Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

• the specification generally relates to novel compounds selectively modulating DNA-dependent protein kinase ( "DNA-PK” ) , and pharmaceutically acceptable salts thereof.
• DNA-PK DNA-dependent protein kinase
• the present disclosure also relates to pharmaceutical compositions comprising one or more of the compounds as an active ingredient, and use of the compounds in the treatment of DNA-PK related disease, including cancer.
• DNA-PK is a nuclear serine/threonine protein kinase complex composed of the catalytic subunit DNA-PKcs and a heterodimer of Ku proteins (Ku70/Ku80) . Functionally, DNA-PK is a crucial component in the repair of DNA double strand breaks (DSBs) , serving to maintain genomic integrity, and in the process of V (D) J recombination, resulting in the highly diverse repertoire of antibodies/immunoglobulins and T cell receptors found on B-and T-cells respectively.
• DSBs DNA double strand breaks
• DNA-PK and its components are involved in a variety of other physiological processes, including modulation of chromatin structure, telomere maintenance, transcriptional regulation, and the response to replication stress (Smith and Jackson, 1999; Goodwin and Knudsen, 2014) .
• ROS reactive oxygen species
• DSBs can occur if previous single-strand breaks occur in close proximity.
• single and single-double strand breaks are caused when the DNA replication fork encounters damaged base patterns.
• foreign influences such as ionizing radiation (eg. Gamma or particle radiation) , and certain anti-cancer drugs (e.g. B. Bleomycin) are able to elicit DSBs.
• DSBs may also occur as intermediates of somatic recombination, a process important to the formation of a functional immune system of all vertebrates.
• DNA-PK DNA non-homologous end-joining
• HR homologous recombination
• DNA-PK inhibitors were demonstrated to be useful in oncology could include targeting tumors with high levels of replication stress (Lin et al., 2014; Ashley et al., 2014; Buisson et al., 2015) , either as a monotherapy or in combination with other agents in prostate (Goodwin et al., 2013) and breast (Medunjanin et al., 2010) cancers.
• the present disclosure provides a compound represented by Formula (I) :
• the present disclosure provides a compound represented by Formula (Ia) :
• the present disclosure provides a compound represented by Formula (Ib) :
• the present disclosure provides a compound represented by Formula (Ic) :
• the present disclosure provides a compound represented by Formula (Id) :
• the present disclosure provides a compound represented by Formula (Ie) :
• the present disclosure provides a pharmaceutical composition
• a pharmaceutical composition comprising one or more compounds of Formula (I) , Formula (Ia) , Formula (Ib) , Formula (Ic) , Formula (Id) , Formula (Ie) , or a pharmaceutically acceptable salts thereof, as an active ingredient.
• the present disclosure further provides a compound of Formula (I) , or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of one or more of the foregoing, for use in inhibiting DNA-PK kinase.
• the present disclosure provides use of the compounds of Formula (I) , or a pharmaceutically acceptable salts thereof, or a pharmaceutical composition of one or more of the foregoing in the manufacture of a medicament for inhibiting DNA-PK kinase in a subject.
• the present disclosure provides a method for inhibiting DNA-PK kinase, by using one or more compounds of Formula (I) , or a pharmaceutically acceptable salts thereof or the pharmaceutical composition of one or more of the foregoing.
• the present disclosure provides a method for treating a DNA-PK related disorder (e.g., cancer) , by using the compounds of Formula (I) , or a pharmaceutically acceptable salts thereof or the pharmaceutical composition of one or more of the foregoing.
• a DNA-PK related disorder e.g., cancer
• the present disclosure provides a compound of Formula (I) , or a pharmaceutically acceptable salt thereof, in combination with a second therapeutic agent, preferably an anti-tumor agent.
• the present disclosure provides a combined use of a compound of Formula (I) , or a pharmaceutically acceptable salt thereof, and a second therapeutic agent, preferably an anti-tumor agent.
• the present disclosure provides compounds of Formula (I) :
• X 1 , X 2 , and X 3 are each independently C or N, provided that at least one of X 1 , X 2 , and X 3 is N and at least one of X 1 , X 2 , and X 3 is C;
• dash line means the bond between X 1 and X 2 , and between X 2 and X 3 can be single or double bonds, provided that at least one of the bonds between X 1 and X 2 , and between X 2 and X 3 is single bond;
• R 1 is absent, halogen, or C 1-6 alkyl, wherein said C 1-6 alkyl can be optionally mono-or independently multi-substituted by hydroxyl, halogen, or deuterium;
• each R 2 , R 3 and R 4 is independently selected from absent, halogen, hydroxyl, cyano, C 1-6 alkyl, C 1-6 alkoxyl, - (CH 2 ) n -Q, which can be optionally mono-or independently multi-substituted by deuterium, hydroxyl, amino, cyano, halogen, C 1-6 alkyl, C 1-6 haloalkyl, (C ⁇ N) -C 1-6 alkyl, C 1-6 alkoxyl, C 1-6 haloalkoxyl, C 3-8 cycloalkyl, C 3-8 cycloalkoxyl, 3-8 membered aryl, or 3-8 membered heterocyclyl,
• n 0, 1 or 2
• Q is 3-8 membered saturated or unsaturated carbocyclyl, or 3-8 membered saturated or unsaturated heterocyclyl;
• Ring A is 5-12 membered aryl, 5-12 membered heteroaryl having 1-5 ring heteroatoms chosen from oxygen, sulfur and nitrogen, 8-10 membered bicyclic ring having 0-5 ring heteroatoms chosen from oxygen, sulfur and nitrogen, wherein Ring A is not phenyl.
• R 2 is selected from methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, oxetanyl, cyclopentanyl, tetrahydrofuryl, cyclohexanyl, tetrahydropyranyl, cycloheptanyl, piperidinyl, phenyl, pyridinyl, pyridonyl, oxocanyl, tetrahydropyranyl, dihydropyranyl, spiro [3.3] heptanyl, spiro [2.5] octanyl, bicyclo [1.1.1] pentanyl, bicyclo [3.2.1] octanyl, 8-oxa bicyclo [3.2.1] octan-3-yl, which can be optionally mono-or independently multi-substituted by hydroxyl, cyano, halogen, C 1-6 alkyl, C 1-6
• R 2 is selected from:
• R 2 is cyclohexanyl, or tetrahydropyranyl, which can be optionally mono-or independently multi substituted by halogen, C 1-6 alkyl or C 1-6 alkoxyl.
• R 1 is methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, or isobutyl, which can be optionally mono-or independently multi-substituted by hydroxyl, halogen, or deuterium.
• R 1 is methyl, ethyl, tri-fluoro-methyl, or tri-deuterium-methyl.
• Ring A is 6 membered heteroaryl having 1 ring heteroatoms nitrogen, 9 membered bicyclic ring having 2-3 ring heteroatoms chosen from oxygen, sulfur and nitrogen, optionally, the 9 membered bicyclic ring is a phenyl-or pyridinyl-fused bicyclic ring, optionally, Ring A is selected from
• each R 3 and R 4 is independently selected from absent, halogen, hydroxyl, cyano, C 1-6 alkyl, CN-C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyl, C 1-6 haloalkoxyl, 3 to 8 membered saturated or unsaturated heterocyclyl, wherein said heterocyclyl can optionally be further mono-or independently multi-substituted by C 1-3 alkyl.
• Ring A is each R 3 and R 4 is independently selected from absent, methyl, cyano, methoxyl, chloro, cyano-methyl, pyrazolyl, oxazolyl, wherein said pyrazolyl or oxazolyl can optionally be further mono-or independently multi-substituted by C 1-3 alkyl.
• the compounds provided herein have a structure of Formula Ia
• R 1 , R 2 , R 3 , R 4 , Ring A are as herein defined.
• the compounds provided herein have a structure of Formula Ib
• R 1 , R 2 , R 3 , R 4 , Ring A are as herein defined.
• the compounds provided herein have a structure of Formula Ic
• R 1 , R 2 , R 3 , R 4 , Ring A are as herein defined.
• the compounds provided herein have a structure of Formula Id
• R 1 , R 2 , R 3 , R 4 , Ring A are as herein defined.
• the compounds provided herein have a structure of Formula Ie
• dash line means the bond between X 1 and N , and between N and X 3 can be single or double bonds, provided that at least one of the bonds between X 1 and N , and between N and X 3 is single bond;
• R 1 is C 1-3 alkyl
• R 2 is cyclopentyl, cyclohexanyl, tetrahydropyranyl or 8-oxabicyclo [3.2.1] octan-3-yl, which can be optionally mono-or independently multi substituted by halogen or C 1-3 alkoxyl,
• Y 1 , Y 2 , and Y 3 are each independently C or N, provided that at least one of Y 1 , Y 2 , and Y 3 is N;
• R 5 is halogen or C 1-3 alkyl
• R 6 is C 1-3 alkyl.
