US20200055883A1 - Cyclic di-nucleotides derivative for the treatment of cancer - Google Patents

Cyclic di-nucleotides derivative for the treatment of cancer Download PDF

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US20200055883A1
US20200055883A1 US16/486,954 US201816486954A US2020055883A1 US 20200055883 A1 US20200055883 A1 US 20200055883A1 US 201816486954 A US201816486954 A US 201816486954A US 2020055883 A1 US2020055883 A1 US 2020055883A1
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Dae-Shik Kim
Frank Fang
Atsushi Endo
Hyeong-Wook Choi
Ming-Hong Hao
Xingfeng BAO
Kuan-Chun Huang
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Eisai R&D Management Co Ltd
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Eisai R&D Management Co Ltd
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    • 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
    • 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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
    • C07F9/6581—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and nitrogen atoms with or without oxygen or sulfur atoms, as ring hetero atoms
    • C07F9/6587—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and nitrogen atoms with or without oxygen or sulfur atoms, as ring hetero atoms having two phosphorus atoms as ring hetero atoms in the same ring
    • 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

Definitions

  • STING (stimulator of interferon genes) is a signaling molecule in the innate response to dsDNA in the cytosol. STING deletion has been reported in multiple human cancers. In addition, deregulation of STING signaling in human cancers also has been reported in melanoma (Xia T, et al., “Recurrent Loss of STING Signaling in Melanoma Correlates with Susceptibility to Viral Oncolysis” Cancer Res. 2016) and colon cancer. (Xia T, et al., “Deregulation of STING Signaling in Colorectal Carcinoma Constrains DNA Damage Responses and Correlates With Tumorigenesis” Cell Rep. 2016; 14:282-97).
  • Embodiments may provide a compound of Formula (III):
  • R 1a is selected from the group consisting of —H, halogen, —C 1-6 alkyl, —N 3 , —CH 2 R 2a , —NHR 2a , or —OR 2a ;
  • R 2a is selected from the group consisting of —H, —C 1-6 -alkyl, or R 2a and R 3 are connected and together represent —CH 2 —, —CH 2 CH 2 —, —CH(CH 2 Y 1 )—, or —CH 2 CH(CH 2 Y 1 )—;
  • Y 1 is selected from the group consisting of —H; —OH optionally substituted with —C 1-3 alkyl; —NH 2 optionally substituted with C 1-3 alkyl; allyl optionally substituted with (i) —OH, (ii) —OCH 3 , (iii) C 1-3 alkyl, or (iv) halogen; vinyl, optionally substituted with
  • X 10 , X 1i , X 12 , X 13 , X 14 , and X 15 are independently selected from a bond; —O—; —NH—; —S—; —C(O)—; —CH—, optionally substituted by (i) (ii) —OH, or (iii) —OCH 3 ; and —CH 2 —, optionally substituted by (i) (ii) —OH, (iii) or —OCH 3 ; and wherein any two adjacent or three contiguous members of the group including X 10 , X 11 , X 12 , X 13 , X 14 , and X 15 may optionally form, with additional atoms, a C 3-6 cycloalkyl, optionally substituted with one or more of (i) C 1-3 alkyl, (ii) —OH, or (iii) —OMe; C 5-6 cycloaryl, optionally substituted with one
  • X 16 is selected from the group consisting of —CH 2 —, —O—, —NH—, —S—, and —C(O)—, optionally substituted with C 1-3 alkyl, —OH, or —OCH 3 ; wherein R 13 , R 14 , R 15 , R 16 , and R 17 , when present, are independently selected from the group consisting of —H, —F, —Cl, —I, —Br, —OH, —SH, —NH 2 , C 1-3 alkyl, C 3-6 cycloalkyl, —O(C 1-3 alkyl), —O(C 3-6 cycloalkyl), —S(C 1-3 alkyl), —S(C 3-6 cycloalkyl), —NH(C 1-3 alkyl), —NH(C 3-6 cycloalkyl), —N(C 1-3 alkyl) 2 , and —N(C 3-6 cyclo
  • R 23 and R 24 are the same or different and are selected from the group consisting of —H and —C 1-3 alkyl.
  • R 1a is selected from the group consisting of —H, halogen, —C 1-6 alkyl, —N 3 , —CH 2 R 2a , —NHR 2a , or —OR 2a ;
  • R ea is selected from the group consisting of —H and —C 1-6 -alkyl;
  • R 1b is selected from the group consisting of —H, halogen, —C 1-6 alkyl, —OR 2b , —NHR 2b , or, with R 11 , forms C 4-6 cycloalkyl or C 4-6 heterocycloalkyl;
  • R 2b is selected from the group consisting of —H, or —C 1-6 alkyl;
  • R 3 is —H; —F; —C 1-6 alkyl, optionally substituted by (i) C 1-3 alkyl, (ii) —OH, (iii) halogen, or (iv) —OCH 3
  • X 10 , X 11 , X 12 , X 13 X 14 , and X 15 are independently selected from a bond; —O—; —NH—; —S—; —C(O)—; —CH—, optionally substituted by (i) C 1 -C 3 alkyl, (ii) —OH, or (iii) —OCH 3 ; and —CH 2 —, optionally substituted by (i) C 1-3 alkyl, (ii) —OH, (iii) or —OCH 3 ; and wherein any two adjacent or three contiguous members of the group including X 10 , X 11 , X 12 , X 13 , X 14 , and X 15 may optionally form, with additional atoms, a C 3-6 cycloalkyl, optionally substituted with one or more of (i) C 1-3 alkyl, (ii) —OH, or (iii) —OMe; C
  • X 16 is selected from the group consisting of —CH2-, —O—, —NH—, —S—, and —C(O)—, optionally substituted with C 1-3 alkyl, —OH, or —OCH 3 ; wherein R 13 , R 14 , R 15 , R 16 , and R 17 , when present, are independently selected from the group consisting of —H, —F, —Cl, —I, —Br, —OH, —SH, —NH 2 , C 1-3 alkyl, C 3-6 cycloalkyl, —O(C 1-3 alkyl), —O(C 3-6 cycloalkyl), —S(C 1-3 alkyl), —S(C 3-6 cycloalkyl), —NH(C 1-3 alkyl), —NH(C 3-6 cycloalkyl), —NH(C 1-3 alkyl), —NH(C 3-6 cycloalkyl), —
  • R 23 and R 24 are the same or different and are selected from the group consisting of —H and —C 1-3 alkyl.
  • R 1a is selected from the group consisting of —H, halogen, —C 1-6 alkyl, —N 3 , —CH 2 R 2a , —NHR 2a , or —OR 2a ;
  • R ea is selected from the group consisting of —H and —C 1-6 -alkyl;
  • R 1b is selected from the group consisting of —H, halogen, —C 1-6 alkyl, —OR 2b , —NHR 2b , or, with R 11 , forms C 4-6 cycloalkyl or C 4-6 heterocycloalkyl;
  • R 2b is selected from the group consisting of —H, or —C 1-6 alkyl;
  • R 3 is —H; —F; —C 1-6 alkyl, optionally substituted by (i) C 1-3 alkyl, (ii) —OH, (iii) halogen, or (iv) —OCH 3
  • X 10 , X 11 , X 12 , X 13 , X 14 , and X 15 are independently selected from a bond; —O—; —NH—; —S—; —C(O)—; —CH—, optionally substituted by (i) C 1 -C 3 alkyl, (ii) —OH, or (iii) —OCH 3 ; and —CH 2 —, optionally substituted by (i) C 1-3 alkyl, (ii) —OH, (iii) or —OCH 3 ; and wherein any two adjacent or three contiguous members of the group including X 10 , X 1i , X 12 , X 13 , X 14 , and X 15 may form, optionally with additional atoms, a C 3-6 cycloalkyl, optionally substituted with one or more of (i) C 1-3 alkyl, (ii) —OH, or (iii) —OM
  • X 16 is selected from the group consisting of —CH2-, —O—, —NH—, —S—, and —C(O)—, optionally substituted with C 1-3 alkyl, —OH, or —OCH 3 ; wherein R 13 , R 14 , R 15 , R 16 , and R 17 , when present, are independently selected from the group consisting of —H, —F, —Cl, —I, —Br, —OH, —SH, —NH 2 , C 1-3 alkyl, C 3-6 cycloalkyl, —O(C 1-3 alkyl), —O(C 3-6 cycloalkyl), —S(C 1-3 alkyl), —S(C 3-6 cycloalkyl), —NH(C 1-3 alkyl), —NH(C 3-6 cycloalkyl), —NH(C 1-3 alkyl), —NH(C 3-6 cycloalkyl), —
  • R 23 and R 24 are the same or different and are selected from the group consisting of —H and —C 1-3 alkyl.
  • the pharmaceutically acceptable salt is a diammonium salt.
  • X 1b and X 2b are —SH.
  • R 4a is —F
  • R 4b , R 12 , and R 6 are —H.
  • R 4b is —F, and R 4a , R 12 , and R 6 are —H.
  • R 12 is —F, and R 4a , R 4b , and R 6 are —H.
  • R 6 is —F, and R 4a , R 4b , and R 12 are —H.
  • R 1a is —F
  • R 3 , R 1b , and R 11 are —H.
  • R 3 is —F, and R 1a , R 1b , and R 11 are —H.
  • R 1b is —F, and R 1a , R 3 , and R 11 are —H.
  • R 11 is —F, and R 1a , R 1b , and R 3 are —H.
  • X 1a and X 2a are both ⁇ O.
  • B 1 and B 2 are both
  • X 7 and X 8 are both O.
  • L 1 is
  • X 1a is S
  • X 1b is —SH
  • X 2a is S
  • X 2b is —SH
  • Z 1 and Z 2 are both —O—.
  • X 17 and X 1g are both —O—.
  • Z 1 , Z 2 , X 17 , and X 1g are each —O—.
  • R 25 and R 1g are both —H.
  • Embodiments may provide a compound or pharmaceutically acceptable salt with one or more of (i) an EC 50 value below 100 micromolar in a cell reporter assay expressing STING HAQ genetic variant; (ii) an EC 50 value below 100 micromolar in reporter cells expressing human STING AQ variant; (iii) an EC 50 value below 100 micromolar in reporter cells expressing human STING WT variant; and (iv) an EC 50 value below 100 micromolar in reporter cells expressing human STING REF variant.
  • Embodiments may provide a pharmaceutical composition comprising a compound or pharmaceutically acceptable salt as reported herein and a pharmaceutically acceptable excipient.
  • Embodiments may provide a method of treating cancer, comprising administering to a patient an effective amount of a pharmaceutical composition as reported herein.
  • Embodiments may further provide mixtures of compounds as reported herein, including mixtures of stereoisomers of these compounds.
  • a mixture may be provided of Compound 11 and Compound 12, or a mixture may be provided of Compound 2 and Compound 4.
  • these are not limiting examples and other mixtures are possible.
