WO2022006231A1 - Dpagt1 inhibitors of capuramycin analogues and their antimigratory activities of solid tumors - Google Patents

Dpagt1 inhibitors of capuramycin analogues and their antimigratory activities of solid tumors Download PDF

Info

Publication number
WO2022006231A1
WO2022006231A1 PCT/US2021/039826 US2021039826W WO2022006231A1 WO 2022006231 A1 WO2022006231 A1 WO 2022006231A1 US 2021039826 W US2021039826 W US 2021039826W WO 2022006231 A1 WO2022006231 A1 WO 2022006231A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
group
cancer
formula
pharmaceutically acceptable
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2021/039826
Other languages
French (fr)
Inventor
Michio Kurosu
Katsuhiko MITACHI
William M. CLEMONS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
California Institute of Technology
University of Tennessee Research Foundation
Original Assignee
California Institute of Technology
University of Tennessee Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by California Institute of Technology, University of Tennessee Research Foundation filed Critical California Institute of Technology
Priority to US18/013,403 priority Critical patent/US12570684B2/en
Publication of WO2022006231A1 publication Critical patent/WO2022006231A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/067—Pyrimidine radicals with ribosyl as the saccharide radical
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals

Definitions

  • A/-acetylglucosamine-phosphotransferase1 (DPAGT1) is an enzyme that catalyzes the first step in the dolichol-linked oligosaccharide pathway for glycoprotein biosynthesis.
  • This protein is an integral membrane protein of the endoplasmic reticulum.
  • tunicamycin a nucleoside antibiotic
  • Capuramycin another nucleoside antibiotic, displays narrow spectrum of antibacterial activity by targeting bacterial translocase I (MraY), but not DPAGT1. Capuramycin is less toxic than tunicamycin.
  • a compound of Formula I or a pharmaceutically acceptable salt thereof, wherein the variables are defined herein.
  • the compound of Formula I is a compound of Formula II: or a pharmaceutically acceptable salt thereof.
  • the compound of Formula I is a compound of Formula III: or a pharmaceutically acceptable salt thereof.
  • the compound of Formula I is a compound of Formula IV: or a pharmaceutically acceptable salt thereof.
  • a compound of Formula VI or a pharmaceutically acceptable salt thereof.
  • a method of inhibiting dolichyl-phosphate N- acetylglucosaminephosphotransferase comprising administering to the subject a therapeutically effective amount of any of the compounds disclosed herein, or a pharmaceutically acceptable salt thereof.
  • provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the compounds disclosed herein, or a pharmaceutically acceptable salt thereof.
  • Figure 1 shows DPAGT1 in A/-glycan biosynthesis.
  • Figures 2A, 2B and 2C show phase area confluence of PD002 treated with CPPB in kinetic proliferation assays.
  • Figures 3A and 3B show analyses of migration inhibition of pancreatic cancers, a cervical carcinoma, and a colorectal adenocarcinoma by treatment of CPPB in wound healing (scratch) assays.
  • Figure 4 shows cells migration of PD002 by gemcitabine, tunicamycin, and CPPB via transwell chamber.
  • Figure 5 shows immunofluorescent staining to observe the effect of CPPB on snail in pancreatic cancer cells and a colorectal adenomcarcinoma.
  • Figure 6 shows a western blot for CPPB in cervical cancer.
  • Figure 7 shows immunofluorescent staining to observe the effect of CPPB on E- cadherin in pancreatic cancer cells and a colorectal adenomcarcinoma.
  • Figure 8 shows immunofluorescent staining to observe the effect of CPPB on DPAGT1 expression levels in pancreatic cancer cells and a colorectal adenomcarcinoma.
  • Figure 9 shows RT-qPCR analyses of DPAGT1 expression level in PD002 treated with CPPB.
  • Capuramycin is a nucleoside antibiotic that has a narrow-spectrum of activity against Gram-positive bacteria including Mycobacterium tuberculosis (Mtb) (Siricilla, S., et al. , J. Antibiot. 2015, 68, 271-278).
  • Mtb Mycobacterium tuberculosis
  • New capuramycin analogues have been synthesized to improve bacterial phospho-MurNAc-pentapeptide translocase I (MraY and MurX for Mycobacterium spp.) enzyme as well as antimycobacterial activities (Kurosu, M.
  • Capuramycin is a specific inhibitor of MraY with the IC 50 value of 0.15 mM, however, some other nucleoside antibiotics (e.g., muraymycin A1 and tunicamycins) display activity against MraY, WecA (polyprenyl phosphate-GlcNAc-1- phosphate transferase), and its human homologue, DPAGT1 ( N - acetylglucosaminephosphotransferase 1)-type phosphotransferases (Mitachi, K., et al. J. Am. Chem. Soc. 2016, 138, 12975-12980).
  • nucleoside antibiotics e.g., muraymycin A1 and tunicamycins
  • WecA polyprenyl phosphate-GlcNAc-1- phosphate transferase
  • DPAGT1 N - acetylglucosaminephosphotransferase 1-type phosphotransferases
  • MraY is an essential enzyme for growth of the vast majority of bacteria that catalyzes the transformation from UDP-MurNAc-pentapeptide (Park's nucleotide) to prenyl-MurNAc-pentapeptide (lipid I) (van Heijenoort, J. Microbiol. Mol. Biol. Rev. 2007, 71, 620-635).
  • WecA catalyzes the transformation from UDP-GIcNAc to decaprenyl-P-P-GIcNAc, the first membrane-anchored glycophospholipid that is responsible for the biosynthesis of mycolylarabinogalactan in Mycobacterium tuberculosis (Mtb).
  • WecA is an essential enzyme for the growth of Mtb and some other bacteria.
  • Acute toxicity of tunicamycin due to its narrow therapeutic window (LD50: 2.0 mg/kg, LD100: 3.5 mg/kg mice, IP) discourages scientists from developing tunicamycin for new antibacterial, antifungal, and anti-cancer agents.
  • LD50 2.0 mg/kg
  • LD100 3.5 mg/kg mice, IP
  • DPAGT1 DPAGT1
  • novel DPAGT1 inhibitors that exhibit in vitro anti-invasion and anti-metastasis activity.
  • the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
  • an element means one element or more than one element.
  • use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.
  • “pharmaceutical combination” or “combination” refers to formulations of the separate compounds with or without instructions for combined use or to combination products.
  • the combination compounds may thus be entirely separate pharmaceutical dosage forms or in pharmaceutical compositions that are also sold independently of each other and where just instructions for their combined use are provided in the package equipment, e.g., leaflet or the like, or in other information, e.g., provided to physicians and medical staff (e.g. oral communications, communications in writing or the like), for simultaneous or sequential use for being jointly active.
  • the terms “synergy” or “synergistic effect” refer to the effect achieved when the active ingredients, i.e., DPAGT1 inhibitor and additional therapeutic agent, used together is greater than the sum of the effects that results from using the compounds separately.
  • Ci_ 6 alkyl is specifically intended to individually disclose (without limitation) methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl alkyl.
  • n-membered typically describes the number of ring-forming atoms in a moiety where the number of ring-forming atoms is n.
  • piperidinyl is an example of a 6-membered heterocycloalkyl ring
  • pyrazolyl is an example of a 5-membered heteroaryl ring
  • pyridyl is an example of a 6-membered heteroaryl ring
  • 1 ,2,3,4-tetrahydro-naphthalene is an example of a 10-membered cycloalkyl group.
  • each linking substituent include both the forward and backward forms of the linking substituent.
  • -NR(CR'R") n - includes both -NR(CR'R") n - and -(CR'R") n NR- and is intended to disclose each of the forms individually.
  • 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” or “aryl” then it is understood that the “alkyl” or “aryl” represents a linking alkylene group or arylene group, respectively.
  • substituted means that an atom or group of atoms formally replaces hydrogen as a “substituent” attached to another group.
  • substituted refers to any level of substitution, e.g., mono-, di-, tri-, tetra- or penta-substitution, where such substitution is permitted.
  • the substituents are independently selected, and substitution may be at any chemically accessible position. It is to be understood that substitution at a given atom is limited by valency. It is to be understood that substitution at a given atom results in a chemically stable molecule.
  • the phrase “optionally substituted” means unsubstituted or substituted.
  • substituted means that a hydrogen atom is removed and replaced by a substituent.
  • a single divalent substituent e.g., oxo, can replace two hydrogen atoms.
  • C n-m indicates a range which includes the endpoints, wherein n and m are integers and indicate the number of carbons. Examples include C- , C I-6 and the like.
  • alkyl employed alone or in combination with other terms, refers to a saturated hydrocarbon group that may be straight-chained or branched.
  • C n-m alkyl refers to an alkyl group having n to m carbon atoms.
  • An alkyl group formally corresponds to an alkane with one C-H bond replaced by the point of attachment of the alkyl group to the remainder of the compound.
  • the alkyl group contains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, or 1 to 2 carbon atoms.
  • alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, ferf-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.
  • alkenyl employed alone or in combination with other terms, refers to a straight-chain or branched hydrocarbon group corresponding to an alkyl group having one or more double carbon-carbon bonds.
  • An alkenyl group formally corresponds to an alkene with one C-H bond replaced by the point of attachment of the alkenyl group to the remainder of the compound.
  • Cn- m alkenyl refers to an alkenyl group having n to m carbons. In some embodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.
  • Example alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n- butenyl, sec-butenyl and the like.
  • alkynyl employed alone or in combination with other terms, refers to a straight-chain or branched hydrocarbon group corresponding to an alkyl group having one or more triple carbon-carbon bonds.
  • An alkynyl group formally corresponds to an alkyne with one C-H bond replaced by the point of attachment of the alkyl group to the remainder of the compound.
  • C n-m alkynyl refers to an alkynyl group having n to m carbons.
  • Example alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl and the like. In some embodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.
  • alkoxy refers to a group of formula -O-alkyl, wherein the alkyl group is as defined above.
  • C n-m alkoxy refers to an alkoxy group, the alkyl group of which has n to m carbons.
  • Example alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), f-butoxy and the like.
  • the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
  • C n-m dialkoxy refers to a linking group of formula -0-(C n -m alkyl)-0-, the alkyl group of which has n to m carbons.
  • Example dialkyoxy groups include -OCH2CH2O- and OCH2CH2CH2O-.
  • the two O atoms of a C n-m dialkoxy group may be attached to the same B atom to form a 5- or 6- membered heterocycloalkyl group.
  • halo refers to fluoro, chloro, bromo and iodo.
  • halo refers to a halogen atom selected from F, Cl, or Br.
  • halo groups are F.
  • haloalkyl refers to an alkyl group in which one or more of the hydrogen atoms has been replaced by a halogen atom.
  • C n-m haloalkyl refers to a C n-m alkyl group having n to m carbon atoms and from at least one up to ⁇ 2(n to m)+1 ⁇ halogen atoms, which may either be the same or different.
  • the halogen atoms are fluoro atoms.
  • the haloalkyl group has 1 to 6 or 1 to 4 carbon atoms.
  • Example haloalkyl groups include CF3, C2F5, CHF2, CH2F, CCb, CHCI2, C2CI5 and the like.
  • the haloalkyl group is a fluoroalkyl group.
  • aromatic refers to a carbocycle or heterocycle having one or more polyunsaturated rings having aromatic character (i.e ., having (4n + 2) delocalized p (pi) electrons where n is an integer).
  • aryl refers to an aromatic hydrocarbon group, which may be monocyclic or polycyclic (e.g., having 2 fused rings).
  • C n-m aryl refers to an aryl group having from n to m ring carbon atoms.
  • Aryl groups include, e.g., phenyl, naphthyl, and the like. In some embodiments, aryl groups have from 6 to about 10 carbon atoms. In some embodiments, aryl groups have 6 carbon atoms.
  • aryl groups have 10 carbon atoms.
  • the aryl group is phenyl.
  • the aryl group is naphthyl.
  • the heteroaryl ring has 1 , 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen.
  • any ring-forming N in a heteroaryl moiety can be an N-oxide.
  • the heteroaryl has 5-14 ring atoms including carbon atoms and 1 , 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl has 5-10 ring atoms including carbon atoms and 1 , 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl has 5-6 ring atoms and 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl is a five-membered or six-membered heteroaryl ring. In other embodiments, the heteroaryl is an eight-membered, nine-membered or ten-membered fused bicyclic heteroaryl ring.
  • Example heteroaryl groups include, but are not limited to, pyridinyl (pyridyl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, pyrazolyl, azolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, furanyl, thiophenyl, quinolinyl, isoquinolinyl, naphthyridinyl (including 1 ,2-, 1 ,3-, 1 ,4-, 1 ,5-, 1,6-, 1 ,7-, 1 ,8-, 2,3- and 2,6-naphthyridine), indolyl, isoindolyl, benzothiophenyl, benzofuranyl, benzisoxazolyl, imidazo[1 ,2-b]thiazolyl, purinyl, and the like.
  • the heteroaryl group is pyri
  • a five-membered heteroaryl ring is a heteroaryl group having five ring atoms wherein one or more (e.g., 1 , 2 or 3) ring atoms are independently selected from N, O and S.
  • Exemplary five-membered ring heteroaryls include thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1 ,2,3-triazolyl, tetrazolyl, 1 ,2,3- thiadiazolyl, 1 ,2,3-oxadiazolyl, 1 ,2,4-triazolyl, 1 ,2,4-thiadiazolyl, 1 ,2,4-oxadiazolyl, 1 ,3,4- triazolyl, 1 ,3,4-thiadiazolyl and 1 ,3,4-oxadiazolyl.
  • a six-membered heteroaryl ring is a heteroaryl group having six ring atoms wherein one or more (e.g., 1 , 2 or 3) ring atoms are independently selected from N, O and S.
  • Exemplary six-membered ring heteroaryls are pyridyl, pyrazinyl, pyrimidinyl, triazinyl, isoindolyl, and pyridazinyl.
  • the compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated.
  • One method includes fractional recrystallization using a chiral resolving acid which is an optically active, salt-forming organic acid.
  • Suitable resolving agents for fractional recrystallization methods are, e.g., optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids such as b- camphorsulfonic acid.
  • resolving agents suitable for fractional crystallization methods include stereoisomerically pure forms of a-methylbenzylamine (e.g., S and R forms, or diastereomerically pure forms), 2-phenylglycinol, norephedrine, ephedrine, N- methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane and the like.
  • Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine).
  • an optically active resolving agent e.g., dinitrobenzoylphenylglycine
  • Suitable elution solvent composition can be determined by one skilled in the art.
  • the compounds provided herein have the ( Reconfiguration.
  • the compounds have the (S)-configuration.
  • each of the chiral centers in the compound may be independently (R) or (S), unless otherwise indicated.
  • Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton.
  • Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge.
  • Example prototropic tautomers include 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, e.g., 1/-/- and 3/-/-imidazole, 1/-/-, 2 H- and 4 H- 1 ,2,4-triazole, 1/-/- and 2 H- isoindole and 1/-/- and 2/-/-pyrazole.
  • Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.
  • Compounds provided herein can also include all isotopes of atoms occurring in the intermediates or final compounds.
  • Isotopes include those atoms having the same atomic number but different mass numbers.
  • isotopes of hydrogen include tritium and deuterium.
  • One or more constituent atoms of the compounds provided herein can be replaced or substituted with isotopes of the atoms in natural or non-natural abundance.
  • the compound includes at least one deuterium atom.
  • one or more hydrogen atoms in a compound of the present disclosure can be replaced or substituted by deuterium.
  • the compound includes two or more deuterium atoms.
  • the compound includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • compound as used herein is meant to include all stereoisomers, geometric isomers, tautomers and isotopes of the structures depicted.
  • the term is also meant to refer to compounds provided herein, regardless of how they are prepared, e.g., synthetically, through biological process (e.g., metabolism or enzyme conversion), or a combination thereof.
  • All compounds, and pharmaceutically acceptable salts thereof can be found together with other substances such as water and solvents (e.g., hydrates and solvates) or can be isolated.
  • solvents e.g., hydrates and solvates
  • the compounds described herein and salts thereof may occur in various forms and may, e.g., take the form of solvates, including hydrates.
  • the compounds may be in any solid state form, such as a polymorph or solvate, so unless clearly indicated otherwise, reference in the specification to compounds and salts thereof should be understood as encompassing any solid state form of the compound.
  • the compounds provided herein, or salts thereof are substantially isolated.
  • substantially isolated is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected.
  • Partial separation can include, e.g., a composition enriched in the compounds provided herein.
  • Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compounds provided herein, or salt thereof.
  • phrases “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions and/or dosage forms that 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.
  • the present disclosure also includes pharmaceutically acceptable salts of the compounds described herein.
  • pharmaceutically acceptable salts refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form.
  • examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • the pharmaceutically acceptable salts of the present disclosure include the non-toxic salts of the parent compound formed, e.g., from non-toxic inorganic or organic acids.
  • the pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.
  • such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, iso-propanol or butanol) or acetonitrile (MeCN) are preferred.
  • non-aqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, iso-propanol or butanol) or acetonitrile (MeCN) are preferred.
  • suitable salts are found in Remington's Pharmaceutical Sciences,
  • the compounds described herein include the N-oxide forms.
  • the phrase “pharmaceutically acceptable carrier or excipient” refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material.
  • Excipients or carriers are generally safe, non-toxic and neither biologically nor otherwise undesirable and include excipients or carriers that are acceptable for veterinary use as well as human pharmaceutical use.
  • each component is “pharmaceutically acceptable” as defined herein. See, e.g., Remington: The Science and Practice of Pharmacy, 21st ed.; Lippincott Williams & Wilkins: Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al .
  • treating refers to inhibiting a disease; for example, inhibiting a disease, condition, or disorder in an individual who is experiencing or displaying the pathology or symptomology of the disease, condition, or disorder ( i.e ., arresting further development of the pathology and/or symptomology) or ameliorating the disease; for example, ameliorating a disease, condition, or disorder in an individual who is experiencing or displaying the pathology or symptomology of the disease, condition, or disorder ( i.e ., reversing the pathology and/or symptomology) such as decreasing the severity of the disease.
  • prevent comprises the prevention of at least one symptom associated with or caused by the state, disease or disorder being prevented.
  • X is NH, O, or -(CH 2 )m-;
  • Y is CH or N
  • Z is CH or N
  • A is absent, O, or NH
  • R 1 is OR 7 or -(CH 2 )nNHR 7 ;
  • R 2 is H or halo
  • R 3 is selected from the group consisting of H, halo, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 1 -C6 alkoxy, and C 1 -C6 haloalkyl;
  • R 4 is H or halo
  • R 5 and R 6 are each independently selected from the group consisting of H, C 1 -C 6 alkyl, and C(0)CrC 6 alkyl;
  • R 7 is selected from the group consisting of H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 6 -C 10 aryl, and 5-10 membered heteroaryl; wherein aryl and heteroaryl are each optionally substituted one, two, or three times with OC 1 -C 6 haloalkyl; and m and n are, independently at each occurrence, 0, 1, 2, or 3.
  • the compound of Formula I is a compound of Formula II:
  • the compound of Formula I is a compound of Formula III: or a pharmaceutically acceptable salt thereof.
  • the compound of Formula I is a compound of Formula IV: or a pharmaceutically acceptable salt thereof.
  • R 1 is OR 7 .
  • R 1 is -(CH2) n NHR 7 .
  • R 7 is selected from the group consisting of O-I-O Q alkyl, O-I-O Q haloalkyl, and phenyl, wherein phenyl is optionally substituted one time with OO-I-O Q haloalkyl.
  • R 7 is selected from the group consisting of methyl, ethyl, C3 alkyl, C4 alkyl, CF3, and phenyl-OCF3.
  • R 2 is H.
  • R 2 is fluoro.
  • R 3 is selected from the group consisting of H, fluoro, methyl, methoxy, C2 alkenyl, and C2 alkynyl. In still another embodiment, R 3 is H.
  • R 4 is H. In another embodiment, R 4 is fluoro.
  • R 5 is methyl.
  • R 6 is H or methyl. In an embodiment, R 6 is H.
  • the compound of Formula I is CPPB: or a pharmaceutically acceptable salt thereof.
  • the compound of Formula I is CPPB1 :
  • X and Y are independently O or NH;
  • R 1 is selected from the group consisting of H, OH, C(0)NH 2 , CO2H, C(0)H, and C(0)halo; and R 2 is selected from the group consisting of H, O- I -O Q alkyl, O- I -O Q haloalkyl, C -C aryl, and 5-10 membered heteroaryl.
  • X is O. In another embodiment, X is NH. In yet another embodiment, Y is O. In still another embodiment, Y is NH. In an embodiment, R 1 is C(0)NH 2 . In another embodiment, R 2 is C -C haloalkyl.
  • the compound of Formula V is or a pharmaceutically acceptable salt thereof.
  • X and Y are independently O or NH;
  • R 1 and R 2 are each independently selected from H and OH; and R 3 is selected from the group consisting of H, O- I -O Q alkyl, O- I -O Q haloalkyl, C -C aryl, and 5-10 membered heteroaryl.
  • X and Y are NH. In another embodiment, X is NH and Y is O. In yet another embodiment, X is O and Y is NH.
  • R 3 is O- I -O Q haloalkyl.
  • the compound of Formula VI is selected from the group consisting of: or a pharmaceutically acceptable salt thereof.
  • composition comprising a compound disclosed herein, and at least one pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises an additional therapeutic agent.
