WO2009141657A1 - Depsipeptides and their therapeutic use - Google Patents

Depsipeptides and their therapeutic use Download PDF

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WO2009141657A1
WO2009141657A1 PCT/GB2009/050553 GB2009050553W WO2009141657A1 WO 2009141657 A1 WO2009141657 A1 WO 2009141657A1 GB 2009050553 W GB2009050553 W GB 2009050553W WO 2009141657 A1 WO2009141657 A1 WO 2009141657A1
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PCT/GB2009/050553
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Stephen Joseph Shuttleworth
Franck Alexandre Silva
Cyrille Davy Tomassi
Alexander Richard Liam Cecil
Arasu Ganesan
Thomas James Hill
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Karus Therapeutics Limited
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Priority claimed from GB0809330A external-priority patent/GB0809330D0/en
Priority claimed from GB0809326A external-priority patent/GB0809326D0/en
Application filed by Karus Therapeutics Limited filed Critical Karus Therapeutics Limited
Priority to EP09750135A priority Critical patent/EP2293845A1/en
Publication of WO2009141657A1 publication Critical patent/WO2009141657A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/15—Depsipeptides; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/10—Anti-acne agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K11/00—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K11/02—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof cyclic, e.g. valinomycins ; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to depsipeptides which act as inhibitors of histone deacetylase (HDAC) and therefore have therapeutic utility.
  • HDACs are zinc metalloenzymes that catalyse the hydrolysis of acetylated lysine residues. In histones, this returns lysines to their protonated state and is a global mechanism of eukaryotic transcriptional control, resulting in tight packaging of DNA in the nucleosome. Additionally, reversible lysine acetylation is an important regulatory process for non-histone proteins. Thus, compounds that are able to modulate HDAC have important therapeutic potential.
  • FK228 (Structure I) and Spiruchostatin A (Structure II) are depsipeptides that have been reported to have potential as HDAC inhibitors.
  • depsipeptide describes a class of oligopeptides or polypeptides that have both ester and peptide links the chain.
  • FK228 is a cyclic depsipeptide containing 4 monomer units together with a cross-ring bridge. This compound, under the trade name of Romidepsin®, has been tested as a therapeutic in human trials and shown that it has valuable effects on a number of diseases.
  • Spiruchostatin A is a cyclic depsipeptide that is structurally related to FK228: it is a cyclic depsipeptide containing a tri-peptide, a statine unit and a cross-ring bridge.
  • Analogues of Spiruchostatin A are disclosed in WO2008/062232. They may have improved HDAC inhibitory properties with respect to Spiruchostatin A or FK228 or other drug-like properties that make them more useful as medicines.
  • R 1 , R 5 , R 7 and R 9 are the same or different and represent hydrogen or an amino acid side chain moiety (from either a natural or an unnatural amino acid), each Ri 0 is the same or different and represents hydrogen or d-C 6 alkyl, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl, Pr 1 and Pr 2 are the same or different and represent hydrogen or a thiol protecting group and Pr 3 is hydrogen or an alcohol protecting group.
  • Structure III Structure IV Analogues of FK228 are disclosed in WO2006/129105. They may have improved HDAC inhibitory properties with respect to FK228 or other drug-like properties that make them more useful as medicines.
  • These compounds have the general structures shown in Structures V & Vl wherein R 1 , R 5 , R 7 and R 9 are the same or different and represent hydrogen or an amino acid side chain moiety (from either a natural or an unnatural amino acid), each R 10 is the same or different and represents hydrogen or C 1 -C 6 alkyl, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl, and Pr 1 and Pr 2 are the same or different and represent hydrogen or a thiol protecting group.
  • Structures VII and VIII are formed inside the cell from Structures I & Il respectively, by reduction of the disulfide bond, and that the 4-thio-butyl-1-ene so formed is a critical part of the mechanism of action of the compound, forming a metallophile capable of binding Zinc in the active site of HDAC.
  • the present invention provides derivatives of Structures Il & VIII in which either the -CH(CH 3 ) 2 (at position 12 on the depsipeptide macrocycle - IUPAC nomenclature) and/or the -CH 3 (at position 6 on the depsipeptide macrocycle - IUPAC nomenclature) is replaced by a groups such as a carbon-linked amide, sulfonamide, functionalised aryl or functionalised or unfunctionalised heteroaryl group.
  • HDACs Hetero-Sidechain Depsipeptides
  • the present invention is a compound of formula IX or X:
  • R 7 and R 9 are the same or different and represent hydrogen or a natural or unnatural amino acid side chain moiety; each R 10 is the same or different and represents hydrogen, C 1 -C 6 alkyl,
  • Pr 1 and Pr 2 are the same or different and represent hydrogen or a thiol protecting group
  • Pr 3 is hydrogen or an alcohol protecting group
  • R 1 and R 5 are the same or different and represent Y or a natural or unnatural amino acid side-chain moiety, provided that R 1 and R 5 are not both a natural or unnatural amino acid side chain moiety;
  • Y is -(CR 11 R 11 )X-NR 11 C(O)NR 11 R 11 , -(CR 11 R 11 ) X -NR 11 C(O)NR 11 R 13 , -(CR 11 R 11 ) X -NR 11 C(O)R 14 , -(CR 11 R 11 ) X -NR 11 C(O)R 13 , -(CR 11 Rn) x -NR 11 SO 2 NR 11 R 11 , -(CR 11 R 11 ) X -NR 11 SO 2 NR 11 R 13 , -(CR 11 R 11 ) X -NR 11 SO 3 R 14 , -(CR 11 R 11 ) X -NR 11 SO 2 R 14 , -(CR 11 R 11 ) X -NR 11 SO 2 R 13 , -(CR 11 R 11 ) X -C(O)NR 11 R 11 , -(CR 11 R 11 ) X -C(O)NR 11 R 13 ,
  • R 13 is NR 11 -C(O)R 14 or NR 11 -SO 2 Ri 4 ;
  • R 14 is C 1 -C 6 alkyl, aryl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl or heteroaryl;
  • Ar is a heteroaryl or substituted aryl, with the proviso that: the compound is none of the compounds shown below:
  • the present invention further provides the use of the compounds defined above, as an inhibitor of HDAC.
  • Synthesis of compounds of Structures IX and X is typically conducted using amino acids of which -CO-CR-NH- forms part of the macrocycle and R is a side-chain moiety.
  • R 1 , R 5 and R 9 may be introduced in this way.
  • R 7 may be an amino acid side chain moiety but may not have been derived directly or indirectly from an amino acid, as such.
  • amino acid side chain moiety refers to any side chain that may be present in a natural or unnatural amino acid.
  • amino acid side chain moieties derived from natural or unnatural amino acids are -(CH 2 ) 2 - C(O)-O-C(CH 3 ) 3 (glutamic acid f-butyl ester), -(CH 2 ) 4 -NH-C(O)-O-C(CH 3 ) 3 (N 6 - (tert-butoxycarbonyl)-lysine), -(CH 2 ) 3 -NH-C(O)NH 2 (citrulline), -CH 2 -CH 2 OH (homoserine) and -(CH 2 ) 3 NH 2 (ornithine).
  • Examples can also include hydrogen, CrC 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, aryl, saturated and unsaturated heterocycles, which can be functionalized or unfunctionalized.
  • a C 1 -C 6 alkyl group or moiety can be linear or branched. Typically, it is a
  • CrC 4 alkyl group or moiety for example methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl and t-butyl.
  • Preferred examples include methyl, i-propyl and t-butyl.
  • a C 2 -C 6 alkenyl group or moiety can be linear or branched. Typically, it is a C 2 -C 4 alkenyl group or moiety. It is preferred that the alkenyl radicals are mono- or di-unsaturated, more preferably monounsaturated. Examples include vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl and 3-butenyl.
  • a C 2 -C 6 alkynyl group or moiety can be linear or branched. Typically, it is a C 2 -C 4 alkynyl group or moiety.
  • aryl means a monocyclic, bicyclic, or tricyclic monovalent aromatic radical, such as phenyl, biphenyl, naphthyl, anthracenyl, which can be optionally substituted (“substituted aryl") with up to five substituents independently selected from the group of CrC 6 alkyl, hydroxy, CrC 3 hydroxyalkyl, CrC 3 alkoxy, CrC 3 haloalkoxy, amino, CrC 3 mono alkylamino, CrC 3 bis alkylamino, CrC 3 acylamino, CrC 3 aminoalkyl, mono (CrC 3 alkyl) amino CrC 3 alkyl, bis (CrC 3 alkyl) amino C r C 3 alkyl, Ci-C 3 -acylamino, CrC 3 alkyl sulfonylamino, halo, nitro, cyano, trifluoromethyl, carboxy, CrC 3 alkoxycarbonyl, aminocarbon
  • heteroaryl means a monocyclic, bicyclic or tricyclic monovalent aromatic radical containing up to four heteroatoms selected from oxygen, nitrogen and sulfur, such as thiazolyl, tetrazolyl, imidazolyl, oxazolyl, isoxazolyl, thienyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, indolyl, quinolyl, isoquinolyl, said radical being optionally substituted with up to three substituents independently selected from the group of Ci-C 6 alkyl, hydroxy, C 1 -C 3 hydroxyalkyl, d-C 3 alkoxy, d-C 3 haloalkoxy, amino, CrC 3 mono alkylamino, Ci-C 3 bis alkylamino, C 1 -C 3 acylamino, Ci-C 3 aminoalkyl, mono (C 1 -C 3 alkyl) amino Ci-C 6 alkyl, hydroxy
  • Y is a carbon-linked amide, sulfonamide, heteroaryl or substituted aryl.
  • examples include thiazole, tetrazole, imidazole, oxazole, isoxazole, thiophene, pyrazole and functionalized derivatives.
  • the amino acid side chain moiety is derived from a natural amino acid.
  • these with the amino acids from which they are derived shown in brackets, are -H (Glycine), -CH 3 (Alanine), -CH(CH 3 ) 2 (Valine), -CH 2 CH(CHa) 2 (Leucine), -CH(CH 3 )CH 2 CH 3 (Isoleucine), -(CH 2 ) 4 NH 2 (Lysine),
  • each amino acid side chain is an amino acid side chain moiety present in a natural amino acid or is -(CH 2 ) 2 -C(O)-O-C(CH 3 ) 3 (glutamic acid f-butyl ester), -(CH 2 ) 4 -NH-C(O)-O-C(CH 3 ) 3 (N ⁇ -(tertbutoxycarbonyl)-lysine), - (CH 2 ) 3 -NH-C(O)NH 2 (citrulline), -CH 2 -CH 2 OH (homoserine) or -(CH 2 ) 2 -CH 2 NH 2
  • the groups Pr 1 and Pr 2 represent hydrogen or a thiol-protecting group.
  • Said thiol-protecting group is typically:
  • a protecting group that forms a thioether to protect a thiol group for example a benzyl group which is optionally substituted by C 1 -C 6 alkoxy (for example methoxy), C 1 -C 6 acyloxy (for example acetoxy), hydroxy and nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamantyl, C 1 -C 6 acyloxymethyl (for example pivaloyloxym ethyl, tertiary butoxycarbonyloxymethyl); (b) a protecting group that forms a monothio, dithio or aminothioacetal to protect a thiol group, for example CrC 6 alkoxymethyl (for example methoxymethyl, isobutoxym ethyl), tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl,
  • a protecting group that forms a thioester to protect a thiol group such as tertiary-butyloxycarbonyl (BOC), acetyl and its derivatives, benzoyl and its derivatives; or
  • a protecting group that forms a carbamic acid thioester to protect a thiol group such as carbamoyl, phenylcarbamoyl, C 1 -C 6 alkylcarbamoyl (for example methylcarbamoyl and ethylcarbamoyl).
  • Pr 1 and Pr 2 are the same or different and each represent hydrogen or a protecting group that forms a thioether, a monothio, dithio or aminothioacetal, a thioester or a carbamic acid thioester to protect a thiol group.
  • Pr 1 and Pr 2 are the same or different and each represent hydrogen or a protecting group selected from a benzyl group which is optionally substituted by C 1 -C 6 alkoxy (for example methoxy), C 1 -C 6 acyloxy (for example acetoxy), hydroxy and nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamantyl, C 1 -C 6 acyloxymethyl (for example pivaloyloxymethyl, tertiary-butyloxycarbonyloxym ethyl), C 1 -C 6 alkoxymethyl (for example methoxymethyl, isobutoxym ethyl), tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidine, acetamidemethyl, benzamidomethyl, tertiary- butyloxycarbonyl (
  • Pr 3 represents hydrogen or a protecting group that forms an ether, an acetal or aminoacetal, an ester or a carbamic acid ester to protect a hydroxyl group.
  • Pr 3 represents hydrogen or a protecting group selected from a benzyl group which is optionally substituted by C 1 -C 6 alkoxy (for example methoxy), C 1 -C 6 acyloxy (for example acetoxy), hydroxy and nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamantyl, CrC 6 acyloxymethyl (for example pivaloyloxymethyl, tertiary butoxycarbonyloxymethyl), CrC 6 alkoxymethyl (for example methoxymethyl, isobutoxymethyl), tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidine, acetamidemethyl, benza
  • X is -CH(OPr 3 ) and the compounds of the invention have Structures IXa and Xa:
  • Preferred embodiments include Compounds Xl to XXXV: A compound according to claim 6, which is a compound shown below:
  • a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base.
  • Pharmaceutically acceptable acids include both inorganic acids such as hydrochloric, sulfuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as citric, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic, methanesulfonic, ethanesulfonic, benzenesulfonic or p-toluenesulfonic acid.
  • Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides and organic bases such as alkyl amines, aralkyl amines or heterocyclic amines.
  • isostere refers to a compound resulting from the exchange of an atom or a group of atoms with another, broadly similar, atom or group of atoms.
  • the moieties which contain isosteric groups are preferably - NR 10 -CHR 1 -CO-, -NR 10 -CHR 9 -CO-O- and -NR 10 -CO-CHR 5 -NR 10 -CO-CHR 7 -.
  • the present invention also embraces pro-drugs which react in vivo to give a compound of the present invention or an isostere or pharmaceutically acceptable salt thereof.
  • Said pharmaceutical composition typically contains up to 85 wt% of a compound of the invention. More typically, it contains up to 50 wt% of a compound of the invention.
  • Preferred pharmaceutical compositions are sterile and pyrogen-free.
  • the pharmaceutical compositions provided by the invention typically contain a compound of the invention which is a substantially pure optical isomer.
  • the pharmaceutical composition comprises a pharmaceutically acceptable salt of a compound of Structure IX or X or an isostere thereof.
  • step (a) an ⁇ /-protected amino acid bearing the side-chain Ri is condensed with an ester enolate bearing the side chain R 9 and the resulting intermediate 1 ,3-diketoester is then reduced to furnish a statine unit, wherein Pr 3 is H or a removable alcohol-protecting group.
  • step (b) the ⁇ /-protecting group is removed, and the statine is coupled to a protected cysteine derivative to furnish a peptide isostere.
  • step (c) the ⁇ /-protecting group is removed, and the peptide isostere is coupled with an /V-protected amino acid bearing the side chain R 5 .
  • step (d) the ⁇ /-protecting group is removed, and the resulting intermediate is coupled with a functionalised ⁇ -hydroxy acid derivative wherein R 15 is a temporary blocking group which can be removed to produce a compound wherein R 15 is H, and X is a chiral auxiliary as reported in Yurek- George, A.; Habens, F.; Brimmell, M.; Packham, G.; Ganesan, A. J. Am. Chem. Soc.2004, 126, 1030-1031.
  • step (e) the ester is hydrolysed, and cyclization is facilitated in step (f), to provide a compound of the invention wherein X is -CH(OPr 3 ) - of Structure Xa.
  • Disulfide bond formation occurs in step (g) to provide a compound of the invention wherein X is -CH(OPr 3 ) - of Structure IXa.
  • step (a) an amino acid ester bearing the side-chain R 9 is coupled with another, ⁇ /-protected amino acid bearing the side chain R 1 (where PG represents a conventional protecting group) to furnish the /V-protected dipeptide ester.
  • step (b) the ⁇ /-protecting group is removed, and the resulting dipeptide ester is coupled to a protected cysteine.
  • step (c) the ⁇ /-protecting group is removed, and the resulting tripeptide is coupled with an amino acid bearing the side chain R 5 to liberate an ⁇ /-protected tetrapeptide ester.
  • step (d) the ⁇ /-protecting group is removed and the resulting tetrapeptide ester is coupled with a functionalized ⁇ -hydroxy acid derivative wherein R 15 is a temporary blocking group which can be removed to produce a compound wherein R 15 is H, and X is a chiral auxiliary as reported in Yurek-George, A.; Habens, F.; Brimmell, M.; Packham, G.; Ganesan, A. J. Am. Chem. Soc. 2004, 126, 1030-1031.
  • Compounds of the invention of Structures IX and X in which R 10 is other than hydrogen can be obtained either by alkylating a corresponding compound of the invention or intermediate in which R 10 is hydrogen or by using appropriately substituted starting materials.
  • Compounds of Structure X may be obtained by reaction of the product of step (g) of the above Schemes 1 and 2, i.e. a compound of Structure IX, to cleave the disulfide bond.
  • the cleavage of the disulfide bond is typically achieved using a thiol compound generally used for a reduction treatment of a protein having a disulfide bond, for example mercaptoethanol, thioglycol acid, 2- mercaptoethylamine, benzenethiol, parathiocresol and dithiothreitol.
  • a thiol compound generally used for a reduction treatment of a protein having a disulfide bond for example mercaptoethanol, thioglycol acid, 2- mercaptoethylamine, benzenethiol, parathiocresol and dithiothreitol.
  • mercaptoethanol and dithiothreitol are used.
  • An excess thiol compound can be removed by for example dialysis or gel filtration.
  • electrolysis, sodium tetrahydroborate, lithium aluminum hydride or sulfite may, for example, be used to cleave the disul
  • Compounds of Structure X in which Pn and/or Pr 2 is other than hydrogen may be prepared by introducing a thiol-protecting group into a corresponding compound in which Pr 1 and/or Pr 2 is/are hydrogen.
  • a suitable agent for introducing thiol-protecting group to be used in this reaction is appropriately determined depending on the protecting group to be introduced.
  • Examples include chlorides of the corresponding protecting group (for example benzyl chloride, methoxybenzyl chloride, acetoxybenzyl chloride, nitrobenzyl chloride, picolyl chloride, picolyl chloride-N-oxide, anthryl methyl chloride, isobutoxym ethyl chloride, phenylthiomethyl chloride) and alcohols of the corresponding protecting group (for example diphenylmethyl alcohol, adamanthyl alcohol, acetam idem ethyl alcohol, benzamidomethyl alcohol), dinitrophenyl, isobutylene, dimethoxymethane, dihydropyran and t-butyl chloroformate.
  • chlorides of the corresponding protecting group for example benzyl chloride, methoxybenzyl chloride, acetoxybenzyl chloride, nitrobenzyl chloride, picolyl chloride, picolyl chloride-N-oxide, anthryl
  • Ri, R 5 , R 7 , R 9 , Ri 0 carries a functional group such as -OH, -SH, -NH 2 or -COOH
  • that group it may be preferred for that group to be protected for one or more of the reaction steps following its introduction.
  • the group in question could be protected in a separate step after its introduction, or, it could be protected already at the time it is introduced.
  • suitable protecting groups that can be used in this regard.
  • the compounds of the invention thus obtained may be salified by treatment with an appropriate acid or base. Racemic mixtures obtained by any of the above processes can be resolved by standard techniques, for example elution on a chiral chromatography column.
  • various assays are suitable for testing for HDAC inhibition and may be used to measure the activity of a compound obtained from Scheme 1 compared to that of the known HDAC inhibitor SAHA.
  • the IC 50 of a test compound against HDAC can, for example, be determined in an in vitro assay, and compared with the IC 50 of SAHA under the same assay conditions. If a test compound has an IC 50 value equal to or lower than that of SAHA it should be understood as having an HDAC inhibitory activity which is at least equal to that exhibited by SAHA.
  • the present invention provides a process for selecting a compound which has an HDAC inhibitory activity which is at least equal to that exhibited by SAHA as defined above, wherein following completion of Scheme 1 , the next step is a an in vitro HDAC assay.
  • said assay comprises contacting a test compound and SAHA, at various concentrations, with diluted HeLa Nuclear Extract to determine the IC 50 of the test compound and of SAHA against HeLa Nuclear Extract.
  • a test compound which has an IC 50 value measured against HeLa Nuclear Extract which is equal to, or lower than, the IC 50 of SAHA under the same assay conditions should be understood as having an inhibitory activity which is at least equal to that exhibited by SAHA.
  • said assay is performed using a HDAC fluorescent activity assay kit (Biomol, UK) and the test compounds are reduced prior to analysis.
  • the present invention provides a process for selecting a compound which has a human cancer cell growth inhibitory activity which is at least equal to that exhibited by SAHA, which process comprises preparing a compound of Structure IX or X via Scheme 1 as defined above followed by screening the thus obtained compound to measure its activity as a human cancer cell growth inhibitor.
  • IC 50 of a test compound against human cancer cell growth can, for example, be determined in an in vitro assay, and compared with the IC 50 of SAHA under the same assay conditions. If a test compound has an IC 50 value equal to or lower than that of SAHA it should be understood as having an inhibitory activity which is at least equal to that exhibited by SAHA.
  • this step comprises an in vitro assay which comprises contacting a test compound and SAHA, at various concentrations, with an MCF7 breast, HUT78 T-cell leukaemia, A2780 ovarian, PC3 or LNCAP prostate cancer cell line to determine the IC 50 of the test compound and of SAHA against the cell line.
  • a test compound which has an IC 50 value measured against any of these cell lines which is equal to, or lower than, the IC 50 of SAHA under the same assay conditions should be understood as having an inhibitory activity at least equal to that of SAHA.
  • said assay is performed using the CyQuantTM assay system (Molecular Probes, Inc. USA).
  • the present invention provides a process for selecting a compound which has an anti-inflammatory activity which is at least equal to that exhibited by SAHA, which process comprises preparing a compound of Structure IX or X via Scheme 1 as defined above followed by screening the thus obtained compound to measure its anti-inflammatory activity.
  • the anti-inflammatory activity of a test compound relative to SAHA may, for example, be determined by measuring the activity of a compound in inhibiting the production of TNF ⁇ from peripheral blood mononuclear cells (PBMCs) relative to SAHA.
  • PBMCs peripheral blood mononuclear cells
  • the ability of a test compound to inhibit the production of TNF ⁇ from PBMCs can, for example, be determined in an assay, and compared with the activity of SAHA under the same assay conditions. If a test compound has an in vitro inhibitory activity of TNF ⁇ production which is equal to or higher than that of SAHA under the same assay conditions it should be understood as having an antiinflammatory activity which is at least equal to that exhibited by SAHA.
  • this step is performed using the Quantikine® Human- ⁇ assay kit (R&D systems, Abingdon UK).
  • the anti-inflammatory activity of a test compound relative to SAHA may be determined by assessing the activity of a compound in inhibiting inflammation in Balb/c mice relative to SAHA. If a test compound has an in vivo inhibitory activity which is equal to or higher than that of SAHA under the same test conditions it should be understood as having an anti-inflammatory activity which is at least equal to that exhibited by SAHA. Typically, in this embodiment this step is performed by assessing the in vivo activity of a test compound and of SAHA in inhibiting inflammation in Balb/c mice induced by a chemical challenge. Typically, said chemical challenge involves the topical administration to the mice of oxalazone or acetone. In this embodiment, the compounds under investigation may be applied before or after the chemical challenge.
  • the present invention provides a process for selecting a compound which has an activity in inducing a predominant G2/M phase arrest or cell death in MCF7 cells which is at least equal to that exhibited by SAHA, which process comprises preparing a compound of Structure I or X via Scheme 1 as defined above followed by screening the thus obtained compound to measure activity in inducing a predominant G2/M phase arrest or cell death in MCF7 cells relative to SAHA.
  • the compounds of the present invention are found to be inhibitors of HDAC.
  • the compounds of the present invention are therefore therapeutically useful.
  • a pharmaceutical composition comprising a compound of the invention may be formulated in a format suitable for oral, rectal, parenteral, intranasal or transdermal administration or administration by inhalation or by suppository. Typical routes of administration are parenteral, intranasal or transdermal administration or administration by inhalation.
  • the compounds of the invention can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules.
  • Preferred pharmaceutical compositions of the invention are compositions suitable for oral administration, for example tablets and capsules.
  • the compounds of the invention may also be administered parenterally, whether subcutaneously, intravenously, intramuscularly, intrasternally, transdermal ⁇ or by infusion techniques.
  • the compounds may also be administered as suppositories.
  • the compounds of the invention may also be administered by inhalation.
  • inhaled medications are their direct delivery to the area of rich blood supply in comparison to many medications taken by oral route. Thus, the absorption is very rapid as the alveoli have an enormous surface area and rich blood supply and first pass metabolism is bypassed.
  • a further advantage may be to treat diseases of the pulmonary system, such that delivering drugs by inhalation delivers them to the proximity of the cells which are required to be treated.
  • the present invention also provides an inhalation device containing such a pharmaceutical composition.
  • said device is a metered dose inhaler (MDI), which contains a pharmaceutically acceptable chemical propellant to push the medication out of the inhaler.
  • MDI metered dose inhaler
  • the compounds of the invention may also be administered by intranasal administration.
  • the nasal cavity's highly permeable tissue is very receptive to medication and absorbs it quickly and efficiently, more so than drugs in tablet form.
  • Nasal drug delivery is less painful and invasive than injections, generating less anxiety among patients. By this method absorption is very rapid and first pass metabolism is usually bypassed, thus reducing inter-patient variability.
  • the present invention also provides an intranasal device containing such a pharmaceutical composition.
  • the compounds of the invention may also be administered by transdermal administration.
  • the present invention therefore also provides a transdermal patch containing a compound of the invention, or a pharmaceutically acceptable salt thereof.
  • the compounds of the invention may also be administered by sublingual administration.
  • the present invention therefore also provides a sub-lingual tablet comprising a compound of the invention or a pharmaceutically acceptable salt thereof.
  • a compound of the invention is typically formulated for administration with a pharmaceutically acceptable carrier or diluent.
  • a compound of the invention may also be formulated with an agent which reduces degradation of the substance by processes other than the normal metabolism of the patient, such as anti-bacterial agents, or inhibitors of protease enzymes which might be the present in the patient or in commensural or parasite organisms living on or within the patient, and which are capable of degrading the compound.
  • Liquid dispersions for oral administration may be syrups, emulsions and suspensions.
  • Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
  • the suspension or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
  • Solutions for injection or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
  • the compounds of the present invention are therapeutically useful in the treatment or prevention of conditions mediated by HDAC. Accordingly, the present invention provides the use of a compound of the Structure IX or X, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment or prevention of a condition materially affected by the activity of an HDAC. Also provided is a method of treating a patient suffering from or susceptible to a condition mediated by HDAC, which method comprises administering to said patient an effective amount of a compound of Structure IX or X, an isostere thereof or a pharmaceutically acceptable salt thereof.
  • the compounds of the present invention may be used in combination with another known inhibitor of HDAC, such as SAHA.
  • the combination product may be formulated such that it comprises each of the medicaments for simultaneous, separate or sequential use.
  • the present invention therefore also provides the use of compounds according to Structure IX or X or an isostere or pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for use in co-administration with another known inhibitor of HDAC, such as SAHA.
  • the compounds of the present invention can be used in both the treatment and prevention of cancer and can be used in a monotherapy or in a combination therapy.
  • the compounds of the present invention are typically used together with small chemical compounds such as platinum complexes, anti-metabolites, DNA topoisomerase inhibitors, radiation, antibody-based therapies (for example herceptin and rituximab), anti-cancer vaccination, gene therapy, cellular therapies, hormone therapies or cytokine therapy.
  • a compound of the invention is used in combination with another chemotherapeutic or antineoplastic agent in the treatment of a cancer.
  • chemotherapeutic or antineoplastic agents include mitoxantrone, vinca alkaloids for example vincristine and vinblastine, anthracycline antibiotics for example daunorubicin and doxorubicin, alkylating agents for example chlorambucil and melphalan, taxanes for example paclitaxel, antifolates for example methotrexate and tomudex, epipodophyllotoxins for example etoposide, camptothecins for example irinotecan and its active metabolite SN 38 and DNA methylation inhibitors for example the DNA methylation inhibitors disclosed in WO 02/085400.
  • products which contain a compound of the invention and another chemotherapeutic or antineoplastic agent as a combined preparation for simultaneous, separate or sequential use in alleviating a cancer.
  • a compound of Structure IX or X as defined above or an isostere thereof or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the alleviation of cancer by co-administration with another chemotherapeutic or antineoplastic agent.
  • the compound of the invention and the said other agent may be administrated in any order. In both these cases the compound of the invention and the other agent may be administered together or, if separately, in any order as determined by a physician.
  • HDAC is believed to contribute to the pathology and/or symptomology of several different diseases such that reduction of the activity of HDAC in a subject through inhibition of HDAC may be used to therapeutically address these disease states. Examples of various diseases that may be treated using the HDAC inhibitors of the present invention are described herein, and the use of compounds of the present invention described by Structure IX or X are included herein. It is noted that additional diseases beyond those disclosed herein may be later identified as applications of the compounds of the present invention, as the biological roles that HDAC play in various pathways becomes more fully understood.
  • HDAC inhibitors of the present invention may be used to treat are those involving undesirable or uncontrolled cell proliferation.
  • Such indications include benign tumours, various types of cancers such as primary tumours and tumour metastasis, restenosis (e.g. coronary, carotid, and cerebral lesions), abnormal stimulation of endothelial cells (atherosclerosis), insults to body tissue due to surgery, abnormal wound healing, abnormal angiogenesis, diseases that produce fibrosis of tissue, repetitive motion disorders, disorders of tissues that are not highly vascularized, and proliferative responses associated with organ transplants.
  • More specific indications for HDAC inhibitors include, but are not limited to prostate cancer, lung cancer, acute leukaemia, multiple myeloma, bladder carcinoma, renal carcinoma, breast carcinoma, colorectal carcinoma, neuroblastoma and melanoma.
  • a method for treating diseases associated with undesired and uncontrolled cell proliferation.
  • the method comprises administering to a subject suffering from uncontrolled cell proliferation a therapeutically effective amount of a HDAC inhibitor according to the present invention, such that said uncontrolled cell proliferation is reduced.
  • a therapeutically effective amount of a HDAC inhibitor according to the present invention is administered to a subject suffering from uncontrolled cell proliferation a therapeutically effective amount of a HDAC inhibitor according to the present invention, such that said uncontrolled cell proliferation is reduced.
  • the particular dosage of the inhibitor to be used will depend on the severity of the disease state, the route of administration, and related factors that can be determined by the attending physician. Generally, acceptable and effective daily doses are amounts sufficient to effectively slow or eliminate uncontrolled cell proliferation.
  • HDAC inhibitors according to the present invention may also be used in conjunction with other agents to inhibit undesirable and uncontrolled cell proliferation.
  • anti-cell proliferation agents include, but are not limited to, retinoid acid and derivatives thereof, 2-methoxyestradiol, ANGIOSTATIN(TM) protein, ENDOSTATIN(TM) protein, suramin, squalamine, tissue inhibitor of metalloproteinase-l, tissue inhibitor of metalloproteinase-2, plasminogen activator inhibitor-1 , plasminogen activator inhibitor-2, cartilage- derived inhibitor, paclitaxel, platelet factor 4, protamine sulfate (clupeine), sulfated chitin derivatives (prepared from queen crab shells), sulfated polysaccharide peptidoglycan complex (sp-pg), staurosporine, modulators of matrix metabolism, including for example, proline analogs ((1-azetidine-2-
  • anti- angiogenesis agents include antibodies, preferably monoclonal antibodies against these angiogenic growth factors: bFGF, aFGF, FGF-5, VEGF isoforms, VEGF-C, HGF/SF and Ang-1/Ang-2.
  • bFGF vascular endothelial growth factor
  • aFGF vascular endothelial growth factor
  • FGF-5 vascular endothelial growth factor
  • VEGF isoforms VEGF-C
  • HGF/SF Ang-1/Ang-2.
  • Ferrara N. and Alitalo, K. "Clinical application of angiogenic growth factors and their inhibitors" (1999) Nature Medicine 5: 1359-1364.
  • a benign tumour is usually localized and nonmetastatic.
  • Specific types of benign tumours that can be treated using HDAC inhibitors of the present invention include hemangiomas, hepatocellular adenoma, cavernous haemangioma, focal nodular hyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bile duct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas, myxomas, nodular regenerative hyperplasia, trachomas and pyogenic granulomas.
  • Malignant tumors In the case of malignant tumors, cells become undifferentiated, do not respond to the body's growth control signals, and multiply in an uncontrolled manner. Malignant tumors are invasive and capable of spreading to distant sites (metastasizing). Malignant tumors are generally divided into two categories: primary and secondary. Primary tumors arise directly from the tissue in which they are found. Secondary tumors, or metastases, are tumors that originated elsewhere in the body but have now spread to distant organs. Common routes for metastasis are direct growth into adjacent structures, spread through the vascular or lymphatic systems, and tracking along tissue planes and body spaces (peritoneal fluid, cerebrospinal fluid, etc.).
  • cancers or malignant tumors include, but are not limited to, leukaemia, breast cancer, skin cancer, bone cancer, prostate cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor, gallstones, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms
  • the HDAC inhibitors of the present invention may also be used to treat abnormal cell proliferation due to insults to body tissue during surgery. These insults may arise as a result of a variety of surgical procedures such as joint surgery, bowel surgery, and cheloid scarring. Diseases that produce fibrotic tissue include emphysema. Repetitive motion disorders that may be treated using the present invention include carpal tunnel syndrome. An example of a cell proliferative disorder that may be treated using the invention is a bone tumor.
  • Proliferative responses associated with organ transplantation that may be treated using HDAC inhibitors of the invention include proliferative responses contributing to potential organ rejections or associated complications. Specifically, these proliferative responses may occur during transplantation of the heart, lung, liver, kidney, and other body organs or organ systems.
  • Abnormal angiogenesis that may be may be treated using this invention include those abnormal angiogenesis accompanying rheumatoid arthritis, ischemic-reperfusion related brain edema and injury, cortical ischemia, ovarian hyperplasia and hypervascularity, (polycystic ovary syndrome), endometriosis, psoriasis, diabetic retinopathy, and other ocular angiogenic diseases such as retinopathy of prematurity (retrolental fibroplastic), macular degeneration, corneal graft rejection, neuroscular glaucoma and Oster Webber syndrome.
  • abnormal angiogenesis accompanying rheumatoid arthritis, ischemic-reperfusion related brain edema and injury, cortical ischemia, ovarian hyperplasia and hypervascularity, (polycystic ovary syndrome), endometriosis, psoriasis, diabetic retinopathy, and other ocular angiogenic diseases
  • Examples of diseases associated with uncontrolled angiogenesis include, but are not limited to retinal/choroidal neovascularization and corneal neovascularization.
  • Examples of retinal/choroidal neovascularization include, but are not limited to, Bests diseases, myopia, optic pits, Stargarts diseases, Pagets disease, vein occlusion, artery occlusion, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum carotid apo structive diseases, chronic uveitis/vitritis, mycobacterial infections, Lyme's disease, systemic lupus erythematosus, retinopathy of prematurity, Eales disease, diabetic retinopathy, macular degeneration, Bechets diseases, infections causing a retinitis or chroiditis, presumed ocular histoplasmosis, pars planitis, chronic retinal detachment, hyperviscos
  • corneal neovascularization examples include, but are not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, Mooren ulcer, Terrien's marginal degeneration, marginal keratolysis, polyarteritis, Wegener sarcoidosis, Scleritis, periphigoid radial keratotomy, neovascular glaucoma and retrolental fibroplasia, syphilis, Mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections and Kaposi sarcoma
  • Chronic inflammatory diseases associated with uncontrolled angiogenesis may also be treated using HDAC inhibitors of the present invention.
  • Chronic inflammation depends on continuous formation of capillary sprouts to maintain an influx of inflammatory cells. The influx and presence of the inflammatory cells produce granulomas and thus maintains the chronic inflammatory state. Inhibition of angiogenesis using a HDAC inhibitor alone or in conjunction with other anti-inflammatory agents may prevent the formation of the granulosmas and thus alleviate the disease.
  • Examples of chronic inflammatory diseases include, but are not limited to, inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, psoriasis, sarcoidosis, and rheumatoid arthritis.
  • Inflammatory bowel diseases such as Crohn's disease and ulcerative colitis are characterized by chronic inflammation and angiogenesis at various sites in the gastrointestinal tract.
  • Crohn's disease occurs as a chronic transmural inflammatory disease that most commonly affects the distal ileum and colon but may also occur in any part of the gastrointestinal tract from the mouth to the anus and perianal area.
  • Patients with Crohn's disease generally have chronic diarrhea associated with abdominal pain, fever, anorexia, weight loss and abdominal swelling.
  • Ulcerative colitis is also a chronic, nonspecific, inflammatory and ulcerative disease arising in the colonic mucosa and is characterized by the presence of bloody diarrhea.
  • inflammatory bowel diseases are generally caused by chronic granulomatous inflammation throughout the gastrointestinal tract, involving new capillary sprouts surrounded by a cylinder of inflammatory cells. Inhibition of angiogenesis by these inhibitors should inhibit the formation of the sprouts and prevent the formation of granulomas. Inflammatory bowel diseases also exhibit extra intestinal manifestations, such as skin lesions. Such lesions are characterized by inflammation and angiogenesis and can occur at many sites other the gastrointestinal tract. Inhibition of angiogenesis by HDAC inhibitors according to the present invention can reduce the influx of inflammatory cells and prevent lesion formation.
  • Sarcoidosis another chronic inflammatory disease, is characterized as a multisystem granulomatous disorder.
  • the granulomas of this disease can form anywhere in the body. Thus, the symptoms depend on the site of the granulomas and whether the disease is active.
  • the granulomas are created by the angiogenic capillary sprouts providing a constant supply of inflammatory cells.
  • HDAC inhibitors according to the present invention to inhibit angiogenesis, such granulomas formation can be inhibited.
  • Psoriasis also a chronic and recurrent inflammatory disease, is characterized by papules and plaques of various sizes. Treatment using these inhibitors alone or in conjunction with other anti-inflammatory agents should prevent the formation of new blood vessels necessary to maintain the characteristic lesions and provide the patient relief from the symptoms.
  • Rheumatoid arthritis is also a chronic inflammatory disease characterized by non-specific inflammation of the peripheral joints. It is believed that the blood vessels in the synovial lining of the joints undergo angiogenesis. In addition to forming new vascular networks, the endothelial cells release factors and reactive oxygen species that lead to pannus growth and cartilage destruction. The factors involved in angiogenesis may actively contribute to, and help maintain, the chronically inflamed state of rheumatoid arthritis. Treatment using HDAC inhibitors according to the present invention alone or in conjunction with other anti-RA agents may prevent the formation of new blood vessels necessary to maintain the chronic inflammation.
  • the compounds of the present invention can further be used in the treatment of cardiac/vasculature diseases such as hypertrophy, hypertension, myocardial infarction, reperfusion, ischemic heart disease, angina, arrhythmias, hypercholesterolemia, atherosclerosis and stroke.
  • the compounds can further be used to treat neurodegenerative disorders/CNS disorders such as acute and chronic neurological diseases, including stroke, Huntington's disease, Amyotrophic Lateral Sclerosis and Alzheimer's disease.
  • the compounds of the present invention can also be used as antimicrobial agents, for example antibacterial agents.
  • the invention therefore also provides a compound for use in the treatment of a bacterial infection.
  • the compounds of the present invention can be used as anti-infectious compounds against viral, bacterial, fungal and parasitic infections. Examples of infections include protozoal parasitic infections (including Plasmodium, Cryptosporidium parvum, toxoplasma gondii, sarcocystis neurona and Eimeria sp.)
  • the compounds of the present invention are particularly suitable for the treatment of undesirable or uncontrolled cell proliferation, preferably for the treatment of benign tumours/hyperplasias and malignant tumors, more preferably for the treatment of malignant tumors and most preferably for the treatment of CCL, breast cancer and T-cell lymphoma.
  • the compounds of the invention are used to alleviate cancer, cardiac hypertrophy, chronic heart failure, an inflammatory condition, a cardiovascular disease, a haemoglobinopathy, a thalassemia, a sickle cell disease, a CNS disorder, an autoimmune disease, diabetes, osteoporosis, MDS, benign prostatic hyperplasia, oral leukoplakia, a genentically related metabolic disorder, an infection, Rubens-Taybi, fragile X syndrome, or alpha-1 antitrypsin deficiency, or to accelerate wound healing, to protect hair follicles or as an immunosuppressant.
  • said inflammatory condition is a skin inflammatory condition (for example psoriasis, acne and eczema), asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), Crohn's disease or colitis.
  • COPD chronic obstructive pulmonary disease
  • RA rheumatoid arthritis
  • IBD inflammatory bowel disease
  • Crohn's disease or colitis a skin inflammatory condition
  • said cancer is chronic lymphocytic leukaemia, breast cancer, prostate cancer, ovarian cancer, mesothelioma or T-cell lymphoma.
  • said cardiovascular disease is hypertension, myocardial infarction (Ml), ischemic heart disease (IHD) (reperfusion), angina pectoris, arrhythmia, hypercholesterolemia, hyperlipidaemia, atherosclerosis, stroke, myocarditis, congestive heart failure, primary and secondary i.e. dilated (congestive) cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, peripheral vascular disease, tachycardia, high blood pressure or thrombosis.
  • Ml myocardial infarction
  • IHD ischemic heart disease
  • said genentically related metabolic disorder is cystic fibrosis (CF), peroxisome biogenesis disorder or adrenoleukodystrophy.
  • CF cystic fibrosis
  • peroxisome biogenesis disorder or adrenoleukodystrophy.
  • the compounds of the invention are used as an immunosuppressant following organ transplant.
  • said infection is a viral, bacterial, fungal or parasitic infection, in particular an infection by S aureus, P acne, Candida or aspergillus.
  • said CNS disorder is Huntingdon's disease, Alzheimer's disease, multiple sclerosis or amyotrophic lateral sclerosis.
  • the compounds of the invention may be used to alleviate cancer, cardiac hypertrophy, chronic heart failure, an inflammatory condition, a cardiovascular disease, a haemoglobinopathy, a thalassemia, a sickle cell disease, a CNS disorder, an autoimmune disease, diabetes or osteoporosis, or are used as an immunosuppressant.
  • the compounds of the invention may also be used to alleviate chronic lymphocytic leukaemia, breast cancer, prostate cancer, ovarian cancer, mesothelioma, T-cell lymphoma, cardiac hypertrophy, chronic heart failure or a skin inflammatory condition, in particular psoriasis, acne or eczema.
  • the compounds of the present invention can be used in the treatment of animals, preferably in the treatment of mammals and more preferably in the treatment of humans.
  • the compounds of the invention may, where appropriate, be used prophylactically to reduce the incidence of such conditions.
  • a therapeutically effective amount of a compound of the invention is administered to a patient.
  • a typical dose is from about 0.001 to 50 mg per kg of body weight, according to the activity of the specific compound, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration.
  • MeCN (8 ml_) was then added to the mixture via cannula, and the resulting mixture was left to warm to rt overnight before being concentrated in vacuo.
  • reaction mixture was quenched with a solution of Na 2 S 2 Oa (0.1 M, 25OmL) and brine (25mL), and the aqueous layer was extracted with CH 2 CI 2 (2 x 10OmL) and EtOAc (10OmL). The combined organic extracts were dried over MgSO 4 and the solvent was removed in vacuo. Purification by column chromatography on silica using CH 2 CI 2 /MeOH (32:1) as eluant yielded compound XIV (118mg, 0.21 mmol, 77%) as a white solid.
  • FmocHN (1) (R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-5-oxo-5-(toluene-4- sulfonylamino)-pentanoic acid tert-butyl ester
  • H 2 N- D-3(2-pyridyl)alanine-D-Cys(STrt)-D-Val-Gly-OMe (2) (153mg, 0.224 mmol, 1.1 eq, purchased from GL Biochem (Shanghai) Ltd, Shanghai 200241 , China) was added and the reaction mixture was then left to warm to rt over 2h before being concentrated in vacuo. Purification by flash column chromatography (eluant 1-4% MeOH/CH 2 CI 2 ) gave the product 5 (135mg, 0.125 mmol, 56%) as a white solid.
  • reaction mixture was concentrated under reduced pressure, and was purified by silica gel column chromatography, eluting with CH 2 CI 2 /Me0H (100:3), and then by solid phase extraction (SPE ISOLUTE SCX-3) with CH 2 CI 2 /MeOH (100:1 to 100:3) to furnish compound XXXVII as a white solid (18.7mg, 66%).
  • reaction mixture was concentrated under reduced pressure, and the residue purified by silica gel column chromatography, eluting with CH 2 CI 2 /MeOH (100:2 to 100:3), and then further purified by solid phase extraction (SPE ISOLUTE SCX-3) with CH 2 CI 2 /MeOH (100:1 to 100:3) to yield compound XL as a white solid (12.9mg, 47%).
  • the mixture was then concentrated in vacuo and then purified by flash column chromatography on silica (eluant 1 :0-98:2-96:4-94:6 CH 2 CI 2 /MeOH).
  • the isolated material was passed through an SCX-3 Isolute ® column (eluant 99:1- 97:3-95:5 CH 2 CI 2 /MeOH) to give XLII (16.8mg, 0.028 mmol, 60%) as a white solid.
  • reaction mixture was stirred for 6 min, and was then added to 5 (472mg, 0.64 mmol) in CH 2 CI 2 (2OmL). After 16h stirring at rt, the mixture was concentrated in vacuo, and was purified by flash column chromatography using EtOAc/Hexane (6:4-7:3-8:2) as eluant to give 7 (540mg, 0.48 mmol, 74%) as a white solid.
  • reaction mixture was stirred for 5.25h, and was then concentrated in vacuo before being purified by flash column chromatography on silica (eluant 98:2-96:4-94:6 CH 2 CI 2 /MeOH) and then by ion exchange chromatography using an SCX-3 Isolute ® column (eluant 98:2-96:4-94:6 CH 2 CI 2 /MeOH).
  • Compound XLVII was isolated as a white solid (10mg, 0.0162 mmol, 63%).
  • reaction mixture was then left to warm to rt overnight, was then concentrated in vacuo, and purified by silica gel column chromatography with hexane/EtOAc (1 :9 -> 0:1 + 0.5% MeOH) to give 2 as a white solid (286mg, 96%).

