 2' position. This class comprises the following compounds:
Figure imgf000008_0002
CL604 CL609 c-(dAMP-dlMP) c-(dAMP-2'FdlMP)
Figure imgf000008_0003
CL614 CL656 c-(2'FdAMP-2'FdlMP) c-[2'FdAMP(S)-2'FdlMP(S)]
Figure imgf000009_0001
CL647 CL626
- AMP-2'FdlMP) 'FdlMP)
Figure imgf000009_0002
CL629 CL603
c-di(2'FdGMP) c-(2'FdGMP-2'FdAMP)
Figure imgf000009_0003
CL632 CL633
c-[2'FdGM P(S)-2'Fd AM P(S)] c-[2'FdGMP(S)-2'FdAMP(S)](POM)2
Figure imgf000009_0004
CL659
c-[2'FdAMP(S)-2'FdlMP(S)](POM)2 We identified a structurally unprecedented subset of these CDNs that showed surprising biological activity and that have never previously been reported as STING agonists. These CDNs form the basis for the present invention and are distinct from previously reported CDN STING agonists (see, for example: US/2014/0329889 and WO/2014/189805). Thus, in one aspect, the present invention provides CDNs that are defined by all of the following structural criteria: firstly, unlike in naturally occurring CDNs, in which the sugar moiety of each nucleotide is a ribose, in the CDNs of the present invention the sugar moiety of each nucleotide is a 2'-deoxyribose; secondly, in the CDNs of the present invention, either the 2' position of the sugar moiety in both nucleotides must be substituted with a fluorine atom, or the 2' position of the sugar moiety of one nucleotide must be substituted with a fluorine atom while the 2' position of the sugar moiety of the other nucleotide must be substituted with a hydrogen atom; thirdly, in the CDNs of the present invention, the base in each nucleotide is preferably chosen from guanine, adenine or hypoxanthine, with the proviso that the two nucleotides of the CDN cannot contain the same base.
Thus, in one embodiment, the present invention provides CDNs of Formula (I) according to Claim 1 , i.e. compounds of Formula (I):
Figure imgf000010_0001
Formula (I)
wherein:
• Xi is H or F;
• X2 is H or F;
• At least one among Xi and X2 is a fluorine atom;
• Z is OH, ORi, SH or SRi, wherein:
Ri is Na or NH4, or
II Ri is an enzyme-labile group which provides OH or SH in vivo such as pivaloyloxymethyl;
• Bi and B2 are bases chosen from:
Figure imgf000010_0002
Adenine, Hypoxanthine, or
Figure imgf000011_0001
Guanine,
and Bi is a different base than B2, or a pharmaceutically acceptable salt thereof.
The expression "Bi is a different base than B2" includes Bi is Guanine and B2 is Adenine or Bi is Guanine and B2 is Hypoxanthine or Bi is Adenine and B2 is Guanine or Bi is Adenine and B2 is Hypoxanthine or Bi is Hypoxanthine and B2 is Guanine or Bi is Hypoxanthine and B2 is Adenine.
A particular class of cyclic dinucleotides of Formula (I) as defined above is when Bi and B2 are different and both Xi and X2 are F. A particular class of cyclic dinucleotides of Formula (I) as defined above is when Bi and B2 are different, both Xi and X2 are F and Z is OH.
A particular class of cyclic dinucleotides of Formula (I) as defined above is when Bi and B2 are different, both Xi and X2 are F and Z is ORi as defined above.
A particular class of cyclic dinucleotides of Formula (I) as defined above is when Bi and B2 are different, both Xi and X2 are F and Z is SH as defined above.
A particular class of cyclic dinucleotides of Formula (I) as defined above is when Bi and B2 are different, both Xi and X2 are F and Z is SRi as defined above.
A particular class of cyclic dinucleotides of Formula (I) as defined above is when Bi and B2 are different and Xi and X2 are different (i.e. Xi = H and X2 = F or Xi = F and Xi = H). A particular class of cyclic dinucleotides of Formula (I) as defined above is when Bi and B2 are different, Xi and X2 are different and Z is OH.
A particular class of cyclic dinucleotides of Formula (I) as defined above is when Bi and B2 are different, Xi and X2 are different and Z is ORi as defined above.
A particular class of cyclic dinucleotides of Formula (I) as defined above is when Bi and B2 are different, Xi and X2 are different and Z is SH as defined above.
A particular class of cyclic dinucleotides of Formula (I) as defined above is when Bi and B2 are different, Xi and X2 are different and Z is SRi as defined above. The cyclic dinucleotides of the present invention induce Type I interferons and/or pro-inflammatory cytokines in vitro in human cells, an imal cells and human blood. The cytokine-induction activity of these cyclic dinucleotides requires the presence of STING, as confirmed by in vitro experiments in human or animal cells.
The cyclic dinucleotides of the invention are agonists of the receptor STING. The term "agonist" refers to any substance that activates a biologic receptor in vitro or in vivo to provoke a physiologic response.
"STING" is an abbreviation of "stimulator of interferon genes", which is also known as "endoplasmic reticulum interferon stimulator (ERIS)", "mediator of IRF3 activation (MITA)", "MPYS" or "transmembrane protein 173 (TM173)". STING is a transmembrane receptor protein that in humans is encoded by the gene TMEM173. Activation of STING by cyclic dinucleotides (CDN) leads to activation of the IRF3 and NF-κΒ pathways and consequently, to induction of Type I interferons and of pro-inflammatory cytokines, respectively. In response to viral infection, STING activates STAT6 (signal transducer and activator of transcription 6) to induce (Th2-type), increase (IL-12) or decrease (IL-10) production of various cytokines, including the chemokines CCL2, CCL20, and CCL26 (Chen et al., 201 1 ). The term "STING agonist" herein refers to a substance that activates the receptor STING in vitro or in vivo. According to the invention, a compound is deemed to be a STING agonist if:
- it induces Type I interferons in vitro in human or animal cells that contain active STING; and
- it does not induce Type I interferons in vitro in human or animal cells that do not contain active STING. A typical test to ascertain whether a ligand is a STING agonist is to incubate the ligand in a wild-type human or animal cell line and in the corresponding cell line in which the STING coding gene has been genetically inactivated by small or long base deletions (e.g. a homozygous STING knockout cell line). An agonist of STING will induce Type I interferons in the wild-type cells but will not induce Type I interferons in the cells in which the STING coding gene has been inactivated. The cyclic dinucleotides of the invention induce Type I interferons in vitro in human or animal cells that contain active STING. However, they do not induce Type I interferons in vitro in human or animal cells that do not contain active STING.
The present invention is concerned with fluorinated deoxyribo-cyclic dinucleotides (CDNs). Specifically, it is concerned with (3',3')-2'(mono- or di-fluorinated)-2'-deoxyribo-(CDNs). The cyclic dinucleotides of Formula (I), (II) or (III) wherein at least one among Xi and X2 is a fluorine atom, in particular Xi and X2 are both a fluorine atom, and Z is OH induce more Type I interferon and NF-κΒ activity than do their non-fluorinated counterparts in human and murine cell lines.
The cyclic dinucleotides of Formula (I), (II) and (III) wherein at least one among Xi and X2 is a fluorine, in particular Xi and X2 are both a fluorine atom, and Z is OH or SH exhibit slower elimination from blood after intravenous injection in mice, i.e. longer kinetics of clearance, and exhibit greater resistance to enzymatic cleavage in vitro; as compared to their corresponding non-fluorinated CDNs. Salts or Prodrug Formulations of the Cyclic Dinucleotides
Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among numerous other acids well known in the pharmaceutical art. The term "pharmaceutically acceptable prodrugs" refers to a compound that is metabolized, for example hydrolyzed or oxidized, in the host (i.e. the human or animal subject that receives the compound) to form the compound of the present invention. Typical examples of prodrugs include compounds that have biologically labile protecting groups on functional moieties of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated or dephosphorylated to produce the active compound.
The CDN prodrugs described herein can be administered to additionally increase the activity, bioavailability or stability, or otherwise alter the properties of the CDN monophosphate.
A number of CDN prodrug ligands are known. In general, alkylation, acylation or other lipophilic modifications on the phosphate moiety, or the use of other analog of the nucleoside, will increase the stability of the nucleotide. Examples of substituent groups that can replace one or more hydrogens on the phosphate moiety are alkyl, aryl, steroids, carbohydrates, including, but not limited to, sugars, 1 ,2-diacylglycerol and alcohols. Many are described in (Jones, 1995).
Use of the compounds of the invention
Another object of the present invention is the cyclic dinucleotides of Formula (I), (II) or (III) for use in a therapeutic treatment in humans or animals.
In particular, the compounds of the present invention may be used for therapeutic or diagnostic applications in human or animal health. The following examples serve to illustrate different possible applications of the present invention; however, these examples are not intended to limit the scope of the invention.
The term "therapeutic agent" refers to one or more substances that are administered to a human or animal in order to achieve some kind of therapeutic effect in that human or animal, including to prevent, cure, or mitigate the effects of, infection or disease, and/or to otherwise improve the health of that human or animal.
The term "monotherapy" refers to the use of a single substance and/or strategy to treat a human or animal in any clin ical or medical context, as opposed to the use of multiple substances and/or strategies to treat a human or animal in the same clinical or medical context, regardless of whether the multiple substances and/or strategies are used sequentially in any order or concurrently.
The term "chemotherapeutic agent" herein refers to one or more chemical substances that are administered to a human or animal in order to kill tumors, or slow or stop the growth of tumors, and/or slow or stop the division of cancerous cells and/or prevent or slow metastasis. Chemotherapeutic agents are often administered to treat cancer, but are also indicated for other diseases. The term "chemotherapy" refers to medical treatment of a human or animal with one or more chemotherapeutic agents (see definition above).
The term "chemoimmunotherapy" refers to the combined use, whether sequentially in any order or concurrently, of chemotherapy substances and/or strategies, and immunotherapy substances and/or strategies. Chemoimmunotherapy is often employed to treat cancer, but can also be employed to treat other diseases.
The term "immune system" refers to the ensemble, or to any one or more components, of the molecules, substances (e.g. bodily fluids), anatomic structures (e.g. cells, tissue and organs) and physiologic processes involved in preventing infection in the body, in protecting the body during infection or during disease, and/or in helping the body to recuperate after infection or disease. A complete definition of "immune system" is beyond the scope of this patent; however, this term should be understood by any ordinary practitioner in the field.
The term "immune agent" refers to any endogenous or exogenous substance that can interact with any one or more components of the immune system. The term "immune agent" includes antibodies, antigens, vaccines and their constituent components, nucleic acids, synthetic drugs, natural or synthetic organic compounds, cytokines, natural or modified cells, synthetic analogs thereof, and/or fragments thereof. The term "immunotherapy" refers to any medical treatment in which one or more components of a human's or animal's immune system is deliberately modulated in order to directly or indirectly achieve some therapeutic benefit, including systemic and/or local effects, and preventative and/or curative effects. Immunotherapy can involve administering one or more immune agents (see definition above), either alone or in any combination, to a human or animal subject by any route (e.g. orally, intravenously, dermally, by injection, by inhalation, etc.), whether systemically, locally or both. "Immunotherapy" can involve provoking, increasing, decreasing, halting, preventing, blocking or otherwise modulating the production of cytokines, and/or activating or deactivating cytokines or immune cells, and/or modulating the levels of immune cells, and/or delivering one or more therapeutic or diagnostic substances to a particular location in the body or to a particular type of cell or tissue, and/or destroying particular cells or tissue. Immunotherapy can be used to achieve local effects, systemic effects or a combination of both. The term "immunosuppressed" describes the state of any human or animal subject whose immune system is functionally diminished, deactivated or otherwise compromised, or in whom one or more immune components is functionally diminished, deactivated or otherwise compromised. "Immunosuppression" can be the cause, consequence or byproduct of disease, infection, exhaustion, malnutrition, medical treatment or some other physiologic or clinical state. The terms "immunomodulating substance", "immunomodulatory substance", "immunomodulatory agent" and "immunomodulator", used here synonymously, refer to any substance that, upon administration to a human or animal, directly influences the functioning of the immune system of that human or animal. Examples of common immunomodulators include, but are not limited to, antigens, antibodies and small-molecule drugs.
The term "vaccine" refers to a biological preparation administered to a human or animal in order to elicit or enhance a specific immune system response and/or protection against one or more antigens in that human or animal. The term "vaccination" refers to treatment of a human or animal with a vaccine or to the act of administering a vaccine to a human or animal.
The term "adjuvant" refers to a secondary therapeutic substance that is administered together (either sequentially in any order, or concurrently) with a primary therapeutic substance to achieve some kind of complimentary, synergic or otherwise beneficial effect that could not be achieved through use of the primary therapeutic substance alone. An adjuvant can be used together with a vaccine, chemotherapy, or some other therapeutic substance. Adjuvants can enhance the efficacy of the primary therapeutic substance, reduce the toxicity or side effects of the primary therapeutic substance, or provide some kind of protection to the subject that receives the primary therapeutic substance, such as, but not limited to, improved functioning of the immune system. In one embodiment, the cyclic dinucleotide of Formula (I), (II) or (III) can be administered as immunotherapy to a human or an animal to induce in vivo production of one or more cytokines that are therapeutically beneficial to that human or animal. This type of immunotherapy could be used alone or in combination with other treatment strategies, whether sequentially in any order, or concurrently. It could be used to prevent, cure, and/or mitigate the effects of, infection or disease in that human or animal, and/or to modulate the immune system of that human or animal to achieve some other therapeutic benefit.
In one particular embodiment, the cyclic dinucleotides of the present invention can be used for cytokine induction immunotherapy of immunosuppressed individuals.
In this example, a cyclic dinucleotide of Formula (I) (II) or (III) would be administered to an immunosuppressed human or animal subject to induce in vivo production of one or more cytokines that directly or indirectly enhance the immune system of that human or animal. Subjects that might benefit from such treatment include those suffering from autoimmune disorders, immune system deficiencies or defects, microbial or viral infections, infectious diseases, or cancer.
The present invention thus discloses a method for inducing cytokine in immunosuppressed individuals, said method comprising administering to a patient in need thereof a cyclic dinucleotide of Formula (I), (II) or (III) or a pharmaceutically acceptable salt or prodrug thereof.
In another embodiment, the cyclic dinucleotides of the present invention can be used for cytokine induction immunotherapy in combination with chemotherapy
In this example, a cyclic dinucleotide of Formula (I) (II) or (III) would be administered together with one or more chemotherapeutic agents, sequentially in any order or concomitantly, to a cancer patient to stop the growth of, shrink and/or destroy tumors in that patient. The chemoimmunotherapy resulting from the combination of cytokine induction, provided by the compound(s) of the present invention, and cytotoxicity, provided by the chemotherapeutic agent(s), might be less toxic to the patient, cause fewer side effects in the patient and/or exhibit greater anti-tumor efficacy than would the chemotherapeutic agent(s) when used as monotherapy.
The present invention thus discloses a method for treating cancer, said method comprising administering to a patient in need thereof:
a chemotherapeutic agent; and a cyclic dinucleotide of Formula (I), (II) or (III) or a pharmaceutically acceptable salt or prodrug thereof.
In another embodiment, the cyclic dinucleotides of the present invention can be used for cytokine induction immunotherapy as vaccine adjuvant therapy.
In this example, a cyclic dinucleotide of Formula (I) (II) or (III) would be administered to a human or animal subject that has received, is receiving or will receive a vaccination. The benefits provided by the present invention might include enhanced efficacy of the vaccination against the target antigen, reduced toxicity of the vaccination, reduced adverse side effects of the vaccination, or enhanced immune protection of the human or animal subject.
Another object of the present invention is the cyclic dinucleotides of Formula (I), (II) or (III) for use in the treatment of a bacterial infection, a viral infection or a cancer.
As used herein, "cancer" refers to the physiological condition in subjects that is characterized by unregulated or dysregulated cell growth or death. The term "cancer" includes solid tumors and blood-born tumors, whether malignant or benign. In a preferred embodiment, the cancer is from the following group: bladder cancer, breast cancer, cholangiocellular cancer, leukemia, lung cancer, lymphoma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer and urothelial cancer.
In one particular embodiment, the cancer is a solid pancreatic tumor. The present invention thus discloses a method for treating a bacterial infection, a viral infection or a cancer, said method comprising administering to a patient in need thereof a cyclic dinucleotide of Formula (I), (III) or (III) or a pharmaceutically acceptable salt or prodrug thereof.
Another object of the present invention is the cyclic dinucleotides of Formula (I), (II) or (III) for use in the treatment of a pathology that may be alleviated by the induction of an immune response via the STING pathway. Another object of the present invention is a kit-of-parts comprising a cyclic dinucleotide of Formula (I), (II) or (III) and a chemotherapeutic agent for use in the treatment of solid pancreatic tumors.
The term "kit-of-parts" herein refers to a combined preparation wherein the active ingredients are physically separated for use in a combined therapy by simultaneous administration or sequential administration to the patient.
Hence, according to the present invention, the chemotherapeutic agent and the cyclic dinucleotide or a pharmaceutically acceptable salt or prodrug thereof are administered to the patient in a separate form, either simultaneously, separately or sequentially in any order, for the treatment of cancer.
In one embodiment, said chemotherapeutic agent is gemcitabine.
Deoxynucleosides for the synthesis of cyclic dinucleotides Natural nucleosides, 2'-deoxy-Adenosine (dA), 2'-deoxy-Guanosine (dG), or 2'-deoxy-lnosine (dl) can be used for the synthesis of CDNs of the Formulas (I) and (II).
The 3'-deoxy-Adenine, 3'-deoxy-Guanosine, or 3'-deoxy-lnosine for CDNs of the Formula (II) and (III) refers to a nucleoside unit having a sugar moiety, for example a ribosyl moiety or a xylosyl moiety that is modified at the 3' position such that the hydroxyl group (3'-OH) is replaced by an hydrogen group.
Fluoro-substituted nucleosides for the synthesis of CDNs
Since STING is located in the endoplasmic reticulum and detects cyclic d inucleotides in the cytoplasm, any STING agonist destined for therapeutic use must be able to penetrate into cells. Furthermore, greater cellular uptake of a compound translates to higher bioavailability, which is a desirable property for clinical use. In the present invention, the fluorinated compounds were designed to explore the possibility that greater cellular uptake conferred by one or two fluorine atoms would lead to greater Type I interferon induction activity than that of the reference compound, c- AIMP, which does not contain any fluorine atoms.