  • the additional therapeutic agent is selected from the group consisting of paclitaxel, tunicamycin, capuramycin, erlotinib, capecitabine, fluorouracil, and gemcitabine.
  • the additional therapeutic agent is paclitaxel.
  • a method of inhibiting dolichyl-phosphate N- acetylglucosaminephosphotransferase (DPAGT1) in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound disclosed herein or a pharmaceutical composition comprising a compound disclosed herein.
  • a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure or a pharmaceutical composition comprising a compound disclosed herein.
  • the cancer is selected from hematological cancers, sarcomas, lung cancers, brain cancers, gastrointestinal cancers, genitourinary tract cancers, liver cancers, bone cancers, nervous system cancers, gynecological cancers, and skin cancers.
  • the lung cancer is selected from non-small cell lung cancer (NSCLC), small cell lung cancer, bronchogenic carcinoma, squamous cell bronchogenic carcinoma, undifferentiated small cell bronchogenic carcinoma, undifferentiated large cell bronchogenic carcinoma, adenocarcinoma, bronchogenic carcinoma, alveolar carcinoma, bronchiolar carcinoma, bronchial adenoma, chondromatous hamartoma, mesothelioma, pavicellular and non-pavicellular carcinoma, bronchial adenoma, and pleuropulmonary blastoma.
  • NSCLC non-small cell lung cancer
  • small cell lung cancer bronchogenic carcinoma, squamous cell bronchogenic carcinoma, undifferentiated small cell bronchogenic carcinoma, undifferentiated large cell bronchogenic carcinoma, adenocarcinoma, bronchogenic carcinoma, alveolar carcinoma, bronchiolar carcinoma, bronchial adenoma, chondromatous hamartoma, mesot
  • the lung cancer is non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • the lung cancer is adenocarcinoma.
  • the gastrointestinal cancer is selected from esophagus squamous cell carcinoma, esophagus adenocarcinoma, esophagus leiomyosarcoma, esophagus lymphoma, stomach carcinoma, stomach lymphoma, stomach leiomyosarcoma, exocrine pancreatic carcinoma, pancreatic ductal adenocarcinoma, pancreatic insulinoma, pancreatic glucagonoma, pancreatic gastrinoma, pancreatic carcinoid tumors, pancreatic vipoma, small bowel adenocarcinoma, small bowel lymphoma, small bowel carcinoid tumors, Kaposi's sarcoma, small bowel leiomyoma, small bowel hemangioma, small bowel lipoma, small bowel neurofibroma, small bowel fibroma, large bowel adenocarcinoma, large bowel tubular a
  • the gastrointestinal cancer is colorectal cancer.
  • the brain cancer is selected from acoustic neuroma, astrocytoma, choroid plexus carcinoma, craniopharyngioma, embryonal tumors, ependymoma, glioblastoma, glioma, medulloblastoma, meningioma, oligodendroglioma, pineoblastoma, mixed glioma, mixed glial and neuronal tumors, and primitive neuroectodermal tumors.
  • the cancer is a carcinoma.
  • the carcinoma is selected from pancreatic carcinoma, colorectal carcinoma, lung carcinoma, bladder carcinoma, gastric carcinoma, esophageal carcinoma, breast carcinoma, head and neck carcinoma, cervical skin carcinoma, and thyroid carcinoma.
  • the cancer is a hematopoietic malignancy.
  • the hematopoietic malignancy is selected from multiple myeloma, acute myelogenous leukemia, and myeloproliferative neoplasms.
  • the cancer is a neoplasm.
  • the neoplasm is glioblastoma or sarcomas.
  • the cancer is selected from the group consisting of hematological cancers, sarcomas, lung cancers, gastrointestinal cancers, genitourinary tract cancers, liver cancers, bone cancers, nervous system cancers, gynecological cancers, and skin cancers.
  • the cancer is selected from the group consisting of pancreatic cancer, cervical cancer, colon cancer, ovarian cancer, breast cancer, pancreatic cancer, carcinoma, and adenocarcinoma.
  • the cancer is pancreatic cancer. In yet another embodiment, the cancer is a solid tumor.
  • the subject is human.
  • the term “individual,” “subject,” or “patient,” used interchangeably, refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.
  • the phrase “therapeutically effective amount” refers to the amount of active compound or pharmaceutical agent such as an amount of any of the solid forms or salts thereof as disclosed herein that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.
  • An appropriate “effective” amount in any individual case may be determined using techniques known to a person skilled in the art.
  • the (S)-cyanohydrin (13) was subjected to NIS-AgBF 4 promoted a-selective mannosylation with the thioglycoside (14) to yield the a-mannosylated cyanohydrin (15) in 78% yield.
  • the cyano group of 15 was hydrated using HgCh-aldoxime in aq. EtOH, and the BOM and chloroacetyl groups of the generated amide were deprotected stepwisely: dechloro- acetylation with thiourea followed by hydrogenation with 10% Pd-C in AcOH-'PrOH-THF provided the C6”-free alcohol 16 in 53% overall yield.
  • DM-CAP dimethyl-capuramycin
  • a solution of the amide (0.21 g, 0.26 mmol) in a 1:1 mixture of THF and MeOH (2.6 ml_) was added thiourea (0.059 g, 0.77 mmol). After being stirred for 11 h at 50 °C, the reaction mixture was concentrated in vacuo. The residue was diluted with H2O and extracted with CHCb. The combined organic extracts were dried over Na2SC>4 and concentrated in vacuo.
  • the carboxylic acid intermediate 32 for CAP (1) was subjected to peptide coupling reaction with (((trifluormethoxy)phenoxy) piperidin-1-yl)phenyl)methylamine (34), providing the protected CPPB, 37. Saponification of 37 and purification by reverse HPLC yielded CPPB (5) in 95% overall yield from 37. Similarly, l-CPPB (6), OM-CPPB (7), and DM-CPPB (8) were synthesized from the cyanohydrins 18, 33, and 31 in 24-34% overall yield.
  • Capuramycin phenoxypiperidinbenzylamine analogues, CPPA (9) and l-CPPA (10), were synthesized via reductive aminations of the aldehydes 29 and 18 with the amine 34, furnishing the desired products in 63-65% overall yield from 26 and 16 after saponification.
  • CAP (1) and its three analogues, OM-CAP (2), l-CAP (3), and DM-CAP (4) were evaluated in enzyme inhibitory assays against bacterial phosphotransferases, MraY and WecA, and archaeal and human dolichyl-phosphate GlcNAc-1 -phosphotransferases, AgIH and DPAGT 1.
  • CAP is a selective MraY inhibitor that does not display inhibitory activity against WecA, AgIH, and DPAGT1 (Table 1).
  • OM-CAP (2) was identified as a selective WecA inhibitor (IC 50 0.060 mM) that does not possess MraY inhibitory activity (IC 50 >50 mM (entry 2 in Table 1).
  • OM-CAP (2) was shown to have inhibitory activity against AgIH and DPAGT1 with the IC 50 values of 13.5 and 3.5 mM, respectively (entry 2).
  • I-CAP (3) showed enzyme inhibitory activity against MraY, WecA, AgIH, and DPAGT1 with the IC 50 between 7.5-20 mM concentrations (entry 3).
  • DM-CAP (4) showed only a weak WecA inhibitory activity (IC 50 35.0 mM) (entry 4).
  • MraY, WecA, AgIH, and DPAGT1 enzyme inhibitory activity assays for the six ((trifluormethoxy)phenoxy) piperidin-1-yl)phenyl)methylamine analogues revealed that CPPB (5) and l-CPPB (6) are high nM range DPAGT1 inhibitors (entries 5 and 6 in Table 1), however, the O-methylation and demethylation analogues (OM-CPPB (7) and DM-CPPB (8)) turned out to be very low- or no-DPAGT1 inhibitor (entries 7 and 8).
  • CPPA The secondary amine analogues, CPPA (9) and l-CPPA (10), did not inhibit all phosphotransferases tested in Table 1 at 50 mM concentration (entries 9 and 10).
  • CPPB was determined to be three times stronger DAPGT1 inhibitor (IC 50 O.2O mM) than l-CPPB (entry 5 vs. 6).
  • I-CPPB did not display MraY inhibitory activity, but showed a very weak WecA and AgIH enzyme inhibitory activity (entry 6).
  • CPPB possessing MraY/WecA inhibitory activity killed Myocobacterium tuberculosis H37Rv, Mycobacterium avium 2285, Mycobacterium smegmatis (ATCC607) with the MIC values 6.25-12.5 pg/mL.
  • HyMraY The gene mraY of Hydrogenivirga spp. 128-5- R1-1 was cloned with a /V-terminal His6 tag into a pET22b vector. The plasmid was transformed and expressed in E. coli NiCo21 (DE3) pLEMO competent cells. The proteins were purified using a nickel, cation exchange, and size exclusion chromatography. The final storage buffer was 20 mM HEPES pH 7.5, 100 mM NaCI, 10% glycerol, 5 mM 3ME, 0.15% decyl 3-D-maltopyranoside (DM).
  • DM decyl 3-D-maltopyranoside
  • MjAglH The gene mj1113 of Methanocaldococcus jannaschii DSM 2661 was cloned with a /V-terminal His9 tag into a pET33b-derived vector. The plasmid was transformed and expressed in E. coli NiCo21(DE3) pLEMO competent cells. The proteins were purified using cobalt and size exclusion chromatography. The final storage buffer was 20 mM Tris-HCI, pH 7.5, 100 mM NaCI, 5% glycerol, 5 mM 3ME, 0.15% DM.
  • the washed cell pellets were suspended in homogenization buffer and disrupted by probe sonication on ice (5s on and 2s off for 1 min, then cool down for 1.5min., 5 cycles, cool down for 15 min, and then, 5s on and 2s off for 1min., cool down for 1.5min, 5 cycles).
  • the resulting suspension was centrifuged at 4,700 x g for 15min. at 4 °C to remove unbroken cells.
  • the lysate was centrifuged at 25,000 x g for 20min. at 4 °C.
  • the supernatant was subjected to ultracentrifugation at 60,000 x g for 1 h at 4 °C.
  • DPAGT1 expression and purification DPAGT1 was expressed in suspended Expi293 cells for 36h. The cells were lysed by drawing through a 26g needle (10 times) and membrane protein was extracted using buffer containing 1% decyl f3-D-maltopyranoside detergent. DPAGT1 was purified using HA-agarose resin and a superdex 200 size exclusion column.
  • MraY assay MraY assay substrates, Park's nucleotide-N £ -C6-dansylthiourea, neryl phosphate, were chemically synthesized according to the reported procedures. 7 Park's nucleotide-N £ -C6-dansylthiourea (2 mM stock solution, 1.88 jiL), MgCh (0.5 M, 5 jiL), KCI (2 M, 5 jiL), Triton X-100 (0.5%, 5.63 jiL), Tris buffer (pH 8.0, 50 mM), neryl phosphate (0.1 M, 2.25 jiL), and inhibitor molecule (0 - 50 .ig/mL in Tris buffer) were placed in a 1.5 mL Eppendorf tube.
  • IC50 values were calculated from plots of the percentage product inhibition versus the inhibitor.
  • UDP-Glucosamine-C6-FITC was chemically synthesized according to the reported procedures. 9 ' 11 ⁇ 58 UDP-Glucosamine-C6-FITC (UDP- GlcN-C6-FITC, 2 mM stock solution, 0.56 mI_), MgCh (0.5 M, 4 mI_), b-mercaptoethanol (50 mM, 5 pL), CHAPS (5%, 11.3 mI_), Tris buffer (pH 8.0, 50 mM), undecaprenyl phosphate (4 mM, 1.4 mI_), and inhibitor molecule (0 - 50 pg/mL in Tris buffer) were place in a 1.5 mI_ Eppendorf tube.
  • reaction mixture P-60 (10 mI_) was added (total volume of reaction mixture: 50 mI_ adjust with Tris buffer). The reaction mixture was incubated for 2h at 37 °C and quenched with n-butanol (150 mI_). Two phases were mixed via vortex and centrifuged at 10,000 xg for 3 min. The upper organic phase was assayed via reverse-phase HPLC. The organic phase (30 mI_) was injected into HPLC (solvent: gradient elution of 85:15 to 95:5 MeOH/0.05 M aq.
  • IC50 values were calculated from plots of the percentage product inhibition versus the inhibitor concentration.
  • AgIH assays were performed as the procedure described for WecA assays, but used MyAglH and a-dihydroundecaprenyl phosphate (Css-dolichyl phosphate) instead of WecA and undecaprenyl phosphate.
  • UDP-GlcN-C 6 -FITC (2 mM stock solution, 0.56 pL), MgCh (0.5 M, 4 pL), b-mercaptoethanol (50 mM, 5 pL), CHAPS (5%, 11.3 pL), Tris buffer (pH 8.0, 50 mM), Css-dolichyl phosphate (4 mM, 1.4 pL), and inhibitor molecule (0 - 50 pg/mL in Tris buffer) were place in a 1.5 pL Eppendorf tube. To a reaction mixture, AgIH solution (10 pL) was added (total volume of reaction mixture: 50 pL adjust with Tris buffer).
  • the reaction mixture was incubated for 2 h at 37 °C and quenched with n-butanol (150 pL). Two phases were mixed via vortex and centrifuged at 10,000 xg for 3 min. The upper organic phase was assayed via reverse-phase HPLC. The organic phase (30 pL) was injected into HPLC (solvent: gradient elution of 85:15 to 95:5 MeOH/0.05 M aq. NH4HCO3; UV: 485 nm; flow rate: 0.5 mL/ min; column: Kinetex 5 pm C8, 100 A, 150 x 4.60 mm), and the area of the peak for Css-P-P-GIcN-Cs-FITC was quantified to obtain the IC50 value. The IC50 values were calculated from plots of the percentage product inhibition versus the inhibitor concentration.
  • DPAGT1 assay DPAGT1 assay. DPAGT1 assays were performed as the procedure described for AgIH assays, but used DPAGT1.
  • the degree of MraY inhibitory activity correlates with their antimycobaterial activity.
  • Antimycobacterial capuramycin analogues display low in vitro cytotoxicity against mammalian cells, and have been recognized as safe drug leads that have acceptable tolerability in animal models.
  • the toxicity of tunicamycin has been studied extensively in vitro : tunicamycin inhibits growth in many cancer cell lines without selectivity, and has a narrow therapeutic window demonstrated in in vivo studies using mice.
  • the toxicity of tunicamycin is believed to be attributable to its ability to inhibit DPAGT1 enzyme function. Tunicamycin's toxicity, however, cannot be explained solely by its inhibition of DPAGT1.
  • the DPAGT1 inhibitors, CPPB and l-CPPB did not show antiproliferative activity against L1210 (a leukemia cell), HPNE (a normal pancreatic ductal cell), and Vero (a normal kidney cell) at 50 mM.
  • KB HeLa, a cervix carcinoma
  • SiHa a cervical squamous cell carcinoma
  • HCT-116 a colorectal adenocarcinoma
  • DLP-1 a colorectal adenocarcinoma
  • Capan-2 a pancreatic ductal adenocarcinoma
  • PANC-1 a pancreatic ductal carcinoma
  • AsPC-1 a pancreatic adenocarcinoma
  • PD002 a patient-derived pancreatic adenocarcinoma
  • the human primary PDAC cell lines PANC-1 (ATCC CRL 1469); Capan-2 (ATCC HTB-80) and metastatic PDAC cell lines Capan-1 (ATCC HTB-79), AsPC-1 (ATCC CRL-1682), L1210 (ATCC CCL-219), KB (ATCC CCL-17), HeLa, SiHa (ATCC HTB-35), HCT-116 (ATCC CCL-247), DLD-1 (ATCC CCL221), hTERT-HPNE (ATCC CRL-4023), Vero (ATCC CCL-81 ) were purchased from the ATCC. The cell lines were cultured and maintained in the media as recommended by the supplier. The cell lines were regularly tested for mycoplasma contamination and authenticated.
  • Dr. Glazer's lab generated patient-derived PDAC cell line PD002.
  • PD002 Cells were maintained and cultured in DMEM medium supplemented with 20% FBS and 100 lll/mL penicillin and incubated at 37°C with 5% CO2.
  • MTT cytotoxicity assay To study the effect of treatment with DPAGT1 inhibitors on the growth and proliferation of cells, a fixed number of cells (5 x 10 4 cells/well, 196 mI_) were plated in a 96 well plate. Into each well 5 mI_ of drug concentration was added. After 72 h of incubation with drugs at 37 °C, 5% CO2, 10 mI_ of MTT solution (5 mg/ml_ in PBS) was added and incubated another 3 h at 37 °C, 5% CO2. The medium was removed, and DMSO (200 pL/well) was added. Viability was assessed on the basis of cellular conversion of MTT into a purple formazan product. The absorbance of the colored formazan product was measured at 570 nm by BioTek Synergy HT Spectrophotometer.
  • a confluent monolayer was formed in 24-well plates. The monolayer was scratched by a sterile 200 pL pipette tip and washed with PBS to remove cell debris. Complete medium with CPPB (5) (0, 0.05, 0.1 , 0.2 mM) were added and scratched areas were photographed with microscope. The scratched cells were incubated at 37 °C, 5%
  • DPAGT1 catalyzes the first step in A/-glycan biosynthesis of mammalian cells ( Figure 1 ).
  • Aberrant A/-glycosylation is common in many solid cancers and important for the epithelial to mesenchymal transition program (EMT, a mechanism of metastases).
  • EMT epithelial to mesenchymal transition program
  • High levels of DPAGT1 protein expression were found in a series of pancreatic cancers (e.g., Panc-1 ,
  • DPAGT1 DPAGT1 epithelial to mesenchymal transition
  • EMT epithelial to mesenchymal transition
  • these processes increase migratory and invasive capabilities of malignant neoplasms that are the initiation of metastasis in cancer progression, especially pancreatic cancer.
  • the wound-healing rate of the untreated PD002 cells was 63% in 24 h.
  • CPPB treated cells inhibited the wound-healing effectively at its IC50 level against DPAGT1 (0.2 mM): the wound-healing rate was approximately 20% ( Figure 3B).
  • the migration inhibition ability of CPPB was thoroughly evaluated compared to gemcitabine, one of the main chemotherapy drugs used to treat pancreatic cancer, and tunicamycin using PD002 cells.
  • Gemcitabine shows a wound-healing rate of 43% at 0.2 mM, and tunicamycin shows 35% at 0.2 mM.
  • CPPB is more effective in inhibiting cancer cell migration than gemcitabine and tunicamycin.
  • PD002 cells ( ⁇ 10 4 cells/mL) were seeded in 96- wells plate (Corning, HTS Transwell-96 Well Permeable Supports, pore size: 8 pm) in FBS free media and the lower chambers were filled with 10% FBS medium.
  • CPPB, tunicamycin, or gemcitabine (0.1 mM) was added to the upper chamber.
  • the cells were incubated for 18 h at 37 °C under 5% CO2.
  • the cells were fixed using 4% paraformaldehyde for 0.5 h and stained with 0.05% crystal violet (300 pL/well), after 0.5 h, images were captured via 10X magnification microscopy.
  • Example 5 Inhibition of a zinc-finger transcription factor, SnaiH (Snail) in the selected cancer cell lines by CPPB
  • Snail protein is one of the most important transcription factors that induces epithelial to mesenchymal transition (EMT), which converts epithelial cells into migratory mesenchymal cells that are more efficient at metastasizing.
  • EMT epithelial to mesenchymal transition
  • Snail produces cancer stem-like properties in a number of solid organ cancers.
  • Aberrant expression of Snail leads to loss of expression of E-cadherin, a cell-cell adhesion glycoprotein.
  • suppression of Snail expression or inhibition of Snail functions represents a potent targeted therapeutic strategy for many cancers.
  • Immunofluorescence assays using an anti-Snail antibody revealed that the fluorescence intensity of Snail was strong in a series of pancreatic cancer cells (PANC-1, AsPC-1 , Capan-2, and PD002), and the expression of Snail was decreased by the treatment with CPPB in a concentration dependent manner.
  • pancreatic cancer cell lines only the data for PD002 and PANC-1 are shown in Figure 5.
  • the Snail expression level of a nonmetastatic pancreatic cancer, PANC-1 was much lower than metastatic pancreatic cancers (e.g., PD002 and Capan-2).
  • HCT-116 colorectal cancer
  • SiHa cervical cancer
  • Figures 5 and 6 The Snail expression in SiHa was inhibited by treatment of CPPB in a concentration dependent manner; at the IC50 concentration (0.2 mM against DPAGT1), CPPB effectively inhibited the Snail expression ( Figure 6).
  • the Snail expression level in HCT-116 was not noticeably changed by the treatment of CPPB between 0.05 and 2.0 mM concentrations.
  • CPPB decreased the DPAGT1 expression level in all pancreatic cancer cell lines examined in Figure 3: the DPAGT1 expression was apparently inhibited by the IC50 concentration of CPPB (0.2 mM) ( Figure 8). However, the DPAGT1 expression of the pancreatic cancer cell lines could not completely be inhibited at a high concentration of CPPB (2-20 mM). In MTT assays, all pancreatic cancers tested remained viable at 20 mM concentration of CPPB (Table 2). In contrast, the DPAGT1 expression levels of a colorectal adenocarcinoma, HCT-116, remained high even at a high concentration of CPPB (20 mM) ( Figure 8).
  • the FOLFIRINOX (a combination of folinic acid, 5-fluorouracil, irinotecan, and oxaliplatin) and nab-paclitaxel (albumin-bound paclitaxel)-gemcitabine regimens have been adopted into clinical practice for patients with metastatic pancreatic cancers. Median progression-free survival was reported in one study of patients with metastatic pancreatic cancer to be 6.4 months in the FOLFIRINOX group and 3.3 months in the gemcitabine group. Over the past years, the clinical data have not supported that FOLFIRINOX is associated with any better (or worse) survival rates compared to the nab-paclitaxel-gemcitabine regimen as there have been no head-to-head trials. However, the inclusion of paclitaxel and its derivatives in combination regimens remains an important therapeutic strategy in pancreatic cancer chemotherapy since nab-paclitaxel-gemcitabine is associated with less adverse effects (toxicity in patients) than FOLFIRINOX.
  • CPPB synergistic or antagonistic activities were assessed in vitro via checkerboard technique.
  • CPPB displayed strong synergistic effects with paclitaxel in a wide range of concentrations against PD002.
  • Table 3 summarizes the results of FIC index analyses for selected combinations of CPPB (IC5035.0 mM) plus paclitaxel (IC50 1.25 mM) that showed synergistic combination ( ⁇ FIC ⁇ 0.5).
  • the FIC index below 0.50 was observed for 20 combinations of two molecules out of 96 different concentrations (see Supporting Information).
  • the IC50 value of paclitaxel against PD002 was lowered (0.024-0.61 mM) in combination with CPPB (0.1-2.0 mM).
  • Table 3 summarizes the results of FIC index analyses for selected combinations of CPPB (IC5035.0 mM) plus paclitaxel (IC50 1.25 mM) that showed synergistic combination ( ⁇ FIC ⁇ 0.5).
  • the FIC index below 0.50 was
  • CPPB showed ⁇ 7.5 times stronger DPAGT1 inhibitory activity than tunicamycin. However, unlike tunicamycin, CPPB did not inhibit growth of cancer cell lines at the IC50 values observed for tunicamycin (0.45 -7.5 mM).
  • CPPB is a cell-permeable molecule which was demonstrated by IncuCyte ® live cell analyses and immunofluorescence assays. The effectiveness of CPPB on the expression levels of Snail, E-cadherin, and DPAGT1 primary in pancreatic cancers has been shown in the present disclosure.
  • CPPB decreased the Snail expression in commercially available pancreatic cancer cell lines (PANC-1, AsPC-1, and Capan-2) and a patient-derived pancreatic ductal adenocarcinoma cell line (PD002) in a dose dependent manner.
  • PANC-1 pancreatic cancer cell lines
  • PD002 patient-derived pancreatic ductal adenocarcinoma cell line
  • the E-cadherin expression level was not noticeably changed in PD002 or slightly increased in Panc-1 at between 0.05-0.2 mM of CPPB.
  • a lower DPAGT1 expression cell a colorectal adenocarcinoma (HCT-116) and a higher DPAGT1 expression cell, a cervical carcinoma (SiHa) were examined.
  • CPPB did not inhibit migration of HCT-116, but strongly inhibited migration of SiHa in scratch assays at its IC50 concentration (0.2 mM against DPAGT1).
  • Snail plays an important role in cancer progression.
  • the accumulated evidence on Snail indicates that over-expression of Snail promotes drug resistance, tumor recurrence and metastasis.
  • Snail inhibitory activity of CPPB suggests that selective DPAGT1 inhibitors have the potential to develop into less toxic anticancer therapeutics than current anticancer agents that are cytotoxic to all dividing cells in the body.
  • CPPB is not cytotoxic against a series of cancer and healthy cell lines at 10 mM or higher concentrations. However, it showed a strong synergistic effect with paclitaxel; cytostatic activity of paclitaxel is improved over 250-times against PD002 in combination with CPPB (0.2-2.0 mM).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Provided herein are compounds and methods of using these compounds to treat disorders related to DPAGT1 function, including cancer and bacterial infections.