  • a compound as reported herein is provided as a free acid.
  • the compound is provided as an NH 4 salt. In other embodiments it is a bis-TEA salt.
  • Embodiments may provide a method of treating cancer in a patient in need thereof, including administering to the patient a therapeutically effective amount of a compound as reported herein or a pharmaceutically acceptable salt thereof, as recited above.
  • the compound is administered as a free acid. In some embodiments, the compound is administered as a diammonium salt (NH 4 ).
  • Pharmaceutical compositions including a compound as reported herein, or a pharmaceutically acceptable salt thereof, as well as a pharmaceutically acceptable excipient, are contemplated. Embodiments may provide a compound according to Formula I in which n is 1; geometry about the double bond is trans; X 1 and X 2 are each SH; stereochemistry at P 1 is S; and stereochemistry at P 2 is R; or a pharmaceutically acceptable salt thereof.
  • Embodiments may provide a method of treating cancer in a patient comprising administering to said patient a compound or pharmaceutically acceptable salt thereof, or a pharmaceutical composition, as reported herein.
  • Cancers treated as reported herein may be metastatic cancers. They may be selected from, for example, melanoma, colorectal cancer, breast cancer, acute myeloid leukemia, colon cancer, liver cancer, and glioma. Uses of compounds, salts, and pharmaceutical compositions for treatment of cancer and/or preparation of a medicament for treatment of cancer are also contemplated.
  • Embodiments may provide a method of treating cancer including identifying an individual having a cancer treatable by a compound, pharmaceutically acceptable salt, or pharmaceutical composition as reported herein, and administering to the individual a compound, pharmaceutically acceptable salt, or pharmaceutical composition by which the patient has been identified as treatable.
  • the individual is identified as having a cancer treatable by a compound, pharmaceutically acceptable salt, or pharmaceutical composition as reported herein by presence of a human REF STING variant allele in the patient.
  • Embodiments may provide a method of treating cancer in a patient having REF STING allele comprising administering to said patient a compound or pharmaceutically acceptable salt thereof, or pharmaceutical composition as reported herein.
  • Embodiments may provide a method of treating cancer in a patient having WT STING allele comprising administering to said patient a compound or pharmaceutically acceptable salt thereof, or pharmaceutical composition as reported herein.
  • Embodiments may provide a method of treating cancer in a patient having AQ STING allele comprising administering to said patient a compound or pharmaceutically acceptable salt thereof, or pharmaceutical composition as reported herein.
  • Embodiments may provide a method of treating cancer in a patient having HAQ STING allele comprising administering to said patient a compound or pharmaceutically acceptable salt thereof, or pharmaceutical composition as reported herein.
  • Embodiments may relate to C 4 -C 6 linkers that may be covalently bound at either end to purine or pyrimidine bases that form part of a cyclic dinucleotide.
  • the linkers are butene, pentene or hexene linkers bound at either end to purine bases.
  • the linkers are butene linkers bound at either end to purine bases; in another embodiment, the linkers are transbutene linkers with the double bond located between the center two carbons.
  • a 1 and A 2 are sugar moieties and may be the same or different;
  • B 3 and B 4 are purine or pyrimidine bases that may be the same or different and that form nucleotides with, respectively, A 1 and A 2 ;
  • L is a alkyl linker
  • X 19 and X 20 are the same or different and are selected from the group consisting of —O—, —CH—, —NH—, and —S—, wherein —CH— and —NH— may be substituted or unsubstituted.
  • a 1 , A 2 , B 3 , and B 4 may be further substituted with, for example, hydroxyl, halogen, or methoxy.
  • Each phosphorous in Formula (X) may be substituted, for example, with —SH, —OH, ⁇ O, or ⁇ S until its valence has been satisfied.
  • cyclic dinucleotide analogs that may benefit from linkers of the present invention include but are not limited to those identified in US 2014/0205653 A1; US 2014/0329889 A1; US 2014/0341976 A1; US 2015/0056224 A1; US 2016/0362441 A1; US 2017/0158724 A1; US 2017/044206 A1; U.S. Pat. Nos.
  • FIG. 1 shows a synthesis of Compound 1a and Compound 2a.
  • FIG. 2A and FIG. 2B show an alternate synthesis of Compound 1 and Compound 1a. That alternate synthesis is also shown in FIG. 2C .
  • FIG. 3 shows an 1 H NMR spectrograph for Compound 1.
  • FIG. 4A , FIG. 4B , and FIG. 4C show X-ray crystallography results (ORTEP drawings) for, respectively, an asymmetric crystal of Compound 1, a first molecule from the asymmetric crystal, and a second molecule from the asymmetric crystal.
  • FIG. 4D shows X-Ray crystallography results (ORTEP drawing) for a crystal of Compound 2.
  • FIG. 5A and FIG. 5B shows a synthesis route for Compounds 18, 19, and 20.
  • FIG. 6 shows an expression vector map for WT STING (pLenti-WT human STING-Puro).
  • FIG. 7 and FIG. 8 accompany Example 108 and show curative activity of Compound 1a in a CT26 dual tumor model.
  • FIG. 9 accompanies Example 109 and shows a tumor volume plot for treated tumors and survival curve.
  • FIG. 10 accompanies Example 110 and shows a tumor volume plot for treated tumors and survival curve.
  • FIG. 11 shows a picture of the X-ray crystal structure of human WT STING in complex with Compound 1.
  • FIG. 12 shows human REF STING C-terminal Domain in complex with Compound 1.
  • FIG. 13 shows an example of synthesis of Compound 38 and Compound 39.
  • the compounds may activate stimulator of interferon genes (STING).
  • the compound is provided as a free acid. In some embodiments, the compound is provided as, for example, an NH 4 salt or TEA salt. Reference to a compound number followed by an “a” will indicate a diammonium salt of the given compound. For example, “Compound 1a” refers to the diammonium salt of Compound 1.
  • Embodiments may provide a method of treating cancer in a patient in need thereof, including administering to the patient a therapeutically effective amount of a Compound of Formula I, Formula II, Formula III, Formula IV, or Formula V, or pharmaceutically acceptable salt thereof.
  • the compound is administered as a free acid. In some embodiments, the compound is administered as, for example, a diammonium salt (NH 4 ).
  • Pharmaceutical compositions for treating cancer may also be provided including a compound reported herein or a pharmaceutically acceptable salt thereof, as well as a pharmaceutically acceptable excipient.
  • Embodiments as reported herein may be used to treat cancer or to prepare medicaments useful for treatment of cancer.
  • “Cancer” may include, but is not limited to, colon cancer, liver cancer, melanoma, colorectal cancer, breast cancer, acute myeloid leukemia, and glioma.
  • the compound selected from the group consisting of the compounds reported herein is provided as a free acid or a pharmaceutically acceptable salt thereof. In some embodiments, the compound selected from the group consisting of the compounds reported herein is provided as an NH 4 salt, which may be a diammonium salt.
  • C 1-6 alkyl or “C 1 -C 6 ” alkyl is intended to include C 1 , C 2 , C 3 , C 4 , C 5 or C 6 straight chain (linear) saturated aliphatic hydrocarbon groups and C 3 , C 4 , C 5 or C 6 branched saturated aliphatic hydrocarbon groups.
  • C 1-6 alkyl is intended to include C 1 , C 2 , C 3 , C 4 , C 5 and C 6 alkyl groups.
  • alkyl examples include moieties having from one to six carbon atoms, such as, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl or n-hexyl.
  • C1-3alkyl or “C1-C3alkyl” is intended to include C 1 , C 2 , or C 3 straight chain (linear) saturated aliphatic hydrocarbon groups and C 3 branched saturated aliphatic hydrocarbon groups.
  • C 3-6 cycloalkyl or “C 3 -C 6 cycloalkyl” refers to a saturated or unsaturated nonaromatic hydrocarbon ring having 3 to 6 carbon atoms (e.g., C 3 -C 6 ).
  • Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, and cyclohexenyl.
  • C 5-6 cycloalkyl or “C 5 -C 6 cycloalkyl” refers to a saturated or unsaturated nonaromatic hydrocarbon ring having 5 or 6 carbon atoms (e.g., C 5 -C 6 ).
  • C 5-6 heterocycloalkyl or “C 5 -C 6 heterocycloalkyl” refers to a saturated or unsaturated nonaromatic 5-6 membered monocyclic having one or more heteroatoms (such as O, N, or S), unless specified otherwise.
  • C 4-6 heterocycloalkyl or “C 4 -C 6 heterocycloalkyl” refers to a saturated or unsaturated nonaromatic 4-6 membered monocyclic having one or more heteroatoms (such as O, N, or S), unless specified otherwise.
  • heterocycloalkyl groups include, but are not limited to, piperidinyl, piperazinyl, pyrrolidinyl, dioxanyl, tetrahydrofuranyl, isoindolinyl, indolinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl, oxiranyl, azetidinyl, oxetanyl, thietanyl, 1,2,3,6-tetrahydropyridinyl, tetrahydropyranyl, tetrahydrothiophene, dihydropyranyl, pyranyl, morpholinyl, 1,4-diazepanyl, 1,4-oxazepanyl, and the like.
  • heterocycloalkyl groups include, but are not limited to, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl,
  • C 5-6 aryl or “C 5 -C 6 aryl” refers to an aromatic hydrocarbon ring having 5 to 6 carbon atoms (e.g., C 5 -C 6 ) that does not contain any heteroatom in the ring structure.
  • C 3-6 heteroaryl or “C 3 -C 6 heteroaryl” refers to an aromatic 3-6 membered monocyclic having one or more heteroatoms (such as O, N, or S), unless specified otherwise, except that a heteroaryl ring will include no more than one oxygen atom or one sulfur atom.
  • C 2-6 alkenyl includes unsaturated aliphatic groups having 2, 3, 4, 5, or 6 carbons and that contain at least one double bond.
  • the term “C 2-6 alkenyl” includes straight chain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl), and branched alkenyl groups.
  • a straight chain or branched alkenyl group has six or fewer carbon atoms in its backbone (e.g., C 2 -C 6 for straight chain, C 3 -C 6 for branched chain).
  • C 2-6 alkenyl includes alkenyl groups containing two to six carbon atoms.
  • C 2-6 alkynyl includes unsaturated aliphatic groups having 2, 3, 4, 5, or 6 carbon atoms, but which contain at least one triple bond.
  • alkynyl includes straight chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl), and branched alkynyl groups.
  • a straight chain or branched alkynyl group has six or fewer carbon atoms in its backbone (e.g., C 2 -C 6 for straight chain, C 3 -C 6 for branched chain).
  • C 2-6 alkynyl includes alkynyl groups containing two to six carbon atoms.
  • Embodiments may provide a method of treating cancer in a patient in need thereof, including administering to the patient a therapeutically effective amount of a compound reported herein or a pharmaceutically acceptable salt thereof.