In the present invention, we surprisingly found that the subject CDNs, all of which contain at least one fluorine atom, are more active as STING agonists than are their corresponding non-fluorinated analogs. Specifically, the CDNs of the present invention exhibit greater STING-dependent cytokine induction activity in human cells, animal cells and human blood than do their corresponding non-fluo nated analogs
The more quickly a drug is enzymatically degraded in the body, the shorter will be its half-life and consequently, the lesser will be its activity. Thus, a desirable property for compounds intended for therapeutic use is resistance to enzymatic degradation. There are in vitro enzymatic cleavage tests that can provide some indication of the resistance of a given compound to common enzymes that degrade compounds that are structurally similar to the one being tested. In our work, we were very surprised to discover yet another distinguishing characteristic of the subject CDNs of the present invention: these fluorinated deoxyribo-CDNs consistently showed superior resistance to cleavage by snake-venom phosphodiesterase (SVPD) or nuclease P1 (NP1 ), compared to their corresponding non-fluo nated ribo-CDNs.
Monofluoro-nucleoside
The 2'-deoxy-2'-fluoro of Adenosine, Guanosine or Inosine for CDNs of Formula (I) and (II) refers to a nucleoside with a modification at the 2' position, such that, the hydroxyl group (2'-OH) is replaced by a fluoro group (2'-F).
The 2'-fluoronucleoside derivatives for the synthesis of CDNs of Formula (I) and (II) may be prepared by any of the methods known in the art (see, for example: (Herdewijna, 1989; Thomas, 1994)and(Ross, 1997)).
The 3'-deoxy-3'-fluoro of Adenosine, Guanosine or Inosine for CDN of Formula (II) and (III) refers to a nucleoside with a modification at the 3' position, such that the hydroxyl group (3'-OH) is replaced by a fluoro group (3'-F).
Phosphorothioate internucleotide linkage The phosphorothioate internucleotide linkage refers to the replacement of a P=0 group with a P=S group, and includes phosphorodithioate internucleoside linkages. One or both of the internucleotide linkages that are present in the cyclic dinucleotides can be phosphorothioate internucleotide linkages.
The phosphorous atom in a phosphodiester linkage of CDNs can be described as being "pro-chiral." Once a non- bonding oxygen atom of the phosphodiester linkage is replaced or modified, a chiral sugar-phosphate linkage is generated. The resulting intersugar linkage is either an Sp intersugar linkage or an Rp intersugar linkage. Replacement of a non-bonding oxygen atom in the natural phosphodiester linkage, with a sulfur atom to obtain a phosphorothioate linkage generates a chiral center and consequently, affords Sp and Rp diastereomers. Molecules wherein substantially all of the phosphorous atoms in the sugar backbone are either Sp or Rp are referred to herein as "chirally pure".
Cyclic dinucleotides are enzymatically degraded by nucleases and/or phosphodiesterases (see, for example: (Li et al., 2014) (Diner et al., 2013) (Danilchanka & Mekalanos, 2013) (Shanahan, Gaffney, Jones, & Strobel, 2013) (Simm, Morr, Kader, Nimtz, & Romling, 2004)) and therefore, when used as therapeutic agents, these compounds can suffer from diminished half-life. The compounds CL655 and CL656 were chosen to enable maximal half-life, and possibly higher activity, in vivo, as they contain phosphorothioate (also known as "P(S)" or "thiophosphate") internucleotide linkages. The use of such linkages is a known strategy to circumvent enzymatic hydrolysis (see, for example: US 2014/0205653 A1 ). An example of a phosphorothioate compound that is more resistant to enzymatic hydrolysis than its phosphodiester analog is (2',3')c-GsAsMP or (2',3') c-GAMP(S) (InvivoGen catalog code: tlrl-scga; Li, 2014). The phosphorothioate linkage introduces an additional chiral center on the phosphorus atom, which yields a diastereoisomer pair ([Rp] and [Sp]) at each phosphorothioate linkage. In the present invention, CL655 and CL656 were obtained and tested as racemic mixtures.
General Schemes for Preparing Active Compounds
Methods for the easy preparation of cyclic dinucleotides or prodrugs thereof disclosed herein can be prepared as described in detail below, or by other methods known to those skilled in the art. It will be understood by one of ordinary skill in the art that these schemes are in no way limiting and that variations of detail can be made without departing from the spirit and scope of the present invention.
The term "protecting group" as used herein, and unless otherwise defined, refers to a chemical functional group that is attached to an oxygen, nitrogen or phosphorus atom to prevent further reaction of that atom, or for other purposes. A wide variety of protecting groups for oxygen and for nitrogen are known to those skilled in the art of organic synthesis, and are described, for example, in (Wuts, Greene, & Greene, 2014).
Generally, cyclic dinucleotides or prodrugs thereof of the Formulas (I), (II) or (III) are prepared by first preparing the corresponding nucleoside, then preparing the 5'-hydroxy group, the functional group (OH) at the 2' position or 3' position, and then, if necessary, protecting the exocyclic amine of the purine base. Then, the appropriately protected nucleosides are converted to the corresponding 3'-phosphoramidite, 2'-phosphoramidite, 3'-H-phosphonate or 2'-H- phosphonate, which constitute the starting material for the preparation of the cyclic dinucleotides described herein.
The reaction schemes shown below apply to the synthesis of CDNs of Formula I and can be employed for the synthesis of CDNs of Formulas (II) and (III):
Figure imgf000019_0001
Scheme 1 is a non-limiting example of the synthesis of active compounds of the present invention, and in particular, a synthetic approach to CDNs of Formula (I), where Bi, B2, Xi , X∑, and Z are defined above. Str is a trityl derivative such as dimethoxytrityl (DMTr), p is a protecting group, R∑ is an alkyl group such as an isopropyl group, R3 is a protecting group such as cyanoethyl, R4 is a protecting group such as allyl, and X3 is O or S.
Figure imgf000020_0001
Formula I
Scheme 2 is a non-limiting example of the synthesis of active compounds of the present invention, and in particular, an alternate synthetic approach to CDNs of Formula (I), where Bi, B2, Xi, X2, and Z are defined above. Str is a trityl derivative such as dimethoxytrityl (DMTr), p is a protecting group, R2 is an alkyl group such as an isopropyl group, R4 is a protecting group such as cyanoethyl, R5 is a protecting group such as allyl, and X3 is O or S.
CDNs of Formulas (I) and (II) are prepared by first preparing appropriately protected phosphoramidite of Formula (lA) or H-phosphonate of Formula (lF):
Figure imgf000020_0002
Scheme 3 is a non-limiting example of the synthesis of phosphoramidites of Formula (lA) and of H-phosphonates of Formula (lF), where B2, Yp, Xp, R7, Re, and Str are defined above. Phosphoramidites of the Formula ( ) and H-phosphonates of the Formula (IF) can be synthesized by first preparing the appropriately protected nucleosides of the Formula (IV), which can be accomplished by one of ordinary skill in the art. Firstly, the base, which has an exocyclic amine, is protected, and then the 3'- and 5'-hydroxyl groups are simultaneously or selectively protected. The substitutions X2 are eventually protected, using protecting groups such as TBDMS or tetrahydropyran (Thp) when X2 is hydroxyl. Finally, the protecting groups at the 3'- and 5'-hydroxyls are cleaved, and the 5'-hydroxyl is subsequently protected with a trityl derivative to give the nucleoside of the Formula (IV):
Figure imgf000021_0001
B2"
Str— O
OH X2p OH X2p
Formula IV
Scheme 4 is a non-limiting example of the synthesis of appropriately protected nucleoside of Formula (IV), where B2, X2, p, and Str are defined above. R5 and R6 are protecting groups.
Nucleosides of the Formula (IVA) can be prepared by methods outlined in: (Chu, 2002; Rajagopalan, 2003; Schinazi, 2004; Vorbriiggen, 2001 ).
Brief description of the figures FIGURE 1. STING signaling in the cell. Activation of STING by cyclic dinucleotides (CDN) leads to activation of the IRF3 and NF-κΒ pathways and consequently, to induction of Type I interferons and of pro-inflammatory cytokines, respectively.
FIGURE 2. In vitro Type I interferon induction activity in THP1 -Dual™ cell cultures: non-fluorinated vs. fluorinated cyclic dinucleotides.
FIGURE 3. In vitro NF-κΒ pathway induction in THP1 -Dual™ cell cultures: non-fluorinated vs. fluorinated cyclic dinucleotides. FIGURE 4. In vitro Type I interferon induction activity in wild-type vs. STING knockout B16 cells. It shows relative ISG54 activity (as an indirect measurement of Type I interferon induction) of non-fluorinated vs. fluorinated cyclic dinucleotides incubated in cultures of wild-type (left-side of graph) or STING-knockout (right-side of graph) B16 cells for 24 h. WT: wild-type; SKO: STING knockout (homozygous). FIGURE 5. In vitro Type I interferon induction activity in wild-type vs. STING-knockout RAW cells. It shows relative ISG54 activity (as an indirect measurement of Type I interferon induction) of non-fluorinated vs. fluorinated cyclic dinucleotides incubated in cultures of wild-type (left-side of graph) or STING-knockout (right-side of graph) RAW cells for 24 h. WT: wild-type; SKO: STING knockout (homozygous).
FIGURE 6. Type I interferon induction activity of cyclic dinucleotides in mice. Measurement of Type I interferon induction in sera from mice at 4 h post-treatment. FIGURE 7. IL-6 induction activity of cyclic dinucleotides in mice. Measurement of IL-6 induction in sera from mice at 4 h post-treatment.
FIGURE 8. In vivo elimination of cyclic dinucleotides in mice. Temporal change in plasma concentration of cyclic dinucleotides in treated mice.
FIGURE 9. Cytokine induction activity of CDNs in human whole blood.
FIGURES 10 A-D. Resistance of distinct CDNs to cleavage by the enzymes SVPD and NP1 over time, as monitored by HPLC. Compare CL614 and CL656 to c-AIMP, for resistance to SVPD (A) or NP1 (B). Compare CL603 and CL656 to c-GAMP, for resistance to SVPD (C) or NP1 (D).
FIGURE 11. In vitro activity of distinct CDNs in THP-1 Dual™ cells before and after incubation with the enzymes SVPD and NP1. The invention will be illustrated by the non-limiting following examples. EXAMPLES
Specific compounds that are representative of this invention were prepared as per the following examples and are offered by way of illustration to aid in the understanding of the invention. They should not be construed to limit in any way the invention set forth in the claims that follow thereafter. The present compounds can also be used as intermediates in subsequent examples to produce additional compounds of the present invention. No attempt has necessarily been made to optimize the yields obtained in any of the reactions. One skilled in the art would know how to increase such yields through routine variations in reaction times, temperatures, solvents, reagents and chemical synthesis other parameters. The present invention is further illustrated in Examples 1 , which shows preparative methods for synthesizing CDNs, and in Example 2, which shows methods for the biological evaluation of these CDNs. It will be understood by one of ordinary skill in the art that these examples are in no way limiting and that variations of detail can be made without departing from the spirit and scope of the present invention.
The terms used in describing the invention are commonly used and known to those skilled in the art. As used herein, the following abbreviations have the indicated meanings: °C for degrees Celsius; A for adenosine; ACN for acetonitrile; aq. for aqueous; CDCI3 for deuterated chloroform; Cis for octadecyl carbon chain bonded silica; d for doublet; dA for deoxyadenosine; dd for doublet of doublets; d l for deoxyinosine; D20 for deuterium oxide; DCA for dichloroacetic acid; DCM for dichloromethane; DMSO-c/6 for deuterated dimethylsulfoxide; DMTrCI for 4;4'-dimethoxytrityl chloride; equiv. for equivalent; ES for electrospray ionization; Et20 for diethyl ether; EtOAc ethyl acetate; EtOH for ethanol; Et3N -3HF for triethylamine trihydrofluoride; g for grams; 1H for proton; h for hours; Hz for Hertz; HPLC for high-performance liquid chromatography; I for inosine; IFN for interferon; IFN-a for interferon alpha; IFN-β for interferon beta; /PrOH for isopropanol; IRF3 for interferon regulatory factor 3; ISG (or ISG54) for interferon-stimulated gene; ISRE for interferon-stimulated response element; i.v. for intravenous; LC for liquid chromatography; m for multiplet; M for molar; m/z for mass-to-charge ratio; MeOH for methanol; mg for milligrams; MgS04 for magnesium sulfate; MHz for megahertz; min for Minutes; mL (or ml) for milliliters; mmol for millimoles; mol/L for mole/liter; MS for mass spectrometry; NaHC03 for sodium bicarbonate; NaHSC>3 for sodium thiosulfate; NH4OH for ammonium hydroxide; NF-κΒ for nuclear factor kappa-light-chain- enhancer of activated B cells; NMR for nuclear magnetic resonance; PADS for phenylacetyl disulfide; ppm for parts per million; PPTS for pyridinium p-toluenesulfonate; rt for room temperature; SEAP for secreted embryonic alkaline phosphatase; s for singlet; si for large singlet; t for triplet; SKO for homozygous STING knockout; STING for stimulator of interferon genes; TBAF for tetra-n-butylammonium fluoride; THF for tetrahydrofuran; TBDMSCI for tert- butyldimethylsilyl chloride; TEAA for triethylammonium acetate; TFA for trifluoroacetic acid; TIPSCI2 for 1 ,3-dichloro- 1 ,1 ,3,3-tetraisopropyldisiloxane; WT for wild type; μg for microgram; μί (or μΙ) for microliter; μιη for micrometer; δ for chemical shift. Anhydrous solvents and reagents suitable for nucleoside and nucleotide synthesis were purchased and were handled under dry argon or nitrogen using anhydrous technique. Amidite coupling reactions and cyclizations were performed in anhydrous acetonitrile or pyrid ine under dry argon or nitrogen. The starting materials for all reactions in dry pyridine were dried by concentration (three times) from pyrid ine. Preparative silica-gel flash chromatography was performed using Fluka 60 A high-purity grade or Merck Grade 9385 silica using gradients of methanol in dichloromethane. Analytical LC/ES MS was performed on an Agilent 1290 Infinity UHPLC system coupled to a diode array detector (DAD) Agilent 1260 Infinity and an Agilent 6130 Quadrupole mass spectrometer equipped with an electrospray ionization source (ESI) and controlled by Chemstation software. The LC system was equipped with an Aquity CSH C18 50x2.1 m m 1 .7 μιη column using gradients of 10 mM ammonioum formate and acetonitrile at 300 μΙ/min flow. The UV detection wavelength was 254 nm. The mass spectrometer was operated in positive and negative ESI modes Preparative HPLC was performed on a Waters preparative 150Q HPLC system monitoring at 254 nm on a SunFire Prep C18 5 μιη OBD 30 x 150mm column using gradients of 10 mM ammonium formate and acetonitrile at a flow rate 60 mL/min. The 1H NMR spectra were acquired on either a Bruker 300 MHz (Fourrier 300) at room temperature and reported in ppm downfield. Molecular sieves (MS) 3A were employed after drying the commercially supplied product at 250 °C for 12 h under vacuum. The commercial nucleoside phosphoramidites were supplied from Chemgenes.
The following examples serve to illustrate the present invention. These examples are in no way intended to limit the scope of the invention.
EXAMPLE 1 : Synthesis of the compounds of the invention Example 1.A: General protocol for preparation of phosphotriester: The appropriately protected phosphoramidite or commercially available phosphoramidite was coevaporated three times with dry ACN, and the resulting solid was dissolved in a solution of Activator42® (0.1 mol/L, 2 equiv.) in the presence of molecular sieves 3A. To the solution was added allyl alcohol (2 equiv.) and the resulting mixture was stirred for 30 min.
For phosphotriester linkage:
Tert-butyl hydroperoxide in decane (5.5 M, 2 equiv.) was added to the mixture, which was stirred for 40 min. The solution was filtered and the molecular sieves were washed with DCM. The filtrate was concentrated in vacuo.
For phosphorothioate triester linkage:
The mixture was concentrated in vacuo and the residue was dissolved in a solution of PADS (2.5 equ) 0.2 M in dry pyridine. The mixture was stirred at rt for 45min. The solution was filtered and the molecular sieves were washed with DCM. The filtrate was concentrated in vacuo and coevaporated three times with ACN.
The residue was treated with a solution of DCA/DCM (3%) in the presence of water (10 equiv.) for 15 min. The reaction was quenched with addition of MeOH and pyridine. The solvents were removed in vacuo and the residue was purified by silica-gel column chromatography, using DCM/MeOH as eluent. The structure of the compound was confirmed by LC-ES/MS analysis with ions at [M-H]" and/or [M+H]+.
Example 1.B: General protocol for preparation of H-phosphonate:
The appropriately protected phosphoramidite or commercial phosphoramidite was dissolved in a solution of ACN. To the solution were added water (2 equiv.) and pyridium TFA (1.2 equiv.), and the mixture was stirred for 15 min. Then, the solvents were removed in vacuo. The residue was treated with a solution of DCA/DCM (3%) in the presence of water (10 equiv.) for 15 min. The reaction was quenched with addition of MeOH and pyridine. The solvents were removed in vacuo and the residue was purified by silica-gel column chromatography, using DCM/MeOH as eluent. The structure of the compound was confirmed by LC-ES/MS analysis with ions at [M-H]" and/or [M+H]+.
Example 1.C: Protocol for the di nucleotide synthesis:
To a solution of compound of Example 1.A or appropriately protected compound in a solution of Activator42® (0.1 mol/L, 2 equiv.) in the presence of molecular sieves 3A was added in one portion an appropriately protected phosphoramidite or commercial phosphoramidite. The mixture was stirred for 30 min.
For phosphotriester linkage:
Tert-butyl hydroperoxide in decane (5.5 M, 2 equiv.) was added to the mixture, which was stirred for 40 min. The solution was filtered and the molecular sieves were washed with DCM. The filtrate was concentrated in vacuo.
For phosphorothioate triester linkage:
The mixture was concentrated in vacuo and the residue was dissolved in a solution of PADS (2.5 equ) 0.2 M in dry pyridine. The mixture was stirred at rt for 45min. The solution was filtered and the molecular sieves were washed with DCM. The filtrate was concentrated in vacuo and coevaporated three times with ACN.
The residue was treated with a solution of DCA/DCM (3%) in the presence of water (10 equiv.) for 15 min. The reaction was quenched by addition of MeOH and pyrid ine. The solvents were removed in vacuo and the residue was purified by silica-gel column chromatography, using DCM/MeOH as eluent. The structure of the compound was confirmed by LC-ES/MS analysis with ions at [M-H]" and/or [M+H]+.
Example 1.D: Alternative protocol for the dinucleotide synthesis: To a solution of compound of Example 1.B in a solution of Activator42® (0.1 mol/L, 2 equiv.) in the presence of molecular sieves 3A was added in one portion an appropriately protected phosphoramidite or commercial phosphoramidite. The mixture was stirred for 30 min.
For phosphotriester linkage:
Tert-butyl hydroperoxide in decane (5.5 M, 2 equiv.) was added to the mixture, which was stirred for 40 min. The solution was filtered and the molecular sieves were washed with DCM. The filtrate was concentrated in vacuo.
For phosphorothioate triester linkage:
The mixture was concentrated in vacuo and the residue was dissolved in a solution of PADS (2.5 equ) 0.2 M in dry pyridine. The mixture was stirred at rt for 45min. The solution was filtered and the molecular sieves were washed with DCM. The filtrate was concentrated in vacuo and coevaporated three times with ACN.
The residue was treated with a solution of DCA DCM (3%) in the presence of water (10 equiv.) for 15 min. The reaction was quenched with addition of MeOH and pyridine. The solvents were removed in vacuo and the residue was purified by silica-gel column chromatography, using DCM/MeOH as eluent. The structure of the compound was confirmed by LC-ES/MS analysis with ions at [M-H]" and/or [M+H]+. Example 1.E: Protocol to remove ally I group:
To a solution of dinucleotide from Example 1 .C in acetone was added sodium iodide (10 equiv.), and the resulting suspension was stirred under reflux for 2 h. The resulting colorless precipitate was collected by filtration and washed with chilled acetone. This precipitate is highly hygroscopic and thus, was immediately used in the next procedure. The structure of the compound was confirmed by LC-ES/MS analysis with ions at [M-H]" and/or [M+H]+. Example 1.F: Alternative protocol to remove allyl group:
To a solution of dinucleotide from Example 1.C in dry THF was added N-methyl aniline (3 equiv.) and tetrakis(triphenylphosphine)palladium(0) (0.2 equiv.). The resulting suspension was stirred at rt for 15 min. Then, the solvent was removed in vacuo and the residue was triturated with diethyl ether. The resulting colorless precipitate was collected by filtration and washed with chilled diethyl ether. This precipitate was purified by silica-gel column chromatography, using DCM/MeOH as eluent. The structure of the compound was confirmed by LC-ES/MS analysis with ions at [M-H]" and/or [M+Hf.