Description

DPAGT1 INHIBITORS OF CAPURAMYCIN ANALOGUES AND THEIR ANTIMIGRATORY ACTIVITIES OF SOLID TUMORS
RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 63/046,103 filed June 30, 2020, the content of which is hereby incorporated by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
This invention was made with government support under Grant No. GM114611 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND
A/-acetylglucosamine-phosphotransferase1 (DPAGT1) is an enzyme that catalyzes the first step in the dolichol-linked oligosaccharide pathway for glycoprotein biosynthesis. This protein is an integral membrane protein of the endoplasmic reticulum. Several solid cancers, including pancreatic cancers, require high DPAGT1 expression in order for cancer progression. Whereas tunicamycin, a nucleoside antibiotic, has been shown to inhibit DPAGT1, its non-selective activity is toxic to healthy cells. Capuramycin, another nucleoside antibiotic, displays narrow spectrum of antibacterial activity by targeting bacterial translocase I (MraY), but not DPAGT1. Capuramycin is less toxic than tunicamycin.
Therefore, there remains a need for DPAGT1 -selective inhibitors to prevent cancer progression.
SUMMARY
Provided herein are compounds and methods of using these compounds to treat disorders related to DPAGT1 function, including cancer and bacterial infections.
In an aspect, provided herein is a compound of Formula I:
Figure imgf000002_0001
or a pharmaceutically acceptable salt thereof, wherein the variables are defined herein.
In an embodiment, the compound of Formula I is a compound of Formula II:
Figure imgf000003_0001
or a pharmaceutically acceptable salt thereof.
In another embodiment, the compound of Formula I is a compound of Formula III:
Figure imgf000003_0002
or a pharmaceutically acceptable salt thereof.
In yet another embodiment, the compound of Formula I is a compound of Formula IV:
Figure imgf000003_0003
or a pharmaceutically acceptable salt thereof.
In another aspect, provided herein is a compound of Formula V:
Figure imgf000004_0001
or a pharmaceutically acceptable salt thereof.
In yet another aspect, provided herein is a compound of Formula VI:
Figure imgf000004_0002
or a pharmaceutically acceptable salt thereof.
In another aspect, provided herein is a method of inhibiting dolichyl-phosphate N- acetylglucosaminephosphotransferase (DPAGT1) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the compounds disclosed herein, or a pharmaceutically acceptable salt thereof.
In still another aspect, provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the compounds disclosed herein, or a pharmaceutically acceptable salt thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows DPAGT1 in A/-glycan biosynthesis.
Figures 2A, 2B and 2C show phase area confluence of PD002 treated with CPPB in kinetic proliferation assays.
Figures 3A and 3B show analyses of migration inhibition of pancreatic cancers, a cervical carcinoma, and a colorectal adenocarcinoma by treatment of CPPB in wound healing (scratch) assays.
Figure 4 shows cells migration of PD002 by gemcitabine, tunicamycin, and CPPB via transwell chamber.
Figure 5 shows immunofluorescent staining to observe the effect of CPPB on snail in pancreatic cancer cells and a colorectal adenomcarcinoma.
Figure 6 shows a western blot for CPPB in cervical cancer. Figure 7 shows immunofluorescent staining to observe the effect of CPPB on E- cadherin in pancreatic cancer cells and a colorectal adenomcarcinoma.
Figure 8 shows immunofluorescent staining to observe the effect of CPPB on DPAGT1 expression levels in pancreatic cancer cells and a colorectal adenomcarcinoma.
Figure 9 shows RT-qPCR analyses of DPAGT1 expression level in PD002 treated with CPPB.
DETAILED DESCRIPTION
Capuramycin is a nucleoside antibiotic that has a narrow-spectrum of activity against Gram-positive bacteria including Mycobacterium tuberculosis (Mtb) (Siricilla, S., et al. , J. Antibiot. 2015, 68, 271-278). To date, medicinal chemistry efforts on capuramycin have been focused on developing a new tuberculosis (TB) drug. New capuramycin analogues have been synthesized to improve bacterial phospho-MurNAc-pentapeptide translocase I (MraY and MurX for Mycobacterium spp.) enzyme as well as antimycobacterial activities (Kurosu, M. et al., Heterocycles 2009, 77, 217-225). Somewhat recently, the anti -Clostridioides difficile (formerly Clostridium difficile) activity of a capuramycin analogue has been reported (Moore, J. H., et al., J. Antimicrob Chemother. 2016, 71, 1300-1306). Capuramycin is a specific inhibitor of MraY with the IC50 value of 0.15 mM, however, some other nucleoside antibiotics (e.g., muraymycin A1 and tunicamycins) display activity against MraY, WecA (polyprenyl phosphate-GlcNAc-1- phosphate transferase), and its human homologue, DPAGT1 ( N - acetylglucosaminephosphotransferase 1)-type phosphotransferases (Mitachi, K., et al. J. Am. Chem. Soc. 2016, 138, 12975-12980). MraY is an essential enzyme for growth of the vast majority of bacteria that catalyzes the transformation from UDP-MurNAc-pentapeptide (Park's nucleotide) to prenyl-MurNAc-pentapeptide (lipid I) (van Heijenoort, J. Microbiol. Mol. Biol. Rev. 2007, 71, 620-635). WecA catalyzes the transformation from UDP-GIcNAc to decaprenyl-P-P-GIcNAc, the first membrane-anchored glycophospholipid that is responsible for the biosynthesis of mycolylarabinogalactan in Mycobacterium tuberculosis (Mtb). WecA is an essential enzyme for the growth of Mtb and some other bacteria.
Biochemical studies of WecA enzyme are hampered by lack of selective inhibitor molecules. Tunicamycin shows inhibitory activity against these phosphotransferases with the IC50 values of 3.38 mM (MraY/MurX), 0.15 mM (WecA), and 2.5 mM (DPAGT1). CPZEN-45, an antimycobacterial MraY inhibitor, was reported to exhibit WecA inhibitory activity (IC50 -0.058 pg/mL). It has been shown that 2'O-methyl capuramycin does not exhibit MraY/MurX inhibitory activity, but displays a strong WecA inhibitory activity (IC500.060 mM). In vitro cytotoxicity of tunicamycin has been documented in a number of articles (Zhong, J-T., et al. Tumor Biol.
2017, 39). Acute toxicity of tunicamycin due to its narrow therapeutic window (LD50: 2.0 mg/kg, LD100: 3.5 mg/kg mice, IP) discourages scientists from developing tunicamycin for new antibacterial, antifungal, and anti-cancer agents. A large number of scientists believe that cytotoxicity of tunicamycin is attributable to its interaction with DPAGT1 , which catalyzes the first and rate limiting step in the dolichol-linked oligosaccharide pathway in A/-linked glycoprotein biosynthesis (Figure 1).
Provided herein are novel DPAGT1 inhibitors that exhibit in vitro anti-invasion and anti-metastasis activity.
Definitions
As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.
As used herein, “pharmaceutical combination” or “combination” refers to formulations of the separate compounds with or without instructions for combined use or to combination products. The combination compounds may thus be entirely separate pharmaceutical dosage forms or in pharmaceutical compositions that are also sold independently of each other and where just instructions for their combined use are provided in the package equipment, e.g., leaflet or the like, or in other information, e.g., provided to physicians and medical staff (e.g. oral communications, communications in writing or the like), for simultaneous or sequential use for being jointly active.
As used herein, the terms “synergy” or “synergistic effect” refer to the effect achieved when the active ingredients, i.e., DPAGT1 inhibitor and additional therapeutic agent, used together is greater than the sum of the effects that results from using the compounds separately.
At various places in the present specification, certain features of the compounds are disclosed in groups or in ranges. It is specifically intended that such a disclosure include each and every individual subcombination of the members of such groups and ranges. For example, the term “Ci_6 alkyl” is specifically intended to individually disclose (without limitation) methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl
Figure imgf000006_0001
alkyl.
The term “n-membered,” where n is an integer, typically describes the number of ring-forming atoms in a moiety where the number of ring-forming atoms is n. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridyl is an example of a 6-membered heteroaryl ring and 1 ,2,3,4-tetrahydro-naphthalene is an example of a 10-membered cycloalkyl group.
At various places in the present specification, variables defining divalent linking groups may be described. It is specifically intended that each linking substituent include both the forward and backward forms of the linking substituent. For example, -NR(CR'R")n- includes both -NR(CR'R")n- and -(CR'R")nNR- and is intended to disclose each of the forms individually. Where the structure 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” or “aryl” then it is understood that the “alkyl” or “aryl” represents a linking alkylene group or arylene group, respectively.
The term “substituted” means that an atom or group of atoms formally replaces hydrogen as a “substituent” attached to another group. The term “substituted,” unless otherwise indicated, refers to any level of substitution, e.g., mono-, di-, tri-, tetra- or penta-substitution, where such substitution is permitted. The substituents are independently selected, and substitution may be at any chemically accessible position. It is to be understood that substitution at a given atom is limited by valency. It is to be understood that substitution at a given atom results in a chemically stable molecule. The phrase “optionally substituted” means unsubstituted or substituted. The term “substituted” means that a hydrogen atom is removed and replaced by a substituent. A single divalent substituent, e.g., oxo, can replace two hydrogen atoms.
The term “Cn-m” indicates a range which includes the endpoints, wherein n and m are integers and indicate the number of carbons. Examples include C- , CI-6 and the like.
The term “alkyl” employed alone or in combination with other terms, refers to a saturated hydrocarbon group that may be straight-chained or branched. The term “Cn-m alkyl,” refers to an alkyl group having n to m carbon atoms. An alkyl group formally corresponds to an alkane with one C-H bond replaced by the point of attachment of the alkyl group to the remainder of the compound. In some embodiments, the alkyl group contains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, or 1 to 2 carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, ferf-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.
The term “alkenyl” employed alone or in combination with other terms, refers to a straight-chain or branched hydrocarbon group corresponding to an alkyl group having one or more double carbon-carbon bonds. An alkenyl group formally corresponds to an alkene with one C-H bond replaced by the point of attachment of the alkenyl group to the remainder of the compound. The term “Cn-m alkenyl” refers to an alkenyl group having n to m carbons. In some embodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms. Example alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n- butenyl, sec-butenyl and the like. The term “alkynyl” employed alone or in combination with other terms, refers to a straight-chain or branched hydrocarbon group corresponding to an alkyl group having one or more triple carbon-carbon bonds. An alkynyl group formally corresponds to an alkyne with one C-H bond replaced by the point of attachment of the alkyl group to the remainder of the compound. The term “Cn-m alkynyl” refers to an alkynyl group having n to m carbons. Example alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl and the like. In some embodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.
The term “alkoxy,” employed alone or in combination with other terms, refers to a group of formula -O-alkyl, wherein the alkyl group is as defined above. The term “Cn-m alkoxy” refers to an alkoxy group, the alkyl group of which has n to m carbons. Example alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), f-butoxy and the like. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. The term “C n-m dialkoxy” refers to a linking group of formula -0-(Cn-m alkyl)-0-, the alkyl group of which has n to m carbons. Example dialkyoxy groups include -OCH2CH2O- and OCH2CH2CH2O-. In some embodiments, the two O atoms of a C n-m dialkoxy group may be attached to the same B atom to form a 5- or 6- membered heterocycloalkyl group.
The terms “halo” or “halogen,” used alone or in combination with other terms, refers to fluoro, chloro, bromo and iodo. In some embodiments, “halo” refers to a halogen atom selected from F, Cl, or Br. In some embodiments, halo groups are F.
The term “haloalkyl” as used herein refers to an alkyl group in which one or more of the hydrogen atoms has been replaced by a halogen atom. The term “Cn-m haloalkyl” refers to a Cn-m alkyl group having n to m carbon atoms and from at least one up to {2(n to m)+1} halogen atoms, which may either be the same or different. In some embodiments, the halogen atoms are fluoro atoms. In some embodiments, the haloalkyl group has 1 to 6 or 1 to 4 carbon atoms. Example haloalkyl groups include CF3, C2F5, CHF2, CH2F, CCb, CHCI2, C2CI5 and the like. In some embodiments, the haloalkyl group is a fluoroalkyl group.
The term “aromatic” refers to a carbocycle or heterocycle having one or more polyunsaturated rings having aromatic character ( i.e ., having (4n + 2) delocalized p (pi) electrons where n is an integer).
The term “aryl,” employed alone or in combination with other terms, refers to an aromatic hydrocarbon group, which may be monocyclic or polycyclic (e.g., having 2 fused rings). The term “Cn-m aryl” refers to an aryl group having from n to m ring carbon atoms. Aryl groups include, e.g., phenyl, naphthyl, and the like. In some embodiments, aryl groups have from 6 to about 10 carbon atoms. In some embodiments, aryl groups have 6 carbon atoms.
In some embodiments, aryl groups have 10 carbon atoms. In some embodiments, the aryl group is phenyl. In some embodiments, the aryl group is naphthyl. The term “heteroaryl” or “heteroaromatic,” employed alone or in combination with other terms, refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen and nitrogen. In some embodiments, the heteroaryl ring has 1 , 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, any ring-forming N in a heteroaryl moiety can be an N-oxide. In some embodiments, the heteroaryl has 5-14 ring atoms including carbon atoms and 1 , 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl has 5-10 ring atoms including carbon atoms and 1 , 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl has 5-6 ring atoms and 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl is a five-membered or six-membered heteroaryl ring. In other embodiments, the heteroaryl is an eight-membered, nine-membered or ten-membered fused bicyclic heteroaryl ring. Example heteroaryl groups include, but are not limited to, pyridinyl (pyridyl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, pyrazolyl, azolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, furanyl, thiophenyl, quinolinyl, isoquinolinyl, naphthyridinyl (including 1 ,2-, 1 ,3-, 1 ,4-, 1 ,5-, 1,6-, 1 ,7-, 1 ,8-, 2,3- and 2,6-naphthyridine), indolyl, isoindolyl, benzothiophenyl, benzofuranyl, benzisoxazolyl, imidazo[1 ,2-b]thiazolyl, purinyl, and the like. In some embodiments, the heteroaryl group is pyridone (e.g., 2- pyridone).
A five-membered heteroaryl ring is a heteroaryl group having five ring atoms wherein one or more (e.g., 1 , 2 or 3) ring atoms are independently selected from N, O and S. Exemplary five-membered ring heteroaryls include thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1 ,2,3-triazolyl, tetrazolyl, 1 ,2,3- thiadiazolyl, 1 ,2,3-oxadiazolyl, 1 ,2,4-triazolyl, 1 ,2,4-thiadiazolyl, 1 ,2,4-oxadiazolyl, 1 ,3,4- triazolyl, 1 ,3,4-thiadiazolyl and 1 ,3,4-oxadiazolyl.
A six-membered heteroaryl ring is a heteroaryl group having six ring atoms wherein one or more (e.g., 1 , 2 or 3) ring atoms are independently selected from N, O and S. Exemplary six-membered ring heteroaryls are pyridyl, pyrazinyl, pyrimidinyl, triazinyl, isoindolyl, and pyridazinyl.
The compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds provided herein that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically inactive starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present disclosure. C/'s and trans geometric isomers of the compounds of the present disclosure are described and may be isolated as a mixture of isomers or as separated isomeric forms.
Resolution of racemic mixtures of compounds can be carried out by any of numerous methods known in the art. One method includes fractional recrystallization using a chiral resolving acid which is an optically active, salt-forming organic acid. Suitable resolving agents for fractional recrystallization methods are, e.g., optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids such as b- camphorsulfonic acid. Other resolving agents suitable for fractional crystallization methods include stereoisomerically pure forms of a-methylbenzylamine (e.g., S and R forms, or diastereomerically pure forms), 2-phenylglycinol, norephedrine, ephedrine, N- methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane and the like.
Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent composition can be determined by one skilled in the art.
In some embodiments, the compounds provided herein have the ( Reconfiguration.
In other embodiments, the compounds have the (S)-configuration. In compounds with more than one chiral centers, each of the chiral centers in the compound may be independently (R) or (S), unless otherwise indicated.
Compounds provided herein also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge.
Example prototropic tautomers include 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, e.g., 1/-/- and 3/-/-imidazole, 1/-/-, 2 H- and 4 H- 1 ,2,4-triazole, 1/-/- and 2 H- isoindole and 1/-/- and 2/-/-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.
Compounds provided herein can also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. One or more constituent atoms of the compounds provided herein can be replaced or substituted with isotopes of the atoms in natural or non-natural abundance. In some embodiments, the compound includes at least one deuterium atom. For example, one or more hydrogen atoms in a compound of the present disclosure can be replaced or substituted by deuterium. In some embodiments, the compound includes two or more deuterium atoms. In some embodiments, the compound includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10,
11 or 12 deuterium atoms. Synthetic methods for including isotopes into organic compounds are known in the art (Deuterium Labeling in Organic Chemistry by Alan F. Thomas (New York, N.Y., Appleton-Century-Crofts, 1971; The Renaissance of H/D Exchange by Jens Atzrodt, Volker Derdau, Thorsten Fey and Jochen Zimmermann, Angew. Chem. Int. Ed. 2007, 7744-7765; The Organic Chemistry of Isotopic Labelling by James R. Hanson, Royal Society of Chemistry, 2011). Isotopically labeled compounds can used in various studies such as NMR spectroscopy, metabolism experiments, and/or assays.
Substitution with heavier isotopes such as deuterium, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances. (A. Kerekes et.al. J. Med. Chem. 2011, 54, 201-210; R. Xu et.al. J. Label Compd. Radiopharm. 2015, 58, 308-312).