Abstract

A compound of formula (IX) or (X): including pharmaceutically acceptable salts thereof; wherein: X is -C(=O)N(R10)- or -CH(OPr3) -; R7 and R9 are the same or different and represent hydrogen or a natural or unnatural amino acid side chain moiety; each R10 is the same or different and represents hydrogen, C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl; Pr1 and Pr2 are the same or different and represent hydrogen or a thiol protecting group; Pr3 is hydrogen or an alcohol protecting group; R1 and R5 are the same or different and represent Y or a natural or unnatural amino acid side-chain moiety, provided that R1 and R5 are not both a natural or unnatural amino acid side chain moiety; and Y is as defined in the application.

Description

DEPSIPEPTIDES AND THEIR THERAPEUTIC USE
This application claims priority from UK patent applications GB 0809326.2 entitled "Depsipeptides and their therapeutic use" filed May 22, 2008 and GB 0809330.4 entitled "Depsipeptides and their therapeutic use", filed May 22, 2008, which are incorporated herein by reference in their entirety. Field of the Invention
The present invention relates to depsipeptides which act as inhibitors of histone deacetylase (HDAC) and therefore have therapeutic utility. Background of the Invention HDACs are zinc metalloenzymes that catalyse the hydrolysis of acetylated lysine residues. In histones, this returns lysines to their protonated state and is a global mechanism of eukaryotic transcriptional control, resulting in tight packaging of DNA in the nucleosome. Additionally, reversible lysine acetylation is an important regulatory process for non-histone proteins. Thus, compounds that are able to modulate HDAC have important therapeutic potential.
The natural products FK228 (Structure I) and Spiruchostatin A (Structure II) are depsipeptides that have been reported to have potential as HDAC inhibitors. The term depsipeptide describes a class of oligopeptides or polypeptides that have both ester and peptide links the chain. FK228 is a cyclic depsipeptide containing 4 monomer units together with a cross-ring bridge. This compound, under the trade name of Romidepsin®, has been tested as a therapeutic in human trials and shown that it has valuable effects on a number of diseases.
Spiruchostatin A is a cyclic depsipeptide that is structurally related to FK228: it is a cyclic depsipeptide containing a tri-peptide, a statine unit and a cross-ring bridge.
Figure imgf000002_0001
Structure I Structure Il However, because both FK228 and Spiruchostatin A are natural products, they are not amenable to optimization for use as a therapeutic agent.
Analogues of Spiruchostatin A are disclosed in WO2008/062232. They may have improved HDAC inhibitory properties with respect to Spiruchostatin A or FK228 or other drug-like properties that make them more useful as medicines.
These compounds have the general structures shown in Structures III&IV wherein R1, R5, R7 and R9 are the same or different and represent hydrogen or an amino acid side chain moiety (from either a natural or an unnatural amino acid), each Ri0 is the same or different and represents hydrogen or d-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, Pr1 and Pr2 are the same or different and represent hydrogen or a thiol protecting group and Pr3 is hydrogen or an alcohol protecting group.
R r,
Figure imgf000003_0001
Structure III Structure IV Analogues of FK228 are disclosed in WO2006/129105. They may have improved HDAC inhibitory properties with respect to FK228 or other drug-like properties that make them more useful as medicines. These compounds have the general structures shown in Structures V & Vl wherein R1, R5, R7 and R9 are the same or different and represent hydrogen or an amino acid side chain moiety (from either a natural or an unnatural amino acid), each R10 is the same or different and represents hydrogen or C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, and Pr1 and Pr2 are the same or different and represent hydrogen or a thiol protecting group.
Figure imgf000004_0001
Structure V Structure Vl
Without being constrained by theory, it is believed that Structures VII and VIII are formed inside the cell from Structures I & Il respectively, by reduction of the disulfide bond, and that the 4-thio-butyl-1-ene so formed is a critical part of the mechanism of action of the compound, forming a metallophile capable of binding Zinc in the active site of HDAC.
Figure imgf000004_0002
Structure VII Structure VIII
Summary of the Invention
The present invention provides derivatives of Structures Il & VIII in which either the -CH(CH3)2 (at position 12 on the depsipeptide macrocycle - IUPAC nomenclature) and/or the -CH3 (at position 6 on the depsipeptide macrocycle - IUPAC nomenclature) is replaced by a groups such as a carbon-linked amide, sulfonamide, functionalised aryl or functionalised or unfunctionalised heteroaryl group.
The present invention also provides derivatives of structures I & VII, in which either the =CH-CH3 (at position 6 on the depsipeptide macrocycle p IUPAC nomenclature) and/or the -CH(CH3)2 (at position 12 on the depsipeptide macrocycle - IUPAC nomenclature) is replaced by groups such as a carbon- linked amide, sulfonamide, functionalised aryl or functionalised or unfunctionalised heteroaryl group. These compounds, surprisingly, are found to be effective inhibitors of HDAC enzymes, and have properties which indicate that they may have potential as treatments for human disease. These compounds are hereinafter designated members of the class of compounds called Hetero-Sidechain Depsipeptides (HSDs).
The present invention is a compound of formula IX or X:
Figure imgf000005_0001
including pharmaceutically acceptable salts thereof; wherein:
X is -C(=O)N(R10)- or -CH(OPr3) -;
R7 and R9 are the same or different and represent hydrogen or a natural or unnatural amino acid side chain moiety; each R10 is the same or different and represents hydrogen, C1-C6 alkyl,
C2-C6 alkenyl or C2-C6 alkynyl;
Pr1 and Pr2 are the same or different and represent hydrogen or a thiol protecting group;
Pr3 is hydrogen or an alcohol protecting group; R1 and R5 are the same or different and represent Y or a natural or unnatural amino acid side-chain moiety, provided that R1 and R5 are not both a natural or unnatural amino acid side chain moiety; and
Y is -(CR11R11)X-NR11C(O)NR11R11, -(CR11R11)X-NR11C(O)NR11R13, -(CR11R11)X-NR11C(O)R14, -(CR11R11)X-NR11C(O)R13, -(CR11Rn)x -NR11SO2NR11R11, -(CR11R11)X-NR11SO2NR11R13, -(CR11R11)X-NR11SO3R14, -(CR11R11)X-NR11SO2R14, -(CR11R11)X-NR11SO2R13, -(CR11R11)X-C(O)NR11R11, -(CR11R11)X-C(O)NR11R13, -(CR11R11)X-C(O)R13, -(CR11R11)X-SO2NR11R11, -(CR11R11)X-SO2NR11R13, -(CR11R11)X-SO2R13, -(CR11Rn)x-Ar; x is an integer between 1 and 10; R11 is hydrogen, Ci-C6 alkyl, aryl, C2-C6 alkenyl, C2-C6 alkynyl or heteroaryl;
R13 is NR11-C(O)R14 or NR11-SO2Ri4;
R14 is C1-C6 alkyl, aryl, C2-C6 alkenyl, C2-C6 alkynyl or heteroaryl; and
Ar is a heteroaryl or substituted aryl, with the proviso that: the compound is none of the compounds shown below:
Figure imgf000006_0001
Figure imgf000006_0002
O) (T)
The present invention further provides the use of the compounds defined above, as an inhibitor of HDAC.
Description of the Invention
Synthesis of compounds of Structures IX and X is typically conducted using amino acids of which -CO-CR-NH- forms part of the macrocycle and R is a side-chain moiety. R1, R5 and R9 may be introduced in this way. R7 may be an amino acid side chain moiety but may not have been derived directly or indirectly from an amino acid, as such. As used herein, the term "amino acid side chain moiety" refers to any side chain that may be present in a natural or unnatural amino acid. Examples of amino acid side chain moieties derived from natural or unnatural amino acids, with the amino acids from which they are derived shown in brackets, are -(CH2)2- C(O)-O-C(CH3)3 (glutamic acid f-butyl ester), -(CH2)4-NH-C(O)-O-C(CH3)3 (N6- (tert-butoxycarbonyl)-lysine), -(CH2)3-NH-C(O)NH2 (citrulline), -CH2-CH2OH (homoserine) and -(CH2)3NH2 (ornithine). Examples can also include hydrogen, CrC6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, saturated and unsaturated heterocycles, which can be functionalized or unfunctionalized. A C1-C6 alkyl group or moiety can be linear or branched. Typically, it is a
CrC4 alkyl group or moiety, for example methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl and t-butyl. Preferred examples include methyl, i-propyl and t-butyl.
A C2-C6 alkenyl group or moiety can be linear or branched. Typically, it is a C2-C4 alkenyl group or moiety. It is preferred that the alkenyl radicals are mono- or di-unsaturated, more preferably monounsaturated. Examples include vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl and 3-butenyl.
A C2-C6 alkynyl group or moiety can be linear or branched. Typically, it is a C2-C4 alkynyl group or moiety.
As used herein "aryl" means a monocyclic, bicyclic, or tricyclic monovalent aromatic radical, such as phenyl, biphenyl, naphthyl, anthracenyl, which can be optionally substituted ("substituted aryl") with up to five substituents independently selected from the group of CrC6 alkyl, hydroxy, CrC3 hydroxyalkyl, CrC3 alkoxy, CrC3 haloalkoxy, amino, CrC3 mono alkylamino, CrC3 bis alkylamino, CrC3 acylamino, CrC3 aminoalkyl, mono (CrC3 alkyl) amino CrC3 alkyl, bis (CrC3 alkyl) amino CrC3 alkyl, Ci-C3-acylamino, CrC3 alkyl sulfonylamino, halo, nitro, cyano, trifluoromethyl, carboxy, CrC3 alkoxycarbonyl, aminocarbonyl, mono d- C3 alkyl aminocarbonyl, bis CrC3 alkyl aminocarbonyl, -SO3H1 CrC3 alkylsulfonyl, aminosulfonyl, mono CrC3 alkyl aminosulfonyl and bis CrC3-alkyl aminosulfonyl.
As used herein "heteroaryl" means a monocyclic, bicyclic or tricyclic monovalent aromatic radical containing up to four heteroatoms selected from oxygen, nitrogen and sulfur, such as thiazolyl, tetrazolyl, imidazolyl, oxazolyl, isoxazolyl, thienyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, indolyl, quinolyl, isoquinolyl, said radical being optionally substituted with up to three substituents independently selected from the group of Ci-C6 alkyl, hydroxy, C1-C3 hydroxyalkyl, d-C3 alkoxy, d-C3 haloalkoxy, amino, CrC3 mono alkylamino, Ci-C3 bis alkylamino, C1-C3 acylamino, Ci-C3 aminoalkyl, mono (C1-C3 alkyl) amino Ci-C3 alkyl , bis (CrC3 alkyl) amino C1-C3 alkyl, CrC3-acylamino, C1-C3 alkyl sulfonyiamino, halo, nitro, cyano, trifluoromethyl, carboxy, C1-C3 alkoxycarbonyl, aminocarbonyl, mono d-C3 alkyl aminocarbonyl, bis C1-C3 alkyl aminocarbonyl, -SO3H, C1-C3 alkylsulphonyl, aminosulfonyl, mono CrC3 alkyl aminosulfonyl and bis CrC3-alkyl aminosulfonyl.
Preferably Y is a carbon-linked amide, sulfonamide, heteroaryl or substituted aryl. Examples include thiazole, tetrazole, imidazole, oxazole, isoxazole, thiophene, pyrazole and functionalized derivatives.
Preferably, the amino acid side chain moiety is derived from a natural amino acid. Examples of these, with the amino acids from which they are derived shown in brackets, are -H (Glycine), -CH3 (Alanine), -CH(CH3)2 (Valine), -CH2CH(CHa)2 (Leucine), -CH(CH3)CH2CH3 (Isoleucine), -(CH2)4NH2 (Lysine),
-(CH2)3NHC(=NH)NH2 (Arginine), -CH2-(5-1 H-imidazolyl) (Histidine),
-CH2CONH2 (Asparagine), -CH2CH2CONH2 (Glutamine), -CH2COOH (Aspartic acid), -CH2CH2COOH (Glutamic acid), -CH2-phenyl (Phenylalanine),
-CH2-(4-OH-phenyl) (Tyrosine), -CH2-(3-1 H-indolyl) (Tryptophan), -CH2SH (Cysteine), -CH2CH2SCH3 (Methionine), -CH2OH (Serine), and -CH(OH)CH3
(Threonine).
More preferably, each amino acid side chain is an amino acid side chain moiety present in a natural amino acid or is -(CH2)2-C(O)-O-C(CH3)3 (glutamic acid f-butyl ester), -(CH2)4-NH-C(O)-O-C(CH3)3 (Nε-(tertbutoxycarbonyl)-lysine), - (CH2)3-NH-C(O)NH2 (citrulline), -CH2-CH2OH (homoserine) or -(CH2)2-CH2NH2
(ornithine).
The groups Pr1 and Pr2 represent hydrogen or a thiol-protecting group. Said thiol-protecting group is typically:
(a) a protecting group that forms a thioether to protect a thiol group, for example a benzyl group which is optionally substituted by C1-C6 alkoxy (for example methoxy), C1-C6 acyloxy (for example acetoxy), hydroxy and nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamantyl, C1-C6 acyloxymethyl (for example pivaloyloxym ethyl, tertiary butoxycarbonyloxymethyl); (b) a protecting group that forms a monothio, dithio or aminothioacetal to protect a thiol group, for example CrC6 alkoxymethyl (for example methoxymethyl, isobutoxym ethyl), tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidine, acetamidemethyl, benzamidomethyl;
(c) a protecting group that forms a thioester to protect a thiol group, such as tertiary-butyloxycarbonyl (BOC), acetyl and its derivatives, benzoyl and its derivatives; or
(d) a protecting group that forms a carbamic acid thioester to protect a thiol group, such as carbamoyl, phenylcarbamoyl, C1-C6 alkylcarbamoyl (for example methylcarbamoyl and ethylcarbamoyl).
Typically, Pr1 and Pr2 are the same or different and each represent hydrogen or a protecting group that forms a thioether, a monothio, dithio or aminothioacetal, a thioester or a carbamic acid thioester to protect a thiol group. Preferably, Pr1 and Pr2 are the same or different and each represent hydrogen or a protecting group selected from a benzyl group which is optionally substituted by C1-C6 alkoxy (for example methoxy), C1-C6 acyloxy (for example acetoxy), hydroxy and nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamantyl, C1-C6 acyloxymethyl (for example pivaloyloxymethyl, tertiary-butyloxycarbonyloxym ethyl), C1-C6 alkoxymethyl (for example methoxymethyl, isobutoxym ethyl), tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidine, acetamidemethyl, benzamidomethyl, tertiary- butyloxycarbonyl (BOC), acetyl and its derivatives, benzoyl and its derivatives, carbamoyl, phenylcarbamoyl and C1-C6 alkylcarbamoyl (for example methylcarbamoyl and ethylcarbamoyl). Most preferably, Pr1 and Pr2 are hydrogen.
The group Pr3 represents hydrogen or a protecting group that forms an ether, an acetal or aminoacetal, an ester or a carbamic acid ester to protect a hydroxyl group. Preferably, Pr3 represents hydrogen or a protecting group selected from a benzyl group which is optionally substituted by C1-C6 alkoxy (for example methoxy), C1-C6 acyloxy (for example acetoxy), hydroxy and nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamantyl, CrC6acyloxymethyl (for example pivaloyloxymethyl, tertiary butoxycarbonyloxymethyl), CrC6alkoxymethyl (for example methoxymethyl, isobutoxymethyl), tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidine, acetamidemethyl, benzamidomethyl, tertiary-butyloxycarbonyl (BOC), acetyl and its derivatives, benzoyl and its derivatives, carbamoyl, phenylcarbamoyl and Ci-C6alkylcarbamoyl (for example methylcarbamoyl and ethylcarbamoyi). Most preferably, Pr3 is hydrogen.
Preferably, X is -CH(OPr3) and the compounds of the invention have Structures IXa and Xa:
Figure imgf000010_0001
Structure IXa Structure Xa
Preferred embodiments include Compounds Xl to XXXV: A compound according to claim 6, which is a compound shown below:
Figure imgf000010_0002
Compound XIII Compound XIV
Figure imgf000011_0001
Compound XVIII
Figure imgf000011_0002
Figure imgf000011_0003
Compound XIX Compound XX
Figure imgf000011_0004
Compound XXI Compound XXII
Figure imgf000012_0001
Compound XXV Compound XXVI
Figure imgf000012_0002
Compound XXVII Compound XXVIII
Figure imgf000012_0003
Compound XXIX Compound XXX
Figure imgf000013_0001
Compound XXXV
Prederably, X is -C(=O)N(R10)- and the compounds of the invention have Structures IXb and Xb:
Figure imgf000013_0002
Structure IXb Structure Xb
Preferred embodiments include Compounds XXXVI to LI
Figure imgf000014_0001
Compound XXXVI Compound XXXVII
Figure imgf000014_0002
Compound XXXVIII Compound XXXIX
Figure imgf000014_0003
Compound XL Compound XLI
Figure imgf000014_0004
Compound XLII Compound XLIII
Figure imgf000015_0001
Compound XLIV
Figure imgf000015_0002
Compound XLVII Compound XLVIII
Figure imgf000015_0003
Compound XLIX
Figure imgf000016_0001
Compound L Compound LI
As used herein, a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include both inorganic acids such as hydrochloric, sulfuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as citric, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic, methanesulfonic, ethanesulfonic, benzenesulfonic or p-toluenesulfonic acid. Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides and organic bases such as alkyl amines, aralkyl amines or heterocyclic amines.
As used herein, the term "isostere" refers to a compound resulting from the exchange of an atom or a group of atoms with another, broadly similar, atom or group of atoms. In the compounds of Structures IX or X, the moieties which contain isosteric groups are preferably - NR10-CHR1-CO-, -NR10-CHR9-CO-O- and -NR10-CO-CHR5-NR10-CO-CHR7-. Examples of such isosteres are compounds of Structures IX or X wherein the moiety -NH- has been replaced by -CH2-, -O- or -S-, the moiety -CO- has been replaced by -CS- or -C(=NH)- and the moiety -O- has been replaced by -S-, -CH2- or -NH-.
For the avoidance of doubt, the present invention also embraces pro-drugs which react in vivo to give a compound of the present invention or an isostere or pharmaceutically acceptable salt thereof. Said pharmaceutical composition typically contains up to 85 wt% of a compound of the invention. More typically, it contains up to 50 wt% of a compound of the invention. Preferred pharmaceutical compositions are sterile and pyrogen-free. Further, the pharmaceutical compositions provided by the invention typically contain a compound of the invention which is a substantially pure optical isomer. Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable salt of a compound of Structure IX or X or an isostere thereof.
The compounds of the invention wherein X is -CH(OPr3) - of Structure IXa and Xa can be prepared by conventional routes, for example using the following Scheme 1 wherein the functional groups are as defined above and PG represents a nitrogen protective group:
R1 R9 R10 O Rt R9
R1. N-J-γOH N PG N ",Y A" . N. A A .,OR PG O R10 OPr3 O s, R10 OPr3 O
Figure imgf000017_0001
Scheme 1 In Scheme 1 step (a), an Λ/-protected amino acid bearing the side-chain Ri is condensed with an ester enolate bearing the side chain R9 and the resulting intermediate 1 ,3-diketoester is then reduced to furnish a statine unit, wherein Pr3 is H or a removable alcohol-protecting group. In step (b), the Λ/-protecting group is removed, and the statine is coupled to a protected cysteine derivative to furnish a peptide isostere. In step (c), the Λ/-protecting group is removed, and the peptide isostere is coupled with an /V-protected amino acid bearing the side chain R5. In step (d), the Λ/-protecting group is removed, and the resulting intermediate is coupled with a functionalised β-hydroxy acid derivative wherein R15 is a temporary blocking group which can be removed to produce a compound wherein R15 is H, and X is a chiral auxiliary as reported in Yurek- George, A.; Habens, F.; Brimmell, M.; Packham, G.; Ganesan, A. J. Am. Chem. Soc.2004, 126, 1030-1031. In step (e), the ester is hydrolysed, and cyclization is facilitated in step (f), to provide a compound of the invention wherein X is -CH(OPr3) - of Structure Xa. Disulfide bond formation occurs in step (g) to provide a compound of the invention wherein X is -CH(OPr3) - of Structure IXa. The compounds of the invention wherein X is -C(=O)N(R10)- of Structures IXb and Xb may be prepared by conventional routes, for example using the following Scheme 2 wherein the functional groups are as defined above:
Figure imgf000018_0001
Scheme 2
In Scheme 2 step (a), an amino acid ester bearing the side-chain R9 is coupled with another, Λ/-protected amino acid bearing the side chain R1 (where PG represents a conventional protecting group) to furnish the /V-protected dipeptide ester. In step (b), the Λ/-protecting group is removed, and the resulting dipeptide ester is coupled to a protected cysteine. In step (c), the Λ/-protecting group is removed, and the resulting tripeptide is coupled with an amino acid bearing the side chain R5 to liberate an Λ/-protected tetrapeptide ester. In step (d), the Λ/-protecting group is removed and the resulting tetrapeptide ester is coupled with a functionalized β-hydroxy acid derivative wherein R15 is a temporary blocking group which can be removed to produce a compound wherein R15 is H, and X is a chiral auxiliary as reported in Yurek-George, A.; Habens, F.; Brimmell, M.; Packham, G.; Ganesan, A. J. Am. Chem. Soc. 2004, 126, 1030-1031. In step (e), the ester is hydrolysed, and cyclization is facilitated in step (f) to provide a compound wherein X is -C(=O)N(R10)- of the Structure Xb. Disulfide bond formation occurs in step (g) to complete the synthesis of a compound wherein X is -C(=O)N(R10)- of the Structure IXb.
Compounds of the invention of Structures IX and X in which R10 is other than hydrogen can be obtained either by alkylating a corresponding compound of the invention or intermediate in which R10 is hydrogen or by using appropriately substituted starting materials. Compounds of Structure X may be obtained by reaction of the product of step (g) of the above Schemes 1 and 2, i.e. a compound of Structure IX, to cleave the disulfide bond. The cleavage of the disulfide bond is typically achieved using a thiol compound generally used for a reduction treatment of a protein having a disulfide bond, for example mercaptoethanol, thioglycol acid, 2- mercaptoethylamine, benzenethiol, parathiocresol and dithiothreitol. Preferably, mercaptoethanol and dithiothreitol are used. An excess thiol compound can be removed by for example dialysis or gel filtration. Alternatively, electrolysis, sodium tetrahydroborate, lithium aluminum hydride or sulfite may, for example, be used to cleave the disulfide bond.
Compounds of Structure X in which Pn and/or Pr2 is other than hydrogen may be prepared by introducing a thiol-protecting group into a corresponding compound in which Pr1 and/or Pr2 is/are hydrogen. In this aspect a suitable agent for introducing thiol-protecting group to be used in this reaction is appropriately determined depending on the protecting group to be introduced. Examples include chlorides of the corresponding protecting group (for example benzyl chloride, methoxybenzyl chloride, acetoxybenzyl chloride, nitrobenzyl chloride, picolyl chloride, picolyl chloride-N-oxide, anthryl methyl chloride, isobutoxym ethyl chloride, phenylthiomethyl chloride) and alcohols of the corresponding protecting group (for example diphenylmethyl alcohol, adamanthyl alcohol, acetam idem ethyl alcohol, benzamidomethyl alcohol), dinitrophenyl, isobutylene, dimethoxymethane, dihydropyran and t-butyl chloroformate.
As the skilled person will appreciate, when one of Ri, R5, R7, R9, Ri0 carries a functional group such as -OH, -SH, -NH2 or -COOH, then it may be preferred for that group to be protected for one or more of the reaction steps following its introduction. In this case the group in question could be protected in a separate step after its introduction, or, it could be protected already at the time it is introduced. The skilled person will be aware of suitable protecting groups that can be used in this regard.
The compounds of the invention thus obtained may be salified by treatment with an appropriate acid or base. Racemic mixtures obtained by any of the above processes can be resolved by standard techniques, for example elution on a chiral chromatography column. The skilled person will appreciate that various assays are suitable for testing for HDAC inhibition and may be used to measure the activity of a compound obtained from Scheme 1 compared to that of the known HDAC inhibitor SAHA. Thus, the IC50 of a test compound against HDAC can, for example, be determined in an in vitro assay, and compared with the IC50 of SAHA under the same assay conditions. If a test compound has an IC50 value equal to or lower than that of SAHA it should be understood as having an HDAC inhibitory activity which is at least equal to that exhibited by SAHA.
In a preferred embodiment the present invention provides a process for selecting a compound which has an HDAC inhibitory activity which is at least equal to that exhibited by SAHA as defined above, wherein following completion of Scheme 1 , the next step is a an in vitro HDAC assay. Typically, said assay comprises contacting a test compound and SAHA, at various concentrations, with diluted HeLa Nuclear Extract to determine the IC50 of the test compound and of SAHA against HeLa Nuclear Extract. A test compound which has an IC50 value measured against HeLa Nuclear Extract which is equal to, or lower than, the IC50 of SAHA under the same assay conditions should be understood as having an inhibitory activity which is at least equal to that exhibited by SAHA. Typically said assay is performed using a HDAC fluorescent activity assay kit (Biomol, UK) and the test compounds are reduced prior to analysis.
In another embodiment the present invention provides a process for selecting a compound which has a human cancer cell growth inhibitory activity which is at least equal to that exhibited by SAHA, which process comprises preparing a compound of Structure IX or X via Scheme 1 as defined above followed by screening the thus obtained compound to measure its activity as a human cancer cell growth inhibitor.
The skilled person will appreciate that various assays are suitable for testing for human cancer cell growth inhibition and may be used to measure the activity of a compound obtained via Scheme 1 compared to that of SAHA. Thus, the IC50 of a test compound against human cancer cell growth can, for example, be determined in an in vitro assay, and compared with the IC50 of SAHA under the same assay conditions. If a test compound has an IC50 value equal to or lower than that of SAHA it should be understood as having an inhibitory activity which is at least equal to that exhibited by SAHA. Typically in this embodiment this step comprises an in vitro assay which comprises contacting a test compound and SAHA, at various concentrations, with an MCF7 breast, HUT78 T-cell leukaemia, A2780 ovarian, PC3 or LNCAP prostate cancer cell line to determine the IC50 of the test compound and of SAHA against the cell line. A test compound which has an IC50 value measured against any of these cell lines which is equal to, or lower than, the IC50 of SAHA under the same assay conditions should be understood as having an inhibitory activity at least equal to that of SAHA. Typically in this embodiment, said assay is performed using the CyQuantTM assay system (Molecular Probes, Inc. USA).
In another preferred embodiment the present invention provides a process for selecting a compound which has an anti-inflammatory activity which is at least equal to that exhibited by SAHA, which process comprises preparing a compound of Structure IX or X via Scheme 1 as defined above followed by screening the thus obtained compound to measure its anti-inflammatory activity.
The skilled person will appreciate that various assays are suitable for assessing the anti-inflammatory activity of a compound. The anti-inflammatory activity of a test compound relative to SAHA may, for example, be determined by measuring the activity of a compound in inhibiting the production of TNFα from peripheral blood mononuclear cells (PBMCs) relative to SAHA. Thus, the ability of a test compound to inhibit the production of TNFα from PBMCs can, for example, be determined in an assay, and compared with the activity of SAHA under the same assay conditions. If a test compound has an in vitro inhibitory activity of TNFα production which is equal to or higher than that of SAHA under the same assay conditions it should be understood as having an antiinflammatory activity which is at least equal to that exhibited by SAHA. Typically this step is performed using the Quantikine® Human-α assay kit (R&D systems, Abingdon UK).
In another aspect of this embodiment, the anti-inflammatory activity of a test compound relative to SAHA may be determined by assessing the activity of a compound in inhibiting inflammation in Balb/c mice relative to SAHA. If a test compound has an in vivo inhibitory activity which is equal to or higher than that of SAHA under the same test conditions it should be understood as having an anti-inflammatory activity which is at least equal to that exhibited by SAHA. Typically, in this embodiment this step is performed by assessing the in vivo activity of a test compound and of SAHA in inhibiting inflammation in Balb/c mice induced by a chemical challenge. Typically, said chemical challenge involves the topical administration to the mice of oxalazone or acetone. In this embodiment, the compounds under investigation may be applied before or after the chemical challenge.
In another preferred embodiment the present invention provides a process for selecting a compound which has an activity in inducing a predominant G2/M phase arrest or cell death in MCF7 cells which is at least equal to that exhibited by SAHA, which process comprises preparing a compound of Structure I or X via Scheme 1 as defined above followed by screening the thus obtained compound to measure activity in inducing a predominant G2/M phase arrest or cell death in MCF7 cells relative to SAHA.
The compounds of the present invention are found to be inhibitors of HDAC. The compounds of the present invention are therefore therapeutically useful.
The compounds of the invention and compositions comprising them may be administered in a variety of dosage forms. In one embodiment, a pharmaceutical composition comprising a compound of the invention may be formulated in a format suitable for oral, rectal, parenteral, intranasal or transdermal administration or administration by inhalation or by suppository. Typical routes of administration are parenteral, intranasal or transdermal administration or administration by inhalation.
The compounds of the invention can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. Preferred pharmaceutical compositions of the invention are compositions suitable for oral administration, for example tablets and capsules. The compounds of the invention may also be administered parenterally, whether subcutaneously, intravenously, intramuscularly, intrasternally, transdermal^ or by infusion techniques. The compounds may also be administered as suppositories.
The compounds of the invention may also be administered by inhalation. An advantage of inhaled medications are their direct delivery to the area of rich blood supply in comparison to many medications taken by oral route. Thus, the absorption is very rapid as the alveoli have an enormous surface area and rich blood supply and first pass metabolism is bypassed. A further advantage may be to treat diseases of the pulmonary system, such that delivering drugs by inhalation delivers them to the proximity of the cells which are required to be treated.
The present invention also provides an inhalation device containing such a pharmaceutical composition. Typically said device is a metered dose inhaler (MDI), which contains a pharmaceutically acceptable chemical propellant to push the medication out of the inhaler.
The compounds of the invention may also be administered by intranasal administration. The nasal cavity's highly permeable tissue is very receptive to medication and absorbs it quickly and efficiently, more so than drugs in tablet form. Nasal drug delivery is less painful and invasive than injections, generating less anxiety among patients. By this method absorption is very rapid and first pass metabolism is usually bypassed, thus reducing inter-patient variability. Further, the present invention also provides an intranasal device containing such a pharmaceutical composition. The compounds of the invention may also be administered by transdermal administration. The present invention therefore also provides a transdermal patch containing a compound of the invention, or a pharmaceutically acceptable salt thereof.
The compounds of the invention may also be administered by sublingual administration. The present invention therefore also provides a sub-lingual tablet comprising a compound of the invention or a pharmaceutically acceptable salt thereof.
A compound of the invention is typically formulated for administration with a pharmaceutically acceptable carrier or diluent. A compound of the invention may also be formulated with an agent which reduces degradation of the substance by processes other than the normal metabolism of the patient, such as anti-bacterial agents, or inhibitors of protease enzymes which might be the present in the patient or in commensural or parasite organisms living on or within the patient, and which are capable of degrading the compound.
Liquid dispersions for oral administration may be syrups, emulsions and suspensions. Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspension or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
Solutions for injection or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
The compounds of the present invention are therapeutically useful in the treatment or prevention of conditions mediated by HDAC. Accordingly, the present invention provides the use of a compound of the Structure IX or X, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment or prevention of a condition materially affected by the activity of an HDAC. Also provided is a method of treating a patient suffering from or susceptible to a condition mediated by HDAC, which method comprises administering to said patient an effective amount of a compound of Structure IX or X, an isostere thereof or a pharmaceutically acceptable salt thereof.
In one embodiment the compounds of the present invention may be used in combination with another known inhibitor of HDAC, such as SAHA. In this embodiment, the combination product may be formulated such that it comprises each of the medicaments for simultaneous, separate or sequential use. The present invention therefore also provides the use of compounds according to Structure IX or X or an isostere or pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for use in co-administration with another known inhibitor of HDAC, such as SAHA.
The compounds of the present invention can be used in both the treatment and prevention of cancer and can be used in a monotherapy or in a combination therapy. When used in a combination therapy, the compounds of the present invention are typically used together with small chemical compounds such as platinum complexes, anti-metabolites, DNA topoisomerase inhibitors, radiation, antibody-based therapies (for example herceptin and rituximab), anti-cancer vaccination, gene therapy, cellular therapies, hormone therapies or cytokine therapy.
In one embodiment of the invention a compound of the invention is used in combination with another chemotherapeutic or antineoplastic agent in the treatment of a cancer. Examples of such other chemotherapeutic or antineoplastic agents include mitoxantrone, vinca alkaloids for example vincristine and vinblastine, anthracycline antibiotics for example daunorubicin and doxorubicin, alkylating agents for example chlorambucil and melphalan, taxanes for example paclitaxel, antifolates for example methotrexate and tomudex, epipodophyllotoxins for example etoposide, camptothecins for example irinotecan and its active metabolite SN 38 and DNA methylation inhibitors for example the DNA methylation inhibitors disclosed in WO 02/085400.
According to the invention, therefore, products are provided which contain a compound of the invention and another chemotherapeutic or antineoplastic agent as a combined preparation for simultaneous, separate or sequential use in alleviating a cancer. Also provided according to the invention is the use of a compound of Structure IX or X as defined above or an isostere thereof or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the alleviation of cancer by co-administration with another chemotherapeutic or antineoplastic agent.
The compound of the invention and the said other agent may be administrated in any order. In both these cases the compound of the invention and the other agent may be administered together or, if separately, in any order as determined by a physician. HDAC is believed to contribute to the pathology and/or symptomology of several different diseases such that reduction of the activity of HDAC in a subject through inhibition of HDAC may be used to therapeutically address these disease states. Examples of various diseases that may be treated using the HDAC inhibitors of the present invention are described herein, and the use of compounds of the present invention described by Structure IX or X are included herein. It is noted that additional diseases beyond those disclosed herein may be later identified as applications of the compounds of the present invention, as the biological roles that HDAC play in various pathways becomes more fully understood. One set of indications that HDAC inhibitors of the present invention may be used to treat are those involving undesirable or uncontrolled cell proliferation. Such indications include benign tumours, various types of cancers such as primary tumours and tumour metastasis, restenosis (e.g. coronary, carotid, and cerebral lesions), abnormal stimulation of endothelial cells (atherosclerosis), insults to body tissue due to surgery, abnormal wound healing, abnormal angiogenesis, diseases that produce fibrosis of tissue, repetitive motion disorders, disorders of tissues that are not highly vascularized, and proliferative responses associated with organ transplants. More specific indications for HDAC inhibitors include, but are not limited to prostate cancer, lung cancer, acute leukaemia, multiple myeloma, bladder carcinoma, renal carcinoma, breast carcinoma, colorectal carcinoma, neuroblastoma and melanoma.
In one embodiment, a method is provided for treating diseases associated with undesired and uncontrolled cell proliferation. The method comprises administering to a subject suffering from uncontrolled cell proliferation a therapeutically effective amount of a HDAC inhibitor according to the present invention, such that said uncontrolled cell proliferation is reduced. The particular dosage of the inhibitor to be used will depend on the severity of the disease state, the route of administration, and related factors that can be determined by the attending physician. Generally, acceptable and effective daily doses are amounts sufficient to effectively slow or eliminate uncontrolled cell proliferation.
HDAC inhibitors according to the present invention may also be used in conjunction with other agents to inhibit undesirable and uncontrolled cell proliferation. Examples of other anti-cell proliferation agents that may be used in conjunction with the HDAC inhibitors of the present invention include, but are not limited to, retinoid acid and derivatives thereof, 2-methoxyestradiol, ANGIOSTATIN(TM) protein, ENDOSTATIN(TM) protein, suramin, squalamine, tissue inhibitor of metalloproteinase-l, tissue inhibitor of metalloproteinase-2, plasminogen activator inhibitor-1 , plasminogen activator inhibitor-2, cartilage- derived inhibitor, paclitaxel, platelet factor 4, protamine sulfate (clupeine), sulfated chitin derivatives (prepared from queen crab shells), sulfated polysaccharide peptidoglycan complex (sp-pg), staurosporine, modulators of matrix metabolism, including for example, proline analogs ((1-azetidine-2- carboxylic acid (LACA), cishydroxyproline, d,l-3,4-dehydroproline, thiaproline), beta.- minopropionitrile fumarate, 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; methotrexate, mitoxantrone, heparin, interferons, 2 macroglobulin-serum, chimp- 3, chymostatin, beta.-cyclodextrin tetradecasulfate, eponemycin; fumagillin, gold sodium thiomalate, d-penicillamine (CDPT), beta.-1-anticollagenase-serum, alpha.2-antiplasmin, bisantrene, lobenzarit disodium, n-(2-carboxyphenyl-4- chloroanthronilic acid disodium or "CCA", thalidomide; angostatic steroid, carboxyaminoimidazole; metalloproteinase inhibitors such as BB94. Other anti- angiogenesis agents that may be used include antibodies, preferably monoclonal antibodies against these angiogenic growth factors: bFGF, aFGF, FGF-5, VEGF isoforms, VEGF-C, HGF/SF and Ang-1/Ang-2. Ferrara N. and Alitalo, K. "Clinical application of angiogenic growth factors and their inhibitors" (1999) Nature Medicine 5: 1359-1364.
Generally, cells in benign tumours retain their differentiated 5 features and do not divide in a completely uncontrolled manner. A benign tumour is usually localized and nonmetastatic. Specific types of benign tumours that can be treated using HDAC inhibitors of the present invention include hemangiomas, hepatocellular adenoma, cavernous haemangioma, focal nodular hyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bile duct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas, myxomas, nodular regenerative hyperplasia, trachomas and pyogenic granulomas.
In the case of malignant tumors, cells become undifferentiated, do not respond to the body's growth control signals, and multiply in an uncontrolled manner. Malignant tumors are invasive and capable of spreading to distant sites (metastasizing). Malignant tumors are generally divided into two categories: primary and secondary. Primary tumors arise directly from the tissue in which they are found. Secondary tumors, or metastases, are tumors that originated elsewhere in the body but have now spread to distant organs. Common routes for metastasis are direct growth into adjacent structures, spread through the vascular or lymphatic systems, and tracking along tissue planes and body spaces (peritoneal fluid, cerebrospinal fluid, etc.).
Specific types of cancers or malignant tumors, either primary or secondary, that can be treated using the HDAC inhibitors of the present invention include, but are not limited to, leukaemia, breast cancer, skin cancer, bone cancer, prostate cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor, gallstones, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms, intestinal ganglioneuromas, hyperplastic corneal nerve tumor, marfanoid habitus tumor, Wilms' tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcoma, malignant hypercalcemia, renal cell tumor, polycythemia vera, adenocarcinoma, glioblastoma multiforme, leukemias, lymphomas, malignant melanomas, epidermoid carcinomas, and other carcinomas and sarcomas.
The HDAC inhibitors of the present invention may also be used to treat abnormal cell proliferation due to insults to body tissue during surgery. These insults may arise as a result of a variety of surgical procedures such as joint surgery, bowel surgery, and cheloid scarring. Diseases that produce fibrotic tissue include emphysema. Repetitive motion disorders that may be treated using the present invention include carpal tunnel syndrome. An example of a cell proliferative disorder that may be treated using the invention is a bone tumor. Proliferative responses associated with organ transplantation that may be treated using HDAC inhibitors of the invention include proliferative responses contributing to potential organ rejections or associated complications. Specifically, these proliferative responses may occur during transplantation of the heart, lung, liver, kidney, and other body organs or organ systems. Abnormal angiogenesis that may be may be treated using this invention include those abnormal angiogenesis accompanying rheumatoid arthritis, ischemic-reperfusion related brain edema and injury, cortical ischemia, ovarian hyperplasia and hypervascularity, (polycystic ovary syndrome), endometriosis, psoriasis, diabetic retinopathy, and other ocular angiogenic diseases such as retinopathy of prematurity (retrolental fibroplastic), macular degeneration, corneal graft rejection, neuroscular glaucoma and Oster Webber syndrome.
Examples of diseases associated with uncontrolled angiogenesis that may be treated according to the present invention include, but are not limited to retinal/choroidal neovascularization and corneal neovascularization. Examples of retinal/choroidal neovascularization include, but are not limited to, Bests diseases, myopia, optic pits, Stargarts diseases, Pagets disease, vein occlusion, artery occlusion, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum carotid apo structive diseases, chronic uveitis/vitritis, mycobacterial infections, Lyme's disease, systemic lupus erythematosus, retinopathy of prematurity, Eales disease, diabetic retinopathy, macular degeneration, Bechets diseases, infections causing a retinitis or chroiditis, presumed ocular histoplasmosis, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma and post-laser complications, diseases associated with rubesis (neovascularization of the angle) and diseases caused by the abnormal proliferation of fibrovascular or fibrous tissue including all forms of proliferative vitreoretinopathy. Examples of corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, Mooren ulcer, Terrien's marginal degeneration, marginal keratolysis, polyarteritis, Wegener sarcoidosis, Scleritis, periphigoid radial keratotomy, neovascular glaucoma and retrolental fibroplasia, syphilis, Mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections and Kaposi sarcoma.
Chronic inflammatory diseases associated with uncontrolled angiogenesis may also be treated using HDAC inhibitors of the present invention. Chronic inflammation depends on continuous formation of capillary sprouts to maintain an influx of inflammatory cells. The influx and presence of the inflammatory cells produce granulomas and thus maintains the chronic inflammatory state. Inhibition of angiogenesis using a HDAC inhibitor alone or in conjunction with other anti-inflammatory agents may prevent the formation of the granulosmas and thus alleviate the disease. Examples of chronic inflammatory diseases include, but are not limited to, inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, psoriasis, sarcoidosis, and rheumatoid arthritis.
Inflammatory bowel diseases such as Crohn's disease and ulcerative colitis are characterized by chronic inflammation and angiogenesis at various sites in the gastrointestinal tract. For example, Crohn's disease occurs as a chronic transmural inflammatory disease that most commonly affects the distal ileum and colon but may also occur in any part of the gastrointestinal tract from the mouth to the anus and perianal area. Patients with Crohn's disease generally have chronic diarrhea associated with abdominal pain, fever, anorexia, weight loss and abdominal swelling. Ulcerative colitis is also a chronic, nonspecific, inflammatory and ulcerative disease arising in the colonic mucosa and is characterized by the presence of bloody diarrhea. These inflammatory bowel diseases are generally caused by chronic granulomatous inflammation throughout the gastrointestinal tract, involving new capillary sprouts surrounded by a cylinder of inflammatory cells. Inhibition of angiogenesis by these inhibitors should inhibit the formation of the sprouts and prevent the formation of granulomas. Inflammatory bowel diseases also exhibit extra intestinal manifestations, such as skin lesions. Such lesions are characterized by inflammation and angiogenesis and can occur at many sites other the gastrointestinal tract. Inhibition of angiogenesis by HDAC inhibitors according to the present invention can reduce the influx of inflammatory cells and prevent lesion formation.
Sarcoidosis, another chronic inflammatory disease, is characterized as a multisystem granulomatous disorder. The granulomas of this disease can form anywhere in the body. Thus, the symptoms depend on the site of the granulomas and whether the disease is active. The granulomas are created by the angiogenic capillary sprouts providing a constant supply of inflammatory cells. By using HDAC inhibitors according to the present invention to inhibit angiogenesis, such granulomas formation can be inhibited. Psoriasis, also a chronic and recurrent inflammatory disease, is characterized by papules and plaques of various sizes. Treatment using these inhibitors alone or in conjunction with other anti-inflammatory agents should prevent the formation of new blood vessels necessary to maintain the characteristic lesions and provide the patient relief from the symptoms.
Rheumatoid arthritis (RA) is also a chronic inflammatory disease characterized by non-specific inflammation of the peripheral joints. It is believed that the blood vessels in the synovial lining of the joints undergo angiogenesis. In addition to forming new vascular networks, the endothelial cells release factors and reactive oxygen species that lead to pannus growth and cartilage destruction. The factors involved in angiogenesis may actively contribute to, and help maintain, the chronically inflamed state of rheumatoid arthritis. Treatment using HDAC inhibitors according to the present invention alone or in conjunction with other anti-RA agents may prevent the formation of new blood vessels necessary to maintain the chronic inflammation.
The compounds of the present invention can further be used in the treatment of cardiac/vasculature diseases such as hypertrophy, hypertension, myocardial infarction, reperfusion, ischemic heart disease, angina, arrhythmias, hypercholesterolemia, atherosclerosis and stroke. The compounds can further be used to treat neurodegenerative disorders/CNS disorders such as acute and chronic neurological diseases, including stroke, Huntington's disease, Amyotrophic Lateral Sclerosis and Alzheimer's disease. The compounds of the present invention can also be used as antimicrobial agents, for example antibacterial agents. The invention therefore also provides a compound for use in the treatment of a bacterial infection. The compounds of the present invention can be used as anti-infectious compounds against viral, bacterial, fungal and parasitic infections. Examples of infections include protozoal parasitic infections (including Plasmodium, Cryptosporidium parvum, toxoplasma gondii, sarcocystis neurona and Eimeria sp.)
The compounds of the present invention are particularly suitable for the treatment of undesirable or uncontrolled cell proliferation, preferably for the treatment of benign tumours/hyperplasias and malignant tumors, more preferably for the treatment of malignant tumors and most preferably for the treatment of CCL, breast cancer and T-cell lymphoma.
In a preferred embodiment of the invention, the compounds of the invention are used to alleviate cancer, cardiac hypertrophy, chronic heart failure, an inflammatory condition, a cardiovascular disease, a haemoglobinopathy, a thalassemia, a sickle cell disease, a CNS disorder, an autoimmune disease, diabetes, osteoporosis, MDS, benign prostatic hyperplasia, oral leukoplakia, a genentically related metabolic disorder, an infection, Rubens-Taybi, fragile X syndrome, or alpha-1 antitrypsin deficiency, or to accelerate wound healing, to protect hair follicles or as an immunosuppressant. Typically, said inflammatory condition is a skin inflammatory condition (for example psoriasis, acne and eczema), asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), Crohn's disease or colitis.
Typically, said cancer is chronic lymphocytic leukaemia, breast cancer, prostate cancer, ovarian cancer, mesothelioma or T-cell lymphoma.
Typically, said cardiovascular disease is hypertension, myocardial infarction (Ml), ischemic heart disease (IHD) (reperfusion), angina pectoris, arrhythmia, hypercholesterolemia, hyperlipidaemia, atherosclerosis, stroke, myocarditis, congestive heart failure, primary and secondary i.e. dilated (congestive) cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, peripheral vascular disease, tachycardia, high blood pressure or thrombosis.
Typically, said genentically related metabolic disorder is cystic fibrosis (CF), peroxisome biogenesis disorder or adrenoleukodystrophy. Typically, the compounds of the invention are used as an immunosuppressant following organ transplant.
Typically, said infection is a viral, bacterial, fungal or parasitic infection, in particular an infection by S aureus, P acne, Candida or aspergillus.
Typically, said CNS disorder is Huntingdon's disease, Alzheimer's disease, multiple sclerosis or amyotrophic lateral sclerosis.