Example 1.G: Protocol for cyclization of the dinucleotide:
The solid obtained in Example 1.E or 1.F was coevaporated three times with dry pyrid ine and then, dry ACN. The residue was suspended in THF, and to the resulting heterogeneous mixture were successively added N- methylimidazole (10 equiv.) and 2,4,6-triisopropylbenzenesulfonyl chloride (10 equiv.). The resulting mixture was stirred at 25 °C for 3 h to 36 h. Then, the solvent was removed in vacuo and the residue was triturated with EtOAc. The resulting colorless precipitate was collected by filtration and washed with chilled EtOAc. This precipitate was used in the next step without any further purification. The structure of the compound was confirmed by LC-ES/MS analysis with ions at [M-H]" and/or [M+H]+. Example 1.H: Alternative protocol for cyclization of the dinucleotide:
The solid obtained from Example 1 .E or 1.F was coevaporated three times with dry pyridine. The residue was suspended in dry pyridine, and to the resulting solution was added 1 -(Mesitylene-2-sulfonyl)-3-nitro-1 ,2,4-triazole (MSNT) (5 equiv.). The resulting mixture was stirred at 25 °C for 3 h to 18 h. Then, the solvent was removed in vacuo, and the resulting product used in the next step without any further purification. The structure of the compound was confirmed by LC-ES/MS analysis with ions at [M-H]" and/or [M+H]+.
Example 1.1: Alternative protocol for cyclization of the dinucleotide:
The solid obtained in Example 1 .D or appropriately protected compound was coevaporated three times with dry pyridine. The residue was suspended in dry pyridine, and to the resulting solution was added 5,5-dimethyl-2-oxo-2- chloro-1 ,3,2-dioxaphosphinane (DMOCP) (3 equiv.). The resulting mixture was stirred at 25 °C for 3 h to 18 h.
For phosphodiester linkage:
Iodine (1.3 equiv.) and water (30 equiv.) were added to the mixture. After 10 min aq. NaHS03 (0.15%) was added until complete decoloration was observed, and then aq. NaHC03 was added. The aqueous layer was extracted three times with a 1 :1 (v/v) mixture of EtOAc/Et20. The organic layers were pooled, dried over MgS04, filtered, and then concentrated in vacuo.
For phosphorothioate triester linkage:
Elemental sulfur (5 equiv.) was added. The mixture was stirred at rt for 45 min. Then the mixture was concentrated in vacuo and coevaporated three times with toluene, precipitated in ACN to removed excess of sulfur and concentrated in dryness.
The residue was used in the next step without any further purification. The structure of the compound was confirmed by LC-ES/MS analysis with ions at [M-H]" and/or [M+H]+. Example 1.J: Protocol for the deprotection and purification of cyclic dinucleotides:
The protected cyclic dinucleotide of Example 1 .G, 1.H or 1.1 was treated with a solution of methylamine in EtOH (33%), and the resulting mixture was stirred at 50 °C for 4 h. The reaction mixture was concentrated, and the resulting residue was dried in vacuo. The dried material was mixed with Et3N -3HF (25 equiv.) and stirred at 25 °C for 6 h. To this mixture was added a 1 M ammonium formate buffer solution, and the mixture was vigorously stirred at 30 °C to 40 °C for 10 min. The resulting precipitate was filtered, and the filtrate was subjected to preparative HPLC using a Ci8 Sunfire column (19 x 150 mm, 5 μιη) and ammonium formate/ACN as eluent. The fractions containing the desired compound were pooled and lyophilized. The structure of the compound was confirmed by LC-ES/MS analysis with ions at [M-H]" and/or [M+H]+.
Example 1.K: Protocol for the deprotection and purification of cyclic dinucleotides:
The protected cyclic dinucleotide of Example 1 .G, 1.H or 1.1 was treated with a solution of methylamine in EtOH (33%), and the resulting mixture was stirred at 50 °C for 4 h. The reaction mixture was concentrated, and the resulting residue was subjected to preparative HPLC using a Cis Sunfire column (19 x 150 mm, 5μιη) and ammonium formate/ACN as eluent. The fractions containing the desired compound were pooled and lyophilized. The structure of the compound was confirmed by LC-ES/MS analysis with ions at [M-H]" and/or [M+H]+.
Intermediate 1: 2'-0(TBDMS)-3'-0(H-phosphonate)-
Figure imgf000026_0001
Intermediate 1 was prepared from the commercially available phosphoramidite of Adenosine using a similar procedure to that described in Example 1.B to provide 6.20 g (93% yield) of intermediate 1 . LC-MS: Rt = 4.41 min, m/z = 550 [M+Hf, m/z = 548 [M-H]".
Intermediate 2: [2'-0(TBDMS)-3'-0(CE)phosphotriester- lnosine]-(3',5')-[2'-0(TBDMS)-3'-0(H- phosphonate)-N6(Bz)Adenosine]
Figure imgf000027_0001
Intermediate 2 was prepared from intermediate 1 and the commercially available phosphoramidite of Inosine using a sim ilar procedure to that described in Example 1.D to provide 8.04 g (68% yield) of intermediate 2. LC-MS: Rt = 5.22 min, m/z = 1048 [M+Hf, m/z = 1046 [M-H]".
Intermediate 3: (3',3')Cyclic-[2'-0(TBDMS)-3'-
0(phosphodiester)-N6(Bz)Adenosine]-[2'-
0(TBDMS)-3'-0(CE)phosphotriester-lnosine]
Figure imgf000027_0002
Intermediate 3 was prepared from intermediate 2 using a similar procedure to that described in Example 1.1 to provide 8.04 g (68% yield) of intermediate 3. LC-MS: Rt = 5.41 min, m/z = 1046 [M+Hf, m/z = 1044 [M-H]".
Intermediate 4: (5',3')-0(TIPS)lnosine
Figure imgf000027_0003
To a solution of Inosine (10.0 g, 37.2 mmol) in dry pyrid ine (200 mL) was added TIPSCI2 (14.1 g, 44.7 mmol). The solution was stirred for 18 h at rt. Then, the reaction was quenched by addition of MeOH (50 mL) and the solvents were removed in vacuo. The residue was dissolved in EtOAc and washed with saturated aq. NaHC03, water and brine. The organic layer was dried over MgS04, filtered, and then concentrated in vacuo. The crude compound was purified by silica-gel column chromatography, using DCM/MeOH as eluent, to give 13.5 g (70% yield) intermediate 4. LC-MS: Rt = 5.32 min, m/z = 51 1 [M+Hf, m/z = 509 [M-H]". 1H NMR (CDCI3-c/r, 300 MHz) δ (ppm) 13.04 (s, 1 H), 8.10 (s, 1 H), 8.03 (s, 1 H), 5.99 (s, 1 H), 4.93 (t, 1 H), 4.50 (d, 1 H), 4.1 1 (m, 3H), 1.83 (m, 4H), 1.09 (m, 32H).
Intermediate 5: (5',3')-0(TIPS)-2'-0(Thp)lnosine
Figure imgf000027_0004
To a solution of intermediate 4 (5 g, 9.79 mmol) in dry DCM (75 mL) were added 3,4-dihydro-2H-pyran (24.7 g, 293.7 mmol) and PPTS (7.38 g, 29.37 mmol). The solution was stirred for 18 h at rt. Then, the reaction was quenched by a saturated solution of NaHC03. The different layers were separated and the organic layer was washed with water and brine, dried over MgS04, filtered, and then concentrated in vacuo. The crude compound was purified by silica-gel column chromatography, using DCM/MeOH as eluent, to give 4.71 g (80% yield) of intermediate 5. LC-MS: Rt = 6.72 min, m/z = 595 [M+H]+, m/z = 593 [M-H]". 1H NMR (CDCI3-cf,, 300 MHz) δ (ppm) 12.89 (d, 1 H), 8.12 (s, 1 H), 8.09 (s, 1 H), 8.04 (d, 1 H), 6.05 (d, 1 H), 5.07 (t, 1 H), 4.76 (m, 1 H), 4.53 (m, 2H), 4.40^.05 (m, 5H), 1.85-1.55 (m, 10H), 1.04 (m, 32H).
Intermediate 6: 2 -0(Thp)lnosine
Figure imgf000028_0001
To a solution of intermediate 5 (4.71 g, 7.92 mmol) in THF (100 mL) was added TBAF on silica gel (10.56 g, 15.84 mmol). The solution was stirred for 3 h at rt. Then, the reaction was filtered, the silica gel was washed with THF, and the filtrate was concentrated in vacuo. The crude compound was purified by silica-gel column chromatography, using DCM/MeOH as eluent, to give 2.7 g (96% yield) of intermediate 6. LC-MS: Rt = 4.32 min, m/z = 353 [M+Hf, m/z = 351 [M-H]".
Intermediate 7: 5'-0(DMTr)-2'-0(Thp)lnosine
Figure imgf000028_0002
To a solution of intermediate 6 (2.70 g, 7.66 mmol) in dry pyridine (40 mL) was added dropwise a solution of DMTrCI (2.17 g, 6.42 mmol) in DCM (5 mL). The solution was at rt for 2 h. Then, the reaction was quenched by addition of 5% aq. NaHC03 (1 10 mL), the aqueous layer was extracted three times with DCM. The organic layers were pooled and dried over MgS04, filtered, and then concentrated in vacuo. The residue was purified by silica-gel column chromatography, using 1 % pyridine in DCM/MeOH as eluent, to give 4.78 g (95% yield) of intermediate 7. LC-MS: Rt = 6.80 min, m/z = 655 [M+Hf, m/z = 653 [M-H]". 1H NMR (CDCI3-cf,, 300 MHz) δ (ppm) 12.80 (s, 1 H), 8.02 (s, 1 H), 8.00 (s, 1 H), 7.71 (m, 2H), 7.73 (m, 2H), 7.24 (m, 9H), 6.20 (d, 1 H), 5.07 (t, 1 H), 4.83 (m, 1 H), 4.35 (m, 2H), 3.78 (s, 6H), 3.45-3.30 (m, 4H), 1.77-1 .56 (m, 6H).
Intermediate 8: 5'-0(DMTr)-3'-0(TBDMS)-2'-0(Thp)lnosine
Figure imgf000028_0003
To a solution of intermediate 7 (4.78 g, 7.30 mmol) in dry pyridine (40 mL) were added imidazole (1.29 g, 18.98 mmol) and TBDMSCI (1.43 g, 9.49 mmol), the reaction was stirred for 18 h at rt. Then, the reaction was diluted with DCM (100 mL), the solution was washed with saturated aq. NaHC03, water and brine. The organic layer was dried over MgS04, filtered, and then concentrated in vacuo. The residue was purified by silica-gel column chromatography, using 1 % pyridine in DCM/MeOH as eluent, to give ((5.12 g (91 % yield) of intermediate 8. LC-MS: Rt = 8.15 min, m/z = 769 [M+Hf, m/z = 767 [M-H]". 1 H NMR (CDCI3-cf,, 300 MHz) δ (ppm) 12.70 (s, 1 H), 8.02 (s, 1 H), 7.97 (s, 1 H), 7.40 (m, 2H), 7.26 (m, 1 1 H), 6.09 (d, 1 H), 4.80 (m, 1 H), 4.36 (m, 1 H), 4.16 (m, 2H), 3.72 (s, 6H), 3.45-3.26 (m, 4H), 1.61 -1.37 (m, 8H), 0.81 (s, 9H), 0.09 (dd, 6H).
Intermediate 9: 3'-0(TBDMS)-2'-0(Thp)lnosine
Figure imgf000029_0001
The intermediate 8 (4.93 g, 6.41 mmol) was dissolved in a solution of ZnBr2 (0.5 M) in DCIW/PrOH (1/1 ) (40 mL, 19.3 mmol). The solution was stirred for 40 min at rt. The reaction was neutralized with 1 N NaHC03 solution. The different layers were separated and the organic layer was washed with water and brine, dried over MgSC filtered, and then concentrated in vacuo. The residue was purified by silica-gel column chromatography, using DCM/MeOH as eluent to give 2.68 g (90% yield) of intermediate 9. LC-MS: Rt = 6.22 min, m/z = 467 [M+Hf, m/z = 465 [M-H]\
Intermediate 10: [2'-0(TBDMS)-3'-0(CE)phosphotriester-
N6(Bz)Adenosine]-(3',5')-[3'-0(TBDMS)-2'-
0(Thp)lnosine]
Figure imgf000029_0002
Intermediate 10 was prepared from intermediate 9 and the commercially available phosphoramidite of Adenosine using a similar procedure to that described in Example 1.C to give 1.9 g (64% yield) of intermediate 10. Rt = 6.99 min, m/z = 1068 [M+Hf, m/z = 1066 [M-H]".
Intermediate 11: [5'-0(H-phosphonate)-2'-0(TBDMS)-3'-
0(CE)phosphotriester-N6(Bz)Adenosine]-
(3,,5')-[3'-0(TBDMS)-2,-0(Thp)lnosine]
Figure imgf000029_0003
To a solution of intermediate 10 (1 .9 g, 1.78 mmol) in dry pyridine (20 mL) was added diphenylphosphite (1.25 g, 5.34 mmol). The reaction was stirred for 2 h at rt. To the reaction was added a solution of TEAA 0.1 M (53 mL, 5.34 mmol). The reaction was stirred for 45 min at rt. Then, the solvents were removed in vacuo and the residue was dissolved in DCM (100 mL). The organic layer was washed with aq. NaHC03 (5%) solution, water and brine, dried over MgS04, filtered, and then concentrated in vacuo to g ive 2.0 g (99% yield) of crude intermediate 1 1 . This intermediate was used in the next step without any further purification. LC-MS: Rt = 6.06 min, m/z = 1 132 [M+H]+, m/z = 1 130 [M-H]". Intermediate 12: [5'-0(H-phosphonate)-2'-0(TBDMS)-3'-
0(CE)phosphotriester-N6(Bz)Adenosine]-
(3,,5')-[3'-0(TBDMS)lnosine]
Figure imgf000030_0001
The intermediate 1 1 (2.0 g, 2.12 mmol) was treated with a solution of DCA 10% in DCM (50 mL). The reaction was stirred for 2 h at rt. Then, the reaction was neutralized by addition of pyridine (17 mL). The solvents were removed in vacuo to give 2.2 g (100% yield) of crude intermediate 12. This intermediate was used in the next step without any further purification. LC-MS: Rt = 7.38 min, m/z = 1048 [M+Hf, m/z = 1046 [M-H]\
Intermediate 13: (3',2')Cyclic-[2'-0(TBDMS)-3'-
0(CE)phosphotriester-N6(Bz)Adenosine]-[2'-
0(phosphodiester)-3'-0(TBDMS)lnosine]
Figure imgf000030_0002
Intermediate 13 was prepared from intermediate 12 using a similar procedure to that described in Example 1 .1 to provide 1.73 g (83% yield) of intermediate 13. LC-MS: Rt = 7.20 min, m/z = 1046 [M+Hf, m/z = 1044 [M-H]\
Intermediate 14: [3'-0(TBDMS)-2'-0(CE)phosphotriester-
N6(Bz)Adenosine]-(3',5')-[3'-0(TBDMS)-2'-
0(Thp)lnosine]
Figure imgf000030_0003
Intermediate 14 was prepared from intermediate 9 and the commercially available phosphoramidite of adenosine using a similar procedure to that described in Example 1 .C to provide 1.9 g (60% yield) of intermediate 14. Rt = 7.08 min, m/z = 1068 [M+H]+, m/z = 1066 [M-H]".
Intermediate 15: [5'-0(H-phosphonate)-3'-0(TBDMS)-2'-
0(CE)phosphotriester-N6(Bz)Adenosine]-
(3,,5')-[3'-0(TBDMS)-2,-0(Thp)lnosine]
Figure imgf000030_0004
To a solution of intermediate 14 (1 .9 g, 1.78 mmol) in dry pyridine (20 mL) was added diphenylphosphite (1.25 g, 5.34 mmol). The reaction was stirred at rt for 2 h. To the reaction was added a solution of TEAA 0.1 M (53 mL, 5.34 mmol). The reaction was stirred for 45 min at rt. Then, the solvents were removed in vacuo and the residue was dissolved in DCM (100 mL). The organic layer was washed with aq. NaHC03 (5%) solution, water and brine, dried over MgS04, filtered, and then concentrated in vacuo to g ive 2.0 g (99% yield) of crude intermediate 15. This intermediate was used in the next step without any further purification. LC-MS: Rt = 6.1 1 min, m/z = 1 132 [M+H]+, m/z = 1 130 [M-H]".
Intermediate 16: [5'-0(H-phosphonate)-2'-0(TBDMS)-3'-
0(CE)phosphotriester-N6(Bz)Adenosine]-
(3,,5')-[3'-0(TBDMS)lnosine]
Figure imgf000031_0001
The intermediate 15 (2.0 g, 1 .76 mmol) was treated with a solution of DCA 10% in DCM (50 mL, 85.0 mmol). The reaction was stirred at rt for 2 h. Then, the reaction was neutralized by addition of pyridine (17 mL, 177.0 mmol). The solvents were removed in vacuo to give 1.8 g (100% yield) of crude intermediate 16. This intermediate was used in the next step without any further purification. LC-MS: Rt = 5.39 min, m/z = 1048 [M+H]+, m/z = 1046 [M-H]".
Intermediate 17: (2',2')Cyclic-[3'-0(TBDMS)-2'-
0(CE)phosphotriester-N6(Bz)Adenosine]-[2'-
0(phosphodiester)-3'-0(TBDMS)lnosine]
Figure imgf000031_0002
Intermediate 17 was prepared from intermediate 16 using a similar procedure to that described in Example 1 .1 to provide 1.5 g (75% yield) of intermediate 17. LC-MS: Rt = 5.41 min, m/z = 1046 [M+Hf, m/z = 1044 [M-H]".
Intermediate 18: 3'-0(TBDMS)-2'-0(H-phosphonate)- N6(Bz)Adenosine
Figure imgf000031_0003
Intermediate 18 was prepared from the commercially available phosphoramidite of Adenosine using a similar procedure to that described in Example 1.B to provide 1.1 g (94% yield) of intermediate 18. LC-MS: Rt = 4.47 min, m/z = 550 [M+Hf, m/z = 548 [M-H]". Intermediate 19: [2'-0(TBDMS)-3'-0(CE)phosphotriester- lnosine]-(3',5')-[3'-0(TBDMS)-2'-0(H- phosphonate)-N6(Bz)Adenosine]
Figure imgf000032_0001
Intermediate 19 was prepared from intermediate 18 and commercially available phosphoramidite of Inosine using a sim ilar procedure to that described in Example 1.D to provide 410 mg (44% yield) of intermediate 19. Rt = 4.70 min, m/z = 1048 [M+Hf, m/z = 1046 [M-H]".
Intermediate 20: (2',3')cyclic-[3'-0(TBDMS)-2'-
0(phosphodiester)-N6(Bz)Adenosine]-[2'-
0(TBDMS)-3'-0(phosphodiester)-lnosine]
Figure imgf000032_0002
Intermediate 20 was prepared from intermediate 19 using a similar procedure to that described in Example 1 .1 to provide 364 mg (89% yield) of intermediate 20. LC-MS: Rt = 5.66 min, m/z = 1046 [M+H]+, m/z = 1044 [M-H]".
Intermediate 21: [2'-0(TBDMS)-3'-0(CE)phosphorothioate- triester-lnosine]-(3',5')-[2'-0(TBDMS)-3'-0(H- phosphonate)-N6(Bz)Adenosine]
Figure imgf000032_0003
Intermediate 21 was prepared from intermediate 1 and the commercially available phosphoramidite of Inosine using a sim ilar procedure to that described in Example 1 .D to provide 1.05 g (65% yield) of intermediate 21. LC-MS: Rt = 5.42 and 5.52 min, m/z = 1064 [M+H]+, m/z = 1062 [M-H]".
Intermediate 22: (3',3')Cyclic-[2'-0(TBDMS)-3'-
O(phosphorothioate-diester)- N6(Bz)Adenosine]-[2'-0(TBDMS)-3'- 0(CE)phosphorothioate-triester-lnosine]
Figure imgf000032_0004
Intermediate 22 was prepared from intermediate 21 using a similar procedure to that described in Example 1 .1 to provide 579 mg (55% yield) of intermediate 22. LC-MS: Rt = 5.61 and 5.71 min, m/z = 1078 [M+Hf, m/z = 1076 [M-H]" Intermediate 23: 3'-0[(Allyl,CE)phosphotriester]-Ne(Bz)-2'- deoxyAdenosine
Figure imgf000033_0001
Intermediate 23 was prepared from the commercially available phosphoramidite of 2'-deoxyadenosine using a similar procedure to that described in Example 1 .A to provide 1.84 g (95% yield) of intermediate 23. LC-MS: Rt = 4.31 min, m/z = 529 [M+Hf, m/z = 527 [M-H]".
Intermediate 24:
Figure imgf000033_0002
Intermediate 24 was prepared from intermediate 23 and commercially available phosphoramidite of 2'-deoxyinosine using a similar procedure to that described in Example 1.C to provide 500 mg (32% yield) of intermediate 24. Rt = 5.28 min, m/z = 896 [M+H]+, m/z = 894 [M-H]".
Intermediate 25:
Figure imgf000033_0003
Intermediate 25 was prepared from intermediate 24 using a similar procedure to that described in Example 1.E to provide 390 mg (83% yield) of intermediate 25. LC-MS: Rt = 3.35 min, m/z = 856 [M+H]+, m/z = 854 [M-H]".
Intermediate 26: (3',3,)Cyclic-[3'-0(CE)phosphothester-Ne(Bz)-
2'-deoxyAdenosine]-[3'-0(CE)phosphotriester-
2'-deoxylnosine]
Figure imgf000033_0004
Intermediate 26 was prepared from intermediate 25 using a similar procedure to that described in Example 1.G to provide 0.38 g (99% yield) of intermediate 26. LC-MS: Rt = 3.91 min, m/z = 838 [M+Hf, m/z = 836 [M-H]". Intermediate 27: [3'-0(CE)phosphotriester-2'-deoxy-2'- fluorolnosine]-(3',5')-[3'- 0(Allyl,CE)phosphotriester-N6(Bz)-2'- deoxyAdenosine]
Figure imgf000034_0001
Intermediate 27 was prepared from intermediate 23 and commercially available phosphoramidite of 2'-deoxy-2'- fluorolnosine using a similar procedure to that described in Example 1.C to provide 3.16 g (62% yield) of intermediate 27. Rt = 4.27 min, m/z = 914 [M+H]+, m/z = 912 [M-H]".
Intermediate 28: [3'-0(CE)phosphotriester-2'-deoxy-2'- fluorolnosine]-(3',5')-[3'-0(CE)phosphodiester-
N6(Bz)-2'-deoxyAdenosine]
Figure imgf000034_0002
Intermediate 28 was prepared from intermediate 27 using a similar procedure to that described in Example 1.E to provide 1.10 g (69% yield) of intermediate 28. LC-MS: Rt = 3.52 min, m/z = 874 [M+H]+, m/z = 872 [M-H]".
Intermediate 29:
Figure imgf000034_0003
Intermediate 29 was prepared from intermediate 28 using a similar procedure to that described in Example 1.H to provide 1.01 g (99% yield) of intermediate 29. LC-MS: Rt = 4.23 and 4.07 min, m/z = 856 [M+H]+, m/z = 854 [M-H]".
Intermediate 30: 3'-0(Allyl,CE)phosphotriester-NB(Bz)-2'-deoxy- 2'-fluoroAdenosine
Figure imgf000034_0004
Intermediate 30 was prepared from the commercially available phosphoramidite of 2'-deoxy-2'-fluoroAdenosine using a similar procedure to that described in Example 1.A to provide 2.68 g (85% yield) of intermediate 30. LC-MS: Rt = 4.50 min, m/z = 547 [M+H]+, m/z = 545 [M-H]". Intermediate 31: [3'-0(CE)phosphotriester-2'-deoxy-2'- fluorolnosine]-(3',5')-[3'- 0(Allyl,CE)phosphotriester-N6(Bz)-2'-deoxy-2'- fluoroAdenosine]
Figure imgf000035_0001
Intermediate 31 was prepared from intermediate 30 and commercial phosphoramidite of 2'-deoxy-2'-fluorolnosine using a similar procedure to that described in Example 1.C to provide 2.53 g (55% yield) of intermediate 31. Rt = 4.39 min, m/z = 932 [M+H]+, m/z = 930 [M-H]".
Intermediate 32: [3'-0(CE)phosphotriester-2'-deoxy-2'- fluorolnosine]-(3',5')-[3'-0-(CE)phosphodiester-
N6(Bz)-2'-deoxy-2'-fluoroAdenosine]
Figure imgf000035_0002
Intermediate 32 was prepared from intermediate 31 using a similar procedure to that described in Example 1.E to provide 4.4 g (92% yield) of intermediate 32. LC-MS: Rt = 3.58 and 3.59 min, m/z = 892 [M+H]+, m/z = 891 [M-H]".
Intermediate 33: (3,,3,)Cyclic-[3,-0(CE)phosphothester-Ne(Bz)2'- deoxy-2'-fluoroAdenosine]-[3'-0-
(CE)phosphotriester-2'-deoxy-2'-fluorolnosine]
Figure imgf000035_0003
Intermediate 33 was prepared from intermediate 32 using a similar procedure to that described in Example 1.G to provide 1.40 g (99% yield) of intermediate 33. LC-MS: Rt = 4.21 and 4.42 min, m/z = 874 [M+Hf, m/z = 872 [M-H]".
Intermediate 34: 3'-0(Allyl,CE)phosphorothioate-triester-Ne(Bz>
2'-deoxy-2'-fluoroAdenosine
Figure imgf000035_0004
Intermediate 34 was prepared from the commercially available phosphoramidite of 2'-deoxy-2'-fluoroAdenosine using a similar procedure to that described in Example 1.A to provide 647 mg (55% yield) of intermediate 34. LC-MS: Rt = 5.06 min, m/z = 563 [M+Hf, m/z = 561 [M-H]". Intermediate 35: [3'-0(CE)phosphorothioate-triester-2'-deoxy-2'- fluorolnosine]-(3',5')-[3'- 0(Allyl,CE)phosphorothioate-triester-N6(Bz)-2'- deoxy-2'-fluoroAdenosine]
Figure imgf000036_0001
Intermediate 35 was prepared from intermediate 34 and commercially available phosphoramidite of 2'-deoxy-2'- fluorolnosine using a similar procedure to that described in Example 1.C to provide 447 mg (35% yield) of intermediate 35. Rt = 6.20 min, m/z = 964 [M+Hf, m/z = 962 [M-H]".