The term “compound” as used herein is meant to include all stereoisomers, geometric isomers, tautomers and isotopes of the structures depicted. The term is also meant to refer to compounds provided herein, regardless of how they are prepared, e.g., synthetically, through biological process (e.g., metabolism or enzyme conversion), or a combination thereof.
All compounds, and pharmaceutically acceptable salts thereof, can be found together with other substances such as water and solvents (e.g., hydrates and solvates) or can be isolated. When in the solid state, the compounds described herein and salts thereof may occur in various forms and may, e.g., take the form of solvates, including hydrates. The compounds may be in any solid state form, such as a polymorph or solvate, so unless clearly indicated otherwise, reference in the specification to compounds and salts thereof should be understood as encompassing any solid state form of the compound.
In some embodiments, the compounds provided herein, or salts thereof, are substantially isolated. By “substantially isolated” is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, e.g., a composition enriched in the compounds provided herein. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compounds provided herein, or salt thereof.
The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions and/or dosage forms that 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.
The present disclosure also includes pharmaceutically acceptable salts of the compounds described herein. The term “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present disclosure include the non-toxic salts of the parent compound formed, e.g., from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, iso-propanol or butanol) or acetonitrile (MeCN) are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences,
17th Ed., (Mack Publishing Company, Easton, 1985), p. 1418, Berge ef a/., J. Pharm. Sci., 1977, 66(1 ), 1-19 and in Stahl ef a/., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, (Wiley, 2002). In some embodiments, the compounds described herein include the N-oxide forms.
As used herein, the phrase “pharmaceutically acceptable carrier or excipient” refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients or carriers are generally safe, non-toxic and neither biologically nor otherwise undesirable and include excipients or carriers that are acceptable for veterinary use as well as human pharmaceutical use. In one embodiment, each component is “pharmaceutically acceptable” as defined herein. See, e.g., Remington: The Science and Practice of Pharmacy, 21st ed.; Lippincott Williams & Wilkins: Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al . , Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC:
Boca Raton, Fla., 2009.
As used herein, the term “treating” or “treatment” refers to inhibiting a disease; for example, inhibiting a disease, condition, or disorder in an individual who is experiencing or displaying the pathology or symptomology of the disease, condition, or disorder ( i.e ., arresting further development of the pathology and/or symptomology) or ameliorating the disease; for example, ameliorating a disease, condition, or disorder in an individual who is experiencing or displaying the pathology or symptomology of the disease, condition, or disorder ( i.e ., reversing the pathology and/or symptomology) such as decreasing the severity of the disease.
The term “prevent,” “preventing,” or “prevention” as used herein, comprises the prevention of at least one symptom associated with or caused by the state, disease or disorder being prevented.
Compounds
In an aspect, provided herein is a compound of Formula I:
Figure imgf000013_0001
or a pharmaceutically acceptable salt thereof; wherein
X is NH, O, or -(CH2)m-;
Y is CH or N;
Z is CH or N;
A is absent, O, or NH;
R1 is OR7 or -(CH2)nNHR7;
R2 is H or halo;
R3 is selected from the group consisting of H, halo, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, and C1-C6 haloalkyl;
R4 is H or halo;
R5 and R6 are each independently selected from the group consisting of H, C1-C6 alkyl, and C(0)CrC6 alkyl;
R7 is selected from the group consisting of H, C1-C6 alkyl, C1-C6 haloalkyl, C6-C10 aryl, and 5-10 membered heteroaryl; wherein aryl and heteroaryl are each optionally substituted one, two, or three times with OC1-C6 haloalkyl; and m and n are, independently at each occurrence, 0, 1, 2, or 3.
In an embodiment, the compound of Formula I is a compound of Formula II:
Figure imgf000014_0001
or a pharmaceutically acceptable salt thereof.
In another embodiment, the compound of Formula I is a compound of Formula III:
Figure imgf000014_0002
or a pharmaceutically acceptable salt thereof.
In yet another embodiment, the compound of Formula I is a compound of Formula IV:
Figure imgf000014_0003
or a pharmaceutically acceptable salt thereof.
In still another embodiment, R1 is OR7. In an embodiment, R1 is -(CH2)nNHR7. In another embodiment, R7 is selected from the group consisting of O-I-OQ alkyl, O-I-OQ haloalkyl, and phenyl, wherein phenyl is optionally substituted one time with OO-I-OQ haloalkyl. In yet another embodiment, R7 is selected from the group consisting of methyl, ethyl, C3 alkyl, C4 alkyl, CF3, and phenyl-OCF3.
In an embodiment, R2 is H. In another embodiment, R2 is fluoro. In yet another embodiment, R3 is selected from the group consisting of H, fluoro, methyl, methoxy, C2 alkenyl, and C2 alkynyl. In still another embodiment, R3 is H.
In an embodiment, R4 is H. In another embodiment, R4 is fluoro.
In yet another embodiment, R5 is methyl. In still another embodiment, R6 is H or methyl. In an embodiment, R6 is H.
In another embodiment, the compound of Formula I is CPPB:
Figure imgf000015_0001
or a pharmaceutically acceptable salt thereof.
In yet another embodiment, the compound of Formula I is CPPB1 :
Figure imgf000015_0002
(CPPB1) or a pharmaceutically acceptable salt thereof.
In another aspect, provided herein is a compound of Formula V:
Figure imgf000015_0003
or a pharmaceutically acceptable salt thereof; wherein
X and Y are independently O or NH;
R1 is selected from the group consisting of H, OH, C(0)NH2, CO2H, C(0)H, and C(0)halo; and R2 is selected from the group consisting of H, O-I-OQ alkyl, O-I-OQ haloalkyl, C -C aryl, and 5-10 membered heteroaryl.
In an embodiment, X is O. In another embodiment, X is NH. In yet another embodiment Y is O. In still another embodiment, Y is NH. In an embodiment, R1 is C(0)NH2. In another embodiment, R2 is C -C haloalkyl.
In another embodiment, the compound of Formula V is
Figure imgf000016_0002
or a pharmaceutically acceptable salt thereof.
In yet another aspect, provided herein is a compound of Formula VI:
Figure imgf000016_0001
or a pharmaceutically acceptable salt thereof; wherein
X and Y are independently O or NH;
R1 and R2 are each independently selected from H and OH; and R3 is selected from the group consisting of H, O-I-OQ alkyl, O-I-OQ haloalkyl, C -C aryl, and 5-10 membered heteroaryl.
In an embodiment, X and Y are NH. In another embodiment, X is NH and Y is O. In yet another embodiment, X is O and Y is NH.
In still another embodiment, R3 is O-I-OQ haloalkyl. In an embodiment, the compound of Formula VI is selected from the group consisting of:
Figure imgf000017_0001
or a pharmaceutically acceptable salt thereof.
In another aspect, provided herein is a pharmaceutical composition comprising a compound disclosed herein, and at least one pharmaceutically acceptable carrier.
In an embodiment, the pharmaceutical composition further comprises an additional therapeutic agent.
In another embodiment, the additional therapeutic agent is selected from the group consisting of paclitaxel, tunicamycin, capuramycin, erlotinib, capecitabine, fluorouracil, and gemcitabine. In yet another embodiment, the additional therapeutic agent is paclitaxel.
Methods of Treatment
In yet another aspect, provided herein is a method of inhibiting dolichyl-phosphate N- acetylglucosaminephosphotransferase (DPAGT1) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein or a pharmaceutical composition comprising a compound disclosed herein. In still another aspect, provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure or a pharmaceutical composition comprising a compound disclosed herein.
In an embodiment, the cancer is selected from hematological cancers, sarcomas, lung cancers, brain cancers, gastrointestinal cancers, genitourinary tract cancers, liver cancers, bone cancers, nervous system cancers, gynecological cancers, and skin cancers.
In another embodiment, the lung cancer is selected from non-small cell lung cancer (NSCLC), small cell lung cancer, bronchogenic carcinoma, squamous cell bronchogenic carcinoma, undifferentiated small cell bronchogenic carcinoma, undifferentiated large cell bronchogenic carcinoma, adenocarcinoma, bronchogenic carcinoma, alveolar carcinoma, bronchiolar carcinoma, bronchial adenoma, chondromatous hamartoma, mesothelioma, pavicellular and non-pavicellular carcinoma, bronchial adenoma, and pleuropulmonary blastoma.
In yet another embodiment, the lung cancer is non-small cell lung cancer (NSCLC).
In still another embodiment, the lung cancer is adenocarcinoma.
In an embodiment, the gastrointestinal cancer is selected from esophagus squamous cell carcinoma, esophagus adenocarcinoma, esophagus leiomyosarcoma, esophagus lymphoma, stomach carcinoma, stomach lymphoma, stomach leiomyosarcoma, exocrine pancreatic carcinoma, pancreatic ductal adenocarcinoma, pancreatic insulinoma, pancreatic glucagonoma, pancreatic gastrinoma, pancreatic carcinoid tumors, pancreatic vipoma, small bowel adenocarcinoma, small bowel lymphoma, small bowel carcinoid tumors, Kaposi's sarcoma, small bowel leiomyoma, small bowel hemangioma, small bowel lipoma, small bowel neurofibroma, small bowel fibroma, large bowel adenocarcinoma, large bowel tubular adenoma, large bowel villous adenoma, large bowel hamartoma, large bowel leiomyoma, colorectal cancer, gall bladder cancer, and anal cancer.
In an embodiment, the gastrointestinal cancer is colorectal cancer.
In another embodiment, the brain cancer is selected from acoustic neuroma, astrocytoma, choroid plexus carcinoma, craniopharyngioma, embryonal tumors, ependymoma, glioblastoma, glioma, medulloblastoma, meningioma, oligodendroglioma, pineoblastoma, mixed glioma, mixed glial and neuronal tumors, and primitive neuroectodermal tumors.
In another embodiment, the cancer is a carcinoma. In yet another embodiment, the carcinoma is selected from pancreatic carcinoma, colorectal carcinoma, lung carcinoma, bladder carcinoma, gastric carcinoma, esophageal carcinoma, breast carcinoma, head and neck carcinoma, cervical skin carcinoma, and thyroid carcinoma. In still another embodiment, the cancer is a hematopoietic malignancy. In an embodiment, the hematopoietic malignancy is selected from multiple myeloma, acute myelogenous leukemia, and myeloproliferative neoplasms.
In another embodiment, the cancer is a neoplasm. In yet another embodiment, the neoplasm is glioblastoma or sarcomas.
In an embodiment, the cancer is selected from the group consisting of hematological cancers, sarcomas, lung cancers, gastrointestinal cancers, genitourinary tract cancers, liver cancers, bone cancers, nervous system cancers, gynecological cancers, and skin cancers.
In an embodiment, the cancer is selected from the group consisting of pancreatic cancer, cervical cancer, colon cancer, ovarian cancer, breast cancer, pancreatic cancer, carcinoma, and adenocarcinoma.
In another embodiment, the cancer is pancreatic cancer. In yet another embodiment, the cancer is a solid tumor.
In an embodiment of the methods, the subject is human.
As used herein, the term “individual,” “subject,” or “patient,” used interchangeably, refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.
As used herein, the phrase “therapeutically effective amount” refers to the amount of active compound or pharmaceutical agent such as an amount of any of the solid forms or salts thereof as disclosed herein that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. An appropriate “effective” amount in any individual case may be determined using techniques known to a person skilled in the art.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment (while the embodiments are intended to be combined as if written in multiply dependent form). Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.
EXAMPLES
Example 1: Synthetic Procedures
Scheme 1
A: Synthesis of rse-captiratnycin (t-CAP)
Figure imgf000020_0001
a> 14, MS, AsSf* SrCO^ aA MS, CHja^CC, 1» b) HaCS^.aicOKitse, ίIOH-H,O.; tj MeOH-THf:. »¾.; 4! Hj, 10¾. W-C. AsCH-’PiOH-rHf:,:
The (S)-cyanohydrin (13) was subjected to NIS-AgBF4 promoted a-selective mannosylation with the thioglycoside (14) to yield the a-mannosylated cyanohydrin (15) in 78% yield. The cyano group of 15 was hydrated using HgCh-aldoxime in aq. EtOH, and the BOM and chloroacetyl groups of the generated amide were deprotected stepwisely: dechloro- acetylation with thiourea followed by hydrogenation with 10% Pd-C in AcOH-'PrOH-THF provided the C6”-free alcohol 16 in 53% overall yield. Oxidation-elimination reaction of 16 with S03*pyridine in a solvent system (ChhCh/EtsN/DMSO = 10/2/1) provided the a,b - unsaturated aldehyde 17 in quantitative yield (determined by 1H NMR analysis). After all volatiles were removed, the aldehyde 17 was oxidized to the corresponding carboxylic acid 18 by using NaCIC^ in the presence of NahhPC^ and 2-methyl-2-butene. The resulting carboxylic acid 18 was coupled with (2S)-aminocaprolactam by using a standard peptideforming reaction condition (HOBt, EDCI, and NMM) to yield the coupling product 19 in 70% overall yield from 16. Saponification of 19 by using Et3N in MeOH provided l-Cap (3) in quantitative yield. Similarly, dimethyl-capuramycin (DM-CAP) was synthesized in 31% overall yield from the cyanohydrin-acetonide 20.
(2S,3S,4S,5R,6R)-2-((1S)-((2R,5R)-3-Acetoxy-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl)(cyano)methoxy)-6-((2- chloro- acetoxy)methyl)-tetrahydro-2H-pyran-3,4,5-triyl triacetate (15). To a stirred suspension of 13 (0.17 g, 0.38 mmol), 14 (0.37 g, 0.75 mmol), MS 3A (0.50 g) and SrC03 (0.28 g, 1.88 mmol) in CH2CI2 (9.4 ml_) were added AgBF4 (0.037 g, 0.19 mmol) and NIS (0.25 g, 1.13 mmol) at 0 °C. After being stirred for 19h, the reaction mixture was added Et3N (1.0 ml_) and passed through a silica gel pad (hexanes/EtOAc = 1/4). The filtrate was concentrated in vacuo. The crude mixture was purified by silica gel column chromatography (hexanes/EtOAc = 2/1 to 1/2) to afford 15 (0.24 g, 78%): 1H NMR (400 MHz, Chloroform-c/) d 7.40 - 7.27 (m, 6H), 6.07 (d, J = 4.7 Hz, 1H), 6.00 (d, J = 8.2 Hz, 1H), 5.51 (d, J = 9.8 Hz, 1H), 5.47 (d, J = 9.6 Hz, 1H), 5.37 (dd, J= 3.4, 2.0 Hz, 1H), 5.28 (d, J = 10.2 Hz, 1H), 5.22 - 5.15 (m, 2H), 4.84 (d, J = 3.2 Hz, 1H), 4.71 (s, 2H), 4.41 (dd, J = 5.5, 3.2 Hz, 1H), 4.39 - 4.35 (m, 1H), 4.33 (d, J = 6.0 Hz, 1H), 4.27 (d, J = 2.3 Hz, 1H), 4.14 (d, J = 1.7 Hz, 2H), 4.03 - 4.01 (m, 1H), 3.93 (ddd, J = 8.9, 5.9, 2.4 Hz,
1H), 3.47 (s, 3H), 2.20 (s, 3H), 2.19 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H); 13C NMR (101 MHz, CDCI3) d 170.20, 169.84, 169.50, 166.92, 162.19, 150.97, 137.75, 137.46, 128.32 (2C), 127.72, 127.69 (2C), 113.96, 103.58, 96.29, 88.68, 80.93, 80.09, 72.30, 70.42, 69.60, 68.46, 67.94, 65.16, 64.35, 63.58, 59.26, 40.61 , 31.58, 20.70, 20.66, 20.62, 20.57; HRMS (ESI+) m/z calcd for C35H4iCIN3017 [M + H] 810.2125, found: 810.2151.
(2R,3S,4S,5R,6R)-2-((1R)-1-((2S,5R)-3-Acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)- 4-methoxy-tetrahydrofuran-2-yl)-2-amino-2-oxoethoxy)-6-(hydroxymethyl)tetrahydro-2H- pyran-3,4,5-triyl triacetate (16). To a stirred solution of 15 (0.24 g, 0.29 mmol) in a 9:1 mixture of EtOH and H2O (2.9 ml_) were added HgCh (0.16 g, 0.59 mmol) and acetaldoxime (0.18 ml_, 2.9 mmol). After being stirred for 13h at r.t., the reaction mixture was concentrated in vacuo. The residue was quenched with aq. NaHC03, and extracted with CHCI3. The combined organic extracts were dried over Na2SC>4 and concentrated in vacuo. The crude product was purified by silica gel column chromatography (hexanes/EtOAc = 1/2 to CHCI3/MeOH = 96/4) to afford the amide (0.21 g, 87%). To a solution of the amide (0.21 g, 0.26 mmol) in a 1:1 mixture of THF and MeOH (2.6 ml_) was added thiourea (0.059 g, 0.77 mmol). After being stirred for 11 h at 50 °C, the reaction mixture was concentrated in vacuo. The residue was diluted with H2O and extracted with CHCb. The combined organic extracts were dried over Na2SC>4 and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHCla/MeOH = 98/2 to 97/3 to 96/4) to afford the primary alcohol (0.15 g, 75%). To a stirred solution of the primary alcohol (0.15 g, 0.19 mmol) and AcOH (0.040 mL) in a 1 :1 mixture of THF and 'PrOH (2.0 mL) was added 10% Pd/C (0.12 g) under N2. H2 gas was introduced and the reaction mixture was stirred under H2 atmosphere for 3h, the solution was filtered through Celite and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHCh/MeOH = 97/3 to 92/8) to afford 16 (0.10 g, 81%): 1H NMR (400 MHz, Chloroform-c/) d 9.08 (brs, 1H), 7.60 (d, J = 8.2 Hz, 1H), 6.87 (brs, 1H), 6.12 (brs, 1H), 6.00 (d, J = 8.1 Hz, 1H), 5.87 (d, J = 3.6 Hz, 1 H), 5.55 - 5.52 (m, 1 H), 5.26 - 5.22 (m, 2H), 5.20 (t, J = 6.0 Hz, 1 H), 5.00 (d, J = 1.7 Hz, 1H), 4.53 (dd, J = 6.5, 2.3 Hz, 1 H), 4.46 (d, J = 2.3 Hz, 1H), 4.06 (dd, J = 5.5, 3.7 Hz, 1H), 3.76 - 3.71 (m, 1 H), 3.69 - 3.57 (m, 2H), 3.44 (s, 3H), 2.17 (s, 3H), 2.15 (s, 3H), 2.09 (s, 3H), 2.04 (s, 3H); 13C NMR (101 MHz, CDCI3) d 171.42, 170.46, 170.46, 170.40, 163.10, 150.03, 139.50, 103.61 , 97.15, 96.95, 88.50, 81.26, 80.96, 75.33, 72.91 , 69.67, 68.89, 65.54, 61.27, 59.05, 50.86, 20.82, 20.75, 20.67, 20.59; HRMS (ESI+) mlz calcd for 025^4^0^ [M + H] 632.1939, found: 632.1963.
(2S,3S,4S)-3,4-Diacetoxy-2-((1R)-1-((2S,5R)-3-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin- 1(2H)-yl)-4-methoxytetrahydrofuran-2-yl)-2-amino-2-oxoethoxy)-3,4-dihydro-2H-pyran-6- carboxylic acid (18). To a stirred solution of 16 (0.10 g, 0.16 mmol) and DMSO (0.11 mL, 1.57 mmol) in a 5:1 mixture of CH2CI2 and EtaN (0.80 mL) was added SCVpyridine (0.25 g, 1.57 mmol). After being stirred for 3h at r.t., the reaction mixture was added H2O (0.16 mL) and passed through a silica gel pad (CHCla/MeOH = 92/8) to provide the crude 17. To a stirred solution of the crude mixture in BuOH (1.0 mL) and 2-methyl-2-butene (0.5 mL) was added a solution of NaCIC>2 (0.071 g, 0.78 mmol) and Na^PCL^^O (0.12 g, 0.78 mmol) in H2O (1.0 mL). After being stirred for 4h at r.t., the reaction was quenched with H20 and extracted with CHCla/MeOH (9/1). The combined organic extracts were dried over Na2SC>4 and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHCh/MeOH = 9/1) to afford 18 (0.078 g, 85%): 1H NMR (400 MHz, Methanol-c/4) d 7.77 (d, J = 8.1 Hz, 1H), 5.94 (d, J = 11.7 Hz, 1H), 5.94 (s, 1H), 5.78 (t, J = 2.1 Hz, 1H), 5.66 (dd, J = 4.5, 2.5 Hz, 1H), 5.56 (ddd, J = 4.7, 3.2, 1.6 Hz, 1H), 5.32 (d, J = 3.3 Hz, 1 H), 4.99 (t, J = 5.2 Hz, 1H), 4.85 (d, J = 2.1 Hz, 1 H), 4.60 (dd, J = 5.2, 2.0 Hz, 1H), 3.95 (t, J = 5.0 Hz, 1H), 3.39 (s, 3H), 2.13 (s, 3H), 2.08 (s, 3H), 2.05 (s, 3H); 13C NMR (101 MHz, MeOD) 0 172.79, 172.06, 171.60,
167.79, 166.01 , 152.05, 148.16, 140.97, 104.23, 103.98, 97.91 , 88.13, 83.38, 82.91 , 76.56, 71.95, 65.17, 65.06, 59.41 , 20.75, 20.63, 20.57; HRMS (ESI+) mlz calcd for CzaHzsNaChs [M + H] 586.1520, found: 586.1549. (2S,3S,4S)-2-((1 R)-1 -((2S,5R)-3-Acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1 (2H)-yl)-4- methoxy-tetrahydrofuran-2-yl)-2-amino-2-oxoethoxy)-6-(((S)-2-oxoazepan-3- yl)carbamoyl)-3,4-dihydro-2H-pyran-3,4-diyl diacetate (19). To a stirred solution of 18 (31 mg, 0.053 mmol), 2-(S)-aminocaprolactam (26 mg, 0.16 mmol), HOBt (21 mg, 0.16 mmol) and NMM (58 pL, 0.53 mmol) in DMF (0.26 mL) was added EDCI (51 mg, 0.26 mmol). After being stirred for 6h at r.t., the reaction was quenched with aq. NaHCCh, and extracted with CHCh/MeOH (9/1). The combined organic extracts were dried over Na SC and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHCh/MeOH = 95/5) to afford 19 (30 mg, 82%): 1H NMR (400 MHz, Methanol-c/4) 0 7.68 (d, J = 8.2 Hz,