In this embodiment, the compounds of the invention may be used to alleviate cancer, cardiac hypertrophy, chronic heart failure, an inflammatory condition, a cardiovascular disease, a haemoglobinopathy, a thalassemia, a sickle cell disease, a CNS disorder, an autoimmune disease, diabetes or osteoporosis, or are used as an immunosuppressant.
The compounds of the invention may also be used to alleviate chronic lymphocytic leukaemia, breast cancer, prostate cancer, ovarian cancer, mesothelioma, T-cell lymphoma, cardiac hypertrophy, chronic heart failure or a skin inflammatory condition, in particular psoriasis, acne or eczema. The compounds of the present invention can be used in the treatment of animals, preferably in the treatment of mammals and more preferably in the treatment of humans.
The compounds of the invention may, where appropriate, be used prophylactically to reduce the incidence of such conditions. A therapeutically effective amount of a compound of the invention is administered to a patient. A typical dose is from about 0.001 to 50 mg per kg of body weight, according to the activity of the specific compound, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration. EXAMPLES
Compound Xl: (EH1 S,5S.6R.9S.20R)-5-Hvdroxy-6-isopropyl-20-thiazol-4- ylmethyl^-oxa-H.^-dithia-y.ig^Σ-triaza-bicvclor/.y.βidocos-iδ-ene- 3,8,18,21 -tetraone
Figure imgf000033_0001
(2): (3S,4R)-4-{(S)-2-[(R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3- thiazol-4-yl-propionylamino]-3-tritylsulfanyl-propionylamino}-3-hydroxy-5-methyl- hexanoic acid allyl ester To a solution of 1 (410mg, 0.533 mmol, 1.0eq, prepared according to the procedure in Doi, T. et al, Tet. Lett. 2006, 47, 1177) in MeCN (5.4ml_) was added Et2NH (10%v/v, 0.6ml_) dropwise at rt under Ar(g). The solution was stirred at rt for 2h, and solvent was then removed in vacuo. The excess of amine was co-evaporated with MeCN (3 x 1OmL) before the reaction mixture was dried under high vacuum for 2h.
To a solution of Fmoc-D-4-thiazoylalanine (231 mg, 0.586 mmol, 1.1 eq) in CH2CI2 (5mL) at O0C was added PyBOP (305mg, 0.586 mmol, 1.1eq) and DIPEA (0.23ml_, 1.3 mmol, 2.5eq) under Ar(g). The crude amine, dissolved in MeCN (5mL), was added to the mixture via cannula. The reaction mixture was then left to warm to rt overnight before being concentrated in vacuo. Purification by flash column chromatography, using EtOAc/Hexane as eluant, furnished the intermediate 2 (406mg, 0.440 mmol, 83%) as a white solid.
1H-NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 8.68 (s, 1H), 7.69 (d, J=7.5Hz, 2H), 7.41-7.54 (m, 2H), 7.00-7.38 (m, 20H), 5.68-5.86 (ddt, J=16.8, 11.0, 5.8Hz, 1 H), 5.20 (d, Λ=17.1Hz, 1H), 5.10 (d, J=10.3Hz, 1H), 4.42-4.54 (m, 2H), 4.23- 4.34 (m, 2H), 4.12-4.21 (m, 1H), 4.00-4.09 (m, 2H), 3.88-4.00 (m, 2H), 3.62-3.72 (m, 1H), 3.17 (dd, J=15.6, 5.3Hz, 1H), 3.07 (dd, J=14.8, 7.0Hz, 1 H), 2.60 (dd, J=12.5, 7.0Hz, 1H), 2.47-2.55 (m, 1 H), 2.41 (dd, J=12.5, 5.8Hz, 1 H), 2.29 (dd, J=16.1 , 10.0Hz, 1 H), 1.94-2.06 (m, 1H), 0.78 (d, J=6.5Hz, 6H). MS (ES+) 945
(100%, [M+Hf).
(4): (E)-(S)-3-Hydroxy-7-tritylsulfanyl-hept-4-enoic acid
To a solution of 3 (934mg, 1.66 mmol, prepared according to the procedure in Yurek-George, A. et al, J. Am. Chem. Soc. 2004, 126, 1030) in THF (3OmL) at 0sC was added a solution of LiOH (196.1mg, 8.19 mmol) in H2O (1OmL). The reaction mixture was allowed to warm to rt over 1h whereupon 1 M HCI was added until pH=2 was reached, and EtOAc (3OmL) was added and the layers were separated. The aqueous layer was extracted with EtOAc (2OmL), the organic layers were combined, dried over MgSO4, and concentrated in vacuo. Purification by flash column chromatography using EtOAc/Hexane as eluant furnished 4 as a white solid (600mg, 1.43 mmol, 86%).
1H-NMR (300MHz, CDCI3) δH: 7.48-7.38 (m, 6H), 7.35-7.18 (m, 9H), 5.60 (m, 1 H), 5.43 (m, 1 H), 4.46 (q, J=6.28Hz, 1 H), 2.59-2.51 (m, 2H), 2.28-2.19 (m, 2H), 2.09 (q, Λ=6.47Hz, 2H). MS (ES") 417 (100%, [M-H]"). R, 0.52 EtOAc+2 drops AcOH; [α]D 27-4.15 (c 0.975, CH2CI2).
(5): (3S,4R)-3-Hydroxy-4-{(S)-2-[(R)-2-((E)-(S)-3-hydroxy-7-tritylsulfanyl-hept-4- enoylamino)-3-thiazol-4-yl-propionylamino]-3-tritylsulfanyl-propionylamino}-5- methyl-hexanoic acid allyl ester To a solution of 2 (405mg, 0.439 mmol, 1eq) in MeCN (5.4mL) was added Et2NH (10%v/v, 0.6mL) dropwise at rt under Ar(g). The solution was stirred at rt for 2h, and the solvent was subsequently removed in vacuo. The excess of amine was co-evaporated with MeCN (3 x 1OmL) before the reaction mixture was dried under high vacuum for 2h. To a solution of the β-hydroxy acid 4 (193mg, 0.461 mmol, 1.05eq) in
CH2CI2 (6mL) at O5C was added PyBOP (251 mg, 0.483 mmol, 1.1 eq) and DIPEA (200 μL, 1.1 mmol, 2.5eq) under Ar(g). The crude amine, dissolved in MeCN (8mL), was then added to the mixture via cannula. The reaction mixture was left to warm to rt over 2h before being concentrated in vacuo. Purification by flash column chromatography using 1-8% IPAZCH2CI2 as eluant, furnished 5 (482mg, 0.438 mmol, 99%) as a white solid.
1H-NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 8.59 (d, J=2.3Hz, 1 H), 6.98-7.36 (m, 31H), 5.66-5.82 (ddt, J=17.3, 10.5, 5.5Hz, 1H), 5.40 (dt, J=15.8, 6.3Hz, 1 H), 5.28 (dd, ^5.3, 6.3Hz, 1H), 5.16 (dq, JM 7.1 , 1.5Hz, 1 H), 5.07 (dq, J=10.5, 1.3Hz, 1 H), 4.52 (dd, J=6.5, 4.8Hz, 1H), 4.40-4.46 (m, 2H), 4.19 (d, J=6.6Hz, 1 H), 3.68-3.80 (m, 1 H), 3.47-3.61 (m, 3H), 3.16 (dd, J=14.8, 6.5Hz, 1H), 3.09 (dd, J=14.8, 4.5Hz, 1 H), 2.44 (dd, JM 5.8, 2.5Hz, 1H), 2.29-2.36 (m, 2H), 2.14- 2.28 (m, 3H), 2.06-2.14 (m, 2H), 1.87-2.02 (m, 3H), 0.65-0.76 (m, 6H). MS (ES+) 1124 (100%, [M+Na]+). (7): (2S,6R,9S,12R,13S)-13-Hydroxy-12-isopropyl-6-thiazol-4-ylmethyl-2-
((£)-4-tritylsulfanyl-but-1 -enyl)-9-tritylsulfanylmethyl-1 -oxa-5,8,11 -triaza- cyclopentadecane-4,7, 10, 15-tetraone
To a solution of 5 (460mg, 0.418 mmol, 1eq) in anhydrous MeOH (6ml_) was added morpholine (55 μl_, 0.627 mmol, 1.5eq) and Pd(PPh3)4 (10mg, 0.01 mmol, 0.02eq) at rt under Ar(g). The reaction mixture was stirred for 1h, and was then concentrated in vacuo. Purification by flash column chromatography, using 1-10% MeOH/CH2CI2 as eluant, furnished 6 (426mg, 0.401 mmol, 96%) as a white solid, which was used immediately in the next step. Rf 0.4 (10% MeOH/CH2CI2 + 2 drops AcOH). MS (ES+) 1083 (100%, [M+Na]+). To a solution of MNBA (152mg, 0.442 mmol) and DMAP (108mg, 0.883 mmol) in CH2CI2 (8OmL) was added dropwise a solution of acid 6 (390mg, 0.367 mmol) in CH2CI2/THF (245:5, 25OmL) over 3h under Ar(g). After 14h, the reaction mixture was concentrated in vacuo to give a yellow solid. Purification by column chromatography on silica gel, using 1-3.5% MeOH/CH2CI2 as eluant, furnished a 1 :1 inseparable mixture of 7 (120mg, 0.115 mmol, 31%) and the uncyclized methyl ester (120mg, 0.116 mmol, 30%) as a white solid.
1H-NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 8.61 (d, J=2.0Hz, 1 H), 8.55 (d, J=2.0Hz, 1H), 6.97-7.28 (m, 62H), 5.13-5.51 (m, 5H), 4.46 (dd, J=6.0, 4.8Hz, 1H), 4.34 (dd, J=7.8, 6.3Hz, 1H), 4.14 (q, J=6.0Hz, 1H), 3.86-3.96 (m, 1 H), 3.77- 3.85 (m, 1 H), 3.68-3.75 (m, 1 H), 3.47-3.59 (m, 3H), 3.47 (s, 3H), 2.94-3.16 (m, 6H), 2.81 (dd, JM 2.8, 9.8Hz, 1 H), 2.52 (dd, J=12.8, 5.5Hz, 1 H), 1.81-2.45 (m, 18H), 0.72 (d, J=6.5Hz, 3H), 0.70 (d, J=6.8Hz, 3H), 0.69 (d, J=6.0Hz, 3H), 0.66 (d, J=6.3Hz, 3H). MS (ES+) 1098 (100%, [M+Na]+), 1066 (100%, [M+Na]+). Compound Xl: (E)-(I S,5S,6R,9S,20R)-5-Hydroxy-6-isopropyl-20-thiazol-4- ylmethyl-2-oxa-11 ,12-dithia-7,19,22-triaza-bicyclo[7.7.6]docos-15-ene-3,8,18,21- tetraone
To a solution of I2 (572mg, 2.25 mmol, 10eq) in CH2CI2 (45OmL) and MeOH (5OmL) was added dropwise a solution of the 1 :1 mixture containing 7 and the uncyclized methyl ester (235mg, 0.225 mmol, 1eq) in CH2CI2 (225mL) and MeOH (25mL) over 3h at rt under Ar(g). The reaction mixture was further stirred for 20min, and a solution of sodium thiosulfate (0.1 M, 5OmL) and brine (25m L) was added. The organic layer was subsequently separated and the aqueous layer was extracted with EtOAc (3 x 5OmL). The combined organic extracts were dried over MgSO4 and the solvent was removed in vacuo. Purification by column chromatography on silica gel, using 1-5% MeOH/CH2CI2 as eluant, furnished compound Xl (42mg, 0.075 mmol, 67%) as a white solid.
1H-NMR (400MHz, CDCI3) δH: 8.76 (d, J=2.0Hz, 1 H), 7.47 (d, J=7.0Hz, 1 H), 7.23 (d, J=1.8Hz, 1H), 6.76 (d, J=9.5Hz, 1 H), 6.09-6.23 (m, 1 H), 5.75 (d, J=15.3Hz, 1 H), 5.46 (br. s, 1 H), 4.80 (td, J=Q.0, 3.5Hz, 1 H), 4.46 (dt, J=9.0, 3.8Hz, 1 H), 4.35 (dd, J=8.0, 5.3Hz, 1H), 3.19-3.37 (m, 3H), 3.11 (dd, J=12.8, 7.0Hz, 1 H), 2.77-2.98 (m, 2H), 2.73 (dt, J=8.8, 7.0Hz, 1 H), 2.48-2.64 (m, 6H), 2.21-2.42 (m, 3H), 0.93 (d, J=6.8Hz, 3H), 0.82 (d, J=6.5Hz, 3H). 13C NMR (100MHz, 9:1 CDCI3/CD3OD) δc: 171.6, 170.5, 169.1 , 153.9, 152.1 , 132.6, 129.3, 117.2, 70.8, 68.8, 63.3, 56.8, 54.7, 41.7, 40.6, 39.9, 33.7, 30.3, 29.6, 20.5, 19.4. MS (ES+) 1136 (40%, [2M+Na]+, 579 (100%, [M+Na]+).
Compound XII: (B-(1S.5S.6R.9S.20R)-5-Hvdroxy-6-isopropyl-20-(2- trifluoromethyl-benzyl)-2-oxa-11,12-dithia-7.19.22-triaza- bicvclorz.y.eidocos-IS-ene-a.β.iβ^i-tetraone
Figure imgf000036_0001
(2): (3S,4R)-4-{(S)-2-[(R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-(2- trifluoromethyl-phenyl)-propionylamino]-3-trityl sulfanyl-propionylamino}-3- hydroxy-5-methyl-hexanoic acid allyl ester
To a solution of 1 (410mg, 0.533 mmol, 1eq, prepared according to the procedure in Doi, T. et al, Tet. Lett. 2006, 47, 1177) in MeCN (5.4 mL) was added Et2NH (10%v/v, 0.6 mL) dropwise at rt under Ar(g). The solution was stirred at rt for 2h, and the solvent was subsequently removed in vacuo. The excess of amine was co-evaporated with MeCN (3 x 1OmL) before the reaction mixture was dried under high vacuum for 2h. To a solution of Fmoc-2-(trifluoromethyl)-D-phenylalanine (267 mg,
0.586 mmol, 1.1eq) in CH2CI2 (7 mL) at O9C was added PyBOP (305 mg, 0.586 mmol, 1.1 eq) and DIPEA (0.23 mL, 1.3 mmol, 2.5eq) under Ar(g). A solution of the crude amine in MeCN (8 mL) was added to the reaction mixture via cannula, and the resulting mixture was left to warm to rt overnight before being concentrated in vacuo. Purification by flash column chromatography using 10- 50% EtOAc/Hexane as eluant furnished the intermediate 2 (474 mg, 0.482 mmol, 90%) as a white solid.
1H-NMR (300MHz, 9:1 CDCI3/CD3OD) δH: 7.69 (d, J=7.5Hz, 2H), 7.56 (d, J=7.7Hz, 1H), 7.05-7.47 (m, 25H), 5.69-5.86 (ddt, J=U.2, 10.8, 5.9Hz, 1H), 5.21 (dd, J=17.0, 1.1Hz, 1 H), 5.12 (dd, J=10.4, 0.9Hz, 1H), 4.42-4.57 (m, 2H), 4.30- 4.41 (m, 1H), 4.14-4.27 (m, 1H), 4.03-4.14 (m, 1H), 3.84-4.02 (m, 3H), 3.62-3.73 (m, 1H), 3.21 (dd, JM4.5, 4.6Hz, 1 H), 2.96 (dd, J=14.1 , 10.4Hz, 1 H), 2.41-2.65 (m, 3H), 2.32 (dd, J^l 6.1 , 9.7Hz, 1H), 1.97-2.11 (m, 1H), 0.79 (t, J=6.2Hz, 6H). MS (ES+) 1006 (100%, [M+Na]+). (4): (£)-(S)-3-Hydroxy-7-trityl sulfanyl-hept-4-enoic acid
To a solution of 3 (934 mg, 1.66 mmol, prepared according to the procedure in Yurek-George, A. et al., J. Am. Chem. Soc. 2004, 126, 1030) in THF (30 mL) at 0eC was added a solution of LiOH (196.1 mg, 8.19 mmol) in H2O (10 mL). The reaction mixture was allowed to warm to rt over 1h, whereupon 1 M HCI was added until the solution reached pH2. EtOAc (30 mL) was then added, and the resulting layers were separated. The aqueous layer was extracted with EtOAc (20 mL) the organic layers were combined, dried over MgSO4, and concentrated in vacuo. Purification by flash column chromatography using EtOAc/Hexane as eluant furnished 4 as a white solid (600 mg, 1.43 mmol, 86%).
1H-NMR (300MHz, CDCI3) δH: 7.48-7.38 (m, 6H), 7.35-7.18 (m, 9H), 5.60 (m, 1 H), 5.43 (m, 1 H), 4.46 (q, J=6.28, 1H), 2.59-2.51 (m, 2H), 2.28-2.19 (m, 2H), 2.09 (q, J=6.47Hz, 2H); MS (ES ) 417 (100%, [M-H]"). Rf 0.52 EtOAc+2 drops AcOH; [α]D 27-4.15 (c 0.975, CH2CI2). (5): (3S,4R)-3-Hydroxy-4-{(S)-2-[(R)-2-((E)-(S)-3-hydroxy-7-trityl sulfanyl-hept-4- enoyiamino)-3-(2-trifluoromethyl-phenyl)-propionylamino]-3-trityl sulfanyl- propionylamino}-5-methyl-hexanoic acid allyl ester
To a solution of 2 (470 mg, 0.478 mmol, 1eq) in MeCN (5.4 ml_) was added Et2NH (10% v/v, 0.6 ml_) dropwise at rt under Ar(g). The solution was stirred at rt for 2h, and the solvent was then removed in vacuo. The excess of amine was co-evaporated with MeCN (3 x 1OmL), and the reaction mixture was dried under high vacuum for 2h.
To a solution of the β-hydroxy acid 4 (210 mg, 0.500 mmol, 1.05 eq) in CH2CI2 (7mL) at 0sC was added PyBOP (274 mg, 0.526 mmol, 1.1eq) and
DIPEA (210 μL, 1.1 mmol, 2.5eq) under Ar(g). As solution of the crude amine in
MeCN (8 ml_) was then added to the mixture via cannula, and the resulting mixture was left to warm to rt overnight before being concentrated in vacuo.
Purification by flash column chromatography using 10-70% EtOAc/Hexane as eluant furnished 5 (421 mg, 0.362 mmol, 76%) as a white solid.
1H-NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 7.57 (d, J=7.5Hz, 1H), 7.11- 7.41 (m, 35H), 6.71 (d, J=9.8Hz, 1 H), 5.74-5.87 (ddt, JM 7.1 , 10.3, 5.8Hz, 1 H),
5.40 (dt, J=15.3, 6.5Hz, 1H), 5.18-5.31 (m, 2H), 5.14 (dd, J=10.5, 1.0Hz, 1 H), 4.63 (dd, J=9.8, 4.5Hz, 1H), 4.46-4.54 (m, 2H), 4.17-4.25 (m, 1 H), 3.87-3.96 (m, 2H), 3.60-3.70 (m, 1 H), 3.29-3.39 (m, 1H), 2.98 (dd, JM 5.1 , 10.0Hz, 1 H), 2.42- 2.59 (m, 3H), 2.32 (dd, J=16.1 , 10.0Hz, 1 H), 1.93-2.21 (m, 7H), 0.80 (d, J=6.8Hz, 2H), 0.78 (d, J=7.0Hz, 3H). MS (ES+) 1185 (100%, [M+Na]+). (6): (3S,4R)-3-Hydroxy-4-{(S)-2-[(R)-2-((£)-(S)-3-hydroxy-7-trityl sulfanyl-hept-4- enoylam ino)-3-(2-trif luoromethyl-phenyl)-propionylam ino]-3-trityl sulf anyl- propionylaminoJ-5-methyl-hexanoic acid
To a solution of 5 (418mg, 0.356 mmol, 1eq) in anhydrous MeOH (6mL) was added morpholine (47 μL, 0.54 mmol, 1.5eq) and Pd(PPh3)4 (8.3 mg, 0.007 mmol, 0.02eq) at rt under Ar(g). The reaction mixture was stirred for 2h, and was then concentrated in vacuo. Purification by flash column chromatography using 1-10% MeOH/CH2CI2 as eluant furnished 6 (382 mg, 0.340 mmol, 96%) as a white solid.
1H-NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 7.57 (d, J=7.7Hz, 1 H), 7.08-
7.41 (m, 35H), 6.70 (d, J=9.9Hz, 1 H), 5.39 (dt, JM 5.4, 6.5Hz, 1 H), 5.19-5.30 (m, 1 H), 4.62 (dd, J=9.9, 4.8Hz, 1 H), 4.16-4.23 (m, 1 H), 3.86-3.99 (m, 2H), 3.61- 3.68 (m, 1 H), 3.13 (d, J=6.3Hz, 1H), 2.99 (dd, JM 5.0, 9.9Hz, 1 H), 2.58 (br. s, 2H), 2.42-2.55 (m, 3H), 2.25 (dd, J=16.0, 9.6Hz, 1 H), 1.93-2.19 (m, 7H), 0.78 (t, J=6.3Hz, 6H). MS (ES+) 1145 (100%, [M+Na]+).
(7): (2S,6R,9S,12R,13S)-13-Hydroxy-12-isopropyl-6-(2-trifluoromethyl-benzyl)-2- ((E)-4-trityl sulfanyl-but-1 -enyl)-9-trityl sulfanylmethyl-1-oxa-5,8,11-triaza- cyclopentadecane-4,7, 10, 15-tetraone
To a solution of MNBA (140 mg, 0.407 mmol) and DMAP (99 mg, 0.81 mmol) in CH2CI2 (8OmL) was added dropwise a solution of acid 6 (380 mg, 0.339 mmol) in CH2CI2ZTHF (245:5, 250 mL) over 3h under Ar(g). After a further 14h, the reaction mixture was concentrated in vacuo to give an off-white solid. Purification by column chromatography on silica gel using1-5% IPA/CH2CI2 as eluant gave 7 (191 mg, 0.173 mmol, 51%) as a white solid.
1H-NMR (300MHz, 9:1 CDCI3/CD3OD) δH: 7.49 (d, J=7.2Hz, 1 H), 7.22 (s, 34H), 5.49 (dt, JM 5.1 , 6.6Hz, 1 H), 5.18-5.38 (m, 2H), 4.34 (t, J=7.9Hz, 1H), 3.81-3.90 (m, 1 H), 3.67-3.74 (m, 1H), 3.08-3.25 (m, 2H), 2.84-3.06 (m, 2H), 2.70-2.82 (m, 2H), 2.35 (dd, J=14.3, 11.1 Hz, 1 H), 2.18-2.28 (m, 2H), 1.87-2.14 (m, 5H), 0.75 (d, Λ=6.8Hz, 3H), 0.74 (d, J=6.6Hz, 3H). MS (ES+) 1127 (100%, [M+Na]+).
Compound XII: (E)-(I S,5S,6R,9S,20R)-5-Hydroxy-6-isopropyl-20-(2- trif luoromethyl-benzyl)-2-oxa-11 , 12-dithia-7, 19,22-triaza-bicyclo[7.7.6]docos-15- ene-3,8,18,21-tetraone
To a solution of I2 (425mg, 1.68 mmol, 10eq) in CH2CI2 (360 mL) and MeOH (40 mL) was added dropwise a solution of 7 (185mg, 0.168 mmol, 1eq) in CH2CI2 (180 mL) and MeOH (20 mL) over 3h at rt under Ar(g). The reaction mixture was further stirred for 20 min, and was then treated with a solution ofς sodium thiosulfate (0.1 M, 50 mL) and brine (25 mL). The organic layer was isolated, and the aqueous layer was extracted with EtOAc (3 x 50 mL). The combined organic extracts were dried over MgSO4 and the solvent was removed in vacuo. Purification by column chromatography on silica gel using 1-3.5% MeOH/CH2CI2 gave compound XII (96 mg, 0.155 mmol, 93%) as a white solid.0 1H-NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 7.57 (d, Λ=8.0Hz, 1 H), 7.40-
7.50 (m, 2H), 7.22-7.38 (m, 3H), 6.86 (d, J=9.0Hz, 1H), 6.00-6.14 (m, 1 H), 5.62 (d, J=15.3Hz, 1 H), 5.39 (br. s, 1 H), 4.70-4.81 (m, 1 H), 4.45 (dt, J=9.3, 3.5Hz, 1 H), 4.31-4.39 (m, 1 H), 3.06-3.34 (m, 5H), 3.02 (dd, J=13.1 , 7.0Hz, 1H), 2.51- 2.75 (m, 5H), 2.47 (d, J=14.1 Hz, 1 H), 2.17-2.40 (m, 2H), 0.90 (d, J=7.0Hz, 3H),5 0.79 (d, J=6.8Hz, 3H).13C NMR (100MHz, 9:1 CDCI3/CD3OD) δc: 171.9, 171.3, 170.4, 169.0, 134.3, 132.4, 131.2, 129.5, 128.5 (q, 2 JC-F =29.3HZ), 127.5, 126.1 (q, 3Jc-F =5.8Hz,), 124.6 (q, 1Jc-F=273.0Hz,), 70.6, 68.3, 62.9, 58.0, 55.2, 41.2, 40.1 , 39.9, 32.7, 32.2, 29.5, 20.4, 19.1. MS (ES+) 618 (100%, [M+Na]+). Compound XIII: (EH1 S,5S,6R,9S,20R)-20-(4-Fluoro-benzvn-5-hvdroxy-β- isopropyl-2-oxa-11,12-dithia-7,19,22-triaza-bicyclor7.7.61docos-15-ene- 3,8,18,21 -tetraone
Figure imgf000040_0001
(2): (3S,4R)-4-{(S)-2-[(R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-(4-fluoro- phenyl)-propionylamino]-3-trityl sulfanyl-propionylamino}-3-hydroxy-5-methyl- hexanoic acid allyl ester
To a solution of 1 (0.31 g, 0.40 mmol, 1eq) in MeCN (8 ml.) was added diethylamine (0.8 ml_, 10%v/v) dropwise at rt. After 1h, the solution was concentrated in vacuo and co-evaporated with MeCN (2 x 5 ml_), then CH2Cl2/hexane (5mL). The resultant oil was then dried under high vacuum for 3h.
To a solution of Fmoc-D-4-F-PheAla-OH (180mg, 0.44 mmol, 1.1 eq) and PyBOP (231 mg, 0.44 mmol, 1.1 eq) in MeCN (7 mL) was added DIPEA (0.19 mL, 1.1 mmol, 2.5eq) at O9C in dropwise fashion. After 5 min, a solution of the crude amine in CH2CI2 (7 mL) was added in dropwise fashion, and the resulting solution was then warmed to rt overnight. The solvent was removed in vacuo. Purification by column chromatography on silica using hexane/EtOAc (1 :1) as eluant yielded 2 (0.37g, 0.40 mmol, 99%) as a white solid.
1H-NMR (300MHz, CDCI3 + MeOD (10%)) δH: 7.78 (d, J=7.5Hz, 2H), 7.51 (d, J=7.0Hz, 2H), 7.36-7.45 (m, 9H), 7.16-7.34 (m, 12H), 7.06-7.15 (m, 2H), 6.91 (t, J=8.5Hz, 2H), 6.73 (d, J=9.7Hz, 1H), 5.78-5.94 (ddt, J=M. Q, 10.7, 5.7Hz, 1 H), 5.29 (d, J=17.2Hz, 1H), 5.20 (d, J=10.6Hz, 1 H), 4.56 (br. s, 2H), 4.38 (br. s, 2H), 4.20-4.31 (m, 1H), 4.13 (s, 1 H), 3.96-4.08 (m, 2H), 3.03 (dd, J=UA, 5.2Hz, 1 H), 2.88 (dd, JM 3.3, 7.9Hz, 1 H), 2.51-2.70 (m, 3H), 2.41 (dd, JM 6.0, 9-7Hz, 1 H), 2.07-2.20 (m, 1 H), 0.84-0.96 (m, 6H). MS (ES+) 956.9 (100%, [M+Na]+). Rf (hexanes/EtOAc, 1 :1) = 0.40. (4): (3S,4R)-4-{(S)-2-[(R)-3-(4-Fluoro-phenyl)-2-((E)-(S)-3-hydroxy-7-trityl sulfanyl-hept-4-enoylamino)-propionylamino]-3-trityl sulfanyl-propionylamino}-3- hydroxy-5-methyl-hexanoic acid allyl ester
To a solution of 2 (0.38 g, 0.40 mmol, 1eq) in MeCN (8 ml_) at rt was added diethylamine (0.8 mL, 10%v/v) in dropwise fashion. After 1h, the solution was concentrated in vacuo and co-evaporated with MeCN (2 x 5 mL), and then CH2CI2/hexane (5 mL). The resultant oil was then dried under high vacuum 3h.
To a solution of (E)-(S)-3-hydroxy-7-trityl sulfanyl-hept-4-enoic acid, 3, (179 mg, 0.43 mmol, 1.05eq) and PyBOP (233 mg, 0.45 mmol, 1.1 eq) in MeCN (7 mL) at O9C was added DIPEA (0.18 mL, 1.0 mmol, 2.5eq) in dropwise fashion. After 5 min, the crude amine solution in CH2CI2 (7mL) was added dropwise to the reaction mixture. The solution was then warmed to rt overnight, the solvent was removed in vacuo, and the crude product was purified by column chromatography on silica using hexane/EtOAc (2:3) as eluant; 4 (0.41 g, 0.37 mmol, 91 %) was furnished as a white solid. 1H-NMR (300MHz, CDCI3 + MeOD (10%)) δH: 7.03-7.48 (m, 31H), 6.73-
6.94 (m, 3H), 5.71-5.94 (m, 1 H), 5.10-5.51 (m, 4H), 4.46-4.59 (m, 3H), 4.21-4.30 (m, 1H), 3.89-4.01 (m, 2H), 3.61-3.75 (m, 1 H), 3.00-3.12 (m, 1 H), 2.80-2.92 (m, 1H), 2.53-2.64 (m, 1H), 2.42-2.52 (m, 2H), 2.27-2.41 (m, 1 H), 2.13-2.23 (m, 3H), 1.98-2.09 (m, 2H), 0.80 (t, J=6.0Hz, 6H). MS (ES+) 1134.5 (100%, [M+Na]+). Rf (hexanes/EtOAc, 1 :1) = 0.20.
(5): (3S,4R)-4-{(S)-2-[(R)-3-(4-Fluoro-phenyl)-2-((E)-(S)-3-hydroxy-7-trityl sulfanyl-hept-4-enoylamino)-propionylamino]-3-trityl sulfanyl-propionylamino}-3- hydroxy-5-methyl-hexanoic acid
To a solution of 4 (0.41 g, 0.37 mmol, 1eq) in THF (1.OmL) and MeOH (7.4 mL) was added at rt Pd(PPh3)4 (8.6 mg, 7.4 μmol, 0.02eq) and morpholine (48 μL, 0.55 mmol, 1.5eq). After 2h, the solvent was removed in vacuo, and the crude product was purified by column chromatography on silica using CH2CI2/MeOH (19:1)/AcOH (0.1%); 5 (0.36 g, 0.33 mmol, 89%) was isolated as a white solid. 1H-NMR (300MHz, CDCI3) δH: 6.60-7.69 (m, 34H), 5.08-5.56 (m, 3H), 4.51 (br. s, 2H), 3.81-4.05 (m, 2H), 3.57-3.76 (m, 3H), 2.77-3.20 (m, 3H), 2.35- 2.68 (m, 5H), 1.94-2.30 (m, 8H), 0.61-0.91 (m, 6H). MS (ES+) 1094.7 (100%, [M+Na]+). Rf (CH2CL2/Me0H, 19:1) = 0.35. (6): (2S.6R.9S, 12R, 13S)-6-(4-Fiuoro-benzyl)-13-hydroxy-12-isopropyl-2-((E)-4- trityl sulfanyl-but-1 -enyl)-9-trityl sulfanylmethyl-1-oxa-5,8,11-triaza- cyclopentadecane-4,7, 10, 15-tetraone
To a solution of MNBA (135 mg, 0.39 mmol, 1.2eq) and DMAP (96 mg, 0.79 mmol, 2.4eq) in CH2CI2 (270 ml_) was added a solution of 4 (0.36 g, 0.33 mmol, 1eq) in CH2CI2 (90 ml_) dropwise over 3h. The reaction mixture was stirred at rt overnight, and the solvent was subsequently removed in vacuo. Purification by column chromatography on silica using CH2CI2/Me0H (19:1) as eluant yielded 6 (220 mg, 0.21 mmol, 66%) as a white solid.
1H-NMR (300MHz, CDCI3) δH: 6.70-7.55 (m, 34H), 5.30-5.71 (m, 3H), 4.21-4.47 (m, 1 H), 3.86-4.09 (m, 1 H), 3.22-3.42 (m, 1 H), 2.76-3.05 (m, 4H), 2.26-2.52 (m, 4H), 1.96-2.25 (m, 5H), 0.84-0.96 (m, 6H). MS (ES+) 1076.9 (100%, [M+Na]+). Rf (CH2CL2:MeOH, 19:1) = 0.40.
Compound XIII: (E)-(I S,5S,6R,9S,20R)-20-(4-Fiuoro-benzyl)-5-hydroxy-6- isopropyl-2-oxa-11 ,12-dithia-7,19,22-triaza-bicyclo[7.7.6]docos-15-ene- 3,8,18,21 -tetraone
To a solution of I2 (0.52 g, 2.1 mmol, 10eq) in CH2CI2/Me0H (9:1 , 750 ml_) was added a solution of 6 (220 mg, 0.21 mmol, 1eq) dropwise over 2h at rt. The mixture was then quenched with a solution of Na2S2O3 (0.1 M, 80 ml_) and brine (25 ml_), and the aqueous layer was extracted with CH2CI2 (2 x 50 mL) and EtOAc (50 mL). The combined organic extracts were dried over MgSO4 and the solvent was removed in vacuo. Purification by column chromatography on silica using CH2CI2/'PrOH (32:1) as eluant furnished compound XIII (81 mg, 0.14 mmol, 70%) as a white solid.
1H-NMR (300MHz, CDCI3) δH: 7.32 (d, J=7.3Hz, 1H), 7.12-7.20 (m, 2H), 7.04 (t, J=8.6Hz, 2H), 6.83 (d, J=9.1Hz, 1H), 6.24-6.39 (m, 1H), 6.12-6.20 (m, 1H), 5.56 (d, J=15.3Hz, 1H), 5.41-5.49 (m, 1 H), 4.87 (td, J=8.7, 3.4Hz, 1 H), 4.39-4.57 (m, 2H), 3.33-3.48 (m, 1 H), 3.13-3.31 (m, 3H), 2.97-3.09 (m, 2H), 2.75-2.85 (m, 2H), 2.60-2.73 (m, 4H), 2.51 (d, J=13.0Hz, 1H), 2.26-2.43 (m, 2H), 1.00 (d, J=6.8Hz, 3H), 0.90 (d, J=6.7Hz, 3H); 13C NMR (75MHz, CDCI3) δc: 171.5, 171.0, 169.8, 168.9, 162.2 (d, 1J0-F =247.0Hz), 133.3, 131.1 (d, 4J0-F =3.2Hz), 130.2 (d, 3Jc-F =8.0Hz, 2C), 128.5, 116.2 (d, 2JC-F=21.3Hz, 2C), 70.5,
68.9, 63.2, 56.9, 54.9, 40.4, 40.2, 39.5, 35.6, 29.6, 25.3, 20.5, 19.5. 19F NMR
(282MHz, CDCI3) δF-: 114.29 (s, 1 F). MS (ES+) 590.6 (100%, [M+Na]+). R,
(CH2CL2/Me0H, 19:1) = 0.40.
Compound XIV: ( EH1 S.5S.6R.9S,20R>-5-Hvdroxy-6-isopropyl-20-(4-methyl- benzyl)-2-oxa-11.12-dithia-7,19,22-triaza-bicvclor7.7.61docos-15-ene-
3,8,18,21 -tetraone
Figure imgf000043_0001
(2): (3S,4fl)-4-{(S)-2-[(f?)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-p-tolyl- propionylamino]-3-trityl sulfanyl-propionylaminoJ-S-hydroxy-δ-methyl-hexanoic acid allyl ester
To a solution of 1 (0.4Og, 0.52 mmol, 1eq) in MeCN (1OmL) was added diethylamine (1.OmL, 10%v/v) dropwise at rt. After 1h, the solution was concentrated in vacuo and co-evaporated with MeCN (2 x 5m L), and then with CH2Cl2/hexane (5mL). The resultant oil was then dried under vacuum for 3h.
To a solution of Fmoc-D-4-MePheAla-OH (219mg, 0.55 mmol, 1.05eq) and PyBOP (298mg, 0.57 mmol, 1.1eq) in MeCN (9mL) was added DIPEA
(0.23mL, 1.3 mmol, 2.5eq) dropwise at O9C. After 5 min, the crude amine solution in CH2CI2 (9mL) was added in dropwise fashion, and the solution was subsequently warmed to rt overnight. The solvent was removed in vacuo.
Purification by column chromatography on silica (hexanes/EtOAc, 1:1) yielded
2a (0.42 g, 0.45 mmol, 87%) as a white solid.
1H-NMR (300MHz, CDCI3 + 10% MeOD) δH: 7.72 (d, J=7.5Hz, 2H), 6.93-
7.51 (m, 26H), 5.70-5.88 (m, 1 H), 5.09-5.27 (m, 2H), 4.46-4.53 (m, 2H), 4.22- 4.36 (m, 2H), 4.09-4.20 (m, 1 H), 3.88-4.00 (m, 2H), 3.63-3.72 (m, 1 H), 2.73-3.04
(m, 2H), 2.26-2.63 (m, 4H), 2.22 (s, 3H), 1.97-2.11 (m, 1H), 0.74-0.89 (m, 6H).
MS (ES+) 953.1 (100%, [M+Na]+). R, (hexanes/EtOAc, 1 :1) = 0.25. (3): (3S,4R)-3-Hydroxy-4-{(S)-2-[(R)-2-((E)-(S)-3-hydroxy-7-trityl sulfanyl-hept-4- enoylamino)-3-p-tolyl-propionylamino]-3-trityl sulfanyi-propionylamino}-5-methyl- hexanoic acid allyl ester
To a solution of 2 (0.42 g, 0.45 mmol, 1eq) in MeCN (8ml_) at rt was added diethylamine (O.δmL, 10% v/v) in dropwise fashion. After 1h, the solution was concentrated in vacuo and co-evaporated with MeCN (2 x 5ml_), and then with CH2CI2/hexane (5mL). The resultant oil was then dried under vacuum for
3h.
To a solution of (E)-(S)-3-Hydroxy-7-trityl sulfanyl-hept-4-enoic acid (199mg, 0.48 mmol, 1.05eq) and PyBOP (259mg, 0.50 mmol, 1.1 eq) in MeCN (7ml_) was added DIPEA (0.2OmL, 1.1 mmol, 2.5eq) in dropwise fashion at 0QC. After 5 min, a solution f the crude amine in CH2CI2 (7mL) was added in dropwise fashion, and the reaction mixture was then warmed to rt overnight. The solvent was removed in vacuo. Purification by column chromatography on silica using hexane/EttOAc (1 :1) as eluant yielded 3 (0.43 g, 0.39 mmol, 86%) as a white solid.
1H-NMR (400MHz, CDCI3 + 10% MeOD) δH: 6.92-7.46 (m, 27H), 5.76- 5.88 (m, 1 H), 5.36-5.47 (m, 1 H), 5.12-5.33 (m, 3H), 3.89-3.99 (m, 1 H), 3.61-3.70 (m, 1H), 3.02 (dd, J=14.3, 4.9Hz, 1H), 2.84 (dd, JM4.2, 8.5Hz, 1 H), 2.43-2.62 (m, 3H), 2.28-2.37 (m, 1 H), 2.23 (s, 3H), 2.12-2.18 (m, 4H), 1.97-2.08 (m, 3H), 0.80 (at, J=7.1 Hz, 6H). MS (ES+) 1131.2 (100%, [M+Na]+). R, (hexanes/EtOAc, 1 :1) = 0.20.
(4): (3S,4fl)-3-Hydroxy-4-{(S)-2-[(f?)-2-((E)-(S)-3-hydroxy-7-trityl sulfanyl-hept-4- enoylamino)-3-p-tolyl-propionylamino]-3-trityl sulfanyl-propionylamino}-5-methyl- hexanoic acid
To a solution of 3 (0.43g, 0.39 mmol, 1eq) in THF (1.3mL) and MeOH (7.8mL) was added Pd(PPh3)4 (9mg, 7.8μmol, 0.02eq) and morpholine (51 μL, 0.58 mmol, 1.5eq) at rt. After 2h, the solvent was removed in vacuo. Purification by column chromatography on silica using CH2CI2/MeOH (19:1)/AcOH (0.1%) as eluant yielded 4 (0.40 g, 0.36 mmol, 86%) as a white solid.
1H NMR (400MHz, CDCI3+10% MeOD) δH: 6.95-7.48 (m, 32H), 6.45 (d, J=9.3Hz, 1 H), 6.19 (d, J=6.3Hz, 1 H), 5.20-5.43 (m, 2H), 4.48-4.56 (m, 1 H), 4.22- 4.32 (m, 1 H), 4.03-4.16 (m, 2H), 3.73-3.82 (m, 1 H), 3.04-3.13 (m, 1 H), 2.95 (dd, J=14.3, 8.1 Hz, 1 H), 2.66 (dd, J=12.6, 7.3Hz, 1 H), 2.52-2.60 (m, 1H), 2.40-2.51 (m, 2H), 2.29 (s, 3H), 2.16-2.26 (m, 3H), 1.99-2.08 (m, 3H), 0.89 (t, Λ=6.7Hz, 6H). MS (ES+) 1091.0 (100%, [M+Na]+). R, (CH2CI2/Me0H, 19:1) = 0.25. (5): (2S,6R,9S, 12R, 13S)-13-Hydroxy-12-isopropyl-6-(4-methyl-benzyl)-2-((£)-4- trityl sulfanyl-but-1 -enyl)-9-trityl sulfanylmethyl-1-oxa-5,8,11-triaza- cyclopentadecane-4,7, 10,15-tetraone
To a solution of MNBA (149mg, 0.43 mmol, 1.2eq) and DMAP (106mg, 0.87 mmol, 2.4eq) in CH2CI2 (27OmL) was added a solution of 4 (0.40 g, 0.36 mmol, 1eq) in CH2CI2 (13OmL) in dropwise fashion over 3h. The reaction mixture was then stirred at rt overnight. The solvent was removed in vacuo, and purification by column chromatography on silica using CH2CI2/Me0H (49:1 -> 32:1) as eluant yielded 5 (286mg, 0.27 mmol, 75%) as a white solid.
1H NMR (400MHz, CDCI3+10% MeOD) δH: 7.09-7.38 (m, 30H), 6.93 (d, J=8.0Hz, 2H), 6.85 (d, J=8.0Hz, 2H), 5.46-5.57 (m, 1H), 5.32-5.41 (m, 1H), 5.21- 5.29 (m, 1H), 2.84-2.91 (m, 2H), 2.63-2.82 (m, 3H), 2.20-2.41 (m, 4H), 2.15 (s, 3H), 2.06-2.13 (m, 3H), 1.92-2.02 (m, 2H), 0.79 (dd, J=17.3, 6.8Hz, 6H). MS (ES+) 1073.2 (100%, [M+Na]+). Rf (CH2CL2:MeOH, 19:1) = 0.50. Compound XIV: (E)-(I S,5S,6/:?,9S,20f?)-5-Hydroxy-6-isopropyl-20-(4-methyl- benzyl)-2-oxa-11 , 12-dithia-7, 19,22-triaza-bicyclo[7.7.6]docos-15-ene-3,8, 18,21- tetraone To a solution of I2 (0.69 g, 2.72 mmol, 10eq) in CH2CI2/MeOH (9:1 , 1.0L) was added at rt a solution of 5 (286mg, 0.27 mmol, 1eq) over 2h in dropwise fashion. The reaction mixture was quenched with a solution of Na2S2Oa (0.1 M, 25OmL) and brine (25mL), and the aqueous layer was extracted with CH2CI2 (2 x 10OmL) and EtOAc (10OmL). The combined organic extracts were dried over MgSO4 and the solvent was removed in vacuo. Purification by column chromatography on silica using CH2CI2/MeOH (32:1) as eluant yielded compound XIV (118mg, 0.21 mmol, 77%) as a white solid.
1H NMR (400MHz, CDCI3) δH: 7.46 (d, J=7.0Hz, 2H), 7.01 (d, Λ=8.0Hz, 2H), 6.98 (d, J=8.0Hz, 2H), 6.74 (d, J=9.0Hz, 1H), 6.04 (dd, J=13.8, 11.6Hz, 1 H), 5.59 (d, J=15.4Hz, 1 H), 5.33-5.40 (m, 1H), 4.68-4.76 (m, 1 H), 4.40 (t, J=3.6Hz, 1 H), 4.25-4.33 (m, 1H), 2.85-3.06 (m, 4H), 2.65-2.83 (m, 2H), 2.41- 2.55 (m, 5H), 2.20-2.24 (m, 1H), 2.19 (S1 3H), 0.88 (d, J=6.8Hz, 3H), 0.77 (d, J=6.7Hz, 3H); 13C NMR (100MHz, CDCI3+10% MeOD) δc: 170.0, 169.1 , 169.0, 167.0, 134.9, 130.2, 130.0, 127.4 (2C), 127.2, 126.5 (2C), 68.4, 66.2, 60.7, 55.3, 52.7, 38.4, 37.8, 33.4, 31.1 , 29.3, 27.4, 20.0, 18.6, 18.2, 17.0;. MS (ES+) 586.9 (100%, [M+Na]+). fl, (CH2CLaZMeOH, 19:1) = 0.30.
Compound XV: (EH1 S,5S.6R,9S,20RV5-H vdroxy-β-isopropyl-20-(3-methyl- benzyl)-2-oxa-11 ,12-dithia-7,19,22-triaza-picvclof7.7.β1docos-15-ene- 3,8,18,21 -tetraone
Figure imgf000046_0001
(2): (3S,4/:?)-4-{(S)-2-[(f?)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-m-tolyl- propionylamino]-3-trϊtyl sulfanyl-propionylaminoJ-S-hydroxy-δ-methyl-hexanoic acid ally! ester To a solution of 1 (0.40 g, 0.52 mmol, 1eq) in MeCN (1OmL) was added diethylamine (1.OmL, 10% v/v) in dropwise fashion at rt. After 1h, the solution was concentrated in vacuo and co-evaporated with MeCN (2 x 5mL), and then with CH2CI2/hexane (5mL). The resultant oil was then dried under vacuum for 3h. To a solution of Fmoc-D-3-MePheAla-OH (219mg, 0.55 mmol, 1.05eq) and PyBOP (298mg, 0.57 mmol, 1.1 eq) in MeCN (9mL) was added diisopropylethylamine (0.23mL, 1.3 mmol, 2.5eq) in dropwise fashion at O9C. After 5 min, a solution of the crude amine in CH2CI2 (9mL) was added dropwise to the reaction mixture. The solution was then warmed rt overnight, and the solvent was subsequently removed in vacuo. Purification by column chromatography on silica using hexane/EtOAc (3:2) as eluant yielded 2 (0.39 g, 0.45 mmol, 81%) as a white solid.