Intermediate 36: [3'-0(CE)phosphorohtioate-triester-2'-deoxy-2'- fluorolnosine]-(3',5')-[3'-0- (CE)phosphorothioate-diester-N6(Bz)-2'-deoxy- 2'-fluoroAdenosine]
Figure imgf000036_0002
Intermediate 36 was prepared from intermediate 35 using a similar procedure to that described in Example 1.E to provide 232 mg (43% yield) of intermediate 36. LC-MS: Rt = 4.25 and 4.55 min, m/z = 924 [M+H]+, m/z = 922 [M-H]".
Intermediate 37: (3',3')Cyclic-[3'-0(CE)phosphorothioate- triester-N6(Bz)2'-deoxy-2'-fluoroAdenosine]-[3'- 0-(CE)phosphorothioate-triester-2'-deoxy-2'- fluorolnosine]
Figure imgf000036_0003
Intermediate 37 was prepared from intermediate 36 using a similar procedure to that described in Example 1.G to provide 35 mg (36% yield) of intermediate 37. LC-MS: Rt = 5.00 and 5.32 min, m/z = 906 [M+H]+, m/z = 904 [M-H]".
Intermediate 38: 2'-0-(H-phosphonate)-3'-0-TBDMS-Ne(Bz>
Adenosine
Figure imgf000036_0004
Intermediate 38 was prepared from the commercially available 2'-phosphoramidite of Adenosine using a similar procedure to that described in Example 1.B to provide 1.81 g (95% yield) of intermediate 38. LC-MS: Rt = 4.53 min, m/z = 550 [M+Hf, m/z = 548 [M-H]". Intermediate 39: [3'-0-(CE)phosphotriester-2'-deoxy-2'- fluorolnosine]-(3',5')-[2'-0-(H-phosphonate)-3'-
0-TBDMS-N6(Bz)-Adenosine]
Figure imgf000037_0001
Intermediate 39 was prepared from intermediate 38 and commercially available phosphoramidite of 2'-deoxy-2'- fluorolnosine using a similar procedure to that described in Example 1.D to provide 340 mg (55% yield) of intermediate 39. Rt = 4.28 min, m/z = 935 [M+H]+, m/z = 933 [M-H]".
Intermediate 40: Cyclic-(2',3')-[2'-0-(CE)phosphotriester-3'-0-
TBDMS-N6(Bz)Adenosine]-[3'-0-
(CE)phosphotriester-2'-deoxy-2'-fluorolnosine]
Figure imgf000037_0002
Intermediate 40 was prepared from intermediate 39 using a similar procedure to that described in Example 1 .1 to provide 340 mg (95% yield) of intermediate 40. LC-MS: Rt = 4.44 min, m/z = 933 [M+Hf, m/z = 931 [M-H]".
Intermediate 41: 3'-0-(Allyl,CE)phosphotriester-2'-deoxy-2'- fluorolnosine
Figure imgf000037_0003
Intermediate 41 was prepared from the commercially available phosphoramidite of 2'-deoxy-2'-fluorolnosine using a sim ilar procedure to that described in Example 1.A to provide 600 mg (98% yield) of intermediate 41. LC-MS: Rt = 3.78 min, m/z = 444 [M+H]+, m/z = 442 [M-H]".
Intermediate 42: [3'-0-(CE)phosphotriester-2'-deoxy- 2'fluorolnosine]-(3',5')-[3'-0- (Allyl,CE)phosphotriester-2'-deoxy-2'- fluorolnosine]
Figure imgf000037_0004
Intermediate 42 was prepared from intermediate 41 and commercially available phosphoramidite of 2'-deoxy-2'- fluorolnosine using a similar procedure to that described in Example 1.C to provide 610 mg (55% yield) of intermediate 42. Rt = 5.50 min, m/z = 829 [M+Hf, m/z = 827 [M-H]". Intermediate 43: [3'-0-(CE)phosphotriester-2'-deoxy-
2'fluorolnosine]-(3',5')-[3'-0-
(CE)phosphodiester-2'-deoxy-2'-fluorolnosine]
Figure imgf000038_0001
Intermediate 43 was prepared from intermediate 42 using a similar procedure to that described in Example 1.F to provide 580 mg (90% yield) of intermediate 43. LC-MS: Rt = 3.40 min, m/z = 789 [M+H]+, m/z = 787 [M-H]".
Intermediate 44: Cyclic-(3',3')-[3'-0-(CE)phosphotriester-2'- deoxy-2'-fluorolnosine]-[3'-0-
(CE)phosphotriester-2'-deoxy-2'-fluorolnosine]
Figure imgf000038_0002
Intermediate 44 was prepared from intermediate 43 using a similar procedure to that described in Example 1.H to provide 500 mg (99% yield) of intermediate 44. LC-MS: Rt = 3.86 min, m/z = 771 [M+Hf, m/z = 769 [M-H]".
Intermediate 45: 3'-0-(Allyl,CE)phosphotriester-Nii(iB)-2'-deoxy- 2'-fluoroGuanosine
Figure imgf000038_0003
Intermediate 45 was prepared from the commercially available phosphoramidite of 2'-deoxy-2'-fluoroGuanosine using a similar procedure to that described in Example 1.A to provide 3.75 g (60% yield) of intermediate 45. LC-MS: Rt = 4.25 min, m/z = 529 [M+Hf, m/z = 527 [M-H]".
Intermediate 46: [3'-0-(CE)phosphotriester-N2(iB)-2'-deoxy- 2'fluoroGuanosine]-(3',5')-[3'-0- (Allyl,CE)phosphotriester-N2(iB)-2'-deoxy-2'- fluoroGuanosine]
Figure imgf000038_0004
Intermediate 46 was prepared from intermediate 45 and commercially available phosphoramidite of 2'-deoxy-2'- fluoroGuanosine using a similar procedure to that described in Example 1.C to provide 1 .07 g (67% yield) of intermediate 46. Rt = 4.80 min, m/z = 999 [M+H]+, m/z = 997 [M-H]". Intermediate 47: [3'-0-(CE)phosphotriester-N2(iB)-2'-deoxy- 2'fluoroGuanosine]-(3',5')-[3'-0- (CE)phosphodiester-N2(iB)-2'-deoxy-2 - fluoroGuanosine]
Figure imgf000039_0001
Intermediate 47 was prepared from intermediate 46 using a similar procedure to that described in Example 1.F to provide 610 mg (99% yield) of intermediate 47. LC-MS: Rt = 4.05 min, m/z = 959 [M+H]+, m/z = 957 [M-H]".
Intermediate 48: Cyclic-(3',3')-[3'-0-(CE)phosphotriester- N2(iB)-2'-deoxy-2'-fluoroGuanosine]-[3'-0- (CE)phosphotriester-N2(iB)-2'-deoxy-2'- fluoroGuanosine]
Figure imgf000039_0002
Intermediate 48 was prepared from intermediate 47 using a similar procedure to that described in Example 1.H to provide 580 mg (96% yield) of intermediate 48. LC-MS: Rt = 4.77 min, m/z = 941 [M+Hf, m/z = 939 [M-H]".
Intermediate 49: [3'-0-(CE)phosphotriester-Ne(Bz)2'-deoxy- 2'fluoroAdenosine]-(3',5')-[3'-0- (Allyl,CE)phosphotriester-N2(iB)-2'-deoxy-2'- fluoroGuanosine]
Figure imgf000039_0003
Intermediate 49 was prepared from intermediate 45 and commercially available phosphoramidite of 2'-deoxy-2'- fluoroAdenosine using a similar procedure to that described in Example 1.C to provide 4.96 g (68% yield) of intermediate 49. Rt = 4.98 min, m/z = 1017 [M+H]+, m/z = 1015 [M-H]".
Intermediate 50: [3'-0-(CE)phosphotriester-NB(Bz)2'-deoxy- 2'fluoroAdenosine]-(3',5')-[3'-0- (CE)phosphodiester-N2(iB)-2'-deoxy-2 - fluoroGuanosine]
Figure imgf000039_0004
Intermediate 50 was prepared from intermediate 47 using a similar procedure to that described in Example 1.E to provide 4.4 g (92% yield) of intermediate 50. LC-MS: Rt = 4.36 min, m/z = 977 [M+H]+, m/z = 975 [M-H]".
Intermediate 51: Cyclic-(3',3')-[3'-0-(CE)phosphotriester- N6(Bz)2'-deoxy-2'fluoroAdenosine]-[3'-0- (CE)phosphotriester-N2(iB)-2'-deoxy-2'- fluoroGuanosine]
Figure imgf000040_0001
Intermediate 51 was prepared from intermediate 50 using a similar procedure to that described in Example 1.G to provide 4.30 g (99% yield) of intermediate 51. LC-MS: Rt = 5.72 min, m/z = 838 [M+H]+, m/z = 836 [M-H]".
Intermediate 52: 3'-0-(Allyl,CE)phosphorothioate-triester-Nii(iB)- 2'-deoxy-2'-fluoroGuanosine
Figure imgf000040_0002
Intermediate 52 was prepared from the commercially available phosphoramidite of 2'-deoxy-2'-fluoroGuanosine using a similar procedure to that described in Example 1.A to provide 632 mg (50% yield) of intermediate 52. LC-MS: Rt = 5.86 min, m/z = 545 [M+Hf, m/z = 543 [M-H]".
Intermediate 53: [3'-0-(CE)phosphorothioate-triester-NB(Bz)2'- deoxy-2'fluoroAdenosine]-(3',5')-[3'-0- (Allyl,CE)phosphorothioate-triester-N2(iB)-2'- deoxy-2'-fluoroGuanosine]
Figure imgf000040_0003
Intermediate 53 was prepared from intermediate 52 and commercially available phosphoramidite of 2'-deoxy-2'- fluoroAdenosine using a similar procedure to that described in Example 1.C to provide 310 mg (25% yield) of intermediate 53. Rt = 6.60 min, m/z = 1049 [M+H]+, m/z = 1047 [M-H]". Intermediate 54: [3'-0-(CE)phosphorothioate-triester-Ne(Bz)2'- deoxy-2'fluoroAdenosine]-(3',5')-[3'-0- (CE)phosphorothioate-diester-N2(iB)-2'-deoxy- 2'-fluoroGuanosine]
Figure imgf000041_0001
Intermediate 54 was prepared from intermediate 53 using a similar procedure to that described in Example 1.E to provide 100 mg (33% yield) of intermediate 54. LC-MS:Rt = 4.58 and 4.70 min, m/z = 1009 [M+Hf, m/z = 1007 [M-H]\
Intermediate 55: Cyclic-(3',3')-[3'-0-(CE)phosphorothioate- triester-N6(Bz)2'-deoxy-2'fluoroAdenosine]-[3'- 0-(CE)phosphorothioate-triester-N2(iB)-2'- deoxy-2'-fluoroGuanosine]
Figure imgf000041_0002
Intermediate 55 was prepared from intermediate 54 using a similar procedure to that described in Example 1.G to provide 75 mg (76% yield) of intermediate 55. LC-MS: Rt = 5.32 and 5.54 min, m/z = 991 [M+H]+, m/z = 989 [M-H]".
Intermediate 56: lodomethyl pivalate
Figure imgf000041_0003
A solution of chloromethyl pivalate (1.0 g, 6.64 mmol) in dry ACN (15 mL) was treated with sodium iodide (1.9 g, 13.28 mmol). The mixture was stirred at RT overnight in the dark. Then the solvent was removed in vacuo and the residue was dissolved in DCM. The solution was washed with water, 5% NaHS03 solution and brine. The organic layer was dried over MgS04, filtered and concentrated in vacuo to provide 1.22 g of crude intermediate 53 which was used for the next step without any further purification. 1H NMR (CDCI3-di, 300 MHz) δ (ppm) 5.86 (s, 2H), 1.12 (s, 9H).
Example 1.1 : c-AIMP
Figure imgf000041_0004
Example 1.1 was prepared from intermediate 3 using a similar procedure to that described in Example 1.J to provide 2.4 g (60% yield) of example 1.1. LC-MS: Rt = 2.72 min, m/z = 660 [M+H]+, m/z = 658 [M-H]". 1H NMR (D20, 300 MHz) δ (ppm) 8.34 (s, 1 H), 8.27 (s, 1 H), 8.10 (s, 1 H), 7.86 (s, 1 H), 5.94 (s, 2H), 5.06^.80 (m, 4H), 4.42 (m, 4H), 4.03 (m, 2H).
Figure imgf000042_0001
Example 1.2 was prepared from intermediate 13 using a similar procedure to that described in Example 1 J to provide 22.5 mg (21% yield) of example 1.2. LC-MS: Rt = 2.46 min, m/z = 660 [M+Hf, m/z = 658 [M-H]".1H NMR (D20, 300 MHz) δ (ppm) 8.27 (s, 1H), 8.15 (s, 1H), 8.04 (s, 1H), 7.88 (s, 1H), 5.99 (s, 2H), 4.95 (m, 2H), 4.86 (m, 1H), 4.42 (m, 4H), 4.05 (m,2H).
Example 1.3: -AIMP
Figure imgf000042_0002
Example 1.3 was prepared from intermediate 17 using a similar procedure to that described in Example 1.J to provide 17.5 mg (22% yield) of example 1.3. LC-MS: Rt = 1.35 min, m/z = 660 [M+H]+, m/z = 658 [M-H]".1H NMR (D20, 300 MHz) δ (ppm) 8.44 (s, 1H), 8.13 (s, 1H), 8.08 (s, 1H), 8.07 (s, 1H), 6.20 (d, 1H), 6.07 (d, 1H), 5.22 (m, 1H), 4.91 (m, 1H), 4.79 (m, 2H), 4.63 (m, 1H), 4.56 (m, 1H), 4.22 (m, 2H), 4.07 (m, 2H).
Example 1.4: -AIMP
Figure imgf000042_0003
Example 1.4 was prepared from intermediate 20 using a similar procedure to that described in Example 1 J to provide 50 mg (19% yield) of example 1.4. LC-MS: Rt = 1.53 min, m/z = 660 [M+H]+, m/z = 658 [M-H]".1H NMR (D20, 300 MHz) δ (ppm) 8.50 (s, 1H), 8.14 (s, 1H), 8.13 (s, 1H), 8.09 (s, 1H), 6.24 (d, 1H), 6.08 (s, 1H), 5.20 (m, 1H), 4.83-4.73 (m, 2H), 4.58 (d, 1H), 4.40 (m, 2H), 4.26 (m, 2H), 4.04 (m, 2H).
Example 1.5: (S)
Figure imgf000042_0004
Example 1.5 was prepared from intermediate 22 using a similar procedure to that described in Example 1 J to provide 7mg (20% yield) of example 1.5. LC-MS: Rt = 3.45 min, m/z = 692 [M+Hf, m/z = 690 [M-H]".1H NMR (D20, 300 MHz) δ (ppm) 8.54 (s, 1H), 8.37 (s, 1H), 8.22 (s, 1H), 7.97 (s, 1H), 6.65 (dd, 1H), 6.15 (dd, 1H), 4.60^.50 (m, 4H), 4.45 (m, 4H), 4.05 (m,2H). Example 1.6: PdIMP
Figure imgf000043_0001
Example 1.6 was prepared from intermediate 26 using a similar procedure to that described in Example 1.K to provide 90 mg (31 % yield) of Example 1 .6. LC-MS: Rt = 2.47 min, m/z = 628 [M+H]+, m/z = 626 [M-H]". 1H NMR (DMSO-c/6, 300 MHz) δ (ppm) 8.37 (s, 1 H), 8.32 (s, 1 H), 8.13 (s, 1 H), 8.05 (s, 1 H), 7.26 (si, 2H), 6.28 (m, 2H), 4.68 (m, 2H), 4.08 (m, 2H), 3.85 (m, 2H), 2.83 (m, 2H).
Example 1.7: P-2'FdlMP]
Figure imgf000043_0002
Example 1.7 was prepared from intermediate 29 using a similar procedure to that described in Example 1.K to provide 40 mg (21 % yield) of Example 1 .7. LC-MS: Rt = 1.97 min, m/z = 646 [M+Hf, m/z = 644 [M-H]". 1H NMR (D20, 300 MHz) δ (ppm) 8.29 (s, 1 H), 8.12 (s, 1 H), 7.98 (s, 1 H), 7.88 (s, 1 H), 6.25 (m, 2H), 5.54 (m, 1 H), 5.07 (m, 2H), 4.37 (m, 4H), 4.06 (m, 2H).
Example 1.8: AMP)-(2'FdlMP)
Figure imgf000043_0003
Example 1.8 was prepared from intermediate 33 using a similar procedure to that described in Example 1.K to provide 104 mg (10% yield) of Example 1.8. LC-MS: Rt = 2.78 min, m/z = 664 [M+Hf, m/z = 662 [M-H]". 1H NMR (D20, 300 MHz) δ (ppm) 8.37 (s, 1 H), 8.35 (s, 1 H), 8.14 (s, 1 H), 7.95 (s, 1 H), 6.23 (m, 2H), 5.45 (m, 2H), 5.39 (m, 1 H), 4.95 (m, 2H), 4.50 (m, 2H), 4.06 (m, 2H). dAMP(S)-2'FdlMP(S)]
Figure imgf000043_0004
Example 1.9 was prepared from intermediate 37 using a similar procedure to that described in Example 1.K to provide 10 mg (18% yield) of Example 1 .9. LC-MS: Rt = 3.41 min, m/z = 696 [M+Hf, m/z = 694 [M-H]". 1H NMR (D20, 300 MHz) δ (ppm) 8.55 (s, 1 H), 8.38 (s, 1 H), 8.20 (s, 1 H), 7.99 (s, 1 H), 6.63 (dd, 1 H), 6.15 (dd, 1 H), 5.16^.95 (m, 4H), 4.52 (m, 4H), 4.07 (m, 2H).
Example 1.10: -(AMP-2'FdlMP)
Figure imgf000044_0001
Example 1.10 was prepared from intermediate 40 using a similar procedure to that described in Example 1.J to provide 50 mg (19% yield) of Example 1 .10. LC-MS: Rt = 2.25 min, m/z = 662 [M+H]+, m/z = 660 [M-H]". 1H NMR (D20, 300 MHz) δ (ppm) 8.58 (s, 1 H), 8.35 (s, 1 H), 8.12 (s, 1 H), 7.97 (s, 1 H), 6.65 (d, 1 H), 6.16 (d, 1 H), 4.50 (m, 2H), 5.15 (m, 2H), 4.41 (m, 4H), 4.02 (m, 1 H), 3.73 (m, 1 H).
Example 1.11 : dlMP)
Figure imgf000044_0002
Example 1.1 1 was prepared from intermediate 44 using a similar procedure to that described in Example 1.K to provide 157 mg (35% yield) of Example 1.1 1. LC-MS: Rt = 1 .48 min, m/z = 665 [M+Hf, m/z = 663 [M-H]". 1H NMR (D20, 300 MHz) δ (ppm) 8.34 (s, 2H), 8.08 (s, 2H), 7.85 (s, 2H), 6.21 (m, 2H), 5.66 (m, 1 H), 5.49 (m, 1 H), 5.20 (m, 2H), 4.97 (m, 4H), 4.06 (m, 2H).
Example 1.12: dGMP)
Figure imgf000044_0003
Example 1.12 was prepared from intermediate 48 using a similar procedure to that described in Example 1.K to provide 15 mg (5% yield) of Example 1 .12. LC-MS: Rt = 1.58 min, m/z = 695 [M+Hf, m/z = 693 [M-H]". 1H NMR (D20, 300 MHz) δ (ppm) 8.70 (s, 2H), 6.05 (d, 2H), 5.06^.05 (m, 8H), 4.08 (m, 2H).
Example 1.13: GMP-2'FdAMP)
Figure imgf000044_0004
Example 1.13 was prepared from intermediate 51 using a similar procedure to that described in Example 1.K to provide 320 mg (31 % yield) of Example 1.13. LC-MS: Rt = 2.48 min, m/z = 679 [M+Hf, m/z = 677 [M-H]". 1H NMR (D20, 300 MHz) δ (ppm) 8.17 (s, 1 H), 7.83 (s, 1 H), 7.63 (s, 1 H), 6.09 (d, 1 H), 5.94 (d, 1 H), 5.62 (m, 1 H), 5.50 (m, 1 H), 5.15 (m, 2H), 4.42 (m, 4H), 4.03 (m, 2H).
Example 1.14: GMP(S)-2'FdAMP(S)]
Figure imgf000045_0001
Example 1.14 was prepared from intermediate 55 using a similar procedure to that described in Example 1.K to provide 15 mg (28% yield) of Example 1 .14. LC-MS: Rt = 2.66 min, m/z = 711 [M+H]+, m/z = 709 [M-H]". 1H NMR (D20, 300 MHz) δ (ppm) 8.58 (s, 1 H), 8.35 (s, 1 H), 7.97 (s, 1 H), 6.65 (d, 1 H), 6.19 (d, 1 H), 4.94 (d, 1 H), 4.62^.50 (m, 4H), 4.42 (m, 4H), 4.03 (m, 2H).
Example 1.15: GMP(S)-2'FdAMP(S)](POM)2
Figure imgf000045_0002
To a solution of Example 1.14 (7 mg, 9.85 μιηοΙ) in water (200 μΙ_) was added dropwise a solution of intermediate 56 (19 mg, 79.0 μιηοΙ) in acetone (500 μΙ_). The mixture was stirred overnight in the dark. Then the mixture was neutralized with a saturated solution of Na2S203 (15 μΙ_) and subsequently diluted with water (10 mL). The aqueous layer was extracted three times with EtOAc (3 x 10mL). The organic layers were pooled, dried over MgS04, filtered and concentrated in vacuo to provide 4 mg (45% Yield) of Example 1 .15. LC-MS: Rt = 4.1 1 min, m/z = 907 [M+H]+, m/z = 905 [M-H]". 1H NMR (DMSO-d6, 300 MHz) δ (ppm) 8.61 (s, 1 H), 8.37 (s, 1 H), 8.00 (s, 1 H), 6.70 (d, 1 H), 6.22 (d, 1 H), 4.70-4.50 (m, 4H), 4.42 (m, 4H), 4.03 (m, 2H), 3.77 (s, 4H), 1.20 (s, 18H).
Example 1.16: lMP(S)-2'FdAMP(S)](POM)2
Figure imgf000045_0003
To a solution of Example 1.9 (5 mg, 7.19 μιηοΙ) in water (200 μί) was added dropwise a solution of intermediate 56 (17 mg, 70 μιηοΙ) in acetone (500 μί). The mixture was stirred overnight in the dark. Then the mixture was neutralized with a saturated solution of Na2S203 (15 μί) and subsequently diluted with water (10 mL). The aqueous layer was extracted three times with EtOAc (3 x 10mL). The organic layers were pooled, dried over MgS04, filtered and concentrated in vacuo to provide 5 mg (78% Yield) of Example 1.16. LC-MS: Rt = 4.55 min, m/z = 892 [M+H]+, m/z = 890 [M-H]". 1H NMR (DMSO-d6, 300 MHz) δ (ppm) 8.61 (s, 1 H), 8.37 (s, 1 H), 8.00 (s, 1 H), 6.70 (d, 1 H), 6.22 (d, 1 H), 4.70^.50 (m, 4H), 4.42 (m, 4H), 4.03 (m, 2H), 3.77 (s, 4H), 1.20 (s, 18H).
EXAMPLE 2: Biological assays We have determined that several cyclic d inucleotides of the present invention induce the production of multiple cytokines in human or animal cells. Specifically, these cyclic dinucleotides induce the production of Type I interferons and/or pro-inflammatory cytokines. The in vitro cytokine-induction activity of a representative set of these cyclic dinucleotides is reported here to require the presence of the eukaryotic cellular receptor stimulator of interferon genes (STING).
In vitro cytokine induction
The cytokine-induction activities of non-fluorinated vs. fluorinated cyclic dinucleotides disclosed in this invention have been demonstrated by using different reporter cell lines. The cell lines and experiments are explained below.
Cell lines
All the cell lines were obtained from InvivoGen. They are described here and provided with their corresponding InvivoGen catalog code.
THP1 -Dual™ (InvivoGen catalog code: thpd-nfis): These cells were derived from the human monocytic cell line THP- 1 by stable integration of two inducible reporter constructs. They enable simultaneous study of the two main signaling pathways for STING: the NF-κΒ pathway, by monitoring the activity of secreted embryonic alkaline phosphatase (SEAP); and the IRF pathway, by assessing the activity of a secreted luciferase (Lucia).
Both reporter proteins are readily measurable in the cell culture supernatant when using QUANTI-Blue™ (InvivoGen catalog code: rep-qb1 ), a SEAP detection reagent that turns purple/blue in the presence of SEAP (quantified by measuring the optical density from 620 nm to 655 nm), and QUANTI-Luc™ (InvivoGen; catalog code: rep-qld ), a luminometric enzyme assay that measures luciferase expression to report on ISG54 expression (as an indicator of IFN-α/β production).
Lucia ISG cell lines: Each of the following two cell lines expresses a secreted luciferase (Lucia) reporter gene under control of an IRF-inducible promoter. This composite promoter comprises five IFN-stimulated response elements (ISREs) fused to a m inimal promoter of the human ISG54 gene, which is unresponsive to activators of the NF-κΒ or AP-1 pathways. Hence, these cells enable monitoring of the IRF pathway based on luciferase (Lucia) activity. In the present invention, monitoring of the IRF pathway is used to measure the STING agonist activity of the subject cyclic dinucleotides.
1 . RAW-Lucia™ ISG (InvivoGen catalog code: rawl-isg): These cells were generated from the murine RAW 264.7 macrophage cell line.
2. RAW-Lucia™ ISG-KO-STING (InvivoGen catalog code: rawl-kostg): These cells were generated from the RAW-Lucia™ ISG54 cell line (see above), through stable homozygous knockout of the STING gene.
B16 Blue™ ISG54 cell lines: Each of the following two cell lines expresses a SEAP reporter gene under a promoter: the I-ISG54 reporter, which comprises the IFN-inducible ISG54 promoter enhanced by a multimeric ISRE. Stimulation of these cells with interferons, or inducers of type I interferons or of the NF-κΒ pathway, triggers activation of the I- ISG54 promoter (and consequently, production of SEAP) or of the IFN-β minimal promoter (and consequently, production of TNF-a). The levels of SEAP in the supernatant can be easily determined using QUANTI- Blue™ (InvivoGen catalog code: rep-qb1 ), a reagent that turns purple/blue in the presence of SEAP, by measuring the optical density from 620 nm to 655 nm. 1 . B16-Blue™ ISG (InvivoGen catalog code: bb-ifnabg): These cells are derived from the murine B16 F1 melanoma cell line. Production of Type I interferons in these cells is measured using QUANTI-Blue™.
2. B16-Blue™ ISG-KO-STING (InvivoGen catalog code: bb-kostg): These cells were generated from the B16- Blue™ ISG cell line (see above), through stable homozygous knockout of the STING gene. Production of Type I interferons in these cells is measured using QUANTI-Blue™.
HEK-Blue™ cell lines: The following three cell lines also were used for biological evaluation of the CDNs.
1 . HEK-Blue IFN-a/p-KO-STING: These cells are derived from HEK-Blue IFN-α/β cells (InvivoGen catalog code: hkb-ifnab), in which the STING gene has been inactivated. HEK-Blue™ IFN-α/β cells enable detection of bioactive human type I IFNs through monitoring of activation of the ISGF3 pathway. These cells were generated by stable transfection of HEK293 cells with the human STAT2 and IRF9 genes to obtain a fully active type I IFN signaling pathway. The other genes of the pathway (IFNAR1 , IFNAR2, JAK1 , TyK2 and STAT1 ) are naturally expressed in sufficient amounts. The cells were further transfected with a SEAP reporter gene under control of an IFN-a/p-inducible ISG54 promoter. Stimulation of HEK-Blue™ IFN-α/β cells with human IFN-a or IFN-β activates the JAK/STAT/ISGF3 pathway and subsequently induces production of SEAP. Production of Type I interferons in these cells is measured using QUANTI-Blue™.