1 H), 6.02 (t, J = 2.1 Hz, 1H), 5.91 (d, J = 4.2 Hz, 1H), 5.89 (d, J = 6.3 Hz, 1H), 5.73 (dd, J = 4.4, 2.5 Hz, 1H), 5.68 - 5.65 (m, 1H), 5.51 (d, J = 3.0 Hz, 1 H), 5.01 (d, J = 4.6 Hz, 1H), 4.76 (d, J = 2.0 Hz, 1H), 4.62 (d, J = 12.5 Hz, 1H), 4.60 - 4.57 (m, 1H), 3.98 (d, J = 6.1 Hz, 1 H), 3.35 (s, 3H), 2.11 (s, 3H), 2.08 (s, 3H), 2.05 (s, 3H), 2.03 - 1.98 (m, 2H), 1.91 - 1.83 (m, 2H), 1.65 - 1.56 (m, 2H), 1.44 - 1.37 (m, 2H); 13C NMR (101 MHz, MeOD) 0 176.12, 172.32, 171.66, 171.29, 171.21 , 165.96, 160.96, 151.90, 144.61 , 141.42, 106.25, 103.54, 98.81 , 88.77, 83.41 , 79.28, 77.39, 74.79, 64.91 , 64.49, 59.26, 57.36, 53.25, 42.36, 32.19, 29.75, 28.94, 20.54, 20.41 , 20.32; HRMS (ESI+) mlz calcd for CzgHasNsO^ [M + H] 696.2364, found: 696.2391.
(2S,3S,4S)-2-((1R)-2-Amino-1-((2S,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3- hydroxy-4-methoxytetrahydrofuran-2-yl)-2-oxoethoxy)-3,4-dihydroxy-N-((S)-2-oxo- azepan-3-yl)-3,4-dihydro-2H-pyran-6-carboxamide (3). A solution of 19 (30 mg, 0.043 mmol) in a 5:1 mixture of MeOH and EtaN (0.60 mL) was stirred for 5h at r.t., and concentrated in vacuo. The crude mixture was purified by reverse-phase HPLC [column: Luna® (C18, 10 pm, 100 A, 250 x 10 mm), solvents: 15:85 MeOhbhhO, flow rate: 3.0 mL/min, UV: 254 nm, retention time: 20 min] to afford l-CAP (3, 24 mg, 98%): 1H NMR (400 MHz, Methanol-c/4) d 7.92 (d, J = 8.1 Hz, 1H), 6.02 (d, J = 3.8 Hz, 1 H), 5.88 (d, J = 5.1 Hz, 1H), 5.74 (d, J = 8.1 Hz, 1H), 5.23 (d, J = 5.6 Hz, 1 H), 4.67 (d, J = 2.0 Hz, 1 H), 4.59 - 4.54 (m,
2H), 4.38 (t, J = 4.2 Hz, 1H), 4.29 (t, J = 5.1 Hz, 1H), 3.98 (t, J = 5.0 Hz, 1H), 3.84 (t, J = 4.6 Hz, 1H), 3.43 (s, 3H), 2.06 - 1.99 (m, 2H), 1.89 - 1.81 (m, 2H), 1.62 - 1.45 (m, 2H), 1.44 - 1.33 (m, 2H); 13C NMR (101 MHz, MeOD) d 176.27, 173.46, 166.15, 161.85, 152.32, 144.23, 141.91 , 109.37, 102.82, 101.22, 90.27, 83.49, 81.02, 78.93, 74.54, 68.51 , 63.53, 58.67,
53.35, 42.48, 32.36, 29.91 , 29.06; HRMS (ESI+) mlz calcd for 023^2^0^ [M + H] 570.2048, found: 570.2071. (2S,3S,4S,5R,6R)-2-((S)-((3aR,4R,6R,6aR)-6-(3-((Benzyloxy)methyl)-2,4-dioxo-3, 4- dihydro- pyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)(cyano)methoxy)-6- ((2-chloroacetoxy)-methyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (21). To a stirred suspension of 20 (0.24 g, 0.56 mmol), 14 (0.55 g, 1.12 mmol), MS 3A (0.72 g), and SrCCh (0.41 g, 2.80 mmol) in CH2CI2 (14.0 ml_) were added AgBF4 (0.055 g, 0.28 mmol) and NIS (0.25 g,
1.12 mmol) at 0 °C. After being stirred for 12h, the reaction mixture was added EtsN (1.0 ml_), and passed through a silica gel pad (hexanes/EtOAc = 1/4). The filtrate was concentrated in vacuo. The crude mixture was purified by silica gel column chromatography (hexanes/EtOAc = 6/4 - 4/6) to afford 21 (0.39 g, 88%): 1H NMR (400 MHz, Chloroform-c/) d 7.37 - 7.28 (m, 5H), 7.20 (d, J = 8.1 Hz, 1H), 5.82 (d, J = 8.1 Hz, 1H), 5.61 (d, J = 1.6 Hz, 1H), 5.50 (d, J = 9.8 Hz, 1H), 5.42 (d, J = 9.8 Hz, 1H), 5.35 - 5.27 (m, 1H), 5.23 (dd, J = 10.0, 3.3 Hz, 1H), 5.07 (d, J =
1.9 Hz, 1H), 5.00 (d, J = 3.5 Hz, 1H), 4.98 (dd, J = 6.4, 1.6 Hz, 1H), 4.84 (d, J = 7.1 Hz, 1H), 4.69 (s, 2H), 4.43 (dd, J = 7.1 , 3.6 Hz, 1H), 4.20 (dd, J = 12.2, 4.3 Hz, 1H), 4.17 - 4.07 (m, 2H), 4.05 (d, J = 1.6 Hz, 2H), 3.97 - 3.92 (m, 1H), 2.16 (s, 3H), 2.02 (s, 3H), 1.98 (s, 3H), 1.59 (s, 3H),
1.39 (s, 3H); 13C NMR (101 MHz, CDCI3) d 169.80, 169.57, 169.47, 166.80, 162.08, 150.84, 140.89, 137.67, 128.33 (2C), 127.91, 127.77, 127.63 (2C), 115.23, 114.72, 102.84, 96.56,
96.30, 86.55, 84.15, 80.95, 72.42, 70.41, 69.41, 68.39 (2C), 66.04, 65.35, 62.90, 40.52, 27.03, 25.20, 20.73, 20.64, 20.59; HRMS (ESI+) m/z calcd for CasH^CINaO^ [M + H] 794.2175, found: 794.2198.
(2R,3S,4S,5R,6R)-2-((R)-2-Amino-1-((3aR,4S,6R,6aR)-6-(2,4-dioxo-3,4-dihydropyrimidin- 1-(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-2-oxoethoxy)-6- (hydroxymethyl)-tetrahydro-2H-pyran-3,4,5-triyl triacetate (22). To a stirred solution of 21 (0.39 g, 0.49 mmol) in a 9:1 mixture of EtOH and H2O (4.9 mL) were added HgCh (0.27 g,
0.98 mmol) and acetaldoxime (0.30 mL, 4.9 mmol) at rt. After being stirred for 12h at r.t., the reaction mixture was concentrated in vacuo. The residue was diluted with aq. NaHCCh, and extracted with CHCI3. The combined organic extracts were dried over Na2SC>4 and concentrated in vacuo. The crude product was purified by silica gel column chromatography (hexanes/EtOAc = 1/2 to CHCI3/MeOH = 97/3) to afford the amide (0.36 g, 91%). To a solution of the amide (0.36 g, 0.45 mmol) in a 1:1 mixture of THF and MeOH (4.5 mL) was added thiourea (0.10 g, 1.34 mmol). After being stirred for 11 h at 50 °C, the reaction mixture was concentrated in vacuo. The residue was diluted with H2O, and extracted with CHCI3. The combined organic extracts were dried over Na2S04 and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHCh/MeOH = 98/2 to 97/3 to 96/4) to afford the primary alcohol (0.25 g, 76%). To a stirred solution of the alcohol (0.25 g, 0.34 mmol) and AcOH (0.08 mL) in a 1 :1 mixture of THF and 'PrOH (4.0 mL) was added 10% Pd/C (0.20 g) under N2. H2 gas was introduced and the reaction mixture was stirred under H2 atmosphere at rt. After being stirred for 4h, the reaction mixture was filtered through Celite and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHCia/MeOH = 96/4 to 92/8) to afford 22 (0.17 g, 80%): 1H NMR (400 MHz, Chloroform-c/) d
9.45 (brs, 1H), 7.34 (d, J = 8.0 Hz, 1H), 6.72 (brs, 1H), 6.17 (brs, 1 H), 5.78 (d, J = 8.0 Hz,
1 H), 5.60 (d, J = 1.7 Hz, 1H), 5.43 (dd, J = 10.1 , 3.5 Hz, 1H), 5.33 (dd, J = 3.5, 1.7 Hz, 1 H), 5.20 (t, J = 10.1 Hz, 1H), 5.12 - 5.05 (m, 1 H), 5.03 (dd, J = 6.4, 1.7 Hz, 1H), 4.98 (s, 1 H),
4.46 (d, J = 5.8 Hz, 1H), 4.33 (t, J = 5.1 Hz, 1H), 3.88 (dt, J = 10.1 , 4.0 Hz, 1H), 3.60 - 3.53 (m, 2H), 2.14 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H), 1.55 (s, 3H), 1.35 (s, 3H); 13C NMR (101 MHz, CDCI3) d 170.68, 170.40, 170.28, 170.24, 163.27, 150.13, 142.81 , 114.72, 102.85, 96.39, 87.34, 84.26, 80.35, 71.92, 69.35, 68.64, 66.03, 61.16, 27.25, 25.42, 20.83, 20.77, 20.74; HRMS (ESI+) mlz calcd for 025^4^0^ [M + H] 616.1990, found: 616.2018.
(2S,3S,4S)-2-(( ?)-2-Amino-1-((3a ?,4S,6 ?,6a ?)-6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)- 2,2-dimethyltetrahydrofuro[3,4-c(][1,3]dioxol-4-yl)-2-oxoethoxy)-6-(((S)-2-oxoazepan-3-yl)- carbamoyl)-3,4-dihydro-2H-pyran-3,4-diyl diacetate (23). To a stirred solution of 22 (0.17 g, 0.27 mmol) and DMSO (0.19 mL, 2.72 mmol) in a 5:1 mixture of CH2CI2 and EtaN (1.4 ml_) was added SCVpyridine (0.43 g, 2.72 mmol). After being stirred for 3h at r.t., the reaction mixture was added H2O (0.27 mL) and passed through a silica gel pad (CHCh/MeOH = 92/8). To a stirred solution of the crude mixture in *BuOH (1.0 mL) and 2-methyl-2-butene (0.5 mL) was added a solution of NaCIC>2 (0.12 g, 1.36 mmol) and NaH2P04*2H20 (0.21 g, 1.36 mmol) in H2O (1.0 mL). After being stirred for 5h at r.t., the reaction was extracted with CHCla/MeOH (9/1). The combined organic extracts were dried over Na2SC>4 and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHC /MeOH = 9/1) to afford the acid (0.13 g, 81%). To a stirred solution of the acid (28 mg, 0.049 mmol), 2-(S)- aminocaprolactam (24 mg, 0.15 mmol), HOBt (20 mg, 0.15 mmol) and NMM (54 pL, 0.49 mmol) in DMF (0.25 mL) was added EDCI (47 mg, 0.25 mmol). After being stirred for 14h at r.t., the reaction was quenched with aq. NaHCCh, and extracted with CHCh/MeOH (9/1). The combined organic extracts were dried over Na2S04 and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHCh/MeOH = 95/5) to afford 23 (30 mg, 89%): 1H NMR (400 MHz, Methanol-c/4) d 7.79 (d, J = 8.0 Hz, 1H), 5.98 (t, J = 2.0 Hz, 1H), 5.84 (d, J = 2.8 Hz, 1H), 5.78 (d, J = 8.0 Hz, 1H), 5.62 (dd, J = 4.5, 2.6 Hz, 1H), 5.51 - 5.48 (m, 1H), 5.45 (d, J = 3.1 Hz, 1H), 4.74 (d, J = 2.8 Hz, 1H), 4.71 (dd, J = 6.2, 2.9 Hz, 1H), 4.65 (dd, J = 6.2, 3.3 Hz, 1 H), 4.59 - 4.55 (m, 2H), 3.76 - 3.67 (m, 4H), 2.09 (s, 3H), 2.04 (s, 3H), 1.86 (d, J = 13.4 Hz, 3H), 1.63 - 1.52 (m, 3H), 1.51 (s, 3H), 1.45 - 1.34 (m, 3H), 1.25 (s, 3H); 13C NMR (101 MHz, MeOD) d 176.35, 172.24, 171.62, 171.28, 166.24, 161.14, 151.99, 144.75, 142.76, 115.34, 106.38, 102.91 , 98.75, 93.55, 87.12, 85.94, 82.02, 78.13, 67.16, 64.99, 64.46, 56.04, 53.46, 46.10, 42.52, 42.43, 32.25, 29.89, 29.08, 27.47, 25.47, 20.61, 20.52; HRMS (ESI+) mlz calcd for C29H38N5O14 [M + H] 680.2415, found: 680.2432.
(2S,3S,4S)-2-((1 R)-2-Amino-1 -((2S,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1 (2H)-yl)-3,4- dihydroxy-tetrahydrofuran-2-yl)-2-oxoethoxy)-3,4-dihydroxy-N-((S)-2-oxoazepan-3-yl)-3,4- dihydro-2H-pyran-6-carboxamide (4). A solution of 23 (30 mg, 0.044 mmol) in a 4:1 mixture of TFA and H2O (0.88 ml_) was stirred for 4h at r.t., and concentrated in vacuo. A solution of the crude diol in a 5:1 mixture of MeOH and Et3N (0.88 ml_) was stirred for 9h at r.t., filtered, and concentrated in vacuo. The crude mixture was purified by reverse-phase HPLC [column: Luna® (C18, 10 pm, 100 A, 250 x 10 mm), solvents: 15:85 MeOH:H20, flow rate: 3.0 mL/min, UV: 254 nm, retention time: 16 min] to afford DM-CAP (4, 22 mg, 90%): 1H NMR (400 MHz, Methanol-c/4) 57.94 (d, J = 8.1 Hz, 1H), 6.03 (d, J = 4.5 Hz, 1H), 5.85 (d, J = 3.7 Hz, 1H), 5.73 (d, J = 8.2 Hz, 1H), 5.19 (d, J = 6.7 Hz, 1H), 4.68 (d, J = 1.7 Hz, 1H), 4.61 (dd, J = 11.2, 1.4 Hz, 1H), 4.54 (dd , J = 5.8, 1.7 Hz, 1H), 4.36 (t, J = 4.5 Hz, 1H), 4.30 (t, J = 5.5 Hz, 1H), 4.12 (t, J = 4.4 Hz, 1H), 3.97 (t, J= 5.7 Hz, 1H), 3.90 (t, J = 6.5 Hz, 1 H), 3.43 - 3.38 (m, 1H), 3.03 (t, J = 7.0 Hz, 1H), 2.70 -
2.64 (m, 1H), 2.07 - 1.97 (m, 2H), 1.81 (d, J = 15.2 Hz, 2H), 1.62 (q, J = 11.9, 10.7 Hz, 1H), 1.39 (s, 1H); 13C NMR (101 MHz, MeOD) 5 176.85, 174.05, 166.21, 162.02, 152.21, 144.60, 141.91, 108.72, 102.50, 101.60, 90.76, 84.77, 79.50, 75.68, 71.40, 63.63, 60.06, 53.44, 42.50, 31.89, 29.82, 29.14; HRMS (ESI+) mlz calcd for C22H3oN5012 [M + H] 556.1891 , found: 556.1912.
Scheme 2.
A; SjHttais «f sapia-amycisi rL*»iί*^rίrbiί¾ίίί L*¥ί»M¾ίb atMfee»**, CWM#, Jse^PPS ji-iPPi&j, anti ttetntlb^CP a fOM-CPPSJ
Figure imgf000027_0001
The carboxylic acid intermediate 32 for CAP (1) was subjected to peptide coupling reaction with (((trifluormethoxy)phenoxy) piperidin-1-yl)phenyl)methylamine (34), providing the protected CPPB, 37. Saponification of 37 and purification by reverse HPLC yielded CPPB (5) in 95% overall yield from 37. Similarly, l-CPPB (6), OM-CPPB (7), and DM-CPPB (8) were synthesized from the cyanohydrins 18, 33, and 31 in 24-34% overall yield. Capuramycin phenoxypiperidinbenzylamine analogues, CPPA (9) and l-CPPA (10), were synthesized via reductive aminations of the aldehydes 29 and 18 with the amine 34, furnishing the desired products in 63-65% overall yield from 26 and 16 after saponification.
(25.35.45)-2-((1 R)-1 -((2S,5R)-4-Acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1 (2H)-yl)-3- methoxy-tetrahydrofuran-2-yl)-2-amino-2-oxoethoxy)-6-((4-(4-(4-(trifluoromethoxy)- phenyl)-piperidin-1-yl)benzyl)carbamoyl)-3,4-dihydro-2H-pyran-3,4-diyl diacetate (37). To a stirred solution of 32 (55 mg, 0.094 mmol), 34 (0.10 g, 0.28 mmol), HOBt (38 mg, 0.28 mmol) and NMM (95 mI_, 0.94 mmol) in DMF (0.47 mL) was added EDCI (90 mg, 0.47 mmol). After being stirred for 7h at r.t., the reaction mixture was quenched with aq. NaHCCh, and extracted with CHCla/MeOH (9/1). The combined organic extracts were dried over Na2S04 and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHCh/MeOH = 95/5) to afford 37 (76 mg, 87%): Ή NMR (400 MHz, Methanol-c/4) d 7.71 (d, J = 8.1 Hz, 1H), 7.23 (d, J = 8.5 Hz, 2H), 7.19 (d, J = 8.8 Hz, 2H), 7.03 (d, J = 9.1 Hz, 2H), 6.97 (d, J = 8.7 Hz, 2H), 5.97 (t, J = 2.1 Hz, 1H), 5.91 (d, J = 8.1 Hz, 1H), 5.87 (d, J = 3.8 Hz, 1H), 5.74 (dd, J = 4.5, 2.4 Hz, 1H), 5.67 (ddd, J = 4.5, 2.8, 1.7 Hz, 1H), 5.43 (d, J = 2.8 Hz, 1H), 5.28 (dd, J = 5.1 , 3.8 Hz, 1 H), 4.70 (d, J = 1.9 Hz, 1 H), 4.54 (dq, J = 7.5, 3.8 Hz, 1 H), 4.42 (d, J = 14.5 Hz, 1H), 4.38 (dd, J = 6.2, 1.9 Hz, 1H), 4.33 (d, J = 14.4 Hz, 1H), 3.82 - 3.79 (m, 1H), 3.48 (td, J = 9.0, 6.8, 3.5 Hz, 3H), 3.13 - 3.06 (m, 2H), 3.05 (s, 3H), 2.09 (s, 3H), 2.09 (s, 3H), 2.06 (s, 3H), 1.87 (dtd, J = 12.3, 8.2, 3.6 Hz, 3H); 13C NMR (101 MHz, MeOD) d 172.85, 171.84, 171.43, 171.17, 166.07, 162.39, 157.61, 152.11 , 151.92, 144.94, 143.92, 141.57, 130.89, 130.02 (2C), 123.59 (2C), 118.04 (2C), 117.98 (2C), 106.19, 103.75, 98.26, 89.36, 83.17, 78.98, 76.60,
74.71 , 73.98, 64.75, 59.35, 48.18, 48.16, 31.43 (2C), 20.70, 20.52, 20.49; HRMS (ESI+) mlz calcd for C42H47F3N5015 [M + H] 918.3021 , found 918.3049.
(25.35.45)-2-((1R)-2-Amino-1-((2S,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1 (2H)-yl)-4- hydroxy-3-methoxytetrahydrofuran-2-yl)-2-oxoethoxy)-3,4-dihydroxy-N-(4-(4-(4- (trifluoromethoxy)-phenyl)piperidin-1-yl)benzyl)-3,4-dihydro-2H-pyran-6- carboxamide (5). A solution of 37 (76 mg, 0.082 mmol) in a 5:1 mixture of MeOH and EtaN (1.6 mL) was stirred for 6h at r.t., and concentrated in vacuo. The crude mixture was purified by reverse-phase HPLC [column: Luna® (C18, 10 pm, 100 A, 250 x 10 mm), solvents: 65:35 MeOH:H20, flow rate: 3.0 mL/min, UV: 254 nm, retention time: 18 min] to afford CPPB (5, 63 mg, 95%): 1H NMR (400 MHz, Methanol-c/4) d 7.86 (d, J = 8.1 Hz, 1H), 7.20 (dd, J = 10.9,
8.6 Hz, 4H), 7.02 (d, J = 9.1 Hz, 2H), 6.97 (d, J = 8.7 Hz, 2H), 5.98 (dd, J = 3.3, 1.0 Hz, 1H), 5.80 (d, J = 4.4 Hz, 1H), 5.76 (d, J = 8.2 Hz, 1H), 5.21 (d, J = 4.6 Hz, 1H), 4.68 (d, J = 2.0 Hz, 1H), 4.54 (dp, J = 7.3, 3.6 Hz, 1H), 4.48 (dd, J = 5.2, 2.0 Hz, 1H), 4.44 (d, J = 14.5 Hz,
1H), 4.39 (t, J = 3.9 Hz, 1H), 4.33 (d, J = 14.6 Hz, 1H), 4.20 (t, J = 4.7 Hz, 1H), 4.06 - 4.02 (m, 1H), 3.66 (t, J = 5.1 Hz, 1H), 3.52 - 3.45 (m, 3H), 3.19 (s, 3H), 3.08 (ddd, J = 12.3, 8.6, 3.4 Hz, 3H), 2.15 - 2.07 (m, 2H), 1.86 (dtd, J = 12.2, 8.3, 3.5 Hz, 3H); 13C NMR (101 MHz, MeOD) d 173.68, 166.16, 163.31 , 157.62, 152.18, 152.08, 144.15, 141.83, 130.91, 129.76 (2C), 123.58 (2C), 118.03 (4C), 109.58, 102.74, 100.64, 90.83, 83.32, 80.35, 77.51 , 74.27, 74.01 , 67.61 , 63.48, 58.61 , 48.20 (2C), 43.51 , 31.47 (2C); HRMS (ESI+) m/z calcd for C36H41 F 3N5O12 [M + H] 792.2704, found 792.2733.
(2S,3S,4S)-2-((1 R)-1 -((2S,5R)-3-Acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1 (2H)-yl)-4- methoxytetrahydrofuran-2-yl)-2-amino-2-oxoethoxy)-6-((4-(4-(4-(trifluoromethoxy)phenyl)- piperidin-1-yl)benzyl)carbamoyl)-3,4-dihydro-2H-pyran-3,4-diyl diacetate (35). To a stirred solution of 18 (33 mg, 0.056 mmol), 34 (62 mg, 0.17 mmol), HOBt (23 mg, 0.17 mmol) and NMM (62 mI_, 0.56 mmol) in DMF (0.28 mL) was added EDCI (54 mg, 0.28 mmol). After being stirred for 14h at r.t., the reaction was quenched with aq. NaHCCh, and extracted with CHCh/MeOH (9/1). The combined organic extracts were dried over Na2SC>4 and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHCL/MeOH = 95/5) to afford 35 (43 mg, 82%): 1H NMR (400 MHz, Methanol-c/4) d 7.69 (d, J = 8.2 Hz, 1H), 7.22 (d, J = 8.5 Hz, 2H), 7.19 (d, J = 8.6 Hz, 2H), 7.02 (d, J = 9.0 Hz, 2H), 6.98 (d, J = 8.4 Hz, 2H), 5.99 - 5.97 (m, 1H), 5.95 (d, J = 5.8 Hz, 1H), 5.91 (d, J = 8.1 Hz, 1H), 5.74 - 5.71 (m, 1H), 5.66 - 5.63 (m, 1H), 5.46 (d, J= 3.2 Hz, 1H), 5.05 (t, J = 4.7 Hz, 1H), 4.78 (d, J = 2.0 Hz, 1H), 4.57 - 4.52 (m, 1H), 4.49 (d, J= 14.5 Hz, 1 H), 4.39 - 4.34 (m, 1H), 4.30 (d, J = 14.7 Hz, 1H), 3.84 - 3.80 (m, 1H), 3.64 (s, 3H), 3.53 - 3.45 (m, 2H), 3.13 - 3.06 (m, 2H), 2.83 (t, J = 6.5 Hz, 1H), 2.10 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 1.87 (dtd, J = 12.7, 8.5, 3.5 Hz, 3H); 13C NMR (101 MHz, MeOD) d 172.92, 171.74, 171.36, 170.39, 166.18, 162.23, 157.56, 152.06, 151.81 , 145.05, 140.90, 130.78, 129.96 (2C), 123.52 (2C), 117.96 (2C), 117.92 (2C), 105.75, 103.47, 98.22, 88.92, 82.72, 78.21 , 76.44, 73.95, 67.92, 64.42, 61.21 , 58.65, 58.20, 48.10 (2C), 43.57, 31.38 (2C), 20.60, 20.44, 20.39; HRMS (ESI+) mlz calcd for C42H47F3N5O15 [M + H] 918.3021 , found 918.3038.
(2S,3S,4S)-2-((1R)-2-Amino-1-((2S,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1 (2H)-yl)-3- hydroxy-4-methoxytetrahydrofuran-2-yl)-2-oxoethoxy)-3,4-dihydroxy-N-(4-(4-(4- (trifluoro-methoxy)phenyl)-piperidin-1-yl)benzyl)-3,4-dihydro-2H-pyran-6-carboxamide (6). A solution of 35 (43 mg, 0.046 mmol) in a 5:1 mixture of MeOH and EtsN (0.92 mL) was stirred for 6h at r.t., and concentrated in vacuo. The crude mixture was purified by reverse- phase HPLC [column: Luna® (C18, 10 pm, 100 A, 250 x 10 mm), solvents: 65:35 MeOH:H20, flow rate: 3.0 mL/min, UV: 254 nm, retention time: 19 min] to afford l-CPPB (6, 35 mg, 93%):
1H NMR (400 MHz, Methanol-c/4) d 7.79 (d, J = 8.2 Hz, 1H), 7.19 (d, J = 8.6 Hz, 4H), 7.02 (d, J = 9.1 Hz, 2H), 6.98 (d, J = 8.5 Hz, 2H), 5.98 (d, J = 3.6 Hz, 1H), 5.94 (d, J = 5.1 Hz, 1H), 5.77 (d, J = 8.0 Hz, 1H), 5.36 (d, J = 5.8 Hz, 1H), 4.67 - 4.65 (m, 1H), 4.61 (d, J = 2.8 Hz, 1H), 4.48 (dd, J = 8.6, 4.6 Hz, 1H), 4.36 - 4.33 (m, 1H), 4.27 (dd, J = 5.4, 2.7 Hz, 1H), 3.71 - 3.65 (m, 2H), 3.51 (d, J = 4.4 Hz, 2H), 3.48 (s, 3H), 3.25 - 3.18 (m, 2H), 2.13 - 2.00 (m, 3H), 1.92 - 1.76 (m, 3H); 13C NMR (101 MHz, MeOD) d 173.68, 166.15, 163.30, 157.62, 152.18, 152.08, 144.15, 141.83, 130.90, 129.76 (2C), 123.58 (2C), 118.10 (2C), 118.02 (2C), 109.57, 102.73, 100.63, 90.83, 83.32, 80.35, 77.51 , 74.27, 74.01, 67.61 , 63.48, 58.61 , 48.20 (2C), 43.51 ,
31.47 (2C); HRMS (ESI+) m/z calcd for CaeH^FaNsO^ [M + H] 792.2704, found 792.2728.
(2S,3S,4S)-2-((1 R)-2-Amino-1 -((2S,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1 (2H)-yl)-3,4-di- methoxytetrahydrofuran-2-yl)-2-oxoethoxy)-6-((4-(4-(4-(trifluoromethoxy)phenyl)piperidin- 1- yl)benzyl)-carbamoyl)-3,4-dihydro-2H-pyran-3,4-diyl diacetate (38). To a stirred solution of 33 (38 mg, 0.068 mmol), 34 (75 mg, 0.20 mmol), HOBt (28 mg, 0.20 mmol) and NMM (75 mI_, 0.68 mmol) in DMF (0.34 mL) was added EDCI (65 mg, 0.34 mmol). After being stirred for 7h at r.t., the reaction was quenched with aq. NaHCCh, and extracted with CHCla/MeOH (9/1). The combined organic extracts were dried over Na2SC>4 and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHC /MeOH = 95/5) to afford 38 (53 mg, 86%): 1H NMR (400 MHz, Methanol-c/4) d 7.75 (d, J = 8.2 Hz, 1 H), 7.24 (d, J = 8.6 Hz, 2H), 7.19 (d, J = 8.8 Hz, 2H), 7.02 (d, J = 9.1 Hz, 2H), 6.98 (d, J = 8.5 Hz, 2H), 5.98 (d, J = 2.0 Hz, 1H), 5.96 (d, J = 8.1 Hz, 1H), 5.85 (d, J = 2.9 Hz, 1H), 5.71 - 5.66 (m, 2H), 5.43 (d, J = 2.3 Hz, 1H), 4.70 (d, J = 1.9 Hz, 1H), 4.57 - 4.51 (m, 1H), 4.45 - 4.40 (m, 2H), 4.34 (d, J = 14.4 Hz, 1 H), 3.86 (dd, J = 4.9, 2.9 Hz, 1 H), 3.79 (t, J = 6.5 Hz, 1 H), 3.60 (dd, J = 6.9, 4.9 Hz, 1 H), 3.50 (td, J = 7.6, 7.0, 3.3 Hz, 2H), 3.46 (s, 3H), 3.11 (dd, J = 8.9, 3.6 Hz, 1 H), 3.08 (s, 3H), 2.83 (t, J = 6.5 Hz, 1H), 2.14 (dd, J = 8.7, 5.4 Hz, 1H), 2.10 (s, 3H), 2.07 (s, 3H), 1.87 (dtd, J = 12.5, 8.2, 3.5 Hz, 2H); 13C NMR (101 MHz, MeOD) d 172.98, 171.81 , 171.42, 166.22, 162.30, 157.62, 152.12, 151.87, 145.11 , 140.96, 130.85, 130.02 (2C), 123.58 (2C), 118.03 (2C), 117.98 (2C), 105.81, 103.54, 98.28, 88.98, 82.79, 82.54, 78.28, 76.50, 74.02, 64.59, 64.49, 61.28, 58.72, 58.27, 48.17 (2C), 43.64, 42.08, 31.45 (2C), 20.67, 20.51; HRMS (ESI+) mlz calcd for C41H47F3N5O15 [M + H] 906.3021 , found 906.3045.