• R 2 of Formula Ie is un-substituted cyclopentyl, cyclohexanyl, tetrahydropyranyl or 8-oxabicyclo [3.2.1] octan-3-yl, which optionally can be mono-or independently multi substituted by halogen or C 1-3 alkoxyl.
• Y 3 of Formula Ie is N and at least one of Y 1 and Y 2 is N.
• R 5 of Formula Ie is methyl
• linking substituents are described. Where the structure clearly requires a linking group, the Markush variables listed for that group are understood to be linking groups. For example, if the structure requires a linking group and the Markush group definition for that variable lists “alkyl” , then it is understood that the “alkyl” represents a linking alkylene group.
• substituted when refers to a chemical group, means the chemical group has one or more hydrogen atoms that is/are removed and replaced by substituents.
• substituted has the ordinary meaning known in the art and refers to a chemical moiety that is covalently attached to, or if appropriate, fused to, a parent group.
• optionally substituted or optionalally...substituted means that the chemical group may have no substituents (i.e. unsubstituted) or may have one or more substituents (i.e. substituted) . It is to be understood that substitution at a given atom is limited by valency.
• C i-j indicates a range of the carbon atoms numbers, wherein i and j are integers and the range of the carbon atoms numbers includes the endpoints (i.e. i and j) and each integer point in between, and wherein j is greater than i.
• C 1-6 indicates a range of one to six carbon atoms, including one carbon atom, two carbon atoms, three carbon atoms, four carbon atoms, five carbon atoms and six carbon atoms.
• the term “C 1-12 ” indicates 1 to 12, including 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 carbon atoms.
• alkyl refers to a saturated or unsaturated hydrocarbon chain, while the latter may be further subdivided into hydrocarbon chain having at least one double or triple bonds (alkenyl or alkynyl) .
• alkyl refers to a saturated hydrocarbon chain.
• the hydrocarbon chain mentioned above may be straight-chain or branched-chain.
• C i-j alkyl refers to an alkyl having i to j carbon atoms.
• saturated alkyl group examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1, 2, 2-trimethylpropyl, and the like.
• Examples of unsaturated alkyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, ethynyl, propyn-1-yl, propyn-2-yl, and the like.
• Examples of “C 1-6 alkyl” include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl and tert-butyl.
• Examples of “C 1-3 alkyl” include, but are not limited to, methyl, ethyl, propyl, and isopropyl.
• alkylene groups include, but are not limited to, methylene, 1, 1-ethylene, 1, 2-ethylene, 1, 1-propylene, 1, 2-propylene, 1, 3-propylene, 2, 2-propylene, tertbutanylene and the like.
• amino refers to the group of formula “-NH 2 ” .
• cyano refers to the group of formula “-C ⁇ N” .
• halo and “halogen” refer to fluoro, chloro, bromo or iodo groups.
• hydroxyl refers to the group of formula “-OH” .
• alkoxy refers to a group of formula -O-alkyl.
• C i-j alkoxy means that the alkyl moiety of the alkoxy group has i to j carbon atoms.
• alkoxy groups include, but are not limited to, methoxyl, ethoxyl, propoxyl (e.g. n-propoxy and isopropoxy) , t-butoxy, and the like.
• Examples of “C 1-12 alkoxyl” are methoxyl, ethoxyl and propoxyl.
• hydroxyC 1-12 alky refers to a group of formula “-C 1-12 alkyl-OH” , wherein the alkyl moiety of the group has 1 to 12 carbon atoms, and one or more hydroxyl groups may be linked to any carbon atoms in the alkyl moiety.
• “C i-j alky-OH” has one hydroxyl group.
• Examples of “C 1-12 alkyl-OH” are hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl and 1-hydroxyisopropyl.
• C i-j haloalkyl refers to a halogen substituted (mono-or multi-substituted) C i-j alkyl group.
• C 1-12 haloalkyl are fluoromethyl, difluoromethyl, trifluoromethyl, fluoroethyl, difluoroethyl, trifluoroethyl, chloroethyl and bromoisopropyl.
• difluoroethyl are 1, 1-difluoroethyl.
• trifluoroethyl are 2, 2, 2-trifluoroethyl and 1, 2, 2-trifluoroethlyl.
• C i-j haloalkoxyl are fluoromethoxyl, difluoromethoxyl, or tri-fluoromethoxyl.
• trifluoroethoxy are 2, 2, 2-trifluoroethoxy and 1, 2, 2-trifluoroethoxy.
• aryl or “aromatic” , whether as part of another term or used independently, refers to a ring system with alternating double and single bonds between atoms forming rings.
• aryl or “aromatic” also intends to include pseudoaromatic.
• pseudoaromatic refers to a ring system which is not strictly aromatic, but which is stabilized by means of delocalization of electrons and behaves in a similar manner to aromatic rings.
• An aryl or an aromatic group may have mono-or poly-ring (s) . Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl and the like.
• heteroaryl refers to aryl which contains at least one ring forming heteroatom selected from O, S, N, P, and the like. Heteroaryl includes but are not limited to, furyl, thienyl, pyridinyl, triazinyl, pyridyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, indolizinyl, indolyl, isoindolyl, indolinyl, 1, 2, 3-oxadiazolyl, 1, 2, 4-oxadiazolyl, 1, 2, 4-oxadiazol-5-one, 1, 2, 3-triazolyl, 1, 3, 4-thiadiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, quinazolinyl, isoquinazolinyl, 1, 3, 5-tri
• the term “carbocyclyl” refers to any ring, including mono-or poly-cyclic ring (s) (e.g. having 2 or 3 fused, bridged or spiro rings) , in which all the ring atoms are carbon and which contains at least three ring forming carbon atoms.
• spiro refers to ring systems having two rings connected through one single common atom; the term “fused” rings refers to ring systems having two rings sharing two adjacent atoms; and the term “bridged” rings refers to ring systems with two rings sharing three or more atoms.
• the carbocyclyl may contain 3 to 12 ring forming carbon atoms (i.e. 3-12 membered carbon atoms) , 3 to 10 ring forming carbon atoms, 3 to 9 ring forming carbon atoms or 3 to 8 ring forming carbon atoms.
• Carbocyclyl groups may be saturated, partially unsaturated or fully unsaturated.
• the carbocyclyl group may be a saturated cyclic alkyl group.
• the carbocyclyl group may be an unsaturated cyclic alkyl group that contains at least one double bond in its ring system.
• an unsaturated carbocyclyl group may contains one or more aromatic rings.
• one or more ring forming -CH 2 -group of the saturated or unsaturated carbocyclyl may be replaced by a -C (O) -group.
• the carbocyclyl group is a monocyclic alkyl group. In some embodiments, the carbocyclyl group is a saturated monocyclic alkyl group. Examples of saturated monocyclic alkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, and the like.
• a 3-8 “membered saturated or unsaturated carbocyclyl” is a saturated, partially unsaturated or fully unsaturated mono-or poly-cyclic ring system having 3 to 8, 3 to 6, or 5 to 8 ring forming carbon atoms respectively, wherein one or more ring forming -CH 2 -group can optionally be replaced by a -C (O) -group.
• Examples of “3-8 membered saturated or unsaturated carbocyclyl” are C 3-6 cycloalkyl, cyclohexyl, cyclohexenyl, cyclopentyl, phenyl, naphthyl and bicyclo [1.1.1] pentan-1-yl.
• Examples of “C 3-8 cycloalkyl” are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
• the term “C 3-8 cycloalkoxyl” refers to the group of formula “C 3- 8 cycloalkyl-O-” .
• heterocyclyl refers to a carbocyclyl group, wherein one or more (e.g. 1, 2 or 3) ring atoms are replaced by heteroatoms, which include, but are not limited to, O, S, N, P, and the like.
• the heterocyclyl is a saturated heterocyclyl.
• the heterocyclyl is an unsaturated heterocyclyl having one or more double bonds in its ring system.
• the heterocyclyl is a partially unsaturated heterocyclyl.
• the heterocyclyl is a fully unsaturated heterocyclyl.
• an unsaturated heterocyclyl group may contain one or more aromatic rings.
• one or more ring forming -CH 2 -group of the heterocyclyl can optionally be replaced by a -C (O) -, a -S-, a -S (O) -, or a -S (O) 2 -group.
• said ring forming sulphur atom may be optionally oxidised to form the S-oxides.
• the heterocyclyl is linked to the other portion of a compound through its ring forming carbon. In some embodiments, the heterocyclyl is linked to the other portion of a compound through its ring forming nitrogen.