1H NMR (300MHz, CDCI3+10% MeOD) δH: 7.71 (d, J=7.5Hz, 2H), 6.83- 7.51 (m, 26H), 5.70-5.88 (m, 1 H), 5.09-5.26 (m, 2H), 4.45-4.53 (m, 2H), 4.20- 4.36 (m, 2H), 4.09-4.18 (m, 1 H), 3.88-3.99 (m, 2H), 3.63-3.73 (m, 1 H), 2.71-3.05 (m, 2H), 2.42-2.64 (m, 3H), 2.26-2.39 (m, 1H), 2.22 (s, 3H), 2.00-2.10 (m, 1 H), 0.75-0.84 (m, 6H). MS (ES+) 952.9 (100%, [M+Na]+). Rf (hexanes/EtOAc, 1 :1) = 0.35.
(3): (3S,4F7)-3-Hydroxy-4-{(S)-2-[(fl)-2-((£)-(S)-3-hydroxy-7-trityl sulfanyl-hept-4- enoylam ino)-3-/77-tolyl-propionylam ino]-3-trityl suit anyl-propionylam ino}-5-methyl- hexanoic acid allyl ester
To a solution of 2 (0.39g, 0.42 mmol, 1eq) in MeCN (8mL) was added at rt diethylamine (0.8OmL, 10% v/v) in dropwise fashion. After 1h, the solution was concentrated in vacuo and co-evaporated with MeCN (2 x 5mL), then CH2CI2/hexane (5ml_). The resultant oil was then dried under vacuum for 3h. To a solution of (E)-(S)-3-hydroxy-7-trityl sulfanyl-hept-4-enoic acid
(184mg, 0.44 mmol, 1.05eq) and PyBOP (240mg, 0.46 mmol, 1.1 eq) in MeCN (7ml_) was added at 00C diisopropylethylamine (0.18mL, 1.1 mmol, 2.5eq) dropwise. After 5 min, a solution of the crude amine in CH2CI2 (7ml_) was added dropwise to the reaction mixture. The solution was then warmed to rt overnight, and the solvent was then removed in vacuo. Purification by column chromatography on silica using hexane/EtOAc (1 :1) furnished 3 (0.40 g, 0.36 mmol, 86%) as a white solid.
1H NMR (400MHz, CDCI3+10% MeOD) δH: 7.12-7.39 (m, 29H), 7.01-7.08 (m, 1H), 6.87-6.98 (m, 3H), 6.66-6.73 (m, 1 H), 5.75-5.87 (m, 1H), 5.35-5.44 (m, 1H), 5.11-5.31 (m, 3H), 4.46-4.53 (m, 3H), 4.17-4.25 (m, 1H), 3.89-3.95 (m, 2H), 3.61-3.69 (m, 1 H), 3.04 (dd, Jt=14.2, 4.9Hz, 1 H), 2.80 (dd, JM4.2, 8.8Hz, 1 H), 2.55 (dd, J=15.9, 2.4Hz, 1 H), 2.48 (d, J=IA Hz, 1 H), 2.32 (dd, J=15.9, 9.9Hz, 1 H), 2.23 (S, 3H), 2.11-2.18 (m, 4H), 1.97-2.06 (m, 2H), 0.79 (t, Λ=6.5Hz, 6H). MS (ES+) 1131.3 (100%, [M+Na]+). Rf (hexanes/EtOAc, 1 :1) = 0.30. (4): (3S,4f?)-3-Hydroxy-4-{(S)-2-[(H)-2-((£)-(S)-3-hydroxy-7-trityl sulfanyl-hept-4- enoylamino)-3-m-tolyl-propionylamino]-3-trityl sulfanyl-propionylamino}-5-methyl- hexanoic acid
To a solution of 3 (0.40 g, 0.36 mmol, 1eq) in THF (1.2mL) and MeOH (7.2mL) was added Pd(PPh3)4 (8mg, 7.2 μmol, 0.02eq) and morpholine (47 μL, 0.54 mmol, 1.5eq) at rt. After 2h, the solvent was removed in vacuo. Purification by column chromatography on silica using CH2CI2/MeOH (19:1->9:1)/AcOH (0.1%) as eluant furnished 4 (0.47 g, 0.44 mmol, 90%) as a white solid.
1H NMR (400MHz, CDCI3)δH: 6.84-7.40 (m, 34H), 5.23-5.41 (m, 2H), 5.11-5.20 (m, 1H), 4.37-4.47 (m, 1 H), 4.14-4.24 (m, 1 H), 3.95-4.10 (m, 2H), 3.84-3.93 (m, 2H), 3.66-3.78 (m, 1H), 3.03 (dd, J=14.2, 4.8Hz, 1H), 2.83 (dd, J=UA, 8.6Hz, 2H), 2.61 (dd, J=12.6, 7.2Hz, 1 H), 2.45-2.53 (m, 1 H), 2.29-2.42 (m, 2H), 2.21 (s, 3H), 2.08-2.17 (m, 3H), 1.91-1.99 (m, 3H), 0.77-0.80 (m, 6H). MS (ES+) 1091.2 (100%, [M+Naf). fl, (CH2Cla/Me0H, 19:1) = 0.20. (5): (2S,6fl,9S,12R, 13S)-13-Hydroxy-12-isopropyi-6-(3-methyl-benzyl)-2-((£)-4- trityl sulfanyl-but-1 -enyl)-9-trityl sulfanylmethyl-1-oxa-5,8,11-triaza- cyclopentadecane-4,7, 10, 15-tetraone
To a solution of MNBA (154mg, 0.37 mmol, 1.2eq) and DMAP (110mg, 0.90 mmol, 2.4eq) in CH2CI2 (28OmL) was added a solution of 4 (0.40 g, 0.37 mmol, 1eq) in CH2CI2 (15OmL) in dropwise fashion over 3h. The reaction mixture was then stirred at rt overnight, and the solvent was subsequently removed in vacuo. Purification by column chromatography on silica using CH2CI2/Me0H (32:1) as eluant yielded 5 (268mg, 0.26 mmol, 68%) as a white solid.
1H NMR (400MHz, CDCI3 + 10% MeOD) δH: 7.12-7.39 (m, 30H), 6.84- 6.97 (m, 4H), 5.50-5.61 (m, 1 H), 5.36-5.43 (m, 1 H), 5.25-5.34 (m, 1 H)1 2.90-3.03 (m, 2H), 2.76-2.86 (m, 3H), 2.42 (dd, JM 4.2, 11.5Hz, 1 H), 2.26-2.34 (m, 2H), 2.22 (S, 3H), 2.10-2.19 (m, 4H), 1.96-2.04 (m, 2H), 0.78-0.87 (m, 6H). MS (ES+) 1073.2 (100%, [M+Na]+). F?, (CH2CL2/MeOH, 19:1) = 0.50. Compound XV: (E)-(I S,5S,6R,9S,20R)-5-Hydroxy-6-isopropyl-20-(3-methyl- benzyl)-2-oxa-11 ,12-dithia-7,19,22-triaza-bicyclo[7.7.6]docos-15-ene-3,8, 18,21 - tetraone
To a solution of I2 (0.65 g, 2.55 mmol, 10eq) in CH2CI2/MeOH (9:1 , 1.0L) was added a solution of 5 (268mg, 0.26 mmol, 1eq) dropwise over 2h at rt. The mixture was then quenched with a solution of Na2S2O3 (0.1 M, 25OmL) and brine (25mL), and the aqueous layer was extracted with CH2CI2 (2 x 10OmL) and EtOAc (10OmL). The combined organic extracts were dried over MgSO4 and the solvent was removed in vacuo. Purification by column chromatography on silica using CH2CI2/MeOH (19:1) yielded compound XV (104mg, 0.18 mmol, 68%) as a white solid.
1H NMR (400MHz, CDCI3) δH: 7.45 (d, J=7.1 Hz, 1 H); 7.18 (t, J=7.5Hz, 1 H), 7.04 (d, J=7.6Hz, 1 H), 6.92-6.99 (m, 2H), 6.75 (d, J=9.2Hz, 1 H), 6.20 (t, J=13.1Hz, 1 H), 5.56 (d, J=15.4Hz, 1H), 5.41 (br. s., 1 H), 4.85 (td, J=8.7, 3.3Hz, 1H), 4.48 (dd, J=8.2, 4.0Hz, 1 H), 4.42 (dd, J=8.8, 5.4Hz, 1 H), 3.33-3.44 (m, 1 H), 3.21 (br. s., 2H), 3.12 (td, J=13.4, 6.2Hz, 2H), 2.98 (dd, JM4.5, 8.9Hz, 2H), 2.70-2.78 (m, 2H), 2.48-2.62 (m, 5H), 2.30-2.35 (m, 1H), 2.27-2.30 (m, 3H), 0.96 (d, J=6.8Hz, 3H), 0.86 (d, J=6.8Hz, 3H); 13C NMR (100MHz, CDCI3+10% MeOD) δc: 171.7, 171.5, 170.7, 169.1 , 139.0, 135.4, 132.7, 128.9, 128.8, 128.4, 125.8,
70.5, 68.9, 63.2, 57.0, 56.9, 54.6, 40.6, 40.3, 39.8, 35.94, 35.90, 33.7, 29.5,
21.3, 20.5, 19.3. MS (ES+) 586.8 (100%, [M+Na]+). R, (CH2CI2/Me0H, 19:1) =
0.20.
Compound XVI: (EH1S.6S.7R.10S.21 R)-6-Hvdroxy-7-isopropyl-21-pyridin- a-vimethyl-Σ.a-dioxa-IΣJS-dithia-β^O^S-triaza-bicvcioFβJ.βitricos-iβ-ene-
4,9,19,22-tetraone
Figure imgf000049_0001
(2): (3S,4H)-4-{(S)-2-[(H)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-pyridin-3- yl-propionylamino]-3-trityl sulfanyl-propionylaminoJ-S-hydroxy-δ-methyl-hexanoic acid allyl ester.
To a solution of 1 (598mg, 0.84 mmol) in MeCN (35mL) was added diethylamine in dropwise fashion (3.5mL, 10% v/v), and the resulting mixture was stirred at rt for 30 min. The reaction mixture was then concentrated in vacuo; hexane (3 x 2OmL) was added, the mixture was concentrated in vacuo, and was then placed on the high vacuum for 4h. DIPEA (0.4mL, 2.3 mmol) was then added dropwise to PyBOP (458mg, 0.88 mmol) and (fl)-/V-Fmoc-(3-pyridyl) alanine (347mg, 0.89 mmol) in CH2CI2 (25mL) at O0C .After 5 min, the reaction mixture was added to the crude amine of 1 in MeCN (25ml_) and the resulting mixture was allowed to stir for 16h at rt Purification by flash column chromatography on silica using EtOAc/hexane (4:1-1 :0) as eluant furnished 2 as a white solid (542mg, 0.59 mmol, 70%). 1H NMR (400MHz, CDCI3+5% MeOD) δH: 8.34-8.24 (m, 2H), 7.68 (d, J=7.53Hz, 2H), 7.49-7.05 (m, 23H), 6.35 (d, J=9.41Hz, 1 H), 5.83-5.70 (m, 1 H), 5.24-5.09 (m, 2H), 4.54 -4.42 (m, 2H), 4.33-4.23 (m, 2H), 4.16(m, 1H), 4.02 (t, J=6.71 Hz, 1H), 3.91 (t, J=7.40Hz, 1H), 3.83 (t, J=6.59Hz, 1H), 3.66 (m, 1H), 2.98 (m, 1 H), 2.82 (d, J=7.91 Hz, 1 H), 2.61-2.38 (m, 6H), 2.31 (dd, JM 6.12, 9.72Hz, 1H), 1.95(m, 1H), 0.84-0.69 (m, 6H). MS (ES+) 939.6 (100%, [M+Na]+). (3): (3S,4R)-4-{(S)-2-[(R)-2-(9H-Fluoren-9-ylmethoxycarbonylaιτiino)-3-pyridin-3- yl-propionylam ino]-3-trityl sulf anyl-propionylam ino}-5-methyl-3- triisopropylsilanyloxy-hexanoic acid allyl ester To 2 (542mg, 0.59 mmol) in CH2CI2/MeCN (13mL: 3mL) at O0C was added 2,6-lutidine (411 μL, 3.5 mmol) followed by TIPSOTf (634 μL, 2.4 mmol) in dropwise fashion. The reaction mixture was stirred for 1h 25 min, and 1 M HCI (15ml_) was then added. The layers were then separated, washed with 5% NaHCO3 (15ml_) and then with saturated brine (15ml_). After drying over MgSO4, the crude material was concentrated in vacuo and was purified by flash column chromatography on silica using EtOAc/hexane (6:4-8:2) as eluant to give 3 as a white solid (411mg, 0.38 mmol, 65%).
1H NMR (400MHz, CDCI3) δH: 8.41 (d, J=4.89Hz, 2H), 7.76 (d, J=7.40Hz, 2H), 7.53-7.05 (m, 26H), 6.18 (br s, 2H), 5.88 (m, 1 H), 5.30 (m, 1 H), 5.23 (dd, J=10.54, 1.13Hz, 1H), 5.12 (br s, 1 H), 4.52 (dd, J=11.61 , 5.83Hz, 2H), 4.38 (br s, 3H), 4.28 (br s, 1H), 4.12 (t, J=6.59Hz, 1 H), 3.98 (br s, 1 H), 3.84 (m, 1 H), 3.05 (br s, 1H), 2.95 (br s, 1H), 2.74 (br s, 1H), 2.65-2.47 (m, 3H), 1.82 (br s, 1H), 1.03 (s, 18H), 0.85 (dd, JM 0.29, 7.03Hz, 6H). MS (ES+) 1096.6 (100%, [M+Na]+). (5): (3S,4fl)-4-{(S)-2-[(fl)-2-((E)-(S)-3-Hydroxy-7-trityl sulfanyl-hept-4- enoylam ino)-3-pyridin-3-yl-propionylam ino]-3-trityl sulf anyl-propionylam ino}-5- methyl-3-triisopropylsilanyloxy-hexanoic acid allyl ester
To 3 (411mg, 0.38 mmol) in MeCN (2OmL) was added diethylamine in dropwise fashion (2mL, 10% v/v) and the reaction mixture was stirred at rt for 1 h 35 min. The reaction mixture was then concentrated in vacuo, and hexane (3x20ml_) was added. The resulting mixture was then concentrated in vacuo and then placedunder high vacuum. DIPEA (0.2ml_, 1.1 mmol) was added in dropwise fashion to PyBOP (217.2mg, 0.42 mmol) and the chiral acid 4 (175.3mg, 0.42 mmol) in CH2CI2 (15ml_) at O0C. After 2 min, the reaction mixture was added to the crude amine derivative of 3 in MeCN (15ml_), and the mixture was allowed to stir for 16h at it Purification by flash column chromatography on silica using 4:1 EtOAc/Hexane as eluant gave 5 as a white solid (415.5mg, 0.33 mmol, 87%).
1H NMR (400MHz, CDCI3+5% MeOD) δH:: 8.38-8.33 (m, 2H), 7.59 (ddd,
5 J=7.94, 1.79, 1.57Hz, 1H), 7.43-7.17 (m, 36H), 7.13 (m, 1H), 6.48 (d, J=10.04Hz, 1H), 5.89 (m, 1 H), 5.46 (m, 1 H), 5.36 -5.21 (m, 2H), 4.66 (dd, J=9.03, 4.64Hz, 1 H), 4.56-4.47 (m, 2H), 4.40 (q, J=5.44Hz, 1H), 4.25 (m, 1H), 3.98 (t, Λ=7.40Hz, 1 H), 3.77 (m, 1 H), 3.39 (dt, J=3.17, 1.62Hz, 1 H), 3.17 (dd, JL14.37, 4.45Hz, 1 H), 2.91 (dd, JM 4.31 , 9.03Hz, 1H), 2.56(m, 1 H), 2.48 (m,
10 1 H), 2.23-2.14 (m, 1 H), 2.10-2/02 (m, 2H), 1.77 (m, 1 H), 1.01 (s, 18H), 0.86 (d, J=6.65Hz, 1H), 0.80 (d, J=6.65Hz, 3H). MS (ES+) 1273.9 (100%, [M+Na]+). R, 0.23 (4:1 EtOAc/Hexane).
(6): (3S,4fl)-4-{(S)-2-[(fO-2-((£)-(S)-3-Hydroxy-7-trityl sulfanyl-hept-4- enoylam ino)-3-pyridin-3-yl-propionylam ino]-3-trityl sulf anyl-propionylam ino}-5-
15 methyl-3-triisopropylsilanyloxy-hexanoic acid
To 5 (410mg, 0.33 mmol) and Pd(PPh3)4 (39.07mg, 0.034 mmol) in dry MeOH (1OmL) was added morpholine (58 μL, 0.66 mmol) in dropwise fashion, and the reaction mixture was stirred for 3h. The mixture was then concentrated in vacuo, and was then purified by flash column chromatography on silica using
20 CH2CI2/MeOH (100:0-98:2-96:4-90:10) to furnish 6 as a white solid (460mg, 1.15 mmol, >99%), with some impurity still present.
1H NMR (400MHz, CDCI3+5% MeOD) δH: 8.78 (s, 1H), 8.45 (d, J=5.40Hz, 1H)1 8.41 (d, J=7.91 Hz, 1 H), 7.42-7.16 (m, 36H), 5.47 (m, 1 H), 5.35 (m, 1H), 4.77 (dd, J=8.91 , 4.27Hz, 1H), 4.35-4.24 (m, 2H), 3.97(m, 1H), 3.77 (t,
?5 J=6.15Hz, 1 H), 3.43-3.36 (m, 2H), 3.21 (dd, J=14.12, 9.10Hz, 1 H), 2.78-2.59 (m, 4H), 2.53-2.46 (m, 2H), 2.35-2.24 (m, 2H), 2.23-2.17 (m, 2H), 2.11-2.03 (m, 2H), 1.86 (dt, JM 3.24, 6.68Hz, 1 H), 1.03 (s, 18H), 0.83 (d, J=6.78Hz, 3H), 0.80 (d, J=6.78Hz, 3H). MS (ES") 1210.1 (100%, [M-H]"). (7): (3SJR, 10S, 13fl, 14S)-13-lsopropyl-7-pyridin-3-ylmethyl-14-triisopropyl
30 silanyloxy-3-((E)-4-trityl sulfanyl-but-1 -enyl)-10-trityl sulfanylmethyl-1 ,2-dioxa- 6,9, 12-triaza-cyclohexadecane-5,8, 11 ,16-tetraone
To a solution of MNBA (135mg, 0.39 mmol) and DMAP (96mg, 0.79 mmol) in CH2CI2 (65.5mL) was added a solution of 6 (433mg, 0.36 mmol) in CH2CI2 (268ml_) in dropwise fashion over 3h. After a further 16h, the reaction
35 mixture was concentrated in vacuo. Purification by flash column chromatography on silica using CH2CI2/Me0H (1 :0-99:1-98:2-96:4-94:66) gave 7 (279.4mg, 0.23 mmol, 65%) as a yellow solid.
1H NMR (400MHz, CDCI3+5% MeOD) δH: 8.38-8.31 (m, 2H), 7.41-7.15 (m, 35H), 6.85 (d, J=9.91 Hz, 1 H), 5.57 (m, 1H), 5.48 (m, 1H), 5.28 (m, 1 H), 4.33 (m, 1H), 4.02 (m, 1H), 3.71 (t, J=8.72Hz, 1 H), 3.46 -3.37 (m, 2H), 3.27-3.18 (m, 2H), 3.07 (dd, J=14.31 , 10.04Hz, 1 H), 2.85 (dd, J=12.86, 4.71 Hz, 1 H), 2.80-2.63 (m, 6H), 2.46(m, 1H), 2.38-2.26 (m, 3H), 2.08-1.99 (m, 2H), 1.03 (s, 18H), 0.86 (d, J=6.78Hz, 3H), 0.82 (d, J=6.78Hz, 3H). MS (ES+) 1216.5 (100%, [M+Na]+). Rf 0.63 CH2CI2/MeOH (90:10). (8): (£)-(1 S,6S,7fl,10S,21 /^-Isopropyl^i-pyridin-S-ylmethyl-θ-triisopropyl silanyloxy-2,3-dioxa-12, 13-dithia-8,20,23-triaza-bicyclo[8.7.6]tricos-16-ene- 4,9,19,22-tetraone
To a solution of I2 (596mg, 2.35 mmol) in CH2CI2/MeOH (796ml_, 9:1) was added dropwise 7 (279mg, 0.23 mmol) in CH2CI2/MeOH (370.5mL, 9:1) over 50 min. After a further 30 min, Na2S2O3 (325ml_, 0.05M) was added; the layers were then separated and the product extracted with EtOAc (3 x 45OmL). The organic layers were combined and dried (MgSO4), and then concentrated in vacuo. Purification by flash column chromatography on silica using CH2CI2/MeOH (1 :0- 99:1-97:3-95:5) gave 8 (72mg, O.IOmmol, 44%) as a white solid: 1H NMR (400MHz, CDCI3 + 5% MeOD) δH: 8.45 (d, Λ=4.52Hz, 1 H), 8.42
(s, 1H), 7.67 (d, J=7.91Hz, 1 H), 7.36-7.29 (m, 2H), 6.87 (d, Λ=9.41 Hz, 1H), 6.07 (m, 1H), 5.78 (d, J=15.43Hz, 1 H), 5.59 (br s, 1 H), 5.01-4.90 (m, 2H), 4.53 (dd, J=8.97, 5.46Hz, 1 H), 3.36-3.21 (m, 2H), 3.15-2.91 (m, 9H), 2.70-2.55 (m, 5H), 2.42 -2.25 (m, 2H), 1.18-1.05 (m, 18H), 0.98 (d, J=6.90Hz, 3H), 0.91 (d, J=6.90Hz, 3H). MS (ES+) 729.7 (100%, [M+Na]+). Rf 0.52 (eluant 1 :9 MeOH/CH2CI2).
Compound XVI : (E)-(I S,6S,7R, 10S,21 R)-6-Hydroxy-7-isopropyl-21 -pyridin-3- ylmethyl-2,3-dioxa-12,13-dithia-8,20,23-triaza-bicyclo[8.7.6]tricos-16-ene- 4,9,19,22-tetraone To 8 (59mg, 0.083 mmol) was added TFA (1.5mL) in dropwise fashion.
The resulting reaction mixture was allowed to stir for 4h, and was subsequently concentrated in vacuo and purified by flash column chromatography on silica using MeOH/CH2CI2 (0:1-5:95-7:93-9:91) as eluant to give a white solid, which was passed through a PE-AX Isolute® column to remove remaining traces of TFA to furnish compound XVI (22mg, 0.04 mmol, 48%). Some starting material was still present by tic.
1H NMR (400MHz, CDCI3+5% MeOD) δH: 8.46 (dd, J=4.83, 1.44Hz, 1 H), 8.43 (d, J=1.88Hz, 1 H), 7.67 (d, ^=8.03Hz, 1 H), 7.56 (d, Λ=7.28Hz, 1H), 7.34 (m, 1H), 6.91 (d, J=9.03Hz, 1 H), 6.09 (m, 1 H), 5.80 (d, J=15.31Hz, 1 H), 5.48(m, 1H), 4.85 (dt, J=8.63, 6.85Hz, 1H), 4.56- 4.45 (m, 2H), 3.26-2.96 (m, 8H), 2.82 (m, 1 H), 2.71-2.58 (m, 5H), 2.42-2.28 (m, 2H), 1.03 (d, J=6.90Hz, 3H), 0.92 (d, J=6.78Hz, 3H); 13C NMR (100MHz, CDCI3+5% MeOD) δc: 172.1 (C), 171.6 (C), 170.4 (C), 169.0 (C), 149.5 (CH), 148.2 (CH), 137.2 (CH), 132.4 (C), 132.3 (CH), 129.9 (CH), 124.0 (CH), 70.9 (CH), 69.0 (CH)1 63.4 (CH), 57.1 (CH), 55.5 (CH), 41.2 (CH), 39.9 (CH), 41.3 (CH2), 39.9 (2 x CH2), 33.1 (2xCH2), 32.6 (CH2), 29.6 (CH), 20.6 (CH3), 19.6 (CH3). MS (ES+) 573.8 (100%, [M+Na]+). R, 0.1 (eluant 1 :9 MeOHZCH2CI2). Compound XVII: 3-((a-(1S.5S.6R.9S.20m-5-Hvdroxy-6-isopropyl-3.8.18.21- tetraoxo-2-oxa-11.12-dithia-7.19.22-triaza-bicvclol7.7.61docos-15-en-20-vD- propionic acid
Figure imgf000053_0001
(2): (3S,4R)-4-{(S)-2-[(R)-4-tert-Butoxycarbonyl-2-(9H-fluoren-9-ylmethoxy carbonylamino)-butyrylamino]-3-trityl sulfanyl-propionylamino}-3-hydroxy-5- methyl-hexanoic acid allyl ester
To a solution of 1 (700mg, 0.91 mmol, 1eq, prepared according to the procedure in Doi, T, et al, Tet. Lett. 2006, 47, 1177) in MeCN (18ml_) was added Et2NH (10% v/v, 2ml_) dropwise at rt under Ar(g). The solution was stirred at rt for 2h, and the solvent was removed in vacuo. The excess of amine was co- evaporated with MeCN (3 x 1OmL) before the reaction mixture was dried under high vacuum for 2h. To a solution of Fmoc-D-Glu(OtBu)-OH (426mg, 1.0 mmol, 1.1 eq) in
CH2CI2 (15mL) at 00C was added PyBOP (521mg, 1.0 mmol, 1.1eq) and DIPEA (0.4OmL, 2.3 mmol, 2.5eq) under Ar(g). The crude amine, dissolved in MeCN (15mL), was added via cannula, and the reaction mixture was then left to warm to rt overnight before being concentrated in vacuo. Purification by flash column chromatography (eluant 10-50% EtOAc/Hexane) gave the product 2 (604mg, 0.633 mmol, 70% yield) as a white solid.
1H NMR (300MHz, CDCI3) δH: 0.87 (d, J=6.7Hz, 6H), 1.42 (s, 9H), 2.19- 2.58 (m, 5H), 2.92-3.03 (m, 1H), 3.19 (d, J=5.4Hz, 1H), 3.74-4.25 (m, 7H), 4.51 (dd, J=5.6, 1.2Hz, 2H), 5.08-5.27 (m, 2H), 5.72-5.88 (m, 1 H), 6.27-6.54 (m, 2H), 6.99-7.76 (m, 25H). MS (ES+) 976.1 (100%, [M+Na]+). R, (hexanes/EtOAc, 1 :1) = 0.50. (4): (£)-(S)-3-Hydroxy-7-trityl sulfanyl-hept-4-enoic acid
At O0C, to a solution of 3 (934mg, 1.66 mmol, prepared according to the procedure in Yurek-George, A. et al, J. Am. Chem. Soc. 2004, 126, 1030) in THF (3OmL) was added a solution of LiOH (196.1mg, 8.19 mmol) in H2O (1OmL). The reaction mixture was allowed to warm to rt over 1 h whereupon 1 M HCI was added until the pH reached 2. EtOAc (3OmL) was added, the layers were separated, and the aqueous layer was extracted with EtOAc (2OmL). The organic layers were combined, dried (MgSO4) and concentrated in vacuo. Purification by flash column chromatography (eluant 3:7-1 :1-1 :0 EtOAc/Hexane) gave the product 4 as a white solid (600mg, 1.43 mmol, 86%).
1H NMR (300MHz, CDCI3) δH: 7.48-7.38 (m, 6H), 7.35-7.18 (m, 9H), 5.60 (m, 1 H), 5.43 (m, 1 H), 4.46 (q, J=6.28, 1 H), 2.59-2.51 (m, 2H), 2.28-2.19 (m, 2H), 2.09 (q, J=6.47Hz, 2H). MS (ES") 417 (100%, [M-H]'). R, 0.52 EtOAc + 2 drops AcOH. [α]D 27-4.15 (c 0.975, CH2CI2). (5): (3S,4R)-4-{(S)-2-[(R)-4-tert-Butoxycarbonyl-2-((E)-(S)-3-hydroxy-7-trityl sulfanyl-hept-4-enoylamino)-butyrylamino]-3-trityl sulfanyl-propionylamino}-3- hydroxy-5-methyl-hexanoic acid allyl ester
To a solution of 2 (600mg, 0.63 mmol, 1eq) in MeCN (13mL) was added Et2NH (10% v/v, 1.3ml_) in dropwise fashion at rt under Ar(g). The solution was stirred at rt for 2h, and the solvent was then removed in vacuo. The excess of amine was co-evaporated with MeCN (3 x 1OmL), and the resulting mixture was dried under high vacuum for 2h.
To a solution of the Dhydroxy acid 4 (276mg, 0.66 mmol, 1.05eq) in CH2CI2 (1OmL) at 00C was added PyBOP (360mg, 0.69 mmol, 1.1 eq) and DIPEA (274 μL, 1.57 mmol, 2.5eq) under Ar(g). The crude amine, dissolved in MeCN (1OmL), was added via cannula, and the reaction mixture was left to warm to rt overnight before being concentrated in vacuo. Purification by flash column chromatography (eluant 10-65% EtOAc/Hexane) gave the product 5 (582mg, 0.514 mmol, 82% yield) as a white solid.
1H NMR (300MHz, 9:1 CDCI3/CD3OD) δH: 0.80 (dd, J=6.8, 1.7Hz, 5H) 1.35 (s, 9H) 2.05-2.64 (m, 10H) 3.25 (d, J=5.6Hz, 1 H) 3.67-3.79 (m, 1H) 3.92- 4.16 (m, 3H) 4.20-4.33 (m, 1H) 4.49 (dd, J=5.6, 1.2Hz, 2H) 5.07-5.43 (m, 4H) 5.72-5.88 (m, 1 H) 6.18 (d, J=9.7Hz, 1 H) 7.06-7.42 (m, 30H). MS (ES+) 1154.5 (100%, [M+Na]+). Rf (hexane/EtOAc, 1 :1) = 0.25.
(6): (3S,4R)-4-{(S)-2-[(R)-4-tert-Butoxycarbonyl-2-((E)-(S)-3-hydroxy-7-trityl sulf anyl-hept-4-enoylam ino)-butyrylam ino]-3-trityl suit anyl-propionylam ino}-3- hydroxy-5-methyl-hexanoic acid
To a solution of 5 (580mg, 0.51 mmol, 1eq) in anhydrous MeOH (1OmL) and THF (1.7mL) was added morpholine (67 μL, 0.77 mmol, 1.5eq) and Pd(PPh3)4 (11.6mg, 0.01 mmol, 0.02eq) at rt under Ar(g). The reaction mixture was stirred for 1h, and was then concentrated in vacuo. Purification by flash column chromatography (eluant 1-6% MeOH/CH2CI2, then + 0.1% AcOH) gave the product 6 (555mg, 0.51 mmol, 99% yield) as a white solid. 1H NMR (300MHz, CDCI3) δH: 0.82 (dd, J=6.6, 3.8Hz, 6H), 1.35 (s, 9H),
2.07-2.67 (m, 10H), 3.04 (d, J=6.7Hz, 1 H), 3.63-3.76 (m, 3H), 3.94-4.31 (m, 3H), 5.10-5.43 (m, 3H), 6.42 (d, J=9.8Hz, 1 H), 7.03-7.48 (m, 30H). MS (ES+) 1115.4 (100%, [M+Na]+). a (CH2CL2/Me0H, 19:1) = 0.35. (7): 3-[(2S,6R,9S,12R, 13S)-13-Hydroxy-12-isopropyl-4,7,10, 15-tetraoxo-2-((E)- 4-trityl sulfanyl-but-1-enyl)-9-trityl sulfanylmethyl-1-oxa-5,8,11-triaza- cyclopentadec-6-yl]-propionic acid tert-butyl ester
To a solution of MNBA (215.6mg, 0.63 mmol, 1.2eq) and DMAP (153.0mg, 1.25 mmol, 2.4eq) in CH2CI2 (14OmL) was added a solution of acid 6 (555mg, 0.51 mmol, 1eq) in CH2CI2 (43OmL) in dropwise fashion over 3h under Ar(g). After a further 14h the reaction mixture was concentrated in vacuo to give an off white solid. Purification by column chromatography on silica gel (1-3% IPA/CH2CI2) gave a 7 (380mg, 0.354 mmol, 69% yield) as a white solid. 1H NMR (400MHz, 9:1 CDCI3ZCD3OD) δH: 7.27-7.37 (m, 14H), 7.08-7.24
(m, 18H), 5.54 (dt, J=15.3, 6.8Hz, 1H), 5.39-5.47 (m, 1H), 5.27 (dd, J=15.4, 6.7Hz, 1 H), 4.06-4.14 (m, 1 H), 3.97-4.05 (m, 1H), 3.84-3.95 (m, 1H), 3.01-3.17 (m, 2H), 2.53 (dd, J=12.7, 5.0Hz, 1 H), 2.18-2.46 (m, 7H), 2.05-2.17 (m, 3H), 1.86-2.04 (m, 3H), 1.73-1.85 (m, 1 H), 1.33 (s, 9H), 0.79 (d, J=6.9Hz, 3H), 0.75 (d, J=6.8Hz, 3H). MS (ES+) 1097 (100%, [M+Na]+).
(8): 3-((E)-(1S,5S,6R,9S,20R)-5-Hydroxy-6-isopropyl-3,8,18,21-tetraoxo-2-oxa- 11 ,12-dithia-7,19,22-triaza-bicyclo[7.7.6]docos-15-en-20-yl)-propionic acid tert- butyl ester
To a solution of I2 (900mg, 3.52 mmol, 10eq) in CH2CI2 (765mL) and MeOH (85mL) was added a solution of 7 (380mg, 0.35 mmol, 1eq) in CH2CI2 (40OmL) and MeOH (4OmL) in dropwise fashion over 2.5h at rt under Ar(g). The reaction mixture was further stirred for 20 min, and a solution of sodium thiosulfate (0.1 M, 14OmL) and brine (4OmL) were added. The organic layer was isolated, and the aqueous layer was extracted with EtOAc (3 x 15OmL). The combined organic extracts were dried over MgSO4 and the solvent was removed in vacuo. Purification by column chromatography on silica gel (1-5% IPA/CH2CI2) gave 8 (120mg, 0.204 mmol, 58% yield) as a white solid.
1H NMR (400MHz, 9:1 CDCI3ZCD3OD) δH: 7.42 (d, J=7.0Hz, 1 H), 6.73 (d, J=9.3Hz, 1H), 6.06 (t, J=13.5Hz, 1 H), 5.69 (d, J=15.4Hz, 1H), 5.40 (br. s., 1 H), 4.68 (td, J=9.0, 3.5Hz, 1 H), 4.41 (dt, J=9.2, 3.6Hz, 1 H), 3.89 (dd, J=8.4, 6.7Hz, 1 H), 3.08-3.24 (m, 1 H), 2.99 (dd, JM 3.1 , 7.0Hz, 2H), 2.88-2.99 (m, 1H), 2.12- 2.71 (m, 11H), 1.85-1.98 (m, 2H), 1.31 (s, 9H), 0.85 (d, J=6.9Hz, 3H), 0.75 (d, J=6.8Hz, 3H). 13C NMR (100MHz, 9:1 CDCI3ZCD3OD) δc: 174.2, 171.7, 171.3, 171.1 , 169.1 , 132.0, 129.7, 81.9, 70.5, 68.2, 62.8, 57.0, 54.6, 40.9, 40.7, 40.3, 39.9, 33.1 , 32.3, 29.5, 27.7, 24.7, 20.3, 18.9. MS (ES+) 1198 (95%, [2M+Na]+), 610 (100%, [M+Na]+).
Compound XVII: 3-((£)-(1S,5S,6R,9S,20R)-5-Hydroxy-6-isopropyl-3,8, 18,21 - tetraoxo-2-oxa-11 ,12-dithia-7,19,22-triaza-bicyclo[7.7.6]docos-15-en-20-yl)- propionic acid
To 8 (80.2mg, 0.136 mmol, 1eq) was added Et3SiH (65 μL, 0.41 mmol, 3eq) followed by TFA (1mL, 13.6 mmol, 100eq) at 00C under Ar(g). The reaction mixture was stirred for 1h, and was then concentrated in vacuo. Purification by column chromatography on silica gel (1-10% MeOH/CH2CI2) gave compound XVII (58mg, 0.109 mmol, 80% yield) as a white solid.
1H NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 7.39 (d, J=7.2Hz, 1 H), 6.69 (d, J=9.3Hz, 1H), 5.94 (t, J=12.6Hz, 1H), 5.63 (d, J=15.4Hz, 1 H), 5.32 (br. s., 1H), 4.59 (td, Λ=9.2, 3.5Hz, 1H), 4.33 (dt, J=9.3, 3.8Hz, 1H), 3.86 (dd, J=7.8, 6.8Hz, 1H), 2.98-3.17 (m, 2H), 2.87 (dd, J=12.8, 6.8Hz, 2H), 2.56-2.66 (m, 1H), 2.17- 2.56 (m, 9H), 2.04-2.16 (m, 1 H), 1.88 (s, 2H), 0.77 (d, J=6.9Hz, 3H), 0.67 (d, J=6.8Hz, 3H). 13C NMR (100MHz, 9:1 CDCVCD3OD) δc: 176.0, 171.9, 171.5, 170.8, 169.0, 131.7, 129.8, 70.5, 67.9, 62.5, 56.7, 54.7, 40.9, 40.7, 40.0, 39.9, 32.8, 30.6, 29.4, 24.5, 20.1 , 18.6. MS (ES+) 1086 (95%, [2M+Na]+), 554 (100%, [M+Na]+). Compound XVIII: N-tert-Butyl-3-((EH1S.5S,6R.9S.20R)-5-hvdroxy-6- isopropyl-3,8.18,21 -tetraoxo-2-oxa-11.12-dithia-7.19,22-triaza- bicvclor7.7.β1docos-15-en-20-yl)-propionamide
Figure imgf000057_0001
XVIII To a solution of compound XVII (15mg, 0.028 mmol, 1eq) in MeCN
(1mL) was added PyBOP (16.0mg, 0.031 mmol, 1.1 eq) and DIPEA (12.2 μL, 0.071 mmol, 2.5eq) at 0QC under Ar (g). The reaction mixture was stirred for 5 min, and was then treated with a solution of fe/f-butylamine (3.1 μL, 0.029 mmol, 1.05eq) in CH2CI2 (1mL) in dropwise fashion. The solution was left to warm to rt overnight, and the solvent was subsequently removed in vacuo. Purification by column chromatography on silica using CH2CI2/Me0H (1 :0 -> 19:1) as eluant furnished compound XVIII (14.5mg, 88%) as a white solid.
1H-NMR (400MHz, CDCI3 + 10% MeOD) δH: 7.30 (d, J=7.2Hz, 1 H), 6.76 (S, 1H), 6.59 (d, J=9.2Hz, 1 H), 5.87 (t, J=12.5Hz, 1H), 5.62 (d, J=15.2Hz, 1H), 5.17 -5.27 (m, 1H), 4.48 (td, J=8.8, 2.9Hz, 1 H), 4.22 (ddd, J=9.0, 3.9, 3.7Hz, 1 H), 3.67 (t, J=7.5Hz, 1H), 2.95-3.05 (m, 3H), 2.73-2.85 (m, 4H), 2.44-2.54 (m, 2H), 2.12-2.35 (m, 7H), 1.89-2.06 (m, 2H), 1.67-1.75 (m, 2H), 0.96-1.01 (s, 9H), 0.67 (d, J=6.8Hz, 3H), 0.57 (d, J=6.8Hz, 3H). 13C NMR (100MHz, CDCI3+10% MeOD) δc: 173.9, 172.6, 172.5, 171.8, 169.7, 132.4, 130.7, 71.4, 68.8, 63.4, 57.8, 55.3, 51.9, 43.3, 41.1 , 40.5, 33.9, 30.1 , 28.8, 25.9, 20.9, 19.5. MS (ES+) 609.5 (100%, [M+Na]+). Rf (CH2CI2/MeOH, 19:1) = 0.30.
Compound XIX: (EW1 S,5S.9S.20R)-5-Hvdroxy-6-isopropyl-20-r3-(4-methyl- piperazin-1-yl)-3-oxo-propyπ-2-oxa-11,12-dithia-7,19,22-triaza- bicyclorz.y.βidocos-IS-ene-S^iβ^i-tetraone
Figure imgf000058_0001
XIX
Compound XVII (225.3mg, 0.424 mmol) was treated with EDC (89.53mg, 0.467 mmol) and HOBt (63.32mg, 0.469 mmol) MeCN/CHCI3 (1 :1 , 18ml_) and the solution was stirred for 5 min. 1 -Methylpiperazine (70 μL, 0.631 mmol) was added, and the reaction mixture was stirred for 16h. The solvent was removed in vacuo, and the resulting crude material was purified by flash column chromatography on silica using MeOH/CH2CI2 (8:92-9:91-1 :9-12:88) as eluant to furnish compound XIX (174.4mg, 0.284 mmol, 67%) as a white solid.
1H-NMR (400MHz, CDCI3 +10% MeOD) δH: 7.52 (d, J=7.03Hz, 1H), 6.80 (d, J=9.16Hz, 1H), 6.09 (t, J=12.49Hz, 1 H), 5.79 (d, JM 5.31 Hz, 1H), 5.44 (br S, 1H), 4.75 (td, J=8.75, 3.45Hz, 1 H), 4.43-4.51 (m, 1H), 3.97 (dd, J=9.03, 4.64Hz, 1H), 3.56 (d, J=3.14Hz, 2H), 3.39-3.47 (m, 2H), 3.19 (br s, 2H), 3.07 (dd, J=12.92, 6.90Hz, 1 H), 3.01 (br s, 1 H), 2.21-2.77 (m, 19H), 2.07-2.19 (m, 1 H), 1.94-2.05 (m, 1H), 0.92 (d, J=6.78Hz, 3H), 0.81 (d, J=6.65Hz, 3H). MS (ES+)
614.2 (100%, [M+H]+). R, 0.40 CH2CI2/Me0H (4:1).
Compound XX: 3-((1 S.5S.6R9S.20/?.£)-5-hvdroxy-6-isopropyl-3.8.18.21- tetraoxo-2-oxa-11.12-dithia-7.19.22-triazabicvclor7.7.61docos-15-en-20-vD-
Λ/-(2-morpholinoethyl)propanamide
Figure imgf000059_0001
XX
Compound XX was prepared using a similar synthetic procedure to that employed for XIX (12.7mg, 55%) 1H NMR (400 MHz, CDCI3) δH: 9.60 (s, 1 H), 7.60 (d, J=6.8 Hz, 1 H), 6.82
(d, J=9.6 Hz, 1 H), 6.55 (s, 1 H), 6.29 - 6.37 (m, 1 H), 5.95 (d, J=15.2 Hz, 1 H), 5.51 (br. s., 1 H), 4.88 - 4.96 (m, 1 H), 4.57 - 4.61 (m, 1 H), 4.10 - 4.19 (m, 1 H), 3.70 - 3.81 (m, 4 H), 3.36 - 3.50 (m, 3 H), 3.30 (dd, J=13.2, 6.8 Hz, 1 H), 3.14 (br. s., 2 H), 2.36 - 2.85 (m, 17 H), 2.15 - 2.22 (m, 2 H), 1.04 (d, Λ=6.8 Hz, 3 H), 0.94 (d, J=6.8 Hz, 3 H). MS (ES+) 644 (100%, [M+H]+).
Compound XXi: ΛH2-(D8methylamino)ethvt)-3-((1S,5S,6fl.9S.20fl.a-5- hvdroxy-β-isopropyl-3,8,18,21-tetraoxo-2-oxa-11.12-dithia-7,19,22- triazabicvclof7.7.β1docos-15-en-20-vQpropanamide
Figure imgf000059_0002
XXI Compound XXI was prepared using a similar synthetic procedure to that employed for XIX (16.7mg, 73%).
1H NMR (400 MHz, CD3OD) δH: 6.02 - 6.12 (m, 2 H), 5.64 (br. s., 1 H), 4.70 - 4.80 (m, 1 H), 4.59 (br. d, J=9.2 Hz, 1 H), 4.09 (t, J=4.0 Hz, 1 H), 3.70 (dt, J=UA, 5.6 Hz, 1 H), 3.56 (dt, JM0.8, 5.4 Hz, 1 H), 3.15 - 3.51 (m, 4 H), 2.75 - 3.08 (m, 5 H), 2.97 (s, 6 H), 2.08 - 2.70 (m, 9 H), 1.04 (d, J=6.8 Hz, 3 H), 0.95 (d, J=6.8 Hz, 3 H). MS (ES+) 602 (100%, [M+H]+).
Compound XXII: 3-((1 S.SS.βfl.gS.ΣOfl.a-S-Hvdroxy-β-isopropyl-a.S.iβ.ΣI- tetraoxo-2-oxa-11.12-dithia-7.19,22-triazabicvclor7.7.61docos-15-en-20-vD- ΛH2-methoxyethyl)propanamide
Figure imgf000060_0001
XXII
Compound XXII was prepared using a similar synthetic procedure to that employed for XIX (10.6mg, 52%).
1H NMR (400 MHz, CDCI3) δH: 9.60 (s, 1 H), 7.60 (d, J=7.2 Hz, 1 H), 6.81 (d, J=9.6 Hz, 1 H), 6.30 - 6.42 (m, 1 H), 6.14 (br. s., 1 H), 5.93 (d, JM 5.2 Hz, 1 H), 5.51 (br. s., 1 H), 4.89 - 5.00 (m, 1 H), 4.59 (br. s, 1 H), 4.11 - 4.20 (m, 1 H), 3.49 - 3.58 (m, 4 H), 3.48 (s, 3 H), 3.30 (dd, J=13.2, 7.2 Hz, 1 H), 2.98 - 3.20 (m, 3 H), 2.10 - 2.80 (m, 13 H), 1.05 (d, J=6.8 Hz, 3 H), 0.94 (d, J=6.8 Hz, 3 H). MS (ES+) 611 (20%, [M+Na]+), 589 (100%, [M+H]+).