(2S,3S,4S)-2-((1R)-2-Amino-1-((2S,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4- dimethoxy-tetrahydrofuran-2-yl)-2-oxoethoxy)-3,4-dihydroxy-N-(4-(4-(4- (trifluoromethoxy)-phenyl)piperidin-1-yl)benzyl)-3,4-dihydro-2H-pyran-6-carboxamide (7). A solution of 38 (53 mg, 0.059 mmol) in a 5:1 mixture of MeOH and EtaN (1.2 mL) was stirred for 6h at r.t., and concentrated in vacuo. The crude mixture was purified by reverse- phase HPLC [column: Luna® (C18, 10 pm, 100 A, 250 x 10 mm), solvents: 65:35 MeOH:H20, flow rate: 3.0 mL/min, UV: 254 nm, retention time: 21 min] to afford OM-CPPB (7, 46 mg, 96%): 1H NMR (400 MHz, Methanol-c/4) d 7.89 (d, J = 8.1 Hz, 1H), 7.22 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 8.7 Hz, 2H), 7.02 (d, J = 9.1 Hz, 2H), 6.96 (d, J = 8.5 Hz, 2H), 5.98 (dd, J = 3.0,
1.2 Hz, 1H), 5.85 (d, J = 3.3 Hz, 1H), 5.76 (d, J = 8.1 Hz, 1H), 5.22 (d, J = 4.1 Hz, 1H), 4.67 (d, J = 1.9 Hz, 1H), 4.57 - 4.51 (m, 1H), 4.46 (dd, J = 6.8, 2.0 Hz, 1H), 4.43 (d, J = 14.9 Hz, 1H), 4.40 (d, J = 3.7 Hz, 1H), 4.32 (d, J = 14.5 Hz, 1H), 4.08 (t, J = 4.2 Hz, 1H), 3.90 (t, J = 4.1 Hz, 1H), 3.70 (t, J = 5.5 Hz, 1H), 3.52 - 3.47 (m, 2H), 3.45 (s, 3H), 3.13 (s, 3H), 3.08 (td, J = 9.1 , 4.6 Hz, 2H), 2.15 - 2.09 (m, 2H), 1.91 - 1.82 (m, 2H); 13C NMR (101 MHz, MeOD) d 173.61 , 166.17, 163.21 , 157.62, 152.09, 151.92, 144.02, 141.57, 130.94, 129.89 (2C), 123.59 (2C), 118.03 (2C), 117.98 (2C), 109.78, 102.61 , 100.76, 88.94, 83.23, 82.71, 78.58, 77.02, 74.02, 67.27, 63.42, 60.07, 58.70, 58.37, 48.18, 47.87, 45.76, 43.53, 31.46 (2C); HRMS (ESI+) mlz calcd for CarH^FsNsOia [M + H] 822.2809, found 822.2838.
(2S,3S,4S)-2-((R)-2-Amino-1-((3aR,4S,6R,6aR)-6-(2,4-dioxo-3,4-dihydropyrimidin-1 (2H)- yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-2-oxoethoxy)-6-((4-(4-(4-(trifluoro- methoxy)phenyl)piperidin-1-yl)benzyl)carbamoyl)-3,4-dihydro-2H-pyran-3,4-diyl diacetate (36). To a stirred solution of 31 (27 mg, 0.047 mmol), 34 (52 mg, 0.14 mmol), HOBt (19 mg, 0.14 mmol) and NMM (52 m|_, 0.47 mmol) in DMF (0.24 mL) was added EDCI (45 mg, 0.24 mmol). After being stirred for 11 h at r.t., the reaction was quenched with aq. NaHCCh, and extracted with CHCk/MeOH (9/1). The combined organic extracts were dried over Na2SC>4 and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHCh/MeOH = 95/5) to afford 36 (37 mg, 84%): 1H NMR (400 MHz, Methanol-c/4) d 7.69 (d, J = 8.1 Hz, 1H), 7.20 (t, J = 9.5 Hz, 4H), 7.02 (d, J = 9.2 Hz, 2H), 6.98 (d, J = 8.5 Hz, 2H), 5.99 (t, J = 2.0 Hz, 1H), 5.87 (d, J = 3.0 Hz, 1H), 5.83 (d, J = 8.1 Hz, 1H), 5.66 (dd, J = 4.4, 2.5 Hz, 1H), 5.57 - 5.54 (m, 1H), 5.41 (d, J= 3.0 Hz, 1H), 4.78 (d, J = 2.3 Hz, 1H), 4.70 (dd, J = 6.3, 3.1 Hz,
1 H), 4.63 (dd, J = 6.4, 3.5 Hz, 1 H), 4.57 - 4.52 (m, 1 H), 4.39 (s, 2H), 3.79 (t, J = 6.5 Hz, 1 H), 3.64 (s, 1H), 3.49 (ddd, J = 10.9, 6.4, 3.6 Hz, 2H), 3.09 (ddd, J = 12.4, 8.6, 3.4 Hz, 2H), 2.83 (t, J = 6.5 Hz, 1H), 2.16 - 2.09 (m, 2H), 2.08 (s, 3H), 2.05 (s, 3H), 1.87 (dtd, J = 12.1 , 8.1 , 3.5 Hz, 2H), 1.53 (s, 3H), 1.21 (s, 3H); 13C NMR (101 MHz, MeOD) d 172.59, 171.76, 171.36, 166.16, 162.38, 157.61, 152.04, 151.92, 145.03, 142.46, 130.80, 129.66 (2C), 123.59 (2C), 118.05 (2C), 118.02 (2C), 115.67, 106.20, 103.33, 98.57, 92.78, 86.55, 85.61 , 81.72, 77.88, 73.99, 64.84, 64.58, 61.28, 48.20 (2C), 43.59, 31.47 (2C), 27.52, 25.45, 20.66, 20.51 ; HRMS (ESI+) mlz calcd for C42H47F3N5014 [M + H] 902.3072, found 902.3098.
(2S,3S,4S)-2-((1 R)-2-Amino-1 -((2S,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1 (2H)-yl)-3,4-di- hydroxy-tetrahydrofuran-2-yl)-2-oxoethoxy)-3,4-dihydroxy-N-(4-(4-(4-(trifluoromethoxy)- phenyl)-piperidin-1-yl)benzyl)-3,4-dihydro-2H-pyran-6-carboxamide (8). A solution of 35 (37 mg, 0.040 mmol) in a 4:1 mixture of TFA and H2O (0.80 mL) was stirred for 4h at r.t., and concentrated in vacuo. A solution of the crude alcohol in a 5:1 mixture of MeOH and EtsN (0.80 mL) was stirred for 10h at r.t., and concentrated in vacuo. The crude mixture was purified by reverse-phase HPLC [column: Luna® (C18, 10 pm, 100 A, 250 x 10 mm), solvents: 35:65 MeOhbhhO, flow rate: 3.0 mL/min, UV: 254 nm, retention time: 15 min] to afford DM-CPPB (8, 29 mg, 91%): 1H NMR (400 MHz, Methanol-c/4) d 7.92 (d, J = 8.1 Hz, 1H), 7.20 (d, J = 3.1 Hz, 2H), 7.18 (d, J = 3.5 Hz, 2H), 7.02 (d, J = 9.1 Hz, 2H), 6.96 (d, J = 8.6 Hz, 2H), 6.01 (d, J = 4.4 Hz, 1H), 5.80 (d, J = 3.0 Hz, 1H), 5.73 (d, J = 8.1 Hz, 1H), 5.14 (d, J = 6.7 Hz, 1H), 4.66 (d, J = 1.8 Hz, 1H), 4.57 - 4.51 (m, 1H), 4.50 (dd, J = 6.7, 1.7 Hz, 1H), 4.41 - 4.31 (m, 3H), 4.16 (dd, J = 6.6, 5.0 Hz, 1 H), 4.04 (dd, J = 4.9, 3.0 Hz, 1 H), 3.88 (dd, J = 6.7, 4.5 Hz, 1 H), 3.47 (t, J = 6.2 Hz, 2H), 3.27 (d, J = 7.2 Hz, 1H), 3.07 (ddd, J = 17.0, 8.1 , 4.0 Hz, 2H), 2.37 - 2.29 (m, 1H), 2.16 - 2.07 (m, 2H), 1.86 (dtd, J = 12.3, 8.2, 3.5 Hz, 2H); 13C NMR (101 MHz, MeOD) d 174.07, 170.14, 166.22, 163.14, 157.62, 152.12, 151.99, 144.89, 141.84, 131.07, 129.64 (2C), 123.58 (2C), 118.08 (2C), 118.03 (2C), 116.78, 108.52, 102.45, 101.32, 91.15, 84.20, 78.81 , 75.49, 74.02, 71.19, 69.27, 63.69, 60.09, 47.81 (2C), 31.46 (2C); HRMS (ESI+) mlz calcd for C35H39F3N5O12 [M + H] 778.2547, found 778.2573.
(2R)-2-(((2S,3S,4S)-3,4-Dihydroxy-6-(((4-(4-(4-(trifluoromethoxy)phenyl)piperidin-1- yl)benzyl)-amino)-methyl)-3,4-dihydro-2H-pyran-2-yl)oxy)-2-((2S,5R)-5-(2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)acetamide (9). To a stirred solution of 16 (5.8 mg, 0.0092 mmol) and DMSO (0.065 mL, 0.92 mmol) in a 5:1 mixture of CH2CI2 and EtsN (0.5 mL) was added SCVpyridine (15 mg, 0.092 mmol). After being stirred for 2h at r.t., the reaction mixture was added H2O (0.1 mL) and passed through a silica gel pad (CHCh/MeOH = 93/7) to afford the crude 17: this was used without purification. To a stirred solution of the crude 17 and 34 (17 mg, 0.046 mmol) in CH3CN (0.5 mL) was added NaB(CN)H3 (5.8 mg, 0.092 mol). After being stirred for 3h at r.t., the reaction was quenched with aq. NaHCCh, and extracted with CHC /MeOH (9/1). The combined organic extracts were dried over Na2SC>4 and concentrated in vacuo. The crude product was passed through a silica gel pad (CHCh/MeOH = 9/1). The solution of the crude product in a 5:1 mixture of MeOH and EtsN (0.5 mL) was stirred for 8h at r.t., and concentrated in vacuo. The crude mixture was purified by reverse-phase HPLC [column: Luna® (C18, 10 pm, 100 A, 250 x 10 mm), solvents: 65:35 MeOH:H20, flow rate: 3.0 mL/min, UV: 254 nm, retention time: 15 min] to afford CPPA (9, 4.6 mg, 65% for 3 steps): 1H NMR (400 MHz, Methanol-c/4) d 8.02 (d, J = 8.1 Hz, 1H), 7.24 (d, J = 8.5 Hz, 2H), 7.19 (d, J = 8.5 Hz, 2H), 7.03 (d, J = 8.9 Hz, 2H), 6.99 (d, J = 8.5 Hz, 2H), 5.90 (d, J = 5.4 Hz, 1H), 5.76 (d, J = 7.8 Hz, 1H), 5.73 (d, J = 8.1 Hz, 1H), 5.06 (d, J = 6.6 Hz,
1 H), 4.57 (dd, J = 6.4, 3.0 Hz, 1 H), 4.52 (td, J = 4.4, 1.7 Hz, 1 H), 4.33 (t, J = 5.2 Hz, 1 H), 4.26 (d, J = 26.3 Hz, 1H), 4.18 (d, J = 1.6 Hz, 1H), 3.90 - 3.77 (m, 3H), 3.55 - 3.50 (m, 2H), 3.49 (s, 3H), 3.15 - 3.06 (m, 3H), 2.16 - 2.07 (m, 3H), 1.86 (m, J = 8.8, 4.3 Hz, 3H); 13C NMR (101 MHz, MeOD) 5 174.07, 165.86, 163.68, 157.58, 151.26, 144.88, 142.07, 131.25, 129.37 (2C), 123.22 (2C), 118.03 (4C), 109.57, 102.60, 100.40, 90.92, 83.37, 80.46, 77.56, 74.63, 73.77, 73.15, 67.78, 63.30, 58.33, 48.19 (2C), 43.21 , 30.74 (2C); HRMS (ESI+) mlz calcd for C36H43N3F5O12 [M + H] 794.2860, found 794.2877.
(2R)-2-(((2S,3S,4S)-3,4-dihydroxy-6-(((4-(4-(4-(trifluoromethoxy)phenyl)piperidin-1- yl)-benzyl)amino)methyl)-3,4-dihydro-2H-pyran-2-yl)oxy)-2-((2S,5R)-5-(2,4-dioxo-3,4- dihydro-pyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)acetamide (10).
To a stirred solution of 26 (4.6 mg, 0.0073 mmol) and DMSO (0.052 mL, 0.73 mmol) in a 5:1 mixture of CH2CI2 and EtsN (0.5 mL) was added S03*pyridine (12 mg, 0.073 mmol). After being stirred for 2h at r.t., the reaction mixture was added H2O (0.1 mL) and passed through a silica gel pad (CHCh/MeOH = 93/7) to afford the crude 29. To a stirred solution of the crude 29 and 34 (13 mg, 0.036 mmol) in CH3CN (0.5 mL) was added NaB(CN)H3 (4.6 mg, 0.073 mol). After being stirred for 3h at r.t., the reaction was quenched with aq. NaHCCh, and extracted with CHCh/MeOH (9/1). The combined organic extracts were dried over Na2S04 and concentrated in vacuo. The crude product was passed through a silica gel pad (CHCh/MeOH = 9/1). A solution of the crude product in a 5:1 mixture of MeOH and EtsN (0.5 mL) was stirred for 8h at r.t., and concentrated in vacuo. The crude mixture was purified by reverse-phase HPLC [column: Luna® (C18, 10 pm, 100 A, 250 x 10 mm), solvents: 65:35 MeOH:H20, flow rate: 3.0 mL/min, UV: 254 nm, retention time: 15 min] to afford l-CPPA (10, 3.6 mg, 63% for 3 steps): 1H NMR (400 MHz, Methanol-c/4) d 8.04 (d, J = 8.1 Hz, 1 H), 7.23 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 8.7 Hz, 2H), 7.03 (d, J = 9.1 Hz, 2H), 6.98 (d, J = 8.5 Hz,
2H), 5.94 (d, J = 3.6 Hz, 1H), 5.74 (d, J = 8.1 Hz, 1H), 5.72 (d, J = 8.1 Hz, 1H), 5.06 (s, 1H), 4.59 - 4.52 (m, 2H), 4.43 - 4.38 (m , 1 H), 4.28 (t, J = 5.4 Hz, 1 H), 4.25 (t, J = 4.4 Hz, 1 H),
4.21 (d, J = 1.5 Hz, 1H), 4.14 (dd, J = 8.3, 4.9 Hz, 1 H), 3.88 - 3.85 (m, 2H), 3.82 (d, J = 8.2 Hz, 2H), 3.53 (s, 3H), 3.13 (ddd, J = 12.3, 8.7, 3.3 Hz, 2H), 2.11 (d, J = 9.7 Hz, 3H), 1.87 (ddt, J = 13.3, 8.9, 4.1 Hz, 3H); 13C NMR (101 MHz, MeOD) d 173.92, 165.98, 162.46,
157.87, 151.93, 143.90, 140.73, 130.53, 129.43 (2C), 123.29 (2C), 118.10 (2C), 117.87 (2C), 109.98, 103.08, 100.53, 91.27, 83.47, 80.53, 78.03, 74.70, 73.54, 67.84, 63.75,
58.42, 57.46, 48.20, 43.48 (2C), 31.49 (2C); HRMS (ESI+) mlz calcd for C36H43N3F5O12 [M + H] 794.2860, found 794.2885. Scheme 3.
Figure imgf000034_0001
Previously, a natural product A-5003659E was used to develop novel capuramycin analogues with strong MraY inhibitory activity (Hotoda et. al. 2003). Its free-carboxylic acid analogue, A-500359F was also isolated from the capuramycin-producing strain, Streptomyces griseus Sank 60196. Although, the currently available synthetic schemes for capuramycin analogues (e.g., Scheme 2) include a relatively short number of chemical steps, a semi-synthetic approach is more feasible to deliver large quantities of CPPB for pharmacological studies. To establish semi-synthesis of CPPB, A-500359F was first synthesized from the CAP-synthetic intermediate 32 (Scheme 2) in a single step. Amideforming reaction of synthetic A-500359F with (((trifluormethoxy)-phenoxy) piperidin-1- yl)phenyl)methylamine (34) was performed under an optimized condition using EDCI, glyceroacetonide-Oxyma, and NMM in DMF. All coupling reagents could be removed by partitions between CHCI3 and water and evaporation. The crude product was passed through DOWEX 50Wx4 column (MeOH:NH4OH = 4:1) to provide CPPB with >95% purity, which was further purified by C18-reverse HPLC (MeOFkhhO = 65:35) to yield pure CPPB (Scheme 3).
(2S,3S,4S)-2-((1R)-2-amino-1-((2S,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4- hydroxy-3-methoxytetrahydrofuran-2-yl)-2-oxoethoxy)-3,4-dihydroxy-3,4-dihydro-2H- pyran-6-carboxylic acid (A-500359F). A solution of 32 (18 mg, 29 mΐtioI) in a 5:1 mixture of MeOH and EtaN (0.5 mL) was stirred for 5h at r.t., and concentrated in vacuo. The crude mixture was purified by reverse-phase HPLC [column: Luna® (C18, 10 pm, 100 A, 250 x 10 mm), solvents: 15:85 MeOH:H20, flow rate: 3.0 mL/min, UV: 254 nm, retention time: 14 min] to afford A-500359F (12 mg, 96%): 1H NMR (400 MHz, Deuterium Oxide) d 7.75 (d, J = 8.1 Hz, 1H), 5.88 (d, J = 8.1 Hz, 1H), 5.80 (dd, J = 2.5, 1.6 Hz, 1H), 5.78 (d, J = 4.0 Hz, 1H), 5.25 (d, J = 3.2 Hz, 1H), 4.71 (d, J = 2.1 Hz, 1H), 4.48 (dd, J = 5.7, 2.1 Hz, 1H), 4.43 (dd, J = 4.5, 2.5 Hz, 1H), 4.30 (dd, J = 5.1 , 4.0 Hz, 1H), 4.12 (ddd, J = 4.8, 3.3, 1.7 Hz, 1H), 3.73 (t, J = 5.4 Hz, 1H), 3.35 (s, 3H); 13C NMR (101 MHz, D20) d 173.32, 168.26, 166.06, 151.29, 144.11 , 140.79, 108.44, 101.93, 98.62, 89.42, 81.99, 78.20, 75.00, 72.10, 64.43, 62.19, 57.93; HRMS (ESI+) mlz calcd for C17H22N3012 [M + H] 460.1204, found 460.1215.
Synthesis of CPPB (5). To a stirred solution of A-500359F (11 mg, 0.024 mmol), 34 (26 mg, 0.072 mmol), Glyceroacetonide-Oxyma (16 mg, 0.072 mmol) and NMM (26 mL, 0.24 mmol) in DMF (0.49 mL) was added EDCI (23 mg, 0.12 mmol). After being stirred for 5h at r.t., the reaction mixture was filtered, and diluted with water. The product was extracted with CHCla/MeOH (9/1). The combined extracts were derived over Na SC> , and concentrated in vacuo. The crude mixture was purified by DOWEX 50Wx4 (MeOH : NFLOH = 4 : 1) followed by reverse-phase HPLC [column: Luna (C18, 10 mm, 100 A, 250 x 10 mm), solvents: 65:35 MeOH : H O, flow rate: 3.0 mL/min, UV: 254 nm, retention time: 18 min] to afford 5 (17 mg, 92%). All physical data were identical to that of CPPB synthesized in Scheme 2.
((2R,4R,5R,6S)-6-(((2R,3S,4S,5S,6S)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-3,4-dihydroxy-5-(3-(4-(4-(4- (trifluoromethoxy)phenoxy)piperidin-1-yl)benzyl)ureido)tetrahydro-2H-pyran-2-yl)methyl ((1R)-2-amino-1-((2R,3R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4- dihydroxytetrahydrofuran-2-yl)-2-oxoethyl)carbamate (Compound A)
1H NMR (400 MHz, Methanol-c/4) d 7.60 (d, J = 6.4 Hz, 1H), 7.23 (d, J = 8.1 Hz, 2H), 7.19 (d, J = 8.8 Hz, 2H), 7.02 (d, J = 9.5 Hz, 4H), 5.80 (d, J = 3.9 Hz, 1 H), 5.69 (d, J = 8.0 Hz, 1 H), 5.36 (d, J = 6.2 Hz, 1H), 4.93 (d, J = 4.7 Hz, 1H), 4.84 (d, J = 8.1 Hz, 1H), 4.60 (d, J = 11.2 Hz, 1H), 4.53 (dt, J = 7.8, 4.0 Hz, 1 H), 4.49 - 4.07 (m, 4H), 3.91 - 3.89 (m, 1 H), 3.88 - 3.85 (m, 1 H), 3.83 - 3.79 (m, 2H), 3.77 - 3.70 (m, 2H), 3.66 - 3.46 (m, 8H), 3.25 (t, J = 7.3 Hz, 1H), 3.03 (ddd, J = 12.1, 8.7, 3.2 Hz, 2H), 2.17 - 2.08 (m, 2H), 1 .89 (s, 3H), 1.92 - 1.83 (m, 2H).
13C NMR (101 MHz, MeOD) d 176.7, 175.6, 170.7, 157.6, 155.9, 154.1 , 153.0, 152.0, 143.9, 142.1, 131.3, 129.6 (2C), 123.6 (2C), 118.1 (2C), 118.1 (2C), 105.1 , 103.8, 102.7, 101.3, 91.7, 84.9, 80.2, 76.7, 75.4, 74.0, 73.2, 72.5, 71.3, 69.4, 66.0, 65.0, 62.2, 60.5, 54.5, 45.5 (2C), 43.7, 31.5 (2C), 27.3.
((2R,4R,5R,6S)-6-(((2R,3S,4S,5S,6S)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-3,4-dihydroxy-5-(3-(4-(4-(4- (trifluoromethoxy)phenoxy)piperidin-1-yl)benzyl)ureido)tetrahydro-2H-pyran-2-yl)methyl ((1S)-2-amino-1-((2R,3R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4- dihydroxytetrahydrofuran-2-yl)-2-oxoethyl)carbamate (Compound B)
1H NMR (400 MHz, Methanol-d,) d 7.61 (d, J = 7.3 Hz, 1H), 7.23 (d, J = 8.1 Hz, 2H), 7.19 (d, J = 8.8 Hz, 2H), 7.02 (d, J = 9.5 Hz, 4H), 5.81 (d, J = 4.3 Hz, 1H), 5.74 (d, J = 8.0 Hz, 1H), 5.42 (d, J = 3.6 Hz, 1H), 4.94 (d, J = 5.1 Hz, 1H), 4.80 (d, J = 8.1 Hz, 1H), 4.57 (d, J = 11.3 Hz, 1H), 4.53 (dt, J = 7.8, 4.0 Hz, 1 H), 4.49 - 4.07 (m, 4H), 3.91 - 3.89 (m, 1 H), 3.88 - 3.85 (m, 1 H), 3.83 - 3.79 (m, 2H), 3.77 - 3.70 (m, 2H), 3.66 - 3.46 (m, 8H), 3.25 (t, J = 7.3 Hz, 1H), 3.03 (ddd, J = 12.1, 8.7, 3.2 Hz, 2H), 2.17 - 2.08 (m, 2H), 1 .97 (s, 3H), 1.92 - 1.83 (m, 2H).
13C NMR (101 MHz, MeOD) d 177.3, 175.6, 170.7, 157.6, 156.7, 154.1 , 153.0, 152.0, 143.9, 142.1, 131.3, 129.6 (2C), 123.6 (2C), 118.1 (2C), 118.1 (2C), 105.1 , 103.8, 102.7, 101.3, 91.7, 84.9, 80.2, 77.5, 75.4, 74.0, 73.2, 72.5, 71.3, 70.6, 66.0, 65.0, 62.2, 60.5, 56.8, 45.5 (2C), 43.7, 31.5 (2C), 27.3.
Example 2: Enzymatic Inhibition
CAP (1) and its three analogues, OM-CAP (2), l-CAP (3), and DM-CAP (4) were evaluated in enzyme inhibitory assays against bacterial phosphotransferases, MraY and WecA, and archaeal and human dolichyl-phosphate GlcNAc-1 -phosphotransferases, AgIH and DPAGT 1. CAP is a selective MraY inhibitor that does not display inhibitory activity against WecA, AgIH, and DPAGT1 (Table 1). Previously, OM-CAP (2) was identified as a selective WecA inhibitor (IC500.060 mM) that does not possess MraY inhibitory activity (IC50 >50 mM (entry 2 in Table 1). OM-CAP (2) was shown to have inhibitory activity against AgIH and DPAGT1 with the IC50 values of 13.5 and 3.5 mM, respectively (entry 2). I-CAP (3) showed enzyme inhibitory activity against MraY, WecA, AgIH, and DPAGT1 with the IC50 between 7.5-20 mM concentrations (entry 3). On the contrary, DM-CAP (4) showed only a weak WecA inhibitory activity (IC5035.0 mM) (entry 4).
MraY, WecA, AgIH, and DPAGT1 enzyme inhibitory activity assays for the six (((trifluormethoxy)phenoxy) piperidin-1-yl)phenyl)methylamine analogues revealed that CPPB (5) and l-CPPB (6) are high nM range DPAGT1 inhibitors (entries 5 and 6 in Table 1), however, the O-methylation and demethylation analogues (OM-CPPB (7) and DM-CPPB (8)) turned out to be very low- or no-DPAGT1 inhibitor (entries 7 and 8). The secondary amine analogues, CPPA (9) and l-CPPA (10), did not inhibit all phosphotransferases tested in Table 1 at 50 mM concentration (entries 9 and 10). CPPB was determined to be three times stronger DAPGT1 inhibitor (IC50 O.2O mM) than l-CPPB (entry 5 vs. 6). I-CPPB did not display MraY inhibitory activity, but showed a very weak WecA and AgIH enzyme inhibitory activity (entry 6). Interestingly, difference in these phosphotransferase inhibitory profiles between CPPB and l-CPPB correlate with their antimycobacterial activity: CPPB possessing MraY/WecA inhibitory activity killed Myocobacterium tuberculosis H37Rv, Mycobacterium avium 2285, Mycobacterium smegmatis (ATCC607) with the MIC values 6.25-12.5 pg/mL.
In contrast, l-CPPB, which does not have MraY inhibitory activity, did not show growth inhibitory activity against these Mycobacterium spp. at 50 pg/mL. Table 1.
Figure imgf000037_0001
Expression and purification of HyMraY. The gene mraY of Hydrogenivirga spp. 128-5- R1-1 was cloned with a /V-terminal His6 tag into a pET22b vector. The plasmid was transformed and expressed in E. coli NiCo21 (DE3) pLEMO competent cells. The proteins were purified using a nickel, cation exchange, and size exclusion chromatography. The final storage buffer was 20 mM HEPES pH 7.5, 100 mM NaCI, 10% glycerol, 5 mM 3ME, 0.15% decyl 3-D-maltopyranoside (DM).
Expression and purification of MjAglH. The gene mj1113 of Methanocaldococcus jannaschii DSM 2661 was cloned with a /V-terminal His9 tag into a pET33b-derived vector. The plasmid was transformed and expressed in E. coli NiCo21(DE3) pLEMO competent cells. The proteins were purified using cobalt and size exclusion chromatography. The final storage buffer was 20 mM Tris-HCI, pH 7.5, 100 mM NaCI, 5% glycerol, 5 mM 3ME, 0.15% DM.
Preparation of membrane fraction P-60 containing WecA. E. coli B21 wecA strain were harvested by centrifugation (4,700 rpm) at 4 °C followed by washing with 0.9% saline solution (thrice). The washed cell pellets were washed with homogenization buffer (containing 50 mM K2HP04, 5 mM MgCI2, 5 mM 1 ,4-dithio-DL-threitol, and 10% glycerol, pH = 7.2) (thrice), and approximately 5 g of pellet (wet weight) was collected. The washed cell pellets were suspended in homogenization buffer and disrupted by probe sonication on ice (5s on and 2s off for 1 min, then cool down for 1.5min., 5 cycles, cool down for 15 min, and then, 5s on and 2s off for 1min., cool down for 1.5min, 5 cycles). The resulting suspension was centrifuged at 4,700 x g for 15min. at 4 °C to remove unbroken cells. The lysate was centrifuged at 25,000 x g for 20min. at 4 °C. The supernatant was subjected to ultracentrifugation at 60,000 x g for 1 h at 4 °C. The supernatant was discarded, and the membrane fraction containing WecA enzyme (P-60) was suspended in the Tris-HCI buffer (pH = 7.5). Total protein concentrations were approximately 8 to 10 mg/ml_. Aliquots were stored in Eppendorf tubes at -80 °C.
DPAGT1 expression and purification. DPAGT1 was expressed in suspended Expi293 cells for 36h. The cells were lysed by drawing through a 26g needle (10 times) and membrane protein was extracted using buffer containing 1% decyl f3-D-maltopyranoside detergent. DPAGT1 was purified using HA-agarose resin and a superdex 200 size exclusion column.