  • the administered compound is provided as a free acid or a pharmaceutically acceptable salt thereof. In some embodiments, the administered compound is provided as an NH 4 salt a free acid or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is provided as an NH 4 salt.
  • isotopes include those atoms having the same atomic number but different mass numbers.
  • isotopes of 1 H hydrogen include tritium and deuterium
  • isotopes of 12 C carbon include 13 C and 14 C.
  • the optimal dose for treatment of cancer can be determined empirically for each individual using known methods and will depend upon a variety of factors, including the activity of the agents; the age, body weight, general health, gender and diet of the individual; the time and route of administration; and other medications the individual is taking. Optimal dosages may be established using routine testing and procedures that are well known in the art. Administration of the above compounds may be by any suitable route.
  • “Pharmaceutically acceptable salt” as used herein refers to acid addition salts or base addition salts of the compounds in the present disclosure.
  • a pharmaceutically acceptable salt is any salt which retains the activity of the parent compound and does not impart any unduly deleterious or undesirable effect on a subject to whom it is administered and in the context in which it is administered.
  • Pharmaceutically acceptable salts include, but are not limited to, metal complexes and salts of both inorganic and carboxylic acids.
  • Pharmaceutically acceptable salts also include metal salts such as aluminum, calcium, iron, magnesium, manganese and complex salts.
  • salts include, but are not limited to, acid salts such as acetic, aspartic, alkylsulfonic, arylsulfonic, axetil, benzenesulfonic, benzoic, bicarbonic, bisulfuric, bitartaric, butyric, calcium edetate, camsylic, carbonic, chlorobenzoic, citric, edetic, edisylic, estolic, esyl, esylic, formic, fumaric, gluceptic, gluconic, glutamic, glycolic, glycolylarsanilic, hexamic, hexylresorcinoic, hydrabamic, hydrobromic, hydrochloric, hydrochloride, hydroiodic, hydroxynaphthoic, isethionic, lactic, lactobionic, maleic, malic, malonic, mandelic, methanesulfonic, methylnitric,
  • Embodiments may be diammonium salts.
  • Pharmaceutically acceptable salts may be derived from amino acids including, but not limited to, cysteine. Methods for producing compounds as salts are known to those of skill in the art (see, e.g., Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH; Verlag Helvetica Chimica Acta, Zurich, 2002; Berge et al., J. Pharm. Sci. 66: 1, 1977).
  • an “effective amount” of a therapeutic agent is an amount sufficient to provide an observable therapeutic benefit compared to cancer left untreated in a subject or patient.
  • Active agents as reported herein can be combined with a pharmaceutically acceptable carrier to provide pharmaceutical formulations thereof.
  • a pharmaceutically acceptable carrier to provide pharmaceutical formulations thereof.
  • the particular choice of carrier and formulation will depend upon the particular route of administration for which the composition is intended.
  • “Pharmaceutically acceptable carrier” refers to a nontoxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated.
  • Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of this invention include, but are not limited to, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium tri silicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene glycol and wool fat.
  • compositions of the present invention may be suitable for parenteral, oral, inhalation spray, topical, rectal, nasal, buccal, vaginal or implanted reservoir administration, etc.
  • the formulation comprises ingredients that are from natural or non-natural sources.
  • the formulation or carrier may be provided in a sterile form.
  • Non-limiting examples of a sterile carrier include endotoxin-free water or pyrogen-free water.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • the compounds are administered intravenously, orally, subcutaneously, or via intramuscular administration.
  • Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • Fatty acids and their glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents that are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
  • a compound or salt may be provided in an acceptable oral dosage form, including, but not limited to, capsules, tablets, aqueous suspensions or solutions.
  • carriers commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, may also be added.
  • useful diluents include lactose and dried cornstarch.
  • the active ingredient may be combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. In addition preservatives may also be added.
  • Suitable examples of pharmaceutically acceptable preservatives include, but are not limited to, various antibacterial and antifungal agents such as solvents, for example ethanol, propylene glycol, benzyl alcohol, chlorobutanol, quaternary ammonium salts, and parabens (such as methyl paraben, ethyl paraben, propyl paraben, etc.).
  • solvents for example ethanol, propylene glycol, benzyl alcohol, chlorobutanol, quaternary ammonium salts, and parabens (such as methyl paraben, ethyl paraben, propyl paraben, etc.).
  • immediate-release is meant to include a conventional release, in which release of the drug starts immediately after administration.
  • immediate release includes dosage forms that allow the drug to dissolve in the gastrointestinal contents, with no intention of delaying or prolonging the dissolution or absorption of the drug. The objective is for the drug to be released rapidly after administration, for example for it to be possible to release at least 80% of the drug within approximately 30 minutes after commencement of dissolution in a dissolution test.
  • “Sustained-release” or “extended-release” includes dosage forms whose drug-release characteristics of time course and/or location are chosen to accomplish therapeutic or convenience objectives not offered by conventional dosage forms such as a solution or an immediate release dosage form.
  • the term “steady-state” means that a plasma level for a given active agent has been achieved and which is maintained with subsequent doses of the active agent at a level which is at or above the minimum effective therapeutic level and is below the minimum toxic plasma level for a given active agent.
  • single formulation refers to a single carrier or vehicle formulated to deliver effective amounts of both therapeutic agents to a patient.
  • the single vehicle is designed to deliver an effective amount of each of the agents along with any pharmaceutically acceptable carriers or excipients.
  • the vehicle is a tablet, capsule, pill, or a patch.
  • unit dose is used herein to mean simultaneous administration of both agents together, in one dosage form, to the patient being treated.
  • the unit dose is a single formulation.
  • the unit dose includes one or more vehicles such that each vehicle includes an effective amount of an agent along with pharmaceutically acceptable carriers and excipients.
  • the unit dose is one or more tablets, capsules, pills, or patches administered to the patient at the same time.
  • dose range refers to an upper and a lower limit of an acceptable variation of the amount of agent specified. Typically, a dose of an agent in any amount within the specified range can be administered to patients undergoing treatment.
  • treat is used herein to mean to relieve, reduce or alleviate at least one symptom of a disease in a subject.
  • the term “treat” may mean to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease or symptom of a disease) and/or reduce the risk of developing or worsening a symptom of a disease.
  • protect is used herein to mean prevent delay or treat, or all, as appropriate, development or continuance or aggravation of symptoms of the disease in a subject.
  • subject or “patient” is intended to include animals, which are capable of suffering from or afflicted with cancer.
  • subjects or patients include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals.
  • the subject is a human, e.g., a human suffering from, at risk of suffering from, or potentially capable of suffering from cancer.
  • the term “about” or “approximately” usually means within 20%, more preferably within 10%, and most preferably still within 5% of a given value or range. Alternatively, especially in biological systems, the term “about” means approximately within a log (i.e., an order of magnitude) preferably within a factor of two of a given value.
  • Exemplary cell proliferative disorders that may be treated using one or more compounds disclosed herein include, but are not limited to cancer, a precancer or precancerous condition, and metastatic lesions in tissue and organs in the body.
  • Cell proliferative disorders may include hyperplasia, metaplasia, and dysplasia.
  • a compound disclosed herein, or a pharmaceutically acceptable salt thereof may be used to treat or prevent a cell proliferative disorder, or to treat or prevent cancer, in a subject having an increased risk of developing cancer relative to the population at large, or used to identify suitable candidates for such purposes.
  • compositions comprising a compound or pharmaceutically acceptable salt thereof for the treatment of cancer.
  • the pharmaceutical formulations may additionally comprise a carrier or excipient, stabilizer, flavoring agent, and/or coloring agent.
  • the compound or pharmaceutically acceptable salt thereof may be administered using a variety of routes of administration known to those skilled in the art. Routes of administration include oral administration. In certain embodiments, a pharmaceutical formulation comprising the compound or pharmaceutically acceptable salt thereof may be taken orally in the form of liquid, syrup, tablet, capsule, powder, sprinkle, chewtab, or dissolvable disk. Alternatively, pharmaceutical formulations of the present invention can be administered intravenously or transdermally. Additional routes of administration are known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, Gennaro A. R., Ed., 20.sup.th Edition, Mack Publishing Co., Easton, Pa.).
  • the compound or pharmaceutically acceptable salt is formulated as a paste, jelly, or suspension.
  • the drug is dissolved, entrapped or suspended in the form of drug particles, microencapsulated particles, or drug-polymer particles in a gelatinous solution or semi-solid.
  • An advantage of an oral jelly formulation is that it is easier to administer the drug to patients who have difficulty swallowing tablets, capsules or pills.
  • the compound is thoroughly mixed and suspended in an appropriate medium to form a paste or a gel. Additional agents can optionally be mixed to provide flavor during oral administration. Peanut butter or alginate, flavored with raspberry and a sweetener are examples of the many suitable taste masking agents.
  • the paste or jelly can also be formulated with suitable binders or excipients known in the art for topical administration.
  • the sustained release formulation is prepared by coating the active ingredient of the drug with a polymer, preferably a water-insoluble polymer.
  • a polymer preferably a water-insoluble polymer.
  • a water-insoluble polymer used in the pharmaceutical field as a sustained release coating agent, an enteric coating agent, or a gastric coating agent.
  • the water-insoluble polymer can include, for example, ethyl cellulose, purified shellac, white shellac, aminoalkyl methacrylate copolymer RS, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, carboxymethylethyl-cellulose, cellulose acetate phthalate, methacrylic acid copolymer L, methacrylic acid copolymer LD, methacrylic acid copolymer S, aminoalkyl methacrylate copolymer E, or polyvinyl acetal diethylaminoacetate.
  • the type, degree of substitution and molecular weight of the water-insoluble polymers can depend on solubility of the active ingredient in water or an alcohol, the desired sustained release level and the like.
  • the water-insoluble polymers can be used either alone or in combination.
  • the sustained release formulation is a matrix-type tablet or granule.
  • the active ingredient can be coated with up to 3 different types of polymers. These three different types of polymers can include: 1) a water insoluble polymer, such as ethylcellulose; 2) a pH independent gelling polymer, such as hydroxypropyl methylcellulose; and 3) a pH dependent gelling polymer, such as sodium alginate. These three different types of polymers can be used together to attenuate the release rate of the drugs.
  • Sustained-release formulations can achieve a degree of sustained effect.
  • the exposure and/or the bioavailability of the active ingredient may vary based on a variety of factors, such as for example, the absorption window, the carriers or excipients used in the formulation, the mode of delivery of the formulation, and/or the transit time of the active ingredient through the gastrointestinal tract of the patient.
  • a therapy can contain at least one sustained-release portion for performing a sustained-release function and one immediate release portion for performing an immediate release function.
  • the therapy when it is in a single dosage form, it can be in the form of tablets formed from a mixture of sustained-release granules constituting a sustained-release portion and immediate-release granules constituting an immediate-release portion, a capsule preparation obtained by filling a capsule with sustained-release granules and immediate-release granules, or press-coated tablets in which an outer layer constituting an immediate-release portion is formed on an inner core constituting a sustained-release portion.