• a 3-8 “membered saturated or unsaturated heterocyclyl” is a saturated, partially unsaturated or fully unsaturated mono-or poly-cyclic ring (s) (e.g. having 2 or 3 fused, bridged or spiro rings) system having 3 to 8 ring forming atoms respectively, of which at least one ring forming atom is chosen from nitrogen, sulphur or oxygen, which may, unless otherwise specified, linked to the other portion of a compound through its ring forming carbon or nitrogen, wherein one or more ring forming -CH 2 -group of the saturated or unsaturated heterocyclyl may be replaced by a -C (O) -, a -S-, a -S (O) -, or a -S (O) 2 -group, and wherein when the heterocyclyl contains a sulphur in its ring system, said ring sulphur atom may be optionally oxidised to form the S-oxides.
• s mono
• Exemplary monocyclic heterocyclyl groups include, but are not limited to oxetanyl, pyranyl, 1, 1-dioxothietanylpyrrolidyl, tetrahydrofuryl, tetrahydrothienyl, pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, thiazolyl, piperidyl, piperidyl, piperazinyl, morpholinyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, pyridonyl, pyrimidonyl, pyrazinonyl, pyrimidonyl, pyridazonyl, triazinonyl, and the like.
• spiro heterocyclyl examples include, but are not limited to, spiropyranyl, spirooxazinyl, and the like.
• fused heterocyclyl examples include, but are not limited to, phenyl fused ring or pyridinyl fused ring, such as quinolinyl, isoquinolinyl, quinoxalinyl, quinolizinyl, quinazolinyl, azaindolizinyl, pteridinyl, chromenyl, isochromenyl, indolyl, isoindolyl, indolizinyl, indazolyl, purinyl, benzofuranyl, isobenzofuranyl, benzimidazolyl, benzothienyl, benzothiazolyl, carbazolyl, phenazinyl, phenothiazinyl, phenanthridinyl, imidazo [1, 2-a] pyridin
• bridged heterocyclyl examples include, but are not limited to, morphanyl, hexamethylenetetraminyl, 8-aza-bicyclo [3.2.1] octane, 1-aza-bicyclo [2.2.2] octane, 1, 4-diazabicyclo [2.2.2] octane (DABCO) , and the like.
• stereoisomer refers to any of the various stereoisomeric configurations (e.genantiomers, diastereomers and racemates) of an asymmetric compound (e.g. those having one or more asymmetrically substituted carbon atoms or “asymmetric centers” ) .
• asymmetric compound e.g. those having one or more asymmetrically substituted carbon atoms or “asymmetric centers”
• Compounds of the present disclosure that contain asymmetric centers can be isolated in optically active (enantiomers or diastereomers) or optically inactive (racemic) forms.
• enantiomer includes pairs of stereoisomers that are non-superimposable mirror images of each other.
• a 1: 1 mixture of a pair of enantiomers is a “racemic mixture” .
• diastereomers or “diastereoisomers” include stereoisomers that have at least two asymmetric atoms, but which are not mirror images of each other. Certain compounds containing one or more asymmetric centers may give rise to enantiomers, diastereomers or other stereoisomeric forms that may be defined, in terms of absolute configuration, as (R) -or (S) -at each asymmetric center according to the Cahn-Ingold-Prelog R-Ssystem. Resolved compounds whose absolute configuration is unknown can be designated using the term “or” at the asymmetric center. Methods on how to prepare optically active forms from racemic mixtures are known in the art, such as resolution by HPLC or stereoselective synthesis.
• geometric isomers or “cis and trans isomers” refer to compounds with same formula but their functional groups are rotated into a different orientation in three-dimensional space.
• tautomers include prototropic tautomers that are isomeric protonation states of compounds having the same formula and total charge.
• prototropic tautomers include, but are not limited to, ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, 1H-and 3H-imidazole, 1H-, 2H-and 4H-1, 2, 4-triazole, 1H-and 2H-isoindole, and 1H-and 2H-pyrazole.
• Tautomers can be in equilibrium or sterically locked into one form by appropriate substitution.
• Compounds of the present disclosure identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified.
• the “compound” of the present disclosure is also intended to encompass all isotopes of atoms in the compounds.
• Isotopes of an atom include atoms having the same atomic number but different mass numbers.
• hydrogen, carbon, nitrogen, oxygen, phosphorous, sulphur, fluorine, chlorine, bromide or iodine in the “compound” of present disclosure are meant to also include their isotopes such as but are not limited to: 1 H, 2 H, 3 H, 11 C, 12 C, 13 C, 14 C, 14 N, 15 N, 16 O, 17 O, 18 O, 31 P, 32 P, 32 S, 33 S, 34 S, 36 S, 17 F, 19 F, 35 Cl, 37 Cl, 79 Br, 81 Br, 127 I and 131 I.
• hydrogen includes protium, deuterium and tritium.
• carbon includes 12 C and 13 C.
• “compound” of the present disclosure only encompasses the isotopes of hydrogen in the compound. In some embodiments, “compound” of the present disclosure only encompasses the isotopes of atoms in natural abundance.
• the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• compounds, materials, compositions, and/or dosage forms that are pharmaceutically acceptable refer to those approved by a regulatory agency (such as U.S. Food and Drug Administration, China Food and Drug Administration or European Medicines Agency) or listed in generally recognized pharmacopoeia (such as U.S. Pharmacopoeia, China Pharmacopoeia or European Pharmacopoeia) for use in animals, and more particularly in humans.
• “pharmaceutically acceptable salts” refers to derivatives of the compounds of present disclosure wherein the parent compound is modified by converting an existing acidic moiety (e.g. carboxyl and the like) or base moiety (e.g. amine, alkali and the like) to its salt form.
• compounds of present disclosure are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.
• the pharmaceutically acceptable salts are acid and/or base salts that retain biological effectiveness and properties of the parent compound, which typically are not biologically or otherwise undesirable.
• Suitable pharmaceutically acceptable salts of a compound of the present disclosure includes, for example, an acid-addition salt, which can be derived from for example an inorganic acid (for example, hydrochloric, hydrobromic, sulfuric, nitric, phosphoric acid and the like) or organic acid (for example, formic, acetic, propionic, glycolic, oxalic, maleic, malonic, succinic, fumaric, tartaric, trimesic, citric, lactic, phenylacetic, benzoic, mandelic, methanesulfonic, napadisylic, ethanesulfonic, toluenesulfonic, trifluoroacetic, salicylic, sulfosalicylic acids and the like) .
• the pharmaceutically acceptable salt of the compound of the present disclosure is a formic acid salt.
• the pharmaceutically acceptable salt of the compound of the present disclosure is a TFA salt.
• Suitable pharmaceutically acceptable salts of a compound of the present disclosure also include, for example, an base-addition salt, which can be derived from for example an inorganic bases (for example, sodium, potassium, ammonium salts and hydroxide, carbonate, bicarbonate salts of metals from columns I to XII of the periodic table such as calcium, magnesium, iron, silver, zinc, copper and the like) or organic bases (for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like) .
• an inorganic bases for example, sodium, potassium, ammonium salts and hydroxide, carbonate, bicarbonate salts of metals from columns I to XII of the periodic table such as calcium, magnesium, iron, silver, zinc, copper and the like
• organic bases for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines,
• organic amines include but are not limited to isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine and tromethamine.
• acids or bases for forming acid/base-addition salts other than those shown in the examples may also be possible. Lists of additional suitable salts can be found, e.g. in “Remington's Pharmaceutical Sciences” , 20th ed., Mack Publishing Company, Easton, Pa., (1985) ; and in “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002) .
• Suitable pharmaceutically acceptable salts of a compound of the present disclosure is inorganic bases salt.
• the present disclosure also includes active intermediates, active metabolites and prodrugs of the compounds of present disclosure.
• an “active intermediate” refer to intermediate compound in the synthetic process, which exhibits the same or essentially the same biological activity as the final synthesized compound.
• an “active metabolite” refers to a break-down or end product of a compound of the present disclosure or its salt or prodrug produced through metabolism or biotransformation in the animal or human body, which exhibits the same or essentially the same biological activity as the specified compound. Such metabolites may result from, for example, oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, and the like, of the administered compound or salt or prodrug.
• prodrugs refer to any compounds or conjugates which release the active parent drug when administered to an animal or human subject.
• Prodrugs can be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleavable, either in routine manipulation or in vivo, from the parent compounds.
• Prodrugs include compounds wherein hydroxyl, amino, sulfhydryl, or carboxyl group is bonded to any group that, when administered to a mammalian subject, is cleavable to form a free hydroxyl, amino, sulfhydryl, or carboxyl group respectively.
• prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups in the compounds of the present disclosure. Preparation and use of prodrugs is discussed in THiguchi and V. Stella, “Pro-drugs as Novel Delivery Systems” , Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are hereby incorporated by reference in their entirety.
• novel compounds or pharmaceutically acceptable salts which can selectively inhibit DNA-PK.
• the compounds of the presend disclosure, or a pharmaceutically acceptable salt thereof when compared with other clinically available DNA-PK inhibitors, exhibit certain improved properties e.g. higher BBB penetration (thus making them potentially useful for the treatment of cancers that have metastasised to the CNS, in particular brain metastases and leptomeningeal metastases) , better potency etc. They may also possess favourable toxicity profiles, and/or favourable metabolic or pharmacokinetic profiles, in comparison with known DNA-PK inhibitors.
• such compounds, or a pharmaceutically acceptable salt thereof may be especially useful in the treatment of cancer, especially those with brain metastasis.
• the reactions for preparing compounds of the disclosure can be carried out in suitable solvents, which can be readily selected by one skilled in the art of organic synthesis.
• suitable solvents can be substantially non-reactive with the starting materials (reactants) , the intermediates, or products at the temperatures at which the reactions are carried out, e.g., temperatures that can range from the solvent's freezing temperature to the solvent's boiling temperature.
• a given reaction can be carried out in one solvent or a mixture of more than one solvent.
• suitable solvents for a particular reaction step can be selected by a skilled artisan.
• Preparation of compounds of the disclosure can involve the protection and deprotection of various chemical groups.
• the need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art.
• the chemistry of protecting groups can be found, for example, in T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3rd Ed., Wiley &Sons, Inc., New York (1999) , which is incorporated herein by reference in its entirety.
• Reactions can be monitored according to any suitable method known in the art.
• product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) , infrared spectroscopy, spectrophotometry (e.g., UV-visible) , mass spectrometry, or by chromatographic methods such as high performance liquid chromatography (HPLC) , liquid chromatography-mass spectroscopy (LCMS) , or thin layer chromatography (TLC) .
• HPLC high performance liquid chromatography
• LCMS liquid chromatography-mass spectroscopy
• TLC thin layer chromatography
• Compounds can be purified by those skilled in the art by a variety of methods, including high performance liquid chromatography (HPLC) ( “Preparative LC-MS Purification: Improved Compound Specific Method Optimization” Karl F. Blom, Brian Glass, Richard Sparks, Andrew P. Combs J. Combi. Chem. 2004, 6 (6)
• g for equivalent orequivalents, “g” for gram or grams, “mg” for milligram or milligrams, “L” for liter or liters, “mL” or “ml” for milliliter or milliliters, “ ⁇ L” for microliter or microliters, “N” for normal, “M” for molar, “mmol” for millimole or millimoles, “min” for minute or minutes, “h” or “hr” for hour or hours, “r. t. ” or “rt” for room temperature, “atm” for atmosphere, “psi” for pounds per square inch, “conc.
• the present disclosure provides pharmaceutical compositions comprising at least one compound of the present disclosure.
• the pharmaceutical composition comprises more than one compounds of the present disclosure.
• the pharmaceutical composition comprises one or more compounds of the present disclosure, and a pharmaceutical acceptable carrier.
• the pharmaceutically acceptable carriers are conventional medicinal carriers in the art which can be prepared in a manner well known in the pharmaceutical art.
• the compounds of the present disclosure may be admixed with pharmaceutically acceptable carrier for the preparation of pharmaceutical composition.
• pharmaceutically acceptable carrier refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound provided herein from one location, body fluid, tissue, organ (interior or exterior) , or portion of the body, to another location, body fluid, tissue, organ, or portion of the body.
• Pharmaceutically acceptable carriers can be vehicles, diluents, excipients, or other materials that can be used to contact the tissues of an animal without excessive toxicity or adverse effects.
• Exemplary pharmaceutically acceptable carriers include, sugars, starch, celluloses, malt, tragacanth, gelatin, Ringer’s solution, alginic acid, isotonic saline, buffering agents, and the like.
• Pharmaceutically acceptable carrier that can be employed in present disclosure includes those generally known in the art, such as those disclosed in “Remington Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991) , which is incorporated herein by reference.
• materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydrox
• compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pHadjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
• compositions depends on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
• the pharmaceutical compositions can be formulated for oral, nasal, rectal, percutaneous, intravenous, or intramuscular administration.
• dosage forms for nasal administration may conveniently be formulated as aerosols, solutions, drops, gels or dry powders; dosage forms for intranasal administration, may be formulated as a fluid formulation.
• the pharmaceutical compositions can be formulated in the form of tablets, capsule, pill, dragee, powder, granule, sachets, cachets, lozenges, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium) , spray, omintment, paste, cream, lotion, gel, patche, inhalant, or suppository.
• the pharmaceutical compositions can also be formulated to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
• the pharmaceutical composition is formulated in a sustained released form.
• sustained released form refers to release of the active agent from the pharmaceutical composition so that it becomes available for bio-absorption in the subject, primarily in the gastrointestinal tract of the subject, over a prolonged period of time (extended release) , or at a certain location (controlled release) .
• the prolonged period of time can be about 1 hour to 24 hours, 2 hours to 12 hours, 3 hours to 8 hours, 4 hours to 6 hours, 1 to 2 days or more.
• the prolonged period of time is at least about 4 hours, at least about 8 hours, at least about 12 hours, or at least about 24 hours.
• the pharmaceutical composition can be formulated in the form of tablet.
• release rate of the active agent can not only be controlled by dissolution of the active agent in gastrointestinal fluid and subsequent diffusion out of the tablet or pills independent of pH, but can also be influenced by physical processes of disintegration and erosion of the tablet.
• polymeric materials as disclosed in “Medical Applications of Controlled Release, ” Langer and Wise (eds. ) , CRC Pres., Boca Raton, Florida (1974) ; “Controlled Drug Bioavailability, ” Drug Product Design and Performance, Smolen and Ball (eds.
• the pharmaceutical compositions comprise about 0.0001mg to about 100mg of the compounds of the present disclosure (e.g. about 0.0001mg to about 10mg, about 0.001mg to about 10mg, about 0.01mg to about 10mg, about 0.1mg to about 10mg, about 0.1mg to about 5mg, about 0.1mg to about 4mg, about 0.1mg to about 3mg, about 0.1mg to about 2mg, about 0.1mg to about 1mg, about 0.1mg to about 0.5mg, about 1mg to about 10mg, about 1mg to about 5mg, about 5mg to about 10mg, about 5mg to about 20mg, about 5mg to about 30mg, about 5mg to about 40mg, about 5mg to about 50mg, about 10mg to about 100mg, about 20mg to about 100mg, about 30mg to about 100mg, about 40mg to about 100mg
• the pharmaceutical compositions can be formulated in a unit dosage form, each dosage containing from about 0.0001mg to about 10mg, about 0.001mg to about 10mg, about 0.01mg to about 10mg, about 0.1mg to about 10mg, about 0.1mg to about 5mg, about 0.1mg to about 4mg, about 0.1mg to about 3mg, about 0.1mg to about 2mg, about 0.1mg to about 1mg, about 0.1mg to about 0.5mg, about 1mg to about 10mg, about 5mg to about 10mg, about 5mg to about 20mg, about 5mg to about 30mg, about 5mg to about 40mg, about 5mg to about 50mg, about 10mg to about 100mg, about 20mg to about 100mg, about 30mg to about 100mg, about 40mg to about 100mg, about 50mg to about 100mg of the compounds of the present disclosure.
• the pharmaceutical compositions comprise one or more compounds of the present disclosure as a first active ingredient, and further comprise a second active ingredient.
• the second active ingredient can be any immunomodulator or anti-tumour agent known in the art, including without limitation, chemotherapeutics, immunotherapeutics, cell signal transduction inhibitors, cell signal transduction inhibitors, alkylating agents, topoisomerase inhibitors, mitosis inhibitors, antihormonal agents, etc.