MraY assay. MraY assay substrates, Park's nucleotide-N£-C6-dansylthiourea, neryl phosphate, were chemically synthesized according to the reported procedures.7 Park's nucleotide-N£-C6-dansylthiourea (2 mM stock solution, 1.88 jiL), MgCh (0.5 M, 5 jiL), KCI (2 M, 5 jiL), Triton X-100 (0.5%, 5.63 jiL), Tris buffer (pH 8.0, 50 mM), neryl phosphate (0.1 M, 2.25 jiL), and inhibitor molecule (0 - 50 .ig/mL in Tris buffer) were placed in a 1.5 mL Eppendorf tube. To a reaction mixture, P-60 (1 OjiL) was added (total volume of reaction mixture: 50 jiL adjust with Tris buffer). The reaction mixture was incubated for 2h at r.t. (26 °C) and quenched with CHCI3 (100 jiL). Two phases were mixed via vortex and centrifuged at 25,000 xg for 10 min. The aqueous phase was assayed via reverse-phase HPLC. The water phase (10 jiL) was injected into HPLC (solvent: CH3CN/O.O5 M aq. NH4HCO3 =
25:75; UV: 350 nm; flow rate: 0.5 mL/min; column: Kinetex 5 mM C8, 100 A, 150 x 4.60 mm), and the area of the peak for lipid l-neryl derivative was quantified to obtain the IC50 value. The IC50 values were calculated from plots of the percentage product inhibition versus the inhibitor.
WecA assay. WecA assay substrate, UDP-Glucosamine-C6-FITC was chemically synthesized according to the reported procedures.9'11·58 UDP-Glucosamine-C6-FITC (UDP- GlcN-C6-FITC, 2 mM stock solution, 0.56 mI_), MgCh (0.5 M, 4 mI_), b-mercaptoethanol (50 mM, 5 pL), CHAPS (5%, 11.3 mI_), Tris buffer (pH 8.0, 50 mM), undecaprenyl phosphate (4 mM, 1.4 mI_), and inhibitor molecule (0 - 50 pg/mL in Tris buffer) were place in a 1.5 mI_ Eppendorf tube. To a reaction mixture, P-60 (10 mI_) was added (total volume of reaction mixture: 50 mI_ adjust with Tris buffer). The reaction mixture was incubated for 2h at 37 °C and quenched with n-butanol (150 mI_). Two phases were mixed via vortex and centrifuged at 10,000 xg for 3 min. The upper organic phase was assayed via reverse-phase HPLC. The organic phase (30 mI_) was injected into HPLC (solvent: gradient elution of 85:15 to 95:5 MeOH/0.05 M aq. NH4HCO3; UV: 485 nm; flow rate: 0.5 mL/ min; column: Kinetex 5 pm C8, 100 A, 150 x 4.60 mm), and the area of the peak for Css-P-P-GIcN-Ce-FITC was quantified to obtain the IC50 value. The IC50 values were calculated from plots of the percentage product inhibition versus the inhibitor concentration.
AgIH assay. AgIH assays were performed as the procedure described for WecA assays, but used MyAglH and a-dihydroundecaprenyl phosphate (Css-dolichyl phosphate) instead of WecA and undecaprenyl phosphate. UDP-GlcN-C6-FITC (2 mM stock solution, 0.56 pL), MgCh (0.5 M, 4 pL), b-mercaptoethanol (50 mM, 5 pL), CHAPS (5%, 11.3 pL), Tris buffer (pH 8.0, 50 mM), Css-dolichyl phosphate (4 mM, 1.4 pL), and inhibitor molecule (0 - 50 pg/mL in Tris buffer) were place in a 1.5 pL Eppendorf tube. To a reaction mixture, AgIH solution (10 pL) was added (total volume of reaction mixture: 50 pL adjust with Tris buffer). The reaction mixture was incubated for 2 h at 37 °C and quenched with n-butanol (150 pL). Two phases were mixed via vortex and centrifuged at 10,000 xg for 3 min. The upper organic phase was assayed via reverse-phase HPLC. The organic phase (30 pL) was injected into HPLC (solvent: gradient elution of 85:15 to 95:5 MeOH/0.05 M aq. NH4HCO3; UV: 485 nm; flow rate: 0.5 mL/ min; column: Kinetex 5 pm C8, 100 A, 150 x 4.60 mm), and the area of the peak for Css-P-P-GIcN-Cs-FITC was quantified to obtain the IC50 value. The IC50 values were calculated from plots of the percentage product inhibition versus the inhibitor concentration.
DPAGT1 assay. DPAGT1 assays were performed as the procedure described for AgIH assays, but used DPAGT1.
Example 3: Cytotoxicity of new capuramycin analogues, CPPB (5) and l-CPPB ( 6 )
In the capuramycin analogue series, the degree of MraY inhibitory activity correlates with their antimycobaterial activity. Antimycobacterial capuramycin analogues display low in vitro cytotoxicity against mammalian cells, and have been recognized as safe drug leads that have acceptable tolerability in animal models. The toxicity of tunicamycin has been studied extensively in vitro : tunicamycin inhibits growth in many cancer cell lines without selectivity, and has a narrow therapeutic window demonstrated in in vivo studies using mice. The toxicity of tunicamycin is believed to be attributable to its ability to inhibit DPAGT1 enzyme function. Tunicamycin's toxicity, however, cannot be explained solely by its inhibition of DPAGT1.
Capuramycin-based DPAGT1 inhibitors, CPPB (5) and l-CPPB (6) inhibited DPAGT1 enzyme with the IC50 values of 0.2 and 0.6 mM, respectively. Unlike the MraY- antimycobacterial activity relationship observed for CAP analogues, the DPAGT1 inhibitors, CPPB and l-CPPB, did not show antiproliferative activity against L1210 (a leukemia cell), HPNE (a normal pancreatic ductal cell), and Vero (a normal kidney cell) at 50 mM. They showed various levels of growth inhibitory activity several against solid cancer cell lines such as KB (HeLa, a cervix carcinoma), SiHa (a cervical squamous cell carcinoma), HCT-116 (a colorectal adenocarcinoma), DLP-1 (a colorectal adenocarcinoma), Capan-2 (a pancreatic ductal adenocarcinoma), PANC-1 (a pancreatic ductal carcinoma), AsPC-1 (a pancreatic adenocarcinoma), PD002 (a patient-derived pancreatic adenocarcinoma) in MTT assays (IC50 15-40 mM, Table 2). A lower DPAGT1 inhibitor, tunicamycin (11), showed growth inhibition of all cell lines in Table 2 with the IC50 values of 0.78-7.5 mM concentrations (entry 5 in Table 2). Cellular behavior and morphological changes of PD002 treated with CPPB were monitored over time via IncuCyte® live cell analysis imaging system (Figure 2A).
Interestingly, 10-13% of phase area confluent of PD002 culture (time Oh) remained the same after 72h for the CPPB-treated cells (50 mM), whereas, ca. 70% of confluence was reached for the control PD002 culture (PBS) (Figures 2B and 2C). Although morphological changes were subtle over time (0-72h), cell viability assessed by the MTT reduction assay revealed that PD002 cells treated with CPPB (50 mM) was significantly decreased (Table 2). Exposure of CPPB (0.2-20 mM) inhibited cell proliferation of PD002: ca. 20% of cell proliferation was inhibited at time 72h. These results indicate that DPAGT1 inhibitors may have cytostatic effect again certain cancerous tumors that require DPAGT1 overexpression for their growth.
Table 2.
Figure imgf000040_0001
Figure imgf000041_0001
Cell lines. The human primary PDAC cell lines PANC-1 (ATCC CRL 1469); Capan-2 (ATCC HTB-80) and metastatic PDAC cell lines Capan-1 (ATCC HTB-79), AsPC-1 (ATCC CRL-1682), L1210 (ATCC CCL-219), KB (ATCC CCL-17), HeLa, SiHa (ATCC HTB-35), HCT-116 (ATCC CCL-247), DLD-1 (ATCC CCL221), hTERT-HPNE (ATCC CRL-4023), Vero (ATCC CCL-81 ) were purchased from the ATCC. The cell lines were cultured and maintained in the media as recommended by the supplier. The cell lines were regularly tested for mycoplasma contamination and authenticated. In addition to ATCC cell lines, Dr. Glazer's lab generated patient-derived PDAC cell line, PD002. PD002 Cells were maintained and cultured in DMEM medium supplemented with 20% FBS and 100 lll/mL penicillin and incubated at 37°C with 5% CO2.
MTT cytotoxicity assay. To study the effect of treatment with DPAGT1 inhibitors on the growth and proliferation of cells, a fixed number of cells (5 x 104 cells/well, 196 mI_) were plated in a 96 well plate. Into each well 5 mI_ of drug concentration was added. After 72 h of incubation with drugs at 37 °C, 5% CO2, 10 mI_ of MTT solution (5 mg/ml_ in PBS) was added and incubated another 3 h at 37 °C, 5% CO2. The medium was removed, and DMSO (200 pL/well) was added. Viability was assessed on the basis of cellular conversion of MTT into a purple formazan product. The absorbance of the colored formazan product was measured at 570 nm by BioTek Synergy HT Spectrophotometer.
Kinetic Proliferation Assay. To study the effect of treatment with DPAGT1 inhibitors on the growth and proliferation of cells, a fixed number of cells (2 x 104) were plated in multiple wells of a 96 well plate, incubated for 24 h to let cells settle down. Then, CPPB (5) was added (0-50 mM). Images were obtained every 4 h using an IncuCyte Live-Cell Imaging System (Essen BioScience, Ann Arbor, Ml). After 72 h, cell proliferation was quantified using the metric phase object confluence (POC), a measurement of the area of the field of view that is covered by cells, which is calculated by the integrated software.
Scratch Assay. A confluent monolayer was formed in 24-well plates. The monolayer was scratched by a sterile 200 pL pipette tip and washed with PBS to remove cell debris. Complete medium with CPPB (5) (0, 0.05, 0.1 , 0.2 mM) were added and scratched areas were photographed with microscope. The scratched cells were incubated at 37 °C, 5%
CO2. After 24 h, medium was removed and cells were stained with a 1 :1 mixture of crystal violet and PBS for 5 min., washed with PBS twice, and photographed with microscope. Wound areas were measured and recovered areas were calculated. Example 4: Cell migratory inhibition by CPPB (5)
DPAGT1 catalyzes the first step in A/-glycan biosynthesis of mammalian cells (Figure 1 ). Aberrant A/-glycosylation is common in many solid cancers and important for the epithelial to mesenchymal transition program (EMT, a mechanism of metastases). High levels of DPAGT1 protein expression were found in a series of pancreatic cancers (e.g., Panc-1 ,
CAP AN-2, and AsPC-1). Dysregulation of DPAGT1 enzyme leads to disturbances in cell-cell adhesions and may increase epithelial to mesenchymal transition (EMT): these processes increase migratory and invasive capabilities of malignant neoplasms that are the initiation of metastasis in cancer progression, especially pancreatic cancer. Interestingly, there is significant crosstalk between DPAGT1 and the Wnt/p-catenin and Snail pathway where DPAGT1 overexpression leads to 1) accumulation of b-catenin in the cytoplasm and then translocation into the nucleus, and 2) reduction of the Snail expression levels, preventing epithelial-mesenchymal transition.
As such, aberrations in these pathways occur in numerous cancers, thus, discovery of small molecules directed towards inhibition of the Wnt and Snail pathways represents an important area of anticancer therapeutics. The degree of cell migration in several commercially available cell lines (Capan-2, PANC-1 , and AsPC-1) were observed when treated with the compounds provided herein to determine the effects on cellular motility. After 24 h of three treatment doses (0.05, 0.1 , and 0.2 mM) with CPPB (5), inhibition of migration (closing the gap) was measured in a scratch assay (wound healing assay) (Figures 3A and 3B). The pancreatic cancer cell lines treated with CPPB migrated far less than the PBS treated (control) cells (Figure 3A). In these assays, the wound-healing rate of the untreated PD002 cells was 63% in 24 h. In sharp contrast, CPPB treated cells inhibited the wound-healing effectively at its IC50 level against DPAGT1 (0.2 mM): the wound-healing rate was approximately 20% (Figure 3B). The migration inhibition ability of CPPB was thoroughly evaluated compared to gemcitabine, one of the main chemotherapy drugs used to treat pancreatic cancer, and tunicamycin using PD002 cells.
Gemcitabine shows a wound-healing rate of 43% at 0.2 mM, and tunicamycin shows 35% at 0.2 mM. Thus, it was concluded that CPPB is more effective in inhibiting cancer cell migration than gemcitabine and tunicamycin. These trends were further confirmed by an endpoint migration assays via Boyden chambers for PD002. In these assays, the cell migrations of PD002 treated with CPPB (0.1 mM) were inhibited on a higher level compared to those with tunicamycin and gemcitabine at the same concentration (0.1 mM) (Figure 4). Cell migration assays using Transwell chamber. Cell migration assay was performed using Boyden chamber (thermo fisher). PD002 cells (~104 cells/mL) were seeded in 96- wells plate (Corning, HTS Transwell-96 Well Permeable Supports, pore size: 8 pm) in FBS free media and the lower chambers were filled with 10% FBS medium. CPPB, tunicamycin, or gemcitabine (0.1 mM) was added to the upper chamber. The cells were incubated for 18 h at 37 °C under 5% CO2. The cells were fixed using 4% paraformaldehyde for 0.5 h and stained with 0.05% crystal violet (300 pL/well), after 0.5 h, images were captured via 10X magnification microscopy.
Example 5: Inhibition of a zinc-finger transcription factor, SnaiH (Snail) in the selected cancer cell lines by CPPB
Snail protein is one of the most important transcription factors that induces epithelial to mesenchymal transition (EMT), which converts epithelial cells into migratory mesenchymal cells that are more efficient at metastasizing. EMT induced by overexpression of Snail produces cancer stem-like properties in a number of solid organ cancers. Aberrant expression of Snail leads to loss of expression of E-cadherin, a cell-cell adhesion glycoprotein. Thus, suppression of Snail expression or inhibition of Snail functions represents a potent targeted therapeutic strategy for many cancers.
Immunofluorescence assays using an anti-Snail antibody revealed that the fluorescence intensity of Snail was strong in a series of pancreatic cancer cells (PANC-1, AsPC-1 , Capan-2, and PD002), and the expression of Snail was decreased by the treatment with CPPB in a concentration dependent manner. Among pancreatic cancer cell lines, only the data for PD002 and PANC-1 are shown in Figure 5. The Snail expression level of a nonmetastatic pancreatic cancer, PANC-1 , was much lower than metastatic pancreatic cancers (e.g., PD002 and Capan-2). A few other types of cancer cells such as a colorectal cancer (HCT-116) and a cervical cancer (SiHa) were examined by similarly designed immunofluorescence assays or Western blot assays (Figures 5 and 6). The Snail expression in SiHa was inhibited by treatment of CPPB in a concentration dependent manner; at the IC50 concentration (0.2 mM against DPAGT1), CPPB effectively inhibited the Snail expression (Figure 6). In contrast, the Snail expression level in HCT-116 was not noticeably changed by the treatment of CPPB between 0.05 and 2.0 mM concentrations.
Interestingly, cell migration of HCT-116 was not inhibited by CPPB demonstrated in the wound healing (scratch) assays (Figure 3). At the concentrations tested in the scratch assays (0.05-2.0 mM), the E-cadherin expression levels of PD002, PANC-1 , and HCT-116 were not changed significantly (Figure 7). Example 6: Inhibition of DPAGT1 by CPPB
CPPB decreased the DPAGT1 expression level in all pancreatic cancer cell lines examined in Figure 3: the DPAGT1 expression was apparently inhibited by the IC50 concentration of CPPB (0.2 mM) (Figure 8). However, the DPAGT1 expression of the pancreatic cancer cell lines could not completely be inhibited at a high concentration of CPPB (2-20 mM). In MTT assays, all pancreatic cancers tested remained viable at 20 mM concentration of CPPB (Table 2). In contrast, the DPAGT1 expression levels of a colorectal adenocarcinoma, HCT-116, remained high even at a high concentration of CPPB (20 mM) (Figure 8). These data imply that the inhibitory effect of CPPB on cell migration varies depending on degree of inhibition of the DPAGT1 expression: at the IC50 of CPPB (0.2 mM against DPAGT1), the degree of DPAGT1 expression was decreased by the following order: PANC-1 > PD002 » HCT-116.
Migration inhibition observed in the scratched assays (Figure 3) is well-correlated with the degree of the DPAGT1 expression inhibition. Although CPPB decreased the protein expression of DPAGT1 without significantly decreasing its gene ( DPAGT1 ) expression, tunicamycin decreased both the gene expression of DPAGT1 and DPAGT1 protein expression (Figure 9). These down-regulation in DPAGT1 gene expression by tunicamycin may be attributable to its high cytotoxicity against mammalian cells without selectivity.
Example 7: Synergistic effect of CPPB with paclitaxel
The FOLFIRINOX (a combination of folinic acid, 5-fluorouracil, irinotecan, and oxaliplatin) and nab-paclitaxel (albumin-bound paclitaxel)-gemcitabine regimens have been adopted into clinical practice for patients with metastatic pancreatic cancers. Median progression-free survival was reported in one study of patients with metastatic pancreatic cancer to be 6.4 months in the FOLFIRINOX group and 3.3 months in the gemcitabine group. Over the past years, the clinical data have not supported that FOLFIRINOX is associated with any better (or worse) survival rates compared to the nab-paclitaxel-gemcitabine regimen as there have been no head-to-head trials. However, the inclusion of paclitaxel and its derivatives in combination regimens remains an important therapeutic strategy in pancreatic cancer chemotherapy since nab-paclitaxel-gemcitabine is associated with less adverse effects (toxicity in patients) than FOLFIRINOX.
The synergistic or antagonistic activities of CPPB were assessed in vitro via checkerboard technique. In these experiments, CPPB displayed strong synergistic effects with paclitaxel in a wide range of concentrations against PD002. Table 3 summarizes the results of FIC index analyses for selected combinations of CPPB (IC5035.0 mM) plus paclitaxel (IC50 1.25 mM) that showed synergistic combination (åFIC<0.5). The FIC index below 0.50 was observed for 20 combinations of two molecules out of 96 different concentrations (see Supporting Information). The IC50 value of paclitaxel against PD002 was lowered (0.024-0.61 mM) in combination with CPPB (0.1-2.0 mM). Table 3.
Figure imgf000045_0001
CPPB showed ~7.5 times stronger DPAGT1 inhibitory activity than tunicamycin. However, unlike tunicamycin, CPPB did not inhibit growth of cancer cell lines at the IC50 values observed for tunicamycin (0.45 -7.5 mM). CPPB is a cell-permeable molecule which was demonstrated by IncuCyte® live cell analyses and immunofluorescence assays. The effectiveness of CPPB on the expression levels of Snail, E-cadherin, and DPAGT1 primary in pancreatic cancers has been shown in the present disclosure. CPPB decreased the Snail expression in commercially available pancreatic cancer cell lines (PANC-1, AsPC-1, and Capan-2) and a patient-derived pancreatic ductal adenocarcinoma cell line (PD002) in a dose dependent manner. On the other hand, the E-cadherin expression level was not noticeably changed in PD002 or slightly increased in Panc-1 at between 0.05-0.2 mM of CPPB. These biochemical data support that a selective DPAGT1 inhibitor, CPPB, is effective in inhibiting migrations of the pancreatic cancer cells in migration (scratch) assays. Other than pancreatic cancers, a lower DPAGT1 expression cell, a colorectal adenocarcinoma (HCT-116) and a higher DPAGT1 expression cell, a cervical carcinoma (SiHa) were examined. CPPB did not inhibit migration of HCT-116, but strongly inhibited migration of SiHa in scratch assays at its IC50 concentration (0.2 mM against DPAGT1).
These observations were supported by the biochemical analyses of the Snail and E- cadherin expression levels. Snail plays an important role in cancer progression. The accumulated evidence on Snail indicates that over-expression of Snail promotes drug resistance, tumor recurrence and metastasis. Snail inhibitory activity of CPPB suggests that selective DPAGT1 inhibitors have the potential to develop into less toxic anticancer therapeutics than current anticancer agents that are cytotoxic to all dividing cells in the body. CPPB is not cytotoxic against a series of cancer and healthy cell lines at 10 mM or higher concentrations. However, it showed a strong synergistic effect with paclitaxel; cytostatic activity of paclitaxel is improved over 250-times against PD002 in combination with CPPB (0.2-2.0 mM).
Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference, including without limitation all patent, patent applications, and publications, cited in the present application is incorporated herein by reference in its entirety.