  • the compound may be dispersed uniformly in the composition, immediate release portion or sustained release portion, or may be contained in only one part of the composition, immediate-release portion or sustained-release portion, or may be contained such that there is a concentration gradient.
  • a sustained-release portion in the composition according to the present invention can contain at least one non-pH-dependent polymeric substance or pH-dependent polymeric substance for controlling drug release.
  • a non-pH-dependent polymeric substance used herein can comprise a polymeric substance whose charge state hardly changes under pH conditions generally found in the gastrointestinal tract, specifically from pH 1 to pH 8. This means, for example, a polymeric substance that does not have functional groups whose charge state changes depending on the pH such as basic functional groups such as amino groups or acidic functional groups such as carboxylic acid groups.
  • the non-pH-dependent polymeric substance can be included for giving the composition according to the present invention a sustained-release function, but may also be included for another purpose.
  • the non-pH-dependent polymeric substance used in the present invention may be water-insoluble, or may swell in water or dissolve in water to form a gel.
  • water-insoluble non-pH-dependent polymeric substances include, but are not limited to, cellulose ethers, cellulose esters, and methacrylic acid-acrylic acid copolymers (trade name Eudragit, manufactured by Rohm GmbH & Co. KG, Darmstadt, Germany).
  • Examples include, but are not limited to, cellulose alkyl ethers such as ethylcellulose (trade name Ethocel, manufactured by Dow Chemical Company, USA), ethyl methylcellulose, ethyl propylcellulose or isopropylcellulose, and butylcellulose, cellulose aralkyl ethers such as benzyl cellulose, cellulose cyanoalkyl ethers such as cyanoethylcellulose, cellulose organic acid esters such as cellulose acetate butyrate, cellulose acetate, cellulose propionate or cellulose butyrate, and cellulose acetate propionate, ethyl acrylate-methyl methacrylate copolymers (trade name Eudragit NE, manufactured by Rohm GmbH & Co.
  • cellulose alkyl ethers such as ethylcellulose (trade name Ethocel, manufactured by Dow Chemical Company, USA), ethyl methylcellulose, ethyl propylcellulose or isopropylcellulose
  • mean particle diameter of a water-insoluble polymer used in the present invention usually the lower this mean particle diameter the better the performance, with the mean particle diameter preferably being from 0.1 to 100 ⁇ m, more preferably from 1 to 50 ⁇ m, particularly preferably from 3 to 15 ⁇ m, most preferably from 5 to 15 ⁇ m.
  • water-soluble or water-swelling non-pH-dependent polymeric substances include, but are not limited to, polyethylene oxide (trade name Polyox, manufactured by Dow Chemical Company, molecular weight 100,000 to 7,000,000), low-substituted hydroxypropyl cellulose (trade name L-HPC, manufactured by Shin-Etsu Chemical, Japan), hydroxypropyl cellulose (trade name HPC, manufactured by Nippon Soda, Co., Ltd, Japan), hydroxypropyl methylcellulose (trade names Metolose 60SH, 65SH, 90SH, manufactured by Shin-Etsu Chemical, Japan), and methylcellulose (trade name Metolose SM, manufactured by Shin-Etsu Chemical, Japan).
  • polyethylene oxide trade name Polyox, manufactured by Dow Chemical Company, molecular weight 100,000 to 7,000,000
  • L-HPC low-substituted hydroxypropyl cellulose
  • HPC manufactured by Nippon Soda, Co., Ltd, Japan
  • Metolose 60SH, 65SH, 90SH manufactured by Shin
  • a single non-pH-dependent polymeric substance may be contained in the composition, or a plurality of the non-pH-dependent polymeric substances may be contained.
  • the non-pH-dependent polymeric substance if used in embodiments reported herein, may be a water-insoluble polymeric substance, more preferably ethylcellulose, an ethyl acrylate-methyl methacrylate copolymer (trade name Eudragit NE), or an aminoalkyl methacrylate copolymer RS (trade name Eudragit RL, Eudragit RS). Particularly preferable is at least one of ethylcellulose and an aminoalkyl methacrylate copolymer RS. Most preferable is ethylcellulose.
  • the amount of the non-pH-dependent polymeric substance contained in the composition this amount can be adjusted as appropriate in accordance with the purpose such as controlling sustained drug release.
  • a pH-dependent polymeric substance that can be used in embodiments reported herein may be a polymeric substance whose charge state changes under pH conditions generally found in the gastrointestinal tract, specifically from pH 1 to pH 8. This means, for example, a polymeric substance having functional groups whose charge state changes depending on the pH such as basic functional groups such as amino groups or acidic functional groups such as carboxylic acid groups.
  • the pH-dependent functional groups of the pH-dependent polymeric substance are preferably acidic functional groups, with the pH-dependent polymeric substance most preferably having carboxylic acid groups.
  • a pH-dependent polymeric substance used in the present invention may be water-insoluble, or may swell in water or dissolve in water to form a gel.
  • pH-dependent polymeric substances used in the present invention include, but are not limited to, enteric polymeric substances.
  • enteric polymeric substances include, but are not limited to, methacrylic acid-methyl methacrylate copolymers (Eudragit L100, Eudragit S100, manufactured by Rohm GmbH & Co. KG, Darmstadt, Germany), methacrylic acid-ethyl acrylate copolymers (Eudragit L100-55, Eudragit L30D-55, manufactured by Rohm GmbH & Co.
  • hydroxypropyl methylcellulose phthalate HP-55, HP-50, manufactured by Shin-Etsu Chemical, Japan
  • hydroxypropyl methylcellulose acetate succinate AQOAT, manufactured by Shin-Etsu Chemical, Japan
  • carboxymethyl ethylcellulose CMEC, manufactured by Freund Corporation, Japan
  • cellulose acetate phthalate HP-55, HP-50, manufactured by Shin-Etsu Chemical, Japan
  • AQOAT hydroxypropyl methylcellulose acetate succinate
  • CMEC carboxymethyl ethylcellulose
  • a single pH-dependent polymeric substance may be contained in the composition, or a plurality of pH-dependent polymeric substances may be contained.
  • the pH-dependent polymeric substance used in the present invention is preferably an enteric polymeric substance, more preferably a methacrylic acid-ethyl acrylate copolymer, a methacrylic acid-methyl methacrylate copolymer, hydroxypropyl methylcellulose phthalate, or hydroxypropyl methylcellulose acetate succinate, particularly preferably a methacrylic acid-ethyl acrylate copolymer.
  • a commercially available product of a powder type or a granular type, or a suspension type in which the pH-dependent polymeric substance has been dispersed in a solvent in advance can be used as is, or such a commercially available product can be used dispersed in water or an organic solvent.
  • an example is Eudragit L100-55.
  • the mean particle diameter of a pH-dependent polymeric substance used in the present invention is preferably from 0.05 to 100 ⁇ m, more preferably from 0.05 to 70 ⁇ m, most preferably from 0.05 to 50 ⁇ m.
  • the amount of the pH-dependent polymeric substance for example, in the case of an enteric polymeric substance, the amount is generally from 0.1 to 90 parts by weight, preferably from 1 to 70 parts by weight, more preferably from 5 to 60 parts by weight, particularly preferably from 10 to 50 parts by weight, based on 100 parts by weight of the composition.
  • a therapy according to embodiments reported herein may further contain any of various additives, such as any of various pharmacologically acceptable carriers such as diluents, lubricants, binders and disintegrants, as well as preservatives, colorants, sweeteners, plasticizers, film coating agents and so on, as necessary.
  • diluents include, but are not limited to, lactose, mannitol, dibasic calcium phosphate, starch, pregelatinized starch, crystalline cellulose, light silicic anhydride, synthetic aluminum silicate, magnesium aluminate metasilicate or the like.
  • Examples of lubricants include, but are not limited to, magnesium stearate, calcium stearate, talc, sodium stearyl fumarate or the like.
  • Examples of binders include, but are not limited to, hydroxypropyl cellulose, methylcellulose, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone or the like.
  • Examples of disintegrants include, but are not limited to, carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose or the like.
  • preservatives include, but are not limited to, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid or the like.
  • colorants include, but are not limited to, water-insoluble lake pigments, natural pigments (e.g., .beta.-carotene, chlorophyll, red ferric oxide), yellow ferric oxide, red ferric oxide, black ferric oxide or the like.
  • sweeteners include, but are not limited to, sodium saccharin, dipotassium glycyrrhizate, aspartame, stevia or the like.
  • plasticizers include, but are not limited to, glycerol fatty acid esters, triethyl citrate, propylene glycol, polyethylene glycol or the like.
  • film coating agents include, but are not limited to, hydroxypropyl methylcellulose, hydroxypropyl cellulose or the like.
  • a single conventional method, or a combination of conventional methods can be used.
  • granulation is the main operation, but this may be combined with other operations such as mixing, drying, sieving, and classification.
  • a wet granulation method in which a binder and a solvent are added to the powder and granulation is carried out
  • a dry granulation method in which the powder is compressed and granulation is carried out
  • a molten granulation method in which a binder that melts on heating is added and heating and granulation are carried out, or the like can be used.
  • an operating method such as a mixing granulation method using a planetary mixer, a screw mixer or the like, a high-speed mixing granulation method using a Henschel mixer, a Super mixer or the like, an extruding granulation method using a cylindrical granulator, a rotary granulator, a screw extruding granulator, a pellet mill type granulator or the like, a wet high-shear granulation method, a fluidized-bed granulation method, a compression granulation method, a crushing granulation method, or a spraying granulation method can be used.
  • a granulation solvent may be used when preparing the composition according to the present invention.
  • a granulation solvent which may be water or any of various organic solvents, for example, water, a lower alcohol such as methanol or ethanol, a ketone such as acetone or methyl ethyl ketone, methylene chloride, or a mixture thereof.
  • sustained-release granules contained in embodiments at least one drug and at least one selected from non-pH-dependent polymeric substances and pH-dependent polymeric substances are mixed together, a diluent and a binder are added as necessary, and granulation is carried out to obtain granular matter.
  • the granular matter obtained is dried using a tray dryer, a fluidized bed dryer or the like, and sieving is carried out using a mill or an oscillator, whereby the sustained-release granules can be obtained.
  • sustained-release granules in the present invention it is possible to add at least one drug, at least one selected from non-pH-dependent polymeric substances and pH-dependent polymeric substances, and as necessary a diluent and a binder using a dry compactor such as a roller compactor or a slug tabletting machine, and carry out compression-molding while mixing, and then carry out granulation by cracking down to a suitable size.
  • a dry compactor such as a roller compactor or a slug tabletting machine
  • the granular matter prepared using such a granulator may be used as is as granules or fine granules according to the present invention, or may be further cracked using a power mill, a roll granulator, a rotor speed mill or the like, and sieved to obtain sustained-release granules.