• immunomodulators or anti-tumour agents are, platinum based chemotherapeutics (e.g., Cisplatin (DDP) , Carboplatin (CBP) , Sulfato-1, 2-diaminocyclohexane platinum (SHP) , Nedaplatin, Oxaliplatin (OXA) , Laboplatin) , Docetaxel, Paclitaxel, Doxorubicin, Etoposide, Mitoxantrone, CTLA-4 inhibitors, anti-CTLA-4 antibodies, PD-1 inhibitors, PD-L1 inhibitors, anti-PD-1/PD-L1 antibodies, CD39 inhibitors, anti-CD39 antibodies, CD73 inhibitors, anti-CD73 antibodies, CCR2 inhibitors, anti-CCR2 antibodies, EGFR inhibitors, CDK 4/6 inhibitors, MELK inhibitors, OX40 agonists, antiandrogen inhibitors, IgG4 isotype antibodies, tyrosine kinase inhibitors,
• anti tumour agents for treating cancers or tumors may include, but are not limited to, cisplatin, carboplatin, SHP, nedaplatin, oxaliplatin, laboplatin, docetaxel, paclitaxel, doxorubicin, etoposide, mitoxantrone, vincristine, vinblastine, gemcitabine, cyclophosphamide, chlormabucil, carmustine, methotrexate, fluorouracil, actinomycin, epirubicin, anthracycline, bleomycin, mitomycin-C, irinotecan, topotecan, teniposide interleukin, interferon, tremelimumab, ipilimumab, pembrolizumab, nivolumab, avelumab, durvalumab, atezolizumab, IPH 52, IPH 53, CPI-006, plozaliz
• anti-tumour agent examples can also be found in Cancer Principles and Practice of Oncology by V.T. Devita and S. Hellman (editors) , 6th edition (Feb. 15, 2001) , Lippincott Williams &Wilkins Publishers. A person of ordinary skill in the art would also be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the cancer involved.
• a combination suitable for use in the treatment of cancer comprising a compound of formula (I) as defined hereinbefore or a pharmaceutically acceptable salt thereof and any one of the immunomodulators or anti tumour agents listed above.
• “combination” refers to simultaneous, separate or sequential administration. In some embodiments, “combination” refers to simultaneous administration. In another aspect of the present disclosure, “combination” refers to separate administration. In a further aspect of the present disclosure, “combination” refers to sequential administration. Where the administration is sequential or separate, the delay in administering the second component should not be such as to lose the beneficial effect of the combination.
• a pharmaceutical composition which comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with an immunomodulator or anti-tumour agent selected from those listed above, in association with a pharmaceutically acceptable diluent or carrier.
• a pharmaceutical composition which comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with an immunomodulator or anti-tumour agent selected from one listed above, in association with a pharmaceutically acceptable diluent or carrier for use in producing an immunomodulating or anti-cancer effect.
• a pharmaceutical composition which comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with an immunomodulator or anti-tumour agent selected from one listed above, in association with a pharmaceutically acceptable diluent or carrier for use in treating DNA-PK related disorder, for example, NSCLC, RCC, prostate cancer, or breast cancer etc.
• kits comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with an immunomodulator or anti-tumour agent selected from one listed above.
• a kit comprising:
• the compounds of formula (I) are also useful as pharmacological tools in the development and standardisation of in vitro and in vivo test systems for the evaluation of the activity or the expression of DNA-PK in laboratory animals such as cats, dogs, rabbits, monkeys, rats and mice, as part of the search for new therapeutic agents.
• the present disclosure provides a method of treating DNA-PK related disorders, comprising administering to a subject an effective amount of one or more compounds, pharmaceutically acceptable salts thereof or the pharmaceutical composition of the present disclosure.
• the present disclosure also provides a method of treating DNA-PK related disorders.
• the method comprises administering to a subject an effective amount of one or more compounds, pharmaceutically acceptable saltsthereof or the pharmaceutical composition of the present disclosure.
• DNA-PK related disorders refers to diseases whose onset or development or both are associated with the expression or activity of DNA-PK. Examples include but are not limited to, hyperproliferative disorder (e.g., cancer) .
• the DNA-PK related disorders is cancer, preferably a DNA-PK overexpressing cancer.
• a “DNA-PK overexpressing cancer” is one which has significantly higher levels of DNA-PK protein in a cancer or tumor cell, compared to a noncancerous cell of the same tissue type. Such overexpression may be caused by gene amplification or by increased transcription or translation. DNA-PK overexpression may be determined in a diagnostic or prognostic assay by evaluating increased levels of the DNA-PK proteins present in a cell (e.g. via an immunohistochemistry assay; IHC) . Alternatively, or additionally, one may measure levels of DNA-PK encoding nucleic acid in the cell, e.g.
• FISH fluorescent in situ hybridization
• PCR polymerase chain reaction
• RT-PCR real time quantitative PCR
• the cancers include but are not limited to, lung cancer (e.g. non-small cell lung cancer (NSCLC) , small cell lung cancer, lung adenocarcinoma, large cell lung cancer, squamous cell lung cancer) , renal cell carcinoma (RCC) , prostate cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bone cacner, uterine cancer, colon cancer, leukemia, glioblastoma, melanoma, chondrosarcoma, brain cancer, cholangiocarcinoma, osteosarcoma, lymphoma, adenoma, myeloma, hepatocellular carcinoma, adrenocortical carcinoma, pancreatic cancer, bladder cancer, liver cancer, gastric cancer, colorectal cancer, esophageal cancer, testicular cancer, skin cancer, kidney cancers, mesothelioma, neuroblastoma, thyroid cancer, head and neck cancers, esophage
• the cancer is NSCLC, RCC, prostate cancer, or breast cancer.
• the cancer as mentioned herein can be at any stage, unless otherwise specified.
• the cancer is early stage cancer.
• the cancer is locally advanced cancer.
• the cancer is locally advanced and/or metastatic cancer.
• the cancer is invasive cancer.
• the cancer is a cancer resistant to existing therapies.
• treatment refers to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein.
• treatment may be administered after one or more symptoms have developed.
• treatment may be administered in the absence of symptoms.
• treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors) . Treatment may also be continued after symptoms have resolved, for example to present or delay their recurrence.
• the one or more compounds, pharmaceutically acceptable salts thereof or the pharmaceutical composition provided herein is administered via a parenteral route or a non-parenteral route.
• the one or more compounds pharmaceutically acceptable salts, hydrates, solvates or stereoisomers thereof or the pharmaceutical composition is administered orally, enterally, buccally, nasally, intranasally, transmucosally, epidermally, transdermally, dermally, ophthalmically, pulmonary, rectally, sublingually, vaginally, topically, subcutaneously, intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intracardiacally, intradermally, intraperitoneally, transtracheally, subcuticularly, intra-articularly, subcapsularly, intraspinally, subarachnoidly, or intrasternally.
• the compounds provided herein can be administrated in pure form, in a combination with other active ingredients or in the form of pharmaceutically compositions of the present disclosure.
• the compounds provided herein can be administered to a subject in need concurrently or sequentially in a combination with one or more anticancer or anti-inflammatory agent (s) known in the art.
• the individual compounds of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical compositions.
• the individual compounds will be administered simultaneously in a combined pharmaceutical composition.
• Appropriate doses of known therapeutic agents will be readily appreciated by those skilled in the art.
• the administration is conducted once a day, twice a day, three times a day, or once every two days, once every three days, once every four days, once every five days, once every six days, once a week.
• the therapeutically effective amount of a compound or a pharmaceutically acceptable salts thereof as provided herein will depend on various factors known in the art, such as body weight, age, past medical history, present medications, state of health of the subject and potential for cross-reaction, allergies, sensitivities and adverse side-effects, as well as the administration route and extent of disease development. Dosages may be proportionally reduced or increased by one skilled in the art (e.g. physician or veterinarian) as indicated by these and other circumstances or requirements.
• the one or more compounds, pharmaceutically acceptable salts thereof or the pharmaceutical composition provided herein is administered orally.
• any dose is appropriate that achieves the desired goals.
• suitable daily dosages are between about 0.001-100mg, preferably between 0.1mg and 5g, more preferably between 5mg and 1g, more preferably between 10mg and 500mg, and the administration is conducted once a day, twice a day, three times a day, every day, or 3-5 days a week.
• the dose of the one or more compounds, pharmaceutically acceptable salts thereof or the pharmaceutical composition provided herein ranges between about 0.0001mg, preferably, 0.001mg, 0.01mg, 0.1mg, 0.2mg, 0.3mg, 0.4mg, 0.5mg, 0.6mg, 0.7mg, 0.8mg, 0.9mg, 1mg, 2mg, 3mg, 4mg, 5mg, 6mg, 7mg, 8mg, 9mg, 10mg per day.
• the present disclosure provides use of the compounds, pharmaceutically acceptable salts thereof, or pharmaceutical composition of the present disclosure in the manufacture of medicaments for treating DNA-PK related disorders.
• the DNA-PK related disorders includes cancers.
• the compounds and pharmaceutical compositions thereof in the present disclosure can be used in the prevention or treatment of the onset or development of any of DNA-PK related disorders (expression or activities) in mammals especially in human.
• the present disclosure also provides a method of screening patient suitable for treating with the compounds or pharmaceutical composition of the present disclosure alone or combined with other ingredients (e.g. a second active ingredient, e.g. anti-inflammatory or anticancer agent) .