Claims

1. A compound of Formula I:
Figure imgf000047_0001
or a pharmaceutically acceptable salt thereof; wherein
X is NH, O, or -(CH2)m-; Y is CH or N;
Z is CH or N;
A is absent, O, or NH;
R1 is OR7 or -(CH2)nNHR7;
R2 is H or halo; R3 is selected from the group consisting of H, halo, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, and C C6 haloalkyl;
R4 is H or halo;
R5 and R6 are each independently selected from the group consisting of H, C1-C6 alkyl, and C(0)CrC6 alkyl; R7 is selected from the group consisting of H, CrC6 alkyl, CrC6 haloalkyl, C6-C10 aryl, and 5-10 membered heteroaryl; wherein aryl and heteroaryl are each optionally substituted one, two, or three times with OC1-C6 haloalkyl; and m and n are, independently at each occurrence, 0, 1, 2, or 3.
2. The compound of claim 1, wherein the compound of Formula I is a compound of Formula II:
Figure imgf000048_0001
or a pharmaceutically acceptable salt thereof.
3. The compound of claim 1 , wherein the compound of Formula I is a compound of Formula III:
Figure imgf000048_0002
or a pharmaceutically acceptable salt thereof.
4. The compound of claim 1 , wherein the compound of Formula I is a compound of Formula IV:
Figure imgf000048_0003
or a pharmaceutically acceptable salt thereof.
5. The compound of any one of claims 1-4, wherein R1 is OR7.
6. The compound of any one of claims 1-4, wherein R1 is -(CH2)nNHR7.
7. The compound of any one of claims 1-6, wherein R7 is selected from the group consisting of CrC6 alkyl, CrC6 haloalkyl, and phenyl, wherein phenyl is optionally substituted one time with OO-I-OQ haloalkyl.
8. The compound of any one of claims 1-7, wherein R7 is selected from the group consisting of methyl, ethyl, C3 alkyl, C4 alkyl, CF3, and phenyl-OCF3.
9. The compound of any one of claims 1-8, wherein R2 is H.
10. The compound of any one of claims 1-8, wherein R2 is fluoro.
11. The compound of any one of claims 1-10, wherein R3 is selected from the group consisting of H, fluoro, methyl, methoxy, C2 alkenyl, and C2 alkynyl.
12. The compound of any one of claims 1-11 , wherein R3 is H.
13. The compound of any one of claims 1-12, wherein R4 is H.
14. The compound of any one of claims 1-12, wherein R4 is fluoro.
15. The compound of any one of claims 1-14, wherein R5 is H or methyl.
16. The compound of any one of claims 1-15, wherein R5 is methyl.
17. The compound of any one of claims 1-16, wherein R6 is H or methyl.
18. The compound of any one of claims 1-17, wherein R6 is H.
19. The compound of claim 1 or 2, wherein the compound of Formula I is CPPB:
Figure imgf000050_0001
or a pharmaceutically acceptable salt thereof.
20. The compound of claim 19, wherein the compound of Formula I is CPPB1:
Figure imgf000050_0002
(CPPB1) or a pharmaceutically acceptable salt thereof.
21. A compound of Formula V:
Figure imgf000050_0003
or a pharmaceutically acceptable salt thereof; wherein
X and Y are independently O or NH;
R1 is selected from the group consisting of H, OH, C(0)NH2, CO2H, C(0)H, and C(0)halo; and
R2 is selected from the group consisting of H, C1-C6 alkyl, C1-C6 haloalkyl, C6-C10 aryl, and 5-10 membered heteroaryl.
22. The compound of claim 21 , wherein X is NH.
23. The compound of claim 21 or 22, wherein Y is O.
24. The compound of any one of claims 21-23, wherein R1 is C(0)NH2.
25. The compound of any one of claims 21-24, wherein R2 is CrC6 haloalkyl.
26. The compound of any one of claims 21-25, wherein the compound of Formula V is
Figure imgf000051_0002
or a pharmaceutically acceptable salt thereof.
27. A compound of Formula VI:
Figure imgf000051_0001
or a pharmaceutically acceptable salt thereof; wherein X and Y are independently O or NH;
R1 and R2 are each independently selected from H and OH; and R3 is selected from the group consisting of H, C C6 alkyl, C1-C6 haloalkyl, OQ-O-IO aryl, and 5-10 membered heteroaryl.
28. The compound of claim 27, wherein X and Y are NH.
29. The compound of claim 27, wherein X is NH and Y is O.
30. The compound of claim 27, wherein X is O and Y is NH.
31. The compound of any one of claims 27-30, wherein R3 is CrC6 haloalkyl.
32. The compound of any one of claims 27-31 , wherein the compound of Formula VI is selected from the group consisting of
Figure imgf000052_0001
or a pharmaceutically acceptable salt thereof.
33. A pharmaceutical composition comprising a compound according to any one of claims 1-32, and at least one pharmaceutically acceptable carrier.
34. The pharmaceutical composition of claim 33, further comprising an additional therapeutic agent.
35. The pharmaceutical composition of claim 34, wherein the additional therapeutic agent is selected from the group consisting of paclitaxel, tunicamycin, capuramycin, erlotinib, capecitabine, fluorouracil, and gemcitabine.
36. The pharmaceutical composition of claim 34 or 35, wherein the additional therapeutic agent is paclitaxel.
37. A method of inhibiting dolichyl-phosphate A/-acetylglucosaminephosphotransferase (DPAGT1) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1-32 or the pharmaceutical composition of any one of claims 33-36.
38. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1-32 or the pharmaceutical composition of any one of claims 33-36.
39. The method of claim 38, wherein the cancer is selected from the group consisting of pancreatic cancer, cervical cancer, brain cancer, colon cancer, ovarian cancer, breast cancer, pancreatic cancer, carcinoma, and adenocarcinoma.
40. The method of claim 38 or 39, wherein the cancer is pancreatic cancer.
41. The method of claim 38, wherein the cancer is a solid tumor.
PCT/US2021/039826 2020-06-30 2021-06-30 Dpagt1 inhibitors of capuramycin analogues and their antimigratory activities of solid tumors Ceased WO2022006231A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US18/013,403 US12570684B2 (en) 2020-06-30 2021-06-30 DPAGT1 inhibitors of capuramycin analogues and their antimigratory activities of solid tumors

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202063046103P 2020-06-30 2020-06-30
US63/046,103 2020-06-30

Publications (1)

Publication Number Publication Date
WO2022006231A1 true WO2022006231A1 (en) 2022-01-06

Family

ID=79315518

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/039826 Ceased WO2022006231A1 (en) 2020-06-30 2021-06-30 Dpagt1 inhibitors of capuramycin analogues and their antimigratory activities of solid tumors

Country Status (2)

Country Link
US (1) US12570684B2 (en)
WO (1) WO2022006231A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030171330A1 (en) * 1999-08-20 2003-09-11 Sankyo Company, Limited Antibacterial compound

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030171330A1 (en) * 1999-08-20 2003-09-11 Sankyo Company, Limited Antibacterial compound

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MITACHI KATSUHIKO, KANSAL RITA G., HEVENER KIRK E., GILLMAN CODY D., HUSSAIN SYED M., YUN HYUN GI, MIRANDA-CARBONI GUSTAVO A., GLA: "DPAGT1 Inhibitors of Capuramycin Analogues and Their Antimigratory Activities of Solid Tumors", JOURNAL OF MEDICINAL CHEMISTRY, vol. 63, no. 19, 8 October 2020 (2020-10-08), US , pages 10855 - 10878, XP055897324, ISSN: 0022-2623, DOI: 10.1021/acs.jmedchem.0c00545 *
MITACHI KATSUHIKO, KUROSU SHOU M., ESLAMIMEHR SHAKIBA, LEMIEUX MADDIE R., ISHIZAKI YOSHIMASA, CLEMONS WILLIAM M., KUROSU MICHIO: "Semisynthesis of an Anticancer DPAGT1 Inhibitor from a Muraymycin Biosynthetic Intermediate", ORGANIC LETTERS, vol. 21, no. 4, 15 February 2019 (2019-02-15), US , pages 876 - 879, XP055843656, ISSN: 1523-7060, DOI: 10.1021/acs.orglett.8b03716 *
SIRICILLA SHAJILA; MITACHI KATSUHIKO; WAN BAJOIE; FRANZBLAU SCOTT G; KUROSU MICHIO: "Discovery of a capuramycin analog that kills nonreplicatingand its synergistic effects with translocase I inhibitors", THE JOURNAL OF ANTIBIOTICS, vol. 68, no. 4, 1 October 2014 (2014-10-01), London, pages 271 - 278, XP036973714, ISSN: 0021-8820, DOI: 10.1038/ja.2014.133 *

Also Published As

Publication number Publication date
US12570684B2 (en) 2026-03-10
US20230340006A1 (en) 2023-10-26

Similar Documents

Publication Publication Date Title
KR102793825B1 (en) (S)-7-(1-Acryloylpiperidin-4-yl)-2-(4-Phenoxyphenyl)-4,5,6,7-tetra-Hydrazolo[1,5-a]Pyrimidine-3-Carboxamide, Preparation, and Uses Thereof
KR102493364B1 (en) Fusion bicyclic inhibitor of the MENIN-MLL interaction
US11198699B2 (en) Compounds targeting PRMT5
US9701681B2 (en) Fused ring heteroaryl compounds and their use as Trk inhibitors
EP2970133B1 (en) Pyrazole derivatives as prmt1 inhibitors and uses thereof
KR101982912B1 (en) Fused heterocyclic compound, preparation method therefor, pharmaceutical composition, and uses thereof
Tabakmakher et al. Monanchomycalin C, a new pentacyclic guanidine alkaloid from the Far-Eastern marine sponge Monanchora pulchra
WO2014100323A1 (en) Polycyclic-carbamoylpyridone compounds and their pharmaceutical use
ES3014225T3 (en) Rapamycin derivatives
CN101711154A (en) carbamate compound
WO2020188467A1 (en) Condensed tricyclic compound used as kinase inhibitor
JP2017522273A (en) Use of antagonists of apoptotic protein inhibitors (IAPs) in the treatment of HIV
EP4353724A1 (en) Compound as cdk kinase inhibitor and use thereof
CN111171020A (en) Hexa-membered and hexa-membered heterocyclic compounds and application thereof as protein receptor kinase inhibitors
EP4074699A1 (en) Compound as cyclin-dependent kinase 9 inhibitor and use thereof
Tabakmakher et al. Monanchoxymycalins A and B, new hybrid pentacyclic guanidine alkaloids from the Far-Eastern marine sponge Monanchora pulchra
TWI795706B (en) Polycyclic compounds as kinase inhibitors
US12570684B2 (en) DPAGT1 inhibitors of capuramycin analogues and their antimigratory activities of solid tumors
AU2014365915C1 (en) Tetrahydro-tetrazolo[1,5-a]pyrazines as ROR-gamma inhibitors
WO2020247679A1 (en) Wdr5 inhibitors and modulators
WO2023202687A1 (en) Pyrazinone derivative and use thereof in medicine
WO2022257047A1 (en) Diazaspirobicylic compounds as protein-protein interaction inhibitors and applications thereof
CN111377854A (en) CXCR4 inhibitors and uses thereof
CN103814025A (en) Compounds useful as inhibitors of choline kinase
US9175030B2 (en) Derivatives of avicin D and methods of making and using thereof

Legal Events

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

Ref document number: 21833468

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21833468

Country of ref document: EP

Kind code of ref document: A1

WWG Wipo information: grant in national office

Ref document number: 18013403

Country of ref document: US