  • immediate-release granules can also be manufactured as for the sustained-release granules.
  • a compression-molded product can be manufactured as a drug-containing sustained-release portion or immediate-release portion, or as a composition reported herein using a single conventional method, or a combination of conventional methods.
  • at least one drug at least one selected from non-pH-dependent polymeric substances and pH-dependent polymeric substances, a diluent such as mannitol or lactose, a binder such as polyvinylpyrrolidone or crystalline cellulose, a disintegrant such as carmellose sodium or crospovidone, and a lubricant such as magnesium stearate or talc are used, and tableting is carried out using an ordinary method, whereby the compression-molded product can be obtained.
  • tabletting is the main operation in the method of manufacturing the compression-molded product, but this may be combined with other operations such as mixing, drying, sugar coating formation, and coating.
  • Examples of the method for the tabletting include, but are not limited to, direct compression molding in which at least one drug and pharmacologically acceptable additives are mixed together and then the mixture is directly compression-molded into tablets using a tabletting machine, and dry granule compression or wet granule compression in which sustained-release granules or immediate-release granules according to the present invention are subjected to compression-molding after adding a lubricant or a disintegrant as necessary.
  • the tabletting machine used in the compression molding for example, a single-punch tabletting machine, a rotary tabletting machine, or a press-coated tabletting machine can be used.
  • Drug-containing sustained-release granules or immediate-release granules, or compression-molded product according to embodiments herein can be used as is in the form of granules or a tablet as the composition, but may also be subjected to further processing to manufacture the composition.
  • the compression-molded product or granules can be given a film coating using a film base material such as ethylcellulose, casein, methylcellulose, hydroxypropyl methylcellulose, methacrylic acid copolymer L, cellulose acetate phthalate, shellac or the like, or given a sugar coating using a sugar coating liquid containing saccharose, sugar alcohol, gum arabic powder, talc or the like, thus producing film-coated tablets or sugar-coated tablets.
  • a film base material such as ethylcellulose, casein, methylcellulose, hydroxypropyl methylcellulose, methacrylic acid copolymer L, cellulose acetate phthalate, shellac or the like
  • a sugar coating liquid containing saccharose, sugar alcohol, gum arabic powder, talc or the like thus producing film-coated tablets or sugar-coated tablets.
  • One solvent in this coating technique may be purified water, but an organic solvent such as an alcohol, a ketone, an ether
  • ethanol for example, ethanol, acetone, methylene chloride or the like can be used as an organic solvent.
  • an apparatus ordinarily used in coating techniques for manufacturing medicines can be used, with examples including a spray coating apparatus in which the coating is carried out by spraying a coating liquid or the like, and a rotor fluidized bed granulator for layering.
  • capsule preparations can be manufactured by filling sustained-release granules or immediate-release granules as above, or mini-tablets into hard gelatin capsules or HPMC capsules using an automatic capsule filling machine.
  • sustained-release granules or immediate-release granules as above can be mixed with a thickener or a dispersant so as to disperse these granules, the mixture then being made into granules or tablets.
  • a liquid or jelly can be made using water, and substances selected from dispersants, emulsifiers, thickeners, preservatives, pH adjustors, sweeteners, flavorings, fragrances and so on.
  • dispersants emulsifiers, thickeners, preservatives, pH adjustors, sweeteners, flavorings, fragrances and so on.
  • DMOCP 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide
  • FIG. 1 A full scheme of this synthesis is available in FIG. 1 .
  • the crude mixture was dissolved in DCM (45.0 ml) and treated with water (0.118 ml, 6.55 mmol, 2.0 eq) and NaHSO 4 —SiO 2 (1.18 g, 6.55 mmol, 2 eq) at ambient temperature.
  • the reaction mixture was filtered and rinsed twice with DCM/MeOH (9/1, 20 ml).
  • the combined filtrates were concentrated in vacuo and treated with 1:1 mixture of n-heptane/toluene ( ⁇ 30 ml). The top layer was removed by decantation.
  • reaction mixture Upon complete sulfurization (approximately 40 minutes), the reaction mixture was partially concentrated in vacuo to approximately 15 ml and poured into a mixture of saturated aqueous NaHCO 3 (50 ml) and water (30 ml). After 10 min stirring at ambient temperature, the mixture was extracted with 1:1 mixture of EtOAc/MTBE (60 ml ⁇ 3 times). The organic layers were combined, washed with brine (25 ml), dried over Mg 2 SO 4 and concentrated in vacuo. The residue was purified by silica gel column chromatography (0-20% MeOH in DCM) to give Compound 104 (3.31 g, 3.20 mmol, 100% theoretical yield assumed) as a brown oil. The product was used in the next step without further purification.
  • FIG. 2A and FIG. 2B An alternative synthetic route for Compound 1a is set out in FIG. 2A and FIG. 2B , as well as in FIG. 2C and reported below.
  • the organic layer was washed with water (2.0 L, 3.5 wt, 3.5 vol). Solid began slowly precipitating out of the organic layer. The water layer was separated. The organic layer was then concentrated to approx. 1 vol. The crude product was slurried with a mixture of n-heptane (2.00 L, 2.40 wt, 3.51 vol) and toluene (0.50 L, 0.76 wt, 0.88 vol). After stirring for 15 minutes, the pale yellow solid was collected by vacuum filtration.
  • the filter cake was sequentially rinsed with: (1) a mixture of n-heptane (0.60 L, 0.72 wt, 1.05 vol) and toluene (0.30 L, 0.46 wt, 0.53 vol), and then (2) n-heptane (3.00 L, 3.6 wt, 5.26 vol).
  • the solid was dried with no heat for 30 minutes and then transferred to trays for drying at 50° C. in a vacuum oven overnight to give Compound 130 as pale yellow solid (996.7 g, 1.47 mol, 1.75 wt, 97% yield).
  • DIAD (0.320 L, 1.65 mol, 333 g, 0.333 wt, 0.320 vol, 1.3 eq) was added slowly over 20 minutes while keeping T-internal below 5° C. The reaction was stirred at 0-5° C. for 1 h and monitored by LCMS. The ice bath was removed and the mixture was allowed to warm up to rt. After overnight stirring (17h), an triphenylphosphine (83 g, 0.32 mol, 0.083 wt, 0.25 eq) and DIAD (62 ml, 0.32 mol, 64 g, 0.064 wt, 0.062 vol, 0.25 eq) were added.
  • reaction mixture was diluted with MTBE (10 L, 10 vol), washed twice with half-saturated NaCl (18 wt % solution in water; 2 ⁇ 4 L) and concentrated in vacuo to a thick oil.
  • the mixture was re-dissolved in a mixture of MTBE (4.00 L, 4 vol) and n-heptane (0.50 L, 0.5 vol) and then cooled to 0° C.
  • a seed crystal of triphenylphosphine oxide was added to the solution. Solids slowly began precipitating out of solution and was stirred overnight. The white solid was collected by vacuum filtration and rinsed with MTBE (2 L, 2 vol) to isolate 540 g of triphenylphosphine oxide.
  • the filtrate was concentrated and purified via Biotage 150L KP-Sil (SiO 2 5 kg; preteated with 1% TEA in Hep/etOAc; eluents: heptane/EtOAc (48 L of 33% EtOAc with 1% TEA, 24 L of 50% EtOAc with 1% TEA, 24 L of 66% EtOAc with 1% TEA) ⁇ 100% EtOAc with 1% TEA).
  • the column was monitored by TLC (2:1 EtOAc/n-heptane).
  • the filtrate was concentrated under vacuum and purified via Biotage 150 L KP-Sil (SiO2 5 kg; preteated with 1% TEA; loaded sample by dissolving in toluene eluents: 9:1 heptane/EtOAc (16 L) and 15 TEA, 3.6:1 (46 L), 2:1 (20 L) and 1% TEA, 1:1 (30 L) and 1% TEA, and 100% EtOAc (16 L) and 1% TEA).
  • the combined clean product fractions were concentrated under vacuum to give Compound 134 as off white solid foam (662.2 g).
  • the mixture fractions were combined and concentrated under vacuum (480 g).
  • a white insoluble solid formed by dilution with toluene (300 ml) prior to loading on Biotage 150L was removed by vacuum filtration.
  • the material soluble in toluene was purified via Biotage 150M HP-Sphere (SiO 2 2.5 kg (preteated with 1% TEA); sample loading with toluene; eluents: 2:1 heptane/EtOAc (26 L) w/1% TEA, 1:1 (25 L) w/1% TEA, 1:4 (34 L) w/1% TEA).
  • the column was monitored by TLC (1:1 heptane/EtOAc).
  • the crude Compound 135 was dissolved in dichloromethane (3.08 L, 4.07 kg, 9.9 wt, 7.5 vol) at ambient temperature. Water (55.7 ml, 0.136 vol, 10 eq) was added followed by a solution of dichloroacetic acid (77 ml, 120 g, 0.93 mol, 0.29 wt, 0.19 vol, 3.0 eq) in DCM (3.08 L, 7.5 vol) while keeping the internal T below 25° C. (Turned into an orange solution).
  • the mixture was concentrated to 1.55 kg (3.8 wt), redissolved in EtOAc (6.2 L, 5.5 kg, 14 wt, 15 vol), sequentially washed with: (1) water (1.0 L, 2.5 vol) and saturated NaHCO 3 (9 wt % solution in water, 0.82 L, 2.0 vol).
  • the crude product EtOAc solution was stored at ⁇ 20° C. over night; 0.82 L, 2.0 vol) and in next day, the solution was concentrated in vacuo at 25° C.
  • the crude mixture thus obtained (654 g) was triturated with: (1) n-heptane (3.01 L, 7.5 vol), (2) a mixture of n-heptane (2.46 L, 6.0 vol) and toluene (0.82 L, 2.0 vol).
  • TEA 129 ml, 927 mmol, 94 g, 0.28 wt, 0.38 vol, 3.0 eq
  • water 100 ml, 5.56 mol, 0.30 wt, 0.30 wt, 18 eq
  • sulfur 34.7 g, 1.08 mol, 0.10 wt, 3.5 eq
  • the filtrate was concentrated the mixture in vacuo, diluted with MTBE (5.1 L, 15 vol), and wash twice with NaCl (30 wt % solution in water; 2 ⁇ 1.35 L, 2 ⁇ 4 vol). Insoluble solids were filtered off and the filtrate was concentrated in vacuo and azeotroped with toluene (4.0 L, 12 vol).
  • the combined organic layers were sequentially washed with: (1) a mixture of NaCl (36 wt % solution in water; 300 ml, 2 vol) and water (300 ml, 2 vol) and (2) water (600 ml, 4 vol).
  • the organic layer was then concentrated under vacuum and azeotroped with n-heptane (1.50 L, 10 vol).
  • MTBE (0.95 L, 6.3 vol) was added to the crude solid and the mixture was heated at 40° C.