2. HEK-Blue™ IL-1 R (InvivoGen catalog code: hkb-il1 r): HEK-Blue™ IL-1 R cells were designed to detect bioactive human and murine IL- β through monitoring of activation of the NF-κΒ and AP-1 pathways.
Additionally, these cells detect bioactive IL-1 β from cynomolgus monkeys, dogs and rats. In fact, HEK-Blue™ IL-1 R cells can detect IL-1a and IL-1 β, as these cytokines bind to the same receptor, IL-1 R. These cells derive from HEK-Blue™ IL-1 β cells (InvivoGen catalog code: hkb-il1 b), in which the TNF-a response is blocked. Therefore, HEK-Blue™ IL-1 R cells respond specifically to IL-1 . These cells endogenously express the human IL-1 receptor and were stably transfected with the murine IL-1 receptor, rendering them very sensitive to both human and murine IL-1 β. HEK-Blue™ IL-1 R cells express a SEAP reporter gene under control of an IFN-β minimal promoter fused to five NF-κΒ and five AP-1 binding sites. Binding of IL-1 β to IL- 1 R on the surface of HEK-Blue™ IL-1 R cells triggers a signaling cascade that leads to the activation of NF- KB and subsequent production of SEAP. Production of IL-1 β in these cells is measured using QUANTI- Blue™.
3. HEK-Blue TNF-a (InvivoGen catalog code: hkb-tnfdmyd): HEK-Blue™ TNF-a cells enable detection of bioactive human and murine TNF-a through monitoring of activation of the NF-κΒ pathway. These cells were generated by stable transfection of HEK293 cells with a SEAP reporter gene under control of an IFN-β minimal promoter fused to five NF-κΒ and five AP-1 binding sites. They were further rendered unresponsive to IL-1 β by knocking out the MyD88 gene. Stimulation of HEK-Blue™ TNF-a cells with TNF-a triggers activation of the NF-KB-inducible promoter and production of SEAP. Production of TNF-a in these cells is measured using QUANTI-Blue™. Quantification of IL-6 in experiments
lnterleukin-6 was quantified using an enzyme-linked immunoassay (ELISA) according to the manufacturer's instructions (R&D Systems).
In cell cultures
In various experiments in which different cell cultures were separately incubated with a cyclic dinucleotide, the cyclic dinucleotide induced production of Type I interferons and/or pro-inflammatory cytokines in those cells, as indirectly determined by an ISG54 (interferon-stimulated gene) reporter assay (Fensterl, White, Yamashita, & Sen, 2008). These experiments were performed as described below.
Example 2.1 : Measuring cytokine induction in treated cell cultures
• Cytokine reporter cell lines used: THP1 -Dual
· Cyclic dinucleotides tested: CL609, CL614, CL656, CL647, CL629, CL626, CL603, CL632 and CL633
• Reference compound: c-AIMP (manufactured by InvivoGen), c-diGMP (InvivoGen catalog code: tlrl-cdg), c- dilMP (InvivoGen catalog code: tlrl-cdi) and c-GAMP (InvivoGen catalog code: tlrl-cga)
• Activities evaluated: Type I IFN induction and NF-κΒ pathway induction. To each well of a flat-bottom 96-well plate were added 20 μί_ of a solution a cyclic dinucleotide (100 μg mL in sterile water), followed by 180 μί_ of a suspension of a single cell line (THP1 -Dual™: ca. 100,000 cells per well). The plate was incubated for 18 h to 24 h at 37 °C in 5% C02. The level of IFN-α/β was indirectly quantified using QUANTI- Luc™, which was prepared and used according to the manufacturer's instructions. The NF-κΒ activity was indirectly quantified using QUANTI-Blue™, which was prepared and used according to the manufacturer's instructions.
The results from this experiment are shown in Figures 2 and 3, which reveal three important findings: firstly, that all of the tested cyclic dinucleotides induce Type I interferons in THP1 -Dual™ cells (Fig. 2); secondly, that all of them induce the NF-κΒ pathway in these cells (Fig. 3); and lastly, that for both of these activities the majority of the fluorinated cyclic dinucleotides are more active than are the corresponding reference compounds (c-AIMP, c-diGMP, c-diIMP and c-GAMP).
Cytokine induction activity is STING-dependent
The cyclic dinucleotides disclosed in the present invention do not induce cytokine production in vitro in the supernatant of cells that lack the receptor STING.
In an experiment in which wild-type (WT) reporter cells and homozygous STING knockout (STING KO) reporter cells were each separately incubated with the cyclic dinucleotide for 18 h to 24 h, the cyclic dinucleotide induced production of Type I interferons in the WT cells but not in the STING KO cells. This finding demonstrated that STING is required for the cytokine-induction activity of the cyclic dinucleotide in vitro in cells. These experiments were performed as described below:
Example 2.2: Measuring cytokine induction in CDN-treated wild-type or STING knockout cells Cytokine reporter cell lines used: B16-Blue ISG and RAW-Lucia ISG
Cyclic dinucleotides tested: CL609, CL614, CL656, CL647, CL629, CL626, CL603, CL632 and CL633 Reference compound c-AIMP (manufactured by InvivoGen), c-diGMP (InvivoGen catalog code: tlrl-cdg), c-diIMP (InvivoGen catalog code: tlrl-cdi) and c-GAMP (InvivoGen catalog code: tlrl-cga)
Cell lines used: RAW-LuciaI M ISG, RAW-Lucia ' M ISG-KO-STING, B16-BlueI M ISG, and B16-BlueI M ISG- KO-STING (depending on experiment).
These Experiments were performed as described in Example 2.1. The results from this experiment are shown in Figures 4 and 5, which reveal three important findings. Firstly, each one of the tested cyclic dinucleotides induces production of Type I interferons in WT B16 (Fig. 4) and WT RAW (Fig. 5) cells. Secondly, none of the compounds exhibits this activity in STING knockout B16 (Fig. 4) or STING knockout RAW (Fig. 5) cells, thereby indicating that this activity requires the presence of STING. Lastly, the majority of the fluorinated cyclic dinucleotides are more active than are the corresponding reference compounds (c-AIMP, c-diGMP, c-diIMP and c-GAMP).
In vivo cytokine induction
The cyclic dinucleotides disclosed in the present invention induce cytokines in vivo in mice. Example 2.3: Measuring cytokine induction in CDN-treated mice
Species evaluated: mouse
Cyclic dinucleotides tested: CL604, CL606, CL609, CL61 1 and CL614
Reference compound: c-AIMP and saline
Cytokines evaluated: IFN-α/β (using RAW ISG54 reporter cells) and IL-6 (by ELISA).
Twenty-one mice (Swiss; female; mean age: 8 weeks) were divided into seven groups of three: one group served as control (saline) and the other six groups were each treated with a cyclic dinucleotide (either c-AIMP, CL604, CL606, CL609, CL61 1 or CL614). On Day -7, blood samples for basal cytokine levels were collected from all mice and stored at -20 °C until analysis. On Day 1 , the mice were treated with either 200 μί of physiologic serum (containing 0.9% NaCI) or 200 μί of a solution of a cyclic dinucleotide (dose: 10 mg/kg) in physiologic serum (containing 0.9% NaCI), by intravenous (i.v.) injection. Blood samples were collected from the mice at 4 h post-injection, and then stored at -20 °C until analysis. Cytokine induction was measured in the sera from the blood samples.
The results from this experiment are shown in Figures 6 and 7, which reveal two important findings: firstly, at the indicated dose, within 4 h post-treatment, all of the tested cyclic dinucleotides except CL61 1 strongly induced Type I interferons (Fig. 6) in mice; and secondly, all of the cyclic dinucleotides except CL61 1 induced IL-6 (Fig. 7).
Example 2.4: Measuring in vivo elimination of CDNs in mice
We have measured the in vivo elim ination of representative cyclic dinucleotides of the present invention in mice.
Cyclic dinucleotides tested: CL603, CL609, CL614, CL626 and CL656 Reference compounds: c-AIMP (manufactured by InvivoGen).
Thirty m ice (C57BL/6) were divided into six groups of five. Within each group, each mouse was treated with a different cyclic dinucleotide (50 mg/kg; i.v. bolus). Each group of mice was sacrificed at a different time point post-treatment: either 2 min, 5 min, 15 min, 30 min or 1 h. Just before sacrifice, a sample of blood (500 μί) was collected. The blood samples were collected in heparin tubes, and then centrifuged. The supernatant (plasma) was stored at - 20 °C until analysis. Before analysis by high performance liquid chromatography-mass spectrometry (HLPC/MS), the plasma samples were processed as follows:
Plasma: each sample was treated with methanol at a ratio of 1 :4, shaken and filtered (0.22 μιη). One μί_ of the processed sample was injected onto the HPLC/MS column. The following HPLC gradient was used (A: 10 mM ammonium formate; B: acetanitrile; total time: 6 min): 100% A for 1 min; followed by 100% A to 100% B in 4 min; followed by 100% B for 1 min. Each cyclic dinucleotide eluted at a different time and was detected by measuring the absorbance at 254 nm.
The results of this experiment are shown in Figure 8, which reveals that the fluorinated cyclic dinucleotides were retained in the blood of mice longer than were the non-fluorinated cyclic dinucleotides. Example 2.5: Comparison of fluorinated and non-fluorinated CDNs for their ability to induce cytokines in vitro in whole blood from healthy human donors
• Reporter cell lines used: HEK-Blue™ IFN-a/p-KO-STING, HEK-Blue™ IL-1 R and HEK-Blue™ TNF- α
• Subject CDNs tested: CL603, CL632, CL614 and CL656
· Reference (non-fluorinated) CDNs tested: c-GAMP, 2',3'-c-GAMP,c-AIMP and c-AIMP(S)
• Activities evaluated: Type I IFN induction (HEK-Blue™ IFN-a/p-KO-STING), IL-1 induction (HEK- Blue IL-1 R) and TNF-a induction (HEK-Blue TNF-a).
Acquisition and handling of human blood samples
Twenty human blood samples were acquired from heathy donors at the San Diego Blood Bank (3636 Gateway Center Ave, Suite 100; San Diego, CA 92102; USA; www.sandiegobloodbank.org). Briefly, the samples were collected by venipuncture into sodium heparin (green-cap) tubes at the time of donation, and then stored at 4 °C until pick-up. The tubes were picked up on the day of collection, stored on ice during transport, and subsequently tested with the CDNs on the same day.
Treatment and testing of human blood samples Each blood sample was diluted (1 :1 ) in RPMI medium and aliguoted into 96-well plates (180-μΙ. wells) containing each CDN at six different concentrations (30 μg mL, 10 μg mL, 3 μg mL, 1 μg mL, 0.3 μg mL and 0.1 μg mL). The plates were incubated at 37 °C in a C02 incubator for 18 to 20 hours. The next day the supernatants were collected, transferred into the corresponding wells of round-bottom 96-well plates, and stored at -80 °C. On the following day, a new 96-well plate was prepared for each of the three reporter cell lines tested, as follows: 10 μί of supernatant from the previous plate (containing the incubated CDNs and plasma) were added to the corresponding well in the new reporter cell plate. Then, a 180-μΙ. aliquot of cells of the desired reporter cell line, previously harvested in medium containing heat-inactivated serum and counted, was added to each well (approximately 50,000 cells/well), and the plate was incubated for approximately 20 hours. The desired cytokine induction activity was determined using the QUANTI-Blue™ Assay, as previously described. Briefly, 20 μL of supernatant from the previously incubated plate was transferred to the corresponding well of a new 96-well plate in which 180 μL of QUANTI-Blue™ reagent had previously been added.
The results from this experiment are summarized in Figure 9, which illustrates three cytokine induction activities (Type I IFN, IL-1 and TNF-a) for representative fluorinated CDNs of the present invention and for related reference CDNs. The values shown are total averages for all 20 donors over the full concentration range tested (30 μg mL, 10 μg mL, 3 μg mL, 1 μg mL, 0.3 μg mL, 0.1 μg mL and 0.03 μg mL). The activity of the CDNs is expressed in terms of "active concentration" (defined here as the CDN giving a SEAP intensity value of at least 0.5 at the indicated concentration), from the lowest of the seven tested concentrations (0.03 μg mL) up to the highest tested concentration (30 μg mL). The Figure reveals two principal findings: firstly, that with in the tested concentration range, each one of the CDNs tested induces each one of the cytokines assessed; and secondly, that for each cytokine induction activity, each fluorinated CDN is more active than is its corresponding non-fluorinated analog (compare CL603 with c-GAMP; CL614 with c-AIMP; and CL656 with c-AIMP[s]).
Example 2.6: Comparison of fluorinated deoxyribo-CDNs and non-fluorinated ribo-CDNs for their resistance to enzymatic cleavage by SVPD or NP1 , as monitored by UHPLC-MS
• Enzymes used: snake-venom phosphodiesterase (SVPD) and nuclease P1 (NP1 )
• Subject CDNs tested: CL603, CL632, CL614 and CL656
• Reference (non-fluorinated) CDNs tested: c-GAMP and c-AIMP
• Property evaluated: resistance to enzymatic cleavage
We incubated each CDN with either of two enzymes known to cleave nucleic acids and CDNs, and then looked for signs of degradation by UHPLC over time, by measuring the decrease in the area of the peak corresponding to the CDN (identified according to MS). Specifically, we assessed the resistance of the CDNs to snake-venom phosphodiesterase (SVPD) and nuclease P1 (NP1 ), both of which can degrade CDNs by cleaving the phosphodiester nucleotide linkages. The experiment was performed as follows:
Each CDN (7 μg) was separately incubated with a solution (21 μί) of either enzyme (either 160 μg SVPD in PBS buffer containing 0.6 mM MgCI2; or 2.5 mU NP1 in 30 mM acetate buffer containing 2 mM ZnCI2 [pH 5.3]), or with water (as control) in a water bath at 37 °C. Aliquots of the reaction mixture were collected at various time points from 0 to 120 hours, heated at 100 °C for 2 min, and then frozen at 0 °C. Finally, 10 μL of each aliquot was injected directly into the HPLC (Agilent 1290 Infinity UHPLC equipped with a UV-detector; column: Waters Acquity UPLC CSH CiS 1 .7 μΐη [2.1 mm x 50 mm; flow rate: 0.3 mL/min]; detection at 254 nm; autosampler temperature: 25 °C) for analysis. The following gradient was used: 100% A (10 mM aq. ammonium formate) for 1 minute; then 100% A to 100% B (acetonitrile) over 5 minutes. The percentage of absorbance of CDNs at each time point was calculated by dividing the peak area corresponding to the parent CDN, by the sum of all the peak areas in the chromatogram, and then multiplying by 100.
The results from this experiment are shown in Figures 10 A-D, which reveal two important findings: firstly, that the fluorinated deoxyribo-CDNs of the present invention are more resistant to enzymatic degradation by either enzyme than are their corresponding non-fluorinated ribo-CDN analogs (in Figures 10A and 10B compare CL656 or CL614 to c-AIMP, and in Figures 10C and 10D compare CL603 or CL632 to c-GAMP); secondly; that among the fluorinated deoxyribo-CDNs of the present invention, those containing two phosphorothioate diester linkages (CL656 and CL632) are more resistant to SVPD than are their corresponding analogs containing two phosphodiester linkages (CL614 and CL603, respectively). Example 2.7: Comparison of the in vitro activity of fluorinated deoxyribo-CDNs and non-fluorinated ribo-CDNs before and after exposure of these CDNs to SVPD or NP1
• Reporter cell lines used: THP1 -Dual™
• Subject CDNs tested: CL603, CL632, CL614 and CL656
• Reference (non-fluorinated) CDNs tested: c-GAMP and c-AIMP
· Activities evaluated: Type I IFN induction
In this experiment four representative fluorinated deoxyribo-CDNs of the present invention (CL603, CL632, CL614 and CL656) and their corresponding non-fluorinated ribo-CDN analogs (c-GAMP and c-AIMP, respectively) were compared for their relative stability to cleavage by two known CDN-cleaving enzymes: snake-venom phosphodiesterase (SVPD) and nuclease P1 (NP1 ). It was performed as described below. Each CDN (7 μg) was incubated with SVPD, NP1 or water (as control), as described in Example 2.6, in a water bath at 37 °C for 2 hours, and then for 10 min at 100 °C. The resulting solutions were cooled down to room temperature. An aliquot (20 μί) of each solution was then separately incubated with THP1 -Dual™ cells (180 μΙ_; concentration: 100,000 cells/well), as described in Example 2.1. ISG54 expression (as an indicator of IFN-α/β production) was quantified with QUANTI-Luc™, as explained above. The results from this experiment are shown in Figure 11 , which reveals two important findings: firstly, before incubation with either enzyme, or after incubation with water alone (control), all of the CDNs induced ISG54 expression in the cells; and secondly, after incubation with either enzyme, the solutions corresponding to the non- fluorinated ribo-CDNs completely lost this activity, whereas those corresponding to the fluorinated deoxyribo-CDNs greatly retained this activity. These findings indicates that the fluorinated deoxyribo-CDNs are more resistant to enzymatic cleavage by SVPD or NP1 than are their corresponding non-fluo nated ribo-analogs.
Class effects of fluorinated deoxyribo-cyclic dinucleotides
We determined that, compared to their corresponding non-fluorinated ribo-CDNs, the fluorinated deoxyribo-CDNs of the present invention surprisingly exhibit unique, unobvious and previously unreported class effects that could be exploited for therapeutic, diagnostic and research applications that involve manipulation of STING activity. For example, the fluorinated CDNs are more active, as we have ascertained in vitro by measuring cytokine induction in different cell lines and in whole blood. Moreover, they are eliminated more slowly in vivo, as we have determined in mice. Lastly, they exh ibit superior resistance to enzymatic cleavage.
References
Patent references:
1 . US 2015/0056224 A1
2. US 2014/0329889 A1
3. WO 2015/077354 A1
4. WO 2014/189805 A1
Non-patent references:
1 . Ablasser, A., Goldeck, M., Caviar, T., Deimling, T., Witte, G., Rohl, I., Homung, V. (2013). cGAS produces a 2'-5'-linked cyclic dinucleotide second messenger that activates STING. Nature, 498(7454), 380-384.
2. Chen, H., Sun, H., You, F., Sun, W., Zhou, X., Chen, L, Jiang, Z. (201 1 ). Activation of STAT6 by STING is critical for antiviral innate immunity. Cell, 147(2), 436^46.
3. Chu, C. K. (2002). Recent Advances in Nucleosides: Chemistry and Chemotherapy: Elsevier.
4. Danilchanka, O., & Mekalanos, J. J. (2013). Cyclic dinucleotides and the innate immune response. Cell, 154(5), 962-970.
5. Diner, E. J., Burdette, D. L, Wilson, S. C, Monroe, K. M., Kellenberger, C. A., Hyodo, M., Vance, R. E.
(2013). The innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING. Cell Rep, 3(5), 1355-1361.
6. Dubensky, T. W., Jr., Kanne, D. B., & Leong, M. L. (2013). Rationale, progress and development of vaccines utilizing STING-activating cyclic dinucleotide adjuvants. Ther Adv Vaccines, 1(4), 131 -143.
7. Fensterl, V., White, C. L., Yamashita, M., & Sen, G. C. (2008). Novel characteristics of the function and induction of murine p56 family proteins. J Virol, 82(22), 11045-1 1053.
8. Gomelsky, M. (201 1 ). cAMP, c-di-GMP, c-di-AMP and now cGMP: bacteria use them all! Mol Microbiol, 79(3), 562-565.
9. Hecker, S. J., & Erion, M. D. (2008). Prodrugs of phosphates and phosphonates. J Med Chem, 51(8), 2328- 2345.
10. Herdewijna, P. V. A. A. a. K. L. (1989). Synthesis of Nucleosides Fluorinated in the Sugar Moiety. The Application of Diethylaminosulfur Trifluoride to the Synthesis of Fluorinated Nucleosides. Nucleosides and Nucleotides, 8(1 ), 65-96.
1 1. Jones, R. B., N. (1995). Nucleotide prodrugs. Antiviral Research, 27(1 -2), 1 -17.
12. Li, L, Yin, Q., Kuss, P., Maliga, Z., Millan, J. L., Wu, H., & Mitchison, T. J. (2014). Hydrolysis of 2'3'-cGAMP by ENPP1 and design of nonhydrolyzable analogs. Nat Chem Biol, 70(12), 1043-1048.
13. Rajagopalan, P. B., F. D; Chu, C. K.; Tennant, B. C; Baldwin, B. H. (2003). Antiviral Nucleosides: Chiral Synthesis and Chemotherapy: Elsevier.
14. Rautio, J., Kumpulainen, H., Heimbach, T., Oliyai, R., Oh, D., Jarvinen, T., & Savolainen, J. (2008).
Prodrugs: design and clinical applications. Nat Rev Drug Discov, 7(3), 255-270.
15. Ross, B. S. S., R.H.; Sprankle, K.G.; & Vasquez, G. (1997). An Efficient and Scalable Synthesis of Arabinosylguanine and 2'-Deoxy-2'-Fluoro-guanosine. Nucleosides and Nucleotides, 16(7-9), 1645-1647.
16. Schinazi, R. F. L. D. C. (2004). Frontiers in Nucleosides & Nucleic Acids: IHL Press. 17. Shanahan, C. A., Gaffney, B. L, Jones, R. A., & Strobel, S. A. (2013). Identification of c-di-GMP derivatives resistant to an EAL domain phosphodiesterase. Biochemistry, 52(2), 365-377.
18. Simm, R., Morr, M., Kader, A., Nimtz, M., & Romling, U. (2004). GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessility to motility. Mol Microbiol, 53(4), 1123-1 134.
19. Thomas, H. J., Tiwaria, K.N.; Claytona, S.J.; Secrist J.A.; & Montgomery J. A. (1994). Synthesis and Biologic
Activity of Purine 2'-Deoxy-2'-fluoro-ribonucleosides. Nucleosides and Nucleotides, 73(1 -3), 309-323.
20. Vorbriiggen, H., & Ruh-Pohlenz, C. . (2001 ). Handbook of Nucleoside Synthesis. New York: Wiley.
21. Wuts, P. G. M., Greene, T. W., & Greene, T. W. (2014). Greene's protective groups in organic synthesis (Fifth edition / ed.). Hoboken, New Jersey: John Wiley & Sons, Inc.
22. Zhang, X., Shi, H., Wu, J., Zhang, X., Sun, L., Chen, C, & Chen, Z. J. (2013). Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Mol Cell, 51(2), 226-235.