Compound XXHI: (1 S.5S,6fl,9S,20/?,£)-5-Hvdroxy-β-isopropyl-20-(3- morpholino-3-oxopropyl)-2-oxa-11 ,12-dithia-7,19,22- triazabicvcloiTJ.eidocos-iδ-ene-S.S.IS^I-tetraone
Figure imgf000061_0001
XXIlI
Compound XXIII was prepared using a similar synthetic procedure to that employed for XIX (6.7mg, 55%).
1H NMR (400 MHz, CDCI3) δH: 9.35 (s, 1 H), 7.59 (d, J=7.2 Hz, 1 H), 6.80 (d, J=9.6 Hz, 1 H), 6.27 - 6.50 (m, 1 H), 5.85 (d, JM 6.4 Hz, 1 H), 5.50 (br. s., 1 H), 4.89 - 4.98 (m, 1 H), 4.56 - 4.63 (m, 1 H), 4.11 - 4.20 (m, 1 H), 3.62 - 3.80 (m, 6 H), 3.48 - 3.52 (m, 2 H)1 3.10 - 3.47 (m, 3 H), 3.32 (dd, JM 2.8, 6.8 Hz, 1 H), 2.62 - 2.80 (m, 6 H), 2.15 - 2.58 (m, 7 H), 1.05 (d, J=6.8 Hz, 3 H), 0.95 (d, J=6.8 Hz, 3 H). MS (ES+) 601 (100%, [M+H]+).
Compound XXIV: 3-K1 S.5S.6fl.9S.20fl.a-5-hvdroxy-β-isopropyl-3,8.18.21- tetraoxo^-oxa-II.IΣ-dithia-y.iQ.ΣΣ-triazabicvclor/.y.βidocos-IS-en^O-yl)- /V-methylpropanamide
Figure imgf000061_0002
XXIV
Compound XXIV was prepared using a similar synthetic procedure to that employed for XIX (14.1mg, 69%).
1H NMR (400 MHz, CDCI3) δH: 9.52 (br. s., 1 H), 7.59 (d, J=7.2 Hz, 1 H), 6.88 (d, J=8.8 Hz, 1 H)1 6.29 - 6.39 (m, 1 H), 6.18 (br. s., 1 H), 5.98 (d, JM 5.2 Hz, 1 H)1 5.53 (br. S., 1 H), 4.87 - 4.96 (m, 1 H)1 4.53 - 4.61 (m, 1 H)1 4.11 - 4.19 (m, 1 H), 3.38 - 3.52 (m, 1 H), 3.31 (dd, JM 2.8, 6.8 Hz, 1 H), 3.05 - 3.28 (m, 2 H), 2.96 (d, J=4.8 Hz1 3 H), 2.32 - 2.82 (m, 10 H), 2.14 - 2.25 (m, 3 H), 1.05 (d, J=6.8 Hz, 3 H), 0.95 (d, J=6.8 Hz, 3 H). MS (ES+) 545 (100%, [M+H]+). Compound XXV: 3-((EH1S,5S,9S,20R)-5-Hvdroxy-6-isopropyl-3.8.18.21 - tetraoxo-2-oxa-11 ,12-dithia-7.19,22-triaza-bicvclo|7.7.61docos-15-en-20-vD- N-(2,2,2-trifluoro-ethyl)propionamide
Figure imgf000062_0001
XXV
To compound XVII (67.5mg, 0.127 mmol), EDC (97.6mg, 0.509 mmol) and HOBt (19.6mg, 0.145 mmol) was added THF (1.5mL) followed by CHCI3 (6mL), and the resulting mixture was stirred for 5 min. 2,2,2-Trifluoroethyiamine (50 μL, 0.628 mmol) was then added, and the reaction mixture was allowed to stir for 16h. The mixture was subsequently washed with 1 M HCI (5mL), and the layers were separated, dried (MgSO4), and the solvent was removed in vacuo. Purification by flash column chromatography on silica using MeOH/CH2CI2 (0:1 - 2:98-4:96) as eluant furnished compound XXV (59.9mg, 0.0978 mmol, 77%) as a white solid. 1H-NMR (400MHz, CDCI3) δH: 8.99 (d, J=2.89Hz, 1 H), 7.50 (d, J=7.15Hz,
1 H), 6.87 (d, J=9.41 Hz, 1 H), 6.73 (t, J=6.34Hz, 1 H), 6.31 (t, J=12.55Hz, 1 H), 5.87 (d, J=15.31 Hz, 1 H), 5.50 (br s, 1 H), 4.87 (m, 1 H), 4.56 (m, 1 H), 4.12 (m, 1 H), 4.06-3.82 (m, 2H), 3.37 (br s, 1 H), 3.29 (dd, Jti3.11 , 6.84Hz, 1 H), 3.21 (br S, 1 H), 3.1 1 (d, JM 0.16Hz, 2H), 2.83-2.33 (m, 10H), 2.28-2.12 (m, 2H), 1.03 (d, J=6.78Hz, 3H), 0.92 (d, J=6.65Hz, 3H). MS (ES+) 635.1 (100%, [M+Na]+). R, 0.35 CH2CI2/MeOH (9:1 ).
Compound XXVI: (1 S,5S.6fl,9S,20ff,a-5-Hvdroxv-6-isopropvl-20-(3-oxo-3- (piperidin-1-yl)propyl)-2-oxa-11 ,12-dithia-7,19,22-triazabicycto[7.7.6]docos-15- ene-3,8,18,21 -tetraone
Figure imgf000063_0001
XXVI
To a solution of compound XVII (230mg, 0.43 mmol, 1eq) in CHCI3 (15ml_) was added HOBT (64.6mg, 0.48 mmol, 1.1 eq) and EDCI (332mg, 1.73 mmol, 4eq) under Ar(g). Then, piperidine (129ml_, 1.30 mmol, 3eq) was added slowly. The reaction mixture was stirred at rt overnight. The mixture was then concentrated in vacuo, then re-dissolved in CH2CI2 (3mL) and washed with 1 N HCI (2 x 1OmL). The organics were dried over MgSO4. After filtration, solvent was removed in vacuo and the residue was further purified by silica gel column chromatography with CH2CI2/MeOH (32:1) to yield the product as a white solid (217mg, 84%).
1H NMR (400 MHz, CDCI3) δH: 9.55 (br s, 1 H), 7.59 (d, J=6.8Hz, 1 H), 6.79 (d, J=9.6Hz, 1H), 5.87 (d, J=15.2Hz, 1H), 5.47 (br s, 1H)1 4.86 - 4.99 (m, 1 H), 4.58 (br s, 1H), 4.12 (dt, J-=I 0.1 , 3.2Hz, 1H), 3.49 - 3.69 (m, 2H), 3.38 - 3.44 (m, 2H)1 3.28 (dd, J=13.0, 6.8Hz, 1 H), 2.12 - 2.79 (m, 14H), 1.52 - 1.73 (m, 9H), 1.04 (d, J=6.8Hz, 3H), 0.93 (d, J=6.7Hz, 3H); MS (ES+) 621.1 (100%, [M+Na]+). Compound XXVII: ΛKcvclopropylmethyl)-3-((1 S,5S,6ft9S.20fl.a-5- hvdroxy-6-isopropyl-3.8,18.21-tetraoxo-2-oxa-11.12-dithia-7.19.22- triazabicyclo|7.7.61docos-15-en-2u-yl)propanamide
Figure imgf000063_0002
XXVII
Compound XXVII was prepared using a similar synthetic procedure to that employed for XIX (15.7mg, 54%). 1H NMR (400 MHz, CDCI3) δH: 9.68 (s, 1 H), 7.60 (d, J=I.2 Hz, 1 H)1 6.83 (d, J=9.2 Hz, 1 H), 6.27 - 6.43 (m, 1 H), 6.06 (br. s., 1 H), 5.94 (d, JM 5.2 Hz, 1 H), 5.51 (br. s., 1 H), 4.87 - 4.96 (m, 1 H), 4.55 - 4.62 (m, 1 H), 4.10 - 4.19 (m, 1 H), 3.40 - 3.50 (br. s., 1 H), 3.30 (dd, JM 2.4, 7.2 Hz, 1 H), 3.06 - 3.23 (m, 4 H), 2.50 - 2.82 (m, 9 H), 2.32 - 2.48 (m, 2 H), 2.15 - 2.24 (m, 2 H), 1.05 (d, J=6.8 Hz, 3 H), 0.88 - 1.00 (m, 1 H), 0.94 (d, J=6.8 Hz, 3 H) 0.52 - 0.62 (m, 2 H), 0.19 - 0.28 (m, 2 H). MS (ES+) 585 (100%, [M+H]+).
Compound XXVIII: 3-((EW1S.5S.6R.9S.20R)-5-Hvdroxy-6-isopropyl- 3,8,18.21 -tetraoxo-2-oxa-11,12-dithia-7,19,224riaza-bicvcloi7.7.61docos-15- en-20-yl)-N,N-dimethyl-propionamide
Figure imgf000064_0001
XXVIII
To a solution of compound XVII (13.5mg, 0.039 mmol, 1eq) in MeCN (1mL) at 00C under Ar(g), PyBOP (22.3 mg, 0.043mmnol, 1.1 eq) and diisopropylethylamine (17DL, 0.098mmnol, 2.5eq) were added. The reaction mixture was stirred for 5 min, then a solution of dimethylamine HCI salt (6.4mg, 0.078mmnol, 2eq) and DIPEA (13.6DL, 0.078 mmol, 2eq) in CH2CI2 (ImL) was added dropwise to the mixture. The solution was left warming to rt overnight. Solvents were then evaporated in vacuo, and the resulting material was purified by alumina column chromatography (CH2CI2/MeOH, 1 :0 -> 19:1) to furnish compound XXVIII (14. 2 mg; 65%).
1H NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 9.32 (d, J=2.4Hz, 1H), 7.55 (d, J=7.2Hz, 1H), 6.80 (d, J=9.5Hz, 1 H), 6.04-6.19 (m, 1 H), 5.81 (d, J=15.3Hz, 1 H), 5.43 (br. s., 1H), 4.77 (td, J=9.2, 3.4Hz, 1H), 4.47 (dt, J=9.1 , 3.7Hz, 1H), 3.98 (dt, J=9.3, 3.6Hz, 1 H), 3.00-3.26 (m, 4H), 2.96 (s, 3H), 2.90 (s, 3H), 2.49-2.76 (m, 7H), 2.35-2.49 (m, 2H), 2.27 (spt, J=6.8Hz, 1 H), 2.08-2.21 (m, 1H), 1.96- 2.07 (m, 1 H), 0.93 (d, J=6.8Hz, 3H), 0.83 (d, J=6.7Hz, 3H). MS (ES+) 559 (100%, [M+H]+). Compound XXIX: N-f3-((EH1 S,5S,6R,9S,20R)-5-Hvdroxy-β-isopropyl- 3.8.18.21 -tetraoxo-2-oxa-11,12-dithia-7,19.22-triaza-bicvclor7.7.61docos-15- en-20-yl)-propionyll-4-methyl-benzenesulfonamide
FmocHN
Figure imgf000065_0001
(1): (R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-5-oxo-5-(toluene-4- sulfonylamino)-pentanoic acid tert-butyl ester
To FmOC-D-GIuO1Bu (747.6mg, 1.76 mmol) in dry THF (7.5mL) was added Et3N (0.26ml_, 1.86 mmol) in dropwise fashion, followed by p- toluenesulfonylisocyanate (0.29mL, 1.90 mmol) and the solution was stirred for 2h at rt. The reaction mixture was then quenched with 1Pr2NEt (0.15mL) and stirred for a further 10 min. CHCI3 (10OmL) was then added followed by 1 M HCI (aq) (10OmL). The layers were separated washed with saturated brine (10OmL), dried (MgSO4), filtered and concentrated in vacuo. Purification by flash column chromatography on silica (eluant 1 :99-2:98 MeOH/CH2CI2) gave 1 (953mg, 1.65 mmol, 94%) as a white solid.
1H NMR (400MHz, CDCI3) δH: 9.44 (br s, 1 H), 7.96 (d, J=8.03Hz, 2H), 7.77 (dd, J=7.47, 3.45Hz, 2H), 7.60 (d, J=7.28Hz, 2H), 7.41 (q, J=7.28Hz, 2H), 7.36-7.25 (m, 5H), 5.47 (d, J=7.40Hz, 1 H), 4.55 (m, 1H), 4.44 (m, 1 H), 4.21 (t, J=6.46Hz, 1H), 4.11 (m, 1H), 2.41 (s, 3H), 2.28-2.07 (m, 2H), 1.75 (m, 1H), 1.43 (s, 9H). MS (ES+) 601.7 (100%, [M+Na]+). Rf 0.72 CH2CI2/MeOH (9:1). (2): (R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-5-oxo-5-(toluene-4- sulfonyiamino)-pentanoic acid
To 1 (945mg, 1.63 mmol) in CH2CI2 (25mL) at O0C was added triethylsilane (0.4OmL, 2.50 mmoi)/TFA (5mL, 20% v/v) and the reaction mixture was stirred for 1h 10 min. Triethylsilane (0.8OmL, 5.01 mmol)/TFA (5mL, 40% v/v) was then added, and the reaction mixture was stirred at O0C for 20 min before being warmed to rt. After stirring for a further 50 min, TFA (1OmL) was added, and the reaction mixture was stirred for a further 45 min. The mixture was then concentrated in vacuo and placed under high vacuum. Purification by flash column chromatography on silica (eluant 0:1-2:98 MeOH/CH2CI2) gave 2 (271 mg, 0.519 mmol, 32%) as a white solid.
1H NMR (400MHz, CDCI3+ 10% MeOD) δH: 9.58 (br s, 1H), 7.91 (d, J=8.03Hz, 2H), 7.74 (d, J=7.40Hz, 2H), 7.57 (d, J=7.28Hz, 1H), 7.51 (br S, 1H), 7.37 (t, J=7.34Hz, 2H), 7.32-7.22 (m, 4H), 6.60 (br s, 1H), 5.73 (d, J=7.53Hz, 1 H), 4.58-4.26 (m, 3H), 4.18 (t, J=6.53Hz, 1 H), 3.99 (d, J=3.14Hz, 1 H), 2.41- 2.30 (m, 4H), 2.15 (br s, 1 H), 2.03-1.93 (m, 1H); MS (ES") 521.8 (100%, [M-H]"). Rf 0.38 CH2CI2/MeOH (9:1) + AcOH (3 drops).
(3): (3S,4R)-4-{(S)-2-[(R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-5-oxo-5- (toluene-4-sulf onylam ino)-pentanoylam ino]-3-tritylsulf anyl-propionylam ino}-3- hydroxy-5-methyl-hexanoic acid allyl ester
To a solution of (3S,4fl)-allyl 4-((S)-2-(((9H-fluoren-9- yl)methoxy)carbonylamino)-3-(tritylthio)propanamido)-3-hydroxy-5- methylhexanoate (380.15mg, 0.494 mmol) in MeCN (2OmL) under Ar(g) was added diethylamine (2mL, 10% v/v) and the reaction mixture was stirred at rt for 1 h 20min. The solvent was removed in vacuo and the residue was co- evaporated with MeCN (3 x 2OmL). The crude amine was then dried under high vacuum for 3h. To a solution of PyBOP (271 mg, 0.521 mmol) and the protected amino acid 2 (271 mg, 0.521 mmol) in MeCN (2OmL) at O0C was added DIPEA (0.35mL, 2.01 mmol) under argon with stirring for 2 mins. A solution of the crude amine in MeCN (2OmL) was then added, and the reaction mixture was allowed to stir at rt for 16h. The solvent was then removed in vacuo, and the residue was purified by flash column chromatography on silica (eluant 1 :99-3:97-5:95 MeOH/CH2CI2) to furnish 3 (428.6mg, 0.408 mmol, 83%) as a white solid.
1H NMR (400MHz, CDCI3+ 10% MeOD) δH: 7.82 (d, J=8.16Hz, 2H), 7.70 (d, J=7.53Hz, 2H), 7.49 (d, J=6.90Hz, 2H), 7.38-7.09 (m, 22H), 6.49 (d, J=9.41Hz, 1 H), 6.21 (d, J=7.65Hz, 1H), 5.78 (m, 1 H), 5.22 (dd, JM 7.25, 1.44Hz, 1H), 5.14 (dd, J=10.42, 1.25Hz, 1 H), 4.52-4.45 (m, 2H), 4.30-4.17 (m, 2H), 4.07 (t, J=6.65Hz, 1H), 3.98 (m, 1H)1 3.90 (m, 1H), 3.81 (t, J=6.90Hz, 1 H), 3.67 (ddd, J=9.76, 7.69, 4.89Hz, 1 H), 3.33 (dt, J=3.23, 1.58Hz, 1 H), 2.56-2.46 (m, 3H), 2.36-2.28 (m, 4H), 2.17-1.87 (m, 5H), 1.66 (qq, 1 H), 0.79 (d, J=6.78Hz, 3H), 0.77 (d,
Figure imgf000067_0001
3H). MS (ES+) 1074.3 (100%, [M+Na]+). Rf 0.20 MeOH/CH2CI2 (1 :9).
(5): (3S,4R)-3-Hydroxy-4-{(S)-2-[(R)-2-((E)-(S)-3-hydroxy-7-tritylsulfanyl-hept-4- enoylam ino)-5-oxo-5-(toluene-4-sulf onylam ino)-pentanoylam ino]-3-tritylsulf anyl- propionylamino}-5-methyl-hexanoic acid allyl ester
To a solution of 3 (421.9mg, 0.401 mmol) in MeCN (2OmL) under Ar(g) was added diethylamine (2.OmL, 10% v/v) and the reaction mixture was allowed to stir at rt for 1.5h. The solvent was removed in vacuo, the residue was co- evaporated with MeCN (3 x 2OmL), and the crude amine was then dried under high vacuum. To a solution of PyBOP (212.6mg, 0.409 mmol) and the carboxylic acid 4 (169.5mg, 0.405 mmol) in MeCN/CH2CI2 (20mL_/2mL) was added DIPEA (0.28mL, 1.61 mmol) under Ar(g). A solution of the resultant deprotected amine of 3 in CH2CI2/CH2CI2 (2OmL/ 2m L) was added and the reaction was allowed to stir at room temperature for 16h. The solvent was then removed in vacuo and the crude product was purified by flash column chromatography on silica (eluant 2:98-4:96-6:94 MeOH/CH2CI2) to give 5 (241.Omg, 0.196 mmol, 49%) as a white solid.
1H NMR (400MHz, CDCI3+ 10% MeOD) δH: 7.79 (d, J=8.28Hz, 2H), 7.36- 7.30 (m, 12H), 7.22 (td, J=7Λ7, 1.51Hz, 14H), 7.18-7.12 (m, 6H), 6.51 (d, J=9.79Hz, 1 H), 5.78 (m, 1H), 5.49 (m, 1 H), 5.33 (dd, J=15.43, 6.53Hz, 1 H), 5.21 (dq, J=M.22, 1.41 Hz, 1H), 5.13 (dq, J=10.42, 1.21 Hz, 1 H), 4.50-4.45 (m, J=5.46, 3.80, 1.40, 1.40Hz, 2H), 4.31 (m, 1 H), 4.16 (dd, J=8.66, 5.02Hz, 1 H), 3.88 (ddd, J=9.79, 7.65, 2.51Hz, 1 H), 3.81 (t, J=6.96Hz, 1 H), 3.65 (ddd, J=9.88, 7.75, 4.71Hz, 1H), 3.32 (dt, J=3.26, 1.63Hz, 2H), 2.57-2.44 (m, 3H), 2.35 (s, 3H), 2.34-2.26 (m, 2H), 1.86-2.24 (m, 12H), 1.71 (m, 1H), 0.76 (d, J=6.7Hz, 3H), 0.74 (d, J=6.7Hz, 3H). MS (ES+) 1252.1 (100%, [M+Na]+). Rf 0.31 MeOH/CH2CI2 (1 :9).
(6): (3S,4R)-3-Hydroxy-4-{(S)-2-[(R)-2-((E)-(S)-3-hydroxy-7-tritylsulfanyl-hept-4- enoylam ino)-5-oxo-5-(toluene-4-sulf onylam ino)-pentanoylam ino]-3-tritylsulf anyl- propionylamino}-5-methyl-hexanoic acid
To a solution of 5 (236.9mg, 0.193 mmol), Pd(PPh3)4 (22.7mg, 0.0196 mmol) in dry MeOH (5.8mL) under Ar(g) was added morpholine (50μL, 0.574 mmol). After stirring for 1.5h, the reaction mixture was concentrated in vacuo and purified by flash column chromatography on silica (eluant 5:95-7:93-9:91- 1 :9-15.85 MeOH/CH2CI2) to furnish 6 an orange/yellow solid (191.4mg, 0.161 mmol, 83%).
1H NMR (400MHz, CDCI3+ 10% MeOD) δH: 7.78 (d, J=8.28Hz, 2H), 7.37- 7.30 (m, 12H), 7.26-7.18 (m, 14H)1 7.18-7.11 (m, 6H), 6.59 (d, J=9.79Hz, 1H), 5.48 (m, 1 H), 5.33 (m, 1 H), 4.31 (m, 1H), 4.13 (dd, J=7.78, 6.15Hz, 1 H), 3.92- 3.82 (m, 2H), 3.65 (br s, 1H), 3.32 (dt, J=3.14, 1.57Hz, 2H), 2.57 (m, 1 H), 2.47 (m, 1 H), 2.40 (dd, J=16.63, 1.94Hz, 1 H)1 2.35 (s, 3H), 2.28-1.86 (m, 14H), 1.77 (m, 1 H), 0.76 (d, J=6.7Hz, 3H), 0.74 (d, J=6.7Hz, 3H). MS (ES") 1188.3 (100%, [M-H]'). Rf 0.26 MeOH/CH2CI2 (1 :9). (7): N-{3-[(2S,6R,9S, 12R, 13S)-13-Hydroxy-12-isopropyl-4,7, 10, 15-tetraoxo-2- ((E)-4-tritylsulfanyl-but-1-enyl)-9-tritylsulfanylmethyl-1-oxa-5,8,11-triaza- cyclopentadec-6-yl]-propionyl}-4-methyl-benzenesulfonamide
To a solution of MNBA (64.66mg, 0.188 mmol) and DMAP (65.7mg, 0.538 mmol) in CH2CI2 (36.5mL) was added dropwise a solution of the acid 6 (187mg, 0.157 mmol) in CH2CI2 (146ml_) over 5h 10 min. The resulting mixture was stirred at rt for 16h, and was then concentrated in vacuo to give a white solid. Purification by flash column chromatography on silica (eluant 1 :99-2:98- 3:97 MeOH/CH2CI2) gave 7 (130.9mg, 0.112 mmol, 71%) as a white solid.
1H NMR (400MHz, CDCI3+ 10% MeOD) δH: 7.83 (d, J=8.03Hz, 2H), 7.39- 7.29 (m, 13H), 7.28-7.11 (m, 25H), 5.50 (m, 1H), 5.40 (br s, 1 H), 5.30 (m, 1H), 4.23 (br s, 1H)1 3.95 (m, 1 H), 3.63 (m, 1 H), 3.41 (br s, 1 H), 3.32 (br s, 1 H), 2.70 (m, 1 H), 2.37 (s, 3H), 2.33 (br s, 2H), 2.19 (br s, 1 H), 2.12 (t, J=7.03Hz, 3H), 2.03-1.95 (m, 4H), 1.76 (br S, 1 H), 1.49 (br S, 1 H), 0.78 (d, J=6.53Hz, 3H), 0.72 (d, J=6.65Hz, 3H). MS (ES+) 1194.2 (100%, [M+Na]+). Rf 0Λ2 MeOH/CH2CI2 (1 :9).
Compound XXIX: N-[3-((E)-(1 S,5S,6R,9S,20R)-5-Hydroxy-6-isopropyl- 3,8, 18,21 -tetraoxo-2-oxa-11 , 12-dithia-7, 19,22-triaza-bicyclo[7.7.6]docos-15-en- 20-yl)-propionyl]-4-methyl-benzenesulfonamide
To a solution of iodine (280.5mg, 1.11 mmol) in CH2CI2/MeOH (9:1 , 371 ml_) was added dropwise a solution of 7 (126mg, 0.108 mmol) in CH2CI2/MeOH (9:1 , 182.5mL) over 50 min. The reaction mixture was then allowed to stir for a further 30 min, after which sodium thiosulfate (156mL, 0.05M) was added. The layers were separated and the product was extracted with EtOAc (3 x 12OmL); after drying over MgSO4, the solvent was removed in vacuo. Purification was then performed using flash column chromatography on silica (eluant 1 :99-2:98- 4:96-6:94-8:92-1 :9 MeOH/CH2CI2) to give compound XXIX (23.5mg, 0.0343 mmol, 32%) as a white solid.
1H NMR (400MHz, CDCI3+ 15% MeOD) δH: 8.94 (d, J=4.89Hz, 1 H), 7.91 (d, J=6.53Hz, 1 H)1 7.80 (d, J=8.41 Hz, 2H), 7.24 (d, J=8.03Hz, 2H), 5.88 (d, J=4.14Hz, 2H), 5.81 (d, J=9.91 Hz, 1 H), 5.01 (br S, 1 H), 4.10 (t, J=8.28Hz, 1 H), 4.03 (d, J=2.01 Hz, 1 H), 3.80 (td, J=9.41 , 3.64Hz, 1 H), 3.65-3.36 (m, 5H), 3.17 (m, 1 H), 3.06-2.80 (m, 3H), 2.76-2.21 (m, 9H), 2.11 (m, 1 H), 1.94 (m, 1 H), 1.75 (dd, JM 5.00, 8.97Hz, 1 H), 0.84 (d, J=6.7Hz, 3H), 0.82 (d, J=6.7Hz, 3H); MS (ES+) 707.9 (100%, [M+Na]+). Rf 0.38 CH2CI2/MeOH (95:5). Compound XXX: (2S,6R,9S,12R,13S)-13-Hydroxy-12-isopropyl-2-((E)-3- mercapto-propenyl)-9-methylsulfanylmethyI-6-thiophen-3-ylmethyl-1-oxa- 5,8,11 -triaza-cyclopentadecane-4,7,10,15-tetraone
Figure imgf000069_0001
4
Figure imgf000069_0002
XXX 6 5
(2): (3S,4R)-4-{(S)-2-[(R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-thiophen- 3-yl-propionylamino]-3-tritylsulfanyl-propionylamino}-3-hydroxy-5-methyl- hexanoic acid allyl ester
To a solution of tripeptide 1 (450mg, 0.58 mmol, 1eq) in MeCN (12mL) was added 1.2mL of Et2NH (10% v/v) dropwise at rt under Ar(g). The solution was stirred at rt for 2h, then the solvent was removed in vacuo. The excess of amine was co-evaporated with MeCN (3 x 1OmL), then with a 1 :5 mixture of
CH2Ci2/hexane (15mL). A white solid was obtained which was dried under vacuum for 2h. To a solution of Fmoc-D-4-ThienylAla (253mg, 0.64 mmol, 1.1 eq) in MeCN (1OmL) at 00C was added PyBOP (335mg, 0.64 mmol, 1.1 eq) and
DIPEA (255 μL, 1.46 mmol, 2.5eq) under Ar(g). The crude amine, dissolved in CH2CI2 (1OmL) was added to the mixture in dropwise fashion, and the reaction mixture was left to warm to rt overnight. The mixture was then concentrated in vacuo and the residue was further purified by silica gel column chromatography using hexane/EtOAc (3:2) as eluant to yield 2 as a pale yellow solid (468mg, 86%).
1H NMR (400MHz, CDCI3) δH: 7.77 (d, J=7.5Hz, 2H), 7.48 (t, J=7.0Hz, 2H), 7.35-7.44 (m, 10H), 7.28-7.33 (m, 3H), 7.22-7.26 (m, 4H), 7.15-7.22 (m, 4H), 6.98 (br. s., 1 H), 6.83-6.91 (m, 1H), 6.35 (br. s., 1 H), 6.08 (br. s., 1H), 5.88 (dd, 1 H), 5.31 (dd, J=M.2, 1.4Hz, 1H), 5.22 (dd, JM 0.4, 1 -1Hz, 1 H), 5.05 (br. s., 1H), 4.51-4.64 (m, 2H), 4.44 (dd, J=10.7, 7.0Hz, 1 H), 4.27 (br. S., 2H), 4.12 (t, J=6.7Hz, 1 H), 3.99 (br. s., 2H), 3.82 (br. s., 1H), 3.05 (br s., 2H), 2.78 (br. s., 1 H), 2.59 (dd, J=16.5, 2.0Hz, 1H), 2.50 (dd, J=13.0, 5.8Hz, 1H), 2.44 (dd, J=16.6, 9.3Hz, 1 H), 2.07-2.17 (m, 1 H), 0.83-0.94 (m, 6H). MS (ES+) 945.2 (100%, [M+Na]+).
(4): (3S,4R)-3-Hydroxy-4-{(S)-2-[(R)-2-((E)-(S)-3-hydroxy-7-tritylsulfanyl-hept-4- enoylamino)-3-thiophen-3-yl-propionylamino]-3-tritylsulfanyl-propionylamino}-5- methyl-hexanoic acid allyl ester
To a solution of 2 (468mg, 0.51 mmol, 1eq) in MeCN (1OmL) was added 1.OmL of Et2NH (10% v/v) in dropwise fashion at rt under Ar(g). The solution was stirred at rt for 2h, and the solvent was then removed in vacuo. The excess of amine was co-evaporated with MeCN (3 x 5mL), then with a 1 :5 mixture of CH2CI2/hexane (1OmL). A white solid was obtained, which was dried under high vacuum for 2h. To a solution of the chiral 3 -hydroxy acid 3 (234mg, 0.56 mmol, 1.05eq) in MeCN (1OmL) at 00C was added PyBOP (372mg, 0.56 mmol, 1.1 eq) and N-ethyldiisopropylamine (221 μL, 1.27 mmol, 2.5eq) under Ar(g). The crude amine, dissolved in CH2CI2 (1OmL) was added in dropwise fashion, and the reaction mixture was left to warm to rt overnight. The mixture was then concentrated in vacuo and the residue was further purified by silica gel column chromatography with hexane/EtOAc (1 :1 -> 0:1) to yield 4 as a pale yellow solid (583mg, 99%).
1H NMR (400MHz, CDCI3 + 10% MeOH) δH: 7.28-7.36 (m, 13H), 7.18- 7.25 (m, 13H), 7.11-7.17 (m, 6H), 7.08 (dd, J=5.0, 2.9Hz, 1 H), 6.95 (d, J=1.8Hz, 1 H), 6.81 (dd, J=4.9, 1.2Hz, 1H) ,6.57 (d, J=9.9Hz, 1 H), 5.73-5.85 (ddt, J=17.2, 10.5, 5.7Hz, 1 H), 5.34-5.44 (m, 1H), 5.22-5.30 (m, 1 H), 5.22 (dd, J=17.2, 1.4Hz, 1 H), 5.13 (dd, J=10.4, 1.3Hz, 1 H), 4.45-4.52 (m, 3H), 4.18-4.25 (m, 1 H), 3.86- 3.97 (m, 2H), 3.64 (ddd, J=9.7, 7.4, 5.3Hz, 1H), 2.93-3.06 (m, 2H), 2.50 (dd, J=16.1 , 2.4Hz, 1 H), 2.44 (d, J=7.2Hz, 2H), 2.31 (dd, J=16.1 , 9.9Hz, 1 H), 2.18 (dd, J=13.7, 3.3Hz, 1 H), 2.07-2.16 (m, 4H), 1.93-2.04 (m, 4H), 0.78 (dd, J=6.8, 3.1 Hz, 6H). MS (ES+) 1123.6 (100%, [M+Na]+).
(5): (3S,4R)-3-Hydroxy-4-{(S)-2-[(R)-2-((E)-(S)-3-hydroxy-7-tritylsulfanyl-hept-4- enoylamino)-3-thiophen-3-yl-propionyiamino]-3-tritylsulfanyl-propionylamino}-5- methyl-hexanoic acid
To a solution of 4 (583mg, 0.53 mmol, 1eq) in anhydrous MeOH (11mL) and THF (2mL) was added morpholine (70 μL, 0.80 mmol, 1.5eq) and Pd(PPh3)4 (12.2mg, 0.01 mmol, 2 mol%) at rt under Ar(g). The reaction mixture was stirred for 1h, concentrated in vacuo, and the resulting residue was purified by silica gel column chromatography with CH2CI2/MeOH (49:1 -> 9:1)/AcOH (0.1%) to yield 5 as a pale yellow solid (580mg, 99%).
1H NMR (300MHz, CDCI3 + 10% MeOH) δH: 7.28-7.38 (m, 14H), 7.19- 7.27 (m, 12H), 7.12-7.19 (m, 7H), 7.10 (dd, J=4.9, 2.9Hz, 1H), 6.95-7.01 (m, 1 H), 6.84 (dd, J=4.9, 1.2Hz, 1H), 6.69 (d, Λ=9.7Hz, 1H), 5.33-5.46 (m, 1 H), 5.27 (dd, J=15.4, 6.0Hz, 1H), 4.47 (br. s., 1H), 4.22 (br. s., 1 H), 3.88-3.98 (m, 2H), 3.81-3.87 (m, 3H), 3.60-3.70 (m, 1H), 3.11-3.18 (m, 2H), 2.92-3.09 (m, 1 H), 2.46 (d, J=7.2Hz, 2H), 2.21-2.32 (m, 1 H), 2.11-2.20 (m, 4H), 1.98-2.07 (m, 2H), 0.76- 0.81 (m, 6H). MS (ES+) 1083.9 (100%, [M+Na]+). (6): (2S,6R,9S, 12R, 13S)-13-Hydroxy-12-isopropyl-6-thiophen-3-ylmethyl-2-((E)- 4-tritylsulfanyl-but-1 -enyl)-9-tritylsulfanylmethyl-1 -oxa-5,8,11 -triaza- cyclopentadecane-4,7, 10,15-tetraone
To a solution of MNBA (226mg, 0.66 mmol, 1.2eq) and DMAP (160.0mg, 1.31 mmol, 2.4eq) in CH2CI2 (13OmL) under Ar(g) was added in dropwise fashion a solution of 5 (580mg, 0.55 mmol, 1eq) in CH2CI2 (45OmL) and THF (2OmL) over 3h. The reaction mixture was left to stir at rt overnight, and was subsequently concentrated in vacuo. The resulting residue was purified by silica gel column chromatography with CH2CI2/MeOH (49:1 -> 19:1) to yield 6 as a white solid (219mg, 40%).
1H NMR (400MHz, CDCI3) δH: 7.37-7.45 (m, 12H), 7.27-7.33 (m, 12H), 7.24-7.27 (m, 3H), 7.18-7.24 (m, 6H), 7.15 (dd, J=4.9, 2.9Hz, 1 H), 6.99 (d, J=2.0Hz, 1 H), 6.89 (dd, J=4.9, 1.0Hz, 1 H), 5.60 (dd, ^=15.2, 6.6Hz, 1H), 5.46- 5.53 (m, 1 H), 5.32 (dd, J=17.8, 6.7Hz, 2H), 4.53 (q, J=7.0Hz, 1 H), 4.24-4.32 (m, 1H), 3.32-3.50 (m, 2H), 3.09 (dd, J=14.6, 6.0Hz, 1 H), 2.87-3.00 (m, 2H), 2.83 (dd, J=12.6, 7.0Hz, 1H), 2.42-2.52 (m, 2H), 2.39 (d, Λ=10.9Hz, 1 H), 2.35 (dd, J=10.8, 4.1 Hz, 1 H), 2.18 (t, Λ=7.2Hz, 3H), 2.00-2.09 (m, 3H), 0.93 (d, J=6.7Hz, 3H), 0.88 (d, J=6.7Hz, 3H). MS (ES+) 1065.1 (100%, [M+Na]+). Compound XXX: (2S,6R,9S, 12R, 13S)-13-Hydroxy-12-isopropyl-2-((E)-3- mercapto-propenyl)-9-methylsulfanylmethyl-6-thiophen-3-ylmethyl-1 -oxa-5,8,11 - triaza-cyclopentadecane-4,7, 10, 15-tetraone
To a solution of I2 (533mg, 2.10 mmol, 10eq) in CH2CI2 (45OmL) and MeOH (5OmL) was added dropwise a solution of 6 (219mg, 0.21 mmol, 1 eq) in CH2CI2 (225mL) and MeOH (25mL) over 2h at rt under Ar(g). The reaction mixture was further stirred for 30 mins, and was then treated with a solution of sodium thiosulfate (0.1 M, 25OmL) and brine (3OmL). The organic layer was isolated and the aqueous layer was extracted with CH2CI2 (2 x 10OmL) and EtOAc (2 x 10OmL). The combined organic extracts were dried over MgSO4 and the solvent was removed in vacuo. The residue was purified by silica gel column chromatography with CH2CI2/MeOH (49:1 -> 19:1) to yield compound XXX as a white solid (100mg, 81%).
1H NMR (400MHz, CDCI3) δH: 7.41 (dd, J=4.9, 2.9Hz, 1 H), 7.36 (d, J=7.1 Hz, 1 H), 7.10 (d, J=2.QHz, 1 H), 6.98 (d, J=4.9Hz, 1 H), 6.72 (d, J=9.5Hz, 1 H), 6.44 (t, J=13.0Hz, 1 H), 5.89 (d, J=3.6Hz, 1 H)1 5.51 (d, J=15.4Hz, 1 H), 5.43- 5.48 (m, 1 H), 4.94-5.01 (m, 1 H), 4.50-4.61 (m, 2H), 3.44-3.71 (m, 2H), 3.27-3.36 (m, 2H), 3.17 (dd, J=15.0, 9.5Hz, 1 H), 3.01 (d, J=13.7Hz, 1 H), 2.63-2.78 (m, 5H), 2.48 (d, J=13.2Hz, 1 H), 2.32-2.44 (m, 2H), 1.04 (d, J=6.8Hz, 3H), 0.94 (d, J=6.7Hz, 3H). MS (ES+) 578.8 (100%, [M+Na]+).
Compound XXXl: (EH1S,5S,6R,9S,20R)-5-Hvdroxy-6-isopropyl-20- thiophen-2-ylmethyl-2-oxa-11.12-dithia-7.19,22-triaza-bicvclor7.7.61docos- 15-ene-3,8, 18,21 -tetraone
Figure imgf000072_0001
Figure imgf000072_0002
(2): (3S,4R)-4-{(S)-2-[(R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-thiophen- 2-yl-propionylamino]-3-tritylsulfanyl-propionylamino}-3-hydroxy-5-methyl- hexanoic acid allyl ester
To a solution of 1 (481.3mg, 0.626 mmol (prepared according to the procedure in Doi, T. et al, Tet. Lett. 2006, 47, 1177)) in MeCN (2OmL) under Ar(g) was added diethylamine (2mL, 10% v/v), and the reaction mixture was stirred at rt for 1h 50 min. The solvent was removed in vacuo, and the residue co-evaporated with MeCN (3 x 2OmL). The crude amine was then dried under high vacuum for 1h 25 min. To a solution of PyBOP (332.6mg, 0.639 mmol) and Fmoc-β-(2-thienyl)-D-alanine (249.3mg, 0.633 mmol) in CH2CI2 (2OmL) was added DIPEA (0.3OmL, 1.72 mmol) at O0C under Ar(g) and the mixture was stirred for 2min. A solution of the crude amine derivative of 1 in MeCN (2OmL) was then added, and the reaction mixture was allowed to stir at O0C for 30 min before being allowed to warm to rt over 16h. The solvent was then removed in vacuo, and purification using flash column chromatography on silica (eluant 3:7- 4:6-1 :1 EtOAc/Hexane) gave 2 (455.2mg, 0.494 mmol, 79%) as a white solid.
1H NMR (300MHz, CDCI3) δH: 7.77 (d, J=7.23Hz, 2H), 7.49 (dd, J=6.77, 5.76Hz, 2H), 7.45-7.11 (m, 22H), 6.89 (dd, J=4.67, 3.66Hz, 1 H), 6.81 (m, 1 H), 6.39 (br s, 1 H), 6.08 (m, 1H), 5.87 (m, 1 H), 5.36-5.12 (m, 3H), 4.58 (dd, J=5.67, 1.37Hz, 2H), 4.42 (m, 1 H), 4.28 (br s, 2H), 4.12 (t, Λ=6.68Hz, 1H), 3.99 (dd, J=16.60, 7.09Hz, 2H), 3.83 (br s, 1 H), 3.24 (br s, 2H), 2.82 (br s, 1H), 2.63-2.37 (m, 3H), 0.88 (d, J=6.50Hz, 6H). MS (ES+) 945.2 (100%, [M+Na]+). Rf 0.56 EtOAc/Hexane (6:4). (4): 3S,4R)-3-Hydroxy-4-{(S)-2-[(R)-2-((E)-(S)-3-hydroxy-7-tritylsulfanyl-hept-4- enoylamino)-3-thiophen-2-yl-propionylamino]-3-tritylsulfanyl-propionylamino}-5- methyl-hexanoic acid allyl ester
To a solution of 2 (450mg, 0.488 mmol) in MeCN (2OmL) under Ar(g) was added diethylamine (2.OmL, 10% v/v). The reaction mixture was stirred at rt for 1h 25 min, the solvent then removed in vacuo, and the residue co-evaporated with MeCN (3 x 2OmL). The crude amine was then isolated and dried under high vacuum for 3h. Then to a solution of PyBOP (271.5mg, 0.522 mmol) and the carboxylic acid 3 (215.8mg, 0.516 mmol) in CH2CI2 (2OmL) was added DIPEA (0.23mL, 1.32 mmol) under Ar(g). A solution of the crude amine derivative of 2 in MeCN (2OmL) was then added, and the reaction mixture stirred at room temperature for 16h. The solvent was removed in vacuo and the crude product was purified by flash column chromatography on silica (eluant 4:6-1 :1-6:4 EtOAc/Hexane) to give 4 (427.6mg, 0.389 mmol, 80%) as a white solid.
1H NMR (400MHz, CDCI3) δH: 7.50 (d, J=7.91 Hz, 1 H), 7.38 (dd, Jt 14.62, 7.59Hz, 12H), 7.31-7.16 (m, 19H), 7.06 (dd, J=4.64, 1.51 Hz, 1 H), 6.89 (d, J=5.77Hz, 1H), 6.85-6.80 (m, 2H), 6.58 (d, J=9.79Hz, 1 H), 5.94 (m, 1H), 5.45 (m, 1 H), 5.35 (d, J=6.15Hz, 1 H), 5.28 (dd, J=17.32, 1.38Hz, 1H), 5.19 (dd, Jt 10.42, 1.13Hz, 1 H), 4.55 (dd, J=5.71 , 1.32Hz, 2H), 4.29 (br s, 1 H), 3.98 (dt, Jt14.27, 7.23Hz, 2H), 3.71 (m, 1 H), 3.25 (d, J=6.02Hz, 2H), 3.01 (br S, 3H), 2.59-2.49 (m, 2H), 2.37 (dd, J=16.06, 9.79Hz, 1 H), 2.27 (m, 1H), 2.23-2.17 (m, 3H), 2.10-1.97 (m, 3H), 0.84 (dd, J=6.71 , 2.45Hz, 6H). MS (ES+) 1122.9 (100%, [M+Na]+). a 0.41 EtOAc/Hexane (6:4).
(5): (3S,4R)-3-Hydroxy-4-{(S)-2-[(R)-2-((E)-(S)-3-hydroxy-7-tritylsulfanyl-hept-4- enoylamino)-3-thiophen-2-yl-propionylamino]-3-tritylsulfanyl-propionylamino}-5- methyl-hexanoic acid To a solution of 4 (425mg, 0.386 mmol), and Pd(PPh3)4 (44.1mg, 0.0382 mmol) in dry MeOH (11.5mL) under Ar(g) was added morpholine (55 μl_, 0.629 mmol). After stirring for 2h 10 min, the reaction mixture was concentrated in vacuo and was purified by flash column chromatography on silica (eluant 2:98- 4:96-6:94-8:92-1.9 MeOH/CH2CI2) to give 5 as a white solid (348mg, 0.328 mmol, 85%): R, 0.10 MeOH/CH2CI2 (7:93).
1H NMR (400MHz, CDCI3+10% MeOD) δH: 7.41 (d, J=7.65Hz, 5H), 7.38- 7.34 (m, 5H), 7.32-7.19 (m, 20H), 6.86 (m, 1 H), 6.37 (m, 1 H), 5.40 (m, 1 H), 5.28 (m, 1 H), 4.54 (q, J=5.98Hz, 1H), 4.30 (br s, 1 H), 4.11 (m, 1 H), 4.05 (br s, 1 H), 3.89 (t, J=4.71Hz, 1H), 3.78 (m, 1 H), 3.37 (d, J=7.40Hz, 1 H), 3.29 (d, J=5.40Hz, 2H), 2.85 (br s, 1H), 2.66 (dd, JM 2.42, 7.15Hz, 1H), 2.55 (dd, JM 6.00, 3.20Hz, 2H), 2.48-2.39 (m, 3H), 2.31 (dd, JM3.24, 2.45Hz, 2H), 2.23-2.15 (m, 3H), 2.09- 2.00 (m, 3H), 0.93-0.86 (m, 6H). MS (ES") 1059.0 (100%, [M-H]"). (6): (2S,6R,9S, 12R, 13S)-13-Hydroxy-12-isopropyl-6-thiophen-2-ylmethyl-2-((E)- 4-tritylsulfanyl-but-1 -enyl)-9-tritylsulfanylmethyl-1 -oxa-5,8,11 -triaza- cyclopentadecane-4,7, 10, 15-tetraone
To a solution of MNBA (134.18mg, 0.390 mmol) and DMAP (95.4mg, 0.781 mmol) in CH2CI2 (75ml_) was added dropwise a solution of the acid 5 (344.53mg, 0.325 mmol) in CH2CI2 (301 ml.) over 5h 20 min. The resulting reaction mixture was stirred at rt for 16h, and was then concentrated in vacuo and purified by flash column chromatography on silica (eluant 0.5-99.5:1-99- 1.5:98.5 MeOH/CH2CI2) to furnish 6 (172mg, 0.165 mmol, 51%) as a white solid. 1H NMR (400MHz, CDCI3) δH: 7.46-7.37 (m, 12H), 7.34-7.18 (m, 20H), 7.10 (dd, J=3.95, 2.32Hz, 1H), 6.96 (d, J=9.04Hz, 1 H), 6.84-6.80 (m, 2H), 6.15 (d, J=6.40Hz, 1 H), 5.60 (m, 1H), 5.50 (br s, 1H), 5.33 (m, 1 H), 4.50 (q, J=6.65Hz, 1 H), 4.29 (m, 1H), 3.39 (dd, J=14.37, 8.09Hz, 2H), 3.27 (m, 1 H), 3.14 (dd, J=15.00, 7.34Hz, 1 H)1 2.90 (qd, J=12.72, 7.15Hz, 2H), 2.54-2.32 (m, 4H), 2.22-2.15 (m, 2H), 2.09-2.00 (m, 2H), 1.92 (m, 1H), 0.91 (dd, J=16.69, 6.65Hz, 6H); MS (ES+) 1064.7 (100%, [M+Naf). f?,0.48 MeOH/CH2CI2 (8:92). Compound XXXI: (E)-(I S,5S,6R,9S,20R)-5-Hydroxy-6-isopropyl-20-thiophen-2- ylmethyl-2-oxa-11 , 12-dithia-7, 19,22-triaza-bicyclo[7.7.6]docos-15-ene-3,8, 18,21 - tetraone
To a solution of iodine (418.6mg, 1.65 mmol) in CH2CI2/Me0H (9:1) (560 ml_) was added dropwise a solution of 6 (170mg, 0.163 mmol) in CH2CI2/Me0H (9:1) (276m L) over 1h. The reaction mixture was then allowed to stir for a further 35 min, after which sodium thiosulfate (235mL, 0.05M) was added. The layers were separated and the product was extracted with EtOAc (3 x 15OmL); the organic layers were isolated, dried (MgSO4) and the solvent was removed in vacuo. Purification was performed using flash column chromatography on silica (eluant 0:1-1 :99-3:97 MeOH/CH2CI2) to furnish compound XXXI (84.8mg, 0.153 mmol, 94%) as a white solid.