  • the mixture was diluted with EtOAc (300 ml, 2 vol) and slowly cooled to 0° C. The dense solid was allowed to settle and the supernatant was pumped off through a filter frit tube.
  • the resulting reaction mixture was stirred at 0° C. for 10 h, warmed to 10° C. over 2 h, held at 10° C. for 10 h and warmed up to rt over 2h.
  • the reaction was monitored by LCMS and TLC (EtOAc with 0.5% TEA). After 18 h, anhydrous acetonitrile (0.73 L, 10 vol) was added and the mixture was stored at ⁇ 20° C. over 3 days.
  • the mixture from Stage 10 was warmed to ambient temperature and added via a dropping funnel in portions (100 mL every 30 minutes, over 9 h) into a mixture of pyridine trifluoroacetate salt (azetroped in advance with pyridine twice; 41.9 g, 217 mmol, 0.57 wt, 3.0 eq) and acetonitrile (5.85 L, 80 vol).
  • the reaction was monitored by LCMS.
  • a solution of 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (5.8 mL, 18 mmol, 0.25 eq) in acetonitrile (24 mL) was added over 4h.
  • Amount of the additional reagent was determined based on the reamining Compound 140 ( ⁇ 30% based on LCMS). More conversion of the diol was observed after 6h.
  • the resulting aqueous layer was treated with HCl (1.0 M solution in water; 90 ml, 90 mmol, 3.2 eq) over a period of 3.5 hours (pH ⁇ 2).
  • HCl 1.0 M solution in water
  • the mixture stirred for 30 minutes and then the solid precipitate was collected by vacuum filtration.
  • the filter cake was washed with water three times (3 ⁇ 200 ml, 3 ⁇ 9 vol) and dried in vacuo overnight.
  • Ammonia 2.0 M solution in MeOH; 250 ml, 500 mmol, 17.6 eq
  • ethanol 100 ml
  • a colorless block single crystal (0.1 ⁇ 0.1 ⁇ 0.1 mm) found in crystallization solution was dispersed in liquid Parabar 10312 and was mounted on a Dual-Thickness MicroMountsTM (MiTeGen). Diffraction data was collected at ⁇ 160° C. on XtaLAB PRO P200 MM007HF (Rigaku) with w axis oscillation method using multi-layer mirror monochromated Cu-K ⁇ radiation.
  • FIG. 4A shows an ORTEP figure of Compound 1 molecules in an asymmetric unit, along with a number of disordered water molecules.
  • FIG. 4B shows the crystal structure of one of the Compound 1 molecules from FIG. 4A .
  • FIG. 4C shows the crystal structure of the other molecule of Compound 1 shown in FIG. 4A .
  • the crystal structure of Compound 1 was solved with a final R-factor of 0.1354.
  • the Flack parameter was nearly zero (0.083(17)), indicating that the absolute configuration of Compound 1 is (R, S).
  • the crystal structure analysis also indicated that many water molecules were present in the large channel of Compound 1, which indicated that water molecules were able to easily slip out from the channel.
  • the analysis also confirmed that the conformations of both crystallographically independent molecules the asymmetric unit were almost the same.
  • Compound 2 (0.5 mg) was weighed and dissolved in acetonitrile/28% ammonia solution. Then, this solution was stored under room temperature with loosely fixed cap. After 2 weeks rod shape crystal appeared.
  • a colorless block single crystal (0.1 ⁇ 0.1 ⁇ 0.5 mm) found in crystallization solution was dispersed in liquid Parabar 10312 and was mounted on a Dual-Thickness MicroMountsTM (MiTeGen). Diffraction data was collected at ⁇ 160° C. on XtaLAB PRO P200 MM007HF (Rigaku) with ⁇ axis oscillation method using multi-layer mirror monochromated Cu-K ⁇ radiation.
  • FIG. 4D shows an ORTEP figure of a Compound 2 molecule.
  • Compound 16 was obtained from compound B (RR isomer of compound A) via the same sequences as described in compound 15.
  • FIG. 3 A route for this synthesis is shown in FIG. 3 .
  • the reaction mixture was transferred into a flask containing acetonitrile (20.8 ml), water (104 ⁇ l, 5.776 mmol) and pyridine trifluoroacetate salt (421 mg, 2.178 mmol). The mixture was stirred for 10 min. UPLC-MS indicated the desired product was formed.
  • the reaction mixture was mixed with EtOAc and washed with HCl [0.1N]/brine and then sat. NaHCO 3 /Brine to prevent DMT deprotection.
  • the aqueous layer was back extracted with EtOAc (1 ⁇ ).
  • the combined organic layer was dried over Na 2 SO 4 and then filtered. Solvents and volatiles in the filtrate were removed on rotavapor to afford the crude product which was used for the next step without further purification.
  • the crude hydrogen phosphonate intermediate was dissolved in DCM (2635 40.946 mmol) and water (29.5 1 1.1, 1.638 mmol) before Dichloroacetic acid (135 1 1.1, 1.638 mmol) in DCM (2635 40.946 mmol) was added at 20° C. The mixture was stirred at 20° C. for 5 min. UPLC-MS indicated that the reaction was completed. The reaction was neutralized with pyridine (510 ⁇ l, 6.308 mmol). The volatiles were removed on rotavapor and then highvac rotavapor. The residue was azeotroped with pyridine one more time before used for cyclization.
  • the reaction was stirred at RT for 1 hr. No change was found on UPLC-MS.
  • the reaction mixture was diluted with EtOAc and washed with sat. NaHCO 3 and brine. The organic layer was dried over Na 2 SO before filtered. The residue after solvent evaporation was dissolved in MeOH before NH 3 —H 2 O was added. The mixture was stirred for 5 min before all the solvent and volatiles were removed on rotavapor and highvac rotavapor.
  • the CDN intermediate was dissolved in acetonitrile (1484 ⁇ l, 28.417 mmol) and 1,4-dioxane (496 ⁇ l, 5.797 mmol) before TEA (39.6 0.284 mmol) and 1-(bromomethyl)-2-nitrobenzene (49.1 mg, 0.227 mmol) was added at 20° C. The mixture was stirred at 20° C. for 16 hr. UPLC-MS indicated that the reaction was incomplete. Neither TLC (EtOAc) nor HPLC indicated any o-nitrobenzyl bromide left.
  • Prep-TLC (EtOAc) was used to isolate the most polar isomer which leads to the final product Compound 18.
  • Compound 27 was prepared from Compound 159, a byproduct in Stage 11b of a route for Compound 1 shown in FIG. 2B .
  • Compound 28 was prepared from Compound 160, a product in Stage 11 of the route for Compound 1 shown in FIG. 2B , using the same method for Compound 27.
  • the resulting mixture was treated with water (0.80 ml) and extracted three times with a mixture of n-heptane/toluene (1/1, 0.5 mL each time) and then toluene (0.3 ml).
  • the aqueous layer was concentrated in vacuo at 40-50° C. and treated with water (1 mL).
  • the resulting solid was filtered off, rinsing with water (0.3 ml).
  • the combined filtrates were treated with 1.0 M HCl (0.07 ml, 0.07 mmol).
  • the resulting slurry was filtered and rinsed with water (1 mL).
  • the filter cake was dissolved with a 2.0 M solution of ammonia (1.0 ml, 2.0 mmol) in MeOH. The resulting solution was concentrated in vacuo to 0.5 mL and treated with EtOH (0.5 ml). The same operation was repeated two more times. To the resulting solution was added EtOAc (0.9 mL) dropwise. Precipitation occurred. The resulting solid was collected by filtration, rinsed with a 4/1 mixture of EtOAc/EtOH (0.4 mL) and dried in vacuo overnight at ambient temperature. 12 mg of Compound 29 was obtained.
  • Compound 25 was prepared via the same reaction sequence (Stage 12-13) as described in FIG. 2A and FIG. 2B .
  • the resulting mixture was stirred at ambient temperature while the reaction was monitored by LCMS Upon complete sulfurization, the reaction mixture was treated with a sat′d aqueous NaHCO 3 (27 ml) and water (10 mL), and concentrated in vacuo. To the resulting residue were added a mixture of MTBE/EtOAc (34/34 ml), a sat′d aqueous NaHCO 3 solution (27 ml) and water (20 mL). The layers were separated and the aqueous layer was extracted with a mixture of EtOAc/MTBE (34/34 mL).
  • Compound 181 was prepared via the same reaction sequence (Stage 5-11) as described in FIG. 2A and FIG. 2B .
  • reaction mixture was cooled to ambient temperature and treated with DMSO (2.54 ml, 35.8 mmol). After 3h stirring, the reaction mixture was concentrated in vacuo and purified by silicagel column chromatography (SiO 2 50 g, 0% to 15% MeOH in EtOAC) to give a mixture of Compound 198 and Compound 199.
  • the mixture was dissolved in dichloromethane (2 mL) and treated with water (0.021 ml, 1.2 mmol) and 6% dichloroacetic acid (0.098 ml, 1.2 mmol) in dichloromethane (2 mL). After 10 min, the reaction was quenched with pyridine (1 ml) and concentrated in vacuo.
  • the slower eluting isomer of Compound 190 (60 mg) was processed through the same reaction sequence as described in compound Compound 191 to give 14 mg of Compound 192 and 7 mg of Compound 191.
  • FIG. 13 shows an example of synthesis of Compound 38 and Compound 39.
  • Compound 217 was processed separately through Step 11 to give Compound 39 and Compound 222.
  • THP1-DualTM Cells (InvivoGen, Cat # thpd-nfis) were applied for EC 50 determination.
  • THP1 DualTM Cells have been characterized to carry the HAQ STING genotype by the vendor Invivogen (Insight 201402-1). Cells were grown and maintained under conditions as recommended by manufacturer. The interferon regulatory factor (IRF) pathway induction described in manufacturer's manual was followed for EC 50 determination. In brief, cells were seeded and treated with different concentrations of compound for 20 hrs while incubated at 37° C., 5% CO 2 . Cells were resuspended and QUANTI-LucTM solution (Cat. #: rep-q1c1) was added. Resulting light emission was measured by luminometer (Envision, Perkin Elmer). Obtained signals were plotted and EC 50 was calculated with GraphPad Prism? software.
  • EC 50 values are reported in Tables 4-8 below.
  • the EC 50 values may be from a single assay or an average of multiple assays. Preceding each table is a structure used for review of that table.
  • Human STING has 4 major variants, including WT, HAQ, REF, and AQ variants.
  • REF-STING also referred to as R232H, for example, occurs in about 14% of the human population. Compared to the wild-type allele, R232H has decreased response to bacterial and metazoan cyclic dinucleotides. Details of these 4 major variants as well as other rare variants are reported by Yi G, et al., “Single nucleotide polymorphisms of human STING can affect innate immune response to cyclic dinucleotides” PLoS One 2013; 8:e77846.