• the method includes sequencing the tissue samples from patients and detecting the accumulation of DNA-PK in the patient.
• the compounds of the present disclosure may be prepared by the methods known in the art.
• the following illustrates the detailed preparation methods of the preferred compounds of the present disclosure. However, they are by no means limiting the preparation methods of the compounds of the present disclosure.
• Synthesis of the compounds provided herein, including pharmaceutically acceptable salts thereof, are illustrated in the synthetic schemes in the examples.
• the compounds provided herein can be prepared using any known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes, and thus these schemes are illustrative only and are not meant to limit other possible methods that can be used to prepare the compounds provided herein. Additionally, the steps in the Schemes are for better illustration and can be changed as appropriate.
• the embodiments of the compounds in examples were synthesized for the purposes of research and potentially submission to regulatory agencies.
• the reactions for preparing compounds of the present disclosure can be carried out in suitable solvents, which can be readily selected by one skilled in the art of organic synthesis.
• suitable solvents can be substantially non-reactive with the starting materials (reactants) , the intermediates, or products at the temperatures at which the reactions are carried out, e.g. temperatures that can range from the solvent's freezing temperature to the solvent's boiling temperature.
• a given reaction can be carried out in one solvent or a mixture of more than one solvent.
• suitable solvents for a particular reaction step can be selected by a skilled artisan.
• Preparation of compounds of the present disclosure can involve the protection and deprotection of various chemical groups.
• the need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art.
• the chemistry of protecting groups can be found, for example, in T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3 rd Ed., Wiley &Sons, Inc., New York (1999) , which is incorporated herein by reference in its entirety.
• Reactions can be monitored according to any suitable method known in the art.
• product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g. 1 H or 13 C) , infrared spectroscopy, spectrophotometry (e.g. UV-visible) , mass spectrometry, or by chromatographic methods such as high performance liquid chromatography (HPLC) , liquid chromatography-mass spectroscopy (LCMS) , or thin layer chromatography (TLC) .
• HPLC high performance liquid chromatography
• LCMS liquid chromatography-mass spectroscopy
• TLC thin layer chromatography
• Compounds can be purified by those skilled in the art by a variety of methods, including high performance liquid chromatography (HPLC) ( “Preparative LC-MS Purification: Improved Compound Specific Method Optimization” Karl F. Blom, Brian Glass, Richard Sparks, Andrew P. Combs J. Combi. Chem. 2004, 6 (6) ,
• the structures of the compounds in the examples are characterized by nuclear magnetic resonance (NMR) or/and liquid chromatography-mass spectrometry (LC-MS) .
• NMR chemical shift ( ⁇ ) is given in the unit of 10 -6 (ppm) .
• 1 H-NMR spectra is recorded in dimethyl sulfoxide-d6 (DMSO-d6) or CDCl 3 or CD 3 OD or D 2 O or Acetone_d 6 or CD 3 CN (from Innochem or Sigma-Aldrich or Cambridge Isotope Lab., Inc. ) on Bruker AVANCE NMR (300 MHz or 400 MHz) spectrometers using ICON-NMR (under TopSpin program control) with tetramethylsilane as an internal standard.
• MS measurement is carried out using Shimadzu 2020 Mass Spectrometer with an electrospray source at positive and negative ion mode.
• High Performance Liquid Chromatography (HPLC) measurement is carried out on Shimadzu LC-20AD systems or Shimadzu LC-20ADXR systems or Shimadzu LC-30AD systems using Shim-pack XR-ODS C18 column (3.0 ⁇ 50mm, 2.2 ⁇ m) , or Ascentis Express C18 column (2.1 ⁇ 50mm, 2.7 ⁇ m) , or Agilent Poroshell HPH-C18 column (3.0 ⁇ 50mm, 2.7 ⁇ m) .
• Thin layer chromatography is carried out using Sinopharm Chemical Reagent Beijing Co., Ltd. and Xinnuo Chemical silica gel plates.
• the silica gel plates used for thin layer chromatography (TLC) are 175-225 ⁇ m.
• the silica gel plates used for separating and purifying products by TLC are 1.0 mm.
• Purified chromatographic column uses the silica gel as the carrier (100 ⁇ 200, 200 ⁇ 300 or 300 ⁇ 400 mesh, produced by Rushanshi Shangbang Xincailiao Co., Ltd. or Rushan Taiyang Desiccant Co., Ltd. etc. ) , or flash column (reversed phase C18 column 20-45 ⁇ m, produced by Agela Technologies) in Agela Technologies flash system. The size of columns are adjusted according to the amount of compounds.
• the known starting materials of the present disclosure can be synthesized by using or according to the known methods in the art, or can be purchased from Alfa Aesar, TCI, Sigma-Aldrich, Bepharm, Bide pharmatech, PharmaBlock, Enamine, Innochem and JW&Y PharmLab etc.
• the reactions are all carried out under argon or nitrogen atmosphere.
• Argon or nitrogen atmosphere refers to that the reaction flask is connected to an argon or nitrogen balloon with a volume of about 1 L. Hydrogenation is usually carried out under pressure.
• the reaction temperature in the examples is ambient temperature, which is 10°C ⁇ 30°C.
• the reaction progress is monitored by TLC or/and LC-MS.
• the eluent systems used for the reactions include dichloromethane-methanol system and petroleum ether-ethyl acetate system. The volume ratios of the solvents are adjusted according to the different polarities of compounds.
• the elution system of column chromatography used for purifying compounds and eluent system of TLC include dichloromethane-methanol system and petroleum ether-ethyl acetate system.
• the volume ratios of the solvents are adjusted according to the different polarities of compounds.
• a small amount of alkaline or acidic agents (0.1% ⁇ 1%) such as formic acid, or acetic acid, or TFA, or ammonia can be added for adjustment.
• the crude product (120 mg) was purified by Prep-HPLC with the following conditions (Column: XBridge Prep OBD C18 Column, 30 ⁇ 150mm 5um; Mobile Phase A: Water (0.05%NH 3 ⁇ H 2 O) , Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 25 B to 51 B in 7 min; ) to afford 1-methyl-N- [7-methyl- [1, 2, 4] triazolo [1, 5-a] pyridin-6-yl] -3- (oxan-4-yl) -1H-pyrazolo [4, 3-d] pyrimidin-5-amine (55 mg, 45.12%) as a white solid.
• Desired product could be detected by LCMS.
• the resulting mixture was filtered, the filter cake was washed with DCM (3x50 mL) .
• the filtrate was concentrated under reduced pressure.
• the crude product (50mg) was purified by Prep-HPLC with the following conditions (Column: XBridge Prep OBD C18 Column, 19*250mm, 5um; Mobile Phase A: Water (0.05%NH 3 ⁇ H 2 O) , Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 22 B to 33 B in 7 min; RT1: 6.63) to afford 1-methyl-N- [7-methylimidazo [1, 2-a] pyridin-6-yl] -3- (oxan-4-yl) pyrazolo [4, 3-d] pyrimidin-5-amine (20 mg, 24.61%) as a yellow solid.
• Step 2. 7- (3, 6-dihydro-2H-pyran-4-yl) -5-methyl-N- [7-methyl- [1, 2, 4] triazolo [1, 5-a] pyridin-6- yl] pyrrolo [3, 2-d] pyrimidin-2-amine
• the crude product (100 mg) was purified by Prep-HPLC with the following conditions (Column: XBridge Prep OBD C18 Column, 19*250mm, 5um; Mobile Phase A: Water (0.05%NH 3 ⁇ H 2 O) , Mobile Phase B: ACN; Flow rate: 25 mL/min; Gradient: 30 B to 45 B in 7 min; RT1: 6.02) to afford 5-methyl-N- [7-methyl- [1, 2, 4] triazolo [1, 5-a] pyridin-6-yl] -7- (oxan-4-yl) pyrrolo [3, 2-d] pyrimidin-2-amine (29 mg, 16.96%) as a white solid.
• Desired product could be detected by LCMS.
• the resulting mixture was concentrated under reduced pressure.
• LCMS: m/z (ESI) , [M+H] + 321.3.
• Desired product could be detected by LCMS.
• the resulting mixture was concentrated under reduced pressure.
• LCMS: m/z (ESI) , [M+H] + 361.3.
• the crude product (150 mg) was purified by Prep-HPLC with the following conditions (Column: XBridge Prep OBD C18 Column, 30 ⁇ 150mm 5um; Mobile Phase A: Water (0.05%NH 3 ⁇ H 2 O) , Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 40 B to 50 B in 7 min; RT1: 6.55) to afford 3-cyclohexyl-1-methyl-N- [7-methyl- [1, 2, 4] triazolo [1, 5-a] pyridin-6-yl] pyrazolo [4, 3-d] pyrimidin-5-amine (17.41 mg, 10.82%) as a white solid.