1H NMR (400MHz, CDCI3+10% MeOD) δH: 7.39 (d, J=7.15Hz, 1 H), 7.19 (dd, J=5.14, 1.00Hz, 1H), 6.95 (dd, J=5.08, 3.45Hz, 1H), 6.89-6.84 (m, 2H), 6.77 (d, J=9.29Hz, 1 H), 6.26 (t, Λ=13.05Hz, 1H), 5.60 (d, J=15.43Hz, 1 H), 5.44 (d, J=4.02Hz, 1 H), 4.86 (td, J=8.66, 3.39Hz, 1H), 4.51-4.41 (m, 2H), 3.41 (m, 1 H), 3.36-3.31 (m, 2H), 3.16 (dd, J=13.11 , 6.96Hz, 1H), 2.92 (d, J=12.67Hz, 1H), 2.84-2.70 (m, 4H), 2.64-2.53 (m, 5H), 2.38-2.25 (m, 2H), 0.96 (d, J=6.90Hz, 3H), 0.86 (d, ^=6.65Hz, 3H); 13C NMR (100MHz, CDCI3+10% MeOD) δc: 171.6 (C), 171.5 (C), 170.1 (C), 169.1 (C), 137.4 (C), 132.9 (CH), 128.7 (CH), 127.7 (CH), 126.8 (CH), 125.4 (CH), 70.6 (CH), 68.7 (CH), 63.3 (CH), 57.0 (CH), 54.7 (CH), 41.7 (CH2), 40.4 (CH2), 40.3 (CH2), 39.7 (CH2), 33.8 (CH2), 30.2 (CH2), 29.6 (CH), 20.6 (CH3), 19.4 (CH3). MS (ES+) 578.8 (100%, [M+Na]+). R, 0.21 CH2CI2/MeOH (95:5). Compound XXXII: (EW1S.5S,6R.9S,20R)-5-Hvdroxy-6-isopropyl-20-(3- trif luoromethyl-benzyl)-2-oxa-11 ,12-dithia-7,19,22-triaza- bicvclorT.y.βidocos-IS-ene-S.S.IS.ΣI-tetraone
Figure imgf000076_0001
(2): (3S,4R)-4-{(S)-2-[(R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-(3- trifluoromethyl-phenyl)-propionylamino]-3-tritylsulfanyl-propionylamino}-3- hydroxy-5-methyl-hexanoic acid allyl ester
To a solution of 1 (410mg, 0.533 mmol, 1eq (prepared according to the procedure in Doi, T. et al, Tet. Lett. 2006, 47, 1177)) in MeCN (5.4mL) was added Et2NH (10% v/v, 0.6ml_) dropwise at rt under Ar(g). The solution was stirred at rt for 2h, then the solvent was removed in vacuo. The excess of amine was co-evaporated with MeCN (3 x 1OmL) before the reaction mixture was dried under high vacuum for 2h.
To a solution of Fmoc-3-(trifluoromethly)-D-phenylalanine (267mg, 0.586 mmol, 1.1 eq) in CH2CI2 (7mL) at O0C was added PyBOP (305mg, 0.586 mmol, 1.1 eq) and DIPEA (0.23mL, 1.3 mmol, 2.5eq) under Ar. The crude amine, dissolved in MeCN (8ml_), was added via cannula and the reaction mixture was then left to warm to rt overnight before being concentrated in vacuo. Purification by flash column chromatography (eluant 5-50% EtOAc/Hexane) gave the product 2 (479mg, 0.487 mmol, 91%) as a white solid.
1H NMR (300MHz, 9:1 CDCI3/CD3OD) δH: 7.69 (d, J=7.5Hz, 2H), 7.05- 7.51 (m, 26H), 5.68-5.87 (m, J=M.2, 10.6, 5.6, 5.6Hz, 1H), 5.22 (dd, J=17.2, 1.4Hz, 1H), 5.13 (dd, J=10.5, 1.0Hz, 1H), 4.40-4.58 (m, 2H), 4.21-4.39 (m, 1H), 4.09-4.21 (m, 1 H), 4.04 (q, J=5.9Hz, 1 H), 3.81-3.96 (m, 2H), 3.61-3.74 (m, 1 H), 3.06 (dd, J=13.7, 4.7Hz, 1H), 2.84 (dd, J=14.5, 9.5Hz, 1 H), 2.41-2.60 (m, 3H), 2.32 (dd, JM 5.9, 9.6Hz, 1 H), 1.95-2.07 (m, 1 H), 0.78 (t, J=6.3Hz, 6H). MS (ES+) 1007 (100%, [M+Na]+).
(4): 3S,4R)-3-Hydroxy-4-{(S)-2-[(R)-2-((E)-(S)-3-hydroxy-7-tritylsulfanyl-hept-4- enoylamino)-3-(3-trifluoromethyl-phenyl)-propionylamino]-3-tritylsulfanyl- propionylamino}-5-methyl-hexanoic acid allyl ester
To a solution of 2 (475mg, 0.483 mmol, 1eq) in MeCN (5.4mL) was added Et2NH (10% v/v, 0.6mL) dropwise at rt under Ar(g). The solution was stirred at rt for 2h, then the solvent was removed in vacuo. The excess of amine was co-evaporated with MeCN (3 x 1OmL) and the crude product was dried under high vacuum for 2h.
To a solution of the carboxylic acidacid 3 (212mg, 0.510 mmol, 1.05eq) in CH2CI2 (7ml_) at 00C was added PyBOP (276mg, 0.530 mmol, 1.1 eq) and DIPEA (210 μL, 1.2 mmol, 2.5eq) under Ar(g). The crude amine, dissolved in MeCN (8mL) was added via cannula, and the reaction mixture was left to warm to rt overnight before being concentrated in vacuo. Purification by flash column chromatography (eluant 10-60% EtOAc/Hexane) gave 4 (375mg, 0.323 mmol, 67%) as a white solid.
1H NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 7.07-7.44 (m, 34H), 6.57 (d, J=9.8Hz, 1H), 5.69-5.85 (m, 1H), 5.38 (dt, JM 5.3, 6.5Hz, 1 H), 5.16-5.30 (m, 2H), 5.08-5.15 (m, 1 H), 4.41-4.58 (m, 3H), 4.13-4.23 (m, 1 H), 3.78-3.95 (m, 2H), 3.56-3.67 (m, 1 H), 3.13 (dd, JM4.3, 4.5Hz, 1 H), 2.82 (dd, JM4.3, 9.4Hz, 1 H), 2.37-2.56 (m, 3H), 2.29 (dd, JM 6.1 , 9.9Hz, 1 H), 2.06-2.17 (m, 4H), 1.92-2.04 (m, 3H), 0.75 (d, J=6.9Hz, 6H). MS (ES+) 1185 (100%, [M+Na]+). (5): (3S,4R)-3-Hydroxy-4-{(S)-2-[(R)-2-((E)-(S)-3-hydroxy-7-tritylsulfanyl-hept-4- enoylamino)-3-(3-trifluoromethyl-phenyl)-propionylamino]-3-tritylsulfanyl- propionylamino}-5-methyl-hexanoic acid
To a solution of 4 (375mg, 0.323 mmol, 1eq) in anhydrous MeOH (5mL) was added morpholine (42 μL 0.48 mmol, 1.5eq) and Pd(PPh3)4 (7.5mg, 0.007 mmol, 0.02eq) at rt under Ar(g). The reaction mixture was stirred for 2h, then concentrated in vacuo, and purified flash column chromatography (eluant 1-10% MeOH/CH2CI2) to furnish 5(362mg, 0.323 mmol, 99%) as a white solid.
1H NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 7.52 (d, J=7.9Hz, 1H), 7.12- 7.46 (m, 34H), 6.53 (d, J=9.7Hz, 1H), 5.41 (dd, JM 5.1 , 6.7Hz, 1H), 5.28 (dd, JM 5.4, 6.2Hz, 1H), 4.57 (dd, J=8.9, 4.6Hz, 1 H), 4.19-4.29 (m, 1 H), 3.80-3.97 (m, 2H), 3.63-3.74 (m, 1H), 3.19 (dd, JM4.4, 4.7Hz, 1 H), 2.91 (dd, JM4.3, 9.1Hz1 1 H), 2.42-2.70 (m, 5H), 2.30 (dd, J=16.3, 9.2Hz, 1 H), 1.96-2.23 (m, 5H), 0.81 (d, J=6.7Hz, 3H), 0.80 (d, J=6.8Hz, 3H). MS (ES+) 1145 (100%, [M+Na]+). (6): (2S,6R,9S, 12R, 13S)-13-Hydroxy-12-isopropyi-6-(3-trif luoromethyl-benzyl)-2- ((E)-4-tritylsulfanyl-but-1-enyl)-9-tritylsulfanylmethyl-1-oxa-5,8,11-triaza- cyclopentadecane-4,7, 10, 15-tetraone
To a solution of MNBA (134mg, 0.388 mmol) and DMAP (95mg, 0.78 mmol) in CH2CI2 (8OmL) was added dropwise a solution of acid 5 (362mg, 0.323 mmol) in CH2CI2/THF (245:5, 25OmL) over 5h under Ar(g). After a further 14h stirring, the reaction mixture was concentrated in vacuo to give an off white solid. Purification by column chromatography on silica gel (1-5% IPA/CH2CI2) gave 6 (196mg, 0.177 mmol, 55%) as a white solid.
1H NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 7.08-7.42 (m, 34H), 5.51 (dt, JM 5.2, 6.8Hz, 1H), 5.31-5.39 (m, 1 H), 5.25 (dd, JM 5.2, 6.5Hz, 1 H), 4.31 (dd, J=8.5, 6.9Hz, 1 H), 3.88-3.97 (m, 2H), 3.21 (dd, J=8.2, 5.0Hz, 1 H), 3.03 (dd, J=13.9, 6.5Hz, 1H), 2.83-2.95 (m, 2H), 2.67-2.77 (m, 1 H), 2.19-2.42 (m, 4H), 1.89-2.15 (m, 5H), 0.79 (d, J=7.0Hz, 3H), 0.77 (d, J=7.0Hz, 3H). MS (ES+) 1127 (100%, [M+Na]+).
Compound XXXII: (E)-(I S,5S,6R,9S,20R)-5-Hydroxy-6-isopropyl-20-(3- trif iuoromethyl-benzyl)-2-oxa-11 , 12-dithia-7, 19,22-triaza-bicyclo[7.7.6]docos-15- ene-3,8,18,21-tetraone
To a solution of I2 (450mg, 1.77 mmol, 10eq) in CH2Ci2 (36OmL) and MeOH (4OmL) was added dropwise a solution of 6 (196mg, 0.177 mmol, 1eq) in CH2CI2 (18OmL) and MeOH (2OmL) over 3h at rt under Ar(g). The reaction mixture was stirred for a further 20 min, and was then treated with a solution of sodium thiosulfate (0.1 M, 5OmL) and brine (25mL). The organic layer was isolated and the aqueous layer was extracted with EtOAc (3 x 5OmL). The combined organic extracts were dried over MgSO4 and the solvent was removed in vacuo. Purification by column chromatography on silica gel (1-3.5% MeOH/CH2CI2) furnished compound XXXII (109mg, 0.176 mmol, 99%) as a white solid.
1H NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 7.79 (br. s., 1 H), 7.50 (d, J=7.3Hz, 1H), 7.31-7.47 (m, 4H), 6.86 (d, J=8.8Hz, 1 H), 5.91-6.05 (m, 1 H), 5.69 (d, J=15.4Hz, 1 H), 5.40 (br. s., 1 H), 4.72 (td, J=9.0, 4.0Hz, 1 H), 4.42 (dt, J=8.8, 3.6Hz, 1H), 4.37 (d, J=6.9Hz, 1 H), 2.90-3.20 (m, 6H), 2.75 (q, J=8.5Hz, 1H), 2.46-2.61 (m, 5H), 2.15-2.32 (m, 2H), 0.92 (d, J=6.8Hz, 3H), 0.81 (d, Λ=6.7Hz, 3H). MS (ES+) 640 (100%, [M+Na]+).
Compound XXXIII: (EH1S.5S.βR.9S.20m-20-(3-Chloro-benzyl)-5-hvdroxy-β- isopropyl-2-oxa-11 ,12-dithia-7,19,22-triaza-bicvclol7.7.61docos-15-ene- 3,8,18.21 -tetraone
Figure imgf000079_0001
Figure imgf000079_0002
(2): (3S,4R)-4-{(S)-2-[(R)-3-(3-Chloro-phenyl)-2-(9H-fluoren-9- ylmethoxycarbonylamino)-propionylamino]-3-tritylsulfanyl-propionylamino}-3- hydroxy-5-methyl-hexanoic acid allyl ester To a solution of 1 (360mg, 0.468 mmol, 1eq (prepared according to the procedure in Doi, T. et al, Tet. Lett. 2006, 47, 117)) in MeCN (5.4ml_) was added Et2NH (10% v/v, 0.6mL) dropwise at rt under Ar(g). The solution was stirred at rt for 2h, and the solvent was removed in vacuo. The excess of amine was co- evaporated with MeCN (3 x 1OmL) and thereaction mixture was dried under high vacuum for 2h.
To a solution of Fmoc-3-chloro-D-phenylalanine (231 mg, 0.49 mmol, 1.1 eq) in CH2CI2 (1OmL) at O0C was added PyBOP (268mg, 0.51 mmol, 1.1 eq) and DIPEA (0.2mL, 1.2 mmol, 2.5eq) under Ar(g). The crude amine, dissolved in MeCN (1OmL) was added to the mixture via cannula. The reaction mixture was then left to warm to rt overnight before being concentrated in vacuo. Purification by flash column chromatography (eluant 10-50% EtOAc/Hexane) furnished 2 (380mg, 0.400 mmol, 85%) as a white solid.
1H NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 7.72 (d, J=7.4Hz, 2H), 6.92- 7.57 (m, 26H), 5.80 (ddt, J=MA , 10.5, 5.8Hz, 1 H), 5.24 (dq, J=17.3, 1.4Hz, 1H), 5.15 (dq, JM0.4, 1.3Hz, 1 H), 4.45-4.57 (m, 2H), 4.24-4.44 (m, 3H), 4.12-4.21 (m, 1H), 3.84-3.96 (m, 2H), 3.65-3.75 (m, 1 H), 2.77-3.01 (m, 2H), 2.44-2.62 (m, 3H), 2.35 (dd, JM 6.2, 9.7Hz, 1 H), 1.98-2.08 (m, 1 H), 0.77-0.84 (m, 6H). MS (ES+) 973 (100%, [M+Na]+).
(4): (3S,4R)-4-{(S)-2-[(R)-3-(3-Chloro-phenyi)-2-(9H-fluoren-9- ylmethoxycarbonylamino)-propionylamino]-3-tritylsulfanyl-propionylamino}-3- hydroxy-5-methyl-hexanoic acid allyl ester
To a solution of compound 2 (376mg, 0.40 mmol, 1eq) in MeCN (1OmL) was added Et2NH (10% v/v, 1mL) dropwise at rt under Ar(g). The solution was stirred at rt for 1 h 20 min, and the solvent was removed in vacuo. The excess of amine was co-evaporated with MeCN (3 x 1OmL) and the reaction mixture was then dried under high vacuum for 3h.
To a solution of the carboxylic acid 3 (174mg, 0.41 mmol, 1.05eq) in CH2CI2 (1OmL) at 00C was added PyBOP (229mg, 0.44 mmol, 1.1 eq) and DIPEA (170 μL, 1.0 mmol, 2.5eq) under Ar(g). The crude amine, dissolved in MeCN (1OmL) was added via cannula, and the reaction mixture was then left to warm to rt overnight before being concentrated in vacuo. Purification by flash column chromatography (eluant 20-70% EtOAc/hexane) gave 4 (387mg, 0.343 mmol, 86%) as a white solid.
1H NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 6.87-7.34 (m, 35H), 6.58 (d, J=9.9Hz, 1 H), 5.70 (ddt, J=17.2, 10.5, 5.7Hz, 1 H), 5.32 (dt, J=15.4, 6.8Hz, 1 H), 5.14-5.22 (m, 1 H), 5.13 (dq, JM 7.2, 1.5Hz, 1 H), 5.04 (dq, J=I 0.5, 1.3Hz, 1 H), 4.33-4.50 (m, 3H), 4.08-4.17 (m, 1 H), 3.83 (ddd, JM 0.1 , 8.2, 2.4Hz, 1 H), 3.77 (t, J=7.0Hz, 1H), 3.49-3.59 (m, 1H), 2.96 (dd, JM4.2, 4.8Hz, 1H), 2.69 (dd, JM4.3, 9.1Hz, 1H), 2.29-2.48 (m, 3H), 2.21 (dd, JM 5.9, 9.9Hz, 1 H), 2.00-2.10 (m, 4H), 1.86-1.96 (m, 3H), 0.69 (d, 3H), 0.67 (d, J=7.0Hz, 3H). MS (ES+) 1151 (100%, [M +Na]+).
(5): (3S,4R)-4-{(S)-2-[(R)-3-(3-Chloro-phenyl)-2-((E)-(S)-3-hydroxy-7- tritylsulfanyl-hept-4-enoylamino)-propionylamino]-3-tritylsulfanyl- propionylamino}-3-hydroxy-5-methyl-hexanoic acid allyl ester
To a solution of 4 (384mg, 0.34 mmol, 1eq) in anhydrous MeOH (5mL) was added morpholine (45 μL, 0.50 mmol, 1.5eq) and Pd(PPh3)4 (8mg, 0.007 mmol, 0.02eq) at rt under Ar(g). The reaction mixture was stirred for 2h, then concentrated in vacuo, and purified by flash column chromatography (eluant 2- 10% MeOH/CH2CI2) to give 5 (350mg, 0.321 mmol, 95%) as a white solid.
1H NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 7.53 (d, J=8.0Hz, 1H), 6.91- 7.36 (m, 33H), 6.61 (d, J=9.9Hz, 1 H), 5.35 (dt, JM 5.7, 6.7Hz, 1 H), 5.21 (dd, JM 5.4, 6.2Hz, 1 H), 4.44 (dd, J=8.8, 4.8Hz, 1H), 4.16 (dt, J=9.2, 4.9Hz, 1 H), 3.81-3.95 (m, 3H), 3.54-3.63 (m, 2H), 3.32 (d, J=12.2Hz, 1 H), 3.00 (dd, J=14.3, 4.8Hz, 1 H), 2.80-2.90 (m, 1H), 2.74 (dd, JM4.2, 9.0Hz, 1 H), 2.37-2.46 (m, 3H), 2.19 (dd, J=16.2, 9.6Hz, 1H), 2.03-2.13 (m, 4H), 1.90-2.00 (m, 3H), 0.72 (t, J=6.7Hz, 6H). MS (ES+) 1145 (100%, [M+Na]+).
(6): (2S,6R,9S, 12R, 13S)-6-(3-Chloro-benzyl)-13-hydroxy-12-isopropyl-2-((E)-4- tritylsulfanyl-but-1 -enyl)-9-tritylsulfanylmethyl-1 -oxa-5,8,11 -triaza- cyclopentadecane-4,7,10,15-tetraone
To a solution of MNBA (131mg, 0.38 mmol) and DMAP (93mg, 0.76 mmol) in CH2CI2 (8OmL) was added dropwise a solution of acid 5 (345mg, 0.317 mmol) in CH2CI2 (25OmL) over 5h under Ar(g). After a further 14h the reaction mixture was concentrated in vacuo to give an off white solid. Purification by column chromatography on silica gel (1-31/2% IPA/CH2CI2) gave 6 (245mg, 0.229 mmol, 72%) as a white solid. 1H NMR (400MHz, 9:1 CDCI3ZCD3OD) δH: 6.88-7.39 (m, 36H), 5.49 (dt,
J=15.2, 6.6Hz, 1H), 5.33 (ddd, J=10.7, 7.3, 3.9Hz, 1 H), 5.24 (dd, J=15.2, 6.6Hz, 1 H), 4.27 (t, J=7.8Hz, 1 H), 3.84-3.92 (m, 1 H), 3.61-3.73 (m, 1H), 3.19 (dd, J=8.7, 5.0Hz, 1 H), 2.68-2.94 (m, 5H), 2.36 (dd, J=14.2, 11.3Hz, 1H), 2.17-2.32 (m, 3H), 2.02-2.13 (m, 3H), 1.89-2.01 (m, 2H), 0.76 (d, J=6.9Hz, 3H), 0.74 (d, J=6.9Hz, 3H). MS (ES+) 1094 (100%, [M+Na]+).
Compound XXXIII: (E)-(I S,5S,6R,9S,20R)-20-(3-Chloro-benzyl)-5-hydroxy-6- isopropyl-2-oxa-11 ,12-dithia-7,19,22-triaza-bicyclo[7.7.6]docos-15-ene-
3,8,18,21 -tetraone
To a solution of I2 (581 mg, 2.3 mmol, 10eq) in CH2CI2 (45OmL) and MeOH (5OmL) was added dropwise a solution of 6 (245mg, 0.23 mmol, 1eq) in CH2CI2 (225mL) and MeOH (25mL) over 2h at rt under Ar(g). The reaction mixture was stirred for a further 20 min, and was themn treated with sodium thiosulfate (0.1 M, 5OmL) and brine (25mL). The organic layer was isolated, and the aqueous layer was extracted with EtOAc (3 x 5OmL). The combined organic extracts were dried over MgSO4 and the solvent was removed in vacuo. Purification by column chromatography on silica gel (1-41/2% MeOH/CH2CI2) gave compound XXXIII (82mg, 0.140 mmol, 61%) as a white solid.
1H NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 7.46 (d, J=7.2Hz, 1 H), 7.21 (d, J=5.0Hz, 4H), 7.07 (dt, J=6.8, 1.7Hz, 1 H), 6.80 (d, J=9.1Hz, 1 H), 6.05-6.19 (m, 1H), 5.65 (d, J=15.3Hz, 1H), 5.43 (br. s., 1 H), 4.81 (td, J=9.0, 3.6Hz, 1 H), 4.47 (dt, J=8.8, 4.0Hz, 1H), 4.41 (dd, J=8.5, 5.6Hz, 1 H), 3.27 (d, J=5.2Hz, 1H), 3.07-
3.16 (m, 2H), 2.95-3.07 (m, 2H), 2.86 (br. s., 1H), 2.75 (dt, J=8.7, 7.2Hz, 1 H),
2.51-2.64 (m, 5H), 2.21-2.37 (m, 2H), 0.96 (d, J=6.8Hz, 3H), 0.85 (d, J=6.7Hz,
3H). MS (ES+) 1190 (20%, [2M+Na]+), 606 (100%, [M+Na]+).
Compound XXXlV: 3-((EH1S.5S.6R.9S.20R)-5-Hvdroxy-6-isopropyl- a.β.iS^I-tetraoxo-a-oxa-ii.ia-dithia-y.ig.a∑-triaza-bicvclorr.y.βidocos-iδ- en-20-ylmethyl)-benzonitrile
Figure imgf000082_0001
Figure imgf000082_0002
(2): (3S,4R)-4-{(S)-2-[(R)-3-(3-Cyano-phenyl)-2-(9H-fluoren-9- ylmethoxycarbonylamino)-propionylamino]-3-tritylsulfanyl-propionylamino}-3- hydroxy-5-methyl-hexanoic acid ailyl ester
To a solution of tripeptide 1 (316mg, 0.41 mmol, 1eq) in MeCN (8mL) was added O.δmL of Et2NH (10% v/v) dropwise at rt under Ar(g). The solution was stirred at rt for 2 h, then the solvent was removed in vacuo. The excess of amine was co-evaporated with MeCN (3 x 5mL), then a 1 :5 mixture of CH2CI2/hexane (1OmL). A white solid was obtained and the flask was dried under high vacuum for 2h. To a solution of Fmoc-D-4-CNPheAla (186mg, 0.45 mmol, 1.1 eq) in MeCN (8mL) at 00C was added PyBOP (235mg, 0.45 mmol, 1.1 eq) and DIPEA (179 μL, 1.02 mmol, 2.5eq) under Ar(g). The crude amine, dissolved in CH2CI2 (1OmL) was added to the mixture in dropwise fashion. The reaction mixture was then left to warm to rt overnight, and was subsequently concentrated in vacuo, and the residue was purified by silica gel column chromatography with hexane/EtOAc (3:2) to furnish 2 s a white solid (301 mg, 78% yield). 1H NMR (400MHz, CDCI3) δH: 6.83-7.61 (m, 30H), 5.69-5.80 (m, 1H)1
5.19-5.25 (m, 3H), 4.49-4.59 (m, 3H), 4.16-4.25 (m, 2H), 4.11-4.18 (m, 1 H),
3.98-4.10 (m, 2H), 3.63-3.75 (m, 1 H), 2.68-3.00 (m, 2H), 2.40-2.60 (m, 3H),
2.24-2.38 (m, 1 H), 1.95-2.09 (m, 1 H), 0.75-0.80 (m, 6H). MS (ES+) 963.9 (100%, [M+Na]+).
(4): (3S,4R)-4-{(S)-2-[(R)-3-(3-Cyano-phenyl)-2-((E)-(S)-3-hydroxy-7- tritylsulfanyl-hept-4-enoylamino)-propionylamino]-3-tritylsulfanyl- propionylamino}-3-hydroxy-5-methyl-hexanoic acid allyl ester
To a solution of 2 (301 mg, 0.32 mmol, 1eq) in MeCN (7mL) was added Et2NH (0.7mL, 10% v/v) dropwise at rt under Ar(g). The solution was stirred at rt for 2h, and the solvent was removed in vacuo. The excess of amine was co- evaporated with MeCN (3 x 5mL), then with a 1 :5 mixture of CH2Cl2/hexane (5mL). A white solid was obtained which was dried under high vacuum for 2h. To a solution of the chiral fihydroxy acid 3 (141mg, 0.34 mmol, 1.05eq) in MeCN (5mL) at 00C was added PyBOP (183mg, 0.35 mmol, 1.1 eq) and DIPEA (140 μL, 0.80 mmol, 2.5eq) under Ar(g). The crude amine, dissolved in CH2CI2 (5ml_) was added in dropwise fashion, and the reaction mixture was then left to warm to rt overnight, after which it was concentrated in vacuo and the resulting residue was purified by silica gel column chromatography using hexane/EtOAc (2:3); 4 was isolated as a white solid (327mg, 91% yield).
1H NMR (400MHz, CDCI3) δH: 7.22-7.49 (m, 32H), 6.99-7.18 (m, 5H), 6.56-6.63 (m, 1 H), 5.72-5.82 (m, 1H), 5.35-5.44 (m, 1H), 5.24-5.30 (m, 3H), 4.49-4.60 (m, 3H), 4.20-4.28 (m, 1 H), 4.00-4.12 (m, 2H), 3.65-3.82 (m, 2H), 3.10-3.20 (m, 1H), 2.82 (m, 1H), 2.56 (m, 1H), 2.46 (m, 1H), 2.33-2.40 (m, 3H), 2.12-2.17 (m, 4H), 1.95-2.09 (m, 2H), 0.78-0.81 (m, 6H). MS (ES+) 1142.2 (100%, [M+Na]+).
(5): (3S,4R)-4-{(S)-2-[(R)-3-(3-Cyano-phenyl)-2-((E)-(S)-3-hydroxy-7- tritylsulfanyl-hept-4-enoylamino)-propionylamino]-3-tritylsulfanyl- propionylamino}-3-hydroxy-5-methyl-hexanoic acid To a solution of 4 (327mg, 0.29 mmol, 1eq) in anhydrous MeOH (6ml_) and THF (1mL) was added morpholine (38 μL, 0.44 mmol, 1.5eq) and Pd(PPh3)4 (6.7mg, 0.006 mmol, 2 mol%) at rt under Ar(g). The reaction mixture was stirred for 1h, and was then concentrated in vacuo. The residue was further purified by silica gel column chromatography using CH2CI2/MeOH (19:1 -> 9:1)/AcOH (0.1%) to furnish 5 as a pale yellow solid (305mg, 97% yield). 1H NMR (400MHz, CDCI3) δH: 7.27-7.50 (m, 27H), 7.16-7.26 (m, 10H), 6.44-6.54 (m, 2H), 5.35-5.44 (m, 1H), 5.24-5.30 (m, 1 H), 4.49-4.60 (m, 1H)-4.30 (m, 1H), 4.02-4.14 (m, 2H), 3.70-3.92 (m, 2H), 3.21 (dd, J=14.6, 4.2Hz, 1H), 2.97 (dd, J=14.0, 9.1 Hz, 1 H), 2.66 (dd, J=12.5, 7.8Hz, 1 H), 2.39-2.58 (m, 3H), 2.16-2.31 (m, 4H), 1.94-2.08 (m, 2H), 0.88-0.93 (m, 6H). MS (ES+) 1101.9 (100%, [M+Na]+).
(6): 3-[(2S,6R,9S, 12R, 13S)-13-Hydroxy-12-isopropyl-4,7, 10, 15-tetraoxo-2-((E)- 4-tritylsulfanyl-but-1 -enyl)-9-tritylsulfanylmethyl-1 -oxa-5,8,11 -triaza- cyclopentadec-6-ylmethyl]-benzonittϊle To a solution of MNBA (117mg, 0.34 mmol, 1.2eq) and DMAP (83mg,
0.68 mmol, 2.4eq) in CH2CI2 (20OmL) under Ar(g) was added dropwise a solution of 5 (305mg, 0.28 mmol, 1eq) in CH2CI2 (12OmL) and THF (2OmL) over 3h. The reaction mixture was left to stir at rt overnight. It was then concentrated in vacuo and the residue was further purified by silica gel column chromatography with CH2CI2/Me0H (49:1 -> 19:1) to furnish 6 as a white solid (197mg, 66% yield).
1H NMR (400MHz, CDCI3) δH: 7.36-7.44 (m, 10H), 7.25-7.34 (m, 19H), 7.17-7.25 (m, 7H), 6.91 (d, J=9.5Hz, 1H), 6.18 (d, J=5.3Hz, 1H), 5.55-5.65 (m, 1H), 5.39-5.47 (m, 1H), 5.33 (dd, J=15.4, 6.4Hz, 1 H), 4.55-4.65 (m, 1H), 4.12- 4.23 (m, 1 H), 3.44-3.56 (m, 1H), 2.97-3.06 (m, 1 H), 2.91 (dd, J=13.9, 7.7Hz, 1 H), 2.78 (dd, J=12.7, 6.6Hz, 1H), 2.31-2.48 (m, 4H), 2.15-2.24 (m, 3H), 2.00- 2.09 (m, 4H), 0.83-0.93 (m, 5H). MS (ES+) 1083.6 (100%, [M+Na]+). Compound XXXiV: 3-((E)-(1 S,5S,6R,9S,20R)-5-Hydroxy-6-isopropyl-3,8, 18,21 - tetraoxo-2-oxa-11 , 12-dithia-7, 19,22-triaza-bicyclo[7.7.6]docos-15-en-20- ylmethyl)-benzonitrile
To a solution of I2 (463mg, 1.83 mmol, 10eq) in CH2CI2 (40OmL) and MeOH (4OmL) was added in dropwise fashion a solution of 6 (194mg, 0.18 mmol, 1eq) in CH2CI2 (20OmL) and MeOH (2OmL) over 2h at rt under Ar(g). The reaction mixture was further stirred for 30 min, amd was then treated with a solution of sodium thiosulfate (0.1 M, 25OmL) and brine (3OmL). The organic layer was isolated and the aqueous layer was extracted with CH2CI2 (2 x 10OmL) and EtOAc (2 x 10OmL). The combined organic extracts were dried over MgSO4 and the solvent was removed in vacuo. The residue was further purified by silica gel column chromatography with CH2CI2/MeOH (19:1) to yield compound XXXIV as a white solid (77mg, 73% yield). 1H NMR (400MHz, CDCI3) δH: 7.55 (d, JM 1.8Hz, 2H), 7.42-7.49 (m, 1 H)1 7.31 (d, J=6.6Hz, 1 H), 7.09 (d, J=6.8Hz, 1 H), 7.02 (br. s., 1H), 6.03-6.21 (m, 1H), 5.78 (d, J=15.4Hz, 1H), 5.49 (br. s., 1 H), 4.68-4.85 (m, 1 H), 4.39-4.55 (m, 2H), 3.04-3.36 (m, 7H), 2.88-3.00 (m, 1 H), 2.61-2.81 (m, 6H), 2.36-2.52 (m, 2H), 2.20-2.32 (m, 1H), 0.98 (d, J=6.4Hz, 3H), 0.89 (d, J=6.3Hz, 3H). MS (ES+) 597.6 (100%, [M+Na]+).
Compound XXXV: fEH1S,5S,6R.9S.20R)-5-Hvdroxy-6-isopropyl-20-(1- methyl-1H-imidazol-4-ylmethvh-2-oxa-11 ^-dithia^.iQ^-triaza- bicvclolT^.eidocos-IS-ene-a.S.IS^I-tetraone
Figure imgf000085_0001
Figure imgf000085_0002
S 6 XXXV
(2): (3S,4R)-4-{(S)-2-[(R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-(1- methyl-1 H-imidazol-4-yl)-propionylamino]-3-tritylsulfanyl-propionylamino}-3- hydroxy-5-methyl-hexanoic acid allyl ester
To a solution of commercially-available D-His(1 -Me)-OH (385mg, 2.28 mmol) and NaHCO3 (479mg, 5.7 mmol, 2.5eq) in H2O (5.5mL) was added a solution of Fmoc-CI (1.18 g, 4.55 mmol, 2eq) in THF (7ml_) to give a biphasic mixture. Acetone (3mL) was then added until the solution became homogeneous, and the mixture was allowed to stir overnight. H2O (2OmL) and EtOAc (3OmL) were subsequently added, and the phases separated. The organic phase was extracted twice more with H2O (1OmL). The combined aqueous phases were acidified to pH 5-6 before being extracted with CH2CI2 (5 x 4OmL). The combined CH2CI2 phases were then dried (MgSO4) and concentrated in vacuo to give Fmoc-D-(1 -Me)-HiS-OH (642mg, 1.64 mmol, 72%) as a white solid that was used straight away in the next reaction [MS (ES+) 414 (100%, [M+Na]+), 392(40%, [M+H]+)]. To a solution of 1 (749mg, 0.97 mmol, 1eq, prepared according to the procedure in Doi, T. et al, Tet. Lett. 2006, 47, 1177)) in MeCN (1OmL) was added Et2NH (10% v/v, 1mL) dropwise at rt under Ar(g). The solution was stirred at rt for 2h, the solvent was removed in vacuo, the excess of amine was co- evaporated with MeCN (3 x 1OmL), and the crude amine was dried under high vacuum for 2h. To a solution of Fmoc-D-(1 -Me)-HiS-OH (400mg, 1.0 mmol, 1.05eq) in CH2CI2 (1OmL) at 00C was added PyBOP (555mg, 1.07 mmol, 1.1 eq) and DIPEA (0.51 mL, 2.91 mmol, 3eq) under Ar(g). The crude amine, dissolved in MeCN (1OmL) was added via cannula, and the resulting reaction mixture was then left to warm to rt overnight before being concentrated in vacuo. Purification by flash column chromatography using 50-90% EtOAc/Hexane then 2-5% MeOH/EtOAc as eluant furnished 2 (683mg, 0.74 mmol, 77%) as a white solid.
1H NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 7.67 (d, J=7.5Hz, 2H), 7.48 (t, J=8.0Hz, 2H), 7.00-7.36 (m, 20H), 6.66 (s, 1H), 5.76 (ddt, JM 7.1 , 10.5, 5.7Hz, 1 H), 5.18 (dq, J=M.2, 1.4Hz, 1 H), 5.08 (dq, JM 0.4, 1.3Hz, 1H), 4.38-4.52 (m, 2H), 4.24 (dd, JM 0.4, 7.6Hz, 1 H), 4.05-4.17 (m, 2H), 3.91-4.05 (m, 2H), 3.74- 3.82 (m, 1 H), 3.64-3.70 (m, 1H), 3.39 (s, 3H), 2.75-2.90 (m, 2H), 2.64 (dd, JM 2.8, 7.0Hz, 1 H), 2.46 (dd, J=15.6, 1.6Hz, 1 H), 2.40 (dd, JM 2.6, 5.0Hz, 1 H), 2.26 (dd, JM 5.7, 10.0Hz, 1 H), 1.89-1.95 (m, 1H), 0.79 (d, J=6.7Hz, 6H). MS (ES+) 930 (40%, [M+H]+), 908 (100%, [M+Na]+).
(4): (3S,4R)-3-Hydroxy-4-{(S)-2-[(R)-2-((E)-(S)-3-hydroxy-7-tritylsulfanyl-hept-4- enoylamino)-3-(1-methyl-1H-imidazol-4-yl)-propionylamino]-3-tritylsulfanyl- propionylamino}-5-methyl-hexanoic acid allyl ester
To a solution of 2 (735mg, 0.80 mmol, 1eq) in MeCN (1OmL) was added Et2NH (10% v/v, 1mL) dropwise at rt under Ar(g). The solution was stirred at rt for 2h, the solvent subsequently removed in vacuo, the excess of amine co- evaporated with MeCN (3 x 1OmL), and the resulting reaction mixture dried under high vacuum for 3h. To a solution of the carboxylic acid 3 (351 mg, 0.84 mmol, 1.05eq) in CH2CI2 (1OmL) at 00C was added PyBOP (458mg, 0.88 mmol, 1.1eq) and DIPEA (0.35mL, 2.0 mmol, 2.5eq) under Ar(g). The crude amine, dissolved in MeCN (1OmL) was added via cannula, and the reaction mixture was then left to warm to rt overnight before being concentrated in vacuo. Purification by flash column chromatography (eluant 50-90% EtOAc/Hexane then 1-41/2% MeOH/CH2CI2) gave 4 (618mg, 0.56 mmol, 70%) as a white solid. 1H NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 8.10 (s, 1H), 7.12-7.46 (m, 30H), 7.09 (S, 1H), 6.40 (d, J=9.7Hz, 1 H), 5.85 (ddt, JM 7.2, 10.5, 5.7Hz, 1 H), 5.48 (dt, J=15.3, 5.8Hz, 1H), 5.41 (dd, JM 5.4, 5.8Hz, 1 H), 5.27 (dq, J=M.3, 1.5Hz, 1 H), 5.18 (dq, J=10.4, 1.3Hz, 1H), 4.47-4.61 (m, 2H), 4.49 (dd, J=7.1 , 4.5Hz, 1 H), 4.21-4.30 (m, 1 H), 3.96-4.05 (m, 1 H), 3.77 (dd, J=8.2, 6.1Hz, 1 H), 3.60-3.73 (m, 2H), 3.57 (s, 3H), 3.13-3.23 (m, 1 H), 3.10 (q, J=7.4Hz, 1H), 2.43- 2.63 (m, 3H), 2.12-2.41 (m, 5H), 1.90-2.11 (m, 3H), 0.81 (d, J=6.7Hz, 3H), 0.81 (d, J=6.7Hz, 3H). MS (ES+) 1121 (60%, [M +Na]+), 1099 (100%, [M +H]+). (5): (3S,4R)-3-Hydroxy-4-{(S)-2-[(R)-2-((E)-(S)-3-hydroxy-7-tritylsulfanyl-hept-4- enoylamino)-3-(1 -methyl-1 H-imidazol-4-yl)-propionylamino]-3-tritylsulfanyl- propionylamino}-5-methyi-hexanoic acid
To a solution of 4(616mg, 0.56 mmol, 1eq) in anhydrous MeOH (1OmL) was added morpholine (73 μL, 0.84 mmol, 1.5eq) and Pd(PPh3)4 (13mg, 0.011 mmol, 0.02eq) at rt under Ar(g). The reaction mixture was stirred for 1h 20 min, then concentrated in vacuo. Purification by flash column chromatography (eluant 2-12% MeOH/CH2CI2) gave the 5 (570mg, 0.54 mmol, 96%) as a white solid.
1H NMR (400MHz, 9:1 CDCI3ZCD3OD) δH: 7.71 (br. s., 1 H), 7.07-7.41 (m, 30H), 6.89 (S, 1H), 5.42 (dt, J=15.5, 6.0Hz, 1 H), 5.34 (dd, JM5.5, 6.1 Hz, 1 H), 4.39 (dd, J=6.5, 5.4Hz, 1 H), 4.17-4.24 (m, 1H), 3.84-3.95 (m, 2H), 3.77 (dd, J=9.2, 5.2Hz, 1H), 3.44 (s, 3H), 3.09-3.13 (m, 1 H), 2.97-3.03 (m, 1H), 2.52 (dd, J=12.9, 9.3Hz, 1 H), 2.37-2.47 (m, 2H), 2.33 (dd, J=13.4, 10.5Hz, 1 H), 2.09-2.26 (m, 3H), 1.90-2.06 (m, 4H), 0.76 (d, J=6.7Hz, 3H), 0.75 (d, J=6.8Hz, 3H). MS (ES+) 1081 (40%, [M+Na]+), 1059 (100%, [M +H]+); (ES") 1057 (100%, [M -H]"). (6): (2S,6R,9S,12R,13S)-13-Hydroxy-12-isopropyl-6-(1-methyl-1H-imidazol-4- ylmethyl)-2-((E)-4-tritylsulfanyl-but-1 -enyl)-9-tritylsulfanylmethyl-1 -oxa-5,8, 11 - triaza-cyclopentadecane^J.IO.Iδ-tetraone
To a solution of MNBA (219mg, 0.63 mmol) and DMAP (155mg, 0.1.27 mmol) in CH2CI2 (10OmL) was added dropwise a solution of acid 5 (560mg, 0.53 mmol) in CH2CI2 (40OmL) over 5h under Ar(g). After a further 14 h, the reaction mixture was concentrated in vacuo to give an off white solid. Purification by column chromatography on silica gel (1-15% MeOH/CH2CI2) gave recovered starting material (450mg, 0.43 mmol) and the desired macrocycle 6 (82mg, 0.079 mmol, 15%) as a white solid.
1H NMR (400MHz, 9:1 CDCI3ZCD3OD) δH: 7.97 (br. s., 1 H), 7.90 (d, J=8.2Hz, 1H), 7.48 (d, J=7.7Hz, 1 H), 7.10-7.42 (m, 29H), 7.04 (s, 1H), 6.94 (d, J=8.7Hz, 1H), 5.43-5.59 (m, 2H), 5.26 (dd, J=15.4, 6.7Hz, 1 H), 4.32 (dd, J=7.9, 5.7Hz, 1 H), 4.12 (dt, J=8.9, 4.6Hz, 1 H), 3.58 (s, 3H), 3.50-3.60 (m, 1H), 2.88- 3.17 (m, 3H), 2.65 (dd, J=13.0, 4.6Hz, 1H), 2.53 (dd, J=14.6, 11.6Hz, 1 H), 2.25- 2.43 (m, 3H), 2.06-2.20 (m, 3H), 1.92-2.03 (m, 3H), 0.80 (d, J=6.8Hz, 3H), 0.77 (d, J=6.8Hz, 3H). MS (ES+) 1063 (100%, [M+Na]+), 1041 (80%, [M +H]+).
Compound XXXV: (E)-(I S,5S,6R,9S,20R)-5-Hydroxy-6-isopropyl-20-(1 -methyl- 1 H-imidazol-4-ylmethyl)-2-oxa-11 ,12-dithia-7,19,22-triaza-bicyclo[7.7.6]docos- 15-ene-3,8, 18,21 -tetraone
To a solution of I2 (199mg, 0.77 mmol, 10eq) in CH2CI2 (18OmL) and MeOH (2OmL) was added dropwise a solution of 6 (80mg, 0.077 mmol, 1eq) in CH2CI2 (9OmL) and MeOH (1OmL) over 2h at rt under Ar(g). The reaction mixture was stirred for a further 20 min, then a solution of sodium thiosulfate (0.1 M, 2OmL) and brine (1OmL) were added to the reaction mixture. The organic layer was isolated and the aqueous layer was extracted with EtOAc (3 x 3OmL). The combined organic extracts were dried over MgSO4 and the solvent was removed in vacuo. Purification by column chromatography on silica gel (using 1-8% MeOH/CH2CI2 as eluant) furnished compound XXXV (2mg, 0.004 mmol, 5%) as a white solid.
1H NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 7.60 (br. s., 1 H), 6.86 (s, 1H), 5.94-6.05 (m, 1H), 5.90 (d, J=15.6Hz, 1 H), 5.39-5.47 (m, 1H), 4.71-4.79 (m, 1H), 4.42 (dt, J=8.6, 3.8Hz, 1H), 4.28 (dd, J=7.7, 4.5Hz, 1 H), 3.63 (s, 3H), 3.53-3.62 (m, 1H), 2.91-3.14 (m, 5H), 2.69-2.82 (m, 2H), 2.43-2.64 (m, 4H), 2.19-2.36 (m, 2H), 0.93 (d, J=6.8Hz, 3H), 0.83 (d, J=6.7Hz, 3H). MS (ES+) 576 (100%, [M+Na]+), 554 (95%, [M+H]+). Compound XXXVI: (EH1S.7R.10S.21 RV7-lsopropyl-21-pyridin-2-ylmethyl-2- oxa-12,13-d ithia-S^^O^S-tetraaza-bicyclorβ.y.βitricos-iβ-ene-S^.gj 9,22- pentaone
Figure imgf000089_0001
(4): (E)-(S)-3-Hydroxy-7-tritylsulfanyl-hept-4-enoic acid
At 0QC, to a solution of 3 (934mg, 1.66 mmol (prepared according to the procedure in Yurek-George, A. et al, J. Am. Chem. Soc. 2004, 126, 1030)) in THF (3OmL) was added a solution of LiOH (196.1mg, 8.19 mmol) in H2O (1OmL). The reaction mixture was allowed to warm to rt over 1h whereupon 1 M HCI was added until the pH reached 2. EtOAc (3OmL) was then added and the layers were separated. The aqueous layer was extracted with EtOAc (2OmL) the organic layers were combined, dried (MgSO4) and concentrated in vacuo. Purification by flash column chromatography (using 3:7-1 :1-1 :0 EtOAc/hexane as eluant) gave 4 as a white solid (600mg, 1.43 mmol, 86%).