  • STING variant specific reporter cell lines were established by using THP1-DualTM KO-STING cells (InvivoGen, Cat # thpd-kostg) and three STING variant protein expression vectors.
  • the expression vector map for WT STING is shown in FIG. 6 .
  • different STING variant sequences were used in that vector, with the WT STING replaced by the appropriate nucleotide sequence.
  • STING variant-expressing vectors for WT-STING, REF-STING, and AQ-STING were prepared and stably transfected into THP1-DualTM KO-STING cells to prepare STING variant-specific reporter assays for WT-STING, REF-STING and AQ-STING, respectively.
  • EC 50 values were determined as described above in Example 103 for the HAQ STING agonist activity reporter assay. Results are shown below in Table 9.
  • the DNA sequences used for these STING variants are shown in SEQ ID NO: 1 (Nucleotide Sequence of WT Human STING), SEQ ID NO: 2 (Nucleotide Sequence of REF Human STING), and SEQ ID NO: 3 (Nucleotide Sequence of AQ Human Sting).
  • RAW-LuciaTM ISG Cells (InvivoGen, Cat # rawl-isg) were used for a mouse STING agonist reporter assay. EC 50 values were determined as described above in Example 103 in the HAQ STING agonist activity reporter assay. Results are shown below in Table 9.
  • a DSF assay was employed to measure the physical interaction between compound and recombinant STING protein.
  • Truncated recombinant STING protein (a.a.155-341) (SEQ ID NO: 4) was expressed in E. coli and isolated for the assay, as described below.
  • Assay matrix was prepared in 384-well plates to a final volume of 10 ⁇ L per well consisting of 1 ⁇ M recombinant STING protein (a.a. 155-341) (SEQ ID NO: 4), 100 mM PBS pH 7.4, supplemented with 100 mM KCl, 5 ⁇ SYPRO orange dye and 50 ⁇ M compound (final DMSO conc. 0-1%).
  • Example 107 Ex Vivo Human PBMC Stimulation Assay
  • PBMC Peripheral blood mononuclear cell isolation was done using SIGMA ACCUSPIN 50 ml Tubes (cat # A2055) and sigma ACCUSPIN System-HISTOPAQUE-1077 (cat # A7054) using protocol provided by manufacturer. PBMC layer was harvested and washed with 1 ⁇ Phosphate Buffered Saline (PBS) as suggested by Sigma.
  • PBS Phosphate Buffered Saline
  • PBMC peripheral blood mononuclear cells
  • RPMI fetal bovine serum
  • FBS fetal bovine serum
  • 1 ml of cell (1 ⁇ 10e6) were transferred into Falcon 5 mL Round Bottom Polypropylene Test Tube (cat #352063) and stimulated with different concentrations (0, 0.1, 1, 10 uM) for 24 hours in 5% CO2 incubator at 37° C.
  • IFN ⁇ measurement was done using Human IFN- ⁇ Base Kit (Meso Scale Diagnostics cat # K151ADA) and protocol provided by manufacturer was used. IFN-beta estimation was done by reading assay plate at MESO SECTOR Imager 2400 and using MSD Discovery Workbench 4.0 program. After 24 hours IFN ⁇ protein was analyzed. The results showed that compound 1a can induce primary human PBMC IFN ⁇ protein production in a dose-dependent manner.
  • Results shown in Table 10 reflect an average of measurements conducted using five different donors.
  • RNA-specific primers for IFNB1 Hs01077958_s1
  • GAPDH Hs99999905_m1
  • Compound 1a was tested for its anti-cancer activity in CT26 dual tumor model, which is a mouse colon cancer model.
  • Female of 5-6 week old Balb/cJ mice (Jackson Labs, Bar Harbor, Me.) were implanted subcutaneously with CT26 tumor cells on both sides of each animals, 10 5 cells for each side.
  • treatment was started 5 days (1.25 mg/kg, 2.5 mg/kg and 5 mg/kg) after the tumor implantation, when the average tumors reached approximately 100 mm 3 .
  • treatment was started 8 days (0.6 mg/kg, and 10 mg/kg) after the tumor implantation, when the average tumors reached approximately 120 mm 3 .
  • the treatment scheme is described in Table 12 and Table 13.
  • mice in the study have two subcutaneous CT26 tumors.
  • the “treated tumor” indicates the tumor with compound direct administration, while “untreated tumor” indicates the tumor without direct compound administration.
  • Tumor volume was followed throughout the experiment. Tumor volume is measured two times weekly after the start of treatment. Tumor burden is calculated from caliper measurements by the formula for the volume of a prolate ellipsoid (L ⁇ W 2 )/2 where L and W are the respective orthogonal length and width measurements (mm).
  • Compound 1a showed potent and curative activity in CT26 dual tumor model ( FIG. 7 and FIG. 8 ).
  • a cure rate of 20% was detected even at the lowest dose tested in the study ( FIG. 8 , 0.6 mg/kg dose).
  • the highest dose (10 mg/kg) cured 100% of animals of that tumor at the end of study.
  • the top dose group (10 mg/kg) showed 80% curative effects; all the lower doses also showed tumor growth inhibition activity.
  • Compound 1a was tested for its anti-cancer activity in a CT26 liver metastatic model.
  • Anesthetized female 5-6 week-old BALB/cJ mice (Jackson Labs, Bar Harbor, Me.) were implanted intra-splenically with luciferase-expressing CT26 tumor cells (5 ⁇ 10 5 cells per mouse).
  • a subsequent ten minute waiting period allowed tumor cells to circulate into the animals' livers.
  • Spleens were then removed and animals were sutured and allowed to recover.
  • CT26 tumor cells (10 5 cells per mouse) were again implanted, this time subcutaneously (sc) under the right forelimb area, to enable development of a tumor mass for compound administration.
  • compound (10 mg/kg) was administered intratumorally, a single time, into the sc tumor.
  • the local anti-cancer effect of compound was measured through its effect on the sc tumor, while the compound's abscopal effect was assessed by the overall survival of treated mice compared with vehicle-treated control mice, based on the detrimental effect of the growing tumor mass in each mouse liver.
  • Compound 1a showed both potent activity towards the local sc tumors and also curative systemic activity in 9 of 10 treated animals ( FIG. 9 ). These results indicate that local administration of compound 1a can induce both local and systemic (abscopal) anti-cancer activity including deep lesion such as in the liver.
  • GL261 is a murine glioma cell line. Luciferase expressing GL261 mouse glioma cells (2 ⁇ 10 4 cells/mouse) were intra-cranially implanted into female 5-6 week-old B6 albino mice (Jackson Labs, Bar Harbor, Me.). Three to 4 days later, GL261 cells were implanted subcutaneously (10 6 cells/mouse) under the right forelimb area to allow development of a tumor mass for compound administration. Ten days after intra-cranial tumor cell implantation, compound (10 mg/kg) was administered intratumorally, a single time, into the sc tumor.
  • the local anti-cancer effect of compound was measured through its effect on the sc tumor, while the compound's abscopal effect was assessed by the overall survival of treated mice compared with vehicle-treated control mice, based on the detrimental effect of the growing tumor mass in each mouse brain.
  • Compound 1a showed both potent activity at local sc tumors and showed curative systemic activity in 5 of 8 treated animals ( FIG. 10 ). These results indicate that local administration of compound 1a can induce both local and systemic (abscopal) anti-cancer activity including deep lesion such as in the brain.
  • DNA sequence encoding human WT STING protein from amino acid 155 to 341 was cloned into the pET21b vector, following a His-TEV-Sumo tag at its N-terminus (SEQ ID NO: 5).
  • the sequence of the pET21b has been deposited in addgene and is available here: addgene.org/vector-database/2550/; that sequence is incorporated by reference herein.
  • E. coli BL21 (DE3) codon plus cells were transformed with this plasmid, and the expression of recombinant protein was induced with 0.1 mM Isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG).
  • IPTG Isopropyl ⁇ -D-1-thiogalactopyranoside
  • Protein was purified from the soluble fraction of cell lysate by Ni-NTA affinity chromatography. The His-TEV-Sumo tag was removed by sumo protease, and was separated from tag-free WT STING 155-341, using a second Ni-NTA affinity column. The protein was further purified by anion-exchange and size-exclusion chromatography, and was stored in buffer containing 20 mM Tris-HCl pH 7.5, and 150 mM NaCl at 35 mg/ml concentration.
  • the WT STING protein was diluted to 10 mg/ml using the storage buffer (20 mM Tris-HCl pH 7.5, and 150 mM NaCl) and mixed with Compound 1 (100 mM stock in DMSO) in molar ratio 1:5. The mixture was incubated for 4 hours at 4° C., and centrifuged at 13,000 rpm for 20 min before crystallization. Crystallization screen trays were set up using the hanging-drop vapor-diffusion method at 18° C.
  • Crystals were grown by mixing 1 ⁇ L of WT STING/Compound 1 solution with an equal volume of well solution, containing 100 mM HEPES pH 7.5, 200 mM CaCl2, and 15% (wt/vol) PEG 8000. 20% (wt/vol) PEG 400 was used as cryoprotectant reagent when crystals were flash-frozen in liquid nitrogen. Diffraction datasets were collected with a Pilatus detector at SSRF BL19U1 beamline, and processed with HKL3000 and program SCALEPACK2MTZ in CCP4 software suite.
  • WT STING 155-341 bound to Compound 1 was determined by molecular replacement using program PHASER (Maximum Likelihood Molecular Replacement), with PDB ID 4F9E as the initial search model.
  • PHASER Maximum Likelihood Molecular Replacement
  • PDB ID 4F9E PDB ID 4F9E
  • the presence of Compound 1 between the dimer interface of WT STING was confirmed in a Fo-Fc difference map calculated with model phases.
  • the model was built and completed manually with Coot program and refined with Refmac5 program in CCP4 software suite.
  • Two copies of WT STING 155-341 were identified in each asymmetric unit binding to one molecule of Compound 1 at the dimer interface.
  • FIG. 11 shows a picture of the X-ray crystal structure of human WT STING in complex with Compound 1.
  • the compound binds at an interface pocket formed by a dimer of WT STING protein.
  • the two faces of adenine base of the compound form 7C-7C stacking interaction with Tyr240 and the guanidine group of Arg238, respectively.
  • the trans olefin linker forms van der Waals interaction with aliphatic portion of the side chain of Arg 238.
  • the fluorine substituent at the C2′ position of ribose group of compound nests in a hydrophobic hole defined by Thr263, Pro264 and Tyr163.
  • the negatively charged thiophosphate group of the compound forms salt bridge with Arg238 and H-bond interactions with Ser162 and Thr267, respectively.
  • the thiophosphate group also forms electrostatic interaction with guanidine group of Arg 232.
  • the LID loop region of WT STING consisting of residue 226 to 243, wraps around the two base groups and the trans olefin linker.