Claims

Claims
1 compound of Formula (I):
Figure imgf000055_0001
Formula (I)
wherein:
• Xi is H or F;
• X2 is H or F;
• At least one among Xi and X2 is a fluorine atom;
• Z is OH, ORi, SH or SRi, wherein:
i) Ri is Na or NH4, or
ii) Ri is an enzyme-labile group which provides OH or SH in vivo such as pivaloyloxymethyl;
• Bi and B2 are bases chosen from:
Figure imgf000055_0002
Guanine, and Bi is a different base than B2, or a pharmaceutically acceptable salt thereof.
2. The cyclic dinucleotide of Formula (I) according to claim 1 , wherein said cyclic dinucleotide is chosen from the following compounds: P-2'FdlMP) AMP-2'FdlMP) AM P(S)-2' Fd IM P(S)] MP-2'FdAMP) M P(S )-2 ' FdAM P(S)]
Figure imgf000056_0001
GMP(S)-2' FdAM P(S)](POM)2
AM P(S)-2' Fd IM P(S)](P0M)2
Figure imgf000057_0001
3. The cyclic dinucleotide of Formula (I) according to any one of claims 1 -2, wherein said cyclic dinucleotide is an agonist of the receptor called "stimulator of interferon genes" (STING).
4. A pharmaceutical composition comprising a cyclic dinucleotide of Formula (I) according to any one of claims 1 -3, and a pharmaceutically acceptable excipient.
5. A cyclic dinucleotide of Formula (I) according to any one of claims 1 -4, for use in a therapeutic treatment in humans or animals.
6. A cyclic dinucleotide of Formula (I), according to any one of claims 1 -5, for use in the treatment of a cancer.
7. A cyclic dinucleotide of Formula (I), according to any one of claims 1 -5, for use in the treatment of a bacterial infection.
8. A cyclic d inucleotide of Formula (I), according to any one of claims 1 -5, for use in the treatment of a viral infection.
9. A cyclic dinucleotide of Formula (I) according to any one of claims 1 -5, for use in the treatment of solid pancreatic tumors.
10. A cyclic dinucleotide of Formula (I) according to any one of claims 1 -9, for use in the treatment of a pathology that may be alleviated by the induction of an immune response via the STING pathway.
1 1. A kit-of-parts comprising a cyclic dinucleotide of Formula (I) according to any one of claims 1 -10, and a chemotherapeutic agent, for use in the treatment of solid pancreatic tumors.
12. The kit-of-parts according to claim 1 1 , wherein said chemotherapeutic agent is gemcitabine.
PCT/EP2015/070635 2014-12-16 2015-09-09 Fluorinated cyclic dinucleotides for cytokine induction WO2016096174A1 (en)

Priority Applications (10)

Application Number Priority Date Filing Date Title
CN201580057976.5A CN107148424B (en) 2014-12-16 2015-09-09 Cyclic dinucleotides for inducing cytokines
ES15762583T ES2764178T3 (en) 2014-12-16 2015-09-09 Fluorinated cyclic dinucleotides for cytokine induction
DK15762583.1T DK3233882T3 (en) 2014-12-16 2015-09-09 FLUORINATED CYCLEIC DINUCLEOTIDES FOR CYTOKININ INDUCTION
PL15762583T PL3233882T3 (en) 2014-12-16 2015-09-09 Fluorinated cyclic dinucleotides for cytokine induction
US15/034,335 US10011630B2 (en) 2014-12-16 2015-09-09 Cyclic dinucleotides for cytokine induction
EP15762583.1A EP3233882B1 (en) 2014-12-16 2015-09-09 Fluorinated cyclic dinucleotides for cytokine induction
EP19174902.7A EP3546473A1 (en) 2014-12-16 2015-09-09 Cyclic [(2',5')p(3',5')p]-dinucleotides for cytokine induction
US16/006,465 US10562929B2 (en) 2014-12-16 2018-06-12 Cyclic dinucleotides for cytokine induction
US16/781,757 US11053272B2 (en) 2014-12-16 2020-02-04 Cyclic dinucleotides for cytokine induction
US17/365,066 US11667664B2 (en) 2014-12-16 2021-07-01 Cyclic dinucleotides for cytokine induction

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP14307054 2014-12-16
EP14307054.8 2014-12-16

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US15/034,335 A-371-Of-International US10011630B2 (en) 2014-12-16 2015-09-09 Cyclic dinucleotides for cytokine induction
US16/006,465 Continuation US10562929B2 (en) 2014-12-16 2018-06-12 Cyclic dinucleotides for cytokine induction

Publications (2)

Publication Number Publication Date
WO2016096174A1 true WO2016096174A1 (en) 2016-06-23
WO2016096174A8 WO2016096174A8 (en) 2016-09-29

Family

ID=52338943

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2015/070635 WO2016096174A1 (en) 2014-12-16 2015-09-09 Fluorinated cyclic dinucleotides for cytokine induction

Country Status (8)

Country Link
US (4) US10011630B2 (en)
EP (2) EP3546473A1 (en)
CN (1) CN107148424B (en)
DK (1) DK3233882T3 (en)
ES (1) ES2764178T3 (en)
PL (1) PL3233882T3 (en)
PT (1) PT3233882T (en)
WO (1) WO2016096174A1 (en)

Cited By (100)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016145102A1 (en) 2015-03-10 2016-09-15 Aduro Biotech, Inc. Compositions and methods for activating "stimulator of interferon gene" -dependent signalling
WO2017027646A1 (en) 2015-08-13 2017-02-16 Merck Sharp & Dohme Corp. Cyclic di-nucleotide compounds as sting agonists
WO2017093933A1 (en) 2015-12-03 2017-06-08 Glaxosmithkline Intellectual Property Development Limited Cyclic purine dinucleotides as modulators of sting
WO2017186711A1 (en) * 2016-04-25 2017-11-02 Invivogen Novel complexes of immunostimulatory compounds, and uses thereof
WO2018020476A1 (en) 2016-07-29 2018-02-01 Aduro Biotech Holdings, Europe B.V. Anti-pd-1 antibodies
WO2018025178A1 (en) 2016-08-02 2018-02-08 Aduro Biotech Holdings, Europe B.V. Antibodies against human ctla-4
WO2018060323A1 (en) 2016-09-30 2018-04-05 Boehringer Ingelheim International Gmbh Cyclic dinucleotide compounds
WO2018065360A1 (en) 2016-10-07 2018-04-12 Biolog Life Science Institute Forschungslabor Und Biochemica-Vertrieb Gmbh Cyclic dinucleotides containing benzimidazole, method for the production of same, and use of same to activate stimulator of interferon genes (sting)-dependent signaling pathways
WO2018100558A2 (en) 2016-12-01 2018-06-07 Takeda Pharmaceutical Company Limited Cyclic dinucleotide
US10047115B2 (en) 2015-01-29 2018-08-14 Glaxosmithkline Intellectual Property Development Limited Cyclic dinucleotides useful for the treatment of inter alia cancer
WO2018156625A1 (en) * 2017-02-21 2018-08-30 Board Of Regents,The University Of Texas System Cyclic dinucleotides as agonists of stimulator of interferon gene dependent signalling
WO2018172206A1 (en) 2017-03-22 2018-09-27 Boehringer Ingelheim International Gmbh Modified cyclic dinucleotide compounds
WO2018189382A1 (en) 2017-04-14 2018-10-18 Solstice Biologics, Ltd. Immunomodulating polynucleotides, antibody conjugates thereof, and methods of their use
WO2018190719A2 (en) 2017-04-13 2018-10-18 Aduro Biotech Holdings, Europe B.V. Anti-sirp alpha antibodies
WO2018198076A1 (en) 2017-04-28 2018-11-01 Aduro Biotech, Inc. Bis 2'-5'-rr-(3'f-a)(3'f-a) cyclic dinucleotide compound and uses thereof
WO2018200812A1 (en) 2017-04-28 2018-11-01 Novartis Ag Antibody conjugates comprising sting agonist
WO2018198084A1 (en) * 2017-04-27 2018-11-01 Lupin Limited Cyclic di-nucleotide compounds with tricyclic nucleobases
WO2018222711A2 (en) 2017-05-30 2018-12-06 Bristol-Myers Squibb Company Compositions comprising a combination of an anti-lag-3 antibody, a pd-1 pathway inhibitor, and an immunotherapeutic agent
WO2019023459A1 (en) * 2017-07-28 2019-01-31 Bristol-Myers Squibb Company Cyclic dinucleotides as anticancer agents
CN109475570A (en) * 2016-03-18 2019-03-15 免疫传感器公司 Ring dinucleotides compound and application method
KR20190032408A (en) * 2016-07-06 2019-03-27 스페로비 바이오사이언시즈 인코포레이티드 Compounds, compositions and methods for the treatment of disease
WO2019069275A1 (en) * 2017-10-05 2019-04-11 Glaxosmithkline Intellectual Property Development Limited Methods for administering sting agonists
WO2019084060A1 (en) 2017-10-24 2019-05-02 Silverback Therapeutics, Inc. Conjugates and methods of use thereof for selective delivery of immune-modulatory agents
WO2019092660A1 (en) 2017-11-10 2019-05-16 Takeda Pharmaceutical Company Limited Sting modulator compounds, and methods of making and using
US10322177B2 (en) 2015-10-07 2019-06-18 Nant Holdings Ip, Llc Activation of immune-related signaling pathways in cells via optofection
EP3505527A1 (en) 2017-12-29 2019-07-03 Invivogen Cyclic dinucleotides for cytokine induction
US10414747B2 (en) 2016-10-04 2019-09-17 Merck Sharp & Dohme Corp. Benzo[b]thiophene compounds as sting agonists
WO2019180683A1 (en) 2018-03-23 2019-09-26 Takeda Pharmaceutical Company Limited Sting modulator compounds with sulfamate linkages, and methods of making and using
WO2019183578A1 (en) 2018-03-23 2019-09-26 Codiak Biosciences, Inc. Extracellular vesicles comprising sting-agonist
WO2019185477A1 (en) 2018-03-27 2019-10-03 Boehringer Ingelheim International Gmbh Cyclic dinucleotide compounds containing 2-aza-hypoxanthine or 6h-pytazolo[1,5-d][1,2,4]triazin-7-one as sting agonists
WO2019185476A1 (en) 2018-03-27 2019-10-03 Boehringer Ingelheim International Gmbh Modified cyclic dinucleotide compounds
WO2019195124A1 (en) 2018-04-03 2019-10-10 Merck Sharp & Dohme Corp. Benzothiophenes and related compounds as sting agonists
US10450341B2 (en) 2014-06-04 2019-10-22 Glaxosmithkline Intellectual Property Development Limited Cyclic di-nucleotides as modulators of STING
CN110446503A (en) * 2016-12-22 2019-11-12 马福制药公司 Phosphodiesterase inhibitors and antimicrobial treatments method
WO2020041720A1 (en) 2018-08-24 2020-02-27 Codiak Biosciences, Inc. Extracellular vesicles targeting dendritic cells and uses thereof
WO2020056008A1 (en) 2018-09-12 2020-03-19 Silverback Therapeutics, Inc. Compositions for the treatment of disease with immune stimulatory conjugates
EP3507367A4 (en) * 2016-07-05 2020-03-25 Aduro BioTech, Inc. Locked nucleic acid cyclic dinucleotide compounds and uses thereof
US10604542B2 (en) 2016-01-11 2020-03-31 Innate Tumor Immunity, Inc. Cyclic dinucleotides for treating conditions associated with sting activity such as cancer
WO2020065408A1 (en) 2018-09-27 2020-04-02 Pierre Fabre Medicament Sulfomaleimide-based linkers and corresponding conjugates
CN111032672A (en) * 2017-08-31 2020-04-17 百时美施贵宝公司 Cyclic dinucleotides as anticancer agents
WO2020092633A1 (en) 2018-10-30 2020-05-07 Vanderbilt University Graft copolymers, methods of forming graft copolymers, and methods of use thereof
WO2020092617A1 (en) 2018-10-31 2020-05-07 Novartis Ag Dc-sign antibody conjugates comprising sting agonists
US10696985B1 (en) 2016-06-06 2020-06-30 Vanderbilt University Reversibly crosslinked endosomolytic polymer vesicles for cytosolic drug delivery
CN111423483A (en) * 2019-01-10 2020-07-17 南开大学 Cyclic dinucleotide prodrug molecule and preparation method and application thereof
EP3484504A4 (en) * 2016-07-15 2020-07-29 Sperovie Biosciences, Inc. Compounds, compositions, and methods for the treatment of disease
EP3558324A4 (en) * 2016-12-20 2020-08-05 Merck Sharp & Dohme Corp. Cyclic dinucleotide sting agonists for cancer treatment
WO2020191377A1 (en) 2019-03-21 2020-09-24 Codiak Biosciences, Inc. Extracellular vesicle conjugates and uses thereof
WO2020191369A1 (en) 2019-03-21 2020-09-24 Codiak Biosciences, Inc. Process for preparing extracellular vesicles
WO2020191361A2 (en) 2019-03-21 2020-09-24 Codiak Biosciences, Inc. Extracellular vesicles for vaccine delivery
WO2020205688A1 (en) 2019-04-04 2020-10-08 Merck Sharp & Dohme Corp. Inhibitors of histone deacetylase-3 useful for the treatment of cancer, inflammation, neurodegeneration diseases and diabetes
WO2021003445A1 (en) 2019-07-03 2021-01-07 Codiak Biosciences, Inc. Extracellular vesicles targeting t cells and uses thereof
WO2021030251A1 (en) 2019-08-12 2021-02-18 Purinomia Biotech, Inc. Methods and compositions for promoting and potentiating t-cell mediated immune responses through adcc targeting of cd39 expressing cells
WO2021062290A1 (en) 2019-09-25 2021-04-01 Codiak Biosciences, Inc. Methods of producing extracellular vesicles
WO2021062060A1 (en) 2019-09-25 2021-04-01 Codiak Biosciences, Inc. Sting agonist comprising exosomes combined with il-12 displaying exosomes for treating a tumour
WO2021062317A1 (en) 2019-09-25 2021-04-01 Codiak Biosciences, Inc. Extracellular vesicle compositions
WO2021062058A1 (en) 2019-09-25 2021-04-01 Codiak Biosciences, Inc. Sting agonist comprising exosomes for treating neuroimmunological disorders
US10966999B2 (en) 2017-12-20 2021-04-06 Institute Of Organic Chemistry And Biochemistry Ascr, V.V.I. 3′3′ cyclic dinucleotides with phosphonate bond activating the sting adaptor protein
WO2021067644A1 (en) 2019-10-01 2021-04-08 Silverback Therapeutics, Inc. Combination therapy with immune stimulatory conjugates
WO2021074695A1 (en) 2019-10-16 2021-04-22 Avacta Life Sciences Limited PD-L1 INHIBITOR - TGFβ INHIBITOR BISPECIFIC DRUG MOIETIES.
WO2021113679A1 (en) 2019-12-06 2021-06-10 Mersana Therapeutics, Inc. Dimeric compounds as sting agonists
WO2021168274A1 (en) 2020-02-21 2021-08-26 Silverback Therapeutics, Inc. Nectin-4 antibody conjugates and uses thereof
US11110106B2 (en) 2018-10-29 2021-09-07 Venenum Biodesign, LLC Sting agonists for treating bladder cancer and solid tumors
WO2021184017A1 (en) 2020-03-13 2021-09-16 Codiak Biosciences, Inc. Extracellular vesicles for treating neurological disorders
WO2021189047A2 (en) 2020-03-20 2021-09-23 Codiak Biosciences, Inc. Extracellular vesicles for therapy
WO2021206158A1 (en) 2020-04-10 2021-10-14 小野薬品工業株式会社 Method of cancer therapy
US11149052B2 (en) 2018-04-06 2021-10-19 Institute Of Organic Chemistry And Biochemistry Ascr, V.V.I. 2′3′-cyclic dinucleotides
EP3692048A4 (en) * 2017-08-30 2021-10-20 Beijing Xuanyi Pharmasciences Co., Ltd. Cyclic di-nucleotides as stimulator of interferon genes modulators
EA038805B1 (en) * 2017-11-21 2021-10-21 Такеда Фармасьютикал Компани Лимитед Cyclic dinucleotides as sting (stimulator of interferon genes) agonists
US11161864B2 (en) 2018-10-29 2021-11-02 Venenum Biodesign, LLC Sting agonists
WO2021232019A1 (en) 2020-05-15 2021-11-18 Immunesensor Therapeutics, Inc. Sting agonist combination treatments with immune checkpoint inhibitors
WO2021237100A1 (en) 2020-05-21 2021-11-25 Codiak Biosciences, Inc. Methods of targeting extracellular vesicles to lung
US11203610B2 (en) 2017-12-20 2021-12-21 Institute Of Organic Chemistry And Biochemistry Ascr, V.V.I. 2′3′ cyclic dinucleotides with phosphonate bond activating the sting adaptor protein
WO2022006327A1 (en) 2020-07-01 2022-01-06 Silverback Therapeutics, Inc. Anti-asgr1 antibody conjugates and uses thereof
US11285131B2 (en) 2017-08-04 2022-03-29 Merck Sharp & Dohme Corp. Benzo[b]thiophene STING agonists for cancer treatment
WO2022066934A2 (en) 2020-09-23 2022-03-31 Codiak Biosciences, Inc. Process for preparing extracellular vesicles
WO2022066883A1 (en) 2020-09-23 2022-03-31 Codiak Biosciences, Inc. Extracellular vesicles comprising kras antigens and uses thereof
WO2022066898A2 (en) 2020-09-23 2022-03-31 Codiak Biosciences, Inc. Methods of producing extracellular vesicles
WO2022066928A2 (en) 2020-09-23 2022-03-31 Codiak Biosciences, Inc. Process for preparing extracellular vesicles
US11292812B2 (en) 2018-04-06 2022-04-05 Institute Of Organic Chemistry And Biochemistry Ascr, V.V.I. 3′3′-cyclic dinucleotides
US11312772B2 (en) 2017-08-04 2022-04-26 Merck Sharp & Dohme Corp. Combinations of PD-1 antagonists and benzo [b] thiophene STING agonists for cancer treatment
WO2022133288A1 (en) 2020-12-17 2022-06-23 Trustees Of Tufts College Fap-activated radiotheranostics, and uses related thereto
TWI769195B (en) * 2016-12-01 2022-07-01 日商武田藥品工業股份有限公司 Cyclic dinucleotide
RU2776060C2 (en) * 2017-12-15 2022-07-13 Янссен Байотек, Инк. Cyclic dinucleotides as sting agonists
EP3868773A4 (en) * 2018-10-12 2022-07-13 Shanghai Jemincare Pharmaceuticals Co., Ltd. Cyclic dinucleotide compound and uses thereof
US11453697B1 (en) 2015-08-13 2022-09-27 Merck Sharp & Dohme Llc Cyclic di-nucleotide compounds as sting agonists
US11466047B2 (en) 2017-05-12 2022-10-11 Merck Sharp & Dohme Llc Cyclic di-nucleotide compounds as sting agonists
WO2022234003A1 (en) 2021-05-07 2022-11-10 Avacta Life Sciences Limited Cd33 binding polypeptides with stefin a protein
WO2023004440A2 (en) 2021-07-23 2023-01-26 Immunesensor Therapeutics, Inc. Sting agonist combination treatments with cytokines
US11584774B2 (en) 2017-09-11 2023-02-21 F-star Therapeutics, Inc. Compounds, compositions, and methods for the treatment of disease
WO2023056468A1 (en) 2021-09-30 2023-04-06 Codiak Biosciences, Inc. Extracellular vesicle comprising cholesterol tagged sting-agonist
WO2023057567A1 (en) 2021-10-07 2023-04-13 Avacta Life Sciences Limited Pd-l1 binding affimers
US11685761B2 (en) 2017-12-20 2023-06-27 Merck Sharp & Dohme Llc Cyclic di-nucleotide compounds as sting agonists
US11702430B2 (en) 2018-04-03 2023-07-18 Merck Sharp & Dohme Llc Aza-benzothiophene compounds as STING agonists
US11707531B2 (en) 2017-09-11 2023-07-25 F-star Therapeutics, Inc. Compounds, compositions, and methods for the treatment of disease
US11725024B2 (en) 2020-11-09 2023-08-15 Takeda Pharmaceutical Company Limited Antibody drug conjugates
WO2023170606A1 (en) 2022-03-08 2023-09-14 Alentis Therapeutics Ag Use of anti-claudin-1 antibodies to increase t cell availability
US11787833B2 (en) 2019-05-09 2023-10-17 Aligos Therapeutics, Inc. Modified cyclic dinucleoside compounds as sting modulators
US11795463B2 (en) 2020-02-28 2023-10-24 Tallac Therapeutics, Inc. Transglutaminase-mediated conjugation
WO2023218243A1 (en) 2022-05-12 2023-11-16 Avacta Life Sciences Limited Lag-3/pd-l1 binding fusion proteins
US11939343B2 (en) 2019-08-02 2024-03-26 Mersana Therapeutics, Inc. Sting agonist compounds and methods of use