• Step 3 3- ( (1r, 4r) -4-methoxycyclohexyl) -1-methyl-N- (7-methyl- [1, 2, 4] triazolo [1, 5-a] pyridin- 6-yl) -1H-pyrazolo [4, 3-d] pyrimidin-5-amine (Ex. 7) and 3- ( (1s, 4s) -4-methoxycyclohexyl) -1- methyl-N- (7-methyl- [1, 2, 4] triazolo [1, 5-a] pyridin-6-yl) -1H-pyrazolo [4, 3-d] pyrimidin-5- amine (Ex. 8)
• Desired product could be detected by LCMS.
• the resulting mixture was concentrated under reduced pressure.
• the residue was purified by Prep-TLC (hexane/EtOAc 1: 2) to afford 5-chloro-3- (3, 6-dihydro-2H-pyran-4-yl) -1- (methyl-d 3 ) -1H-pyrazolo [4, 3-d] pyrimidine (100 mg, 43.43%) as a pink solid.
• LCMS: m/z (ESI) , [M+H] + 254.2.
• Desired product could be detected by LCMS.
• the resulting mixture was concentrated under reduced pressure.
• Step 4 1- (methyl-d 3 ) -N- (7-methyl- [1, 2, 4] triazolo [1, 5-a] pyridin-6-yl) -3- (tetrahydro-2H- pyran-4-yl) -1H-pyrazolo [4, 3-d] pyrimidin-5-amine. (Ex. 10)
• the crude product (40 mg) was purified by Prep-HPLC with the following conditions (Column: XBridge Prep OBD C18 Column, 19*250mm, 5um; Mobile Phase A: Water (0.05%NH 3 ⁇ H 2 O) , Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 15 B to 35 B in 7 min; RT1: 6.4) to afford 1- (methyl-d 3 ) -N- (7-methyl- [1, 2, 4] triazolo [1, 5-a] pyridin-6-yl) -3- (tetrahydro-2H-pyran-4-yl) -1H-pyrazolo [4, 3-d] pyrimidin-5-amine (10 mg, 9.85%) as a white solid.
• Step 1 4- (5-chloro-1-methyl-1H-pyrazolo [4, 3-d] pyrimidin-3-yl) cyclohex-3-en-1-ol
• Step 3 3- (4- (difluoromethoxy) cyclohex-1-enyl) -1-methyl-N- (7-methyl- [1, 2, 4] triazolo [1, 5- a] pyridin-6-yl) -1H-pyrazolo [4, 3-d] pyrimidin-5-amine
• Step 4 Preparation of 1-methyl-N- [7-methyl- [1, 2, 4] triazolo [1, 5-a] pyridin-6-yl] -3- [ (1s, 4s) -4- (difluoromethoxy) cyclohexyl] pyrazolo [4, 3-d] pyrimidin-5-amine (Ex. 11) and 1-methyl-N- [7- methyl- [1, 2, 4] triazolo [1, 5-a] pyridin-6-yl] -3- [ (1r, 4r) -4- (difluoromethoxy) cyclohexyl] pyrazolo [4, 3-d] pyrimidin-5-amine (Ex. 12)
• the filtrate was concentrated under reduced pressure to afford crude solid.
• the crude product was purified by Prep-HPLC with the following conditions (Column: XBridge Prep OBD C18 Column, 30 ⁇ 150mm 5um; Mobile Phase A: Water (0.05%NH 3 ⁇ H 2 O) , Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 34 B to 54 B in 7 min; RT1: 5.93) to afford solid.
• the crude product was purified by Prep-HPLC with the following conditions (Column: X select CSH OBD Column 30 x 150 mm 5 um n; Mobile Phase A: Water (0.05%TFA ) , Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 18%B to 29%B in 7 min; t R : 6.30 min) to afford 3-methyl-N- [7-methyl- [1, 2, 4] triazolo [1, 5-a] pyridin-6-yl] -1- (oxan-4-yl) -1H-pyrazolo [3, 4-d] pyrimidin-6-amine (74 mg, 25.66%) as a white solid.
• the crude product was purified by Prep-HPLC to afford crude solid.
• the crude product was purified by Prep-HPLC with the following conditions (Column: XBridge Prep OBD C18 Column, 30 ⁇ 150mm 5um; Mobile Phase A: Water (0.05%NH 3 ⁇ H 2 O) , Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 17 B to 37 B in 7 min; RT1: 6.75) to afford 3-methyl-N- [7-methylimidazo [1, 2-a] pyridin-6-yl] -1- (oxan-4-yl) pyrazolo [3, 4-d] pyrimidin-6-amine (140 mg, 60.84%) as off-white solid.
• the crude product (70 mg) was purified by Prep-HPLC with the following conditions (Column: XBridge Prep OBD C18 Column, 30 ⁇ 150mm 5um; Mobile Phase A: Water (0.05%NH 3 ⁇ H 2 O) , Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 28 B to 48 B in 7 min; RT1: 5.80) to afford 2-chloro-5-methyl-7- (oxan-4-yl) pyrrolo [2, 3-d] pyrimidine (40 mg, 6.66%) as a yellow solid.
• Step 1 4-difluorocyclohexyl) -3-methyl-N- (7-methylimidazo [1, 2-a] pyridin-6-yl) -1H- pyrazolo [3, 4-d] pyrimidin-6-amine (Ex. 22)
• Step 1 1- (4-methoxybenzyl) -3-methyl-N- (7-methylimidazo [1, 2-a] pyridin-6-yl) -1H-pyrazolo [3, 4-d] pyrimidin-6-amine
• Step 3 1- ( (1r, 4r) -4-methoxycyclohexyl) -3-methyl-N- (7-methylimidazo [1, 2-a] pyridin-6-yl) - 1H-pyrazolo [3, 4-d] pyrimidin-6-amine (Ex. 26)
• Step 3 1- ( (1s, 4s) -4-fluorocyclohexyl) -3-methyl-N- (7-methyl- [1, 2, 4] triazolo [1, 5-a] pyridin-6- yl) -1H-pyrazolo [3, 4-d] pyrimidin-6-amine (Ex. 27) and 1- ( (1r, 4r) -4-fluorocyclohexyl) -3- methyl-N- (7-methyl- [1, 2, 4] triazolo [1, 5-a] pyridin-6-yl) -1H-pyrazolo [3, 4-d] pyrimidin-6- amine (Ex. 28)
• the crude product (60 mg) was purified by Prep-HPLC with the following conditions (Column: XBridge Prep OBD C18 Column, 19*250mm, 5um; Mobile Phase A: Water (0.05%NH 3 ⁇ H 2 O) , Mobile Phase B: MeOH ; Flow rate: 25 mL/min; Gradient 58 B to 70 B in 7 min) to afford 3-methyl-N- [7-methyl- [1, 2, 4] triazolo [1, 5-a] pyridin-6-yl] -1- [spiro [2.5] octan-6-yl] pyrazolo [3, 4-d] pyrimidin-6-amine (30 mg, 14.29%) as a white solid.
• the crude product (40 mg) was purified by Prep-HPLC with the following conditions (Column: XBridge Shield RP18 OBD Column, 19*250mm, 10um; Mobile Phase A: Water (10MMOL/L NH 4 HCO 3 +0.1%NH 3 ⁇ H 2 O) , Mobile Phase B: ACN; Flow rate: 20 mL/min; Gradient: 36 B to 46 B in 7 min; RT1: 5.73) to afford 4- [3-methyl-6- ( [7-methylimidazo [1, 2-a] pyridin-6-yl] amino) pyrazolo [3, 4-d] pyrimidin-1-yl] benzonitrile (8.8mg, 4.31%) as a white solid.
• Step 1 Preparation of 1- (3-methoxycyclopentyl) -3-methyl-N- (7-methyl- [1, 2, 4] triazolo [1, 5- a] pyridin-6-yl) -1H-pyrazolo [3, 4-d] pyrimidin-6-amine (mixtures of 108/109) and Preparation of 1- (3-methoxycyclopentyl) -3-methyl-N- (7-methyl- [1, 2, 4] triazolo [1, 5-a] pyridin-6-yl) -1H- pyrazolo [3, 4-d] pyrimidin-6-amine (mixtures of 110/111)
• the crude product (150 mg) was purified by Prep-HPLC with the following conditions (Column: YMC-Actus Triart C18, 30*250, 5um; Mobile Phase A: Water (0.05%NH 3 H 2 O) , Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 34 B to 46 B in 8.5 min) to afford 1- (3-methoxycyclopentyl) -3-methyl-N- (7-methyl- [1, 2, 4] triazolo [1, 5-a] pyridin-6-yl) -1H-pyrazolo [3, 4-d] pyrimidin-6-amine (mixtures of Ex. 108/109, 25 mg, 16.67%) as a white solid.