  • DNA sequence encoding human REF STING protein from amino acid 155 to 341 was cloned into the pET21b vector, following a His-TEV-Sumo tag at its N-terminus (SEQ ID NO: 7).
  • the sequence of the pET21b has been deposited in addgene and is available here: addgene.org/vector-database/2550/; that sequence is incorporated by reference herein.
  • E. coli BL21 (DE3) codon plus cells were transformed with this plasmid, and the expression of recombinant protein was induced with 0.1 mM Isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG).
  • IPTG Isopropyl ⁇ -D-1-thiogalactopyranoside
  • Protein was purified from the soluble fraction of cell lysate by Ni-NTA affinity chromatography. The His-TEV-Sumo tag was removed by sumo protease, and was separated from tag-free REF STING_155-341 using a second Ni-NTA affinity column. The protein was further purified by anion-exchange and size-exclusion chromatography, and was stored in buffer containing 20 mM Tris-HCl pH 7.5, and 150 mM NaCl at 24 mg/ml concentration.
  • REF STING protein was diluted to 10 mg/ml using the storage buffer (20 mM Tris-HCl pH 7.5, and 150 mM NaCl) and mixed with Compound 1 (100 mM stock in DMSO) in molar ratio 1:5. The mixture was incubated for 4 hours at 4° C., and centrifuged at 13,000 rpm for 20 min before crystallization. Crystallization screen trays were set up using the hanging-drop vapor-diffusion method at 18° C.
  • Crystals were grown by mixing 1 ⁇ L of REF STING/Compound 1 solution with an equal volume of well solution, containing 100 mM HEPES pH 7.5, 200 mM CaCl2, and 15% (wt/vol) PEG 8000. 20% (wt/vol) PEG 400 was used as cryoprotectant reagent when crystals were flash-frozen in liquid nitrogen. Diffraction datasets were collected with a Pilatus detector at SSRF BL18U1 beamline, and processed with HKL3000 and program SCALEPACK2MTZ in CCP4 software suite. This structure is shown in FIG. 12 .
  • REF STING 155-341 bound to Compound 1 was determined by molecular replacement using program PHASER (Maximum Likelihood Molecular Replacement), using previously determined WT STING 155-341 structure (as described above) as the initial search model.
  • PHASER Maximum Likelihood Molecular Replacement
  • Two copies of REF STING 155-341 were identified in each asymmetric unit binding to one molecule of Compound 1 at the dimer interface.
  • FIG. 12 shows the X-ray crystal structure of human REF STING in complex with Compound 1, which was co-crystallized from a sample of Compound 1a.
  • the compound binds at an interface pocket formed by a dimer of STING protein.
  • the two faces of adenine base of the compound form 7C-7C stacking interaction with Tyr240 and the guanidine group of Arg238, respectively.
  • the trans olefin linker forms van der Waals interaction with the aliphatic portion of the side chain of Arg238 while the guanidine portion of the side chain of Arg238 forms 7C-7C stacking interaction with the imidazole group of the side chain of His232 from outside.
  • the olefin linker is in contact with the interacting pair of the side chains of Arg238 and His232.
  • the fluorine substituent at the C2′ position of ribose group of compound nests in a hydrophobic hole defined by Thr263, Pro264 and Tyr163.
  • the negatively charged thiophosphate group of the compound forms salt bridge with Arg238 and H-bond interactions with Ser162 and Thr267, respectively.
  • the LID loop region of REF STING consisting of residue 226 to 243, wraps around the two base groups and the trans olefin linker.
  • EC50 values were calculated for human STING assays of WT STING, HAQ STING, AQ STING, and REF STING, in head-to-head comparisons using Compound 1a of the present disclosure, a natural STING ligand (2′3′ cGAMP), and the purported STING agonist ML RR-S2 CDA, as reported in Corrales, et al., “Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity,” Cell Reports (2015) 11:1018-1030, which is incorporated by reference herein. Assays were conducted as described in examples set forth above. Note that reported assay values in Table 14 were limited to the assays conducted in the head-to-head comparisons and may not reflect averaged values determined over a greater number of trials as reported in Table 5 or elsewhere.
  • Table 14 also reports dissociation binding constants (Kd) for the binding of human WT STING to each of the three tested compounds, as measured by isothermal titration calorimetry (ITC).
  • ITC isothermal titration calorimetry
  • ITC is a microcalorimetic titration technique that measures thermodynamic properties associated with intermolecular interactions. Based on these tests, Compound 1a appears to form the strongest bond with WT STING of the tested compounds.
  • Recombinant human wild type STING (aa, 139-379, H232R) protein was generated by expressing a construct in E. coli encoding a cytosolic domain of human WT STING comprising amino acids 139-379.
  • STING protein was stored at ⁇ 60° C. in 90 ⁇ L and 100 ⁇ L aliquots each at a concentration of 3.0 mg/mL and 20 mg/mL, respectively in PBS, pH 7.5 containing 5% glycerol.
  • aliquots of the protein were thawed, diluted to 400 uL and buffer-exchanged into PBS using an Amicon Ultra centrifugal filter unit (10 k MW cutoff, 0.5 mL) with at least four 10 min 14000 ⁇ g centrifugations with an Eppendorf microcentrifuge and then finally diluted to 20 ⁇ M to 30 ⁇ M (experiment-dependent) with 1 ⁇ PBS.
  • Protein concentration was determined using a Nanodrop 2000 spectrophotometer and a protein extinction coefficient of 22140 (M ⁇ 1 cm ⁇ 1 ).
  • Assays were performed on an Affinity ITC unit (TA Instruments no. 609003.901) equipped with the ITC cleaning accessory (TA Instruments no 601800.901).
  • the STING protein solution approximately 400 ⁇ L containing 20 ⁇ M to 30 ⁇ M STING protein, was pipetted into the 185 ⁇ L calorimeter cell allowing some acceptable overload.
  • the reference cell contained an equivalent amount of Milli-Q water. Incubation was carried out at 25° C. with 20 ⁇ 2.5 ⁇ L injections of 100 ⁇ M to 300 ⁇ M compounds.
  • the control software was ITC Run Ver. 3.3.0.0 (TA Instruments) was used to obtain the thermograms consisting of multiple peaks of raw heat ( ⁇ cal/sec) representing the heat rate at each injection.
  • the Kds and n values were derived and reported. Optimal conditions for the concentrations of protein and ligand were derived from preliminary experiments.
  • Compound 1a was incubated in hepatocytes of CD-1 mouse, Sprague Dawley rat, Beagle dog, Cynomolgus monkey, and human, to evaluate the formation of major metabolites.
  • Cryopreserved pooled hepatocytes were purchased from ThermoFisher Scientific (Waltham, Mass.), Xenotech, LLC (Kansas City, Kans.) and In Vitro ADMET Laboratories (Columbia, Md.), and the appropriate media were purchased from In Vitro ADMET Laboratories (Columbia, Md.) and Life Technologies (Carlsbad, Calif.).
  • AOPI staining solution and phosphate buffer were obtained from Corning Life Sciences (Tewksbury, Mass.) and Nexcelom Bioscience (Lawrence, Mass.), respectively. All chemicals, reagents, and solvents used in analysis were of either analytical or HPLC grade.
  • Compound 1a was weighed and dissolved in HPLC-water containing 0.12% formic acid PBS to make 1020 mmol/L. The solution was then diluted 2.5-fold individually to 4 mmol/L and then further diluted 21000-fold with the Williams' E medium containing 0.1% human serum albumin and 2 mmol/L L-glutamine to make the working stock solution with concentration of 20 ⁇ mol/L.
  • cryopreserved hepatocytes Prior to incubations, the cryopreserved hepatocytes were thawed in a water bath at 37° C. One tube of cryopreserved hepatocytes was added to each 50-mL conical tube of cryopreserved hepatocyte recovery medium (UCRM) obtained from In Vitro ADMET Laboratories (Columbia, Md.). The cells were spun in a Beckman centrifuge (Brea, Calif.) with a GH 3.8 rotor at 740 rpm for 10 minutes at room temperature 4° C. The supernatant was removed and the cells were re-suspended in plating media for counting.
  • UCRM cryopreserved hepatocyte recovery medium
  • the hepatocyte suspension (50 ⁇ L/well) was added into a 48-well plate. Fifty microliters of working stock solution containing Compound 1a (20 ⁇ mon) were added to start the reaction. The plate was placed into a tissue culture incubator (5% CO2/95% air humidified atmosphere and 37° C.), and the reactions were terminated with 200 ⁇ L of stop solution consisting of 100% methanol/acetonitrile (1/1, v/v) with 2010 ng/mL furosemide and 0.2 ⁇ mol/L (R)-propranolol at 5, 30, 60, 120, 180 and 240 minutes. The mixture was centrifuged and filtered, and the supernatant was collected for analysis. The final concentrations of cryopreserved hepatocytes were 1 ⁇ 106 cells/mL. The final incubation concentration of Compound 1a was 10 ⁇ mon.
  • the LC-MS/MS system was composed of a Shimadzu HPLC and an AB-SCIEX TripleTOF 5600 hybrid quadrupole and TOF mass spectrometer (Framingham, Mass.).
  • the Shimadzu HPLC (Kyoto, Japan), consisted of a communications bus module (CBM-20A), an auto-sampler (SIL-30AC) with an attached rack changer (Rack Changer II) two pumps (LC-30AD) and a column oven (CTO-30A).
  • the mass spectrometer was calibrated using the AB-SCIEX APCI both Negative and Positive Calibration Solutions (Framingham, Mass.). The samples obtained from incubations with hepatocytes were analyzed under both negative and positive scan modes.
  • the basic analytical method and instrumental conditions are summarized below. Modification of the spectrometer settings was dependent on the necessity of the analyte.
  • Mass spectrometry data were acquired using AB-Sciex Analyst TF (Version 1.5.1; Framingham, Mass.). Chromatograms and spectra were obtained using AB-Sciex PeakView (Version 2.2.0.1; Framingham, Mass.). The comparison of relative peak areas for extracted ion chromatograms were based on ⁇ 0.0002 Da of the expected exact mass-to-charge ratio (m/z) for each analyte of interest.
  • Compound 1a Under positive scan mode, Compound 1a showed the protonated molecular ion m/z 747 (C 24 H 27 F 2 N 10 O 8 P 2 S 2 + ) and the major MS/MS product ions with m/z 651 (C 24 H 26 F 2 N 10 O 6 PS + ), m/z 252 (C 10 H 11 FN 5 O 2 + ), and m/z 188 (C 9 H 10 N 5 + ).
  • the MS and MS/MS data are confirmative to the structure of Compound 1a.
  • Compound 1a was stable in the incubations with hepatocytes of mouse, rat, dog, monkey, and human. No apparent metabolite of Compound 1a was identified in this study. In the samples obtained from incubations with hepatocytes, only Compound 1a itself could be detected and confirmed by the fragments of tandem mass spectrometry (MS/MS).

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