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR112015013440B1 (en) 2012-12-13 2020-12-08 Aduro Biotech, Inc compositions comprising cyclic purine dinucleotides with stereochemistry
WO2014179335A1 (en) 2013-04-29 2014-11-06 Memorial Sloan Kettering Cancer Center Compositions and methods for altering second messenger signaling
ES2754269T3 (en) 2013-05-18 2020-04-16 Aduro Biotech Inc Compositions and Activation Methods of "Interferon Gene Stimulator-dependent" Signaling
EP3546473A1 (en) * 2014-12-16 2019-10-02 Kayla Therapeutics Cyclic [(2',5')p(3',5')p]-dinucleotides for cytokine induction
JOP20170188A1 (en) * 2016-11-25 2019-01-30 Janssen Biotech Inc Cyclic dinucleotides as sting agonists
US20200055883A1 (en) 2017-02-17 2020-02-20 Eisai R&D Management Co., Ltd. Cyclic di-nucleotides derivative for the treatment of cancer
US10859566B2 (en) 2017-03-20 2020-12-08 Genocea Biosciences, Inc. Treatment methods
WO2019079261A1 (en) * 2017-10-16 2019-04-25 Bristol-Myers Squibb Company Cyclic dinucleotides as anticancer agents
TWI818943B (en) * 2018-01-04 2023-10-21 中央研究院 Cell-associating immunologic adjuvants for treatment enhancement
EP3801561A1 (en) 2018-06-01 2021-04-14 Eisai R&D Management Co., Ltd. Methods for the treatment of bladder cancer
BR112021001349A2 (en) 2018-08-16 2021-04-20 Eisai R&D Management Co., Ltd. compound salts and crystals thereof
CN113272015A (en) 2018-10-29 2021-08-17 健诺西生物科学公司 Method of treatment
MA55805A (en) 2019-05-03 2022-03-09 Flagship Pioneering Innovations V Inc METHODS OF MODULATING IMMUNE ACTIVITY
WO2021216572A1 (en) 2020-04-20 2021-10-28 Massachusetts Institute Of Technology Lipid compositions for delivery of sting agonist compounds and uses thereof
US20230210968A1 (en) 2020-06-11 2023-07-06 Massachusetts Institute Of Technology Ribonucleoprotein approach to boost the sting signaling for cancer immunotherapy
CN111909223A (en) * 2020-07-17 2020-11-10 清华大学 Cyclic dinucleotide covalent modifier and preparation method and application thereof
JP2023546278A (en) * 2020-10-20 2023-11-01 タイリガンド バイオサイエンス（シャンハイ）リミテッド Multifunctional cyclic dinucleotide and its use
KR102466750B1 (en) 2020-10-23 2022-11-15 아주대학교산학협력단 Pharmaceutical composition comprising indolizine derivative for activating stimulator of interferon gene dependent signalling

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140066436A1 (en) 2007-09-28 2014-03-06 Cyclacel Limited Pyrimidine derivatives as protein kinase inhibitors
US20140205653A1 (en) 2012-12-13 2014-07-24 Aduro Biotech, Inc. Compositions comprising cyclic purine dinucleotides having defined stereochemistries and methods for their preparation and use
WO2014179335A1 (en) * 2013-04-29 2014-11-06 Memorial Sloan Kettering Cancer Center Compositions and methods for altering second messenger signaling
US20140329889A1 (en) 2013-05-03 2014-11-06 The Regents Of The University Of California Cyclic di-nucleotide induction of type i interferon
WO2014189805A1 (en) 2013-05-18 2014-11-27 Auro Biotech, Inc. Compositions and methods for activating "stimulator of interferon gene"-dependent signalling
WO2015077354A1 (en) 2013-11-19 2015-05-28 The University Of Chicago Use of sting agonist as cancer treatment

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7592326B2 (en) 2004-03-15 2009-09-22 Karaolis David K R Method for stimulating the immune, inflammatory or neuroprotective response
US9789202B2 (en) 2011-09-05 2017-10-17 Hanmi Science Co., Ltd. Method for treating cancer using an interferon alpha conjugate
CA2876150A1 (en) 2012-06-08 2013-12-12 The Johns Hopkins University Compostions and methods for cancer immunotherapy
EP2674170B1 (en) 2012-06-15 2014-11-19 Invivogen Novel compositions of TLR7 and/or TLR8 agonists conjugated to lipids
PL2934598T3 (en) * 2012-12-19 2018-10-31 Board Of Regents, The University Of Texas System Pharmaceutical targeting of a mammalian cyclic di-nucleotide signaling pathway
DE102013019468B4 (en) 2013-11-19 2022-07-07 Audi Ag Battery with a plurality of battery cells
US10092644B2 (en) * 2013-11-22 2018-10-09 Brock University Use of fluorinated cyclic dinucleotides as oral vaccine adjuvants
CN103908468B (en) 2014-04-21 2017-02-08 上海捌加壹医药科技有限公司 Application of cyclic dinucleotide cGAMP in preparing anti-tumor medicaments
EP3546473A1 (en) * 2014-12-16 2019-10-02 Kayla Therapeutics Cyclic [(2',5')p(3',5')p]-dinucleotides for cytokine induction
EP3233191A1 (en) * 2014-12-16 2017-10-25 Invivogen Combined use of a chemotherapeutic agent and a cyclic dinucleotide for cancer treatment

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140066436A1 (en) 2007-09-28 2014-03-06 Cyclacel Limited Pyrimidine derivatives as protein kinase inhibitors
US20140205653A1 (en) 2012-12-13 2014-07-24 Aduro Biotech, Inc. Compositions comprising cyclic purine dinucleotides having defined stereochemistries and methods for their preparation and use
WO2014179335A1 (en) * 2013-04-29 2014-11-06 Memorial Sloan Kettering Cancer Center Compositions and methods for altering second messenger signaling
US20140329889A1 (en) 2013-05-03 2014-11-06 The Regents Of The University Of California Cyclic di-nucleotide induction of type i interferon
WO2014189805A1 (en) 2013-05-18 2014-11-27 Auro Biotech, Inc. Compositions and methods for activating "stimulator of interferon gene"-dependent signalling
US20150056224A1 (en) 2013-05-18 2015-02-26 Aduro Biotech, Inc. Compositions and methods for activating stimulator of interferon gene-dependent signalling
WO2015077354A1 (en) 2013-11-19 2015-05-28 The University Of Chicago Use of sting agonist as cancer treatment

Non-Patent Citations (26)

* Cited by examiner, † Cited by third party
Title
ABLASSER, A.; GOLDECK, M.; CAVLAR, T.; DEIMLING, T.; WITTE, G.; ROHL, I.; HOMUNG, V.: "cGAS produces a 2'-5'-linked cyclic dinucleotide second messenger that activates STING", NATURE, vol. 498, no. 7454, 2013, pages 380 - 384
BENJAMIN T. ROEMBKE ET AL: "A cyclic dinucleotide containing 2-aminopurine is a general fluorescent sensor for c-di-GMP and 3',3'-cGAMP", MOLECULAR BIOSYSTEMS, vol. 10, no. 6, 28 March 2014 (2014-03-28), pages 1568, XP055172186, ISSN: 1742-206X, DOI: 10.1039/c3mb70518h *
CARLY A. SHANAHAN ET AL: "Identification of c-di-GMP Derivatives Resistant to an EAL Domain Phosphodiesterase", BIOCHEMISTRY, vol. 52, no. 2, 15 January 2013 (2013-01-15), pages 365 - 377, XP055172476, ISSN: 0006-2960, DOI: 10.1021/bi301510v *
CHEN, H.; SUN, H.; YOU, F.; SUN, W.; ZHOU, X.; CHEN, L.; JIANG, Z.: "Activation of STAT6 by STING is critical for antiviral innate immunity", CELL, vol. 147, no. 2, 2011, pages 436 - 446
CHU, C. K.: "Recent Advances in Nucleosides: Chemistry and Chemotherapy", 2002, ELSEVIER
DANILCHANKA, 0.; MEKALANOS, J. J.: "Cyclic dinucleotides and the innate immune response", CELL, vol. 154, no. 5, 2013, pages 962 - 970
DINER, E. J.; BURDETTE, D. L.; WILSON, S. C.; MONROE, K. M.; KELLENBERGER, C. A.; HYODO, M.; VANCE, R. E.: "The innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING", CELL REP, vol. 3, no. 5, 2013, pages 1355 - 1361
DUBENSKY, T. W., JR.; KANNE, D. B.; LEONG, M. L.: "Rationale, progress and development of vaccines utilizing STING-activating cyclic dinucleotide adjuvants", THERADV VACCINES, vol. 1, no. 4, 2013, pages 131 - 143
FENSTERL, V.; WHITE, C. L.; YAMASHITA, M.; SEN, G. C.: "Novel characteristics of the function and induction of murine p56 family proteins", J VIROL, vol. 82, no. 22, 2008, pages 11045 - 11053
GOMELSKY, M.: "cAMP, c-di-GMP, c-di-AMP and now cGMP: bacteria use them all!", MOL MICROBIOL, vol. 79, no. 3, 2011, pages 562 - 565
HECKER, S. J.; ERION, M. D.: "Prodrugs of phosphates and phosphonates", J MED CHEM, vol. 51, no. 8, 2008, pages 2328 - 2345
HERDEWIJNA, P. V. A. A. A. K. L.: "Synthesis of Nucleosides Fluorinated in the Sugar Moiety. The Application of Diethylaminosulfur Trifluoride to the Synthesis of Fluorinated Nucleosides", NUCLEOSIDES AND NUCLEOTIDES, vol. 8, no. 1, 1989, pages 65 - 96
JONES, R. B., N.: "Nucleotide prodrugs", ANTIVIRAL RESEARCH, vol. 27, no. 1-2, 1995, pages 1 - 17
LI, L.; YIN, Q.; KUSS, P.; MALIGA, Z.; MILLAN, J. L.; WU, H.; MITCHISON, T. J.: "Hydrolysis of 2'3'-cGAMP by ENPP1 and design of nonhydrolyzable analogs", NAT CHEM BIOL, vol. 10, no. 12, 2014, pages 1043 - 1048
LINGYIN LI ET AL: "Hydrolysis of 2'3'-cGAMP by ENPP1 and design of nonhydrolyzable analogs", NATURE CHEMICAL BIOLOGY, vol. 10, no. 12, 26 October 2014 (2014-10-26), pages 1043 - 1048, XP055172172, ISSN: 1552-4450, DOI: 10.1038/nchembio.1661 *
RAJAGOPALAN, P. B., F. D; CHU, C. K.; TENNANT, B. C; BALDWIN, B. H.: "Antiviral Nucleosides: Chiral Synthesis and Chemotherapy", 2003, ELSEVIER
RAUTIO, J.; KUMPULAINEN, H.; HEIMBACH, T.; OLIYAI, R.; OH, D.; JARVINEN, T.; SAVOLAINEN, J.: "Prodrugs: design and clinical applications", NAT REV DRUG DISCOV, vol. 7, no. 3, 2008, pages 255 - 270
ROSS, B. S. S., R.H.; SPRANKLE, K.G.; VASQUEZ, G.: "An Efficient and Scalable Synthesis of Arabinosylguanine and 2'-Deoxy-2'-Fluoro-guanosine", NUCLEOSIDES AND NUCLEOTIDES, vol. 16, no. 7-9, 1997, pages 1645 - 1647
SCHINAZI, R. F. L. D. C.: "Frontiers in Nucleosides & Nucleic Acids", 2004, IHL PRESS
SHANAHAN, C. A.; GAFFNEY, B. L.; JONES, R. A.; STROBEL, S. A.: "Identification of c-di-GMP derivatives resistant to an EAL domain phosphodiesterase", BIOCHEMISTRY, vol. 52, no. 2, 2013, pages 365 - 377
SIMM, R.; MORR, M.; KADER, A.; NIMTZ, M.; ROMLING, U.: "GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessility to motility", MOL MICROBIOL, vol. 53, no. 4, 2004, pages 1123 - 1134
TAKAFUMI TEZUKA ET AL: "Synthesis of 2'-Modified Cyclic Bis(3'-5')diadenylic Acids (c-di-AMPs) and Their Promotion of Cell Division in a Freshwater Green Alga", CHEMISTRY LETTERS, vol. 41, no. 12, 8 December 2012 (2012-12-08), pages 1723 - 1725, XP055172477, ISSN: 0366-7022, DOI: 10.1246/cl.2012.1723 *
THOMAS, H. J.; TIWARIA, K.N.; CLAYTONA, S.J.; SECRIST J.A.; MONTGOMERY J.A: "Synthesis and Biologic Activity of Purine 2'-Deoxy-2'-fluoro-ribonucleosides", NUCLEOSIDES AND NUCLEOTIDES, vol. 13, no. 1-3, 1994, pages 309 - 323
VORBRUGGEN, H.; RUH-POHLENZ, C.: "Handbook of Nucleoside Synthesis", 2001, WILEY
WUTS, P. G. M.; GREENE, T. W.; GREENE, T. W.: "Greene's protective groups in organic synthesis", 2014, JOHN WILEY & SONS, INC
ZHANG, X.; SHI, H.; WU, J.; ZHANG, X.; SUN, L.; CHEN, C.; CHEN, Z. J.: "Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING", MOL CELL, vol. 51, no. 2, 2013, pages 226 - 235