1H NMR (300MHz, CDCI3) δH: 7.48-7.38 (m, 6H), 7.35-7.18 (m, 9H), 5.60 (m, 1 H), 5.43 (m, 1 H), 4.46 (q, J=6.28, 1H), 2.59-2.51 (m, 2H), 2.28-2.19 (m, 2H), 2.09 (q, J=6.47Hz, 2H). MS (ES') 417 (100%, [M-H]"). Rf 0.52 EtOAc+2 drops AcOH. [α]D 27-4.15 (c 0.975, CH2CI2).
(5): ((R)-2-{(S)-2-[(R)-2-((E)-(S)-3-Hydroxy-7-tritylsulfanyl-hept-4-enoylamino)-3- pyridin-2-yl-propionylamino]-3-tritylsulfanyl-propionylamino}-3-methyl- butyrylamino)-acetic acid methyl ester
To a solution of the carboxylic acid acid 4 (99mg, 0.236 mmol, 1.05eq) in CH2CI2 (1OmL) and MeCN (1OmL) at 00C was added PyBOP (128mg, 0.246 mmol, 1.1eq) and DIPEA (120 μL, 0.672 mmol, 2.5eq) under Ar(g). Then, H2N- D-3(2-pyridyl)alanine-D-Cys(STrt)-D-Val-Gly-OMe (2) (153mg, 0.224 mmol, 1.1 eq, purchased from GL Biochem (Shanghai) Ltd, Shanghai 200241 , China) was added and the reaction mixture was then left to warm to rt over 2h before being concentrated in vacuo. Purification by flash column chromatography (eluant 1-4% MeOH/CH2CI2) gave the product 5 (135mg, 0.125 mmol, 56%) as a white solid.
1H NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 8.38 (d, J=4.3Hz, 1 H), 7.14- 7.57 (m, 35H), 7.02 (dd, J=7.3, 5.0Hz, 1H), 5.52 (dt, JM 5.3, 6.5Hz, 1H), 5.39 (dd, JM 5.3, 6.0Hz, 1 H), 4.70 (dd, J=6.3, 5.0Hz, 1H), 4.27-4.38 (m, 1 H), 4.12- 4.21 (m, 1H), 3.84-3.97 (m, 2H), 3.67 (s, 3H), 3.22 (dd, JM 4.8, 5.0Hz, 1 H), 3.15 (dd, J=14.6, 6.8Hz, 1H), 2.43-2.57 (m, 3H), 2.14-2.32 (m, 7H), 2.08 (q, J=7.0Hz, 3H), 0.90 (t, J=7.3Hz, 5H). MS (ES+) 1083 (100%, [M+H]+). (7): (6R,9S, 12R, 16S)-6-lsopropyl-12-pyridin-2-ylmethyl-16-((E)-4-tritylsulfanyl- but-1 -enyl)-9-tritylsulfanylmethyl-1 -oxa-4,7, 10, 13-tetraaza-cyclohexadecane- 2,5,8,11 ,14-pentaone
At 00C, to a solution of methyl ester 5 (135mg, 0.125 mmol) in THF (6ml_),= was added a solution of LiOH (4.5mg, 0.187 mmol) in H2O (1.5mL). After 1.5 h the reaction was quenched by addition of 1M HCI (1OmL). EtOAc (5OmL) and brine (1OmL) were added and the organic phase was separated, re- extracting with EtOAc (2 x 2OmL). The organic phases were combined and washed with brine (15mL), dried (MgSO4) and concentrated in vacuo to furnish acid 6 (125mg, 0.117 mmol, 94%) as a white solid that was used immediately in the next step [MS (ES") m/z 1067 (100%, [M-H]")]. To a solution of MNBA (48mg, 0.14 mmol) and DMAP (34mg, 0.28 mmol) in CH2CI2 (3OmL) was added dropwise a solution of acid 6 (125mg, 0.117 mmol) in CH2CI2/THF (90:10, 10OmL) over 3h under Ar(g). After a further 14h, the reaction mixture was concentrated in vacuo to give a yellow solid. Purification by column chromatography on silica gel (using 1-10% IPA/CH2CI2 as eluant) gave 7 (62mg, 0.059 mmol, 50%) as a white solid.
1H NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 8.28 (d, J=4.0Hz, 1H), 7.45 (td, J=7.5, 1.5Hz1 1 H), 7.07-7.38 (m, 35H), 7.00 (dd, J=7.3, 5.8Hz, 1 H), 5.50 (dt, JM4.3, 6.8Hz, 1H), 5.20-5.34 (m, 2H), 4.30-4.43 (m, 2H), 4.17 (d, J=4.8Hz, 1 H)1 3.48 (dd, J=8.8, 5.0Hz, 1 H), 3.41 (dd, JM6.8, 2.5Hz, 1 H), 3.20 (dd, JM4.1 , 6.8Hz, 1 H), 3.06 (dd, JM4.3, 7.5Hz, 1H), 2.88 (dd, JM2.5, 9-OHz, 1 H), 2.52 (dd, JM2.5, 5.0Hz, 1 H), 2.34-2.45 (m, 2H), 2.27 (dd, JM5.1 , 9.5Hz, 1 H), 2.09 (t, J=7.3Hz, 2H), 1.87-2.02 (m, 2H), 0.83 (d, J=6.8Hz, 3H), 0.77 (d, J=7.0Hz, 3H). MS (ES+) 1051 (100%, [M+H]+). Compound XXXVI: (E)-(I S,7R,10S,21 R)-7-lsopropyl-21-pyridin-2-ylmethyl-2- oxa-12, 13-dithia-5,8,20,23-tetraaza-bicyclo[8.7.6]tricos-16-ene-3,6,9, 19,22- pentaone
To a solution of I2 (145mg, 0.57 mmol, 10eq) in CH2CI2 (144ml_) and MeOH (16mL) was added dropwise a solution of 7 (60mg, 0.057 mmol, 1eq) in CH2CI2 (72mL) and MeOH (8mL) over 1h at rt under Ar(g). The reaction mixture was further stirred for 20 min, and was then treated with a solution of sodium thiosulfate (0.1 M, 2OmL) and brine (1OmL). The organic layer was isolated, and the aqueous layer was extracted with EtOAc (3 x 2OmL). The combined organic extracts were dried over MgSO4 and the solvent was removed in vacuo. Purification by column chromatography on silica gel (1-4% MeOH/CH2CI2) furnished compound XXXVI (30mg, 0.053 mmol, 93%) as a white solid.
1H NMR (400MHz, 9:1 CDCI3/CD3OD) δH: 8.42 (br. s., 1H), 7.69 (td, J=7.8, 1.5Hz, 1H), 7.45-7.57 (m, 1H), 7.14-7.40 (m, 3H), 5.75-5.90 (m, 1 H), 5.59-5.73 (m, 2H), 4.66-4.81 (m, 1H), 4.42 (dd, J=9.5, 3.8Hz, 1H), 4.18 (dt, J=17.8, 3.8Hz, 1 H), 3.82-3.94 (m, 1 H), 3.82 (dd, J=17.6, 4.3Hz, 1H), 3.08-3.34 (m, 4H), 2.47-2.95 (m, 7H), 0.87 (d, J=6.5Hz, 6H)). 13C NMR (100MHz, 9:1 CDCI3/CD3OD) δc: 171.9, 170.9, 170.6, 170.0, 168.1 , 156.8, 148.4, 138.0, 130.3, 129.5, 124.4, 122.8, 70.0, 64.4, 56.4, 55.4, 41.7, 38.6, 38.1, 37.0, 36.7, 31.9, 27.0, 20.2, 19.7. MS (ES+) 564 (100%, [M+H]+).
Compound XXXVII: (E)-(I S.7R.10S.21 RK7-lsopropyl-21-(3-morpholin-4-yl-3- oxo-propyO-2-oxa-12,1 S-dithia-δ.β^O^-tetraaza-bicvclorβ.y.βitricos-i 6- ene-3.6,9,19,22-pentaone
Figure imgf000091_0001
(1)
To a solution of 1 (25mg, 0.046 mmol (prepared according to patent WO 2006/129105)), in CH2CI2/MeCN (1.5mL, 1 :1 , v/v) at 00C was added PyBOP (27mg, 0.05 mmol, 1.1 eq) followed by DIPEA (20 μL, 0.45 mmol, 2.5eq) under Ar(g). After 5 min stirring at 00C, morpholine (4.2 μL, 0.048 mmol, 1.05eq) was added and the mixture was left to warm to rt. After 2h the reaction mixture was concentrated under reduced pressure, and was purified by silica gel column chromatography, eluting with CH2CI2/Me0H (100:3), and then by solid phase extraction (SPE ISOLUTE SCX-3) with CH2CI2/MeOH (100:1 to 100:3) to furnish compound XXXVII as a white solid (18.7mg, 66%).
1H NMR (400MHz, CDCI3) δH: 9.11 (d, J=2.8Hz, 1H)1 7.39 (d, J=6.4Hz, 1H), 7.22 (t, J=5.1Hz, 1H), 7.12 (d, J=8.9Hz, 1H), 5.93-6.04 (m, 1 H), 5.73-5.84 (m, 2H), 4.98 (td, J=8.7, 3.6Hz, 1 H), 4.02-4.17 (m, 3H), 3.43-3.76 (m, 9H), 3.24 (dd, Λ=10.5, 6.5Hz, 1 H), 2.80-2.99 (m, 4H), 2.59-2.80 (m, 4H), 2.41-2.58 (m, 2H), 2.24-2.37 (m, 1H), 2.12-2.23 (m, 1 H), 0.98 (d, J=6.7Hz, 3H), 0.92 (d, J=6.7Hz, 3H). 13C NMR (100MHz, CDCI3) δc: 172.8 (C), 172.2 (C), 172.1(C), 171.1(C), 170.0(C), 168.6 (C), 130.1 (CH), 130.0 (CH), 69.8 (CH), 67.0 (CH2), 66.6 (CH2), 65.6 (CH), 57.8 (CH), 54.2 (CH), 46.4 (CH2), 42.8 (CH2), 42.7 (CH2), 40.5 (CH2), 38.7 (CH2), 32.6 (CH2), 31.3 (CH2), 30.1 (CH2), 27.6 (CH), 24.9 (CH2), 20.9 (CH3), 20.5 (CH3). MS (ES): 636.6 (100%, [M+Na]+), 1249.6 (30%, [2M+Na]+). Compound XXXVIII: 3-((a-(1S.7R,10S.21R)-7-lsopropyl-3.6.9.19.22- pentaoxo-2-oxa-12,13-dithia-5,8,20,23-tetraaza-bicvclof8.7.61tricos-16-βn- 21-yl)-N-(2-morpholin-4-yl-ethvD-propionamide
Figure imgf000092_0001
To a solution of carboxylic acid (1) as outlined for compound XXXVII above (25mg, 0.046 mmol) in CH2CI2/MeCN (1.5mL, 1 :1 , v/v) at O0C was added
PyBOP (27mg, 0.05 mmol, 1.1eq) followed by DIPEA (20 μL, 0.45 mmol, 2.5eq) under Ar(g). After 5 min stirring at 00C, 4-(2-aminoethyl)morpholine (6.3 μL,
0.048 mmol, 1.05eq) was added and the mixture was left to warm to rt. After 2h the reaction mixture was concentrated under reduced pressure, and the residue purified by silica gel column chromatography, eluting with CH2CL2/MeOH (100:2 to 100:6), and further purified by solid phase extraction (SPE ISOLUTE SCX-3) with CH2CI2/Me0H (100:2 to 80:20) then MeOH/H2O/NH4OH (9:1 :0.1) to yield compound XXXVIII as a white solid (17.2mg, 57%).
1H NMR (400MHz, CDCI3) δH: 9.25 (d, J=2.8Hz, 1 H), 7.46 (d, J=5.5Hz, 1H), 7.27-7.38 (m, 2H), 7.18 (d, J=8.8Hz, 1H), 6.87 (br. s., 1H), 5.97-6.06 (m, 1 H), 5.83 (d, J=16.2Hz, 1 H), 5.77-5.79 (m, 1H), 4.93 (td, J=8.7, 3.5Hz, 2H), 4.01-4.19 (m, 4H), 3.75 (t, J=4.5Hz, 3H), 3.68 (dt, J=13.4, 6.7Hz, 1 H), 3.53 (dd, J=15.0, 8.7Hz, 1 H), 3.43 (q, J=5.4Hz, 2H), 3.25 (dd, Λ=10.3, 6.7Hz, 1 H), 3.09 (q, J=7.4Hz, 1H), 2.76-2.92 (m, 5H), 2.75 (d, J=6.1Hz, 1H), 2.61-2.70 (m, 4H), 2.58 (dd, J=7.1 , 4.5Hz, 1H), 2.53 (t, J=5.6Hz, 1H), 2.42 (t, J=5.8Hz, 1 H), 2.38 (dd, J=7.6, 4.2Hz, 1 H), 0.97 (d, J=6.7Hz, 3H), 0.91 (d, J=6.5Hz, 3H). 13C NMR (100MHz, CDCI3) δc: 174.7 (C), 172.8 (C), 172.5 (C), 171.6 (C), 170.9 (C), 169.0 (C), 130.6 (2xCH), 70.5 (CH), 66.9 (2xCH2), 65.8 (CH), 57.8 (CH2), 57.7 (CH), 54.9 (CH), 53.6 (2xCH2), 43.0 (2xCH2), 40.2 (CH2), 39.2 (CH2), 36.3 (CH2), 34.1 (CH2), 33.9 (CH2), 28.0 (CH), 25.9 (CH2), 21.3 (CH3), 20.8 (CH3). MS (ES): 679.7 (100%, [M+Na]+).
Compound XXXIX: 3-((a-(1S.7R.10S,21R)-7-lsopropyl-3,β,9,19,22- pentaoxo-2-oxa-12,1 a-dithia-S.β^O.∑a-tetraaza-bicvclore.y.βitricos-i 6-en- 21-yl)-N-(2,2,2-trifluoro-ethyl)-propionamide
Figure imgf000093_0001
To a solution of carboxylic acid (1) as outlined for compound XXXVII above (25mg, 0.046 mmol) in CH2CI2/MeCN (1.5ml_, 1 :1 , v/v) at 00C was added PyBOP (27mg, 0.05 mmol, 1.1 eq) followed by DIPEA (20 μL, 0.45 mmol, 2.5eq) under Ar(g). After 5 min stirring at 00C, 2,2,2-trifluoroethylamine (3.8 μL, 0.048 mmol, 1.05eq) was added and the mixture was left to warm to rt. After 2h the reaction mixture was concentrated under reduced pressure, and the residue purified by silica gel column chromatography, eluting with CH2CI2/MeOH (100:2 to 100:3), and then further purified by solid phase extraction (SPE ISOLUTE SCX-3) with CH2CI2/MeOH (100:1) to yield compound XXXIX as a white solid (11.6mg, 40%). 1H NMR (400MHz, CDCI3) δH: 8.39 (d, J=3.4Hz, 1 H), 7.50 (t, J=5.6Hz, 1 H), 7.31 (d, J=6.5Hz, 1 H), 7.14 (t, J=6.5Hz, 1 H), 7.10 (d, J=9.2Hz, 1H), 6.03- 6.14 (m, 1 H), 5.73-5.82 (m, 2H), 5.00 (td, J=BA, 3.5Hz, 1H), 4.21 (dd, J=17.5, 5.7Hz, 1H), 4.06-4.13 (m, 1H), 3.84-4.01 (m, 3H), 3.64 (dd, JM 3.6, 8.5Hz, 1 H), 3.21 (dd, J=10.9, 6.6Hz, 1 H), 3.16 (dt, J=6.5, 3.3Hz, 1 H), 2.83-2.94 (m, 3H), 2.80 (dd, JM 5.1 , 3.1 Hz, 1 H), 2.70-2.76 (m, 1 H), 2.65-2.70 (m, 3H), 2.62 (dd, J=13.4, 6.0Hz, 1 H), 2.48 (dd, JM 3.2, 1.3Hz, 1 H), 1.86 (ddd, J=GA, 3.5, 3.3Hz, 1 H), 0.97 (d, J=6.6Hz, 3H), 0.94 (d, J=6.6Hz, 3H). 13C NMR (100MHz, CDCI3) δc: 175.5 (C), 172.9 (C), 172.3 (C), 171.6 (C), 170.2 (C), 168.4 (C), 130.5 (CH), 129.7 (CH), 69.9 (CH), 66.4 (CH), 57.6 (CH), 53.8 (CH), 46.7 (CH2), 46.6 (CH2), 42.5 (CH2), 38.9 (CH2), 33.1 (CH2), 27.7 (CH), 26.8 (CH2), 26.7 (CH2), 24.9 (CH2), 20.7 (CH3), 20.5 (CH3), (CF3 not observed). 19F NMR (282MHz, CDCI3) δF: -72.3 (S). MS (ES): 648.5 (100%, [M+Na]+), 1273.7 (60%, [2M+Na]+). Compound XL: ΛH2-Cvano-ethvn-3-((EH1S.7R.10S,21R)-7-isopropyl- 3,6,9.19,22-pentaoxo-2-oxa-12.13-dithia-5.8.20,23-tetraaza- bicvclorβ.7.61tricos-16-en-21 -yl)-propionamide
Figure imgf000094_0001
To a solution of carboxylic acid (1) as outlined for compound XXXVII above (25mg, 0.046 mmol) in CH2CI2/MeCN (1.5mL, 1 :1 , v/v) at 00C was added PyBOP (27mg, 0.05 mmol, 1.1 eq) followed by DIPEA (20 μL, 0.45 mmol, 2.5eq) under Ar(g). After 5 min stirring at 00C, 3-aminopropionitrile (3.8 μL, 0.048 mmol, 1.05eq) was added and the mixture was left to warm to rt. After 2h the reaction mixture was concentrated under reduced pressure, and the residue purified by silica gel column chromatography, eluting with CH2CI2/MeOH (100:2 to 100:3), and then further purified by solid phase extraction (SPE ISOLUTE SCX-3) with CH2CI2/MeOH (100:1 to 100:3) to yield compound XL as a white solid (12.9mg, 47%).
1H NMR (400MHz, CDCI3 + 10% MeOD) δH: 5.84-5.96 (m, 1H), 5.73-5.81 (m, 2H), 4.76-4.84 (m, 1 H), 4.25 (d, J=MJHz, 1H), 4.09 (dd, J=7.7, 6.5Hz, 1 H), 3.95 (d, J=MJHz, 1H), 3.49 (dddd, JM 7.1 , 7.0, 6.8, 6.7Hz, 2H), 3.28-3.36 (m, 1 H), 3.25 (s, 1H), 2.93-3.03 (m, 3H), 2.87 (dt, JM 0.5, 6.6Hz, 1 H), 2.71-2.76 (m, 2H), 2.59-2.70 (m, 4H), 2.49-2.58 (m, 1 H), 2.36-2.47 (m, 1 H), 2.09-2.17 (m, 2H), 0.95 (d, J=6.7Hz, 3H), 0.92 (d, J=6.7Hz, 3H). 13C NMR (100MHz, CDCI3 + 10% MeOD) δc: 174.9 (C), 172.4 (C), 171.1 (C), 170.5 (C), 168.6 (C), 130.4 (C), 130.1 (C), 118.3 (C), 70.4 (CH), 65.2 (CH), 56.7 (CH), 55.7 (CH), 42.3 (CH2), 39.4 (CH2), 38.6 (CH2), 37.5 (CH2), 35.9 (CH2), 32.8 (CH2), 32.4 (CH2), 27.5 (CH), 25.7 (CH2), 20.7 (CH3), 20.3 (CH3), 18.4 (CH2). MS (ES): 619.8 (100%, [M+Na]+). Compound XLI: /V-Cvclopropylmethyl-3-((EH1S.7R,10S,21 R)-7-isopropyl- 3.6.9.19.22-pentaoxo-2-oxa-12,13-dithia-5,8,20,23-tetraaza- bicvclorβ.7.β1tricos-16-en-21 -yl)-propionamide
Figure imgf000095_0001
To a solution of carboxylic acid (1) as outlined for compound XXXVII above (23mg, 0.042 mmol) in CH2CI2/MeCN (1.5mL, 1 :1 , v/v) and Λ/-(3- dimethylaminopropyl)-/V-ethylcarbodiimide hydrochloride (24mg, 0.12 mmol, 3eq) in CH2CI2 (1.5mL) under Ar(g) at rt was added (aminomethyl)-cyclopropane (7.3 μL, 0.084 mmol, 2eq) followed by DIPEA (18 μL, 0.105 mmol, 2.5eq). After 2h the reaction mixture was diluted with CH2CI2 (15ml_), and aqueous 0.1 M HCI (15ml_) was added. The phases were separated and the aqueous phase was extracted with CH2CL2 (2x1 Om L). The organic phases were combined, dried over MgSO4, filtered then concentrated under reduced pressure. The residue was purified by silica gel column chromatography with CH2CI2/MeOH (100:2 to 100:3) as eluant to furnish compound XLI as a white solid (6mg, 25%). 1H NMR (400MHz, CDCI3) δH: 9.41 (d, J=3.6Hz, 1 H), 7.42 (d, J=6.4Hz,
1 H), 7.22 (t, J=5.6Hz, 1H), 7.11 (d, J=9.0Hz, 1 H), 5.98-6.07 (m, 1 H), 5.92 (t, J=5.6Hz, 1 H), 5.79-5.88 (m, 2H), 4.98 (td, J=8.5, 3.8Hz, 1 H), 4.09-4.17 (m, 2H), 3.99-4.09 (m, 1H), 3.60 (dd, JM4.7, 8.0Hz, 1 H), 3.24 (dd, JM0.5, 6.6Hz, 1 H), 3.11 (dd, J=6.9, 5.8Hz, 2H), 2.79-2.90 (m, 4H), 2.75 (dd, JM3.3, 6.3Hz, 1 H), 2.68 (br. s., 3H), 2.60-2.63 (m, 1 H), 2.56-2.60 (m, 1H), 2.39-2.49 (m, 1 H), 2.15- 2.21 (m, 2H), 0.99 (d, J=6.7Hz, 3H), 0.92 (d, J=6.7Hz, 3H), 0.52-0.59 (m, 2H), 0.22 (q, J=5.1 Hz, 2H). 13C NMR (100MHz, CDCI3) δc: 177.0 (C), 172.3 (C), 172.3 (C), 171.2 (C), 170.5 (C), 168.6 (C), 130.1 (CH), 130.0 (CH), 69.9 (CH), 65.6 (CH), 57.6 (CH), 54.1 (CH), 45.4 (2xCH2), 42.7 (CH2), 40.3 (CH2), 38.9 (CH2), 34.3 (CH2), 33.7 (CH2), 27.6 (CH), 25.2 (CH2), 20.9 (CH3), 20.5 (CH3), 10.9 (CH), 4.0 (CH2), 3.9 (CH2). MS (ES): 620.3 (100%, [M+Na]+). Compound XLII: 3-((£)-(1S,7/?.10S,21/7)-7-lsopropyl-3.6,9,19,22-pentaoxo-2- oxa-12.13-dithia-5.8.20,23-tetraaza-bicvclorβ.7.6ltricos-16-en-21-yl)-ΛK2- methoxy-ethvD-propionamide
Figure imgf000096_0001
To a solution of carboxylic acid (1) as outlined for compound XXXVII above (25.4mg, 0.047 mmol) and PyBOP (25.4mg, 0.049 mmol) was added CH2CI2 (0.75mL) followed by MeCN (0.75mL). DIPEA (25 μL, 0.143 mmol) was then added dropwise, the solution was allowed to stir for 5 min, and 2- methoxyethylamine (4 μL, 0.036 mmol) was added. An additional volume of CH2CI2 (0.3ml_) was added and the reaction mixture was stirred for 1h 30 min. The mixture was then concentrated in vacuo and then purified by flash column chromatography on silica (eluant 1 :0-98:2-96:4-94:6 CH2CI2/MeOH). The isolated material was passed through an SCX-3 Isolute® column (eluant 99:1- 97:3-95:5 CH2CI2/MeOH) to give XLII (16.8mg, 0.028 mmol, 60%) as a white solid.
1H NMR (400MHz, CDCI3+5% MeOD) δH: 5.89 (m, 1H), 5.72 (br s, 1H), 4.76 (m, 1H), 4.16 (d, J=17.69Hz, 1 H), 4.06 (dd, J=8.09, 5.71 Hz, 1H), 3.93 (d, J=17.69Hz, 1H), 3.45-3.35 (m, 5H), 3.32 (s, 3H), 3.30-3.19 (m, 2H), 2.94-2.87 (m, 3H), 2.79 (m, 1H), 2.73-2.69 (m, 2H), 2.61 (br s, 2H), 2.54-2.37 (m, 2H), 2.09 (t, J=6.27Hz, 2H), 0.89 (t, J=6.59Hz, 6H); 13C NMR (100MHz, CDCI3+5% MeOD) δc: 174.3 , 172.6 (C), 170.8 (C), 170.3 (C), 168.3 (C), 130.2, (CH) 129.8 (CH), 70.6 (CH2), 70.2 (CH), 64.8 (CH), 58.8 (CH3), 56.4 (CH), 55.2 (CH), 42.0 (CH2),
39.7 (CH2), 39.0 (CH2), 38.3 (CH2), 37.3 (CH2), 32.8 (CH2), 32.3 (CH2), 27.2 (CH), 25.5 (CH2), 20.4 (CH3), 20.0 (CH3). MS (ES+) 625.0 (100%, [M+Na]+). Compound XLIII: 3-KEH1 S.7R10S.21 m-7-lsopropyl-3,β.9.19.22-pentaoxo- 2-oxa-12.13-dithia-5.8.20,23-tetraaza-bicvclor8.7.61tricos-16-en-21-yl)-Λ/- methyl-propionamide
Figure imgf000097_0001
To a solution of carboxylic acid (1) as outlined for compound XXXVII above (14.6mg, 0.027 mmol) and PyBOP (15.7mg, 0.030 mmol) was added CH2CI2 (1mL) followed by MeCN (1mL). DIPEA (30 μL, 0.18 mmol) was added dropwise, the solution was allowed to stir for 5 min, then methylamine (0.1 μL, 0.2 mmol, 2.0M in THF) was added. The reaction mixture was stirred for 55 min and then concentrated in vacuo. Purification by flash column chromatography on silica (eluant 1 :0-98:2-96:4-94:6 CH2CI2/MeOH) gave the product contaminated with traces of PyBOP The crude material was then passed through a SCX-3 column Isolute® (eluant 99:1-97:3-95:5 CH2CI2/MeOH) to furnish XLIII (9mg, 0.016 mmol, 59%) as a white solid.
1H NMR (400MHz, CDCI3+5% MeOD) δH: 5.83 (m, 1 H), 5.75 -5.67 (m, 2H), 4.73 (m, 1 H), 4.16 (d, J=17.69Hz, 1 H), 4.01 (dd, J=8.41 , 5.27Hz, 1H), 3.89 (d, J=17.69Hz, 1 H), 3.36-3.17 (m, 3H), 2.91-2.53 (m, 10H), 2.41 (m, 1 H), 2.33- 2.24 (m, 1 H), 2.09-2.01 (m, 2H), 0.87 (dd, J=6.53, 2.76Hz, 6H); 13C NMR (100MHz, CDCI3+5% MeOD) δc: 174.3 (C), 172.2 (C), 172.1 (C), 170.9 (C), 170.3 (C), 168.3 (C), 130.1 (CH), 129.8 (CH), 70.1 (CH), 64.9 (CH), 56.5 (CH), 55.4 (CH), 42.0 (CH2), 39.2 (CH2), 38.2 (CH2), 37.2 (CH2), 32.7 (CH2), 32.1 (CH2), 27.2 (CH), 26.4 (CH3), 25.4 (CH2), 20.4 (CH3), 20.0 (CH3). MS (ES+) 580.9 (100%, [M+Na]+).
Compound XLIV: N-r3-((EH1S.7R.10S,21R)-7-lsopropyl-3,6,9.19,22- pentaoxo-2-oxa-12,1 S-dithia-S.S^O^S-tetraaza-bicyclorS^.θitricos-i 6-en- 21-yl)-propionyll-4-methyl-benzenesulfonamide
Figure imgf000098_0001
6
(2): ((^^-{(SJ-a-tC^^-CgH-Fluoren-g-ylmethoxycarbonylaminoJ-δ-oxo-δ-
(toluene-4-sulfonylamino)-pentanoylamino]-3-tritylsulfanyl-propionylamino}-3- methyl-butyrylamino)-acetic acid methyl ester
To a solution of 1 (288.1 mg, 0.381 mmol) in MeCN/CH2CI2 (10mL/16mL) under Ar(g) was added diethylamine (2.6ml_, 10% v/v) and the reaction mixture was stirred at rt for 1h 50 min. The solvent was removed in vacuo and the residue co-evaporated with MeCN (3 x 2OmL). The crude amine was then dried under high vacuum for 3h. To a solution of PyBOP (202.25mg, 0.389 mmol) and Fmoc-D-Glu(NTs)-OH (198.1mg, 0.379 mmol) in MeCN (2OmL) was added DIPEA (0.26mL, 1.49 mmol) under Ar(g) with stirring for 2 min at O0C. A solution of the crude amine in MeCN (2OmL) was added; the reaction mixture was stirred at 0aC for 1h, and then at rt for 16h. The solvent was then removed in vacuo and purification was performed by flash column chromatography on silica (eluant 1 :99-3:97-5:95 MeOH/CH2CI2) to give XXLIV (152mg, 0.146 mmol, 38%) as a white solid.
1H NMR (400MHz, CDCI3+10% MeOD) δH: 7.83 (d, J=7.91 Hz, 2H), 7.69 (d, J=7.53Hz, 2H), 7.48 (d, J=6.90Hz, 3H), 7.36-7.29 (m, 8H), 7.28-7.12 (m, 17H), 6.99 (d, J=8.91 Hz, 1 H), 4.31-4.25 (m, 1 H), 4.23-4.12 (m, 2H), 4.07 (t, J=6.15Hz, 1 H), 3.99 (dd, J=8.85, 4.96Hz, 1H), 3.86-3.78 (m, 2H), 3.62 (s, 3H), 3.32 (dt, J=3.26, 1.63Hz, 2H), 2.67-2.60 (m, 1H), 2.56-2.50 (m, 1H), 2.18-2.07 (m, 4H), 1.96-1.86 (m, 1H), 1.70-1.61 (m, 1H), 0.85 (dd, J=9.35, 6.84Hz, 6H). MS (ES+) 1060.0 (100%, [M+Na]+). Rf 0.43 MeOH/CH2CI2 (1 :9). (4): ((R)-2-{(S)-2-[(R)-2-((E)-(S)-3-Hydroxy-7-tritylsulfanyl-hept-4-enoylamino)-5- oxo-5-(toluene-4-sulfonylamino)-pentanoylamino]-3-tritylsulfanyl- propionylamino}-3-methyl-butyrylamino)-acetic acid methyl ester To a solution of 2 (148.2mg, 0.143 mmol) in MeCN (2OmL) under Ar(g) was added diethylamine (2.OmL, 10% v/v) and the reaction mixture was allowed to stir at room temperature for 1h 30 min. The solvent was removed in vacuo, and this was then repeated again with MeCN (3 x 2OmL). The crude amine was then dried under high vacuum for 3h. To a solution of PyBOP (77.6mg, 0.149 mmol) and the carboxylic acid 3 (62.9mg, 0.150 mmol) in MeCN/CH2CI2 (10ml_/5mL) was added DIPEA (0.1OmL, 0.574 mmol) under Ar(g). A solution of the crude amine derivative from 2 in MeCN/CH2CI2 (10mL/5mL) was added, and the reaction mixture was allowed to stir at rt for 16h. The solvent was then removed in vacuo and purification was performed using flash column chromatography on silica (eluant 1 :99-2:98-3:97-4:96 MeOH/CH2CI2) to give 4 (100mg, 0.0822 mmol, 57%) as a white solid.
1H NMR (300MHz, CDCI3+10% MeOD) δH: 7.76 (d, J=8.38Hz, 2H), 7.36- 7.27 (m, 12H), 7.25-7.10 (m, 23H), 6.96 (d, J=8.10Hz, 1H), 5.47 (m, 1H), 5.31 (m, 1H), 4.28 (m, 1H), 4.13 (dd, J=8.62, 5.42Hz, 1H), 3.99 (m, 1 H), 3.84 (m, 1 H), 3.77 (d, J=5.84Hz, 1H), 3.72 (d, J=5.75Hz, 1H), 3.60 (S, 3H), 2.62 (m, 1H), 2.49 (m, 1 H), 2.24-2.18 (m, 2H), 2.11 (dt, JM 4.15, 7.10Hz, 5H), 2.04-1.94 (m, 4H), 1.87 (m, 1 H), 1.78-1.66 (m, 2H), 1.31 (dd, J=6.59, 3.39Hz, 1H), 1.11 (m, 1 H), 0.81 (t, J=6.69Hz, 6H). MS (ES+) 1238.4 (100%, [M+Na]+). R, 0.27 MeOH/CH2CI2 (1 :9).
(5): ((R)-2-{(S)-2-[(R)-2-((E)-(S)-3-Hydroxy-7-tritylsulfanyl-hept-4-enoylamino)-5- oxo-5-(toluene-4-sulfonylamino)-pentanoylamino]-3-tritylsulfanyl- propionylamino}-3-methyl-butyrylamino)-acetic acid
To 4 (100mg, 0.850 mmol) in THF (1.3mL) at O0C was added LiOH (12.8mg, 0.534 mmol) in water (0.13mL) and the reaction mixture was stirred for 1h 40 min. An additional volume of LiOH (9.3mg, 0.388 mmol) was subsequently added, and the reaction mixture was then quenched with 1 M HCI (aq) (1OmL), diluted with water (1OmL), and treated with EtOAc (2OmL). The layers were separated and the product was extracted with EtOAc (3x 25mL). The organic layers were combined, dried (MgSO4) concentrated in vacuo to give the product 5 (96mg, 96%) as a white solid. The product was used without further purification [MS (ES ) 1201.2 (100%, [M-H]')].
(6): N-{3-[(6R,9S,12R,16S)-6-lsopropyl-2,5,8,11 ,14-pentaoxo-16-((E)-4- tritylsulfanyl-but-1 -enyl)-9-tritylsulfanylmethyl-1 -oxa-4,7, 10, 13-tetraaza- cyclohexadec-12-yl]-propionyl}-4-methyl-benzenesulfonamide To a solution of MNBA (33.7mg, 0.0979 mmol) and DMAP (33.9mg, 0.277 mmol) in CH2CI2 (18.7mL) was added dropwise a solution of the acid 5 (95.5mg, 0.0795 mmol) in CH2CI2/THF (74.5mU 16mL) over 4h. The resulting reaction mixture was stirred overnight at rt, and was then concentrated in vacuo to give a brown solid. Purification by flash column chromatography on silica (eluant 1 :99-3:97-5:95 MeOH/CH2CI2) gave 6 (18.3mg, 0.0155 mmol, 19%) as a white solid.
1H NMR (400MHz, CDCI3+10% MeOD) δH: 8.00 (d, J=8.16Hz, 3H), 7.92 (d, J=7.91Hz, 1 H), 7.59-7.53 (m, 4H), 7.48 (t, J=7.84Hz, 4H), 7.44-7.35 (m, 7H), 7.33-7.16 (m, 15H), 5.64-5.48 (m, 2H), 5.40 (m, 1 H), 4.53 (br s, 1 H), 4.37 (m, 1 H), 4.25-4.06 (m, 2H), 3.92 (br s, 1H), 3.54 (d, J=16.06Hz, 1 H), 3.40 (m, 1H), 3.31 (dt, J=7.12, 3.65Hz, 1 H), 2.97 (dd, JM 1.73, 7.09Hz, 1 H), 2.39 (s, 3H), 2.62-1.88 (m, 5H), 1.67 (m, 1 H), 1.21-1.11 (m, 2H), 0.90 (dd, J=6.59, 4.71 Hz, 6H). MS (ES+) 1206.6 (100%, [M+Na]+). Rf0Λ4 MeOH/CH2CI2 (1 :9). Compound XLIV: N-[3-((E)-(1S,7R,10S,21 R)-7-lsopropyl-3,6,9,19,22-pentaoxo- 2-oxa-12, 13-dithia-5,8,20,23-tetraaza-bicyclo[8.7.6]tricos-16-en-21 -yl)- propionyl]-4-methyl-benzenesulfonamide
To a solution of iodine (39.45 g, 0.155 mmol) in CH2CI2/MeOH (9:1) (50 ml_) was added dropwise a solution of 6 (17.3mg, 0.0146 mmol) in CH2CI2/MeOH (9:1) (24.5ml_) over 55 min. The reaction mixture was then allowed to stir for a further 30 min, and was treated with a solution of sodium thiosulfate (21 ml_, 0.05 M). The layers were separated and the crude product was extracted with EtOAc (3 x 15mL), and the layers separated. The organic layers were combined, dried (MgSO4) and the solvent was removed in vacuo. Purification was performed using flash column chromatography on silica (eluant 1 :99-2:98-3:97-5:95 MeOH/CH2CI2) to furnish compound XLIV (2.07mg, 0.00229 mmol, 16%) as a yellow solid.
1H NMR (400MHz, CDCI3+10% MeOD) δH: 8.17 (dd, JM 6.38, 6.46Hz, 1 H), 8.04 (d, J=8.03Hz, 1 H), 7.92 (d, J=8.03Hz, 1 H), 7.84 (d, J=8.41Hz, 2H), 7.50 (d, J=7.65Hz, 1H), 7.39 (m, 1 H), 7.29 (s, 1H), 6.57 (d, J=9.54Hz, 1 H), 5.69 (m, 1 H), 5.58 (m, 1H), 5.43 (br s, 1H), 4.59-4.47 (m, 2H), 4.16 (m, 1H), 3.74 (dd, JM 6.75, 2.70Hz, 1 H), 3.64 (m, 1 H), 3.48-3.30 (m, 4H), 3.05 (dd, JM 7.50, 4.58Hz, 1H), 2.92 (ddd, JM 3.83, 6.62, 2.38Hz, 1 H), 2.64-2.25 (m, 8H), 2.03 (m, 1 H), 1.80 (m, 1 H), 0.92 (d, J=6.78Hz, 3H), 0.85 (d, J=6.78Hz, 3H). MS (ES+) 720.7 (100%, [M+H]+). R, 0.13 CH2CI2/MeOH (9:1). Compound XLV: 3-((a-(i S,7fi,10S,21 /:?)-7-tert-Butvl-3,6,9,19,22-pentaoxo-2- oxa-12, 13-dithia-5,8,20,23-tetraaza-bicyclo[8.7.6]tricos-16-en-21 -yl)-propionic acid tert-butyl ester
Figure imgf000101_0001
Figure imgf000101_0002
XLV (2): [(f?)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3,3-dimethyl-butyrylamino]- acetic acid methyl ester
To Fmoc-D-α-t-butylglycine 1 (867mg, 2.45mmol) and PyBOP (1.277g,
2.45mmol) in CH2CI2 (5OmL) was added DIPEA (1.7ml_, 9.76mmol) dropwise at
O0C. The activated acid was then added to Gly-OMe.HCI (308mg, 2.45mmol) in MeCN (5OmL) and the reaction was stirred for 16h. The solvent was removed in vacuo and the crude material was purified by flash column chromatography on silica (eluant 3:7-1 :1 EtOAc/Hexane) to give 2 (947.9mg, 2.32mmol, 95%) as a white solid.
1H NMR (400MHz, CDCI3) δH: 7.75 (d, J=7.40Hz, 2H), 7.60-7.53 (m, 2H), 7.38 (td, J=7.34, 3.01 Hz, 2H), 7.33-7.24 (m, 2H), 6.68 (br s, 1 H), 5.68 (d,
J=8.91Hz, 1 H), 4.40 (m, 1H), 4.30 (m, 1 H), 4.20 (t, J=6.09Hz, 1H), 4.16-4.06 (m, 2H), 3.86 (dd, J=17.94, 4.52Hz, 1 H), 3.69 (s, 3H), 1.04 (s, 9H). MS (ES+) 447.6 (100%, M+Na+). Rf 0.4 EtOAc/Hexane (1 :1).
(3) {(f?)-2-[(S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-
3tritylsulfanylpropionylamino]-3,3-dimethyl-butyrylamino}-acetic acid methyl ester To a solution of 2 (983mg, 2.3 mmol) in MeCN (5OmL) was added diethylamine (5ml_, 10% v/v) under Ar(g). After 45 min stirring the solvent was removed in vacuo and the residue was co-evaporated with hexane (3 x 25m L) and then placed under a high vacuum. At O0C, to Fmoc-D-Cys-OH (1.4035g, 2.4 mmol) and PyBOP (1.2486g, 2.4 mmol) in CH2CI2 (5OmL) was added dropwise DIPEA (1.3mL, 7.5 mmol). After 3 min stirring, the solution was then added to the crude amine in MeCN (5OmL). The reaction mixture was then allowed to stir at rt for 16h. The solvent was subsequently removed in vacuo and the resulting solid was purified by column chromatography on silica (eluant 3:7-1 :1 EtOAc/Hexane) to give 3 (1.72 g, 2.23 mmol, 99%) as a white solid. 1H NMR (400MHz, CDCI3) δH: 7.74 (t, J=7.34Hz, 2H), 7.57 (d, J=7.28Hz,
2H), 7.43-7.17 (m, 21H), 6.90 (d, J=8.91Hz, 1 H), 4.37 (d, J=6.90Hz, 2H), 4.19 (t, J=6.713Hz, 1 H), 4.12 (m, 1H), 3.94 (m, 1H), 3.88-3.74 (m, 2H), 3.68 (s, 3H), 2.68-2.56 (m, 2H), 0.95 (s, 9H). MS (ES+) 792.7 (100%, M+Na). Rf 0.13 EtOAc/Hexane (4:6). (4): (/:?)-4-(9H-Fluoren-9-ylmethoxycarbonylamino)-4-{(S)-1 -[(R)-I -
(methoxycarbonylmethyl-carbamoyl)-2,2-dimethyl-propylcarbamoyl]-2- tritylsulfanyl-ethyicarbamoyl}-butyric acid tert-butyl ester
To a solution of 3 (1.158 g, 1.5 mmol) in MeCN/CH2CI2 (30mL:13mL) was added diethylamine (4.3mL, 10% v/v) under Ar(g). After 1h 55 min, the solvent was removed in vacuo and the excess of amine co-evaporated with hexane (3 x 15mL) after which time the crude material was placed under high vacuum. At O0C to Fmoc-D-Glu(OtBu)-OH (643.6mg, 1.5 mmol) and PyBOP (825mg, 1.59 mmol) in CH2CI2 (3OmL) was added DIPEA in dropwise fashion (0.7mL, 4.0 mmol). After 5 min, this solution was then added to the crude amine of 3 in MeCN (3OmL). The reaction mixture was then allowed to stir overnight at rt. The solvent was removed in vacuo and the crude product purified by column chromatography on silica (eluant 3:7-4:6-1 :1-6:4-8:2-1 :0 EtOAc/Hexane) to give 4 (1.243 g, 1.30 mmol, 86%) as a white solid.
1H NMR (400MHz, CDCI3+5% MeOD) δH: 7.76 (d, J=7.53Hz, 2H), 7.56 (t, J=6.84Hz, 2H), 7.42-7.13 (m, 19H), 6.98 (d, ^=8.66Hz, 1 H), 4.36 (m, 1H), 4.25 (m, 1 H), 4.19-4.06 (m, 4H), 4.00-3.91 (m, 2H), 3.86 (m, 1 H), 3.68 (s, 3H), 2.78 (dd, J=12.799, 7.027Hz, 1H), 2.52 (dd, J= 12.862, 5.709Hz, 1H), 2.41-2.23 (m, 2H), 1.99 (m, 1 H), 1.83 (m, 1H), 1.44 (s, 9H), 0.99 (s, 9H). MS (ES+) 977.7 (100%, M+Na+). Rf 0.44 EtOAc/Hexane (6:4). (5): (fl)-4-Amino-4-{(S)-1 -[(R)-1 -(methoxycarbonylmethyl-carbamoyl)-2,2- dimethylpropylcarbamoyll^-tritylsulfanyl-ethyicarbamoylj-butyric acid tert-butyl ester
To a solution of 4 (1.215 g, 1.27 mmol) in MeCN/CH2CI2 (10mL:40mL) was added diethylamine (1OmL, 10% v/v) under Ar(g). After 3.5h, additional diethylamine (5ml_) was added. After a further 1h stirring, the solvent was removed in vacuo and the excess of amine was co-evaporated with hexane (3 x 3OmL) after which time the crude material was placed under high vacuum for 1h.. The resulting amine was purified by flash column chromatography using MeOH/CH2CI2 (5:95-10:90) as eluant to give 5 (472mg, 0.64 mmol, 50%) a white solid.
1H NMR (400MHz, CDCI3) δH: 7.52-7.48 (m, 1 H), 7.42 (d, J=8.03Hz, 5H), 7.33-7.19 (m, 10H), 6.65 (d, J=8.28Hz, 1H), 6.40 (br s, 1 H), 4.13 (d, J=8.91 Hz, 1 H), 3.98 (m, 1 H), 3.87 (m, 1H), 3.76-3.68 (m, 4H), 3.42 (br s, 1H), 3.18 (q, J=7.40Hz, 2H), 2.72 (m, 1 H), 2.59 (dd, JM 3.05, 5.40Hz, 1 H), 2.34 (t, J=6.96Hz, 1H), 2.07-1.98 (m, 2H), 1.79 (m, 1 H), 1.42 (s, 9H), 0.97 (s, 9H). MS (ES+) 755.9 (100%, [M+Na]+). Rf 0.35 MeOH/CH2CI2 (6:94).