• Step 2 Preparation of 1- (3-methoxycyclopentyl) -3-methyl-N- (7-methyl- [1, 2, 4] triazolo [1, 5- a] pyridin-6-yl) -1H-pyrazolo [3, 4-d] pyrimidin-6-amine (Ex. 108, isomer 1) /1- (3- methoxycyclopentyl) -3-methyl-N- (7-methyl- [1, 2, 4] triazolo [1, 5-a] pyridin-6-yl) -1H- pyrazolo [3, 4-d] pyrimidin-6-amine (Ex. 109, isomer 2)
• Step 4 1- (3-methoxycyclopentyl) -3-methyl-N- (7-methyl- [1, 2, 4] triazolo [1, 5-a] pyridin-6-yl) - 1H-pyrazolo [3, 4-d] pyrimidin-6-amine (Ex. 110, isomer 3) /1- (3-methoxycyclopentyl) -3- methyl-N- (7-methyl- [1, 2, 4] triazolo [1, 5-a] pyridin-6-yl) -1H-pyrazolo [3, 4-d] pyrimidin-6- amine (Ex. 111, isomer 4)
• the crude product (170 mg) was purified by Chiral-Prep-HPLC with the following conditions (Column: CHIRALPAK-AD-H-UL001, 20*250mm, 5 um; Mobile Phase A: Hex (8mmol/L NH 3 .
• Exemplary compounds disclosed herein have been characterized in one or more of the following biological assays.
• the inhibitory activity of compounds against DNA-PK was determined by TR-FRET measuring a fluorescent labelled peptide substrate converting to a phosphorylated product. All assays were performed in black Greiner 384 well low volume plates (Greiner) , in a total reaction volume of 6 ⁇ L and 0.5 % (v/v) final DMSO concentration.
• Full length human DNAPK protein, Fluorescein-P53 (Ser15) Peptide Substrate (Fluorescein-EPPLSQEAFADLWKK) and LanthaScreen TM Tb-anti-phospho-p53 [pSer15] Antibody Kit were purchased from Thermo Fisher Scientific.
• DNA-PK protein was incubated with compound for 30 minutes at room temperature in reaction buffer (50 mM HEPES pH 7.5, 0.01%Brij-35, 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 10 ⁇ g/ml Calf Thymus DNA) .
• reaction buffer 50 mM HEPES pH 7.5, 0.01%Brij-35, 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 10 ⁇ g/ml Calf Thymus DNA
• the reaction was then initiated by the addition of ATP and Fluorescein-P53 (Ser15) Peptide Substrate.
• the kinase reaction (10 ⁇ ATP, 1.6 ⁇ peptide substrate) was quenched after 60 minutes by the addition of 6 ⁇ l of stop buffer containing 20 mM EDTA, 4 nM Tb anti-phospho-p53 [Serl5] Antibody.
• Viability of cryopreserved hepatocytes was determined using trypan blue and the cell conc. was adjusted to 106 cells per mL with buffer. 1 ⁇ M compound (in Acetonitrile; 0.01%DMSO) was incubated with 250 ⁇ L of hepatocytecells (1 million cells per mL) in a 96 deep well plate. Reaction was stopped at different time points (0, 0.5, 5, 15, 30, 45, 60, 80, 100 and 120 min) by addition of 3 volumes of chilled acetonitrile to 20 ⁇ L of reaction mixture and centrifuged at 4°C for 15 min. 40 ⁇ L of supernatant was diluted to 200 ⁇ L with pure water and analyzed using LC-MS/MS.
• In vitro hepatocyte clearance was estimated based on determination of elimination half-life (T 1/2 ) of compounds disappearance from their initial concentrations. Peak area ratios of each compound (test or control) to IS was calculated. Drug elimination rate constant k (min-1) , T 1/2 (min) , and in vitro intrinsic clearance CL int ( ⁇ L/min/E6) was calculated according to the following equations:
• C hep (cells ⁇ L -1 ) is the cell concentration in the incubation system.
• 1 ⁇ M compound was incubated with 1 mg/mL of microsomes (Pooled HLM with 20 mg/ml protein cone) at 37°C in 250 ⁇ L of buffer (100 mM phosphate buffer, pH-7.4) containing 1 mM NADPH solution. 20 ⁇ L of incubation mix was quenched with 5 volumes chilled acetonitrile at different time points 0, 0.5, 5, 10, 15, 20 and 30 min in a fresh 96 well plate. The quench plate was centrifuged at 4000 rpm for 15 min. 40 ⁇ L of supernatant was diluted to 200 ⁇ L with pure water and analyzed using LC-MS/MS.
• A-B and basolateral-to-apical (B-A) transport of compound in HBSS was measured across MDCKII-MDR1-BCRP cell monolayers.
• Incubations were performed at approximately 37°C for 120 min, with functionality of the test system being confirmed using 5 ⁇ M digoxin as a positive control substrate.
• Transport of 5 ⁇ M compound and control compound were determined by quantifying substrate concentration in the incubation medium of donor compartment at the beginning of the incubation period, and both donor and receiver compartments at the end of the incubation period. The data was used to calculate the apparent permeability (Papp) . All incubations were performed in triplicate and integrity of the cell monolayers was confirmed using the marker Lucifer yellow.
• dCr/dt is the cumulative concentration of compound in the receiver chamber as a function of time ( ⁇ M/s) ;
• Vr is the solution volume in the receiver chamber (0.1 mL on the apical side, 0.3 mL on the basolateral side) ;
• A is the surface area for the transport, i.e. 0.11 cm2 for the area of the monolayer;
• C0 is the initial concentration in the donor chamber ( ⁇ M) .
• Table 6 Data are shown in table 6 below.
• Kp uu
• uu the relationship between concentrations of unbound drug in brain and in plasma, is the key to prediction of CNS action and should be the main parameter measured and optimized for in drug discovery (Di L et al., Journal of Medicinal Chemistry [2013] , 56: 2-12) .
• Plasma and diluted brain homogenate (1: 4 with DPBS pH7.4) were spiked with 5 ⁇ M test compound (in triplicate) and dialyzed against equal volume of 150 ⁇ L 100 mM PBS buffer (pH7.4) at 37°C for 18 hours in a slowly rotated plate. At the end of incubation, a 50 ⁇ L aliquot from the receiver side and a 5 ⁇ L from the donor chamber were taken. The 5 ⁇ L sample was further diluted with 45 ⁇ L of blank plasma or brain homogenate.
• Paired samples were matrix-matched with either buffer or blank plasma/brain homogenate and mixed for 2 min, and then precipitated with 150 ⁇ L cold acetonitrile with 100 ng/mL tolbutamide as internal standard. After centrifuging at 4000 rpm for 20 min, supernatant was diluted with 0.1%formic acid aqueous solution and analyzed for LC/MS/MS (API 4000, Applied Biosystems, Foster City) .
• a Short oral absorption (SOA) model is an in-vivo screening model to identify brain penetration of a compound.
• Six male Han Wistar rats purchased from Beijing Vital River were orally dosed with the compound at 10 mg/kg in 1%methylcellulose
• CSF cerebral spinal fluid
• Plasma samples will be processed for plasma by centrifugation at approximately 4°C, 3,000g within half an hour of collection. Plasma samples will be removed to a labeled tube and stored at -80 degree until analysis.
• Brain tissue was harvested and homogenized in 3X volume of 100mM phosphate buffered saline (pH7.4) . All samples were stored at ⁇ -70°C prior to LC/MS/MS analysis.
• Standards were prepared by spiking blank plasma, brain homogenate and artificial CSF covering 0.5 to 500 ng/mL. Homogenized brain tissue along with plasma samples were precipitated by adding 3-fold volume of cold acetonitrile containing internal standard (40 ng/mL Dexamethasone and 40 ng/mL Diclofenac) , and 10 ⁇ L of CSF samples were precipitated with 100 ⁇ L of cold acetonitrile containing internal standard. After 2 min vortex and 5 min centrifugation at 14,000 rpm, supernatant was analyzed by LC/MS/MS (API 4000, Applied Biosystems, Foster City) . Two sets of standard curves were run at the beginning and end of each batch from plasma sample analysis. For brain and CSF samples, one standard curve was analyzed along with test samples.
• internal standard 40 ng/mL Dexamethasone and 40 ng/mL Diclofenac
• 10 ⁇ L of CSF samples were precipitated with 100 ⁇ L of cold ace
• Kp brain/plasma ratio

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