Cited By (186)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10450341B2 (en) 2014-06-04 2019-10-22 Glaxosmithkline Intellectual Property Development Limited Cyclic di-nucleotides as modulators of STING
US10047115B2 (en) 2015-01-29 2018-08-14 Glaxosmithkline Intellectual Property Development Limited Cyclic dinucleotides useful for the treatment of inter alia cancer
US11040053B2 (en) 2015-03-10 2021-06-22 Chinook Therapeutics, Inc. Compositions and methods for activating “stimulator of interferon gene”13 dependent signalling
US10449211B2 (en) 2015-03-10 2019-10-22 Aduro Biotech, Inc. Compositions and methods for activating “stimulator of interferon gene”—dependent signalling
EP3268035A4 (en) * 2015-03-10 2018-10-31 Aduro Biotech, Inc. Compositions and methods for activating "stimulator of interferon gene" -dependent signalling
EP3268035A1 (en) 2015-03-10 2018-01-17 Aduro BioTech, Inc. Compositions and methods for activating "stimulator of interferon gene" -dependent signalling
WO2016145102A1 (en) 2015-03-10 2016-09-15 Aduro Biotech, Inc. Compositions and methods for activating "stimulator of interferon gene" -dependent signalling
US10738074B2 (en) 2015-08-13 2020-08-11 Merck Sharp & Dohme Corp. Cyclic di-nucleotide compounds as STING agonists
KR20180030926A (en) * 2015-08-13 2018-03-26 머크 샤프 앤드 돔 코포레이션 As a STING agonist, the cyclic di-nucleotide compound
US10759825B2 (en) 2015-08-13 2020-09-01 Merck Sharp & Dohme Corp. Cyclic di-nucleotide compounds as STING agonists
WO2017027646A1 (en) 2015-08-13 2017-02-16 Merck Sharp & Dohme Corp. Cyclic di-nucleotide compounds as sting agonists
US11453697B1 (en) 2015-08-13 2022-09-27 Merck Sharp & Dohme Llc Cyclic di-nucleotide compounds as sting agonists
US10106574B2 (en) 2015-08-13 2018-10-23 Merck Sharp & Dohme Corp. Cyclic di-nucleotide compounds as sting agonists
US10766919B2 (en) 2015-08-13 2020-09-08 Merck Sharp & Dohme Corp. Cyclic di-nucleotide compounds as sting agonists
KR102222186B1 (en) 2015-08-13 2021-03-03 머크 샤프 앤드 돔 코포레이션 Cyclic di-nucleotide compounds as STING agonists
US10322177B2 (en) 2015-10-07 2019-06-18 Nant Holdings Ip, Llc Activation of immune-related signaling pathways in cells via optofection
WO2017093933A1 (en) 2015-12-03 2017-06-08 Glaxosmithkline Intellectual Property Development Limited Cyclic purine dinucleotides as modulators of sting
US10730907B2 (en) 2015-12-03 2020-08-04 Glaxosmithkline Intellectual Property Development Limited Compounds
EP3322713B1 (en) * 2015-12-03 2021-01-20 GlaxoSmithKline Intellectual Property Development Limited Cyclic purine dinucleotides as modulators of sting
JP2018516903A (en) * 2015-12-03 2018-06-28 グラクソスミスクライン、インテレクチュアル、プロパティー、ディベロップメント、リミテッドＧｌａｘｏｓｍｉｔｈｋｌｉｎｅ Ｉｎｔｅｌｌｅｃｔｕａｌ Ｐｒｏｐｅｒｔｙ Ｄｅｖｅｌｏｐｍｅｎｔ Ｌｉｍｉｔｅｄ Cyclic purine dinucleotides as regulators of STING
US9994607B2 (en) 2015-12-03 2018-06-12 Glaxosmithkline Intellectual Property Development Limited Compounds
US9718848B2 (en) 2015-12-03 2017-08-01 Glaxosmithkline Intellectual Property Development Limited Compounds
US10364266B2 (en) 2015-12-03 2019-07-30 Glaxosmithkline Intellectual Property Development Limited Compounds
US11505571B2 (en) 2016-01-11 2022-11-22 Innate Tumor Immunity, Inc. Compounds and compositions for treating conditions associated with sting activity
US10961270B2 (en) 2016-01-11 2021-03-30 Innate Tumor Immunity, Inc. Compounds and compositions for treating conditions associated with sting activity
US10604542B2 (en) 2016-01-11 2020-03-31 Innate Tumor Immunity, Inc. Cyclic dinucleotides for treating conditions associated with sting activity such as cancer
EP3692996A1 (en) * 2016-03-18 2020-08-12 Immune Sensor, LLC Cyclic di-nucleotide compounds and methods of use
EP3429596A4 (en) * 2016-03-18 2020-04-08 Immune Sensor, LLC Cyclic di-nucleotide compounds and methods of use
CN109475570A (en) * 2016-03-18 2019-03-15 免疫传感器公司 Ring dinucleotides compound and application method
US10519188B2 (en) 2016-03-18 2019-12-31 Immunesensor Therapeutics, Inc. Cyclic di-nucleotide compounds and methods of use
US11299512B2 (en) 2016-03-18 2022-04-12 Immunesensor Therapeutics, Inc. Cyclic di-nucleotide compounds and methods of use
WO2017186711A1 (en) * 2016-04-25 2017-11-02 Invivogen Novel complexes of immunostimulatory compounds, and uses thereof
US10696985B1 (en) 2016-06-06 2020-06-30 Vanderbilt University Reversibly crosslinked endosomolytic polymer vesicles for cytosolic drug delivery
EP3507367A4 (en) * 2016-07-05 2020-03-25 Aduro BioTech, Inc. Locked nucleic acid cyclic dinucleotide compounds and uses thereof
US11098077B2 (en) 2016-07-05 2021-08-24 Chinook Therapeutics, Inc. Locked nucleic acid cyclic dinucleotide compounds and uses thereof
KR102569881B1 (en) 2016-07-06 2023-08-22 에프-스타 테라퓨틱스 인코포레이티드 Compounds, compositions and methods for treating disease
EP3481402A4 (en) * 2016-07-06 2020-01-22 Sperovie Biosciences, Inc. Compounds, compositions, and methods for the treatment of disease
JP2022106989A (en) * 2016-07-06 2022-07-20 エフ－スター・セラピューティクス・インコーポレイテッド Compounds, compositions and methods for the treatment of disease
KR20190032408A (en) * 2016-07-06 2019-03-27 스페로비 바이오사이언시즈 인코포레이티드 Compounds, compositions and methods for the treatment of disease
JP7100227B2 (en) 2016-07-06 2022-07-13 エフ－スター・セラピューティクス・インコーポレイテッド Compounds, compositions, and methods for the treatment of diseases
IL264049B1 (en) * 2016-07-06 2023-07-01 Sperovie Biosciences Inc Compounds, compositions, and methods for the treatment of disease
US20190262372A1 (en) * 2016-07-06 2019-08-29 Sperovie Biosciences, Inc. Compounds,compositions, and methods for the treatment of disease
US11033569B2 (en) * 2016-07-06 2021-06-15 Sperovie Biosciences, Inc. Compounds,Compositions, and methods for the treatment of disease
AU2017293781B2 (en) * 2016-07-06 2022-12-22 F-star Therapeutics, Inc. Compounds, compositions, and methods for the treatment of disease
JP2019532084A (en) * 2016-07-06 2019-11-07 スペロビー・バイオサイエンシズ・インコーポレイテッド Compounds, compositions and methods for the treatment of disease
CN109843302A (en) * 2016-07-06 2019-06-04 斯必乐维生物科学公司 For treating the compound, composition and method of disease
US11744845B2 (en) 2016-07-06 2023-09-05 F-star Therapeutics, Inc. Compounds,compositions, and methods for the treatment of disease
EP3484504A4 (en) * 2016-07-15 2020-07-29 Sperovie Biosciences, Inc. Compounds, compositions, and methods for the treatment of disease
WO2018020476A1 (en) 2016-07-29 2018-02-01 Aduro Biotech Holdings, Europe B.V. Anti-pd-1 antibodies
US10494436B2 (en) 2016-07-29 2019-12-03 Aduro Biotech Holdings, Europe B.V. Anti-PD-1 antibodies
WO2018025178A1 (en) 2016-08-02 2018-02-08 Aduro Biotech Holdings, Europe B.V. Antibodies against human ctla-4
US10808030B2 (en) 2016-08-02 2020-10-20 Aduro Biotech Holdings, Europe B.V. Anti-HCTLA-4 antibodies
AU2017333933B2 (en) * 2016-09-30 2022-01-06 Boehringer Ingelheim International Gmbh Cyclic dinucleotide compounds
CN109843903A (en) * 2016-09-30 2019-06-04 勃林格殷格翰国际有限公司 Ring dinucleotides compound
KR20190057131A (en) * 2016-09-30 2019-05-27 베링거 인겔하임 인터내셔날 게엠베하 The cyclic dinucleotide compound
TWI774697B (en) * 2016-09-30 2022-08-21 德商百靈佳殷格翰國際股份有限公司 Cyclic dinucleotide compounds
US10537590B2 (en) 2016-09-30 2020-01-21 Boehringer Ingelheim International Gmbh Cyclic dinucleotide compounds
EA037030B1 (en) * 2016-09-30 2021-01-28 Бёрингер Ингельхайм Интернациональ Гмбх Cyclic dinucleotide compounds
KR102536447B1 (en) * 2016-09-30 2023-05-26 베링거 인겔하임 인터내셔날 게엠베하 Cyclic Dinucleotide Compound
JP2018536695A (en) * 2016-09-30 2018-12-13 ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング Cyclic dinucleotide compound
CN109843903B (en) * 2016-09-30 2023-01-24 勃林格殷格翰国际有限公司 Cyclic dinucleotide compounds
WO2018060323A1 (en) 2016-09-30 2018-04-05 Boehringer Ingelheim International Gmbh Cyclic dinucleotide compounds
US10730849B2 (en) 2016-10-04 2020-08-04 Merck Sharp & Dohme Corp. Benzo[b]thiophene compounds as STING agonists
US10703738B2 (en) 2016-10-04 2020-07-07 Merck Sharp & Dohme Corp. Benzo[b]thiophene compounds as STING agonists
US10414747B2 (en) 2016-10-04 2019-09-17 Merck Sharp & Dohme Corp. Benzo[b]thiophene compounds as sting agonists
US11001605B2 (en) 2016-10-07 2021-05-11 Biolog Life Science Institute Gmbh & Co. Kg Cyclic dinucleotides containing benzimidazole, method for the production of same, and use of same to activate stimulator of interferon genes (sting)-dependent signaling pathways
WO2018065360A1 (en) 2016-10-07 2018-04-12 Biolog Life Science Institute Forschungslabor Und Biochemica-Vertrieb Gmbh Cyclic dinucleotides containing benzimidazole, method for the production of same, and use of same to activate stimulator of interferon genes (sting)-dependent signaling pathways
JP2020504085A (en) * 2016-12-01 2020-02-06 武田薬品工業株式会社 Cyclic dinucleotides as STING (stimulator of interferon genes) agonists
WO2018100558A2 (en) 2016-12-01 2018-06-07 Takeda Pharmaceutical Company Limited Cyclic dinucleotide
US11666594B2 (en) 2016-12-01 2023-06-06 Takeda Pharmaceutical Company Limited Antibody-drug conjugates comprising a cyclic dinucleotide
WO2018100558A3 (en) * 2016-12-01 2018-07-19 Takeda Pharmaceutical Company Limited Cyclic purine dinucleotides as sting (stimulator of inteferon genes) agonists
JP2020055860A (en) * 2016-12-01 2020-04-09 武田薬品工業株式会社 Cyclic dinucleotide as sting (stimulator of interferon genes) agonist
US10980825B2 (en) 2016-12-01 2021-04-20 Takeda Pharmaceutical Company Limited Cyclic dinucleotide
CN110325543B (en) * 2016-12-01 2023-03-31 武田药品工业株式会社 Cyclic dinucleotides as STING (stimulator of interferon genes) agonists
TWI769195B (en) * 2016-12-01 2022-07-01 日商武田藥品工業股份有限公司 Cyclic dinucleotide
CN110325543A (en) * 2016-12-01 2019-10-11 武田药品工业有限公司 Cyclic annular dinucleotides as STING (interferon gene stimulating factor) agonist
KR20190113768A (en) * 2016-12-01 2019-10-08 다케다 야쿠힌 고교 가부시키가이샤 Cyclic dinucleotides as STING (stimulators of the interferon gene) agonists
EP3868384A1 (en) 2016-12-01 2021-08-25 Takeda Pharmaceutical Company Limited Cyclic dinucleotides as sting (stimulator of interferon genes) agonists
KR102640802B1 (en) 2016-12-01 2024-02-26 다케다 야쿠힌 고교 가부시키가이샤 Cyclic dinucleotides as STING (stimulator of interferon genes) agonists
IL267020A (en) * 2016-12-01 2019-07-31 Takeda Pharmaceuticals Co Cyclic dinucleotides as sting (stimulator of interferon genes) agonists
US20240066047A1 (en) * 2016-12-01 2024-02-29 Takeda Pharmaceutical Company Limited Antibody-drug conjugates comprising a cyclic dinucleotide
JP7229907B2 (en) 2016-12-01 2023-02-28 武田薬品工業株式会社 Cyclic dinucleotides as STING (stimulator of interferon genes) agonists
EP3558324A4 (en) * 2016-12-20 2020-08-05 Merck Sharp & Dohme Corp. Cyclic dinucleotide sting agonists for cancer treatment
CN110446503A (en) * 2016-12-22 2019-11-12 马福制药公司 Phosphodiesterase inhibitors and antimicrobial treatments method
US11400108B2 (en) 2017-02-21 2022-08-02 Board Of Regents, The University Of Texas System Cyclic dinucleotides as agonists of stimulator of interferon gene dependent signalling
WO2018156625A1 (en) * 2017-02-21 2018-08-30 Board Of Regents,The University Of Texas System Cyclic dinucleotides as agonists of stimulator of interferon gene dependent signalling
US10933078B2 (en) 2017-02-21 2021-03-02 Board Of Regents, The University Of Texas System Cyclic dinucleotides as agonists of stimulator of interferon gene dependent signalling
US10392419B2 (en) * 2017-03-22 2019-08-27 Boehringer Ingelheim International Gmbh Modified cyclic dinucleotide compounds
WO2018172206A1 (en) 2017-03-22 2018-09-27 Boehringer Ingelheim International Gmbh Modified cyclic dinucleotide compounds
WO2018190719A2 (en) 2017-04-13 2018-10-18 Aduro Biotech Holdings, Europe B.V. Anti-sirp alpha antibodies
WO2018189382A1 (en) 2017-04-14 2018-10-18 Solstice Biologics, Ltd. Immunomodulating polynucleotides, antibody conjugates thereof, and methods of their use
WO2018198084A1 (en) * 2017-04-27 2018-11-01 Lupin Limited Cyclic di-nucleotide compounds with tricyclic nucleobases
WO2018200812A1 (en) 2017-04-28 2018-11-01 Novartis Ag Antibody conjugates comprising sting agonist
US10975114B2 (en) 2017-04-28 2021-04-13 Chinook Therapeutics, Inc. Bis 2′-5′-RR-(3′F-A)(3′F-A) cyclic dinucleotide compound and uses thereof
WO2018198076A1 (en) 2017-04-28 2018-11-01 Aduro Biotech, Inc. Bis 2'-5'-rr-(3'f-a)(3'f-a) cyclic dinucleotide compound and uses thereof
US11466047B2 (en) 2017-05-12 2022-10-11 Merck Sharp & Dohme Llc Cyclic di-nucleotide compounds as sting agonists
WO2018222711A2 (en) 2017-05-30 2018-12-06 Bristol-Myers Squibb Company Compositions comprising a combination of an anti-lag-3 antibody, a pd-1 pathway inhibitor, and an immunotherapeutic agent
EP4245375A2 (en) 2017-05-30 2023-09-20 Bristol-Myers Squibb Company Compositions comprising a combination of an anti-lag-3 antibody, a pd-1 pathway inhibitor, and an immunotherapeutic agent
JP7186764B2 (en) 2017-07-28 2022-12-09 ブリストル－マイヤーズ スクイブ カンパニー Cyclic dinucleotides as anticancer agents
CN110997691A (en) * 2017-07-28 2020-04-10 百时美施贵宝公司 Cyclic dinucleotides as anticancer agents
CN110997691B (en) * 2017-07-28 2023-11-07 百时美施贵宝公司 Cyclic dinucleotides as anticancer agents
WO2019023459A1 (en) * 2017-07-28 2019-01-31 Bristol-Myers Squibb Company Cyclic dinucleotides as anticancer agents
JP2020529400A (en) * 2017-07-28 2020-10-08 ブリストル−マイヤーズ スクイブ カンパニーＢｒｉｓｔｏｌ−Ｍｙｅｒｓ Ｓｑｕｉｂｂ Ｃｏｍｐａｎｙ Cyclic dinucleotide as an anticancer drug
US10744150B2 (en) 2017-07-28 2020-08-18 Bristol-Myers Squibb Company Cyclic dinucleotides as anticancer agents
US11312772B2 (en) 2017-08-04 2022-04-26 Merck Sharp & Dohme Corp. Combinations of PD-1 antagonists and benzo [b] thiophene STING agonists for cancer treatment
US11285131B2 (en) 2017-08-04 2022-03-29 Merck Sharp & Dohme Corp. Benzo[b]thiophene STING agonists for cancer treatment
EP3692048A4 (en) * 2017-08-30 2021-10-20 Beijing Xuanyi Pharmasciences Co., Ltd. Cyclic di-nucleotides as stimulator of interferon genes modulators
TWI808092B (en) * 2017-08-30 2023-07-11 中國大陸商北京軒義醫藥科技有限公司 Cyclic di-nucleotides as stimulator of interferon genes modulators
US11773132B2 (en) 2017-08-30 2023-10-03 Beijing Xuanyi Pharmasciences Co., Ltd. Cyclic di-nucleotides as stimulator of interferon genes modulators
CN111032672A (en) * 2017-08-31 2020-04-17 百时美施贵宝公司 Cyclic dinucleotides as anticancer agents
US11707531B2 (en) 2017-09-11 2023-07-25 F-star Therapeutics, Inc. Compounds, compositions, and methods for the treatment of disease
US11584774B2 (en) 2017-09-11 2023-02-21 F-star Therapeutics, Inc. Compounds, compositions, and methods for the treatment of disease
WO2019069275A1 (en) * 2017-10-05 2019-04-11 Glaxosmithkline Intellectual Property Development Limited Methods for administering sting agonists
CN111194214A (en) * 2017-10-05 2020-05-22 葛兰素史密斯克莱知识产权发展有限公司 Methods of administering STING agonists
WO2019084060A1 (en) 2017-10-24 2019-05-02 Silverback Therapeutics, Inc. Conjugates and methods of use thereof for selective delivery of immune-modulatory agents
WO2019092660A1 (en) 2017-11-10 2019-05-16 Takeda Pharmaceutical Company Limited Sting modulator compounds, and methods of making and using
US11542293B2 (en) 2017-11-10 2023-01-03 Takeda Pharmaceutical Company Limited Sting modulator compounds, and methods of making and using
EA038805B1 (en) * 2017-11-21 2021-10-21 Такеда Фармасьютикал Компани Лимитед Cyclic dinucleotides as sting (stimulator of interferon genes) agonists
RU2776060C2 (en) * 2017-12-15 2022-07-13 Янссен Байотек, Инк. Cyclic dinucleotides as sting agonists
US10966999B2 (en) 2017-12-20 2021-04-06 Institute Of Organic Chemistry And Biochemistry Ascr, V.V.I. 3′3′ cyclic dinucleotides with phosphonate bond activating the sting adaptor protein
US11203610B2 (en) 2017-12-20 2021-12-21 Institute Of Organic Chemistry And Biochemistry Ascr, V.V.I. 2′3′ cyclic dinucleotides with phosphonate bond activating the sting adaptor protein
US11685761B2 (en) 2017-12-20 2023-06-27 Merck Sharp & Dohme Llc Cyclic di-nucleotide compounds as sting agonists
EP3505527A1 (en) 2017-12-29 2019-07-03 Invivogen Cyclic dinucleotides for cytokine induction
JP7337086B2 (en) 2018-03-23 2023-09-01 武田薬品工業株式会社 STING MODULATOR COMPOUNDS HAVING SULFAMATE LINKS AND METHODS OF MAKING AND USING
WO2019180683A1 (en) 2018-03-23 2019-09-26 Takeda Pharmaceutical Company Limited Sting modulator compounds with sulfamate linkages, and methods of making and using
EP4242212A3 (en) * 2018-03-23 2023-11-15 Takeda Pharmaceutical Company Limited Sting modulator compounds with sulfamate linkages, and methods of making and using
WO2019183578A1 (en) 2018-03-23 2019-09-26 Codiak Biosciences, Inc. Extracellular vesicles comprising sting-agonist
JP2021518400A (en) * 2018-03-23 2021-08-02 武田薬品工業株式会社 STING modulator compounds with sulfamate bonds, and methods of manufacture and use
EP4242212A2 (en) 2018-03-23 2023-09-13 Takeda Pharmaceutical Company Limited Sting modulator compounds with sulfamate linkages, and methods of making and using
WO2019185477A1 (en) 2018-03-27 2019-10-03 Boehringer Ingelheim International Gmbh Cyclic dinucleotide compounds containing 2-aza-hypoxanthine or 6h-pytazolo[1,5-d][1,2,4]triazin-7-one as sting agonists
WO2019185476A1 (en) 2018-03-27 2019-10-03 Boehringer Ingelheim International Gmbh Modified cyclic dinucleotide compounds
US11702430B2 (en) 2018-04-03 2023-07-18 Merck Sharp & Dohme Llc Aza-benzothiophene compounds as STING agonists
WO2019195124A1 (en) 2018-04-03 2019-10-10 Merck Sharp & Dohme Corp. Benzothiophenes and related compounds as sting agonists
US10793557B2 (en) 2018-04-03 2020-10-06 Merck Sharp & Dohme Corp. Sting agonist compounds
US11292812B2 (en) 2018-04-06 2022-04-05 Institute Of Organic Chemistry And Biochemistry Ascr, V.V.I. 3′3′-cyclic dinucleotides
US11149052B2 (en) 2018-04-06 2021-10-19 Institute Of Organic Chemistry And Biochemistry Ascr, V.V.I. 2′3′-cyclic dinucleotides
WO2020041720A1 (en) 2018-08-24 2020-02-27 Codiak Biosciences, Inc. Extracellular vesicles targeting dendritic cells and uses thereof
WO2020056008A1 (en) 2018-09-12 2020-03-19 Silverback Therapeutics, Inc. Compositions for the treatment of disease with immune stimulatory conjugates
WO2020065408A1 (en) 2018-09-27 2020-04-02 Pierre Fabre Medicament Sulfomaleimide-based linkers and corresponding conjugates
EP3868773A4 (en) * 2018-10-12 2022-07-13 Shanghai Jemincare Pharmaceuticals Co., Ltd. Cyclic dinucleotide compound and uses thereof
US11110106B2 (en) 2018-10-29 2021-09-07 Venenum Biodesign, LLC Sting agonists for treating bladder cancer and solid tumors
US11161864B2 (en) 2018-10-29 2021-11-02 Venenum Biodesign, LLC Sting agonists
US11883420B2 (en) 2018-10-29 2024-01-30 Venenum Biodesign, LLC Sting agonists for treating bladder cancer and solid tumors
WO2020092633A1 (en) 2018-10-30 2020-05-07 Vanderbilt University Graft copolymers, methods of forming graft copolymers, and methods of use thereof
WO2020092617A1 (en) 2018-10-31 2020-05-07 Novartis Ag Dc-sign antibody conjugates comprising sting agonists
JP7224688B2 (en) 2019-01-10 2023-02-20 南▲開▼大学 Cyclic dinucleotide prodrug molecules, methods of making and uses thereof
JP2022517592A (en) * 2019-01-10 2022-03-09 南▲開▼大学 Cyclic dinucleotide prodrug molecule, its production method and use
CN111423483A (en) * 2019-01-10 2020-07-17 南开大学 Cyclic dinucleotide prodrug molecule and preparation method and application thereof
WO2020191377A1 (en) 2019-03-21 2020-09-24 Codiak Biosciences, Inc. Extracellular vesicle conjugates and uses thereof
WO2020191361A2 (en) 2019-03-21 2020-09-24 Codiak Biosciences, Inc. Extracellular vesicles for vaccine delivery
WO2020191369A1 (en) 2019-03-21 2020-09-24 Codiak Biosciences, Inc. Process for preparing extracellular vesicles
WO2020205688A1 (en) 2019-04-04 2020-10-08 Merck Sharp & Dohme Corp. Inhibitors of histone deacetylase-3 useful for the treatment of cancer, inflammation, neurodegeneration diseases and diabetes
US11787833B2 (en) 2019-05-09 2023-10-17 Aligos Therapeutics, Inc. Modified cyclic dinucleoside compounds as sting modulators
WO2021003445A1 (en) 2019-07-03 2021-01-07 Codiak Biosciences, Inc. Extracellular vesicles targeting t cells and uses thereof
US11939343B2 (en) 2019-08-02 2024-03-26 Mersana Therapeutics, Inc. Sting agonist compounds and methods of use
WO2021030251A1 (en) 2019-08-12 2021-02-18 Purinomia Biotech, Inc. Methods and compositions for promoting and potentiating t-cell mediated immune responses through adcc targeting of cd39 expressing cells
WO2021062058A1 (en) 2019-09-25 2021-04-01 Codiak Biosciences, Inc. Sting agonist comprising exosomes for treating neuroimmunological disorders
WO2021062290A1 (en) 2019-09-25 2021-04-01 Codiak Biosciences, Inc. Methods of producing extracellular vesicles
WO2021062060A1 (en) 2019-09-25 2021-04-01 Codiak Biosciences, Inc. Sting agonist comprising exosomes combined with il-12 displaying exosomes for treating a tumour
WO2021062317A1 (en) 2019-09-25 2021-04-01 Codiak Biosciences, Inc. Extracellular vesicle compositions
WO2021067644A1 (en) 2019-10-01 2021-04-08 Silverback Therapeutics, Inc. Combination therapy with immune stimulatory conjugates
WO2021074695A1 (en) 2019-10-16 2021-04-22 Avacta Life Sciences Limited PD-L1 INHIBITOR - TGFβ INHIBITOR BISPECIFIC DRUG MOIETIES.
WO2021113679A1 (en) 2019-12-06 2021-06-10 Mersana Therapeutics, Inc. Dimeric compounds as sting agonists
US11179473B2 (en) 2020-02-21 2021-11-23 Silverback Therapeutics, Inc. Nectin-4 antibody conjugates and uses thereof
WO2021168274A1 (en) 2020-02-21 2021-08-26 Silverback Therapeutics, Inc. Nectin-4 antibody conjugates and uses thereof
US11795463B2 (en) 2020-02-28 2023-10-24 Tallac Therapeutics, Inc. Transglutaminase-mediated conjugation
US11920136B2 (en) 2020-02-28 2024-03-05 Tallac Therapeutics, Inc. Transglutaminase-mediated conjugation
WO2021184017A1 (en) 2020-03-13 2021-09-16 Codiak Biosciences, Inc. Extracellular vesicles for treating neurological disorders
WO2021189047A2 (en) 2020-03-20 2021-09-23 Codiak Biosciences, Inc. Extracellular vesicles for therapy
WO2021206158A1 (en) 2020-04-10 2021-10-14 小野薬品工業株式会社 Method of cancer therapy
WO2021232019A1 (en) 2020-05-15 2021-11-18 Immunesensor Therapeutics, Inc. Sting agonist combination treatments with immune checkpoint inhibitors
WO2021237100A1 (en) 2020-05-21 2021-11-25 Codiak Biosciences, Inc. Methods of targeting extracellular vesicles to lung
WO2022006327A1 (en) 2020-07-01 2022-01-06 Silverback Therapeutics, Inc. Anti-asgr1 antibody conjugates and uses thereof
US11541126B1 (en) 2020-07-01 2023-01-03 Silverback Therapeutics, Inc. Anti-ASGR1 antibody TLR8 agonist comprising conjugates and uses thereof
WO2022066898A2 (en) 2020-09-23 2022-03-31 Codiak Biosciences, Inc. Methods of producing extracellular vesicles
WO2022066883A1 (en) 2020-09-23 2022-03-31 Codiak Biosciences, Inc. Extracellular vesicles comprising kras antigens and uses thereof
WO2022066928A2 (en) 2020-09-23 2022-03-31 Codiak Biosciences, Inc. Process for preparing extracellular vesicles
WO2022066934A2 (en) 2020-09-23 2022-03-31 Codiak Biosciences, Inc. Process for preparing extracellular vesicles
US11725024B2 (en) 2020-11-09 2023-08-15 Takeda Pharmaceutical Company Limited Antibody drug conjugates
WO2022133288A1 (en) 2020-12-17 2022-06-23 Trustees Of Tufts College Fap-activated radiotheranostics, and uses related thereto
WO2022234003A1 (en) 2021-05-07 2022-11-10 Avacta Life Sciences Limited Cd33 binding polypeptides with stefin a protein
WO2023004440A2 (en) 2021-07-23 2023-01-26 Immunesensor Therapeutics, Inc. Sting agonist combination treatments with cytokines
WO2023056468A1 (en) 2021-09-30 2023-04-06 Codiak Biosciences, Inc. Extracellular vesicle comprising cholesterol tagged sting-agonist
WO2023057567A1 (en) 2021-10-07 2023-04-13 Avacta Life Sciences Limited Pd-l1 binding affimers
WO2023170606A1 (en) 2022-03-08 2023-09-14 Alentis Therapeutics Ag Use of anti-claudin-1 antibodies to increase t cell availability
WO2023218243A1 (en) 2022-05-12 2023-11-16 Avacta Life Sciences Limited Lag-3/pd-l1 binding fusion proteins

Also Published As

Publication number Publication date
US20180354983A1 (en) 2018-12-13
US11667664B2 (en) 2023-06-06
US20200255469A1 (en) 2020-08-13
US11053272B2 (en) 2021-07-06
EP3546473A1 (en) 2019-10-02
DK3233882T3 (en) 2020-01-27
PT3233882T (en) 2020-01-21
US10562929B2 (en) 2020-02-18
US20160362441A1 (en) 2016-12-15
WO2016096174A8 (en) 2016-09-29
US20220017561A1 (en) 2022-01-20
ES2764178T3 (en) 2020-06-02
EP3233882A1 (en) 2017-10-25
CN107148424A (en) 2017-09-08
EP3233882B1 (en) 2019-10-30
PL3233882T3 (en) 2020-04-30
CN107148424B (en) 2021-01-08
US10011630B2 (en) 2018-07-03

Similar Documents

Publication Publication Date Title
US11667664B2 (en) Cyclic dinucleotides for cytokine induction
US11001605B2 (en) Cyclic dinucleotides containing benzimidazole, method for the production of same, and use of same to activate stimulator of interferon genes (sting)-dependent signaling pathways
Lioux et al. Design, synthesis, and biological evaluation of novel cyclic adenosine–inosine monophosphate (cAIMP) analogs that activate stimulator of interferon genes (STING)
AU2012223012B2 (en) Phosphoramidate derivatives of 5 - fluoro - 2 ' - deoxyuridine for use in the treatment of cancer
US9351989B2 (en) Substituted purine nucleosides, phosphoroamidate and phosphorodiamidate derivatives for treatment of viral infections
AU2003217863B9 (en) Nucleotide mimics and their prodrugs
EP1778251B1 (en) Nucleoside phosphonate conjugates as anti hiv agents
KR20090007569A (en) 2'-fluoronucleoside phosphonates as antiviral agents
AU780879B2 (en) Anti-viral pyrimidine nucleoside analogues
EA028974B1 (en) Hcv polymerase inhibitors
KR20050059975A (en) β-2'- or 3'-halonucleosides
KR20140068245A (en) Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
KR20050040912A (en) Compounds with the bicyclo[4.2.1]nonane system for the treatment of flaviviridae infections
WO2004080466A1 (en) Cytidine analogs and methods of use
JP2008532950A (en) Bicyclic nucleosides and bicyclic nucleotides as therapeutic agents
KR20110115601A (en) Purine nucleoside monophosphate prodrugs for treatment of cancer and viral infections
JP2018507261A (en) Β-D-2'-deoxy-2'-α-fluoro-2'-β-C-substituted-2-modified-N6-substituted purine nucleotides for HCV treatment
EP3505527A1 (en) Cyclic dinucleotides for cytokine induction
Lim et al. Novel applications of biocatalysis to stereochemistry determination of 2′ 3′-cGAMP bisphosphorothioate (2′ 3′-cGSASMP)
Slusarczyk et al. Synthesis and biological evaluation of 6-substituted-5-fluorouridine ProTides
Stazzoni Design and synthesis of clickable nucleic acid analogues for cancer therapy and diagnosis
KR101323698B1 (en) Modified 5'-phosphonates of AZT potential antiviral drugs
Xie et al. Design, synthesis, and cell-based in vitro assay of deoxyinosine-mixed SATE-dCDN prodrugs that activate all common STING variants
US20040158054A1 (en) Di-ribonucleotides as specific viral RNA-polymerase inhibitors for the treatment or prevention of viral infections
Xie et al. Design, Synthesis, and Biological Evaluation of Deoxyinosine-Mixed SATE-dCDN Prodrugs that Activate All Different STING Variants

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 15034335

Country of ref document: US

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15762583

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

REEP Request for entry into the european phase

Ref document number: 2015762583

Country of ref document: EP