(7): (fl)-4-((E)-(S)-3-Hydroxy-7-tritylsulfanyl-hept-4-enoylamino)-4-{(S)-1 -[(fl)-1 - (methoxycarbonylmethyl-carbamoyl)-2,2-dimethyl-propylcarbamoyl]-2- tritylsulfanyl-ethylcarbamoyl}-butyric acid tert-butyl ester Carboxylic acid 6 (269.7mg, 0.64 mmol) and PyBOP (339.0mg, 0.65 mmol) were dissolved in CH2CI2 (2OmL), and DIPEA (0.3mL, 1.72 mmol) was added in dropwise afashion. The reaction mixture was stirred for 6 min, and was then added to 5 (472mg, 0.64 mmol) in CH2CI2 (2OmL). After 16h stirring at rt, the mixture was concentrated in vacuo, and was purified by flash column chromatography using EtOAc/Hexane (6:4-7:3-8:2) as eluant to give 7 (540mg, 0.48 mmol, 74%) as a white solid.
1H NMR (400MHz, CDCI3) δH: 7.44-7.36 (m, 12H), 7.32-7.24 (m, 14H), 7.24-7.16 (m, 6H), 7.10 (d, J=6.40Hz, 1H), 6.70 (t, J=5.46Hz, 1 H), 6.62 (d, J=8.66Hz, 1 H), 5.47 (m, 1H), 5.35 (m, 1 H), 4.35 (ddd, J=9.60, 6.40, 2.70Hz, 1 H), 4.26 (ddd, J=8.78, 6.40, 4.39Hz, 1 H), 4.16 (d, J=8.66Hz, 1H), 3.93-3.88 (m, 3H), 3.68 (S, 3H), 2.65 (dd, JM2.86, 7.72Hz, 1 H), 2.50 (dd, JM 2.92, 5.65Hz, 1 H), 2.42(m, 1H), 2.33 (d, J=3.39Hz, 1 H), 2.29 (d, J=2.89Hz, 1H), 2.24-2.16 (m, 3H), 2.11-2.01 (m, 3H), 1.91 (m, 1 H), 1.41 (s, 9H), 0.99 (s, 9H). MS (ES+) 1151.1 (100%, [M+Na]+). f?, 0.35 MeOH/CH2CI2 (6:94). (8): (/^-4-{(S)-1-[(/^-1-(Carboxymethyl-carbamoyi)-2,2-dimethyl- propylcarbamoyl]-2-tritylsulfanyl-ethylcarbamoyl}-4-((£)-(S)-3-hydroxy-7- tritylsulfanyl-hept-4-enoylamino)-butyric acid tert-butyl acid
To a solution of 7 (920mg, 0.81 mmol) in THF (13mL) at O0C was added dropwise LiOH (28.7mg, 1.20 mmol) in water (3.4mL), and stirring was continued at O0C. After 2.5h, additional LiOH (9.2mg, 0.38 mmol) was added, and after a further 30 min, HCI (aq) (1OmL, 1 M) was added and then water (2OmL). EtOAc was added (4 x 25mL) and the layers were separated. The organic extracts were combined, washed with saturated brine (25mL), dried (MgSO4) and concentrated in vacuo to give 8 as a white solid (756mg, 0.68 mmol, 83%) which was used in the next step without further purification [MS (ES-) 1117.8 (100%, [M-H'])].
(9): 3-[(6fl,9S,12fl,16S)-6-tert-Butyl-2,5,8,11 ,14-pentaoxo-16-((£)-4- tritylsulfanyl-but-1 -enyl)-9-tritylsulfanylmethyl-1 -oxa-4,7,10,13-tetraaza- cyclohexadec-12-yl]-propionic acid tert-butyl ester
To a solution of MNBA (280.6mg, 0.82 mmol) and DMAP (197.46mg, 1.62 mmol) in CH2CI2 (123mL) was added dropwise a solution of acid 8 (756mg, 0.675 mmol) in CH2CI2 (502mL) over 5h 20 min. After a further 16h the reaction mixture was concentrated in vacuo. Purification by flash column chromatography on silica (eluant 1:0-99:1-98:2-97:3 CH2CI2/MeOH) gave 9 (343mg, 0.31 mmol, 46%) as a yellow solid. 1H NMR (400MHz, CDCI3) δH: 8.08 (d, J=7.654Hz, 1 H), 7.44-7.16 (m,
30H), 7.02 (br s, 1 H), 6.82 (br s, 1 H), 6.73 (d, J=7.65Hz, 1H), 6.57 (d, J=7.15Hz, 1 H), 5.58 (m, 1 H), 5.45 (t, J=6.96Hz, 1 H), 5.32 (m, 1H), 4.24-4.11 (m, 2H), 4.06 (d, J=8.28Hz, 1H), 3.68-3.51 (m, 2H), 3.22 (s, 2H), 3.01 (m, 1H), 2.60 (dd, J=12.61 , 5.58Hz, 1H), 2.51-2.28 (m, 3H), 2.22-2.14 (m, 2H), 2.07-1.94 (m, 4H), 1.41 (s, 9H), 0.99 (s, 9H). MS (ES+) 1124.2 (100%, [M+Na]+). Rf 0.36 CH2CI2/MeOH (94:6).
Compound XLV: 3-((£)-(1 S,7H,10S,21 fl)-7-tert-Butyl-3,6,9,19,22-pentaoxo-2- oxa-12, 13-dithia-5,8,20,23-tetraaza-bicyclo[8.7.6]tricos-16-en-21 -yl)-propionic acid tert-butyl ester To a solution of I2 (747mg, 2.94 mmol) in CH2CI2/Me0H (1001ml_, 9:1) was added dropwise 9 (343mg, 0.31 mmol) in CH2CI2/Me0H (467mL, 9:1) over 1 h. After a further 30 min, Na2S2O3 (438mL, 0.05M) was added, the layers were separated, and the product extracted with EtOAc (3 x 60OmL). The organic layers were combined, dried (MgSO4) and concentrated in vacuo. Purification by flash column chromatography on silica (eluant 1 :0-98:2-95:5 CH2CI2/MeOH) gave compound XLV (138mg, 0.225 mmol, 72%) as a yellow solid.
1H NMR (400MHz, CDCI3+5% MeOD) δH: 8.22 (d, J=4.39Hz, 1 H), 7.39 (dd, J=5.65, 3.14Hz, 1 H), 7.28 (m, 1 H), 5.86 (m, 1H), 5.81-5.73 (m, 2H), 4.84 (m, 1H) 4.36 (dd, JM 7.38, 6.34Hz, 1 H), 4.15 (m, 1H), 3.82 (dd, J=17.32, 3.14Hz, 1 H), 3.57 (1H, m) 3.31 (dd, JM 5.37, 11.10Hz, 1H), 3.03 (t, J=5.77Hz, 2H), 2.96-2.88 (m, 3H), 2.83-2.68 (m, 2H), 2.65-2.53 (m, 3H), 2.43 (m, 1H), 2.17- 2.09 (m, 2H) 1.47 (s, 9H), 1.07 (s, 9H); 13C NMR (400MHz, CDCI3+5% MeOD) δc: 174.6 (C), 171.9 (C), 169.8 (C), 169.8 (C), 169.3 (C), 168.4 (C), 130.5 (CH), 130.3 (CH), 82.0 (C), 69.6 (CH), 63.2 (CH), 56.2 (CH), 55.2 (CH), 42.5 (CH2), 38.5 (CH2), 38.2 (CH2), 35.4 (CH2), 34.6 (CH2), 32.5 (CH2), 31.6 (CH2), 28.0 (CH3), 27.3 (CH3), 24.8 (C). MS (ES+) 637.8 (100%, [M+Na]+). Rf 0.35 CH2CI2/MeOH (90:10). Compound XLVI: 3-((a-(1 S7fl.10S.21 fl)-7-tert-Butyl-3.6.9.19.22-pentaoxo- 2-oxa-12.13-dithia-5.8.20,23-tetraaza-bicvclor8.7.6ltricos-1β-en-21-vn- propionic acid
Figure imgf000105_0001
To compound XLV above (130.7mg, 0.21 mmol) at 00C was added Et3SiH (0.17ml_, 1.06 mmol) and TFA (1.5ml_) dropwise. The reaction mixture was stirred for 3h 20min, was concentrated in vacuo, and was placed under high vacuum for 1h. Toluene was added to the crude material twice, and was then removed in vacuo before being placed back under high vacuum for a further 10 min. Purification by flash column chromatography on silica (eluant 100:1-98:2- 96:4-94:6-92:8-90:10-85:15 CH2CI2:MeOH) gave compound XLVI (105.7mg, 0.189 mmol, 89%) as a pale yellow solid. 1H NMR (400MHz, CDCI3+5% MeOD) δH: 7.28 (d, J=8.41 Hz, 1H), 5.90- 5.70 (m, 3H), 4.90 (m, 1H), 4.26 (d, J=17.32Hz, 1H), 4.20 (dd, J=8.78, 4.77Hz, 1 H), 3.96 (S, 1H), 3.82 (d, J=17.32Hz, 1 H), 3.41-3.27 (m, 4H), 3.06 (t, J=4.27Hz, 2H), 2.97 (dd, JM 4.87, 3.20Hz, 1 H), 2.79-2.65 (m, 3H), 2.64-2.51 (m, 3H), 2.23- 2.05 (m, 2H), 1.02 (s, 9H); 13C NMR (100MHz, CDCI3+5% MeOD) δc: 172.0 (C), 171.0 (C), 170.3 (C), 169.8 (C), 168.5 (2xC), 130.3 (2xCH), 70.3 (CH), 62.6 (CH), 55.8 (CH), 50.1 (CH), 42.2 (CH2), 42.1 (CH2), 38.8 (CH2), 38.0 (CH2), 34.7 (CH2), 33.6 (CH2), 31.2 (CH2), 26.8 (CH3), 24.9 (C). MS (ES+) 557.5 (100%, [M+Na]+). Rf 0.23 CH2CI2/MeOH (85:15)+ 3 drops AcOH. Compound XLVII: 3-((E)-(I S,7fl.10S,21 fn-7-tert-Butyl-3,6.9,19,22-pentaoxo- 2-oxa-12.13-dithia-5,8,20,23-tetraaza-bicvclor8.7.61tricos-1β-en-21-yl)-N-(2- methoxy-ethvD-propionamide
Figure imgf000106_0001
To compound XLVI (14.5mg, 0.026 mmol) and PyBOP (15.4mg, 0.030 mmol) was added CH2CI2 (1mL) followed by MeCN (1mL). DIPEA (15 μL, 0.086 mmol) was then added dropwise, the solution was stirred for 3 min, and 2- methoxyethylamine (7.5 μL, 0.067 mmol) was then added. The reaction mixture was stirred for 5.25h, and was then concentrated in vacuo before being purified by flash column chromatography on silica (eluant 98:2-96:4-94:6 CH2CI2/MeOH) and then by ion exchange chromatography using an SCX-3 Isolute® column (eluant 98:2-96:4-94:6 CH2CI2/MeOH). Compound XLVII was isolated as a white solid (10mg, 0.0162 mmol, 63%).
1H NMR (300MHz, CDCI3+10% MeOD) δH: 5.75 (m, 1H), 5.71-5.60 (m, 2H), 4.69 (dd, J=11.40, 3.49HZ, 1H), 4.20 (d, J=17.33Hz, 1H), 3.69-3.57 (m, 2H), 3.39-3.19 (m, 8H), 3.13 (dd, JM 5.07, 11.49Hz, 2H), 2.92 (t, J=5.56Hz, 2H), 2.79 (dd, JM 5.16, 3.58Hz, 1 H), 2.71-2.46 (m, 5H), 2.43-2.25 (m, 3H), 2.09-1.95 (m, 4H), 0.92 (s, 9H); 13C NMR (100MHz, CDCI3+5% MeOD) δc: 175.3 (C), 171.8 (C), 170.5 (C), 170.4 (C), 169.1 (C), 168.4 (C), 130.8 (CH), 130.4 (CH), 70.2 (CH), 70.2 (CH2), 62.9 (CH), 58.8 (CH3), 56.0 (CH), 55.5 (CH), 42.3 (CH2), 40.3 (CH2), 38.4 (CH2), 38.1 (CH2), 35.2 (CH2), 33.9 (CH2), 32.4 (CH2), 31.3
(CH2), 27.1 (CH3), 25.9 (C). MS (ES+) 650.9 (100%, [M+Na]+).
Compound XLVHI: (EH1 S.7R10S.21 fl)-7-tert-Butyl-21-(3-morpholin-4-yl-3- oxo-propyD-a-oxa-ia.iS-dithia-S.β^O^S-tetraaza-bicvciorβ^.βitricos-iβ- ene-3,6,9,19,22-pentaone
Figure imgf000107_0001
To compound XLVI (16mg, 0.029 mmol) and PyBOP (16.4mg, 0.032 mmol) was added CH2CI2 (1mL) followed by MeCN (1mL). DIPEA (14 μL, 0.080 mmol) was then added dropwise, and the solution was allowed to stir for 4 min. Morpholine (3 μL, 0.034 mmol) was added , the resulting mixture stirred for 1h 35min, and following removal of the solvent by evaporationuder reduced pressure, the crude product was purified by flash column chromatography on silica (eluant 98:2-96:4-94:6 CH2CI2/MeOH) and then by ion exchange chromatography using an SCX-3 Isolute® column (eluant 98:2-96:4-94:6 CH2CI2/MeOH). Compund XLVII was isolated as a white solid (10mg, 0.0159 mmol, 55%).
1H NMR (400MHz, CDCI3+5% MeOD) δH: 5.85-5.71 (m, 2H), 4.82 (m, 1 H), 4.31 (d, J=17.32Hz, 1 H), 4.15 (dd, J=8.60, 4.20Hz, 1 H), 3.77 (d, J=17.32Hz, 1H), 3.70-3.62 (m, 3H), 3.61-3.54 (m, 2H), 3.45 (t, J=4.83Hz, 2H), 3.23 (dd, JM 5.31, 11.29Hz, 1 H), 2.98 (m, 1 H), 2.89 (dd, JM 5.31 , 3.76Hz, 1 H), 2.73 (m, 1H), 2.70-2.43 (m, 4H), 2.40-2.21 (m, 7H), 1.02 (s, 9H); 13C NMR (100MHz, CDCI3+5% MeOD) δc: 172.4 (C), 172.0 (C), 170.0 (C), 169.8 (C), 169.3 (C), 168.5 (C), 130.4 (CH), 130.1 (CH), 69.7 (CH), 66.6 (CH2), 66.30 (CH2), 63.3 (CH), 56.3 (CH), 55.4 (CH), 46.1 (CH2), 42.6 (CH2), 42.5 (CH2), 42.4 (CH2), 38.8 (CH2), 38.0 (CH2), 35.4 (CH2), 34.5 (CH2), 31.4 (CH2), 27.3 (CH3), 24.6 (C). MS (ES+) 639.9 (100%, [M+Na]+).
Compound XLIX: (EH1S.7R.10S.21R)-7-lsopropyl-21-r3-(4-methyl- piperazin-1 -yl)-3-oxo-propyπ-2-oxa-12,13-dithia-5,8,20.23-tetraaza- bicvcloF8.7.61tricos-16-ene-3,β.9.19.22-pentaone
Figure imgf000108_0001
To carboxylic acid (1) as outlined for compound XXXVII above (14.6mg, 0.0268 mmol) and PyBOP (14.95mg, 0.029 mmol) was added CH2CI2 (1mL) followed by MeCN (1mL). DIPEA (15 μL, 0.0861 mmol) was then added and the reaction mixture was stirred for 5min. 1 -Methylpiperazine (5 μL, 0.0451 mmol) was added, the resulting mixture was stirred for 1h 20min, after which time the solvent was removed in vacuo. Purification by flash column chromatography on silica (eluant 2:98-4:96-6:94-8:92-1 :9 MeOH/CH2CI2) followed by SCX-3 ion exchange column furnished compound XLIX (2.99mg, 18%, 0.00477 mmol) as a white solid.
1H NMR (400MHz, CDCI3+ 5% MeOD) δH: 9.05 (br s, 1 H), 7.46 (dd, J=6.09, 4.58Hz1 1 H), 7.33 (d, J=6.27Hz, 1 H), 7.21 (d, J=8.53Hz, 1 H), 5.83 (m, 1H), 5.76-5.65 (m, 2H), 4.79 (td, J=9.16, 3.76Hz, 1H), 4.19 (dd, J=M.69, 6.90Hz, 1H), 4.07 (dt, J=8.44, 4.25Hz, 1 H), 3.90 (dd, JM 7.63, 4.20Hz, 1H), 3.60 (br s, 2H), 3.49 (br s, 2H), 3.39-3.17 (m, 3H), 2.99-2.77 (m, 3H), 2.74-2.55 (m, 5H), 2.52-2.27 (m, 8H), 2.20 (m, 1H), 2.09 (m, 1 H), 0.93-0.88 (m, 6H). MS (ES+) 650.0 (100%, [M+Na]+).
Compound L: (EH1S,7R,10S,21 R)-7-isopropyl-21-thiazol-4-ylmethyl-2-oxa- 12.1 a-dithia-S.S.ZO^a-tetraaza-bicvclorβ^.eitricos-iβ-ene-a.e.g.i 9.22- pentaone
Figure imgf000109_0001
Figure imgf000109_0002
(2): ((R)-2-{(S)-2-[(R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-thiazol-4-yl- propionylam ino]-3-tritylsulf anyl-propionylam ino}-3-methyl-butyry lam ino)-acetic acid methyl ester
Et2NH (5ml_) was added to ((R)-2-{(S)-2-[(R)-2-(9H-fluoren-9- ylmethoxycarbonylamino)-3-thiazol-4-yl-propionylamino]-3-tritylsulfanyl- propionylamino}-3-methyl-butyrylamino)-acetic acid methyl ester 1 (3g, 3.97 mmol (prepared according to WO 2006/129105)) in MeCN (25ml_) at rt under Ar(g). After 1.5h of stirring the solvent was removed under reduced pressure, then the residue was redissolved, evaporated with MeCN (3 x 2OmL) and hexane (2 x 2OmL). The crude product was then dried under high vacuum at least 3h. DIPEA (1.66mL, 9.92 mmol) was added to (R)-2-(9H-fluoren-9- ylmethoxycarbonylamino)-3-thiazol-4-yl-propionic acid (1.723mg, 4.37 mmol) and PyBOP (2.272 g, 4.37 mmol) in CH2CI2 (8OmL) at O0C under Ar(g). After 10min of stirring, the mixture was transferred to the crude amine outlined above, solubilised in MeCN (8OmL) at O0C under Ar(g). The reaction mixture was warmed to rt and, after 16h stirring, was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography, eluting with hexane/EtOAc (50:50 to 0:100) to yield 2 as a white solid (2.97 g, 75%).
1H NMR (400MHz, 9/1 CDCI3ZCD3OD) δ H: 8.69 (s, 1 H), 7.72 (d, J=7.5Hz, 2H), 7.47-7.55 (m, 2H), 7.09-7.44 (m, 19H), 7.05 (s, 1 H), 4.36 (m, J=7.2Hz, 1 H), 4.33 (d, J=7.0Hz, 1 H), 4.18-4.24 (m, 1 H), 4.12-4.17 (m, 1 H), 4.09-4.12 (m, 1 H), 4.01 -4.05 (m, 1 H), 3.89 (dd, JM 7.6, 4.5Hz, 1 H), 3.76 (dd, JM 8.9, 4.9Hz, 1 H), 3.64 (s, 3H), 3.21 (dd, J=14.6, 4.8Hz, 1 H), 3.12 (dd, J=15.3, 6.4Hz, 1H), 2.73 (dd, J=12.3, 6.3Hz, 1 H), 2.44 (dd, J=12.7, 5.9Hz, 1 H), 2.19 (spt, J=7.0Hz, 1 H), 0.89 (d, J=6.7Hz, 3H), 0.85 (d, J=6.7Hz, 3H). MS (ES): 932.3 (100%, [M+Na]+). (4): ((R)-2-{(S)-2-[(R)-2-((E)-(S)-3-hydroxy-7-tritylsulfanyl-hept-4-enoylamino)-3- thiazol-4-yl-propionylamino]-3-tritylsulfanyl-propionylamino}-3-methyl- butyrylamino)-acetic acid methyl ester
Et2NH (1OmL) was added to 2 (2.704 g, 2.97 mmol) in MeCN (9OmL) at rt under Ar(g). After 2.5h of stirring, the solvent was removed under reduced pressure, then the residue was redissolved and evaporated with MeCN (3x20mL) and hexane (2x20mL). The crude amine was dried under high vacuum for at least 3h. DIPEA (1.3mL, 7.42 mmol) was added to a solution 3 (1.306mg, 3.12 mmol) and PyBOP (1.623 g, 3.12 mmol) in CH2CI2 (8OmL) at 00C under Ar(g). After 10min of stirring, the mixture was transferred to the crude amine outlined above, dissolved in MeCN (8OmL), at O0C under Ar(g), and the resulting reaction mixture was left to warm to rt. After 48h, the mixture was concentrated under reduced pressure, the residue purified by silica gel column chromatography, eluting with hexane/EtOAc (2:1 , 2:3 then 0:1) to yield 4 as a white solid (2.135 g, 72%).
1H NMR (400MHz, CD3OD) δH: 8.86 (d, ^ .9Hz, 1 H), 7.14-7.42 (m, 32H), 5.51 (dt, JM 7.1 , 6.5Hz, 1 H), 5.38 (dd, J=15.4, 6.3Hz, 1 H), 4.70 (dd, J=7.9, 4.9Hz, 1H), 4.32 (dd, J=12.8, 6.1 Hz, 1H), 4.16 (d, J=6.9Hz, 1 H), 3.84 (d, J=17.7Hz, 1 H), 3.79 (d, J=17.6Hz, 1H), 3.66 (s, 3H), 3.25 (d, J=4.9Hz, 1H), 3.14 (dd, J=14.6, 8.1 Hz, 1H), 2.56-2.61 (m, 1 H), 2.50-2.56 (m, 1 H), 2.34 (dd, J=13.9, 8.4Hz, 1 H), 2.28 (dd, J=13.9, 5.0Hz, 1 H), 2.15-2.23 (m, 2H), 2.10 (td, J=13.6, 6.9Hz, 1 H), 2.02-2.05 (m, 2H), 0.93 (d, J=6.8Hz, 3H), 0.90 (d, J=6.8Hz, 3H). MS (ES): 1111.4 (100%, [M+Na]+).
(5): ((R)-2-{(S)-2-[(R)-2-((E)-(S)-3-hydroxy-7-tritylsulfanyl-hept-4-enoylamino)-3- thiazol-4-yl-propionylamino]-3-tritylsulfanyl-propionylamino}-3-methyl- butyrylamino)-acetic acid LiOH (70.4mg, 2.94 mmol) in water (1OmL) was added to 4 (2.135 g, 1.96 mmol) in THF (45m L) at 00C. After 2h stirring at 00C, the reaction mixture was neutralized (pH=7) with aqueous 2N HCI, and then treated with brine (5OmL) and EtOAc (5OmL). The phases were separated and the aqueous phase was extracted with EtOAc (2x50mL). The organic phases were combined, dried over MgSO4, filtered, then concentrated under reduced pressure. The crude product, 5,was dried under high vacuum, and was used in the next step without any additional purification.
1H NMR (400MHz, CD3OD) δ H: 8.86 (d, 1 H), 7.32-7.42 (m, 12H), 7.16- 7.32 (m, 19H), 5.51 (dt, JM3.1 , 6.5Hz, 1 H), 5.38 (dd, J=15.4, 6.3Hz, 1H), 4.71 (dd, J=7.8, 4.8Hz, 1 H), 4.32 (dd, J=13.1 , 5.9Hz, 1H), 4.17 (d, J=6.8Hz, 1 H), 4.10-4.15 (m, 1 H), 3.77 (s, 2H), 3.23-3.29 (m, 1 H), 3.14 (dd, J=15.1 , 7.9Hz, 1 H), 2.49-2.62 (m, 2H), 2.34 (dd, J=13.7, 8.2Hz, 1 H), 2.28 (dd, JS=14.1 , 5.0Hz, 1 H), 2.16-2.22 (m, 2H), 2.12 (sxt, J=6.5Hz, 1 H), 2.02-2.07 (m, 2H), 0.93 (d, J=6.8Hz, 3H), 0.90 (d, J=6.8Hz, 3H). MS (ES): 1096.2 (100%, [M+Na]+). (6):(6R,9S,12R,16R)-6-isopropyl-12-thiazol-4-yimethyl-16-((E)-4-thtylsuifanyl- but- 1 -enyl)-9-tritylsulf anylm ethyl- 1 -oxa-4, 7, 10,13-tetraaza-cyclohexadecane- 2,5,8,11 ,14-pentaone
A solution of 5 in CH2CI2/THF (54OmL, 12:1 , v/v) was added dropwise over a period of 3h to 2-methyl-6-nitrobenzoic anhydride (810mg, 2.35 mmol) and 4-(dimethylamino)pyridine (575mg, 4.70 mmol) in CH2CI2 (1.7 L) at rt under
Ar(g). After 23h, the reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography, eluting with
CH2CI2/isopropanol (100:3 then 100:5), to yield 6 as a white solid (809mg, 40%)
1H NMR (400MHz, CD3OD) δH: 8.81 (d, J=2.0Hz, 1H), 7.15-7.42 (m, 31H), 5.55-5.67 (m, 1 H), 5.35-5.44 (m, 2H), 4.55 (t, J=7ΛHz, 1H), 4.31 (d, J=16.9Hz, 1 H), 4.15 (d, J=5.0Hz, 1 H), 3.56 (d, J=16.9Hz, 1 H), 3.51 (dd, J=9.5, 5.8Hz, 1 H), 3.44 (br. s., 2H), 2.92 (dd, J=12.7, 9.5Hz, 1H), 2.65 (dd, ^=12.7, 5.8Hz, 1 H), 2.50 (dd, J=I 5.1 , 2.9Hz, 1 H), 2.37-2.46 (m, 2H), 2.13-2.23 (m, 2H), 2.02 (q, J=7.2Hz, 2H), 0.88 (d, J=7.0Hz, 3H), 0.85 (d, Λ=6.9Hz, 3H). MS (ES): 1078.1 (100%, [M+Naf).
Compound L: (E)-(I S,7R,10S,21 R)-7-isopropyl-21-thiazol-4-ylmethyl-2-oxa-
12, 13-dithia-5,8,20,23-tetraaza-bicyclo[8.7.6]tricos-16-ene-3,6,9, 19,22-pentaone
Compound 6 (805 mg, 0.76 mmol) in CH2CI2/CH3OH (500 mL, 9:1 , v/v) was added dropwise over a period of 3h to I2 (1.934 mg, 7.62mmol) in CH2CI2/ CH3OH (1.25 L, 9:1 , v/v) at rt under Ar(g). After 4h stirring, 0.1 M Na2S2O3 (1 L) was added. The phases were separated, and the aqueous phase was extracted with CH2CI2 (2x400 mL) and EtOAc (400 mL). The organic phases were then combined, dried over MgSO4, filtered, then concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with CH2CI2/ CH3OH (100:1 to 100:4) to yield compound L as a white solid (297mg, 68%).
1H NMR (400MHz, CD3OD) δH: 9.22 (d, JM .8Hz, 1H), 7.63 (s, 1 H), 6.03- 6.13 (m, 1H), 5.90-6.01 (m, 2H), 4.90 (d, J=4.0Hz, 1H), 4.87 (dd, J=9.0, 4.8Hz, 1 H), 4.51 (d, J=17.4Hz, 1 H), 4.01 (d, J=17.4Hz, 1 H), 3.60-3.73 (m, 2H), 3.50 (dd, J=19.2, 11.3Hz, 1 H), 3.43 (d, JM 1.3Hz, 1H), 3.33 (dd, JM 5.4, 3.9Hz, 1H), 3.28 (t, J=4.5Hz, 1H), 3.21 (dd, JM 0.4, 3.9Hz, 1 H), 3.18 (dd, JM 0.7, 4.0Hz, 1 H),3.13 (t, J=6.5Hz, 1 H), 3.07 (dd, JM 3.4, 1.6Hz, 1 H), 2.94 (dd, JM 3.6, 5.9Hz, 1H), 2.84-2.90 (m, 1H), 1.21 (d, J=6.7Hz, 3H), 1.16 (d, J=6.7Hz, 3H). MS (ES): 570.1 (15%, [M+H]+), 592.0 (100%, [M+Na]+).
Compound Ll: (E)-(I S.10S.21 ff)-7-isopropyl-21-pyridin-3-ylmethyl-2-oxa- 12.1 S-dithia-δ.δ^O^a-tetraaza-bicvclorS^.eitricos-ie-ene-S.β.Q.19,22- pentaone
Figure imgf000112_0001
Figure imgf000112_0002
U 5 (2): (2-{(S)-2-[(R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-pyridin-3-yl- propionylamino]-3-tritylsulfanyl-propionylamino}-3-methyl-butyrylamino)-acetic acid methyl ester
To a solution of 1 (250mg, 0.33 mmol, 1eq) in MeCN (7mL) was added Et2NH (0.7mL, 10% v/v) dropwise at rt under Ar(g). The solution was stirred at rt for 2h, then the solvent was removed in vacuo. The excess of amine was co- evaporated with MeCN (3 x 5ml_), and then with a 1 :5 mixture of CH2CI2/hexane (5mL). A white solid was obtained which was then dried under vacuum for 2h. To a solution of Fmoc-D-3-PyridylAlanine (142mg, 0.36 mmol, 1.1 eq) in MeCN (6ml_) at 00C was added PyBOP (172mg, 0.36 mmol, 1.1 eq) and N- ethyldiisopropylamine (144 μL, 0.83 mmol, 2.5eq) under Ar(g). The crude amine, dissolved in CH2CI2 (6ml_) was added to the mixture in dropwise fashion. The reaction mixture was then left to warm to rt overnight, was then concentrated in vacuo, and purified by silica gel column chromatography with hexane/EtOAc (1 :9 -> 0:1 + 0.5% MeOH) to give 2 as a white solid (286mg, 96%).
1H NMR (400MHz, CDCI3) δH: 8.39-8.45 (m, 2H), 7.76 (d, J=7.5Hz, 2H), 7.46-7.52 (m, 3H), 7.38 (m, 9H), 7.28-7.31 (m, 2H), 7.22-7.26 (m, 5H), 7.13-7.21 (m, 4H), 6.95-7.02 (m, 1H), 6.65-6.73 (m, 1 H), 6.46 (d, J=7.8Hz, 1 H), 5.46 (d, J=6.3Hz, 1 H), 4.35-4.49 (m, 1H), 4.20-4.34 (m, 1H), 4.08-4.15 (m, 1H), 3.92 (dd, J=18.1 , 5.9Hz, 1H), 3.75 (m, 2H), 3.66 (s, 3H), 3.02-3.11 (m, 1 H), 2.89-3.00 (m, 1 H), 2.70-2.79 (m, 1 H), 2.58 (dd, J=13.2, 6.5Hz, 1 H), 2.03-2.10 (m, 3H), 0.90- 0.95 (m, 6H). MS (ES+) 926.9 (100%, [M+Na]+). (4): (2-{(S)-2-[(f?)-2-((E)-(S)-3-Hydroxy-7-tritylsulfanyl-hept-4-enoylamino)-3- pyridin-3-yl-propionylamino]-3-tritylsulfanyl-propionylamino}-3-methyl- butyrylamino)-acetic acid methyl ester
To a solution of 2 (286 mg, 0.32 mmol, 1eq) in MeCN (7mL) was added Et2NH (0.7mL, 10% v/v) dropwise at rt under Ar(g). The solution was stirred at rt for 2h, and the solvent was removed in vacuo. The excess of amine was co- evaporated with MeCN (3 x 5mL), and then with a 1 :5 mixture of CH2CI2/hexane (5mL). A white solid was obtained and was dried under high vacuum for 2h. To a solution of carboxylic acid 3 (140mg, 0.33 mmol, 1.1eq) in MeCN (5mL) at O0C was added PyBOP (181mg, 0.35 mmol, 1.1eq) and N-ethyldiisopropylamine (140 μL, 0.79 mmol, 2.5eq) under Ar(g). The crude amine, dissolved in CH2CI2 (5mL) was added in dropwise fashion, and the reaction mixture was then left to warm to rt overnight before being concentrated in vacuo and purified by silica gel column chromatography, eluting with EtOAc/MeOH (1 :0 -> 9:1); 4 was isolated as a white solid (254mg, 83%). 1H NMR (400MHz, CDCI3) δH: 8.39-8.44 (m, 2H), 7.54-7.59 (m, 1 H), 7.34-
7.43 (m, 13H), 7.27-7.31 (m, 8H), 7.25-7.27 (m, 4H), 7.18-7.24 (m, 6H), 7.14 (dd, J=7.8, 5.0Hz, 1 H), 7.00 (t, J=5.6Hz, 1 H), 6.55 (d, J=8.5Hz, 1 H), 6.42 (d, J=7.4Hz, 1 H), 5.39-5.48 (m, 1H), 5.29 (dd, J=15.4, 6.3Hz, 1 H), 4.66 (td, J=7.7, 5.2Hz, 1 H), 4.27-4.34 (m, 1H), 4.19 (dd, J=8.3, 6.1 Hz, 1H), 3.94 (dd, J=18.1 , 5.9Hz, 1 H), 3.84-3.90 (m, 1 H)1 3.78 (dd, J=18.1 , 5.6Hz, 1 H), 3.67 (s, 3H), 3.13 (dd, J=14.3, 4.8Hz, 1H), 2.99 (dd, J=14.3, 8.3Hz, 1 H), 2.62 (dd, J=13.0, 7.6Hz, 1H), 2.55 (dd, J=12.9, 6.4Hz, 1 H), 2.24-2.30 (m, 2H), 2.16-2.22 (m, 3H), 2.00- 2.07 (m, 3H), 0.91 (dd, J=6.6, 4.2Hz, 6H). MS (ES+) 1105.2 (100%, [M+Na]+). (5): (9S, 12fl,16S)-6-lsopropyl-12-pyridin-3-yimethyl-16-((E)-4-tritylsulfanyl-but-1 - enyl)-9-tritylsulf anylmethyl-1 -oxa-4,7, 10,13-tetraaza-cyclohexadecane- 2,5,8,11 ,14-pentaone
To a solution of 4 (254mg, 0.23 mmol, 1eq) in THF (8mL) at 00C was added a solution of LiOH (8.4mg, 0.35 mmol, 1.5eq) in H2O (2ml_) dropwise. The mixture was stirred for 2h, then quenched with 1 N HCI (2mL) and brine (1OmL). The organic layer was separated and the resulting aqueous layer was further extracted with EtOAc (2 x 15mL) and with CH2CI2 (15mL). The combined organic extracts were dried over MgSO4 and the solvent was removed in vacuo. The resulting carboxylic acid was then dried under high vacuum for 2h. To a solution of MNBA (97mg, 0.28mmol, 1.2eq) and DMAP (69mg, 0.56mmol, 2.4eq) in CH2CI2 (16OmL) was added a solution of the crude carboxylic acid in CH2CI2 (7OmL) and THF (1OmL) in dropwise fashion over 3h. The reaction mixture was then left to stir at rt overnight. The solvent was subsequently removed in vacuo and the resulting residue was purified by silica gel column chromatography with CH2CI2/MeOH (24:1 -> 19:1) to yield 5 as a white solid (172mg, 70%).
1H NMR (400MHz, CDCI3) δH: 8.36-8.43 (m, 2H), 7.28-7.43 (m, 24H), 7.17-7.26 (m, 12H), 7.07-7.13 (m, 1 H), 5.50-5.59 (m, 1 H), 5.35-5.42 (m, 1H), 5.29 (dd, J=15.3, 6.6Hz, 1 H), 4.29-4.43 (m, 2H), 3.56-3.65 (m, 1 H), 3.40 (dd, J=16.6, 2.1 Hz, 1 H), 2.91-3.09 (m, 3H), 2.63 (dd, J=12.6, 5.7Hz, 1 H), 2.46-2.53 (m, 1 H), 2.28-2.43 (m, 2H), 2.16 (t, J=7.2Hz, 2H), 1.94-2.06 (m, 2H), 0.93 (dd, J=17.8, 6.8Hz, 6H). MS (ES+) 1073.2 (100%, [M+Na]+).
Compound Ll: (E)-(I S,10S,21 F0-7-lsopropyl-21-pyridin-3-ylmethyl-2-oxa-12,13- dithia-5,8,20,23-tetraaza-bicyclo[8.7.6]tricos-16-ene-3,6,9, 19,22-pentaone To a solution of I2 (415mg, 1.64mmol, 10eq) in CH2CI2/MeOH (40OmL,
9:1) was added a solution of 5 (172mg, 0.16mmol, 1eq) dropwise over 2h at rt. The mixture was quenched with a solution of Na2S2O3 (0.1 M, 20OmL) and brine (1OmL). The organic layer was separated and the resulting aqueous layer was further extracted with extracted with CH2CI2 (2 x 5OmL) and EtOAc (5OmL). The combined organic extracts were dried over MgSO4 and the solvent was removed in vacuo. Purification by silica gel column chromatography using CH2CI2/MeOH (12:1 -> 9:1) as eluant furnished compound LI (44.0mg, 48%) as a white solid.
1H NMR (400MHz, CDCI3) δH: 8.45 (d, J=3.8Hz, 1 H), 8.39 (br. s., 1 H), 7.56 (d, J=7.3Hz, 1 H), 7.39 (d, J=6.3Hz, 1H), 7.27-7.34 (m, 1 H), 7.20-7.25 (m, 1H), 6.55 (br. s., 1 H), 5.77-5.91 (m, 1 H), 5.57-5.71 (m, 2H), 4.83 (t, J=6.6Hz, 1H)1 4.47-4.58 (m, 1 H), 3.96-4.17 (m, 2H), 3.41 (dd, JM5.0, 10.3Hz, 1 H), 3.14- 3.28 (m, 2H), 3.00-3.11 (m, 1 H), 2.58-2.97 (m, 7H), 2.54 (d, J=12.7Hz, 2H), 0.93 (d, J=6.3Hz, 3H), 0.86 (d, J=6.3Hz, 3H). MS (ES+) 586.8 (100%, [M+Na]+).

Claims

1. A compound of formula IX or X:
Figure imgf000116_0001
including pharmaceutically acceptable salts thereof; wherein:
X is -C(=O)N(R10)- or -CH(OPr3) -;
R7 and R9 are the same or different and represent hydrogen or a natural or unnatural amino acid side chain moiety; each Rio is the same or different and represents hydrogen, C1-C6 aikyl, C2-C6 alkenyl or C2-C6 alkynyl;
Pr1 and Pr2 are the same or different and represent hydrogen or a thiol protecting group; Pr3 is hydrogen or an alcohol protecting group;
R1 and R5 are the same or different and represent Y or a natural or unnatural amino acid side-chain moiety, provided that R1 and R5 are not both a natural or unnatural amino acid side chain moiety;
Y is -(CR11R11)X-NR11C(O)NR11R11, -(CR11Rn)x-NR11C(O)NR11R13, -(CR1 1 R1OX-NR1 1C(O)R14, -(CR11R11)X-NR11C(O)R13, -(CR11R11)X
-NR11SO2NR11R11, -(CR11R11)X-NR11SO2NR11R13, -(CR11R11)X-NR11SO3R14, -(CR11R11)X-NR11SO2R14, -(CR11R11)X-NR11SO2R13, -(CR11R11)X-C(O)NR11R11, -(CR11R11)X-C(O)NR11R13, -(CR11R11)X-C(O)R13, -(CR11R11)X-SO2NR11R11, -(CR11R11)X-SO2NR11R13, -(CR11R11)X-SO2Ri3, -(CR11Rn)x-Ar; x is an integer between 1 and 10;
R11 is hydrogen, C1-C6 aikyl, aryl, C2-C6 alkenyl, C2-C6 alkynyl or heteroaryl;
R13 is NR11-C(O)R14 or NR11-SO2R14;
R14 is C1-C6 aikyl, aryl, C2-C6 alkenyl, C2-C6 alkynyl or heteroaryl; and Ar is a heteroaryl or substituted aryl, with the proviso that: the compound is none of the compounds shown below:
Figure imgf000117_0001
Figure imgf000117_0002
(7) (7')
2. A compound according to claim 1 , wherein Y is a carbon-linked amide, sulfonamide, heteoaryl or substituted aryl,
3. A compound according to claim 1 or claim 2, wherein R1 is a natural or unnatural amino acid side-chain moiety, and wherein R5 is Y, wherein Y is as defined in claim 1or claim 2.
4. A compound according to any preceding claim, wherein the natural or unnatural amino acid side chain moiety is -CH3 (Alanine), -CH(CH3)2 (Valine), -CH2CH(CHa)2 (Leucine), -CH(CH3)CH2CH3 (Isoleucine), -(CH2)4NH2 (Lysine), -(CH2)3NHC(=NH)NH2 (Arginine), -CH2-(5-1 H-imidazolyl) (Histidine), -CH2CONH2 (Asparagine), -CH2CH2CONH2 (Glutamine), -CH2COOH (Aspartic acid), -CH2CH2COOH (Glutamic acid), -CH2--phenyl (Phenylalanine), -CH2-(4- OH-phenyl) (Tyrosine), -CH2-(3-1 H-indolyl) (Tryptophan), -CH2SH (Cysteine), -CH2CH2SCH3 (Methionine), -CH2OH (Serine), and -CH(OH)CH3 (Threonine), -(CH2)2-C(O)-O-C(CH3)3 (glutamic acid f-butyl ester), -(CH2J4-NH-C(O)-O- C(CH3)3 (Nε-(fert-butoxycarbonyl)-lysine), -(CH2)3-NH-C(O)NH2 (citruliine), -CH2- CH2OH (homoserine) and -(CH2)3NH2 (ornithine), -H(Glycine), C1-C6 aikyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or a saturated or unsaturated heterocycle which can be functionalized or unfunctionalized.
5. A compound according to claim 4, wherein the natural or unnatural amino acid side chain moiety is -H (Glycine), -CH3 (Alanine), -CH(CH3)2 (Valine), -CH2CH(CHg)2 (Leucine), -CH(CH3)CH2CH3 (Isoleucine), -(CH2)4NH2 (Lysine), -(CH2)3NHC(=NH)NH2 (Arginine), -CH2-(5-1 H-imidazolyl) (Histidine), -CH2CONH2 (Asparagine), -CH2CH2CONH2 (Glutamine), -CH2COOH (Aspartic acid), -CH2CH2COOH (Glutamic acid), -CH2-phenyl (Phenylalanine), -CH2-(4- OH-phenyl) (Tyrosine), -CH2-(3-1 W-indolyl) (Tryptophan), -CH2SH (Cysteine), -CH2CH2SCH3 (Methionine), -CH2OH (Serine), or -CH(OH)CH3 (Threonine).
6. A compound according to any preceding claim, wherein X is -CH(OPr3)-.
7. A compound according to claim 6, which is a compound shown below:
Figure imgf000118_0001
Compound XIII Compound XIV
Figure imgf000119_0001
Compound XV Compound XVI
Figure imgf000119_0002
Compound XVIII
Figure imgf000119_0003
Compound XIX Compound XX
Figure imgf000119_0004
Compound XXI Compound XXII
Figure imgf000120_0001
Compound XXIIi Compound XXIV
Figure imgf000120_0002
Compound XXV Compound XXVI
Figure imgf000120_0003
Compound XXVII Compound XXVIII
Figure imgf000120_0004
Compound XXIX Compound XXX
Figure imgf000121_0001
Compound XXXI Compound XXXII
Figure imgf000121_0002
Compound XXXIII Compound XXXIV
Figure imgf000121_0003
Compound XXXV
8. A compound according to any of claims 1 to 5, wherein X is -C(=O)N(R10)-.
9. A compound according to claim 8, which is a compound shown below:
Figure imgf000121_0004
Compound XXXVI Compound XXXVII
Figure imgf000122_0001
Compound XXXVIII Compound XXXIX
Figure imgf000122_0002
Compound XLII Compound XLIII
Figure imgf000122_0003
Compound XLIV
Figure imgf000123_0001
Compound XLV Compound XLVI
Figure imgf000123_0002
Compound XLVII Compound XLVIII
Figure imgf000123_0003
Compound XLIX
Figure imgf000123_0004
Compound L Compound LI
10. A compound according to any preceding claim, for use in therapy.
11. An compound according to any preceding claim, for use in the treatment or prevention of a condition mediated by histone deacetylate (HDAC).
12. A compound analogue according to claim 11 , wherein the condition is cancer, cardiac hypertrophy, chronic heart failure, an inflammatory condition, a cardiovascular disease, a haemoglobinopathy, a thalassemia, a sickle cell disease, a CNS disorder, an autoimmune disease, diabetes, osteoporosis, MDS, benign prostatic hyperplasia, endometriosis, oral leukoplakia, a genentically related metabolic disorder, an infection, Rubens-Taybi, fragile X syndrome, or alpha- 1 antitrypsin deficiency.
13. A compound according to claim 11 or claim 12, wherein the condition is chronic lymphocytic leukaemia, breast cancer, prostate cancer, ovarian cancer, mesothelioma, T-cell lymphoma, cardiac hypertrophy, chronic heart failure or a skin inflammatory condition, in particular psoriasis, acne or eczema.
14. A compound according to any of claims 1 to 9, for use in accelerating wound healing, protecting hair follicles, or as an immunosuppressant.
15. A pharmaceutical composition comprising a compound according to any of claims 1 to 9 and a pharmaceutically acceptable carrier or diluent.
16. A composition according to claim 15, which is in a form suitable for oral, rectal, parenteral, intranasal or transdermal administration or administration by inhalation or by suppository.
17. A composition according to claim 16, which is in the form of granules or a tablet, e.g. a sub-lingual tablet, capsule, troche, lozenge, aqueous or oily suspension, or dispersible powder.
18. A product containing (a) a compound according to any of claims 1 to 9, and (b) another inhibitor of HDAC, for simultaneous, separate or sequential use in therapy.
19. A product according to claim 18, wherein the therapy is of a condition mediated by HDAC.
20. A product containing (a) a compound according to any of claims 1 to 10, and (b) a chemotherapeutic or antineoplastic agent, for simultaneous, separate or sequential use in therapy.
21. A product according to claim 20, wherein the therapy is of cancer.
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WO2010116173A1 (en) 2009-04-06 2010-10-14 Karus Therapeutics Limited Depsipeptides and their therapeutic use
WO2012045804A1 (en) 2010-10-08 2012-04-12 Vib Vzw Hdac inhibitors to treat charcot-marie-tooth disease
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WO2017122822A1 (en) * 2016-01-13 2017-07-20 国立大学法人東北大学 Production intermediate of depsipeptide compound, and method for producing same
CN113861267A (en) * 2021-10-25 2021-12-31 深圳湾实验室坪山生物医药研发转化中心 Depsipeptide compound LZG-PKU-H and synthesis method and application thereof
CN113861267B (en) * 2021-10-25 2023-06-27 深圳湾实验室坪山生物医药研发转化中心 Depsipeptide compound LZG-PKU-H and synthetic method and application thereof

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